Novel anti-viability ceramide analogs: Design, synthesis, and structure-activity relationship studies of substituted (S)-2-(benzylideneamino)- 3-hydroxy-N-tetradecylpropanamides

Article abstract of DOI:10.1016/j.bmc.2010.05.044

A group of novel l-serinamides, substituted (S)-2-(benzylideneamino)-3- hydroxy-N-tetradecylpropanamides (3a-o) and substituted (S)-2-(benzylamino)-3- hydroxy-N-tetradecyl propanamides (4c, 4i, 4l, and 4o), were synthesized as potential anti-tumor lead compounds. In vitro cell viability assay results indicate treatment with 3a-o compounds resulted in significant inhibition of cell viability in the chemoresistant breast cancer cell line, MCF-7TN-R. Compounds 3i and 3l, both ortho-substituted analogs, show the greatest efficacy with IC₅₀ values of 10.3 μM and 12.5 μM, respectively. The SAR analysis indicate that the imine functional group of 3a-o is critical for the cellular anti-viability effect, and the partial atomic charge (PAC) value of imine C atom is a valuable structural parameter for predicting the activity of these ceramide analogs.

Full text of DOI:10.1016/j.bmc.2010.05.044

Bioorganic & Medicinal Chemistry 18 (2010) 5316–5322
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel anti-viability ceramide analogs: Design, synthesis, and structure–activity
relationship studies of substituted (S)-2-(benzylideneamino)-3-hydroxy-N-
tetradecylpropanamides
à
à
à
,
*
Jiawang Liu , James W. Antoon , Adharsh Ponnapakkam , Barbara S. Beckman , Maryam Foroozesh
Department of Chemistry, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125, USA
Department of Pharmacology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of novel L-serinamides, substituted (S)-2-(benzylideneamino)-3-hydroxy-N-tetradecylpropana-
Received 15 December 2009
Revised 14 May 2010
Accepted 16 May 2010
Available online 8 June 2010
mides (3a–o) and substituted (S)-2-(benzylamino)-3-hydroxy-N-tetradecyl propanamides (4c, 4i, 4l,
and 4o), were synthesized as potential anti-tumor lead compounds. In vitro cell viability assay results
indicate treatment with 3a–o compounds resulted in significant inhibition of cell viability in the chemo-
resistant breast cancer cell line, MCF-7TN-R. Compounds 3i and 3l, both ortho-substituted analogs, show
the greatest efficacy with IC50 values of 10.3 lM and 12.5 lM, respectively. The SAR analysis indicate that
Keywords:
Ceramide
Sphingosine-1-phosphate
MTT viability assay
Partial atomic charge
Resonance
the imine functional group of 3a–o is critical for the cellular anti-viability effect, and the partial atomic
charge (PAC) value of imine C atom is a valuable structural parameter for predicting the activity of these
ceramide analogs.
Ó 2010 Elsevier Ltd. All rights reserved.
Inductive effects
Structure–activity relationship
1. Introduction
L
-Serinamides,14,15 serinol,16 and aminoethanol amide17,18 have
been previously investigated as anti-cancer ceramide analogs. We
have recently reported that a series of ceramide analogs based on
Recent research suggests that bioactive sphingolipids play an
important role in the regulation of cancer pathogenesis and pro-
vide novel clues towards the discovery of new chemotherapeutic
agents.1–3 The central molecule in the sphingolipid metabolism,
ceramide, is known to induce apoptosis,4 senescence5,6 and
growth arrest7 in many human cancers. Ceramide could be metab-
olized into sphingosine-1-phosphate (S-1-P) by the successive
actions of the enzymes ceramidase (CDase) and sphingosine ki-
nase (SK). This metabolite, however, causes cellular transforma-
tion, decreased apoptosis, and induction of angiogenesis.1,2
Therefore, many researchers have focused on designing ceramide
analogs with the same apoptotic activity as ceramide but with
greater inhibitory effect on the enzymes involved in ceramide
metabolism, leading to the synthesis of dozens of active molecules
recently reported.8–13 In fact, decreased synthesis and increased
metabolism of ceramide has already become an important target
of anti-cancer agents. Figure 1 illustrates the probable mechanism
of action of ceramide analogs.
the structure of
L-serine amide, particularly (S)-2-(benzylideneami-
no)-3-hydroxy-N-tetradecylpropanamide (analog 3, IC50, 7.67
lM),
exhibit increased efficacy as anti-proliferative agents in both che-
mosensitive and chemoresistant breast cancer cell lines compared
to the parent compound (C8-ceramide, IC50, 8.59 l
M).9 This com-
pound is able to alter the sphingolipid metabolism profile of both
chemosensitive and chemoresistant breast cancer cells causing a
significant increase in sphinganine levels while diminishing intra-
cellular S1P protein concentrations.
Using (S)-2-(benzylideneamino)-3-hydroxy-N-tetradecylpro-
panamide (Analog 3) as the lead, we incorporated five functional
groups (–NO2, –CH3, –Cl, –OCH3, and –OH) into each position
(ortho-, meta-, and para-) of the benzyl group in order to alter elec-
tronic properties of the imine group, the metabolism-relevant
functional group. The position of the substituent (o-, m-, p-), Log K
(representing lipid–water partition coefficient), electron-with-
drawing or electron-releasing ability, and molecular weight
(molecular size) were taken in consideration as parameters for
the investigation of the structure–activity relationships. Nineteen
analogs were synthesized and assayed for their effects on cell via-
bility in chemo-resistant (MCF-7TN-R) breast cancer cell line. The
MCF-7TN-R cell line was derived from MCF-7 cells grown in
* Corresponding author. Tel.: +1 504 520 5078.
E-mail address: mforooze@xula.edu (M. Foroozesh).
Xavier University of Louisiana, Department of Chemistry, USA.
Tulane University, School of Medicine, USA.
à
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.044

J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 5316–5322
5317
M)
Table 2
OH
OH
HO
O
OH
O
IC50 values of 3a–o in a non-tumorigenic human kidney cell line (HEK294)
P
HO
C
13H27 CDase
SK
C
13H27
Compd
IC50 (l
HN
R
NH2
S-1-P
Ceramide
3a
3b
3c
3d
3e
3f
49.51
82.66
82.61
47.32
51.11
81.06
77.69
76.96
94.88
81.65
77.26
79.15
71.68
77.52
80.78
O
Ceramide
Analogs
Anti-proliferation
Proliferation
3g
3h
3i
Figure 1. The expected mechanism of action of ceramide analogs. Ceramidase
(CDase), sphingosine kinase (SK), sphingosine-1-phosphate (S-1-P).
