Pharmaceutical composition for treating and preventing ductal adenocarcinoma

Abstract

The present invention relates to a pharmaceutical composition for treating and preventing pancreatic ductal adenocarcinoma, 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. The compound of Formula 1 of the present invention simultaneously has a mechanism blocking calcium overexpressed in pancreatic ductal adenocarcinoma 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 pancreatic ductal adenocarcinoma and shows an anticancer activity through apoptosis mechanism in the tumoral tissues of mice.

Description

PHARMACEUTICAL COMPOSITION FOR TREATING AND PREVENTING DUCTAL ADENOCARCINOMA

[0001] The present invention relates to the pharmaceutical use of a compound of the following Formula 1, disclosed in WO 2014 / 021591 A2 and WO 2015 / 178608 A2, for treating and preventing pancreatic ductal adenocarcinoma as a type of pancreatic cancer.

[0002] [Formula 1]

[0003]

[0004] The prior art WO 2008 / 136631 A1 discloses 3,4-dihydroquinazoline derivative of the following Formula A, and also describes that the compound of the following Formula A 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.

[0005] [Formula A]

[0006]

[0007] WO 2014 / 021591 A2 discloses the hydroquinazoline derivative of the following Formula B, which comprises the compound of Formula 1 used in the present invention. It discloses that the compound of Formula B as a T-type calcium channel antagonist is capable of inhibiting the flow of calcium through the calcium channels and the growth of various cancer cells, and thereby, the compound of Formula B 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.

[0008] [Formula B]

[0009]

[0010] Moreover, WO 2015 / 178608 A2 discloses 3,4-dihydroquinazoline derivative of the following Formula C, which comprises the compound of Formula 1 used in the present invention. It discloses that the compound of 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 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.

[0011] [Formula C]

[0012]

[0013] The pancreatic ductal adenocarcinoma, as a subject-matter of the pharmaceutical use of the present invention, is a type of pancreatic cancer as an adenocarcinoma.

[0014] Adenocarcinoma refers to a tumor developed in epithelial tissue, including glandular tissue, and may be developed in various parts of organs in the body including epithelial tissue. The adenocarcinoma mainly develops in colon, lung, prostate, ureters, vagina and esophagus, breasts, stomach, and pancreas. Especially, tumors developed in the pancreas vary in type, from benign tumors that can be treated with surgical resection to malignant tumors, namely, cancer, with a very poor prognosis (prospect of the future state of the disease, or the course of the disease after treatment).

[0015] Pancreatic cancer is one of world-famous cancers and is notorious as one of the worst cancer along with glioblastoma and anaplastic thyroid cancer. As with other cancers, by the time symptoms are recognized, pancreatic cancer is often in stage 3 or 4 when it metastasizes to other parts of the body and thus greatly reduces the survival rate; however, pancreatic cancer has an extremely low survival rate even among the cancers. Such an extremely low survival rate is because the pancreas is located so deep in the body, which makes it common to think that when symptoms appear, there is a problem with something other than the pancreas. Moreover, since the pancreas is located dorsally, it is difficult to detect early as the lump is rarely palpable, and if the cancer can be detected early and treated with local excision, it can be truly called a blessing. Given such a situation, ironically, pancreatic cancer has a good prognosis when developed in a location that causes symptoms quickly. For example, when cancer develops in the head of the pancreas, the bile duct is easily blocked, easily resulting in jaundice or pain. Thus, pancreatic head cancer has a better prognosis compared to pancreatic tail cancer, which causes the least symptoms. On the other hand, when cancer develops in the tail of the pancreas, it is detected the latest, as symptoms appear only after the cancer has grown. In addition, there are cases where only the stomach hurts, and due to the nature of the pancreas located dorsally, it is less painful when the body is bent. If the pain is severe, it is likely too late as the tumor already has grown very large or has invaded the surrounding nerves. As some specific symptoms, migratory thrombophlebitis may occur in about 10% of patients with pancreatic cancer, and palpable gallbladder (Courvoisier sign) may be found in patients with cancer located in the head of the pancreas. In addition, nonspecific symptoms such as weight loss, loss of appetite, and indigestion, which occur in most cancers, may appear. Recently, as CT scans are increasingly used, the number of cases where early-stage pancreatic cancer is discovered accidentally is also increasing. However, it is not recommended to receive regular CT scans to prevent pancreatic cancer because CT scans for pancreatic cancer require high resolution, which results in high radiation exposure. Hence, many pancreatic cancers that have recently been discovered by CT scans are often discovered accidentally when CT scans are performed for other reasons.

