STAT3 small molecule inhibitors and their applications

Abstract

The present invention relates to the field of medical technology, and in particular to STAT3 small molecule inhibitors and their applications. The present invention screens a large number of compounds in an existing compound library and finds that several compounds described in the present invention have an inhibitory effect on STAT3 activation and on the activity of multiple tumor cells. 50 The value can reach 1.9μmol / L. Therefore, it can be used to prepare anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to STAT3 small molecule inhibitors and applications thereof. Background Art

[0002] Signal transducer and activator of transcription 3 (STAT3) is a member of the STATs family. It is widely expressed in various tissues and organs of the human body and has the dual role of transmitting cell signals and regulating gene expression. It is the convergence point of multiple signal pathways such as JAK, Src, and EGFR, and can sense a variety of signals including cytokines and growth factors. The main structure of STAT3 includes the C-terminal transcriptional activation region, SH2 domain, linker domain, DNA binding domain, and N-terminal conserved domain. After receiving extracellular signal stimulation, STAT3 is tyrosine phosphorylated in the SH2 region and interacts with the SH2 region of another molecule to form a homodimer, enters the cell nucleus to regulate downstream gene transcription, and participates in various physiological activities such as cell proliferation, differentiation, apoptosis, and immunity. Under normal circumstances, the activation of STAT3 is short-lived and controllable. However, in the case of cancer, studies have shown that excessive and continuously activated STAT3 signals can be detected in many diseased tissues, which plays an important role in tumor proliferation, angiogenesis, and immunosuppression. The development of related inhibitors targeting STAT3 is expected to develop a new generation of targeted anticancer drugs.

[0003] There are three main types of STAT3 direct inhibitors under development: peptides, antisense nucleotides and small molecules. The former two have the risk of instability in the body and poor bioavailability. Although the latter also has the problem of low inhibitory activity, it has the largest number of studies, the longest research time, and the fastest clinical progress. STAT3-SH2 region small molecule inhibitors represented by Napabucasin (BBI608) have reached Phase 3 clinical trials. Among them, the clinical trial of advanced colorectal cancer has failed, and the Phase 3 trial of metastatic pancreatic cancer is still in progress. Other STAT3 small molecule inhibitors such as TTI-101 and WP1066 are still in Phase 1 clinical trials, so the field of STAT3 small molecule inhibitors is in urgent need of new structural attempts. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide more compounds for inhibiting STAT3 activity and further serving as anti-tumor drugs.

[0005] The present invention provides the use of a compound in the preparation of a STAT3 inhibitor; the compound is:

[0006]

[0007] Wherein, X1, X2, X3, and X4 are independently selected from C, N, O, or S;

[0008] R1 and R2 are independently selected from H, carboxamido, acetamido, amino, aminomethyl, hydroxyl or hydroxymethyl;

[0009] R3, R4, R5, R6 are independently selected from H, methyl or halogen;

[0010] Ar Among them, R7, R8, R9, R 10 , R 11、 R 12 Independently selected from H, methyl or halogen; n=1-3, m=1-2.

[0011] Preferably, the compound used as STAT3 inhibitor is:

[0012]

[0013] In the present invention, the analog is a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or inclusion compound of any one of the five compounds.

[0014] In the present invention, the STAT3 inhibited by the compound is STAT3 in tumor cells. The inhibition refers to inhibiting the activation of STAT3 in tumor cells and / or reducing the level of STATS.

[0015] In some embodiments, the tumor cells are from breast cancer or liver cancer. The tumor cells are MCF-7, MDA-MB-231 and / or HepG2.

[0016] The present invention also provides the use of the compound in the preparation of anti-tumor drugs; the compound is:

[0017]

[0018] Wherein, X1, X2, X3, and X4 are independently selected from C, N, O, or S;

[0019] R1 and R2 are independently selected from H, carboxamido, acetamido, amino, aminomethyl, hydroxyl or hydroxymethyl;

[0020] R3, R4, R5, R6 are independently selected from H, methyl or halogen;

[0021] Ar Among them, R7, R8, R9, R 10 , R 11 , R 12 Independently selected from H, methyl or halogen; n=1-3, m=1-2.

