CN111171053A

CN111171053A - Application of compound in preparation of medicine for treating pancreatic cancer

Info

Publication number: CN111171053A
Application number: CN202010011128.3A
Authority: CN
Inventors: 邵初晓; 涂朝勇; 李卓锴; 吴亦峰; 吴逸超
Original assignee: Lishui Central Hospital
Current assignee: Lishui Central Hospital
Priority date: 2020-01-06
Filing date: 2020-01-06
Publication date: 2020-05-19
Anticipated expiration: 2040-01-06
Also published as: CN111171053B

Abstract

The invention provides a compound of formula (I), formula (II) and formula (III), and also relates to a preparation method of the compound, wherein the synthesis route is simple, the raw materials are easy to obtain, and the industrial large-scale production can be conveniently realized. The compound provided by the invention has an inhibiting effect on pancreatic cancer tumors, has high survival rate and small weight reduction amplitude of mice in a mouse test, has small toxic and side effects, and is a compound medicine for treating pancreatic cancer, which is expected to be industrialized. The invention also comprises a pharmaceutical composition and a preparation of the compound, and application of the compound in preparing a medicament for treating pancreatic cancer.

Description

Application of compound in preparation of medicine for treating pancreatic cancer

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of a compound in preparation of a medicine for treating pancreatic cancer.

Background

Pancreatic cancer is a common clinical malignant tumor, has high malignancy, high mortality and poor prognosis, is diagnosed in middle and advanced stages due to occult disease, and is called as 'Wan cancer king'. At present, the 5-year survival time of pancreatic cancer is only about 5%, and the mortality rate is high worldwide, 90.8% in China, 78.5% in the United states and 95.0% in Canada. According to the Chinese epidemiological survey, pancreatic cancer is the seventh fatal tumor disease ranked in China.

Pancreatic cancer takes a long time to naturally occur, the pancreatic epithelium usually takes 12 years from intraepithelial neoplasia to tumor acquisition of invasive characteristics, 7 years after tumor invasion to metastasis, and an average of 3 years from metastasis to death. However, the clinical time span of pancreatic cancer is much shorter due to the hidden location of pancreatic cancer, the survival time of a pancreatic cancer patient who is not treated is only about 4 months, the survival time of a pancreatic cancer patient who is treated by bypass surgery is about 7 months, and the survival time of a pancreatic cancer patient who is treated by resection surgery is generally 16 months. However, in the present situation, only 10-15% of patients have surgical indications after definite diagnosis, and most of patients cannot be subjected to radical surgical resection at the time of diagnosis. The average survival time of the patients receiving the surgical resection is 16.9-20.2 months after the surgery, and the survival time can be prolonged to 20.1-23.6 months if the patients receive the postoperative adjuvant therapy. The survival time of the locally advanced unresectable pancreatic cancer is usually only 6-11 months, if the resection is successfully completed after the new adjuvant therapy, the survival time can reach 20.5 months, and the average survival time of the metastatic pancreatic cancer is 5-9 months.

In spite of great efforts in the past 50 years on the study of pancreatic cancer treatment methods and drugs, no major progress has been made in improving pancreatic cancer survival. Surgical statistical data in China show that the 5-year survival rate of pancreatic cancer patients in China is only about 5%. Therefore, early diagnosis and early treatment are the key to improving and improving prognosis of pancreatic cancer, and data show that tumors are radically cured at early stage, and 5-year survival rate can be more than 20%.

