Synthesis and biological evaluation of bufalin-3-yl nitrogen-containing-carbamate derivatives as anticancer agents

Article abstract of DOI:10.1016/j.steroids.2016.01.011

A series of bufalin-3-yl nitrogen-containing-carbamate derivatives 3 were designed, synthesized, and evaluated for their proliferation inhibition activities against human cervical epithelial adenocarcinoma (HeLa) cell line. The structure-activity relationships (SARs) of this new series are described in this paper. Cytotoxicity data revealed that the C3 moiety had an important influence on cytotoxic activity. Compound 3i-HCl exhibited significant in vitro antiproliferative activity against the ten tested tumor cell lines, with IC₅₀ values ranging from 0.30 to 1.09 nM. Furthermore, 3i-HCl can significantly inhibit tumor growth by 100% at the dose 2 mg/kg by iv, or 4 mg/kg by ig.

Full text of DOI:10.1016/j.steroids.2016.01.011

Accepted Manuscript
Synthesis and biological evaluation of bufalin-3-yl nitrogen-containing-carba-
mate derivatives as anticancer agents
Min Lei, Zhiyong Xiao, Biao Ma, Yijia Chen, Miao Liu, Junhua Liu, De’an
Guo, Xuan Liu, Lihong Hu
PII:
S0039-128X(16)00012-X
http://dx.doi.org/10.1016/j.steroids.2016.01.011
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DOI:
Reference:
Steroids
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Accepted Date:
28 September 2015
5 January 2016
19 January 2016
Please cite this article as: Lei, M., Xiao, Z., Ma, B., Chen, Y., Liu, M., Liu, J., Guo, D., Liu, X., Hu, L., Synthesis
and biological evaluation of bufalin-3-yl nitrogen-containing-carbamate derivatives as anticancer agents, Steroids
(2016), doi: http://dx.doi.org/10.1016/j.steroids.2016.01.011
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Synthesis and biological evaluation of bufalin-3-yl
nitrogen-containing-carbamate derivatives as anticancer agents
,
Min Lei a,b*, Zhiyong Xiao a, Biao Ma a, Yijia Chen a, Miao Liu a, De'an Guo a, Xuan
Liu a,* and Lihong Hu a,*
a Shanghai Research Center for the Modernization of Traditional Chinese Medicine,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China.
b State Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing 210009, China.
Corresponding authors. Tel.: +86 21 20231965; E-mail address: E-mail:
mlei@simm.ac.cn (M. Lei), xuanliu@simm.ac.cn (X. Liu), lhhu@simm.ac.cn (L. Hu).
1

Abstract: A series of bufalin-3-yl nitrogen-containing-carbamate derivatives 3 were
designed, synthesized, and evaluated for their proliferation inhibition activities against
human cervical epithelial adenocarcinoma (HeLa) cell line. The structure-activity
relationships (SARs) of this new series are described in this paper. Cytotoxicity data
revealed that the C3 moiety had an important influence on cytotoxic activity.
Compound 3i-HCl exhibited significant in vitro antiproliferative activity against the
ten tested tumor cell lines, with IC50 values ranging from 0.30 to 1.09 nM.
Furthermore, 3i-HCl can significantly inhibit tumor growth by 100% at the dose 2
mg/kg by iv, or 4 mg/kg by ig.
Keywords: Cardiac glycosides, Bufalin (BF), Natural product, Bufalin-3-yl carbamate,
Cytotoxicity.
2

