Synthesis and mechanistic studies of novel spin-labeled combretastatin derivatives as potential antineoplastic agents

Article abstract of DOI:10.1016/j.bmc.2012.12.046

Two series (14a-d and 21a-h) of novel spin-labeled combretastatin derivatives were synthesized and evaluated for cytotoxicity against four tumor cell lines (K562, SGC-7901, Hela and HepG-2). Simultaneously, a representative compound 21a was selected to investigate the antitumor mechanisms of these synthetic compounds. The results indicated that some of the compounds showed significant cytotoxicity against four tumor cell lines in vitro and were more active than etoposide, a clinically available anticancer drug. Among the newly synthesized compounds, 21a, 21b and 21c displayed the greatest cytotoxicity against three tested tumor cell lines (HEPG-2, BGC-832 and Hela), with IC ₅₀ values ranging from 0.15 to 1.05 μM, compared with values of 0.014-0.403 μM for 3-amino-deoxycombretastatin A-4 (3). In addition, the mechanistic analysis revealed that compound 21a effectively interfered with tubulin dynamics to prevent mitosis in cancer cells, leading to cell cycle arrest and, eventually, dose dependent apoptosis.

Full text of DOI:10.1016/j.bmc.2012.12.046

Bioorganic & Medicinal Chemistry 21 (2013) 1248–1256
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and mechanistic studies of novel spin-labeled
combretastatin derivatives as potential antineoplastic agents
a
a
a
a
b
Ying-Qian Liu a, , Xiao-Jing Li , Chun-Yan Zhao , Xiang Nan , Jing Tian , Susan L. Morris-Natschke ,
⇑
Zhi-Jun Zhang a, Xiao-Ming Yang b, Liu Yang d, Lin-Hai Li a, Xing-Wen Zhou a, Kuo-Hsiung Lee b,c,
⇑
a School of Pharmacy, Lanzhou University, Lanzhou 730000, PR China
b Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, United States
c Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
d Environmental and Municipal Engineering School, Lanzhou Jiaotong University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series (14a–d and 21a–h) of novel spin-labeled combretastatin derivatives were synthesized and
evaluated for cytotoxicity against four tumor cell lines (K562, SGC-7901, Hela and HepG-2). Simulta-
neously, a representative compound 21a was selected to investigate the antitumor mechanisms of these
synthetic compounds. The results indicated that some of the compounds showed significant cytotoxicity
against four tumor cell lines in vitro and were more active than etoposide, a clinically available anticancer
drug. Among the newly synthesized compounds, 21a, 21b and 21c displayed the greatest cytotoxicity
against three tested tumor cell lines (HEPG-2, BGC-832 and Hela), with IC50 values ranging from 0.15
Received 10 October 2012
Revised 10 December 2012
Accepted 20 December 2012
Available online 9 January 2013
Keywords:
Combretastatin
Spin-labeled
Nitroxide
Cytotoxicity
Mechanistic analysis
to 1.05 lM, compared with values of 0.014–0.403 lM for 3-amino-deoxycombretastatin A-4 (3). In addi-
tion, the mechanistic analysis revealed that compound 21a effectively interfered with tubulin dynamics
to prevent mitosis in cancer cells, leading to cell cycle arrest and, eventually, dose dependent apoptosis.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
activity and (b) a substituent at the 30-position of the B-ring was
almost always necessary for significant cytotoxic activity.6 These
Combretastatin A-4 (CA-4, 1), isolated from the bark of the
South African tree Combretum caffrum, is a highly effective natural
tubulin-binding stilbenoid. It affects microtubule dynamics by
binding to the colchicine site, and shows potent cytotoxicity
against a wide variety of human cancer cell lines, including those
that are multidrug resistant.1–3 The relative molecular simplicity
of this compound coupled with its strong potency against cancer
cell lines has stimulated significant interest in a numerous diverse
ligands designed to mimic 1. In recent years, such efforts have been
devoted to detailed studies of the structure–activity relationships
(SAR) of variously substituted stilbenes, as well as water-soluble
prodrugs. Among the research highlights, the sodium phosphate
prodrug (CA-4P, 2) and other derivatives, including 3-amino-
deoxycombretastin A-4 (AVE8063, 3), the amino acid derivative
AC-7739 (4), the dihydroxy derivative CA-1 (5), and its sodium
diphosphate prodrug CA-1P (6, OXI-4503) (Fig. 1), represent prom-
ising drugs in various clinical studies. These compounds can
potently disrupt vasculature and significantly reduce a tumor’s
blood flow.4,5 From these investigations, we noticed that (a) the
(Z)-configuration of the olefin bridge is essential for biological
critical SAR findings prompted us to select 30-amino-deoxycombre-
tastatin A-4 (AVE8063, 3) as a logical starting point in our contin-
uing pursuit of novel anticancer chemotherapeutics.
