Synthesis and biological evaluation of 6H-pyrido[2′,1′:2,3] imidazo[4,5-c]isoquinolin-5(6H)-ones as antimitotic agents and inhibitors of tubulin polymerization

Article abstract of DOI:10.1016/j.bmc.2013.12.004

A series of 6H-pyrido[2′,1′:2,3]imidazo[4,5-c]isoquinolin-5(6H) -ones have been synthesized and evaluated for their antiproliferative activities. Among them, compounds 2j and 4d displayed potent cytotoxic activities in vitro against HeLa cell line with IC₅₀ values of 0.07 and 0.06 μM, respectively. In general, the antiproliferative activities are correlated with the inhibitory effect on tubulin polymerization and binding property of the colchicine binding site. In addition, flow cytometry and immunofluorescence analysis revealed selected compounds caused G2/M phase arrest of the cell cycle and disruption of the mitotic spindle assembly, which had correlation with proliferation inhibitory activity.

Full text of DOI:10.1016/j.bmc.2013.12.004

Bioorganic & Medicinal Chemistry 22 (2014) 848–855
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 6H-pyrido[20,10:2,3]
imidazo[4,5-c]isoquinolin-5(6H)-ones as antimitotic agents
and inhibitors of tubulin polymerization
a
b,
Tao Meng a, , Wei Wang b, , Zhixiang Zhang b, Lanping Ma a, , Yongliang Zhang , Zehong Miao
⇑
⇑
,
Jingkang Shen a,
⇑
a Division of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic
of China
b Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s
Republic of China
a r t i c l e i n f o
a b s t r a c t
A series of 6H-pyrido[20,10:2,3]imidazo[4,5-c]isoquinolin-5(6H)-ones have been synthesized and evalu-
ated for their antiproliferative activities. Among them, compounds 2j and 4d displayed potent cytotoxic
activities in vitro against HeLa cell line with IC50 values of 0.07 and 0.06 lM, respectively. In general, the
antiproliferative activities are correlated with the inhibitory effect on tubulin polymerization and binding
property of the colchicine binding site. In addition, flow cytometry and immunofluorescence analysis
revealed selected compounds caused G2/M phase arrest of the cell cycle and disruption of the mitotic
spindle assembly, which had correlation with proliferation inhibitory activity.
Ó 2014 Published by Elsevier Ltd.
Article history:
Received 19 August 2013
Revised 20 October 2013
Accepted 3 December 2013
Available online 11 December 2013
Keywords:
6H-Pyrido[20,10:2,3]imidazo[4,5-c]
isoquinolin-5(6H)-ones
Antitumor agents
Inhibitors of tubulin polymerization
Colchicine binding site
1. Introduction
window. In the past few years, small molecular colchicines site
binders based on combretastatin A4 were actively pursued as anti-
Attacking the microtubule system is a common strategy to
inhibit tumor cell proliferation. The microtubule constituted by
mitotic agents which exerted potent cytotoxic activities.8 How-
ever, up until now, no representative members of this class have
been employed as cancer chemotherapeutic agents in the market.
Therefore, great efforts have recently been made to develop novel
small-molecular tubulin binders derived not only from natural
sources but also by screening compound libraries in combination
with traditional medicinal chemistry, such as ABT-751, BNC105,
ELR510444 and IRC-083927 showed good efficacy.9–12
a
,b-tubulin heterodimers, is regarded as one of the most important
targets to treat many types of malignancies.1–3 A still expanding
family of antimitotic drugs displaying a wide structural heteroge-
neity has been identified to act on tubulin, and they usually bind
to tubulin at three distinct sites: the taxane site, the vinca site
and the colchicine site.4,5
A large number of structurally diverse antimitotic agents have
been identified as inhibitors of the polymerization of tubulin
(e.g., vinblastine and vincristine) and stabilizers of the microtubule
structure (e.g., paclitaxel and docetaxel), which are widely used
clinically as important chemotherapeutic drugs for the treatment
of many malignancies.6,7 Colchicine is another significant antimi-
totic agent, played an important role in the elucidation of the prop-
erties and functions of tubulin and microtubules. However, it has not
been applied in anticancer therapy due to its narrow therapeutic
We have earlier described a series of 6H-pyrido[20,10:2,3]imi-
dazo[4,5-c]isoquinolin-5(6H)-ones-derived tubulin polymerization
inhibitors with in vitro antitumor activity, which was generated by
using a three-component reaction.13 One of the compounds, MT-7
(1a, Table 1), exhibited cytotoxic activity in the cell based screen,
with an averaged IC50 of 2.58
lM ranging from 0.85 to
5.01
l
M.13,14 More importantly, MT-7 was found to be able to in-
duce cell cycle arrest at G2/M phase. Continuing our search strat-
egy for novel potent tubulin-targeting compounds, we conducted
further investigation on the structural parameters of
6H-pyrido[20,10:2,3]imidazo[4,5-c]isoquinolin-5(6H)-one deriva-
tives associated with their antiproliferative activity. Some of them
demonstrated the antiproliferative ability against HeLa cell line.
Moreover, 4d inhibited the growth of various tumor cell lines.15
⇑
Corresponding authors. Tel.: +86 21 50806600/5407; fax: +86 21 50807088.
E-mail addresses: lpma@simm.ac.cn (L. Ma), zhm@jding.dhs.org (Z. Miao),
jkshen@simm.ac.cn (J. Shen).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2014 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.12.004

T. Meng et al. / Bioorg. Med. Chem. 22 (2014) 848–855
849
Table 1
with a solution of BBr3 in CH2Cl2 to afford the phenolic compound
2k. Derivatives 4a–h were synthesized by microwave reaction of
the bromo derivative 3 with corresponding amines in the presence
Antiproliferative activity of 1a–f against HeLa cells
11
N
10
of CuI, -proline and K2CO3 in DMSO at 150 °C while irradiating at
250 W for 30 min (Scheme 2).
L
R2
N
9
8
N
O
2.1.1. In vitro antiproliferative activity assays
The compounds’ antiproliferative activities were evaluated
in vitro against the HeLa cell line (Tables 1 and 2). The preliminary
SAR information in Table 1 of various R2 substitutions showed that
only compounds 1b out of five compounds (1b–f) exerted IC50 of
H3CO
Compd
R2 substitution
HeLa IC50 (l
M)a
6.6 lM (Table 1), which indicated that modifications at C-9 posi-
tion might be tolerated compare to positions 8, 10 or 11. The anti-
proliferative activity was closely related to the 4-methoxybenzyl at
R1 group according to our previous SAR studies,13 which could sug-
gest the existence of the H-bonding interaction with the amino
acid side chains of the putative biological target. Thus various func-
tional groups containing H-bond acceptors were introduced by
reaction with the corresponding isocyanides (2a–l, Table 2).
