Design, synthesis and biological evaluation of colchicine derivatives as novel tubulin and histone deacetylase dual inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.03.035

A new class of colchicine derivatives were designed and synthesized as tubulin-HDAC dual inhibitors. Biological evaluations of these hybrids included the inhibitory activity of HDAC, tubulin polymerization analysis, in vitro cell cycle analysis in HCT-116 cells and cytotoxicity against different cancer cell lines. Hybrid 6d behaved as potent HDAC-tubulin dual inhibitor and showed comparable cytotoxicity with colchicine. Compound 11a exhibited powerful tubulin inhibitory activity, moderate anti-HDAC activity and the most potent cytotoxicity (IC₅₀ Combining double low line 2-105 nM).

Full text of DOI:10.1016/j.ejmech.2015.03.035

European Journal of Medicinal Chemistry 95 (2015) 127e135
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of colchicine derivatives as
novel tubulin and histone deacetylase dual inhibitors
Xuan Zhang a, Yannan Kong b, Jie Zhang c, Mingbo Su d, Yubo Zhou d, Yi Zang d, Jia Li d,
,
, *
Yi Chen c, Yanfen Fang b *, Xiongwen Zhang b, Wei Lu a
a Institute of Drug Discovery and Development, East China Normal University, Shanghai 200062, PR China
b Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, East China Normal University, Shanghai 200062, PR China
c State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
d National Center for Drug Screening, State Key Laboratory of Drug Research, 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 new class of colchicine derivatives were designed and synthesized as tubulineHDAC dual inhibitors.
Biological evaluations of these hybrids included the inhibitory activity of HDAC, tubulin polymerization
analysis, in vitro cell cycle analysis in HCT-116 cells and cytotoxicity against different cancer cell lines.
Hybrid 6d behaved as potent HDACetubulin dual inhibitor and showed comparable cytotoxicity with
colchicine. Compound 11a exhibited powerful tubulin inhibitory activity, moderate anti-HDAC activity
and the most potent cytotoxicity (IC50 ¼ 2e105 nM).
Received 11 January 2015
Received in revised form
14 March 2015
Accepted 17 March 2015
Available online 18 March 2015
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Colchicine
Dual inhibitor
HDAC
Tubulin
Hybrid
1. Introduction
chemotherapy can simultaneously block several key signaling
pathways and create synergistic antitumor effects [3e11]. These
Though cancer pharmacotherapy has a long history of more
than 70 years, it is still a great challenge to cure cancer. In 2012,
there were more than 14 million new cancer cases around the
world and the number is expected to continuously increase in the
next 20 years [1]. Traditional antitumor drugs including cytotoxic
agents have been widely used for years. However, the usage of
these drugs is hindered by severe toxicity and other undesirable
side effects. The development of targeted molecularly anti-cancer
drugs has made significant achievement over the past decade. But
only part patients show positive response. In addition, the acquired
drug resistance may limit the use of these agents. As a matter of
fact, diseases with linear pathways might be well treated with
single target agents. However, cancer is a disease with complex
signaling networks, which means it is difficult to treat most cancers
by using single classical targeted drug [2]. Combination
regimens are widely used and can improve therapeutic efficacy and
reduce drug toxicity.
Recently, multi-target drug design has become an active
research field [12e14]. These agents can create synergistic anti-
cancer effects. Compared with combination chemotherapy, they
always exhibit simpler pharmacokinetics. Histone deacetylase
(HDAC) involved multi-target drug design is one of the hotspots in
this area. HDAC plays an important role in the regulation of gene
expression. HDAC inhibitors can cause growth arrest, differentia-
tion and apoptosis in cancer cells [15e18]. In general, a HDAC in-
hibitor consists of a capping group, a zinc-binding group (ZBG) and
an appropriate linker (Fig. 1). The effectiveness in oncotherapy
together with the simple SAR has attracted many oncologists into
the exploration of HDAC-involved multi-target agents [19e26].
Three HDAC-targeting drugs, vorinostat (SAHA), romidepsin and
belinostat have been approved by FDA for the treatment of cuta-
neous T-cell lymphoma and peripheral T-cell lymphoma [27e30].
Over 12 HDAC inhibitors are currently in clinical trials for different
cancers [31,32]. Zinc-binding groups play an important role in the
binding efficiency between HDAC inhibitors and enzyme. Among
* Corresponding authors.
E-mail addresses: yffang@sat.ecnu.edu.cn (Y. Fang), wlu@chem.ecnu.edu.cn
(W. Lu).
http://dx.doi.org/10.1016/j.ejmech.2015.03.035
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

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X. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 127e135
of these compounds with TFA gave the target molecules 6aed.
As shown in Scheme 2, preparation of dual inhibitors with
amine linkage started from hydroxymethyl benzoic acids 7 or 8.
These acids were treated with SOCl2 followed by the corresponding
amines in the presence of triethylamine to give compounds 9aed in
good yields. Subsequently, these molecules were treated with de-
Ac-colchicine to afford compounds 10aed. Target hybrids 11aed
were obtained after the removal of protecting groups.
2.2. Functional assay
2.2.1. In vitro HDAC inhibitory activity
We first tested the HDAC inhibition activity of the hybrids
against recombinant human HDAC1, HDAC2 and HDAC3 enzymes,
using mocetinostat as the positive control compound. As shown in
Table 1, all of the hybrids exhibited inhibitory activity against
HDAC1, HDAC2 and HDAC3 except compound 11c. The ZBG type
had a great influence on anti-HDAC activity. Compared with hy-
brids equipped with unsubstituted benzamide ZBG, their analogs
with bis(aryl)-type ZBG showed better potency against HDAC1-3,
which was consistent with the reported literature (6a vs 6b, 6c vs
6d, 11a vs 11b and 11c vs 11d) [38]. Among these conjugates,
compound 6d showed the most potent anti-HDAC activity against
Fig. 1. Representative structures of HDAC inhibitors.
all types of ZBGs, hydroxamate and benzamide are most widely
studied [23].
