CN114751859B

CN114751859B - Cyclopentapyridine compound, and preparation method and application thereof

Info

Publication number: CN114751859B
Application number: CN202210308419.8A
Authority: CN
Inventors: 王跃飞; 张潇予; 柴欣; 董雪媛; 孟宁; 崔英; 于海洋; 于卉娟; 张瑞虎
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: 2023-06-30
Anticipated expiration: 2042-03-23
Also published as: CN114751859A

Abstract

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

Description

Cyclopentapyridine compound, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of chemical medicines, and particularly relates to a cyclopentapyridine compound, and a preparation method and application thereof.

Background

Colon cancer is a common malignant tumor which is primary on colon mucosa epithelium, and the incidence rate in China tends to rise year by year. The early colon cancer is asymptomatic, the prognosis effect is good after treatment, and the patient has a longer survival time. After colon cancer develops to middle and late stages, symptoms such as hematochezia, abdominal pain and the like can appear, and the cure rate and the survival time of patients are obviously reduced. At present, the operation is a main method for treating colon cancer, and combines radiotherapy, chemotherapy and targeted therapy, so that partial patients can be completely cured or the survival time of the patients can be prolonged.

At present, few medicines capable of effectively treating colon cancer have side effects with different degrees. Therefore, obtaining more compounds that can be used to treat colon cancer is of great importance for both drug development and clinical treatment of colon cancer.

Disclosure of Invention

Aiming at the problems, the invention provides a cyclopentapyridine compound, a preparation method and application thereof, and the compound can inhibit proliferation of colon cancer cells and can be used for preparing antitumor drugs.

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

in a first aspect, an embodiment of the present invention provides a cyclopentapyridine compound having a structural formula shown in formula I:

experiments prove that the compound has remarkable inhibition effect on proliferation of colon cancer cells. No report on the cyclopentapyridine compounds is found at present.

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

step A, genipin and methylamine are dissolved in non-nitrogenous buffer solution with pH of 6-8, and react for more than 4 hours at 30-80 ℃ to obtain reaction solution;

and B, adding the reaction solution into a small-pore resin chromatographic column, sequentially adopting ethanol-water solvents with volume ratios of 0:10, 1:9, 2:8, 3:7, 4:6, 5:5 and 6:4 for gradient elution, and collecting eluent obtained by eluting the ethanol-water solvents with volume ratios of 5:5-6:4 to obtain the cyclopentapyridine compound.

The method utilizes genipin and methylamine to react in a specific solution at a specific temperature to obtain a reaction solution containing the cyclopentapyridine compound, and then the cyclopentapyridine compound can be separated from other compounds in the reaction solution through small-hole resin column chromatography, so that the cyclopentapyridine compound can be obtained in eluent.

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

Preferably, the chromatographic conditions of the preparative liquid chromatograph 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 (40:60) - (60:40), and the elution is performed isocratically.

Under the chromatographic conditions, the chromatographic column can select Waters

C18 OBD ^TM Prep Column, specification 10X 250mm,5 μm; 0.1% (v/v) formic acid water-methanol with optional volume ratio of mobile phase of 50:50; the flow rate of isocratic elution can be 4mL/min; the sample injection amount can be 2mL.

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

With reference to the first aspect, the pH of the buffer solution is preferably 6.8.

With reference to the first aspect, the temperature of the reaction in step A is preferably 45 to 55 ℃.

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

In a second aspect, the embodiment of the invention also provides application of the cyclopentapyridine compound in preparation of antitumor drugs.

Experiments prove that the compound can obviously inhibit 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 preparation of antitumor drugs.

With reference to the second aspect, the antitumor drug is an antitumor drug.

