New compounds could treat cancer, other diseases

Abstract

This patent provides a method for synthesizing new medicinal compounds. The new medicinal compounds can treat cancer, and the cancers include breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain cancer, leukemia, lung cancer, colon cancer, central nervous system cancer (CNS), melanoma, renal duct cancer, cervical cancer, esophageal cancer, testicular cancer, spleen cancer, kidney cancer, lymphoma, pancreatic cancer, gastric cancer and thyroid cancer. This patent provides an anti-adhesion therapy, that is, using these compounds as regulatory agents or inhibitors of adhesion proteins and angiogenin, inhibiting excessive adhesion and cell attachment, and regulating angiogenesis. These compounds can also be used as cell adhesion receptors, cell circulation and movement, and regulatory agents for inflammatory diseases.

Description

[0001] This application is a divisional application of the invention patent application with application number 201180043561.4, application date July 15, 2011, and invention name “New compounds for treating cancer and other diseases”.

[0002] This patent claims the benefit of International Patent Application No. PCT / US20100042240, filed on July 16, 2010, U.S. Patent Application No. 12 / 856322, filed on August 13, 2010, U.S. Patent Application No. 12 / 541,713, filed on August 14, 2009, U.S. Patent Application No. 61 / 226,043, filed on July 16, 2009, and International Patent Application No.

[0003] PCT / US09 / 34115 filed on February 13, 2009. This patent also claims the rights or priority of U.S. Patent Application No. 61 / 038,277 filed on March 20, 2008, U.S. Patent Application No. 61 / 054,308 filed on May 19, 2008, and International Patent Application No.

[0004] PCT / US2008 / 002086 was filed on February 15, 2008, International Patent Application No.

[0005] PCT / US2007 / 077273 filed on August 30, 2007, U.S. Patent Application No.

[0006] 60 / 890,380 filed on February 16, 2007, U.S. Patent Application No. 60 / 947,705 filed on July 3, 2007 and U.S. Patent Application No. 11 / 683,198 filed on March 7, 2007, which also claims U.S. Patent Application No. USSerial No. Nos. 60 / 795,417, filed April 27, 2006; 60 / 841,727, filed September 1, 2006 and 60 / 890,380, filed February 16, 2007) and International Patent Application No. PCT / US2006 / 016158, filed April 27, 2006, which in turn claim the benefit of or priority to the following patent applications: (1) U.S. Patent Application No. 11 / 289,142, filed November 28, 2005 and U.S. Patent Application No. 11 / 267,523, filed November 4, 2005 filed on September 7, 2005, (2) International Patent Application No. PCT / US2005 / 031900, filed on September 7, 2005, which application claims the rights or priority claimed in U.S. Patent Application No. 60 / 617,379, filed on October 8, 2004, U.S. Patent Application No. 60 / 613,811, filed on September 27, 2004, and U.S. Patent Application No. 60 / 607,858, filed on September 7, 2004, (3) U.S. Patent Application No. 11 / 131,551, filed on May 17, 2005 and (4) U.S. Patent Application No. 11 / 117,760, filed on April 27, 2005. The present invention claims the benefit of or priority to U.S. Patent Application No. 11 / 412,659, filed April 27, 2006; U.S. Patent Application No. 10 / 906,303, filed February 14, 2005, and U.S. Patent Application No. 12 / 344,682, filed December 29, 2008. The contents of these pending patent applications are hereby fully incorporated into this patent application. Technical Field

[0007] This patent provides compounds, compositions, extracts and methods that can inhibit cancer invasion, cell invasion or cancer cell invasion. Background Art

[0008] This patent provides a method for synthesizing new compounds for pharmaceutical use, and provides compounds, compositions, extracts and methods for treating cancer, inhibiting cancer invasion, cell invasion or cancer cell invasion. Here, cancer includes breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain cancer, leukemia, lung cancer, colon cancer, central nervous system cancer (CNS), melanoma, renal duct cancer, cervical cancer, esophageal cancer, testicular cancer, spleen cancer, kidney cancer, lymphoma, pancreatic cancer, stomach cancer and thyroid cancer, etc. Summary of the invention

[0009] This patent provides a method for synthesizing new compounds for pharmaceutical use. The compounds provided by this patent can be used as regulators or inhibitors of adhesion proteins or angiopoietin, which can inhibit excessive adhesion and connection of adhesion cells, regulate angiopoietin, and can be used as regulators of cell adhesion receptors.

[0010] The compound or composition containing the compound provided by this patent can treat cancer, inhibit cancer growth, inhibit viruses, prevent brain aging, improve memory and brain function, treat enuresis, frequent urination and urgency, dementia, Parkinson's disease, or other diseases caused by brain dysfunction, arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's disease, angina pectoris, heart dysfunction, coronary heart disease, headache, dizziness and kidney dysfunction, cardiovascular disease, inhibit NF-Kappa B activation, cerebral edema, respiratory distress syndrome, respiratory viral diseases; chronic venous insufficiency; hypotension; chronic venous disease; edema; anti-inflammatory; hemorrhoids, peripheral edema; varicose veins; influenza; post-traumatic edema; postoperative swelling; Inhibit thrombosis, inhibit ethanol absorption; lower blood sugar; adjust the level of corticotropin and corticosterone. The compounds provided by this patent can be AntiMS, anti-aneurysm, anti-asthma, vasodilation, anti-capillary bleeding, anti-headache, anti-cervical and arm pain, eclampsia, edema, encephalitis, epiglottitis, exudation, influenza, fracture, gingivitis, hematoma, herpes, histamine shock, joint effusion, meningitis, antioxidant effect, periodontitis, phlebitis, pleurisy, hoarseness, rhinitis, tonsillitis, ulcer, varicose veins, vertigo, cancer metastasis, cause corticosterone, diuretic, antifungal, hemolytic effect, hyaluronidase inhibitor, lymphatic agent, natriuretic effect, insecticide, mucolytic agent, thymic, protect blood vessels and veins. Inhibit Leishmaniasis, inhibit cancer cell adhesion or angiopoietin, antiparasitic; enhance the expression of the following genes: ANGPT2, DDIT3, LIF and NFKB1Z; manufacture auxiliary compositions and use for venous resection treatment.

[0011] This patent provides compounds, compositions and methods that can cure cancer, inhibit cancer cell growth, invasion and metastasis. It is characterized in that the compounds contain chemical structures in the molecular formula provided in this patent, and these chemical structures contain triterpenes, pentacyclic triterpenes and other chemical functional groups, which can be artificially synthesized or extracted. Here, the cancers include breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain cancer, leukemia, lung cancer, colon cancer, central nervous system cancer (CNS), melanoma, renal duct cancer, cervical cancer, esophageal cancer, testicular cancer, spleen cancer, kidney cancer, lymphoma, pancreatic cancer, gastric cancer and thyroid cancer, etc. The cells described herein are breast cells, leukocytes, liver cells, ovarian cells, bladder cells, prostate cells, skin cells, bone cells, brain cells, leukemia cells, lung cells, colon cells, central nervous system cells (CNS), melanoma cells, renal duct cells, cervical cells, esophageal cells, testicular cells, spleen cells, kidney cells, lymphocytes, pancreatic cells, stomach cells and thyroid cells.

[0012] The present invention provides compounds that can treat cancer, inhibit cancer cell growth, invasion and metastasis, cell circulation in the body, and cell adhesion. These compounds are triterpenes, pentacyclic triterpenoid saponin compounds, containing tigloyl, angelica acyl, crotonyl, senecioyl, acetyl, 3,3-dimethylacryloyl, cinnamoyl, pentenoyl, hexenoyl, benzyl, ethylbutyryl, dibenzoyl, alkanoyl, alkenoyl, benzylalkyl substituted alkanoyl, alkanoyl substituted phenyl, alkenoyl substituted phenyl, aromatic group, acyl, heterocyclic compound, heterocyclic aromatic compound, sugar chain or sugar chain with diangelica acyl.

[0013] The present invention discloses pentacyclic triterpenes, triterpenes, triterpenoid saponin compounds which can inhibit cancer growth and invasion, cell invasion and cancer cell invasion, and contain cis-1, 2, 24 and / or 28 carbonyl groups, such as angeloyl, acetyl, crotonoyl, 3,3-dimethylacryloyl, senecioyl, cinnamoyl, pentenoyl, hexenoyl, benzoyl, ethylbutyryl, dibenzoyl, alkanoyl, alkenoyl, alkanoyl substituted with benzylalkyl, alkanoyl substituted with phenyl, alkenoyl substituted with phenyl, aromatic groups, acyl, heterocyclic compounds, heterocyclic aromatic compounds, and sugar chains with diangeloyl groups. The present invention discloses that the pentacyclic triterpenes, triterpenes, triterpenoid saponin compounds provided by the present invention contain cis-1-yl, angeloyl, acetyl, crotonoyl, 3,3-dimethylacryloyl, senecioyl, cinnamoyl, pentenoyl, hexenoyl, benzoyl, ethylbutyryl, dibenzoyl, alkanoyl, alkenoyl, alkanoyl substituted with benzylalkyl, alkanoyl substituted with phenyl, alkenoyl substituted with phenyl, aromatic, acyl, heterocyclic compound, heterocyclic aromatic compound, or sugar chain with diangeloyl at carbon 3, 8, 15, 21, 22, 24 and / or 28 positions, and these compounds have the ability to inhibit cancer invasion, cell invasion and cancer cell invasion, and cell adhesion. In an embodiment, the saponin compound has a functional group at carbon 24 and has biological activity. In an embodiment, the saponin compound has a functional group at carbon 24 and 28 and has biological activity. In embodiments, saponin compounds have biological activity when they have functional groups at carbon 24, and 21. In embodiments, saponin compounds have biological activity when they have functional groups at carbon 24, 28, and 21. In embodiments, saponin compounds have biological activity when they have functional groups at carbon 24, 28, and 22. In embodiments, saponin compounds have biological activity when they have functional groups at carbon 24, 28, and 3. Here, saponin compounds have biological activity when they have functional groups at carbon 24, and 3. In embodiments, saponin compounds have biological activity when they have functional groups at carbon 28, and 3. In embodiments, saponin compounds have biological activity when they have functional groups at carbon 3. In embodiments, saponin compounds have biological activity when they have functional groups at carbon 21, and 22.

[0014] This patent provides a method for artificially synthesizing a biologically active compound. The method described herein is characterized in that an active chemical group is connected to a parent core compound. The active chemical groups described herein include cis-zhioyl, angelicoyl, acetyl, crotonoyl, 3,3-dimethylacryloyl, senecioyl, cinnamoyl, pentenoyl, hexenoyl, benzoyl, ethylbutyryl, dibenzoyl, alkanoyl, alkenoyl, alkanoyl substituted with benzylalkyl, alkanoyl substituted with phenyl, alkenoyl substituted with phenyl, aromatic, acyl, heterocyclic compound, heterocyclic aromatic compound. The parent core compound described in the embodiment here is a pentacyclic triterpene. The parent core compound described in the embodiment may also be a tetracyclic terpene. The parent core compound described in the embodiment may also be a tricyclic terpene. The parent core compound described in the embodiment may also be a bicyclic terpene. The parent core compound described in the embodiment may also be a monocyclic terpene. The artificially synthesized biologically active compounds provided by this patent can treat cancer, inhibit cancer growth, inhibit viruses, prevent brain aging, improve memory and brain function, treat enuresis, frequent urination and urgency, dementia, Parkinson's disease, or other diseases caused by brain dysfunction, arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's disease, angina pectoris, heart dysfunction, coronary heart disease, headache, dizziness and kidney dysfunction, cardiovascular disease, inhibit NF-Kappa B activation, cerebral edema, dyspnea syndrome, respiratory viral diseases; chronic venous insufficiency; hypotension; chronic venous disease; anti-edema; anti-inflammatory; hemorrhoids, peripheral edema; varicose veins; influenza; post-traumatic edema; postoperative swelling; inhibit thrombosis, inhibit ethanol absorption; lower blood sugar; adjust the levels of corticotropin and corticosterone. The compounds provided by this patent can be AntiMS, anti-aneurysm, anti-asthma, Vasodilation, anti-capillary bleeding, anti-headache, anti-cervical pain, eclampsia, edema, encephalitis, epiglottitis, exudation, influenza, fracture, gingivitis, hematoma, herpes, histamine shock, joint effusion, meningitis, antioxidant effect, periodontitis, phlebitis, pleurisy, hoarseness, rhinitis, tonsillitis, ulcer, varicose veins, vertigo, cancer metastasis, corticosterone production, diuretic, antifungal, hemolytic effect, hyaluronidase inhibitor, lymphatic agent, natriuretic effect, insecticide, mucolytic agent, thymic, vascular and venous protection. Inhibit leishmaniasis, inhibit cancer cell adhesion or angiopoietin, antiparasitic; enhance the expression of the following genes: ANGPT2, DDIT3, LIF and NFKB1Z; manufacture auxiliary compositions and for phlebotomy treatment.

