Design, synthesis, and biological evaluation of novel γ-carboline ketones as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2011.01.057

A series of novel γ-carboline ketones were designed, synthesized and evaluated for their cytotoxic activity in vitro against six human cancer cell lines (A549, SGC, HCT116, MCF-7, K562 and K562R). Biological evaluation revealed that almost all of the new

Full text of DOI:10.1016/j.ejmech.2011.01.057

European Journal of Medicinal Chemistry 46 (2011) 1343e1347
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 novel g-carboline ketones as
anticancer agents
,
c
c
b
c
c
b,
*
Jing Chen a, Tao Liu b , Rui Wu , Jianshu Lou , Xiaowu Dong , Qiaojun He , Bo Yang , Yongzhou Hu
*
a College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053 Zhejiang, China
b ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058 Zhejiang, China
c Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058 Zhejiang, 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
g-carboline ketones were designed, synthesized and evaluated for their cytotoxic
Received 16 August 2010
Received in revised form
26 January 2011
Accepted 26 January 2011
Available online 20 February 2011
activity in vitro against six human cancer cell lines (A549, SGC, HCT116, MCF-7, K562 and K562R). Bio-
logical evaluation revealed that almost all of the new compounds displayed moderate to potent cytotoxic
activities against the tested cells. Among them, seven of the fourteen new compounds show more potent
cytotoxic activities against K562R cell line than that of the positive control, taxol. Primary mechanism
research on the most potent compound 6f indicated that it was a potent tubulin polymerization inhibitor,
with IC50 value of 4.3
m
M, equivalent to that of CA-4, and arresting cell cycle in G2/M phase.
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
g-Carboline ketones
Synthesis
Anticancer
Antitubulin
1. Introduction
Three series of g-carboline derivatives with some displaying
potent cytotoxicities have been reported in our previously studies
on anticancer agents (1e3, Fig. 1.) [7]. We supposed that these
compounds match well with the common structure of CSIs: the
Microtubules, the key components of cytoskeleton of eukaryotic
cells, are involved in numerous cellular functions, including cell
motility, cell division and vesicle transport. Microtubules are in
dynamic equilibrium with tubulin dimmers. Disruption of the
dynamic equilibrium blocks the cell division machinery at mitosis
and leads to cell death. Therefore, microtubule has attracted
considerable attention as being a promising target of antitumor
drugs [1]. Reported antitubulin agents can be divided into three
groups, namely, the vinca-, the taxus-, and the colchicine-binding
site inhibitors (CSIs) [2]. Although vinca alkaloids, taxanes are used
for the treatment of cancer, their adverse effects, difficulty in
synthesis and high cost limited their use [3]. So, the search for new
antitubulin agents with better activity is still in progress. Recent
years, CSIs have attracted more and more attention because of their
simple structures and potent activities [4]. The common structural
features of CSIs have been described [5,6]. It is reported that most
CSIs bear two hydrophobic groups (ring A and B) and a cis-bridge
between them. It is also reported that the existence of hydrogen-
bond acceptor at ring A and hydrophobic groups or polar functional
groups at ring B is beneficial for binding affinity.
planar g-carboline acts as the hydrophobic ring A of CSIs, and the
N2 position of carboline contributes as a hydrogen-bond acceptor;
the aromatic ring connected to the linker is the hydrophobic ring B
of CSIs. Further mechanism study showed they could inhibit
tubulin polymerization (data haven’t been revealed yet). Enlight-
ened by these results and the fact that many CSIs bearing a carbonyl
group, such as BPR0L075 (4) [8,9] and Phenstatin (5) [10], showed
potent activity, we introduced a carbonyl group as the linker in
designed compounds. We also introduced hydrophobic substitu-
ents, such as halogen and methoxy group, as well as polar func-
tional group, such as amino group, on ring B. Here, we reported
the synthesis and biological evaluation of this series of novel
g-carboline ketone compounds, with the aim to better understand
the structureeactivity relationships (SARs) of the g-carboline series
and to discover more potent antitubulin agents.