[0016] Cystic tumors, namely cysts, are the most common type of pancreatic cancer, and there are several types of them. Most cystic tumors are benign, but occasionally, some of them are malignant from the beginning or were benign at the time of diagnosis but later turned malignant. Cystic tumors include tumors such as serous and mucinous cystic neoplasm, intraductal papillary mucinous neoplasm, solid pseudopapillary tumors, lymphoepithelial cysts, and cystic teratomas. Moreover, malignant tumors include exocrine pancreatic tumors such as pancreatic ductal adenocarcinoma, acinar cell carcinoma, and neuroendocrine tumors.

[0017] Pancreatic cystic neoplasms account for approximately 1% of pancreatic tumors, but their incidence has been increasing recently. Moreover, pancreatic cystic neoplasms do not show symptoms, and even when they do, their symptoms are nonspecific and, therefore, often discovered incidentally during X-ray examinations for other diseases. Serous cystadenomas are a type of pancreatic cystic tumors, which are more common in women and have a very low risk of developing into cancer. Mucinous cystic neoplasms, which are common in women, develop mostly in the tail of the pancreas and have a tendency to become malignant, which makes it important to have an accurate diagnosis. Intraductal papillary mucinous neoplasms (IPMN) have a variety of findings from benign to malignant, and even when malignant, the prognosis is often better than that of general pancreatic ductal adenocarcinoma. Solid pseudopapillary tumors, accounting for less than 5% of pancreatic cystic tumors, develop mostly in young women, and since the possibility of their malignancy cannot be ruled out, surgical treatment is considered a priority. Among malignant pancreatic tumors, pancreatic ductal adenocarcinoma refers to adenocarcinoma that develops in the pancreatic duct that transports pancreatic juice, and in terms of incidence, when there is a death from pancreatic cancer, the cause is usually this subtype. Though the exact cause of pancreatic ductal adenocarcinoma is yet unknown, compared to colon, stomach, bile duct, and the like, where cancer usually develops from adenocarcinoma, it has more cases where the differentiation is bad than good, and since the surrounding structure is complex, even if it develops only a little, it can invade the surrounding structures, resulting in poor prognosis even given that 'they are hard to detect.' If found only by metastasis, pancreatic ductal adenocarcinoma is often not distinguished from adenocarcinoma originating from other organs, and particularly, it is near impossible to distinguish it from bile duct cancer, gastric cancer (long type), and the like without clinical information. Pancreatic adenosquamous carcinoma is a malignant tumor that develops in the exocrine pancreas, accounting for 1-2% of pancreatic malignancies, and histologically shows a mixture of glandular structures and clear squamous cell carcinoma forms. Acinar cell carcinoma is a rare tumor, accounting for less than 1-2% of exocrine pancreatic tumors. It develops often in middle-aged and elderly men, shows nonspecific clinical symptoms such as weight loss, abdominal pain, nausea, vomiting, and diarrhea, and progresses relatively quickly and has a poor prognosis due to distant metastasis. Neuroendocrine tumors (NETs) are not considered ordinary pancreatic cancers, and they are slow-growing tumors that develop from neuroendocrine cells. NETs develop often in the intestines, but can also develop in the stomach, lungs, pancreas, and other parts of the body. Although NETs are categorized as malignant, there are many cases having a very good prognosis, resulting in a much better survival rate than that of pancreatic ductal adenocarcinoma. Since there are many types of cells, the types of tumors are also diverse, and because they originate from endocrine cells, they may show systemic endocrine symptoms. For example, in the case of a gastrinoma secreting gastrin, it may show symptoms where excessive secretion of gastric acid occurs; and in the case of an insulinoma secreting insulin, symptoms of excessive blood sugar drop may appear. However, when observing actual patients, there are many tumors that do not cause symptoms.

