Application of dihydroisoflavone derivative

Abstract

The invention discloses an application of a dihydroisoflavone derivative. Specifically, the invention provides application of a compound shown as a formula I or pharmaceutically acceptable salt thereof in preparation of an STAT3 inhibitor. The compound and STAT3 protein have high affinity, and the compound shows good activity on human cancer cells, especially on one or more of prostate cancer, head and neck cancer and human tongue squamous cell carcinoma at the cellular level, has the potential of being prepared into novel antitumor drugs and has good market prospects. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine. Specifically, the present invention relates to the use of a class of dihydroisoflavone derivatives, and more specifically, to the use of a class of dihydroisoflavone derivatives in the preparation of STAT3 inhibitor drugs. Background Art

[0002] STAT3 is an important member of the STAT transcription factor family, participating in the regulation of multiple important signaling pathways and mediating the transcription of downstream genes. Abnormal activation of STAT3 expression is closely related to different biological behaviors such as human tumor cell proliferation, anti-apoptosis, invasion, angiogenesis, and immune escape. The research on using STAT3 inhibitors for treating tumors has great development potential. Many studies have shown that STAT3 is abnormally activated in tumors such as melanoma, colorectal cancer, breast cancer, prostate cancer, and head and neck squamous cell carcinoma, promoting the proliferation, differentiation, migration, and survival of tumor cells. Although a large number of STAT3 inhibitors have been reported, they are still in the clinical stage and there are no marketed drugs yet. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the defect in the prior art of lacking STAT3 inhibitors, thereby providing the use of dihydroisoflavone derivatives.

[0004] The present invention provides the application of a compound represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of a STAT3 inhibitor,

[0005]

[0006] R is hydrogen or hydroxy;

[0007] R 1 is hydroxy or protected hydroxy;

[0008] R 2 is hydrogen or

[0009] In one embodiment, the protecting group of the protected hydroxy is an ester protecting group, a silyl ether protecting group, an alkyl ether protecting group, or an alkoxyalkyl ether protecting group; for example, a benzyl protecting group.

[0010] In one embodiment, the compound represented by Formula I is any of the following structures:

[0011]

[0012] The present invention also provides the application of a compound represented by Formula I or a pharmaceutically acceptable salt thereof as described above in the preparation of a STAT3 inhibitor.

[0013] In the applications described above, the STAT3 inhibitor can be used in mammalian organisms; it can also be used in vitro, mainly for experimental purposes, such as providing comparison as a standard sample or control sample, or being made into a kit according to conventional methods in the art to provide a rapid detection of the inhibitory effect of STAT3.

[0014] The present invention also provides an application of a compound of formula I or a pharmaceutically acceptable salt thereof as described above in the preparation of a drug, wherein the drug is for preventing and / or treating cell proliferative diseases; and / or, the drug is for preventing and / or treating diseases or disorders related to STAT3.

[0015] The diseases or disorders related to STAT3 may be cell proliferative diseases. The drug can prevent and / or treat cell proliferative diseases by regulating STAT3 expression and / or activity.

[0016] The cell proliferative diseases as described above include diseases such as head and neck cancer, multiple myeloma, human tongue squamous cell carcinoma, esophageal cancer, prostate cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma or leukemia.

[0017] The drug for preventing and / or treating diseases or disorders related to STAT3 and the drug for preventing and / or treating cell proliferative diseases are preferably drugs having inhibitory proliferative activity against one or more of prostate cancer (DU145 cells), head and neck cancer (CAL33 cells) and human tongue squamous cell carcinoma (HN6 cells).

[0018] The present invention also provides an application of a compound of formula I or a pharmaceutically acceptable salt thereof as described above in the preparation of a drug, wherein the drug is for preventing and / or treating one or more of prostate cancer (DU145 cells), head and neck cancer (CAL33 cells) and human tongue squamous cell carcinoma (HN6 cells).

