Pharmaceutical composition for treating and preventing triple-negative breast cancer

Abstract

The present invention relates to a pharmaceutical composition for treating and preventing triple-negative breast cancer, comprising a compound of the following Formula 1. The compound of Formula 1 of the present invention may exist in the forms of a pharmaceutically acceptable salt, hydrate, solvate, clathrate, prodrug, and polymorphic isomer. [Formula 1] The compound of Formula 1 of the present invention simultaneously has a mechanism blocking calcium overexpressed in triple-negative breast cancer cells essential for cancer cell growth and a mechanism inhibiting the target protein STAT3. Moreover, the present invention shows that the compound of Formula 1 inhibits tumoral growth in xenograft mouse models induced to have triple-negative breast cancer and shows an anticancer activity through apoptosis mechanism in the tumoral tissues of mice.

Description

PHARMACEUTICAL COMPOSITION FOR TREATING AND PREVENTING TRIPLE-NEGATIVE BREAST CANCER

[0001] The present invention relates to a pharmaceutical composition for treating and preventing triple-negative breast cancer, comprising a compound of the following Formula 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier thereof.

[0002] [Formula 1]

[0003]

[0004] Adenocarcinoma is a tumor in epithelial tissues, including glandular tissue. Adenocarcinoma can arise in various organs of the human body having epithelial tissues and mainly occur in organs such as the colon, lungs, prostate, ureter, vagina and esophagus, breast, stomach, pancreas, and the like.

[0005] Especially, breast cancer collectively refers to every type of malignant tumor that, unlike a benign tumor that stays therein, occurs and spreads outside of the breast and may develop to be life-threatening. Breast cancer is a fatal disease where an abnormal tissue keeps growing inside the breast or even spreads to other organs. Moreover, since there are various types of cells in the breast and any type of cell may turn into cancer cells, there are many types of cancer that may occur in the breast. However, the majority of breast cancers occur from the cells of lactiferous ducts and lobules (especially lactiferous duct cells), and thus term "breast cancer" normally indicates cancers that occur in the epithelial cells (cells covering the body surface or inner surface of internal organs) of lactiferous ducts or lobules. The cancer occurring in the lactiferous ducts and lobules can be divided into invasive breast cancer and non-invasive breast cancer, depending on the level of invasion (into the adjacent cells or tissues) of the cancer cell.

[0006] Triple-negative breast cancer, which stands for ER-negative PR-negative HER2 / neu-negative breast cancer, is a type of invasive breast cancer where, unlike most types of breast cancer, three hormone receptors, namely, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), helping the growth and proliferation of cancer, are missing. This type of breast cancer does not show expression which makes it hard to react with the anti-hormones or targeted agents and has a poor prognosis.

[0007] Approximately 15% of all breast cancer patients suffer from triple-negative breast cancer. Whereas the other types of breast cancer are positive for all three estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptors 2 (HER2) and usually occurs in patients over their 40s, triple-negative breast cancer is known to be occurring more from the young women of 20s to 30s or women of other ages having a mutation in breast cancer gene 1 (BRCA1). Triple-negative breast cancer is highly aggressive even among the types of breast cancers and progresses rapidly so that 50% of the early-diagnosed patients suffer a recurrence within 1-2 years. When diagnosed at stages 3-4, the five-year survival rate of patients is relatively low at 30% and it is known to have a poor prognosis because it often spreads to the brain (30%) or lungs (40%).

[0008] Tumor removal surgery is common in triple-negative breast cancer treatment, and radiotherapy or anticancer therapy is performed to prevent the recurrence of the tumor after the surgery. However, since the estrogen receptors and progesterone receptors are not expressed, hormone therapy is ineffective. Moreover, targeted therapy is also ineffective because HER2 receptors are also not expressed. For these reasons, cytotoxic chemotherapy is used as a main treatment method. Currently, doxorubicin, docetaxel, paclitaxel, and the like are used for triple-negative breast cancer treatment. However, they have undesirable disadvantages such as that recurrence rate is high, progression-free survival (length of time for cancer to progress anew after the treatment) is low at 6 months on average when recurred as metastatic triple-negative breast cancer, and overall survival is limited to about a year.

[0009] Recently, PARP inhibitor Lynparza, checkpoint inhibitor Keytruda, and antibody-drug conjugate (ADC) Trodelvy targeting Trop-2 protein have been developed as new therapeutic agents for triple-negative breast cancer.

[0010] The compound of Formula 1 used in the present invention is a compound known in WO 2014 / 021591 A2 and WO 2015 / 178608 A2 and has anticancer activity due to T-type calcium channel antagonism.

[0011] Calcium is known to play an important role as an intracellular messenger and is involved in cell growth by regulating various cell activities (Berridge, M.J. et al., Nat. Rev. Mol. Cell Biol. 2003; 4:517-529). For example, calcium signals are required during cell mitosis phases to proceed from the G1 phase to the S phase of the cell cycle, and if calcium ions in the cell are depleted, the cell cycle stops in the middle of the G0 / G1 phases and the S phase (Clapham, D. E., Cell 1995, 80, 259-268). The amount of calcium in a cell is regulated through the T-type calcium channel, and its basis is reported in the recent papers reporting that the T-type calcium channel blocker inhibits cell differentiation (McCalmont, W. F. et al., Bioorg. Med. Chem. Lett. 2004, 14, 3691-3695; McCalmont, W. F. et al., Bioorg. Med. Chem. 2005, 13, 3821-3839).

[0012] It is known that the regulation of T-type calcium channels can act as a treatment for various diseases; brain conditions such as epilepsy, hypertension, and the like; heart conditions such as angina and the like; pain conditions such as chronic pain, neurogenic pain, and the like, or as an anticancer agent. T-type calcium channels are also known to consist of three subtypes of a1G (Cav3.1), a1H (Cav3.2), and a1I (Cav3.3), and to regulate the growth and metastasis of cancer cells by regulating the amount of calcium influx into the cell and thereby regulating the activation of CaMKII protein.

[0013] Meanwhile, though still controversial, L-type calcium channel blockers as a hypertension treatment have the potential to cause cancer in elderly people, unlike T-type calcium channel blockers. For example, research has reported that the L-type calcium channel blockers inhibited cell death and thereby promoted the growth of existing cancer cells in the body (La Vecchia, C. et al., Eur. J. Cancer 2003, 39, 7-8). Therefore, the selective T-type calcium channel blocker may be a new drug for treating cancer cells having an abnormal cell cycle.

[0014] WO 2008 / 136631 A1 discloses 3,4-dihydroquinazoline derivative of Formula A, which may be used as a new chemotherapy drug capable of treating hyperproliferative diseases such as cancer by T-type calcium channel blocking, which is a different action mechanism from the existing drugs.

[0015] [Formula A]

[0016]

[0017] WO 2014 / 021591 A2 discloses the hydroquinazoline derivative of Formula B as a T-type calcium channel antagonist, which is useful for treating and preventing disorders and diseases related to the functions of T-type calcium channels.

[0018] [Formula B]

[0019]

[0020] According to WO 2014 / 021591 A2, the compound shown above as Formula B is capable of inhibiting the flow of calcium through the calcium channels and thereby can be used as a calcium channel blocker and anticancer agent, and is capable of showing significant effects for, preferably, prostate cancer, breast cancer, lung cancer, prostate cancer, colorectal cancer, pancreatic cancer, skin cancer, or uterine cancer.

[0021] WO 2015 / 178608 A2 discloses 3,4-dihydroquinazoline derivative of Formula C capable of enhancing the efficacy of existing anticancer agents by blocking the intracellular influx of calcium ions as the essential second messenger for the proliferation and growth of cancer cells and thereby inducing the cell cycle block in cancer cells.