Analysis of the substitution pattern indicated that the presence
of para-methoxy group at phenyl ring was essential to the bioactiv-
ity. The replacement of the methoxy group of 1a by the 4-trifluoro-
methoxy (2e) or N-methylmethanesulfonamide (2i) resulted with
the complete loss of activity. 3,4-Dimethoxy substitution at phenyl
ring produced inactive derivative 2f, while 2,4-dimethoxy substi-
1a (MT-7)
H
9-CH3
8-CH3
11-CH3
8-CH3, 9-Br
9-Br, 10-CH3
1.813,14
6.613
>10
>10
>10
1b
1c
1d
1e
1f
>10
a
IC50 values are mean SD from three independent tests.
We also found that these analogues exerted the antiproliferative
function by arresting tumor cells at mitosis and inhibition of tubu-
lin polymerization. Antitubulin activities of the most active com-
pounds were comparable to the compound vincristine (VCR) and
colchicine (COL). This study also helps us to dissect the struc-
ture–activity relationship of this antimitotic library and will guide
our ongoing research.
tution gave derivative (2g) with fair activity (IC50 = 2.2 lM),
whereas cleavage of the ether groups (2k) resulted in loss of potency.
The aliphatic esters (2a–c) and furanmethyl substitution (2d) were
also completely inactive. Noteworthy, the dependence of antipro-
liferative activity upon spacer length between phenyl ring and
the rest of the molecule is crucial, increasing the number of meth-
ylene units to two resulted in a 25-fold increase of activity (2j vs
MT-7). Further chain elongation led to loss of activity in antiprolif-
erative activity (2l).
Encouraged by further SARs of R1 group, keeping the preferred
2-(4-methoxyphenyl)ethyl unchanged, attention shifted to explo-
ration of the 9-position of the molecule 2j. Furthermore, we tried
to increase the water solubility of the highly lipophilic parent
compounds by the introduction of hydrophilic side chains (entries
4a–h, Scheme 2). As expected, this region of the molecule
allows various structural modifications with diverse hydrophilic
2. Results and discussion
2.1. Chemistry
The general method used for the synthesis of the
6H-pyrido[20,10:2,3]imidazo[4,5-c]isoquinolin-5(6H)-one deriva-
tives 1a–f and 2a–l is depicted in Scheme 1 as reported previously
by our group.13 The preparation of 6H-pyrido[20,10:2,3]imi-
dazo[4,5-c]isoquinolin-5(6H)-ones (compounds 1a–f, 2a–j, 2l and
intermediate 3) were readily achieved by three component conden-
sation of various commercial or synthetic isocyanides, 2-aminopyri-
dines and phthaldehydic acid. The compound 2j was demethylated
R2
R2
N
N
CHO
i
+
R1 NC
+
1a-f
R
1 = p-methoxybenzyl,
R2 = listed in Table 1;
2a-l R2 = H;
N
NH2
N
COOH
R1
O
O
∗
COOCH3
OAc
∗
COOCH3
2a
∗
∗
∗
OCF3
2b
2c
2d
2e
OCH3
O
O
∗
∗
OCH3
∗
∗
Ms
N
R1
OCH3
OH
OCH3
CH3
2g
2h
2i
2f
OCH3
∗
OCH3
∗
∗
2l
2j
2k
ii
Scheme 1. Reagents and conditions: (i) MeOH, 55 °C, overnight; (ii) BBr3, dry CH2Cl2, ꢀ78 to ꢀ25 °C;

850
T. Meng et al. / Bioorg. Med. Chem. 22 (2014) 848–855
N
O
N
O
R2
Br
N
N
i
N
N
OCH3
OCH3
3
4a-h
N
H
H
H
N
N
N
N
N
N
N
∗
∗
∗
∗
N
4a
4b
4c
4d
R2
H
N
H
N
N
H
N
H
N
∗
∗
N
N
∗
OH
∗
O
4f
4g
-proline, K2CO3, DMSO,
4h
4e
Scheme 2. Reagents and conditions: (i) CuI,
L
lW(150 °C, 250 W), 30 min.
Table 2
microscopes, we found that HeLa cells became round after the
treatment of compounds 2a–l and 4a–h. Naturally, we speculated
that the antiproliferative activities of these compounds were re-
lated to the interference with cell cycle progression.16 Based on
their antiproliferative activity in vitro, 2j and 4d were selected as
the representative compounds to analyse the effect on cell cycle
profile in HeLa cells. Cells were cultured for 12 h in the presence
of each compound at the indicated concentration, and in the flow
cytometry results, the selected compounds 2j and 4d caused a
markable increase in the percentage of cells blocked in the G2-M
phase of the cell cycle (37% and 41%, respectively), with a simulta-
neous decrease of cells in S and G0-G1 phases, which was stronger
than the effect of MT-7 (Fig. 1).14 These data suggested that this
class of molecules inhibited the proliferation of HeLa cells through
cell cycle arrest.
Antiproliferative activity of 2a–l and 4a–h against HeLa
cells
Compd
HeLa IC50 (l
M)a
2a
2b
2c
2d
2e
2f
2g
2h
2i
>10
>10
>10
>10
>10
>10
2.24 0.57
>10
>10
2j
2k
2l
0.07 0.01
>10
>10
4a
4b
4c
4d
4e
4f
4g
4h
VCR
0.12 0.01
0.77 0.20
0.24 0.12
0.06 0.01
0.10 0.02
2.28 0.33
1.20 0.62
0.35 0.08
2.3. In vitro tubulin polymerization and colchicine site binding
assays
It has been well documented that microtubule inhibitors prefer-
entially arrest cells at the G2/M phase, so we investigated whether
microtubule system could be a potential target of these com-
pounds.17–19 Compounds 2j and 4a–h were evaluated for their di-
rect inhibitory effects on tubulin polymerization in vitro, as
presented in Figure 2A. Purified tubulins were polymerized to stea-
dy state in the presence of GTP at 37 °C in a control sample and as
expected, treatment with these compounds inhibited tubulin
0.015 0.003
a
IC50 values are mean SD from three independent
tests.
nitrogen-contained heterocyclic ring systems such as imidazole
(4a, 4b), pyridine (4c–e), morpholine (4g) or polar substitution
group as N,N-diethylethanediamine (4f) and ethanolamine (4h).
