Design, synthesis, and evaluations of the antiproliferative activity and aqueous solubility of novel carbazole sulfonamide derivatives as antitumor agents

Article abstract of DOI:10.1016/j.bioorg.2020.103766

Optimization of IG-105 (1) on the carbazole ring provided five series of new carbazole sulfonamides derivatives, 7a–e, 8a–g, 9a–g, 10a–e, and 11a–g. All of the compounds were evaluated against HepG2, MCF-7, MIA PaCa-2, and Bel-7402 cells for antiproliferative activity. Each series of compounds was 2–5 times more active against HepG2 cells (IC₅₀: 1.00–10.0 μM) than the other three tumor cell lines. Several representative compounds, selected from each series, showed aqueous solubility (13.4–176.5 μg/mL at pH 7.4 and 2.0) better than 1, with the aqueous solubility of corresponding salts > 30 mg/mL. From the results of evaluating the effects of the compounds 7b, 8c, 9c, 10c and 11c on tubulin in vitro, we speculated that their targets were different from those of 1 and CA-4P. We tested the antitumor activity of the representative compound 7b·HCl (10 mg/kg) in an in vivo study and found that its tumor growth inhibition rate was 41.1%. The tumor growth inhibition rate of 7b·HCl (20 mg/kg) was 54.6%, whereas the tumor growth inhibition rate of CA-4P (50 mg/kg) was 48.3%. And in another batch of in vivo antitumor activity testing, 9c·HCl and 11c·HCl at doses of 10 mg/kg resulted in 61.1% and 50.0% inhibition, respectively. These promising results warrant further development of the derivatives, which may use a novel mechanism and show potential potency as antitumor drug candidates.

Full text of DOI:10.1016/j.bioorg.2020.103766

Journal Pre-proofs
Design, Synthesis, and Evaluations of the Antiproliferative Activity and
Aqueous Solubility of Novel Carbazole Sulfonamide Derivatives as Antitu-
mor Agents
Lianqi Sun, Chenxi Wang, Xinyue Hu, Yanbin Wu, Zhi Jiang, Zhuorong Li,
Xiaofang Chen, Laixing Hu
PII:
S0045-2068(19)32001-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103766
YBIOO 103766
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
29 November 2019
13 March 2020
15 March 2020
Please cite this article as: L. Sun, C. Wang, X. Hu, Y. Wu, Z. Jiang, Z. Li, X. Chen, L. Hu, Design, Synthesis,
and Evaluations of the Antiproliferative Activity and Aqueous Solubility of Novel Carbazole Sulfonamide
Derivatives as Antitumor Agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2
020.103766
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier Inc.

Design, Synthesis, and Evaluations of the Antiproliferative Activity
and Aqueous Solubility of Novel Carbazole Sulfonamide Derivatives
as Antitumor Agents
Lianqi Sun, Chenxi Wang, Xinyue Hu, Yanbin Wu, Zhi Jiang, Zhuorong Li,
Xiaofang Chen*, Laixing Hu*
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing 100050, People’s Republic of China
*
Corresponding author. Tel. /fax: +86 10 63132113.
E-mail address: chenchemistry@hotmail.com, hulaixing@hotmail.com
Abstract:
Optimization of IG-105 (1) on the carbazole ring provided five series of new
carbazole sulfonamides derivatives, 7a–e, 8a–g, 9a–g, 10a–e, and 11a–g. All of the
compounds were evaluated against HepG2, MCF-7, MIA PaCa-2, and Bel-7402 cells
for antiproliferative activity. Each series of compounds was 2–5 times more active
against HepG2 cells (IC : 1.00–10.0 M) than the other three tumor cell lines.
5
0
Several representative compounds, selected from each series, showed aqueous
solubility (13.4–176.5 µg/mL at pH 7.4 and 2.0) better than 1, with the aqueous
solubility of corresponding salts > 30 mg/mL. From the results of evaluating the
effects of the compounds 7b, 8c, 9c, 10c and 11c on tubulin in vitro, we speculated
that their targets were different from those of 1 and CA-4P. We tested the antitumor
activity of the representative compound 7b
found that its tumor growth inhibition rate was 41.1%. The tumor growth inhibition
rate of 7b HCl (20 mg/kg) was 54.6%, whereas the tumor growth inhibition rate of
CA-4P (50 mg/kg) was 48.3%. And in another batch of in vivo antitumor activity
testing, 9c HCl and 11c HCl at doses of 10 mg/kg resulted in 61.1% and 50.0%
•HCl (10 mg/kg) in an in vivo study and
•
•
•
inhibition, respectively. These promising results warrant further development of the
derivatives, which may use a novel mechanism and show potential potency as
antitumor drug candidates.

Keywords: Carbazole sulfonamide derivatives; Synthesis; Antiproliferative activity;
Aqueous solubility.
1
. Introduction
MeO
MeO
A
R1
R2
B
OMe
OMe
CA-4 : R = H; R = OH
1
2
CA-4P:R = H; R = OPO Na
2
1
2
3
Fig. 1 Structures of CA-4 and CA-4P
Microtubules, as key components of cytoskeleton, have intrigued scientists since
their discovery because of their critical importance to the cellular processes such as
regulation of motility, cell signaling, formation and maintenance of cell shape,
secretion, and intracellular transport [1]. Microtubule-active drugs generally bind to
one of three main classes of sites on tubulin, the paclitaxel site, the Vinca domain and
colchicine domain [2]. The compounds like colchicine and podophyllotoxin (podo),
discovered in the early days irreversibly bind at the colchicine domain thus inducing
great toxicities and limiting the use in clinical [3]. The reversibly binding inhibitors
discovered in later, like Combretastatin A4 (CA-4), a novel small molecule tubulin
binding agent which was isolated from the South African bush willow Combretum
caffrum. (Fig. 1) [4]. CA-4 strongly inhibits the polymerization of tubulin by binding
to the colchicine binding site, and can be served as clinical candidates for further
development [5]. However, the low water solubility of this compound has limited its
efficacy in vivo. Its water-soluble prodrug, CA-4 phosphate disodium (CA-4P) has
good water solubility, stability and cytostatic activity and is in phase Ⅱ/Ⅲ clinical
trials both as a single drug and in combination for anticancer therapy [6]. It appears to
act by a unique irreversible and selective antivascular effect that disrupts immature
endothelial cell’s cytoskeleton in vivo [7]. In recent years, a large number of
structure-activity relationship (SAR) studies on CA-4 have been investigated. In brief,
it is thought that the presence of the cis-orientation of the two aromatic rings, the
3
,4,5-trimethoxy group on ring A, and the para-methoxy group on ring B were
essential for optimal cytotoxic activity [8]. However, in later studies found that CA-4

and other olefinic analogues are prone to isomerize into their inactive trans-forms
during storage and administration. To overcome this problem, varieties of
conformationally restricted cis-locked analogues have been designed through the
replacement of the olefinic bond with heterocyclic moieties. We also were interested
in exploring the structure transformation of CA-4. We replaced the cis-olefin linker
with sulfonamide group, the 3,4,5-trimethoxyphenyl with 2,6-dimethoxylpridine, the
B ring with carbazole-ring to synthesize IG-105 (1) [9].
O
6
5
O
O
4
A
O
7
S
N
N
3
2
B
H
8
N
9
1
1
IG-105
Fig. 2 Structure of IG-105 (1)
IG-105 (1, Fig. 2), exhibited potent activity against human leukemia and solid
tumors in the breast, liver, prostate, lung, skin, colon, and pancreas, with IC values
5
0
between 0.012 and 0.298 µM. However, only modest antitumor activity was observed
in vivo when 1 was administered intraperitoneally to BDF1 mice bearing Lewis lung
carcinoma cells at 40 mg/kg/day. The tumor mass inhibition rate of 1 was 59%
compared to 71% produced by etoposide, a clinical anticancer drug that was used as a
positive control [9]. The unexpectedly low antitumor activity was considered a result
of its low bioavailability, which was caused, at least in part, by its poor aqueous
solubility. Thus, improving aqueous solubility allows the administration dose to be
increased, potentially increasing the therapeutic value of this antitumor agent.
To improve the aqueous solubility and bioavailability of 1, the 6- and 7-positions
of the carbazole rings were modified, yielding several antitumor-active compounds
with water solubility (0.11–19.60 µg/mL at pH 7.4 and 2.0) better than that of 1 [10].。
The 6-position and 7-position are preferred for modification because the two positions
on the carbazole ring are the most active, especially the 6-position, and the target
product can be synthesized easily. At the same time, we learned to optimize the
quinoline ring of receptor tyrosine kinase inhibitors (Fig. 3) [11]. Introduction of
ionizable groups (nitrogen-containing heterocyclic groups) into compound structures

is a common approach to improving aqueous solubility. In this study, we introduced
ionizable groups (benzylamines and phenyl ether alkylamines) into the 6-position of
the carbazole ring of 1, so as to significantly increase the water-solubility and may
improve the oral bioavailability while maintaining or increasing the anti-tumor
activity.
F
F
O
HN
N
Cl
O
HN
N
Cl
N
N
O
O
N
N
O
O
geftinib
canertinib
N
HN
N
N
HN
N
N
O
N
N
N
N
H
H
N
N
initinib
AEE788
Fig. 3 Structures of receptor tyrosine kinase inhibitors
. Results and Discussion
.1 Chemistry
As shown Scheme 1, compounds 5 and 6 were synthesized using our previously
2
2
reported approach [10].
OHC
OHC
OHC
O
O
O
b
c
a
SO Cl
d
S
N
2
N
H
N
N
O
H
N
N
4
N
2
3
5
HO
O
e
O
O
S
N
N
H
O
N
6
Scheme 1. Reagents and conditions: a: CH I, NaOH, DMF, 4 h; b: POCl , DMF, 0–80 °C, 3 h; c:
3
3
ClSO H, 0 °C, 10 min; d: 3-amino-2, 6-dimethoxypyridine, K CO , DMF, rt.1 h; e: CH OH/DCM,
3
2
3
3
H O , c H SO , 5 h.
2
2
2
4
2
.1.1 Benzylamine compounds：

