Antiproliferative activity of synthetic fatty acid amides from renewable resources

Article abstract of DOI:10.1016/j.bmc.2014.11.019

In the work, the in vitro antiproliferative activity of a series of synthetic fatty acid amides were investigated in seven cancer cell lines. The study revealed that most of the compounds showed antiproliferative activity against tested tumor cell lines, mainly on human glioma cells (U251) and human ovarian cancer cells with a multiple drug-resistant phenotype (NCI-ADR/RES). In addition, the fatty methyl benzylamide derived from ricinoleic acid (with the fatty acid obtained from castor oil, a renewable resource) showed a high selectivity with potent growth inhibition and cell death for the glioma cell line - the most aggressive CNS cancer.

Full text of DOI:10.1016/j.bmc.2014.11.019

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiproliferative activity of synthetic fatty acid amides
from renewable resources
Daiane S. dos Santos a, Luciana A. Piovesan a, Caroline R. Montes D’Oca a, Carolina R. Lopes Hack a,
Tamara G. M. Treptow a, Marieli O. Rodrigues a, Débora B. Vendramini-Costa b, Ana Lucia T. G. Ruiz b,
João Ernesto de Carvalho ,b, Marcelo G. Montes D’Oca a, ,
⇑
a Escola de Química e Alimentos, Universidade Federal do Rio Grande, Rio Grande, RS, Brazil
b Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Unicamp, Campinas, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In the work, the in vitro antiproliferative activity of a series of synthetic fatty acid amides were investi-
gated in seven cancer cell lines. The study revealed that most of the compounds showed antiproliferative
activity against tested tumor cell lines, mainly on human glioma cells (U251) and human ovarian cancer
cells with a multiple drug-resistant phenotype (NCI-ADR/RES). In addition, the fatty methyl benzylamide
derived from ricinoleic acid (with the fatty acid obtained from castor oil, a renewable resource) showed a
high selectivity with potent growth inhibition and cell death for the glioma cell line—the most aggressive
CNS cancer.
Received 22 August 2014
Revised 1 November 2014
Accepted 14 November 2014
Available online xxxx
Keywords:
Glioma
Ovarian cancer
Multidrug resistance
Ricinoleic acid
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
atom may be responsible for differences in antiproliferative pro-
files.9–11 The effect of endocannabinoids and fatty acid amides in
In recent years, considerable efforts have been made to develop
novel drugs for cancer treatment. Cancer is a major public health
problem in both developed and developing countries, and it is
the second leading cause of death in the United States. Annual esti-
mates show an alarming increase of new cases worldwide.1 The
disease is characterized by the uncontrolled division and prolifer-
ation of cells, which can be caused, among other mechanisms, by
DNA mutations, cell cycle defects, and deregulated apoptosis; com-
pounds that induce apoptosis may be useful as targeted cancer
therapies.2,3 In addition, multidrug resistance (MDR) is the princi-
pal mechanism by which many cancers, such as breast, ovarian,
lung among others, develop resistance to antitumor drugs,4,5 and
new drugs that can overcome this obstacle are important goals of
anticancer studies.
Fatty acid amides (Fig. 1) are considered a new family of biolog-
ically important lipids as shown by different biochemical and phar-
macological studies,6,7 and they are part of the endocannabinoid
family.8 Studies with synthetic fatty acid amides showed antipro-
liferative activity against several tumor cells, suggesting that vari-
ation in the fatty acid moieties on groups attached to the nitrogen
cancer cell proliferation was reported by the binding to cannabi-
noids and vanilloids receptors.2,12,13
Previously, we reported the synthesis and antituberculosis
activity of new fatty acid amides and the fatty acid isoniazid, and
we demonstrated that the fatty acid chain is of fundamental
importance to biological activity, most likely by facilitating its per-
meability in bacterial cells.14,15 To continue our efforts to discover
fatty acid molecules with improved therapeutic potential, we eval-
uated the in vitro antiproliferative activity of a series of amide
derivatives of long-chain fatty acids in different tumor cell lines.
