Synthesis and structure-activity relationships study of cytotoxic bufalin 3-nitrogen-containing-ester derivatives

Article abstract of DOI:10.1016/j.steroids.2013.02.007

A series of bufalin 3-nitrogen-containing-ester derivatives (2-6) were designed, synthesized, and evaluated for their proliferation inhibition activities against human cervical epithelial adenocarcinoma (HeLa) and non-small-cell lung cancer (A549) cell lines. The structure-activity relationships (SARs) of this new series were described in this paper. Cytotoxicity data revealed that C3 moiety had important influence on cytotoxic activity. On two cell lines, the bufalin-3-piperidinyl-4-carboxylate compound 2 (IC₅₀ values on HeLa and A549 cell lines were 0.76 nM and 0.34 nM, respectively) displayed a significant cytotoxic potency compared to the parent compound bufalin.

Full text of DOI:10.1016/j.steroids.2013.02.007

Steroids 78 (2013) 508–512
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and structure–activity relationships study of cytotoxic bufalin
3-nitrogen-containing-ester derivatives
⇑
⇑
Biao Ma, Zhi-Yong Xiao, Yi-Jia Chen, Min Lei, Yu-Hui Meng, De-An Guo, Xuan Liu , Li-Hong Hu
Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of bufalin 3-nitrogen-containing-ester derivatives (2–6) were designed, synthesized, and evalu-
ated for their proliferation inhibition activities against human cervical epithelial adenocarcinoma (HeLa)
and non-small-cell lung cancer (A549) cell lines. The structure–activity relationships (SARs) of this new
series were described in this paper. Cytotoxicity data revealed that C3 moiety had important influence on
cytotoxic activity. On two cell lines, the bufalin-3-piperidinyl-4-carboxylate compound 2 (IC50 values on
HeLa and A549 cell lines were 0.76 nM and 0.34 nM, respectively) displayed a significant cytotoxic
potency compared to the parent compound bufalin.
Received 24 October 2012
Received in revised form 16 January 2013
Accepted 11 February 2013
Available online 26 February 2013
Keywords:
Bufalin
Bufalin 3-nitrogen-containing-ester
Structure–activity relationships
Cytotoxicity
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
associated downstream signaling pathways such as phospholipase
C (PLC) signaling, mitogen-activated protein kinase (MAPK) signal-
Bufadienolides are a type of natural cardiac steroids with potent
antitumor activities, originally isolated from the Traditional Chi-
nese Medicine Chan’Su. Bufalin (Fig. 1) is one of major components
of Chan’Su, and its content in the crude drug can be as high as 1–5%
of the dry weight. It also is one of the most potent bufadienolides
against cancer cells, such as human prostate carcinoma cells (PC3,
DU145) [1], human leukemia cells (U937, HL60) [2,3] and human
cervical carcinoma cells (HeLa) [4], with IC50 values of 10ꢀ9 to
10ꢀ8 mol/L. Furthermore, bufalin can inhibit the growth of human
HepG2 cell-transplanted tumor in nude mice and prolong the sur-
vival of host significantly [5]. The mechanism of the anti-cancer ef-
fects of bufalin is still subject to study. While, similar to other
cardiac glycosides, bufalin could also bind to Na+/K+-ATPase and
inhibit the Na+/K+ pump [6,7]. Therefore, present knowledge about
the anti-cancer mechanisms of other cardiac glycosides such as
ouabain or digitoxin might provide useful information for the
understanding of the mechanism of bufalin. By inhibiting the
Na+/K+ pump, cardiac glycosides could increase the intracellular
concentration of Ca2+ [(Ca2+) intra], which would lead to cardio-
tonic effect in cardiomyocytes but cause endoplasmic reticulum
stress that eventually become lethal in cancer cells expressing par-
ticular Na+/K+-ATPase subunits [8]. Furthermore, at lower dose that
would not exhibit inhibiting effect on Na+/K+ pump, cardiac glyco-
sides could activate Na+/K+-ATPase signalosome and thus activate
ing, phosphatidyl-inositol-3-kinase (PI3 K) signaling, and Src ki-
nase signaling. The activation of Na+/K+-ATPase signalosome
would influence cellular activities including apoptosis, cell prolif-
eration, cell motility, and tight junctions [9]. Similar results were
also found in the study of bufalin. For example, the cytotoxicity
and anti-invasion effects of bufalin on cancer cells were also found
to be related to its inhibition on MAPK pathway [10,11].
