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

Article abstract of DOI:10.1016/j.ejmech.2012.12.063

A series of novel aliphatic amido-quaternary ammonium salts were synthesized and evaluated for their anticancer effects involving induction of RhoB. Most of these compounds, featuring open-ring forms of aliphatic amido-quaternary ammonium salts, exhibited potent anti-proliferative activities in human cancer cell lines, including PC-3, NUGC-3, MDA-MB-231, ACHN, HCT-15, and NCI-H23. In further evaluation, the representative compound N,N-diethyl-N-(2-(N-methyltetradecanamido)ethyl)prop-2-en-1-aminium bromide (3b) exhibited potent pro-apoptotic activity, through RhoB activation, in HeLa cells.

Full text of DOI:10.1016/j.ejmech.2012.12.063

European Journal of Medicinal Chemistry 63 (2013) 621e628
Contents lists available at SciVerse 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 II
Jee Sun Yang a, Doona Song a, Won Jin Ko a, Bunyea Kim a, Bo-Kyung Kim c, Song-Kyu Park b,
**
,
a,f,
*
Misun Won c, Kiho Lee b, Kyeong Lee d, Hwan Mook Kim 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, Sejong City 339-700, Republic of Korea
c Medical Genomics Research Center, KRIBB, Daejeon 305-806, Republic of Korea
d College of Pharmacy, Dongguk University-Seoul, Seoul 100-715, Republic of Korea
e College of Pharmacy, Gachon University, Incheon 406-799, 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:
A series of novel aliphatic amido-quaternary ammonium salts were synthesized and evaluated for their
anticancer effects involving induction of RhoB. Most of these compounds, featuring open-ring forms of
aliphatic amido-quaternary ammonium salts, exhibited potent anti-proliferative activities in human
cancer cell lines, including PC-3, NUGC-3, MDA-MB-231, ACHN, HCT-15, and NCI-H23. In further evalu-
ation, the representative compound N,N-diethyl-N-(2-(N-methyltetradecanamido)ethyl)prop-2-en-1-
aminium bromide (3b) exhibited potent pro-apoptotic activity, through RhoB activation, in HeLa cells.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 25 September 2012
Received in revised form
28 December 2012
Accepted 29 December 2012
Available online 14 March 2013
Keywords:
Aliphatic amido-quaternary ammonium
salts
Open-ring form of aliphatic amido-
quaternary ammonium salts
RhoB
GTPase RAt Sarcoma (Ras)
Perifosine
NSC126188
1. Introduction
The Rho family includes RhoA, -B, and -C, Rac1 and -2, and
Cdc42. The RhoA-related subgroup contains RhoA, RhoB and RhoC.
The Ras genes, the first oncogenes to be identified, are found in
approximately 30% of all human cancers: in adenocarcinomas of
the pancreas (90%), colon (50%), and lung (30%); in thyroid tumors
(50%); and in myeloid leukemia (30%) [1]. The Rho proteins are
members of the Ras superfamily. The Rho family itself contains
more than 20 members, which regulate diverse cellular functions
including cell growth, morphogenesis, cell motility, cytokinesis,
cytoskeletal organization, transcriptional activation, and mem-
brane trafficking. They are also essential for Ras transformation,
metastasis and apoptosis [2e4].
These three isoforms share w85% protein sequence identity, with
the greatest divergence close to the C-terminus. Despite the high
sequence similarity among RhoA-like proteins, they have different
activities with respect to cancer: RhoB is a tumor suppressor
that promotes growth inhibition and induces apoptosis in cancer
cells, whereas RhoA and RhoC promote oncogenesis, invasion, and
metastasis [5,6]. Recent studies suggest that RhoB determines the
stability and nuclear trafficking of Akt in endothelial cells, where it
plays roles in cell survival [7]. Many other reports have demon-
strated suppression of RhoB during tumor progression in multiple
cancers; consistent with this finding, cancer cell proliferation can
be inhibited by restoration of RhoB [8e11]. Therefore, induction of
RhoB expression represents a promising target for cancer therapy.
Several studies showed that apoptosis can be induced by acti-
vating RhoB protein by treating cells with small-molecule drugs
such as farnesyltransferase inhibitors and inhibitors of Akt and
PI3K. Ras proteins must be farnesylated in order to exert oncogenic
* Corresponding author. Translational Research Center for Protein Function
Control, Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of
Korea. Tel.: þ82 2 21232882; fax: þ82 2 3627265.
** Corresponding author. Tel.: þ82 32 8204825; fax: þ82 32 8204829.
E-mail addresses: hwanmook@gachon.ac.kr (H.M. Kim), 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.2012.12.063

622
J.S. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 621e628
activity, and RhoB contains farnesyl as well as geranylgeranyl
groups [12]. BMS-186511 is a farnesyltransferase inhibitor that in-
hibits Ras signaling and the transformed growth of spontaneous
tumors in H-Ras transgenic mice with minimal toxicity [13]. The
protein kinase B (Akt) pathway, which is generally activated
in cancer cells, has several downstream targets that regulate tumor-
associated cell processes; therefore, blocking this pathway
can induce apoptosis and growth inhibition in cancer cells [14].
Furthermore, downregulation of RhoB inhibits transformation, in-
vasion, and metastasis through an Akt-dependent mechanism [15].
Perifosine is a representative Akt inhibitor that targets cellular
membranes and is orally bioactive. Furthermore, we have synthe-
sized a series of piperazine alkyl derivatives based on NSC126188,
which induces apoptosis through the PI3K/Akt pathway and/or the
RhoB mediated pathway [16]. All of these RhoB-induced anticancer
agents share common structural features, including a long aliphatic
chain (Fig. 1).
Here, we present the synthesis and biological activities of a
novel series of aliphatic amido-quaternary ammonium salts, based
on our previous study of NSC126188 derivatives. We performed a
structureeactivity relationship (SAR) study on this series of com-
pounds, based on RhoB activation level and anti-proliferative ac-
tivities in several human cancer cells. Our results suggest that novel
aliphatic amido-quaternary ammonium salts in the open-ring form
represent more promising chemotherapeutic anticancer agents
than compounds with a piperazine moiety.
series EA chemical analyzer. The results of elemental analyzes (C, H,
N) were within ꢀ0.4% of the calculated values. All quaternary
ammonium salt analogs were characterized and evaluated for
in vitro biological activities.
3. Results and discussion
All newly synthesized compounds were assayed for growth
inhibitory activity in six human cancer cell types: PC-3 (prostate
cancer), NUGC-3 (stomach cancer), MDA-MB-231 (breast cancer),
ACHN (renal cancer), HCT15 (colon cancer), and NCI-H23 (non-
small cell lung cancer). Results are tabulated as GI50 values in the
micromolar range (Tables 1e4). To evaluate the effect of these
compounds on RhoB induction, we conducted RhoB promoter as-
says. Perifosine and NSC126188 were used as positive references for
comparison of in vitro activities.
