Molecular design of anti-MRSA agents based on the anacardic acid scaffold

Article abstract of DOI:10.1016/j.bmc.2007.06.022

A series of anacardic acid analogues possessing different side chains viz. phenolic, branched, and alicyclic were synthesized and their antibacterial activity tested against methicillin-resistant Staphylococcus aureus (MRSA). The maximum activity against

Full text of DOI:10.1016/j.bmc.2007.06.022

Bioorganic & Medicinal Chemistry 15 (2007) 6236–6241
Molecular design of anti-MRSA agents based
on the anacardic acid scaffold
Ivan R. Green,a,* Felismino E. Tocoli,a Sang Hwa Lee,b Ken-ichi Niheib and Isao Kubob,*
aDepartment of Chemistry, University of the Western Cape, Bellville 7530, South Africa
bDepartment of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA
Received 21 November 2006; revised 31 May 2007; accepted 12 June 2007
Available online 14 June 2007
Abstract—A series of anacardic acid analogues possessing different side chains viz. phenolic, branched, and alicyclic were synthe-
sized and their antibacterial activity tested against methicillin-resistant Staphylococcus aureus (MRSA). The maximum activity
against this bacterium occurred with the branched side-chain analogue, 6-(40,80-dimethylnonyl)salicylic acid, and the alicyclic
side-chain analogue, 6-cyclododecylmethyl salicylic acid, with the minimum inhibitory concentration (MIC) of 0.39 lg/mL, respec-
tively. This activity was superior to that of the most potent antibacterial anacardic acid isolated from the cashew Anacardium occi-
dentale (Anacardiaceae), apple and nut, that is, the 6-[80(Z),110(Z),140-pentadecatrienyl]salicylic acid.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
these scaffolds mainly by selecting appropriate head
portions.3
In our previous reports on structure–antimicrobial
activity relationship (SAR) studies on a series of ali-
phatic alcohols, their maximum antimicrobial activity
appeared to be an interactive function of the hydropho-
bic alkyl (tail) chain length and the hydrophilic hydroxyl
group (head). It was proposed that the hydrophilic head
moiety binds with an intermolecular hydrogen bond like
a ‘hook’ attaching itself to a hydrophilic portion of the
membrane, after which the hydrophobic tail portion of
the molecule is then able to enter into the membrane
lipid bilayer. As a result, disorder in the fluid bilayer
of the membrane is created and this conclusion may
be explained by the alcohols’ non-ionic surfactant prop-
erties as well as by their non-specificity in the activity. It
also appears that biophysical processes are a major con-
tributor to the antimicrobial activity of amphipathic
alkanols.1,2 The hydrophilic hydroxyl group can be re-
placed by any other hydrophilic groups as long as there
is a balance in the ‘head and tail’ structure. Hence, var-
ious additional biological activities can be introduced in
Staphylococcus aureus is one of the main bacteria that
cause suppuration, food poisoning, and toxic shock syn-
drome4 and is thus the focus of this work. Strains of
methicillin-resistant S. aureus (MRSA) are known to
be resistant to many antibiotics and currently represent
a serious problem to hospitalized patients as well as their
caretakers.5 Among the important biological activities
known about anacardic acids is their antibacterial activ-
ity and this has been most extensively studied.1,2,6–10 In
addition, due to its adaptability, S. aureus can easily
develop resistance to the commonly used antibiotics.
This resistance involves both an enzymatic inactivation
as well as the presence of an altered penicillin binding
protein in the resistant bacteria.11 Such resistance genes
are then often transferred to other bacteria by a variety
of gene transfer mechanisms12 which exacerbates the
problem further. It is thus imperative that the search
for new anti-MRSA agents continues since it is crucial
to the future maintenance of public health.13 Addition-
ally, there is a great need for more effective antibacterial
agents having new modes of action and consequently the
ideal antimicrobial agent should best be produced by ra-
tional design.14 Antibacterial agents that primarily act
as surface-active ones viz. surfactants may have the po-
tential of addressing this need, since they target the
extracytoplasmic region and thus do not need to enter
Keywords: Methicillin-resistant Staphylococcus aureus (MRSA); Anti-
bacterial; Anacardic acid analogue; 6-(40,80-Dimethylnonyl)salicylic
acid.