At present, the basic treatment principle of pancreatic cancer is still mainly surgical treatment combined with radiotherapy, chemotherapy and other comprehensive treatments. Pancreatic cancer has a high malignancy, a low surgical resection rate, and a poor prognosis. Although surgery remains the primary treatment, the chance of radical cure is lost as pancreatic cancer is often found late. Therefore, chemotherapy is mainly used for pancreatic cancer patients at present, people try to improve the curative effect of chemotherapy by various means, and the survival time of the patients is prolonged, and meanwhile, the survival quality of the patients is influenced by more and more toxic and side effects caused by radiotherapy and chemotherapy. Gemcitabine is the most commonly used drug for pancreatic cancer, and in recent years, oral fluorouracil (tegafur) is found to have similar curative effect to gemcitabine, and when three drugs (fluorouracil + irinotecan + oxaliplatin) are used in combination, pancreatic cancer patients can obtain 11.1 months of survival time, but the incidence of toxic and side effects such as III-IV level myelosuppression and the like is also high. In addition, gemcitabine and nano-paclitaxel albumin are treated to form a new treatment mode, and the average survival time of patients is 8.5 months, which is 6.7 months better than that of pure gemcitabine. Since pancreatic cancer treatment has progressed slowly without breaking through new advances for a long time, the U.S. NCCN pancreatic cancer guideline preferentially recommends patients to receive scientifically designed clinical trials for each stage of pancreatic cancer. It can also be seen that there is a need to develop or develop a new drug for treating pancreatic cancer that improves patient survival while reducing the toxic side effects of treatment.

Patent document CN201310370956.6 discloses an application of a compound Nardoaristolones a in preparing a medicament for treating pancreatic cancer, belonging to a new application of medicaments. The compound Nardoaristolones A is proved to have a protective effect on myocardial damage induced by hydrogen peroxide in the current clinic, and the Nardoaristolones A has a remarkable inhibiting effect on the growth of human pancreatic cancer cell strains PANC-1 and BXPC-3 through the evaluation experiment of cell activity in vitro. However, although the patent literature uses MTT experiments to prove that the compound has an inhibitory effect on pancreatic cancer cells, the toxic and side effects of the compound on the body are not further verified, and those skilled in the art know that the toxic and side effects of the drug are important factors of research and development personnel in the process of drug development in the tumor field.

Disclosure of Invention

In view of the current situation that the type of pancreatic cancer treatment drugs in the prior art is single and the toxic and side effects are large, the invention aims to provide a compound with a general formula I or a pharmaceutically acceptable salt thereof; it is another object of the present invention to provide a process for the preparation of the compounds of formula I and compositions comprising the compounds of formula I; it is a further object of the present invention to provide the use of a compound of formula I for the preparation of a medicament for the treatment of pancreatic cancer.

The above object of the present invention is achieved by the following technical solutions:

a compound of the general formula (I) or a pharmaceutically acceptable salt thereof:

wherein R is₁Selected from H, hydroxyl, halogen atom, nitro, optionally substituted alkyl, optionally substituted alkoxy, 1-furyl, 1-tetrahydrofuryl, R₂And R₄Independently selected from H or alkyl, R₃Selected from H, alkyl, alkoxy, halogen atom and aryl.

The alkyl group is a straight or branched chain alkyl group having 1 to 10 carbon atoms, preferably having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl; the alkoxy group is a linear or branched alkoxy group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, butoxy.

Optionally substituted means that the alkyl or alkoxy group may be substituted with some conventional groups which do not interfere with the biological activity of the compounds of the present invention, such as halogen atoms, hydroxy, amino, lower alkyl, lower alkoxy, lower alkenyl, lower alkynyl groups and the like. The term "lower" according to the present invention means a group having 1 to 4 carbon atoms. Examples of substituents optionally substituted on alkyl or alkoxy include, but are not limited to: trifluoromethyl, amino, hydroxy, methyl, ethyl, propyl, butyl, methoxy, ethoxy, hydroxymethyl.

In a preferred embodiment of the present invention, there is provided a compound represented by the general formula (II) or the general formula (III):

wherein R is₂Selected from methyl, ethyl, propyl, R3 is selected from H, F, Cl, Br, hydroxyl, methyl, ethyl, propyl, methoxy, ethoxy, phenyl, trifluoromethyl.

