Hydrazine-substituted phenylquinazoline compounds, methods of making and antitumor applications thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a hydrazine-substituted phenyl quinazoline compound, a preparation method thereof and anti-tumor application, which has a structure as shown in formula I, and has high affinity, high membrane permeability and high selectivity. It is verified through experiments that the compound has the effects of inhibiting breast cancer cell proliferation and migration and promoting apoptosis at a cell level. The application provides a theoretical basis for developing new STAT3 allosteric inhibitor anti-breast cancer drugs. Formula I.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, this invention relates to a hydrazine-substituted phenylquinazoline compound, its preparation method, and its antitumor applications. Background Technology

[0002] Cancer is a major challenge facing humanity, and breast cancer is one of the leading causes of malignant tumors. Despite aggressive interventions, recurrence and metastasis are unavoidable. Signal transduction and transcription factor 3 (STAT3), belonging to the STAT family, initiates the intracellular JAK-STAT3 signaling pathway by binding extracellular cytokines and growth factors to their corresponding receptors on the cell surface. Compared to normal STAT signaling pathways, STAT3 is abnormally activated in many human solid tumors and hematologic malignancies. Persistently activated STAT3 participates in abnormal tumor growth, survival, angiogenesis, and suppression of host immune surveillance escape, indicating that persistently activated STAT3 is a key molecule leading to carcinogenesis and tumor development. Studies have shown that abnormal STAT3 activation was first discovered in breast cancer cells. Activated STAT3 upregulates the expression of downstream target genes such as C-Myc and Cyclin D1, thereby disrupting the cell cycle and causing abnormal proliferation in breast cancer cells, leading to the occurrence and development of breast cancer. Therefore, inhibiting STAT3 activity can effectively control or reverse tumor occurrence and development, making the research on STAT3 inhibitors a foundation for clinical anti-tumor drugs. The STAT3 signaling pathway plays a crucial role in maintaining normal cellular physiological activities. In normal cells and tissues, STAT3 activation is instantaneous and precisely regulated. When extracellular cytokines and growth factors bind to their corresponding receptors on the cell surface, downstream kinases are activated. Activated kinases phosphorylate non-phosphorylated STAT3 monomers. The phosphorylated STAT3 monomers interact to form dimers, which then cross the nuclear membrane, enter the nucleus, and bind to the corresponding DNA promoter region, initiating the expression of downstream target genes. Ultimately, STAT3 is dephosphorylated by tyrosine phosphatase and returns to the cytoplasm. However, in tumor cells, STAT3 is overactivated, leading to abnormal expression of downstream target genes. These target genes are involved in cell growth, proliferation, and apoptosis. Therefore, inhibiting the activity of the STAT3 signaling pathway is critical, making STAT3 particularly important. Consequently, research on STAT3-targeted tumor drugs has been valued for many years because STAT3 plays a vital role in tumor development and progression.

[0003] Currently, inhibitors targeting STAT3 face various challenges, broadly categorized into three types: One type involves phosphorylated peptide analogs and peptide-like compounds that bind to the SH2 domain and inhibit STAT3 dimerization. However, the phosphorylated tyrosine residues required for binding the SH2 domain of STAT3 are negatively charged, making the synthesis of such analogs and their acceptance by cells difficult. Due to the crucial role of STAT3 in tumorigenesis and development, many research teams are still developing phosphopeptide inhibitors targeting STAT3 dimers as anti-tumor drugs. Examples include soluble peptide analogs, replacing the pY residue on pYLPQ with phosphorylated cinnamamide derivatives to reduce protease degradation, and synthesizing pYLPQTV analogs by adding a long hydrocarbon chain to improve cell permeability. While these findings are constructive, the application of STAT3 phosphopeptide analogs to cells still faces physicochemical challenges. The second category consists of small-molecule inhibitors targeting the SH2 domain of STAT3; however, these inhibitors have low affinity and low bioavailability, limiting their clinical application. Another category comprises tetrameric oligonucleotides that inhibit the binding of dimerized STAT3 to DNA. By binding to STAT3 and interfering with its binding to DNA, they downregulate the expression of cyclin D1, Bcl-XL, VEGF, etc. However, these inhibitors suffer from membrane permeability issues, making it difficult to achieve inhibitory effects at low concentrations, thus hindering their development into clinical drugs.

