20(S)-Protopanaxadiol (PPD) analogues chemosensitize multidrug-resistant cancer cells to clinical anticancer drugs

Article abstract of DOI:10.1016/j.bmc.2013.04.067

Novel 20(S)-protopanoxadiol (PPD) analogues were designed, synthesized, and evaluated for the chemosensitizing activity against a multidrug resistant (MDR) cell line (KBvcr) overexpressing P-glycoprotein (P-gp). Structure-activity relationship analysis showed that aromatic substituted aliphatic amine at the 24-positions (groups V) effectively and significantly sensitized P-gp overexpressing multidrug resistant (MDR) cells to anticancer drugs, such as docetaxel (DOC), vincristine (VCR), and adriamycin (ADM). PPD derivatives 12 and 18 showed 1.3-2.6 times more effective reversal ability than verapamil (VER) for DOC and VCR. Importantly, no cytotoxicity was observed by the active PPD analogues (5 μM) against both non-MDR and MDR cells, suggesting that PPD analogues serve as novel lead compounds toward a potent and safe resistance modulator. Moreover, a preliminary mechanism study demonstrated that the chemosensitizing activity of PPD analogues results from inhibition of P-glycoprotein (P-gp) overexpressed in MDR cancer cells.

Full text of DOI:10.1016/j.bmc.2013.04.067

Bioorganic & Medicinal Chemistry 21 (2013) 4279–4287
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
20(S)-Protopanaxadiol (PPD) analogues chemosensitize
multidrug-resistant cancer cells to clinical anticancer drugs q
a,
Junhua Liu a, , Xu Wang b, , Peng Liu a, Rongxin Deng b, Min Lei a, Wantao Chen b, , Lihong Hu
⇑
⇑
a Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
b Department of Oral and Maxillofacial—Head and Neck Oncology, Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200011, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 20(S)-protopanoxadiol (PPD) analogues were designed, synthesized, and evaluated for the chemo-
sensitizing activity against a multidrug resistant (MDR) cell line (KBvcr) overexpressing P-glycoprotein
(P-gp). Structure–activity relationship analysis showed that aromatic substituted aliphatic amine at
the 24-positions (groups V) effectively and significantly sensitized P-gp overexpressing multidrug resis-
tant (MDR) cells to anticancer drugs, such as docetaxel (DOC), vincristine (VCR), and adriamycin (ADM).
PPD derivatives 12 and 18 showed 1.3–2.6 times more effective reversal ability than verapamil (VER) for
Received 2 April 2013
Accepted 25 April 2013
Available online 3 May 2013
Keywords:
20(S)-Protopanoxadiol
Multidrug resistant
P-glycoprotein
Chemosensitizer
Structure–activity relationship
DOC and VCR. Importantly, no cytotoxicity was observed by the active PPD analogues (5 lM) against both
non-MDR and MDR cells, suggesting that PPD analogues serve as novel lead compounds toward a potent
and safe resistance modulator. Moreover, a preliminary mechanism study demonstrated that the chemo-
sensitizing activity of PPD analogues results from inhibition of P-glycoprotein (P-gp) overexpressed in
MDR cancer cells.
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
1. Introduction
experiments as positive controls. Since then, several potent and
selective second- and third-generation chemosensitizers were
Chemotherapeutic is an important tool in the treatment of can-
cers. However, chemotherapy usually fails due to the development
of tumor cell resistance to multiple drugs, a phenomenon known as
multidrug resistance (MDR),1,2 and essentially all cancer-related
deaths are considered to be a result of chemotherapy failure.3
MDR may originate from several biochemical mechanisms, but
the major mechanism of drug resistance is the overexpression of
active drug efflux transporters, such as ABCB1 (also known as P-
glycoprotein, P-gp) and ABCC1 (multidrug resistance associate pro-
tein 1, MRP 1).4,5 In order to surmount MDR, a considerable re-
search efforts have been put into developing clinically usable
chemosensitizing agents over the past 2 decades. Verapamil
(VER) and cyclosporine A (CsA) are examples of first-generation
chemosensitizers that inhibit the activity of P-gp and were evalu-
ated clinically as adjuvants for chemotherapy.6,7 Both compounds
were precluded from clinical use because of significant toxicity at
the dose required to attenuate P-gp funiction,8 but are used in
developed and investigated. However, unsatisfactory toxicity and
pharmacokinetic complications still impeded drug candidate
development. Although several third-generation P-gp inhibitors,
including tariquidar, are now in phase II cancer clinical trials,9 their
clinical efficiencies are not yet clear.10 So far, no chemosensitizer
has been approved for therapeutic use. Therefore, the discovery
of safe and effective MDR modulators is still urgently needed to
overcome the MDR of tumors.
Ginsenosides are the active ingredients of Panax ginseng, the
root of which has been used in traditional herbal remedies/medi-
cine in Eastern Asia for over 2000 years.11 It exhibits various phar-
macological and physiological effects, including antioxidation,12
immunostimulation,13 antistress,14 anticancer,15 as well as stimu-
lative effects on the central nervous system.16 Within the ginseno-
side family, ginsenoside Rg3 (Fig. 1) has been reported to reverse
P-gp mediated multidrug resistance.17 20(S)-Protopanoxadiol
(PPD, Fig. 1), an important metabolite of Rg3 produced by intesti-
nal bacteria,18 has been paid more attention for its distinguished
anti-cancer activity and significant inhibition of P-gp in tumor
cells,19 and extremely low toxicity.20–22 Given extremely low
toxicity of the compound, PPD is a potential candidate of chemo-
sensitizer for treatment of multidrug resistant tumors. However,
so far PPD analogues have not been prepared and reported, and
their MDR reversal abilities and structure–activity relationship
(SAR) studies have not been extensively performed. To explore
more potent nontoxic MDR reversal analogues with lower effective
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding authors. Tel.: +86 21 63138341x5211; fax: +86 21 63135412
(W.C.); tel./fax: +86 21 20231965 (L.H.).
