Efficient synthesis of anacardic acid analogues and their antibacterial activities

Article abstract of DOI:10.1016/j.bmcl.2013.01.074

Anacardic acid derivatives exhibit a broad range of biological activities. In this report, an efficient method for the synthesis of anacardic acid derivatives was explored, and a small set of salicylic acid variants synthesised retaining a constant hydrophobic element (a naphthyl tail). The naphthyl side chain was introduced via Wittig reaction and the aldehyde installed using directed ortho-metalation reaction of the substituted o-anisic acids. The failure of ortho-metalation using unprotected carboxylic acid group compelled us to use directed ortho-metalation in which a tertiary amide was used as a strong ortho-directing group. In the initial route, tertiary amide cleavage during final step was challenging, but cleaving the tertiary amide before Wittig reaction was beneficial. The Wittig reaction with protected carboxylic group (methyl ester) resulted in side-products whereas using sodium salt resulted in higher yields. The novel compounds were screened for antibacterial activity and cytotoxicity. Although substitution on the salicylic head group enhanced antibacterial activities they also enhanced cytotoxicity.

Full text of DOI:10.1016/j.bmcl.2013.01.074

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1667–1670
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis of anacardic acid analogues and their antibacterial
activities
⇑
Sreeman K. Mamidyala, Soumya Ramu, Johnny X. Huang, Avril A. B. Robertson, Matthew A. Cooper
Institute for Molecular Bioscience, University of Queensland, St Lucia QLD 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Anacardic acid derivatives exhibit a broad range of biological activities. In this report, an efficient method
for the synthesis of anacardic acid derivatives was explored, and a small set of salicylic acid variants syn-
thesised retaining a constant hydrophobic element (a naphthyl tail). The naphthyl side chain was intro-
duced via Wittig reaction and the aldehyde installed using directed ortho-metalation reaction of the
substituted o-anisic acids. The failure of ortho-metalation using unprotected carboxylic acid group com-
pelled us to use directed ortho-metalation in which a tertiary amide was used as a strong ortho-directing
group. In the initial route, tertiary amide cleavage during final step was challenging, but cleaving the ter-
tiary amide before Wittig reaction was beneficial. The Wittig reaction with protected carboxylic group
(methyl ester) resulted in side-products whereas using sodium salt resulted in higher yields. The novel
compounds were screened for antibacterial activity and cytotoxicity. Although substitution on the sali-
cylic head group enhanced antibacterial activities they also enhanced cytotoxicity.
Received 20 December 2012
Accepted 16 January 2013
Available online 29 January 2013
Keywords:
Anacardic acid
Antibiotic
Directed ortho-metalation reaction
Wittig reaction
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
The advent of bacterial resistance due to global gene transfer
(metallo-b-lactamase NDM-1),1 and excessive use of antibiotics is
life threatening posing a loss of therapeutic options.2,3 Hence, there
is an urgent need to develop new antibacterial agents to combat
bacterial resistance. Anacardic acid and its derivatives were first
isolated from the cashew Anacardium occidentale (Anacardiaceae)4
and exhibited a broad range of biological activities such as antiox-
idant, anti-inflammatory, anti-cancer and antibacterial, including
activity against MRSA.5 During the course of our work6 on the dis-
covery of new antibiotics, we have synthesised a number of ana-
cardic acid analogues and were interested to further explore
their potential as antimicrobial agents. Naturally occurring anacar-
dic acid is comprised of a salicylic acid group substituted with alkyl
chains of 15–17 carbon length. Previous antibacterial studies based
on this simple scaffold,7,8 revealed a number of structure–activity
relationships (SAR) (Figs. 1 and 2); (a) the salicylic acid moiety is
important for antibacterial activity and protection leads to inactive
compounds,7 (b) modifying the carboxylic acid group to benzyl
amine and its derivatives does not improve the activity,9 (c) the
hydrophobic ‘tail’ makes a significant contribution to the antimi-
crobial activity where an aliphatic alkyl chain or cyclo-octane were
the most active and substituted phenolic groups were inactive, (d)
protection of phenolic hydroxyl (ethyl or isopropyl ether) and
benzamide derivatives of salicylic group are cytotoxic.10 It is as-
sumed that antibacterial activity of these molecules might be
due to disorder in the fluid bilayer of the membrane or surfactant
properties and non-specific activity. In this report, we describe an
efficient method for the synthesis of anacardic acid derivatives and
explore their antibacterial and cytotoxic activities. Our analogues
modify the salicylic acid core structure by modulating the electron
density of the aromatic ring and acidity of the protons while
retaining a consistent 2-carbon linker to a naphthyl group.
A small set of salicylic acid variants were synthesised while
retaining a constant hydrophobic naphthyl tail and short two car-
bon linker. The naphthyl side chain was introduced via Wittig reac-
tion and aldehyde was installed using ortho-metalation of the
substituted o-anisic acids (Scheme 1).
The initial synthetic route started from 2-methoxybenzoic acid
(1a) that was subjected to an ortho-metalation reaction11,12 with
s-BuLi and in situ electrophilic substitution by formyl group (using
two acidic hydrogens are important
OAc
not active
OH
CO2Me
not active
CO2H
Core
Tail
linker
lipophilic tail
C-C bond with one or two carbon
chain linker and hydrophobic bulky
cyclic ring (6-8) group is good
long chain aliphatic
group is good
⇑
Corresponding author. Tel.: +61 7 3346 2044; fax: +61 7 3346 2090.
E-mail addresses: m.cooper@uq.edu.au, m.cooper@imb.uq.edu.au (M.A. Cooper).
Figure 1. Preliminary SAR of anacardic acid analogues.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.074