[0015] The patent experiment proves that the compound AKOH does not have the ability to treat and inhibit cancer growth, cancer invasion, cell or cancer cell invasion. The compound AKOH is obtained by removing the angelica acyl groups at carbon 21 and 22 of the active compound Xanifolia Y (Y3). The patent experiment proves that after the angelica acyl groups at carbon 21 and 22 of the active compound Xanifolia Y (Y3) are removed, the ability to treat and inhibit cancer growth, cancer invasion, cell or cancer cell invasion is lost.

[0016] The patent experiment proves that the parent compound E4A, E5A, Xanifolia Y-core does not have the ability to treat and inhibit cancer growth, cancer invasion, cell or cancer cell invasion. Removing the angelica acyl group from the carbon 21 and 22 positions of the compound Xanifolia Y (Y3) and removing the sugar chain from the carbon 3 position will give the parent compound Xanifolia Y-core. Removing the active groups at carbon 3, 21, and 22 positions of the active compound Escin will give the compound E4A (EIVA). Removing the active groups at carbon 3, 21, and 22 positions of the active compound Escin II will give the compound E5A (EVA).

[0017] The patent experiment proves that the parent core compounds E4A, E5A, Xanifolia Y-core and AKOH have no hemolytic effect and no anticancer activity.

[0018] The patent test proves that the compounds Tig-N, Tig-Q, Tig-R, Tig-T Tig-S and Tig-V have no hemolytic effect at a concentration below 20ug / ml. The original compound ES can dissolve all red blood cells at a concentration of 5ug / ml. Compared with compound Y3, ACH-Y3 has a smaller hemolytic effect. Compound Tig-R has no hemolytic effect. The patent test proves that the compounds Tig-N, Tig-Q, Tig-R, Tig-T Tig-S and Tig-V have anti-cancer effects.

[0019] The patent experiment proves that after the sugar chain at the carbon 3 position of the pentacyclic triterpenoid, triterpenoid, and triterpenoid saponin compound is removed, the ability to treat and inhibit cancer growth, cancer invasion, cell or cancer cell invasion is still retained. The patent experiment proves that the compound ACH-Y3 has the ability to treat and inhibit cancer growth, cancer invasion, cell or cancer cell invasion. The compound Xanifolia Y (Y3) is obtained by removing the sugar chain at the carbon 3 position of the active compound Xanifolia Y (Y3). The patent experiment proves that after the sugar chain at the carbon 3 position of Xanifolia Y (Y3) is removed, the ability to treat and inhibit cancer growth, cancer invasion, cell or cancer cell invasion is still retained.

[0020] A compound that has the activity of therapeutically inhibiting cancer growth, cancer invasion, cell or cancer cell invasion is called an active compound.

[0021] This patent provides compounds, compositions and methods of making drugs for treating cancer, inhibiting cancer invasion, cell invasion or cancer cell invasion, these compounds are synthesized or extracted, and there are pentacyclic triterpenes in these chemical structures. Here, cancer includes breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain cancer, leukemia, lung cancer, colon cancer, central nervous system cancer (CNS), melanoma, renal duct cancer, cervical cancer, esophageal cancer, testicular cancer, spleen cancer, kidney cancer, lymphoma, pancreatic cancer, gastric cancer and thyroid cancer, etc. The drug concentration used to treat cancer, inhibit cancer invasion, cell invasion or cancer cell invasion will not produce cytotoxicity, and will not affect cell morphology.

[0022] The patent provides a method for inhibiting cancer invasion, cell invasion and cancer cell invasion and metastasis, characterized in that the method affects gene expression, including stimulating gene expression, or inhibiting gene expression, or adjusting the compounds provided by the patent, depending on the effective dosage of the composition or extract. The specific method is to contact the above cells with the following compounds: A1-18, A20-32, B1-18, B20-32, C1-18, C20-32, D1-18, D20-32, D1-18, D20-32, D1-18, D20-32, D1-18, D20-32, E1-18, E20-32, G1-18, G20-32, H1-18, H20-32, I1-18, I20-32, J1-18, J20-32, K1-18, K20-32, Xanifolia Y0, Y1, Y2, Y(Y3), Y5, Y7, Y8, Y9, Y10, Xanifolia(x), M10, Escin(bES), Aescin, ACH-Y(Y3), ACH-Y10, ACH-Y2, ACH-Y8, ACH-Y7, ACH-Y0, ACH-X, ACH-Z4, ACH-Z1, ACH-Escin(bES), ACH-M10 and their salts, esters, and metabolites, and compounds selected from the group consisting of formula 2A and K.

[0023] The results of laboratory research in this patent show that compounds with a 2A or K chemical structure can inhibit cell adhesion to the wall of the culture bottle and have the effect of preventing these adhesion molecules from attaching to cells. The cells mentioned here are cancer cells and mesothelial cells. This patent provides an anti-adhesion treatment method, which is characterized in that the method uses the above-mentioned compounds as regulators or inhibitors of adhesion proteins and angiopoietin. That is, excessive adhesion and cell adhesion are inhibited. As regulators, these compounds regulate cell circulation, cell movement and anti-inflammatory diseases. Here, the compounds can bind to adhesion cells (attached to the adhesion cell membrane) to prevent adhesion proteins and their receptors from acting. Here, the compounds act on the adhesion cell membrane to affect the function of the adhesion protein membrane, directly or indirectly removing the adhesion of cancer cells.

[0024] (For our separation and purification methods and biological assays, including MTT assays, see International Patent Application No. PCT / US2005 / 031900, filed September 7, 2005, U.S. Patent Application No. 11 / 289142, filed November 28, 2005, and U.S. Patent Application No. 11 / 131551, filed May 17, 2005, and Patent Application No. PCT / US2008 / 002086,1188-ALA-PCT, filed February 15, 2008. For cell invasion assays, see International Patent Application No. PCT / US2010 / 0042240, filed July 16, 2010, and the contents of these pending patent applications are hereby fully incorporated into this patent application).

[0025] This patent provides compounds or methods that can inhibit cancer invasion, cell invasion, cancer cell invasion and metastasis, characterized in that the method is a process and method of using a drug (composition), and the method of using the drug here is intravenous injection, intravenous drip, intraperitoneal injection or oral administration. For intravenous drip, the compound is dissolved in 250 ml of 10% glucose solution or 250 ml of 0.9% NaCl saline, with a dose of 0.003-0.03 mg / kg; for intravenous injection, 0.003-0.03 mg / kg / day is dissolved in 10-20 ml of 10% glucose solution or 0.9% NaCl saline; or 0.01-0.03 mg / kg is dissolved in 10-20 ml of 10% glucose solution or 0.9% NaCl saline; or the intravenous drip dose is 0.01-0.03 mg / kg / day, dissolved in 250 ml of 10% glucose solution or 250 ml of 0.9% NaCl saline; or 0.01-0.05 mg / kg is dissolved in 250 ml of 10% glucose solution or 250 ml of 0.9% NaCl Saline; intravenous injection 0.01-0.05 mg / kg / day, dissolved in 10-20 ml of 10% glucose solution or 0.9% NaCl saline; or 0.05-0.2 mg / kg, dissolved in 250 ml of 10% glucose solution or 250 ml of 0.9% NaCl saline; intravenous injection 0.05-0.2 mg / kg / day, dissolved in 10-20 ml of 10% glucose solution or 0.9% NaCl saline; intravenous drip 0.1-0.2 mg / kg / day, Dissolved in 250ml of 10% glucose solution or 250ml of 0.9% NaCl saline; intravenous injection 0.1-0.2mg / kg / day, dissolved in 10-20ml of 10% glucose solution or 0.9% NaCl saline; peritoneal injection (IP) dose 2.5mg / kg / day, dissolved in 10% glucose solution or 0.9% NaCl saline; or oral, the dose for mammals is 1-10mg / Kg, 10-30mg / Kg, 30-60mg / Kg, or 60-90mg / Kg; for intravenous injection or intravenous drip, the dose for mammals is 0.01-0.1mg / Kg, 0.1-0.2mg / Kg, 0.2- 0.4mg / Kg, or 0.4–0.6mg / Kg; for peritoneal injection (IP) the dose for mammals is 1-3mg / Kg, 3-5mg / Kg, 4-6mg / Kg, or 6-10mg / Kg.

[0026] The patent provides compounds or methods that can inhibit cancer invasion, cell invasion, cancer cell invasion and metastasis, characterized in that the composition is a drug composition containing the compound provided by the patent, or a pharmaceutically acceptable salt, or a pharmaceutically acceptable carrier or diluent. The concentration of the compound in the above composition is 0.01ug / ml to 65ug / ml, or 0.01 ug / ml to 40ug / ml, or 0.01ug / ml to 30ug / ml, or 0.01ug / ml to 10ug / ml, or 0.01ug / ml to 5ug / ml, or 5ug / ml to 10ug / ml, or 0.1ug / ml to5ug / ml, or 0.1ug / ml to 7.5ug / ml, or 0.1ug / ml to 10ug / ml, or 0.1ug / ml to 15ug / ml, or 0.1ug / ml to 20ug / ml, or 0.1ug / ml to 30ug / ml, or 1ug / ml to 5ug / ml, or 1ug / ml to7.5ug / ml, or 1ug / ml to 10ug / ml, or 1ug / ml to 15ug / ml, or 1ug / ml to 20ug / ml, or 1ug / ml to 30ug / ml, or 3ug / ml to 5ug / ml, or 3ug / ml to 7.5ug / ml, or 3ug / ml to 10ug / ml, or 3ug / ml to 15ug / ml, or 3ug / ml to 20ug / ml, or 3ug / ml to 30ug / ml, or 4ug / ml to 5ug / ml, or 4ug / ml to 7.5ug / ml, or 4ug / ml to 10ug / ml, or 4ug / ml to 15ug / ml, or 4ug / ml to 20ug / ml, or 4ug / ml to 30ug / ml, or 5ug / ml to 8ug / ml, or 5ug / ml to 9ug / ml, or 5ug / ml to10ug / ml, or 5ug / ml to 15ug / ml, or 5ug / ml to 20ug / ml, or 5ug / ml to 30ug / ml, or 7ug / mlto 8ug / ml, or 7ug / ml to 9ug / ml, or 7ug / ml to 10ug / ml, or 7ug / ml to 15ug / ml, or 7ug / mlto 20ug / ml, or 7ug / ml to 30ug / ml.

[0027] The patent provides compounds or methods that can inhibit cancer invasion, cell invasion, cancer cell invasion and metastasis, characterized in that the composition is a drug composition containing the compound provided by the patent, or a pharmaceutically acceptable salt, or a pharmaceutically acceptable carrier or diluent. The concentration of the compound in the above composition is 0.008uM to 80uM, or 0.01uM to 60uM, or 0.01uM to 50uM, or 0.01uM to 40uM, or 0.01uM to 30uM, or 0.01uM to 20uM, or 0.01uM to 10uM, or 5uM to 10uM, or 0.1uM to5uM, or 0.1uM to 7.5uM, or 0.1uM to 10uM, or 0.1uM to 15uM, or 0.1uM to 20uM, or 0.1uM to 30uM, or 0.1uM to 40uM, or 0.1uM to 50uM, or 0.1uM to 60uM, or 0.1uM to 80uM, or 1uM to 5uM, or 1uM to 7.5uM, or 1uM to 10uM, or 1uM to 15uM, or 1uM to 20uM, or 1uM to30uM, or 1uM to 40uM, or 1uM to 50uM or 1uM to 60uM, or 1uM to 80uM, or 3uM to 5uM, or 3uM to 7.5uM, or 3uM to 10uM, or 3uM to 15uM, or 3uM to 20uM, or 3uM to 30uM, or 3uM to 40uM, or 3uM to 50uM, or 3uM to 60uM, or 3uM to 80uM, or 5uM to 8uM, or 5uM to 10uM, or 5uM to 15uM, or 5uM to 20uM, or 5uM to 30uM, or 5uM to 40uM, or 5uM to 50uM, or 5uMto 60uM, or 5uM to 80uM, or 7uM to 8uM, or 7uM to 10uM, or 7uM to 15uM, or 7uM to20uM, or 7uM to 30uM, or 7uM to 40uM, or 7uM to 50uM, or 7uM to 60uM, or 7uM to 80uM.

[0028] The information disclosed in this patent can be better understood through the following experimental details, however, the details provided by these special experiments are limited, and the information provided by this patent is not limited to the details provided by these experiments, but is broader in scope, as described in detail in the following claims section.

[0029] Some references are cited in this patent application in order to better describe the details of this patent.

[0030] It should be noted that the word "comprise" is synonymous with including, comprising or its characteristics, and the content described is open and does not exclude the inclusion of contents not mentioned. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 HPLC spectra of the esterification products of E4A with Tigloyl chloride (A) at different times of the esterification reaction, every 5 seconds, 1 minute, 2 minutes, 5 minutes and 10 minutes. The spectra were prepared based on the elution time and the optical density of the components. The esterification reactions were carried out at room temperature (upper row) and 0°C (lower row).

[0032] Figure 2 HPLC spectra of the esterification products of E4A with 3,3-dimethylacryloyl chloride (B) at different times of the esterification reaction, every 5 seconds, 1 minute, 2 minutes, 5 minutes and 10 minutes. The spectra were prepared based on the elution time and the optical density of the components. The esterification reactions were carried out at room temperature (upper row) and 0°C (lower row).

