CN116874519B - Andrographolide modified compound H4 and preparation method and application thereof - Google Patents

Andrographolide modified compound H4 and preparation method and application thereof Download PDF

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CN116874519B
CN116874519B CN202311141800.0A CN202311141800A CN116874519B CN 116874519 B CN116874519 B CN 116874519B CN 202311141800 A CN202311141800 A CN 202311141800A CN 116874519 B CN116874519 B CN 116874519B
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andrographolide
compound
cells
hours
reforming
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CN116874519A (en
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胡元会
崔翰明
李全
吕梦
朱雪萍
尤雅萍
张秋燕
吴华芹
杜柏
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Guanganmen Hospital of CACMS
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Abstract

The invention relates to the technical field of pharmaceutical chemistry, in particular to an andrographolide modified compound H4, a preparation method and application thereof. The andrographolide modified compound H4 provided by the invention has the effect of treating cancers, and the research of the invention shows that compared with andrographolide, the andrographolide modified compound H4 has more remarkable effect of inhibiting tumors, and can be used for preparing medicaments for treating cancers.

Description

Andrographolide modified compound H4 and preparation method and application thereof
Technical Field
The invention relates to the technical field of pharmaceutical chemistry, in particular to an andrographolide modified compound H4, a preparation method and application thereof.
Background
The traditional Chinese medicine herba Andrographitis is dry aerial parts of herba Andrographitis of Acanthaceae, and has effects of clearing heat, removing toxic substances, cooling blood and detumescence, and inducing heart, lung, large intestine and bladder meridian. Andrographolide is white prismatic crystal, and is the main active ingredient of medicinal plant Andrographis paniculata Nees. Because andrographolide is a diterpenoid lactone compound, the andrographolide has the defects of poor water solubility, poor in vivo stability and the like, so that the in vivo bioavailability is low, the clinical use is limited to a certain extent, and based on the nature defect of andrographolide and the relatively active structural characteristics of the andrographolide, researchers in recent years do a large number of structural modification and activity screening work so as to find more effective candidate compounds.
The research finds that the anti-tumor effect of andrographolide is closely related to the apoptosis of tumor cells mediated by the andrographolide, and the andrographolide can promote the apoptosis of various cancer cells such as liver cancer, prostate cancer and the like, and the tumor inhibition effect of the andrographis paniculata reconstruction compound is to be discussed.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide an andrographolide modified compound H4, a preparation method and application thereof.
For this purpose, the invention provides the following technical scheme:
an andrographolide-modified compound H4 has the following structural formula:
。
The preparation method of the andrographolide modified compound H4 comprises the following steps:
S1, in a weak alkaline reaction system, andrographolide and tert-butyl dimethyl chlorosilane are mixed to generate nucleophilic substitution reaction, so as to obtain an andrographolide modified compound E1;
S2, mixing an andrographolide reforming compound E1 with acetic anhydride, heating to 120-180 ℃, and carrying out nucleophilic substitution reaction on acyl carbon to modify hydroxyl groups at positions 3 and 14 of the andrographolide reforming compound E1 into acetyl groups to obtain an andrographolide reforming compound H4; the synthetic route is as follows:
。
Optionally, primary hydroxyl at 19 th position of andrographolide is protected by tert-butyldimethylsilyl (TBS), and secondary hydroxyl at 3 rd and 14 th positions are protected by acetyl to obtain andrographolide modified compound H4.
。
Optionally, in the step S1, the molar ratio of andrographolide to tert-butyldimethyl chlorosilane is 0.2:1-0.3:6; and/or
In the step S1, stirring and reacting for 1-4 hours at room temperature under the nucleophilic substitution reaction condition; and/or
In step S1, the method further comprises the steps of quenching, extracting, washing, drying, filtering, concentrating under reduced pressure and/or purifying the obtained andrographolide derivative compound E1.
Optionally, in the step S2, the mass-volume ratio of the andrographolide-modified compound E1 to acetic anhydride is less than or equal to 93:4, and the proportional relationship is mg/ml; and/or
In the step S2, the nucleophilic substitution reaction time is 1-4 hours; and/or
In the step S2, the method further comprises the steps of extracting, washing, drying, filtering, concentrating under reduced pressure and/or purifying the obtained andrographolide derivative compound H4.
