Discovery, synthesis, and structure-activity relationships of 20(S)-protopanaxadiol (PPD) derivatives as a novel class of AMPKα2β 1γ1 activators

Article abstract of DOI:10.1016/j.ejmech.2014.04.010

Adenosine 5′-monophosphate-activated protein kinase (AMPK) has been demonstrated as a promising drug target due to its regulatory function in glucose and lipid metabolism. 20(S)-protopanoxadiol (PPD) was firstly identified from high throughput screening as a small molecule activator of AMPK subtype α2β1γ1. In order to enhance its potency on AMPK, a series of PPD derivatives were synthesized and evaluated. Structure-activity relationship study showed that the amine derivatives at the 24-position (groups I-VI) can improve the potency (EC₅₀: 0.7-2.3 μM) and efficacy (fold: 2.5-3.8). Among them, compounds 12 and 13 exhibited the best potency (EC ₅₀: 1.2 and 0.7 μM) and efficacy (fold: 3.7 and 3.8). Further study suggested the mechanism of AMPK activation may functioned at the allosteric position, resulting the inhibition of the lipid synthesis in HepG2 cell model.

Full text of DOI:10.1016/j.ejmech.2014.04.010

European Journal of Medicinal Chemistry 79 (2014) 340e349
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery, synthesis, and structureeactivity relationships of
20(S)-protopanaxadiol (PPD) derivatives as a novel class of
AMPK
a
2
b1g
1 activators
,
,
,
a,
*
Junhua Liu a 1, Dakai Chen a 1, Peng Liu b, Mengna He a, Jia Li a, Jingya Li a , Lihong Hu
*
a Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
b School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China
a r t i c l e i n f o
a b s t r a c t
Adenosine 50-monophosphate-activated protein kinase (AMPK) has been demonstrated as a promising
drug target due to its regulatory function in glucose and lipid metabolism. 20(S)-protopanoxadiol (PPD)
was firstly identified from high throughput screening as a small molecule activator of AMPK subtype
Article history:
Received 13 January 2014
Received in revised form
30 March 2014
Accepted 4 April 2014
Available online 4 April 2014
a
2b
1
g
1. In order to enhance its potency on AMPK, a series of PPD derivatives were synthesized and
evaluated. Structureeactivity relationship study showed that the amine derivatives at the 24-position
(groups IeVI) can improve the potency (EC50: 0.7e2.3 M) and efficacy (fold: 2.5e3.8). Among them,
compounds 12 and 13 exhibited the best potency (EC50: 1.2 and 0.7 M) and efficacy (fold: 3.7 and 3.8).
m
m
Keywords:
20(S)-Protopanoxadiol
AMPK
AMPK heterotrimer
Activators
Further study suggested the mechanism of AMPK activation may functioned at the allosteric position,
resulting the inhibition of the lipid synthesis in HepG2 cell model.
Ó 2014 Elsevier Masson SAS. All rights reserved.
a2b1g1
Structureeactivity relationship
1. Introduction
two first-class antidiabetic drugs, biguanides and thiazolidine-
diones, were proved to be related to the AMPK activation [11,12].
Adenosine 50-monophosphate-activated protein kinase (AMPK)
highly-reserved serine/threonine protein kinase that is
Although the treatment with the root of Panax ginseng C.A.
Meyer (Araliaceae) has been practiced as an omnipotent remedy in
eastern Asia over 2000 years [13], it is still questionable that eating
ginseng itself relates to cure any diseases from the viewpoint of
modern medicine. However, ginsenosides, the major active con-
stituents in ginseng, appear to be mostly responsible for the ac-
tivities of ginseng, including antihyperlipidemic [14,15],
antidiabetic effects [16], antioxidation [17], immunostimulation
[18], antistress [19], anticancer [20], as well as the learning and
memory ameliorating agents in the central nervous system [21].
Specific to the antihyperlipidemic and antidiabetic effects, ginse-
nosides Rb1, Re, Rg3, Rb2, Rh2, and compound K have displayed
positive effects in animal models [14,22e24]; besides, some studies
argued that these effects are ascribed to the AMPK activation
[15,24e26]. Since 20(S)-protopanoxadiol (PPD, Fig. 1) is the main
metabolite of protopanoxadiol type ginsenosides in rats [27], we
suspected whether PPD could have positive effect on AMPK
pathway. Our ensuing screen verified that PPD activated the AMPK
is
composed by a catalytic
and . These subunits include multiple isoforms (
2,
a
a
subunit and two regulatory subunits,
1, 2; 1, 2;
b
g
a
a
b
b
g
1,
g
g
3) and are expressed in various tissues and subcellular loca-
tions [1,2]. The major function of AMPK is the cellular energy
regulation and maintains the energy balance in the whole body.
Upon activation, AMPK stimulates ATP generation, glucose uptake
and fatty acid oxidation, while it simultaneously down-regulates
the ATP-consuming anabolic pathways and inhibits the syntheses
of hepatic triacylglycerol, cholesterol, protein and glycogen [3e7].
Due to its central role in the regulation of body weight, systemic
glucose homeostasis, lipid metabolism, and mitochondrial
biogenesis [8,9], recent study postulated that whether the activa-
tion of AMPK would be a promising therapeutics against type 2
diabetes as well as other metabolic syndromes [10]. For example,
heterotrimer a2b1g1 by 3.2 fold with an EC50 of 2.5 mM. Based on
this result and our previous finding in new AMPK activators from
medicinal plants [28,29], we herein report a series of PPD de-
rivatives and their biological evaluations on AMPK related pathway.
* Corresponding authors.
E-mail addresses: jyli@mail.shcnc.ac.cn (J. Li), lhhu@simm.ac.cn (L. Hu).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.04.010
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

J. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 340e349
341
filterassays [33]. We also got similar EC50 valuesofA769662andAMP
on AMPK activation using parallel HTRF and radioactive filter assays
(See supporting information s1). However, HTRF assays showed
higher sensitivity (For A769662 and AMP, AMPK activity increased
4.3-fold and 3.2-fold in HTRF assays, but 3.5-fold and 2-fold in Filter
assays) and better adaptability in 384-wells plate high-throughput
screening. Therefore, HTRF assay was employed in the evaluation
of PPD derivatives (1e31). The EC50 and efficacy values (fold) were
shown in Table 1. PPD (1) activated the AMPK heterotrimer
a2b1g1
by 3.2 fold with an EC50 of 2.5 M. PPD derivatives, including the
m
reduced product 4, lactone 5, and amide-substituted PPD derivatives
(6e8) had no activity. On contrary, the major metabolites 2e3 and
amine-substituted PPD 9-31 showed activation of the AMPK heter-
otrimer
(a) the 24,25-double bond is significant for the activation of the
AMPK heterotrimer 1, since the saturated product 4 totally lost
a2b1g1. The screening results suggested the following SAR:
a2b1g
Fig. 1. The major metabolites of 20(S)-protopanaxadiol in humans.
activity; (b) amide and lactone substitutions at the 23 position (6e8)
also resulted in activity loss; (c) amine substitutions, including
aliphatic acyclic amine (group I, 9e13), aliphatic cyclic amine (group
II, 14), heterocycle-aliphatic amine (group III, 15e16), polar aliphatic
amine (group IV, 17), and aromatic substituted aliphatic amine
2. Design and syntheses
On the basis of previous literature [30e32], the predominant
metabolic pathway of PPD was the epoxidation and cyclization of
the 24,25-double bond to 2 and 3 (Fig. 1). Therefore, the 24,25-
double bond was primarily investigated in two methods. The ma-
jor metabolites of 20(S)-protopanaxadiol (2 and 3) were firstly
(group VI, 19e31) improved the potency (0.7e2.3 vs 3.2
efficacy (fold: 2.5e3.8 vs 2.5); (d) aniline (group V) at the 24 position
(18) slightly decreased the potency (EC50: 5.3 vs 3.2 M) but
mM) and
m
enhanced efficacy (fold: 3.1 vs 2.5); (e) the electrical properties of the
aromatic ring (group VI,19e31) had no significant impact on activity.