[0019] When used as a drug, the compound of formula I or a pharmaceutically acceptable salt thereof as described above can be administered in the form of a pharmaceutical composition.

[0020] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered indeterminate or unclear without a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding product or its active ingredient.

[0021] As used herein, the term "treatment" refers to therapeutic or palliative measures. Beneficial or desired clinical outcomes include, but are not limited to: alleviation of symptoms associated with a disease or disorder or condition, in whole or in part, reduction of the extent of the disease, stabilization (i.e., not worsening) of the disease state, delay or slowing of disease progression, remission or alleviation of the disease state (e.g., one or more symptoms of the disease), and detectable or undetectable remission (whether partial or complete). "Treatment" can also refer to an extension of survival as compared to expected survival without receiving treatment.

[0022] The term "pharmaceutical excipient" or "excipient" refers to a pharmaceutically acceptable chemical substance, such as a reagent known to those of ordinary skill in the pharmaceutical art for use in assisting the administration of a medicament. It is a compound that can be used in the preparation of a pharmaceutical composition and is generally safe, non-toxic, and biologically or otherwise undesirable. It includes excipients acceptable for veterinary and human drugs. Common excipients include binders, surfactants, diluents, disintegrants, and lubricants.

[0023] As used herein, the term "pharmaceutically acceptable" pertains to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0024] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared with relatively non-toxic, pharmaceutically acceptable acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the parent compounds with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent.

[0025] Without departing from the common general knowledge in the art, the above preferred conditions can be arbitrarily combined to obtain various preferred examples of the present invention.

[0026] The reagents and raw materials used in the present invention are all commercially available.

[0027] The positive and progressive effects of the present invention are as follows: The compounds of the present invention have a high affinity for STAT3 protein. At the cellular level, the compounds of the present invention show good activity against human cancer cells, especially one or more of prostate cancer, head and neck cancer, and human tongue squamous cell carcinoma, and have the potential to be developed into novel anti-tumor drugs, with good market prospects. Detailed Description of the Invention

[0028] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0029] Example 1 Synthesis of Compound 3

[0030]

[0031] Weigh compound 12 (M = 790.65, 157 mg, 0.2 mmol), compound 15 (M = 282.23, 112 mg, 0.4 mmol), PPh 3 (triphenylphosphine, M = 262.29, 2.1 mg, 0.008 mmol), Pd(OAc) 2 (palladium acetate, M = 224.51, 1.8 mg, 0.008 mmol) and t-BuOLi (lithium tert-butoxide, M = 80.05, 32 mg, 0.4 mmol) and add them into the microwave tube in turn. Then dissolve with anhydrous DMF (4 ml), remove the air in the microwave tube with nitrogen, quickly seal the microwave tube under nitrogen atmosphere, place it at 90 °C for reaction for 4 h, cool to room temperature, monitor by TLC (PE:EA = 2:1) spotting plate. After the reaction is completed, add ethyl acetate and water for extraction, combine the organic layers, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and separate and purify by column chromatography (PE:EA = 4:1). After drying, it is a pale yellow solid product 3 (M = 819.01, 112.0 mg, 68.5%).

