Pharmaceutical composition targeting mutants P53 and HER2

Abstract

The invention relates to a pharmaceutical composition with anticancer activity. More specifically, the present invention relates to compositions and methods for treating cancer. In addition, the present invention relates to pharmaceutical compositions targeting P53 and HER2.

Description

Technical Field

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

[0002] Cancer is a disease that is currently considered incurable. Furthermore, chemotherapy is extremely expensive and beyond the means of most people due to its toxicity and other side effects (which also kill living cells). Patients experience weight loss, decreased appetite, and numerous physical and psychological problems. Scientists worldwide are working tirelessly, investing significant funds and resources to find a cure for cancer.

[0003] Nature is an unlimited and inexhaustible reserve for the development of new and effective drugs and therapies (R. Nabi et al., 2019). In recent years, herbal remedies have become increasingly important in the treatment of disease globally. Since the last century, biological and natural compounds have played a significant role in the treatment and prevention of a wide range of diseases, forming the backbone of traditional medicine and treatment systems.

[0004] The use of natural compounds as a basis for developing new drugs or as complementary or alternative drugs in the field of oncology is already widely adopted. Therefore, several novel cytotoxic compounds are isolated from plants each year, representing new potential anticancer agents.

[0005] Natural compounds, as drugs for treating cancer, have the benefits of reducing side effects and influencing multiple signal transduction pathways involved in the carcinogenesis process.

[0006] Of course, nature is great; it has solutions to all problems, and the goal of this invention is to provide an anticancer composition without side effects.

[0007] Therefore, this study focuses on the anticancer potential of compounds isolated from barley extracts. In this study, compounds isolated from barley extracts as potential anticancer agents were also investigated, including their structural characterization and anticancer activity. Summary of the Invention

[0008] Invention Objective

[0009] The main objective of this invention is to provide a pharmaceutical composition (SACC) comprising the main compounds represented by chemical formulas (I), (II), (III) and (IV) obtained from barley extract.

[0010] Another objective of this invention is to provide compounds isolated from barley extract.

[0011] Another objective of this invention is to provide an HPLC method for detecting all compounds present in barley extract.

[0012] Another objective of this invention is to provide a method for separating compounds from barley extract.

[0013] Another objective of this invention is to provide ESI-MS analysis of compounds and to determine the structures of some compounds isolated from barley extract using NMR and IR spectroscopy.

[0014] Another objective of the present invention is to provide a pharmaceutical composition comprising the compounds described herein for treating cancer.

[0015] Another objective of the present invention is to provide a method for preventing or treating cancer in a subject in need of it, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV).

[0016] Another objective of the present invention is to provide the use of pharmaceutical compositions comprising compounds shown in chemical formulas (I), (II), (III), and (IV) in the production of medicaments for treating cancer.

[0017] Another objective of the present invention is to provide a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV) for use in treating cancer.

[0018] Another objective of the present invention is to provide the use of the compounds shown in chemical formulas (I), (II), (III) and (IV) for the production of medicaments for treating cancer.

[0019] Another objective of the present invention is to provide a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV) as an agent for treating prostate cancer, triple-negative breast cancer, invasive carcinoma, lung cancer, sarcoma, serous carcinoma, pancreatic cancer, and HER2-positive breast cancer in subjects. Invention Overview

[0021] This disclosure provides a pharmaceutical composition comprising at least one compound selected from the following chemical formulas obtained from barley extract.

[0022] a)

[0023]

[0024] Chemical formula (I) for 6-hydroxy-3H-isochromene-3,8(4H)-dione

[0025] b)

[0026]

[0027] 5-Methyl-1H-indole-3-carboxylic acid

[0028] Molecular formula: C 10 H9NO3 chemical formula (II)

[0029] c)

[0030]

[0031] 6-Hydroxy-3-methyl-2H-pyran-2-one

[0032] Molecular formula = C6H6O3 Chemical formula (III)

[0033] d)

[0034]

[0035] 2-Methyl-4H-pyran-4-one

[0036] Molecular formula = C6H6O2 Chemical formula (IV)

[0037] Or a combination thereof.

[0038] This disclosure provides an HPLC method for detecting compounds of chemical formulas (I), (II), (III) and (IV) present in barley extracts.

[0039] This disclosure provides a method for isolating the compound from barley extract.

[0040] This disclosure provides ESI-MS analysis of the compounds and uses NMR and IR spectroscopy to determine the structures of the compounds isolated from barley extract.

[0041] This disclosure provides pharmaceutical compositions comprising compounds of formulas (I), (II), (III), and (IV) obtained from barley extract, said pharmaceutical compositions for (i) inducing apoptosis and inhibiting the growth of pancreatic cancer cells unrelated to 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 treating prostate cancer, triple-negative breast cancer (TNBC), invasive carcinoma, lung cancer, sarcoma, serous carcinoma, pancreatic cancer, and HER2-positive breast cancer in subjects.

[0042] This disclosure provides the use of a pharmaceutical composition comprising barley extract as an active ingredient in the manufacture of a pharmaceutical agent for (i) inducing apoptosis and inhibiting the growth of pancreatic cancer cells unrelated to mutant p53 in subjects, (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, invasive carcinoma, lung cancer, sarcoma, serous carcinoma, pancreatic cancer, and HER2-positive breast cancer.

[0043] This disclosure provides methods for inducing apoptosis and inhibiting the growth of pancreatic cancer cells unrelated to 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 treating prostate cancer, triple-negative breast cancer, invasive carcinoma, lung cancer, sarcoma, serous carcinoma, pancreatic cancer, and HER2-positive breast cancer in subjects.

[0044] This disclosure provides pharmaceutical compositions that can be used as medicaments for treating cancer in subjects.

[0045] This disclosure provides for the use of the pharmaceutical composition in the treatment of cancer.

[0046] This disclosure provides a pharmaceutical composition for treating pancreatic cancer.

[0047] This disclosure provides pharmaceutical compositions comprising compounds of chemical formulas (I), (II), (III), and (IV) obtained from barley extracts for the inhibition and treatment of prostate cancer, triple-negative breast cancer, invasive carcinoma, lung cancer, sarcoma, serous carcinoma, pancreatic cancer, and HER2-positive breast cancer. Attached Figure Description

[0048] To facilitate understanding and practical application of this disclosure, exemplary embodiments will now be described with reference to the accompanying drawings. These drawings, together with the following detailed description, are incorporated in and form part of this specification and serve to further illustrate the embodiments and explain the various principles and advantages of this disclosure, wherein:

[0049] Figure 1 The HPLC chromatograms of barley extract (crude, B. No: KWO 037) at different UV wavelengths (nm) are shown.

[0050] Figure 2 The figure shows a typical preparative HPLC chromatogram.

[0051] Figure 3The typical maximum absorbance plot (200-400 nm) HPLC chromatogram is shown, and peak 2 is indicated.

[0052] Figure 4 A typical HPLC chromatogram at 215 nm is shown, and peak 2 is indicated.

[0053] Figure 5 The IR spectrum of peak 2 is shown.

[0054] Figure 6 The ESI mass spectrum of peak 2 is shown.

[0055] Figure 7 Peak 2 is shown. 1 H NMR spectrum.

[0056] Figure 8 The typical maximum absorbance (200-400 nm) HPLC chromatogram is shown, and peak 4 is indicated.

[0057] Figure 9 A typical HPLC chromatogram at 274 nm is shown, and peak 4 is indicated.

[0058] Figure 10 The IR spectrum of peak 4 is shown.

[0059] Figure 11 The ESI mass spectrum of peak 4 is shown.

[0060] Figure 12 Peak 4 is shown. 1 H NMR spectrum.

[0061] Figure 13 The typical maximum absorbance (200-400 nm) HPLC chromatogram is shown, and peak 5 is indicated.

[0062] Figure 14 A typical HPLC chromatogram at 291 nm is shown, and peak 5 is indicated.

[0063] Figure 15 The IR spectrum of peak 5 is shown.

[0064] Figure 16 The ESI mass spectrum of peak 5 is shown.

[0065] Figure 17 Peak 5 is shown. 1 H NMR spectrum.

[0066] Figure 18 The bar chart shows the kinase response (1 / 30 final dilution SACC).

[0067] Figure 19 The bar chart shows the kinase response (1 / 120 final dilution SACC).

[0068] Figure 20 The diagram shows the method used to determine the IC. 50 Complete inhibitor titration.

[0069] Figure 21 The graph shows the cell viability of the test samples after treatment with the A549 cell line.

[0070] Figure 22 The graph shows the cell viability of the standard (doxorubicin) after treatment with the A549 cell line.

[0071] Figure 23 The graph shows the cell inhibition of the test sample after treatment with A549 cells in the MTT assay.

[0072] Figure 24 The inhibitory activity and IC50 of the tested samples against A549 cells were shown in the MTT assay. 50 A graph of value.

[0073] Figure 25 A graph showing the cell inhibition of A549 cells after treatment with the standard (doxorubicin) in the MTT assay.

[0074] Figure 26 The results showed that the standard (doxorubicin) exhibited inhibitory activity and IC50 against A549 cells in the MTT assay. 50 A graph of value.

[0075] Figure 27 A. A representative line graph showing tumor volume at specified time points in the control group and the SACC treatment group. The values ​​in the figure are the average values ​​of n=8 mice in the control group and n=9 mice in the SACC treatment group. SACC was administered 5 days a week for 6 consecutive weeks.

[0076] Figure 27 B. A representative bar chart showing tumor weight in control and SACC mice at week 9. The values ​​in the chart are the mean values ​​for n=8 and n=9 mice in the control and SACC treatment groups, respectively.

[0077] Figure 28 Images from a 12-week study in control and SACC-treated mice.

[0078] Figure 29 The image shows a line graph of xenografted tumors from AsPC1 cells in control mice.

[0079] Figure 30The image shows a line graph of xenografted tumors of AsPC1 cells in the SACC group mice.

[0080] Figure 31 A. Images of control and SACC-treated mice with xenograft tumors. The photographs in the lower plate show the resected xenograft tumors in control and SACC-treated mice.

[0081] Figure 31 B. Shows the tumor volume of xenograft tumors in the control and SACC-treated mice at specified weeks, with the blue line representing the control and the red line representing the SACC. Figure 31 C. Shows the weight of excised xenograft tumors in the control and SACC-treated mice at week 5.

[0082] Figure 32 Histopathological results of Pan-CSC-derived xenograft tumors in control and SACC-treated mice.

[0083] Figure 33 Effects of SACC on the expression of transcription factor GLi-1 and chemokine receptor CXCR4. Representative immunohistochemical images of GLi-1 and CXCR4 expression in control and SACC xenograft tumors.

[0084] Figure 34 The effect of SACC on pancreatic cancer cell viability and apoptosis:

[0085] A. The effect of SACC on the cell growth of AsPc1 cells, as determined by MTT assay.

[0086] B. Effects of control and SACC on apoptosis induction in MiaPaCa-2 cells, as evaluated by flow cytometry.

[0087] Figure 35 Effects of SACC on the expression of HER-2 / p53 and apoptosis proteins in pancreatic cancer cells.

[0088] Figure 36 The percentage of peak area is displayed in the HPLC chromatogram.

[0089] Figure 37 The roles of SA1 (peak 5) and SA2 (peak 4) in the human breast cancer cell line MDA-MB-231, as shown by the growth curve, compared with the control (doxorubicin).

[0090] Figure 38 The role of SACC in the human breast cancer cell line MDA-MB-231, as shown by growth curves, compared with the control (doxorubicin).

[0091] Figure 39The roles of SA1 (peak 5) and SA2 (peak 4) in the human lung cancer cell line A-549, as shown by the growth curve, compared with the control (doxorubicin).

[0092] Figure 40 The role of SACC in the human lung cancer cell line A-549, as shown by growth curves, compared with the control (doxorubicin).

[0093] Figure 41 The roles of SA1 (peak 5) and SA2 (peak 4) in the human pancreatic cancer cell line Mia-Pa-Ca-2, as shown by the growth curve, compared with the control (doxorubicin).

[0094] Figure 42 The role of SACC in the human pancreatic cancer cell line Mia-Pa-Ca-2, as shown by growth curves, compared with the control (doxorubicin).

[0095] Figure 43 The image shows a plot of relative tumor volume (RTV) in the human tumor xenograft model MDA-MB-231 from the AD group.

[0096] Figure 44 A plot showing the T / C values ​​from RTV data in the human tumor xenograft model MDA-MB-231 of the AD group.

