Anti-HIV and NO production inhibition activities of epi-aleuritolic acid derivatives

Article abstract of DOI:10.1080/10286020.2013.787990

Fifteen epi-aleuritolic acid derivatives were synthesized and evaluated for anti-HIV activity in 293 T cells and NO production inhibition activity. Of the derivatives, 1, 2, 3, 4, 11, and 13 showed relatively potent anti-HIV activity with EC₅₀ values ranging from 5.80 to 13.30 μM. The most potent compound, 3α-2′,2′-dimethylsuccinic acyl epialeuritolic acid (11), displayed significant anti-HIV activity with an EC₅₀ value of 5.80 μM. Compounds 1, 3, 4, and 11 showed NO inhibition activity, with IC₅₀ values ranging from 3.40 to 7.10 μM and compound 1 inhibited NO production with an IC₅₀ value of 3.40 μM.

Full text of DOI:10.1080/10286020.2013.787990

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Anti-HIV and NO production inhibition
activities of epi-aleuritolic acid
derivatives
Jia-Bao Liu a , Ying Zhang a , Bao-Song Cui a , Ying-Li Cao a , Shao-
Peng Yuan a , Ying Guo a , Qi Hou a & Shuai Li a
a State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences and Peking Union Medical College , Beijing ,
100050 , China
Published online: 03 May 2013.
To cite this article: Jia-Bao Liu , Ying Zhang , Bao-Song Cui , Ying-Li Cao , Shao-Peng Yuan , Ying
Guo , Qi Hou & Shuai Li (2013): Anti-HIV and NO production inhibition activities of epi-aleuritolic
acid derivatives, Journal of Asian Natural Products Research, 15:5, 515-524
To link to this article: http://dx.doi.org/10.1080/10286020.2013.787990
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Journal of Asian Natural Products Research, 2013
Vol. 15, No. 5, 515–524, http://dx.doi.org/10.1080/10286020.2013.787990
Anti-HIV and NO production inhibition activities of epi-aleuritolic
acid derivatives
Jia-Bao Liu, Ying Zhang, Bao-Song Cui, Ying-Li Cao, Shao-Peng Yuan, Ying Guo,
Qi Hou and Shuai Li*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100050, China
(Received 20 November 2012; final version received 18 March 2013)
Fifteen epi-aleuritolic acid derivatives were synthesized and evaluated for anti-HIV
activity in 293 T cells and NO production inhibition activity. Of the derivatives, 1, 2, 3,
4, 11, and 13 showed relatively potent anti-HIV activity with EC50 values ranging from
5.80 to 13.30 mM. The most potent compound, 3a-20,20-dimethylsuccinic acyl epi-
aleuritolic acid (11), displayed significant anti-HIV activity with an EC50 value of
5.80 mM. Compounds 1, 3, 4, and 11 showed NO inhibition activity, with IC50 values
ranging from 3.40 to 7.10 mM and compound 1 inhibited NO production with an IC50
value of 3.40 mM.
Keywords: epi-aleuritolic acid derivatives; anti-HIV; NO production inhibition
1. Introduction
continuing search for new anti-HIV drug
candidates, we report that modification of
epi-aleuritolic acid yielded extremely
potent anti-HIV compounds. To explore
the role of the acyl substitution along with
other functional groups, we developed a
method to hydrolyze myricarin A (1),
which was separated and enriched from the
stems of Myricaria bracteata [6], to obtain
epi-aleuritolic acid. Then we acylate
the acid at the C-3 hydroxy position,
and convert the C17-carboxyl to the
sodium salt. As a traditional Tibetan herb,
M. bracteata has been reported to cure
rheumatism and arthritis [7]. Myricarin
A is one of the active constituents
found in this herb; therefore, we also
examined the inhibitory effect of these epi-
aleuritolic acid derivatives on nitric oxide
production.