2. Chemistry
The synthetic routes of
g-carboline ketones 6aen are summa-
rized in Scheme 1. -Carboline (7a) was prepared using our recently
g
developed synthetic protocol [11]. Treatment of 7a with various
alkylating reagents (1.2 equiv.) and NaH in dry THF-DMF at room
* Corresponding authors. Tel./fax: þ86 571 88208460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.057

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J. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1343e1347
Table 1
In vitro cytotoxic activities of the synthesized compounds (6aen) against six human
cancer cell lines.
Compd.
Cytotoxicity (IC50
,
m
M)a,b
HCT116
A549
SGC
MCF-7
K562
4.82
K562R
6a
6b
6c
ec
e
e
e
e
23.22
e
11.79
e
8.63
e
8.36
e
4.96
6.46
9.09
e
6d
6e
6f
6g
6h
6i
6j
6k
6l
20.52
21.37
24.15
27.18
41.43
35.15
46.81
29.13
49.31
17.80
18.66
24.86
62.49
38.59
33.96
35.59
>100
>100
41.43
>100
3.34
70.22
34.30
37.75
62.58
42.29
24.26
35.94
59.22
40.65
3.85
9.99
3.28
6.39
6.42
5.26
4.69
2.09
1.57
0.66
1.58
7.08
1.58
1.45
3.68
23.34
30.62
21.03
>100
4.74
3.21
5.04
11.95
56.00
2.11
13.71
2.11
12.19
16.79
13.41
>100
>100
1.16
6m
6n
Taxol
>100
>100
2.46
>100
>100
4.37
>100
>100
5.63
Fig. 1. Structure of BPR0L075 (4), Phenstatin (5), and g-carboline derivatives (1e3, 6).
a
IC50, compound concentration required to inhibit tumor cell proliferation by
temperature gave the corresponding compounds 7bed [12].
Increasing the amount of alkylating reagent (for example C2H5Br)
to 2.4 equiv. led to the yield of quaternary g-carboline 7e. Then,
50%.
b
c
Values are means of three experiments.
NT, not tested.
compounds 7bee were treated with various benzoyl chlorides in
dry CH2Cl2 in the presence of AlCl3 at room temperature to afford
target compounds 6aek, 6m and 6n. In addition, reduction of
compound 6k by Raney Ni in EtOH yielded the amino compound
6l [13].
revealed that introducing methyl, ethyl, or isopropyl group at N9
position of -carboline would do little effect on their cytotoxicities
g
against K562 cell line. The influence of various substitutions on the
B ring was also examined. Compound 6b with an unsubstituted B
ring exhibited the most potent activity against SGC and HCT116 cell
lines, while compound 6f bearing methylsulphonyl group at 4-
position was the most potent one against MCF-7 and K562R cell
lines. Besides, we also evaluated the effect of substitution on N2
position. It was obviously that introduction of ethyl group at the N2
position of 6b and 6i afforded the corresponding quaternary
3. Biological studies and discussion
The synthesized g-carboline ketones 6aen were tested for their
cytotoxic activities in vitro against several human cancer cell lines
including human non-small lung cancer cell A549, human gastric
adenocarcinoma cell SGC, human colon cancer cell HCT116, human
breast carcinoma cell MCF-7, human myeloid leukemia cell K562
and K562R (a multidrug resistant cell) by MTT assay. Taxol was
employed as the positive control. The results are summarized in
Table 1.
By a scrutiny of the MTT assay results, it showed that almost all
of the new compounds displayed moderate to potent cytotoxic
activities against the tested cells. Most of the g-carboline ketones
g-carboline salts 6m and 6n with no activity. This was in agreement
with the evaluated results of sulfonates 2 [7].