E-mail addresses: chenwantao2003@sohu.com (W. Chen), lhhu@simm.ac.cn
(L. Hu).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.067

4280
J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4279–4287
dosing and to study SAR, we designed and synthesized a series of
OH
PPD analogues. Herein, we report the chemosensitizing effects of
newly synthesized PPD analogues.
OH
OH
O
HO
HO
2. Design and syntheses
O
O
On the basis of previous literature, PPD had obvious effects on
inhibition of P-gp and activation of MDR MCF-7/Adr.23–25 Further-
more, the predominant metabolic pathway of PPD observed was
the oxidation of the 24,25-double bond.26,27 Therefore, the 24,25-
double bond was selected to modify in order to block the pathway
of predominant metabolic. For the sake of more effective structural
modifications at 24,25-double bond, we carefully analysed the
structural properties of various known P-gp inhibitors, especially
the first-, second-, and third-generation chemosensitizers (Fig. 2),
and found that most of P-gp inhibitors identified common features:
(i) high hydrophobicity; (ii) two or more aromatic rings; (iii) a
methoxy group on the aromatic ring (hydrogen bond acceptor);
(iv) one or two protonatable nitrogens.28,29 Building on this idea,
we designed various amine-substituted PPD analogues 2–26
(Scheme 1) in order to insert the nitrogen and the lipophilic moie-
ties. The amine groups (R in Scheme 1) were selected by consider-
ing size, hydrophobicity, and electron density. The diverse set
included aliphatic acyclic amine (group I), aliphatic cyclic amine
O
OH
OH
HO
OH
Rg3
OH
24
21
26
OH
20
25
27
23
16
13
11
19
17
18
1
4
9
2
15
10
30
7
HO
6
29
28
PPD
Figure 1. Structures of Rg3 and PPD.
First Generation
NO2
O
O
O
O
O
O
O
O
O
N
NC
N
H
Verapamil
Nimodipine
Second Generation
O
O
N
O
O
O
MeO
MeO
O
O
N
N
N
NC
O
OMe
dexverapamil
biricodar
Third Generation
H
N
N
O
O
H
N
OMe
OMe
N
N
H
O
N
OMe
OMe
H
OMe
O
N
H
OMe
Elacridar
Tariquidar
OMe
F
F
Br
H
O
N
N
N
O
NH2
N
N
Br
N
O
Cl
OH
Zosuquidar
Lonafarnib
Figure 2. Selected P-gp inhibitors.

J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4279–4287
4281
(group II), heterocycle-aliphatic amine (group III), polar aliphatic
amine (group IV), and aromatic substituted aliphatic amine (group
V) groups, which could be transformed into water-soluble salts, if
necessary. PPD were firstly treated with O3, then the related amine
(RNH2), and sodium triacetoxyborohydride (STAB) to produce 2–26
(Scheme 1).
moid carcinoma of the nasopharynx), and a resistant subline, KBvcr
(overexpression of P-gp selected using increasing concentrations of
vincristine). Although compounds 13, 15, 16, 19, 20 and 23 were
slightly cytotoxic, most of the PPD-derived compounds did not ex-
hibit significant cytotoxicity, which implied low toxicity of these
analogues.
For evaluating chemosensitizing activity, KBvcr cells were co-
treated with test compounds at 5
Docetaxel (DOC) (Fig. 3). As shown in Figure 3, PPD (1) did exhibit
a weak MDR reversal effect at a concentration of 5 M in KBvcr
cells. PPD analogues with aliphatic acyclic amine (group I), ali-
phatic cyclic amine (group II), heterocycle-aliphatic amine (group
III), and polar aliphatic amine (group IV) did show an inactive
activity (Fig. 3). However, PPD analogues with aromatic–aliphatic
amine substituents (group V) did show potent activity (Fig. 3).
The screening results suggested a rough SAR, including (a) the aro-
lM and the anticancer drug
3. Results and discussion
l
3.1. Evaluation of cytotoxicity and preliminary MDR reversal
activity screening
Based on the preliminary experiment, PPD (5
bit cytotoxicity, but exhibit a weak MDR reversal effect in KBvcr
cells. Thus, all synthesized compounds (5 M) were evaluated in
lM) did not exhi-
l
a cytotoxic activity assay using two tumor cell lines, KB (epider-
R
Cmpd
Aliphatic
9
17
Acyclic Group (I)
Me
Et
2
3
Polar Group (IV)
18
19
-(CH2)3OH
10
Pr
4
Aromatic-aliphatic Group (V) 20
iPr
5
6
11
12
21
22
tBu
Aliphatic
13
14
23
Cyclic Group (II)
7
8
24
25
26
Heterocycle-aliphatic Group (III) 15
16
Scheme 1. Syntheses of amines derivatives 2–26.a aReagents and conditions: (a) O3, CH2Cl2, À78 °C; (b) RNH2, NaBH(OAc)3, 0 °C.