Initially, in order to assess the effects of the open-ring moiety
on amido-quaternary ammonium salts, we synthesized 11 com-
pounds (Table 1) based on our previous study. The N,N-dimethyl
moiety of NSC126188 and the carbon-chain length (14e18 car-
bons) of the compounds were fixed based on the results of the
previous study, and the piperazine ring was replaced with the
open-ring form. Multiple N,N-dimethyl-diamines, such as N,N-
dimethylethane-1,2-diamine, N,N-dimethylpropane-1,3-diamine,
or N,N,2,2-tetramethylpropane-1,3-diamine, were introduced at
the open-ring position.
The overall activities of the newly synthesized compounds are
displayed in Table 1. The compounds significantly inhibited growth,
especially in gastric and prostate cancer cell lines. Of note, most of
the 11 initially prepared compounds inhibited proliferation in
gastric cancer cell lines more effectively than perifosine. Moreover,
three analogs, 3a, 3h, and 3r, all synthesized with N,N-dimethyl-
ethane-1,2-diamine, exhibited remarkably potent growth inhibi-
tion than NSC126188 with GI50 values of 0.22, 0.17, and 0.13 mM,
respectively, in the NUCG-3 cell line. In particular, 3h also illus-
trated strong anti-proliferative activity in the PC-3 cell line
2. Chemistry
Based on the amido-piperazinium structures of the compounds
described in our previous work, we designed and synthesized 25
analogs with one major modification, namely, introduction of the
open-ring form in place of the piperazine moiety. As illustrated in
Schemes 1 and 2, the final compounds, 3aes and 5aef, were syn-
thesized from various fatty acids by two-step procedures based on
design. Reactions of the fatty acids 1 with 1-(3-dimethylamino-
propyl)-3-ethyl-carbodiimide hydrochloride (EDCI) and 4-dimeth-
ylaminopyridine (DMAP) in the presence of dichloromethane
resulted in the in situ formation of activated acyl compounds which
on further addition of substituted N-alkyl diamines, produces the
desired amides 2aej and 4aec, respectively in good yields. Syn-
thesized amides 2 and 4 were converted to desired amido-
quaternary ammonium salts 3aes and 5aef by N-alkylation using
various alkyl halides in the presence of anhydrous acetonitrile. The
structures of amido-quaternary ammonium salts 3 and 5 were
confirmed by spectral data. The N-alkylation was detected by
(GI50 ¼ 0.37
mM).
For the synthesis of compounds containing N,N-dimethylpro-
pane-1,3-diamine, we used two different reagents, N,N,N’-trime-
thylpropane-1,3-diamine and N,N-dimethylpropane-1,3-diamine.
Comparisons between 3m and 3n, and between 3d and 3e, confirm
that a methyl group at the N’ position increases growth inhibitory
activity. Furthermore, an N’-methyl seems to be important for in-
duction of RhoB expression, as shown in Fig. 2. According to the re-
sults of the RhoB promoter assay, all compounds listed in Table 1
induced RhoB expression, although less potently than perifosine in
most cases. This result also suggests that the anti-proliferative ac-
tivities of these compounds are mediated through the RhoB pathway.
We assessed the effect of carbon-chain length on activity, using
three groups of compounds classified by N,N-dimethyl-diamines
with carbon-chains of lengths between 14 and 18 (n ¼ 12e16).
studying the NMR spectra. The eCH2Nþ(CH2)CH2e protons (
3.94 ppm) are distinctive (downfield) compared to the eCH2N(CH2)
CH2e protons (
2.19e2.92 ppm). The 1H NMR spectra also revealed
a triplet signal at 0.79e0.91 ppm due to CH3CH2-proton. Elemental
analyzes were obtained from a Thermo Finnigan model Flash 1112
d 3.03e
d
d
Fig. 1. Chemical structures of farnesyltransferase inhibitors and PI3K/Akt inhibitors.

J.S. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 621e628
623
Scheme 1. Reaction protocol for the synthesis of aliphatic amido-quaternary ammonium salts (3aes). Reagents and conditions: (a) DMAP, EDCI, N-alkyl-diamine, CH2Cl2, overnight;
(b) alkyl halide, CH3CN at w140e150 ꢁC, 7 h.
Among the first group of compounds (N,N-dimethylethane-1,2-
diamine derivatives, 3a, 3h, and 3r), 3h (n ¼ 14) showed the
most striking inhibition of growth in the PC-3 and NUGC-3 cell lines
as stated above. Among the second group of compounds synthe-
sized with N,N,N’-trimethylpropane-1,3-diamine (3e, 3n and 3s), 3e
(n ¼ 12) displayed the most potent growth inhibition. Of note,
whereas the other group of compounds inhibited RhoB expression
to a lesser extent than NSC126188 and perifosine, 3n (n ¼ 14)
induced RhoB expression to a greater extent than the other com-
pounds in its group. Growth inhibition by compounds containing
N,N,2,2-tetramethylpropane-1,3-diamine (5aec) was moderate in
In our previous study, A671 (1-allyl-1-ethyl-4-palmitoylpi-
perazin-1-ium bromide), which promotes apoptosis through the
RhoB pathway, emerged as the most promising chemotherapeutic
agent. To evaluate the effect of the open-ring moiety on the activ-
ities of amido-quaternary ammonium salts, we compared N-ethyl-
N-allyl substituted compounds (3b and 3j) with A671. Except in
colon cancer cells, 3b (n ¼ 12) and 3j (n ¼ 14) displayed anti-
proliferative activities higher than that of A671, which contains a
piperazine ring moiety (Table 2). Furthermore, the result of RhoB
promoter activation level shows that compounds with the open-
ring moiety induced RhoB expression level more than the previ-
ously reported compounds (Fig. 2). These results confirm that
replacing the piperazine ring with an open-ring diamine is a valid
approach to increasing anti-proliferative activity via the RhoB
mediated pathway.
Because we had validated that compounds with N’-methyl
inhibited growth more effectively than compounds with hydrogen
at the N’ position, we retained the N’-methyl and fixed carbon-
chain length at 14 or 16 in further syntheses. As shown in
Table 3, growth inhibition by compounds in this series was not as
impressive as inhibition by compounds in Table 2. Most of the
compounds in this series displayed equally effective inhibition in
the two human cancer cell lines described above. Of interest,
compounds with carbon-chain length of 16 (3o and 3p) elevated
the expression level of RhoB considerably; however, the RhoB
expression level was not proportional to the inhibitory activity of
N,N-dimethylpropane-1,3-diamine derivatives. Instead, com-
pounds with low RhoB expression exhibited stronger anti-
proliferative activity than compounds that induced high levels of
RhoB expression. This result indicates that RhoB mediated
apoptosis might not represent the main mechanism underlying the
efficacies of this series of compounds.
gastric cancer cell line, with GI50 from 0.32 to 0.45
with a carbon-chain length of 18, exhibited striking activity in a
M). These results suggest that
mM. However, 5c,
prostate cancer cell line (GI50 ¼ 0.24
m
carbon-chain length is not a major factor in determining anti-
proliferative activity, but it might be important in regulating
RhoB promoter expression.