In comparison with the parent compound 2j, by introduction of
the hydrophilic substitutions, compounds 4a–h showed moderate
to potent antiproliferative activity. Among those, the 3-picolylami-
no substitution (4d) displayed strong antiproliferative activity
(IC50 HeLa: 0.06
assay. Similar activity was found for the constitutional isomers 4c
and 4e (IC50 HeLa: 0.24 and 0.10 M, respectively). In addition, the
lM), and it was the most active compound in this
l
imidazole (4a, 4b) and ethanolamine (4h) substituted analogues
also showed activities in the submicromolar range, with IC50 values
ranging from 0.12 to 0.77 lM. Analysis of the above SAR results at-
tracted our interest for further studies on their mechanisms of
action.
2.2. Cell-cycle progression analysis
When entering the mitosis phase, cells are tuning round and
then split into two daughter cells. In light of the observations with
Figure 1. Cell cycle distribution of HeLa cells was analysed after treatment of
0.5 lM MT7, 2j and 4d.

T. Meng et al. / Bioorg. Med. Chem. 22 (2014) 848–855
851
polymerization to varying degrees consistent with their in vitro
antiproliferative activity (Fig. 2B). Further analysis revealed that
compounds 2j (76%) and 4d (74%) were as active as the reference
78% inhibition, respectively, exhibited the ability to compete for
the colchicine binding site about as potent as the positive reference
inhibitor colchicine 77% (Fig. 2C). The colchicine site binding
capacity for 2j and 4a–h may contribute to their antitubulin poly-
merization activity and further antiproliferative ability.
inhibitor 20 lM VCR (77%) and 20 lM colchicines (76%), although
both compounds were less active in their effects on cell prolifera-
tion. To evaluate if the compounds interacted with tubulin at the
colchicine site, we determined whether 2j and 4a–h inhibited
2.4. Disruption of mitotic spindle assembly
binding of 5
l
M [3H]-colchicine to 3
lM tubulin. In the colchicine
site binding studies, 20
lM compounds 2j and 4d, with 74% and
Proper tubulin polymerization is essential for the assembly of
mitotic spindle, guaranteeing the precise segregation of chromo-
somes.20,1 Since these compounds could bind to the colchicine site
to disrupt the polymerization of tubulin, we further examined their
effect on the bipolar spindle formation. Immunofluorescence anal-
ysis was performed to evaluate the selected compounds 2j and 4d
on the cellular mitotic spindle assembly. As shown in Figure 3A,
the mitotic spindle exhibited normal bipolar orientation with chro-
mosomes ordered alignment in HeLa cells without treatment. In
contrast, 12 h of exposure to 0.5 lM selected compounds caused
multipolar mitotic spindle, with disordered chromosome organiza-
tion. Then the percentage of cells with abnormal spindle assembly
in total mitotic cells was calculated. As expected, compounds 2j
and 4d disrupting mitotic spindle assembly in accordance with
their anti-proliferation and anti-tubulin activities (Fig. 3B).
3. Conclusion
We have presented
a
series of 6H-pyrido[20,10:2,3]imi-
dazo[4,5-c]isoquinolin-5(6H)-ones compounds, in which 2j and
4a–h exerted antiproliferative activity and proved as inhibitors
of tubulin polymerization through binding to colchicine site. Se-
lected compounds 2j and 4d were efficacious inhibiting tumor
cell proliferation with IC50 values at the low nanomolar level.
Our studies revealed that a 4-methoxy substitution pattern in
the terminal phenyl ring is critical for strong inhibition of tumor
cell proliferation and inhibition of tubulin polymerization. The
compounds described in this report are structurally simpler than
the well-known colchicine or combretastatin A-4, chemically sta-
ble, and easily accessible.21 Due to their promising in vitro anti-
proliferative activities, we believe that the compounds of this
structural class are attractive for further structural modifications
and may provide a useful template for the design of new antitu-
moral agents. Structure–activity relationship studies concerning
the pharmacophore requirements for activity are in progress
and will be reported in due course.
4. Experimental section
4.1. Chemistry
4.1.1. General methods
Reagents were purchased from Aldrich or Lancaster and were
used as received, isocyanides were purchased or prepared accord-
ing to the procedure reported in the literature. The solvents were
not dried prior to use. Melting points were determined in open
capillary tubes on an Electrothermal melting point apparatus and
are uncorrected. 1H (300 MHz) and 13C NMR (100 MHz) spectra
were measured at 25 °C on compounds in solution in CDCl3 or
DMSO-d6 on a Varian spectrometer. Chemical shifts (d) are given
in ppm downfield from tetramethylsilane, an internal standard.
The following abbreviations were used to explain the multiplici-
ties: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of
doublets, m = multiplet, br = broad. The high resolution mass spec-
tra were measured on Finnigan MAT 95 and MicroMass Q-Tof
ultima™ mass spectrometers. Microwave reaction was conducted
with a Biotage microwave reactor.
Figure 2. (A) The inhibitory activity of 2j and 4a–h against tubulin polymerization
was examined in cell-free system; (B) The inhibition percent of tubulin
polymerization for 2j and 4a–h in the above cell-free system; (C) The capacities
of 2j and 4a–h to bind to the colchicine site on tubulins were evaluated by site
competitive binding assays.
a

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T. Meng et al. / Bioorg. Med. Chem. 22 (2014) 848–855
Figure 3. (A) Immunofluorescent microscope was used to observe the spindle assembly after treatment with compounds for 12 h; (B) The number of cells with the
disorganized spindle was calculated in each sample.
4.1.2. General procedure a for synthesis of compounds 1c–1f
and 2a–2j and 2l
4.1.2.5. Methyl (5-oxopyrido[20,10:2,3]imidazo[4,5-c]isoquino-
lin-6(5H)-yl)acetate (2a). Yellow solid (67% yield, HPLC purity:
To a mixture of 2-formylbenzoic acid (100 mg, 0.67 mmol),
2-amino-pyridine type amine (1 equiv) in MeOH (1 mL) was added
with isocyanide (1 equiv). The reaction mixture was stirred at 55 °C
and monitored by TLC, typically the reaction time is 4–12 h. After
the reaction was completed, the precipitate that formed was iso-
lated by filtration and washed with MeOH (1 mL ꢁ 3) to give the
desired product.
100); mp 212–214 °C; 1H NMR (300 MHz, CDCl3)
d 8.51(d,
J = 8.1 Hz, 1H), 8.46(d, J = 7.3 Hz, 1H), 8.19(d, J = 8.1 Hz, 1H),
7.85(t, J = 7.7 Hz, 1H), 7.45(d, J = 9.2 Hz, 1H), 7.59(t, J = 7.5 Hz,
1H), 7.20(dd, J = 9.3, 6.8 Hz, 1H), 6.86(t, J = 7.0 Hz, 1H), 5.50(s,
2H), 3.84(s, 3H); HRMS(EI) m/z calcd for C17H13N3O3, 307.0957;
found, 307.0960.
