N-Thiolated β-lactam antibacterials: Effects of the N-organothio substituent on anti-MRSA activity

Article abstract of DOI:10.1016/j.bmc.2006.01.029

A study on the structure-activity profiles of N-thiolated β-lactams 1 is reported which demonstrates the importance of the N-organothio moiety on antibacterial activity. Our results indicate that elongation of the N-alkylthio residue beyond two carbons, or extensive branching within the organothio substituent, diminishes antibacterial effects. Of the derivatives we examined, the N-sec-butylthio β-lactam derivative 5g possesses the strongest growth inhibitory activity against methicillin-resistant Staphylococcus aureus strains. Sulfur oxidation state is important, as the N-sulfenyl and N-sulfinyl groups provide for the best antibacterial activity, while lactams bearing the N-sulfonyl or N-sulfonic acid functionalities have much weaker or no anti-MRSA properties. Stereochemistry within the organothio chain does not seem to be a significant factor, although for N-sec-butylthio β-lactams 15a-d, the 3R,4S-lactams 15c, d are more active than the 3S,4R-stereoisomers 15a, b in agar diffusion experiments. The N-methylthio lactams are the most sensitive to the presence of glutathione, followed by N-ethylthio and N-sec-butylthio lactams, which indicates that bioactivity and perhaps bacterial selectivity of the lactams may be related to the amount of organothiols in the bacterial cell. These results support the empirical model for the mechanism of action of the compounds in which the lactam transverses the bacterial membrane to deliver the organothio moiety to its cellular target.

Full text of DOI:10.1016/j.bmc.2006.01.029

Bioorganic & Medicinal Chemistry 14 (2006) 3775–3784
N-Thiolated b-lactam antibacterials: Effects of the
N-organothio substituent on anti-MRSA activity
Bart Heldreth,a Timothy E. Long,a Seyoung Jang,a G. Suresh Kumar Reddy,a
Edward Turos,a,* Sonja Dickeyb and Daniel V. Limb
aDepartment of Chemistry, 4202 East Fowler Avenue, SCA 400, University of South Florida, Tampa, FL 33620, USA
bDepartment of Biology, University of South Florida, Tampa, FL 33620, USA
Received 19 October 2005; revised 13 January 2006; accepted 16 January 2006
Available online 15 February 2006
Abstract—A study on the structure–activity profiles of N-thiolated b-lactams 1 is reported which demonstrates the impor-
tance of the N-organothio moiety on antibacterial activity. Our results indicate that elongation of the N-alkylthio residue
beyond two carbons, or extensive branching within the organothio substituent, diminishes antibacterial effects. Of the deriv-
atives we examined, the N-sec-butylthio b-lactam derivative 5g possesses the strongest growth inhibitory activity against
methicillin-resistant Staphylococcus aureus strains. Sulfur oxidation state is important, as the N-sulfenyl and N-sulfinyl groups
provide for the best antibacterial activity, while lactams bearing the N-sulfonyl or N-sulfonic acid functionalities have much
weaker or no anti-MRSA properties. Stereochemistry within the organothio chain does not seem to be a significant factor,
although for N-sec-butylthio b-lactams 15a–d, the 3R,4S-lactams 15c, d are more active than the 3S,4R-stereoisomers 15a, b
in agar diffusion experiments. The N-methylthio lactams are the most sensitive to the presence of glutathione, followed by
N-ethylthio and N-sec-butylthio lactams, which indicates that bioactivity and perhaps bacterial selectivity of the lactams may
be related to the amount of organothiols in the bacterial cell. These results support the empirical model for the mechanism
of action of the compounds in which the lactam transverses the bacterial membrane to deliver the organothio moiety to its
cellular target.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
drug-resistant microbes such as MRSA is attributable
to the stability these compounds have toward bacterial
penicillinases.1b The compounds are inert to hydrolytic
degradation by these enzymes, a property highly desir-
able for a b-lactam drug, and do not inhibit the
hydrolytic cleavage of penicillin G by b-lactamases.
Additionally, the lactams possess promising anticancer
properties, yet are not cytotoxic to normal mammalian
(human fibroblast) cells at concentrations well beyond
that needed to inhibit bacterial growth.2 Our studies
also indicate that these N-thiolated lactams display
different structure–activity profiles to those of tradi-
tional b-lactam antibacterials and exert their growth
inhibitory effects in a manner which is unique to those
of other b-lactams. For instance, the C3 and C4 ring
substituents (R1–R4) have only a marginal effect on
anti-MRSA activity, and neither relative nor absolute
chirality at these two centers seems to significantly
perturb activity.3These preliminary structure–activity
studies also indicate that the N-organothio group
may play a more important role, however, since
N-Thiolated b-lactams 1 are a new family of antibac-
terial agents active against Staphylococcus bacteria,
including methicillin-resistant strains of Staphylococcus
aureus (MRSA).1 Although these compounds exhibit
antigrowth properties against other Staphylococcus
species, including S. epidermidis, S. simulans, S. sap-
rophyticus, and a few other genera such as Micrococ-
cus luteus and Neisseria gonorrhoeae, a wide range of
other Gram-positive and Gram-negative microbes
appear to be unaffected.1b This observed selectivity
for certain bacteria is rather interesting and undoubt-
edly relates to the mode of action, which has not yet
been completely defined. Moreover, the ability of lac-
tams 1 to retain their full antibacterial activity against
Keywords: N-Thiolated b-lactams; MRSA; SAR; Antibiotics.
Corresponding author. Tel.: +1 813 974 7312; fax: +1 813 974
1733; e-mail: eturos@shell.cas.usf.edu
*
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.029