1. Introduction
Cardiac glycosides are a class of natural compounds with prominent cardiotonic
effects. In clinical research, compounds such as digoxin, digitoxin, lanatosaide C,
strophanthin K, and divarcosise, are used widely as drugs for treatment of heart
disease. In 1960s, Shiratori [1] and Hartwell et. al. [2] reported that cardiac glycosides
exhibited significant antitumor activities in vitro, respectively. From then on, many
papers have been published on studies of cardiac glycosides as antitumor therapeutic
agents [3–8]. In recent years, several cardiac glycosides, such as Anvirzel [9],
PBI-05204 [10], and UNBS-1450 [11], are being tested in phase I and II clinical trials
as new anti-cancer agents. Cardiac glycosides as novel drugs for treatment of tumor
might be a promising research area.
Chansu is a Traditional Chinese Medicine made from the dried skin and parotid
venom glands of Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider.
Huachansu injection, the water-soluble extract of the dried skin of Bufo bufo
gargarizans Cantor, has been successfully used in the clinic for treatment of various
cancers in China. Bufalin (Fig. 1), a cardiac glycoside, is one of the major active
components of Chansu. As is known in the literature, bufalin exhibits remarkable
cytotoxic activity with IC50 values of 10-8 to 10-9 mol/L for various tumor cells
including human prostate carcinoma cells (PC3, DU145) [12], human leukemia cells
(U937, HL60) [13–14], human cervical carcinoma cells (HeLa) [15] and
non-small-cell lung cancer cells (A549) [16]. Although the antitumor mechanism of
3

bufalin has not been reported, bufalin, as with other cardiac glycosides, could bind to
Na+/K+-ATPase and inhibit the Na+/K+ pump [17–20].
Fig. 1. The structure of bufalin (BF).
Through years of research, the antitumor activity of cardiac glycosides in vivo and
in vitro has been widely recognized. However, these compounds exhibite high
toxicity which severely limits their clinical application. Hence, reducing toxicity,
enhancing activity and improving drug-like properties of cardiac glycosides might be
an important strategy for the structural modification. Bufalin, just as other cardiac
glycosides, has the toxicity problems. In our previous study, we found that
bufalin-3-yl piperidin-4-carboxylate (IC50 value on HeLa 0.76 nM) displayed
significant cytotoxic potency compared to the parent bufalin (IC50 value on HeLa 26.3
nM) [16]. These results suggest that the hydroxyl on C3 of bufalin is a suitable site for
the structural modification.
4

Fig. 2. The design of bufalin-3-yl carbamate derivatives.
Although bufalin-3-yl piperidin-4-carboxylate showed significant cytotoxic
potency in vitro, this compound was inactive in vivo. We speculated that the ester was
prone to metabolism in vivo because ester groups are susceptible to hydrolysis by
esterases in the blood. Therefore, we designed bufalin-3-yl piperazine-1-carbamate by
bioisosteric replacement (Figure 2), and the activity test indicated that this compound
could maintain cytotoxic potency (IC50 (HeLa) = 0.77 nM). In order to find new
compounds with better cytotoxic potency,
a
series of bufalin-3-yl
nitrogen-containing-carbamate derivatives were designed, synthesized, and evaluated
for their proliferation inhibition activities.
2. Experimental
A two-step reaction route was designed for the synthesis of bufalin-3-yl carbamate
derivatives as shown in Scheme 1. In the first step, bufalin was reacted with
4-nitrophenyl chloroformate in DCM using pyridine as the base to obtain the
intermediate 1. Then, 1 was reacted with amine 2 in the presence of Et3N to obtain the
5

desired products 3.
Scheme 1. The synthesis of bufalin-3-yl carbamate derivatives 3.
All reactions were performed under a nitrogen atmosphere 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
specified. All solvents were redistilled under argon atmosphere. The progress of
reactions were monitored by thin layer chromatography (TLC) plates (silica gel 60GF,
Yantai jiangyou company) visualized with 254-nm UV light and/or by staining with
vanillin solution (2.7 g vanillin + 100 mL H2O + 35 mL concentrated H2SO4 diluted
6

