Anacardic acid derivatives as inhibitors of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Article abstract of DOI:10.1016/j.bmc.2008.08.057

Chagas' disease, a parasitic infection caused by the flagellate protozoan Trypanosoma cruzi, is a major public health problem affecting millions of individuals in Latin America. On the basis of the essential role in the life cycle of T. cruzi, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been considered an attractive target for the development of novel antitrypanosomatid agents. In the present work, we describe the inhibitory effects of a small library of natural and synthetic anacardic acid derivatives against the target enzyme. The most potent inhibitors, 6-n-pentadecyl- (1) and 6-n-dodecylsalicilic acids (10e), have IC₅₀ values of 28 and 55 μM, respectively. The inhibition was not reversed or prevented by the addition of Triton X-100, indicating that aggregate-based inhibition did not occur. In addition, detailed mechanistic characterization of the effects of these compounds on the T. cruzi GAPDH-catalyzed reaction showed clear noncompetitive inhibition with respect to both substrate and cofactor.

Full text of DOI:10.1016/j.bmc.2008.08.057

Bioorganic & Medicinal Chemistry 16 (2008) 8889–8895
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anacardic acid derivatives as inhibitors of glyceraldehyde-3-phosphate
dehydrogenase from Trypanosoma cruzi
Junia M. Pereira a, Richele P. Severino a, Paulo C. Vieira a, João B. Fernandes a, M. Fátima G.F. da Silva a,
b
a,
*
Aderson Zottis b, Adriano D. Andricopulo b, , Glaucius Oliva , Arlene G. Corrêa
*
a Departamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos, SP, Brazil
b Instituto de Física de São Carlos, Universidade de São Paulo, 13560-970 São Carlos, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Chagas’ disease, a parasitic infection caused by the flagellate protozoan Trypanosoma cruzi, is a major pub-
lic health problem affecting millions of individuals in Latin America. On the basis of the essential role in
the life cycle of T. cruzi, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has
been considered an attractive target for the development of novel antitrypanosomatid agents. In the pres-
ent work, we describe the inhibitory effects of a small library of natural and synthetic anacardic acid
derivatives against the target enzyme. The most potent inhibitors, 6-n-pentadecyl- (1) and 6-n-dodecyl-
Received 21 July 2008
Revised 25 August 2008
Accepted 26 August 2008
Available online 29 August 2008
Keywords:
salicilic acids (10e), have IC50 values of 28 and 55 lM, respectively. The inhibition was not reversed or
Anacardic acids
Trypanosoma cruzi
GAPDH
prevented by the addition of Triton X-100, indicating that aggregate-based inhibition did not occur. In
addition, detailed mechanistic characterization of the effects of these compounds on the T. cruzi GAP-
DH-catalyzed reaction showed clear noncompetitive inhibition with respect to both substrate and
cofactor.
Noncompetitive inhibition
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
leishmaniasis, respectively.5–7 On the basis of the essential role in
the life cycle of trypanosomes, GAPDH has been considered an
Chagas’ disease, caused by the protozoan parasite Trypanosoma
cruzi, is a major cause of illness, morbidity, long-term disability,
and death in Latin America.1 Currently, there are over 9 million
people infected with T. cruzi, resulting in a variety of adverse health
events such as heart failure, with more than 50,000 deaths each
year. It is thought that another 100 million people are at risk of
infection. In spite of the alarming health, economic, and social con-
sequences of this parasitic infection, the limited existing drug ther-
apy (nifurtimox and benznidazole) suffers from a combination of
drawbacks including poor efficacy, and serious side effects. There-
fore, there is an urgent need for new safe and effective therapy
against Chagas’ disease.2,3
important target for drug development. Considering that intracellu-
lar amastigotes depend on glycolysis for ATP production, the inhibi-
tion of the glycosomal GAPDH would prevent T. cruzi from being
infective.2,8 In addition, the enzyme GAPDH from the pathogenic
parasites T. cruzi,9 T. brucei,10 and Leishmania mexicana11 are closely
related (about 90% sequence identity) and possess important struc-
tural differences for drug design when compared to the homologue
protein of the mammalian host (about 45% sequence identity).
Several natural products and synthetic compounds have been
evaluated against T. cruzi GAPDH using a standard biochemical as-
say.12–15 Among these, a mixture of anacardic acids, isolated from
the Brazilian cashew-nut shell liquid, presented promising re-
sults.16 Chemically, anacardic acids feature a convenient salicylic
acid system and a long side chain at the 6-position, in which a dou-
ble bond is found at C-8 in the monoene, diene and triene compo-
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC
1.2.1.12) is a key enzyme involved in the parasite’s glycolytic
pathway. This homotetrameric enzyme catalyzes the oxidative
phosphorylation of
D
-glyceraldehyde-3-phosphate (G-3-P) to 1,3-
nents. These compounds exhibit a wide range of biological
diphosphoglycerate (1,3DPGA) in presence of NAD+ and inorganic
activities (e.g., antimicrobial, antitumoral, molluscicide, antifungal,
insecticide),17–21 stimulating the search for new derivatives with
improved properties.22–29,18 A variety of synthetic methods for
the preparation of anacardic acids,30–37 as well as for converting
these materials to other useful compounds, has been reported.37,38
As part of our research program aimed at discovering novel T. cruzi
GAPDH inhibitors and in order to identify the mode of action of
new inhibitors, we have synthesized and evaluated a small library
of anacardic acid analogues.
phosphate.4
GAPDH plays a central role in controlling ATP production in
pathogenic parasites such as T. cruzi, T. brucei, and Leishmania sp.,
the causative agents of Chagas’ disease, sleeping sickness, and
* Corresponding authors. Tel.: +55 16 33518215; fax: +55 16 33518281.
E-mail addresses: aandrico@if.sc.usp.br (A.D. Andricopulo), agcorrea@ufscar.br
(A.G. Corrêa).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.057

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J. M. Pereira et al. / Bioorg. Med. Chem. 16 (2008) 8889–8895
quenching with a large excess of ethyl chloroformate afforded 5
2. Results and discussion
that after reaction with triphenylphosphine provided the fosfoni-
um salt 6. The Wittig reaction was carried out in order to extend
the side chain at the C-6 position; thus, the phosphonium salt 6
was treated with lithium bis(trimethylsilyl)amide (LHMDS) at
ꢀ78 °C and quenched with different aldehydes to afford the prod-
ucts with the correspondent side chains (n = 4ꢀ9, R = OTHP/CH3).
The next steps were reduction of the double bound using 5%
palladium on carbon as catalyst to give 8a–e and demethylation
with AlCl3 (8a–c) or BBr3 (8d,e) to furnish 9a–e. Finally, an alkaline
hydrolysis in refluxing aqueous DMSO provided the anacardic
acids analogs. Derivative 11,40 containing a terminal aldehyde
group, was obtained through Swern oxidation of 10. The acids were
obtained in overall yields of 1.8–12.2% (6% average) from amine
intermediate 4.
