A bipyridine carboxylate derivative and use thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a dipicolinate ester derivative and application thereof. The dipicolinate ester derivative has broad-spectrum antitumor activity, can significantly inhibit the proliferation activity of lung cancer cells, pancreatic cancer cells and prostate cancer cells, and has more excellent inhibitory activity on lung cancer cells, and can significantly inhibit the growth of mouse subcutaneous small cell lung cancer transplanted tumors. In addition, the dipicolinate ester derivative has no toxic effect on normal cells of the body, can be applied to preparation of anticancer drugs, and has broad application prospects in the aspect of anticancer.

Description

A bispyridine carboxylate derivative and its application Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a bispyridine carboxylate derivative and its applications. Background Technology

[0002] In recent years, cancer has become the second leading cause of death after heart disease. In 2020, the top five cancers in terms of mortality were: lung cancer (1.8 million deaths), colorectal cancer (916,000 deaths), liver cancer (830,000 deaths), stomach cancer (769,000 deaths), and breast cancer (685,000 deaths). Cancer is characterized by the uncontrolled and rapid proliferation of cells, leading to tumor formation. These cells grow beyond their normal boundaries and can invade adjacent parts of the body and spread to other organs; this is called metastasis, and widespread metastasis is a major cause of cancer death. The 5-year relative survival rate for the ten most common cancers detected early and occurring at the primary lesion site is approximately 34.2-100%, with a local recurrence rate of <16.1% after surgery or radiotherapy / chemotherapy. However, for advanced cancers with frequent recurrence or metastasis, the 5-year relative survival rate drops to 2.5-30.2%.

[0003] Currently, the main treatments for cancer include surgery, chemotherapy, radiation therapy, immunotherapy, and targeted therapy. Surgery and chemotherapy are the most commonly used methods, and have successfully cured millions of patients to date. However, due to varying patient conditions, chemotherapy drugs can cause many complications and are largely unsuitable for treating advanced cancer cases. Radiation therapy, which uses high-energy beams (such as protons, electrical energy, or X-rays) to target and destroy cancer cells, has been used to treat many types of cancer; however, the absorption of X-ray nanoparticles by the specific tumor microenvironment often causes severe damage to normal tissues. Immunotherapy, which treats cancer by modulating the immune system, is used as a monotherapy or in combination with many other treatment regimens to enhance its efficacy. Advances in bioinformatics and the invention of various high-throughput genomic methods have supported gene therapy, which can identify specific mutations in gene delivery to achieve targeted treatment of cancer cells.

[0004] In clinical treatment, there is still a relative lack of drugs for cancer patients. Therefore, there is an urgent need to explore more new and effective drugs for cancer patients in order to improve the effectiveness of clinical treatment. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiency and inadequacy of existing cancer treatment drugs, and to provide the application of bispyridine carboxylate derivatives and their pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, stereoisomers, prodrugs or isotopic compounds in the preparation of anticancer drugs.

[0006] The purpose of this invention is to provide a bispyridine carboxylate derivative, which has broad-spectrum antitumor activity and good inhibitory activity against a variety of tumor (cancer) cells.

[0007] Another object of the present invention is to provide a pharmaceutical composition.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention protects the use of a bispyridine carboxylate derivative and its pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, stereoisomers, prodrugs, or isotopic compounds in the preparation of anticancer drugs, wherein the cancer is one or more of lung cancer, pancreatic cancer, and prostate cancer;

[0010] The bispyridine carboxylate derivatives have the structure shown in formula (I):

[0011]

[0012] In equation (I), R 1 R 2 Each is independently selected from hydrogen and C. 1～6 Alkyl, C 3～6 Heterocyclic alkyl, phenyl, or heteroaryl; wherein C 1～6 Alkyl groups that are unsubstituted or have one or more substituents R 3 replace;

[0013] The R 3 Selected from C 3～6 Heterocyclic alkyl, hydroxyl, halogen, or amino;

[0014] The C 3～6 Heterocyclic alkyl groups that are unsubstituted or have one or more substituents R 4 replace;

[0015] The R 4 Selected from C 1～6 Alkyl, hydroxyl, halogen, or amino;

[0016] X is either nitrogen or carbon;

[0017] n is any integer from 1 to 8;

[0018] L is selected from C≡C or CH2;

[0019] The C 3～6The heterocyclic alkyl group comprises 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, with the remaining ring atoms being carbon; the heteroaryl group is a 5- to 6-membered aromatic monocyclic ring or an 8- to 10-membered aromatic bicyclic ring, comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, with the remaining ring atoms being carbon; at least one ring in the 8- to 10-membered aromatic bicyclic ring is a benzene ring.

