Synthesis and biological evaluation of novel aliphatic amido-quaternary ammonium salts for anticancer chemotherapy: Part i

Article abstract of DOI:10.1016/j.ejmech.2011.04.009

We synthesized novel aliphatic amido-quaternary ammonium salts in an effort to discover anticancer agents that increase Ras homolog gene family, member B, (RhoB) levels. These compounds exert anti-proliferative activities against several human cancer cell types. Seventeen compounds, varying in aliphatic carbon chain length and N-substituents, were synthesized and their biological activities were evaluated. Of these 17 compounds, compound 3i emerged as the most promising anticancer compound by promoting apoptosis through the RhoB mediated pathway. Potent biological activities observed for these novel aliphatic amido-quaternary ammonium salt analogues support their potential as anticancer, chemotherapeutic agents.

Full text of DOI:10.1016/j.ejmech.2011.04.009

European Journal of Medicinal Chemistry 46 (2011) 2861e2866
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel aliphatic amido-quaternary
ammonium salts for anticancer chemotherapy: Part I
Jee Sun Yang a, Doona Song a, Boah Lee a, Won Jin Ko a, Song-Kyu Park b, Misun Won c, Kiho Lee d,
,e,
Hwan Mook Kim d, Gyoonhee Han a
*
a Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seongsan-ro, Seodaemun-gu, 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 Bio-Evaluation Center, KRIBB, Ochang 363-883, 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:
We synthesized novel aliphatic amido-quaternary ammonium salts in an effort to discover anticancer
agents that increase Ras homolog gene family, member B, (RhoB) levels. These compounds exert anti-
proliferative activities against several human cancer cell types. Seventeen compounds, varying in
aliphatic carbon chain length and N-substituents, were synthesized and their biological activities were
evaluated. Of these 17 compounds, compound 3i emerged as the most promising anticancer compound
by promoting apoptosis through the RhoB mediated pathway. Potent biological activities observed for
these novel aliphatic amido-quaternary ammonium salt analogues support their potential as anticancer,
chemotherapeutic agents.
Received 26 January 2011
Received in revised form
23 March 2011
Accepted 3 April 2011
Available online 14 April 2011
Keywords:
Aliphatic amido-quaternary ammonium
salts
Ó 2011 Elsevier Masson SAS. All rights reserved.
RhoB
GTPase RAt Sarcoma (Ras)
Perifosine
Piperazine alkyl derivatives
NSC126188
1. Introduction
phosphatidylinositol 3-kinase (PI3K), were discovered through
studies of mutated Ras [4].
Cancer occurs when regulation of normal cell growth and
differentiation is compromised due to defects in the proliferation
related signaling mechanisms. Ras genes were the first-identified
oncogenes, and they are observed to be mutated in 20e30% of all
human tumors [1,2]. Disruption of the regulation of Ras and Ras-
relatedproteins in cancerleads toincreased invasion andmetastasis,
and decreased apoptosis in cancer cells. Activated Ras interacts with
several effector proteins in ways that stimulate catalytic activities,
and it also controls the activity of crucial signaling that regulates
normal cellular proliferation [3]. Mutations in downstream targets
of Ras signaling, such as v-raf murine sarcoma viral oncogene
homolog B1 (B-Raf), phosphatase and tensin homolog (PTEN), and
Ras is a low-molecular-weight GTP/GDP binding GTPase that
activates two major pathways that are deduced to play a critical
role in human cancer progression [5]: 1) the PI3K/Akt pathway, and
2) the mitogen-activated Mek/extracellular signal-related kinase
(Erk) pathway. Each of these pathways can induce uncontrolled
proliferation and tumor cell survival by activating genes that
stimulate cellular proliferation and survival. This type of carcino-
genesis could be reversed in order to suppress proliferation, and/or
induce apoptosis, by antagonistic effects on these two pathways.
The protein kinase B (Akt) pathway is generally activated in
cancer cells and has a wide range of downstream targets that
regulate tumor-associated cell processes, such as cell growth, cell
cycle progression, survival, migration, epithelialemesenchymal
transition, and angiogenesis [6]. By blocking the Akt signaling
pathway, apoptosis and growth inhibition in tumor cells were
observed. In the last several years, a number of inhibitors of the Akt
pathway have been identified by high-throughput, and virtual
screening using combinatorial or medicinal chemical libraries.
Phosphatidylinositol (PI) analogues are one class of Akt inhibitors
* Corresponding author. Translational Research Center for Protein Function
Control, Department of Biotechnology, Yonsei University, Seongsan-ro, Seodaemun-
gu, Seoul 120-749, Republic of Korea. Tel.: þ82 2 21232882; fax: þ82 2 3627265.