[0022] [Formula C]

[0023]

[0024] According to WO 2015 / 178608 A2, the compound of the above Formula C shows activity blocking the cell cycle in cancer cells and also shows the effect of increasing the cytotoxicity of the existing anticancer agents against the cancer cells. Moreover, it is also disclosed that the complex comprising the compound of the above Formula C and the other anticancer agent selected from the group consisting of platinum coordination complex compound as an anti-tumor agent, anthracycline, topoisomerase inhibitor, and tyrosine kinase inhibitor can be used for treating and preventing cancer such as lung cancer, sarcoma, malignant melanoma, pleural mesothelioma, bladder cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer, esophageal cancer, adrenal cancer, parotid carcinoma, head and neck cancer, cervical cancer, mesothelioma, leukemia, and lymphoma.

[0025] The present inventors, while analyzing the potency of the compound of Formula 1 against various cancers, discovered that the compound has excellent cell viability (IC50), especially against breast cancer, and conducted a further study to find out that the compound of Formula 1 of the present invention is capable of treating and preventing triple-negative breast cancer by double-target anticancer effect on MDA-MB-231, MDA-MB-453, Hs 578T, and BT20 cell line tumors, corresponding to the cell lines for triple-negative breast cancer. Namely, the present inventors found out that the compound of Formula 1 of the present invention simultaneously has a mechanism blocking overexpressed calcium essential for cancer cell growth in cancer cells and a mechanism inhibiting the target protein STAT3 (Signal Transducer and Activator of Transcription 3) in cancer cells, thereby completed the present invention.

[0026] An object of the present invention is to provide a pharmaceutical composition capable of treating and preventing triple-negative breast cancer as a type of invasive breast cancer.

[0027] Another object of the present invention is to provide a medical use of a compound of Formula 1 capable of treating and preventing triple-negative breast cancer as a double-target anticancer agent.

[0028] To achieve the above objectives, the present invention provides a pharmaceutical composition for treating and preventing triple-negative breast cancer, comprising a compound of Formula 1.

[0029] [Formula 1]

[0030]

[0031] The above compound of Formula 1 used in the present invention is known in WO 2014 / 021591 A2 and WO 2015 / 178608 A2 and may exist in the forms of pharmaceutically acceptable salts, hydrates, solvates, clathrates, prodrugs, and polymorphic isomers.

[0032] First, certain terms are defined below for a better understanding of the present invention.

[0033] The term 'pharmaceutically acceptable' used in the present invention refers to non-toxic substances substantially not hindering the effects in the biological activity of the compound of Formula 1.

[0034] The term 'preventing' used in the present invention refers to treating subjects at risk of triple-negative breast cancer occurrence preventively and thereby decreasing the probability of the occurrence of the conditions in the subjects. Moreover, the term 'treating' refers to both therapeutic treatment and preventive treatment, wherein the purpose thereof is to mitigate the diseases or disorders by relieving or mitigating at least one physical parameter, including the one unidentifiable to the patients (i.e., slowing or halting or reducing at least one of the diseases occurrence or clinical symptoms thereof).

[0035] The term 'treating or preventing' used in the present invention includes administering the compound of Formula 1 arbitrarily in combination with one or more additional treatments to prevent or to delay the occurrence of the symptoms of disease (e.g., lung cancer or pancreatic cancer), complications, or biochemical signals, and thereby mitigating the symptoms of diseases, conditions, or disorders, or preventing or inhibiting the additional occurrence of diseases, conditions, or disorders. The treatment may be the preventive inhibition (to prevent or delay the occurrence of the disease or to prevent the occurrence of the clinical or subclinical symptoms thereof) or the therapeutic inhibition or mitigation of the symptoms after the disease.

[0036] The term 'effective amount' used in the present invention refers to the amount sufficient to achieve a beneficial result or desired result. For example, the therapeutic amount is to achieve the desired therapeutic effect. The amount may be the same as or different from the preventive effective dose required for preventing the occurrence of the disease or symptoms of the disease. The effective dose can be administered by more than one time of injection, application, or oral administration. The therapeutically effective amount (i.e., effective dose) varies depending on the selected therapeutic compound. The composition may be administered more than one time every day to more than one time per week or may include less frequent administration, for example, as disclosed in the present application. A skilled person would understand that the dosage and the timing required for effectively treating the subject may be affected by the prescribed factors including, but not limited to, the following: severity of disease or disorder; previous　treatments; overall health and / or age of the subject; and other existing diseases. In addition, the treatment of the subject with the therapeutically effective amount of the therapeutic compound disclosed in the present application may include one-time treatment or a series of treatments. The term 'therapeutically effective amount' used in the present application refers to the amount wherein the compound disclosed herein induces the biological or medical response of the subject, for example, the amount mitigating symptoms, relieving conditions, slowing or delaying the progression of diseases, preventing the diseases, or the like. In one un-limiting embodiment, the term 'therapeutically effective amount' refers to the amount effective for at least partially mitigating, inhibiting, preventing, and / or relieving triple-negative breast cancer when the compound of Formula 1 according to the present invention is administered to the subject.

[0037] The compound of Formula 1 used in the present invention is, not limited by the theory, 2-(3-(4-Cyclohexylphenyl)-2-((3-(3,3-dimethylureido)propyl)(methyl)amino)-3,4-dihydroquinazolin-4-yl)-N-(4-fluorobenzyl)acetamide having the following structure.

[0038] In addition, the above compound is disclosed in various studies as 4-(4-fluorobenzylcarbamoylmethyl)-3-(4-cyclohexylphenyl)-2-[3-(N,N-dimethylureido)-N'-methylpropylamino]-3,4-dihydroquinazoline.

[0039] The term 'pharmaceutically acceptable salt' used in the present invention refers to salts prepared from non-toxic acids and bases including pharmaceutically acceptable inorganic acids and bases and organic acids and bases. The pharmaceutically acceptable base addition salts suitable for the compound of the present invention include metal salts prepared with aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc; or organic salt prepared with lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. The suitable non-toxic acids include, but are not limited to, acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, and p-toluenesulfonic acid.

[0040] Unless instructed otherwise, the term 'prodrug' used herein refers to the derivative of a compound capable of hydrolysis, oxidation, or other response under biological conditions (in-vivo or ex-vivo) for providing the compound. The examples of the prodrug include, but are not limited to, biohydrolyzable moieties such as biohydrolyzable amide, biohydrolyzable ester, biohydrolyzable carbamate, biohydrolyzable carbonate, biohydrolyzable ureide, and biohydrolyzable phosphate analogue. The prodrug can be prepared using a commonly disclosed method.

[0041] Unless used otherwise, the terms 'biohydrolyzable amide', 'biohydrolyzable ester', 'biohydrolyzable carbamate', 'biohydrolyzable carbonate', 'biohydrolyzable ureide', and 'biohydrolyzable phosphate' used herein respectively refer to amide, ester, carbamate, carbonate, ureide, and phosphate of a compound capable of 1) giving good effects such as uptake rate, duration of the activity, or sign of activity, in-vivo, though not being hindered by the biological activity of the compound; or capable of 2) being converted into a compound biologically inactive but biologically active in-vivo. Examples of biohydrolyzable esters include, but are not limited to, lower　alkyl　ester, alkoxyacyloxy ester, alkyl acylamino alkyl ester, and choline ester. Examples of biohydrolyzable amide include, but are not limited to, lower　alkyl　amide, α-amino acid amide, alkoxyacyl amide, and alkylaminoalkyl carbonyl amide. Examples of biohydrolyzable carbamate include, but are not limited to, alkylamine, substituted ethylenediamine, amino acid, hydroxyalkylamine, heterocyclic amine, heteroaromatic amine, and polyetheramine.