Our previous findings reported that the
natural products myricarins A and B (1 and
2) (Figure 1), epi-aleuritolic acid deriva-
tives isolated from the stems of Myricaria
paniculata (Tamaricaceae) [1], have struc-
tures similar to pentacyclic triterpene
compounds in the literature [2] that are
reported to be potent inhibitors of HIV-1
reverse transcriptase. After further litera-
ture review, we found that pentacyclic
triterpene derivatives, such as betulinic
acid, oleanolic acid, and ursolic acid, have
been widely studied for their anti-HIV
activity [3–5]. We found that both 1 and 2
showed anti-HIV activity, with EC50 values
of 7.80 and 11.30 mM, respectively, similar
to those of the reported compounds [2] and
betulinic acid in the same experiment,
which has an EC50 value of 8.37 mM. Based
on these findings, epi-aleuritolic acid was
considered as a potential lead for the
synthesis of anti-HIV triterpenoids. In our
Overall, fifteen 3-O-acyl-epi-aleurito-
lic acid derivatives were obtained, includ-
ing seven new compounds (3, 7, 8, 9, 10,
*Corresponding author. Email: lishuai@imm.ac.cn
q 2013 Taylor & Francis

516
J.-B. Liu et al.
COOH
COOH
HO
O
O
HO
HO
O
O
2
1
Figure 1. The structures of epi-aleuritolic acid derivatives 1 and 2.
11, and 12) and six known compounds (4,
5 [1,8], 6 [9], 13 [10], 14 [11], and 15). The
known compounds’ structures were ident-
ified based on comparison of spectroscopic
data with literature values. We also
examined the anti-HIV and NO production
inhibition activities of each compound.
of the 3-hydroxy ester analogs, showed
that the C-3 acyl chain was crucial for HIV
inhibition.
Among the monocarboxylic fatty acid
derivatives of epi-aleuritolic acid, the
butyric acid derivative 7 had a slightly
greater inhibitory effect (IR ¼ 15.0%) than
the acetic acid derivative 6 (IR ¼ 11.4%).
Similarly, isobutyrylic derivative
8
2. Results and discussion
(IR ¼ 32.3%) displayed stronger inhibition
than 7. This suggested that the anti-HIV
activity was enhanced with the carbon chain
length and side chain in some monocar-
boxylic fatty acid derivatives. The opposite
result was observed with the isovaleric
derivative 9 (IR ¼ 17.2%), which suggests
that the anti-HIV activity is not enhanced
with chains .5 carbons in length for
monocarboxylic fatty acid derivatives. Of
the dicarboxylic fatty acid derivatives of
epi-aleuritolic acid, the succinic acid deriva-
tive 10 showed a slightly stronger inhibitory
effect (IR ¼ 22.4%) than the butyric deriva-
tive 7. Compound 11, the 2,2-dimethyl
succinic acid derivative, had a stronger
The 30,40-diacetyl derivative 3 and sodium
salt of myricarin A (4) (Scheme 1) had only
slightly lower inhibition rate values than
the unsubstituted compound myricarin
A (1) ranging from 53.8% to 56.8%. The
derivative compounds had lower anti-HIV
activity, with EC50 values ranging from
11.4 to 13.3 mM (Table 1). Thus, the free
hydroxyl and carboxyl groups in the
structure of epi-aleuritolic acid derivatives
are necessaryto maintain anti-HIV activity.
The hydrolysis product 5 showed almost
no anti-HIV activity relative to the 3-
hydroxy acylated compounds (1, 2, and 6–
12) (Scheme 2). Thisresult, along withthose
COOH
O
O
O
O
O
O
3
4
COOH
COONa
O
O
HO
HO
HO
O
O
HO
1
DMAP (4–dimethylaminopyridine)
Scheme 1. Modification of epi-aleuritolic acid derivative 1.

Journal of Asian Natural Products Research
517

518
J.-B. Liu et al.
COOH
O
HO
HO
O
1
O
6 R=
75.9%
2% NaOH, MeOH, rt, 24h
O
O
7 R=
8 R=
COOH
O
9 R=
HO
5
O
10 R=
DMAP
Pyridine
110˚C
HOOC
R
Cl
O
11 R=
12 R=
HOOC
O
COOH
RO
6-12
Scheme 2. 3-O-Esterification derivatives of epi-aleuritolic acid.
inhibitory effect than both 10 and the other
derivatives of epi-aleuritolic acid. Thus, the
dimethylgroupsatthe secondpositionin the
dicarboxylic fatty acid enhanced anti-HIV
activity. Compound 11 showed the most
potent anti-HIV activity (EC50 ¼ 5.80mM)
and had a remarkable IR (69.3%) at a
concentration of 1 £ 1025 M. The dicar-
boxylic acid acylate of 5 showed a
stronger inhibitory effect than compounds
6–9 and 12.