Based on compound 3h, which showed potent anti-proliferative
activity in gastric and prostate cancer cell lines and induced the
strongest reporter expression in the RhoB promoter-based assay,
we prepared six more analogs (Table 2). To evaluate the effect of
N,N-dialkyl substituents, we synthesized multiple N,N-dialkyl
substituents using N,N-dimethylethane-1,2-diamine. Compounds
3b, with an allyl group at R3, displayed a more robust effect on
growth inhibition in the NUGC-3 cell line (GI50 ¼ 0.14
PC-3 cell line (GI50 ¼ 0.21 M). Other compounds from this series
also inhibited the growth of these two cancer cell lines, with GI50
under 0.40 M. Furthermore, the induction of RhoB expression by
mM) and the
m
m
all of these compounds was far superior to induction by amido-
quaternary ammonium salts from other groups. Therefore, we
conclude that these compounds inhibit proliferation of cancer cells
by upregulating RhoB (Table 2).
Scheme 2. Reaction protocol for the synthesis of aliphatic amido-quaternary ammonium salts (5aef). Reagents and conditions: (a) DMAP, EDCI, N,N,2,2-tetramethylpropane-1,3-
diamine, CH2Cl2, overnight; (b) alkyl halide, CH3CN at w140e150 ꢁC, 7 h.

624
J.S. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 621e628
Table 1
Cell growth-inhibitory activities of compounds substituted with N,N-dimethyl-diamine.
Cell line
Tissue
Growth inhibition GI50 (m
M)a
3a
3d
3e
3h
3m
3n
3r
3s
5a
5b
5c
NSC126188
Perifosine
PC-3
MDA-MB-231
ACHN
NUGC-3
HCT15
NCI-H23
Prostate
Breast
Kidney
Gastric
Colon
0.41
0.80
0.26
0.22
1.47
0.92
0.39
1.48
1.56
0.43
0.79
1.02
0.32
1.78
1.01
0.32
1.72
0.56
0.37
1.14
0.45
0.17
1.31
1.54
0.60
2.18
3.36
1.07
2.56
2.15
0.49
1.42
0.76
0.19
1.98
0.96
0.42
1.07
0.36
0.14
0.80
1.52
0.52
2.41
3.40
0.97
0.79
1.68
0.51
1.31
3.12
0.84
3.70
1.44
0.44
1.54
2.60
0.43
NAb
1.34
0.24
1.21
2.30
0.45
1.73
1.71
0.48
1.44
1.04
0.29
0.58
2.34
0.44
2.86
4.56
0.54
1.25
4.21
Lung
a
Growth inhibition was measured by SRB (sulforhodamine B) assay. The values given are means of three experiments.
(NA) not active.
b
Finally, among the compounds containing N,N,2,2-tetramethyl-
and 75 MHz, at 400 and 100 MHz, or at 500 and 125 MHz. Proton
and carbon chemical shifts are expressed in ppm relative to internal
tetramethylsilane, and coupling constants (J) are expressed in
Hertz. Liquid chromatographyemass spectrometry (LC/MS) spectra
were recorded by electrospray ionization (ESI) on Shimazu LC/MS
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 (Mꢂz)/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) e positive mode on the Micromass Q-TOF (Waters
Corp., USA; the capillary and sample cone voltages 4000 V & 30 V,
the desolvation gas flow 600 L/h at 200 ꢁC, and the source tem-
perature 100 ꢁC) High Resolution Tandem Mass Spectrometer at
Yonsei University in Seoul, Korea.
propane-1,3-diamine (5aec), compound 5c was the most potent in
prostate cancer cells. Therefore, in subsequent syntheses, only the
N,N-dialkyl moiety of 5c was modified (Table 4). Allyl, benzyl, and
ethyl groups were introduced at the N-position. No compound
performed better than 5c, but the reporter levels in the RhoB
promoter-based assay of this series of compounds were moderately
good.
Farnesyltransferase inhibitors, perifosine, NSC126188 and
amido-quaternary ammonium salts share structural features,
including long aliphatic carbon-chains and electronegative atoms
(specifically, nitrogen). Furthermore, these compounds control
growth inhibition through the RhoB mediated pathway. We have
designed and synthesized 25 analogs of novel amido-quaternary
ammonium salts with an open-ring form, based on our previous
work and the structures of farnesyltransferase inhibitors. The
newly synthesized compounds were evaluated for their anticancer
effects in six human cancer cell lines. Among them, compound 3b
emerged as the most promising anticancer agent; it was more
effective than all other compounds, including the piperazine ring-
substituted compounds in our previous study, in terms of potent
inhibition of cancer cell growth and stimulation of apoptosis. The
effects of 3b in RhoB promoter-based reporter assays indicate that
this compound may act through RhoB mediated signaling. Taken
together, the data presented here suggest that novel aliphatic
amido-quaternary ammonium salts in the open-ring form repre-
sent promising chemotherapeutic anticancer agents that act
through the RhoB pathway, and that these compounds are more
effective than those containing the piperazine moiety.
4.1. General procedures for the synthesis of 2aej and 4aec
Carboxylic acid 1 (2.9 mmol) was dissolved in anhydrous
dichloromethane (0.2 M) at room temperature under Argon atmo-
sphere. N,N-alkyl-diamine (4.3 mmol), EDC (1-ethyl-3-[3-dimethyl-
aminopropyl]carboimide hydrochloride, 4.3 mmol), and DMAP (4-
dimethylaminopyridine, 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
(3ꢃ). 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
4.1.1. N-(2-(Dimethylamino)ethyl)-N-methyltetradecanamide (2a)
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), and spots were visualized under
UV light (254 nm). Flash column chromatography was performed
with silica (Merck EM9385, 230e400 mesh). 1H and 13C nuclear
magnetic resonance (NMR; Varian) spectra were recorded at 300
1H NMR (DMSO-d6, 300 MHz)
d 3.36e3.31 (m, 2H), 2.93e2.79 (br
m, 3H), 2.37e2.21 (m, 4H), 2.16e2.13 (br m, 6H), 1.46 (br m, 2H), 1.24
(br m, 20H), 0.85 (t, 3H, J ¼ 6.3 Hz); ESI-MS: m/z ¼ 313 [MþH].
4.1.2. N-(2-(Diethylamino)ethyl)-N-methyltetradecanamide (2b)
1H NMR (CDCl3, 300 MHz)
d
3.43e3.27 (m, 2H), 2.98e2.89
(m, 3H), 2.56e2.46 (m, 6H), 2.31e2.21 (m, 2H), 1.59e1.55 (m, 2H),
Table 2
Cell growth-inhibitory activities of derivatives of compound 3h.
Cell line
Tissue
Growth inhibition GI50
(
m
M)
3b
3c
3h
3i
3j
3k
3l
A671
NSC126188
perifosine
PC-3
MDA-MB-231
ACHN
NUGC-3
HCT15
NCI-H23
Prostate
Breast
Kidney
Gastric
Colon
0.21
0.30
0.10
0.14
NA
0.26
0.36
0.23
0.16
4.33
0.28
0.37
1.14
0.45
0.17
1.31
1.54
0.25
0.66
0.58
0.41
1.01
0.58
0.20
0.44
0.35
0.27
1.98
0.17
0.31
1.13
0.60
0.36
NA
0.23
0.36
0.27
0.16
0.61
1.11
0.37
0.68
0.50
0.27
1.33
0.88
0.48
1.44
1.04
0.29
0.58
2.34
0.44
2.86
4.56
0.54
1.25
4.21
Lung
0.24
1.22

J.S. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 621e628
625
Table 3
Cell growth-inhibitory activities of derivatives of compound 3e.