3j
3k
3l
3m
3n
3o
increasing concentrations of tumor necrosis factor
a (TNF-a) until
resistance was established.19 Using in vitro assays, 3i and 3l, both
o-substituted analogs, showed the greatest efficacy with IC50 val-
ues of 10.3 and 12.5 lM, respectively. The partial atomic charges
(PAC)20 of metabolism-relevant N and C atoms of the imine group
were calculated in CERIUS2, and the results indicate that the PAC of
the C atom of the imine functional group is relevant to the viability
inhibition of MCF-7TN-R cell line (Table 1). Given the need for
experimental therapeutics to exhibit at least a modest selectivity
for cancer rather than normal cells, we analyzed the effect of these
ceramide analogs on the non-tumorigenic HEK294 kidney cell line
(Table 2). Most analogs showed higher potency in the cancer cell
line, with the exceptions of analog 3a, 3b, 3d, 3e, and 3f. Interest-
ingly, the analogs 3i and 3l which have shown the highest potency
against breast cancer cells also show the highest degree of
selectivity.
provided (S)-2-(benzylamino)-3-hydroxy-N-tetradecyl propana-
mides 4c, 4i, 4l, and 4o in 73–78% yields. All of these reactions oc-
curred in mild conditions, and ceramide analogs were produced in
acceptable yields.
2.2. Anti-viability effects of compounds 3a–o in MCF-7TN-R
chemoresistant breast cancer cells and HEK294 non-tumorigenic
kidney cells
The effects of compounds 3a–o on the viability of MCF-7TN-R
cells were assessed by means of MTT assays according to previ-
ously published methods.9 Among the fifteen imine analogs, 3i
and 3l, with o-chloro- and o-methoxy-substituents, showed the
2. Results and discussion
2.1. Synthesis
greatest anti-viability effect with IC50 values of 10.3 and 12.5 lM,
respectively. (Table 1, Fig. 2). Most analogs showed higher potency
The synthetic route of ceramide analogs is depicted in Scheme 1.
in the cancer cell line, with the exceptions of analogs 3a, 3b, 3d, 3e,
Boc-
mide in 92% yield. After removing the Boc protecting group with tri-
fluoroacetic acid in dichloromethane, -serinamide was formed in
L
-serine was coupled with tetradecylamine giving Boc-
L
-serina-
and 3f. IC50s ranged from 47.32 to 94.88 lM (Table 2).
L
2.3. Structure–activity relationship studies of compounds 3a–o
66% yield. In the presence of sodium hydroxide, 3a–o were formed
in methanol, and crystals were obtained by recrystallization from
petroleum ether/ethyl acetate (3a–l) and petroleumether/methanol
(3m–o) solvent systems. Reduction of 3c, 3i, 3l and 3o by NaBH4
The structural parameters of 3a–o are listed in Table 1. Due to
the structural similarity of these compounds, molecular weight
could be treated as a parameter of molecular size. Partial atomic
Table 1
Structural parameters and IC50 values of 3a-o and 4c,4i,4l,4o
Compd
Group
Position
MW
Log K
Log P (predicted)
PAC (N)
PAC (C)
IC50 (lM)
C8-Cera
8.59c
3b
7.67c
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
4c
–NO2
–NO2
–NO2
–CH3
–CH3
–CH3
–Cl
–Cl
–Cl
–OCH3
–OCH3
–OCH3
–OH
–OH
–OH
–NO2
–Cl
–OCH3
–OH
para-
meta-
ortho-
para-
meta-
ortho-
para-
meta-
ortho-
para-
meta-
ortho-
para-
meta-
ortho-
ortho-
ortho-
ortho-
ortho-
433.5
433.5
433.5
402.6
402.6
402.6
423.0
423.0
423.0
418.6
418.6
418.6
404.5
404.5
404.5
435.5
425.0
420.6
406.8
0.86
0.86
0.88
1.09
1.09
1.09
1.09
1.08
1.12
0.91
0.92
0.89
0.73
0.76
0.89
0.28
0.27
0.27
0.22
5.59
5.59
5.56
6.37
6.37
6.20
6.59
6.59
6.43
5.94
5.94
5.96
5.67
5.67
5.70
4.92
5.79
5.32
5.06
ꢀ0.2768
ꢀ0.2768
ꢀ0.2767
ꢀ0.2768
ꢀ0.2768
ꢀ0.2768
ꢀ0.2768
ꢀ0.2768
ꢀ0.2768
ꢀ0.2768
ꢀ0.2768
ꢀ0.2767
ꢀ0.2768
ꢀ0.2768
ꢀ0.2767
ꢀ0.2997
ꢀ0.2998
ꢀ0.2997
ꢀ0.2997
0.0295
0.0297
0.0337
0.0295
0.0295
0.0298
0.0295
0.0296
0.0310
0.0295
0.0296
0.0322
0.0295
0.0296
0.0322
0.0255
0.0288
0.0239
0.0239
216.5
103.9
21.71
101.5
82.94
87.71
27.48
24.10
10.26
28.13
31.25
12.50
19.33
32.63
18.97
29.35
43.69
100.4
75.29
4i
4l
4o
a
b
c
C8-ceramide.
Analog 3.
Data of clonogenic survival assay previously, Ref. 9.