OHC
R
O
O
7
a R= H2C N
7b R= H2C N
c R= H2C N
d R= H2C N
e R= H2C N
O
N
O
O
O
O
S
N
a
S
N
N
N
H
H
O
O
N
N
7
7
5
7
7
NH
Scheme 2. Reagents and conditions: a: amines, THF, Ti(OiPr) , NaBH CN, 3 h.
4
3
In Scheme 2, a Borch reduction reaction of aldehyde 5 with different
heterocyclic amines in Ti(OiPr) was catalyzed by a sodium cyanoborohydride
4
reduction to 40–70% yields of 6-modified benzylamine carbazole sulfonamide
compounds 7a–g [12]. A hydrochloric acid-methanol solution was added to 7a–g in
methanol and stirred for 1 h at 0 °C to produce the corresponding hydrochlorides.
2
.1.2 Phenylethylamine compounds：
Cl
R
HO
O
O
O
O
O
n
n
O
O
O
O
O O
a
b
S
N
S
N
S
N
N
N
N
H
H
H
O
O
O
N
N
N
6
8
a n=2
a n=3
8b n=2 R=
c n=2 R=
d n=2 R=
e n=2 R=
f n=2 R=
g n=2 R=
N
N
O
N
9b n=3 R=
N
O
N
9
8
9c n=3 R=
9d n=3 R=
N
8
N
N
N
N
N
N
8
9e n=3 R=
9f n=3 R=
8
NH
NH
8
N
N
9g n=3 R=
N
N
Scheme 3. Reagents and conditions: a: 1-bromo-3-chloropropane or 1-bromo-2- chloroethane,
ACE (acetone), K CO , reflux, 5-10h; b: amine, CH CN, reflux, 24-48h.
2
3
3
2
.1.3 Double substituted alkanines：
Cl
R
HO
O
O
O
O
O
n
n
O
S
O
O
S
O
O O
a
b
N
N
S
N
N
N
N
H
O
O
O
N
N
N
Cl
nR
1b n=3 R=
11c n=3 R=
1d n=3 R=
1e n=3 R=
n
1
N
N
O
N
6
10a n=2
10b n=2 R=
0c n=2 R=
0d n=2 R=
0e n=2 R=
N
O
N
11a n=3
1
N
N
N
1
N
N
N
1
1
1
11f n=3 R=
NH
11g n=3 R=
N
N
Scheme 4. Reagents and conditions: a: 1-bromo-3-chloropropane or 1-bromo-2-chloroethane,
ACE (acetone), K CO , reflux, 5-10 h; b: amine, CH CN, reflux, 24-48h.
2
3
3
In Scheme 3, Compound 6 reacted with 1-bromo-3-chloropropane or

1
-bromo-2-chloroethane and potassium carbonate under reflux in acetone for 5–10 h
to produce ether compounds 8a and 9a [13]. The ether compounds were refluxed with
different heterocyclic amines in acetonitrile to give carbazole sulfonamide compounds
8
b-g and 9b-g [14]. The nitrogen on the sulfonamide also underwent a coupling
reaction with 1-bromo-3-chloropropane or 1-bromo-2-chloroethane during the
formation of 8a and 9a to obtain compounds 10a and 11a, in which the hydroxyl
group and sulfonamide were disubstituted as shown in Scheme 4. And then react with
different heterocyclic amines to obtain disubstituted alkylamine compounds 10b-e
and 11b-g.
A hydrochloric acid-methanol solution was added to the final compounds having
nitrogen-containing heterocycles in methanol, and the new solution was stirred for 1 h
at 0 °C to produce the corresponding hydrochloride.
2
.2 In vitro antitumor activity
All of the target compounds were evaluated against HepG2 (hepatoma cancer),
MCF-7 (breast cancer), MIA PaCa-2 (pancreatic cancer), and Bel-7402
hepatoma/liver cancer) cells for antiproliferative activity using the standard
(
sulforhodamine B (SRB) assay [15-17]. The data are summarized in Table 1, Table 2,
and Table 3.
R1
O
O
O
S
N
N
R2
O
N
Table 1. Antiproliferative activities of compounds 7a-e against four tumor cell lines
IC50(µM)a
Comp.
R
1
R
2
HepG2
>25
MIA PaCa-2
NTb
MCF-7
NTb
Bel-7402
NTb
7
a
6-CH N(CH CH ) O
H
H
H
H
H
H
2
2
2 2
7
b
6-CH N(CH CH ) NCH
3
3.98
10.48
15.70
8.57
5.38
2
2
2 2
7
c
6-CH N(CH )
2.24
5.28
10.06
18.90
7.27
2
2 5
7
d
6-CH N(CH )
7.17
12.10
15.32
9.85
2
2 4
7
e
6-CH N(CH CH ) NH
2.62
6.34
2
2
2 2
1
H
0.095
0.003
0.002
0.18
0.68
1.07
Podo
CA-4
0.012
0.020
0.005
0.016
0.020
0.003
a: IC values, concentrations required to inhibit 50% proliferation of human tumor cells after 48 h
5
0
of treatment.

b: NT: Not tested.
Table 2. Antiproliferative activities of compounds 8a-g and 9a-g against four tumor cell lines
IC50(µM)a
Comp.
R
1
R
2
HepG2
1.10
MIA PaCa-2
2.15
MCF-7
2.87
Bel-7402
2.64
8
a
6-O(CH
2
)
2
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8
b
6-O(CH N(CH
2
)
2
2
CH
2
)
2
O
4.28
5.27
6.86
6.19
8
c
6-O(CH
2
)
2
N(CH
2
CH
2
)
2
NCH
3
4.55
4.25
8.88
4.59
8
d
6-O(CH
6-O(CH
2
2
)
)
2
2
N(CH
N(CH
2
2
)
)
5
4
5.50
5.34
6.15
6.37
8
e
4.95
5.13
6.00
6.26
8
f
6-O(CH
2
)
2
N(CH
2
CH
2
)
2
NH
5.93
6.25
7.85
9.11
8
9
g
a
6-O(CH
2
)
2
N(CH
2
CH
2
)
2
CHN(CH
2
)
4
2.76
4.59
5.10
4.76
6-O(CH
2
)
3
Cl
1.69
2.56
3.21
3.14
9
b
6-O(CH
6-O(CH
2
)
3
N(CH
2
CH
2
)
2
O
8.12
9.31
9.55
10.26
5.67
9
c
2
)
3
N(CH CH
2
2
)
2
NCH
3
5.68
6.00
6.73
9
d
6-O(CH
6-O(CH
2
2
)
)
3
3
N(CH
N(CH
2
2
)
)
5
4
5.79
6.01
5.98
6.32
9
e
4.94
5.40
6.34
5.76
9
f
6-O(CH
2
)
3
N(CH
2
CH
2
)
2
NH
10.91
5.15
11.13
5.78
15.44
6.05
20.08
5.93
9
g
6-O(CH N(CH
2
)
3
2
CH
2
)
2
CHN(CH )
2 4
1
H
0.095
0.003
0.002
0.18
0.68
1.07
Podo
CA-4
0.012
0.003
0.020
0.005
0.016
0.020
a: IC values, concentrations required to inhibit 50% proliferation of human tumor cells after 48 h
5
0
of treatment.
Table 3. Antiproliferative activities of compounds 10a-e and 11a-g against four tumor cell lines
IC50(µM)a
Comp.
R
1
R
2
HepG2 MIA PaCa-2 MCF-7 Bel-7402
1
0a
6-O(CH
2
)
2
Cl
(CH
2
)
2
Cl
CH
1.78
5.41
5.60
5.23
4.89
2.50
7.41
4.22
1.53
3.32
10.35
6.12
0.095
0.003
0.002
2.54
5.79
5.94
5.38
5.05
3.03
6.86
8.15
2.00
5.10
11.11
6.50
0.18
0.012
0.003
3.17
6.33
6.21
6.24
6.52
3.45
7.54
20.54
2.57
5.63
14.36
6.94
0.68
0.020
0.005
2.96
5.85
5.90
5.90
6.19
3.18
7.47
4.75
2.32
4.95
13.75
6.20
1.07
0.016
0.020
1
0b
6-O(CH N(CH
2
)
2
2
CH
2
)
2
O
(CH
2
)
2
N(CH
2
2 2
) O
1
0c
6-O(CH
2
)
2
N(CH
2
CH
2
)
2
NCH
3
(CH
2
)
2
N(CH
2
CH
2
)
2
NCH
3
1
0d
0e
6-O(CH
6-O(CH
2
2
)
)
2
2
N(CH
N(CH
2
2
)
)
5
4
(CH
2
2
)
)
2
2
N(CH
N(CH
2
2
)
5
4
1
(CH
)
1
1a
1b
1c
6-O(CH
6-O(CH N(CH
6-O(CH N(CH CH
2
)
3
Cl
(CH
N(CH
N(CH CH
2
)
3
Cl
CH
NCH
1
2
)
3
2
CH
2
)
2
O
(CH
2
)
3
2
2
)
2
O
1
2
)
3
2
2
)
2
NCH
3
(CH
2
)
3
2
2
)
2
3
1
1d
1e
6-O(CH
6-O(CH
2
2
)
)
3
3
N(CH
N(CH
2
2
)
)
5
4
(CH
2
2
)
)
3
3
N(CH
N(CH
2
2
)
5
4
1
(CH
)
1
1f
6-O(CH
2
)
3
N(CH
2
CH
2
)
2
NH
2 3 2 2 2
(CH ) N(CH CH ) NH
1
1g
1
6-O(CH
2
)
3
N(CH
2
CH
2
)
2
CHN(CH
2
)
4
2 3 2 2 2 2 4
(CH ) N(CH CH ) CHN(CH )
H
H
Podo
CA-4
a: IC values, concentrations required to inhibit 50% proliferation of human tumor cells after 48 h
5
0