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J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4279–4287
Figure 3. Screening of reversal abilities against KBvcr. Note: (a) Concentration of compounds: 5 lM; (b) survival rate (%) was measured by MTT method using KBvcr cells in
the presence (+) or absence (À) of docetaxel (DOC). Compounds with cell viability below 20% were considered very potent and moved to further experiments; (c) Group: (I)
aliphatic acyclic group, (II) aliphatic cyclic group, (III) heterocycle-aliphatic group, (IV) polar group, (V) aromatic–aliphatic group.
matic substituted aliphatic amine (group V) tended to enhance the
reversal ability of PPD analogues, but aliphatic acyclic amine
(group I), aliphatic cyclic amine (group II), heterocycle-aliphatic
amine (group III), and polar aliphatic amine (group IV) led to re-
duced activity; (b) one- or two-carbon linear chain in the aromatic
substituted aliphatic amine was effective in reversing MDR. Com-
pounds (12, 14, 17, 18, 21, 22, and 24–26) which contained one-
or two-carbon linear chain displayed the most significant activity.
But compound with 4-methoxy-benzenamine 11 was inactive, and
compound 23 containing three-carbon linear chain showed low
toxicity; (c) The electronic effect from the aromatic ring of the aro-
matic substituted aliphatic amine had a significant impact on
activity. For example, compounds with aromatic ring carrying elec-
tron-withdrawing (15, 16, 19, and 20) showed low toxicity, but
compounds with aromatic ring carrying electron-donating substit-
uents (14, 17, 21, and 22) displayed the most significant activity;
(d) Compounds with hetero-aromatic ring (24–26) also showed
potent activity.
culated, and fold reversal was determined by dividing the IC50 of
anticancer drug alone by the IC50 of anticancer drugs plus PPD ana-
logue (Table 1). Most of the tested analogues, including VER,
showed great reversal against DOC, VCR, and ADM resistance.
Especially, compounds 12 and 18 were 1.3–2.6 times more potent
than the positive control VER for chemoreversal ability against
DOC, VCR, and ADM resistance. The following SAR correlations
were proposed based on the chemosensitizing effects against
DOC, VCR, and ADM. Compounds with aromatic ring carrying no
substituents (12 and 18) were more potent activity than com-
pounds with hetero-aromatic ring (25) and aromatic ring carrying
electron-donating substituents (21).
On the basis of the above results, compounds 12 and 18 were
selected for further evaluation of chemosensitizing efficacy. The
dose–response proliferation inhibitory effects of DOC, VCR, and
ADM at 5 lM were analyzed against KB and KBvcr cells (Fig. 4).
In the absence of PPD compound or VER, KBvcr cells were resistant
to all three anticancer drugs, resulting in IC50 values over 1000 nM.
From these results, the most active compounds (12, 18, 21, and
25) were selected for further investigation.
When 5 lM of compound 12 or 18 or VER was added, the sensitiv-
ity of KBvcr cells to each anticancer drug was dramatically
increased. The chemosensitizing efficacy of 12 or 18 was either
similar or better than that of VER. These results demonstrated that
3.2. Chemoreversal ability of PPD analogues with docetaxel
(DOC), vincristine (VCR), and adriamycin (ADM)
5 lM of 12 and 18 effectively chemosensitized MDR cells.
A quantitative evaluation of the reversal ability of PPD ana-
logues 12, 18, 21, and 25 was performed using MDR KBvcr cells
with various concentrations of DOC, VCR, and ADM, which are clin-
ically used and known as significant P-gp substrates, partly
accounting for their resistance. The IC50 value of anticancer drugs
3.3. Dose–response effect of compounds 12 and 18 on
sensitization of KBvcr to DOC
To evaluate the reversal activity of 12 and 18 in a dose–
response manner, KBvcr cells were cultured with nontoxic concen-
tration of DOC (100 nM) in the presence of various concentrations
in the presence of test compounds at 5 lM concentration was cal-
Table 1
Effect of compounds on the cytotoxicity of DOC, VCR and ADM in the P-gp overexpressing KBvcr cancer cells
Compda
IC50 of DOCb (nM)
Foldc
IC50 of VCR (nM)
Fold
—
IC50 of ADM (nM)
Fold
5628 (DOC alone)
—
5091 (VCR alone)
13120 (ADM alone)
—
VER
PPD
12
18
21
75.9
525.5
50.9
28.7
142.6
117.7
74.2
10.7
110.7
196.1
39.5
47.8
85.2
645.1
64.9
48.0
201.8
188.3
66.1
8.7
86.7
117.3
27.9
29.9
301.1
935.6
233.2
211.8
452.1
278.5
43.6
14.0
56.3
61.9
29.0
47.1
25
a
b
c
Concentration of compound: 5
SD is shown in Supplementary data.
The reversal fold values were calculated as the following: reversal fold = IC50 (anticancer drug alone)/IC50 (anticancer drug + test compound).
lM.

J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4279–4287
4283
Figure 5. Dose–response effect of compounds 12 and 18 on sensitization of KBvcr
to DOC. Multidrug-resistant KBvcr cells were treated with various concentrations of
compounds 12 or 18 in the presence of 100 nM DOC, an absolutely nontoxic
concentration for KBvcr. Data are expressed as mean SD of three independent
experiments. Calculated median effective concentration (EC50) of compounds 12,
18, or VER was also listed.
Cellular accumulation of Rho123
15
12
18
VER
10
5
0
0
1
2
3
4
Compound(µM)
Figure 6. Effect of compounds on P-gp function in KBvcr cells. KBvcr cells were
pretreated with compounds followed by addition of Rhodamine123. The cellular
accumulation of Rhodamine123 is represented by the relative fluorescent unit
(Â104 RFU). Cellular accumulation of Rhodamine123 demonstrates inhibition of
efflux activity of P-gp. Data are shown with mean SD of three independent
experiments.