Furthermore, the introduction of a stable nitroxyl radical into
pharmaceutical molecules can cut down the toxicity and potenti-
ate the antitumor effects to a certain degree. Some studies have
shown that the introduction of nitroxyl moiety can lead to fast
decomposition, higher alkylating and lower carbamoylating activ-
ity, better antimelanomic activity, lower general toxicity, and the
ability to transport through cell membranes, while the nitroxyl free
radicals themselves possess low toxicity and are not mutagenic or
carcinogenic.7–11 In our prior studies, we prepared numerous spin-
labeled compounds that displayed significant antineoplastic activ-
ity with markedly decreased toxicity compared with their parent
compound.12–18 Among the tested compounds, GP-11 (8), a deriv-
ative of podophyllotoxin (7) (Fig. 1), was reported as a low immu-
nosuppressive antitumor agent, which increased the mitotic index
and resulted in G2/M and, to a lesser extent, S arrest. GP-11 may
possibly become a new antitumor agent with improved bioactivity
and low toxicity. More recently, we also found the introduction of
nitroxyl radical into the molecule of combretastatin A-4 potentiate
its cytotoxic effect and some of these derivatives exhibited more
potent activity than the clinical drug irinotecan.19 Inspired by this
⇑
Corresponding authors. Tel.: +1 919 962 0066; fax: +1 919 966 3893.
E-mail addresses: yqliu@lzu.edu.cn (Y.-Q. Liu), khlee@unc.edu (K.-H. Lee).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.046

Y.-Q. Liu et al. / Bioorg. Med. Chem. 21 (2013) 1248–1256
1249
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
O
P
ONa
ONa
OCH3
OCH3
O
OCH3
OH
OCH3
NH2
OCH3
OCH3
CA-4P (2)
CA-4 (1)
AVE8063 (3)
O
P
H3CO
H3CO
H3CO
H3CO
ONa
ONa
O
OH
OH
O
H3CO
H3CO
O
NH2
OH
ONa
ONa
OCH3
OCH3
OCH3
N
H
P
O
OCH3
OCH3
OCH3
CA-1 (5)
AC-7739 (4)
CA-1P (6)
OH
OH
O
O
O
O
OH
NHNHCNH
O
O
N
O
O
O
H3CO
OCH3
OCH3
OCH3
OCH3
podophyllotoxin (7)
H3CO
GP-11 (8)
Figure 1. Structures of CA-4 (1), CA4P (2), AVE8063 (3), AC-7739 (4), CA-1 (5), CA-1P (6), podophyllotoxin (7) and GP-11(8).
prior work, we herein describe the synthesis and mechanistic stud-
ies of two novel series of derivatives of AVE8063 (3) as part of our
continuing search for promising natural product-derived antican-
cer agents.
followed by decarboxylation of the corresponding acrylic acid 11
using copper and quinoline. In this reaction, only the pure cis-
isomer was obtained in 52% yield after purification of the crude
precipitate by fractional crystallization. The resulting compound
12 was reduced with zinc powder and acetic acid to afford the
key intermediate 3. Compound 3 was then condensed with the
appropriate piperidine (pyrroline) nitroxyl acids (13a–d) in
the presence of N,N-dicyclohexylcarbodiimide (DCC) and
4-dimethylaminopyridine (DMAP) to provide target compounds
14a–d.
2. Results and discussion
2.1. Chemistry
As shown in Figure 2, compound 12 was obtained via the Perkin
condensation20,21 of commercially available 3,4,5-trimethoxyphe-
nylacetic acid (9) and 4-methoxy-3-nitrobenzaldehyde (10),
Based on previous work,16,17 as well as the facts that
L-amino
acids are actively transplanted into mammalian tissues, have good
COOH
CHO
H3CO
H3CO
H3CO
COOH
+
i
H3CO
H3CO
ii
H3CO
NO2
OCH3
OCH3
NO2
OCH3
NO2
OCH3
12
OCH3
OCH3
9
10
11
H3CO
H3CO
H3CO
H3CO
O
iii
iv
+
O
R
OH
OCH3
OCH3
N
H
R
NH2
OCH3
13a-d
OCH3
3
14a-d
R=
N
O
N
O
a
N
O
N
O
b
d
c
Figure 2. Synthesis of compounds 14 a–d. Reagents and conditions: (i) Ac2O/Et3N, 140 °C; (ii) Cu/quinoline, 230 °C; (iii) Zn/HOAc, 24 h; (iv) DCC/DMAP/CH2Cl2.

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Y.-Q. Liu et al. / Bioorg. Med. Chem. 21 (2013) 1248–1256
O
O
TosO
NH+
OH
O
N
OH
O
N
N
ii
iii
i
iv
N
O
N
H
N
O
N
O
15
18
17
16
O
O
R
a
R=H
OH
b
c
d
R=Me
O
N3
O
N
H
R=CHMe2
R=CH2CHMe2
O
v
e
R=CH(Me)CH2Me
R=Proline
N
O
N
O
f
g
h
R=CH2Ph
R=CH2(C6H4)OH
20a-h
19
Figure 3. Synthesis of compounds 20a–h. Reagents and conditions: (i) Na2WO4/H2O2/EDTA; (ii) N,N0-carbonyl-diimidazole/THF; (iii) p-toluenesulfonic acid monohydrate;
(iv) NaN3/H2O; (v) amino acids/MgO, 24 h.