With reference to the second aspect, the antitumor drug is a targeting agent 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 is a diagram of Genimethylamine B in example 1 of the present invention ¹ H-NMR spectrum;

FIG. 2 is a diagram of Genimethylamine B in example 1 of the present invention ¹³ C-NMR spectrum;

FIG. 3 is a diagram of Genimethylamine B in example 1 of the present invention ¹ H- ¹ H COSY profile;

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

FIG. 5 is a graph showing the HMBC spectra of Genimethylamine B in example 1 of the present invention;

FIG. 6 shows the in vitro inhibition of proliferation activity of SW480 cells of the human colorectal cancer cell line of Genimethylamine B of example 8 of the present invention;

FIG. 7 shows the in vitro inhibition of CT26 cell proliferation activity of the mouse colorectal cancer cell line of Genimethylamine B in example 8 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The incidence rate of colon cancer is higher, the cure rate and survival rate of middle and late stages are reduced, and few medicines capable of effectively treating colon cancer at present are available. The embodiment of the invention provides a cyclopentapyridine compound (named Genimethylamine B), which has a structural formula shown in formula I:

experiments show that Genimethylamine B can inhibit 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, 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 B, adding the reaction solution into a small-pore resin chromatographic column, sequentially adopting ethanol-water solvents with volume ratios of 0:10, 1:9, 2:8, 3:7, 4:6, 5:5 and 6:4 for gradient elution, and collecting eluent obtained by eluting the ethanol-water solvents with volume ratios of 5:5-6:4, thus Genimethylamine B can be obtained in the eluent.

In the preparation method, the eluent obtained in the step B can be further purified by preparative liquid chromatography to obtain a pure product of Genimethylamine B. The purification method may be selected from the following operations: and B, separating the eluent obtained in the step B by using a preparative liquid chromatograph, 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 (40:60) - (60:40), and performing isocratic elution. Wherein the chromatographic column is selected from Waters

C18OBD ^TM Prep Column, specification 10X 250mm,5 μm; 0.1% (v/v) formic acid water-methanol with optional volume ratio of mobile phase of 50:50; the flow rate of isocratic elution was selectable at 4mL/min. Under the condition, collecting the elution fraction for 12.0-12.5 min according to the peak-exiting time, concentrating and drying to obtain the Genimethylamine B pure product.

In the preparation method, the mass ratio of genipin to methylamine is not limited, and the cyclopentapyridine compound can be prepared 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 represent that genipin and methylamine can all participate in the reaction at any ratio, and the raw materials with large feeding amount can be 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 methylamine is preferably 8-12:3, for example, 10:3 can be selected.

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

The pH of the buffer solution is preferably 6.8. Other conditions were the same, and when the pH was 6.8, the yield of the cyclopentapyridine compound could be improved.

The reaction temperature of the preparation method is preferably 45-55 ℃ and the reaction time is 48 hours. More target products Genimethylamine B can be produced at the preferred raw material mass ratio, reaction temperature, pH and reaction time.

In the process of small-pore resin column chromatography elution, the dosage of each eluting solvent is 3 column volumes, so as to improve the separation effect of Genimethylamine B and other compounds and enrich Genimethylamine B. The small-pore resin chromatographic column can adopt CHP20/P120 small-pore resin as filler.

The following describes the embodiments of the present invention by way of specific examples.

Example 1

The embodiment provides a cyclopentapyridine compound Genimethylamine B and a preparation method thereof.

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

step A, respectively taking 150mg of genipin and 45mg of methylamine, precisely weighing, respectively placing in a 150mL beaker, respectively adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with pH=6.8 to fully dissolve and prepare stock solutions with the concentration of 4.45mmol/L respectively; respectively transferring 150mL of the stock solution, placing in a beaker, adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with pH=6.8 to 900mL, shaking uniformly, placing in a water bath with the temperature of 50 ℃, reacting for 48h, and taking out to obtain a reaction solution;

step B, separating the reaction solution by adopting CHP20/P120 macroporous resin through column chromatography, and performing gradient elution (3 column volumes are eluted by each elution system) by adopting an ethanol-water (0:10, 1:9, 2:8, 3:7, 4:6, 5:5 and 6:4) solvent system to obtain a series of eluents, combining the eluents of the ethanol-water (5:5-6:4), and concentrating for preparing the eluents for liquid chromatography separation and purification;

step C, taking concentrated solution of ethanol-water (5:5-6:4) eluent, and separating and purifying by adopting preparative liquid chromatography, wherein a chromatographic column is Waters

C18OBD ^TM Prep Column (10×250mm,5 μm), mobile phase 0.1% (v/v) formic acid water-methanol (50:50), isocratically eluting at a flow rate of 4mL/min, detection wavelength 290nm, sample injection amount 2mL, collecting eluate of 12.0-12.5 min according to the peak-out time, concentrating, drying to obtain Genimethylamine B, yield 0.54% (yield= Genimethylamine B weight/genipin and total weight×100% of methylamine, the same applies hereinafter), purity 99.2% (purity=fraction collected from the prepared liquid phase is detected in an ultra-high performance liquid chromatograph, peak area of Genimethylamine B obtained is ≡sum of peak areas in all ultra-high performance liquid chromatographs×100%, the same applies hereinafter).