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J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 5316–5322
O
O
O
C
H
i
14 29
ii
HO
Boc
OH
C
H
HO
Boc
N
H
14 29
C
H NH
14 29 2
HO
N
H
NH
NH
Tetradecylamine
NH
3
Boc-L-serine
1 (92%)
2 (66%)
CF COO
3
R = - NO2
R = - CH3
R = - Cl
R = - OCH3
R = - OH
3a-c & 4c
3d-f
O
O
C
H
14 29
C H
14 29
3g-i & 4i
3j-l & 4l
3m-o & 4o
HO
N
H
HO
N
H
iv
iii
N
NH
3a,3d,3g,3j,3m
3b,3e,3h,3k,3n
3c,3f,3i,3l,3o
4c,4i,4l,4o
R = para-
R = meta-
R = ortho-
R = ortho-
4c,4i,4l,4o
(73-78%)
3a-o (37-89%)
R
R
Scheme 1. Synthesis of 3a–o and 4c, 4i, 4l, 4o. Reagents and conditions: (i) DCC/HOBT in THF 0 °C; (ii) TFA/CH2Cl2 (0 °C); (iii) NaOH in methanol, and various substituted
benzaldehydes; (iv) NaBH4/methanol.
charge (PAC) as an electronic descriptor, quantifies the charges in a
local electric field surrounding the nucleus of an atom. The PAC
values of atoms N and C of the imine functional group were calcu-
lated in Cerius2 (Accelrys Software Inc., San Diego, California), since
the imine group is the key site of hydrolysis in ceramide metabo-
lism. The 3D structures of 3a–o were sketched in ISIS Draw (Symyx
Technologies, Inc.), and energetically minimized in Cerius2 4.11.
The partial atomic charges were assigned using the Gasteiger
method.21–23 The data show that almost no difference exists in
PAC (N) values among 3a–o, however, a significant rise in electrop-
ositivity of the carbon atom occurs in the o-substituted analogs ex-
cept the compound possessing an o-methyl group 3f.
It is well known that Log K value of a compound is highly corre-
lated with the corresponding Log P value, and it is treated as a
parameter that represents the lipid/water partition coefficient.
Log K values of 3a–o were calculated using retention times ob-
tained from RP-HPLC analysis. Predicted Log P values are also dis-
played in Table 1, which were calculated in the QSAR
(quantitative structure–activity relationship) module of Cerius2,
after regular energy minimization, and charge distribution
employing Merck Molecular Force Field. As is shown in Table 1,
the analogs with the same substituent possess the similar Log K
values except hydroxy-substituted compounds. The Log K value
of o-hydroxy-substituted analog 3o is significantly higher than
the values of its p- and m-substituted counterparts 3m and 3n,
which may be attributed to the formation of intramolecular hydro-
gen bond in 3o. However, no correlation was observed between
Log K and anti-viability effect in this study.
2.5. o-Substitution versus partial atomic charge of the imine
carbon
We further compared the analogs containing the same type of
substituents and found a notable increase of anti-viability activity
in o-substituted compounds, except for the analog with methyl
group 3f. At the same time, a similar increase in electro positivity
of the carbon atom was observed in these analogs. As described
above, anti-viability effect correlates with electron-withdrawing ef-
fect by induction at o-position. Analogs, 3c (o-nitro), 3i (o-chloro), 3l
(o-methoxy), and 3o (o-hydroxy) with EWGs by inductive effect,
withdraw electrons from the carbon atom of the imine group and
subsequently increase the PAC (C) value as well as the anti-viability
activity, while analog 3f (o-methyl) with EDG by inductive effect
works in an opposite manner. This finding suggests that the
electronic charge distribution of the imine functional group is criti-
cal for increased activity and PAC (C) value could be used as a useful
indicator of activity in future studies.
2.6. The effect of 4c, 4i, 4l, and 4o on the cell viability in
MCF-7TN-R cells
In order to investigate the importance of carbon–nitrogen double
bond on anti-viability activity, the four most effective o-substituted
analogs 3c, 3i, 3l, and 3o were reduced to form compounds 4c, 4i, 4l,
and 4o. The effects of compounds 4c, 4i, 4l, and 4o on the viability of
MCF-7TN-RcellswerealsodeterminedbyMTTassays. Aseriesofrel-
atively high IC50 values were obtained with 29.35
lM, 43.69 lM,
100.4 M, 75.29 M for 4c, 4i, 4l, and 4o, respectively (Table 1).
l
l
The decline of activity brought by introducing carbon–nitrogen sin-
gle bond indicates that the conjugated system composed by the
imine functional group (C@N) and phenyl ring is critical for the ob-
served anti-viability effect. In addition, the relatively low PAC (C)
values of 4c, 4i, 4l, and 4o may also account for the decrease in activ-
ity compared to their imine counterparts.
2.4. Activity of the analogs with same substituted position
Among the p-substituted analogs, 3g (27.48
and 3m (19.33 M) were more effective than 3a (216.5
3d (101.5 M) in IC50 values. Analogs 3g, 3j, 3m, respectively,
lM), 3j (28.13
lM),
l
lM) and
l
contain chloro, methoxy, and hydroxy groups which donate elec-
trons by resonance but withdraw electrons by inductive effect.
However, the substituent of analog 3a (nitro), is an electron-with-
drawing group (EWG) both by inductive effect and resonance, and
substituent of analog 3d (methyl), is an electron-donating group
(EDG) only by inductive effect. This observation indicates that
the electron-donating effect by resonance accounts for the
relatively high activity of these compounds. A similar trend was
observed among the m-substituted analogs 3b, 3e, 3h, 3k, and
3n. However, in the o-substituted analogs, 3f (o-methyl) contain-
ing EDG only by inductive effect, showed less efficacy than the
other compounds, 3c (o-nitro), 3i (o-chloro), 3l (o-methoxy),
and 3o (o-hydroxy). This suggests that electron-withdrawing by
inductive effect, rather than by resonance, will enhance the anti-
viability activity of o-substituted analogs.
3. Conclusions
Nineteen ceramide analogs were designed and synthesized
using fifteen benzaldehydes as starting material in acceptable
yields. Five different substituents were incorporated into each po-
sition of the phenyl ring (o, m, p) in order to determine the most
effective combination of substituent and position. These analogs
were tested as potential anti-viability agents in MCF-7TN-R cells,
and the structure–activity relationships were studied using several
parameters. The results indicate that all analogs containing chloro-,
methoxy-, and hydroxyl- substituents show great efficacy. In
analogs containing a methyl group, a significant increase of the
PAC (C) in the o-substituted compound was detected, which may

J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 5316–5322
5319
we found that the relatively high activity of the imine functional
group is most likely due to the stability of the conjugation
p–P
at the hydrolytic site of the enzymes involved. These analogs were
also tested as potential anti-viability agents in non-tumorigenic
human kidney cell line HEK294, and the results showed that the
most potent analogs in breast cancer cells are also the most selec-
tive. In conclusion, our study illustrates that targeting ceramide
analogs for chemotherapeutic drug design has potential in treating
drug resistant breast cancer.