Optionally, in step S1, the purification step is to separate the crude product of andrographolide derivative compound E1 by silica gel column chromatography, wherein the eluent is petroleum ether and ethyl acetate according to the volume ratio of (40-60): (0.5-2) → (1-20) → (0.1-2) for gradient elution, and preferably 50:1→10:1, gradient elution is carried out; and/or
In the step S2, the purification step is to separate the andrographolide reconstruction compound H4 crude product by silica gel column chromatography, wherein the eluent is petroleum ether and ethyl acetate according to the volume ratio of (40-80): (0.5-2) → (1-20): (0.1-2) for gradient elution, and the preferable ratio is 50:1→10:1 gradient elution.
The andrographolide modified compound H4 is prepared by the preparation method of the andrographolide modified compound H4.
The andrographolide modified compound H4 or the derivative thereof has application in preparing medicaments for treating cancers.
Alternatively, the cells of the cancer include gastric cancer, renal cancer or melanoma.
Optionally, the cells of the gastric cancer are selected from gastric cancer MKN45 cells; and/or
The cells of kidney cancer are selected from kidney cancer 786O cells; and/or
The melanoma cells are selected from melanoma A375 cells.
Alternatively, the derivative of andrographolide reforming compound H4 includes its cis-trans isomer, its diastereoisomer, its geometric isomer, its racemate, its solvate, its pharmaceutically acceptable salt or its prodrug.
A pharmaceutical composition for the treatment of cancer comprising a therapeutically effective amount of said andrographolide modifying compound H4 or a derivative thereof.
Optionally, a pharmaceutically acceptable carrier is also included, including water, oil, vegetables, minerals, a cream base, a lotion base, or an ointment base.
Optionally, the cream, lotion or ointment base comprises a suspending agent, a viscosity increasing agent and/or a transdermal enhancer.
Definition and description
Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are present in the present invention, it is intended to refer to their corresponding commercial products or active ingredients thereof.
The andrographolide reforming compound H4 of the present invention may exist in unsolvated forms or solvated forms, including hydrated forms. In general, solvated forms, which are equivalent to unsolvated forms, are intended to be encompassed within the scope of the present invention.
The andrographolide-modified compound H4 of the present invention may have an asymmetric carbon atom (optical center) or a double bond. Racemates, diastereomers, geometric isomers and individual isomers are all included within the scope of the present invention.
Pharmaceutically acceptable carriers for andrographolide-modifying compound H4 refer to any formulation or carrier medium representative of carriers including water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like capable of delivering an effective amount of the active substances of the present invention, without interfering with the biological activity of the active substances and without toxic or side effects to the host or patient. Such matrices include suspending agents, viscosity enhancers, transdermal enhancers, and the like.
The technical scheme of the invention has the following advantages:
1. The andrographolide modified compound H4 provided by the invention has the effect of treating cancers, and the research of the invention shows that compared with andrographolide, the andrographolide modified compound H4 has more remarkable effect of inhibiting tumors, and can be used for preparing medicaments for treating cancers.
2. The invention provides a preparation method of an andrographolide modified compound H4, which comprises the following steps: s1, under a weak alkaline environment, andrographolide and tert-butyl dimethyl chlorosilane are mixed to generate nucleophilic substitution reaction, so as to obtain an andrographolide modified compound E1; s2, mixing an andrographolide reforming compound E1 with excessive acetic anhydride, heating to 120-180 ℃, and carrying out nucleophilic substitution reaction on acyl carbon to modify hydroxyl groups at positions 3 and 14 of the andrographolide reforming compound E1 into propionyl groups to obtain an andrographolide reforming compound H4; the preparation method is simple and quick, the types of the reaction substrates required to be added are fewer, the subsequent purification steps are facilitated, and the propionic anhydride can be used as a reactant or a reactant, so that the preparation method has no toxic or side effect.
