ACS Medicinal Chemistry Letters
Letter
the membrane fluidity and decreases its integrity, while the
phenolic function is capable of disrupting the cation gradient
across the membrane leading to osmolysis.
Table 1. Results from Paraben Derivative Synthesis and
Antimicrobial Screening against S. aureus
Alternatively, parabens have been linked to breast cancer
through their xenoestrogen properties.3 Many phenols can bind
the estrogen receptor (ERα) leading to upregulation of gene
expression similar to estrogen.16 Admittedly, the system is
complex such that the genes expressed by xenoestrogen
compounds of similar structures (even differing parabens) can
lead to varying gene expression profiles.17 This activity requires
a specific binding interaction between the paraben and the ER-
estrogen binding site.18 From known xenoestrogen activities, it
has been shown that mimicking the phenolic function of
estrogen is most important for ERα binding and thus is the
most likely pharmacophore in parabens.19
The nonspecific (lacking enzymatic binding) antimicrobial
activities associated with parabens should lend greater flexibility
in their structure−activity relationship (SAR). Alternatively, the
specific binding of the phenolic function to the ERα is likely to
result in a less amenable SAR associated with their
xenoestrogen properties. A plethora of molecules capable of
activating the estrogen receptor have been reviewed, which
does indicate a potential flexibility in the binding pocket.
However, Terasaki et al. examined the activation of the ERα by
chlorinated parabens produced during the chlorination of
wastewater and found that mono- and dichlorinated parabens
tend to be less active or entirely inactive.20 This work is
encouraging to the prospects that dissociation of the two
activities is possible.
Herein, it can be seen that paraben activities can be
dissociated given that the antimicrobial properties result from a
nonspecific binding to the cell wall, while the precancerous
activity results from a specific binding of the ER. To show that
the disassociation of the SARs are related to a general blocking
of access to the phenolic function, a multitude of substituted
paraben was synthesized and screened. Thus, we report 3,5-
substitutions to butyl paraben derivatives that are equivalent if
not improved antimicrobial compounds that lack the
competitive ER binding. To confirm the continued antimicro-
bial properties of these compounds a microdilution assay was
used to determine minimum inhibitory concentrations (MIC).
Alternatively, an estradiol competition enzyme-linked immu-
nosorbent assay (ELISA) and cell proliferation assay were used
to illustrate the efficacy of these potential precancerous
activities. The affinity for the ERα was then determined by a
time-resolved fluorescence resonance energy transfer (TR-
FRET) binding assay.
Chemistry. To prevent alkylation of the phenolic function,
Fisher esterification, illustrated in Table 1, was used to convert
various 3,5-substituted p-hydroxybenzoic acid derivatives 1 to
their corresponding butyl or octyl esters, 2 and 3, respectively.
Catalytic amounts of sulfuric acid were used to enhance the
reaction rates, while tosylic acid failed to enhance conversion
despite being devoid of water. Ester formation often required
overnight (∼18 h) reflux but failed to show complete
conversion by thin layer chromatography (TLC). Electron
withdrawing groups such as halides and nitro functions allowed
for faster reaction times.
rxn
time
(h)
rxn
yield
(%)
S. aureus MIC
μg/mL (μM)
X, Y, Z
F, F, F
ROH
compd
BuOH
BuOH
BuOH
BuOH
BuOH
BuOH
BuOH
BuOH
BuOH
BuOH
BuOH
OctOH
OctOH
OctOH
OctOH
2a
2b
2c
2d
2e
2f
18
18
18
18
1
35
65
53
66
75
42
70
67
50
88
56
59
71
40
82
128 (483)
64 (245)
32 (91)
Cl, Cl, H
Br, Br, H
Br, H, H
I, I, H
64 (236)
16 (36)
I, H, H
18
18
18
18
18
3
64 (199)
a
CH3, CH3, H
2g
2h
2i
>256 (>1045)
a
t
tBu, Bu, H
>256 (834)
OH, OH, H
OMe, OMe, H
NO2, NO2, H
I, I, H
512 (2055)
a
2j
>256 (924)
2k
3e
3g
3i
512 (1809)
16 (31.8)
64 (213)
1
CH3, CH3, H
OH, OH, H
NO2, NO2, H
18
18
18
64 (210)
3k
16 (47)
b
butyl paraben
256 (1320)
0.25 (0.749)
penicillin G
a
Not soluble at the higher 512 μg/mL concentration and therefore
was not tested. Equivalent to butyl ester (2) with X, Y, Z = H, H, H.
b
by staining TLCs with KMnO4. Moderate yields could be
achieved even with these extra purification steps and can be
seen in Table 1.
Biology. Antimicrobial activity was determined by finding
the MIC via dilution assays in 96-well plates. Reproducibility
was confirmed through trials run in duplicate across three
separate days for a total of six biological replicates. S. aureus was
used as a representative Gram-positive bacteria. Each paraben
derivative was dissolved in biological grade DMSO to generate
stock solutions at both 128 and 16 mg/mL. Serial dilution of
the paraben derivatives then began at 512 μg/mL and occurred
in half-fold dilutions with the exception of those not soluble
above 256 μg/mL. Some samples reported with MICs at or
greater than 256, most notably 2h, illustrate the first
concentrations that appear visibly cloudy. Due to the assay
pushing the limits of these compounds solubility, active
compounds were tested with a minimum of one trial beginning
at 128 μg/mL (∼500 μM), including the parent butyl paraben,
to confirm dilution errors are not present. Butyl paraben and
penicillin G were used to compare activities. The results of
these studies can be found in Table 1.
Screening against S. aureus allowed for a SAR that could be
compared directly to the commercially available paraben. In all
derivatives we saw comparable if not improved antimicrobial
activity from prepared 3,5-substituted parabens. The increased
activity is likely a result of a combined increase in hydro-
phobicity and decrease in phenolic pKa. Halogenated parabens,
2a−2f, provided the greatest increase in activity against S.
aureus; however, such changes in phenols have been
documented before.20 Aliphatic substitutions, 2g and 2h,
initially appeared to have lost all activity; however, increasing
This process proved reliable, but product purification often
required multiple flash chromatography purifications. Through-
1
out purification, the purity was confirmed by H NMR to
ensure that the reactant alcohol was removed. The purity of
individual column fractions could be qualitatively determined
B
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX