CA2209222A1 - Retrovirus protease inhibitors - Google Patents

Abstract

A composition and method of treating a retrovirus infection therapeutically effective is described. In particular, isolated triterpenes have been shown to have significant inhibitory activity against HIV1 protease.

Description

B&P File No. 9158-003/MG
Title: Retrovims Protease Inhibitors FIELD OF THE INVENTION
The invention relates to triterpenes that are protease inhibitors and are useful in treating retroviral infections such as HIV and HTLV.
BACKGROUND OF THE INVENTION
Retroviruses are a family of viruses that contain an RNA
genome and reverse transcriptase activity. Many classes of retroviruses have been identified, one of the major classes being the T-lymphotropic viruses which include HTLV-I which appears to be causative of adult T-cell leukemia-lymphoma; HTLV-II which appears to be related to a variant of hairy cell leukemia; and HIV-1(HTLV-III) and HIV-2 which are linked to Acquired Immune Deficiency Syndrome (AIDS). Patients that test positive for HIV may be initially asymptomatic but typically develop AIDS related complex (ARC) followed by AIDS. The AIDS virus attacks and depletes the T lymphocytes causing severe immunosuppression in infected individuals which predisposes them to debilitating and ultimately fatal opportunistic infections.
No treatment is currently available to prevent or reverse the immunodeficiency of AIDS and ARC caused by the HIV virus.
However, a number of compounds that interfere with viral replication have been developed to treat AIDS, including inhibitors of HIV reverse transcriptase (RT), such as nucleoside analogs including 3'-azido-3'-deoxythymidine (AZT, zidovidine) and 2', 3'-dideoxy nucleosides such as 2', 3'-dideoxyinosine (DDI, ddI) and 2', 3'-dideoxycytidine (DDC, ddC).
AZT is in use for early treatment of HIV positive individuals as well as individuals with ARC and AIDS. Unfortunately, AZT is frequently toxic, causing bone marrow suppression resulting in anemia, leucopenia and thrombocytopenia. Also, AZT-resistant HIV
strains have been observed after six months of AZT treatment. DDI and DDC have been approved for treatment of individuals with ARC and AIDS only in combination with AZT.
Consequently, the search for effective and non-toxic treatments for AIDS continues. Due to the toxicity associated with 5 synthetic compounds, some groups are looking to natural sources in the search for a treatment for AIDS.
Some natural compounds have been reported to show anti-HIV activity. One compound is glycyrrhizin which is isolated from the aqueous extract of licorice root (Glycyrrhiza radix) [1], and is known as an anti-inflammatory substance in Chinese medicine. This compound consists of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid. Ito et al [1] investigated its antiviral action on the human immunodeficiency virus in vitro, using cytopathic effect and plaque forming assay system in MT-4 cells. Cloned Molt-4 cells, which are sensitive to HIV and fused to giant cells after infection, were used as a parameter for cytopathic effect of HIV. Glycyrrhizin inhibited HIV-induced plaque formation in MT-4 cells at a concentration of 0.6 mM, the 50% inhibitory dose being 0.15 mM. Glycyrrhizin inhibited the cytopathic effect of HIV and the HIV-specific antigen expression in MT-4 cells at a concentration of 0.3 and 0.6 mM, respectively. Furthermore, glycyrrhizin inhibited giant cell formation of HIV-infected Molt-4 clone No. 8 cells.
Wang et al [2] have reported that fifteen novel non-peptide HIV-1 protease inhibitors were identified by flexible 3D database pharmacophore searching of NCI DIS 3D database. The pharmacophores they used in the search were derived directly from the X-ray determined structures of protease/inhibitor complexes. These 15 inhibitors, belonging to nine different chemical classes, are promising leads for further development. The two best inhibitors found, the 4-hydroxycoumarin dimer and the 2-hydroxynapthoquinone tetramer, had ID50 values of 0.32 and 0.75 ~lM, respectively, for HIV-1 protease inhibition.
Ardisia japonica (Thunb.) B1. (Myrsinaceae), in the form of a decoction of the roots, is widely used in traditional Chinese medicine to stop cough and uterine bleeding. In vitro anti-HIV activity studies, norbergenin and bergenin were found with moderate anti-HIV activity [3]. The most active compound was norgergenin, which exhibited an EC50 value of 20 (llg/ml) and a IC50 value of over 500 (~g/ml) with a selectivity index above 25.
From Chrysanthemum morifolium Ramar (Compositae), acacetin-7-O-~-D-galactopyranoside [4] was found to show significant anti-HIV activity. It has IC50 37 ',lM, EC50 8 ~M and therapeutic index 5.
Fujioka et al [5] found the MeOH extract of the leaves of Syzigium claviflorum (Roxb.) WALL (Myrtaceae) to show significant anti-HIV
activity. Subsequent bioactivity-directed fractionation has resulted in the isolation of betulinic acid, platanic acid and dihydrobetulinic acid. These compounds were also potent inhibitors of HIV replication.
Geum is a genus of 65 species of rhizomatous herbs and subshrubs with simple or pinnately lobed leaves and regular flowers such as G. bortsii, G. chiloense, G. coccineum, G. macrophyllum, G.
montanum, G. reptans, G. rivale, G. triflorum, G. urbanum and G.
japonicum etc.
Geum japonicum Thunb. is a perennial herb and the flowering plant of the Rosaceae family. The whole plant of G e u m japonicum Thunb. has been used as a diuretic in traditional Chinese medicine [6]. The plants of Geum species are known to be rich in tannins. Several hydrolyzable tannins such as gemin A, gemin B, gemin C, gemin D, gemin E and gemin F have been isolated from G e u m japonicum [7, 8]. In the continuing investigation of the plant, some triterpenoids including 2-hydroxyoleanolic acid, 2-hydroxyursolic acid, 2,19-dihydroxy-ursolic acid, 2,3,19, 23-tetrahydroxyurs-12-en-28-oic acid 28-O--D-glucopyranoside were isolated by Shigenaga et al [9]. Xu et al [10]
isolated geponin, 1, 2, 3-tri-O-galloyl--D-glucopyranside, blumenol A, gallic aldehyde, 3, 4-dihydroxybenzoic acid, caffeic acid and kampferol-3-O-glucopyranside from G. japonicum aqueous extract. Of these isolated compounds, geponin and gallic aldehyde showed strong anti- HSV
activity.
One potential target for inhibiting the HIV virus is to inhibit the aspartyl protease that is encoded for by HIV(HIV PR). The function of the HIV PR is essential for proper virion assembly and maturation. Inactivation of HIV PR by either mutation or chemical inhibition leads to the production of immature, non-infectious viral particles [11, 12]. HIV PR inhibitors represent a new class of therapeutic agents that complements existing approaches to antiviral therapy that targets another enzyme, the HIV reverse transcriptase, as discussed above.
To date, most inhibitors of the HIV aspartic acid protease have been transition state mimetics. These have included reduced amides (Moore et al., Biochem. Biophys. Res. Commun. 159:420-425 (1989); Dreyer et al., Proc. Natl. Acad. Sci. 86:9752-9756 (1989)), hydroxyethylene isosteres (Dreyer et al., Proc. Natl. Acad. Sci. 86:9752-9756 (1989); Vacca et al., J. Med. Chem. 34:1225-1228 (1991); Tomasselli et al., J.
Biol. Chem. 265: 14675-14683 (1990); Roberts et al., Science 248:358-361 (1990)), statine analogs (Dreyer et al., 1989), phosphinic acid derivatives (Grobelny et al., Biochem. Biophys. Res. Commun. 169:1111-1116 (1990)) and difluoroketone derivatives (Dreyer et al., 1989; Sham et al., Biochem.
Biophys. Res. Commun. 175: 914-916 (1991)) .
Some natural compounds have been reported to be inhibitory to HIV-1 protease [13]. Thus, traditional medicines utilizing natural products appear to be useful sources for discovery of new anti-AlDS agents.

