Design, synthesis, and quantitative structure-activity relationship of cytotoxic γ-carboline derivatives

Article abstract of DOI:10.1016/j.bmc.2009.03.037

Three series of γ-carboline derivatives were designed and synthesized. All the compounds were tested for their cytotoxic activities in vitro against five human tumor cell lines (A549, SGC, HCT116, MCF-7, K562) and one multi-drug resistant subline (K562R).

Full text of DOI:10.1016/j.bmc.2009.03.037

Bioorganic & Medicinal Chemistry 17 (2009) 3324–3331
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and quantitative structure–activity relationship of cytotoxic
c-carboline derivatives
Jing Chen a, Xiaowu Dong a, Tao Liu a, Jianshu Lou b, Chaoyi Jiang a, Wenhai Huang a, Qiaojun He b,
Bo Yang b, Yongzhou Hu a,
*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China
b 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:
Three series of c-carboline derivatives were designed and synthesized. All the compounds were tested for
Received 17 February 2009
Revised 19 March 2009
Accepted 20 March 2009
Available online 26 March 2009
their cytotoxic activities in vitro against five human tumor cell lines (A549, SGC, HCT116, MCF-7, K562)
and one multi-drug resistant subline (K562R). Most compounds showed moderate to potent cytotoxic
activities against the tested cell lines. Sulfonate 11f exhibited more potent cytotoxic activities against
almost all of the tested cells in comparison with the positive control, taxol, with IC50 values ranging from
0.15 to 4.5 lM. The structure–activity relationships were discussed and a statistically reliable QSAR
Keywords:
model (r2 = 0.936, q2 = 0.581) was established by the CoMFA analysis performed using the cytotoxic data
against K562 cell line as a template.
c
-Carboline derivatives
Cytotoxicity
CoMFA
Ó 2009 Elsevier Ltd. All rights reserved.
3D-QSAR
1. Introduction
potent topoisomerase inhibitory activity.21,22 And the presence of
sulfonamide, sulfonate, or amide group in many potent tubulin
Combretastatin A-4 (CA-4, 1), a cis-stilbene natural product iso-
lated by Pettit and co-workers in 1989 from the bark of the South
African bush willow tree Combretum caffrum,1,2 is one of the well-
known natural tubulin-binding molecules affecting microtubule
dynamics. It displays potent antitumor effect in a variety of pre-
clinical tumor models3–8 as well as substantial antivascular activity
in tumor blood flow while causing no significant blood flow reten-
tion in normal tissues.9–15 However, it does not show in vivo effi-
cacy due to its low aqueous solubility.16 Thus, several prodrugs
were investigated to improve its solubility and pharmacokinetics,
leading to the development of disodium phosphate analog CA-4P
(2) which is currently under phase II and III clinical trials on
advanced cancers based on the vascular shutdown mechanism of
action17 and AVE8062 (3) which is currently under clinical evalua-
tion as tumor vascular targeting agent.18,19 The promising pharma-
cological and clinical profiles of 2 and 3 have drawn a lot of
attention for medicinal chemists to develop a variety of derivatives
or analogues of CA-4 in effort to obtain better compounds.20 Since
the SAR of CA-4 derivatives demonstrated that the great impor-
tance of the 3,4,5-trimethyloxyphenyl group (ring A), most of the
studies were focused on the modification of the linking group
polymerization inhibitors is also a noteworthy point (e.g., 4–7,
Fig. 1).23–30 Based on the above insights, we replaced the B ring
of CA-4 with a
c-carboline moiety, and introduced sulfonamide,
sulfonate, and amide group as the linker between ring A and B.
We expected the resulting compounds would possess improved
and the ring B to get more potent compounds. Besides, c-carboline
skeleton has been identified as a superior scaffold structure with
* Corresponding author. Tel./fax: +86 571 88208460.
E-mail addresses: huyz@zju.edu.cn (Y. Hu).
Figure 1. Structures of CA-4 and selected analogues of CA-4.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.037

J. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3324–3331
3325
Scheme 1. Reagents and conditions: (a) C2H5Br, K2CO3, DMF, rt 12 h; (b) ClSO3H, dry CH3CN, 0 °C to rt 12 h; (c) POCl3, PCl5, 180 oC, 4 h; (d) R2NH2, Et3N, rt 0.5 h.
antitumor activities by inhibition both tubulin polymerization and
topoisomerase. Herein, three series of new CA-4 analogues (10, 11
and 14) were synthesized and tested for their cytotoxic activities
in vitro against six human tumor cell lines. In addition, a CoMFA
analysis was performed using the cytotoxic data against K562 cell
line as a template to probe the QSAR of these compounds.
2.2. Biological activity and SAR
The synthesized -carboline derivatives 10a–k, 11a–i and 14a–
h were tested for their cytotoxic activities in vitro against several
human cancer cell lines including human non-small lung cancer
cells A549, human gastric adenocarcinoma SGC, human colon can-
cer cell HCT116, human breast carcinoma cell MCF-7, human mye-
loid 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.
c
2. Results and discussion
2.1. Chemistry
By a scrutiny of the MTT assay results, it showed that almost all of
the sulfonates showed moderate to potent cytotoxic activities
against the tested cells. Compound 11f, which bore 3,4,5-trimethyl-
oxyphenyl group as ring A, was the most promising one among the
tested compounds. It exhibited more potent cytotoxic activities
against almost all of the tested cells compared with taxol, with IC50
The synthetic routes of
c-carboline sulfonamides 10a–k were
c-Carboline (8a) was prepared using
summarized in Scheme 1.
our recently developed synthetic protocol.31 Treatment of 8a with
C2H5Br and K2CO3 in dimethylformamide (DMF) at room tempera-
ture gave compound 8b.32 The
c-carboline-6-sulfonyl chlorides 9
values ranging from 0.15 to 4.5 lM. Decreasing the number of meth-
were prepared from 8 following the method described by Mitsu-
mori et al.33 Then reaction of 9 and various amines in the presence
of triethylamine (TEA) in DMF at room temperature afforded the
target compounds 10a–i. In addition, compound 10h could be fur-
ther transformed into 10j or 10k by N-alkylation with different
amount of C2H5Br (Scheme 1).
On the other hand, treatment of c-carboline-6-sulfonyl chloride
(9a) with various phenols in the presence of pyridine and dimeth-
ylamino pyridine (DMAP) in dry CH2Cl2 at room temperature pro-
oxy group on ring A of compound 11f led to compound 11d or 11e
with lower cytotoxicities. Replacing the 3,4,5-trimethoxy group on
phenyl ring with other substituents, such as chlorine atom (11b,
c), nitro group (11g), amino group (11h) or introducing naphthaline
(11i) instead of the substituted phenyl also reduced the activities.