[0042] In addition, the present invention provides a medical use of the compound of Formula 1 capable of treating and preventing triple-negative breast cancer as a double-target anticancer agent.

[0043] According to the present invention, the subjects being administered with the compound of Formula 1 are MDA-MB-231, MDA-MB-453, Hs 578T, and BT20 cell line tumors as the cell lines for triple-negative breast cancer.

[0044] In the present invention, the compound of Formula 1 may exist in the forms of pharmaceutically acceptable salts, hydrates, solvates, clathrates, prodrugs, and polymorphic isomers for treating or preventing triple-negative breast cancer.

[0045] The present invention found the method of treating or preventing triple-negative breast cancer by regulating T-type calcium channel and STAT3 activation in vitro and in vivo with the compound of Formula 1. Namely, in an Example, it is confirmed that the compound of Formula 1 inhibits the calcium influx into the cell and regulates the activation of CaMKII protein in the cell lines for triple-negative breast cancer through a T-type calcium channel. Moreover, another Example of the present invention confirmed the inhibition of the formation of STAT3 dimer and STAT3 phosphorylation regulation through the binding of the compound of Formula 1 with the SH2 domain.

[0046] The present invention found in another Example that the compound of Formula 1 regulates the expression of the regulatory protein of the Bcl-2 family and thereby induces cell death through a caspase-mediated apoptosis mechanism. Namely, the present invention regulates the expression of the regulatory protein of the Bcl-2 family and thereby shows an anticancer effect against triple-negative breast cancer through the apoptosis mechanism by the caspase activation.

[0047] Another Example of the present invention confirmed that the compound of Formula 1 inhibits the growth of tumors in the triple-negative breast cancer-induced mouse model and induces cell growth inhibition and apoptosis from the tumoral tissue obtained from the mouse model and thereby showed a medical use showing an anti-cancer effect.

[0048] The present invention includes the compound of Formula 1 and may be prepared as a single unit administration form as a pharmaceutical composition useful for preventing and treating triple-negative breast cancer.

[0049] The compound of Formula 1 may exist in the forms of pharmaceutically acceptable salts, hydrates, solvates, clathrates, prodrugs, and polymorphic isomers to be prepared as the single unit administration form according to the present invention.

[0050] As described above, the compound of Formula 1 as an active ingredient of the present invention can be used for treating and preventing the disease and condition of triple-negative breast cancer. The preventive or therapeutic dose of the compound of Formula 1 of the present invention varies. The dose also varies depending on the age, weight, and individual response of patients. An appropriate administration regimen can be appropriately selected by a skilled person. In general, the recommended daily dosage range for the condition described below is approximately 0.01mg / kg to 100mg / kg. More specifically, the above daily dosage is equally distributed and is administered 1 to 3 times a day. In some cases, it may be necessary to use a dosage beyond the range disclosed herein as widely known to the skilled people. Moreover, clinical therapists or therapeutic practitioners need to understand how and when to intervene, adapt, and conclude treatment based on the individual responses of patients.

[0051] The term 'dosage' used herein includes both the above-described dosage and the administration frequency schedule. As known to the skilled people, the other therapeutically effective amounts may be applied to other diseases and conditions. Similarly, the amount sufficient for treating or preventing such disorders is the amount insufficient to cause side effects related to the compound of Formula 1 but sufficient to reduce such side effects.

[0052] The respective administration form of the present invention may be performed by oral, mucosal (including rectal, nasal, or vaginal), parenteral (subcutaneous, intramuscular, bolus injection, intra-arterial, or intravenous), sublingual, and skin administration, and the like.

[0053] The pharmaceutical composition of the present invention and the administration form of the invention include the compound of Formula 1 or the pharmaceutically acceptable salts, hydrates, solvates, clathrates, prodrugs, and polymorphic isomers thereof, and generally include pharmaceutically acceptable excipients.

[0054] The single unit administration form of the present invention may be administered to the patient by oral, mucosal (e.g., nasal, sublingual, vaginal, palatal mucosal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intra-arterial), or percutaneous administration.

[0055] Examples of administration forms include tablets; couplets; hard or soft capsules; cachets; troches; lozenges; spraying agents; suppositories; salves; poultices; pastes; powders; dressing agents; creams; ointments; solutions; patches; aerosols (e.g., nasal nebulizer or inhaler); gels; liquid administration forms suitable for oral or mucosal administration to the patients including solution administration forms suitable for oral or mucosal administration (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid administration forms suitable for parenteral administration to the patients; and sterile solids.

[0056] The types of the composition, form, and administration form of the present invention vary depending on the usage form thereof. For example, the administration form used in the short-term treatment of triple-negative breast cancer may comprise more than one active ingredient than the administration form used in the long-term treatment of the same disease.

[0057] Similarly, the parenteral administration form may comprise more than one active ingredient less than the oral administration form for treating triple-negative breast cancer. The specific administration forms included in the present invention may be performed variously by the embodiment selected by a skilled person. For example, please refer to Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).

[0058] The pharmaceutical composition and the administration form of the present invention include more than one excipient. Appropriate excipients are well known to the skilled people in the pharmaceutical industry, and the non-limiting examples of the suitable excipients are disclosed herein. Whether a specific excipient is suitable to be bound as a pharmaceutical composition or administration form is determined by various factors, such as the administration form administered to the patient.

[0059] For example, the oral administration form of the present invention is prepared by binding active ingredient(s) in a mixture close to at least any excipient according to conventional pharmaceutical mixing techniques. The excipients may take various forms in accordance with the forms prepared to be administered. For example, the excipients suitable for use in the administration of oral liquid or aerosol include, but are not limited to, water, glycol, oil, alcohol, flavoring agent, preservative, and coloring agent. The examples of excipients suitable for use in the solid oral administration form (e.g., powder, tablet, capsule, and couplet) include, but are not limited to, starch, sugar, microcrystalline cellulose, diluents, granulation agent, lubricant, binder, and disintegrant. For ease of administration, the tablets and capsules are represented as the most advantageous oral administration form. In this case, solid excipients are applied. If desired, tablets may be coated by the standard aqueous or non-aqueous techniques. The above-described administration form can be prepared by any method of preparation. In general, the pharmaceutical composition and the administration form of the present invention are prepared by mixing liquid carrier, finely divided solid carrier, or both with the active ingredient evenly and intimately. If required, the above product is molded in the above-described desirable forms. For example, the tablets may be prepared by compression or molding. Compressed tablets may be prepared by compressing active ingredients in a suitable machine in a free-flowing form, such as small grains or powders, selectively mixed with excipients. Molded tablets may be prepared by molding a mixture of a compound as powder damped with the inactive liquid diluent in a suitable machine. The excipients that may be used in the oral administration form of the present invention include, but are not limited to, binders, fillers, disintegrants, and lubricants.

[0060] The binders suitable for the use in the pharmaceutical composition and the administration of the present invention include, but are not limited to, corn starch, potato starch, gelatin, acacia gum, alginic acid, tragacanth gum, guar gum, cellulose and derivatives thereof (e.g., ethyl cellulose, cellulose acetate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose), polyvinyl pyrrolidine, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and the combination thereof. The examples of filler suitable for use in the pharmaceutical composition and the administration of the present invention include, but are not limited to, talc, calcium carbonate, microcrystalline cellulose, dextrate, kaolinite, mannitol, silicic acid, sorbitol, starch, and pregelatinized starch. In the above pharmaceutical composition of the present invention, binder or filler typically exists in about 50% to 99% of the weight of the pharmaceutical composition or the administration form. Suitable forms of the microcrystalline cellulose include, but are not limited to, AVICELEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105, and the combination thereof. The specific binder is a combination of sodium carboxymethyl cellulose and the marketed microcrystalline cellulose such as AVICELRC-581.