[0022] In the present invention, the anti-tumor compound is:

[0023]

[0024] In the present invention, the analog is a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or inclusion compound of any one of the five compounds.

[0025] The anti-tumor effect is to inhibit the growth and / or proliferation of tumor cells.

[0026] In the present invention, the tumor cells are from: bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, external genital cancer, urogenital tract cancer, head cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, muscle tissue cancer, cervical cancer, oral or nasal mucosal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer and / or thyroid cancer.

[0027] In some embodiments, the tumor cells are breast cancer cells and / or liver cancer cells.

[0028] The present invention also provides an anti-tumor drug, which comprises:

[0029]

[0030] Wherein, X1, X2, X3, and X4 are independently selected from C, N, O, or S;

[0031] R1 and R2 are independently selected from H, carboxamido, acetamido, amino, aminomethyl, hydroxyl or hydroxymethyl;

[0032] R3, R4, R5, R6 are independently selected from H, methyl or halogen;

[0033] Ar Among them, R7, R8, R9, R 10 , R 11 , R 12 Independently selected from H, methyl or halogen; n=1-3, m=1-2.

[0034] The medicine of the present invention also includes pharmaceutically acceptable excipients.

[0035] The medicine of the present invention may also include other medicines with anti-tumor function.

[0036] In some embodiments provided by the present invention, the pharmaceutically acceptable excipient is one or a mixture of two or more of a flavoring agent, an osmotic pressure regulator, a filler, a lubricant, a preservative, a suspending agent, a food coloring, a diluent, an emulsifier, a disintegrant or a plasticizer.

[0037] Preferably, the drug is an oral preparation or an injection. The dosage form of the oral preparation is tablets, pills, oral liquids, capsules, syrups, pellets or granules. Preferably, the capsule is a hard capsule or a soft capsule. The tablet is an oral tablet or an oral tablet. The oral tablet refers to a tablet for oral administration. Most of the drugs in such tablets are absorbed through the gastrointestinal tract to exert their effects, and some of the drugs in the tablets exert their effects locally in the gastrointestinal tract. In some embodiments provided by the present invention, the oral tablet is a common compressed tablet, a dispersible tablet, an effervescent tablet, a chewable tablet, a coated tablet or a sustained-release tablet. The injection includes an injection powder or an injection solution.

[0038] The present invention also provides an anti-tumor method, which comprises administering the drug of the present invention.

[0039] The drug of the present invention can be used in combination therapy, i.e., in combination with one or more other agents, wherein the combination includes administering the drug of the present invention and the other agents together, or administering them sequentially. For example, the other agent can be administered before, during, or after the administration of the drug of the present invention. The mode of administration can be the same or different, for example, the drug of the present invention and the other agent can both be administered in the form of injection, can both be administered orally, or can one be administered in the form of injection and the other in the form of oral administration.

[0040] The present invention screened a large number of compounds in the existing compound library and found that several compounds described in the present invention have an inhibitory effect on STAT3 activation and have an inhibitory effect on the activity of multiple tumor cells. 50 The value can reach 1.9μmol / L. Therefore, it can be used to prepare anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The results show that Napabucasin has an inhibitory effect on tumor cells;

[0042] Figure 2 It shows the inhibitory effect of DPP021-191028-1-1-00036 on tumor cells;

[0043] Figure 3 It shows the inhibitory effect of DPP021-191028-1-1-00066 on tumor cells;

[0044] Figure 4It shows the inhibitory effect of DPP021-191028-1-1-00101 on tumor cells;

[0045] Figure 5 It shows the inhibitory effect of DPP021-191028-1-1-00110 on tumor cells;

