Pharmaceutical compositions targeting P53 and HER2

Abstract

This invention relates to pharmaceutical compositions having anticancer activity. More specifically, this invention relates to compositions and methods for treating cancer. Furthermore, this invention relates to pharmaceutical compositions targeting P53 and HER2.

Description

[Technical Field]

[0001] This invention relates to pharmaceutical compositions having anticancer activity. More specifically, this invention relates to compositions and methods for treating cancer. [Background technology]

[0002] Cancer is currently considered an incurable disease. Furthermore, the chemotherapy currently administered to cancer patients is extremely expensive and usually difficult to access due to the toxicity of the chemotherapy system and other side effects (which also kill living cells). Patients lose weight and appetite and suffer a great deal of physical and mental distress. Scientists around the world are diligently researching and investing a great deal of money and effort to find a cure for cancer. The natural world contains an unlimited and inexhaustible stockpile for advancements in new and effective drugs and therapies (Non-Patent Literature 1). In recent years, medicinal plants have been recognized worldwide for their essential importance in treating diseases. Biological and natural compounds have long played a vital role in the treatment and prevention of various diseases and have functioned as a major system of conventional medicine and therapy. The use of natural compounds as a basis for new drug development or as drugs themselves is widely adopted in the field of oncology as a complementary or alternative option. Therefore, each year, several novel cytotoxic compounds are isolated from plants, forming potential cancer treatments. Natural compounds used as drugs to treat cancer have the advantage of having reduced side effects and the ability to influence multiple signaling pathways involved in the carcinogenic process. Of course, nature is great and offers solutions to all challenges, so the objective of this invention is to provide a composition that acts as an anticancer agent without side effects. Therefore, this study has focused on the anticancer effects of compounds isolated from barley extract. This study has also investigated the isolation of compounds from barley extract as potential anticancer agents (including their structural characterization and anticancer function). [Prior art documents]

Non-Patent Literature

[0003]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] The main object of the present invention is to provide a pharmaceutical composition (SACC) containing the main compounds represented by formula (I), formula (II), formula (III) and formula (IV) derived from barley extract. Another object of the present invention is to provide a compound isolated from barley extract. Another object of the present invention is to provide an HPLC method for detecting all compounds present in barley extract. Another object of the present invention is to provide a method for isolating compounds from barley extract. Another object of the present invention is to provide ESI-MS analysis of compounds and to determine the structures of some of the compounds isolated from barley extract using NMR and IR spectroscopy. Another object of the present invention is to provide a pharmaceutical composition containing the compounds of the present invention for the treatment of cancer. Another object of the present invention is to provide a method for preventing or treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition containing the compounds represented by formula (I), formula (II), formula (III) and formula (IV). Another object of the present invention is to provide the use of pharmaceutical compositions comprising compounds represented by formulas (I), (II), (III), and (IV) for the manufacture of pharmaceuticals for the treatment of cancer. Another object of the present invention is to provide the use of pharmaceutical compositions comprising compounds represented by formulas (I), (II), (III), and (IV) for the treatment of cancer. Another object of the present invention is to provide the use of compounds represented by formulas (I), (II), (III), and (IV) for the manufacture of pharmaceuticals for the treatment of cancer. Another object of the present invention is to provide a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) for use as a pharmaceutical for the treatment of prostate cancer, triple-negative breast cancer, invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer in a subject. [Means for solving the problem]

[0005] The present invention relates to the following formula derived from barley extract: (i)

[0006] [ka] Equation (I): 6-hydroxy-3H-isochromene-3,8(4H)-dione (ii)

[0007] [ka] Formula (II): 5-methyl-1H-indole-3-carboxylic acid (iii)

[0008] [ka] Formula (III): 6-hydroxy-3-methyl-2H-pyran-2-one, and (iv)

[0009] [ka] Formula (IV) 2-methyl-4H-pyran-4-one Disclosed are pharmaceutical compositions comprising at least one compound selected from the compounds represented by or combinations thereof. This disclosure provides an HPLC method for detecting compounds represented by formulas (I), (II), (III), and (IV) present in barley extract. This disclosure provides a method for isolating the compound from barley extract. This disclosure provides a method for determining the structure of compounds isolated from barley extract using ESI-MS analysis of compounds, as well as NMR and IR spectroscopy. This disclosure provides pharmaceutical compositions comprising compounds represented by formulas (I), (II), (III), and (IV) derived from barley extract, for (i) inducing apoptosis and inhibiting proliferation of pancreatic cancer cells regardless of mutant p53, (ii) inhibiting HER-2 expression, (iii) inducing the expression of the pro-apoptotic protein Bax and inhibiting the anti-apoptotic protein Bcl2, (iv) inducing PARP protein cleavage, and (v) for the treatment of prostate cancer, triple-negative breast cancer (TNBC), invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer in subjects. The present invention provides the use of a pharmaceutical composition containing barley extract as an active ingredient in the manufacture of a pharmaceutical for the treatment of prostate cancer, triple-negative breast cancer (TNBC), invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer in a subject, for (i) inducing apoptosis and inhibiting proliferation of pancreatic cancer cells regardless of mutant p53, (ii) inhibiting HER-2 expression, (iii) inducing the expression of the pro-apoptotic protein Bax and inhibiting the anti-apoptotic protein Bcl2, (iv) inducing PARP protein cleavage, and (v) for the treatment of prostate cancer, triple-negative breast cancer (TNBC), invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer in a subject. This disclosure provides a method for inducing apoptosis and inhibiting proliferation of pancreatic cancer cells, regardless of mutant p53, for the treatment of prostate cancer, triple-negative breast cancer (TNBC), invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer in subjects, by (ii) inhibiting HER-2 expression, (iii) inducing the expression of the pro-apoptotic protein Bax to inhibit the anti-apoptotic protein Bcl2, and (iv) inducing PARP protein cleavage. This disclosure provides a pharmaceutical composition for use as a pharmacopoeia in a subject for the treatment of cancer. The present invention provides the use of the aforementioned pharmaceutical composition for the treatment of cancer. The present invention provides a pharmaceutical composition for the treatment of pancreatic cancer. The present invention provides pharmaceutical compositions for the suppression and treatment of prostate cancer, triple-negative breast cancer, invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer, comprising compounds represented by formulas (I), (II), (III), and (IV) derived from barley extract. Exemplary embodiments shown herein, with reference to the accompanying drawings, are provided hereby referenced to facilitate understanding and practical implementation of this disclosure. The drawings, together with the following detailed description, are incorporated herein and form part of this specification, and are useful in further illustrating embodiments and illustrating various principles and advantages in accordance with this disclosure. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows the HPLC chromatogram of barley extract (crude: B.No: KWO 037). [Figure 2] This figure shows a typical preparative HPLC chromatogram. [Figure 3] This figure shows a typical maximum plot (200-400 nm) HPLC chromatogram, representing peak 2. [Figure 4] This figure shows a typical HPLC chromatogram at 215 nm, representing peak 2. [Figure 5]This figure shows the IR spectrum of peak 2. [Figure 6] This figure shows the ESI mass spectrum of peak 2. [Figure 7] This figure shows the 1H NMR spectrum of peak 2. [Figure 8] This figure shows a typical maximum plot (200-400 nm) HPLC chromatogram, representing peak 4. [Figure 9] This figure shows a typical HPLC chromatogram at 274 nm, representing peak 4. [Figure 10] This figure shows the IR spectrum of peak 4. [Figure 11] This figure shows the ESI mass spectrum of peak 4. [Figure 12] This figure shows the 1H NMR spectrum of peak 4. [Figure 13] This figure shows a typical maximum plot (200-400 nm) HPLC chromatogram, representing peak 5. [Figure 14] This figure shows a typical HPLC chromatogram at 291 nm, representing peak 5. [Figure 15] This figure shows the IR spectrum of peak 5. [Figure 16] This figure shows the ESI mass spectrum of peak 5. [Figure 17] This figure shows the 1H NMR spectrum of peak 5. [Figure 18] This figure shows a histogram of the kinase reaction (1 / 30 final dilution SACC). [Figure 19] This figure shows a histogram of the kinase reaction (1 / 120 final dilution SACC). [Figure 20] This figure shows the complete inhibitor titration for IC50 determination. [Figure 21] This graph shows the cell viability of the A549 cell line after treatment of the test sample. [Figure 22] This graph shows the cell viability of the A549 cell line after standard (doxorubicin) treatment. [Figure 23] This graph shows the cell inhibition after treatment of the test sample in the MTT assay for A549 cells. [Figure 24] This graph shows the inhibitory activity and IC50 value of the test samples in the MTT assay against A549 cells. [Figure 25] This graph shows the cell inhibition after treatment with the standard substance (doxorubicin) in the MTT assay for A549 cells. [Figure 26] This graph shows the inhibitory activity and IC50 value of the standard substance (doxorubicin) in the MTT assay against A549 cells. [Figure 27] A: This is a representative line graph showing the tumor volume at the indicated time point for the control group and the SACC-treated group. The values ​​in the graph are the averages of n=8 mice in the control group and n=9 mice in the SACC-treated group. SACC was administered 5 days / week for 6 consecutive weeks. B: This is a representative bar graph showing the tumor mass of the control and SACC-treated mice at week 9. The values ​​in the graph are the averages of n=8 and n=9 mice in the control and SACC-treated groups, respectively. [Figure 28] These are images from a 12-week study of control mice and SACC-treated mice. [Figure 29] This is a line graph of the AsPC1 cell xenograft tumors in the control group mice. [Figure 30] This is a line graph of xenograft tumors of SACC group mice using AsPC1 cells. [Figure 31] A: Images of control mice and SACC-treated mice with xenograft tumors. The photographs in the lower panel show the excised xenograft tumors of control and SACC-treated mice. B: The figure shows the tumor volume of xenograft tumors in control and SACC-treated mice at the indicated weeks, where the blue line represents the control and the red line represents SACC. C: Figure shows the excised xenograft tumor mass of control and SACC-treated mice at 5 weeks. [Figure 32] This figure shows the histopathological results of Pan-CSC-derived xenograft tumors from control and SACC-treated mice. [Figure 33]This figure shows the effect of SACC on the expression of the transcription factor GLI-1 and the chemokine receptor CXCR4. Representative immunohistochemical images of GLi-1 and CXCR4 expression in control and SACC xenograft tumors are shown. [Figure 34] Effect of SACC on pancreatic cancer cell survival and apoptosis: A: Figure showing the effect of SACC on cell proliferation of AsPc1 cells as determined by the MTT assay. B: Figure showing the effect of control and SACC on apoptosis induction in MiaPaCa-2 cells as evaluated by flow cytometry. [Figure 35] This figure shows the effect of SACC on the expression of HER-2 / p53 and apoptotic proteins in pancreatic cancer cells. [Figure 36] This figure shows the area percentage of peaks in an HPLC chromatogram. [Figure 37] This figure shows the effects of SA1 (peak 5) and SA2 (peak 4) in the human breast cancer cell line MDA-MB-231, as indicated by the growth curve compared to the control (adriamycin). [Figure 38] This is a growth curve comparing the effect of SACC on human breast cancer cell line MDA-MB-231 with that of a control (adriamycin). [Figure 39] This figure shows the effects of SA1 (peak 5) and SA2 (peak 4) in human lung cancer cell line A-549, as indicated by the growth curve, compared to the control (adriamycin). [Figure 40] This is a growth curve comparing the effect of SACC on human lung cancer cell line A-549 with that of a control (adriamycin). [Figure 41] This is a growth curve comparing the effects of SA1 (peak 5) and SA2 (peak 4) in the human pancreatic cancer cell line Mia-Pa-Ca-2 with the control (adriamycin). [Figure 42] This is a growth curve comparing the effect of SACC on human pancreatic cancer cell line Mia-Pa-Ca-2 with that of a control (adriamycin). [Figure 43]This graph shows the relative tumor volume (RTV) in the human tumor xenograft model MDA-MB-231 for groups A through D. [Figure 44] This graph shows the T / C ratio derived from RTV data in the human tumor xenograft model MDA-MB-231 for groups A to D. [Figure 45] This graph shows the survival rates for groups A through D in the human tumor xenograft model MDA-MB-231. [Figure 46] Figure 46 shows graphs of mean animal body weight in the human tumor xenograft model MDA-MB-231 for groups A to D. [Figure 47] This is a graph of tumor volume in the human tumor xenograft model MDA-MB-231 from group A. [Figure 48] This is a graph of tumor volume in the human tumor xenograft model MDA-MB-231 from group B. [Figure 49] This is a graph of tumor volume in the human tumor xenograft model MDA-MB-231 from group C. [Figure 50] This is a graph of tumor volume in the human tumor xenograft model MDA-MB-231 from group D. [Modes for carrying out the invention]

