CN114149440B - Preparation and application of isoquinoline alkaloid with benzoquinone reductase induction activity - Google Patents

Preparation and application of isoquinoline alkaloid with benzoquinone reductase induction activity Download PDF

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CN114149440B
CN114149440B CN202111390216.XA CN202111390216A CN114149440B CN 114149440 B CN114149440 B CN 114149440B CN 202111390216 A CN202111390216 A CN 202111390216A CN 114149440 B CN114149440 B CN 114149440B
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induction activity
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benzoquinone
benzoquinone reductase
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周志望
王勇
刘运来
王肖娜
江甜甜
唐圆圆
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Nanchang University
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Abstract

The invention discloses preparation and application of isoquinoline alkaloid with benzoquinone reductase induction activity, and relates to the technical field of medicines. The preparation method and the application of isoquinoline alkaloid with benzoquinone reductase induction activity comprise a compound 1 and a compound 2, and the preparation steps are as follows: pulverizing dried Cryptocarya wrayi, extracting with ethanol by soaking method to obtain extractive solution, concentrating under reduced pressure, and recovering ethanol to obtain crude extract. The compound 1 and the compound 2 provided by the invention belong to new compounds and are not disclosed, meanwhile, the results of benzoquinone reductase induction activity tests on the compound 1 and the compound 2 show that the compound 1 and the compound 2 have medium strength benzoquinone reductase induction activity on liver cancer cells of a Hepa 1c1c7 mouse, and the experimental results show that the compound 1 and the compound 2 provided by the invention can be used for preparing a benzoquinone reductase inducer and preparing an anti-tumor medicament.