Previously, we reported a series of colchicineeSAHA hybrids as
novel antitumor agents based on the synergistic antitumor effect
between HDAC inhibitors and tubulin inhibitors [25,33e35].
Among these hybrids, compound 1 behaved as tubulineHDAC dual
inhibitor and displayed the best in vitro antiproliferative activity.
This work suggested that the colchicine moiety is an appropriate
capping group for HDAC inhibitors. For HDAC-involved dual in-
hibitors, ZBG might contribute to the binding activities of both
targets. To further explore this kind of tubulineHDAC dual in-
hibitors, we designed and synthesized a new series of colchicine
derivatives with benzamide moiety as HDAC ZBG (Fig. 2). The
biological evaluations of these molecules include the inhibitory
activity of HDAC, tubulin polymerization analysis, in vitro cell cycle
analysis in HCT-116 cells and cytotoxicity against different cancer
cell lines.
HDAC1 (IC50 ¼ 1.50
mM), HDAC2 (IC50 ¼ 0.19
mM) and HDAC3
(IC50 ¼ 1.49 M).
m
2.2.2. Effect of hybrids on the tubulin polymerization
To probe the impact of these hybrids on tubulin polymerization
dynamics, we followed the kinetics of tubulin polymerization in the
presence of colchicine and hybrids. As shown in Fig. 3, colchicine
can block the polymerization of tubulin into microtubules while
mocetinostat had very little effect [39]. Most of the hybrids caused
inhibition on tubulin polymerization when compared to control
group. Among these tested molecules, compounds 6d, 11a and 11c
displayed comparable tubulin inhibitory activity with the positive
control colchicine.
2. Results and discussion
2.1. Chemistry
2.2.3. Effect of compounds 6d and 11a on cell cycle
Cell cycle analysis of the best HDAC inhibitor 6d and the most
powerful tubulin polymerization inhibitor 11a was investigated.
Fig. 4 shows a significantly increased G2/M peak after treatment of
HCT-116 cells with colchicine at 20 nM. In consistence with the
tubulin polymerization inhibition effects, 6d and 11a have also
exhibited the cell cycle arrest effects. Compared with colchicine,
cells treated with 6d or 11a at 10 nM has already showed a striking
G2/M phase arrest.
The general route for the synthesis of colchicine derivatives with
amide linkage is depicted in Scheme 1. The benzoic acid 2 or 3 was
treated with SOCl2 followed by the corresponding amines in the
presence of triethylamine to afford compounds 4aed. Hydrolysis of
these esters with LiOH gave the corresponding carboxylic acids.
Subsequent amidation of these intermediates with de-Ac-
colchicine [36,37] afforded compounds 5aed. Finally, treatment
Fig. 2. Design of novel tubulin-HDAC dual inhibitors.

X. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 127e135
129
Scheme 1. Synthesis of tubulin-HDAC dual inhibitors with amide linkage. Reagents and conditions: a) i. SOCl2, toluene, 60 ꢀC; ii. triethylamine, DCM; b) i. LiOH, MeOH, H2O; ii. de-
Ac-colchicine, HATU, triethylamine, DCM; c) TFA, DCM.
2.2.4. In vitro anticancer activity
osteosarcoma (SJSA-1). As shown in Table 3, HDAC inhibitor
mocetinostat showed moderate cytotoxicity while tubulin inhibitor
colchicine displayed excellent growth inhibitory activity against
these cancer cells. The antiproliferative activity of dual inhibitor 6d
is comparable with colchicine. Hybrid 11a showed improved
cytotoxicity toward most of tested cancer cell lines. Especially on
HCT-116 cells, the growth inhibitory activity is 8- fold more potent
than the parent compound colchicine.
Evaluation of the antiproliferative activity of designed hybrids
on A549 human lung adenocarcinoma cell line was performed
using the MTT assay. As shown in Table 2, all of the conjugates can
cause cell growth inhibition and showed submicromolar-
micromolar IC50 values. Compound 11a showed the best cytotox-
icity which is comparable with colchicine (IC50 ¼ 0.106
mM). Sur-
prisingly, compound 6d with powerful HDAC and tubulin
inhibitory activity displayed moderate antiproliferative activity
against A549 cell line (IC50 ¼ 2.790
mM). The difference between
their cytotoxicity could be due to the different cell membrane
penetration.
3. Conclusion
In conclusion, as a continuation of our previous work, we pre-
pared a new series of colchicine derivatives. Benzamide type HDAC
zinc binding group was introduced into designed hybrids to regu-
late HDAC and tubulin inhibitory activity. Biological evaluation
displayed that most of these hybrids with colchicine moiety as
HDAC capping group exhibited potent HDAC inhibitory activity.
Subsequently, we selected compounds 6d and 11a to evaluate
their antiproliferative activity against a panel of human cancer cell
lines including colorectal cancer (HCT-116, SW620), hepatocellular
carcinoma (Hep3B, HeoG2 and MHCC97H), gastric cancer (SNU-5,
SNU-16 AND MKN-45), pancreatic carcinoma (PANC-1) and
Scheme 2. Synthesis of tubulin-HDAC dual inhibitors with amine linkage. Reagents and conditions: a) i. SOCl2, toluene, 60 ꢀC; ii. triethylamine, DCM; b) de-Ac-colchicine, NaOAc, KI,
THF, reflux; c) TFA, DCM.

