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

Abstract

The invention discloses the preparation and application of a class of isoquinoline alkaloids with benzoquinone reductase inducing activity, and relates to the technical field of medicine. Preparation and application of a class of isoquinoline alkaloids with benzoquinone reductase inducing activity, including compound 1 and compound 2, the preparation steps are as follows, step 1: after crushing the dried Cryptocarya wrayi, extract it with ethanol by dipping method, The extract is obtained, and the extract is concentrated under reduced pressure to recover ethanol to obtain a crude extract. Compound 1 and compound 2 proposed in the present invention all belong to new compounds, which have not been disclosed. At the same time, by carrying out the benzoquinone reductase inducing activity test on compound 1 and compound 2, the results show that compound 1 and compound 2 have an effect on Hepa 1c1c7 mouse liver cancer cells have moderately strong benzoquinone reductase inducing activity. Experimental results show that the compounds 1 and 2 proposed in the present invention can be used to prepare benzoquinone reductase inducers and antitumor drugs.

Description

Preparation of a class of isoquinoline alkaloids with benzoquinone reductase inducing activity and its use

technical field

The invention belongs to the technical field of medicine, and in particular relates to the preparation and application of a class of isoquinoline alkaloids with quinone reductase inducing effect isolated from the plant Cryptocaryawrayi of the family Lauraceae.

Background technique

Benzoquinone reductase is a flavoprotease ubiquitous in various tissues of the human body. It can reduce quinones to hydroquinone, thereby preventing oxidative damage mediated by quinones. The induction and activation of benzoquinone reductase reflects the cellular response to internal Ability to suppress oxidative stress in response to exogenous or exogenous injury. As a reductive enzyme, benzoquinone reductase is related to chemoprevention and anticancer activities. It has dual functions of detoxifying quinone toxic substances and activating bioreductive antitumor drugs, so it can play the role of tumor prevention and treatment respectively. Therefore, the induction and activation of benzoquinone reductase is often used as a strategy to protect cells.

And Cinnamon genus plants are rich in alkaloid secondary metabolites with various structures and wide activity, involving types such as benzylisoquinoline, phenanthrene and indolishidine, former apophen and pavines, the present invention starts from Cryptocarya The isoquinoline alkaloids with quinone reductase inducing activity extracted from wrayi have important significance and prospects in the research of antitumor drugs. Therefore, a class of isoquinoline alkaloids with quinone reductase inducing activity is proposed preparation and use thereof.

Contents of the invention

The object of the present invention is to provide the preparation and application of a class of isoquinoline alkaloids with benzoquinone reductase inducing activity, so as to solve the problems raised in the above-mentioned background technology.

In order to achieve the above object, the present invention provides the following technical scheme: the preparation and application of a class of isoquinoline alkaloids with benzoquinone reductase inducing activity, and the isoquinoline alkaloids with benzoquinone reductase inducing activity include compounds 1 and compound 2, the chemical structural formulas of said compound 1 and compound 2 are as follows respectively:

Its preparation steps are as follows:

Step 1: Grinding the dried Cryptocarya wrayi and extracting it with ethanol according to the dipping method to obtain an extract, concentrating the extract under reduced pressure to recover ethanol to obtain a crude extract;

Step 2: dispersing the crude extract obtained in step 1 in water to form a suspension, extracting the suspension with ethyl acetate, and concentrating the obtained ethyl acetate phase to obtain ethyl acetate extraction extract;

Step 3: Extract the extract with ethyl acetate and perform MCI column chromatography, elute with methanol/water gradient, and combine similar fractions according to TLC color development to obtain 7 components, which are respectively Fr.1-Fr.7, and The Fr.5 component in it was separated on a SephadexLH-20 (EtOH) gel column to obtain 4 subcomponents, which were respectively Fr.7A-Fr.7D, and the Fr.7C subcomponent was passed through a silica gel column layer Four subcomponents were obtained through analysis and separation, which were respectively Fr.7C ₁ -Fr.7C ₄ ;

Step 4: further purify the Fr.7C ₂ subfraction by semi-preparative HPLC to obtain compound 1 and compound 2 as claimed in claim 1.

Preferably in the technical solution, the temperature of the vacuum concentration in the step 1 is ≤45°C.

Preferably in the technical solution, the purity of the ethanol used in the first step is 95%.

