New synthetic inhibitors of fatty acid synthase with anticancer activity

Article abstract of DOI:10.1021/jm2016045

Fatty acid synthase (FASN) is a lipogenic enzyme that is highly expressed in different human cancers. Here we report the development of a new series of polyphenolic compounds 5-30 that have been evaluated for their cytotoxic capacity in SK-Br3 cells, a hu

Full text of DOI:10.1021/jm2016045

Article
pubs.acs.org/jmc
New Synthetic Inhibitors of Fatty Acid Synthase with Anticancer
Activity
Carlos Turrado,† Teresa Puig,‡ Javier García-Carceles,† Marta Artola,† Bellinda Benhamu,†
́
́
Silvia Ortega-Gutierrez,† Joana Relat,§ Gloria Oliveras,‡ Adriana Blancafort,‡ Diego Haro,§
́
Pedro F. Marrero,§ Ramon Colomer,∥ and María L. Lopez-Rodríguez*,†
́
́
†Departamento de Química Organ
́
ica I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain
‡Oncology, Girona Institute for Biomedical Research, E-17001 Girona, Spain, and School of Medicine, University of Girona, E-17071
Girona, Spain
§Biochemistry and Molecular Biology, School of Pharmacy and Institute of Biomedicine of the University of Barcelona (IBUB), 08028
Barcelona, Spain
∥Spanish National Cancer Research Center (CNIO), Clinical Research Program, Melchor Fernan
́
dez Almagro, 3, E-28029 Madrid,
Spain
S
* Supporting Information
ABSTRACT: Fatty acid synthase (FASN) is a lipogenic enzyme that is highly expressed in different human cancers. Here we
report the development of a new series of polyphenolic compounds 5−30 that have been evaluated for their cytotoxic capacity in
SK-Br3 cells, a human breast cancer cell line with high FASN expression. The compounds with an IC50 < 50 μM have been tested
for their ability to inhibit FASN activity. Among them, derivative 30 blocks the 90% of FASN activity at low concentration (4
μM), is highly cytotoxic in a broad panel of tumor cells, induces apoptosis, and blocks the activation of HER2, AKT, and ERK
pathways. Remarkably, 30 does not activate carnitine palmitoyltransferase-1 (CPT-1) nor induces in mice weight loss, which are
the main drawbacks of other previously described FASN inhibitors. Thus, FASN inhibitor 30 may aid the validation of this
enzyme as a therapeutic target for the treatment of cancer.
aggressive tumors.3 Thus, FASN has been considered as a
INTRODUCTION
■
metabolic oncogene.4 In addition, FASN inhibition induces
apoptosis selectively in human cancer cells both in vitro and in
vivo, with minimal effect on normal cells.3 This fact, directly
related to the high dependence of the cancer cells on de novo
fatty acid synthesis and to the differential expression of FASN,
makes this enzyme a suitable target for cancer treatment with a
potential good therapeutic index. Indeed, pharmacological
inhibitors of FASN that block tumor cell proliferation, elicit
tumor cell death, and prevent tumor growth in animal models
have been identified.1,3 These studies confirmed the proposed
use of FASN inhibitors as novel antitumor therapeutics. The
Altered lipogenesis is a hallmark of many types of cancer, and
there is evidence that links overexpression and overactivation of
the lipogenic enzymes that are involved in de novo lipid
biosynthesis with different human tumors.1 A key step in
elucidating the relationship between cancer and lipogenesis was
the identification of the oncogenic antigen-519 (OA-519), a
protein linked to poor prognosis in breast cancer patients, as
fatty acid synthase (FASN).2 Since that initial finding, increased
FASN expression level has been observed in a wide variety of
solid human tumors, including cancers of breast, prostate,
colon, lung, bladder, ovary, stomach, endometrium, kidney,
skin, esophagus, tongue, and soft tissues. In addition, FASN
overexpression occurs early in tumor progression and is
associated with the unfavorable outcomes of the most
Received: November 28, 2011
Published: May 4, 2012
© 2012 American Chemical Society
5013
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023

Journal of Medicinal Chemistry
Article
Chart 1. Reported FASN Inhibitors Cerulenin (1), C75 (2), Orlistat (3), and (−)-Epigallocatechin 3-Gallate (EGCG, 4), and
New Compounds 5−30
most representative FASN inhibitors are shown in Chart 1.
Cerulenin [(2R,3S)-3-[(4E,7E)-nona-4,7-dienoyl]oxirane-2-car-
boxamide, 1], a metabolite produced by the fungus
Cephalosporum caerulens, was the first characterized FASN
inhibitor.5 It induced selective cytotoxicity in various types of
cancer cells and showed promising effects in different cancer
xenograft models. However, the presence of the reactive
epoxide ring makes cerulenin susceptible to other nonspecific
ring-opening reactions, a feature that has limited any potential
clinical application.1,3,6 To overcome this drawback, related
analogue C75 (trans-4-carboxy-5-octyl-3-methylenebutyrolac-
tone, 2, Chart 1) was synthesized.7 As expected from its
capacity to inhibit FASN activity, 2 showed significant
antitumor effects on several cancer cell lines and xenograft
models.3 However, it has been suggested that 2 could be
sensitive to the presence of endogenous thiols.8 In addition,
both 1 and 2 induce a profound weight loss, which is far from
ideal for cancer patients undergoing chemotherapy. This side
effect is apparently due to their activity in increasing fatty acid
oxidation through direct activation of carnitine palmitoyltrans-
ferase-1 (CPT-1).9−11 Also, they reduce food intake by
blocking the production of hypothalamic neuropeptide-Y.11
These effects clearly hindered the further clinical development
of 1 and 2. In 2004, orlistat [(−)-tetrahydrolipstatin, 3, Chart
1], marketed as antiobesity drug, was found to inhibit FASN,12
and more recently, green tea polyphenols, especially highly
abundant catechins such as (−)-epigallocatechin 3-gallate
(EGCG, 4, Chart 1), have received much attention on the
grounds of their reported capacity to block the formation and
development of tumors in a variety of animal models.13
Furthermore, EGCG has been found to inhibit FASN and to
induce selective apoptosis in human breast and prostate cancer
cells14−16 without producing concomitant weight loss.17
Unfortunately, its effectiveness in vivo is quite limited due to
its high IC50 value, poor oral bioavailability, and low serum
stability.18 In our research group, we are involved in a project
aimed at the development of new FASN inhibitors devoid of
CPT-1 stimulation as a side effect with the objective of
advancing toward the validation of FASN as a new therapeutic
target for the treatment of cancer. On the basis of our
promising preliminary results with some structurally simple
molecules initially inspired on the polyphenolic nature of
EGCG that showed significant in vitro and in vivo antitumor
properties,19,20 in the present work we report a new series of
gallate derivatives in which the A subunit has been modified
(compounds 5−30, Chart 1). Among them, derivative 30
stands out as a highly cytotoxic compound in a broad panel of
tumor cells of different tissue origins (IC50 values ranging from
1.4 to 9 μM). Furthermore, compound 30 almost completely
abrogates FASN activity at a concentration of 4 μM in breast
cancer SK-Br3 cells, induces apoptosis of tumor cells, and
interferes with the mitogen-activated protein kinase (MAPK)
pathway. Importantly, all these sought-after effects occur
without parallel CPT-1 stimulation or weight loss induction.
Overall, the possibility of inhibiting FASN without concomitant
weight loss induction clearly highlights the potential of this
enzyme as a new target in oncology. Accordingly, FASN
inhibitors could become a new class of drugs that aid in the
challenging endeavor of handling those types of tumors
resistant to the current therapies or those lacking adequate
treatments.
RESULTS AND DISCUSSION
■
Synthesis of Compounds 5−30. In a previous study, and
initially inspired by the polyphenolic nature of EGCG, we
designed a small series of compounds structurally simpler than
this natural product and with a straightforward synthetic
accessibility. Encouraged by the interesting results,20 in the
present work we have expanded the structural variability and we
have studied the influence of the A subunit in the cytotoxicity
and FASN inhibition of compounds 5−30 (Chart 1).