Cell line
Tissue
Growth inhibition GI50 (mM)
3e
3f
3g
3o
3p
3q
NSC126188
Perifosine
PC-3
MDA-MB-231
ACHN
NUGC-3
HCT15
NCI-H23
Prostate
Breast
Kidney
Gastric
Colon
0.32
1.78
1.01
0.32
1.72
0.56
0.31
1.35
1.89
0.28
NA
0.53
1.41
1.89
0.43
2.83
0.66
0.43
0.80
0.47
0.36
NA
0.85
1.35
1.30
0.52
NA
0.49
1.38
1.02
0.22
3.42
0.78
0.48
1.44
1.04
0.29
0.58
2.34
0.44
2.86
4.56
0.54
1.25
4.21
Lung
0.46
0.68
1.11
1.24e1.16 (br m, 20H), 1.02e0.94 (m, 6H), 0.85e0.78 (m, 3H); ESI-
MS: m/z ¼ 341 [MþH].
4.1.10. N-(3-(Dimethylamino)propyl)stearamide (2j)
1H NMR (CDCl3, 500 MHz)
7.05 (s, 1H), 3.47e3.33 (m, 2H), 2.81
(t, 2H, J ¼ 7.2 Hz), 2.62 (s, 6H), 2.21 (t, 2H, J ¼ 7.3 Hz), 1.95 (s, 2H),
1.62 (s, 2H), 1.27 (d, 28H, J ¼ 17.3 Hz), 0.89 (t, 3H, J ¼ 6.8 Hz); ESI-
MS: m/z ¼ 369 [MþH].
d
4.1.3. N-(3-(Dimethylamino)propyl)tetradecanamide (2c)
1H NMR (CDCl3, 500 MHz)
d 6.95 (br s, 1H), 3.35 (q, 2H,
J ¼ 5.5 Hz), 2.50 (t, 2H, J ¼ 6.5 Hz), 2.33 (s, 6H), 2.16 (t, 2H,
J ¼ 7.5 Hz), 1.74e1.71 (m, 2H), 1.61 (br m, 2H), 1.30e1.26 (br m, 20H),
0.88 (t, 3H, J ¼ 7.0 Hz); ESI-MS: m/z ¼ 313 [MþH].
4.1.11. N-(3-(Dimethylamino)-2,2-dimethylpropyl)tetradecanamide
(4a)
1H NMR (DMSO-d6, 500 MHz)
d
7.57 (t, 1H, J ¼ 5.7 Hz), 2.92 (d,
4.1.4. N-(3-(Dimethylamino)propyl)-N-methyltetradecanamide (2d)
2H, J ¼ 6.0 Hz), 2.20 (s, 6H), 2.08 (t, 2H, J ¼ 7.3 Hz), 2.04 (s, 2H),
1.48e1.46 (m, 2H), 1.27e1.23 (m, 20H), 0.85 (t, 3H, J ¼ 6.8 Hz), 0.78
(s, 6H); ESI-MS: m/z ¼ 341 [MþH].
1H NMR (CDCl3, 500 MHz)
d 3.43e3.32 (m, 2H), 2.96 (ds, 3H,
J ¼ 46.0 Hz), 2.48 (t, 2H, J ¼ 7.7 Hz), 2.39 (s, 3H), 2.35e2.26 (m, 2H),
2.24 (s, 3H), 1.85e1.71 (m, 2H), 1.65e1.61 (m, 2H), 1.30e1.26 (m,
20H), 0.88 (t, 3H, J ¼ 7.0 Hz); ESI-MS: m/z ¼ 327 [MþH].
4.1.12. N-(3-(Dimethylamino)-2,2-dimethylpropyl)palmitamide (4b)
1H NMR (DMSO-d6, 400 MHz)
d 7.56 (s, 1H), 2.91 (d, 2H,
4.1.5. N-(2-(Dimethylamino)ethyl)-N-methylpalmitamide (2e)
J ¼ 4.8 Hz), 2.19 (s, 6H), 2.08e2.03 (m, 4H), 1.47e1.46 (m, 2H), 1.22
(s, 24H), 0.84 (t, 3H, J ¼ 5.4 Hz), 0.77 (s, 6H); ESI-MS: m/z ¼ 369
[MþH].
1H NMR (DMSO-d6, 300 MHz)
d 3.35e3.33 (m, 2H), 2.94e2.79
(br m, 3H), 2.37e2.21 (m, 4H), 2.21e2.13 (br m, 6H), 1.47e1.45 (br
m, 2H), 1.24 (br m, 24H), 0.85e0.83 (m, 3H); ESI-MS: m/z ¼ 341
[MþH].
4.1.13. N-(3-(Dimethylamino)-2,2-dimethylpropyl)stearamide (4c)
1H NMR (CDCl3, 500 MHz)
2.87e2.43 (m, 8H), 2.26 (s, 2H),1.62 (d, 2H, J ¼ 7.2 Hz),1.34e1.20 (m,
28H), 1.18e1.01 (m, 6H), 0.87 (t, 4H, J ¼ 7.0 Hz); ESI-MS: m/z ¼ 397
[MþH].
d
7.27 (d, 1H, J ¼ 2.2 Hz), 3.26 (s, 2H),
4.1.6. N-(2-(Diethylamino)ethyl)-N-methylpalmitamide (2f)
1H NMR (DMSO-d6, 300 MHz)
d 3.29e3.27 (m, 2H), 2.95e2.79
(br m, 3H), 2.49e2.40 (m, 6H), 2.29e2.20 (m, 2H), 1.46 (br m, 2H),
1.24 (br m, 24H), 0.93 (t, 6H, J ¼ 7.9 Hz), 0.85 (t, 3H, J ¼ 6.5 Hz); ESI-
MS: m/z ¼ 369 [MþH].
4.2. General procedures for the synthesis of 3aes and 5aef
4.1.7. N-(3-(Dimethylamino)propyl)palmitamide (2g)
Alkyl halide (1.6 mmol) was added to a solution of 2aej and 4ae
c (0.8 mmol) in anhydrous acetonitrile (0.3 M) at room tempera-
ture, and 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, 400 MHz)
d 8.92 (s, 1H), 3.15e3.08 (m, 2H),
2.31 (t, 2H, J ¼ 7.2 Hz), 2.20 (s, 6H), 1.66e1.59 (m, 2H), 1.47e1.45 (m,
2H), 1.20 (s, 26H), 0.81 (t, 3H, J ¼ 6.6 Hz); ESI-MS: m/z ¼ 341 [MþH].