[0061] The compound of Formula 1 of the present invention has a double-target anticancer effect against triple-negative breast cancer. Namely, the present invention found that the compound of Formula 1 blocks T-type calcium channels to the triple-negative breast cancer cell to inhibit the calcium influx into the cell and regulates the formation of STAT3 dimer and phosphorylation through the binding with STAT3 SH2 domain, thereby inducing the inhibition of the overgrowth of the cancer cell, and the apoptosis by the caspase activation. The compound of Formula 1 has mechanisms that simultaneously block overexpressed calcium, which is essential for cancer cell growth, and inhibit the target protein STAT3 in cancer cells.

[0062] Moreover, the present invention shows that the compound of Formula 1 inhibits the growth of tumors in the triple-negative breast cancer-induced xenograft mouse model and shows antitumoural activity through the apoptosis mechanism in the tumoral tissue of the mouse.

[0063] Therefore, the present invention provides a pharmaceutical composition for treating and preventing triple-negative breast cancer comprising a compound of Formula 1.

[0064] Figure 1 confirms the expression level of T-type calcium channel and STAT3, targeted by the compound of Formula 1 according to the present invention, through Western blotting in the cell lines of breast cancer including triple-negative breast cancer.

[0065] Figure 2 shows the expression of phosphorylation of CaMKII as an important kinase for the calcium signal transduction and the change in calcium influx into the cell through the T-type calcium channel blockade by the compound of Formula 1 in the triple-negative breast cancer cell line MDA-MB-231, wherein A shows the change in the expression of phosphorylation of CaMKII by the compound of Formula 1 through Western blotting, and wherein B confirms the amount of calcium influx from outside to inside of the cell through Fura-2am.

[0066] Figure 3 shows the effect of the compound of Formula 1 in accordance with the present invention on the formation of STAT3 dimer by STAT3 binding, wherein A shows the formation of the dimer by reacting recombinant STAT3 protein with the fluorescence-labeled peptide binding thereto, wherein B shows the results for the compound of Formula 1 in accordance with the present invention, wherein C shows the control group S31-201, and wherein D shows the control group Stattic.

[0067] Figure 4 confirms the phosphorylation of STAT3 by the treatment of the compound of Formula 1 in accordance with the present invention in the triple-negative breast cancer cell lines, wherein A shows the result of treating 20μM of compound of Formula 1 by time (30, 60, 120 minutes), wherein B shows the result of treating compound of Formula 1 by concentration, and wherein C shows the confocal microscope photomicrograph of the expression of STAT3 phosphorylated within the cell after one hour of treatment of the compound of Formula 1 in concentration of 20μM.

[0068] Figure 5 shows the effect of the compound of Formula 1 in accordance with the present invention on inhibiting STAT3 phosphorylation induced by IL-6 in triple-negative breast cancer cell lines.

[0069] Figure 6 confirms the induction of apoptosis in the triple-negative breast cancer cell lines by the compound of Formula 1 in accordance with the present invention through the TUNEL fluorescent staining method, wherein A shows the apoptosis by time, and wherein B shows apoptosis after 12 hours after the treatment by concentration.

[0070] Figure 7 shows the effect of apoptosis in the triple-negative breast cancer cell lines by the treatment of the compound of Formula 1 in accordance with the present invention using early apoptosis and late apoptosis assay.

[0071] Figure 8 confirms the increase of cleavages of caspase-3, caspase-9, and PARP protein by the treatment of the compound of Formula 1 in accordance with the present invention in the triple-negative breast cancer cell lines, wherein A shows the increase of cleavages by time, and wherein B shows the increase of cleavages by concentration.

[0072] Figure 9 confirms the change in the expression of the Bcl-2 family protein by the treatment of the compound of Formula 1 in accordance with the present invention in the triple-negative breast cancer cell lines, wherein A shows the change by time, and wherein B shows the change by concentration (5, 10, 20 μM).

[0073] Figure 10 shows the tumor growth inhibition effect of the compound of Formula 1 in accordance with the present invention against triple-negative breast cancer in a xenograft mouse model using the triple-negative breast cancer cell line MDA-MB-231, wherein A and B show the tumor size at different concentrations, wherein C shows the tumor weight at different concentrations, and wherein D shows changes in body weight at different concentrations.

[0074] Figure 11 shows the expression and activation of p-STAT3, survivin, Bcl-2, and PARP-1 cleavage, which are apoptosis-related mechanism markers, by the compound of Formula 1 in accordance with the present invention in a tumor isolated from a xenograft mouse model induced by the triple-negative breast cancer cell line MDA-MB-231.

[0075] Figure 12 confirms the expression of p-STAT3 and PCNA by the compound of Formula 1 in accordance with the present invention in a tumoral tissue from a xenograft mouse model using the triple-negative breast cancer cell line MDA-MB-231 through immunohistochemistry.

[0076] The present invention is further described below with Examples and Experimental Examples. The Examples and Experimental Examples below are provided to further describe the present invention in detail and shall not be construed as limiting the scope of the present invention.

[0077] <Example 1>Screening Evaluation for Various Cancer Cells

[0078] Cell viability assay has been performed for the 6 carcinoma cell lines (35 types) including breast carcinoma. The culture medium was prepared by adding 10% fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine, 1 mM Na pyruvate, or Special medium to RPMI 1640. The cells were dispensed in a 384 plate well and were incubated for 72 hours in an incubator maintained at 95% humidity, 5% CO2, and 37°C after being treated with the compound of Formula 1 at 24 hours of incubation. The Cell Titer-GLo (Promega) detection reagent was treated thereafter to measure the bioluminescence with a PerkinElmer EnVision microplate reader to derive a compound concentration (IC50) value inhibiting 50% of cell viability. The results are shown in Table 1 below.

[0079]

[0080]

[0081] As can be seen in Table 1 above, according to the results of cell viability (IC50) assay in 35 types of cell lines of 6 carcinomas, including breast carcinoma, the triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-453, Hs 578T, and BT20 showed difference in efficacy of at least 2 to up to 7 times or more compared to other carcinoma cell lines, showing that they can have an excellent cytotoxic effect.

[0082] Thus, it can be seen that the compound of Formula 1 of the present invention has a distinctive effect against triple-negative breast cancer among breast cancer types than other cancer types.

[0083] <Example 2>Evaluation of Target Expression in Breast Cancer Cell Lines

[0084] 11 types of breast cancer cells including the triple-negative breast cancer cells (MDA-MB-231, MDA-MB-453, Hs 578T, BT20) were seeded at a density of 5x105cells in a 100mm culture dish and cultured for 2-3 days. Cells were harvested at a density of 1Х106cells and washed with PBS to remove media components. Cell lysis was performed using RIPA buffer and then centrifuged (4°C, 13,000 rpm, 15 min) to transfer the supernatant to a new tube. Proteins were quantified by Bradford Assay, and a sample to be loaded was prepared by boiling at 100°C to perform Western blotting. The sample was loaded into the SDS polyacrylamide gel (8% concentration), electrophoresed at 80V, then converted to 100-120V (when the loaded sample moves from the stacking gel to the running gel), and electrophoresed. The polyacrylamide gel, after electrophoresis, was transferred to a membrane (transfer buffer was diluted with the ratio of 10X transfer buffer: methanol: D.W = 1:2:7 and maintained in a cold state at 280 mA for 2 hours). The membrane was then blocked for 30 minutes with 5% skim milk and washed with PBS-T (phosphate-buffered saline with Tween 20) buffer. The primary antibody was diluted in 5% BSA + NaN3solution to react with the membrane for more than 12 hours, washed with PBS-T, and reacted with 5% skim milk + secondary antibody for 1 hour and 30 minutes. After washing with PBS-T, the ECL solution was reacted to the membrane, and the band was detected using an imaging device to confirm T-type calcium channel (Cav), STAT3, and p-STAT3 protein expression, and the relative expression levels are shown in Table 2 and Figure 1.