[0004] Therefore, it is necessary to invent or optimize a new type of STAT3 inhibitor. Summary of the Invention

[0005] One of the objectives of this invention is to provide a hydrazine-substituted phenylquinazoline compound.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned hydrazine-substituted phenylquinazoline compounds.

[0007] Another object of the present invention is to provide a composition containing the above-mentioned hydrazine-substituted phenylquinazoline compounds.

[0008] Another object of the present invention is to provide the use of the above-mentioned hydrazine-substituted phenylquinazoline compounds as STAT3 inhibitors.

[0009] Another object of the present invention is to provide the use of the above-mentioned hydrazine-substituted phenylquinazoline compounds in the preparation of antitumor drugs.

[0010] Terminology Definition

[0011] The terminology used in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The nomenclature used herein and the laboratory procedures described herein in organic chemistry, medicinal chemistry, and biology are well-known and commonly used in the art. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0012] As used herein, the term "treatment" aims to alleviate or eliminate a targeted disease state or symptom. A subject is successfully "treated" if, in accordance with the methods described herein, a therapeutic amount of a compound or its pharmaceutically acceptable salt, isomer, or pharmaceutical composition thereof is received, and the subject exhibits an observable and / or detectable reduction or improvement in one or more indications and symptoms. It should also be understood that treatment of the disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

[0013] Technical Topic 1

[0014] This invention provides hydrazine-substituted phenylquinazoline compounds having the structure of Formula I or as shown:

[0015]

[0016] Technical Theme Two

[0017] The present invention also provides a method for synthesizing the compound shown in Formula I, comprising the following steps:

[0018]

[0019] Reaction conditions:

[0020] Compound a was dissolved in anhydrous ethanol, and compound b was added dropwise at room temperature. The mixture was stirred for 0.5–3 h, filtered, washed with n-hexane, and dried to obtain the final product.

[0021] Technical Theme 3

[0022] The present invention provides a composition comprising the substituted phenylquinazoline compound of Formula I.

[0023] Furthermore, the "pharmaceutical composition" may also contain one or more pharmaceutically acceptable carriers or excipients, and be prepared in the form of tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, tinctures, decoctions, lozenges, mixtures, suppositories, injections, inhalants, or sprays.

[0024] As used herein, “pharmaceutically acceptable carriers or excipients” include: diluents, fillers, binders, disintegrants, lubricants, flow aids, granulators, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffers. Those skilled in the art will understand that some pharmaceutically acceptable excipients may be used for more than one function and for alternative functions, depending on the amount of said excipient present in the formulation and what other ingredients are present in the formulation.

[0025] For example, when intended for oral administration, it can be formulated into oral preparations such as tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, and pills, containing fillers (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, and sodium carboxymethyl cellulose); Arabica... Gum; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (e.g., gelatin, polyvinylpyrrolidone, and polyethylene glycol), disintegrants (e.g., cellulose derivatives such as sodium carboxymethyl cellulose and polyvinylpyrrolidone), lubricants (e.g., talc, calcium stearate, magnesium stearate, cetyl, boric acid, sodium benzoate, leucine), stabilizers (methylparaben, propylparaben, etc.), flavoring agents (e.g., commonly used sweeteners, acidulants, and flavorings, etc.).

[0026] When used parenterally, the drug can be formulated as an injectable preparation, including sterile powder for injection and solvent for injection. The carrier or excipients used may include sterile water, Ringer's solution, and isotonic sodium chloride solution. Appropriate excipients such as antioxidants, buffers, antibacterial agents, solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators may be added depending on the properties of the drug. Solubilizers or co-solvents may include poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters may include phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; and osmotic pressure regulators may include sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder for injection, mannitol, glucose, etc., may also be added as a support agent. When used rectally, the drug can be formulated as suppositories, etc.

