TITLE OF INVENTION

Asiatic acid derivatives, its manufacturing method and dermatological agent containing it

TECHINICAL FIELD

This invention relates to asiatic acid derivatives expressed by the following chemical formula, and its pharmaceutically acceptable salt or eεter, its manufac¬ turing method and dermatological agent containing it.

Wherein;

R, is selected from the group consisting of hydrogen, hydroxy which may be protected by acetyl or bentyl, methyl, ethyl, methoxy, ethoxy, vinyl, ethynyl, cyano, azaide, methanesulfonyloxy, phenylthio, or (methylthio) thiocarbonyloxy;

R_ is selected from the group consisting of

hydrogen, hydroxy which may be protected by acetyl or benzyl, methoxy, or ethoxy ;

R ₁ and R ₂ may form oxo altogether;

R ₃ is selected from the group consisting of hydrogen, hydroxy which may be protected by acetyl or benzyl, vinyl, methyl, or ethyl ;

R ₄ is selected from the group consisting of hydrogen, methyl, ethyl, vinyl, or hydroxy which may be protected by acetyl or benzyl ;

R ₂ and R ₄ may form epoxy altogether;

R ₃ and ₄ may form oxo altogether;

R ₅ is selected from the group consisting of methyl, hydroxymethyl where hydroxy may be protected by acetyl or benzyl, tert-butyldimethylsilyloxymethyl, carboxyl, carboxylester , carboxylamide, or aldehyde;

R ₄ and R ₅ may form -0C(CH ₃ ) ₂ OCH _∑ - altogether;

R, is selected from the group consisting of hydrogen, or

methyl ;

R ₇ is selected from the group consisting of -CH ₂ COOR or -COOR [hence, R is hydrogen, methyl, and CH(OR _1l )CH ₂ R ₁₀ (R _g is selected from the group consisting of hydrogen, methyl or ethyl ;

is selected from the group consisting of methyl, ethyl, octyl, benzyl, methoxymethyl, or methoxyethyl ;

R ₁₀ is selected from the group consisting of hydrogen, methyl or ethyl ;

R ₁₀ and R _n may be associated to form -CH ₂ CH-,CH ₂ -, glucosyl or rhamnosyl where hydroxy may be protected by acetyl or benzyl ] , hydroxymethyl where hydroxy may be protected by acetyl or benzyl, ethanesulfony-loxymethyl, or cyano ethyl;

R ₂ and R ₁₃ represent hydrogen, respectively, or oxo altogether. [When R., R ₂ , R ₃ , R _H , R ₅ and R ₆ represent hydroxy, hydrogen, hydrogen, hydroxy, hydroxymethyl and methyl, respectively, R is not hydrogen or methyl and R _£ is not hydrogen; In case where R ₁ is hydroxy, R _∑ is hydrogen, R ₃ _. R _k form -OC(CH ₃ )-,0CH_,- together with R ₅ and R ₆ is

methyl, R is not methyl. ]

BACKGROUND ART

Asiatic acid, its trisaccharide asiaticoside and madecassic acid are extracted from Centella asiatica. They were first separated by Bontems et al. in 1941 [J.E. Bontems, Bull. Sci. Pharmacol., 49, 186-91 (1941)] and their structures were also disclosed by Polonsky et al. [J. Polonsky, Compt. Rend., 232, 1878-80(1951); J. Polonsky, Bull. Soc. Chim. , 173-80(1953)]. Centella asiatica extracts containing asiatic acid and asiaticoside have been for a long time used in the management of skin scars and chronic ulcers. Said extracts have also used in the treatment of skin deformity owing to tuberculosis and leprosy [P. Boiteau, A. Buzaε, E. Lederer and J. Polonsky, Bull. Soc. Chim., 31, 46-51 (1949)]. The pharmacological mode of action related to said substances' wound-healing properties has been reportedly said to activate malpighean cells and induce keratinization [May. Anne, Eur. J. Pharmacol., 4(3), 331-9 (1968) 1.

Kadecassol, one of the currently marketed dermatological agents is also a mixture of three

compounds containing asiaticoside (40%) plus asiatic acid and madecassic acid (60%) . Among them, asiaticoside, trisaccharide of asiatic acid, has reportedly demonstrated main efficacy while asiatic acid itself has produced no efficacy (Kiesswetter, Wien. Med. Wochschr. , 114(7), 124-6 (1964)]. However, there have been some reports that since the efficacy mechanism of said substances depends on their absorption in the body, asiatic acid itself proved to have exhibited the actual efficacy [L.F. Chasseaud, B.J. Fry, D.R. Hawkins, J.D. Lewis, T. Taylor ard D.E. Hathway, Arzneim-Forsch, 21(9), 179-84 (1971)]. Thus, the synthesis and pharmacological mechanism of asiatic acid derivatives have drawn considerable interest. However, the total synthesis of asiatic acid from simple starting material has recognized some disadvantages in that a lot of process steps are inevitably made, thus require significant manufacturing costs.

DISCLOSURE OF INVENTION

To overcome these shortcomings, the inventor et al. have successfully synthesized various asiatic acid derivatives by using asiatic acid obtained from Centella asiatica as a starting material and noted that said

derivatives have excellent wound-healing properties so that the present invention was completed.

The process of manufacturing asiatic acid derivatives according to the present invention is described as set forth hereunder.

Process 1

To perform the molecular modification on the OH at 2 position of asiatic acid, asiatic acid is treated with diazomethane to yield methylasiatate (2b) quantitatively and again treated with p-toluenesulfonic acid (PTSA) in acetone solvents, to prepare methyl 3,23-O-isopropylidene asiatate (3) where 3,23-OH is protected. The unreacted starting material is recovered. Further, the protected methylasiatate (3) is oxidized by piridinium dichromate (PDC) and acetic anhydride to afford methyl 2-oxo-3,23- O-isopropylidene asiatate (4, R=methyl) . The resulting compound is treated with PTSA in methanol solvents, to yield methyl 2-oxoaεiatate (5,R=methyl)

[ Scheme 1 ]

In the same synthesis procedure using asiatic acid (2a) , 3 , 23-0-isopropylidene asiatic acid (3, R=H) and -OXO-3, 23-0-iεopropylidene asiatic acid (4, R=H) is

yielded, respectively. Said compound (4, R=H) is treated with PTSA to prepare methyl 2-oxo-asiatic acid (5, R=H) [Scheme 1] .

When R [said Scheme 1] is hydrogen and alkyl, respectively, both oxidation and deprotecting reactions show that the yield in R=hydrogen is lower than R=alkyl. Accordingly, in case of manufacturing a compound (R=hydrogen) , it is rather desirable that a compound (R=alkyl) should be prepared and hydrolyzed.

Process 2

Further, said prepared compound (4, R=alkyl) is reduced with sodium borohydride to prepare methyl 2/3, hydroxy-3/3, 23-isopropylidendioxyurs-12-ene-28-oate (6, R=alkyl) , a novel compound of the present invention. Then, said compound (6, R=alkyl) is treated with PTSA to prepare methyl 2/3, 3/3, 23-trihydroxyurε-12-ene-28-oate(7, R=alkyl) , another novel compound of the present invention ^Scheme 2]. Even in case of a compound (4, R=H) , the same reaction iε made εo that a compound of the preεent invention (6,7) repreεenting R=hydrogen iε yielded.

[Scheme 2]

R=H, Alkyl

Process 3

Also, 2o:-alkyl-3,23-0-isopropylidene asiatic acid (17) is prepared by Grignard reaction of the compound (4, R=H) of the present invention with R'MgBr(R'=methyl, ethyl, vinyl, ethynyl, and cyano) , followed by deprotecting reaction of compound(17) to yield 2α-alkylasiatic acid (16) , another compound of the preεent invention [Scheme 3].

[ Scheme 3 ]

RMgBr

Process 4

Meantime, in order to introduce various substituents at 2,3 positions of asiatic acid, the 2,3-hydroxy iε converted to 2,3-epoxy and via reactions with various nucleophiles for the clevage of epoxy, a series of novel compounds of the present invention may be made available. In other words, methanesulfonylchloride and triethylamine are added to said prepared methyl 3 , 23-0-isopropylidene aεiatate (3) to give methyl 2-methanesulfonyl-3 ,23-0-isopropylidene asiatate (8) , one compound(8) of the present invention. Then, said compound is treated with PTSA to prepare methyl 2-methanesulfonyl

asiatate (9) , one compound of the present invention. The resulting compound is again treated with potasεiu carbonate in methanol solvents to synthesize methyl 2/3,3/3-epoxy-23-hydroxyurs-12-ene-28-oate(10) , one compound of the preεent invention [Scheme 4].

[Scheme 4]

Process 5

Also, the inventor et al. discover that when epoxy- cyclohexane, having the same strong bonding forces as said

prepared compound (10) , is reacted with metal hydrides, especially lithium aluminum hydride(LAH) , LAH proceeded generally via the axial attack by the stereoelectronic controlling or after its preferential bonding with surrounding hydroxy groups, LAH proceeded via intramolecular attack to form 2/3-alcohol. Accordinlgy, compound (10) is treated with LAH to prepare 2/3, 3/3-epoxyurs-12-ene-23 , 28-diol (11) where ester sites are reduced and 3-deoxyasiatic alcohol (12) where ester sites and epoxy groups are reduced. Through the prolonged time of such reaction, the compound(12) is prepared in high yield [Scheme 5].

[Scheme 5]

LAH,THF 12

Process 6

Said compoundε (11,12) are alεo made available by the following process: Compound (10) is treated with tert-butyldimethylεilyl chloride and i idazole in

dimethylformamide solvents to give the intermediate compound (13) quantitatively. Said compound is refluxed to reduce epoxide and is again deεilylated with tetrabutylammonium fluoride [Scheme 6].

[Scheme 6]

13

Process 7

Also, the compound (10) is reacted with diborane and catalytic amountε of sodium borohydride to prepare methyl 2-deoxyasiatate (14) and methyl 3-deoxyasiatate (15) , one compound of the present invention [scheme 7] .

[Scheme 7]

Process 8

Also, under the same reduction condition as described in said Scheme 7, reduction of the compound (13) is made and then followed by desilylation with tetrabutylammonium fluoride, thus further enhancing the ratio of compound (14) among the product [Scheme 8].

[Scheme 8]

Process 9

Treatment of said prepared compound (14) with LAH may give of 2-deoxyasiatic alcohol (16) in high yield, one compound of the present invention [Scheme 9].

Scheme 9]

Process 10

The 2-OH poεition of methyl 3,23-0-isopropylidene asiatates (3) is treated with sodium hydride and imidazole for its conversion into alkoxide. Said compound is refluxed with the addition of carbon disulfide and then treated with methyl iodide to give xantate (19) . Said xantate (19) is treated with tributyltin hydride and small amounts of AIBN to prepare methyl 2-deoxy-3,23-0- iεopropylidene aεiatate (20) and via the deprotecting reaction, methyl 2-deoxyasiatate (21) is prepared. Said compound (21) iε hydrolyzed with lithium iodide in 2,4,6-collidine εolvents to prepare 2-deoxyasiatic acid (22) [Scheme 10] .