Moreover, removing all of the substituents on ring A (11a) dimin-
ishedthecytotoxicitiesdramatically. Thus, itsuggestedthe3,4,5-tri-
methoxyphenyl group was essential for cytotoxicity in the sulfonate
series. This was in agreement with the results reported in other liter-
atures about CA-4 analogues.36 But the above results were not appli-
cable to the sulfonamide or amide series. It was found that the
sulfonamides 10h, 10i and the amide 14c, also with 3,4,5-trime-
thoxyphenyl as ring A, did not exhibited prominent activities. Intro-
ducing halogen atom instead of the 3,4,5-trimethoxy (10c, 14g),
decreasing the number of methoxy group (10b) or replacing the
substituted phenyl with other aromatic heterocycles (10g) would
retain or even improve the activities. Further more, the cytotoxicites
vided
-Carboline amides 14a–h were synthesized as shown in
Scheme 3. -Carboline (8a) was nitrified with concd HNO3–
H2SO4 to provide 6-nitro-
-carboline (12a),34 which was
c-carboline sulfonates 11a–i (Scheme 2).
c
c
c
alkylated with C2H5Br to give nitro compound 12b. Reduction
of compound 12b using Raney Ni in EtOH yielded the amino
compound 13.35 Condensation of compound 13 with correspond-
ing acyl chlorides in i-C3H7OH under reflux afforded the target
compounds 14a–h.
Scheme 2. Reagents and conditions: (e) R2OH, Pyridine, DMAP, dry CH2Cl2, rt 12 h.

3326
J. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3324–3331
Scheme 3. Reagents and conditions: (f) concd HNO3–H2SO4, rt 4 h (75%); (g) C2H5Br, K2CO3, DMF, rt 12 h (63%); (h) Raney Ni, H2, EtOH, rt 2 h; (i) R2COCl, i-C3H7OH, reflux 2 h.
spectrum of these two series were not as wide as that of the sulfo-
nates. The sulfonamides were more sensitive to K562, HCT116 and
MCF-7 cell lines, while the amides were more potent against K562,
K562R, MCF-7 and SGC cell lines.
In addition, we also evaluated the effect of substituent at differ-
ent nitrogen atom (N-3, N-9 and linker) of sulfonamides. The data
in Table 1 showed that the ethylated products (10b, i) at the N-9
position of 10a and 10h displayed a drastic loss of activities against
A549 and K562R cell lines. But it would not affect or even a little
improved the cytotoxicities against the other cell lines. However,
the N-ethylation product (10j) of compound 10i, with a second
ethyl substituted at the nitrogen atom of the linker, promoted
Table 1
In vitro cytotoxic activities of the synthesized compounds and taxol against six human cancer cell lines
Compound
R1
R2
Cytotoxicity (IC50
HCT116
,
l
M)a,b
K562
K562R
MCF-7
A549
SGC
Sulfonamides
10a
10b
10c
10d
H
C2H5
H
Ph
Ph
21.03
20.74
16.43
33.87
92.09
>100
NT
35.16
13.74
NT
60.12
0.88
NT
78.39
>100
NTc
>100
8.00
NT
4-Br-Ph
4-OMe-Ph
H
NT
NT
NT
NT
NT
10e
10f
C2H5
H
>100
>100
>100
>100
>100
>100
N
12.71
18.46
30.82
23.74
22.81
95.37
N
N
10g
C2H5
12.16
16.84
23.24
4.99
73.45
73.21
CH3
10h
10i
10j
H
3,4,5-OMe-Ph
3,4,5-OMe-Ph
3,4,5-OMe-Ph
3,4,5-OMe-Ph
11.32
6.09
1.02
73.41
>100
3.02
16.91
32.32
10.20
>100
19.16
5.80
2.07
48.16
>100
33.88
>100
>100
>100
13.80
>100
C2H5
C2H5
C2H5
10k
>100
>100
>100
Sulfonates
11a
11b
11c
11d
11e
11f
11g
11h
H
H
H
H
H
H
H
H
Ph
4-Cl-Ph
3-Cl-Ph
2-OMe-Ph
4-OMe-Ph
3,4,5-OMe-Ph
4-NO2-Ph
4-NH2-Ph
77.39
20.46
13.21
14.11
6.60
4.50
27.86
18.45
48.90
13.88
8.42
15.27
5.70
2.62
8.34
5.95
45.23
32.02
42.73
64.14
23.87
0.77
38.60
26.34
47.52
24.72
16.00
0.86
>100
47.30
43.62
31.98
22.55
NT
>100
39.72
5.82
54.26
21.36
0.15
21.01
20.89
10.75
16.56
17.49
10.14
7.15
2.12
11i
H
25.91
10.68
39.37
14.32
33.36
23.26
Amides
14a
14b
14c
14d
C2H5
C2H5
C2H5
C2H5
Ph
26.22
11.32
13.57
12.74
10.62
5.15
NT
>100
50.73
NT
11.83
4.92
NT
>100
72.02
15.88
>100
26.54
21.34
16.13
19.16
4-OMe-Ph
3,4,5-OMe-Ph
2-OH-Ph
22.94
>100
20.85
14e
C2H5
14.51
40.59
87.24
18.11
73.49
50.89
N
14f
14g
14h
Taxol
C2H5
C2H5
C2H5
4-SO2CH3-Ph
2-Cl-Ph
4-Br-Ph
22.90
8.58
13.24
1.16
96.58
22.93
15.58
5.63
>100
2.63
>100
4.37
22.80
6.52
11.87
2.11
>100
37.33
>100
2.46
36.04
20.78
71.24
3.34
a
IC50, compound concentration required to inhibit tumor cell proliferation by 50%.
Values are means of three experiments.
NT, not tested.
b
c

J. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3324–3331
3327
the cytotoxicities against all of the tested cell lines. This result
implied that the introduction of ethyl at the nitrogen atom of the
linker was beneficial. But further introduction of ethyl at the N-3
position of c-carboline afforded the quaternary N-ethylpyridinium
salt 10k with no activity.
Table 2
Predicted activities from CoMFA models compared with the experimental activities
and the residues
Compound
IC50
(l
M)
pIC50
CoMFA
Pred. pIC50
Figure 2. CoMFA calculated versus actual pIC50 values.
Res.
