Sulfonamide 18 beta-glycyrrhetinic acid derivative and preparation method and application thereof
阅读说明：本技术 磺酰胺类18β-甘草次酸衍生物及其制备方法和应用 (Sulfonamide 18 beta-glycyrrhetinic acid derivative and preparation method and application thereof ) 是由 怀其勇 李伊 于 2021-09-17 设计创作，主要内容包括：本发明提供一种磺酰胺类18β-甘草次酸衍生物及其制备方法和应用,属于医药技术领域。本发明设计并合成了磺酰胺基修饰的18β-甘草次酸的系列衍生物。通过引入活性磺酰胺基,能够显著改善化合物的水溶性和生物利用度,以提高相关衍生物的抗肿瘤生物活性。经试验证明,化合物6a对3种人类癌细胞(MCF-7,A549,HEPG2)均具有极佳的细胞毒活性,且远远高于母体药物GA,同时化合物6a相比阳性对照抗癌药物吉非替尼的活性亦有显著地提高。表明该化合物有望成为替代吉非替尼的前药,可以应对癌症患者对吉非替尼的耐药性。因此具有良好的实际应用之价值。(The invention provides a sulfonamide 18 beta-glycyrrhetinic acid derivative and a preparation method and application thereof, belonging to the technical field of medicines. The invention designs and synthesizes a series of sulfamide modified 18 beta-glycyrrhetinic acid derivatives. By introducing active sulfonamide, the water solubility and bioavailability of the compound can be obviously improved, so that the antitumor bioactivity of the related derivative is improved. Experiments prove that the compound 6a has excellent cytotoxic activity on 3 human cancer cells (MCF-7, A549 and HEPG2), is far higher than that of a parent drug GA, and simultaneously, the activity of the compound 6a is remarkably improved compared with that of a positive control anti-cancer drug gefitinib. The compound is expected to be a prodrug for replacing gefitinib, and can cope with the drug resistance of cancer patients to gefitinib. Therefore, it has good practical application value.)
1. A sulfonamide 18 beta-glycyrrhetinic acid derivative is characterized in that the structural formula is shown as a formula I:
wherein R is1Independently selected from H, CH3；
R2Independently selected from CH3，CH2CH2CH3，CH2CH2CH2CH3。
2. The sulfonamide 18 β -glycyrrhetinic acid derivatives according to claim 1, wherein the sulfonamide 18 β -glycyrrhetinic acid derivatives are selected from the group consisting of:
Benzyl-3-((N,N-dimethylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate；
Benzyl-3-((N-propylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate；
Benzyl-3-((N-butylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate。
3. the process for preparing the sulfonamide 18 β -glycyrrhetinic acid derivatives according to claim 1 or 2, characterized in that the synthetic route is as follows:
4. the method of claim 3, wherein the reaction reagents and conditions comprise:
(a) benzyl bromide, K2CO3DMF, room temperature, overnight;
(b) PCC, DCM, silica gel, room temperature, 4 h;
(c)CH3COONH4，NaCNBH3MeOH, room temperature, overnight;
(d) triethylamine, DCM, room temperature, overnight;
(e) triethylamine, DCM, room temperature, overnight.
5. The method of claim 3, further comprising isolating and purifying the product.
6. The method according to claim 5, wherein the separation and purification specifically comprises: carrying out column chromatography separation on the crude product of the reaction product; preferably, silica gel is used as a stationary phase, and n-hexane and ethyl acetate are used as eluents for elution; mixing the organic phases, concentrating, and drying.
7. A pharmaceutical composition comprising the sulfonamide 18 β -glycyrrhetinic acid derivatives of claim 1 or 2.
8. A pharmaceutical preparation, characterized in that it comprises the sulfonamide 18 β -glycyrrhetinic acid derivatives according to claim 1 or 2 and at least one pharmaceutically acceptable adjuvant or carrier.