to 300 mL with 95% ethanol). Melting points were measured by a WRS-1B
1
micromelting point apparatus and are uncorrected. H and 13C NMR spectra were
recorded on a Varian Mercury-VX300 Fourier transform spectrometer or a Bruker
AM-400 spectrometer, and chemical shifts (δ) were reported in ppm; the
hydrogenated residues of deuterated solvent were used as internal standard CDCl3:
1
7.26 ppm for H NMR and 77.00 ppm for 13C NMR. ESIMS was run on a Bruker
Esquire 3000 plus spectrometer in MeOH. HRESIMS were determined on a
Micromass Q-TOF Global mass spectrometer.
2.1. General synthesis of bufalin-3-yl carbamate derivatives
To a stirred solution of bufalin (100 mg, 0.28 mmol) and 4-nitrophenyl
chloroformate (129 mg, 0.64 mmol, 2.3 eq) in anhydrous DCM (2 mL) at room
temperature under nitrogen gas was added pyridine (50 mg, 0.64 mmol, 2.3 eq), and
stirring continued for 2 h. After completion (by TLC), DCM (10 mL), and H2O (10
mL) were added. The organic phase was separated, washed with aq. Na2CO3, H2O
and brine, dried over Na2SO4, and concentrated in vacuo to afford yellow residue 2.
To a solution of the residue in DCM (2.5 mL) was added amine (3 eq) and Et3N (3 eq),
and stirring continued at room temperature for 2 h. 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, and the residue was subjected to chromatographic separation on silica gel (30
7

g) with petroleum ether/acetone/triethylamine (100:30:1 v/v/v).
2.1.1. Compound 3a
3a (77 mg, 55%) mp 199–201 oC; 1H NMR (300 MHz, CDCl3): δ 7.83 (dd, 1H, J =
9.6, 2.4), 7.22 (s, 1H), 6.25 (d, 1H, J = 9.6), 4.98 (brs, 1H), 3.27 (q, 2H, J = 6.0, 5.4),
2.75 (t, 2H, J = 6.0), 2.65 (q, 2H, J = 6.9), 1.10 (t, 3H, J = 7.2), 0.93 (s, 3H), 0.69 (s,
3H); 13C NMR (75 MHz, CDCl3): δ 162.6, 156.7, 148.7, 147.0, 122.9, 115.5, 85.5,
70.8, 51.5,49.1, 48.6, 44.0, 42.5, 41.1, 40.8, 37.0, 36.0, 35.3, 33.0, 30.9, 29.9, 28.9,
26.6, 25.5, 21.6, 23.9, 21.5, 16.7, 15.4; MS (ESI) m/z: 501.4 [M+H]+; HRMS (ESI):
calcd for C29H45N2O5 [M+H]+ 501.3323, found 501.3310.
2.1.2. Compound 3b
3b (89 mg, 60%) mp 198–200 oC; 1H NMR (300 MHz, CDCl3): δ 7.84 (dd, 1H, J =
9.9, 2.4), 7.22 (d, 1H, J = 2.4), 6.25 (d, 1H, J = 9.6), 4.98 (br s, 1H), 3.23 (br d, 2H, J
= 5.4), 2.55 (m, 6H), 1.02 (t, 6H, J = 7.2), 0.94 (s, 3H), 0.69 (s, 3H); 13C NMR (75
MHz, CDCl3): δ 162.6, 156.6, 148.7, 147.1 (2C), 122.9, 115.4, 85.5, 70.7, 52.1, 51.4,
48.5, 47.1, 42.5, 41.0, 38.7, 36.9, 36.0, 35.3, 32.9, 30.9, 29.9, 28.9, 26.6, 25.5, 23.9,
21.6, 21.5, 16.7, 11.8 (2C); MS (ESI) m/z: 529.7 [M+H]+; HRMS (ESI): calcd for
C31H49N2O5 [M+H]+ 529.3636, found 529.3649.
2.1.3. Compound 3c
3c (89 mg, 60%) mp 202–204 oC; 1H NMR (300 MHz, CDCl3): δ 7.83 (dd, 1H, J =
9.6, 2.4), 7.23 (d, 1H, J = 2.1), 6.25 (d, 1H, J = 9.6), 5.01 (br s, 1H), 3.34 (m, 2H),
2.67 (q, 2H, J = 7.2), 1.11 (m, 6H), 0.95 (s, 3H), 0.70 (s, 3H); 13C NMR (75 MHz,
8