In the present study, we have investigated the activity of Ana-
cardium occidentale (cashew, a tree in the flowering plant family
Anacardiaceae) extracts against T. cruzi GAPDH. Cashew nut shell
liquid is a rich source of anacardic acids, and the access to these
compounds was possible through the utilization of rotation locular
counter-current chromatography (RLCC).39 A mixture of anacardic
acids containing different degrees of unsaturation in the alkenyl
side chain was obtained, and produced about 80% GAPDH inhibi-
tion at a dose of 1 mg/mL. Next, we have reduced by hydrogenation
the double bonds of the different components of the mixture,
obtaining a single derivative, as shown in theFigure 1. This strategy
allowed the evaluation of the pure anacardic acid derivative 1,
which has an IC50 value of 28 lM, and can be considered one of
Among the synthesized anacardic acids are compounds contain-
ing 6–12 carbons in the alkyl side chain, with the presence or ab-
sence of a polar group at the end, and also substituted at the
carboxyl or methoxyl groups. Twenty-one derivatives were pre-
pared (Table 1) and then submitted to biochemical evaluation
against T. cruzi GAPDH (Table 2).
the most potent inhibitor of T. cruzi GAPDH yet described.
Based on these preliminary results, we have employed the
methodology described by Yamagiwa et al.30 in order to prepare
a series of anacardic acid derivatives starting from a common inter-
mediate (6) (Scheme 1). The reduction of m-anisaldehyde with
NaBH4 furnished alcohol 2 that was treated with SOCl2 to give
compound 3, which when reacting with dimethylamine led to
amine 4. Lithiation of this with n-BuLi in THF at 0 °C, followed by
The percentage of inhibition was calculated according to the fol-
lowing equation:
% Inhibition ¼ 100 ꢁ ð1 ꢀ Vi=V0Þ
CO2H
HO
where Vi and V0 are the initial velocities (enzyme activities) deter-
mined in the presence and in the absence of inhibitor, respectively.
Values of IC50 were independently determined by making rate mea-
surements for at least five inhibitor concentrations. The IC50 values
correspond to the concentration of compound required for 50% inhi-
bition of GAPDH, and were determined from the collected data by
nonlinear regression analysis.
1
As can be seen in Table 2, the two most potent inhibitors,
among the 21 derivatives evaluated against the parasite enzyme,
Figure 1. Chemical structure of anacardic acid derivative 1, a potent inhibitor of
T. cruzi GAPDH.
O
H
Cl
OH
NMe2
NaBH4
99%
NHMe2
94%
SOCl2
96%
OMe
OMe
OMe
OMe
m-anisaldehyde
3
4
2
1)
LHMDS
Cl
CO2Et
PPh3+Cl -
R(CH2)nCHO
( )n
CO2Et
R
n-BuLi
PPh3
82%
18-51%
ClCO2CH2CH3
42%
2) H2, Pd /C
95-98%
CO2Et
OMe
OMe
OMe
5
6
8a-c: n = 4,6,8/R = OTHP
8d,e: n = 6,9/R = CH3
O
AlCl3 (a-c)
42-64%
( )n
CO2Et
R
( )n
CO2H
R
( )6
H
NaOH
68-85%
(ClCO)2
or BBr3 (d,e)
82%
DMSO
73%
CO2H
OH
OH
OH
11
9a-c: n = 4,6,8/R = OH
9d,e: n = 6,9/R = CH3
10a-c: n = 4,6,8/R = OH
10d,e: n = 6,9/R = CH3
Scheme 1. Synthetic route for the preparation of anacardic acid derivatives.

J. M. Pereira et al. / Bioorg. Med. Chem. 16 (2008) 8889–8895
8891
Table 1
spect to both substrates. Noncompetitive inhibitors have binding
affinity for both free enzyme and the enzyme–substrate complex.
In this situation, dissociation constants must be defined for the
binary enzyme-inhibitor complex (Ki) and the ternary ESI complex
Anacardic acid analogues synthesized
R1
n-1
R1
( )n
(a
Ki). Data for the most potent T. cruzi GAPDH inhibitors 1 and 10e
are shown in the Lineweaver–Burk (or double-reciprocal) plots in
Figure 2. Ki and Ki values obtained for these noncompetitive inhib-
CO2R2
CO2R2
a
OR3
OR3
itors are in the low micromolar range (Table 3), and were deter-
mined from the collected data employing the Sigma-Plot enzyme
kinetics module. Lineweaver–Burk plots showed intercepts of all
lines (obtained at different inhibitor concentrations) in the upper
left quadrant for the cofactor NAD+ (similar plots were observed
for the substrate G-3-P). Both the slope and the y intercept of the
double-reciprocal plot were affected by the presence of the non-
competitive inhibitors. The pattern of lines seen when the plots
for varying inhibitor concentrations are overlaid depend essen-
7 a-e
8-11
Compound
n
R1
R2
R3
7a–c
7d–e
8a–c
8d–e
9a–c
9d–e
10a–c
10d–e
11
4,6,8
6,9
4,6,8
6,9
4,6,8
6,9
4,6,8
6,9
5
OTHP
CH3
OTHP
CH3
OH
CH3
OH
CH3
CHO
Et
Et
Et
Et
Et
Et
H
Me
Me
Me
Me
H
H
H
H
H
tially on the value of a. It can be seen in Figure 2 that the lines
intersected at a value of 1/[S] less than zero and a value of 1/v
greater than zero.
H
H
Therefore, when
a > 1, the inhibitor preferentially binds to the
free enzyme, as confirmed by the dissociation constants depicted
in Table 3. It is worth noting that noncompetitive inhibitors do
not compete with substrate for binding to the free enzyme, they
bind to the enzyme at a site distinct from the active site. When
the inhibitor displays finite but unequal affinity for the two en-
zyme forms, they are also sometimes called mixed inhibitors, but
this term should be avoided in inhibitors design.42
The inhibitors in the present study represent a new chemical
diversity for the target enzyme, and have been shown to act non-
competitively in the presence of both physiological substrates. Ta-
ken together our data indicate that potency and selectivity can be
achieved by inhibitors that bind differentially to the enzyme-sub-
strate complex and free enzyme.
Table 2
IC50 values for a series of synthetic anacardic acid derivatives as inhibitors of T. cruzi
GAPDH
a
Compound
IC50
(
lM)
1
28
3
7a–e
8a–e
9a–d
9e
10a
10b
10c
10d
10e
11
>300
>250
>250
215 17
>250
>250
240 22
200 17
55
5
>250
a
3. Experimental
The values represent means of at three individual experiments SD.
