CN114751906A - Cyclopentenoquinolizine imidazole compound and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of chemical drugs, and particularly relates to a cyclopentenoquinolizine imidazole compound as well as a preparation method and application thereof. The cyclopentenoquinolizinimidazole compound is prepared by taking genipin and histidine as raw materials, reacting under specific conditions, and separating by column chromatography and liquid chromatography. Experiments prove that the compound has a remarkable inhibiting effect on proliferation of colon cancer cells and can be used for preparing antitumor drugs.

Description

Cyclopentenoquinolizine imidazole compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of chemical drugs, and particularly relates to a cyclopentenoquinolizine imidazole compound as well as a preparation method and application thereof.

Background

Colon cancer is a malignant tumor of the digestive tract occurring in the colon part of a human body, is a common disease, often occurs in the junction part of the rectum and the sigmoid colon, and patients with chronic colitis, colon polyp, male obesity and the like are susceptible people.

Surgery is the first method for treating colon cancer, but residual focus is left, so that relapse and metastasis are easy to occur; radiotherapy and chemotherapy are common methods for treating colon cancer, but radiotherapy has great side effects, can cause serious adverse reactions such as physical weakness, immunologic function reduction, bone marrow suppression, digestive tract disorder and the like of a patient, and can also damage main organs such as liver and kidney of the patient; chemotherapy also causes many toxic and side effects, which are likely to cause complications, sequelae, etc. Therefore, obtaining more compounds capable of being used for colon cancer is of great significance to drug development and clinical treatment of colon cancer.

Disclosure of Invention

Aiming at the problems, the invention provides a cyclopentenoquinolizinidazole compound, a preparation method and application thereof.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

in a first aspect, an embodiment of the present invention provides a cyclopentenoquinolizinio imidazole compound, which has a structural formula shown in formula I:

experiments prove that the compound has a remarkable inhibiting effect on the proliferation of colon cancer cells. At present, no report related to the cyclopentenoquinolizinimidazole compound is found.

The invention also provides a preparation method of the cyclopentenoquinolizine imidazole compound, which comprises the following steps:

a, dissolving genipin and histidine in a non-nitrogen buffer solution with the pH value of 4-10, and reacting at 30-80 ℃ for more than 2 hours to obtain a reaction solution;

and step B, adding the reaction solution into a small-hole resin chromatographic column, performing gradient elution by sequentially adopting methanol-water solvents with volume ratios of 0:100, 5:95, 1:9, 2:8 and 4:6, and collecting eluent obtained by elution of the methanol-water solvents with the volume ratio of 4:6 to obtain the cyclopentenoquinolizine imidazole compound.

The cyclopentenooquinazine imidazole compound is obtained by reacting genipin and histidine in a specific solution at a specific temperature, and then the cyclopentenooquinazine imidazole compound is separated from other compounds in the reaction solution through a small-hole resin column chromatography, so that the cyclopentenooquinazine imidazole compound can be obtained from an eluent.

In combination with the first aspect, the preparation method may further include separating the eluate by preparative liquid chromatography, collecting an eluted fraction according to a peak appearance time of a chromatographic peak of the cyclopentenoquinolizinimidazole compound, concentrating, and drying to obtain a pure product of the cyclopentenoquinolizinimidazole compound in a specific eluted fraction.

Preferably, the chromatographic conditions of the preparative liquid chromatography are: the chromatographic column is a carbon-eighteen bonded silica gel chromatographic column, the mobile phase is 0.05-0.20% (v/v) formic acid water-methanol with the volume ratio of (80:20) - (60:40), and isocratic elution is carried out.

Under the chromatographic condition, the chromatographic column can be selected from Zorbax PrepHTXDB C18 with the specification of 21.2X 250mm and 7 μm; the mobile phase can be 0.1% (v/v) formic acid water-methanol with the volume ratio of 65: 35; the flow rate of isocratic elution can be selected from 10 mL/min; the sample size can be 1 mL.

With reference to the first aspect, the buffer solution is a potassium dihydrogen phosphate-sodium hydroxide buffer solution, a PBS buffer solution, a sodium acetate-glacial acetic acid, or a borax-sodium carbonate buffer solution.

In combination with the first aspect, the pH of the buffer solution is preferably 7.35.

In combination with the first aspect, the temperature of the reaction in step A is preferably 32 to 42 ℃.

In connection with the first aspect, the amount of each elution solvent used in step B may be selected from 3 column volumes to ensure separation of the target compound from other compounds and to obtain a better enrichment of the target compound.

In a second aspect, the embodiment of the invention also provides an application of the cyclopentenoquinolizinimidazole compound in preparing an antitumor drug.

Experiments prove that the compound can obviously inhibit the proliferation activity (P <0.05) of SW480 cells of a human colon cancer cell line and CT26 cells of a mouse colon cancer cell line, so that the compound can be applied to preparing antitumor drugs.

In combination with the second aspect, the anti-tumor drug is an anti-colon cancer drug.

In combination with the second aspect, the antitumor drug is a targeted preparation against colon cancer. The compound is prepared into a targeting preparation, so that the drug effect can be improved, and the influence on other tissues can be reduced.

Drawings

FIG. 1 shows Genihistidine B in example 1 of the present invention¹H-NMR spectrum;

FIG. 2 is a drawing showing Genihistidine B in example 1 of the present invention¹³C-NMR spectrum;

FIG. 3 is a drawing showing Genihistidine B in example 1 of the present invention¹H-¹H COSY spectrogram;

FIG. 4 is a HSQC spectrum of Genihistidine B in example 1 of the present invention;

FIG. 5 is an HMBC chromatogram of Genihistidine B in example 1 of the present invention;

FIG. 6 shows that Genihistidine B inhibits the proliferative activity of SW480 cells of human colorectal cancer cell line in vitro in example 8

FIG. 7 shows that Genithidine B inhibits the cell proliferation activity of the mouse colorectal cancer cell line CT26 in vitro in example 8.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The incidence of colon cancer is high, the cure rate and survival rate of middle and late stages are reduced, and in order to obtain more compounds for treating colon cancer, the embodiment of the invention provides a cyclopentenoquinolizinimidazole compound (named as Genihistidine B), the structural formula of which is shown as formula I:

Experiments show that Genihistidine B can inhibit the proliferation activity of SW480 cells of a human colon cancer cell line and CT26 cells of a mouse colon cancer cell line (P <0.05), and has important significance for treating colon cancer.

The embodiment of the invention also provides a preparation method of the compound, which comprises the following steps:

a, dissolving genipin and histidine in a non-nitrogen buffer solution with the pH value of 4-10, and reacting at 30-80 ℃ for more than 2 hours to obtain a reaction solution;

and step B, adding the reaction solution into a small-pore resin chromatographic column, performing gradient elution by sequentially adopting methanol-water solvents with volume ratios of 0:100, 5:95, 1:9, 2:8 and 4:6, and collecting eluent obtained by elution of the methanol-water solvents with the volume ratio of 4:6, so as to obtain Genihistidine B in the eluent.

In this preparation, the eluate obtained in step B can be further purified by preparative liquid chromatography to obtain a pure product of Genihistidine B. The purification method can select the following operations: and D, separating the eluent obtained in the step B by using preparative liquid chromatography, wherein the chromatographic column is a carbon-eighteen bonded silica gel chromatographic column, and the mobile phase is 0.05-0.20% (v/v) formic acid water-methanol with the volume ratio of (80:20) - (60:40), and isocratic elution is carried out. Wherein the chromatographic column can be Zorbax PrepHTXDB C18 with specification of 21.2 × 250mm and 7 μm; the mobile phase can be 0.1% (v/v) formic acid water-methanol with the volume ratio of 65: 35; the flow rate of isocratic elution may be selected to be 10 mL/min. Under the condition, collecting the elution fraction for 12.0-13.0 min according to the peak appearance time, concentrating and drying to obtain a pure product of Genihistidine B.