[0085] As can be seen in Table 2 and Figure 1 below, it can be seen that the levels of expression of T-type calcium channel and STAT3 targets, targeted by the compound of Formula 1 according to the present invention, were high in cell lines MDA-MB-231, MDA-MB-453, Hs578T, and BT20. Moreover, in MDA-MB-231, the expression of p-STAT3 (active form of STAT3) was also high.

[0086] Surprisingly, however, the expression of T-type calcium channel (Cav), STAT3, and p-STAT3 protein was not shown in the breast cancer cell lines T47D, AU565, MDA-MB-415, EFM-19, ZR-75-1, CAMA-1, and MCF7.

[0087] Thus, it can be seen that the compound of Formula 1 according to the present invention has a distinctive effect against triple-negative breast cancer cell lines among breast cancer cell lines.

[0088]

[0089]

[0090] As a result of Examples 1 and 2, a follow-up study was conducted with the MDA-MB-231 cell line in consideration of the level of expression of the target to confirm the anticancer effect and mechanism of the compound of Formula 1 according to the present invention.

[0091] <Example 3>Confirmation of Influence on Calcium Signal Transduction in the Cells by T-Type Calcium Channel Blockade

[0092] In this Example, the expression level of calcium-calmodulin-dependent protein kinase 2 (CaMKII), which is one of the important kinases belonging to the calcium signaling pathway, and intracellular calcium concentration were evaluated to confirm the influence of T-type calcium channel blockade by the compound of Formula 1 according to the present invention.

[0093] A triple-negative breast cancer cell (MDA-MB-231) was seeded at a density of 2x106cells in a 100mm culture dish and cultured for more than 12 hours. After removing the media, the compound of Formula 1 was treated by time (3, 6, 12 hours) at a concentration of 20 μM. Cells were then harvested to perform cell lysis using RIPA buffer and then centrifuged (4°C, 13,000 rpm, 15 min) to transfer the supernatant to a new tube. Proteins were quantified by Bradford Assay, and a sample to be loaded was prepared by boiling at 95°C to perform Western blotting. The sample was loaded into the SDS polyacrylamide gel (8% concentration), electrophoresed at 80V, then converted to 100-120V (when the loaded sample moves from the stacking gel to the running gel), and electrophoresed. The polyacrylamide gel, after electrophoresis, was transferred (transfer buffer was diluted with the ratio of 10X transfer buffer: methanol: D.W = 1:2:7 and maintained in a cold state at 280 mA for 2 hours). The membrane was then blocked for 30 minutes with 5% skim milk and washed with PBS-T (phosphate-buffered saline with Tween 20) buffer. The primary antibody was diluted in 5% BSA + NaN3solution to react with the membrane for more than 12 hours, washed with PBS-T, and reacted with 5% skim milk + secondary antibody for 1 hour and 30 minutes. After washing with PBS-T, the ECL solution was reacted to the membrane, and the band was detected using an imaging device to confirm the expression of T-type calcium channel Cav and the change in expression of phosphorylation of CaMKII by calcium. In addition, after treating the triple-negative breast cancer cell line with the compound of Formula 1, the cells were stained with fura-2 am, and the amount of calcium in the cells was measured, and then Thapsigargin 2 μM was treated to block the amount of calcium flowing from the cell ER, and the amount of calcium moving from the outside to the inside of the cell was measured using a PTI calcium analyzer and showed as A and B of Figure 2, respectively.

[0094] As can be seen in Figure 2 below, when the compound of Formula 1 according to the present invention was treated with triple-negative breast cancer (MDA-MB-231) cell line at a concentration of 20 μM by time (3, 6, 12 hours), there was no change in the expression of the T-type calcium channel, and when the compound of Formula 1 was treated for more than 3 hours, CaMKII phosphorylation was reduced, and the amount of calcium influx from the outside to the inside of the cell was reduced.

[0095] Thus, it can be seen from the present invention that the compound of Formula 1 reduces the amount of calcium in cells by blocking the T-type calcium channel, and regulates the activation of CaMKII protein thereby showing an effect against triple-negative breast cancer.

[0096] <Example 4>Confirmation of Target Protein STAT3 Dimer Formation Inhibition Rate

[0097] STAT3 is a transcription factor that phosphorylates by receiving a higher signal to form a dimer and moves to the nucleus to regulate the expression of various genes. The present Example is to confirm the STAT3 dimer formation inhibition rate by the compound of Formula 1 according to the present invention by FP assay (fluorescence polarization).

[0098] The fluorescence polarization (FP assay) is a method of measuring the polarization emitted by small fluorescence-labeled molecules during excitation / emission, wherein a substance having a high molecular weight by combination with proteins has a higher polarization, while a substance with a lower molecular weight has a lower polarization.

[0099] The recombinant STAT3 protein was added with Assay buffer and -10 mM of HEPEs (N-2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), and pH7.5 buffer was added with 50 mM of sodium chloride, 1 mM of ethylenediamine tetraacetic acid (EDTA), 2 mM of dithiothreitol (DTT), and 0.01% of Triton X100. The compound of Formula 1 according to the present invention was treated by concentration (0.01~100μM), reacted at room temperature for 1 hour, treated with a fluorescence-labeled peptide (10nM) binding to the recombinant STAT3 protein, and then reacted for 30 minutes. A millipolrization (mp) was measured by using an FP Fluorescein Dual module to derive a dimer formation inhibition rate and showed it in A, B, C, and D of Figure 3. As a comparative compound of the compound of Formula 1 according to the present invention, two kinds of STAT3 inhibiting compounds, S31-201 and Stattic were used.

[0100] As can be seen in Figure 3 below, the increase of mP value by combining the two proteins of GST-STAT3 protein and peptide can be confirmed by comparing mP values for GST-STAT3 protein and peptide single substance (Figure 3A); the binding of GST-STAT3 and peptide is inhibited at IC50of 14.2±2.6 uM when treating the compound of Formula 1 according to the present invention by concentration (0.01~100 μM) (Figure 3B); the binding is inhibited at IC50of 493.1±32.8 uM when the control group S31-201 is reacted by concentration (0.01~1000 μM) (Figure 3C); and the binding is inhibited at IC50of 739±18.6 uM when Stattic is reacted by concentration (0.01~1000 μM) (Figure 3D).

[0101] Thus, it can be seen from the present invention that the compound of Formula 1 has an effect against triple-negative breast cancer by inhibiting the formation of STAT3 dimer.

[0102] <Example 5>Confirmation of Target Protein STAT3 Activation Inhibition

[0103] STAT3 is a protein (transcription factor) that promotes the expression of multiple genes involved in the growth, proliferation, metastasis, and drug tolerance　formation of cancer cells and inhibits apoptosis. Although it has been known in a number of studies that suppressing STAT3 is expected to have strong anticancer effects, the research to develop STAT3-targeted anticancer drugs is globally insufficient.

[0104] The present Example is to confirm whether the compound of Formula 1 according to the present invention can regulate the activation of the target protein STAT3 related to the growth of the triple-negative breast cancer cell line.