[0011] In providing a detailed explanation, it should be understood that the following description merely illustrates specific embodiments of the present invention. However, these specific embodiments are merely exemplary and do not imply any limitation of the scope of the present invention. Therefore, the description should be understood as exemplary embodiments and teachings of the present invention and should not be interpreted restrictively. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, and preferred methods and materials are also described. For the purposes of the present invention, the following terms are defined below. The articles "a" and "an" (original text) are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements.

[0012] As used herein, the term "compound" includes the compounds disclosed in this invention. As used herein, the term SACC refers to a composition comprising compounds represented by formulas (I), (II), (III), and (IV) derived from barley extract. As used herein, the terms “comprises” or “comprising” are used generally to mean “include,” that is, to allow the presence of one or more features or components. As used herein, the terms "optional" or "optionally" mean that the event or situation described thereafter may or may not occur, and that the description includes both cases in which the event or situation occurs and cases in which it does not occur. As used herein, the terms “prevent,” “prevention,” and “prevention” refer to methods of preventing the onset of a disease and / or its associated symptoms, or methods of preventing a subject from contracting the disease. As used herein, the terms “prevent,” “prevention,” and “prevention” also include delaying the onset of a disease and / or its associated symptoms, and reducing the risk to a subject from contracting the disease. As used herein, the term “therapeutic dose” means the amount of active ingredient administered that is sufficient to prevent or alleviate to some extent the onset of one or more symptoms of the condition or disorder being treated. As used herein, the terms “to treat,” “to treat,” and “treatment” refer to methods of alleviating or suppressing a disease and / or its associated symptoms. As used herein, the terms "composition" and "formulation" are interchangeable and have the same meaning. Each embodiment is provided for illustrative purposes of the invention and is not intended to limit the invention. In fact, it will be apparent to those skilled in the art that various modifications and changes can be made to the compounds and methods described herein without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be applied to other embodiments, resulting in further embodiments. Thus, the invention is intended to include such modifications and variations and their equivalents. Other objects, features and aspects of the invention are disclosed in or apparent from the following detailed description. It will be understood to those skilled in the art that the description of the invention is merely a description of exemplary embodiments and should not be construed as limiting broader aspects of the invention.

[0013] In one embodiment, the present disclosure is based on the following formula: (i)

[0014] [ka] Equation (I): 6-hydroxy-3H-isochromene-3,8(4H)-dione (ii)

[0015] [ka] Formula (II): 5-methyl-1H-indole-3-carboxylic acid (iii)

[0016] [ka] Formula (III): 6-hydroxy-3-methyl-2H-pyran-2-one, and (iv)

[0017] [ka] Formula (IV) 2-methyl-4H-pyran-4-one The present invention provides a pharmaceutical composition (SACC) comprising at least one compound selected from the compounds represented by or combinations thereof.

[0018] In one embodiment, the present disclosure is based on the following formula: (i)

[0019] [ka] Equation (I): 6-hydroxy-3H-isochromene-3,8(4H)-dione (ii)

[0020] [ka] Formula (II): 5-methyl-1H-indole-3-carboxylic acid (iii)

[0021] [ka] Formula (III): 6-hydroxy-3-methyl-2H-pyran-2-one, and (iv)

[0022] [ka] Formula (IV) 2-methyl-4H-pyran-4-one The present invention provides a pharmaceutical composition (SACC) containing a compound represented by [formula].

[0023] In one embodiment, the amount of compound of formula (I) in the pharmaceutical composition is in the range of 6% to 18% by mass. In other embodiments, the amount of the compound of formula (I) in the pharmaceutical composition is 12.80% by mass. In one embodiment, the amount of the compound of formula (II) in the pharmaceutical composition is in the range of 8% to 22% by mass. In other embodiments, the amount of the compound of formula (II) in the pharmaceutical composition is 15.41% by mass. In one embodiment, the amount of the compound of formula (III) in the pharmaceutical composition is in the range of 2% to 10% by mass. In other embodiments, the amount of the compound of formula (III) in the pharmaceutical composition is 6.48% by mass. In one embodiment, the amount of the compound of formula (IV) in the pharmaceutical composition is in the range of 1% by mass to 10% by mass. In other embodiments, the amount of the compound of formula (IV) in the pharmaceutical composition is 5.89% by mass.

[0024] In one embodiment, the present disclosure is based on the following formula (I):

[0025] [ka] Equation (I): 6-hydroxy-3H-isochromene-3,8(4H)-dione The present invention provides a compound represented by the following: In one embodiment, the present disclosure is based on the following formula (II):

[0026] [ka] Formula (II): 5-methyl-1H-indole-3-carboxylic acid The present invention provides a compound represented by the following: In one embodiment, the present disclosure is based on the following formula (III):

[0027] [ka] Formula (III): 6-hydroxy-3-methyl-2H-pyran-2-one The present invention provides a compound represented by the following: In one embodiment, the present disclosure is based on the following formula (IV):

[0028] [ka] Formula (IV) 2-methyl-4H-pyran-4-one The present invention provides a compound represented by the following:

[0029] In one embodiment, the present disclosure relates to the following formula derived from barley extract: (i)

[0030] [ka] Equation (I): 6-hydroxy-3H-isochromene-3,8(4H)-dione (ii)

[0031] [ka] Formula (II): 5-methyl-1H-indole-3-carboxylic acid (iii)

[0032] [ka] Formula (III): 6-hydroxy-3-methyl-2H-pyran-2-one, and (iv)

[0033] [ka] Formula (IV) 2-methyl-4H-pyran-4-one A method for isolating a compound represented by, wherein: (i) Perform chromatography of the barley extract by preparative HPLC; (ii) Elute peaks 2, 3, 4, and 5; (iii) Manually isolate the peak obtained in (ii) above by repeated injection to obtain a fraction; (iv) pooling and concentrating the fraction; and (v) A method is provided which includes analyzing peaks and determining the structure of the compound represented by formulas (I), (II), (III), and (IV).

[0034] In other embodiments, the preparative HPLC method of (i) includes mobile phase A and mobile phase B. In other embodiments, the elution of peaks 2, 3, 4, and 5 in (ii) occurs at approximately 14.3, 15.5, 16.4, and 23.0 minutes, respectively. In other embodiments, in (iv), the fractions are pooled and concentrated at a temperature in the range of 25°C to 45°C. In other embodiments, in (iv) above, the fractions are pooled and concentrated at a temperature of 35°C. In other embodiments, in (v) above, the structure is determined by IR, mass, and NMR spectroscopy.

[0035] In one embodiment, the disclosure provides an HPLC method for the analysis of barley extract and a preparative HPLC method for the isolation of required peaks corresponding to compounds represented by formulas (I), (II), (III), and (IV). Elution of the band corresponding to the compound from thin-layer chromatography confirmed that it was a single band. HPLC analysis of the isolated compound showed a single peak at retention times (Rt) of 14.3, 15.5, 16.4, and 23.0 minutes. The purity of the compound was also confirmed by HPLC, yielding a single peak with a purity in the range of 85% to 100%, preferably 87.9% or higher, or 98.1% or higher, or 99.7% or higher. The structure of the isolated compound was determined by LC-MS, ESI-MS, IR spectroscopy, and 1 This was determined using 1H NMR.

[0036] In one embodiment, the present invention provides an HPLC method for the analysis of barley extracts and a preparative HPLC method for the isolation of necessary peaks corresponding to compounds represented by formulas (I), (II), (III), and (IV). In one embodiment, the barley extract is obtained by a method comprising mixing barley flour with distilled water to obtain a mixture, stirring the mixture at ambient temperature, and distilling the mixture to obtain an extract. In some examples of the extraction process, the mixture is maintained at a temperature of about 27 ± 3°C for about 16 ± 2 hours. In one embodiment, the mixture is distilled at a temperature of about 110 ± 30°C to obtain an extract. The extract thus obtained is maintained at about 10 ± 3°C for about 1 hour.

[0037] In one embodiment, the disclosure provides a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) extracted from barley extract as an active ingredient, and optionally mixed with a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment, the present disclosure provides an anticancer composition comprising at least one compound selected from those represented by formulas (I), (II), (III), and (IV) or combinations thereof, and a pharmaceutically acceptable carrier / excipient. In one embodiment, the present disclosure provides an anticancer composition comprising compounds represented by formulas (I), (II), (III), and (IV), and pharmaceutically acceptable carriers and / or excipients. In one embodiment, the disclosure provides a pharmaceutical formulation comprising a compound represented by formula (I), formula (II), formula (III), and formula (IV) or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers and optionally one or more other therapeutic agents. In other embodiments, the therapeutic agent is an anticancer drug. The carrier must be "acceptable" in the sense that it is compatible with the other components of the formulation and is not harmful to its recipient. The appropriate formulation depends on the chosen route of administration. Any known technology, carrier, or excipient may be used as appropriate and as understood in the art. In one embodiment, the present disclosure provides a method for formulating a composition disclosed for pharmaceutical administration. In one embodiment, administration includes intravenous, intrathecal, intramuscular, oral, and any other acceptable route of administration.

[0038] In one embodiment, the present disclosure provides a pharmaceutical composition for inducing apoptosis and inhibiting the proliferation of pancreatic cancer cells, regardless of mutant p53, comprising compounds represented by formulas (I), (II), (III), and (IV) derived from barley extract. In one embodiment, the present disclosure provides a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) derived from barley extract for inhibiting HER-2 expression. In one embodiment, the present disclosure provides a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) derived from barley extract for inducing the expression of the pro-apoptotic protein Bax and inhibiting the anti-apoptotic protein Bcl2. In one embodiment, the present disclosure provides a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) derived from barley extract for inducing PARP protein cleavage. In one embodiment, the present disclosure provides a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) derived from barley extract for the treatment of prostate cancer, triple-negative breast cancer, invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer in a subject. In one embodiment, the present disclosure provides a pharmaceutical composition for use in the manufacture of a drug for inducing apoptosis and inhibiting the proliferation of pancreatic cancer cells, regardless of the mutant p53. In one embodiment, the present disclosure provides a pharmaceutical composition for use in the manufacture of a pharmacopoeia for inhibiting HER-2 expression. In one embodiment, the present disclosure provides a pharmaceutical composition for use in the manufacture of a pharmacopoeia for inducing the expression of the pro-apoptotic protein Bax and inhibiting the anti-apoptotic protein Bcl2. In one embodiment, the present disclosure provides a pharmaceutical composition for use in the manufacture of a pharmaceutical for inducing PARP protein cleavage.

