2-Hydroxy-substituted cinnamic acids and acetanilides are selective growth inhibitors of Mycobacterium tuberculosis

Article abstract of DOI:10.1039/c3md00251a

Selective chemical hits are required for feeding the initial discovery phase of the anti-tuberculosis therapeutics pipeline. These chemical entities should ideally target novel mechanisms of action in order to tackle drug resistance in Mycobacterium tuberculosis. In this work, hydroxycinnamic acid and acetamidophenol skeleta were employed for assessing the effects of constitutional isomerism on in vitro anti-TB activity. The whole cell evaluation of minimum inhibitory concentration values of different substituted cinnamic acids and acetamidophenols showed that the free ortho hydroxyl group conferred both potency and selectivity. Both 2-coumaric acid and 2-acetamidophenol showed minimum inhibitory concentration below 150 μM against M. tuberculosis H ₃₇Rv and selectivity index higher than 30.

Full text of DOI:10.1039/c3md00251a

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CONCISE ARTICLE
2-Hydroxy-substituted cinnamic acids and
acetanilides are selective growth inhibitors of
Mycobacterium tuberculosis†
Cite this: Med. Chem. Commun., 2014,
5, 47
Juan D. Guzman, abc Parisa N. Mortazavi,a Tulika Munshi,a Dimitrios Evangelopoulos,d
*
Timothy D. McHugh,d Simon Gibbons,b John Malkinsonb and Sanjib Bhaktaa
Selective chemical hits are required for feeding the initial discovery phase of the anti-tuberculosis
therapeutics pipeline. These chemical entities should ideally target novel mechanisms of action in order
to tackle drug resistance in Mycobacterium tuberculosis. In this work, hydroxycinnamic acid and
acetamidophenol skeleta were employed for assessing the effects of constitutional isomerism on in vitro
anti-TB activity. The whole cell evaluation of minimum inhibitory concentration values of different
substituted cinnamic acids and acetamidophenols showed that the free ortho hydroxyl group conferred
both potency and selectivity. Both 2-coumaric acid and 2-acetamidophenol showed minimum inhibitory
concentration below 150 mM against M. tuberculosis H37Rv and selectivity index higher than 30.
Received 3rd September 2013
Accepted 27th October 2013
DOI: 10.1039/c3md00251a
www.rsc.org/medchemcomm
pathogens.6 In fact, historically all of the antibiotics and current
drugs in the pipeline against TB have been developed based on
Introduction
their whole–cell activity.
Tuberculosis (TB) continues to be a great scourge for humanity.
According to the last report from the WHO, in 2009 almost 10
million children were orphaned as a consequence of parental
death caused by TB.1 Drug-resistant strains of the causal path-
ogen Mycobacterium tuberculosis, are rising everywhere and
there were recently reports of isolates resistant to all the tested
drugs in Iran, Italy and India, which once again triggered
alarm.2 These drug-resistant strains of M. tuberculosis are
extremely difficult to treat. In HIV-prevalent regions, infection
by drug-resistant TB almost always has fatal consequences.3
Certainly there is an urgent need for new anti-TB drugs that are
effective in reducing the length of treatment and for combatting
drug-resistant strains. Target-based exploration efforts have
been unfruitful in antimicrobial drug discovery.4,5 The reasons
for this failure have been attributed to the difficulty in crossing
the bacterial cell wall to reach their intracellular targets, or to
the physicochemical properties of the members of the
screening libraries which are not ideal for targeting microbial
Cinnamic acid, which is typically found in cinnamon (Cin-
namomum verum) and storax (Liquidambar orientalis), was used in
the 19th century for treating TB.7 Substituted cinnamic acids
have been recently reported as novel scaffolds for the develop-
ment of selective anti-TB agents.8–10 In particular, O-prenylation
and O-geranylation of 4-hydroxycinnamic acids have been found
to accord increased anti-TB activity in a series of thioester, amide
and azide derivatives.9 In this work we evaluated different posi-
tional isomers of hydroxycinnamic (coumaric) acid with the aim
of extending the study of O-prenylation and O-geranylation of
coumarates on anti-TB activity. In addition the positional
isomers of 4-acetamidophenol, a widely used and relatively safe
analgesic known as paracetamol,11 were also included in this
study to examine a possible trend of anti-TB activity among
positional isomers of aromatic compounds. This class has never
been reported to display anti-mycobacterial properties.