of treatment.
Compounds 7a–e are derivatives of aldehydes with antitumor activity at the
same levels and IC values against HepG2 at 1–10 μM, which are at least 10 times
5
0
lower than that of compound 1. Compounds 8a-g, 9a-g, 10a-e, and 11a-g are
hydroxyl group derivatives, with 10a-e and 11a-g having the same groups introduced
at hydroxyls and sulfonamides. The antitumor activity of each compound was
generally reduced. In addition to 8a, 9a, 10a, and 11a, the four compounds used
originally as key intermediates against HepG2 cells had IC values of approximately
5
0
1
–2 μM, other compounds containing water-soluble groups IC at 5–10 μM were
50
tested. The antitumor activities of compounds containing two C atoms in the ether
chain were equivalent to those of compounds containing three C atoms, and the IC50
values against HepG2 were 1–10 μM. The antitumor activity of compounds 10a–e and
1
1a–g which introduce group on the hydroxy and sulfonamide were equivalent to that
of the compounds 8a–g and 9a–g which only introduce the same group on the
hydroxyl; the IC against HepG2 was generally 5–10 μM. The antitumor activity of
5
0
all derivatives against the other three tumor cells (IC : 5–20 μM) was generally
5
0
weaker or equivalent to their activity against HepG2.
Table 4. Antitumor activities of compounds and their corresponding hydrochlorides against four
tumor cell lines
IC50(µM)a
HepG2 MIA PaCa-2 MCF-7 Bel-7402
Comp.
R
1
R
2
7
b
6-CH
2
N(CH
N(CH
2
CH
2
)
2
NCH
NCH
3
3
H
H
3.98
5.76
5.93
10.17
2.76
8.28
5.68
5.41
5.79
8.65
5.41
6.37
5.60
10.48
NTb
6.25
NTb
4.59
NTb
6.00
NTb
6.01
NTb
5.79
NTb
5.94
15.70
NTb
7.85
NTb
5.10
NTb
6.73
NTb
5.98
NTb
6.33
NTb
6.21
5.38
NTb
9.11
NTb
4.76
NTb
5.67
NTb
6.32
NTb
5.85
NTb
5.90
7
b•HCl
6-CH
2
2
CH
2
)
2
8
f
6-O(CH
6-O(CH
2
2
)
)
2
2
N(CH
N(CH
2
2
CH
CH
2
2
)
)
2
2
NH
NH
H
8
f•HCl
H
8
g
6-O(CH
6-O(CH
2
)
)
2
N(CH
N(CH
2
CH
CH
2
)
)
2
CHN(CH
CHN(CH
2
)
)
4
4
H
8
g•HCl
2
2
2
2
2
2
H
9
c
6-O(CH
6-O(CH
2
2
)
)
3
3
N(CH
N(CH
2
2
CH
CH
2
2
)
)
2
2
NCH
NCH
3
3
H
9
c•HCl
H
9
d
6-O(CH
6-O(CH
2
2
)
)
3
3
N(CH
N(CH
2
2
)
)
5
5
H
9
d•HCl
0b
0b•HCl
0c
H
1
6-O(CH
6-O(CH
2
2
)
)
2
2
N(CH
N(CH
2
2
CH
CH
2
)
)
2
2
O
O
(CH
2
2
)
)
2
2
N(CH
N(CH
2
2
CH
CH
2
2
)
)
2
2
O
O
1
2
(CH
1
6-O(CH
2
)
2
N(CH
2
CH
2
)
2
NCH
3
2 2 2 2 2 3
(CH ) N(CH CH ) NCH

1
0c•HCl
0d
0d•HCl
0e
0e•HCl
1c
1c•HCl
1d
1d•HCl
6-O(CH
2
)
2
N(CH
2
CH
2
)
2
NCH
3
(CH
2
)
2
N(CH
2
CH
2
)
2
NCH
3
6.16
5.23
5.87
4.89
5.17
4.22
14.64
1.53
2.37
3.32
2.15
5.08
5.38
NTb
5.05
NTb
8.15
NTb
2.00
NTb
5.10
NTb
8.17
6.24
NTb
6.52
NTb
20.54
NTb
2.57
NTb
5.63
NTb
4.96
5.90
NTb
6.19
NTb
4.75
NTb
2.32
NTb
4.95
NTb
1
6-O(CH
6-O(CH
6-O(CH
6-O(CH
2
2
2
2
)
)
)
)
2
2
2
2
N(CH
N(CH
N(CH
N(CH
2
2
2
2
)
)
)
)
5
5
4
4
(CH
(CH
(CH
(CH
2
2
2
2
)
)
)
)
2
2
2
2
N(CH
N(CH
N(CH
N(CH
2
2
2
2
)
5
5
4
1
1
1
)
)
)
1
4
1
6-O(CH
6-O(CH
2
)
)
3
N(CH
N(CH
2
CH
CH
2
)
)
2
2
NCH
NCH
3
3
(CH
2
2
)
)
3
3
N(CH
N(CH
2
2
CH
CH
2
2
)
)
2
2
NCH
3
3
2
3
2
2
(CH
NCH
1
6-O(CH
6-O(CH
6-O(CH
6-O(CH
2
2
2
2
)
)
)
)
3
3
3
3
N(CH
N(CH
N(CH
N(CH
2
2
2
2
)
)
)
)
5
5
4
4
(CH
(CH
(CH
(CH
2
2
2
2
)
)
)
)
3
3
3
3
N(CH
N(CH
N(CH
N(CH
2
2
2
2
)
5
5
4
4
1
)
)
)
1
1e
1
1e•HCl
a: IC values, concentrations required to inhibit 50% proliferation of human tumor cells after 48 h
5
0
of treatment.
b: NT: not tested.
Because the introduced ionizable groups contained free amines, the
corresponding hydrochloride compounds were synthesized. We selected the
hydrochloride of the representative compound from each series, and tumor cell
inhibitory activity of the hydrochloride compounds was evaluated. The data in Table
4
shows that the in vitro antitumor activities of most compounds, except 8g and 11c,
were equivalent to those of their corresponding hydrochloride salts.
2
.3 The aqueous solubility
An important goal of this research was to increase the aqueous solubility of the
new compounds compared to that of 1. To assess this property, we measured the
aqueous solubility of several representative compounds in each series using an
high-performance liquid chromatography/ultraviolet (HPLC/UV) method (method A)
under two conditions [18], pH = 2.0 and pH = 7.4, as shown in Table 5. Each tested
compound showed improved solubilities at both pH 2.0 and pH 7.4 compared with
those of 1. The water solubility of the polar group-containing compounds were 10–
1
00 times higher than those of 1 under at pH = 2.0 and pH = 7.4, especially with the
introduction of an ionizable group (i.e., a nitrogen-containing heterocycle). Most of
the compounds showed comparable water solubilities under both conditions, whereas
amino acids and most ionizable groups have water solubilities at pH = 2.0 that are
significantly better than those at pH = 7.4. However, there were exceptions.
Compounds 9e, 9g, 11e, and 11g had water solubilities at pH = 7.4 that were 2–3

times higher than those at pH = 2.0.
The aqueous solubilities of the corresponding hydrochloride salts of compounds
7
a–e, 8b–g, 9b–g, 10b–e, and 11b-g, were measured by adding excess quantities the
hydrochlorides to a predetermined volume of distilled water (method B) [19]. Due to
limited quantities of the hydrochlorides, no further effort was made to measure the
maximum solubility of these compounds. Their aqueous solubilities ( > 30 mg/mL at
pH 7.0) were improved 300,000 times relative to that of 1, so they may exhibit more
antitumor activity in vivo than in vitro.
Table 5. Aqueous solubilities of representative compounds
Aqueous solubility (µg/mL)
Comp.
R1
R2
pH = 2.0
pH = 7.4
6
-CH N(CH CH ) O
7
a
2
2
2
2
H
H
13.4
43.4
30.2
7
b
6-CH N(CH CH ) NCH
118.8
2
2
2
2
3
6
-CH N(CH )
2 2 5
7
c
H
H
H
75.7
112.8
122.0
21
6
-O(CH ) N(CH )
2 2 2 5
8
d
36.6
59.4
6
-O(CH ) N(CH CH ) NH
2 2 2 2 2
8
f
6
-O(CH ) N(CH CH ) NCH
2 3 2 2 2 3
9
9
c
e
H
H
52.9
22.4
76.4
66.3
6
-O(CH ) N(CH )
2 3 2 4
9
g
6-O(CH ) N(CH CH ) CHN(CH )
H
36.9
91.3
2
3
2
2
2
2
4
6
-O(CH ) N(CH )
(CH ) N(CH )
1
0d
2
2
2
5
2
2
2
5
70.8
71.2
55.3
85.5
6
-O(CH ) N(CH CH ) NCH
(CH ) N(CH CH ) NCH
2 3 2 2 2 3
1
1
1c
1e
2
3
2
2
2
3
6
-O(CH ) N(CH )
(CH ) N(CH )
2 3 2 4
2
3
2
4
35.7
76.2
62.7
6
-O(CH ) N(CH CH ) CHN(CH )
(CH ) N(CH CH ) CHN(CH )
2 4
176.5
1
1g
2
3
2
2
2
2
4
2
3
2
2
2
1
H
H
0.10
< 0.10
2
.4 Inhibition of tubulin polymerization in vitro
, CA-4P inhibited tubulin polymerization in a colchicine-like manner [20]. We
1
further evaluated the effects of the compounds 7b, 8c, 9c, 10c and 11c, which selected
from each series and their 6-position have the same derived group, on tubulin in vitro.
The effects on the assembly of purified porcine tubulin were evaluated by measuring
an increase in absorbance at 340 nm at 37 ℃, by using 1, CA-4P and Taxol as
comparative agents.
As depicted in Fig.4 and Table 6, Compounds 7b, 8c, 9c, 10c and 11c showed
little inhibition of tubulin polymerization at concentrations of 10 μM. At a

concentration of 100 μM, the inhibition of tubulin polymerization was improved, but
was significantly weaker than that of CA-4P and 1, which was basically consistent
with the results of anti-tumor activity in vitro. At 100 μM, the inhibition rate of all
compounds was lower than 50% except compound 9c, which reached 84%, indicating
that these compounds had little effect on tubulin. According to the above results,
although these compounds were derived from 1, their targets were different from
those of 1 and CA-4P.
Table 6. Inhibition of tubulin polymerization
Inhibition of tubulin polymerization (%)a
Comp.
R
1
R
2
1
0 μM
100 μM
7
b
6-CH N(CH CH ) NCH
H
H
H
0
3
27
49
84
8
2
2
2
2
3
8
9
c
c
6-O(CH
2
2
2
2
)
)
)
)
2
3
2
3
N(CH
2
2
2
2
CH
CH
CH
CH
2
2
2
2
)
)
)
)
2
2
2
2
NCH
3
3
3
3
6-O(CH
6-O(CH
6-O(CH
N(CH
NCH
NCH
NCH
4
1
1
0c
1c
N(CH
(CH
2
2
)
)
2
3
N(CH
2
CH
CH
2
2
)
)
2
2
NCH
NCH
3
3
3
N(CH
(CH
N(CH
2
6
12
94
94
-
1
-
-
-
-
-
-
91
95
-
CA-4P
Taxol
a: inhibition% = 100 - activity%, activity% = A /A
test control
Fig. 4. Effects of test compounds on tubulin polymerization in vitro. Test compounds, IG-105,
CA-4P, Taxol and DMSO were added in the plates and then polymerizations were tested as an
increase in absorbance at 340 nm over 40 min at 37 ℃. (Fig. 4A 10 μM and Fig. 4B 100 μM)
2
.5 In vivo antitumor activity
The antitumor activities of novel derivatives (IC : 1–20 μM) were significantly
5
0