2. Results and discussion
The fatty acid amides were readily obtained by the synthetic
pathway shown in Figure 214–17 and were subjected to an
in vitro antiproliferative study using a panel of seven human can-
cer cell lines—glioma (U251), breast epithelial carcinoma (MCF-7),
ovarian with a phenotype of multiple drug resistance (NCI-ADR/
RES), kidney (786-0), non-small cell lung cancer (NCI-H460), pros-
tate (PC-3), ovarian (OVCAR-3)—as well as one non-tumor cell line,
human keratinocyte (HaCaT), and kidney epithelial cells from the
African green monkey (VERO). The chemotherapeutic agent
doxorubicin was used as a standard positive control.
⇑
Corresponding author. Tel.: +55 53 32336961; fax: +55 53 32336960.
E-mail address: dqmdoca@furg.br (Marcelo G. Montes D’Oca).
These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.bmc.2014.11.019
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: dos Santos, D. S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.019

2
D.S. dos Santos et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
O
OH
( )4
( )3
N
H
AEA
O
OH
N
H
OEA
O
OH
N
H
PEA
Figure 1. Fatty acid amides biologically actives.
O
1) MeOH, H2SO4 or
DCC, DMAP, Et3N
O
amines
H2N
NR1R2
R
OH
R
1
2) amine
Fatty acid
Fatty acid amide
1-7a-f
H2N
H2N
3
5
2
4
R =
( )14
a
( )16
b
( )7
( )7
OH
c
HN
H2N
HN
( )4
( )7
OH
e
( )5
( )7
( )7
HN
O
( )7
d
f
6
7
Figure 2. Fatty acid amides evaluated for antiproliferative activity on several cell lines.
Table 1
GI50 values of fatty acid amides against several cell lines
Amide
Tumor cell line
Mean GI50
Non-tumor cell lineb
U251
MCF-7
NCI-ADR/RES
786-0
NCI-H460
PC-3
OVCAR-3
g mLÀ1
)
a
GI50
(l
1a
1c
—
55.4
8.6
160.6
6.1
—
5.1
3.6
0.7
—
—
116.9
27.3
3.6
4.6
8.0
3.8
1.5
—
53.4
37.8
0.06
6.1
—
7.17
5.1
5.3
110.2
—
129.5
25.9
6.5
4.6
7.4
5.2
4.4
4.2
7.6
6.3
—
50.3
8.6
25.7
3.0
—
0.3
1.9
3.0
—
—
—
106.9
122.6
—
46.9
—
13.7
11.7
6.8
—
—
75.6
16.9
6.3
—
103.2
31.6
—
56.9
—
10.5
11.1
3.5
21.7
—
157.7
35.4
4.1
6.3
7.3
—
—
92.0
30.8
—
104.4
32.3
—
26.8
—
10.3
6.7
3.2
—
—
—
35.4
6.6
5.5
14.0
5.2
4.1
4.7
10.6
24.2
58.4
33.1
41.1
4.5
94.9
32.3
—
36.6
—
15.2
8.3
6.1
—
—
—
36.5
4.1
(R)-1d
(R)-2b
(R)-2c
(S)-3b
(S)-3c
(R,S)-3d
(S)-3e
(S)-3f
4a
4b
4c
(R)-4d
5a
5b
5c
(R)-5d
5e
6c
(R)-6d
6e
7a
7b
7c
(R)-7d
39.1
—
26.5
—
8.8
6.9
4.0
—
10.3c
69.5
6.4
9.7
1.3
—
24.6
—
117.7
14.4
3.4c
4.8
157.7
157.7
9.1
2.8
4.6
7.1
0.2
4.0
4.7
—
—
26.6
4.8
5.2
9.1
12
4.4
5.0
10.5
16.9
45.6
24.1
42.0
4.7
37
5.9
4.7
9.4
13.0
6.2
4.7
10.8
45.6
6.9
6.3
11.5
4.3
5.7
3.9c
4.0c
13.7c
7.7
4.2
6.7
7.0
7.7
23.0
10.7
29.1
34.1
53.1
7.9
8.7
9.3
14.8
44.2
25
39.9
2.8
22.7
63.2
6.2
33.1
3.3
23.3
53.6
22.2
41.1
5.2
16.6
48.9
33.1
43.3
4.9
2.6
4.2
28.9
41.5
5.3
22.1
15.4
42.8
4.9
33.1
41.8
29.4
36.0
49.1
38.5
31.0
41.3
a
b
c
GI50 = 50% inhibition of cell growth.
