Synthesis, molecular modeling and biological evaluation of guanidine derivatives as novel antitubulin agents

Article abstract of DOI:10.1016/j.bmc.2010.10.008

A series of novel chalcone guanidine derivatives (4a-4q) have been designed and synthesized, and their biological activity were also evaluated as potential antiproliferative and antitubulin polymerization inhibitors. Compound 4q showed the most potent biological activity (IC₅₀ = 0.09 ± 0.01 μM for MCF-7 and IC₅₀ = 8.4 ± 0.6 μM for tubulin), which is comparable to the positive controls. Docking simulation was performed to position compound 4q into the colchicine binding site to determine the probable binding model, which suggested probable inhibition mechanism.

Full text of DOI:10.1016/j.bmc.2010.10.008

Bioorganic & Medicinal Chemistry 18 (2010) 8218–8225
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular modeling and biological evaluation of guanidine
derivatives as novel antitubulin agents
⇑
Yong Qian, Hong-Jia Zhang, Peng-Cheng Lv, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel chalcone guanidine derivatives (4a–4q) have been designed and synthesized, and their
Received 31 August 2010
Revised 2 October 2010
Accepted 5 October 2010
Available online 29 October 2010
biological activity were also evaluated as potential antiproliferative and antitubulin polymerization
inhibitors. Compound 4q showed the most potent biological activity (IC50 = 0.09 0.01
lM for MCF-7
and IC50 = 8.4 0.6 M for tubulin), which is comparable to the positive controls. Docking simulation
l
was performed to position compound 4q into the colchicine binding site to determine the probable bind-
ing model, which suggested probable inhibition mechanism.
Published by Elsevier Ltd.
Keywords:
Chalcone
Guanidine
Molecular modeling
Antitubulin polymerization
1. Introduction
the human tumor cells. For example, Methyl-GAG (methylgly-
oxal-bisguanylhydrazone, MAGA) as a single agent has been used
Microtubules are key cytoskeletal filaments and play essential
roles in numerous cellular functions, such as cell motility, cell divi-
sion, shape maintenance and vesicle transport. Microtubules are in
dynamic equilibrium with tubulin dimers.1 Inhibiting tubulin poly-
merization or interfering with microtubule disassembly will inter-
rupt the dynamic equilibrium, leading to cell cycle arrest or cell
apoptosis induction. Given their significant role in the cellular
functions, microtubule has been a proven molecular target for can-
cer chemotherapeutic agents. Several natural and synthetic agents
capable of interfering with the polymerization or depolymerization
of microtubulin have been discovered and currently used in pa-
tient.2–4 More recently, new tubulin targeting agents have been
intensively investigated and developed, such as combretastatin
A-4 phosphate, which target the colchicine binding site of the
tubulin and disrupt the endothelial cell morphology to stop blood
flow through tumor capillaries, and consequently, leading to tumor
cell death.5–7
in clinical for malignant lymphoma, carcinoma of the head, neck,
and esophageal and non-small-cell lung cancer.15 Recently, Chen
and Cai groups designed and synthesized two series of guanidine
analogs as antitubulin polymerization agents which can cause pro-
liferation inhibition and apoptosis induction in tumor models.16,17
As a part of our research for novel antitubulin polymerization
agents, we designed and synthesized a series of aminoguanidine
analogs based on chalcone scaffold. We chose chalcone moiety as
it was an important pharmacophore of natural products and the
synthetic precursors that were found to possess various biologi-
cally activity, including antimitotic, and antiproliferative activi-
ties.18,19 Particularly, chalcone derivatives as tubulin binding
agents are also explored and developed.20 Herein, we described
the synthesis and the SAR of the novel series of guanidine deriva-
tives, and the biological activity evaluation indicated that some of
these compounds are potent inhibitors of tubulin polymerization.
Docking simulations were also performed using the X-ray crystal-
lographic structure of the tubulin in complex with an inhibitor to
explore the binding modes of these compounds at the active site.
Guanidine derivatives have been intensively reported to possess
potent antiviral and anticancer activites.8–10 Guanidine derivatives
have attracted considerable attention as they have been shown
a
tendency to inhibit S-adenosylmethionine decarboxylase
2. Results and discussion
2.1. Chemistry
(SAMDC),11 ribonucleotide reductase (RR),12 and can accumulate
in the mitochondria to damage mitochondrial and inhibit
respiratory chain,13 and other can bind DNA minor groove,14
leading to cell proliferation inhibition and apoptosis induction in
The synthetic route for the novel aminoguanidine derivatives
4a–4v is outlined in Scheme 1. These compounds were prepared
according to the modified procedure of Fukumoto et al.21 by
reaction of chalcones with aminoguanidine in the presence of
⇑
Corresponding author. Tel./fax: +86 25 8359 2572.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.10.008

Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 8218–8225
8219
Scheme 1. Synthesis of chalcone aminoguanidine derivatives.