Adopting NMR ¹ H-NMR、 ¹³ C-NMR、 ¹ H- ¹ H COSY, HSQC, HMBC) technique characterizes the chemical structure of Genimethylamine B, ¹ the H-NMR spectrum is shown in FIG. 1, ¹³ the C-NMR spectrum is shown in FIG. 2, ¹ H- ¹ the H COSY spectrum is shown in FIG. 3, the HSQC spectrum is shown in FIG. 4, and the HMBC spectrum is shown in FIG. 5.

¹ There are 7 sets of proton signals in the H-NMR spectrum, including 4 sets of olefin proton signals at δ7.00, 7.83, 8.22, 9.00. ¹³ The C NMR spectrum shows 12 carbon signals, where δ 167.0 is the ester carbonyl carbon signal, and 8 olefinic carbons are shown at δ 133.2, 116.5, 135.2, 109.0, 143.3, 127.4, 120.9, 136.5. In the HSQC spectrum, 7 carbons were shown to be associated with proton signal presence, where δ52.9 methoxy carbon atom and δ46.6 aminomethyl carbon atom were associated with δ3.94 and 4.12 proton signals, respectively, δ184.3 aldehyde carbon was associated with δ9.81 proton signal, and δ133.2 (C-3), 136.5 (C-1), 143.3 (C-7), 109 (C-6) 4 olefin carbons were associated with δ8.22, 9.00, 7.83, 7.00 proton signals, respectively. ¹ H- ¹ The H COSY spectrum shows H-6 and H-7, H-3 and H-1, and azomethine hydrogen atom and HThere is a correlation between-3, H-1. The substitution position of the azomethine can be determined by the correlation signals between the azomethine carbon and H-1 and H-3 in the HMBC spectrum, the correlation of the signals between the aldehyde group hydrogen and C-8 and C-9 determines the position of the aldehyde group, and in addition, the correlation information of the signals of H-3 and carbonyl carbon, H-7, C-5 and the like is displayed.

As can be seen from the NMR spectrum, the structural formula of the compound is shown as formula I:

example 2

The embodiment provides a preparation method of a cyclopentapyridine compound Genimethylamine B.

The method specifically comprises the following steps:

step A, respectively weighing 120mg of genipin and 45mg of methylamine, respectively placing in a 150mL beaker, respectively adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with pH=6.8 to fully dissolve the genipin and the methylamine to prepare stock solutions with the concentrations of 3.5 and 4.4mmol/L respectively; respectively transferring 150mL of the stock solution, placing in a beaker, adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with pH=6.8 to 900mL, shaking uniformly, placing in a water bath with the temperature of 45 ℃, reacting for 60h, and taking out to obtain a reaction solution;

C18OBD ^TM Prep Column (10X 250mm,5 μm), mobile phase 0.2% (v/v) of formic acid water-methanol (60:40), isocratic elution at a flow rate of 4mL/min, detection wavelengthCollecting elution fraction of 12.5-13.5 min according to peak time at 290nm and sample injection amount of 2mL, concentrating, and drying to obtain Genimethylamine B with yield of 0.36% and purity of 80.5%.

Example 3

The method specifically comprises the following steps:

step A, respectively taking 180mg of genipin and 45mg of methylamine, precisely weighing, respectively placing in a 150mL beaker, respectively adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with pH=6.8 to fully dissolve and prepare stock solutions with the concentration of 5.3 and 4.4mmol/L respectively; respectively transferring 150mL of the stock solution, placing in a beaker, adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with pH=6.8 to 900mL, shaking uniformly, placing in a 55 ℃ water bath, reacting for 36h, and taking out to obtain a reaction solution;

C18OBD ^TM Prep Column (10×250mm,5 μm), mobile phase 0.05% (v/v) formic acid water-methanol (40:60), isocratically eluting at a flow rate of 4mL/min, detecting wavelength 290nm, sampling 2mL, collecting eluate of 10.5-12 min according to peak-out time, concentrating, drying to obtain Genimethylamine B with yield 0.41% and purity 76.2%.