[0097] Figure 45 The graph shows the survival percentage in the human tumor xenograft model MDA-MB-231 in the AD group.

[0098] Figure 46 The figure shows the average animal body weight in the MDA-MB-231 human tumor xenograft model in the AD group.

[0099] Figure 47 The figure shows the tumor volume in the human tumor xenograft model MDA-MB-231 from group A.

[0100] Figure 48 The figure shows the tumor volume in the human tumor xenograft model MDA-MB-231 in group B.

[0101] Figure 49 The figure shows the tumor volume in the human tumor xenograft model MDA-MB-231 in group C.

[0102] Figure 50 The figure shows the tumor volume in the human tumor xenograft model MDA-MB-231 in group D. Detailed Implementation

[0103] At the outset of the detailed description, it is understood that the following description merely illustrates specific forms of the invention. However, these specific forms are merely exemplary embodiments and are not intended to limit the scope of the invention in any way. Therefore, this description should be understood as exemplary embodiments and teachings of the invention, and not as limiting.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods and materials are described. For the purposes of this invention, the following terms are defined.

[0105] The article “a (one)” is used in this text to refer to one (one) or more (more than one) (i.e., at least one (at least one)) of the grammatical objects of the article. For example, “an element” means one element or more than one element.

[0106] As used herein, the term "compound" includes the compounds disclosed in this invention.

[0107] As used herein, SACC refers to a composition comprising compounds of formulas (I), (II), (III), and (IV) obtained from barley extracts.

[0108] As used herein, the terms “comprising” or “including” are generally used in the sense of inclusion, that is, allowing the presence of one or more features or components.

[0109] As used herein, the terms “optional” or “optionally” mean that the event or situation described below may or may not occur, and the description includes both the scenario in which the event or situation occurs and the scenario in which it does not occur.

[0110] As used herein, the term “prevention” refers to methods for preventing the onset of a disease and / or its accompanying symptoms or for preventing a subject from becoming ill. As used herein, “prevention” also includes delaying the onset of a disease and / or its accompanying symptoms and reducing the risk of a subject becoming ill.

[0111] As used herein, the term "therapeutic effective amount" refers to an amount of active ingredient applied that is sufficient to prevent the development of one or more symptoms of the condition or symptom to be treated, or to alleviate said symptoms to a certain extent.

[0112] As used in this article, the term "treatment" refers to methods for alleviating or eliminating a disease and / or its accompanying symptoms.

[0113] As used herein, the terms “composition” and “formulation” are interchangeable and have the same meaning.

[0114] Each embodiment is provided by way of explanation, not limitation. Indeed, it will be apparent to those skilled in the art that various modifications and alterations can be made to the compounds and methods described herein without departing from the scope or spirit of the invention. For example, features shown or described as part of one embodiment may be adapted to another embodiment to produce yet another embodiment. Therefore, the invention is intended to include these modifications and alterations, as well as their equivalents. Other objects, features, and aspects of the invention are disclosed in, or may be apparent from, the following detailed description. Those skilled in the art will understand that the discussion of the invention is merely a description of exemplary embodiments and should not be construed as limiting the broader aspects of the invention.

[0115] In one embodiment, this disclosure provides a pharmaceutical composition (SACC) comprising at least one compound selected from the following chemical formulas:

[0116] (i)

[0117]

[0118] Chemical formula (I) for 6-hydroxy-3H-isochromene-3,8(4H)-dione.

[0119] (ii)

[0120]

[0121] 5-Methyl-1H-indole-3-carboxylic acid

[0122] Molecular formula: C 10 H9NO3 has the chemical formula (II).

[0123] (iii)

[0124]

[0125] 6-Hydroxy-3-methyl-2H-pyran-2-one

[0126] Molecular formula = C6H6O3 Chemical formula (III)

[0127] (iv)

[0128]

[0129] 2-Methyl-4H-pyran-4-one

[0130] Molecular formula = C6H6O2 Chemical formula (IV)

[0131] Or a combination thereof.

[0132] In one embodiment, this disclosure provides a pharmaceutical composition (SACC) comprising compounds represented by the following chemical formulas (I), (II), (III), and (IV):

[0133] (i)

[0134]

[0135] Chemical formula (I) for 6-hydroxy-3H-isochromene-3,8(4H)-dione.

[0136] (ii)

[0137]

[0138] 5-Methyl-1H-indole-3-carboxylic acid

[0139] Molecular formula: C 10 H9NO3 has the chemical formula (II).

[0140] (iii)

[0141]

[0142] 6-Hydroxy-3-methyl-2H-pyran-2-one

[0143] Molecular formula = C6H6O3 Chemical formula (III)

[0144] (iv)

[0145]

[0146] 2-Methyl-4H-pyran-4-one

[0147] Molecular formula = C6H6O2 Chemical formula (IV).

[0148] In one embodiment, the amount of the compound represented by Formula I in the pharmaceutical composition is in the range of 6 wt% to 18 wt%.

[0149] In another embodiment, the amount of the compound represented by Formula I in the pharmaceutical composition is 12.80 wt%.

[0150] In one embodiment, the amount of the compound represented by chemical formula II in the pharmaceutical composition is in the range of 8 wt% to 22 wt%.

[0151] In another embodiment, the amount of the compound represented by chemical formula II in the pharmaceutical composition is 15.41 wt%.

[0152] In one embodiment, the amount of the compound represented by Formula III in the pharmaceutical composition is in the range of 2 wt% to 10 wt%.

[0153] In another embodiment, the amount of the compound represented by Formula III in the pharmaceutical composition is 6.48 wt%.

[0154] In one embodiment, the amount of the compound represented by chemical formula IV in the pharmaceutical composition is in the range of 1 wt% to 10 wt%.

[0155] In another embodiment, the amount of the compound represented by chemical formula IV in the pharmaceutical composition is 5.89 wt%.

[0156] In one embodiment, this disclosure provides a compound represented by chemical formula I:

[0157]

[0158] 6-Hydroxy-3H-isochrome-3,8(4H)-dione.

[0159] In one embodiment, this disclosure provides a compound represented by chemical formula II:

[0160]

[0161] 5-Methyl-1H-indole-3-carboxylic acid

[0162] Molecular formula: C 10 H9NO3.

[0163] In one embodiment, this disclosure provides a compound represented by chemical formula III:

[0164]

[0165] 6-Hydroxy-3-methyl-2H-pyran-2-one

[0166] Molecular formula = C6H6O3.

[0167] In one embodiment, this disclosure provides a compound represented by chemical formula IV:

[0168]

[0169] 2-Methyl-4H-pyran-4-one

[0170] Molecular formula = C6H6O2.

[0171] In one embodiment, this disclosure provides a method for separating compounds of formula I, formula II, formula III or formula IV from barley extracts:

[0172] (i)

[0173]

[0174] Chemical formula (I) for 6-hydroxy-3H-isochromene-3,8(4H)-dione.

[0175] (ii)

[0176]

[0177] 5-Methyl-1H-indole-3-carboxylic acid

[0178] Molecular formula: C 10 H9NO3 has the chemical formula (II).

[0179] (iii)

[0180]

[0181] 6-Hydroxy-3-methyl-2H-pyran-2-one

[0182] Molecular formula = C6H6O3 Chemical formula (III)

[0183] (iv)

[0184]

[0185] 2-Methyl-4H-pyran-4-one

[0186] Molecular formula = C6H6O2 Chemical formula (IV)

[0187] The method includes the following steps:

[0188] • Barley extract was chromatographically analyzed using a preparative HPLC method;

[0189] • Elution peaks 2, 3, 4, and 5;

[0190] • The peaks obtained in step (ii) are manually separated by repeated injections to obtain fractions;

[0191] • Combined and concentrated at 35°; and

[0192] • Analyze the peaks and determine the structures of the compounds represented by chemical formulas I, II, III, and IV.

[0193] In another embodiment, in step (i), the preparative HPLC method includes mobile phase A and mobile phase B.

[0194] In another embodiment, in step (ii), the elution of peaks 2, 3, 4 and 5 is performed at approximately 14.3, 15.5, 16.4 and 23.0 minutes, respectively.

[0195] In another embodiment, in step (iv), the fractions are combined and concentrated at a temperature ranging from 25°C to 45°C.

[0196] In another embodiment, in step (iv), the fractions are combined and concentrated at a temperature of 35°C.

[0197] In another embodiment, in step (v), the structure is determined by IR, mass spectrometry, and NMR spectroscopy.

[0198] In one embodiment, this disclosure provides an HPLC method for analyzing barley extracts and a preparative HPLC method for separating desired peaks corresponding to compounds represented by chemical formulas (I), (II), (III), and (IV).

[0199] The bands corresponding to the compounds were eluted from thin-layer chromatography and confirmed to be single bands. HPLC analysis of the separated compounds showed single peaks with retention times (Rt) of 14.3, 15.5, 16.4, and 23.0 minutes. Furthermore, the purity of the compounds was examined by HPLC, which provided single peaks with a purity ranging from 85% to 100%, preferably with a purity greater than or equal to 87.9%, or greater than or equal to 98.1%, or greater than or equal to 99.7%. LC-MS, ESI-MS, IR spectroscopy, and... 1 The structure of the isolated compound was determined by 1H NMR.

[0200] In one embodiment, the present invention provides an HPLC method for analyzing barley extracts and a preparative HPLC method for separating desired peaks corresponding to compounds represented by chemical formula I, II, III or IV.

[0201] In one embodiment, barley extract is obtained by a method comprising the following steps: 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 cases of the extraction process, the mixture is maintained at a temperature of about 27 ± 3°C for about 16 ± 2 hours. In some embodiments, 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.

[0202] In one embodiment, this disclosure provides a pharmaceutical composition comprising a compound of formula I, II, III or IV extracted from barley extract as an active ingredient, optionally mixed with a pharmaceutically available carrier or excipient or diluent.

[0203] In one embodiment, this disclosure provides an anticancer composition comprising at least one compound selected from chemical formulas (I), (II), (III) and (IV) or a combination thereof and a pharmaceutically available carrier / excipient.

[0204] In one embodiment, this disclosure provides an anticancer composition comprising compounds of chemical formulas (I), (II), (III) and (IV) and a pharmaceutically available carrier and / or excipient.

[0205] In one embodiment, this disclosure provides a pharmaceutical formulation comprising a compound of chemical formulas (I), (II), (III) and (IV) or a pharmaceutically available salt thereof, as well as one or more pharmaceutically available carriers and optionally one or more other therapeutic agents.

[0206] In another embodiment, the therapeutic agent is an anticancer agent.

[0207] The carrier must be "acceptable" in the sense that it is compatible with the other components of the formulation and harmless to its receptors. Appropriate formulation depends on the chosen route of administration. Any well-known technology, carrier, and excipient may be used where suitable and understood in the art.

[0208] In one embodiment, this disclosure provides a method for formulating the disclosed composition for drug administration.

[0209] In one aspect of this implementation, administration includes intravenous, intrathecal, intramuscular, oral, and any other acceptable route of administration.

[0210] In one embodiment, this disclosure provides a pharmaceutical composition comprising compounds of formulas (I), (II), (III), and (IV) obtained from barley extract for inducing apoptosis and inhibiting the growth of pancreatic cancer cells unrelated to mutant p53.

[0211] In one embodiment, this disclosure provides a pharmaceutical composition comprising compounds of formulas (I), (II), (III), and (IV) obtained from barley extract for inhibiting HER-2 expression.

[0212] In one embodiment, this disclosure provides a pharmaceutical composition comprising compounds of chemical formulas (I), (II), (III), and (IV) obtained from barley extract for inducing the expression of the pro-apoptotic protein Bax and inhibiting the anti-apoptotic protein Bcl2.

[0213] In one embodiment, this disclosure provides a pharmaceutical composition comprising compounds of formulas (I), (II), (III), and (IV) obtained from barley extract for inducing PARP protein cleavage.

[0214] In one embodiment, this disclosure provides a pharmaceutical composition comprising compounds of formulas (I), (II), (III), and (IV) obtained from barley extract for the treatment of prostate cancer, triple-negative breast cancer, invasive carcinoma, lung cancer, sarcoma, serous carcinoma, pancreatic cancer, and HER2-positive breast cancer in subjects.

[0215] In one embodiment, this disclosure provides a pharmaceutical composition for producing agents that induce apoptosis and inhibit the growth of pancreatic cancer cells unrelated to mutant p53.