In the preparation method, the mass ratio of genipin to histidine is not limited, and the cyclopentenoquinolizinimidazole compound can be prepared from both the genipin and the histidine by the preparation method under any mass ratio. However, the mass ratio of the raw materials is not limited in the preparation method, and the preparation method does not mean that all genipin and histidine can participate in the reaction at any ratio, and the raw materials with large feeding amount are remained in the reaction process. In order to ensure the yield of the product and reduce the waste of raw materials, the mass ratio of genipin to histidine is preferably 1: 3-4, and more preferably 1: 3.4-3.5.

The type of the buffer solution is not limited in the invention, and a non-nitrogen-containing common buffer solution can be used, such as potassium dihydrogen phosphate-sodium hydroxide buffer solution, PBS buffer solution, sodium acetate-glacial acetic acid or borax-sodium carbonate buffer solution.

The pH of the buffer solution is preferably 7.35. Under otherwise identical conditions, the cyclopentenoquinolizinimidazoles produced in the highest yields at a pH of 7.35.

The reaction temperature of the preparation method is preferably 32-42 ℃, and the reaction time is 60 hours. At the preferred raw material mass ratio, reaction temperature, pH value and reaction time, more Genihistidine B as the target product can be produced.

During the elution of the small pore resin column chromatography, each of the elution solvents was used in 3 column volumes to improve the separation of Genihistidine B from other compounds and to enrich Genihistidine B. The small-pore resin chromatographic column can use CHP20/P120 small-pore resin as packing.

The embodiment of the present invention will be described below with reference to specific examples.

Example 1

This example provides cyclopentenoquinolizinimidazole compound Genihistidine B and a method for preparing the same.

The preparation method of Genihistidine B specifically comprises the following steps:

and step A, precisely weighing 113mg of genipin and 387.5mg of histidine, respectively placing the genipin and the histidine in 100mL measuring bottles, adding a PBS (phosphate buffer solution) with the pH value of 7.35 to dissolve, fixing the volume to a scale, and uniformly mixing to obtain stock solutions with the concentrations of 5 and 25mmol/L respectively. Respectively transferring 90mL of the stock solutions, placing the stock solutions into a beaker, adding a PBS (phosphate buffer solution) with the pH value of 7.35 to 450mL, uniformly mixing, reacting in a water bath at 37 ℃ for 60 hours, and taking out to obtain a reaction solution;

step B, separating the reaction solution by using CHP20/P120 small-pore resin through column chromatography, and performing gradient elution by using methanol-water (0:100, 5:95, 1:9, 2:8 and 4:6) solvent systems in sequence (each elution system elutes 3 column volumes) to obtain a series of eluents; mixing 40% methanol water eluate (methanol: water is 4:6), concentrating, and separating and purifying the prepared liquid phase;

And step C, taking a 40% methanol water eluent concentrated solution, separating and purifying by using preparative liquid chromatography, wherein a chromatographic column is Zorbax PrepHTXDB C18(21.2 x 250mm, 7 mu m), a mobile phase is 0.1% (v/v) formic acid water-methanol (65:35), isocratic elution is carried out at a flow rate of 10mL/min, a detection wavelength is 254nm, the sample inlet amount is 1mL, 12.0-13.0 min of elution fractions are collected according to peak emergence time, concentration and drying are carried out, and Genihistidine B is obtained, wherein the yield is 4.3% (the yield is the weight of the Genihistidine B divided by the total weight of genipin and histidine multiplied by 100%, the same below), and the purity is 99.2% (the peak area is the peak area of the fraction collected from the preparative liquid phase, which is detected in an ultra-high performance liquid chromatograph, and the peak area of the Genihistidine B is divided by the sum of all ultra-high performance liquid chromatography multiplied by 100%, the peak area, and the same below).