The aromatic acid derivative 12
showed very weak activity (IR ¼ 3.4%),
much lower than the cinnamic acid
derivatives of 1 and 2 (IR ¼ 57.3% and
55.4%, respectively). This suggested
that the hydroxyl group in the aromatic
ring was necessary to retain anti-HIV
activity.
Compound 13 (Scheme 3), the C-3-
carbonyl derivative of 5, showed slightly
lower anti-HIV activity than
1
(IR ¼ 51.8%), but did not show a dose-
dependent relationship upon further
screening. Compound 14, a 3b-configur-
ation isomer of 5, showed a slightly
stronger inhibitory effect (IR ¼ 23.4%)
than 5 (3a-configuration). This result
suggests that the anti-HIV activities of
epi-aleuritolic acid derivatives are influ-
enced by the C-3 hydroxyl configuration.
Modifying the 3b-configurational isomer
of epi-aleuritolic acid may enhance anti-
HIV activity. Compound 15, the sodium
salt of 5, had no anti-HIV activity.
The above results indicated that
compounds with acyl side chains at the
C-3 position showed significant activity.
The activity was affected by the type of

Journal of Asian Natural Products Research
519
PCC, CH3COCH3–CH2Cl2, 6h;
COOH
COOH
88%
5
PCC (pyridinium chlorochromate
HO
O
13
HOOC(CH3)2CCH2COOH,
DEAD, TPP, rt, 24h;
COOH
COOH
DEAD (dicthyl azodicarboxylat)
TPP (tetraphenylporphine)
14
HO
HO
5%NaOH, CH3COCH3–
CH2Cl2(1:1), rt, 30min
COOH
COONa
15
HO
HO
Scheme 3. Modification of epi-aleuritolic acid.
side chain linkage; the ester linkage and
3b-configuration resulted in the largest IR.
Incorporating a non-carboxylic linkage
gave a less active compound.
when compared with the other test
compounds (IR ¼ 5.2–7.5%).
3. Experimental
In our studies, compound 11, with a
20,20-dimethyl-glutaryl group, demon-
strated optimal activity and the highest
inhibitory rate. This compound represents
the discovery of another promising potent
anti-HIV agent through modification of a
suitable lead.
3.1 General experimental procedures
Melting points were measured on an XT-
5B micro-melting point apparatus (Beijing
Keyi Electro-Optic Instrument Plant, Beij-
ing, China). Optical rotations were
measured on a JASCO P-2000 polarimeter
(JASCO Corporation, Tokyo, Japan).
NMR spectra were obtained at 500 MHz
for 1H and 125 MHz for 13C on an INOVA-
500 NMR spectrometer using solvent peaks
as the references (Varian, Inc., Palo Alto,
CA, USA). ESI mass spectra were obtained
with an Agilent 1100 Series LC/MSD-Trap
(Agilent Technologies, Santa Clara, CA,
USA). Column chromatography was done
using silica gel (200–300 mesh, Qingdao
Marine Chemical, Inc., Qingdao, China).
Thin-layer chromatography (TLC) was
carried out on precoated silica gel GF254
plates. The spots were visualized under
UV light or by spraying with 10% sulfuric
acid in EtOH followed by heating.
4-Dimethylaminopyridine (DMAP), diethyl
We also investigated these compounds
for their inhibitory effect on nitric oxide
production.
The
dexamethasone
(1 £ 1026 M) positive control showed an
IR of 88.4%. Compounds 1, 3, 4, and 11
(1 £ 1025 M) significantly decreased the
NO production to the levels from 55.7% to
81.5% (see Table 2), with IC50 values of
3.40, 5.80, 5.90, and 7.10 mM. Compounds
8, 10, and 13 (1 £ 1025 M) had low
inhibitory rate values, ranging from
41.4% to 48.6%. Compounds 2, 5, 6, 7,
10, 12, and 15 (1 £ 1025 M) showed slight
inhibitory effects (IR ¼ 14.5–31.1%).
