Design, synthesis and biological evaluation of novel aliphatic amido/sulfonamido-quaternary ammonium salts as antitumor agents

Article abstract of DOI:10.1016/j.bmc.2012.11.027

RhoB, one of the upstream signaling proteins of the phosphatidylinositol-3- kinase (PI3K)/Akt pathway, is frequently mutated in human cancer. Based on a piperazine alkyl derivative that induced apoptosis via up-regulation of RhoB, we synthesized novel aliphatic amido/sulfonamido-quaternary ammonium salts and evaluated their biological activities using an in vitro growth inhibition assay and RhoB promoter assay in human cancer cells. Compound 3a was the most promising anticancer agent in the series, based upon its potent growth inhibition via RhoB-mediated signaling. These novel aliphatic amido/sulfonamido-quaternary ammonium salts may be useful as a platform for development of anticancer chemotherapeutic agents.

Full text of DOI:10.1016/j.bmc.2012.11.027

Bioorganic & Medicinal Chemistry 21 (2013) 788–794
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel aliphatic
amido/sulfonamido-quaternary ammonium salts as antitumor agents
Doona Song a, Jee Sun Yang a, Seo Joong Kim a, Bo-Kyung Kim c, Song-Kyu Park b, Misun Won c, Kiho Lee b,
a,f,
Hwan Mook Kim d, Kang-Yell Choi a, Kyeong Lee e, , Gyoonhee Han
⇑
⇑
a Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
b College of Pharmacy, Korea University, Chochiwon, Chungnam 339-700, Republic of Korea
c Medical Genomics Research Center, KRIBB, Daejeon 305-806, Republic of Korea
d College of Pharmacy, Gachon University, Incheon 406-799, Republic of Korea
e College of Pharmacy, Dongguk University-Seoul, Seoul 100-715, Republic of Korea
f 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, one of the upstream signaling proteins of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, is
frequently mutated in human cancer. Based on a piperazine alkyl derivative that induced apoptosis via
up-regulation of RhoB, we synthesized novel aliphatic amido/sulfonamido-quaternary ammonium salts
and evaluated their biological activities using an in vitro growth inhibition assay and RhoB promoter
assay in human cancer cells. Compound 3a was the most promising anticancer agent in the series, based
upon its potent growth inhibition via RhoB-mediated signaling. These novel aliphatic amido/sulfon-
amido-quaternary ammonium salts may be useful as a platform for development of anticancer chemo-
therapeutic agents.
Received 25 September 2012
Revised 1 November 2012
Accepted 15 November 2012
Available online 1 December 2012
Keywords:
RhoB
GTPase rat sarcoma (Ras)
Perifosine
Ó 2012 Elsevier Ltd. All rights reserved.
NSC126188
Aliphatic amido/sulfonamido-quaternary
ammonium salts
1. Introduction
The small GTPase Ras, one of the proteins involved in the up-
stream pathway of PI3K/Akt signaling, is also frequently mutated
Carcinogenesis can be the result of changes in proteins and sig-
naling pathways which regulate cell growth, differentiation, and
development. In these pathways, PI3K/Akt signaling and Ras sig-
naling are often stimulated in human cancers via several different
mechanisms.1–5
Phosphatidylinositol-3-kinase (PI3K) is a lipid kinase which
generates phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3].
PI(3,4,5)P3 acts as a second messenger and is necessary for the
translocation of Akt to the plasma membrane where it is phosphor-
ylated and activated by phosphoinositide-dependent kinase
(PDK)1 and PDK2.6 Activated Akt plays a crucial role in cell prolif-
eration and survival by phosphorylation of a variety of substrates.6
With similar structural features as PI(3,4,5)P3, phosphatidylinosi-
tol (PI) analogues are one class of Akt inhibitors represented by
perifosine [octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate]
(Fig. 1), which is synthetic and orally bioavailable.7–9
in human cancer.10 Mutated Ras results in an altered PI3K, which
is a downstream target of Ras signaling.11 Rho (Ras homologous
small GTPase) are members of the Ras-superfamily which is
responsible for proliferation, survival, invasion, metastasis, as
well as overall angiogenic capacity of cancer cells.12–15 Rho is
composed of subgroups which show 86% sequence homology.
Of note is RhoB, a subgroup member with different activities
from the RhoA-related subgroups (RhoA and RhoC). RhoB has a
tumor suppressive role, whereas most other Rho proteins have
promoting roles in cancer.16,17 Because RhoB expression is fre-
quently suppressed in tumor progression, it is believed that
RhoB overexpression prevents oncogenic signaling by modifying
oncogene proteins such as EGFR, ErbB2, Ras, and Akt resulting
in changes in their localizations.17 Recently, it was reported that
a piperazine alkyl derivative (NSC126188, Fig. 1) induced apop-
tosis via up-regulation of RhoB in HeLa cells.18 Structural fea-
tures of this compound are similar to perifosine. Therefore, it
was envisioned that compounds with a cognate structure like
perifosine or NSC126188 can trigger apoptosis through the RhoB
mediated pathway in cancer.19,20
⇑
Corresponding authors. Tel.: +82 31 961 5214; fax: +82 31 961 5206 (K.L.); tel.:
+82 2 2123 2882; fax: +82 2 362 7265 (G.H.).
E-mail addresses: kaylee@dongguk.edu (K. Lee), gyoonhee@yonsei.ac.kr (G. Han).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.027

D. Song et al. / Bioorg. Med. Chem. 21 (2013) 788–794
789
O
O
O
O
O
O
O
O
c
d
O
P
O-
I-
N+
S
S
S
I-
Br-
O- +Na
N
N
O
O
n
N
n
n
N
17
14
14
N
N+
N+
N+
6
7a
8a
n=11
n=11
perifosine
A1012
NSC126188
7b n=13
7c n=15
8b n=13
8c n=15
Figure 1. Chemical structures of PI analogue (perifosine), NSC126188 (4-hexa-
decanoyl-1,1-dimethyl-piperazin-1-ium iodide), and A10127 (a representative
analogue of the aliphatic amido-quaternary ammonium salt series).