hydrochloric acid with good yields of 70–80%. All the aminoguani-
dine derivatives were isolated and purified by column chromatog-
raphy and then recrystallized by acetone/MeOH/Et2O, and their
structure were shown in Table 1. All of the synthetic compounds
gave satisfactory analytical and spectroscopia data, which were
in full accordance with their depicted structures.
human breast carcinoma cells. The results were summarized in
Table 2. A number of guanidine compounds exhibited remarked ef-
fects on antiproliferative activities. Among them, compound 4q
displayed the most potent inhibitory activity (IC50 = 0.09 0.01
for MCF-7 and IC50 = 8.4 0.6 M for tubulin), comparable to the
positive control colchicine (IC50 = 0.017 0.01 M for MCF-7 and
IC50 = 3.2 0.4 M for tubulin, respectively).
lM
l
l
l
2.2. Biological activity
Structure–activity relationships in these guanidine derivatives
demonstrated that compounds with substitution at the para (4a–
4c, 4e, and 4f) position showed more potent activities than those
with substitution at the ortho (4l–4o) and meta (4i–4k) position
in the A-ring. A comparison of the para position substitution on
the A-ring demonstrated that a para halogen group (4a–4c) have
slightly improved antiproliferative activity and the potency order
is F < Cl < Br, whereas a methyl (4g) and methoxy (4h) group
All the synthesized guanidine compounds 4a–4v were evalu-
ated for their ability to antiproliferative activities against MCF-7
Table 1
Structure of chalcone aminoguanidine derivatives
Table 2
Inhibition of tubulin polymerization and inhibition (IC50) of MCF-7 cells
proliferation by compound 4a–4v
Compound
MCF-7a
Tubulinb
IC50 SD (lM)
IC50 SD (lM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
41
4m
4n
4o
4p
4q
2.5 0.12
0.7 0.1
0.25 0.04
28
14
10
140 40
12
16
170 30
140 20
85
150 20
130 40
42
4
2
3
Compound
R1
R2
R3
R4
R5
4a
4b
4c
4d
4e
41
4g
4h
4i
H
H
H
H
H
H
H
H
H
H
H
F
CI
Br
OMe
H
H
H
F
CI
Br
H
Ph
OCH2Ph
Me
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N02
F
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CI
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
17
2
0.8 0.05
1.2 0.02
3
2
16
14
4
2
6.5 0.7
8
16
13
3
3
4j
3.9 0.15
4.2 0.45
4.2 0.5
7.2 0.6
0.35 0.04
0.09 0.01
5
4k
41
4m
4n
4o
4p
4q
4r
55 10
52
84
15
8.4 0.6
150 20
130 15
24
80 10
18
3.2 0.4
2.2 0.2
8
7
4
CI
Br
Me
OMe
CI
OMe
4r
4s
4t
4u
14
13
3
2
H
H
H
H
1.9 0.7
8.1 0.4
1.6 0.2
0.017 0.01
0.013 0.003
6
4s
4t
4u
H
H
CI
H
H
H
4v
3
Colchicine
CSA-4
4v
H
H
a
Inhibition the growth of MCF-7 human breast carcinoma cells.
Inhibition of tubulin polymerization.
b

8220
Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 8218–8225
substituent had minimal effects compared with 4d. Meanwhile, a
significant loss of activity was observed when the halogen substi-
tuent was moved from the para position to the ortho (4j) or meta
(4l–4n) position in the A-ring. A methoxy group at para (4h), meta
(4k), and ortho (4o) in the A-ring all led to a noteworthy poor activ-
ity. Considering the steric effect of the A-ring, we designed and
evaluated the compounds with p-phenyl (4e), p-benzyloxy (4f)
group substituent in the A-ring, moreover, variation of the aro-
matic A-ring with a naphthyl moiety substituent (4v), which all
exhibited good antiproliferative activity. The compounds with para
halogen substituted (4p, 4q) in the B-ring exhibited excellent anti-
proliferative activity, 4q with substituent of p-Br showed the most
potent activity in these compounds. However, compound 4r
(p-Me) and 4s (p-OMe) displayed a poor activity. Interesting, com-
pound 4t (with 3,4-Cl group in the B-ring) and compound 4u (with
a p-Cl group in the A-ring and a p-OMe group in the B-ring) also
showed the moderate activity.
evaluated as potent antitubulin polymerization inhibitors and
antiproliferative activity against MCF-7 human breast carcinoma
cells. Compound 4q demonstrated the most potent inhibitory
activity that inhibited the growth of MCF-7 cells with IC50 of
0.09 0.01
lM and inhibited the polymerization of tubulin with
IC50 of 8.4 0.6
lM, which was compared with the positive control.
Docking simulation was performed to study the probable binding
model. Analysis of the compound 4q’s binding conformation in
the colchicine binding site demonstrated that compound 4q was
stabilized by hydrogen bonding interaction with THR 179 and
ASN 349. Moreover, the binding of compound 4q to tubulin was
also stabilized by hydrophobic interaction. Antitubulin polymeri-
zation and antiproliferative assay results showed the compound
4q was a potential anticancer agent.