[0018] The present inventors, while analyzing the potency of the compound of Formula 1, disclosed in WO 2014 / 021591 A2 and WO 2015 / 178608 A2, against various cancers, discovered that the compound has excellent cell viability (IC50) against pancreatic ductal adenocarcinoma cell lines, Mia PaCa-2, PANC-1, PSN-1, YAPC and HPAF-II cell lines, among pancreatic cancers, and conducted a further study to find out that the compound of Formula 1 of the present invention is capable of treating and preventing pancreatic ductal adenocarcinoma by double-target anticancer effect. 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 cell growth in pancreatic ductal adenocarcinoma cells and a mechanism inhibiting the target protein STAT3 (Signal Transducer and Activator of Transcription 3) in pancreatic ductal adenocarcinoma cells, thereby completed the present invention.

[0019] An object of the present invention is to provide a pharmaceutical composition capable of treating and preventing pancreatic ductal adenocarcinoma as a type of pancreatic cancers.

[0020] Another object of the present invention is to provide a pharmaceutical use of a compound of Formula 1 capable of treating and preventing pancreatic ductal adenocarcinoma as a double-target anticancer agent.

[0021] To achieve the above objectives, the present invention provides a pharmaceutical composition for treating and preventing pancreatic ductal adenocarcinoma, comprising a compound of Formula 1.

[0022] [Formula 1]

[0023]

[0024] 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.

[0025] First, certain terms are defined below for a better understanding of the present invention.

[0026] 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.

[0027] The term 'preventing' used in the present invention refers to treating subjects at risk of pancreatic ductal adenocarcinoma 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).

[0028] 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., breast 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.

[0029] 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 pancreatic ductal adenocarcinoma when the compound of Formula 1 according to the present invention is administered to the subject.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In addition, the present invention provides a pharmaceutical use of the compound of Formula 1 capable of treating and preventing pancreatic ductal adenocarcinoma as a double-target anticancer agent.

[0036] According to the present invention, the subjects being administered with the compound of Formula 1 are Mia PaCa-2, PANC-1, PSN-1, YAPC, and HPAF-II cell line tumors as the cell lines for pancreatic ductal adenocarcinoma.

[0037] 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 pancreatic ductal adenocarcinoma.

[0038] The present invention found the method of treating or preventing pancreatic ductal adenocarcinoma 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 pancreatic ductal adenocarcinoma 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 SH2domain.

[0039] 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 pancreatic ductal adenocarcinoma through the apoptosis mechanism by the caspase activation.

[0040] Another Example of the present invention confirmed that the compound of Formula 1 inhibits the growth of tumors in the pancreatic ductal adenocarcinoma-induced mouse model and induces cell growth inhibition and apoptosis from the tumoral tissue obtained from the mouse model and thereby showed a pharmaceutical use showing an anti-cancer effect.

[0041] 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 pancreatic ductal adenocarcinoma.

[0042] 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.

[0043] 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 pancreatic ductal adenocarcinoma. 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] Similarly, the parenteral administration form may comprise more than one active ingredient less than the oral administration form for treating pancreatic ductal adenocarcinoma. 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).

[0051] 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.

[0052] 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, disintegrants, and lubricants.

[0053] 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, diluent typically exists in about 50% to 90% 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 diluent is a combination of sodium carboxymethyl cellulose and the marketed microcrystalline cellulose such as AVICELRC-581.

[0054] The compound of Formula 1 of the present invention has a double-target anticancer effect against pancreatic ductal adenocarcinoma. Namely, the present invention found that the compound of Formula 1 blocks T-type calcium channels to the pancreatic ductal adenocarcinoma cell to inhibit the calcium influx into the cell and regulates the formation of STAT3 dimer and phosphorylation through the binding with STAT3 SH2domain, 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.

[0055] Moreover, the present invention shows that the compound of Formula 1 inhibits the growth of tumors in the pancreatic ductal adenocarcinoma-induced xenograft mouse model and shows antitumoural activity through the apoptosis mechanism in the tumoral tissue of the mouse.

[0056] Therefore, the present invention provides a pharmaceutical composition for treating and preventing pancreatic ductal adenocarcinoma comprising a compound of Formula 1, and pharmaceutical use for treating and preventing pancreatic ductal adenocarcinoma as a double-target anticancer agent.

[0057] 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 pancreatic cancer including pancreatic ductal adenocarcinoma.

[0058] 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 pancreatic ductal adenocarcinoma cell lines Mia paca-2 and Panc-1, 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.

[0059] 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.