Among them, the most potent compounds (12 and 13) in both
potency (EC50: 1.2 and 0.7 mM) and efficacy (fold: 3.7 and 3.8) were
selected for further investigation.
synthesized to test their effects on AMPKa2b1g1 activation
(Scheme 1). Second, the double bond reduction product 4, amide-
substituted PPD derivatives (6e8), and amine-substituted PPD
derivative (9) were synthesized to block the predominant meta-
bolic pathway (Scheme 1). The preliminary results showed that
3.2.2. Activation of AMPK by 12 and 13 in HepG2 cells
amine-substituted PPD derivative 9 showed better potency (EC50
1.5 M) and efficacy (fold: 3.3) on the activation of AMPK hetero-
trimer 1 than PPD (EC50: 3.2 M, fold: 2.5). We then designed
:
After observing the allosteric activation of AMPK, we examined
whether these compounds were able to activate AMPK in HepG2
cells and consistent with the profile shown on molecular level. The
results exhibited the increased phosphorylation level of both AMPK
and acetyl-CoA carboxylase (ACC) in a dose-dependent manner
after the treatment of 12 and 13 for 1 h in HepG2 cells (Fig. 2A).
Compounds the 12 and 13 presented a more potent activation of
AMPK than PPD, which is consistent with their EC50 values of AMPK
activation. The cellular results also showed that the AMPK activa-
m
a2
b1
g
m
a set of amine-substituted PPD derivatives 9e31 (Scheme 2) to
further understand the structureeactivity relationships. According
to the chemical structure, the set is divided into six groups,
including aliphatic acyclic amine (group I), aliphatic cyclic amine
(group II), heterocycle-aliphatic amine (group III), polar aliphatic
amine (group IV), aromatic amine (group V), and aromatic
substituted aliphatic amine (group VI) groups.
tion effects occurred at 20
mM, whereas, 13 had a higher level of
AMPK phosphorylation than 12 and A769662 at 40
m
M (Fig. 2B).
3. Results and discussion
The equilibrium of AMP and ATP in cell is regulated by mito-
chondria and reflected by the mitochondrial membrane potential.
Elevated AMP level leads to surplus AMP binding to the allosteric
3.1. Chemistry
site of AMPK
g subunit, which is followed by AMPK activation. In
The synthetic route of PPD derivatives (2e8) has been shown in
Scheme 1. Compounds 2 and 3 were obtained via the epoxidation
with mCPBA. The relative configuration of 2 and 3 were elucidated
by NMR spectroscopy with comparison of literature data [25].
Compound 4 was prepared by catalytic hydrogenation, while
lactone 5 was achieved by oxidative cleavage and carboxylation
with KMnO4 and NaIO4. Continuous aminolysis afforded the cor-
responding amide-substituted PPD derivatives 6e8. Amine-
substituted PPD derivatives 9e31 were conceived by ozonolysis
and reducts amination with different amines.
our assay, PPD, 12 and 13 showed little influence on mitochondrial
membrane potential (Fig. 2C), indicating that these compounds
may directly binding to the AMPK allosteric position rather than
affect mitochondrial function and the AMP/ATP ratio.
3.2.3. 12 and 13 inhibit lipid synthesis in HepG2 cells
As ACC plays a key role in the regulation of fatty acid syntheses
(FAS), activation of AMPK can down-regulated the level of malonyl-
CoA by ACC phosphorylation and circuitously inhibited FAS. Alter-
natively speaking, the inhibition of lipid synthesis is correlated
with the increased ACC phosphorylation. Since 12 and 13 were able
to activate AMPK and promote ACC phosphorylation, we next
confirmed they had better FAS inhibition than the parent com-
pound 1 in HepG2 cells as well (Fig. 2D).
3.2. Biochemistry
3.2.1. Allosteric activation of AMPK
compounds
a2b1g1 by PPD serial
Two major AMPK assays were reported, homogeneous time
resolved fluorescence (HTRF) and radiometric AMPK filter assay. As
shown in the literature, HTRF assay is based on antigeneantibody
interaction and its AMP titration curves were almost identical to the
4. Conclusion
In summary, thirty PPD derivatives were synthesized and eval-
uated on the activation of AMPKa2b1g1. Improved AMPK activation

342
J. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 340e349
Scheme 1. Synthesis of compounds 2e9. Reagents and conditions: (a) mCPBA, CH2Cl2, rt; (b) Pd/C, H2, CH3OH, rt; (c) Ac2O, Py, rt; (d) KMnO4, NaIO4, acetone, rt; (e) RNH2, THF, rt; (f)
2 N NaOH, THF; (g) O3, CH2Cl2, -78 ꢁC; (h) isopropylamine, NaBH(OAc)3, 0 ꢁC.
level was observed by introducing an amine group at 24 position. A
detailed SAR studies suggested that aliphatic acyclic amine (group I,
9e13), aliphatic cyclic amine (group II, 14), heterocycle-aliphatic
amine (group III, 15, 16), polar aliphatic amine (group IV, 17), and
aromatic substituted aliphatic amine (group VI, 19e31) enhanced
the activation of AMPK heterotrimer. Among all tested compounds,
compounds 12 and 13 exhibited the best potency (EC50: 1.2 and
Shanghai Chemical Reagent Co. and used without further purifi-
cation. Nuclear magnetic resonance (NMR) spectroscopy was per-
formed on Bruker AMX-300 or AMX-400 NMR (IS as TMS).
Chemical shifts were reported in parts per million (ppm,
d)
downfield from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broad (br). HRESIMS were determined on a Micromass Q-
Tif Global mass spectrometer and ESIMS were run on a Bruker
Esquire 3000 Plus Spectrometer. All reactions were monitored by
thin-layer chromatography (TLC) on HSGF254 silica gel plates
0.7
mM) and efficacy (fold: 3.7 and 3.8). Further cellular experi-
ments proved 12 and 13 obviously promoted AMPK activation and
sequentially inhibited lipid synthesis in HepG2 cells. The unaffected
mitochondrial potential suggested that their function may be
possibly introduced to allosteric site of AMPK.
(150ꢀ200
mm thickness; Yantai Huiyou Co., China). Ozone was
produced with a BGF-YQ ozone generator (2.0 L/min O2, 100 V;
Beijing ozone Co., China). Anhydrous solvents were purchased from
commercial suppliers.
5. Experimental section
5.1. Chemistry
5.1.2. General procedure for the synthesis of 2 and 3
To a stirred solution of 1 (0.10 g, 0.22 mmol) in CH2Cl2 (5 mL),
mCPBA (0.44 g, 0.26 mmol) was added. The resulting
mixture was stirred at room temperature for 6 h. After comple-
tion of the reaction (TLC monitoring, PE/EtoAc, 1/2, V/V, on silica
5.1.1. General information
All final compounds are >95% pure based on HPLC. The reagents
(chemicals) were purchased from Lancaster, Alfa Aesar, and

J. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 340e349
343
Scheme 2. Syntheses of amines derivatives 9e33. Reagents and conditions: (a) O3, CH2Cl2, e 78 ꢁC, (b) RNH2, NaBH(OAc)3, 0 ꢁC.
gel plate), the reaction mixture was diluted with CH2Cl2
5.1.3. General procedure for the synthesis of 4
(10 ml) and the mixture was washed by saturated Na2S2O3 so-
lution, saturated Na2CO3 solution, water, and brine. The
organic layer was dried over anhydrous magnesium sulfate and
the solvent removed under reduced pressure. The residue
was purified by column chromatography (silica gel 60, 70e
230 mesh ASTM, Merck) to afford 2 (41 mg, 40%) and 3 (46 mg,
45%).
Compound 1 (0.10 g, 0.22 mmol) was dissolved in methanol
(10 mL) followed by the addition of 10% PdeC (0.020 g) at ambient
temperature. The mixture was stirred under hydrogen at 1 atm for
3 h and then filtered, concentrated under reduced pressure to give
crude solid. The crude solid was purified by column chromatog-
raphy (silica gel 60, 70e230 mesh ASTM, Merck) to afford 96 mg
(95%) of 4 as a white powder.