Results and discussion
Chemistry
aDepartment of Biological Sciences, Institute of Structural and Molecular Biology,
Birkbeck, Malet Street, London, WC1E 7HX, UK
The synthesis of 4-, 3- and 2-O-prenyl-substituted coumaric
acids 4a–c was carried out by a slight modication of the
bDepartment of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy,
29-39 Brunswick Square, London, WC1N 1AX, UK
Departamento de Quımica y Biologıa, Universidad del Norte, Km 5 vıa Puerto synthetic route reported by De et al. in 2011.9 Firstly, coumaric
c
´
´
´
Colombia, Barranquilla, Colombia. E-mail: jguzmand@uninorte.edu.co; Fax: +57 5
acids 1a–c (Fig. 1) were esteried under Fischer conditions, to
3598852; Tel: +57 5 3509509 ext. 4284
yield the corresponding methyl coumarates 2a–c (Scheme 1).
dCentre for Clinical Microbiology, Department of Infection, Royal Free Campus,
The O-prenylated methyl coumarates 3a–c were obtained by
University College London, London, NW3 2QG, UK
reuxing the esters in acetone with 3,3-dimethylallyl bromide
† Electronic supplementary information (ESI) available: Materials, synthetic
and potassium carbonate. Several attempts to deprotect the
procedures, experimental data of compounds 2a–c, 3a–c, 4a–c, 5a–b and
biological evaluation methods can be found as ESI. See DOI: 10.1039/c3md00251a methyl esters 3a–c to yield the carboxylic acids using potassium
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Fig. 2 Structures of the geranylated 4-coumaric derivatives. Chemical
structures of methyl O-geranyl-4-coumarate (5a) and O-geranyl-4-
coumaric acid (5b).
(MIC ¼ 366 mM) against M. tuberculosis H37Rv. The same trend
of activity was observed against M. bovis BCG. The three posi-
tional isomers 1a–c showed comparable cytotoxicity with GIC50
values between 4560 and 5294 mM. The most selective coumaric
acid was the 2-hydroxycinnamic acid (1c), achieving an SI value
higher than 37.
Cinnamic acid (1d) completely inhibited M. tuberculosis
H37Rv growth at 270 mM (Table 1), showing a GIC50 of 6547 mM
against the macrophage cell line. Its selectivity index (SI) was 24.
3,4-Methylenedioxycinnamic acid (1e) showed an MIC value of
312 mM, however it was the most cytotoxic of the commercially
available cinnamic acids with a GIC50 value of 2774 mM,
attaining an SI less than 10.
Fig. 1 Structure of the commercially available cinnamic acids and
acetamidophenols used in this study. Chemical structures of 4-cou-
maric acid (1a), 3-coumaric acid (1b), 2-coumaric acid (1c), cinnamic
acid (1d), 3,4-methylenedioxycinnamic acid (1e), 4-acetamidophenol
(6a), 3-acetamidophenol (6b) and 2-acetamidophenol (6c).
carbonate under different conditions were unsuccessful.
Saponication was successfully performed by treating 3a–c with
an excess of lithium hydroxide in a methanol/water (3 : 1)
mixture at room temperature for 16 h which yielded the O-
prenylated coumaric acids 4a–c in satisfactory yields (Scheme
1). The O-geranyl derivatives of methyl 4-coumarate and 4-cou-
maric acid, 5a and 5b (Fig. 2), were prepared following the same
procedure described in Scheme 1, using geranyl bromide
instead of prenyl bromide. For a description of experimental
procedures and characterisation data refer to Supplementary
Information.
Methyl coumarates 2a–c displayed MIC values (Table 1)
comparable to the corresponding carboxylic acids 1a–c, with
values ranging from 112 to 224 mM. However the esters were
signicantly more cytotoxic than the free carboxylic acids having
GIC50 values between 609 and 1125 mM. Among the methyl
coumarates, the ortho-substituted 2c was the most potent and
selective, with an MIC value of 112 mM and an SI of 10.