To investigate whether the cytotoxic activities of these
compounds were related to an interaction with the microtubule
system, compound 6f, the most promising compound against MCF-
7 and K562R cell lines, was evaluated for its tubulin polymerization
inhibition. The IC50 value of compound 6f was 4.3
with that of CA-4 (1.5 M).
mM, comparable
m
Then, flow cytometry experiments were carried out to deter-
mine whether compound treatment would lead to cell cycle arrest
exhibited similar activities against MCF-7, K562, and K562R cell
lines in comparison with taxol. It was interesting that seven of the
fourteen new compounds showed more potent cytotoxic activities
against K562R cell line than that of taxol, with IC50 values ranging
at G2/M phase. A549 cells were treated with 2
M CA-4 was used as a positive control. It was shown that
compound 6f caused a marked increase in the percentage of cells
mM 6f for 48 h, and
1
m
from 0.66 to 3.68
mM. Comparing the cytotoxicities of 6aec
Scheme 1. Reagents and conditions: (a) R1X (1.2 equiv.), NaH, dry THFeDMF, r.t 0.5 h; (b) R1X (2.4 equiv.), NaH, dry THFeDMF, r.t 0.5 h; (c) AlCl3, dry CH2Cl2, N2, reflux 3 h; (d) Raney
Ni, EtOH, H2, r.t 2 h.

J. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1343e1347
1345
Fig. 2. Effect of compound 6f (2 mM) and CA-4 (1 mM) on the cell cycle of A549 cells.
blocked in the G2/M phase of the cell cycle, with a simultaneous
decrease of cells in G0/G1 and S phase (Fig. 2). It was in the similar
trend as CA-4.
7.62 (t, 2H, J ¼ 7.2 Hz), 3.97 (s, 3H, CH3). 13C NMR (
d
, DMSO-d6):
196.30, 144.85, 144.18, 143.72, 142.97, 136.74, 132.38, 131.66, 130.36,
129.98, 125.69, 121.46, 120.79, 120.25, 113.38, 109.92, 37.53. ESI-MS:
m/z ¼ 287 [Mþ1]þ. Anal. calcd for C19H14N2O: C, 79.70; H, 4.93; N,
9.78. Found: C, 79.74; H, 5.01; N, 9.76.
4. Conclusion
In summary,
a
series of novel
g
-carboline ketones were
5.1.1.2. 8-Benzoyl-5-ethyl-
carboline (7c, 196 mg, 1 mmol), benzoyl chloride (280 mg,
2 mmol). White solid (51%), mp: 80e82 ꢁC. 1H NMR (
, DMSO-d6):
g-carboline (6b). Reagent: 5-ethyl-g-
synthesized and tested for their cytotoxic activities in vitro against
six human tumor cell lines. Most of the compounds showed
moderate to potent cytotoxic activities against all the tested cells,
especially for MCF-7, K562, and K562R cell lines. Primary mecha-
nism research indicated that the synthesized compounds could
inhibit tubulin polymerization. The IC50 value of compound 6f was
d
9.46 (s, 1H), 8.74 (s, 1H), 8.55 (d, 1H, J ¼ 6.0 Hz), 7.96 (d, 1H,
J ¼ 8.8 Hz), 7.85 (d, 1H, J ¼ 8.8 Hz), 7.79 (d, 2H, J ¼ 7.2 Hz), 7.73 (m,
2H), 7.61 (t, 2H, J ¼ 7.2 Hz), 4.54 (q, 2H, J ¼ 6.8 Hz), 1.36 (t, 3H,
J ¼ 6.8 Hz). 13C NMR (
d, DMSO-d6): 195.60, 145.74, 144.74, 143.62,
4.3
mM, equivalent to that of CA-4. Moreover, it could also lead to
142.29, 138.33, 132.34, 129.80, 129.54, 129.04, 128.71, 123.84,
120.78, 119.32, 109.80, 105.35, 37.77, 13.93. ESI-MS: m/z ¼ 301
[Mþ1]þ. Anal. calcd for C20H16N2O: C, 79.98; H, 5.37; N, 9.33.
