Anacardic acid derived salicylates are inhibitors or activators of lipoxygenases

Article abstract of DOI:10.1016/j.bmc.2012.06.019

Lipoxygenases catalyze the oxidation of unsaturated fatty acids, such as linoleic acid, which play a crucial role in inflammatory responses. Selective inhibitors may provide a new therapeutic approach for inflammatory diseases. In this study, we describe

Full text of DOI:10.1016/j.bmc.2012.06.019

Bioorganic & Medicinal Chemistry 20 (2012) 5027–5032
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anacardic acid derived salicylates are inhibitors or activators of lipoxygenases
⇑
Rosalina Wisastra, Massimo Ghizzoni, André Boltjes, Hidde J. Haisma, Frank J. Dekker
Department of Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipoxygenases catalyze the oxidation of unsaturated fatty acids, such as linoleic acid, which play a crucial
role in inflammatory responses. Selective inhibitors may provide a new therapeutic approach for inflam-
matory diseases. In this study, we describe the identification of a novel soybean lipoxygenase-1 (SLO-1)
inhibitor and a potato 5-lipoxygenase (5-LOX) activator from a screening of a focused compound collec-
tion around the natural product anacardic acid. The natural product anacardic acid inhibits SLO-1 with an
Received 22 March 2012
Revised 4 June 2012
Accepted 9 June 2012
Available online 21 June 2012
IC50 of 52 lM, whereas the inhibitory potency of the novel mixed type inhibitor 23 is fivefold enhanced.
Keywords:
Anacardic acid
Soybean lipoxygenase-1
Potato 5-LOX
In addition, another derivative (21) caused non-essential activation of potato 5-LOX. This suggests the
presence of an allosteric binding site that regulates the lipoxygenase activity.
Ó 2012 Elsevier Ltd. All rights reserved.
Enzyme inhibition
Enzyme activation
1. Introduction
often associated with anti-inflammatory, anti-tumor, mollusci-
cidal, and anti-microbial activities.7–9 Previous studies reported
Lipoxygenases (LOXs) are a family of non-heme iron-contain-
ing enzymes that catalyze the oxygenation of cis,cis-1,4-pentadi-
ene moieties in lipids. In general, lipoxygenases (LOXs), which
are widely distributed in both the plant and animal kingdom,
are categorized into 5-LOX, 8-LOX-, 12-LOXand 15-LOX based
on the position of oxygenation of their substrates. Lipoxygenases
convert their natural substrates, arachidonic acid, to hydroperoxy
eicosatetraenoic acids (HPETEs) by a radical mechanism.1 Lipoxy-
genases found in plant oxygenate linoleic acid to generate 13-
hydroperoxy-9(Z),11(E)-octadecadienoic acid (13-HPOD). 5-HPETE
is transformed into Leukotriene A4, which contains an unstable
epoxide. Subsequently, Leukotriene A4 is converted either into
Leukotriene B4 by an enzymatic hydrolysis or into Leukotriene
C4 by glutathione S-transferase. The resulting Leukotriene C4 is
further metabolized to Leukotriene D4 and Leukotriene E4.2 It
has been reported that activities of lipoxygenases and their re-
lated products play an important role in numerous inflammatory
and proliferative diseases such as asthma, atherosclerosis, and
cancer.3–5 Leukotriene B4 activates inflammatory cells such as
neutrophils and macrophages.6 This broad range of biological
that anacardic acid inhibits peroxidation of linoleic acid by soy-
bean lipoxygenase-1 (SLO-1) with an IC50 of 85
l
M.10 In addition,
anacardic acid and its derivatives inhibit histone acetyl transfer-
ases (HATs) and cyclooxygenases, which are also involved in
inflammation and cancer.11–13
In this study, we investigate the affinity and selectivity of ana-
cardic acid derived inhibitors for SLO-1 and potato 5-LOX as rep-
resentatives of the lipoxygenase family. Although lipoxygenases
are well-known drug targets, relatively little inhibitors with high
selectivity and potency have been described (reviewed by Pergola
and Werz14). Currently, the selective 5-LOX inhibitor Zileuton
(IC50 = 0.5 l
M) is marketed for treatment of asthma.15 Although
there are no 15-LOX inhibitors available for the clinic, recent
studies identified inhibitors with nanomolar affinity.16,17 Never-
theless, their efficacy in cell-based studies remains limited. There-
fore, development of inhibitors of new-structural classes remains
necessary in order to explore these enzymes further as drug
targets.