[ Scheme 10 ]

nBujSnH, AIBN 1N-HC

(the same as the compound 14)

Process 11

The 2-hydroxy of methyl 3 , 23-0-isopropylidene asiatate (3) iε protected with the benzyl and reduction of the eεter sites with LAH produced 2-0-benzyl-3 , 23-0-isopropylidene asiaticol (24) . Said compound (24) is mesylated with methanesulfonyl chloride and then εubεtituted with sodium cyanide to prepare 2α-benzyloxy-28-cyano-3/3 ,

23-isopropylidenedioxyurε-12-ene (26) . Said compound (26) is treated with hydrochloric acid/ tetrahydrofuran to give 2α-benzyloxy-28-cyano-3/3 ,23- dihydroxyurs-12-ene (27) . Said compound (27) is treated with Pd/C to prepare 28-cyano-2 ,3 , 23-trihydroxyurs-12-ene(2δ) [Scheme 11].

[Scheme 11]

1N-HG, THF 1C"- ⁷ - Pd-CH-

Process 12

By treating pyridinium dichromate (PDC) of the compound of the present invention (14=21) , a compound (29) , where the hydroxy at 3 position is oxidized to the oxo and hydroxymethyl at 4 position is converted to the methyl , is prepared. By reducing said compound (29) with sodium borohydride, the oxo at 3 position may be converted to the hydroxy . Further, by the Grignard reaction of the said compound, the alkyl or alkenyl may be introduced at 3 position (compound 31,32) [Scheme 12].

[Scheme 12]

PDC

¹ NaBP

Process 13

By treating the compound of the present invention

(4, R=methyl) with KHMDS and dimethylsulfate, the methoxy may be introduced at 2 position (compound 33) .

When this compound iε treated with dilute hydrochloric acid and itε protecting are removed, methyl 2-methoxyurs- 12-ene-3-one-28-oate(34) is prepared [Scheme 13].

[ Scheme 13 ]

4 (R= e ; 33

1 G

Process 14 , 15

Meantime, the corresponding esters (two compounds of the present invention (35, 37) are prepared by the following Schemes, [14] and [15]:

After direct hydrolysis of Centella asiatica extracts, crude products are prepared via their neutralization and freeze-dried process. Triacetyl asiatic acid produced by direct acetylation of said crude products is reacted with alkoxymethyl chloride derivatives using base [Scheme 14], or

Triacetyl asiatic acid is reacted with vinyl ether derivatives using catalytic amounts of acid [Scheme 15].

By treating said prepared triacetyl esters (35,37) with potassium carbonate/methanol, the selective deacetylation with presevation of ester sites in saccharide proceeded so that asiaticoside derivatives (36, 38) where a cyclic saccharide is combinded in ester linkage are prepared.

[Scheme 14]

AcO ^'

35

36 R, = CHa.CzHs.Octyl.Bn.CHjOCHz f Scheme 15] X = Cl, Br, I, OMε, OTs

ac. VIeCh

Process 16

Also, asiatic acid derivatives of the present invention contains their glycosides. In case of manufacturing said glycoεide compoundε, the selective and effective protection of hydroxy of saccharide is important. According to the present invention, the target glycosides could be prepared by protecting saccharide in acetylation, which has the following advantages: a) reaction may be easy, b) proceεs iε simple, c) high yield is expected, and d) deprotected reaction is also easy.

Scheme 16 shows that asiatic acid (or triacetyl asiatic acid) , a starting material, is reacted with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosylbromide to prepare 2,3,4,6-tetra-0-acetyl-/3-D-glucopyranosyl asiatate compound (39) .

[Scheme 16]

39 Y=H, Acetyl

In a similar process as above, α-L-rhamnoεe iε acetylated to εyntheεize 1,2,3,4-tetra-0-acetyl-α-L- rhamnopyranose, followed by bromination with hydrobromic acid, to prepare 1,2,3,4-tetra-0-acetyl rhamnopyranoεyl bromide. Then, said bromide is reacted with triacetyl asiatic acid to prepare tetraacetylrhamnopyranosyl triacetylasiatate (40) .

From said prepared glycoside compounds (39, 40), acetyl combined with εaccharides iε easily hydrolyzed under extremely mild conditions. Accordingly, in case of using said glycoside compound as pharmaceuticals, it is conceivable that the acetyl in the body may be hydrolyzed into hydroxy . In case of said compoundε (35,40) whose saccharide sites contain several acetyl ε, their lipophilicity becomes highly increasing and it iε expected that their trandermal absorption may be further facilitated. And after transder al absoption,

their saccharide sites are hydrolyzed by skin eεterase and this may facilitate the hydrolysis in ester sites, whereby asiatic acid may be easily freed in the body.

Process 17

Methyl 3 ,23-0-isopropylidene asiatate (3, R=methyl) is reacted with sodium hydride and alkyl iodide to alkylate the hydroxy at 2 position. By deprotecting acetonide with hydrochloric acid and methanol, methyl 2-0-alkylaεiatate (41) iε prepared. The hydroxy s at 3 , 23 positions of said compound is acetylated for protection and then oxidized with sodium dichromate using acetic acid to prepare methyl 3,23-0-diacetyl -2-0-alkyl-ll-oxoasiatate (42) where the oxo at is introduced at 11 position.

Said compound is treated with potassium carbonate to deprotect the acetyl so that methyl 2-0-alkyl-ll- oxoasiatate (43) iε synthesized.

[Scheme 17]

Process 18

The hydroxy at 2 position of methyl 3,23-0-isopropy- lidene aεiatate (3, R=methyl) iε acetylated with acetic anhydride for protection and then acetonide only iε εelectively deprotected with 0.IN HCl to syntheεize methyl 2-0-acetylaεiatate (44). Said compound (44) iε oxidized with l equivalent of pyridinium dichromate at room temperature so that methyl 2-acetyloxy-3-hydroxyurs-12 -ene-23-al-28- oate (45) is prepared. Said compound (45) iε again oxidized by refluxing with 2 equivalent of pyridinium dichromate in dichloromethane to obtain methyl 2-acetyloxyurs-12- ene-23-al- 3-one-28-oate (46).

[ Scheme 18 ]

^■ HO /

44

PDC.CH-G-

46

Process 19

Aε manufactured in εaid Procesε ll,methyl-2-0-benzyl- 3,23-0-iεopropylidene asiatate (23) is treated with hydrochloric acid for deprotecting acetonide only and then oxidized with pyridinium dichromate to synthesize methyl 2-0-benzyl- 3-hydroxyurs-12-ene-23-al- 28-oate (48) .

[Scheme 19]

e

48

Process 20

As manufactured in said Procesε 11, 2-benzyloxy-28- cyano-3 , 23-dihyroxyurε-12-ene (27) iε hydrolyzed with 80% potassium hydride to synthesize 2 -benzyloxy-3 ,23- dihydroxyurs-12-ene-28-carboxylic acid (49) . Said compound iε contacted with Pd/C catalyεt for reduction to prepare 2/3, 3/3, 23-trihydroxyurε-12-ene-28-carboxylic acid (homoaεiatic acid) .

[ Scheme 20 ]

4 9

x

H O Process 21

3,23-0-isopropylidene-2-oxoasiatic acid (51) is reacted with chloro ethylethyl ether using diisopropylethylamine as Hunig base to εyntheεize ethoxymethyl 3,23-isopropyl- idenedioxyurs-12-ene-2- one-28-oate (52) . Under the same condition, εaid compound (51) iε reacted with chloromethyloctyl ether to synthesize octyloxymethyl 3,23-isopropylidenedioxyurε- 12-ene-2-one-28-oate (53).

[ Scheme 21 ]

1

σ -:;θ(CH:)-c ^ή ₃ . ctyl

Diisosrrovieώv! nt

Process 22

The hydroxy at 2 position of 3,23-0-isopropylidne aεiatic acid (3,R=H) iε acetylated for protection. Then, compound (54) so obtained iε reacted with chloromethyletyl ether and chloromethyloctyl ether, reεpectively, uεing diiεopropylethylamine aε Hunig baεe to εyntheεize both ethoxymethyl-2-α.-acetyloxy-3/3 ,23-iεopropylidenedioxyurε -12-ene-28-oate (55) and octyloxymethyl 2-α-acetyloxy-3/3 , 23-isopropylidenedioxyurs-12-ene-28-oate (56). Said prepared compounds (55) , (56) are treated with potassium carbonate, respectively, for deprotecting acetyl, whereby ethoxymethyl 2 -hydroxy-3/3, 23- iεopropylidenedioxyurε -12-ene-28-oate (58) and

oc _t y _l oxymethyl ₂ α -hydroxy-3/3, 2._-isopropyli ^d ened ⁱ oxyurs -12-ene-28-oate (59) are synthesized.

₂ -acetyl- ₃ ,2 ₃ - ₀ -isopropylidene asiatic acid ⁽ 5 ⁴⁾ is reacted with dihydropyran using pyridinium p-toluenesulfonate (PPTS) to synthesize 2-

tetrahydropyranyl 3/3, 23 - i sopropyl - idenedioxyurs

-12-ene-28-oate (57).

[ Scheme 22 ]

t-υ o Ui UI

Process 23

The hydroxy s at 3, 23 poεitionε of 2-deoxyasiatic acid (22) are acetylated for protection, to prepare 2-deoxy-3,23-diacetyl asiatic acid (60). Said compound (60) is reacted with dihydropyran using pyridinium p-toluenesulfonate to prepare 2-tetrahydropyranyl 3/3, 23-diacetyloxyurs -12-ene- 28-oate (61) .

Said compound (60) is reacted with chloromethylethyl ether and chloromethyloctyl ether, respectively, using diiεopropylethylamine to synthesize both ethoxymethyl 3/3,23- diacetyloxyurs-12-ene-28-oate (62) and octyloxymethyl 33, 23-diacetyloxyurs-12-ene-28-oate (63). Said prepared compounds(62) , (63) are treated with potassium carbonate, respectively, and after hydrolyzing their acetyl s, both ethoxymethyl 3/3, 23-dihydroxyurε-12 -ene-28-oate (64) and octyl-oxymethyl 3/3, 23- dihydroxyurε-12-ene-28-oate (65) are prepared.