Figure 4. Reversal of chemosensitivity of KBvcr by 12 or 18. Chemoresistant KBvcr
cells were incubated with various concentrations of anticancer drugs DOC (A), VCR
(B), or ADM (C) in the presence of test compounds, as indicated, for 72 h to evaluate
the effect on chemosensitization. Chemosensitization of KBvcr cells was observed
drugs in MDR cells, the effect of compounds 12 and 18 on P-gp
function was investigated by using Rhodamine123 as a fluorogenic
P-gp substrate (Fig. 6). Dose-dependent intracellular accumulation
of Rhodamine123 was observed in the presence of these com-
pounds. Although 18 was slightly less potent than VER, 12 was
more potent than VER, especially at concentrations around the
when the cells were cotreated with 5 lM of 12, 18, or VER.
of compounds (Fig. 5). As we expected, compounds 12 and 18
exhibited reversal activity in a dose-dependent manner. The effec-
EC50 value (1.367 lM) of 12. Therefore, these results clearly indi-
tive concentration (EC50
)
values of 12 (1.367
l
M) and 18
cated that PPD analogues, especially 12, are effective P-gp
(1.310 M) were similar to that of VER (1.253
l
lM). These results
inhibitors.
demonstrate that the effect of 12 and 18 could be similar to that
of VER in chemosensitizing the MDR cells to DOC.
To demonstrate the effective efflux inhibition of anticancer
drugs, direct measurement of cellular accumulation of ADM in
KBvcr cells was studied as the intensity of intrinsic fluorescence
of ADM (Fig. 7). KBvcr cells were pretreated with compounds fol-
lowed by addition of ADM. Intracellular accumulation of ADM
was measured as the fluorescence intensity and standardized as
fold ratio. All PPD analogues induced ADM accumulation in KBvcr
3.4. The effect of PPD analogues on P-gp function in KBvcr cells
To confirm our hypothesis that PPD analogues inhibit efflux
activity of P-gp resulting in elevated concentration of anticancer

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J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4279–4287
were 6.10, 6.17 and 4.6, which is close to that of VER. This fact im-
plied that hydrophobicity is an important parameter in P-gp
inhibition.
4. Conclusion
In summary, we selected PPD as a lead, and twenty-five new
PPD analogues were newly designed and synthesized. All synthe-
sized analogues were evaluated for MDR chemosensitizing effects
on clinically used anticancer drugs, such as DOC, VCR, and ADM.
We succeeded to improve chemoreversal action by introducing
an aromatic substituted aliphatic amine group to PPD, and SAR
studies are summarized. The aromatic substituted aliphatic amine
(group V) tended to enhance the reversal ability of PPD analogues,
but aliphatic acyclic amine (group I), aliphatic cyclic amine (group
II), heterocycle-aliphatic amine (group III), and polar aliphatic
amine (group IV) led to reduce activity. Among all tested com-
pounds, PPD derivatives 12 and 18 were 1.3–2.6 times more effec-
tive than VER for DOC and VCR reversal ability. Importantly, active
Figure 7. Recovered ADM accumulation in PPD analogue-treated drug resistant
KBvcr cells. KBvcr cells were incubated in ADM medium (final concentration
10 lM) for 3 h in the presence of 5 lM PDD analogues, and then cellular
PPD analogues (5 lM) displayed no cytotoxicity against tumor
accumulation of ADM was measured as the intrinsic fluorescence intensity of
ADM. The fluorescence intensity of ADM was expressed as the ratio of effect of
compound to negative control (ADM). Intracellular accumulation of ADM was
clearly observed in the presence of PPD-derived compounds. Data are represented
cells, suggesting that our novel PPD analogues are significant lead
compounds for further clinical development to overcome the MDR
phenotype. Intracellular accumulation studies using calcein-AM
and ADM in KBvcr cells clearly demonstrated that PPD analogues
interfere with the P-gp drug efflux pump. To conclude, newly syn-
thesized PPD analogues were identified as P-gp inhibitors possess-
ing a new scaffold for nontoxic chemosensitizer drug development.
as mean SD, n = 3. P-gp inhibitor VER (5 lM) was used as a positive control.
cells at 1.3- to 2.1-fold. The cellular accumulation of ADM by PPD
analogues was consistent with sensitization of KBvcr cells to
ADM (Table 1). Thus, these data further support that PPD-derived
chemosensitizers function as P-gp inhibitors resulting in cellular
accumulation of anticancer drugs.
5. Experimental
5.1. General
The reagents (chemicals) were purchased from Lancaster, Alfa
Aesar, and Shanghai Chemical Reagent Co. and used without fur-
ther purification. Nuclear magnetic resonance (NMR) spectroscopy
was performed on a Bruker AMX-300 NMR (IS as TMS). Chemical
shifts were reported in parts per million (ppm, d) downfield from
tetramethylsilane. Proton coupling patterns were described as sin-
glet (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 chroma-
3.5. Hydrophobicity evaluation of active PDD analogues
P-gp is a large polytopic membrane protein which contains a
large drug-binding pocket including predominantly hydrophobic
and aromatic residues.30,31 Because of similarity, hydrophobic
and aromatic substrates would bind to the hydrophobic and
aromatic residues. Thus, it is possible that hydrophobicity of PPD
analogues could influence their MDR reversal activity. The clogP
values of synthesized compounds are showed in Table 2. Although
a few exceptions were present, the chemosensitizing effects of
compounds were moderately correlated with their clogP values.
Active compounds had clogP values of 4–7. The clogP values of
12, 18, and 25, which were significantly active as described above,
tography (TLC) on HSGF254 silica gel plates (150–200 lm thick-
ness; Yantai Huiyou Co., China). Ozone was produced with a
BGF-YQ ozone generator (2.0 L/min O2, 100 V; Beijing ozone Co.,
China). All final compounds are >95% pure based on HPLC.