[0039] In one embodiment, the present disclosure provides a pharmaceutical composition for use in the manufacture of pharmaceuticals for the treatment of prostate cancer, triple-negative breast cancer, invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer in a subject. In one embodiment, the present disclosure provides the use of a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) for the manufacture of a pharmacopoeia for the treatment of cancer.

[0040] In one embodiment, the present disclosure provides a method for preventing or treating cancer in a subject requiring treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV). In one embodiment, the present disclosure provides a method for inducing apoptosis and inhibiting the growth of pancreatic cancer cells in a subject requiring treatment, regardless of the variant p53, by administering a therapeutically effective amount of a pharmaceutical composition comprising a compound represented by formula (I), formula (II), formula (III), and formula (IV). In one embodiment, the present disclosure provides a method for inhibiting HER-2 expression in a subject requiring treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV). In one embodiment, the present disclosure provides a method for inducing the expression of the pro-apoptotic protein Bax and inhibiting the anti-apoptotic protein Bcl2 in a subject requiring treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a compound represented by formula (I), formula (II), formula (III), and formula (IV). In one embodiment, the present disclosure provides a method for inducing PARP protein cleavage in a subject requiring such cleavage, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV).

[0041] In one embodiment, the present disclosure provides a method of using a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV), which includes inducing apoptosis and inhibiting the proliferation of pancreatic cancer cells. In one embodiment, the present disclosure provides a method of using a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV), the method comprising inhibiting the expression of HER-2. In one embodiment, the present disclosure provides a method of using a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV), the method comprising inducing the expression of the pro-apoptotic protein Bax and inhibiting the anti-apoptotic protein Bcl2. In one embodiment, the present disclosure provides a method for using a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV), the method comprising inducing PARP protein cleavage. In one embodiment, the present disclosure provides a method of using a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV), the method of treatment of prostate cancer, triple-negative breast cancer, invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer in a subject. In one embodiment, the present disclosure provides a compound of formula (I) for use in the treatment of cancer. In one embodiment, the present disclosure provides a compound of formula (III) for use in the treatment of cancer. In one embodiment, the present disclosure provides the use of a compound of formula (I) for the manufacture of a pharmaceutical for the treatment of cancer. In one embodiment, the present disclosure provides the use of a compound of formula (III) for the manufacture of a pharmaceutical product for the treatment of cancer. In one embodiment, the present disclosure provides a pharmaceutical composition for use in treating a subject in which such treatment is required. In one embodiment, the present disclosure provides a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) for use as a pharmacopoeia for the treatment of cancer. In one embodiment, the present disclosure provides the use of a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) in the treatment of cancer.

[0042] In one embodiment, the present disclosure provides a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) for use as a pharmacopoeia for inducing apoptosis, inducing the expression of the pro-apoptosis protein Bax, and inducing polyadenosine diphosphate-ribose polymerase (PARP) protein cleavage. In one embodiment, the present disclosure provides a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) for use as a pharmacopoeia for inhibiting the expression of human epidermal growth factor receptor-2 (HER-2), inhibiting the anti-apoptotic protein Bcl2, inhibiting the proliferation of pancreatic cancer cells, degrading p53 mutations, and normalizing wild-type p53. In one embodiment, the present disclosure provides a pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) for use as a pharmaceutical for the treatment of prostate cancer, triple-negative breast cancer, invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer in a subject.

[0043] In one aspect of this disclosure, the pharmaceutical composition includes oral, parenteral, nasal, subcutaneous, intradermal, intramuscular, intravenous, intra-articular, and intramedullary, intraperitoneal, transmucosal, transdermal, rectal, and topical compositions.

[0044] In one embodiment, the present disclosure provides a method for inducing apoptosis, inducing the expression of the pro-apoptotic protein Bax, and inducing PARP protein cleavage in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a compound represented by formulas (I), (II), (III), and (IV). In one embodiment, the present disclosure provides a method for inhibiting HER-2 expression, inhibiting the anti-apoptotic protein Bcl2, inhibiting the proliferation of pancreatic cancer cells, degrading p53 mutations, and normalizing wild-type p53 in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition containing a compound represented by formulas (I), (II), (III), and (IV).

[0045] In other embodiments, the present disclosure provides pharmaceutical compositions in which the subject is human or a non-human mammal. When administered to animals such as humans, the composition or barley extract is preferably administered as a pharmaceutical composition comprising, for example, the barley extract of this disclosure and / or a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiological buffer saline, or other solvents such as glycol or glycerol, or vehicles and oils such as olive oil or injectable organic esters. In other embodiments, the Disclosure provides pharmaceutical compositions for human administration, particularly for invasive routes of administration (i.e., routes such as injection or transplantation that avoid transport or diffusion through the epithelial barrier), wherein the aqueous solutions are pyrogenic or substantially pyrogenic. Excipients can be selected, for example, to delay the release of the drug or to selectively target one or more cells, tissues, or organs. In other embodiments, the pharmaceutical composition may be in unit dosage forms such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, and lyophilized products for reconstitution, powders, liquids, syrups, suppositories, and injections. The composition may also be present in transdermal delivery systems, such as skin patches. The composition may also be present in solutions suitable for topical administration, such as eye drops. The dosage of the active ingredient (barley extract) of the present invention varies depending on the patient's age, weight, symptoms, the potency or therapeutic effect of the compound, the administration plan, and / or the treatment time. Generally, suitable routes of administration include, for example, oral, ophthalmic, rectal, transmucosal, topical, or intestinal administration; parenteral delivery (including intramuscular, subcutaneous, intrathecal injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection). The barley extract of the present invention can be administered in amounts ranging from 0.2 mg, 0.5 mg, or 1 mg to 500 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, or 10 g per single dose. Doses may be administered at frequencies of once a week, once every three days, once every two days, once a day, twice a day, three times a day, or more. In alternative embodiments, in certain adults, the barley extract may be administered continuously by intravenous administration over a period specified by a physician. Since the dosage is affected by various conditions, in some cases, amounts lower or higher than the expected dosage range may be administered. Physicians can easily determine the appropriate dosage for patients undergoing treatment. The pharmaceutical compositions of this disclosure can be manufactured by methods known in themselves, for example, by conventional mixing, dissolution, granulation, sugar-coating, polishing, emulsification, encapsulation, encapsulation, or compression processes. Formulations suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intra-articular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal, and topical (including cutaneous, buccal, sublingual, and intraocular) administration may vary, but the most appropriate route may depend, for example, on the recipient's condition and impairment.

[0046] The pharmaceutical compositions of this disclosure may be used in diagnostic imaging methods. The pharmaceutical compositions of this disclosure are preferably administered to a patient (e.g., a human) by intravenous injection. The formulations may preferably be provided in unit dosage forms and may be prepared by any method well known in the pharmaceutical field. All methods involve associating a compound of the present invention or a pharmaceutically acceptable salt thereof ("active ingredient") with a carrier constituting one or more auxiliary components. Generally, the formulations are prepared by homogeneously and closely associating the active ingredient with a liquid carrier or a fine solid carrier or both, and then, if necessary, shaping the product into a desired formulation.

[0047] As used herein, the term “pharmaceutically acceptable salt” refers to a prepared salt of a pharmaceutically acceptable, nontoxic acid, including inorganic and organic acids. Examples of physiologically or pharmaceutically acceptable salts of the compounds disclosed herein include salts derived from suitable bases such as alkali metals (e.g., sodium), alkaline earth metals (e.g., magnesium), ammonium, and NX4+ (wherein X is a C1-C4 alkyl group). Examples of physiologically acceptable salts of hydrogen atoms or amino groups include organic carboxylic acids such as acetic acid, benzoic acid, lactic acid, fumaric acid, tartaric acid, maleic acid, malonic acid, malic acid, isethionic acid, lactobionic acid, and succinic acid; organic sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; and salts of inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and sulfamic acid. Examples of physiologically acceptable salts of compounds with a hydroxyl group include anions of the said compounds combined with a suitable cation such as Na+ and NX / (wherein X is independently selected from H or C1-C4 alkyl groups).

[0048] For therapeutic purposes, salts of the active ingredients of the compounds disclosed herein are typically physiologically acceptable, meaning they are salts derived from physiologically acceptable acids or bases. However, salts of physiologically unacceptable acids or bases may still be used, for example, in the preparation or purification of physiologically acceptable compounds. All salts, whether derived from physiologically acceptable acids or bases or not, are within the scope of this disclosure. Salts of parent compounds comprising one or more amino acids are also included within the scope of this disclosure. Natural or non-natural amino acids are suitable, particularly naturally occurring amino acids found as protein components, which typically have a side chain with a basic or acidic group, e.g., lysine, arginine, or glutamic acid, or a neutral group, e.g., glycine, serine, threonine, alanine, isoleucine, or leucine.

[0049] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. Some examples of substances that can act as pharmaceutically acceptable carriers are: (1) sugars (e.g., lactose, glucose, and sucrose); (2) starches (e.g., corn starch and potato starch); (3) cellulose and its derivatives (e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate); (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients (e.g., cocoa butter and suppository wax); (9) oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and sesame oil). (10) Soybean oil; (11) Glycols (e.g., propylene glycol); (12) Polyols (e.g., glycerin, sorbitol, mannitol, and polyethylene glycol); (13) Esters (e.g., ethyl oleate and ethyl laurate); (14) Agar; (15) Buffers (e.g., magnesium hydroxide and aluminum hydroxide); (16) Alginic acid; (17) Water free of pyrogens; (18) Isotonic saline; (19) Ringer's solution; (20) Ethyl alcohol; (21) Phosphate buffer; and (21) Other non-toxic, suitable substances used in pharmaceutical formulations.