[0046] Figure 6 The results show the inhibitory effect of DPP021-191028-1-1-00077 on tumor cells. DETAILED DESCRIPTION

[0047] The present invention provides STAT3 small molecule inhibitors and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0048] middle, In the present invention, the positions of the two connecting bonds are not fixed, and they can be both on the benzene ring side of the indole ring, or both on the pyrrole ring side, or one on the benzene ring and the other on the pyrrole ring. 11 )n or -(R 12 )m represents a substituent on the indole ring. When n=2-3, the substituent on the benzene ring is independently selected from H, methyl or halogen; when m=2, the substituent on the pyridine ring is independently selected from H, methyl or halogen. In the present invention, the halogen is fluorine, chlorine, bromine or iodine.

[0049] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0050] The compounds and their numbers in the examples are:

[0051] DPP021-191028-1-1-00036:

[0052] DPP021-191028-1-1-00066:

[0053] DPP021-191028-1-1-00101:

[0054] DPP021-191028-1-1-00110:

[0055] DPP021-191028-1-1-00077:

[0056] IC50: (halfmaximal inhibitory concentration) refers to the half inhibitory concentration of the measured antagonist. It can indicate the half amount of a drug or substance (inhibitor) in inhibiting certain biological processes (or certain substances contained in this process, such as enzymes, cell receptors or microorganisms). In terms of cell proliferation, it can be understood as the drug concentration required when the number of surviving cells after drug administration is reduced by half.

[0057] The present invention verifies the inhibition of STAT3 activity and tumor growth by the compound through IC50 determination. The present invention is further described in conjunction with the following examples:

[0058] Example 1: Detection of anticancer activity of STAT3 small molecule inhibitors at the cellular level

[0059] Experimental principle: Exogenous MTT detection reagent can be reduced to water-insoluble blue-purple crystalline formazan by succinate dehydrogenase in the mitochondria of living cells. The light absorption value at a wavelength of 570nm after formazan is dissolved in dimethyl sulfoxide (DMSO) can indirectly reflect the number of living cells.

[0060] Experimental materials: MCF-7 (human breast cancer cells, low expression of STAT3), MDA-MB-231 (human breast cancer cells, high expression of STAT3), HepG2 (human liver cancer cells, high expression of STAT3), cell viability detection kit (MTT method), positive drug Napabucasin.

[0061] Experimental steps:

[0062] 1. Plate: Cells in the logarithmic growth phase were seeded in 96-well plates, with approximately 1×10 cells per well. 4 The cells were cultured at 37°C and 5% CO2 for 24 h.

[0063] 2. Adding drugs: Add different concentrations of test compounds, positive drugs and control solvents to the cell culture plate, set up 3 replicate wells for each concentration, and continue culturing for 48 hours.

[0064] 3. Detection: Add MTT detection solution to each well and react at 37°C and 5% CO2 for 4 hours. Then remove the culture medium and add DMSO solvent to dissolve the crystals at the bottom. Use an enzyme-labeled instrument to detect the OD value at a wavelength of 570 nm.

[0065] Cell viability = test well OD / control well OD × 100%

[0066] Table 1 Inhibitory effect of compounds on tumor cells - IC 50 (μM)

[0067] Compound No. MCF-7 HepG2 MDA-MB-231 Napabucasin 0.5985 0.6426 2.352 DPP021-191028-1-1-00036 2.86 4.681 1.9 DPP021-191028-1-1-00066 27.26 15.04 3.445 DPP021-191028-1-1-00101 37.04 3.521 5.629 DPP021-191028-1-1-00110 24.78 9.678 7.759 DPP021-191028-1-1-00077 33.95 22.86 14.69

[0068] The experimental results are as follows Figures 1 to 6 As shown in Table 1, the five compounds had good growth inhibition effects on STAT3 high-expressing cells MDA-MB-231 and HepG2, among which DPP021-191028-1-1-00036 had a strong inhibitory effect on MDA-MB-231 cells with the positive drug Napabucasin IC 50 In addition, the activities of the five compounds on STAT3 low-expressing MCF-7 cells were relatively low, indicating good selectivity.

