New synthetic aliphatic sulfonamido-quaternary ammonium salts as anticancer chemotherapeutic agents

Article abstract of DOI:10.1016/j.ejmech.2013.09.022

RhoB is expressed during tumor cell proliferation, survival, invasion, and metastasis. In malignant progression, the expression levels of RhoB are commonly attenuated. RhoB is known to be linked to the regulation of the PI3K/Akt survival pathways. Based on aliphatic amido-quaternary ammonium salts that induce apoptosis via up-regulation of RhoB, we synthesized novel aliphatic sulfonamido-quaternary ammonium salts. These new synthetic compounds were evaluated for their biological activities using an in vitro RhoB promoter assay in HeLa cells, and in a growth inhibition assay using human cancer cell lines including PC-3, NUGC-3, MDA-MB-231, ACHN, HCT-15, and NCI-H23. Compound 5b (ethyl-dimethyl-{3-[methyl-(tetradecane-1-sulfonyl)-amino]-propyl}-ammonium; iodide) was the most promising anticancer agent in the series, based upon the potency of growth inhibition and RhoB promotion. These new aliphatic sulfonamido-quaternary ammonium salts could be a valuable series for development of new anticancer chemotherapeutic agents.

Full text of DOI:10.1016/j.ejmech.2013.09.022

European Journal of Medicinal Chemistry 69 (2013) 670e677
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New synthetic aliphatic sulfonamido-quaternary ammonium salts as
anticancer chemotherapeutic agents
,
Doona Song a, Jee Sun Yang a, Changmok Oh a, Shuolin Cui a e, Bo-Kyung Kim b,
, e,
Misun Won b, Jang-ik Lee c, Hwan Mook Kim d, Gyoonhee Han a
*
a Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
b Medical Genomics Research Center, KRIBB, Daejeon 305-806, Republic of Korea
c College of Pharmacy, Yonsei University, Incheon 406-840, Republic of Korea
d College of Pharmacy, Gachon University, Incheon 406-799, Republic of Korea
e Department of Integrated OMICS for Biomedical Sciences (WCU Program), Yonsei University, Seoul 120-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
RhoB is expressed during tumor cell proliferation, survival, invasion, and metastasis. In malignant pro-
gression, the expression levels of RhoB are commonly attenuated. RhoB is known to be linked to the
regulation of the PI3K/Akt survival pathways. Based on aliphatic amido-quaternary ammonium salts that
induce apoptosis via up-regulation of RhoB, we synthesized novel aliphatic sulfonamido-quaternary
ammonium salts. These new synthetic compounds were evaluated for their biological activities using
an in vitro RhoB promoter assay in HeLa cells, and in a growth inhibition assay using human cancer cell
lines including PC-3, NUGC-3, MDA-MB-231, ACHN, HCT-15, and NCI-H23. Compound 5b (ethyl-
dimethyl-{3-[methyl-(tetradecane-1-sulfonyl)-amino]-propyl}-ammonium; iodide) was the most
promising anticancer agent in the series, based upon the potency of growth inhibition and RhoB pro-
motion. These new aliphatic sulfonamido-quaternary ammonium salts could be a valuable series for
development of new anticancer chemotherapeutic agents.
Received 16 July 2013
Received in revised form
9 August 2013
Accepted 9 September 2013
Available online 21 September 2013
Keywords:
Aliphatic sulfonamido-quaternary
ammonium salts
Anticancer chemotherapeutic agents
Open-ring moiety of quaternary ammonium
salts
RhoB
Ó 2013 Elsevier Masson SAS. All rights reserved.
Perifosine
NSC126188
1. Introduction
The role of RhoB in human cancer is equivocal. Some studies
suggest that RhoB acts as a suppressor or negative modifier in
Cancer cells acquire a variety of characteristic changes during
carcinogenesis [1]. There is a change in expression of proteins and
signaling pathways which regulate cell growth, differentiation, and
development. Many of these proteins and signaling pathways
currently are under investigation as possible targets for cancer
therapy. Among different cancers, gastric cancer is a common
malignancy in many countries, and increasing evidence has re-
ported that gastric carcinogenesis displays multiple genetic alter-
ations including activation of oncogenes, inactivation of tumor
suppressor genes, loss of heterozygosity in chromosomes, and er-
rors in DNA replication [2,3].
cancer, as evidenced by the down-regulation of RhoB protein
expression in head and neck carcinoma and lung cancer [4e6]. Loss
of RhoB was found to be associated with an increased susceptibility
to chemical carcinogenesis in mice [7]. In addition, targeted dele-
tion of RhoB in mice increased tumor formation initiated by a Ras
mutation [1]. In gastric cancer cell lines, RhoB expression sup-
pressed the growth and migration of these cells, and enhanced the
chemosensitivity [8]. Conversely, RhoB was found to be overex-
pressed in breast tumors [9]. Overall, it is reported that RhoB is
associated with cell proliferation, survival, invasion, and metastasis
curing carcinogenesis, and its levels are commonly attenuated
during malignant progression.
The RhoB gene is a short-lived, early response inducible protein
involved in receptor endocytosis, apoptosis, and gene expression
[10]. In addition, it is rapidly up-regulated by a number of stimuli
such as UV irradiation, cytokines, growth factors, and steroid and
toxin treatments [11e13]. Expression of this gene is reduced by Ras
* Corresponding author. Department of Integrated OMICS for Biomedical Sciences
(WCU Program), Yonsei University, Seoul 120-749, Republic of Korea. Tel.: þ82 2
21232882; fax: þ82 2 3627265.
E-mail addresses: gyoonhee@yonsei.ac.kr, gyoonhee@gmail.com (G. Han).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.022

D. Song et al. / European Journal of Medicinal Chemistry 69 (2013) 670e677
671
via Akt/PKB, EGFR, and ErbB2 [14]. The function of RhoB also has
been linked to the regulation of PI3K/Akt survival pathways [15,16].
PI3K/Akt signaling is essential for tumor progression, because
numerous genetic lesions have been discovered in Akt signaling
components in human breast cancers and other solid tumors [17].
Furthermore, some studies reported that loss of RhoB decreases Akt
phosphorylation and blocks its nuclear translocation in stromal
endothelial cells [15]. In tumor cells, regulation of the Akt signaling
axis by RhoB controls invasion and migration [18,19].
3, 5, and 7 were confirmed by 1H and 13C NMR spectra and high
resolution mass spectrometry (HRMS). The newly prepared qua-
ternary ammonium salt analogs were characterized and evaluated
for in vitro biological activities and RhoB promoter activities.
3. Results and discussion
Twenty-four compounds synthesized using Schemes 1e3 were
assayed for their growth inhibitory activity against the human
cancer cell lines PC-3 (prostate cancer), NUGC-3 (gastric cancer),
MDA-MB-231 (breast cancer), ACHN (renal cancer), HCT-15 (colon
cancer), and NCI-H23 (non-small cell lung cancer). The GI50 values
from the sulforhodamine B (SRB) assay for the compounds are
listed in Table 1, showing that the synthesized compounds signifi-
cantly inhibited the gastric cancer cell line. Perifosine (Fig. 1), a
known PI3K/Akt inhibitor, and NSC126188 were used as positive
references to compare in vitro growth inhibitory activities of the
synthesized compounds. Structurally, perifosine and NSC126188
share a long aliphatic chain and the same quaternary ammonium
salt moiety with N,N-dimethyl substituent [24]. According to our
previous study, aliphatic amido-quaternary ammonium salt com-
pounds with 14 and 16 carbon chain lengths usually showed good
inhibitory activity [25]. To identify compounds with better activity
than known compounds (perifosine, NSC126188, and A913), a
structureeactivity relationship study was conducted with
emphasis on the sulfonyl group, diamine, and/or N-substituent of
novel aliphatic sulfonamido-quaternary ammonium salts.