SUMMARY OF THE INVENTION
The present inventors have isolated several triterpenes from natural sources which inhibit a retroviral protease, such as HIV
protease.
In one aspect, the present invention relates to an isolated triterpene that can inhibit a retroviral protease. Preferably, the triterpene is of the Formula I shown in Figure 11 where R1 and R2 are identical or different and are independently selected from H,OH,=O, Cl-C10 unbranched alkoxy group, C1-Cl0 branched alkoxy group, C1-Cl0 unbranched acyloxyl group and C1-Cl0 branched acyloxy group; R3, R4, R5 and R8 are identical or different and are independently selected from H, C1-C10 branched alkyl group, C1-Cl0 unbranched alkyl group, C1-Cl0 branched alkoxy group, C1-C10 unbranched alkoxy group, a C1-C10 branched acyloxy group, a C1-Cl0 unbranched acyloxy group and a =O
group; R6, R7 and R9 are identical or different and are independently selected from a COOH group, a -COOCH3 group, a -CH20H group and a C1-C10 unbranched alkyl group. Also included are pharmaceutically acceptable salts and modified derivatives of the compounds of Formula I
such as the 28-beta-D-glucoside derivatives.
The triterpene may be isolated from a variety of plants including Geum jeponicum, Anemarrhena asphodeloides, Brainia insignis, Epimedium sagittatum, Magnolia fargesii, Oldenlandia diffusa, Phellodendron amurense, Platycodon grandiflorum, Prunus mume, Punica granatum, Rhus javanica, Scutellaria baicalensis, Sophora japonica, Taraxacum mongolicum, Terminalia arjuna, Terminalia chebula, Woodfordia floribunda, Woodwardia orientalis.
Anti-retroviral activity has been demonstrated in a methanol extract of Geum jeponicum. The present inventors have characterized the methanol abstract and have isolated nine triterpenes from the methanol extract and have determined that some of these have anti-HIV activity. The nine compounds are:

(1) 2-alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid (2) ursolic acid (3) epimolic acid (4) maslinic acid (5) euscaphic acid (6) tormentic acid (7) 19-alpha-hydroxyasiatic acid (8) 28-beta-D-glucoside of tormentic acid (9) 28-beta-D-glucoside-19-alpha-hydroxyasiatic acid.
Consequently, the present invention provides a composition for treating a retroviral infection comprising at least one triterpene selected from the group consisting of (1) 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid (2) ursolic acid; (3) epimolic acid; (4) maslinic acid; (5) euscaphic acid; (6) tormentic acid; (7) 10-alpha-hydroxyasiatic acid; (8) 28-beta-D-glucoside of tormentic acid; and (9) 28-beta-D-glucoside-19-alpha-hydroxyasiatic acid or a pharmaceutically acceptable salt thereof, in admixture with a physiologically acceptable diluent or carrier. The composition can also include other agents that are useful in the treatment of retrovirus infections such as other HIV
protease inhibitors as well as reverse transcriptase inhibitors. In a preferred embodiment the composition includes the triterpene 2-alpha, 19-alpha-dihydroxy-3-oxo 12 ursen-28-oic acid.
The present invention also includes a method for treating a retroviral infection comprising administering a therapeutically effective amount of an isolated triterpene to a mammal in need of such a treatment. The triterpene is preferably of the Formula I shown in Figure 11 as defined above.
In one embodiment, the present invention provides a method of treating a retroviral infection comprising administering a therapeutically effective amount of at least one triterpene selected from the group consisting of (1) 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid; (2) ursolic acid; (3) epimolic acid; (4) maslinic acid; (5) euscaphic acid; (6) tormentic acid; (7)19-alpha-hydroxyasiatic acid; (8) 28-beta-D-glucoside of tormentic acid; and (9) 28-beta-D-glucoside-19-alpha-hydroxyasiatic acid to a mammal in need of such a treatment.
In a preferred embodiment, the retroviral infection is Human Immunodeficiency Virus (HIV).
Other objects, features and advantages of the present invention will become apparent from the following detailed description.
It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DR~WINGS
The invention will now be described in relation to the drawings in which:
Figure 1 shows the chemical formula of compound 1.
Figure 2 shows the chemical formula of compound 2.
Figure 3 shows the chemical formula of compound 3.
Figure 4 shows the chemical formula of compound 4.
Figure 5 shows the chemical formula of compound 5.
Figure 6 shows the chemical formula of compound 6.
Figure 7 shows the chemical formula of compound 7.
Figure 8 shows the chemical formula of compound 8.
Figure 9 shows the chemical formula of compound 9.
Figure 10 shows the chemical formula of compounds 10 and 11.
Figure 11 shows a general chemical formula of a triterpene of the invention.
Figure 12 is a schematic diagram showing the fractionation of the methanol extract from G.japonicum.
Figure 13 is a schematic diagram showing the fractionation of the ethyl acetate soluble extract from G.japonicum.

Figure 14 is a schematic diagram showing the mass fragmentations of compound 1.
DETAILED DESCRIErIlON OF THE INVENTION
As hereinbefore mentioned, the present invention relates to isolated triterpenes that are useful in treating infections caused by a retrovirus such as Human Immunodeficiency Virus (HIV) or human T-cell leukemia virus (HTLV). The triterpenes act by inhibiting the protease activity of the retrovirus.
Broadly stated, the present invention relates to a composition for treating a retroviral infection comprising an isolated triterpene in admixture with a physiologically acceptable carrier or diluent.
Preferably, the triterpene is of the Formula I shown in Figure 11 wherein Rl and R2 are identical or different and are independently selected from H,OH,=O, C1-Cl0 unbranched alkoxy group, Cl-Cl0 branched alkoxy group, Cl-Cl0 unbranched acyloxyl group and Cl-Cl0 branched acyloxy group; R3, R4, R5 and R8 are identical or different and are independently selected from H, C1-Cl0 branched alkyl group, C1-Cl0 unbranched alkyl group, Cl-C10 branched alkoxy group, C1-C10 unbranched alkoxy group, a Cl-Cl0 branched acyloxy group, a Cl-C10 unbranched acyloxy group and a =O group; R6, R7 and Rg are identical or different and are independently selected from a COOH group, a -COOCH3 group, a -CH2OH group and a Cl-Cl0 unbranched alkyl group.
The triterpene may be isolated from a variety of plants including Geum jeponicum, Anemarrhena asphodeloides, Brainia insignis, Epimedium sagittatum, Magnolia fargesii, Oldenlandia diffusa, Phellodendron amurense, Platycodon grandiflorum, Prunus mume, Punica granatum, Rhus javanica, Scutellaria baicalensis, Sophora japonica, Taraxacum mongolicum, Terminalia arjuna, Terminalia chebula, Woodfordia floribunda, Woodwardia orientalis.

In one embodiment, triterpenes isolated from the methanol extract of the whole plant of Geum japonicum were found to show significant inhibitory activity against HIV-1 protease. The triterpenes that have been isolated from the methanol extract are as follows:
(1) 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid (2) ursolic acid (3) 3-alpha-19-dihydroxy-12-ursen-28-oic acid or epimolic acid (4) maslinic acid (5) 2-alpha-3-alpha-19-alpha-trihydroxy-12-ursen-28-oic acid or euscaphic acid (6) 2-alpha-3-beta-19-alpha-trihydroxy-12-ursen-28-oic or tormentic acid (7) 2-alpha-3-beta-19-alpha-23-tetrahydroxy-12-ursen-28-oic acid or 19-alpha-hydroxyasiatic acid (8) 28-beta-D-glucoside of tormentic acid (9) 28-beta-D-glucoside-19-alpha-hydroxyasiatic acid.
For ease of referral, the compounds have been given the following abbreviations and the terms may be used interchangeably throughout the application:
"2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid" is also referred to as compound 1.
"ursolic acid" is also referred to as compound 2.
"3-alpha-19-dihydroxy-12-ursen-28-oic acid" is also "epimolic acid" and these are also referred to as compound 3.
"maslinic acid" is also referred to as compound 4.
"2-alpha-3-alpha-19-alpha-trihydroxy-12-ursen-28-oic acid"
is also "euscaphic acid" and these are also referred to as compound 5.
"2-alpha-3-beta-19-alpha-trihydroxy-12-ursen-28-oic" is also "tormentic acid" and these are also referred to as compound 6.
"2-alpha-3-beta-19-alpha-23-tetrahydroxy-12-ursen-28-oic acid" is also "19-alpha-hydroxyasiatic acid" and these are also referred to as compound 7.