E-mail address: gyoonhee@yonsei.ac.kr (G. Han).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.009

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J.S. Yang et al. / European Journal of Medicinal Chemistry 46 (2011) 2861e2866
that is represented by perifosine [octadecyl-(1,1-dimethyl-piper-
idinio-4-yl)-phosphate] and phosphatidylinositol ether lipid
analogues (PIAs) (Fig.1). Perifosine is a synthetic, orally bioavailable
alkylphospholipid that is structurally similar to naturally occurring
phospholipids. It is known to represent a new class of antitumor
agent that targets cell membranes and inhibits Akt phosphoryla-
tion, and it has exhibited significant anti-proliferative activity
in vitro and in vivo in several human tumor model systems [7e11].
Not by MEK/ERK pathway but by PI3K/Akt pathway, oncogenic
Ras downregulates RhoB which is a suppressor of transformation,
invasion and metastasis [12]. RhoB is 86% identical at the amino
acid sequence level to the RhoA-related subgroups (RhoA and
RhoC), but the activities of these different proteins that relate to
oncogenesis are quite different [13]. While RhoA and RhoC promote
oncogenesis, invasion and metastasis, RhoB appears to be required
for stress-induced apoptosis and anti-neoplastic activity, and
performs as a negative moderator of cell survival [14,15]. Interest-
ingly, it was recently reported that a piperazine alkyl derivative
(NSC126188, Fig. 1), which is structurally similar to perifosine,
induces apoptosis via up-regulation of RhoB in HeLa cells [16]. This
prompted us to suggest that piperazine alkyl derivatives can induce
apoptosis through the PI3K/Akt pathway, the RhoB mediated
pathway, or both pathways.
Scheme 1. Reaction protocol for the synthesis of aliphatic amido-quaternary ammo-
nium salts (3aeq). Reagents and conditions: (a) i) SOCl2, CH2Cl2 at 60 ꢀC; ii) 1-alkyl-
piperazine, CH2Cl2 at 0 ꢀC, or DMAP, EDCI, 1-alkylpiperazine, CH2Cl2; (b) alkyl halide,
CH3CN at w95e100 ꢀC or alkyl halide, toluene, w110e115 ꢀC.
3. Results and discussion
We present the synthesis of novel aliphatic amido-quaternary
ammonium salts based on the structural characteristics of lead
compound NSC126188, and describe the biological activities of these
derivatives. We performed a closely related structureeactivity
relationship (SAR) study of these compounds based on their RhoB
activation using promoter assays and via assessment of anti-
proliferative activities in several human cancer cell types. Our
results suggest that such novel aliphatic amido-quaternary
ammonium salts may be promising chemotherapeutic anticancer
agents.
Compounds 3aeq were assayed for growth inhibitory activity
against six human cancer cell types: 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). Results are tabulated as GI50 values in Tables 1e3. Peri-
fosine, known PI3K/Akt inhibitors, and NSC126188 were each used
as positive references for comparison of in vitro activities. Inter-
estingly, perifosine, NSC126188, and other PI analogues share
a common structural motif consisting of a long aliphatic chain and
a six-membered ring. Among these known compounds, perifosine
and NSC126188 also share the same amido-quaternary ammonium
salt moiety with variable N,N-dimethyl substituents.
2. Chemistry
To assess the effects of carbon chain length, eight compounds
with diversified chain length (from 10 to 24 carbons), were
prepared. N,N-Dialkyl substituents of compounds were fixed to the
dimethyl group just as the known inhibitors were. Among the eight
compounds, 3cef (carbon chain length 14e20) exhibited similar
levels of anti-proliferative activity. In particular, 3c and 3f exhibited
increased growth inhibitory activity compared to perifosine and
NSC126188 (re-synthesized as 3d) in prostate cancer cell types.
Compounds 3cef also showed impressive growth inhibitory activ-
ities, especially in gastric cancer cells. Compound 3d exhibited the
most potent inhibitory profiles, which were more robust than per-
ifosine. For compounds with a relatively shorter or longer carbon
chain (n ¼ 8 or n ¼ 22), cell growth inhibition was either observed
weakly, or not observed at all, irrespective of cell type. Every
compound having a carbon chain length in the range of 14e18
(n ¼ 12e16) generally exhibited a more robust effect than perifosine
on growth inhibition against PC-3 and NUGC-3 cell types.
Based on the amidopiperazinium structure of the lead
compound NSC126188, we designed and synthesized 17 analogues
with two major modification points: 1) diversification of aliphatic
carbon chain length, and 2) introduction of various N-substituents.
As illustrated in Scheme 1, molecules were synthesized from the
various fatty acids by two-step procedures based on design. Fatty
acids 1 were reacted with thionyl chloride, and the resultant
intermediate, acid chloride was amidated with 1-methylpiperazine
or 1-ethylpiperazine to yield amides 2aei. Amides were converted
to amido-quaternary ammonium salts (3) by N-alkylation using
diverse alkyl halides. All quaternary ammonium salt analogues
were fully characterized and evaluated for in vitro biological
activities.
On the topic of optimal chain length, we were interested in
exploring the structural effects of the 6-membered ring and the N-
alkyl substituents on potency. Therefore, carbon chain length was
fixed at 16 or 22 carbons, which is same chain length of NSC126188
or perifosine, respectively. Replacements of N-alkyl substituents,
including: methyl, ethyl, allyl, and benzyl, were investigated (3ieq).