From the 1990s, a lot of bufalin analogues have been prepared
by isolation, and chemical or biological transformation, and these
compounds are evaluated in vitro and in vivo for their cytotoxici-
ties [12–16]. The essential structural requirements of bufalin for
increasing cytotoxicities have been indentified: a steroidal C/D
cis ring junctures with a C14b-hydroxyl group and a C17b-2-pyr-
one ring [17]. Unfortunately, these efforts have provided few
new more active bufalin derivatives. Therefore, new ideas and ap-
proaches are needed to extend investigations of the use of these
bufadienolides as anticancer agents. Recently, O’Doherty et al
found that introduction of mono-sugars at C3 site of digitoxigenin
(Fig. 1) enhanced its cytotoxicity [18–20]. Henrik group reported
that ethylene glycol linked digitoxin mimics were comparable with
digitoxigenin for cytotoxicity [21]. However, both synthesis of the
sugar and sugar-mimics coupling with bufalin are laborious, which
hinder further analogue development.
Acknowledging the limited SARs for bufalin 3-O-glycosides and
its cumbersome preparation, it is obvious that structurally simpler
polar substituents could be advantageously grafted on C3 of bufa-
lin to increase activity while simultaneously increasing solubility.
⇑
Corresponding authors. Tel.: +86 21 20231965.
E-mail addresses: xuanliu@mail.shcnc.ac.cn (X. Liu), simmhulh@mail.shcnc.
N-Heterocycles are found in
a variety of biologically active
ac.cn (L.-H. Hu).
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.02.007

B. Ma et al. / Steroids 78 (2013) 508–512
509
protecting group was removed with p-toluenesulfonic acid in
methanol to give desired products 2–5. Of note, TBS protecting
group was also hydrolyzed in the same condition to afford com-
pound 6. For a further insight into the impact of C40-nitrogen atom
in piperidine ring on their cytotoxicities, we synthesized different
substituents at C40-nitrogen atom. The N-acetylated derivative 2b
was synthesized from 2 via acylation reaction with acetic anhy-
dride (Scheme 2). N-alkylated derivatives 2c–e was achieved via
reductive amination of
2 and the corresponding aldehyde
(Scheme 3). All reactions were performed under a nitrogen atmo-
sphere with dry solvents in oven-baked or flame-dried glassware,
unless otherwise noted. All reagents were commercially available,
and were used without further purification unless otherwise spec-
ified. All solvents were redistilled under argon atmosphere. The
progress of reactions were monitored by thin layer chromatogra-
phy (TLC) plates (silica gel 60GF, Yantai jiangyou company) visual-
ized with 254-nm UV light and/or by staining with Vanillin
solution (2.7 g Vanillin + 100 mL H2O + 35 mL concentrated H2SO4
diluted to 300 mL with 95% ethanol). 1H and 13C NMR spectra were
recorded on a Varian Mercury-VX300 Fourier transform spectrom-
eter or a Bruker AM-400 spectrometer, and chemical shifts (d) were
reported in ppm; the hydrogenated residues of deuterated solvent
were used as internal standard CDCl3: 7.26 ppm for 1H NMR,
77.00 ppm for 13C NMR; CD3OD: 3.31 ppm for 1H NMR; DMSO-
d6: 2.50 ppm for 1H NMR, 39.52 ppm for 13C NMR. ESIMS was
run on a Bruker Esquire 3000 plus spectrometer in MeOH. HRE-
Fig. 1. Structures of bufalin, digitoxigenin and digitoxin mono-O-digitoxoside.
compounds and are easily to form water-soluble salts with organic
or inorganic acids [22]. Therefore, we designed and synthesized a
series of nitrogen-contained carbocycle esters, and the structures
of these compounds were identified using MS, 1H NMR and 13C
NMR spectroscopy. Meanwhile, their biological activities (IC50 val-
ues) were compared using a MTT assay with two human cancer cell
lines including human lung cancer cells A549 and human cervical
carcinoma cells HeLa. Bufalin was used as reference.