Preferably in this technical solution, the eluent in the step 3 is petroleum ether/acetone, wherein the volume ratio of petroleum ether and acetone is 8:1-2:1.

Preferably in the technical scheme, in the step 4, semi-preparative HPLC is used to obtain compound 1 at a retention time of 40.2min with acetonitrile and water volume ratio of 40:60, a flow rate of 3.0mL/min, and compound 2 at a retention time of 41.0min. .

Its application method is as follows, applying any one or a mixture of the two compounds 1 and 2 to the benzoquinone reductase-inducing activity in liver cancer cells.

Compared with prior art, the beneficial effect of the present invention is:

Compound 1 and compound 2 proposed in the present invention belong to new compounds, which have not been disclosed. At the same time, by carrying out the benzoquinone reductase induction activity test on compound 1 and compound 2, the results show that compound 1 and compound 2 have the effect on Hepa1c1c7 Mouse liver cancer cells have moderately strong benzoquinone reductase inducing activity. The experimental results show that the compound 1 and compound 2 proposed in the present invention can be used to prepare benzoquinone reductase inducers for the preparation of antitumor drugs. It has important significance and prospects in the research of tumor drugs.

Description of drawings

Figure 1 is the ¹ H-NMR spectrum of compound 1 proposed by the present invention (deuterated reagent: CD ₃ OD);

Figure 2 is the ¹³ C-NMR spectrum of compound 1 proposed by the present invention (deuterated reagent: CD ₃ OD);

Fig. 3 is the HSQC spectrum of compound 1 proposed by the present invention (deuterated reagent: CD ₃ OD);

Figure 4 is the HMBC spectrum of compound 1 proposed by the present invention (deuterated reagent: CD ₃ OD);

Figure 5 is the ¹ H- ¹ H COZY spectrum of compound 1 proposed by the present invention (deuterated reagent: CD ₃ OD);

Figure 6 is the ROESY spectrum of compound 1 proposed by the present invention (deuterated reagent: CD ₃ OD);

Fig. 7 is the (+)-HRESIMS spectrum of compound 1 proposed by the present invention;

Fig. 8 is the IR spectrum of compound 1 proposed by the present invention;

Figure 9 is the ¹ H-NMR spectrum of compound 2 proposed by the present invention (deuterated reagent: CDCl ₃ );

Figure 10 is the ¹³ C-NMR spectrum of compound 2 proposed by the present invention (deuterated reagent: CDCl ₃ );

Figure 11 is the HSQC spectrum of compound 2 proposed by the present invention (deuterated reagent: CDCl ₃ );

Figure 12 is the HMBC spectrum of compound 2 proposed by the present invention (deuterated reagent: CDCl ₃ );

Figure 13 is the ROESY spectrum of compound 2 proposed by the present invention (deuterated reagent: CDCl ₃ );

Figure 14 is the (+)-HRESIMS spectrum of compound 2 proposed by the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts 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", The orientation or positional relationship indicated by "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, not to indicate or imply the referred device Or elements must have a particular orientation, be constructed and operate in a particular orientation, and therefore should not be construed as limiting the invention.

In addition, it should be understood that, for the convenience of description, the dimensions of the various components shown in the drawings are not drawn according to the actual scale relationship, for example, the thickness or width of some layers may be exaggerated relative to other layers.

It should be noted that similar reference numerals and letters denote similar items in the following drawings, therefore, once an item is defined or described in one drawing, it will not need to be redefined in the description of subsequent drawings. It is further discussed and described in detail.

The invention provides a technical solution: preparation and application of a class of isoquinoline alkaloids with benzoquinone reductase inducing activity, the isoquinoline alkaloids with benzoquinone reductase inducing activity, including compound 1 and compound 2 , the chemical structural formulas of the compound 1 and the compound 2 are respectively as follows:

该化学结构式中的阿拉伯数字是化学结构中碳原子的标位。

The Arabic numerals in the chemical structural formula are the positions of the carbon atoms in the chemical structure.