Non-tumor cell line = HaCat.
Non-tumor cell line = VERO.
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D.S. dos Santos et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
The antitumor activity (
l
g mLÀ1) of tested fatty acid amides
dose-dependent manner. The compounds with the lowest values
were 5c (GI50 = 0.2
g mLÀ1) and (S)-3c (GI50 = 0.3 g mLÀ1), fol-
lowed by compound (R,S)-3d (GI50 = 1.9
g mLÀ1).
To find a structure–activity relationship (SAR), we evaluated the
fatty acid amides in terms of the TGI values. Here, the compounds
were arranged into three sets according to the fragment attached
to the nitrogen atom: (1) benzylamine derivatives (Table 2), (2)
ethanolamine derivatives (Table 3) and (3) heterocyclic amine
derivatives (Table 4).
was given by three parameters for each cell line: GI50 (concentra-
tion which inhibits 50% cell growth), TGI (concentration for total
inhibition of cell growth) and LC50 (concentration which leads to
50% cell death), calculated by origin 9.1 software. The dose–
response curves for all synthesized compounds against the tested
cell lines are given in the Supplementary material.
l
l
l
First, the data were analyzed in terms of the GI50 values
(Table 1). For this parameter, compounds with GI50 values lower
than 30
l
g mLÀ1 were considered to be active. Most of the com-
In Table 2, the antiproliferative activities of the benzylamine
derivatives are shown. The derivatives encompass benzylamine
pounds were active against most of the cell lines tested, in a
Table 2
Growth inhibition effect of the fatty acid benzylamide series 1–3 on several cell lines
Amide
Tumor cell line
NCI-ADR/RES 786-0
TGIa
Mean TGI
Non-tumor cell lineb
U251
>250
>250
MCF-7
>250
NCI-H460
>250
PC-3
>250
>250
122.6
OVCAR-3
>250
(l
g mLÀ1
)
O
(
(
(
)
)
N
H
14
>250
>250
>250
>250
>250
95.5
>250
>250
66.5
1a
O
(
)
7
N
H
7
>250
>250
>250
>250
1c
OH
O
)
(
)
7
N
H
5
47.5
104.5
46.6
71.5
178.8
86.0
(R)-1d
O
(
)
)
N
H
16
>250
>250
>250
>250
>250
>250
>250
>250
>250
(R)-2b
O
(
(
(
(
( )
7
N
H
7
28.3
40.8
>250
43.0
35.3
84.8
105.6
>250
49.3
161.6
>250
47.1
>250
>250
45.5
>100.9
>250
43.0c
>250
36.8
(R)-2c
O
)
)
)
N
H
16
>250
>250
>250
(S)-3b
O
(
)
7
N
H
7
17.8
7.1
45.8
44.5
(S)-3c
OH
O
(
)
7
N
H
5
13.9
5.1
84.5
>250
60.1
7.7
132.1
141.9
>250
>133.2
59.1
(R,S)-3d
O
(
)
( )
7
N
H
4
38.6
16.4
38.9
30.0
20.0
22.4
77.2
(S)-3e
O
( )7
( )6
N
H
>250
>250
3.3
>250
>250
>250
4.9
>250
11.7
>250
7.6
>250
7.9
>250
(S)-3f
Doxorubicin
0.92
1.6
>25
4.1c
2.3
a
b
c
TGI = total growth inhibition.
Non-tumor cell line = HaCat.
Non-tumor cell line = VERO.
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4
D.S. dos Santos et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 3
Growth inhibition effect of the fatty acid ethanolamides 4a–d on several cell lines
Amide
Tumor cell line
NCI-ADR/RES 786-0
TGIa
Mean TGI
Non-tumor cell lineb
U251
MCF-7
NCI-H460
PC-3
OVCAR-3
(l
g mLÀ1
)
O
OH
OH
( )14
N
H
4a
>250
>250
>250
>250
66.7
>250
>250
>250
>250
>250
81.0
>250
>250
>250
>250
71.7
>250
>250
59.9
>250
>250
53.5
7.6c
O
( )16
N
H
4b
>250
O
OH
( )7
( )7
N
H
51.3
39.5
64.6
44.2
4c
OH
( )5
O
OH
( )7
N
H
6.5
3.7
3.3
5.4
1.6
20.4
9.8
4.9
23.5
11.7
8.8
7.6
11.2
7.9
(R)-4d
Doxorubicin
0.92
>25
4.1c
2.3
a
b
c
TGI = total growth inhibition.