3. The application of the andrographolide reforming compound H4 or the derivative thereof in preparing medicaments for treating cancers including gastric cancer, renal cancer or melanoma is verified by the invention, and compared with the anti-tumor effect of the non-reforming andrographolide, the andrographolide reforming compound H4 or the derivative thereof has more remarkable inhibition effect on gastric cancer, renal cancer or melanoma.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a HRMS of andrographolide derivative H4 obtained in example 1;
FIG. 2 is a 1 H NMR chart of andrographolide derivative H4 obtained in example 1;
FIG. 3 is a 13 C NMR chart of andrographolide derivative H4 obtained in example 1;
FIG. 4 is an HPLC chart of andrographolide modified compound H4 obtained in example 1;
FIG. 5 shows the proliferation inhibition of gastric cancer MKN45 cells by andrographolide modifying compound H4 at various concentrations for 24 hours;
FIG. 6 shows the proliferation inhibition of gastric cancer MKN45 cells by andrographolide modifying compound H4 at different concentrations at 48 hours;
FIG. 7 shows the proliferation inhibition of gastric cancer MKN45 cells by andrographolide modifying compound H4 at various concentrations for 72 hours;
FIG. 8 shows the effect of andrographolide modifying compound H4 on gastric cancer MKN45 cells;
FIG. 9 shows the compound concentrations at which gastric cancer MKN45 cells reached half inhibition 24 hours after administration of andrographolide reforming compound H4;
FIG. 10 shows the half-inhibitory compound concentrations of gastric cancer MKN45 cells 48 hours after administration of andrographolide reforming compound H4;
FIG. 11 shows the compound concentrations at which gastric cancer MKN45 cells reached half inhibition when andrographolide reforming compound H4 was administered for 72 hours;
FIG. 12 shows the proliferation inhibition of 786O cells of renal carcinoma by andrographolide reforming compound H4 at various concentrations for 24 hours;
FIG. 13 shows the proliferation inhibition of 786O cells of renal carcinoma by andrographolide reforming compound H4 at various concentrations at 48 hours;
FIG. 14 shows the proliferation inhibition of 786O cells of renal carcinoma by andrographolide reforming compound H4 at various concentrations for 72 hours;
FIG. 15 shows the effect of andrographolide-modifying compound H4 on renal carcinoma 786O cells;
FIG. 16 shows the half-inhibitory compound concentrations of andrographolide-modified compound H4 administered 24 hours to renal carcinoma 786O cells;
FIG. 17 shows the half-inhibitory compound concentrations of andrographolide-modified compound H4 administered 48 hours for renal carcinoma 786O cells;
FIG. 18 shows the half-inhibitory compound concentrations of andrographolide-modified compound H4 administered for 72 hours to renal carcinoma 786O cells;
FIG. 19 shows the proliferation inhibition of melanoma A375 cells by andrographolide structuring compound H4 at various concentrations over 24 hours;
FIG. 20 shows the proliferation inhibition of melanoma A375 cells by andrographolide modifying compound H4 at various concentrations at 48 hours;
FIG. 21 shows the proliferation inhibition of melanoma A375 cells by andrographolide structuring compound H4 at various concentrations over 72 hours of administration;
FIG. 22 shows the effect of andrographolide modifying compound H4 on melanoma A375 cells;
FIG. 23 shows the compound concentrations at which half inhibition of melanoma A375 cells was achieved 24 hours after administration of andrographolide reforming compound H4;
FIG. 24 shows the compound concentrations at which half inhibition of melanoma A375 cells was achieved 48 hours after administration of andrographolide reforming compound H4;
FIG. 25 shows the compound concentrations at which melanoma A375 cells reached half inhibition 72 hours after administration of andrographolide reforming compound H4;
FIG. 26 shows the proliferation inhibition of gastric cancer MKN45 cells at various concentrations of AG at 24 hours of administration;
FIG. 27 shows the proliferation inhibition of gastric cancer MKN45 cells at various concentrations of AG at 48 hours of administration;