Preferably, the present invention provides the following specific compounds:

the second purpose of the invention is to provide a preparation method of the compound shown in the general formula (I), and the synthetic route is as follows:

the method specifically comprises the following steps:

(i) mixing the compound of formula 1 with methanol as solvent, and introducing N₂After air is removed, liquid ammonia is slowly added to react for 4 hours at the temperature of 40-60 ℃ to obtain an ammoniated product, namely a compound shown in the formula 2, wherein the molar ratio of the compound shown in the formula 1 to the liquid ammonia is 1: 1-2;

(ii) after the compound of the formula 2 and N-tert-butylcarbylpiperazine are mixed, CDI (N, N-carbonyl-diimidazole) is added, dichloromethane is used as a solvent, and the reaction is carried out for 2 to 3 hours at the temperature of between 20 and 40 ℃ to obtain a product, namely a compound of the formula 3, wherein the molar ratio of the compound of the formula 2 to the N-tert-butylcarbylpiperazine to the CDI is 1:1 to 1.5:2 to 3.

(iii) Removing Boc from the compound of formula 3 under the action of TFA to obtain a product, namely a compound of formula 4, wherein the ratio of the compound of formula 3 to TFA is 1mol:1-1.5 mL;

(iv) compounds of formula 4 and R₁The substitution reaction of-Br under potassium carbonate to obtain the product of the formula (I)Compound of formula 4, R₁The molar ratio of-Br to potassium carbonate is 1: 1-2: 1-2.

The third purpose of the invention is to provide a pharmaceutical composition, which comprises the compound of the general formula I or the pharmaceutically acceptable salt thereof, and also comprises pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials are selected from: carrier, diluent, adhesive, lubricant and wetting agent.

Preferably, the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula i. In certain embodiments, the pharmaceutical compositions may be used alone or in combination with other types of pharmaceutical formulations and/or methods of treatment.

Such other classes of pharmaceutical formulations and/or methods of treatment include, but are not limited to: immunosuppressants, targeted antineoplastic drugs, glucocorticoids, non-steroidal anti-inflammatory drugs, anti-tumor vaccines, CDK4/6 and/or FGFR4 kinase inhibitors, adoptive cellular immunotherapy or radiotherapy.

The invention further aims to provide the application of the compound with the general formula I or the pharmaceutically acceptable salt thereof in preparing a medicament for treating pancreatic cancer.

The mode of administration and dosage of the invention are selected according to the particular stage of the disease being treated, the age and condition of the patient, the duration, the particular mode of administration, and the like, as is practical and desired, and within the knowledge and time frame of one skilled in the art. Generally, an effective amount is sufficient to cause a favorable phenotypic change, such as alleviation or elimination of symptoms, permanent cessation of progression, or delay of onset of the disease. In general, the dosage of the active compounds according to the invention is from about 0.01mg/kg to 10mg/kg per day, preferably orally, once or several times a day.

Optionally, the active compounds of the present invention may be formulated into pharmaceutical formulations with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to one or more compatible solids, liquids, diluents, and the like, suitable for administration to the human body. The carrier does not destroy the efficacy of the active compound.

Optionally, the pharmaceutical formulation may contain suitable buffer solutions including acetate, citrate, borate or phosphate.

Various modes of administration may be employed so long as they achieve effective levels of the active compound without causing clinically unacceptable side effects. Modes of administration include oral, rectal, topical, intranasal, intradermal, transdermal or parenteral. Specific modes of administration include tablets, capsules, powders, suppositories, injections and nasal sprays.

The active compounds according to the invention have the following advantages:

the compound provided by the invention has the advantages of simple preparation method, good physicochemical property and stable quality, and is suitable for large-scale industrial production.

Secondly, the compound has the activity of obviously inhibiting pancreatic cancer cells, and animal experiments prove that the compound has small toxic and side effects and strong safety.