I-
O
N+
O
c
d
N
S
O
O
S
N
H
O- +Na
O
O
n
S
N
H
n
n
We previously reported synthesis and biological activity of the
aliphatic amido-quaternary ammonium salt series as represented
by A1012 (Fig. 1) as a potential anticancer agent.7 Herein, design
and synthesis of novel aliphatic amido/sulfonamido-quaternary
ammonium salts through isosteric replacement of the functional
group of NSC126188 and A1012, and biological evaluation of these
derivatives are described. We performed a structural–activity rela-
tionship (SAR) study of these synthesized compounds, based on
their estimation of growth inhibitory activities and RhoB promoter
assays in six human cancer cell types. The results suggested that
these novel aliphatic amido/sulfonamido-quaternary ammonium
salts may be useful lead compounds for development of effective
anticancer chemotherapeutic agents.
6
9a
n=11
10a
n=11
9b n=13
10b n=13
Scheme 2. Reaction protocol for the synthesis of aliphatic sulfonamide-quaternary
ammonium salts (8a–8c, 10a–10b). Reagents and conditions: (a) (i) SOCl2, CH2Cl2 at
60 °C (ii) 1-methylhomopiperazine or N,N-dimethyl-1,4-phenylenediamine, CH2Cl2
at 0 °C, or DMAP, EDCI, 1-methylhomopiperazine or N,N-dimethyl-1,4-phenylene-
diamine, CH2Cl2; (b) methyl iodide, CH3CN at 95–100 °C or methyl iodide, toluene,
110–115 °C.
and 9a–b were prepared from various sulfonate sodium salts 6 in
a similar fashion. Sulfonamides were further converted to sulfon-
amide-quaternary ammonium salts 8a–8c and 10a–10b by N-
alkylation using methyl iodide. The newly prepared quaternary
ammonium salt analogues were characterized and evaluated for
their biological activities.
2. Chemistry
Based on structural characteristics of NSC126188, we designed
and synthesized 19 analogues, which included three major modifi-
cations. First, the piperazine structure was substituted with a phe-
nyl ring or homopiperazine. Second, the carbonyl group was
replaced with a sulfonyl group. Third, a variety of N-substituents
were introduced. As illustrated in Schemes 1 and 2, compounds
were synthesized from various fatty acids by two step procedures.
Fatty acids 1 were reacted with thionyl chloride to produce an acid
chloride intermediate, which was coupled with 1-methylhomopip-
erazine or N,N-dimethyl benzene-1,4-diamine to yield amides 2a–
d and 4a–d (Scheme 1). Amides were converted to amido-quater-
nary ammonium salts 3a–3j and 5a–5d by N-alkylation using di-
verse alkyl halides. As shown in Scheme 2, sulfonamides 7a–c
3. Results and discussion
Nineteen compounds synthesized using Schemes 1 and 2 were
assayed for their growth inhibitory activity against six human can-
cer cell lines: PC-3 (prostate cancer), NUGC-3 (stomach cancer),
MDA-MB-231 (breast cancer), ACHN (renal cancer), HCT-15 (colon
cancer), and NCI-H23 (non-small cell lung cancer). The GI50 data
for the compounds are listed in Table 1. Perifosine, a known
PI3K/Akt inhibitor, was used as a positive reference to compare
in vitro growth inhibitory activities of the synthesized compounds.
Structurally, perifosine and NSC126188 share a long aliphatic chain
O
O
O
a
b
X-
N
OH
N
n
n
n
N+
N
R
1
3a
3b
3c
n=12, R=Me
n=12, R=Bn
n=12, R=Allyl
2a
n=12
X=I
2b n=14
2c n=16
X=Br
X=Br
X=Br
X=Br
X=I
2d
3d n=12, R=3-NitroBn
3e n=12, R=4-FluoroBn
3f n=12, R=Et
n=18
3g n=14, R=Me
X=I
3h n=14, R =3- CF3-o-Bn
3i n=16, R=Me
X=Br
X=I
3j
n=18, R=Me
X=I
I-
O
N+
a
b
N
O
O
OH
n
N
N
n
n
H
H
4a
n=12
5a
n=12
1
4b n=14
4c n=16
5b n=14
5c n=16
4d
n=18
5d
n=18
Scheme 1. Reaction protocol for the synthesis of aliphatic amido-quaternary ammonium salts (3a–3j, 5a–5d). Reagents and conditions: (a) (i) SOCl2, CH2Cl2 at 60 °C (ii) 1-
methylhomopiperazine or N,N-dimethyl-1,4-phenylenediamine, CH2Cl2 at 0 °C, or DMAP, EDCI, 1-methylhomopiperazine or N,N-dimethyl-1,4-phenylenediamine, CH2Cl2; (b)
alkyl halide, CH3CN at 95–100 °C or alkyl halide, toluene, 110–115 °C.

790
D. Song et al. / Bioorg. Med. Chem. 21 (2013) 788–794
Table 1
group, and homopiperazine, but different N-substituents. Six com-
Growth inhibition of synthesized compounds
pounds exhibited good anti-proliferative activity on PC-3 and
NUGC cells. The compound 3e (4-fluorobenzyl) showed selective
activity on PC-3 cells. Among the six compounds, the compound
3a, which had an N-methyl group, had the highest growth inhibi-
tion of NUGC cells.
Compound
Growth inhibition (
lM)
PC-3 NUGC-3 MDA-MB-231 ACHN HCT-15 NCI-H23
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
5a
5b
5c
5d
8a
8b
8c
0.36 0.08
0.45 0.31
0.18 0.26
0.18 0.46
0.45 0.94
0.27 0.22
0.98
1.16
0.45
0.71
1.48
0.68
0.94
1.34
0.67
2.22
3.56
2.83
4.34
3.68
0.84
0.83
0.59
NA
0.49
1.00
0.44
0.82
1.10
1.61
0.40
1.93
0.57
2.78
4.54
3.15
1.87
1.76
1.16
1.33
2.01
4.50
NA
1.70
NA
NA
NA
NA
1.86
0.93
NA
0.40
0.99
1.36
2.37
1.22
3.02
0.96
0.38
0.77
NA
1.36
1.07
0.28
0.89
1.48
1.18
2.38
1.45
1.16
3.84
4.14
1.80
3.29
1.93
0.96
0.80
3.40
3.94
0.73
4.21
2.34
For this series, the RhoB promoter activity was also determined
using the luciferase reporter gene assay as described in Figure 2.