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X. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 127e135
Table 1
Chemical shifts were reported as
d (ppm) and spinespin coupling
In vitro HDAC Inhibition.a
constants as J (Hz) values. The mass spectra (MS) were recorded on
a Finnigan MAT-95 mass spectrometer. The purity of all tested
compounds was established by HPLC to be >95.0%. HPLC analysis
was performed at room temperature using an Agilent Eclipse XDB-
C18 (250 mm ꢁ 4.6 mm) and as a mobile phase gradient from 10%
Compound
IC50 (m
M) SDa
HDAC1
HDAC2
HDAC3
Mocetinostat
Colchicine
6a
6b
6c
0.39 0.03
0.17 0.00
e
0.36 0.00
e
b
-
MeOH/H2O (1
60% MeOH/H2O (1
to 90% MeOH/H2O (1
‰
AcOH) for 1 min, 10% MeOH/H2O (1
AcOH) for 6 min, 60% MeOH/H2O (1
AcOH) for 6 min and 90% MeOH/H2O (1
‰
AcOH) to
15.92 2.62
2.24 0.08
9.62 1.49
1.50 0.36
12.50 1.13
3.32 0.71
e
10.42 0.20
0.38 0.00
4.91 0.31
0.19 0.00
6.73 0.25
0.27 0.03
e
12.10 2.40
1.97 0.08
10.78 1.16
1.49 0.13
11.23 0.46
1.47 0.06
e
‰
‰
AcOH)
‰
‰
6d
AcOH) for 5 min more, a flow rate of 1.0 mL/min and plotted at
254 nm.
11a
11b
11c
11d
25.50 1.49
5.41 0.28
19.68 2.02
4.2. Representative procedure for 4aed
a
Each value was reproduced in three experiments.
b
IC50 > 20 mg/mL.
Acid 2/3 (1.0 equiv), SOCl2 (6.0 equiv) and DMF (1 drop) were
stirred in toluene at 60 ꢀC for 2 h. Then the solvent was removed
under reduced pressure. The acyl chloride was dissolved in DCM
and added dropwise into a solution of corresponding aromatic
amine (1.0 equiv) and triethylamine (2.0 equiv). After the addition,
the mixture was stirred at room temperature for 1 h and extracted
with EtOAc. The organic layer was washed with 1 N HCl (aq) fol-
lowed by 10% Na2CO3 (aq) and dried over Na2SO4. Concentrated in
vacuo gave the crude product, which was further purified by
crystallization from EtOAc-light petroleum.
Compared with molecules equipped with unsubstituted benza-
mide ZBG, hybrids with bis(aryl)-type ZBG showed better anti-
HDAC activity. Compound 6d displayed the most efficient anti-
HDAC activity which is comparable with mocetinostat. On the
other hand, though it is difficult to conclude the SAR for their
tubulin inhibitory activity, the introduction of HDAC pharmaco-
phore into colchicine core kept its anti-tubulin activity. Compounds
6d, 11a and 11c displayed similar anti-tubulin activity with
colchicine. Potent HADCetubulin dual inhibitor 6d exhibited
comparable in vitro antiproliferative activity with colchicine.
Compound 11a with powerful tubulin inhibitory activity and
moderate anti-HDAC activity showed superior cytotoxicity to the
positive control compound (IC50 ¼ 2e105 nM). The present
inspiriting results give a promising future for the optimization of
the series. This finding could be of interest in the search for efficient
anticancer multi-target agents.
4.2.1. Methyl 4-((2-((tert-butoxycarbonyl)amino)phenyl)
carbamoyl)benzoate (4a)
Starting from acid 2 and tert-butyl (2-aminophenyl)carbamate,
87% of compound 4a was obtained as white solid. mp 142e143 ꢀC,
1H NMR (400 MHz, d6-DMSO)
d 9.98 (s, 1H), 8.71 (s, 1H), 8.15e8.03
(m, 4H), 7.55 (dd, J ¼ 13.6, 7.8 Hz, 2H), 7.27e7.08 (m, 2H), 3.91 (s,
3H), 1.44 (s, 9H); HRMS (ESI): m/z calcd for C20H22N2NaO5
(M þ Naþ): 393.1421, found: 393.1412.
4. Experimentals
4.2.2. Methyl 4-((2-((tert-butoxycarbonyl)amino)-5-(thiophen-2-
yl)phenyl)carbamoyl)benzoate (4b)
4.1. General
Starting from acid 2 and tert-butyl (2-amino-4-(thiophen-2-yl)
phenyl)carbamate, 75% of compound 4a was obtained as pale yel-
Melting points were taken on a FishereJohns melting point
apparatus, uncorrected and reported in degrees Centigrade. 1H
NMR spectra and 13C NMR were recorded in CDCl3 and DMSO-d6 on
a Bruker DRX-400 (400 MHz) using TMS as internal standard.
low solid. mp 206e208 ꢀC, 1H NMR (400 MHz, d6-DMSO)
d 10.07 (s,
1H), 8.77 (s, 1H), 8.22e8.08 (m, 4H), 7.81 (d, J ¼ 1.7 Hz, 1H), 7.65 (d,
J ¼ 8.5 Hz, 1H), 7.58e7.50 (m, 2H), 7.46 (d, J ¼ 3.1 Hz, 1H), 7.13 (dd,
J ¼ 5.0, 3.7 Hz, 1H), 3.90 (d, J ¼ 9.4 Hz, 3H), 1.45 (s, 9H); HRMS (ESI):
m/z calcd for C24H24N2NaO5S (M þ Naþ): 475.1298, found: 475.1297.