2. Experimental
The derivatives 2–6 were synthesized from bufalin by the meth-
od as shown in Scheme 1. Boc-masked amino acids were reacted
with bufalin to give nitrogen-contained bufalin esters, then Boc
SIMS were determined on
spectrometer.
a Micromass Q-Tof Global mass
Scheme 1. Reagents and conditions: (a) R1COOH, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, DCM, 12 h, rt and (b) TsOH, MeOH, 2 d, rt.
Scheme 2. Reagents and conditions: (a) AcCl, 4-Dimethylaminopyridine, DCM, 6 h, rt.

510
B. Ma et al. / Steroids 78 (2013) 508–512
Scheme 3. Reagents and conditions: (a) R3CHO, NaBH3CN, AcOH, MeOH, 12 h, rt.
2.1. General synthesis of bufalin 3-nitrogen-containing-ester
derivatives
2.1.2. Compound 3
Synthesis of compound 3 with method A to yield a white, amor-
phous powder. Yield: 53%; 1H NMR (300 MHz, CDCl3): d 7.83 (d,
J = 9.0 Hz, 1H, H22), 7.22 (s, 1H, H21), 6.24 (d, J = 9.0 Hz, 1H,
H23), 5.08 (s, 1H, H3), 3.66 (s, 1H, NH), 3.19–3.15 (m, 1H, H20a),
2.93–2.91 (m, 1H, H20b), 2.79–2.75 (m, 1H, H40a), 2.63–2.61 (m,
1H, H40b), 2.47–2.42 (m, 2H, H10), 2.23–1.16 (m, 26H), 0.95 (s,
3H, H19), 0.70 (s, 3H, H18); 13C NMR (75 MHz, CDCl3): d 173.6,
162.4, 148.5, 146.8, 122.6, 115.3, 85.3, 70.2, 51.2, 48.4, 46.2, 42.6,
42.3, 40.8, 36.9, 35.8, 35.1, 32.7, 30.5, 30.4, 28.7, 27.4, 26.4, 25.4,
25.0, 23.8, 23.7, 21.4, 21.3, 16.5; MS (ESI) m/z: 498 [M + H]+; HRMS
(ESI): calcd for C30H44NO5 [M + H]+ 498.3214, found 498.3198.
Method A: To a stirred solution of bufalin (200 mg, 0.52 mmol),
4-dimethylaminopyridine (63 mg, 0.52 mmol, 1 eq), and N-pro-
tected acid (1.56 mmol, 3 eq) in anhydrous DCM (2 mL) at room
temperature under nitrogen gas was added 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride (300 mg, 1.56 mmol,
3 eq), and stirring continued for 12 h. After completion (by TLC),
DCM (10 mL), and H2O (10 mL) were added. The organic phase
was separated, washed with H2O and brine, dried over Na2SO4,
and concentrated in vacuo to afford yellow residue. To a solution
of the residue in MeOH (2.5 mL) was added a solution of TsOH in
MeOH (2.5 mL, 4%), and stirring continued at room temperature
for 2d. After completion (by TLC), DCM (15 mL) and H2O (15 mL)
were added. The organic phase was separated, washed with aq.
Na2CO3 and brine, dried over Na2SO4, and concentrated in vacuo
to give yellow residue. Dissolved in minimal DCM, the crude prod-
uct was subjected to purify on silica gel.
