Preparation of analogues of Territrem B, a potent AChE inhibitor

Article abstract of DOI:10.1016/S0040-4020(00)00817-6

The synthesis of analogues of the potent acetylcholinesterase inhibitor, Territrem B, was carried out starting from the naturally occurring jujubogenin glycosides. Dihydrojujubogenin-2-en-1-one (1), a dammarane derivative that possesses a skeleton and pharmacophore partially similar to those of Territrem B, was synthesized via three different paths. The derivative 21, which contains two potential pharmacophores, was also synthesized. The anti-AChE activity of the analogues was measured. The aromatic ring moiety seemed to be less important when compared with the 2-en-1-one pharmacophore. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00817-6

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 8901±8913
Preparation of Analogues of Territrem B,
a Potent AChE Inhibitor
b
a
b,
Yu Zhao,a,b, Yuan-Ling Ku, Xiao Jiang Hao and Shoei-Sheng Lee
*
*
aLaboratory of Phytochemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China
bSchool of Pharmacy, College of Medicine, National Taiwan University, Taipei 10018, China
Received 5 June 2000; revised 23 August 2000; accepted 7 September 2000
AbstractÐThe synthesis of analogues of the potent acetylcholinesterase inhibitor, Territrem B, was carried out starting from the naturally
occurring jujubogenin glycosides. Dihydrojujubogenin-2-en-1-one (1), a dammarane derivative that possesses a skeleton and pharmacophore
partially similar to those of Territrem B, was synthesized via three different paths. The derivative 21, which contains two potential
pharmacophores, was also synthesized. The anti-AChE activity of the analogues was measured. The aromatic ring moiety seemed to be
less important when compared with the 2-en-1-one pharmacophore. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
the preliminary SAR investigation of Terretrim B deriva-
tives suggested that the 2-en-1-one moiety was essential for
its inhibitory activity on AChE.7 So we utilized a naturally
occuring compound which has a similar skeleton and stereo-
chemistry at the A and B rings to Territrem B as starting
material and tried to build up the 2-en-1-one pharmacophore
and assayed their anti-AChE activity. Based on this design,
the method of preparation of a jujubogenin-2-en-1-one
analog 1 from the triterpenoid jujubogenin glycosides,
present in the subtropical plant Colubrina asiatica, was
investigated.8,9 Furthermore, to search and generalize new
SAR, we have designed another analogue 21, which
contains an aromatic ring moiety attached to the terpenoid
skeleton as well as the existence of a 2-en-1-one pharmaco-
phore in the molecule. The syntheses and pharmaceutical
activities of these two compounds are reported below.
The past two decades have been marked by dramatically
increased public and scienti®c attention to late-life cognitive
disorders, especially Alzheimer's Disease (AD). One of the
strategies of overcoming this disorder is focused on
cholinergic compounds based on the cholinergic hypothesis
by Summers and his co-workers.1 The low level of acetyl-
choline (ACh) in the brain is considered to be one of main
reasons for memory loss. Thereby, methods for elevating
the ACh level in the brain and enhance the cognition have
become major issues.2,3 One of the approaches is to investi-
gate acetylcholinesterase (AChE) inhibitors, such as THA,
physostigmine, oxotremorine, as well as the newly
developed Huperzine A,4 which can prevent the hydrolysis
of ACh in the brain. Territrem B is a newly found potent
AChE inhibitor (IC50 47 nM, H. Zea AChE)5 and, most
interestingly, its inhibitory mechanism is totally different
to the known AChE inhibitors.6 A previous investigation
found that Territrem B block the entrance of acetylcholine
into AChE by its hydrophobic interaction with the lipophilic
amino acid, which entered the binding site channel of
AChE.6 This makes Territrem B a new lead compound
and this prompted us to study its analogues, since the source
of Territrem B is heavily restricted from the fermentation
liquid of Aspergillus terrus5 and thus restricts further
investigation into this type of AChE inhibitor. Furthermore,
Results and Discussion
The n-BuOH extract from the aerial part of Colubrina
asiatica which contained jujubogenin glycosides was
oxidatively cleaved under strongly alkaline conditions
(O2, NaOBu, BuOH, 908C, overnight)10 to afford the
common aglycone±jujubogenin 28 in 0.1% overall yield.
Catalytic hydrogenation of 2 yielded dihydrojujubogenin
3. This procedure was aimed to avoid any interference
caused by D24 in the following reactions. Selective O-mesyl-
ation at the 3-OH, using a large excess of mesyl chloride due
to the steric hindrance caused by 4,4-dimethyl groups,
afforded 4.11 Treatment of 4 with DBU afforded the 2-ene
product 5 in a 65% yield.11 Allylic oxidation on C-1 of 5,
however, suffered a lot of problems, attributable to the steric
hindrance of the 10-methyl group (see Table 1). Finally, we
found that reaction of 5 with chromic trioxide in acetic acid
Keywords: chemotherapy; enzyme inhibitors; terpenes and terpenoids;
structure±activity.
* Corresponding authors. Laboratory of Phytochemistry, Kunming Insti-
tute of Botany, Chinese Academy of Sciences, Kunming 650204, China.
Tel.: 186-871-5150660, ext. 3242; fax: 186-871-5150227; e-mail:
zhaoyu@ms26.hinet.net Tel.: 1886-2-23916127; Fax: 1886-2-
23919098; e-mail: shoeilee@ha.mc.ntu.edu.tw
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00817-6

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Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
Table 1. Comparison of the yields of 1 and 6 by different allylic oxidative
conditions (1: could not be detected)
the structure for 1. These two methyl signals were distin-
guished by another NOE difference experiment, which
enhanced the signals of 10-Me singlet (d1.17) and H-3
upon irradiation at the methyl singlet at d 1.07 (Scheme 1).
Condition
Yield of 1 (%)
Yield of 6 (%)
PDC, Celite, t-BuOOH,
toluene, re¯ux
PDC, Celite, t-BuOOH,
benzene, re¯ux
PDC, CH2Cl2, 808C, 20 h
PDC, CH2Cl2, sealed tube, 8 h
SeO2/MnO2 (1:1), CH2Cl2, rt,
10 days
SeO2/MnO2 (1:1), t-BuOOH,
CH2Cl2, rt, 10 days
8
4
7
4
Another synthetic route as depicted in Scheme 2 was
explored to improve the low yield obtained with the
previous route. Jujubogenin 2 was ®rst oxidized by PDC in
DMF to a 3-one product 7, followed by hydrogenation of the
23,24-double bond to afford 8.13 Treatment of the 3-one 8
with LDA and phenylselenium bromide, and then NaIO4
oxidation, afford the D1,3-one product 6.14,15 Epoxidation of
the 1,2-double bond in 6 by hydrogen peroxide in an ice bath
afforded 9.16 Wharton rearrangement16,17 of the epoxide 9 led
to the production of the allylic alcohol 10. PDC oxidation of
10 led to the expected product 1 in a high yield (82%).
7
5
8
4
4
1
10
10
24
1
1
HgBr2, hn, t-BuOH/
Cyclohexane (1:1), 2 days
CrO3, HOAc, 808C, 24 h
22
afforded the highest yield of 24% of 1 and 22% of 6.12 The
ESI±MS of 1 showed [M1H]1 at m/z 471 and its 1H NMR
spectrum (CDCl3) showed a characteristic AX system for
H-2 (d 5.62, d) and H-3 (d 6.26, d), Jaxˆ10.1 Hz. The EIMS
In another procedure, the 2-ene product 5 was ®rst epoxi-
dized by MPCBA (meta-perchlorobenzoic acid) to form an
epoxide 11, which was further cleaved by the phenyl-
selenide anion and followed by an oxidation by 30% hydro-
gen peroxide to allow the consequent elimination of a
PhSeOH. This produced an allylic alcohol 12,18 which
1
spectrum of 6 showed [M]1 at m/z470 and its H NMR
spectrum (CDCl3) showed a characteristic AX system for
H-1 (d 7.07, d) and H-2 (d 5.79, d), Jaxˆ10.2 Hz. The
structures for 1 and 6 were distinguished by NOE difference
experiments. Irradiation at the doublet of the down®eld
ole®nic proton (H-3, d 6.26) in 1 enhanced two methyl
singlets at d 1.07 (4b-Me) and 1.03 (4a-Me), supporting
could easily be subsequently oxidized to enone
6
(Scheme 3). NOE difference experiments disclosed that
the epoxide of 11 adopted an a-orientation. Furthermore,
this base-induced rearrangement proceeds via a cyclic syn
elimination mechanism,19 leading to the C-1 syn product of
Scheme 1. (a) NaOBun, O2, n-NuOH, 908C, 24 h; (b) Pd-C, H2, rt, 48 h (91%); (c) MsCl, py, 08C, 1 h (82%); (d) DBU, toluene, re¯ux, 34 h (65%); (e) CrO3,
HOAc, 808C, 24 h (1, 24%; 6, 22%).
Scheme 2. (a) PDC, DMF, 08C, 10 h (93%); (b) Pd-C, H2, rt, 48 h (90%); (c) (i) LDA, PhSeBr, THF, 2788C, 0.5 h (ii) NaIO4, MeOH-H2O (1:1), 12 h (50% in
two steps); (d) 30% H2O2, 10% NaOH, MeOH, 08C, 4 h (78%); (e) N2H4H2O, MeOH, AcOH, 08C to re¯ux (0.5 h113 h) (40%); (f) PDC, DMF, re¯ux 60 h
(82%).

Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
8903
Scheme 3. (a) Pd-C, H2, rt, 48 h (91%); (b) MsCl, py, 08C, 1 h (82%); (c) DBU, toluene, re¯ux, 34 h (65%); (d) MCPBA, CH2Cl2, 08C-rt, 8 h (77%); (e) (i)
PhSeSePh, NaBH4, EtOH, rt, 2.5 h (ii) exces H2O2, 08C-rt, 8 h, (77% in two steps); (f) MnO2, Na2CO3, CH2Cl2, 08C, 3 h (90%); (g) 30% H2O2, 10% NaOH,
MeOH, 08C, 4 h (78%); (h) N2H4H2O, MeOH, AcOH, 08C to re¯ux (0.5 h113 h) (40%); (i) PDC, DMF, re¯ux 60 h (82%).
the 2a, 3a-epoxy. Therefore the 3-hydroxy group of the
allylic alcohol 12 should also be an axial one (3a-OH).
acidic hydrolysis, the glycosides were not only hydrolyzed
but also cleaved on the acidic-sensitive ketal group and
afforded trans-Ebelin lactone 13 and cis-Ebelin lactone
14.21,22 The mixture of 13 and 14 was mesylated and
subjected to ozonolysis, which formed three aldehydes
including the totally ozone-oxidized product, 15, as well
as a pair of partial ozone-oxidized aldehydes, 16 and 17.23
The formation of the unexpected aldehydes 16 and 17 is
possibly due to the steric hinderince of C-20 as well as
the nonuniformity of the electric density of the conjugated
triene. DBU treatment of 15 afforded 2-en-Ebehyde 18,11
which was subsequently reacted with phenyl magnesium
bromide, under kinetic control. The expected alcohol 19
was obtained in 28% yield as well as a rearranged alcohol
20 isolated in 23% yield, 16% of unreacted aldehyde 18.
The enone products, 2-en-1-one 1, 6, and 5 were assayed
against eel AChE.20 The preliminary results showed that the
IC50 of compounds 1 and 5 was about 50 mM, at such
concentration 6 inhibited AChE only about 10%. The
inhibitory activities should be higher if the problem of the
poor solubility of the samples get solved. This study demon-
strated at least the 2-en or 2-en-1-one moiety was essential
for anti-AChE activity.
To further mimic Territrem B by constructing an aromatic
moiety in the molecule, which is possibly another pharma-
cophore, jujubogenin glycosides were utilized. After an
Figure 1. Energy-minimized con®guration analysis of the Territrem B analogues