For the 16-carbon chain length series, 3i (N-ethyl-N-allyl) exhibited
remarkably potent growth inhibitory activity toward most of the
human cancer cell types used in this study, except HCT-15 cell
types. N-Methyl substituted compounds, such as 3k, showed no
activity at all. More growth inhibition tended to coincide with
bulkier substituents. However, contrary to expectations benzyl
Fig. 1. Chemical structures of PI analogues (PIA5 and perifosine) and NSC126188 (4-
hexadecanoyl-1,1-dimethyl-piperazin-1-ium iodide).

J.S. Yang et al. / European Journal of Medicinal Chemistry 46 (2011) 2861e2866
2863
Table 1
Cell growth inhibitory activities of compounds 3aeh.a,b
Cell line
Tissue
Growth inhibition (mM)
3a
3b
3c
3d
3e
3f
3g
3h
Perifosine
PC-3
Prostate
Gastric
Breast
Kidney
Colon
NA
NA
NA
NA
NA
NA
0.58
1.73
3.53
NA
NA
NA
0.29
0.56
0.89
1.01
0.85
1.58
0.48
0.29
1.44
1.04
0.58
2.34
0.53
0.56
1.62
2.74
0.81
2.7
0.31
0.42
1.63
1.39
0.54
1.74
0.99
1.15
3.51
2.39
2.41
3
4.79
NA
NA
NA
NA
NA
0.44
0.54
2.86
4.56
1.25
4.21
NUGC-3
MDA-MB-231
ACHN
HCT-15
NCI-H23
Lung
a
Growth inhibition was measured by SRB (sulforhodamine B) assay.
NA not active.
b
group substituted compounds, such as 3j and 3l, exhibited poor
inhibitory activities. Such poor activities with benzyl group
substituted compounds could be due to solubility problems. As
shown in Table 4, benzyl group substituted compounds increased
the log D value, which was calculated using Discovery Studio
(Accelrys, San Diego, CA, USA).
In the series of diverse N-alkyl substituted compounds (3iem)
that have 16 carbon chains, anti-proliferative activities against both
prostate and gastric cancer cell types were observed. Similarly,
corresponding RhoB promoter-based reporter expression levels
were generally high. The N-ethyl-N-allyl substituted compound 3i
exhibited the most robust reporter induction and the most
impressive overall growth inhibitory effects. The same N-ethyl-N-
allyl substituted characteristic in the context of the 22-carbon chain
compounds resulted in similar ability to induce reporter gene
expression. However, 3k (N-methyl-N-allyl) lacked anti-
proliferative activities, but induced reporter levels quite highly.
This may indicate that some compounds, including 3k, mediate
other signaling pathways that offset the regulation of apoptosis by
RhoB. As mentioned above, compounds with 22-carbon chain
length are highly lipophilic that the compounds are hardly dis-
solved to penetrate cell membranes. Thus, the compounds showed
conspicuously low RhoB expression level, except 3n which has N-
ethyl-N-allyl substituent same as the most potent analogue, 3i.
The substituent effect may be explained through comparison of
the differences in reporter levels versus anti-proliferative activity
between three sets of compounds: 1) comparison of 3i (N-ethyl-N-
allyl) to 3d (N,N-dimethyl), 2) comparison of 3n (N-ethyl-N-allyl) to
3g (N,N-dimethyl), and 3) comparison of 3n to perifosine (N,N-
dimethyl). Based on the reporter outputs for first two sets of
compounds, N-ethyl-N-allyl substituted analogues exhibited
almost twice the reporter induction ability of that observed from
N,N-dimethyl substituted compounds. The last set of compounds
(3n compared with perifosine) illustrated similar levels of induc-
tion control. In regards to effects on cell growth, 3i showed greater
anti-proliferative activities than 3d in human cancer cell types, and
these activities are in conjunction with reporter induction through
the RhoB promoter. The growth inhibitory activity of 3n, however,
was inferior to that of perifosine, even though they induce similar
levels of reporter expression through the RhoB promoter. Such
results could be also explained by the lipophilic properties of these
compounds, as mentioned above. While carbon chain length in
these compounds is the same, the lipophilicity of perifosine could
be offset by four hydrogen bond acceptors. Consequently, peri-
fosine might be more soluble in vitro and more easily access the
Perifosine structural analogues having a chain length of 22
carbons (n ¼ 20) were also prepared with diverse N-alkyl substit-
uents. As previously stated, compounds having long carbon chains
exhibit relatively poor activities (Table 1), and the overall anti-
proliferative activities of 3neq were less impressive than that of
perifosine. Moreover 3g, which is strikingly similar in structure to
perifosine, with the exception of the piperazine ring moiety, also
exhibited poor growth inhibitory activity. It is possible that this
poor level of activity is due to lipophilicity, which is supported by
the A log P98 value. A log P98 values increase with carbon chain
length. The 22-carbon chain length series is characterized by
A log P98 values of 7 or greater, which is too lipophilic to reveal
inhibitory activities in cellular assays. Moreover, the large number
of rotatable bonds may prevent these compounds from penetrating
the cell membrane. Perifosine has a relatively long carbon chain,
but it has an A log P98 value of 5.75. While other compounds
possess only one hydrogen bond acceptor, the carbonyl group
adjacent to the piperazine ring, perifosine has four hydrogen bond
acceptors, and this might impact the A log P98 value in a way that
counter balances the effect of the long carbon chain (Table 4).