O-Prenylated methyl 4-coumarate 3a inhibited the growth of
M. tuberculosis H37Rv at a concentration of 81.2 mM, whereas the
meta and ortho derivatives, 3b and 3c, were less active with MIC
values of 244 and >406 mM respectively. However 3a displayed
high cytotoxicity with a GIC50 value of only 487 mM, achieving a
low SI of 6. O-Prenylation increased the toxicity of the para
isomer (from 609 to 487 mM) whereas the meta and ortho
Biological activity
Minimum inhibitory concentration (MIC) values were deter- isomers reduced their cytotoxicity (from 850 to 2972 mM, and
mined against both M. tuberculosis H37Rv and M. bovis BCG from 1125 to 2598 mM respectively) compared to the non-
using the spot culture growth inhibition assay (SPOTi) in 24 or prenylated compounds. O-Geranylation of 2a yielded 5a which
96 well-plates.12,13 Half growth inhibitory concentrations (GIC50
)
was found to be the most cytotoxic of its class, with a GIC50 of
against RAW264.7 murine macrophages enabled the evaluation 159 mM, without signicant gain in anti-mycobacterial activity.
of cytotoxicity and anti-TB selectivity. Among the coumaric These results led us to hypothesise that the para-O-prenyl/
acids 1a–c, clearly the most active was the ortho substituted 1c geranyl derivatives may act by a different biological mecha-
with an MIC value of 122 mM (Table 1), followed by the para nism compared to the meta and ortho-O-prenyl derivatives,
substituted 1a (MIC ¼ 244 mM) and then the meta substituted 1b because O-prenylation of the para derivative increased
Scheme 1 Synthetic route for prenylation of the structural isomers of coumaric acid. Reagents and conditions: (i) H2SO4, MeOH, reflux, 16 h,
>97%; (ii) 3,3-dimethylallyl bromide, K2CO3, acetone, reflux, 24 h, 37–91%; (iii) LiOH, MeOH/H2O, rt, 16 h, 42–96%.
48 | Med. Chem. Commun., 2014, 5, 47–50
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Table 1 Anti-tubercular activity and cytotoxicity of substituted cin-
namic acids (1a–e, 2a–c, 3a–c, 4a–c, 5a–b) and acetamidophenols
(6a–c) against M. tuberculosis H37Rv, M. bovis BCG and murine
macrophage RAW264.7 cell line. Isoniazid (INH) and rifampin (RIF)
were included as positive controls
MIC
(mM) H37Rv
MIC
(mM) BCG
GIC50
(mM) RAW264.7
Selectivity
index (SI)
Entry
1a
1b
1c
1d
1e
2a
2b
2c
3a
3b
3c
4a
4b
4c
244
366
122
270
312
224
224
112
81.2
244
>406
86.1
172
366
609
244
405
416
337
561
112
60.9
244
>406
86.1
258
258
254
134
4560 ꢀ 160
5294 ꢀ 226
4563 ꢀ 310
6547 ꢀ 205
2774 ꢀ 178
609 ꢀ 194
850 ꢀ 148
1125 ꢀ 247
487 ꢀ 183
2972 ꢀ 110
2598 ꢀ 151
1339 ꢀ 172
1296 ꢀ 302
1683 ꢀ 124
159 ꢀ 105
210 ꢀ 82
18.7
14.5
37.4
24.2
8.80
2.72
3.80
10.0
6.00
12.2
<6.40
15.6
7.53
6.52
1.25
3.14
<1.52
<8.08
31.8
30 000
14 000
Fig. 3 Chemical structures of 1c, 6c, PAS, and zosteric acid. Chemical
structures of 2-coumaric acid (1c), 2-acetamidophenol (6c) and para-
aminosalycilic acid (PAS) and zosteric acid.