[0216] In one embodiment, this disclosure provides a pharmaceutical composition for producing an agent that inhibits HER-2 expression.

[0217] In one embodiment, this disclosure provides a pharmaceutical composition for producing an agent that induces the expression of the pro-apoptotic protein Bax and inhibits the anti-apoptotic protein Bcl2.

[0218] In one embodiment, this disclosure provides a pharmaceutical composition for producing an agent that induces PARP protein cleavage.

[0219] In one embodiment, this disclosure provides a pharmaceutical composition for producing an agent for treating prostate cancer, triple-negative breast cancer, invasive carcinoma, lung cancer, sarcoma, serous carcinoma, pancreatic cancer, and HER2-positive breast cancer in subjects.

[0220] In one embodiment, this disclosure provides the use of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV) for the production of a medicament for treating cancer.

[0221] In one embodiment, this disclosure provides a method for preventing or treating cancer in a subject in need, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV).

[0222] In one embodiment, this disclosure provides a method for inducing apoptosis and inhibiting the growth of pancreatic cancer cells unrelated to mutant p53 in subjects in need of such treatment, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV).

[0223] In one embodiment, this disclosure provides a method for inhibiting HER-2 expression in a subject in need of it, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV).

[0224] In one embodiment, this disclosure provides a method for inducing the expression of the pro-apoptotic protein Bax and inhibiting the anti-apoptotic protein Bcl2 in a subject in need of it, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV).

[0225] In one embodiment, this disclosure provides a method for inducing PARP protein cleavage in a subject in need of it, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV).

[0226] In one embodiment, this disclosure provides a method of using a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV), wherein the method includes inducing apoptosis and inhibiting the growth of pancreatic cancer cells.

[0227] In one embodiment, this disclosure provides a method of using a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV), wherein the method includes inhibiting HER-2 expression.

[0228] In one embodiment, this disclosure provides a method of using a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV), wherein the method comprises inducing the expression of the pro-apoptotic protein Bax and inhibiting the anti-apoptotic protein Bcl2.

[0229] In one embodiment, this disclosure provides a method of using a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV), wherein the method includes inducing PARP protein cleavage.

[0230] In one embodiment, this disclosure provides a method of using a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV), wherein the method comprises treating a subject with prostate cancer, triple-negative breast cancer, invasive carcinoma, lung cancer, sarcoma, serous carcinoma, pancreatic cancer, and HER2-positive breast cancer.

[0231] In one embodiment, this disclosure provides a compound of chemical formula I for treating cancer.

[0232] In one embodiment, this disclosure provides a compound of chemical formula III for the treatment of cancer.

[0233] In one embodiment, this disclosure provides the use of the compound represented by Chemical Formula I for the production of a medicament for treating cancer.

[0234] In one embodiment, this disclosure provides the use of the compound represented by Formula III for the production of a medicament for treating cancer.

[0235] In one embodiment, this disclosure provides a pharmaceutical composition for treating a subject in need of treatment.

[0236] In one embodiment, this disclosure provides a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV) for use as a medicament for treating cancer.

[0237] In one embodiment, this disclosure provides the use of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV) in the treatment of cancer.

[0238] In one embodiment, this disclosure provides a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV) as an agent for inducing apoptosis, inducing the expression of the pro-apoptotic protein Bax, and inducing the cleavage of poly(ADP-ribose) polymerase (PARP) protein.

[0239] In one embodiment, this disclosure provides a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV) for use as an agent for inhibiting human epidermal growth factor receptor-2 (HER-2) expression, inhibiting the anti-apoptotic protein Bcl2, inhibiting the growth of pancreatic cancer cells, and degrading p53 mutations and normalizing wild-type p53.

[0240] In one embodiment, this disclosure provides a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV) as an agent for treating prostate cancer, triple-negative breast cancer, invasive carcinoma, lung cancer, sarcoma, serous carcinoma, pancreatic cancer, and HER2-positive breast cancer in a subject.

[0241] In one aspect of this disclosure, the pharmaceutical composition is an oral, parenteral, nasal, subcutaneous, intradermal, intramuscular, intravenous, intra-articular and intramedullary, intraperitoneal, transmucosal, transdermal, rectal and topical composition.

[0242] In one embodiment, this disclosure provides a method for inducing apoptosis in a subject, inducing expression of the pro-apoptotic protein Bax, and inducing cleavage of PARP protein, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV).

[0243] In one embodiment, this disclosure provides a method for inhibiting HER-2 expression, inhibiting the anti-apoptotic protein Bcl2, inhibiting the growth of pancreatic cancer cells, and degrading p53 mutations and normalizing wild-type p53 in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV).

[0244] In another embodiment, this disclosure provides a pharmaceutical composition wherein the subject is a human or a non-human mammal.

[0245] 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 disclosed herein and / or a pharmaceutically available carrier. Pharmaceutically available carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or media such as glycols, glycerols, and oils such as olive oil or injectable organic esters.

[0246] In another embodiment, this disclosure provides pharmaceutical compositions for human administration, particularly for invasive administration routes (i.e., routes that bypass transport or diffusion across the epithelial barrier, such as injection or implantation), wherein the aqueous solution is pyrogen-free or substantially pyrogen-free. For example, excipients may be selected to achieve delayed release of the drug or selective targeting of one or more cells, tissues, or organs.

[0247] In another embodiment, the pharmaceutical composition may be in the form of dosage units, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, and lyophilized products for reconstitution, powders, solutions, syrups, suppositories, injections, etc. 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 application, such as eye drops.

[0248] The dosage of the active ingredient (barley extract) disclosed herein varies depending on the patient's age, weight, or symptoms, as well as the potency or therapeutic efficacy of the compound, the dosage administration regimen, and / or the duration of treatment. Generally, suitable routes of administration may include, for example, oral, ophthalmic, rectal, mucosal, local, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0249] The barley extract of this disclosure can be administered in doses of 0.2 mg, 0.5 mg, or 1 mg up 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 dosage regimen. This dosage can be administered once a week, once every three days, once every two days, once a day, twice a day, three times a day, or more frequently. In alternative embodiments, in certain adults, the barley extract can be administered intravenously for a period of time as prescribed by a physician. Because the dosage is affected by various conditions, doses less than or greater than the expected dose range may be administered in some cases. Physicians can easily determine the appropriate dosage for patients receiving therapeutic treatment.

[0250] The pharmaceutical compositions disclosed herein can be produced in ways known per se, for example by conventional methods of mixing, dissolving, granulating, dressing-making, levigating, emulsifying, encapsulating, embedding, or compressing. Formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intra-articular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal, and topical (including skin, oral (buccal), sublingual, and intraocular) administration, although the most suitable route may depend, for example, on the condition and disease of the recipient.

[0251] The pharmaceutical compositions disclosed herein can be used in diagnostic imaging methods. The pharmaceutical compositions disclosed herein are preferably administered to a patient (e.g., a human) via intravenous injection. The formulations can be readily available in unit dose form and can be prepared by any method well known in the pharmaceutical field. All methods include the step of binding the compound of the invention or a medicamentous salt thereof (“active ingredient”) to a carrier constituting one or more auxiliary ingredients. Typically, formulations are prepared by uniformly and tightly binding the active ingredient to a liquid carrier or a finely fragmented solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0252] As used herein, the phrase "pharmaceutical-available salt" refers to the preparative salt of a pharmaceutically available non-toxic acid (including inorganic and organic acids). Examples of physiologically or pharmaceutically-available 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+ (where X is a C1-C4 alkyl group). Physiologically available salts of hydrogen atoms or amino groups include salts of organic carboxylic acids, such as acetic acid, benzoic acid, lactic acid, fumaric acid, tartaric acid, maleic acid, malonic acid, malic acid, ethanesulfonic acid, lactobionic acid, and succinic acid; organic sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; and inorganic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, and aminosulfonic acid. Physiologically available salts of hydroxyl compounds include anions of the compounds combined with suitable cations, such as Na+ and NX / (where X is independently selected from H or C1-C4 alkyl groups).

[0253] For therapeutic uses, the salts of the active ingredients of the compounds disclosed herein will generally be physiologically usable, i.e., they will be salts derived from physiologically usable acids or bases. However, salts of physiologically incompatible acids or bases may also be used, for example, for the preparation or purification of physiologically usable compounds. All salts, whether or not derived from physiologically usable acids or bases, are within the scope of this disclosure.

[0254] Salts of the parent compound and one or more amino acids are also included within the scope of this disclosure. Any natural or non-natural amino acid is suitable, especially naturally occurring amino acids present as protein components, although said amino acids are generally amino acids with side chains having basic or acidic groups, such as lysine, arginine, or glutamic acid, or amino acids with side chains having neutral groups, such as glycine, serine, threonine, alanine, isoleucine, or leucine.

[0255] As used herein, the phrase “drug-available carrier” refers to a drug-available material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other components of the formulation and harmless to the patient. Some examples of materials that can be used as drug-available carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered astragalus gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes. (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl lauryl ester (ethyl lauryl ester); (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic and compatible substances used in pharmaceutical preparations.

[0256] The pharmaceutical composition (formulation) can be administered to the subject via any of a number of routes of administration, including, for example, oral (e.g., as infusions, tablets, capsules (including powdered capsules and gelatin capsules), pills, powders, granules, or ointments applied to the tongue); absorption via the oral mucosa (e.g., sublingual); rectally, rectally, or vaginally (e.g., as vaginal suppositories, creams, or foams); parenteral (including intramuscular, intravenous, subcutaneous, or intrathecal administration, as, for example, a sterile solution or suspension); nasal; intraperitoneal; subcutaneous; percutaneously (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 can also be formulated for inhalation. In yet another embodiment, the barley extract can be simply dissolved or suspended in sterile water or double-distilled water (DDW).

[0257] The formulation can be conveniently present in unit dose form and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dose form will vary depending on the host to be treated and the specific route of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dose form will generally be the amount of the compound that produces the therapeutic effect. Typically, in 100%, this amount will be 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%.

[0258] Formulations of this disclosure suitable for oral administration may be in the following forms: capsules (including powdered capsules and gelatin capsules), flat capsules, pills, tablets, sugar tablets (using flavoring bases, typically sucrose and gum arabic 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 tablets (using inert bases, such as gelatin and glycerin or sucrose and gum arabic) and / or mouthwashes, each containing a predetermined amount of the barley extract of this disclosure as an active ingredient.

[0259] To prepare solid dosage forms (capsules (including powdered capsules and gelatin capsules), tablets, pills, sugar-coated pills, powders, granules, etc.) for oral administration, the active ingredient is mixed with one or more pharmaceutically available carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following substances: (1) fillers or supplements (extenders), such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) (3) Humectants, such as glycerin; (4) Disintegrants, such as agar, calcium carbonate, potato or cassava starch, alginic acid, certain silicates and sodium carbonate; (5) Solution blockers, such as paraffin; (6) Absorption promoters, such as quaternary ammonium compounds; (7) Wetting agents, such as cetyl alcohol and glyceryl monostearate; (8) Adsorbents, such as kaolin and bentonite; (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.

[0260] In the case of capsules (including powdered capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also contain a buffer.

[0261] Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose or milksugar and high molecular weight polyethylene glycol.

[0262] Useful liquid dosage forms for oral administration include emulsions, lyophilized products for reconstitution, microemulsions, suspensions, solutions, syrups, and elixirs available for the drug. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrins and their derivatives, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters and mixtures thereof of sorbitol.

[0263] In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, aroma agents and preservatives.

[0264] In addition to the active ingredients, suspensions may also contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and tragacanth gum and mixtures thereof.

[0265] Formulations of pharmaceutical compositions for rectal, vaginal, or urethral administration may be available as suppositories, which can 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 salicylates, which are solid at room temperature but liquid at body temperature and thus melt in the rectal or vaginal cavity, releasing the active compound.

[0266] Formulations of pharmaceutical compositions for oral application may exist as mouthwashes, oral sprays, or oral ointments.

[0267] Alternatively or otherwise, compositions can be formulated for delivery via catheters, stents, wires, or other endoluminal devices. Delivery via these devices is particularly useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

[0268] The pharmaceutical compositions disclosed herein can be used alone (monotherapy) or in combination with one or more other methods / compositions (combination therapy).