4. Experiments
To examine whether the compounds interact with tubulin and
inhibit tubulin polymerization in vitro, we performed the tubulin
assembly assay. As shown in Table 2, compounds 4c, 4e, and 4q
showed strong inhibitory effect and their 50% tubulin polymeriza-
4.1. Materials and measurements
All chemicals and reagents used in current study were of analyt-
ical grade. All the 1H NMR spectra were recorded on a Bruker DRX
500 or DPX 300 model Spectrometer in CDCl3 or DMSO-d6 and
chemical shifts was reported in ppm (d). ESI-MS spectra were re-
corded on a Mariner System 5304 Mass spectrometer. Elemental
analyzes were performed on a CHN–O–Rapid instrument. TLC
was performed on the glass-backed silica gel sheets (silica gel
60 Å GF254) and visualized in UV light (254 nm). Column chroma-
tography was performed using silica gel (200–300 mesh) eluting
with ethyl acetate and petroleum ether.
tion inhibition about 10
3
lM, 12
4 lM, and 8.4 0.6 lM,
respectively. Compound 4q displayed the most potent antitubulin
polymerization activity. The result confirmed that the antiprolifer-
ative effect was mediated by direct interaction of compounds with
tubulin.
To further address the death pattern, the effect of compound 4q
on the morphology of MCF-7 human breast carcinoma cells was
further tested using the fluorescent nuclear DNA stain DAPI, which
is sensitive to DNA. Results of DAPI staining assay showed that
cells demonstrated apoptotic features, including chromatin aggre-
gation, nuclear and cytoplasmic condensation, nuclear fragmenta-
tion, and nucleus into membrane bound vesicles (apoptotic bodies)
after treatment with the compound 4q for 24 h. (Fig. 2)
Compound 4q was also performed cell cycle analysis using flow
cytometry. Cell cycle analysis (Fig. 3) showed that the compound
4q strongly induced G2/M arrest in MCF-7 cells, and this effect
was observed in a dose-independent manner after treatment for
24 h increasing amounts of the compound 4q (treatment with
50 nm and 500 nm, respectively). About 54.12% of the cells were
arrested in the G2/M phase after treatment with 500 nM 4q for
24 h. These findings indicated a continuing impairment of cell divi-
sion and confirmed compound 4q was a potent antitubulin agent.
In order to gain more understanding of the structure–activity
relationships observed at the tubulin, molecular docking of the
most potent inhibitor 4q into the colchicine binding site of tubulin
was performed on the binding model based on the tubulin struc-
ture (PDB code: 1SA0). The binding model of compound 4q and
tubulin is depicted in Figures 4A and B. In the binding model, com-
pound 4q is nicely bound to the colchicine site of tubulin via
hydrogen bond with THR 179 (angle OꢀꢀꢀH–N = 146.729°, dis-
tance = 2.249 Å), ASN 349 (angle OꢀꢀꢀH–N = 161.980°, dis-
tance = 1.584 Å). As depicted in Figure 2B, more details revealed
that the hydrophobic pockets of binding site were also occupied
by compound 4q. The bromophenyl moiety of B-ring occupied a
pocket bounded by LEU 248, ALA 250 and LEU 255, the phenyl moi-
ety of A-ring was embedded in a hydrophobic pocket constructed
by the residues of SER 178 and LYS 352. The assay data and the
molecular docking results showed that compound 4q was a poten-
tial antitubulin inhibitor.
4.2. General procedure for synthesis of chalcones
To a stirred solution of acetophenone derivatives or acetone
(1 mmol) and a benzlaldehyde derivatives (1 mmol) in MeOH
(30 mL) was added 6 M KOH (4 mL) and the reaction mixture
was stirred until the solids formed. The products were filtrated
and washed carefully with ice water and cool MeOH; the resulting
chalcones were purified by crystallization from MeOH in refrigera-
tor. (Scheme 1)
4.3. General procedure for the preparation of aminoguanidine
derivatives of chalcones
Chalcone (1 mmol), aminoguanidine hydrochloride (1 mmol)
and concentrated HCl (0.2 mL) in EtOH (6 mL) were refluxed for
24 h. The solvent was evaporated under reduced pressure. The res-
idue was washed by water several times, then washed by Et2O, and
the residue was purified by column chromatography and then
recrystallized by acetone/MeOH/Et2O to give the compounds
(4a–4x). (Scheme 1)
4.3.1. 2-(3-(4-Fluorophenyl)-1-phenylallylidene) hydrazinecarb-
oximidamide (4a)
1H NMR (500 MHz, DMSO-d6, d ppm): 6.87(d, J = 16.0 Hz, 1H, –
CH@), 7.24 (t, J = 8.5 Hz 2H, ArH), 7.46–7.48 (m, 3H, ArH), 7.62 (d,
J = 16 Hz, 1H, –CH@), 7.63–7.66 (m, 2H, ArH), 7.77 (br s, 3H), 7.86
(dd, J = 6.0, 8.0 Hz, 2H, ArH), 11.9 (s, 1H). ESI-MS: 283.1
(C16H16FN4, [M+H]+). Anal. Calcd for C16H15FN4: C, 68.07%; H,
5.36%; N, 19.85%. Found: C, 68.38%; H, 5.79%; N, 20.17%.