Example 4

step A, respectively taking 150mg of genipin and 45mg of methylamine, precisely weighing, respectively placing in a 150mL beaker, respectively adding PBS buffer solution with pH=7.35 to fully dissolve the genipin and the methylamine to prepare stock solutions with the concentration of 4.4mmol/L respectively; respectively transferring 150mL of the stock solution, placing in a beaker, adding PBS buffer solution with pH of 7.35 to 900mL, shaking uniformly, placing in a water bath with the temperature of 50 ℃, reacting for 48h, and taking out to obtain a reaction solution;

C18 OBD ^TM Prep Column (10×250mm,5 μm), mobile phase 0.1% (v/v) formic acid water-methanol (50:50), isocratically eluting at a flow rate of 4mL/min, detecting wavelength of 290nm, sampling amount of 2mL, collecting eluate of 12.0-12.5 min according to peak-out time, concentrating, drying to obtain Genimethylamine B with yield 0.48% and purity of 97.6%.

Example 5

step A, respectively taking 150mg of genipin and 45mg of methylamine, precisely weighing, respectively placing in a 150mL beaker, respectively adding sodium acetate-glacial acetic acid buffer solution with pH=6.2 to fully dissolve and prepare stock solutions with the concentration of 4.4mmol/L respectively; respectively transferring 150mL of the stock solution, placing in a beaker, adding a sodium acetate-glacial acetic acid buffer solution with pH of 6.2 to 900mL, shaking uniformly, placing in a water bath with the temperature of 50 ℃, reacting for 48h, and taking out to obtain a reaction solution;

C18OBD ^TM Prep Column (10×250mm,5 μm), mobile phase 0.1% (v/v) formic acid water-methanol (50:50), isocratically eluting at a flow rate of 4mL/min, detecting wavelength of 290nm, sampling amount of 2mL, collecting eluate of 12.0-12.5 min according to peak-out time, concentrating, drying to obtain Genimethylamine B with yield of 0.49% and purity of 98.3%.

Example 6

step A, respectively taking 150mg of genipin and 45mg of methylamine, precisely weighing, respectively placing in a 150mL beaker, respectively adding sodium acetate-glacial acetic acid buffer solution with pH=6.0 to fully dissolve and prepare stock solutions with the concentration of 4.4mmol/L respectively; respectively transferring 150mL of the stock solution, placing in a beaker, adding a sodium acetate-glacial acetic acid buffer solution with pH of 6.0 to 900mL, shaking uniformly, placing in a water bath with the temperature of 50 ℃, reacting for 48h, and taking out to obtain a reaction solution;

C18OBD ^TM Prep Column (10×250mm,5 μm), 0.1% (v/v) formic acid water-methanol (50:50) as mobile phase, isocratic eluting at a flow rate of 4mL/min, detecting wavelength of 290nm, sampling amount of 2mL, collecting eluate of 12.0-12.5 min according to peak time, concentrating, drying to obtain Genimethylamine B with yield of 0.47% and purity of 99.0%.

Example 7

step A, respectively taking 150mg of genipin and 45mg of methylamine, precisely weighing, respectively placing in a 150mL beaker, respectively adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with pH=8.0 to fully dissolve and prepare stock solutions with the concentration of 4.4mmol/L respectively; respectively transferring 150mL of the stock solution, placing in a beaker, adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with pH=8.0 to 900mL, shaking uniformly, placing in a water bath with the temperature of 50 ℃, reacting for 48h, and taking out to obtain a reaction solution;

C18OBD ^TM Prep Column (10X 250mm,5 μm), 0.1% (v/v) formic acid water-methanol (50:50) as mobile phase, isocratic elution at a flow rate of 4mL/min, detection wavelength of 290nm, sample injection amount of 2mL, collecting elution fraction of 12.0-12.5 min according to peak-out time, concentrating, drying to obtain Genimethylamine B with yield of 0.31% and purity of 98.3%.