[ Scheme 23 ]

y

AcO

61

AcO

62

HO'

64

63

Octyl

y

HO

Process 24

65

Methyl 2/3, 23-dihydroxyurs-12-ene-28-oate (66) , together with lithium iodide, are heated for refluxed uεing 2,4,6- trimethylpyridine (collidine) and then hydrolyzed to synthesize 23, 23-dihydroxyurε- 12-ene-28-oic acid (67) . The hydroxy at 23, 23 poεitionε of said prepared compound (67) are acetylated to prepare 2/3, 23-diacetyloxyurs-12- ene-28-oic acid (68). Said compound (68) iε reacted with dihydropyran uεing pyridinium p-toluenesulfonate aε a catalyεt to prepare 2- tetrahydropyranyl 2/3, 23-diacetyloxyurε-12-ene-28-oate(71) .

Said compound (68) iε reacted with chloromethylethyl ether and chloromethyloctyl ether, respectively, uεing diiεopropylethylamine, reεpectively, to εyntheεize both ethcxy-methyl 2/3, 23-diacetyloxyurs-12-ene-28-oate (69) and octyloxymetyl 2/3, 23-diactyloxyurε-12-ene-28-oate (70). Said prepared compoundε (69) , (70) are deproteced with KOH, respectively, to synthesize both ethoxymethyl 2/3, 23-dihydroxyurs-12-ene-28-oate(72) and octyloxymethyl 2/3, 23-dihydroxyurs-12-ene-2δ-oate (73) .

7819

[Scheme 24]

66 HO y

61

i

HO'

72

• J

Process 25

Methyl 2/3, 3/3 epoxy-23-hydroxyurs-12-ene-28-oate(10) εyntheεized from Process 4 is reacted with tri ethylεilylazide or thiophenol aε nuclophile uεing Ti(0-i-Pr)4, thuε obtaining 2-azidoasiatic acid ester (74) and 2-thiopenoxyasiatic acid eεter (75) , respectively.

[Scheme 25]

. _>

To increase their lipophilicity of each hydroxymethyl related to 2-deoxyasiatic acid ester (14) prepared from Process 7 and methyl 2/3, 3/3-epoxy-hydroxyurs-12-ene-28-oate (10) prepared from Procesε 4, undecylenic acid is reacted to synthesize the corresponding esters (76) and (77) , respectively.

[Scheme 26]

noesvl

^" 6

Me

J T2£ S -'

Process 27

The hydroxymethyl of methyl 2/3, 3/3-epoxy-23- hydroxyurs-12-ene-28-oate(10) of Process 4 is oxidized with Jones reagent to prepare epoxy acid (78) . Said methyl 2/3, 3/3-epoxyurs-12-ene-28-oate-23 oic acid (78) iε treated with diazomethane to εynthesize epoxy ester or methyl 2/3, 3/3-epoxyurs-12-ene-23-(N-phenyl) amido-28- oate(80) by reacting said methyl 2/3, 3/3-epoxyurs-12-ene-28- oate-23-oic acid (78) with aniline.

[Scheme 27]

10

O^OH

As a result of investigating the wound-healing property of asiatic acid derivatives of the present invention, when rats are given wounds, their efficacious are equivalent to 1% TECA (Titrated Extracted Centella asiatica) , a control drugs or more remarkable.

BE8T MODE FOR CARRYING OUT THE INVENTION

The present invention is described in more detail by Examples and Experiments as shown below but is not confined to said scopeε.

Example l ; Separation and purification of asiaticoside asiatic acid in mass-scale

An extract (5g) of Centella asiatica was directly separated on silica gel chromatography (silical gel, 230-400 mesh, dichloromethane: methanol = 10:1) to give aεiatic acid (1.5g), madecassic acid (1.4 g) and a mixture (2.0g) containing asiaticoεide and madecasoεide. The mixture, so obtained, was disεolved in catalylic amounts of 60% methanol on water bath at 100°C and then cooled at room temperature to yield pure asiaticoside as needle cryεtal (mp. : 230-240°C) .Aside from that, εaid extract (20g) is dissolved in methanol (500ml) and then hydrolyzed with 5N-NaOH. This is purified with column chromatography to give pure asiatic acid (7-8g) aε white

solid (mp: 300-310 °C ) .

TLC (methanol/dichloromethane= 1:8) R _f 0.32

Example 2;Preparation of methyl 3,23-O-isopropylidene asiatate(3)

Methyl asiatate 2 (27.7mg, 0.055 mmole) was diεsolved in anhydrous acetone (3ml) , added with p-toluenesulfonic acid (27.7mg) and then refluxed. Water was added to the reaction mixture, and the solution was neutralized with 5% potassium carbonate and then extracted with ethyl acetate. The organic layer was washed with water and brine, dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. The was chromatographed with benzene and ethyl acetate (3:2) to give pure deεired compound (18mg, 60%) .

Η NMR (CDCl _j ) 6 5.25(lH,m), 3.78(lH,m), 3.60(3H,s), 3.51(IH,d,J=10.5Hz) , 3.47 (lH,d,J=10.5Hz) , 3.32(IH,d,J=9.5Hz) , 1.46(3H,s), 1.45(3H,ε), 1.09(3H,s) 1.07(3H,s), 1.04(3H,ε), 0.9 (3H,d,J=6.0Hz) , 0.86(3H,d,J=7.0Hz) , 0.73(3H,ε)

Example 3 : Preparation of methyl 3,23-Q-isopropyliden-2

-oxoasiatate (4, R=methyl)

Compound 3 (1.25g, 2.31 mmole) was diεsolved in

dichloromethane (8ml) , added with pyridinium dichromate

(0.61g, 1.62 mmole) and acetic anhydride (0.71g, 6.93 mmole) and then refluxed under nitrogen atmosphere. The reaction mixture was added with ethyl acetate (50ml) and then filtered. The organic layer was washed with brine solution and dried over anhydrous magnesium sulfate.

After filtration, the remaining solution was concentrated under reduced preεεure. The waε chromatographed with hexane and ethyl acetate (2:1) to give pure deεired compound (1.107g, 89%) aε white εolid.

¹ H NMR (CDC1 ₃ )(5

5.25(lH,m), 4.40(1H,S), 3.69 (IH,d,J=10.5Hz) , 3.60(3H,ε), 3.59(lH,d,J=10.5Hz) , 2.40(2H,d,J=12.5Hz) , 1.52(3H,s), 1.45(3H,s), 1.08(3H,ε), 1.05(3H,s), 1.02(3H,ε), 0.95(3H,d,J=6.0Hz) , 0.86(3H,d,J=6.5Hz) , 0.74(3H,ε)

3,23-0-isopropyliden-2-oxoasiatic acid (4, R=H)

¹ H NMR (CDC1 ₃ ) δ 5.23(lH,m) , 4.39(lH,bs) , 3.62 (lH,bs) , 3.49(lH,bε) , 2.62(lH,ε) , 1.50(3H,ε) , 1.44(3H,ε) , 1.24(3H,ε) , 1.13(3H,ε) 1.01(3H,ε) , 0.95-0.85(6H,m) , 0.75(3H,ε)

Example 4 : Preparation of methyl 2-oxoasiatate (5, R=methγl.

Compound 4 (R=methyl ; 448.3mg, 0.83 mmole) waε diεεolved in methanol (25ml) , added with p-tolueneεulfonic

acid (179.3mg, 0.94 mmole) and then refluxed under nitrogen atmosphere. Water (500ml) was added to the reaction mixture, and the solution was neutralized with 5% potassium carbonate and then extracted with ethyl acetate (50ml x 3) . The organic layer was washed with brine, dried over anhydrous magnesium sulfate and then concentrated under reduced preεsure. The residue was chromatographed with hexane and ethyl acetate (2:1) to yield pure desired compound (340mg, 82%) as white solid.

¹ H NMR (CDC1 ₃ )<S

5.29(lH,m), 4.37(lH,m), 3.74(lH,m), 3.60(3H,s),

3.52(lH,m), 1.14(3H,ε), 1.08(3H,ε), 0.91(9H,bε),

0.76(3H,ε)

2-oxoasiatic acid (R=H)

^] H NMR (CDC1 ₃ )

5.27(lH,m), 4.29(1H,S), 3.53-3.42(2H,m) , 1.13(3H,s), 1.02(3H,S), 0.96(3H,bs), 0.88(3H,S), 0.87 (3H,d,J=7.0Hz) , 0.76(3H,d,J=3.8Hz)

Example 5 : Preparation of methyl 2/3- ydroxy-3/3, 23- isopropγlideneoxγurs-12-ene-28-oate (6)

Methyl 3,23-isopropyliden-2-oxoaεiatate (4, R=H? 619.2mg, 1.15 mmole) waε dissolved in methanol (40ml), added with sodium borohydride (21.9mg, 0.58 mmole)

and then stirred overnight under nitrogen atmosphere at room temperature. After completion of the reaction, the solvent was removed and the reaction mixture waε extracted with ethyl acetate (50ml x 3). The organic layer waε waεhed with water and brine solution, dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. The residue was purified with column chromatography (hexane:ethyl acetate=2:l) to give pure desired compound (606.6mg, 98%) as white solid.

¹ H NMR (CDC1 ₃ )<S

5.29(lH,m), 4.01(lH,bs), 3.59(3H,s), 3.49(3H,m), 1.44(6H,bs), 1.31(6H,bs), 1.08(3H,s), 0.91(3H,d,J=4.0Hz) , 0.85(3H,d,J=6.0Hz) , 0.75(3H,ε)

Example 6 ; Preparation of methyl 2/3, 3/3.23- trihydroxyurs-12-ene-28-oate 7, R=methyl)

Compound 6 (557.2mg, 1.02 mmole), so obtained from said Example 5, was dissolved in methanol (15ml) , added with p-toluenesulfonic acid (223mg, 1.17 mmole) and then refluxed under nitrogen atmosphere for 10 minε. Water (50ml) waε added to the reaction mixture, and the solution was neutralized with a solution of 5% potassium carbonate and then extracted with ethyl acetate (50ml x 3). The organic layer waε waεhed with brine, dried over anhydrous magnesium sulfate and then concentrated

under reduced pressure. The residue was purified with column chromatography(hexane:ethyl acetate=l:l) to give pure desired compound (407.3mg, 79%) as white solid.

¹ H NMR (CDCl _j )δ

5.26(lH,m) , 4.11(lH,m) , 3.75(lH,m) , 3.72 (IH,d,J=10.0Hz) , 3.60(3H,S) , 3.42(lH,d,J=10.0Hz) , 1.30(3H,S) , 1.11(3H,S), 1.06(3H,ε), 0.94(3H,d,J=6.0Hz) , 0.85 (3H, d, J-6.0Hz) , 0.76 (3H, S)

2)5, 33, 23-trihydroxyurs-12-ene-28-oic acid (l , R=H.

¹ H NMR (CDCl _j ) 6

5.25 (lH,m) , 4.13 (lH,m) , 3.63 ( IH , d , =4. IHz ) ,

3.52 (lH,d,J=l0.9Hz) , 3.29 (IH, d,J=10.9Hz) ,1.28 (3H,s) , 1.11 (3H, S) , 0.97 (6H,S) , 0.88 ( 3H , d , J=6.5HZ ) , 0.86(3H,d,J=4.1Hz)

Example 7 : Preparation of methyl 2-methanesulfonyl-3 ,23

-0-isopropγlidene asiatate (8)

Methyl 3 , 23-O-isopropylidene asiatate 3 (354.7mg, 0.65 mmole) was dissolved in dichloromethane (15ml), added with triethyl amine (82.4mg, 0.72 mmole) and methanesulfonyl chloride (99.2mg, 0.98 mmole) and then stirred under nitrogen atmosphere at 0°C for 3 hrs. After completion of the reaction, the solvent was removed and the reaction mixture waε extracted with ethyl acetate (50ml x 3) . The organic layer was

waεhed with water and brine, dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. The residue was purified with column chromatography (hexane: ethyl acetate= 2:1) to give pure desired compound (380mg, 93%) aε white solid.

¹ H NMR (CDC1 ₃ )<S

5.24(lH,m), 4.69-4.62 (lH,m) , 3.60(3H,s),

3.57 (lH,d,J=10.5Hz) , 3.53 (lH,d,J=10.5HZ) , 3.49(lH,d,J=10.5Hz) 3.01(3H,s), 2.26-2.20(lH,m) , 2.23(lH,bs), 1.44(3H,ε), 1.40(3H,ε), l.ll(3H,ε), 1.09(3H,ε), 1.07(3H,s), 0.94(3H, d,J=6.0Hz), 0.85(3H,d,J=7.0Hz) , 0.72(3H,s)

Example 8 : Preparation of methyl 2-methanesulfonyl asiatate (9)

Compound 8 (1.2g, 1.92 mmole) was dissolved in methanol (30ml) , added with p-toluenesulfonic acid (480mg, 2.52 mmole) and then refluxed under nitrogen atmosphere for 10 mins. Water (100ml) waε added to the reaction mixture, and the εolution waε neutralized with a εolution of 5% potaεεiu carbonate and then extracted with ethyl acetate (100ml x 3). The organic layer waε waεhed with brine, dried over anhydrouε magnesium sulfate and then concentrated under reduced presεure. The reεidue waε purified with column chromatography (hexane:ethyl acetate=l:1) to give pure deεired compound

(1.06g, 94%) as a colorless oil.

^ NMR (CDC1 ₃ )<5

5.24(lH,m) , 4.77-4.74(lH,m) , 3.69 ( IH, d, J=10.5Hz) , 3.61(3H,S) , 3.44 (lH,d,J=10.5Hz) , 3.20(lH,bs) , 3.10(3H,S) , 1.08(3H,s) , 1.07(3H,S) , 0.95(3H,ε) , 0.94 (3H, d, J=5.1Hz) , 0.85(3H,d,J=6.5Hz) , 0.74(3H,ε)

Example 9 ; Preparation of methyl 2α_ 3β-epoxy-23- hydroxγurs-12-ene-28-oate(10_

Compound 9 (2.78g ,4.77 mmole) was dissolved in methanol (60ml), added with potassium carbonate (1.32g, 9.53 mmole) and then stirred under nitrogen atmosphere at room temperature for 3 days. After completion of the reaction, the solvent was removed and the reaction mixture waε extracted with ethyl acetate (100ml x 3) . The organic layer was washed with 5% dilute hydrochloric acid, water and brine, dried over anhydrous magnesium εulfate and then concentrated under reduced preεεure. The residue was purified with column chromatography (hexane:ethyl acetate=2:1) to give pure desired compound (2.05g, 89%) as white solid.

Η NMR (CDC1 ₃ ) 6

5.27(lH,m), 3.60(3H,s), 3.56(lH,m), 3.31(3H,m), 3.27 (lH,m) , 3.11 (IH,d,J= .OH∑) , 1.12(3H,ε), 1.06(3H,ε), 0.S6(3H,ε), 0.9 (3H,d.J=5.1Hz) , 0.86(2H,d,J=6.4Hz) ,

0 . 74 ( 3H , s )

Example 10 :Preparation of 2c__ 3g-»epoχyυrs-l2-ene-23, 28 -diol(ll) and 3-deoχyasiatic alcohol (12)

Compound 10 (140.9mg, 0.29 mmole) was dissolved in tetrahydrofuran (5ml) and added with lithium aluminum hydride (ll.Omg, 0.29 mmole). While agitating it under nitrogen atmosphere at room temperature, the reaction mixture was added with catalylic amounts of LAH until a εtarting material waε entirely annihilated (about 2 dayε) . The reaction waε stopped by adding a mixture containing water and tetrahydrofuran (1:1) to the reaction mixture at 0°C. The reaction mixture was added with 5% dilute hydrochloric acid (10ml) and extracted with ethyl acetate (50ml x 3) . The organic layer waε washed with a solution of saturated sodium carbonate, water and brine and dried over anhydrous magnesium sulfate and then concentrated under reduced presεure. The residue waε purified with column chromatography (hexane:ethyl acetate=2:l) to give both pure compound 11 (44.1mg, 33%) and 12 (63.3mg, 47%) aε white εolid, reεpectively.

2α, 3/3-epoxyurs-12-ene-23,28-diol (11)

¹ H NMR (CDCl _j ) δ

5.16(lH,m), 3.56-3.48 (3H,m) , 3.27(lH,bε) ,

3.19 (lH,d,J=10.3HZ) , 3.11 (IH,d , =4.0Hz) , l.Ϊ5(3H,ε),

1.09(3H,s) , 0.99(3H,ε) , 0.98 (3H, d, J=6. Hz) , 0.93(3H,ε) , 0.81(3H,d,J=5.6Hz)

3-deoxyasiatic alcohol (12) ¹ H NMR (CDC1 ₃ )<5

5.08(lH,m) , 4.04-3.97(lH,m) , 3.47 (IH,d,J=ll. OHz) , 3.25(lH,d,J=11.0Hz) , 3.00(lH,d, 10.9Hz) ,

2.94 (lH,d,J=ll. OHz) , 1.17(3H,s), 1.05(3H,ε), 0.95(3H,s), 0.84(6H,bε), 0.74 (3H,d,J=5.9Hz)

Example 11 : Preparation of 3-deoxγasiatic alcohol (12)

Compound 10 (87.4mg, 0.18 mmole), so obtained from Example 9, was dissolved in tetrahydrofuran (5ml), added with lithium aluminum hydride (13.6mg,0.36 mmole) and then refluxed under nitrogen atmosphere for 1 hr. The reaction was stopped by adding a mixture containing water and tetrahydrofuran (1:1) in small portions to the reaction mixture at 0°C. The reaction mixture waε added with 5% dilute hydrochloric acid (10ml) and extracted with ethyl acetate (50ml x 3) . The organic layer was waεhed with a εolution of εaturated εodium carbonate, water and brine and dried over anhydrouε magnesium εulfate and then concentrated under reduced pressure. The reεidue waε purified with column chromatography (hexane:ethyl acetate= 2:1) to give pure desired compound (SO.Omg, 97%) aε white εolid.

Example 12 : Preparation of methyl 2-deoxy-23-tert- butyldimethylsilyl asiatate (13)

Compound 10 (200mg, 0.41 mmole) , so obtained from Example 9, waε dissolved in dimethylform amid (5ml) , added with tert-butyldimethylsilyl chloride (68.5mg, 0.45 mmole) and imidazole (61.9mg, 0.91 mmole) and then stirred under nitrogen atmosphere at room temperature for 1 day. The reaction was stopped by adding a solution of saturated ammonium chloride to the reaction mixture at 0°C and the reaction mixture was extracted with ethyl acetate (50ml x 3) . The organic layer was washed with 5% dilute hydrochloric acid, a solution of saturated εodium carbonate, water and brine and dried over anhydrouε magneεium sulfate and then concentrated under reduced preεεure. The reεidue waε purified with column chromatography (hexane: ethyl acetate=4 : 1) to yield pure deεired compound (273.5mg, >100%) aε colorleεε oil.

'H NMR (CDC1 ₃ ) δ

5.28-5.26(lH,m) , 3.60(3H,s) , 3.49 (IH, d,J=9.7Hz) , 3.32 (lH,d,J=9.6Hz) , 3.21(lH,m) , 3.04 (IH,d,J=4.1Hz) , 1.10(3H,ε) , 1.05(3H,ε) , 0.95(3H,ε) , 0.92-0.85 (15H,m) , 0.74(3H,ε) , 0.05(3H,ε) , 0.04(3H,s) .

Example 13 : Preparation of methyl 2-deoxyasiatate (14 ) and methyl 3-deoxyasiatate (15)

Compound 10 (77.5mg, 0.16 mmolee) , so obtained from Example 9, was dissolved in tetrahydrofuran(2ml) , added with di ethylsulfide (0.11ml, 2M εolution) and εodium borohydride (1.6mg, 0.04 mmole) and then refluxed under nitrogen atmosphere for 1 day. The reaction was stopped by adding a mixture (1ml) containing l.OM sulfuric acid and tetrahydrofuran (1:1) at 0°C to the reaction mixture, and the reaction mixture was extracted with ethyl acetate (50ml x 3) . The organic layer waε waεhed with a εolution of εaturated sodium carbonate, water and brine and dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. The residue was purified with column chromatography (hexane: ethyl acetate=2: 1) to give both pure compound 14 (48.4mg, 62%) and pure compound 15 (16.3mg, 21%) aε white εolid, respectively.

Methyl 2-deoxyasiatate (14) mp. : 250 - 254°C ^"* H NMR (CDC1 ₃ ) δ

5.22(lH,m) , 3 , 69 (lH,d,J=10.3HZ) , 3.61-3.59 ( IH, m) , 3.58(3H,S) , 3.39(lH,d,J=10.3Hz) , 2.18 (IH,d,J=ll.5Hz) , 1.05(3H,s) , 0.94 (3H,ε) ,0.91(3H,d,J=5. OHZ) , 0.86(3H,ε) , 0.83 i 3H _r d,J=6.5Hz) , 0.72(3H,ε) .

Methyl 2/3, 23-dihydroxyurs-12-ene-28-oate (15)

^* 'H NMR (CDCl _j ) 6

5.28 ( 1 H , IT. ) , 4.22-4.16 ( lH , m) , 3.61 ^" ( 3 H , s

3.42 (lH,d,J=10.8Hz) ,

3.18(lH,d,J=10.8Hz) , l,24(3H,s) , 1.08(3H,s) , 0.98(3H,s) ,

0.94 (3H,d,J=5.5Hz) , 0.86 (3H, d, 3=6.5Hz) , 0.77(3H,s) .