[0020] To develop a more efficient, safe, and low-toxicity novel anticancer drug with broad-spectrum antitumor activity, the applicant conducted further research based on previous patent CN117126105A, devoting considerable effort to the research. They creatively discovered that newly synthesized bispyridine carboxylate derivatives have significant inhibitory effects on various cancer cells, including lung cancer cells, pancreatic cancer cells, and prostate cancer cells. They exhibit particularly superior inhibitory activity against lung cancer cells, significantly inhibiting the growth of small cell lung cancer xenografts in mouse subcutaneous tissue. Furthermore, these bispyridine carboxylate derivatives have no toxic effects on normal cells in the body.

[0021] In the above applications, preferably, the bispyridine carboxylate derivative has the structure shown in formula (II):

[0022]

[0023] In equation (II), R 1 R 2 Each is independently selected from hydrogen and C. 1～6 Alkyl, C 3～6 Heterocyclic alkyl, phenyl, or heteroaryl; wherein C 1～6 Alkyl groups that are unsubstituted or have one or more substituents R 3 replace;

[0024] The R 3 Selected from C 3～6 Heterocyclic alkyl, hydroxyl, halogen, or amino;

[0025] The C 3～6 Heterocyclic alkyl groups that are unsubstituted or have one or more substituents R 4 replace;

[0026] The R 4 Selected from C 1～6 Alkyl, hydroxyl, halogen, or amino;

[0027] X is either nitrogen or carbon;

[0028] n is any integer from 1 to 8;

[0029] L is selected from C≡C or CH2;

[0030] The C3～6 The heterocyclic alkyl group comprises 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, with the remaining ring atoms being carbon; the heteroaryl group is a 5- to 6-membered aromatic monocyclic ring or an 8- to 10-membered aromatic bicyclic ring, comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, with the remaining ring atoms being carbon; at least one ring in the 8- to 10-membered aromatic bicyclic ring is a benzene ring.

[0031] In the above applications, preferably, in formula (II), the R 1 R 2 Each is independently selected from hydrogen or C. 1～6 Alkyl; wherein C 1～6 Alkyl groups that are unsubstituted or have one or more substituents R 3 replace;

[0032] The R 3 Selected from C 3～6 Heterocyclic alkyl, hydroxyl, halogen, or amino;

[0033] The C 3～6 Heterocyclic alkyl groups that are unsubstituted or have one or more substituents R 4 replace;

[0034] The R 4 Selected from C 1～6 Alkyl, hydroxyl, halogen, or amino;

[0035] X is either nitrogen or carbon;

[0036] n is any integer from 1 to 8;

[0037] L is selected from C≡C or CH2.

[0038] In the above applications, preferably, the R 1 R 2 Each is independently selected from hydrogen or C. 1～6 Alkyl; wherein C 1～6 Alkyl groups that are unsubstituted or have one or more substituents R 3 replace;

[0039] The R 3 Selected from C 3～6 Heterocyclic alkyl or hydroxyl groups;

[0040] The C 3～6 Heterocyclic alkyl groups that are unsubstituted or have one or more substituents R 4 replace;

[0041] The R 4 Selected from C 1～6 alkyl;

[0042] X is nitrogen;

[0043] n is any integer from 6 to 8;

[0044] L is selected from C≡C or CH2.

[0045] In the above applications, preferably, the L is selected from CH2.

[0046] More preferably, in the above applications, the bispyridine carboxylate derivative is selected from any of the following structures:

[0047]

[0048] In the above applications, more preferably, the bispyridine carboxylate derivative is selected from any of the following structures:

[0049]

[0050] More preferably, the cancer is lung cancer.

[0051] Furthermore, the lung cancer is caused by small cell lung cancer cell lines and / or non-small cell lung cancer cell lines.

[0052] Furthermore, the small cell lung cancer cell line is one or more of H446, H69, H82, H526, and H146.

[0053] Furthermore, the small cell lung cancer cell line is one or more of H157, H1975, H358, A549, and PC9.

[0054] Furthermore, the pancreatic cancer is caused by the Aspc-1 and / or Bxpc-3 pancreatic cancer cell line.

[0055] Furthermore, the prostate cancer is caused by the Lascpc-01 prostate cancer cell line.

[0056] This invention protects a bispyridine carboxylate derivative and its pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, stereoisomers, prodrugs, or isotopic compounds, characterized in that the bispyridine carboxylate derivative has the structure shown in formula (I):

[0057]

[0058] In equation (I), R 1 R 2 Each is independently selected from hydrogen and C. 1～6 Alkyl, C 3～6Heterocyclic alkyl, phenyl, or heteroaryl; wherein C 1～6 Alkyl groups that are unsubstituted or have one or more substituents R 3 replace;

[0059] The R 3 Selected from C 3～6 Heterocyclic alkyl, hydroxyl, halogen, or amino;

[0060] The C 3～6 Heterocyclic alkyl groups that are unsubstituted or have one or more substituents R 4 replace;

[0061] The R 4 Selected from C 1～6 Alkyl, hydroxyl, halogen, or amino;

[0062] X is either nitrogen or carbon;

[0063] n is any integer from 1 to 8;

[0064] L is selected from C≡C or CH2;

[0065] And it does not contain the following compounds:

[0066] The C 3～6 The heterocyclic alkyl group comprises 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, with the remaining ring atoms being carbon; the heteroaryl group is a 5- to 6-membered aromatic monocyclic ring or an 8- to 10-membered aromatic bicyclic ring, comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, with the remaining ring atoms being carbon; at least one ring in the 8- to 10-membered aromatic bicyclic ring is a benzene ring.