[0060] Figure 4 confirms the phosphorylation of STAT3 by the treatment of the compound of Formula 1 in accordance with the present invention in the pancreatic ductal adenocarcinoma cell lines, wherein A shows the result of Western blotting by treating 20μM of compound of Formula 1 by time (Mia paca-2; 3, 6, 12 hours / Panc-1; 8, 24, 48 hours) or by treating compound of Formula 1 by concentration (Mia paca-2, Panc-1; 5, 10, 15, 20μM), wherein B shows the confocal microscope photomicrograph of the result of treating compound of Formula 1 by concentration (Mia paca-2; 10, 20μM / Panc-1; 5, 10, 20μM).

[0061] 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 pancreatic ductal adenocarcinoma cell lines.

[0062] Figure 6 confirms the induction of apoptosis in the pancreatic ductal adenocarcinoma 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 24 hours after the treatment by concentration.

[0063] Figure 7 shows the effect of apoptosis in the pancreatic ductal adenocarcinoma cell lines by the treatment of the compound of Formula 1 in accordance with the present invention using early apoptosis and late apoptosis assay.

[0064] 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 pancreatic ductal adenocarcinoma cell lines, wherein A shows the result of confirmation under time and concentration conditions in Mia paca-2 cell line, and wherein B shows the result of confirmation under time and concentration conditions in Panc-1 cell line.

[0065] 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 pancreatic ductal adenocarcinoma cell lines, wherein A shows the result of confirmation under time and concentration conditions in Mia paca-2 cell line, and wherein B shows the result of confirmation under time and concentration conditions in Panc-1 cell line.

[0066] Figure 10 shows the tumor growth inhibition effect of the compound of Formula 1 in accordance with the present invention against pancreatic ductal adenocarcinoma in a xenograft mouse model using the pancreatic ductal adenocarcinoma cell lines Mia paca-2 and Panc-1, wherein A, B, and C respectively show the tumor size, tumor weight, and changes in body weight at different concentrations for Mia paca-2, and wherein D, E, and F respectively show the tumor size, tumor weight, and changes in body weight at different concentrations for Panc-1.

[0067] Figure 11 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 induced by the pancreatic ductal adenocarcinoma cell line Mia paca-2 through immunohistochemistry.

[0068] 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 pancreatic ductal adenocarcinoma cell line Panc-1 through immunohistochemistry.

[0069] 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.

[0070] <Example 1>Screening Evaluation for Various Cancer Cells

[0071] Cell viability assay has been performed for the 34 types of the 6 carcinoma cell lines including pancreatic 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 of the present invention 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.

[0072] As can be seen in Table 1 below, according to the results of cell viability (IC50) assay in 34 types of cell lines of 6 carcinomas, including pancreatic carcinoma, the pancreatic carcinoma cell lines have an excellent cytotoxic effect in pancreatic cancer cell lines overall, and in particular, the pancreatic ductal adenocarcinoma cell lines Mia paca-2, Panc-1, PSN-1, YAPC, and HPAF-II showed difference in efficacy of at least 2 to up to 10 times or more compared to other carcinoma cell lines, showing that they can have an excellent cytotoxic effect.

[0073] Thus, it can be seen that the compound of Formula 1 of the present invention has a distinctive effect against pancreatic ductal adenocarcinoma among pancreatic cancer types than other cancer types.

[0074]

[0075] <Example 2>Evaluation of Target Expression in Pancreatic Ductal Adenocarcinoma Cell Lines

[0076] 8 types of pancreatic ductal adenocarcinoma cells 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 1x106cells 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.

[0077]

[0078] As can be seen in Table 2 above and Figure 1, 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 pancreatic ductal adenocarcinoma cell lines Mia paca-2 and Panc-1, whereas the expression of T-type calcium channel (Cav), STAT3, and p-STAT3 protein was relatively significantly low in other pancreatic ductal adenocarcinoma cell lines such as CFPAC-1, SU.86.86, and Capan-1.

[0079] Thus, it can be seen that the compound of Formula 1 according to the present invention has a distinctive effect against pancreatic cancer cell lines showing high levels of T-type calcium channel and STAT3 expression in the pancreatic ductal adenocarcinoma cell lines Mia paca-2 and Panc-1.

[0080] Examples below show the results of a follow-up study conducted with the pancreatic ductal adenocarcinoma cell lines Mia paca-2 and Panc-1 in consideration of the comprehensive results of Examples 1 and 2 to confirm the anticancer effect and mechanism of the compound of Formula 1 according to the present invention against pancreatic ductal adenocarcinoma.