[0105] A triple-negative breast cancer cell (MDA-MB-231) was seeded at a density of 2x106cells in a 100mm culture dish and cultured for more than 12 hours. After removing the media, the compound of Formula 1 was treated by conditions - by time (3, 6, 12 hours) at a concentration of 20 μM, by concentration (5, 10, 20 μM) for 24 hours, or the like. Cells were then harvested to perform cell lysis using RIPA buffer and then centrifuged (4°C, 13,000 rpm, 15 min) to transfer the supernatant to a new tube. Proteins were quantified by Bradford Assay, and a sample to be loaded was prepared by boiling at 95°C to perform Western blotting. The sample was loaded into the SDS polyacrylamide gel (8% concentration), electrophoresed at 80V, then converted to 100-120V (when the loaded sample moves from the stacking gel to the running gel), and electrophoresed. The polyacrylamide gel, after electrophoresis, was transferred (transfer buffer was diluted with the ratio of 10X transfer buffer: methanol: D.W = 1:2:7 and maintained in a cold state at 280 mA for 2 hours). The membrane was then blocked for 30 minutes with 5% skim milk and washed with PBS-T (phosphate-buffered saline with Tween 20) buffer. The primary antibody was diluted in 5% BSA + NaN3solution to react with the membrane for more than 12 hours, washed with PBS-T, and reacted with 5% skim milk + secondary antibody for 1 hour and 30 minutes. After washing with PBS-T, the ECL solution was reacted to the membrane, and the band was detected using an imaging device to show the expression of STAT3 phosphorylation (Tyrosin 705 residue) in A and B of Figure 4. The compound of Formula 1 was treated at a concentration of 20 μM in the triple-negative breast cancer cell line for 1 hour, and the expression of phosphorylated STAT3 in the cell was confirmed by a confocal microscope and shown in C of Figure 4.

[0106] As can be seen in Figure 4 below, when triple-negative breast cancer cell (MDA-MB-231) was treated with the compound of Formula 1 according to the present invention at a concentration of 20 μM for 30 minutes, the phosphorylated form of STAT3 was not shown (Figure 4A); when the compound of Formula 1 was treated by concentration and compared after 24 hours, STAT3 phosphorylation was significantly reduced when the compound of Formula 1 was treated at 20 μM (Figure 4B); when STAT3 phosphorylation in the cell was confirmed as an intracellular image 1 hour after treating the compound of Formula 1, the expression was significantly reduced (Figure 4C).

[0107] Moreover, since IL-6 cytokine is reported to activate the signal transduction by STAT3, the increase of STAT3 induced by IL-6 and the regulation of STAT3 phosphorylation by the compound of Formula 1 can be confirmed by the expression of phosphorylated STAT3, and therefore, the compound of Formula 1 was treated by concentration (5, 10, 20 μM) in the triple-negative breast cancer cell line, treated with IL-6 at a concentration of 10 ng / ml for 30 minutes, thereby confirming the phosphorylation of STAT3 by Western blotting to show it in Figure 5.

[0108] As can be seen in Figure 5 below, the increase of STAT3 phosphorylation by IL-6 is inhibited by the concentration of the compound of Formula 1 according to the present invention, and such abnormal activation of STAT3 is reduced by the inhibition of STAT3 phosphorylation when treated with the compound of Formula 1 according to the present invention.

[0109] Thus, it can be seen from the present invention that the compound of Formula 1 inhibits the growth of cells by inhibiting the STAT3 signaling mechanism when treated to the triple-negative breast cancer cell growing dependently on STAT3 activity.

[0110] As described above, the present invention shows in Examples 4 and 5 that the compound of Formula 1 inhibits STAT3 activation, specifically phosphorylation, in triple-negative breast cancer cells. By binding to the SH2 domain, the compound of Formula 1 suppresses phosphorylation, thereby reducing the activation of STAT3, which acts as a transcription factor in its dimerized form resulting from STAT3 phosphorylation. Consequently, the compound of Formula 1 can be used for the prevention and treatment of triple-negative breast cancer.

[0111] <Example 6>Evaluation of Apoptosis in Triple-Negative Breast Cancer Cell Lines

[0112] 1)Confirmation of apoptosis of cells by TUNEL fluorescent staining method

[0113] The present Example is to confirm the apoptosis of triple-negative breast cancer cells. The occurrence of apoptosis can be confirmed by observing through a microscope using the DAPI fluorescent staining method, which is an experimental method capable of observing DNA condensation, a morphological characteristic of apoptosis, and the TUNEL fluorescent staining method, which is an experimental method capable of observing DNA fragmentation. Cells that have undergone apoptosis can be confirmed through the color development of green fluorescence, and the staining method of the nucleus of the cells is simultaneously observed through blue fluorescence, thereby making it possible to confirm the apoptosis of MDA-MB-231.

[0114] In this experiment, DAPI fluorescence staining and TUNEL assay were performed to confirm the apoptosis of triple-negative breast cancer cells. Cells treated with the compound of Formula 1 according to the present invention by conditions - by concentration (5, 10, 20 μM) for 12 hours or by time (3, 6, 12 hours) at a concentration of 20 μM - were fixed by reacting with a 4% formaldehyde solution for 10 minutes, and TUNEL (Terminal Doxsynucleotidyl Transferase dUTPNick end Labeling) mixture was reacted and stained according to the manufacturer's protocol (in situ cell death detection kit, POD, Roche, Germany), and washed three times with PBS buffer. Moreover, the mixture was stained with DAPI (4',6-diamidino-2-Phenylindole) and reacted for 10 minutes, treated with mounting medium, and measured with a fluorescent microscope to confirm apoptosis, and showed in A and B of Figure 6, respectively.

[0115] As can be seen in Figure 6 below, the breast cancer cell line (MDA-MB-231), treated with the compound of Formula 1 according to the present invention of a concentration of 20 μM, has shown an increase of apoptosis (green fluorescence) over time, wherein in particular, significant apoptosis was indicated by more than 50% of apoptosis proceeded when treated for more than 6 hours at a concentration of 20 μM (Figure 6A), and the higher the concentration of the compound of Formula 1 according to the present invention in the breast cancer cell line (MDA-MB-231), the higher the green fluorescence was increased, wherein in particular, significant apoptosis was shown when treated for more than 12 hours at a concentration of 20 μM (Figure 6B).

[0116] 2)Confirmation of apoptosis of cells by comparing early apoptosis and late apoptosis

[0117] The present experiment is to confirm cell death through early apoptosis and late apoptosis assay.

[0118] The early apoptosis and late apoptosis assays are conducted using the Annexin V / Propidium Iodide (PI) double staining method. Annexin V, a calcium-dependent protein, binds readily to phosphoserine. During early apoptosis, a characteristic feature is the exposure of phosphoserine, which is normally located on the cytoplasmic side of the cell membrane, to the outer surface of the cell membrane. This binding of Annexin V to phosphoserine generates green fluorescence. In late apoptosis, due to cell membrane damage, phosphoserine on the cytoplasmic side of the membrane becomes increasingly accessible to Annexin V, resulting in enhanced binding. Additionally, as the cell membrane is compromised, PI staining also increases. Flow cytometry is then used to distinguish cell death stages based on the degree of Annexin V and PI staining.

[0119] The compound of Formula 1 according to the present invention for the MDA-MB-231 triple-negative breast cancer cell line was washed with PBS after 12 hours of treatment by concentration (5, 10, 20 μM) and mixed with an Annexin V binding buffer - a 10 mM HEPES buffer added with sodium hydroxide (NaOH), 140 mM of sodium chloride (NaCl), and 2.5 mM of calcium chloride (CaCl2), pH 7.4 -, and Annexin V and PI were added to react at room temperature in the dark for 30 minutes and analyzed with a flow cytometer within 1 hour, and shown in Figure 7.