[0067] Preferably, the bispyridine carboxylate derivative has the structure shown in formula (II):

[0068]

[0069] In equation (II), R 1 R 2 Each is independently selected from hydrogen and C. 1～6 Alkyl, C 3～6 Heterocyclic alkyl, phenyl, or heteroaryl; wherein C 1～6 Alkyl groups that are unsubstituted or have one or more substituents R 3 replace;

[0070] The R 3 Selected from C 3～6 Heterocyclic alkyl, hydroxyl, halogen, or amino;

[0071] The C 3～6Heterocyclic alkyl groups that are unsubstituted or have one or more substituents R 4 replace;

[0072] The R 4 Selected from C 1～6 Alkyl, hydroxyl, halogen, or amino;

[0073] X is either nitrogen or carbon;

[0074] n is any integer from 1 to 8;

[0075] L is selected from C≡C or CH2;

[0076] The C 3～6 The heterocyclic alkyl group comprises 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, with the remaining ring atoms being carbon; the heteroaryl group is a 5- to 6-membered aromatic monocyclic ring or an 8- to 10-membered aromatic bicyclic ring, comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, with the remaining ring atoms being carbon; at least one ring in the 8- to 10-membered aromatic bicyclic ring is a benzene ring.

[0077] Preferably, the R 1 R 2 Each is independently selected from hydrogen or C. 1～6 Alkyl; wherein C 1～6 Alkyl groups that are unsubstituted or have one or more substituents R 3 replace;

[0078] The R 3 Selected from C 3～6 Heterocyclic alkyl, hydroxyl, halogen, or amino;

[0079] The C 3～6 Heterocyclic alkyl groups that are unsubstituted or have one or more substituents R 4 replace;

[0080] The R 4 Selected from C 1～6 Alkyl, hydroxyl, halogen, or amino;

[0081] X is either nitrogen or carbon;

[0082] n is any integer from 1 to 8;

[0083] L is selected from C≡C or CH2.

[0084] Preferably, the R 1 R 2 Each is independently selected from hydrogen or C. 1～6 Alkyl; wherein C 1～6 Alkyl groups that are unsubstituted or have one or more substituents R 3 replace;

[0085] The R 3 Selected from C 3～6 Heterocyclic alkyl or hydroxyl groups;

[0086] The C 3～6 Heterocyclic alkyl groups that are unsubstituted or have one or more substituents R 4 replace;

[0087] The R 4 Selected from C 1～6 alkyl;

[0088] X is nitrogen;

[0089] n is any integer from 6 to 8;

[0090] L is selected from C≡C or CH2.

[0091] More preferably, the L is selected from CH2.

[0092] More preferably, the bispyridine carboxylate derivative is selected from any of the following structures:

[0093]

[0094] More preferably, the bispyridine carboxylate derivative is selected from any of the following structures:

[0095]

[0096] This invention protects a pharmaceutical composition comprising one or more of the following: the bispyridine carboxylate derivative, a pharmaceutically acceptable salt thereof, its hydrate, its solvate, its polymorph, its tautomer, its stereoisomer, its prodrug, and its isotopic compound.

[0097] Compared with the prior art, the present invention has the following beneficial effects: The bispyridine carboxylate derivatives provided by the present invention have broad-spectrum antitumor activity, and can significantly inhibit the proliferation activity of lung cancer cells, pancreatic cancer cells and prostate cancer cells, especially exhibiting superior inhibitory activity against lung cancer cells, and can significantly inhibit the growth of mouse subcutaneous small cell lung cancer xenografts; in addition, the bispyridine carboxylate derivatives have no toxic effect on normal cells of the body, and can be used to prepare anticancer drugs, showing broad application prospects in anticancer. Attached Figure Description

[0098] Figure 1 is a statistical graph showing the activity evaluation data of the bispyridine carboxylate derivatives obtained in this invention against the small cell lung cancer cell line (H128).

[0099] Figure 2 is a statistical graph showing the activity evaluation data of compounds 3 and 5 on small cell lung cancer cells.

[0100] Figure 3 is a statistical graph showing the activity evaluation data of compounds 3 and 5 on non-small cell lung cancer cells.

[0101] Figure 4 is a statistical graph showing the activity evaluation data of compounds 3 and 5 on pancreatic cancer cells.

[0102] Figure 5 is a statistical graph showing the activity evaluation data of compounds 3 and 5 on prostate cancer cells.

[0103] Figure 6 shows the appearance of subcutaneous small cell lung cancer tumors collected 19 days after administration during a mouse subcutaneous small cell lung cancer cell (H446 and H128) xenograft model.

