KR20070113185A - Anti-tumor compounds with angeloyl groups - Google Patents

Abstract

Novel compounds such as compounds designated herein as Xanifolia-Y or-Y3,-Y1,-Y2,-Y8,-Y9 and-Y10 are disclosed. These compounds have anticancer activity. The compounds of the present invention are obtainable from plants in the sapindaceae family, such as Xanthoceras sorbifolia, or other natural sources or products. The compounds of the present invention may also be synthesized chemically, Formula (I).

Description

Anti-tumor compounds with angeloyl groups

This application is a CIP application of US Application No. 11 / 131,551, filed May 17, 2005, a CIP application of US Application No. 11 / 117,760, filed April 27, 2005, and a US application of February 14, 2005. CIP Application No. 10 / 906,303, US Application Nos. 60 / 532,101, filed Dec. 23, 2003, and Oct. 9, 2003, filed CIP Application No. PCT / US04 / 33359, filed Oct. 8, 2004; The CIP application of International Application No. PCT / US04 / 43465, filed Dec. 23, 2004, claiming the benefit of U.S. Application No. 60 / 509,851; US Application No. 60 / 617,379, filed Oct. 8, 2004, US Application No. 60 / 613,811, filed September 27, 2004, US Application No. 60 / 607,858, filed September 7, 2004 It claims the advantages of US Application No. 60 / 675,284, filed April 27. The contents of these prior applications are hereby incorporated by reference in their entirety.

Throughout this application, various documents are referred to. The contents of these documents are all incorporated by reference in the present application to more fully describe the related art of the present invention.

The present invention Xanthocera sorbobia ( Xanthoceras sorbifolia ) and novel antitumor compounds obtainable from plants of the sapindaceae family.

Wenguanguo is a member of the family sapindaceae . Its scientific name is Xanthoceras sorbifolia Bunge . Tanguanguo is a common Chinese name. Others are Tianguangguo, Tianguanmu, Tianguanhua, Xylaseden, Goldenhorn and Yellowhorn. Tanguanguo grows in Liaoning, Jilin, Hebei, Shandong, Jiangsu, Henan, Shanxi, Xiangxi, Gansu, Ningxia and Inner Mongolia of China. His seeds, leaves and flowers are edible and have been used as folk or traditional medicine for centuries. Its branches and trees have also been used in folk or traditional medicine. For further information and background or related art of the present invention, pages 1, lines 25-38 to 13 of International PCT Application No. PCT / US04 / 33359, filed Oct. 8, 2004, and filed Feb. 14, 2005 See US Application No. 10 / 906,303. The contents of these prior applications are incorporated herein by reference in their entirety.

Chem. Pharm. Ing, reported in Bull 33 (4) 1387-1394 (1985), describes the study of Xanthoceras sorbifolia Bunge to Chen, Tadahiro Takeda and Yukio Ogi. See section V. Saponins from the fruits of Xanthoceras sorbifolia . Four new saponin compounds were isolated from the fruits of Xanthoceras sorbifolia Bunge . These structures are Bunkancasaponin A, B, C and D. Chem. Pharm. Ing, reported in Bull 33 (4) 1387-1394 (1985), describes another study of the Xanthoceras sorbifolia Bunge to Chen, Tadahiro Takeda and Yukio Ogi. IV. See the structural section of trace prosapogenin. Chem. Pharm. Another study of the Xanthoceras sorbifolia Bunge telling Chen, Tadahiro Takeda and Yukio Ogi to Bully, reported in Bull 33 (1) 127-134 (1985). III. See section Trace Prosapogenin Saponins from Fruits of Xanthoceras sorbifolia Bunge . Other studies focused on saponin compounds include:

The contents of the aforementioned documents are all incorporated by reference.

However, these references do not disclose saponin compounds having the same structure as the compounds of the present invention that can inhibit cancer or tumor cell growth.

Cancer cells are defined by two genetic properties: (1) they regenerate and resist normal arrest upon cell division; (2) They invade and invade regions normally contained in other cells. Cancers require mutations of one to many genes for development and are classified according to the tissue and cell type in which the cancer develops. Cancer resulting from epithelial cells is called carcinoma; Cancer resulting from connective tissue or muscle cells is called sarcoma. There is also a cancer called leukemia derived from blood cells. Cancer may also arise from nervous system cells.

Ovarian cancer is the fifth leading cause of cancer death in women and the first cause of death from gynecological malignancies. In the United States, women are at risk of developing ovarian cancer in 1.4-2.5%, or 1 in 40-60 women, over their lifetime. Older women are most at risk. More than half of ovarian cancer deaths occur in women between 55 and 74 years of age, and nearly one quarter of ovarian cancer deaths occur in women between 35 and 54 years of age. MedlinePlus Encyclopedia on http://www.nlm.nih.gov/medlineplus/ency/aticle/000889.htm ovarian cancer Reference.

Ovarian cancer is fatal for several reasons. First, the symptoms are mild and nonspecific, so women or physicians treat them as more general. By the time cancer is diagnosed, tumors often spread out of the ovaries. Ovarian cancer also emits malignant cells that are commonly implanted on the linings of the uterus, bladder, intestines, and intestinal walls (net membranes). These cells may begin to form new tumor growth before cancer is suspected. Second, because there is no test to detect ovarian cancer cost effectively, more than 50% of women with ovarian cancer are diagnosed with significant progression of ovarian cancer.

It is therefore an object of the present invention to provide compounds and / or compositions having significant efficacy against ovarian cancer, extracted from Xanthoceras sorbifolia or plants or synthesized chemically or biochemically.

Summary of the Invention

According to these and other objects of the present invention, a brief summary of the present invention is provided. The invention may be partially simplified and omitted in the following summary, which is intended to highlight some features of the invention and to introduce some features thereof, but not to limit the scope of the invention. Detailed descriptions of preferred exemplary embodiments suitable for allowing those skilled in the art to make and use the concepts of the present invention are described later.

The present invention provides six novel compounds having the structure (Y1, Y2, Y or Y3, Y8, Y9, Y10) as shown in FIG. As used herein, "structure Y" is also referred to as "structure Y3".

The chemical formula, chemical name and general name of these compounds are listed in Table 1 below.

Table 1. Chemical formula, chemical and general names of six new compounds having structures (Y, Y1, Y2, Y8, Y9, Y10)

designation Chemical formula Chemical name Xanifolia-Y (Y3) C ₅₇ H ₈₈ O ₂₃ 3-O- [β-D-galactopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3) -β-D-glucuronopyranosyl-21,22-O Diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene Xanifolia-Y1 C ₆₅ H ₁₀₀ O ₂₇ 3-O- [β-D-galactopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3) -β-D-glucuronopyranosyl-21-O- ( 3,4-Diangeloyl) -α-L-lamnopyranosyl-22-O-acetyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene Xanifolia-Y2 C ₅₇ H ₈₈ O ₂₄ 3-O- [β-D-glucopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3)-(-D-glucuronopyranosyl-21,22-O- Diangeloyl-3β, 15α, 16α, 21β, 22α, 24β, 28-heptahydroxyolean-12-ene Xanifolia-Y8 C ₅₇ H ₈₇ O ₂₃ 3-O- [β-galactopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-glucuronopyranosyl-21,22-O-diangeloyl- 3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene Xanifolia-Y9 C ₆₅ H ₁₀₀ O ₂₇ 3-O- [β-galactopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-glucuronopyranosyl-21-O- (3,4-diangel Loyl) -α-ramnopyranosyl-28-O-acetyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene Xanifolia-Y10 C ₅₇ H ₈₇ O ₂₂ 3-O- [β-galactopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-glucuronopyranosyl-21,22-O-diangeloyl- 3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene

The six compounds (Y, Y1, Y2, Y8, Y9 and Y10) have anticancer effects. These compounds inhibit the growth of human ovaries and other cancer cells. See FIGS. 2, 3 and 4.

Concensus sub-structures were derived or identified from the bioactive compounds of the invention (Y, Y1, Y2, Y8, Y9 and Y10). The consensus sub-structure of the compounds of the invention is a biangeloyl group located on adjacent carbons. For example, in structures Y, Y2, Y8 and Y10, the nonangeloyl group is located at 21β and 22α of the triterpene backbone. See FIG. 5. For structures Y1 and Y9, the nonangeloyl group is located at C3 and C4 of the sugar ring. See FIG. 6. In one embodiment, the non-angeloyl groups of the bioactive compounds (Y, Y1, Y2, Y8, Y9 and Y10) of the invention are located in the trans position in adjacent carbons of the structure. See FIG. 7. In another embodiment, the sugar residue or sugar chain is located at C3 of the triterpene. In another embodiment, the sugar moiety or sugar chain is D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, alduronic acid, D-glucuronic acid and D-galacturonic acid Selected from the group consisting of.

Studies on the structural and functional relationships of the six compounds of the present invention indicate that alterations or substitutions of functional groups located at C15 and C24 of triterpenes do not affect anticancer activity. Compounds of the invention (Y, Y1, Y2, Y8, Y9 and Y10) show activity in inhibiting tumor growth. See FIGS. 2, 3 and 4. These compounds are prepared by chromatographic purification including FPLC and HPLC as described in Figures 8, 9, 10, 11, 12 and 13. Compound Y is purified using the procedure described in this application. 11A shows NMR peaks of compound Y. FIG. The controls in FIGS. 2 and 14 show that purified Compound Y has a 10-fold higher potency (IC50 = 1.5 μg / ml) compared to the original extract (IC50 = 25 μg / ml). Compound Y has high selectivity for ovarian cancer. See FIG. 15.

Purified compounds Y1, Y2, Y8, Y9 and Y10 also exhibit inhibitory activity against human cancer cells with high efficacy against ovarian carcinoma. See FIGS. 3 and 4. Plant extracts containing compound Ys exhibit inhibitory activity against human cancer cells. For example, studies are conducted on eleven human cancer cell lines, and the plant extracts of the present invention show higher efficacy against ovarian carcinoma. See Figures 14, 15 and 16 and Table 3.1 for contrasting the activity of the plant extracts of the invention among different cancer cell lines. As used herein, "Ys" or "Compound Ys" is a compound Y or Y3, Y1, Y2, Y8, Y9, Y10, or other bioactive obtainable from the Xanthoceras sorbobia extract of the present invention. Used to mean a compound.

The present invention provides an extract of Xanthoceras sorbifolia that can inhibit cancer growth. Cancers include, but are not limited to ovarian cancer, bladder cancer, prostate cancer, leukocyte cancer, and bone cancer. Compounds of the invention that have been found to be effective against cancer are isolated from plants called Xanthoceras sorbifolia , chemically synthesized, or from other biological sources, including plants of the family sapindaceae . Can be extracted.

The present invention provides a method for producing bioactive compounds from husks, leaves, branches or stems, fruit-stems, seed husks and bark of mutuanguo. Extracting the bioactive compounds of the present invention from different parts of the guanguuo plant can be carried out individually or simultaneously. The invention also discloses a method for obtaining the bioactive compounds of the invention. In addition to saponin compounds, the extracts of the present invention contain saccharides, proteins, glycosides, flavonoids, coumarin extracts, alkaloid extracts, organic acid extracts, tannins and other bioactive compounds. Saponin compounds obtained from the extracts of the present invention were collected and found to possess inhibitory activity against cancer growth.

Compounds, extracts or compositions of the invention can modulate a number of cellular pathways, including receptors or components of cells such as G-protein receptors, Fas proteins, receptor tyrosine kinases, mitogens, mitogen receptors. The compounds of the present invention can be isolated from plants called Xanthoceras sorbifolia , chemically synthesized, or extracted from other biological sources, including plants of the family sapindaceae .