*
Corresponding authors. Tel.: +27 21 9592262; fax: +27 21 9593055
(I.R.G.); tel.: +1 510 643 6303; fax: +1 510 643 0215 (I.K.); e-mail
addresses: igreen@uwc.ac.za; ikubo@calmail.berkeley.edu
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.022

I. R. Green et al. / Bioorg. Med. Chem. 15 (2007) 6236–6241
6237
the cell, thereby avoiding most cellular pump-based
resistance mechanisms. Possible structural variations in
the head and tail moieties of the anacardic acid scaffold
suggest that optimization of activity is possible through
a process of selective synthetic design. To date, only the
non-branched aliphatic chains having various lengths
viz. (C5, C8, C10, C12, and C20) have been synthesized.7
It is thus conceivable that optimization of more specific
activity is a strong possibility through the synthetic ap-
proach in which cases the molecular dimensions, to-
gether with the lipophilicity, would have a critical
impact on the biologically active profile of these
molecules.
that of salicylic acid.7 In previous papers, anacardic acid
(C15:3) (1) was described to exhibit bactericidal activity
against S. aureus at any growth stage, and additionally
even when cell division was inhibited by chlorampheni-
col.9 In general anacardic acids are amphipathic mole-
cules, which implies that their hydrophobic properties
dominate the properties of the molecule. This finding
is consistent with the proposed mechanism of their anti-
bacterial activity which involves disordering of the mem-
brane. However, the specific site of action for this
disordering effect is less clear. The primary response
for the antibacterial activity of anacardic acids arises
from their biophysical disruption of the membrane, indi-
cating that the alk(en)yl side chain is an important scaf-
fold to elicit this activity. Furthermore, anacardic acids
were also reported to inhibit the oxidation of NADH
oxidase by a membrane fraction prepared from Micro-
coccus luteus cells, indicating that anacardic acids also
inhibit respiratory chain enzyme activity to demonstrate
their wider activity profile. However, although this bio-
chemical mechanism is not a primary contributor to eli-
cit their antibacterial activity2, it does, however, not
exclude the possibility that anacardic acids first act as
surfactants after which biochemical processes become
involved, a factor which needs to be born in mind when
drawing conclusions about possible mechanisms. The
antibacterial activity of anacardic acids against S. aureus
was previously found to be proportional to the degree of
the side-chain unsaturation.6,17 However, a recent study
indicates that the double bond in the C-6 side chain is
not essential in eliciting the antibacterial activity but is
rather associated with increasing the activity.2 In addi-
tion, anacardic acid (C15:0) (4) has previously been
2. Results and discussion
The anacardic acids viz. 6-[80(Z),110(Z),140-pentadecat-
rienyl]salicylic acid (1) (see Fig. 1 for structures), will
be referred to as anacardic acid (C15:3) for simplicity
hereafter; and 6-[80(Z),110(Z)-pentadecadienyl]salicylic
acid (2), as anacardic acid (C15:2), and 6-[80(Z)-pentade-
cenyl]salicylic acid (3) as anacardic acid (C15:1) are all
isolated from the cashew apple, Anacardium occidentale
(Anacardiaceae) and are well known to exhibit potent
antibacterial activity against Gram-positive bacteria
including MRSA strains.15,16 Since these anacardic acids
are salicylic acid derivatives having a C-6 alk(en)yl side
chain, their inhibitory activity was also compared with
R1
reported to show high selectivity toward Fe2+ and
R2O
Cu2+
.
This finding implies that chelation might also
18
play a role in the antimicrobial activity of anacardic
acids by reducing their bioavailability for bacteria.19
Evidence currently available tends to support this
possibility since anacardic acid (C15:0) (4) did not show
any antibacterial activity against S. aureus up to
800 lg/mL. On the other hand, it has also been shown
that activity is inversely proportional to the length of
the C-6 side chain since the hydrophobicity of molecules
is well known to play a critical role in biological activi-
ties.20 However, the rationale for this, especially the role
of the hydrophobic portion, is still poorly understood,
since this area of study has received scant attention.