344
J. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 340e349
Table 1
Allosteric activation of the AMPK heterotrimer a2b1g
1 by PPD derivatives (1e31).a
Cmpd
EC50
(
mM)
Fold
Cmpd
EC50
(
mM)
Foldb
1(PPD)
2
3
4
5
6
7
8
3.2 ꢂ 0.2
2.7 ꢂ 0.5
3.5 ꢂ 0.1
NAc
NA
NA
2.5
3.2
3.0
NA
NA
NA
NA
NA
3.3
3.2
3.2
3.7
3.8
3.3
Polar Group (IV)
Aromatic Group (V)
Aromatic-aliphatic Group (VI)
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.0 ꢂ 0.3
5.3 ꢂ 0.4
1.2 ꢂ 0.2
1.9 ꢂ 0.2
2.2 ꢂ 0.5
1.7 ꢂ 0.3
1.9 ꢂ 0.4
2.0 ꢂ 0.2
1.7 ꢂ 0.2
1.6 ꢂ 0.2
2.0 ꢂ 0.2
1.7 ꢂ 0.2
1.7 ꢂ 0.2
1.9 ꢂ 0.3
3.3
3.1
3.0
2.9
3.3
3.5
3.1
3.5
2.5
3.1
2.7
3.3
2.5
3.0
Major Metabolites of PPD
Reduction Product
Amide-substituted
PPD Derivatives
NA
NA
Aliphatic
Acyclic Group (I)
9
1.5 ꢂ 0.2
1.8 ꢂ 0.2
2.0 ꢂ 0.4
1.2 ꢂ 0.2
0.7 ꢂ 0.2
2.1 ꢂ 0.2
10
11
12
13
14
Aliphatic
Cyclic Group (II)
Heterocycle-aliphatic Group (III)
15
16
2.1 ꢂ 0.1
1.5 ꢂ 0.2
3.0
3.1
31
AMP
2.3 ꢂ 0.4
1.4 ꢂ 0.1
3.1
3.2
Positive control
a
See experimental section and supporting information S2.
Fold, defined as activation fold relative to negative control.
Not active, defined as Activation fold <1.5.
b
c
5.1.4. General procedure for the synthesis of 5
column chromatography on silica gel (PE/EtOAc, 6/1, V/V) to afford
A solution of 1 (2.0 g, 4.35 mmol) in pyridine (15 mL) was mixed
with Ac2O (4 mL), and the mixture was stirred at room temperature
for 6 h. After evaporation of excess reagent, the residue was sub-
jected to column chromatography on silica gel using PE/EtOAc (1/
10, V/V) to yield white powder (2.25 g, 4.13 mmol, 95%). To a so-
lution of the white powder (2.25 g, 4.13 mmol) in acetone (20 mL)
was added dropwise the mixture of KMnO4 (32.0 mg, 0.20 mmol),
and NaIO4 (2.21 g, 10.32 mmol) in acetone-H2O (7:3, 20 mL). After
the reaction mixture was stirred at room temperature for 20 h, the
solvent mixture was evaporated. The residue was purified by
1.27 g (60%) of 5 as a white solid.
5.1.5. General procedure for the synthesis of 6-8
A solution of 5 (0.10 g, 0.19 mmol) in THF (5 mL) was mixed with
the appropriate amine (RNH2, 0.21 mmol), and the mixture was
stirred at room temperature for 6 h. After completion of the reac-
tion (TLC monitoring, PE/EtoAc, 1/1, V/V, on silica gel plate), 2 N
NaOH solution (5 mL) was added, and the reaction mixture was
stirred at room temperature for 1 h. After evaporation of excess
reagent, the residue was diluted with CH2Cl2 (10 ml) and the
Fig. 2. The effects of PPD serial compounds in HepG2 cells. (A) Comparison of AMPK activation effect between PPD and its derivants in HepG2 cells after 1 h treatment. (B)
Concentration response of the effects on AMPK and ACC phosphorylation due to 12 and 13 in HepG2 cells after 1 h treatment. (C) 12 and 13 did not reduce mitochondrial membrane
potential in HepG2 cells after 1 h treatment (n ¼ 3). (D) PPD and its derivants inhibit lipid synthesis in HepG2 cells after 6 h incubation in HepG2 cells. D is refer to DMSO as negative
control and A is refer to A769662 (40
mM) as positive control (n ¼ 3). *, P < 0.05, **, P < 0.01, ***, P < 0.001 compared with the negative control.

J. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 340e349
345
mixture was washed by water, and brine. The organic layer was
5.1.10. (3S,8R,10R,12R,14R,17S)-4,4,8,10,14-Pentamethyl-17-((R)-2-
methyl-5-oxotetrahydrofuran-2-yl)hexadecahydro-1H-cyclopenta
[a]phenanthrene-3,12-diyl diacetate (5)
dried over anhydrous magnesium sulfate and the solvent removed
under reduced pressure. The residue was purified by column
chromatography (silica gel 60, 70e230mesh ASTM, Merck) to afford
the appropriate product (6e8).
The product was purified by flash chromatography (PE/EtOAc, 6/
1, V/V) to give a white powder (1.27 g, 60%). ESI(þ)-MS (m/z): 517.3
[MþH]þ; 1H NMR (400 MHz, CDCl3):
4.88 (td, J ¼ 10.7, 5.2 Hz, 1H),
d
5.1.6. General procedure for the synthesis of 9-31
4.49 (dd, J ¼ 11.2, 4.8 Hz, 1H), 2.70e2.50 (m, 2H), 2.31 (td, J ¼ 10.3,
4.9 Hz, 1H), 2.08 (s, 3H), 2.05 (s, 3H), 1.39 (s, 3H), 1.27 (s, 3H), 1.01 (s,
3H), 0.98 (s, 3H), 0.89 (s, 3H), 0.86 (s, 3H); 13C NMR (100 MHz,
Into a solution of PPD (1, 100 mg, 0.22 mmol) in CH2Cl2 (15 mL)
and cooled to ꢀ78 ꢁC, ozone was bubbled (at a flow rate of 2.0 L/min
of oxygen containing 5% of ozone) with stirring. The mixture was
maintained at ꢀ78 ꢁC for 5 min. The reaction was monitored by
thin-layer chromatography (TLC). The excess of ozone was elimi-
nated by bubbling nitrogen into the solution. The appropriate
amine (RNH2) (0.1 mL), NaBH(OAc)3 (368.8 mg, 1.7 mmol), and
CH3OH (8 mL) were successively added, and the mixture was
allowed to reach 0 ꢁC. After completion of the reaction (TLC
monitoring, CH2Cl2/CH3OH, 30/1, V/V, on silica gel plate), water was
added, and the product was isolated by extraction with dichloro-
methane. The organic phase was washed with water, brine, dried
over anhydrous sodium sulfate, filtered, and concentrated under
vacuum to obtain the desired crude products. The appropriate
compounds (9e31) were obtained following purification by silica
gel column chromatography.
CDCl3):
d 176.5, 170.9, 170.9, 89.5, 80.5, 74.5, 55.8, 52.5, 49.7, 49.1,
46.8, 39.6, 38.5, 37.9, 37.0, 34.4, 32.5, 31.3, 29.1, 28.5, 27.9, 26.4, 24.2,
23.5, 21.9, 21.3, 18.1, 17.6, 16.5, 16.1, 15.5.
5.1.11. (4S)-4-((3S,8R,10R,12R,14R,17S)-3,12-Dihydroxy-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-
4-hydroxy-N-isopropylpentanamide (6)
The product was purified by flash chromatography (PE/EtOAc, 1/
2, V/V) to give a white powder (69 mg, 75%). ESI(þ)-MS (m/z): 492.6
[MþH]þ; 1H NMR (400 MHz, CD3OD):
3.97 (p, J ¼ 6.6 Hz, 1H), 3.56
d
(td, J ¼ 10.4, 5.2 Hz, 1H), 3.17 (dd, J ¼ 11.1, 5.0 Hz, 1H), 2.35 (ddd,
J ¼ 14.1, 10.4, 5.4 Hz, 1H), 2.24 (ddd, J ¼ 14.1, 10.4, 6.0 Hz, 1H), 2.08
(td, J ¼ 10.7, 7.1 Hz,1H),1.15 (d, J ¼ 1.2 Hz, 3H),1.14 (d, J ¼ 1.2 Hz, 3H),
1.13 (s, 3H), 1.04 (s, 3H), 0.99 (s, 3H), 0.94 (s, 6H), 0.80 (s, 3H); 13C
NMR (100 MHz, CD3OD):
d 174.5, 78.1, 72.5, 70.8, 55.9, 53.8, 51.2,
5.1.7. (3S,8R,10R,12R,14R,17S)-17-((2R,5S)-5-(2-Hydroxypropan-2-
yl)-2-methyltetrahydrofuran-2-yl)-4,4,8,10,14-
50.0, 47.5, 40.9, 39.6, 38.9, 38.6, 36.8, 34.5, 30.7, 30.6, 30.4, 30.3,
27.2, 26.6, 25.9, 24.9, 21.2, 18.0, 15.7, 15.4, 14.8, 14.7.