4. Experimental section
4.1. Chemistry
All the chemicals were purchased from Sigma–Aldrich Chemical
Co. (St. Louis, MO). Chromatography was performed on HPTLC Silica
Gel 60 F254, and RP-HPLC was performed on hp Hewlett Packard Ser-
ies 1050 (Column: phenomenex Gemini-NX 5u C18 110A). The ami-
no acid analysis was determined with a Hitachi 835-50 instrument.
Mass spectral data was determined by Micromass Quattro micro™
API and Agilent 6890 GC with 5973 MS. 1H NMR and 13C NMR spectra
were recorded on Varian 300 MHz NMR spectrometer.
4.1.1. (S)-tert-Butyl 3-hydroxy-1-oxo-1-(tetradecylamino)
propan-2-ylcarbamate (1)
Compound 1 was prepared according to the literature.9
4.1.2. (S)-2-Amino-3-hydroxy-N-tetradecylpropanamide (2)
Compound 2 was prepared according to the literature.9
4.1.3. General procedure for synthesis of nitro-, methyl-, chloro-
and methoxy-substituted (S)-2-(benzylideneamino)-3-hydroxy-
N-tetradecylpropanamide (3a–l)
A mixture of 0.40 g (1.00 mmol) of (S)-2-amino-3-hydroxy-N-
tetradecylpropanamide hydrochloride, 0.04 g (1.00 mmol) of
NaOH, 1.00 mmol of substituted benzaldehyde, and 40 mL of
methanol was stirred at room temperature for eight hours. The sol-
vent was then evaporated under reduced pressure, and the residue
was dissolved in 60 mL of ethyl acetate. After filtration and evapo-
ration, the residue was washed four times with 10 mL of petroleum
ether and 20 mL of cold methanol (4 °C) to get the crude products.
The substituted (S)-2-(benzylideneamino)-3-hydroxy-N-tetrade-
cylpropanamides (3a–l) were obtained by recrystallization from
petroleum ether/ethyl acetate.
4.1.3.1. (S)-2-(4-Nitrobenzylideneamino)-3-hydroxy-N-tetrade-
cylpropanamide (3a). Yield: 0.18 g (42%) as colorless powder.
GC/MS (m/e): 434 [M+1]+, 317 [M +1ꢀOH]+, 1H NMR (DMSO-d6,
300 MHz) d/ppm = 8.416 (s, 1H), 8.297 (d, J = 8.7 Hz, 2H), 8.125
(d, J = 8.7 Hz, 2H), 7.800 (t, J = 5.7 Hz, 1H), 4.913 (t, J = 6 Hz, 1H),
3.866 (m, 2H), 3.504 (m, 1H), 3.084 (m, 2H), 1.391 (m, 2H), 1.176
(m, 22H), 0.826 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz) d/
ppm = 169.996, 161.706, 149.362, 142.104, 131.339, 124.415,
76.466, 63.621, 39.115, 31.964, 29.808, 29.632, 29.686, 29.398,
26.999, 22.763, 14.609. Anal. Calcd for C24H39N3O4: C, 66.48; H,
9.07; N, 9.69; O, 14.76. Found: C 66.44; H, 9.65; N, 9.52; O, 14.34.
Figure 2. Effect of compounds 3a–n on breast cancer viability in MCF-7TN-R cells.
MCF-7TN-R cells were plated at 7.5 ꢁ 105 cells per 96 well plate. The following day
cells were treated with indicated concentrations of compound for 24 h. Data are
presented as percent of vehicle treated samples. Mean values of S.E.M. of four
different experiments in quadruplicate are reported.
4.1.3.2. (S)-2-(3-Nitrobenzylideneamino)-3-hydroxy-N-tetrade-
cylpropanamide (3b). Yield: 0.22 g (51%) as colorless powder.
GC/MS (m/e): 433 M+, 1H NMR (DMSO-d6, 300 MHz) d/
ppm = 8.671 (s, 1H), 8.421 (s, 1H), 8.291 (m, 2H), 7.784 (m, 2H),
4.907 (t, J = 6 Hz, 1H), 3.869 (m, 2H), 3.520 (m, 1H), 3.081 (m,
2H), 1.389 (m, 2H), 1.176 (m, 22H), 0.825 (t, J = 6 Hz, 3H). 13C
NMR (DMSO-d6, 75 MHz) d/ppm = 170.087, 161.539, 148.830,
138.126, 135.545, 130.929, 125.918, 123.003, 76.405, 63.636,
39.100, 31.964, 29.808, 29.671, 29.701, 29.382, 26.999, 22.778,
account for the increased anti-viability activity. Therefore, ortho-
substituted analogs with electron withdrawing groups by induc-
tion seem to be the best choice for further research, and PAC (C)
seems to be a valuable predictor of the structural property of the
molecule to be used in our future QSAR analysis of ceramide
analogs. While comparing imine and amine functional groups,

5320
J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 5316–5322
14.609. Anal. Calcd for C24H39N3O4: C, 66.48; H, 9.07; N, 9.69; O,
14.76. Found: C 66.23; H, 8.93; N, 9.62; O, 14.45.
14.609. Anal. Calcd for C24H39ClN2O2: C, 68.14; H, 9.29; N, 6.62;
O, 7.56. Found: C 67.65; H, 9.76; N, 6.71; O, 7.93.
4.1.3.3. (S)-2-(2-Nitrobenzylideneamino)-3-hydroxy-N-tetrade-
cylpropanamide (3c). Yield: 0.38 g (89%) as yellow powder. GC/
MS (m/e): 434 [M+1]+, 1H NMR (DMSO-d6, 300 MHz) d/
ppm = 8.543 (s, 1H), 8.187 (d, J = 7.5 Hz, 1H), 8.028 (d, J = 7.5 Hz,
1H), 7.805 (t, J = 7.5 Hz, 1H), 7.720 (t, J = 7.5 Hz, 1H), 7.611 (t,
J = 5.7 Hz, 1H), 4.855 (t, J = 6 Hz, 1H), 3.903 (dd, J = 3.9 Hz,
J = 8.1 Hz, 1H), 3.793 (m, 1H), 3.506 (m, 1H), 3.084 (m, 2H), 1.397
(m, 2H), 1.198 (m, 22H), 0.832 (t, J = 6 Hz, 3H). 13C NMR (DMSO-
d6, 75 MHz) d/ppm = 169.905, 159.018, 149.665, 142.104,
133.981, 132.295, 130.656, 124.764, 76.329, 63.636, 39.100,
31.979, 29.808, 29.701, 29.641, 29.382, 26.968, 22.778, 14.624.