Found: C, 80.14; H, 5.42; N, 9.16.
cell cycle arrest at G2/M phase. With all these results, further design
and synthesis of potent antitumor agents are ongoing in our labo-
ratory and the results will be reported in due course.
5. Experiment
5.1.1.3. 8-Benzoyl-5-isopropyl-
-carboline (7d, 210 mg, 1 mmol), benzoyl chloride (280 mg, 2 mmol).
White solid (67%), mp: 134e136 ꢁC. 1H NMR (
, DMSO-d6): 9.48 (s,
1H), 8.75 (s, 1H), 8.53 (d, 1H, J ¼ 4.4 Hz), 7.96 (d, 1H, J ¼ 8.8 Hz), 7.93
(dd, 1H, J ¼ 8.8, 0.8 Hz), 7.80 (m, 3H, J ¼ 7.2 Hz), 7.71 (t, 1H, J ¼ 7.6 Hz),
7.52 (t, 2H, J ¼ 7.6 Hz), 5.24 (m, 1H),1.67 (d, 6H, J ¼ 7.2 Hz). ESI-MS: m/
z ¼ 315 [Mþ1]þ. Anal. calcd for C21H18N2O: C, 80.23; H, 5.77; N, 8.91.
Found: C, 80.26; H, 5.84; N, 9.07.
g-carboline (6c). Reagent: 5-isopropyl-
g
Melting points were obtained on a B-540 Büchi melting-point
apparatus and are uncorrected. 1H NMR and 13C NMR spectra were
recorded on a Brüker AM 400 instrument at 400 MHz (chemical
d
shifts are expressed as
d values relative to TMS as internal stan-
dard). ESI (positive) were recorded on an Esquire-LC-00075 spec-
trometer. Element analyses were performed on an Eager 300
instrument.
5.1.1.4. 5-Ethyl-8-(4-methoxybenzoyl)-
ethyl- -carboline (7c, 196 mg, 1 mmol), 4-methoxybenzoyl chlo-
ride (340 mg, 2 mmol). White solid (29%), mp: 122e124 ꢁC. 1H NMR
g-carboline (6d). Reagent: 5-
5.1. Synthesis
g
5.1.1. General procedure for synthesis of
Benzoyl chloride (8, 2 mmol) and AlCl3 (4.5 mmol) were added
to a suspension of -carboline (7, 1 mmol) in dry CH2Cl2 (5 mL)
g
-carboline ketones 6aen
(
d
, DMSO-d6): 9.41 (s, 1H), 8.64 (s, 1H), 8.49 (d, 1H, J ¼ 5.2 Hz), 7.87
(d, 1H, J ¼ 8.4 Hz), 7.80 (d, 1H, J ¼ 8.4 Hz), 7.76 (d, 2H, J ¼ 8.4 Hz),
g
7.67 (d, 1H, J ¼ 5.2 Hz), 7.07 (d, 2H, J ¼ 8.4 Hz), 4.48 (q, 2H,
under an inert atmosphere at room temperature. The mixture was
stirred under reflux until completion of reaction (monitored by
TLC). After adding ice water (10 mL), the mixture was neutralized
with 10% NaOH, and extracted with AcOEt (3 ꢀ 20 mL). The organic
phase was washed with brine (2 ꢀ 20 mL), dried over anhydrous
Na2SO4, and concentrated under vacuum. The residue was purified
over silica column chromatography using PE: EtOAc: EtOH (10:10:1,
V/V/V) or PE: EtOAc: 95% EtOH (1:1:4, V/V/V) as eluent to afford
6aen.
J ¼ 7.2 Hz), 3.82 (s, 3H), 1.31 (t, 3H, J ¼ 7.2 Hz). 13C NMR (
d, DMSO-
d6): 195.98, 158.31, 146.21, 145.23, 144.43, 143.17, 136.93, 126.74,
125.71, 123.66, 122.91, 115.23, 115.08, 112.81, 111.42, 110.84, 55.92,
40.27,15.73. ESI-MS: m/z ¼ 331 [Mþ1]þ. Anal. calcd for C21H18N2O2:
C, 76.34; H, 5.49; N, 8.48. Found: C, 76.26; H, 5.52; N, 8.72.