CDCl3): δ 162.6, 156.1, 148.7, 147.1, 122.9, 113.4, 85.4, 71.1, 51.4, 48.5, 48.1, 47.0,
44.2, 42.7, 42.5, 41.0, 37.5, 36.0, 35.4, 32.9, 31.0, 29.8, 28.9, 26.7, 25.6, 24.1, 21.6,
21.5, 16.7, 15.4, 14.0; MS (ESI) m/z: 529.6 [M+H]+; HRMS (ESI): calcd for
C31H49N2O5 [M+H]+ 529.3636, found 529.3644.
2.1.4. Compound 3d
o
1
3d (102 mg, 73%) mp 205–206 C; H NMR (400 MHz, CDCl3): δ 7.84 (dd, J =
9.8, 2.6 Hz, 1H), 7.23 (d, J = 2.3 Hz, 1H), 6.26 (dd, J = 9.9, 2.3 Hz, 1H), 5.01 (s, 1H),
3.63–3.27 (m, 4H), 3.02–2.66 (m, 4H), 2.55–2.36 (m, 2H), 0.95 (s, 3H), 0.69 (s, 3H);
13C NMR (100 MHz, CDCl3): δ 163.4, 155.1, 148.4, 147.8, 123.4, 114.6, 84.7, 71.7,
50.9, 48.3, 44.9, 41.6, 40.7, 37.1, 35.6, 35.0, 32.2, 31.7, 30.6, 30.5, 29.4, 28.7, 26.3,
25.1, 23.6, 21.1, 16.4; MS (ESI) m/z: 499.7 [M+H]+; HRMS (ESI): calcd for
C29H43N2O5 [M+H]+ 499.3166, found 499.3177.
2.1.5 Compound 3e
3e (108 mg, 75%) mp 203–205 oC; 1H NMR (400 MHz, CDCl3): δ 7.84 (dd, J = 9.8,
2.6 Hz, 1H), 7.23 (m, 1H), 6.26 (d, J = 9.7 Hz, 1H), 5.02 (s, 1H), 3.50 (dd, J = 6.4, 3.9
Hz, 4H), 2.51–2.42 (m, 1H), 2.37 (t, J = 5.0 Hz, 4H), 2.31 (s, 3H), 0.95 (s, 3H), 0.70
(s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 162.6, 155.1, 148.6, 146.9, 122.8, 115.4,
85.4, 71.3, 54.9, 51.3, 48.5, 46.3, 42.5, 41.0, 37.3, 36.0, 35.3, 32.9, 30.9, 30.8, 29.8,
28.9, 26.6, 25.5, 24.1, 21.6, 21.5, 16.7; MS (ESI) m/z: 513.8 [M+H]+; HRMS (ESI):
calcd for C30H45N2O5 [M+H]+ 513.3323, found 513.3311.
2.1.6. Compound 3f
9

o
1
3f (95 mg, 66%) mp 203–205 C; H NMR (300 MHz, CDCl3): δ 7.84 (m, 1H),
7.23 (s, 1H), 6.26 (d, J = 9.7 Hz, 1H), 5.02 (s, 1H), 3.69–3.29 (m, 4H), 2.94 (t, J = 5.3
Hz, 2H), 2.91–2.85 (m, 2H), 2.59–2.36 (m, 1H), 0.94 (m, 3H), 0.70 (s, 3H); 13C NMR
(100 MHz, CDCl3): δ 163.0, 155.9, 148.6, 147.3, 123.1, 115.2, 85.2, 71.4, 51.2, 49.0,
48.8, 47.9, 47.5, 45.7, 42.2, 40.9, 37.4, 35.9, 35.2, 32.7, 30.9, 30.8, 29.8, 28.8, 26.6,
25.4, 24.0, 21.5, 21.4, 16.6; MS (ESI) m/z: 513.8 [M+H]+; HRMS (ESI): calcd for
C30H45N2O5 [M+H]+ 513.3323, found 513.3316.
2.1.7. Compound 3g
o
1
3g (44 mg, 30%) mp 209–211 C; H NMR (300 MHz, CDCl3): δ 7.84 (d, J = 9.7
Hz, 1H), 7.22 (s, 1H), 6.25 (d, J = 9.8 Hz, 1H), 4.99 (s, 1H), 4.07 (br s, 2H), 2.86 (m,
2H), 2.60–2.47 (m, 2H), 2.44 (s, 3H), 2.26–2.10 (m, 1H), 2.04–1.11 (m, 26H), 0.94 (s,
3H), 0.69 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 162.6, 155.1, 148.6, 147.0, 122.9,
115.4, 85.4, 71.2, 58.4, 56.7, 51.3, 48.5, 42.7, 42.4, 41.0, 37.3, 35.9, 35.3, 33.3, 32.9,
32.0, 30.9, 30.8, 28.9, 26.6, 25.4, 24.1, 21.5, 21.5, 18.5, 16.7; MS (ESI) m/z: 527.5
[M+H]+; HRMS (ESI): calcd for C31H47N2O5 [M+H]+ 527.3479, found 527.3488.
2.1.8. Compound 3h
3h (98 mg, 65%) mp 210–212 oC; 1H NMR (300 MHz, CDCl3): δ 7.83 (dd, J = 9.8,
2.2 Hz, 1H), 7.23 (s, 1H), 6.26 (d, J = 9.6 Hz, 1H), 5.01 (s, 1H), 4.27–4.07 (m, 2H),
2.94–2.63 (m, 2H), 2.54–2.38 (m, 1H), 2.28 (s, 6H), 2.20–1.12 (m, 27H), 0.95 (s, 3H),
0.70 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 162.6, 155.0, 148.6, 147.0, 122.9, 115.4,
85.4, 71.1, 62.2, 51.3, 48.5, 43.2, 42.4, 41.6, 41.0, 37.3, 35.9, 35.3, 32.9, 30.9, 30.8,
10