Anal. Calcd for C24H39N3O4: C, 66.48; H, 9.07; N, 9.69; O, 14.76.
Found: C 66.59; H, 9.08; N, 9.28; O, 14.69.
4.1.3.8. (S)-2-(3-Chlorobenzylideneamino)-3-hydroxy-N-tetrade
cylpropanamide (3h). Yield: 0.35 g (83%) as colorless powder. GC/
MS (m/e): 423 M+, 406 [MꢀOH]+, 1H NMR (DMSO-d6, 300 MHz) d/
ppm = 8.257 (s, 1H), 7.979 (s, 1H), 7.767 (m, 2H), 7.502 (m, 2H),
4.851 (t, J = 6 Hz, 1H), 3.808 (m, 2H), 3.478 (m, 1H), 3.080 (m,
2H), 1.390 (m, 2H), 1.192 (m, 22H), 0.830 (t, J = 6 Hz, 3H). 13C
NMR (DMSO-d6, 75 MHz) d/ppm = 170.239, 161.888, 138.612,
134.254, 131.278, 131.126, 128.272, 128.014, 76.314, 63.742,
39.069, 31.979, 29.838, 29.717, 29.398, 27.014, 22.778, 14.609.
Anal. Calcd for C24H39ClN2O2: C, 68.14; H, 9.29; N, 6.62; O, 7.56.
Found: C 66.55; H, 9.20; N, 6.47; O, 8.81.
4.1.3.9. (S)-2-(2-Chlorobenzylideneamino)-3-hydroxy-N-tetrade
cylpropanamide (3i). Yield: 0.35 g (83%) as colorless powder. GC/
MS (m/e): 423 M+, 1H NMR (DMSO-d6, 300 MHz) d/ppm = 8.603
(s, 1H), 8.185 (d, J = 7.5 Hz, 1H), 7.772 (t, J = 5.7 Hz, 1H), 7.465
(m, 3H), 4.868 (t, J = 6 Hz, 1H), 3.908 (dd, J = 3.9 Hz, J = 8.1 Hz,
1H), 3.802 (m, 1H), 3.478 (m, 1H), 3.083 (m, 2H), 1.390 (m, 2H),
1.194 (m, 22H), 0.834 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6,
75 MHz) d/ppm = 170.117, 159.231, 137.260, 134.998, 133.131,
130.458, 129.487, 127.983, 76.421, 63.545, 39.069, 31.979,
29.717, 29.398, 26.999, 22.778, 14.624. Anal. Calcd for
4.1.3.4. (S)-2-(4-Methylbenzylideneamino)-3-hydroxy-N-tetra-
decylpropanamide (3d). Yield: 0.24 g (60%) as colorless powder.
GC/MS (m/e): 402 M+, 385 [MꢀꢂOH]+, 1H NMR (DMSO-d6,
300 MHz) d/ppm = 8.215 (s, 1H), 7.705 (m, 3H), 7.253 (d,
J = 8.1 Hz, 2H), 4.808 (t, J = 6 Hz, 1H), 3.769 (m, 2H), 3.466 (m,
1H), 3.067 (m, 2H), 2.336 (s, 3H), 1.385 (m, 2H), 1.194 (m, 22H),
0.833 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz) d/
ppm = 170.588, 163.011, 141.405, 134.011, 129.775, 129.061,
76.254, 63.834, 39.024, 31.994, 29.823, 29.732, 29.413, 26.999,
22.778, 21.760, 14.609. Anal. Calcd for C25H42N2O2: C, 74.58; H,
10.51; N, 6.96; O, 7.95. Found: C 72.67; H, 10.41; N, 7.29; O, 8.78.
C24H39ClN2O2: C, 68.14; H, 9.29; N, 6.62; O, 7.56. Found: C,
67.80; H, 9.43; N, 6.69; O, 7.42.
4.1.3.10. (S)-2-(4-Methoxybenzylideneamino)-3-hydroxy-N-tetr
adecylpropanamide (3j). Yield: 0.36 g (86%) as colorless powder.
GC/MS (m/e): 419 [M+H]+, 1H NMR (DMSO-d6, 300 MHz) d/
ppm = 8.179 (s, 1H), 7.775 (d, J = 9.0 Hz, 2H), 7.680 (t, J = 5.7 Hz,
1H), 6.989 (d, J = 9.0 Hz, 2H), 4.814 (t, J = 6 Hz, 1H), 3.792 (s, 3H),
3.769 (m, 2H), 3.443 (m, 1H), 3.074 (m, 2H), 1.381 (m, 2H), 1.192
(m, 22H), 0.832 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz) d/
ppm = 170.755, 162.465, 162.116, 130.732, 129.426, 114.576
76.178, 63.909, 55.984, 39.009, 31.979, 29.808, 29.717, 29.398,
26.983, 22.778, 14.624. Anal. Calcd for C25H42N2O3: C, 71.73; H,
10.11; N, 6.69; O, 11.47. Found: C 71.27; H, 10.10; N, 6.88; O, 11.80.
4.1.3.5. (S)-2-(3-Methylbenzylideneamino)-3-hydroxy-N-tetra
decylpropanamide (3e). Yield: 0.31 g (77%) as colorless powder.
GC/MS (m/e): 402 M+, 385 [MꢀOH]+, 1H NMR (DMSO-d6,
300 MHz) d/ppm = 8.223 (s, 1H), 7.686 (m, 2H), 7.595 (d,
J = 7.2 Hz, 1H), 7.306 (m, 2H), 4.823 (t, J = 6 Hz, 1H), 3.790 (m,
2H), 3.479 (m, 1H), 3.080 (m, 2H), 2.342 (s, 3H), 1.387 (m, 2H),
1.203 (m, 22H), 0.833 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6,
75 MHz) d/ppm = 170.542, 163.330, 138.445, 136.501, 132.250,
129.228, 129.107, 126.586, 76.345, 63.788, 39.024, 31.979,
29.808, 29.701, 29.398, 26.999, 22.778, 21.533, 14.624. Anal. Calcd
for C25H42N2O2: C, 74.58; H, 10.51; N, 6.96; O, 7.95. Found: C 73.37;
H, 10.40; N, 7.05; O, 8.21.