4.2.3. Methyl 3-((2-((tert-butoxycarbonyl)amino)phenyl)
carbamoyl)benzoate (4c)
Starting from acid 3 and tert-butyl (2-aminophenyl)carbamate,
53% of compound 4a was obtained as white solid. mp 129e130 ꢀC,
1H NMR (400 MHz, CDCl3)
d 9.36 (s, 1H), 8.62 (s, 1H), 8.31e8.08 (m,
2H), 7.76 (d, J ¼ 7.6 Hz,1H), 7.56 (t, J ¼ 7.7 Hz,1H), 7.26e7.09 (m, 3H),
6.86 (s, 1H), 3.95 (s, 3H), 1.50 (s, 9H); HRMS (ESI): m/z calcd for
C
20H22N2NaO5 (M þ Naþ): 393.1421, found: 393.1421.
4.2.4. Methyl 3-((2-((tert-butoxycarbonyl)amino)-5-(thiophen-2-
yl)phenyl)carbamoyl)benzoate (4d)
Starting from acid 3 and tert-butyl (2-amino-4-(thiophen-2-yl)
phenyl)carbamate, 59% of compound 4a was obtained as pale yel-
low solid. mp 142e144 ꢀC, 1H NMR (400 MHz, CDCl3)
d 9.29 (br s,
1H), 8.65 (s, 1H), 8.22 (dd, J ¼ 13.6, 7.8 Hz, 2H), 8.09 (d, J ¼ 1.6 Hz,
1H), 7.58 (t, J ¼ 7.7 Hz, 1H), 7.43 (dd, J ¼ 8.3, 2.1 Hz, 1H), 7.33e7.26
(m, 3H), 7.07 (dd, J ¼ 5.1, 3.6 Hz,1H), 6.75 (s,1H), 3.96 (s, 3H),1.52 (s,
9H); HRMS (ESI): m/z calcd for C24H24N2NaO5S (M þ Naþ):
475.1298, found: 475.1300.
Fig. 3. Kinetics of tubulin polymerization in the absence and presence of 10
lution of hybrids. Tubulin (4.0 mg/mL) was polymerized in the absence and presence of
10 M of colchicine and each of hybrids. The change in absorbance at 340 nm was
monitored.
mM so-
m

X. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 127e135
131
Fig. 4. Effect of hybrids 6d and 11a on cell cycle. Colchicine was used as a positive control.
4.3. Representative procedure for 5aed
concentrated in vacuo. The crude acid (1.0 equiv), de-Ac-colchicine
(0.83 equiv), DIPEA (2.5 equiv) and HATU (0.87 equiv) were stirred
in DCM for 2 h. Subsequently, the mixture was extracted with DCM,
washed with 1 N HCl (aq), dried over Na2SO4 and concentrated in
vacuo. The crude product was further purified by column
Ester (1.0 equiv) and LiOH (2.0 equiv) were stirred in a solution
of MeOH and H2O for 3 h at room temperature. Then 1 N HCl (aq)
(2.2 equiv) was added into the flask and the mixture was

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X. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 127e135
Table 2
(m, 1H), 2.46e2.33 (m, 2H), 2.03e1.84 (m, 1H), 1.43 (s, 9H); HRMS
(ESI): m/z calcd for C43H43N3O9SNa (M þ Naþ): 800.2612, found:
800.2606.
In vitro Cell Growth Inhibition against A549 cell line.a
Compound
A549
Compound
A549
Colchicine
0.105
1.620
0.740
3.430
2.790
Mocetinostat
2.080
0.106
1.040
0.354
0.393
4.4. Representative procedure for 6aed
6a
6b
6c
6d
11a
11b
11c
11d
Trifluoroacetic acid (50.0 equiv) was added dropwise into a
solution of compound 5aed (1.0 equiv) in DCM. The mixture was
stirred at room temperature for 3 h and quenched with saturated
Na2CO3 (aq). Subsequently, the solution was extracted with DCM
and the organic layer was dried over Na2SO4 and concentrated in
vacuo. The crude product was further purified by column chro-
matography (DCM: MeOH ¼ 20:1).
a
Each value was reproduced in three experiments.
Table 3
Evaluation of compounds 6d and 11a against a panel of human cancer cell lines.a
Cell line
Mocetinostat (
m
M)
Colchicine (
m
M)
6d (
m
M)
11a (mM)
4.4.1. Compound 6a
HCT-116
SW620
Hep3B
HepG2
MHCC97H
SNU-5
SNU-16
MKN-45
PANC-1
SJSA-1
0.396
0.419
0.823
0.876
4.563
1.009
0.142
0.610
26.774
3.624
0.040
0.005
0.014
0.027
0.110
0.034
0.005
0.002
0.021
0.008
0.039
0.004
0.010
0.020
0.339
0.119
0.005
0.002
0.019
0.027
0.005
0.002
0.005
0.014
0.043
0.014
0.002
0.002
0.005
0.009
Starting from acid 5a, 70% of compound 6a was obtained as
white solid. mp 192e194 ꢀC, 1H NMR (400 MHz, CDCl3)
d 9.32 (s,
1H), 8.50 (d, J ¼ 6.3 Hz, 1H), 7.78 (s, 1H), 7.70 (d, J ¼ 8.0 Hz, 2H), 7.51
(d, J ¼ 7.8 Hz, 2H), 7.37 (d, J ¼ 10.8 Hz,1H), 7.26e7.23 (m,1H), 7.00 (t,
J ¼ 7.4 Hz, 1H), 6.94 (d, J ¼ 11.0 Hz, 1H), 6.84e6.68 (m, 2H), 6.56 (s,
1H), 4.98e4.83 (m, 1H), 4.02 (s, 3H), 3.92 (s, 3H), 3.90 (s, 3H), 3.55
(s, 3H), 2.61e2.47 (m, 1H), 2.42e2.25 (m, 2H), 2.21e2.09 (m, 1H).
HPLC: room temperature; tR ¼ 9.35 min, UV254 ¼ 95.6%; HRMS
(ESI): m/z calcd for C34H33N3O7Na (M þ Naþ): 618.2216, found:
618.2224.
a
Each value was reproduced in three experiments.
chromatography (DCM: MeOH ¼ 20:1).