Here, we describe the identification of lipoxygenase inhibitors
and activators from a focused compound collection based on ana-
cardic acid. Anacardic acid and its derivatives were synthesized
following previous research18 and inhibition of SLO-1 and potato
5-LOX were investigated. We found that derivative 23 inhibits
SLO-1 selectively to potato 5-LOX, whereas derivative 21 shows a
threefold activation on potato 5-LOX. The enzyme kinetics of these
inhibitors indicate the presence of an allosteric site that influences
the activity of the lipoxygenase active site.
effects demonstrates the importance of lipoxygenases as
therapeutic target.
a
6-pentadecyl salicylic acid, commonly known as anacardic acid,
is a natural product found in cashew nut shells. This compound is
⇑
Corresponding author. Tel.: +31 50 3638030; fax: +31 50 3637953.
E-mail address: f.j.dekker@rug.nl (F.J. Dekker).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.019

5028
R. Wisastra et al. / Bioorg. Med. Chem. 20 (2012) 5027–5032
and cyclization.22 Subsequently, 7a was attached to 2 and 7b was
2. Results and discussion
2.1. Design
attached to 5 to give the corresponding alkynyls, which were re-
duced using catalytic hydrogenation to give 24 and 31 (Scheme 1).
Sonogashira coupling of aryl chlorides has been described to
proceed with PdCl2(PPh3)2, P(tBu)3, and 1,8-diazabicyclo [5.4.0]un-
dec-7-ene (DBU) as catalysts, and Cs2CO3 as a base to give >80%
yields.23 However, our attempt to synthesize compounds 24 and
31 under these conditions yielded 20–32% of the desired products.
These relatively low yields are possibly due to electronic properties
of the benzoxazole and/or the limited stability of the ester func-
tionality in 2 or 5 under the basic coupling conditions.
Benzoxazole 9 was prepared from commercially available 2-(4-
bromophenyl)acetic acid (8) and 2-aminophenol following two
reaction steps. Firstly, 2-(4-bromophenyl)acetic acid (8) was con-
verted into the acyl chloride using thionyl chloride followed by
amide bond formation with 2-aminophenol to give the amide with
high yields (70%). Secondly, this amide was cyclized using p-tolu-
enesulfonic acid in toluene at reflux for 4 h to give the product 9
with high yields (78–85%). Subsequently, the arylbromide (9)
was coupled to trimethylsilyl acetylene by a Sonogashira coupling.
The trimethylsilyl protective group was cleaved to give the corre-
sponding alkyne 10. This alkyne was coupled to triflate 1124 using
a Sonogashira coupling to provide 55% yields of the corresponding
alkyne (Scheme 1), which was hydrogenated to yield 25 in a single
step.
The fact that the lipoxygenases are iron-containing enzymes
combined with the fact that salicylates are known iron-binding
compounds justifies the hypothesis that the iron-binding proper-
ties of salicylates are key to their inhibitory potency. In addition,
hydrophobic interactions between the hydrophobic tail of anacar-
dic acid 18 and the lipoxygenase active site are probably impor-
tant. Based on these hypotheses, we expect that the different 6-
alkyl substituents in our focused compound collection can improve
the potency for lipoxygenase inhibition.