5.1.1.5. 5-Ethyl-8-(3-methoxybenzoyl)-
ethyl- -carboline (7c, 196 mg, 1 mmol), 3-methoxybenzoyl chlo-
ride (340 mg, 2 mmol). White solid (24%), mp: 96e98 ꢁC. 1H NMR
g-carboline (6e). Reagent: 5-
g
(
d
, DMSO-d6): 9.49 (s, 1H), 8.76 (s, 1H), 8.56 (d, 1H, J ¼ 6.0 Hz), 7.98
5.1.1.1. 8-Benzoyl-5-methyl-
-carboline (7b, 182 mg, 1 mmol), benzoyl chloride (280 mg,
2 mmol). White solid (23%), mp: 78e80 ꢁC. 1H NMR (
, DMSO-d6):
g
-carboline (6a). Reagent: 5-methyl-
(d, 1H, J ¼ 8.8 Hz), 7.87 (d, 1H), 7.75 (d, 1H, J ¼ 6.0 Hz), 7.53 (t, 1H,
J ¼ 8.0 Hz), 7.34 (d, 1H, J ¼ 8.0 Hz), 7.30 (s, 1H), 7.27 (d,
1H, J ¼ 8.0 Hz), 4.56 (q, 2H, J ¼ 7.2 Hz), 3.83 (s, 3H), 1.38 (t, 3H,
J ¼ 7.2 Hz). ESI-MS: m/z ¼ 331 [Mþ1]þ. Anal. calcd for C21H18N2O2:
C, 76.34; H, 5.49; N, 8.48. Found: C, 76.52; H, 5.76; N, 8.26.
g
d
9.47 (s, 1H), 8.74 (s, 1H), 8.57 (d, 1H, J ¼ 5.2 Hz), 7.98 (dd, 1H, J ¼ 8.8,
1.2 Hz), 7.84 (d, 1H, J ¼ 8.8 Hz), 7.80 (d, 2H, J ¼ 7.2 Hz), 7.72 (m, 2H),

1346
J. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1343e1347
5.1.1.6. 5-Ethyl-8-(4-methylsulfonylbenzoyl)-
5-ethyl- -carboline (7c, 196 mg, 1 mmol), 4-methylsulfonylbenzoyl
chloride (438 mg, 2 mmol). White solid (54%), mp: 161e162 ꢁC. 1H
NMR (
(d, 2H, J ¼ 8.0 Hz), 8.08 (m, 3H), 7.90 (d, 1H, J ¼ 8.0 Hz), 7.76 (d, 1H,
J ¼ 4.8 Hz), 4.55 (q, 2H, J ¼ 7.2 Hz), 3.34 (s, 3H), 1.35 (t, 3H, J ¼ 7.2 Hz).
ESI-MS: m/z ¼ 379 [Mþ1]þ. Anal. calcd for C21H18N2O3S: C, 66.65; H,
4.79; N, 7.40. Found: C, 66.76; H, 4.64; N, 7.47.
g
-carboline (6f). Reagent:
Anal. calcd for C20H17N3O: C, 76.17; H, 5.43; N, 13.32. Found: C,
76.25; H, 5.62; N, 13.26.
g
d
, DMSO-d6): 9.50 (s,1H), 8.78 (s,1H), 8.57 (d,1H, J ¼ 4.8 Hz), 8.15
5.1.1.12. 8-Benzoyl-2, 5-diethyl-
Reagent: 2, 5-diethyl- -carboline-3-ium bromide (7e, 305 mg,
1 mmol), benzoyl chloride (280 mg, 2 mmol). White solid (45%),
mp: 103e105 ꢁC. 1H NMR (
, DMSO-d6): 8.93 (s, 1H), 8.32 (d, 1H,
g-carboline-3-ium bromide (6m).