"28-beta-D-glucoside of tormentic acid" is also "28-beta-D-glucoside of 2-alpha-3-beta-19-alpha-trihydroxy-12-ursen-28-oic" and these are also referred to as compound 8.
"28-beta-D-glucoside-19-alpha-hydroxyasiatic acid" is also referred to as compound 9.
In one aspect, the present invention provides a composition for treating a retroviral infection comprising at least one isolated triterpene selected from the group consisting of (1) 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid; (2) ursolic acid; (3) epimolic acid;
(4) maslinic acid; (5) euscaphic acid; (6) tormentic acid; (7) 10-alpha-hydroxyasiatic acid; (8) 28-beta-D-glucoside of tormentic acid; and (9) 28-beta-D-glucoside-19-alpha-hydroxyasiatic acid; or a pharmaceutically acceptable salt thereof, in admixture with a physiologically acceptable diluent or carrier.
The term "pharmaceutically acceptable salt" as used herein means an organic or inorganic salt of a triterpene of the present invention that is suitable for administration to a mammal such as a human.
The term "physiologically acceptable diluent or carrier"
means vehicles that are suitable for administration to a mammal, such as a human. Suitable diluents or carriers include sterile solutions such as saline, aqueous buffer solutions, glycerol, etc.
In a preferred embodiment, the composition comprises at least one triterpene selected from the group consisting of (1) 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid; (2) ursolic acid; and (4) maslinic acid or a pharmaceutically acceptable salt thereof. In a more preferred embodiment, the composition comprises (1) 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid or a pharmaceutically acceptable salt thereof.
In another aspect, the present invention provides a method of treating a retroviral infection comprising administering a therapeutically effective amount of at least one triterpene selected from the group consisting of (1) 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid; (2) ursolic acid; (3) epimolic acid; (4) maslinic acid; (5) euscaphic acid; (6) tormentic acid; (7)19-alpha-hydroxyasiatic acid; (8) 28-beta-D-glucoside of tormentic acid; and (9) 28-beta-D-glucoside-19-alpha-hydroxyasiatic acid or a pharmaceutically acceptable salt thereof to a mammal in need of such a treatment.
In a preferred embodiment, the method comprises administering a therapeutically effective amount of at least one triterpene selected from the group consisting of (1) 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid; (2) ursolic acid; and (4) maslinic acid or a pharmaceutically acceptable salt thereof. The retroviral infection to be treated is preferably HIV.
Administration of a "therapeutically effective amount" of the compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. The therapeutically effective amount of a triterpene of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The composition may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application or rectal administration. Depending on the route of administration, the triterpene may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
A composition of the present invention can be administered to a subject in an appropriate carrier or diluent, or in an appropriate carrier such as liposomes. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol 7:27). The active compound may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of mlcroorganlsms .
Compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The physiologically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like.
In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the triterpenes in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
When the active compound is suitably protected, as described above, the composition may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the therapeutic treatment of individuals.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES
Example 1 This example describes the characterization of a methanol extract of the whole plant of G. japonicum which was found to show significant inhibitory activity against HIV-1 protease. In particular, a methanol extract of the whole plant of G. japonicum was successively partitioned into hexane, ethyl acetate and n-BuOH soluble fractions (Figure 12). The biological evaluation of the extract and fractions indicated that most of the activity was in the ethyl acetate soluble fraction (Table 1).
MATERIALS AND METHODS
Thin layer chromatography (TLC) was carried out on silica gel pre-coated glass plates a. T. Baker, Si254F, 250~1m layer thickness) and on Cl8 silica pre-coated plates (Whatman, KCl8F, 200,um). Analytical TLC
plates were visualized using UV light and by spraying with ceric sulphate-H2SO4 or methanolic FeCl3. Flash chromatography was carried out using Kieselgel 60 of mesh range 230-400 ASTM (Merck or J. T.
Baker). For gel permeation column chromatography, sephadex LH 20 was used.
Optical rotations were determined on a automatic polarimeter. IR spectra were recorded on a Perkin Elmer 1600 Series FTIR spectrometer. EIMS were recorded on a MICROMASS 7035E mass spectrometer at 70 eV. All NMR spectra (lH, 13C, COSY, NOE, HMQC
and HMBC) were recorded on a Bruker AMX 500 spectrometer (500 MHz for lH and 125 MHz for 13C) in pyridine-d5, the chemical shifts are reported in ppm with TMS as an internal standard and coupling constants(J) are given in Hz.
Plant material The whole plant of Gel~m japonicl~m used in this experiment was collected from China in August, 1994. The material was identified as G. japonicum Thunb. by Dr. Dao-Feng Chen, Department of Pharmacology, Shanghai Medical University, China. A voucher specimen was deposited in Department of Pharmacology, Shanghai University of Traditional Chinese Medicine, China.
Extraction and isolation Dried whole plants (2.6 kg) were chopped into small pieces and extracted three times with MeOH (20 liters) at room temperature for 7 days and the extract was evaporated in vacuo to yield MeOH extract (350 g). The MeOH extract was suspended in distilled water (1l) and successively extracted with hexane (2 l x 5), EtOAc (2 l x 5) and n-BuOH (1 l x 5). The EtOAc soluble fraction was filtered and the filtrate was evaporated under reduced pressure to give a brown oil (120 g).
A portion of the EtOAc extract (80 g) was subjected to silica gel (2.0 kg) chromatography on a 120 mm i. d. column and eluted with a linear gradient CHCl3-MeOH system (100:0 to 70:30). Fractions of 500 ml each were collected and examined on silica gel TLC (CHCl3-MeOH, 9:1 and 7:3). Fractions with similar Rf (TLC) were combined and concentrated to afford fractions 1 to 5 as shown in Figure 13. Fraction 1 (2.5 g) was further purified by rechromatography on silica gel (150 g) column of 50 mm i. d. and eluted with CHCl3-MeOH (95:5) to yield a mixture of compound 1 and 3~-hydroxy-12-ursen-28-oic acid. The mixture was further purified by preparative TLC (CHCl3-MeOH, 95:5) to give 2a, 19a-dihydroxy-3- oxo-12-ursen-28-oic acid (1) (44 mg) and 3~-hydroxy-12-ursen-28-oic acid (2) (70 mg).
NMR and Other Physical Data for Compounds 1-11 2a, 19a-Dihydroxy-3-oxo-12-ursen-28-oic acid (1) A colourless powder, [a]D +1.7~(c 1.0, EtOH); mp 215-217 ~C;
C30H~605; HRMS: 486.3373 (cald. for C3OHl6O5, 486.3345); EIMS m/z: 486 (M), 438, 366, 246, 231, 221, 220, 219, 218, 205, 203, 202, 185, 146; IR KBr (cm-l): 3600-3200, 2928, 2869, 1684, 1650, 1384, 1105; lH-NMR (pyridine-d5, 500 MHz): ~1.01 (3H, s, 24-H3), 1.09 (3H, s, 26-H3), 1.12 (3H, d, J=6.6 Hz, 30-H3), 1.15 (3H, s, 25-H3), 1.22 (3H, s, 23-H3), 1.25 (lH, m, 5-H), 1.37 (lH, m, 1-Ha), 1.42 (3H, s, 29-H3), 1.50 (lH, m, 20-H), 1.64 (3H, s, 27-H3), 1.87 (lH, t, J=8.9 Hz, 9-H), 2.08 (lH, m, 21-Ha), 2.11 (lH, m, 22-Ha), 2.13 (lH, m, 21-Hb), 2.15 (lH, m, 22-Hb), 2.48 (lH, dd, J=12.5, 6.5 Hz, 1-Hb), 3.03 (lH, s, 18-H), 4.80 (lH, dd, J=12.5, 6.5 Hz, 2-H), 5.55 (lH, br s, 12-H). l3C-NMR
(pyridine-d5): ~= 50.10 (t, C1), 69.73 (d, C2), 216.41 (s, C3), 48.10 (s, C4), 57.67 (d, C5), 19.55 (t, C6), 33.14 (t, C7), 40.37 (s, C8), 47,33 (d, C9), 37.90 (s, C10), 24.12 (t, C11), 127.55 (d, C12), 140.07 (s, C13), 42.16 (s, C14), 29.24 (t, C15), 26.31 (t, C16), 48.25 (s, C17), 54.56 (d, C18), 72.67 (s, C19), 42.35 (d, C20), 26.92 (t, C21), 38.45 (t, C22), 25.28 (q, C23), 21.77 (q, C24), 17.30 (q, C25), 16.77 (q, C26), 24.66 (q, C27), 180.65 (s, C28), 27.06 (q, C29), 15.95 (q, C30).
3,~-Hydroxy-12-ursen-28-oic acid (2) A colourless powder, [a]D +65 (c 1.0, MeOH); mp 290-292 ~C;
C30H48O3; ErMS m/z: 456 [M] +, 410, 248, 203, 157; IR KBr (cm-l): 3425, 1689, 1640; 1H-NMR (pyridine-d5, 500 MHz): ~ 0.91 (3H, s, 24-H3), 1.00 (6H, d, J=6.4 Hz, 29, 30-H3), 1.02 (3H, s, 26-H3), 1.06 (3H, s, 25-H3), 1.24 (3H, s, 23-H3), 1.29 (3H, s, 27-H3), 2.65 (lH, s, 18-H), 3.46 (lH, t, J=7.9 Hz, 3-H), 5.50 (lH, br s, 12-H). l3C-NMR (pyridine-d5, 125 MHz): ~ 39.12 (t, C1), 28.13 (t, C2), 78.15 (d, C3), 39.39 (s, C4), 55.85 (d, C5), 18.81 (t, C6), 33.61 (t, C7), 40.00 (s, C8), 48.07 (d, C9), 37.31 (s, C10), 23.65 (t, C11), 125.66 (d, C12), 139.28 (s, C13), 42.53 (s, C14), 28.71 (t, C15), 24.93 (t, C16), 48.07 (s, C17), 53.57 (d, C18), 39.42 (s, C19), 39.51 (d, C20), 31.10 (t, C21), 37.45 (t, C22), 28.82 (q, C23),15.70 (q, C24), 16.57 (q, C25), 17.48 (q, C26), 23.94 (q, C27), 179.87 (s, C28), 17.53 (q, C29), 21.43 (q, C30).
A portion of fraction 2 (3.0 g) was rechromatographed on a 30 mm i. d. column of silica gel (180.0 g) and eluted with CHCl3:MeOH(90:10). Fractions of 15 ml each were collected and examined on silica gel TLC (CHCl3-MeOH, 9:1). Fractions with similar Rf (TLC) were combined and concentrated then by preparative TLC ( CHCl3-MeOH, 90:10) to give 3a, l9a-dihydroxy-12-ursen-28-oic acid (3) (23 mg) and 2a, 3.~-dihydroxy- 12-oleanen-28-oic acid (4) (20 mg).
3a,19a-Dihydroxy-12-ursen-28-oic acid (3) A colourless powder, [a]D +36.5 (c 1.5, Pyridine); mp 302-304 oC; C30H48O4; EIMS m/z: 472 [M]+, 454, 439, 426, 408, 393, 354, 219, 218, 146;
IR KBr (cm~1): 3427, 1695, 1630; lH-NMR (pyridine-d5, 500 MHz): ~ 0.92 (3H, s, 26-H3), 1.03 (3H, s, 23-H3), 1.12 (3H, s, 24-H3), 1.17 (3H, d, J=6.4 Hz,30-H3), 1.24 (3H, s, 25-H3), 1.45 (3H, s, 27-H3), 1.73 (3H, s, 29-H3), 3.07 (lH, s, 18-H), 3.43 (lH, dd, J=3.0 Hz, 3-H), 5.61 (lH, br s, 12-H). 13C-NMR