28.9, 26.6, 25.4, 24.1, 21.5, 21.5, 16.7; MS (ESI) m/z: 541.9 [M+H]+; HRMS (ESI):
calcd for C32H49N2O5 [M+H]+ 541.3636, found 541.3626.
2.1.9. Compound 3i
o
1
3i (79 mg, 55%) mp 210–212 C; H NMR (400 MHz, CDCl3): δ 7.83 (d, J = 9.6
Hz, 1H), 7.23 (d, J = 1.5 Hz, 1H), 6.25 (d, J = 9.6 Hz, 1H), 5.00 (s, 1H), 4.09 (br s,
2H), 2.89–2.75 (m, 3H), 2.51–2.40 (m, 1H), 2.23–1.12 (m, 25H), 0.95 (s, 3H), 0.69 (s,
3H); 13C NMR (75 MHz, CDCl3): δ 164.7, 156.4, 150.5, 149.4, 125.0, 115.4, 86.0,
73.5, 68.8, 52.2, 49.7, 49.6, 43.0, 41.8, 38.8, 36.9, 36.3, 33.2, 31.9, 31.8, 30.9, 29.9,
27.7, 26.2, 24.4, 22.6, 22.5, 17.4; MS (ESI) m/z: 513.5 [M+H]+; HRMS (ESI): calcd
for C30H45N2O5 [M+H]+ 513.3323, found 513.3320.
2.1.10. Compound 3j
o
1
3j (92 mg, 58%) mp 202–204 C; H NMR (400 MHz, CDCl3): δ 7.83 (d, J = 9.8
Hz, 1H), 7.23 (s, 1H), 6.26 (d, J = 9.8 Hz, 1H), 4.99 (s, 1H), 4.43 (d, J = 8.8 Hz, 1H),
3.95–3.91 (m, 1H), 2.54–2.39 (m, 1H), 2.25–1.02 (m, 26H), 1.24 (s, 6H), 1.12 (s, 6H),
0.94 (s, 3H), 0.70 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 162.6, 155.6, 148.5, 147.1,
122.9, 115.2, 85.2, 70.5, 51.3, 51.2, 48.4, 45.5, 44.0, 42.2, 40.8, 36.8, 35.8, 35.1, 34.8,
32.7, 30.7, 30.5, 28.8, 28.3, 26.4, 25.3, 23.8, 21.4, 21.3, 16.6; MS (ESI) m/z: 569.1
[M+H]+; HRMS (ESI): calcd for C34H53N2O5 [M+H]+ 569.3949, found 569.3940.
2.1.11. Compound 3k
3k (37 mg, 25%) mp 204–206 oC; 1H NMR (300 MHz, CDCl3): δ 7.84 (dd, J = 9.7,
2.6 Hz, 1H), 7.22 (s, 1H), 6.25 (d, J = 9.6 Hz, 1H), 5.02 (s, 1H), 3.16 (d, J = 11.7 Hz,
11