4.1.8. N-(3-(Dimethylamino)propyl)-N-methylpalmitamide (2h)
1H NMR (DMSO-d6, 400 MHz)
d
3.23 (q, 2H, J ¼ 8.0 Hz), 2.25e
2.16 (m, 8H), 2.12 (s, 3H), 1.63e1.51 (m, 2H), 1.43 (br s, 2H), 1.20
4.2.1. N,N,N-Trimethyl-2-(N-methyltetradecanamido)ethanaminium
(s, 26H), 0.81 (t, 3H, J ¼ 6.8 Hz); ESI-MS: m/z ¼ 355 [MþH].
iodide (3a)
1H NMR (DMSO-d6, 300 MHz)
d 3.71e3.66 (m, 2H), 3.42e3.38
4.1.9. N-(2-(Dimethylamino)ethyl)-N-methylstearamide (2i)
(m, 2H), 3.08 (br, 9H), 3.00 (s, 3H), 2.29 (t, 2H, J ¼ 7.4 Hz), 1.47 (br
1H NMR (DMSO-d6, 400 MHz)
d 3.44e3.40 (m, 2H), 2.93e2.79
m, 2H), 1.24 (br s, 20H), 0.85 (t, 3H, J ¼ 6.9 Hz); 13C NMR (DMSO-d6,
(ds, 3H), 2.36e2.21 (m, 4H), 2.16 (s, 3H), 2.13 (s, 3H), 1.48e1.39
(m, 2H), 1.23 (m, 28H), 0.85 (t, 3H, J ¼ 8.0 Hz); ESI-MS: m/z ¼ 369
[MþH].
100 MHz)
d 172.89 and 171.99, 61.24 and 60.99, 54.40 and 52.43,
54.36 and 52.40, 54.32 and 52.36, 42.36 and 41.11, 35.19, 33.22 and
32.44, 31.68 and 31.32, 29.10, 29.09, 29.07, 29.05, 29.03, 28.97,
28.81, 28.77, 28.75, 24.75 and 24.40, 22.13, 13.98 ppm; ESI-MS: m/
z ¼ 327 [Mþ]; HRMS (ESI) calcd. for C20H43N2O (Mþ) 327.3375,
found 327.3387.
Table 4
Cell growth-inhibitory activities of derivatives of compound 5c.
Cell line
Tissue
Growth inhibition GI50
5c 5d 5e 5f
Prostate 0.24 0.47 0.51 0.30 0.48
(
m
M)
4.2.2. N,N-Diethyl-N-(2-(N-methyltetradecanamido)ethyl)prop-2-
NSC126188 Perifosine
en-1-aminium bromide (3b)
PC-3
0.44
2.86
4.56
0.54
1.25
4.21
1H NMR (DMSO-d6, 300 MHz)
d 6.09e6.00 (m, 1H), 5.72e5.61
MDA-MB-231 Breast
ACHN
NUGC-3
HCT15
NCI-H23
1.21 1.64 2.15 1.12 1.44
2.30 1.75 2.23 1.95 1.04
0.45 0.77 1.32 0.42 0.29
(m, 2H), 4.04e4.01 (m, 2H), 3.94e3.91 (m, 2H), 3.63 (m, 2H),
3.29e3.19 (m, 4H), 3.01 (s, 3H), 2.29 (t, 2H, J ¼ 7.7 Hz), 1.47 (m, 2H),
1.27e1.15 (m, 26H), 0.87e0.83 (m, 3H); 13C NMR (DMSO-d6,
Kidney
Gastric
Colon
Lung
1.73 NA
NA
2.74 0.58
1.71 0.90 0.91 0.98 2.34
100 MHz) d 172.91 and 171.97,127.21 and 126.80,125.74 and 125.55,

626
J.S. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 621e628
Fig. 2. Detection of RhoB promoter activation by reporter assays. Luciferase activity was determined in cells transfected with either the pGL2 vector or pGL-RhoB-Luc in the
presence of aliphatic amido-quaternary ammonium salts and controls [perifosine, NSC126188, and A671 (1-allyl-1-ethyl-4-palmitoylpiperazin-1-ium bromide)].
59.73 and 59.41, 53.30, 53.19, 52.43, 40.64, 35.72, 33.28 and 32.36,
31.66 and 31.34, 29.11, 29.10, 29.08, 29.06, 29.03, 28.98, 28.76, 24.71
and 24.42, 22.14,13.98, 7.50 and 7.28 ppm; ESI-MS: m/z ¼ 381 [Mþ];
HRMS (ESI) calcd. for C24H49N2O (Mþ) 381.3845, found 381.3841;
EA for C24H49N2OBr calculated: C, 62.45; H, 10.70; N, 6.07; found: C,
62.42; H, 10.68; N, 6.07.
and 24.57, 22.14, 21.26 and 20.32, 13.98 ppm; ESI-MS: m/z ¼ 367
[Mþ]; HRMS (ESI) calcd. for C23H47N2O (Mþ) 367.3688, found
367.3694.
4.2.7. N-Benzyl-N,N-dimethyl-3-(N-methyltetradecanamido)
propan-1-aminium bromide (3g)
1H NMR (DMSO-d6, 500 MHz)
d 7.54e7.51 (m, 5H), 4.57e4.55 (m,
4.2.3. N,N,N-Triethyl-2-(N-methyltetradecanamido)ethanaminium
2H), 3.32 (s, 3H), 3.20e3.18 (m, 2H), 2.96 (s, 6H), 2.81e2.75 (m, 2H),
2.27e2.25 (m, 2H), 1.99 (m, 2H), 1.47 (m, 2H), 1.23 (br m, 20H),
iodide (3c)
1H NMR (DMSO-d6, 300 MHz)
d
4.04e4.01 (m, 2H), 3.61e3.35
0.86e0.83 (m, 3H); 13C NMR (DMSO-d6, 100 MHz)
d 172.36 and
(m, 2H), 3.28e3.17 (m, 6H), 3.01 (s, 3H), 2.29 (t, 2H, J ¼ 7.5 Hz),
171.73, 133.00 and 132.96, 130.27 and 130.23, 128.90 and 128.86,
128.21 and 128.17, 66.60 and 66.10, 61.01 and 60.60 49.18, 43.80 and
43.75, 42.14, 34.96 and 34.86, 32.77 and 32.67, 32.59 and 32.53,
31.98, 31.34, 29.11, 29.09, 29.07, 29.02, 28.88 (2), 28.76, 24.99 and
24.57, 22.14 and 22.03, 21.29 and 20.50, 14.00 ppm; ESI-MS: m/
z ¼ 417 [Mþ]; HRMS (ESI) calcd. for C27H49N2O (Mþ) 417.3845,
found 417.3828.
1.47e1.45 (m, 2H), 1.30e1.11 (m, 29H), 0.85 (t, 3H, J ¼ 6.8 Hz); 13C
NMR (DMSO-d6, 100 MHz)
d 172.87 and 171.92, 52.58, 52.46 (2),
51.87, 40.60, 35.77, 33.33 and 32.37, 31.61 and 31.32, 29.09 (2),
29.07, 29.05, 29.02, 28.96, 28.81, 28.75, 24.67 and 24.40, 22.12,
13.97, 7.42 and 7.21 (3) ppm; ESI-MS: m/z ¼ 369 [Mþ]; HRMS (ESI)
calcd. for C23H49N2O (Mþ) 369.3845, found 369.3858.