Example 14 : Preparation of 2-deoxyasiatic alcohol (16)

Compound 14 (463mg, 0.94 mmole) was disεolved in tetrahydrofuran (15ml) , added with lithium aluminum hydride (71.7mg, 1.89 mmole) and then refluxed under nitrogen atmoεphere for 3 hrs. The reaction was stopped by adding a mixture containing water and tetrahydrofuran (1:1) in small portions at 0°C to the reaction mixture, followed by addition with 5% dilute hydrochloric acid (20ml) and extraction with ethyl acetate (50ml x 3) . The organic layer was waεhed with a εolution of εaturated εodium carbonate, water and brine and dried over anhydrouε magneεium εulfate and then concentrated under reduced preεεure. The residue was purified with column chromatography (hexane: ethyl acetate= 1:1) to give pure deεired compound (415.7mg, 95%) aε white εolid.

'K NMR (CDCl _j ) 6

5.13(lH,m), 3.69(lH,d,J=10.3Hz) , 3.63(lH,m), 3.53(1H, d,J=10.9Hz), 3.40(lH,d,J=10.3Hz) , 3.16(IH,d,J=10.8Hz) , 1.10(3H,s) , 0.99(6H,ε) , 0.93(3H,ε) , 0.8S(3H,s) , u .81(3K,bε)

Example 15 : Preparation of 3, 23-0-isopropyliden-2α

-methylasiatic acid (17. R'-smethγl)

3,23-0-isopropyliden-2-oxoasiatic acid (4, R=H; lOOOmg, 1.90 mmole) waε diεsolved in tetrahydrofuran (40ml), and cooled to -78°C. Methylmagnesium chloride (3.0M THF Solution 1.9ml, 5.60 mmole) was added to the mixture and stirred for 10 mins. The reaction mixture was treated with water (1ml) and concentrated under reduced pressure to remove the solvent. The residue waε added with ethyl acetate (50ml) and a solution of εaturated ammonium chloride (5ml) and extracted. The organic layer waε waεhed with brine solution (5ml) , dried over anhydrouε magnesium sulfate and then concentrated under reduced pressure. The residue was purified with column chromatography (dichloromethane: methanol= 50:1) to give pure desired compound (980mg, 95%) aε white εolid.

[α] ²² _D ;+53.4 (CHCI ₃ ,C=1.19) IR(neat) :3400,1696cm ^*1 Η NMR (CDCl _j ) δ

5.19(lH,bt) , 3.37, 3.43(2H,d) , 3.20(lH,s) , 2.12 (lH,d,J=9.2Hz) , 0.89(3H,d,J=6.OHz) , 0.80(3H,d,J=6.4Hz) , C.72, 1.03, 1.08, 1.21, 1.23, 1.36, 1.40, (each 3H,ε).

Example 16 : Preparation of 3,23-0-isopropyliden-2α- ethγlasiatic acid (17, R'-sethyl)

By the same procedure as described above for the preparation of Example 15, using ethylmagnesium bromide (l.OM THF Solution, 373mg, 2.80 mmole) instead of methylmagnesium chloride, pure desired compound (490mg, 92%) was obtained aε white solid.

'H NMR (CDC1 ₃ )(S

5.26(lH,bt) , 3.42, 3.51 (2H,d,J=10.8Hz) , 3.28(lH,s), 2.18(lH,d) , 1.41, 1.44(each 3H,ε)

Example 17 : Preparation of 2α-methylasiatic acid (18, R'=methyl)

3 ,23-0-isopropyliden-2α-methylasiatic acid (17, R'=methyl; 370mg, 0.68 mmole) was added with methanol (10ml) and IN hydrochloric acid (0.5ml) and εtirred at room temperature for 10 hrs. The reaction mixture was distilled under reduced pressure to remove the solvent. The residue waε purified with column chromatography (dichloromethane: methanol=30: 1) to give pure deεired compound (325mg, 95%) aε white εolid. The crude product was recrystallized from methanol to give needle crystal .

Η NMR (CDC1 ₃ ) δ

5.15(lH,brt) , 3.35(1H,S) , 3.58(2H,AB q,J=10.4Hz) , 3.25, 2.15(lH,d,J=11.2Hz) , 0.79 (3H,d,J=6.4Hz) , 0.73(3H,d, J=6.0Hz) , 1.18, 1.13, 0.93, 0.92, 0.66(3H, each ε)

Example 18:Preparation of 2α-ethylasiatic acid(l8,R =ethyl)

By the same procedure aε described above for the preparation of Example 17, compound (17, R'=ethyl; 260mg, 0.47 mmole) waε deprotected to give white solid

(227mg, 94%) . The residue was recrystallized from methanol to give needle crystal.

¹ H NMR (CDC1 ₃ )<5 5.32(lH,brt,J=3.4, 7.7Hz), 3.53(lH,ε), 3.75, 3.42(2H,AB q, J=10.3Hz), 2.32(lH,d,J=11.7Hz) , 1.29, 1.12, 1.10, 0.86(each 3H,ε)

TϋYwin i<» 19 t Preparation of methyl 3,23-0-isopropyliden- 2-0-f (methγlthio) thiocarbonyll asiatate

Methyl 3,23-0-isopropylidene asiatate 3 (50mg, 0.092 mmole) was added with sodium hydride (60% dispersion in mineral oil; 18.3mg, 0.46 mmole), imidazole (2mg) and tetrahydrofuran (2ml) , and the mixture was stirred for 30 mins. Then, carbon disulfide (0.2ml, excess) was added to the mixture and refluxed for 2 hrs. Methyl iodide (0.1ml, excess) and the mixture was heated for reflux for further 1 hr. The reaction mixture waε treated with water (1ml) and the solvent waε diεtilled and removed under reduced preεεure. The mixture was extracted with ethyl acetate (10ml), waεhed with water (2ml x 3) and brine (2ml x 3) and then dried over anhydrouε magnesium εulfate. The

organic layer was concentrated under reduced presεure.

The reεidue waε purified with column chromatography (hexane:ethyl acetate=10:1) to give white εolid (56mg, 96%) .

[α] ²⁵ _D ;-32.3 (c=1.33,CHCl ₃ ) IR(neat) :1723, 1233, 1057cm ^{"1 1} H NMR (CDC1 ₃ ) δ

5.78(lH,m) , 5.24(lH,bt), 3.80 (IH,d,J=10Hz) , 3.60(3H,ε) , 3.54, 3.58(2H,dd,J=7.2Hz) , 2.51(3H,ε) 2.23 (IH,d,J=ll.2Hz) , 0.94(3H,d,J=5.2HZ) , 0.84 (3H,d,J=6Hz) , 0.73, 1.09, 1.11, 1.14, 1.41, 1.45 (each 3H,S) .

Example 20:Preparation of methyl 2-deoxy-3,23-0- isopropylidene asiatate(20)

Xantate compound 19 (202mg, 0.32 mmole) was added with catalytic amounts of AIBN and benzene (10ml) and heated for reflux. The mixture waε then added with tributyltin hydride(0.26ml, 0.96 mmole) and εtirred for 1.5 hrs. The reaction mixture waε concentrated under reduced pressure to remove the εolvent. The reεidue was purified with column chromatography (hexane:ethyl acetate=10:l) to give white solid (168mg, 100%) . The crude product waε recryεtallized from hexane to give needle cryεtal.

α -' ;+56.2 (c=1.07, CHCl,)

IR(neat) :1724cm ^"1

MS (El) : 527(M ^* +1), 512, 407, 262, 203, 133. ¹ H NMR (CDCl ₃ )δ

5.25(lH,bt) , 3.60(3H,S) , 3.52(lH,t) , 3.44, 3.54 ( 2H , dd , J= 1 OHz ) , 2.23 ( 1H , d , J= 11.2H z ) , 0.94(3H,d,J=5.6Hz) , 0.86(3H,d,J=6.4Hz) , 0.73, 0.97, 1.07, 1.09, 1.42, 1.45(each 3H,ε)

Example 21 : Preparation of methyl 2-deoxyasiatate(21)

Compound 20 (460mg, 0.87 mmole) was added with tetrahydrofuran (10ml) and IN hydrochloric acid (1ml) and stirred at room temperature for 5 hrs. The mixture was distilled under reduced pressure to completely remove the solvent. The reεidue was purified with column chromatography (hexane : ethyl acetate=3:2) to give white solid (402mg, 95%). The crude product was recrystallized from ethyl acetate to give needle crystal.

[α] ,2"5 _D ;+69.6 (c=1.22, CHCl ₃ ) IR(neat) :3400, 1724cm ^'1 MS (El) :486(M ^* ), 426, 262, 203, 133

Example 22 : Preparation of 2-deoxyasiatic acid(22)

Methyl 2-deoxyaεiatate 21 (38mg, 0.78 mmole) waε added with Lil'3H _∑ 0 (450mg, 2.39 mmole) and 2,4, 6-collidine (5mg) and heated for reflux for 10 hrs. During the

reflux, a flask was wrapped with aluminum foil to prevent the light. The reaction mixture was concentrated under reduced pressure to remove collidine. The reεidue was purified with column chromatography (dichloromethane : methanol=20:1) to give pale yellow solid. The crude product was recrystallized from methanol to give needle crystal (280mg, 76%) .

IR (KBr) : 3436,1693cm ^'1 MS (El) : 472(M+), 426, 248, 203, 133 ¹ H NMR (CDC1 ₃ + pyridine-d ₅ )<S

5.21(lH,bt,J=2.8Hz,3.6Hz) , 3.60(IH,t, =1 .2Hz,8.2Hz) , 3.36, 3.70(2H,dd,J=10.0Hz) , 2.21(IH,d,J=ll.2Hz) .

Example 23 : Preparation of methyl 2-0-benzyl-3,23- isopropylidene asiatate (23)

Sodium hydride (60% in mineraloil; 35mg, 0.88 mmole) was washed with anhydrous hexane and added with a εolution of methyl 3 ,23-0-iiεopropylidene aεiatate 3 (240mg, 0.44 mmole) and TBAI (20mg) diεεolved in anhydrouε tetrahydrofuran. The mixture waε εtirred at room temperature for 20 minε, added with benzyl bromide (114mg, 0.67 mmole) and heated for reflux for 2 hrε. After removing the εolvent by diεtillation under reduced preεεure, the mixture waε treated with water (3ml) and extracted with ethyl acetate (20ml) . The organic layer waε waεhed with water (2ml x 3), and a εolution of εaturated ^' εodiu

chloride (3ml x 3) , and dried over anhydrouε magneεium εulfate. The organic layer waε concentrated under reduced pressure. The residue waε purified with column chromatography (hexane : ethyl acetate=10: 1) to give white solid (258mg, 92%) . The crude product was recrystallized from ethyl acetate to give needle crystal.

[α] ²³ _D ;+27.3(c=1.31, CHC1 ₃ ) IR(neat) : 1724cm ^'1

¹ H NMR (CDCl ₃ )ό

7.24-7.33(5H,m) , 5.25(lH,bt) , 4.58, 4.80(2H,dd,J=ll.6Hz) ,

3.62(3H,ε) , 3.60-3.50(4H,m) , 2.23 (IH,d,J=ll.2Hz) , 1.46,

1.47(each 3H,s) .