Non-tumor cell line = HaCat.
Non-tumor cell line = VERO.
(series 1), (R)-methylbenzylamine (series 2) and (S)-methylbenzyl-
amine (series 3). clear structure-activity relationship was
observed, as the various changes in the molecules influence its
activity against the tested cell lines.
six times more active than the hydroxyl analogue 3d (Mean TGI
>133.2
g mLÀ1) and twice as active as the monounsaturated ana-
logue 3c (Mean TGI = 44.5
g mLÀ1).
These data suggest that in the benzylamide series 1, 2, and 3, a
monounsaturated fatty acid along with a methyl group in the ben-
zylic position and a hydroxyl group attached to the fatty chain is
important for antiproliferative activity to a greater or lesser extent,
and an additional unsaturation in the fatty chain caused a substan-
tial drop in mean TGI.
A
l
l
In this set, the structurally simplest compound, a benzylamine
with a saturated fatty chain derivative (1a) was totally inactive
(mean TGI >250 l
g mLÀ1). The introduction of an unsaturation in
the fatty chain (cis-1c) or a methyl group in the benzylic position
(R-2a, and S-3b) had no influence on activity (mean TGI
>250
center.
The derivative 1d, with a hydroxyl functional group in the
monounsaturated chain, showed potential antiproliferative
(mean TGI 95.5
g mLÀ1). It seems that functionalization of mono-
l
g mLÀ1), regardless of the configuration of the stereogenic
Finally, the double bound configuration was evaluated and was
seen that the change of the configuration from cis (3c) to trans (3f)
caused a total loss of antiproliferative activity.
a
Analysis of compounds derived from ethanolamine (Table 2,
series 4) showed that substitution of a saturated fatty chain (com-
pounds 4a, b) with a monounsaturated chain (compound 4c)
improved the antiproliferative activity from undetectable (mean
l
unsaturated benzylamide derivatives is necessary for antiprolifer-
ative activity. Comparing with derivative 1d, compounds (R)-2c
and (S)-3c, with a methyl group in the benzylic position and a
monounsaturated fatty chain, showed a lower to higher potential
TGI >250
TGI = 59.9
a hydroxyl group was introduced to the monounsaturated fatty
chain (mean TGI = 11.2
g mLÀ1, compound 4d). Thus, in this ser-
l
g mLÀ1, 4a) to some potential antiproliferative (mean
l
g mLÀ1, 4c). This activity increased even further when
antiproliferative, respectively (mean TGI >100.9
l
g mLÀ1 and
44.5
l
g mLÀ1, respectively). In this instance, it was observed that
l
the spatial orientation of the methyl group has a great influence
on activity because, in terms of mean TGI, compound (S)-3c was
twice more active than (R)-2c. These results suggest that the unsat-
urated chain was an important fragment only when coupled with a
stereogenic center in the benzylic position or a hydroxyl group in
the fatty chain.
ies, it again appears that the presence of a hydroxyl group is impor-
tant for antiproliferative activity when associated with an
unsaturated chain.