2H), 2.79 (s, 3H), 2.73–2.55 (m, 2H), 2.21–1.12 (m, 26H), 0.94 (s, 3H), 0.69 (s, 3H);
13C NMR (100 MHz, CDCl3): δ 162.6, 148.7, 146.9, 122.8, 115.5, 85.5, 71.1, 51.3,
48.5, 42.5, 41.0, 36.0, 35.3, 32.9, 30.9, 28.9, 26.6, 25.5, 21.6, 21.5, 16.7; MS (ESI)
m/z: 527.9 [M+H]+; HRMS (ESI): calcd for C31H47N2O5 [M+H]+ 527.3479, found
527.3470.
2.1.12. Compound 3l
3l (103 mg, 70%) mp 200–202 oC; 1H NMR (300 MHz, CDCl3): δ 7.84 (dd, J = 9.7,
2.6 Hz, 1H), 7.22 (d, J = 2.5 Hz, 1H), 6.25 (d, J = 9.7 Hz, 1H), 4.99 (d, J = 5.0 Hz,
1H), 4.14 (br s, 2H), 2.74 (br s, 2H), 2.62–2.54 (m, 1H), 2.51–2.38 (m, 1H), 2.21–1.13
(m, 26H), 0.94 (s, 3H), 0.69 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 163.4, 155.1,
148.4, 147.8, 123.5, 114.6, 84.7, 71.3, 51.0, 46.8, 43.5, 41.7, 40.7, 38.4, 37.1, 35.6,
35.0, 32.2, 30.6, 30.5, 29.4, 28.6, 26.3, 25.1, 23.6, 21.3, 21.1, 16.4; MS (ESI) m/z:
527.9 [M+H]+; HRMS (ESI): calcd for C31H47N2O5 [M+H]+ 527.3479, found
527.3474.
2.2. General procedure for synthesis of compound 3i-HCl
To a stirred solution of 1% HCl (5 mL) was added compound 3i (0.51 g, 1 mmol).
The mixture was stirred at rt for 15 min. Then, the precipitate was collected by
filtration and washed by cold 50% aq EtOH (5 mL) to afford 3i-HCl.
2.3. In vitro cytotoxic assay
12

HeLa, HepG2, Daudi, MV-4-11, Jurkat, HT1080, PC-3, AGS, MCF-7 and
NCI-H2228 were obtained from American Type Culture Collection (Rockville,
MD), and were used for the cytotoxicity assay in vitro by the MTT assay method as
reported before [21].
2.4. In vivo antitumor assay
Female 7-week-old specific pathogen free (SPF) BALB/cA-nude mice (weight,
25–27 g) were obtained from the Shanghai Laboratory Animal Center, Chinese
Academy of Sciences. MV-4-11 cells (7 × 107) were subcutaneously implanted into
the axilla region of mice. After tumors reached 100~200 mm3 in size, mice were
randomly divided into 5 groups ( 8–16 mice in each group) and started treatment of
vehicle control (iv., QOD), positive control (sunitinib, 40 mg/kg, ig., QD), bufalin (1
mg/kg, ip., QOD), 3i-HCl (1 mg/kg, iv., QOD), 3i-HCl (2 mg/kg, iv., QOD), 3i-HCl
(2 mg/kg, ig., QOD), 3i-HCl (4 mg/kg, ig., QOD). Tumor sizes were measured every
3–4 days using calipers and their volumes were calculated using a standard formula:
width2×length/2. Body weight was measured daily. The weights of tumors were
measured at the end of the experiment and the inhibition rates of drugs were
calculated.
3. Results and discussion
In our previous study, we found that bufalin-3-yl nitrogen-containing-ester
13

derivatives displayed a significant cytotoxic potency compared to the parent
compound bufalin [16]. However, these compounds showed low activities in vivo. In
order to maintain the activities in vitro and improve the activities in vivo, a series of
bufalin-3-yl nitrogen-containing-carbamate derivatives 3 were designed and
synthesized (Table 1). Of the examples listed in Table 1, the yields of 3g and 3k were
much lower than the others, because when N-methylpiperidin-4-amine was used as
the substrate, both 3g and 3k were formed in 30% and 25% yields, respectively. As
shown in Table 1, although asymmetric diamines were used as substrates (Tabel 1,
entries 9, 10 and 12), the compounds mentioned in Table 1 were the main products,
and the yields of the another isomers were very low.
Table 1. Synthesis of bufalin 3-carbamate derivatives 3.
Entry
1
Yield (%)a
Product 3
55
3a
14