By NMR¹H-NMR、¹³C-NMR、¹H-¹H COSY, HSQC and HMBC) technology for representing the chemical structure of Genihistidine B,¹the H-NMR spectrum is shown in figure 1,¹³the C-NMR spectrum is shown in figure 2,¹H-¹the H COSY spectrum is shown in figure 3, the HSQC spectrum is shown in figure 4, and the HMBC spectrum is shown in figure 5.

In that¹In the H-NMR spectrum, the characteristic proton signals delta 7.69(1H, s, H-2), the three methylene proton signals delta 2.75, 2.16(H-5), delta 3.19, 2.99(H-15), delta 4.24(H-19) are shown. The high field shows 1 methoxy signal, delta 3.67 (H-18). ¹³The C-NMR spectrum shows 17 carbon signals, mainly comprising 1 ester carbonyl signal delta 171.7, 4 aromatic carbon signals delta 149.3, 99.5, 131.8 and 144.6, 2 methylene signals delta 38.6, 59.8, 3 methine signals delta 37.9, 46.9 and 51.5, and also shows a methoxyl signal delta 51.1; the δ 177.3 signal is presumed to be the carboxyl carbon signal, and δ 130.6, 134.6, 124.5 are carbon signals on the imidazole ring. Compound (I)¹H-¹In the H COSY spectrum, the correlation exists between H-6 and H-5, H-4 and H-8, and H-16 and H-15; in HMBC spectrum, H-9 is observed to be related to a C-14 signal, H-16 is related to a C-9 signal, and genipin and histidine can be determined to be connected through C-9 and C-10 phases; in addition, it was observed that H-2 is associated with C-16, and H-16 is associated with C-9.

According to the NMR spectrum, the structural formula of the compound is shown as the formula I:

example 2

This example provides a method for preparing Genihistidine B.

The method specifically comprises the following steps:

and step A, precisely weighing 113mg of genipin and 339mg of histidine, respectively placing the genipin and the histidine into 100mL measuring bottles, adding PBS (phosphate buffer solution) with the pH value of 7.35 to dissolve, fixing the volume to a scale, and uniformly mixing to obtain stock solutions with the concentrations of 5 and 22mmol/L respectively. Respectively transferring 90mL of the stock solutions, placing the stock solutions into a beaker, adding a PBS (phosphate buffer solution) with the pH value of 7.35 to 450mL, uniformly mixing, reacting in a water bath at 32 ℃ for 55 hours, and taking out to obtain a reaction solution;

Step B, separating the reaction solution by using CHP20/P120 pore resin through column chromatography, and performing gradient elution by using methanol-water (0:100, 5:95, 1:9, 2:8 and 4:6) solvent systems in sequence (each elution system elutes 3 column volumes) to obtain a series of eluents; mixing 40% methanol water eluate (methanol: water is 4:6), concentrating, and separating and purifying the prepared liquid phase;

and step C, taking a concentrated solution of 40% methanol water eluent, separating and purifying by adopting a preparative liquid chromatography, wherein a chromatographic column is Zorbax PrepHTXDB C18 (21.2X 250mm, 7 mu m), a mobile phase is 0.2% (v/v) methanol-methanol (60:40), isocratic elution is carried out at a flow rate of 10mL/min, a detection wavelength is 254nm, the sample injection amount is 1mL, an elution fraction of 11-12 min is collected according to the peak time, and concentration and drying are carried out to obtain Genihistidine B, the yield is 38%, and the purity is 90.4%.

Example 3

This example provides a method for preparing Genihistidine B.