4.2.4. N,N,N-Trimethyl-3-tetradecanamidopropan-1-aminium iodide
4.2.8. N,N,N-Trimethyl-2-(N-methylpalmitamido)ethanaminium
(3d)
iodide (3h)
1H NMR (DMSO-d6, 500 MHz)
d
7.87 (t,1H, J ¼ 5.2 Hz), 3.26e3.23
1H NMR (DMSO-d6, 300 MHz)
d
3.69 (t, 2H, J ¼ 6.9 Hz), 3.40 (t,
(m, 2H), 3.10e3.06 (m, 2H), 3.03 (s, 9H), 2.05 (t, 2H, J ¼ 7.3 Hz),
1.83e1.77 (m, 2H), 1.49e1.46 (m, 2H), 1.27e1.23 (br m, 20H), 0.85 (t,
3H, J ¼ 6.5 Hz); ESI-MS: m/z ¼ 327 [Mþ].
2H, J ¼ 7.2 Hz), 3.13e3.06 (m, 9H), 3.00 (s, H), 2.29 (t, 2H, J ¼ 7.5 Hz),
1.53e1.42 (m, 2H), 1.33e1.21 (m, 24H), 0.85 (t, 3H, J ¼ 6.6 Hz); 13C
NMR (DMSO-d6, 100 MHz)
d 172.88 and 171.98, 61.24 and 61.00,
52.43, 52.40, 52.37, 42.36 and 41.11, 35.18, 33.22 and 32.45, 31.68
and 31.33, 29.10 (3), 29.06 (2), 28.99, 28.82, 28.79 (2), 28.76, 24.75
and 24.41, 22.13, 13.98 ppm; ESI-MS: m/z ¼ 355 [Mþ]; HRMS (ESI)
calcd. for C22H47N2O (Mþ) 355.3688, found 355.3694.
4.2.5. N,N,N-Trimethyl-3-(N-methyltetradecanamido)propan-1-ami-
nium iodide (3e)
1H NMR (DMSO-d6, 500 MHz)
d 3.31 (m, 2H), 3.24e3.21 (m, 2H),
3.06 (s, 3H), 3.04 (s, 6H), 2.96 (s, 3H), 2.29e2.25 (m, 2H), 1.94e1.85
(m, 2H), 1.47 (m, 2H), 1.23 (br m, 20H), 0.85 (t, 3H, J ¼ 6.3 Hz); 13
C
4.2.9. N-Ethyl-N,N-dimethyl-2-[((N-methylpalmitamido)ethanami-
NMR (DMSO-d6, 100 MHz)
d
172.27 and 171.70, 63.09 and 62.60,
nium iodide)-ethyl]-dimethyl-ammonium; iodide (3i)
52.35 and 52.24, 52.32 and 52.20, 52.28 and 52.16, 45.77 and 43.64,
34.85, 32.65 and 32.60, 31.99, 31.32, 29.10 (2), 29.08, 29.05, 29.01,
28.86, 28.75, 24.97 and 24.54, 22.13, 21.61, 20.68, 13.98 ppm; ESI-
MS: m/z ¼ 341 [Mþ]; HRMS (ESI) calcd. for C21H45N2O (Mþ)
341.3532, found 341.3534.
1H NMR (DMSO-d6, 300 MHz)
d
3.68e3.63 (t, 2H, J ¼ 7.0 Hz),
3.38e3.33 (m, 4H), 3.05e2.83 (d, 9H), 2.32e2.27 (t, 3H, J ¼ 7.3 Hz),
1.47 (br m, 2H), 1.24 (br m, 27H), 0.86e0.83 (br m, 3H); 13C NMR
(DMSO-d6, 100 MHz)
d 172.85 and 171.98, 59.30 and 58.83, 58.65
and 58.16, 49.80 and 49.51 (2), 42.11 and 40.91, 35.39, 33.25 and
32.41, 31.66 and 31.33, 29.10 (3), 29.06 (2), 28.99, 28.82, 28.78 (2),
28.76, 24.74 and 24.41, 22.13, 13.97, 7.96 and 7.85 ppm; ESI-MS: m/
z ¼ 369 [Mþ]; HRMS (ESI) calcd. for C23H49N2O (Mþ) 369.3845,
found 369.383.
4.2.6. N,N-Dimethyl-N-(3-(N-methyltetradecanamido)propyl)prop-
2-en-1-aminium bromide (3f)
1H NMR (DMSO-d6, 500 MHz)
d 6.06e5.97 (m, 1H), 5.63e5.60
(m, 2H), 3.97e3.93 (m, 2H), 3.22 (m, 2H), 3.18e3.15 (m, 2H), 3.00
(s, 3H), 2.97 (s, 3H), 2.95 (s, 3H), 2.27e2.25 (m, 2H), 1.94e1.88 (m,
2H), 1.47 (m, 2H), 1.23 (br m, 20H), 0.85 (t, 3H, J ¼ 6.0 Hz); 13C NMR
4.2.10. N,N-Diethyl-N-(2-(N-methylpalmitamido)ethyl)prop-2-en-
1-aminium bromide (3j)
(DMSO-d6, 100 MHz)
d
172.29 and 171.73, 127.40 and 127.34, 126.16
1H NMR (DMSO-d6, 300 MHz)
d 6.08e6.02 (m, 1H), 5.73e5.61 (d,
and 126.03, 65.33 and 65.02, 60.74 and 60.22, 49.76 and 49.71 (2),
43.72 and 42.11, 34.90 and 34.85, 32.70 and 32.66, 32.59 and 32.53,
31.96 and 31.34, 29.11 (2), 29.09, 29.06, 29.02, 28.88, 28.76, 24.99
2H), 4.01e3.94 (m, 2H), 3.65e3.61 (m, 2H), 3.35e3.18 (m, 6H),
3.06e2.58 (s, 3H), 2.30e2.27 (t, 2H, J ¼ 7.4 Hz), 1.46 (br m, 2H),
1.26e1.23 (br m, 30H), 0.86e0.83 (t, 3H, J ¼ 6.4 Hz); 13C NMR

J.S. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 621e628
627
(DMSO-d6, 100 MHz)
d
172.91 and 171.96, 127.22 and 126.81, 125.76
4.2.17. N-Ethyl-N,N-dimethyl-3-(N-methylpalmitamido)propan-1-
and 125.54, 59.43, 53.30 and 53.19 (2), 52.43, 40.64, 35.72, 32.70
and 32.37, 31.66 and 31.34, 29.11 (3), 29.07, 29.05, 28.99, 28.83,
28.78 (2), 28.77, 24.72 and 24.42, 22.14, 13.98, 7.49 and 7.27
(2) ppm; ESI-MS: m/z ¼ 410 [Mþ]; HRMS (ESI) calcd. for C26H53N2O
(Mþ) 409.4158, found 409.4151; EA for C26H53N2OBr calculated: C,
63.78; H, 10.91; N, 5.72; found: C, 63.80; H, 11.20; N, 5.50.
aminium iodide (3q)
1H NMR (DMSO-d6, 500 MHz)
d
3.36e3.27 (m, 5H), 3.17e3.13
(m, 2H), 2.97e2.93 (m, 8H), 2.29e2.24 (m, 2H), 1.91e1.79 (m, 2H),
1.46e1.34 (m, 2H), 1.22e1.09 (m, 2H), 0.84 (t, 3H, J ¼ 8.5 Hz); ESI-
MS: m/z ¼ 383 [Mþ]; HRMS (ESI) calcd. for C24H51N2O (Mþ)
383.4001, found 383.4005.