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (2)
5.1.12. (4S)-4-((3S,8R,10R,12R,14R,17S)-3,12-dihydroxy-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-
4-hydroxy-N-isobutylpentanamide (7)
The product was purified by flash chromatography (PE/EtOAc, 1/
1, V/V) to give a white powder (41 mg, 40%). ESI(þ)-MS (m/z): 499.9
[MþNa]þ; 1H NMR (400 MHz, CDCl3):
d
5.74 (s, 1H), 3.87 (dd,
The product was purified by flash chromatography (PE/EtOAc, 1/
2, V/V) to give a white powder (74 mg, 78%). ESI(þ)-MS (m/z): 506.4
J ¼ 10.8, 5.4 Hz, 1H), 3.52 (td, J ¼ 10.3, 4.7 Hz, 1H), 3.19 (dt, J ¼ 10.6,
5.1 Hz, 1H), 2.25 (td, J ¼ 10.4, 4.4 Hz, 1H), 1.27 (s, 3H), 1.23 (s, 3H),
1.10 (s, 3H),1.00 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.77 (s,
3H), 0.73 (dd, J ¼ 10.9, 2.6 Hz, 1H); 13C NMR (125 MHz, CDCl3):
[MþH]þ; 1H NMR (400 MHz, CD3OD):
d
3.56 (td, J ¼ 10.4, 5.1 Hz,
1H), 3.17 (dd, J ¼ 11.1, 5.1 Hz, 1H), 3.01 (dd, J ¼ 6.7, 4.7 Hz, 2H), 2.40
(ddd, J ¼ 14.0, 10.6, 5.3 Hz, 1H), 2.29 (ddd, J ¼ 14.1, 10.5, 5.8 Hz, 1H),
2.09 (td, J ¼ 10.7, 6.9 Hz, 1H), 1.14 (s, 3H), 1.04 (s, 3H), 0.99 (s, 3H),
0.94 (d, J ¼ 2.4 Hz, 6H), 0.93 (s, 3H), 0.92 (s, 3H), 0.80 (s, 3H); 13C
d
87.4, 87.1, 78.8, 70.5, 70.0, 60.4, 55.9, 52.2, 50.2, 48.9, 48.8, 39.7,
38.9, 37.2, 34.8, 32.2, 31.7, 31.6, 28.9, 28.6, 28.0, 27.5, 25.1, 24.2, 18.3,
17.8, 16.3, 15.5, 15.3, 14.2.
NMR (100 MHz, CD3OD):
d 175.5, 78.1, 72.5, 70.7, 55.9, 53.8, 51.2,
50.0, 47.5, 46.6, 39.6, 38.9, 38.6, 36.8, 34.6, 30.7, 30.6, 30.6, 30.3,
28.2, 27.3, 26.6, 25.9, 24.9, 19.2, 18.1, 15.8, 15.4, 14.8, 14.8.
5.1.8. (3S,8R,10R,12R,14R,17S)-17-((2R,5R)-5-(2-hydroxypropan-2-
yl)-2-methyltetrahydrofuran-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (3)
5.1.13. (4S)-N-butyl-4-((3S,8R,10R,12R,14R,17S)-3,12-dihydroxy-
4,4,8,10,14-pentamethylhexadecahydro-1H-cyclopenta[a]
phenanthren-17-yl)-4-hydroxypentanamide (8)
The product was purified by flash chromatography (PE/EtOAc,1/1,
V/V) to give a white powder (46 mg, 45%). ESI(þ)-MS (m/z): 477.9
The product was purified by flash chromatography (PE/EtOAc, 1/
2, V/V) to give a white powder (73 mg, 79%). ESI(þ)-MS (m/z): 505.3
[MþH]þ; 1H NMR (400 MHz, CDCl3):
d
3.84 (dd, J ¼ 8.9, 6.6 Hz, 1H),
3.50(td, J ¼ 10.5, 4.5 Hz,1H), 3.18 (dd, J ¼ 11.3, 4.9 Hz), 2.18 (td, J ¼ 10.3,
9.7, 3.6 Hz,1H),1.27 (s, 3H),1.26 (s, 3H),1.09 (s, 3H), 0.97 (s, 3H), 0.94(s,
3H), 0.89(s, 3H), 0.84(s, 3H), 0.76 (s, 3H), 0.72 (dd, J ¼ 11.2, 2.7 Hz, 1H);
[M þ H]þ; 1H NMR (400 MHz, CD3OD):
d 3.55 (m, 1H), 3.17 (m, 3H),
2.38 (ddd, J ¼ 13.9, 10.6, 5.2 Hz, 1H), 2.26 (ddd, J ¼ 14.0, 10.5, 5.9 Hz,
1H), 2.08 (m, 1H), 1.14 (s, 3H), 1.04 (s, 3H), 0.99 (s, 3H), 0.97 (t,
J ¼ 7.3 Hz, 3H), 0.94 (s, 6H), 0.80 (s, 3H); 13C NMR (100 MHz,
13C NMR (125 MHz, CDCl3):
d 86.5, 85.4, 78.9, 71.0, 70.1, 55.9, 52.0,
50.5, 49.4, 47.9, 39.7, 38.9, 38.9, 37.2, 34.8, 32.6, 31.3, 31.2, 28.6, 28.0,
27.9, 27.6, 27.5, 26.1, 25.0, 18.3, 18.2, 16.3, 15.4, 15.3.
CD3OD): d 175.4, 78.1, 72.5, 70.7, 55.9, 53.8, 51.2, 50.0, 47.5, 39.6,
38.9, 38.8, 38.6, 36.8, 34.6, 31.1, 30.7, 30.6, 30.6, 30.2, 27.3, 26.6, 25.9,
24.9, 19.7, 18.0, 15.7, 15.4, 14.8, 14.7, 12.7.
5.1.9. (3S,8R,10R,12R,14R,17S)-17-((S)-2-hydroxy-6-methylheptan-
2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-cyclopenta[a]
phenanthrene-3,12-diol (4)
5.1.14. (3S,8R,10R,12R,14R,17S)-17-((S)-2-hydroxy-5-
(isopropylamino)pentan-2-yl)-4,4,8,10,14-
The product was purified by flash chromatography (PE/EtOAc, 5/
1, V/V) to give a white powder (96 mg, 95%). ESI(þ)-MS (m/z): 463.4
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (9)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 60/1/0.5, V/V/V) to give a white powder (92 mg, 88%).
[MþH]þ; 1H NMR (400 MHz, CDCl3):
d
3.61 (tt, J ¼ 10.2, 4.6 Hz, 1H),
3.26e3.17 (m, 1H), 2.05 (td, J ¼ 10.7, 7.1 Hz, 1H), 1.94e1.81 (m, 2H),
1.78e1.72 (m, 2H), 1.19 (s, 3H), 0.99 (d, J ¼ 4.0 Hz, 6H), 0.90 (s, 3H),
0.89 (s, 9H), 0.80 (s, 3H), 0.73 (d, J ¼ 8.7 Hz, 1H); 13C NMR (125 MHz,
ESI(þ)-MS (m/z): 478.6 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d 3.57
(td, J ¼ 12.5, 6.0 Hz, 1H), 3.19 (dd, J ¼ 10.8, 5.2 Hz, 1H), 2.95 (d,
J ¼ 12.6 Hz, 1H), 2.82e2.87 (m, 1H), 2.48e2.52 (m, 1H), 2.08e2.10
(m, 1H), 1.14 (s, 3H), 1.13 (d, J ¼ 7.2 Hz, 6H), 0.97 (s, 3H), 0.95 (s, 3H),
0.87 (s, 3H), 0.86 (s, 3H), 0.76 (s, 3H), 0.73 (d, J ¼ 10.9 Hz, 1H); 13C
CDCl3):
d 78.9, 74.4, 70.9, 55.8, 53.5, 51.6, 50.1, 47.7, 39.8, 39.7, 38.9,
38.9, 37.1, 35.1, 34.7, 31.2, 30.9, 28.2, 28.0, 27.4, 27.1, 26.6, 22.7, 22.6,
21.3, 18.3, 16.9, 16.1, 15.6, 15.4.