[0269] abbreviation:

[0270] The following abbreviations are used in this disclosure:

[0271] °C

[0272] HPLC (High Performance Liquid Chromatography)

[0273] h hours

[0274] mL / ml

[0275] THF tetrahydrofuran

[0276] R t Retention time

[0277] TLC thin-layer chromatography

[0278] NMR nuclear magnetic resonance

[0279] MHz

[0280] s single peak

[0281] d double peak

[0282] triple peak

[0283] m multiplet

[0284] H protons

[0285] MS mass spectrometry

[0286] LC-MS (Liquid Chromatography-Mass Spectrometry)

[0287] IV or IV intravenous administration

[0288] HEPES4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid)

[0289] ATP adenosine triphosphate

[0290] DTT dithiothreitol

[0291] EGTA (ethylene glycol bis(2-aminoethyl) ether-N,N,N,N-tetraacetic acid)

[0292] DAPK1 death-related protein kinase

[0293] PKD protein kinase D

[0294] CAMKK2 (calcitonin / calmodulin-dependent protein kinase 2)

[0295] PKCδ protein kinase C-δ

[0296] FTIR Fourier Transform Infrared Spectroscopy

[0297] ppm (parts per million)

[0298] PARP (poly(ADP-ribose) polymerase)

[0299] This disclosure will now be described in general terms, and will be more readily understood by referring to the following embodiments, which are included only for the purpose of illustrating certain aspects and implementations of this disclosure, and are not intended to limit this disclosure in any way.

[0300] Example

[0301] Instruments and materials

[0302] The following instruments were used in this invention:

[0303] Preparative HPLC: Agilent Semi-Preparative HPLC

[0304] NMR: 400 MHz NMR Varian Make (Agilent)

[0305] FTIR: Perkin Elmer 100 series

[0306] HPLC: Waters HPLC, equipped with Empower software.

[0307] LC-MS: Agilent single quadrupole, equipped with ESI

[0308] Balance: Sartorius

[0309] Chromatographic column: HPLC analysis: Inertsil C18 (250 × 4.6 mm, 5 µm), and

[0310] Preparative HPLC: Inertsil C18 (250×19mm, 5µm)

[0311] The following materials were used in this invention:

[0312] Acetonitrile: HPLC grade

[0313] Methanol: HPLC grade

[0314] Trifluoroacetic acid: AR grade

[0315] Dichloromethane (DCM): HPLC grade

[0316] Water: Mili-Q Water

[0317] Preparation of barley extract

[0318] One kilogram of barley flour, obtained from milled barley grains (barley cereal) sourced from India, was placed in a glass container equipped with a standard fitting. Two liters of double-distilled water were added, and the mixture was thoroughly stirred and vigorously shaken. The container was then sealed tightly and placed in an incubator at 27±3°C for 16 hours. After curing, the container lid was removed, and the container was placed on a heating system (a rotating hood with heating elements and a thermostat, insulated with glass wool to prevent direct heat contact with the glass flask). The container was equipped with a distillation apparatus with a suitable cooling system, maintaining the optimal distillation temperature at 110±30°C.

[0319] The liquid obtained during distillation is passed through a cotton bed fitted in a funnel and collected in a flask, typically in 3-4 fractions, each approximately 400 ml. These fractions are sealed and stored at 10 ± 3 °C for 1 hour. Subsequently, the flask is brought to normal room temperature, which may vary depending on the season. The resulting solution constitutes the pharmaceutical preparation for its intended use.

[0320] Example 2: Isolation of compounds from barley extract

[0321] The compound was isolated from barley extract using HPLC. The purity of the compound was determined.

[0322] 1. Detailed information on the fractionation method:

[0323] 1.1 Detailed information on the HPLC method used for the analysis of barley extract (B. No. KWO 037)

[0324] Mobile phase A: 0.1% aqueous solution of TFA

[0325] Mobile phase B: 0.1% TFA solution in methanol:ACN (80:20 v / v)

[0326] Barley extract was analyzed under the following chromatographic conditions:

[0327] Column: Inertsil ODS 3V (250mm × 4.6mm, 5µm)

[0328] Injection volume: 10µL

[0329] Flow rate: 1.0 mL / min

[0330] Column temperature: 25℃

[0331] Detection wavelengths: Maximum absorbance plots, 280, 254, and 230 nm

[0332] Running time: 45 minutes

[0333] Test concentration: original sample

[0334] Elution modes: The gradient modes / procedures are shown in Table 1.

[0335] Table 1: Gradients

[0336]

[0337] exist Figure 1 The figure shows typical HPLC chromatograms at different UV wavelengths (in nm).

[0338] 1.2 Detailed information on preparative HPLC methods for peak separation

[0339] The peaks were separated using the following HPLC method:

[0340] Mobile phase A: 0.1% aqueous solution of TFA

[0341] Mobile phase B: 0.1% TFA solution in methanol:ACN (80:20 v / v)

[0342] The peaks were separated under the following chromatographic conditions:

[0343] Column: Inertsil C18 (250mm × 19mm, 5µm)

[0344] Flow rate: 15.0 mL / min

[0345] Column temperature: 25℃

[0346] Detection wavelength: 230nm

[0347] Running time: 45 minutes

[0348] Test concentration: original sample

[0349] program:

[0350] When using the above method to perform chromatographic separation of barley extract, such as Figure 2 As shown, peaks 2, 3, 4, and 5 were eluted at approximately 14.3, 15.5, 16.4, and 23.0 minutes, respectively. Peaks were manually separated by repeated injections, fractions were combined, and concentrated at 35°C.

[0351] a) Separation and analysis of peak 2

[0352] Peak 2 was manually separated by repeated injections under the chromatographic conditions described in Section 1.2, fractions were combined and concentrated at 35°.

[0353] Detailed information on HPLC methods:

[0354] Section 1.1 provides the chromatographic conditions. HPLC chromatogram ( Figure 3-4The results show an area purity of 98.17% on the maximum absorbance plot. The peak purity angle and peak purity threshold are 5.893 and 6.041, respectively, indicating that the compound has high peak purity and the purity determined by HPLC is reliable.

[0355] The structure of the compound corresponding to peak 2 was determined by infrared spectroscopy (IR), mass spectrometry (MS), and 1H-NMR. All analyses were performed using peak 2, which was separated and concentrated by preparative HPLC in dry form.

[0356] • Infrared spectroscopy (IR):

[0357] IR spectra were generated and recorded by dispersion with KBr. Figure 5 The representative IR spectrum of peak 2 is shown. The IR spectrum of peak 2 is mentioned in Table 2 below:

[0358] Table 2: IR spectrum of peak 2:

[0359]

[0360] • Mass spectrometry (MS):

[0361] The spectroscopic measurements were performed using electrospray ionization (ESI) / APCI and a quadrupole analyzer. Spectra were acquired in positive ion mode. Figure 6 The representative MS spectrum is shown. The MS peak of the 179.1 m / z [M+H] spectrum corresponds to a molecular weight of 178 amu.

[0362] • NMR spectroscopy

[0363] The spectroscopic determination was performed using deuterated methanol (CD3OD) as the solvent. Figure 7 Peak 2 was shown 1 1H NMR spectrum. Peak 2 is mentioned in Table 3 below. 1 1H-NMR (400MHz, CD3OD, ppm) spectrum:

[0364] Table 3: NMR spectrum of peak 2:

[0365]

[0366] The structure of peak 2 has been determined:

[0367] Based on the results obtained by LC-MS, IR, and NMR, the basic structure of the compound corresponding to peak 2 was identified as chemical formula (I). LC-MS analysis of this compound showed that the molecular ion peak (M+1) at 179.1 m / z corresponds to a mass of 178 amu. Figure 6(At 3354.33, 2934.45, 1689.10, 1364.04 and 1220.72cm) -1 The IR absorption at δ indicates that the compound contains OH, CH, C=O, CH-bent, and CO groups, respectively. In the proton NMR spectrum of peak 2, the singlet at δ 4.496 ppm indicates two protons, corresponding to the methylene (-CH2). Signals at δ 6.27–6.286, 6.340–6.352, and 7.438–7.440 ppm confirm the aromatic CH protons. Based on LC-MS, IR, and NMR spectral data, peak 2 is inferred to be 6-hydroxy-3H-isochromene-3,8(4H)-dione, and the structure of peak 2 is as follows:

[0368]

[0369] 6-Hydroxy-3H-isochrome-3,8(4H)-dione

[0370] Chemical formula (I)

[0371] b) Separation and analysis of peak 3

[0372] Peak 3 was manually separated by repeated injections under the chromatographic conditions described in Section 1.2, fractions were combined and concentrated at 35°.

[0373] The structure of peak 3 has been determined:

[0374] Based on the results obtained by LC-MS, IR, and NMR, the basic structure of the compound corresponding to peak 3 was identified as chemical formula (II). LC-MS analysis 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 inferred to be 5-methyl-1H-indole-3-carboxylic acid, and the structure of peak 3 is as follows:

[0375]

[0376] 5-Methyl-1H-indole-3-carboxylic acid

[0377] Molecular formula: C 10 H9NO3

[0378] Chemical formula (II)

[0379] c) Separation and analysis of peak 4

[0380] Peak 4 was manually separated by repeated injections under the chromatographic conditions described in Section 1.2, fractions were combined and concentrated at 35°.

[0381] Detailed information on HPLC methods:

[0382] HPLC chromatogram ( Figure 8-9 The area purity shown in the maximum absorbance plot is 87.98%. The peak purity angle and peak purity threshold are 0.710 and 2.010, respectively, indicating that the compound has high peak purity and the purity determined by HPLC is reliable.

[0383] The structure of the compound corresponding to peak 4 was determined by infrared spectroscopy (IR), mass spectrometry (MS), and 1H-NMR. All analyses were performed using peak 4, which was separated and concentrated by preparative HPLC in dry form.

[0384] • Infrared spectroscopy (IR):

[0385] IR spectra were generated and recorded by dispersion with KBr. Figure 10 The representative IR spectrum of peak 4 is shown. The IR spectrum of peak 4 is mentioned below:

[0386] Table 4: IR assignment of peak 4

[0387]

[0388] • mass spectrometry

[0389] The spectroscopic measurements were performed using electrospray ionization (ESI) / APCI and a quadrupole analyzer. Spectra were acquired in positive ion mode. Figure 11 Representative MS spectra are shown. The MS peak at 127.1 m / z [M+H] corresponds to the molecular weight of 126 amu confirmed in the spectrum.

[0390] • NMR spectroscopy

[0391] The spectroscopy was performed using deuterated methanol (CD3OD) as a solvent. Figure 12 Representative NMR spectra are shown. Table 5 below mentions peak 4. 1 1H-NMR (400MHz, CD3OD, ppm) spectrum:

[0392] Table 5: NMR spectrum of peak 4:

[0393]

[0394] The structure of peak 4 has been determined:

[0395] Based on the results obtained by LC-MS, IR, and NMR, the basic structure of the compound corresponding to peak 4 was identified as chemical formula (III). LC-MS analysis of this compound showed that the molecular ion peak (M+1) at 127.1 m / z corresponds to a mass of 126 amu. Figure 12 (At 3241.03, 3158.86, 1631.74 and 1255.33cm) -1 The IR absorption at [value missing] indicates that the compound contains OH, CH, C=O, and CO groups, respectively. In the proton NMR spectrum of peak 4, the singlet at δ 2.341 ppm indicates three protons, corresponding to a methyl group (-CH3). Signals at δ 6.373–6.387 and 7.922–7.936 ppm confirm the aromatic CH protons. Based on LC-MS / MS, IR, and NMR spectral data, peak 4 is inferred to be 6-hydroxy-3-methyl-2H-pyran-2-one, and the structure of peak 4 is as follows:

[0396]

[0397] 6-Hydroxy-3-methyl-2H-pyran-2-one

[0398] Molecular formula = C6H6O3

[0399] Chemical formula (III)

[0400] d) Separation and analysis of peak 5

[0401] Peak 5 was manually separated by repeated injections under the chromatographic conditions described in Section 1.2, fractions were combined and concentrated at 35°.

[0402] Detailed information on HPLC methods:

[0403] HPLC chromatogram ( Figure 13-14 The area purity shown in the maximum absorbance plot is 99.74%. The peak purity angle and peak purity threshold are 2.244 and 2.512, respectively, indicating that the compound has high peak purity and the purity determined by HPLC is reliable.

[0404] The structure of peak 5 was determined by infrared spectroscopy (IR), mass spectrometry (MS), and 1H-NMR. All analyses were performed using peak 5 in dry form, separated and concentrated by preparative HPLC.