4.1.2.6. Methyl 3-(5-oxopyrido[20,10:2,3]imidazo[4,5-c]isoquino-
lin-6(5H)-yl)propanoate (2b). Yellow solid (60% yield, HPLC pur-
ity: 96.7); mp 205–207 °C; 1H NMR (300 MHz, CDCl3) d 8.57(t,
J = 7.9 Hz, 1H), 8.48(t, J = 7.0 Hz, 1H), 8.42(t, J = 7.7 Hz, 1H),
7.82(m, 1H), 7.73(t, J = 9.2 Hz, 1H), 7.57(m, 1H), 7.20(m, 1H),
6.89(m, 1H), 4.94(t, J = 8.1 Hz, 2H), 3.76(s, 3H), 3.03(t, J = 8.1 Hz,
4.1.2.1.
6-(4-Methoxybenzyl)-8-methylpyrido[20,10:2,3]imi-
dazo[4,5-c]isoquinolin-5(6H)-one (1c). Yellow solid (72% yield,
HPLC purity: 96.7); mp 146–148 °C; 1H NMR (300 MHz, CDCl3) d
8.37(d, J = 5.1 Hz, 1H), 8.35(d, J = 5.1 Hz, 1H), 7.78(m, 1H), 7.55(d,
J = 9.2 Hz, 1H), 7.49(t, J = 7.1 Hz, 1H), 7.13(dd, J = 9.2, 7.0 Hz, 1H),
6.81–6.84(m, 2H), 6.61–6.65(m, 3H), 5.47(s, 2H), 3.66(s, 3H),
2.93(s, 3H); HRMS(EI) m/z calcd for C23H19N3O2, 369.1477; found,
369.1480.
2H); HRMS(EI) m/z calcd for
C18H15N3O3, 321.1113; found,
321.1111.
4.1.2.7.
6(5H)-yl)ethyl acetate (2c). Yellow solid (55% yield, HPLC purity:
99.0); mp 247–250 °C; 1H NMR (300 MHz, CDCl3)
8.80(d,
2-(5-Oxopyrido[20,10:2,3]imidazo[4,5-c]isoquinolin-
4.1.2.2.
6-(4-Methoxybenzyl)-11-methylpyrido[20,10:2,3]imi-
d
J = 7.3 Hz, 1H), 8.49(d, J = 8.4 Hz, 1H), 8.42(d, J = 8.1 Hz, 1H),
7.82(t, J = 7.7 Hz, 1H), 7.71(d, J = 9.5 Hz, 1H), 7.57(t, J = 7.7 Hz,
1H), 7.20(dd, J = 9.2, 6.6 Hz, 1H), 6.90(t, J = 7.0 Hz, 1H), 4.87(t,
J = 6.6 Hz, 2H), 4.58(t, J = 6.6 Hz, 2H), 1.97(s, 3H); HRMS(EI) m/z
calcd for C18H15N3O3, 321.1113; found, 321.1110.
dazo[4,5-c]isoquinolin-5(6H)-one (1d). Yellow solid (70% yield,
HPLC purity: 98.6); mp 197–200 °C; 1H NMR (300 MHz, CDCl3) d
8.58(d, J = 5.1 Hz, 1H), 8.55(d, J = 5.1 Hz, 1H), 8.10(d, J = 7.3 Hz,
1H), 7.85(t, J = 7.9 Hz, 1H), 7.59(t, J = 7.7 Hz, 1H), 7.16–7.18(m,
2H), 6.92(d, J = 7.0 Hz, 1H), 6.86–6.89(m, 2H), 6.56(t, J = 7.0 Hz,
1H), 5.85(s, 2H), 3.76(s, 3H), 2.69(s, 3H); HRMS(EI) m/z calcd for
4.1.2.8.
quinolin-5(6H)-one (2d). Yellow solid (42% yield, HPLC purity:
98.1); mp 206–209 °C; 1H NMR (300 MHz, CDCl3)
8.55(d,
6-(2-Furylmethyl)pyrido[20,10:2,3]imidazo[4,5-c]iso-
C23H19N3O2, 369.1477; found, 369.1480.
d
4.1.2.3.
9-Bromo-6-(4-methoxybenzyl)-8-methylpyri-
J = 7.4 Hz, 1H), 8.51(d, J = 8.5 Hz, 1H), 8.42(d, J = 8.0 Hz, 1H),
7.82(t, J = 7.6 Hz, 1H), 7.68(d, J = 9.1 Hz, 1H), 7.56(t, J = 7.7 Hz,
1H), 7.39(s, 1H), 7.16(dd, J = 8.9, 6.7 Hz, 1H), 6.79(t, J = 6.9 Hz,
1H), 6.34–6.40(m, 2H), 5.83(s, 2H); 13C NMR (100 MHz, CDCl3)
dC: 161.41, 149.00, 143.12, 142.69, 133.27, 131.81, 129.36,
127.20, 125.17, 124.23, 123.86, 123.76, 123.52, 121.93, 118.72,
112.48, 111,00, 109.07, 40.54. HRMS(EI) m/z calcd for
do[20,10:2,3]imidazo[4,5-c]isoquinolin-5(6H)-one (1e). Yellow
solid (49% yield, HPLC purity: 100); mp 205–207 °C; 1H NMR
(300 MHz, CDCl3) d 8.12(d, J = 8.1 Hz, 1H), 7.50(t, J = 7.7 Hz, 1H),
7.43(d, J = 8.8 Hz, 1H), 7.12–7.22(m, 4H), 7.07(d, J = 7.7 Hz, 1H),
6.82–6.85(m, 2H), 4.47(d, J = 6.2 Hz, 2H), 3.79(s, 3H), 2.53(s, 3H);
HRMS(EI) m/z calcd for C23H18BrN3O2, 447.0582; found, 447.0580.
C19H13N3O2, 315.1008; found, 315.1010.
4.1.2.4.
9-Bromo-6-(4-methoxybenzyl)-10-methylpyri-
4.1.2.9.