Although these anacardic acids, vide infra, are available
in quantities, they are rather unstable for practical appli-
cations due to their side-chain unsaturation.
1: R1 = COOH, R2 = H
5: R1 = COOMe, R2 = H
6: R1 = COOH, R2 = Ac
8: R1 = H, R2 = H
COOH
HO
2
COOH
HO
3
COOH
HO
Neither methyl ester (5) nor acetate (6) of anacardic acid
(C15:3) exhibited any antibacterial activity against S. aur-
eus up to 200 lg/mL,21 indicating that the free salicylic
acid moiety is an essential element to elicit the antibac-
terial activity. In addition, anacardic acids are known
to exhibit potent antibacterial activity against MRSA
strains but salicylic acid itself has little effect on these
strains which leads to the conclusion that the hydropho-
bic alk(en)yl side chain is undoubtedly associated with
the antibacterial activity. Hence, it may not be far
fetched to assume that side-chain alkyl salicylates would
demonstrate varying degrees of antibacterial activity
4
OH
O
O
7
Figure 1. Structures of anacardic acid and its related compounds.

6238
I. R. Green et al. / Bioorg. Med. Chem. 15 (2007) 6236–6241
against S. aureus. To this end, nonyl salicylate (7) lack-
ing the C-6 alkyl side chain was synthesized and tested
for antibacterial activity against S. aureus. No antibacte-
rial activity was found again supporting the contention
that the 6-alk(en)yl salicylic acid molecular scaffold is
a prerequisite to elicit the activity. This conclusion is fur-
ther supported by the observation that cardanol (8), an
COOH
HO
R
R2
R1
R3
R4
artifact of the corresponding anacardic acid (C15:3
)
*
n
R =
R =
obtained by pyrolysis, did not show any antibacterial
activity against S. aureus up to 200 lg/mL.22 In addition
to molecules having the anacardic acid scaffold, it
should be further noted that the degree of antimicrobial
activity depends both on the hydrophobic tail portion as
well as the microorganisms being tested.7 Abundant lit-
erature examples have shown that a small change in the
chemical structure of molecules affects their biological
activities to a large extent. This prompted us to synthe-
size a series of anacardic acid analogues possessing
different side chains viz. phenolic, branched, and alicy-
clic in order to gain new insights into their antibacterial
activity on a molecular basis. The introduction of
branching or unsaturation into the hydrophobic moiety
is known to increase the solubility of the surfactant in
water.23 If the hydrophobic portion of the molecule en-
ters into the membrane lipid bilayer and creates disorder
in the fluid bilayer, then it stands to reason that by
increasing the volume of the hydrophobic portion
through synthetic modification, the resulting analogue
may demonstrate an enhancement in its activity.
n
m
9: m = n = 1
18: n = 1, R1 = R3 = R4 = OH, R2 = H
19: n = 1, R1 = R4 = OH, R2 = R3 = H
20: n = 1, R1 = R3 = OH, R2 = R4 = H
21: n = 1, R1 = R2 = H, R3 = R4 = OH
22: n = 1, R1 = R2 = R4 = H, R3 = OH
23: n = 1, R1 = R2 = R3 = R4 = H
10: m = 2, n = 1
11: m = 4, n = 1
12: m = 8, n = 1
13: m = 2, n = 2
24: n = 3, R1 = R2 = R3 = R4 = H
R =
R =
14
15
R =
R =
6
4
16
17
Figure 2. Structures of anacardic acid derivatives.