[0050] The pharmaceutical composition (formulation) can be administered to a subject by any of the following routes of administration, including, for example, oral (e.g., aqueous or non-aqueous solution or suspension of liquid medicine, tablets, capsules (including sprinkle capsules and gelatin capsules), bolus, powder, granules, paste for application to the tongue); absorption via the oral mucosa (e.g., sublingual); anal, rectal or vaginal (e.g., as a pessary, cream or foam); parenteral (intramuscular, intravenous, subcutaneous or intrathecal, e.g., as a sterile solution or suspension); nasal; intraperitoneal; subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment or spray applied to the skin, or as eye drops). Barley extract may be formulated for inhalation. In yet another embodiment, barley extract may simply be dissolved or suspended in sterile water or redistilled water (DDW). The formulation may be conveniently provided in unit dosage forms and may be prepared by any method well known in the field of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the host being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the compound that produces the therapeutic effect. Generally, out of 100%, this amount is in the range of about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%. Preparations of the present disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavoring base, usually sucrose and acacia or tragacanth), lyophilized products, powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as flavored tablets (using an inert base such as gelatin and glycerin or sucrose and acacia), and / or as mouthwashes, etc., each containing a predetermined amount of the barley extract of the present disclosure as an active ingredient. To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, sugar-coated tablets, powders, granules, etc.), the active ingredient may be carried by one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; (3) Moisturizing agents such as glycerol; (4) Disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) Dissolution retarders such as paraffin; (6) Absorption enhancers such as quaternary ammonium compounds; (7) Wetting agents such as cetyl alcohol and glycerol monostearate; (8) Absorbents such as kaolin and bentonite clay; (9) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. (10) Complexing agents, such as modified and unmodified cyclodextrins; and (11) Colorants, to be mixed with any of these. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also include a buffering agent. Similar solid compositions may be used as fillers in soft and hard gelatin capsules, using lactose or milk sugar and excipients such as high molecular weight polyethylene glycol. Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophilized products for reconstitution, microemulsions, suspensions, solutions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may include inert diluents commonly used in the art, such as water or other solvents, cyclodextrins and their derivatives, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, as well as mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants such as humectants, emulsifiers and suspending agents, sweeteners, flavoring agents, colorants, fragrances and preservatives. In addition to the active ingredient, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof. Formulations of pharmaceutical compositions for rectal, vaginal, or urethral administration may be provided as suppositories, which may be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol, suppository wax, or salicylate, which are solid at room temperature but liquid at body temperature, and therefore melt in the rectal or vaginal cavity, releasing the active compounds. Pharmaceutical compositions for oral administration may be provided as mouthwashes, oral sprays, or oral ointments. Alternatively, the composition may be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such device may be particularly useful for delivery to the bladder, urethra, ureter, rectum, or intestine. The pharmaceutical compositions of this disclosure may be used alone (monotherapy) or in combination with one or more other methods / compositions (combination therapy).

[0051] Abbreviation: The following abbreviations are used in this disclosure. ℃: Celsius HPLC: High-Performance Liquid Chromatography h: time mL: milliliter THF: Tetrahydrofuran Rt: retention time TLC: Thin-layer chromatography NMR: nuclear magnetic resonance MHz: Megahertz s: Single Let d: doublet t: triplet m: multiplet H: Proton MS: Mass spectrometry LC-MS: Liquid Chromatography-Mass Spectrometry IV or iv: Intravenous administration HEPES: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) ATP: Adenosine triphosphate DTT: Dithiothreitol EGTA: Ethylene glycol (bis(2-aminoethyl) ether-N,N,N,N-tetraacetic acid) DAPK1: Death-related protein kinase PKD: Protein Kinase D CAMKK2: Calcium / calmodulin-dependent protein kinase kinase 2 PKCδ: Protein kinase C-delta FTIR: Fourier Transform Infrared Spectroscopy ppm: parts per million PARP: Polyadenosine diphosphate-ribose polymerase This disclosure is generally described herein and will be more readily understood by reference to the following examples, which are included solely for illustrative purposes of specific aspects and embodiments of the disclosure and are not intended to limit the disclosure in any way. [Examples]

[0052] Equipment and materials The present invention uses the following equipment. Preparative HPLC: Agilent semi-preparative HPLC NMR: 400 MHz NMR Varian Make(Agilent) FTIR: PerkinElmer 100 Series HPLC: Waters HPLC with Empower software LC-MS: Agilent Single Quadrupole with ESI Balance: Sartorius Columns: Inertsil C18 for HPLC analysis (250 × 4.6 mm, 5 μm) and Inertsil C18 for preparative HPLC (250 × 19 mm, 5 μm) The following materials are used in this invention. Acetonitrile: HPLC grade Methanol: HPLC grade Trifluoroacetic acid: AR grade Dichloromethane (DCM): HPLC grade Water: Mili-Q water Production of barley extract Place 1 kilogram of barley flour derived from ground barley grains of Indian origin into a glass container fitted with a standard joint. Add 2 liters of redistilled water and thoroughly mix the mixture, shaking vigorously. Then, tightly cover the glass container and place it in an incubator at 27±3°C for 16 hours. After maturation, remove the cover from the container and place it on a heating system (a rotamantle equipped with a heating element and thermostat, insulated with glass wool to prevent direct heat contact with the glass flask). The container is equipped with a distillation set featuring a suitable cooling system to maintain an optimal distillation temperature of 110±30°C. The liquid obtained during the distillation process is passed through a cotton bed attached to a funnel and collected in flasks, usually in 3-4 fractions of approximately 400 ml each. These fractions are tightly covered and stored at 10±3°C for 1 hour. After that, the flasks are allowed to return to normal room temperature, which may vary depending on the season. The resulting solution constitutes the pharmaceutical formulation intended for use. [Examples]

[0053] Isolation of compounds from barley extract The compound was isolated from barley extract using HPLC technology. The purity of the compound was determined. 1. Details of the method for fraction isolation : 1.1 Details of the HPLC method for the analysis of barley extract (B.No.KWO 037) Mobile phase A: 0.1% TFA in water Mobile phase B: Methanol:ACN (80:20 v / v) with 0.1% TFA For the analysis of barley extract, follow these chromatography conditions: Column: Inertsil ODS 3 V (250 mm × 4.6 mm, 5 μm) Injection volume: 10μL Flow rate: Flow rate: 1.0 mL / min Column temperature: 25℃ Detection wavelengths: Max plot, 280, 254, and 230 nm Execution time: 45 minutes Test concentration: as such Dissolution mode: Gradient modes / programs shown in Table 1 Table 1: Gradient

[0054] [Table 1] Figure 1 shows typical HPLC chromatograms at different UV wavelengths (nm). 1.2 Details of the preparative HPLC method for peak isolation The following HPLC method is used to isolate the peaks. Mobile phase A: 0.1% TFA in water Mobile phase B: Methanol:ACN (80:20 v / v) with 0.1% TFA Follow the following chromatography conditions to isolate the peaks. Column: Inertsil C18 (250 mm × 19 mm, 5 μm) Flow rate: Flow rate: 15.0 mL / min Column temperature: 25℃ Detection wavelength: 230 nm Execution time: 45 minutes Test concentration: as such procedure: When the barley extract was subjected to chromatography using the method described above, peaks 2, 3, 4, and 5 were eluted at approximately 14.3, 15.5, 16.4, and 23.0 minutes, respectively, as shown in Figure 2. The peaks were manually isolated by repeated injection, the fractions were pooled, and then concentrated at 35°C. a) Isolation and analysis of isolated peak 2 Peak 2 is manually isolated by repeated injection, the fraction is pooled, and concentrated at 35°C according to the chromatographic conditions described in item 1.2. Details of the HPLC method: Chromatographic conditions are provided in Section 1.1. The HPLC chromatograms (Figures 3-4) show an area purity of 98.17% at the maximum plot. The peak purity angles and peak purity thresholds are 5.893 and 6.041, respectively, indicating high purity of the compound peaks and the reliability of the purity measured by HPLC. The structure of the compound corresponding to peak 2 was determined by the following studies: infrared spectroscopy (IR), mass spectrometry (MS), and 1 1H NMR. All analyses were performed using isolated peak 2 in a dry form, isolated and concentrated by preparative HPLC. • Infrared spectroscopy (IR): The IR spectrum is generated and recorded by dispersing with KBr. Figure 5 shows a typical IR spectrum of peak 2. The IR spectrum of peak 2 is shown in Table 2 below. Table 2: IR spectrum of Peak 2

[0055] [Table 2] ·Mass spectrometry (MS): This spectroscopic measurement is performed using electrospray ionization (ESI) / APCI and a quadruple analyzer. The spectrum is acquired in cation mode. Figure 6 shows a typical MS spectrum. The MS peak in the 179.1 m / z [M+H] spectrum corresponds to a molecular weight of 178 amu. ·NMR spectroscopy This spectroscopic measurement is performed in deuterated methanol (CD3OD) as the solvent. Figure 7 shows the peak 2. 1 This shows the 1H NMR spectrum. Peak 2 1 The 1H NMR (400 MHz, CD3OD, ppm) spectra are shown in Table 3 below. Table 3: NMR spectrum of Peak 2

[0056] [Table 3] Determination of the structure of Peak 2: Based on results obtained by LC-MS, IR, and NMR, the basic structure of the compound corresponding to peak 2 was identified as formula (I). LC-MS analysis of the compound showed a molecular ion peak (M+1) at 179.1 m / z, corresponding to a mass of 178 amu (Figure 6). (3354.33, 2934.45, 1689.10, 1364.04, and 1220.72 cm⁻¹) ―1 IR absorption revealed that the compound contains OH, CH, C=O, CH angle-bending, and C-O groups, respectively. In the proton NMR spectrum of peak-2, a singlet appeared at δ4.496 ppm, explaining the two protons corresponding to the methylene group (-CH2). Aromatic CH protons were confirmed from signals at δ6.27-6.286, 6.340-6.352, and 7.438-7.440 ppm. Based on LC-MS, IR, and NMR spectral data, peak-2 is 6-hydroxy-3H-isochromene-3,8(4H)-dione, and the structure of peak-2 is inferred as follows.

[0057] [ka] 6-hydroxy-3H-isochromene-3,8(4H)-dione Equation (I) b) Isolation and analysis of isolated peak 3 Peak 3 is manually isolated by repeated injection, the fraction is pooled, and concentrated at 35°C according to the chromatographic conditions described in Section 1.2. Determination of the structure of Peak 3: Based on the results obtained by LC-MS, IR, and NMR, the basic structure of the compound corresponding to peak 3 is identified as formula (II). The LC-MS analysis data of this compound showed a molecular ion peak (M+1) corresponding to a mass of 191 amu. Based on the LC-MS, IR, and NMR spectral data, peak 3 is presumed to be 5-methyl-1H-indole-3-carboxylic acid, and its structure is as follows.