(pyridine-d5, 125 MHz):~ = 39.03 (t, C1), 28.12 (t, C2), 78.22 (d, C3), 39.39(s,C4), 55.89 (d, C5), 18.96 (t, C6), 33.62 (t, C7), 40.39(s, C8), 47.80 (d, C9), 37.37 (s, C10), 24.04 (t, C11), 128.06 (d, C12), 139.97(s, C13), 42.13(s, C14), 29.33 (t, C15), 26.43(t,C16), 48.33(s,C17), 54.65 (d, C18), 72.72(s,Cl9), 42.38 (d, C20), 26.95(t,C21), 38.50 (t, C22), 28.79(q,C23), 17.24(q,C24), 16.77(q,C25), 16.51 (q,C26), 24.69(q,C27), 180.72(s,C28), 27.14(q,C29), 15.58(q,C30).
Acetylation of 3a,19a-dihydroxy-12-ursen-28-oic acid (3) Compound 3 (15 mg) was dissolved in Ac20 (1 ml) and dried pyridine (1 ml) and the reaction mixture was kept at 80 ~C for 48 h.
The reaction mixture was then poured into cold H20, extracted with EtOAc. The acetate was obtained (3a) (11.5 mg). IR KBr (cm-1):3425, 1735, 1690, 1630;1H-NMR (CD30D, 500 MHz): ~ 0.82 (3H,s, 26-H3), 0.89 (3H,s, 23- H3), 0.90(3H,s, 24-H3), 0.94(3H, d, J=6.7 Hz, 30-H3), 0.99(3H,s, 25- H3), 1.20 (3H, s, 27-H3), 1.36 (3H,s, 29-H3), 2.04 (3H,s, COCH3), 2.51 (lH, s, 18-H), 4.47(lH, m, 3-H), 5.29(lH, br s, 12-H).
2a, 3~-Dihydroxy-12-oleanen-28-oic acid (4) A colourless powder, [a]D +30.5(c 1.2, Pyridine); mp 263-265 ~C; C30H48O4;EIMS m/z:472[M]+, 454, 426, 408, 248, 235, 233, 223, 219, 203, 189, 133;IR KBr (cm~l): 3440, 1690;1H-NMR (pyridine-d5, 500 MHz): ~ 0.95 (3H,s, 26-H3), 1.00(3H,s, 23-H3), 1.01 (3H,s, 24-H3), 1.03(3H, d,J=6.4 Hz, 30-H3), 1.08 (3H,s, 25-H3), 1.27 (3H,s, 27-H3), 2.25 (lH, dd, J=14, 4 Hz, 18-H), 3.40 (lH,d,J=10 Hz, 3-H), 4.10 (lH,dd,J=10, 10, 4.0 Hz, 2-H), 5.25 (lH, br t,J=4 Hz, 12-H).l3C-NMR (pyridine-d~, 125 MHz): ~ = 46.42(t,Cl), 68.55 (d, C2), 83.78(d, C3), 39.80(s, C4), 55.88(d, C5), 18.81 (t, C6), 33.18(t,C7)39.80 (s, C8), 48.13 (d, C9), 38.51(s, C10), 23.73 (t, Cll), 122.43 (d, C12),144.81(s,C13), 41.96(s,C14), 28.24(t,C15), 23.89(t,C16), 47.71(s,C17), 42.18 (d, C18), 46.63 (t, Cl9), 30.91 (s, C20), 34.19 (t, C21), 33.18 (t, C22), 29.30 (q, C23), 17.44 (q, C24), 16.81 (q, C25), 17.62 (q, C26), 26.13 (q, C27), 180.13 (s,C28), 33.18(q,C29), 23.66(q,C30).