4.3.2. 2-(3-(4-Chlorophenyl)-1-phenylallylidene)hydrazinecarb-
oximidamide (4b)
3. Conclusions
1H NMR (500 MHz, CDCl3, d ppm): 3.49 (s, 1H), 6.37 (d, J = 16 Hz,
1H, –CH@), 6.39 (br s, 1H), 7.07 (d, J = 16 Hz, 1H, –CH@), 7.24–7.25
(m, 2H, ArH), 7.29–7.34 (m, 4H, ArH), 7.58–7.59 (m, 3H, ArH), 7.95
A series of chalcone-type guanidine compounds have been
designed and synthesized, and their biological activities were also

Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 8218–8225
8221
(br s, 1H), 10.09 (s, 1H). ESI-MS: 299.1 (C16H16ClN4, [M+H]+). Anal.
Calcd for C16H15ClN4: C, 64.32%; H, 5.06%; N, 18.75%. Found: C,
64.68%; H, 5.41%; N, 19.12%.
4.3.10. 2-(3-(3-Fluorophenyl)-1-phenylallylidene)hydrazine-
carboximidamide (4j)
1H NMR (500 MHz, CDCl3, d ppm): 6.38(d, J = 16.0 Hz, 1H, –
CH@), 6.52 (br s, 1H), 6.99 (t, J = 6.5 Hz, 1H, ArH), 7.05–7.15 (m,
3H), 7.24–7.31 (m, 3H, ArH), 7.42–7.61 (m, 4H), 7.93 (br s, 2H).
ESI-MS: 283.1 (C16H16FN4, [M+H]+). Anal. Calcd for C16H15FN4: C,
68.07%; H, 5.36%; N, 19.85%. Found: C, 68.38%; H, 5.79%; N, 20.17%.
4.3.3. 2-(3-(4-Bromophenyl)-1-phenylallylidene)hydrazinecarb-
oximidamide (4c)
1H NMR (300 MHz, CDCl3, d ppm): 2.49 (s, 1H), 6.34 (s, 1H), 6.36
(d, J = 16.3 Hz, 1H, –CH@), 7.04 (d, J = 16.3 Hz, 1H, –CH@), 7.23–
7.51 (m, 6H), 7.51–7.70 (m, 4H), 8.53 (s, 1H). ESI-MS: 343.0
(C16H16BrN4, [M+H]+). Anal. Calcd for C16H15BrN4: C, 55.99%; H,
4.41%; N, 16.32%. Found: C, 56.35%; H, 4.73%; N, 16.67%.
4.3.11. 2-(3-(3-Methoxyphenyl)-1-phenylallylidene)hydrazine-
carboximidamide (4k)
1H NMR (500 MHz, CDCl3, d ppm): 3.88 (s, 3H, –OCH3), 6.36 (br
s, 1H), 6.85 (d, J = 16.0 Hz, 1H, –CH@), 6.96 (d, J = 8.5 Hz, 2H, ArH),
7.28–7.34 (m, 5H, ArH), 7.50 (d, J = 8.5 Hz, 2H, ArH), 7.54 (d,
J = 16.0 Hz, 1H, –CH@), 7.82 (br s, 2H), 7.99 (br s, 1H). ESI-MS:
295.1 (C17H19N4O, [M+H]+). Anal. Calcd for C17H18N4O: C, 69.37%;
H, 6.16%; N, 19.03%. Found: C, 69.42%; H, 6.49%; N, 19.33%.
4.3.4. 2-(1,3-Diphenylallylidene)hydrazinecarboximidamide (4d)
1H NMR (500 MHz, CDCl3, d ppm): 6.33 (br s, 1H), 6.85 (d,
J = 16.0 Hz, 1H, –CH@), 7.29–7.36 (m, 5H, ArH), 7.52–7.57 (m, 3H,
ArH), 7.62 (d, J = 16.0 Hz, 1H, –CH@), 7.69 (d, J = 7.0 Hz, 2H, ArH),
7.83 (br s, 2H), 11.63 (br s, 1H). ESI-MS: 265.1 (C16H17N4,
[M+H]+). Anal. Calcd for C16H16N4: C, 72.70%; H, 6.10%; N, 21.20%.
Found: C, 72.95%; H, 6.43%; N, 21.51%.