All commercially available reagents were purchased from Al-
drich Chemical Co. Reagents and solvents were purified when nec-
essary according to the usual procedures described in the
literature. 1H and 13C NMR spectra were recorded on a Bruker
ARX-200 (200 and 50 MHz, respectively). The IR spectra refer to
films and were measured on a Bomem M102 spectrometer. Mass
Spectra were recorded on a Shimadzu GCMS-QP5000. Analytical
are compounds 1 and 10e (with IC50 values of 28 and 55 lM,
respectively), which were then selected for further enzyme kinetic
studies. Firstly, these compounds were confirmed to be authentic
inhibitors of the target enzyme, since nonspecific inhibition has
been discarded as enzyme inhibition was not affected in all cases
in the presence of 0.01% Triton X-100 (results not shown). If aggre-
gates formed by a promiscuous compound are responsible for en-
zyme inhibition, removal of the aggregates from a solution of the
compound would decrease inhibition. Therefore, these results
eliminate possible mechanisms of inhibition through the interac-
tion of aggregates of many compounds molecules with T. cruzi
GAPDH, rather than the binding of the individual molecules. This
so called promiscuous inhibition generally involves hits from vir-
tual and high-throughput screening as well as some natural prod-
ucts and their synthetic small molecule derivatives.41
thin-layer chromatography was performed on a 0.25-lm film of
silica gel containing fluorescent indicator UV254 supported on an
aluminum sheet (Sigma–Aldrich). Flash column chromatography
was performed using silica gel (Kieselgel 60, 230–400 mesh,
E. Merck). Gas chromatography was performed in a Shimadzu
GC-17A with H2 as carrier and using a DB-5 column. Melting points
were performed in Microquímica MQAPF—301. The rotation locu-
lar counter-current chromatography was performed using
a
RLCC-100 apparatus (Tokyo Rikakikai, Tokyo, Japan), which con-
sisted of 16 columns (45 cm ꢁ 11 mm ID) divided by centrally per-
forated PFTE disks into 37 loculi each. Injections were made
manually by filling the RLCC loop volume (3 mL). The flow-rate
was 90 mL/h, the rotation was 60–70 rpm and the slope 40o. The
experiment was carried out at 22 °C.
To explore the mechanism of inhibition in more detail, we have
determined Ki values and the type of inhibition with respect to the
physiological substrates G-3-P and NAD+, employing the two com-
pounds (1 and 10e) with the lowest IC50 values for T. cruzi GAPDH
(Table 3 and Fig. 2).
The results shown in Table 3 and Figure 2 indicate that the inhi-
bition of T. cruzi GAPDH was found to be noncompetitive with re-
3.1. Isolation of ancardic acids mixture
Table 3
Values Ki and
a
Ki for the noncompetitive inhibitors of T. cruzi GAPDH.
NAD+
G-3-P
The cashew-nut shell (400 g) was extracted with hexane fol-
lowed by dichloromethane and methanol (1 L, three times for each
solvent, for three days). The dichloromethane extract (32.8 g) was
analyzed by 1H NMR and showed to be rich in anacardic acids,
cardols and cardanols. This extract was fractionated by RLCC. The
system of phases used was hexane/methanol/water (10:9:1). The
Inhibitor
Ki (
lM)
a
Ki (l
M)
Ki (lM)
a
Ki (lM)
1
10e
4
5
6
43
2
4
4
38

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J. M. Pereira et al. / Bioorg. Med. Chem. 16 (2008) 8889–8895
stirring for 2 h the mixture was quenched with water and ex-
A
1000
800
600
400
200
0
tracted with EtOAc. The organic layer was dried with Na2SO4 and
evaporated to yield an oil. The crude oil was purified by flash chro-
matography (SiO2, 6:4 hexane/EtOAc as eluent) to afford 2 (1.89 g,
[1] - 0.0 µM
[1] - 42.5 µM
[1] - 45.0 µM
[1] - 47.5 µM
[1] - 50.0 µM
99%) as a pure yellow oil. IR (m
max/cmꢀ1): 3429, 1596, 1035, 858,
784. 1H NMR (200 MHz, CDCl3): d 7.23 (t, 1H, J 8.0 Hz); 6.90–6.77
(m, 3H); 4.58 (s, 2H); 3.76 (s, 3H); 2.65 (br s, 1H). MS (m/z): 138
(M+) (base peak), 121, 109, 105, 79, 77 and 51.
3.3. 1-Chloromethyl-3-methoxybenzene (3)
To a mixture of 2 (1.50 g, 0.011 mol) and pyridine (0.44 mL,
5.5 mmol) in benzene (9 mL) was added thionyl chloride
(5.62 mL, 0.077 mol) dropwise at 0 °C. After stirring for 2 h the
mixture was quenched with water and saturated sodium bicarbon-
ate solution and extracted with EtOAc. The combined organic
phase was washed with brine, dried (Na2SO4) and evaporated to
yield an oil. The crude oil was purified by flash chromatography
(SiO2, 8:2 hexane/EtOAc as eluent) to afford 3 (1.16 g, 96%) as a
-20
-10
0
10
20
30
pure yellow oil. IR (m
max/cmꢀ1): 2960, 2837, 1602, 1490, 1274,
711. 1H NMR (200 MHz, CDCl3): d 7.27 (t, J 8.0 Hz, 1H); 6.99–6.92
(m, 2H); 6.85 (ddd, 1H, J 1.0, 2.5, 8.0 Hz); 4.56 (s, 2H); 3.81 (s,
3H). MS (m/z): 158 (M+2), 156 (M+), 121 (base peak), 106, 92, 91,
77, 51.
B
400
300
200
100
0
[10e] - 0.0 µM
[10e] - 5.0 µM
[10e] - 6.0 µM
[10e] - 7.5 µM
3.4. 3-Methoxy-N,N-dimethylbenzylamine (4)
To a solution of 3 (2.5 g, 0.016 mol) in CH2Cl2 (12.5 mL) was
added dimethylamine (12.5 mL, 0.24 mol) at 0 °C. The mixture
was stirred for 24 h at room temperature and then quenched with
water and extracted with CH2Cl2. The organic layer was dried
(Na2SO4) and evaporated to yield an oil. Distillation gave 4 (2.5 g,
94%) as a pure yellow oil, bp 43–49 °C/0.12 Torr. IR (m
max/cmꢀ1):
2943, 2775, 1602, 1488, 1269, 1043, 783. 1H NMR (200 MHz,
CDCl3): d 7.22 (t, 1H, J 8.0 Hz); 6.90-6.87 (m, 2H); 6.80 (ddd, 1H,
J 1.0, 2.5, 8.0 Hz); 3.80 (s, 3H); 3.39 (s, 2H); 2.24 (s, 6H). MS (m/
z): 165 (M+), 136, 122, 107, 92, 91, 65, 77, 58 (base peak).