Example 24 : Preparation of 2-0-benzyl-3,23-0- isopropγlidene asaticol (24)

Compound 23 (940mg, 1.49 mmole) waε diεεolved in anhydrouε ether (5ml) , added with lithium aluminum hydride

. l.OM ether εolution; 1.5ml, 1.49 mmole) and heated for stirring for 1 hr. Water in εmall portionε waε added to the reaction mixture and then, produced aluminum hydride waε filtered off. The organic layer waε concentrated under reduced preεsure. The reεidue waε purified with column chromatography (hexane:ethyl acetate=l: 3) to give white εolid (890mg, 99%) .

[α] ^Z5 _D ; + 32.6(c=1.17, CHC1 ₃ ) IR(neat) : 3467cm ^"1

7.25-7.35(5H, m) , 5.14(1H, bt) , 4.82(2H, ABq, J=12.0Hz) , 3.46-3.64(5H, m) , 3.19(1H, d, J=11.2Hz) , 2.09 (IH, d) , 1.47, 1.48(each 3H,s)

RYam i 25 : Preparation of 2α-benzyloxy-3g. 23- isopropylidenedioxyurs-12-ene-28-methanesulfonate (25)

Compound 24 (400mg, 0.66 mmole) and triethyl amine

(0.28ml, 1.98 mmole) were dissolved in anhydrous dichloromethane (5ml) , added with methanesulfonyl chloride (76ul, 0.99 mmole) at 0°C and stirred for 30 mins. The mixture was distilled under reduced pressure to remove the solvent and extracted wrth ethyl acetate (20ml) . The organic layer was washed with water (30ml x 3) and a solution of εaturated εodium chloride (3ml x 3) , and dried over anhydrouε magneεium sulfate. The organic layer waε concentrated under reduced pressure. The residue was purified with column chromatography (hexane: ethyl acetate=2:l) to give white solid (430mg, 95%).

[ ] ²³ _D ;-*-23.4(c=1.28, CHC1 ₃ ) IR(neat): 1361, 1176cm ^"1 Η NMR (CDC1 ₃ )<5

7.25-7.35 (5H,m) , 5.17 (IH,b ,J=2.8Hz , 3.6HZ) , 4.58, 4.81(2H,dd,J=11.6Hz) ,3.74, 4.19 (2H,dd,J=9.2HZ) , 3.48-3.71(4H,m) , 2.96(3H,s), 1.46, 1.47(each 3H,s).

Example 26 : Preparation of 2α-benzyloxγ-28-cγano-3g

,23-isopropγlidenedioxyurs- 12-ene(26)

Compound 25 (500mg, 0.73 mmole) and sodium cyanide (90%, 121mg, 2.22 mmole) were dissolved in anhydrous dimethylform amide and heated for reflux for 12 hrs. The mixture waε distilled under reduced pressure to completely remove the solvent and followed by the addition of ethyl acetate (10ml) to dissolve produced nitrile and remove the salts by filtration. The organic layer was concentrated underr reduced pressure. The residue was purified with column chromatography (hexane: ethyl acetate=5:l) to give white solid (383mg, 85%).

[ ] ²³ _D ;+27.7(c=1.13, CHC1 ₃ )

IR(neat) : 2241cm ^'1

¹ H NMR (CDC1 ₃ )<S

7.22-7.27(5H,m) , 5.20(lH,t), 4.54, 4.78 (2H,dd,J=ll.6Hz) ,

3.47-3.60(4H,m) , 2.38(lH,d) .

Example 27 : Preparation of 2α-benzyloχy-28-cyano-3g

,23-dihydroxyurs-12-ene(27)

By the same procedure aε deεcribed above for the preparation of Example 17 acetonid of compound 26 (95mg, 0155 mmole) waε deprotected to give white solid (85mg, 96%) .

IR(neat) :3436, 2240cm ^"1

MS (El) : 573(M ^* ) , 482, 434, 331, 243, 203, 133, 91 ¹ H NMR (400 MHz, CDC1 ₃ ) δ

5.26(lH,bt) , 4.46, 4.68(2H,AB quartet ,J=11.2Hz) , 3.57(lH,m) , 3.53(lH,d,J=9.2Hz) , 3.41, 3.67(2H,AB quartet, J=l 0.4Hz) , 2.09, 2.42 (2H,AB quartet ,J=16.4Hz) .

Example 28 Preparation of 28-cyano-2α, 3/3,23-trihvdroxyurs- 12-ene(28)

Compound 27 (500mg, 0.873 mmole) and 10% Pd/C (90mg, about 3 mole%) were dissolved in methanol (10ml) and the inside of flask was filled with hydrogen. The mixture was then stirred at ambient pressure for 8 hrs and Pd/C waε filtered off. The reaction mixture was concentrated under reduced pressure to give white solid (420mg, 99.6%).

IR(neat) :3401, 2240cm ^'1

MS (El) : 483(M ^* ), 465,447,435,243,203,199,133 Η NMR (400 MHz, CDC1 ₃ ) δ

5.25(lH,bt) , 3.77(lH,m) 3.69 (IH,d,J=10.4Hz) , 3.43,

3.45(2H,AB quartet) , 2.03, 2.45 (2H,AB quartet,J=10.8Hz) .

Example 29 : Preparation of methyl 24-norurs-l2-ene-3-one -28-oate(29)

Methyl 2-deoxyasiatate 14 (24mg, 0.05 mmole) and pyridinium dichromate (56mg, 0.15 mmole) were placed in a

flask and the air was subεtituted with nitrogen. Then, by the addition of dichloromethane (4ml) , the mixture waε εtirred at room temperature for 5 hrs, filtered off by silica gel pad and concentrated under reduced pressure. The residue was purified with column chromatography to give white foaming solid (I6mg, 71%) .

^ NMR (CDC1 ₃ )<5

5.27(lH,t), 3.62(3H,S), 2.46(IH,dt,J=12Hz, =6.8Hz) , 2.33-2.27 (2H,m) , 2.25(IH,d,J=12Hz) , 1.13, 1.07,

0.827(each 3H,ε), 1.00(3H,d, 3=6.4Hz) , 0.96(3H,d,J=10Hz) , 0.86(3H,d,J=6.8Hz)

Example 30 : Preparation of methyl 3j3-hγdroxy-24-norurs-12 -ene-28-oate

'H NMR (400MHz, CDCl ₃ )<5

5.24(lH,t) , 3.58(3H,S) , 3.06(lH,dt) , 2.21 ( IH , d , J=ll .2Hz ) , 1.24 (3H,ε) , 1.05(3H,s) , 0.95 ( 3H , d , 3=6.4Hz ) , 0.84 (3H,d,J=7Hz) , 0.84 (3H, d, 3=6.6Hz) , 0.75(3H,ε)

Example 31 : Preparation of methyl 33-hydroxy-3α -vinyl-24 -norurs-l2-ene-28-oate

Η NMR (400MKZ, COCI.) δ

5 . 7 9 ( l H , d d , J = 1 2 . 2 H z , J = 2 1 . 4 H z ) , 5.24 (lH,dd,J=1.4Hz J=21.4HZ) , 5.03 ( IH , dd , J=l . Hz ,

J=12.2Hz) , 3.57(3H,ε) , 2.22(lH,d) , 1.08(3H,s) , 0.93(3H,ε) ,

0 . 76 ( 3H , ε )

Example 32 : Preparation of methyl 3/3-hydroxy-3α

-methyl-24-norurs-l2-ene-28-oate

Compound 29 (15mg, 0.034 mmole) was dissolved in anhydrous tetrahydrofuran (4ml) and added with methylmagnesium chloride (34μl, 0.1 mmole) at room temperature. The mixture was stirred at the same temperature for 5 hrs and the reaction was stopped by the addition of water in small portions. The reaction mixture was filtered off and concentrated under reduced pressure. The residue was purified with column chromatography to give desired compound in 98% yield.

H NMR (400MHz, CDC1 ₃ ) 5 ⁶

5.26(lH,t), 3.60(3H,S) , 2.23 (IH,d,J=ll.2Hz) , 1.18(3H,ε) , 1.09(3H,ε) , 0.94 (3H,d,J=6Hz) ,

0.89(3H,d,J=6.8Hz) , 0.85(3H,ε) , 0.77(3H,ε)

Example 33 Preparation of methyl 3,23-isopropylidenedioxy -2-methoxyurs-2.l2-diene-28-oate (33)

¹ H NMR (400MHz, CDCl ₃ ) 5 5.30(lH,t) , 3.62(3H,S), 3.56(IH,d,J=6Hz) , 3.54(3H,ε) , 2.27(lH,d) , 1.53, 1.42, 1.26, 1.10, 1.04, 0.78 (each 3H,s) , 0.95 (3H, d, 3=6. Hz) , 0.88 (3H, d,J=4.4Hz)

Example 34 : Preparation of methyl 23-hvdroχy-2-methoxyurs- 12-ene-3-one-28-oate(34)

¹ H NMR (400MHZ, CDC1 ₃ )<5 5.28(lH,t), 4.25(lH,dd,J=7.2Hz, J=11.2HZ), 3.60(3H,S), 3.40(3H,S), 2.26(lH,d,J=12Hz) , 1.13(3H, s) , 1.07( 3H, S) , 0.94 (3H,d,J=6Hz) , 0.90(3H,S), 0.87 (3H,d, 3=6.4Hz)

Example 35 : Preparation of ethoxymethγl 2,3,23- triacetylasiatate(35, R _a =H, R^s-methyl)

2,3,23-triacetylasiatic acid (300mg, 0.49 mmole) was dissolved in dichloromethane (20ml) , added with diisopropylethyl amine (0.3ml, 1.7 mmole) and then cooled. The mixture waε stirred by the addition of chloromethyl ether (0.07ml, 0.6 mmole) in small portions. After confirming the annihilation of a starting material, the reaction mixture was diluted with ethyl acetate. The organic layer waε washed with 5% hydrochloric acid, water, a solution of saturated sodium bicarbonate and saturated brine in sequence. The solution waε dried over anhydrouε sodium εulfate and filtered off. The remaining εolution waε concentrated under reduced preεsure. The reεidue waε purified with column chromatography with hexane and ethyl acetate (5:1) to give deεired compound as a solid (250mg, 78%) .

Η NMR (300MHZ, CDC1,) δ

5.28 ( IH , t , J=3.6Hz ) , 5.21 ( IH , d , J=6Hz ) ,

5.17 (lH,d, J=6Hz) , 5.18 ( IH , td , J=10.2Hz , J=3.9Hz) ,

5.08 (lH, d,J=10.2Hz) , 3.86 (lH, d,J=11.7Hz) ,

3.58(lH,d,J=11.7Hz) , 3.45(3H,ε) , 2.28 ( IH, d, J=ll .4Hz) , 2.09, 2.03, 1.98, 1.11, 1.09, 0.89, 0.79(each 3H,s) , 0.96(3H,d,J=6.0Hz) , 0.86 (3H, d, J=6.6Hz) , 2.0-1.0 (20H,m)

Example 36-46

By the similar procedure as described in Example 5, compoundε were synthesized or by deprotecting compounds, so obtained, some compounds as shown in the followng table 1 were prepared.