MeO
MeO
MeO
MeO
20a-h
EDCI/HOBt/DIPEA
NH2
O
H
N
OMe
OMe
O
O
N
N
H
OMe
3
O
OMe
R
21a-h
a
b
c
d
R=H
H2C
e
f
g
R=CH(Me)CH2Me
R=Me
h
R=
N
R=CHMe2
R=(CH2)2SCH3
R=Proline
H
R=CH2Ph
Figure 4. Synthesis of compounds 21a–h.
water solubility, and are often used as carrier vehicles for some
drugs, we also used an amino acid spacer as a linkage between
the amino-combretastatin core (3) and the nitroxyl radical moiety.
The starting materials, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidi-
nyl-oxycarbonyl) amino acids 20a–h, for the preparation of the tar-
get compounds 21a–h were synthesized according to our previous
procedure as shown in Figure 3.22 Briefly, 4-hydroxy-2,2,6,6-tetra-
methylpiperidine-1-oxyl (16) was prepared by catalytic oxidation
of 4-hydroxy-2,2,6,6-tetramethylpiperidine (15) with sodium
tungstate–hydrogen peroxide–EDTA in 85% yield. Subsequently,
the reaction of 16 with N,N-carbonyldiimidazole gave N-(1-oxyl-
uenesulfonic acid monohydrate to give its more reactive tosylate
(18). Compound 18 was converted instantaneously into the corre-
sponding alkoxycarbonyl azide (19) when dissolved in an aqueous
solution of sodium azide. Compounds 20a–h were obtained in
good yields by reaction of 19 with various amino acids in the pres-
ence of MgO (Fig. 3). The desired compounds 21a–h were produced
by treating compound 3 with the corresponding [N-(1-oxyl-
2,2,6,6-tetramethyl-4-piperidinyl-oxycarbonyl) amino acids 20a–
h using N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (EDCI) as the coupling agent (Fig. 4). All synthesized target
compounds 14a–d and 21a–h were purified by column chromatog-
raphy and their structures were confirmed from melting point, IR,
ESR and HRMS analyses.
2,2,6,6-tetramethylpiperidinyl
oxycarbonyl)-imidazole
(17).
Without further purification, compound 17 was reacted with p-tol-
2.2. Biological assay
Table 1
In vitro cytotoxicity against four tumor cell lines
2.2.1. Cytotoxicity of compounds 14a–d and 21a–h against four
cancer cell lines
Entry
IC50
(
lM)
Target compounds 14a–d and 21a–h were evaluated for
in vitro cytotoxicity against the four tumor cell lines mentioned
previously using an MTT assay with triplicate experiments.23 Com-
pound 3 and etoposide were used as positive controls. The screen-
ing results are shown in Table 1.
Although compounds 14a–d and 21a–h exhibited lower cyto-
toxicity than 3, the common parent core, many of the synthesized
compounds showed greater cytotoxicity than etoposide, a clini-
cally available drug, against certain tumor cell lines in vitro. Com-
pounds 21a, 21b, and 21c exhibited the greatest cytotoxicity
against three tested tumor cell lines (HEPG-2, BGC-832, and Hela),
K-562
HEPG-2
BGC-832
Hela
3
0.12
0.028
38.76
24.97
10.70
51.25
0.98
0.73
1.05
2.05
2.19
0.014
>100
4.93
6.21
42.54
0.39
0.96
0.43
1.68
3.07
4.71
6.33
3.55
26.85
0.403
42.26
7.60
8.57
59.22
0.41
0.39
0.15
1.16
2.33
3.39
8.37
2.08
17.68
14a
14b
14c
14d
21a
21b
21c
21d
21e
21f
21g
21h
Etoposide
>100
10.38
15.62
16.95
1.09
1.56
11.98
4.15
18.77
5.53
5.80
0.56
2.85
4.48
0.28
2.27
103.81
with IC50 values ranging from 0.15 to 1.05 lM.
From the screening results, conversion of 3 into the spin-labeled
compounds 14a–d increased the IC50 values greatly. These results

Y.-Q. Liu et al. / Bioorg. Med. Chem. 21 (2013) 1248–1256
1251
A
120
100
80
60
40
20
0
B
0
0.5
1
1.5
2
2.5
3
3.5
Concentration (µM)
Figure 5. Effects of compound 21a on the cell cycle distribution of Hela cells. The cells were cultured for 36 h without the compound or with compound 21a at 0.375, 1.5 and
M (A) inhibition of cell growth; (B) quantitative analysis of cell cycle phase.