Among 19 analogues, five compounds containing the homopiper-
azine moiety (3a, 3c, 3f, 3g, and 8b) exhibited good reporter
expression. Of note, the compounds 3c and 3g showed better re-
porter expression than NSC126188 in the RhoB promoter-based as-
say. However, 3b, 3d, 3j, 8a, and 8c showed weak reporter
expression activity, while exhibiting better growth inhibition than
perifosine in cancer cell lines. Overall, the RhoB promoter assay re-
sults did not parallel those of the anti-proliferative activities of the
analogues. To understand the reason of this difference, we ana-
lyzed the molecular and physical properties of the analogues, par-
ticularly AlogP (atomic calculation based the octanol–water
partition coefficient) and FPSA (Fractional Polar Surface Area, total
partially charged and solvent accessible molecular surface area di-
vided by the total molecular surface area) parameters which could
be related to the solubility of compounds and/or cell permeability
(Table 2). With good promoter and anti-proliferative activities, 3a,
3c, 3f, and 3g had similar molecular properties; AlogP value <5 and
FPSA <0.051, respectively. With low growth inhibition and pro-
moter assay value, most compounds showed higher AlogP values
and/or high FPSA than those of compounds which both growth
inhibition and promoter assay value are good. Especially, com-
pound 3d which had very low promoter activity in spite of the best
GI50 showed higher AlogP and FPSA than the compounds with
good values both in growth inhibition and promoter assay. It was
suggested that the different tendency of GI50 value and promoter
assay activity of some compounds could result from their AlogP
and FPSA parameters which are involved in solubility and/or cell
permeability. Overall, 3a (14 carbon chain, carbonyl homopiper-
zine, N,N-dimethyl subsituents) was considered an excellent anti-
cancer agent in the series, with the best inhibitory activity on
NUGC cells and a positive value in the RhoB promoter assay.
Regarding molecular properties, it showed better drug-like values
in terms of AlogP and FPSA than did perifosine.
0.40
0.21
0.63 0.87
0.24 0.26
NA
0.36
0.54 0.74
0.79 0.70
1.20 0.41
1.63 1.28
0.38 0.24
0.62 0.25
0.40
0.38
10a
10b
Perifosine
1.02 1.21
0.90 1.74
0.44 0.54
4.54
2.86
1.44
3.38
1.25
0.58
4.56
1.04
NSC126188 0.48 0.29
and the same quaternary ammonium salt moiety with N,N-di-
methyl substituents. According to our previous study,7 aliphatic
amido-quaternary ammonium salt compounds were better with
carbon chain lengths from 14 to 20 than with carbon lengths under
14 and over 20. Accordingly, we synthesized the analogues which
had carbon chain lengths from 14 to 20. In the case of the sulfon-
amides, chain length, including sulfur, were set to 13–17. To find
compounds with better activity and properties than known com-
pounds, a structure–activity relationship study was conducted
with emphasis on the carbonyl group, piperzine, and/or N-substi-
tuent of NSC126188.
First, we synthesized compounds which were modified in the
piperazine ring of NSC126188, while the N-methyl-N-methyl
group was fixed. To evaluate the effects of the piperazine ring
group, piperazine was substituted with a phenyl group (5a–5d).
Compounds 5a–5d had lower activity than NSC126188 in all cell
lines. Next, compounds 10a and 10b were substituted with sulfo-
nyl (as isostere of carbonyl) and phenyl (analogue of piperazine)
Perifosine inhibited PI3K/Akt, and NSC126188 mediated the
RhoB protein. These two compounds which have structural simi-
larities may be responsible for modulating apoptosis through the
PI3K/Akt pathway and/or RhoB-mediated pathways. Accordingly,
we designed and synthesized a novel series of aliphatic quaternary
ammonium salts and evaluated their activities using the growth
inhibition assay and the RhoB promoter assay. The effects of ring
moiety, carbonyl or sulfonyl group, and N-substituent patterns
on the aliphatic amido/sulfonamide-quaternary ammonium salt
series were further compared with their physical properties. The
groups. Two compounds showed over 0.5 lM in growth inhibition
of all cancer cell lines. In order to assess the effect of the ring moi-
ety, the phenyl ring was replaced with homopiperazine. Generally,
compounds 8a–8c with a sulfonyl group and homopiperazine were
more active than those with the phenyl group. In most of the cell
lines, 8a–8c exhibited better activity than 10a–10b. In addition,
this series of compounds showed better activities slightly than
NSC126188 in some cell lines. It was determined that homopiper-
azine was the more active of the two functional groups, therefore
the ring group played an important role in cell growth inhibitory
activity. Based on the results of 8a–8c and 5a–5d, we synthesized
analogues with a carbonyl group and homopiperazine (3a, 3g, 3i,
and 3j), among which, 3a, 3g, 3i, and 3j showed better or similar
growth inhibition than NSC126188 in most cell line. Most notably,
the compound 3a showed very potent growth inhibition in NUGC-
3 cells. Interestingly, there was a correlation between the activity
and carbon chain length as shown in 5a–d and 3a, 3g, 3i, and 3j,
indicating lipophilicity was one of the key factors for growth inhib-
itory activity.
Second, we introduced diverse N-substituents, including the
ethyl, benzyl, allyl, 3-nitrobenzyl, and 4-fluorobenzyl groups on
an N-methyl group to evaluate the effects of N-substituents. The
compounds 3a–3f had the same 14 carbon chain length, carbonyl
Figure 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 lM). NSC126188 (5 lM) and perifosine (5 lM) were used as positive controls.

D. Song et al. / Bioorg. Med. Chem. 21 (2013) 788–794
791
Table 2
(10%) was added (final pH 13), and the 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 chromatography on silica gel with 5% MeOH/CHCl3 to
give the corresponding amides.
Molecular and physical properties
Entry
Compound
AlogP
FPSA
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
5a
5b
5c
5d
8a
8b
8c
3.929
5.513
4.546
5.969
6.281
4.278
4.842
8.545
5.754
6.666
5.347
6.259
7.172
8.084
3.013
3.925
4.838
4.431
4.607
0.051
0.043
0.048
0.131
0.043
0.049
0.047
0.054
0.044
0.041
0.070
0.065
0.060
0.057
0.115
0.106
0.099
0.131
0.135
4.1.1. 1-(4-Methyl-1,4-diazepan-1-yl)tetradecan-1-one (2a)
Myristic acid was used and 2a (23.1%) was obtained as a yellow-
ish oil. 1H NMR (300 MHz, DMSO-d6) d 3.47–3.41 (m, 4H), 2.45–2.24
(m, 4H), 2.28–2.21 (m, 5H), 1.81–1.69 (m, 2H), 1.48 (br, 2H), 1.24 (m,
20H), 0.85 (t, J = 6.4 Hz, 3H); ESI-MS: m/z = 325 [M+H].