Anhydrous solvents were purchased from commercial suppliers.
5.2. General synthetic procedure for compounds 2–26
Table 2
clogP values of synthesized PPD-derived compounds
Into a solution of PPD (1, 200 mg, 0.435 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 eliminated by bubbling nitrogen into the solution.
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 al-
lowed to reach 0 °C. After completion of the reaction (TLC monitor-
ing, CH2Cl2/CH3OH, 30:1, v/v, on silica gel plate), water was added,
and the product was isolated by extraction with dichloromethane.
The organic phase was washed with water, brine, dried over anhy-
drous sodium sulfate, filtered, and concentrated under vacuum to
obtain the desired crude products. The appropriate compounds
(2–26) were obtained following purification by silica gel column
Compd
clogPa
Compd
clogP
1
2
3
4
5
6
7
8
9
10
11
12
13
6.78
4.07
4.60
5.13
4.91
5.31
4.65
4.38
6.22
3.90
6.33
6.10
6.41
14
15
16
17
18
19
20
21
22
23
24
25
26
VER
6.02
6.24
6.81
6.12
6.17
6.89
5.91
6.27
6.13
6.55
4.6
4.6
4.6
4.58
a
clogP was calculated by ChemDraw Ultra Version 12.0.

J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4279–4287
4285
chromatography
with
dichloromethane/methanol/triethyl-
5.2.7. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((3-(1H-Imidazol-1-yl)
amine = 60:1:0.5 (v/v/v).
propyl)amino)-2-hydroxypentan-2-yl)-4,4,8,10,14-pentame
thylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-diol
(8)
5.2.1. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-(methyl am
ino)pentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-cy
clopenta[a]phenanthrene-3,12-diol (2)
Yield 90% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 7.49 (s, 1H), 7.05 (s, 1H), 6.92 (s, 1H), 4.00
(m, 2H), 3.55 (td, J = 12.7, 6.0 Hz, 1H), 3.19 (dd, J = 11.0, 5.2 Hz,
1H), 2.88 (m, 1H), 2.68 (m, 1H), 2.52 (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 = 10.9 Hz,
1H); HRMS (ESI) m/z calcd for C33H58N3O3 (M+H)+ 544.4473, found
544.4470.
Yield 85% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 4.12 (m, 1H), 3.52 (td, J = 13.0, 6.2 Hz, 1H),
3.21 (dd, J = 10.9, 5.0 Hz, 1H), 2.94 (m, 1H), 2.62 (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 = 11.0 Hz, 1H); HRMS (ESI) m/z calcd for C28H52NO3
(M+H)+ 450.3942, found 450.3945.
5.2.8. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((2-(pipera
zin-1-yl)ethyl)amino)pentan-2-yl)-4,4,8,10,14-pentamethyl
hexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (9)
Yield 80% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 5.31 (d, J = 1.4 Hz, 1H), 3.57 (td, J = 13.0,
5.6 Hz, 1H), 3.20 (dd, J = 10.9, 5.3 Hz, 1H), 2.80 (t, J = 6.0 Hz, 2H),
2.47 (m, 12H), 1.14 (s, 3H), 0.98 (s, 6H), 0.89 (s, 6H), 0.78 (s, 3H),
0.73 (d, J = 10.9 Hz, 1H); HRMS (ESI) m/z calcd for C33H62N3O3
(M+H)+ 548.4786, found 548.4784.
5.2.2. (3S,8R,10R,12R,14R,17S)-17-((S)-5-(Ethylamino)-2-hydrox
ypentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-
cyclopenta[a]phenanthrene-3,12-diol (3)
Yield 86% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 3.55 (td, J = 12.9, 6.3 Hz, 1H), 3.19 (dd,
J = 10.9, 4.9 Hz, 1H), 2.93 (m, 1H), 2.72 (m, 1H), 2.58 (m, 1H),
2.48 (m, 1H), 2.09 (m, 1H), 1.15 (s, 3H), 1.11 (t, J = 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, J = 10.7 Hz, 1H); HRMS (ESI) m/z calcd for C29H54NO3
(M+H)+ 464.4098, found 464.4094.
5.2.9. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((3-hydrox
ypropyl)amino)pentan-2-yl)-4,4,8,10,14-pentamethylhexadeca
hydro-1H-cyclopenta[a]phenanthrene-3,12-diol (10)
5.2.3. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-(propy lam i
no)pentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-cy
clopenta[a]phenanthrene-3,12-diol (4)
Yield 80% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 3.74 (t, J = 5.7 Hz, 2H), 3.54 (td, J = 12.6,
5.8 Hz, 1H), 3.20 (dd, J = 10.8, 5.4 Hz, 1H), 2.89 (m, 2H), 2.77 (m,
1H), 2.60 (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), 0.72 (d, J = 10.7 Hz, 1H); HRMS
(ESI) m/z calcd for C30H56NO4 (M+H)+ 494.4204, found 494.4207.
Yield 87% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 3.55 (td, J = 12.7, 6.2 Hz, 1H), 3.20 (dd,
J = 10.8, 5.2 Hz, 1H), 2.94 (m, 1H), 2.67 (m, 1H), 2.52 (m, 2H),
2.11 (m, 1H), 1.14 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.93 (t,
J = 7.3 Hz, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.78 (s, 3H), 0.73 (d,
J = 10.9 Hz, 1H); HRMS (ESI) m/z calcd for C30H56NO3 (M+H)+
478.4255, found 478.4257.