3
l
indicated that the cytotoxic profile may be sensitive to the size and
electronic density of the nitroxide substituents at the NH2 group of
3. However compounds 21a–h with an amino acid linker between
the nitroxide moiety and the core of 3 showed significantly im-
proved cytotoxic activity against all four tested tumor cell lines
compared to compounds 14a–d. From an SAR consideration, the
structures of the amino acid linkages between the core and nitrox-
ide moieties affected the inhibitory activity against four tumor cell
lines in vitro. Compounds 21a (glycine spacer) and 21b (alanine
active against all cell lines, except for K-562. Compounds 21d
(methionine spacer) and 21e (isoleucine spacer) showed the same
trend, but were slightly less potent than 21c. Compounds 21f (pro-
line spacer), 21g (phenylalanine spacer), and 21h (tryptophan
spacer) generally were less potent, with two exceptions. Com-
pound 21h showed the highest potency (IC50 0.56
lM) against K-
562, and 21g showed the highest potency (IC50 0.28
lM) against
HEPG-2. These differences could be ascribed to a combination of
factors, such as the nature of the substituents (e.g., size, electronic
characteristics, and other factors) or by a different in interaction at
the active site. This investigation highlighted that the identity of
spacer) exhibited IC50 values between 0.39 and 1.56
lM against
all four cell lines. Compound 21c (valine spacer) was significantly

1252
Y.-Q. Liu et al. / Bioorg. Med. Chem. 21 (2013) 1248–1256
A
70
B
60
50
40
30
20
10
0
0
0.375
1.5
3
Concentration (µM)
Figure 6. (A) Apoptotic effects on human leukemia Hela cell line induced by compound 21a. The cells were incubated in media containing various concentration of 21a (0,
0.375, 1.5 and 3 M) for 24 h. Cells and the amount of apoptotic cells were determined by flow cytometry using FITV-Annexin V/PI staining. (B) The rate of apoptotic cells as
detected by flow cytometry. Cells in B1 and B2 quadrants were considered to be apoptotic cells. The experiment was repeated three times.
l
the amino acid spacers had a major impact on cytotoxic activity of
such analogues. Hence, a systemic, predictable correlation could be
made between the nature of the amino acids and antitumor
activity.
and 3 lM of 21a led to accumulation of cells in the G1 phase;
32.85%, 85.82%, and 96.25%, respectively, of the cells were in G1
phase, compared with 47.36% in untreated cultures. In parallel to
the G1 block, the cell cycle analysis showed a clearly increased pro-
portion of sub-G1 cells, which is regarded as a characteristic of
apoptotic cells. In untreated cultures, only 0.20% of the cells were
in sub-G1 phase, whereas 1.14%, 16.71%, and 32.42% of cells were
in the sub-G1 phase after 36 h treatment with 0.375, 1.5, and
2.2.2. Analysis of cell cycle
On the basis of the above results, it was found that compound
21a significantly inhibited the growth of four human cancer cell
lines. To determine whether the antitumor effects of these com-
pounds were caused by cell cycle accumulation at a certain phase,
the effects of 21a on cell cycle progression were also determined
by flow cytometry analysis in Hela cells cultured for 36 h in the
presence of increasing concentrations of compound 21a (0, 0.375,
3 lM of compound 21a, respectively. These data demonstrated
that 21a can induce G1 arrest and apoptosis in Hela cells. In addi-
tion, a simultaneous sharp decrease in S and G2 cells occurred. All
effects were observed in a dose-dependent manner. These results
demonstrated that 21a interferes with cell proliferation by arrest-
ing the cell cycle in a certain phase.
1.5, and 3 lM). As shown in Figure 5, treatment with 0.375, 1.5,

Y.-Q. Liu et al. / Bioorg. Med. Chem. 21 (2013) 1248–1256
1253
Figure 7. (A) Normal interphase microtubules; (B) loss of interphase microtubules induced by compound 21a.
2.2.3. Effects on apoptosis
3. Conclusions
In addition, to determine whether compounds exhibiting high
antitumor activity induce apoptosis of Hela cells, the cells were
treated with the active compound 21a to measure the induction
of cell death. After exposure to compound 21a for 24 h, flow
cytometry using the FITC-Annexin V/PI double staining method
was used to generate an apoptotic cell scatterplot of the control
group and the groups treated with compound 21a. As shown in
Figure 6, B1 quadrant presented the late apoptotic cells, B2 quad-
rant presented the early apoptotic cells, B3 quadrant presented
the living cells, and B4 quadrant presented the damaged cells. With
the increasing concentration of compound 21a, the percentage of
the early apoptotic cells increased from 0.17% to 52.6%, together
with a percentage increase of late apoptotic cells from 0.81% to
13.96%. The cell counts in B1 and B2 quadrants were considered
to be apoptotic cells, and the total percentages were 0.98%,
43.99%, 55.75%, and 66.54%, respectively (Fig. 6b). The results dem-
onstrated that the most active derivative 21a induced cellular
apoptosis and thus, as expected, caused the antitumor effects.
In summary, two series (14a–d and 21a–h) of novel spin-la-
beled derivatives of AVE8063 were synthesized and their in vitro
anticancer activity was tested against four tumor cell lines using
a MTT assay. The results showed that most of the new spin-labeled
compounds exhibited more potent cytotoxicity against four tumor
cell lines compared to etoposide, a clinically available anticancer
drug, although they were less potent than 3. Compounds 21a–c
exhibited the highest potency against three tested tumor cell lines
(HEPG-2, BGC-832, and Hela), with IC50 values ranging from 0.15 to
1.05 lM, compared with values of 0.014–0.403 lM for 3). Com-
pound 21a was further selected as a representative compound to
investigate the antitumor mechanisms of these synthetic com-
pounds. In cell cycle and apoptosis analyses, compound 21a
strongly inhibited the proliferation of Hela cancer cells, resulting
in a massive accumulation of cells in the G2/M phase and inducing
a high level of apoptosis. Compound 21a exhibited strong levels of
tubulin depolymerization in the mictrotubule assembly assay. All
of the above results indicated that these synthesized compounds
effectively interfered with tubulin dynamics and prevented mitosis
in cancer cells, thus, leading to cell cycle arrest and, eventually,
dose-dependent apoptosis. These studies suggest that compounds
21a–c merit further investigation for development as anticancer
clinical trial candidates.