The present invention Xanthocera sorbobia ( Xanthoceras sorbifolia ) and compounds including compounds of structures Y, Y1, Y2, Y8, Y9 and Y10 which can be obtained from the cancer and inhibit cancer growth. In one embodiment, the cancer is not limited to bladder cancer but includes cervical cancer, prostate cancer, lung cancer, breast cancer, leukocyte cancer, colon cancer, liver cancer, bone cancer, brain cancer and ovarian cancer. The present invention provides an oleanene triterpenoid saponin compound comprising a side chain comprising an angeloyl group at carbon 21 and carbon 22 of the compound. In one embodiment, the compound comprises one or more sugars, wherein the C3 and C4 of the sugars are acylated by an angeloyl group. The present invention includes a triterpene backbone and a biangeloyl group, wherein one angeloyl group is attached to 21β of the triterpene backbone and one angeloyl group is attached to 22α and the presence of the antiangeloyl group is anticancer Provided are triterpinoidal saponin compounds exhibiting activity. The present invention includes a triterpene backbone and a sugar residue or rhamnose, wherein the sugar residue or rhamnose is attached to a triterpene backbone, and the sugar residue or rhamnose further comprises a biangeloyl group, and the biangel The presence of a loyl group provides a triterpenoid saponin compound that produces anticancer activity. The present invention includes a triterpene backbone, wherein the triterpene backbone is acylated by sugar residues or sugar chains at one of the 21β or 22α positions or at both 21β and 22α positions, and at least one sugar in the sugar residues or sugar chains is It provides a triterpenoid saponin compound comprising an angeloyl group attached to the C3 and C4 of the sugar. In one embodiment, the two angeloyl groups are in the trans position of the structure, and the presence of the nonangeloyl group produces anticancer activity. In one embodiment, the two angeloyl groups are in the trans position in the adjacent carbon of the structure, and the presence of the biangeloyl group produces anticancer activity. The present invention also provides salts of the aforementioned compounds. The present invention provides a pharmaceutical composition comprising an effective amount of the compound and a pharmaceutically acceptable carrier.

The present invention relates to Xanthoceras by using an appropriate amount of an organic solvent for an appropriate time to obtain an extract. sorbifolia ) extracting the powder; Identifying a bioactive compound in the extract; Purifying the bioactive compound in the extract by FPLC to obtain a bioactive compound fraction; And isolating the individual bioactive compounds purified by preparative HPLC from the fractions of the bioactive compounds. The present invention relates to NMR spectral data derived from proton NMR, carbon NMR, heteronuclear mulitple quantum correlation (HMQC), heteronuclear mulitple bond correlation (HMBC), 2D NMR of NOESY and COSY, and mass spectroscopic data derived from MALDITOF and ESI-MS. It provides a compound having a structure identified by.

The present invention provides compounds and derivatives thereof that are effective against cancer. Compounds or compositions of the present invention can modulate various cellular pathways including, but not limited to: G-protein receptors, Fas proteins, receptors for tyrosine kinases, mitogens, mitogen receptors, TGF beta-smad, FGF, TGF -Receptors or components such as beta and TGF-alpha, ras-GTPase-MAP kinase, jun-fos, Src-fyn, Jak-Jnk-STAT, BMP, Wnt or myc-cell proliferation. Compounds and compositions of the invention derived from xanthoseras sorbibolia can modulate components and receptors associated with cell death and can (re) activate cell death programs or pathways.

Detailed description of the invention

The present invention provides a compound selected from compounds of formula (1) or salts, esters or derivatives thereof:

In which R 1 is an angeloyl group; R 2 is an angeloyl group; R 3 is OH or H; R 4 is CH 3 or CH 2 OH; R 7 is H; And R 5 is D-glucose or D-galactose; And R6 is L-arabinose.

The present invention provides compounds selected from compounds of formula (2) or salts, esters or derivatives thereof:

In which R 1 is an angeloyl group; R 2 is an angeloyl group; R 3 is Ac or H; R 4 is H or Ac; R 5 is CH 3 or CH 2 OH.

 The present invention provides compounds selected from compounds of formula (3) or salts, esters or derivatives thereof:

In which R 1 is an angeloyl group; R 2 is an angeloyl group; R 3 is OH or H; R4 is CH3 or CH2OH or an alkyl group or derivative thereof; R 6 is Ac or H and R 5 is a sugar moiety wherein the sugar moiety is one or more sugars, or D-glucose, or D-galactose, or L-rhamnose, or L-arabinose, or D-k Cellulose, or alduronic acid, or D-glucuronic acid, or D-galacturonic acid, or derivatives thereof or combinations thereof. In one embodiment, R 5 is a compound capable of acting as a sugar moiety. In another embodiment, the sugar moiety comprises L-arabinose, or D-glucose, or D-galactose, or a combination thereof.

The present invention provides compounds selected from compounds of formula (3A) or salts, esters or derivatives thereof:

In which R 1 is an angeloyl group; R 2 is an angeloyl group; R 3 is OH or H; Positions 23, 24, 25, 26, 27, 29, 30 of the compound comprise CH 3, or CH 2 OH or CHO or COOH, an alkyl group, or an acetyl group, or a derivative; R6 is Ac or H; And R 5 is a sugar residue, wherein the sugar residue is one or more sugars, or D-glucose, or D-galactose, or L-rhamnose, or L-arabinose, or D-xylose, or alduronic acid, Or D-glucuronic acid, or D-galacturonic acid, or derivatives thereof or combinations thereof. In another embodiment, R 5 represents a compound capable of acting as a sugar residue. In another embodiment, the sugar moiety comprises L-arabinose, D-glucose and / or D-galactose or combinations thereof. In another embodiment, two of R1, R2 or R6 are angeloyl groups, or one of R1, R2 or R6 is attached to a sugar moiety wherein two angeloyl groups are attached to adjacent carbons of the monosaccharide. It is. In another embodiment, R 1, R 2 and R 6 include angeloyl groups, acetyl groups, tigloyl groups, sensioli groups or acids having 2 to 5 carbons or combinations thereof. In another embodiment, at least one of R1, R2 or R6 is attached to a sugar moiety or rhamnose, wherein the sugar moiety or rhamnose is two angeloyl groups, an acetyl group, a tigloyl group, a sensioli Groups, acids with 2 to 5 carbons or combinations thereof.

The present invention provides a compound selected from compounds of formula (4), or salts, esters or derivatives thereof:

In the formula,

이고;

ego;

S1-S4 = S1 or S2 or S3 or S4; R 1 is an angeloyl group; R 2 is an angeloyl group; R 3 is Ac or H; R 4 is H or OH; R 6 is Ac or H; R7 is a CH3 or CH2OH or alkyl group or derivative thereof and R5 is D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, alduronic acid, D-glucuronic acid, A sugar residue or sugar chain selected from the group consisting of D-galacturonic acid and derivatives thereof. In one embodiment, R 1 is an angeloyl group; R 2 is an angeloyl group; R 3 is Ac or H; R 4 is H or OH; R 6 is Ac or H; Positions 23, 24, 25, 26, 27, 29, 30 of the compound are CH 3, CH 2 OH, CHO, COOH, an alkyl group, an acetyl group, or a derivative thereof; R 5 is a sugar residue, wherein the sugar residue is one or more sugars, or D-glucose, or D-galactose, or L-rhamnose, or L-arabinose, or D-xylose, or alduronic acid, or D-glucuronic acid, or D-galacturonic acid, or derivatives thereof, or combinations thereof. In one embodiment, R 5 represents a compound capable of acting as a sugar residue. In another embodiment, R 1, R 2, R 3, R 6 comprise an angeloyl group, an acetyl group, a tigloyl group, a sensioli group or an acid having 2 to 5 carbons. In another embodiment, the sugar moiety comprises L-arabinose and / or D-glucose and / or D-galactose or combinations thereof.

The present invention provides a compound of formula (5) or a compound selected from salts, esters, metabolites or derivatives thereof:

In which R 1 and R 2 are angeloyl groups; R 3 is H or OH; R4 is CH2OR6; And R 6 is H; R 5 is one or more sugar residues or derivatives thereof. In one embodiment, R 1 and R 2 are angeloyl groups; R 3 is H or OH; R 4 is COOR 6, wherein R 6 is H; R 5 is one or more sugar residues or derivatives thereof. In one embodiment, R 1 is H; R 2 is an angeloyl group; R 3 is H or OH; R 4 is CH 2 OR 6 or COOR 6, wherein R 6 is an angeloyl group; And R 5 is one or more sugar residues or derivatives thereof. In another embodiment, two or more of R1, R2, and R6 comprise an angeloyl group or oxygen having five carbons; R 3 is H or OH; R 4 is CH 2 OR 6 or COOR 6, wherein R 6 is an angeloyl group; R 5 is one or more sugar residues or derivatives thereof. In another embodiment, at least one angeloyl in R 1 or R 2 is substituted by an acetyl group, a tigloyl group, a sensioli group, or an acid having 2 to 5 carbons; R 3 is H or OH; R 4 is CH 2 OR 6 or COOR 6, wherein R 6 is an angeloyl group; And R 5 is one or more sugar residues or derivatives thereof. In another embodiment, at least one of R1, R2, and R6 is a sugar moiety comprising at least two angeloyl groups, acetyl groups, tigloyl groups, sensioli groups, or acids having 2 to 5 carbons Or rhamnose. In another embodiment, positions 23, 24, 25, 26, 29, 30 of the compound include CH 3, CH 2 OH, CHO, COOH, alkyl groups, acetyl groups or derivatives thereof. In another embodiment, R 5 is a sugar moiety comprising glucose, galactose or arabinose. In another embodiment, R 5 is a sugar moiety wherein the sugar moiety is one or more sugars, or D-glucose, or D-galactose, or L-rhamnose, or L-arabinose, or D-xylose, Or alduronic acid, or D-glucuronic acid, or D-galacturonic acid, or derivatives thereof, or combinations thereof. In one embodiment, R 5 represents a compound capable of acting as a sugar residue. In another embodiment, R5 is H. In another embodiment, R 4 is H or OH or CH 3.

Substitutions, deletions and / or additions of any groups in the compounds described above will be apparent to those skilled in the art based on the technical content of the present application. In another embodiment, substitutions, deletions and / or additions of groups in the compounds of the invention do not substantially affect the biological action of the compounds. In another embodiment, the angeloyl group is in a trans position on the structure.

The present invention provides compounds comprising the following structures (A), (B) or (C):

(A)

(A)

(B)

(B)

(C)

(C)

Wherein R 1 is an angeloyl group and R 2 is an angeloyl group. In one embodiment, the nonangeloyl group is acylated in the trans position. In another embodiment, the biangeloyl group is acylated at the trans position on adjacent carbons. In another embodiment, the nonangeloyl group is acylated in the structure. In another embodiment, R 1, R 2 include angeloyl groups, tigloyl groups, sensioli groups, acids with 2 to 5 carbons, or combinations thereof. In another embodiment, the compound comprises two or more angeloyl groups, tigloyl groups, sensioli groups, acids with 2 to 5 carbons, or combinations thereof. In another embodiment, the compound further comprises a sugar moiety. In another embodiment, the sugar moiety comprises glucose, galactose or arabinose or combinations thereof. In another embodiment, the sugar moiety is one or more sugars, or D-glucose, or D-galactose, or L-rhamnose, or L-arabinose, or D-xylose, or alduronic acid, or D- Glucuronic acid, or D-galacturonic acid, or derivatives thereof, or combinations thereof. In another embodiment, structures A, B or C include compounds that can act as sugar residues. Substitutions, deletions and / or additions of any groups in the compounds described above will be apparent to those skilled in the art based on the technical content of the present application. In another embodiment, substitutions, deletions and / or additions of groups in the compounds of the invention do not substantially affect the biological action of the compounds. In another embodiment, the angeloyl group is in a trans position on the structure.

A composition for treating cancer or inhibiting a virus is provided comprising at least two side chains comprising an angeloyl group, the side chains comprising a compound present on adjacent carbons in the trans position. In one embodiment, the side chains are at other carbons in the cis position. In one embodiment, the side chain is on another carbon in the trans position. In one embodiment, the side chains are on nonadjacent carbons in the cis or trans position. In one embodiment, the side chain comprises a functional group capable of acting an angeloyl group.

The present invention provides a composition for inhibiting tumor cell growth comprising the compound. In one embodiment, the composition of the present invention comprises a suitable carrier. In another embodiment, the composition comprises a pharmaceutically suitable carrier. The present invention provides a method of treating ovarian cancer in a patient comprising administering an effective amount of the composition to the ovarian cancer patient.