[0032] 1 H NMR (400 MHz, CDCl 3) δ 7.42 (d, J = 7.3 Hz, 2H), 7.34–7.25 (m, 14H), 7.22–7.20 (m, 2H), 7.18 (d, J = 6.3 Hz, 2H), 7.02 (d, J = 8.7 Hz, 1H), 6.54 (d, J = 8.7 Hz, 1H), 6.06 (d, J = 2.1 Hz, 1H), 5.97 (d, J = 2.1 Hz, 1H), 5.17 (s, 1H), 5.05 (s, 1H), 5.02 (d, J = 2.0 Hz, 1H), 4.97 (d, J = 9.3 Hz, 3H), 4.90 (d, J = 9.1 Hz, 3H), 4.78 (d, J = 11.3 Hz, 1H), 4.40 (s, 1H), 4.13 (d, J = 11.3 Hz, 1H), 3.39 (dd, J = 15.0, 6.2 Hz, 1H), 3.29 (dd, J = 15.1, 5.7 Hz, 1H), 1.85 (t, J = 6.4 Hz, 2H), 1.41 (s, 3H), 1.28 (d, J = 4.5 Hz, 2H), 1.18 (s, 2H), 1.05 (s, 6H). 13 C NMR (101 MHz, CDCl 3 ) δ 192.07, 165.19, 164.53, 160.81, 158.48, 157.16, 137.87, 137.17, 136.34, 135.73, 135.39, 128.75, 128.56, 128.51, 128.44, 128.21, 127.79, 127.71, 127.67, 127.48, 127.41, 127.05, 126.56, 125.19, 125.13, 123.31, 107.27, 104.92, 94.93, 94.50, 76.46, 73.51, 73.36, 70.94, 70.37, 70.20, 69.92, 43.53, 39.91, 29.74, 29.18, 23.70, 22.61, 16.14. HR-ESI-MS: C 53 H 54 O 8 Na[M + Na] + Calculated: 841.3711, Found: 841.3709.

[0033] Example 2 Binding Activity Test of the Target Compound SATT3

[0034] Using microscale thermophoresis, the STAT3 pure protein (Shanghai Jinan Biotechnology Co., Ltd., protein number P40763) was centrifuged at 10,000 rpm for 2 - 3 min, and ultrapure water (ddH 2O) Dilute to a working solution of 5 μM and filter in column A at 1500 g, 4 °C for 2 min.

[0035] Perform protein labeling using a fluorescent dye kit (die yielding approx) and incubate in the dark at room temperature for 30 min. Wash column B with MST buffer, add the labeled protein-dye mixture to the washed column B, and collect the liquid in a 1.5 mL centrifuge tube.

[0036] After the liquid collection is completed, detect the protein fluorescence intensity in a Monolith NT.115 instrument and combine the protein liquids with stronger fluorescence intensity.

[0037] During the formal experiment, serially dilute the drug concentration to be detected into 16 concentrations (ensuring that the total system in the centrifuge tube is 10 μL)

[0038] Add 5 μL of protein to each tube, mix well, insert the capillary for siphoning, and then perform the detection on the machine to fit the Kd value.

[0039] First, we verified the protein binding of the compounds. Investigated whether compounds 1, 2, and 4 could bind to the STAT3 protein. The compound structures are as follows:

[0040]

[0041] We used MST technology to directly detect the direct binding of these three compounds to the STAT3 protein. The experimental results showed that the affinities of compounds 1, 2, and 4 for the STAT3 protein were 71.4 nM, 114 nM, and 17.9 nM, respectively. This indicates that all three compounds can directly bind to the STAT3 protein with strong affinities, further demonstrating that this parent nuclear skeleton has good affinity for the STAT3 protein.

[0042] Example 3 Biological Activity Test of the Target Compound

[0043] 3.1 Cell Culture

[0044] Take out DU145 cells, CAL33 cells or HN6 cells from the liquid nitrogen tank, quickly thaw them in a water bath at 37°C, and then transfer them to a 15 mL centrifuge tube pre-added with 2 times the volume of the culture medium. Centrifuge at 800 rpm for 3 min to obtain cell clumps, resuspend them and transfer them to a culture flask. When the prostate cancer DU145 cells, head and neck squamous cell carcinoma CAL33 cells or HN6 cells adhere and grow to a density of 90%-95% in the culture flask, perform cell passage: First, wash with pre-warmed PBS solution, then add 1-2 mL of trypsin and digest for 4-5 min. After terminating the digestion, centrifuge at 800 rpm for 3 min to obtain a cell suspension. After mixing evenly, passage at a ratio of 1:2. Finally, add 1 mL of cryopreservation solution (no temperature reduction) to the obtained cell clumps above, immediately transfer them to a -80°C refrigerator, and transfer them to the liquid nitrogen tank for long-term preservation the next day.