9
10
11
12
13
14
15
16
17
18
19
4.1.2. 1-(4-Methyl-1,4-diazepan-1-yl)-hexadecan-1-one (2b)
Palmitic acid was used and 2b (95.3%) was obtained as a yellow-
ish oil. 1H NMR (300 MHz, DMSO-d6) d 3.48–3.41 (m, 4H), 2.45–2.40
(m, 4H), 2.28–2.22 (m, 5H), 1.81–1.71 (m, 2H), 1.47 (br s, 2H), 1.24
(m, 24H), 0.85 (t, J = 6.3 Hz, 3H); ESI-MS: m/z = 353 [M+H].
10b
Perifosine
4.1.3. 1-(4-Methyl-[1,4]diazepan-1-yl)-octadecan-1-one (2c)
Stearic acid was used and 2c (98.0%) was obtained as a yellowish
oil. 1H NMR (300 MHz, DMSO-d6) d 3.50–3.41 (m, 4H), 2.47–2.38 (m,
3H), 2.29–2.18 (m, 4H), 1.80–1.70 (m, 2H), 1.50–1.43 (m, 2H), 1.32–
1.21 (m, 30H), 0.85 (t, J = 6.6 Hz, 3H); ESI-MS: m/z = 381 [M+H].
compound 3a was the most promising anticancer agent in the ser-
ies, based upon its potent growth inhibition via RhoB mediated sig-
naling. Together, our results showed that synthetic aliphatic
quaternary ammonium salts are valuable lead compounds for
development of anticancer chemotherapy.
4.2. General procedures 1 (amidation step in Scheme 1)
Carboxylic acid 1 (2.9 mmol) was dissolved in anhydrous
dichloromethane (0.2 M) at room temperature under an Ar atmo-
sphere, and 1-methylhomopiperazine (4.3 mmol), EDC (1-ethyl-
3-[3-dimethylaminopropyl]carboimide hydrochloride, 4.3 mmol)
and DMAP (4-dimethylaminopyridine, 0.9 mmol) were added.
The mixture was stirred for 8 h at room temperature. Saturated
NH4Cl solution was added, and the mixture was extracted with
dichloromethane (3Â). The organic layer was washed with brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The res-
idue was purified by chromatography on silica gel with 5% MeOH/
CH2Cl2 to resolve the amides.
4. Experimental section
All chemicals were obtained from commercial suppliers and
used without further purification. All reactions were monitored
by thin layer chromatography on precoated silica gel 60 F254
(mesh; E. Merck, Mumbai, India) and spots were visualized under
ultraviolet light (254 nm). Flash column chromatography was per-
formed with silica (Merck EM9385, 230–400 mesh). 1H nuclear
magnetic resonance (NMR; Varian) spectra were recorded at 300
and 75 MHz, at 400 and 100 MHz, or at 500 and 125 MHz. Proton
chemical shifts were expressed in ppm relative to internal tetra-
methylsilane, and coupling constants (J) were expressed in Hertz.
Liquid chromatography-mass spectrometry spectra were recorded
by electrospray ionization (ESI) on liquid chromatography-mass
spectrometry instruments (Shimadzu Kyoto, Japan, 10% 0.1% triflu-
oroacetic acid in H2O/90% 0.1% trifluoroacetic acid in acetonitrile),
in scanned mode (from 0 to 600 amu/z), with detected ion peaks
(M+z)/z and (MÀz)/z in positive and negative ion modes, respec-
tively, where M represents the molecular weight of the compound
and z represents the charge (number of protons). High-resolution
mass spectrometry spectrometry (HRMS) were obtained from
ESI-positive mode of the Micromass Q-TOF (Waters Corp, Man-
chester, UK, The capillary and sample cone voltages were 4000 V
and 30 V, the desolvation gas flow was 600 L/h at 200 °C and the
source temperature was 100 °C) High resolution tandem mass
spectrometry was conducted at the Yonsei University Center for
Research Facilities in Seoul, Korea.
4.3. Procedures for 3a–j
Alkyl halide (1.6 mmol) was added to a solution of 2a–d
(0.8 mmol) in anhydrous acetonitrile (0.3 M) at room temperature.
The reaction mixture was heated to 140–150 °C for 7 h. After the
reaction mixture equilibrated to room temperature, the precipitate
was granulated for 1 h at 0 °C. Solids were collected by filtration,
washed with ethyl acetate, and dried in vacuo.
4.3.1. 1,1-Dimethyl-4-tetradecanoyl-[1,4]diazepan-1-ium;
iodide (3a)
Compound 2a was used and 3a (71.4%) was obtained as a white
solid. Mp: 189–191 °C; 1H NMR (300 MHz, DMSO-d6) d 3.80–3.71
(m, 2H), 3.58–3.40 (m, 6H), 3.11 (s, 6H), 2.31 (t, J = 7.5 Hz, 2H),
2.20–2.05 (m, 2H), 1.55–1.46 (m, 2H), 1.34–1.22 (m, 20H), 0.85
(t, J = 6.6 Hz, 3H); ESI-MS: m/z = 339 [M+]; HRMS (ESI) calcd for
C21H43N2OI (MH+) 339.3370, found 339.3384.
4.3.2. 1-Benzyl-1-methyl-4-tetradecanoyl-[1,4]diazepan-1-ium;
bromide (3b)
4.1. Procedures for 2a–d
Compound 2a was used and 3b (93.6%) was obtained as a white
solid. Mp: 151–153 °C; 1H NMR (300 MHz, DMSO-d6) d 7.54(s, 5H),
4.65 (s, 2H), 4.00–3.85 (m, 1H), 3.76–3.60 (m, 1H), 3.59–3.53 (m,
2H), 3.46–3.39 (m, 4H), 2.97(s, 3H), 2.31 (t, J = 7.2 Hz, 2H), 2.28–
2.10 (m, 2H), 1.58–1.41 (m, 2H),1.32–1.22 (m, 20H), 0.85 (t,
J = 6.6 Hz, 3H); ESI-MS: m/z = 415 [M+]; HRMS (ESI) calcd for
Fatty acid 1 (2.9 mmol) was dissolved in anhydrous dichloro-
methane (0.1 M solution), and thionyl chloride (3.0 equiv) was
added under Argon (Ar) atmosphere. The stirred suspension was
heated and refluxed for 4 h. The reaction mixture was cooled and
poured onto crushed ice for 1 h, and 1-methylhomopiperazine
(5.8 mmol) was added dropwise. The reaction mixture was allowed
to warm to room temperature and stirred for 2 h. NaOH solution
C27H47N2OBr (MH+) 415.3680, found 415.3691.