2
3
60
60
3b
3c
4
5
73
75
3d
3e
6
7
66
30
3f
3g
8
9
65
70
3h
3i
HN
10
58
3j
N
H
11
12
25
70
3k
3l
N
H
HN
a Isolated yields based on bufalin.
15

All the bufalin-3-yl nitrogen-containing-carbamate derivatives 3 were evaluated in
vitro for their cytotoxicities against the human cervical epithelial adenocarcinoma
(HeLa) cell line, and the results are presented in Table 2. The screening results
suggested a rough SAR: among the nitrogen-contained carbocycle carbamates, the
piperazinyl, homopiperazinyl and piperidinyl moieties were effective in increasing
cytotoxicity with the IC50 values of 0.59–0.85 nM, which were 8–28 fold more potent
than bufalin. Compounds 3d, 3f and 3i which contained piperazinyl, homopiperazinyl
and piperidinyl moieties displayed the most significant activity. The compounds with
N-methyl substituents (3e, 3g, 3h, 3j and 3k) at the nitrogen-contained carbocycle
demonstrated lower activities.
Table 2. Cytotoxic activity of bufalin-3-yl carbamate derivatives 3.
IC50 (nM)a
HeLa
IC50 (nM)a
HeLa
Compound
Compound
7.27 1.00
3.26 0.45
2.19 0.40
5.99 0.92
0.77 0.11
21.70 5.50
0.85 0.17
16.50 3.25
9.88 1.00
0.59 0.04
0.54 0.04
15.50 1.70
22.33 3.16
> 200
BF
3a
3b
3c
3d
3e
3f
3g
3h
3i
3i-HCl
3j
3k
3l
a
IC50: 50% inhibitory concentration. Values are an average of three separate
experiments.
16

The results showed that both 3i and 3i-HCl displayed strong anti-proliferative
activity, therefore, 3i and 3i-HCl were chosen for further evaluation against a panel of
human tumor cell lines, including HeLa, HepG2, Daudi, MV-4-11, Jurkat, HT1080,
PC-3, AGS, MCF-7 and NCI-H2228. Bufalin was used as a positive control and the
antiproliferative activities of 3i and 3i-HCl are shown in Table 3. As shown in Table
3, 3i and 3i-HCl exhibited significant in vitro anti-proliferative activity against the ten
tested tumor cell lines, with IC50 values ranging from 0.30 to 10.78 nM, and were
more potent than bufalin. Remarkably, 3i-HCl showed the greatest cytotoxicity
against MV-4-11 cell line with the IC50 value of 0.30 nM.
Table 3. Cytotoxic activity of bufalin, 3i and 3i-HCl towards 10 human tumor cell
lines.
IC50 (nM)a
Cell line
BF
3i
3i-HCl
HeLa
7.27 1.00
6.02 0.71
12.56 0.76
6.26 2.22
8.79 2.48
11.31 0.38
6.00 0.45
51.80 1.34
45.87 5.18
16.06 2.51
0.59 0.04
0.51 0.04
2.86 0.21
0.75 0.19
1.09 0.26
2.90 0.33
0.55 0.10
9.62 0.69
9.97 1.45
0.56 0.05
0.54 0.04
0.33 0.02
2.34 0.22
0.30 0.11
0.67 0.10
2.34 0.10
0.38 0.08
9.35 0.53
10.78 1.08
0.67 0.03
HepG2
Daudi
MV-4-11
Jurkat
HT1080
PC-3
AGS
MCF-7
NCI-H2228
17