[0081] <Example 3>Confirmation of Influence on Calcium Signal Transduction in the Cells by T-Type Calcium Channel Blockade

[0082] 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.

[0083] Pancreatic ductal adenocarcinoma cells (Mia paca-2, Panc-1) were 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 (Mia paca-2; 3, 6, 12 hours / Panc-1; 8, 24, 48 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 pancreatic ductal adenocarcinoma cell lines (Mia paca-2, Panc-1) with the compound of Formula 1, the cells were stained with fura-2 am, and the amount of calcium in the cells was measured. After treating with the compound of Formula 1, staining the cells with fura-2 am, and measuring the amount of calcium in the cells, 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.

[0084] As can be seen in Figure 2 below, when the compound of Formula 1 according to the present invention was treated with pancreatic ductal adenocarcinoma cell lines (Mia paca-2, Panc-1) at a concentration of 20 μM by time (Mia paca-2; 3, 6, 12 hours / Panc-1; 8, 24, 48 hours), when the compound of Formula 1 was treated for more than 6 hours in Mia paca-2 cell lines and for 48 hours in Panc-1 cell lines, CaMKII phosphorylation was reduced, and the amount of calcium influx from the outside to the inside of the cell was reduced.

[0085] 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 pancreatic ductal adenocarcinoma.

[0086] <Example 4>Confirmation of Target Protein STAT3 Dimer Formation Inhibition Rate

[0087] 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).

[0088] 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.

[0089] 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 millipolarization (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.

[0090] 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).

[0091] Thus, it can be seen from the present invention that the compound of Formula 1 has an effect against pancreatic ductal adenocarcinoma by inhibiting the formation of STAT3 dimer.

[0092] <Example 5>Confirmation of Target Protein STAT3 Activation Inhibition

[0093] 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.

[0094] 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.

[0095] Pancreatic ductal adenocarcinoma cells (Mia paca-2, Panc-1) were seeded at a density of 2x106cells in a 100mm culture dish and cultured for 12-16 hours. After removing the media, the compound of Formula 1 was treated by conditions - by time (Mia paca-2; 3, 6, 12 hours, Panc-1; 8, 24, 48 hours) at a concentration of 20 μM, by concentration (Mia paca-2, Panc-1; 5, 10, 15, 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 of Figure 4. The compound of Formula 1 was treated by concentration (Mia paca-2; 10, 20 μM / Panc-1; 5, 10, 20 μM) in the pancreatic ductal adenocarcinoma cell lines (Mia paca-2, Panc-1) for 24 hours, and the expression of phosphorylated STAT3 in the cell was confirmed by a confocal microscope and shown in B of Figure 4.

[0096] As can be seen in Figure 4, when pancreatic ductal adenocarcinoma cell lines (Mia paca-2, Panc-1) were treated with the compound of Formula 1 according to the present invention at a concentration of 20 μM, the phosphorylated form of STAT3 decreased after 3 hours in the Mia paca-2 cell line and after 8 hours in the Panc-1 cell line, and when comparing the results after 24 hours of treatment with different concentrations of the compound of Formula 1, STAT3 phosphorylation was significantly decreased from treatment with the compound of Formula 1 at a concentration of 15 μM (Figure 4A). Moreover, when STAT3 phosphorylation in the cell was confirmed as an intracellular image, the expression was significantly reduced from treatment with the compound of Formula 1 at a concentration of 10 μM (Figure 4B).

[0097] 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 condition (time: 1h, concentration: 20 μM) in the pancreatic ductal adenocarcinoma cell lines (Mia paca-2, Panc-1), treated with IL-6 at a concentration of 10 ng / ml for 1 hour, thereby confirming the phosphorylation of STAT3 by Western blotting to show it in Figure 5.

[0098] 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.

[0099] 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 pancreatic ductal adenocarcinoma cell growing dependently on STAT3 activity.

[0100] As described above, the present invention shows in Figures 4 and 5 that the compound of Formula 1 inhibits STAT3 activation, specifically phosphorylation, in pancreatic ductal adenocarcinoma cells. By binding to the SH2domain, 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 pancreatic ductal adenocarcinoma.

[0101] <Example 6>Evaluation of Apoptosis in Pancreatic Ductal Adenocarcinoma Cell Lines

[0102] 1)Confirmation of apoptosis of cells by TUNEL fluorescent staining method

[0103] The present Example is to confirm the apoptosis of pancreatic ductal adenocarcinoma 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 Mia paca-2 and Panc-1.