10a
10ba
10c
10d
10f
21.03
20.74
16.43
33.87
12.71
12.16
11.32
6.09
4.677
4.683
4.784
4.47
4.896
4.915
4.946
5.215
5.991
4.111
4.689
4.879
4.85
4.545
4.865
4.688
4.468
4.876
5.182
5.003
5.305
5.957
4.372
4.519
4.893
4.754
5.223
5.379
4.52
4.569
4.536
4.78
4.909
4.898
4.842
4.804
4.887
5.037
4.807
À0.132
0.182
À0.096
À0.002
À0.02
0.267
2.3. CoMFA analysis
3D-QSAR methods, especially CoMFA, are widely used in drug
design because they allow rapid generation of QSAR models, from
which biological activity of newly designed molecules can be pre-
dicted.37–39 In this study, 26 compounds with available IC50 were
employed for the CoMFA analysis. For 3D-QSAR analyses, 21 com-
pounds (unasterisked molecules in Table 2) were selected as the
training set for model construction, and the remaining 5 com-
pounds (asterisked molecules in Table 2) as the testing set for
model validation. The pIC50 values (pIC50 = ÀlogIC50) of these com-
pounds were used as dependent variables. The CoMFA results are
summarized in Table 3.
As shown in Table 3, a CoMFA model was developed with con-
ventional correlation coefficient r2 = 0.936, cross-validated coeffi-
cient q2 = 0.581, the estimated F = 43.96, and standard error of
0.107. These statistical indexes were reasonably high, indicating
that this new CoMFA model had a strong predictive ability. The
data in Table 3 also showed that the contributions of steric and
electrostatic field were 71.9% and 28.1%, respectively. The plots
of the experimental results versus calculated values of the 21 stud-
ied compounds are shown in Figure 2. To evaluate the predictive
ability of this model, we subsequently calculated the pIC50 values
of 5 compounds in the testing set. As it can be seen in Table 3
and Figure 2, the theoretical results of 5 compounds from the test-
ing set were in good agreement with the experimental values, sug-
gesting that the new CoMFA model was reliable.
10ga
10h
10i
0.057
0.09
10j
1.02
À0.034
11a
11ba
11c
11d
11e
11f
11g
11ha
11i
14a
14b
14c
14d
14e
14fa
14g
14h
77.39
20.46
13.21
14.11
6.60
0.261
À0.17
0.014
À0.096
5.18
0.043
4.50
5.347
4.555
4.734
4.587
4.581
4.946
4.867
4.895
4.838
4.64
0.032
27.86
18.45
25.91
26.22
11.32
13.57
12.74
14.51
22.90
8.58
À0.035
À0.165
À0.051
0.199
À0.037
0.031
À0.053
À0.034
0.247
5.067
4.878
À0.03
13.24
À0.071
a
Compounds of the testing set.
Table 3
Summary of CoMFA analysis
CoMFA model
Result
The steric contour plot is shown in Figure 3A. It was found that a
R2 cross-validated (q2)
Number of components
Non cross-validated r2
Standard error of estimate
F
0.581
5
huge yellow colored contour was near the A ring of the c-carboline
derivatives, suggesting that bulky group in this area would
decrease the cytotoxicity. The green region indicated that the
bulky substitute near the nitrogen atom of the linker was favorable
to the activity. CoMFA electrostatic contour map is shown in Figure
3B. There was a big blue contour near the ethyl substituent of the
sulfonamide linker, indicating that more positive charges in this
0.936
0.107
43.96
71.9%
28.1%
Steric contribution
Electrostatic contribution
Figure 3. Steric and electrostatic CoMFA maps of the compound 10j showing contributions to the inhibitory activities on the K562 cell line. (A) The favorable steric areas with
more bulk are indicated by green isopleths, whereas the disfavorable steric areas are shown by yellow isopleths. (B) The favorable electrostatic areas with positive charges are
indicated by blue isopleths, whereas the favorable electrostatic areas with negative charges are show by red isoplet.

3328
J. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3324–3331
region would result in enhanced activity. There was a red contour
near the C-3 and C-4 of the phenyl ring, suggesting the negatively
charged substituents in this area lead to an increase of cytotoxicity.
This model could well explain the potent activity of compound 10j.
The ethyl group at the nitrogen atom of the sulfonamide linker was
in the green and blue region. Introduction of this group changed
the location of the 3,4,5-trimethoxyphenyl, and made it shift away
from the yellow area. Further more, the oxygen atom of the 3-
methoxy group was in the red area. Thus, the pIC50 value of 10j
reached 5.991.
1H NMR (d, DMSO-d6): 10.26 (br s, 1H), 9.49 (s, 1H), 8.76 (d, 1H,
J = 1.6 Hz), 8.56 (d, 1H, J = 5.6 Hz), 7.91(dd, 1H, J = 8.8, 1.6 Hz),
7.87 (d, 1H, J = 8.8 Hz), 7.72 (d, 1H, J = 5.6 Hz), 7.21 (m, 4H), 6.97
(t, 1H, J = 7.2 Hz), 4.50 (q, 2H, J = 7.2 Hz), 1.32 (t, 3H, J = 7.2 Hz).
ESI-MS: m/z = 352 [M+1]+. Anal. Calcd for C19H17N3O2S: C, 64.94;
H, 4.88; N, 11.96. Found: C, 64.74; H, 4.89; N, 12.01.
4.1.1.3. N-(4-Bromophenyl)-
c-carboline-6-sulfonamide (10c).
Reagent: -carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol),
c
4-bromoaniline (175 mg, 1 mmol). White solid (35%), mp:
>250 °C. 1H NMR (d, DMSO-d6): 12.25 (br s, 1H), 10.41 (br s,
1H), 9.48 (s, 1H), 8.75 (s, 1H), 8.50 (d, 1H, J = 6.0 Hz), 7.84(d, 1H,
J = 8.4), 7.70 (d, 1H, J = 8.4 Hz), 7.56 (d, 1H, J = 6.0 Hz), 7.39 (d,
2H, J = 8.8 Hz), 7.09 (d, 2H, J = 8.8 Hz). ESI-MS: m/z = 402 [M+1]+.
Anal. Calcd for C17H12BrN3O2S: C, 50.76; H, 3.01; N, 10.45. Found:
C, 50.45; H, 2.91; N, 10.24.
3. Conclusion
Three series of
c-carboline derivatives were synthesized and
tested for their cytotoxic activities in vitro against six human
tumor cell lines. The results demonstrated that most compounds
showed moderate to potent cytotoxic activities against all the
tested cell lines and sulfonate 11f exhibited more potent cytotoxic
activities in comparison with taxol, suggesting it might be the
promising lead compound for future investigation. 3D-QSAR anal-
ysis using CoMFA was performed to explore comprehensive struc-
ture–activity relationships and a statistically reliable model with
good predictive power (r2 = 0.936, q2 = 0.581) was established on
the basis of the common substructure-based alignment. According
to the CoMFA contours, further design, synthesis, and biological
evaluation are ongoing in our laboratory and the results will be
reported in due course.