Based on the results of the RhoB promoter assay, the anti-
proliferative activity of the prepared analogues parallels their
ability to induce reporter output (Fig. 2). For the first compound set
(3aeh), molecules with potent anti-proliferative activities robustly
induced reporter expression in the RhoB promoter-based assay. For
example, compounds 3c, 3d and 3f exhibited potent anti-
proliferative activity and showed the ability to induce reporter
expression in the RhoB promoter-based assay, while other
compounds (3a, 3b, 3g, and 3h) with poor anti-proliferative
activities exhibited poor ability to induce reporter expression
through the RhoB promoter. This result suggests that cell growth
inhibition is decreased by apoptosis, which can be induced by
activated RhoB after the treatment with these analogues.
Table 2
Table 3
Cell growth inhibitory activities of compounds 3iem.
Cell growth inhibitory activities of compounds 3neq.
Cell line
Tissue
Growth inhibition (
3i 3j 3k 3l
Prostate 0.37 0.55 NA 0.51 0.8
m
M)
Cell line
Tissue
Growth inhibition (mM)
3m
3d
Perifosine
3n
3o
3p
3q
3g
Perifosine
PC-3
NUGC-3
MDA-MB-231 Breast
ACHN
HCT-15
NCI-H23
0.48 0.44
PC-3
Prostate
Gastric
Breast
Kidney
Colon
1.47
1.54
2.53
3.38
2.78
3.94
0.96
1.53
3.16
2.37
2.13
3.07
1.24
1.9
1.98
4.74
2.53
2.95
0.85
1.22
2.01
1.9
1.69
2.6
0.99
1.15
3.51
2.39
2.41
3
0.44
0.54
2.86
4.56
1.25
4.21
Gastric 0.27 0.46 NA 0.58 0.29 0.54
0.68 1.16 NA 1.45 2.05 1.44 2.86
0.5 0.92 NA 1.98 1.89 1.04 4.56
1.33 NA NA 5.06 1.8 0.58 1.25
0.88 1.76 NA 2.05 3.65 2.34 4.21
1
NUGC-3
MDA-MB-231
ACHN
HCT-15
NCI-H23
Kidney
Colon
Lung
Lung

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J.S. Yang et al. / European Journal of Medicinal Chemistry 46 (2011) 2861e2866
Table 4
thin layer chromatography (TLC) on precoated silica gel 60 F254
Molecular properties of compounds 3aeq.
(mesh) (E. Merck, Mumbai, India) and spots were visualized under
UV light (254 nm). Flash column chromatography was performed
with silica (Merck EM9385, 230e400 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 and
carbon chemical shifts are expressed in parts per million relative to
internal tetramethylsilane, and coupling constants (J) are expressed
in hertz. Liquid chromatography/mass spectrometry (LC/MS)
spectra were recorded by electrospray ionization (ESI) on Shimadzu
LC/MS instruments (10% 0.1% TFA in H2O/90% 0.1% TFA in acetoni-
trile) scan (from 0 to 600 amu/z) mode, and 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
fast atom bombardment (FAB) on the JMS-700 (Jeol, Japan) High
Resolution Tandem Mass Spectrometer at the Korea Basic Science
Institute in Seoul, Korea.
A log P98
log D
Num_rotatable
bonds
Num_H
_acceptors
Num_H
_donors
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
Perifosine
2.042
2.954
3.867
4.779
5.692
6.604
7.516
8.429
5.745
6.712
5.396
6.363
5.128
8.482
8.133
9.100
7.865
5.750
ꢁ1.070
ꢁ0.158
0.755
1.667
2.579
3.492
4.404
5.317
3.327
4.294
2.978
3.945
2.016
6.064
5.715
6.682
4.753
4.607
8
10
12
14
16
18
20
22
17
17
16
16
15
23
22
22
21
20
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4.1. Procedures for 2aei
surface of the cell membrane than 3n, thereby facilitating an ability
to penetrate the membrane.
Fatty acid 1 (2.9 mmol) was dissolved in anhydrous dichloro-
methane (0.1 M solution), and then thionyl chloride (3.0 equiv) was
added under argon (Ar) atmosphere. The stirred suspension was
heated to reflux for 4 h. Then the reaction mixture was cooled and
poured onto crushed ice for 1 h. Piperazine (5.8 mmol) was added
dropwise, and the reaction mixture was then allowed to warm up
to room temperature and stirred for 2 h. NaOH solution (10%) was
added (final pH 13), and the mixture was extracted with chloro-
form. The organic layer was washed with brine, dried over anhy-
drous MgSO4, and then concentrated in vacuo. The residue was
purified by chromatography on silica gel with 5% MeOH/CHCl3 to
afford the corresponding amides.