1668
S. K. Mamidyala et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1667–1670
Figure 2. Known anacardic acid analogues.
the acid 10a, which was in equilibrium with its corresponding lac-
OMe
O
O
OH
OH
O
tol. The compound 10a on reaction with naphthyl ylide using
t-BuOK resulted in a complex mixture and failed to give the ole-
fin.20 Hence, the lactol was methylated using the known procedure
by DBU and MeI,21 to give methyl ester 11a in 95% yield (Scheme 3).
The compound 11a on subjecting to the Wittig olefination reaction
also failed to give the olefin resulting in the isolation of side prod-
ucts. The formation of by-products was explained based on the for-
mation of methylated lactol and may be the formation of alkyne by
ring opening with strong base such as t-BuOK.20
OH
OH
X
O
X
X
R
OH
Head group substrates
Tail
X
OMe O
OH
CO2H
Y
Alternatively, it was evident from the earlier report20 that olef-
ination reaction of the lactol using sodium salt would result in the
formation of olefin in much higher yields than methyl ester. Hence,
the olefination reaction of lactol 10a was performed by the use of
NaH at 50 °C, which resulted in the formation of olefin 12a in quan-
titative yields (see Supplementary data). The olefin 12a on depro-
tection with BBr3 gave compound 13a, which on reduction with
Pd/C gave the compound 14a (Scheme 4). Once the synthetic route
was established we pursued the other 4-substituted 2-methoxy-
benzoic acid derivatives (viz. 4-Cl, 4-OMe, 4-CH3, and 4-NO2)22
by, performing similar reactions that resulted in the formation ana-
logues of 14a (see Table S1 in Supplementary data). The ortho-
metalation reaction 4-nitro-2-methoxybenzoic acid derivative
(4e) failed to give the substituted aldehyde (5e) due to the strong
deactivating nature of nitro group.
Once we accomplished the synthesis of 4-substituted ana-
logues, we focused on sterically bulky groups. Thus, 1-methoxy
and 3-methoxy naphthoic acids 2a, 2b were converted to tertiary
amides 15a, 15b, respectively (Scheme 5). The compound 15a on
subjecting to directed ortho-metalation reaction gave the required
aldehyde 16a whereas the 3-methoxy derivative 15b failed to give
the ortho-substituted product. The aldehyde 16a on deprotection
with HCl/AcOH resulted in the formation of decomposition prod-
ucts. Therefore, the synthetic strategy failed with the bulky naph-
thyl derivatives. In continuing efforts to explore the new
modification, we also explored other head group such as phenolic
head group by performing similar reactions as explored for the pre-
vious compounds (Supplementary data).
X
3
1 a-e
X = H, OMe, Cl, Me, NO2
2a
) X = OCH3, Y = H
2b) X = H, Y = OCH3
Scheme 1. Retrosynthetic analysis.
DMF as a source). The formation of the ortho substituted product
was not observed. A similar reaction with 4-methoxy or 4-chloro
substituted 2-methoxybenzoic acids (1b, 1c) also failed to give
the required product and instead resulted in a complex mixture.
Challenges encountered in performing the ortho-metalation in
the presence of an unprotected carboxylic acid group compelled
us to use directed ortho-metalation (DoM)13 where a tertiary
amide was used as a strong ortho-directing group.14 Thus, 2-meth-
oxybenzoic acid (1a), on reaction with thionyl chloride at reflux,
gave the acid chloride, which on treating with diethyl amine re-
sulted in the formation of diethyl benzamide 4a.15 The compound
4a was subjected to ortho-metalation using s-BuLi followed by
in situ formylation to give the aldehyde 5a in quantitative yield
(Scheme 2).16
The aldehyde 5a on Wittig reaction17 with naphthyl ylide using
t-BuOK in THF resulted in the formation of a mixture of cis/trans
olefins 6a, 7a. The olefins were separated by column chromatogra-
phy and characterised by LCMS, 1H and 13C NMR.18 The double
bond was reduced using 10% Pd/C under H2 atmosphere to give
the compound 8a. The methyl ether of the compound 8a was
deprotected using 48% aq HBr solution at reflux temperature to
give the compound 9a. Similar reactions were performed with
2,4-dimethoxybenzoic acid (1b) to obtain the compound 9b. The
deprotection of diethylamide group was not achieved when con-
ducted using aq HBr, alternative attempts to cleave diethylamide
were also unsuccessful. Hence, we decided to cleave the diethyl
amide group before conducting the Wittig reaction. The compound
5a on treating with AcOH/HCl19 at reflux temperature for 12 h gave
The compounds synthesized were tested for antibacterial
activity and cytotoxicity using standard assay procedures. The
antibacterial MIC were evaluated against sensitive and resistant
Gram-positive strains (Staphylococcus aureus-ATCC25923, MRSA-
ATCC43300,, S. aureus-NRS 19) and Gram-negative organisms
(Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosae)