The synthesis of non-branched 6-alkylsalicylic acids has
previously been described7,27 and thus 15 new anacardic
acid analogues (9–24) were synthesized by a standard
protocol developed in our laboratories and shown in
Scheme 1.28–30 In brief, phosphonate ester 25 and com-
mercially available aldehydes were condensed under ba-
sic Wittig type conditions to afford the corresponding
trans olefins 26 in average yields of 60%. Catalytic
hydrogenation of these olefins afforded the saturated
aryl esters 27 (85%) which were in turn hydrolyzed in
strongly basic dimethylsulfoxide at 110 °C to produce
the corresponding aryl carboxylic acids 28 (85%).
Demethylation of the aryl methoxy group was accom-
plished using boron tribromide in 1,2-dichloroethane
at low temperature to afford the anacardic acid ana-
logues (70%). Thus the average transformation of phos-
phonate ester 25 into the anacardic acid analogues was
26% for the four steps.
In addition to their antibacterial activities, anacardic
acids are also known to act as antioxidants in a variety
of ways including inhibition of various prooxidant
enzymes involved in the production of the reactive oxy-
gen species and additionally chelate divalent metal ions
such as Fe2+ or Cu2+, but do not quench reactive oxygen
species. Recently, their lipoxygenase inhibitory activity
has been reported.24 Lipoxygenases (EC 1.13.11.12)
are suggested to be involved in the early event of athero-
sclerosis by inducing plasma low-density lipoprotein
(LDL) oxidation.25,26 For this and other reasons, the
molecular design of our compounds was dictated to by
keeping in mind these various biological activities. Sec-
ond, we reasoned that since phenolic compounds are
generally considered to possess antibacterial activity
and are used as domestic disinfectants, attaching them
to the side chain of the anacardic acid nucleus would
be a valid area to investigate. Third, it is also well known
that the hydrophobic tail chain length and degree of
unsaturation plays a role in the activity of anacardic
acids1,2 and that some initial attempts to quantify this
have been made.7 Consequently, it was reasoned that
this aspect needed to be broadened and thus we included
some analogues with variations of chain length and
branching to comparatively evaluate these to known
anacardic acids. Finally, the molecular volume of the
hydrophobic side chains at C-6 in anacardic acids has
been speculated to play a role in increasing the antimi-
crobial activity7 and this prompted us to synthesize a
new series of anacardic acid analogues having saturated
hydrophobic carbocyclic ring systems of differing size to
thus represent increasing molecular volumes at C-6 and
evaluate these on a comparative basis (Fig. 2).
The synthesized compounds were tested for their anti-
bacterial activity against S. aureus ATCC 33592
(MRSA) using a 2-fold serial broth dilution method.
The highest concentration tested was 200 lg/mL because
of the solubility limitation of some samples in the water
based medium. Results are listed in Table 1 and the data
obtained were compared with those of anacardic acid
(C15:3) (1).2,10 All synthesized compounds were also
tested for their antibacterial activity against Escherichia
coli ATCC 9637, but none of them exhibited any activity
up to 200 lg/mL.
The alicyclic anacardic acid analogues 9–13 showed
antibacterial activity against S. aureus with MICs rang-
ing from 0.39 to 100 lg/mL. Among these compounds,

I. R. Green et al. / Bioorg. Med. Chem. 15 (2007) 6236–6241
6239
COOEt
COOEt
MeO
MeO
O
MeO
aldehyde or ketone
t-BuOK, THF
H2, 5% Pd/C
MeOH
P
R
OEt
OEt
25
26
COOEt
COOH
COOH
MeO
HO
BBr3
0.5 M NaOH aq.
R
R
R
DMSO
CH2ClCH2Cl
27
28
Scheme 1. Syntheses of anacardic acid derivatives.