4.3.1. Compound 5a
4.4.2. Compound 6b
Starting from acid 5b, 83% of compound 6b was obtained as pale
yellow solid. 191e193 ꢀC, 1H NMR (400 MHz, CDCl3)
d 9.62 (s, 1H),
Starting from acid 4a, 81% of compound 5a was obtained as pale
8.49 (d, J ¼ 5.8 Hz, 1H), 7.80 (d, J ¼ 8.0 Hz, 2H), 7.71 (d, J ¼ 7.8 Hz,
2H), 7.60 (s, 1H), 7.49 (s, 1H), 7.33 (d, J ¼ 10.8 Hz, 1H), 7.20e7.11 (m,
2H), 7.09e7.02 (m, 1H), 7.00e6.94 (m, 1H), 6.88 (d, J ¼ 11.1 Hz, 1H),
6.69 (d, J ¼ 8.2 Hz, 1H), 6.52 (s, 1H), 4.88e4.75 (m, 1H), 4.44 (br s,
2H), 3.98 (s, 3H), 3.90 (s, 6H), 3.62 (s, 3H), 2.53e2.39 (m, 1H),
2.37e2.12 (m, 2H), 2.04e1.88 (m, 1H). HPLC: room temperature;
tR ¼ 10.94 min, UV254 ¼ 97.5%; HRMS (ESI): m/z calcd for
yellow solid. mp 184e186 ꢀC, 1H NMR (400 MHz, CDCl3)
d
9.38 (s,
1H), 7.84e7.67 (m, 6H), 7.63 (s,1H), 7.41e7.35 (m, 2H), 7.23e7.13 (m,
3H), 6.90 (d, J ¼ 10.9 Hz,1H), 6.58 (s, 1H), 4.97e4.81 (m, 1H), 4.00 (d,
J ¼ 12.6 Hz, 3H), 3.98e3.87 (m, 6H), 3.61 (s, 3H), 2.68e2.55 (m, 1H),
2.53e2.31 (m, 2H), 2.20e2.06 (m, 1H), 1.45 (s, 9H); HRMS (ESI): m/z
calcd for C39H41N3O9Na (M þ Naþ): 718.2735, found: 718.2750.
C
34H36N3O7S (M þ Hþ): 678.2268, found: 678.2255.
4.3.2. Compound 5b
Starting from acid 4b, 50% of compound 5b was obtained as pale
4.4.3. Compound 6c
yellow solid. mp 188e190 ꢀC, 1H NMR (400 MHz, CDCl3)
d 9.41 (s,
Starting from acid 5c, 57% of compound 6c was obtained as pale
yellow solid. 178e180 ꢀC, 1H NMR (400 MHz, CDCl3)
d 9.90 (br s,
1H), 7.99 (s, 1H), 7.83e7.71 (m, 5H), 7.53 (s, 1H), 7.40e7.31 (m, 4H),
7.26e7.22 (m, 2H), 7.07e7.04 (m, 1H), 6.90 (d, J ¼ 11.0 Hz, 1H), 6.53
(s, 1H), 4.90e4.78 (m, 1H), 4.02 (s, 3H), 3.93e3.89 (m, 6H), 3.64 (s,
3H), 2.53e2.29 (m, 3H), 2.08e2.01 (m,1H),1.46 (s, 9H); HRMS (ESI):
1H), 8.71 (d, J ¼ 5.0 Hz, 1H), 8.65 (s, 1H), 7.90e7.79 (m, 2H), 7.49 (s,
1H), 7.33 (d, J ¼ 10.8 Hz, 1H), 7.17e7.07 (m, 2H), 7.03 (t, J ¼ 7.5 Hz,
1H), 6.87e6.75 (m, 2H), 6.71 (t, J ¼ 7.5 Hz, 1H), 6.52 (s, 1H),
4.76e4.67 (m, 1H), 4.59 (br s, 2H), 3.94 (s, 3H), 3.92 (s, 3H), 3.90 (s,
3H), 3.74 (s, 3H), 2.52e2.42 (m, 1H), 2.37e2.25 (m, 1H), 2.21e2.09
(m, 1H), 1.85e1.72 (m, 1H). HPLC: room temperature; tR ¼ 9.66 min,
UV254 ¼ 97.0%; HRMS (ESI): m/z calcd for C34H33N3O7Na (M þ Naþ):
618.2216, found: 618.2247.
m/z calcd for
800.2606.
C
43H43N3O9SNa (M
þ
Naþ): 800.2612, found:
4.3.3. Compound 5c
Starting from acid 4c, 46% of compound 5c was obtained as
yellow solid.176e178 ꢀC, 1H NMR (400 MHz, CDCl3)
9.20 (br s,1H),
d
8.51 (br s, 1H), 8.12 (br s,1H), 7.98 (t, J ¼ 7.4 Hz, 2H), 7.67 (s, 1H), 7.57
(d, J ¼ 8.1 Hz, 1H), 7.50e7.31 (m, 4H), 7.21 (t, J ¼ 7.1 Hz, 1H), 7.11 (t,
J ¼ 7.1 Hz, 1H), 6.82 (d, J ¼ 11.0 Hz, 1H), 6.54 (s, 1H), 4.87e4.74 (m,
1H), 3.94 (s, 3H), 3.90 (s, 3H), 3.86 (s, 3H), 3.68 (s, 3H), 2.59e2.50
(m, 1H), 2.46e2.33 (m, 2H), 2.03e1.84 (m, 1H), 1.43 (s, 9H); HRMS
(ESI): m/z calcd for C39H41N3O9Na (M þ Naþ): 718.2735, found:
718.2746.