4.3.12. 2-(3-(2-Fluorophenyl)-1-phenylallylidene)hydrazine-
carboximidamide (4l)
1H NMR (500 MHz, CDCl3, d ppm): 6.41 (br s, 1H), 6.84 (d,
J = 15.5 Hz, 1H, –CH@), 7.20–7.35 (m, 5H, ArH), 7.43–7.55 (m, 3H,
ArH), 7.57–7.59 (m, 2H), 7.84 (br s, 2H), 11.64 (br s, 1H). ESI-MS:
283.1 (C16H16FN4, [M+H]+). Anal. Calcd for C16H15FN4: C, 68.07%;
H, 5.36%; N, 19.85%. Found: C, 68.42%; H, 5.69%; N, 20.16%.
4.3.5. 2-(3-(Biphenyl-4-yl)-1-phenylallylidene)hydrazinecarb-
oximidamide (4e)
1H NMR (500 MHz, CDCl3, d ppm): 6.41 (br s, 1H), 6.84 (d,
J = 16.0 Hz, 1H, –CH@), 7.33 (d, J = 7.5 Hz, 1H, ArH), 7.37–7.42 (m,
4H, ArH), 7.46–7.54 (m, 7H, ArH), 7.67 (d, J = 16.0 Hz, 1H, –CH@),
7.74 (d, J = 8.5 Hz, 2H, ArH), 7.93 (br s, 1H), 8.03 (br s, 1H), 11.67
(br s, 1H). ESI-MS: 341.2 (C22H21N4, [M+H]+). Anal. Calcd for
4.3.13. 2-(3-(2-Chlorophenyl)-1-phenylallylidene)hydrazine-
carboximidamide (4m)
1H NMR (300 MHz, CDCl3, d ppm): 6.32 (br s, 1H), 6.88 (d,
J = 16.3 Hz, 1H, –CH@), 7.03 (d, J = 16.3 Hz, 1H, –CH@), 7.22–7.35
(m, 4H, ArH), 7.51 (s, 2H), 7.56–7.70 (m, 5H, ArH), 8.59 (br s, 1H).
ESI-MS: 299.1 (C16H16ClN4, [M+H]+). Anal. Calcd for C16H15ClN4: C,
64.32%; H, 5.06%; N, 18.75%. Found: C, 64, 68%; H, 5.37%; N, 19.07%.
C22H20N4: C, 77.62%; H, 5.92%; N, 16.46%. Found: C, 77.91%; H,
6.27%; N, 16.71%.
4.3.6. 2-(3-(4-(Benzyloxy)phenyl)-1-phenylallylidene)hydrazin-
ecarboximidamide (4f)
1H NMR (500 MHz, CDCl3, d ppm): 5.07 (s, 2H, –CH2–), 6.37 (d,
J = 16.0 Hz, 1H, –CH@), 6.43 (br s, 1H), 6.93 (d, J = 8.5 Hz, 1H, ArH),
6.97 (d, J = 16.0 Hz, 1H, –CH@), 7.24–7.25 (m, 3H, ArH), 7.33–7.35
(m, 3H, ArH), 7.38–7.43 (m, 4H, ArH), 7.56–7.57 (m, 3H, ArH),
7.91 (br s, 3H). ESI-MS: 371.2 (C23H23N4O, [M+H]+). Anal. Calcd
for C23H22N4O: C, 74.57%; H, 5.99%; N, 15.12%. Found: C, 74.93%;
H, 6.37%; N, 15.45%.
4.3.14. 2-(3-(2-Bromophenyl)-1-phenylallylidene)hydrazine-
carboximidamide (4n)
1H NMR (500 MHz, CDCl3, d ppm): 6.40 (br s, 1H), 6.82 (d,
J = 16.0 Hz, 1H, –CH@), 7.02 (d, J = 16.0 Hz, 1H, –CH@), 7.12–7.17
(m, 1H, ArH), 7.28–7.34 (m, 3H, ArH), 7.45–7.63 (m, 5H, ArH),
7.85 (br s, 2H), 11.65 (br s, 1H). ESI-MS: 343.0 (C16H16BrN4,
[M+H]+). Anal. Calcd for C16H15BrN4: C, 55.99%; H, 4.41%; N,
16.32%. Found: C, 56.32%; H, 4.79%; N, 16.68%.
4.3.7. 2-(1-Phenyl-3-p-tolylallylidene)hydrazinecarboximida-
mide (4g)
4.3.15. 2-(3-(2-Methoxyphenyl)-1-phenylallylidene)hydrazine-
carboximidamide (4o)
1H NMR (500 MHz, CDCl3, d ppm): 2.31 (s, 3H, –CH3), 6.33(br s,
1H), 6.82(d, J = 15.5 Hz, 1H, –CH@), 7.12–7.15 (m, 3H, ArH), 7.28 (d,
J = 7.0 Hz, 1H, ArH), 7.45 (d, J = 5.0 Hz, 2H, ArH), 7.52–7.53 (m, 4H,
ArH), 7.81 (br s, 2H), 11.61(s, 1H). ESI-MS: 279.2 (C17H19N4,
[M+H]+). Anal. Calcd for C17H18N4: C, 73.35%; H, 6.52%; N, 20.13%.