reduced compared with that of compound 1, but the aqueous solubilities of the
compounds were significantly improved. To investigate the effect of the aqueous
solubilities of the compounds on its antitumor activity in vivo, and consider the
antitumor activity and water solubility of the compound comprehensively, we selected
compound 7b
model in BALB/c nude mice [21]. Remarkably, 7b
dose-dependent manner. CA-4P at a dose of 50 mg/kg inhibited tumor growth by
•HCl for in vivo antitumor activity testing, and established a HepG2
•HCl inhibited tumor growth in a
4
5
8.3%, whereas 7b
•
HCl at doses of 10 mg/kg and 20 mg/kg resulted in 41.1% and
4.6% inhibition, respectively. Taxol at a dose of 10 mg/kg inhibited tumor growth
by 78.3% (Fig. 5A).
To investigate in vivo the antitumor activity of other series of hydrochlorides, we
selected compounds 9c
•
HCl and 11c HCl for in vivo antitumor activity testing, and
•
established another HepG2 model in BALB/c nude mice. They were randomized into
4
groups named blank control (saline solution), 7b
1c HCl. 7b HCl at a dose of 20 mg/kg inhibited tumor growth by 63.6%，9c
HCl at doses of 10 mg/kg resulted in 61.1% and 50.0% inhibition,
•
HCl (positive control), 9c
•
HCl and
1
•
•
•HCl
and 11c
•
respectively (Fig. 6A).
The inhibition rate of Taxol (dose 10 mg/kg) reached 78.3%, but the weight of
mice in this group was significantly decreased, which indicated that cytotoxicity of
Taxol was greater than that of CA-4P and 7b•HCl. During the experiments, there
were no deaths and no significant adverse reactions such as lethargy or anorexia, and
the changes in body weight were in an acceptable range (Fig. 5B, Fig.6B).
The tumor inhibition rate of 7b HCl (20 mg/kg) was 54.6% and 63.6%,
•
respectively, which was within the acceptable range considering that the two batches
of animal experiments were conducted by different companies. Although the
antitumor activity against HepG2 of CA-4 (IC : 0.002 µM) was 2000 times higher
5
0
than that of compound 7b (IC : 3.98 µM) in vitro, the inhibition rate of 7b
•
HCl (dose,
0 mg/kg) was higher than that of CA-4P (dose, 50 mg/kg) in vivo. Compounds 9c
IC : 5.68 µM) and 11c (IC : 4.22 µM), which have poor activity in vitro, and the
5
0
2
(
5
0
50
tumor inhibition rate of their hydrochloride salts in vivo can reach 61.1% and 50.0%,

showing efficient in vivo. This is a very interesting result, suggesting that the
derivatives may have novel anti-tumor mechanisms, and we will have further studies
in the future.
Fig 5. In vivo efficacy of 7b•HCl against HepG2 cells. (A) Antitumor effects of 7b•HCl in
BALB/c nude mice bearing HepG2. (B) Body weight changes in HepG2-bearing mice.
Fig 6. In vivo efficacy of 7b•HCl, 9c•HCl and 11c•HCl against HepG2 cells. (A) Antitumor
effects of 7b•HCl, 9c•HCl and 11c•HCl in BALB/c nude mice bearing HepG2. (B) Body weight
changes in HepG2-bearing mice.
4
. Conclusion
Based on our optimization strategy, 31 novel carbazole sulfonamide derivatives
were synthesized by modifying the R group on the carbazole ring of 1. All of the
compounds showed moderate antitumor activity against HepG2, MIA PaCa-2, MCF-7,
and Bel-7402 cell lines, with micromolar IC values (1.00–10.0 µM) higher than 1.
5
0
The water solubility of compounds at both pH levels was higher than that of 1, with

the water solubility of corresponding hydrochlorides > 30 mg/mL. From the results of
evaluating the effects of the compounds 7b, 8c, 9c, 10c and 11c on tubulin in vitro,
we speculated that their targets were different from those of 1 and CA-4P. The in
vitro antitumor activity against HepG2 of compound 7b (IC : 3.98 µM) was
5
0
significantly lower than that of CA-4 (IC : 0.002 µM), but the inhibition rate of
5
0
7
b
•
HCl (20 mg, 54.6%) was higher than that of CA-4P (50 mg, 48.3%) in vivo. And
in another batch of in vivo antitumor activity testing, 9c HCl and 11c HCl at doses of
0 mg/kg resulted in 61.1% and 50.0% inhibition, respectively. Studies have shown
•
•
1
that derivatives with poor in vitro efficacy like 7b
•
HCl, 9c
•
HCl and 11c HCl are
•
efficient in vivo owing to their excellent biodistribution or novel antitumor
mechanism, which will be further explored in future research.
Modifications of the R substituent on the carbazole ring provided the following
new structure-activity relationships (SAR) information: (1) the 6 position on
carbazole rings were active variable sites, and the long chain nitrogen-containing
heterocyclic compounds derived from aldehydes and hydroxyl groups at the
6
-position R , both alkanes and ethers, were associated with a significant decrease in
1
antitumor activity in vitro; (2) the antitumor activities of compounds (8a–g)
containing two C atoms in the ether chain were equivalent to those of compounds
(9a–g) containing three C atoms, the length of the ether chain has little effect on in
vitro antitumor activity; (3) R is also a reactive variable group, and in vitro antitumor
3
activity decreased after introduction of the alkyl chain; (4) the in vitro antitumor
activity of compounds 8a, 9a, 10a and 11a is higher than that of other compounds in
respective series. The introduction of a larger group at the end of the alkyl chain may
decrease the activity in vitro, and the activity of compounds having different
nitrogen-containing heterocycles is equal; (5) at both pH 2.0 and 7.4, the solubilities
of all derivatives were much higher than those of 1, and the structures of
nitrogen-containing heterocycles improved the water solubility of the compounds.
4
4
. Experimental section
.1. Chemistry
Melting points were measured using a Mettler Toledo MP90 melting apparatus

1
13
without correction. The nuclear magnetic resonance ( H-NMR and C-NMR) spectra
were measured on a MERCURY-400 spectrometer using tetramethylsilane (TMS) as
the internal standard. Unless otherwise indicated, d-DMSO was the solvent used.
Mass spectra were obtained on an AutoSpec Ultima-Tof mass spectrometer with
electrospray ionization, and the relative intensity of each ion peak is presented as a
percentage (%). The microwave reactions were performed on a microwave reactor
from CEM Inc. Thin-layer chromatography (TLC) and preparative TLC plates were
used with silica gel GF254 (200-300 mesh) purchased from Qingdao Haiyang
Chemical Company. Medium-pressure column chromatography was performed with a
CombiFlash companion purification system. All chemical reagents and solvents were
obtained from Beijing Chemical Works or J&K.
4
4
.2. Synthesis
.2.1. Final compounds 7a-e
General procedures used to prepare 7a–e. To a solution of 5 (425 mg, 1.0 mmol) in
THF (10 mL), different heterocyclic amines (2 mmol) and Ti(OiPr) (0.36 mL, 1.2
4
mmol) were added at room temperature. After stirring for 1.0 h, sodium
cyanoborohydride (NaBH CN, 252 mg, 4.0 mmol) was added slowly, and the mixture
3
was stirred for 1.0–2.0 h. The reaction was quenched with cold water and subjected to
EtOAc extraction three times. The combined organics were washed with brine (10 mL)
and dried with Na SO . After removing the solvent in vacuo, the crude product was
2
4
purified by column chromatography to obtain the corresponding compounds.
.2.1.1.
4
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(morpholinomethyl)-9H-carbazole-3-s
ulfonamide (7a). Starting with 5 (425 mg, 1.0 mmol), morpholine (0.18 mL, 2.0
mmol) was added to produce 200 mg of 7a in 40.3% yield, white solid, mp 193–
1
1
95°C. HNMR (DMSO-d , 400 MHz) δppm 2.39 (4H, m), 3.43 (3H, s), 3.58 (4H,
6
m), 3.63 (2H, s), 3.72 (3H, s), 3.91 (3H, s), 6.29 (1H, d, J = 8.4 Hz), 7.43 (1H, d, J =
.4 Hz), 7.50 (1H, dd, J = 8.4, 1.2 Hz), 7.62 (1H, d, J = 8.4 Hz), 7.70 (1H, d, J = 8.4
Hz), 7.74 (1H, dd, J = 8.4, 1.2 Hz), 8.12 (1H, d, J = 1.2 Hz), 8.46 (1H, d, J = 1.2 Hz),
.31 (1H, s); 13CNMR (DMSO-d , 101 MHz) δppm 29.83, 53.40, 53.59, 53.85,
8
9
6

6
1
4
7
7
3.10, 66.69, 101.13, 109.59, 110.06, 112.73, 120.23, 121.43, 121.47, 121.91, 124.68,
28.60, 129.83, 130.78, 139.69, 141.18, 142.91, 157.17, 160.64; HRMS (ESI+)
97.18525 calculated for C H O N S 497.18532 [M+H] +.
2
5
29
5
4
a•HCl
a (100 mg, 0.20 mmol) was directly dissolved in methanol (5 mL), 3 M
HCl-methanol (0.04 mL) was added, and the solution was stirred in an ice bath for 1 h.
After removing the solvent, the residue was purified by added anhydrous ether, and
the solution was stirred at room temperature for 0.5 h and filtered to obtain the
corresponding approximately 100 mg of the hydrochloride salt 7a •HCl. MS m/z (%)
4
97 (M+1, 100).
4
.2.1.2.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-((4-methylpiperazin-1-yl)methyl)-9H-
carbazole-3-sulfonamide (7b). Starting with 5 (425 mg, 1.0 mmol), N-methyl
piperazine (0.22 mL, 2.0 mmol) was added to produce 250 mg of 7b in 49.1% yield,
1
white solid, mp 177.5–179.5°C. HNMR (DMSO-d , 400 MHz) δppm 2.17 (3H, s),
6
2
.22-2.46 (8H, m), 3.43 (3H, s), 3.63 (2H, s), 3.72 (3H, s), 3.92 (3H, s), 6.30 (1H, d, J
8.4 Hz), 7.43 (1H, d, J = 8.4 Hz), 7.49 (1H, dd, J = 8.4, 1.2 Hz), 7.62 (1H, d, J = 8.4
Hz), 7.71 (1H, d, J = 8.4 Hz), 7.74 (1H, dd, J = 8.4, 1.2 Hz), 8.11 (1H, d, J = 1.2 Hz),
=
1
3
8
4
1
1
.46 (1H, d, J = 1.2 Hz), 9.32 (1H, s); CNMR (DMSO-d , 101 MHz) δppm 29.83,
6
6.13, 52.87, 53.40, 53.85, 55.18, 62.68, 101.13, 109.57, 110.03, 112.74, 120.20,
21.27, 121.49, 121.89, 124.65, 128.52, 130.34, 130.76, 139.67, 141.15, 142.90,
57.16, 160.63; HRMS (ESI+) 510.21684 calculated for C H O N S 510.21695
2
6
32
4
5
[M+H] +.
7
b•HCl
This compound was synthesized using the same procedure described for 7a with 7b
100 mg, 0.20 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 7b HCl. MS m/z (%) 510 (M+1, 100).
.2.1.3.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(piperidin-1-ylmethyl)-9H-carbazole-3
sulfonamide (7c). Starting with 5 (425 mg, 1.0 mmol), piperidine (0.20 mL, 2.0
(
•
4
-