[0104] Figure 7 is a statistical graph of tumor volume changes during the mouse subcutaneous small cell lung cancer cell (H446 and H128) xenograft model.

[0105] Figure 8 shows the statistical data of subcutaneous tumor weight of small cell lung cancer collected 19 days after drug administration.

[0106] Figure 9 is a statistical chart of mouse weight data measured 19 days after drug administration. Detailed Implementation

[0107] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0108] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0109] Example 1: Structure of bispyridine carboxylate derivatives

[0110] This study synthesized nine bispyridine carboxylate derivatives with novel structures. The structural formulas and molecular weights of these compounds are shown in Table 1.

[0111] Table 1. Structural formulas and molecular weights of bispyridine carboxylate derivatives

[0112]

[0113]

[0114]

[0115] Example 2 Synthesis of bispyridine carboxylate derivative - compound 1

[0116] The synthetic route of dimethyl 5,5'-(deca-1,9-diyne-1,10-diyl)dipicolinate (i.e., compound 1 in Example 1) is shown below:

[0117]

[0118] The specific synthetic method includes the following steps: methyl 5-bromopyridine-2-carboxylic acid (0.5 g, 2.31 mmol), 1,9-decadiyne (0.14 g, 1.05 mmol), Pd(Ph3P)2Cl2 (73.8 mg, 0.1 mmol), and cuprous iodide (30.05 mg, 0.16 mmol) were weighed and dissolved in THF (5 mL) and TEA (5 mL). The mixture was stirred at 100 °C for 12 hours under nitrogen protection. After the reaction was complete, the mixture was filtered through diatomaceous earth. The filter cake was washed with ethyl acetate, and the filtrate was evaporated to dryness. 20% citric acid aqueous solution (10 mL) was added, followed by extraction with ethyl acetate. The organic phase was washed twice with water and once with saturated sodium chloride solution. The washed organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 0.35 g of a white solid product (compound 1), with a yield of 67%.

[0119] The NMR data for compound 1 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.71(dd,J=2.1,0.8Hz,2H),8.06(d,J=0.8Hz,1H),8.04(d,J=0.8Hz,1H),7.80(d,J=2.1H z,1H),7.78(d,J=2.1Hz,1H),4.00(s,6H),2.48(t,J=7.1Hz,4H),1.72–1.63(m,4H),1.53(q,J=3.6Hz,4H).MS(ESI)m / z 405.85[M+H] + .

[0120] The synthetic route of (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl-5-(10-(6-(methoxycarbonyl)pyridin-3-yl)deca-1,9-diyn-1-yl)picolinate (compound 2) is shown below.

[0121] Example 3 Synthesis of bispyridine carboxylate derivative - compound 2

[0122] The synthetic route of (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl-5-(10-(6-(methoxycarbonyl)pyridin-3-yl)deca-1,9-diyn-1-yl)picolinate (compound 2) is shown below:

[0123]

[0124] The specific synthesis method includes the following steps:

[0125] Step 1: Weigh 5.0 g (23.14 mmol) of methyl 5-bromopyridine-2-carboxylic acid and dissolve it in tetrahydrofuran (40 mL), methanol (10 mL), and water (10 mL). Then add lithium hydroxide monohydrate (0.6 g, 25.46 mmol) and stir overnight at room temperature. After the reaction is complete, remove the organic solvent by rotary evaporation, add 30 mL of water, and then adjust the pH to 2-3 with 2M hydrochloric acid. A solid precipitates out. Filter the solid, wash the filter cake with water, and dry it to obtain 3.80 g of white solid product, with a yield of 81.3%.

[0126] Step 2: Dissolve the white solid product obtained in Step 1 (3.80 g, 18.81 mmol) in dichloromethane (30 mL), control the temperature at 0-5 °C, add triphosgene (5.58 g, 18.81 mmol), and stir at room temperature for two hours. After the reaction is complete, remove the organic solvent by rotary evaporation, add dichloromethane again and distill twice to obtain a white solid, then dissolve it in dichloromethane and add it dropwise at 0-5 °C to another reaction flask containing (R)-(+)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane (2.73 g, 20.65 mmol) and triethylamine (2.85 g, 28.16 mmol). After stirring at room temperature for two hours, when the reaction was complete, 30 mL of 20% citric acid aqueous solution was added, followed by extraction with dichloromethane twice, washing the organic phase twice with water and once with saturated sodium chloride solution. The washed organic phase was dried and concentrated with anhydrous sodium sulfate and purified by column chromatography to obtain 4.60 g of oil, with a yield of 77.3%.