Fraction 3 (4.5 g) was purified on a column of silica gel (250 g) and eluted with CHCl3-MeOH=80:20. Fractions of 15 ml each were collected and examined on silica gel TLC (CHCl3-MeOH, 8:2). Fractions with similar Rf (TLC) were combined and concentrated to afford afford two fractions A and B. Fraction A was purified by preparative TLC (Ethyl ether- Acetone, 7:3) to give 2a, 3a, 19a-trihydroxy-12-ursen-28-oic acid (5) (50 mg) and 2a, 3~, 19a-trihydroxy-12-ursen-28-oic acid (6) (30 mg), and fraction B (0.21 g) was purified by further silica gel (20.0 g) chromatography and eluted with Ethyl ether-EtOAc (6:4) to yield 2a, 3~, 19a, 23-tetrahydroxy-12-ursen-28-oic acid (7) (80 mg).
2a, 3a, 19a -Trihydroxy-12-ursen-28-oic acid (5) A colourless powder, [a]D +12.0 (c 1.0, Pyridine); mp 270 ~C;
C30H48O5; EIMS m/z: 488 [M]+, 470, 452, 442, 424, 370, 352, 293, 246, 219, 218, 146; IR KBr (cm~1): 3400, 1690, 1630, 820, 805; 1H-NMR (pyridine-d5, 500 MHz): ~ 0.92 (3H, s, 26-H3), 1.03 (3H, s, 23-H3), 1.12 (3H, s, 24-H3), 1.17 (3H, d, J=6.4 Hz, 30-H3), 1.24 (3H, s, 25-H3), 1.45 (3H, s, 27-H3), 1.73 (3H, s,29-H3), 3.07 (lH, s, 18-H), 3.43 (lH, dd, J=12.1, 5.0 Hz, 2- H), 5.61 (lH, br s,12-H). l3C-NMR (pyridine-d5, 125 MHz): â= 42.20 (t, C1), 66.08 (d, C2), 79.34 (d, C3), 38.68 (s, C4), 48.80 (d, C5), 18.62 (t, C6), 33.55 (t, C7), 40.61 (s, C8), 47.65 (d, C9), 38.80 (s, C10), 24.09 (t, C11), 128.01 (d, C12), 139.96 (s, C13), 42.37 (s C14), 29.44 (t, C15), 26.40 (t, C16), 48.29 (s, C17), 54.61 (d, C18), 72.71 (s, C19), 42.89 (d, C20), 27.09 (t, C21), 38.49 (t, C22), 29.26 (q, C23), 22.28 (q, C24), 16.77 (q, C25), 17.29 (q, C26), 24.64 (q, C27), 180.64 (s, C28), 26.96 (q,C29), 16.65 (q, C30).
2a, 3,~, 19a -Trihydroxy-12-ursen-28-oic acid (6) A colourless powder, [a]D-20 (c 0.8, Pyridine); mp 272-274 oc;
C30H48O5; EIMS m/z: 488 [M]+, 470, 452, 370, 264, 246, 218, 146; IR KBr (cm~l): 3450, 1690; lH-NMR (pyridine-d5, 500 MHz): ~ 0.82 (3H, s, 26-H3), 0.87 (3H, s, 23-H3), 0.93 (3H, s, 24-H3), 1.05 (3H, d, J=6.4 Hz, 30-H3), 1.24 (3H, s, 25-H3), 1.45 (3H, s, 27-H3), 1.73 (3H, s, 29-H3), 3.07 (lH, s, 18- H), 3.43 (lH, dd, J=12.1, 5.0 Hz, 2-H), 5.61 (lH, br s, 12-H). l3C-NMR (pyridine-d5, 125 MHz): ~ = 48.00 (t, Cl), 68.93 (d, C2), 83.91 (d, C3), 38.23 (s, C4), 55.51 (d, C5), 18.92 (t, C6), 33.56 (t, C7), 40.72 (s, C8), 47.85 (d, C9), 39.31 (s, C10), 24.32 (t, Cll). 128.00 (d, C12), 139.95 (s, C13), 42.12 (s, C14), 28.91 (t, C15), 26.52 (t, C16), 48.21 (s, C17), 54.52 (d, C18), 72.21 (s, Cl9), 42.01 (d, C20), 25.95 (t, C21), 38.05 (t, C22), 28.93 (q, C23), 16.41 (q, C24), 16.76 (q, C25), 17.43 (q, C26), 24.52 (q, C27), 180.52 (s, C28), 27.62 (q, C29), 17.15 (q, C30).
2a, 3,~, 19a, 23-Tetrahydroxy-12-ursen-28-oic acid (7) A colourless powder, [a]D +18.6 (c 0.9, Pyridine); mp 301-304 ~C; C30H48O6; EIMS m/z: 504 [M]+, 264, 246, 219, 201; IR KBr (cm-l): 3400, 1700, 1630, 835; lH-NMR (pyridine-d5, 500 MHz): ~ 1.06 (3H, s, 26-H3), 1.09 (3H, s, 24-H3), 1.11 (3H, d, J=6.0 Hz, 30-H3), 1.12 (3H, s, 25-H3), 1.42 (3H, s,27-H3), 1.65 (3H, s, 29-H3), 3.03 (lH, s, 18-H), 3.40 (lH, d, J=9.5, 3-H), 3.71 (lH, d, J=10.1, 23-Ha), 4.17 (lH, d, J=10.1, 23-Hb), 4.24 (lH, t, J=ll.0, 9.5 Hz, 2-H), 5.57 (lH, br s, 12-H). l3C-NMR (pyridine- d5, 125 MHz): ~ = 47.84 (t, Cl), 68.87 (d, C2), 78.34(d, C3), 43.62 (s, C4), 48.04 (d, C5), 18.69 (t, C6), 33.20 (t, C7), 40.47 (s, C8), 47.84 (d, C9), 38.40 (s, C10), 24.17 (t, Cll). 127.95 (d, C12), 140.03 (s, C13), 42.18 (s, C14), 29.29 (t, C15), 26.40 (t, C16), 48.29 (s, C17), 54.61 (d, C18), 72.71 (s, Cl9), 42.36 (d, C20), 26.96 (t, C21), 38.51 (t, C22), 66.66 (t, C23), 14.31 (q, C24), 17.34 (q, C25), 17.40 (q, C26), 24.69 (q, C27), 180.83 (s,C28), 27.12 (q, C29), 16.78 (q, C30).
A portion of fraction 4 (3.0 g) was subjected sephadex LH 20 (50.0 g) and eluted with H2O-MeOH gradient to give 28-D-glucoside of 2a, 3~, l9a- trihydroxy-12-ursen-28-oic acid (8) (25 mg), 28-D-glucoside of 2a, 3~, l9a, 23-tetrahydroxy-12-ursen-28-oic acid (10) (520 mg) and 3~-D-glucoside-~-sitosterol (11) (72 mg).
28-D-glucoside of 2a, 3~, 19a -trihydroxy-12-ursen-28-oic acid (8) Colourless needles, [a]D +36.5 (c 1.5, Pyridine); mp 302-304 ~C;
C36H56O4; EIMS m/z: 488 (M+-C6Hl0O5), 470, 452, 442, 424, 246, 198, 173, 146; IR KBr (cm~l): 3400, 1680, 1640; lH-NMR (pyridine-d5, 500 MHz): ~