[0405] • Infrared spectroscopy (IR):

[0406] IR spectra were generated and recorded by dispersion with KBr. Figure 15 The representative IR spectrum of peak 5 is shown. The IR spectrum of peak 5 is mentioned below:

[0407] Table 6: IR distribution of peak 5

[0408]

[0409] • mass spectrometry

[0410] The spectroscopic measurements were performed using electrospray ionization (ESI) / APCI and a quadrupole analyzer. Spectra were acquired in positive ion mode. Figure 16 Representative MS spectra are shown. The MS peak at 111.1 m / z [M+H] corresponds to the molecular weight of 110 amu confirmed in the spectrum.

[0411] • NMR spectroscopy

[0412] The spectroscopy was performed using deuterated methanol (CD3OD) as a solvent. Figure 17 Representative NMR spectra are shown. Table 7 below mentions peak 5. 1 1H-NMR (400MHz, CD3OD, ppm) spectrum:

[0413] Table 7: NMR spectrum of peak 5:

[0414]

[0415] The structure of peak 5 has been determined:

[0416] Based on the results obtained by LC-MS, IR, and NMR, the basic structure of the compound corresponding to peak 5 was identified as chemical formula (IV). LC-MS analysis of this compound showed that the molecular ion peak (M+1) at 111.1 m / z corresponds to a mass of 111 amu ( Figure 16 (At 2939.39, 1667.53, 1518.75, 1395.41 and 1181.87cm) -l The IR absorption at [value missing] indicates that the compound contains CH, C=O, C=C stretching, CH bending, and CO groups, respectively. In the proton NMR spectrum of peak 5, a singlet at δ 2.256 ppm indicates three protons, corresponding to a methyl group (-CH3). Signals at δ 5.326, 5.968–5.963, and 6.270–6.262 ppm confirm the aromatic CH protons. Based on LC-MS, IR, and NMR spectral data, peak 5 is inferred to be 2-methyl-4H-pyran-4-one, and the structure of peak 5 is determined as follows:

[0417]

[0418] 2-Methyl-4H-pyran-4-one

[0419] Molecular formula = C6H6O2

[0420] Chemical formula (IV)

[0421] Bioactivity of the composition

[0422] Example 3: Preparation of the pharmaceutical composition

[0423] Pharmaceutical compositions comprising compounds of chemical formulas (I), (II), (III), and (IV) derived from barley extract were prepared by means of suitable extraction and purification methods to obtain high-quality barley extract. The obtained barley extract was kept in liquid or dry form for intended use.

[0424] Example 4: Kinase Response Experiment

[0425] Reaction settings:

[0426] 2.5 μL barley extract (SACC extract) (1 / 30 final dilution, 1 / 120 final dilution or titration);

[0427] 5.0 μL enzyme dilution buffer (EDB) (1×) or kinase (5×, in EDB);

[0428] Pre-incubate at room temperature for 10 minutes;

[0429] 17.5 μL of a reaction mixture containing ATP & C(Sx) substrate (pre-incubated at 30 °C for 5 min);

[0430] 25 μL final reaction volume;

[0431] The reaction was carried out at 30°C for 120 or 240 minutes.

[0432] Reaction conditions:

[0433] 54 mM HEPES, pH 7.5

[0434] 1mM ATP

[0435] 1mM DTT (No DTT for ASK1)

[0436] 0.012% Brij-35

[0437] 1% glycerin

[0438] 0.52mM EGTA (no EGTA for CAMKK2, DAPK1, DAPK2, PKCδ, PKD1, PKD2, PKD3)

[0439] 0.4 mM CaCl2, 5 ng / µl calcitonin (CAMKK2, DAPK1, DAPK2, PKD1, PKD2, PKD3 only)

[0440] 3.8 µM diglyceride (diacylglycerol), 140 µM phosphatidylserine (PKCδ only)

[0441] 250µM MnCl2 (HER2 only)

[0442] 10mM MgCl2

[0443] 15 μM C(Sx) substrate

[0444] 0.5-20 nM kinase

[0445] Notice:

[0446] • Enzyme dilution buffer (EDB): 20 mM MEPES, pH 7.5, 0.01% Brij-35, 0.1 mM EGTA (EGTA-free for CAMKK2, DAPK1, DAPK2, PKCδ, PKD1, PKD2, PKD3), 5% glycerol, 1 mg / mL bovine serum albumin, 1 mM DTT (DTT-free for ASK1).

[0447] • After sealing with an optically clear adhesive film [TopSealA-Plus plate seal (PerkinElmer, catalog number #6050185)], the reaction was carried out in a Corning 384-well white round flat-bottom polystyrene NBS microplate (catalog number #3824).

[0448] The tested kinases

[0449]

[0450]

[0451] exist Figure 18 In the bar chart (for the 1 / 30 final dilution SACC), the inhibitory effects of the crude compound at a 1:30 dilution on various kinases are shown. The bars in the bar chart represent the level of inhibition, with higher bars indicating stronger inhibition. Notably, at this dilution, four kinases showed no inhibitory effect, two kinases showed less than 25% inhibition, while a significant number of the 30 kinases (61%) showed significant inhibition, exceeding 50%.

[0452] exist Figure 19In the bar chart (1 / 120 final dilution SACC), the inhibition spectrum of the crude compound at a 1:120 dilution is shown. In this paper, higher bars still indicate higher levels of inhibition. Surprisingly, at this more diluted concentration, only five kinases (10%) showed inhibition exceeding 25%. These include CAMKK2, COT / MAP3K8 / Tpl2, HER2, PKCd, and TNIK / MAP4K7.

[0453] These targets play a crucial role at the interface between inflammation and cancer. Furthermore, according to Reyland et al., 2016, PKCδ is a tumor promoter in mouse models of breast and lung cancer, and increased PKCδ expression is a negative prognostic indicator for HER2+ and other subtypes of human breast cancer.

[0454] Figure 20 Provided for IC 50 A comprehensive view of the determined inhibitor titration. The graph, arranged from left to right, represents the gradient of the crude compound from high to low concentrations. This titration analysis aimed to determine the half-maximum inhibitory concentration (IC50) of the compound at different concentrations. 50 ).

[0455] Example 5: Cell viability or MTT assay

[0456] The effect of SACC fractions (peaks 2, 3, 4, and 5) on lung cell viability was determined by MTT assay.

[0457] program:

[0458] Cells were spaced at 1×10⁶ cells per well. 4 Cells were seeded at a density of 200 μL each into 96-well plates in complete medium containing 10% FBS and 1% antibiotic. After 70% confluence, cells were treated with individual doses (shown in the figure) of SACC and its fractions in complete medium for 24 h. After incubation at a humidified incubator at 37°C for the specified time, MTT dye (5 mg / mL, solution in PBS; diluted in 10 mL serum-free medium) was added to each well and incubated for 2 h. The plates were then centrifuged at 1,000 rpm for 5 min at 4°C. After carefully removing the medium, 0.1 mL of buffered DMSO was added to each well. The absorbance was recorded at 540 nm using a microplate reader. The effect of SACC and its fractions on cell growth inhibition was evaluated as percentage of cell viability, where media-treated cells were considered 100% viable.

[0459] result:

[0460] Of all the SACC fractions, peaks 2, 3, and 4 exhibited the most significant inhibitory effects on lung cancer cell growth, with inhibitory concentrations measured at 1.04 μg, 1.06 μg, and 1.02 μg, respectively. This indicates that these specific fractions contain potent bioactive components that contribute to the observed anticancer effects. Overall, the results suggest that SACC is more effective in treatment compared to individual fractions.

[0461] Example 6: Cell viability test of A549 cell line using trypan blue exclusion assay

[0462] Test sample: SACC composition

[0463] Standard: Doxorubicin

[0464] Preparation of test solution:

[0465] For cytotoxicity studies, the test sample was treated as a 100% stock solution, and a series of 2-fold dilutions were prepared from 10% to 0.156% using F-12K standard medium (basal medium) for treatment. A 10 mM standard doxorubicin stock solution was diluted from 100 μM to 1.56 μM using F-12K standard medium for treatment.

[0466] Cell lines and culture media:

[0467] A549 cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in F-12K medium with appropriate supplementation until confluence. Cell viability was assessed using a standard hemocytometer. Appropriate cell densities were prepared per ml of medium and seeded into 96-well plates, then incubated at 37°C, 5% CO2 for 24 h to evaluate cell viability.

[0468] program:

[0469] 1. Adjust the cell count to 5 × 10⁶ cells using complete culture medium. 5 Cells / ml. Add 100 μL of diluted cell suspension (50,000 cells / well) to each well of a 96-well microtiter plate and incubate at 37°C under a 5% CO2 atmosphere for 24 h.

[0470] 2. After 24 hours, the cells were treated with multiple concentrations of the test compound and incubated at 37°C in a 5% CO2 atmosphere for 24 hours.

[0471] 3. After incubation, discard the test solution in the well.

[0472] 4. Trypsinize the cells and take 20 μL of the cell suspension for viability counting.

[0473] 5. Add 20 μL of trypan blue dye and mix thoroughly.

[0474] 6. Then, with the aid of a pipette, load the mixture onto the notch end of the blood cell counter.

[0475] 7. Place the blood cell counter under an inverted microscope and score it in each quadrant.

[0476] 8. Count and record cells according to Neubauer's rules. Calculate the percentage of cell viability from the obtained data using the formula given below.

[0477] • calculate:

[0478] Live cells = Average live cells * 2 * 10 3 1 cell / 100μl

[0479] Viability % = Live cells / Total cells × 100

[0480] Table 1 shows the evaluation of cell viability of the test samples on the A549 cell line, and Figure 21 The cell viability force map after the test sample was treated is shown.

[0481] Table 1: Evaluation of cell viability of test samples in the A549 cell line

[0482]

[0483] Table 2 shows the evaluation of cell viability of the standard for the A549 cell line, and Figure 22 The results of the standard (doxorubicin) evaluation of the cell viability of the A549 cell line are shown.

[0484] Table 2: Evaluation of cell viability of A549 cell line by standard products

[0485]

[0486] result:

[0487] In experimental analysis, when applied to A549 cells, the test sample treated with SACC and the standard (doxorubicin) showed cell viability of 25.81% and 11.11%, respectively. The role of SACC is as follows: Figure 21 As shown, and the effect of Dorothy stars is as follows: Figure 22 As shown.

[0488] Results Summary

[0489]

[0490] The test sample SACC and the standard doxorubicin showed cell viability of 25.81% and 11.11%, respectively, in A549 cells after treatment. These results indicate that the test sample SACC exhibited higher cell viability than the standard treatment. The standard showed potential toxicity or side effects on cell health.

[0491] Example 7: Evaluation of the cytotoxicity of the test samples to the A549 cell line

[0492] • Preparation of test solution:

[0493] For cytotoxicity studies, the test sample was treated as a 100% stock solution, and a series of two-fold dilutions were prepared from 10% to 0.156% using F-12K standard medium for treatment. A 10 mM standard doxorubicin stock solution was diluted from 100 μM to 1.56 μM using F-12K standard medium for treatment.

[0494] • Cell lines and culture media:

[0495] A549 cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in F-12K medium with appropriate supplementation until confluence. Cell viability was assessed using a standard hemocytometer. Appropriate cell densities were prepared per ml of medium and seeded into 96-well plates, then incubated at 37°C, 5% CO2 for 24 h to evaluate cell viability.

[0496] • program:

[0497] 1. Adjust the cell count to 5 × 10⁶ cells using complete culture medium. 5 Cells / ml. Add 100 μL of diluted cell suspension (50,000 cells / well) to each well of a 96-well microtiter plate and incubate at 37°C under a 5% CO2 atmosphere for 24 h.

[0498] 2. After 24 hours, the cells were treated with multiple concentrations of the test compound and incubated at 37°C in a 5% CO2 atmosphere for 24 hours.

[0499] 3. After incubation, discard the test solution in the wells and add 100 μl of MTT (5 mg / 10 ml MTT in PBS) to each well. Incubate the plate at 37°C in a 5% CO2 atmosphere for 4 h.

[0500] 4. Remove the supernatant, add 100 μL of DMSO, and gently shake the plate to dissolve the formed formazan. Measure the absorbance at 590 nm using a microplate reader.

[0501] 5. Calculate the percentage of growth inhibition using the following formula, and if the sample exhibits significant inhibitory effects, derive the concentration (IC50) of the test compound required to inhibit cell growth by 50% from the dose-response curve of the cell line. 50 ).