FIG. 28 shows the proliferation inhibition of gastric cancer MKN45 cells at various concentrations of AG for 72 hours;
FIG. 29 is a graph showing half-inhibitory compound concentrations of gastric cancer MKN45 cells at 24 hours AG administration;
FIG. 30 is a graph showing half-inhibitory compound concentrations in gastric cancer MKN45 cells at 48 hours AG administration;
FIG. 31 is a graph showing half-inhibitory compound concentrations of gastric cancer MKN45 cells at 72 hours AG administration;
FIG. 32 shows the proliferation inhibition of renal carcinoma 786O cells by AG at various concentrations over 24 hours of administration;
FIG. 33 shows the proliferation inhibition of 786O cells of renal carcinoma by AG at various concentrations for 48 hours;
FIG. 34 shows the proliferation inhibition of 786O cells of renal carcinoma by AG at various concentrations over 72 hours of administration;
FIG. 35 is a graph showing the half-inhibitory concentration of compounds in renal carcinoma 786O cells 24 hours after AG administration;
FIG. 36 shows the compound concentrations to half inhibition of renal carcinoma 786O cells 48 hours after AG administration;
FIG. 37 is a graph showing the half-inhibitory concentration of compounds in renal carcinoma 786O cells at 72 hours of AG administration;
FIG. 38 shows the proliferation inhibition of melanoma A375 cells by AG at various concentrations over 24 hours of administration;
FIG. 39 shows the proliferation inhibition of melanoma A375 cells by AG at various concentrations over 48 hours of administration;
FIG. 40 shows the proliferation inhibition of melanoma A375 cells by AG at various concentrations over 72 hours of administration;
FIG. 41 is a graph showing the half-maximal inhibitory compound concentration of melanoma A375 cells at 24 hours of AG administration;
FIG. 42 is a graph showing the half-maximal inhibitory compound concentration of melanoma A375 cells at 48 hours of AG administration;
FIG. 43 is a graph showing the half-maximal inhibitory compound concentration of melanoma A375 cells at 72 hours of AG administration;
the base of log on the abscissa in the figures above for half-inhibited compound concentrations is 10.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1
The present embodiment provides an andrographolide derivative compound H4, wherein the name is: (3 s,4 e) -4- {2- [ (1 r,4as,5r,6r,8 as) -6- (acetoxy) -5- { [ (tert-butyldimethylchlorosilane) methoxy } -5,8 a-dimethyl-2-ethylene-decahydronaphthalen-1-yl ] ethylene } -5-oxooxolan-3-acetic acid ethyl ester having the following structural formula:
。
The synthetic route is as follows:
。
The preparation method comprises the following steps:
(1) Andrographolide (100.0 mg,0.285 mmol) and pyridine (2 mL) were added to the flask, followed by tert-butyldimethylsilyl (TBDMSCl) (500.0 mg, 3.32 mmol) and magnetically stirred at room temperature (25 ℃) for reaction 3 h. EtOAc (30 mL) was added to the reaction mixture to dilute it, then water (40 mL) was added to quench the reaction, and extracted with ethyl acetate (EtOAc, 80 mL ×3). The combined organic phases were washed sequentially with brine (100 mL), dried over anhydrous Na 2SO4, filtered and the crude organic filtrate concentrated under reduced pressure was separated by column chromatography over silica gel (PE: ea=50:1→10:1, v/v) gradient to give 107 mg as a white solid (compound E1), yield: 81%.
(2) Compound E1 (93.0 mg,0.2 mmol) and acetic anhydride (Ac 2 O) (4 mL) were added to the reaction flask, heated to 145 ℃, and reaction 1 h continued at 145 ℃. After cooling to room temperature, etOAc (100 mL) was added to the reaction mixture to dilute, then saturated NaHCO 3 solution (100 mL) was added to quench, washed with water, and extracted with EtOAc (100 mL ×3). The combined organic phases were washed sequentially with brine (100 mL), dried over anhydrous Na 2SO4, filtered and the crude organic filtrate concentrated under reduced pressure was separated by column chromatography on silica gel (PE: ea=50:1→10:1, v/v) gradient elution to give 81 mg as a white solid (andrographolide derivative compound H4), yield: 74%.