4.1.1.4. N-(4-Methoxyphenyl)-c-carboline-6-sulfonamide (10d).
Reagent: -carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol), 4-
c
methoxyaniline (125 mg, 1 mmol). White solid (57%), mp: >250 °C.
1H NMR (d, DMSO-d6): 12.67 (br s, 1H), 9.91 (br s, 1H), 9.61 (s,
1H), 8.73 (d, 1H, J = 1.6 Hz), 8.57 (d, 1H, J = 6.4 Hz), 7.86 (dd, 1H,
J = 8.4, 1.6 Hz), 7.77 (d, 1H, J = 8.4 Hz), 7.72 (d, 1H, J = 6.4 Hz), 7.00
(d, 2H, J = 8.8 Hz), 6.77 (d, 2H, J = 8.8 Hz), 3.61 (s, 3H). ESI-MS: m/
z = 354 [M+1]+. Anal. Calcd for C18H15N3O3S: C, 61.18; H, 4.28; N,
11.89. Found: C, 61.31; H, 4.26; N, 11.84.
4.1.1.5.
9-Ethyl-N-(pyridine-4-yl)-c-carboline-6-sulfonamide
(10e). Reagent: 9-ethyl-
c
-carboline-6-sulfonyl chloride (9b)
4. Experimental
(295 mg, 1 mmol), 4-aminopyridine (94 mg, 1 mmol). White solid
(51%), mp: >250 °C. 1H NMR (d, DMSO-d6): 9.67 (s, 1H), 8.68 (s,
1H), 8.64 (d, 1H, J = 6.0 Hz), 8.11(d, 1H, J = 6.0 Hz), 8.00 (d, 2H),
7.95 (dd, 1H, J = 8.4, 0.8 Hz), 7.79 (d, 1H, J = 8.4 Hz), 6.79 (d, 2H,
J = 6.0 Hz), 4.58 (q, 2H, J = 7.2 Hz), 1.37 (t, 3H, J = 7.2 Hz). ESI-MS:
m/z = 353 [M+1]+. Anal. Calcd for C18H16N4O2S: C, 61.35; H, 4.58;
N, 15.90. Found: C, 61.63; H, 4.36; N, 16.11.
Melting points were obtained on a B-540 Büchi melting-point
apparatus and are uncorrected. 1H NMR spectra was recorded on
a Brüker AM 400 instrument at 400 MHz (chemical shifts are
expressed as d values relative to TMS as internal standard). ESI
(positive) was recorded on an Esquire-LC-00075 spectrometer. Ele-
ment analyses were performed on an Eager 300 instrument. All of
the CoMFA calculations were performed on a SGI O2 workstation
using the SYBYL 6.91 program.
4.1.1.6. N- (Quinolin-5-yl)-
c-carboline-6-sulfonamide(10f). Reagent:
c-carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol), 5-amino-quino-
line (144 mg, 1 mmol). White solid (80%), mp: >250 °C. 1H NMR (d,
DMSO-d6): 12.16 (br s, 1H), 9.84 (br s, 1H), 9.46 (s, 1H), 8.94 (d, 1H,
J = 2.0 Hz), 8.85 (dd, 1H, J = 4.4, 1.6 Hz), 8.47 (d, 1H, J = 5.6 Hz), 8.31 (dd,
1H, J = 8.0, 1.6 Hz), 8.00 (dd, 1H, J = 8.8, 2.0 Hz), 7.77 (d, 1H, J = 8.0 Hz),
7.59 (d, 2H, J = 8.8 Hz), 7.55 (dd, 1H, J = 8.0, 4.4 Hz), 7.50 (m, 2H). ESI-
MS: m/z = 375 [M+1]+. Anal. Calcd for C20H14N4O2S: C, 64.16; H, 3.77; N,
14.96. Found: C, 64.42; H, 3.86; N, 15.14.
4.1. Synthesis
4.1.1. General procedure for synthesis of
sulfonamides 10a–i
c-carboline
A
mixture of
amines (1 mmol) in DMF (5 mL) was stirred for 5 min at room tem-
perature, TEA (210 L, 1.5 mmol) was added. Then, the mixture
c-carboline-6-sulfonyl chloride 9 (1 mmol),
l
was stirred for an additional 0.5 h. After adding ice water
(10 mL), the mixture was extracted with EtOAc (3 Â 20 mL). The
organic phase was washed with brine (2 Â 20 mL), dried over
anhydrous Na2SO4, and concentrated under vacuum. The residue
was purified over silica column chromatography using petroleum
ether (PE): EtOAc: EtOH (5:5:1, V/V/V) as eluent to afford 10a–i.
4.1.1.7. 9-Ethyl-N-(1-methyl-indol-5-yl)-
c-carboline-6-sulfon-
amide (10g). Reagent: 9-ethyl- -carboline-6-sulfonyl chloride
c
(9b) (295 mg, 1 mmol), 1-methyl-5-amino-indole (prepared as
described in Ref. 40) (146 mg, 1 mmol). White solid (48%), mp:
>250 °C. 1H NMR (d, DMSO-d6): 9.81 (br s, 1H), 9.41 (s, 1H), 8.64
(s, 1H), 8.52 (d, 1H, J = 4.8 Hz), 7.83 (m, 2H), 7.68 (d, 1H,
J = 8.8 Hz), 7.25 (s, 1H), 7.21 (m, 2H), 6.90 (d, 1H, J = 8.0 Hz), 6.26
(d, 1H, J = 2.4 Hz), 4.44 (q, 2H, J = 7.2 Hz), 3.64 (s, 3H), 1.29 (t, 3H,
J = 7.2 Hz). ESI-MS: m/z = 405 [M+1]+. Anal. Calcd for C22H20N4O2S:
C, 65.33; H, 4.98; N, 13.85. Found: C, 65.26; H, 4.74; N, 13.79.
4.1.1.1. N-Phenyl-
carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol), aniline
(92
L, 1 mmol). White solid (54%), mp: >250 °C. 1H NMR (d,
c-carboline-6-sulfonamide (10a). Reagent: c-
l
DMSO-d6): 12.40 (br s, 1H), 9.42 (s, 1H), 8.69 (d, 1H, J = 2.0 Hz),
8.47 (d, 1H, J = 5.6 Hz), 7.85(dd, 1H, J = 8.8, 2.0 Hz), 7.66 (d, 1H,
J = 8.8 Hz), 7.52 (d, 1H, J = 5.6 Hz), 7.14 (m, 4H), 6.87 (t, 1H,
J = 6.8). ESI-MS: m/z = 324 [M+1]+. Anal. Calcd for C17H13N3O2S: C,
63.14; H, 4.05; N, 12.99. Found: C, 63.35; H, 4.21; N, 12.73.