The present invention relates to (a) Xanthoceras using organic solvents. sorbifolia ) or extracting plant powder to obtain an organic extract; (b) collecting the organic extracts; (c) refluxing the organic extract to obtain a second extract; (d) removing the organic solvent from the second extract; (e) drying and sterilizing the second extract to obtain a crude extract powder; (f) fractionating the crude extract powder into components using HPLC and FPLC chromatography using silica gel, C18 and other equivalent solids; (g) monitoring the absorption wavelength at 207 nm or 254 nm; (h) identifying the bioactive component of the crude extract powder; (i) purifying one or more bioactive ingredients of the crude extract powder by FPLC to obtain one or more fractions of the bioactive ingredients; And (j) isolating a desired fraction of the bioactive component by preparative HPLC. The method provides a method for isolating a compound from a plant of xanthoserras sorbobia herb, or a plant of sapindaceae . do.

Compound Y or Y3

In the present invention, the chemical formula is C ₅₇ H ₈₈ O ₂₃ and the chemical name is 3-O- [β-D-galactopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3) -β- D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene and known as xanifolia-Y There is provided a compound comprising a structure (see FIG. 17): This compound can be obtained from plants of the family Xanthocera sorbobia or sapindaceae .

This compound belongs to an oleanene triterpenoid saponin having a trisaccharide chain attached to C-3 of aglycone and two angeloyl groups acylated at C-21 and C-22. This compound has anticancer activity. Evaluation of this structure is supported by spectral data, namely H-NMR, 2D NMR (HMBC, HMQC) and MS (MALDI-TOF, EMS). Thus, this compound has the characteristic properties as shown in FIGS. 18-22 or Table 5.1.

compound Y1

In the present invention, the chemical formula is C ₆₅ H ₁₀₀ O ₂₇ and the chemical name is 3-O- [β-D-galactopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3) -β- D-glucuronopyranosyl-21-O- (3,4-diangeloyl) -α-L-ramnopyranosyl-22-O-acetyl-3β, 16α, 21β, 22α, 28-pentahydride Another compound is provided that is lioolean-12-ene and comprises the following structure known as xanipolya-Y1 (see FIG. 23).

This compound is a bisdesmosidic with a trisaccharide chain at C-3 and a monosaccharide residue at C-21 of the main chain and two angeloyl groups at the C-3 and C-4 positions acylated Polyhydroxyoleanene triterpenoid saponins. This compound has anticancer activity. Evaluation of this structure is supported by spectral data, namely H-NMR, 2D NMR (HMBC, HMQC) and MS (MALDI-TOF, EMS). Thus, this compound has the characteristic properties as shown in FIGS. 24-27.

compound Y2

In the present invention, the chemical formula is C ₅₇ H ₈₈ O ₂₄ and the chemical name is 3-O- [β-D-glucopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3) -β-D Glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 24β, 28-heptahydroxyolean-12-ene and known as xanifolia-Y2 A third compound is provided comprising the following structure (see FIG. 28).

This compound (Y2) belongs to a saponin comprising triterpenes, sugar residues and angeloyl groups linked to the main chain. Angeloyl groups are linked to the main chain at the C21 and C22 positions. This compound has anticancer activity. Evaluation of this structure is supported by spectral data, namely H-NMR, 2D NMR (HMBC, HMQC, TOCSY) and MS (MALDI-TOF). Thus, this compound has the characteristic properties as shown in FIGS. 29-34.

compound Y8

The present invention provides the fourth active compound Y8 whose structure was determined by 1D NMR, 2D NMR and MS analysis. This compound has the formula C ₅₇ H ₈₇ O ₂₃ and has the chemical name 3-O- [β-D-glucopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-D-glu Kuronopyranosyl-21,22-O-diangeloyl-3β, 16α, 21β, 22α, 24β, 28-hexahydroxyolean-12-ene and the following structure known as xanifolia-Y8 (FIG. 35).

Evaluation of the structure is supported by spectral data, namely H-NMR, C13-NMR and 2D NMR (HMQC). Thus, the compound has the characteristic characteristics as shown in FIGS. 36-38.

compound Y9

The present invention provides the fifth active compound Y9, whose structure was determined by 1D NMR, 2D NMR and MS analysis. This compound has the chemical name 3-O- [β-galactopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-glucuronopyranosyl-21-O- (3 , 4-Diangeloyl) -α-lamnopyranosyl-28-O-acetyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene and known as xanifolia-Y9 (Ie, see FIG. 39).

Evaluation of the structure is supported by spectral data, namely H-NMR, 2D NMR (HMQC and HMBC). Thus, the compound has the characteristic characteristics as shown in FIGS. 40-42.

compound Y10

The present invention provides the sixth active compound Y10, whose structure was determined by 1D NMR, 2D NMR and MS analysis. This compound has the formula C ₅₇ H ₈₇ O ₂₂ and has a chemical name 3-O- [β-galactopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-glucurono Pyranosyl-21,22-O-diangeloyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene and includes the following structure known as xanifolia-Y10 (see FIG. 43). do.

Evaluation of the structure is supported by spectral data, namely H-NMR, C13-NMR and 2D NMR (HMQC). Thus, the compound has the characteristic characteristics as shown in FIGS. 44-46.

The present invention provides a compound comprising a sugar moiety and a triterpene or sapogenin, wherein the triterpene or sapogenin is acylated by an angeloyl group at carbon 21 and 22. In one embodiment, the triterpenes or sapogenins are acylated by sugar chains comprising two angeloyl groups on carbon 21 and / or 22. In another embodiment, the compound comprises one or more sugars. In another embodiment, the compound comprises two or more angeloyl groups. In another embodiment, the sugar moiety is one or more sugars, or D-glucose, or D-galactose, or L-rhamnose, or L-arabinose, or D-xylose, or alduronic acid, or D Glucuronic acid or D-galacturonic acid, derivatives thereof, or combinations thereof. In another embodiment, the compound capable of acting as a sugar moiety is attached to triterpene or sapogenin. In another embodiment, the angeloyl group can be substituted by a tigloyl group, a sensioli group, an acid having 2 to 5 carbons, or a combination thereof. Substitution, deletion and / or addition of any group in the compounds will be apparent to those skilled in the art based on the technical content of the present application. In another embodiment, substitutions, deletions and / or additions of groups in the compounds of the invention do not substantially affect the biological action of the compounds. In another embodiment, the angeloyl group is in a trans position on the structure. In another embodiment, the angeloyl group is in an adjacent trans position on the structure.

 An extract with anticancer activity obtained from Xantho ceras sorbibolia is disclosed. Experiments for measuring the anticancer activity of the extracts of the present invention include human cell lines obtained from 11 human organs, namely (HTB-9 (bladder), HeLa-S3 (cervical), DU145 (prostate), H460 (lung), MCF-)). 7 (breast), K562 (leukocyte), HCT116 (large colon), HepG2 (liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary)). The response of the 11 cell lines to the extracts of the present invention can be classified into four groups: (A) highest sensitivity: ovary, see FIG. 14; (B) sensitivity: bladder, bone, (C) semi-sensitive: prostate, leukocyte, liver, breast and brain; And (D) lowest sensitivity: large intestine, cervical and lung. See Figures 16A, B, C. Each IC50 value is listed in Table 3.1 below.

Table 3.1. IC50 Values of Xanthoceras Sorbipolya Extracts Measured on Different Cancer Cells

Cancer Cells from Different Organs IC50 (ug / ml) measured by MTT assay Ovary (highest sensitivity) 15-15 Bladder (sensitive) 45-50 bone 40-55 Prostate (semi-sensitive) 40-50 leukocyte 40-50 liver 45-65 breast 65 brain 70-85 Large intestine (lowest sensitivity) 90 cervix 115 lungs 110

In order to identify the active compounds of Xanthoceras sorbivifolia, extracts were separated from xanthoceras sorbifolia by chromatography, including Fast Protein Liquid Chromatography (FPLC) and High Preferment Liquid Chromatography (HPLC). Multiple fractions were obtained, for example, by the FPLC procedure shown in FIG. 9 and by HPLC as shown in FIG. 8. Analysis of these fractions by HPLC shows that the extract comprises 26 identifiable fractions, denoted a to z, as shown in FIG. 8. The anticancer activity of these fractions was measured by MTT assay.

FPLC fraction 5962 as shown in FIG. 10, corresponding to fraction Y in HPLC shown in FIG. 8, has anticancer activity. Compound Y can be purified from fraction Y. Fraction 5962 may be further separated into four components represented by Y1 to Y4. See FIG. 11. Components Y1-Y4 can be purified from fraction 5962. Fraction 6365 was further separated into 5-6 components represented by Y5-Y10. See FIG. 12. Compound Y5-Y10 can be purified from fraction 6365. Compound Y or Y3, Y1 and Y2 exhibited strong antitumor activity (such as shown in FIGS. 2-3) and was also isolated and purified. Similarly, compounds Y8, Y9 and Y10 also exhibited strong antitumor activity as shown in FIG. 4 and were also isolated and purified. See FIG. 13. Thus, the structures of these active compounds, ie Y, Y1, Y2, Y8, Y9 and Y10 and their use are the subject of the present application.

The inhibitory effect of the compounds of the invention on ovarian cancer cells was evaluated by MTT assay. Compound Y exhibits at least 10-fold higher potency (IC50 = 1.5 μg / ml) compared to the original crude extract (IC50 = 20 μg / ml) as shown in FIG. 14. Selectivity of compound Y against different cell lines was tested and it was found that compound Y has much higher potency against ovarian cancer cells compared to cervical cancer cells. See FIG. 15.

The present invention provides methods for identifying and isolating bioactive compounds from plants, herbs or plant extracts. In one embodiment, the extract comprises an extract of Xanthoseras sorbibolia or an extract of a Safinaceae family of plants. The present invention provides the chemical structure of six bioactive compounds isolated from Xanthoseras sorbibolia or safinaceae family. The structures of these compounds of the invention are shown in FIG. The present invention provides spectral data including H-NMR, C-13-NMR, 2D NMR (HMBC, HMQC, COZY, TOCSY) and MS (MALDI-TOF, ESI-MS) to support the structures shown.

The present invention provides consensus substructures or functional groups derived from bioactive compounds purified from fraction Y. Compounds isolated from fraction Y such as Y or Y3, Y1, Y2, Y8, Y9 and Y10 are collectively referred to as "Ys" and their generic name is referred to as xanifolia-Ys. Consensus substructures or functional groups of these compounds are biangeloyl groups located on adjacent carbons. For example, in compounds Y, Y2, Y8 and Y10, the biangeloyl group is located at 21β and 22α of the triterpene backbone. See FIG. 5. In compounds Y1 and Y9, the biangeloyl group is located at C3 and C4 of the sugar ring. See FIG. 6. Thus, the non-angeloyl groups of the bioactive compounds of the invention are located in the trans position relative to each one on the structure. See FIG. 7. The bioactive functional groups of the compounds of the invention are biangeloyl groups attached in-trans to adjacent carbons located in the structure. See FIG. 7. The present invention also provides salts of these compounds.

The present invention provides a composition comprising the compound and a suitable carrier. The present invention provides a pharmaceutical composition comprising an effective amount of the compound and a pharmaceutically acceptable carrier. The present invention provides an anti-ovarian cancer agent or composition comprising the composition. The present invention provides compositions that are effective against cancer growth. Such cancers include but are not limited to bladder cancer, bone cancer and ovarian cancer. The present invention provides compositions that are effective against skin cancer and KB cancer growth. The present invention provides a composition comprising the compounds and salts, esters, derivatives or metabolites thereof that can inhibit tumor growth. The present invention provides a composition comprising said compounds and salts, esters, derivatives or metabolites thereof which can inhibit viral growth and / or activity.