Example 8

3- (4, 5-dimethyl-2-thiazole) -2, 5-diphenyltetrazolium bromide (MTT) can be reduced by dehydrogenase in mitochondria of living cells to produce formazan as a dark purple crystalline product. The faster the cell proliferation, the higher the dehydrogenase activity, and the more formazan is formed. Formazan can be completely dissolved into blue by DMSO, has a strong absorption peak at 570nm wavelength, and can evaluate the number of living cells and proliferation activity by detecting absorbance value at 570nm wavelength by an enzyme-labeled instrument. This example demonstrates the effect of Genimethylamine B on inhibiting the proliferation activity of colon cancer cells by measuring cell viability using MTT colorimetry.

1. Reagent, instrument and consumable

(1) Reagent: MTT, solarbio; DMSO, solarbio; PBS buffer, bai Si Nuo (Tianjin) Biotech Co.

(2) Instrument, consumable: 450 type enzyme labeling instrument, shanghai Berle company; 96-well plates, nest.

2. Experimental method

Preparation of MTT working solution: 25mg of MTT powder is added into 5mL of PBS buffer solution to prepare 5mg/mL solution, and the solution is separated and then stored at-20 ℃ in a dark place.

Colon cancer cell line SW480 in logarithmic growth phase was selected, the cell mass was digested into single cells with 0.25% (g/mL) trypsin, the digestion was stopped by adding three volumes of DMEM complete medium, centrifuging at 1000rpm for 5min, and the supernatant was discarded. After adding 5mL of DMEM complete medium and mixing well, 10 μl of cell suspension was aspirated, and the cells were counted by injecting cell fluid into a counting plate along the edge of the coverslip. Preparation of 4X 10 with DMEM complete Medium ⁴ Each/mL of the cell suspension was inoculated into a 96-well plate with 200. Mu.L of each well and placed in CO ₂ Culturing in an incubator for 24 hours. Removing culture solution from 96-well plate, and diluting with DMEM complete mediumThe concentrations of (2) are respectively 10, 20, 30, 40 and 50 mu M of Genimethylamine B solution, and 10 compound holes are arranged in each concentration; at the same time, a blank group was established. Continue to be placed in CO ₂ After incubation for 24h 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, after incubation at 37℃for 4h, the crystalline dark purple product was observed microscopically, 100. Mu.L/well DMSO was added to dissolve the dark purple crystals, and absorbance (A) was measured at 570 nm.

Treatment of colon cancer cell line CT26 by the same procedure and preparation of 4X 10 using RPMI 1640 complete medium ⁴ Each/mL of the cell suspension was inoculated into a 96-well plate with 200. Mu.L of each well and placed in CO ₂ Culturing in an incubator for 24 hours. Removing the culture solution from the 96-well plate, adding Genimethylamine B solutions with concentration gradients of 5, 10, 15, 20, 25 and 30 mu M diluted by RPMI 1640 complete culture medium, and setting 10 compound wells for each concentration; at the same time, a blank group was established. Continue to be placed in CO ₂ After incubation in the incubator for 24h, the test drug was removed, 100. Mu.L/well MTT solution (RPMI 1640 was completely dissolved in medium at a concentration of 0.5 mg/mL) was added, after incubation at 37℃for 4h, the crystalline dark purple product was observed microscopically, 100. Mu.L/well DMSO was added to dissolve the dark purple crystals, and absorbance (A) was measured at 570 nm.

The cell viability of colon cancer cells was calculated by the following formula:

cell viability/% = a _{Test drug} /A _{Blank space} ×100％

3. Statistical analysis

Study data were analyzed using Graphpad Prism 8 statistical software. The difference between the blank and experimental groups was analyzed using one-way ANOVA.

4. Experimental results

The results show that: compared with the blank control group, genimethylamine B obviously inhibits proliferation activity (P < 0.001) of SW480 cells of the human colon cancer cell line and CT26 cells of the mouse colon cancer cell line, and a certain dose-dependent relationship exists, and the results are shown in fig. 6 and 7. Panel C is a blank; * P <0.05 and P <0.001.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.

Claims

1. The cyclopentapyridine compound is characterized by having a structural formula shown in formula I:

2. the use of a cyclopentapyridine compound as defined in claim 1 for the preparation of an antitumor drug.

3. The use according to claim 2, wherein the antineoplastic agent is an anti-colon cancer agent.

4. The use according to claim 3, wherein the antineoplastic agent is a targeted formulation against colon cancer.