Compounds 9 and 14 (1 £ 1025 M)
showed almost no inhibitory activity

520
J.-B. Liu et al.
Table 2. Inhibitory rate values of NO production for the compounds in LPS-stimulated
macrophages.
NO inhibitory rate
(%)
MTT cell growth inhibitory rate
(%)
IC50
(mM)
Compound
Concentration
–
LPS
DEX
1
2
3
4
5
6
7
–
1 £ 1026
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
1 £ 1025
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
88.4
81.5
31.1
76.4
78.4
22.5
23.2
16.8
41.4
5.2
42.6
55.7
16.8
48.6
7.5
22.8
24.4
224.3
27.6
214.0
44.3
22.5
221.4
217.5
210.6
219.4
216.2
29.8
10.6
3.4
5.8
5.9
8
9
10
11
12
13
14
15
7.1
7.3
2.8
14.5
Note: Bold numbers represent active compounds and results.
azodicarboxylate
(DEAD),
3.3 General procedure for synthesis of
acyl derivatives (3 and 6–12)
pyridinium chlorochromate (PCC), and
esterification reagents were purchased from
Alfa Aesar (Tianjin, China). Other reagents
and solvents were from Beijing Chemical
Works (Beijing, China).
To a solution of succinic acid (100.0 mg)
in anhydrous chloroform at room tem-
perature was added 2 M oxalyl chloride
solution in anhydrous chloroform
(11 ml). The reaction mixture was stirred
for 48 h at 708C and then concentrated in
vaccum.
3.2 Synthesis of 3a-hydroxy-14-
taraxeren-28-acid (5)
A solution of myricarin A (1) or 3a-
hydroxy-taraxeren-28-acid (5) (20 mg),
A solution of myricarin A (50.0 mg) was
treated with 2% NaOH–MeOH at room
temperature for 24 h. A solution of 1 N
HNO3 was added to adjust pH to 5.0–6.0
until a white precipitate formed. The
mixture was extracted with chloroform,
and the organic layer was washed with
water until the rinses were neutral. The
organic layer was dried over anhydrous
Na2SO4 and was concentrated under
reduced pressure. The residue was chro-
matographed on a silica gel column to give
compound 5 (28 mg). Yield 75.9% [after
chromatography with CHCl3:MeOH
(100:1)]. ESI-MS: m/z 479 [M þ Na]þ,
455 [M 2 H]2.
a
catalytic amount of 4-(dimethyl-
amino)pyridine, and acetic anhydride in
anhydrous pyridine was heated overnight
at 1108C until the starting material was
no longer observed by TLC. The
reaction times ranged from 8 to 48 h.
The reaction mixture was diluted with
brine and extracted three times with
50 ml of chloroform. The organic layer
was dried over anhydrous Na2SO4 and
concentrated under reduced pressure.
The residue was chromatographed on a
silica gel column to give the product
[5,12].

Journal of Asian Natural Products Research
521
3.3.1 3a-(300,400-Diacetyl-trans-
cinnamoyl)-D-friedoolean-14-en-28-oic
acid (3)
0.95 (3H, overlapping, CH3CH2CH2-
COOZ), 0.95 (6H, s, 2 £ CH3), 0.92 (3H,
s, CH3), 0.90 (3H, s, CH3), and 0.82 (3H, s,
CH3). 13C NMR (CDCl3, 125 MHz) d 33.2
(C-1), 22.9 (C-2), 78.3 (C-3), 37.0 (C-4),
50.7 (C-5), 18.9 (C-6), 40.9 (C-7), 39.4 (C-
8), 49.2 (C-9), 38.2 (C-10), 17.5 (C-11),
33.6 (C-12), 37.6 (C-13), 161.1 (C-14),
116.9 (C-15), 31.6 (C-16), 51.8 (C-17),
41.6 (C-18), 35.6 (C-19), 29.5 (C-20), 33.9
(C-21), 30.9 (C-22), 28.1 (C-23), 22.0
(C-24), 15.6 (C-25), 26.5 (C-26), 22.8
(C-27), 184.4 (C-28), 32.1 (C-29), 28.9
(C-30), 173.5 (CH3CH2CH2COOZ), 36.8
(CH3CH2CH2COOZ), 18.9 (CH3CH2CH2-
COOZ), and 14.0 (CH3CH2CH2COOZ).