Its preparation steps are as follows:

Step 1: After crushing 6.0kg of dry Cryptocarya wrayi sprigs, use 30L concentration of 95% ethanol to extract by dipping method, soak for 7 days each time, extract 3 times in total, obtain extract after merging, and extract the extract at a temperature of 40 Under the condition of ℃, concentrate and recover ethanol under reduced pressure to obtain 285.0 g of crude extract;

Step 2: Disperse the crude extract obtained in step 1 in water to form a suspension, extract the suspension 3 times with 1.5 L of ethyl acetate, and concentrate the obtained ethyl acetate phase under reduced pressure to obtain ethyl acetate extraction extract 38.5g;

Step 3: Extract the extract with ethyl acetate and perform MCI column chromatography, elute with methanol/water gradient, and combine similar fractions according to TLC color development to obtain 7 components, which are respectively Fr.1-Fr.7, and Wherein the Fr.5 component of 4.6g is separated on the SephadexLH-20 (EtOH) gel column to obtain 4 subcomponents, which are respectively Fr.7A-Fr.7D, wherein the Fr.7C subcomponent of 476.8mg Four subcomponents were obtained by silica gel column chromatography, which were Fr.7C ₁ -Fr.7C ₄ , wherein the eluent was petroleum ether/acetone, and the volume ratio of petroleum ether and acetone was 4:1;

Step 4: The 104.8 mg Fr.7C ₂ subcomponent obtained in Step 3 was further purified by semi-preparative HPLC with acetonitrile and water volume ratio of 40:60, flow rate 3.0mL/min, and obtained when the retention time was 40.2min 2.0 mg of compound 1 and 1.2 mg of compound 2 were obtained at a retention time of 41.0 min.

The chemical structures of compound 1 and compound 2 have been confirmed by various modern spectroscopic techniques such as NMR, HRESIMS, calculated ECD and UV according to the routine, and their physical and chemical properties are as follows:

(c 0.05,CH₃OH)；UVλ_max(logε)231(4.03),278(3.78),305(3.70)nm；ECD(c 2.0×10^-5gmL^-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；¹H-NMR和¹³C-NMR数据，见表1；(+)HR-ESI-MS m/z372.1440[M+H]⁺(calcd for C₂₀H₂₂NO₆,372.1447).Compound 1: yellow amorphous powder;

(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).

(c 0.02,CH₃OH)；UVλ_max(logε)208(4.40),224(4.43),274(3.98),303(3.89)nm；ECD(c 1.7×10^-4gmL^-1,MeOH)λ(Δε)220(-28.86),237(+32.36),260(+9.52),284(-6.79),301(4.21),321(-7.62)nm；¹H-NMR和¹³C-NMR数据，见表1；(+)HR-ESI-MS m/z370.1288[M]⁺(calcd for C₂₀H₂₀NO₆,370.1285).Compound 2: yellow amorphous powder;

(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/z 370.1288[M] ⁺ (calcd for C ₂₀ H ₂₀ NO ₆ ,370.1285).

The specific proton spectrum and carbon spectrum data of compound 1 and compound 2 are shown in Table 1.

Table 1

Application experiment: Hepa 1c1c7 mouse liver cancer cells (ATCC CRL-2026) were cultured in Eagle's minimal basal medium supplemented with 10% fetal bovine serum at 37°C in 95% air and 5% CO ₂ humidified air , the cells were seeded in 96-well plates and treated with the indicated doses of compounds for 24 h. The medium was removed, and the cells were lysed at 37°C for 15 minutes with 30 µL of cell lysing buffer [0.8% digitalisin and 2 mM EDTA solution (pH 7.8)]. Then, 170 μL containing bovine serum albumin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1.5% Tween 20, 0.5 M trishydrochloride, 7.5mM flavin adenine dinucleotide, 150mM glucose-6-phosphate, 10 units/μL glucose-6-phosphate dehydrogenase, 50mM nicotinamide adenine dinucleotide A complete reaction mixture of glycoside phosphate and 50 mM menadione was added to each well. After incubation for about 4 minutes, a blue color was formed, and the absorbance at 630 nm was recorded on a microplate reader.

Experimental results: Compared with the positive control, sulforaphane showed 1.7-fold induction activity at 2.0 μM, compound 1 and compound 2 both showed moderate-intensity QR induction activity, and showed 1.2-fold and 1.1-fold induction activity at 3.125 μM concentration, respectively.

Through the above application test, it can be known that compound 1 and compound 2 have moderately strong benzoquinone reductase inducing activity on Hepa 1c1c7 mouse liver cancer cells, which can be used to prepare benzoquinone reductase inducers for the preparation of antitumor drugs. It is of great significance and prospect in the research of medicine.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by 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:

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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