8904
Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
Scheme 4. (a) HCl:EtOH:H2O (1:3:3, v/v/v), 808C, 5 h, 13:14 in a 45:55 ratio for total yield of 4.5% of the crude extract; (b) MsCl, Py, )8C, 10 H, 83%; (c) 1.
O3, MeOH, 2788C, ,1 h; 2. thiourea, 08C±rt, 6 h; (d) DBU, toluene, re¯ux, 10 h, 54%; (e) 0.8 equiv. of C6H5MgBr, CH2Cl2, 2788C, 20 min, 28% of 19, 23%
of 20, 19% of unreacted 18; (f) CrO3, HOAc, 808C, 2 h, 69%; (g) Br2, pyridine, 0±808C, 1.5 h, 23:24 in ca 1:1 ratio for total yield of 86%; (h) 1 equiv. of Mg,
THF, rt, 15 min; (I) 0.8 equiv. of 25 and 26, CH2Cl2, 2788C, 20 min, 12% of 27, 16% of 28, 12% of 27, 16% of 28, 12% of 29 and 8% of 30, 17% of unreacted
18; (j) CrO3, HOAc, 808C, 2 h, 84%; (k) excess CrO3 in HOAc, re¯ux 12 h, 56%.
The side reaction producing 20 consumed the expected
product 19 but it can be seen that the structure of 20
possesses more structural similarity to Territrem B (Fig. 1).
Unfortunately, further oxidation of 20 by chromic trioxide
or pyridinium dichromate could only afford 21, which is the
oxidative product of the secondary alcohol 19 under the
same conditions12 (Scheme 4).
phenyl Grignard reagent was also investigated. During
preparation of the trimethoxy phenyl Grignard reagent
from 1,2,3-trimethoxybenzene 22, the monobromide 23
and dibromide 24 were formed in a 1:1 ratio. The dibromide
24 was further converted into Grignard reagents 25 and 26,
which were directly reacted with 2-en-Ebehyde 18. Four
products were isolated after work-up. The expected alcohol
27 was obtained in a 12% yield and three other rearranged
alcohols were obtained: 28 in a 16% yield, 29 in a 12% yield
The combination of 2-en-Ebehyde 18 with a trimethoxy-

Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
8905
as well as 30 in a 8% yield. Furthermore, 18% of the
unreacted 18 was recovered.
(b) Oxidative cleavage of saponins to jujubogenin. To a
stirred solution of the above-mentioned extract (140 g) in
dry BuOH (500 mL) at room temperature was added care-
fully, ®ne sodium bar (58 g) in one hour. Temperature was
controlled by periodically emerging the reaction ¯ask into
an ice bath so as not to exceed 1008C. After another 20 min
stirring, oxygen gas was carefully introduced, the vigorous
reaction must be controlled by an ice bath and by adjusting
the bubble speed of oxygen gas. After 40 min stirring, all the
sodium bar was dissolved and the reaction was calmed down
and oxygen was bubbled in for another 24 h. The reaction
was quenched by water (950 mL) and was washed out by
water (800 mL) to a separating funnel. The mixture was
washed by dist. H2O (500 mL£3). The BuOH layer was
evaporated under vacuum to afford an extract of 85 g,
while the water was combined and extracted with CHCl3
(500 mL£3). The CHCl3 solution was combined and evapo-
rated to give a residue (24 g). The BuOH extract and CHCl3
extract was combined and subjected to a 1500 g silica gel
column, eluting with a gradient of 3±100% Me2CO/CHCl3.
The fractions containing jujubogenin were combined,
evaporated and recrystalized from EtOAc to give ®nally
jujubogenin (2) (8.07 g). [Rf (10% Me2CO/CHCl3) 0.38,
In the ®nal step chromic trioxide was applied to oxidize the
C-1 of 27±30 to form a 2-en-1-one moiety. Alcohol 27 and
the rearranged alcohols 29 and 30 were easily oxidized to
the mono-ketone product 31.12 However, treatment of 31
with much excess of chromic trioxide only gave compound
32, which possesses the 2-en-1-one moiety but lost the
aromatic ring, since the electron-rich trimethoxy phenyl
part is fragile under these oxidative conditions.
The anti-AChE activity of 21, which contains the 2-en-1-
one pharmacophore and a aromatic ring moiety, was also
tested against eel AChE.20 The IC50 was ca. 10 mM, a little
bit stronger than that of dihydrojujubogenin-2-en-1-one 1.
This suggested that the aromatic ring present in Territrem B
is perhaps not the major factor of the anti-AChE activity.
Therefore further investigation into the preparation
Territrem B analogues possessing both 5a-OH and 9a-OH
together with the 2-en-1-one moiety will be designed and
synthesised.
1
its IR, UV, MS, H- and 13C NMR spectra were compared
with the literature,20 and was directly compared with the
authentic sample.]
Experimental
General
23,24-Dihydrojujubogenin (3). To a mixture of juju-
bogenin 2 (2.0 g, 4.24 mmol) and 10% Pd±C (300 mg)
was added EtOAc (35 mL). The suspension was slightly
pressured by hydrogen atmosphere and stirred for 48 h.
The suspension was ®ltered through a celite funnel and
washed by CHCl3 (30 mL£3). The ®ltrate was combined
and evaporated to provide crude product (1.89 g).
Recrystalization from EtOAc afforded ®nally 23,24-
dihydrojujubogenin 3 (1.81 g, 91%) as a white powder,
mp 270±2718C; [Found: C, 75.81; H, 10.50. C30H50O4
requires C, 75.90; H, 10.62%]; Rf (10% Acetone /CHCl3)
0.42; nmax (KBr) 3530 (OH), 2951, 2477, 2455, 1386, 1289,
1029, 1010 cm21; dH (200 MHz, CDCl3) 3.97 (2H, brs,
H-30 and H-300), 3.91 (1H, m, H-23), 3.13 (1H, dd,
Jˆ10.3, 5.9 Hz, H-3), 2.29 (1H, m, H-13), 1.13 (3H, s,
Me-21), 2.0±1.0 (4H, m), 1.06 (3H, s, Me-18), 0.93 (3H,
s, Me-28), 0.85 (3H, d, Jˆ6.5 Hz, Me-26), 0.83 (3H, d,
Jˆ6.6 Hz, Me-27), 0.79 (3H, s, Me-19), 0.73 (3H, s,
Me-29); dC (50 MHz, CDCl3) 110.23 (C-16), 79.26 (C-3),
69.88 (C-20), 69.87 (C-23), 66.28 (C-30), 56.10 (C-5),
54.05 (C-14), 53.37 (C-17), 53.20 (C-9), 45.37 (C-24),
45.14 (C-22), 39.44 (C-4), 39.00 (C-1), 37.87 (C-8), 37.74
(C-10), 37.49 (C-13), 36.44 (C-15), 36.11 (C-7), 30.64
(C-21), 28.48 (C-25), 28.45 (C-12), 27.75 (C-2), 24.56
(C-26), 23.77 (C-27), 22.51 (C-18), 21.88 (C-11), 19.13
(C-28), 18.63 (C-6), 16.63 (C-29), 15.88 (C-19); m/z
(ESI±MS) 475 (4, MH1), 457 (4), 439 (12), 421 (3), 396
(10), 383 (14), 371 (14), 257 (14), 229 (13), 217 (20), 203
(33), 189 (44), 163 (50), 149 (51), 135 (100), 109 (72);
HRMS (EI): M1, found 474.3703. C30H50O4 requires:
474.3711.
Diethyl ether and toluene were dried over sodium; tetra-
hydrofuran was distilled from sodium benzophenone
ketyl. Organic solutions were dried over anhydrous magne-
sium sulfate. Column chromatographies were performed
using Merck silica gel 60 (70±230 Mesh) and TLC were
carried out using Merck Kiesel gel 60 F254 plates. Melting
points were determined on a Fisher±Johns melting point
apparatus (uncorrected). IR spectra were recorded on a
Jasco IR Report-100 infrared spectrometer. NMR spectra
were recorded on a Bruker AMX 400 (400/100 MHz) and/
or a Bruker DPX 200 (200/50 MHz). Selected 1H NMR data
is reported. ESIMS and EIMS data were measured on a
Finnigan Mat TSQ-7000 mass spectrometer while HRMS
was recorded on JEOL-JMS-HX 110 mass spectrometer.
Plant material
Colubrina asiatica (L.). Brongn belongs to the genus
Colubrina and the aerial parts were collected in June 1996
in Tong Sha islands, China. A voucher specimen was
deposited at the School of Pharmacy, College of Medicine,
National Taiwan University.
Preparation of aglycone-jujubogenin (2)
(a) Crude extraction by solvent partition. The stems of
Colubrina asiatica were air-dried (8.48 kg) and chopped
to pieces, which was followed by extraction with 95%
EtOH (30 L£6). The EtOH extract was further extracted
by a mixed solvent composed of MeOH and hexane
(6L:4L, four times) to afford hexane extract (75 g) and
methanol layer (ca. 650 mL), which is dif®cult to be dried
thoroughly. This methanol layer was subjected to partition
between water (1L) and CHCl3 (1 L£2). The CHCl3 extract
(278 g) was subjected to the next step.
3-Methanesulfonyl-23,24-dihydrojujubogenin (4). To a
stirred solution of 3 (1.79 g, 3.81 mmol) in pyridine
(10 mL) at 08C was added, 890 mL of methanesulfonyl-
chloride (11.43 mmol, 3 equiv.) under
a
nitrogen