ESI-MS: m/z 549 [M þ Na]þ, 525
[M 2 H]2; HR-ESI-MS m/z 525.3941
[M 2 H]2 (calcd for C34H53O4, 525.3949).
A white amorphous powder; yield 79%
[after chromatography with CHCl3:MeOH
(50:1)], C43H58O8; 1H NMR (CDCl3,
500 MHz) d 7.60 (1H, d, J ¼ 16.0 Hz, H-
70), 7.40 (1H, d, J ¼ 2.0 Hz, H-20), 7.40
(1H, dd, J ¼ 8.0, 2.0 Hz, H-60), 7.21 (1H,
d, J ¼ 8.0 Hz, H-50), 6.43 (1H, d,
J ¼ 16.0 Hz, H-80), 5.57 (1H, dd, J ¼ 8.0,
3.0 Hz, H-15), 4.76 (1H, br s, H-3),
2.30 (6H, br s, 2 £ CH3CO), 0.99 (6H, s,
2 £ CH3), 0.98 (3H, s, CH3), 0.95 (6H,
s, 2 £ CH3), 0.93 (3H, s, CH3), 0.87 (3H, s,
CH3). ESI-MS m/z 725 [M þ Na]þ, 701
[M 2 H]2.
3.3.2 3a-Acetyl-14-taraxeren-28-acid
(6)
A white amorphous powder; yield 96%
[after chromatography with CHCl3:MeOH
(200:1)]. 13C NMR (CDCl3, 125 MHz) d
32.9 (C-1), 22.6 (C-2), 78.2 (C-3), 36.5
(C-4), 50.3 (C-5), 18.6 (C-6), 40.6 (C-7),
39.2 (C-8), 48.9 (C-9), 38.0 (C-10), 17.2 (C-
11), 33.4 (C-12), 37.3 (C-13), 160.8 (C-14),
116.7 (C-15), 31.3 (C-16), 51.5 (C-17), 41.3
(C-18), 35.3 (C-19), 29.3 (C-20), 33.6 (C-
21), 30.7 (C-22), 27.8 (C-23), 21.7 (C-24),
15.3 (C-25), 26.3 (C-26), 22.6 (C-27), 184.3
(C-28), 31.8 (C-29), 28.7 (C-30), 170.8
(CH3COOZ), and 21.3 (CH3COOZ). ESI-
MS: m/z 521 [M þ Na]þ, 497 [M 2 H]2.
3.3.4 3a-Isobutyryl-14-taraxeren-28-oic
acid (8)
A white amorphous powder; yield 43%
[after chromatography with CHCl3:MeOH
(100:1)]; C34H54O4; m.p. 137–1408C;
1
25
½aꢀD 221.1 (c ¼ 0.17, CHCl3). H NMR
(CDCl3, 500 MHz) d 5.55 (1H, dd,
J ¼ 8.0, 3.0 Hz, H-15), 4.61 (1H, br s, H-
3b), 2.56 [1H, m, (CH3)2CHCOOZ], 2.39
(1H, m, H-16b), 2.29 (1H, m, H-18a), 1.14
[6H, d, J ¼ 7.5 Hz, (CH3)2CHCOOZ],
0.97 (6H, s, 2 £ CH3), 0.95 (6H, s,
2 £ CH3), 0.92 (3H, s, CH3), 0.90 (3H, s,
CH3), 0.83 (3H, s, CH3). 13C NMR
(CDCl3, 125 MHz) d 33.0 (C-1), 22.5 (C-
2), 77.7 (C-3), 37.3 (C-4), 50.6 (C-5), 18.6
(C-6), 40.8 (C-7), 39.2 (C-8), 49.0 (C-9),
38.0 (C-10), 17.2 (C-11), 33.4 (C-12), 36.6
(C-13), 160.9 (C-14), 116.6 (C-15), 31.4
(C-16), 51.5 (C-17), 41.4 (C-18), 35.3 (C-
19), 29.3 (C-20), 33.7 (C-21), 30.7 (C-22),
27.9 (C-23), 21.8 (C-24), 15.3 (C-25), 26.3