[0502] • Calculate suppression:

[0503] Inhibition % = (Sample OD - Control OD / Control OD) × 100

[0504] Table 1 shows the cytotoxicity evaluation of the test samples against the A549 cell line, and Figure 23-24 The bar chart and line graph show the cytotoxicity of the test samples after treatment.

[0505] Table 1: Cytotoxicity evaluation of test samples against A549 cell line

[0506]

[0507] Table 2 shows the cytotoxicity evaluation of the standard (doxorubicin) on the A549 cell line, and Figure 25-26 The bar and line graphs show the cytotoxicity after treatment with the standard (doxorubicin).

[0508] Table 2: Cytotoxicity evaluation of the standard to the A549 cell line

[0509]

[0510] result:

[0511] The test sample SACC and the standard drug doxorubicin showed inhibitory effects on A549 cells, as indicated by their respective IC50 values. 50 The values ​​confirmed this. The IC50 values ​​of the test sample SACC and the standard doxorubicin in A549 cells were [data missing]. 50 The inhibition values ​​were 1.35% (0.65 μM) and 20.3 μM, respectively. Figure 23-26 A comparison of the cytotoxic effects of SACC and doxorubicin is shown.

[0512] Results Summary

[0513]

[0514] Compared to the standard Doxorubicin, SACC has a lower IC. 50 The values ​​indicate that it has a higher efficacy in inhibiting A549 cell growth. These findings highlight the promising anticancer properties of SACC as a potential therapeutic agent.

[0515] Example 8: Therapeutic activity of SACC against pancreatic cancer

[0516] 1. The therapeutic efficacy of SACC in a mouse model of human pancreatic cancer cells (AsPC1) ectopic xenograft was evaluated.

[0517] The results shown in Figures 27-30 confirm the inhibitory effect of SACC on pancreatic tumor growth in a mouse model of pancreatic cancer xenograft. In athymic nude mice with xenograft tumors derived from AsPC1 cells, intraperitoneal administration of 200 μL of SACC significantly inhibited tumor growth.

[0518] Figure 27-A shows the tumor volume at specified time points in both the control and SACC-treated groups. The values ​​in the figure represent the mean values ​​from 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 significant reduction in tumor volume demonstrates that SACC administration highlights its potential as a promising antitumor agent.

[0519] Figure 27B shows the tumor weight of mice in both the control and SACC groups at week 9. The values ​​in the figure represent the mean of 8 mice in the control group and 9 mice in the SACC-treated group. The figure shows a significant reduction in tumor weight in the SACC-treated group. This further supports the antitumor efficacy of SACC in inhibiting pancreatic tumor growth.

[0520] exist Figure 29 In the figure, a line graph shows the growth pattern of AsPC1 cell xenograft tumors in control mice. This graph provides a visual representation of tumor development over time, thus offering insights into tumor dynamics and progression in the absence of SACC treatment.

[0521] exist Figure 30 The line graph depicts the growth pattern of AsPC1 cell xenograft tumors in mice treated with SACC. This figure visually illustrates the effect of SACC administration on tumor growth curves during the experiment.

[0522] Overall, compared with the control group, tumor volume and weight were consistently reduced in the SACC-treated group, indicating that SACC has a significant inhibitory effect on pancreatic tumor growth in this xenograft mouse model. These findings suggest that SACC shows potential as a chemotherapeutic agent against pancreatic cancer.

[0523] 2. SACC inhibits the growth of pancreatic cancer stem cell xenograft tumors in SCID mice.

[0524] The effects of SACC on pancreatic cancer stem cell xenografting in SCID mice have been investigated, with a total of 8 mice used in this study. Briefly, 2000 viable cells (100 μL of 100% viable) were subcutaneously injected into SCID mice. Two weeks later, SACC (250 μL) was administered intraperitoneally 5 days a week for another two weeks. The target tumor volume of 1500 mm² was reached in control mice. 3 At that time, all mice were euthanized.

[0525] Figure 31A Images of mice with xenograft tumors are shown in the control and SACC-treated groups. The figures in the lower panel show the xenograft tumors removed from both groups of mice, allowing for a visual comparison of tumor characteristics between the control and SACC-treated groups.

[0526] Figure 31B shows the tumor volume of xenograft tumors, illustrating the changes in tumor size in the control and SACC-treated groups over the specified weeks. This figure provides quantitative data on the effect of SACC treatment on tumor volume compared to the control group.

[0527] Figure 31C shows the weight of xenograft tumors removed from mice in the control and SACC-treated groups at week 5. The comparison of tumor weight demonstrates an understanding of the inhibitory effect of SACC on tumor growth, thus providing a basis for further research. Figure 31B The findings in [the document] provide additional support.

[0528] Figure 33 The effects of SACC on the expression of transcription factor GLI-1 and chemokine receptor CXCR4 were investigated. Representative immunohistochemical images show the expression levels of GLI-1 and CXCR4 in control and SACC xenograft tumors. The results indicate that SACC partially inhibits both GLI-1 and CXCR4, thus demonstrating a potential antitumor effect.

[0529] Example 9: Molecular mechanism by which SACC inhibits pancreatic cancer growth

[0530] A study on the effects of SACC on MiaPaCa-2 and AsPc-1 cells using MTT assay:

[0531] The anticancer efficacy of SACC in pancreatic cancer cells (i.e., MiaPaCa-2 and AsPc1) was investigated using the previously described MTT method [1]. MiaPaCa-2 and AsPc-1 cells maintained in DMEM and RPMI-1640 media, respectively, were cultured at 5 × 10⁻⁶ cells / year. 3Cells were seeded at a density of 10 cells / well into 96-well plates and incubated overnight at 37°C with 5% CO2 for adhesion. After 24 h, different concentrations (0, 0.625%, and 1.25%, v / v) of SACC were added to these adherent MiaPaCa-2 and AsPc-1 cells, and incubated under similar growth conditions for another 24 h. After incubation, 0.2 mL of MTT (20% v / v, prepared in DMEM or RPMI-1640 medium) was added to these cells, and the cells were incubated at 37°C in the dark for 4 h. The formation of formazan crystals was evaluated by dissolving the formazan in 0.15 mL of DMSO on a shaker and reading the plate at 570 nm.

[0532] Immunoblotting

[0533] MiaPaCa-2 and AsPc-1 cells were fed at a concentration of 1.25 × 10⁻⁶. 6Cells were seeded at the appropriate seeding density in 100 mm culture dishes and incubated at 37 °C with 5% CO2. After 70% confluence, MiaPaCa-2 and AsPc1 cells were treated with 0.625% and 1.25% (v / v) SACC for 24 h, while untreated cells were designated as controls for each cell type. After 24 h of treatment, cells were lysed in ice-cold RIPA buffer (containing protease and phosphatase inhibitors). The lysates were then centrifuged at 14,000 × g for 15 min at 4 °C, followed by protein quantification using the bicinchoninic acid (BCA) method. 20 μg of protein from both untreated and treated cell lysates was denatured in sample buffer containing β-mercaptoethanol at 95 °C for 7 min and separated on a 10% gel at a constant voltage of 75 V. The isolated proteins were transferred onto a PVDF membrane using the Bio rad TransBlot® Turbo™ transfer system (catalog number #1704150EDU). After transfer, the membrane was blocked for 2 hours at 20 rpm on a shaker with 5% skim milk or 5% bovine serum albumin (prepared in 1× TBST), and then incubated overnight at 4°C with primary antibodies against the target proteins, namely HER2 / ErbB2 (Cell Signaling Technology; Catalog No. # 4290S; Dil. 1:1000), pHER2 (Dil. 1:1000), p53 (Santa Cruz Biotechnology; Dil. 1:3000), p21 (abcam; Catalog No. # ab18209; Dil. 1:1000), Bcl2 (Santa Cruz Biotechnology; Catalog No. # Sc-7382; Dil. 1:1000), Bax (Santa Cruz Biotechnology; Catalog No. # sc-65582; Dil. 1:1000), and PARP (Dil. 1:1000). The secondary antibody was prepared by incubating the membrane overnight with the primary antibody (1:1000) and β-actin (Santa Cruz Biotechnology; Catalog No. # sc-47778; Dil. 1:2000). After 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 anti-mouse or anti-rabbit HRP-conjugated antibody at 20 rpm for 1 h. Unbound secondary antibody was washed with 1× TBST as described for the primary antibody. Protein bands were detected using Immobilon Western chemiluminescent HRP substrate (Catalog No. # WBKLS0500), a method based on the high-sensitivity detection of chemiluminescence generated by immunoreactive bands.All antibodies used were validated by generating bands with the expected molecular weight (kDa) of the target protein.

[0534] Results and Discussion:

[0535] SACC possesses the ability to induce apoptosis (programmed cell death) and inhibit the growth of MiaPaCa-2 and AsPc-1 cells. This indicates that SACC has potential anti-cancer properties by inducing cell death mechanisms and limiting the proliferation of these specific cancer cell lines. Observations of apoptosis induction and growth inhibition in these cells highlight the potential therapeutic efficacy of SACC in combating cancer.

[0536] There are different strategies for determining the antiproliferative effect of any treatment regimen on a variety of cancer cells, and the MTT assay for evaluating cell viability is one of the preferred and most important strategies [1]. In the same context, the effect of SACC treatment on the viability of two pancreatic cancer cell lines, namely MiaPaCa-2 and AsPc-1 cells with mutated p53 and WT p53, respectively, was investigated. The findings from the cell viability assay of the present invention confirmed that SACC significantly inhibited the growth of pancreatic cancer cells regardless of p53 mutation. According to the observed IC50 of SACC in AsPC1 cells, 50 It is 0.37% (v / v), which shows IC in MiaPaCa-2. 50 (This experiment continues). Furthermore, the effect of SACC on apoptosis in PanCa cells was evaluated. SACC (1.25% v / v) treatment showed a significant apoptosis-inducing effect in MiaPaCa-2 cells. These results provide evidence for the potential of SACC to kill pancreatic cancer cells by inducing apoptosis. Figure 34 The study demonstrated the effects of SACC on pancreatic cancer cell viability and apoptosis.

[0537] a) SACC targets the HER-2 and p53 signaling pathways in pancreatic cancer cells.

[0538] Human epidermal growth factor receptor-2 (HER-2), also known as c-Neu / ErbB2, has been shown to have a profound impact on the pathophysiology of a variety of cancers, most notably pancreatic cancer; however, recent studies have warned of its undeniable role in other cancers [2-7]. Even genetic defects in HER-2 are associated with cancer severity through enhanced HER-2 function [3], making it a prime therapeutic target for controlling a variety of cancers. Furthermore, HER-2, alone or in combination with MUC4, is also associated with impaired efficacy of currently available chemotherapy, particularly resistance to gemcitabine [7, 8]. In contrast, HER-2-targeting therapies have shown the potential to sensitize gemcitabine-resistant pancreatic cells [9]. Against this backdrop, a suite of anti-HER-2 treatment options has been explored, with tyrosine kinase inhibitors considered the first-line treatment for HER-2-positive cancer phenotypes

[10] . As with other chemotherapy agents, even the best-studied HER-2 blocker, lapatinib, has been associated with serious adverse events, including cardiotoxicity

[11] .

[0539] Therefore, considering these adverse events associated with different HER-2 targeted therapies, the inventors of this disclosure explored whether SACC could target HER-2 in pancreatic cancer cells. In this study, immunoblotting analysis was performed to investigate the effect of SACC on HER-2 expression. The results showed that SACC downregulated HER-2 protein expression in both cell lines in a dose-dependent manner. Interestingly, SACC treatment at higher concentrations enhanced HER-2 phosphorylation in AsPC-1 and MiaPaCa-2 cells. Figure 35 ).

[0540] This effect was more pronounced in AsPC-1 cells expressing WT p53 compared to MiaPaCa-2 cells expressing mutant p53. A recently published study confirmed the results of this invention, showing that treatment with the chemotherapeutic drug gemcitabine enhanced HER-2 phosphorylation in AsPC-1 [9].

[0541] b) SACC regulates the expression of apoptosis proteins in pancreatic cancer cells.

[0542] During apoptosis, PARP cleavage has become a useful marker of cell death in this class of cells. This cleavage has been well studied and is produced by caspases 3 and 7 (proteases that are activated during apoptosis)

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

[14] .