[0027] When intended for pulmonary administration, the drug may be formulated as an inhaler or spray, etc. Numerous resources available to those skilled in the art describe pharmaceutically acceptable excipients and can be used to select appropriate pharmaceutically acceptable excipients, such as books like *Remington's Complete Pharmacy*, *Chinese Pharmaceutical Yearbook*, and *Pharmaceutics*.

[0028] The compounds of this invention and their pharmaceutically acceptable salts can be formulated into conventional formulations, as well as sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0029] Technical Theme 4

[0030] This invention also provides the application of substituted phenylquinazoline compounds with the structure shown in Formula I as STAT3 inhibitors.

[0031] Technology Theme 5

[0032] The present invention also provides the use of the substituted phenylquinazoline compound of Formula I in the preparation of antitumor drugs.

[0033] As a further improvement of the present invention, the antitumor drug is an anti-breast cancer drug.

[0034] The beneficial effects of this invention are as follows:

[0035] This invention develops a hydrazine-substituted phenylquinazoline compound with high affinity, high membrane permeability, and high selectivity. At the cellular level, its ability to inhibit breast cancer cell proliferation and migration and promote apoptosis was verified. This invention provides a theoretical basis for the development of novel STAT3 allosteric inhibitors for the treatment of breast cancer. Attached Figure Description

[0036] Figure 1 This is the mass spectrum of compound K2;

[0037] Figure 2 The graph shows the affinity results between compound K2 and STAT3;

[0038] Figure 3 This is a graph showing the inhibitory effect of compound K2 on breast cancer cell growth.

[0039] Figure 4 The diagram shows the effect of compound K2 on the cell cycle of breast cancer cells; where: A) is the control group; B) is the 1 μM group; C) is the 10 μM group; P3 is the G1 / S phase, and P4 is the G2 / M phase.

[0040] Figure 5The diagram illustrates how compound K2 promotes apoptosis in breast cells; where: A) is the control group; B) is the 1 μM group; C) is the 10 μM group; Q1 represents necrotic cells; Q2 represents secondary necrotic cells; Q3 represents normal cells; and Q4 represents apoptotic cells.

[0041] Figure 6 Figure showing the effect of compound K2 on the migration ability of breast cancer cells;

[0042] Figure 7 This is a graph showing the growth inhibition of compound K2 on normal human cardiomyocytes AC16. Detailed Implementation

[0043] The present invention is illustrated below with reference to specific embodiments. These embodiments are not intended to limit the scope of the invention, but rather to provide guidance for those skilled in the art to prepare and use the compounds and compositions of the present invention.

[0044] Example 1: Preparation of 4-{[2-(2-(2-(2-chloro)phenylquinazolin-4-yl)hydrazine-1-yl]methyl}phenyl-1,3-diol

[0045]

[0046] The synthesis route is as follows:

[0047]

[0048] Compound a (27 g, 100 mmol) was dissolved in 1.8 L of ethanol. Compound b (13.8 g, 100 mmol) of 2,4-dihydroxybenzaldehyde was added dropwise at room temperature. The reaction was stirred for 1 h, filtered, washed with n-hexane, and dried to give 23.3 mg of a yellow solid, yield 59.7%. MS (ESI, positive ion) m / z: 390.92 (M+1). Figure 1 As shown.

[0049] Example 2: Determination of binding affinity to STAT3

[0050] The binding ability of small molecules to STAT3 was detected by fluorescence polarization assay. A fluorescence polarization screening system was constructed, with Ac-pYLPQTV-NH2 as a positive control (peptide), non-phosphorylated peptide Ac-YLPQTV-NH2 as a negative control (CY-252), and FITC-pYLPQTV-NH2 as a binding probe. The inhibitory effect of small molecules was measured by inhibiting the binding of peptides with fluorescent groups to STAT3.

[0051] result Figure 2 The IC shows that K2 inhibits the binding of STAT3 with FITC-pYLPQTV-NH2. 50 It is 13.49 μM.

