Efficient synthesis and biological evaluation of epiceanothic acid and related compounds

Article abstract of DOI:10.1016/j.bmcl.2010.11.004

Epiceanothic acid (1) is a naturally occurring, but very rare pentacyclic triterpene with a unique pentacyclic triterpene (PT) structure. An efficient synthesis of 1 starting from betulin (3) has been accomplished in 12-steps with a total yield of 10% in

Full text of DOI:10.1016/j.bmcl.2010.11.004

Bioorganic & Medicinal Chemistry Letters 21 (2011) 338–341
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis and biological evaluation of epiceanothic acid
and related compounds
Pu Zhang a, Li Xu a, Keduo Qian b, Jun Liu c, Luyong Zhang c, Kuo-Hsiung Lee b,d, , Hongbin Sun a,
⇑
⇑
a Center for Drug Discovery, College of Pharmacy, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, China
b Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
c Jiangsu Center for Drug Screening, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, China
d Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Epiceanothic acid (1) is a naturally occurring, but very rare pentacyclic triterpene with a unique penta-
cyclic triterpene (PT) structure. An efficient synthesis of 1 starting from betulin (3) has been accom-
plished in 12-steps with a total yield of 10% in our study. Compound 1 and selected synthetic
intermediates were further evaluated as anti-HIV-1 agents, inhibitors of glycogen phosphorylase (GP),
and cytotoxic agents. Compound 1 exhibited moderate HIV-1 inhibition. Most importantly, compound
Received 30 August 2010
Revised 27 October 2010
Accepted 1 November 2010
Available online 5 November 2010
5, with an opened A-ring, showed significant GP inhibitory activity with an IC50 of 0.21 lM, suggesting
a potential for development as an anti-diabetic agent. On the other hand, compound 12, with a closed
A-ring, showed potent cytotoxicity against A549 and MCF-7 human tumor cell lines, with IC50 values
Keywords:
Epiceanothic acid
Pentacyclic triterpene
Anti-HIV agents
Glycogen phosphorylase inhibitors
Cytotoxic agents
of 0.89 and 0.33
lM, respectively. These results suggest that the A-ring of PTs is an important pharma-
cophore that could be modified to involve different biological activities.
Ó 2010 Elsevier Ltd. All rights reserved.
Pentacylic triterpenes (PTs), a group of widespread natural com-
pounds, possess several intriguing biological activities, such as anti-
HIV, antitumor, anti-diabetic, anti-inflammatory, antibacterial,
antiviral, antiparasitic, hepatoprotective, wound healing, antioxi-
dant, antipruritic, antiangiogenic, antiallergic, and immunomodula-
tory activities.1–5 In recent years, PTs have been the focus of much
interest due to their significant therapeutic potentials. The anti-
HIV and antitumor activities of PTs have received the most attention,
as several synthetic PT derivatives have advanced into clinical trials
[e.g., PA-457 (DSB, Bevirimat, MPC-4326)6,7 and PA-1050040 for
AIDS therapy, and betulinic acid, CDDO, and CDDO-Me for cancer
therapy]. Our previous investigation also showed that PTs represent
a new class of glycogen phosphorylase (GP) inhibitors, which may be
a key contributing mode of action in their anti-diabetic activity.8–10
Epiceanothic acid (EA, 1) (Fig. 1) is a naturally occurring ceano-
thane-type PT isolated from the seeds of the traditional Chinese
medicine Ziziphus jujuba var. spinosa (Bunge) Hu and the stings of
Gleditsia sinensis Lam.11–13 It is reported to possess strong anti-
HIV-1 replication activity in HIV-1IIIB infected C8166 cell lines
H
H
OH
28
OH
H
H
H
H
R1
R2
O
HO
H
H
Epiceanothic acid (1): R1=β -COOH, R2 =β-OH
Ceanothic acid (2a): R1=α-COOH, R2=β-OH
Isoceanothic acid (2b): R1=β -COOH, R2=α-OH
Betulin (3)
Figure 1. Structures of epiceanothic acid (1) and related PT compounds (2a, 2b, 3).
of the 2-carboxylic acid (2a) and 3-hydroxy group (2b) in the
A-ring. Compound 2a was reported to possess anti-microbial and
cytotoxic activity,20–22 and its derivatives were found to be potent
cancer chemopreventive agents.23
Despite its obvious potential, only limited research has been
reported on 1, because it is very rare in nature. Therefore, it is
highly desirable to establish a reliable access to 1-analogs for bio-
logical evaluation. Herein, we report an efficient synthetic route to
1 in 12-steps with a total yield of 10% starting from betulin (3),
which is easily available at a low price. Compound 1 and the pen-
tacyclic triterpene intermediates24 were then evaluated for anti-
HIV-1, GP inhibitory, and cytotoxic activities.