2.2. Synthesis
A compound collection was assembled around the natural prod-
uct anacardic acid 18. This collection comprises of the previously
published salicylates 15–23, 26–30, and 32 and the newly synthe-
sized salicylates 24, 25, 31, and 33. The synthesis of anacardic acid
and its derivatives 15–23, 26–30, and 32 were described previously
by Ghizzoni et al.18,19 and compounds 24, 25, 31, and 33 were syn-
thesized using similar procedures (Scheme 1). Triflate 1 was syn-
thesized as described by Uchiyama et al.20 Alkynes 221 and 5
were prepared from triflate 1 or 3-iodobenzoic acid 3 by Sonogash-
ira coupling with trimethylsilyl (TMS) acetylene and subsequent
cleavage of the TMS protective group to yield the corresponding
products (Scheme 1). Benzoxazoles 7a and 7b were prepared from
2-amino-5-chlorophenol (6) and benzoic acid through amidation
Acetonide 13 was synthesized from 2,4-dihydroxybenzoic acid
12 as described by Tranchimand et al.24 Furthermore, acetonide
13 was converted to triflate 14 as described previously.20 Sono-
gashira reaction, coupling of 1-ethynyl-4-heptyl benzene with
Cl
OH
OH
OH
O
O
O
O
O
a, b
c,d,e
7a
NH2
6
O
O
O
N
g,i
OTf
24
1
2
Cl
O
N
R
7a R = H
O
O
O
OH
O
O
HO
O
Bn
7b R = OCH3
Bn
a,b
n,d
f
OBz OBz
7b
O
N
I
I
O
O
5
4
31
3
BzO
OTf
11
OH
25
HO
OH
Br
Br
c,d
h,i
O
a,b
11
O
O
N
O
N
HO
O
9
8
10
N
OH
O
O
O
O
O
j,k
OH
l,m
Bn
c,d
OH
O
O
1-ethynyl-4-
Bn
Bn
HO
OH
O
O
OTf
heptylbenzene
33
14
12
13
6
Scheme 1. Reagent and conditions: (a) Trimethylsilylacetylene, CuI, PdCl2(PPh3)2, Et2NH, PPh3, CH3CN, (MW, 120 °C, 95 W), (b) TBAF, THF, 0 °C; (c) CuI, PdCl2(PPh3)2, Et2NH,
HC„CR, CH3CN (MW, 100 °C, 70 W); (d) H2, Pd/C, MeOH, 40 °C; (e) aqueous KOH 5 N, THF, 55 °C; (f) benzyl bromide, K2CO3, DMF, rt; (g) benzoic acid anhydride, pyridine,
DMF, rt; (h) SOCl2, 60 °C followed by 2-aminophenol, 0 °C; (i) p-toluensulfonic acid, toluene, reflux; (j) SOCl2, DMAP, DME, acetone, rt; (k) Bn-Br, K2CO3, DMF, rt; (l) Bn-OꢀNa+,
THF, rt; (m) Tf2O, pyridine, CH2Cl2, 0 °C; (n) HC„CR, aryl chloride, Cs2CO3, PdCl2(PPh3)2, P(tBu)3, DBU, DMF (MW, 150 °C, 95 W).

R. Wisastra et al. / Bioorg. Med. Chem. 20 (2012) 5027–5032
5029
Table 1
Compound collection of anacardic acid and its derivatives
Compounds
R1
H
R2
H
R3
15
16
17
H
H
H
OH
18
19
H
CH3
H
H
20
H
OH
21
H
H
22
23
H
H
H
OH
24
25
26
27
28
H
H
H
H
H
H
OH
H
H
H
29
30
31
H
H
H
H
OH
H
32
33
H
H
H
OH
Figure 2. The IC50 value for inhibition of (A) SLO-1 and (B) potato 5-LOX by
anacardic acid (18). The results were the average of three independent experiments
with error bars ( S.D.).
triflate 14, was performed to give the alkyne with high yield (79%),
which was hydrogenated to give compound 33.
The collection of compounds (Table 1) was screened for inhibi-
tion of the lipoxygenase activity of soybean lipoxygenase-1 (SLO-1)
and potato 5-lipoxygenase (potato 5-LOX). The lipoxygenase
activity was monitored in real time using a spectrophotometric
assay to monitor the formation of conjugated diene 13-hydroper-
oxy-cis-9-trans-11-octadecadienoic acid (13-HPOD) from linoleic
acid.25,26 The residual enzyme activity was monitored after
contrast, we observed an IC50 of 43
(Fig. 2B), whereas literature reports a 30% activity inhibition at
M.12 This difference is probably due to a difference in the type
of substrate that was used. Salicylates 21, 23, 24 and 25 show
50% inhibition or more at 50 M for SLO-1, whereas this is not ob-
served for potato 5-LOX. In contrast, compounds 21 and 22 activate
the potato 5-LOX enzyme at these concentrations. This demon-
strates that these compounds activate or inhibit lipoxygenase
activity and that selectivity between both lipoxygenases can be ob-
tained by variation of the substitution in the 4- and/or 6-position
of the salicylate.
lM for potato lipoxygenase
6
l
l
10 min of pre-incubation with 50 lM of the respective inhibitor.
The enzymatic activity without inhibitor present was taken as ref-
erences and set to 100% and the activity without enzyme present
was set to 0%. The percentages of the residual enzyme activity of
SLO-1 and potato 5-LOX in the presence of the inhibitors are
plotted in Figure 1.