Description

Preparation and application of isoquinoline alkaloid with benzoquinone reductase induction activity
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to isoquinoline alkaloid with a quinone reductase induction function, which is separated from Cryptocarya wrayi of Cinnamomum in Lauraceae, and preparation and application of the compounds.
Background
Benzoquinone reductase is a flavoprotein enzyme universally existing in various tissues of human bodies, and can reduce quinones into hydroquinones so as to prevent oxidative damage mediated by the quinones, and the induced activation of the benzoquinone reductase reflects the ability of cells to inhibit oxidative stress in response to endogenous or exogenous damage. As a reducing enzyme, benzoquinone reductase is related to chemoprevention and anticancer activity, and has double functions of detoxifying quinone toxic substances and activating biological reductive antitumor drugs, so that the benzoquinone reductase can respectively play roles in preventing and treating tumors. Therefore, induction of activated benzoquinone reductase is often used as a strategy to protect cells.
The invention provides a preparation method of isoquinoline alkaloid with quinone reductase induction effect, which is characterized in that the plants of the genus Eichhornia are rich in alkaloid secondary metabolites with various structures and wide activity, and relate to benzyl isoquinoline, phenanthroindolizidine, protoaporphine, pavines and the like.
Disclosure of Invention
The invention aims to provide a preparation method and application of isoquinoline alkaloid with benzoquinone reductase inducing activity, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: the isoquinoline alkaloid with the benzoquinone reductase induction activity comprises a compound 1 and a compound 2, wherein the chemical structural formulas of the compound 1 and the compound 2 are respectively as follows:
Figure GDA0003963398170000021
the preparation method comprises the following steps:
the method comprises the following steps: pulverizing dried Cryptocarya wrayi, extracting with ethanol by soaking method to obtain extractive solution, concentrating the extractive solution under reduced pressure, and recovering ethanol to obtain crude extract;
step two: dispersing the crude extract obtained in the step one in water to form a suspension, extracting the suspension with ethyl acetate, and concentrating the obtained ethyl acetate phase to obtain an ethyl acetate extract;
step three: subjecting the ethyl acetate extract to MCI column chromatography, eluting with methanol/water gradient, combining similar fractions according to TLC color development to obtain 7 fractions, which are Fr.1-Fr.7 respectively, separating Fr.5 fraction on SephadexLH-20 (EtOH) gel column to obtain 4 sub-fractions, which are Fr.7A-Fr.7D respectively, and separating Fr.7C sub-fraction by silica gel column chromatography to obtain four sub-fractions, which are Fr.7C 1 -Fr.7C 4 ；
Step four: mixing Fr.7C 2 Sub-fractions were further purified by semi-preparative HPLC to give compound 1 and compound 2 as claimed in claim 1.
In the technical scheme, the preferable temperature of the decompression concentration in the step one is less than or equal to 45 ℃.
In the technical scheme, preferably, the purity of the ethanol adopted in the step one is 95%.
In the technical scheme, preferably, the eluent in the step three adopts petroleum ether/acetone, wherein the volume ratio of the petroleum ether to the acetone is 8:1-2:1.
Preferably, in the fourth step, semi-preparative HPLC is performed under the conditions that the volume ratio of acetonitrile to water is 40.
The application method comprises the step of applying any one or a mixture of two of the compound 1 and the compound 2 to the benzoquinone reductase induction activity in the liver cancer cells.
Compared with the prior art, the invention has the beneficial effects that:
the compound 1 and the compound 2 provided by the invention belong to new compounds and are not disclosed, meanwhile, the results of benzoquinone reductase induction activity tests on the compound 1 and the compound 2 show that the compound 1 and the compound 2 have medium strength benzoquinone reductase induction activity on liver cancer cells of a Hepa 1c1c7 mouse, and the experimental results show that the compound 1 and the compound 2 provided by the invention can be used for preparing a benzoquinone reductase inducer and preparing an anti-tumor medicament, and have important significance and prospect in the research of the anti-tumor medicament.
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FIG. 1 shows a scheme of Compound 1 according to the present invention 1 H-NMR Spectroscopy (deuterated reagent: CD) 3 OD)；
FIG. 2 shows the preparation of Compound 1 according to the present invention 13 C-NMR Spectroscopy (deuterated reagent: CD) 3 OD)；
FIG. 3 shows HSQC spectra of Compound 1 proposed in the present invention (deuterated reagent: CD) 3 OD)；
FIG. 4 shows the HMBC profile of Compound 1 proposed by the present invention (deuterated agent: CD) 3 OD)；
FIG. 5 shows Compound 1 according to the present invention 1 H- 1 H COSY spectrum (deuterated reagent: CD) 3 OD)；
FIG. 6 shows the ROESY spectrum of Compound 1 according to the present invention (deuterated agent: CD) 3 OD)；
FIG. 7 is a (+) -HRESIMS spectrum of Compound 1 as set forth in the present invention;
FIG. 8 is an IR spectrum of Compound 1 proposed by the present invention;
FIG. 9 shows the preparation of Compound 2 according to the present invention 1 H-NMR Spectroscopy (deuterated reagent: CDCl) 3 )；
FIG. 10 shows the preparation of Compound 2 according to the present invention 13 C-NMR Spectroscopy (deuterated reagent: CDCl) 3 )；
FIG. 11 shows HSQC spectra of Compound 2 proposed by the present invention (deuterated reagent: CDCl) 3 )；
FIG. 12 shows the HMBC profile of Compound 2 proposed by the present invention (deuterated reagent: CDCl) 3 )；
FIG. 13 shows the ROESY spectrum of Compound 2 proposed by the present invention (deuterated reagent: CDCl) 3 )；
FIG. 14 is a (+) -HRESIMS spectrum of Compound 2 as presented herein.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
It should be noted that in the description of the present invention, the terms "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, which are only for convenience of description and simplification of description, and do not indicate or imply that the referred device or element must have a specific orientation, be configured in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.
Further, it will be appreciated that the dimensions of the various elements shown in the figures are not drawn to scale, for ease of description, and that the thickness or width of some layers may be exaggerated relative to other layers, for example.
It should be noted that like reference numerals and letters refer to like items in the following figures, and thus, once an item is defined or illustrated in one figure, it will not need to be further discussed or illustrated in detail in the description of the following figure.
The invention provides a technical scheme that: the isoquinoline alkaloid with the benzoquinone reductase inducible activity comprises a compound 1 and a compound 2, wherein the chemical structural formulas of the compound 1 and the compound 2 are respectively as follows:
Figure GDA0003963398170000041
the Arabic numerals in the chemical structural formula are the mark positions of carbon atoms in the chemical structure.
The preparation method comprises the following steps:
the method comprises the following steps: pulverizing 6.0kg dried Cryptocarya wrayi small branch, extracting with 30L 95% ethanol by soaking method for 7 days each time for 3 times, mixing to obtain extractive solution, concentrating the extractive solution under reduced pressure at 40 deg.C, and recovering ethanol to obtain coarse extract 285.0g;
step two: dispersing the crude extract obtained in the step one in water to obtain a suspension, extracting the suspension for 3 times by using 1.5L ethyl acetate, and concentrating the obtained ethyl acetate phase under reduced pressure to obtain 38.5g of ethyl acetate extract;
step three: subjecting the ethyl acetate extract to MCI column chromatography, eluting with methanol/water gradient, and combining similar fractions according to TLC color development to obtain 7 fractions, which are Fr.1-Fr.7 respectively, separating 4.6g of Fr.5 fraction on SephadexLH-20 (EtOH) gel column to obtain 4 sub-fractions, which are Fr.7A-Fr.7D respectively, and separating 476.8mg of Fr.7C sub-fraction on silica gel column chromatography to obtain four sub-fractions, which are Fr.7C respectively 1 -Fr.7C 4 The eluent is petroleum ether/acetone, wherein the volume ratio of the petroleum ether to the acetone is 4:1;
step four: mixing 104.8mg Fr.7C obtained in step three 2 A subset was further purified by semi-preparative HPLC to give 2.0mg of Compound 1 at a retention time of 40.2min and 1.2mg of Compound 2 at a retention time of 41.0min at a acetonitrile to water volume ratio of 40, 3.0 mL/min.
The chemical structures of the compound 1 and the compound 2 are determined by a plurality of modern spectral techniques such as NMR, HRESIMS, ECD calculation, UV calculation and the like, and the physicochemical properties are as follows:
compound 1 yellow amorphous powder;
Figure GDA0003963398170000051
(c 0.05,CH 3 OH)；UVλ max (logε)231(4.03),278(3.78),305(3.70)nm；ECD(c 2.0×10 -5 gmL -1 ,MeOH)λ(Δε)223(+22.08),243(-3.20),269(+5.95),303(+3.74)nm；IRν max 1716,1641,1607,1515,1471cm -1 ； 1 H-NMR and 13 C-NMR data, see Table 1; (+) HR-ESI-MS M/z372.1440[ M + H] + (calcd for C 20 H 22 NO 6 ,372.1447).
Compound 2 is a yellow amorphous powder;
Figure GDA0003963398170000052
(c 0.02,CH 3 OH)；UVλ max (logε)208(4.40),224(4.43),274(3.98),303(3.89)nm；ECD(c 1.7×10 -4 gmL -1 ,MeOH)λ(Δε)220(-28.86),237(+32.36),260(+9.52),284(-6.79),301(4.21),321(-7.62)nm； 1 H-NMR and 13 C-NMR data, see Table 1; (+) HR-ESI-MS M/z370.1288[ M] + (calcd for C 20 H 20 NO 6 ,370.1285).
Wherein the specific hydrogen spectrum and carbon spectrum data of the compound 1 and the compound 2 are shown in the table 1.
TABLE 1
Figure GDA0003963398170000061
Application experiments: hepa 1c1c7 mouse hepatoma cells (ATCC CRL-2026) were treated in minimal basal medium of eagle supplemented with 10% fetal bovine serum, 95% air and 5% CO at 37% 2 Cells were cultured in humidified air, seeded in 96-well plates, and treated with the indicated dose of compound for 24h. The medium was removed and the cells were lysed with 30. Mu.L of cell lysate [0.8% digitonin and 2mM EDTA solution (pH 7.8)]Cleavage was carried out at 37 ℃ for 15 minutes. Then, 170. Mu.L of the complete reaction mixture containing bovine serum albumin, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium ammonium bromide (MTT), 1.5% Tween 20, 0.5M tris hydrochloride, 7.5mM flavin adenine dinucleotide, 150mM glucose-6-phosphate, 10 units/. Mu.L glucose-6-phosphate dehydrogenase, 50mM nicotinamide adenine dinucleotide phosphate and 50mM menadione was added to each well. After incubation for about 4min, a blue color developed and the absorbance at 630nm was recorded on the microplate reader.
The experimental results are as follows: sulforaphane showed 1.7-fold induction activity at 2.0. Mu.M, compound 1 and compound 2 both showed moderate-intensity QR induction activity, and 1.2-fold and 1.1-fold induction activity at a concentration of 3.125. Mu.M, respectively, as compared to the positive control.
The application tests show that the compound 1 and the compound 2 have medium-strength benzoquinone reductase induction activity on hepatoma cells of a Hepa 1c1c7 mouse, can be used for preparing a benzoquinone reductase inducer and preparing an anti-tumor drug, and have important significance and prospect in the research of the anti-tumor drug.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (2)