5.2.10. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((4-metho
xyphenyl)amino)pentan-2-yl)-4,4,8,10,14-pentamethylhexa
decahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (11)
Yield 90% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 6.76 (d, J = 9.0 Hz, 2H), 6.68 (d, J = 9.0 Hz,
2H), 3.71 (s, 3H), 3.54 (td, J = 12.8, 6.3 Hz, 1H), 3.14 (dd, J = 11.0,
5.2 Hz, 1H), 3.05 (t, J = 6.4 Hz, 2H), 2.04 (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 = 10.6 Hz,
1H); HRMS (ESI) m/z calcd for C34H56NO4 (M+H)+ 542.4204, found
542.4208.
5.2.4. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-(isopropyl
amino)pentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-
1H-cyclopenta[a]phenanthrene-3,12-diol (5)
Yield 88% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 3.57 (td, J = 12.5, 6.0 Hz, 1H), 3.19 (dd,
J = 10.8, 5.2 Hz, 1H), 2.95 (d, J = 12.6 Hz, 1H), 2.85 (m, 1H), 2.50
(m, 1H), 2.10 (m, 1H), 1.14 (s, 3H), 1.13 (d, J = 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 = 10.9 Hz, 1H); HRMS (ESI) m/z calcd for C30H56NO3 (M+H)+
478.4255, found 478.4258.
5.2.11. (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 (12)
5.2.5. (3S,8R,10R,12R,14R,17S)-17-((S)-5-(tert-Butylamino)-2-hy
droxypentan-2-yl)-4,4,8,10,14-pentamethylhexadecahydro-1H-
cyclopenta[a]phenanthrene-3,12-diol (6)
Yield 90% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 7.32 (m, 5H), 3.74 (s, 2H), 3.57 (td, J = 12.9,
6.0 Hz, 1H), 3.20 (dd, J = 10.8, 5.2 Hz, 1H), 2.97 (d, J = 12.3 Hz,
1H), 2.78 (dd, J = 12.3, 7.2 Hz, 1H), 2.52 (t, J = 11.0 Hz, 1H), 2.12
(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 = 10.7 Hz, 1H); HRMS (ESI) m/z calcd
for C34H56NO3 (M+H)+ 526.4255, found 526.4253.
Yield 88% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 5.30 (s, 1H), 3.57 (td, J = 12.3, 6.1 Hz, 1H),
3.19 (dd, J = 10.7, 5.3 Hz, 1H), 2.93 (d, J = 10.8 Hz, 1H), 2.44 (t,
J = 11.5 Hz, 1H), 2.09 (m, 1H), 1.13 (s, 12H), 0.97 (s, 6H), 0.88 (s,
6H), 0.77 (s, 3H), 0.72 (d, J = 10.8 Hz, 1H); HRMS (ESI) m/z calcd
for C31H58NO3 (M+H)+ 492.7966, found 492.7969.
5.2.12. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-(((S)-1-
phenylethyl)amino)pentan-2-yl)-4,4,8,10,14-
5.2.6. (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 (7)
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-
3,12-diol (13)
Yield 86% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 7.29 (m, 5H), 3.82 (q, J = 6.7 Hz, 1H), 3.56
(td, J = 12.8, 5.9 Hz, 1H), 3.19 (dd, J = 10.8, 5.3 Hz, 1H), 2.69 (m,
1H), 2.40 (m, 1H), 2.11 (m, 1H), 1.40 (d, J = 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, J = 10.8 Hz, 1H); HRMS (ESI) m/z calcd for C35H58NO3
(M+H)+ 540.4411, found 540.4414.
Yield 80% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 3.54 (td, J = 12.6, 6.2 Hz, 1H), 3.20 (dd,
J = 10.7, 5.3 Hz, 1H), 3.05 (m, 1H), 2.95 (dd, J = 11.0, 7.2 Hz, 1H),
2.56 (t, J = 11.0 Hz, 1H), 2.11 (m, 1H), 2.07 (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 = 10.9 Hz, 1H); HRMS (ESI) m/z calcd for C30H54NO3 (M+H)+
476.4098, found 476.4095.

4286
J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4279–4287
5.2.13. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((4-metho
xybenzyl)amino)pentan-2-yl)-4,4,8,10,14-pentamethylhexad
ecahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (14)
Yield 91% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 7.21 (dd, J = 8.6, 4.2 Hz, 2H), 6.87 (dd, J = 8.6,
4.2 Hz, 2H), 3.80 (s, 3H), 3.79 (s, 2H), 3.69 (s, 1H), 3.57 (td,
J = 12.8, 6.1 Hz, 1H), 3.20 (dd, J = 10.8, 5.3 Hz, 1H), 2.96 (d,
J = 12.0 Hz, 1H), 2.52 (t, J = 11.0 Hz, 1H), 2.11 (m, 1H), 1.14 (s,
3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.78 (s,
3H), 0.73 (d, J = 10.9 Hz, 1H); HRMS (ESI) m/z calcd for C35H58NO4
(M+H)+ 566.4360, found 566.4362.
5.2.19. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((4-nitroph
enethyl)amino)pentan-2-yl)-4,4,8,10,14-pentamethylhexadeca
hydro-1H-cyclopenta[a]phenanthrene-3,12-diol (20)
Yield 83% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 8.16 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.8 Hz,
2H), 3.47 (m, 2H), 3.18 (m, 2H), 3.08 (m, 4H), 2.80 (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 = 11.0 Hz, 1H); HRMS (ESI) m/z calcd for C35H57N2O5 (M+H)+
585.4262, found 585.4260.