(C-26), 22.6 (C-27), 184.6 (C-28), 31.9
(C-29), 28.7 (C-30), 176.5 [(CH3)2-
CHCOOZ], 34.5 [(CH3)2CHCOOZ],
19.2 [(CH3)2CHCOOZ]. ESI-MS m/z
549 [M þ Na]þ, 525 [M 2 H]2; HR-
3.3.3 3a-Butyryl-14-taraxeren-28-oic
acid (7)
A white amorphous powder; yield 63%
[after chromatography with CHCl3:MeOH
(200:1)], C34H54O4, m.p. 110–1128C;
1
25
½aꢀD 225.7(c ¼ 0.05, CHCl3); H NMR
(CDCl3, 500 MHz) d 5.54 (1H, br d,
J ¼ 8.0 Hz, H-15), 4.63 (1H, br s, H-3b),
2.40 (1H, dd, J ¼ 13.5, 8.0 Hz, H-16b),
2.29(1H, overlapping, H-18a), 2.29 (2H, m,
CH3CH2CH COOZ), 1.64 (2H, m, CH3-
2
CH CH2COOZ), 0.96 (6H, s, 2 £ CH3),
2

522
J.-B. Liu et al.
ESI-MS m/z 525.3923 [M 2 H]2 (calcd
for C34H53O4, 525.3949).
s, CH3), 0.83 (3H, s, CH3). 13C NMR
(CDCl3, 125 MHz) d 32.9 (C-1), 22.6 (C-2),
78.7 (C-3), 36.6 (C-4), 50.4 (C-5), 18.6 (C-
6), 40.7 (C-7), 39.2 (C-8), 48.9 (C-9), 38.0
(C-10), 17.2 (C-11), 33.4 (C-12), 37.3
(C-13), 160.8 (C-14), 116.6 (C-15), 31.4
(C-16), 51.3 (C-17), 41.3 (C-18), 35.3 (C-
19), 29.3 (C-20), 33.6 (C-21), 30.7 (C-22),
27.8 (C-23), 21.7 (C-24), 15.3 (C-25), 26.3
(C-26), 22.4 (C-27), 182.8 (C-28), 31.9 (C-
29), 28.7 (C-30), 171.7 (HOOCCH2CH2-
COOZ), 172.3 (HOOCCH2CH2COOZ),
29.1 (HOOCCH2CH2COOZ), 29.5
(HOOCCH2CH2COOZ). ESI-MS m/z 579
[M þ Na]þ.
3.3.5 3a-Isovaleryl-14-taraxeren-28-oic
acid (9)
A white amorphous powder; yield 42%
[after chromatography with CHCl3:MeOH
(100:1)], C35H56O4, m.p. 237–2398C;
1
25
½aꢀD 237.0 (c ¼ 0.05, CHCl3). H NMR
(CDCl3, 500 MHz) d 5.56 (1H, br d,
J ¼ 10.5 Hz, H-15), 4.64 (1H, br s, H-3b),
2.40 (1H, m, H-16b), 2.29 (1H, m, H-18a),
2.10 [1H, m, (CH3)2CHCH2COOZ], 2.18
[2H, d, J ¼ 5.5 Hz, (CH3)2CHCH2COOZ],
0.96 [6H, overlapping, (CH3)2CHCH2-
COOZ], 0.97 (6H, s, 2 £ CH3), 0.95 (6H,
s, 2 £ CH3), 0.93 (3H, s, CH3), 0.90 (3H, s,
CH3), and 0.83 (3H, s, CH3). 13C NMR
(CDCl3, 125 MHz) d 33.0 (C-1), 22.7 (C-
2), 77.9 (C-3), 37.3 (C-4), 50.4 (C-5), 18.6
(C-6), 40.7 (C-7), 39.2 (C-8), 48.9 (C-9),
38.0 (C-10), 17.2 (C-11), 33.4 (C-12), 36.5
(C-13), 160.9 (C-14), 116.6 (C-15), 31.4
(C-16), 51.4 (C-17), 41.4 (C-18), 35.3 (C-
19), 29.3 (C-20), 33.7 (C-21), 30.7 (C-22),