The present invention provides a composition comprising the compounds and salts, esters, derivatives or metabolites thereof that can exhibit hemolytic activity. See FIG. 73. It has been found that the compounds of the present invention having two angeloyl groups have stronger anticancer activity than the compounds having one angeloyl group. See FIGS. 65, 2, 3. Compounds having two angeloyl groups show higher hemolytic activity compared to compounds having one angeloyl group. See FIG. 73A. The compound loses hemolytic activity when the angeloyl group is removed. See FIG. 73B. The compounds of the present invention or derivatives thereof can be obtained by chemical synthesis or can be isolated from natural or biological sources, including sapinaceae plants. The saponin compounds of the present invention having two angeloyl groups have stronger potency than Escin for treating diseases related to hemolytic activity. See FIG. 73A.

The present invention relates to chronic venous insufficiency, peripheral edema, hyperlipidemia, chronic venous disease, varicose vein disease, varicose veins syndrome, venous congestion, expectoration, cebro-organic cramps, cerebral circulation disease, brain edema, psychosis, dysmenorrhea, To treat hemorrhoids, vulva, hemorrhoids, peripheral edema formation or postoperative swelling; Reduce symptoms of leg pain, itching, leg size, thrombosis, thrombophlebitis; The present invention also provides a composition for preventing stomach ulceration. The present invention is anti-MS, anti-aneurysms, anti-asthma, anti-bradykinin, anti-capillary hemorrhage, anti-headache, anticervicobrachialic, anti- liver, anti-edema, anti-encephalitis, anti-occipitalitis, anti-exudation, anti-influenza (antiflu), anti-fracture, anti-gum disease, anti-hematoma, antihydrathritic, anti-meningitis, antioxidant, anti-periodontics, anti-herpes, anti-histamine, anti-veinitis, anti-pleural, anti-sheep, anti-rhinitis, anti Tonsillitis, anti-ulcer, anti- varicose veins, anti-dizziness, cancer growth inhibition, corticosteroids, diuretics, fungicides, hemolysis, hyaluronidase inhibitors, lymphogenicity, natriuresis, pesticides, pituitary stimulation, thymol soluble, vascular Provided are compositions for protection and venotonic (vein soluble).

Other compounds have also been isolated from fractions R and O of the extracts of Xanthoseras sorbibolia, which are denoted R1 and O54, respectively. Their structure was determined. These compounds are all triterpenoid saponins. These compounds do not have biangeloyl attachment in the triterpene backbone or sugar rings. Preliminary experiments revealed that both R1 and O54 do not have anticancer activity.

compound R1

The structure of compound R1 is shown below and in FIG. 47. Compound R1 has the chemical formula C ₆₅ H ₁₀₆ O ₂₉ and has a chemical name 3-O- [angeloyl- (1 → 3) -β-D-glucopyranosyl- (1 → 6)]-β-D-glucopyra. Nosyl-28-O- [α-L-lamnopyranosyl- (1 → 2) -β-D-glucuronopyranosyl- (1 → 6) -β-D-glucopyranosyl-3β, 21β, 22α, 28-tetrahydroxyolean-12-ene and is known as xanipolya-R1.

Evaluation of the structure is supported by spectral data, namely H-NMR, C13-NMR, 2D NMR (HMBC, HMQC, COZY) and MS (MALDI-TOF, EMS). Thus, the compound has the characteristic characteristics as shown in FIGS. 48-52.

compound- O54

The present invention provides compound O54 isolated from an extract of Xanthoseras sorbibolia. The structure of O54 was measured and the chemical formula is C ₆₀ H ₁₀₀ O ₂₈ . The structure of compound O54 is shown below. See FIG. 53.

The chemical name of compound -O54 is 3-O- [β-D-glucopyranosyl- (1 → 6)]-β-D-glucopyranosyl-28-O- [α-L-rhamnopyranosyl- (1 → 2) -β-D-glucuronopyranosyl- (1 → 6)]-β-D-glucopyranosyl-3β, 21β, 22α, 28-tetrahydroxyolean-12-ene, xanipolya Known as -O54. Evaluation of this structure is supported by spectral data, i.e. 1 H-NMR, 2D NMR (HMBC, HMQC). Thus, the compound has the characteristic characteristics as shown in FIGS. 54-56.

In addition to the compounds Ys, R1 and O54, other compounds are also isolated from fraction X of the extract of Xanthoseras sorbiviola, denoted X. The structure was measured. This compound is a triterpenoid saponin with an angeloyl group attached to C22 of the triterpene.

Compound-X

The present invention provides an active compound isolated from an extract of Xanthoseras sorbibolia and represented by "Compound X". Compound X is an oleanene triterpenoid saponin having a trisacarbide chain attached to C-3 of aglycone and one angeloyl group acylated at C-22. The chemical formula of compound X is C ₅₈ H ₉₂ O ₂₂ , and the chemical name of compound X is 3-O-{[β-D-galactopyranosyl (1 → 2)]-[α-L-arabinofuranosyl (1 → 3)]-β-D-glucuronopyranoside butyl ester} -21-O-acetyl-22-O-angeloyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12 -Yen. The chemical structure of compound X is shown below. See also FIG. 70.

(X)

(X)

The present invention provides a composition comprising a compound of the present invention for the treatment of nocturnal enuresis and frequent urination and for improving the action of the central nervous system by signaling to the bladder to wake up from deep sleep or to relax the bladder to store more urine. To provide. The compounds of the present invention can be used to relax urination sensations caused by aging, stress, neurosis, excessive activity, anxiety, hyper-reflexia, and uncontrolled bladder. In another embodiment, the compound can be used to relax the contracted bladder tissue induced by acetylcholine (Ach). The compound identified and isolated from the extracts of Xanthosera sorbobia can be used as an acetylcholinesterase, an AChE inhibitor to modulate antidiuretic hormone (ADH), which can reduce the amount of urine and can also be used as an anti-inflammatory have.

The compounds of the present invention are used to promote the growth of the bladder, to inhibit in deep sleep, to increase alertness to sleeping persons, to regulate the release, destruction and absorption of antidiuretic hormone (ADH) and its receptors. Acetylcholine (Ach) and its receptors to regulate the release, destruction and uptake of 5-hydroxytryptamine and its receptors, to regulate the secretion, destruction and uptake of the renocorticotropic hormone (ACTH) and its receptors. To regulate the release, destruction and absorption of adrenaline (AD) and its receptors, to regulate the release, destruction and absorption of dopamine (DA) and its receptors, to regulate the release, destruction and absorption of norepinephrine (NE) Regulating the formation, release, destruction and activity of neuropeptides and their receptors in order to control the release, destruction and uptake of the It can be used.

The present invention is for treating cancer; To inhibit the virus; To prevent brain aging; To improve memory and improve brain function; to treat nocturia, frequent urination, incontinence, dementia, Alzheimer's disease, autism, brain trauma, Parkinson's disease or other diseases caused by brain abnormalities; To treat arthritis, rheumatism, poor circulation, atherosclerosis, Raynaud's syndrome, angina, heart disease, coronary heart disease, headache, dizziness, kidney disease, cerebrovascular disease; To inhibit NF-kappa B activation; Brain edema, severe acute respiratory syndrome, respiratory viral disease, chronic venous insufficiency, hypertension, chronic venous disease, anti-laryngitis, anti-inflammatory, hemorrhoids, peripheral edema formation, varicose vein disease, flu, post-traumatic edema and postoperative swelling To treat; To inhibit ethanol absorption; To lower blood sugar; To regulate the amount of adorecorticotropin and corticosterone; The present invention also provides a composition comprising a compound of the present invention for treating erectile dysfunction or premature ejaculation or diabetes. United States Application No. 10 / 906,303, filed February 14, 2005, incorporated herein by reference, International Application No. PCT / US04 / 43465, filed December 23, 2004, filed October 8, 2004 See International Application No. PCT / US04 / 33359, and US Application No. 11/131551, filed May 17, 2005.

The present invention provides a composition capable of modulating the enzymatic activity of bladder cells. Xanthoceras sorbibolia derived compounds and / or compositions of the present invention may modulate various components or receptors of a cell and may enhance cell growth processes. Xanthoceras sorbibolia derived compounds and / or compositions modulate the activity of cellular enzymes. See US Application No. 60 / 675,284, filed April 27, 2005, which is incorporated herein by reference. The present invention provides a compound isolated from Xanthoceras sorbibolia, or a derivative or metabolite thereof capable of modulating the pathways involved in cell proliferation. US Application No. 10 / 906,303, filed February 14, 2005, International Application No. PCT / US04 / 43465, filed December 23, 2004, 8, 2004, the contents of which are incorporated herein by reference. See International Application No. PCT / US04 / 33359, filed one.

The present invention provides a method for the purification and isolation of triterpenoid saponins from plants and their use. The present invention provides a composition comprising a triterpenoid saponin or a derivative thereof that inhibits tumor growth. Compounds of the present invention include angeloyl or thigloyl groups or sensioli groups or combinations thereof attached to carbons 21 and 22 of these sapongenins. In one embodiment, the compound may comprise two angeloyl groups or thigloyl groups or sensioli groups or combinations thereof attached to sugar residues attached to carbon 21 or 22 of their sapogenin . This bioactive compound can be isolated from natural plants, including plants of the Safinaceae family having about 1400 to 2000 species in the genus 140-150. See US Application No. 60 / 675,282, filed April 27, 2005.

summary

The present invention provides a method for identifying and purifying bioactive compounds from plant extracts of Xanthoseras sorbibolia. Eight bioactive compounds have been identified and purified so far, and six of them have been found to have anticancer activity. Consensus sub-structures have been identified or derived from the structures of the bioactive compounds of the invention. Consensus substructures or active functional groups of bioactive compounds are biangeloyl groups present on adjacent carbons. Angeloyl groups are located at 21β and 22α of the triterpene backbone (ie, FIG. 5) or at C3 and C4 of the sugar ring (ie, FIG. 6). Thus, the non-angeloyl groups of these bioactive compounds are acylated at trans positions in adjacent carbons of the structure. See FIG. 7. Compounds having a single angeloyl group have weaker anticancer activity than compounds having two angeloyl groups. See FIGS. 65, 2, 3. Compounds with a single angeloyl group have less hemolytic activity than compounds with two angeloyl groups. See FIG. 73. The structures or derivatives of the compounds of the present invention can be obtained by chemical synthesis or from biological sources.

The invention will be better understood from the following examples. However, one of ordinary skill in the art will appreciate that the particular methods and results discussed are merely illustrative of the invention as they are more fully described in the following claims.

1 shows the structure of six bioactive anticancer saponin compounds of the invention.

2 shows the anticancer activity of purified Compound Y. This experiment was performed on ovarian cancer cells (OCAR-3) and inhibitory activity was measured by MTT assay. See Experiment 3 for details. Abscissa: concentration (μg / ml). Ordinate:% cell growth. IC50 is about 1-1.5 μg / ml. A: Point scale. B: linear scale.

3 shows the inhibitory effect of purified compound Y1 and compound Y2 on ovarian cancer cell growth.

4 shows the anticancer activity of Y, Y8, Y9 and Y10 against ovarian cancer cells as measured by MTT assay.

5 shows consensus structures derived from the four bioactive anticancer saponin compounds of the invention (ie, Y, Y2, Y8 and Y10).

6 shows consensus structures derived from two bioactive anticancer saponin compounds of the invention (ie, Y1 and Y9).

FIG. 7 shows the general formula derived from the consensus structure of the six bioactive compounds of the invention (ie, Y, Y1, Y2, Y8, Y9 and Y10). (A) The consensus active functional group is a biangeloyl group attached to 21β and 22α of the triterpene backbone. (B) The consensus active functional group is a biangeloyl group attached to C3 and C4 of the sugar ring (or rhamnose). In one embodiment, the functionally active structure is a biangeloyl group located in the trans position on the structure.

FIG. 8 shows separation of components of Xanthoceras sorbifolia by HPLC using μbondapak C18 column. Details of this experiment are presented in Experiment 2.

Figure 9 shows the elution characteristics of the extract of Xanthoceras sorbifolia in FPLC using a 10-80% gradient. Ordinate: optical density (at 245 nm). Abscissa: fraction (5 ml / fraction).