4.4.4. Compound 6d
Starting from acid 5d, 60% of compound 6d was obtained as pale
yellow solid. mp 191e192 ꢀC, 1H NMR (400 MHz, CDCl3)
d 9.64 (br s,
1H), 8.80 (br s, 1H), 8.65 (s, 1H), 7.91 (d, J ¼ 7.8 Hz, 1H), 7.83 (d,
J ¼ 7.8 Hz, 1H), 7.60 (s, 1H), 7.40 (s, 1H), 7.32e7.27 (m, 2H), 7.19e7.04
(m, 3H), 7.01e6.94 (m, 1H), 6.76 (d, J ¼ 10.0 Hz, 2H), 6.51 (s, 1H),
4.80e4.53 (m, 3H), 3.93 (s, 3H), 3.90 (s, 3H), 3.79 (s, 3H), 3.77 (s,
3H), 2.54e2.29 (m, 2H), 2.26e2.12 (m,1H),1.89e1.77 (m,1H). HPLC:
room temperature; tR ¼ 11.06 min, UV254 ¼ 96.5%; HRMS (ESI): m/z
calcd for C34H36N3O7S (M þ Hþ): 678.2268, found: 678.2220.
4.3.4. Compound 5d
Starting from acid 4d, 68% of compound 5d was obtained as
yellow solid. 182e183 ꢀC, 1H NMR (400 MHz, CDCl3)
d 9.20 (br s,
1H), 8.51 (br s,1H), 8.12 (br s,1H), 7.98 (t, J ¼ 7.4 Hz, 2H), 7.67 (s,1H),
7.57 (d, J ¼ 8.1 Hz, 1H), 7.50e7.31 (m, 4H), 7.21 (t, J ¼ 7.1 Hz, 1H), 7.11
(t, J ¼ 7.1 Hz, 1H), 6.82 (d, J ¼ 11.0 Hz, 1H), 6.54 (s, 1H), 4.87e4.74 (m,
1H), 3.94 (s, 3H), 3.90 (s, 3H), 3.86 (s, 3H), 3.68 (s, 3H), 2.59e2.50
4.5. Representative procedure for 9aed
Acid 7/8 (1.0 equiv), SOCl2 (6.0 equiv) and DMF (1 drop) were
stirred in toluene at 60 ꢀC for 2 h. Then the solvent was removed

X. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 127e135
133
under reduced pressure. The acyl chloride was dissolved in DCM
and added dropwise into a solution of corresponding aromatic
amine (1.0 equiv) and triethylamine (3.0 equiv). After stirring for
1 h at room temperature, the solution was extracted with EtOAc,
washed with 1 N HCl (aq) followed by brine and dried over Na2SO4.
Concentrated in vacuo gave the crude product, which was further
purified by column chromatography.
3.79e3.74 (m, 1H), 3.59 (s, 3H), 3.52e3.43 (m, 2H), 2.53e2.31 (m,
2H), 2.30e2.14 (m, 1H), 1.70e1.65 (m, 1H), 1.50 (s, 9H); HRMS (ESI):
m/z calcd for C39H43N3NaO8 (M þ Naþ): 704.2942, found: 704.2938.
4.6.2. Compound 10b
Starting from compound 9b, 56% of compound 10b was ob-
tained as pale yellow solid. mp 157e159 ꢀC, 1H NMR (400 MHz,
CDCl3)
d 9.01 (br s, 1H), 8.05 (s, 1H), 7.91e7.80 (m, 3H), 7.44e7.29
4.5.1. tert-Butyl (2-(4-(chloromethyl)benzamido)phenyl)carbamate
(9a)
(m, 6H), 7.25e7.19 (m, 1H), 7.09e7.02 (m, 1H), 6.88 (s, 1H), 6.81 (d,
J ¼ 10.8 Hz, 1H), 6.52 (s, 1H), 3.99 (s, 3H), 3.91 (s, 3H), 3.90 (s, 3H),
3.79 (d, J ¼ 13.8 Hz, 1H), 3.60 (s, 3H), 3.51 (d, J ¼ 13.6 Hz, 1H), 3.45
(dd, J ¼ 10.8, 6.2 Hz, 1H), 2.52e2.34 (m, 2H), 2.28e2.17 (m, 1H),
1.78e1.67 (m, 1H), 1.52 (s, 9H) HRMS (ESI): m/z calcd for
Starting from acid 7 and tert-butyl (2-aminophenyl)carbamate,
82% of compound 9a was obtained as white solid. mp 176e177 ꢀC,
1H NMR (400 MHz, CDCl3)
d
9.24 (s, 1H), 7.97 (d, J ¼ 8.1 Hz, 2H), 7.83
(d, J ¼ 7.8 Hz, 1H), 7.49 (d, J ¼ 8.2 Hz, 2H), 7.26e7.14 (m, 3H), 6.73 (s,
C
43H45N3NaO8S (M þ Naþ): 786.2820, found: 786.2820.
1H), 4.64 (s, 2H), 1.52 (s, 9H); HRMS (ESI): m/z calcd for
C
19H21ClN2O3Na (M þ Naþ): 383.1133, found: 383.1149.
4.6.3. Compound 10c
Starting from compound 9c, 88% of compound 10c was obtained
4.5.2. tert-Butyl (2-(4-(chloromethyl)benzamido)-4-(thiophen-2-
yl)phenyl)carbamate (9b)
as yellow soild. mp 99e101 ꢀC, 1H NMR (400 MHz, CDCl3)
d 9.08 (br
s, 1H), 7.97 (s, 1H), 7.85 (s, 1H), 7.79 (d, J ¼ 7.5 Hz, 2H), 7.44e7.31 (m,
3H), 7.25e7.15 (m, 3H), 7.09 (s, 1H), 6.81 (d, J ¼ 10.7 Hz, 1H), 6.53 (s,
1H), 3.98 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.72 (d, J ¼ 13.4 Hz, 1H),
3.60e3.52 (m, 4H), 3.46 (dd, J ¼ 10.8, 6.2 Hz,1H), 2.52e2.33 (m, 2H),
2.31e2.18 (m, 1H), 1.78e1.72 (m, 1H), 1.47 (s, 9H); HRMS (ESI): m/z
calcd for C39H43N3NaO8 (M þ Naþ): 704.2942, found: 704.2947.