-20
-10
0
10
20
30
3.5. Ethyl 6-chloromethyl-2-methoxybenzoate (5)
To a stirred solution of 4 (1.0 g, 6.5 mmol) in dry THF (20 mL)
was added n-butyllithium (0.91 M hexane solution, 9.3 mL,
8.5 mmol) under nitrogen atmosphere at 0 °C. After the solution
have been stirred at room temperature for 1 h, ethyl chloroformate
(4.25 mL, 34.5 mmol) was added at ꢀ78 °C and the mixture was
stirred at the same temperature for 1 h and was allowed to stand
at room temperature overnight. The mixture was quenched with
water, extracted with CH2Cl2 and the extract was concentrated to
yield an oil. After purification by distillation of byproducts and
flash chromatography (SiO2, 8:1:1 hexane/EtOAc/CH2Cl2 as eluent)
the pure product 5 was obtained (0.58 g, 42%) as a pale yellow oil,
Figure 2. Lineweaver-Burk plots showing that compounds
1 (A) and 10e (B)
inhibits T. cruzi GAPDH noncompetitively with respect to NAD+.
stationary phase was hexane and the mobile phase was methanol/
water. A solution of dichloromethane extract (1.0 g) in system
phases (3 mL) was injected and eluted in the descending mode
with the lipophilic phase. The eluate containing anacardic acids
was divided into five fractions. The aqueous extracts were evapo-
rated to dryness and then each fraction was analyzed by 1H
bp 105–112 °C/0.55 Torr. IR (m
max/cmꢀ1): 2979, 1820, 1735, 1473,
NMR. The fraction containing
a
mixture of anacardic acids
1272, 732. 1H NMR (200 MHz, CDCl3) d: 7.36 (t, 1H, J 8.0 Hz);
7.03 (d, 1H, J 8.0 Hz); 6.92 (d, 1H, J 8.0 Hz); 4.61 (s, 2H); 4.44 (q,
2H, J 8.0 Hz); 3.85 (s, 3H); 1.40 (t, 3H, J 8.0 Hz). MS (m/z): 228
(M+), 193, 185, 184, 183, 182 (base peak), 147, 118, 105, 77, 51.
(200 mg) was hydrogenated using H2 and Pd/C (10%) in methanol
during 36 h. After that, the solution was filtrated in Celite and sat-
urated anacardic acid 1 was obtained. IR (m
max/cmꢀ1): 3175, 1630,
1680. 1H NMR (200 MHz, CDCl3): d 11.05 (s, 1H); 7.35 (t, 1H , J
8.0 Hz); 6.86 (dd, 1H, J 0.9, 8.0 Hz); 6.77 (dd, 1H, J 0.9, 8.0 Hz);
2.97 (d, 2H, J 8.0 Hz); 1.20–1.60 (m, 6H); 0.88 (t, 3 H, J 6.0 Hz).
13C NMR (50 MHz, CDCl3): d 176.0; 163.7; 147.9; 135.4; 122.9;
116.0; 110.8; 36.6; 32.2; 29.7; 22.8; 14.3.
3.6. 2-Ethoxycarbonyl-3-methoxybenzyltriphenylphosphonium
chloride (6)
A solution of 5 (1.43 g, 6.3 mmol) and triphenylphosphine
(2.14 g, 6.3 mmol) in dry acetonitrile (10 mL) was kept under re-
flux for 72 h. The solvent was removed and the residue was tritu-
rated with dry ether, filtered and evaporated under vacuum to
afford 6 (2.52 g, 82%) as a white powder, mp 209–213 °C. IR (nujol,
3.2. (3-Methoxyphenyl)-methanol (2)
To a solution of m-anisaldeyde (2.03 g, 0.015 mol) in dry meth-
anol (10 mL) was added NaBH4 (0.14 g, 3.75 mmol) at 0 °C. After
m
max/cmꢀ1): 542, 798, 1284, 1439, 1712. 1H NMR (200 MHz, CDCl3)

J. M. Pereira et al. / Bioorg. Med. Chem. 16 (2008) 8889–8895
8893
d: 7.83–7.71 (m, 3H); 7.65–7.55 (m, 12H); 7.26 (t, 1H, J 8.0 Hz);
7.00 (dd, 1H, J 2.0, 8.0 Hz); 6,91 (dd, 1H, J 2.0, 8.0 Hz); 5.41 (d,
2H, J 14.0 Hz); 4.06 (q, 2H, J 8.0 Hz); 3.77 (s, 3H); 1,20 (t, 3 H, J
8.0 Hz).
(m, 1H); 6.20 (dt, 1H, J 6.2, 15.6 Hz); 4.40 (q, 2 H, J 7.1 Hz); 3.80
(s, 3H); 2.16 (qui, 2H, J 6.2 Hz); 1.37 (t, 3H, J 7.1 Hz); 1.50–1.15
(m, 10H); 0.93–0.83 (m, 3H). Z isomer: 7.24 (t, 1H, J 8.0 Hz);
6.82–6.73 (m, 2H); 6.40–6.30 (m, 1H); 5.71 (dt, 1H,
J 7.4,
11.5 Hz); 4.35 (q, 2 H, J 7.1 Hz); 3.80 (s, 3H); 2.16 (qui, 2H, J
7.4 Hz); 1.34 (t, 3H, J 7.1 Hz); 1.50–1.15 (m, 10H); 0.93–0.83 (m,
3H). 13C NMR (50 MHz, CDCl3) d: E isomer: 168.1; 156.3; 136.5;
134.5; 130.0; 126.0; 121.5; 117.7; 109.2; 61.1; 55.9; 33.1; 31.8;
29.6; 29.1; 28.5; 22.6; 14.2; 14.0. MS (m/z): 304 (M+), 174 (base
peak), 132, 115, 55, 43, 41.
3.6.1. Compounds 7a–e
To a suspension of the phosphonium salt 6 (500 mg, 1.02 mmol)
in dry THF (3.06 mL) was added dropwise lithium bis(trimethyl-
silyl)amide (1 M hexane solution, 1.53 mL, 1.53 mmol) at ꢀ78 °C
under nitrogen atmosphere. After the solution has been stirred at
ꢀ78 °C for 30 min, a solution of the aldehyde (1.22 mmol) in dry
THF (3.06 mL) was added dropwise and then allowed to warm to
room temperature. After removal of the solvent, the residue was
diluted with hexane and the resulting precipitates were filtered.
The filtrate was concentrated and after purification by distillation
and chromatography on silica gel (95:5 then 1:1 hexane/EtOAc as
eluent) the pure products 7a–e were obtained as a pale yellow oil.
