CN114751906B

CN114751906B - Cyclopentenoquinolizinimidazole compound as well as preparation method and application thereof

Info

Publication number: CN114751906B
Application number: CN202210308458.8A
Authority: CN
Inventors: 柴欣; 张潇予; 王跃飞; 于卉娟; 王丹妮; 房士明; 于海洋; 董雪媛; 李�瑞
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2022-03-23
Filing date: 2022-03-23
Publication date: 2023-03-17
Anticipated expiration: 2042-03-23
Also published as: CN114751906A

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 cyclopentenoquinolizinimidazole 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 choice for the treatment of colon cancer, but leaves residual lesions, leading to relapse and metastasis; 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 cyclopentenoquinolizinimidazole compound, a preparation method and an 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. No report related to the cyclopentenoquinolizinimidazole compound is found at present.

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

step 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 2h 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 a methanol-water solvent with a volume ratio of 0, 5, 95, 1, 2, 8 and 4.

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.

With reference to 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) of formic acid-methanol in a volume ratio of (80.

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 selected from 0.1% (v/v) formic acid water-methanol with the volume ratio of 65; the flow rate of isocratic elution can be selected from 10mL/min; the sample amount can be 1mL.

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 connection 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 anti-tumor medicament.

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.

With reference to the second aspect, the antitumor drug is an anti-colon cancer drug.

In combination with the second aspect, the antitumor drug is a targeting 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 invention ¹ H-NMR spectrum;

FIG. 2 shows Genihistidine B in example 1 of the present invention ¹³ A C-NMR spectrum;

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

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

FIG. 5 is a HMBC spectrum 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 further described in detail with reference to the following 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:

and step B, adding the reaction solution into a small-pore resin chromatographic column, performing gradient elution by using a methanol-water solvent with the volume ratio of 0.

In the preparation method, the eluent obtained in the step B can be further purified by preparative liquid chromatography separation so as to obtain a pure product of Genihistidine B. The purification method can select the following operations: and (B) 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. Wherein, the chromatographic column can be selected from Zorbax PrepHTXDB C18 with the specification of 21.2 × 250mm and 7 μm; the mobile phase can be selected from 0.1% (v/v) formic acid water-methanol with the volume ratio of 65; the flow rate for isocratic elution may be selected to be 10mL/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 the pure Genihistidine B product.

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 preparation method does not limit the mass ratio of the raw materials, and 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.

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 cyclopentenoquinolizinimidazole compound produced the highest amount at a pH of 7.35.

The reaction temperature of the preparation method is preferably 32-42 ℃, and the reaction time is 60h. Under the preferable raw material mass ratio, reaction temperature, pH value and reaction time, more Genihistidine B as a target product can be generated.

During the elution of the small pore resin column chromatography, each of the elution solvents was used in an amount of 3 column volumes to enhance 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 a filler.

The embodiments of the present invention are 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 PBS (phosphate buffer solution) with the pH =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 in a beaker, adding a PBS (phosphate buffer solution) with the pH =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, the reaction solution was separated by column chromatography using CHP20/P120 pore resin, and gradient elution was performed sequentially with a methanol-water (0, 5; mixing 40% methanol water eluate (methanol: water is 4;

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 PrepHTXDDB C18 (21.2 x 250mm,7 mu m), a mobile phase is 0.1% (v/v) formic acid water-methanol (65.

By NMR ¹ H-NMR、 ¹³ C-NMR、 ¹ H- ¹ H COSY, HSQC, HMBC) technology for representing the chemical structure of Genihistidine B, ¹ the H-NMR spectrum is shown in figure 1, ¹³ the C-NMR spectrum is shown in FIG. 2, ¹ H- ¹ the 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 that ¹ In 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). ¹³ 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, 144.6,2 methylene signals delta 38.6, 59.8,3 methine signals delta 37.9, 46.9, 51.5, and also shows a methoxy 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 the carbon signals on the imidazole ring. Compound (I) ¹ H- ¹ In 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 producing 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 in 100mL measuring flasks, adding PBS (phosphate buffer solution) with the pH =7.35 to dissolve, fixing the volume to a scale, and uniformly mixing to obtain stock solutions with the concentrations of 5mmol/L and 22 mmol/L. Respectively transferring 90mL of the stock solutions, placing the stock solutions in a beaker, adding a PBS (phosphate buffer solution) with the pH =7.35 to 450mL, uniformly mixing, reacting in a water bath at 32 ℃ for 55h, and taking out to obtain a reaction solution;

step B, the reaction solution was separated by column chromatography using CHP20/P120 pore resin, gradient eluted sequentially with methanol: water (0, 5, 95, 1, 9, 2, 8, 4) solvent system (3 column volumes eluted per elution system) to give a series of eluents; mixing 40% methanol water eluate (methanol: water is 4);

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 PrepHTXDDB C18 (21.2X 250mm,7 mu m), a mobile phase is 0.2% (v/v) formic acid water-methanol (60).