28.0 (C-23), 21.8 (C-24), 15.3 (C-25), 26.3
(C-26), 22.5 (C-27), 181.1 (C-28), 31.9 (C-
29), 28.7 (C-30), 172.7 [(CH3)2CHCH2-
COOZ], 43.9 [(CH3)2CHCH2COOZ],
3.3.7 3a-20,20-Dimethylsuccinyl-14-
taraxeren-28-acid (11)
A white amorphous powder; C36H56O6,
yield 23.4% [after chromatography with
CHCl3:MeOH (50:1)], m.p. 138–1408C;
1
25
½aꢀD 269.5 (c ¼ 0.04, CHCl3). H NMR
(C5D5N, 500 MHz) d 5.72 (1H, br d,
J ¼ 6.0 Hz, H-15), 4.90 (1H, br s, H-3b),
2.11 [1H, br d, J ¼ 14.5 Hz, HOOCC(CH3)2-
CH COOZ], 2.04 (1H, br d, J ¼ 13.5 Hz,
2
H-16b), 1.97 (1H, m, H-18), 1.50 [3H, s,
HOOCC(CH3)2CH2COOZ], 1.49 [3H,
s, HOOCC(CH3)2CH2COOZ], 1.14 (3H,
s, CH3), 1.08 (6H, s, 2 £ CH3), 0.99 (3H, s,
CH3), 0.94 (3H, s, CH3), 0.84 (3H, s, CH3),
0.80 (3H, s, CH3). 13C NMR (C5D5N,
125 MHz) d 32.9 (C-1), 23.4 (C-2), 78.6 (C-
3), 37.1 (C-4), 51.1 (C-5), 19.1 (C-6), 41.1
(C-7), 39.7 (C-8), 49.5 (C-9), 38.5 (C-10),
18.1 (C-11), 33.6 (C-12), 38.0 (C-13), 160.9
(C-14), 117.4 (C-15), 34.7 (C-16), 51.6
(C-17), 41.4 (C-18), 36.2 (C-19), 29.9
(C-20), 34.3 (C-21), 31.9 (C-22), 28.5
(C-23), 22.2 (C-24), 15.7 (C-25), 26.1
(C-26), 22.8 C-27), 180.6 (C-28), 32.7 (C-
29), 29.6 (C-30), 171.7 [HOOCC(CH3)2-
CH2COOZ], 179.6 [HOOCC(CH3)2CH2-
COOZ], 45.4 [HOOCC(CH3)2CH2COOZ],
42.5 [HOOCC(CH3)2CH2COOZ], 26.8
[HOOCC(CH3)2CH2COOZ]. ESI-MS m/z
583 [M 2 H]2, 619 [M þ Cl]2; HR-ESI-
25.8
[(CH3)2CHCH2COOZ],
22.4
[(CH3)2CHCH2COOZ]. ESI-MS m/z 563
[M þ Na]þ, 539 [M 2 H]2; HR-ESI-MS
m/z 539.4111 [M 2 H]2 (calcd for
C35H55O4, 539.4106).
3.3.6 3a-Succinyl-14-taraxeren-28-oic
acid (10)
A white amorphous powder; C34H52O6,
yield 16.4% [after chromatography with
CHCl3:MeOH (50:1)], m.p. 145–1478C;
1
25
½aꢀD 221.9 (c ¼ 0.59, CHCl3). H NMR
(CDCl3, 500 MHz) d 5.70 (1H, dd, J ¼ 8.0,
3.0 Hz, H-15), 4.63 (1H, br s, H-3b), 2.35
(1H, m, J ¼ 13.5, 8.0 Hz, H-16b), 2.30 (1H,
m, H-18a), 2.02 (4H, br s, HOOCCH2CH2-
COOZ), 1.01 (3H, s, CH3), 0.98 (6H, s,
2 £ CH3), 0.95 (6H, s, 2 £ CH3), 0.93 (3H,

Journal of Asian Natural Products Research
523
MS m/z 583.3984 [M 2 H]2 (calcd for
C36H55O6, 583.4004).
powder; C30H47O3Na; ESI-MS: m/z 457
[M þ H]þ, 479 [M þ Na]þ, 455 [M 2 H]2.