346
J. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 340e349
NMR (100 MHz, CDCl3):
48.3, 47.3, 39.9, 39.1, 37.3, 34.9, 34.3, 31.1, 30.8, 28.2, 27.6, 26.5, 24.0,
22.4, 21.4, 18.5, 16.9, 16.3, 15.9, 15.6.
d
79.1, 72.3, 70.5, 56.0, 53.3, 51.5, 50.2, 49.3,
(td, J ¼ 12.6, 6.2 Hz, 1H), 3.20 (dd, J ¼ 10.7, 5.3 Hz, 1H), 3.05 (m, 1H),
2.95 (dd, J ¼ 11.0, 7.2 Hz, 1H), 2.56 (t, J ¼ 11.0 Hz, 1H), 2.09e2.13 (m,
1H), 2.05e2.09 (m, 1H), 1.11 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.87 (s,
6H), 0.76 (s, 3H), 0.72 (d, J ¼ 10.9 Hz, 1H); 13C NMR (125 MHz,
5.1.15. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-
(methylamino)pentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (10)
CDCl3): d 78.9, 72.4, 70.4, 55.8, 53.0, 51.4, 50.1, 50.1, 48.1, 39.8, 38.9,
37.1, 34.8, 34.1, 30.9, 30.6, 30.6, 28.0, 27.4, 27.4, 26.3, 23.7, 18.3, 16.7,
16.2, 15.7, 15.4, 5.6, 5.5.
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 60/1/0.5, V/V/V) to give a white powder (83 mg, 85%).
ESI(þ)-MS (m/z): 450.7 [M þ H]þ; 1H NMR (300 MHz, CDCl3):
5.1.20. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((3-(1H-imidazol-1-yl)
propyl)amino)-2-hydroxypentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (15)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (83 mg, 70%).
d
4.09e4.16 (m, 1H), 3.52 (td, J ¼ 13.0, 6.2 Hz, 1H), 3.21 (dd, J ¼ 10.9,
5.0 Hz, 1H), 2.92e2.97 (m, 1H), 2.60e2.64 (m, 1H), 2.47 (s, 3H), 1.13
(s, 3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.88 (s, 6H), 0.78 (s, 3H), 0.73 (d,
J ¼ 11.0 Hz, 1H); 13C NMR (100 MHz, CDCl3):
d
78.8, 72.3, 70.7, 55.8,
ESI(þ)-MS (m/z): 544.7 [M þ H]þ; 1H NMR (300 MHz, CDCl3):
d 7.49
53.3, 51.5, 50.9, 50.2, 48.1, 39.8, 38.9, 37.1, 34.8, 34.2, 32.8, 30.9, 30.8,
28.0, 27.4, 26.8, 26.4, 22.2, 18.3, 16.7, 16.2, 15.7, 15.4.
(s, 1H), 7.05 (s, 1H), 6.92 (s, 1H), 4.00e4.04 (m, 2H), 3.55 (td, J ¼ 12.7,
6.0 Hz, 1H), 3.19 (dd, J ¼ 11.0, 5.2 Hz, 1H), 2.86e2.90 (m, 1H), 2.66e
2.70 (m, 1H), 2.50e2.54 (m, 2H), 1.13 (s, 3H), 0.98 (s, 3H), 0.97 (s,
3H), 0.89 (s, 6H), 0.78 (s, 3H), 0.72 (d, J ¼ 10.9 Hz, 1H); 13C NMR
5.1.16. (3S,8R,10R,12R,14R,17S)-17-((S)-5-(ethylamino)-2-
hydroxypentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-
cyclopenta[a]phenanthrene-3,12-diol (11)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 60/1/0.5, V/V/V) to give a white powder (87 mg, 86%).
(100 MHz, CDCl3):
d 137.2, 129.5, 118.8, 78.8, 72.4, 70.5, 55.8, 53.0,
51.4, 50.1, 48.1, 45.9, 44.7, 39.8, 38.9, 37.1, 34.8, 33.8, 31.0, 30.7, 30.1,
28.0, 27.4, 27.3, 26.5, 23.3, 18.3, 16.8, 16.2, 15.7, 15.4.
ESI(þ)-MS (m/z): 464.7 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d
3.55
5.1.21. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((2-
(piperazin-1-yl)ethyl)amino)pentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (16)
(td, J ¼ 12.9, 6.3 Hz,1H), 3.19 (dd, J ¼ 10.9, 4.9 Hz,1H), 2.91e2.95 (m,
1H), 2.68e2.76 (m, 1H), 2.52e2.62 (m, 1H), 2.44e2.50 (m, 1H),
2.07e2.11 (m, 1H), 1.15 (s, 3H), 1.11 (t, J ¼ 7.2 Hz, 3H), 0.99 (s, 3H),
0.98 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.78 (s, 3H), 0.73 (d,
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 45/1/0.5, V/V/V) to give a white powder (85 mg, 71%).
J ¼ 10.7 Hz, 1H); 13C NMR (100 MHz, CDCl3):
d 78.9, 71.9, 70.2, 55.8,
52.9, 51.4, 50.0, 49.7, 48.1, 43.4, 39.8, 38.9, 37.1, 34.8, 34.3, 31.0, 30.6,
28.0, 27.5, 27.4, 26.4, 23.7, 18.3, 16.8, 16.2, 15.7, 15.4, 14.2.
ESI(þ)-MS (m/z): 548.6 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d 5.31
(d, J ¼ 1.4 Hz, 1H), 3.57 (td, J ¼ 13.0, 5.6 Hz, 1H), 3.20 (dd, J ¼ 10.9,
5.3 Hz, 1H), 2.80 (t, J ¼ 6.0 Hz, 2H), 2.46e2.48 (m, 12H), 1.14 (s, 3H),
0.98 (s, 6H), 0.89 (s, 6H), 0.78 (s, 3H), 0.73 (d, J ¼ 10.9 Hz, 1H); 13C
5.1.17. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-(propylamino)
pentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-
cyclopenta[a]phenanthrene-3,12-diol (12)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 60/1/0.5, V/V/V) to give a white powder (91 mg, 87%).
NMR (125 MHz, CDCl3): d 78.9, 72.1, 70.1, 59.6, 59.3, 55.8, 52.8, 52.3,
51.3, 49.9, 48.2, 39.8, 38.9, 38.9, 38.2, 37.1, 34.8, 30.9, 30.5, 28.0, 27.9,
27.4, 26.4, 20.8, 18.3, 16.7, 16.1, 15.7, 15.4.
ESI(þ)-MS (m/z): 478.7 [M þ H]þ; 1H NMR (300 MHz, CDCl3):
d
3.55
5.1.22. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((3-
hydroxypropyl)amino)pentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (17)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 40/1/0.5, V/V/V) to give a white powder (75 mg, 70%).
(td, J ¼ 12.7, 6.2 Hz,1H), 3.20 (dd, J ¼ 10.8, 5.2 Hz,1H), 2.91e2.96 (m,
1H), 2.62e2.71 (m, 1H), 2.48e2.55 (m, 2H), 2.106e2.17 (m, 1H), 1.14
(s, 3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.93 (t, J ¼ 7.3 Hz, 3H), 0.89 (s, 3H),
0.88 (s, 3H), 0.78 (s, 3H), 0.73 (d, J ¼ 10.9 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d 78.9, 72.0, 70.4, 55.8, 53.0, 51.4, 50.8, 50.1, 49.8,
48.1, 39.8, 38.9, 37.1, 34.8, 34.1, 30.9, 30.9, 30.6, 28.0, 27.4, 27.4, 26.4,
23.4, 21.9, 18.3, 16.7, 16.2, 15.7, 15.4, 11.6.
ESI(þ)-MS (m/z): 494.6 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d 3.74 (t,
J ¼ 5.7 Hz, 2H), 3.54 (td, J ¼ 12.6, 5.8 Hz,1H), 3.20 (dd, J ¼ 10.8, 5.4 Hz,
1H), 2.83e2.94 (m, 2H), 2.74e2.80 (m,1H), 2.56e2.63 (m,1H),1.15 (s,
3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H), 0.86 (s, 3H), 0.78 (s, 3H),
5.1.18. (3S,8R,10R,12R,14R,17S)-17-((S)-5-(Tert-butylamino)-2-
hydroxypentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-
cyclopenta[a]phenanthrene-3,12-diol (13)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 60/1/0.5, V/V/V) to give a white powder (95 mg, 88%).