1.07 (3H, d, J=7.5 Hz, 30-H3), 1.05, 1.08, 1.19, 1.25, 1.40, 1.66 (each 3H, 23-29-H3), 2.92 (lH, s, 18-H), 3.38 (lH, d, J=9.3 Hz, 3-H), 4.07 (lH, m, 2-H), 4.12-4.51 (sugar protons), 5.53 (lH, br s, 12-H), -H), 6.26 (lH, d, J=8.1 Hz, l'-H). l3C-NMR (pyridine-d5, 125 MHz): ~ = 47.97 (t, Cl), 68.65 (d, C2), 83.82 (d, C3), 38.48 (s, C4), 55.95 (d, C5), 19.03 (t, C6), 33.49 (t, C7), 40.60 (s, C8), 47.84 (d, C9), 39.82 (s, C10), 24.16 (t, Cll). 128.35 (d, C12), 139.25 (s, C13), 42.12 (s, C14), 29.18 (t, C15), 26.68 (t, C16), 48.62 (s, C17), 54.39 (d, C18), 72.66 (s, Cl9), 42.12 (d, C20), 26.09 (t, C21), 37.69 (t, C22), 29.33 (q, C23), 16.68 (q, C24), 16.96 (q, C25), 17.63 (q, C26), 24.56 (q, C27), 177.03 (s, C28), 27.01 (q,C29), 17.45 (q, C30), 95.80 (d, Cl'), 74.00 (d, C2'), 78.86 (d, C3'), 71.24 (d, C4'), 79.17 (d, C5'), 62.35 (t, C6').
~lk~line hydrolysis of compound 8 Compound 8 (10 mg) was added to a solution prepared from methanol (2.5 ml), water (1.5 ml) and potassium hydroxide (100 mg). The resulting mixture was heated under reflux for 4 h, poured into water, acidified by addition of hydrochloric acid, and extracted with ethyl acetate 30 ml x 3. From the ethyl acetate layer, aglycone (6 mg) was identified as tormentic acid by comparison of lH And l3C-NMR data with the those of compound 6.
28-D-glucoside of 2a, 3~, l9a, 23 -tetrahydroxy-12-ursen-28-oic acid (9) Colourless needles, [a]D +36.5 (c 1.2, Pyridine); mp 235-237 ~C;
C36H58Oll; EIMS m/z: 504 (M+-C6Hl0O5), 264, 246, 219, 201; IR KBr (cm~
3407, 1685, 1620; lH-NMR (pyridine-d5, 500 MHz): ~ 1.06 (3H, d, J=6.5 Hz, 30-H3), 1.09, 1.15, 1.25, 1.37, 1.62 (each 3H, 24-29 H3), 2.92 (lH, s, 18-H), 3.71 (lH, d, J=10.5 Hz, 3-H), 4.07 (lH, m, 2-H), 4.13-4.50 (sugar protons), 5.54 (lH, br s, 12-H), 6.30 (lH, d, J=8.1 Hz, l'-H). l3C- NMR (pyridine-d5, 125 MHz): ~ = 47.98 (t, Cl), 68.95 (d, C2), 78.39 (d, C3), 43.63 (s, C4), 48.03 (d, C5), 18.76 (t, C6), 33.19 (t, C7), 40.68 (s, C8), 47.89 (d, C9), 38.42 (s, C10), 24.24 (t, Cll). 128.41 (d, C12), 139.32 (s, C13), 42.13 (s, C14), 29.23 (t, C15), 26.13 (t, C16), 48.64 (s, C17), 54.44 (d, C18), 72.67 (s, Cl9), 42.20 (d, C20), 26.72 (t, C21), 37.74 (t, C22), 66.66 (t, C23), 14.35 (q, C24), 17.52 (q, C25), 17.56 (q, C26), 24.56 (q, C27), 177.00 (s, C28), 27.00 (q, C29), 16.71 (q, C30), 95.85 (d, C1'), 74.08 (d, C2'), 79.25 (d, C3'), 71.29 (d, C4'), 78.97 (d, C5'), 62.40 (t, C6').
,All~line hydrolysis of compound 9 Compound 9 (20 mg) was added to a solution prepared from methanol (2.5 ml), water (1.5 ml) and potassium hydroxide (150 mg). The resulting mixture was heated under reflux for 4 h, poured into water, acidified by addition of hydrochloric acid, and extracted with ethyl acetate (30 ml x 3). From the ethyl acetate layer, the aglycone (14 mg) was identified as 19-hydroxy-asiatic acid by comparison of lH and 13C-NMR
data with the those of compound 7.
3-D-gluco~ylal~osyl-sitosterol (10) Colourless needles, C35H60O6; 13C-NMR (pyridine-d5, 125 MHz): ~ = 37.47 (t, C1), 30.15 (t, C2), 78.11 (d, C3), 39.97 (t, C4), 140.88 (d, C5), 121.85 (d, C6), 32.16 (t, C7), 32.06 (d, C8), 50.35 (d, C9), 36.89 (s, C10), 21.27 (t, C11). 39.24 (t, C12), 42.49 (s, C13), 56.86 (d, C14), 24.50 (t, C15), 28.52 (t, C16), 56.26 (d, C17), 12.18 (q, C18), 19.42 (q, C19), 36.36 (d, C20), 19.03 (q, C21), 34.21 (t, C22), 26.41 (t, C23), 46.05 (d, C24), 29.47 (d, C25), 19.99 (q, C26), 19.25 (q, C27), 23.39 (d, C28), 12.01 (q, C29), 102.42 (d, C1'), 75.06 (d, C2'), 78.24 (d, C3'), 71.57 (d, C4'), 78.29 (d, C5'), 62.71 (t, C6').
Fraction 5 was chromatographed on a silica gel column (Hexane: EtOAc(8:2) to give ~-sitosterol (11) (25 mg).
Biological Assay Recombinant HIV-1 protease was prepared from the expression vector, PGEX-PR 107, in E. coli DH5a and was purified according to the method established in Dr. Wan's laboratory [14].
The proteolytic activity of HIV-1 PR was measured by Dr.
Wan's group at the Biochemistry Department using the synthetic heptapeptide Ser-Gln-Asn-Tyr-Pro-Ile-Val (SQNYPIV), corresponding to the p24-pl7 cleavage site in the natural gag precursor, as substrate. The products of cleavage were analyzed by reversed-phase high-perforrnance liquid chromatography (RP-HPLC, Ultrasphere ODS, 5 ,u, 4.6 mm x 15 cm, Beckman, CA, USA) with a 7-33% acetonitrile gradient in 0.1%
trifluoroacetic acid (TFA). Product peak areas were integrated by a data system (Chromatopac C-R3A, Shimadzu, Japan) and compared with the standard peptide SQNY, which is expected to be generated from heptapeptide SQNYPIV after HIV-1 protease digestion. The extinction coefficient of the peptide SQNY was used to estimate the rate of proteolysis of the heptapeptide substrate. The assay was perforrned in a volume of 140 ~l containing 90 pmole of substrate, 2 U of HIV-1 protease and compound solution in 0.1 M NaOAc buffer, pH 5.5, with 1 M
(NH4)2SO4, at 37~C. The reaction which proceeded for 2 h was terminated by the addition of 20 ~l of aqueous 10% trifluoroacetic acid.
The supernatant obtained by centrifuging at 12,000 rpm for 3 min was then analyzed by the hplc method. The unit of enzyme activity was defined as the amount of enzyme which yields 1 nmol of tetrapeptide SQNY under the above conditions in 1 min.
The proteolytic activity of HIV-1 protease inhibited by the compound was calculated as the activity of the control subtracting that of the sample, and then divided by itself.
RESULTS
Isolation and structure determination of the compounds from Geum ja ponicum The active EtOAc soluble fraction was subjected to silica gel column chromatography to give five fractions (Figure 13). Further silica gel and sephadex LH 20 column gave a new triterpene acid, 2-alpha-19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid (1) together with eight known triterpenes, ursolic acid (2), epipomolic acid (3), maslinic acid (4), euscaphic acid (5), tormentic acid (6), 19-hydroxyasiatic acid (7), 28-D-glucoside of tormentic acid (8) and 28-D-glucoside- 19-hydroxyasiatic acid (9) as well as 3-D-glucoside--sitosterol (10) and -sitosterol (11). The structures of these compounds were elucidated by various spectroscopic means and are shown in Figures 1-10.
1. 2a, 19a-Dihydroxy-3-oxo-12-ursen-28-oic acid (1) Compound (1) was obtained as a colourless powder. The IR
spectrum indicated the presence of hydroxyl group (3600-3200 cm-l), carboxylic acid group (1684 cm-1), and double bond (1650 cm-l). Its molecular formula was determined to be C30H46O5 by high-resolution mass spectroscopy (M+ 486.3373).
The l3C-NMR together with DEPT spectrum revealed thirty carbon signals including characteristic signals due to a trisubstituted double bond [~ 127.55 (d) and 140.07 (s)], a carboxylic acid group (~ 180.65), a ketone carbonyl group (~ 216.41) and two alcoholic carbons [~ 69.73 (d) and 72.67 (s)]. The lH-NMR spectrum showed the presence of a hydroxyl methine proton at ~ 4.80 (lH, dd, J=12.5, 6.5 Hz), an olefinic proton at 5.55 (lH, br s), six singlets at ~ 1.01 (3H), 1.09 (3H), 1.15 (3H), 1.22 (3H), 1.42 (3H), 1.64 (3H) for six tertiary methyl group, and a doublet at ~ 1.12 (3H, d, J=6.6 Hz) for a secondary methyl group. The secondary methyl signal on ring E provides a most useful indicator for the presence of an urs-12-ene skeleton. In addition, the signals in its 13C- NMR at ~ 127.55 (d) and 140.07 (s) are characteristic for C-12/C-13 double bond in the ursene type structure [15-18]. The presence of the ursene skeleton was further verified using 2D NMR spectroscopy including HMBC
measurement. The 1H-l3C long-range COSY and 1H-1H COSY spectrum of this compound gave very good information for establishing the assignment of the partial structure of C-1 to C-3 carbons. There were long range correlations between H-2 proton at ~ 4.80 and the methylene carbon at ~ 50.10 (C-1) in the HMBC spectrum, and a cross- peak between the signals of H-2 proton and H-1 proton in 1H-1H COSY spectrum, which indicated that the ketone carbonyl group must be either at position C-1 or C-3. Furthermore, the observation of the correlation between the ketone carbonyl group and two methyl carbons (C-23 at ~ 25.28 and C-24 at ~
21.77) suggested the ketone must be at C-3 position. The secondary hydroxyl group was assigned to C-2 due to the correlation between the C-1 methylene carbon at ~ 50.10 and the C-25 methyl group. Splitting patterns for the hydroxyl methine proton (~ 4.80, dd, J=12.5, 6.5 Hz) showed that the hydroxyl group is equatorial. The stereochemistry of the hydroxyl group was further confirmed by NOE experiments. In particular, on irradiation of signal of H-2 proton at ~ 4.80, the NOE effect was observed on the H-24 at ~ 1.01 and H-25 at ~ 1.15 methyl protons.
Furthermore, irradiation of the signal of the H-25 methyl protons gave the NOE enhancements for H-26 methyl protons at ~ 1.09, H-24 methyl protons and H-2 proton. These data clearly suggested the equatorial configuration of the C-2 hydroxyl group, as indicated in structure. On the other hand, a signal at ~ 3.03 (lH, s, H-18) assigned to the bridgehead H-18 suggested the presence of a 19-O-substituted urs-12-ene skeleton [19]. The hydroxyl group at C-19 was assigned to the a position by analogy with the 13C-NMR chemical shifts of similarly substituted triterpene acids [20]. The carboxylic acid group was assigned to C-17 on the basis of a HMBC correlation to H-18.
Furthermore, the structure of compound 1 was supported by analysis of ion peaks in the mass spectrum. The peaks at m/z 246 [264 -H2O]+, 220 [264 -CO2]+, 219 [264 -CO2H]+ and 218 [264 -HCO2H]+
corresponded to the typical retro-Diels-Alder (R. D. A.) cleavage fragments commonly found in the spectra of urs-12-ene derivatives possessing a carboxyl group on C-17 and a hydroxyl substituent on ring D
or E. Besides, there was a base peak at m/z 146 apparently due to the secondary retro- Diels-Alder cleavage of the m/z 218 fragment ion. These fragmentations resemble very closely those of tormentic acid [21]. Other important ions were at m/z 438, 366 and 220 which indicated that a secondary hydroxyl group and a ketone group were located in the A/B
ring portion (Figure 14) .
The complete assignment of all the carbons was determined (Table 2) with the help of DEPT, lH-lH COSY, lH-l3C COSY, NOE and followed by analysis of the HMBC spectral data (Table 3). Based on all above information, compound 1 was found to be a new triterpene acid, and the structure of the natural product was formulated as 2a, 19a-dihydroxy-3- oxo-12-ursen-28-oic acid as shown in Figure 1.
2. Ursolic acid (2) Compound 2 was obtained as a colourless powder mp 290-292 ~C, [alD ~ 65 (MeOH, c 1.0). Its IR spectrum showed the presence of hydroxy group (3425 cm-1), carboxylic acid group (1689 cm-1) and olefinic group (1640 cm-1). The molecular formula of compound 2 was determined to be C30H48O3 by mass spectromscopy.
The 13C-NMR together with DEPT spectrum revealed thirty carbon signals including characteristic signals due to a trisubstituted double bond [ 125.66 (d) and 139.28 (s)], a carboxylic acid group [ 179.87 (s)]
and a hydroxy methine carbon [ 78.15 (d)]. The 1H-NMR spectrum showed the presence of a hydroxy methine proton at 3.46 (lH, t, J=7.9 Hz), an olefinic proton at 5.50 (lH, br s), five singlets at 0.91 (3H), 1.02 (3H), 1.06 (3H), 1.24 (3H), 1.29 (3H) for five tertiary methyl groups and two doublets at 1.00 (6H, d, J=6.0Hz) for two secondary methyl groups. The secondary methyl groups at ring E and the signals in its 13C-NMR at 125.66 (s) and 139.28 (s) are characteristic for C-12/C-13 double bond in ursene-type structure. The splitting pattern of the hydroxy methine proton supported the equatorial 3-hydroxyl group.
The mass spectrum was similar to that of ursolic acid and exhibited peaks at m/z 456 [M]+, 410 [M-HCOOH]+, 248, 203 [248-COOH]+
and 157. By direct comparision of data ( NMR, IR, mp and MS) with those reported in the literature [15], compound 20 was identified to be ursolic acid as shown in Figure 2.