792
D. Song et al. / Bioorg. Med. Chem. 21 (2013) 788–794
4.3.3. 1-Allyl-1-methyl-4-tetradecanoyl-1,4-diazepan-1-ium
bromide (3c)
4.3.10. 4-Icosanoyl-1,1-dimethyl-[1,4]diazepan-1-ium; iodide
(3j)
Compound 2a was used and 3c (71.6%) was obtained as a white
solid. Mp: 189–191 °C; 1H NMR (500 MHz, DMSO-d6) d 6.11–6.00
(m, 1H), 5.67–5.62 (m, 2H), 4.08–4.03 (m, 2H), 3.85–3.73 (m,
2H), 3.55–3.39 (m, 6H), 3.02 (s, 3H), 2.31 (t, J = 7.5 Hz, 2H), 2.17–
2.06 (m, 2H), 1.52–1.45 (m, 2H), 1.30–1.18 (m, 20H), 0.85 (t,
J = 7.0 Hz, 3H); ESI-MS: m/z = 365 [M+]; HRMS (ESI) calcd for
Compound 2d was used and 3j (90.3%) was obtained as a white
solid. Mp: 176–177 °C; 1H NMR (400 MHz, DMSO-d6) d 3.73 (d,
J = 16.0 Hz, 2H), 3.52–3.41 (m, 6H), 3.09 (d, J = 8.0 Hz, 6H), 2.28
(t, J = 8.0 Hz, 2H), 2.07 (d, J = 28.0 Hz, 2H), 1.46 (s, 2H), 1.20 (s,
32H), 0.82 (t, J = 6.0 Hz, 3H); ESI-MS: m/z = 423 [M+]; HRMS (ESI)
calcd for C27H55N2OI (MH+) 423.4309, found 423.4300.
C23H45N20Br (MH+) 365.3526, found 365.3539.
4.4. Procedures for 4a–d
4.3.4. 1-Methyl-1-(3-nitrobenzyl)-3-tetradecanoyl-1,3-
diazepan-1-ium bromide (3d)
Carboxylic acid 1 (2.9 mmol) was dissolved in anhydrous
dichloromethane (0.2 M) at room temperature under an Ar atmo-
sphere. N,N-dimethyl-1,4-phenylenediamine (4.3 mmol), EDC
(4.3 mmol) and DMAP (0.9 mmol) were added, and the mixture
was stirred for 8 h at room temperature. Saturated NH4Cl solution
was added, and the mixture was extracted with dichloromethane
(3Â). The organic layer was washed with brine, dried over anhy-
drous Na2SO4, and concentrated in vacuo. The residue was purified
by chromatography on silica gel with 5% MeOH/CH2Cl2 to resolve
amides.
Compound 2a was used and 3d (91.8%) was obtained as a pale
yellow solid. Mp: 156–158 °C; 1H NMR (500 MHz, DMSO-d6) d 8.47
(d, J = 3.7 Hz, 1H), 8.41–8.37 (m, 1H), 8.03 (t, J = 7.5 Hz, 1H), 7.85–
7.80 (m, 1H), 4.87–4.82 (m, 2H), 4.02–3.84 (m, 1H), 3.74–3.62(m,
1H), 3.61–3.42 (m, 6H), 3.04 (s, 3H), 2.37–2.31 (m, 2H), 2.25–
2.18 (m, 2H), 1.54–1.45 (m, 2H), 1.33–1.19 (m, 20H), 0.85 (t,
J = 6.5 Hz, 3H); ESI-MS: m/z = 460 [M+]; HRMS (ESI) calcd for
C27H46N3O3Br (MH+) 460.3534, found 460.3548.
4.3.5. 1-Methyl-1-(3-nitrobenzyl)-3-tetradecanoyl-1,3-
diazepan-1-ium bromide (3e)
4.4.1. Tetradecanoic acid (4-dimethylamino-phenyl)-amide (4a)
Myristic acid was used and 4a (19.5%) was obtained as a yellow-
ish oil. 1H NMR (500 MHz, DMSO-d6) d 9.52 (s, 1H), 7.38 (d,
J = 8.5 Hz, 2H), 6.66 (d, J = 9.0 Hz, 2H), 2.82 (s, 6H), 2.21 (t,
J = 7.5 Hz, 2H), 1.55 (m, 2H), 1.27–1.23 (m, 20H), 0.86–0.84 (m,
3H); ESI-MS: m/z = 347 [M+H].
Compound 2a was used and 3e (31.0%) was obtained as a white
solid. Mp: 189–191 °C; 1H NMR (500 MHz, DMSO-d6) d 7.66–7.61
(m, 2H), 7.38–7.34 (m, 2H), 4.71–4.66 (m, 2H), 4.01–3.88 (m,
1H), 3.76–3.68 (m, 1H), 3.61–3.38 (m, 6H), 2.98 (s, 3H), 2.34–
2.29 (m, 2H), 2.23–2.13 (m, 2H), 1.52–1.45 (m, 2H), 1.30–1.19
(m, 20H), 0.85 (t, J = 6.5 Hz, 3H); ESI-MS: m/z = 433 [M+]; HRMS
(ESI) calcd for C27H46FN2OBr (MH+) 433.3584, found 433.3574.
4.4.2. Hexadecanoic acid (4-dimethylamino-phenyl)-amide (4b)
Palmitic acid was used and 4b (47.3%) was obtained as a yellow-
ish oil. 1H NMR (400 MHz, DMSO-d6) d 9.52 (s, 1H), 7.38 (d,
J = 6.9 Hz, 2H), 6.66 (d, J = 6.6 Hz, 2H), 2.82 (s, 6H), 2.21 (t,
J = 7.6 Hz, 2H), 1.56–1.53 (m, 2H), 1.27–1.23 (br m, 24H), 0.85 (t,
J = 6.8 Hz, 3H); ESI-MS: m/z = 375 [M+H].