Example 3

This example provides a method for producing 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 =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 in a beaker, adding a PBS (phosphate buffer solution) with the pH =7.35 to 450mL, uniformly mixing, reacting in a water bath at 42 ℃ for 65 hours, and taking out to obtain a reaction solution;

step B, the reaction solution was separated by column chromatography using CHP20/P120 pore resin, and gradient elution was performed sequentially with a methanol-water (0, 5; mixing 40% methanol water eluate (methanol: water is 4);

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 PrepHTXDDB C18 (21.2X 250mm,7 mu m), a mobile phase is 0.05% (v/v) formic acid water-methanol (80).

Example 4

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 pH =9 for dissolving, 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 in a beaker, adding a borax-sodium carbonate buffer solution with the pH =9 to 450mL, uniformly mixing, reacting in a water bath at 37 ℃ for 60 hours, and taking out to obtain a reaction solution;

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 PrepHTXDDB C18 (21.2X 250mm,7 mu m), a mobile phase is 0.1% (v/v) formic acid water-methanol (65).

Example 5

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

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 =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, placing the stock solutions in a beaker, adding a sodium acetate-glacial acetic acid buffer solution with the pH =6 to 450mL, uniformly mixing, reacting in a water bath at 37 ℃ for 60h, and taking out to obtain a reaction solution;

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 water-methanol (65).

Example 6

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 pH =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, placing the stock solutions into a beaker, adding a borax-sodium carbonate buffer solution with the pH =10 to 450mL, uniformly mixing, reacting in a water bath at 37 ℃ for 60 hours, and taking out to obtain a reaction solution;

Example 7

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

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 was 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 with microplate reader. 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) Instruments and consumables: type 450 microplate reader, shanghai berle corporation; 96-well plate, nest.

2. Experimental method

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 and the cell mass was digested with 0.25% (g/mL) trypsinThe cells were singulated, digestion was stopped by adding three volumes of DMEM complete medium, centrifugation was carried out 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 Using DMEM complete Medium ⁴ Cell suspension/mL, seeded in 96-well plates at 200. Mu.L per well and placed in CO ₂ Culturing in an incubator for 24h. Removing culture solution in a 96-well plate, adding Genihistidine B solution with the concentration of 10, 20, 30, 40 and 50 mu M respectively diluted by DMEM complete medium, and setting 10 multiple wells for each concentration; meanwhile, a blank control group was established. Continuously placing in CO ₂ After 24h incubation in an incubator the test drug was removed, 100. Mu.L/well MTT solution (dissolved in DMEM complete medium at a concentration of 0.5 mg/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 colon cancer cell line CT26 was treated in the same manner and prepared in RPMI 1640 complete medium at 4X 10 ⁴ Cell suspension/mL, seeded in 96-well plates at 200. Mu.L per well and placed in CO ₂ Culturing in an incubator for 24h. Removing the culture solution from the 96-well plate, adding Genihistidine B solution with concentration gradient of 5, 10, 15, 20, 25 and 30 mu M respectively diluted by RPMI 1640 complete medium, and setting 10 multiple wells for each concentration; meanwhile, a blank control group was set up. Continuously placing in CO ₂ After 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 0.5 mg/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

The study data were analyzed using Graphpad Prism 8 statistical software. The difference between the blank control group and the experimental group was analyzed by one-way ANOVA.

4. Results of the experiment

The results show that: genihistidine B significantly inhibited the proliferation activity of SW480 cell, the cell of human colon cancer cell line (P < 0.001), CT26 cell of mouse colon cancer cell line (P < 0.001), and there was a dose-dependent relationship, as compared with 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 intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims

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

formula I.

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

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

3. The method for preparing cyclopentenoquinolizinimidazole compounds according to claim 2, further comprising separating the eluate by preparative liquid chromatography, collecting the eluate according to the peak appearance time of the chromatographic peak of the cyclopentenoquinolizinimidazole compounds, concentrating, and drying to obtain pure cyclopentenoquinolizinimidazole compounds.

4. The method for preparing cyclopentenoquinolizinimidazole 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% of the volume ratio of (80) - (20) (60),v/vformic acid-methanol isocratic elution.

5. The method for preparing cyclopentenoquinolizinimidazole compounds according to claim 4, wherein the chromatographic column is Zorbax PrepHT XDB C18 with specification of 21.2X 250mm,7 μm; and/or

The mobile phase is 0.1 percent of 65 by volume,v/vformic acid water-methanol; and/or

Isocratic elution at a flow rate of 10mL/min; and/or

The sample size was 1mL.

6. The method for preparing cyclopentenoquinolizinio imidazole 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 cyclopentenoquinolizinimidoimidazoles as claimed in claim 2, wherein the reaction temperature in step A is 32 to 42 ℃; and/or

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

8. The use of cyclopentenoquinolizinimidazole compounds as claimed in claim 1 in the preparation of anti-colon cancer drugs.