] 0

20

Fγ»ιn ie 47 : Preparation of 2 ^/ -tetrahydropyranyl-2.3,23 -triacetylasiatate (37, R ₁₀ -R ₁ ■■■= -CH-.CH-.CH-,-)

2,3,23-triacetylasiatic acid (1150mg, 1.88 mmole) 5 _. waε dissolved in dichloromethane (15ml) , added with 3,4-dihyro-2H-pyran (0.68ml, 7.52 mmole), and then cooled. The mixture was stirred by the addition of p-toluenesulfonic acid (lOmg) for 12 hrs. The reaction mixture was diluted with ethyl acetate, dried over

10 anhydrous sodium sulfate and filtered off. The remaining solution was concentrated under reduced pressure. The residue was purified with column chromatography with hexane and methanol (20:1) to give deεired compound as white powder (1185mg, 90%).

15 NMR (400MHZ, CDC1 ₃ )<5

5.88 ( 1/2H , m) , 5.84 ( 1/2H , m) , 5.20 (lH,m) ,

5.09(td,J=4Hz,J=10Hz,) , 5.01 (IH, d, J=10Hz) , 3.80(lH,m) ,

3.78 (lH,d, J=12HZ) , 3.60(lH,m) , 3.52 ( IH , d , J = 12Hz ) ,

20 _. 2.22 (l/2H,d, J=11HZ) , 2.19 ( l/2H,d, J=llHz) , 2.02, 1.95,

1.91, 1.03, 1.02, 0.81(each 3H,s) , 0.88 (3H, d, J=6Hz ) ,

0.79 (3H,d,J=6.4Hz) , 0.71 ( 3H, d, J=3.6Hz ) , 2.0-1.0(26H,m)

25 Example 48-54

By the similer procedure aε described in example 47 and by deprotecting the hydroxy s of compoundε, εo

obtained, some compounds (48-52) aε shown in the following table 2 were prepared. The compounds 53 and 54 were prepared based upon said Example 35 or upon deprotecting the hydroxy s of the compoundε, εo obtained, with potassium carbonate.

Table. 2

] <J

o

15

?0

10

l ^r )

20

?X>

.0

Example 55 : Preparation of methyl 2-0-methylasiatate

( 1.R3=methyl)

Η NMR (400MHz, CDC1 ₃ ) δ 5.26(lH,t,J=3.6Hz) , 3.24-3.32 (lH,m) 2.24(d,J=ll.2Hz) , 3.60, 3.39, 1.07, 1.03, 0.92, 0.75, (each 3H,S), 0.94 (3H,d,J=5.8Hz) , 0.85(3H,d,J=6.4Hz)

Example 56 :Preparation of methyl 3,23-diacetγl-2-0- methyl-ll-oxoasiatate(42. R-,=methyl)

Compound 41 (R3=methyl, 400mg) and DMAP (30mg) were placed in a flaεk and the air was substituted into nitrogen. Then, by the addition of tetrahydrofuran (10ml) and acetic anhydride (1ml) in sequence, the mixture waε εtirred at room temperature for 1 hr. After confirming the progreεε of the reaction by TLC, methanol (10ml) waε added to the reaction mixture and followed by concentration under reduced preεsure. Sodium dichromate (460mg) was added to the reaction mixture and followed by the addition of acetic acid (20ml) . The reaction mixture was heated for reflux in oil bath for 2 hrε and the progreεs of the reaction waε confirmed by TLC. Acetic acid was filtered off under reduced preεεure. The residue was extracted with ethyl acetate and then, the organic layer waε waεhed with water five timeε and concentrated under reduced preεsure. A desired compound waε obtained aε white solid.

¹ H NMR (400MHz, COCI.) δ

5.63(lH,ε) , 4.95(lH,d,J=10Hz) , 3.80, 3.56 (2H, ABq, J=ll.6Hz) , 3.40-3.48(lH,m) , 3.61, 3.33, 1.30, 1.23, 0.90, 0.85(each 3H,S) , 2.06(6H,S) , 0.97 (3H, d, 3=6.4Hz) , 0.87 (3H, d, 3=6.8Hz) , 2.43(lH,d,J=11.2Hz)

Exaunple 57 Preparation of methyl 2-O-methγl-ll- oxoasiatate ( 3, R=methγl )

E NMR (400MHz, CDC1 ₃ ) δ

5.63 (IH, s) , 3.62, 3.41,1.31, 1.20, 0.92, 0.89 (each 3H, S) , 2.42 ( IH, d) , 0.98 ( 3H, d, J=7Hz) , 0.87 ( 3H, d, J=6.4Hz)

Example 58 : Preparation of methyl 2-O-ethyl asiatate (43,

R _j ≡ethyJ.

^ NMR (400MHZ, CDC1 ₃ ) δ

5.26(lH,t) , 3.61(3H,ε) , 2.23(lH,d) , 1.20 ( 3H , t , 3=6.8Hz ) , 0.94 (3H,d,J=6.4Hz) , 0.85 (3H , d, 3=6.4Hz ) , 0.75, 0.90, 1.04, 1.56 (each 3H,ε)

Example 59 Preparation of methyl 2-O-ethyl-ll-oxoasiatate ( 3,R-,=ethyl)

¹ H NMR (400MHZ, COCI.) £

5.61 ( IH, ε) , 3.61 (3H, ε) , 2.41 ( IH , d) , 1.19 ( 3H, t, J=6.8HZ) , 0.97, ( 3H, d, J=6.0Hz) , 0.86 ( 3H, d,

J=6.8Hz) , 0.89, 0.91, 1.23, 1.30 (each 3H, ε)

Example 60 : Synthesis of methyl 2-Q-acetyl asiatate(44)

¹ H NMR (400MHz, CDC1 ₃ ) <5

5.24 (lH,t, J=3.6Hz) , 4.97-5.03 ( lH,m) , 3.60(3H,s) ,

2.23(lH,d,J=11.6Hz) , 2.02(3H,S) , 1.08 (3H, d, 3=6. OHz) ,

0.84(3H,d,J=6.4Hz) , 0.75, 0.90, 1.07, 1.09(each 3H,ε)

Example 61 : Synthesis of methyl 2α-acetyl-3i3-hydroxyurs

-l2-ene-23-al-28-oate (45)

Compound 44 (300mg, 0.55 mmole) and pyridinium dichromate (PDC; 413mg, 2 equivalents) were placed in a flask and the air was εubεtituted into nitrogen. Then, said materials were dissolved in dichloromethane (9ml) and stirred at room temperature for 3 hrs. After confirming the end of the reaction by TLC, iεopropanol (1ml) waε added to the reaction mixture and eluted with ethyl acetate on silica gel εhort column to remove the mineral material. The εolution, εo obtained, was dried over anhydrouε magneεium εulfate and concentrated under reduced pressure. The reεidue waε purified with silica gel column chromatography with hexane and ethyl acetate

(10:1) to give deεired compound aε white εolid (150mg,

53%) .

^ NMR (400MHz, COCI.) δ

9.40(1H,S) , 5.25(lH,t,J=4Hz) , 4.99-5.05 ( lH,m) ,

3.60(3H,ε) , 2.24 (IH, d, J=12Hz) , 2.08(3H,ε) ,

0.94 (3H,d,J=6Hz) , 0.85(3H,d,J=6.4Hz) , 0.75, 1.09, 1.11, 1.29(each 3H,s)

τ.v>ιtnpift '2 : Synthesis of methyl 2α-acetylurs-12-ene-23-al- 3-one- 28-oate(45)

¹ H NMR (400MHz, CDC1 ₃ ) δ

9.43(lH,ε) , 5.47-5.55 (lH,m) , 5.27(lH,t) , 3.61(3H,ε) , 2.25 (lH,d,J=llHz) , 2.13(3H,ε) , 0.94 (3Hd, 3 = 6.4Hz) , 0.86(3H,d,J=6.4Hz) , 0.82, 1.10, 1.33, 1.34(each 3H, s)

Example 63 : Synthesis of methyl 2α-benzyl-3g ,23-dihydroxyurs-12-ene-28-oate (47)

^' 'H NMR (400MHz, CDC1 ₃ ) δ

7.27-7.36(5H,m) , 4.68, 4.44 (2H,ABq,J=ll.2HZ) , 3.61(3H,s) , 2.21(lH,d) , 1.08, 1.03, 0.90, 0.75(each 3H,ε) , 0.94 (3H,d,J=5.8Hz) , 0.86 (3H,d, 3=6.4Hz)

Example 64 : Synthesis of methyl 2α-benzyl-3g-hydroxyurs-

12-ene-23-al-28-oate (48)

^' Η NMR (400MHz, CDC1 ₃ ) δ

9.35(lH,ε) , 7.29-7.36 (5H,m) , 5.27(lH,t) , 4.69,

4.46(ABq,2H,J=11.2Hz) , 3.60(3H,s) , 2.25(lH,d) , l. 'l,

1.09, 1.04, 0.75(each 3H,ε) 0.95 (3H, d, 3=6.4Hz) ,

0.91 (3H,d,J=6. OHz)

Example 65 : Preparation of 2α-benzyloxy-3_3,23-hγdroxγurs- 12ene -28-carboxylic acid (49)

¹ H NMR (400MHZ, CDC1 ₃ ) δ

7.30-7.40(5H,m) , 5.21(lH,t), 4.70,

4.47 (2H,ABq,J=11.6HZ) , 3.67, 3.43 (2H,ABq,J=10.8Hz) , 2.28(lH,t,J=13.7Hz) , 1.13, 1.08, 1.05, 0.94(each 3H,s), 0.93 (3H,d,J=8.8Hz) , 0.82 (3H,d, 3=6.4Hz)

Example 66 : Preparation of 2α,30 ,23-trihydroxyurs-12-ene- 28-carboxylic acid (50, 28-homoasiatic acid)

H NMR (400MHZ, CDC1.) δ

5.17(lH,t,J=3.4Hz) , 3.87-3.93 (lH,m) , 3.82,

3.70(2H,ABq,J=10.3Hz) , 3.50 (IH,d, 3=1.8Hz) ,

2.43 (lH,d,J=13.2Hz) , 1.98, 1.89 (2H,ABq,J=13.2Hz) , 1.08, 1.06, 1.02, 0.93(each 3H,ε) , 0.92 (3H,d,J=8.8Hz) , 0.81 (3H,d,J=6.4Hz)

Example 67 Preparation of 3,23-0-isopropylidene-2- oxoasiatic acid (51)

Η NMR (400MHZ, CDCl ₃ ) 6

5.23(lH,m) , 4.39(lH,bε) , 3.62(lH,bε) , 3.49(lH,bε) ,

2.62(lH,s) , 1.50(3H,ε) , 1.44(3H,ε) , 1.24(3H,ε) , 1.13(3H,s)

1.01(3H,S) , 0.95-0.85(6H,m) , 0.75(3H,s)

Example 68-69.71-73 and Example 75-80

By the similar procedure as described in Example 35 or 47, compoundε were synthesized or by deprotecting the hydroxy s of compounds, so obtained, in a common method, compounds as shown in the followng table 3 were prepared.