9. Use of the sulfonamide 18 β -glycyrrhetinic acid derivatives according to claim 1 or 2, the pharmaceutical compositions according to claim 7, and/or the pharmaceutical preparations according to claim 8 for the preparation of antitumor drugs.
10. A method for treating cancer, which comprises administering to a subject a therapeutically effective amount of the sulfonamide 18 β -glycyrrhetinic acid derivative of claim 1 or 2, the pharmaceutical composition of claim 7, and/or the pharmaceutical formulation of claim 8.
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to sulfonamide 18 beta-glycyrrhetinic acid derivatives, and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Glycyrrhetinic acid is the main ingredient of glycyrrhizic acid in traditional Chinese medicine Glycyrrhrizae radix. According to the different conformation of 18-position hydrogen atom in glycyrrhetinic acid, glycyrrhetinic acid is divided into 18 alpha-glycyrrhetinic acid and 18 beta-glycyrrhetinic acid, wherein the 18 beta-glycyrrhetinic acid is the main active component of glycyrrhizic acid. The 18 beta-glycyrrhetinic acid is an oleanane type pentacyclic triterpenoid compound, and has a plurality of excellent pharmacological activities, such as antitumor activity, antiviral activity, anti-inflammatory activity, anti-ulcer activity, liver protection activity, antioxidant activity and the like. In recent years, research on antitumor drugs is very extensive, and 18 beta-glycyrrhetinic acid attracts people's attention with its excellent antitumor biological activities of resisting cancer cell proliferation and promoting cancer cell apoptosis. However, the compound has the defects of low hydrophobicity, low bioavailability and the like, and the bioactivity has a certain distance from the clinical anticancer drug, so that the structural modification of the 18 beta-glycyrrhetinic acid is needed to improve and enhance the antitumor activity of the related compound. According to the research of structural modification in recent decades, the structural modification sites of 18 β -glycyrrhetinic acid are mainly concentrated on the hydroxyl at the 3-position of the A ring, the carbonyl at the 11-position of the C ring and the carboxyl at the 30-position of the E ring.
Sulfonamides are the first class of antibacterial drugs synthesized by humans and have milestone significance in the development history of antibiotics. Further research shows that the sulfonamide compounds also have the activities of resisting tumors, diabetes, inflammation, viruses and the like. In particular, in recent years, many sulfonamide compounds have been reported to have good antitumor activity, and some of the compounds have entered clinical trials. The action mechanisms of the related derivatives are different, and comprise interference on tubulin polymerization, obstruction on normal cell cycle, inhibition on carbonic anhydrase, folic acid dependent enzyme, cyclooxygenase-2, aromatizing enzyme, methionyl aminopeptidase and histone deacetylase, inhibition on vascular endothelial cell growth factor and the like. Therefore, the sulfonamide group becomes an important functional group in organic synthesis and drug design, and has good application potential.
Disclosure of Invention
The invention provides a sulfonamide 18 beta-glycyrrhetinic acid derivative and a preparation method and application thereof, and the invention can remarkably improve the water solubility and bioavailability of a compound by introducing active sulfonamide on 3-position hydroxyl of 18 beta-glycyrrhetinic acid so as to improve the anti-tumor bioactivity of related derivatives, thereby having good value of practical application.
Specifically, the invention relates to the following technical scheme:
the invention provides a sulfonamide 18 beta-glycyrrhetinic acid derivative, which has a structural formula shown in a formula I:
wherein R is1Independently selected from H, CH3；
R2Independently selected from CH3，CH2CH2CH3，CH2CH2CH2CH3。
Specifically, the sulfonamide 18 beta-glycyrrhetinic acid derivative is selected from the following compounds:
Benzyl-3-((N,N-dimethylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate(6a)
Benzyl-3-((N-propylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate(6b)
Benzyl-3-((N-butylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate(6c)
according to experimental research, the compound 6a has excellent cytotoxic activity on 3 human cancer cells (MCF-7, A549 and HEPG2), and is far higher than that of a parent drug GA, and the activity of the compound 6a is remarkably improved compared with that of a positive control anti-cancer drug gefitinib. The compound is expected to be a prodrug for replacing gefitinib, and can cope with the drug resistance of cancer patients to gefitinib.