Method B: To a stirred solution of bufalin-3-piperidinyl-4-car-
boxylate compound 2 (200 mg, 0.52 mmol) and 4-dimethylamino-
pyridine (32 mg, 0.26 mmol, 0.5 eq) in anhydrous DCM (2 mL) at
room temperature under nitrogen gas was added acyl chloride
(0.78 mmol, 1.5 eq) or anhydride (0.78 mmol, 1.5 eq) dropwise,
and stirring continued for 6 h. After completion (by TLC), the mix-
ture solution was extracted with DCM (twice). The organic phase
was combined, washed with aq. Na2CO3 and brine, dried over Na2-
SO4, and concentrated in vacuo to give yellow residue. Dissolved in
minimal DCM, the crude product was subjected to purify on silica
gel.
2.1.3. Compound 4
Synthesis of compound 4 with method A to yield a white, amor-
phous powder. Yield: 55%; 1H NMR (300 MHz, CDCl3): d 7.83 (d,
J = 9.0 Hz, 1H, H22), 7.22 (s, 1H, H21), 6.25 (d, J = 9.0 Hz, 1H,
H23), 5.12 (s, 1H, H3), 3.34–3.31 (m, 1H, H10), 3.10–3.07 (m, 1H,
H30a), 2.68–2.64 (m, 1H, H30b), 2.48–2.43 (m, 1H), 2.24–1.15 (m,
28H), 0.97 (s, 3H, H19), 0.70 (s, 3H, H18); 13C NMR (75 MHz,
CDCl3): d 172.8, 162.4, 148.4, 146.9, 122.7, 115.0, 85.0, 70.7, 58.5,
51.0, 48.2, 45.6, 42.1, 40.7, 36.8, 35.7, 35.0, 32.6, 30.4, 30.2, 29.2,
28.6, 26.3, 25.7, 24.9, 24.0, 23.7, 21.3, 21.1, 16.4; MS (ESI) m/z:
498 [M + H]+; HRMS (ESI): calcd for C30H44NO5 [M + H]+
498.3214, found 498.3223.
2.1.4. Compound 5
Synthesis of compound 5 with method A to yield a white, amor-
phous powder. Yield: 61%; 1H NMR (300 MHz, CDCl3): d 7.84 (d,
J = 9.0 Hz, 1H, H22), 7.23 (s, 1H, H21), 6.26 (d, J = 9.0 Hz, 1H,
H23), 5.13 (s, 1H, H3), 3.76 (dd, J = 9.0, 6.0 Hz, 1H, H10), 3.12–
3.08 (m, 1H, H30a), 2.93–2.90 (m, 1H, H30b), 2.49–2.44 (m, 1H),
2.25–1.18 (m, 26H), 0.96 (s, 3H, H19), 0.70 (s, 3H, H18); 13C NMR
(75 MHz, CDCl3): d 174.8, 162.4, 148.5, 146.8, 122.6, 115.3, 85.2,
77.2, 71.1, 59.9, 51.2, 48.3, 76.0, 42.3, 40.8, 36.8, 35.8, 35.1, 32.7,
30.5, 30.3, 28.7, 26.3, 25.5, 25.0, 23.8, 21.4, 21.3, 16.5; MS (ESI)
m/z: 484 [M + H]+; HRMS (ESI): calcd for C29H42NO5 [M + H]+
484.3057, found 484.3042.
Method C: To a solution of compound bufalin-3-piperidinyl-4-
carboxylate compound 2 (200 mg, 0.40 mmol) in anhydrous MeOH
(2 mL) was added with aldehyde (0.60 mmol, 1.5 eq), AcOH
(0.24 mmol, 0.6 eq) and NaBH3CN (1.20 mmol, 3 eq), then the
resulting solution was stirred at room temperature overnight. After
completion (by TLC), DCM (15 mL) and H2O (15 mL) were added.
The organic phase was separated, washed with H2O and brine,
dried over Na2SO4, and concentrated in vacuo to give yellow resi-
due. Dissolved in minimal DCM, the crude product was subjected
to purify on silica gel.