[0033] Figure 3 HPLC spectra of the esterification products of E4A with 4-pentenoyl chloride (C) at different times of the esterification reaction, every 5 seconds, 1 minute, 2 minutes, 5 minutes and 10 minutes. The spectra were prepared based on the elution time and the optical density of the components. The esterification reaction was carried out at room temperature.

[0034] Figure 4 HPLC spectra of the esterification product of E4A with hexenoyl chloride (D) at different times of the esterification reaction, every 5 seconds, 1 minute, 2 minutes, 5 minutes and 10 minutes. The spectra were prepared based on the elution time and the optical density of the components. The esterification reaction was carried out at 0°C (upper row). Also, HPLC spectra of the esterification product of E4A with 2-ethylbutanoyl chloride (E) at different times of the esterification reaction, every 5 seconds, 1 minute, 2 minutes, 5 minutes and 10 minutes. The spectra were prepared based on the elution time and the optical density of the components. The esterification reaction was carried out at 0°C (lower row).

[0035] Figure 5HPLC spectra of the esterification products of E4A with acetyl chloride (H) at different times of the esterification reaction, every 5 seconds, 1 minute, 2 minutes, 5 minutes and 10 minutes. The spectra were prepared based on the elution time and the optical density of the components. The esterification reaction was carried out at room temperature.

[0036] Figure 6 HPLC profiles of the esterification products of E4A with crotonoyl chloride (I) at different times of the esterification reaction, every 5 seconds, 1 minute, 2 minutes, 5 minutes and 10 minutes. The profiles were prepared based on the elution time and the optical density of the components. The esterification reaction was carried out at room temperature.

[0037] Figure 7 HPLC spectra of the esterification products of E4A with cinnamoyl chloride (J) at different times of the esterification reaction, every 1 minute, 1 hour, 2 hours, 18 hours and 18 hours (heating). The spectra were prepared based on the elution time and the optical density of the components. The esterification reaction was carried out at 75°C.

[0038] Figure 8 HPLC spectra of the esterification products of E4A with benzoyl chloride (K) at different times of the esterification reaction, every 5 seconds, 1 minute, 2 minutes, 5 minutes and 10 minutes. The spectra were prepared based on the elution time and the optical density of the components. The esterification reaction was carried out at 0°C.

[0039] Fig. 9 .MTT toxicity study at room temperature, A: E4A-cis-enoyl; B: E4A-3,3-dimethylacryloyl; C: E4A-4-pentenoyl.

[0040] Fig.10 .MTT toxicity study at 0℃, A: E4A-cis-1-yl; B: E4A-3,3-dimethylacryloyl; C: E4A-4-pentenoyl.

[0041] Fig.11 .Toxicity study, J: E4A-cinnamyl; D: E4A-hexenoyl; E: E4A-2-ethylbutyryl. Control: Tig is cis-azoyl chloride; AC is acetyl chloride; H is E4A and acetyl chloride after esterification for 1 minute.

[0042] Fig.12 .Toxicity studies, H: E4A-acetyl; I: E4A-crotonyl.

[0043] Fig.13 .Toxicity study of E4A-Tig, esterification reaction for 1 min, 15 min, 30 min, 1 h and 2 h.

[0044] Fig.14.HPLC spectra of the esterification products of E4A-Tig at different times of the esterification reaction, 1 minute and 2 hours.

[0045] Fig.15 .E4A-Tig toxicity study results: E4A-Tig esterification reaction is most toxic during 5 seconds to 1 minute, weakens after 1 minute, and is minimal or absent after 10 minutes.

[0046] Fig.16 .HPLC spectra of the esterification products of E4A-Tig, E4A, E4A-ASAP (5 seconds), E4A (1 minute), E4A (2 minutes), E4A (5 minutes), E4A (10 minutes) and E4A (30 minutes) at different times of the esterification reaction.

[0047] Fig.17 .The order of toxicity: M, N, O, P, Q, R, S, T, E4A. M = E4A is not toxic.

[0048] Fig.18 .The results of MTT toxicity study of E4A-Tig-R on cancer cells: A, bone cancer cells (U2OS): IC50 = 4.5ug / ml; B, bladder cancer cells (TB9): IC50 = 2.5ug / ml; C, lung cancer cells (H460): IC50 = 4.8ug / ml; D, ovarian cancer cells (ES2): IC50 = 2.8ug / ml.

[0049] Fig.19 .The results of MTT toxicity study of E4A-Tig-R on cancer cells of different organs: E, colorectal cancer (HCT116) IC50 = 5.2ug / ml; F, pancreatic cancer (Capan) IC50 = 2.4ug / ml; G, ovarian cancer (OVCAR3) IC50 = 5.8ug / ml; H, breast cancer (MCF-7) IC50 = 4.5ug / ml.

[0050] Fig. 20 .The results of MTT toxicity study of E4A-Tig-R on cancer cells of different organs: I, prostate cancer (DU145) IC50 = 3.6ug / ml; J, skin cancer (SK-Mel-5) IC50 = 5.1ug / ml; K, oral cancer (KB) IC 50 = 3ug / ml; L, kidney cancer (A498) IC50 = 3.5ug / ml.

[0051] Fig.21.The results of MTT toxicity study of E4A-Tig-R on cancer cells of different organs: M, liver cancer (HepG2) IC50 = 6ug / ml; N, brain cancer (T98G) IC50 = 8ug / ml; P, melanoma cancer (K562) IC50 = 2ug / ml; Q, cervical cancer (HeLa) IC50 = 5ug / ml.

[0052] Fig. 22 .(A) Tig-N, -Q, -R, -T, -S and -V had no hemolytic effect at a concentration as high as 20ug / ml. The original compound ES lyse 100%red blood cells(RBC)at 5 ug / ml.(B) Compared with compound Y3, ACH-Y3 had less hemolytic effect, and Tig-R had no hemolytic effect.

[0053] Fig.23 (A) HPLC spectrum of the esterification product, multiple fractions were obtained, and each fraction was collected for further study. (B) The results of the purification of E4A-Tig-R.

[0054] Fig.24 .MTT test results of E4A-Tig-R on bone cancer cells (U2OS).

[0055] Fig.25 .HNMR results of E4A-Tig-R.

[0056] Fig.26 .CNMR results of E4A-Tig-R.

[0057] Fig. 27 .HMQC results of E4A-Tig-R.

[0058] Fig.28 .HMBC results of E4A-Tig-R.

[0059] Fig.29 The mass of Tig-R (M+H) is 671.4509, which is consistent with the proposed structure of the compound.

[0060] Fig.30 .The chemical structure of E4A-Tig-R, 24,28-O-cis-1,3β,16α,21β,22α,24β, 28-hexahydroxyolean-12-ene pentacyclic triterpenoid saponin, molecular formula: C40H62O8, molecular weight: 670.91548.

[0061] Fig.31(A) HPLC spectrum of esterification products, each fraction was collected for further study. (B) Results of purification of E4A-Tig-R. (C) Results of purification of E4A-Tig-S. ; (D) Results of purification of E4A-Tig-T.

[0062] Fig.32 .(A) MTT test results of E4A-Tig-N on bone cancer cells (U2OS); (B) MTT test results of E4A-Tig-S on bone cancer cells (U2OS).

[0063] Fig.33 .(A) MTT test results of E4A-Tig-T on bone cancer cells (U2OS); (B) MTT test results of E4A-Tig-V on ovarian cancer (ES2). IC50 = 2ug / ml; (C) E4A-Tig-V purification results.

[0064] Fig.34 .HNMR results of E4A-Tig-V.

[0065] Fig.35 .HMQC results of E4A-Tig-V.

[0066] Fig.36 .HMBC results of E4A-Tig-V.

[0067] Fig.37 .The mass spectrum of E4A-Tig-V, Tig-R (M+H) mass is 753.4924, which is consistent with the assumed molecular formula of the compound (C45H68O9). DETAILED DESCRIPTION

[0068] The following examples are used to further illustrate the present invention, but are not intended to limit the effective scope of the present invention in any way.

[0069] Test 1 doses contain 10 mg, 20 mg, 30 mg of active compound

[0070]

[0071] The active compound, microcrystalline cellulose and corn starch are mixed to form a granular 10% corn starch mass. The granular 10% corn starch mass is sieved, dried, mixed with the remaining corn starch and magnesium stearate, and then compressed into tablets, each containing 1, 5, 10, 20, 30 mg of active ingredient, respectively.

[0072] Test 2 Preparation of intravenous solution

[0073] Active compound 1-10ug

[0074] Sodium citrate 5-50mg

[0075] Citric acid 1-15mg

[0076] Sodium chloride 1-8 mg

[0077] Add water for injection (USP) to 1 mL

[0078] The active compound is dissolved in the above prepared solution of sodium citrate, citric acid, sodium chloride and water for injection (USP) at room temperature.

[0079] Experiment 3 Preparation of intravenous drip solution

[0080] 0.25-2.5 mg of active compound is dissolved in 250 ml of 10% glucose solution or 250 ml of 0.9% NaCl saline.

[0081] Preparation of intravenous drip: 1-2 mg of active compound is dissolved in 250 ml of 10% glucose solution or 250 ml of 0.9% NaCl saline.

[0082] Angelica chloride is produced when angelic acid reacts with one of several standard chlorides, including phosphorus oxychloride, phosphorus trichloride and thionyl chloride. Oxalyl chloride produces angelica chloride and angelica chloride (in a 2:1 ratio).

[0083] The potassium salt was reacted with oxalyl chloride and crystalline DMF in diethyl ether for 2 hours (0°C) to produce pure angelica chloride.

[0084]

[0085]

[0086] The following compounds were hydrolyzed by acid

[0087] a) Xanifolia (Y),

[0088]

[0089] 3-0-[β-D-galactopyranoyl (1→2)]-α-L-arabinofuranoyl (1→3)-β-D-glucuronic acid pyranoyl-21,22-O-biguanylate-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene pentacyclic triterpenoid saponin.

[0090] c) Xanifolia (Y2),

[0091]

[0092] 3-0-[β-D-glucose pyranoyl (1→2)]-α-L-arabinofuranoyl (1→3)β-D-glucuronic acid pyranoyl-21,22-O-biphenyl furanoyl-3β,15α,16α,21β,22α,24β, 28-heptahydroxyolean-12-ene pentacyclic triterpenoid saponin.

[0093] d) Xanifolia (Y8),

[0094]

[0095] 3-0-[β-glucose pyranoyl (1→2)]-α-arabinofuranoyl (1→3)-β-glucuronic acid pyranoyl-21,22-O-biphenyl furanoyl-3β,16α,21β,22α,24β,28-hexahydroxyolean-12-ene pentacyclic triterpenoid saponin.

[0096] f) Xanifolia (Y10),

[0097]

[0098] 3-0-[β-galactopyranoyl (1→2)]-α-arabinofuranoyl (1→3)-β-glucuronic acid pyranoyl-21,22-O-bipyranoyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene pentacyclic triterpenoid saponin.

[0099] j) Compound (M10)

[0100]

[0101] m) Compound (bES):

[0102]

[0103] The product after hydrolysis is the following compound containing (ACH) structure:

[0104]

[0105] The composition provided by this patent contains compounds with biological activity extracted from natural plants or synthesized artificially. For compound purification and biological activity determination (including MTT determination), please refer to the international patent application (PCT / US05 / 31900, filed on September 7, 2005, the US patent application (USSerial No. 11 / 289142, November 28, 2005, USSerial No. 11 / 131551, filed on May 17, 2005) and the international patent application (PCT / US2008 / 002086, 1188-ALA-PCT, 2008 The present invention relates to an international patent application filed on February 15, 2008 (PCT / US2008 / 002086,1188-ALA-PCT, filed on February 15, 2008), and PC ...

[0106] Determination of hemolysis

[0107] Red blood cells (RBC) were randomly collected from human body (EDTA whole blood). 50ul of 10% RBC suspension was dissolved in PBS to prepare 2ml sample solution with concentration ranging from 0.1ug / ml to 400ug / ml. The mixture was slightly shaken, placed at room temperature for 60 minutes, and then shaken (3K) for 10min. A certain amount of dissolved hemoglobin was measured in the supernatant (540nm). The synthetic compounds provided in this patent were tested for hemolysis using this method.

[0108] Hydrolysis of saponins

[0109] 15 mg of Xanifolia-Y was dissolved in 1 ml of methanol, 1 ml of 2N HCl was added, and the mixture was refluxed in a water bath at 80°C for 5 hours, and 2 ml of 1N NaOH was added to neutralize the mixture (the final pH was 4-6). The mixture was extracted twice with 3 ml of ethyl acetate to obtain the saponin ligand. The ligands obtained twice were mixed and further separated by HPLC (80-100% acetonitrile as normal liquid elution) to obtain ACH-Y. Compounds Z4, Y10, Y2, Y8, Y7, Y0, X, M10 and ESCIN (bES) were treated in the same manner to obtain ACH-Z4, ACH-Y10, ACH-Y2, ACH-Y8, ACH-Y7, ACH-Y0, ACH-X, ACH-E, ACH-Z5, ACH-M10 and ACH-bES.

[0110] The test method is described in the international patent application PCT / US2010 / 0042240 filed on July 16, 2010, and therefore, the contents of these pending patent applications should be fully incorporated into this patent application.