6-[4-(Trifluoromethoxy)benzyl]pyrido[20,10:2,3]imi-
do[20,10:2,3]imidazo[4,5-c]isoquinolin-5(6H)-one (1f). Yellow
solid (50% yield, HPLC purity: 96.5); mp 197–200 °C; 1H NMR
(300 MHz, CDCl3) d 8.56(d, J = 8.1 Hz, 1H), 8.45(s, 1H), 8.41(d,
J = 7.3 Hz, 1H), 7.85(m, 1H), 7.60(t, J = 7.5 Hz, 1H), 7.47(s, 1H),
7.17–7.20(m, 2H), 6.88–6.91(m, 2H), 5.83(s, 2H), 3.77(s, 3H),
2.38(s, 3H); HRMS(EI) m/z calcd for C23H18BrN3O2, 447.0582;
found, 447.0579.
dazo[4,5-c]isoquinolin-5(6H)-one (2e). Yellow solid (56% yield,
HPLC purity: 99.1); mp 220–222 °C; 1H NMR (300 MHz, CDCl3) d
8.53(d, J = 8.3 Hz, 1H), 8.45(d, J = 8.0 Hz, 1H), 8.08(d, J = 7.2 Hz,
1H), 7.85(m, 1H), 7.66(d, J = 9.4 Hz, 1H), 7.59(m, 1H), 7.20–
7.32(m, 4H), 7.09(m, 1H), 6.63(m, 1H), 5.89(s, 2H); 13C NMR
(100 MHz, CDCl3) dC: 161.66, 148.78, 143.02, 134.65, 133.41,

T. Meng et al. / Bioorg. Med. Chem. 22 (2014) 848–855
853
131.88, 129.48, 127.33, 127.06 ꢁ 3, 125.19, 124.30, 123.77 ꢁ 2,
122.67, 121.96, 121.89 ꢁ 2, 118.85, 112.77, 46.28; HRMS(EI) m/z
calcd for C22H14F3N3O2, 409.1038; found, 409.1034.
4.1.2.15.
6-[3-(4-Methoxyphenyl)propyl]pyrido[20,10:2,3]imi-
dazo[4,5-c]isoquinolin-5(6H)-one (2l). Yellow solid (61% yield,
HPLC purity: 99.5); mp 170–173 °C; 1H NMR (300 MHz, CDCl3) d
8.43(d, J = 8.0 Hz, 1H), 8.31(d, J = 7.4 Hz, 1H), 7.73(t, J = 8.3 Hz,
1H), 7.47–7.60(m, 3H), 7.18(d, J = 8.4 Hz, 2H), 7.01(m, 1H),
6.89(d, J = 8.4 Hz, 2H), 6.41(m, 1H), 4.40(t, J = 8.1 Hz, 2H), 3.82(s,
3H), 2.81(t, J = 8.1 Hz, 2H), 2.17(m, 2H); 13C NMR (100 MHz, CDCl3)
dC: 161.11, 158.13, 142.70, 132.71, 132.26, 131.34, 129.68 ꢁ 2,
128.96, 126.88, 124.73, 124.13, 123.78, 123.29, 122.72, 121.64,
118.57, 114.00 ꢁ 2, 112.36, 55.24, 42.25, 31.93, 31.13; HRMS(EI)
m/z calcd for C24H21N3O2, 383.1634; found, 383.1626.
4.1.2.10. 6-(3,4-Dimethoxybenzyl)pyrido[20,10:2,3]imidazo[4,5-
c]isoquinolin-5(6H)-one (2f). Yellow solid (44% yield, HPLC pur-
ity: 100); mp 180–183 °C; 1H NMR (300 MHz, CDCl3) d 8.55(d,
J = 7.9 Hz, 1H), 8.45(d, J = 7.6 Hz, 1H), 8.18(d, J = 7.3 Hz, 1H),
7.85(t, J = 7.6 Hz, 1H), 7.65(d, J = 9.2 Hz, 1H), 7.59(t, J = 7.8 Hz,
1H), 7.09(m, 1H), 6.70–6.83(m, 3H), 6.61(t, J = 6.6 Hz, 1H), 5.84(s,
2H), 3.83(s, 3H), 3.82(s, 3H); 13C NMR (100 MHz, CDCl3) dC:
161.52, 149.65, 148.53, 142.85, 133.12, 131.62, 129.31, 128.17,
127.07, 124.75, 124.53, 123.74, 123.22, 121.75, 118.36, 117.36,
112.43, 111.64, 108.60, 55.88, 55.75, 50.40, 46.45; HRMS(EI) m/z
calcd for C23H19N3O3, 385.1426; found, 385.1422.
4.1.2.16.
6-[2-(4-Hydroxyphenyl)ethyl]pyrido[20,10:2,3]imi-
dazo[4,5-c]isoquinolin-5(6H)-one (2k). To a stirred solution of
compound 2j (50 mg, 0.2 mmol) in 4 mL of dry dichloromethane
was added at ꢀ78 °C a solution of 0.04 mL(0.4 mmol) of BBr3 in
1 mL of dichloromethane. After 1 h the mixture was warmed to
ꢀ25 °C. The reaction was monitored by TLC, after completion, the
mixture was quenched with saturated sodium hydrogen carbon-
ate. The usual workup yielded 41 mg of the pure product as yellow
solid (yield 85%, HPLC purity: 98.6). mp 280–283 °C; 1H NMR
(300 MHz, DMSO-d6) d 9.16(s, 1H), 8.51(d, J = 7.0 Hz, 1H), 8.21(d,
J = 7.9 Hz, 1H), 8.17(d, J = 7.9 Hz, 1H), 7.74(t, J = 7.3 Hz, 1H),
7.57(d, J = 9.2 Hz, 1H), 7.47(t, J = 7.5 Hz, 1H), 7.18(m, 1H), 6.87–
6.96(m, 2H), 6.56(d, J = 8.2 Hz, 2H), 4.70(t, J = 7.3 Hz, 2H), 2.86(t,
J = 7.3 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) dC: 160.11, 156.04,
142.29, 132.91, 131.30, 129.65 ꢁ 2, 128.66, 127.55, 126.84,
124.36, 124.22, 124.05, 123.76, 123.42, 121.47, 118.01,
115.28 ꢁ 2, 112.58, 43.69, 34.22. ESI: m/z (relative intensity)
356.1(M+1, 100%).
4.1.2.11. 6-(2,4-Dimethoxybenzyl)pyrido[20,10:2,3]imidazo[4,5-
c]isoquinolin-5(6H)-one (2g). Yellow solid (58% yield, HPLC pur-
ity: 96.3); mp 188–200 °C; 1H NMR (300 MHz, CDCl3) d 8.56(d,
J = 7.4 Hz, 1H), 8.45(d, J = 8.0 Hz, 1H), 8.12(d, J = 7.4 Hz, 1H),
7.83(m, 1H), 7.55–7.63(m, 2H), 7.06(ddd, J = 9.2, 6.6, 1.0 Hz, 1H),
6.78(d, J = 8.5 Hz, 1H), 8.55–6.59(m, 2H), 6.30(dd, J = 8.5, 2.2 Hz,
1H), 5.77(s, 2H), 3.95(s, 3H), 3.73(s, 3H); 13C NMR (100 MHz,
CDCl3) dC: 161.75, 160.54, 156.69, 143.05, 133.08, 131.84, 129.49,
127.07, 127.04, 125.02, 124.76, 124.03, 123.64, 123.45, 121.85,
118.46, 116.19, 112.30, 104.62, 98.88, 55.54, 55.33, 41.92;
HRMS(EI) m/z calcd for C23H19N3O3, 385.1426; found, 385.1426.