0.72 (d, J ¼ 10.7 Hz,1H); 13C NMR (125 MHz, CDCl3):
d 78.9, 72.3, 70.6,
61.7, 55.8, 53.2, 51.5, 50.2, 49.7, 48.1, 47.3, 39.6, 38.9, 37.1, 34.8, 33.2,
31.1, 30.8, 28.0, 27.4, 27.1, 26.5, 23.1, 18.3, 16.8, 16.2, 15.7, 15.4.
ESI(þ)-MS (m/z): 492.7 [M þ H]þ; 1H NMR (300 MHz, CDCl3):
d
5.30
5.1.23. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((4-
methoxyphenyl)amino)pentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (18)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (107 mg, 90%).
(s, 1H), 3.57 (td, J ¼ 12.3, 6.1 Hz, 1H), 3.19 (dd, J ¼ 10.7, 5.3 Hz, 1H),
2.93 (d, J ¼ 10.8 Hz, 1H), 2.44 (t, J ¼ 11.5 Hz, 1H), 2.06e2.12 (m, 1H),
1.13 (s, 12H), 0.97 (s, 6H), 0.88 (s, 6H), 0.77 (s, 3H), 0.72 (d,
J ¼ 10.8 Hz, 1H); 13C NMR (100 MHz, CDCl3):
d
78.9, 71.9, 70.2, 55.8,
53.1, 51.3, 50.0, 48.1, 42.5, 39.8, 38.9, 38.9, 37.2, 34.8, 34.2, 30.9, 30.6,
28.1, 28.0, 27.6, 27.5, 26.3, 24.3, 18.3, 16.7, 16.1, 15.7, 15.4.
ESI(þ)-MS (m/z): 542.6 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d 6.76
(d, J ¼ 9.0 Hz, 2H), 6.68 (d, J ¼ 9.0 Hz, 2H), 3.71 (s, 3H), 3.54 (td,
J ¼ 12.8, 6.3 Hz, 1H), 3.14 (dd, J ¼ 11.0, 5.2 Hz, 1H), 3.05 (t, J ¼ 6.4 Hz,
2H), 2.03e2.05 (m, 1H), 1.14 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H), 0.91 (s,
6H), 0.78 (s, 3H), 0.75 (d, J ¼ 10.6 Hz, 1H); 13C NMR (125 MHz,
5.1.19. (3S,8R,10R,12R,14R,17S)-17-((S)-5-(Cyclopropylamino)-2-
hydroxypentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-
cyclopenta[a]phenanthrene-3,12-diol (14)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 60/1/0.5, V/V/V) to give a white powder (83 mg, 80%).
CDCl3): d 152.8, 141.9, 115.4, 114.9, 78.8, 73.5, 70.9, 55.8, 55.8, 53.5,
51.6, 50.1, 47.8, 46.6, 39.7, 38.9, 38.9, 37.1, 34.8, 32.8, 31.2, 30.9, 28.0,
27.4, 27.0, 26.5, 23.9, 18.3, 16.8, 16.1, 15.7, 15.4.
ESI(þ)-MS (m/z): 476.6 [M þ H]þ; 1H NMR (300 MHz, CDCl3):
d 3.54

J. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 340e349
347
5.1.24. (3S,8R,10R,12R,14R,17S)-17-((S)-5-(Benzylamino)-2-
hydroxypentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-
cyclopenta[a]phenanthrene-3,12-diol (19)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (103 mg, 90%).
(s, 1H), 6.86 (d, J ¼ 8.4 Hz, 1H), 6.84 (d, J ¼ 8.4 Hz, 1H), 5.30 (s, 1H),
3.89 (s, 3H), 3.87 (s, 3H), 3.82 (s, 2H), 3.53 (td, J ¼ 12.8, 6.0 Hz, 1H),
3.20 (dd, J ¼ 10.8, 5.3 Hz, 1H), 2.94e2.98 (m, 1H), 2.67e2.71 (m, 1H),
2.06e2.10 (m, 1H), 1.12 (s, 3H), 0.98 (s, 6H), 0.88 (s, 6H), 0.78 (s, 3H),
0.71 (d, J ¼ 10.8 Hz, 1H); 13C NMR (125 MHz, CDCl3):
d 149.2, 148.9,
ESI(þ)-MS (m/z): 526.5 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d
7.30e
127.2, 121.5, 112.2, 111.1, 78.9, 72.6, 70.8, 55.9, 55.9, 55.8, 53.2, 51.9,
51.5, 50.2, 48.2, 48.0, 39.7, 38.9, 37.1, 34.8, 32.7, 31.0, 30.9, 28.0, 27.4,
26.8, 26.5, 23.5, 22.2, 18.3, 16.7, 16.2, 15.7, 15.4.
7.32 (m, 5H), 3.74 (s, 2H), 3.57 (td, J ¼ 12.9, 6.0 Hz, 1H), 3.20 (dd,
J ¼ 10.8, 5.2 Hz,1H), 2.97 (d, J ¼ 12.3 Hz,1H), 2.78 (dd, J ¼ 12.3, 7.2 Hz,
1H), 2.52 (t, J ¼ 11.0 Hz,1H), 2.10e2.14 (m,1H),1.15 (s, 3H), 0.99 (s, 3H),
0.97 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.78 (s, 3H), 0.73 (d, J ¼ 10.7 Hz,
5.1.29. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-
(phenethylamino)pentan-2-yl)-4,4,8,10,14-
1H); 13C NMR (125 MHz, CDCl3):
d 138.2,128.7,128.4,127.5, 78.9, 72.2,
70.2, 55.9, 53.7, 52.8, 51.4, 50.1, 48.2, 46.2, 39.8, 38.9, 37.2, 34.8, 34.5,
31.1, 30.7, 28.1, 27.7, 27.5, 26.4, 23.8, 18.3, 16.8, 16.2, 15.7, 15.4.
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (24)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (106 mg, 90%).
5.1.25. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-(((S)-1-
phenylethyl)amino)pentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (20)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (106 mg, 90%).
ESI(þ)-MS (m/z): 540.9 [MþH]þ; 1H NMR (300 MHz, CDCl3):
ESI(þ)-MS (m/z): 540.8 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d 7.17e
7.29 (m, 5H), 3.56 (td, J ¼ 13.1, 6.1 Hz, 1H), 3.20 (dd, J ¼ 10.9, 4.9 Hz,
1H), 2.76e2.87 (m, 5H), 2.50e2.57 (m, 1H), 2.00e2.12 (m, 1H), 1.13
(s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.77 (s,
3H), 0.71 (d, J ¼ 10.9 Hz, 1H); 13C NMR (125 MHz, CDCl3):
d 138.7,
128.8, 128.7, 126.5, 78.9, 72.2, 70.4, 55.9, 53.0, 51.4, 50.1, 50.0, 49.7,
48.1, 39.8, 38.9, 37.1, 34.9, 34.8, 34.0, 31.0, 30.7, 28.0, 27.4, 27.3, 26.4,
23.3, 18.3, 16.8, 16.2, 15.7, 15.4.
d
7.23e7.37 (m, 5H), 3.82 (q, J ¼ 6.7 Hz,1H), 3.56 (td, J ¼ 12.8, 5.9 Hz,
1H), 3.19 (dd, J ¼ 10.8, 5.3 Hz, 1H), 2.66e2.71 (m, 1H), 2.36e2.44 (m,
1H), 2.06e2.16 (m, 1H), 1.40 (d, J ¼ 6.7 Hz, 3H), 1.18 (s, 3H), 0.97 (s,
3H), 0.96 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.77 (s, 3H), 0.77 (d,
5.1.30. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((4-
nitrophenethyl)amino)pentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (25)
J ¼ 10.8 Hz, 1H); 13C NMR (125 MHz, CDCl3):
d 142.6, 128.8, 127.6,
126.7, 78.9, 72.4, 70.4, 57.5, 55.9, 52.9, 51.4, 50.1, 48.1, 47.1, 39.8, 38.9,
38.9, 37.1, 34.8, 33.9, 31.0, 30.8, 28.0, 27.6, 27.4, 26.5, 23.2, 22.7, 18.3,
16.8, 16.2, 15.7, 15.4.
