Pyrazolopyridine calnexin ligands, methods of making and using the same

Abstract

The application discloses a pyrazolopyridine calnexin ligand and a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The structure of the pyrazolopyridine calnexin ligand is shown in the following formula: wherein R1 is preferably or R2 is preferably. Experiments prove that the pyrazolopyridine derivative disclosed by the application has a proliferation inhibiting effect on various tumor cells, and has a stronger IC 50 on pancreatic cancer cells; has a significant inhibiting effect on the adipogenic differentiation of fat cells 3T3-L1, has good weight loss and blood lipid reducing activities, and has good long-term drug safety.

Description

Technical Field

[0001] This invention relates to calreticulin ligands, their preparation methods and applications, specifically to pyrazolopyridine calreticulin ligands, their preparation methods and applications, and belongs to the field of medicinal chemistry technology. Background Technology

[0002] Calreticulin (CALR) is an endoplasmic reticulum chaperone protein comprising an N-terminal domain, a central domain, and a C-terminal acidic domain. The N-terminal domain binds to glucocorticoid receptors, integrins, and other receptors, regulating CALR membrane translocation; the central domain binds to Ca2+ receptors with high affinity. 2+ However, its load-bearing capacity is low; the C-terminal acidic domain has low resistance to Ca. 2+ While possessing low affinity, CALRs have a high carrying capacity and can promote apoptosis signaling. After nascent CALRs assemble and mature in the endoplasmic reticulum, some remain intracellularly to function as molecular chaperones, while others are transported to the plasma membrane or secreted into the extracellular space to perform multiple functions. For example, CALRs can bind over 50% of intracellular Ca2+. 2+ To act as a calcium reservoir, allowing the endoplasmic reticulum lumen to store calcium. 2+ The concentration is 2-10 times higher than in the cytoplasm; CALR can promote the folding and maturation of various substrate proteins, including calcium-binding proteins and heat shock proteins; CALR can promote the formation of histocompatibility class I molecules, present endogenous antigens, and activate CD4+. 8+ T cells clear infected and tumor cells; CALRs act as proliferative factors in physiological and pathological mitosis and are closely related to a variety of diseases.

[0003] CALR is highly expressed in various tumor tissues, and clinical research data also indicate that high CALR expression is closely related to low survival rates in patients with lung cancer, liver cancer, and breast cancer. Furthermore, the function and regulation of CALR differ between tumor and normal cells. Chemical targeting or biological intervention of CALR expression can significantly inhibit tumor cell proliferation, making it an important target for chemical intervention. In recent years, various novel CALR ligands have been developed for cancer therapy; however, most molecules exhibit low affinity for CALR, resulting in poor tumor-suppressive activity and intracellular off-target effects. The lack of structural characterization of the CALR ligand design pocket and its complex interactions with substrates further complicate the design and development of specific CALR ligands.

[0004] Normal fat metabolism is a crucial foundation for maintaining human physiological health. Modern people are prone to obesity due to excessive fat intake and insufficient metabolism. Obesity can cause various physiological disorders and seriously endanger human health, thus creating a huge demand for anti-obesity drugs in clinical practice. Currently marketed drugs are mostly appetite suppressants (such as liraglutide), digestive enzyme inhibitors (such as orlistat), and neurotransmitter blockers (such as phentermine-topiramate). These drugs can cause adverse reactions in the central nervous system and cardiovascular system with long-term use. Therefore, the discovery of new targets and the development of new active molecules are of great significance in the field of anti-obesity research.

[0005] Intracellular Ca 2+ Regulating homeostasis is closely related to the catabolism of fat. On the one hand, Ca... 2+ Increased concentration can activate lipases, promoting the hydrolysis of triglycerides (TG) in adipocytes and releasing fatty acids into the bloodstream to accelerate fat breakdown; secondly, Ca... 2+ It can activate calmodulin-dependent protein kinase II (CAMKII) to enhance mitochondrial fatty acid oxidation and promote acetyl-CoA production to accelerate fat metabolism; thirdly, Ca 2+ It can reduce hunger by stimulating calcitonin secretion, thereby decreasing appetite and indirectly affecting energy intake. Simultaneously, it can synergistically affect the secretion of hormones related to fat metabolism with vitamin D; fourthly, Ca... 2+ It can act as an intracellular signaling molecule, participating in adipocyte differentiation, proliferation, and metabolic regulation. In summary, the endoplasmic reticulum (ER) contains Ca2+. 2+ An increase in calcium content can effectively accelerate fat breakdown, while... 2+ The decrease in content will inhibit the activity of lipases. Given that chemically targeting CALR can effectively induce Ca... 2+ The release and increase in cytoplasm content have potential lipid-lowering effects. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a pyrazolopyridine calreticulin ligand with good chemical targeting properties for CALR, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The structure of pyrazolopyridine calreticulin ligands is shown below:

[0008] Where R1 can be any of the following structures: ; R2 can be any of the following structures: .

[0009] The aforementioned method for preparing pyrazolopyridine calreticulin ligands includes the following steps: (1) 5-bromo-2-chloronicotinonitrile was added to anhydrous ethanol, the reaction system was heated to 70°C, and then hydrazine hydrate was added dropwise while maintaining the reaction at 70°C. After the reaction was completed, the product was separated and purified to obtain compound 2. (2) Add compound 2, phenyl or pyridylboronic acid compound and inorganic base to a mixed solvent of organic solvent and water, stir to dissolve, purge with nitrogen for 10 min, then add palladium catalyst, heat the reaction system to 100℃-110℃ and react at this temperature, after the reaction is completed, separate and purify the product to obtain compound A; (3) Dissolve compound A in N,N-dimethylformamide, add acyl chloride, and react at 25°C. After the reaction is complete, separate and purify the product to obtain phenyl or pyridine-substituted pyrazolopyridine derivatives. The phenylboronic acid compounds are selected from 4-(morpholinesulfonyl)phenylboronic acid, 4-(trifluoromethoxy)phenylboronic acid, 2,3-dimethoxyphenylboronic acid, 3,4-dimethoxyphenylboronic acid, methyl carbamate phenylboronic acid, 4-methanesulfonylphenylboronic acid, 4-ethanesulfonylphenylboronic acid, 4-(N,N-dimethylaminosulfonyl)phenylboronic acid, 4-(N-methylaminosulfonyl)phenylboronic acid, 4-fluorophenylboronic acid, 4-(isopropylthio)phenylboronic acid, 2,4-difluorophenylboronic acid, 4-isopropylsulfonylphenylboronic acid, or 2-naphthoboronic acid; the pyridylboronic acid compounds are selected from 6-isopropoxypyridine-3-boronic acid pinacol ester or 2-fluoropyridine-4-boronic acid.

[0010] The aforementioned application of pyrazolopyridine calreticulin ligands in the preparation of antitumor drugs, wherein the application is any one of the following: (1) , , , , , , or Application in the preparation of anti-gastric cancer drugs; (2) , , , , or Application in the preparation of anti-liver cancer drugs; (3) , , or Application in the preparation of drugs for treating non-small cell lung cancer; (4) , , , , , , , , or Application in the preparation of anti-pancreatic cancer drugs.

[0011] The aforementioned application of pyrazolopyridine calreticulin ligands in the preparation of lipid-lowering drugs, wherein the pyrazolopyridine calreticulin ligand is any one of the following compounds: , , , , .