4.1.1.8. N-(3,4,5-Trimethoxyphenyl)-
c-carboline-6-sulfonamide
(10h). Reagent: -carboline-6-sulfonyl chloride (9a) (267 mg,
c
1 mmol), 3,4,5-trimethoxyaniline (185 mg, 1 mmol). White solid
(52%), mp: >250 °C. 1H NMR (d, DMSO-d6): 12.20 (br s, 1H), 10.06
(br s, 1H), 9.49 (s, 1H), 8.77 (d, 1H, J = 1.6 Hz), 8.50 (d, 1H,
J = 5.6 Hz), 7.88 (dd, 1H, J = 8.8, 1.6 Hz), 7.71 (d, 1H, J = 8.8 Hz),
7.55 (d, 1H, J = 5.6 Hz), 6.43 (s, 2H), 3.62 (s, 6H), 3.50 (s, 3H).
4.1.1.2. 9-Ethyl-N-phenyl-
Reagent: 9-ethyl- -carboline-6-sulfonyl chloride (9b) (295 mg,
1 mmol), aniline (92 L, 1 mmol). White solid (65%), mp: >250 °C.
c-carboline-6-sulfonamide (10b).
c
l

J. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3324–3331
3329
ESI-MS: m/z = 414 [M+1]+. Anal. Calcd for C20H19N3O5S: C, 58.10; H,
4.63; N, 10.16. Found: C, 58.14; H, 4.58; N, 10.25.
4.1.3.2. 4-Chlorophenyl
c
-carboline-6-sulfonate (11b). Reagent:
c
-carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol), 4-chloro-
phenol (129 mg, 1 mmol). White solid (45%), mp: 222–224 °C. 1H
NMR (d, DMSO-d6): 9.53 (s, 1H), 8.86 (d, 1H, J = 2.0 Hz), 8.51 (d,
1H, J = 6.4 Hz), 7.84 (d, 1H, J = 8.0 Hz), 7.77 (d, 1H, J = 8.0 Hz), 7.58
(d, 1H, J = 6.4 Hz), 7.41 (d, 2H, J = 8.8 Hz), 7.04 (d, 2H, J = 8.8 Hz).
ESI-MS: m/z = 360 [M+1]+. Anal. Calcd for C17H11ClN2O3S: C, 56.91;
H, 3.09; N, 7.81. Found: C, 57.11; H, 3.28; N, 7.68.
4.1.1.9. 9-Ethyl-N-(3,4,5-trimethoxyphenyl)-
c-carboline-6-sul-
fonamide (10i). Reagent: 9-ethyl- -carboline-6-sulfonyl chloride
c
(9b) (295 mg, 1 mmol), 3,4,5-trimethoxyaniline (185 mg, 1 mmol).
White solid (77%), mp: >250 °C. 1H NMR (d, DMSO-d6): 10.09 (br s,
1H), 9.51 (s, 1H), 8.80 (d, 1H, J = 1.6 Hz), 8.57 (d, 1H, J = 5.6 Hz),
7.94 (dd, 1H, J = 8.8, 1.6 Hz), 7.90 (d, 1H, J = 8.8 Hz), 7.73 (d, 1H,
J = 5.6 Hz), 6.43 (s, 2H), 4.52 (q, 2H, J = 7.2 Hz), 3.62 (s, 6H), 3.30
(s, 3H), 1.33 (t, 3H, J = 7.2 Hz). ESI-MS: m/z = 442 [M+1]+. Anal.
Calcd for C22H23N3O5S: C, 59.85; H, 5.25; N, 9.52. Found: C,
59.67; H, 5.44; N, 9.45.
4.1.3.3. 3-Chlorophenyl
c-carboline-6-sulfonate (11c). Reagent:
c-carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol), 3-chloro-
phenol (129 mg, 1 mmol). White solid (45%), mp: 213–215 °C. 1H
NMR (d, DMSO-d6): 12.85 (br s, 1H) 9.56 (s, 1H), 8.92 (d, 1H,
J = 1.6 Hz), 8.53 (d, 1H, J = 5.6 Hz), 7.90 (dd, 1H, J = 8.4, 1.6 Hz),
7.81 (d, 1H, J = 8.4 Hz), 7.60 (d, 1H, J = 5.6 Hz), 7.37 (m, 2H), 7.20
(s, 1H), 6.96 (d, 1H, J = 6.8 Hz). ESI-MS: m/z = 360 [M+1]+. Anal.
Calcd for C17H11ClN2O3S: C, 56.91; H, 3.09; N, 7.81. Found: C,
56.86; H, 2.86; N, 7.96.
4.1.2. N,9-Diethyl-N-(3,4,5-trimethoxyphenyl)-
sulfonamide (10j) and 3,9-diethyl-6-(N-ethyl-N-(3,4,5-trimetho-
xyphenyl)sulfamoyl)- -carboline-3-ium bromide (10k)
c-carboline-6-
c
To a solution of 10h (41 mg, 0.1 mmol) in DMF (0.5 mL) was
added K2CO3 (55 mg, 0.4 mmol) and the reaction mixture was stir-
red for 30 min at ambient temperature. Then C2H5Br (15
lL,
4.1.3.4. 2-Methoxyphenyl
c-carboline-6-sulfonate (11d). Rea-
0.2 mmol) was added into the reaction mixture and stirred for
4 h. After that, the mixture was concentrated under reduced pres-
sure. Then water (10 mL) added into the residue and extracted
with EtOAc (3 Â 10 mL). The organic phase was washed with brine
(2 Â 10 mL), dried over anhydrous Na2SO4 and concentrated under
vacuum. The product was purified by silica column chromatogra-
phy using PE:EtOAc:EtOH (5:5:1, V/V/V) as eluent to afford 10j.
White solid (34%), mp: 172–174 °C. 1H NMR (d, DMSO-d6): 9.54
(s, 1H), 8.70 (s, 1H), 8.58 (d, 1H, J = 5.6 Hz), 7.92 (d, 1H,
J = 8.4 Hz), 7.76 (d, 1H, J = 5.6 Hz), 7.74 (dd, 1H, J = 8.4, 1.6 Hz),
6.24 (s, 2H), 4.54 (q, 2H, J = 6.8 Hz), 3.64 (s, 3H), 3.60 (q, 2H,
J = 6.8 Hz), 3.54 (s, 6H), 1.35 (t, 3H, J = 6.8 Hz), 1.02 (t, 3H,
J = 6.8 Hz). ESI-MS: m/z = 471 [M+1]+. Anal. Calcd for C24H27N3O5S:
C, 61.39; H, 5.80; N, 8.95. Found: C, 61.35; H, 5.85; N, 9.13.