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (106 mg, 83%).
5.1.26. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((4-Fluorobenzyl)amino)-
2-hydroxypentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-
cyclopenta[a]phenanthrene-3,12-diol (21)
ESI(þ)-MS (m/z): 585.9 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d 8.16
(d, J ¼ 8.8 Hz, 2H), 7.40 (d, J ¼ 8.8 Hz, 2H), 3.44e3.51 (m, 2H), 3.16e
2.22 (m, 2H), 3.05e3.11 (m, 4H), 2.78e2.82 (m, 1H), 1.10 (s, 3H), 1.00
(s, 3H), 0.97 (s, 3H), 0.88 (s, 6H), 0.77 (s, 3H), 0.72 (d, J ¼ 11.0 Hz,1H);
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (105 mg, 88%).
13C NMR (125 MHz, CDCl3):
d 147.0, 145.3, 129.7, 123.9, 78.8, 72.8,
ESI(þ)-MS (m/z): 544.6 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d
7.32
71.3, 55.8, 53.3, 51.6, 50.3, 49.4, 49.1, 48.0, 39.8, 38.9, 38.9, 37.1, 34.7,
33.5, 32.9, 30.9, 30.8, 28.0, 27.4, 26.7, 26.5, 22.1, 18.3, 16.6, 16.2, 15.7,
15.4.
(dd, J ¼ 8.6, 5.4 Hz, 2H), 7.03 (dd, J ¼ 8.6, 5.4 Hz, 2H), 5.30 (s,1H), 3.84
(s, 2H), 3.53 (td, J ¼ 12.9, 6.0 Hz, 1H), 3.20 (dd, J ¼ 11.0, 5.2 Hz, 1H),
2.96e3.00 (m,1H), 2.62e2.67 (m,1H), 2.06e2.11 (m,1H),1.12 (s, 3H),
0.98 (s, 6H), 0.88 (s, 6H), 0.78 (s, 3H), 0.73 (d, J ¼ 10.8 Hz, 1H); 13C
5.1.31. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((3,4-
NMR (125 MHz, CDCl3):
d
163.6 and 161.6 (1JCF ¼ 245 Hz),147.1,130.9
Dimethoxyphenethyl)amino)-2-hydroxypentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (26)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (122 mg, 93%).
and 130.8 (3JCF ¼ 8.7 Hz), 115.9 and 115.7 (2JCF ¼ 21.3 Hz), 78.9, 72.6,
70.8, 55.8, 53.2, 51.7, 51.5, 50.2, 48.5, 48.1, 39.8, 38.9, 37.1, 34.8, 33.0,
31.0, 30.8, 28.0, 27.4, 26.9, 26.5, 23.1, 22.4, 18.3, 16.7, 16.2, 15.7, 15.4.
5.1.27. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((4-chlorobenzyl)amino)-
2-hydroxypentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-
cyclopenta[a]phenanthrene-3,12-diol (22)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (106 mg, 87%).
ESI(þ)-MS (m/z): 600.7 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d 6.80
(d, J ¼ 8.2 Hz, 1H), 6.73 (d, J ¼ 8.2 Hz, 1H), 6.71 (s, 1H), 5.30 (s, 1H),
3.86 (s, 3H), 3.85 (s, 3H), 3.56 (td, J ¼ 12.8, 6.0 Hz, 1H), 3.20 (dd,
J ¼ 10.9, 5.3 Hz, 1H), 2.72e2.87 (m, 5H), 2.52 (t, J ¼ 9 Hz, 1H), 1.13 (s,
3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H),
ESI(þ)-MS (m/z): 560.7 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d
7.29 (d,
0.73 (d, J ¼ 11.0 Hz, 1H); 13C NMR (125 MHz, CDCl3):
d 148.9, 147.6,
J ¼ 6.1 Hz, 2H), 7.20 (d, J ¼ 6.1 Hz, 2H), 3.84 (s, 1H), 3.71 (s, 2H), 3.56
(td, J ¼ 12.9, 5.9 Hz, 1H), 3.19 (dd, J ¼ 10.9, 5.3 Hz, 1H), 2.94 (m, 1H),
2.52 (t, J ¼ 10.1 Hz, 1H), 2.06e2.14 (m, 1H), 1.13 (s, 3H), 0.98 (s, 3H),
0.97 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.77 (s, 3H), 0.74 (d, J ¼ 10.7 Hz,
131.3, 120.6, 111.9, 111.4, 78.9, 72.1, 70.3, 55.9, 55.9, 53.0, 51.4, 50.3,
50.1, 49.9, 48.1, 39.8, 38.9, 37.1, 34.8, 34.7, 34.2, 31.0, 30.7, 28.0, 27.5,
27.4, 26.4, 23.6, 18.3, 16.8, 16.2, 15.7, 15.4.
1H); 13C NMR (125 MHz, CDCl3):
d
136.4, 133.5, 129.8, 128.9, 78.9,
5.1.32. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((2-(Benzo[d][1,3]dioxol-
5-yl)ethyl)amino)-2-hydroxypentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (27)
72.3, 70.3, 55.9, 52.9, 52.8, 51.4, 50.1, 50.0, 48.2, 39.8, 38.9, 37.1, 34.8,
34.3, 31.1, 30.7, 28.0, 27.6, 27.4, 26.5, 23.7, 18.3, 16.8, 16.2, 15.7, 15.4.
5.1.28. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((3,4-dimethoxybenzyl)
amino)-2-hydroxypentan-2-yl)-4,4,8,10,14-pentamethylhexa-
decahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (23)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (117 mg, 92%).
ESI(þ)-MS (m/z): 584.9 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d 6.72
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (118 mg, 92%).
(d, J ¼ 7.8 Hz, 1H), 6.65 (d, J ¼ 1.6 Hz, 1H), 6.61 (dd, J ¼ 7.8, 1.6 Hz,
1H), 5.91 (s, 2H), 3.56 (td, J ¼ 12.8, 5.8 Hz, 1H), 3.19 (dd, J ¼ 10.9,
5.3 Hz, 1H), 2.66e2.91 (m, 6H), 2.44e2.51 (m, 1H), 2.02e2.15 (m,
ESI(þ)-MS (m/z): 586.6 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d 6.93

348
J. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 340e349
1H), 1.13 (s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H),
78.9, 72.5, 70.3, 55.8, 52.8, 52.4, 51.4, 50.2, 50.0, 48.1, 39.8, 38.9, 37.1,
34.8, 34.1, 31.0, 30.7, 28.0, 27.5, 27.4, 26.5, 23.7, 18.3, 16.8, 16.2, 15.7,
15.4.
0.77 (s, 3H), 0.73 (d, J ¼ 10.7 Hz, 1H); 13C NMR (125 MHz, CDCl3):
d
147.8, 146.2, 132.6, 121.7, 109.0, 108.4, 100.9, 78.9, 72.1, 70.3, 55.9,
52.9, 51.4, 50.2, 50.1, 49.9, 48.1, 46.0, 39.8, 38.9, 37.1, 34.9, 34.8, 34.3,
31.0, 30.7, 28.0, 27.5, 27.4, 26.4, 23.6, 18.3, 16.8, 16.2, 15.7, 15.4.
5.2. Biology
5.1.33. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((3-
phenylpropyl)amino)pentan-2-yl)-4,4,8,10,14-
5.2.1. Recombinant AMPK heterotrimers construction, expression
and purification
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (28)
The coding sequences of different
a/b/g subunit that form
AMPK 1 heterotrimers were amplified from cDNA of each
a2b1g
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 50/1/0.5, V/V/V) to give a white powder (109 mg, 90%).
human AMPK subunit and constructed into pET28b vector, and
then expression and purified as previously described [34].