[0012] The advantages of this invention are: (1) The present invention uses commercially available and inexpensive pyridine as a substrate. The target product can be prepared by only three chemical reactions (ring-closing reaction, Suzuki-Miyaura coupling reaction, N-acylation reaction). The reaction conditions are mild, the cost is low, the product is easy to separate, and the synthesis efficiency is high, making it suitable for large-scale industrial production. (2) The pyrazolopyridine derivatives prepared in this invention have good binding affinity for CALR, with the binding site located in the N-terminal domain. These derivatives can upregulate intracellular Ca2+ to varying degrees. 2+ The content and effect on mitochondrial membrane potential; (3) The pyrazolopyridine derivatives prepared in this invention have inhibitory effects on the proliferation of various tumor cells, and generally inhibit the IC50 of human pancreatic cancer cells PANC-1. 50 More potent, compound a1 is preferred for development as a lead molecule against pancreatic cancer; it has a significant inhibitory effect on adipogenic differentiation of adipocytes 3T3-L1, and the EC50 of compound a13 is preferred. 50 The concentration is 1.3 μM, which has good weight loss and lipid-lowering activities, and the long-term administration has good safety. Attached Figure Description

[0013] Figure 1 The pyrazolopyridine derivatives prepared in this invention, after being incubated with human pancreatic cancer cells PANC-1 for 24 hours, resulted in intracellular Ca2+. 2+ Graph showing the calculated content; Figure 2 These are confocal microscopy images of human pancreatic cancer cells PANC-1 before and after administration of compound a1. Figure 3 This is a graph showing the calculated cell cycle distribution ratio of human pancreatic cancer cells PANC-1 after treatment with compound a1. Figure 4 This is a graph showing the calculated apoptosis rate of human pancreatic cancer cells PANC-1 after treatment with compound a1. Figure 5 This is an image of Oil Red O staining results of mouse embryonic fibroblasts 3T3-L1 after treatment with pyrazolopyridine derivatives prepared in this invention. Figure 6 The graph shows the calculated TG inhibition rates of compounds a13, berberine, and resveratrol. Figure 7 This is an image showing the immunofluorescence staining results of mouse embryonic fibroblast 3T3-L1 cells before and after administration of compound a13; Figure 8 This is a graph showing the calculated relative body weight of mice in each group during the drug administration period; Figure 9 This is a graph showing the calculated relative food intake of mice in each group after the last administration of the drug; Figure 10 This is a graph showing the calculated relative liver weight of mice in each group after the last administration. Figure 11 This is a graph showing the calculated relative fat weight of inguinal white adipose tissue (iWAT) in mice after the last administration of the drug in each group; Figure 12 This is a graph showing the calculated relative fat weight of epididymal white adipose tissue (eWAT) in each group of mice after the last administration. Figure 13 This is a graph showing the calculated relative fat weight of brown adipose tissue (BAT) in the scapular region of mice after the last administration of each group of mice. Figure 14 These are HE staining results of the liver, kidney, and fat of mice in each group after the last administration. Figure 15 This is a graph showing the calculated relative body weight of healthy adult ICR mice during a 40-day period of continuous administration of high-dose drugs. Detailed Implementation

[0014] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0015] I. Structure of the novel calreticulin ligand The novel calreticulin ligands provided by this invention are a class of phenyl or pyridine-substituted pyrazolopyridine derivatives, with the following structure:

[0016] Where R1 can be any of the following structures: ; R2 can be any of the following structures: .

[0017] II. Preparation method of novel calreticulin ligands

[0018] Example 1

[0019]

[0020] Step 1: Commercially available 5-bromo-2-chloronicotinonitrile (23 mmol, compound 1) was added to a reaction vessel containing 200 mL of anhydrous ethanol. The reaction system was heated to 70 °C. After the temperature stabilized, hydrazine hydrate (115 mmol) was added dropwise to the reaction vessel. After the addition was complete, the reaction was maintained at 70 °C for 2 hours. After the reaction was complete, heating was stopped, and the mixture was allowed to cool naturally. The mixture was then subjected to low-temperature recrystallization, vacuum filtration, and drying to obtain compound 2, a yellow crystal with a crude yield of 56%.

[0021] Step 2: Compound 2 (14 mmol), 4-(morpholinesulfonyl)phenylboronic acid (16.8 mmol, compound 3), potassium phosphate (28 mmol), and a mixed solvent (80 mL) were added to the reaction vessel. The mixed solvent consisted of 1,4-dioxane and water in a volume ratio of 2:1. The mixture was stirred to dissolve the compounds. Nitrogen gas was purged for 10 min, and then di-, tri-tert-butylphosphine palladium (0.7 mmol) was added. The reaction system was heated to 110 °C and reacted at this temperature for 72 h. After the reaction was completed, the mixture was filtered hot through diatomaceous earth. The filtrate was extracted with ethyl acetate and water. The organic phase was purified by rotary evaporation and then passed through a silica gel column. The mobile phase consisted of dichloromethane and methanol in a volume ratio of 50:1. The eluent was collected and dried to give compound 4 as a yellow solid with a crude yield of 60%.

[0022] Step 3: Compound 4 (0.5 mmol) was dissolved in N,N-dimethylformamide (3 mL), and chloroacetyl chloride (0.6 mmol) was added. The reaction was carried out at 25 °C for 3 h. After the reaction was completed, the mixture was quenched with water, extracted with ethyl acetate and water, and purified by rotary evaporation of the organic phase followed by silica gel column chromatography. The mobile phase consisted of a mixture of petroleum ether and ethyl acetate in a volume ratio of 2:1. The eluent was collected and dried to give compound a1 as a white solid with a yield of 67%.

[0023] Compound a1 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.53 (s, 1H), 11.20 (s, 1H), 8.91 (d, J =2.1Hz, 1H), 8.72 (d, J =2.2Hz, 1H), 8.00 (d,J =8.1Hz, 2H), 7.86 (d, J =8.2Hz, 2H), 4.42 (s, 2H), 3.65 (t, J =4.6Hz, 4H), 2.93 (t, J =4.8Hz, 4H). 13 C NMR (126MHz, DMSO) δ 164.95, 151.61, 148.88, 142.78, 139.60, 133.15, 131.09, 128.56 (2C), 127.83 (2C), 126.95, 107.60, 65.32 (2C), 45.93 (2C), 42.88; HR-MS (ESI): m / z [M+H] + The calculated value is 436.08408, and the measured value is 436.08367.

[0024] Example 2

[0025]

[0026] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0027] Step 2: This step is basically the same as step 2 in Example 1, except that 4-(morpholine sulfonyl)phenylboronic acid (compound 3) is replaced with an equimolar amount of 6-isopropoxypyridine-3-boronic acid pinacol ester (compound 5) to obtain compound 6, a yellow solid with a crude yield of 43%.

[0028] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 6 to obtain compound a2, a white solid with a yield of 70%.

[0029] Compound a2 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.42 (s, 1H), 11.08 (s, 1H), 8.80 (s, 1H), 8.57-8.41 (m, 2H), 7.99 (d, J =11.2Hz, 1H), 6.87 (d, J =8.6Hz, 1H), 5.30 (p, J =6.2Hz, 1H), 4.40 (s, 2H), 1.32 (d,J =6.2Hz, 6H). 13 C NMR (126MHz, DMSO) δ 164.89, 162.37, 151.25, 148.46, 144.86, 139.15, 137.95, 129.63, 126.85, 125.80, 111.42, 107.66, 67.71, 42.85, 21.91 (2C); HR-MS (ESI): m / z [M+H] + The calculated value is 346.10653, and the measured value is 346.10586.