8906
Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
atmosphere. After 30 min stirring, the reaction was
quenched by a Py:H2O (2:1) mixture (4 mL of pyridine
and 2 mL of water) and the solution was stirred for another
15 min at 08C. The solution was then evaporated under
vacuum to a residue, the residue was dissolved in H2O
(50 mL) and was subjected to a partition by dichloro-
methane and water (50 mL£3). The combined organic
extract (1.83 g) was puri®ed by ¯ash chromatography
(7±10% EtOAc/CHCl3) to afford 4 (1.57 g, 82%) as a
white powder, mp 147±1488C (EtOAc); [Found: C, 67.26;
H, 9.49; S, 5.91. C31H52O6S requires C, 67.35; H, 9.48; S,
5.80%]; Rf (10% Acetone /CHCl3) 0.61; nmax (KBr) 3540
(OH), 2950, 1486, 1475, 1406, 1308, 1240, 1201, 1044,
1023, 1002 cm21; dH (400 MHz, CDCl3) 4.27 (1H, dd,
Jˆ11.5, 5.2 Hz, H-3), 3.98 (1H, d, Jˆ7.8 Hz, H-30), 3.95
(1H, d, Jˆ8.0 Hz, H-300), 3.91 (1H, m, H-23), 2.98 (3H, s,
SO2Me), 2.32 (1H, ddd, Jˆ6.4, 5.8, 5.1 Hz, H-13), 1.14 (3H,
s, Me-21), 1.07 (3H, s, Me-18), 0.98 (3H, s, Me-28), 0.86
(3H, d, Jˆ6.6 Hz, Me-26), 0.85 (3H, d, Jˆ6.6 Hz, Me-27),
0.84 (3H, s, Me-19), 0.83 (3H, s, Me-29); dC (100.6 MHz,
CDCl3) 109.6 (C-16), 90.1 (C-3), 69.2 (C-20), 68.4 (C-23),
65.5 (C-30), 55.7 (C-5), 53.3 (C-17), 52.3 (C-9), 44.7
(C-24), 44.4 (C-22), 38.6 (SO2Me), 38.5 (C-4), 38.1 (C-1),
37.1 (C-10), 37.0 (C-8), 36.7 (C-13), 35.8 (C-15), 35.3
(C-7), 32.7 (C-14), 28.0 (C-25), 27.7 (C-29), 27.0 (C-21),
25.0 (C-12), 23.9 (C-2), 23.1 (C-26), 21.8 (C-27), 21.3
(C-11), 18.4 (C-18), 18.0 (C-6), 16.1 (C-28), 15.9 (C-19);
m/z (ESI±MS) 553 (37, MH1), 507 (3), 451 (6), 439 (24),
421 (9), 383 (16), 371 (27), 355 (11), 311 (14), 295 (8), 269
(14), 247 (16), 217 (25), 203 (50), 191 (100), 163 (49), 162
(48), 149 (45), 109 (59), 95 (27), 81 (15); HRMS (EI): M1,
found 552.3475 C31H52O6S requires 552.3486.
m, H-10), 1.16 (3H, s, Me-21), 1.10 (3H, s, Me-18), 0.92
(3H, s, Me-28), 0.87 (3H, d, Jˆ6.6 Hz, Me-26), 0.86 (6H, s,
Me-29), 0.85 (3H, d, Jˆ6.7 Hz, Me-27); dC (100.6 MHz,
CDCl3) 137.9 (C-3), 121.2 (C-2), 109.6 (C-16), 69.4 (C-23),
68.6 (C-20), 65.8 (C-30), 53.5 (C-14), 52.9 (C-5), 52.4
(C-17), 51.4 (C-9), 44.9 (C-24), 44.6 (C-22), 40.9 (C-1),
40.9 (C-4), 37.4 (C-13), 37.1 (C-8), 37.1 (C-10), 36.5
(C-15), 35.9 (C-7), 31.7 (C-21), 30.1 (C-25), 24.0 (C-26),
23.3 (C-29), 22.6 (C-18), 22.0 (C-27), 21.7 (C-11), 19.2
(C-6), 17.9 (C-28), 16.3 (C-19); m/z (ESI±MS) 457 (29,
MH1), 439 (7), 421 (4), 403 (1), 393 (4), 383 (10), 371
(12), 364 (4), 355 (4), 343 (3), 323 (6), 267 (18), 229
(12), 217 (23), 203 (22), 191 (30), 177 (33), 161 (45), 133
(77), 123 (100), 121 (85), 109 (67), 95 (45); HRMS (EI):
M1, found 456.3607. C30H48O3 requires 456.3605.
PDC oxidation of 5 with celite and t-BuOOH
(1) Preparation of 3 M t-BuOOH. To a 70% TBHP (tert-
butylhydroperoxide) solution (43 mL) was added dichloro-
methane (70 mL) at 48C in a cool room, the milk sap was
vortex for 10 min, then transferred to a separating funnel.
After 1 h, the clear organic layer was collected, transferred
to a septum-equipped bottle using cannulation technique,
and stored in a 48C refrigerator until use.
(2) Oxidation of 5. To a stirred solution of 5 (53 mg,
0.12 mmol) was added celite (100 mg) and PDC (150 mg,
0.4 mmol) at 08C, then the 3 M TBHP in CH2Cl2 solution
was added (140 mL, 0.42 mmol). The suspension was stirred
at 08C for 45 min, then re¯ux for 24 h. The mixture was
cooled to room temperature and diethyl ether (20 mL) was
added, followed by a partition with 1 M potassium carbon-
ate solution (25 mL). The aqueous layer was extracted by
diethyl ether (20 mL). The organic layer was combined and
was evaporated to a residue (45 mg), which was subjected to
column chromatography (10±50% EtOAc/hexane) to afford
1 (4.4 mg, 8%) and 6 (3.8 mg, 7%). 5 (36 mg, 9%) was
recovered.
Elimination reaction of 4 by DBU. To a stirred yellow
solution of 4 (0.90 g, 1.79 mmol) in dry toluene (10 mL)
was added DBU (3 mL, 20.10 mmol). After 10 min stirring
at room temperature, the temperature of oil bath was
elevated and the solution was re¯uxed 34 h. The products
mixture was dried and subjected to a silica gel column
chromatography (hexane:EtOAc 20:1±1:1) to afford 5
(0.526 g, 65%).
23,24-Dihydrojujubogenin-2-en-1-one (1). White powder,
mp 262±2638C (EtOAc); [Found: C, 76.68; H, 9.96.
C30H46O4 requires C, 76.55; H, 9.85%]; Rf (33% EtOAc/
hexane) 0.36; nmax (KBr) 3480 (OH), 2955, 1669, 1471,
1458, 1383, 1368, 1285, 1259, 1233, 1220, 1015,
819 cm21; UV 228 nm (e 18200); dH (400 MHz, CDCl3)
6.25 (1H, d, Jˆ10.1 Hz, H-3), 5.62 (1H, d, Jˆ10.1 Hz,
H-2), 4.08 (1H, d, Jˆ7.5 Hz, H-30), 3.94 (1H, dd, Jˆ7.6,
1.8 Hz, H-300), 3.91 (1H, dddd, Jˆ8.7, 8.7, 4.3, 4.3 Hz,
H-23), 2.37 (1H, ddd, Jˆ6.2, 5.8, 5.2 Hz, H-13), 1.16
(6H, s, Me-19 and Me-21), 1.15 (3H, s, Me-28), 1.06 (3H,
s, Me-18), 1.03 (3H, s, Me-29), 0.86 (3H, d, Jˆ6.6 Hz,
Me-26), 0.85 (3H, d, Jˆ6.6 Hz, Me-27); dC (100.6 MHz,
CDCl3) 206.9 (C-1), 154.4 (C-3), 124.2 (C-2), 109.8 (C-16),
69.2 (C-20), 68.6 (C-23), 66.3 (C-30), 53.6 (C-17), 52.7
(C-14), 51.4 (C-5), 48.0 (C-10), 44.8 (C-24), 44.6 (C-22),
43.2 (C-9), 37.5 (C-13), 36.4 (C-15), 36.0 (C-8), 34.8 (C-7),
31.0 (C-21), 30.9 (C-25), 30.2 (C-26), 28.0 (C-12), 24.8
(C-11), 24.1 (C-18), 23.3 (C-29), 22.0 (C-27), 18.6
(C-28), 15.8 (C-19); m/z (ESI±MS) 471 (29, MH1), 453
(6), 435 (28), 417 (8), 385 (18), 367 (14), 337 (10), 325
(17), 283 (18), 231 (38), 217 (100), 187 (48), 173 (95),
Elimination reaction of 4 by NaI/HMPA. To a stirred
solution of 4 (0.15 g, 0.30 mmol) in hexamethylphosphor-
amide (5 mL) was added sodium iodide (0.138 g,
0.87 mmol). The temperature of the oil bath was elevated
to 1308C and keep re¯ux for 8 h. The solvent was evapo-
rated under vacuum (0.01 mmHg) at 1208C. The residue
was washed out by sodium thiosulfate (8 g in 15 mL of
water), and was extracted by diethyl ether (25 mL£4).
The organic layer was combined, dried (MgSO4), evapo-
rated to a residue (0.158 g), which was puri®ed by ¯ash
chromatography to afford 5 (0.082 g, 66%).
2-Ene-23,24-dihydrojujubogenin (5). White powder
(EtOAc), mp 238±2398C; [Found: C, 78.79; H, 10.49;
C30H48O3 requires C, 78.90; H, 10.59%]; Rf (8% Me2CO/
CHCl3) 0.64; nmax (KBr) 3522 (OH), 2953, 2920, 1462,
1454, 1383, 1286, 1222, 1116, 1039, 998 cm21; dH
(400 MHz, CDCl3) 5.38 (1H, ddd, Jˆ10.1, 5.1, 1.0 Hz,
H-2), 5.33 (1H, d, Jˆ10.1, 2.1 Hz, H-3), 4.00 (2H, brs,
H-30 and H-300), 3.93 (1H, m, H-23), 2.33 (1H, ddd,
Jˆ6.2, 5.8, 5.2 Hz, H-13), 1.85 (1H, m, H-1), 1.69 (1H,

Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
8907
149 (75), 109 (45); HRMS (EI): M1, 470.3393. C30H46O4
requires 470.3398.
rated NaHCO3 solution, followed by an extraction of diethyl
ether (20 mL£3). The ether layer was combined and dried
(MgSO4), evaporated, and the residue (16 mg) subjected to
a ¯ash column (10% EtOAc/CHCl3) to afford 1 (2.3 mg,
10%). Repeat this experiment and elevate the reaction
temperature to 808C for 24 h gave ®nally 1 (5.7 mg, 24%)
and 6 (5.2 mg, 22%).
23,24-Dihydrojujubogenin-1-en-3-one (6). Colourless
gum, [Found: C, 76.73; H, 10.03. C30H46O4 requires C,
76.55; H, 9.85]; Rf (33% EtOAc/hexane) 0.22; nmax (KBr)
3446 (OH), 2950, 1668, 1477, 1382, 1290, 1261, 1239,
1220, 1214, 1160, 994 cm21; UV 229 nm (e 16500); dH
(400 MHz, CDCl3) 7.07 (1H, d, Jˆ10.2 Hz, H-1), 5.79
(1H, d, Jˆ10.2 Hz, H-2), 3.98 (2H, brs, H-30 and H-300),
3.94 (1H, dddd, Jˆ8.7, 8.7, 4.4, 4.4 Hz, H-23), 2.42 (1H, m,
H-13), 1.16 (3H, s, Me-28), 1.11 (3H, s, Me-19), 1.07 (3H, s,
Me-18), 1.04 (3H, s, Me-29), 0.88 (3H, d, Jˆ6.6 Hz,
Me-26), 0.86 (1H, d, Jˆ6.6 Hz, Me-27); dC (100.6 MHz,
CDCl3) 206.9 (C-3), 154.4 (C-1), 124.2 (C-2), 109.8 (C-16),
69.2 (C-20), 68.6 (C-23), 65.9 (C-30), 53.6 (C-5), 52.7
(C-17), 51.4 (C-14), 48.0 (C-9), 44.9 (C-24), 44.6 (C-22),
43.2 (C-4), 37.5 (C-13), 37.5 (C-8), 36.4 (C-10), 36.0
(C-15), 34.9 (C-7), 30.2 (C-21), 28.0 (C-12), 28.0 (C-25),
24.8 (C-26), 24.1 (C-29), 23.3 (C-18), 22.0 (C-27), 21.9
(C-11), 19.2 (C-6), 18.9 (C-28), 15.8 (C-19); m/z (EIMS)
470 (100 M1), 452 (46), 422 (41), 395 (80), 377 (42), 343
(13), 285 (8), 237 (18), 203 (23), 159 (18), 150 (25), 109
(48), 91 (36), 69 (66); HRMS (EI): M1, 470.3392. C30H46O4
requires 470.3398.
PDC oxidation of 3. To a stirred solution of PDC (1.316 g,
3.5 mmol) in dry DMF (9 mL) was added, a solution of 3
(260 mg, 0.55 mmol) in dry DMF (5 mL) in an ice bath
under N2 atmosphere. The suspension was stirred for
another 4 h and warmed up to room temperature in 7 h.
The mixture was evaporated to a small volume (4 mL)
and water (50 mL) was added while the mixture was
extracted by diethyl ether (50 mL£4). The combined
organic layer was evaporated to a dryness (233 mg) and
was subjected to ¯ash chromatography (10% EtOAc/
CH2Cl2) to afford 8 (199 mg, 77%) and of 7 (23 mg, 9%).
3-Oxo-jujubogenin (7). White powder (EtOAc), mp 243±
2448C, [Found: C, 76.66; H, 9.99. C30H46O4: C, 76.55; H,
9.85%]; Rf (10% Me2CO/CHCl3) 0.67; nmax (KBr) 3524,
2955, 1705, 1458, 1384, 1288, 1216, 1184, 1018, 1008,
998, 838 cm21; dH (400 MHz, CDCl3) 5.16 (1H, dq,
Jˆ8.2, 1.4 Hz, H-24), 4.62 (1H, ddd, Jˆ10.8, 10.7,
2.9 Hz, H-23), 3.98 (2H, brs, H-30 and H-300), 2.40 (1H,
m, H-13), 1.85 (1H, m, H-20), 1.66 (3H, d, Jˆ1.1 Hz,
Me-26), 1.64 (3H, d, Jˆ1.2 Hz, Me-27), 1.16 (3H, s,
Me-21), 1.12 (3H, s, Me-28), 1.05 (3H, s, Me-18), 1.01
(3H, s, Me-19), 0.90 (3H, s, Me-19); dC (100.6 MHz,
CDCl3) 217.8 (C-3), 135.4 (C-25), 125.1 (C-24), 109.5
(C-16), 69.3 (C-20), 68.0 (C-23), 65.7 (C-30), 55.0 (C-5),
53.3 (C-14), 52.6 (C-17), 52.0 (C-9), 47.3 (C-4), 44.5
(C-22), 39.2 (C-1), 37.2 (C-8), 37.0 (C-10), 37.0 (C-13),
36.1 (C-15), 34.9 (C-7), 33.9 (C-2), 29.9 (C-21), 28.0
(C-12), 26.8 (C-29), 25.6 (C-26), 21.9 (C-11), 21.0 (C-18),
19.5 (C-6), 18.4 (C-27), 18.2 (C-28), 16.0 (C-19); m/z
(ESI±MS) 471 (2 MH1), 453 (100), 435 (10), 407 (3),
393 (14), 385 (22), 367 (35), 339 (29), 325 (6), 321 (5),
247 (8), 219 (10), 205 (20), 191 (14), 188 (16), 133 (8),
109 (9); HRMS (EI): M1, found 470.3407. C30H46O4
requires 470.3398.
PDC oxidation of 5 in CH2Cl2. To a stirred suspension of
pyridinium dichromate (500 mg, 1.33 mmol) in dichloro-
methane (5 mL) was added a solution of 5 (100 mg,
0.22 mmol) in dichloromethane (5 mL) in 5 min. The
suspension was then re¯uxed under a N2 atmosphere for
20 h. The solution was evaporated to dryness, then dissolved
in water (30 mL) and was extracted by diethyl ether
(30 mL£4). The combined ether layer was dried, the
residue (56 mg) was separated by column chromatography
(10±50% EtOAc/hexane) to afford 1 (7.3 mg, 7%) and 6
(4.2 mg, 4%). 5 (42 mg, 42%) was recovered.
Allylic oxidation of 5 to 1 by SeO2/MnO2 mixture. To a
stirred solution of SeO2 (20 mg, 0.32 mmol) and MnO2
(35 mg, 0.4 mmol) in dichloromethane (3 mL) was added,
a solution of 5 (23 mg, 0.05 mmol) in dichloromethane
(2 mL). The suspension was stirred at room temperature
for 10 days. The solvent was ®ltrate and evaporated, while
the residue was further puri®ed by ¯ash chromatography
(7±25% EtOAc/CHCl3) to afford 1 (2.4 mg, 8%). 5
(11 mg, 48%) was recovered.
3-Oxo-23,24-dihydrojujubogenin (8). White powder
(EtOAc), mp 240±2418C; [Found: C, 76.36; H, 10.19.
C30H48O4 requires C, 76.23; H, 10.24%]; Rf (10% Me2CO/
CHCl3) 0.71; nmax (KBr) 3522, 2952, 1710, 1457, 1386,
1376, 1288, 1221, 1082, 1042, 1022, 998, 840 cm21; dH
(400 MHz, CDCl3) 3.98 (2H, brs, H-30 and H-300), 3.94
(1H, m, H-23), 2.40 (1H, m, H-2), 2.37 (1H, m, H-13),
1.84 (1H, m, H-20), 1.14 (3H, s, Me-21), 1.10 (3H, s,
Me-28), 1.04 (3H, s, Me-18), 1.00 (3H, s, Me-29), 0.88
(3H, s, Me-19), 0.86 (3H, d, Jˆ6.6 Hz, Me-26), 0.84 (3H,
d, Jˆ6.5 Hz, Me-27); dC (100.6 MHz, CDCl3) 217.7 (C-3),
109.5 (C-16), 69.3 (C-20), 68.6 (C-23), 65.6 (C-30), 55.0
(C-5), 53.4 (C-14), 52.9 (C-17), 52.0 (C-9), 47.3 (C-4), 44.8
(C-24), 44.6 (C-22), 39.2 (C-1), 37.2 (C-8), 37.0 (C-10),
37.0 (C-13), 35.9 (C-15), 34.8 (C-7), 33.9 (C-2), 30.1
(C-21), 28.0 (C-12), 26.8 (C-25), 24.0 (C-26), 23.3 (C-
29), 22.0 (C-18), 21.9 (C-11), 21.0 (C-27), 19.5 (C-6),
18.1 (C-28), 16.0 (C-19). m/z (ESI±MS) 473 (18 MH1),
455 (7), 437 (8), 419 (2), 409 (3), 399 (13), 387 (9), 377
Photocatalic oxidation of 5 to 1. To a stirred solution of 5
(20 mg, 0.044 mmol) in cyclohexane (5 mL) and tert-buta-
nol (5 mL) was added of mercury (II) bromide (40 mg,
0.1 mmol, 2.3 equiv. of 5). The suspension was stirred
under an UV lamp at 254 nm for 2 days. The solution was
®ltrate, evaporated and the residue was subjected to ¯ash
chromatography (10% EtOAc/CHCl3) to afford 1 (1.9 mg,
10%). 5 (6.6 mg, 33%) was recovered.
Allylic oxidation of 5 by CrO3/HOAc. To a stirred solution
of 5 (23 mg, 0.05 mmol) in acetic acid (5 mL) at 08C was
added, 3 equiv. of chromium trioxide (16 mg, 0.15 mmol) in
acetic acid (3 mL). After 30 min, the solution was allowed
to be warmed up to room temperature, then stirred for
another 6 h. The acidic mixture was neutralized by a satu-