4.1.2.12.
6-(1,3-Benzodioxol-5-ylmethyl)pyrido[20,10:2,3]imi-
dazo[4,5-c]isoquinolin-5(6H)-one (2h). Yellow solid (67% yield,
HPLC purity: 100); mp 216–219 °C; 1H NMR (300 MHz, CDCl3) d
8.52(d, J = 8.3 Hz, 1H), 8.42(d, J = 8.0 Hz, 1H), 8.17(d, J = 7.2 Hz,
1H), 7.83(m, 1H), 7.62(d, J = 9.1 Hz, 1H), 7.57(m, 1H), 7.07(ddd,
J = 9.2, 6.7, 1.1 Hz, 1H), 6.68–6.76(m, 3H), 6.61(m, 1H), 5.92(s,
2H), 5.79(s, 2H); 13C NMR (100 MHz, CDCl3) dC: 161.61, 148.62,
147.25, 142.98, 133.24, 131.83, 129.62, 129.47, 127.19, 125.06,
124.53, 123.87, 123.70, 123.20, 121.88, 118.64, 118.56, 112.55,
108.96, 106.06, 101.26, 46.51; HRMS(EI) m/z calcd for
4.1.3. General procedure B (Ullmann coupling) for synthesis of
compounds 4a–4h
In a 10 mL Biotage vial was charged with the compound 3 (1
equiv, prepared according to the procedure A), the appropriate
amines (10 equiv), copper(I) iodide(0.15 equiv),
L-proline(0.2
equiv), potassium carbonate(2.0 equiv) and 4 mL of DMSO. The vial
was sealed with a septum and the resulting suspension was heated
in the Biotage Initiator Synthesizer under 150 °C (300 W) over
10 min. The reaction mixture was cooled to ambient temperature,
and evaporated in vacuo. The residue was dissolved with CH2Cl2
(30 mL), and the resultant solution was washed sequentially with
water (10 mL), and brine (10 mL). The organic layer was dried, fil-
tered, and evaporated, and the residue was purified by flash chro-
matography on silica gel (CH2Cl2/MeOH = 10:1) to afford the
desired products.
C22H15N3O3, 369.1113; found, 369.1116.
4.1.2.13.
c]isoquinolin-6(5H)-yl)methyl]phenyl}
N-Methyl-N-{4-[(5-oxopyrido[20,10:2,3]imidazo[4,5-
methanesulfonamide
(2i). Yellow solid (34% yield, HPLC purity: 96.7); mp 233–
236 °C; 1H NMR (300 MHz, DMSO-d6) d 8.33–8.41(m, 3H), 7.91(t,
J = 7.5 Hz, 1H), 7.60–7.67(m, 2H), 7.29–7.40(m, 4H), 7.20(dd,
J = 9.1, 6.7 Hz, 1H), 6.82(t, J = 6.9 Hz, 1H), 5.98(s, 2H), 3.19(s, 3H),
2.91(s, 3H); 13C NMR (100 MHz, DMSO-d6) dC: 160.55, 142.35,
140.83, 135.36, 133.29, 131.65, 128.91, 127.07, 126.69 ꢁ 2,
126.42 ꢁ 2, 124.45, 124.16, 124.03, 123.35, 121.64, 118.01,
112.41, 45.28, 37.63, 35.02; HRMS(EI) m/z calcd for C23H20N4O3S,
432.1256; found, 432.1261.
4.1.3.1. 9-(1H-Imidazol-1-yl)-6-[2-(4-methoxyphenyl)ethyl]pyr-
ido[20,10:2,3]imidazo[4,5-c]isoquinolin-5(6H)-one (4a). Brown
solid (61% yield, HPLC purity: 96.8); mp 240–244 °C; 1H NMR
(300 MHz, CDCl3) d 8.53(d, J = 8.3 Hz, 1H), 8.42(d, J = 8.0 Hz, 1H),
8.29(s, 1H), 7.85(m, 1H), 7.75–7.79(d, J = 9.6 Hz, 2H), 7.62(m, 1H),
7.31(br s, 1H), 7.21(dd, J = 9.6, 1.9 Hz, 2H), 7.07(d, J = 8.5 Hz, 2H),
6.71(d, J = 8.5 Hz, 2H), 4.80(t, J = 7.5 Hz, 2H), 3.73(s, 3H), 3.22(t,
J = 7.5 Hz, 2H); 13C NMR (100 MHz, CDCl3) dC: 161.40, 158.84,
141.35, 133.34, 131.32, 131.06, 129.54 ꢁ 3, 129.32, 128.92,
127.79, 126.69, 125.70, 125.24, 124.28, 122.01, 119.84, 119.64
116.48, 114.50 ꢁ 3, 55.27, 44.90, 34.88; HRMS(EI) m/z calcd for
4.1.2.14.
6-[2-(4-Methoxyphenyl)ethyl]pyrido[20,10:2,3]imi-
dazo[4,5-c]isoquinolin-5(6H)-one (2j). Yellow solid (67% yield,
HPLC purity: 96.5); mp 155–157 °C; 1H NMR (300 MHz, CDCl3) d
8.48(d, J = 8.3 Hz, 1H), 8.39(d, J = 7.7 Hz, 1H), 8.35(d, J = 7.2 Hz,
1H), 7.79(m, 1H), 7.68(d, J = 9.4 Hz, 1H), 7.55(m, 1H), 7.21–
7.27(m, 2H), 7.15(ddd, J = 9.3, 6.7, 1.1 Hz, 1H), 6.85–6.88(m, 2H),
6.81(m, 1H), 4.80(t, J = 8.0 Hz, 2H), 3.78(s, 3H), 3.15(t, J = 8.0 Hz,
2H); 13C NMR (100 MHz, CDCl3) dC: 161.24, 158.65, 142.90,
132.96, 131.55, 129.63 ꢁ 2, 129.11, 129.07, 127.11, 125.10,
124.29, 124.01, 123.41, 122.58, 121.82, 119.00, 114.29 ꢁ 2,
112.75, 55.25, 44.45, 34.86; HRMS(EI) m/z calcd for C23H19N3O2,
369.1477; found, 369.1479.
C26H21N5O2, 435.1695; found, 435.1697.