Compound 7e. (110 mg; 31% yield). IR (m
max/cmꢀ1): 2954, 2923,
2854, 1731, 1577, 1469, 1267, 1108, 1066, 730. 1H NMR (200 MHz,
CDCl3) d: E isomer: 7.26 (t, 1H, J 8.0 Hz); 7.10 (d, 1H, J 8.0 Hz); 6.86
(d, 1H. J 8,0 Hz); 6.39–6.30 (m, 1H); 6.19 (dt, 1H, J 6.2, 15.6 Hz);
4.40 (q, 2 H, J 7.1 Hz); 3.82 (s, 3H); 2.16 (qui, 2H, J 6.2 Hz); 1.38
(t, 3H, J 7.1 Hz); 1.39–1.24 (m, 18H); 0.91–0.85 (m, 3H). Z isomer:
7.29 (t, 1H, J 8.0 Hz); 6.83–6.75 (m, 2H); 6.39–6.30 (m, 1H); 5.71
(dt, 1H, J 7.4, 11.5 Hz); 4.35 (q, 2 H, J 7.1 Hz); 3.83 (s, 3H); 2.16
(qui, 2H, J 7.4 Hz); 2.30–2.10 (m, 2H); 1.34 (t, 3H, J 7.1 Hz); 1.39–
1.24 (m, 18H); 0.91–0.85 (m, 3H). 13C NMR (50 MHz, CDCl3) d: E
isomer: 168.1; 156.3; 136.5; 134.5; 130.0; 126.0; 121.5; 117.7;
109.2; 61.1; 55.9; 33.1; 31.9; 29.64; 29.55; 29.3; 29.2; 29.1;
28.5; 22.6; 14.2; 14.0. MS (m/z): 346 (M+), 205, 174 (base peak),
147, 115, 55.
Compound 7a. (65 mg; 18% yield). IR (m
max/cmꢀ1): 2939, 2867,
1731, 1575, 1471, 1365, 1267, 1076, 1022, 869, 736. 1H NMR
(200 MHz, CDCl3) d: E isomer: 7.27 (t, 1H, J 8.0 Hz); 7.10 (d, 1H, J
8.0 Hz); 6.86 (d, 1H, J 8.0 Hz); 6.41–6.31 (m, 1H); 6.20 (dt, 1 H, J
6.2, 15.6 Hz); 4.59–4.53 (m, 1H); 4.41 (q, 2 H, J 7.1 Hz); 3.82 (s,
3H); 3.78–3.62 (m, 2 H); 3.55–3.32 (m, 2 H); 2.24–2.17 (m, 2H);
1.75–1.47 (m, 12H); 1.38 (t, 3H, J 7.1 Hz). Z isomer: 7.29 (t, 1H, J
8.0 Hz); 6.80–6.75 (m, 2H); 6.41–6.31 (m, 1H); 5.72 (dt, 1H, J 7.4,
11.5 Hz); 4.59-4.53 (m, 1H); 4.36 (q, 2 H, J 7.1 Hz); 3.83 (s, 3H);
3.78–3.62 (m, 2H); 3.55–3.32 (m, 2H); 2.24–2.17 (m, 2H); 1.75–
1.47 (m, 12H); 1.34 (t, 3H, J 7.1 Hz). 13C NMR (50 MHz, CDCl3) d:
E isomer: 168.1; 156.3; 136.4; 134.1; 130.0; 126.4; 121.5; 117.7;
109.3; 98.8; 67.3; 62.2; 61.1; 55.9; 32.9; 30.7; 29.2; 25.8; 25.5;
19.6; 14.2. MS (m/z): 278, 232, 187, 115, 85 (base peak), 57.
3.6.2. Compounds 8a–e
A mixture of the olefin 7a–e (0.25 mmol) and 5% Pd/C (5.2 mg;
0.025 mmol) in hexane (5.25 mL) was stirred for 2 h under hydro-
gen atmosphere. The catalyst was filtered through a short column
of Celite and the filtered was concentrated to afford pure 8a-e as a
pale yellow oil.
Compound 7b. (143 mg; 36% yield). IR (
m
max/cmꢀ1): 2935, 2856,
Compound 8a. (90.3 mg; 98% yield). IR (m
max/cmꢀ1): 2937, 2864,
1731, 1585, 1577, 1471, 1267, 1110, 1068, 1033, 811. 1H NMR
(200 MHz, CDCl3) d: E isomer: 7.27 (t, 1H, J 8.0 Hz); 7.09 (d, 1H, J
8.0 Hz); 6.86 (d, 1H, J 8.0 Hz); 6.40–6.30 (m, 1H); 6.19 (dt, 1H, J
6.0, 15.6 Hz); 4.59–4.56 (m, 1H), 4.40 (q, 2 H, J 7.1 Hz); 3.82 (s,
3H); 3.77–3.65 (m, 2H); 3.54–3.33 (m, 2H); 2.20–2.13 (m, 2H);
1.75–1.46 (m, 11H); 1.38 (t, 3H, J 7.1 Hz), 1.42–1.30 (m, 3H). Z iso-
mer: 7.29 (t, 1H, J 8.0 Hz); 6.79–6.75 (m, 2H); 6.40–6.30 (m, 1H);
5.71 (dt, 1H, J 7.4, 11.5 Hz); 4.59–4.56 (m, 1H); 4.35 (q, 2 H, J
7.1 Hz); 3.83 (s, 3H); 3.77–3.65 (m, 2H); 3.54–3.33 (m, 2H);
2.20–2.13 (m, 2H); 1.75–1.46 (m, 11H); 1.34 (t, 3H, J 7.1 Hz);
1.42–1.30 (m, 3H). 13C NMR (50 MHz, CDCl3) d: E isomer: 168.1;
156.4; 136.5; 134.4; 130.0; 126.1; 121.5; 117.7; 109.2; 98.8;
67.6; 62.3; 61.1; 55.9; 33.1; 30.8; 29.7; 29.6; 29.1; 26.1; 25.5;
19.7; 14.3. MS (m/z): 260, 187, 174, 115, 85, 55 (base peak).
1731, 1583, 1471, 1365, 1267, 1118, 1074, 1024, 869. 1H NMR (200
MHz, CDCl3) d: 7.25 (t, 1H, J 8.0 Hz); 6.80 (d, 1H, J 8.0 Hz); 6.75 (d,
1H, J 8.0 Hz); 4.60–4.52 (m, 1H); 4.38 (q, 2H, J 7.1 Hz); 3.81 (s, 3H);
3.74–3.66 (m, 1H); 3.51–3.34 (m, 3H); 2.56 (t, 2H, J 7.6 Hz); 1.69–
1.53 (m, 10H); 1.37 (t, 3H, J 7.1 Hz); 1.40–1.33 (m, 4H). 13C NMR
(50 MHz, CDCl3) d:168.3; 156.2; 141.1; 130.0; 123.8; 121.4;
108.4; 98.8; 67.6; 62.2; 61.0; 55.8; 33.3; 31.1; 30.7; 29.6; 29.3;
26.0; 25.4; 19.6; 14.2. MS (m/z): 280, 234, 161, 121, 85 (base peak),
55.