[0069] Example 2: Molecular level detection of STAT3 small molecule inhibitor activity

[0070] Principle of fluorescence polarization experiment: When a fluorescent small molecule binds to a receptor, its effective volume increases, its rotation slows down, the emitted light in the same plane as the excitation light increases, the depolarization weakens, and the polarization increases.

[0071] Experimental materials: STAT3 protein, fluorescent peptide FITC-pYLPQTV-NH2, strong positive control peptide Ac-pYLPQTV-NH2, weak positive control peptide Ac-YLPQTV-NH2, negative control peptide Ac-Pylktkf, buffer, 96-well plate, multifunctional microplate reader.

[0072] Experimental steps:

[0073] 1. Prepare small molecule solutions of various concentrations and various types of control peptide solutions of various concentrations.

[0074] 2. Use buffer to prepare a mixture of STAT3 and FITC-pYLPQTV-NH2, with a final concentration of STAT3 of 5 μM and a final concentration of FITC-pYLPQTV-NH2 of 1 nM. Store in a dark place.

[0075] 3. Add different concentrations of control peptide solutions and small molecule solutions to a 96-well plate, then add the mixture containing STAT3 and fluorescent peptides to different wells, and react on a horizontal shaker in the dark for 3 hours.

[0076] 4. The fluorescence polarization values ​​of each well of 96 wells were read by a multifunctional microplate reader (excitation wavelength 485 nm, emission wavelength 525 nm), and the IC values ​​of the compounds were calculated using the prism8 software. 50 (μM).

[0077] The results showed that at the molecular level, DPP021-191028-1-1-00036, DPP021-191028-1-1-00066, DPP021-191028-1-1-00101, DPP021-191028-1-1-00110, and DPP021-191028-1-1-00077 had an inhibitory effect on STAT3.

[0078] Example 3: Testing the ability of compounds to inhibit STAT3 activation at the cellular level

[0079] Experimental principle: STAT3 forms a homodimer after phosphorylation and enters the cell nucleus to exert its effect, that is, the STAT3 phosphorylation level can reflect the degree of STAT3 activation.

[0080] Experimental materials: MDA-MB-231 (human breast cancer cells), IL-6 protein, STAT3, p-STAT3 (Tyr-705), β-actin antibodies, Western blotting related instruments (gel electrophoresis instrument, Bio-rad exposure instrument), 24-well plate

[0081] Experimental steps:

[0082] 1. Plating: Inoculate cells in the logarithmic growth phase into 24-well plates and culture at 37°C and 5% CO2 for 24 hours.

[0083] 2. Adding drugs: Add different concentrations of test compounds, positive drugs and control solvents to the cell culture plates and incubate for 1 hour.

[0084] 3. IL-6 induced STAT3 activation: IL-6 (30 ng / ml) was added to different wells and incubated for 30 min.

[0085] 4. Detection: Lyse cells to prepare samples, and detect p-STAT3 and STAT3 levels by Western blotting, with β-actin as an internal reference.

[0086] The results showed that DPP021-191028-1-1-00036, DPP021-191028-1-1-00066, DPP021-191028-1-1-00101, DPP021-191028-1-1-00110, and DPP021-191028-1-1-00077 had an inhibitory effect on the activation of STAT3.

[0087] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of the compound in the preparation of anti-tumor drugs; The tumor is breast cancer and / or liver cancer; The compound is:

2. The use according to claim 1, characterized in that: The tumor cells are MCF-7, MDA-MB-231 and / or HepG2.

3. The use according to claim 1 or 2, characterized in that: The anti-tumor effect is to inhibit the growth and / or proliferation of tumor cells.

4. The use according to claim 1 or 2, characterized in that: The anti-tumor effect includes inhibiting STAT3 levels.

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