[0058] [ka] 5-methyl-1H-indole-3-carboxylic acid Molecular formula C 10 H9NO3 Formula (II) c) Isolation and analysis of isolated peak 4 Peak 4 is manually isolated by repeated injection, the fraction is pooled, and concentrated at 35°C according to the chromatographic conditions described in Section 1.2. Details of the HPLC method: The HPLC chromatogram (Figures 8-9) shows an area purity of 87.98% at the maximum plot. The peak purity angle and peak purity threshold are 0.710 and 2.010, respectively, indicating high purity of the compound peak and the reliability of the purity measured by HPLC. The structure of the compound corresponding to peak 4 was determined by the following studies: infrared spectroscopy (IR), mass spectrometry (MS), and 11H NMR. All analyses were performed using isolated peak - 4 in dry form, isolated and concentrated by preparative HPLC. • Infrared spectroscopy (IR): IR spectra were generated and recorded by dispersion in KBr. Figure 10 shows a representative IR spectrum of peak 4. The IR spectrum of peak 4 is shown below. Table 4: IR assignments of peak 4

[0059]

Table 4

[0060]

Table 5

[0061] [ka] 6-hydroxy-3-methyl-2H-pyran-2-one Molecular formula C6H6O3 Formula (III) d) Isolation and analysis of isolated peak 5 Peak 5 is manually isolated by repeated injection, the fraction is pooled, and concentrated at 35°C according to the chromatographic conditions described in Section 1.2. Details of the HPLC method: The HPLC chromatogram (Figures 13-14) shows an area purity of 99.74% at the maximum plot. The peak purity angle and peak purity threshold are 2.244 and 2.512, respectively, indicating high purity of the compound peak and the reliability of the purity measured by HPLC. The structure of Peak 5 was determined by the following studies: infrared spectroscopy (IR), mass spectrometry (MS), and 1 1H NMR. All analyses were performed using isolated peak-5 in a dry form, isolated and concentrated by preparative HPLC. • Infrared spectroscopy (IR): The IR spectrum is generated and recorded by dispersing with KBr. Figure 15 shows a typical IR spectrum of peak 5. The IR spectrum of peak -5 is shown below. Table 6. IR Attribution for Peak 5

[0062] [Table 6] ·Mass spectrometry This spectroscopic measurement is performed using electrospray ionization (ESI) / APCI and a quadruple analyzer. The spectrum is acquired in cation mode. Figure 16 shows a typical MS spectrum. The MS peak in the 111.1 m / z [M+H] spectrum corresponds to the molecular weight of 110 amu identified in the spectrum. ·NMR spectroscopy This spectroscopic method used deuterated methanol (CD3OD) as the solvent. Figure 17 shows a typical NMR spectrum. Peak 5 1 The 1H NMR (400 MHz, CD3OD, ppm) spectra are shown in Table 7 below. Table 7: NMR spectrum of peak 5.

[0063] [Table 7] Determination of the structure of Peak 5: Based on the results obtained by LC-MS, IR, and NMR, the basic structure of the compound corresponding to peak 5 is identified as formula (IV). LC-MS analysis data of the compound showed a molecular ion peak (M+1) at 111.1 m / z, corresponding to a mass of 111 amu (Figure 16). (2939.39, 1667.53, 1518.75, 1395.41, and 1181.87 cm⁻¹) ―1 IR absorption revealed that the compound contains CH, C=O, C=C stretched, C-H bent, and C-O groups, respectively. In the proton NMR spectrum of peak 5, the single let at δ2.256 ppm, which explains the three protons, corresponds to a methyl group (-CH3). Signals at δ5.326, 5.968-5.963, and 6.270-6.262 ppm confirm aromatic CH protons. Based on LC-MS, IR, and NMR spectral data, peak 5 is presumed to be 2-methyl-4H-pyran-4-one, and the structure of peak 5 is assigned as follows.

[0064] [ka] 2-methyl-4H-pyran-4-one Molecular formula C6H6O2 Formula (IV) Biological activity of the composition [Examples]

[0065] Preparation of pharmaceutical compositions Pharmaceutical compositions containing compounds represented by formulas (I), (II), (III), and (IV) derived from barley extract are prepared by obtaining high-quality barley extract through appropriate extraction and purification processes. The obtained barley extract is stored and used in liquid or dry form. [Examples]

[0066] Kinase reaction experiment Response settings: 2.5 μL of barley extract (SACC extract) (1 / 30 final dilution, 1 / 120 final dilution, or titration); 5.0 μL of enzyme dilution buffer (EDB) (1×) or kinase (5× in EDB); Pre-incubate at room temperature for 10 minutes. 17.5 μL of reaction mixture substrate containing ATP & C(Sx) (pre-incubated at 30°C for 5 minutes); Final reaction volume: 25 μL; The reaction was carried out at 30°C for 120 or 240 minutes. Reaction conditions: 54 mM HEPES, pH 7.5 1 mM ATP 1 mM DTT (No DTT for ASK1) 0.012% Brij - 35 1% glycerol 0.52 mM EGTA (CAMKK2, DAPK1, DAPK2, PKCδ, PKD1, PKD2, PKD3 do not contain EGTA) 0.4 mM CaCl2, 5 ng / μl calmodulin (CAMKK2, DAPK1, DAPK2, PKD1, PKD2, PKD3 only) 3.8 μM diacylglycerol, 140 μM phosphatidylserine (PKCδ only) 250 μM MnCl2 (HER2 only) 10 mM mgCl2 15μM C(Sx) substrate 0.5-20 nM kinase Note: • Enzyme dilution buffer (EDB): 20 mM HEPES, pH 7.5, 0.01% Brij-35, 0.1 mM EGTA (no EGTA for CAMKK2, DAPK1, DAPK2, PKCδ, PKD1, PKD2, PKD3), 5% glycerol, 1 mg / mL bovine serum albumin, 1 mM DTT (no DTT for ASK1). The reaction was carried out in Corning, 384-well, white, flat, round-bottom polystyrene NBS microplates (catalog no. 3824), after sealing with an optically transparent adhesive film [TopSealA-Plus plate seal (PerkinElmer, catalog no. 6050185)]. tested kinases

[0067] [Table 8] In Figure 18 of TIFF2026513637000034.tif149170, the histogram (for the 1 / 30 final dilution of SACC) shows the inhibitory effect of the crude compound at a 1:30 dilution on various kinases. The bars in the histogram represent the level of inhibition, with larger bars indicating greater inhibition. In particular, at this dilution, four kinases showed no inhibition, two kinases showed less than 25% inhibition, while a considerable number of 30 kinases (61%) showed significant inhibition of over 50%. In Figure 19, the histogram (for 1 / 120 final dilution SACC) shows the inhibition profile of the crude compound at a 1:120 dilution. Here, larger bars indicate higher inhibition levels. Surprisingly, at this more diluted concentration, only five kinases constituting 10% show inhibition exceeding 25%. These include CAMKK2, COT / MAP3K8 / Tpl2, HER2, PKCd, and TNIK / MAP4K7. These targets play a crucial role at the interface between inflammation and cancer. Furthermore, according to Reyland (Horton) et al., 2016, PKCδ is a tumor promoter in mouse models of breast and lung cancer cells, and increased PKCδ expression is a negative prognostic indicator in Her2+ and other subtypes of human breast cancer cells. Figure 20 provides a comprehensive diagram of inhibitor titration for IC50 determination. The graph is arranged from left to right and represents the gradient from high to low concentrations of the crude compound. This titration analysis determines the maximum half-molecule inhibitory concentration (IC50) of the compound over various concentrations. 50 The purpose is to determine ). [Examples]

[0068] Cell viability or MTT assay The effect of the SACC fraction (peaks 2, 3, 4, and 5) on lung cell viability was determined by the MTT assay. procedure: Cells were seeded at a density of 1 × 10⁴ per well in 200 μL of complete medium containing 10% FBS and 1% antibiotic in a 96-well plate. After reaching a 70% concentration, cells were treated for 24 hours with the respective doses of SACC and its fraction in the complete medium (shown in the graph). After incubation at 37°C for the specified time in a humidified incubator, the dye MTT (5 mg / mL in PBS; diluted in 10 mL of serum-free medium) was added to each well, and the plates were incubated for 2 hours. The plates were then centrifuged at 1,000 rpm for 5 minutes at 4°C. After carefully removing the medium, 0.1 mL of buffered DMSO was added to each well. Absorbance was recorded on a microplate reader at a wavelength of 540 nm. The effect of SACC and its fraction on inhibiting cell proliferation was evaluated as percentage cell viability, where vehicle-treated cells were considered 100% viable. ·result: Within all SACC fractions, peaks 2, 3, and 4 showed the most significant inhibition of lung cancer cell proliferation, with inhibitory concentrations measured at 1.04 μg, 1.06 μg, and 1.02 μg, respectively. This suggests that these specific fractions contain potent bioactive components that contribute to the observed anticancer effect. Overall, the results indicate that SACC is more therapeutically effective compared to the individual fractions. [Examples]

[0069] Cell viability testing of A549 cell line using trypan blue exclusion method Test sample: SACC composition Standard: Doxorubicin drug Preparation of the test solution: For cytotoxicity studies, test samples were treated as 100% stock, and serial 2-fold dilutions ranging from 10% to 0.156% were prepared using F-12K plain medium for treatment. 10 mM stock of standard doxorubicin was diluted from 100 μM to 1.56 μM using F-12K plain medium for treatment. Cell lines and culture media: A549 cells were cultured in F-12K medium with appropriate supplements at 37°C in a humidified atmosphere of 5% CO2 until confluence. Cell viability was checked using a conventional hemocytometer. Appropriate cell densities were prepared per 1 ml of medium, seeded in 96-well plates, and incubated at 37°C for 24 hours with 5% CO2 to evaluate cell viability. procedure: 1. Measure the number of cells using complete culture medium: 5 × 10 5 The cell concentration was adjusted to cells / ml. 100 μl of diluted cell suspension (50,000 cells / well) was added to each well of a 96-well microtiter plate and incubated at 37°C for 24 hours in a 5% CO2 atmosphere. 2. After 24 hours, the cells were treated with various concentrations of the test compound and incubated at 37°C for 24 hours in a 5% CO2 atmosphere. After incubation, the test solutions in the wells were discarded. 4. The cells were treated with trypsin, and a 20 μl cell suspension was collected for viability counting. 5. Add 20 μl of trypan blue dye to this mixture and mix well. 6. The mixture was then loaded into a hemocytometer using a pipette at the end of the notch. 7. The hemocytometer was maintained under an inverted microscope, and each quarter was scored. 8. Cell counting was performed and recorded according to Neubauer's rules. Cell viability was calculated from the obtained data using the following formula. ·Calculation: Survival cells = average survival cells*2*10 3 cells / 100μl % survival rate = surviving cells / total cells × 100 Table 1 shows the cell viability evaluation of the test samples against the A549 cell line, and Figure 21 shows a graph of the cell viability of the test samples after treatment. Table 1: Evaluation of cell viability of test samples against A549 cell line

[0070] [Table 9] Table 2 shows the standard cell viability evaluation for the A549 cell line, and Figure 22 shows the results of the cell viability evaluation of the standard (doxorubicin) for the A549 cell line. Table 2: Standard cell viability evaluation for the A549 cell line

[0071]

Table 10

[0072]

Table 11

Example

[0073] Evaluation of the cytotoxicity of the test sample against the A549 cell line · Preparation of test solutions: For the cytotoxicity study, the test sample was considered as a 100% stock, and serial 2-fold dilutions from 10% to 0.156% were prepared using F-12K plane medium for treatment. A 10 mM stock of standard doxorubicin was diluted from 100 μM to 1.56 μM using F-12K plane medium for treatment. · Cell line and medium: A549 cells were cultured in F-12K medium with appropriate supplements at 37°C in a humidified atmosphere of 5% CO2 until confluence. Cell viability was checked using a conventional hemocytometer. Appropriate cell densities were prepared per 1 ml of medium, seeded in 96-well plates, and incubated at 37°C for 24 hours with 5% CO2 to evaluate cell viability. ·procedure: 1. Measure the number of cells using complete culture medium: 5 × 10 5 The cell concentration was adjusted to cells / ml. 100 μl of diluted cell suspension (50,000 cells / well) was added to each well of a 96-well microtiter plate and incubated at 37°C for 24 hours in a 5% CO2 atmosphere. 2. After 24 hours, the cells were treated with various concentrations of the test compound and incubated at 37°C for 24 hours in a 5% CO2 atmosphere. 3. After incubation, the test solution in the wells was discarded, and 100 μl of MTT (5 mg / 10 ml of MTT in PBS) was added to each well. The plate was incubated at 37°C in a 5% CO2 atmosphere for 4 hours. 4. Remove the supernatant, add 100 μl of DMSO, and gently shake the plate to solubilize the formed formazan. Absorbance was measured at a wavelength of 590 nm using a microplate reader. 5. Calculate the growth inhibition rate using the following formula. If the sample shows significant inhibition, determine the concentration of the test compound (IC) required to inhibit cell proliferation by 50%. 50 The values ​​are created from the dose-response curve of the cell line. • Calculation of inhibition: % inhibition = (OD of sample - OD of control / OD of control) × 100 Table 1 shows the cytotoxicity evaluation of the test samples against the A549 cell line, and Figures 23-24 show bar graphs and line graphs of the cytotoxicity of the test samples after treatment. Table 1: Evaluation of cytotoxicity of test samples against A549 cell line