2.2.4. Microtubule assembly/depolymerization assay
Combretastatin and its derivatives are recognized as tubulin
binding agents with damaging effects on vasculature.24,25 In Figure
7, the effect of 21a on microtubule assembly was analyzed by
transmission electron microscope studies. Normal interphase
microtubules in dimethylsulfoxide (DMSO) solution exhibited typ-
ical reticular formation (Fig. 7A) of polymerization state at 37 °C. In
4. Experimental section
4.1. General
the presence of a high concentration (3 lM) of 21a, the microtu-
bule polymerization was disturbed (Fig. 7B). The complete depoly-
merization of microtubules is illustrated in Figure 7B, and a
discrete club-shaped tubulin cytoskeleton was observed. The depo-
lymerizing effects of 21a provide evidence that loss of microtubule
function in interphase cells treated with 21a may prevent these
cells from progressing into mitosis. In summary, the correlation be-
tween microtubule depolymerization and cancer cell growth inhi-
bition indicates a tight linkage of antiproliferative effects with
tubulin-dependent mechanisms of action. The antiproliferative
activity of these compounds may derive from their interaction
with tubulin and interference with microtubule assembly. Com-
pounds that effectively interfere with tubulin dynamics prevent
mitosis in cancer cells, leading to cell cycle arrest and, eventually,
apoptosis.
Melting points were taken on a Kofler melting point apparatus
and were uncorrected. Mass spectra were recorded on ZAB-HS
and Bruker Daltonics APEXII49e instruments. NMR spectra were
recorded on a Bruker AM-400 spectrometer at 400 MHz using
TMS as reference (Bruker Company, USA). The electron spin reso-
nance (ESR) spectra were obtained with a Bruker A300 X-band
EPR spectrometer. IR spectra were measured on a Nicolet 5DX-
FT-IR spectrometer on neat samples placed between KBr plates.
The synthetic compounds were purified by flash chromatography
on Merck silica gel (70–230 mesh). Thin-layer chromatography
(TLC) was performed on silica gel plates with a fluorescent indica-
tor (Merck Silica Gel 60 F254 0.25 mm thick). N-(1-Oxyl-2,2,6,6-tet-

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Y.-Q. Liu et al. / Bioorg. Med. Chem. 21 (2013) 1248–1256
ramethyl-4-piperidinyloxycarbonyl)-amino acids were synthe-
sized by employing our previous procedures.22
HRMS: m/z calcd for C
27H35N2O6: 484.2568 [M+H]+, found
484.2556 [M+H]+.
4.4.3. 30-N-(100-Oxyl-200,200,600,600-tetramethyl-400-
4.2. Synthesis of the intermediate 12
tetrahydropyridinylcarbonyl)-amido-AVE8063 (14c)
Red powder, yield: 64%; mp 93–95 °C. IR (KBr) cmꢀ1: 3426,
2970, 2931, 2849, 1679, 1531, 1505, 1459, 1360 (N–O), 1127;
A mixture of 3,4,5-trimethoxyphenylacetic acid (8.84 mmol)
and 3-nitro-4-methoxyenzaldehyde (4.4 mmol), acetic anhydride
(4 mL) and triethylamine (2 mL) were heated under reflux for
3 h. After acidification with concentrated hydrochloric acid
(6 mL), the resulting solid was filtered off and recrystallised from
ethanol to give acrylic acid intermediate 11 as fine yellow needles.
Subsequently the corresponding acrylic acid intermediate
(5.56 mmol) was added to powdered copper (28.8 mmol) in quin-
oline (20 mL), and the resulting mixture was heated at 140 °C for
2 h. Upon cooling, diethyl ether was added, and the copper was fil-
tered off through Celite. The filtrate was washed with 1 M hydro-
chloric acid, and the aqueous layer was separated and extracted
with diethyl ether. The combined organic layers were washed with
saturated aqueous sodium carbonate, water, brine, dried (MgSO4),
and concentrated in vacuo. Flash column chromatography (SiO2
petrol/EtOAc 7:3) and recrystallization from EtOAc and petrol
afforded desired intermediate compound 12 in 52% yields. yellow
crystals, mp 123–124 °C; 1H NMR (CDCl3, 400 MHz) d: 7.79 (1H,
d, J = 2.1), 7.42 (1H, dd, J = 2.1, 8.7), 6.94 (1H, d, J = 8.7), 6.57 (1H,
d, J = 12.9), 6.47 (2H, s), 6.44 (1H, d, J = 12.9), 3.93 (3H, s), 3.85
(3H, s), 3.71 (6H, s); ESI: m/z: 346 [M+H]+; HRMS calcd 345.1212,
found 345.1236.