5.2.20. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((3,4-Dimethoxyph
enethyl)amino)-2-hydroxypentan-2-yl)-4,4,8,10,14-pentamethy
lhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (21)
Yield 93% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 6.80 (d, J = 8.2 Hz, 1H), 6.73 (d, J = 8.2 Hz,
1H), 6.71 (s, 1H), 5.30 (s, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.56 (td,
J = 12.8, 6.0 Hz, 1H), 3.20 (dd, J = 10.9, 5.3 Hz, 1H), 2.80 (m, 5H),
2.52 (t, J = 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), 0.73 (d, J = 11.0 Hz, 1H); HRMS
(ESI) m/z calcd for C37H62NO5 (M+H)+ 600.4623, found 600.4620.
5.2.14. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((4-Fluorobenzyl)
amino)-2-hydroxypentan-2-yl)-4,4,8,10,14-pentamethylhexa
decahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (15)
Yield 88% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 7.32 (dd, J = 8.6, 5.4 Hz, 2H), 7.03 (dd, J = 8.6,
5.4 Hz, 2H), 5.30 (s, 1H), 3.84 (s, 2H), 3.53 (td, J = 12.9, 6.0 Hz,
1H), 3.20 (dd, J = 11.0, 5.2 Hz, 1H), 2.98 (m, 1H), 2.65 (m, 1H),
2.09 (m, 1H), 1.12 (s, 3H), 0.98 (s, 6H), 0.88 (s, 6H), 0.78 (s, 3H),
0.73 (d, J = 10.8 Hz, 1H); HRMS (ESI) m/z calcd for C34H55NO3
(M+H)+ 544.4161, found 544.4165.
5.2.21. (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 (22)
5.2.15. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((4-Chlorobenzyl)
amino)-2-hydroxypentan-2-yl)-4,4,8,10,14-pentamethylhexad
ecahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (16)
Yield 87% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 7.29 (d, J = 6.1 Hz, 2H), 7.20 (d, J = 6.1 Hz,
2H), 3.84 (s, 1H), 3.71 (s, 2H), 3.56 (td, J = 12.9, 5.9 Hz, 1H), 3.19
(dd, J = 10.9, 5.3 Hz, 1H), 2.94 (m, 1H), 2.52 (t, J = 10.1 Hz, 1H),
2.10 (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 = 10.7 Hz, 1H); HRMS (ESI) m/z
calcd for C34H55ClNO3 (M+H)+ 560.3865, found 560.3863.
Yield 92% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 6.72 (d, J = 7.8 Hz, 1H), 6.65 (d, J = 1.6 Hz,
1H), 6.61 (dd, J = 7.8, 1.6 Hz, 1H), 5.91 (s, 2H), 3.56 (td, J = 12.8,
5.8 Hz, 1H), 3.19 (dd, J = 10.9, 5.3 Hz, 1H), 2.78 (m, 6H), 2.47 (m,
1H), 2.09 (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), 0.73 (d, J = 10.7 Hz, 1H); HRMS
(ESI) m/z calcd for C36H58NO5 (M+H)+ 584.4310, found 584.4312.
5.2.22. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((3-phenyl
propyl)amino)pentan-2-yl)-4,4,8,10,14-pentamethylhexadeca
hydro-1H-cyclopenta[a]phenanthrene-3,12-diol (23)
5.2.16. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((3,4-Dimethoxy
benzyl)amino)-2-hydroxypentan-2-yl)-4,4,8,10,14-pentamethy
lhexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (17)
Yield 92% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 6.93 (s, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.84 (d,
J = 8.4 Hz, 1H),5.30 (s, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.82 (s, 2H),
3.53 (td, J = 12.8, 6.0 Hz, 1H), 3.20 (dd, J = 10.8, 5.3 Hz, 1H), 2.96
(m, 1H), 2.69 (m, 1H), 2.08 (m, 1H), 1.12 (s, 3H), 0.98 (s, 6H),
0.88 (s, 6H), 0.78 (s, 3H), 0.71 (d, J = 10.8 Hz, 1H), HRMS (ESI) m/z
calcd for C36H60NO5 (M+H)+ 586.4466, found 586.4468.
Yield 90% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 7.22 (m, 5H), 3.53 (td, J = 13.1, 6.5 Hz, 1H),
3.19 (dd, J = 10.8, 5.3 Hz, 1H), 2.90 (m, 1H), 2.66 (m, 6H), 2.07 (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, 3H), 0.72 (d, J = 10.7 Hz, 1H); HRMS (ESI) m/z calcd
for C36H60NO3 (M+H)+ 554.4568, found 554.4565.
5.2.23. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((pyridin-
2-ylmethyl)amino)pentan-2-yl)-4,4,8,10,14-pentamethylhexa
decahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (24)
Yield 82% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 8.53 (d, J = 4.9 Hz, 1H), 7.64 (t, J = 4.9 Hz,
1H), 7.20 (d, J = 4.9 Hz, 1H), 7.16 (t, J = 4.9 Hz, 1H), 3.85 (s, 1H),
3.83 (s, 1H), 3.55 (d, J = 12.8, 5.9 Hz, 1H), 3.19 (dd, J = 11.0,
5.1 Hz, 1H), 2.92 (m, 1H), 2.47 (m, 1H), 2.06 (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 = 10.9 Hz, 1H); HRMS (ESI) m/z calcd for C33H55N2O3 (M+H)+
527.4207, found 527.4204.
5.2.17. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-
(phenethylamino)pentan-2-yl)-4,4,8,10,14-
pentamethylhexadecahydro-1H-cyclopenta[a]phenanthrene-
3,12-diol (18)
Yield 90% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 7.23 (m, 5H), 3.56 (td, J = 13.1, 6.1 Hz, 1H),
3.20 (dd, J = 10.9, 4.9 Hz, 1H), 2.83 (m, 5H), 2.54 (m, 1H), 2.07 (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 = 10.9 Hz, 1H); HRMS (ESI) m/z calcd
for C35H58NO3 (M+H)+ 540.4411, found 540.4414.