g
d
J ¼ 6.8 Hz), 7.61 (d, 2H, J ¼ 8.4 Hz), 7.22 (m, 2H), 7.11 (t, 1H,
J ¼ 7.2 Hz), 6.85 (t, 2H, J ¼ 7.2 Hz), 6.59 (d, 2H, J ¼ 7.2 Hz), 4.38 (q,
2H, J ¼ 6.8 Hz), 4.02 (q, 2H, J ¼ 6.8 Hz), 1.46 (t, 3H, J ¼ 6.8 Hz),1.09 (t,
3H, J ¼ 6.8 Hz). ESI-MS: m/z ¼ 409 [Mþ1]þ. Anal. calcd for
C22H21BrN2O: C, 64.55; H, 5.17; N, 6.84. Found: C, 64.47; H, 4.94; N,
7.03.
5.1.1.7. 8-(4-Chlorobenzoyl)-5-ethyl-
-carboline (7c, 196 mg, 1 mmol), 4-chlorobenzoyl chloride (350 mg,
2 mmol). White solid (45%), mp: 115e117 ꢁC. 1H NMR (
, DMSO-d6):
g-carboline (6g). Reagent: 5-ethyl-
g
d
9.48 (s, 1H), 8.74 (s, 1H), 8.56 (d, 1H, J ¼ 5.6 Hz), 7.97 (d, 1H, J ¼ 8.0 Hz),
7.87 (d, 1H, J ¼ 8.0 Hz), 7.82 (d, 2H, J ¼ 8.0 Hz), 7.74 (d, 1H, J ¼ 5.6 Hz),
7.67 (d, 2H, J ¼ 8.0 Hz), 4.56 (q, 2H, J ¼ 7.2 Hz), 1.37 (t, 3H, J ¼ 7.2 Hz).
ESI-MS: m/z ¼ 335 [Mþ1]þ. Anal. calcd for C20H15ClN2O: C, 71.75; H,
4.52; N, 8.37. Found: C, 71.73; H, 4.26; N, 8.27.
5.1.1.13. 8-(4-Bromobenzoyl)-2, 5-diethyl-
(6n). Reagent: 2, 5-diethyl- -carboline-3-ium bromide (7e, 305 mg,
1 mmol), 4-bromobenzoyl chloride (440 mg, 2 mmol). White solid
(30%), mp: 121e122 ꢁC. 1H NMR (
, DMSO-d6): 9.23 (s, 1H), 8.50 (d,
g-carboline-3-ium bromide
g
d
5.1.1.8. 8-(2-Chlorobenzoyl)-5-ethyl-
-carboline (7c, 196 mg, 1 mmol), 2-chlorobenzoyl chloride (350 mg,
2 mmol). White solid (25%), mp: 98e100 ꢁC. 1H NMR (
, DMSO-d6):
9.47 (s, 1H), 8.72 (s, 1H), 8.55 (d, 1H, J ¼ 4.4 Hz), 7.85 (d, 1H, J ¼ 8.8 Hz),
7.74 (d, 1H, J ¼ 8.8 Hz), 7.63 (m, 2H), 7.40 (t, 1H, J ¼ 7.2 Hz), 7.29 (m, 2H),
4.51 (q, 2H, J ¼ 6.8 Hz), 1.33 (t, 3H, J ¼ 6.8 Hz). ESI-MS: m/z ¼ 335
[Mþ1]þ. Anal. calcd for C20H15ClN2O: C, 71.75; H, 4.52; N, 8.37. Found:
C, 71.47; H, 4.75; N, 8.47.
g
-carboline (6h). Reagent: 5-ethyl-
1H, J ¼ 6.8 Hz), 8.13 (s, 1H), 7.88 (d, 1H, J ¼ 6.8 Hz), 7.63 (d, 1H,
J ¼ 8.8 Hz), 7.54 (d,1H, J ¼ 8.8 Hz), 7.15 (d, 2H, J ¼ 8.4 Hz), 6.89 (d, 2H,
J ¼ 8.4 Hz), 4.55 (q, 2H, J ¼ 6.8 Hz), 4.34 (q, 2H, J ¼ 6.8 Hz), 1.59 (t,
3H, J ¼ 6.8 Hz), 1.29 (t, 3H, J ¼ 6.8 Hz). ESI-MS: m/z ¼ 487 [Mþ1]þ.