When the amount of C2H5Br was up to 0.4 mmol, compound
10k was afforded using a procedure similar to compound 10j.
White solid (41%), mp: 143–145 °C. 1H NMR (d, DMSO-d6): 10.21
(s, 1H), 9.02 (d, 1H, J = 6.8 Hz), 8.94 (d, 1H, J = 8.0 Hz), 8.47 (d,
1H, J = 6.8), 8.20 (d, 1H, J = 8.8 Hz), 7.98 (t, 1H, J = 8.8 Hz), 6.28 (s,
2H), 4.74 (m, 6H), 3.63 (s, 3H), 3.58 (s, 6H), 1.64 (t, 3H, J = 7.2 Hz)
1.42 (t, 3H, J = 7.2 Hz), 1.08 (t, 3H, J = 7.2 Hz). ESI-MS: m/z = 580
[M+1]+. Anal. Calcd for C26H32BrN3O5S: C, 53.98; H, 5.58; N, 7.26.
Found: C, 54.02; H, 5.35; N, 7.36.
gent: -carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol),
c
2-methoxyphenol (124 mg, 1 mmol). White solid (28%), mp:
229–231 °C. 1H NMR (d, DMSO-d6): 9.54 (s, 1H), 8.83 (s, 1H),
8.52 (d, 1H, J = 6.0 Hz), 7.85 (d, 1H, J = 8.8 Hz), 7.76 (d, 1H,
J = 8.8 Hz), 7.59 (d, 1H, J = 6.0 Hz), 7.23 (t, 1H, J = 8.0 Hz), 7.06 (d,
1H, J = 8.0 Hz), 6.99 (d, 1H, J = 8.0 Hz), 6.91 (t, 1H, J = 8.0 Hz),
3.39 (s, 3H). ESI-MS: m/z = 355 [M+1]+. Anal. Calcd for
C18H14N2O4S: C, 61.01; H, 3.98; N, 7.90. Found: C, 61.23; H,
4.12; N, 7.78.
4.1.3.5. 4-Methoxyphenyl
c-carboline-6-sulfonate (11e). Rea-
gent: -carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol),
c
4-methoxyphenol (124 mg, 1 mmol). White solid (51%), mp:230–
232 °C. 1H NMR (d, DMSO-d6): 12.57 (br s, 1H), 9.56 (s, 1H), 8.87
(s, 1H), 8.54 (d, 1H, J = 6.0 Hz), 7.86 (d, 1H, J = 8.0 Hz), 7.78 (d,
1H, J = 8.0 Hz), 7.59 (d, 1H, J = 6.0 Hz), 6.91 (d, 2H, J = 8.8 Hz),
6.85 (d, 2H, J = 8.8 Hz), 3.67 (s, 3H). ESI-MS: m/z = 355 [M+1]+. Anal.
Calcd for C18H14N2O4S: C, 61.01; H, 3.98; N, 7.90. Found: C, 61.14;
H, 3.90; N, 7.78.
4.1.3.6. 3,4,5-Trimethoxyphenyl
c-carboline-6-sulfonate (11f).
Reagent: -carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol),
c
3,4,5-trimethoxyphenol (180 mg, 1 mmol). White solid (43%),
mp: >250 °C. 1H NMR (d, DMSO-d6): 9.58 (s, 1H), 8.90 (s, 1H),
8.52 (d, 1H, J = 5.2 Hz), 7.93 (d, 1H, J = 8.8 Hz), 7.82 (d, 1H,
J = 8.8 Hz), 7.59 (d, 1H, J = 5.2 Hz), 6.28 (s, 2H), 3.56 (s, 3H), 3.53
(s, 6H). ESI-MS: m/z = 415 [M+1]+. Anal. Calcd for C20H18N2O6S: C,
57.96; H, 4.38; N, 6.76. Found: C, 58.08; H, 4.74; N, 6.87.
4.1.3. General procedure for synthesis of
11a–i
c-carboline sulfonates
Pyridine (0.32 mL, 4 mmol) and DMAP (45 mg, 0.36 mmol)
were added to a mixture of -carboline-6-sulfonyl chloride 9a
c
(1 mmol) and phenols (1 mmol) in dry CH2Cl2 (5 mL). And then,
the mixture was stirred for an additional 12 h at room tempera-
ture. After that, the mixture was concentrated under vacuum.
Water (10 mL) was added into the residue and extracted with
EtOAc (3 Â 20 mL). The organic phase was washed with brine
(2 Â 20 mL), dried over anhydrous Na2SO4, and concentrated
under vacuum. The residue was purified over silica column chro-
matography using PE:EtOAc:EtOH (10:10:1 to 4:4:1, V/V/V) as
eluent to afford 11a–i.
4.1.3.7. 4-Nitrophenyl
c-carboline-6-sulfonate (11g). Reagent:
c
-carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol), 4-nitro-
phenol (109 mg, 1 mmol). Yellow solid (38%), mp:198–200 °C. 1H
NMR (d, DMSO-d6): 12.84 (br s, 1H), 9.57 (s, 1H), 8.96 (s, 1H), 8.54
(d, 1H, J = 5.6 Hz), 8.24 (d, 2H, J = 9.2 Hz), 7.93 (d, 1H, J = 8.4 Hz),
7.81 (d, 1H, J = 8.4 Hz), 7.60 (d, 1H, J = 5.6 Hz), 7.33 (d, 2H,
J = 9.2 Hz). ESI-MS: m/z = 370 [M+1]+. Anal. Calcd for C17H11N3O5S:
C, 55.28; H, 3.00; N, 11.38. Found: C, 55.35; H, 2.97; N, 11.47.