2.1.5. Compound 6
2.1.1. Compound 2
Synthesis of compound 6 with method A to yield a white, amor-
phous powder. Yield: 62%; 1H NMR (300 MHz, CD3OD): d 7.96 (dd,
J = 9.7, 2.5 Hz, 1H, H22), 7.47 (s, 1H), 7.31 (dd, J = 2.5, 1.0 Hz, 1H,
H21), 6.27 (dd, J = 9.7, 1.0 Hz, 1H, H23), 5.12 (s, 1H, H3), 4.40–
4.36 (m, 1H, H4’), 3.96 (t, J = 8.0 Hz, 1H, H1’), 3.16 (dd, J = 11.8,
4.6 Hz, 1H, H30a), 2.86 (d, J = 11.8 Hz, 1H, H3’b), 2.52–2.47 (m,
1H), 2.24–1.17 (m, 24H), 0.96 (s, 3H, H19), 0.70 (s, 3H, H18); 13C
NMR (75 MHz, DMSO-d6): d 174.0, 161.4, 149.2, 147.4, 122.8,
114.2, 83.4, 70.9, 70.2, 58.7, 55.1, 50.1, 48.1, 41.2, 39.9, 39.6,
36.7, 34.9, 34.8, 32.0, 30.3, 29.9, 28.5, 26.3, 24.4, 23.7, 21.1, 21.0,
16.7; MS (ESI) m/z: 500 [M + H]+; HRMS (ESI): calcd for
Synthesis of compound 2 with method A to yield a white, amor-
phous powder. Yield: 57%; 1H NMR (400 MHz, CDCl3): d 7.83 (dd,
J = 9.7, 2.5 Hz, 1H, H22), 7.23 (d, J = 2.5 Hz, 1H, H21), 6.26 (d,
J = 9.7 Hz, 1H, H23), 5.13 (s, 1H, H3), 3.40–3.38 (m, 2H, H30a,
H50a), 3.03 (t, J = 9.8 Hz, 2H, H30b, 50b), 2.60–2.58 (m, 1H, H10),
2.48–2.44 (m, 1H), 2.13–1.21 (m, 24H), 0.95 (s, 3H, H19), 0.69 (s,
3H, H18); 13C NMR (100 MHz, CDCl3): d 171.8, 162.4, 148.5,
146.7, 122.6, 115.3, 85.3, 71.3, 51.2, 48.3, 42.8, 42.3, 40.8, 38.4,
37.1, 35.8, 35.2, 32.7, 30.6, 30.4, 28.7, 26.4, 25.0, 24.8, 24.7, 23.8,
21.4, 21.3, 16.5; MS (ESI) m/z: 498 [M + H]+; HRMS (ESI): calcd
for C29H42NO6 [M + H]+ 498.3214, found 498.3228.
C
29H42NO6 [M + H]+ 500.3007, found 500.3001.

B. Ma et al. / Steroids 78 (2013) 508–512
511
2.1.6. Compound 2a
2.1.10. Compound 2e
To a stirred solution of bufalin (200 mg, 0.52 mmol), 4-dimeth-
ylaminopyridine (63 mg, 0.52 mmol, 1 eq) and 1-Boc-4-piperidine-
carboxylic acid (357 mg, 1.56 mmol, 3 eq) in anhydrous DCM
(2 mL) at room temperature under nitrogen gas was added 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(300 mg, 1.56 mmol, 3 eq), and stirring continued for 12 h. After
completion (by TLC), DCM (10 mL), and H2O (10 mL) were added.