4.3.6. 1-Ethyl-1-methyl-4-tetradecanoyl-[1,4]diazepan-1-ium;
iodide (3f)
Compound 2a was used and 3f (88.7%) was obtained as a pale
yellow solid. Mp: 162–163 °C; 1H NMR (300 MHz, DMSO-d6) d
3.47–3.41 (m, 4H), 2.45–2.24 (m, 4H), 2.28–2.21 (m, 5H), 1.81–
1.69 (m, 2H), 1.48 (br, 2H), 1.24 (m, 20H), 0.85 (t, J = 6.4 Hz, 3H);
ESI-MS: m/z = 353 [M+]; HRMS (ESI) calcd for C26H53N2OI (MH+)
353.3526, found 353.3533.
4.4.3. Octadecanoic acid (4-dimethylamino-phenyl)-amide (4c)
Stearic acid was used and 4c (21.4%) was obtained as a brown-
ish oil. 1H NMR (500 MHz, DMSO-d6) d 10.15 (s, 1H), 7.67 (d,
J = 9.0 Hz, 2H), 7.30 (d, J = 9.0 Hz, 2H), 3.44–3.38 (m, 6H), 2.31 (t,
J = 6.1 Hz, 2H), 1.55 (t, J = 4.5 Hz, 2H), 1.27–1.20 (m, 28H), 0.85 (t,
J = 6.8 Hz, 3H); ESI-MS: m/z = 403 [M+H].
4.3.7. 4-Hexadecanoyl-1,1-dimethyl-[1,4]diazepan-1-ium;
iodide (3g)
Compound 2b was used and 3g (54.9%) was obtained as a white
solid. Mp: 180–181 °C; 1H NMR (300 MHz, DMSO-d6) d 3.80–3.73
(m, 2H), 3.58–3.48 (m, 4H), 3.48–3.40 (m, 2H), 3.12 (s, 6H), 2.31
(t, J = 7.5 Hz, 2H), 2.26–2.03 (m, 2H), 1.57–1.44 (m, 2H), 1.35–
1.17 (m, 24H), 0.85 (t, J = 6.6 Hz, 3H); ESI-MS: m/z = 367 [M+];
HRMS (ESI) calcd for C23H47N2OI (MH+) 367.3680, found 367.3785.
4.4.4. Icosanoic acid (4-dimethylamino-phenyl)-amide (4d)
Arachidic acid was used and 4d (38.4%) was obtained as a
brownish oil. 1H NMR (400 MHz, DMSO-d6) d 7.38 (d, J = 6.8 Hz,
2H), 6.66 (d, J = 6.3 Hz, 2H), 5.76 (s, 1H), 2.82 (s, 6H), 2.21 (t,
J = 7.0 Hz, 2H), 1.97–1.94 (m, 2H), 1.57–1.55 (m, 2H), 1.23 (br m,
30H), 0.85–0.83 (m, 3H); ESI-MS: m/z = 431 [M+H].
4.3.8. 1-Methyl-4-palmitoyl-1-(3-(trifluoromethoxy)benzyl)-
1,4-diazepan-1-ium bromide (3h)
4.5. Procedures for 5a–d
Compound 2b was used and 3h (71.8%) was obtained as a white
solid. Mp: 181–182 °C; 1H NMR (300 MHz, CDCl3) d 7.71–7.66 (m,
2H), 7.49–7.44 (m, 2H), 7.31 (d, J = 8.6 Hz, 1H), 5.42 (br m, 2H),
4.26–3.69 (m, 8H), 3.33 (s, 3H), 2.37–2.18 (m, 4H), 1.56 (br s,
2H), 1.23 (br m, 24H), 0.85 (t, J = 6.7 Hz, 3H); ESI-MS: m/z = 528
[M+]; HRMS (ESI) calcd for C30H50F3N2O2Br (MH+) 527.3819, found
527.3824.
Methyl iodide (1.6 mmol) was added to a solution of 5a–d
(0.8 mmol) in anhydrous acetonitrile (0.3 M) at room temperature.
The reaction mixture was heated to 140–150 °C for 7 h. After the
reaction mixture equilibrated to room temperature, the precipitate
was granulated for 1 h at 0 °C. Solids were collected by filtration,
washed with ethyl acetate, and dried in vacuo.
4.3.9. 1,1-Dimethyl-4-stearoyl-1,4-diazepan-1-ium iodide (3i)
Compound 2c was used and 3i (79.4%) was obtained as a pale
yellow solid. Mp: 188–189 °C; 1H NMR (500 MHz, DMSO-d6) d
3.80–3.72 (m, 2H), 3.55–3.43 (m, 6H), 3.12 (s, 6H), 2.31 (t,
J = 7.5 Hz, 2H), 2.18–2.05 (m, 2H), 1.52–1.46 (m, 2H), 1.31–1.19
(m, 28H), 0.85 (t, J = 7.5 Hz, 3H); ESI-MS: m/z = 395 [M+]; HRMS
(ESI) calcd for C25H51N2OI (MH+) 395.3996, found 395.3995.
4.5.1. Trimethyl-(4-tetradecanoylamino-phenyl)-ammonium;
iodide (5a)
Compound 4a was used and 5a (78.7%) was obtained as a white
solid. Mp: 148–149 °C; 1H NMR (500 MHz, DMSO-d6) d 10.21 (s,
1H), 7.87 (d, J = 9.5 Hz, 2H), 7.76 (d, J = 9.5 Hz, 2H), 3.56 (s, 9H),
2.32 (t, J = 7.5 Hz, 2H), 1.59–1.56 (m, 2H), 1.28–1.24 (br m, 20H),

D. Song et al. / Bioorg. Med. Chem. 21 (2013) 788–794
793
0.85 (t, J = 6.8 Hz, 3H); ESI-MS: m/z = 361 [M+]; HRMS (ESI) calcd
for C23H41N2OI (MH+) 361.3213, found 361.3210.
1.74 (m, 2H), 1.42–1.38 (m, 2H), 1.29–1.27 (m, 26H), 0.89 (t,
J = 6.8 Hz, 3H); ESI-MS: m/z = 403 [M+H].
4.5.2. (4-Hexadecanoylamino-phenyl)-trimethyl-ammonium;
iodide (5b)
4.7. Procedures for 8a–c
Compound 4b was used and 5b (20.5%) was obtained as a pale
gray solid. Mp: 145–146 °C; 1H NMR (500 MHz, DMSO-d6) d 10.18
(s, 1H), 7.88–7.86 (m, 2H), 7.77–7.75 (m, 2H), 3.56 (s, 9H), 2.32 (t,
J = 7.4 Hz, 2H), 1.58 (m, 2H), 1.27–1.23 (m, 24H), 0.85 (t, J = 6.8 Hz,
3H); ESI-MS: m/z = 389 [M+]; HRMS (ESI) calcd for C25H45N2OI
(MH+) 389.3526, found 389.3519.