[0074] [Table 12] Table 2 shows the cytotoxicity evaluation of the standard (doxorubicin) against the A549 cell line, and Figures 25-26 show bar graphs and line graphs of cytotoxicity after treatment with the standard (doxorubicin). Table 2: Standard cytotoxicity assessment of A549 cell line

[0075] [Table 13] ·result: The test sample SACC and the standard drug doxorubicin each underwent IC (Information Confirmation). 50 As demonstrated by the values, the inhibitory effect on A549 cells was observed. Test sample SACC and standard doxorubicin showed IC50 levels of inhibition of 1.35% (0.65 μM) and 20.3 μM, respectively, in A549 cells. 50 The values ​​are shown. Figures 23-26 show a comparison of the cytotoxic effects of SACC and doxorubicin. Summary of results

[0076] [Table 14] SACC IC 50 The lower values ​​suggest a higher efficacy in inhibiting A549 cell proliferation compared to standard doxorubicin. These findings highlight the promising anti-cancer properties of SACC as a potential therapeutic agent. [Examples]

[0077] Therapeutic activity of SACC for pancreatic cancer 1. We evaluated the therapeutic efficacy of SACC in a mouse model of ectopic xenografting of human pancreatic cancer cells (AsPC1). The results shown in Figures 27-30 demonstrate the inhibitory effect of SACC on pancreatic tumor growth in a xenograft mouse model of pancreatic cancer. In athymic nude mice with xenograft tumors derived from AsPC1 cells, intraperitoneal administration of 200 μl of SACC significantly inhibited tumor growth. Figure 27-A shows the tumor volume at the indicated time points for both the control group and the SACC-administered group. The values in the graph represent the average of 8 mice in the control group and 9 mice in the SACC-treated group. SACC was administered 5 days a week for 6 consecutive weeks. The administration of SACC, indicated by a significant decrease in tumor volume, emphasizes its potential as a promising anti-tumor agent. Figure 27 B shows the tumor mass of mice in both the control group and the SACC group at week 9. The values in the graph represent the average of 8 mice in the control group and 9 mice in the SACC-treated group. The graph shows a substantial decrease in tumor mass in the SACC-treated group. This further supports the anti-tumor effect of SACC in inhibiting the growth of pancreatic tumors. In Figure 29, the line graph shows the growth pattern of AsPC1 cell xenograft tumors in control group mice. The graph provides a visual representation of tumor development over time and insights into the dynamics and progression of tumors in the absence of SACC treatment. In Figure 30, the line graph shows the growth pattern of AsPC1 cell xenograft tumors in SACC-treated group mice. This graph visually represents the effect of SACC administration on the tumor growth curve over the experimental period. Overall, the consistent decrease in tumor volume and mass in the SACC-treated group compared to the control group suggests a significant inhibitory effect of SACC on pancreatic tumor growth in this xenograft mouse model. The conclusion drawn from these findings is that SACC shows potential as a chemotherapeutic agent against pancreatic cancer.

[0078] 2. SACC inhibits the proliferation of pancreatic cancer stem cell xenograft tumors in SCID mice. The effect of SACC on pancreatic cancer stem cell xenografts in SCID mice was investigated, and a total of 8 mice were used in the study. Briefly, 2000 viable cells (1:1 ratio of culture medium + Matrigel) in a 100 μl volume were subcutaneously injected into SCID mice. Two weeks later, SACC (250 μl) was administered intraperitoneally 5 days a week for 2 weeks. When the tumors in the control mice reached a target volume of 1500 mm 3 all the mice were sacrificed. Figure 31A shows images of mice with xenograft tumors in both the control and SACC-treated groups. Images in the lower panel show the xenograft tumors removed from both sets of mice, allowing for a visual comparison of tumor features between the control and SACC-treated groups. Figure 31B shows the tumor volume of xenografted tumors, and this graph shows the change in tumor size over the indicated weeks for both the control group and the SACC treatment group. The graph provides quantitative data on the effect of SACC treatment on tumor volume compared to the control group. Figure 31C shows the amount of xenograft tumor removed from mice in both the control and SACC-treated groups at week 5. The comparison of tumor mass provides insight into the inhibitory effect of SACC on tumor growth and further supports the findings in Figure 31B. Figure 33 investigates the effects of SACC on the expression of the transcription factor GLI-1 and the chemokine receptor CXCR4. Representative immunohistochemical images show the expression levels of GLI-1 and CXCR4 in control and SACC xenograft tumors. The results show that SACC partially inhibits both GLI-1 and CXCR4, suggesting a potential antitumor effect. [Examples]

[0079] Molecular mechanism of SACC that inhibits the proliferation of pancreatic cancer Investigation of the effects of SACC on MiaPaCa-2 and AsPc-1 cells using the MTT assay: The anticancer efficacy of SACC was investigated in pancreatic cancer cells, specifically MiaPaCa-2 and AsPc1, using the previously described MTT method (Non-Patent Literature 1). MiaPaCa-2 and AsPc-1 cells maintained in DMEM and RPMI-1640 medium, respectively, were placed in 96-well plates in quantities of 5 × 10⁶. 3Cells were seeded at a cell / well seeding density and left overnight in 5% CO2 and 37°C for adhesion. After 24 hours, SACC at various concentrations (0, 0.625%, and 1.25%, v / v) was added to these adhered MiaPaCa-2 and AsPc-1 cells and incubated for a further 24 hours under similar growth conditions. After incubation, 0.2 mL of MTT (20% v / v, prepared in either DMEM or RPMI-1640 medium) was added to these cells and incubated in the dark at 37°C for 4 hours. Formazan crystal formation was evaluated by solubilizing them in 0.15 mL of DMSO, then placing the plate on a shaker and reading the plate at 570 nm.

[0080] Immunoblotting MiaPaCa-2 and AsPc-1 cells were divided into 1.25 × 10⁻¹⁴ cells. 6MiaPaCa-2 and AsPc1 cells were seeded at a seeding density in 100 mm culture dishes and incubated at 37°C under 5% CO2. After reaching a concentration of 70%, MiaPaCa-2 and AsPc1 cells were treated with 0.625% and 1.25% (v / v) SACC for 24 hours, while untreated cells were used as controls for each cell type. 24 hours after treatment, the cells were lysed in ice-cold RIPA buffer (containing protease and phosphatase inhibitors). The lysates were then centrifuged at 14,000 × g for 15 minutes at 4°C, and protein was subsequently quantified using the bicinchoninic acid (BCA) method. 20 micrograms of protein from both untreated and treated cell lysates were denatured in a sample buffer containing β-mercaptoethanol at 95°C for 7 minutes, and then separated on a 10% gel at a constant voltage of 75 V. The isolated proteins were transferred onto a PVDF membrane using Bio rad's TransBlot® Turbo® Transfer System (catalog number #1704150 EDU). After transcription, the membrane is blocked on a shaker at 20 rpm for 2 hours with either 5% skim milk or 5% bovine serum albumin (prepared in 1×TBST), and then the target proteins, namely HER2 / ErbB2 (Cell Signaling Technology; catalog number #4290S; dilution 1:1000), pHER2 (dilution 1:1000), p53 (Santa Cruz Biotechnology; dilution 1:3000); p21 (abcam; catalog number #ab18209; dilution 1:1000), Bcl2 (Santa Cruz Biotechnology; catalog number #Sc-7382; dilution 1:1000); Bax (Santa Cruz Biotechnology; Cat.#sc-65582; dilution 1:1000), PARP (dilution 1:1000), and β-actin (Santa Cruz Biotechnology; Catalog No. #sc-47778; Dilution 1:2000). After overnight incubation with the primary antibody, the membrane was washed three times with 1×TBST at 60-70 rpm to remove unbound antibody, and then incubated with either anti-mouse or anti-rabbit HRP conjugate antibody at 20 rpm for 1 hour. The unbound secondary antibody was washed with 1×TBST as described for the primary antibody.Protein bands were detected using Immobilon Western Chemiluminescent HRP Substrate (Cat#WBKLS0500), which is based on the highly sensitive detection of chemiluminescence generated from immunoreactive bands. All antibodies used were validated by the generated band representing the predicted molecular weight (kDa) of the target protein.

[0081] Results and Discussion SACC has the ability to induce apoptosis, programmed cell death, and inhibit the proliferation of MiaPaCa-2 and AsPc-1 cells. This means that SACC may have potential anti-cancer properties by inducing cell death mechanisms and limiting the proliferation of these specific cancer cell lines. The observation of apoptosis induction and proliferation inhibition in these cells highlights the potential anti-cancer therapeutic efficacy of SACC. There are distinct strategies for determining the antiproliferative effects of any treatment regimen against various cancer cells, and the MTT assay, used to assess cell viability, is one of the most preferred and primary strategies (Non-Patent Literature 1). In the same context, we investigate the effect of SACC treatment on the viability of two pancreatic cancer cell lines, namely MiaPaCa-2 cells and AsPc-1 cells, which have mutated p53 and WT p53, respectively. Findings from this cell viability assay demonstrate that SACC significantly inhibited the proliferation of pancreatic cancer cells, regardless of p53 mutation. IC of SACC in AsPC1 cells 50 It was observed to be 0.37% (v / v), which corresponds to IC in MiaPaCa-2. 50 This was demonstrated (this experiment will be continued). Furthermore, the effect of SACC on apoptosis in PanCa cells was evaluated. SACC (1.25% v / v) treatment showed significant induction of apoptosis in MiaPaCa-2 cells. These results provide evidence for the potential of SACC to kill pancreatic cancer cells by inducing apoptosis. Figure 34 shows the effect of SACC on the survival rate and apoptosis of pancreatic cancer cells.

[0082] a) SACC targets the HER-2 and p53 signaling pathways in pancreatic cancer cells. Human epidermal growth factor receptor-2 (HER-2), also known as c-Neu / ErbB2, has shown significant effects on the pathophysiology of various cancers, most importantly pancreatic cancer, but recent studies have already warned that its role in a range of other cancers cannot be ruled out [2–7]. Even genetic defects in HER-2 correlate with cancer severity through enhancement of HER-2 function [3], making HER-2 a primary therapeutic target in the management of various cancers. Furthermore, HER-2, alone or in conjunction with MUC4, correlates with reduced efficacy of currently available chemotherapy, particularly resistance to gemcitabine [7, 8]. In contrast, HER-2-targeted therapies have shown potential to sensitize gemcitabine-resistant pancreatic cells [9]. In this context, a range of anti-HER-2 treatment regimens are being explored, among which tyrosine kinase inhibitors are considered a candidate drug for HER-2-positive cancer phenotypes

[10] . Like other chemotherapy drugs, even lapatinib, the most well-studied HER-2 blocker, has been associated with serious adverse events, including cardiotoxicity.