+
ESR: An = 14.62 G, g0 = 2.0055; MS (ESI) m/z: 496 [M+H] ; HRMS:
m/z calcd for C28H35N2O6: 496.2568 [M+H]+, found 496.2559
[M+H]+.
4.4.4. 30-N-(100-Oxyl-200,200,600,600-tetramethyl-400-
piperidinylcarbonyl)-amido-AVE8063 (14d)
Red powder, yield: 76%; mp 146–148 °C. IR (KBr) cmꢀ1: 3334,
2929, 2852, 1683, 1535, 1505, 1458, 1362 (N–O), 1126; ESR:
An = 15.76 G, g0 = 2.0058; MS (ESI) m/z: 498 [M+H]+; HRMS: m/z
calcd for C28H37N2O6: 498.2724 [M+H]+, found 498.2711 [M+H]+.
4.5. General procedure for synthesis of target compounds 21a–h
The corresponding N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidi-
nyl-oxycarbonyl) amino acids 20a–h (0.47 mmol) were dissolved
in 50 mL of anhydrous CH2Cl2 and cooled to 0 °C. 1-Hydroxybenzo-
triazole (HOBt) (0.7 mmol) and EDCI (0.56 mmol) were added, and
the mixture was stirred for 30 min at 0 °C. Subsequently,
0.39 mmol of compound 3 and finally 2.4 mL of N-ethyl diisopro-
pylamine were added. The mixture was stirred for 16 h at ambient
temperature. The solvent was removed under reduced pressure
and the residue was purified by flask-column chromatography
(gradient elution with mixtures of chloroform/methanol) on silica
gel as monitored by TLC. Synthesized target compounds 21a–h
were characterized by mp, IR, ESR and HRMS analyses.
4.3. Synthesis of the intermediate 3 (AVE8063) from 12
To a solution of compound 12 (2.0 mmol) in HOAc (60 mL) was
added zinc powder (32 g). The reaction mixture was stirred at
room temperature for 1 h. The reaction mixture was filtered over
Celite, and the filtrate was evaporated to dryness. After concentra-
tion, the residue was purified by silica gel column chromatography
(CH2Cl2) to give pure product in 45% yields. 1H NMR (CDCl3,
400 MHz) d: 6.69 (1H, s), 6.67 (2H, s), 6.55 (2H, s), 6.45 (1H, d,
J = 12.0), 6.36 (1H, d, J = 12.0), 3.84 (3H, s), 3.82 (3H, s), 3.69 (6H,
s); ESI: m/z: 316 [M+1]; HRMS calcd 315.1471, found 315.1457.
4.5.1. 30-N-[N-(100-Oxyl-200,200,600,600-tetramethyl-400-
piperidinylcarbonyl)-L-glycinyl]-amido-AVE8063 (21a)
Red powder, yield: 72%; mp 77–79 °C; IR (KBr) cmꢀ1: 3397,
3331, 2972, 2934, 2853, 1723, 1696, 1637, 1584, 1535, 1507,
1460, 1363(N–O), 1326, 1239, 1126, 1012; ESR: An = 16.02 G,
g0 = 2.0061; MS m/z: 571 [M+H]+; HRMS: m/z calcd for
C
30H40N3O8: 593.2708 [M+Na]+, found 593.2716 [M+Na]+.
4.4. General procedure for synthesis of target compounds 14a–d
4.5.2. 30-N-[N-(100-Oxyl-200,200,600,600-tetramethyl-400-
piperidinylcarbonyl)- -alaninyl]-amido-AVE8063 (21b)
A mixture of the appropriate piperidine (pyrroline) nitroxyl
acids (0.5 mmol), compound 3 (0.5 mmol) and DMAP (20 mg)
was stirred in anhydrous CH2Cl2 (20 mL) for 5 min at room temper-
ature under nitrogen. DCC (106 mg, 0.5 mmol) was added and the
mixture was stirred for 24 h. The reaction mixture was filtered and
the filtrate was evaporated. The residue was subjected to column
chromatography on silica gel with CH2Cl2–acetone to afford target
compounds 14a–d, and their structures were confirmed from mp,
IR, ESR and HRMS analyses.
L
Red powder, yield: 68%; mp 75–77 °C; IR (KBr) 3400, 3327,
2974, 2936, 2838, 1722, 1699, 1628, 1584, 1533, 1506, 1459,
1360 (N–O), 1326, 1239, 1126, 1028; ESR: An = 16.16 G,
g0 = 2.006; MS m/z: 585 [M+H]+; HRMS: m/z calcd for
C
31H42N3O8: 607.2864 [M+Na]+, found 607.2855 [M+Na]+.