Methyl iodide (1.6 mmol) was added to a solution of 7a-c
(0.8 mmol) in anhydrous acetonitrile (0.3 M) at room tempera-
ture. The reaction mixture was heated to 140–150 °C for 7 h.
After the reaction mixture equilibrated to room temperature,
the precipitate was granulated for 1 h at 0 °C. Solids were col-
lected by filtration, washed with ethyl acetate, and dried in
vacuo.
4.5.3. Trimethyl-(4-octadecanoylamino-phenyl)-ammonium;
iodide (5c)
4.7.1. 4-(Dodecane-1-sulfonyl)-1,1-dimethyl-[1,4]diazepan-1-
ium; iodide (8a)
Compound 4c was used and 5c (36.7%) was obtained as a brown
solid. Mp: 127–128 °C; 1H NMR (500 MHz, DMSO-d6) d 10.11 (s,
1H), 7.71 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 9.0 Hz, 2H), 3.93 (s, 9H),
2.30 (t, J = 4.8 Hz, 2H), 1.57 (t, J = 4.5 Hz, 2H), 1.27–1.23 (m, 28H),
0.85 (t, J = 6.8 Hz, 3H); ESI-MS: m/z = 417 [M+]; HRMS (ESI) calcd
for C27H49N2OI (MH+) 417.3839, found 417.3839.
Compound 7a was used and 8a (47.5%) was obtained as a pale
yellow solid. Mp: 171–172 °C; 1H NMR (500 MHz, CDCl3) d 3.69
(br m, 2H), 3.53 (br m, 2H), 3.10 (br m, 2H), 3.04–3.01 (m, 2H),
2.99–2.93 (m, 2H), 2.67 (s, 3H), 1.80–1.74 (m, 2H), 1.42–1.38 (m,
2H), 1.31–1.27 (m, 18H), 0.89 (t, J = 7.0 Hz, 3H); ESI-MS: m/
z = 347 [M+]; HRMS (ESI) calcd for C19H41N2O2SI (MH+) 361.2883,
found 361.2877.
4.5.4. (4-Icosanoylamino-phenyl)-trimethyl-ammonium; iodide
(5d)
Compound 4d was used and 5d (93.2%) was obtained as a pale
purple solid. Mp: 155–156 °C; 1H NMR (500 MHz, DMSO-d6) d
7.88–7.86 (m, 2H), 7.77–7.75 (m, 2H), 5.75–5.75 (m, 1H), 3.55 (s,
9H), 2.31 (t, J = 7.3 Hz, 2H), 1.26–1.23 (m, 34H), 0.84 (t, J = 6.9 Hz,
3H); ESI-MS: m/z = 445 [M+]; HRMS (ESI) calcd for C29H53N2OI
(MH+) 445.4152, found 445.4152.
4.7.2. 1,1-Dimethyl-4-(tetradecane-1-sulfonyl)-[1,4]diazepan-1-
ium; iodide (8b)
Compound 7b was used and 8b (65.9%) was obtained as a
white solid. Mp: 178–179 °C; 1H NMR (400 MHz, DMSO-d6) d
3.73 (d, J = 16.0 Hz, 2H), 3.52–3.39 (m, 6H), 3.09 (d, J = 7.6 Hz,
6H), 2.28 (t, J = 7.4 Hz, 2H), 2.07 (d, J = 29.6 Hz, 2H), 1.46 (s,
2H), 1.20 (s, 22H), 0.82 (t, J = 6.4 Hz, 3H); ESI-MS: m/z = 389
[M+]; HRMS (ESI) calcd for C21H45N2O2SI (MH+) 389.3196, found
389.3215.
4.6. Procedures for 7a–c
Sulfonate sodium salts 6 (3.8 mmol) were dissolved in anhy-
drous dichloromethane (0.1 M solution), and thionyl chloride
(3.0 equiv) was added under an Ar atmosphere. The stirred suspen-
sion was heated and refluxed for 4 h. The reaction mixture was
cooled and poured onto crushed ice for 1 h. The compound 1-
methylhomopiperazine (5.8 mmol) was added dropwise, and the
reaction mixture was allowed to warm to room temperature and
stirred for 2 h. NaOH solution (10%) was added (final pH 13), and
the mixture was extracted with chloroform. The organic layer
was washed with brine, dried over anhydrous MgSO4, and concen-
trated in vacuo. The residue was purified by chromatography on
silica gel with 5% MeOH/CHCl3 to resolve the corresponding
amides.
4.7.3. 4-(Hexadecane-1-sulfonyl)-1,1-dimethyl-[1,4]diazepan-1-
ium; iodide (8c)
Compound 7c was used and 8c (85.4%) was obtained as a
white solid. Mp: 159–160 °C; 1H NMR (500 MHz, DMSO-d6) d
3.67 (br m, 2H), 3.59–3.54 (m, 4H), 3.42–3.39 (m, 2H), 3.13 (s,
6H), 2.12 (br m, 2H), 1.66–1.60 (m, 2H), 1.36–1.35 (m, 2H),
1.24 (br m, 26H), 0.85 (t, J = 6.8 Hz, 3H); ESI-MS: m/z = 417
[M+]; HRMS (ESI) calcd for C23H49N2O2SI (MH+) 417.3509, found
417.3496.
4.8. Procedures for 9a–b
Sulfonate sodium salts 6 (3.8 mmol) were dissolved in anhy-
drous dichloromethane (0.1 M solution), and thionyl chloride
(3.0 equiv) was added under an Ar atmosphere. The stirred sus-
pension was heated and refluxed for 4 h. The reaction mixture
was cooled and poured onto crushed ice for 1 h. N,N-dimethyl-
benzene-diamine (5.8 mmol) was added dropwise, and the reac-
tion mixture was warmed to room temperature and stirred for
2 h. NaOH solution (10%) was added (final pH 13), and the mix-
ture was extracted with chloroform. The organic layer was
washed with brine, dried over anhydrous MgSO4, and concen-
trated in vacuo. The residue was purified by chromatography
on silica gel with 5% MeOH/CHCl3 to resolve the corresponding
amides.