[0111] Alkaline hydrolysis to remove acyl groups

[0112] 20 mg of Xanifolia-Y was dissolved in 0.5 ml of 1 M NaOH and incubated in a water bath at 80°C for 4 hours. When the solution reached room temperature, 0.5 ml of 1 N HCl was added for neutralization (pH adjustment to 3). Then, the solution was extracted three times with 2 ml of 1-butanol. The butanol extract was collected and freeze-dried in vacuo to obtain the saponin, which was then separated and purified by HPLC (C-18 column, 25% acetonitrile elution).

[0113]

[0114] Compounds AKOH-Y and AKOH-M10 lost their activity in inhibiting cancer invasion, cell invasion, and cancer cell invasion and metastasis.

[0115] Core compound

[0116] Saponins isolated from nature can be hydrolyzed with acid and alkali to obtain parent core compounds, pentacyclic triterpenes and hydroxypentacyclic triterpenes. Pentacyclic triterpenes can also be synthesized artificially. The synthesis method is as follows:

[0117] Beta-escin, compound Y, Y10, Y2, Y8, Y7, Y0, X, or M10 was dissolved in 1M NaOH (20 mg / ml) and incubated at 70°C for 5 hours. The hydrolyzate was neutralized with HCl and water and evaporated to dryness by freeze drying. The resulting product was dissolved in 50% methanol and 1N HCl and incubated at 70°C for 5 hours. The hydrolyzate was neutralized with NaOH. The hydrolyzate was extracted with ethyl acetate and then evaporated to dryness by freeze drying. The hydrolyzate, i.e., the core compound, including (E4A), was further purified by FPLC chromatography (using a C18 column, 70% acetonitrile / TFA, 1 ml / min) to obtain the core compound.

[0118] The parent compound has no activity in inhibiting cancer invasion, cell invasion, and cancer cell invasion and metastasis.

[0119] The structure of the parent compound is as follows:

[0120]

[0121]

[0122] Also known as bES-core, E IV A, ES4A, E4A or (E);

[0123] Also known as ES V, E5A or (F)

[0124]

[0125] Wherein R1, R2, R5, R8 represent OH; R3 represents OH or H; R4, R10 represent CH3 or CH2OH; R9, R11, R12, R13, R14, R15 represent CH3;

[0126]

[0127] Among them, R1, R2, R5, R8, R17, R18 represent OH; R3 represents OH or H; R9, R11, R12, R13, R14, R15 represent CH3.

[0128]

[0129] This is the standard nomenclature for carbon 1 to 30 of a pentacyclic triterpene.

[0130] That is, compound E4A or (E), wherein R1, R2, R5, R8, R17, R18 represent hydrogen, respectively, and R9, R11, R12, R13, R14, R15 represent hydroxyl groups.

[0131] This patent provides a method for artificially synthesizing new active compounds, characterized in that the method connects functional groups to the parent core compound, and the functional groups include but are not limited to (A), (B), (C), (D), (E), (F), (G), and (H). This connection process involves an esterification reaction process of the parent core compound and acyl chloride, cis-acid chloride, angelica acyl chloride, acetyl chloride, crotonyl chloride, 3,3-dimethylacryloyl chloride, senecioyl chloride, cinnamic acid chloride, pentenoyl chloride, hexenoyl chloride, benzoyl chloride or ethylbutyryl chloride (not limited to these functional groups), and the reaction is carried out at 0℃, 25℃ or 75℃ for 5sec, 1min, 2min, 5min, 10min, 30min, 1hr, 2hr, 18hr, 2 days or 3 days. At the end of the reaction, add 5 ml of 2N HCl or 1M NaHCO3, then extract with 10 ml of ether three times, and then vacuum dry and freeze-dry at 45°C. The obtained product is dissolved in 80% acetonitrile-0.005% trifluoroacetic acid. The esterification product is purified by HPLC. The MTT activity of acetyl chloride, the solution after esterification reaction and each component and each compound is determined. These parent core compounds can be synthesized, semi-synthesized or extracted from natural plants. These parent core compounds include terpenes, isoprene, triterpenes and hydroxytriterpenes.

[0132] The MTT activity of these core compounds was studied at 0°C, 25°C or 75°C with different acylation reaction times (5 sec, 1 min, 2 min, 5 min, 10 min, 30 min, 1 hr, 2 hr, 18 hr, 2 days or 3 days). The HPLC spectra of the esterification products of the core compound E4A and acyl chlorides including cis-acid chloride, angelica acid chloride, acetyl chloride, crotonyl chloride, 3,3-dimethylacryloyl chloride, senecioyl chloride, cinnamoyl chloride, pentenoyl chloride, hexenoyl chloride, benzoyl chloride or ethylbutanoyl chloride (not limited to these functional groups) showed that the composition of the compounds changed when the reaction temperature or time was changed (see Figure 1-21 ).

[0133] Peaks, components and compounds are selected based on their activity at different reaction times. HPLC components are selected for separation based on the cytotoxicity of the reaction products at specific time periods. Compounds with activity from strong to weak are selected and separated. Based on the anticancer activity of the compounds studied by MTT, the cancers mentioned here include bone (U2OS), lung (H460), bladder (HTB-9), ovary (ES2), colon (HCT116), pancreas (Capan), ovary (OVCAR3), prostate (DU145), skin (SK-Mel-5), oral cavity (KB), kidney (A498), breast (MCF-7), liver (HepG2), brain (T98G), melanoma (K562), cervix (HeLa.

[0134] After the esterification reaction between the parent compound E4A and Tigloyl chloride, the following compounds were obtained by HPLC separation:

[0135]

[0136]

[0137]

[0138] The following compounds were obtained after esterification of the core compound E4A and Angeloyl chloride and separation by HPLC.

[0139]

[0140]

[0141] The parent compound E4A was subjected to esterification reaction with senecioyl chloride, acetyl chloride, and 3,3-dimethylacryloyl chloride, and the following compounds were obtained by HPLC separation, where Sen = senecioyl.

[0142]

[0143]

[0144] After the esterification reaction between the core compound E4A and 4-pentenoyl chloride, the following compound was obtained by HPLC separation, Pen = pentenoyl.

[0145]

[0146]

[0147]

[0148] After the core compound E4A and hexenoyl chloride were reacted for esterification, the following compound was obtained by HPLC separation, Hex = hexenoyl.

[0149]

[0150]

[0151] After the esterification reaction between the core compound E4A and 2-ethylbutyryl chloride, the following compound was obtained by HPLC separation, where Eth = 2-ethylbutyryl.

[0152]

[0153]

[0154] After the core compound E4A was esterified with acetyl chloride, the following compounds were obtained by HPLC separation: Acy = acetyl.

[0155]

[0156]

[0157] After the core compound E4A and crotonyl chloride were reacted for esterification, the following compound was obtained by HPLC separation, Cro = crotonyl.

[0158]

[0159]

[0160] After the esterification reaction between the core compound E4A and cinnamoyl chloride, the following compound was obtained by HPLC separation, where Cin = cinnamoyl.

[0161]

[0162]

[0163] After the esterification reaction between the core compound E4A and benzoyl chloride, the following compound was obtained by HPLC separation, where Ben = benzoyl.

[0164]

[0165]

[0166] The following compounds were obtained after esterification of the core compound E4A-Tig-N and senecioyl chloride by HPLC separation.

[0167] R1 R2 R5 R8 R17 R18 Cytotoxicity E4A-Tig-N OH OH OH OH O-Tig OH medium Tig-Sen-1 OH OH OH OH O-Tig O-Sen powerful Tig-Sen-2 O-Sen OH OH OH O-Tig O-Sen medium Tig-Sen-3 OH O-Sen OH OH O-Tig O-Sen medium Tig-Sen-4 OH OH O-Sen OH O-Tig O-Sen medium Tig-Sen-5 O-Sen OH OH OH O-Tig OH medium Tig-Sen-6 OH O-Sen OH OH O-Tig OH medium

[0168] After the esterification reaction of the parent compound E4A-Tig-N and crotonyl chloride, the following compounds were obtained by HPLC separation:

[0169]

[0170]

[0171] After the esterification reaction of the parent compound E4A-Tig-N and acetyl chloride, the following compounds were obtained by HPLC separation:

[0172] R1 R2 R5 R8 R17 R18 Cytotoxicity E4A-Tig-N OH OH OH OH O-Tig OH medium Tig-Acy-1 OH OH OH OH O-Tig O-Acy powerful Tig-Acy-2 O-Acy OH OH OH O-Tig O-Acy medium Tig-Acy-3 OH O-Acy OH OH O-Tig O-Acy medium Tig-Acy-4 OH OH O-Acy OH O-Tig O-Acy medium Tig-Acy-5 O-Acy OH OH OH O-Tig OH medium Tig-Acy-6 OH O-Acy OH OH O-Tig OH medium

[0173] After the esterification reaction of the parent compound E4A-Tig-N and pentenoyl chloride, the following compounds were obtained by HPLC separation:

[0174]

[0175]

[0176] After the esterification reaction between the parent compound E4A-Tig-N and hexenoyl chloride, the following compounds were obtained by HPLC separation:

[0177] R1 R2 R5 R8 R17 R18 Cytotoxicity E4A-Tig-N OH OH OH OH O-Tig OH medium Tig-Hex-1 OH OH OH OH O-Tig O-Hex powerful Tig-Hex-2 O-Hex OH OH OH O-Tig O-Hex medium Tig-Hex-3 OH O-Hex OH OH O-Tig O-Hex medium Tig-Hex-4 OH OH O-Hex OH O-Tig O-Hex medium Tig-Hex-5 O-Hex OH OH OH O-Tig OH medium Tig-Hex-6 OH O-Hex OH OH O-Tig OH medium

[0178] After the esterification reaction of the parent compound E4A-Tig-N and cinnamoyl chloride, the following compounds were obtained by HPLC separation:

[0179] R1 R2 R5 R8 R17 R18 Cytotoxicity E4A-Tig-N OH OH OH OH O-Tig OH medium Tig-Cin-1 OH OH OH OH O-Tig O-Cin powerful Tig-Cin-2 O-Cin OH OH OH O-Tig O-Cin medium Tig-Cin-3 OH O-Cin OH OH O-Tig O-Cin medium Tig-Cin-4 OH OH O-Cin OH O-Tig O-Cin medium Tig-Cin-5 O-Cin OH OH OH O-Tig OH medium Tig-Cin-6 OH O-Cin OH OH O-Tig OH medium

[0180] After the esterification reaction of the parent compound E4A-Tig-N and angelica acyl chloride, the following compounds were obtained by HPLC separation:

[0181] R1 R2 R5 R8 R17 R18 Cytotoxicity E4A-Tig-N OH OH OH OH O-Tig OH medium Tig-Ang-1 OH OH OH OH O-Tig O-Ang Strong Tig-Ang-2 O-Ang OH OH OH O-Tig O-Ang Medium Tig-Ang-3 OH O-Ang OH OH O-Tig O-Ang Medium Tig-Ang-4 OH OH O-Ang OH O-Tig O-Ang Medium Tig-Ang-5 O-Ang OH OH OH O-Tig OH Medium Tig-Ang-6 OH O-Ang OH OH O-Tig OH Medium

[0182] After the esterification reaction of the parent compound E4A-Tig-N and ethylbutyryl chloride, the following compounds were obtained by HPLC separation:

[0183] R1 R2 R5 R8 R17 R18 Cytotoxicity E4A-Tig-N OH OH OH OH O-Tig OH Medium Tig-Eth-1 OH OH OH OH O-Tig O-Eth Strong Tig-Eth-2 O-Eth OH OH OH O-Tig O-Eth Medium Tig-Eth-3 OH O-Eth OH OH O-Tig O-Eth Medium Tig-Eth-4 OH OH O-Eth OH O-Tig O-Eth Medium Tig-Eth-5 O-Eth OH OH OH O-Tig OH Medium Tig-Eth-6 OH O-Eth OH OH O-Tig OH Medium

[0184] The compounds produced by the esterification reaction of compounds (A), (B), (C), (D), (E), (F), (G) and (H) with acyl chlorides including citric acid chloride, angelic acid chloride, acetyl chloride, crotonyl chloride, 3,3-dimethylacryloyl chloride, senecioyl chloride, cinnamoyl chloride, pentenoyl chloride, hexenoyl chloride, benzoyl chloride or ethylbutanoyl chloride (not limited to these functional groups) have a composition that changes when the reaction temperature or time changes. Components and compounds are selected based on the changes in time and peaks. Compounds with activities from weak to strong are selected and separated. The anticancer activity of the compounds was determined by MTT studies on the following cancer cells: bone (U2OS), lung (H460), bladder (HTB-9), ovary (ES2), colon (HCT116), pancreas (Capan), ovary (OVCAR3), prostate (DU145), skin (SK-Mel-5), oral cavity (KB), kidney (A498), breast (MCF-7), liver (HepG2), brain (T98G), melanoma (K562), cervix (HeLa). The active esterification products were purified by HPLC. The MTT cytotoxicity test method of each compound is detailed in Experiment 3 of this patent. The second esterification of the compound can produce new active compounds. Some of the esterification products can be selected and esterified for the third time with different acyl chloride compounds to produce new active compounds.