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B. Heldreth et al. / Bioorg. Med. Chem. 14 (2006) 3775–3784
replacement of the N-methylthio moiety for other N-
substituents such as hydrogen, alkyl or alkoxy results
in complete loss of bioactivity.
Given the requirement for a thio transfer from the lac-
tam to its target, we speculated whether the anti-MRSA
properties of the compounds could be enhanced by
altering the structure of the organothio side chain. In
this report, we investigate this in more detail by examin-
ing N-thiolated lactams in which the N-organothio sub-
stituent is systematically varied either in length or in its
degree of chain branching. We also examine the effect of
sulfur oxidation state on anti-MRSA properties. The re-
sults we obtained from this study, in conjunction with
those of our previous findings, enable us to clarify the
role of the ring substituents and the process by which
these compounds may function as antibacterials.
O
SCH3
N
R1
R3
R2 R4
1
2. Results and discussion
2.1. Preliminary model studies
A hypothetical model for the mode of action of these
lactams, as depicted in Figure 1, serves as a starting
point for these investigations. We have evidence that
the antibacterial properties of these N-thiolated lactams
come from the transfer of the organothio moiety onto its
target with consequential loss of the N-protio lactam.
We have been able to isolate this by-product by extrac-
2.2. Synthesis and evaluation of b-lactam analogues
2.2.1. Linear N-alkylthio side-chain derivatives. The first
series of compounds we investigated are N-alkylthio lac-
tams 5a–e, in which the alkylthio side chain is linear and
contains from one to eight carbons (Fig. 3). These deriv-
atives were obtained through synthesis as shown in
Scheme 1. Staudinger coupling of N-(4-methoxyphe-
nyl)-imine 2 with methoxyacetyl chloride afforded exclu-
sively the cis-disubstituted b-lactam 3.4 Following
oxidative cleavage of the N-aryl ring with aqueous ceric
ammonium nitrate, the resulting lactam 4 was N-alkyl-
thiolated using readily prepared sulfenylating reagents5
1
tion of the cultured broth media with ethyl acetate. H
NMR studies of the extract indicate the clean formation
of the N-protio b-lactam, as represented by the appear-
ance of the broad N–H signal and the simultaneous loss
of the N-methylthio substituent (Fig. 2). Antimicrobial
testing of the resulting N–H lactam shows that this
N–S cleavage product possesses no antibacterial proper-
ties. The identity of the cellular target has not yet been
determined but is most likely a cytoplasmic thiol needed
for bacterial survival.
CH3
CH3
O
S
O
S
O
S
CH3
N
N
N
CH3O
CH3O
CH3O
Cl
Cl
5c
Cl
Nu
5a
5b
O
S
O
CH3
R
thio transfer
Cytoplasm
CH3
N
N-
R2
O
S
+ Nu-SR
O
S
N
R1
R2
R1
N
CH3O
CH3O
cell membrane
Cl
Cl
5e
5d
Figure 1. Model depicting the proposed mode of action of N-thiolated
b-lactams.
Figure 3. Linear N-alkylthio-substituted b-lactams 5a–e.
OCH3
a
OCH3
CH3O
O
Ar
S
N
O
N
a
N
CH3
H
CH3O
Cl
2
Cl
3
b
CH3O
Ar
O
H
O
SR
N
N
b
c
N
O
H
CH3O
CH3O
Cl
Cl
4
5a-e
Figure 2. Proton NMR spectra of lactam 5a (a) prior to inoculation
with S. aureus and (b) after inoculation with S. aureus and ethyl acetate
extraction of the growth media.
Scheme 1. Reagents and conditions: (a) AcCl, NEtiPr2, PhMe, 0 °C to
rt; (b) (NH4)2Ce(NO3)6, MeCN–H2O, 0 °C; (c) N-alkylthiophthali-
mide, NEtiPr2, C6H6, reflux.