[0030] Example 3

[0031]

[0032] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0033] Step 2: Compound 2 (14 mmol), 4-(trifluoromethoxy)phenylboronic acid (16.8 mmol, compound 7), cesium carbonate (28 mmol), and a mixed solvent (90 mL) prepared by mixing toluene and water in a volume ratio of 3:1 were added to the reaction vessel and stirred to dissolve. Nitrogen gas was purged for 10 min, and chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium (0.7 mmol) was added. The reaction system was heated to 100 °C and reacted at this temperature for 72 h. After the reaction was completed, the mixture was filtered hot through diatomaceous earth. The filtrate was extracted with ethyl acetate and water. The organic phase was purified by rotary evaporation and silica gel column chromatography. The mobile phase was prepared by mixing dichloromethane and methanol in a volume ratio of 45:1. The eluent was collected and dried to obtain compound 8, a yellow solid, with a crude yield of 45%.

[0034] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 8 to obtain compound a3, a white solid with a yield of 50%.

[0035] Compound a3 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.46 (s, 1H), 11.13 (s, 1H), 8.72 (d, J =114.3Hz, 2H), 7.66 (dd,J =165.0, 8.7Hz, 4H), 4.40 (s, 2H); 13 C NMR (126MHz, DMSO) δ 164.89, 151.40, 148.72, 147.87, 139.37, 137.34, 130.39, 128.88 (3C), 127.36, 121.69 (2C), 107.56, 42.84; HR-MS (ESI): m / z [M+H] + The calculated value is 371.05171, and the measured value is 371.05153.

[0036] Example 4

[0037]

[0038] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0039] Step 2: This step is basically the same as step 2 in Example 1, except that 2,3-dimethoxyphenylboronic acid (compound 9) is used to replace compound 3 in an equal molar amount. The mobile phase is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:1 to obtain compound 10, which is yellow and oily with a crude yield of 80%.

[0040] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 10 to obtain compound a4, a pale yellow solid with a yield of 60%.

[0041] Compound a4 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.39 (s, 1H), 11.09 (s, 1H), 8.60 (s, 1H), 8.45 (s, 1H), 7.21-7.09 (m, 2H), 7.01-6.95 (m, 1H), 4.38 (s, 2H), 3.87 (s, 3H), 3.57 (s, 3H); 13 C NMR (126MHz, DMSO) δ164.79, 152.87, 150.94, 150.15, 146.05, 139.14, 132.35, 132.17, 126.06, 124.43, 122.27, 112.71, 107.24, 60.21, 55.84, 42.81; HR-MS (ESI): m / z [M+H] + The calculated value is 347.09054, and the measured value is 347.09006.

[0042] Example 5

[0043]

[0044] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0045] Step 2: This step is basically the same as step 2 in Example 1, except that compound 3 is replaced with an equal molar amount of 3,4-dimethoxyphenylboronic acid (compound 11) to obtain compound 12, a yellow solid with a crude yield of 65%.

[0046] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 12 to obtain compound a5, a pale yellow solid with a yield of 55%.

[0047] Compound a5 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.36 (s, 1H), 11.05 (s, 1H), 8.82 (s, 1H), 8.50 (s, 1H), 7.27-7.17 (m, 2H), 7.08 (d, J =8.4Hz, 1H), 4.40 (s, 2H), 3.86 (s, 3H), 3.81 (s, 3H); 13 C NMR (126MHz, DMSO) δ 164.87, 151.16, 149.24, 148.84, 148.56, 139.06, 130.73, 129.24, 128.90, 119.29, 112.48, 110.89, 107.66, 55.70, 55.62, 42.83; HR-MS (ESI): m / z [M+H] +The calculated value is 347.09054, and the measured value is 347.09006.

[0048] Example 6

[0049]

[0050] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0051] Step 2: This step is basically the same as step 2 in Example 1, except that compound 3 is replaced with an equal molar amount of methyl 4-carboxylate phenylboronic acid (compound 13), and the organic phase is not purified by silica gel column, but is first dried with anhydrous sodium sulfate, then purified by slurrying with dichloromethane (10 mL), filtered and dried to obtain compound 14, a yellowish-brown solid with a crude yield of 40%.

[0052] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 14 to obtain compound a6, a pale yellow solid with a yield of 55%.

[0053] Compound a6 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.49 (s, 1H), 11.15 (s, 1H), 8.89 (d, J =2.1Hz, 1H), 8.69 (d, J =2.2Hz, 1H), 8.08 (d, J =8.6Hz, 2H), 7.87 (d, J =8.3Hz, 2H), 4.41 (s, 2H), 3.89 (s, 3H); 13 C NMR (126MHz, DMSO) δ 166.04, 164.91, 151.56, 148.80, 142.56, 139.54, 130.67, 130.01, 128.41, 127.39, 127.14, 107.59, 59.76, 52.21, 20.76, 14.09; HR-MS (ESI): m / z [M+H] + The calculated value is 345.07489, and the measured value is 345.07408.

[0054] Example 7

[0055]

[0056] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0057] Step 2: This step is basically the same as step 2 in Example 6, except that compound 13 is replaced with an equimolar amount of 4-methanesulfonylphenylboronic acid (compound 15) to obtain compound 16, a yellow solid with a crude yield of 57%.

[0058] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 16 to obtain compound a7, a pale yellow solid with a yield of 55%.

[0059] Compound a7 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 11.75 (s, 1H), 9.16 (d, J =2.3Hz, 1H), 8.87 (d, J =2.3Hz, 1H), 8.17-7.99 (m, 4H), 5.29 (s, 2H), 4.50 (s, 2H), 3.29 (s, 3H); 13 C NMR (126MHz, DMSO) δ 163.07, 150.98, 150.27, 144.12, 141.61, 140.36, 130.56, 128.16 (2C), 127.95 (2C), 112.14, 44.62, 43.56, 43.00; HR-MS (ESI): m / z [M+H] + The calculated value is 365.04697, and the measured value is 365.04576.

[0060] Example 8

[0061]

[0062] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0063] Step 2: This step is basically the same as step 2 in Example 6, except that compound 13 is replaced with an equimolar amount of 4-ethanesulfonylphenylboronic acid (compound 17) to obtain compound 18, a yellow solid with a crude yield of 59%.

[0064] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 18, and the mobile phase is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:2, to obtain compound a8, a white solid with a yield of 56%.

[0065] Compound a8 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.54 (s, 1H), 11.19 (s, 1H), 8.92 (s, 1H), 8.71 (s, 1H), 8.00 (s, 4H), 4.41 (s, 2H), 3.33 (d, J =7.3Hz, 2H), 1.14 (t, J =7.3Hz, 3H); 13 C NMR (126MHz, DMSO) δ 164.92, 151.61, 148.88, 143.13, 139.60, 137.18, 131.10, 128.69 (2C), 127.79 (2C), 126.96, 107.57, 49.28, 42.85, 7.20; HR-MS (ESI): m / z [M+H] + The calculated value is 379.06262, and the measured value is 379.06210.

[0066] Example 9

[0067]

[0068] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0069] Step 2: This step is basically the same as step 2 in Example 6, except that compound 13 is replaced with an equimolar amount of 4-(N,N-dimethylaminosulfonyl)phenylboronic acid (compound 19) to obtain compound 20, a yellow solid with a crude yield of 53%.

[0070] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 20, and the mobile phase is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:1.5, to obtain compound a9, a white solid with a yield of 58%.