The organic phase was separated, washed with H2O and brine,
dried over Na2SO4, and concentrated in vacuo to afford yellow res-
idue (250 mg). Dissolved in minimal DCM, the crude product was
subjected to purify on silica gel (4:1, petroleum ether–acetone)
to yield a white, amorphous powder (215 mg, 69%); 1H NMR
(400 MHz, CDCl3): d 7.83 (dd, J = 9.7, 2.6 Hz, 1H, H22), 7.23 (d,
J = 2.6 Hz, 1H, H21), 6.26 (d, J = 9.7 Hz, 1H, H23), 5.10 (s, 1H, H3),
4.03 (m, 2H, H30a, 50a), 2.84 (t, J = 9.0 Hz, 2H, H30b, 50b), 2.46–
2.42 (m, 2H), 2.17–1.21 (m, 26H), 1.45 (s, 9H, C(CH3)3), 0.96 (s,
3H, H19), 0.70 (s, 3H, H18); 13C NMR (75 MHz, CDCl3): d 173.9,
162.4, 154.7, 148.5, 146.8, 122.7, 115.2, 85.2, 79.6, 70.4, 51.2,
48.3, 42.3, 41.4, 40.8, 37.0, 35.8, 35.1, 32.7, 30.5, 30.4, 28.7, 28.4,
28.1, 28.0, 26.4, 25.0, 23.8, 21.4, 21.3, 16.5; MS (ESI) m/z: 598
[M + H]+; HRMS (ESI): calcd for C35H52NO7 [M + H]+ 598.3738,
found 598.3733.
Synthesis of compound 2e with method C to yield a white,
amorphous powder. Yield: 73%; 1H NMR (300 MHz, CDCl3): d
7.84 (dd, J = 9.0, 3.0 Hz, 1H, H22), 7.22 (s, 1H, H21), 6.25 (d,
J = 9.0 Hz, 1H, H23), 5.08 (s, 1H, H3), 2.90–2.86 (m, 2H, H30a, 50a),
2.46–2.44 (m, 1H), 2.28–2.24 (q, J = 6.0 Hz, 2H, CH2CH3), 2.02–
1.20 (m, 31H), 0.93 (s, 3H, H19), 0.88 (t, J = 6.0 Hz, 3H,CH2CH3),
0.69 (s, 3H, H18); 13C NMR (75 MHz, CDCl3): d 174.5, 162.4,
148.5, 146.8, 122.7, 115.3, 85.3, 70.1, 61.0, 53.0, 51.2, 48.3, 42.3,
41.7, 40.8, 36.9, 35.8, 35.2, 32.8, 30.6, 30.5, 28.7, 28.4, 28.3, 26.4,
25.1, 23.8, 21.4, 21.3, 20.1, 16.5, 12.0; MS (ESI) m/z: 540
[M + H]+; HRMS (ESI): calcd for C33H50NO5 [M + H]+ 540.3684,
found 540.3669.
2.2. In vitro cytotoxic assay
Human cervix epithelial adenocarcinoma cell line (HeLa) and
non-small-cell lung cancer (A549) cell lines, obtained from Amer-
ican Type Culture Collection (Rockville, MD), were used for the
cytotoxicity assay in vitro by MTT assay as reported before [23].
Briefly, cells were plated in 96-well flat-bottomed plates at density
of 1 ꢁ 103 cells/well in complete medium and incubated over-
night. Then, the media were changed into fresh media containing
various amounts of compounds for 72 h. At the end of the incuba-
tion, 20
razolium bromide, 5 mg/mL), MTT, was added to each well and
the plates were incubated for 3 h at 37 °C. Then, 100 L of lysis buf-
lL of the dye (3,[4,5-dimethylthiazol-2-yl-] diphenyltet-
2.1.7. Compound 2b
Synthesis of compound 2b with method B to yield a white,
amorphous powder. Yield: 91%; 1H NMR (300 MHz, CDCl3): d
7.83 (d, J = 9.0 Hz, 1H, H22), 7.23 (s, 1H, H21), 6.26 (d, J = 9.0 Hz,
1H, H23), 5.11 (s, 1H, H3), 4.42 (d, J = 12.0 Hz, 1H, H30a), 3.78 (d,
J = 12.0 Hz, 1H, H50a), 3.14 (t, J = 9.0 Hz, 1H, H30b), 2.80 (t,
J = 9.0 Hz, 1H, H50b), 2.54–2.43 (m, 2H), 2.09 (s, 3H, CH3COO),
2.24–1.21 (m, 26H), 0.96 (s, 3H, H19), 0.70 (s, 3H, H18); 13C NMR
(75 MHz, CDCl3): d 173.5, 168.9, 162.4, 148.5, 146.7, 122.6, 115.3,
85.3, 70.6, 51.2, 48.3, 45.7, 42.3, 41.3, 40.9, 40.8, 37.0, 35.8, 35.2,
32.7, 30.5, 30.4, 28.7, 28.5, 27.9, 26.4, 25.0, 23.8, 21.5, 21.4, 21.3,
16.5; MS (ESI) m/z: 540 [M + H]+; HRMS (ESI): calcd for
l
fer (20% sodium dodecyl sulfate [SDS] in 50% N,N-dimethylform-
amide, containing 0.5% [v:v] 80% acetic acid and 0.4% [v:v] 1 N
HCl) was added to each well and incubated overnight (16 h). Cell
viability was evaluated by measuring the mitochondrial-depen-
dent conversion of the yellow tetrazolium salt MTT to purple for-
mazan crystals by metabolic active cells. The optical density
(proportional to the number of live cells) was assessed with a
Microplate Reader Bio-Rad 550 at 570 nm. Each experiment was
performed in triplicate. Results of three independent experiments
were used for statistical analysis. IC50 value (half-maximal inhibi-
tory concentration) was calculated by the Logit method.