[0127] Step 3: Weigh the product obtained in Step 2 (0.80 g, 2.54 mmol), methyl 5-bromopyridine-2-carboxylate (0.56 g, 2.59 mmol), 1,9-decadiyne (0.30 g, 2.5 mmol), Pd(Ph3P)2Cl2 (0.18 g, 0.25 mmol), and cuprous iodide (0.12 g, 0.63 mmol), and dissolve them in THF (10 mL) and TEA (10 mL). Stir at 100 °C for 12 hours. After the reaction is complete, filter through diatomaceous earth. Wash the filter cake with ethyl acetate, evaporate the filtrate to dryness, add 20% citric acid aqueous solution (10 mL), and then add ethyl acetate for extraction. Wash the organic phase twice with water and once with saturated sodium chloride solution. Dry the washed organic phase with anhydrous sodium sulfate, concentrate, and then purify by column chromatography to obtain 0.70 g of white solid product (compound 2), with a yield of 54.8%.

[0128] The NMR data for compound 2 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.71(dd,J=5.1,2.0Hz,2H),8.06(dd,J=3.5,0.9Hz,1H) ,8.04(dd,J=3.5,0.8Hz,1H),7.79(ddd,J=8.1,4.4,2.1Hz,2H),4.55–4.39(m,3H),4.1 5(dd,J=8.6,6.2Hz,1H),4.00(s,3H),3.87(dd,J=8.6,5.6Hz,1H),2.48(t,J=7.0Hz,4 H),1.67(q,J=6.8Hz,4H),1.53(p,J=3.6Hz,4H),1.45(s,3H),1.38(s,3H).MS(ESI)m / z 505.99[M+H] + .

[0129] Example 4 Synthesis of bispyridine carboxylate derivative - compound 3

[0130] The synthetic route of (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl-5-(10-(6-(methoxycarbonyl)pyridin-3-yl)decyl)picolinate (compound 3) is shown below:

[0131]

[0132] The specific synthesis method includes the following steps:

[0133] Compound 2 (0.50 g, 0.99 mmol) obtained in Example 3 was dissolved in ethyl acetate (3 mL) and methanol (3 mL), and then 0.1 g of Pd / C was added. The mixture was then purged three times with a hydrogen balloon, and stirred overnight at room temperature. After the reaction was complete, the mixture was filtered through a diatomaceous earth filter, the filter cake was washed with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The resulting product was purified by column chromatography to give 0.25 g of a white solid product (compound 3), with a yield of 60.8%.

[0134] The NMR data for compound 3 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.72(dd,J=5.0,2.0Hz,2H),8.06(d,J=3.1Hz,1H),8.0 4(d,J=3.0Hz,1H),7.79(ddd,J=8.2,4.3,2.1Hz,2H),4.56–4.39(m,3H),4.16(dd,J= 8.6,6.3Hz,1H),4.00(s,3H),3.88(dd,J=8.6,5.7Hz,1H),2.68(t,J=7.7Hz,4H),1.6 4(dd,J=10.3,4.9Hz,4H),1.46(s,3H),1.38(s,3H),1.34–1.23(m,12H).MS(ESI)m / z 514.22[M+H] + .

[0135] Example 5 Synthesis of bispyridine carboxylate derivatives - compounds 4-6

[0136] The synthetic routes for compounds 4-6 are shown below:

[0137]

[0138] The specific synthesis method includes the following steps:

[0139] Step 1: Same as Step 1 in Example 3.

[0140] Step 2: Same as step 2 in Example 3.

[0141] Step 3: Replace the methyl 5-bromopyridine-2-carboxylate from Example 2 with (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl5-bromopyridinecarboxylate obtained in Step 2. Other reagents and parameters are the same as in the synthesis steps of Example 2, yielding 0.36 g of white solid product (compound 4), with a three-step yield of 40.1%.

[0142] The NMR data for compound 4 are as follows: 1 H NMR(400MHz,Chloroform-d)δ8.72(dd,J=2.2,0.8Hz,2H),8.05(d,J=0.9Hz,1H), 8.03(d,J=0.9Hz,1H),7.79(d,J=2.1Hz,1H),7.77(d,J=2.1Hz,1H),4.54–4.40(m, 6H), 4.15 (dd, J=8.6, 6.2Hz, 2H), 3.87 (dd, J=8.6, 5.6Hz, 2H), 2.48 (t, J=7.1Hz, 4H ),1.67(d,J=6.9Hz,4H),1.55–1.50(m,4H),1.45(s,6H),1.38(s,6H).MS(ESI)m / z 605.54 [M+H] + .

[0143] Step 4: Replace compound 2 in Example 4 with compound 4 obtained in Step 3 above. Other reagents and parameters are the same as in the synthesis steps of Example 4. 150 mg of white solid product (compound 5) is obtained, with a yield of 73%.

[0144] The NMR data for compound 5 are as follows: 1 H NMR(500MHz,Chloroform-d)δ8.57(d,J=2.1Hz,2H),8.06(s,1H),8.04(s,1H),7.63(d,J=2.2Hz,1H),7.62(d,J=2.2Hz,1H),4.55–4.40(m,6H),4.1 5(dd,J=8.6,6.3Hz,2H),3.88(dd,J=8.6,5.8Hz,2H),2.68(t,J=7.7Hz,4H ),1.65(s,4H),1.46(s,6H),1.38(s,6H),1.34–1.23(m,12H).MS(ESI)m / z 613.79[M+H] + .