258
127
5a
5b
6a
6b
6c
66.8
>661
>661
132
0.73
60.7 nM
>661
>661
132
0.73
60.7 nM
1002 ꢀ 68
5338 ꢀ 602
4204 ꢀ 265
21 900
However the ortho isomer 6c was a notable inhibitor of myco-
bacterial growth, with an MIC value of 132 mM against both
species. This compound displayed a RAW264.7 GIC50 close to
4200 mM and its SI was calculated to be 32. Surprisingly the para
isomer (which is widely used as analgesic) was far more cyto-
toxic than the other structural isomers.
INH
RIF
850
cytotoxicity whereas the opposite effect was observed for the
meta and ortho methyl coumarates.
Compounds 1a–1e, 2a–c, 3a–c, 4a–c, 5a–b and 6a–c were
tested against the rapid-growing species Mycobacterium smeg-
matis mc2-155, each displaying MIC values higher than 600 mM.
The narrow spectrum of activity of 1c and 6c suggested the
possibility that these compounds operate by interfering with a
unique biological mechanism present specically in members
of the M. tuberculosis complex.
The O-prenyl 4-coumaric acid 4a was equally as active as the
ester 3a displaying an MIC value of 86.1 mM against both M.
tuberculosis H37Rv and M. bovis BCG (Table 1). This free
carboxylic acid derivative showed higher selectivity against
mycobacterial cells than mammalian macrophages with an SI of
15.6. The other prenylated coumaric acids 4b and 4c were less
active, with MIC values between 172 and 258 mM, achieving SI
values lower than 10. The O-geranyl 4-coumaric acid 5b was
more active than the methyl ester 5a, with an MIC value of 66.8
mM, but was toxic, achieving a low SI value of 3.
The presence of unsaturation on the propenyl chain of the
cinnamic acids was found to be essential for anti-TB activity. All
of the ve dihydro derivatives of 1a–e, obtained by catalytic
hydrogenation using Pd/C in methanol (data not shown) were
found to be inactive against M. bovis BCG with MIC values
higher than 600 mM. Moreover the free phenolic OH was also
found to be important for anti-TB activity and selectivity. Zos-
teric acid (Fig. 3), a naturally occurring para-sulfooxy (–OSO3H)
derivative of coumaric acid is known for its potent antifouling
and antimicrobial effects.14 This natural compound and its
ortho and meta constitutional isomers were prepared by treating
1a–c with sulphur trioxide/pyridine complex15 (data not shown).
All of the sulfooxy derivatives were devoid of growth inhibitory
activity against both M. tuberculosis H37Rv and M. bovis BCG.
The constitutional isomers of the acetamidophenols were
also screened for their anti-TB activity and cytotoxicity. The para
The most potent molecules evaluated in this study, 2-cou-
maric acid (1c) and 2-acetamidophenol (6c) share structural
similarity with 4-aminosalicylic acid (PAS), a well known second-
line anti-TB drug (Fig. 3). It can be hypothesised that 2-coumaric
acid and 2-acetamidophenol may act via a similar biological
mechanism to PAS, either by interference with the folate
pathway16 or with iron uptake.17 In addition, the structural
similarity serves to support predictions of potentially more active
anti-TB agents. Introducing amino groups to 1c and 6c in the
meta position to the phenolic hydroxyl (and para with respect to
the other substituent), leading to 4-amino-2-coumaric acid and 2-
acetamido-5-aminophenol respectively, could be the rst step for
obtaining a new generation of anti-tuberculars. It seems that the
minimum structural requirement for anti-TB activity of this
group is the presence of an aromatic nucleus with a conjugated
group in position ortho to a free phenolic hydroxyl. Further
structural requirements for increasing potency on this basic
skeleton will be elucidated in future studies.
Conclusions
and meta isomers 6a–b were inactive, with MIC values higher Among a variety of cinnamic acids, 2-coumaric acid (1c) was
than 661 mM against M. tuberculosis H37Rv and M. bovis BCG. identied as the most potent and selective anti-TB agent with an
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MIC value of 122 mM against M. tuberculosis H37Rv and an SI
value of 37. Methyl esterication and O-prenylation of cou-
maric acids, yielded derivatives usually more toxic and/or less
active than the parent compounds. Structural isomers of
acetamidophenol were also screened for anti-TB activity, and
interestingly 2-acetamidophenol was found to be the most
active, with an MIC value of 132 mM against M. tuberculosis
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