In a previous study, we found that up-regulation of RhoB by
NSC126188 (Fig. 1) is mediated via c-Jun N-terminal kinase (JNK)
signaling [20], which constitutes the principle member of the
mitogen-activated protein kinase (MAPK) family [21,22]. JNK reg-
ulates many cellular processes, including cell proliferation, differ-
entiation, survival, and migration. There is also cross-talk and
integration with other signaling pathways in a cell specific manner
[22]. Recently, it was reported that an alkyl piperazine derivative
(NSC126188, Fig. 1) caused apoptosis via up-regulation of RhoB in
HeLa cells [23]. In addition, we previously reported synthesis and
biological activity of the aliphatic amido-quaternary ammonium
salt series as represented by allyl-diethyl-[2-(methyl-
tetradecanoyl-amino)-ethyl]-ammonium bromide (Fig. 1, A913)
which was synthesized as an anticancer agent, based on NSC126188
[24]. In the present study, novel aliphatic sulfonamido-quaternary
ammonium salts were designed and synthesized by isosteric
replacement of the functional group of NSC126188 and A913. A
structural activity relationship (SAR) study of these synthesized
compounds was also conducted, based on their RhoB promoter
assays in HeLa cells and their growth inhibitory activities in six
human cancer cell lines including PC-3, NUGC-3, MDA-MB-231,
ACHN, HCT-15, and NCI-H23. The results demonstrated that these
new aliphatic sulfonamido-quaternary ammonium salts could be
new compounds for development of effective anticancer chemo-
therapeutic agents.
First, based on A913 (14 carbon chain length, N,N-diethyl-N0-
methylethane-1,2-diamine, allyl group, GI50 ¼ 0.14
mM for the
NUGC-3 cancer cell line, GI50 ¼ 0.21 M for the prostate cancer cell
m
line) which showed the most potent inhibitory activity in the
previous study, analogs 3ae3f were designed and synthesized. We
fixed the carbon chain length at 14 and 16, which resulted in good
anti-proliferation activity. The carbonyl group of A913 was changed
to the sulfonyl group because sulfonyl group is more stable than
carbonyl group with regard to the chemistry although sulfonyl and
carbonyl group are isosteric. In addition, its R2 group (described on
Scheme 1) was substituted with the methyl, ethyl, or allyl group. Six
analogs showed lower growth inhibitory activity than A913 for the
NUGC-3 and PC-3 cell lines. However, they showed better anti-
proliferative activity than perifosine, especially for the NUGC-3
cell line. While A913 showed good growth inhibitory activities in
most cancer cell lines, six analogs exhibited selective anti-
proliferative activities for the gastric cancer cell line. In addition,
compounds 3c and 3e exhibited better growth inhibition activities
2. Chemistry
Based on structural characteristics of NSC126188 and A913, we
designed and synthesized 24 analogs with three major modifica-
tions. First, the carbonyl group was substituted with a sulfonyl
group. Second, diverse N-substituents were introduced such as
methyl, ethyl, and ally groups. Third, various aliphatic moieties
were introduced into quaternary ammonium salts. As illustrated in
Schemes 1e3, various sulfonate sodium salts 1 were reacted
with thionyl chloride to produce sulfonyl chlorides in situ, which
were coupled with N,N-diethyl-N0-methylethylenediamine, N,N,N0-
trimethylethylenediamine, N,N-dimethyl-1,3-propanediamine, N,
N,N0-trimethyl-1,3-diaminopropane, or N,N,2,2-tetramethyl-1,3-
propanediamine to yield sulfonamides 2ae2d, 4ae4d, and 6ae6b
(Schemes 1-a, 2-a, 3-a), which were confirmed by 1H nuclear
magnetic resonance (NMR) spectra. Sulfonamides were converted
to sulfonamido-quaternary ammonium salts, 3ae3l, 5ae5h, and
7ae7d, by N-alkylation using diverse alkyl halides (Schemes 1-b, 2-
b, 3-b). The structures of sulfonamido-quaternary ammonium salts
than NSC126188, with GI50 values of 0.21 and 0.22 mM, respectively,
in the NUCG-3 cell line. The results demonstrated that the substi-
tution of a sulfonyl group could increase the specificity of inhibitory
activities for a gastric cancer cell line.
Second, we assessed the effect of N-substituents by diverse alkyl
groups such as methyl, ethyl, and allyl. The effect of N-substituents
were compared among compounds with fixed carbon chain length
and N,N-dialkylethane diamine. The effect of N-substituents was
barely noticeable for compounds with N,N-diethylethane diamine
(3ae3f). In contrast, analogs with N,N-dimethylethane diamine
(3ge3l) displayed the effect of the N-substituent on the gastric
cancer cell line. Compounds with an ally group (3i, GI50 ¼ 0.83
m
M;
M) exhibited poorer inhibitory activity than com-
methyl group (3g, GI50 0.39 M; 3j,
M) or with an ethyl group (3h, GI50 ¼ 0.30 M; 3k,
M). When comparing two compounds with either a
3l, GI50 ¼ 0.76
m
pounds with
a
¼
m
m
GI50 ¼ 0.33
m
m
GI50 ¼ 0.36
A913
Perifosine
NSC126188
methyl group or ethyl group (3a and 3b, 3d and 3e, 3g and 3h, 3j
and 3k), most compounds with an ethyl group showed higher anti-
proliferative activity for the NUGC-3 cell line, except 3j and 3k.
These results demonstrated that a simple alkyl group, especially the
Fig. 1. Chemical structures of perifosine, NSC126188 (4-hexadecanoyl-1,1-dimethyl-
piperazin-1-ium iodide), and A913 (a representative analogue of the aliphatic amido-
quaternary ammonium salt series).

672
D. Song et al. / European Journal of Medicinal Chemistry 69 (2013) 670e677
Scheme 1. Reaction protocol for the synthesis of aliphatic sulfonamido-quaternary ammonium salts (3ae3l). Reagents and conditions: (a) i) SOCl2, CH2Cl2 at 60 ꢀC ii) N,N-dialkyl-
N0-methylethylenediamine, CH2Cl2 at 0 ꢀC (b) alkyl halide, CH3CN at 95e100 ꢀC or methyl iodide, toluene, 110e115 ꢀC.
ethyl group, on the N-substituent position induced inhibitory ac-
tivities more effectively.
displayed similar or lower anti-proliferation activities than 5ae5d
and NSC126188. Branched open-ring moieties were introduced to
7ae7d (fourth group, N,N,2,2-tetramethylpropane-1,3-diamine
derivatives), which decreased the inhibitory activity of the ana-
logs. Among four open-ring moiety groups, a group of N,N,N’-tri-
methylpropane-1,3-diamine compounds induced the most positive
effects on activity than any other groups, for both the gastric and
prostate cancer cell lines.
The synthesized compounds significantly suppressed cell pro-
liferation, especially for the gastric cancer cell line (NUGC-3) and
the prostate cancer cell line (PC-3) among the six cancer cell lines
tested. Introduction of a sulfonyl group instead of a carbonyl group
increased the specificity for the cancer cell lines. Analogs with an
introduced sulfonyl group showed more effective growth inhibition
activities for the gastric cancer cell lines than for any other cancer
cell line. In addition, the ethyl group on the N-substituent position
and N,N,N0-trimethylpropane-1,3-diamine, among open-ring moi-
eties, increased inhibitory activities for both the gastric and pros-
tate cancer cell lines. Among 24 compounds, compound 5b (N,N,N0-
trimethylpropane-1,3-diamine, R2 ¼ ethyl group) showed the best
growth inhibitory activity for both the NUGC-3 and PC-3 cancer cell
lines.