4.2.11. N-Benzyl-N,N-diethyl-2-(N-methylpalmitamido)ethanami-
4.2.18. N,N,N-Trimethyl-2-(N-methylstearamido)ethanaminium
nium bromide (3k)
iodide (3r)
1H NMR (DMSO-d6, 300 MHz)
d
7.53 (s, 5H), 4.64 (s, 2H), 4.00e
1H NMR (DMSO-d6, 500 MHz)
d
3.69 (t, 2H, J ¼ 7.3 Hz), 3.41 (t,
3.67 (m, 2H), 3.61e3.50 (m, 2H), 3.50e3.32 (m, 4H), 2.98 (s, 3H),
2.32e2.24 (m, 2H), 2.24e2.15 (m, 2H), 1.52e1.43 (m, 2H), 1.32e1.21
(m, 28H), 0.85 (t, 3H, J ¼ 6.9 Hz); 13C NMR (DMSO-d6, 100 MHz)
2H, J ¼ 7.3 Hz), 3.10e3.09 (m, 9H), 3.00 (s, 3H), 2.31e2.28 (m, 2H),
1.48e1.45 (m, 2H), 1.23e1.19 (m, 28H), 0.85 (t, 3H, J ¼ 7.0 Hz); 13C
NMR (DMSO-d6, 100 MHz)
d 172.89 and 171.98, 61.24 and 61.00,
d
172.15 and 171.90, 133.33 and 133.28 (2), 130.27, 128.90 (2), 127.97
52.44, 52.40, 52.37, 42.36 and 41.12, 35.17, 33.21 and 32.45,
31.68 and 31.34, 29.10 (5), 29.06, 29.00 (2), 28.84, 28.80 (2),
28.76, 24.76 and 24.42, 22.14, 13.98 ppm; ESI-MS: m/z ¼ 383 [Mþ];
HRMS (ESI) calcd. for C24H51N2O (Mþ) 383.4001, found 383.4017.
and 127.95, 67.02 and 66.89, 63.43 and 63.37, 62.10 and 61.59, 47.30
and 47.18, 45.11 and 42.80, 36.82, 32.70 and 32.59, 31.34, 29.11 (3),
29.06 (2), 29.01, 28.85 (2), 28.76, 24.72 and 24.52, 22.14, 21.43,
20.77, 13.99 ppm; ESI-MS: m/z ¼ 459 [Mþ].
4.2.19. N,N,N-Trimethyl-3-stearamidopropan-1-aminium iodide (3s)
4.2.12. N,N,N-Triethyl-2-(N-methylpalmitamido)ethanaminiumiodide
1H NMR (DMSO-d6, 500 MHz)
d 7.88e7.87 (m, 1H), 3.27e3.23 (m,
(3l)
2H), 3.11e3.07 (m, 2H), 3.03 (s, 9H), 2.05 (t, 3H, J ¼ 7.5 Hz), 1.83e1.77
1H NMR (DMSO-d6, 300 MHz)
d 3.61e3.56 (m, 2H), 3.33e3.17
(m, 2H), 1.49e1.46 (m, 2H), 1.27e1.23 (br m, 28H), 0.85 (t, 3H,
(m, 8H), 3.01e2.84 (d, 3H), 3.31e2.26 (t, 2H, J ¼ 7.3 Hz), 1.47 (br
J¼ 7.0 Hz); 13C NMR (DMSO-d6,100MHz)
d 172.32, 63.50, 52.27, 52.24,
m, 2H),1.24e1.19 (br m, 33H), 0.87e0.83 (t, 3H, J ¼ 6.6 Hz); 13C NMR
52.20, 35.45, 31.33, 29.10 (3), 29.05 (2), 29.01 (2), 28.89 (2), 28.80 (2),
28.76 (2), 25.21, 23.02, 22.14, 13.99 ppm; ESI-MS: m/z ¼ 383 [Mþ];
HRMS (ESI) calcd. for C24H51N2O (Mþ) 383.4001, found 383.4001.
(DMSO-d6, 100 MHz)
d 172.88 and 171.93, 52.58 and 52.45 (3),
51.86, 40.60, 35.76, 33.33 and 32.38, 31.61 and 31.33, 29.10 (3),
29.05, 29.03 (2), 28.98, 28.83, 28.77, 28.75, 24.69 and 24.41, 22.13,
13.98, 7.41 and 7.20 (3) ppm; ESI-MS: m/z ¼ 397 [Mþ]; HRMS (ESI)
calcd. for C25H53N2O (Mþ) 397.4158, found 397.4153; EA for
4.2.20. N,N,N,2,2-Pentamethyl-3-tetradecanamidopropan-1-ami-
nium iodide (5a)
C
25H53N2OI calculated: C, 57.24; H,10.18; N, 5.34; found: C, 57.34; H,
1H NMR (DMSO-d6, 500 MHz)
d
7.85 (t, 1H, J ¼ 6.3 Hz), 3.26 (s,
10.12; N, 5.24.
2H), 3.17 (s, 9H), 3.05 (d, 2H, J ¼ 6.5 Hz), 2.13 (t, 2H, J ¼ 7. 5Hz), 1.50
(m, 2H), 1.24 (br m, 20H), 1.07 (s, 6H), 0.85 (t, 3H, J ¼ 6.8 Hz); 13C
4.2.13. N,N,N-Trimethyl-3-palmitamidopropan-1-aminium iodide
NMR (DMSO-d6, 100 MHz) d 172.89, 72.55, 54.78 (3), 48.00, 37.23,
(3m)
35.39, 31.33, 29.11 (2), 29.07, 29.06 (2), 29.01, 28.82, 28.76 (2), 25.33,
24.60, 22.14, 14.00 ppm; ESI-MS: m/z ¼ 355 [Mþ]; HRMS (ESI) calcd.
for C22H47N2O (Mþ) 355.3688, found 355.3689.
1H NMR (DMSO-d6, 500 MHz)
d
8.12e8.11 (d, 1H, J ¼ 7.5 Hz),
3.41e3.38 (m, 2H), 3.30 (t, 2H, J ¼ 7.2 Hz), 3.27 (s, 2H), 3.16 (s, 9H),
2.23 (t, 2H, J ¼ 7.5 Hz), 2.03e1.99 (m, 2H), 1.64e1.61 (m, 2H), 1.34e
1.30 (m, 24H), 0.91 (t, 3H, J ¼ 6.8 Hz); ESI-MS: m/z ¼ 355 [Mþ];
HRMS (ESI) calcd. for C22H47N2O (Mþ) 355.3688, found 355.3691.