Drawings

FIG. 1 statistics of body weights of mice in experimental and control groups after 40 days of administration

FIG. 2 tumor weight statistics of mice in experimental and control groups 40 days after administration

Detailed Description

The compounds of the present invention, methods of preparation and uses thereof are further illustrated below with reference to specific examples, but it should be understood that the scope of the present invention is not limited to the following examples.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Example 1Preparation of Compound 1

(i) 0.1mol of 5-chloro-6-methoxy-7-methylthiazolo [4,5-b ]]Pyridin-2 (3H) -one and 0.3mol methanol were charged to a 250mL four-necked flask and charged with N₂After air is removed, slowly adding liquid ammonia under the condition of heating and stirring, wherein the adding amount of the liquid ammonia is 0.2mol, and reacting for 4 hours at the temperature of 40-50 ℃ to obtain an ammoniated product 5-amino-6-methoxy-7-methylthiazolo [4,5-b ]]Pyridine-2 (3)H) -ketone, yield 83%.

(ii) 0.1mol of 5-amino-6-methoxy-7-methylthiazolo [4,5-b ] pyridin-2 (3H) -one and 0.13mol of N-tert-butylcarbonylpiperazine were added to a reaction vessel in the solvent DMF and 0.2mol of CDI (N, N-carbonyl-diimidazole) in the solvent dichloromethane was added. Reaction at 20-40 deg.c for 2-3 hr to obtain N- (6-methoxy-7-methyl-2-oxo-2, 3-dihydrothia [4,5-b ] pyridin-5-yl) -4-tert-butylcarbonylpiperazine-1-carboxamide in 70% yield.

(iii)0.1mol of N- (6-methoxy-7-methyl-2-oxo-2, 3-dihydrothia [4,5-b ] pyridin-5-yl) -4-methylpiperazine-1-carboxamide and 250mL of dichloromethane were added dropwise slowly in ice bath to react for 2h with removal of the protecting group Boc to give the product N- (6-methoxy-7-methyl-2-oxo-2, 3-dihydrothia [4,5-b ] pyridin-5-yl) -4-methylpiperazine-1-carboxamide in 88% yield.

(iv)0.1mol N- (6-methoxy-7-methyl-2-oxo-2, 3-dihydrothia [4, 5-b)]Dissolving pyridine-5-yl) -4-methylpiperazine-1-formamide and 0.15mol of 2-bromofuran in a solvent dichloromethane, adding 0.1mol of potassium carbonate, stirring and reacting at room temperature for 4 hours to obtain a compound 14- (furan-2-yl) -N- (6-methoxy-7-methyl-2-oxo-2, 3-dihydrothia-ne And [4,5-b ]]Pyridin-5-yl) piperazine-1-carboxamide in 82% yield. ¹H-NMR(CD₃OD):2.36(3H，s)，3.25-3.32(4H，m),3.46-3.49(4H，m)，3.83(3H，s)，6.67(1H,m)，7.02(1H,m)，7.90(1H,m)。

The molecular formula is as follows: c₁₇H₁₉N₅O₄S, molecular weight: 389.43, Mass Spectrometry (M + H): 390.12.

examples 2 to 10Preparation of Compounds 2-10

Following the synthetic route, compounds 2-10 were each synthesized by a method similar to that of example 1, and the data are shown in Table 1.

TABLE 1

The beneficial effects of the active compounds according to the invention are further illustrated below by in vitro pharmacological activity experiments, but this should not be understood as having the following beneficial effects on the compounds according to the invention:

application example 1

The purpose of the test is as follows: the inhibitory effect of the compound prepared by the invention on pancreatic cancer cells is detected.

The test method comprises the following steps: the pancreatic cancer cell line Panc-1 used in the present invention was purchased from ATCC (American Type CurtureColection, Manassas, Va.).