Anticipating the need of a synthetic route that enabled for us
both the quick generation of a variety of compounds as well as
their synthesis on the gram-scale needed for in vivo studies, we
set up a methodology for the esterification reaction based on
5014
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Final Compounds 5−30
a
Reagents and conditions: (a) PS-HOBt, DIC, DMAP, DCM, rt, quantitative; (b) (COCl)2, DMF, toluene, 55 °C, quantitative; (c) DIEA, THF, rt,
20−77%; (d) TEA, THF, rt, 24−53%; (e) H2, Pd(OH)2/C, EtOH, DCM (P = Bn), rt, 54−95%; (f) HF·py, pyridine, THF (P = TBS), rt, 55−88%.
the use of polymer supported (PS) reagents (Scheme 1,
method A). For this purpose, different commercially available
PS reagents were tested, including Mukaiyama (PS-Mukaiya-
ma), dicyclohexyl- and 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (PS-DCC and PS-EDC, respectively), and a
N,N′-dimethylaminopyridine surrogate bound to polystyrene
(PS-DMAP). However, all these reagents led to different and
nondesired outcomes, such as the isolation of mixtures of
esterification product and the corresponding acid anhydride
(with PS-Mukaiyama), of the activated alcohol intermediate
(PS-DMAP), or a lack of reactivity of the protected gallic acid
with the resin (PS-DCC and PS-EDC). Finally, positive results
were obtained with the use of PS-1-hydroxybenzotriazole (PS-
HOBt). Thus, protected gallic acid 55 or 56 was loaded on the
resin in the presence of diisopropylcarbodiimide (DIC) and
DMAP, and the resulting activated acid 57 or 58 linked to the
resin was filtered, washed, and dried (Scheme 1, method A). It
is important to note that this intermediate can be stored at least
one month at 4 °C without significant reactivity loss. In a
second step, the reaction of 57 or 58 with the corresponding
diol in the presence of diisopropylethylamine (DIEA) afforded
all protected target esters except 38, 39, 46, and 50 that could
not be obtained using this PS-based methodology possibly due
to steric hindrance. Therefore, they were synthesized by
classical condensation reaction between the corresponding acyl
chloride 59 or 6021 and the adequate diol in the presence of
triethylamine (TEA) (Scheme 1, method B). Final removal of
the protecting groups either by catalytic hydrogenation (in the
case of benzyl groups) or by treatment with a hydrofluoride
acid-pyridine (HF·py) complex (for the TBS-protected
intermediates) afforded the desired final compounds 5−30.
In the case of benzyl intermediates 43 and 44, in which the A
subunit is naphthalene, catalytic hydrogenation for 3 to 5 h
gave naphthalene compounds 20 and 21, respectively, whereas
exhaustive hydrogenation overnight afforded tetrahydronaph-
thalene analogues 17 and 18, respectively.
The synthesis of the starting carboxylic acid 5522 involved
the perbenzylation of the commercial methyl gallate followed
by saponification of the ester, whereas 5623 was obtained by
reaction of gallic acid with tert-butyldimethylsilyl chloride
(TBSCl) and subsequent selective cleavage of the silyl ester.
Noncommercial intermediate diols trans- and cis-1,2,3,4-
tetrahydronaphthalene-1,4-diol,24 naphthalene-1,2-diol,25 meth-
yl 1,4-dihydroxy-2-naphthoate,26 and naphthalene-1,8-diol27
were obtained following previously described procedures.
Cytotoxicity in SK-Br3 Cells. All the new final compounds
were first screened for their in vitro cytotoxic activity against
SK-Br3 human breast cancer cell line, selected because of its
high FASN expression. The IC50 values are shown in Table 1,
where the reference compound 4 has also been included for
comparative purposes. These data have allowed us to carry out
a SAR study on the influence of the modifications of the A
subunit in the cytotoxic capacity of the compounds. The main
conclusions can be summarized as follows: (i) Most of the final
compounds are highly cytotoxic in SK-Br3 cells (IC50 < 50
μM), being more potent than 4 (IC50 = 149 20 μM); (ii) in
general, the presence of at least one aromatic ring in the A
subunit is required for cytotoxicity, as suggested by derivatives
5−7, inactive in SK-Br3 cells (IC50 > 150 μM); (iii) for those
compounds where the A subunit is a phenyl ring (8−14),
cytotoxicity increases as the distance between the two galloyl
moieties augments. Thus, all the compounds with a relative 1,4-
5015
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023

Journal of Medicinal Chemistry
Article
substitution in the phenyl ring show lower IC50 values than
analogues 8 and 9 (with 1,2- and 1,3-substitution, respectively).
Among them, derivative 14 deserves special attention as being
the most cytotoxic one (IC50 = 8 μM); (iv) replacement of the
benzene ring in the A subunit for different tetrahydronaph-
thalenes (derivatives 15−18) did not yield any significant
improvement in cytotoxicity, with compounds showing IC50
values between 22 and 42 μM; (v) introduction of a
naphthalene ring as A subunit (compounds 19−29) yielded
some interesting derivatives with good cytotoxicity values.
Among them, polyphenols 20, 23, 28, and 29 with IC50 values
between 5 and 26 μM, deserve special attention. Importantly,
this naphthalene series suggests that, as observed for benzene
derivatives 8−14, cytotoxicity increases as the distance between
the galloyl moieties augments, 28 being the most potent
compound (relative 2,6-orientation of galloyl groups and IC50 =
5 μM), and compounds 19, 26, and 27 the less active ones
(relative 1,2-, 1,8-, and 2,3-orientation of galloyl groups, and
IC50 values of 74, 78, and 76 μM, respectively). These data
prompted us to introduce a biphenyl system as A subunit with
the two galloyl groups as distant as possible (i.e., in a relative
4,4′-position, see Table 1). This modification led to the
identification of analogue 30, the most potent derivative in the
series in terms of cytotoxicity, with an IC50 value of 4 μM.
FASN Inhibition. Considering our objective of identifying
FASN inhibitors with antitumor properties, those compounds
that inhibited more effectively the proliferation of the SK-Br3
cells (IC50 value lower than 50 μM) were assessed for their
capacity to block FASN activity. Toward this end, SK-Br3 cells
were incubated for 24 h in the presence of a concentration of
the compound equal to its cytotoxic IC50 value in these cells,
and FASN activity was measured in particle-free supernatants.
The inhibition is expressed as the percentage of inactivation
compared to a 100% activity control sample (Table 1). The
obtained results show that some of the cytotoxic compounds
(13, 16, 20, 21, 23, 27, and 30) are indeed FASN inhibitors
able to block between 30% and 90% of its total activity. Among
them, derivatives 20 and 30 stand out as being not only highly
cytotoxic in SK-Br3 cells (IC50 values of 21 and 4 μM,
respectively) but also the most potent FASN inhibitors
identified within the series (90% of FASN inhibition, Table
1). Prompted by these results, we previously disclosed the
pharmacological profile of derivative 20, which turned out to
induce apoptosis in SK-Br3 cells without affecting CPT-1
activity.20 Considering that both 20 and 30 block FASN activity
in a similar extent (up to 90%) but 30 does it at a 5-fold lower
concentration (21 μM for 20 vs 4 μM for 30), this latter
compound is clearly worthy of an in-depth pharmacological
study because it could be a good tool-compound for ensuing in
vivo studies and validation of FASN as a target with therapeutic
interest. Among the obtained results, it is important to note
that compound 28 had a cytotoxic potency in SK-Br3 cells
comparable to compound 30 but did not inhibit FASN
significantly (Table 1), which suggests that there are additional
mechanisms apart from inhibition of FASN activity that may
derive in cytotoxic effects. Conversely, an effect of derivative 30
on an additional target different from FASN cannot be ruled
out. Accordingly, we sought to rule out general cytotoxicity of
compound 30 and to confirm that FASN was the main
mechanism mediating the cell death induced by this derivative.
With respect to the former, we determined the capacity of
compound 30 to inhibit cell proliferation in non cancer human
mammary epithelial HMEpC and hepatic HL-7702 cells. In
Table 1. Cytotoxicity in SK-Br3 Breast Cancer Tumor Cells
and Inhibition of FASN Activity of Compounds 5−30
a
The values are the mean SEM from two independent experiments
b
performed in triplicate. FASN inhibition values are determined at a
concentration equal to the IC50 value of the cytotoxicity of the
c
compound in SK-Br3 cells. Data are from ref 20. N.D., not
determined. N.I., no significant inhibition of FASN activity at the
tested concentration.
5016
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023

Journal of Medicinal Chemistry
Article
both cell lines, derivative 30 induced little cytotoxicity, with
IC50 values from 8- to 10-fold higher than in SK-Br3 cells [IC50
(30, SK-Br3) = 4 μM; IC50 (30, HMEpC) = 31 μM; IC50 (30,
HL-7702) = 41 μM]. To study the actual contribution of FASN
to the cytotoxic effects, its expression was knocked down in SK-
Br3 cells using a FASN specific siRNA and cellular viability was
determined in parallel for transfected and nontransfected cells.