[0543] Based on these previous observations, the downregulation of mutant p53 in MiaPaCa-2 cells in the current study indicates the antiproliferative efficacy of SACC. Interestingly, SACC treatment induces 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 multiple p53-targeting genes (i.e., p21) has also been associated with tumor suppression and growth inhibition / cell arrest

[15] . Our findings are particularly interesting because we report p21 overexpression in AsPC-1 cells, which can be attributed to the induction of WT p53 by SACC treatment. Unlike AsPC-1 cells, p21 expression was not altered in SACC-treated MiaPaCa-2 cells. Furthermore, the process of apoptosis is highly complex and closely regulated by the coordination of various mediators, including (but not limited to) Bcl-2 and Bcl-2-associated X, apoptosis regulator (BAX)

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

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

[0544] in conclusion

[0545] Regardless of mutant p53, SACC induces apoptosis and inhibits the growth of pancreatic cancer cells. SACC inhibits HER-2 expression but increases its phosphorylation at higher doses. SACC induces expression of wild-type p53 protein but reduces the level of mutant p53 protein. SACC induces the expression of the pro-apoptotic protein Bax and inhibits the expression of the anti-apoptotic protein Bcl2. SACC induces PARP protein cleavage.

[0546] Effect of SACC on WT p53 expression in CaPan-2 cells. These cells express WT p53. p53 has been reported to be in a truncated form in AsPC-1 cells. However, many studies have reported WT p53 expression in these cells.

[0547] The SACC composition was also investigated in prostate xenograft and TNBC. SACC was also studied in CWR22Rv1 cell xenograft tumors in athymic nude mice.

[0548] Oral administration of Satcon inhibited xenograft tumors derived from CWR22v1 cells. In short, a total of 8 athymic mice were used in this study. 4 × 10 6 One cell was subcutaneously implanted into the lateral dorsal surface of each mouse. Mice were divided into two groups. One week later, one group of mice received oral SACC (300 μl, 5 days a week) for 3 weeks, while the other group received saline. Tumor volume was recorded at weeks 3 and 4. The tumor volume in the control mice reached approximately 2000 mmHg. 3 At the designated time, all mice in both groups were sacrificed. Blood was collected, tumors were removed, and weighed on an electronic balance. At the end of the experiment, the tumor volume treated with SACC was 400 mmHg. 3 The tumor weight decreased from 3.3g to 1gm. Significant activity was observed. It is a potent inhibitor of TNBC xenograft tumors.

[0549] Example 10: Therapeutic activity of SA1 (peak 5) and SA2 (peak 4) against human breast cancer cell line MDA-MB-231

[0550] The therapeutic effects of SA1 (peak 5) and SA2 (peak 4) on the human lung cancer cell line MDA-MB-231 were determined by calculating the percentage of growth inhibition, specifically the drug concentration (GI) of the test compound required to inhibit cell growth by 50%. 50 It is evaluated based on its value.

[0551] Table 1 shows the percentage of cell growth and drug concentration:

[0552]

[0553] Table 2 shows the effects of SA1 (peak 5) and SA2 (peak 4) on cell viability and tumor cell growth inhibition:

[0554]

[0555] *GI 50 Value ≤ 10 -6 A mole (i.e., 1 micromole) or ≤10 μg / ml is considered to confirm activity in the case of the pure compound. For extracts, GI 50 A value ≤20 μg / ml was considered to confirm activity.

[0556] *GI 50 The test values ​​listed below represent activity.

[0557] LC 50 =Drug concentration that causes 50% cell killing

[0558] GI 50 =Drug concentration that causes 50% cell growth inhibition

[0559] TGI = Drug concentration that causes complete inhibition of cell growth

[0560] ADR = Doxorubicin, positive control compound

[0561] NE = Data that cannot be evaluated. The experiment needs to be repeated using different drug concentration groups.

[0562] Unstable data = Data may be unstable due to the low solubility of the compound.

[0563] •result:

[0564] Test samples SA1 (peak 5) and SA2 (peak 4) confirmed their inhibitory effect on MDA-MB-231 cells, as evidenced by their respective GI levels. 50 This is confirmed by the TGI value. Figure 37 The inhibitory effects of SA1 (peak 5), SA2 (peak 4), and doxorubicin on cell growth were confirmed.

[0565] Example 11: Therapeutic activity of SACC against human breast cancer cell line MDA-MB-231

[0566] The therapeutic effect of SACC on the human lung cancer cell line MDA-MB-231 was determined by calculating the percentage of growth inhibition, specifically the drug concentration (GI) of the test compound required to inhibit cell growth by 50%. 50 It is evaluated based on its value.

[0567] Table 1 shows the percentage of cell growth and drug concentration:

[0568]

[0569] SACCRT: SACC can be stored at 20℃ for 6 months.

[0570] SACCD / RT: Fresh SACC samples

[0571] A6 / 2023: SACC should be stored at 6°C for 6 months.

[0572] Table 2 shows the effects of SACC on cell viability and tumor cell growth inhibition:

[0573]

[0574] *GI 50 Value ≤ 10 -6 A mole (i.e., 1 micromole) or ≤10 μg / ml is considered to confirm activity in the case of the pure compound. For extracts, GI 50 A value ≤20 μg / ml was considered to confirm activity.

[0575] *GI 50 The test values ​​listed below represent activity.

[0576] •result:

[0577] The test sample SACC and the control drug doxorubicin showed inhibitory effects on MDA-MB-231 cells, as indicated by their respective IC50 values. 50 GI 50 And as evidenced by the TGI value. Figure 38 The study demonstrated the inhibitory effect of SACC and doxorubicin on cell growth.

[0578] Example 12: Therapeutic activity of SA1 (peak 5) and SA2 (peak 4) against human lung cancer cell line A-549

[0579] The therapeutic effects of SA1 (peak 5) and SA2 (peak 4) on the human lung cancer cell line A-549 were determined by calculating the percentage of growth inhibition, specifically the drug concentration (GI) of the test compound required to inhibit cell growth by 50%. 50 It is evaluated based on its value.

[0580] Table 1 shows the percentage of cell growth and drug concentration:

[0581]

[0582] Table 2 shows the effects of SA1 (peak 5) and SA2 (peak 4) on cell viability and tumor cell growth inhibition:

[0583]

[0584] *GI 50 Value ≤ 10 -6 A mole (i.e., 1 micromole) or ≤10 μg / ml is considered to confirm activity in the case of the pure compound. For extracts, GI 50 A value ≤20 μg / ml was considered to confirm activity.

[0585] *GI 50 The test values ​​listed below represent activity.

[0586] NE = Data that cannot be evaluated. The experiment needs to be repeated using different drug concentration groups.

[0587] Unstable data = Data may be unstable due to the low solubility of the compound.

[0588] •result:

[0589] Both the test sample SA2 (peak 4) and the control drug doxorubicin showed inhibitory effects on A-549. This was confirmed by their GI... 50 The values ​​show the inhibitory effect of SA2, while the inhibitory effect of doxorubicin is mediated through its individual LC values. 50 GI 50 Supports TGI values. Figure 39 The inhibitory effects of SA1 (peak 5), SA2 (peak 4), and doxorubicin on cell growth were confirmed.

[0590] Example 13: Therapeutic activity of SACC against human lung cancer cell line A-549

[0591] The therapeutic effect of SACC on the human lung cancer cell line A-549 is determined by calculating the percentage of growth inhibition, specifically the drug concentration (GI) of the test compound required to inhibit cell growth by 50%. 50 It is evaluated based on its value.

[0592] Table 1 shows the percentage of cell growth and drug concentration:

[0593]

[0594] Table 2 shows the effects of SACC on cell viability and tumor cell growth inhibition:

[0595]

[0596] *GI 50 Value ≤ 10 -6 A mole (i.e., 1 micromole) or ≤10 μg / ml is considered to confirm activity in the case of the pure compound. For extracts, GI 50 A value ≤20 μg / ml was considered to confirm activity.

[0597] *GI 50 The test values ​​listed below represent activity.

[0598] •result:

[0599] The test sample SACC and the control drug doxorubicin showed inhibitory effects on A549 cells, as indicated by their respective LC50 values. 50 and GI50 And as evidenced by the TGI values. The SACC and doxorubicin values ​​of the test samples showed LC... 50 The values ​​are 46.9 and 72, respectively. Figure 40 The study demonstrated the inhibitory effect of SACC and doxorubicin on cell growth.

[0600] Example 14: Therapeutic activity of SA1 (peak 5) and SA2 (peak 4) against human pancreatic cancer cell line Mia-Pa-Ca-2

[0601] The therapeutic effects of SA1 (peak 5) and SA2 (peak 4) on the human pancreatic cancer cell line Mia-Pa-Ca-2 were determined by calculating the percentage of growth inhibition, specifically the drug concentration (GI) of the test compound required to inhibit cell growth by 50%. 50 It is evaluated based on its value.

[0602] Table 1 shows the percentage of cell growth and drug concentration:

[0603]

[0604] Table 2 shows the effects of SA1 (peak 5) and SA2 (peak 4) on cell viability and tumor cell growth inhibition:

[0605]

[0606] *GI 50 Value ≤ 10 -6 A mole (i.e., 1 micromole) or ≤10 μg / ml is considered to confirm activity in the case of the pure compound. For extracts, GI 50 A value ≤20 μg / ml was considered to confirm activity.

[0607] *GI 50 The test values ​​listed below represent activity.

[0608] NE = Data that cannot be evaluated. The experiment needs to be repeated using different drug concentration groups.

[0609] •result:

[0610] Test samples SA1 (peak 5), SA2 (peak 4), and the control drug doxorubicin showed inhibitory effects on Mia-Pa-Ca-2. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 50 The TGI values ​​show the inhibitory effects of SA1 and SA2. Furthermore, through their individual LC... 50 GI 50 The TGI values ​​indicate the inhibitory effect of doxorubicin. Figure 41 The inhibitory effects of SA1 (peak 5), SA2 (peak 4), and doxorubicin on cell growth were confirmed.

[0611] Example 15: Therapeutic activity of SACC against human pancreatic cancer cell line Mia-Pa-Ca-2

[0612] The therapeutic effect of SACC on the human lung cancer cell line Mia-Pa-Ca-2 was determined by calculating the percentage of growth inhibition, specifically the drug concentration (GI) of the test compound required to inhibit cell growth by 50%. 50 It is evaluated based on its value.

[0613] Table 1 shows the percentage of cell growth and drug concentration:

[0614]

[0615] Table 2 shows the effects of SACC on cell viability and tumor cell growth inhibition:

[0616]

[0617] *GI 50 Value ≤ 10 -6 A mole (i.e., 1 micromole) or ≤10 μg / ml is considered to confirm activity in the case of the pure compound. For extracts, GI 50 A value ≤20 μg / ml was considered to confirm activity.

[0618] *GI 50 The test values ​​listed below represent activity.

[0619] •result:

[0620] The test sample SACC and the control drug doxorubicin showed inhibitory effects on Mia-Pa-Ca-2 cells, as indicated by their respective LC50 values. 50 and GI 50 And as evidenced by the TGI value. Figure 42 The study demonstrated the inhibitory effect of SACC and doxorubicin on cell growth.

[0621] Example 16: Evaluation of tumor volume and relative tumor volume (RTV) in human tumor xenograft MDA-MB- Evaluating the therapeutic effect of SACC on the triple-negative breast cancer cell line MDA-MB-231 in the 231 model.

[0622] The therapeutic effect of SACC on the triple-negative breast cancer cell line MDA-MB-231 was evaluated by analyzing tumor volume and relative tumor volume. Six mice were used in this study. These mice were treated according to the following groups:

[0623]

[0624] Table 1: Relative tumor volume in groups A, B, C, and D

[0625]

[0626] *RTV = Relative Tumor Volume = Tumor volume measured on the day of measurement / Tumor volume on day 1

[0627] •result:

[0628] The test sample SACC and the control drug doxorubicin showed reduced tumor volume in the human tumor xenograft MDA-MB-231 model, as shown in their relative tumor volume. Figure 43 The results showed a decrease in relative tumor volume for SACC and doxorubicin.

[0629] Table 2: Treatment / Control (T / C) values ​​of RTV data from groups A, B, C, and D

[0630]

[0631] The portion with T / C ≤ 0.42 (highlighted in blue) was considered to have confirmed activity.

[0632] •result:

[0633] The test sample SACC and the control drug doxorubicin showed tumor volume reduction in the human tumor xenograft MDA-MB-231 model, as shown by their T / C values ​​derived from RTV data. Figure 44 The decrease in T / C values ​​for SACC and doxorubicin was observed.