a
IC50: 50% inhibitory concentration. Values are an average of three separate
experiments.
The water solubility of bufalin, 3i and 3i-HCl were tested, and the solubilities were
0.04, 0.10, and 2.0 mg/mL, respectively. The result indicated that the solubility of
3i-HCl was about 50 times greater than that of bufalin.
On the basis of its potent anti-proliferative effect, bufalin and 3i-HCl were selected
for in vivo studies using sunitinib as a positive control. Xenograft tumors were
generated by subcutaneous implantation of MV-4-11 cells into nude mice. Mice were
treated ig once a day with sunitinib, or ip once in two days with bufalin, or iv or ig
once in two days with 3i-HCl for 3 weeks. At the end of the experiment, animals were
euthanized and the weights of tumors were measured.
Table 4. Anti-tumor efficacy of bufalin, sunitinib and 3i-HCl against MV-4-11
xenografts in nude mice.
Dosage Administration Mice (n)
Tumor weight (g) Inhibition
Compound
(mg/kg)
route
Initial/end
16/16
8/8
X
SD
rate (%)
Solvent
Sunitinib
bufalin
3i-HCl
3i-HCl
3i-HCl
3i-HCl
/
40
1
iv, QOD × 21d
ig, QD × 21d
ip, QOD × 21d
iv, QOD × 21d
iv, QOD × 21d
ig, QOD × 21d
ig, QOD × 21d
1.9508 0.1202
-
0.0000 0.0000* 100.00%
8/8
2.4146 0.2589
2.2448 0.4022
-23.77%
-15.07%
1
8/8
2
8/8
0.0000 0.0000* 100.00%
1.5118 0.2714 39.04%
0.0000 0.0000* 100.00%
2
8/8
4
8/8
18

a The in vivo experiment was carried out in nude mice bearing MV-4-11 xenografts.
As shown in Table 4, sunitinib at a dose of 40 mg/kg/d by ig significantly inhibited
tumor growth. According to a previous report, the LD50 value of bufalin (ip.) in mice
was about 2.2 mg/kg [22]. The result indicated that both bufalin and 3i-HCl showed
no inhibitive effects on tumor growth in vivo at the dose of 1 mg/kg/d, while 3i-HCl
inhibited tumor growth by 100% at the dose 2 mg/kg by iv (P < 0.01). 3i-HCl also
showed strong inhibitive effects on tumor growth at a dose of 4 mg/kg/d by ig and the
tumor inhibition rate of 3i-HCl was also 100% (P < 0.01).
In conclusion, we have synthesized
a
series of new bufalin-3-yl
nitrogen-containing-carbamate derivatives from bufalin by a two-step reaction route,
and evaluated their cytotoxicity against Hela cells. Among them, 3i-HCl had a strong
cytotoxic effect on various tumor cells with IC50 = 0.30–10.78 nM. Furthermore,
3i-HCl significantly inhibited tumor growth by 100% at a dose of 2 mg/kg by iv, or 4
mg/kg by ig. These bufalin-3-yl nitrogen-containing-carbamate derivatives will
provide better insight into the effect of the C3 site on cytotoxic activity for designing
potential cardiac glycoside antitumor agents.
Acknowledgments
This work was supported by the Chinese National Science & Technology Major
19

Project “Key New Drug Creation and Manufacturing Program” (grants
2013ZX09508104, 2014ZX09301-306-003), the Open Project Program of State Key
Laboratory of Natural Medicines, China Pharmaceutical University (No.
SKLNMKF201413), and Shanghai Science & Technology Support Program
(13431900401).
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23

Graphical abstract
Synthesis and biological evaluation of bufalin-3-yl nitrogen-containing-carbamate
derivatives as anticancer agents
Min Lei, Zhiyong Xiao, Biao Ma, Yijia Chen, Miao Liu, Junhua Liu, De'an Guo,
Xuan Liu, Lihong Hu
24

Highlights
► Highly efficient method to synthesize bufalin-3-yl nitrogen-containing-carbamate
►
derivatives.
Bufalin-3-yl nitrogen-containing-carbamate derivatives show
significant cytotoxicity. ► 3i-HCl significantly inhibits tumor growth in vivo by 100%
at the dose 2 mg/kg by iv, or 4 mg/kg by ig.
25