[0052] Example 3: Effects on the growth and proliferation of breast cancer cells

[0053] The effect of small molecules on breast cancer cell survival was detected using the CCK8 assay. Four T1 cells were seeded in each 96-well plate, with 5 x 10⁶ cells per well. 3 Cells were cultured overnight and then added to the culture vessel at a K2 concentration gradient (200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.56 μM, 0.78 μM, 0.39 μM). The cells were incubated at 37°C with three wells. After 24 hours, 10 μl of CCK8 was added to each well, and the OD value was read by a microplate reader after 3 hours.

[0054] The results are as follows Figure 3 As shown, the effect of K2 on the growth and proliferation of breast cancer cells was detected by the CCK8 assay. Adding a predetermined concentration of K2 resulted in a concentration-dependent inhibition of breast cancer cell proliferation, with an IC50 value of [missing information]. 50 It is 16.44 μM.

[0055] Example 3: Effects on the breast cancer cell cycle

[0056] The experiment was set up with three groups: a control group, a 1 μM inhibitor group, and a 10 μM inhibitor group. Four T1 cells were seeded in each 6-well plate, with 4 × 10⁶ cells per well. 5 Cells were divided into two groups: 0.2 μl of DMSO was added as the control group, and 1 μM and 10 μM of K2 were added as the inhibitor groups. After 24 hours, the cells were harvested and the cell cycle was analyzed by flow cytometry.

[0057] The results are as follows Figure 4 As shown, K2 alters the cell cycle of breast cancer cells. 10 μM K2 significantly arrested 4T1 cells in the S and G2 / M phases, suggesting that it has an inhibitory effect on the growth of breast cancer cells.

[0058] Example 4: Small molecules promote apoptosis in breast cancer cells

[0059] The experiment was set up with three groups: a control group, a 1 μM inhibitor group, and a 10 μM inhibitor group. Four T1 cells were seeded in each 6-well plate, with 4 × 10⁶ cells per well. 5 Cells were divided into two groups: 0.2 μl of DMSO was added as the control group, and 1 μM and 10 μM of K2 were added as the inhibitor groups. After 24 hours, the cells were harvested and the cell apoptosis was analyzed by cell flow cytometry.

[0060] like Figure 5 As shown, compared with the control group, 1 μM K2 did not change the effect of 4T1 cell apoptosis, but 10 μM K2 treatment of 4T1 cells could significantly promote 4T1 cell apoptosis.

[0061] Example 5: Effects of small molecules on the migration ability of breast cancer cells

[0062] The experiment was set up with three groups: a control group, a 1 μM inhibitor group, and a 10 μM inhibitor group. Four T1 cells were seeded in each 6-well plate, with 4 × 10⁶ cells per well. 5 Cells were divided into control groups and inhibitor groups. 0.2 μl of DMSO was added to each cell. 1 μM and 10 μM of K2 were added to each cell. Cells were harvested after 24 hours and their migration ability was detected by trans-well assay.

[0063] from Figure 6 It can be seen that 10 μM K2 can inhibit the migration ability of 4T1 breast cancer cells.

[0064] Example 6 Safety Evaluation

[0065] AC16 is a normal cardiomyocyte. The effect of K2 on AC16 cell viability was detected using the CCK8 assay. AC16 cells were seeded in 96-well plates with 5 x 10 cells per well. 3 Cells were cultured overnight and then added to the culture vessel at a K2 concentration gradient (200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.56 μM, 0.78 μM, 0.39 μM). The cells were incubated at 37°C with three wells. After 24 hours, 10 μl of CCK8 was added to each well, and the OD value was read by a microplate reader after 3 hours.

[0066] Figure 7 As can be seen, the K2 small molecule has almost no effect on the growth of AC16 cells, indicating that this inhibitor has the advantage of low toxicity at the cellular level.

Claims

1. Application of a hydrazine-substituted phenylquinazoline compound of Formula I in the preparation of STAT3 inhibitor drugs ； Formula I.

2. The use of a hydrazine-substituted phenylquinazoline compound of Formula I in the preparation of an antitumor drug, characterized in that, Anti-tumor drugs are drugs that inhibit breast cancer. ； Formula I.

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