Carrying out mass spectrum and nuclear magnetic resonance detection on the andrographolide modified compound H4 obtained in the above way:
Hi-Res MS (ESI) M/z [ M+Na ] +：C30H48O7 SiNa 571.3055 (calculated 571.3062). As shown in fig. 1.
1H NMR (400 MHz, CDCl3) δ 7.01 (t,J= 6.6 Hz, 1H), 5.91 (d,J= 6.4 Hz, 1H), 4.87 (s, 1H), 4.62-4.50 (m, 2H), 4.48 (s, 1H), 4.24 (d,J= 12.8 Hz, 1H), 3.81 (d,J= 10.4 Hz, 1H), 3.58 (d,J= 10.4 Hz, 1H), 2.50-2.33 (m, 3H), 2.11 (s, 3H), 2.04 (s, 3H), 1.90-1.78 (m, 3H), 1.76-1.65 (m, 3H), 1.34-1.21 (m, 3H), 0.93 (s, 3H), 0.87 (s, 9H), 0.80 (s, 3H), 0.02 (s, 6H) . As shown in fig. 2.
13C NMR (101 MHz, CDCl3) δ 170.90, 170.65, 169.22, 150.82, 147.40, 123.90, 108.48, 80.21, 71.72, 67.95, 63.81, 56.18, 55.63, 42.54, 39.14, 38.36, 37.47, 25.99, 25.41, 24.49, 23.40, 21.41, 20.86, 18.35, 14.47, -5.52, -5.59. As shown in fig. 3.
HPLC test is carried out on the andrographolide as a starting material, and the chromatographic conditions are as follows: the mobile phase is methanol-water (95:5, v/v); the elution procedure is isocratic elution; the chromatographic column is RPC18 column; the detection wavelength is 230 nm; the column temperature was room temperature (27 ℃); the results of the detection are shown in Table 1 below, and the purity of andrographolide is 98.8%.
TABLE 1
Carrying out HPLC detection on the obtained andrographolide modified compound E1, wherein the chromatographic conditions are as follows: the mobile phase is methanol-water (90:10, v/v); the elution procedure is isocratic elution; the chromatographic column is RPC18 column; the detection wavelength is 230 nm; the column temperature was room temperature (27 ℃); the results of the detection are shown in the following table, and the purity of the andrographolide derivative compound E1 is 98.1%.
TABLE 2
Carrying out HPLC detection on the obtained andrographolide modified compound H4, wherein the chromatographic conditions are as follows: the mobile phase is methanol-water (90:10, v/v); the elution procedure is isocratic elution; the chromatographic column is RPC18 column; the detection wavelength is 230 nm; the column temperature was room temperature (27 ℃); the results of the tests are shown in the following table and in FIG. 4, the purity of Andrographis paniculata Nees modified compound H4 is 98.8%.
TABLE 3 Table 3
Example 2
The preparation method of the andrographolide modified compound H4 comprises the following steps:
(1) Andrographolide (0.2 mmol) and pyridine (2 mL) were added to the flask, and tert-butyldimethylsilyl (TBDMSCl) (1 mmol) was added thereto, followed by magnetic stirring at room temperature (20deg.C) for 1 hour. EtOAc (30 mL) was added to the reaction mixture to dilute it, then water (40 mL) was added to quench the reaction, and extracted with ethyl acetate (EtOAc, 80 mL ×3). The combined organic phases were washed successively with brine (100 mL), dried over anhydrous Na 2SO4, filtered and the crude organic filtrate concentrated under reduced pressure was separated by column chromatography over silica gel (PE: ea=40:0.5→1:0.1, v/v) gradient elution to give a white solid (compound E1).
(2) Compound E1 and acetic anhydride (Ac 2 O) (mass to volume ratio of compound E1 to propionic anhydride 93:5, ratio relationship mg/ml) were added to the reaction flask, heated to 120 ℃, and reacted for 3 hours at 120 ℃. After cooling to room temperature, etOAc (100 mL) was added to the reaction mixture to dilute, then saturated NaHCO 3 solution (100 mL) was added to quench, washed with water, and extracted with EtOAc (100 mL ×3). The combined organic phases were washed successively with brine (100 mL), dried over anhydrous Na 2SO4, filtered and the crude organic filtrate concentrated under reduced pressure was separated by column chromatography on silica gel (PE: ea=40:0.5→1:0.1, v/v) gradient elution to give a white solid (andrographolide derivative compound H4).