Compound 8b. (94 mg; 97% yield). IR (m
max/cmꢀ1): 2935, 2858,
2360, 2337, 1731, 1585, 1469, 1268, 1118, 1076, 1033. 1H NMR
(200 MHz, CDCl3) d: 7.26 (t, 1H, J 8.0 Hz); 6.81 (d, 1H, J 8.0); 6.75
(d, 1H, J 8.0 Hz); 4.59–4.55 (m, 1H); 4.39 (q, 2H, J 7.1 Hz); 3.81
(s, 3H); 3.78–3.66 (m, 2H); 3.55–3.31 (m, 2H); 2.55 (t, 2H, J
7.6 Hz); 1.37 (t, 3H, J 7.1 Hz); 1.49–1.23 (m, 18H). 13C NMR (50
MHz, CDCl3) d: 168.3; 156.2; 141.1; 130.0; 123.8; 121.4; 108.4;
98.8; 67.6; 62.3; 61.0; 55.8; 33.4; 31.2; 30.8; 29.7; 29.5;
29.3(2C); 26.2; 25.5; 19.6; 14.2. MS (m/z): 392, 262,194,175,161
(base peak), 121, 85, 55.
Compound 7c. (105 mg; 24% yield). IR (m
max/cmꢀ1): 2929, 2854,
1731, 1575, 1471, 1268, 1110, 1068, 1031, 810, 734. 1H NMR (200
MHz, CDCl3) d: E isomer: 7.26 (t, 1H, J 8.0 Hz); 7.09 (d, 1H, J 8.0 Hz);
6.85 (d, 1H, J 8.0 Hz); 6.39–6.30 (m, 1H); 6.19 (dt, 1H, J 6.2,
15.6 Hz); 4.60–4.53 (m, 1H), 4.40 (q, 2 H, J 7.1 Hz); 3.81 (s, 3H);
3.75–3.66 (m, 2H); 3.54–3.31 (m, 2H); 2.16 (qui, 2H, J 6.2 Hz);
1.78–1.47 (m, 8H); 1.38 (t, 3H, J 7.1 Hz); 1.41–1.22 (m, 10H). Z iso-
mer: 7.29 (t, 1H, J 8.0 Hz); 6.79–6.74 (m, 2H); 6.39–6.30 (m, 1H);
5.71 (dt, 1 H, J 7.4, 11.5 Hz); 4.60–4.53 (m, 1H); 4.35 (q, 2 H, J
7.1 Hz); 3.82 (s, 3H); 3.75–3.66 (m, 2H); 3.54–3.31 (m, 2H); 2.16
(qui, 2H, J 7.4 Hz); 1.78–1.47 (m, 8H); 1.34 (t, 3H, J 7.1 Hz), 1.41–
1.22 (m, 10H). 13C NMR (50 MHz, CDCl3) d: E isomer: 168.1;
156.3; 136.5; 135.1; 130.0; 126.1; 121.5; 117.7; 109.2; 98.8;
67.6; 62.3; 61.1; 55.9; 33.1; 30.8; 29.7; 29.4 (2C); 29.1; 28.5;
26.2; 25.5; 19.6; 14.2. MS (m/z): 334, 288, 187, 174 (base peak),
115, 91, 55.
Compound 8c. (103 mg; 98% yield). IR (m
max/cmꢀ1): 2927, 2854,
1731, 1583, 1469, 1267, 1117, 1074, 1033, 763. 1H NMR (200 MHz,
CDCl3) d: 7.25 (t, 1H, J 8.0 Hz); 6.80 (d, 1H, J 8.0 Hz); 6.75 (d, 1H, J
8.0 Hz); 4.59–4.55 (m, 1H); 4.39 (q, 2H, J 7.1 Hz); 3.80 (s, 3H);
3.75–3.58 (m, 2H); 3.54–3.35 (m, 2H); 2.55 (t, 2H, J 7.6 Hz);
1.65–1.47 (m, 9H); 1.37 (t, 3H, J 7.1 Hz); 1.42–1.24 (m, 13H). 13C
NMR (50 MHz, CDCl3) d: 168.3; 156.2; 141.1; 130.0; 123.8;
121.4; 108.4; 98.8; 67.6; 62.2; 60.9; 55.8; 33.4; 31.1; 30.7; 29.7;
29.5 (2C); 29.39 (2C); 29.36; 26.2; 25.5; 19.6; 14.2. MS (m/z):
334, 288, 187, 174 (base peak), 115, 67, 55.
Compound 8d. (72 mg; 94% yield). IR (m
max/cmꢀ1): 2954, 2925,
Compound 7d. (160 mg; 51% yield). IR (
2854, 1733, 1583, 1469, 1365, 1267, 1107, 1072, 721 and
541 cmꢀ1 1H NMR (200 MHz, CDCl3) d: E isomer: 7.25 (t, 1H, J
8.0 Hz); 7.09 (d, 1H, J 8.0 Hz); 6.86 (d, 1 H, J 8.0 Hz); 6.40–6.30
m
max/cmꢀ1): 2952, 2925,
2854, 1731, 1583, 1469, 1267, 1107, 1074, 748. 1H NMR (200
MHz, CDCl3) d: 7.23 (t, 1H, J 8.0 Hz); 6.80 (d, 1H, J 8.0 Hz); 6.73
(d, 1H, J 8.0 Hz); 4.38 (q, 2H, J 7.1 Hz); 3.78 (s, 3H); 2.55 (t, 2H, J
7.6 Hz); 1.64–1.55 (m, 2H); 1.36 (t, 3H, J 7.1 Hz); 1.40–1.20 (m,
.

8894
J. M. Pereira et al. / Bioorg. Med. Chem. 16 (2008) 8889–8895
12H); 0.92–0.84 (m, 3H). 13C NMR (50 MHz, CDCl3); d: 168.2;
156.1; 141.0; 129.9; 123.8; 121.3; 108.3; 60.8; 55.7; 33.3; 31.7;
31.1; 29.41; 29.36; 29.3; 29.1; 22.5; 14.1; 13.9. MS (m/z): 306
(M+), 261, 194, 161 (base peak), 121, 91, 77, 55.
Compound 9e. (33.7 mg; 83% yield). IR (m
max/cmꢀ1): 2923, 2853,
1663, 1607, 1576, 1448, 1372, 1312, 1249, 1212, 1119, 1020, 816.
1H NMR (200 MHz, CDCl3) d: 7.28 (t, 1H, J 7.9 Hz); 6.83 (dd, 1H, J
1.2, 8.3 Hz); 6.71 (dd, 1H, J 1.2, 7.5 Hz); 4.43 (q, 2H, J 7.1 Hz);
2.92–2.88 (m, 2 H); 1.58–1.51 (m, 2H); 1.43 (t, 3H, J 7.1 Hz);
1.33–1.22 (m, 18H); 0.88 (t, 3H, J 6.8 Hz). 13C NMR (50 MHz, CDCl3)
Compound 8e. (83.1 mg; 95% yield). IR (m
max/cmꢀ1): 2923, 2852,
1731, 1585, 1469, 1267, 1107, 1074, 746. 1H NMR (200 MHz,
CDCl3) d: 7.25 (t, 1H, J 8.0 Hz); 6.81 (d, 1H, J 8.0 Hz); 6.75 (d, 1H,
J 8.0 Hz); 4.39 (q, 2H, J 7.1 Hz); 3.81 (s, 3H); 2.55 (t, 2H, J 7.6 Hz);
1.65–1.55 (m, 2H); 1.37 (t, 3H, J 7.1 Hz); 1.40–1.22 (m, 20H);
0.91–0.84 (m, 3H). 13C NMR (50 MHz, CDCl3) d: 168.4; 156.2;
141.2; 130.0; 123.9; 121.5; 108.4; 61.0; 55.8; 33.4; 31.9; 31.2;
29.62; 29.54; 29.5 (2C); 29.4; 29.3; 22.6; 14.2; 14.0. MS (m/z):
348 (M+); 303; 175; 161 (base peak); 147; 121; 55.
d: 171.6; 162.7; 146.1; 134.0; 122.4; 115.6; 112.0; 61.6; 36.7; 32.3;
31.9; 29.9; 29.7 (3C); 29.6 (3C); 29.3; 22.7; 14.1. MS (m/z): 334
(M+), 288, 262, 288, 180, 161, 147 (base peak), 134, 105, 91, 77, 55.