The second aspect of the present invention provides a preparation method of the above sulfonamide 18 β -glycyrrhetinic acid derivative, specifically, the synthetic route is as follows:
wherein, the reaction reagent and the conditions are as follows:
(a) benzyl bromide, K2CO3DMF, room temperature, overnight;
(b) PCC, DCM, silica gel, room temperature, 4 h;
(c)CH3COONH4，NaCNBH3MeOH, room temperature, overnight;
(d) triethylamine, DCM, room temperature, overnight;
(e) triethylamine, DCM, room temperature, overnight.
In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the sulfonamide 18 β -glycyrrhetinic acid derivatives as described in the first aspect above.
In a fourth aspect of the present invention, the present invention provides a pharmaceutical preparation, which comprises the sulfonamide 18 β -glycyrrhetinic acid derivatives described in the first aspect above and at least one pharmaceutically acceptable adjuvant or carrier.
The pharmaceutical carrier may be a liquid or a solid; the pharmaceutical preparation can be oral preparation and parenteral preparation, and can be tablets, pills, capsules or injections.
In a fifth aspect of the present invention, the present invention provides an application of the sulfonamide 18 β -glycyrrhetinic acid derivative of the first aspect, the pharmaceutical composition of the third aspect, or the pharmaceutical preparation of the fourth aspect in preparing an antitumor drug.
In a sixth aspect of the present invention, there is provided a method for treating cancer, which comprises administering to a subject a therapeutically effective amount of the sulfonamide 18 β -glycyrrhetinic acid derivatives according to the above first aspect of the present invention or a pharmaceutical composition or pharmaceutical preparation comprising the same. The cancer is selected from breast cancer, lung cancer and liver cancer.
The beneficial technical effects of one or more technical schemes are as follows:
the technical proposal designs and synthesizes the serial derivatives of the 18 beta-glycyrrhetinic acid modified by the sulfonamide group. The water solubility and bioavailability of the compound can be obviously improved by introducing the active sulfamide, so that the anti-tumor bioactivity of the related derivatives is improved, tests prove that the compound 6a has excellent cytotoxic activity on 3 human cancer cells (MCF-7, A549 and HEPG2) and is far higher than that of a parent drug GA, and simultaneously, the activity of the compound 6a is obviously improved compared with that of a positive control anti-cancer drug gefitinib. The compound is expected to be a prodrug for replacing gefitinib, and can cope with the drug resistance of cancer patients to gefitinib. Therefore, it has good practical application value.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. The experimental procedures, if specific conditions are not indicated in the following detailed description, are generally in accordance with conventional procedures and conditions of molecular biology within the skill of the art, which are fully explained in the literature.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.
In a typical embodiment of the present invention, a sulfonamide 18 β -glycyrrhetinic acid derivative is provided, which has a structural formula shown in formula I:
wherein R is1Independently selected from H, CH3；
R2Independently selected from CH3，CH2CH2CH3，CH2CH2CH2CH3。
In one embodiment of the present invention, the sulfonamide 18 β -glycyrrhetinic acid derivative is selected from the following compounds:
Benzyl-3-((N,N-dimethylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate(6a)
Benzyl-3-((N-propylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate(6b)
Benzyl-3-((N-butylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate(6c)
according to experimental research, the compound 6a has excellent cytotoxic activity on 3 human cancer cells (MCF-7, A549 and HEPG2), and is far higher than that of a parent drug GA, and the activity of the compound 6a is remarkably improved compared with that of a positive control anti-cancer drug gefitinib. The compound is expected to be a prodrug for replacing gefitinib, and can cope with the drug resistance of cancer patients to gefitinib.