5.2.24. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((pyridin-
3-ylmethyl)amino)pentan-2-yl)-4,4,8,10,14-pentamethylhexa
decahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (25)
Yield 81% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 8.52 (s, 1H), 8.43 (d, J = 4.7 Hz, 1H), 7.85 (d,
J = 7.4 Hz, 1H), 7.41 (dd, J = 7.4, 4.7 Hz, 1H), 3.79 (s, 2H), 3.53 (d,
J = 13.1, 5.7 Hz, 1H), 3.14 (dd, J = 10.8, 5.2 Hz, 1H), 2.63 (m, 2H),
2.02 (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, J = 10.7 Hz, 1H); HRMS (ESI) m/z calcd for
5.2.18. (3S,8R,10R,12R,14R,17S)-17-((S)-5-((4-Chlorophenethyl)
amino)-2-hydroxypentan-2-yl)-4,4,8,10,14-pentamethyl
hexadecahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (19)
Yield 84% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 7.29 (d, J = 6.2 Hz, 2H), 7.17 (d, J = 6.2 Hz,
2H), 3.58 (td, J = 12.7, 5.9 Hz, 1H), 3.19 (dd, J = 10.8, 5.2 Hz, 1H),
2.73 (m, 4H), 2.38 (m, 1H), 2.11 (m, 1H), 1.16 (s, 3H), 0.98 (s,
3H), 0.97 (s, 3H), 0.88 (s, 3H), 0.87 (s, 3H), 0.77 (s, 3H), 0.72 (d,
J = 10.9 Hz, 1H); HRMS (ESI) m/z calcd for C35H57ClNO3 (M+H)+
574.4022, found 574.4025.
C
33H55N2O3 (M+H)+ 527.4207, found 527.4205.

J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4279–4287
4287
5.2.25. (3S,8R,10R,12R,14R,17S)-17-((S)-2-Hydroxy-5-((pyridin-
4-ylmethyl)amino)pentan-2-yl)-4,4,8,10,14-pentamethylhexa
decahydro-1H-cyclopenta[a]phenanthrene-3,12-diol (26)
Yield 81% (starting from 200 mg of 1); white power; 1H NMR
(300 MHz, CDCl3): d 8.56 (dd, J = 4.4, 1.6 Hz, 2H), 7.23 (dd, J = 4.4,
1.6 Hz, 2H), 3.77 (s, 2H), 3.58 (td, J = 12.9, 5.8 Hz, 1H), 3.21 (dd,
J = 11.0, 5.3 Hz, 1H), 2.96 (m, 1H), 2.56 (m, 1H), 2.13 (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, J = 10.8 Hz, 1H); HRMS (ESI) m/z calcd for
The excitation and emission wavelengths of ADM were 485 and
590 nm, respectively. The excitation and emission wavelengths of
Rhodamine123 were 494 and 517 nm, respectively. Protein con-
centrations were measured by the Lowry method using a Bio-Rad
BCA protein assay kit with bovine serum albumin as the standard.
Accumulation ratios were calculated using the accumulation of
Rhodamine123 in cells incubated without treatment as a control.
Acknowledgments
C
33H55N2O3 (M+H)+ 527.4207, found 527.4203.
This work was supported by the National Natural Science Foun-
dation of China (Grants 30925040, 81102329, 81273397), the Chi-
nese National Science Technology Major Project ‘Key New Drug
Creation and Manufacturing Program’ (Grants 2013ZX09508104,
2012ZX09301001-001, 2011ZX09307-002-03), and the Science
Foundation of Shanghai (Grant 12XD1405700).
5.3. Cell culture
The human oral squamous cell carcinoma cell line KB and its
drug-resistant variant KBvcr were obtained from the American
Type Culture Collection. These cell lines were maintained in Dul-
becco’s Minimum Essential Medium (Invitrogen) supplemented
with 10% fetal bovine serum, 100 units/ml penicillin and 100 lg/
ml streptomycin. The cells were incubated in a humidified atmo-
Supplementary data
sphere with 5% CO2 at 37 °C.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.04.067.
5.4. Cell viability
References and notes
Cell viability was assessed with an MTT assay, which utilizes the
ability of viable cells to reduce the MTT reagent to colored forma-
zan products. In brief, cells were seeded in 96-well microculture
plates for 12 h to attach and then incubated for 72 h with various
concentrations of compounds. MTT was added to each well, and
incubated for 4 h.32 The colored formazan product was quantified
photometrically at 490 nm in a multiwell plate reader (Bio-Rad
Laboratories).
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presence of 5 lM test compound. IC50 values were calculated by
log–linear interpolation of data points. VRP, as a known P-gp inhib-
itor/modulator, was used as a positive control in all experiments.
The reversal fold value, as the potency parameter of test com-
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5.6. Measurement of the cellular accumulation of
Rhodamine123 and ADM
The accumulation of two fluorescent substrates of P-gp were
measured and the effects of tested compounds were determined
as described previously.33 Briefly, KBvcr cells were plated at
1 Â 105 cells/well in 24-well plates and incubated with 10
lM Rho-
damine123 or ADM in the presence or absence of tested com-
pounds for 2 h in a CO2 incubator at 37 °C. After incubation, the
medium was removed by aspiration, and the cells were washed
with ice-cold phosphate-buffered saline (PBS) and lysed with 1%
sodium dodecyl sulfate (SDS) in PBS. Fluorescence intensity was
measured with a microplate fluorometer (Fluoroskan Ascent).
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