4.5.3. 30-N-[N-(100-Oxyl-200,200,600,600-tetramethyl-400-
piperidinylcarbonyl)-
L
-valinyl]-amido-AVE8063 (21c)
Red powder, yield: 75%; mp 75–77 °C; IR (KBr) cmꢀ1: 3327,
2968, 2931, 2851, 1717, 1685, 1629, 1581, 1533, 1505, 1463,
1363(N–O), 1325, 1238, 1127, 1025; ESR: An = 16.04 G,
g0 = 2.0061; MS m/z: 613 [M+H]+; HRMS: m/z calcd for
4.4.1. 30-N-(100-Oxyl-200,200,500,500-tetramethyl-300-
pyrrolinylcarbonyl)-amido-AVE8063 (14a)
Yellow powder, yield: 33%; mp 126–127 °C. IR (KBr) cmꢀ1
:
3424, 2972, 2928, 2852, 1675, 1530, 1504, 1459, 1360 (N–O),
33H46N3O8: 635.3177 [M+Na]+, found 635.3188 [M+Na]+.
1126; ESR: An = 14.70 G, g0 = 2.0055; MS (ESI) m/z: 482 [M+H]+,
C
HRMS: m/z calcd for
C
27H33N2O6: 482.2411 [M+H]+, found
4.5.4. 30-N-[N-(100-Oxyl-200,200,600,600-tetramethyl-400-
piperidinylcarbonyl)-
482.2418 [M+H]+.
L
-methioninyl]-amido-AVE8063 (21d)
Red powder, yield: 60%; mp 72–73 °C; IR (KBr) cmꢀ1: 3397,
3324, 2971, 2934, 2840, 1718, 1688, 1583, 1534, 1507, 1459,
1363 (N–O), 1326, 1238, 1126, 1030; ESR: An = 15.78 G,
g0 = 2.0061; MS m/z 645 [M+H]+; HRMS: m/z calcd for
4.4.2. 30-N-(100-Oxyl-200,200,500,500-tetramethyl-300-
pyrrolidinylcarbonyl)-amido-AVE8063 (14b)
Yellow powder, yield: 39%; mp 128–129 °C. IR (KBr) cmꢀ1
:
3420, 2972, 2930, 2846, 1687, 1532, 1504, 1460, 1365 (N–O),
33H46N3SO8: 667.2898 [M+Na]+, found 667.2880 [M+Na]+.
1127; ESR: An = 15.28 G, g0 = 2.0061; MS (ESI) m/z: 484 [M+H]+;
C

Y.-Q. Liu et al. / Bioorg. Med. Chem. 21 (2013) 1248–1256
1255
4.5.5. 30-N-[N-(100-Oxyl-200,200,600,600-tetramethyl-400-
piperidinylcarbonyl)-
fixation, these cells were incubated with RNase A (0.1 mg/mL) at
37 °C for 30 min, and then stained with propidium iodide (50 g/
mL) for 30 min on ice in the dark. Cells were harvested by centri-
fugation and further stained with DNA staining solution (10 mg).
The DNA contents were then detected by flow cytometer and the
cell cycle profile was analyzed from the DNA content histograms.26
L
-isoleucinyl]-amido-AVE8063 (21e)
l
Red powder, yield: 70%; mp 62–64 °C; IR (KBr) cmꢀ1: 3397,
3324, 2968, 2934, 2878, 1709, 1684, 1627, 1583, 1535, 1506,
1461, 1362 (N–O), 1236, 1127, 1026; ESR: An = 16.10 G,
g0 = 2.006; MS m/z: 627 [M+H]+; HRMS: m/z calcd for
C
34H48N3O8: 649.3334 [M+Na]+, found 649.3318 [M+Na]+.
4.6.3. Flow cytometry with FITC-Annexin V/PI double staining
Trypsin (Sigma) without EDTA was used to digest and collect
the control group and the cells treated with 0.375, 1.5, and 3 lM
4.5.6. 30-N-[N-(100-Oxyl-200,200,600,600-tetramethyl-400-
piperidinylcarbonyl)-
L
-prolinyl]-amido-AVE8063 (21f)
Red powder, yield: 64%; mp 51–52 °C; IR (KBr) cmꢀ1: 3402,
3327, 2973, 2936, 2838, 1697, 1583, 1534, 1503, 1461, 1417,
1366 (N–O), 1327, 1241, 1124, 1027; ESR: An = 15.88G,
g0 = 2.006; MS m/z: 611 [M+H]+; HRMS: m/z calcd for
of compound 21a. Flow cytometry was performed according to
the apoptosis detection kit procedures. The Hela cells were washed
twice with PBS and centrifuged at 300ꢁg for 5 min. 5 ꢁ 105 cells
were collected. Binding buffer suspension (500
lL) was added to
C
33H44N3O8: 633.3021 [M+Na]+, found 633.3037 [M+Na]+.
the cells, and then 5
ll of the FITC-Annexin V mix was added. Next,
5
l
L of the PI mix was added, and the suspension was mixed and
4.5.7. 30-N-[N-(100-Oxyl-200,200,600,600-tetramethyl-400-
piperidinylcarbonyl)-
kept at room temperature in the dark for 15 min of reaction. Flow
cytometry was performed using a FACSC alibur Xow cytometer (BD
Biosciences, CA, USA).
L
-phenylalaninyl]-amido-AVE8063 (21g)
Red powder, yield: 70%; mp 78–80 °C; IR (KBr) cmꢀ1: 3400,
3324, 2972, 2935, 2838, 1717, 1690, 1627, 1583, 1535, 1504,
1458, 1361(N–O), 1326, 1239, 1125, 1029; ESR: An = 15.98 G,
g0 = 2.006; MS m/z: 661[M+H]+; HRMS: m/z calcd for C37H46N3O8:
683.3177 [M+Na]+, found 683.3166 [M+Na]+.