Perifosine inhibits PI3K/Akt, and NSC126188 modulates RhoB.
These two compounds exhibit structural similarities that may be
responsible for regulating apoptosis through PI3K/Akt pathway
and/or RhoB mediated pathways. Therefore, we have designed and
synthesized 17 aliphatic amido-quaternary ammonium salts for
anticancer chemotherapeutic agents based on the structural
moieties common to both compounds. We then evaluated the
effects of diversity in carbon chain length and N-substituent
patterns on the growth of six human cancer cell types. Compound
3i emerged as the most promising anticancer agent, exhibiting
potent inhibition of cancer cell growth and stimulation of
apoptosis. The effects of 3i in RhoB promoter-based reporter assays
indicate that this compound may act through RhoB mediated
signaling. Compound 3i showed impressive parallels in activities
based on the ability to prevent cell proliferation by gastric and
prostate cancer cells and the ability to induce reporter output in the
RhoB promoter-based assay. This study is the first to identify
aliphatic amido-quaternary ammonium salts that have potential
chemotherapeutic properties and act through the RhoB mediated
pathway.
General procedures 1 (amidation step in Scheme 1)
Carboxylic acid 1 (2.9 mmol) was dissolved in anhydrous
dichloromethane(0.2 M)atroomtemperatureunderAratmosphere.
Piperazine (4.3 mmol), EDC (1-ethyl-3-[3-dimethylaminopropyl]
carboimide hydrochloride, 4.3 mmol) and DMAP (4-dimethylami-
nopyridine, 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 (3ꢂ). The organic
layer was washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by chromatography
on silica gel with 5% MeOH/CH2Cl2 to afford amides.
4. Experimental section
All chemicals were obtained from commercial suppliers and
used without further purification. All reactions were monitored by
4.1.1. 1-(4-Methylpiperazin-1-yl)decan-1-one (2a)
12
11
10
9
8
7
1H NMR (CDCl3, 500 MHz)
d 3.65e3.58 (m, 2H), 3.51e3.45 (m,
2H), 2.46e2.36 (m, 4H), 2.30 (s, 3H), 2.23 (t, J ¼ 7.5 Hz, 2H),
1.57e1.50 (m, 2H), 1.35e1.10 (m, 12H), 0.80 (t, J ¼ 6.7 Hz, 3H); ESI-
MS: m/z ¼ 255 [MþH].
6
5
4
3
2
1
0
4.1.2. 1-(4-Methylpiperazin-1-yl)dodecan-1-one (2b)
1H NMR (CDCl3, 300 MHz)
d 3.72e3.67 (m, 2H), 3.53 (t,
J ¼ 4.5 Hz, 2H), 2.50e2.41 (m, 4H), 2.36 (s, 3H), 2.30 (t, J ¼ 7.8 Hz,
2H), 1.65e1.54 (m, 2H), 1.36e1.22 (m, 16H), 0.87 (t, J ¼ 6.6 Hz, 3H);
ESI-MS: m/z ¼ 283 [MþH].
4.1.3. 1-(4-Methylpiperazin-1-yl)tetradecan-1-one (2c)
1H NMR (CDCl3, 500 MHz)
d 3.65e3.55 (m, 2H), 3.46 (t,
(HeLa, 5µM)
J ¼ 4.5 Hz, 2H), 2.39 (t, J ¼ 5.0 Hz, 4H), 2.29 (s, 3H), 2.23 (t, J ¼ 5.0 Hz,
2H), 1.57e1.51 (m, 2H), 1.28e1.13 (m, 20H), 0.80 (t, J ¼ 6.7 Hz, 3H);
ESI-MS: m/z ¼ 311 [MþH].
Fig. 2. Detection of RhoB promoter activation by reporter assay. Luciferase activity was
determined in the cells transfected with either the pGL2 vector or pGL-RhoB-Luc in the
presence of aliphatic amido-quaternary ammonium salts.

J.S. Yang et al. / European Journal of Medicinal Chemistry 46 (2011) 2861e2866
2865
4.1.4. 1-(4-Methylpiperazin-1-yl)hexadecan-1-one (2d)
1H NMR (DMSO-d6, 500 MHz)
3.96e3.84 (m, 4H), 3.17 (m, 4H),
4.2.4. 1,1-Dimethyl-4-palmitoylpiperazin-1-ium iodide (3d)
1H NMR (DMSO-d6, 500 MHz)
3.79e3.76 (m, 4H), 3.41e3.39
d
d
2.68 (s, 3H), 2.31 (t, J ¼ 7.5 Hz, 2H), 1.64e1.58 (m, 2H), 1.29e1.21 (m,
(m, 4H), 3.14 (s, 6H), 2.34 (t, J ¼ 7.5 Hz, 2H), 1.49e1.47 (m, 2H),
24H), 0.87 (t, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 339 [MþH].