For compounds in Table 3, it was observed that the presence of
a five-member heterocyclic compound (series 5) resulted in signif-
icantly greater antiproliferative activity compared with saturated
chain analogues (series 4). Moreover, changing the fatty chain from
C16:0 (5a) to C18:0 (5b) resulted in a reduction in the mean anti-
Compound 3d contains all the structural requirements for activ-
ity observed so far: an unsaturated chain coupled with a stereogen-
ic center with S configuration in the benzylic position, and a
hydroxyl group in the monounsaturated fatty chain. Comparing
the derivative 3d to its analogue 3c, which lacks a hydroxyl group,
reveals a mean TGI for 3c that is three times lower than that of 3d
proliferative activity (from 19.9 mg mLÀ1 for 5a, to 46.5 g mLÀ1
l
for 5b), whereas replacing the saturated chain with a monounsat-
urated chain (5c) further reduced the mean antiproliferative activ-
ity (from 46.5
presence of a second double bond in the fatty acid chain (5e)
resulted in increased potency (mean TGI = 24.6
g mLÀ1), and the
l l
g mLÀ1 for 5b, to >67.7 g mLÀ1 for 5c). The
(Mean TGI >133.2 l l
g mLÀ1 vs 44.5 g mLÀ1, respectively). How-
ever, comparing the derivative 3d with the analogue 1d, which
l
lacks a methyl group, showed a smaller decrease in mean TGI
presence of a hydroxyl group in the monounsaturated fatty chain
(mean TGI >133.2 l l
g mLÀ1 vs 95.5 g mLÀ1, respectively).
resulted in the best observed mean antiproliferative activity (mean
The compound 3e contains one additional unsaturation in the
TGI = 4.5 l
g mLÀ1 for 5d).
fatty chain, substituting the hydroxyl group (3d), and this com-
To evaluate the influence of heterocyclic ring size on antiprolif-
erative activity, we changed the five-member ring (Table 3, series
pound showed the best mean TGI value (22.4 l
g mLÀ1), which is
Please cite this article in press as: dos Santos, D. S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.019

D.S. dos Santos et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Table 4
Growth inhibition effect of the fatty acid heterocyclic amide series 5–7 on several cell lines
Amide
Tumor cell line
786-0
Mean TGI
Non-tumor cell lineb
U251
MCF-7
NCI-ADR/RES
NCI-H460
PC-3
OVCAR-3
TGIa g mLÀ1
(l )
O
( )14
N
N
11.4
32.5
5.8
13.2
39.9
30.0
43.5
5.6
16.1
48.4
23.3
33.5
56.8
12.2
36.0
21.9
22.9
68.5
14.0
19.9
46.5
12.9
27.4
10.6c
5a
O
( )16
5b
O
( )7
( )7
N
)
>250
153.1
>67.7
5c
OH
O
(
)
(
4.4
1.5
4.0
7.7
6.9
6.2
4.3
4.1
4.5
6.8c
N
5
7
(R)-5d
O
( )7
( )4
45.1c
N
12.0
27.5
21.9
36.6
32.6
33.9
24.6
5e
O
( )7
N
( )7
25.0
6.3
33.3
14.8
44.2
47.4
22.7
63.2
40.7
10.7
29.1
40.7
23.3
53.6
31.8
16.6
48.9
35.4
24.2
58.4
36.3
16.9
45.6
47.9
12.8
30.5
6c
OH
( )5
O
( )7
N
(R)-6d
O
( )7
( )4
N
22.1
6e
O
( )14
N
55.9
82.6
6.6
54.7
81.7
13.1
70.3
86.9
10.6
69.9
85.6
11.2
75.7
91.0
160.8
34.7
36.7
102.4
14.8
72.7
111.1
13.9
64.5
101.6
15.0
73.7
94.2
23.2
97.4
O
7a
O
( )16
N
O
O
7b
( )7
( )7
N
O
N
7c
OH
( )5
O
( )7
54.5
0.92
115.6
3.3
142.8
1.6
152.4
4.9
72.6
11.7
62.2
7.6
96.5
7.9
O
(R)-7d
Doxorubicin
>25
4.1c
2.3
a
b
c
TGI = Total Growth Inhibition.
Non-tumor cell line = HaCat.
Non-tumor cell line = VERO.
5) to a six-member ring (Table 3, series 6). However, a consequent
pounds showed that the presence of one or two double bonds in
the fatty acid chain resulted in a similar antiproliferative profile
(mean TGI = 36.3 and 45.6
In addition, the presence of a hydroxyl group in the monounsatu-
improvement in mean antiproliferative activity was only observed
with the monounsaturated compound (mean TGI >67.7
l
g mLÀ1
l
g mLÀ1 for 6c and 6e, respectively).
for 5c compared with 36.3
l
g mLÀ1 for 6c). Analysis of the com-
Please cite this article in press as: dos Santos, D. S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.019

6
D.S. dos Santos et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
rated fatty chain resulted in improved antiproliferative activity
(mean TGI 16.9
g mLÀ1, 6d).