1. An isoquinoline alkaloid with benzoquinone reductase induction activity is characterized in that the isoquinoline alkaloid is a compound 1 or a compound 2, and the chemical structural formulas of the compound 1 and the compound 2 are respectively as follows:
Figure FDA0003963398160000011
2. the use of isoquinoline alkaloids with benzoquinone reductase inducing activity according to claim 1, wherein the compound 1 or compound 2 is used in the preparation of antitumor drugs.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107082780A (en) * 2017-04-14 2017-08-22 山东省医学科学院药物研究所 A kind of alkaloid with the parallel isoquinoline structure of pyrroles and preparation method and application
CN109206429A (en) * 2017-07-07 2019-01-15 中国科学院西北高原生物研究所 A kind of isoquinoline alkaloid compound and its preparation method and application
CN113105428A (en) * 2021-03-10 2021-07-13 宁波大学 Xanthone compound and preparation method and application thereof

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US7495100B2 (en) * 2004-05-07 2009-02-24 Purdue Research Foundation Synthesis of indenoisoquinolines

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
CN107082780A (en) * 2017-04-14 2017-08-22 山东省医学科学院药物研究所 A kind of alkaloid with the parallel isoquinoline structure of pyrroles and preparation method and application
CN109206429A (en) * 2017-07-07 2019-01-15 中国科学院西北高原生物研究所 A kind of isoquinoline alkaloid compound and its preparation method and application
CN113105428A (en) * 2021-03-10 2021-07-13 宁波大学 Xanthone compound and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
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Molecular Modeling of 3-Arylisoquinoline Antitumor Agents Active Against A-549. A Comparative Molecular Field Analysis Study;Won-Jea Cho，等;《Bioorganic & Medicinal Chemistry》;20021231;第10卷;第2953-2961页 *
Novel phenanthrene and isocoumarin from the rhizomes of Dioscorea nipponica Makino subsp. rosthornii (Prain et Burkill) C. T. Ting (Dioscoreaceae);Xuejiao Li，等;《Bioorganic & Medicinal Chemistry Letters》;20170401;第27卷;第3595-3601页 *

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