In a specific embodiment of the present invention, a preparation method of the above sulfonamide 18 β -glycyrrhetinic acid derivative is provided, and specifically, a synthetic route thereof is as follows:
wherein, the reaction reagent and the conditions are as follows:
(a) benzyl bromide, K2CO3DMF, room temperature, overnight;
(b) PCC, DCM, silica gel, room temperature, 4 h;
(c)CH3COONH4，NaCNBH3MeOH, room temperature, overnight;
(d) triethylamine, DCM, room temperature, overnight;
(e) triethylamine, DCM, room temperature, overnight.
It should be noted that, in this documentThe biggest difficulty encountered in the synthesis process of the compound is that the 3-hydroxyl structure of the 18 beta-glycyrrhetinic acid is transformed into a part of amino. The invention firstly adopts the reaction of the 3-carbonyl of the compound 3 and hydroxylamine hydrochloride to prepare the oxime compound. The oxime compound is then reacted in methanol solution with ammonium acetate, 15% TiCl3The aqueous solution and sodium cyanoborohydride are reduced to produce compound 4. However, the results of TLC monitoring of the reaction are not ideal, the product is rich in impurities and the reaction is unstable.
Therefore, the synthesis method is improved, firstly, the 3-bit carbonyl of the compound 3 and ammonium acetate are subjected to nucleophilic addition reaction to obtain a key intermediate product with 3-bit substituted by imine. However, this intermediate is unstable and the imine is reduced to the amine group directly by sodium cyanoborohydride in a one-pot process. In the imine reduction process, compound 2 can also be isolated by silica gel column chromatography.
In addition, the invention also explores a synthetic route of the ethylamine sulfonamide derivative. As only ethylamine aqueous solution is commercially available, under the reaction condition d, water in the ethylamine aqueous solution can hydrolyze sulfonyl chloride reagent in the reaction system to decompose the sulfonyl chloride reagent to obtain hydrochloric acid and sulfuric acid. TLC monitoring of the reaction also showed no target product formation.
In another embodiment of the present invention, the preparation method further comprises the step of separating and purifying the product.
The separation and purification specifically comprises: and carrying out column chromatography separation on the crude product of the reaction product. Specifically, silica gel is used as a stationary phase, and n-hexane and ethyl acetate are used as eluents for elution; mixing the organic phases, concentrating, and drying.
Wherein the ratio of the n-hexane to the ethyl acetate is 6-10: 1, such as 8: 1.
In a specific embodiment of the present invention, there is provided a pharmaceutical composition comprising the sulfonamide 18 β -glycyrrhetinic acid derivatives as described in the first aspect above.
In a specific embodiment of the present invention, the present invention provides a pharmaceutical preparation, which comprises the sulfonamide 18 β -glycyrrhetinic acid derivatives described in the first aspect above and at least one pharmaceutically acceptable adjuvant or carrier.
The pharmaceutical carrier may be a liquid or a solid; the pharmaceutical preparation can be oral preparation and parenteral preparation, and can be tablets, pills, capsules or injections.
The compounds of the present invention may be formulated into pharmaceutical compositions or pharmaceutical formulations using techniques well known to those skilled in the art. In addition to those mentioned herein, suitable pharmaceutical excipients are known in the art, see for example the 2005 edition handbook of pharmaceutical excipients (fourth edition original works).
In a specific embodiment of the present invention, the present invention provides an application of the sulfonamide 18 β -glycyrrhetinic acid derivative described in the first aspect, or the pharmaceutical composition described in the third aspect, or the pharmaceutical preparation described in the fourth aspect, in preparing an antitumor drug.
It is noted that tumors are used in the present invention as known to those skilled in the art, and include benign tumors and/or malignant tumors. Benign tumors are defined as cellular hyperproliferation that fails to form aggressive, metastatic tumors in vivo. Conversely, a malignant tumor is defined as a cell with various cellular and biochemical abnormalities capable of forming a systemic disease (e.g., forming tumor metastases in distant organs).