Third, to evaluate the effects of the open-ring moiety on
sulfonamido-quaternary ammonium salts, we introduced four
aliphatic groups such as N,N-dialkyl-N0-methylethylenediamine
(N,N-diethyl-N0-methylethylenediamine, N,N,N0-trimethylethane-
1,2-diamine), N,N-dimethylpropane-1,3-diamine, N,N,N0-trime-
thylpropane-1,3-diamine, and N,N,2,2-tetramethylpropane-1,3-
diamine (3ae3l, 5ae5h, 7ae7d). The first group of compounds
(N,N-dialkyl-N0-methylethylenediamine derivatives, 3ae3l), which
were synthesized with the same diamine moiety as A913, exhibited
similar or better growth inhibitory activity than A913. Compound
3i showed specific anti-proliferative activity for the PC-3 cancer cell
line and compound 3j displayed specificity for the NUGC-3 cancer
cell line. 5ae5d (second group, N,N,N0-trimethylpropane-1,3-
diamine derivatives) had longer carbon length of the open-ring
moiety than the first group of compounds. These compounds dis-
played remarkably more potent inhibitory activity than the refer-
ence compounds, NSC126188 and perifosine. Among them,
compound 5b (R2 ¼ ethyl group, described in Scheme 2) displayed
the most potent growth inhibition of all synthesized analogs for
both gastric and prostate cancer cell lines. 5ee5g (third group, N,N-
dimethylpropane-1,3-diamine derivatives), which had hydrogen at
the R3 position instead of a methyl group in the second group,
For this series of compounds, the activity of RhoB was deter-
mined using the luciferase reporter gene assay. The results are
Scheme 2. Reaction protocol for the synthesis of aliphatic sulfonamido-quaternary ammonium salts (5ae5h). Reagents and conditions: (a) i) SOCl2, CH2Cl2 at 60 ꢀC ii) N,N-
dimethyl-N0-alkylpropanediamine, CH2Cl2 at 0 ꢀC (b) alkyl halide, CH3CN at 95e100 ꢀC or methyl iodide, toluene, 110e115 ꢀC.

D. Song et al. / European Journal of Medicinal Chemistry 69 (2013) 670e677
673
Scheme 3. Reaction protocol for the synthesis of aliphatic sulfonamido-quaternary ammonium salts (7ae7d). Reagents and conditions: (a) i) SOCl2, CH2Cl2 at 60 ꢀC ii) N,N,2,2-
tetramethylpropane-1,3-diamine, CH2Cl2 at 0 ꢀC (b) alkyl halide, CH3CN at 95e100 ꢀC or methyl iodide, toluene, 110e115 ꢀC.
shown in Fig. 2. Most of the synthesized compounds displayed
better RhoB promoter activity than NSC126188. To assess the effects
on RhoB expression based on N-substituents, the analogs with an
N-ethyl group on the quaternary ammonium salts were compared
with analogs with an N-methyl group. In a manner similar to the
growth inhibition results, most analogs with an ethyl group
showed better expression than analogs with a methyl group. In
addition, RhoB promoter activity based on open-ring moieties also
displayed a similar tendency as the growth inhibitory activity.
Compounds with N,N-diethyl-N0-methylethylenediamine and
N,N,N0-trimethylpropane-1,3-diamine generally induced high RhoB
promoter activity. These results demonstrated that diamine and N-
substituents of new aliphatic sulfonamido-quaternary ammonium
salts showed the same qualitative effects for both RhoB expression
and growth inhibition.
assay. Using the growth inhibition assay, the sulfonyl group of an-
alogs was generally more effective for the gastric cancer cell line
than other tested cancer cell lines. Among 24 synthesized com-
pounds, the analogs with N-ethyl substituent and N,N,N0-trime-
thylpropane-1,3-diamine showed effective results on both the anti-
proliferative assay and the RhoB promoter assay. The compound 5b
was the most promising anticancer agent among this series, based
upon growth inhibition as well as activation of RhoB expression.
Together, the results show that synthetic aliphatic sulfonamido-
quaternary ammonium salts are remarkably potent reagents for
further development of anti-cancer chemotherapy.
4. Experimental section
All chemicals were obtained from commercial suppliers and
used without further purification. All reactions were monitored by
thin layer chromatography (TLC) on pre-coated silica gel 60 F254
(mesh) (E. Merck, Mumbai, India). Spots were visualized under UV
light (254 nm). Flash column chromatography was performed with
silica (E. Merck, EM9385, 230e400 mesh). 1H and 13C nuclear
magnetic resonance (NMR, Varian) spectra were recorded at 300
and 75 MHz, at 400 and 100 MHz, or at 500 and 125 MHz,
respectively. Proton and carbon chemical shifts were expressed in
ppm relative to the internal standard tetramethylsilane, and
coupling constants (J) were expressed in Hertz. Liquid chroma-
tographyemass spectrometry (LC/MS) spectra were recorded by
electrospray ionization (ESI) on a Shimazu LC/MS (Dong-il SHI-
MADZU Corp., Seoul, Korea) instruments (10% 0.1% TFA in H2O/90%
0.1% TFA in acetonitrile) in scan mode (from 0 to 600 amu/z). The
detected ion peaks are (Mþz)/z and (Mꢁz)/z in positive and negative
ion modes, respectively, where M represents the molecular weight
of the compound and z represents the charge (number of protons).
High resolution mass spectrometry (HRMS) spectra were obtained
from electrospray ionization (ESI) in positive mode using the
Micromass Q-TOF (Waters Corp., Seoul, Korea). The capillary and
sample cone voltages were 4000 V and 30 V, respectively, the
desolvation gas flow was 600 L/h at 200 ꢀC, and the source tem-
perature was 100 ꢀC, using the high resolution tandem mass
spectrometer at Yonsei University, Seoul, Korea.
Based on NSC126188 and A913 which share structural features,
we designed and synthesized
a new series of aliphatic
sulfonamido-quaternary ammonium salts and evaluated their ac-
tivities using the anti-proliferative assay and the RhoB promoter
Table 1
Growth inhibition of synthesized compounds.
Compound
Growth inhibition (
NUGC-3 PC-3 MDA-MB-231 ACHN HCT-15 NCI-H23
m
M)a
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
5a
5b
5c
5d
5e
5f
5g
5h
7a
7b
7c
0.52
0.32
0.21
0.39
0.22
0.39
0.39
0.30
0.83
0.33
0.36
0.76
0.15
0.13
0.21
0.20
0.38
0.29
0.56
0.32
0.32
0.51
0.43
0.57
0.54
0.82
0.52
0.53
0.56
0.52
0.76
0.47
0.34
0.46
0.62
0.57
0.62
0.30
0.22
0.44
0.36
0.45
0.30
0.58
0.56
0.35
0.46
0.56
0.79
0.44
0.48
0.21
1.22
0.87
0.71
1.12
1.17
1.34
0.92
0.99
1.80
0.93
0.54
1.35
1.26
0.75
1.04
0.69
1.40
1.17
1.39
0.91
2.11
0.72
1.52
2.79
2.86
1.44
0.30
1.74
1.46
1.35
1.29
1.31
1.45
1.40
1.30
1.39
1.60
1.75
3.48
1.01
0.54
1.00
0.95
2.04
1.64
1.89
1.29
2.60
2.52
2.21
2.27
4.56
1.04
0.10
2.35
3.46
2.03
1.49
1.61
1.81
0.75
1.61
1.71
0.70
1.26
4.73
0.68
1.27
0.78
0.78
0.94
1.14
0.86
1.20
1.70
NAb
0.56
1.35
1.25
0.58
NAb
0.71
0.47
0.30
0.62
0.55
0.45
2.04
0.59
0.73
1.29
1.81
0.89
0.21
0.38
0.91
0.43
0.46
0.33
1.56
0.91
0.31
0.73
0.74
0.69
4.21
2.34
0.24
4.1. General procedures for the synthesis of 2aed, 4aed, and 6aeb
Sulfonate sodium salts 1 (2.9 mmol) were dissolved in anhy-
drous dichloromethane (0.1 M solution), and thionyl chloride (3.0
equivalents) was added under an argon atmosphere. The stirred
suspension was heated and refluxed for 4 h. The reaction mixture
was cooled and poured onto crushed ice for 1 h. The compound
N,N,N0-alkyl-diamine (5.8 mmol) was added dropwise, and the re-
action mixture was allowed to warm to room temperature and
stirred for 2 h. NaOH solution (10%) was added (final pH 13), and the
7d
Perifosine
NSC126188 0.29
A913
0.14
a
Growth inhibition was measured by the SRB (sulforhodamine B) assay. The
values are means of three experiments.
b
NA, not active.