Our results show that decreasing FASN expression in an 80%
(quantified by Western blot band intensity densitometry)
eliminates the cytotoxicity of compound 30, which does not
induce any significant cell death up to a concentration of 10 μM
(Figure 1). Hence, derivative 30 was selected for a
Table 2. Cytotoxicity of Compound 30 in Different Human
Cancer Cell Lines
a
tissue origin
cell line
HCT116
IC50 (μM)
colon
pancreas
skin
4
2
PANC-1
A431
2.1 0.5
1.4 0.1
liver
Bel-7402
SK-Hep1
HepG2
SKOV3
SK-Br3
MCF-7
ZR-75−1
MDA-MB-231
BT-20
40
56
3
5
70 21
ovary
breast
5
4
2
1
3.8 0.8
1.9 0.3
5
17
24
14
9
3
2
5
8
1
AU565
lung
HCC827
H460
A549
17 10
a
The values are the mean
SEM from at least two independent
experiments performed in duplicate or triplicate.
Figure 1. (A) Effect of siRNA-induced silencing of FASN gene
expression on cytotoxicity induced by compound 30. Cytotoxicity of
compound 30 at different concentrations (white bars) is eliminated by
siRNA-induced reduction of FASN expression levels (black bars). (B)
SK-Br3 breast cancer cells were transfected with siRNA-targeting
FASN for 72 h. Equal amounts of total protein from SK-Br3 cells
transfected with siRNA-targeting FASN or with the control were
subjected to immunoblotting analyses with antibodies against FASN or
β-actin. Results shown in (A) are the average SEM of three different
experiments performed in duplicate. Gels shown in (B) are
representative of those obtained in two independent experiments. *p
< 0.01 (Student’s t test) vs the same concentration of compound 30 in
control-transfected cells.
Figure 2. FASN expression levels in different breast (A) and liver (B)
cancer cell lines. Equal amounts of total protein were subjected to
immunoblotting analysis with a monoclonal antibody against FASN.
Immunoreactive bands in the figure are from one representative
experiment. Equivalent results were found in 2−4 independent
experiments. Blots were reprobed with an antibody for β-actin to
control for protein loading and transfer (not shown for the sake of
clarity).
sensitivity of cells toward compound 30, as observed in BT-20
and AU565 breast cancer cell lines (IC50 values of 17 and 24
μM, respectively, Table 2 and Figure 2A). This correlation
between FASN levels and cytotoxicity of derivative 30 is also
observed in the hepatic cancer cells Bel-7402, SK-Hep1, and
HepG2 (Figure 2B), with IC50 values of 40, 56, and 70 μM,
respectively (Table 2). Taken together, these results clearly
support the potential of the new FASN inhibitor 30 as an
antitumor compound with wide application and prompted us to
gain insights on the cell death mechanism and molecular
pathways involved in this effect.
Cell Death Mechanism and Signaling Pathways. To
establish the mechanism of action of compound 30, we first
evaluated whether it induced apoptosis and caspase activity by
Western blotting analysis showing cleavage of poly(ADP-
ribose) polymerase (PARP). Treatment of SK-Br3 cells for 48 h
induced a marked increase in the levels of the PARP cleavage
product (89 kDa band) in a time-dependent manner (Figure
3A). In light of the previously reported effect of FASN
inhibitors not only in apoptosis but also in the HER2 receptor
and its associated pathways,17,20,28 we assessed the effects of 30
on HER2 activation as well as on its downstream signal
comprehensive biological characterization including the study
of its cytotoxicity in a panel of different tumor cell lines, its
molecular mechanism of action as well as its selectivity vs CPT-
1 enzyme.
Cytotoxicity of FASN Inhibitor 30 in a Panel of Tumor
Cells. The capacity of derivative 30 to induce cytotoxicity was
further evaluated in a panel comprising a variety of tumor cells
of different tissue origins, including colon, pancreas, skin, liver,
ovary, breast, and lung. The IC50 values obtained for the
different cell lines are shown in Table 2. In general, compound
30 showed a remarkable cytotoxicity in the tumor cell line
panel, being especially relevant its potency in the HCT116,
PANC-1, A431, SKOV3, MCF-7, ZR-75−1, MDA-MB-231,
and H460 cells in which the IC50 values were below 10 μM. In
general, breast cancer cells with high FASN levels, such as SK-
Br3, MCF-7, ZR-75−1, and MDA-MB-231 cells (Figure 2A),
are more sensitive to compound 30, with IC50 values between 2
and 5 μM (Table 2). Lower FASN levels decrease the
5017
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023

Journal of Medicinal Chemistry
Article
Figure 3. Compound 30 (A) induces apoptosis as assessed by PARP
cleavage (note intact PARP at 116 kDa and its cleavage product at 89
kDa), and (B) blocks the activation of HER2, AKT, and ERK1/2
signaling pathways. SK-Br3 cells were treated with 30 for 6, 12, or 24
h, and equal amounts of lysates were immunoblotted with the
corresponding antibody. Blots were reprobed for β-actin as loading
control. In all cases, shown gels are representative of those obtained
from at least two independent experiments.
Figure 4. Compound 30 (A) does not modulate CPT-1 activity in
vitro and (B) does not induce weight loss in vivo in a similar manner
to EGCG (4) and in sharp contrast to 2, both included in the figure
for comparative purposes. CPT-1 activity was determined in intact
yeast mitochondria as a source of enzyme in the absence or presence
of the corresponding compound. For determination of the effect of the
compounds in body weight, mice were fasted for 12 h, treated with a
single ip (30, 50, or 75 mg/kg) dose of the compound under study or
vehicle as indicated, refed for 24 h, and body weight was registered.
transduction pathways ERK1/2 and PI3K/AKT. Incubation of
cells with 30 dramatically decreased the levels of the
phosphorylated form of HER2 (p-HER2) that occurred as
soon as 2 h after treatment (data not shown) and became
complete at 6 h after treatment (Figure 3B). During this period,
there was no significant change in the total level of HER2
protein, as assessed by either Western blotting analysis (Figure
3B) or HER2-specific ELISA (data not shown). Phosphorylated
forms of ERK1/2 (p-ERK1/2) and AKT (p-AKT) were also
noticeably decreased after 6 h of exposure to compound 30
(Figure 3B). Overall, the derivative 30 induced a sustained
blockade of the HER2 signaling pathway, at least throughout
the whole time interval analyzed (24 h). During this period, no
significant changes in the total level of the corresponding
proteins (HER2, AKT, and ERK1/2) were detected.
Remarkably, under the same culture conditions, EGCG did
not induce apoptosis nor blocked the activation of HER2
oncogene and its downstream signal transduction pathways,
ERK1/2 and PI3K/AKT.20
CPT-1 Activation and in Vivo Weight Loss. Given the
importance of inhibition of FASN without a cross-activation of
CPT-1, which is relevant to avoid the side effect of weight loss,
compound 30 was analyzed both in vitro and in mice.
Importantly, 30 did not stimulate CPT-1 activity (Figure 4A),
in a similar manner to what had been previously described for
the natural FASN inhibitor 4 and in sharp contrast to
compound 2 which, as previously reported, produced a
significant activation of CPT-1 activity. Both reference FASN
inhibitors 2 and 4 are included in Figure 4A for comparative
purposes. Consistent with these in vitro results, healthy mice
treated with a single intraperitoneal (ip) dose of 30, 50, or 75
mg/kg of compound 30 did not show any significant weight
loss vs the control group (Figure 4B). Food intake was similar
Results shown are the average
SEM of a least three different
experiments with independent mitochondrial preparations (A) or the
average SEM, n = 4 (B).
to controls, and altered behavior or signs of suffering or distress
were not observed in mice treated with 30 (data not shown).
These effects are especially significant when compared with
animals treated with a 30 mg/kg ip dose of 2, in which a 21%
weight loss, as well as signs of suffering, were observed.
Altogether, our findings indicate that the new synthetic FASN
inhibitor 30 does not induce weight loss in vivo.
CONCLUSION
■
In this work, we report the synthesis of a new series of
polyphenolic derivatives that have been evaluated for their
cytotoxic capacity in SK-Br3 cells, a human breast cancer cell
line with high FASN expression. The most cytotoxic
compounds have been further assessed as FASN inhibitors.
Among them, derivative 30 stands out as a potent FASN
inhibitor which blocks 90% of FASN activity at low
concentration (4 μM) and is highly cytotoxic in a broad
panel of tumor cells of different tissue origins, with IC50 values
below 10 μM. At the molecular level, compound 30 induces
apoptosis of tumor cells and blocks activation of the HER2
receptor and its downstream proteins AKT and ERK1/2.