4.1.3.11. (S)-2-(3-Methoxybenzylideneamino)-3-hydroxy-N-tetr
adecylpropanamide (3k). Yield: 0.36 g (86%) as colorless powder.
GC/MS (m/e): 419 [M+H]+, 1H NMR (DMSO-d6, 300 MHz) d/
ppm = 8.234 (s, 1H), 7.719 (t, J = 5.7 Hz, 1H), 7.422 (s, 1H), 7.354
(m, 2H), 7.048 (m, 1H), 4.857 (t, J = 6 Hz, 1H), 3.790 (m, 5H),
3.480 (m, 1H), 3.081 (m, 2H), 1.364 (m, 2H), 1.192 (m, 22H),
0.833 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz) d/
ppm = 170.482, 163.148, 160.142, 137.974, 130.306, 121.986,
117.659, 113.286, 76.284, 63.742, 55.893, 39.039, 31.979, 29.701,
29.398, 26.999, 22.778, 14.609. Anal. Calcd for C25H42N2O3: C,
71.73; H, 10.11; N, 6.69; O, 11.47. Found: C 71.21; H, 9.91; N,
6.95; O, 11.97.
4.1.3.6. (S)-2-(2-Methylbenzylideneamino)-3-hydroxy-N-tetra-
decylpropanamide (3f). Yield: 0.30 g (74%) as colorless powder.
GC/MS (m/e): 402 M+, 385 [MꢀOH]+, 1H NMR (DMSO-d6,
300 MHz) d/ppm = 8.527 (s, 1H), 7.949 (d, J = 7.5 Hz, 1H), 7.642
(d, J = 5.7 Hz, 1H), 7.261 (m, 3H), 4.814 (t, J = 6 Hz, 1H), 3.825 (m,
2H), 3.475 (m, 1H), 3.092 (m, 2H), 2.466 (s, 3H), 1.391 (m, 2H),
1.196 (m, 22H), 0.831 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6,
75 MHz) d/ppm = 170.573, 161.918, 138.399, 134.436, 131.384,
131.096, 128.272, 126.526, 76.694, 63.803, 39.024, 31.994,
29.838, 29.747, 29.428, 27.014, 22.793, 19.604, 14.594. Anal. Calcd
for C25H42N2O2: C, 74.58; H, 10.51; N, 6.96; O, 7.95. Found: C 73.10;
H, 10.46; N, 6.89; O, 9.11.
4.1.3.12. (S)-2-(2-Methoxybenzylideneamino)-3-hydroxy-N-tetr
adecylpropanamide (3l). Yield: 0.30 g (72%) as colorless powder.
GC/MS (m/e): 418 M+, 1H NMR (DMSO-d6, 300 MHz) d/
ppm = 8.576 (s, 1H), 8.014 (d, J = 7.8 Hz, 1H), 7.698 (t, J = 5.7 Hz,
1H), 7.443 (t, J = 7.8 Hz, 1H), 7.089 (d, J = 7.8 Hz, 1H), 6.983 (t,
J = 7.8 Hz, 1H), 4.796 (t, J = 6 Hz, 1H), 3.835 (s, 3H), 3.784 (m, 2H),
3.451 (m, 1H), 3.073 (m, 2H), 1.384 (m, 2H), 1.196 (m, 22H),
0.834 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz) d/
ppm = 170.603, 159.291, 158.289, 133.161, 127.892, 124.355,
121.014, 112.375, 76.709, 63.818, 56.272, 39.009, 31.979, 29.808,
29.717, 29.398, 26.983, 22.778, 14.624. Anal. Calcd for
4.1.3.7. (S)-2-(4-Chlorobenzylideneamino)-3-hydroxy-N-tetrade
cylpropanamide (3g). Yield: 0.37 g (87%) as colorless powder. GC/
MS (m/e): [422ꢀH] M+, 1H NMR (DMSO-d6, 300 MHz) d/
ppm = 8.264 (s, 1H), 7.870 (d, J = 7.8 Hz, 2H), 7.741 (t, J = 5.7 Hz,
1H), 7.506 (d, J = 7.8 Hz, 2H), 4.870 (t, J = 6 Hz, 1H), 3.809 (m,
2H), 3.463 (m, 1H), 3.065 (m, 2H), 1.382 (m, 2H), 1.188 (m, 22H),
0.829 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz) d/
ppm = 170.360, 162.040, 136.198, 135.393, 130.747, 129.304,
76.193, 63.712, 39.054, 31.979, 29.732, 29.398, 26.983, 22.778,
C25H42N2O3: C, 71.73; H, 10.11; N, 6.69; O, 11.47. Found: C
71.33; H, 10.09; N, 6.68; O, 11.72.

J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 5316–5322
5321
4.1.4. General procedure for synthesis of hydroxy-substituted
(S)-2-(benzylideneamino)-3-hydroxy-N-tetradecylpropanamide
(3m–o)
d/ppm = 7.926 (d, J = 3.6 Hz, 1H), 7.720 (m, 3H), 7.500 (t,
J = 8.4 Hz, 1H), 4.720 (t, J = 5.4 Hz, 1H), 3.977 (d, J = 15.3 Hz, 1H),
3.847 (d, J = 15.3 Hz, 1H), 3.462 (m, 2H), 3.021 (m, 3H), 1.359 (m,
2H), 1.209 (m, 24H), 0.833 (t, J = 6.6 Hz, 3H). 13C NMR (DMSO-d6,
75 MHz) d/ppm = 172.243, 137.245, 135.803, 133.844, 131.415,
128.834, 124.947, 64.608, 63.120, 48.741, 38.963, 31.979, 29.732,
29.671, 29.382, 27.014, 22.778, 14.609. Anal. Calcd for
A mixture of 0.40 g (1.00 mmol) of (S)-2-amino-3-hydroxy-N-
tetradecylpropanamide hydrochloride, 0.04 g (1.00 mmol) of
NaOH, 1.00 mmol of hydroxybenzaldehyde, and 40 mL of methanol
was stirred at room temperature for eight hours. The solvent was
then evaporated under reduced pressure, and the residue was
washed with cold methanol to yield hydroxy-substituted (S)-2-
(benzylideneamino)-3-hydroxy-N-tetradecylpropanamide as pow-
der. Recrystallization was performed using petroleum ether/
methanol.