C
32H46NO6 [M + H]+ 540.3320, found 540.3314.
2.1.8. Compound 2c
3. Results and discussion
Synthesis of compound 2c with method C to yield a white,
amorphous powder. Yield: 77%; 1H NMR (300 MHz, CDCl3): d
7.83 (d, J = 9.0 Hz, 1H, H22), 7.22 (s, 1H,H21), 6.25 (d, J = 9.0 Hz,
1H, H23), 5.08 (s, 1H, H3), 2.83–2.79 (m, 2H, H30a, 50a), 2.46–2.44
(m, 1H), 2.26 (s, 3H, NCH3), 2.20–1.15 (m, 29H), 0.94 (s, 3H,
H19), 0.69 (s, 3H, H18); 13C NMR (75 MHz, CDCl3): d 174.4,
162.4, 148.5, 146.8, 122.7, 115.2, 85.2, 70.1, 54.9, 51.1, 48.4, 48.3,
46.3, 42.2, 41.0, 40.8, 36.9, 35.7, 35.1, 32.7, 30.5, 30.4, 28.7, 28.3,
28.2, 26.4, 25.0, 23.7, 21.4, 21.2, 16.5; MS (ESI) m/z: 512
[M + H]+; HRMS (ESI): calcd for C31H46NO5 [M + H]+ 512.3371,
found 512.3357.
All the bufalin 3-nitrogen-containing-ester derivatives (2–6)
were evaluated in vitro for their cytotoxicities against human cer-
vical epithelial adenocarcinoma (HeLa) and non-small-cell lung
cancer (A549) cell lines, and the results are presented in Table 1.
Among three bufalin-3-piperidinyl-ester derivatives (2–4), the
piperidinyl-4-carboxylate (2) exhibited greater potency (IC50 val-
ues on HeLa and A549 cell lines are 0.76 and 0.34 nM, respectively)
than 3-carboxylate (3) or 2-carboxylate (4) against both cell lines.
For example, against the HeLa cell line, compound 2 was 32-time
more active than 3 (IC50 = 24.8 nM), and 101-time more active than
compound 4 (IC50 = 77.1 nM), respectively. These results indicated
that the location of the nitrogen was important for cytotoxicity. In
the five-membered ester series (5 and 6), L-prolinyl ester 5 showed
less activity (IC50 = 42.7 nM) than bufalin against HeLa cell line,
whereas L-hydroxyprolinyl ester 6 with one additional C40-hydro-
xyl group demonstrated higher activity (IC50 = 9.9 nM) than bufalin
(IC50 = 26.3 nM). Similar SARs results of these N-heterocycles ana-
logues (2–6) also could be found against the A549 cell line, and in
general, all these compounds were more sensitive against A549
than HeLa cell line.