3. 3, 19-Dihydroxy-12-ursen-28-oic acid (3) Compound 3 was obtained as a colourless powder, [a]D
+36.5. Its IR spectrum showed bands for hydroxyl (3427 cm-1), carbonyl (1695 cm-l), and olefin (1630 cm-1) absorptions. On acetylation with acetic anhydride, it formed a mono-acetate (3a). The molecular formula of compound 3 was determined to be C30H48O4 by mass spectroscopy (M+
472.3). Compound 3 showed diagnostically important peaks at m/z 246, 218, 207, 146 due to retro-Diels-Alder fragmentation around ring C, which are consistent with the fragmentation pattern characteristic for the amyrin series [22]. From the above mass spectral fragments, it was also evident that the secondly hydroxyl group was present in the A/B ring portion and its location at C-3 was highly probable on a biogenetic basis [23]. The lH- NMR spectrum of 3 exhibited signals for six tertiary methyl groups, one proton singlet at 3.06 (18-H), and one proton multiplet at 5.61 (12-H), as expected for an 12-ursen skeleton bearing an with alpha-hydroxy at C-19 [24-25]. Also, it showed a triplet-like signal centered at 3.43 (lH, t, J= 3 Hz, 3-H), whose chemical shift and splitting pattern were typical of a 3-alpha-hydroxy group [26].
The 13C-NMR together with DEPT spectrum revealed thirty carbon signals including characteristic signals due to a trisubstituted double bond [~ 128.06 (d) and 139.98 (s)], a carboxylic acid group (~ 180.72) and two alcoholic carbons [~ 78.22 (d) and 72.72 (s)]. By direct comparison of data ( NMR, IR, mp and MS) with those reported in the literature [27], compound 3 was identified to be 3, 19-dihydroxy-12-ursen-28-oic acid as shown in Figure 3.
4. Maslinic acid (4) Compound 4 was obtained as a colourless powder, mp 263-265~C, [a]D +42.5 (pyridine, c 1.2), C30H48O4 [M]+, Its IR spectrum showed bands for hydroxyl (3440 cm-1), carboxylic acid (1690 cm-1), and olefinic (1630 cm- 1) absorptions.

The lH-NMR spectrum of 4 exhibited signals for seven tertiary methyl groups, one proton multiplet at 2.25 (lH, dd, J=14, 4 Hz, 18-H) and one proton multiplet at 5.25 (lH, br t, J=4 Hz, 12-H), as expected for an 12- oleanene skeleton. The l3C-NMR together with DEPT
spectrum revealed thirty carbon signals including characteristic signals due to a trisubstituted double bond [o 122.43 (d) and 144.81 (s)], a carboxylic acid group (~ 180.13) and two alcoholic carbons [~ 68.55 (d) and 83.78 (s)].
By direct comparison of data ( NMR, IR, mp and MS) with those reported in the literature [28], compound 4 was identified to be maslinic acid as shown in Figure 4.
5. 2, 3, 19-Trihydroxy-12-ursen-28-oic acid (5) Compound 5 is a colourless powder, mp 270~C, [a]D +12 (c 1.0, MeOH). Its IR spectrum showed a hydroxyl group (3400 cm-l), carboxylic acid group (1690 cm-l) and a trisubstituted double bond (1630, 820, 805 cm-l). Its mass spectrum showed characteristic fragments at m/z 264 (a), 219 (b), 146 (c), 370 (d). These observations indicated that the basic skeleton of compound 5 was a 12-ursen-28-oic acid possessing one hydroxyl group at C-19.
The l3C-NMR together with DEPT spectrum revealed thirty carbon signals including characteristic signals due to a trisubstituted double bond [~ 128.01 (d) and 139.96 (s)], a carboxylic acid group (~ 180.64) and two alcoholic carbons [~ 66.08 (d) and 79.34 (s)]. By direct comparison of data ( NMR, IR, mp and MS) with those reported in the literature [29], compound 5 was identified to be 2, 3, 19-trihydroxy-12-ursen-28-oic acid or euscaphic acid as shown in Figure 5.
6. 2, 3 19-Trihydroxy-12-ursene-28-oic acid (6) Compound 6 is a colourless powder with mp. 272-2740C and [a]D -20 (pyridine, c 0.8). Its IR spectrum showed a hydroxyl group (3450 cm-l) and carboxylic acid group (1690 cm-l). Its mass spectrum showed m/z 488 [M]+ and characteristic fragments at m/z 264, 219, 201 and 370 as compound 5. The difference between compound 6 and compound 5 was mainly in the l3C-NMR signals at C-2 ( 68.93 and 66.08 respectively) and C-3 ( 83.91 and 79.34 respectively). From the comparison of the 13C-NMR
of 27 with tormentic acid [30, 31], the structure of compound 6 was deduced as 2, 3, 19-trihydroxy-12- ursene-28-oic acid or tormentic acid.
7. 2, 3, 19, 23-Tetrahydroxy-12-ursen-28-oic acid (7) Compound 7 was obtained from fraction 3 with mp 301-304 ~C, [a]D +18.6. Its EI mass spectrum showed the molecular formula C30H48O6 corresponding to the molecular weight 504 [M]+. The characteristic fragmentations at m/z 264, 246, 219, 201, 146 are due to the retro-Diels- Alder cleavage of urs-12-en-28-oic acid derivatives bearing one free hydroxyl group in D or E ring [32]. The 13C-NMR spectrum of 7 was the same as that of 2, 3, 19, 23-tetrahydroxy-12-ursene-28-oic acid [33].
The 1H-NMR spectrum of 7 showed six methyl groups including a secondary methyl group in ring E for 12-ursen skeleton, a hydroxy methylene at 3.71 (lH, d, J =10.2, 23-Ha) and 4.17 (lH, d, J =10.2, 23- Hb).
By direct comparison of data ( NMR, IR, mp and MS) with those reported in the literature [33,34], compound 7 was identified to be 2, 3, 19, 23-tetrahydroxy-12-ursen-28-oic acid or 19-alphahydroxyasiatic acid as shown in Figure 7.
8. 28--D-glucoside of 2, 3, 19-trihydroxy-12-ursen-28-oic-acid (8) A series of conventional separations including repeated silica gel chromatography column and sephadex LH 20 chromatography column of fraction 5 yielded compounds 8-10. Compound 8 was obtained as colourless needles. The 13C-NMR spectrum of 8 showed a signal at 95.80 for an anomeric carbon confirming the presence of a sugar moiety, and a triterpene aglycone with an ursolic acid type skeleton [C-12 and C-13 at 128.35 (d) and 139.25 (s), respectively]. Its 1H-NMR spectrum also displayed signals at 6.26 (lH, d, J= 8.1 Hz, 1'-H) for an anomeric proton and seven methyl groups including a secondary methyl group between 1.07 and 1.66 for the triterpene aglycone methyl protons. Basic hydrolysis of 8 provided glucose and 8, indicating that the glycosidation site on the aglycone is at the carboxyl function group. The identity of aglycone was confirmed as tormentic acid by comparison with an authentic sample as well as by cochromatography with compound 6. Thus, the structure of 8 5 is identified to be tormentic acid ester glucoside (28--D-glucoside of 2, 3, 19-trihydroxy-12-ursen-28-oic-acid) [35-36] as shown in Figure 8.
9. 28-D-glucoside of 2,3,19a,23-tetrahydroxy-12-ursen-28-oic acid (9) The 13C-NMR spectrum of 9 was quite similar to that of 8 with the only major difference arising from the appearance of a -CH2OH
carbon signal at 66.66 in 9 and the absence of one of the signals corresponding to a methyl group in 8. Compound 9 underwent basic hydrolysis to yield glucose, indicating that the sugar is attached to the aglycone through ester linkage. The site of attachment for the-COOH
group was established by comparison of the 13C-NMR spectral data of 9 with those reported in the literature for 23-hydroxyl tormentic acid glucoside [9]. The aglycone was identified to be asiatic acid by comparison with compound 7. Thus, compound 9 was identified as 23-hydroxyl-tormentic acid glucoside (28-D-glucoside of 2, 3, 19a, 23-tetrahydroxy-12-ursen-28-oic acid) as shown in Figure 9.
10. 3-D-glucopyranosvl sitosterol (10) The 13C-NMR spectrum of 10 showed a signal at 102.42 for an anomeric carbon confirming the presence of a sugar moiety, and a -sitosterol aglycone skeleton [C-5 and C-6 at 140.88 (s) and 121.85 (d) respectively]. The 1H-NMR spectrum also showed an anomeric proton at 4.94 (lH, d, J=7.7 Hz, 1'-H) and signals between 0.68 and 1.00 for six methyl group protons. Compound 1 0 was identified to be 3-D-glucopyranosyl sitosterol by comparison of data (13C- and 1H-NMR data) with those reported in the literature [37-39] as shown in Figure 10.
Compound 1 1 was identified to be ,~-sitosterol by comparison with an authentic sample.