Starting from acid 7 and tert-butyl (2-amino-4-(thiophen-2-yl)
phenyl)carbamate, 84% of compound 9b was obtained as pale yel-
low solid. mp 167e169 ꢀC, 1H NMR (400 MHz, CDCl3)
d 9.23 (br s,
1H), 8.09 (d, J ¼ 1.6 Hz, 1H), 7.98 (d, J ¼ 8.2 Hz, 2H), 7.50 (d,
J ¼ 8.2 Hz, 2H), 7.40 (dd, J ¼ 8.3, 2.1 Hz, 1H), 7.31e7.28 (m, 1H),
7.28e7.26 (m, 1H), 7.26e7.23 (m, 1H), 7.06 (dd, J ¼ 5.0, 3.6 Hz, 1H),
6.76 (s, 1H), 4.64 (s, 2H), 1.52 (s, 9H); HRMS (ESI): m/z calcd for
4.6.4. Compound 10d
C
23H23ClN2O3SNa (M þ Naþ): 465.1010, found: 465.1001.
Starting from compound 9d, 56% of compound 10d was ob-
tained as yellow soild. mp 146e148 ꢀC, 1H NMR (400 MHz, CDCl3)
4.5.3. tert-Butyl (2-(3-(chloromethyl)benzamido)phenyl)carbamate
(9c)
d 9.13 (br s, 1H), 8.12e7.99 (m, 2H), 7.91e7.78 (m, 2H), 7.45e7.30 (m,
6H), 7.25e7.14 (m, 2H), 7.10e7.03 (m, 1H), 6.82 (d, J ¼ 10.8 Hz, 1H),
6.53 (s, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.71 (d,
J ¼ 12.5 Hz, 1H), 3.61 (d, J ¼ 13.3 Hz, 1H), 3.56 (s, 3H), 3.51e3.44 (m,
1H), 2.52e2.33 (m, 2H), 2.31e2.19 (m, 1H), 1.78e1.70 (m, 1H), 1.48
(s, 9H); HRMS (ESI): m/z calcd for C43H45N3NaO8S (M þ Naþ):
786.2820, found: 786.2822.
Starting from acid 8 and tert-butyl (2-aminophenyl)carbamate,
95% of compound 9c was obtained as white solid. mp 50e52 ꢀC, 1H
NMR (400 MHz, CDCl3)
d 9.25 (br s, 1H), 7.99 (s, 1H), 7.92 (d,
J ¼ 7.8 Hz, 1H), 7.82 (d, J ¼ 7.9 Hz, 1H), 7.59 (d, J ¼ 7.7 Hz, 1H), 7.48 (t,
J ¼ 7.7 Hz, 1H), 7.26e7.15 (m, 3H), 6.74 (s, 1H), 4.64 (s, 2H), 1.52 (s,
9H); HRMS (ESI): m/z calcd for C19H21ClN2O3Na (M þ Naþ):
383.1133, found: 383.1129.
4.7. Representative procedure for 11aed
4.5.4. tert-Butyl (2-(3-(chloromethyl)benzamido)-4-(thiophen-2-
yl)phenyl)carbamate (9d)
Trifluoroacetic acid (50.0 equiv) was added dropwise into a
solution of compound 10aed (1.0 equiv) in DCM. The mixture was
stirred for 3 h at room temperature and quenched with saturated
Na2CO3 (aq). The solution was extracted with DCM and the organic
layer was dried over Na2SO4 and concentrated in vacuo. The crude
product was further purified by column chromatography (DCM:
MeOH ¼ 20:1).
Starting from acid 8 and tert-butyl (2-amino-4-(thiophen-2-yl)
phenyl)carbamate, 94% of compound 9d was obtained as brown
soild. mp 146e148 ꢀC, 1H NMR (400 MHz, CDCl3)
d 9.27 (br s, 1H),
8.07 (d, J ¼ 1.8 Hz, 1H), 8.01 (s, 1H), 7.93 (d, J ¼ 7.8 Hz, 1H), 7.60 (d,
J ¼ 7.7 Hz, 1H), 7.48 (t, J ¼ 7.7 Hz, 1H), 7.40 (dd, J ¼ 8.3, 2.1 Hz, 1H),
7.29 (dd, J ¼ 3.6, 1.0 Hz, 1H), 7.26e7.21 (m, 2H), 7.05 (dd, J ¼ 5.1,
3.6 Hz, 1H), 6.80 (s, 1H), 4.64 (s, 2H), 1.52 (s, 9H); HRMS (ESI): m/z
calcd for C23H23ClN2O3SNa (M þ Naþ): 465.1010, found: 465.1001.
4.7.1. Compound 11a
Starting from compound 10a, 56% of compound 11a was ob-
tained as white solid. mp 128e129 ꢀC, 1H NMR (400 MHz, CDCl3)
4.6. Representative procedure for 10aed
d
8.04 (s, 1H), 7.83 (s, 1H), 7.76 (d, J ¼ 6.9 Hz, 2H), 7.41e7.29 (m, 3H),
7.23 (d, J ¼ 10.7 Hz, 1H), 7.07 (t, J ¼ 7.4 Hz, 1H), 6.91e6.75 (m, 3H),
6.52 (s, 1H), 3.96 (s, 3H), 3.91 (s, 3H), 3.90 (s, 3H), 3.73 (d,
J ¼ 13.5 Hz, 1H), 3.60 (s, 3H), 3.54 (d, J ¼ 13.5 Hz, 1H), 3.47e3.41 (m,
1H), 2.50e2.34 (m, 2H), 2.28e2.15 (m,1H),1.77e1.65 (m,1H). HPLC:
room temperature; tR ¼ 7.89 min, UV254 ¼ 100.0%; HRMS (ESI): m/z
calcd for C34H35N3NaO6 (M þ Naþ): 604.2418, found: 604.2454.