4.1.3.2. 9-{[3-(1H-Imidazol-1-yl)propyl]amino}-6-[2-(4-
methoxyphenyl)ethyl]pyrido[20,10:2,3]imidazo[4,5-c]isoquino-
lin-5(6H)-one (4b). Brown solid (70% yield, HPLC purity: 100);
mp 180–182 °C; 1H NMR (300 MHz, CDCl3) d 8.49(d, J = 7.6 Hz,
1H), 8.36(d, J = 7.9 Hz, 1H), 7.78(t, J = 7.5 Hz, 1H), 7.48–7.56(m,
3H), 7.39(s, 1H), 7.21(d, J = 9.0 Hz, 2H), 7.09(s, 1H), 6.84–6.88(m,

854
T. Meng et al. / Bioorg. Med. Chem. 22 (2014) 848–855
3H), 6.7(dd, J = 9.8, 1.5 Hz, 1H), 4.86(m, 2H), 4.01(t, J = 6.9 Hz, 2H),
3.79(s, 3H), 3.22(m, 2H), 2.82(t, J = 6.6 Hz, 2H), 1.26(m, 2H); 13C
NMR (100 MHz, CDCl3) dC: 161.45, 158.70, 140.19, 136.18,
132.97, 131.97, 129.5 ꢁ 5, 129.20, 126.74, 124.91, 123.74, 121.58,
119.50, 119.11, 118.71, 114.29 ꢁ 3, 101.67, 55.36, 44.39, 43.73,
J = 7.0 Hz, 1H), 7.84(m, 1H), 7.49–7.57(m, 3H), 7.25(d, J = 8.6 Hz,
2H), 7.02(m, 1H), 6.87–6.90(m, 2H), 4.88(m, 2H), 3.74(s, 3H),
3.59(m, 3H), 3.00–3.14(m, 6H), 2.41(m, 3H); 13C NMR (100 MHz,
DMSO-d6) dC: 160.21, 158.00, 139.34, 136.88, 132.82, 131.80,
129.40 ꢁ 3, 128.63, 126.16, 123.50, 122.90, 121.04, 120.54,
117.86, 113.93 ꢁ 3, 100.12, 65.99 ꢁ 3, 54.98, 53.21 ꢁ 2, 43.22,
34.40 ꢁ 2; HRMS(EI) m/z calcd for C29H31N5O3, 497.2427; found,
497.2418.
41.12, 34.71, 30.33; HRMS(EI) m/z calcd for
C29H28N6O2,
492.2274; found, 492.2275.
4.1.3.3. 6-[2-(4-Methoxyphenyl)ethyl]-9-[(pyridin-2-ylmethyl)-
amino]pyrido[20,10:2,3]imidazo[4,5-c]isoquinolin-5(6H)-one
(4c). Brown solid (52% yield, HPLC purity: 96.9); mp 190–193 °C;
4.1.3.8.
9-[(2-Hydroxyethyl)amino]-6-[2-(4-methoxyphenyl)-
ethyl]pyrido[20,10:2,3]imidazo[4,5-c]isoquinolin-5(6H)-one
1H NMR (300 MHz, CDCl3)
d 8.57(d, J = 4.9 Hz, 1H), 8.47(d,
(4h). Brown amorphous solid (80% yield, HPLC purity: 99.1); 1H
J = 7.6 Hz, 1H), 8.35(d, J = 7.9 Hz, 1H), 7.75(m, 1H), 7.67(td, J = 7.8,
1.8 Hz, 1H), 7.47–7.55(m, 3H), 7.27–7.30(m, 2H), 7.22(dd, J = 7.0,
5.5 Hz, 1H), 7.13(d, J = 7.9 Hz, 1H), 6.86–6.89(m, 3H), 4.86(m, 2H),
4.14(s, 2H), 3.78(s, 3H), 3.20(m, 2H); 13C NMR (100 MHz, DMSO-
d6) dC: 161.39, 158.57, 156.07, 149.12, 140.14, 136.72, 136.29,
132.84, 131.94, 129.63 ꢁ 2, 129.47, 129.11, 126.54, 124.95,
124.82, 123.63, 122.57, 121.89, 121.48, 119.94, 118.82,
114.18 ꢁ 2, 101.51, 55.24, 48.84, 43.80, 34.83; HRMS(EI) m/z calcd
for C29H25N5O2, 475.2008; found, 475.2026.
NMR (300 MHz, DMSO-d6)
d 8.24(d, J = 7.9 Hz, 1H), 8.16(d,
J = 7.9 Hz, 1H), 7.74(t, J = 7.5 Hz, 1H), 7.39–7.48(m, 3H), 7.18(d,
J = 8.5 Hz, 2H), 6.98(d, J = 9.7 Hz, 1H), 6.84(d, J = 8.5 Hz, 2H),
5.61(br s, 1H), 4.85(br s, 1H), 4.72(t, J = 7.8 Hz, 2H), 3.70(s, 3H),
3.62(t, J = 4.8 Hz, 2H), 2.95–3.04(m, 4H); 13C NMR (100 MHz,
DMSO-d6) dC: 160.20, 158.05, 139.33, 137.19, 132.74, 131.75,
129.52 ꢁ 2, 129.40, 128.61, 126.12, 124.49, 123.44, 122.88,
121.06, 120.60, 117.80, 113.99 ꢁ 2, 99.87, 59.39, 55.03, 46.11,
43.49, 34.52; HRMS(EI) m/z calcd for C25H24N4O3, 428.1848; found,
428.1844.
4.1.3.4. 6-[2-(4-Methoxyphenyl)ethyl]-9-[(pyridin-3-ylmethyl)-
amino]pyrido[20,10:2,3]imidazo[4,5-c]isoquinolin-5(6H)-one
(4d). Brown solid (58% yield, HPLC purity: 96.7); mp 188–190 °C;
4.2. Sulforhodamine B (SRB) assays
1H NMR (300 MHz, DMSO-d6)
d 8.44–8.45(m, 3H), 8.34(d,
J = 8.0 Hz, 1H), 7.76(t, J = 7.4 Hz, 1H), 7.48–7.56(m, 3H), 7.40(s,
1H), 7.26(m, 1H), 7.16-7.19(d, J = 8.5 Hz, 2H), 6.78–6.83(m, 3H),
4.76(t, J = 8.0 Hz, 2H), 3.71(s, 3H), 3.12(t, J = 8.0 Hz, 2H); 13C NMR
(100 MHz, CDCl3) dC: 161.29, 158.62, 149.17, 148.98, 140.08,
135.87, 135.18, 133.08, 132.90, 131.79, 129.43 ꢁ 3, 129.14,
126.66, 125.05, 124.76, 123.60, 121.47, 119.51, 118.99,
114.17 ꢁ 3, 101.88, 55.19, 45.90, 40.90, 34.64; HRMS(EI) m/z calcd
for C29H25N5O2, 475.2008; found, 475.2010.
HeLa cells were purchased from the American Type Culture
Collection (Manassas, VA). HeLa cells were treated with indicated
compounds for 72 h and then were fixed with 10% pre-cooled
trichloroacetic acid (TCA), washed with distilled water, and
stained with SRB (Sigma–Aldrich, St. Louis, MO) in 1% acetic acid.