mmol) was added to produce 300 mg of 7c in 60.7% yield, white solid, mp 213–
1
2
15°C. HNMR (DMSO-d , 400 MHz) δppm 1.39 (2H, m), 1.47-1.50 (4H, m), 2.36
6
(
4H, m), 3.43 (3H, s), 3.59 (2H, s), 3.72 (3H, s), 3.91 (3H, s), 6.29 (1H, d, J = 8.4 Hz),
.43 (1H, d, J = 8.4 Hz), 7.49 (1H, d, J = 8.4Hz), 7.61 (1H, d, J = 8.4 Hz), 7.71 (1H, d,
7
1
3
J = 8.4 Hz), 7.74 (1H, d, J = 8.4Hz), 8.09 (1H, s), 8.46 (1H, s), 9.32 (1H, s); CNMR
(
DMSO-d , 101 MHz) δppm 24.55, 26.04, 29.83, 53.40, 53.84, 54.25, 63.45, 101.13,
6
1
1
09.55, 109.96, 112.75, 120.19, 121.21, 121.51, 121.86, 124.63, 128.52, 130.62,
30.74, 139.65, 141.11, 142.89, 157.15; HRMS (ESI+) 495.20596 calculated for
C H O N S 495.20605 [M+H] +.
2
6
31
4
4
7
c
•HCl
This compound was synthesized using the same procedure described for 7a with 7c
100 mg, 0.20 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 7c HCl. MS m/z (%) 495 (M+1, 100).
.2.1.4.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(pyrrolidin-1-ylmethyl)-9H-carbazole-
-sulfonamide (7d). Starting with 5 (425 mg, 1.0 mmol), pyrrolidine (0.17 mL, 2.0
(
•
4
3
mmol) was added to produce 200 mg of 7d in 41.7% yield, white solid, mp 182–
1
1
84°C. HNMR (DMSO-d , 400 MHz) δppm 1.71 (4H, m), 2.48 (4H, m), 3.43 (3H,
6
s), 3.72 (3H, s), 3.75 (2H, s), 3.91 (3H, s), 6.29 (1H, d, J = 8.4 Hz),7.43 (1H, d, J =
.4 Hz), 7.50 (1H, dd, J = 8.4, 1.2 Hz), 7.60 (1H, d, J = 8.4 Hz), 7.70 (1H, d, J = 8.4
Hz), 7.74 (1H, dd, J = 8.4, 1.2 Hz), 8.11 (1H, s), 8.45 (1H, d, J = 1.2Hz), 9.31 (1H, s);
8
1
3
CNMR (DMSO-d , 100 MHz) δppm 23.55, 29.84, 53.39, 53.83, 53.84, 60.04,
6
1
1
01.12, 109.59, 110.01, 112.76, 120.20, 120.91, 120.95, 121.53, 121.92, 124.65,
28.30, 130.78, 139.64, 141.11, 142.92, 157.16, 160.64; HRMS (ESI+) 481.19019
calculated for C H O N S 481.19040 [M+H] +.
2
5
29
4
4
7
d
•
HCl
This compound was synthesized using the same procedure described for 7a with 7d
100 mg, 0.21 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 7d HCl. MS m/z (%) 481 (M+1, 100).
.2.1.5.
(
•
4

N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(piperazin-1-ylmethyl)-9H-carbazole-
-sulfonamide (7e). Starting with 5 (425 mg, 1.0 mmol), piperazine (172 mg, 2.0
3
mmol) was added to produce 250 mg of 7e in 50.5% yield, white solid, mp 168–
1
1
70°C. HNMR (DMSO-d , 400 MHz) δppm 2.41, 2.95 (4H, m), 3.34 (4H, m), 3.44
6
(
3H, s), 3.68 (2H, s), 3.73 (3H, s), 3.92 (3H, s), 6.30 (1H, d, J = 8.4 Hz), 7.43 (1H, d,
J = 8.4 Hz), 7.50 (1H, dd, J = 8.4Hz, 1.2 Hz), 7.63 (1H, d, J = 8.4 Hz), 7.72 (1H, d, J
8.4 Hz), 7.76 (1H, dd, J = 8.8 Hz, 1.4 Hz), 8.14 (1H, d, J = 1.2 Hz), 8.46 (1H, d, J =
=
1
3
1
6
1
4
7
.2 Hz); CNMR (DMSO-d , 100 MHz) δppm 29.86, 44.53, 51.49, 53.42, 53.87,
6
2.70, 101.13, 109.66, 110.08, 112.76, 120.24, 121.44, 121.49, 121.94, 124.73,
28.61, 129.61, 130.84, 139.72, 141.22, 142.91, 157.22, 160.65; HRMS (ESI+)
96.20117 calculated for C H O N S 496.20130 [M+H] +.
2
5
30
4
5
e•HCl
This compound was synthesized using the same procedure described for 7a with 7e
100 mg, 0.20 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 7e HCl. MS m/z (%) 496 (M+1, 100).
(
•
4
4
.2.2. Final compounds 8a-g
.2.2.1.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-chloroethoxy)-9H-carbazole-3-sulfo
namide (8a). To a solution of 6 (413 mg, 1.0 mmol) and 1-bromo-2-chloroethane
(
0.17 mL, 2.0 mmol) in ACE, K O (276 mg, 2.0 mmol) was added, and the mixture
2 3
was refluxed for 5 h. The reaction mixture was concentrated under reduced pressure,
quenched with cold water, and then subjected to EtOAc extraction three times. Then,
the combined organics were washed with brine (10 mL) and dried with Na SO . After
2
4
removing the solvent in vacuo, the crude product was purified by column
chromatography (PE/EtOAc = 3/1) to give 354 mg of 8a in 74.5% yield, white solid,
1
mp 118–120°C. HNMR (DMSO-d , 400 MHz) δppm 3.29 (3H, s), 3.61 (2H, t, 6.0
6
Hz), 3.79 (2H, t, 6.0 Hz), 3.81 (3H, s), 3.88 (3H, s), 6.40 (1H, d, J = 8.4 Hz), 7.05 (1H,
dd, J = 8.8, 2.4 Hz), 7.49 (1H, d, J = 8.8 Hz), 7.50 (1H, d, J = 8.4 Hz), 7.56 (1H, d, J
=
2.4 Hz), 7.60 (1H, dd, J = 8.8, 1.8 Hz), 7.65 (1H, d, J = 8.8 Hz), 8.33 (1H, d, J = 1.8
1
3
Hz), 9.20 (1H, s); CNMR (DMSO-d , 101 MHz) δppm 29.81, 43.29, 51.19, 53.43,
6

5
1
4
4.07, 101.62, 105.97, 109.42, 110.84, 113.70, 116.70, 120.80, 121.63, 122.91,
24.81, 128.45, 136.00, 143.30, 144.84, 152.10, 159.21, 162.32; HRMS (ESI+)
76.10407 calculated for C H O N ClS 476.10415 [M+H] +.
2
2
23
5
3
General procedure to prepare 8b-g
Sodium iodide and different heterocyclic amines were added to 8a in DMF (15 mL),
and the mixture was refluxed overnight. The reaction was quenched with cold water,
and subjected to EtOAc extraction three times. Then, the combined organics were
washed with brine (10 mL) and dried with Na SO . After removing the solvent in
2
4
vacuo, the crude product was purified by column chromatography to obtain the
corresponding compounds.
4
.2.2.2.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-morpholinoethoxy)-9H-carbazole-3
sulfonamide (8b). Starting with 8a (475 mg, 1.0 mmol), sodium iodide (1.5 g, 10
-
mmol) and morpholine (0.87 mL, 10 mmol) were added to produce 300 mg of 8b in
1
5
7.0% yield, white solid, mp 88–90°C. HNMR (DMSO-d , 600 MHz) δppm 2.25
6
(4H, m), 2.31 (2H, t, J = 6.4 Hz), 3.41 (3H, s), 3.43 (4H, m), 3.59 (2H, t, J = 6.4 Hz),
3
7
.83 (3H, s), 3.88 (3H, s), 6.36 (1H, d, J = 8.4 Hz), 7.06 (1H, dd, J = 8.8 Hz, 2.4 Hz),
.45 (1H, d, J = 8.4 Hz), 7.49 (1H, d, J = 8.8 Hz), 7.57 (1H, d, J = 2.4 Hz), 7.66 (2H,
1
3
m), 8.36 (1H, s), 9.20 (1H, s); CNMR (DMSO-d , 150 MHz) δppm 29.80, 46.34,
6
5
1
1
8
3.44, 53.56, 54.01, 57.20, 66.54, 101.30, 105.95, 109.37, 110.80, 114.35, 116.64,
20.72, 121.59, 122.92, 124.86, 128.96, 135.98, 143.17, 144.49, 152.05, 159.54,
61.99; HRMS (ESI+) 527.19568 calculated for C H O N S 527.19588 [M+H] +.
2
6
31
6
4
b•HCl
This compound was synthesized using the same procedure described for 7a with 8b
100 mg, 0.19 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 8b HCl. MS m/z (%) 527 (M+1, 100).
.2.2.3.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-(4-methylpiperazin-1-yl)ethoxy)-9H
(
•
4
-carbazole-3-sulfonamide (8c). Starting with 8a (475 mg, 1.0 mmol), sodium iodide
(1.5 g, 10 mmol) and N-methyl piperazine (1.1 mL, 10 mmol) were added to produce