8908
Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
(6), 369 (11), 351 (8), 343 (10), 324 (15), 313 (12), 257 (14);
HRMS (EI): M1, found 472.3550. C30H48O4 requires
472.3554.
44.9 (C-24), 44.8 (C-4), 44.6 (C-22), 38.6 (C-10), 37.6
(C-8), 37.0 (C-13), 36.0 (C-15), 34.6 (C-7), 30.9 (C-21),
27.7 (C-25), 27.0 (C-12), 24.1 (C-26), 23.3 (C-29), 22.3
(C-11), 22.0 (C-18), 20.1 (C-27), 18.9 (C-6), 18,4 (C-28),
15.2 (C-19); m/z(ESI±MS) 487 (60 M1), 469 (10), 440 (8),
412 (11), 403 (10), 304 (10), 355 (18), 343 (16), 311 (32),
268 (23), 233 (62), 199 (34), 189 (57), 175 (73), 147 (100),
123 (46), 69 (51); HRMS (EI): M1, found 486.3348.
C30H46O5 requires 486.3347.
Catalytic hydrogenation of 7 to 8. The mixture of 7
(20 mg, 0.043 mmol) and Pd±C (10%) (5 mg) was
dissolved in EtOAc (6 mL), then pressured by H2 gas for
overnight. Filtration through Celite and removal of solvent
afforded 8 (18.2 mg, 91%).
Preparation of 8 from jujubogenin 2. To a stirred suspen-
sion of PDC (5.414 g, 14.4 mmol) in dry DMF (9 mL) in an
ice bath was added, a solution of 2 (0.98 g, 2.1 mmol) in
DMF (8 mL) under a nitrogen atmosphere. The solution was
stirred at 08C for 1 h, then another 7 h in room temperature.
Evaporation of part of the solvent to a 7 mL volume, water
(50 mL) was added and the mixture was extracted by ether
(60 mL£4). The combined organic layer was evaporated
and the dryness was subjected to recrystallization and afford
7 (903 mg, 93%). The pure 7 was hydrogenated by H2 gas
under Pd±C catalization as above and afforded ®nally 8
(814 mg, 90%).
1a-Hydroxy-2-ene-23,24-dihydrojujubogenin (10). To a
stirred solution of 9 (96 mg, 0.2 mmol) in dry MeOH
(15 mL) at 08C was added, hydrazine hydrate (150 mL,
0.3 mmol) dropwise. After 0.5 h, the solution was allowed
to warm up to room temperature. Acetic acid (20 mL) was
added and the solution was stirred for another 1 h, then
re¯uxed for12 h. The cooled solution was evaporated to a
dryness and the residue was partitioned by water (30 mL)
and diethyl ether (30 mL£3). The organic layer was
combined, dried and evaporated. Puri®cation by ¯ash
column chromatography (10±50% EtOAc/CHCl3) afford
of 10 (38 mg, 40%) as white powder (EtOAc), mp 228±
2298C, [Found: C, 76.32; H, 10.12. C30H48O4 requires C,
76.23; H, 10.24%]; Rf (12.5% EtOAc/CHCl3) 0.28; nmax
(KBr) 3481 (OH), 2951, 1462, 1453, 1385, 1289, 1218,
1033, 1018, 992 cm21; dH (400 MHz, CDCl3) 5.68 (1H,
dd, Jˆ9.9, 5.8 Hz, H-2), 5.51 (1H, d, Jˆ9.9 Hz, H-3),
4.09 (1H, d, Jˆ7.6 Hz, H-30), 4.03 (1H, dd, Jˆ7.6,
1.7 Hz, H-300), 3.96 (1H, ddddd, Jˆ8.7, 8.7, 4.4, 4.4,
1.4 Hz, H-23), 3.59 (1H, d, Jˆ5.8 Hz, H-1), 2.34 (1H, m,
H-13), 1.16 (3H, s, Me-21), 1.12 (3H, s, Me-18), 0.96 (3H, s,
Me-28), 0.87 (3H, d, Jˆ6.7 Hz, Me-26), 0.86 (3H, d,
Jˆ6.6 Hz, Me-27), 0.85 (3H, s, Me-19), 0.79 (3H, s,
Me-29); dC (100.6 MHz, CDCl3) 142.1 (C-3), 124.1 (C-2),
110.2 (C-16), 69.9 (C-23), 69,0 (C-1), 68.7 (C-20), 66.4
(C-30), 53.4 (C-14), 53.3 (C-17), 45.7 (C-5), 45.3 (C-24),
45.1 (C-22), 42.4 (C-9), 40.8 (C-10), 37.6 (C-4), 37.5
(C-13), 36.6 (C-15), 35.6 (C-8), 34.9 (C-7), 32.0 (C-21),
30.6 (C-25), 28.3 (C-12), 24.5 (C-26), 23.7 (C-29), 22.9
(C-18), 22.9 (C-27), 22.2 (C-11), 19.0 (C-6), 18.5 (C-28),
16.6 (C-19); m/z(ESI±MS) 473 (19 MH1), 455 (2), 437
(11), 419 (5), 391 (2), 377 (8), 313 (14), 253 (14), 215
(29), 201 (51), 175 (42), 147 (45), 133 (100), 109 (60);
HRMS (EI): M1, found 472.3549. C30H48O4 requires
472.3554.
a-Dehydrogenation of 8. To a stirred solution of diiso-
propylamine (17 mL, 0.12 mmol) in dry THF (2 mL) at
2788C was added, 1.6 M n-Buli in hexane (75 mL,
0.12 mmol) under a nitrogen atmosphere. After 10 min, 8
(47 mg, 0.1 mmol) in THF (2 mL) was added dropwise.
After another 10 min, phenylselenylbromide (28.2 mg,
0.12 mmol) in THF (1 mL) was added quickly. 1.6 M
n-BuLi in hexane (150 mL, 0.24 mmol) was added and the
solution was stirred for 20 min. Then a solution of NaIO4
(36 mg, 0.72 mmol) in a mixture of MeOH (1.4 mL) and
H2O (0.7 mL) was added carefully and the mixture was
allowed to warm up to room temperature in 2 h then
re¯uxed for 10 h. The cooled solution was partitioned by
water (10 mL) and ether (25 mL£2). The organic layer was
washed and dried, and evaporated to dryness. Puri®cation
by column chromatography (5±20% EtOAc/CHCl3)
provided 6 (23 mg, 50%).
1a,2a-Epoxy-3-oxo-23,24-dihydrojujubogenin (9). To a
stirred solution of 6 (160 mg, 0.4 mmol) in MeOH (4 mL)
at 08C was added 30% aqueous hydrogen peroxide (300 mL,
2.4 mmol). After stirring for 4 h, the solution was evapo-
rated to a 2 mL volume and was partitioned between water
(10 mL) and dichloromethane (20 mL£2). The organic
layer was dried and evaporated to afford a residue
(155 mg), which was subjected to ¯ash chromatography
(33% EtOAc/hexane) to provide 9 (129 mg, 78%) as color-
less gum; [Found: C, 73.96; H, 9.46. C30H46O5 requires C,
74.03; H, 9.53%]; Rf (12.5% EtOAc/CHCl3) 0.61; nmax
(KBr) 3524, 2952, 1712, 1458, 1385, 1371, 1288, 1080,
1025, 998, 836 cm21; dH (400 MHz, CDCl3) 4.06 (1H, d,
Jˆ8.0 Hz, H-30), 4.00 (1H, dd, Jˆ7.8, 1.5 Hz, H-300), 3.94
(1H, dddd, Jˆ8.8, 8.8, 4.3, 4.3 Hz, H-23), 3.54 (1H, d,
Jˆ4.7 Hz, H-2), 3.33 (1H, d, Jˆ4.7 Hz, H-1), 2.41 (1H,
m, H-13), 1.17 (3H, s, Me-21), 1.13 (3H, s, Me-28), 1.07
(3H, s, Me-28), 1.01 (3H, s, Me-21), 0.88 (3H, d, Jˆ6.6 Hz,
Me-26), 0.86 (3H, s, Me-19), 0.85 (3H, d, Jˆ6.6 Hz,
Me-27); dC (100.6 MHz, CDCl3) 212.4 (C-3), 109.5
(C-16), 69.4 (C-20), 68.6 (C-23), 65.6 (C-30), 63.4 (C-2),
56.8 (C-1), 53.4 (C-14), 52.8 (C-17), 46.1 (C-5), 45.6 (C-9),
PDC oxidation of 10
(1) To a mixture of 10 (10 mg, 0.021 mmol) and PDC
(80.4 mg, 0.21 mmol) was added dichloromethane (5 mL).
The solution was stirred at room temperature for 8 h and
re¯ux for 60 h. After evaporation, the residue was puri®ed
by ¯ash chromatography (10±33% EtOAc/CHCl3) to afford
1 (4.5 mg, 45%).
(2) Similar experiment was carried out using DMF as
solvent. Utilizing 10 (10 mg, 0.021 mmol) provided ®nally
1 (8.2 mg, 82%).
2a,3a-Epoxy-23,24-dihydrojujubogenin (11). To a stirred
solution of 5 (57 mg, 0.13 mmol) in dichloromethane
(5 mL) at 08C was added dropwise, MCPBA (35 mg,
0.2 mmol) in ice-cooled dichloromethane (2 mL). After

Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
8909
2 h, the solution was allowed to warm up to room tempera-
ture and stirred for another 8 h. Saturated NaHCO3 (5 mL)
solution was added, the dichloromethane layer was
separated while the water layer was extracted by dichloro-
methane (10 mL£2). The organic layer was combined
and dried (MgSO4). Evaporation followed by ¯ash
chromatography (12.5% EtOAc/hexane) provided 11
(55 mg, 80%) as white powder (EtOAc), mp 236±2378C,
[Found: C, 76.05; H, 10.12. C30H48O4 requires C, 76.23; H,
10.24%]; Rf (50% EtOAc/hexane) 0.55; nmax (KBr) 3519
(OH), 2949, 1458, 1386, 1286, 1219, 999, 834 cm21; dH
(400 MHz, CDCl3) 3.94 (2H, brs, H-30 and H-300), 3.91
(1H, ddddd, Jˆ8.7, 8.7, 4.4, 4.4, 1.4 Hz, H-23), 3.14 (1H,
dd, Jˆ12.0, 7.8 Hz, H-2), 2.75 (1H, d, Jˆ7.6 Hz, H-3), 2.32
(1H, m, H-13), 1.13 (3H, s, Me-21), 1.06 (3H, s, Me-18),
1.04 (3H, s, Me-19), 0.98 (3H, s, Me-28), 0.85 (3H, d,
Jˆ6.5 Hz, Me-26), 0.84 (3H, d, Jˆ6.6 Hz, Me-27), 0.82
(3H, s, Me-29); dC (100.6 MHz, CDCl3) 109.7 (C-16),
69.4 (C-20), 68.6 (C-23), 65.7 (C-30), 61.6 (C-2), 61.6
(C-3), 53.3 (C-14), 52.4 (C-17), 51.3 (C-9), 47.1 (C-5),
44.9 (C-24), 44.6 (C-22), 40.2 (C-1), 37.2 (C-4), 37.0
(C-13), 36.2 (C-15), 35.9 (C-8), 34.7 (C-7), 32.8 (C-10),
29.7 (C-21), 28.1 (C-25), 28.0 (C-12), 24.1 (C-26), 23.3
(C-29), 22.0 (C-18), 22.0 (C-27), 21.7 (C-11), 18.6
(C-28), 18.4 (C-6), 17.6 (C-19); m/z (ESI±MS) 473 (17
MH1), 455 (1), 437 (4), 419 (2), 409 (1), 392 (1), 371 (3),
325 (3), 277 (2), 213 (14), 200 (15), 187 (31), 159 (34), 133
(100), 119 (44); HRMS (EI): M1, found 472.3553.
C30H48O4 requires 472.3554.
to a small volume (3 mL), then water (10 mL) was added to
dilute the solution, which was subjected to a extraction with
diethyl ether (20 mL£2). The Organic layer was combined
and washed twice with aqueous potassium carbonate, and
dried by MgSO4. Evaporation of the solvent afford the crude
allylic alcohol 12 (13 mg). Recrystallization in EtOAc
provide ®nally 12 (11.5 mg, 77%).
1-Ene-3a-hydroxy-23,24-dihydrojujubogenin (12). White
powder (EtOAc), mp 250±2518C, Rf (50% EtOAc/hexane)
0.27; nmax (KBr) 3448 (OH), 2952, 2915, 1455, 1382, 1368,
1287, 1219, 1168, 1022, 998 cm21; dH (400 MHz, CDCl3)
5.98 (1H, d, Jˆ10.2 Hz, H-1), 5.59 (1H, dd, Jˆ10.2, 4.8 Hz,
H-2), 3.97 (2H, brs, H-30 and H-300), 3.93 (m, 1H, H-23),
3.50 (1H, d, Jˆ4.8 Hz, H-3), 2.34 (1H, ddd, Jˆ6.8, 6.0,
5.9 Hz, H-13), 1.16 (3H, s, Me-21), 1.11 (3H, s, Me-18),
0.95 (3H, s, Me-28), 0.91 (3H, s, Me-19), 0.87 (3H, d,
Jˆ6.5 Hz, Me-26), 0.86 (3H, d, Jˆ6.7 Hz, Me-27), 0.80
(3H, s, Me-29); dC (100.6 MHz, CDCl3) 140.1 (C-2),
124.2 (C-1), 109.7 (C-16), 73.6 (C-3), 69.4 (C-20), 68.6
(C-23), 65.8 (C-30), 53.7 (C-17), 52.9 (C-14), 49.2 (C-5),
49.2 (C-9), 44.9 (C-24), 44.6 (C-22), 39.5 (C-4), 37.7
(C-10), 37.0 (C-8), 37.0 (C-13), 36.0 (C-15), 35.9 (C-7),
30.2 (C-21), 27.8 (C-12), 26.4 (C-25), 24.1 (C-26), 23.5
(C-27), 23.3 (C-29), 22.0 (C-18), 21.6 (C-11), 19.3
(C-28), 17.6 (C-6), 17.5 (C-9); m/z(ESIMS) 473 (12
MH1), 455 (6), 437 (16), 419 (22), 404 (6), 378 (4), 377
(10), 313 (23), 255 (9), 253 (10), 215 (20), 201 (34), 175
(53), 147 (54), 133 (100), 109 (57); HRMS (EI): M1, found
472.3549. C30H48O4 requires 472.3554.
Base-induced rearrangement of epoxide 11. To a stirred
solution of butyllithium (1.3 mL of the 1.6 M solution in
hexane, 2.1 mmol) in dry THF (5 mL) at 2788C was
added diisopropylamine (0.32 mL, 4.4 mmol). After
20 min, a solution of 11 (24 mg, 0.05 mmol) in dry THF
(5 mL) was added dropwise at 2788C. After 12 h, the
solution was allowed to warm up to room temperature and
stirred for another 24 h. Water (10 mL) was added and the
solution was extracted by ether (20 mL£3). The organic
layer was combined and dried. Evaporation followed by
¯ash column chromatography (20% EtOAc/hexane)
afforded 12 (5.8 mg, 24%).
PDC oxidation of allylic alcohol 12. To a stirred solution
of PDC (258 mg, 0.7 mmol) in CH2Cl2 (2 mL) at 08C was
added 12 (47 mg, 0.1 mmol) in CH2Cl2 (2 mL). After 0.5 h,
the solution was stirred at room temperature for 24 h. The
solution was evaporated to dry, and was partitioned by ether
(20 mL£3) and water (15 mL). The ether layer was
combined and evaporated to a dryness (46 mg), followed
by ¯ash chromatography (10% EtOAc/CH2Cl2) to afford 6
(42 mg, 89%).
MnO2 oxidation of the allylic alcohol 12. To a stirred
solution of magnesium dioxide (90%, Merck) (17.4 mg,
0.02 mmol) and sodium carbonate (10.6 mg, 0.1 mmol)
was added the allylic alcohol 12 (10 mg, 0.02 mmol) at
08C. The solution was stirred overnight while the tempera-
ture was warmed up to room temperature. Filtration through
celite, the CH2Cl2 ®ltrate was evaporated and afford 9.2 mg
of crude residue. Recrystallization from EtOAc afforded
®nally 6 (9.0 mg, 90%).
Eliminative ring opening of epoxide 11
(1) Preparation of diphenyldiselenide. The diphenyldisele-
nide was freshly prepared by Sharpless method.17
(2) To a stirred solution of diphenyldiselenide (12 mg,
0.04 mmol) in absolute ethanol (5 mL) under a nitrogen
atmosphere at 08C was added sodium borohydride (3 mg,
0.08 mmol) in three batches. After the bright yellow color
changed to colorless in 10 min, the epoxide 11 (15 mg, ca.
0.035 mmol) was added in 5 min. The clear solution was
stirred for another 20 min at room temperature, then was
re¯uxed for 2 h. Absolute ethanol (5 mL) was added to
the bottle and the re¯ux was kept overnight. The solution
was cooled to 08C and THF (10 mL) was added. 30% hydro-
gen peroxide (200 mL, ca. 2.4 mmol) was added in 10 min
while the solution was vigorously stirred and the tempera-
ture was kept below 108C. After 8 h the yellow color faded
to colorless which indicated the reaction went thoroughly.
The gray±white suspension was evaporated under vacuum
Preparation of trans-Ebelin lactone (13) and cis-Ebelin
lactone (14). To a stirred solution of crude chloroform
extract of jujubogenin glucosides (30 g) in ethanol
(90 mL) was added, dist. Water (90 mL) and HCl (5 mL).
The solution was heated at 808C for 5 h and recovered to
room temperature. Concentrated under vacuum to remove
ethanol, the aqueous solution was neutralized by 1 M potas-
sium carbonate solution, and extracted by chloroform
(150 mL£4). The chloroform layer was dried (MgSO4),
solution evaporated off, and puri®ed by ¯ash column
(50% EtOAc/hexane) to afford mixture of trans-Ebelin
lactone (13) and cis-Ebelin lactone (14) (1.36 g, 4.5%