10 shows the results of screening cell growth activity using fractions obtained from FPLC chromatography. The assay was performed on bladder cells. Fractions obtained from FPLC were used as shown in FIG. 9. As shown in this figure, different components or xanthoceras sorbibolia extracts induce growth or inhibitory effects in cells. Only fraction 5962 (fraction Y) causes cell inhibition. Fractions 610, 1116, and 1724 cause a slight cell growth simulation. Abscissa: concentration (μg / ml). Ordinate:% cell growth (as measured by MTT assay).

FIG. 11 shows the HPLC characteristics of Fraction Y by 45% acetonitrile cosolvent elution on preparative C18 column (Delta Pack C18). Under these conditions, the fractions Y (Y3), Y1 and Y2 are each well separated from each other and they are purified. A and B show the purity of Y3 and Y2 collected by HPLC under the same conditions.

FIG. 12 shows the separation characteristics of Y8, Y9 and Y10 by 45% acetonitrile cosolvent elution in preparative C18 column (delta pack C18).

13 shows the HPLC characteristics of purified Y8, Y9 and Y10.

FIG. 14 shows the growth curves of ovarian cancer cells after treatment with crude extracts of Xanthoceras sorbobia as measured by MTT assay. The two curves in the figure are the results of two experiments. This study measured the efficacy of xanthoceras sorbibolia extract on cancer cells. In these experiments, cancer cell lines from 11 different human organs were used. This figure shows that ovarian cancer cells are the most sensitive cancer cells with respect to the xanthoceras sorbibolia extract of the present invention. The results of the other cancer cells are shown in Figures 16A, 16B and 16C. Abscissa: concentration (μg / ml). Ordinate:% cell growth (as measured by MTT assay).

15 shows a comparison of the efficacy of Compound Y in ovarian cancer cells and cervical cancer cells. Ovarian cancer cells are much more sensitive than cervical cancer cells. The IC 50 for Compound Y in ovarian cells is about 1.5 μg / ml, whereas in cervical cancer cells the IC 50 is greater than 20 μg / ml.

FIG. 16A depicts the growth curves of susceptible groups of bladder and bone cancer cells as measured by MTT assay. The curve in this figure is a result of repeated experiments. Abscissa: concentration (μg / ml). Ordinate:% cell growth (as measured by MTT assay).

FIG. 16B depicts the growth curve of semi-sensitive groups of leukocyte cancer, liver cancer, prostate cancer, breast cancer and brain cancer cells as measured by MTT assay. The curve in this figure is a result of repeated experiments. Abscissa: concentration (μg / ml). Ordinate:% cell growth (as measured by MTT assay).

FIG. 16C shows the growth curves of the least susceptible group of colorectal, cervical and lung cancer cells as measured by MTT assay. The curve in this figure is a result of repeated experiments. Abscissa: concentration (μg / ml). Ordinate:% cell growth (as measured by MTT assay).

17 is a chemical formula C ₅₇ H ₈₈ O ₂₃ and has a chemical name 3-O- [β-D-galactopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3) -β- The structure of compound Y, which is D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene, is shown.

18 shows the spectrum of proton NMR of compound Y. FIG.

FIG. 19 shows 2D NMR (HMQC) results of Compound Y.

FIG. 20 shows 2D NMR (HMBC) results of Compound Y.

FIG. 21 shows the mass spectrum of Compound Y by MALDI-TOF (high mass): Y + matrix (CHCA) + angiotensin 1 "2-point calibration".

22 shows the mass spectrum of Compound Y by ESI-MS.

23 is a chemical formula C ₆₅ H ₁₀₀ O ₂₇ and a chemical name 3-O- [β-D-galactopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3) -β- D-glucuronopyranosyl-21-O- (3,4-diangeloyl) -α-L-ramnopyranosyl-22-O-acetyl-3β, 16α, 21β, 22α, 28-pentahydride The structure of the compound Y1 which is a roxyolean-12-ene is shown.

24 shows the spectrum of proton NMR of compound Y1.

25 shows the 2D NMR (HMQC) results of compound Y1.

FIG. 26 shows 2D NMR (HMBC) results of compound Y1.

27 depicts COSY-NMR characteristics of compound Y1.

28 shows the chemical structures and chemical names of compound Y2.

29 shows the spectrum of proton NMR of compound Y2.

30 shows 2D NMR spectra of Y 2 (HMQC) -level-1.

FIG. 31 shows the C13 NMR spectrum of Compound Y2.

32 shows the 2D NMR (HMBC) -level-1 spectrum of compound Y2.

FIG. 33 shows the 2D NMR HOHAHA (TOCSY) -Level-1 spectrum of Compound Y2.

34 shows the mass spectra of Compound Y2 + Matrix + Standard.

35 shows the chemical structure of Y8.

36 shows the H-NMR spectrum of Y8.

37 shows the C13-NMR spectra of Y8.

38 shows the 2D NMR HMQC (Level 1) spectrum of Y8.

39 shows the chemical structure of Y9.

40 shows the H-NMR spectrum of Y9.

FIG. 41 shows 2D NMR HMQC (Level 1) spectrum of Y9.

FIG. 42 shows 2D NMR HMBC (Level 1) spectrum of Y9.

43 shows the chemical structure of Y10.

44 shows the H-NMR spectrum of Y10.

45 shows the C13-NMR spectra of Y10.

FIG. 46 shows the 2D NMR HMQC (Level 1) spectrum of Y10.

FIG. 47 shows the chemical structure and chemical name of compound R1.

FIG. 48 shows proton-NMR spectrum of Compound R1. FIG.

FIG. 49 shows 2D NMR (HMQC) spectra of Compound R1.

FIG. 50 shows the 2D NMR (HMBC) spectrum of Compound R1.

FIG. 51 shows the 2D NMR (COSY) spectrum of Compound R1.

FIG. 52 shows the C13 NMR spectrum of Compound R1.

53 shows the chemical structure of compound O54.

54 shows the proton-NMR spectrum of compound O54.

FIG. 55 shows the 2D NMR (HMQC) spectrum of Compound O54.

FIG. 56 shows the 2D NMR (HMBC) spectrum of Compound 054.

Figure 57 shows the absorption spectrum of Xanthoceras sorbobia extract of the present invention. Abscissa: wavelength nm. Ordinate: optical density. The extract has three maximum absorptions at 207 nm, 278 nm and 500 nm.

58 shows the proton NMR spectrum of Y4.

59 shows 2D NMR (HMQC) spectra of Y4.

60 shows the purification of Component-R by HPLC. A: The extract from fraction # 10 of FPLC (iso-30) was isolated by HPLC. B: Rechromatogram of the main components under the same conditions as in A.

Figure 61 Fractionation of Fraction-O by HPLC with 20% acetonitrile cosolvent elution (iso-20).

62 shows the rechromatography of O54, O28 and O34 (from iso-20).

63A shows R 1 is an angeloyl group; R 2 is an angeloyl group; R 3 is OH or H; R 4 is H or OH or CH 3 or CH 2 OR 6 or COOR 6; R6 = H or acetyl or sugar residues; Positions 23, 24, 25, 26, 27, 29, 30 of the compound are CH3 or CH2OH or CHO or COOH or an alkyl group or an acetyl group or derivatives thereof; R6 is Ac or H and R5 is a sugar moiety wherein the sugar moiety is one or more sugars, or D-glucose, or D-galactose, or L-rhamnose, or L-arabinose, or D-xyllo The chemical structure of a compound comprising oss, or alduronic acid, or D-glucuronic acid, or D-galacturonic acid, or derivatives thereof, or combinations thereof is shown. In one embodiment, R 5 represents a compound capable of performing sugar residues.

63B shows R 1 is an angeloyl group; R 2 is an angeloyl group; R 3 is Ac or H; R 4 is H or OH; R 6 is Ac or H; R 7 is H or OH or CH 3 or CH 2 OR 6 or COOR 6, wherein R 6 = H or acetyl or sugar residues; Positions 23, 24, 25, 26, 27, 29, 30 of the compound are CH3 or CH2OH or CHO or COOH or an alkyl group or an acetyl group or a derivative thereof and R5 is a sugar moiety wherein the sugar residue is at least one sugar Or D-glucose, or D-galactose, or L-rhamnose, or L-arabinose, or D-xylose, or alduronic acid, or D-glucuronic acid, or D-galacturonic acid, or The chemical structures of the compounds, including their derivatives or combinations thereof, are shown. In one embodiment, R 5 represents a compound capable of performing sugar residues.

64 shows the HPLC characteristics of FPLC (iso-45) fraction # 5657.

Figure 65 depicts the effect of Compound X on the growth of OCAR-3 cells.

66 depicts H-MNR of compound X.

FIG. 67 shows 2D NMR (HMQC) of Compound X.

FIG. 68 shows 2D NMR (HMBC) of Compound X.

69 depicts C13-NMR of compound X.

FIG. 70 shows the chemical structure of Compound X.

71 shows Spectrum 1 (WALDI-TOF) of compound X. FIG.

72 shows Spectrum 2 (WALDI-TOF) of compound X. FIG.

FIG. 73 shows the contrast of MTT and hemolytic activity of saponin compounds and compounds Ys of the present invention. FIG. A and B show hemolytic activity. C and D show MTT activity.

74A shows a compound of the present invention having no angeloyl group, and B shows a compound of the present invention having no sugar residue.

75 depicts saponin compounds of the invention.

Experiment Details

Experiment 1: Herb Extraction

(a) Xanthocera sorbobia outer husks or branches or stems or leaves or whole grains or roots or bark powder were extracted 4-5 times in a 1: 2 ratio using an organic solvent for 4-5 times for 20-35 hours. To form an extract; (b) collecting the organic extracts; (c) refluxing the organic extract 2-3 times at 80 ° C. to form a second extract; (d) removing the organic solvent from the second extract; In addition, (e) the second extract is dried and sterilized to form Xanthoceras sorbibolia extract powder.

Experiment 2: HPLC  By chromatography Xanthosras Sorbipolia  Analysis of the extract

Way

HPLC. C-18 reversed phase μbondapak column (Water P / N 27324) is equilibrated with 10% acetonitrile, 0.005% trifluoroacetic acid (equilibration solution). Extracts of xanthoseras sorbobia prepared using the method described in Experiment 1 were dissolved in equilibrium solution (1 mg / ml) prior to application to the column. 20 μg of sample was applied to the column. Elution conditions: Fractions were eluted (flow rate 0.5 ml / min) in 70 minutes using an acetonitrile gradient of 10% to 80% and then held at 80% for 10 minutes. The acetonitrile concentration was then reduced to 10% and maintained at 10% for 25 minutes. These fractions were monitored at 207 nm and recorded using a chart rate of 0.25 cm / min and OD full scale 0.128.

Instrument. Waters Model 510 Solvent Delivery System; Waters 484 tunable gmqtn detector (Absorbance Detector); Waters 745 / 745B Data Module.

Absorption analysis. Absorption characteristics of Xanthoceras sorbibolia extract were measured at various wavelengths. The extract of xanthoseras sorbobia of the present invention was dissolved in 10% acetonitrile / TFA and spectrophotometer at 200-700 nm [Spectronic Ins. Scanning using Model Gene Sys2].

result

HPLC. About 60-70 peaks can be counted for each profile. Four of these are the main peaks, ten are medium in size and the rest are small fractions. These peaks are labeled az with increasing concentrations of acetonitrile elution. See FIG. 8.

Absorption. Three maximal uptakes were identified for the Xanthorasas sorbibolia plant extract; 207 nm, 278 nm and 500 nm. See FIG. 57.

Experiment 3: MTT  Essays for Cancer Cells Derived from Different Human Organs Xanthosras Sorbipolia  Measurement of Cell Growth Activity by Extracts

Method and material

cell. Human cancer cell lines were obtained from the American type culture collection: HTB-9 (bladder cancer), HeLa-S3 (cervical cancer), DU145 (prostate cancer), H460 (lung cancer), MCF-7 (breast cancer), K562 (leukocyte cancer), HCT116 (Colon cancer), HepG2 (liver cancer), U2OS (bone cancer), T98G (brain cancer) and OVCAR-3 (ovarian cancer). 5% CO2 wetted incubator, cell medium supplemented with 10% fetal calf serum, glutamine and antibiotics at 37 ° C. (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in Eagle) (RPI); RPMI Cells were grown in HTB-9, H460, K562, OVCAR-3 in H-1640; HCT-116 in McCoy-5A, U2OS).