Importantly, these effects occur without parallel CPT-1
stimulation or in vivo weight loss. Overall, our results clearly
indicate that compound 30 is a promising new FASN inhibitor
that could contribute to the validation of this enzyme as a
therapeutic target for the treatment of different types of cancer.
5018
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023

Journal of Medicinal Chemistry
Article
General Procedure for the Synthesis of Protected Esters
31−37, 40−45, 47−49, and 51−54 (Method A). A suspension of
57 or 58 (2.14 equiv), the adequate diol (1 equiv), and DIEA (2.86
equiv) in anhydrous THF (20 mL/mmol of diol) was stirred at room
temperature under an argon atmosphere overnight. The reaction
mixture was filtered through a plug of silica gel, washed with saturated
NH4Cl, water and brine, dried over Na2SO4, and evaporated under
vacuum. The title compound was obtained as a white solid and was
used in the following reaction without further purification.
1,2-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)ethane (31). 31 was
prepared from 57 and ethylene glycol. Yield 71%.
cis-1,2-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)cyclohexane
(32). 32 was prepared from 57 and cis-cyclohexane-1,2-diol. Yield 64%.
trans-1,2-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)cyclohexane
(33). 33 was prepared from 57 and trans-cyclohexane-1,2-diol. Yield
50%.
EXPERIMENTAL SECTION
■
Chemistry. Melting points (mp, uncorrected) were determined on
a Stuart Scientific electrothermal apparatus. Infrared (IR) spectra were
measured on a Shimadzu-8300 or Bruker Tensor 27 instrument
equipped with a Specac ATR accessory of 5200−650 cm−1
transmission range; frequencies (ν) are expressed in cm−1. Nuclear
magnetic resonance (NMR) spectra were recorded on a Bruker
Avance 300-AM (1H, 300 MHz; 13C, 75 MHz) or Bruker 200-AC
spectrometer (1H, 200 MHz; 13C, 50 MHz) at the UCM’s NMR
facilities. Chemical shifts (δ) are expressed in parts per million relative
to internal tetramethylsilane; coupling constants (J) are in hertz (Hz).
The following abbreviations are used to describe peak patterns when
appropriate: s (singlet), d (doublet), t (triplet), m (multiplet), br
(broad). Mass spectrometry (MS) was carried out on a Bruker LC-
Esquire in electrospray mode (ESI). High pressure liquid chromatog-
raphy−mass spectrometry (HPLC-MS) analysis was performed using
an Agilent 1200LC-MSD VL. LC separation was achieved with an
Eclipse XDB-C18 column (5 μm, 4.6 mm × 150 mm) together with a
guard column (5 μm, 4.6 mm × 12.5 mm). The gradient mobile
phases consisted of A (95:5 water/MeOH) and B (5:95 water/
MeOH) with 0.1% ammonium hydroxide and 0.1% formic acid as the
solvent modifiers. MS analysis was performed with an ESI source. The
capillary voltage was set to 3.0 kV, and the fragmentor voltage was set
at 72 eV. The drying gas temperature was 350 °C, the drying gas flow
was 10 L/min, and the nebulizer pressure was 20 psi. Spectra were
acquired in negative ionization mode from 200 to 800 m/z and in UV
mode at four different wavelengths (210, 230, 254, and 280 nm).
Satisfactory HPLC-MS traces confirmed a purity of at least 95% for all
tested compounds, and their retention times are provided in the
Supporting Information. Hydrogenations were carried out in a Parr
hydrogenator under an initial pressure of hydrogen of 40−50 psi at
room temperature for 1 h. Analytical thin-layer chromatography
(TLC) was run on Merck silica gel plates (Kieselgel 60 F-254) with
detection by UV light (254 nm), ninhydrin solution, or 10%
phosphomolybdic acid solution in ethanol. Flash chromatography
was performed on glass column using silica gel type 60 (Merck,
particle 230−400 mesh) or on a Varian 971-FP flash purification
system using silica gel cartridges (Varian, particle size 50 μm). Unless
stated otherwise, starting materials, reagents, and solvents were
purchased as high-grade commercial products from Sigma-Aldrich,
Lancaster, Scharlab, or Panreac and were used without further
purification. Tetrahydrofuran (THF) was distilled from sodium
benzophenone ketyl and used immediately. Dichloromethane
(DCM) was distilled from calcium hydride.
1,2-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)benzene (34). 34
was prepared from 57 and cathechol. Yield 37%.
1,3-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)benzene (35). 35
was prepared from 57 and resorcinol. Yield 68%.
1,4-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)benzene (36). 36
was prepared from 57 and hydroquinone. Yield 24%.
2-Methyl-1,4-bis([3,4,5-tris(benzyloxy)benzoyl]oxy)benzene
(37). 37 was prepared from 57 and 2-methylbenzene-1,4-diol. Yield
52%.
2-Methoxy-1,4-bis([3,4,5-tris(benzyloxy)benzoyl]oxy)-
benzene (40). 40 was prepared from 57 and 2-methoxybenzene-1,4-
diol. Yield 30%; mp 197−198 °C. IR (KBr) ν 1735, 1585, 1506, 1115.
1H NMR (200 MHz, CDCl3) δ 3.90 (s, 3H, CH3), 5.24 (s, 4H, 4CH2
Bn), 5.26 (s, 8H, 4CH2 Bn), 6.88−6.96 (m, 2H, benzene), 7.25 (d, J =
8.4, 1H, benzene), 7.34−7.57 (m, 30H, Bn), 7.62 (s, 2H, gal), 7.65 (s,
2H, gal). 13C NMR (50 MHz, CDCl3) δ 56.1 (CH3), 71.2, 71.3
(4CH2), 75.1 (2CH2), 106.8 (CH), 109.7, 109.8 (4CH), 113.5, 123.1
(2CH), 124.2 (2C), 127.5, 127.6, 128.0, 128.2, 128.5 (30CH), 136.5,
137.3, 137.4, 137.6 (7C), 143.0, 143.1, 149.3, 151.9 (4C), 152.6, 152.7
(4C), 164.1, 164.5 (2CO). ESI-MS 1007.1 (M + Na)+.
cis-1,4-Bis[(3,4,5-tris(tert-butyldimethylsilyloxy)benzoyl)-
oxy]-1,2,3,4-tetrahydronaphthalene (41). 41 was prepared from
58 and cis-1,2,3,4-tetrahydronaphthalene-1,4-diol. Yield 20%.
trans-1,4-Bis[(3,4,5-tris(tert-butyldimethylsilyloxy)benzoyl)-
oxy]-1,2,3,4-tetrahydronaphthalene (42). 42 was prepared from
58 and trans-1,2,3,4-tetrahydronaphthalene-1,4-diol. Yield 53%.
1,3-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)naphthalene (43).
43 was prepared from 57 and naphthalene-1,3-diol. Yield 47%.
1,4-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)naphthalene (44).
44 was prepared from 57 and naphthalene-1,4-diol. Yield 64%.
1,2-Bis[(3,4,5-tris(tert-butyldimethylsilyloxy)benzoyl)oxy]-
naphthalene (45). 45 was prepared from 58 and naphthalene-1,2-
diol. Yield 60%.
1,5-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)naphthalene (47).
47 was prepared from 57 and naphthalene-1,5-diol. Yield 42%.
1,6-Bis[(3,4,5-tris(tert-butyldimethylsilyloxy)benzoyl)oxy]-
naphthalene (48). 48 was prepared from 58 and naphthalene-1,6-
diol. Yield 24%.
1,7-Bis[(3,4,5-tris(tert-butyldimethylsilyloxy)benzoyl)oxy]-
naphthalene (49). 49 was prepared from 58 and naphthalene-1,7-
diol. Yield 24%.
2,3-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)naphthalene (51).
51 was prepared from 57 and naphthalene-2,3-diol. Yield 37%.
2,6-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)naphthalene (52).
52 was prepared from 57 and naphthalene-2,6-diol. Yield 39%.