(EC50 <0.064 l
g/mL).12,13 Compound 1 has two natural configura-
tional isomers, ceanothic acid (2a)14–18 and isoceanothic acid
(2b).19 Their structures differ from that of 1 only in the orientations
⇑
Corresponding authors. Tel.: +86 25 83271198; fax: +86 25 83271198 (H.B.S.);
tel.: +1 919 962 0066; fax: +1 919 966 3893 (K.H.L.).
E-mail addresses: khlee@unc.edu (K.-H. Lee), hbsun2000@yahoo.com (H. Sun).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.004

P. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 338–341
339
H
H
H
H
H
OTr
H
OTr
MeI, K2CO3
KOH, H2O2
Ref.24
HO
O
HOOC
HOOC
3
MeOH, reflux
70%
DMF, rt, 83%
H
H
5
4
H
H
H
H
H
H
H
H
OTr
OTr
t-BuOK
OTr
H
MeOOC
HO
MeOOC
NaBH4, THF
MeOOC
MeOOC
2
Toluene, reflux
EtOH, rt
62% 2 steps
O
H
H
H
8
7: a mixture rich in 2β−isomer 7b
6
H
H
H
H
H
H
H
OTr
PPTs, EtOH
H
OH
PCC
CH2Cl2, rt, 91%
MeOOC
AcO
MeOOC
AcO
Ac2O
pyridine, rt, 78%
reflux, 89%
9
10
H
H
H
H
H
OH
OH
H
H
H
H
H
NaClO2
NaH2PO4
H
MeOOC
HOOC
MeOOC
AcO
O
O
KOH
O
t-BuOH, THF
MeOH, reflux
69%
HO
AcO
H
H
isopentene, 0ºC-rt
96%
1
12
11
Scheme 1. Synthesis of epiceanothic acid (1) from betulin (3).
Table 1
As shown in Scheme 1, crude dione compound 4, which was
readily prepared from 3 in 67% yield,25 was treated with KOH
and H2O2 in MeOH26 to give the dicarboxylate compound 5
(70%). Methylation of 5 with iodomethane afforded compound 6
(83%). Dieckmann condensation of 6 (t-BuOK/toluene)27 gave
Anti-HIV-1 replication activity of PT compounds in HIV-1NL4–3 infected MT-4 cell lines
a
a
Compound
EC50
(lM)
IC50
(lM)
TI
PA-457
1
0.082
15.6
15.2
38.9
185.4
2.49
crude five-membered keto ester 7 as a mixture of 2a-ester (7a)
a
Results are averaged from two experiments. Compounds 3 and 9–12 were also
and 2b-ester (7b). This mixture could not be separated by column
chromatography, as isomerization could be observed during the
purification process. The major component was likely 7b, based
on a comparison with our related work on oleanane-type PTs
(unpublished data) and identification of the reduction product 8.
Reduction of the crude 7 in the presence of NaBH4 in THF and EtOH
gave 2b-methoxycarbonyl-3b-hydroxy compound 8 as the only
product (62% over two-steps). Acetylation of the 3b-hydroxy group
was accomplished with acetic anhydride in pyridine in 78% yield.
Deprotection of 9 with pyridinium p-toluenesulfonate (PPTs) in
EtOH gave primary alcohol 10 (89%). Oxidation of 10 with pyridini-
um chlorochromate (PCC) gave aldehyde 11 in good yield (91%),
which was further oxidized with NaClO2 and NaH2PO4 in a mixture
of t-BuOH/THF/2-methyl-2-butene25 to afford carboxylic acid 12 in
high yield (96%). Hydrolysis of 12 afforded epiceanothic acid (1) in
69% yield.