S. K. Mamidyala et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1667–1670
OMe O
1669
OMe O
OMe
i, ii
iii
CO2H
NEt2
CHO
5c)
NEt2
X
X
X
5a)
X = H
X = Cl
5d) X = CH3
4c)
4a)
X = Cl
4d) X = CH3
4e)
1c)
X = H
X = Cl
1a)
X = H
5b) X = OMe
4b) X = OMe
1b) X = OMe 1d) X = CH3
5e)
X = NO2
X = NO2
1e)
X = NO2
O
OMe
NEt2
OMe O
BrPh3P
X
NEt2
+
D
X
5a, 5b
iv
7a
6a
) X = H
) X = H
7b) X = OMe
6b) X = OMe
OH
O
OMe O
) X = H
v
vi
NEt2
NEt2
X
X
9a
) X = H
9b) X = OMe
8a
8b) X = OMe
Scheme 2. Reagents and conditions: (i) SOCl2, reflux, 3 h; (ii) Et2NH, CH2Cl2, 0 °C–rt, 12 h; (ii) s-BuLi/TMEDA, À78 °C, DMF; (iv) t-BuOK, THF, 0 °C–rt, 12 h; (v) Pd/C (10%) H2
atm, rt, 1 h; (vi) 48% aq HBr, reflux, 5 h.
OMe O
OMe
X
X
O
Y
O
i
CO2H
OH
CHO
i, ii
NEt2
5a, 5b
O
X
X
Y
10a
OH
) X = H
15a
) X = OMe, Y = H
15b) X = H, Y = OMe
2a
) X = OMe, Y = H
2b) X = H, Y = OMe
10b) X = OMe
iii
iii
iii
16a
) X = OMe, Y = CHO
16b) X = CHO, Y = OMe
X
OMe O
Scheme 5. Reagents and conditions: (i) SOCl2, reflux, 3 h; (ii) Et2NH, CH2Cl2, 0 °C–rt,
12 h; (iii) s-BuLi/TMEDA, À78 °C, DMF.
OMe
X
X
CHO
11a
) X = H
11b) X = OCH3
with tertiary amide did not show any activity against G-positive
bacteria (Supplementary data). The compounds with free phenolic
and carboxylic groups were active against Gram-positive bacteria.
Among the compounds containing carbon–carbon double bond
with free carboxylic acid, methyl ether compound 12c showed
Scheme 3. Reagents and conditions: (i) AcOH/HCl, reflux, 12 h; (ii) DBU-MeI,
MeCN, rt; (iii) D, t-BuOK, THF, 0 °C–rt, 12 h.
using CLSI standards23 by twofold serial broth micro dilution. Of
the compounds tested for antibacterial MIC, no compounds
showed activity against G-negative bacteria and the compounds
64
lg/mL against MRSA. The compounds containing carbon–car-
bon double bond linker with free carboxylic acid and phenolic
group showed 16–64
lg/mL activities against Gram-positive
OMe O
OMe
O
i
OH
CHO
ii
5a d
−
O
X
X
OH
10a) X = H
10c)
X = Cl
10d) X = CH3
10b
) X = OMe
OH
O
OH
O
OMe O
iv
iii
OH
OH
OH
X
X
X
14a)
14b)
X = H
X = OMe
14c)
X = Cl
14d) X = CH3
13a) X = H
13b)
13c) X = Cl
13d)
12a) X = H
12b)
12c) X = Cl
12d)
X = OMe
X = OMe
X = CH3
X = CH3
Scheme 4. Reagents and conditions: (i) AcOH/HCl, reflux, 12 h; (ii) D, NaH, THF, 50 °C, 2 h; (iii) BBr3, CH2Cl2; (iv) Pd/C (10%), H2 atm., rt, 1 h.