Table 1. Minimal inhibitory concentrations of natural anacardic acids and their analogues for methicillin-resistant Staphylococcus aureus ATCC
33592
Compounds tested
MIC (lg/mL)
logP
6-[80(Z),110(Z),140-Pentadecatrienyl]salicylic acid (1)
6-[80(Z),110(Z)-Pentadecadienyl]salicylic acid (2)
6-[80(Z)-Pentadecenyl]salicylic acid (3)
6.25a
12.5a
100a
>800a
>800
>800
100
6.62
6.89
7.21
7.53
6.89
6.60
3.19
3.61
4.44
6.11
4.02
5.69
4.52
3.69
7.21
2.54
2.93
2.93
2.93
3.32
3.71
4.55
6-(Pentadecenyl)salicylic acid (4)
6-[80(Z),110(Z),140-Pentadecatrienyl]salicylic acid methyl ester (5)
Acetyl 6-[80(Z),110(Z),140-pentadecatrienyl]salicylic acid (6)
6-Cyclopentylmethyl salicylic acid (9)
6-Cyclohexylmethyl salicylic acid (10)
6-Cyclooctylmethyl salicylic acid (11)
25
12.5
0.39
12.5
0.39
6.25
50
6-Cyclododecylmethyl salicylic acid (12)
6-Cyclohexylethyl salicylic acid (13)
6-(40,80-Dimethylnonyl)salicylic acid (14)
6-(20-Ethylheptyl)salicylic acid (15)
6-(20-Methylhexyl)salicylic acid (16)
6-(80E-Pentadecaenyl)salicylic acid (17)
6.25
>200
100
6-[20-(200,400,500-Trihydroxyphenyl)ethyl]salicylic acid (18)
6-[20-(200,500-Dihydroxyphenyl)ethyl]salicylic acid (19)
6-[20-(200,400-Dihydroxyphenyl)ethyl]salicylic acid (20)
6-[20-(300,400-Dihydroxyphenyl)ethyl]salicylic acid (21)
6-[20-(400-Hydroxyphenyl)ethyl]salicylic acid (22)
6-(20-Phenylethyl)salicylic acid (23)
>200
100
50
100
25
6-(40-Phenylbutyl)salicylic acid (24)
a The data were extracted from previous papers.2,10
the maximum activity was found for 6-cyclododecylm-
ethyl salicylic acid (12) with an MIC of 0.39 lg/mL. In
contrast, 6-cyclopentylmethyl salicylic acid (9) repre-
senting the smallest side-chain ring molecule in the
series was the least active analogue with an MIC of
100 lg/mL. In general the activity of compounds 9–13
increased in direct proportion to the number of C-atoms
in the cyclic ring at C-6.
In the aliphatic side chain series, the maximum activity
of C-6 branched side-chain anacardic acid analogues
against MRSA was found with 6-(40,80-dimethylno-
nyl)salicylic acid 14 with an MIC of 0.39 lg/mL. The
activity of 14 against MRSA was also 16 times stronger
than that of natural anacardic acid (C15:3) (1). With an
MIC of 50 lg/mL, 6-(20-methylhexyl)salicylic acid 16
was the least active branched side-chain anacardic acid
analogue and also incidentally represented the shortest
side chain in this series of analogues. The MIC of ana-
logue 15 with a C9H19 branched side chain was
6.25 lg/mL. It would thus appear that the more hydro-
phobic the C-6 side chains of anacardic acids are, the
more readily they should be able to disrupt membrane
integrity.
The activity of 12 increased 16 times compared to that of
anacardic acid (C15:3) (1). Compound 10 was 50% less
active than 13, with the essential difference between
them being an additional C-atom separating the phenyl
and cyclohexyl rings. The MIC of 6-cyclohexylethyl sal-
icylic acid 13 was 12.5 lg/mL for MRSA. 6-Cyclooc-
tylmethyl salicylic acid 11 exhibited stronger activity
than those of cyclopentylmethyl and cyclohexylmethyl
salicylic acid indicating that the molecular volume of
the side chain is an important contributing factor to
improved activity a fact very clearly demonstrated by
analogue 12 having the dodecyl ring. The MIC of 11
for MRSA was 12.5 lg/mL.
When tested against MRSA compound 15 exhibited the
same antibacterial activity as the natural anacardic acid
(C15:3) (1) with an MIC of 6.25 lg/mL. It seems that a
branched side chain increases antibacterial activity of
6-alkylsalicylic acids and that the presence of a double
bond in the C-6 hydrocarbon side chain may not

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I. R. Green et al. / Bioorg. Med. Chem. 15 (2007) 6236–6241
contribute all that significantly to the activity of these
types of analogues.
