excellent (≤0.50 g/mL), very good (1-2 g/mL), and good (3.9-
7.8 g/mL) activity against 5 (D, E, J, U, and X), 16 (A, C, F, H,
I, K, M-Q, S, V, W, Y, and Z), and 7 (B, G, L, R, T, AA, and
AB) of these S. aureus strains, respectively. We then investigated
the replacement of the selenium atom by sulfur by synthesizing
and testing ebsulfur (2a). Compound 2a was found to have
similar MIC values to those of ebselen against all S. aureus
strains tested. As replacement of the selenium atom by sulfur
could result in decreased toxicity, we decided to use compound
2a as our model compound for further derivatization and
evaluation.
MIC values (15.6 to 2 g/mL) relative to the parent compound
2a, suggesting that introduction of a nitrogen atom into the
phenyl ring may not be the way to pursue.
We next investigated the effect of replacing the phenyl ring
by linear alkyl chains, which were previously shown to improve
the antibacterial activity of another class of antibiotics, the
aminoglycosides.6-8,25,26 Compounds 3a-d were synthesized to
contain n-pentyl, n-hexyl, n-octyl, and n-dodecyl side chains
instead of the typical phenyl ring found in 2a. Among these
compounds, the n-octyl analogue (3c) displayed the best MIC
values (3.9 to ≤0.25 g/mL). Compounds 3a, 3b, and 3d
displayed MIC values (62.5 to 0.25 g/mL) that were similar to
that of the parent compound 2a. Inspired by our best compounds
in series 2 containing an isopropyl moiety, compounds 2e and 2h,
we decided to explore the effect of branched alkyl chains by
synthesizing and testing compounds 3e-g. These compounds
displayed mostly good to moderate activity (3.9 to 15.6 g/mL).
However, they were definitely inferior when comparing to
compounds 2e and 2h. Additionally, we were also able to isolate
the sulfoxide analogues of these compounds (4e and 4f) and
evaluated them. We were surprised to find that these oxidized
compounds completely lost their antibacterial activity against S.
aureus. This finding was consistent with previous reports that the
S-N bond is essential for biological activity by covalently
binding to cysteine residues of targeted enzymes.17,19
Previous literature on the antibacterial properties of ebselen
suggested that the 1,2-benzisothiazol-3(2H)-one core could be
required for antibacterial activity.16,19 We also thought of possibly
removing the annulated benzene ring of the core to generate 1,2-
isothiazolin-3-one analogues. However, we shied away from
these analogues once we realized that there were reports
suggesting that these compounds could be allergenic and
neurotoxic against humans.22,23 Thus, we decided to keep the 1,2-
benzisothiazol-3(2H)-one core intact and hypothesized that the
phenyl group adjacent to the core scaffold would be a good site
for our investigation. In search of chemical modifications to
increase the biological activity of 2a, we first replaced the phenyl
ring by the following moieties: substituted phenyl and other
aromatic rings (2b-o), alkyl chains (3a-g), alkyl chains with a
terminal phenyl group (3h-j), and aliphatic rings (3k-o). In
general, all of our analogues displayed moderate to excellent
activity against S. aureus (15.6 to ≤0.25 g/mL), except for
compounds 4e, 4f, and 4n, which were found to be completely
inactive (>125 g/mL) against all S. aureus strains tested. In
addition, compounds 2i, 2n, 2o, and 3o were not evaluated due to
solubility issues in liquid Mueller-Hinton medium.
In an attempt to further understand the SAR of the phenyl
ring, we explored whether having this ring directly attached to
(compound 2a) or at a distance from the 1,2-benzisothiazol-
3(2H)-one core made any difference. We synthesized compounds
3h-j with 1-3 carbon linkers separating the phenyl ring and the
core. These compounds displayed good to excellent activity (3.9
to ≤0.25 g/mL). Lastly, to confirm that the aromaticity of the
substituent is not required for antibacterial activity, we
synthesized a series of compounds containing different-sized
aliphatic rings (3k-o). We found that compound 3l with a
cyclohexyl ring displayed very similar MIC values (7.8 to 0.5
g/mL) to its aromatic counterpart 2a. We noted that all of these
compounds retained good to excellent antibacterial activity
against S. aureus, with the exception of the adamantyl derivative
3o, which could not be tested due to solubility issues.
Most of the compounds with mono- (2b, 2c, 2d, 2f, and 2g)
and disubstitutions (2i and 2j) at the 3- and 4-positions of the
phenyl ring were found to display good to excellent activity (3.9
to ≤0.25 g/mL), but the activity was very similar to that of 2a.
The three overall best compounds of this series of analogues (2e,
2h, and 2k) were found to have very good to excellent activity
(≤2.0 g/mL) across all S. aureus strains tested (Table 1). Among
these three, the relatively bulkier 4-isopropylphenyl (2e) and 3-
isopropylphenyl (2h) analogues were our two best compounds
and displayed up to 16-fold improvement in MIC values when
compared to the parent compound 2a. We also noticed that the
substitution pattern (p- vs m-isopropylphenyl (2e vs 2h) or p- vs
m-bromophenyl (2d vs 2g)) did not have a substantial effect on
the MIC values of these compounds (mostly within 2 fold
dilutions). Intrigued by this result, we synthesized and tested
more analogues with bulky substituents on the phenyl ring.
Surprisingly, the 2,3-dimethoxyphenyl analogue (2k) also
yielded very good to excellent MIC values (2 to ≤0.25 g/mL).
We decided to add even more bulkiness to the scaffold by
synthesizing the naphthyl analogue (2l), which displayed very
good to excellent MIC values (2 to ≤0.25 g/mL). This result
further suggested that adding different bulky groups to the phenyl
ring is a favorable strategy to increase activity.
2.2.2. Evaluation of compounds 2a-4n against various non-S.
aureus strains
To further examine the antibacterial spectrum of compounds
2a-4n, we also tested them against a panel of non-S. aureus
strains. Overall, we found that many of our ebsulfur (2a)
analogues were more specific towards S. aureus strains. We did
observe that a few analogues actually displayed moderate to
excellent activity (15.6 to ≤0.25 g/mL) against certain non-S.
aureus strains (Table 2). Interestingly, we found that ebselen still
displayed good to excellent activity against many non-S. aureus
strains (7.8 to ≤0.25 g/mL), in contrary to what was previously
observed when ebselen was tested against a different panel of
bacterial strains.16 We were especially enlightened to find that
our best compounds, 2e and 2h, still retained their excellent
activity (1 to ≤0.25 g/mL) against selected strains such as S.
epidermidis (AC), VRE (AG), L. monocytogenes (AH), and M.
smegmatis (AI).
To further understand the structure-activity-relationship
(SAR) of our ebsulfur scaffold, we decided to introduce a
heteroatom into the benzene ring. Previously, the 3-chloropyridyl
replacement of the phenyl ring in the ebselen scaffold was
reported to greatly reduce toxicity in mammalian HEK293T cell
line (IC50 >160 M).24 We applied this knowledge to our ebsulfur
scaffold and synthesized the pyridyl (2m), 3-chloropyridyl (2n),
as well as the bulky quinolinyl (2o) analogues. However, only
2m was soluble enough for biological testing, but yielded inferior
To gain a better understanding of the antibacterial spectrum
of our ebsulfur (2a) analogues, we decided to test them against a
biofilm-forming S. epidermidis (AC), another Gram-positive
bacterium of the genus Staphylococcus. As expected, other than
the previously inactive oxidized compounds 4e, 4f, and 4n, we
found that our entire library of analogues still retained moderate