Example 3
The preparation method of the andrographolide modified compound H4 comprises the following steps:
(1) Andrographolide (0.3 mmol) and pyridine (4 mL) were added to the flask, and tert-butyldimethylsilyl (TBDMSCl) (6 mmol) was added thereto, followed by magnetic stirring at room temperature (20deg.C) for 24 hours. EtOAc (30 mL) was added to the reaction mixture to dilute it, then water (40 mL) was added to quench the reaction, and extracted with ethyl acetate (EtOAc, 80 mL ×3). The combined organic phases were washed successively with brine (100 mL), dried over anhydrous Na 2SO4, filtered and the crude organic filtrate concentrated under reduced pressure was separated by column chromatography over silica gel (PE: ea=60:2→20:2, v/v) gradient elution to give a white solid (compound E1).
(2) Compound E1 and acetic anhydride (Ac 2 O) (mass to volume ratio of compound E1 to propionic anhydride is 93:4, ratio is mg/ml) were added to the reaction flask, heated to 180 ℃, and reacted for 4 hours at 180 ℃. After cooling to room temperature, etOAc (100 mL) was added to the reaction mixture to dilute, then saturated NaHCO 3 solution (100 mL) was added to quench, washed with water, and extracted with EtOAc (100 mL ×3). The combined organic phases were washed successively with brine (100 mL), dried over anhydrous Na 2SO4, filtered and the crude organic filtrate concentrated under reduced pressure was separated by column chromatography on silica gel (PE: ea=80:2→20:2, v/v) gradient elution to give a white solid (andrographolide remodelling compound H4).
EXAMPLE 4 action of Andrographolide modification Compound H4 in inhibiting human gastric cancer
1. Experimental materials
Cell origin and culture: human gastric cancer cell line MKN45 was purchased from the institute of synergetics cell resource center (which is the headquarters of the national infrastructure for cell line resources, NSTI) and cells were cultured in 1640 medium containing 10% fetal bovine serum, 100U/ml penicillin and 100 μg/ml streptomycin in a 5% co 2 incubator at 37 ℃.
Test sample: andrographolide modified compound H4 prepared in example 1, commercially available Andrographolide (AG) and a commercially available anti-gastric cancer positive drug cyclophosphamide.
2. Experimental method
And the influence of the andrographis paniculata compound H4 on the gastric cancer cell viability is detected by adopting a CCK8 experimental method. The specific operation is as follows: MKN45 cells were seeded at a density of 5000 cells/well in 96-well plates for culture; the cells were grouped as follows: andrographolide-modified Compound H4 different final concentration gradient drug administration groups (1. Mu.M, 5. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M, 75. Mu.M, 100. Mu.M); AG different final concentration gradient dosing groups (1. Mu.M, 5. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M); cyclophosphamide group (final concentration 3.583 mM), an anti-gastric cancer positive drug; the control group is 1640 culture medium group without adding medicine; a blank group without cells and without medicine is additionally arranged.
Drug intervention 24 hours after cell plating, and after 24, 48 and 72 hours after drug administration, CCK8 reagent (purchased from japan homozygote) was added to measure tumor cell viability, and IC 50 was calculated.
As shown in fig. 5-11 and 26-31, the andrographis paniculata compound H4 can significantly inhibit the proliferation of gastric cancer MKN45 cells, exert an antitumor effect, have an IC 50 (24H) of 18.46 μm, an IC 50 (48H) of 12.22 μm, an IC 50 (72H) of 13.48 μm, an IC 50 (24H) of AG of 47.16 μm, an IC 50 (48H) of 15.52 μm, and an IC 50 (72H) of 12.52 μm, and thus it can be seen that the IC 50 of the andrographis paniculata compound H4 after administration is significantly lower than that of AG, indicating that the andrographis paniculata compound H4 has a more significant effect of inhibiting the proliferation of gastric cancer MKN45 cells than AG, and also indicating that the andrographis paniculata compound H4 inhibits the proliferation of gastric cancer MKN45 cells more rapidly.