[0045] 3.2 Antitumor activity detection

[0046] (1) Drug treatment

[0047] Precisely weigh the target compounds 1-4 and the positive drug BP-1-102 using an analytical balance, dissolve them in DMSO to prepare a stock solution of 2 mM, aliquot and store in a -80°C refrigerator.

[0048] (2) Cell seeding

[0049] Prostate cancer DU145 cells, head and neck cancer CAL33 cell lines or human tongue squamous cell carcinoma HN6 cell lines are respectively inoculated on 96-well plates at a density of 5×104 cells / mL, 100 μL per well, with 3 replicates per group. The outermost wells are filled with culture medium to eliminate edge effects.

[0050] (3) Drug administration

[0051] The next day, when the cell confluence reaches 50%, add a low-serum (2.5% FBS) culture medium containing different concentrations of the target compounds 1-4.

[0052] (4) Detection

[0053] After drug administration, discard the original culture medium, and replace it with freshly prepared CCK-8 working solution (the volume ratio of CCK-8 reagent to basal medium is 1:9). After 1 h of incubation, measure the absorbance (OD) at a wavelength of 450 nm in an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the antitumor activity of IVB9 according to the following formula, and calculate the half-maximal inhibitory concentration (IC 50 ): Cell viability (%) = (OD value detected in the drug administration group - OD value detected in the blank group) / (OD value detected in the control group - OD value detected in the blank group) × 100%. The results are shown in Table 1.

[0054] Table 1 IC of target compounds 1-4 inhibiting the proliferation of DU-145, CAL33 and HN6 cells50 Value

[0055]

[0056]

[0057] The results of the in vitro cell proliferation inhibition test showed that the target compounds of the present invention exhibited good inhibitory activities against prostate cancer DU145 cells, head and neck cancer CAL33 cells, and human tongue squamous cell carcinoma HN6 cells with continuously activated STAT3 activity.

Claims

1. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a STAT3 inhibitor, R is hydrogen or hydroxyl; R 1 is a hydroxyl group or a protected hydroxyl group; R 2 is hydrogen or 2. The use according to claim 1, wherein, the protecting group of the protected hydroxyl group is an ester protecting group, a silyl ether protecting group, an alkyl ether protecting group or an alkoxyalkyl ether protecting group.

3. The use according to claim 1, wherein, the protecting group of the protected hydroxyl group is a benzyl protecting group.

4. The use according to claim 1, wherein, the compound of formula I is any of the following structures:

5. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug, the drug being for preventing and / or treating cell proliferative diseases; and / or, the drug being for preventing and / or treating diseases or disorders related to STAT3; R is hydrogen or hydroxyl; R 1 is a hydroxyl group or a protected hydroxyl group; R 2 is hydrogen or 6. The use according to claim 5, wherein, the diseases or disorders related to STAT3 are cell proliferative diseases; and / or, the drug for preventing and / or treating diseases or disorders related to STAT3 and the drug for preventing and / or treating cell proliferative diseases are drugs having inhibitory proliferative activity against one or more of prostate cancer, head and neck cancer, and human tongue squamous cell carcinoma; and / or, the compound of formula I is as described in any one of claims 2-4.

7. The use according to claim 6, wherein, the cells of the prostate cancer are DU145 cells; and / or, the cells of the head and neck cancer are CAL33 cells; and / or, the cells of the human tongue squamous cell carcinoma are HN6 cells.

8. The use according to claim 6, wherein, the cell proliferative diseases are head and neck cancer, multiple myeloma, human tongue squamous cell carcinoma, esophageal cancer, prostate cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma or leukemia.

9. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug, the drug being for preventing and / or treating one or more of prostate cancer, head and neck cancer, and human tongue squamous cell carcinoma; R is hydrogen or hydroxyl; R 1 is a hydroxyl group or a protected hydroxyl group; R 2 is hydrogen or 10. The use according to claim 9, wherein, the compound of formula I is as described in any one of claims 2-4.

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