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D. Song et al. / European Journal of Medicinal Chemistry 69 (2013) 670e677
1
1
2.0
0.0
8.0
6.0
4.0
2.0
0.0
Fig. 2. Activation of RhoB promoter was determined by luciferase activity in cells transfected with the pGL2-RhoB-Luc in the presence of various compounds (5
(5 M) was used as a positive control.
mM). NSC126188
m
mixture was extracted with chloroform. The organic layer was
washed with brine, dried over anhydrous MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
silica gel with 5% MeOH/CHCl3 to resolve the corresponding
amides.
4.1.6. Hexadecane-1-sulfonic acid (3-dimethylamino-propyl)-
methyl-amide (4b)
Sodium 1-hexadecanesulfonate was used and 4b (54.2%) was
obtained. 1H NMR (500 MHz, DMSO-d6)
d
3.09 (t, J ¼ 7.3 Hz, 2H),
2.75 (s, 3H), 2.22 (t, J ¼ 6.7 Hz, 2H), 2.13 (s, 6H), 1.64e1.58 (m, 4H),
1.35 (br s, 2H), 1.24 (s, 26H), 0.85 (t, J ¼ 7.8 Hz, 3H); ESI-MS: m/
z ¼ 405 [MþH].
4.1.1. Tetradecane-1-sulfonic acid (2-diethylamino-ethyl)-methyl-
amide (2a)
Sodium 1-tetradecanesulfonate was used and 2a (53.8%) was
4.1.7. Tetradecane-1-sulfonic acid (3-dimethylamino-propyl)-
amide (4c)
obtained. 1H NMR (500 MHz, DMSO-d6)
d
3.14 (t, J ¼ 7.0 Hz, 2H),
3.06 (t, J ¼ 7.5 Hz, 2H), 2.84 (s, 3H), 2.52e2.44 (m, 6H),1.63e1.60 (m,
2H), 1.36e1.24 (m, 22H), 0.94 (t, J ¼ 7.5 Hz, 6H), 0.85 (t, J ¼ 6.8 Hz,
3H); ESI-MS: m/z ¼ 391 [MþH].
Sodium 1-tetradecanesulfonate was used and 4c (83.0%) was
obtained. 1H NMR (500 MHz, CDCl3)
d 7.03e7.01 (m, 1H), 2.97e2.91
(m, 4H), 2.42e2.40 (m, 2H), 2.25 (s, 6H), 1.64e1.58 (m, 2H), 1.24 (br
s, 22H), 0.85 (t, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 363 [MþH].
4.1.2. Hexadecane-1-sulfonic acid (2-diethylamino-ethyl)-methyl-
amide (2b)
4.1.8. Hexadecane-1-sulfonic acid (3-dimethylamino-propyl)-
amide (4d)
Sodium 1-hexadecanesulfonate was used and 2b (50.1%) was
obtained. 1H NMR (500 MHz, CDCl3)
d
3.58 (br m, 2H), 3.00e2.94
Sodium 1-hexadecanesulfonate was used and 4d (72.4%) was
(m, 9H), 2.22e2.10 (m, 2H), 1.82e1.76 (m, 2H), 1.42e1.38 (m, 4H),
obtained. 1H NMR (400 MHz, DMSO-d6)
d
6.96 (t, J ¼ 6.8 Hz, 1H),
1.26 (m, 28H), 0.88 (t, J ¼ 6.5 Hz, 3H); ESI-MS: m/z ¼ 419 [MþH].
2.93e2.86 (m, 4H), 2.21 (t, J ¼ 7.0 Hz, 2H), 2.08 (s, 6H), 1.61 (br m,
4H), 1.31 (br s, 2H), 1.20 (br s, 24H), 0.82 (t, J ¼ 6.6 Hz, 3H); ESI-MS:
m/z ¼ 391 [MþH].
4.1.3. Tetradecane-1-sulfonic acid (2-dimethylamino-ethyl)-
methyl-amide (2c)
Sodium 1-tetradecanesulfonate was used and 2c (88.3%) was
4.1.9. Tetradecane-1-sulfonic acid (3-dimethylamino-2,2-dimethyl-
propyl)-amide (6a)
obtained. 1H NMR (400 MHz, DMSO-d6)
d
3.16 (t, J ¼ 6.4 Hz, 2H),
3.03 (t, J ¼ 7.6 Hz, 2H), 2.78 (s, 3H), 2.36 (t, J ¼ 6.2 Hz, 2H), 2.14 (s,
6H), 1.65e1.55 (m, 2H), 1.38e1.30 (m, 2H), 1.29e1.18 (m, 20H), 0.84
(t, J ¼ 6.6 Hz, 3H); ESI-MS: m/z ¼ 363 [MþH].
Sodium 1-tetradecanesulfonate was used and 6a (94.0%) was
obtained. 1H NMR (500 MHz, CDCl3)
d 2.98e2.95 (m, 4H), 2.28e2.27
(m, 8H), 1.82e1.76 (m, 2H), 1.44e1.38 (m, 2H), 1.28e1.25 (m, 20H),
0.87 (t, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 391 [MþH].
4.1.4. Hexadecane-1-sulfonic acid (2-dimethylamino-ethyl)-
methyl-amide (2d)
4.1.10. Hexadecane-1-sulfonic acid (3-dimethylamino-2,2-
dimethyl-propyl)-amide (6b)
Sodium 1-hexadecanesulfonate was used and 2d (52.5%) was
obtained. 1H NMR (500 MHz, DMSO-d6)
d
3.17 (t, J ¼ 6.5 Hz, 2H),
Sodium 1-hexadecanesulfonate was used and 6b (91.2%) was
3.04 (t, J ¼ 8.0 Hz, 2H), 2.79 (s, 3H), 2.37 (t, J ¼ 6.5 Hz, 2H), 2.15 (s,
6H), 1.66e1.60 (m, 2H), 1.36e1.32 (m, 2H), 1.27e1.25 (m, 24H), 0.85
(t, J ¼ 6.8 Hz, 3H); ESI-MS: m/z ¼ 319 [MþH].
obtained. 1H NMR (500 MHz, DMSO-d6)
d 6.88e6.86 (m,1H), 2.94 (t,
J ¼ 8.0 Hz, 2H), 2.50e2.49 (m, 2H), 2.21 (s, 6H), 2.08 (s, 2H), 1.65e
1.59 (m, 2H), 1.24 (m, 26H), 0.85 (t, J ¼ 7.0 Hz, 3H), 0.82 (s, 6H); ESI-
MS: m/z ¼ 419 [MþH].