[0145] Step 5: Dissolve compound 5 (0.10 g, 0.99 mmol) obtained in Step 4 in acetone (3 mL), then add 2 M HCl and stir at 50 °C for 3 hours. After the reaction is complete, remove the acetone by rotary evaporation, adjust the pH to 8-9 with 20% sodium bicarbonate aqueous solution, extract with ethyl acetate, dry and concentrate the organic phase with anhydrous sodium sulfate, and then purify by column chromatography to obtain 60 mg of white solid product (compound 6), with a yield of 69.3%.

[0146] The NMR data for compound 6 are as follows: 1H NMR(500MHz,Chloroform-d)δ8.50(d,J=2.1Hz,2H),8.04(d,J=8.0Hz,2H),7.63 (dd,J=8.0,2.2Hz,2H),4.49(dd,J=11.4,3.8Hz,2H),4.41(dd,J=11.4,6.7Hz,2H ),4.16(dd,J=6.1,3.8Hz,1H),4.02–3.99(m,1H),3.77(qd,J=11.4,4.9Hz,4H), 2.66(t,J=7.6Hz,4H),1.61(d,J=8.2Hz,5H),1.27(d,J=7.1Hz,11H).MS(ESI)m / z 533.68 [M+H] + .

[0147] Example 6 Synthesis of bispyridine carboxylate derivative - compound 7

[0148] bis(((R)-2,2-dimethyl-1,3-dioxolan-4-yl)methyl)

[0149] The synthetic route of 5,5'-(nonane-1,9-diyl)dipicolinate (compound 7) is shown below:

[0150]

[0151] The specific synthesis method includes the following steps:

[0152] Step 1: Same as Step 1 in Example 3.

[0153] Step 2: Same as step 2 in Example 3.

[0154] Step 3: Replace 1,9-decadiyne with 1,8-nonadiyne. Other reagents and parameters are the same as in Step 3 of Example 5. 1.1 g of white solid product is obtained, with a yield of 79%.

[0155] Step 4: Replace compound 2 in Example 4 with the white solid product obtained in Step 3 above. Other reagents and parameters are the same as in the synthesis steps of Example 4. 150 mg of white solid product (compound 7) is obtained, with a yield of 80%.

[0156] The NMR data for compound 7 are as follows: 1H NMR (400MHz, Chloroform-d) δ8.57(d,J=2.2Hz,2H),8.06(s,1H),8.04(s,1H),7.63(d,J=2.3Hz,1H),7.61(d,J=2.2Hz,1H),4.56–4.42(m,6H),4.15(dd ,J=8.5,6.3Hz,2H),3.88(dd,J=8.6,5.7Hz,2H),2.68(t,J=7.7Hz,4H),1.63 (p,J=7.2Hz,4H),1.46(s,6H),1.38(s,6H),1.35–1.27(m,10H).MS(ESI)m / z 599.80[M+H] + .

[0157] Example 7 Synthesis of bispyridine carboxylate derivatives - compounds 8-9

[0158] The synthetic routes for compounds 8 and 9 are shown below:

[0159]

[0160] The specific synthesis method includes the following steps:

[0161] Step 1: Same as Step 1 in Example 3.

[0162] Step 2: Replace (R)-(+)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane with (S)-(-)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane. Other reagents and parameters are the same as in Step 2 of Example 3.

[0163] Step 3: Replace (R)-(2,2-dimethyl-1,3-dioxolane-4-yl)methyl5-bromopyridine carboxylate from Example 5 with the product obtained in Step 2 above. Other reagents and parameters are the same as in Step 3 of Example 5.

[0164] Step 4: Replace compound 2 from Example 4 with the product obtained in Step 3. Other reagents and parameters are the same as in the synthesis steps of Example 4. 152 mg of white solid product (compound 8) is obtained, with a yield of 78%.

[0165] The NMR data for compound 8 are as follows: 1H NMR(500MHz,Chloroform-d)δ8.57(d,J=2.1Hz,2H),8.06(s,1H),8.04(s,1H),7.63(d,J=2.2Hz,1H),7.62(d,J=2.2Hz,1H),4.55–4.40(m,6H),4.1 5(dd,J=8.6,6.3Hz,2H),3.88(dd,J=8.6,5.8Hz,2H),2.68(t,J=7.7Hz,4H ),1.65(s,4H),1.46(s,6H),1.38(s,6H),1.34–1.23(m,12H).MS(ESI)m / z 614.25[M+H] + .

[0166] Step 5: Replace compound 5 with compound 8, and keep the other reagents and parameters the same as in step 5 of Example 5 to obtain 51 mg of white solid product (compound 9), with a yield of 66%.