[0634] Table 3: Survival percentage in groups A, B, C, and D

[0635]

[0636] •result:

[0637] The test sample SACC and the control drug doxorubicin showed good survival rates in the human tumor xenograft MDA-MB-231 model, as shown in their survival percentages. Figure 45 The results showed that for SACC and doxorubicin, the survival rate remained 100% up to 25 days.

[0638] Table 4: Animal weight data for groups A, B, C, and D

[0639]

[0640] * Mortality and weight loss of ≥4 g / mice are considered indicators of toxicity.

[0641] •result:

[0642] The test sample SACC and the control drug doxorubicin showed minimal changes in body weight in the human tumor xenograft MDA-MB-231 model, as indicated by their average body weight. Figure 46 A graph showing the average animal weight for SACC and doxorubicin.

[0643] Table 5: Tumor volume in group A

[0644]

[0645] Table 5 and Figure 47 This represents the tumor volume in control group A.

[0646] Table 6: Tumor volume in group B

[0647]

[0648] Table 6 and Figure 48 This indicates the tumor volume in the positive control ADR (2.5 mg / Kg) group B.

[0649] •result:

[0650] The positive control drug doxorubicin showed a reduction in tumor volume in the human tumor xenograft MDA-MB-231 model.

[0651] Table 7: Tumor volume in group C

[0652]

[0653] Table 7 and Figure 49 This indicates the tumor volume in group C of SACC (4 mL / Kg).

[0654] •result:

[0655] SACC administered at a dose of 4 mL / Kg showed a reduction in tumor volume in the human tumor xenograft MDA-MB-231 model.

[0656] Table 8: Tumor volume in group D

[0657]

[0658] Table 8 and Figure 50 This indicates the tumor volume in group D of SACC (4 mL / Kg).

[0659] •result:

[0660] 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.

[0661] This document describes certain embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Of course, changes to these described embodiments will become apparent to those skilled in the art upon reading the above description. The inventors intend that those skilled in the art will use these changes where appropriate, and the inventors intend to practice the invention in ways other than those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter listed in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, the invention covers any combination of the foregoing elements with all possible variations.

[0662] Furthermore, numerous references have been made to patents and print publications throughout this specification. Each of the above-cited references and print publications is incorporated herein by reference in its entirety.

[0663] Finally, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be used are also within the scope of the invention. Therefore, alternative constructions of the invention may be used, by way of example and not by way of limitation, in accordance with the teachings herein. Thus, the invention is not limited to what is precisely shown and described.

[0664] References:

[0665] [1] R. Nabi, SS Alvi, A. Shah, CP Chaturvedi, D. Iqbal, S. Ahmad,MS Khan, Modulatory role of HMG-CoA reductase inhibitors and ezetimibe onLDL-AGEs-induced ROS generation and RAGE-associated signaling in HEK-293Cells, Life Sci. 235 (2019) 116823.

[0666] [2] MM Moasser, The oncogene HER2: Its signaling and transformingfunctions and its role in human cancer pathogenesis, Oncogene. 26 (2007)6469–6487.

[0667] [3] D.M. Hyman, S.A. Piha-Paul, H. Won, J. Rodon, C. Saura, G.I.Shapiro, D. Juric, D.I. Quinn, V. Moreno, B. Doger, I.A. Mayer, V. Boni, E.Calvo, S. Loi, A.C. Lockhart, J.P. Erinjeri, M. Scaltriti, G.A. Ulaner, J.Patel, J. Tang, H. Beer, S. Duygu Selcuklu, A.J. Hanrahan, N. Bouvier, M.Melcer, R. Murali, A.M. Schram, L.M. Smyth, K. Jhaveri, B.T. Li, A. Drilon,J.J. Harding, G. Iyer, B.S. Taylor, M.F. Berger, R.E. Cutler, F. Xu, A.Butturini, L.D. Eli, G. Mann, C. Farrell, A.S. Lalani, R.P. Bryce, C.L.Arteaga, F. Meric-Bernstam, J. Baselga, D.B. Solit, HER kinase inhibition inpatients with HER2-and HER3-mutant cancers, Nature. 554 (2018) 189–194.

[0668] [4] A. Majumder, M. Sandhu, D. Banerji, V. Steri, A. Olshen, M.M.Moasser, The role of HER2 and HER3 in HER2-amplified cancers beyond breastcancers, Sci. Rep. 11 (2021) 9091.

[0669] [5] S.K. Joshi, J.M. Keck, C.A. Eide, D. Bottomly, E. Traer, J.W.Tyner, S.K. McWeeney, C.E. Tognon, B.J. Druker, ERBB2 / HER2 mutations aretransforming and therapeutically targetable in leukemia, Leukemia. 34 (2020)2798–2804. https: / / doi.org / 10.1038 / s41375-020-0844-7.

[0670] [6] W. Wen, W.S. Chen, N. Xiao, R. Bender, A. Ghazalpour, Z. Tan, J.Swensen, S.Z. Millis, G. Basu, Z. Gatalica, M.F. Press, Mutations in thekinase domain of the HER2 / ERBB2 gene identified in a wide variety of humancancers, J. Mol. Diagnostics. 17 (2015) 487–495.

[0671] [7] P. Chaturvedi, A.P. Singh, S. Chakraborty, S.C. Chauhan, S.Bafna, J.L. Meza, P.K. Singh, M.A. Hollingsworth, P.P. Mehta, S.K. Batra,MUC4 Mucin Interacts with and stabilizes the HER2 Oncoprotein in HumanPancreatic Cancer Cells, Cancer Res. 68 (2008) 2065–2070.

[0672] [8] S. Bafna, S. Kaur, N. Momi, S.K. Batra, Pancreatic cancer cellsresistance to gemcitabine: the role of MUC4 mucin, Br. J. Cancer. 101 (2009)1155–1161.

[0673] [9] H. Yao, W. Song, R. Cao, C. Ye, L. Zhang, H. Chen, J. Wang, Y.Shi, R. Li, Y. Li, X. Liu, X. Zhou, R. Shao, L. Li, An EGFR / HER2-targetedconjugate sensitizes gemcitabine-sensitive and resistant pancreatic cancerthrough different SMAD4-mediated mechanisms, Nat. Commun. 13 (2022) 5506.https: / / doi.org / 10.1038 / s41467-022-33037-x.

[0674]

[10] I. Schlam, S.M. Swain, HER2-positive breast cancer and tyrosinekinase inhibitors: the time is now, Npj Breast Cancer. 7 (2021) 1–12.https: / / doi.org / 10.1038 / s41523-021-00265-1.

[0675]

[11] B. Moy, P.E. Goss, Lapatinib-Associated Toxicity and PracticalManagement Recommendations, Oncologist. 12 (2007) 756–765. https: / / doi.org / 10.1634 / theoncologist.12-7-756.

[0676]

[12] S. Gobeil, C.C. Boucher, D. Nadeau, G.G. Poirier,Characterization of the necrotic cleavage of poly (ADP-ribose) polymerase(PARP-1): Implication of lysosomal proteases, Cell Death Differ. 8 (2001)588–594. https: / / doi.org / 10.1038 / sj.cdd.4400851.

[0677]

[13] R. Gradiz, H.C. Silva, L. Carvalho, M.F. Botelho, A. Mota-Pinto,MIA PaCa-2 and PANC-1-Pancreas ductal adenocarcinoma cell lines withneuroendocrine differentiation and somatostatin receptors, Sci. Rep. 6 (2016)1–14. https: / / doi.org / 10.1038 / srep21648.

[0678]

[14] C. Fiorini, M. Cordani, C. Padroni, G. Blandino, S. Di Agostino,M. Donadelli, Mutant p53 stimulates chemoresistance of pancreaticadenocarcinoma cells to gemcitabine, Biochim. Biophys. Acta - Mol. Cell Res.1853 (2015) 89–100.

[0679]

[15] A.M. Boutelle, L.D. Attardi, p53 and Tumour Suppression: ItTakes a Network, Trends Cell Biol. 31 (2021) 298–310.

[0680]

[16] T. Yamazaki, L. Galluzzi, BAX and BAK dynamics controlmitochondrial DNA release during apoptosis, Cell Death Differ. 29 (2022)1296–1298. https: / / doi.org / 10.1038 / s41418-022-00985-2。

Claims

1. A pharmaceutical composition comprising at least one compound selected from the following chemical formulas: （i） Chemical formula (I) for 6-hydroxy-3H-isochromene-3,8(4H)-dione. (ii) 5-Methyl-1H-indole-3-carboxylic acid Molecular formula: C 10 H9NO3 Chemical formula (II) (iii) 6-Hydroxy-3-methyl-2H-pyran-2-one Molecular formula = C6H6O3 Chemical formula (III) (iv) 2-Methyl-4H-pyran-4-one Molecular formula = C6H6O2 Chemical formula (IV) Or a combination thereof.

2. The composition according to claim 1, wherein the composition further comprises a drug-use carrier.

3. The composition according to claim 1, wherein the amount of the compound represented by chemical formula I is in the range of 6 wt% to 18 wt%.

4. The composition according to claim 3, wherein the amount of the compound represented by chemical formula I is 12.60 wt%.

5. The composition according to claim 1, wherein the amount of the compound represented by chemical formula II is in the range of 8 wt% to 22 wt%.

6. The composition according to claim 5, wherein the amount of the compound represented by chemical formula II is 15.41 wt%.

7. The composition according to claim 1, wherein the amount of the compound represented by chemical formula III is in the range of 2 wt% to 10 wt%.

8. The composition according to claim 7, wherein the amount of the compound represented by chemical formula III is 6.48 wt%.

9. The composition according to claim 1, wherein the amount of the compound represented by chemical formula I is in the range of 1 wt% to 10 wt%.

10. The composition according to claim 9, wherein the amount of the compound represented by chemical formula IV is 5.89 wt%.

11. The pharmaceutical composition according to the preceding claims, wherein the composition further comprises one or more other 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 medicament in treating cancer in a subject.

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

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

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

17. A pharmaceutical composition comprising compounds shown in chemical 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, inhibiting the anti-apoptotic protein Bcl2, and inhibiting the growth of pancreatic cancer cells.

18. A pharmaceutical composition comprising compounds represented by chemical formula (I), chemical formula (II), chemical formula (III) and chemical formula (IV) for use as a pharmaceutical agent in subjects for treating prostate cancer, triple-negative breast cancer, invasive carcinoma, lung cancer, sarcoma, serous carcinoma, pancreatic cancer and HER2-positive breast cancer.

19. A compound represented by chemical formula I: 6-Hydroxy-3H-isochrome-3,8(4H)-dione.

20. A compound represented by chemical formula III: 6-Hydroxy-3-methyl-2H-pyran-2-one Molecular formula = C6H6O3.

21. A compound for treating cancer, selected from chemical formula (I), chemical formula (II), chemical formula (III), chemical formula (IV) or chemical formula (V).

22. A method for separating compounds of formula I, formula II, formula III or formula IV from barley extract: （i） Chemical formula (I) for 6-hydroxy-3H-isochromene-3,8(4H)-dione. (ii) 5-Methyl-1H-indole-3-carboxylic acid Molecular formula: C 10 H9NO3 Chemical formula (II) (iii) 6-Hydroxy-3-methyl-2H-pyran-2-one Molecular formula = C6H6O3, chemical formula (III), or (iv) 2-Methyl-4H-pyran-4-one Molecular formula = C6H6O2 Chemical formula (IV). in, The method includes the following steps: i. Barley extract was subjected to chromatography using a preparative HPLC method; ii. Elution peaks 2, 3, 4, and 5; iii. Fractions are obtained by manually separating the peaks obtained in step (ii) through repeated injections; iv. Merging and condensing the fractions; and v. Analyze the peaks and determine the structures of the compounds represented by chemical formulas I, II, III, and IV; In step (i), the preparative HPLC method includes mobile phase A and mobile phase B; In step (ii), the elution of peaks 2, 3, 4 and 5 is performed at approximately 14.3, 15.5, 16.4 and 23.0 minutes, respectively.

23. The pharmaceutical composition according to claim 1, wherein the composition is 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, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III), and (IV).

25. A method for inducing apoptosis in a subject, inducing expression of the pro-apoptotic protein Bax and inducing cleavage of PARP protein, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising compounds represented by chemical formulas (I), (II), (III) and (IV).

26. A method for inhibiting HER-2 expression, inhibiting the anti-apoptotic protein Bcl2, inhibiting the growth 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 comprising compounds represented by chemical formulas (I), (II), (III), and (IV).

Images