4.1.5. Tetradecane-1-sulfonic acid (3-dimethylamino-propyl)-
methyl-amide (4a)
4.2. General procedures for the synthesis of 3ael, 5aeh, and 7aed
Sodium 1-tetradecanesulfonate was used and 4a (37.7%) was
obtained. 1H NMR (400 MHz, DMSO-d6)
d
3.08 (t, J ¼ 6.8 Hz, 2H),
Alkyl halide (1.6 mmol) was added to a solution of 2aed, 4aed,
and 6aeb (0.8 mmol) in anhydrous acetonitrile (0.3 M) at room
temperature, and the reaction mixture was heated to w140e150 ꢀC
for 7 h. After the reaction mixture equilibrated to room temperature,
2.98 (t, J ¼ 7.0 Hz, 2H), 2.74 (s, 3H), 2.24 (t, J ¼ 6.4 Hz, 2H), 2.15 (s,
6H), 1.67e1.55 (m, 4H), 1.40e1.30 (m, 2H), 1.30e1.15 (m, 20H), 0.84
(t, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 377 [MþH].

D. Song et al. / European Journal of Medicinal Chemistry 69 (2013) 670e677
675
the precipitate was granulated for 1 h at 0 ꢀC. Solids were collected
by filtration, washed with ethyl acetate, and dried in vacuo.
2H), 1.24e1.18 (m, 20H), 0.85 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6) d 127.7, 125.8, 60.0, 54.3, 53.7, 48.5, 46.8, 43.3, 35.9, 31.7,
29.5, 29.5, 29.5, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.2, 23.0,
22.5, 14.4, 8.8, 7.7 ppm; ESI-MS: m/z ¼ 459 [Mþ]; HRMS (ESI) calcd.
for C26H55N2O2S (Mþ) 459.3979 found 439.3965.
4.2.1. Diethyl-methyl-{2-[methyl-(tetradecane-1-sulfonyl)-amino]-
ethyl}-ammonium; iodide (3a)
2a was used and 3a (78.9%) was obtained. 1H NMR (500 MHz,
DMSO-d6)
d
3.52 (t, J ¼ 7.3 Hz, 2H), 3.41 (t, J ¼ 7.3 Hz, 2H), 3.40e3.34
4.2.7. Trimethyl-{2-[methyl-(tetradecane-1-sulfonyl)-amino]-
ethyl}-ammonium; iodide (3g)
(m, 4H), 3.15e3.12 (m, 2H), 2.98 (s, 3H), 2.86 (s, 3H), 1.66e1.63 (m,
2H), 1.37e1.34 (m, 2H), 1.24e1.21 (m, 26H), 0.85 (t, J ¼ 7.0 Hz, 3H);
2c was used and 3g (68.7%) was obtained. 1H NMR (500 MHz,
DMSO-d6) 4.14 (br s, 2H), 3.83 (br s, 2H), 3.48 (s, 9H), 3.04 (t,
J ¼ 8.0 Hz, 2H), 1.83e1.80 (m, 2H), 1.45e1.42 (m, 2H), 1.31e1.27 (m,
13C NMR (100 MHz, DMSO-d6)
d 56.9, 56.5, 56.5, 48.5, 47.4, 43.6,
35.7, 31.7, 29.5, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.1, 23.0, 22.5,
14.4, 8.0, 8.0 ppm; ESI-MS: m/z ¼ 405 [Mþ]; HRMS (ESI) calcd. for
22H), 0.90 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 61.9,
C
22H49N2O2S (Mþ) 405.3509, found 405.3494.
53.0, 48.5, 48.5, 48.5, 44.3, 35.3, 31.7, 29.5, 29.5, 29.5, 29.5, 29.4,
29.3, 29.2, 29.0, 28.2, 23.0, 22.5, 14.4 ppm; ESI-MS: m/z ¼ 377 [Mþ];
HRMS (ESI) calcd. for C19H43N2O3S (Mþ) 377.3196, found 377.3182.
4.2.2. Triethyl-{2-[methyl-(tetradecane-1-sulfonyl)-amino]-ethyl}-
ammonium; iodide (3b)
2a was used and 3b (82.1%) was obtained. 1H NMR (500 MHz,
4.2.8. Ethyl-dimethyl-{2-[methyl-(tetradecane-1-sulfonyl)-amino]-
ethyl}-ammonium; iodide (3h)
DMSO-d6)
d
3.48 (t, J ¼ 7.3 Hz, 2H), 3.37e3.34 (m, 2H), 3.30e3.27
(m, 6H), 3.14 (t, J ¼ 7.8 Hz, 2H), 2.87 (s, 3H), 1.66e1.63 (m, 2H), 1.37e
1.34 (m, 2H), 1.24 (m, 20H), 1.19 (t, J ¼ 7.0 Hz, 9H), 0.85 (t, J ¼ 6.8 Hz,
2c was used and 3h (83.9%) was obtained. 1H NMR (500 MHz,
DMSO-d6) 3.55 (t, J ¼ 6.5 Hz, 2H), 3.47 (t, J ¼ 6.5 Hz, 2H), 3.39e3.32
(m, 2H), 3.13 (t, J ¼ 8.0 Hz, 2H), 3.04 (s, 6H), 2.85 (s, 3H), 1.65 (m,
2H), 1.37e1.24 (m, 22H), 0.85 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz,
3H); 13C NMR (100 MHz, DMSO-d6)
d 53.7, 53.0, 53.0, 53.0, 48.5,
43.3, 36.0, 31.7, 29.5, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.2,
23.0, 22.5, 14.4, 7.7, 7.7, 7.7 ppm; ESI-MS: m/z ¼ 419 [Mþ]; HRMS
(ESI) calcd. for C23H51N2O2S (Mþ) 419.3666, found 419.3647.
DMSO-d6)
d 59.6, 59.4, 50.3, 48.5, 48.5, 44.0, 35.5, 31.7, 29.5, 29.5,
29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.2, 23.0, 22.5, 14.4, 8.3 ppm; ESI-
MS: m/z ¼ 391 [Mþ]; HRMS (ESI) calcd. for C21H47N2O3S (Mþ)
391.3353, found 391.3341.
4.2.3. Allyl-diethyl-{2-[methyl-(tetradecane-1-sulfonyl)-amino]-
ethyl}-ammonium; bromide (3c)
2a was used and 3c (45.8%) was obtained. 1H NMR (500 MHz,
4.2.9. Allyl-dimethyl-{2-[methyl-(tetradecane-1-sulfonyl)-amino]-
ethyl}-ammonium; bromide (3i)
DMSO-d6)
d 6.05e5.97 (m, 1H), 5.72e5.62 (m, 2H), 3.95 (d,
J ¼ 7.0 Hz, 2H), 3.54e3.51 (m, 2H), 3.37e3.34 (m, 2H), 3.31e3.28 (m,
4H), 3.15e3.12 (m, 2H), 2.86 (s, 3H), 1.66e1.63 (m, 2H), 1.37e1.34
(m, 2H), 1.24e1.22 (m, 26H), 0.85 (t, J ¼ 6.8 Hz, 3H); 13C NMR
2c was used and 3i (51.5%) was obtained. 1H NMR (500 MHz,
DMSO-d6) 6.04e5.96 (m, 1H), 5.64e5.53 (m, 2H), 4.01e3.99 (d,
J ¼ 7.0 Hz, 2H), 3.57e3.42 (m, 2H), 3.27 (t, J ¼ 20.3 Hz, 2H), 3.12e
2.96 (m, 6H), 2.83e2.71 (m, 3H), 1.61e1.55 (m, 2H), 1.30e1.27 (m,
2H), 1.21e1.13 (m, 22H), 0.83e0.73 (m, 3H); 13C NMR (100 MHz,
(100 MHz, DMSO-d6) d 127.7, 125.8, 60.0, 54.3, 53.7, 53.7, 48.5, 43.3,
35.9, 31.7, 29.5, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.2, 23.0,
22.5, 14.4, 7.7, 7.7 ppm; ESI-MS: m/z ¼ 431 [Mþ]; HRMS (ESI) calcd.
for C24H51N2O2S (Mþ) 431.3666, found 431.3646.