[0071] Compound a9 1 H NMR,13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.53 (s, 1H), 11.19 (s, 1H), 8.91 (s, 1H), 8.70 (s, 1H), 7.93 (dd, J =56.2, 8.1Hz, 4H), 4.41 (s, 2H), 2.66 (s, 6H); 13 C NMR (126MHz, DMSO) δ 164.92, 151.59, 148.84, 142.41, 139.58, 133.49, 131.03, 128.41 (2C), 127.72 (2C), 127.01, 107.57, 42.86, 37.60 (2C); HR-MS (ESI): m / z [M+H] + The calculated value is 394.07352, and the measured value is 394.07260.

[0072] Example 10

[0073]

[0074] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0075] Step 2: This step is basically the same as step 2 in Example 1, except that compound 3 is replaced with an equimolar amount of 4-(N-methylaminosulfonyl)phenylboronic acid (compound 21) to obtain compound 22, a yellow solid with a crude yield of 50%.

[0076] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 22, and the mobile phase is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:2, to obtain compound a10, a white solid with a yield of 78%.

[0077] Compound a10 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.52 (s, 1H), 11.18 (s, 1H), 8.90 (s, 1H), 8.69 (s, 1H), 8.01-7.85 (m, 4H), 7.53 (q, J=5.0Hz, 1H), 4.41 (s, 2H), 2.46 (d, J =5.0Hz, 3H); 13 C NMR (126MHz, DMSO) δ 164.91, 151.56, 148.85, 141.82, 139.53, 138.04, 130.83, 127.64 (2C), 127.55 (2C), 127.18, 107.57, 42.85, 28.68; HR-MS (ESI): m / z [M+H] + The calculated value is 380.05787, and the measured value is 380.05695.

[0078] Example 11

[0079]

[0080] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0081] Step 2: Compound 2 (14 mmol), 4-fluorophenylboronic acid (16.8 mmol, compound 23), potassium phosphate (28 mmol), and a mixed solvent (80 mL) were added to the reaction vessel. The mixed solvent was composed of 1,4-dioxane and water in a volume ratio of 2:1. The mixture was stirred to dissolve. Nitrogen gas was purged for 10 min, and 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (0.7 mmol) was added. The reaction system was heated to 110 °C and reacted at this temperature for 72 h. After the reaction was completed, the mixture was filtered hot through diatomaceous earth. The filtrate was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate, then purified by slurrying with dichloromethane (10 mL). After filtration and drying, compound 24 was obtained as a yellowish-brown solid with a crude yield of 35%.

[0082] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 24, and the mobile phase is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:1.5 to obtain compound a11, a white solid with a yield of 52%.

[0083] Compound a11 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.43 (s, 1H), 11.13 (s, 1H), 8.80 (s, 1H), 8.55 (d, J=2.3Hz, 1H), 7.80-7.66 (m, 2H), 7.41-7.30 (m, 2H), 4.40 (s, 2H); 13 C NMR (126MHz, DMSO) δ 164.88, 162.87, 160.92, 151.29, 148.71, 139.26, 134.47, 134.44, 130.02, 129.06, 128.99, 127.82, 116.06, 115.89, 107.57, 42.85; HR-MS (ESI): m / z [M+H] + The calculated value is 305.05999, and the measured value is 305.05936.

[0084] Example 12

[0085]

[0086] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0087] Step 2: This step is basically the same as step 2 in Example 11, except that 4-(isopropylthio)phenylboronic acid (compound 25) is used to replace compound 23 in an equal molar amount to obtain compound 26, a yellow solid with a crude yield of 40%.

[0088] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 26, and the mobile phase is a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1 to obtain compound a12, a white solid with a yield of 80%.

[0089] Compound a12 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.43 (s, 1H), 11.13 (s, 1H), 8.83 (s, 1H), 8.58 (s, 1H), 7.58 (dd, J =87.7, 8.4Hz, 4H), 4.40 (s, 2H), 3.55 (p, J =6.6Hz, 1H), 1.27 (d, J =6.8Hz, 6H); 13 C NMR (126MHz, DMSO) δ164.87, 151.33, 148.62, 139.28, 135.96, 134.67, 131.15 (2C), 129.83, 128.02, 127.45 (2C), 107.62, 42.85, 36.91, 22.82 (2C); HR-MS (ESI): m / z [M+H] + The calculated value is 361.08844, and the measured value is 361.08741.

[0090] Example 13

[0091]

[0092] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0093] Step 2: This step is basically the same as step 2 in Example 11, except that compound 23 is replaced with an equimolar amount of 2-fluoropyridine-4-boronic acid (compound 27) to obtain compound 28, a white solid with a crude yield of 41%.

[0094] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 28 to obtain compound a13, a white solid with a yield of 65%.

[0095] Compound a13 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.61 (s, 1H), 11.21 (s, 1H), 8.99 (s, 1H), 8.80 (s, 1H), 8.34 (d, J =5.3Hz, 1H), 7.74 (d, J =5.3Hz, 1H), 7.60 (s, 1H), 4.42 (s, 2H); 13 C NMR (126MHz, DMSO) δ 164.98, 163.11, 151.97, 151.27, 148.77, 148.39, 148.26, 139.81, 131.40, 124.65, 119.76, 107.56, 106.84, 42.85; HR-MS (ESI): m / z [M+H] + The calculated value is 306.05524, and the measured value is 306.05480.

[0096] Example 14

[0097]

[0098] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0099] Step 2: This step is basically the same as step 2 in Example 11, except that compound 23 is replaced with an equimolar amount of 2,4-difluorophenylboronic acid (compound 29) to obtain compound 30, a pale yellow solid with a crude yield of 57%.

[0100] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 30, and the mobile phase is a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1, to obtain compound a14, a white solid with a yield of 67%.

[0101] Compound a14 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.49 (s, 1H), 11.14 (s, 1H), 8.65 (s, 1H), 8.52 (s, 1H), 7.75-7.60 (m, 1H), 7.50-7.36 (m, 1H), 7.32-7.18 (m, 1H), 4.39 (s, 2H); 13 C NMR (126MHz, DMSO) δ 164.88, 160.94, 160.27, 158.30, 158.20, 151.11, 149.69, 139.30, 132.54, 132.26, 132.21, 132.18, 122.60, 122.37, 112.40, 112.37, 112.23, 107.26, 104.84, 104.63, 104.42, 42.83; HR-MS (ESI): m / z [M+H] + The calculated value is 323.05057, and the measured value is 323.05001.

[0102] Example 15

[0103]

[0104] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0105] Step 2: This step is basically the same as step 2 in Example 1, except that compound 3 is replaced with an equimolar amount of 2-naphthoboric acid (compound 31) to obtain compound 32, a yellow solid with a crude yield of 63%.

[0106] Step 3: This step is basically the same as step 3 in Example 1, except that compound 4 is replaced with an equimolar amount of compound 32 to obtain compound a15, a white solid with a yield of 59%.

[0107] Compound a15 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.46 (s, 1H), 11.15 (s, 1H), 8.98 (d, J =2.3Hz, 1H), 8.72 (d, J =2.3Hz, 1H), 8.26 (s, 1H), 8.11-7.84 (m, 4H), 7.63-7.46 (m, 2H), 4.43 (s, 2H); 13 C NMR (126MHz, DMSO) δ 164.91, 151.38, 149.10, 139.33, 135.34, 133.37, 132.12, 130.26, 128.79, 128.62, 128.13, 127.58, 126.59, 126.24, 125.48, 125.30, 107.73, 42.88; HR-MS (ESI): m / z [M+H] + The calculated value is 337.08507, and the measured value is 337.08437.