The medicine of the present invention may be used in treating malignant tumor. Examples of malignant tumors that can be treated with the drug of the present invention include solid tumors and hematological tumors. Solid tumors may be tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (such as thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testis, stomach, skin (such as melanoma), ureter, vagina and vulva. Malignant tumors include hereditary cancers such as retinoblastoma and Wilmstumor. Furthermore, malignant tumors include primary tumors in the organs and corresponding secondary tumors in distant organs (tumor metastases). Hematological tumors can be aggressive and indolent forms of leukemia and lymphoma, i.e., non-hodgkin's disease, chronic and acute myeloid leukemia (CML/AML), Acute Lymphocytic Leukemia (ALL), hodgkin's disease, multiple myeloma, and T-cell type lymphoma. Also included are myelodysplastic syndromes, plasmacytomas, carcinoid syndromes, and cancers of unknown primary site and AIDS-related malignancies.
In a specific embodiment of the present invention, the present invention provides a method for treating cancer, which comprises administering to a subject a therapeutically effective amount of the sulfonamide 18 β -glycyrrhetinic acid derivatives according to the above first aspect of the present invention or a pharmaceutical composition or pharmaceutical preparation comprising the same. The cancer is selected from breast cancer, lung cancer and liver cancer.
The subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
By "therapeutically effective amount" is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.
The range of therapeutically effective amounts that can be used will be known to the researcher, veterinarian, medical doctor or other medical professional in the art based on clinical trials or other means known in the art.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
First, compound design and synthesis
1. Structure and synthesis route of compound
3 kinds of 6a-6c sulfonamide 18 beta-glycyrrhetinic acid derivatives are designed and synthesized. The relevant synthetic routes are as follows:
wherein the names of the compounds 6a-6c are respectively as follows:
6a：Benzyl-3-((N,N-dimethylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate；
6b：Benzyl-3-((N-propylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate；
6c：Benzyl-3-((N-butylsulfamoyl)amino)-11-oxo-olean-12-en-30-oate；
reaction reagents and conditions:
(a) benzyl bromide, K2CO3DMF, r.t., overnight;
(b) PCC, DCM, silica gel, r.t., 4 h;
(c)CH3COONH4，NaCNBH3MeOH, r.t., overnight;
(d) triethylamine, DCM, r.t., overnight;
(e) triethylamine, DCM, r.t., overnight.
2. Laboratory apparatus and reagent
(1) The instrument comprises the following steps: a circulating water type multi-purpose vacuum pump (SHK-III), a three-purpose ultraviolet analyzer (ZF-1), a constant temperature heating magnetic stirrer (DF-101Z), a rotary evaporator (N-1001), an electronic balance (WT-2002), a nuclear magnetic resonance instrument (Bruker) and a GF254 silica gel plate (Xincheng silica gel materials Co., Ltd.).
(2) Experimental reagent: glycyrrhetinic acid (beta form) 97% AR; benzyl bromide 98% AR; PCC 98% AR; 99% AR ammonium acetate; sodium cyanoborohydride 95% AR; n, N-dimethylaminosulfonyl chloride 97% AR; propylamine 98% AR; butylamine 98% AR; sulfonyl chloride 98% AR; potassium carbonate 99% AR; anhydrous sodium sulfate 99% AR; sodium hydroxide 98% AR; DMF 99.8% AR; pyridine 99.5% AR; triethylamine 99% AR; methanol 99.5% AR; dichloromethane 99.5% AR; ethyl acetate 99.5% AR; n-hexane 97% AR.
3. Method for synthesizing compound
The following compounds were synthesized according to the above synthetic route starting from 18 β -Glycyrrhetinic Acid (GA), compound 1.