DMSO-d6) d 128.3, 126.2, 66.1, 60.0, 50.4, 48.5, 48.5, 43.9, 35.5, 31.7,
29.5, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.2, 23.0, 22.5,
14.4 ppm; ESI-MS: m/z ¼ 403 [Mþ]; HRMS (ESI) calcd. for
4.2.4. Diethyl-{2-[(hexadecane-1-sulfonyl)-methyl-amino]-ethyl}-
methyl-ammonium; iodide (3d)
C22H47N2O2S (Mþ) 403.3353, found 403.3338.
2b was used and 3d (85.3%) was obtained. 1H NMR (500 MHz,
4.2.10. Trimethyl-{2-[methyl-(tetradecane-1-sulfonyl)-amino]-
ethyl}-ammonium; iodide (3j)
DMSO-d6)
d
3.52 (t, J ¼ 7.0 Hz, 2H), 3.42 (t, J ¼ 7.3 Hz, 2H), 3.40e3.35
(m, 4H), 3.13 (t, J ¼ 7.8 Hz, 2H), 2.98 (s, 3H), 2.86 (s, 3H), 1.66e1.63
(m, 2H), 1.37e1.34 (m, 2H), 1.23e1.21 (m, 30H), 0.85 (t, J ¼ 6.8 Hz,
2d was used and 3j (27.3%) was obtained. 1H NMR (500 MHz,
DMSO-d6)
d
7.56e7.51 (m, 5H), 4.59 (s, 2H), 3.68 (t, J ¼ 7.0 Hz, 2H),
3H); 13C NMR (100 MHz, DMSO-d6)
d
56.9, 56.5, 56.5, 48.5, 47.4,
3.52 (t, J ¼ 6.8 Hz, 2H), 3.14 (t, J ¼ 8.0 Hz, 2H), 3.02 (s, 6H), 2.86 (s,
3H), 1.65 (t, J ¼ 7.5 Hz, 2H), 1.36 (t, J ¼ 7.3 Hz, 2H), 1.23 (s, 25H), 0.85
43.6, 35.7, 31.7, 29.5, 29.5, 29.5, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2,
29.0, 28.2, 23.0, 22.5, 14.4, 8.0, 8.0 ppm; ESI-MS: m/z ¼ 433 [Mþ];
HRMS (ESI) calcd. for C24H53N2O2S (Mþ) 433.3822, found 433.3811.
(t, J ¼ 6.5 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 62.0, 53.0, 48.5,
48.5, 48.5, 44.3, 35.3, 31.7, 29.5, 29.5, 29.5, 29.5, 29.5, 29.5, 29.5,
29.4, 29.3, 29.1, 29.0, 23.0, 22.5, 14.4 ppm; ESI-MS: m/z ¼ 406 [Mþ];
HRMS (ESI) calcd. for C23H51N2O2S (Mþ) 405.3509, found 405.3495.
4.2.5. Triethyl-{2-[(hexadecane-1-sulfonyl)-methyl-amino]-ethyl}-
ammonium; iodide (3e)
2b was used and 3e (29.0%) was obtained. 1H NMR (500 MHz,
4.2.11. Ethyl-{2-[(hexadecane-1-sulfonyl)-methyl-amino]-ethyl}-
dimethyl-ammonium; iodide (3k)
DMSO-d6)
d
3.48 (t, J ¼ 7.3 Hz, 2H), 3.37e3.27 (m, 6H), 3.14 (t,
J ¼ 8.0 Hz, 2H), 1.66e1.63 (m, 2H), 1.36e1.34 (m, 2H), 1.24 (br, 24H),
2d was used and 3k (47.5%) was obtained. 1H NMR (500 MHz,
1.19 (t, J ¼ 7.0 Hz, 6H), 0.85 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6)
d
3.55 (t, J ¼ 5.5 Hz, 2H), 3.32 (s, 2H), 3.14e3.11 (m, 9H),
DMSO-d6)
d
53.7, 53.0, 53.0, 53.0, 48.5, 43.3, 36.0 31.7, 29.5, 29.5,
2.85 (s, 3H), 1.65 (t, J ¼ 7.5 Hz, 2H), 1.35 (t, J ¼ 7.5 Hz, 3H), 1.23 (s,
29.5, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.2, 23.0, 22.5, 14.4, 7.6,
23H), 0.85 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 59.1,
7.6, 7.6 ppm; ESI-MS: m/z ¼ 445 [Mþ]; HRMS (ESI) calcd. for
58.9, 49.8, 48.0, 43.4, 35.0, 31.3, 29.0, 29.0, 29.0, 29.0, 29.0, 29.0,
29.0, 28.9, 28.8, 28.7, 28.5, 27.7, 22.5, 22.1, 13.9, 7.8 ppm; ESI-MS: m/
z ¼ 420 [Mþ]; HRMS (ESI) calcd. for C22H49N2O2S (Mþ) 419.3666,
found 419.3646.
C
25H55N2O2S (Mþ) 447.3979, found 447.3966.
4.2.6. Allyl-diethyl-{2-[(hexadecane-1-sulfonyl)-methyl-amino]-
ethyl}-ammonium; bromide (3f)
2b was used and 3f (38.1%) was obtained. 1H NMR (500 MHz,
4.2.12. Allyl-{2-[(hexadecane-1-sulfonyl)-methyl-amino]-ethyl}-
dimethyl-ammonium; bromide (3l)
DMSO-d6)
d 6.05e5.97 (m, 1H), 5.72e5.62 (m, 2H), 3.95 (d,
J ¼ 7.0 Hz, 2H), 3.53 (t, J ¼ 7.3 Hz, 2H), 3.44 (t, J ¼ 6.8 Hz, 2H), 3.30e
2d was used and 3l (78.1%) was obtained. 1H NMR (500 MHz,
3.10 (m, 6H), 2.84e2.82 (m, 3H), 1.68e1.62 (m, 2H), 1.37e1.34 (m,
DMSO-d6)
d
6.06 (m, 1H), 5.65 (t, J ¼ 6.5 Hz, 2H), 4.02e4.01 (d,

676
D. Song et al. / European Journal of Medicinal Chemistry 69 (2013) 670e677
J ¼ 7.2 Hz, 2H), 3.59e3.58 (m, 2H), 3.50e3.48 (m, 2H), 3.13 (t,
J ¼ 7.8 Hz, 2H), 3.05 (s, 6H), 2.84 (s, 3H), 1.65 (m, 2H), 1.37 (m, 2H),
1.23 (m, 26H), 0.85 (t, J ¼ 6.9 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
4.2.18. Ethyl-dimethyl-[3-(tetradecane-1-sulfonylamino)-propyl]-
ammonium; iodide (5f)
4c was used and 5f (58.2%) was obtained. 1H NMR (500 MHz,
d
128.3, 126.2, 66.1, 60.0, 50.4, 48.5, 48.5, 43.9, 35.5, 31.7, 29.5, 29.5,
DMSO-d6) d 7.15e7.13 (m, 1H), 3.34e3.33 (m, 2H), 3.28e3.24 (br m,
29.5, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.2, 23.0, 22.5,
2H), 3.02e2.98 (br m, 10H), 1.86e1.80 (m, 2H), 1.65e1.59 (m, 2H),
14.4 ppm; ESI-MS: m/z ¼ 431 [Mþ]; HRMS (ESI) calcd. for
1.23 (br s, 25H), 0.85 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-
C
24H51N2O2S (Mþ) 431.3666, found 431.3649.
d6) d 60.63, 59.16, 51.13, 50.13, 50.13, 50.13, 42.98, 39.86, 31.74,
29.50, 29.47, 29.41, 29.27, 29.16, 29.06, 28.04, 23.63, 23.34, 22.54,
14.40, 8.25 ppm; ESI-MS: m/z ¼ 391 [Mþ]; HRMS (ESI) calcd. for
4.2.13. Trimethyl-{3-[methyl-(tetradecane-1-sulfonyl)-amino]-
propyl}-ammonium; iodide (5a)
C21H47N2O2S (Mþ) 391.3353, found 391.3339.