ESI(þ)-MS (m/z): 554.9 [MþH]þ; 1H NMR (300 MHz, CDCl3):
d 7.14e
7.29 (m, 5H), 3.53 (td, J ¼ 13.1, 6.5 Hz, 1H), 3.19 (dd, J ¼ 10.8, 5.3 Hz,
1H), 2.88e2.93 (m, 1H), 2.49e2.80 (m, 6H), 2.04e2.09 (m, 1H), 1.10
(s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H), 0.87 (s, 3H), 0.72 (s,
5.2.2. Phosphorylation and activation of AMPK
AMPK protein was completely phosphorylated by incubation
with CaMKKb
at 30 ꢁC for 4 h as previously described [35]. The
3H), 0.72 (d, J ¼ 10.7 Hz, 1H); 13C NMR (125 MHz, CDCl3):
d
141.2,
AMPK activity and the activation of compounds on AMPK were test
with the STK substrate 1-biotin, XL-665 and STK-Antibody of HTRFÒ
KinEASEÔ-STK1 Kit. The reaction was carried out in 384-well plates
128.4, 128.3, 126.0, 78.9, 72.2, 70.4, 55.8, 53.0, 51.4, 50.1, 49.8, 48.5,
48.1, 46.1, 39.8, 38.9, 37.1, 34.8, 33.9, 33.4, 31.0, 30.7, 30.1, 28.0, 27.4,
27.3, 26.4, 23.2, 18.3, 16.7, 16.2, 15.7, 15.4.
in a reaction volume of 10
7.5, 4 mmol/l MgCl2, 0.8 mmol/l DTT,160
and 4 mol/l ATP. The reaction was initiated by addition of 1.6 nmol/
mL containing 32 mmol/l TriseHCl, pH
mmol/l substrate-1 peptide
5.1.34. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((pyridin-2-
ylmethyl)amino)pentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (29)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 30/1/0.5, V/V/V) to give a white powder (94 mg, 82%).
m
l recombinant AMPK into the well, followed by incubation for
45 min at 30 ꢁC. The reaction were terminated by addition of
detection reagent contains 57.5 nmol/l XL-665 and STK-Antibody
labeled with Eu3þ-Cryptate. The fluorescence is measured at
615 nm (Cryptate) and 665 nm (XL665). A ratio is calculated (665/
620) for each well and represents the AMPK activity.
ESI(þ)-MS (m/z): 527.8 [M þ H]þ; 1H NMR (300 MHz, CDCl3):
d 8.53
(d, J ¼ 4.9 Hz, 1H), 7.64 (t, J ¼ 4.9 Hz, 1H), 7.20 (d, J ¼ 4.9 Hz, 1H), 7.16
(t, J ¼ 4.9 Hz, 1H), 3.85 (s, 1H), 3.83 (s, 1H), 3.55 (d, J ¼ 12.8, 5.9 Hz,
1H), 3.19 (dd, J ¼ 11.0, 5.1 Hz, 1H), 2.87e2.99 (m, 1H), 2.45e2.49 (m,
1H), 2.02e2.10 (m, 1H), 1.15 (s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.88 (s,
6H), 0.77 (s, 3H), 0.73 (d, J ¼ 10.9 Hz, 1H); 13C NMR (125 MHz,
5.2.3. Cell culture and compound treatment
Cell culture was performed as previously described [36]. Before
compound incubation, the cells were deprived of serum for 2 h,
then incubated with compounds of indicated concentration for
indicated time. Before Western blotting analysis, the cells were
washed with phosphate-buffered saline and lysed using SDS-PAGE
loading buffer.
CDCl3): d 156.3, 149.4, 136.9, 122.9, 122.7, 78.9, 72.3, 70.5, 55.8, 53.6,
53.1, 51.4, 50.1, 49.7, 48.1, 39.8, 38.9, 37.1, 34.8, 33.9, 31.0, 30.7, 28.0,
27.4, 27.3, 26.4, 23.3, 18.3, 16.7, 16.2, 15.7, 15.4.
5.1.35. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((pyridin-3-
ylmethyl)amino)pentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (30)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 30/1/0.5, V/V/V) to give a white powder (93 mg, 81%).
5.2.4. Western blot
Samples were subjected to 8% SDS-PAGE, and proteins were
transferred onto polyvinylliidene difluoride membranes. The
amount of protein and the level of phosphorylation level were
detected by the respective antibodies.
ESI(þ)-MS (m/z): 527.8 [M þ H]þ; 1H NMR (300 MHz, CDCl3):
d
8.52
5.2.5. Lipid synthesis
(s, 1H), 8.43 (d, J ¼ 4.7 Hz, 1H), 7.85 (d, J ¼ 7.4 Hz, 1H), 7.41 (dd,
J ¼ 7.4, 4.7 Hz,1H), 3.79 (s, 2H), 3.53 (d, J ¼ 13.1, 5.7 Hz,1H), 3.14 (dd,
J ¼ 10.8, 5.2 Hz, 1H), 2.60e2.65 (m, 2H), 1.99e2.05 (m, 1H), 1.14 (s,
3H), 1.00 (s, 3H), 0.96 (s, 3H), 0.91 (s, 6H), 0.77 (s, 3H), 0.74 (d,
The experimental procedure is adaptation of the method
described previously [37]. HepG2 cells were plated at 4 ꢃ 104 cells
per well on white-walled 96-well plates in HG-DMEM supple-
mented with 10% FBS. Cells were deprived of serum for 2 h followed
by a 6hr compound incubation with serum-free medium contain-
J ¼ 10.7 Hz, 1H); 13C NMR (125 MHz, CDCl3):
d 149.6, 149.1, 136.1,
133.8, 123.8, 78.9, 72.4, 70.2, 55.9, 52.8, 51.4, 51.1, 50.3, 50.0, 48.2,
39.8, 38.9, 37.1, 34.8, 34.4, 31.1, 30.7, 28.0, 27.7, 27.4, 26.5, 23.9, 18.3,
16.8, 16.2, 15.7, 15.4.
ing 14C acetic sodium (0.1
mCi/ml, Perkin Elmer). The plates were
rinsed with cold PBS, The final wash was replaced with 0.25 M
NaOH, after protein concentration measurement, the Microscint20
was added into wells and radioactivity incorporated into lipid was
monitored on a Wallac Microbeta plate reader and amended with
protein concentration.
5.1.36. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((pyridin-4-
ylmethyl)amino)pentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-
diol (31)
The product was purified by flash chromatography (CH2Cl2/
CH3OH/TEA, 30/1/0.5, V/V/V) to give a white powder (93 mg, 81%).
5.2.6. Mitochondria membrane potential assay
HepG2 cells were plated at 4 ꢃ 104 cells per well on black-walled
96-well plates in HG-DMEM supplemented with 10% FBS, Cells
were deprived of serum for 2 h followed by a 1hr compound in-
ESI(þ)-MS (m/z): 527.7 [M þ H]þ; 1H NMR (300 MHz, CDCl3):
d 8.56
(dd, J ¼ 4.4, 1.6 Hz, 2H), 7.23 (dd, J ¼ 4.4, 1.6 Hz, 2H), 3.77 (s, 2H),
3.58 (td, J ¼ 12.9, 5.8 Hz, 1H), 3.21 (dd, J ¼ 11.0, 5.3 Hz, 1H), 2.94e
2.98 (m, 1H), 2.52e2.60 (m, 1H), 2.08e2.18 (m, 1H), 1.17 (s, 3H), 0.99
(s, 3H), 0.98 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.78 (s, 3H), 0.78 (d,
cubation with serum-free medium, after incubation, 100
medium containing 0.2 g JC-1 was added to each well and incu-
bated for another 30 min, the plates were rinsed three times with
100 L KrebseRinger phosphate HEPES buffer. Fluorescence was
measured as described previously.
mL fresh
m
m
J ¼ 10.8 Hz, 1H); 13C NMR (100 MHz, CDCl3):
d 150.1, 146.9, 123.1,

J. Liu et al. / European Journal of Medicinal Chemistry 79 (2014) 340e349
349
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5.2.7. Statistical analysis
The results are presented as the mean ꢂ SEM. The differences
between the two groups were analyzed using paired student’s t-
test. P < 0.05 was regarded as statistically significant.
Acknowledgments
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some related triterpenoid compounds, Journal of Korean Medical Sciences 16
(2001) 28e37.
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This work was supported by the National Natural Science Foun-
dation of China (grants 81273397, 81125023 and 81273566), the
Chinese National Science & Technology Major Project “Key New Drug
Creation and Manufacturing Program” (grants 2013ZX09508104,
2012ZX09103-101-018, 2014ZX09301-306-03), and the Science
Foundation of Shanghai (grants 12XD1405700 and 13DZ2290300).
Appendix A. Supplementary data
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and pharmacological diversity, Phytochemistry 72 (2011) 689e699.
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Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2014.04.
010. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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