This salicylate compound collection shows interesting struc-
ture–activity relationships for SLO-1. The 6-pentadecyl substituent
in 18 is important for inhibition, because inhibitors 15 and 16 with
a 6-pentyl or 6-decyl substituents are less potent. The same is
Anacardic acid 18 inhibits both potato 5-LOX and SLO-1 about
50% at 50
lM and the IC50 value for SLO-1 was 52 lM (Fig. 2A
and Table 2), which is in line with literature for SLO-1.10 In
Figure 1. Residual enzyme activity that was observed for the screening of the salicylate compound collection for inhibition of the lipoxygenase activity. The bars show the
residual activity of potato LOX-5 (light gray) and soybean lipoxygenase-1 (SLO1) (dark gray) in the presence of 50 M of the respective inhibitor. The percentage activity for
l
each compound was calculated in comparison with the blank in which no inhibitor was present. The results were the average of three independent experiments with the
standard deviations.

5030
R. Wisastra et al. / Bioorg. Med. Chem. 20 (2012) 5027–5032
Table 2
IC50 values for inhibition of SLO-1 by
anacardic acid derivatives
Compounds
IC50 (lM)
18
21
23
25
51.9 5.0
55.4 6.3
11.1 0.8
58.5 3.6
Table 3
Enzyme kinetics parameter for SLO-1 inhibition
Figure 3. Conversion of the substrate linoleic acid by the enzyme SLO-1 with no
[23] (lM)
R2
app
max
V
(lM/s)
Kampp
(lM)
inhibitor present (ꢀ), after preincubation with 6
l
M inhibitor 23 (N), and with
11 M inhibitor 23 (j). The results were the average of three independent
l
0
6
11
0.437
0.321
0.251
12.4
14.3
15.5
0.963
0.967
0.979
experiments with error bars ( S.D.).
Km
kcat
E + S
+
E + P
ES
+
observed for the salicylates 26 and 27 without an aliphatic substit-
uents in the 6-position. These results demonstrate that the side
chain length of the 6-alkyl chain is important for the SLO-1 inhib-
itory potency. This is possibly due to the hydrophobic interactions
between the inhibitor and the binding pocket of the enzyme. In
addition, a complete loss of inhibition was observed if the carbox-
ylate of anacardic acid 18 was converted into a methyl ester 19,
which demonstrates that the free carboxylate is also important
for binding. Despite the presence of a 6-pentadecyl substitutent,
compound 20 is slightly less active for SLO-1 and inactive for pota-
to 5-LOX. Apparently, the extra hydroxyl in the para-position influ-
ences both the steric and electronic properties in such a way that
binding is impaired. Furthermore, the importance of the 2-hydro-
xyl functionality of the salicylate is demonstrated by the inactivity
of the inhibitors 31–33, which show less than 50% inhibition
I
I
Ki
K
'
i
Km'
ESI
EI + S
Scheme 2. Kinetic model for mixed enzyme inhibition.
Figure 4. Equation for the enzyme kinetics according to the model in Scheme 2. v is
the reaction velocity, Vmax is the maximal reaction velocity, [S] is the substrate
concentration and Km is the Michaelis–Menten constant.
50 lM. These results demonstrate that the carboxylate and the hy-
a
and a0, respectively, are
droxyl of the salicylate core are both crucial for inhibition of SLO-1.
Salicylates 24 and 25 with a benzoxazole functionality in their
side chain maintain their inhibition of SLO-1, whereas no inhibi-
tion of potato 5-LOX was observed. This demonstrates that the
selectivity between potato 5-LOX and SLO-1 can be obtained by
variation of the 6-alkyl substituent in anacardic acid.
the parameters to describe the change of substrate binding affinity to the enzyme
and the change of the maximum velocities. Ki is the dissociation constant of
inhibitor to the free enzyme and K0i is the dissociation constant of inhibitor to the
enzyme–substrate complex.
each inhibitor 23 concentration were calculated in the same man-
ner as in the absence of inhibitor. a0 and a0 values, which are the
a/
Inhibitor 23 shows an IC50 of 11 lM (Table 2) for SLO-1, which
change in Vmax and Km, are derived respectively from Vapp divided
is fivefold improved compared to anacardic acid 18, whereas no
inhibition of potato 5-LOX was observed. Furthermore, inhibitor
23 also shows its selectivity towards HATs, in which inhibitor 23
gives no inhibitory effect on the activity of p300, and PCAF and
max
by Vmax and from Kampp divided by Km. The inhibitor binding con-
stant, Ki and K0i values, were calculated from the Eqs. 1.2 and 1.3
(Fig. 4) as described in Supplementary data. The binding constant
modest effect on Tip60 at 200 l
M.19 In comparison, the lower
binding for inhibition of the free enzyme (Ki) is 9.8
l
M and the
binding constant to the substrate bound enzyme (Ki0) is 15.7
lM.