[11] Therefore, considering these adverse events associated with different HER-2 targeted therapies, the inventors of this disclosure investigated whether SACC could target HER-2 in pancreatic cancer cells. In this study, Western blot analysis was performed to examine the effect of SACC on HER-2 expression. The results revealed that SACC dose-dependently downregulated HER-2 protein expression in both cell lines. Interestingly, SACC treatment enhanced HER-2 phosphorylation in both AsPC-1 and MiaPaCa-2 cells at higher concentrations (Figure 35). This effect was greater in WT p53-expressing AsPC-1 cells compared to mutant p53-expressing MiaPaCa-2 cells. This result is supported by a recently published study that showed enhanced HER-2 phosphorylation in AsPC-1 cells treated with the chemotherapeutic drug gemcitabine [9].

[0083] b) SACC regulates the expression of apoptotic proteins in pancreatic cancer cells. During apoptosis, PARP cleavage is a useful feature of this type of cell death. This cleavage has been well studied and is mediated by caspases 3 and 7, proteases that are activated during apoptosis

[12] . Similarly, in this study, it has been reported that the dose-dependent cytotoxicity of SACC was mediated by PARP protein cleavage and subsequent apoptotic events in MiaPaCa-2 and AsPc1 cells. Most interestingly, SACC treatment resulted in a marked suppression of mutant p53 in MiaPaCa-2 cells, and mutant p53 (exons 3, 6, and 7; R89W, R116W, R209W, R248W, C265T, C346T, C625T, and C742T) are known to exist [9,13], and the presence of these mutant p53s is also associated with gemcitabine resistance in pancreatic cancer cells, particularly MiaPaCa-2

[14] . Based on these previous observations, the downregulation of mutant p53 in MiaPaCa-2 cells in this study demonstrates the antiproliferative efficacy of SACC. Interestingly, SACC treatment induced p53 expression in AsPC-1 cells [9]. These results suggest that SACC induces apoptosis-mediated cell death in pancreatic cells by targeting mutant p53, WT p53, and HER-2. Similarly, overexpression of various p53 target genes, namely p21, is also associated with tumor suppression and growth inhibition / cell arrest

[15] . The findings from our study are particularly interesting because we reported overexpression of p21 in AsPC-1 cells, which may be due to the induction of WT p53 by SACC treatment. Unlike in AsPC-1 cells, p21 expression was not altered in SACC-treated MiaPaCa-2 cells. Furthermore, the apoptotic process is extremely complex and is tightly regulated by the coordinated action of distinct mediators, including but not limited to Bcl-2, Bcl-2-related X, and apoptotic regulators (BAX)

[16] . BAX is known to induce permeabilization of the mitochondrial outer membrane, while Bcl-2 plays a negative role in the phenomenon of apoptosis

[16] . In the same context, treatment with SACC significantly upregulated the BAX / Bcl-2 ratio in a dose-dependent manner in both AsPc-1 and MiaPaCa-2 cells. These findings clearly suggest that SACC exerts its antiproliferative capacity against pancreatic cancer by targeting the HER-2 / p53 / BAX-Bcl-2 axis.

[0084] conclusion SACC induces apoptosis and inhibits the proliferation of pancreatic cancer cells, regardless of mutant p53. SACC inhibits HER-2 expression but increases its phosphorylation at higher doses. SACC induces the expression of wild-type p53 protein but reduces the levels of mutant p53 protein. SACC induces the expression of the pro-apoptotic protein Bax and inhibits the anti-apoptotic protein Bcl2. SACC induces PARP protein cleavage. The effect of SACC on WT p53 expression in CaPan-2 cells. These cells express WT p53. The p53 state in AsPC-1 cells has been reported to be cleaved. However, many studies have reported that these cells express WT p53. SACC compositions are also being studied for prostate xenografts and TNBC. SACC is also being studied for CWR22Rv1 cell xenograft tumors in athymic nude mice. Oral feeding of Satcon inhibits xenograft tumors derived from CWR22v1 cells. Briefly, a total of 8 athymic mice were used in the study. 4 × 10 6 Cells were subcutaneously transplanted onto the dorsal surface of the flank of each mouse. The mice were divided into two groups. After one week, one group of mice was orally administered SACC for three weeks (300 μl, 5 days a week), while the other group was administered physiological saline. Tumor volume was recorded at weeks 3 and 4. The tumor volume of the control mice was approximately 2000 mm². 3 When the tumor reached a certain point, all mice in both groups were sacrificed. Blood was collected, the tumors were excised, and their mass was measured using an electronic balance. At the end of the experiment, the SACC-treated tumor volume was 400 mm³. 3 The tumor mass decreased from 3.3 g to 1 gm. Significant activity was observed. This is a potent inhibitor of TNBC xenograft tumors. [Examples]

[0085] Therapeutic activity of SA1 (peak 5) and SA2 (peak 4) against the human breast cancer cell line MDA-MB-231 The therapeutic effects of SA1 (peak 5) and SA2 (peak 4) were evaluated based on the drug concentration (GI) of the test compound required to inhibit cell proliferation by 50%. 50 The growth inhibition rate, which is a value, is used to evaluate the human lung cancer cell line MDA-MB-231. Table 1 shows the percentage of cell proliferation and drug concentration.

[0086] [Table 15] Table 2 shows the effects of SA1 (peak 5) and SA2 (peak 4) on cell viability and inhibition of tumor cell proliferation.

[0087] [Table 16] *≦10 ―6 GI in moles (i.e., 1 μmol) or ≤ 10 μg / ml 50 The value is thought to indicate activity in the case of the pure compound. For extracts, GI 50 A value of ≤20 μg / ml is considered to indicate activity. *GI 50 These test values ​​in the column indicate activity. LC 50 = 50% concentration of a drug that causes cell death GI 50 =Concentration of a drug that causes a 50% inhibition of cell proliferation TGI = the concentration of a drug that causes complete inhibition of cell proliferation. ADR = Adriamycin, positive control compound NE = Data that cannot be evaluated. The experiment needs to be repeated using different sets of drug concentrations. Unstable data = Unstable data can occur due to the low solubility of the compound. ·result: Test samples SA1 (peak 5) and SA2 (peak 4) have their respective GI values. 50 Furthermore, as evidenced by the TGI values, inhibitory effects were observed on MDA-MB-231 cells. Figure 37 shows the inhibition of cell proliferation for SA1 (peak 5), SA2 (peak 4), and Adriamycin. [Examples]

[0088] Therapeutic activity of SACC against human breast cancer cell line MDA-MB-231 The therapeutic effect of SACC is determined by the drug concentration (GI) of the test compound required to inhibit cell proliferation by 50%. 50 The growth inhibition rate, which is a value, is used to evaluate the human lung cancer cell line MDA-MB-231. Table 1 shows the percentage of cell proliferation and drug concentration.

[0089] [Table 17] SACCRT: SACC stored at 20°C for 6 months. SACCD / RT: Fresh SACC samples A6 / 2023: SACC stored at 6℃ for 6 months Table 2 shows the effects of SACC on cell viability and inhibition of tumor cell proliferation.

[0090] [Table 18] *≦10 ―6 GI in moles (i.e., 1 μmol) or ≤ 10 μg / ml 50 The value is thought to indicate activity in the case of the pure compound. For extracts, GI 50 A value of ≤20 μg / ml is considered to indicate activity. *GI 50 These test values ​​in the column indicate activity. ·result: The test sample SACC and the control drug Adriamycin each received IC (Information Confirmation). 50 GI 50 As evidenced by the TGI values, these drugs showed inhibitory effects on MDA-MB-231 cells. Figure 38 shows the inhibition of cell proliferation by SACC and Adriamycin. [Examples]

[0091] Therapeutic activity of SA1 (peak 5) and SA2 (peak 4) against human lung cancer cell line A-549 The therapeutic effects of SA1 (peak 5) and SA2 (peak 4) were evaluated based on the drug concentration (GI) of the test compound required to inhibit cell proliferation by 50%. 50 The human lung cancer cell line A-549 is evaluated by calculating the proliferation inhibition rate, which is a value of ). Table 1 shows the percentage of cell proliferation and drug concentration.

[0092] [Table 19] Table 2 shows the effects of SA1 (peak 5) and SA2 (peak 4) on cell viability and inhibition of tumor cell proliferation.

[0093] [Table 20] *≦10 ―6 GI in moles (i.e., 1 μmol) or ≤ 10 μg / ml 50 The value is thought to indicate activity in the case of the pure compound. For extracts, GI 50 A value of ≤20 μg / ml is considered to indicate activity. *GI 50 These test values ​​in the column indicate activity. NE = Data that cannot be evaluated. The experiment needs to be repeated using different sets of drug concentrations. Unstable data = Unstable data can occur due to the low solubility of the compound. ·result: Both the test sample SA2 (peak 4) and the control drug Adriamycin showed inhibitory effects against A-549. The inhibitory effect of SA2 was due to its GI. 50 This is indicated by the value, while the inhibition of Adriamycin is shown by its respective LC 50 GI 50 This is supported by the TGI value. Figure 39 shows the inhibition of cell proliferation for SA1 (peak 5), SA2 (peak 4), and Adriamycin. [Examples]

[0094] Therapeutic activity of SACC against human lung cancer cell line A-549 The therapeutic effect of SACC is determined by the drug concentration (GI) of the test compound required to inhibit cell proliferation by 50%. 50 The human lung cancer cell line A-549 is evaluated by calculating the proliferation inhibition rate, which is a value of ). Table 1 shows the percentage of cell proliferation and drug concentration.

[0095] [Table 21] Table 2 shows the effects of SACC on cell viability and inhibition of tumor cell proliferation.

[0096] [Table 22] *≦10 ―6 GI in moles (i.e., 1 μmol) or ≤ 10 μg / ml 50 The value is thought to indicate activity in the case of the pure compound. For extracts, GI 50 A value of ≤20 μg / ml is considered to indicate activity. *GI 50 These test values ​​in the column indicate activity. ·result: The test sample SACC and the control drug Adriamycin were each subjected to LC (Low-Cooling) testing. 50 and GI 50 Furthermore, as evidenced by the TGI values, the inhibitory effect on A549 cells was observed. The test samples SACC and Adriamycin showed LC values ​​of 46.9 and 72, respectively. 50 The values ​​are shown. Figure 40 shows the inhibition of cell proliferation by SACC and Adriamycin. [Examples]

[0097] Therapeutic activity of SA1 (peak 5) and SA2 (peak 4) against the human pancreatic cancer cell line Mia-Pa-Ca-2 The therapeutic effects of SA1 (peak 5) and SA2 (peak 4) were evaluated based on the drug concentration (GI) of the test compound required to inhibit cell proliferation by 50%. 50 The human pancreatic cancer cell line Mia-Pa-Ca-2 is evaluated by calculating the proliferation inhibition rate, which is a value of ). Table 1 shows the percentage of cell proliferation and drug concentration.

[0098] [Table 23] Table 2 shows the effects of SA1 (peak 5) and SA2 (peak 4) on cell viability and inhibition of tumor cell proliferation.