Compound 1, betulin (3), four selected pentacylic triterpene
intermediates (9–12) and PA-457 (as a positive control) were
tested in acutely HIV-1NL4–3 infected MT-4 cells, according to the
literature methods.28–31 However, 3 and 9–12 did not exhibit sig-
nificant antiviral activity in our assay. In addition, despite the pre-
vious report, compound 1 demonstrated only moderate anti-HIV
activity with an EC50 value of 15.6 lM and a therapeutic index
(TI) of 2.49 (Table 1). Esterification or acylation of the free carbox-
ylic acid and hydroxy functionalities in A-ring may be a contribut-
tested, but were not active.
ing factor to the loss of potency, as seen with 1 versus 9, 10, 11, and
12. Interestingly, 2a, which has a 2a-COOH rather than the
2b-COOH in 1, showed no anti-HIV-1 activity (unpublished data),
suggesting that the configuration of the 2-carboxylic acid group
has an impact on the anti-HIV-1 activity. Compared with the re-
ported data,12,13 our results also suggested that different HIV viral
strains may have different sensitivity to 1. The molecular mecha-
nisms underlying this phenomenon remain to be elucidated.
Type 2 diabetes mellitus is a severe disease with great economic
consequences. Hepatic glucose output is elevated in type 2 diabetic
patients, and GP is the enzyme that catalyzes glycogenolysis
(release of monomeric glucose from the glycogen polymer storage
form, resulting in abnormally high glucose production). GP inhibi-
tors lower glucose level acutely and chronically in diabetic animal
models, representing promising new hypoglycemic agents for the
treatment of type 2 diabetes mellitus.
In our continuing efforts to find potent GP inhibitors from PT
compounds, all of the related compounds (1, 3, 5–6, and 8–12) in
our study were evaluated for inhibitory activity against rabbit
muscle glycogen phosphorylase a (RMGPa).32 As described previ-
ously, the activity of RMGPa was measured by detecting the
amount of phosphates released from glucose-1-phosphates in the

340
P. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 338–341
Table 2
Noticeably, natural product 1 showed the greatest cytotoxicity
against gastric carcinoma BGC-823 with an IC50 value of 2.41 M.
The potency against this cell line was at least 10-fold higher than
against all the remaining four tested cancer cell lines, thus, demon-
strating selective sensitivity. Compound 12, with ester-protected
carboxylic acid and hydroxy groups in the A-ring relative to 1,
showed three-fold decreased activity with an IC50 of 7.64 lM. All
of the remaining PTs showed moderate to little cytotoxic activity
against BGC-823.
Inhibition of RMGPa by synthesized PT compounds
l
a
a
Compound
IC50
(l
M) SD
Compound
IC50
NIb
(lM) SD
3
5
6
8
9
41.5 3.2
0.21 0.1
2.87 0.1
20.1 1.1
15.2 0.7
10
11
12
53.8 4.9
46.4 3.6
194.1 17.5
75.3 6.6
1
Caffeinec
a
b
c
Values are means of three experiments.
NI = no inhibition.
Caffeine was used as a positive control.
Generally, the A549, MCF-7, and Hela cancer cell lines were
insensitive to the tested PT analogs. However, compound 12
showed selective potent cytotoxicity against A549 and MCF-7 with
IC50 values of 0.89 and 0.33 lM, respectively. Interestingly, the free
direction of glycogen synthesis.33 The assay results are summa-
rized in Table 2. Most of the newly synthesized PTs exhibited
inhibitory activity against RMGPa.
As shown in Table 2, opening of the A-ring of lupine-type PTs
significantly improved the GP inhibitory potency. The A-ring
opened compound 6 showed potent GP inhibitory activity with
carboxylic acid and hydroxy groups in the A-ring of 1 decreased its
cytotoxic activity against A549 and MCF-7 by more than 20-fold
compared with 12, which is opposite to the activity profile against
BGC-823. The activity of 12 against MCF-7 was slightly better than
that of adriamycin, suggesting that it merits further SAR and mech-
anism of action study.
In summary, an efficient access to epiceanothic acid (1) starting
from betulin (3) has been developed in 12-steps with an overall
yield of 10%. Because 3 is readily available, this preparation gives
practical access to the very rare natural PT epiceanothic acid and
enables further pharmacological research and drug development.