3.6.5. Compounds 10a–e
A
solution of 9a–e (0.10 mmol) and 20% NaOH solution
(0.20 mL) in DMSO (0.20 mL) was kept under reflux at 120 °C for
5 h. The mixture was acidified with 10% HCl and extracted with
EtOAc. The organic layer was washed with water, dried (Na2SO4)
and evaporated to give a pale yellow oil. The crude oil was chro-
matographed on silica gel (9:1:0.2 hexane/EtOAc/acetic acid as elu-
ent) to afford the anacardic acids and analogues as white powders.
3.6.3. Compounds 9a–c
To a solution of 8a–c (0.25 mmol) in dry CH2Cl2 (10 mL) was
added AlCl3 (1.30 g; 9.7 mmol) under nitrogen atmosphere. After
stirring for 14 h at room temperature the mixture was
quenched with water and extracted with EtOAc. The organic
layer was dried (Na2SO4) and evaporated to give 9a–c as a pale
yellow oil.
Compound 10a. (16 mg; 68% yield). mp 78–79 °C; IR (mmax
/
cmꢀ1): 3421, 2933, 2858, 1718, 1654, 1602, 1452, 1315, 1247,
1216, 1020, 709. 1H NMR (200 MHz, CDCl3) d: 7.22 (t, 1H, J
7.8 Hz); 6.73–6.70 (m, 2H); 5.48 (s, 1H); 3.53 (t, 2H, J 6.6 Hz);
2.92–2.88 (m, 2H); 1.62–1.49 (m, 5H); 1.39–1.36 (m, 3H). 13C
NMR (50 MHz, CDCl3) d: 174.2; 162.2; 146.7; 133.9; 122.8;
115.7; 115.6; 63.0; 36.6; 33.1; 33.2; 30.6; 26.7. MS (m/z): 220,
194, 147, 120, 108 (base peak), 91, 77, 55.
3.6.4. Compounds 9d–e
To a solution of 8d–e (0.12 mmol) in dry CH2Cl2 (0.03 mL) was
added BBr3 (1 M solution in CH2Cl2, 5.2
l
L) dropwise at ꢀ40 °C un-
der nitrogen atmosphere. After stirring for 2 h at room tempera-
ture the mixture was poured into ice water and extracted with
CH2Cl2. The organic layer was dried (Na2SO4) and evaporated to
give 9d-e as a pale yellow oil.
Compound 10b. (18.1 mg; 72% yield). mp 79–81 °C; IR (mmax
/
cmꢀ1): 3467, 2923, 2850, 2534, 1708, 1662, 1604, 1454, 1261,
1214, 1091, 802, 732. 1H NMR (200 MHz, CDCl3) d: 7.09 (dd, 1H,
J 7.6, 8.3 Hz); 6.57 (ddd, 2H, J 1.2, 7.6, 8.3 Hz); 3.39 (t, 2H, J
6.6 Hz); 2.75–2.71 (m, 2H); 1.46–1.34 (m, 4H); 1.27–1.13 (m,
8H). 13C NMR (50 MHz, CDCl3) d: 174.3; 162.2; 146.7; 134.0;
122.9; 115.7; 115.6; 62.9; 36.7; 33.6; 33.2; 30.8; 30.5 (2C); 26.9.
MS (m/z): 222, 147, 120, 108 (base peak), 77, 55.
Compound 9a. (26 mg; 42% yield). IR (m
max/cmꢀ1): 3406, 2933,
2858, 1728, 1654, 1606, 1450, 1373, 1249, 1213, 1058, 1027,
709. 1H NMR (200 MHz, CDCl3) d: 11.22 (s, 1H); 7.28 (t, 1H, J
7.6 Hz); 6.83 (dd, 1H, J 1.2, 8.3 Hz); 6.71 (dd, 1H, J 1.2, 7.5 Hz);
4.43 (q, 2H, J 7.1 Hz); 3.64 (t, 2 H, J 6.4 Hz); 2.95–2.87 (m, 2H);
1.60–1.54 (m, 5H); 1.43 (t, 3H, J 7.1 Hz); 1.48–1.35 (m, 3H). 13C
NMR (50 MHz, CDCl3) d: 171.5; 162.6; 145.9; 134.0; 122.4;
115.6; 112.0; 62.9; 61.6; 36.5; 32.7; 32.1; 29.6; 25.7; 14.1. MS
(m/z): 266 (M+), 220, 147, 134 (base peak), 105, 91, 77, 55.
Compound 10c. (22 mg; 77% yield). mp 80–82 °C; IR (mmax
/
cmꢀ1): 3474, 3420, 2919, 2849, 1652, 1609, 1448, 1325, 1247,
1215, 736. 1H NMR (200 MHz, CDCl3) d: 7.23 (t, 1H, J 7.8 Hz);
7.74–6.69 (m, 2H); 3.52 (t, 2H, J 6.6 Hz); 2.88–2.85 (m, 2H);
1.59–1.48 (m, 4H); 1.39–1.25 (m, 12H). 13C NMR (50 MHz, CDCl3)
d: 174.3; 162.2; 146.7; 134.1; 122.9; 115.7; 115.6; 63.0; 36.7;
33.6; 33.2; 30.8; 30.7; 30.6 (2C); 30.5; 26.9. MS (m/z): 276, 250,
161, 147, 120, 108 (base peak), 77, 55.
Compound 9b. (47 mg; 64% yield). IR (
m
max/cmꢀ1): 3444, 2927,
2854, 1658, 1608, 1450, 1249, 1064, 813 cmꢀ1
.
1H NMR (200
MHz, CDCl3) d: 11.22 (s, 1H); 7.28 (dd, 1H, J 7.6, 8.3 Hz); 6.83
(dd, 1H, J 1.2, 8.3 Hz); 6.71 (dd, 1H, J 1.2, 7.6 Hz); 4.44 (q, 2H, J
7.1 Hz); 3.64 (t, 2 H, J 6.6 Hz); 2.92–2.88 (m, 2H); 1.58–1.51 (m,
4H); 1.43 (t, 3H, J 7.1 Hz); 1.37–1.33 (m, 8H). 13C NMR (50 MHz,
CDCl3) d: 171.5; 162.6; 146.1; 134.0; 122.4; 115.6; 112.0; 63.0;
61.6; 36.7; 32.7; 32.2; 29.8; 29.5; 29.4; 25.7; 14.1. MS (m/z): 294
(M+), 248, 197, 152, 147 (base peak), 134, 105, 77, 55.