The nitro compound 11g (148 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 2 h. Then, the mixture was fil-
tered over Celite, and the filtrate was evaporated to dryness. The
residue was purified by silica gel column chromatography (PE:E-
tOAc:EtOH, 3:3:1, V/V/V), yielded pure compound 4-aminophenyl
4.1.3.1. Phenyl c-carboline-6-sulfonate (11a). Reagent: c-carbo-
line-6-sulfonyl chloride (9a) (267 mg, 1 mmol), phenol (94 mg,
1 mmol). White solid (28%), mp: >250 °C. 1H NMR (d, DMSO-d6):
9.54 (s, 1H), 8.87 (s, 1H), 8.52 (d, 1H, J = 5.6 Hz), 7.87(d, 1H,
J = 8.4 Hz), 7.78 (d, 1H, J = 8.4 Hz), 7.59 (d, 1H, J = 5.6 Hz), 7.35 (t,
2H, J = 7.2 Hz), 7.28 (t, 1H, J = 7.2 Hz), 7.01 (d, 2H, J = 7.2 Hz). ESI-
MS: m/z = 325 [M+1]+. Anal. Calcd for C17H12N2O3S: C, 62.95; H,
3.73; N, 8.64. Found: C, 62.76; H, 3.75; N, 8.35.
c
-carboline-6-sulfonate (11h) as a white solid (98%), mp:
>250 °C. 1H NMR (d, DMSO-d6): 12.69 (br s, 1H), 9.55 (s, 1H),
8.82 (s, 1H), 8.53 (d, 1H, J = 5.6 Hz), 7.82 (d, 1H, J = 8.0 Hz), 7.77

3330
J. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3324–3331
(d, 1H, J = 8.0 Hz), 7.59 (d, 1H, J = 5.6 Hz), 6.59 (d, 2H, J = 8.4 Hz),
6.39 (d, 2H, J = 8.4 Hz), 5.19 (s, 2H). ESI-MS: m/z = 340 [M+1]+. Anal.
Calcd for C17H13N3O3S: C, 60.17; H, 3.86; N, 12.38. Found: C, 60.14;
H, 4.02; N, 12.34.
4.1.4.5. 9-Ethyl-6-nicotinamido-
c-carboline (14e). Reagent: 6-
amino-9-ethyl- -carboline (13) (211 mg, 1 mmol), nicotinoyl chlo-
c
ride (142 mg, 1 mmol). White solid (21%), mp: 135–137 °C. 1H
NMR (d, DMSO-d6): 10.71 (br s, 1H), 9.30 (s, 1H), 9.20 (s, 1H), 8.78
(d, 1H, J = 4.0 Hz), 8.69 (s, 1H), 8.49 (d, 1H, J = 5.6 Hz), 8.42 (d, 1H,
J = 7.2 Hz), 7.88 (d, 1H, J = 8.8 Hz), 7.73 (d, 1H, J = 8.8 Hz), 7.65 (t,
1H, J = 5.6 Hz), 7.61 (m, 1H), 4.49 (q, 2H, J = 7.2 Hz), 1.35 (t, 3H,
J = 7.2 Hz). ESI-MS: m/z = 317 [M+1]+. Anal. Calcd for C19H16N4O: C,
72.13; H, 5.10; N, 17.71. Found: C, 72.42; H, 5.22; N, 17.57.
4.1.3.8. Naphthalen-1-yl
c-carboline-6-sulfonate (11i). Reagent:
c-carboline-6-sulfonyl chloride (9a) (267 mg, 1 mmol), 1-naphthol
(144 mg, 1 mmol). White solid (27%), mp: >250 °C. 1H NMR (d,
DMSO-d6): 9.53 (s, 1H), 8.96 (d, 1H, J = 1.2 Hz), 8.51 (d, 1H,
J = 5.6 Hz), 7.94 (m, 3H), 7.88 (d, 1H, J = 8.4 Hz), 7.77 (d, 1H,
J = 8.4 Hz),7.58 (d, 1H, J = 5.6 Hz), 7.53 (m, 2H), 7.46 (t, 1H,
J = 8.1 Hz), 7.14 (d, 1H, J = 8.4 Hz). ESI-MS: m/z = 375 [M+1]+. Anal.
Calcd for C21H14N2O3S: C, 67.37; H, 3.77; N, 7.48. Found: C, 67.25;
H, 3.43; N, 7.38.
4.1.4.6. 9-Ethyl-6-(4-methylsulfonyl)-
c-carboline (14f). Reagent:
6-amino-9-ethyl- -carboline (13) (211 mg, 1 mmol),4-(methylsulfo-
c
nyl)benzoyl chloride (219 mg, 1 mmol). White solid (57%), mp:
110–112 °C. 1H NMR (d, DMSO-d6): 8.80 (s, 1H), 8.13 (d, 1H,
J = 6.8 Hz), 7.91 (s, 1H), 7.50 (m, 5H), 7.39 (d, 1H, J = 8.8 Hz), 7.32
(d, 1H, J = 8.8 Hz), 4.06 (q, 2H, J = 7.2 Hz), 3.02 (s, 3H), 1.19 (t, 3H,
J = 7.2 Hz). ESI-MS: m/z = 394 [M+1]+. Anal. Calcd for C21H19N3O3S:
C, 64.10; H, 4.87; N, 10.68. Found: C, 64.13; H, 5.03; N, 10.67
4.1.4. Generalprocedureforsynthesisof
c-carboline amides 14a–h
A mixture of 6-amino-9-ethyl- -carboline (1 mmol) 13, acyl
c
chlorides (1 mmol) was refluxed in i-C3H7OH (5 mL) for 2 h. After
cooling to room temperature, the mixture was concentrated under
vacuum. Then water (10 mL) was added into the residue, neutral-
ized with aq. Na2CO3, and extracted with AcOEt (3 Â 20 mL). The
organic phase was washed with brine (2 Â 20 mL), dried over
anhydrous Na2SO4, and concentrated under vacuum. The residue
was purified over silica column chromatography using PE:EtOAc:E-
tOH (10:10:1 to 3:3:1, V/V/V) as eluent to afford 14a–h.
4.1.4.7. 9-Ethyl-6-(2-chlorobenzamido)-
c-carboline (14g). Rea-
gent: 6-amino-9-ethyl- -carboline (13) (211 mg, 1 mmol), 2-chlo-
c
robenzoyl chloride (175 mg, 1 mmol). White solid (46%), mp:
77–79 °C. 1H NMR (d, DMSO-d6): 10.64 (br s, 1H), 9.30 (s, 1H),
8.69 (s, 1H), 8.48 (d, 1H, J = 4.0 Hz), 7.76 (d, 1H, J = 8.4 Hz), 7.71
(d, 1H, J = 8.4 Hz), 7.64 (d, 1H, J = 6.4 Hz), 7.98 (d, 1H, J = 8.0 Hz),
7.54 (m, 3H), 4.48 (q, 2H, J = 7.2 Hz), 1.34 (t, 3H, J = 7.2 Hz). ESI-
MS: m/z = 351 [M+1]+. Anal. Calcd for C20H16ClN3O: C, 68.67; H,
4.61; N, 12.01. Found: C, 68.36; H, 4.74; N, 12.24.