C24H41N3O4: C, 66.17; H, 9.49; N, 9.65; O, 14.69. Found: C, 66.60;
H, 9.69; N, 9.54; O, 14.33.
4.1.5.2. (S)-2-(2-Chlorobenzylamino)-3-hydroxy-N-tetradecyl-
propanamide (4i). Yield: 73%, colorless powder. GC/MS (m/e):
424 M+, 1H NMR (CDCl3, 300 MHz) d/ppm = 7.404–7.233 (m, 5H),
3.899 (s, 2H), 3.794 (t, J = 5.1 Hz, 2H), 3.237 (t, J = 5.7 Hz, 3H),
1.633 (m, 2H), 1.492 (t, J = 6.9 Hz, 2H), 1.257 (m, 24H), 0.882 (t,
J = 6.9 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d/ppm = 136.319,
130.641, 130.048, 129.168, 127.270, 114.971, 63.180, 62.922,
50.639, 39.312, 32.145, 29.868, 29.762, 29.579, 29.504, 27.150,
22.899, 14.335. Anal. Calcd for C24H41ClN2O2: C, 67.82; H, 9.72; N,
6.59; O, 7.53. Found: C, 67.58; H, 9.93; N, 6.69; O, 9.72.
4.1.4.1. (S)-2-(4-Hydroxybenzylideneamino)-3-hydroxy-N-tetra
decylpropanamide (3m). Yield: 0.20 g (50%) as colorless powder.
GC/MS (m/e): 404 M+, 387 [MꢀOH]+, 1H NMR (DMSO-d6,
300 MHz) d/ppm = 8.114 (s, 1H), 7.639 (m, 3H), 6.806 (d,
J = 8.1 Hz, 2H), 4.768 (t, J = 6 Hz, 1H), 3.739 (m, 2H), 3.429 (m,
1H), 3.070 (m, 2H), 1.381 (m, 2H), 1.201 (m, 22H), 0.834 (t,
J = 6 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz) d/ppm = 170.846,
162.617, 160.734, 130.868, 127.953, 115.958, 76.132, 63.955,
39.994, 31.979, 29.797, 29.717, 29.382, 26.983, 22.778, 14.624.
Anal. Calcd for C24H40N2O3: C, 71.25; H, 9.97; N, 6.92; O, 11.86.
Found: C 71.22; H, 10.24; N, 6.86; O, 11.66.
4.1.5.3. (S)-2-(2-Methyoxybenzylamino)-3-hydroxy-N-tetrade-
cylpropanamide (4l). Yield: 78%, colorless powder. GC/MS (m/e):
319 [MꢀH]+, 1H NMR (CDCl3, 300 MHz) d/ppm = 7.359–6.906 (m,
5H), 4.076 (dd, J = 3.0 Hz, J = 11.1 Hz, 1H), 3.855 (s, 3H), 3.758 (m,
2H), 3.590 (m, 1H), 3.250 (m, 2H), 2.247 (m, 1H), 1.501 (m, 2H),
1.254 (m, 24H), 0.880 (t, J = 6.9 Hz, 3H). 13C NMR (CDCl3,
75 MHz) d/ppm = 136.319, 130.641, 130.048, 129.168, 127.270,
114.971, 63.180, 62.922, 52.203, 50.639, 39.312, 32.145, 29.868,
29.762, 29.579, 29.504, 27.150, 22.899, 14.335. Anal. Calcd for
4.1.4.2. (S)-2-(3-Hydroxybenzylideneamino)-3-hydroxy-N-tetra
decylpropanamide (3n). Yield: 0.21 g (52%) as colorless powder.
GC/MS (m/e): 387 [MꢀOH]+, 1H NMR (DMSO-d6, 300 MHz) d/
ppm = 8.166 (s, 1H), 7.635 (t, J = 5.7 Hz, 1H), 7.235 (m, 3H), 6.860
(m, 1H), 3.783 (m, 2H), 3.464 (m, 1H), 3.075 (m, 2H), 1.385 (m,
2H), 1.202 (m, 22H), 0.834 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6,
75 MHz) d/ppm = 170.497, 163.406, 158.365, 137.883, 130.170,
120.255, 118.782, 115.123, 76.223, 63.773, 39.039, 31.994,
29.853, 29.732, 29.413, 27.014, 22.793, 14.624. Anal. Calcd for
C25H44N2O3: C, 71.39; H, 10.54; N, 6.66; O, 11.41. Found: C,
71.30; H, 9.82; N, 6.90; O, 11.81.
4.1.5.4. (S)-2-(2-Hydroxybenzylamino)-3-hydroxy-N-tetradecyl-
propanamide (4o). Yield: 75%, colorless powder. GC/MS (m/e): 405
[MꢀH]+, 389 [MꢀOH]+,1H NMR (CDCl3, 300 MHz) d/ppm = 7.188 (t,
J = 7.5 Hz, 1H), 6.985 (d, J = 7.5 Hz, 1H), 6.846 (d, J = 7.5 Hz, 1H),
6.799 (t, J = 7.5 Hz, 1H), 6.196 (br, 1H), 4.090 (d, J = 14.1 Hz, 1H),
3.839 (m, 2H), 3.757 (dd, J = 5.4 Hz, J = 11.1 Hz, 1H), 3.281 (m,
2H), 3.142 (t, J = 5.4 Hz, 1H), 1.513 (t, J = 6.6 Hz, 2H), 1.262 (m,
24H), 0.882 (t, J = 6.6 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d/
ppm = 159.003, 129.396, 129.198, 122.335, 119.815, 116.702,
62.953, 61.283, 50.578, 39.859, 32.145, 29.868, 29.807, 29.716,
29.579, 29.473, 27.089, 22.899, 14.335. Anal. Calcd for
C24H40N2O3: C, 71.25; H, 9.97; N, 6.92; O, 11.86. Found: C 71.20;
H, 10.21; N, 6.83; O, 11.70.
4.1.4.3. (S)-2-(2-Hydroxybenzylideneamino)-3-hydroxy-N-tetra
decylpropanamide (3o). Yield: 0.15 g (37%) as yellow powder.