(1) Synthesis of Compound 2:
compound 1(5mmol, 2.35g), anhydrous potassium carbonate (15mmol, 2.50g), DMF (10mL), and troche were added to a 50mL round bottom flask, stirred at room temperature for 30min, then benzyl bromide (7.5mmol, 1.0mL) was added with stirring, and the reaction was allowed to react at room temperature for 8h, and the progress of the reaction was monitored by TLC. After the reaction was complete, it was diluted with ethyl acetate (30mL) and washed 3 times with deionized water. And drying the organic phase by using anhydrous sodium sulfate, and then carrying out suction filtration and rotary evaporation to obtain a crude product of the compound 2. And (3) carrying out column chromatography separation on the crude product, taking silica gel as a stationary phase, and taking n-hexane: ethyl acetate 8:1/4:1, compound 2 at 4: 1. And combining organic phases, concentrating and drying to obtain the compound 2.
(2) Synthesis of Compound 3:
compound 2(4mmol, 2.24g), silica gel (1.92g), dichloromethane (15mL), and a rotor were charged into a 50mL round-bottomed flask, and the mixture was stirred at 0 ℃ to dissolve the compound, and then a suspension of PCC (6mmol, 1.28g) and dichloromethane (10mL) was added dropwise to the reaction system in an ice bath, followed by reaction at room temperature for 5 hours, and the progress of the reaction was monitored by TLC. After the reaction is finished, removing the silica gel in the reaction system by vacuum filtration, and washing the organic phase obtained by the vacuum filtration with deionized water for 3 times. And drying the organic phase by using anhydrous sodium sulfate, and then carrying out suction filtration and rotary evaporation to obtain a crude product of the compound 3. And (3) carrying out column chromatography separation on the crude product, taking silica gel as a stationary phase, and taking n-hexane: eluent of ethyl acetate ═ 8: 1. And combining organic phases, concentrating and drying to obtain a compound 3.
(3) Synthesis of Compound 4:
compound 3(4mmol, 2.24g), ammonium acetate (40mmol, 3.12g), methanol (15mL) were added to a 50mL round bottom flask, stirred at room temperature for 2h, followed by addition of sodium cyanoborocyanide (20mmol, 1.28g), and reacted under nitrogen at room temperature overnight with TLC to monitor the progress of the reaction. After the reaction is finished, diluting with 3 times of water, adjusting the pH value of the solution to 9-10 by using 2N sodium hydroxide, filtering and drying to obtain a crude product of the compound 4. And (3) carrying out column chromatography separation on the crude product, taking silica gel as a stationary phase, and taking n-hexane: ethyl acetate 8:1/6:1/4: 1. And combining organic phases, concentrating and drying to obtain a compound 4.
(4) Synthesis of compounds 5a, 5b, 5 c:
compound 5a is commercially available as a relevant reagent and therefore is not synthesized anymore.
Sulfonyl chloride (8mmol, 0.64mL), dichloromethane (5mL) and a rotator were added to a 25mL round bottom flask, a mixture of propylamine (8mmol, 0.66mL) or butylamine (8mmol, 0.79mL) and triethylamine (8mmol, 1.1mL) was slowly dropped into the reaction system under ice-water bath, and the reaction was carried out overnight at room temperature, and progress of the reaction was monitored by TLC. After the reaction is finished, the solvent is removed by rotary evaporation to obtain a compound 5b or 5 c.
(5) Synthesis of compounds 6a, 6b, 6 c:
compound 4(4mmol, 2.24g), compounds 5a-5c (4mmol), dichloromethane (10mL), and the rotor were added to a 50mL round bottom flask, stirred for a while, triethylamine (8mmol, 1.12mL) was added, the reaction was allowed to proceed overnight at room temperature, and the progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with dichloromethane (20mL) and washed 3 times with deionized water. The organic phase is dried by anhydrous sodium sulfate, and then is filtered, filtered and rotary evaporated to obtain the crude products of the compounds 6a-6 c. And (3) carrying out column chromatography separation on the crude product, taking silica gel as a stationary phase, and taking n-hexane: eluent of ethyl acetate ═ 8: 1. And combining organic phases, concentrating and drying to obtain the target compounds 6a-6 c.