[0108] Example 16

[0109]

[0110] Step 1: The procedure is exactly the same as Step 1 in Example 1, and will not be repeated here. Compound 2 is obtained.

[0111] Step 2: This step is basically the same as step 2 in Example 6, except that compound 13 is replaced with an equimolar amount of 4-isopropylsulfonylphenylboronic acid (compound 33) to obtain compound 34, a yellow solid with a crude yield of 63%.

[0112] Step 3: Compound 34 (0.5 mmol) was dissolved in N,N-dimethylformamide (3 mL), and dichloroacetyl chloride (0.6 mmol) was added. The reaction was carried out at 25 °C for 3 h. After the reaction was completed, the mixture was quenched with water, extracted with ethyl acetate and water, and purified by rotary evaporation of the organic phase followed by silica gel column chromatography. The mobile phase consisted of a mixture of petroleum ether and ethyl acetate in a volume ratio of 1.5:1. The eluent was collected and dried to give compound b1 as a white solid with a yield of 59%.

[0113] Compound b1 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.69 (s, 1H), 11.61 (s, 1H), 8.94 (s, 1H), 8.70 (s, 1H), 8.04-7.93 (m, 4H), 6.74 (s, 1H), 3.47 (h, J =6.7Hz, 1H), 1.20 (d, J =6.8Hz, 6H); 13 C NMR (126MHz, DMSO) δ 162.08, 151.60, 149.17, 143.10, 138.72, 135.61, 130.71, 129.53 (2C), 127.83 (2C), 127.31, 107.33, 66.65, 54.26, 15.2 (2C); HR-MS (ESI): m / z [M+H] + The calculated value is 427.03929, and the measured value is 427.03842.

[0114] Example 17

[0115]

[0116] Compound 34 was used as the starting material. Compound 34 (0.5 mmol) was dissolved in N,N-dimethylformamide (3 mL), and acetoxyacetyl chloride (0.6 mmol) was added. The reaction was carried out at 25 °C for 3 h. After the reaction was complete, the reaction solution was treated with the same post-treatment method as in step 3 of Example 16 to obtain compound b2, a white solid, in 63% yield.

[0117] Compound b2 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1H NMR (500MHz, DMSO) δ 13.50 (s, 1H), 11.02 (s, 1H), 8.91 (s, 1H), 8.67 (s, 1H), 8.03-7.87 (m, 4H), 4.79 (s, 2H), 3.46 (hept, J =6.4Hz, 1H), 2.13 (s, 3H), 1.19 (d, J =6.9Hz, 6H). 13 C NMR (126MHz, DMSO) δ 170.10, 165.89, 151.62, 148.85, 143.25, 139.60, 135.48, 131.15, 129. 49 (2C), 127.73 (2C), 126.86, 107.66, 62.12, 54.24, 20.51, 15.24 (2C); HR-MS (ESI): m / z [M+H] + The calculated value is 417.12272, and the measured value is 417.12167.

[0118] Example 18

[0119]

[0120] Using compound b2 as a starting material, compound b2 (0.5 mmol) was dissolved in methanol (4 mL) at 0 °C, and sodium hydroxide (1.5 mmol) was dissolved in ice water. The alkali solution was added dropwise to the methanol solution with stirring in an ice bath. After the addition was complete, the reaction system was heated to room temperature and reacted for 3 h. After the reaction was complete, the mixture was quenched with water, extracted with ethyl acetate and water, and the organic phase was purified by rotary evaporation followed by silica gel column chromatography. The mobile phase consisted of a mixture of dichloromethane and methanol in a volume ratio of 50:1. The eluent was collected and dried to give compound b3 as a white solid, with a yield of 64%.

[0121] Compound b3 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.47 (s, 1H), 10.36 (s, 1H), 8.92 (s, 1H), 8.71 (s, 1H), 8.03-7.94 (m, 4H), 5.64 (s, 1H), 4.13 (s, 1H), 3.47 (p, J =6.8Hz, 1H), 1.19 (d, J=6.8Hz, 6H); 13 C NMR (126MHz, CDCl3) δ 171.21, 151.67, 148.68, 143.24, 139.66, 135.46, 131.03, 129.47 (2C), 127.66 (2C), 126.76, 108.00, 61.44, 54.23, 15.24 (2C); HR-MS (ESI): m / z [M+H] + The calculated value is 375.11215, and the measured value is 375.11112.

[0122] Example 19

[0123]

[0124] The method was essentially the same as in Example 17, except that acetoxyacetyl chloride was replaced with an equimolar amount (0.6 mmol) of oxaloyl chloride monomethyl ester, and the mobile phase was a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:1, to obtain compound b4, a white solid with a yield of 73%.

[0125] Compound b4 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.68 (s, 1H), 11.53 (s, 1H), 8.95 (d, J =2.2Hz, 1H), 8.63 (d, J =2.3Hz, 1H), 8.05-7.94 (m, 4H), 3.89 (s, 3H), 3.46 (p, J =6.8Hz, 1H), 1.19 (d, J =6.8Hz, 6H); 13 C NMR (126MHz, DMSO) δ 160.65, 156.02, 151.58, 148.95, 143.07, 138.35, 135.58, 130.30, 129.48 (2C), 127.78 (2C), 127.22, 108.36, 54.28, 53.22, 15.26 (2C); HR-MS (ESI): m / z [M+H] +The calculated value is 403.10707, and the measured value is 403.10635.

[0126] Example 20

[0127]

[0128] The method was essentially the same as in Example 17, except that acetoxyacetyl chloride was replaced with an equimolar amount (0.6 mmol) of 2-chloropropionyl chloride, and the mobile phase was a mixture of petroleum ether and ethyl acetate in a volume ratio of 2:1, to obtain compound b5, a white solid with a yield of 45%.

[0129] Compound b5 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.55 (s, 1H), 11.23 (s, 1H), 8.91 (s, 1H), 8.73 (s, 1H), 8.03-7.94 (m, 4H), 4.85 (q, J =6.7Hz, 1H), 3.46 (p, J =6.8Hz, 1H), 1.68 (d, J =6.7Hz, 3H), 1.20 (d, J =6.8Hz, 6H); 13 C NMR (126MHz, DMSO) δ 167.59, 151.62, 148.94, 143.24, 139.63, 135.51, 131.19, 129.51 (2C), 127.77 (2C), 127.00, 107.48, 54.26, 53.89, 21.11, 15.25 (2C); HR-MS (ESI): m / z [M+H] + The calculated value is 407.09392, and the measured value is 407.09243.

[0130] Example 21

[0131]

[0132] The method was essentially the same as in Example 17, except that acetoxyacetyl chloride was replaced with an equimolar amount (0.6 mmol) of thiophene-3-yl-acetyl chloride, and the mobile phase was a mixture of petroleum ether and ethyl acetate in a volume ratio of 2:1, to obtain compound b6, a white solid with a yield of 45%.

[0133] Compound b6 1 H NMR, 13 The specific C NMR and HR-MS data are as follows: 1 H NMR (500MHz, DMSO) δ 13.45 (s, 1H), 11.08 (s, 1H), 8.89 (s, 1H), 8.71 (d, J =2.2Hz, 1H), 7.97 (d, J =2.3Hz, 4H), 7.40 (d, J =5.1Hz, 1H), 7.06-6.97 (m, 2H), 4.02 (s, 2H), 3.45 (h, J =6.7Hz, 1H), 1.19 (d, J =6.8Hz, 6H); 13 C NMR (126MHz, DMSO) δ 168.27, 151.64, 148.80, 143.29, 140.23, 136.81, 135.45, 131.44, 129.50 (3 C), 127.70 (2C), 126.76, 126.64, 125.21, 107.56, 54.25, 36.36, 15.25 (2C); HR-MS (ESI): m / z [M+H] + The calculated value is 441.10496, and the measured value is 441.10406.