A mixture of de-Ac-colchicine (1.0 equiv), compound 9aed
(1.5 equiv), NaOAc (5.0 equiv) and KI (1.2 equiv) were refluxed in
THF for 48 h. Then the mixture was cooled to room temperature
and extracted with EtOAc. The organic layer was washed with 10%
Na2S2O3 (aq) followed by brine and dried over Na2SO4. Concen-
trated in vacuo gave the crude product, which was further purified
by column chromatography (DCM: MeOH ¼ 20:1).
4.7.2. Compound 11b
Starting from compound 10b, 57% of compound 11b was ob-
4.6.1. Compound 10a
tained as white solid. mp 143e145 ꢀC, 1H NMR (400 MHz, CDCl3)
Starting from compound 9a, 80% of compound 10a was obtained
d
8.02 (br s, 1H), 7.82 (s, 1H), 7.78 (d, J ¼ 8.1 Hz, 2H), 7.65 (s, 1H),
as pale yellow solid. mp 139e141 ꢀC, 1H NMR (400 MHz, CDCl3)
d
7.38e7.31 (m, 3H), 7.25e7.16 (m, 3H), 7.02 (dd, J ¼ 4.9, 3.8 Hz, 1H),
6.82 (dd, J ¼ 19.4, 9.5 Hz, 2H), 6.52 (s, 1H), 3.96 (s, 3H), 3.92 (s, 3H),
3.90 (s, 3H), 3.74 (d, J ¼ 13.6 Hz,1H), 3.61 (s, 3H), 3.57 (d, J ¼ 13.6 Hz,
1H), 3.43 (dd, J ¼ 10.8, 6.2 Hz, 1H), 2.51e2.32 (m, 2H), 2.27e2.16 (m,
9.10 (s, 1H), 7.89e7.80 (m, 3H), 7.75 (dd, J ¼ 7.8, 1.3 Hz, 1H),
7.36e7.29 (m, 3H), 7.25e7.11 (m, 3H), 6.99 (s, 1H), 6.80 (d,
J ¼ 11.0 Hz, 1H), 6.52 (s, 1H), 3.98 (s, 3H), 3.91 (s, 3H), 3.90 (s, 3H),

134
X. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 127e135
1H), 1.75e1.70 (m, 1H). HPLC: room temperature; tR ¼ 9.85 min,
UV254 ¼ 95.0%; HRMS (ESI): m/z calcd for C38H38N3O6S (M þ Hþ):
664.2476, found: 664.2513.
4.10. Cell cycle analysis
Cells treated with test compounds for 24 h were collected and
cell cycle distribution was determined by flow cytometry. In brief,
the cellular DNA content was measured on an easyCyte 6HT-2L
(Millipore) after cells were incubated with RNase A (10 mg/mL) and
propidium iodide (50 mg/mL). The percentage of each population
was analysed using ModFIT software (BD Biosciences). At least
20 000 cells were analyzed for each data point.
4.7.3. Compound 11c
Starting from compound 10c, 38% of compound 11c was ob-
tained as pale yellow solid. mp 120e122 ꢀC, 1H NMR (400 MHz,
CDCl3)
d
8.36 (br s, 1H), 7.97 (s, 1H), 7.88 (s, 1H), 7.76 (d, J ¼ 6.9 Hz,
1H), 7.44 (d, J ¼ 7.6 Hz, 1H), 7.38e7.28 (m, 2H), 7.25e7.20 (m, 1H),
7.07 (t, J ¼ 7.3 Hz, 1H), 6.91e6.77 (m, 3H), 6.53 (s, 1H), 3.97 (s, 3H),
3.91 (s, 3H), 3.90 (s, 3H), 3.72e3.58 (m, 2H), 3.57 (s, 3H), 3.50e3.43
(m, 1H), 2.51e2.44 (m, 1H), 2.43e2.34 (m, 1H), 2.30e2.22 (m, 1H),
1.78e1.69 (m, 1H). HPLC: room temperature; tR ¼ 8.09 min,
UV254 ¼ 95.9%; HRMS (ESI): m/z calcd for C34H35N3NaO6 (M þ Naþ):
604.2418, found: 604.2464.
Acknowledgments
This work was supported by the grants of National Natural
Science Foundation of China (No. 81172936 and No. 81173033).
Appendix A. Supplementary data
4.7.4. Compound 11d
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.03.035.
Starting from compound 10d, 31% of compound 11d was ob-
tained as yellow solid. mp 136e138 ꢀC, 1H NMR (400 MHz, CDCl3)
d
8.46 (br s, 1H), 8.01 (s, 1H), 7.91 (s, 1H), 7.84e7.69 (m, 2H),
References
7.41e7.30 (m, 3H), 7.26e7.13 (m, 3H), 7.07e6.99 (m, 1H), 6.88e6.75
(m, 2H), 6.54 (s,1H), 3.96 (s, 3H), 3.92 (s, 3H), 3.90 (s, 3H), 3.71e3.63
(m, 2H), 3.58 (s, 3H), 3.52e3.44 (m, 1H), 2.51e2.34 (m, 2H),
2.31e2.21 (m, 1H), 1.77e1.72 (m, 1H). HPLC: room temperature;
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