The method specifically comprises the following steps:

and step A, precisely weighing 113mg of genipin and 452mg of histidine, respectively placing the genipin and the histidine in 100mL measuring bottles, adding PBS (phosphate buffer solution) with the pH value of 7.35 to dissolve, fixing the volume to a scale, and uniformly mixing to obtain stock solutions with the concentrations of 5 and 29mmol/L respectively. Respectively transferring 90mL of the stock solutions, placing the stock solutions into a beaker, adding a PBS (phosphate buffer solution) with the pH value of 7.35 to 450mL, uniformly mixing, reacting in a 42 ℃ water bath for 65 hours, and taking out to obtain a reaction solution;

Step B, separating the reaction solution by using CHP20/P120 pore resin through column chromatography, and performing gradient elution by using methanol-water (0:100, 5:95, 1:9, 2:8 and 4:6) solvent systems in sequence (each elution system elutes 3 column volumes) to obtain a series of eluents; mixing 40% methanol water eluate (methanol: water is 4:6), concentrating, and separating and purifying the prepared liquid phase;

and step C, taking a concentrated solution of 40% methanol water eluent, separating and purifying by adopting a preparative liquid chromatography, wherein a chromatographic column is Zorbax PrepHTXDB C18 (21.2X 250mm, 7 mu m), a mobile phase is 0.05% (v/v) methanol-methanol (80:20), isocratic elution is carried out at a flow rate of 10mL/min, a detection wavelength is 254nm, the sample injection amount is 1mL, elution fractions of 14-16 min are collected according to peak time, and are concentrated and dried to obtain Genihistidine B, the yield is 3.8%, and the purity is 99.0%.

Example 4

This example provides cyclopentenoquinolizinimidazole compound Genihistidine B and a method for preparing the same.

The preparation method of Genihistidine B specifically comprises the following steps:

and step A, precisely weighing 113mg of genipin and 387.5mg of histidine, respectively placing the genipin and the histidine in 100mL measuring bottles, adding borax-sodium carbonate buffer solution with the pH value of 9 to dissolve, fixing the volume to a scale, and uniformly mixing to obtain stock solutions with the concentrations of 5 and 25mmol/L respectively. Respectively transferring 90mL of the stock solutions, putting the stock solutions into a beaker, adding a borax-sodium carbonate buffer solution with the pH value of 9 to 450mL, uniformly mixing, reacting in a water bath at 37 ℃ for 60 hours, and taking out to obtain a reaction solution;

Step B, separating the reaction solution by using CHP20/P120 pore resin through column chromatography, and performing gradient elution by using methanol-water (0:100, 5:95, 1:9, 2:8 and 4:6) solvent systems in sequence (each elution system elutes 3 column volumes) to obtain a series of eluents; mixing 40% methanol water eluate (methanol: water is 4:6), concentrating, and separating and purifying the prepared liquid phase;

and step C, separating and purifying a concentrated solution of 40% methanol water eluent by adopting a preparative liquid chromatography, wherein a chromatographic column is Zorbax PrepHTXDB C18(21.2 x 250mm, 7 mu m), a mobile phase is 0.1% (v/v) methanol-methanol (65:35), isocratic elution is carried out at a flow rate of 10mL/min, the detection wavelength is 254nm, the sample inlet amount is 1mL, elution fractions of 12.0-13.0 min are collected according to the peak-out time, and are concentrated and dried to obtain Genihistidine B, the yield is 4.0%, and the purity is 98.9%.

Example 5

This example provides a cyclopentenoquinolizinimidazole Genihistidine B compound and a method for preparing the same.

The preparation method of Genihistidine B specifically comprises the following steps:

and step A, precisely weighing 113mg of genipin and 387.5mg of histidine, respectively placing the genipin and the histidine in 100mL measuring bottles, adding a sodium acetate-glacial acetic acid buffer solution with the pH value of 6 to dissolve, fixing the volume to a scale, and uniformly mixing to obtain stock solutions with the concentrations of 5 and 25mmol/L respectively. Respectively transferring 90mL of the stock solutions, putting the stock solutions into a beaker, adding a sodium acetate-glacial acetic acid buffer solution with the pH value of 6 to 450mL, uniformly mixing, reacting in a water bath at 37 ℃ for 60 hours, and taking out to obtain a reaction solution;