Anti-HIV-1 Protease Activity Of The Isolated Compounds Six of the compounds isolated from the EtOAc soluble fraction were tested for their anti-HIV-1 protease activity by HPLC assay [14]. As shown in Table 4, 2a, 19a-dihydroxy-3-oxo-12-ursen-28-oic acid (1) (72%), ursolic acid (2) (85%) and maslinic acid (4) (100%) exhibited stronger activity than the others [epipomolic acid (3) (42%) and tormentic acid (6) (49 %), respectively] at the concentration of 17.9 ~g/ml. Maslinic acid (4) was the most active, while euscaphic acid (5) did not show activity at the tested concentration. This is the first report of anti-HIV-1 activity of this plant. The results of the experiments obtained in the present study indicate that compounds 1, 2 and 4 may contribute towards the anti-HIV-1 protease activity of the EtOAc soluble fraction of G.
japonicum .
While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent compounds included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Table 1 Anti-HIV-1 protease activity of fractions from G. japonicum Fractions ~hibition(%) 0.0179 mg/ml MeOH extract 100.00 Hexane soluble fraction 4.86 EtOAc soluble fraction 100.00 H2O soluble fraction 56.99 Table 2 13C-NMR spectral data of compound 1 (~ relative to TMS in pyridine-d5, 125 MHz) C ~ C ~ C

50.10 t 11 24.12 t 21 26.9 2 t 2 69.73d 12 127.55d 22 38.45 t 3 216.41 s 13 140.07 s 23 25.28 q 4 48.10 s 14 42.16 s 24 21.77 q 57.67 d 15 29.26 t 25 17.30 q 6 19.55 t 16 26.31 t 26 16.77 q 7 33.14 t 17 48.25 s 27 24.66 q 8 40.37 s 18 54.56 d 28 180.65 s 9 47.33 d 19 72.67 s 29 27.06 q 37.90s 20 42.35d 30 15.95q Table 3 H-13C Long-range correlation detected in the HMBC spectrum of compound 1 (~ relative to TMS in pyridine-d5) Proton~ Correlated C

1.01 (H-24) 216.41 (C-3),48.10 (C-4),57.67 (C-5) 1.09 (H-26) 33.14 (C-7),40.37 (C-8),47.33 (C-9),42.16 (C-14) 1.12 (H-30) 72.67 (C-19),26.92 (C-21) 1.15 (H-25) 50.10 (C-1),57.67 (C-5),47.33 (C-9) 1.22 (H-23) 216.41 (C-3),48.10 (C-4),57.67 (C-5) 1.42 (H-29) 54.56 (C-18),72.67 (C-19),42.35 (C-20) 1.64 (H-27) 40.37 (C-8),140.07 (C-13),42.16 (C-14),29.26 (C-15) 1.87 (H-9) 50.10 (C-l), 40.37 (C-8), 37.90 (C-10), 17.30 (C-25), 16.77 (C-26) 2.48 (H-l) 69.73 (C-2),216.41 (C-3),37.90 (C-10),17.30 (C-25) 3.03 (H-18) 127.55 (C-12), 140.07 (C-13), 42.16 (C-14), 26.31 (C- 16), 48.25 (C-17),72.67 (C-19),42.35 (C-20),180.65 (C-28) 4.80 (H-2) 50.10 (C-l) 5.55 (H-12) 47.33 (C-9),42.16 (C-14),54.56 (C-18) a ~ ppm.

Table 4 Inhibitory activity of compounds against HIV-l protease using HPLC
assay Compound Inhibition (%) 72.19 2 85.07 3 42.18 4 100.0 6 49.47 FULL CITATIONS FOR REFERENCES REFERRED TO IN THE
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Claims (12)

1. A composition for treating a retroviral infection comprising an isolated triterpene in admixture with a physiologically acceptable diluent or carrier.

2. A composition according to claim 1 wherein said triterpene is of the Formula I

wherein R1 and R2 are identical or different and are independently selected from H,OH,=O, C1-C10 unbranched alkoxy group, C1-C10 branched alkoxy group, C1-C10 unbranched acyloxyl group and C1-C10 branched acyloxy group; R3, R4, R5 and R8 are identical or different and are independently selected from H, C1-C10 branched alkyl group, C1-C10 unbranched alkyl group, C1-C10 branched alkoxy group, C1-C10 unbranched alkoxy group, a C1-C10 branched acyloxy group, a C1-C10 unbranched acyloxy group and a =O group; R6, R7 and R9 are identical or different and are independently selected from a COOH group, a -COOCH3 group, a -CH2OH group and a C1-C10 unbranched alkyl group.

3. A composition according to claim 1 comprising at least one triterpene is selected from the group consisting of 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid; ursolic acid; epimolic acid; maslinic acid; tormentic acid; 19-alpha-hydroxyasiatic acid; 28-beta-D-glucoside of tormentic acid; and 28-beta-D-glucoside-19-alpha-hydroxyasiatic acid and pharmaceutically acceptable salts thereof.

4. A composition according to claim 1 wherein said triterpene is 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid.

5. A composition according to claim 4 further including ursolic acid and maslinic acid.

6. A method of treating a retroviral infection comprising administering a therapeutically effective amount of a triterpene to a mammal in need of such a treatment.

7. A method according to claim 6 wherein said triterpene is of the Formula I

wherein R1 and R2 are identical or different and are independently selected from H,OH,=O, C1-C10 unbranched alkoxy group, C1-C10 branched alkoxy group, C1-C10 unbranched acyloxyl group and C1-C10 branched acyloxy group; R3, R4, R5 and R8 are identical or different and are independently selected from H, C1-C10 branched alkyl group, C1-C10 unbranched alkyl group, C1-C10 branched alkoxy group, C1-C10 unbranched alkoxy group, a C1-C10 branched acyloxy group, a C1-C10 unbranched acyloxy group and a =O group; R6, R7 and R9 are identical or different and are independently selected from a COOH group, a -COOCH3 group, a -CH2OH group and pharmaceutically acceptable salts thereof.

8. A method according to claim 6 comprising administering at least one triterpene selected from the group consisting of 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid; ursolic acid; epimolic acid;
maslinic acid; tormentic acid; 19-alpha-hydroxyasiatic acid;
28-beta-D-glucoside of tormentic acid; and 28-beta-D-glucoside-19-alpha-hydroxyasiatic acid and pharmaceutically acceptable salts thereof.

9. A method according to claim 6 comprising administering at least one triterpene selected from the group consisting of 2- alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid; ursolic acid; and maslinic acid.

10. A method according to claim 6 wherein said triterpene is 2-alpha,19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid.

11. A method according to claim 6 wherein said retroviral infection is Human Immunodeficiency Virus (HIV).

12. A compound 2-alpha, 19-alpha-dihydroxy-3-oxo-12-ursen-28-oic acid.