4a was used and 5a (86.9%) was obtained. 1H NMR (500 MHz,
DMSO-d6)
d
3.30e3.27 (m, 2H), 3.17 (t, J ¼ 6.5 Hz, 2H), 3.07e3.04
4.2.19. [3-(Hexadecane-1-sulfonylamino)-propyl]-trimethyl-
ammonium; iodide (5g)
(m, 9H), 2.98 (s, 3H), 1.98e1.93 (m, 2H), 1.65e1.60 (m, 2H), 1.36e
1.33 (m, 2H), 1.27e1.24 (m, 22H), 0.85 (t, J ¼ 6.8 Hz, 3H); 13C NMR
4d was used and 5g (84.4%) was obtained. 1H NMR (500 MHz,
(100 MHz, DMSO-d6)
d
63.5, 52.9, 52.8, 52.8, 48.3, 47.0, 34.8, 31.7,
DMSO-d6)
d
3.30 (t, J ¼ 7.5 Hz, 2H), 3.05 (s, 9H), 3.01e2.97 (m, 4H),
29.5, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.2, 23.1, 22.5, 21.4,
1.88e1.84 (m, 2H), 1.65e1.59 (m, 2H), 1.36e1.23 (m, 26H), 0.83 (t,
14.4 ppm; ESI-MS: m/z ¼ 391 [Mþ]; HRMS (ESI) calcd. for
J ¼ 7.5 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 63.77, 63.74, 63.71,
C
21H47N2O2S (Mþ) 391.3353, found 391.3336.
52.84, 52.80, 52.76, 51.15, 39.87, 31.74, 29.50, 29.50, 29.50, 29.45,
29.42, 29.27, 29.15, 29.07, 28.05, 23.77, 23.61, 22.54, 14.39 ppm; ESI-
MS: m/z ¼ 405 [Mþ]; HRMS (ESI) calcd. for C22H49N2O2S (Mþ)
405.3509, found 405.3497.
4.2.14. Ethyl-dimethyl-{3-[methyl-(tetradecane-1-sulfonyl)-
amino]-propyl}-ammonium; iodide (5b)
4a was used and 5b (56.4%) was obtained. 1H NMR (500 MHz,
DMSO-d6)
d
3.25e3.21 (m, 2H), 3.17 (t, J ¼ 6.5 Hz, 2H), 3.05 (t,
4.2.20. Ethyl-[3-(hexadecane-1-sulfonylamino)-propyl]-dimethyl-
ammonium; iodide (5h)
J ¼ 8.0 Hz, 2H), 3.00 (s, 6H), 2.80 (s, 3H), 1.94e1.90 (m, 2H), 1.64e
1.60 (m, 2H), 1.38e1.33 (m, 2H), 1.24e1.21 (m, 25H), 0.83 (t,
4d was used and 5h (82.8%) was obtained. 1H NMR (500 MHz,
J ¼ 6.8 Hz); 13C NMR (100 MHz, DMSO-d6)
d
60.6, 59.2, 50.1, 48.4,
DMSO-d6)
d
7.14 (m, 1H), 3.33e3.31 (m, 4H), 3.24 (t, J ¼ 7.5 Hz, 2H),
47.1, 35.0 and 34.9, 31.7, 29.5, 29.5, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2,
29.0, 28.2, 23.1, 22.5, 21.1 and 20.8, 14.4, 8.3 and 8.0 ppm; ESI-MS:
3.01e2.98 (m, 8H), 1.83 (m, 2H), 1.62 (m, 2H), 1.36e1.23 (m, 26H),
0.83 (t, J ¼ 7.5 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 60.62, 59.15,
m/z
¼
405 [Mþ]; HRMS (ESI) calcd. for C22H49N2O3S (Mþ)
51.11, 50.15, 50.12, 50.09, 39.85, 31.74, 29.50, 29.50, 29.50, 29.50,
29.45, 29.42, 29.27, 29.15, 29.07, 28.05, 23.63, 23.33, 22.54, 14.39,
8.24 ppm; ESI-MS: m/z ¼ 419 [Mþ]; HRMS (ESI) calcd. for
C23H51N2O2S (Mþ) 419.3666, found 419.3654.
405.3509, found 405.3497.
4.2.15. {3-[(Hexadecane-1-sulfonyl)-methyl-amino]-propyl}-
trimethyl-ammonium; iodide (5c)
4b was used and 5c (98.0%) was obtained. 1H NMR (500 MHz,
4.2.21. [2,2-Dimethyl-3-(tetradecane-1-sulfonylamino)-propyl]-
trimethyl-ammonium; iodide (7a)
DMSO-d6)
d 3.31e3.26 (m, 2H), 3.26e3.15 (m, 2H), 3.06e3.02 (m,
9H), 2.79 (s, 3H), 1.94 (m, 2H), 1.63 (m, 2H), 1.36e1.23 (m, 26H),
6a was used and 7a (80.7%) was obtained. 1H NMR (500 MHz,
0.86e0.83 (t, J ¼ 7.5 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d
63.5,
DMSO-d6) d 7.18e7.15 (m, 1H), 3.28 (s, 2H), 3.15 (s, 9H), 3.0 (t,
52.9, 52.8, 52.8, 48.3, 47.0, 34.8, 31.7, 29.5, 29.5, 29.5, 29.5, 29.5,
29.5, 29.4, 29.3, 29.2, 29.0, 28.2, 23.1, 22.5, 21.4, 14.4 ppm; ESI-MS:
m/z ¼ 419 [Mþ]; HRMS (ESI) calcd. for C23H51N2O2S (Mþ) 419.3666,
found 419.3657.
J ¼ 7.8 Hz, 2H), 2.86 (d, J ¼ 7.0 Hz, 2H), 1.66e1.60 (m, 2H), 1.35e1.34
(m, 2H), 1.24 (br s, 20H), 1.11 (s, 6H), 0.85 (t, J ¼ 7.0 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6) d 72.68, 55.27, 55.27, 55.27, 52.74, 51.45, 36.92,
31.74, 29.50, 29.50, 29.50, 29.50, 29.50, 29.50, 29.47, 29.24, 29.03,
28.05, 25.04, 23.51, 22.54, 14.40 ppm; ESI-MS: m/z ¼ 405 [Mþ];
HRMS (ESI) calcd. for C22H49N2O2S (Mþ) 405.3509, found 405.3496.
4.2.16. Ethyl-{3-[(hexadecane-1-sulfonyl)-methyl-amino]-propyl}-
dimethyl-ammonium; iodide (5d)
4b was used and 5d (87.8%) was obtained. 1H NMR (500 MHz,
4.2.22. [2,2-Dimethyl-3-(tetradecane-1-sulfonylamino)-propyl]-
ethyl-dimethyl-ammonium; iodide (7b)
DMSO-d6)
d
3.34e3.33 (m, 2H), 3.21e3.17 (t, J ¼ 12.5 Hz, 2H), 3.17e
3.15 (t, J ¼ 5.0 Hz, 2H), 3.05e3.02 (t, J ¼ 7.5 Hz, 2H), 2.99 (s, 6H), 2.8
6a was used and 7b (85.9%) was obtained. 1H NMR (500 MHz,
(s, 3H), 1.91 (m, 2H), 1.63 (m, 2H), 1.36e1.21 (m, 29H), 0.86e0.83 (t,
DMSO-d6)
d 7.20 (m, 1H), 3.43e3.41 (m, 2H), 3.21 (s, 2H), 3.02 (s,
J ¼ 7.5 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d
60.6, 59.2, 50.0,
6H), 2.98 (m, 2H), 2.86e2.85 (m, 2H) 1.29e1.24 (m, 27H), 1.12 (s,
48.4, 48.4, 47.1, 34.9, 31.7, 29.5, 29.5, 29.5, 29.5, 29.5, 29.5, 29.4, 29.3,
29.2, 29.0, 28.2, 23.1, 22.5, 21.1, 14.4, 8.3 ppm; ESI-MS: m/z ¼ 433
[Mþ]; HRMS (ESI) calcd. for C24H53N2O2S (Mþ) 433.3822, found
433.3805.