[0134] III. Evaluation of the compound's ability to inhibit tumor cell proliferation Human gastric cancer cells MKN-45, human liver cancer cells HepG2, human non-small cell lung cancer cells A549, and human pancreatic cancer cells PANC-1 in logarithmic growth phase were seeded at a density of 5000 cells / well in 96-well plates and cultured at 37℃ in a 5% CO2 incubator for 24 h to allow for full cell adhesion. The cultured cells were then divided into two groups: an experimental group and a positive control group. The old culture medium was discarded, and 200 μL of different concentrations (0 μM-50 μM) of the test compound was added to the experimental group. 200 μL of different concentrations (0 μM-50 μM) of the positive control compound 2a (a reported CALR ligand, structure shown below) was added to the positive control group. Each group was divided into three replicates, and the cells were cultured for 48 h. After culture, 20 μL of 2.5 mg / mL MTT solution was added to each well and incubated for 4 h. The supernatant was then discarded, and 100 μL of DMSO was added to each well, and the cells were mixed by micro-oscillation. The absorbance of each well was measured at 490 nm using a microplate reader. The inhibitory rate of each compound on cell proliferation was calculated, and the half-maximal inhibitory concentration (IC50) was calculated using Non-Linear Curve Fit. 50 ).

[0135]

[0136] The half-maximal inhibitory concentrations (IC50) of each compound against human gastric cancer cells MKN-45, human liver cancer cells HepG2, human non-small cell lung cancer cells A549, and human pancreatic cancer cells PANC-1 were observed. 50 The calculation results are shown in Tables 1-1 and 1-2.

[0137] Table 1-1 Detection of antitumor activity of various compounds by MTT colorimetric method (Part 1)

[0138] Table 1-2 Detection of antitumor activity of various compounds by MTT colorimetric method (II)

[0139] As shown in Tables 1-1 and 1-2, most of the compounds provided by this invention exhibit good antitumor activity. Among them, the compounds most effective against human pancreatic cancer cells PANC-1 (for which there is currently no effective therapeutic molecule) are the most numerous, with compound a1 showing the best activity and its IC50 value being [missing information]. 50 The concentration is 1.2 ± 0.4 μM, which makes it a preferred compound for further study.

[0140] IV. Determination of the affinity of the compound for CALR A gene fragment (SEQ ID NO: 1) encoding the functional domain of human CALR (amino acid sequence S40-K272) was cloned into the pET-28a expression vector and subsequently transformed into *E. coli* BL21(DE3) competent cells. After IPTG induction, lysozyme digestion, and sonication, the recombinant protein carrying the His tag was purified using a Ni-NTA His-Tag agarose gel column and eluted with gradient concentrations of imidazole to obtain CALR with a purity of approximately 95%. The binding affinity of each compound to CALR was detected using a Biacore molecular interaction analyzer: first, the chip was activated using an EDC / NHS system with sodium acetate solution at pH 5.0 as the coupling medium; then, CALR (5.0 μg-10 μg) was injected into the flow path and covalently immobilized on the chip surface; finally, unreacted active sites were blocked using ethanolamine hydrochloride solution; simultaneously, an unimmobilized flow cell was set up as a reference channel to subtract background signals and buffer effects. The compounds were diluted to different concentrations (0 μM-50 μM) and sequentially flowed through a chip surface immobilized with CALR under constant temperature (25 °C) and flow rate conditions. Changes in binding and dissociation signals were monitored. After each cycle, the chip surface was washed with buffer to restore the baseline. The results were analyzed using a dual-reference channel data processing method. Binding and dissociation curves were fitted using a 1:1 Langmuir binding model, and the binding rate constant of each compound with CALR was calculated. K on ) and dissociation rate constant ( K off ), and finally according to the formula K D = K off / K on The equilibrium dissociation constant was calculated.

[0141] Equilibrium dissociation constants of each compound with CALR ( K D The calculation results are shown in Table 2.

[0142] Table 2. Binding affinity of compounds to CALR as determined by SPR technology.

[0143] As shown in Table 2, the compounds provided by this invention generally have good binding affinity for CALR. Among them, compound a1 has a binding affinity for CALR of 1.2 μM, which is better than the reported positive control compound 2a.

[0144] V. The effect of compounds on intracellular calcium 2+ Effect of content Human pancreatic cancer cells (PANC-1) in logarithmic growth phase were grafted onto black 96-well plates at a density of 5000 cells / well and cultured at 37°C in a 5% CO2 incubator for 24 h to achieve 60%-70% confluence. After cell attachment, each compound (3.0 μM) was added to the cells for 24 h, followed by detection using the Fluo-4 calcium ion detection kit. 100 μL of fluorescent probe was added to each well, and the cells were incubated at 37°C in the dark for 40 min. Changes in fluorescence intensity (excitation wavelength 494 nm, emission wavelength 516 nm) were detected using a multi-mode microplate reader. The experiment was repeated three times to comprehensively evaluate the effects of the compounds on intracellular calcium. 2+ The effect of content.

[0145] After incubation of each compound with human pancreatic cancer cells PANC-1 for 24 hours, intracellular Ca2+ was reduced. 2+ The calculation results of the content are shown in Figure 1 .Depend on Figure 1 It can be seen that: after incubation with human pancreatic cancer cells PANC-1 for 24 hours, most of the compounds provided by this invention can significantly increase intracellular calcium levels. 2+ The levels of these compounds may be related to their targeted binding to CALR. Specifically, compound a1 targets intracellular Ca2+. 2+ The upregulation effect was most pronounced after treatment with compound a1, resulting in increased intracellular Ca2+ levels. 2+ The content increased to 371.3±3.1%.

[0146] VI. Preferred compound a1 for its effect on intracellular Ca 2+ The influence of distribution Human pancreatic cancer cells (PANC-1) in logarithmic growth phase were seeded at an appropriate density in confocal culture dishes to ensure a uniform monolayer distribution. After cell attachment, the preferred compound a1 (3.0 μM) was added for 24 h, with the solvent prepared simultaneously. The original culture medium was removed, and a mixture of Fluo-4 calcium ion fluorescent probe and Hoechst 33342 working solution was added to the confocal culture dish. The dish was incubated at 37°C in a 5% CO2 incubator for 40 min in the dark. The culture dish was then placed on the stage of a confocal microscope for imaging to evaluate the effect of the preferred compound a1 on intracellular calcium levels before and after administration. 2+ The influence of distribution and content.

[0147] The results of the confocal microscope images are shown below. Figure 2 .Depend on Figure 2 It can be seen that the preferred compound a1 (3.0 μM) can significantly increase the Ca2+ level in human pancreatic cancer cells PANC-1. 2+ Content, and Ca 2+ Widely distributed in the cytoplasm and nucleus, suggesting that the preferred compound a1 may induce calcium overload in tumor cells.