2,7-Bis[(3,4,5-tris(tert-butyldimethylsilyloxy)benzoyl)oxy]-
naphthalene (53). 53 was prepared from 58 and naphthalene-2,7-
diol. Yield 39%; mp 66−67 °C. IR (neat) ν1741, 1578, 1082. 1H NMR
(300 MHz, CDCl3) δ 0.19 (s, 12H, 2Me2SiO), 0.29 (s, 24H,
4Me2SiO), 0.99 (s, 36H, 4t-BuSiO), 1.03 (s, 18H, 2t-BuSiO), 7.36 (dd,
J = 8.9, 2.2, 2H, naph), 7.42 (s, 4H, gal), 7.67 (d, J = 2.2, 2H, naph,),
7.92 (d, J = 7.9, 2H, naph). 13C NMR (75 MHz, CDCl3) δ −3.9, −3.6
(12CH3), 18.5, 18.8 (6C), 26.1, 26.2 (18CH3), 116.1 (4CH), 118.4
(2CH), 121.1 (2C), 121.2, 129.1 (4CH), 129.3, 134.4 (2C), 144.0,
148.7 (6C), 149.5 (2C), 164.8 (2 CO). ESI-MS 1171.6 (M + Na)+.
Compounds 55,22 56,23 59,21 and 6021 were synthesized according
to described procedures, and their spectroscopic data are in agreement
with those previously reported. Spectral characterization data of all
described compounds were consistent with the proposed structures.
Satisfactory HPLC chromatograms were also obtained for final
compounds 5−30. We include here the data of protected esters 39,
40, and 53 and final compounds 16, 17, 23, 29, and 30. All other
compounds are fully characterized in the Supporting Information.
General Procedure for the Preparation of the Resin-Bound
Intermediates 57 and 58. According to a previously optimized
methodology,29 the following anhydrous DCM solutions were
sequentially added to 1 equiv of PS-HOBt: 0.6 equiv of DMAP in
20 mL/mmol, 1.5 equiv of 55 or 56 in 2.5 mL/mmol, and 4.4 equiv of
DIC in 0.6 mL/mmol. The reaction was stirred under an argon
atmosphere at room temperature for 3 h, and the resulting solid was
filtered and washed with DMF, DCM, and THF. The residue was
dried under vacuum to give 57 or 58 in quantitative yield.
3,4,5-Tris(benzyloxy)benzoic Acid Bound to PS-HOBt (57).
57 was prepared from 475 mg of PS-HOBt, 39 mg of DMAP dissolved
in 6.4 mL of DCM, 350 mg of 55 dissolved in 2.0 mL of DCM and 1.2
mL of DMF, and 0.36 mL of DIC dissolved in 1.4 mL of DCM.
3,4,5-Tris(tert-butyldimethylsilyloxy)benzoic Acid Bound to
PS-HOBt (58). 58 was prepared from 150 mg of PS-HOBt, 12 mg of
DMAP dissolved in 2 mL of DCM, 128 mg of 56 dissolved in 0.6 mL
of DCM and 0.3 mL of DMF, and 0.11 mL of DIC dissolved in 0.44
mL of DCM.
5019
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023

Journal of Medicinal Chemistry
Article
4,4′-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)-1,1′-biphenyl
(54). 54 was prepared from 57 and 1,1′-biphenyl-4,4′-diol. Yield 77%.
General Procedure for the Synthesis of Protected Esters 38,
39, 46, and 50 (Method B). To a solution of 59 or 60 (2.2 equiv)
and TEA (2.2 equiv) in anhydrous THF (10 mL/mmol), the
corresponding diol (1 equiv) dissolved in THF (2 mL/mmol) was
added. The reaction was stirred under an argon atmosphere at room
temperature overnight. Afterward, the reaction mixture was washed
with saturated NaHCO3, dried over Na2SO4, and evaporated under
reduced pressure. The residue was purified by column chromatog-
raphy using the appropriate eluent to give pure title compound as a
white solid.
120.8 (CH), 128.4 (C), 140.6, 140.7, 141.1 (3C), 146.7, 146.8 (4C),
150.3, 151.0 (2C), 166.6, 167.0 (2CO). ESI-MS 466.9 (M − H)−.
1,4-Bis[(3,4,5-trihydroxybenzoyl)oxy]-5,6,7,8-tetrahydro-
naphthalene (18). 18 was prepared from intermediate 44. Yield 88%.
1,3-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (20). 20
was prepared intermediate 43. Yield 65%.
1,4-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (21). 21
was prepared from intermediate 44. Yield 88%.
1,5-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (23). 23
was prepared from intermediate 47. Yield 30%; mp 265 °C (dec).
IR (KBr) ν 3404, 1718, 1618, 1209. 1H NMR (300 MHz, CD3OD) δ
7.35 (s, 4H, gal), 7.40 (d, J = 7.5, 2H, naph), 7.58 (t, J = 7.9, 2H,
naph), 7.84 (d, J = 8.2, 2H, naph). 13C NMR (50 MHz, DMSO-d6) δ
109.2 (4CH), 117.5 (2CH), 119.3 (2C), 127.6, 127.8 (4CH), 128.4
(2C), 139.6 (2C), 145.8 (2C), 146.7 (4C), 164.5 (2CO). ESI-MS
462.7 (M − H)−.
2,3-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (27). 27
was prepared from intermediate 51. Yield 60%.
2,6-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (28). 28
was prepared from intermediate 52. Yield 54%.
2,5-Bis([3,4,5-tris(benzyloxy)benzoyl]oxy)-1,1′-biphenyl
(38). 38 was prepared from 59 and 1,1′-biphenyl-2,5-diol.
Chromatography DCM/hexane 7:3 (Rf 0.26); yield 57%.
Methyl 2,5-Bis[(3,4,5-tris(tert-butyldimethylsilyloxy)-
benzoyl)oxy]benzoate (39). 39 was prepared from 60 and methyl
2,5-dihydroxybenzoate. Chromatography DCM/hexane 2:8 (Rf 0.27,
DCM/hexane 4:6); yield 53%; mp 191−192 °C. IR (neat) ν 1741,
1
1576, 1081. H NMR (300 MHz, CDCl3) δ 0.18 (s, 12H, 2Me2SiO),
0.28 (s, 24H, 4Me2SiO), 0.98 (s, 36H, 2t-BuSiO), 1,02 (s, 18H, 2t-
BuSiO), 3.73 (s, 3H, CH3), 7.28 (d, J = 8.9, 1H, benzene), 7.38 (s, 2H,
gal), 7.41 (s, 2H, gal), 7.45 (dd, J = 8,7, 2,9, 1H, benzene), 7.89 (d, J =
2.8, 1H, benzene). 13C NMR (75 MHz, CDCl3) δ −3.5, −3.4, −3.2
(12CH3), 18.9, 19.2 (6C), 26.5, 26.6 (18CH3), 52.6 (CH3), 116.5,
116.7 (4CH), 121.1, 121.4 (2C), 124.9 (C), 125.3, 125.4, 127.4
(3CH), 144.4, 144.6 (2C), 148.6, 148.7 (2C), 149.0, 149.1 (4C),
164.8, 164.9, 165.4 (3CO). ESI-MS 1179.6 (M + Na)+.
Methyl 1,4-Bis[(3,4,5-tris(tert-butyldimethylsilyloxy)-
benzoyl)oxy]-2-naphthoate (46). 46 was prepared from 60 and
methyl 1,4-dihydroxy-2-naphthoate. Chromatography DCM/hexane
2:8 (Rf 0.42); yield 41%.
1,8-Bis[(3,4,5-tris(tert-butyldimethylsilyloxy)benzoyl)oxy]-
naphthalene (50). 50 was prepared from 60 and naphthalene-1,8-
diol. Chromatography DCM/hexane 1:1 (Rf 0.24); yield 24%.
General Procedure for the Synthesis of Final Compounds
5−12, 14, 17, 18, 20, 21, 23, 27, 28, and 30. To a solution of
benzyl derivative 31−38, 40, 43, 44, 47, 51, 52, or 54 in a mixture of
DCM/ethanol (270 mL/mmol), 20% Pd(OH)2 on carbon (980 mg/
mmol) was added and the mixture was hydrogenated (initial hydrogen
pressure: 50 psi) at room temperature for 3−5 h or overnight (for
compounds 17 and 18). Then, the catalyst was filtered, the solvents
were evaporated under reduced pressure, and the resulting solid was
recrystallized from DCM/methanol to afford pure title compound.
1,2-Bis[(3,4,5-trihydroxybenzoyl)oxy]ethane (5). 5 was pre-
pared from intermediate 31. Yield 95%.
cis-1,2-Bis[(3,4,5-trihydroxybenzoyl)oxy]cyclohexane (6). 6
was prepared from intermediate 32. Yield 70%.
trans-1,2-Bis[(3,4,5-trihydroxybenzoyl)oxy]cyclohexane (7).
7 was prepared from intermediate 33. Yield 75%.
1,2-Bis[(3,4,5-trihydroxybenzoyl)oxy]benzene (8). 8 was
prepared from intermediate 34. Yield 60%.