MTT Essay. The MTT assay process was carried out with a slight modification of the method described in 1987, Carmichael et al. These cells were 10,000 / well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000 / well (DU145, MCF-7, HepG2, U2OS) or 40,000 / in 96-well plates prior to drug treatment. Sowing was carried out at a concentration of wells (K562) for 24 hours. Cells were then exposed to drug for 48 hours (72 hours for HepG2, U2OS, 96 hours for MCF-7). After drug treatment, MTT (0.5 mg / ml) was added to the culture for 1 hour. Formazan (reducing product of tetrazolium by viable cells) was lysed by DMSO and O.D. at 490 nm was obtained by ELISA reader [Dynatech. Model MR700]. MTT amounts of cells prior to drug treatment were also measured (T0). % Cell growth is calculated as follows:

(1)

(One)

In the formula, TC or TD is the O.D. of control or drug treated cells. Indicates reading. If TO> TD, cytotoxicity (LC), expressed as% of control, is calculated as follows:

(2)

(2)

result

In eleven cell line studies, cell growth inhibition was observed after exposure of plant extracts. However, their susceptibility to xanthoceras sorbibolia extracts was different. The sensitivity of the cell line to xanthoseras extract can be distinguished into four groups: highest sensitivity, ie ovary; Sensitivity, ie bladder, bone; Semi-sensitive, ie prostate, leukocytes, liver, breast, and brain; And lowest sensitivity, ie large intestine, cervical, and lung. See FIGS. 14, 15 and 16 A-D. Their IC50 values are listed in Table 3.1 above.

In addition to inhibition of cell growth, Xanthoceras sorbiviola plant extract also stimulates trace cell growth at low concentrations in bladder, bone and lung cells. The results indicate that there is a cell or tissue stimulating component present in the extract. See FIGS. 16A and 16D.

In order to investigate the inhibitory components of the Xanthoceras sorbibolia plant extract, the plant extracts were fractionated. 10 shows the results of the screening of fractions obtained after FPLC chromatography to measure cell growth inhibitory activity. This assay was carried out with bladder cells. Fractions obtained from FPLC as shown in FIG. 9 were used. As shown in FIG. 9, different components of xanthosera sorbobia extract induced growth or inhibitory effects on cells. Only fraction 5962, represented by fraction Y, caused cell growth inhibition. Abscissa: concentration (μg / ml). Ordinate:% cell growth (as measured by MTT assay).

Experiment 4: Xanthosras Sorbipolia  Purification of Inhibitory Components from Extracts

(A) FPLC Fraction of plant extracts by

Way

column. Octadecyl functionalized silica gel. Column size: 2 cm x 28 cm; Equilibrated with 10% acetonitrile-0.005% TFA before use.

Sample loading: 1-2 ml, concentration: 100 mg / ml in 10% acetonitrile / TFA.

Gradient elution conditions: 10-80% acetonitrile in 500 ml total volume.

Absorption Wavelength Monitor: 254 nm.

Fraction collector: 5 ml / fraction (collected from 10% to 72% acetonitrile)

Apparatus: AKTA-FPLC, P920 Pump; Monitor UPC-900; Frac-900.

result

Elution characteristics of the chromatography show 4-5 broad fractions. See FIG. 9. These fractions were analyzed by HPLC. Specific components corresponding to a to z are shown in these FPLC fractions as shown in FIG. 8. FPLC fractions were divided into 7 pools and analyzed for cell growth activity in bladder cells by MTT assay. See Experiment 3. It was found that only pool # 5962, corresponding to fraction Y in HPLC, contained inhibitory activity. See FIG. 10. In later experiments it was found that fractions above 62 show inhibitory activity. The component isolated from fractions 63-65 showed inhibitory activity. See FIGS. 4, 12 and 13.

(B) for manufacturing HPLC Ingredients by Ys Isolation

Way

Column: preparative HPLC column (Waters Delta Pak C18-300A);

Elution conditions: Elution of 45% acetonitrile cosolvent at flow rate 1 ml / min

Fractions were monitored at 207 nm, collected and lyophilized.

result

Final separation of the Y fraction was achieved by HPLC using a preparative column. See FIGS. 11 and 12. These fractions containing compounds Y1, Y2, Y or Y3 and Y4 were collected. Rechromatography of compound Y showed a single peak in HPLC using a C18 reversed phase column. See FIGS. 11A and 11B. Rechromatography of compounds Y8, Y9 and Y10 showed a single peak in HPLC using a C18 reversed phase column. See FIG. 13.

(C) appearance and solubility

Pure compound Ys is an amorphous white powder soluble in aqueous alcohol, ie methanol or ethanol, 50% acetonitrile and 100% pyridine.

(D) MTT  Compound by Essay Ys Inhibition Analysis of

 Inhibition assay of Compound Y was determined by MTT assay. FIG. 2 shows that compound Y has activity against ovarian cancer cells (OCAR-3) due to IC 50 value of 1.5 μg / ml 10-15 times stronger than the crude extract shown in FIG. 14. 15 shows the selectivity of Compound Y for ovarian cancer cells compared to cervical cancer cells (HeLa). 3 shows the inhibitory activity of compounds Y1 and Y2 on the growth of ovarian cancer cells (OCAR-3). 4 shows the inhibitory activity of compounds Y, Y8, Y9 and Y10 on the growth of ovarian cancer cells (OCAR-3).

Experiment 5: Measurement of Chemical Structure

Way

NMR analysis. Pure Compound Y of Xanthoceras sorbiviola was dissolved in pyridine-D5 with 0.05% w / w TMS. All NMR spectra were obtained using a Bruker Avance 600 MHz NMR spectrometer at 298K using a QXI probe (1H / 13C / 15N / 31P). The scan count for 1D 1H is 16 to 128 depending on the sample concentration. 2D HMQC spectra were recorded with spectral width 6000 × 24,000 Hz and data points 2024 × 256 for t2 and t1 dimensions. The number of scans was 4 to 128. 2D HMQC spectra were recorded with a spectral width of 6000 x 30,000 Hz and data points 2024 x 512 for t2 and t1 dimensions. The number of scans was 64. 2D data were zero-filled at t1 dimensions to double the data points, multiplied by the cosine-square-bell window function at t1 and t2 dimensions, and then fore-transformed using software XWIN-NMR. The final actual matrix size of these 2D spectra is 2048 x 256 and 2048 x 512 data points (F2xF1) for HMQC and HMBC.

Mass spectral analysis. The mass of the sample was analyzed by (A) MALDI-TOF mass spectrometer and (B) ESI-MS mass spectrometer. (A) A sample for MALDI-TOF was dissolved in acetonitrile followed by matrix CHCA, alpha-cyano-4-hydroxycinnamic acid, 10 mg CHCA / mL in 50:50 water / acetonitrile and 0.1 in final concentration. Mixed with% TFA. The molecular weight was determined by high resolution mass spectrometry according to the standard. (B) For ESI, analysis was performed with an LCQ DECA XP Plus machine manufactured by Thermo Finnigan. It is ionized by the ESI source and the solvent for the compound is acetonitrile.

result

The characteristics of proton NMR are shown in FIG. 18. 2D NMR characteristics of HMQC and HMBC are shown in FIGS. 19 and 20, respectively. Table 5.1 summarizes the 2D NMR chemical shift data and functional group assignments derived from these data. Based on these data and the analysis, the structure of compound Y (Y3) is shown below.

Structure of Compound Y (see FIG. 17)

The chemical name of compound Y is 3-O- [β-D-galactopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3) -β-D-glucuronopyranosyl- 21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene. The mass spectrum of compound Y as measured by MALDI-TOF and ESI-MS (ie, see FIGS. 21, 22) indicates that the mass of compound Y is 1140.57, which is consistent with the theoretical mass of compound Y.

conclusion

Active compound Y, isolated from extracts of Xanthosera sorbobia, is an oleane triter having a trisaccharide chain attached to C-3 of aglycone and two angelloyl groups acylated at C-21 and C-22 Phenoidal saponins. The chemical formula of Y is C ₅₇ H ₈₈ O ₂₃ and the chemical name of compound Y is 3-O- [β-D-galactopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3)- β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene.

Experiment 6: Xanthosras Sorbipolia  Compound of extract Y1 Of chemical structure of

Way

NMR and MS analytical methods for compound Y1 are similar to those described in Experiment 5.

result

The spectrum of H-NMR is shown in FIG. 24. 2D NMR spectra of HMQC, HMBC and COSY are shown in FIGS. 25, 26 and 27, respectively. Table 6.1 summarizes chemical shift data and functional group assignments derived from these data. Based on these data and analysis, the structure of compound Y1 is designated as follows and shown below.

Y1 Structure (see Figure 23)

The chemical name of Y1 is 3-O- [β-D-galactopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3) -β-D-glucuronopyranosyl-21 -O- (3,4-diangeloyl) -α-L-lamnopyranosyl-22-O-acetyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene.

conclusion

Compound Y1 isolated from xanthoseras sorbobia extract has a trisaccharide chain at C-3 of the main chain and a monosaccharide residue at C-21, with two angeloyl groups at the C-3 and C-4 positions Acylated bisdesmosidic polyhydroxyoleanene triterpenoid saponins. The chemical formula of Y 1 is C ₆₅ H ₁₀₀ O ₂₇ .

Experiment 7: Xanthosras Sorbipolia  Compound of extract Y2 Of chemical structure of

Way

NMR and MS assays for Compound Y2 are similar to those described in Experiment 5.

result

1D and 2D NMR spectra of H-NMR, C-13 NMR, HMQC, HMBC and (TOCSY) and MS (MALDI-TOF) of Y2 are shown in FIGS. 29-34. Table 7.1 summarizes the 1D and 2D NMR chemical shift data and the assignment of functional groups derived from these data.

conclusion

Based on these data and analyzes, compound Y2 isolated from xanthoseras sorbobia extract is a trisaccharide chain in C-3 of the main chain and two angeloyl groups acylated in C-21 and C-22. Is an oleanene triterpenoid saponin. The chemical structure of Y2 is shown below. See FIG. 28.

The chemical formula of Y2 is C ₅₇ H ₈₈ O ₂₄ and the chemical name of compound Y2 is 3-O- [β-D-glucopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3)- β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 24β, 28-heptahydroxyolean-12-ene.

Experiment 7B: Y4 Chemical structure analysis

Y4  Analysis

The proton NMR characteristic of Y4 is shown in FIG. The characteristic of 2D NMR (HMQC) of Y4 is shown in FIG.

Experiment 8: Xanthosras Sorbipolia  Inhibitory Ingredient in Extracts Y8 - Y10 Tablets

(A) FPLC  By way Xanthosras Sorbipolia  Classification of Extract Ingredients

Way

The method of this experiment is similar to the method described in Experiment 4 parts (A) and (B).

result

Elution characteristics represent 4-5 broad fractions. See FIG. 9. These fractions were analyzed by HPLC. FPLC fractions 63, 64 and 65 were further separated in 45% cosolvent analysis and 4-5 major components were separated. See FIG. 12. These fractions are referred to as Y8, Y9 and Y10. These fractions were collected. Rechromatography of compounds Y8, Y9 and Y10 showed a single peak in HPLC using a C18 reversed phase column. See FIG. 13.

(B) MTT  Inhibition analysis by essay

Inhibition assay of the purified compounds was determined by MTT assay. The results show that compounds Y8, Y9 and Y10 are active against ovarian cancer cells (OCAR-3) by IC50 values of 3, 4 and 1.5 μg / ml. See FIG. 4.