The synthesized PT derivatives were evaluated biologically as
anti-HIV-1 agents, inhibitors of glycogen phosphorylase, and cyto-
toxic agents. The results showed that 1 has moderate potency
against HIV-1NL4–3 virus strains. In addition, compound 5 with
two free carboxylic acids in an opened A-ring showed potent GP
an IC50 value of 2.87
(3, IC50 41.5 M). Another A-ring opened compound 5 showed the
most potent GP inhibitory activity with an IC50 value of 0.21 M.
lM, and was 14-fold more potent than betulin
l
l
Compound 5 was 14-fold more potent than its methylated parent
compound 6 and almost 200-fold more potent than 3. The trityl
ether compounds 8 and 9 were slightly more potent than 3, and
compounds 11 and 12 showed comparable potency to 3. However,
1 exhibited only weak GP inhibitory activity with an IC50 value of
194.1 lM. Overall, the A-ring was proven to be a very important
pharmacophore for modifying PTs’ GP inhibition activity, and the
preliminary SAR analysis showed that opening of the A-ring of lu-
pine-type PTs may enhance GP inhibition. The most potent GP
inhibitory activity with an IC50 value of 0.21 lM. To our knowl-
edge, it is the most potent PT derived GP inhibitor thus far. On
the other hand, compound 12, with a closed A-ring and protected
carboxylic acid and hydroxy groups, exhibited potent cytotoxic
activity against A549 and MCF-7 cancer cell lines with IC50 values
inhibitor, 5 (IC50 0.21
lM), merits further development as a poten-
tial anti-diabetic agent.
The cytotoxic activity of 1 and its synthetic intermediates (5, 6,
8–12) was tested in vitro using the MTT cytotoxicity assay,34,35 and
the results are summarized in Table 3. Five different cancer cell
lines were used including PC3 (human prostate cancer), A549 (hu-
man lung carcinoma), MCF-7 (human breast cancer), HeLa (human
epithelial carcinoma), and BGC-823 (human gastric carcinoma).
Adriamycin was used as the reference standard.
of 0.89 and 0.33 lM, respectively, which were comparable or bet-
ter than those of adriamycin. These results suggest that the A-ring
is an important pharmacophore for both GP inhibitory and cyto-
toxic activity of PTs. Different modifications on the A-ring could
change the bioactivity of epiceanothic acid derivatives. Further
mechanistic and pharmacologic studies of these compounds are
currently ongoing.
Overall, different cancer cell lines showed different sensitivity to
the PT compounds. The trityl ether compounds 8 and 9 were inactive
against the PC3 cell line, while aldehyde 11 (IC50 = 8.51
boxylic acid 12 (IC50 = 10.8 M) were more cytotoxic than the corre-
sponding primary alcohol 10 (IC50 = 66.3 M). With a free carboxylic
acid and hydroxy group in the A-ring, 1 (IC50 = 19.7 M) showed
slightly decreased activity compared with 12. The A-ring opened
compounds 5 and 6 exhibited more potent cytotoxicity against
lM) and car-
Acknowledgments
l
l
H.B. Sun acknowledges the following agencies for funding:
European Foundation for the Study of Diabetes (2007 Grant
Awards for Collaborative Diabetes Research between China and
Europe), National Natural Science Foundation of China (grants
30672523 and 90713037) and Ministry of Education of China
l
PC3 with IC50 values of 6.75 and 5.32 lM, respectively.
Table 3
Cytotoxic activity of synthesized PT compounds
a
Compound
IC50
(lM) SD for cancer cell lines
PC3
A549
MCF-7
HeLa
BGC-823
5
6
8
9
10
11
12
1
6.75 0.57
5.32 0.43
NAb
136.1 10.1
996.5 80.7
770.1 65.6
892.5 46.8
62.9 5.69
194.2 10.6
0.89 0.07
16.8 2.13
0.54 0.04
559.6 45.5
373.6 23.7
397.5 15.8
565.3 22.4
603.5 44.5
32.5 2.79
0.33 0.04
87.2 8.46
1.65 0.11
N/Ab
N/Ab
9.54 0.78
19.3 1.66
NAb
25.9 1.49
12.7 0.85
7.64 0.19
2.41 0.22
1.33 0.09
17.1 0.9
NAb
NAb
NAb
66.3 5.56
8.51 0.73
10.8 0.08
19.7 1.55
0.68 0.07
63.1 2.8
105.4 11.2
56.7 4.9
19.1 2.1
1.35 0.08
Adriamycinc
a
b
c
Values are means of three experiments.