[0185] One of the esterification methods: 1) dissolving a core compound, such as a triterpene core, a hydroxytriterpene core, in pyridine. 2) adding an acyl chloride compound. 3) stirring at different temperatures for 5 sec, 1 min, 2 min, 5 min, 10 min, 30 min, 1 hr, 2 hr, 18 hr, 2 days or 3 days. 4) after the reaction, adding an acidic or alkaline aqueous solution or water. 5) extracting with ether and freeze drying. 6) dissolving the reaction product in acetonitrile with trifluoroacetic acid or DMSO. 7) determining the cytotoxicity of the reaction product and each component by MTT method. 8) selecting a certain component according to the cytotoxicity of the reaction product obtained at a specific time, separating and extracting it by HPLC. 10) purifying the separated active component by HPLC. 11) collecting the product. 12) detecting the products. The core compound mentioned here is terpene, isoprene, triterpene or hydroxytriterpene. Here, the core compound is dissolved in pyridine. The acid chloride compounds mentioned here include cis-acid chloride, angelica acid chloride, acetyl chloride, crotonyl chloride, 3,3-dimethylacryloyl chloride, senecioyl chloride, cinnamoyl chloride, pentenoyl chloride, hexenoyl chloride, benzoyl chloride or ethylbutyryl chloride. Here, the stirring is 5sec, 1min, 2min, 5min, 10min, 30min, 1hr, 2hr, 18hr, 2 days or 3 days. Here, the reaction temperature is 0℃, 25℃, 50 or 75℃. Here, the acid is HCl and the base is NaHCO3. Here, the extraction with ether and freeze drying are three times. Here, the reaction product is dissolved in 80% acetonitrile with 0.005% trifluoroacetic acid or DMSO. Here, the different reaction times are 5sec, 1min, 2min, 5min, 10min, 30min, 1hr, 2 hr, 18hr, 2 days or 3 days, and the reaction temperature is 0℃, or 75℃.

[0186] The anticancer activity of compound Tig-R, i.e. IC50, is as follows: bone (U2OS) 4.5ug / ml, lung (H460) 4.8ug / ml, bladder (HTB-9) 2.5ug / ml, ovary (ES2) 2.8ug / ml, colon (HCT116) 5.2ug / ml, pancreas (Capan) 2.4ug / ml, ovary (OVCAR3) 5.8ug / ml, prostate (DU145) 3.6ug / ml, skin (SK-Mel-5) 5.1ug / ml, oral cavity (KB) 3ug / ml, kidney (A498) 3.5ug / ml, breast (MCF-7) 4.5ug / ml, liver (HepG2) 6ug / ml, brain (T98G) 8ug / ml, melanoma (K562) 2ug / ml, cervix (HeLa) 5ug / ml.

[0187] The anticancer activity of compound Tig-V, i.e. IC50, is as follows: bone (U2OS) 7ug / ml, lung (H460) 6.8ug / ml, bladder (HTB-9) 4ug / ml, ovary (ES2) 2.0ug / ml, colon (HCT116) 8 ug / ml, pancreas (Capan) 5ug / ml, ovary (OVCAR3) 9ug / ml, prostate (DU145) 4 ug / ml, skin (SK-Mel-5) 5.1ug / ml, oral cavity (KB) 3ug / ml, kidney (A498) 3.5ug / ml, breast (MCF-7) 4.56ug / ml, liver (HepG2) 12ug / ml, brain (T98G) 14ug / ml, melanoma (K562) 4ug / ml, cervix (HeLa) 7ug / ml.

[0188] The anticancer activity of compound Tig-N, i.e. IC50, is as follows: bone (U2OS) 15ug / ml, lung (H460) 13ug / ml, bladder (HTB-9) ug / ml, ovary (ES2) 9ug / ml, colon (HCT116) 15ug / ml, pancreas (Capan) 8ug / ml, ovary (OVCAR3) 18ug / ml, prostate (DU145) 4.8ug / ml, skin (SK-Mel-5) 15ug / ml, oral cavity (KB) 9ug / ml, kidney (A498) 11ug / ml, breast (MCF-7) 13ug / ml, liver (HepG2) 18ug / ml, brain (T98G) 19ug / ml), melanoma (K562) 6ug / ml, cervix (HeLa) 15ug / ml.

[0189] The anticancer activity of compound Tig-Q, i.e. IC50, is as follows: bone (U2OS) 20ug / ml, lung (H460) 18ug / ml, bladder (HTB-9) 10ug / ml, ovary (ES2) 12ug / ml, colon (HCT116) 22ug / ml, pancreas (Capan) 9ug / ml, ovary (OVCAR3) 23ug / ml, prostate (DU145) 15ug / ml, skin (SK-Mel-5) 20ug / ml, oral cavity (KB) 12ug / ml, kidney (A498) 13ug / ml, breast (MCF-7) 18ug / ml, liver (HepG2) 24ug / ml, brain (T98G) 29ug / ml), melanoma (K562) 6ug / ml, cervix (HeLa) 20ug / ml.

[0190] The anticancer activity of compound Tig-T, i.e. IC50, is as follows: bone (U2OS) 20ug / ml, lung (H460) 21ug / ml, bladder (HTB-9) 12ug / ml, ovary (ES2) 14ug / ml, colon (HCT116) 23ug / ml, pancreas (Capan) 11ug / ml, ovary (OVCAR3) 25ug / ml, prostate (DU145) 16ug / ml, skin (SK-Mel-5) 22ug / ml, oral cavity (KB) 13ug / ml, kidney (A498) 15ug / ml, breast (MCF-7) 20ug / ml, liver (HepG2) 26ug / ml, brain (T98G) 26ug / ml), melanoma (K562) 9ug / ml, cervix (HeLa) 18ug / ml.

[0191] The anticancer activity of compound Tig-S, i.e. IC50, is as follows: bone (U2OS) 5.2ug / ml, lung (H460) 5.6ug / ml, bladder (HTB-9) 3.5ug / ml, ovary (ES2) 4.2ug / ml, colon (HCT116) 6.6ug / ml, pancreas (Capan) 2.9ug / ml, ovary (OVCAR3) 6.5ug / ml, prostate (DU145) 4.3ug / ml, skin (SK-Mel-5) 5.8ug / ml, oral cavity (KB) 4ug / ml, kidney (A498) 4.8ug / ml, breast (MCF-7) 6.3ug / ml, liver (HepG2) 8.5ug / ml, brain (T98G) 9ug / ml), melanoma (K562) 4.3ug / ml, cervix (HeLa) 7ug / ml.

[0192] The anticancer activity of compound Tig-U, i.e. IC50, is as follows: bone (U2OS) 23ug / ml, lung (H460) 19ug / ml, bladder (HTB-9) 15ug / ml, ovary (ES2) 17ug / ml, colon (HCT116) 26ug / ml, pancreas (Capan) 9ug / ml, ovary (OVCAR3) 27ug / ml, prostate (DU145) 15ug / ml, skin (SK-Mel-5) 15ug / ml, oral cavity (KB) 16ug / ml, kidney (A498) 18ug / ml, breast (MCF-7) 25ug / ml, liver (HepG2) 23ug / ml, brain (T98G) 22ug / ml), melanoma (K562) 10ug / ml, cervix (HeLa) 17ug / ml.

[0193] The patent provides a compound or method that can inhibit cancer invasion, cell invasion and cancer cell invasion and metastasis, and adjust cell adhesion and attachment, characterized in that the composition has a molecular formula (2A):

[0194]

[0195] R1, R2, R3, R4, R5, R8, R9, R10, R11, R12, R13, R14, and R15 are hydrogen, hydroxy, methyl, O-angeloyl, O-cis-noyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutanoyl, O-alkanoyl, alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl, CHO-angeloyl, CHO-cis-chiorhoyl, CHO-senecioyl, CHO-acetyl, CHO-crotonyl, CHO-3,3-dimethylacryloyl, CHO-cinnamoyl, CHO-pentenoyl, CHO-hexenoyl, CHO-benzoyl, CHO-ethylbutyryl, methyl, hydroxymethyl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl-substituted alkanoyl, O-alkanoyl-substituted phenyl, O-alkenoyl-substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl, alkane, alkene and sugar chain and its derivatives. The at least two functional groups described herein are: O-angeloyl, O-cisinoyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl-substituted alkanoyl, O-alkanoyl-substituted phenyl, O-alkenoyl-substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl; or R1 herein and R2 is O-angeloyl, O-cis-oyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl; in one embodiment, R1 and R2 are linked to OH;In one embodiment, R4 and R10 are connected to CHO-angelic acid, CHO-cisinoyl, CHO-senecioyl, CHO-acetyl, CHO-crotonoyl, CHO-3,3-dimethylacryloyl, CHO-cinnamoyl, CHO-pentenoyl, CHO-hexenoyl, CHO-benzoyl, CHO-ethylbutanoyl; In one embodiment, R3 and R8 are hydrogen or hydroxyl; In one embodiment, R9, R11, R12, R13, R14, R15 are connected to a methyl group respectively; In one embodiment, R4 represents CH3, CHO, CH2R6 or COR6, where R6 is one of the following functional groups: hydroxyl, O-angelic acid, O-cisinoyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compounds, heterocyclic aromatic compounds, O-alkenecarbonyl, and derivatives thereof; in one embodiment, R3 is H or OH. In one embodiment, R8 is H or OH; In one embodiment, R16 is H, CH3, OH or R4, R16 may be CH2-X-, CH(OH)-X- or C(=O)-X-, where –X- can be O, NH or S, and R16 is absent if carbon C12-13 on the triterpene ring is a double bond; In one embodiment, R10 represents CH3, CHO or CH2R6, where R6 is one of the following functional groups: hydroxyl, O-angeloyl, O-cisanoyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, O-heterocyclic aromatic compound, O-alkenecarbonyl, and derivatives thereof. In one embodiment, R5 is hydrogen, hydroxyl, heterocyclic or O-sugar chain, wherein the sugar chain is composed of the following sugars: glucose, galactose, rhamnose, arabinose, xylose, fucose, allose, altrose, gulose, idose, lyxose, mannose, ribose, sorbose, tagatose, talose, fructose, or uronic acid, glucuronic acid, galacturonic acid, or derivatives thereof;Here, R9, R10, R11, R12, R13, R14, R15 are CH3, CH2OH, CHO, COOH, COO-alkane, COO-aromatic, COO-heterocyclic compound, COO-heterocyclic aromatic compound, CH2O-aromatic, CH2O-heterocyclic compound, CH2O-heterocyclic aromatic compound, alkyl, hydroxyl, acetyl. Here, R4 and R16 form a divalent group: CH2O, CH(OR7)O, or COOR7, where R7 is hydrogen, alkyl, angelic acid, cis-chiayl, senecioyl, dibenzoyl, benzoyl, alkanoyl, alkenoyl, benzylalkyl substituted alkanoyl, aromatic, acyl, heterocyclic compound, heterocyclic aromatic compound, and derivatives thereof, where, At least two of R1, R2 and R6 are connected to the following functional groups: O-angeloyl, O-cisinoyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl, and derivatives thereof. Or at least one of R1, R2 and R4 is a sugar chain, and the sugar chain is connected with at least two of the following functional groups: angelica acyl, acetyl, cis-acyl, senecioyl, crotonoyl, 3,3-dimethylacryloyl, cinnamoyl, pentenoyl, hexenoyl, benzoyl, ethylbutyryl, dibenzoyl, alkanoyl, alkenoyl, benzylalkyl-substituted alkanoyl, aromatic, acyl, heterocyclic compound, heterocyclic aromatic compound, and derivatives thereof;Alternatively, R4 is CH2R6, wherein R6 is a functional group selected from the group consisting of hydroxy, O-angeloyl, O-cis-noyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl-substituted alkanoyl, O-alkanoyl-substituted phenyl, O-alkenoyl-substituted phenyl, O-aryl, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl, and derivatives thereof. Wherein R5 is a sugar chain, which is composed of the following sugars and uronic acid: glucose, galactose, rhamnose, arabinose, xylose, fucose, allose, altrose, gulose, idose, lyxose, mannose, ribose, sorbose, tagatose, talose, fructose, or uronic acid, glucuronic acid, galacturonic acid, or a derivative thereof; in one embodiment, wherein R5 is hydroxy, O-angeloyl, O-cis-oyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl-substituted alkanoyl, O-alkanoyl-substituted phenyl, O-alkenoyl-substituted phenyl, O-aryl, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl, and derivatives thereof. In one embodiment, R1, R2, R3, R4, R5, R8, R9, R10, R11, R12, R13, R14 or R15 consists of one or more sugar chains; In one embodiment, R1, R2, R3, R4, R5, R8, R9, R10, R11, R12, R13, R14 or R15 consists of one or more acids; In one embodiment, at least one of 1, 2, 3 or 4 of R1, R2, R3, R4, R5, R8, R9, R10, R11, R12, R13, R14 and R15 is a hydroxyl group;In one embodiment, at least one of 2, 3, 4, 5, 6, or 7 of R1, R2, R3, R4, R5, R8, R9, R10, R11, R12, R13, R14, and R15 is connected to one of the following functional groups: O-acetyl, O-angeloyl, O-cis-noyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bis-benzoyl, O- Benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl, and derivatives thereof; wherein these functional groups are directly connected to the triterpene or the sugar chain; wherein at least one of 1, 2, 3, 4, 5, 6, or 7 of R1, R2, R3, R4, R5, R8, and R10 is connected to one of the following functional groups: O-angeloyl, O-cis-noyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenoyl, CHO-angeloyl, CHO-cisenoyl, CHO-senecioyl, CHO-acetyl, CHO-crotonoyl, CHO-3,3-dimethylacryloyl, CHO-cinnamoyl, CHO-pentenoyl, CHO-hexenoyl, CHO-benzoyl, CH2O-ethylbutyryl, CH3, CH2OH, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl, and derivatives thereof, wherein these functional groups are directly attached to the triterpene or to the sugar chain;In one embodiment, the cancer includes breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain cancer, leukemia, lung cancer, colon cancer, central nervous system cancer (CNS), melanoma, renal duct cancer, cervical cancer, esophageal cancer, testicular cancer, spleen cancer, kidney cancer, lymphoma, pancreatic cancer, stomach cancer and thyroid cancer, etc. The cells described here are breast cells, leukocytes, liver cells, ovarian cells, bladder cells, prostate cells, skin cells, bone cells, brain cells, leukemia cells, lung cells, colon cells, central nervous system cells (CNS), melanoma cells, renal duct cells, uterine cells, esophageal cells, testicular cells, spleen cells, kidney cells, lymphocytes, pancreatic cells, stomach cells and thyroid cells, etc.;