1.26e1.24 (br m, 24H), 0.85 (t, J ¼ 7.0 Hz, 3H); 13C NMR (DMSO-d6,
500 MHz)
d 171.76, 60.90, 60.76, 51.1, 40.39, 39.73, 32.00, 29.72,
4.1.5. 1-(4-Methylpiperazin-1-yl)octadecan-1-one (2e)
29.42, 25.24, 22.80, 14.66; MS (FAB) m/z 353 (MHþ), HRMS (FAB)
1H NMR (CDCl3, 400 MHz)
d
3.63 (t, J ¼ 4.8 Hz, 2H), 3.47 (t,
calcd. for C22H45N2OI (MHþ) 353.3532, found 353.3537.
J ¼ 4.8 Hz, 2H), 2.41e2.35 (m, 4H), 2.30 (t, J ¼ 8.0 Hz, 5H), 1.64e1.59
(m, 2H), 1.34e1.23 (m, 28H), 0.88 (t, J ¼ 6.8 Hz, 3H); ESI-MS: m/
z ¼ 367 [MþH].
4.2.5. 1,1-Dimethyl-4-stearoylpiperazin-1-ium iodide (3e)
1H NMR (DMSO-d6, 400 MHz)
d
3.77 (t, J ¼ 5.4 Hz, 4H), 3.39 (t,
J ¼ 4.8 Hz, 2H), 3.32e3.30 (m, 2H), 3.14 (s, 6H), 2.33 (t, J ¼ 7.2 Hz,
2H), 1.50e1.45 (m, 2H), 1.25e1.20 (m, 28H), 0.85 (t, J ¼ 5.6 Hz, 3H);
4.1.6. 1-(4-Methylpiperazin-1-yl)icosan-1-one (2f)
1H NMR (CDCl3, 300 MHz)
d
3.72 (br m, 2H), 3.58e3.55 (m, 2H),
13C NMR (DMSO-d6, 500 MHz)
d 171.72, 62.31, 60.96, 60.83, 51.22,
2.51e2.49 (m, 4H), 2.40 (s, 3H), 2.32 (t, J ¼ 7.7 Hz, 2H), 1.65e1.60 (m,
2H), 1.30e1.26 (m, 32H), 0.89 (t, J ¼ 6.7 Hz, 3H); ESI-MS: m/z ¼ 395
[MþH].
40.45, 39.79, 31.99, 29.74, 29.40, 25.23, 22.78, 14.62; MS (FAB) m/z
381 (MHþ). HRMS (FAB) calcd. for C24H49N2OI (MHþ) 381.3845,
found 381.3843.
4.1.7. 1-(4-Methylpiperazin-1-yl)docosan-1-one (2g)
4.2.6. 4-Icosanoyl-1,1-dimethylpiperazin-1-ium iodide (3f)
1H NMR (CDCl3, 400 MHz)
d
3.64 (t, J ¼ 5.0 Hz, 2H), 3.48 (t,
1H NMR (DMSO-d6, 400 MHz)
d 3.78 (m, 4H), 3.39 (m, 4H), 3.14
J ¼ 5.0 Hz, 2H), 2.41e2.36 (m, 4H), 2.33 (t, J ¼ 7.8 Hz, 5H), 1.65e1.58
(m, 2H), 1.30e1.25 (m, 36H), 0.88 (t, J ¼ 6.8 Hz, 3H); ESI-MS: m/
z ¼ 423 [MþH].
(s, 6H), 2.33 (t, J ¼ 7.6 Hz, 2H), 1.48 (m, 2H), 1.23 (m, 32H), 0.85 (t,
J ¼ 6.4 Hz, 3H); MS (FAB) m/z 409 (MHþ). HRMS (FAB) calcd. for
C26H53N2O I (MHþ) 409.4158, found 409.5097.
4.1.8. 1-(4-Methylpiperazin-1-yl)tetracosan-1-one (2h)
4.2.7. 4-Docosanoyl-1,1-dimethylpiperazin-1-ium iodide (3g)
1H NMR (CDCl3, 300 MHz)
d
3.75 (br m, 2H), 3.60 (m, 2H), 2.55
1H NMR (DMSO-d6, 300 MHz)
d 3.76 (br, 4H), 3.37 (br, 4H), 3.12
(m, 4H), 2.43 (s, 3H), 2.32 (t, J ¼ 7.6 Hz, 2H), 1.65e1.58 (m, 2H),
1.31e1.20 (m, 40H), 0.89 (t, J ¼ 6.6 Hz, 3H); ESI-MS: m/z ¼ 451
[MþH].
(s, 6H), 2.32 (t, J ¼ 7.4 Hz, 2H), 1.43 (m, 2H), 1.22 (m, 36H), 0.84 (t,
J ¼ 5.9 Hz, 3H); MS (FAB) m/z 437 (MHþ). HRMS (FAB) calcd. for
C28H57N2OI (MHþ) 437.4471, found 437.4470.