4.6.4. Separation of active microtubule protein
Goat brain (1.8 kg) was homogenized in a Waring blender and
then centrifuged in the cold at 32,800 rpm for 1 h. The supernatant
was decanted and divided into four aliquots. ATP and GTP were
added to the aliquots at 37 °C, incubated, and centrifuged. The
supernatants were discarded, and the pellets were homogenized.
After 30 min on ice, the viscous suspension was centrifuged in
the cold for 30 min. The pellet was re-homogenized sequentially
two times and the cold centrifugation was repeated each time.
All supernatants were pooled and the cold pellets were set aside.
To each 100 mL of the combined supernatant were added 55.3 g
of glycerol (4 M) and 5 mL of a solution containing Mes (pH 6.4),
EGTA, MgC12, 2-mercaptoethanol, and EDTA to maintain the con-
centrations of these components and ATP and GTP for final concen-
trations of 1 and 0.3 mM. The mixture was incubated at 37 °C for
1 h and centrifuged with warming for 1 h at 32,000 rpm, and the
warm supernatant was set aside. The suspension was left on ice
for 30 min and centrifuged in the cold for 30 min. Very small cold
pellets were obtained and were combined with the prior cold
pellets.27
4.5.8. 30-N-[N-(100-Oxyl-200,200,600,600-tetramethyl-400-
piperidinylcarbonyl)-L-tyrosinyl]-amido-AVE8063 (21h)
Red powder, yield: 55%; mp 96–97 °C; IR (KBr) cmꢀ1: 3404,
3329, 2970, 2931, 2849, 1713, 1690, 1626, 1582, 1533, 1504,
1458, 1364 (N–O), 1327, 1238, 1125, 1028; ESR: An = 15.78 G,
g0 = 2.0061; MS m/z: 700 [M+H]+; HRMS: m/z calcd for
C
39H47N4O8: 722.3286 [M+Na]+, found 722.3279 [M+Na]+.
4.6. Biological assay
4.6.1. Cell culture and inhibition of cell growth
Four independent human tumor cell lines, K562 leukemia, SGC-
7901 gastric cancer, HeLa cervical carcinoma and HepG2 hepato-
cellular liver carcinoma, were obtained from Key Lab of Preclinical
Study for New Drugs, Lanzhou University, Gansu Province. All test
compounds were dissolved in DMSO at 1 mg/mL immediately be-
fore use and diluted in the medium before addition to the cells.
All cell lines were cultured in an RPMI 1640 medium (GIBCO) sup-
4.6.5. In vitro tubulin polymerization analysis
plemented with 10% bovine fetal calf serum, 2 mM
L
-glutamine
Electron micrographs were used to observe the depolymeriza-
tion state of the tubulin protein. Tubulins were incubated with or
without compound 21a in assay buffer (80 mM PIPES (pH 6.9),
1 mM MgCl2, and 1 mM EDTA) containing 30% glycerol and 1 mM
GTP for 30 min at 37 °C. Samples were fixed by dilution (1:20- to
1:50-fold) into assay buffer containing 30% glycerol and 0.5% glu-
taraldehyde at 37 °C, and then 10 mL of each sample were spotted
onto Formavar-coated grids and stained with uranyl acetate for
30 s. Electron micrographs were obtained using a Philips CM-12
transmission electron microscope with an accelerating voltage of
100 kV.
(GIBCO), 10 mM b-mercaptoethanol, 100 U/mL of penicillin and
100
l
g/mL of streptomycin. Cells (2 ꢁ 105 cells in 100 mL medium)
in their log phase of growth were seeded in 96-well microtitre
plates. After 24 h of incubation at 37 °C and 5% CO2 to allow cell
attachment, cultures were treated with varying concentrations
(0–100 lM) of test compounds. For each concentration, five repli-
cate wells were used. Considering the possible antiproliferative ef-
fects of DMSO, the concentration of DMSO in the array was less
than 0.1% and would not affect the cell growth of the cell lines.
The cell growth was calculated by subtracting the mean OD value
of the respective blank from the mean OD value of the experimen-
tal set. Percentage of growth in the presence of test compounds
was calculated considering the growth in the absence of any test
compounds as 100%, and the results are reported in terms of IC50
(concentration causing 50% inhibition relative to untreated con-
trols) values.
Acknowledgments
This work was financially supported by the National Natural
Science Foundation of China (30800720); Project supported by
the Young Scholars Science Foundation of Lanzhou Jiaotong Uni-
versity (2011011); the Post-Doctor Research Foundation
(20090450142); the Fundamental Research Funds for the Central
Universities (lzujbky-2009-98).
4.6.2. Cell cycle analysis and apoptosis detection
Flow cytometry was used to measure cell cycle profile and
apoptosis. For cell cycle analysis, HeLa cells (1 ꢁ 105) treated with
compounds 21a with various concentrations (0, 0.375, 1.5, and
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