3. Conclusion
Since the head portion of the anacardic acids studied in
this series is the same, data are interpreted to signify that
changes in the hydrophobic tail portions may be corre-
lated and are indeed responsible for the specific activity.
As far as anacardic acids (1–4) are concerned, their anti-
bacterial activity can be optimized by selecting a suitable
side chain length to give the appropriate partition coef-
ficient (logP) as a standard. Our current study has dem-
onstrated that other factors viz. branching and
volumetric size introduced into the hydrophobic moiety
which is known to increase the solubility of the mole-
cules in water and in this way cause looser packing of
the surfactant molecules at the interface is also a major
contributing factor associated with the activity.23 The
surfactant molecules showed significant toxicity at high-
er concentrations. It is thus a strong possibility that ana-
cardic acids and their derivatives might be non-specific
cytotoxic agents. However, it has been demonstrated
that anacardic acid (C15:3) does not induce chromosome
damage in vivo,35 indicating that their analogues may
thus only possess low genotoxic properties. In view of
the increasing importance of controlling specific bacteria
such as MRSA, studies directed at both the branched
and alicyclic side chain anacardic acid analogues have
demonstrated that they do have the potential to pave
the way for a new generation of drugs with anti-MRSA
activity.
The anacardic acid analogues 18–24 having phenolic
moieties in their side chains at C-6 exhibited antibacte-
rial activities in which the MICs ranged between 25
and >200 lg/mL. The maximum activity occurred with
6-(40-phenylbutyl)salicylic acid (24) that had an MIC
of 25 lg/mL which is superior to that of 6-phenylethyl-
salicylic acid (23) that had an MIC of 100 lg/mL for
MRSA. It thus seems that both the length of the linker
alkyl side chain C4 in (24) versus C2 in (23) and the pres-
ence or absence of hydroxyl groups in the aryl ring viz.
4-OH in (22) versus 4-H in (23) may also play an impor-
tant role in increasing the activity of these molecules.
However, the absolute activity of 24 was no better than
that of 6-[80(Z),11(Z),140-pentadecatrienyl]salicylic acid
(1) that had an MIC 6.25 lg/mL.
One of the more important observations made from this
work was that the antibacterial activity was generally
lower for those anacardic acid analogues having a phe-
nolic and not an alkyl side chain at C-6. For example,
the MICs of compounds 18–22 that contain between
one to three hydroxyl groups on the aromatic ring of
the side chain varied between 50 and >200 lg/mL for
MRSA.
It is possible that the presence of hydrophilic groups
on both phenyl rings separately and collectively in
the molecule may lower the antibacterial activity
against MRSA.1 On the other hand, the MICs of
the compounds 18–21 suggest that the activity against
S. aureus is only affected by the number and position
of the hydroxyl groups on the phenyl ring of the side
chain. For instance, the activity of compound 18
(MIC >200 lg/mL) which contains three hydroxyl
groups was lower than that of either 19 or 21 (MIC’s
of 100 lg/mL) both of which possess two hydroxyl
groups on the side-chain phenyl ring. On the other
hand, compound 20 which also possesses two hydro-
xyl groups on the phenyl ring but at different posi-
tions, that is, a different regioisomer did not show
any activity against MRSA up to 200 lg/mL. When
tested against MRSA the MIC of 18 was comparable
with that of 20. It is thus clear that in these cases the
actual substitution pattern of the hydroxyl groups on
the phenyl rings has a role to play in the activity. It is
too early to speculate on the possible reasons for this
with the information at hand.
4. Experimental
4.1. Chemicals
Anacardic acids and their analogues were available from
previous works,1,7,15,27 and synthesis of 6-(80E-pentade-
caenyl)salicylic acid 17 was previously described.28–30
Salicylic acid and 2,2-diphenyl-1-picrylhydrazyl (DPPH)
were purchased from Sigma Chemical Co. (St. Louis,
MO). N,N-Dimethyl formamide (DMF) was obtained
from EM Science (Gibbstown, NJ). Other reagents were
purchased from commercial suppliers and used as
received, unless otherwise noted.