GC/MS (m/e): 403 [MꢀH]+, 387 [MꢀOH]+, 1H NMR (DMSO-d6,
300 MHz) d/ppm = 8.468 (s, 1H), 7.924 (t, J = 5.7 Hz, 1H), 7.473 (d,
J = 7.5 Hz, 1H), 7.322 (t, J = 7.5 Hz, 1H), 6.882 (m, 2H), 4.926 (br,
1H), 3.918 (dd, J = 4.5 Hz, J = 7.8 Hz, 1H), 3.777 (dd, J = 4.5 Hz,
J = 10.8 Hz, 1H), 3.569 (m, 1H), 3.050 (m, 2H), 1.373 (m, 2H),
1.195 (m, 22H), 0.835 (t, J = 6 Hz, 3H). 13C NMR (DMSO-d6,
75 MHz) d/ppm = 169.571, 167.278, 161.022, 133.115, 132.478,
119.587, 119.238, 117.127, 75.115, 63.408, 39.176, 31.979,
29.717, 29.656, 29.398, 26.983, 22.778, 14.624. Anal. Calcd for
C24H42N2O3: C, 70.89; H, 10.41; N, 6.89; O, 11.80. Found: C, 70.29;
H, 10.99; N, 6.86; O, 11.92.
4.2. Bioassays
C24H40N2O3: C, 71.25; H, 9.97; N, 6.92; O, 11.86. Found: C 71.12;
H, 10.06; N, 6.78; O, 12.00.
4.2.1. Cell culture
Human cancer cell lines derived from breast, MCF-7TN-R were
cultured in 75 cm2 culture flasks in DMEM (Invitrogen, Co., Carls-
bad, CA) supplemented with 10% FBS (Life Technologies, Inc., Gai-
thersburg, MD), basic minimum MEM essential (50ꢁ, Invitrogen
Co.) and MEM non-essential (100ꢁ, Invitrogen, Co.) amino acids,
sodium pyruvate (100ꢁ Invitrogen Co.), antimycotic–antibiotic
4.1.5. General procedure for synthesis substituted (S)-2-
(benzylamino)-3-hydroxy-N-tetradecylpropanamide (4c, 4l,
4i, and 4o)
To the solution of 0.20 mmol substituted (S)-2-(benzylidenea-
mino)-3-hydroxy-N-tetradecylpropanamides 3c, 3l, 3i, and 3o in
10 mL of methanol, 20 mg (0.54 mmol) of NaBH4 was added. The
reaction mixture was stirred at room temperature for 10 h, and
evaporated under vacuum. The residue was purified by thin layer
chromatography to give substituted (S)-2-(benzylamino)-3-hydro-
xy-N-tetradecylpropanamides 4c, 4l, 4i, and 4o as colorless powder
in 73–78% yields.
(10,000 U/mL penicillin G sodium; 10,000
lg/mL streptomycin sul-
fate; 25
l
g/mL amphotericin B as FungizoneÒ), and human recom-
binant insulin (4 mg/mL. Invitrogen Co). Culture flasks were
maintained in a tissue culture incubator in a humidified atmo-
sphere of 5% CO2 and 95% air at 37 °C.
4.2.2. MTT viability assay
4.1.5.1. (S)-2-(2-Nitrobenzylamino)-3-hydroxy-N-tetradecylpro-
panamide (4c). Yield: 75%, colorless powder. GC/MS (m/e):
435 M+, 418 [MꢀOH]+, 1H NMR (DMSO-d6, 300 MHz)
MCF-7TN-R cells were plated at 7.5 ꢁ 105 cells per well in a
96-well plate in phenol-free DMEM supplemented with 5% FBS
and allowed to adhere overnight. Cells were then treated with

5322
J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 5316–5322
analogs 3a–o (ranging from 0.1 to 100 lM) for 24 h. Following
Acknowledgements
treatment, 20 mL of MTT (5 mg/mL) reagent was incubated in each
well for 4 h. Cells were then lysed with 20% SDS in 50% dimethyl-
formamide. The pH and absorbances are read on an ELx808 Micro-
tek plate reader (Winooski, VT) at 550 nm, with a reference
wavelength of 630 nm. The values are the mean of SE of four inde-
pendent experiments. The same protocol was used for the MTT as-
says on HEK294 non-tumorigenic human kidney cell line.
This research was supported by a grant from the Louisiana Can-
cer Research Consortium (#631324). We thank Ms. Xiaoyi Zhang
and Beijing Area Major Laboratory of Peptide and Small Molecular
Drugs (IHLB, KZ200810025010) for their assistance in computa-
tional chemistry.
References and notes
4.2.3. Statistical analysis
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Statistical analysis of IC50 values were calculated from concen-
tration-response curves using GraphPad Prism 5.0 (Graphpad Soft-
ware, San Diego, CA), using with the equation:
Y ¼ Bottom þ ðTop ꢀ BottomÞ=1 þ 10LogEC50 ꢀ X:
Assuming a standard slope, where the response goes from 10%
to 90% of maximal as X increases over two log units. Differences in
IC50 were compared using Student’s unpaired t-test with p<0.05 as
the limit of statistical significance. Experiments comparing
multiple concentrations to the control will be tested with one-
way ANOVA with Bonferroni post-test to compare individual
concentrations. All statistical analysis will be done using GraphPad
Prism 5.0.
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4.3. Determination of Log K of 3a–o
Substituted (S)-2-(benzylideneamino)-3-hydroxy-N-tetradecyl-
propanamides 3a-o were dissolved in aqueous to prepare sample
solutions of 15
Hewlett Packard Series 1050 (phenomenex Gemini-NX 5u C18
110A). After 5 L of the sample was loaded, the column was eluted
lM. The RP-HPLC analysis was carried out on hp
l
with a solution consisting of CH3OH/H2O 85:15 (v/v) as the mobile
phase for 30 min. The flow rate was 1 mL/min. The peak of 3a–o in
the sample was monitored with UV detector at 254.40 nm
(550.10 nm for reference) and the retention time (tR) corresponding
to its peak was recorded. With the same HPLC conditions, the reten-
tion time of acetone peak was recorded as dead time t0. In order to
offset the influence of the solvent on the appearance time of the peak
of 3a–o, the appearance time of acetone peak (t0, 1.556 min) was
used as a control. As an alternative representation of tR, Log K was
defined based on the equation Log K = Log [(tR ꢀ t0)/t0].
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