EXAMPLE 5 action of Andrographis paniculata modification Compound H4 in inhibiting human renal carcinoma
1. Experimental materials
Cell origin and culture: human renal cancer cell line 786O was purchased from the institute of Association and medicine cell resource center (which is the national infrastructure of cell line resources, NSTI headquarters) and cells were cultured in 1640 medium containing 10% fetal bovine serum, 100U/ml penicillin and 100. Mu.g/ml streptomycin in a 5% CO 2 incubator at 37 ℃.
Test sample: andrographolide modified compound H4 prepared in example 1, commercially available Andrographolide (AG) and a commercially available renal cancer positive drug fluorouracil.
2. Experimental method
The experimental method comprises the following steps: the effect of andrographis paniculata compound H4 on the viability of kidney cancer cells was examined by CCK8 assay. The specific operation is as follows: 786O cells were seeded at a density of 5000 cells/well in 96-well plates for culture; the cells were grouped as follows: andrographolide-modified Compound H4 different concentration gradient administration groups (1. Mu.M, 5. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M, 75. Mu.M, 100. Mu.M); AG different concentration gradient dosing groups (1. Mu.M, 5. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M); fluorouracil group (38.4. Mu.M, 192. Mu.M) as positive drug for renal cancer; the control group is 1640 culture medium group without adding medicine; a blank group without cells and without medicine is additionally arranged.
Drug intervention 24 hours after cell plating, and after 24, 48 and 72 hours after drug administration, CCK8 reagent (purchased from japan homozygote) was added to measure tumor cell viability, and IC 50 was calculated.
As shown in fig. 12-18 and fig. 32-37, andrographolide reforming compound H4 can significantly inhibit proliferation of kidney cancer 786-O cells, and exert antitumor effect, and it was revealed that IC 50 (24H) was 6.406 μm, IC 50 (48H) was 3.462 μm, IC 50 (72H) was 3.349 μm, IC 50 (24H) of AG was 22.37 μm, IC 50 (48H) was 11.11 μm, and IC 50 (72H) was 6.397 μm, and that IC 50(24h)、IC50(48h)、IC50 (72H) of andrographolide reforming compound H4 was significantly lower than AG, indicating that andrographolide reforming compound H4 was significantly more effective in inhibiting proliferation of kidney cancer 786-O cells than AG.
EXAMPLE 6 Andrographolide modification Compound H4 inhibition of human melanoma
1. Experimental materials
Cell origin and culture: human melanoma A375 was purchased from the institute of Kogyo cell resource center (which is the national infrastructure of cell line resources, NSTI headquarters) and cells were cultured in high-sugar DMEM medium containing 10% fetal bovine serum, 100U/ml penicillin and 100. Mu.g/ml streptomycin at 37℃in a 5% CO 2 incubator.
Test sample: andrographolide modified compound H4 prepared in example 1, commercially available Andrographolide (AG) and commercially available anti-melanoma positive drug paclitaxel.
2. Experimental method
The effect of compound H4 on melanoma cell viability was examined using CCK8 assay. The specific operation is as follows: a375 cells were seeded at a density of 5000 cells/well in 96-well plates for culture; the cells were grouped as follows: compound H4 different final concentration gradient dosing groups (1 μm,5 μm,10 μm,25 μm,50 μm,75 μm,100 μm); AG different final concentration gradient dosing groups (1. Mu.M, 5. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M); the anti-melanoma positive drug paclitaxel group (final concentration 5. Mu.M, final concentration 10. Mu.M); the control group is 1640 culture medium group without adding medicine; a blank group without cells and without medicine is additionally arranged.
Drug intervention 24 hours after cell plating, and after 24, 48 and 72 hours after drug administration, CCK8 reagent (purchased from japan homozygote) was added to measure tumor cell viability, and IC 50 was calculated.