[0196] In one embodiment, the compound has the following structure:

[0197]

[0198] Among them, R1, R2, R3, R4, R5, R8, R9, R10, R11, R12, R13, R14, and R15 are CH3, CH2OH, hydrogen, hydroxyl, methyl, O-angelicyl, O-cis-yl, O-senecioyl, O-acetyl, O-crotonyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O- Hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl, CHO-angeloyl, CHO-cis-chiorinoyl, CHO-senecioyl, CHO-acetyl, CHO-crotonoyl, CHO-3,3-dimethylacryloyl, CHO-cinnamoyl, CHO-pentenoyl, CHO-hexenoyl, CHO-benzoyl, CHO-ethylbutyryl, alkane, alkene and sugar chain, and derivatives thereof. wherein any one of 1, 2, 3 or 4 of R1, R2, R3, R4, R5, R8 and R10 is connected to one of the following functional groups: O-angeloyl, O-cisinoyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutanoyl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl-substituted alkanoyl, O-alkanoyl-substituted phenyl, O-alkenoyl-substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl, CH2O-angeloyl, CHO-cis-azoyl, CHO-senecioyl, CHO-acetyl, CHO-crotonoyl, CHO-3,3-dimethylacryloyl, CHO-cinnamoyl, CHO-pentenoyl, CHO-hexenoyl, CHO-benzoyl, CHO-ethylbutanoyl, CH3, CH2OH, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl-substituted alkanoyl, O-alkanoyl-substituted phenyl, O-alkenoyl-substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, O-heterocyclic aromatic compound, O-alkenecarbonyl, and derivatives thereof. R9, R11, R12, R13, R14, R15 are each connected to a CH3.or wherein R10 is connected to O-angeloyl, O-cisinoyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl-substituted alkanoyl, O-alkanoyl-substituted phenyl, O-alkenoyl-substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl, CHO-angeloyl, CHO-cisinoyl, CHO-senecioyl, CHO-acetyl, CHO-crotonoyl, CH2O-3,3-dimethylacryloyl, CH2O-cinnamoyl, CH2O-pentenoyl, CH2O-hexenoyl, CH2O-benzoyl, CH2O-ethylbutyryl, CH3,CH2OH, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compounds, O-heterocyclic aromatic compounds, O-alkenecarbonyl, and derivatives thereof, CH2O-alkyl, CHO-bisbenzoyl, CHO-benzoyl, CHO-alkanoyl, CHO-alkenoyl, CHO-benzylalkyl-substituted alkanoyl, CHO-alkanoyl-substituted phenyl, CHO-alkenoyl-substituted phenyl, CHO-aromatic, CHO-acyl, CHO-heterocyclic compound, CHO-heterocyclic aromatic compound, CHO-alkenecarbonyl.or wherein R4 and R10 are linked to O-angeloyl, O-cis-noyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl-substituted alkanoyl, O-alkanoyl-substituted phenyl, O-alkenoyl-substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, O-heterocyclic aromatic compound, O-alkenecarbonyl, CH2O- Angelica acyl, CHO-cisanoyl, CHO-senecioyl, CHO-acetyl, CHO-crotonyl, CHO-3,3-dimethylacryloyl, CHO-cinnamoyl, CHO-pentenoyl, CHO-hexenoyl, CHO-benzoyl, CHO-ethylbutanoyl, CH3, CH2OH, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl-substituted alkanoyl, O-alkanoyl-substituted phenyl, O-alkenoyl-substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, O-heterocyclic aromatic compound, O-alkenecarbonyl. Wherein, R3 is OH or H, or absent. Wherein, R1, R2, R3, R5, R8 are OH or H, or absent. wherein R9, R11, R12, R13, R14 and R15 are CH3. or wherein R1, R2, R5, R8 are OH, R3 is OH or H, or absent. R4, R10 are CHO-angelicyl, R9, R11, R12, R13, R14, R15 are CH3. or wherein R1, R2, R5, R8 are OH or O-angelicyl, R3 is OH or H, or absent. R4, R10 are CHO-angelicyl, R9, R11, R12, R13, R14, R15 are CH3. Among them, the functional group connected to the parent core compound is one of the following functional groups: acetyl, angelica acyl, cis-acyl, senecioyl, alkyl, crotonyl, dimethyl acryloyl, cinnamoyl, pentenoyl, hexenoyl, benzoyl, ethyl butyryl, alkyl, dibenzoyl, benzoyl, alkanoyl, alkenoyl, alkanoyl substituted with benzyl alkyl, alkanoyl substituted with phenyl, alkenoyl substituted with phenyl, aromatic, acyl, heterocyclic compound, heterocyclic aromatic compound, alkene carbonyl. These functional groups can be the same or different in combination. Substitution, removal, or addition of any functional group on the above compounds is one of the means of synthesizing new active compounds of this patent. In a further embodiment, substitution, removal, or addition of any functional group on the compound provided by this patent will not significantly affect the activity of the compound.

[0199] In one embodiment, the compound has the following structure:

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209] This patent provides a composition, which contains a certain amount of the above-mentioned compound, which has the above-mentioned structure, or its salts, esters, metabolites, or derivatives. The composition can be used to prepare anticancer drugs that inhibit cancer invasion, cell invasion and cancer cell invasion and metastasis. The cancer described here is breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain cancer, leukemia, lung cancer, colon cancer, central nervous system cancer (CNS), melanoma, renal duct cancer, cervical cancer, esophageal cancer, testicular cancer, spleen cancer, kidney cancer, lymphoma, pancreatic cancer, gastric cancer and thyroid cancer, etc. The cells described here are breast cells, leukocytes, liver cells, ovarian cells, bladder cells, prostate cells, skin cells, bone cells, brain cells, leukemia cells, lung cells, colon cells, central nervous system cells (CNS), melanoma cells, renal duct cells, cervical cells, esophageal cells, testicular cells, spleen cells, kidney cells, lymphocytes, pancreatic cells, gastric cells and thyroid cells, etc.

[0210] The present invention provides a composition, which contains the compound provided by the present invention. The composition can treat cancer, inhibit viruses, prevent brain aging, improve memory and brain function, treat enuresis, frequent urination and urgency, dementia, Parkinson's disease, or other diseases caused by brain dysfunction, arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's disease, angina pectoris, heart dysfunction, coronary heart disease, headache, dizziness and kidney dysfunction, cardiovascular disease, inhibit NF-Kappa B activation, cerebral edema, respiratory distress syndrome, respiratory viral diseases; chronic venous insufficiency; hypotension; chronic venous disease; anti-edema; anti-inflammatory; hemorrhoids, peripheral edema; varicose veins; influenza; post-traumatic edema; postoperative swelling; inhibit thrombosis, inhibit ethanol absorption; lower blood sugar; adjust the levels of corticotropin and corticosterone. The compound provided by the present invention can be AntiMS, anti-aneurysm, anti-asthma, vasodilation, anti-capillary bleeding, anti-headache, anti-cervical and brachial pain, eclampsia, Edema, encephalitis, epiglottitis, exudate, influenza, fracture, gingivitis, hematoma, herpes, histamine shock, joint effusion, meningitis, antioxidant effect, periodontitis, phlebitis, pleurisy, hoarseness, rhinitis, tonsillitis, ulcer, varicose veins, vertigo, cancer metastasis, corticosterone production, diuretic, antifungal, hemolytic effect, hyaluronidase inhibitor, lymphatic agent, natriuretic effect, insecticide, mucolytic, thymolytic, vascular and venous protection. Inhibit leishmaniasis, inhibit cancer cell adhesion or angiopoietin, antiparasitic; enhance the expression of the following genes: ANGPT2, DDIT3, LIF and NFKB1Z; manufacture auxiliary compositions and for phlebotomy treatment.

[0211] Alkenyl refers to unsaturated straight or branched chain structures and combinations thereof having the formula R2C=CR2, one or more doublets. Examples of alkenyl include ethenyl, propenyl, isopropenyl, butenyl, S- and T-butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, and hexadienyl.

[0212] An aromatic group is a functional group derived from an aromatic compound such as benzene. An aromatic ring system consisting of 6-14 carbon atoms containing 1-3 benzene rings. If two or more aromatic rings are together, adjacent rings share a common bond and are connected together. Examples of such compounds are phenol and naphthol. Aromatic groups can be replaced by one or more other groups such as halogens, alkyl groups or alkoxy groups.

[0213] An acyl group is a functional group that is derived from an organic acid by removing the carboxyl group. Acyl compounds can be written as -COR, where there is a double bond between the carbon and oxygen atoms. The names of acyl compounds end with -yl, such as formyl, acetyl, propionyl, butyryl, and benzoyl. Benzoyl, C6H5COR, is derived from benzoic acid by removing the carboxyl group.

[0214] Heterocyclic compounds are compounds containing heterocycles. The so-called heterocycle refers to a non-aromatic ring, that is, a single ring containing 1-4 heteroatoms or connected to a second ring. The second ring is a heteroaliphatic ring of 3-7 carbon atoms and contains 0-4 heteroatoms, aromatic groups, or heteroaromatic groups. The heteroaliphatic groups referred to here include pyrrolidine acyl, pyrazinyl, morpholinyl, tetrahydrofuranyl, imidazolyl and thiomorpholinyl.

[0215] Heteroaliphatic acyl groups are obtained by removing hydrogen from heterocyclic aromatic hydrocarbons.

[0216] Alkanoyl is a general term for organic compounds containing RCO-functional groups, wherein R is hydrogen or alkyl. Preferred alkanoyl groups include acetyl, propionyl, butyryl, isobutyryl, valeryl and hexanoyl.

[0217] Alkenyl is an alkene hydroxyl group (alkene carbonyl), such as pentenyl carbonyl (cis-ene carbonyl) and hexenyl carbonyl (angeloyl).

[0218] Alkyl is a group containing only hydrogen and carbon atoms, in a chain, branched, cyclic, bicyclic, or a combination thereof, containing 1-18 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

[0219] Benzoylalkyl substituted chain alkyl refers to a group in which a straight chain or branched chain alkyl is substituted by at least one benzoyl group and an alkyl group, wherein the benzyl group is connected to the straight chain or branched chain alkyl group. Benzoylmethyl isobutyryl is an example of benzoylalkyl substituted chain alkyl in this patent.

[0220] A sugar chain is a molecular fragment of a saponin compound that contains multiple sugars or uronic acids.

[0221] Isobutyryl is a synonym for 2-methylpropionyl.

[0222] Y and Y3 are the same compound.

[0223] YM and (ACH-Y) are the same compound.

[0224] The linked sugar chain is a substructure or group that connects the active group to the parent nucleus compound, such as angelica acyl group connected to the triterpene nucleus through a sugar chain.

[0225] The chemical building blocks used to construct the active compounds in this patent include triterpenoids, hydroxytriterpenoids, acetyl, angeloyl, cis-enoyl, senecioyl, alkyl, crotonoyl, O-3,3-dimethylacryloyl, cinnamoyl, pentenoyl, hexenoyl, benzoyl, ethylbutyryl, alkyl, dibenzoyl, benzoyl, alkanoyl, alkenoyl, benzylalkyl substituted alkanoyl, alkanoyl substituted phenyl, alkenoyl substituted phenyl, aromatic, acyl, heterocyclic compounds, heterocyclic aromatic compounds, alkenecarbonyl, acetyl chloride, cis-enoyl chloride, angeloyl chloride, crotonoyl chloride, senecioyl chloride, 3,3-dimethylacryloyl chloride, cinnamoyl chloride, pentenoyl chloride, hexenoyl chloride, benzoyl chloride or ethylbutyryl chloride.