[0148] VII. Lip-MS Detection of the Binding Mode of Optimal Compound a1 with CALR Human pancreatic cancer cells PANC-1 were inoculated into cell culture dishes until the cell density reached 60%-70%. The experimental group was treated with the preferred compound a1 (1.0 μM) for 24 h during the logarithmic growth phase, while the control group was treated with an equal volume of solvent for 24 h during the logarithmic growth phase. Total protein from the PANC-1 cells was then extracted, and a protease inhibitor was added to maintain the samples in a non-denatured state. Proteinase K was added to the samples for 10 min of enzymatic digestion. Proteins bound to the preferred compound a1 were locally protected, and their binding peptides were not easily cleaved. Excess trypsin was added for complete cleavage (4℃, 16 h). The samples were then subjected to denaturation, reduction, and alkylation treatments, followed by desalting and purification using a C18 column to obtain a peptide mixture. The digested peptide samples were analyzed by liquid chromatography-mass spectrometry (LC-MS). The mass spectra of the experimental and control groups were compared to identify peptides whose abundance changed significantly in the presence of the preferred compound a1. These differentially expressed peptides indicated potential binding sites of the preferred compound a1 on the CALR.

[0149] Lip-MS assays showed that the CALR polypeptide sequence (SEQ ID NO: 2) could be identified in cell lysates from both the control and experimental groups. The abundance of the SQDARFYALSASFEPF peptide at positions 69-84 was 18.4% in the experimental group and 100% in the control group. The significantly reduced abundance of this peptide in the experimental group suggests that the preferred compound a1 may bind to the N-terminal peptide at positions 69-84 of CALR.

[0150] VIII. Effects of preferred compound a1 on cell cycle and apoptosis Logarithmic growth phase human pancreatic cancer cells PANC-1 were seeded in 6-well plates (20,000 cells / well) and cultured at 37°C and 5% CO2 for 24 h to allow cell adhesion. An experimental group (preferably compound a1, 1.0 μM) and a solvent control group were set up, with 3 replicates per group. Treatment with the drug lasted 24 h. Cell cycle detection: Cells were collected by trypsin digestion, washed with PBS, resuspended, and fixed with pre-chilled 70% ethanol (-20°C) overnight at 4°C. In the formal experiment, ethanol was removed by washing with PBS, followed by incubation with propidium iodide (PI, 50 μg / mL) containing RNase A (100 μg / mL) at room temperature in the dark for 30 min. Apoptosis detection: Cells were resuspended in Binding Buffer and stained using an Annexin V-FITC / PI double staining kit at room temperature in the dark for 15 min. Flow cytometry was used for detection, with 10,000 cell signals collected for each sample. The experiment was independently repeated three times. Fluorescence signals were analyzed using FlowJo software. The changes in cell cycle distribution ratio and apoptosis rate were compared between the experimental group and the solvent control group to evaluate the effect of the preferred compound a1.

[0151] The calculation results of the cell cycle distribution ratio between the experimental group and the solvent control group are shown in the figure. Figure 3 The results of the apoptosis rate calculation are shown in [link to calculation]. Figure 4 .Depend on Figure 3 and Figure 4 It can be seen that the preferred compound a1 can significantly induce G2 / M phase arrest and apoptosis in human pancreatic cancer cells PANC-1. After treatment with 1.0 μM for 24 h, the percentage of cells in cell cycle arrest increased from 29.5±3.8% to 59.5±4.1%, while the percentage of apoptotic cells increased from 4.4±1.8% to 31.3±3.6%.

[0152] IX. Oil Red O staining to detect the effects of compounds on lipid formation Logarithmic growth phase mouse embryonic fibroblasts (3T3-L1) were grafted into 6-well plates at a density of 20,000 cells / well and cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2 until complete confluence. The medium was then replaced with adipogenic differentiation-inducing medium (0.5 mmol / L IBMX, 1 μmol / L dexamethasone, 10 μg / mL insulin) for 48 h of further induction, followed by maintenance culture with 10 μg / mL insulin. In the experimental group, the test compound (10 μM) was added during differentiation induction, administered every three days, with berberine and resveratrol used as positive controls. On day 8 of induction, the medium was aspirated, and the cells were washed twice with PBS, fixed with 4% paraformaldehyde solution at room temperature for 30 min, stained with Oil Red O, and protected from light at room temperature for 20 min, followed by washing with PBS until the background was colorless. The staining results were observed using an inverted optical microscope. Lipid droplets were stained red or orange-red by Oil Red O. Images were acquired by randomly selecting fields of view, and the number, size, and distribution of lipid droplets were recorded. After Oil Red O staining and thorough washing, 200 μL of isopropanol was added to each well for extraction. The mixture was shaken at room temperature for 15 min until the dye was completely dissolved. The OD value was detected at 520 nm using a microplate reader to obtain the TG quantification value for each group. The lipid-lowering rate was calculated using the following formula:

[0153] The results of Oil Red O staining for each group are shown below. Figure 5 .Depend on Figure 5 It can be seen that the cells treated with compounds a1, a11, and a14 died significantly, and the decrease in lipid content was due to the toxicity of the compounds; the cells treated with compound a13 were in good condition, and the neutral lipid content was significantly reduced, suggesting that compound a13 has good lipid-lowering activity, which is better than that of the positive control compound.

[0154] The calculated lipid-lowering rates for each group are shown in Table 3.

[0155] Table 3. Calculation results of the lipid-lowering rate of the compounds.

[0156] As shown in Table 3, the lipid-lowering rate of compound a13 (10 μM) was 99.4%, which was significantly higher than that of berberine and resveratrol.

[0157] After expanding the concentration gradient, the calculated TG inhibition rates of compounds a13, berberine, and resveratrol are shown in the table below. Figure 6 .Depend on Figure 6 It can be seen that compound a13 inhibits the EC of adipogenesis. 50 The concentration was 1.3 ± 0.2 μM, which was also significantly better than the positive control compound.

[0158] 10. Immunofluorescence detection of the effect of the preferred compound a13 on mitochondria Log-phase 3T3-L1 cells were seeded in confocal culture dishes. After adhesion, 3.0 μM of the preferred compound a13 was added for 24 h. After incubation, the culture medium was removed, and mitochondrial-specific fluorescent probes (JC-1 probe for membrane potential detection and MitoSO Red probe for reactive oxygen species detection) were added. The cells were incubated in the dark for 20 min. After staining, the cells were washed with PBS to remove unbound dye, and images were acquired using a confocal microscope.

[0159] Immunofluorescence staining results are shown in Figure 7 .Depend on Figure 7 It can be seen that the preferred compound a13 can moderately increase intracellular calcium in 3T3-L1 cells. 2+ The content (fluorescence value 156±4.3%) and the significantly increased mitochondrial membrane potential (fluorescence value 213±14%) indicate that the preferred compound a13 can promote Ca 2+ Releases and enhances mitochondrial activity.

[0160] XI. In vivo anti-obesity activity of preferred compound a13 Five-week-old C57BL / 6J mice were selected and fed a high-fat diet continuously for 8-12 weeks until their body weight was significantly higher than that of age-matched mice on a normal diet, accompanied by significant abdominal fat accumulation. The successfully modeled obese mice were randomly divided into a model group (HFD group), an orlistat group (HFD-orlistat group), and a preferred compound a13 group (HFD-a13 group). Age-matched mice on a normal diet served as a negative control group (ND). Mice in the orlistat group and the preferred compound a13 group were administered orlistat or preferred compound a13 by gavage. The dosage in the orlistat group was 20 mg / kg, and the dosage in the preferred compound a13 group was 10 mg / kg or 20 mg / kg, every two days for 4 weeks. During this period, all groups of mice maintained their original diet (the negative control group was given a normal diet, and the other groups were given a high-fat diet), had free access to water, and their body weight and food intake were recorded regularly. Following the last administration, blood was collected from the ocular rims of mice and deposited in anticoagulant tubes containing sodium heparin. The blood was stored at 4°C and centrifuged to collect the supernatant. Subsequently, an automated biochemical analyzer was used to detect serum levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (UREA), and creatinine (CREA) to evaluate the effects of the preferred compound a13 on obesity-related indicators. Mice in each group were dissected, and major adipose tissues, including inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and scapular brown adipose tissue (BAT), as well as the liver, were accurately weighed to assess the lipid-lowering effect of the preferred compound a13.