NA = no activity.
Adriamycin was used as a positive control.

P. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 338–341
341
47.5, 47.7, 47.8, 48.9, 50.2, 51.1, 59.5, 59.8, 62.1, 83.6, 85.8, 109.4, 126.8, 127.7,
(grants 706030, 20050316008 and NCET-05-0495). Thanks are due
to Dr. Chin-Ho Chen at Duke University for anti-HIV replication
screening. This research was also partially supported by Grant
AI-077417 from the National Institute of Allergies and Infectious
Diseases awarded to K.H. Lee.
128.8, 144.5, 150.7, 170.5,171.6; ESI-MS m/z: 793.7 [M+Na]+; HRMS for
D
C
52H66O5+Na calcd 793.48025, found 793.47867. Compound 10: [
a]
À0.67 (c
0.06, CH2Cl2); IR (film, cmÀ1): 3355, 2945, 2866, 1749, 1458, 1376, 1242, 1159,
1026, 873, 739; 1H NMR (CDCl3) d 0.76 (3H, s), 0.97 (3H, s), 1.02 (3H, s), 1.11
(3H, s), 1.18 (3H, s), 1.67 (3H, s), 2.00 (3H, s), 0.76–1.67 (18H, m), 2.32–2.41
(1H, m), 2.44 (1H, d, J = 7.7 Hz), 3.33 (1H, d, J = 10.8 Hz), 3.57 (3H, s), 3.79 (1H,
d, J = 10.7 Hz), 4.58 (1H, d, J = 1.4 Hz), 4.67 (1H, d, J = 1.8 Hz), 5.17 (1H, d,
J = 7.6 Hz); 13C NMR (CDCl3) d 13.4, 14.8, 16.4, 17.8, 19.0, 19.1, 20.8, 23.8, 24.8,
27.3, 29.3, 29.8, 31.1, 34.0, 34.1, 37.1, 41.7, 42.4, 42.9, 47.76, 47.81, 47.85, 48.8,
References and notes
50.3, 51.2, 59.9, 60.6, 62.2, 83.6, 109.7, 150.4, 170.5, 171.7; HRMS for
1. Ovesna, Z.; Vachalkova, A.; Horvathova, K.; Tothova, D. Neoplasma 2004, 51,
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72, 1414.
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12. Li, W.; Zhao, W. 101062936, 2007, 15.
D
C
33H52O5+Na calcd 551.37070, found 551.37012. Compound 11: [
a
]
À4.67 (c
0.03, CH2Cl2); IR (film, cmÀ1): 2946, 2867, 1747, 1452, 1377, 1360, 1241, 1061,
1042, 738; 1H NMR (CDCl3) d 0.81 (3H, s), 0.91 (3H, s), 0.97 (3H, s), 1.10 (3H, s),
1.18 (3H, s), 1.69 (3H, s), 2.00 (3H, s), 0.85–2.11 (20H, m), 2.44 (1H, d,
J = 7.5 Hz), 2.79–2.89 (1H, m), 3.58 (3H, s), 4.63 (1H, s), 4.75 (1H, s), 5.17 (1H, d,
J = 7.5 Hz) 9.68 (1H, s); 13C NMR (CDCl3) d 13.5, 14.3, 16.3, 17.8, 19.00, 19.04,
20.9, 23.7, 25.1, 29.0, 29.3, 30.0, 31.1, 33.4, 34.2, 38.5, 41.7, 42.4, 42.8, 47.6,
47.9, 48.2, 50.4, 51.2, 59.3, 60.0, 62.2, 83.6, 110.3, 149.6, 170.5, 171.6, 206.6;
ESI-MS m/z: 549.0 [M+Na]+; HRMS for C33H50O5+Na calcd 549.35505, found
549.35424. Compound 12: [
D
a]
À10.67 (c 0.09, CH3OH); IR (film, cmÀ1): 3346,
2948, 2868, 1748, 1701, 1457, 1377, 1241, 1194, 1158, 1029, 736, 638; 1H NMR
(C5H5N) d 0.87 (3H, s), 1.03 (3H, s), 1.06 (3H, s), 1.15 (3H, s), 1.37 (3H, s), 1.76
(3H, s), 2.03 (3H, s), 1.19–1.95 (16H, m), 2.23–2.28 (2H, m), 2.60–2.76 (2H, m),
2.80 (1H, d, J = 7.7 Hz), 3.46–3.53 (1H, m), 3.58 (3H, s), 4.73 (1H, s), 4.91 (1H, s),
5.47 (1H, d, J = 7.7 Hz); 13C NMR (C5H5N) d 13.8, 14.6, 16.5, 17.8, 19.0, 19.2,
20.4, 24.2, 25.4, 30.2, 30.7, 31.0, 32.7, 34.3, 37.3, 38.1, 41.6, 42.3, 42.8, 47.6,
47.9, 49.5, 50.4, 50.8, 56.3, 59.9, 62.0, 83.8, 109.7, 150.9, 170.1, 171.5, 178.5;
ESI-MS m/z: 565.0 [M+Na]+; HRMS for C33H50O6ÀH calcd 541.35346, found
D
541.35249. Compound 1 (Epiceanothic acid): [
a]
À14.5 (c 0.076, CH3OH) [lit.4,
13. Li, W.-H.; Zhang, X.-M.; Tian, R.-R.; Zheng, Y.-T.; Zhao, W.-M.; Qiu, M.-H. J. Asian
Nat. Prod. Res. 2007, 9, 551.