Step B, separating the reaction solution by using CHP20/P120 pore resin through column chromatography, and performing gradient elution by using methanol-water (0:100, 5:95, 1:9, 2:8 and 4:6) solvent systems in sequence (each elution system elutes 3 column volumes) to obtain a series of eluents; mixing 40% methanol water eluate (methanol: water is 4:6), concentrating, and separating and purifying the prepared liquid phase;

and step C, taking a concentrated solution of 40% methanol water eluent, separating and purifying by adopting preparative liquid chromatography, wherein a chromatographic column is Zorbax PrepHTXDB C18 (21.2X 250mm, 7 mu m), a mobile phase is 0.1% (v/v) methanol-methanol (65:35), isocratic elution is carried out at a flow rate of 10mL/min, the detection wavelength is 254nm, the sample injection amount is 1mL, an eluted fraction of 12.0-13.0 min is collected according to the peak time, concentrating and drying are carried out, so that Genihistidine B is obtained, the yield is 2.5%, and the purity is 98.3%.

Example 6

This example provides cyclopentenoquinolizinimidazole compound Genihistidine B and a method for preparing the same.

The preparation method of Genihistidine B specifically comprises the following steps:

and step A, precisely weighing 113mg of genipin and 387.5mg of histidine, respectively placing the genipin and the histidine in 100mL measuring bottles, adding borax-sodium carbonate buffer solution with the pH value of 10 to dissolve, fixing the volume to a scale, and uniformly mixing to obtain stock solutions with the concentrations of 5 and 25mmol/L respectively. Respectively transferring 90mL of the stock solutions, putting the stock solutions into a beaker, adding a borax-sodium carbonate buffer solution with the pH value of 10 to 450mL, uniformly mixing, reacting in a water bath at 37 ℃ for 60 hours, and taking out to obtain a reaction solution;

Step B, separating the reaction solution by using CHP20/P120 small-pore resin through column chromatography, and performing gradient elution by using methanol-water (0:100, 5:95, 1:9, 2:8 and 4:6) solvent systems in sequence (each elution system elutes 3 column volumes) to obtain a series of eluents; mixing 40% methanol water eluate (methanol: water: 4:6), and concentrating for separating and purifying the prepared liquid phase;

and step C, separating and purifying a concentrated solution of 40% methanol water eluent by adopting a preparative liquid chromatography, wherein a chromatographic column is Zorbax PrepHTXDB C18(21.2 x 250mm, 7 mu m), a mobile phase is 0.1% (v/v) methanol-methanol (65:35), isocratic elution is carried out at a flow rate of 10mL/min, the detection wavelength is 254nm, the sample inlet amount is 1mL, elution fractions of 12.0-13.0 min are collected according to the peak-out time, and are concentrated and dried to obtain Genihistidine B, the yield is 1.2%, and the purity is 97.3%.

Example 7

This example provides a cyclopentenoquinolizinimidazole Genihistidine B compound and a method for preparing the same.

The preparation method of Genihistidine B specifically comprises the following steps:

and step A, precisely weighing 113mg of genipin and 387.5mg of histidine, respectively placing the genipin and the histidine in 100mL measuring bottles, adding a sodium acetate-glacial acetic acid buffer solution with pH of 4 to dissolve, fixing the volume to a scale, and uniformly mixing to obtain stock solutions with the concentrations of 5 and 25mmol/L respectively. Respectively transferring 90mL of the stock solutions, putting the stock solutions into a beaker, adding a sodium acetate-glacial acetic acid buffer solution with the pH value of 4 to 450mL, uniformly mixing, reacting in a water bath at 37 ℃ for 60 hours, and taking out to obtain a reaction solution;