SRB in the cells was dissolved in 10 mM Tris–HCl and was mea-
sured at 515 nm with spectraMAX190 (Molecular Devices, Sun-
nyvale, CA). The cell proliferation inhibition rate was calculated
as: proliferation inhibition (%) = [1ꢀ(A515 treated/A515 control)] ꢁ 100%.
4.1.3.5. 6-[2-(4-Methoxyphenyl)ethyl]-9-[(pyridin-4-ylmethyl)-
amino]pyrido[20,10:2,3]imidazo[4,5-c]isoquinolin-5(6H)-one
(4e). Brown amorphous solid (56% yield, HPLC purity: 100); 1H
NMR (300 MHz, DMSO-d6) d 8.17–8.24(m, 3H), 7.75–7.80(m, 2H),
7.46–7.53(m, 3H), 7.34(s, 1H), 6.99–7.06(m, 3H), 6.75–6.78(d,
J = 8.2 Hz, 2H), 6.42(t, J = 6.5 Hz, 1H), 4.67(t, J = 7.3 Hz, 2H),
4.23(s, 2H), 3.66(s, 3H), 2.81(t, J = 7.3 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) dC: 160.24, 158.00, 149.66, 148.01, 139.39,
136.39, 132.92, 131.71, 129.48 ꢁ 4, 129.28, 128.72, 126.34,
124.51, 123.68, 122.96, 121.16, 120.28, 118.22, 113.87 ꢁ 3,
100.98, 55.03, 45.84, 42.94, 34.13; HRMS(EI) m/z calcd for
The average IC values were determined with the Logit method
50
from at least three independent tests.
4.3. In vitro tubulin polymerization assays
Purified tubulin was purchased from Cytoskeleton (Denver, CO,
USA). 20 lM indicated compounds were mixed with tubulin in a
96-well plate on ice, respectively. The plate was put into a 37 °C
incubator and tubulin polymerization was monitored at 340 nm
with spectraMAX190 (Molecular Devices, Sunnyvale, CA). The reac-
tion buffer for this assay contained 80 mM PIPES pH6.9, 0.5 mM
EGTA, 2 mM MgCl2 and 1 mM GTP.
C29H25N5O2, 475.2008; found, 475.2010.
4.1.3.6.
9-{[2-(Diethylamino)ethyl]amino}-6-[2-(4-methoxy-
phenyl)ethyl]pyrido[20,10:2,3] imidazo[4,5-c]isoquinolin-5(6H)-
one (4f). Brown amorphous solid (71% yield, HPLC purity: 98.3);
1H NMR (300 MHz, DMSO-d6) d 8.31(d, J = 8.2 Hz, 1H), 8.23(d,
J = 7.9 Hz, 1H), 7.83(m, 1H), 7.47–7.64(m, 3H), 7.21-7.26(m, 2H),
7.01(d, J = 9.8 Hz, 1H), 6.84–6.89(m, 2H), 4.84(m, 2H), 4.49(m,
2H), 3.72(s, 3H), 2.89–3.09(m, 4H), 2.73(m, 2H), 2.60(m, 2H),
1.07(m, 6H); 13C NMR (100 MHz, DMSO-d6) dC: 160.30, 158.06,
139.43, 136.95, 132.88, 131.81, 129.45 ꢁ 2, 129.36, 128.67,
126.25, 124.57, 123.56, 122.95, 121.11, 120.59, 117.89,
113.98 ꢁ 2, 100.33, 55.01, 50.82, 46.41ꢁ2, 43.34, 41.11, 34.46,
11.20 ꢁ 2; HRMS(EI) m/z calcd for C29H33N5O2, 483.2634; found,
483.2632.
4.4. Colchicine competitive binding assays
Tubulin (3
pounds for 1 h, respectively, following which added [3H]-colchicine
(5
M). After 30 min of incubation at 37 °C, the bound [3H]-colchi-
lM) was pre-incubated with 20 lM indicated com-
l
cine of each sample was determined using DE81 filter assays de-
scribed by Gary G. Borisy.22
4.5. Cell cycle progression analysis
HeLa cells were treated with 0.5
12 h, then harvested and washed with PBS, fixed with pre-cooled
70% ethanol at 4 °C. After being stained with 10 g/ml propidium
lM indicated compounds for
l
4.1.3.7. 6-[2-(4-Methoxyphenyl)ethyl]-9-[(2-morpholin-4-yleth-
yl)amino]pyrido[20,10:2,3]imidazo [4,5-c]isoquinolin-5(6H)-one
(4g). Brown amorphous solid (68% yield, HPLC purity: 98.8); 1H
iodide in the dark for 30 min, cells were collected with FACS Cali-
bur (BD Biosciences, Franklin Lakes, NJ) and analysed by using the
CELLQUEST software (BD Biosciences, Franklin Lakes, NJ).
NMR (300 MHz, DMSO-d6)
d 8.32(d, J = 7.6 Hz, 1H), 8.24(d,

T. Meng et al. / Bioorg. Med. Chem. 22 (2014) 848–855
855
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4.6. Mitotic spindle assembly assays
After 0.5 M compounds treatments, HeLa cells cultured on
glass coverslips were fixed with 4% paraformaldehyde, permeabili-
zed with 0.2% TritonX-100 and saturated with 3% bovine serum
albumin. Then the cells were incubated sequentially with the pri-
l
mary antibody against
a-Tubulin (1:200, Invitrogen, Carlsbad,
CA) and with Alexa FluorVR 488-conjugated goat anti-mouse IgG
(1:100, Invitrogen, Carlsbad, CA) for 1 h, respectively. Finally, cells
were stained with DAPI and imaged under an Olympus BX51 fluo-
rescence microscope system (Olympus, Tokyo, Japan) and a Nikon
Eclipse C1 Plus confocal microscope (Nikon, Tokyo, Japan).
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Acknowledgments
This work was supported by the National Science & Technology
Major Project ‘Key New Drug Creation and Manufacturing Pro-
gram’, China (no.: 2012ZX09103-101-026).
14. Zhang, Z.; Meng, T.; He, J.; Li, M.; Tong, L. J.; Xiong, B.; Lin, L.; Shen, J.; Miao, Z.
H.; Ding, J. Invest. New Drugs 2010, 28, 715.
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Supplementary data
16. Viola, G.; Bortolozzi, R.; Hamel, E.; Moro, S.; Brun, P.; Castagliuolo, I.; Ferlin, M.
G.; Basso, G. Biochem. Pharmacol. 2012, 83, 16.
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Supplementary data (copies of proton and carbon NMR for all
the compounds) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.004.
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