[0226] Acetyl chloride Angelicacid chloride Cis-1-chloro-1-yl chloride Senecioyl chloride Crotonyl chloride 3,3-Dimethylacryloyl chloride Cinnamyl chloride Pentenoyl chloride Hexenoyl chloride Benzoyl chloride Ethyl butyryl chloride

[0227] In the experiments provided in this patent, the concentration of the drug that can inhibit cell growth by 15% or less compared to the control without drug (DMSO) (85% or higher) is considered to be a non-cytotoxic concentration. In another embodiment of the present invention, the concentration of the drug that can inhibit cell growth by 10% or less compared to the control without drug (90% or higher) is considered to be a non-cytotoxic concentration. In another embodiment of the present invention, the concentration of the drug that can inhibit cell growth by 5% or less compared to the control without drug (95% or higher) is considered to be a non-cytotoxic concentration. In another embodiment of the present invention, the concentration of the drug that can inhibit cell growth by 20% or less compared to the control without drug (80% or higher) is considered to be a non-cytotoxic concentration. In another embodiment of the present invention, the concentration of the drug that can inhibit cell growth by 25% or less compared to the control without drug (75% or higher) is considered to be a non-cytotoxic concentration. In another embodiment of the present invention, a concentration of the drug that inhibits cell growth by 45% or less compared to a control without the drug (DMSO) is considered to be non-cytotoxic. (DMSO)

[0228] Some of the triterpenoid compounds provided by this patent can be used for medical purposes, characterized in that the medical purposes include inhibiting cancer, or inhibiting the occurrence of cancer or killing cancer or tumor cells as an auxiliary treatment, and preventing cancer cell invasion. In another embodiment of the present invention, these compounds can inhibit the activation of nuclear factor-kB. The inhibition here refers to inhibiting localization, or inhibiting binding to DNA. In another embodiment of the present invention, these compounds can induce apoptosis of cancer cells.

[0229] Table 1-12 Effects of Compounds Y and YM on Gene Expression (For details, see Tables 1-12 in the patent application PCT / US2008 / 002086,1188-ALA-PCT filed on February 15, 2008). Table 13-19 Effects of Compounds Y and YM on Gene Expression Effects of Glypican on Expression in Cancer Cell ES2 (For details, see Tables 13-19 in the patent application PCT / US2008 / 002086,1188-D-PCT filed on February 13, 2009).

[0230] Gene expression was measured by real-time PCR (Brilliant QPCR, Agilent Technologies):

[0231] The results obtained by microarray analysis were further confirmed by real-time PCR. Real-time PCR results (see below) confirmed that compounds Y3 and YM can enhance the expression of the following genes: ANGPT2, DDIT3, LIF and NFKB1Z (see Tables 19-21 in patent application PCT / US09 / 34115 filed on February 13, 2009).

[0232] The products of the partial hydrolysis of the saponin compounds can be separated by HPLC. The specific partial hydrolysis of the saponin compounds can be achieved by enzymes. Glucosidase can catalyze the hydrolysis of glucosidic bonds. Beta-galactosidase can catalyze the hydrolysis of galactosides. Beta-glucosidase can disconnect glucose from saponin. Other hydrolases include xylitase, lactase, amylase, chitinase, fructase, maltase and neuraminidase.

[0233] The sugar chains of triterpenoid saponins (such as Xanifolia Y) can be removed by acid hydrolysis. The synthetic compound ACH-Y is obtained in this way. The compound ACH-Y is a triterpene attached to an acyl group, but without a sugar chain. The acyl group of triterpene saponins (such as Xanifolia Y) can also be removed by alkaline hydrolysis. The synthetic compound AKOH-Y is obtained in this way. The compound AKOH-Y is a pentacyclic triterpene attached to a sugar chain. Pentacyclic triterpenes can be obtained by acid and alkaline hydrolysis of natural saponins. Pentacyclic triterpenes can also be obtained by synthetic methods. (Reference: Surendra et al., Rapid and Enantioselective Synthetic Approches to Germanicol and Other Pentacyclic Triterpenes, Journal of the American Chemical Society, 2008, 130 (27), 8865-8869). Pentacyclic triterpenes with sugar chains can be obtained by synthetic methods (Reference: Ple et al., Synthesis of L-arabinopyranose containing hederagenin saponins, Tetrahedron 61 (2005) 4347-4362). Acetylation is the process of adding an acetyl group to a compound. The so-called Friedel-Crafts reaction is an example of acetylation. The active compound can be obtained by acetylation of a triterpene. In one embodiment of the present invention, it is obtained by acetylation of a triterpene or a hydroxy triterpene compound. In one embodiment, acylation of C24, C28, C21 and C22 of pentacyclic triterpenoids, or hydroxylation of triterpenoids produces compounds that inhibit cancer cell growth, cancer cell invasion, cell invasion, tumor cell invasion, cell adhesion, or cell cycle. In one embodiment, the acyl group may be attached at the C3 carbon position. In one embodiment, the sugar moiety is C21, 22, 24 or 28, wherein the sugar moiety is attached with two acyl groups. In another embodiment of the present invention, compounds (A), (B), (C), (D), (F), (G), (H) are acetylated to obtain compounds that inhibit cancer invasion, cell invasion, and cancer cell invasion. This patent provides chemical building blocks for constructing active saponins.

[0234] The acetylated compound (G) has angeloyl and crotonyl groups to produce the following compounds:

[0235]

[0236] Wherein R1, R2, R5, R8 represent OH or O-angeloyl; R3 represents OH, H or O-angeloyl; R4, R10 represent CH3, CH2OH or CH2O angeloyl; R9, R11, R12, R13, R14, R15 represent CH3; or R1, R2, R5, R8 represent OH orO-angeloyl; R3 represents OH, H or O-angeloyl; R4, R10 represent CH3, CH2OH or CH2O angeloyl; R9, R11, R12, R13, R14, R15 represent CH3; the compounds here can inhibit cancer invasion, cell nuclear cancer cell invasion.

[0237] Compound (G) is acylated with the following functional groups to produce compound (K), wherein the functional groups are acetyl, angelica acyl, cis-acyl, senecioyl, alkyl, crotonoyl, O-3,3-dimethylacryloyl, cinnamoyl, pentenoyl, hexenoyl, benzoyl, ethylbutyryl, alkyl, dibenzoyl, benzoyl, alkanoyl, alkenoyl, alkanoyl substituted with benzylalkyl, alkanoyl substituted with phenyl, alkenoyl substituted with phenyl, aromatic, acyl, heterocyclic compound, heterocyclic aromatic compound, CHO-alkenecarbonyl, alkane, alkene. Wherein, R1, R2, R5, R8 represent, respectively, O-angeloyl, O-cisinoyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl-substituted alkanoyl, O-alkanoyl-substituted phenyl, O-alkenoyl-substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, O-heterocyclic aromatic compound, O-alkenecarbonyl; R4, R10 represent, respectively, CH3, CH2OH, CHO-angeloyl, CHO-cisinoyl, CHO-senecioyl, CHO-acetyl, CHO-crotonoyl, CHO-3,3-dimethylacryloyl, CHO-cinnamoyl, CHO-pentenoyl, CHO-hexenoyl, CHO-benzoyl, CHO-ethylbutyryl, CHO-alkyl, CHO-bisbenzoyl, CHO-benzoyl, CHO-alkanoyl, CHO-alkenoyl, CHO-benzylalkyl-substituted alkanoyl, CHO-alkanoyl-substituted phenyl, CHO-alkenoyl-substituted phenyl, CHO-aromatic, CHO-acyl, CHO-heterocyclic compound, CHO-heterocyclic aromatic compound, CHO-alkenecarbonyl, alkane, alkene. R3 is absent or represents OH, H, O-angeloyl, O-cisinoyl, O-senecioyl, O-acetyl, O-crotonoyl, O-3,3-dimethylacryloyl, O-cinnamoyl, O-pentenoyl, O-hexenoyl, O-benzoyl, O-ethylbutyryl, O-alkyl, O-bisbenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzylalkyl substituted alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substituted phenyl, O-aromatic, O-acyl, O-heterocyclic compound, heterocyclic aromatic compound, O-alkenecarbonyl. Wherein R9, R11, R12, R13, R14, R15 are CH3. Herein, these compounds inhibit cancer growth, invasion, cell invasion or cancer cell invasion.These compounds can be used as regulators or inhibitors of adhesion proteins or angiogenin. These compounds can be used as regulators to regulate the secretion, expression or synthesis of adhesion proteins, reduce fibronectin to inhibit cell adhesion or cell circulation. The adhesion proteins include fibronectin, integrin, myosin, vitronectin, collagen, laminin, polysaccharide, cadherin, heparin, tendon, CD. 54 , and CAM. These compounds can be used as anti-adhesion therapy or therapy targeting adhesion molecules.

[0238] The patent applicant further states that anti-adhesion therapy or therapy targeting adhesion molecules is a new medical direction. Among clinical anti-adhesion drugs, Efalizumab, Odulimomab, Alicaforsen, Aselizumab, etc. are good representative drugs, which target different adhesion proteins. Please refer to the textbook, "Adhesion Molecules: Function and Inhibition" edited by Klaus Ley, page 289-291, 297 (reference 2), Adhesion molecules in inflammatory disease (Adhesion molecules in inflammatory disease) (reference 4), abstract line 7-8: "Blocking the expression of CAM has become a new therapeutic target for the treatment of inflammatory diseases." The anti-adhesion therapy proposed in this patent is a new application of these active compounds, that is, to be used as a regulator or inhibitor of adhesion proteins or angiogenin, which can inhibit excessive adhesion and cell attachment.

[0239] In this patent application, the applicant uses the compound having structure (2A) as a regulator or inhibitor for anti-adhesion therapy to inhibit excessive adhesion and cell attachment.

[0240] Experimental details

[0241] Regarding the extraction and separation of compounds, the components of the extracted compounds were analyzed by HPLC, the activity of compound Xanifolia Y in affecting the growth of various human organ cells was tested by MTT, and then the active ingredients were separated and purified by FPLC, and compound Ys were separated by HPLC, and their chemical structures were determined. Cell and animal experiments are detailed in the following patent applications: International patent applications (PCT / US05 / 31900, PCT / US05 / 31900, US Serial No. 11 / 289142, US Serial 10 / 906303, US Serial No. 11 / 131551 and US Serial Nos. 11 / 683198, March 7, 2007, PCT / US2007 / 077273, August 30, 2007, US Serial No. 60 / 890380, 2007 16, 2007, US Nos. 60 / 947,705, 3 July 2007, PCT / US2008 / 002086,1188-ALA-PCT, 15 February 2008, App'l No. PCT / US09 / 34115, 13 February 2009, PCT / US2008 / 002086,1188-ALA-PCT, 15 February 2008. The contents of these pending patent applications are hereby fully incorporated into this patent application.

[0242] Experiment 1: Saponin acid hydrolysis to remove sugar chains

[0243] 15 mg of Xanifolia-Y was dissolved in 1 ml of methanol, 1 ml of 2N HCl was added, and the mixture was refluxed in a water bath at 80°C for 5 hours, and 2 ml of 1N NaOH was added to neutralize the mixture (the final pH value was 4-6). The mixture was extracted twice with 3 ml of ethyl acetate to obtain the saponin ligand, and the ligands obtained by mixing the two times were further separated by HPLC (80-100% acetonitrile as normal liquid elution).

[0244] Experiment 2: Alkaline hydrolysis to remove acyl groups

[0245] 20 mg of Xanifolia-Y was dissolved in 0.5 ml of 1 M NaOH and incubated in a water bath at 80°C for 4 hours. When the solution reached room temperature, 0.5 ml of 1 N HCl was added for neutralization (pH adjustment to 3). Then, the solution was extracted three times with 2 ml of 1-butanol. The butanol extract was collected and freeze-dried in vacuo to obtain the saponin, which was then separated and purified by HPLC (C-18 column, 25% acetonitrile elution).

[0246] Experiment 3: Adding acyl groups to triterpenes by esterification

[0247] Method: 40 mg of the triterpene mother ring (a part of compound IV) was dissolved in 1 ml of pyridine in a 50 ml test tube, and 0.2 ml of acyl chloride (crotonyl chloride, angelyl chloride, cisinoyl chloride, senecioyl chloride, acetyl chloride, crotonyl chloride, O-3,3-dimethylacryloyl chloride, cinnamyl chloride, pentenoyl chloride, hexenoyl chloride, phenylformyl chloride, or ethylbutyryl chloride) was added and stirred at 25°C or 75°C for 5 sec, 1 min, 2 min, 5 min, 10 min, 30 min, 1 hr, 2 hr, 18 hr, 2 days or 3 days. At the end of the reaction, 3 ml of NaHCO3 was slowly added to the mixture. Then, it was extracted with 10 ml of ethyl acetate three times, and the ethyl acetate was recovered at 45 degrees and freeze-dried. The recovered extract was dissolved in 80% acetonitrile-0.005% trifluoroacetic acid or DMSO, and the activated esterification product was separated by HPLC. Peaks, components and compounds are selected based on the activity at different reaction times. HPLC components are selected for separation based on the cytotoxicity of the reaction products at a specific time period. Compounds with strong to weak activity are selected and separated. The activated esterification products are purified by HPLC. The anticancer activity of each component and compound is studied based on MTT (see Figures 1-12 for details).