10

-J

1

20

10

15

20

BvwTnpift 70Preparation of 2-Q-acetyl-3 ,23-o-isopropγlidene asiatic acid (54)

'H NMR (400MHz, CDC1 ₃ ) δ

5.23(t,lH,J=3.6Hz) , 4.96-5.03 (m, IH) , 3.67, 3.42(AB 5 q,2H,J=10.8Hz) , 3.65(d, IH, 3=9.8Hz) , 2.18 (d, IH,J«ll.7Hz) ,

2.09, 1.09, 1.08, 0.88, 0.78(each s, 3H) , 0.94(d,

3H,J=6Hz) , 0.84 (d,3H,J=6.8Hz)

Example 74Preparation of 2-deoxγ-3 ,23-0-diacetγlasiatic acid (60)

10 ^ NMR (400MHz, CDC1 ₃ ) 6

5.25(1H,S) , 4.79(lH,t), 3.88, 3.70(AB q, 2H,J=ll ^' .2Hz) , 2.19(lH,d,J=10.8Hz) , 2.03, 2.06, 1.08, 0.99, 0.86, 0.78(each s, 3H) , 0.86 (d, 3H,J=5.6Hz)

Example 78 : Preparation of 2, 23-diacetylurs-12-ene-28-oic 15 acid(68)

^" 'H NMR (400MHZ, CDC1 ₃ ) δ

5.26 ( IH , t ) , 5.17 ( lH, t , J= 9.2Hz ) , 3 , 89 ,

3.69 (2H,ABq,J=10.8Hz) , 2.19 (IH,d,J==10.8Hz) , 2.08, 2.02, 1.20, 1.08, 0.99, 0.79(each 3H,ε) , 0.95 (3H,d,J=6. Hz) , 2.0 0.85(3K,d,J=6.8Hz)

Example 81 : Preparation of methyl 2α-azidoasiatate (74)

¹ H NMR (400MHz, CDC1 ₃ ) <S

5.25(lH,m) , 3.65-3.39(4H,m) , 3.55(3H,ε) , 1.18(3H,ε) ,

1.05(3H, s) , 0.98(3H,S) , 0.94 (3H,m, J=5. OHz) ,

0.86(3H,m,J=8.0Hz) , 0.75 (3H,s) .

Example 82 Preparation of methyl 2α-thiophenoxyasiatate(75)

m.p.: 229-233°C

Example 83 : Preparation of methyl 3g-hydroxγ-23- undecylenyloxyurs-12-ene-28-oate (76)

Compound 14 (200mg, 0.41 mmole), εo obtained from Example 13, and DCC (93.4mg, 0.45 mmole) were dissolved in dichloromethane (15ml). Then, undecylenic acid (83.4 mg, 0.45 mmole) and dimethylaminopyridine (40.2mg, 0.33 mmole) were added to the mixture at 0°C and stirred under nitrogen atmosphere at room temperature for 2 hrs. The reaction mixture waε filtered on celite. The remaining solution was concentrated under reduced presεure and followed by the addition of water. The organic layer waε extracted with ethyl acetate, waεhed with 10% hydrochloric acid, a εolution of εaturated εodium carbonate, water, and a solution of εaturated sodium chlcride, and then, dried over anhydrouε magneεium εulfate. The remaining solution was concentrated under reduced presεure. The reεidue was purified with column chromatography with hexane and ethyl acetate

(2:1) to give desired compound as an oil (225mg, 85%).

Example 84 : Preparation of methyl 2.3, 3/3-epoχy-23- undecγlenγloxγurs-12-ene-28-oate(77)

¹ H NMR (400MHZ, CDC1 ₃ ) <S 5.80(lH,m) , 5.27(lH,m) , 4.99 ( IH, d, J=17. OHz) ,

4.93(lH,d, -74 J=12.3Hz) , 4.10 (IH, d, J=ll.1Hz) ,

3.88 (IH, d, J=ll .1Hz) , 3.60(3H,s) , 3.25(lH,bs) ,

3.04(lH,d,J=3.2 Hz) .

Example 85 Preparation of dimethyl 2/3. 3β-epoχyurs-12-ene- 23,28-dioate(79)

¹ H NMR (400MHz, CDCl ₃ ) <5

5.26(lH,m) , 3.75(3H,ε) , 3.60(3H,s) , 3.30 (IH, d, J=3.8Hz) , 3.19(lH,d,J= 3.9Hz) , 1.25(3H,s) , 1.09(3H,ε) , 1.06(3H,ε) , 0.94 (3H,d,J=5.2 Hz) , 0.86 ( 3H, d, J=8. OHz) , 0.72(3H,ε) .

Preparation of crude product (compound 78)

Η NMR (400MHZ, CDCl ₃ ) δ

5.27(lH,m) , 3.60(3H,S) , 3.32 ( IH , d , J=3.8Hz ) ,

3.28-3.27(lH,m) , 1.24 (3H,s) , 1.10(3H,ε) , 1.06(3H,s) , 0.9 (3H,d,J=5.3Hz) , 0.86 ( 3H , d , J=6.4 Hz) , 0.73(3H,s) .

Example 86 Preparation of methyl 2/3. 30-epoxyurs-l2-ene-

23-N-phenylamido-28-oate (80)

¹ H NMR (400MHz, CDC1.) δ

8.10(lH,d,J=8.5Hz) , 7.61-7.55 (lH,m) , 7.49-7.43 (IH, m) ,

7.35(lH,d,J= 8.5HZ) , 5.29(lH,m) , 3.62-3.60 (lH,m) ,

3.60(3H,S) , 3.50-3.47(lH,m) , 1.25(3H,s) , 1.19(3H,s) , 1.11(3H,S) , 0.94 (3H, d, J=6.0Hz) , 0.86 (3H,d, 3= 6.4 Hz) , 0.78(3H,S) .

Experiment l : Wound healinσ properties of the present invention

Preparation of an ointment

200mg of a compound of the present invention, accurately weighed, was put into a syringe (20ml) . Then, propylene glycol (6g) , glycol stearate (3g) and white petriatum (lg) , accurately weighed, were put into said syringe. The syringe was immersed in water bath 80°C to completely melt the contents. It was agitated for about 5 mins εo that active ingredientε may be homogeneouεly diεperεed to εaid 3 bases. Another syringe to put purified water (lOg) heated at 80°C waε prepared. By connection of two εyringeε to a threeway connector, the input at both εideε waε repeated about 20 timeε εo that the contentε were homogenized. The homogenized contentε were put into a container and εolidified εlowly at room temperature.

Method

To evaluate the wound healing effects related to newly synthesized asiaticoside derivatives and naturally separated asiaticoside, aεiatic acid and madecassic acid, rats were given wounds. Among several 5. methods to measure the wound healing effects based upon a rationale that the wound leεionε associated with trauma or necroεiε are cured by tissue regeneration such as exuberant granulation, etc. , the tensile strength method is derived from the fact that tensile strength iε 0 evenly increaεed until a recovered site of wounded tissue is to be recleaved and under pulling at both sides, the force until the wound εite iε cleaved is measured. Meantime, it has been noted that the tensile strength method in cleaved wounds reflects the quality and speed of 5 regeneration very well.

The following table, using said tensile strength method, compared the wound healing effects between TECA

(titrated extracted Centella asiatica) , one of the active ingredients of currently marketed madecassol ointment and 0 newly syntheεized aεiaticoεide derivatives.

0 Compound 7 (R=H) 345.00 ± 29.74 -0.3

Compound 7(R=CH ₃ ) 383.00 ± 28.78 10.7

Compound 5(R=CH ₃ ) 357.00 ± 21.54 3.2

TECA 346.00 ± 22.64 5 Compound 14 520.00 ± 46.94 7.7

Compound 10 321.25 ± 26.66 -3.3

Compound 15 506.25 ± 41.57 4.9

TECA 482.50 ± 42.71 0 Compound 12 306.25 ± 26.69 -20.7 Compound 16 303.75 ± 32.01 -21.4 Compound 74 316.25 ± 25.24 -18.1 Compound 41 (R ₃ =CH ₃ ) 331.25 ± 20.19 -14.2 Compound 43 (R ₃ =CH ₃ ) 392.50 ± 27.83 1.6 TECA 386.25 ± 41.11

Compound 41 (R ₃ =C ₂ H ₅ ) 355.00 ± 52.35 4.8

Compound 43 (R ₃ =C ₂ H ₅ ) 405.00 ± 43.59 19.6

Compound 2a 416.25 ± 23.58 22.9 0 Compound 2b 452.50 ± 43.86 33.6

TECA 338.75 ± 30.38

Compound 75 418.75 ± 32.51 -2.9

Compound 78 (R ₅ =0H) 368.57 ± 30.85 -14.5 _5 Compound 79 (R ₅ =0Me) 367.50 ± 56.12 -14.8

Compound 77 362.50 ± 32.90 -15.9

TECA 431.11 ± 42.09

Compound 76 391.25 ± 40.75 4.7 _0 Compound 18 (R'=CH ₃ ) 277.78 ± 14.72 -25.7

Compound 18 (R'=C ₂ H ₅ ) 297.50 ± 25.66 -20.4

TECA 373.75 ± 23.45

Compound 45 287.50 ± 2.83 -4.2 _5 Compound 53 351.15 ± 49.51 17.1

Compound 52 303.75 ± 17.49 1.3

Compound 64 336.25 ± 31.02 12.1

Compound 61 360.00 ± 21.58 20.0

Compound 59 323.75 ± 47.37 7.9 0 Compound 58 327.50 ± 40.96 9.2

Compound 57 320.00 ± 28.50 6.7

TECA 300.00 + 34.68

Test Tensile Strength % increase from

Compounds (g ± S.E) TECA Compound 38 (R ₁₀ -R ₁₁ =-CH ₂ CH ₂ CH ₂ -) 488.89 ± 33.05 21.55

Compound 36(R _g =H, R ₉ =octyl) 396.00 ± 26.43 -1.55

Compound 36(R ₈ =H, R ₉ =ethyl) 410.00 ± 32.32 1.93

Compound 37 (R^-R^-CH-.CH^CH-,-) 426.67 ± 22.50 6.08

Compound 35(R ₈ =H, R ₉ =ethyl) 538.00 ± 38.75 33.76 TECA 402.22 + 27.48