4. Structural characterization of target Compounds 6a-6c
(1) Melting point, hydrogen spectrum and carbon spectrum data of compound 6a
mp:150.0～151.8℃.1H NMR(400MHz,CDCl3):δ(ppm)7.52-7.31(m,5H,H-Ar),5.54(s,1H,H-12),5.19(d,1H,Bn-CH2,J＝12.2Hz),5.08(d,1H,Bn-CH2,J＝12.2Hz),4.31-4.28(m,1H,N-H),3.07(d,1H,H-3,J＝9.5Hz),2.75(s,6H,N(CH3)2) 2.42(s,1H, H-9),1.39,1.15,1.13,1.09,1.00,0.94,0.72(s,21H, in order H-27, 28, 25, 26, 23, 24, 29),2.06-1.51,1.37-1.27,0.89-0.85(m,20H).
13C NMR(101MHz,CDCl3):δ(ppm)200.2,176.2,169.5,136.1,128.5,128.5,128.3,128.2,128.2,128.2,66.1,61.5,59.5,50.0,48.2,45.4,43.9,43.3,41.0,38.0,38.0,37.6,37.0,37.0,34.1,32.4,31.7,31.1,29.6,29.1,28.4,28.2,26.3,23.6,23.3,23.2,18.6,17.2,16.6.
(2) Melting Point, Hydrogen Spectroscopy, carbon Spectroscopy data of Compound 6b
mp:100.5～102.6℃.1H NMR(400MHz,CDCl3):δ(ppm)7.38-7.31(m,5H,H-Ar),5.53(s,1H,H-12),5.19(d,1H,Bn-CH2,J＝12.2Hz),5.07(d,1H,Bn-CH2,J＝12.2Hz),4.33(t,1H,NH-CH2J6.2 Hz),4.20(d,1H, N-H, J6.2 Hz),3.00(q,2H, J6.9 Hz),3.94-2.88(m,1H, H-3),2.31(s,1H, H-9),1.37,1.15,1.11,1.09,1.02,0.76,0.72(s,21H, in order H-27, 28, 25, 26, 23, 24, 29),2.03-1.37,1.31-1.16,0.96-0.80(m,24H).
13C NMR(101MHz,CDCl3):δ(ppm)200.0,176.2,169.2,136.1,128.6,128.6,128.5,128.3,128.3,128.3,66.2,62.2,61.7,56.0,48.2,45.3,45.1,44.0,43.2,41.1,39.9,38.3,37.7,36.8,32.7,31.8,31.6,31.2,28.3,26.4,26.3,23.3,22.9,22.65,18.7,18.0,16.5,16.2,14.1,11.3.
(3) Melting Point, Hydrogen Spectroscopy, carbon Spectroscopy data of Compound 6c
mp:99.2～102.4℃.1H NMR(400MHz,CDCl3):δ(ppm)7.45–7.28(m,5H,H-Ar),5.54(s,1H,H-12),5.19(d,1H,Bn-CH2,J＝12.3Hz),5.08(d,1H,Bn-CH2,J＝12.2Hz),4.25(t,1H,NH-CH2J ═ 6.2Hz),4.15(d,1H, N-H, J ═ 9.9Hz),2.32(s,1H, H-9),2.01,1.62,1.56, 1.42-1.34, 1.26,1.15,1.10,1.02,0.89,0.74, as saturated C — H moieties.
13C NMR(101MHz,CDCl3):δ199.98,176.20,169.15,136.13,128.61,128.46,128.30,128.25,66.23,62.17,61.65,55.97,48.22,45.29,43.99,43.17,43.11,41.06,39.88,38.33,37.65,36.82,32.69,31.78,31.66,31.59,28.42,28.33,28.28,26.44,26.39,26.27,23.29,22.65,19.94,18.65,17.96,16.45,16.22,14.13,13.71.
Second, compound cytotoxic activity assay
MTT screening assay
1. The purpose is as follows: IC of test sample on human breast cancer cell strain, human lung cancer cell strain, human liver cancer cell strain and human normal liver cell50And (6) primary screening.