Taking out the freezing tube for freezing the Panc-1 cells from the liquid nitrogen tank, rapidly placing the tube into a 37 ℃ water bath tank for oscillation to rapidly dissolve the cells, transferring the dissolved cell sap into a centrifuge tube with a fresh culture medium added in advance to avoid influence on cell growth caused by DMSO added during cell freezing, placing the centrifuge tube into a centrifuge at 1000rpm/min for 5min, taking out the tube, discarding the supernatant, transferring the cells into a culture bottle, adding the culture medium, shaking up, and keeping the tube at 37 ℃ and containing 5% CO₂The cells were cultured in the incubator of (1) at 1 passage every 2 days, and used when the cells were well grown.

The experimental steps are as follows: 1, inoculating cells, designing control wells and zero setting wells, and adding 200 μ L sterile PBS per well on the periphery of a 96-well plate. Dispersing Panc-1 cells in culture medium to obtain single cell suspension, counting cells under microscope, inoculating about 5000 cells per well onto 96-well plate with volume of 200 μ L per well, placing at 37 deg.C and containing 5% CO₂And culturing for 24h in an incubator with saturated humidity. 2, preparing experimental medicines, namely preparing the compound 1 prepared in the example 1 into solutions with Au concentrations of 0.1, 0.3, 0.5, 1, 2, 5, 10 and 50 mu M by adopting a fresh culture medium, filtering the solutions by using a 0.22 mu M microporous filter membrane, and storing the filtered solutions under the aseptic condition for later use. And 3, performing drug action, sucking out the old culture medium of each hole by using a pipette after the cells are cultured for 24 hours, respectively adding the drugs with corresponding concentrations, adding fresh culture medium into the control hole and the zero setting hole, and continuously culturing for 24 hours. 4, coloring, weighing MTT, preparing a solution with the concentration of 5mg/mL by using sterile PBS, taking out a 96-well plate after the medicine acts for 24 hours, adding 20 mu L of MTT solution into each well, and adding the MTT solution into the wellIncubate for 4h in the incubator, aspirate the old media, add 150 μ L DMSO per well, incubate for 10 min. 5, colorimetric, selecting 490nm wavelength, measuring the light absorption value of each pore on an enzyme-linked immunosorbent detector, and recording IC₅₀The value is obtained.

The compound 2-10 has the same concentration and experimental method, and the compound IC is obtained₅₀The results are shown in Table 2.

TABLE 2

Sample (I)	IC₅₀(μM)	Sample (I)	IC₅₀(μM)
				Compound 1	1.1±0.3	Compound 2	0.8±0.4
Compound 3	1.2±0.2	Compound 4	0.9±0.7
				Compound 5	1.1±0.7	Compound 6	1.3±0.1
Compound 7	1.7±0.4	Compound 8	1.6±0.9
				Compound 9	1.9±0.3	Compound 10	1.6±0.6

As can be seen from the data in the table above, the liquid medicines prepared from the compounds 1-10 prepared by the invention have obvious inhibition effect on pancreatic cancer cells, wherein the pancreatic cancer cells have the highest sensitivity to the compounds 2 and 4.

Application example 2

The purpose of the test is as follows: the treatment and toxic and side effects of the compound prepared by the invention on tumor-bearing mice are detected.

The test method comprises the following steps: 1, SPF-level immunosuppression nude mice are selected for the experiment, the age of the mice is 4-6 weeks, the mice are female, and the weight of the mice is 18-20g, and all animal experiment operation schemes conform to the instruction of experimental animal nursing and use; 2, inoculating a pancreatic cancer cell line (KP3L) to the back of a nude mouse by subcutaneous injection; 3, feeding for 3-4 weeks under conventional conditions, and starting the experiment when the tumor grows to about 6X 9 mm; 4, preparing the compound 1-10 into a liquid medicine with the concentration of 1mg/mL by using physiological saline, filtering the liquid medicine by using a 0.22 mu m microporous filter membrane, and storing the liquid medicine under the aseptic condition for later use; randomly dividing the nude mice into 11 groups, namely a blank control group and a compound 1-10 group, and carrying out intraperitoneal injection administration every 2 days (the blank control group is physiological saline, the compound 1-10 group is liquid medicine 1-10, and the concentration is 1 mg/kg); observing the survival condition of the mouse every day, and recording the survival rate; the weight of each group of mice was recorded on day 40, and the tumors were weighed after the mice were sacrificed.