8910
Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
yield of the crude extracts). (1H NMR showed the integral
area of 13 and 14 in a 45:55 ratio.)
H-20), 5.80 (1H, d, Jˆ10.8 Hz, H-24), 4.44 (1H, d,
Jˆ10.6 Hz, H-17), 4.36 (1H, d, Jˆ10.6 Hz, H-170), 4.31
(1H, dd, Jˆ11.8 Hz, 5.0, H-3), 2.99 (3H, s, SO2Me), 2.54
(1H, d, Jˆ18.4 Hz, H-15), 1.92 (1H, d, J 18.4 Hz, H-150),
1.77 (3H, d, J 1.1 Hz, Me-23), 1.06 (3H, s, Me-18), 0.89
(3H, s, Me-19), 0.85 (3H, s, Me-25); dC (100.6 MHz,
CDCl3) 195.2 (C-22), 175.8 (C-16), 151.8 (C-20), 141.6
(C-21), 90.0 (C-3), 69.6 (C-17), 55.8 (C-5), 52.9 (C-9),
51.3 (C-14), 40.6 (C-13), 39.9 (C-4), 39.3 (SO2Me), 38.8
(C-8), 38.7 (C-1), 36.9 (C-10), 35.2 (C-15), 34.5 (C-7), 28.6
(C-25), 28.4 (C-12), 25.6 (C-2), 20.2 (C-11), 18.6 (C-18),
18.4 (C-6), 16.7 (C-24), 16.4 (C-19), 10.5 (C-23); HRMS
(EI): M1, found 480.3491. C26H40O6S requires 480.3486.
Mesylation of Ebelin lactones 13/14. To a stirred solution
of Ebelin lactones (Z/E mixture, 1.39 g, 3.1 mmol) in dry
pyridine (20 mL) at 08C was added, mesyl chloride (4 mL),
and the temperature was kept at 08C for 10 h. A mixture of
pyridine (5 mL) and water (5 mL) was added to the solution,
which was then evaporated under vacuum. The residue was
partitioned between water (20 mL) and chloroform
(30 mL£3). The organic layer was combined, dried
(MgSO4) and solvent evaporated off. The residue (2.1 g)
was puri®ed by ¯ash column and afforded ®nally mesylated
ebelin lactone (MsEbelactone) (1.39 g, 2.5 mmol, 83%) as
oil.
MsEbehyde C (17). Colourless gum, Rf (33% EtOAc/hexane)
0.33; nmax (KBr) 2952, 2675, 1781, 1693, 1417, 1405, 1366,
1194, 966 cm21; UV: 238 nm (e 17800); dH (400 MHz,
CDCl3) 10.08 (1H, s, H-22), 6.16 (1H, dd, Jˆ10.1,
1.2 Hz, H-20), 4.39 (1H, d, Jˆ10.6 Hz, H-17), 4.32 (1H,
d, Jˆ10.4 Hz, H-170), 4.31 (1H, dd, Jˆ11.8, 4.6 Hz, H-3),
3.00 (3H, s, SO2Me), 2.56 (1H, d, Jˆ18.2 Hz, H-15), 2.06
(1H, d, Jˆ18.3 Hz, H-150), 1.80 (1H, d, Jˆ1.2 Hz, Me-23),
1.04 (3H, s, Me-18), 1.02 (3H, s, Me-24), 0.88 (3H, s,
Me-19), 0.86 (3H, s, Me-25); HRMS (EI): M1, found
480.3489. C26H40O6S requires 480.3486.
General procedure of ozonolysis of MsEbelactone
To a stirred solution of MsEbelactone (300 mg, 0.55 mmol)
in anhydrous methanol (15 mL) at 2788C was introduced,
ozone ¯uid (1 cc/s at 125 mA) for a different time (Table 2),
and then nitrogen gas for another 10 min. The temperature
of the solution was then recovered to 08C (ice bath), while
thiourea (150 mg) in dry MeOH (3 mL) was added portion-
wise, and the solution stirred for another 6 h. The white
suspension was ®ltered and washed by cooled dry MeOH
(2 mL). The elute was concentrated under vacuum and was
washed out by ether (25 mL) and 1% Na2CO3 (10 mL).
Extracted by ether (20 mL), the ether layer were combined
and washed by distilled water (10 mL), dried (MgSO4), and
the solvent evaporated off. Puri®cation by ¯ash column
(33±50% EtOAc/hexane) afforded different yields of 15,
16 and 17 (see Table 2).22
2-EneEbehyde (18). To a stirred solution of 15 (50 mg,
0.11 mmol) in toluene (5 mL) was added DBU (1 mL).
The brown solution was heated under re¯ux for 10 h. The
reaction was quenched by addition of acetic acid (1 mL) in
water (8 mL), stirred for 5 min. The solution was extracted
by diethyl ether (20 mL£3). The aqueous layer was then
basi®ed to pHˆ8 and partitioned by diethyl ether
(20 mL). The ether layer was combined, dried (MgSO4),
puri®ed by ¯ash column (33±50% EtOAc/hexane) to
afforded ®nally 18 (21.1 mg, 54%) as colorless needles
(acetone), mp 203±2048C; Rf (50% EtOAc/hexane) 0.40;
nmax (KBr) 3021, 2778, 1775, 1736, 1657, 1415, 962,
710 cm21; dH (400 MHz, CDCl3) 9.63 (1H, d, Jˆ1.2 Hz,
H-20), 5.37 (1H, m, H-2), 5.36 (1H, m, H-3), 4.19 (2H, d,
Jˆ10.3 Hz, H-17 & H-170), 2.99 (3H, s, SO2Me), 2.78 (1H,
m, H-13), 2.60 (1H, d, Jˆ18.3 Hz, H-15), 2.46 (1H, d,
Jˆ18.6 Hz, H-150), 0.98 (3H, s, Me-18), 0.96 (3H, s,
Me-24), 0.93 (3H, s, Me-21), 0.86 (3H, s, Me-19), 0.85
(3H, s, Me-22); dC (100.6 MHz, CDCl3) 203.2 (C-20),
176.4 (C-16), 137.9 (C-3), 120.8 (C-2), 70.2 (C-17), 52.7
(C-5), 52.2 (C-13), 51.1 (C-9), 49.2 (C-14), 41.3 (C-5), 52.2
(C-13), 51.1 (C-9), 49.2 (C-14), 41.3 (C-1), 40.8 (C-4), 39.2
(SO2Me), 36.3 (C-8), 34.7 (C-15), 34.5 (C-10), 31.9 (C-7),
31.6 (C-22), 23.3 (C-12), 22.6 (C-18), 19.7 (C-11), 18.7
(C-6), 16.3 (C-19); HRMS (EI): M1, found 344.2351.
C22H32O3 requires 344.2353.
Table 2. The relationship of ozonolysis time and the yields of Ebehydes
T (min)
15 (%)
16 (%)
17 (%)
10
25
40
60
5
35
12
10
0
34
10
0
26
22
18
0
MsEbehyde (15). Colourless gum, Rf (33% EtOAc/hexane)
0.29; nmax (KBr) 2950, 1778, 1735, 1403, 1364, 1203, 1196,
977 cm21; dH (400 MHz, CDCl3) 9.61 (1H, d, Jˆ1.1 Hz,
H-20), 4.35 (1H, d, Jˆ10.2 Hz, H-17), 4.30 (1H, dd,
Jˆ11.8, 4.6 Hz, H-3), 4.20 (1H, d, Jˆ10.2 Hz, H-170),
3.00 (3H, s, SO2Me), 2.60 (1H, d, Jˆ18.6 Hz, H-15), 2.46
(1H, d, Jˆ18.7 Hz, H-150), 1.01 (3H, s, Me-18), 0.97 (3H, s,
Me-21), 0.88 (3H, s, Me-19), 0.85 (3H, s, Me-22); dC
(100.6 MHz, CDCl3) 203.2 (C-20), 176.4 (C-16), 89.8
(C-3), 70.5 (C-17), 55.7 (C-5), 53.0 (C-13), 52.8 (C-9),
49.6 (C-14), 40.6 (C-4), 39.2 (SO2Me), 38.9 (C-8), 38.6
(C-1), 37.3 (C-10), 35.0 (C-7), 33.1 (C-15), 28.5 (C-22),
25.5 (C-2), 23.5 (C-12), 19.9 (C-11), 18.1 (C-18), 18.1
(C-6), 16.6(C-21), 16.4 (C-19); HRMS (EI): M1, found
440.3175. C23H36O6S requires 440.3173.
First Grignard reaction of 18
(a) Preparation of Grignard reagent. To a stirred solution
of dry diethyl ether (2 mL) including magnesium pieces
(70 mg, 2.92 mmol) and one piece of iodine was added,
bromobenzene (250 mL) in dry ether (1 mL) under vigorous
stirring for 20 min until the magnesium pieces disappeared.
MsEbehyde B (16). Colourless gum, Rf (33% EtOAc/hexane)
0.32; nmax (KBr) 2953, 2677, 1776, 1699, 1418, 1405, 1365,
1194, 969 cm21; UV 239 nm (e 18600); dH (400 MHz,
CDCl3) 9.36 (1H, s, H-22), 6.23 (1H, dd, Jˆ10.4, 1.2 Hz,
(b) Grignard reaction. To a stirred solution of 2-en-
Ebehyde 18 (17 mg, 0.05 mmol) in ether (1 mL) at 2608C

Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
8911
was added dropwise, the above-prepared Grignard reagent
(45 mL) under nitrogen. The solution was stirred for 20 min
and quenched by 0.5% HCl (0.5 mL) in water (3 mL) at
2188C. Stirred for another 10 h, the solution was extracted
by diethyl ether (20 mL£2). The aqueous layer was basi®ed
to pHˆ8 and extracted by diethyl ether (10 mL£2). The
ether layer was combined, washed by brine (10 mL), dried
(MgSO4), and the solvent evaporated off. The residue
(22 mg) was puri®ed by ¯ash column (17% EtOAc/hexane)
to afford 19 (5.8 mg, 28%) and 20 (4.8 mg, 23%), as well as
unreacted 18 (3.3 mg, 19%).
HRMS (EI): M1, found 434.2453. C28H34O5 requires
434.2458.
Preparation of trimethoxy bromobenzene. To a stirred
solution of of trimethoxybenzene (1.076 g, 6.4 mmol) in
hexane (10 mL) was added, dry pyridine (30 mL) and,
after 5 min of stirring, quickly Bromine (1.8 mL). The reac-
tion was heated at 808C for 45 min, until the red vapor
ceased and no more HBr gas created. The solution was
washed by water (300 mL), and basi®ed by 1 M NaOH
solution (20 mL£3). The aqueous layer was further
extracted by EtOAc (20 mL£2). The organic layer were
combined and puri®ed by ¯ash column (10% EtOAc/
hexane) to afford, 1,5-dibromo-2,3,4-trimethoxybenzene
(880 mg, 42%) and 2,3,4-trimethoxybromobenzene (696
mg, 44%).
2-Ene-Ebelacol A (19). Colourless gum, Rf (50% EtOAc/
hexane) 0.42; IR (KBr): 3633 (OH), 3066, 3022, 1776,
1608, 1512, 1417, 1360, 1105, 1027, 712 cm21; dH
(400 MHz, CDCl3) 7.31 (2H, m, H-30 and H-50), 7.28 (2H,
m, H-20 and H-60), 7.27 (1H, m, H-40), 5.36 (1H, m, H-2),
5.35 (1H, m, H-3), 4.48 (1H, dd, Jˆ8.9 Hz, 1.5, H-20),
4.40 (1H, d, Jˆ10.4 Hz, H-17), 4.32 (1H, d, Jˆ10.3 Hz,
H-170), 3.15 (1H, d, Jˆ18.3 Hz, H-15), 2.64 (1H,
d, Jˆ18.5 Hz, H-150), 1.03 (3H, s, Me-18), 0.94 (3H,
s, Me-21), 0.86 (3H, s, Me-22), 0.79 (3H, s, Me-19);
HRMS (EI): M1, found 422.2819. C28H38O3 requires:
422.2823.
Grignard reaction of 18 with 1,5-dibromo-2,3,4-tri-
methoxybenzene (24)
(a) Preparation of Grignard reagent. To a nitrogen
atmosphere bottle including of magnesium pieces (70 mg,
2.92 mmol) and one piece of iodine was added, a solution of
1,5-dibromotrimethoxybenzene 24 (720 mg, 2.2 mmol) in
dry THF (3 mL) under vigorous stirring. After the reaction
was initiated (in 5 min), the solution was continued stirring
for another 20 min.
2-Ene-Ebelacol B (20). Colourless gum, Rf (50% EtOAc/
hexane) 0.48; nmax (KBr) 3599 (OH), 3060, 1752, 1611,
1515, 1346, 1334, 1031, 1023, 708 cm21; dH (400 MHz,
CDCl3) 7.33 (2H, m, H-30 and H-50), 7.27 (2H, m, H-20
and H-60), 7.26 (1H, m, H-40), 5.39 (1H, m, H-2), 5.36
(1H, m, H-3), 4.99 (1H, d, Jˆ9.8 Hz, H-17), 4.96 (1H, d,
Jˆ3.6 Hz, H-20), 4.41 (1H, d, Jˆ18.6 Hz, H-150), 0.94 (6H,
s, Me-18 and Me-21), 0.86 (3H, s, Me-22), 0.78 (3H, s,
Me-19); dC (100.6 MHz, CDCl3) 178.5 (C-16), 144.2
(C-10), 137.9 (C-3), 128.6 (C-30 and C-50), 128.1 (C-40),
126.9 (C-20), 121.0 (C-2), 78.5 (C-20), 69.6 (C-17), 61.1
(OMe), 60.7 (OMe), 56.0 (OMe), 51.6 (C-5), 51.4 (C-9),
45.1 (C-14), 40.8 (C-1), 40.6 (C-4), 36.2 (C-8), 34.6
(C-10), 33.4 (C-7), 31.6 (C-13), 29.7 (C-22), 26.8 (C-12),
22.6 (C-18), 20.1 (C-11), 19.1 (C-6), 17.6 (C-21), 16.1
(C-19); HRMS (EI): M1, found 422.2824. C28H38O3
requires 422.2823.
(b) Grignard Reaction. To a stirred solution of 2-en-
Ebehyde 18 (35 mg, 0.1 mmol) in dry THF (3 mL) at
2708C was added slowly, the gray Grignard reagent
above-mentioned (81 mL) and stirred for another 0.5 h.
The reaction was quenched by 0.5% HCl (0.5 mL) in H2O
(3 mL) (Caution!), and the solution was basi®ed by
NaHCO3 to pHˆ8, and then extracted by diethyl ether
(10 mL£2) followed by EtOAc (10 mL). The organic
layer were combined, dried (MgSO4), solvent evaporated
off to afford a residue (55 mg). Puri®ed by column chroma-
tography to afford 27 (5.8 mg, 12%), 28 (9.1 mg, 16%), 29
(6.5 mg, 12%), 30 (4.3 mg, 8%), and 18 (5.9 mg, 17%).
2-EneEbelacol C (27). Colourless gum, Rf (50% EtOAc/
hexane) 0.36; nmax (KBr) 3626 (OH), 3045, 3019, 1775,
1611, 1518, 1422, 1355, 1107, 1020, 708 cm21; dH
(400 MHz, CDCl3) 6.97 (1H, d, Jˆ8.6 Hz, H-50), 6.65
(1H, d, Jˆ8.6 Hz, H-60), 5.38 (1H, m, H-2), 5.35 (1H, m,
H-3), 4.80 (1H, dd, Jˆ8.6, 3.8 Hz, H-20), 4.41 (1H, d,
Jˆ10.1 Hz, H-17), 4.37 (1H, d, Jˆ10.3 Hz, H-170), 3.90
(3H, s, OMe), 3.84 (s, 3, OMe), 3.83 (s, 3, OMe), 3.23
(1H, d, Jˆ18.5 Hz, H-15), 2.61 (1H, d, Jˆ18.5 Hz,
H-150), 1.01 (3H, s, Me-18), 0.94 (3H, s, Me-21), 0.86
(3H, s, Me-22), 0.80 (3H, s, Me-19); dH (100.6 MHz,
CDCl3) 178.6 (C-16), 153.3 (C-30), 151.1 (C-40), 141.7
(C-20), 137.9 (C-3), 129.6 (C-10), 121.0 (C-2), 107.4
(C-50), 72.0 (C-20), 70.1 (C-17), 60.7 (OMe), 60.6 (OMe),
56.0 (OMe), 51.7 (C-5), 51.3 (C-9), 45.4 (C-14), 40.8 (C-1),
40.6 (C-4), 36.2 (C-8), 34.7 (C-10), 33.2 (C-7), 34.7 (C-15),
31.6 (C-13), 29.3 (C-22), 26.4 (C-12), 22.5 (C-18), 20.3
(C-11), 19.0 (C-6), 17.4 (C-21), 16.1 (C-19); HRMS (EI):
M1, found 526.2934. C31H42O6 requires 510.9826.
1-Oxo-2-en-Ebelacone (21). To a stirred solution of 19
(2.2 mg, 5.2 mmol) in acetic acid (2 mL) was added, chro-
mium trioxide (10 mg, 10 mmol) in acetic acid (1 ml). The
solution was heated in an 808C oil bath for 2 h, and the
temperature was allowed to recover to room temperature
and stirred for another 8 h. Solvent was evaporated under
vacuum in a 508C water bath and, residue was partitioned
between CH2Cl2 (20 mL£3) and water (10 mL). The
organic layer was combined, dried (MgSO4) and puri®ed
by ¯ash column (50% EtOAc/hexane) to afford of 21
(1.55 mg, 69%) as colorless oil, Rf (50% EtOAc/hexane)
0.31; nmax (KBr) 3063, 2994, 1773, 1690, 1668, 1566,
1487, 1218, 1186 cm21; UV: 251 nm (e 18800), 225 nm
(e 18600); dH (400 MHz, CDCl3) 7.86 (2H, dd, Jˆ8.6,
1.3 Hz, H-20 and H-60), 7.56 (1H, t, Jˆ8.6 Hz, H-40), 7.47
(2H, m, H-30 and H-50), 6.32 (1H, d, Jˆ10.1 Hz, H-3), 5.67
(1H, d, Jˆ10.1 Hz, H-2), 4.96 (1H, d, Jˆ10.7 Hz, H-17),
4.44 (1H, d, Jˆ10.2 Hz), 2.52 (1H, d, Jˆ18.5 Hz, H-15),
2.15 (1H, d, Jˆ18.5 Hz, H-150), 1.19 (3H, s, Me-19), 1.18
(3H, s, Me-18), 1.10 (3H, s, Me-21), 1.07 (3H, s, Me-22);
2-EneEbelacol D (28). Colourless gum, Rf (50% EtOAc/
hexane) 0.37; nmax (KBr) 3589 (OH), 3038, 1750, 1612,