1,3-Bis[(3,4,5-trihydroxybenzoyl)oxy]benzene (9). 9 was
prepared from intermediate 35. Yield 60%.
1,4-Bis[(3,4,5-trihydroxybenzoyl)oxy]benzene (10). 10 was
prepared from intermediate 36. Yield 80%.
2-Methyl-1,4-bis[(3,4,5-trihydroxybenzoyl)oxy]benzene
(11). 11 was prepared from intermediate 37. Yield 67%.
4,4′-Bis[(3,4,5-trihydroxybenzoyl)oxy]-1,1′-biphenyl (30). 30
was prepared from intermediate 54. Yield 70%; mp 268 °C (dec). IR
1
(KBr) ν 3396, 1701, 1608, 1192. H NMR (300 MHz, CD3OD) δ
7.24 (s, 4H, gal), 7.24−7.28 (m, 4H, biphenyl), 7.67−7.71 (m, 4H,
biphenyl). 13C NMR (50 MHz, CD3OD) δ 110.8 (4CH), 120.8 (2C),
123.5, 129.2 (8CH), 139.4 (2C), 140.6 (2C), 146.9 (4C), 152.4 (2C),
167.2 (2CO). ESI-MS 488.8 (M − H)−.
General Procedure for the Synthesis of Final Compounds
13, 15, 16, 19, 22, 24−26, and 29. To a solution of tert-
butyldimethylsilyl derivative 39, 41, 42, 45, 46, 48−50, and 53 in a
mixture of pyridine (5.8 mL/mmol) and THF (11.5 mL/mmol),
HF·py complex (5.8 mL/mmol) was added, and the reaction was
stirred at room temperature. After the complete disappearance of
starting material (TLC), the reaction mixture was diluted with water
(50 mL/mmol) and the crude was extracted with ethyl acetate. The
organic layers were then combined, washed with saturated aqueous
CuSO4, dried over MgSO4, and concentrated under reduced pressure.
The obtained off-white solid was recrystallized from DCM/methanol,
to afford pure title compound.
Methyl 2,5-Bis[(3,4,5-trihydroxybenzoyl)oxy]benzoate (13).
13 was prepared from intermediate 39. Yield 70%.
cis-1,4-Bis[(3,4,5-trihydroxybenzoyl)oxy]-1,2,3,4-tetrahydro-
naphthalene (15). 15 was prepared from intermediate 41. Yield 88%.
trans-1,4-Bis[(3,4,5-trihydroxybenzoyl)oxy]-1,2,3,4-tetrahy-
dronaphthalene (16). 16 was prepared from intermediate 42. Yield
62%; mp 225 °C (dec). IR (neat) ν 3351, 1683, 1612, 1214. 1H NMR
(300 MHz, acetone-d6) δ 2.09−2.15 (m, 2H, tetrahydronaph), 2.43−
2.47 (m, 2H, tetrahydronaph), 6.23 (s, 2H, tetrahydronaph), 7.12 (s,
4H, gal), 7.37−7.47 (m, 4H, tetrahydronaph), 8.07 (br s, 2H, OH),
8.20 (br s, 4H, OH). 13C NMR (75 MHz, acetone-d6) δ 25.8 (2CH2),
69.8 (2CH), 109.9 (4CH), 122.0 (2C), 129.6, 130.5 (4CH), 136.6,
139.9 (4C), 146.1 (4C), 166.2, 167.0 (2CO). ESI-MS 467.0 (M −
H)−.
1,2-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (19). 19
was prepared from intermediate 45. Yield 80%.
Methyl 1,4-Bis[(3,4,5-trihydroxybenzoyl)oxy]-2-naphthoate
(22). 22 was prepared from intermediate 46. Yield 70%.
1,6-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (24). 24
was prepared from intermediate 48. Yield 60%.
1,7-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (25). 25
was prepared from intermediate 49. Yield 69%.
1,8-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (26). 26
was prepared from intermediate 50. Yield 78%.
2,5-Bis[(3,4,5-trihydroxybenzoyl)oxy]-1,1′-biphenyl (12). 12
was prepared from intermediate 38. Yield 75%.
2-Methoxy-1,4-bis[(3,4,5-trihydroxybenzoyl)oxy]benzene
(14). 14 was prepared from intermediate 40. Yield 77%.
1,3-Bis[(3,4,5-trihydroxybenzoyl)oxy]-5,6,7,8-tetrahydro-
naphthalene (17). 17 was prepared from intermediate 43. Yield
92%; mp 213 °C (dec). IR (neat) ν 3356, 1705, 1615, 1200. 1H NMR
(200 MHz, CD3OD) δ 1.80 (m, 4H, tetrahydronaph), 2.59 (m, 2H,
tetrahydronaph), 2.83 (m, 2H, tetrahydronaph), 6.78 (d, J = 2.3, 1H,
tetrahydronaph), 6.84 (d, J = 2.3, 1H, tetrahydronaph), 7.18 (s, 2H,
gal), 7.21 (s, 2H, gal). 13C NMR (50 MHz, CD3OD) δ 23.6, 23.8,
24.3, 30.6 (4CH2), 110.7 (4CH), 114.4 (CH), 120.4, 120.6 (2C),
2,7-Bis[(3,4,5-trihydroxybenzoyl)oxy]naphthalene (29). 29
was prepared from intermediate 53. Yield 55%; mp 273 °C (dec).
IR (KBr) ν 3389, 1687, 1610, 1146. 1H NMR (300 MHz, CD3OD) δ
7.26 (s, 4H, gal), 7.35 (dd, J = 8.9, 2.1, 2H, naph), 7.70 (d, J = 2.1, 2H,
naph), 7.99 (d, J = 8.9, 2H, naph). 13C NMR (75 MHz, CD3OD) δ
109.6 (4CH), 118.6 (2CH), 119.5 (2C), 121.5, 129.3 (4CH), 129.8,
5020
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023

Journal of Medicinal Chemistry
Article
134.9 (2C), 139.6, 145.7 (6C), 150.0 (2C), 166.1 (2CO). ESI-MS
487.0 (M + Na)+.
HCl, pH 7.5) and kept at 4 °C while they were routinely mixed every 2
min on the vortex during 30 min. A sample was taken for
measurement of protein content by the Lowry-based BioRad assay.
Equal amounts of protein were heated in sodium dodecylsulphate
(SDS) sample buffer (Laemmli) for 5 min at 95 °C, separated on a 3−
8% SDS-polyacrylamide gel (p185HER2/neu, phospho-p185HER2/neu) or
4−12% SDS-polyacrylamide gel (AKT, phospho-AKT, ERK1/2,
phospho-ERK1/2, and PARP), and transferred onto nitrocellulose
membranes. Membranes were incubated for 1 h at room temperature
in blocking buffer (2.5% powdered-skim milk in tris-buffered solution
with 0.05% Tween-20; TBS-T [10 mM Tris-HCl pH 8.0, 150 mM
NaCl, and 0.05% Tween-20]) to prevent nonspecific antibody binding,
and incubated with the corresponding primary antibody diluted in
blocking buffer overnight at 4 °C. After 3 × 5 min washes in TBS-T,
blots were incubated for 1 h with corresponding peroxidase conjugated
secondary antibody and developed employing a commercial kit (West
Pico chemiluminescent substrate). Blots were reprobed with an
antibody against β-actin as control of protein loading and transfer.
Measurement of CPT-1 Activity. CPT-1 activity was assayed by
the forward exchange method using L-[3H]carnitine as we previously
described.10 Briefly, reactions (total volume of 0.5 mL) consisted of
the standard enzyme assay mixture with 200 μM of the corresponding
FASN inhibitor, 2 mM L-[3H]carnitine (∼5000 dpm/nmol), 80 μM
palmitoyl-CoA, 20 mM HEPES (pH 7.0), 1% fatty acid-free albumin,
and 40−75 mM KCl. Reactions were initiated by addition of isolated
intact yeast mitochondria. The reaction was linear up to 4 min, and all
incubations were done at 30 °C for 3 min. Reactions were stopped by
addition of 6% perchloric acid and were then centrifuged at 2500 rpm
for 5 min. The resulting pellet was suspended in water and the product
[3H]palmitoylcarnitine was extracted with butanol at low pH. After
centrifugation at 2300 rpm for 3 min, an aliquot of the butanol phase
was transferred to a vial and counted by liquid scintillation. To
perform the assay, mitochondria from GS115 Pichia pastoris
transformants that express Rat CTPIA were isolated by disrupting
the yeast cells with glass beads as previously described.10
In Vivo Experiments. Male mice C57BL/6J (12 weeks, 23−25 g)
were purchased from Harlan Laboratories (France), fed ad libitum
with a standard rodent chow, and housed in a light/dark 12 h/12 h
cycle at 22 °C for one week. Animals were randomized into four
groups (treated with vehicle or compounds 2, 4, or 30) of four animals
each. All experiments were done in accordance with guidelines on
animal care and use established by the University of Barcelona School
of Farmacia Institutional Ethic and Scientific Committee (Barcelona,
Spain). Treatments were done as previously described.11,30 Briefly,
mice were fasted for 12 h during the dark cycle before treatment. Each
group received a single ip injection (0.5 mL) of FASN inhibitor at the
corresponding dose or vehicle alone (DMSO), dissolved in RPMI
1640 medium. After ip injections, mice were given free access to
rodent chow for 24 h. At this time, the experiment finished and body
weight was registered.