[0161] Using the initial average body weight of mice in the negative control group as a baseline (100%), the relative body weight (%) of mice in each group during the drug administration period is calculated as follows: Figure 8 .Depend on Figure 8 It can be seen that after successfully modeling obese mice, gavage administration of the preferred compound a13 (10 mg / kg or 20 mg / kg) or orlistat (20 mg / kg) for 4 weeks reduced their body weight to be comparable to that of the ND group, indicating that compound a13 has anti-obesity activity comparable to orlistat.

[0162] Using the food intake of the negative control group mice after the last administration as a baseline (100%), the relative food intake (%) of the other groups after the last administration is calculated as follows: Figure 9 .Depend on Figure 9 It can be seen that there was no significant difference in food intake among the groups of mice.

[0163] Using the liver or adipose tissue (iWAT, eWAT, BAT) weight of the negative control group mice as a baseline (100%), the calculation results of the relative liver weight (%) and relative fat weight (%) of the other groups of mice are shown below. Figures 10 to 13 .Depend on Figures 10 to 13 It can be seen that there was no significant difference in liver weight among the groups of mice, but the weight of adipose tissue in the group with the preferred compound a13 was significantly reduced, with the reduction being comparable to that in the orlistat group.

[0164] The biochemical indicators of mice in each group are shown in Table 4.

[0165] Table 4 Effects of preferred compound a13 on biochemical indicators in mice

[0166] As shown in Table 4, compared with the HFD group, the preferred compound a13 reduced TG by about 35.3%, TC by about 53%, increased HDL-C by about 169%, and reduced LDL-C by about 39% in obese mice. After administration, there were no significant changes in the liver function indicators ALT and AST levels, and the kidney function indicators CREA and UREA levels, indicating that compound a13 can effectively reduce blood lipids and increase high-density lipoprotein content, and has no significant effect on liver and kidney function.

[0167] XII. Safety evaluation of the preferred compound a13 Representative mice from the ND group, HFD group, and HFD-a13 (20 mg / kg) group were selected, and their liver, kidney, and fat tissues were stained with hematoxylin and eosin (HE) to observe changes in tissue structure in order to assess the safety of the preferred compound a13.

[0168] HE staining results of liver, kidney, and fat in mice of each group are shown in the figure. Figure 14 .Depend on Figure 14 It can be seen that after continuous administration, there were no significant changes in cell morphology of fat metabolism-related organs and tissues in mice, but the fat volume was significantly reduced, indicating that compound a13 has good safety.

[0169] Healthy adult ICR mice (weighing 18-22g, half male and half female) were selected and randomly divided into a solvent control group (ND group) and a preferred compound a13 administration group (ND-a13 group). The preferred compound a13 administration group was administered the preferred compound a13 by gavage at a dose of 40mg / kg once a day, while the solvent control group was administered an equal amount of water by gavage once a day for 40 consecutive days. During this period, all animals had free access to food and water, and their body weight, food intake, and behavioral performance were recorded regularly.

[0170] Using the initial average body weight of mice in the solvent control group as a baseline (100%), the relative body weight (%) of mice in each group during the drug administration period is calculated as follows: Figure 15.Depend on Figure 15 It can be seen that the preferred compound a13 can reduce the weight of mice by about 10%, and there are no significant changes in the mice's behavior, food intake, and mental state, indicating that the preferred compound a13 will not cause significant toxic reactions after continuous administration at high doses.

[0171] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description.

Claims

1. A pyrazolopyridine calreticulin ligand, characterized in that, The structure of the pyrazolopyridine calreticulin ligand is shown below: ； Where R1 can be any of the following structures: ； R2 can be any of the following structures: 。 2. The method for preparing the pyrazolopyridine calreticulin ligand according to claim 1, characterized in that, Includes the following steps: (1) 5-bromo-2-chloronicotinonitrile was added to anhydrous ethanol, the reaction system was heated to 70°C, and then hydrazine hydrate was added dropwise while maintaining the reaction at 70°C. After the reaction was completed, the product was separated and purified to obtain compound 2. (2) Add compound 2, phenyl or pyridylboronic acid compound and inorganic base to a mixed solvent of organic solvent and water, stir to dissolve, purge with nitrogen for 10 min, then add palladium catalyst, heat the reaction system to 100℃-110℃ and react at this temperature, after the reaction is completed, separate and purify the product to obtain compound A; (3) Dissolve compound A in N,N-dimethylformamide, add acyl chloride, and react at 25°C. After the reaction is complete, separate and purify the product to obtain phenyl or pyridine-substituted pyrazolopyridine derivatives. The phenylboronic acid compounds are selected from 4-(morpholinesulfonyl)phenylboronic acid, 4-(trifluoromethoxy)phenylboronic acid, 2,3-dimethoxyphenylboronic acid, 3,4-dimethoxyphenylboronic acid, methyl carbamate phenylboronic acid, 4-methanesulfonylphenylboronic acid, 4-ethanesulfonylphenylboronic acid, 4-(N,N-dimethylaminosulfonyl)phenylboronic acid, 4-(N-methylaminosulfonyl)phenylboronic acid, 4-fluorophenylboronic acid, 4-(isopropylthio)phenylboronic acid, 2,4-difluorophenylboronic acid, 4-isopropylsulfonylphenylboronic acid, or 2-naphthaleneboronic acid; The pyridylboronic acid compounds are selected from 6-isopropoxypyridine-3-boronic acid pinacol ester or 2-fluoropyridine-4-boronic acid.

3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of 5-bromo-2-chloronicotinonitrile and hydrazine hydrate is 1:

5.

4. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of compound 2, phenyl or pyridylboronic acid compound, inorganic base and palladium catalyst is 14:16.8:28:0.

7.

5. The preparation method according to claim 2, characterized in that, In step (2), the palladium catalyst is selected from 2,3-tert-butylphosphine palladium, chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium or 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride.

6. The preparation method according to claim 2, characterized in that, In step (2), the inorganic base is selected from potassium phosphate or cesium carbonate.

7. The preparation method according to claim 2, characterized in that, In step (3), the molar ratio of compound A to acyl chloride is 5:

6.

8. The preparation method according to claim 2, characterized in that, In step (3), the acyl chloride is selected from chloroacetyl chloride, dichloroacetyl chloride, acetoxyacetyl chloride, oxalyl chloride monomethyl ester, 2-chloropropionyl chloride or thiophene-3-yl-acetyl chloride.

9. The use of the pyrazolopyridine calreticulin ligand according to claim 1 in the preparation of antitumor drugs, characterized in that, The application is any one of the following: (1) , , , , , , or Application in the preparation of anti-gastric cancer drugs; (2) , , , , or Application in the preparation of anti-liver cancer drugs; (3) , , or Application in the preparation of drugs for treating non-small cell lung cancer; (4) , , , , , , , , or Application in the preparation of anti-pancreatic cancer drugs.

10. The use of the pyrazolopyridine calreticulin ligand according to claim 1 in the preparation of lipid-lowering drugs, characterized in that, The pyrazolopyridine calreticulin ligand is any one of the following compounds: 、 、 、 、 。

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