Step B, separating the reaction solution by using CHP20/P120 pore resin through column chromatography, and performing gradient elution by using methanol-water (0:100, 5:95, 1:9, 2:8 and 4:6) solvent systems in sequence (each elution system elutes 3 column volumes) to obtain a series of eluents; mixing 40% methanol water eluate (methanol: water is 4:6), concentrating, and separating and purifying the prepared liquid phase;

and step C, separating and purifying a concentrated solution of 40% methanol water eluent by adopting a preparative liquid chromatography, wherein a chromatographic column is Zorbax PrepHTXDB C18(21.2 x 250mm, 7 mu m), a mobile phase is 0.1% (v/v) methanol-methanol (65:35), isocratic elution is carried out at a flow rate of 10mL/min, the detection wavelength is 254nm, the sample inlet amount is 1mL, elution fractions of 12.0-13.0 min are collected according to the peak-out time, and are concentrated and dried to obtain Genihistidine B, the yield is 1.9%, and the purity is 98.6%.

Example 8

3- (4, 5-dimethyl-2-thiazole) -2,5 diphenyl tetrazole bromide (MTT) can be reduced by dehydrogenase in mitochondria of living cells to generate a dark purple crystalline product formazan. The faster the cell proliferated, the higher the dehydrogenase activity and the more formazan formed. Formazan can be completely dissolved into blue by DMSO, has strong absorption peak at 570nm wavelength, and can be used for evaluating living cell number and proliferation activity by detecting absorbance value at 570nm wavelength by enzyme labeling instrument. This example demonstrates the effect of Genihistidine B on inhibiting colon cancer cell proliferation activity using MTT colorimetric assays for cell viability.

1. Reagent, instrument and consumable

(1) Reagent: MTT, Solarbio; DMSO, Solarbio; PBS buffer, Bai Si Nuo (Tianjin) Biotech limited.

(2) Instrument, consumable: type 450 microplate reader, shanghai berle corporation; 96-well plate, Nest.

2. Experimental methods

Preparation of MTT working solution: adding 25mg MTT powder into 5mL PBS buffer solution to prepare 5mg/mL solution, subpackaging, and storing at-20 ℃ in the dark.

The colon cancer cell line SW480 in the logarithmic growth phase was selected, the cell mass was digested into single cells with 0.25% (g/mL) of trypsin, digestion was stopped by adding three volumes of DMEM complete medium, centrifugation was performed at 1000rpm for 5min, and the supernatant was discarded. 5mL of DMEM complete medium is added, 10 mu L of cell suspension is sucked after uniform mixing, and cell liquid is injected into a counting plate along the edge of a cover slip to count the cells. Preparation of 4X 10 with DMEM complete Medium⁴Cell suspension/mL, seeded in 96-well plates at 200. mu.L per well and placed in CO₂Culturing in an incubator for 24 h. Removing culture solution from a 96-well plate, and adding Genihistidine B solution with the concentration of 10, 20, 30, 40 and 50 mu M respectively diluted by DMEM complete medium, wherein 10 multiple wells are arranged at each concentration; meanwhile, a blank control group was set up. Continuously placing in CO ₂After 24h incubation in the incubator the test drug was removed, 100. mu.L/well MTT solution (dissolved in DMEM complete medium at a concentration of 0.5mg/mL) was added, after incubation for 4h at 37 ℃ the crystalline dark purple product was observed microscopically, 100. mu.L/well DMSO was added to dissolve the dark purple crystals and the absorbance value (A) was measured at 570 nm.

The colon cancer cell line CT26 was treated in the same manner and completely cultured in RPMI 1640Preparation of nutrient 4X 10⁴Cell suspension of cell/mL, seeded in 96-well plates at 200. mu.L per well and placed in CO₂Culturing in an incubator for 24 h. Removing the culture solution from the 96-well plate, adding Genihistidine B solution with concentration gradient of 5, 10, 15, 20, 25, 30 μ M diluted by RPMI 1640 complete medium, and setting 10 multiple wells for each concentration; meanwhile, a blank control group was established. Continuously placing in CO₂After 24h incubation in an incubator the test drug was removed, 100. mu.L/well MTT solution (dissolved in RPMI 1640 complete medium at a concentration of 0.5mg/mL) was added, incubation at 37 ℃ for 4h and microscopic observation of the crystalline dark purple product, 100. mu.L/well DMSO was added to dissolve the dark purple crystals and the absorbance value (A) was measured at 570 nm.