Found: C, 73.69%; H, 6.88%; N, 20.47%.
1H NMR (500 MHz, CDCl3, d ppm): 3.38 (s, 3H, –OCH3), 6.41 (br
s, 1H), 6.70 (d, J = 16.5 Hz, 1H, –CH@), 6.88–6.97 (m, 2H, ArH), 7.13
(d, J = 16.5 Hz, 1H, –CH@), 7.21–7.28 (m, 2H, ArH), 7.45–7.51 (m,
5H, ArH), 7.74 (br s, 2H), 11.48 (br s, 1H). ESI-MS: 295.1
(C17H19N4O, [M+H]+). Anal. Calcd for C17H18N4O: C, 69.37%; H,
6.16%; N, 19.03%. Found: C, 69.98%; H, 6.51%; N, 19.37%.
4.3.8. 2-(3-(4-Methoxyphenyl)-1-phenylallylidene) hydrazine-
carboximidamide (4h)
4.3.16. 2-(1-(4-Chlorophenyl)-3-phenylallylidene) hydrazine-
carboximidamide (4p)
1H NMR (300 MHz, DMSO-d6, d ppm): 3.79 (s, 1H, –OCH3), 6.79
(d, J = 9.6 Hz, 1H, –CH@), 6.96 (d, J = 5.4 Hz, 2H, ArH), 7.47–7.49 (m,
3H, ArH), 7.62–7.64 (m, 3H), 7.75–7.79 (m, 5H), 12.0 (s, 1H). ESI-
MS: 295.1 (C17H19N4O, [M+H]+). Anal. Calcd for C17H18N4O: C,
69.37%; H, 6.16%; N, 19.03%. Found: C, 69.68%; H, 6.49%; N, 19.40%.
1H NMR (300 MHz, DMSO-d6, d ppm): 6.88(d, J = 9.6 Hz, 1H, –
CH@), 7.39–7.44 (m, 3H, ArH), 7.53–7.55 (m, 2H, ArH), 7.64 (d,
J = 9.6 Hz, 1H, –CH@), 7.71–7.80 (m, 7H), 11.83 (br s, 1H). ESI-MS:
299.1 (C16H16ClN4, [M+H]+). Anal. Calcd for C16H15ClN4: C,
64.32%; H, 5.06%; N, 18.75%. Found: C, 64.71%; H, 5.42%; N, 19.10%.
4.3.9. 2-(3-(3-Nitrophenyl)-1-phenylallylidene) hydrazinecarb-
oximidamide (4i)
4.3.17. 2-(1-(4-Bromophenyl)-3-phenylallylidene)hydrazine-
carboximidamide (4q)
1H NMR (500 MHz, DMSO-d6, d ppm): 7.06(d, J = 16.0 Hz, 1H, –
CH@), 7.47–7.50 (m, 3H, ArH), 7.69–7.79 (m, 4H, ArH), 7.77–7.79
(br s, 3H), 8.21 (d, J = 8.5 Hz, 1H, ArH), 8.32 (d, J = 7.0 Hz, 1H,
ArH), 8.57 (s, 1H), 11.8 (s, 1H). ESI-MS: 310.3 (C16H16N5O2,
[M+H]+). Anal. Calcd for C16H15N5O2: C, 62.13%; H, 4.89%; N,
22.64%. Found: C, 62.49%; H, 5.23%; N, 22.91%.
1H NMR (300 MHz, DMSO-d6, d ppm): 6.88 (d, J = 16.3 Hz, 1H, –
CH@), 7.32–7.43 (m, 3H), 7.47–7.53 (m, 1H), 7.62–7.72 (m, 4H),
7.80–7.85 (m, 5H), 12.05 (s, 1H). ESI-MS: 343.0 (C16H16BrN4,
[M+H]+). Anal. Calcd for C16H15BrN4: C, 55.99%; H, 4.41%; N,
16.32%. Found: C, 56.31%; H, 4.78%; N, 16.65%.

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Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 8218–8225
295.1 (C17H19N4O, [M+H]+). Anal. Calcd for C17H18N4O: C, 69.37%;
H, 6.16%; N, 19.03%. Found: C, 69.68%; H, 6.49%; N, 19.40%.
4.3.20. 2-(1-(3,4-Dichlorophenyl)-3-phenylallylidene)hydra-
zinecarboximidamide (4t)
1H NMR (500 MHz, CDCl3, d ppm): 6.39 (br s, 1H), 6.75 (d,
J = 15.0 Hz, 1H, –CH@), 7.40 (d, J = 8.0 Hz, 1H, ArH), 7.52–8.05 (m,
8H), 8.16 (br s, 1H), 8.73 (br s, 2H). ESI-MS: 332.1 (C16H15Cl2N4,
[M+H]+). Anal. Calcd for C16H14Cl2N4: C, 57.67%; H, 4.23%; N,
16.81%. Found: C, 57.93%; H, 4.58%; N, 17.16%.