4.2.21. N-Ethyl-N,N,2,2-tetramethyl-3-palmitamidopropan-1-ami-
nium iodide (5b)
1H NMR (DMSO-d6, 500 MHz)
d
7.83 (t, NH, J ¼ 6.0 Hz), 3.24 (s, 2H),
4.2.14. N,N,N-Trimethyl-3-(N-methylpalmitamido)propan-1-aminium
3.15 (br s, 8H), 3.05 (d, 2H, J ¼ 6.4 Hz), 2.13 (t, 2H, J ¼ 7.4 Hz),1.51e1.48
(m, 2H),1.23 (br m, 27H),1.07 (s, 6H), 0.85 (t, 3H, J ¼ 6.6 Hz); ESI-MS:
m/z ¼ 397 [Mþ].
iodide (3n)
1H NMR (DMSO-d6, 500 MHz)
d 3.33e3.28 (m, 3H), 3.22 (t, 2H,
J ¼ 8.2 Hz), 3.06 (s, 3H), 3.04 (s, 6H), 2.96 (s, 2H), 2.27 (t, 2H,
J ¼ 7.2 Hz), 1.89e1.85 (m, 2H), 1.47 (m, 2H), 1.24 (m, 24H), 0.85 (t,
3H, J ¼ 6.5 Hz); ESI-MS: m/z ¼ 370 [Mþ]; HRMS (ESI) calcd. for
4.2.22. N,N,N,2,2-Pentamethyl-3-stearamidopropan-1-aminium
iodide (5c)
C
23H49N2O (Mþ) 369.3845, found 369.3849.
1H NMR (DMSO-d6, 500 MHz)
d
7.84 (t, 1H, J ¼ 6.4 Hz), 3.30 (s,
9H), 3.23 (s, 2H), 3.14 (s, 2H), 2.12 (t, 2H, J ¼ 7.4 Hz), 1.52e1.46 (m,
2H), 1.23 (s, 28H), 1.07 (s, 6H), 0.85 (t, 3H, J ¼ 6.9 Hz); ESI-MS: m/
z ¼ 411 [Mþ]; HRMS (ESI) calcd. for C26H55N2O (Mþ) 411.4314,
found 411.4318; EA for C26H55N2OI calculated: C, 57.98; H, 10.29; N,
5.20; found: C, 57.97; H, 10.16; N, 5.09.
4.2.15. N,N-Dimethyl-N-(3-(N-methylpalmitamido)propyl)prop-2-
en-1-aminium bromide (3o)
1H NMR (DMSO-d6, 500 MHz)
d 6.02e5.90 (m, 1H), 5.57e5.53
(m, 2H), 3.91e3.85 (m, 2H), 3.33e3.22 (m, 5H), 3.13e3.08 (m,
2H), 2.93e2.89 (m, 8H), 2.22e2.18 (m, 2H), 1.92e1.78 (m, 2H), 1.40e
1.32 (m, 2H), 1.19e1.00 (m, 24H), 0.79 (t, 3H, J ¼ 8.5 Hz); ESI-MS: m/
z ¼ 395 [Mþ]; HRMS (ESI) calcd. for C25H51N2O (Mþ) 395.4001,
found 395.3984.
4.2.23. N-(2,2-Dimethyl-3-stearamidopropyl)-N,N-dimethylprop-
2-en-1-aminium bromide (5d)
1H NMR (DMSO-d6, 500 MHz)
d 7.85 (s, 1H), 6.07e6.01 (m, 1H),
5.64e5.60 (m, 2H), 4.00 (d, 2H, J ¼ 7.5 Hz), 3.17 (s, 2H), 3.04 (d, 2H,
J ¼ 6.0 Hz), 2.13 (t, 2H, J ¼ 7.5 Hz), 1.51e1.50 (m, 2H), 1.27e1.19 (m,
28H), 1.10 (s, 6H), 0.85 (t, 3H, J ¼ 7.0 Hz); 13C NMR (DMSO-d6,
4.2.16. N-Benzyl-N,N-dimethyl-3-(N-methylpalmitamido)propan-
1-aminium bromide (3p)
1H NMR (DMSO-d6, 500 MHz)
d
3.34e3.33 (m, 5H), 3.18 (t, 2H,
100 MHz) d 172.93, 127.68, 126.24, 70.22, 68.75, 51.33, 48.33, 46.16
J ¼ 8.0 Hz), 2.98e2.95 (m, 8H), 2.26 (t, 2H, J ¼ 7.5 Hz), 2.00e1.97
(m, 2H), 1.47e1.46 (m, 2H), 1.23 (m, 24H), 0.85 (t, 3H,
J ¼ 6.5 Hz); ESI-MS: m/z ¼ 445 [Mþ]; HRMS (ESI) calcd. for
and 45.71, 37.24, 35.38, 31.36, 29.12 (3), 29.08 (2), 29.06, 28.90 (2),
28.85, 28.81, 28.78 (2), 25.41, 24.84, 23.77 and 22.15 (2), 13.98 ppm;
ESI-MS: m/z ¼ 437 [Mþ]; HRMS (ESI) calcd. for C28H57N2O (Mþ)
437.4471, found 437.4451.
C
29H53N2O (Mþ) 445.4158, found 445.4144.

628
J.S. Yang et al. / European Journal of Medicinal Chemistry 63 (2013) 621e628
4.2.24. N-Benzyl-N,N,2,2-tetramethyl-3-stearamidopropan-1-ami-
Appendix A. Supplementary data
nium bromide (5e)
1H NMR (DMSO-d6, 500 MHz)
d
7.85 (s, 1H), 7.58e7.47 (m, 5H),
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.12.063.
4.56 (s, 2H), 3.31 (s, 6H), 3.27 (d, 2H, J ¼ 4.2 Hz), 3.06 (d, 2H,
J ¼ 9.5 Hz), 2.08 (dd, 2H, J ¼ 13.2, 5.7 Hz),1.47 (d, 2H, J ¼ 7.0 Hz),1.22
(s, 28H), 1.13 (s, 6H), 0.84 (t, 3H, J ¼ 6.9 Hz); 13C NMR (DMSO-d6,
References
100 MHz)
d 172.90, 133.29 (2), 130.28, 128.83 (2), 128.18, 71.20,
69.97, 50.73 (2), 48.68, 37.27, 35.36, 31.34, 29.09 (5), 29.02, 28.85
(2), 28.79 (2), 28.76 (2), 25.32, 25.02, 23.80, 22.15, 14.02 ppm; ESI-
MS: m/z ¼ 487 [Mþ]; HRMS (ESI) calcd. for C32H59N2O (Mþ)
487.4627, found 487.4622.
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1.51 (s, 2H), 1.28e1.19 (m, 31H), 1.08 (s, 6H), 0.85 (t, 3H, J ¼ 7.0 Hz);
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Acknowledgments
This research was supported by National Research Foundation
(2012-0000884); Korean Research WCU grant (R31-2008-000-
10086-0); and the Brain Korea 21 Project of the Republic of
Korea.
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