Compound 5e
l
100
50
The exchange of a carbon atom with an oxygen atom in the six-
member ring (Table 3, series 7) caused a worsening in mean anti-
proliferative activity when compounds 7a, 7b, and 7d were com-
pared with compounds 5a, 5b, 5d, and 6d. However, when this
oxygenated six-member ring was associated with a monounsatu-
rated fatty chain, its mean antiproliferative activity improved
g mLÀ1 for 7c) compared with that of 5c (mean
0
(mean TGI = 15.0
l
TGI >67.7
l
g mLÀ1) and 6c (mean TGI = 36.3
l
g mLÀ1).
These data suggest that the antiproliferative activity is influ-
enced by the structural variation in the fatty acid amides, including
both the fatty acid derivative moiety and the moiety attached to
the nitrogen atom.
The data in Tables 2–4 shows that most of the fatty acid amides
studied showed activity against most of the cell lines, with a broad
spectrum inhibition profile, and there were varied significant differ-
ences in cell selectivity and safety. Among the benzylamide deriva-
tives (series 1–3), based on the mean TGI, five compounds showed a
potential antiproliferative: 1d, (R)-2c, (S)-3c, (R,S)-3d, and (S)-3e.
Among these, compounds (S)-3c and (S)-3e were noted for exhibit-
ing the lowest mean TGI values. Furthermore, compound (S)-3c
showed high activity and selectivity for ovarian cancer cells with
U251
MCF7
-50
-100
NCI/ADR-RES
786-0
NCI-H460
PC-3
OVCAR-3
Vero
-3
-2
-1
250
100
101
102
0,25
2,5
25
10
10
10
-1
Concentration (µg.mL
)
Figure 4. Dose–response analysis of cell growth inhibition by compound 5e against
several cell lines.
was observed when a comparison was made with a non-tumor cell
line (VERO, TGI = 7.6 l
g mLÀ1).
a
phenotype of multiple drug resistance (NCI-ADR/RES,
TGI = 7.1
g mLÀ1) and glioma cell lines (U251, TGI = 17.8 g mLÀ1),
as well as a favorable safety profile when compared with non-
tumor cells (HaCat, TGI = 36.8
g mLÀ1). In contrast, compound
(S)-3e showed high activity for glioma cells (U251, TGI =
5.1
g mLÀ1), prostate cancer cells (PC-3, TGI = 7.7 g mLÀ1) and
the resistant ovarian cancer cells (NCI-ADR/RES, TGI =
16.4
g mLÀ1) cell lines but with relative selectivity and a favorable
safety profile when compared with a non-tumor cell line (HaCat,
TGI = 77.2
g mLÀ1). However, compound (R,S)-3d, derived from
Among the heterocyclic derivatives, two compounds were effi-
cacious in inhibiting the proliferation of more than one cell line:
compound 5c, which demonstrated specificity of action in glioma
l
l
l
(U251, TGI = 5.8
notype (NCI-ADR/RES, TGI = 5.6
cer cell line (OVCAR-3, TGI = 14.0
death to glioma cells (U251, LC50 = 14.2); and compound 5e, which
was specific for glioma (U251, TGI = 12.0
g mLÀ1), and ovarian
cancer cells with an MDR phenotype (NCI-ADR/RES, TGI =
7.7 and caused cell death to glioma cells (U251,
g mLÀ1
l
g mLÀ1), ovarian cancer cells with an MDR phe-
l
g mLÀ1) and another ovarian can-
g mLÀ1), as well as causing cell
l
l
l
l
l
l
l
)
ricinoleic acid and methyl benzylamine, showed good growth inhi-
LC50 = 44.4). Although there was loss of both potency and specific-
ity of action with an additional unsaturation in the fatty acid chain,
compound 5e appeared to have a more favorable safety profile
than that of compound 5c, as it yielded the greatest difference of
activity between tumor and non-tumor cell lines (VERO,
bition and cell death for the glioma cell line (U251, TGI =
13.9
favorable safety profile when compared with a non-tumor cell line
(VERO, TGI = 59.1
g mLÀ1). The dose–response analysis of cell
l
g mLÀ1, LC50 = 63.6), with a high degree of selectivity and a
l
growth inhibition by compound (R,S)-3d is displayed in Figure 3.