Anal. calcd for C22H20Br2N2O: C, 54.12; H, 4.13; N, 5.74. Found: C,
54.26; H, 3.97; N, 5.47.
g
d
5.2. Cytotoxic assay
5.1.1.9. 8-(4-Bromobenzoyl)-5-ethyl-
-carboline (7c, 196 mg, 1 mmol), 4-bromobenzoyl chloride (440 mg,
2 mmol). White solid (18%), mp: 139e141 ꢁC. 1H NMR (
, DMSO-d6):
g
-carboline (6i). Reagent: 5-ethyl-
The tumor cell lines (A549, SGC, HCT116, MCF-7, K562, K562R)
were obtained from Shanghai Institute of Pharmaceutical Industry.
The cytotoxic activity in vitro was measured using the MTT assay
g
d
9.43 (s, 1H), 8.68 (s, 1H), 8.50 (d, 1H, J ¼ 6.0 Hz), 7.91 (d, 1H, J ¼ 8.0 Hz),
7.82 (d, 1H, J ¼ 8.0 Hz), 7.75 (d, 2H, J ¼ 8.0 Hz), 7.68 (m, 3H), 4.48 (q, 2H,
J ¼ 7.2 Hz), 1.30 (t, 3H, J ¼ 7.2 Hz). ESI-MS: m/z ¼ 379 [Mþ1]þ. Anal.
calcd for C20H15BrN2O: C, 63.34; H, 3.99; N, 7.39. Found: C, 63.36; H,
4.04; N, 7.52.
[7]. MTT solution (10.0 mL/well) in RPMI-1640 (Sigma, St. Louis, MO)
was added after cells were treated with drug for 48 h, and cells
were incubated for a further 4 h at 37 ꢁC. The purple formazan
crystals were dissolved in 100.0 mL DMSO. After 5 min, the plates
were read on an automated microplate spectrophotometer (Bio-Tek
Instruments, Winooski, VT) at 570 nm. Assays were performed in
triplicate in three independent experiments. The concentration
required for 50% inhibition of cell viability (IC50) was calculated
using the software ‘Dose-Effect Analysis with Microcomputers’. The
tumor cell line panel consisted of A549, SGC, HCT116, MCF-7, K562,
and K562R. In all of these experiments, three replicate wells were
used to determine each point.
5.1.1.10. 5-Ethyl-8-(2-fluorobenzoyl)-
-carboline (7c, 196 mg, 1 mmol), 2-fluorobenzoyl chloride (318 mg,
2 mmol). White solid (39%), mp: 143e145 ꢁC. 1H NMR (
, DMSO-d6):
g-carboline (6j). Reagent: 5-ethyl-
g
d
9.47 (s, 1H, H-4), 8.75 (s, 1H, H-5), 8.55 (d, 1H, J ¼ 4.4 Hz), 7.94 (d, 1H,
J ¼ 8.8 Hz), 7.82 (d, 1H, J ¼ 8.8 Hz), 7.71 (m, 2H), 7.64 (t, 1H, J ¼ 7.2 Hz),
7.43 (m, 2H), 4.51 (q, 2H, J ¼ 6.8 Hz), 1.34 (t, 3H, J ¼ 6.8 Hz). ESI-MS: m/
z ¼ 319 [Mþ1]þ. Anal. calcd for C20H15FN2O: C, 75.46; H, 4.75; N, 8.80.
Found: C, 75.64; H, 4.84; N, 8.58.