4.3.21. 2-(3-(2-Chlorophenyl)-1-(4-methoxyphenyl)allylidene)-
hydrazinecarboximidamide (4u)
1H NMR (300 MHz, CDCl3, d ppm): 3.90 (s, 3H, –OCH3), 6.35 (br
s, 1H), 6.90–6.99 (m, 2H), 7.05–7.11 (m, 2H), 7.21–7.26 (m, 4H),
7.32–7.36 (m, 3H), 7.62–7.65 (m, 1H), 8.65 (s, 1H). ESI-MS: 329.1
(C17H18ClN4O, [M+H]+). Anal. Calcd for C17H17ClN4O: C, 62.10%; H,
5.21%; N, 17.04%. Found: C, 62.43%; H, 5.58%; N, 17.31%.
4.3.22. 2-(3-(Naphthalen-2-yl)-1-phenylallylidene)hydrazine-
carboximidamide (4v)
1H NMR (500 MHz, CDCl3, d ppm): 6.36 (br s, 1H), 7.41–7.49 (m,
4H), 7.51 (d, J = 7.5 Hz, 2H, ArH), 7.54 (d, J = 7.0 Hz, 1H, ArH), 7.60
(d, J = 6.5 Hz, 2H, ArH), 7.65 (d, J = 9.5 Hz, 1H, ArH), 7.73–7.79 (m,
3H), 8.43 (d, J = 7.5 Hz, 1H, ArH), 7.70 (br s, 3H), 11.69 (s, 1H).
ESI-MS: 315.2 (C20H19N4, [M+H]+). Anal. Calcd for C20H18N4: C,
76.41%; H, 5.77%; N, 19.40%. Found: C, 76.72%; H, 6.08%; N, 19.73%.
Figure 1. Crystal structure diagrams of compound 4i. Molecule structure diagram
with displacement ellipsoids being at the 30% probability level and H atoms are
shown as small spheres of arbitrary radii (Cl anion is omitted for clarity).
4.3.18. 2-(3-Phenyl-1-p-tolylallylidene)hydrazinecarboximida-
mide (4r)
4.4. Crystal structure determination
1H NMR (500 MHz, CDCl3, d ppm): 2.44 (s, 3H, –CH3), 6.40 (br s,
1H), 7.09 (d, J = 16.5 Hz, 1H, –CH@), 7.14 (d, J = 7.5 Hz, 2H, ArH),
7.31–7.41 (m, 7H, ArH), 7.46 (d, J = 16.5 Hz, 1H, –CH@), 7.78 (br
s, 1H), 8.04 (br s, 1H), 10.12 (br s, 1H). ESI-MS: 279.2 (C17H19N4,
[M+H]+). Anal. Calcd for C17H18N4: C, 73.35%; H, 6.52%; N, 20.13%.
Found: C, 73.63%; H, 6.81%; N, 20.50%.
Crystal structure determination of compound 4i were carried
out on a Nonius CAD4 diffractome0ter equipped with graphite-
monochromated MoKa (k = 0.71073 ÅA) radiation (Fig. 1). The struc-
ture was solved by direct methods and refined on F2 by full-matrix
least-squares methods using SHELX–97.22 All the non-hydrogen
atoms were refined anisotropically. All the hydrogen atoms were
placed in calculated positions and were assigned fixed isotropic
thermal parameters at 1.2 times the equivalent isotropic U of the
atoms to which they are attached and allowed to ride on their
respective parent atoms. The contributions of these hydrogen
atoms were included in the structure-factors calculations. The
crystal data, data collection, and refinement parameter for the
compound 4i are listed in Table 3.
4.3.19. 2-(1-(4-Methoxyphenyl)-3-phenylallylidene)hydrazine-
carboximidamide (4s)
1H NMR (500 MHz, CDCl3, d ppm): 3.89 (s, 3H, –OCH3), 6.35 (br
s, 1H), 6.87 (d, J = 15.5 Hz, 1H, –CH@), 6.97 (d, J = 8.0 Hz, 1H, ArH),
7.10 (d, J = 16.5 Hz, 1H, ArH), 7.33–7.36 (m, 2H, ArH), 7.44–7.45 (m,
1H, ArH), 7.51–7.52 (m, 2H, ArH), 7.67–7.71 (m, 2H, ArH), 7.82 (br
s, 1H), 7.83 (d, J = 15.5 Hz, 1H, –CH@), 11.55 (br s, 1H). ESI-MS:
Figure 2. The morphological changes of DAPI-stained MCF-7 human breast carcinoma cells after treatment with compound 4q (1.0
microscope. (A). cells were treated without the compound; (B) cells were treated with the compound at 1.0 M.
lM) as observed under a fluorescent
l

Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 8218–8225
8223
Figure 3. Cell cycle analysis of MCF-7 cells treated with compound 4q using flow cytometry. Cells were harvested after treatment with 4q for 24 h and subjected to cell cycle
analysis. The percentage of cells in each cell cycle phase was indicated.