Experiment 9. Xanthosras Sorbipolia  Compound of extract Y8 Of chemical structure of

Way

NMR and MS analytical methods for compound Y8 are similar to those described in Experiment 5.

result

The spectral characteristics of H-NMR, C-13 NMR and 2D NMR (HMQC) of compound Y8 are shown in FIGS. 36 to 38. Table 9.1 summarizes the 1D and 2D NMR chemical shift data and the assignment of functional groups derived from these data. Based on these data and analyzes, compound Y8 isolated from xanthoseras sorbobia extract was converted into trisaccharide chain attached to aglycone C-3 and two angelylated acyls at C-21 and C-22. Oleanene triterpenoid saponins having one group. The chemical formula of compound Y8 is C ₅₇ H ₈₇ O ₂₃ and the chemical name of Y8 is 3-O- [β-D-glucopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β Glucuronopyranosyl-21,22-O-diangeloyl-3β, 16α, 21β, 22α, 24β, 28-hexahydroxyolean-12-ene. The chemical structure of compound Y8 is shown below. See FIG. 35.

Experiment 10. Xanthosras Sorbipolia  Compound of extract Y9 Of chemical structure of

Way

NMR and MS assays for Compound Y9 are similar to those described in Experiment 5.

result

The spectral characteristics of H-NMR and 2D NMR of Y9, ie HMQC and HMBC, are shown in FIGS. 40-42. Table 10.1 summarizes the 1D and 2D NMR chemical shift data and the assignment of functional groups derived from these data. Based on these data and analyzes, compound Y9 isolated from xanthoseras sorbobia extract has a trisaccharide chain at C-3 of the main chain and a monosaccharide residue at C-21 and two angeloyl groups are present. Bisdesmocidic polyhydroxyoleanene triterpenoid saponins which are acylated at the -3 and C-4 positions. The chemical formula of compound Y9 is C ₆₅ H ₁₀₀ O ₂₇ and the chemical name of Y9 is 3-O- [β-galactopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-glu Curonopyranosyl-21-O- (3,4-diangeloyl) -α-ramnopyranosyl-28-O-acetyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12 -Yen. The chemical structure of compound Y9 is shown below. See FIG. 39.

Experiment 11. Xanthosras Sorbipolia  Compound of extract Y10 Of chemical structure of

Way

NMR and MS assays for Compound Y10 are similar to those described in Experiment 5.

result

The spectral characteristics of H-NMR, C13-NMR and 2D NMR (HMQC) of Y10 are shown in FIGS. 44 to 46. Table 11.1 summarizes the 1D and 2D NMR chemical shift data and the assignment of functional groups derived from these data. Based on these data and analyzes, compound Y10 isolated from xanthoseras sorbobia extract is a trisaccharide chain attached to C-3 of aglycone and two acylated at positions C-21 and C-22. Triterpenoid saponins with angeloyl groups. The chemical formula of compound Y10 is C ₅₇ H ₈₇ O ₂₂ and the chemical name of Y10 is 3-O- [β-galactopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-glu Kuronopyranosyl-21,22-O-diangeloyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene. The chemical structure of compound Y10 is shown below. See FIG. 43.

Experiment 12. Xanthosras Sorbipolia  Purification of Component R from Extracts

(A) FPLC  And HPLC On by Xanthosras Sorbipolia  Purification of Extract Ingredients

Way

The method used is similar to the method described in Experiment 4, (A) and (B), except that 30% acetonitrile cosolvent elution was used in HPLC for the isolation of Compound R.

result

Fractions 39-41 from the gradient elution of FPLC were collected and further purified on an open ODS-C18 column using a cosolvent 30% acetonitrile elution. Six identifiable fractions from two groups were collected. Fractions 6-13 were characterized by HPLC. These fractions were further separated into 4-5 components by 30% acetonitrile cosolvent elution on a DeltaPak column. The fraction designated as "R1" is the main component. See FIG. 60A. Further pure R1 was collected from the column elution. See FIG. 60B.

(B) Appearance and Solubility

Pure R1 appears as an amorphous white powder soluble in aqueous alcohol, ie methanol or ethanol, 50% acetonitrile and 100% pyridine.

(C) R1 Of chemical structure of

Way

NMR and MS analysis of R1 is similar to the method described in Experiment 5.

result

NMR spectra of pure R1 are shown in FIGS. 48-52. Based on chemical shift analysis, Compound R1 isolated from xanthoseras sorbobia extract is a triterpenoid saponin having five sugars and one angeloyl group attached to this sugar residue. The chemical structure of R1 is shown below. See FIG. 47.

The chemical formula of compound R1 is C ₆₅ H ₁₀₆ O ₂₉ and the chemical name of R1 is 3-O- [angeloyl- (1 → 3) -β-D-glucopyranosyl- (1 → 6)]-β-D -Glucopyranosyl-28-O- [α-L-rhamnopyranosyl- (1 → 2) -β-D-glucopyranosyl- (1 → 6) -β-D-glucopyranosyl-3β, 21β, 22α, 28-tetrahydroxyolean-12-ene.

Experiment 13. Xanthosras Sorbipolia  Purification of Component O from Extract

(A) FPLC  And HPLC On by Xanthosras Sorbipolia  Classification of Extract Ingredients

Way

The method used is similar to the method described in Experiment 4, (A) and (B), except that 20% acetonitrile cosolvent elution was used in HPLC for the isolation of Compound O.

result

Fractions obtained from FPLC were analyzed by HPLC. Specific component, fraction O, was identified by comparison with the characteristics of the original sample (# 28-30). Fraction O was collected and further purified. Sixteen identifiable HPLC fractions were observed in the elution characteristics. See FIG. 61. Fractions 28, 34 and 54 were further purified. See FIG. 62. These purified components are named compounds O28, O34 and O54, respectively.

(B) Appearance and Solubility

Purified compounds O23 and O34 are pale yellow amorphous powders soluble in aqueous alcohols, ie methanol or ethanol, 50% acetonitrile and 100% pyridine. Purified compound O54 is a white amorphous powder soluble in aqueous alcohol, ie methanol or ethanol, 50% acetonitrile and 100% pyridine.

(C) compound O54 Structural analysis

Way

NMR and MS analysis of O54 is similar to the method described in Experiment 5.

result

NMR spectra of compound O54 are shown in FIGS. 54-56. Based on the chemical shift analysis, Compound O54 isolated from Xanthoceras sorbibolia extract is a disaccharide chain [β-D-glucopyranosyl- (1 → 6) -β-D- attached to C-3. Glucopyranoside] and trisaccharide chain attached to C-28 [α-L-rhamnopyranosyl- (1 → 2) -β-D-glucopyranosyl- (1 → 6) -β-D-glucose Pyranosyl ester] bisdesmosidic polyhydroxyoleanene triterpenoid glycosides. The chemical structure of compound O54 is shown below. See FIG. 53.

The chemical formula of compound O54 is C ₆₀ H ₁₀₀ O ₂₈ and the chemical name of O54 is 3-O- [β-D-glucopyranosyl- (1 → 6)]-β-D-glucopyranosyl-28-O- [ α-L-lamnopyranosyl- (1 → 2) -β-D-glucopyranosyl- (1 → 6) -β-D-glucopyranosyl-3β, 21β, 22α, 28-tetrahydroxyolean- 12-yen.

Experiment 14. Xanthosras Sorbipolia  Purification of Component-X from the Extract

(A) FPLC  And HPLC On by Xanthosras Sorbipolia  Classification of Extract Ingredients

Way

The method used is similar to the method described in Experiment 4, (A) and (B), except that the collected FPLC fractions 56 and 57 containing Compound X were further separated by preparative HPLC.

result

Five major and seven minor peaks were observed in the chromatogram. Compound X eluted near the middle of the elution characteristics (FIG. 64). Compound X was collected and lyophilized.

(B) Appearance and Solubility

Purified compound X is a white powder and soluble in 50% acetonitrile.

(C) MTT   Inhibition Assay of Compound X by Essay

MTT analysis of Compound X in OVCAR-3 cells shows some inhibitory activity. The IC 50 of compound X is about 27 μg / ml. See FIG. 65.

Experiment 15. Xanthosras Sorbipolia  Determination of Chemical Structure of Compound X from Extract

Way

NMR and MS assays for Compound X are similar to those described in Experiment 5.

result

Proton and C13 NMR characteristics are shown in FIGS. 66 and 69, respectively. 2D NMR characteristics of HMQC and HMBC are shown in FIGS. 67 and 68. Table 15.1 summarizes the 2D NMR chemical shift data and the assignment of functional groups derived from these data. Based on the data and analysis, the structure of compound X is designated as follows. Mass spectrum of compound X (see FIGS. 71 and 72) measured by MALDI-TOF indicates that the mass of compound Y is 1140.60 consistent with the theoretical mass of compound X.

conclusion

Active compound X isolated from xanthoseras sorbibolia extract is an oleoene triterpenoid with trisaccharide chain attached to C-3 of aglycone and one angeloyl group acylated at position C-22 Saponin. The chemical formula of compound X is C ₅₈ H ₉₂ O ₂₂ and the chemical name of compound X is 3-O-{[β-D-galactopyranosyl (1 → 2)]-[α-L-arabinofuranosyl (1 → 3) -β-D-glucuronopyranoside butyl ester} -21-O-acetyl-22-O-angeloyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12- Yen. The chemical structure of compound X is shown below. See FIG. 70.

Experiment 16. Xanthosras Sorbipolia  Determination of Hemolytic Activity of Compound Y

Way:

Human whole blood was obtained from the Houston Gulf Coast Blood Center.

Erythrocytes were isolated by the following method: Human blood (in EDTA) was diluted 1: 1 using PBS, spread under 4 ml of Histopaque-1077 (Sigma) and centrifuged at 400 g for 30 minutes.

Red blood cells (RBCs) were collected and washed three times with PBS.

10% RBC suspension was prepared using PBS before use.

50 μl of the RBC suspension was added to 2 ml of saponin at different concentrations.

The suspension was mixed by vortex treatment and left at room temperature for 60 minutes.

The suspension was centrifuged for 5 minutes at 3000 rpm. The absorption of the supernatant was measured at 540 nm.

result:

In this experiment, hemolytic activity of human erythrocyte cells by Xanifolia-Y (# 63Y), Essin and Sigma saponin standards was compared. Y contains two angeloyl groups, esin has one angeloyl group and the sigma saponin standard is a saponin mixture obtained from the Quilaia bark. The results show that # 63Y (Compound Y) has higher hemolytic activity (IC50 = 1 μg / ml) compared to the Essin or Sigma saponin standard (IC50 = 5 μg / ml). See FIG. 73A.

Experiment 17. By Alkali or Acid Hydrolysis Angeloyl  Or per Residues  Hemolytic activity of Compound Y after removal and MTT  Measurement of activity

Way:

(A) Alkali hydrolysis of xanipolya-Y. 20 mg of xanifolia-Y was dissolved in 0.5 ml of 1M NaOH. This solution was incubated for 4 hours in a water bath at 80 ℃. Cool to room temperature before neutralization with 0.5 ml 1N HCl (adjust pH to about 3). This mixture was extracted three times with 2 ml 1-butanol. Butanol fractions were collected and lyophilized. Hydrolyzed saponins were further purified by HPLC with a C-18 column eluted with 25% acetonitrile. (B) Acid hydrolysis of xanipolya-Y. 15 mg of xanifolia-Y was dissolved in 1 ml of methanol. 1 ml of 2N HCl was added. The mixture was refluxed for 5 hours in an 80 DEG C water bath. The solution was neutralized by adding 2 ml of 1N NaOH (final pH 3-4). Aglycone was extracted using 3 ml × 3 of ethyl acetate. Extracts were collected and collected. Further isolation of aglycone (sugar-depleted xanipolya-Y) was achieved by HPLC by cosolvent elution of 80% acetonitrile.

result:

Angeloyl groups or sugar residues of compound Y were removed by alkali or acid hydrolysis. The elution activity of the hydrolyzed product was analyzed. The results of these studies indicate that elimination of glucose reduces hemolytic activity, but elimination of angeloyl groups does not eliminate hemolytic activity. This suggests that sugar is useful but not essential for hemolytic activity. See FIG. 73B. The results show that Compound-Y loses MTT activity when the angeloyl group is removed. However, MTT activity was very weak when the sugar moiety of the compound was removed. See FIGS. 73C, 73D. A comparison of hemolytic activity between saponin standards of Compound Y, Essin and Sigma is shown in FIGS. 73A and 73B. A comparison of hemolytic activity between Compound Y, Compound Y without a sugar residue or Angeloyl group is shown in FIG. 73B. The chemical structures of Compound Y without sugar residues or angeloyl groups are shown in FIGS. 74A and 74B, respectively.