The ethanolamine derivative (R)-4d showed an excellent mean
TGI, with growth inhibition and death for most of the cell lines, but
with relative selectivity. Furthermore, no favorable safety profile
TGI = 45.1 and 10.6 l
g mLÀ1, respectively). The dose–response
analysis of cell growth inhibition by compound 5e is displayed in
Figure 4. Ovarian cancer is difficult to diagnose, and recurrence
may occur through multidrug resistance. Both compounds 5c and
5e showed a better potency for ovarian cancer cells with an MDR
phenotype than for native ovarian cancer cells, a very relevant
result given the importance of the search of new drugs for cancers
with MDR.
Among all tested compounds, compound (R)-5d showed the
best mean TGI value, exhibiting growth inhibition and cell death
for most of the lines but without selectivity. This compound
appears to have a general cytotoxic effect.
Compound(R,S)-3d
100
50
0
The morfolyl amine derivative 7c showed a good mean TGI,
with growth inhibition for the most of cell lines, but with relatively
good selectivity and a moderately favorable safety profile when
comparing tumor and non-tumor cell lines.
U251
3. Conclusions
MCF7
-50
NCI/ADR-RES
786-0
NCI-H460
PC-3
In conclusion, the work presented herein demonstrates that
several fatty acid amides exhibited potent and selective antiprolif-
erative activity in the cancer cell lines tested. In general, benzyla-
mide derivatives showed a clear structure-activity relationship,
with an interesting activity profile when there was a hydroxyl
group in the fatty chain and/or a methyl group in the benzylamino
moiety, as well as an unsaturated chain. For the other derivatives,
the results of the growth inhibition assay demonstrate that TGI is
OVCAR-3
Vero
-100
-3
-2
-1
0,25
100
101
102
250
2,5
25
10
10
10
Concentration (µg.mL )
-1
Figure 3. Dose–response analysis of cell growth inhibition by compound (R,S)-3d
against several cell lines
Please cite this article in press as: dos Santos, D. S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.019

D.S. dos Santos et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
greatly influenced by structural modifications of the fatty acid
amides, in particular, both the fatty acyl chain and the amino-con-
taining moieties of the molecule. Compound (R,S)-3d showed a
high selectivity with potent growth inhibition and cell death for
the glioma cell line—the most aggressive CNS cancer. Also, com-
pound 5e, which was specific for glioma cells and ovarian cancer
cells with an MDR phenotype, with good antiproliferative poten-
tial. Both compounds showed a favorable safety profile when ana-
lyzing their effects on a non-tumor cell line, and they can serve as
templates for the development of candidate antitumor drugs,
including those for cancers with MDR. Future studies with fatty
acid amides are needed to confirm these important observations.
2H), 1.98 (m, 4H), 1.64 (m, 1H), 1.51 (d, 3H), 1.29 (m, 23H), 0.9
(t, 3H). 13C NMR (75 MHz, CDCl3) d 172.5 (C@O), 143.2; 130.2;
128.6; 127.3; 76.7; 48.7; 36.7; 32.6; 31.9; 28.9; 25.8; 22.7; 21.7;
14.2. MS [m/z(%)] 385 (M+, 5), 163 [–CH2C(OH)NHCH(CH3)Ph, 40],
120 [–NHCH(CH3)Ph, 40], 105 [–C(CH3)Ph, 100].