And (3) test results: the survival rates of the nude mice in the experimental group and the control group are shown in the following table, and the body weights and tumor weights of the nude mice in each group on the 40 th day of the experiment are shown in fig. 1 and 2.

TABLE 3

According to the results in the table above, the compound prepared by the invention has an improvement effect on the survival rate of the pancreatic tumor-bearing mice compared with the blank control group, wherein the survival rate of the mice after the compound 2, the compound 4, the compound 6 and the compound 7 are administered for 40 days is more than 90%, especially the survival rate of the mice acted by the compound 2 is 100%, which shows that the toxic and side effects of the compound 2 are minimum.

According to the statistical weight chart of mice shown in fig. 1, the weight of mice administered with the compound prepared in the present application varied in a smaller range than that of the blank control group, which had no administration of any drug and had a greater invasion and consumption of tumors to the mice. There was also a phenomenon that there was no significant difference in the body weight of the mice that finally survived in the compound 1-10 groups, and the reason for the analysis was probably that the compounds 1-10 did have an inhibitory effect on pancreatic cancer tumors, the survival rate of the mice was high and the reduction in the body weight of the mice was small compared to the control group, but there was no significant difference in the body weight of the mice that finally survived 40 days after administration because most of the mice that had no tumor inhibitory effect by administration of the drug had died.

As can be seen from the statistical graph of tumor weights of the mice shown in fig. 2, the blank control group finally resulted in a significantly higher tumor weight than the other groups due to no drug administration; in addition, statistical graphs show that the weight of the tumor of the mice administered with the compounds 2 and 6 is the smallest, and the tumor is obviously different from other groups, which shows that the pancreatic cancer inhibition effect of the compounds 2 and 6 is better than that of other compounds, so that the compound has good application prospect and is suitable for further research and development.

Therefore, the above description is only a preferred embodiment of the present invention, and is not intended to limit the embodiments of the present invention, and those skilled in the art can easily make various changes or modifications according to the main concept and spirit of the present invention, and some modifications and alterations of the present invention should fall into the protection scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. A compound of the general formula (I) or a pharmaceutically acceptable salt thereof:

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the alkyl group is a linear or branched alkyl group having 1 to 10 carbon atoms, preferably having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl; the alkoxy group is a linear or branched alkoxy group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, butoxy.

3. A compound of formula (II) or formula (III) or a pharmaceutically acceptable salt thereof:

4. The following compounds or pharmaceutically acceptable salts thereof:

5. a process for the preparation of a compound according to any one of claims 1 to 4, which is prepared by the following scheme:

6. the method of claim 5, comprising the steps of:

(iv) compounds of formula 4 and R₁The substitution reaction of-Br under potassium carbonate to obtain the product of the compound of formula (I), the compound of formula 4 and R₁The molar ratio of-Br to potassium carbonate is 1: 1-2: 1-2.

7. Use of a compound according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment of pancreatic cancer.

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4.

9. The pharmaceutical composition of claim 8, further comprising a pharmaceutically acceptable excipient selected from the group consisting of: carrier, diluent, adhesive, lubricant and wetting agent.

10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition can be used alone or in combination with other types of pharmaceutical agents and/or treatments including, but not limited to: immunosuppressants, targeted antineoplastic drugs, glucocorticoids, non-steroidal anti-inflammatory drugs, anti-tumor vaccines, CDK4/6 and/or FGFR4 kinase inhibitors, adoptive cellular immunotherapy or radiotherapy.