SLO-1 inhibition of 22 and 33 demonstrate that both hydroxyl
groups of 23 are important for inhibition. In addition, the reduced
potency of 20 shows that also the 1-ethyl-4-heptylbenzene substi-
tuent in 23 is important for inhibition.
The fact that inhibitor 23 binds to the free as well as the substrate
bound enzyme in the mixed type inhibition suggests the presence
of an allosteric binding site.28–30
In an attempt to improve the potency of inhibitor 23, we de-
signed a novel compound 25 in which the aliphatic chain was re-
placed for a heterocycle. However, this derivative did not provide
a better inhibition of SLO-1 (Table 2).
The influence of 23 on the Michaelis–Menten kinetics for con-
version of the substrate linoleic acid was determined in order to
Interestingly, inhibitor 21, in which the 6-aliphatic side chain is
replaced by an ether functionality, shows equal inhibition com-
pared to anacardic acid 18 on SLO-1 (Table 2), whereas this inhib-
itor (and also inhibitor 22) shows about threefold activation of
potato 5-LOX. Compound 21 provided a concentration dependent
activation with a maximal activation that is about threefold higher
establish the mechanism of SLO-1 inhibition. Inhibitor 23 causes
than the control activity at concentrations higher than 50
(Fig. 5).
lM
app
max
both a reduction of V
and an increase of Kampp (Table 3), which
indicates a mixed type of inhibition (Fig. 3). The enzyme activity
is expected to obey the model in Scheme 2, in which the inhibitor
can bind to the free enzyme as well as to the substrate bound
enzyme.
We decided to study the enzyme kinetics for the activation of
potato 5-LOX by 21 in order to investigate the binding mechanism.
The concentration dependent effect of 21 on the Lineweaver–Burk
plot of potato 5-LOX activity was investigated (Fig. 6A). Activator
21 causes an increase of the Vmax and a decrease of the Km values.
This behavior, in which the reaction can occur in the presence or in
the absence of the activator, indicates non-essential activation of
The values for Vmax and Km were determined from the Linewe-
aver–Burk plot using Eq. 1.1 (Fig. 4) in the absence of inhibitor 23,
app
max
respectively from the y-intercept and x-intercept. V
and Kampp for

R. Wisastra et al. / Bioorg. Med. Chem. 20 (2012) 5027–5032
5031
kcat
Km
E + S
+
E + P
E + P
ES
+
A
A
KA
αKA
αKm
βkcat
ESA
EA + S
Scheme 3. Kinetic model for non-essential activation.
the affinity of the activator for the substrate bound enzyme is
24 M, which explains the observed activation at concentrations
of 25 M and higher. In addition, activator binding increases the
catalysis rate by 1.4-fold. Taking this together, activator 21 shows
non-essential activation and binds with relatively high affinity to
substrate bound lipoxygenase and enhances the enzymatic conver-
sion of the substrate.
l
Figure 5. Compound 21 activates potato 5-LOX. The experiments were performed
in the absence (control) or the presence of compound 21 with various concentra-
tions. The results were the average of three independent experiments with error
bars ( S.D.)