[0248] Experiment 4: Preparation of E4A

[0249] 7. Beta-Escin was dissolved in 1 M NaOH (20 mg / ml) and incubated at 75°C for 5 hours.

[0250] 8. The hydrolyzate was neutralized with HCl and freeze-dried to remove water.

[0251] 9. The obtained product was dissolved in 50% methanol and 1N HCl and incubated at 75°C for 5 hours.

[0252] 10. The resulting solution was neutralized with HCl.

[0253] 11. The hydrolyzate was extracted with acetonitrile and then freeze-dried to remove water.

[0254] 12. The hydrolyzate was further purified by (E4A) FPLC chromatography (C18 column, eluted with 70% acetonitrile, 1 ml / min).

[0255] Experiment 5: Esterification of E4A with Cis-1-Chloroyl

[0256] 10. Dissolve 50 mg of E4A in 1 ml of pyridine and gently stir in a 50 ml test tube.

[0257] 11. Add 200ul of cis-1-yl chloride and esterify at 25°C.

[0258] 12. Stir for 1 minute and immediately add 5 ml of 2N HCl.

[0259] 13. Stir for 1 hour and let stand at room temperature overnight.

[0260] 14. Extract the esterification product with 10 ml of acetonitrile.

[0261] 15. Evaporate the acetonitrile.

[0262] 16. Dissolve the sample in 1 ml DMSO.

[0263] 17. Separate the reaction products by HPLC.

[0264] 18. Collect samples.

[0265] Experiment 6: Separation of active compound E4A-Tig by HPLC

[0266] 4. Column: ZORBAX ODS 9.4x250 mm, 5um.

[0267] 5. Solvent: A: 45% AN / TFA; B: 100% AN / TFA.

[0268] 6. Chromatographic conditions: a) Elution: solvent A into B within 80 min; then solvent B for 40 min; b) Flow rate: 1 ml / min. c) Monitoring OD: at 207 nm.

[0269] Experiment 7: MTT assay

[0270] Cancer cells: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (melanoma), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain), SK-MEL-5 (skin) and OVCAR 3, ES2 (ovary), pancreas (Capan), oral cavity (KB), kidney (A498).

[0271] MTT Assay The MTT assay was performed according to Carmichael et al. (1987) with slight modifications. The cancer cells were seeded in a 96-well culture dish, incubated for 24 hours, and then treated with drugs for 48, 72, or 96 hours. Then, MTT (0.5 mg / mL) was added and incubated for 1 hour. Formazan (a product of tetrazolium reduction by living cells) was formed, dissolved in DMSO, and the OD (at 490 nm) was measured by ELISA. The MTT value of the cancer cells before drug treatment was also measured (T0). The growth of the cancer cells (%G) was calculated as follows:

[0272] %G = (TD-T0 / TC-T0) x 100 (1), where TC or TD is the OD value after control or drug treatment.

[0273] When T0>TD, the cytotoxicity (LC) of the control was calculated as follows: %LC=(TD-T0 / T0)x 100(2).

[0274] Experiment 8: Chemical synthesis, isolation and characterization of E4A-Tig-R

[0275] Chemical synthesis of E4A-Tig-R: 1. Preparation of E4A; 2. Esterification of E4A with crotonyl chloride; 3. Separation of E4A-Tig-R by HPLC.

[0276] Cytotoxicity assay: MTT method.

[0277] Chemical structure identification: 1. NMR analysis; 2. Mass spectrometry analysis.

[0278] See Figure 23 - 30

[0279] See Table 1

[0280] Compound E4A-Tig-R

[0281] 24,28-O-cis-1,3-diol-3β,16α,21β,22α,24β,28-hexahydroxyolean-12-ene

[0282] Experiment 9: Chemical synthesis, isolation and identification of E4A-Tig-N

[0283] Chemical synthesis of E4A-Tig-N: 1. Preparation of E4A; 2. Esterification of E4A with crotonyl chloride; 3. Separation of E4A-Tig-N by HPLC.

[0284] Cytotoxicity assay: MTT method.

[0285] Chemical structure identification: 1. NMR analysis; 2. Mass spectrometry analysis.

[0286]

[0287] Experiment 10: Chemical synthesis, isolation and identification of E4A-Tig-Q

[0288] Chemical synthesis of E4A-Tig-Q: 1. Preparation of E4A; 2. Esterification of E4A with crotonyl chloride; 3. Separation of E4A-Tig-Q by HPLC.

[0289] Cytotoxicity assay: MTT method.

[0290] Chemical structure identification: 1. NMR analysis; 2. Mass spectrometry analysis.

[0291]

[0292] Experiment 11: Chemical synthesis, isolation and identification of E4A-Tig-V

[0293] Chemical synthesis of E4A-Tig-V: 1. Preparation of E4A; 2. Esterification of E4A with crotonyl chloride; 3. Separation of E4A-Tig-V by HPLC.

[0294] Cytotoxicity assay: MTT method.

[0295] Chemical structure identification: 1. NMR analysis; 2. Mass spectrometry analysis.

[0296]

[0297] Experiment 12: Chemical synthesis, isolation and identification of E4A-Tig-T

[0298] Chemical synthesis of E4A-Tig-T: 1. Preparation of E4A; 2. Esterification of E4A with crotonyl chloride; 3. Separation of E4A-Tig-T by HPLC.

[0299] Cytotoxicity assay: MTT method.

[0300] Chemical structure identification: 1. NMR analysis; 2. Mass spectrometry analysis.

[0301]

[0302] Experiment 13: Chemical synthesis, isolation and identification of E4A-Tig-U

[0303] Chemical synthesis of E4A-Tig-U: 1. Preparation of E4A; 2. Esterification of E4A with crotonyl chloride; 3. Separation of E4A-Tig-U by HPLC.

[0304] Cytotoxicity assay: MTT method.

[0305] Chemical structure identification: 1. NMR analysis; 2. Mass spectrometry analysis.

[0306]

[0307] Experiment 14: Chemical synthesis, isolation and identification of E4A-Tig-S

[0308] Chemical synthesis of E4A-Tig-S: 1. Preparation of E4A; 2. Esterification of E4A with crotonyl chloride; 3. Separation of E4A-TigS by HPLC.

[0309] Cytotoxicity assay: MTT method.

[0310] Chemical structure identification: 1. NMR analysis; 2. Mass spectrometry analysis.

[0311]

[0312] Experiment 15: In the same manner as Experiment 8, E4A was esterified with acetyl, crotonyl, angelicyl, cisinoyl, senecioyl, crotonyl, cinnamoyl, pentenoyl, hexenoyl, or ethylbutyryl to give the following compounds:

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319] Experiment 16: Esterification of E4A-Tig-N with Senecioyl Chloride

[0320] Chemical synthesis of E4A-Tig-Sen-1: 1. Preparation of E4A-Tig-N; 2. Esterification of E4A-Tig-N with senecioyl; 3. Separation of E4A-Tig-Sen by HPLC.

[0321] Cytotoxicity assay: MTT method.

[0322] Chemical structure identification: 1. NMR analysis; 2. Mass spectrometry analysis.

[0323]

[0324] Test 17: E4A-Tig-N was esterified with the following compounds: acetyl chloride, angelicyl chloride, cisinoyl chloride, senecioyl chloride, O-3,3-dimethylacryloyl chloride, cinnamyl chloride, pentenoyl chloride, hexenoyl chloride, phenylformyl chloride, or ethylbutyryl chloride. E4A-Tig-N was separated by HPLC; cytotoxicity was detected by MTT method.

[0325] Chemical structure identification: 1. NMR analysis; 2. Mass spectrometry analysis.

[0326] The following compounds were obtained:

[0327]

[0328] Test 18: Inhibition of cell adhesion

[0329] Methods and Results Cancer cells ES2 or Hey8A were placed in a T25 flask containing culture medium containing 5ug / ml of a compound containing the chemical structure (2A), including E4A-Tig-R, E4A-Tig-V, E4A-Tig-S, E4A-Tig-N, E4A-Tig-Q and E4A-Tig-T, and cultured for 5 hours. The adhered cells were removed by trypsin treatment and weighed. Compared with the control, under this condition, 80±4% of ES2 cells and 60±4% of Hey8A cells adhered to the flask. In the treatment of the above compounds at a concentration of 5ug / ml, more than 90% of the non-adherent cells survived (determined by trypan blue exclusion and the ability of surviving cells to adhere to the flask when placed in culture medium without compound treatment). However, at a concentration of 10ug / ml, less than 40% of the cells adhered to the flask, and many of them were dead. These results indicate that the tested compounds have an effect of inhibiting adhesion.

[0330] Test 19: Fibronectin secretion test

[0331] In this patent, Western blot is used to detect specific proteins in cells treated or not treated with active compounds, wherein the cells are bladder, cervix, prostate, lung, breast, melanoma, colon, liver, bone, brain, skin, ovary, pancreas (Capan), oral cavity (KB), and kidney.

[0332] Cells: The cells to be tested were cultured in RPMI medium, 1.5 million cells were seeded in a T25 flask and cultured for 24 hours.

[0333] Drug treatment: The cultured cells were re-placed in fresh RPMI medium containing 2.5ul of DMSO (as a control) [D]; or 10, 20, 30, 40, 80ug / ml of the test compound.

[0334] After 24 hours, two aliquots of the culture were used for fibronectin secretion assay (Western blot method).

[0335] Cell survival (at 24 hours) was determined using MTT assay. The culture was incubated in RPMI medium (5 ml) containing MTT for 1 hour. The formazan formed was dissolved in 10 ml of DMSO and the OD (at 570 nm) (MTT units) was measured.

[0336] Western Blot: The spent culture medium was mixed with SDS sample buffer, boiled for 3 minutes, and then loaded on a 6-10% SDS gel and electrophoresed for 2 hours (100 watts). The protein was electrophoresed onto a nitrocellulose membrane and incubated with the primary antibody and the secondary antibody (AP conjugated, Promega S3721). Thus, the immune band was displayed in the BCIP / NBT color display system.

[0337] Western band intensity: The band-images of Western blot were captured using a digital camera and the intensity of the bands was determined using “Image J” software.

[0338] The results showed that compounds E4A-Tig-R, E4A-Tig-V, E4A-Tig-S, E4A-Tig-N, E4A-Tig-Q, and E4A-Tig-T inhibited the secretion of fibronectin by 20-40% in the following cancer cells: bladder, cervix, prostate, lung, breast, melanoma, colon, liver, bone, brain, skin, ovary, pancreas (Capan), oral (KB), and kidney.

[0339] Table 1. 13C and 1H NMR data of E4A-Tig-R (in DMSO-d6)a

[0340]

[0341]

[0342]

[0343] Table 2. 13C and 1H NMR data of E4A-Tig-V (in DMSO-d6)a

[0344]

[0345]

Claims

1. A method for preparing a compound without hemolytic activity, having the following structure: Chemical name: 21,24-O-cis-1,3-diol-3β,16α,21β,22α,24β,28-hexahydroxyolean-12-ene; Its characteristics are Beta-escin bES was hydrolyzed with acid and alkali to obtain the core compound E4A, and E4A was esterified with cis-chloridized cis-chlorid to obtain the compound 21,24-O-cis-chlorid-3β,16α,21β,22α,24β,28-hexahydroxyolean-12-ene; Its Beta-escin bES: Its E4A: The compound was prepared by the following method: (A) Preparation of E4A 1) Beta-escin bES was dissolved in 1M 20mg / ml NaOH and incubated at 75°C for 5 hours; 2) The hydrolyzate is neutralized with HCl and then freeze-dried to remove water; 3) The obtained product was dissolved in 50% methanol and 1N HCl and incubated at 75°C for 5 hours; 4) neutralizing the resulting solution with HCl; 5) The hydrolyzate is extracted with acetonitrile and then freeze-dried to remove water; 6) The hydrolyzed product E4A was further purified by FPLC chromatography using a C18 column, eluted with 70% acetonitrile, 1 ml / min, and the product E4A was collected; (B) Esterification of E4A with cis-1-yl chloride 1) Dissolve 50 mg of E4A in 1 ml of pyridine and gently stir in a 50 ml test tube; 2) Add 200ul of cis-1-yl chloride and esterify at 25°C; 3) Stir for 1 minute and immediately add 5 ml of 2N HCl; 4) Stir for 1 hour and let stand at room temperature overnight; 5) Extract the esterification product with 10 ml of acetonitrile; 6) Evaporating acetonitrile; 7) Dissolve the sample in 1 ml DMSO; 8) Separating the reaction products by HPLC to separate the sample 21,24-O-zhioyl-3β,16α,21β,22α,24β,28-hexahydroxyolean-12-ene, the conditions are: ① Column: ZORBAX ODS 9.4x250 mm, 5um; ②Solvent: A: 45% AN / TFA; B: 100% AN / TFA; ③ Chromatographic conditions: a) Elution: solvent A enters B within 80 min; then solvent B for 40 min; b) Flow rate: 1 ml / min. c) Monitoring OD: at 207 nm; 9) Collect the sample 21,24-O-zhioyl-3β,16α,21β,22α,24β,28-hexahydroxyolean-12-ene.

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