3.6 Oxidation of 3a-hydroxy-14-
taraxeren-28-oic acid (13)
3.3.8 3a-Benzoyl-14-taraxeren-28-acid
(12)
A mixture of 3a-hydroxy-taraxeren-28-oic
acid (5) (20.5 mg) and PCC (20.0 mg) in
anhydrous CH2Cl2 (5 ml) was stirred at
room temperature for 24 h. The solid was
filtered and discarded, and the filtrate
was evaporated [5]. The reaction product
was chromatographed on a silica gel
column to give 3-oxo-14-taraxeren-28-oic
acid (13) (18.0 mg). Yield 88% (after
chromatography with CHCl3). ESI-MS:
m/z 453 [M 2 H]2.
A white amorphous powder; yield 20%
[after chromatography with CHCl3:MeOH
(50:1)], C37H52O4. 1H NMR (CDCl3,
500 MHz) d 8.03 (2H, br d, J ¼ 7.5 Hz,
H-20, 60), 7.56 (1H, br t, J ¼ 7.5 Hz, H-40),
7.45 (2H, br t, J ¼ 7.5 Hz, H-30, 50), 5.59
(1H, dd, J ¼ 8.0, 3.0 Hz, H-15), 4.88 (1H,
br s, H-3b), 2.42 (1H, dd, J ¼ 14.0, 8.0 Hz,
H-16b), 2.30 (1H, dd, J ¼ 8.0, 3.0 Hz, H-
18), 1.25 (3H, s, CH3), 1.00 (3H, s, CH3),
0.98 (3H, s, CH3), 0.97 (3H, s, CH3), 0.95
(3H, s, CH3), 0.93 (6H, s, 2 £ CH3). ESI-
MS m/z 559 [M 2 H]2.
3.7 Preparation of 3b-hydroxy-14-
taraxeren-28-oic acid (14)
A mixture of 3a-hydroxy-taraxeren-28-oic
acid (5) (20.0 mg), triphenylphosphine
(3.0 eq), DEAD (2.0 eq), and 2,2-
dimethylsuccinic acid (2.0 eq) in dry
THF (5 ml) was stirred in an ice bath
under an argon atmosphere at room
temperature for 24 h [14]. The reaction
solution was evaporated, and the reaction
product was purified by silica gel column
chromatography using CHCl3 as eluent to
give 3b-hydroxy-taraxeren-28-oic acid
(14) (8.0 mg). Yield 40% (after chroma-
tography with CHCl3). ESI-MS: m/z 455
[M 2 H]2.
3.4 Preparation of the sodium salt of
myricarin A
A solution of myricarin A (20.0 mg) was
treated with 5% NaHCO3 in Me2CO:H2O
(1:1) at room temperature for 30 min. After
removal of Me2CO by evaporation, the
resulting aqueous solution was chromato-
graphed through an MCI-gel column and
washed with H2O to remove excess
amounts of NaHCO3 [13]. Subsequent
elution with MeOH gave the sodium salt of
myricarin A (4) (17.0 mg) as a white
powder; C39H52O6Na; m.p. . 3008C;
ESI-MS: m/z 641 [M þ Na]þ.
3.8 HIV-1 and NO inhibition activity of
obtained compounds
3.5 Preparation of sodium salt of 3a-
hydroxy-14-taraxeren-28-acid
The biological evaluation of HIV-1
inhibition for the epi-aleuritolic acid
derivatives was carried out according to
established methods [15]. The results are
represented as inhibitory rates and EC50
values in Table 1.
A solution of 5 (15.0 mg) was treated with
5% NaOH in Me2CO:H2O (1:1) at room
temperature for 30 min. After removing
Me2CO by evaporation, the resulting
aqueous solution was passed through an
MCI-gel column, and washed with H2O to
remove excess amounts of NaHCO3 [13].
Subsequent elution with MeOH gave the
sodium salt of 5 (15) (11.0 mg) as a white
The biological evaluation of NO
inhibition for the epi-aleuritolic acid
derivatives was carried out according to
established methods [16].

524
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phages were then seeded in 96-well plates
(Costar, Tewksbury, MA, USA) at a
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Acknowledgment
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