Figure 4A. Compound 4q (Carbon atom are green) docked into the tubulin pocket. Both side chains of important active site amino acids and hydrogen bonds are shown: THR
179 (angle OꢀꢀꢀH–N = 146.729°, distance = 2.249 Å), ASN–349 (angle OꢀꢀꢀH–N = 161.980°, distance = 1.584 Å).
4.5. Antiproliferation assay
was added to each well of 96-well culture plates. The subsequent
incubation was permitted at 37 °C, 5% CO2 atmosphere for 24 h
before the cytotoxicity assessments. Tested samples at pre-set
concentrations were added to 6-wells with colchicine and CSA-4
co-assayed as positive reference. After 48 h exposure period,
The antiproliferative activity of the prepared compounds
against MCF-7 human breast carcinoma cells was evaluated as de-
scribed elsewhere23 with some modifications. Target tumor cell
line was grown to log phase in RPMI 1640 medium supplemented
with 10% fetal bovine serum. After diluting to 2 ꢁ 104 cells mLꢂ1
40
zol-2-yl)-2,5-diphenyltetrazolium bromide)) was added to each
well. Four hours later, 100 L extraction solution (10% SDS–5%
l
L of PBS containing 2.5 mg mLꢂ1 of MTT (3-(4,5-dimethylthia-
with the complete medium, 100
l
L of the obtained cell suspension
l

8224
Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 8218–8225
Figure 4B. 3D model of the interaction between compound 4q and the colchicine binding site. The protein is represented by molecular surface. Compound 4q is depicted by
sticks and balls.
with natural double-stranded DNA. This method was used to
Table 3
observe the apoptosis morphological changes. Briefly, MCF-7 hu-
man breast carcinoma cells were cultured on 6-well plates before
treatment. After treatment with 0, 1.0 lM compound 4q for 24 h,
MCF-7 cells were fixed with ice-cold 4% paraformaldehyde for
20 min and washed with ice-cold PBS, then the cell suspension
were permeabilized by incubation in 0.1% sodium citrate contain-
ing 0.1% Triton X-100 for 2 min at 4 °C and washed with ice-cold
PBS, stained with fluorochrome dye DAPI (300 nM). After labeling,
samples were observed under a fluorescence microscope.
Crystallographical and experimental data for compound 4i
Compounds
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
4i
C
16H15ClN5O2
344.78
Monoclinic
P2(1)/n
11.127(2)
11.932(2)
25.889(5)
90.00
91.71(3)
90.00
b (Å)
c (Å)
a
(°)
b (°)
(°)
c
4.8. Cell cycle assay
V (Å)
Z
3435.7(12)
8
1.333
1.57–25.28
1432
Dcalc (gꢂ1 cmꢂ3
h range (°)
F(0 0 0)
)
MCF-7 human breast carcinoma cells were treated with com-
pound 4q at 50 nM and 500 nM concerntrations for 24 h. Then cells
were collected and fixed in 70% cold ethanol (ꢂ20 °C) overnight.
Cells were resuspended in PBS after washing twice with PBS, RNase
A (0.5 mg/mL) and PI (2.5 lg/mL) were then added to the fixed
cells for 30 min. The DNA content of cells was then analyzed with
Reflections collected/unique
Data/restraints/parameters
Absorption coefficient (mmꢂ1
3126/3120 [Rint = 0.0287]
3120/0/217
0.241
)
R1; wR2 [I >2
R1; wR2 (all data)
GOOF
r
(I)]
0.0760/0.1817
0.1439/0.2244
1.038
flow cytometer.
4.9. Molecular docking modeling
Molecular docking of compound 4q into the three-dimensional
X-ray structure of tubulin (1SA0 PDB, downloaded from the PDB)
was carried out using the Auto-Dock software package (version
4.0) as implemented through the graphical user interface Auto-
Dock Tool Kit (ADT 1.4.6).
isobutyl alcohol–0.01 M HCl) was added. After an overnight incu-
bation at 37 °C, the optical density was measured at a wavelength
of 570 nm on an ELISA microplate reader. In all experiments three
replicate wells were used for each drug concentration. Each assay
was carried out at least three times. The results were summarized
in Table 2.
Acknowledgements
4.6. Effects on tubulin polymerization
The work was financed by a grant (no. BK2009239) from the
Jiangsu National Science Foundation.
Bovine brain tubulin was purified as described previously.24 To
evaluate the effect of the compounds on tubulin assembly
in vitro,25 varying concentrations were preincubated with 10
lM
References and notes
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recording spectrophotometer and warmed-up to 30 °C and the
assembly of tubulin was observed turbidimetrically. The IC50 was
defined as the compound concentration that inhibited the extent
of assembly by 50% after 20 min incubation.
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