As shown in fig. 19-25 and 38-43, andrographis paniculata compound H4 can significantly inhibit proliferation of melanoma a375 cells, exert antitumor effect, have IC 50 (24H) of 5.021 μm, IC 50 (48H) of 6.368 μm, IC 50 (72H) of 3.044 μm, IC 50 (24H) of AG of 18.6 μm, IC 50 (48H) of 14.11 μm, and IC 50 (72H) of 12.06 μm, and thus it can be seen that the andrographolide reforming compound H4 has significantly lower IC 50(24h)、IC50(48h)、IC50 (72H) than AG, indicating that andrographolide reforming compound H4 has more significant effect in inhibiting proliferation of melanoma a375 cells than AG, and also has a faster effect.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (4)

1. The application of an andrographolide-modified compound H4 in preparing a medicament for treating gastric cancer MKN45 cells, renal cancer 786O cells or melanoma A375 cells is characterized in that the andrographolide-modified compound H4 has the following structural formula:
。
2. The use of andrographolide reforming compound H4 according to claim 1 for preparing a medicament for treating cancer, further comprising a preparation method of the andrographolide reforming compound H4, comprising the following steps:
S1, in a weak alkaline reaction system, andrographolide and tert-butyl dimethyl chlorosilane are mixed to generate nucleophilic substitution reaction, so as to obtain an andrographolide modified compound E1;
S2, mixing an andrographolide reforming compound E1 with acetic anhydride, heating to 120-180 ℃, performing nucleophilic substitution reaction on acyl carbon, and modifying hydroxyl groups at positions 3 and 14 of the andrographolide reforming compound E1 into acetyl groups to obtain an andrographolide reforming compound H4; the synthetic route is as follows:
。
3. Use of andrographolide modifying compound H4 according to claim 2, in the preparation of a medicament for the treatment of cancer, characterized in that in step S1 the molar ratio of andrographolide to t-butyldimethylchlorosilane is 0.2:1-0.3:6; and/or
In the step S1, stirring and reacting for 1-24 hours at room temperature under the nucleophilic substitution reaction condition; and/or
In the step S1, the weakly alkaline reaction system is pyridine; and/or
In the step S1, the method further comprises the steps of quenching, extracting, washing, drying, filtering, concentrating under reduced pressure and/or purifying the obtained andrographolide modified compound E1; and/or
In the step S2, the mass volume ratio of the andrographolide modified compound E1 to acetic anhydride is less than or equal to 93:4, and the proportional relationship is mg/ml; and/or
In the step S2, the nucleophilic substitution reaction time is 1-4 hours; and/or
In the step S2, the method further comprises the steps of extracting, washing, drying, filtering, concentrating under reduced pressure and/or purifying the obtained andrographolide derivative compound H4.
4. The use of andrographolide modifying compound H4 as claimed in claim 3, for the preparation of a medicament for the treatment of cancer,
In the step S1, the purification step is to separate the crude andrographolide reconstruction compound E1 by silica gel column chromatography, wherein the eluent is petroleum ether and ethyl acetate according to the volume ratio of (40-60): (0.5-2) → (1-20): (0.1-2) for gradient elution; and/or
In the step S2, the purification step is to separate the andrographolide reconstruction compound H4 crude product by silica gel column chromatography, and the eluent is petroleum ether and ethyl acetate according to the volume ratio of (40-80)/(0.5-2) → (1-20)/(0.1-2) and carry out gradient elution.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114053265A (en) * 2020-08-02 2022-02-18 上海中医药大学附属龙华医院 Medical application of NEDDylation blocking agent

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114053265A (en) * 2020-08-02 2022-02-18 上海中医药大学附属龙华医院 Medical application of NEDDylation blocking agent

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Rada Bunthawong et al..Synthesis and cytotoxic activity of new 7-acetoxy-12-amino-14-deoxy andrographolide analogues.《Bioorg. Med.Chem.Lett.》.2020,第33卷第127741页. *
Synthesis and cytotoxic activity of new 7-acetoxy-12-amino-14-deoxy andrographolide analogues;Rada Bunthawong et al.;《Bioorg. Med.Chem.Lett.》;第33卷;第127741页 *

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