[0120] In Figure 7, apoptosis occurred when the compound of Formula 1 according to the present invention was treated on the breast cancer cell line (MDA-MB-231) with a concentration of 5 μM, compared with the control groups, and a significant result was confirmed that the higher the concentration was, the apoptosis increased.

[0121] Thus, the compound of Formula 1 according to the present invention can increase cytotoxicity against breast cancer cells and induce cell death by apoptosis, thereby preventing and treating triple-negative breast cancer.

[0122] <Example 7>Caspase Signal Transduction Mechanism of Triple-Negative Breast Cancer Cell Apoptosis

[0123] To elucidate the signal transduction mechanism inducing apoptosis by the compound of Formula 1 according to the present invention, the activation of the caspase-dependent pathway known as the major signal transduction mechanism for the occurrence of apoptosis was confirmed. Specifically, the cleavage of caspase-3, caspase-9, and PARP proteins was observed by Western blotting. A triple-negative breast cancer cell (MDA-MB-231) was seeded at a density of 2x106cells in a 100mm culture dish and cultured for more than 12 hours. After removing the media, the compound of Formula 1 was treated by conditions - by time (3, 6, 12 hours) at a concentration of 10 μM, by concentration (5, 10, 20 μM) for 12 hours, or the like. Cells were then harvested to perform cell lysis using RIPA buffer and then centrifuged (4°C, 13,000 rpm, 15 min) to transfer the supernatant to a new tube. Proteins were quantified by Bradford Assay and a sample to be loaded was prepared by boiling at 95°C to perform Western blotting. The sample was loaded into the SDS polyacrylamide gel (8% concentration), electrophoresed at 80V, then converted to 100-120V (when the loaded sample moves from the stacking gel to the running gel), and electrophoresed. The polyacrylamide gel, after electrophoresis, was transferred (transfer buffer was diluted with the ratio of 10X transfer buffer: methanol: D.W = 1:2:7 and maintained in a cold state at 280 mA for 2 hours). The membrane was then blocked for 30 minutes with 5% skim milk and washed with PBS-T (phosphate-buffered saline with Tween 20) buffer. The primary antibody was diluted in 5% BSA + NaN3solution to react with the membrane for more than 12 hours, washed with PBS-T, and reacted with 5% skim milk + secondary antibody for 1 hour and 30 minutes. After washing with PBS-T, the ECL solution was reacted to the membrane, and the band was detected using an imaging device to show the expression of cleavages of caspase-3, caspase-9, and PARP protein and showed it in A and B of Figure 8, respectively.

[0124] As can be seen in Figure 8 below, when 20 μM of the compound of Formula 1 according to the present invention was treated on the MDA-MB-231 cell line, the cleavage form of PARP-1 increased after 3 hours of treatment, whereas caspase activation significantly increased after 6 hours for caspase-9, and 12 hours for caspase-3 (Figure 8A), and in addition, when the compound of Formula 1 according to the present invention was treated on the MDA-MB-231 cell line by concentration, the cleavage forms of PARP-1, caspase-9, and caspase-3 were confirmed at 10 uM concentration (Figure 8B).

[0125] Thus, it can be seen from the present invention that the compound of Formula 1 can induce apoptosis through the caspase activation pathway and thereby prevent and treat triple-negative breast cancer.

[0126] <Example 8>Mechanism of Mitochondria-Dependent Bcl-Family Protein

[0127] The present Example is to confirm the mechanism of the mitochondria-dependent Bcl-family protein by the compound of Formula 1 according to the present invention.

[0128] From the caspase-dependent apoptosis signaling mechanism, the expression of the Bcl-2 family (Bcl-2, Bax, Bak) protein regulating the dislocation of the membrane of mitochondria by intrinsic pathway in triple-negative breast cancer cell lines was confirmed and thereby confirmed the apoptosis signal transduction mechanism. A triple-negative breast cancer cell (MDA-MB-231) was seeded at a density of 2x106cells in a 100mm culture dish and cultured for more than 12 hours. After removing the media, the compound of Formula 1 was treated by conditions - by time (3, 6, 12 hours) at a concentration of 20 μM, by concentration (5, 10, 20 μM) for 24 hours. Cells were then harvested to perform cell lysis using RIPA buffer and then centrifuged (4°C, 13,000 rpm, 15 min) to transfer the supernatant to a new tube. Proteins were quantified by Bradford Assay, and a sample to be loaded was prepared by boiling at 95°C to perform Western blotting. The sample was loaded into the SDS polyacrylamide gel (8% concentration), electrophoresed at 80V, then converted to 100-120V (when the loaded sample moves from the stacking gel to the running gel), and electrophoresed. The polyacrylamide gel, after electrophoresis, was transferred (transfer buffer was diluted with the ratio of 10X transfer buffer: methanol: D.W = 1:2:7 and maintained in a cold state at 280 mA for 2 hours). The membrane was then blocked for 30 minutes with 5% skim milk and washed with PBS-T (phosphate-buffered saline with Tween 20) buffer. The primary antibody was diluted in 5% BSA + NaN3solution to react with the membrane for more than 12 hours, washed with PBS-T, and reacted with 5% skim milk + secondary antibody for 1 hour and 30 minutes. After washing with PBS-T, the ECL solution was reacted to the membrane, and the band was detected using an imaging device to confirm the change in the expression of the Bcl-2 family (Bcl-2, Bax, Bak) protein and showed it in A and B of Figure 9, respectively.

[0129] As can be seen in Figure 9 below, the expression of Bcl-2 protein decreased as the time for treating the compound of Formula 1 according to the present invention on the triple-negative breast cancer cell (MDA-MB-231) at a concentration of 20 μM increased, whereas the expression of Bax / Bak increased at 12 hours of the treatment of the compound of Formula 1 (Figure 9A), and the expression of Bcl-2 protein decreased and the expression of Bax / Bak increased as the concentration of the compound of Formula 1 increased (Figure 9B).

[0130] Thus, it can be seen from the present invention that the compound of Formula 1 can regulate the expression of the Bcl-2 family protein so that the expression of Bcl-2 inhibiting the apoptosis decreases, the expression of Bax promoting the apoptosis increases, that is, the expression of Bax over Bcl-2 increases, and thereby induce the apoptosis of breast cancer cells to prevent and treat triple-negative breast cancer.

[0131] <Example 9>Inhibition of Tumoral Growth in Xenograft Mouse Models

[0132] The present Example confirmed the anticancer effect of the compound of Formula 1 according to the present invention against the triple-negative breast cancer cells in animal models to confirm the anticancer effect against the triple-negative breast cancer.

[0133] Tumor-derived animal models (MDA-MB-231 derived xenograft mouse models) were prepared by subcutaneously injecting triple-negative breast cancer cell lines MDA-MB-231 at a density of 1.5 x 107cells / mouse into BALB / c / nu / nu old nude mice to transplant the cell line and induce tumor formation. After 12 days, the models were divided into five groups with the compound of Formula 1 according to the present invention at concentrations of 15 mg / kg, 30 mg / kg, and 60 mg / kg (oral administration, 5 times / week, 66 days), a control group, and a paclitaxel 10 mg / kg (intraperitoneal administration, 2 times / week, 66 days) administration control group. The drugs were then administered accordingly. The weight and tumor size of the models were measured twice a week from the start date of administration of the test substance to the date of autopsy. The tumor size was determined by measuring the long and short axes of the tumor using calipers, and calculated with the formula: tumor size = ab2 / 2 (a: long axis length, b: short axis length). The results are shown in A, B, C, and D of Figure 10.