[0104] In this experiment, DAPI fluorescence staining and TUNEL assay were performed to confirm the apoptosis of pancreatic ductal adenocarcinoma cells Mia paca-2 and Panc-1. Cells treated with the compound of Formula 1 according to the present invention by conditions - Mia paca-2; by time (3, 6, 12 hours for 10 μM of compound of Formula 1), by concentration (5, 10, 20 μM of the compound of Formula 1 for 24 hours) / Panc-1; by time (12, 24 hours for 10 μM of compound of Formula 1), by concentration (10, 20 μM of the compound of Formula 1 for 24 hours) - 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.

[0105] As can be seen in Figure 6 below, the pancreatic ductal adenocarcinoma cell line, treated with the compound of Formula 1 according to the present invention of a concentration of 10 μM, has shown an increase of apoptosis (green fluorescence) over time, and significant apoptosis was indicated after 12 hours of treatment. In case of Mia paca-2, more than 50% of apoptosis was proceeded when treated for more than 12 hours at a concentration of 10 μM, particularly (Figure 6A). Moreover, a significant apoptosis (green fluorescence) was indicated at a concentration over 10 μM from the Mia paca-2 cell line and at a concentration of 20 μM from the Panc-1 cell line, when each cell lines were treated with the compound of Formula 1 for 24 hours (Figure 6B).

[0106] 2)Confirmation of apoptosis of cells by comparing early apoptosis and late apoptosis

[0107] The present experiment is to confirm cell death through early apoptosis and late apoptosis assay.

[0108] 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.

[0109] The compound of Formula 1 according to the present invention for the pancreatic ductal adenocarcinoma cell lines (Mia paca-2, Panc-1) was washed with PBS after 12 or 36 hours of treatment by concentration (Mia paca-2; 5, 10, 20 μM / Panc-1; 5, 10 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 A and B of Figure 7.

[0110] In Figure 7, when the compound of Formula 1 according to the present invention was treated on the pancreatic ductal adenocarcinoma cell line by concentration, apoptosis was induced. Moreover, on pancreatic ductal adenocarcinoma cell line Mia paca-2, compared with the control groups, apoptosis occurred when treated with compound of Formula 1 over concentration of 10 μM, and a significant result was confirmed that the higher the concentration was, the apoptosis increased (Figure 7A). Furthermore, on cell line Panc-1, compared with the control groups, apoptosis occurred when treated with compound of Formula 1 over concentration of 5 μM, and a significant result that the higher the concentration was, the apoptosis increased was also confirmed (Figure 7B).

[0111] Thus, the compound of Formula 1 according to the present invention can increase cytotoxicity against pancreatic ductal adenocarcinoma cells and induce cell death by apoptosis, thereby preventing and treating pancreatic ductal adenocarcinoma.

[0112] <Example 7>Caspase Signal Transduction Mechanism of Pancreatic Ductal Adenocarcinoma Cell Apoptosis

[0113] 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.

[0114] Pancreatic ductal adenocarcinoma cells (Mia paca-2, Panc-1) were seeded at a density of 2x106cells in a 100mm culture dish and cultured for more than 12-16 hours. After removing the media, the compound of Formula 1 was treated by conditions - by time (Mia paca-2; 3, 6, 12 hours, Panc-1; 8, 24, 48 hours) at a concentration of 20 μM, by concentration (Mia paca-2; 5, 10, 15, 20 μM / Panc-1; 5, 10, 15, 20 μM) for 24 or 48 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.

[0115] 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 pancreatic ductal adenocarcinoma cell line Mia paca-2, the cleavage form of PARP-1 increased after 12 hours of treatment, whereas caspase activation significantly increased, and when the compound of Formula 1 was treated by concentration for 24 hours, the cleavages of PARP-1 and caspase-3 were confirmed at concentration over 10 uM (Figure 8A). In addition, when 20 μM of the compound of Formula 1 was treated on the pancreatic ductal adenocarcinoma cell line Panc-1, the cleavages of PARP-1 and caspase-3 significantly increased after 48 hours of treatment, whereas when the compound of Formula 1 was treated by concentration for 48 hours, the cleavages of PARP-1 and caspase-3 significantly increased at concentration of 20 uM (Figure 8B).