2. The main apparatus is as follows: enzyme mark instrument (VERSA max microplate reader, MD, USA)
3. Cell line: human breast cancer cells: MCF-7
Human lung cancer cells: a549
Human liver cancer cell: HEPG-2
Human normal hepatocytes: LO2
4. Compounding of liquid medicine and reagent
(1) A compound: 10mM stock solution, subpackaged, and stored at-20 ℃ for later use.
(2) Positive control: gefitinib. The stock was dissolved in cell-grade DMSO to 10mM and stored at-20 ℃ until use.
(3) Other reagents: MTT (M2128, Sigma, USA), DMSO (cell culture grade CAS-NO:67-68-5, Applichem, Germany), DMSO (analytical grade, batch number: 20151102, national drug group, China); fetal bovine serum (Gibco, South America); medium (Hyclone, USA);
5. the experimental method comprises the following steps:
inoculating cells in 96-well plate at cell density of 3-4 × 103A hole. Placing in 5% CO2Culturing in a 37 ℃ cell culture box, adding a sample to be detected with specified concentration after the cells adhere to the wall, wherein the negative control group is DMSO with the same concentration, and the drug with the same concentration is provided with three parallel holes. Adding drug for 48h, adding 20 μ L MTT (5mg/mL) per well, culturing for 4h, removing supernatant by suction pump, adding 150 μ L DMSO, detecting OD value of each well at 570nm with microplate reader, and using IC50Software (Prism 8.0) computing IC50The value is obtained. The experiment was repeated three times.
6. Statistical analysis
IC with 3 times of independent calculation50Statistics were performed and expressed as mean soil standard deviation (s.d).
7. The results are shown in Table 1.
TABLE 1 cytotoxicity (IC) of samples against six cell lines50/μM)
Third, experimental results and discussion
1. Synthetic part of the Compound
In the synthetic part of the experiment, the greatest difficulty encountered was the structural modification of the hydroxyl group at position 3 of 18 β -glycyrrhetinic acid to the part of the amine group. The experimental group firstly adopts the reaction of the 3-carbonyl of the compound 3 and hydroxylamine hydrochloride to prepare the oxime compound. The oxime compound is then reacted in methanol solution with ammonium acetate, 15% TiCl3The aqueous solution and sodium cyanoborohydride are reduced to produce compound 4. However, the results of TLC monitoring of the reaction are not ideal, the product is rich in impurities and the reaction is unstable.
Therefore, the experimental group improves the synthesis method, firstly, the 3-bit carbonyl of the compound 3 and ammonium acetate undergo nucleophilic addition reaction to obtain a key intermediate product with 3-bit substituted by imine. However, this intermediate is unstable, so the experimental group directly used a one-pot method to reduce imine to amine group by sodium cyanoborohydride. In the imine reduction process, compound 2 can also be isolated by silica gel column chromatography.
In addition, the experimental group also explored the synthetic route of the ethylamine sulfonamide derivatives. As only ethylamine aqueous solution is commercially available, under the reaction condition d, water in the ethylamine aqueous solution can hydrolyze sulfonyl chloride reagent in the reaction system to decompose the sulfonyl chloride reagent to obtain hydrochloric acid and sulfuric acid. TLC monitoring of the reaction also showed no formation of the target product, and the synthesis of this compound was left to further study.
2. Active part of compound
The experimental group tests the cytotoxic activity of 6a-6c and 3 sulfonamide 18 beta-glycyrrhetinic acid derivatives on MCF-7, A549 and HEPG2 and 3 human cancer cells and normal human liver cells LO2, and compares the cytotoxic activity with the activity of a positive control anti-cancer drug gefitinib.
The results show that compound 6a has the best activity and excellent cytotoxic activity against all 3 human cancer cells tested. The activity of the compound 6a is far higher than that of the parent drug GA, and more importantly, the activity of the compound 6a is obviously improved compared with that of the positive control anticancer drug gefitinib. The compound is expected to be a prodrug for replacing gefitinib, and can cope with the drug resistance of cancer patients to gefitinib.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.