The cell viability of colon cancer cells was calculated as follows:

Cell viability/% ═ a_{Testing drugs}/A_{Blank space}×100％

3. Statistical analysis

Data from the study were analyzed using Graphpad Prism 8 statistical software. Analysis of differences between blank control and experimental groups was performed using one-way ANOVA.

4. Results of the experiment

The results show that: genihistidine B significantly inhibited the proliferation activity of SW480 cell, the mouse CT26 cell, the human colon cancer cell line (P <0.001), and the dose-dependent relationship between the cell and the human colon cancer cell line (P <0.001), compared to the blank control group, and the results are shown in FIGS. 6 and 7. In the figure, C is a blank control group; denotes P < 0.001.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. A cyclopentenoquinolizinimidazole compound is characterized in that the structural formula is shown as a formula I:

2. a process for the preparation of cyclopentenoquinolizinimidazoles as claimed in claim 1, comprising the steps of:

step A, dissolving genipin and histidine in a non-nitrogen-containing buffer solution with the pH value of 4-10, and reacting at 30-80 ℃ for more than 2 hours to obtain a reaction solution;

And step B, adding the reaction solution into a small-pore resin chromatographic column, performing gradient elution by sequentially adopting methanol-water solvents with volume ratios of 0:100, 5:95, 1:9, 2:8 and 4:6, and collecting eluent obtained by elution of the methanol-water solvents with the volume ratio of 4:6 to obtain the cyclopentenoquinolizine imidazole compound.

3. The method according to claim 2, characterized in that the method further comprises separating the eluate by preparative liquid chromatography, collecting the eluate according to the peak appearance time of the chromatographic peak of the cyclopentenoquinolizinimaimidazoles, concentrating, and drying to obtain the pure cyclopentenoquinolizinimaimidazoles.

4. The method for preparing cyclopentenoquinolizinio imidazole compounds according to claim 3, wherein the chromatographic conditions of the preparative liquid chromatography are as follows: the chromatographic column is a carbon-eighteen bonded silica gel chromatographic column, the mobile phase is 0.05-0.20% (v/v) formic acid water-methanol with the volume ratio of (80:20) - (60:40), and isocratic elution is carried out.

5. The method for preparing cyclopentenoquinolizinio imidazoles according to claim 2, wherein the chromatographic column is Zorbax PrepHTXDB C18 with specification of 21.2 x 250mm, 7 μm; and/or

The mobile phase is 0.1% (v/v) formic acid water-methanol with a volume ratio of 65: 35; and/or

Isocratic elution is carried out at the flow rate of 10 mL/min; and/or

The sample size was 1 mL.

6. The method for preparing cyclopentenoquinolizinimidazole compounds according to claim 2, wherein the buffer solution is potassium dihydrogen phosphate-sodium hydroxide buffer solution, PBS buffer solution, sodium acetate-glacial acetic acid buffer solution or borax-sodium carbonate buffer solution; and/or

The pH of the buffer solution was 7.35.

7. The method for preparing cyclopentenoquinolizinimidazole compounds according to claim 2, wherein the reaction temperature in step A is 32-42 ℃; and/or

The amount of each eluting solvent used in step B was 3 column volumes.

8. Use of the cyclopentenoquinolizinimidazole compound of claim 1 in the preparation of an antitumor drug.

9. The use according to claim 8, wherein the anti-neoplastic drug is an anti-colon cancer drug.

10. The use according to claim 9, wherein the anti-tumor drug is a targeted agent against colon cancer.

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