[0099] [Table 24] *≦10 ―6 GI in moles (i.e., 1 μmol) or ≤ 10 μg / ml 50 The value is thought to indicate activity in the case of the pure compound. For extracts, GI 50 A value of ≤20 μg / ml is considered to indicate activity. *GI 50 These test values ​​in the column indicate activity. NE = Data that cannot be evaluated. The experiment needs to be repeated using different sets of drug concentrations. ·result: The test samples SA1 (peak 5), SA2 (peak 4), and the control drug Adriamycin showed inhibitory effects on Mia-Pa-Ca-2. The inhibitory effects of SA1 and SA2 were compared to their GI. 50 and is indicated by the TGI value. Inhibition of Adriamycin is indicated by its respective LC 50 GI 50 This is indicated by the TGI value. Figure 41 shows the inhibition of cell proliferation for SA1 (peak 5), SA2 (peak 4), and Adriamycin. [Examples]

[0100] Therapeutic activity of SACC against the human pancreatic cancer cell line Mia-Pa-Ca-2 The therapeutic effect of SACC is determined by the drug concentration (GI) of the test compound required to inhibit cell proliferation by 50%. 50 The human lung cancer cell line Mia-Pa-Ca-2 is evaluated by calculating the proliferation inhibition rate, which is a value of ). Table 1 shows the percentage of cell proliferation and drug concentration.

[0101] [Table 25] Table 2 shows the effects of SACC on cell viability and inhibition of tumor cell proliferation.

[0102] [Table 26] *≦10 ―6 GI in moles (i.e., 1 μmol) or ≤ 10 μg / ml 50 The value is thought to indicate activity in the case of the pure compound. For extracts, GI 50 A value of ≤20 μg / ml is considered to indicate activity. *GI 50 These test values ​​in the column indicate activity. ·result: The test sample SACC and the control drug Adriamycin underwent their respective LC tests. 50 and GI 50 Furthermore, as evidenced by the TGI values, it showed an inhibitory effect on Mia-Pa-Ca-2 cells. Figure 42 shows the inhibition of cell proliferation by SACC and Adriamycin. [Examples]

[0103] Evaluation of the therapeutic efficacy of SACC against the triple-negative breast cancer cell line MDA-MB-231 in a human tumor xenograft MDA-MB-231 model using tumor volume and relative tumor volume (RTV) assessment. The therapeutic effect of SACC was evaluated against the triple-negative breast cancer cell line MDA-MB-231 by analyzing tumor volume and relative tumor volume. This study involved the use of six mice. These mice were treated according to the following groups:

[0104] [Table 27] Table 1: Relative tumor volume in groups A, B, C, and D

[0105] [Table 28] *RTV = Relative Tumor Volume = Tumor Volume on Measurement Day / Tumor Volume on Day 1 ·result: The test sample SACC and the control drug Adriamycin showed a reduction in tumor volume in the human tumor xenograft MDA-MB-231 model, as indicated by their relative tumor volumes. Figure 43 shows the relative reduction in tumor volume for SACC and Adriamycin. Table 2: Processing / Control (T / C) values ​​from RTV data in Group A, Group B, Group C, and Group D

[0106] [Table 29] *The portion with T / C ≤ 0.42 (highlighted in blue) is considered to indicate activity. ·result: The test sample SACC and the control drug Adriamycin showed a reduction in tumor volume in the human tumor xenograft MDA-MB-231 model, as indicated by their T / C values ​​obtained from RTV data. Figure 44 shows the decrease in T / C values ​​for SACC and Adriamycin. Table 3: Survival rates in Group A, Group B, Group C, and Group D

[0107] [Table 30] ·result: The test sample SACC and the control drug Adriamycin showed favorable survival rates in the human tumor xenograft MDA-MB-231 model, as indicated by their survival rates. Figure 45 shows 100% survival rates up to day 25 for SACC and Adriamycin. Table 4: Animal body weight data for groups A, B, C, and D.

[0108] [Table 31] *Mortality and weight loss ≥ 4 grams / mouse are considered to indicate toxicity. ·result: The test sample SACC and the control drug Adriamycin showed the smallest change in body weight in the human tumor xenograft MDA-MB-231 model, as indicated by their mean body weight. Figure 46 shows graphs of mean animal body weight for SACC and Adriamycin. Table 5

[0109] [Table 32] Table 5 and Figure 47 show the tumor volume in control group A. Table 6

[0110] [Table 33] Table 6 and Figure 48 show the tumor volume in the positive control ADR (2.5 mg / kg) group B. ·result: The positive control drug adriamycin showed a reduction in tumor volume in the human tumor xenograft MDA-MB-231 model. Table 7

[0111] [Table 34] Table 7 and Figure 49 show the tumor volume in the SACC (4 mL / Kg) C group. ·result: SACC administered at a dose of 4 mL / kg showed a reduction in tumor volume in the human tumor xenograft MDA-MB-231 model. Table 8

[0112] [Table 35] Table 8 and Figure 50 show the tumor volume in the SACC (4 mL / Kg) D group. ·result: SACC administered at a dose of 1.2 mL / kg showed a significant reduction in tumor volume in the human tumor xenograft MDA-MB-231 model.

[0113] Specific embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Naturally, variations of these described embodiments will be apparent to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will use such variations as appropriate, and the inventors intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is encompassed by the Invention unless otherwise shown herein or unless it is clearly inconsistent with the context. Furthermore, numerous references to patents and printed publications are made throughout this specification. Each of the above references and publications is incorporated herein by reference, individually or in whole. Finally, it should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be used are within the scope of the invention. Therefore, alternative configurations of the present invention may be used in accordance with the teachings herein, not as examples but as examples. Accordingly, the present invention is not limited to those precisely illustrated and described. References

[0114] [Table 36] TIFF2026513637000063.tif225170 TIFF2026513637000064.tif42170

Claims

1. The following formula: (i) 【Chemistry 1】 Equation (I): 6-Hydroxy-3H-Isochromene-3,8(4H)-dione (ii) 【Chemistry 2】 Formula (II): 5-methyl-1H-indole-3-carboxylic acid (iii) 【Transformation 3】 Formula (III): 6-hydroxy-3-methyl-2H-pyran-2-one, and (iv) 【Chemistry 4】 Formula (IV) 2-methyl-4H-pyran-4-one A pharmaceutical composition comprising at least one compound selected from the compounds represented by or combinations thereof.

2. Furthermore, the pharmaceutical composition according to claim 1, comprising a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 1, wherein the amount of the compound of formula (I) is in the range of 6% by mass to 18% by mass.

4. The pharmaceutical composition according to claim 3, wherein the amount of the compound of formula (I) is 12.60% by mass.

5. The pharmaceutical composition according to claim 1, wherein the amount of the compound of formula (II) is in the range of 8% by mass to 22% by mass.

6. The pharmaceutical composition according to claim 5, wherein the amount of the compound of formula (II) is 15.41% by mass.

7. The pharmaceutical composition according to claim 1, wherein the amount of the compound of formula (III) is in the range of 2% by mass to 10% by mass.

8. The pharmaceutical composition according to claim 7, wherein the amount of the compound of formula (III) is 6.48% by mass.

9. The pharmaceutical composition according to claim 1, wherein the amount of the compound of formula (I) is in the range of 1% by mass to 10% by mass.

10. The pharmaceutical composition according to claim 9, wherein the amount of the compound of formula (IV) is 5.89% by mass.

11. Furthermore, the pharmaceutical composition according to any one of claims 1 to 10, comprising one or more further therapeutic agents.

12. The pharmaceutical composition according to claim 11, wherein the therapeutic agent is an anticancer agent.

13. The pharmaceutical composition according to claim 1, for use as a medicine for treating cancer in a subject.

14. Use of the pharmaceutical composition according to claim 1 for the treatment of cancer.

15. The pharmaceutical composition according to any one of claims 1 to 10, wherein the cancer is selected from pancreatic cancer, prostate cancer, triple-negative breast cancer, invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, or HER2-positive breast cancer.

16. A pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV) for use as a pharmaceutical agent for inducing apoptosis, for inducing the expression of the apoptosis-promoting protein Bax, and for inducing polyadenosine diphosphate-ribose polymerase (PARP) protein cleavage.

17. A pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV), for use as a pharmaceutical agent for inhibiting the expression of human epidermal growth factor receptor-2 (HER-2) positive breast cancer, for inhibiting the anti-apoptotic protein Bcl2, and for inhibiting the proliferation of pancreatic cancer cells.

18. A pharmaceutical composition comprising compounds represented by formulas (I), (II), (III), and (IV), for use as a pharmaceutical for the treatment of prostate cancer, triple-negative breast cancer, invasive cancer, lung cancer, sarcoma, serous cancer, pancreatic cancer, and HER2-positive breast cancer in a subject.

19. The following formula: (i) 【Transformation 5】 Equation (I): 6-Hydroxy-3H-Isochromene-3,8(4H)-dione A compound represented by the formula.

20. The following formula: (iii) 【Transformation 6】 Formula (III): 6-hydroxy-3-methyl-2H-pyran-2-one, A compound represented by the formula.

21. A compound selected from the compounds represented by formulas (I), (II), (III), and (IV) for use in the treatment of cancer.

22. The following formula is derived from barley extract: (i) 【Transformation 7】 Equation (I): 6-Hydroxy-3H-Isochromene-3,8(4H)-dione (ii) 【Transformation 8】 Formula (II): 5-methyl-1H-indole-3-carboxylic acid (iii) 【Chemistry 9】 Formula (III): 6-hydroxy-3-methyl-2H-pyran-2-one, or (iv) 【Chemistry 10】 Formula (IV) 2-methyl-4H-pyran-4-one A method for isolating a compound represented by, where: (i) Perform chromatography of the barley extract by preparative HPLC; (ii) Elute peaks 2, 3, 4 and 5; (iii) The peaks obtained in (ii) above are manually isolated by repeated injection to obtain a fraction; (iv) pooling and concentrating the fraction; and (v) Analyze the peaks and determine the structure of the compound represented by formula (I), formula (II), formula (III), and formula (IV). Including, here, The preparative HPLC method in (i) above includes mobile phase A and mobile phase B; The elution of peaks 2, 3, 4, and 5 in (ii) above occurs at approximately 14.3, 15.5, 16.4, and 23.0 minutes, respectively. method.

23. A pharmaceutical composition according to claim 1, selected from oral, parenteral, nasal, subcutaneous, intradermal, intramuscular, intravenous, intra-articular, intramedullary, intraperitoneal, transmucosal, transdermal, rectal, and topical compositions.

24. A method for preventing or treating cancer in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition containing the compounds of formula (I), formula (II), formula (III), and formula (IV).

25. A method for inducing apoptosis in a subject, for inducing the expression of the apoptosis-promoting protein Bax, and for inducing PARP protein cleavage, comprising administering a therapeutically effective amount of a pharmaceutical composition containing the compounds of formula (I), formula (II), formula (III), and formula (IV).

26. A method for inhibiting HER-2 expression in a subject, inhibiting the anti-apoptotic protein Bcl2, inhibiting the proliferation of pancreatic cancer cells, degrading p53 mutations, and normalizing wild-type p53, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the compounds of formula (I), formula (II), formula (III), and formula (IV).

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