1
3
05 mg of 8c in 56.5% yield, white solid, mp 84–86°C. HNMR (CDCl , 400 MHz)
3
δppm 2.07 (3H, s), 2.21-2.25 (4H, m), 2.30 (2H, m), 3.33-3.37 (4H, m), 3.41 (3H, s),
3
2
7
2
1
1
5
8
.57 (2H, m), 3.82 (3H, s), 3.88 (3H, s), 6.35 (1H, d, J = 8.4 Hz),7.05 (1H, dd, J = 8.8,
.4 Hz), 7.42 (1H, d, J = 8.4 Hz), 7.49 (1H, d, J = 8.8 Hz), 7.57 (1H, d, J = 2.4 Hz),
1
3
.66 (2H, m), 8.35 (1H, s), 9.20 (1H, s); CNMR (DMSO-d , 101 MHz) δppm
6
9.79, 46.16, 46.65, 52.96, 53.43, 53.99, 55.06, 56.84, 101.29, 105.96, 109.36, 110.78,
14.40, 116.63, 120.71, 121.59, 122.92, 124.86, 128.98, 135.98, 143.17, 144.42,
52.04, 159.55, 161.95; HRMS (ESI+) 540.22742 calculated for C H O N S
2
1
228
5
3
40.22752 [M+H] +.
HCl
c
•
This compound was synthesized using the same procedure described for 7a with 8c
100 mg, 0.18 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 8c HCl. MS m/z (%) 540 (M+1, 100).
.2.2.4.
(
•
4
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-(piperidin-1-yl)ethoxy)-9H-carbazo
le-3-sulfonamide (8d). Starting with 8a (475 mg, 1.0 mmol), sodium iodide (1.5 g, 10
mmol) and piperidine (0.99 mL, 10 mmol) were added to produce 300 mg of 8d in
1
5
7.3% yield, white solid, mp 93–95°C. HNMR (DMSO-d , 400 MHz) δppm 1.30
6
(
2H, m), 1.36 (4H, m), 2.22 (4H, m), 2.29 (2H, m), 3.41 (3H, s), 2.31 (2H, m), 3.82
3H, s), 3.89 (3H, s), 6.36 (1H, d, J = 8.4 Hz), 7.06 (1H, dd, J = 8.8 Hz, 2.0 Hz), 7.41
1H, d, J = 8.4 Hz), 7.50 (1H, d, J = 8.8 Hz), 7.57 (1H, d, J = 2.0 Hz), 7.66 (2H, s),
(
(
1
3
8
4
1
1
8
.35 (1H, s), 9.21 (1H, s); CNMR (DMSO-d , 101 MHz) δppm 24.32, 25.89, 29.80,
6
6.75, 53.43, 54.01, 54.38, 57.57, 101.28, 105.95, 109.37, 110.80, 114.41, 116.63,
20.72, 121.58, 122.93, 124.87, 128.98, 135.98, 143.18, 144.47, 152.06, 159.53,
61.97; HRMS (ESI+) 525.21637 calculated for C H O N S 525.21662 [M+H] +.
2
7
33
5
4
d•HCl
This compound was synthesized using the same procedure described for 7a with 8d
100 mg, 0.19 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 8d HCl. MS m/z (%) 525 (M+1, 100).
.2.2.5.
(
•
4

N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-(pyrrolidin-1-yl)ethoxy)-9H-carbaz
ole-3-sulfonamide (8e). Starting with 8a (475 mg, 1.0 mmol), sodium iodide (1.5 g,
1
5
0 mmol) and pyrrolidine (0.85 mL, 10 mmol) were added to produce 300 mg of 8e in
1
8.8% yield, white solid, mp 98–100°C. HNMR (DMSO-d , 400 MHz) δppm 1.60
6
(
4H, m), 2.36 (4H, m), 2.43 (2H, t, 6.0 Hz), 3.40 (3H, s), 3.58 (2H, t, 6.0 Hz), 3.82
3H, s), 3.86 (3H, s), 6.37 (1H, d, J = 8.4 Hz), 7.06 (1H, dd, J = 8.8 Hz, 2.0 Hz), 7.43
1H, d, J = 8.4 Hz), 7.50 (1H, d, J = 8.8 Hz), 7.57 (1H, d, J = 2.0 Hz), 7.66 (2H, s),
(
(
1
3
8
5
1
.35 (1H, s), 9.21 (1H, s); CNMR (DMSO-d , 101 MHz) δppm 23.56, 29.79, 48.30,
6
3.45, 53.89, 54.03, 54.55, 101.39, 105.94, 109.38, 110.81, 114.20, 116.64, 120.73,
21.58, 122.93, 124.85, 128.91, 135.98, 143.19, 144.49, 152.07, 159.50, 162.05;
HRMS (ESI+) 511.20088 calculated for C H O N S 511.20097 [M+H] +.
2
6
31
5
4
8
e
•
HCl
This compound was synthesized using the same procedure described for 7a with 8e
100 mg, 0.20 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 8e HCl. MS m/z (%) 511 (M+1, 100).
.2.2.6.
(
•
4
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-(piperazin-1-yl)ethoxy)-9H-carbazo
le-3-sulfonamide (8f). Starting with 8a (475 mg, 1.0 mmol), sodium iodide (1.5 g, 10
mmol) and piperazine (860 mg, 10 mmol) were added to produce 318 mg of 8f in 60.6%
1
yield, white solid, mp 130–132°C. HNMR (DMSO-d , 400 MHz) δppm 2.27 (4H,
6
m), 2.31 (2H, t, J = 6.4 Hz), 2.66 (4H, m), 3.40 (3H, s), 3.57 (2H, t, J = 6.4 Hz), 3.82
(3H, s), 3.88 (3H, s), 6.36 (1H, d, J = 8.4 Hz), 7.05 (1H, dd, J = 8.8, 2.4 Hz), 7.44 (1H,
d, J = 8.4 Hz), 7.49 (1H, d, J = 8.8 Hz), 7.56 (1H, d, J = 2.4 Hz), 7.64-7.66 (2H, m),
1
3
8
5
1
.34 (1H, s), 9.22 (1H, s); CNMR (DMSO-d , 101 MHz) δppm 29.33, 44.59, 46.00,
6
2.35, 52.97, 53.55, 56.69, 100.85, 105.48, 108.91, 110.35, 113.87, 116.18, 120.24,
21.12, 122.44, 124.38, 128.46, 135.51, 142.71, 144.07, 151.60, 159.05, 161.53;
HRMS (ESI+) 526.21179 calculated for C H O N S 526.21187 [M+H] +.
2
6
32
5
5
8
f
•
HCl
This compound was synthesized using the same procedure described for 7a with 8f
100 mg, 0.19 mmol) instead of 7a to obtain approximately 100 mg of the
(

corresponding hydrochloride salt 8f•HCl. MS m/z (%) 526 (M+1, 100).
4
.2.2.7.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-(4-(pyrrolidin-1-yl)piperidin-1-yl)et
hoxy)-9H-carbazole-3-sulfonamide (8g). Starting with 8a (475 mg, 1.0 mmol),
sodium iodide (1.5 g, 10 mmol) and 4-(1-pyrrolidinyl) piperidine (1.54 g, 10 mmol)
were added to produce 325 mg of 8g in 54.8% yield, white solid, mp 123–125°C.
1
HNMR (DMSO-d , 400 MHz) δppm 1.17-1.23 (2H, m), 1.61-1.62 (4H, m), 1.65
6
(2H, m), 1.79-1.84 (4H, m), 2.06 (1H,s), 2.27 (2H, m), 2.37 (4H, m), 3.41 (3H, s),
3
2
7
2
1
1
.57 (2H, m), 3.82 (3H, s), 3.88 (3H, s), 6.36 (1H, d, J = 8.4 Hz), 7.05 (1H, dd, J = 8.8,
.4 Hz), 7.42 (1H, d, J = 8.4 Hz), 7.48 (1H, d, J = 8.8 Hz), 7.56 (1H, d, J = 2.4 Hz),
1
3
.64-7.66 (2H, m), 8.35 (1H, s), 9.25 (1H, s); CNMR (DMSO-d , 101 MHz) δppm
6
3.33, 29.79, 31.27, 45.63, 46.88, 51.17, 52.27, 53.45, 54.01, 61.40,101.29, 105.92,
09.38, 110.79, 114.35, 116.63, 120.71, 121.61, 122.93, 124.86, 129.02, 135.98,
43.18, 144.46, 152.07, 159.56, 161.98; HRMS (ESI+) 594.27466 calculated for
C H O N S 594.27447 [M+H] +.
3
1
40
5
5
8
g
•HCl
This compound was synthesized using the same procedure described for 7a with 8g
100 mg, 0.017 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 8g HCl. MS m/z (%) 594 (M+1, 100).
(
•
4
4
6
.2.3. Final compounds 9a-g
.2.3.1.
-(3-chloropropoxy)-N-(2,6-dimethoxypyridin-3-yl)-9-methyl-9H-carbazole-3-sul
fonamide (9a). To a solution of 6 (413 mg, 1.0 mmol) and 1-bromo-3-chloropropane
0.20 mL, 2.0 mmol) in ACE (10 mL), K O (276 mg, 2.0 mmol) was added, and the
(
2
3
mixture was refluxed for 5 h. The reaction mixture was concentrated under reduced
pressure, quenched with cold water, and then subjected to EtOAc extraction three
times. The combined organics were washed with brine (10 mL) and dried with
Na SO . After removing the solvent in vacuo, the crude product was purified by
2
4
column chromatography (PE/EtOAc = 4/1) to give 355 mg of 9a in 72.6% yield,
1
white solid, mp 148–150°C. HNMR (DMSO-d , 400 MHz) δppm 1.78 (2H, m),
6

3
.44 (3H, s), 3.60 (2H, t, J = 6.4 Hz), 3.66 (2H, t, J = 6.4 Hz), 3.83 (3H, s), 3.89 (3H,
s), 6.37 (1H, d, J = 8.4 Hz), 7.06 (1H, dd, J = 8.8, 2.4 Hz), 7.41 (1H, d, J = 8.4 Hz),
.50 (1H, d, J = 8.8 Hz), 7.56 (1H, d, J = 2.4 Hz), 7.63 (1H, dd, J = 8.8, 2.0 Hz), 7.68
7
1
3
(
1H, d, J = 8.8 Hz), 8.32 (1H, d, J = 2.0 Hz), 9.21 (1H, s); CNMR (DMSO-d , 151
6
MHz) δppm 29.81, 31.86, 42.82, 47.14, 53.56, 54.06, 101.64, 105.91, 109.50, 110.85,
1
1
4
14.12, 116.71, 120.77, 121.63, 122.90, 124.82, 128.40, 136.00, 143.25, 143.95,
52.10, 159.71, 162.14;HRMS (ESI+) 490.11980 calculated for C H O N ClS
2
3
25
5
3
90.11980 [M+H] +.
General procedure to prepare 9b–g:
To a solution of 9a (1.0 mmol) in CH CN or DMF (15 mL), added different
3
heterocyclic amines were added, and the mixture was refluxed for 3–24 h. The
reaction was quenched with cold water and subjected to EtOAc extraction three times.
The combined organics were washed with brine (10 mL) and dried with Na SO .
2
4
After removing the solvent in vacuo, the crude product was purified by column
chromatography to obtain the corresponding compounds.
4
.2.3.2.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(3-morpholinopropoxy)-9H-carbazole
3-sulfonamide (9b). Starting with 9a (489 mg, 1.0 mmol), morpholine (0.87 mL, 10
-
mmol) was added to produce 290 mg of 9b in 53.7% yield, white solid, mp 85–87°C.
1
HNMR (CDCl , 400 MHz) δppm 1.45 (2H, m), 2.19 (4H, m), 2.24 (2H, t, J = 6.0
3
Hz), 3.44 (4H, m), 3.46 (3H, s), 3.49 (2H, t, J = 6.0 Hz), 3.83 (3H, s), 3.88 (3H, s),
6
.38 (1H, d, J = 8.4 Hz), 7.06 (1H, dd, J = 8.4, 2.0 Hz), 7.43 (1H, d, J = 8.4 Hz), 7.50
1H, d, J = 8.8 Hz), 7.55 (1H, d, J = 2.0 Hz), 7.64 (1H, dd, J = 8.4, 1.2 Hz), 7.67 (1H,
d, J = 8.8 Hz), 7.95 (1H, s), 8.33 (1H, d, J = 1.2 Hz), 9.21 (1H, s); 13CNMR
DMSO-d , 101 MHz) δppm 25.57, 29.80, 47.54, 53.50, 53.59, 54.04, 55.44, 66.55,
(
(
6
1
1
01.50, 105.86, 109.46, 110.85, 114.24, 116.68, 120.76, 121.57, 122.89, 124.86,
28.68, 135.99, 143.19, 144.06, 152.09, 159.70, 162.02; HRMS (ESI+) 541.21143
calculated for C H O N S 541.21253 [M+H] +.
2
7
33
6
4
9
b•HCl
This compound was synthesized using the same procedure described for 7a with 9b