[0116] 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 pancreatic ductal adenocarcinoma.

[0117] <Example 8>Mechanism of Mitochondria-Dependent Bcl-Family Protein

[0118] 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 on pancreatic ductal adenocarcinoma cell lines.

[0119] 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 pancreatic ductal adenocarcinoma cell lines was confirmed and thereby confirmed the apoptosis signal transduction mechanism. Pancreatic ductal adenocarcinoma cells (Mia paca-2, Panc-1) were seeded at a density of 2x106cells in a 100mm culture dish and cultured for more than 12-16 hours. After removing the media, the compound of Formula 1 was treated by conditions - by time (Mia paca-2; 3, 6, 12 hours, Panc-1; 8, 24, 48 hours) at a concentration of 20 μM, by concentration (Mia paca-2; 5, 10, 15, 20 μM / Panc-1; 5, 10, 15, 20 μM) for 24 or 48 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 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.

[0120] As can be seen in Figure 9 below, when the compound of Formula 1 was treated on the pancreatic ductal adenocarcinoma cell line Mia paca-2 at a concentration of 20 μM, the expression of Bak and Bax proteins increased at 12 hours of the treatment, as the time increased. When the compound of Formula 1 was treated at different concentration for 24 hours, the expression of Bak and Bax proteins increased at a concentration over 15 μM, whereas the expression of Bcl-2 protein decreased at a concentration of 20 μM (Figure 9A). Furthermore, as the time for treating the compound of Formula 1 on the pancreatic ductal adenocarcinoma cell line Panc-1 at a concentration of 20 μM increased, the expression of Bak and Bax proteins increased at 48 hours of the treatment. In addition, when the compound of Formula 1 was treated by concentration for 48 hours, the expression of Bak and Bax proteins was confirmed at a concentration over 15 μM (Figure 9B).

[0121] 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 pancreatic ductal adenocarcinoma cells to prevent and treat pancreatic ductal adenocarcinoma.

[0122] <Example 9>Inhibition of Tumoral Growth in Xenograft Mouse Models

[0123] The present Example confirmed the anticancer effect and mechanism of the compound of Formula 1 in accordance with the present invention in the pancreatic ductal adenocarcinoma cell line Mia paca-2-induced xenograft mouse and Panc-1-induced xenograft mouse to confirm its anticancer effect against the pancreatic ductal adenocarcinoma in animal models.

[0124] Tumor-derived animal models (Mia paca-2 derived xenograft mouse models) were prepared by subcutaneously injecting pancreatic ductal adenocarcinoma cell line Mia paca-2 at a density of 5 x 106cells / mouse into BALB / c / nu / nu old nude mice to transplant the cell line and induce tumor formation. The models were divided into five groups with 3 groups administering compound of Formula 1 at concentrations of 15 mg / kg, 30 mg / kg, and 60 mg / kg (oral administration, 5 times / week, 53 days), a control group, and a gemcitabine 20 mg / kg (intraperitoneal administration, 2 times / week, 53 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, and C of Figure 10.

[0125] Moreover, tumor-derived animal models (Panc-1 derived xenograft mouse models) were prepared by subcutaneously injecting pancreatic ductal adenocarcinoma cell line Panc-1 at a density of 2 x 106cells / mouse into BALB / c / nu / nu old nude mice to transplant the cell line and induce tumor formation. The models were divided into five groups with 3 groups administering compound of Formula 1 at concentrations of 15 mg / kg, 30 mg / kg, and 45 mg / kg (oral administration, 5 times / week, 56 days), a control group, and a gemcitabine 20 mg / kg (intraperitoneal administration, 2 times / week, 56 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 D, E, and F of Figure 10.

[0126] As can be seen in A, B, and C of Figure 10, it was confirmed that administration of the compound of Formula 1 in Mia paca-2-derived tumors reduced tumoral growth in the groups treated with the compound of Formula 1 and in the group treated with gemcitabine. In contrast, the control group exhibited a significant increase in tumor size after 30 days. Notably, administration of the compound of Formula 1 at a low concentration of 15 mg / kg reduced tumor growth by approximately 46% compared to the control group. Administration at a concentration of 30 mg / kg reduced the tumor growth by 66%. Furthermore, administration at a concentration of 60 mg / kg reduced the tumor growth by 78%. The group treated with gemcitabine, a previously established anticancer agent, showed a tumor growth reduction comparable to that of the group treated with compound of Formula 1 at a low concentration of 15 mg / kg. Furthermore, as can be seen in Figure 10C, it is confirmed that the compound of Formula 1 inhibits tumoral growth but does not cause a change such as weight loss in mice.