4.2. Synthesis
The synthetic work was previously published.28–30 In
brief, to a stirred solution of phosphonate ester 257
(2.0 g; 6.6 mM) in dry THF (20 mL) at 0 °C was added
a solution of potassium t-butoxide (1.25 g; 11.2 mM) in
THF (10 mL) under an atmosphere of nitrogen. The
resulting mixture was stirred at 0 °C for a further
Anacardic acids are known to act as multifunctional
agents with the two most important being antibacte-
rial and antioxidant.24,31,32 All the compounds tested
are assumed to possess chelation ability since this
activity originates from the salicylic acid moiety (the
functional unit in anacardic acid).33,34 In the cases
of compounds 18–24, none of these analogues exhib-
ited notable antibacterial activity, but some showed
good antioxidant activity. For example, 18 and 19
were found to scavenge DPPH and 19 has been
described to demonstrate weak inhibitory activity of
potato lipoxygenase.18
30 min after which
a solution of the aldehyde
(6.6 mM) in THF (15 mL) was added. Stirring was con-
tinued at 0 °C for a further 10 min and the solution then
allowed to warm up to 25 °C and stirred for another
12 h. The reaction mixture was quenched with saturated
aqueous ammonium chloride (100 mL) and extracted
with ether to afford a residue that was purified by col-
umn chromatography using ethyl acetate–hexane (1/4–
2/3) as eluent to yield the trans olefins 26. Catalytic
hydrogenation of the olefins 26 over Pd(0) (5% Pd/C)

I. R. Green et al. / Bioorg. Med. Chem. 15 (2007) 6236–6241
6241
yielded the corresponding saturated aryl esters 27. A
References and notes
solution of the ester 27 (2 mM) in DMSO (5 mL) was
treated with aqueous sodium hydroxide (5 mL of a
0.5-M solution, 2.5 mM) and stirred in an oil bath at
110 °C for 12 h after which the cooled solution was acid-
ified (litmus) with aqueous hydrogen chloride (1 M) and
then diluted with water to a volume of 20 mL and then
extracted with ethyl acetate. The residue obtained was
recrystallized from ethyl acetate–hexane to afford the
corresponding methoxy benzoic acids 28. Finally, to a
stirred solution of the methoxy benzoic acids 28 (1
mM) in 1,2-dichloroethane (10 mL) at between 25 and
ꢀ78 °C was added boron tribromide (1.1 mM per meth-
oxy group present) under nitrogen and stirring was con-
tinued for 3 h. The temperature of the reaction mixture
was allowed to rise to 25 °C after which water (100 mL)
was added and the solution acidified (litmus) by the
addition of aqueous hydrogen chloride (1 M). Extrac-
tion with ethyl acetate afforded the anacardic acids.
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broth and yeast extract were purchased from BD
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DMF, and 30 lL of each dilution was added to 3 mL
of NYG broth, which consisted of 0.8% nutrient broth,
0.5% yeast extract, and 0.1% glucose. These were inocu-
lated with 30 lL of an overnight culture of the test bac-
terium. After incubation of the cultures at 37 °C for
48 h, the MIC was determined as the lowest concentra-
tion of the test compound that demonstrated no visible
growth. Because of solubility limitation of the samples
in the water based medium, the highest concentration
tested in the assay was 200 lg/mL, unless otherwise
specified. The MIC of each compound was determined
at least in triplicate on separate occasions.
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using Crippen’s fragmentation.36
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Acknowledgments
´
35. Acevedo, H. R.; Rojas, M. D.; Arceo, S. D. B.; Hernan-
dez, M. S.; Vazquez, M. M.; Terrazas, T.; del Toro, G. V.
´
The authors are grateful to Dr. M. Himejima and Dr. H.
Muroi for performing the antibacterial assay at an ear-
lier stage of the work.
Mutat. Res. 2006, 609, 43.
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