Experiment 18.Compound Y Anti-virus  Measurement of activity

The main procedure for measuring the antiviral activity of Compound Y is as follows:

A. HIV virus production is measured after a non-lethal dose of Compound Y is added to the virus culture system.

B. Growth of the HIV virus is measured after contact with compound Y. The steps for these experiments are as follows:

1. Pretreat HIV virus with different doses of Compound Y for various times.

2. Remove compound Y from the virus.

3. Mix the treated virus with the cells.

4. Measure virus production.

5. Negative Control: No virus in the cells.

6. Positive Control: Untreated virus mixed with cells.

Results: Virus growth will be inhibited after treatment of compound Y.

Table of Experimental Results:

While the present invention has been described in detail with particular preferred embodiments, it should be understood that different embodiments are possible and details thereof may be varied in various ways. As will be apparent to those skilled in the art, modifications and variations are possible as long as they are within the spirit and scope of the invention. Accordingly, the above disclosure, description, and drawings are exemplary only and do not limit the invention as defined by the claims.

references:

Claims (18)

A compound of formula (5) or a salt, ester or metabolite thereof:

(5) Wherein R 1 comprises H, an acetyl group, a tigloyl group, a sensioli group or an acid comprising 2 to 5 carbons, or a sugar residue or rhamnose; The sugar moiety or rhamnose comprises at least two groups selected from the group consisting of angeloyl groups, acetyl groups, tigloyl groups, sensioli groups and acids comprising 2 to 5 carbons; R2 comprises H, an acetyl group, a tigloyl group, a sensioli group or an acid comprising from 2 to 5 carbons, or a sugar residue or rhamnose; The sugar moiety or rhamnose comprises at least two groups selected from the group consisting of angeloyl groups, acetyl groups, tigloyl groups, sensioli groups and acids comprising 2 to 5 carbons; R 4 comprises CH 2 OR 6 or COOR 6, wherein R 6 is H or an acid comprising an angeloyl group, an acetyl group, a tigloyl group, a sensioli group and 2 to 5 carbons; Carbon positions 23, 24, 25, 26, 29 30 of the compounds independently include CH 3, CH 2 OH, CHO, COOH, alkyl groups, acetyl groups or derivatives thereof; R 5 is H or a sugar residue, wherein the sugar residue is glucose, or galactose, or rhamnose, or arabinose, or xylose, or alduronic or glucuronic acid, or galacturonic acid or a derivative thereof, or Including combinations thereof. The compound of claim 1, wherein one of R 1, R 2, R 6 comprises an angeloyl group or an acid having 5 carbons and the other two or more of R 1, R 2, R 6 are angeloyl groups, acetyl groups, A tigloyl group, a sensioli group and an acid comprising from 2 to 5 carbons; Or at least one of R1, R2, R6 comprises a sugar moiety or rhamnose, wherein the sugar moiety or rhamnose is an angeloyl group, an acetyl group, a tigloyl group, a sensioli group and 2 to 5 carbons At least two groups selected from the group consisting of acids comprising; Or two or more of R1, R2, R6 comprise an angeloyl group or an acid having five carbons. A compound of formula (1), (2), (3) or (4) or a salt or ester thereof:

(One) In which R 1 comprises an angeloyl group; R2 contains an angeloyl group; R 3 comprises OH or H; R 4 comprises CH 3 or CH 2 OH; R 7 comprises H; And R5 comprises glucose or galactose; And R6 contains arabinose,

(2) In which R 1 comprises an angeloyl group; R2 contains an angeloyl group; R 3 comprises Ac or H; R 4 comprises H or Ac; R5 comprises CH3 or CH2OH,

(3) In which R 1 comprises an angeloyl group; R2 contains an angeloyl group; R 3 comprises OH or H; R4 comprises a CH3 or CH2OH or an alkyl group or derivative thereof; R 6 comprises Ac or H and R 5 comprises a sugar moiety wherein the sugar residue is at least one sugar or glucose, or galactose or rhamnose or arabinose, or xylose, or alduronic acid, or glut Curonic acid, or galacturonic acid, or derivatives or combinations thereof, or

(4) In the formula,

ego; S1-S4 = S1 or S2 or S3 or S4; R 1 comprises an angeloyl group; R2 contains an angeloyl group; R 3 comprises Ac or H; R 4 comprises H or OH; R6 comprises Ac or H; R7 comprises a CH3 or CH2OH or an alkyl group or derivative thereof and R5 comprises a sugar moiety wherein the sugar moiety is one or more sugars or D-glucose, D-galactose, L-rhamose, L-arabino Os, D-xylose, alduronic acid, D-glucuronic acid, D-galacturonic acid, or derivatives thereof or combinations thereof. The compound of claim 3 having the structure

(a) In which R 1 comprises an angeloyl group; R2 contains an angeloyl group; R1 and R2 comprise an angeloyl group, an acetyl group, a tigloyl group, a sensioli group or an acid having 2 to 5 carbons; The structure comprises a sugar moiety wherein the sugar moiety is one or more sugars, or glucose, or galactose, or rhamnose, or arabinose, or xylose, or alduronic acid, or glucuronic acid, or galacto Lonic acid, or derivatives thereof or combinations thereof,

(b) or

Wherein R 1 comprises an angeloyl group and R 2 comprises an angeloyl group;

(c) Wherein R 1 contains an angeloyl group and R 2 contains an angeloyl group. A compound according to claim 1, 2, 3 or 4 comprising the structure: (a)

Having the structure (Y3) or having a chemical name of Glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene, or xanipolya-Y; (b)

Or have the chemical name 3-O- [β-D-galactopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3) -β-D-glu Kuronopyranosyl-21-O- (3,4-diangeloyl) -α-L-lamnopyranosyl-22-O-acetyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean -12-ene, Y1 or xanipolya-Y1; (c)

Or have the chemical name 3-O- [β-D-glucopyranosyl (1 → 2)]-α-L-arabinofuranosyl (1 → 3)-(-D-glucu Lonofyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α, 24β, 28-heptahydroxyolean-12-ene, or xanipolya-Y2; (d)

Or have a chemical name of 3-O- [β-glucopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-glucuronopyranosyl-21 , 22-O-diangeloyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene, or xanipolya-Y8; (e)

Or have the chemical name 3-O- [β-galactopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-glucuronopyranosyl -21-O- (3,4-diangeloyl) -α-lamnopyranosyl-28-O-acetyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene, or xani Polya-Y9; (f)

Or have the chemical name 3-O- [β-galactopyranosyl (1 → 2)]-α-arabinofuranosyl (1 → 3) -β-glucuronopyranosyl- 21,22-O-diangeloyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene, or xanipolya-Y10; or (g) Compound (X)

Contains the chemical structure of (X) or has a chemical name of 3-O-{[β-D-galactopyranosyl (1 → 2)]-[α-L-arabinofuranosyl (1 → 3)]-β -D-glucuronopyranoside butyl ester} -21-O-acetyl-22-O- angeloyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene. The compound of claim 5, wherein the compound comprises a triterpene backbone, wherein the triterpene backbone is acylated by an angeloyl group or a sugar moiety at the 21β or 22α position or at both 21β and 22α positions, wherein 1 The sugars include angeloyl groups attached to the C3 and C4 positions of the sugar; The triterpene backbone comprises a sugar moiety; The sugar moiety comprises one or more sugars, or glucose, or galactose, or rhamnose, or arabinose, or xylose, or alduronic acid or glucuronic acid, or galacturonic acid or derivatives thereof, or combinations thereof. Compound. The compound according to any one of claims 1 to 6, which is in salt form. 7. A compound according to any one of claims 1 to 6 which is a structure synthesized or isolated from natural sources. The compound according to any one of claims 1 to 6, which is a medicament or a health food. A composition comprising an effective amount of another compound according to any one of claims 1 to 6 as a medicament for inhibiting tumor or cancer cell growth. The composition of claim 10 wherein the cancer is breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone cancer or brain cancer, in particular ovarian cancer. To inhibit cancer; To inhibit viruses, HIV, fungi or bacteria; To prevent brain aging; To improve memory and improve brain function; to treat nocturia, frequent urination, incontinence, dementia, Alzheimer's disease, autism, brain trauma, Parkinson's disease or other diseases caused by brain abnormalities; To treat arthritis, rheumatism, poor circulation, atherosclerosis, Raynaud's syndrome, angina, heart disease, coronary heart disease, headache, dizziness, kidney disease, cerebrovascular disease; To inhibit NF-kappa B activation; Brain edema, severe acute respiratory syndrome, respiratory viral disease, chronic venous insufficiency, hypertension, chronic venous disease, anti-laryngitis, anti-inflammatory, hemorrhoids, peripheral edema formation, varicose vein disease, influenza, post-traumatic edema and postoperative swelling To; To inhibit ethanol absorption; To reduce thrombus formation; To lower blood sugar; To regulate the amount of adorecorticotropin and corticosterone; To modulate the release of ACTH, PGF2, antagonistic action against 5-HT or histamine; To reduce metabolism of tissue mucopolysaccharides; A composition comprising a compound according to any one of claims 1 to 6 for the treatment of erectile dysfunction or premature ejaculation or diabetes. The functional group according to any one of claims 1 to 6, for carrying out the biological activity of at least two adjacent angeloyl groups or acids having 5 carbons or an acid or angeloyl group having two or more carbons. A compound comprising or a composition for inhibiting a cancer or a virus comprising a saponenin or a triterpene. The method of claim 13, wherein the two angeloyl groups are in the side chain of the trans position; Or two angeloyl groups are in the side chain of the cis position; Or two angeloyl groups are present in the side chain of the carbon having a trans or cis position in the structure. A triterpene or sapogenin backbone, said backbone comprising a sugar moiety, wherein said sugar moiety is glucose, or galactose, or rhamnose, or arabinose, or xylose, or alduronic acid or glucuronic acid, Or galacturonic acid or a derivative thereof, or a combination thereof; The main chain comprises two groups selected from the group comprising an acid having 5 carbons attached to the main chain, an angeloyl group, a tigloyl group, a sensioli group, and combinations thereof, wherein the two side chains Is a compound that produces anticancer activity or antiviral activity. A non-angeloyl group attached to the structure, wherein the presence of the non-angeloyl group produces anticancer or antiviral activity. (a) extracting Xanthoceras sorbifolia or plant powder using an organic solvent to obtain an organic extract, wherein the powder is obtained from the skins, branches, stems, leaves, grains, roots of herbs or plants. Prepared from bark or seed shells; (b) collecting the organic extracts; (c) refluxing the organic extract to obtain a second extract; (d) removing the organic solvent from the second extract; (e) drying and sterilizing the second extract to obtain a crude extract powder; (f) fractionating the crude extract powder into components using FPLC chromatography using silica gel, C18 and other equivalent solid materials; (g) monitoring the absorption wavelength at 207 nm or 254 nm; (h) identifying the bioactive component of the crude extract powder; (i) purifying the bioactive component of the crude extract powder by FPLC to obtain a fraction of the bioactive component; And (j) A method for isolating a compound from a plant with xanthoserras sorbobia herb, or sapindaceae , comprising isolating the compound from the fraction of the bioactive component by preparative HPLC. 18. The compound of claim 17, wherein the compound is 18, 29, 20, 24, 25, 26, 27, 29, 30, 31, 32, 33, 36, 37, 38, 40, 41, 42, 45, 46, 48 Or NMR spectral data shown at 49, 50, 51, 52, 54, 55, 56, 58 or 59; Or the compound is a compound as shown in FIG. 17, 23, 28, 35, 39 or 43.

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