4.1.9. 1-(4-Ethylpiperazin-1-yl)hexadecan-1-one (2i)
4.2.8. 1,1-Dimethyl-4-tetracosanoylpiperazin-1-ium iodide (3h)
1H NMR (CD3OD, 500 MHz)
d
3.62e3.57 (m, 4H), 2.51e2.44 (m,
1H NMR (DMSO-d6, 300 MHz)
d 3.69(m, 4H), 3.26 (m, 4H), 3.06
6H), 2.39 (t, J ¼ 7.7 Hz, 2H), 1.59 (br m, 2H), 1.33e1.29 (m, 24H), 1.13
(s, 6H), 2.25 (t, J ¼ 7.3 Hz, 2H), 1.39 (m, 2H), 1.14 (m, 40H), 0.76 (t,
(t, J ¼ 7.2 Hz, 3H), 0.91 (t, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 353 [MþH].
J ¼ 6.0 Hz, 3H); 13C NMR (DMSO-d6, 500 MHz)
d 171.82, 61.05, 51.53,
51.20, 40.59, 40.25, 39.92, 32.66, 29.64, 29.32, 25.22, 22.72, 14.54;
MS (FAB) m/z 465 (MHþ). HRMS (FAB) calcd. for C30H61N2OI (MHþ)
465.4784, found 465.4783.
4.2. Procedures for 3aeq
4.2.9. 1-Allyl-1-ethyl-4-palmitoylpiperazin-1-ium bromide (3i)
Alkyl halide (1.6 mmol) was added to a solution of 2aei
(0.8 mmol) in anhydrous acetonitrile (0.3 M) at room temperature.
Then, the reaction mixture was heated to w140e150 ꢀ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.
1H NMR (DMSO-d6, 500 MHz)
d 6.04e5.97 (m, 1H), 5.71e5.63
(m, 2H), 4.08 (d, J ¼ 7.5 Hz, 2H), 3.80e3.77 (m, 4H), 3.44e3.38 (m,
4H), 3.36e3.35 (m, 2H), 2.34 (t, J ¼ 7.5 Hz, 2H), 1.50e1.47 (m, 2H),
1.26e1.24 (br m, 27H), 0.85 (t, J ¼ 7.0 Hz, 3H); 13C NMR (DMSO-d6,
500 MHz)
d 171.84, 128.27, 125.78, 59.45, 57.17, 57.00, 54.12, 40.47,
39.87, 32.61, 29.72, 29.41, 25.19, 22.80, 14.67, 7.55; MS (FAB) m/z 393
(MHþ). HRMS (FAB) calcd. for C25H49N2OBr (MHþ) 393.3845, found
393.3847.
4.2.1. 4-Decanoyl-1,1-dimethylpiperazin-1-ium iodide (3a)
1H NMR (DMSO-d6, 300 MHz)
d 3.78e3.75 (m, 4H), 3.40e3.34
(m, 4H), 3.12 (s, 6H), 2.32 (t, J ¼ 7.2 Hz, 2H), 1.50e1.42 (m, 2H),
1.30e1.21 (m, 12H), 0.85 (t, J ¼ 6.6 Hz, 3H); MS (FAB) m/z 269(MHþ).
HRMS (FAB) calcd. for C16H33N2OI (MHþ) 269.2593, found
269.2600.
4.2.10. 1-Benzyl-1-ethyl-4-palmitoylpiperazin-1-ium bromide (3j)
1H NMR (DMSO-d6, 500 MHz)
d 7.56e7.52 (m, 5H), 4.64 (s, 2H),
4.13e4.09 (m, 1H), 3.97e3.94 (m, 1H), 3.74e3.71 (m, 1H), 3.54e3.49
(m, 1H), 3.40e3.36 (m, 4H), 3.30e3.27 (m, 2H), 2.36e2.30 (m, 2H),
1.49e1.46 (m, 2H), 1.37 (t, J ¼ 7.2 Hz, 3H), 1.24e1.20 (br m, 24H),
4.2.2. 4-Dodecanoyl-1,1-dimethylpiperazin-1-ium iodide (3b)
0.85 (t, J ¼ 7.5 Hz, 3H); 13C NMR (DMSO-d6, 500 MHz)
d 171.84,
1H NMR (CDCl3, 300 MHz)
d
4.02e3.83 (m, 5H), 3.65e3.55 (m,
133.68, 131.07, 129.74, 127.91, 62.55, 56.09, 51.64, 40.55, 35.36,
29.72, 29.41, 25.17, 22.80, 14.67, 9.87, 7.91; MS (FAB) m/z 443 (MHþ).
HRMS (FAB) calcd. for C29H51N2OBr (MHþ) 443.4001, found
443.3998.
8H), 2.39 (t, J ¼ 7.5 Hz, 2H), 1.68e1.55 (m, 5H), 1.36e1.22 (m, 16H),
0.87 (t, J ¼ 6.6 Hz, 3H); 13C NMR (DMSO-d6, 500 MHz)
d 171.78,
60.91, 60.91, 60.76, 55.26, 51.16, 46.58, 40.48, 39.72, 29.75, 29.42,
25.23, 22.80, 14.67; MS (FAB) m/z 297 (MHþ). HRMS (FAB) calcd. for
C18H37N2OI (MHþ) 297.2906, found 297.2908.