6H), 0.85 (t, J ¼ 6.9 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 69.84,
62.94, 53.16, 51.75, 51.44, 36.82, 31.74, 29.50, 29.50, 29.50, 29.50,
29.50, 29.47, 29.40, 29.25, 29.16, 29.04, 28.05, 25.09, 23.52, 22.54,
14.40, 8.67 ppm; ESI-MS: m/z ¼ 419 [Mþ]; HRMS (ESI) calcd. for
C23H51N2O2S (Mþ) 419.3666, found 419.3652.
4.2.17. Trimethyl-[3-(tetradecane-1-sulfonylamino)-propyl]-
ammonium; iodide (5e)
4.2.23. [3-(Hexadecane-1-sulfonylamino)-2,2-dimethyl-propyl]-
trimethyl-ammonium; iodide (7c)
4c was used and 5e (52.7%) was obtained. 1H NMR (500 MHz,
DMSO-d6)
d
7.13 (t, J ¼ 6.1 Hz, 1H), 3.10e2.95 (m, 13H), 2.20e1.96
6b was used and 7c (87.3%) was obtained. 1H NMR (500 MHz,
(m, 3H), 1.91e1.82 (m, 2H), 1.61 (d, J ¼ 7.6 Hz, 2H), 1.36 (s, 3H), 1.24
DMSO-d6) d 7.19e7.16 (m, 1H), 3.28 (s, 2H), 3.15 (s, 9H), 3.00 (t,
(s, 21H), 0.85 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
J ¼ 7.7 Hz, 2H), 2.85 (d, J ¼ 7.0 Hz, 2H), 1.62 (br s, 2H), 1.35 (br s, 2H),
d
63.77, 63.74, 63.71, 52.83, 52.79, 52.76, 51.13, 36.87, 31.73, 29.50,
1.23 (br s, 24H), 1.11 (s, 6H), 0.84 (t, J ¼ 6.5 Hz, 3H); 13C NMR
29.47, 29.41, 29.26, 29.15, 29.06, 28.04, 23.77, 23.61, 22.53,
(100 MHz, DMSO-d6) d 72.68, 55.27, 52.75, 51.46, 36.92, 31.74, 29.49,
14.40 ppm; ESI-MS: m/z ¼ 377 [Mþ]; HRMS (ESI) calcd. for
29.49, 29.49, 29.49, 29.49, 29.49, 29.49, 29.45, 29.41, 29.25, 29.15,
29.04, 28.06, 25.05, 26.51, 22.54, 14.39 ppm; ESI-MS: m/z ¼ 433
C
20H45N2O2S (Mþ) 377.3196, found 377.3184.

D. Song et al. / European Journal of Medicinal Chemistry 69 (2013) 670e677
677
[Mþ]; HRMS (ESI) calcd. for C24H53N2O2S (Mþ) 433.3822, found
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433.3809.
4.2.24. Ethyl-[3-(hexadecane-1-sulfonylamino)-2,2-dimethyl-
propyl]-dimethyl-ammonium; iodide (7d)
6b was used and 7d (78.9%) was obtained. 1H NMR (500 MHz,
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development, but it modifies susceptibility to tumor formation as well as cell
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DMSO-d6) d 7.22e7.20 (m, 1H), 3.42e3.41 (m, 2H), 3.33 (s, 3H), 3.21
(s, 2H), 3.07 (s, 3H), 3.00 (t, J ¼ 8.0 Hz, 2H), 2.85 (d, J ¼ 6.5 Hz, 2H),
1.62 (br s, 2H), 1.29e1.23 (m, 29H), 1.11 (s, 6H), 0.84 (t, J ¼ 6.8 Hz,
3H); 13C NMR (100 MHz, DMSO-d6)
d 69.83, 62.94, 55.26, 53.17,
51.74, 51.44, 36.92, 36.82, 31.74, 29.50, 29.50, 29.50, 29.50, 29.46,
29.41, 29.26, 29.15, 29.04, 28.06, 25.09, 25.04, 23.52, 22.54, 14.40,
8.67 ppm; ESI-MS: m/z ¼ 447 [Mþ]; HRMS (ESI) calcd. for
C
25H55N2O2S (Mþ) 447.3979, found 447.3961.
4.3. SRB assay
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Growth inhibition of cancer cell lines in the presence of
NSC126188 and perifosine was determined using the SRB assay.
After an incubation period, cells were fixed with 10% formalin for
30 min, washed with distilled water, and then stained for 30 min
with 0.4% sulforhodamine B. The excess dye was removed by
washing with 0.1% acetic acid. The amount of SRB dye that bound to
the cell matrix was dissolved in 10 mM Tris (pH 10.5) and was
quantified using a microplate reader at 540 nm. The concentration
of NSC126188 required to inhibit growth of 50% of cells (GI50) was
determined after 48 h of drug treatment [26].
4.4. Luciferase assay
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Transactivation of RhoB was determined by a reporter assay
using the dual-luciferase reporter assay system (Promega, Madison,
WI, USA), as previously described [27]. HeLa cells at 75e90% con-
fluency were transiently cotransfected with a plasmid encoding
pGL2-RhoB-firefly luciferase under control of the RhoB promoter
and pRL-SV40-Renilla luciferase. Luciferase activity was integrated
over a 10 s period and measured using a luminometer (Victor X
Light; Perkin Elmer, Waltham, MA, USA). The results were
normalized to the levels of Renilla luciferase.
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Acknowledgments
[20] B.K. Kim, H.M. Kim, K.S. Chung, D.M. Kim, S.K. Park, A. Song, K.J. Won, K. Lee,
Y.K. Oh, K.B. Song, J.A. Simon, G. Han, M. Won, Upregulation of RhoB via c-
Jun N-terminal kinase signaling induces apoptosis of the human gastric
carcinoma NUGC-3 cells treated with NSC12618, Carcinogenesis 32 (2011)
254e261.
[21] G.L. Johnson, R. Lapadat, Mitogen-activated protein kinase pathways medi-
ated by ERK, JNK, and p38 protein kinases, Science 298 (2002) 1911e1912.
[22] E.F. Wagner, A.R. Nebreda, Signal integration by JNK and p38 MAPK pathways
in cancer development, Nat. Rev. Cancer 9 (2009) 537e549.
This research was supported by a grant from the National
Research Foundation (grant 2012-0000884), the Translational
Research Center for Protein Function Control (NRF-2009-0083522),
the Basic Science Research Program (NRF-2013R1A1A2008165), the
Korea Health21 R&D Project, Ministry of Health
& Welfare
(A120478), and the Brain Korea 21 Program, Republic of Korea.
[23] B.K. Kim, D.M. Kim, K.S. Chung, S.K. Park, S.J. Choi, A. Song, K. Lee, C.W. Lee,
K.B. Song, G. Han, J. Simon, H.M. Kim, M. Won, NSC126188, a piperazine alkyl
derivative, induces apoptosis via upregulation of RhoB in HeLa cells, Invest.
New Drugs 29 (2011) 853e860.
Appendix A. Supplementary data
[24] J.S. Yang, D. Song, W.J. Ko, B. Kim, B.K. Kim, S.K. Park, M. Won, K. Lee, H.M. Kim,
G. Han, Synthesis and biological evaluation of novel aliphatic amido-
quaternary ammonium salts for anticancer chemotherapy: part II, Eur. J.
Med. Chem. 63 (2013) 621e628.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.022.
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