4.6.1. 1-(Dodecane-1-sulfonyl)-4-methyl-[1,4]diazepane (7a)
Sodium 1-dodecanesulfonate was used and 7a (94.3%) was ob-
tained as a yellowish oil. 1H NMR (500 MHz, CDCl3) d 3.69 (br m,
2H), 3.53 (br m, 2H), 3.10 (br m, 2H), 3.04–3.01 (m, 2H), 2.99–
2.93 (m, 2H), 2.67 (s, 3H), 1.80–1.74 (m, 2H), 1.42–1.38 (m, 2H),
1.31–1.27 (m, 18H), 0.89 (t, J = 7.0 Hz, 3H); ESI-MS: m/z = 347
[M+H].
4.6.2. 1-Methyl-4-(tetradecane-1-sulfonyl)-[1,4]diazepane (7b)
Sodium 1-tetradecanesulfonate was used and 7b (79.9%) was
obtained as a yellowish oil. 1H NMR (400 MHz, DMSO-d6) d 3.32
(t, J = 6.0 Hz, 4H), 3.01 (t, J = 8.0 Hz, 2H), 2.74 (s, 1H), 2.53 (t,
J = 5.0 Hz, 4H), 2.25 (s, 3H), 1.77–1.74 (m, 2H), 1.61–1.57 (m, 2H),
1.34–1.32 (m, 2H), 1.24 (br s, 20H), 0.85 (t, J = 6.8 Hz, 3H); ESI-
MS: m/z = 375[M+H].
4.8.1. Dodecane-1-sulfonic acid (4-dimethylamino-phenyl)-
amide (9a)
Sodium 1-dodecanesulfonate was used and 9a (83.3%) was ob-
tained as a brownish oil. 1H NMR (500 MHz, DMSO-d6) d 7.32–7.25
(m, 2H), 7.23–7.15 (m, 2H), 3.07–2.94 (m, 8H), 2.82 (s, 1H), 1.87–
1.78 (m, 2H), 1.42–1.28 (m, 18H), 0.90 (t, J = 7.0 Hz, 3H); ESI-MS:
m/z = 369 [M+H].
4.6.3. 1-(Hexadecane-1-sulfonyl)-4-methyl-[1,4]diazepane (7c)
1-Hexadecanesulfonyl chloride was used and 7c (49.0%) was
obtained as a yellowish oil. 1H NMR (500 MHz, CDCl3) d 3.65 (br
m, 2H), 3.52 (br m, 2H), 2.99–2.96 (m, 6H), 2.62 (s, 3H), 1.81–

794
D. Song et al. / Bioorg. Med. Chem. 21 (2013) 788–794
4.8.2. Tetradecane-1-sulfonic acid (4-dimethylamino-phenyl)-
amide (9b)
luciferase plasmid containing the RhoB promoter, and pRL-SV40-
renilla luciferase. Luciferase activity was integrated over a 10 s per-
iod and measured using a luminometer (Victor X Light; Perkin El-
mer, Waltham, MA, USA). The results were normalized to the levels
of renilla luciferase.
Sodium 1-tetradecanesulfonate was used and 9b (73.7%) was
obtained as a brownish oil. 1H NMR (500 MHz, DMSO-d6) d 9.13
(s, 1H), 7.03 (d, J = 7.9 Hz, 2H), 6.67 (d, J = 8.3 Hz, 2H), 2.85 (t,
J = 8.1 Hz, 9H), 1.63 (s, 2H), 1.29 (s, 3H), 1.21 (d, J = 15.7 Hz, 23H),
0.84 (t, J = 6.5 Hz, 3H); ESI-MS: m/z = 397 [M+H].
Acknowledgments
4.9. Procedures for 10a–b
This research was supported by National Research Foundation
(2012-0007275), Translational Research Center for Protein Func-
tion Control, NSF (2012-0000884), and the Brain Korea 21 project,
of the Republic of Korea.
Methyl iodide (1.6 mmol) was added to a solution of 9a–b
(0.8 mmol) in anhydrous acetonitrile (0.3 M) at room temperature.
The reaction mixture was heated to 140–150 °C for 7 h. After the
reaction mixture equilibrated to room temperature, the precipitate
was granulated for 1 h at 0 °C. Solids were collected by filtration,
washed with ethyl acetate, and dried in vacuo.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.027.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
4.9.1. [4-(Dodecane-1-sulfonylamino)-phenyl]-trimethyl-
ammonium; bromide (10a)
Compound 9a was used and 10a (33.3%) was obtained as a gray
solid. Mp: 119–120 °C; 1H NMR (500 MHz, DMSO-d6) d 7.16 (t,
J = 5.8 Hz, 1H), 3.33 - 3.30 (m, 1H), 3.06 (s, 6H), 3.02–2.97 (m,
4H), 1.89–1.83 (m, 2H), 1.65–1.59 (m, 2H), 1.37–1.33 (m, 2H),
1.28–1.20 (m, 20H), 0.85 (t, J = 7.0 Hz, 3H); ESI-MS: m/z = 397
[M+]; HRMS (ESI) calcd for C21H39N2O2SI (MH+) 383.2727, found
383.2738.
References and notes
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4.9.2. Trimethyl-[4-(tetradecane-1-sulfonylamino)-phenyl]-
ammonium; iodide (10b)
Compound 9b was used and 10b (86.7%) was obtained as a
white solid. Mp: 140–141 °C; 1H NMR (500 MHz, DMSO-d6) d
10.25 (s, 1H), 7.91–7.89 (d, J = 9.0 Hz, 2H), 7.34–7.32 (d,
J = 9.0 Hz, 2H), 3.56 (s, 9H), 3.16 (t, J = 7.5 Hz, 2H), 1.65–1.63 (m,
2H), 1.32–1.21 (m, 22H), 0.85 (t, J = 6.8 Hz, 3H); ESI-MS: m/
z = 411 [M+]; HRMS (ESI) calcd for C23H43N2O2SI (MH+) 411.3040,
found 411.3047.
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4.10. Sulforhodamine (SRB) assay
Growth inhibition of cancer cell lines in the presence of
NSC126188 was determined using the SRB assay as previously de-
scribed.10 SRB dye bound to the cell matrix was quantified using a
spectrophotometer at 530 nm.
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4.11. Luciferase assay
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Transactivation of RhoB was determined by reporter assay
using the dual-luciferase reporter assay system (Promega, Madi-
son, WI, USA), as previously described.11 HeLa cells at 75–90% con-
fluency were transiently cotransfected with the pGL2-RhoB-firefly