(
100 mg, 0.19 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 9b HCl. MS m/z (%) 541 (M+1, 100).
.2.3.3.
•
4
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(3-(4-methylpiperazin-1-yl)propoxy)-9
H-carbazole-3-sulfonamide (9c). Starting with 9a (489 mg, 1.0 mmol), N-methyl
piperazine (1.1 mL, 10 mmol) was added to produce 280 mg of 9c in 50.6% yield,
1
white solid, mp 96–98 °C. HNMR (CDCl , 400 MHz) δppm 1.43 (2H, m), 2.07 (3H,
3
s), 2.17 (4H, m), 2.19 (4H, m), 2.22 (2H, t, J = 6.8 Hz), 3.46 (3H, s), 3.47 (2H, t, J =
6
.8 Hz), 3.83 (3H, s), 3.88 (3H, s), 6.37 (1H, d, J = 8.4 Hz), 7.06 (1H, dd, J = 8.4, 2.2
Hz), 7.42 (1H, d, J = 8.4 Hz), 7.49 (1H, d, J = 8.8 Hz), 7.55 (1H, d, J = 2.2 Hz), 7.64
1H, dd, J = 8.8, 1.2 Hz), 7.67 (1H, d, J = 8.8 Hz), 8.32 (1H, d, J = 1.2 Hz), 9.24 (1H,
(
1
3
s); CNMR (DMSO-d , 101 MHz) δppm 25.95, 29.80, 46.16, 47.55, 52.93, 53.49,
6
5
1
4.04, 54.98, 55.14, 101.48, 105.88, 109.46, 110.82, 114.26, 116.67, 120.78, 121.57,
22.90, 124.85, 128.67, 135.98, 143.19, 144.04, 152.10, 159.69, 161.99; HRMS
(
ESI+) 554.24298 calculated for C H O N S 554.24317 [M+H] +.
28 36 5 5
9
c•HCl
This compound was synthesized using the same procedure described for 7a with 9c
100 mg, 0.18 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 9c HCl. MS m/z (%) 554 (M+1, 100).
.2.3.4.
(
•
4
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(3-(piperidin-1-yl)propoxy)-9H-carba
zole-3-sulfonamide (9d). Starting with 9a (489 mg, 1.0 mmol), piperidine (0.99 mL,
1
1
0 mmol) was added to produce 280 mg of 9d in 52.0% yield, white solid, mp 125–
1
27°C. HNMR (DMSO-d , 400 MHz) δppm 1.39 (8H, m), 2.18 (6H, m), 3.45 (3H,
6
s), 3.48 (2H, m), 3.83 (3H, s), 3.88 (3H, s), 6.38 (1H, d, J = 8.4 Hz), 7.06 (1H, dd, J =
.8, 2.4 Hz), 7.42 (1H, d, J = 8.4 Hz), 7.49 (1H, d, J = 8.8 Hz), 7.54 (1H, d, J = 2.4
Hz), 7.63 (1H, dd, J = 8.8, 2.4 Hz), 7.67 (1H, d, J = 8.8 Hz), 8.32 (1H, d, J = 2.4 Hz),
8
1
3
9
5
1
.20 (1H, s); CNMR (CDCl , 101 MHz) δppm 23.87, 25.11, 25.33, 29.49, 47.77,
3
3.09, 53.82, 54.41, 56.29, 101.24, 106.22, 107.97, 109.68, 113.46, 116.23, 120.95,
21.89, 123.16, 125.08, 128.93, 136.43, 143.20, 143.72, 150.56, 159.47, 162.30;

HRMS (ESI+) 539.23197 calculated for C H O N S 539.23227 [M+H] +.
2
8
35
5
4
9
d
•
HCl
This compound was synthesized using the same procedure described for 7a with 9d
100 mg, 0.190 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 9d HCl. MS m/z (%) 539 (M+1, 100).
.2.3.5.
(
•
4
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(3-(pyrrolidin-1-yl)propoxy)-9H-carb
azole-3-sulfonamide (9e). Starting with 9a (489 mg, 1.0 mmol), pyrrolidine (0.85 mL,
1
9
0 mmol) was added to produce 290 mg of 9e in 55.3% yield, white solid, mp 88–
1
0°C. HNMR (DMSO-d , 400 MHz) δppm 1.45 (2H, m), 1.58 (4H, m), 2.26 (4H,
6
m), 2.34 (2H, t, J = 6.4 Hz), 3.45 (3H, s), 3.51 (2H, t, J = 6.4 Hz), 3.84 (3H, s), 3.88
3H, s), 6.38 (1H, d, J = 8.4 Hz), 7.06 (1H, dd, J = 8.4, 2.4 Hz), 7.41 (1H, d, J = 8.4
Hz), 7.50 (1H, d, J = 8.4 Hz), 7.55 (1H, d, J = 2.4 Hz), 7.63 (1H, dd, J = 8.4, 1.6 Hz),
(
1
3
7
1
1
1
.67 (1H, d, J = 8.4 Hz), 8.32 (1H, d, J = 1.6 Hz), 9.21 (1H, s); CNMR (DMSO-d ,
6
01 MHz) δppm 23.47, 27.91, 29.79, 47.79, 53.04, 53.47, 53.87, 54.04, 101.49,
05.88, 109.44, 110.83, 114.16, 116.66, 120.72, 121.57, 122.90, 124.84, 128.71,
35.98, 143.19, 144.08, 152.08, 159.70, 162.02; HRMS (ESI+) 525.21619 calculated
for C H O N S 525.21662 [M+H] +.
2
7
33
5
4
9
e
•
HCl
This compound was synthesized using the same procedure described for 7a with 9e
100 mg, 0.19 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 9e HCl. MS m/z (%) 525 (M+1, 100).
.2.3.6.
(
•
4
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(3-(piperazin-1-yl)propoxy)-9H-carba
zole-3-sulfonamide (9f). Starting with 9a (489 mg, 1.0 mmol), piperazine (860 mg,
1
1
0 mmol) was added to produce 280 mg of 9f in 51.9% yield, white solid, mp 118–
1
20°C. HNMR (DMSO-d , 400 MHz) δppm 1.40-1.47 (2H, m), 2.12 (4H, m), 2.20
6
(2H, m), 2.57 (4H, m), 3.46 (3H, s), 3.50 (2H, m), 3.84 (3H, s), 3.89 (3H, s), 6.31 (1H,
d, J = 8.4 Hz), 7.06 (1H, dd, J = 8.8 Hz, 2.2 Hz), 7.42 (1H, d, J = 8.4 Hz), 7.50 (1H, d,
J = 8.8 Hz), 7.55 (1H, d, J = 2.2 Hz), 7.64 (1H, dd, J = 8.8 Hz, 1.6 Hz), 7.67 (1H, d, J

1
3
=
8.8 Hz), 8.33 (1H, d, J = 1.6 Hz), 9.24 (1H, s); CNMR (DMSO-d , 101 MHz)
6
δppm 25.75, 29.80, 45.82, 47.63, 53.48, 54.04, 54.19, 55.75, 101.48, 105.86, 109.44,
1
1
10.83, 114.27, 116.67, 120.73, 121.58, 122.89, 124.85, 128.70, 135.98, 143.19,
44.05, 152.09, 159.68, 162.00 ； HRMS (ESI+) 540.22751 calculated for
C H O N S 540.22752 [M+H] +.
2
7
34
5
5
9
f
•
HCl
This compound was synthesized using the same procedure described for 7a with 9f
100 mg, 0.18 mmol) instead of 7a, to obtain approximately 100 mg of the
corresponding hydrochloride salt 9f HCl. MS m/z (%) 540 (M+1, 100).
.2.3.7.
(
•
4
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(3-(4-(pyrrolidin-1-yl)piperidin-1-yl)p
ropoxy)-9H-carbazole-3-sulfonamide (9g). Starting with 9a (489 mg, 1.0 mmol),
4
4
-(1-pyrrolidinyl) piperidine (1.54 g, 10 mmol) was added to produce 280 mg of 9g in
1
6.1% yield, white solid, mp 110–112°C. HNMR (DMSO-d , 400 MHz) δppm 1.20
6
(
2H, m), 1.42 (2H, m),1.62-1.66 (6H, m), 1.69-1.76 (2H, m), 1.80 (1H, m), 2.19 (2H,
m),2.38 (4H, m), 2.58 (2H, m), 3.47 (2H, m), 3.48 (3H, s),3.84 (3H, s), 3.89 (3H, s),
6
7
.38 (1H, d, J = 8.4 Hz), 7.06 (1H, dd, J = 8.8 Hz, 2.0 Hz), 7.44 (1H, d, J = 8.4 Hz),
.50 (1H, d, J = 8.8 Hz), 7.55 (1H, d, J = 2.0 Hz),7.67 (2H, s), 8.34 (1H, s), 9.25 (1H,
1
3
s); CNMR (DMSO-d , 101 MHz) δppm 23.35, 26.15, 29.79, 31.54, 47.62, 51.21,
6
5
1
1
9
2.27, 53.50, 54.03, 55.10, 61.64, 101.49, 105.87, 109.46, 110.81, 114.25, 116.65,
20.78, 121.58, 122.91, 124.86, 128.69, 135.98, 143.19, 144.04, 152.11, 159.71,
61.99; HRMS (ESI+) 608.28839 calculated for C H O N S 608.28839 [M+H] +.
3
2
42
5
5
g•HCl
This compound was synthesized using the same procedure described for 7a with 9g
100 mg, 0.16 mmol) instead of 7a to obtain approximately 100 mg of the
corresponding hydrochloride salt 9g HCl. MS m/z (%) 608 (M+1, 100).
(
•
4
4
6
.2.4. Final compounds 10a-e
.2.4.1.
-(2-chloroethoxy)-N-(2-chloroethyl)-N-(2,6-dimethoxypyridin-3-yl)-9-methyl-9H
-carbazole-3-sulfonamide (10a). To a solution of 6 (413 mg, 1.0 mmol) and