[0134] As can be seen in A, B, and C of Figure 10 below, it was confirmed that administration of the compound of Formula 1 in MDA-MB-231-derived tumors reduced tumoral growth in the groups treated with the compound of Formula 1 according to the present invention and in the group treated with paclitaxel. In contrast, the control group exhibited a significant increase in tumor size after 40 days. Notably, administration of the compound of Formula 1 at a low concentration of 15 mg / kg reduced tumor growth by approximately 50% compared to the control group. At a concentration of 30 mg / kg, the results were comparable to those of the group treated with paclitaxel, a previously established anticancer agent. Furthermore, administration at 60 mg / kg effectively inhibited tumor growth.

[0135] Moreover, as can be seen in D of Figure 10 below, it is confirmed that the compound of Formula 1 according to the present invention inhibits tumoral growth but does not cause a change such as weight loss in mice.

[0136] Thus, the compound of Formula 1 according to the present invention not only can inhibit tumoral growth by inducing apoptosis of triple-negative breast cancer tumoral tissue but also can reduce tumor growth without affecting body weight.

[0137] <Example 10>Tumoral Growth Inhibition Mechanism in Xenograft Mouse Models

[0138] In order to confirm tumoral growth inhibition mechanism of the compound of Formula 1 according to the present invention against the triple-negative breast cancer cell lines, tumoral tissue was obtained from xenograft mice induced to have triple-negative breast cancer cell line MDA-MB-231, added with lysis buffer (20 mM Tris-HCl pH 7.4, 1% Trion X-100, 1mM DTT, 1mM EDTA, 1mM EGTA, 1mM PMSF, 150mM NaCl), and then centrifuged (4°C, 15,000 rpm, 10 min) to transfer the supernatant to a new tube. Proteins were quantified by Bradford Assay, and a sample to be loaded was prepared by boiling at 95°C to perform Western blotting. The sample was loaded into the SDS polyacrylamide gel (8% concentration), electrophoresed at 80V, then converted to 100-120V (when the loaded sample moves from the stacking gel to the running gel), and electrophoresed. The polyacrylamide gel, after electrophoresis, was transferred (transfer buffer was diluted with the ratio of 10X transfer buffer: methanol: D.W = 1:2:7 and maintained in a cold state at 280 mA for 2 hours). The membrane was then blocked for 30 minutes with 5% skim milk and washed with PBS-T (phosphate-buffered saline with Tween 20) buffer. The primary antibody was diluted in 5% BSA + NaN3solution to react with the membrane for more than 12 hours, washed with PBS-T, and reacted with 5% skim milk + secondary antibody for 1 hour and 30 minutes. After washing with PBS-T, the ECL solution was reacted to the membrane, and the band was detected using an imaging device to confirm the change in the expression of p-STAT3, survivin, Bcl-2, and cleaved PARP-1 which are markers related to the tumor production and apoptosis mechanisms. Moreover, tumor tissue obtained from xenograft mice induced to have triple-negative breast cancer cell line MDA-MB-231, were fixed with 10% formalin and embedded in paraffin blocks. The sections were incubated with a primary antibody for p-STAT3 and PCNA at 4°C for over 12 hours, followed by washing with PBS (phosphate-buffered saline). They were then incubated with a secondary antibody at room temperature for 2 hours, and washed again with PBS. Staining is performed using DAB (3,3' diaminobenzidine) to confirm by an optical microscope. The results are shown in Figures 11 and 12, respectively.

[0139] In Figure 11, in tumor tissue obtained from xenograft mice induced to have triple-negative breast cancer cell line MDA-MB-231, proteins related to cell growth and apoptosis were confirmed, and as a result, it is confirmed that, when the compound of Formula 1 according to the present invention was administered at concentrations of 15 mg / kg, 30 mg / kg, and 60 mg / kg, the phosphorylation of STAT3 was significantly reduced, and when administered at 30 mg / kg, 60 mg / kg, the expression of survivin and Bcl-2 is reduced, but PAPR-1 activation involved in apoptosis is increased.

[0140] In Figure 12, the expression of the target protein was confirmed in the paraffin blocks prepared from tumor tissue obtained from MDA-MB-231-induced xenograft mice, wherein the triple-negative breast cancer cell line, MDA-MB-231 was treated with the compound of Formula 1 according to the present invention. Moreover, it is also confirmed that the expression of p-STAT3, which is well expressed in the control group, was significantly reduced when the compound of Formula 1 according to the present invention was treated. Furthermore, when the expression of PCNA (proliferating cell nuclear antigen), used as an important marker for cell division and cancer occurrence, was confirmed, the expression was significantly reduced in tissues treated with the compound of Formula 1 according to the present invention compared to the control group.

[0141] Therefore, it can be seen that the compound of Formula 1 according to the present invention inhibits the activation of p-STAT3 involved in tumoral growth and induces apoptosis in the tumoral tissues derived from triple-negative breast cancer through a typical apoptosis mechanism as a result of inhibiting the expression of PARP activation, Bcl-2, and survivin, used as markers of apoptosis mechanism.

[0142] Thus, in the present invention, the compound of Formula 1 inhibits the calcium influx into the cells through the T-type calcium channel in triple-negative breast cancer cells, suppresses the STAT3 dimerization by binding to the STAT3 SH2 domain, and regulates phosphorylation, regulates the expression of the regulatory proteins of the typically mitochondrial-dependent Bcl-2 family to kill them through apoptosis mechanism by caspase activation, inhibits tumor growth in mouse models induced to have triple-negative breast cancer, and has anti-tumor activity through apoptosis mechanism in mouse tumor tissues, thereby is capable to be developed as an anticancer treatment for triple-negative breast cancer.

Claims

1.A pharmaceutical composition for treating and preventing triple-negative breast cancer comprising a compound of the following Formula 1 or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, prodrug, or polymorphic isomer thereof as an active ingredient, and comprising a pharmaceutically acceptable carrier.[Formula 1]2.The pharmaceutical composition of claim 1, wherein the triple-negative breast cancer is a tumor of MDA-MB-231, MDA-MB-453, Hs 578T, or BT20 cell lines.3.The pharmaceutical composition of claim 2, wherein the triple-negative breast cancer is a tumor of MDA-MB-231 cell line.4.The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated in parenteral, percutaneous, topical, mucosal, nasal, palatal mucosal, sublingual, or oral administration form.5.The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is formulated in oral administration form.6.The pharmaceutical composition of claim 5, wherein the oral administration form is in the form of a tablet or a capsule.7.The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is prepared to have a daily dosage of 0.01mg / kg to 100mg / kg of the compound of Formula 1.8.A method for treating or preventing triple-negative breast cancer in a subject in need thereof, comprising a step of administering a therapeutically effective amount of the compound of Formula 1 of claim 1 or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, prodrug, or polymorphic isomer thereof to the subject.9.The method of claim 8, wherein the subject is triple-negative breast cancer of MDA-MB-231, MDA-MB-453, Hs 578T, or BT20 cell line tumors.10.The method of claim 8, wherein the method for treating or preventing triple-negative breast cancer in a subject in need thereof is characterized by a dual-target anticancer effect on the triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-453, Hs 578T, and BT20.11.The method of claim 10, wherein the double-target refers to simultaneously having a mechanism blocking overexpressed calcium essential for cancer cell growth and a mechanism inhibiting the target protein STAT3, in triple-negative breast cancer cells.12.The method of any one of claims 8 to 11, wherein the method for treating or preventing triple-negative breast cancer in a subject in need thereof additionally includes an apoptosis mechanism by caspase activation by regulating the expression of the regulatory protein of the Bcl-2 family.

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