4.1.4.1. 9-Ethyl-6-benzamido-
c-carboline (14a). Reagent: 6-ami-
no-9-ethyl- -carboline (13) (141 mg, 1 mmol), benzoyl chloride
c
(141 mg, 1 mmol). White solid (72%), mp: 199–201 °C. 1H NMR
(d, DMSO-d6): 10.63 (br s, 1H), 9.81 (s, 1H), 9.01 (d, 1H,
J = 0.8 Hz), 8.73 (d, 1H, J = 6.8 Hz), 8.25 (d, 1H, J = 6.8 Hz), 8.07 (d,
2H, J = 8.0 Hz), 8.02 (m, 2H), 7.65 (m, 1H), 7.59 (m, 2H), 4.69 (q,
2H, J = 7.2 Hz), 1.42 (t, 3H, J = 7.2 Hz). ESI-MS: m/z = 316 [M+1]+.
Anal. Calcd for C20H17N3O: C, 76.17; H, 5.43; N, 13.32. Found: C,
76.24; H, 5.26; N, 13.11.
4.1.4.8. 9-Ethyl-6-(4-bromobenzamido)-
c-carboline (14h). Rea-
gent: 6-amino-9-ethyl- -carboline (13) (211 mg, 1 mmol), 4-bro-
c
mobenzoyl chloride (220 mg, 1 mmol). White solid (90%), mp:
205–207 °C. 1H NMR (d, DMSO-d6): 10.73 (br s, 1H), 9.81 (s, 1H),
8.98 (d, 1H, J = 1.2 Hz), 8.73 (d, 1H, J = 6.4 Hz), 8.25 (d, 1H,
J = 6.4 Hz), 8.03 (m, 4H), 7.79 (d, 2H, J = 8.8 Hz), 4.68 (q, 2H,
J = 7.2 Hz), 1.42 (t, 3H, J = 7.2 Hz). ESI-MS: m/z = 394 [M+1]+. Anal.
Calcd for C20H16BrN3O: C, 60.93; H, 4.09; N, 10.66. Found: C,
60.74; H, 4.25; N, 10.45.
4.1.4.2. 9-Ethyl-6-(4-methoxybenzamido)-c-carboline
(14b). Reagent: 6-amino-9-ethyl- -carboline (13) (211 mg,
c
1 mmol), 4-methoxybenzoyl chloride (171 mg, 1 mmol). White so-
lid (65%), mp: 153–155 °C. 1H NMR (d, DMSO-d6): 10.46 (br s, 1H),
9.79 (s, 1H), 8.98 (s, 1H), 8.72 (d, 1H, J = 6.4 Hz), 8.24 (d, 1H,
J = 6.4 Hz), 8.07 (d, 2H, J = 8.8 Hz), 7.97 (m, 2H), 7.10 (d, 2H,
J = 8.8 Hz), 4.66 (q, 2H, J = 7.2 Hz), 3.86 (s, 3H), 1.42 (t, 3H,
J = 7.2 Hz). ESI-MS: m/z = 346 [M+1]+. Anal. Calcd for C21H19N3O2:
C, 73.03; H, 5.54; N, 12.17. Found: C, 73.32; H, 5.85; N, 12.42.
4.2. Cytotoxic assay
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.41 MTT solution (10.0
lL/well) in RPMI-1640 (Sigma, St.
Louis, MO) was added after cells were treated with drug for 48 h,
4.1.4.3. 9-Ethyl-6-(3, 4, 5-trimethoxybenzamido)-
c
-carboline
and cells were incubated for a further 4 h at 37 °C. The purple for-
(14c). Reagent: 6-amino-9-ethyl- -carboline (13) (211 mg,
c
mazan crystals were dissolved in 100.0 lL DMSO. After 5 min, the
1 mmol), 3, 4, 5-trimethoxybenzoyl chloride (231 mg, 1 mmol).
White solid (62%), mp: 117–119 °C. 1H NMR (d, DMSO-d6):
10.31 (br s, 1H), 9.30 (s, 1H), 8.59 (d, 1H, J = 1.2 Hz), 8.49 (d,
1H, J = 5.6 Hz), 7.86 (d, 1H, J = 8.8 Hz), 7.73 (d, 1H, J = 8.8 Hz),
7.64 (d, 1H, J = 5.6 Hz), 7.38 (s, 2H), 4.48 (q, 2H, J = 7.2 Hz), 3.99
(s, 6H), 3.75 (s, 3H), 1.36 (t, 3H, J = 7.2 Hz). ESI-MS: m/z = 406
[M+1]+. Anal. Calcd for C23H23N3O4: C, 68.13; H, 5.72; N, 10.36.
Found: C, 68.25; H, 5.56; N, 10.62.
plates were read on an automated microplate spectrophotometer
(Bio-Tek Instruments, Winooski, VT) at 570 nm. Assays were per-
formed in triplicate in three independent experiments. The con-
centration required for 50% inhibition of cell viability (IC50) was
calculated using the software ‘Dose-Effect Analysis with Micro-
computers’. 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.
4.1.4.4. 9-Ethyl-6-(2-hydroxybenzamido)-c-carboline (14d). Rea-
4.3. Molecular modeling and alignment
gent: 6-amino-9-ethyl- -carboline (13) (211 mg, 1 mmol), 2-metho-
c
xybenzoyl chloride (171 mg, 1 mmol). White solid (29%), mp:
143–145 °C. 1H NMR (d, DMSO-d6): 10.64 (br s, 1H), 9.30 (s, 1H), 8.69
(s, 1H), 8.48 (d, 1H, J = 4.0 Hz), 7.76 (d, 1H, J = 8.4 Hz), 7.71 (d, 1H,
J = 8.4 Hz), 7.64 (d, 1H, J = 6.4 Hz), 7.98 (d, 1H, J = 8.0 Hz), 7.54 (m,
3H), 4.48 (q, 2H, J = 7.2 Hz), 1.34 (t, 3H, J = 7.2 Hz). ESI-MS: m/z = 332
[M+1]+. Anal. Calcd for C20H17N3O2: C, 72.49; H, 5.17; N, 12.68. Found:
C, 72.63; H, 5.26; N, 12.56.
The 3D structures of all the compounds were built and mini-
mized by using SYBYL 6.91. The geometries of all molecules involved
in this study were optimized by Powell’s method using the tripos
force field. Conformation analyses were also carried out using the
SYBYL/Grid search module. The lowest-energy conformations were
considered as the bioactive conformations. It was noticed that all
the 21 molecules in the training set had the same c-carboline skel-

J. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3324–3331
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