1670
S. K. Mamidyala et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1667–1670
Table 1
Biological assay results
Compounds
Vancomycin
12a
13a
14a
12b
13b
14b
12c
13c
14c
12d
13d
14d
G-positive (MIC
S. aureusa
MRSAb
l
1
2
1
g/mL)
>128
>128
>128
64
64
64
>128
>128
>128
>128
>128
>128
>128
128
128
64
64
128
>128
64
128
16
16
16
16
16/32
128
>128
>128
>128
16
16
32
32
64
128
S. aureusc
Cytotoxicity (CC50 lM)
HEK293
HepG2
Nd
Nd
>100
>100
97.0
71
>100
>100
>100
>100
65
79
65
71
93
104
38
25
35
35
119.2
117
52
30
40
38
a
b
c
ATCC25923.
ATCC43300.
NRS 19 (GISA, glycopeptide-intermediate S. aureus).
bacteria. The compounds containing carbon–carbon single bond
linker with free carboxylic acid and phenolic group showed 16–
128 lg/mL activities against Gram-positive bacteria. The cytotoxic-
of biological assays) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
01.074.
ity of the compounds was evaluated against HepG2 and HEK293
cell lines and is detailed in the Supplementary data. Compared to
o-anisic acid derivatives the substituted o-anisic acid derivatives
gave no significant improvement in antimicrobial activities but
were found to be more cytotoxic (Table 1).
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In summary we have developed an efficient method for the syn-
thesis of anacardic acid derivatives. The failure of ortho-metalation
in the presence of an unprotected carboxylic acid group compelled
us to use directed ortho-metalation where a tertiary amide was
used as a strong ortho-directing group. In the initial route using
tertiary amide cleavage during the final step was challenging and
cleaving the tertiary amide before Wittig reaction was beneficial.
The Wittig reaction with protected carboxylic group (methyl ester)
resulted in side-products and using sodium salt resulted in higher
yields. The new compounds synthesized were screened for anti-
bacterial activity and cytotoxicity. Although substitution on the
salicylic head group improved antibacterial activities the com-
pounds were cytotoxic and were not pursued as antibacterial leads.
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The authors are thankful to National Health and Medical Re-
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ing. The GISA isolates was obtained through the Network on
Antimicrobial Resistance in Staphylococcus aureus (NARSA) pro-
gram:
supported
under
NIAID,
NIH
Contract
HHSN272200700055C.
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Supplementary data
Supplementary data (experimental details for the synthesis of
new compounds, analytical data including NMR spectra and details