8912
Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
1502, 1351, 1299, 1057, 890, 656 cm21; dH (400 MHz,
CDCl3) 7.35 (s, 1H, H-60), 5.39 (1H, m, H-3), 5.06 (1H, d,
Jˆ3.9 Hz, H-20), 4.95 (1H, d, Jˆ9.8 Hz, H-17), 4.39 (1H,
d, Jˆ9.8 Hz, H-170), 3.89 (6H, s, 2£OMe), 3.87 (3H, s,
OMe), 2.74 (1H, d, Jˆ18.6 Hz, H-15), 2.58 (1H, d,
Jˆ17.8 Hz, H-150), 0.95 (3H, s, Me-18), 0.93 (3H, s,
Me-21), 0.79 (3H, s, Me-22), 0.73 (3H, s, Me-19); HRMS
(EI): M1, found 587.8845. C31H41O6Br requires 587.8852.
(C-15), 34.7 (C-10), 31.8 (C-7), 31.6 (C-13), 29.3 (C-22),
27.1 (C-12), 22.7 (C-18), 22.5 (C-11), 18.7 (C-6), 16.3
(C-21), 15.7 (C-19); HRMS (EI): M1, found 508.2821.
C31H40O6 requires 508.2826.
1-Oxo-2-en-Ebelactonic acid (32). Colourless gum, Rf
(33% EtOAc/hexane) 0.25; nmax (KBr) 3507, 3056, 2987,
1774, 1718, 1668, 1221, 1187, 820 cm21; UV: 225 nm (e
16900); dC (400 MHz, CDCl3) 6.35 (1H, d, Jˆ9.9 Hz, H-3),
5.68 (1H, d, Jˆ9.9 Hz, H-2), 4.90 (1H, d, Jˆ10.2 Hz,
H-17), 4.43 (1H, d, Jˆ10.2 Hz, H-170), 2.54 (1H, d,
Jˆ18.6 Hz, H-15), 2.18 (1H, d, Jˆ18.6 Hz, H-150), 1.06
(3H, s, Me-19), 1.04 (3H, s, Me-18), 1.01 (3H, s, Me-21),
0.96 (s, 3H, Me-22); dC (100.6 MHz, CDCl3) 205.6 (C-1),
180.7 (C-20), 124.1 (C-2), 153.8 (C-3), 41.2 (C-4), 51.6
(C-5), 18.7 (C-6), 32.6 (C-7), 36.1 (C-8), 45.5 (C-9), 48.2
(C-10), 24.2 (C-11), 27.8 (C-12), 35.6 (C-13), 51.1 (C-14),
35.9 (C-15), 178.0 (C-16), 70.4 (C-17), 18.0 (C-21), 24.4
(C-22); HRMS (EI): M1, found 374.2097. C22H30O5
requires 374.2094.
2-EneEbelacol E (29). Colourless gum, Rf (50% EtOAc/
hexane) 0.38; nmax (KBr) 3593 (OH), 3060, 1748, 1616,
1511, 1348, 1305, 1067, 884 cm21
; dH (400 MHz,
CDCl3): 6.94 (1H, d, Jˆ8.6 Hz, H-50), 6.63 (1H, d,
Jˆ8.6 Hz, H-60), 5.38 (1H, m, H-3), 5.36 (1H, m, H-3),
5.11 (1H, d, Jˆ2.4 Hz, H-20), 4.96 (1H, d, Jˆ9.7 Hz,
H-17), 4.40 (1H, d, Jˆ9.5 Hz, H-170), 3.84 (3H, s, OMe),
3.83 (3H, s, OMe), 3.75 (s, 3H, OMe), 2.73 (1H, d,
Jˆ18.6 Hz, H-15), 2.63 (1H, d, Jˆ18.5 Hz, H-150), 0.95
(3H, s, Me-18), 0.93 (3H, s, Me-21), 0.87 (3H, s, Me-22),
0.78 (3H, s, Me-19); dC (100.6 MHz, CDCl3) 178.0 (C-16),
150.3 (C-30), 148.8 (C-40), 146.0 (C-20), 137.8 (C-3), 134.9
(C-10), 121.1 (C-20), 111.2 (C-50), 70.3 (C-17), 66.8 (C-20),
60.9 (OMe), 60.7 (OMe), 51.6 (OMe), 51.5 (C-9), 51.2
(C-5), 44.9 (C-14), 40.9 (C-1), 40.7 (C-4), 36.3 (C-8),
34.7 (C-15), 34.3 (C-10), 33.2 (C-7), 31.6 (C-22), 29.3
(C-13), 22.6 (C-18), 20.2 (C-11), 19.6 (C-12), 19.2 (C-6),
17.7 (C-21), 16.6 (C-19); HRMS (EI): M1, found 510.9825.
C31H42O6 requires 510.9826.
Enzyme: Electrophorus acetylcholinesterase (1000±2000/
mgprotein) was purchased from Sigma.
Assay of inhibition ration of compounds 1, 5, 6 and 21
(a) The sample was dissolved in 0.1 M phosphate buffer at
pH 7.0.
2-EneEbelacol F (30). Colourless gum, Rf (50% EtOAc/
hexane) 0.41; nmax (KBr) 3615 (OH), 3047, 1749, 1618,
1510, 1344, 1301, 1053, 1019 cm21; dH (400 MHz,
CDCl3) 7.08 (1H, d, Jˆ8.6 Hz, H-50), 6.64 (1H, d,
Jˆ8.6 Hz, H-60), 5.39 (1H, m, H-2), 5.36 (1H, m, H-3),
5.09 (1H, brs, H-20), 4.98 (1H, d, Jˆ9.8 Hz, H-17), 4.39
(1H, d, Jˆ9.7 Hz, H-170), 3.91 (s, 3H, OMe), 3.84 (s, 3H,
OMe), 3.83 (s, 3H, OMe), 2.73 (1H, d, Jˆ18.7 Hz, H-15),
2.65 (1H, d, Jˆ18.6 Hz, H-150), 0.94 (3H, s, Me-18), 0.93
(3H, s, Me-21), 0.86 (3H, s, Me-22), 0.78 (3H, s, Me-19); dC
(100.6 MHz, CDCl3) 178.1 (C-16), 149.4 (C-40), 137.8
(C-30), 137.8 (C-3), 130.5 (C-10), 120.7 (C-2), 120.6
(C-20), 106.5 (C-50), 70.4 (C-17), 67.2 (C-20), 60.9
(OMe), 60.7 (OMe), 51.7 (C-9), 51.6 (OMe), 51.3 (C-5),
45.1 (C-14), 40.9 (C-1), 40.7 (C-4), 36.2 (C-8), 34.7
(C-15), 34.2 (C-10), 33.2 (C-10), 31.7 (C-22), 29.3
(C-13), 22.7 (C-18), 20.3 (C-11), 19.8 (C-12), 17.7
(C-21), 16.1 (C-19); HRMS (EI): M1, found 510.9830.
C31H42O6 requires 510.9826.
(b) A cellulose dialysis tube, purchased from Visking Co.,
was boiled for 30 min in 1% Na2CO3/1 mM EDTA solution
and washed thoroughly with distilled water before use. To
this freshly prepared cellulose tube was added 50 mL of
0.1% BSA in 0.1 M phosphate buffer at pH 7.0, 5 units
AChE and 1£1028 M samples. 1% MeOH was used in the
controlled experiment to replace the inhibitor. The tube was
made to stand at room temperature for 1 h, and subjected to
a dialyzation against 10 L of 0.1 M phosphate buffer at pH
7.0 in a 48C cool room. At predetermined time points during
dialysis, 1.5 mL of the solution was taken from the tube for
assay of AChE activity.
(c) The AChE activity was determined by the colorimetric
method of Ellman et.al.20 An aliquot (20±40 mL) of the
working AChE assay system containing 4.8£1024M acetyl-
thiocholine and 3.2£10-4 M DTNB in 0.1 M phosphate
buffer at pH 8.0. The initiate rate of substrate hydrolysis
was determined at 412 nm by a double-beam spectrometer
(Hitachi model 100±50 with a model 056 recorder) at 258C.
2-EneEbelacone A (31). Colourless gum, Rf (50% EtOAc/
hexane) 0.65; nmax (KBr) 3015, 2942, 2918, 1776, 1689,
1644, 1621, 1505, 1465, 1382, 1182, 1100, 858,
697 cm21; UV: 282 nm (e 19,200); dH (400 MHz, CDCl3)
7.29 (1H, d, Jˆ8.8 Hz, H-60), 6.68 (1H, d, Jˆ9.8 Hz, H-17),
4.41 (1H, d, Jˆ9.6 Hz, H-170), 3.96 (3H, s, OMe), 3.88 (3H,
s, OMe), 3.85 (3H, s, OMe), 2.54 (1H, d, Jˆ18.7 Hz, H-15),
2.19 (1H, d, Jˆ18.7 Hz, H-150), 1.06 (3H, s, Me-18), 0.98
(3H, s, Me-21), 0.88 (3H, s, Me-22), 0.86 (3H, s, Me-19); dC
(100.6 MHz, CDCl3) 204.1 (C-20), 177.2 (C-16), 157.3
(C-30), 152.7 (C-40), 141.8 (C-20), 137.9 (C-3), 125.5
(C-10), 120.9 (C-2), 107.5 (C-50), 70.6 (C-17), 61.8
(OMe), 60.9 (OMe), 56.1 (OMe), 51.7 (C-5), 50.9 (C-9),
49.8 (C-14), 41.0 (C-1), 40.8 (C-4), 36.2 (C-8), 35.2
Acknowledgements
This work was ®nancially supported by National Science
Council, China (Taipei) under grant NSC 87-2314-002-
005. The authors appreciate Professor Chao-Chou Kang
and Mr Huan-Wen Fang for the acetylcholinesterase inhibi-
tion assay. One of us (Y. Zhao) would like to express his
thankfulness to NSC for offering the postdoctoral research
fellowship and to National Taiwan University for their
affording the visiting professor employment.

Y. Zhao et al. / Tetrahedron 56 (2000) 8901±8913
8913
13. Corey, E. J.; Schmidt, G. Tetrahedron Lett. 1979, 20, 399±
402.
References
14. Sharpless, K. B.; Lauer, R. F.; Teranishi, A. Y. J. Am. Chem.
Soc. 1973, 95, 6137±6139.
1. Summer, W. K.; Majovski, L. V.; Marsh, G. M.; Tachiki, K.;
Kling, A. N. Engl. J. Med. 1986, 315, 1241±1245.
2. Hallak, M.; Giacobini, E. Neuropharmacology 1989, 28, 199±
206.
15. Reich, H. J.; Reich, I. L.; Renga, J. M. J. Am. Chem. Soc. 1973,
95, 5813±5815.
3. Rupniak, N. M. J.; Field, M. J.; Samson, N. A.; Steventon, M. J.;
Iversen, S. D. Neurobiol. Aging 1990, 11, 609±613.
4. Kozikowski, A. P.; Thiels, E.; Tang, X. C.; Hanin, I. Adv. Med.
Chem. 1992, 1, 175±205.
16. Zoretic, P. A.; Chambers, R. J. J. Org. Chem. 1985, 50, 2981±
2987.
17. Stork, G.; Williard, P. G. J. Am. Chem. Soc. 1977, 99, 7067±
7068.
5. Dowd, P. F.; Peng, F. C.; Chen, J. W.; Ling, K. H. Entomol.
Exp. Appl. 1992, 65, 57±64.
18. Sharpless, K. B.; Lauer, R. F. J. Am. Chem. Soc. 1975, 95,
2697±2699.
6. Chen, J. W.; Ling, K. H. J. Biomed. Sci. 1996, 3, 54±58.
7. Peng, F. C. J. Nat. Prod. 1995, 58, 54±58.
8. Kawai, K. I.; Akiyama, T.; Ogihara, Y.; Shibata, S.
Phytochemistry 1974, 13, 2829±2832.
19. Thummel, R. P.; Rickborn, B. J. Am. Chem. Soc. 1970, 92,
2064±2067.
20. Ellman, G. L.; Courtney, K. D.; Andres Jr., V.; Featherstone,
R. M. Biochem. Pharmacol. 1961, 7, 88±95.
21. Kulshreshtha, M. J.; Rastogi, R. P. Ind. J. Chem. 1972, 10,
152±154.
9. Wagner, H.; Sticher, O.; Jurcic, K.; Morton, J.; Neszmelyi, A.
Planta Med. 1983, 48, 136±141.
10. Cui, J. F.; Bystrm, S. B.; Eneroth, P.; Berkhem, I. J. J. Org.
Chem. 1994, 59, 8251±8255.
22. Eade, R. A.; Ellis, J.; Shannon, J. S.; Simes, H. V.; Simes, J. J.
H. Aust. J. Chem. 1970, 23, 2085±2090.
23. Gupta, D.; Soman, R.; Dev, S. Tetrahedron 1982, 38, 3013±
3018.
11. Williams, R. M.; Maruyama, L. K. J. Org. Chem. 1987, 52,
4044±4047.
12. Rosenthal, D.; Grabowich, P.; Sabo, E. F.; Fried, J. J. Am.
Chem. Soc. 1963, 85, 3971±3979.