4.3. In vitro antiproliferative activity assay
The in vitro antiproliferative activity assay was performed as
described by Monks et al. (1991).18 Seven human tumor cell lines
[U251 (glioma), MCF-7 (breast), NCI-ADR/RES (multiple drug-resis-
tant ovarian cancer cells), 786-0 (renal), NCI-H460 (non-small cell
lung cancer cells), PC-3 (prostate) and OVCAR-03 (ovarian)] were
kindly provided by Frederick Ma (National Cancer Institute,
Bethesda, MD, USA). Additionally, the HaCaT (human keratino-
cytes) cell line was used, a kind donation from Dr. Ricardo Della
Coletta (FOP, UNICAMP). Stock and experimental cultures were
grown in medium containing 5 mL RPMI 1640 (GIBCO BRL) supple-
mented with 5% fetal bovine serum (GIBCO BRL). Penicillin/strep-
4. Experimental
4.1. Apparatus and chemistry
Reagents and fatty acids were purchased from Aldrich Chemical
Co. and used without further purification. Ricinoleic acid (cis-
C18:1,12-OH) was obtained from castor oil or castor oil biodiesel
hydrolysis. Column chromatography was performed with Silica
Gel 60 A (ACROS Organics 0.035–0.070 mesh). Analytical thin-layer
chromatography was performed with plates containing silica gel
(Merck 60GF245), and the spots were visualized using iodine.
Yields refer to chromatographically and spectroscopically homoge-
neous materials. Melting points were obtained with a Fisatom
430D apparatus and are uncorrected. The NMR spectra were
tomycin mixture (1000 U/mL:1000
added to the experimental cultures. Cells in 96-well plates
(100
L cells wellÀ1) were exposed to sample concentrations in
DMSO/RPMI (0.25, 2.5, 25, 250
g mLÀ1) at 37 °C and incubated
lg/mL, 1 mL/L RPMI) was
l
l
in a 5% CO2 atmosphere for 48 h. The final DMSO concentration
did not affect cell viability. Before (T0 plate) and after the sample
addition (T1 plates), cells were fixed with 50% trichloroacetic acid,
and cell proliferation was determined by spectrophotometric
quantification (540 nm) of cellular protein using the sulforhoda-
mine B assay. Using the dose–response curve for each cell line,
total growth inhibition (TGI, the concentration that produces total
growth inhibition or cytostatic effects) was determined through
non-linear regression analysis using ORIGIN software version 8.0
(OriginLab Corporation) (Shoemaker, 2006).19
recorded on
a
Varian VNMRS 300 MHz spectrometer (1H,
300 MHz and 13C, 75.5 MHz—Universidade Federal do Rio Grande
do Sul, UFRGS, Brazil) in deuterochloroform (CDCl3) solution. The
chemical shift data are reported in units of d (ppm) downfield from
tetramethylsilane (TMS), which was used as an internal standard.
Coupling constants (J) are reported in hertz (Hz) and refer to appar-
ent peak multiplicities. GC/MS analysis was used to ascertain the
purity of all compounds for which biological data was determined
using a GCMS-QP2010Plus chromatographic system (Shimadzu)
equipped with a split/splitless injector coupled with a mass
detector.
Acknowledgments
The authors are thankful for the financial support and fellow-
ships from the Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES/Brazil), the Fundação de Apoio à Pesquisa
do Estado do Rio Grande do Sul (PRONEM-FAPERGS/Brazil), and
the Conselho Nacional de Desenvolvimento Científico e Tecnológi-
co (CNPq/Brazil).
4.2. Synthesis
All compounds used in this work were synthesized according to
a published method. Amides 1–3 were synthesized by reacting the
respective fatty acid (0.3 mmol) with amines (0.3 mmol), triethyl-
amine (0.3 mmol), and a catalytic amount of (dimethylamino)pyr-
idine (DMAP), and dicyclohexyl carbodiimide (DCC, 0.3 mmol) in
CH2Cl2 was added dropwise to the above reaction mixture, which
was then stirred at room temperature for 24 h. The solid dicyclo-
hexylurea formed was removed by filtration and the solvent was
evaporated under reduced pressure. Amides 4–7 were synthesized
from fatty acid methyl esters (FAMEs) obtained via esterification of
the respective fatty acid. The aminolysis reaction of FAMEs
(0.3 mmol) was realized in the presence of the amines (1.8 mmol)
and acetonitrile for 24 h. The progress of the reactions was moni-
tored by silica gel TLC. The raw products were purified via column
chromatography on silica gel (n-hexane/ethyl acetate, 7:3) and
analyzed by proton and carbon NMR IR, and ESI–MS/MS. The pre-
sented spectroscopic data are in agreement with the literature.14,15
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.11.019.
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Please cite this article in press as: dos Santos, D. S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.019