1
-bromo-2-chloroethane (0.33 mL, 4.0 mmol) in ACE (10 mL), K O (276 mg, 2.0
2 3
mmol) was added, and the mixture was refluxed for 8 h. The reaction mixture was
concentrated under reduced pressure, quenched with cold water, and then subjected to
EtOAc extraction three times. The combined organics were washed with brine (10 mL)
and dried with Na SO . After removing the solvent in vacuo, the crude product was
2
4
purified by column chromatography（PE/EtOAc = 5/1）to obtain 340 mg of 10a in
1
6
3.2% yield, white solid, mp 130–132°C. HNMR (CDCl , 400 MHz) δppm 3.29
3
(3H, s), 3.63 (2H, t, 5.8 Hz), 3.81 (3H, s), 3.81 (2H, t, 5.8 Hz), 3.93 (3H, s), 4.01 (2H,
t, J = 5.2 Hz), 4.38 (2H, t, J = 5.2 Hz), 6.41 (1H, d, J = 8.4 Hz), 7.22 (1H, dd, J = 8.8,
2
8
4
1
1
.0 Hz), 7.52 (1H, d, J = 8.4 Hz), 7.61 (1H, s), 7.63 (1H, s), 7.70 (1H, d, J = 8.8 Hz),
1
3
.04 (1H, d, J = 2.0 Hz), 8.57 (1H, s); CNMR (DMSO-d , 101 MHz) δppm 29.89,
6
3.25, 43.68, 51.22, 53.43, 54.07, 69.13, 101.67, 105.36, 109.64, 111.19, 113.66,
17.18, 121.32, 121.87, 122.73, 124.98, 128.91, 136.99, 143.32, 144.90, 153.06,
59.19, 162.34;HRMS (ESI+) 539.0998 calculated for C H Cl N O S 539.09967
2
5
25
2
3
5
[M+H] +.
General procedure to prepare 10b–e
Sodium iodide and different heterocyclic amines were added to 10a in DMF (15 mL),
and the mixture was refluxed overnight. The reaction was quenched with cold water
and subjected to EtOAc extraction three times. The combined organics were washed
with brine (10 mL) and dried with Na SO . After removing the solvent in vacuo, the
2
4
crude product was purified by column chromatography (DCM/MeOH/TEA = 50/1/1)
to obtain the corresponding compounds.
4
.2.4.2.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-morpholinoethoxy)-N-(2-morpholin
oethyl)-9H-carbazole-3-sulfonamide (10b). Starting with 10a (475 mg, 1.0 mmol),
sodium iodide (1.5 g, 10 mmol) and morpholine (0.87 mL, 10 mmol) were added to
1
produce 300 mg of 10b in 46.9% yield, white solid, mp 62–64°C. HNMR
(
DMSO-d , 400 MHz) δppm 2.25 (4H, m), 2.32 (2H, t, J = 6.4 Hz), 2.53 (4H, m),
6
2
3
.76 (2H, t, J = 6.4 Hz), 3.36, 3.58 (2H, t, J = 4.8 Hz), 3.39 (3H, s), 3.44 (4H, m),
.60 (4H, m), 3.82 (3H, s), 3.92 (3H, s), 4.21 (2H, t, J = 6.0 Hz), 6.36 (1H, d, J = 8.4

Hz), 7.18 (1H, dd, 8.8 Hz, 2.2 Hz), 7.45 (1H, d, J = 8.4 Hz), 7.58 (1H, d, J = 8.8 Hz),
.66 (1H, d, J = 8.8 Hz), 7.69 (1H, d, J = 8.8 Hz), 8.00 (1H, d, J = 2.2 Hz), 8.56 (1H,
7
1
3
s); CNMR (DMSO-d , 101 MHz) δppm 29.86, 46.36, 53.43, 53.57, 54.01, 54.14,
6
5
1
1
1
7.22, 57.66, 66.44, 66.54, 66.66 , 101.34, 104.98, 109.56, 111.04, 114.30, 117.09,
21.18, 121.86, 122.75, 124.94, 129.34, 136.73, 143.15, 144.59, 153.57, 159.55,
62.01; HRMS (ESI+) 640.27977 calculated for C H O N S 640.27995 [M+H] +.
3
2
42
7
5
0b HCl
•
This compound was synthesized using the same procedure described for 7a with 10b
100 mg, 0.16 mmol) instead of 7a, to obtain approximately 100 mg of the
corresponding hydrochloride salt 10b HCl. MS m/z (%) 640 (M+1, 100).
.2.4.3.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-(4-methylpiperazin-1-yl)ethoxy)-N-
2-(4-methylpiperazin-1-yl)ethyl)-9H-carbazole-3-sulfonamide (10c). Starting with
0a (475 mg, 1.0 mmol), sodium iodide (1.5 g, 10 mmol) and N-methyl piperazine
(
•
4
(
1
(1.1 mL, 10 mmol) were added to produce 325 mg of 10b in 48.9% yield, white solid,
1
mp 58–60°C. HNMR (DMSO-d , 400 MHz) δppm 2.07 (3H, s), 2.16 (3H, s),
6
2
.18-2.23 (4H, m), 2.26-2.31 (4H, m), 2.32-2.41 (4H, m), 2.51-2.58 (4H, m), 2.74 (2H,
t, J = 6.0 Hz), 3.36 (2H, t, J = 6.0 Hz), 3.39 (3H, s), 3.55 (2H, t, J = 6.0 Hz), 3.82 (3H,
s), 3.91 (3H, s), 4.18 (2H, t, J = 6.0 Hz), 6.35 (1H, d, J = 8.4 Hz), 7.17 (1H, dd, J =
8
8
.8 Hz, 2.2 Hz), 7.42 (1H, d, J = 8.4 Hz), 7.58 (1H, d, J = 8.8 Hz), 7.65 (1H, dd, J =
.8 Hz, 2.2 Hz), 7.69 (1H, d, J = 8.8 Hz), 8.00 (1H, d, J = 2.2 Hz), 8.56 (1H, s);
1
3
CNMR (DMSO-d , 101 MHz) δppm 29.86, 46.12, 46.19, 46.67, 52.93, 53.42,
6
5
1
1
6
1
3.51, 53.99, 55.04, 55.20, 56.85, 57.27, 66.73, 101.33, 104.99, 109.53, 111.01,
14.36, 117.09, 121.18, 121.88, 122.76, 124.92, 129.35, 136.71, 143.14, 144.52,
53.60, 159.55, 161.97; HRMS (ESI+) 666.34308 calculated for C H O N S
3
4
48
5
7
66.34321 [M+H] +.
0c HCl
•
This compound was synthesized using the same procedure described for 7a with 10c
100 mg, 0.15 mmol) instead of 7a, to obtain approximately 100 mg of the
corresponding hydrochloride salt 10c HCl. MS m/z (%) 666 (M+1, 100).
(
•

4
.2.4.4.
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-(piperidin-1-yl)ethoxy)-N-(2-(piperi
din-1-yl)ethyl)-9H-carbazole-3-sulfonamide (10d). Starting with 10a (475 mg, 1.0
mmol), sodium iodide (1.5 g, 10 mmol) and piperidine (0.99 mL, 10 mmol) were
1
added to produce 300 mg of 10d in 47.2% yield, white solid, mp 64–66°C. HNMR
(
DMSO-d , 400 MHz) δppm 1.39-1.42 (4H, m), 1.46-1.50 (8H, m), 1.57-1.62 (8H,
6
m), 2.27 (2H, m), 3.27 (2H, m), 3.38 (3H, s), 3.58 (2H, t, J = 6.0 Hz), 3.81 (3H, s),
.91 (3H, s), 4.19 (2H, t, J = 6.0 Hz), 6.35 (1H, d, J = 8.4 Hz), 7.17 (1H, dd, J = 8.8
3
Hz, 2.2 Hz), 7.42 (1H, d, J = 8.4 Hz), 7.58 (1H, d, J = 8.8 Hz), 7.65 (1H, dd, J = 8.8
Hz, 2.2 Hz), 7.69 (1H, d, J = 8.8 Hz), 8.00 (1H, d, J = 2.2 Hz), 8.56 (1H, J = 2.2 Hz);
1
3
CNMR (DMSO-d , 101 MHz) δppm 24.25, 25.85, 29.87, 46.73, 53.42, 54.00,
6
5
1
4.36, 54.70, 54.73, 57.56, 101.33, 105.03, 109.56, 111.03, 114.36, 117.11, 121.19,
21.86, 122.76, 124.95, 129.35, 136.75, 143.15, 144.56, 153.50, 159.51, 162.00;
HRMS (ESI+) 636.32111 calculated for C H O N S 636.32142 [M+H] +).
3
4
46
5
5
1
0d
This compound was synthesized using the same procedure described for 7a with 10d
100 mg, 0.16 mmol) instead of 7a, to obtain approximately 100 mg of the
corresponding hydrochloride salt 10d HCl. MS m/z (%) 636 (M+1, 100).
.2.4.5.
•
HCl
(
•
4
N-(2,6-dimethoxypyridin-3-yl)-9-methyl-6-(2-(pyrrolidin-1-yl)ethoxy)-N-(2-(pyrr
olidin-1-yl)ethyl)-9H-pyrido[2,3-b]indole-3-sulfonamide (10e). Starting with 10a
(475 mg, 1.0 mmol), sodium iodide (1.5 g, 10 mmol) and pyrrolidine (0.85 mL, 10
mmol) were added to produce 310 mg of 10e in 51.1% yield, white solid, mp 68–
1
7
0°C. HNMR (DMSO-d , 400 MHz) δppm 1.59 (4H, m), 1.69-1.72 (4H, m), 2.34
6
(
4H, m), 2.42 (2H, t, J = 6.4 Hz), 2.57 (4H, m), 2.85 (2H, t, J = 6.0 Hz), 3.22,3.44 (2H,
t, J = 6.4 Hz), 3.39 (3H, s), 3.82 (3H, s), 3.92 (3H, s), 4.19 (2H, t, J = 6.0 Hz), 6.36
1H, d, J = 8.4 Hz), 7.17 (1H, dd, 8.8 Hz, 2.2 Hz), 7.43 (1H, d, J = 8.4 Hz), 7.58 (1H,
d, J = 8.8 Hz), 7.65 (1H, d, J = 8.8 Hz), 7.67 (1H, d, J = 8.8 Hz), 7.80 (1H, d, J = 2.2
(
1
3
Hz), 8.57 (1H, s); CNMR (DMSO-d , 101 MHz) δppm 23.57, 23.60, 29.86, 43.13,
6
4
5.81, 48.34, 53.44, 53.90, 54.51, 54.92, 67.74, 101.41, 104.90, 109.53, 111.04,