[0127] As can be seen in D, E, and F of Figure 10, it was confirmed that administration of the compound of Formula 1 in Panc-1-derived tumors reduced tumoral growth in the groups treated with the compound of Formula 1 and in the group treated with gemcitabine. In contrast, the control group exhibited a significant increase in tumor size after 30 days. Notably, administration of the compound of Formula 1 at a concentration of 30 mg / kg reduced tumor growth by approximately 53% compared to the control group. Administration at a concentration of 45 mg / kg reduced the tumor growth by 70%. The group treated with gemcitabine, a previously established anticancer agent, merely showed a tumor reduction effect of approximately 34%. Furthermore, as can be seen in Figure 10F, it is confirmed that the compound of Formula 1 inhibits tumoral growth but does not cause a change such as weight loss in mice.

[0128] Thus, the compound of Formula 1 according to the present invention not only can inhibit tumoral growth by inducing apoptosis of pancreatic ductal adenocarcinoma tumoral tissue but also can reduce tumor growth without affecting body weight.

[0129] <Example 10>Tumoral Growth Inhibition Mechanism in Xenograft Mouse Models

[0130] In order to confirm tumoral growth inhibition mechanism of the compound of Formula 1 according to the present invention against the pancreatic ductal adenocarcinoma cell lines, tumor tissues obtained from xenograft mice, induced to have pancreatic ductal adenocarcinoma cell lines Mia paca-2 and Panc-1, 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.

[0131] In Figure 11, the expression of the target protein was confirmed in the paraffin blocks prepared from tumor tissue obtained from Mia paca-2-induced xenograft mice, wherein the pancreatic ductal adenocarcinoma cell line Mia paca-2 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 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 compared to the control group.

[0132] In Figure 12, the expression of the target protein was confirmed in the paraffin blocks prepared from tumor tissue obtained from Panc-1-induced xenograft mice, wherein the pancreatic ductal adenocarcinoma cell line Mia paca-2 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 was treated at a concentration of 45 mg / kg. Furthermore, the expression of PCNA (proliferating cell nuclear antigen), used as an important marker for cell division and cancer occurrence, was also significantly reduced in tissues treated with the compound of Formula 1 compared to the control group.

[0133] 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 pancreatic ductal adenocarcinoma.

[0134] Thus, in the present invention, the compound of Formula 1 inhibits the calcium influx into the cells through the T-type calcium channel in pancreatic ductal adenocarcinoma cells, suppresses the STAT3 dimerization by binding to the STAT3 SH2domain, and regulates phosphorylation, regulates the expression of the regulatory proteins of the typically mitochondria-dependent Bcl-2 family to kill them through apoptosis mechanism by caspase activation, inhibits tumor growth in mouse models induced to have pancreatic ductal adenocarcinoma, and has anti-tumor activity through apoptosis mechanism in mouse tumor tissues, thereby is capable to be developed as an anticancer treatment for pancreatic ductal adenocarcinoma as a type of pancreatic cancer.

Claims

1.A pharmaceutical composition for treating and preventing pancreatic ductal adenocarcinoma 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 pancreatic ductal adenocarcinoma is a tumor of Mia PaCa-2, PANC-1, PSN-1, YAPC, or HPAF-II cell line.3.The pharmaceutical composition of claim 2, wherein the pancreatic ductal adenocarcinoma is a tumor of Mia PaCa-2 cell line or PANC-1 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 pancreatic ductal adenocarcinoma 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 pancreatic ductal adenocarcinoma of Mia PaCa-2, PANC-1, PSN-1, YAPC, or HPAF-II cell line tumor.10.The method of claim 8, wherein the method for treating or preventing pancreatic ductal adenocarcinoma in a subject in need thereof is characterized by a dual-target anticancer effect on the pancreatic ductal adenocarcinoma cell line Mia PaCa-2, PANC-1, PSN-1, YAPC, or HPAF-II.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 pancreatic ductal adenocarcinoma cells.12.The method of any one of claims 8 to 11, wherein the method for treating or preventing pancreatic ductal adenocarcinoma 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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