Compound 10d. (20 mg; 85% yield). mp 78–79 °C; IR (mmax
/
cmꢀ1): 3429, 3056, 2923, 2854, 2596, 1652, 1606, 1450, 1301,
1245, 1211, 707. 1H NMR (200 MHz, CDCl3) d: 11.09 (br s, 1H);
7.34 (t, 1H, J 7.9 Hz); 6.85 (d, 1H, J 8.3 Hz); 6.76 (d, 1H, J 7.5 Hz);
3.01–2.93 (m, 2H); 1.65–1.53 (m, 2H); 1.40–1.10 (m, 14H); 0.93–
0.80 (m, 3H). 13C NMR (50 MHz, CDCl3) d: 175.3; 163.5; 147.6;
135.0; 122.6; 115.7; 110.7; 36.4; 32.0; 31.9; 29.8; 29.6; 29.5;
29.3; 22.6; 14.0. MS (m/z): 220, 121, 108 (base peak), 77, 55.
Compound 9c. (32 mg; 42% yield). IR (
2852, 1726, 1654, 1606, 1450, 1373, 1249, 1213, 1103, 1058,
1022, 817, 709 cmꢀ1 1H NMR (200 MHz, CDCl3) d: 11.23 (s, 1H);
m
max/cmꢀ1): 3402, 2923,
.
7.28 (t, 1H, J 7.6 Hz); 6.83 (dd, 1H, J 1.2, 8.3 Hz); 6.71 (dd, 1H, J
1.2, 7.6 Hz); 4.44 (q, 2H, J 7.1 Hz); 3.64 (t, 2 H, J 6.6 Hz); 2.92–
2.88 (m, 2H); 1.57–1.53 (m, 4H); 1.43 (t, 3H, J 7.1 Hz); 1.36–1.29
(m, 12H). 13C NMR (50 MHz, CDCl3) d: 171.6; 162.6; 146.1;
134.0; 122.5; 115.6; 112.0; 63.0; 61.6; 36.7; 32.8; 32.2; 29.9;
29.6 (2C); 29.5; 29.4; 25.7; 14.1. MS (m/z): 322 (M+), 276, 161,
147 (base peak), 134, 105, 55.
Compound 10e. (24 mg; 85% yield). mp 90–92 °C; IR (mmax/
cmꢀ1): 3453. 3415, 3084, 3055, 2916, 2848, 1652, 1603, 1445,
1307, 1247, 1215, 896, 732. 1H NMR (200 MHz, CDCl3) d: 7.23 (t,
1H, J 7.8 Hz); 6.73–6.69 (m, 2H); 2.89–2.85 (m, 2H); 1.59–1.52
(m, 2H); 1.38–1.24 (m, 18H); 0.88 (t, 3H, J 6.9 Hz). 13C NMR
(50 MHz, CDCl3) d: 174.3; 162.2; 146.7; 134.0; 122.9; 115.7;
115.6; 36.7; 33.2; 33.0; 30.9; 30.8; 30.7 (3C); 30.5; 30.4; 23.7;
14.4. MS (m/z): 262, 149, 121, 108 (base peak), 77, 55.
Compound 9d. (28.3 mg; 82% yield). IR (
m
max/cmꢀ1): 2954, 2925,
2854, 1660, 1606, 1448, 1373, 1311, 1249, 1211, 1118, 709 cmꢀ1
.
1H NMR (200 MHz, CDCl3) d: 11.23 (s, 1H); 7.28 (t, 1H, J 7.9 Hz);
6.83 (dd, 1H, J 1.2, 8.3 Hz); 6.71 (dd, 1H, J 1.2, 7.5 Hz); 4.43 (q,
2H, J 7.1 Hz); 2.94–2.86 (m, 2 H); 1.43 (t, 3H, J 7.1 Hz); 1.48–1.23
(m, 14H); 0.91–0.85 (m, 3H). 13C NMR (50 MHz, CDCl3) d: 171.5;
162.6; 146.2; 134.0; 122.4; 115.6; 112.0; 61.6; 36.7; 32.3; 31.9;
29.9; 29.6; 29.5; 29.3 (2C); 22.6; 14.0. MS (m/z): 292 (M+), 246,
175, 161, 147, 134 (base peak), 133, 107, 105, 91, 77, 55.
3.7. 2-Hydroxy-6-(8-oxooctyl) benzoic acid (11)41
To a solution of oxalyl chloride (17.1 mL, 0.19 mmol) in dry
CH2Cl2 (0.38 mL) at ꢀ78 °C was added DMSO (25.0 mL, 0.35 mmol)
in dry CH2Cl2 (0.38 mL) under nitrogen atmosphere. After stirring
for 5 min a solution of alcohol 10b (35.0 mg, 0.13 mmol) in dry
CH2Cl2/DMSO (0.38/0.025 mL) was added dropwise. After stirring

J. M. Pereira et al. / Bioorg. Med. Chem. 16 (2008) 8889–8895
8895
for more 5 min, triethylamine was added (0.13 mL, 0.97 mmol) and
the mixture was allowed to warm to room temperature. Finally,
the solvent was removed, the residue was diluted with ethyl ace-
tate and the resulting precipitates were filtered. The filtrate was
concentrated and after a quick purification by chromatography
on silica gel (8:2:0.25 hexane/EtOAc/acetic acid as eluent) the pure
product was obtained as pale yellow oil (0.025 mg, 73%). mp 78–
lowed by Triton X-100 and then enzyme. In both cases, the de-
gree of inhibition was kept constant independently if there was
Triton or if the mixtures were or not incubated (data not
shown), which proved a nonpromiscuous inhibition.
Acknowledgements
79 °C; IR (
m
max/cmꢀ1): 3421, 2928, 2854, 1716, 1651, 1605, 1575,
The authors gratefully acknowledge the financial support from
FAPESP, CAPES and CNPq.
1450, 1243, 1221, 1043, 741 cmꢀ1
.
1H NMR (200 MHz, CDCl3) d:
9.76 (t, 1H, J 1.7 Hz); 7.34 (t, 1 H, J 8.0 Hz); 6.86 (dd, 1H, J 1.0,
8.0 Hz); 6.75 (dd, 1H, J 1.0, 7.6 Hz); 3.00–2.92 (m, 2H); 2.44 (dt,
2H, J 1.7, 7.1 Hz); 1.70–1.51 (m, 4H); 1.45–1.26 (m, 6H). MS (m/
z): 220, 207, 147, 121, 108 (base peak), 77, 55.
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