D
[a
]
À16.3 (c 0.08, CH3OH)]; IR (film, cmÀ1): 3476, 2951, 2868, 1696, 1643,
1459, 1377, 1320, 1237, 1187, 1058, 884; 1H NMR (C5H5N) d 1.08 (3H, s), 1.13
(3H, s), 1.15 (3H, s), 1.20 (3H, s), 1.67 (3H, s), 1.74 (3H, s), 1.13–2.22 (18H, m),
2.60–2.64 (1H, m), 2.73–2.80 (1H, m), 2.89 (1H, d, J = 7.2 Hz), 3.43–3.49 (1H,
m), 4.66 (1H, d, J = 7.4 Hz), 4.69 (1H, s), 4.86 (1H, s); 13C NMR (C5H5N) d14.6,
15.0, 16.9, 18.4, 19.5, 19.9, 24.6, 25.9, 30.5, 31.2, 32.1, 33.0, 34.8, 37.6, 38.5,
42.0, 42.9, 43.1, 47.8, 48.1, 49.8, 51.1, 56.5, 62.7, 63.1, 83.1, 110.0, 151.1, 175.7,
178.8; ESI-MS m/z: 485.0 [MÀH]À; HRMS for C30H46O5ÀH calcd 485.32725,
found 485.32683.
14. Boyer, J. P.; Eade, R. A.; Locksley, H.; Simes, J. J. H. Aust. J. Chem. 1958, 11, 236.
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17. Roitman, J. N.; Jurd, L. Phytochemistry 1978, 17, 491.
18. Sharma, S. C.; Kumar, R. Pharmazie 1983, 38, 65.
19. Merkuza, V. M.; Mascaretti, O. A.; Crohare, R.; Ruveda, E. A. Phytochemistry
1971, 10, 908.
20. Lee, S.-S.; Chen, W.-C.; Huang, C.-F.; Su, Y. J. Nat. Prod. 1998, 61, 1343.
21. Jou, S.-J.; Chen, C.-H.; Guh, J.-H.; Lee, C.-N.; Lee, S.-S. J. Chin. Chem. Soc. (Taipei,
Taiwan) 2004, 51, 827.
25. Hao, J.; Zhang, P.; Wen, X.; Sun, H. J. Org. Chem. 2008, 73, 7405.
26. Urban, M.; Sarek, J.; Klinot, J.; Korinkova, G.; Hajduch, M. J. Nat. Prod. 2004, 67,
1100.
22. Suksamrarn, S.; Panseeta, P.; Kunchanawatta, S.; Distaporn, T.; Ruktasing, S.;
Suksamrarn, A. Chem. Pharm. Bull. 2006, 54, 535.
27. Konoike, T.; Takahashi, K.; Kitaura, Y.; Kanda, Y. Tetrahedron 1999, 55, 14901.
28. HIV-1NL4–3 replication inhibition assay in MT-4 lymphocytes:
A previously
23. Nakagawa-Goto, K.; Yamada, K.; Taniguchi, M.; Tokuda, H.; Lee, K.-H. Bioorg.
Med. Chem. Lett. 2009, 19, 3378.
described HIV-1 infectivity assay was used.29–31 A 96-well microtiter plate
was used to set up the HIV-1NL4–3 replication screening assay. NL4–3 variants at
a multiplicity of infection (MOI) of 0.01 were used to infect MT-4 cells. Culture
supernatants were collected on day four post-infection for the p24 antigen
capture using an ELISA kit from ZeptoMetrix Corporation (Buffalo, NY).