2.1.9. Compound 2d
Synthesis of compound 2d with method C to yield a white,
amorphous powder. Yield: 71%; 1H NMR (300 MHz, CDCl3): d
7.83 (d, J = 9.0 Hz, 1H, H22), 7.22 (s, 1H, H21), 6.25 (d, J = 9.0 Hz,
1H, H23), 5.08 (s, 1H, H3), 2.92–2.87 (m, 2H, H30a, 50a), 2.46–2.44
(m, 1H), 2.38 (q, J = 6.0 Hz, 2H, NCH2CH3), 2.24–1.12 (m, 29H),
1.07 (t, J = 6.0 Hz, 3H, NCH2CH3), 0.94 (s, 3H, H19), 0.69 (s, 3H,
H18); 13C NMR (75 MHz, CDCl3): d 174.4, 162.4, 148.5, 146.8,
122.6, 115.3, 85.3, 70.1, 52.6, 52.5, 51.2, 48.3, 42.3, 41.7, 40.8,
36.9, 35.8, 35.1, 32.7, 30.5, 30.4, 29.7, 28.7, 28.3, 28.2, 26.4, 25.0,
23.8, 21.4, 21.3, 16.5, 12.0; MS (ESI) m/z: 526 [M + H]+; HRMS
(ESI): calcd for C32H48NO5 [M + H]+ 526.3527, found 526.3534.
We further investigated the impact of different substituents at
C40-nitrogen atom in piperidine ring on cytotoxicities. In Table 1,
we observed that incorporation of the Boc (2a) and Ac (2b) groups
resulted in a significant loss of potency against two cell lines,

512
B. Ma et al. / Steroids 78 (2013) 508–512
Table 1
In summary, we synthesize a new series of bufalin 3-nitrogen-
Cytotoxicity of compounds 2–6.
containing-ester derivatives via a two-step simple and highly effi-
cient method, and find that C3 moiety has important influence on
cytotoxic activity. The bufalin-3-piperidinyl-4-carboxylate com-
pound 2 (IC50 values on HeLa and A549 cell lines are 0.76 and
0.34 nM, respectively) displays a significant cytotoxic potency
compared to the parent bufalin. Moreover, these bufalin 3-nitro-
gen-containing-ester derivatives will provide better insight into
the effect of the C3 site on cytotoxic activity for designing potential
cardiac glycoside antitumor agents. For the compounds found in
the present study to possess stronger cytotoxicity than bufalin, fur-
ther study checking their effects on Na+/K+-ATPase signalosome
and downstream signaling pathways would be conducted.
No.
R
IC50 (nM)a
HeLa
A549
6.2 0.4
Acknowledgments
1
2
26.3 3.7
0.76 0.06
0.34 0.02
This work was supported by the National Natural Science
Foundation of China (grants: 30925040, 81102329), the Chinese
National Science & Technology Major Project ‘‘Key New Drug
Creation and Manufacturing Program’’ (grants: 2012ZX09301001-
001, 2011ZX09307-002-03), and the Science Foundation of
Shanghai (grant 12XD14057).
3
4
24.8 3.0
77.1 6.4
2.2 0.3
7.9 0.6
Appendix A. Supplementary data
5
6
42.7 5.1
9.9 1.0
21.3 3.8
7.7 0.8
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2013.
02.007.
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whereas introduction of the methyl (2c) group displayed moderate
potency (IC50 values on HeLa and A549 cell lines are 28.0 and
4.4 nM, respectively) compared to bufalin. Among three alkyl pipe-
ridinyl-4-carboxylates (2c–e), we observed a significant loss of
cytotoxicity against HeLa and A549 cell lines with increasing the
size alkyl groups compared to 2. These results clearly indicated
that C40-NH was favorable to its cytotoxic activity. In conclusion,
a series of bufalin derivatives were investigated in their cytotoxic-
ities against human lung tumor cells A549 and human cervical car-
cinoma cells HeLa in the current study. Some derivatives
demonstrated good cytotoxic activities, in some cases are better
than bufalin. The most promising compounds in this series of
synthetic compounds were 2, 3, 6 and 2c, which showed strong
cytotoxic activities.