Biochemistry. 4, 3-(4,5-dimethyl-1,3-thiazol-2-yl)-2,5-diphenyl-
2H-tetrazol-3-ium bromide (MTT), EDTA, dithiotreitol, acetyl-CoA,
malonyl-CoA, and NADPH were purchased from Sigma (St. Louis,
MO, USA), and 2 was from Alexis Biochemicals (San Diego, CA,
USA). Monoclonal anti-β-actin mouse antibody (C-4), monoclonal
anti-FASN, polyclonal anti-β-actin, and polyclonal Her-2 (Neu-C18)
rabbit antibody were from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). Antibody against phospho-Her-2 (C-erb-2 Ab18) mouse
monoclonal was from Thermo Fisher (Fremont, CA, USA). Rabbit
polyclonal antibodies against PARP, AKT, phospho-AKTSer473, ERK-1
and -2, and mouse monoclonal antibodies against phospho-ERK-1 and
-2 were from Cell Signaling Technology (Denvers, MA, USA).
Peroxidase-conjugated secondary antibodies were from Calbiochem
(San Diego, CA, USA). CPT-1 activity assay was done using
palmitoyl-CoA lithium salt from Sigma (St. Louis, MO, USA), fatty
acid-free bovine serum albumin from Roche (Manheim, Germany), L-
carnitine hydrochloride from Sigma (St. Louis, MO, USA), and L-
[methyl-3H] carnitine hydrochloride (82 Ci/mmol) from Amersham
Biosciences.
Cell Lines and Cell Culture. Cells were routinely incubated at 37
°C with 5% CO2. MCF-7 and MDA-MB-231 breast cancer cells were
obtained from the American Type Culture Collection (ATCC,
Rockville, MD, USA) and were routinely grown in Dulbecco’s
Modified Eagle’s Medium (Gibco, Berlin, Germany) containing 10%
heat-inactivated fetal bovine serum (FBS, HyClone Laboratories, UT,
USA), 1% L-glutamine, 1% sodium pyruvate, 50 U/mL penicillin, and
50 μg/mL streptomycin (Gibco). SK-Br3 breast cancer cells were
obtained from Eucellbank (Barcelona, Spain) and were passaged in
McCoy’s 5A medium containing 10% FBS, 1% L-glutamine, 1%
sodium pyruvate, 50 U/mL penicillin, and 50 μg/mL streptomycin.
Cytotoxicity Assay. Drug sensitivity was determined using a
standard colorimetric MTT assay. Briefly, cells were plated out at a
density of 5 or 7 × 103 cells/100 μL/well in 96-well microtiter plates
and allowed an overnight period for attachment. Then the medium
was removed and cells were incubated for 48 h with fresh medium
containing different concentrations of EGCG or the corresponding
compound 5−30. Following treatment, drug-free medium (100 μL/
well) and 10 μL of a 5 mg/mL MTT solution were added and cells
were incubated for 3 h at 37 °C. After careful removal of the
supernatants, the MTT-formazan crystals formed by metabolically
viable cells were dissolved in DMSO (100 μL/well) and absorbance
was measured at 570 nm in a multiwell plate reader (Model Anthos
Labtec 2010 1.7) or at 540 nm on a FlexStation 3 instrument.
FASN Activity Assay. After 6, 12, or 24 h of exposure to
compound under study, cells were harvested by treatment with
trypsin-EDTA solution, pelleted by centrifugation, washed twice, and
resuspended in cold phosphate buffered solution (PBS). Cells were
sonicated during 30 min at 4 °C (PSelecta Ultrasons) and centrifuged
for 15 min at 4 °C to obtain particle-free supernatants. FASN activity,
expressed in nmol NADPH oxidized min−1·protein mg−1, was assessed
in particle-free supernatant samples of equal protein content
(measured by the Lowry-based BioRad assay) by spectrophotometri-
cally monitoring oxidation of NADPH at 340 nm as described.17
RNA Interference-Mediated Silencing of the FASN Gene.
FASN siRNA (h), a pool of 3 target-specific 19−25 nt siRNAs,
commercially available from Santa Cruz Biotechnology, was used
following the manufacturer’s instructions to knock down FASN gene
expression in SK-Br3 cells. To determine cytotoxicity in these cells, the
MTT protocol indicated above was followed except for the fact that
the percentage of cell viability was measured after 24 h incubation in
the presence of compound 30.
Statistical Analysis for in Vivo Studies. Results were analyzed
by Student’s t test or by one-way ANOVA using a Tukey test as a post-
test. P < 0.05 was considered statistically significant. All data are means
SD. All observations were confirmed by at least three independent
experiments.
ASSOCIATED CONTENT
■
S
* Supporting Information
Spectral characterization data of protected esters 31−38, 41−
52, and 54 and final compounds 5−15, 18−22, 24−28, as well
as HPLC-MS analysis of purity for all final compounds 5−30.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Immunoblot Analysis of p185HER2/neu, Phospho-p185HER2/neu
,
ERK1/2, Phospho-ERK1/2, AKT, Phospho-AKTSer473, and PARP.
Following treatment of SK-Br3 cells with EGCG or FASN inhibitor 30
at the corresponding concentration and for the indicated time, cells
were harvested using trypsin-EDTA, washed twice with PBS, and
stored at −80 °C. Cells were lysed in lysis buffer (1 mM EDTA, 150
mM NaCl, 100 μg/mL phenylmethylsulfonyl fluoride 50 mM Tris-
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (+34) 913944239. Fax: (+34) 913944103. E-mail:
mluzlr@quim.ucm.es.
5021
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023

Journal of Medicinal Chemistry
Article
sensitive and insensitive components of the CPT system. Biochem.
Biophys. Res. Commun. 2004, 325, 660−664.
Notes
The authors declare no competing financial interest.
(11) Thupari, J. N.; Landree, L. E.; Ronnett, G. V.; Kuhajda, F. P.
C75 increases peripheral energy utilization and fatty acid oxidation in
diet-induced obesity. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9498−9502.
(12) Kridel, S. J.; Axelrod, F.; Rozenkrantz, N.; Smith, J. W. Orlistat is
a novel inhibitor of fatty acid synthase with antitumor activity. Cancer
Res. 2004, 64, 2070−2075.
ACKNOWLEDGMENTS
■
This work was supported by grants from the Spanish Ministerio
de Economıa y Competitividad (MINECO, SAF2010-22198-
́
C02-01, M.L.L.-R.; SAF2010-15217, D.H.; CIT090000-2008-
10, T.P., and predoctoral fellowship to C.T.), the Spanish
Instituto de Salud Carlos III (FIS PI082031, R.C. and T.P.), the
Catalan government (2009SGR163, D.H.), Comunidad de
Madrid (S2010/BMD-2353, M.L.L.-R.), and the Spanish
Society of Medical Oncology (SEOM08, T.P.). S.O.-G. is a
Ramon y Cajal Scholar funded by MINECO and the European
Social Fund.
(13) Yang, C. S.; Wang, X.; Lu, G.; Picinich, S. C. Cancer prevention
by tea: animal studies, molecular mechanisms and human relevance.
Nature Rev. Drug Discovery 2009, 9, 429−439.
(14) Wang, X.; Tian, W. Green tea epigallocatechin gallate: a natural
inhibitor of fatty-acid synthase. Biochem. Biophys. Res. Commun. 2001,
288, 1200−1206.
(15) Brusselmans, K.; De Schrijver, E.; Heyns, W.; Verhoeven, G.;
Swinnen, J. V. Epigallocatechin-3-gallate is a potent natural inhibitor of
fatty acid synthase in intact cells and selectively induces apoptosis in
prostate cancer cells. Int. J. Cancer 2003, 106, 856−862.