29. Huang, L.; Ho, P.; Lee, K. H.; Chen, C. H. Bioorg. Med. Chem. Lett. 2006, 14, 2279.
30. Yu, D. L.; Sakurai, Y.; Chen, C. H.; Chang, F. R.; Huang, L.; Kashiwada, Y.; Lee, K.
H. J. Med. Chem. 2006, 49, 5462.
24. All newly synthesized compounds provided satisfactory MS, 1H NMR, and 13C
NMR spectra without any discernable impurities. Selected analytical and
D
spectroscopic data are shown as follows. Compound 5: [
a
]
+25.0 (c 0.18,
CH2Cl2); IR (film, cmÀ1): 2950, 2869, 1714, 1693, 1560, 1489, 1449, 1265, 1228,
1063, 883, 743, 706, 632; 1H NMR (CDCl3) d 0.51 (3H, s), 0.86 (3H, s), 0.89 (3H,
s), 1.16 (3H, s), 1.24 (3H, s), 1.64 (3H, s), 0.96–2.63 (23H, m), 2.92 (1H, d,
J = 8.9 Hz), 3.13 (1H, d, J = 8.8 Hz), 4.52 (1H, s), 4.57 (1H, s), 7.21–7.33 (9H, m),
7.48–7.51 (6H, m); 13C NMR (CDCl3) d 14.8, 15.9, 19.1, 19.3, 20.7, 21.2, 25.0,
27.0, 29.4, 29.9, 30.1, 33.7, 35.2, 37.1, 40.5, 40.9, 41.8, 42.9, 45.7, 47.6, 47.8,
48.4, 48.9, 59.5, 85.9, 109.5, 126.8, 127.7, 128.8, 144.5, 150.8, 178.3, 187.3; ESI-
31. Qian, K.; Yu, D.; Chen, C. H.; Huang, L.; Morris-Natschke, S. L.; Nitz, T. J.;
Salzwedel, K.; Reddick, M.; Allaway, G. P.; Lee, K. H. J. Med. Chem. 2009, 52,
3248.
MS m/z: 753.5 [M+Na]+; HRMS for C49H62O5+Na calcd 753.44895, found
32. Enzyme assay: The inhibitory activity of the test compounds against rabbit
D
753.4505. Compound 6: [a]
À5.0 (c 0.14, CH2Cl2); IR (film, cmÀ1): 2947, 2868,
muscle glycogen phosphorylase
a (GPa) was monitored using microplate
reader (BIO-RAD) based on the published method.1 In brief, GPa activity was
measured in the direction of glycogen synthesis by the release of phosphate
from glucose-1-phosphate. Each test compound was dissolved in DMSO and
diluted at different concentrations for IC50 determination. The enzyme was
added into 100 L of buffer containing 50 mM Hepes (pH 7.2), 100 mM KCl,
2.5 mM MgCl2, 0.5 mM glucose-1-phosphate, 1 mg/mL glycogen and the test
compound in 96-well microplates (Costar). After the addition of 150 L of 1 M
HCl containing 10 mg/mL ammonium molybdate and 0.38 mg/mL malachite
green, reactions were run at 22 °C for 25 min, and then the phosphate
absorbance was measured at 655 nm. The IC50 values were estimated by fitting
the inhibition data to a dose-dependent curve using a logistic derivative
equation.