[0167] 1 H NMR(500MHz,Chloroform-d)δ8.50(d,J=2.1Hz,2H),8.04(d,J=8.0Hz,2H),7.63 (dd,J=8.0,2.2Hz,2H),4.49(dd,J=11.4,3.8Hz,2H),4.41(dd,J=11.4,6.7Hz,2H ),4.16(dd,J=6.1,3.8Hz,1H),4.02–3.99(m,1H),3.77(qd,J=11.4,4.9Hz,4H), 2.66(t,J=7.6Hz,4H),1.61(d,J=8.2Hz,5H),1.27(d,J=7.1Hz,11H).MS(ESI)m / z 533.65 [M+H] + .

[0168] Example 8: Evaluation of the in vitro anti-small cell lung cancer activity of bispyridine carboxylate derivatives

[0169] (1) Cell Culture

[0170] The small cell lung cancer cell line H128 was cultured in RPMI 1640 medium containing double antibiotics (streptomycin / penicillin, 1%) and fetal bovine serum (10%), and incubated at 37°C in a 5% CO2 incubator.

[0171] (2) Cell counting

[0172] 1) Discard the culture medium in the culture dish containing the cells to be counted, wash gently with 2 mL PBS, discard the PBS, add 2 mL trypsin and digest in a constant temperature incubator for 2-5 min, add 5 mL culture medium to stop digestion, mix thoroughly and then aspirate 1 mL of cell suspension into a 1.5 mL EP tube;

[0173] 2) After thoroughly mixing the cell suspension with a vortex mixer, take 10 μL and place it on a cell counting plate. Gently blot the remaining culture medium outside the slide with a paper towel, and then observe it under a microscope (10×10). Based on the principle of "counting the top but not the bottom, counting the left but not the right", calculate the number of cells per milliliter.

[0174] (3) CCK-8 cell viability assay

[0175] 1) After cell counting, according to the cell growth rate, seed 96-well plates with 500-5000 cells / well and 100μL of culture medium / well, and incubate in an incubator;

[0176] 2) After culturing for 24 hours, the dipyridine carboxylate derivatives were 10 μM as the initial concentration and then diluted 50 μL / well. Three replicates were set for each concentration, and then the wells were incubated in a constant temperature incubator.

[0177] 3) After culturing for 96 hours, CCK-8 reagent was added to each well using a multi-channel pipette under dark conditions, at a rate of 10 μL / well. After 30 minutes, air bubbles were gently blown away with a blower. The absorbance of each well was measured using a microplate reader at a wavelength of 450 nm. Cell viability was calculated based on the results.

[0178] (4) Experimental Results

[0179] The experimental results are shown in Figure 1. Compounds 1-9 can inhibit the proliferation of small cell lung cancer cells (H128) in a dose-dependent manner (compound 1: IC50). 50 =6.385μM; Compound 2: IC 50 =4.338 μM; Compound 3: IC 50 =0.04035μM; Compound 4: IC 50 =1.862 μM; Compound 5: IC 50 =0.02158 μM; Compound 6: IC 50 =1.247 μM; Compound 7: IC 50 =1.327 μM; Compound 8: IC 50 =0.07536 μM; Compound 9: IC 50 =0.2463 μM;), among which compounds 3, 5 and 8 have excellent inhibitory effects.

[0180] Example 9: Evaluation of the in vitro anticancer activity of bispyridine carboxylate derivatives

[0181] Using compounds 3 and 5 as examples, the antitumor activity of the bispyridine carboxylate derivatives provided by this invention in different cancers was evaluated.

[0182] (1) Cell Culture

[0183] 1) Small cell lung cancer cell lines H446, H69, H82, H526 and H146 were cultured in RPMI 1640 medium containing double antibiotics (streptomycin / penicillin, 1%) and fetal bovine serum (10%).

[0184] 2) Non-small cell lung cancer cell lines H157, H1975, H358, A549, and PC9 were cultured in RPMI 1640 medium containing double antibiotics (streptomycin / penicillin, 1%) and fetal bovine serum (10%).

[0185] 3) Pancreatic cancer cell lines Aspc-1 and Bxpc-3 were cultured in RPMI 1640 medium containing double antibiotics (streptomycin / penicillin, 1%) and fetal bovine serum (10%), while Miapaca-2 was cultured in high-glucose DMEM medium (1% double antibiotics, 10% fetal bovine serum).

[0186] 4) The prostate cancer cell line Lascpc-01 was cultured in RPMI 1640 medium containing double antibiotics (streptomycin / penicillin, 1%), fetal bovine serum (10%), insulin (0.005 mg / ml), transferrin (0.01 mg / ml), sodium nitrite (30 nM), hydrocortisone (10 nM), and estradiol (10 nM);

[0187] All the cells were cultured in a 37°C, 5% CO2 incubator.

[0188] (2) The cell counting method is the same as in Example 9.

[0189] (3) The method for measuring CCK-8 cell viability is the same as in Example 9.