l
Furthermore, the critical micelle concentrations (CMCs) for both
anacardic acid (18) and 23 at the assay conditions (0.2 M borate
buffer, pH 9.0, rt) are, respectively, 390
whereas for compound 21 (in 0.1 M phosphate buffer pH 6.3, rt),
the CMC value is 208 M (Fig. 8B). These results indicate that mi-
lM and 436 lM (Fig. 8A,C)
l
celle formation does not occur at concentrations employed for inhi-
bition or activation. In addition, we found a CMC value for linoleic
acid of 163
concentration in the inhibition studies (100
l
M (Fig. S2),27 which demonstrates that the substrate
lM) was below the
CMC. Thus the enzyme kinetics were evaluated in a homogeneous
system. We also performed a docking simulation of 18 and 23 to
SLO-1 to generate plausible binding poses of the inhibitor in the
SLO-1 active site (Fig. S3)
The kinetic studies on both potato 5-LOX and SLO-1 indicate the
presence of an allosteric binding site that influences substrate bind-
ing and conversely is influenced by binding of the substrate. For po-
tato 5-LOX we demonstrated activation of the enzyme activity and
for SLO-1 we demonstrated inhibition. These indications for an allo-
steric binding pocket are in line with previous studies. The observa-
tion of substrate inhibition of soybean lipoxygenase by linoleic acid
indicates the presence of an allosteric binding site.32 It can be pre-
sumed that the salicylate based inhibitors resemble the lipid sub-
strate linoleic acid and binds in a similar way to the enzyme,
thereby inhibiting the enzyme activity via an allosteric binding
pocket. In addition, mixed inhibition has been described previously
for soybean lipoxygenase by the inhibitor oleyl sulfate, which also
indicates the presence of an allosteric binding site.28 Non-essential
activation has been described previously for 5-LOX by compound
Figure 6. (A) Lineweaver–Burk plot of the potato 5-LOX activity with no activator
present (ꢀ), after preincubation with 12.5
21 (j), and with 50 M activator 21 (d). The results were the average of three
independent experiments with error bars ( S.D.) (B) Re-plot of 1/
1/ intercept (ꢀ).
lM activator 21 (N), with 25 lM activator
l
D
slope (d) and the
D
(R,S)-2-hydroxy-2-trifluoromethyl-trans-n-octadec-4-enoic
acid
(HTFOA) and phosphatidic acid.33,34 These findings also indicate
the existence of allosteric site as enzyme activity regulator. Never-
theless, the location and structural properties of the allosteric bind-
ing pocket in these enzymes remain elusive.35 This demonstrates
that structural characterization of the allosteric binding pocket re-
mains a major challenge in lipoxygenase research.
Table 4
Enzyme kinetics parameter for potato 5-LOX activation
R2
app
[21] (
lM)
V
(lM/s)
max
Kampp (mM)
0
12.5
25
1.597
1.754
1.852
1.949
1.160
0.733
0.537
0.370
0.998
0.999
0.995
0.992
50
potato 5-LOX31 (Table 4). The enzyme kinetics for the activation of
potato 5-LOX obeys the model shown in Scheme 3. The activator
dissociation constants (KA), the change in the affinity of substrate
binding (
a
value) and the change in the catalytic constant (b value)
slope and the 1/ inter-
were determined from the re-plot of 1/
D
D
cept (Fig. 6B) according to Eq. 2 (Fig. 7) as described in Supplemen-
tary data.
Figure 7. Equation for the enzyme kinetics according to the model in Scheme 3.31
is the reaction velocity, Vmax is the maximal reaction velocity, [S] is the substrate
concentration and Km is the Michaelis–Menten constant, [A] is the activator
v
The kinetic analysis shows that
1.8 mM. The value indicates that the substrate and the activator
mutually enhance their binding by close to 100-fold. Consequently,
a is 0.013, b is 1.4 and KA is
a
concentration.
a and b, respectively, are the parameters to describe the change in
the affinity of substrate binding and the change in the catalytic constant.

5032
R. Wisastra et al. / Bioorg. Med. Chem. 20 (2012) 5027–5032
an activator 21 of potato 5-lipoxygenase was identified for which
KA is 1.8 mM, is 0.013 and b is 1.4 (Scheme 3). The affinity of
21 for the substrate bound enzyme ( KA) is 24 M. To our knowl-
a
a
l
edge, this is the first activator with micromolar potency for potato
lipoxygenase. These inhibitors might provide valuable starting
points for development of inhibitors that target lipoxygenases,
which is relevant for inflammatory diseases.
Supplementary data
Supplementary data (synthetic procedures and compounds
analytical data as well as kinetic studies, assay description and
molecular docking) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.06.019.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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This study identifies salicylate based molecules as allosteric
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mains to be investigated. It is encouraging that the anacardic acid
derived salicylates show clear structure–activity relationships,
which indicates that further optimization of the affinity and selec-
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3. Conclusions
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This study demonstrates that anacardic acid 18 and its deriva-
tives are lipoxygenase inhibitors and that their potency depends
on the substitution pattern in the 4- and 6-position of the salicylate
core. A novel inhibitor 23 was identified for soybean lipoxygenase-
1 with a fivefold improved IC50 value compared to anacardic acid
18. Enzyme kinetics reveal a mixed type inhibition pattern for 23
with a Ki of 9.8 l lM (Scheme 2). In addition,
M and a Ki0 of 15.7