1726, 1597, 1449, 1275, 1151, 1064, 764, 706, 632; 1H NMR (CDCl3) d 0.52 (3H,
s), 0.83 (3H, s), 0.92 (3H, s), 1.21 (3H, s), 1.22 (3H, s), 1.62 (3H, s), 0.76–1.74
(16H, m), 2.11–2.40 (7H, m), 2.90 (1H, d, J = 8.8 Hz), 3.12 (1H, d, J = 8.9 Hz), 3.59
(3H, s), 3.61 (3H, s), 4.50 (1H, s), 4.56 (1H, s), 7.19–7.32 (9H, m), 7.46–7.49 (6H,
m); 13C NMR (CDCl3) d 14.7, 15.9, 19.1, 19.6, 20.8, 21.8, 23.8, 25.3, 27.0, 27.8,
29.9, 30.0, 33.1, 35.2, 37.5, 40.5, 41.6, 41.8, 41.9, 42.9, 46.3, 47.6, 47.7, 48.4,
48.8, 50.7, 51.7, 59.6, 85.9, 109.3, 126.8, 127.7, 128.8, 144.5, 150.8, 171.9,
179.9; ESI-MS m/z: 781.5 [M+Na]+; HRMS for C51H66O5+Na calcd 781.48025,
D
found 781.48348. Compound 8: [
a]
À12.3 (c 0.105, CH2Cl2); IR (film, cmÀ1):
3456, 2945, 2866, 1733, 1632, 1597, 1449, 1375, 1160, 1063, 1030, 775, 741,
706, 632; 1H NMR (CDCl3) d 0.48 (3H, s), 0.88 (3H, s), 0.91 (3H, s), 1.00 (3H, s),
1.05 (3H, s), 1.63 (3H, s), 0.79–1.68 (18H, m), 2.17–2.32 (4H, m), 2.69 (1H, br s),
2.91 (1H, d, J = 8.9 Hz), 3.11 (1H, d, J = 8.8 Hz), 3.65 (3H, s), 4.01 (1H, d,
33. Martin, W. H.; Hoover, D. J.; Armento, S. J.; Stock, I. A.; McPherson, R. K.;
Danley, D. E.; Stevenson, R. W.; Barrett, E. J.; Treadway, J. L. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 1776.
J = 7.3 Hz), 4.51 (1H, s), 4.57 (1H, s), 7.19–7.32 (9H, m), 7.46–7.49 (6H, m); 13
C
NMR (CDCl3) d 13.9, 14.7, 16.5, 18.0, 18.8, 19.2, 23.4, 24.8, 27.1, 30.0, 30.2, 31.7,
34.1, 35.2, 37.0, 41.6, 42.7, 43.0, 47.6, 47.7, 48.9, 49.3, 50.2, 51.4, 59.5, 60.1,
62.0, 82.4, 85.8, 109.4, 126.8, 127.7, 128.8, 144.5, 150.7, 174.2; HRMS for
34. MTT assay: The MTT assay was carried out as described previously.31 Cells were
seeded in 96-well plates and incubated in the CO2 incubator at 37 °C. When the
cells adhered, compounds at different concentrations (0.0001, 0.0005, 0.001,
0.005, 0.01, 0.05 or 0.1 mmol LÀ1) were added to every well. After incubation
D
C
50H64O4+Na calcd 751.46968, found 751.47246. Compound 9: [
a]
À16.7 (c
0.06, CH2Cl2); IR (film, cmÀ1): 3382, 2946, 2866, 1747, 1449, 1375, 1240, 1153,
1063, 764, 705, 632; 1H NMR (CDCl3) d 0.50 (3H, s), 0.80 (3H, s), 0.88 (3H, s),
1.08 (3H, s), 1.11 (3H, s), 1.62 (3H, s), 1.99 (3H, s), 0.76–1.67 (18H, m), 2.16–
2.23 (3H, m), 2.40 (1H, d, J = 7.7 Hz), 2.90 (1H, d, J = 8.8 Hz), 3.12 (1H, d,
J = 8.8 Hz), 3.54 (3H, s), 4.50 (1H, s), 4.56 (1H, s), 5.14 (1H, d, J = 7.5 Hz), 7.19–
7.32 (9H, m), 7.46–7.49 (6H, m); 13C NMR (CDCl3) d 13.3, 14.7, 16.2, 17.8, 19.0,
19.1, 20.8, 23.7, 24.7, 27.1, 29.9, 30.2, 31.1, 34.0, 35.2, 37.0, 41.4, 42.3, 42.6,
for another 48 h, 20 lL MTT (5%) was added to each well and incubated for an
additional 4 h. The viable cells were stained with MTT and scanned with an
electrophotometer at 570 nm. Each concentration treatment was done in
triplicate wells. The IC50 values were estimated by fitting the inhibition data to
a dose-dependent curve using a logistic derivative equation.
35. INVITTOX. Protocol 17, 1980.