[0190] (4) Experimental Results

[0191] The experimental results are shown in Figures 2-5. Compounds 3 and 5, exhibiting the strongest activity, were selected to evaluate their antitumor activity in different cancers. Figure 2 shows that compounds 3 and 5 can dose-dependently inhibit the proliferation of small cell lung cancer cells; Figure 3 shows that compounds 3 and 5 can dose-dependently inhibit the proliferation of non-small cell lung cancer cells; Figure 4 shows that compounds 3 and 5 can dose-dependently inhibit the proliferation of pancreatic cancer cells; and Figure 5 shows that compounds 3 and 5 can dose-dependently inhibit the proliferation of prostate cancer cells. These results indicate that the bispyridine carboxylate derivatives provided in this application possess broad-spectrum tumor-suppressive activity and exhibit inhibitory effects against various cancers.

[0192] Example 10: Activity evaluation of bispyridine carboxylate derivatives in an in vivo model of small cell lung cancer.

[0193] Using compounds 3 and 5 as examples, the antitumor activity of the bispyridine carboxylate derivatives provided in this invention in an in vivo model of small cell lung cancer was evaluated.

[0194] (1) Establishment of a subcutaneous xenograft model of small cell lung cancer cells H446 / H128

[0195] 1) Remove the pre-amplified and healthy H446 / H128 cells from the incubator, add trypsin for digestion, stop digestion with culture medium, centrifuge to remove the supernatant, wash the cells twice with pre-cooled PBS, count the cells, and add an appropriate amount of pre-prepared Matrigel / PBS premix (Matrigel:PBS = 1:1) to make a concentration of 5*102 cells. 6 Cell suspension of 100 μL cells;

[0196] 2) Inject 100 μL of cell suspension into each side of the subcutaneous tissue on the ventral and dorsal sides of the nude mouse using a 1 mL syringe;

[0197] 3) When the tumor size increases to 50mm 3 Mice were randomly divided into three groups as required. The groups and drug administration settings were as follows: control group, which received intraperitoneal injection of placebo (i.e., the solvent used to dissolve the drug: 15% castor oil + 85% sterile PBS) daily; compound group 3, which received intraperitoneal injection of 10 mg / kg daily; and compound group 5, which received intraperitoneal injection of 10 mg / kg daily.

[0198] 4) Administer the drug continuously for 19 days, measuring and recording the mouse's body weight and tumor size every three days. Tumor volume was calculated as π / 6 (length × width). 2 )calculate;

[0199] 5) After the experiment, the mice were euthanized, and the tumor tissue was collected, photographed, and weighed.

[0200] (2) Experimental Results

[0201] The experimental results are shown in Figures 6-9. Figure 6 shows the appearance of subcutaneous tumors of small cell lung cancer collected 19 days after administration. Figure 7 shows the changes in tumor volume during the mouse subcutaneous small cell lung cancer cell (H446 and H128) xenograft model. The results show that both compound 3 and compound 5 can inhibit the growth of subcutaneous tumors in mice. Figure 8 shows the weight of subcutaneous tumors of small cell lung cancer collected 19 days after administration. The results show that both compound 3 and compound 5 can significantly reduce the weight of subcutaneous tumors of small cell lung cancer. Figure 9 shows the weight of mice weighed 19 days after administration. The results show that compound 3 and compound 5 have no significant effect on the weight of mice, indicating that compound 3 and compound 5 are non-toxic or have very low toxicity and will not harm the normal cells of the body.

[0202] In summary, the bispyridine carboxylate derivatives provided by this invention have novel structures, good activity, and low toxicity, exhibiting good antitumor activity. They show good inhibitory activity against various tumor cells (such as lung cancer, pancreatic cancer, and prostate cancer). Compounds 3 and 5, in particular, show inhibitory activity against various small cell lung cancer cells, especially exhibiting excellent inhibitory activity against the H128 small cell lung cancer cell line, with an IC50 value of [missing information]. 50 The concentrations were 40.35 nM and 21.58 nM, respectively. Compounds 3 and 5 also dose-dependently inhibited the proliferation of pancreatic and prostate cancer cells. In in vivo experiments, compounds 3 and 5 significantly inhibited the growth of subcutaneous tumors of small cell lung cancer and reduced the weight of these tumors.

[0203] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a bispyridine carboxylate derivative and its pharmaceutically acceptable salt in the preparation of an anti-small cell lung cancer drug, characterized in that, The bispyridine carboxylate derivatives are selected from any one of the following structures: 。 2. The use of a bispyridine carboxylate derivative and its pharmaceutically acceptable salt in the preparation of anticancer drugs, characterized in that, The cancer is one or more of non-small cell lung cancer, pancreatic cancer, and prostate cancer; the bispyridine carboxylate derivative is selected from any of the following structures: 。 3. A bispyridine carboxylate derivative and its pharmaceutically acceptable salt, characterized in that, The bispyridine carboxylate derivatives are selected from any one of the following structures: 。 4. A pharmaceutical composition, characterized in that, It includes one or more of the bispyridine carboxylate derivatives of claim 3 and their pharmaceutically acceptable salts.

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