4.2.11. 1-Allyl-1-methyl-4-palmitoylpiperazin-1-ium bromide (3k)
1H NMR (DMSO-d6, 500 MHz)
d 6.10e6.02 (m, 1H), 5.66 (d,
4.2.3. 1,1-Dimethyl-4-tetradecanoylpiperazin-1-ium iodide (3c)
J ¼ 12.5 Hz, 2H), 4.10 (d, J ¼ 7.5 Hz, 2H), 3.94e3.86 (m, 2H),
3.77e3.74 (m, 1H), 3.65e3.62 (m, 1H), 3.39e3.37 (m, 4H), 3.07 (s,
3H), 2.34 (t, J ¼ 7.5 Hz, 2H), 1.49e1.47 (m, 2H), 1.24 (br m, 24H), 0.85
1H NMR (DMSO-d6, 300 MHz)
d 3.76 (s, 4H), 3.39e3.34 (m, 4H),
3.12 (s, 6H), 2.31 (t, J ¼ 7.2 Hz, 2H), 1.50e1.41 (m, 2H), 1.30e1.20 (m,
20H), 0.83 (t, J ¼ 6.6 Hz, 3H); MS (FAB) m/z 325 (MHþ). HRMS (FAB)
calcd. for C20H41N2OI (MHþ) 325.3219, found 325.3224.
(t, J ¼ 6.8 Hz, 3H); 13C NMR (DMSO-d6, 500 MHz)
d 171.80, 156.23,
128.75, 125.94, 65.24, 59.00, 47.06, 40.64, 40.21, 39.29, 29.77, 29.42,

2866
J.S. Yang et al. / European Journal of Medicinal Chemistry 46 (2011) 2861e2866
25.23, 22.80, 14.66; MS (FAB) m/z 379 (MHþ). HRMS (FAB) calcd. for
4.4. Luciferase assay
C24H47N2O Br (MHþ) 379.3688, found 379.3695.
Transactivation of RhoB was determined by reporter assay using
the dual-luciferase reporter assay system (Promega, Madison, WI,
USA), as previously described [18]. HeLa cells at 75e90% confluence
were transiently cotransfected with the pGL2-RhoB-firefly lucif-
erase plasmid containing 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.
4.2.12. 1-Benzyl-1-methyl-4-palmitoylpiperazin-1-ium bromide
(3l)
1H NMR (DMSO-d6, 500 MHz)
d 7.56e7.53 (m, 5H), 4.66 (s, 2H),
4.20e4.17 (m, 1H), 4.01e3.98 (m,1H), 3.73e3.69 (m, 2H), 3.49e3.48
(m, 2H), 3.39e3.37 (m, 2H), 3.02 (s, 3H), 2.35 (q, J ¼ 7.5 Hz, 2H),
1.50e1.47 (m, 2H), 1.27e1.24 (br m, 24H), 0.85 (t, J ¼ 7.0 Hz, 3H); 13C
NMR (DMSO-d6, 500 MHz)
d 171.78, 133.89, 131.04, 129.61, 128.02,
67.44, 59.23, 59.01, 45.46, 40.21, 39.41, 32.63, 29.78, 29.43, 25.23,
22.81, 14.66; MS (FAB) m/z 429 (MHþ). HRMS (FAB) calcd. for
C28H49N2OBr (MHþ) 429.3845, found 429.3846.
Acknowledgments
4.2.13. 1-Ethyl-1-methyl-4-palmitoylpiperazin-1-ium iodide (3m)
1H NMR (DMSO-d6, 500 MHz)
d 3.95e3.84 (m, 2H), 3.75e3.71
This research was supported by the 21st Century Frontier for
Functional Analysis of the Human Genome, National Research
Foundation (2009-0092966), Korean Research WCU Grant (R31-
2008-000-10086-0) and the Brain Korea 21 Project, of the Republic
of Korea.
(m, 1H), 3.60e3.56 (m, 1H), 3.46 (q, J ¼ 7.0 Hz, 4H), 3.40 (m, 2H),
3.06 (s, 3H), 2.34 (t, J ¼ 7.5 Hz, 2H), 1.49e1.47 (m, 2H), 1.26e1.20 (br
m, 27H), 0.85 (t, J ¼ 7.0 Hz, 3H); 13C NMR (DMSO-d6, 500 MHz)
d
171.75, 59.28, 59.13, 58.81, 45.99, 40.41, 39.34, 35.49, 29.79, 29.64,
29.44, 25.22, 22.81, 14.85, 7.84; MS (FAB) m/z 367 (MHþ). HRMS
(FAB) calcd. for C23H47N2OI (MHþ) 367.3688, found 367.3690.
References
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1H NMR (CDCl3, 300 MHz)
d 5.90e6.00 (m, 2H), 5.76e5.80 (m,
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1H NMR (DMSO-d6, 300 MHz)
d 6.03 (m, 1H), 5.66 (m, 2H), 4.09
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d 171.82, 133.88,
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