5.3. Microtubule polymerization assay [14]
5.1.1.11. 5-Ethyl-8-(4-nitrobenzoyl)-
(4-aminobenzoyl)- -carboline (6l). Reagent: 5-ethyl-
(7c, 196 mg, 1 mmol), 4-nitrobenzoyl chloride (372 mg, 2 mmol).
Yellow solid (67%), mp: 174e176 ꢁC. 1H NMR (
, DMSO-d6): 9.50 (s,
g
-carboline (6k) and 5-ethyl-8-
In vitro tubulin polymerization assays were conducted with
reagents as described by the manufacturer (Cytoskeleton, Inc.). In
g
g-carboline
brief, compound 6f with series concentrations (3
mM, 0.75 mM,
d
0.1875 M) was incubated with purified bovine tubulin and buffer
m
1H), 8.79 (s, 1H), 8.58 (d, 1H, J ¼ 6.0 Hz), 8.44 (d, 2H, J ¼ 8.4 Hz), 8.03
(m, 3H), 7.91 (d, 1H, J ¼ 8.4 Hz), 7.77 (d, 1H,1, J ¼ 6.0 Hz), 4.56 (q, 2H,
J ¼ 6.8 Hz), 1.38 (t, 3H, J ¼ 6.8 Hz). ESI-MS: m/z ¼ 346 [Mþ1]þ. Anal.
calcd for C20H15N3O3: C, 69.56; H, 4.38; N, 12.17. Found: C, 69.63; H,
4.36; N, 12.26.
containing 20% glycerol, 1 mM GTP, 80 mM PIPES (pH 6.9), 2.0 mM
MgCl2, and 0.5 mM EGTA at 37 ꢁC and the effect of compound 6f on
tubulin polymerization was monitored kinetically using a fluores-
cent plate reader. The IC50 value is defined as the concentration of
product which inhibits the rate of polymerization by 50%.
The nitro compound 6k (345 mg) was dissolved in EtOH (5 mL)
and Raney Ni (74 mg) was added. The reaction mixture was stirred
at room temperature under H2 for 2h. Then, the mixture was
filtered over Celite, and the filtrate was evaporated to dryness. The
residue was purified by silica gel column chromatography (PE:
5.4. Flow cytometry analysis [15]
For flow cytometry analysis of DNA content. A549 cells in
exponential growth were treated with 6f (2
mM) for 48 h. Cells were
EtOAc: EtOH, 3:3:1, V/V/V), yielded 5-ethyl-8-(4-aminobenzoyl)-
g-
washed twice with PBS and fixed in 75% ethanol. The cell pellet was
carboline (6l ) as a yellow solid (69%), mp: 200e202 ꢁC. 1H NMR (
d,
resuspended in 100.0 mL of PBS containing 200.0 mg/mL RNase
DMSO-d6): 9.46 (s, 1H), 8.61 (s, 1H), 8.54 (d, 1H, J ¼ 5.6 Hz), 7.86 (d,
1H, J ¼ 8.4 Hz), 7.81 (d, 1H, J ¼ 8.4 Hz), 7.71 (d, 1H, J ¼ 5.6 Hz), 7.61
(d, 2H, J ¼ 8.0 Hz), 6.66 (d, 2H, J ¼ 8.0 Hz), 6.11 (s, 2H, 40-NH2), 4.54
(q, 2H, J ¼ 7.2 Hz),1.38 (t, 3H, J ¼ 7.2 Hz). ESI-MS: m/z ¼ 316 [Mþ1]þ.
(Amersco, Solon, OH), then incubated at 37 ꢁC for 0.5 h. After
incubation, the cells were stained with 20.0 mL/L propidium iodide
(PI, Sigma, St. Louis, MO) for 15 min. The fluorescence cell was
measured with FACSCalibur (BectoneDickinson, Lincoln Park, NJ).

J. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1343e1347
1347
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Acknowledgments
This study was financially supported by the National Key Tech
Project for Major Creation of New drugs (NO. 2009ZX09501-003)
and the Fundamental Research Funds for the Central Universities
(NO. KYJD09038).
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