(16) Vergote, D.; Cren-Olive, C.; Chopin, V.; Toillon, R. A.;
Rolando, C.; Hondermarck, H.; Le Bourhis, X. (−)-Epigallocatechin
(EGC) of green tea induces apoptosis of human breast cancer cells but
not of their normal counterparts. Breast Cancer Res. Treat. 2002, 76,
195−201.
(17) Puig, T.; Vazquez-Martin, A.; Relat, J.; Petriz, J.; Menendez, J.
A.; Porta, R.; Casals, G.; Marrero, P. F.; Haro, D.; Brunet, J.; Colomer,
R. Fatty acid metabolism in breast cancer cells: differential inhibitory
effects of epigallocatechin gallate (EGCG) and C75. Breast Cancer Res.
Treat. 2008, 109, 471−479.
ABBREVIATIONS USED
■
CPT-1, carnitine palmitoyltransferase-1; DIC, diisopropylcar-
bodiimide; DIEA, diisopropylethylamine; EDC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; EGCG, (−)-epigallocate-
chin 3-gallate; FASN, fatty acid synthase; FBS, fetal bovine
serum; HOBt, 1-hydroxybenzotriazole; ip, intraperitoneal;
MTT, 3-(4,5-dimethyl-1,3-thiazol-2-yl)-2,5-diphenyl-2H-tetra-
zol-3-ium bromide; PARP, poly(ADP-ribose) polymerase;
PBS, phosphate buffered solution; PS, polymer supported;
SDS, sodium dodecylsulphate; SEM, standard error of the
mean; TBS-T, Tris-buffered solution with 0.05% Tween 20;
TEA, triethylamine
(18) Nakagawa, K.; Miyazawa, T. Chemiluminescence-high-perform-
ance liquid chromatographic determination of tea catechin, (−)-epi-
gallocatechin 3-gallate, at picomole levels in rat and human plasma.
Anal. Biochem. 1997, 248, 41.
REFERENCES
(19) Colomer, R; Puig, T.; Brunet, J.; Lop
Benhamu, B.; Ortega-Gutierrez, S.; Turrado, C. PCT/EP2008/
058099, WO 2009/000864 A1, 2009.
(20) Puig, T.; Turrado, C.; Benhamu, B.; Aguilar, H.; Relat, J.;
́
ez-Rodríguez, M. L.;
■
́
́
(1) Abramson, H. N. The lipogenesis pathway as a cancer target. J.
Med. Chem. 2011, 54, 5615−5638.
́
(2) Kuhajda, F. P.; Jenner, K.; Wood, F. D.; Hennigar, R. A.; Jacobs,
L. B.; Dick, J. D.; Pasternack, G. R. Fatty acid synthesis: a potential
selective target for antineoplastic therapy. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 6379−6383.
(3) Liu, H.; Liu, J.-Y.; Wu, X.; Zhang, J.-T. Biochemistry, molecular
biology, and pharmacology of fatty acid synthase, an emerging
therapeutic target and diagnosis/prognosis marker. Int. J. Biochem. Mol.
Biol. 2010, 18, 69−89.
(4) Migita, T.; Ruiz, S.; Fornari, A.; Fiorentino, M.; Priolo, C.; Zadra,
G.; Inazuka, F.; Grisanzio, C.; Palescandolo, E.; Shin, E.; Fiore, C.; Xie,
W.; Kung, A. L.; Febbo, P. G.; Subramanian, A.; Mucci, L.; Ma, J.;
Signoretti, S.; Stampfer, M.; Hahn, W. C.; Finn, S.; Loda, M. Fatty acid
synthase: a metabolic enzyme and candidate oncogene in prostate
cancer. J. Natl. Cancer Inst. 2009, 101, 519−532.
(5) Omura, S. The antibiotic cerulenin, a novel tool for biochemistry
as an inhibitor of fatty acid synthesis. Bacteriol. Rev. 1976, 40, 681−
697.
Ortega-Gutier
́
rez, S.; Casals, G.; Marrero, P. F.; Urruticoechea, A.;
ez-Rodríguez, M. L.; Colomer, R. Novel inhibitors of
Haro, D.; Lop
́
fatty acid synthase with anticancer activity. Clin. Cancer Res. 2009, 15,
7608−7615.
(21) Ren, Y.; Himmeldirk, K.; Chen, X. Synthesis and structure−
activity relationship study of antidiabetic penta-O-galloyl-D-glucopyr-
anose and its analogs. J. Med. Chem. 2006, 49, 2829−2837.
(22) Nakazono, M.; Ma, L.; Zaitsu, K. Synthesis of poly(3,4,5-
trihydroxybenzoate ester)dendrimers and their chemiluminescence.
Tetrahedron Lett. 2002, 43, 8185−8189.
(23) Feldman, K. S.; Sambandam, A.; Bowers, K. E.; Appel, H. M.
Probing the role of polyphenol oxidation in mediating insect-pathogen
interactions. Galloyl-derived electrophilic traps for the Lymantria
dispar nuclear polyhedrosis virus matrix protein polyhedrin. J. Org.
Chem. 1999, 64, 5794−5803.
(24) Kundig, E. P.; Enriquez-Garcia, A. Diastereoselective and
̈
enantioselective reduction of tetralin-1,4-dione. Beilstein J. Org. Chem.
(6) Lupu, R.; Menendez, J. A. Pharmacological inhibitors of fatty acid
synthase (FASN)-catalyzed endogenous fatty acid biogenesis: a new
family of anti-cancer agents? Curr. Pharm. Biotechnol. 2006, 7, 483−
493.
2008, 4, doi:10.3762/bjoc.4.37.
(25) Fieser, L. F.; Fieser, M. The synthesis from β-naphthohy-
droquinone of a tautomer of 4-benzyl-1,2-naphthoquinone. J. Am.
Chem. Soc. 1939, 61, 596−608.
(7) Kuhajda, F. P.; Pizer, E. S.; Li, J. N.; Mani, N. S.; Frehywot, G. L.;
Townsend, C. A. Synthesis and antitumor activity of an inhibitor of
fatty acid synthase. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3450−3454.
(8) Rendina, A. R.; Cheng, D. Characterization of the inactivation of
rat fatty acid synthase by C75: inhibition of partial reactions and
protection by substrates. Biochem. J. 2005, 388, 895−903.
(9) Yang, N.; Kays, J. S.; Skillman, T. R.; Burris, L.; Seng, T. W.;
Hammond, C. C75 [4-methylene-2-octyl-5-oxo-tetrahydro-furan-3-
carboxylic acid] activates carnitine palmitoyltransferase-1 in isolated
mitochondria and intact cells without displacement of bound malonyl
CoA. J. Pharmacol. Exp. Ther. 2005, 312, 127−133.
(26) Adams, S. P.; Whitlock, H. W., Jr. Synthesis, conformation, and
complexation behavior of 2,9,18,25-tetraoxa[8.8](1,4)-
naphthalenophane. J. Org. Chem. 1971, 46, 3474−3478.
(27) Ono, K.; Yamaguchi, H.; Taga, K.; Saito, K.; Nishida, J.;
Yamashita, Y. Synthesis and properties of BF2 complexes to
dihydroxydiones of tetracene and perylene: novel electron acceptors
showing n-type semiconducting behavior. Org. Lett. 2009, 11, 149−
152.
(28) Menendez, J. A.; Vellon, L.; Mehmi, I.; Oza, B. P.; Ropero, S.;
Colomer, R.; Lupu, R. Inhibition of fatty acid synthase (FAS)
suppresses HER2/neu (erbB-2) oncogene overexpression in cancer
cells. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10715−10720.
(10) Nicot, C.; Napal, L.; Relat, J.; Gonzalez, S.; Llebaria, A.;
Woldegiorgis, G.; Marrero, P. F.; Haro, D. C75 activates malonyl-CoA
5022
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023

Journal of Medicinal Chemistry
Article
(29) Gooding, O. W.; Vo, L.; Bhattacharyya, S.; Labadie, J. W. Use of
statistical design of experiments in the optimization of amide synthesis
utilizing polystyrene-supported N-hydroxybenzotriazole resin. J. Comb.
Chem. 2002, 4, 576−583.
(30) Loftus, T. M.; Jaworsky, D. E.; Frehywot, G. L.; Townsend, C.
A.; Ronnett, G. V.; Lane, M. D.; Kuhajda, F. P. Reduced food intake
and body weight in mice treated with fatty acid synthase inhibitors.
Science 2000, 288, 2379−2381.
5023
dx.doi.org/10.1021/jm2016045 | J. Med. Chem. 2012, 55, 5013−5023