B. Heldreth et al. / Bioorg. Med. Chem. 14 (2006) 3775–3784
3777
to give N-thiolated lactams 5a–e. These five analogues
35
30
25
20
15
10
5
V 4mm
were tested for antibacterial activity against MRSA iso-
lates using the Kirby–Bauer method of disk diffusion on
agar plates (Table 1). Previously, we have demonstrated
that the growth inhibition zone sizes for N-methylthio
lactams done in this manner correlate well with their
minimum inhibitory concentrations (MICs) from broth
dilution experiments, and thus represent a reliable way
to gauge bioactivity within a closely related series of
analogues.6 For each compound, the microbiological
data from these agar plate assays are plotted out graph-
ically in Figure 4, with the vertical bars indicating the
average diameter (from triplicate trials) of the growth
inhibition zones observed. Nine clinical strains of
MRSA were tested against each derivative, including
eight clinical isolates and one ATCC strain. All MRSA’s
were b-lactamase-producing strains.
V 3mm
V 3mm
V 3mm
V 2mm
0
Pen G
5a
5b
5c
5d
5e
Figure 4. Antimicrobial activities of lactams 5a–e against MRSA.
Penicillin G (Pen G) is used as a control. The vertical bars measured
along the y axis indicate the average diameter in mm of the growth
inhibition zones produced against nine different MRSA strains (in
triplicate). Margin of error for these triplicate measurements is 1 mm.
The number (V) above each vertical bar indicates the range of
variation observed among the nine MRSA strains, taken from Table 1.
While the lead compound, N-methylthio lactam 5a,
shows inhibition zones of around 28 mm, the N-ethyl-
thio analogue 5b is about 15% more active. Bioactivity
then drops off steadily as the N-alkylthio chain length
is further increased, with N-octylthio lactam 5e retaining
less than half the activity of 5a. This suggests that the
increase of lipophilicity of the N-alkylthio chain through
elongation of the alkyl residue beyond two carbons
causes a systematic drop in bioactivity.
smaller zone sizes could simply be the result of dimin-
ished water solubility.
Therefore, broth minimum inhibitory concentration
(MIC) values were determined for lactams 5a, b, and e
against S. aureus (ATCC 25923) and MRSA (ATCC
43300) (Table 2). The MICs further validate the trend
observed in the Kirby–Bauer tests, with anti-MRSA
activity of the lactams increasing in the order
5e < 5a < 5b. The MIC data also show that these
We considered the possibility that the reason why
antibacterial activity of lactams 5a–e diminishes as a
function of the N-alkylthio chain length could be
artifactual, due to the decreasing ability of the different
analogues to diffuse through the agar media. Thus,
Table 1. Kirby–Bauer well-diffusion assays of N-thiolated b-lactams against nine different MRSA strains
(O)n
O
SR
N
Cl
MeO
Lactam
n
R
MRSA
652
MRSA
653
MRSA
654
MRSA
655
MRSA
656
MRSA
657
MRSA
658
MRSA
659
ATCC
43300
5a
5b
5c
5d
5e
5f
0
0
0
0
0
0
0
0
0
0
0
1
2
3
CH3
30
33
30
25
13
33
44
10
24
27
28
22
11
0
29
33
30
24
12
32
41
10
24
24
27
21
10
0
28
29
28
24
13
31
39
10
23
25
28
21
9
27
29
28
22
12
29
39
10
23
22
27
19
9
27
30
27
23
12
30
39
10
22
23
26
19
8
28
30
27
24
14
31
40
10
23
24
27
20
10
0
27
31
28
25
13
32
41
10
26
25
29
21
9
27
29
27
24
13
30
39
10
24
24
27
19
8
28
31
28
25
14
31
40
10
24
25
28
20
10
0
CH2CH3
CH2CH2CH3
CH2(CH2)2CH3
CH2(CH2)6CH3
CH(CH3)2
CH(CH3)CH2CH3
C(CH3)3
c-C6H11
5g
5h
5i
5j
Ph
CH2Ph
5k
6i
c-C6H11
c-C6H11
7i
9a
H
0
0
0
0
0
The values indicate the average diameters (of three trials) in mm for the zone of growth inhibition. For each test, 20 lg of the lactam in DMSO
solution (1 mg/mL) was applied to a 6-mm well prior to inoculation. Incubation was for 24 h at 37 °C. All of the microbes are b-lactamase-producing
strains. MRSAs 652–659 are clinical isolates obtained from Lakeland Regional Medical Center (Lakeland, Florida). Error values for each mea-
surement are typically within 1 mm.

3778
B. Heldreth et al. / Bioorg. Med. Chem. 14 (2006) 3775–3784
Table 2. Minimum inhibitory concentrations, MIC (lg/mL), of
lactams 5a, b, e, and g versus penicillin G (Pen G)
ment of the N-ethylthio substituent of lactam 5b with an
N-isopropylthio group (lactam 5f) led to a 7% increase
in anti-MRSA activity based on agar diffusion testing.
Thus, enhancement of steric hindrance around the sulfur
atom does not necessarily diminish activity, but in this
case, seems to enhance bacterial growth inhibition. Fur-
ther lengthening of the alkyl chain from this branch
point, as shown for sec-butyl analogue 5g, continues
to increase potency. MIC testing concurred with these
results, yielding a value of less than 0.25 lg/mL for
100% growth inhibition of the MRSA (ATCC 43300)
compared to 8 lg/mL for N-methylthio compound 5b
(Table 1).
Lactam
S. aureus (ATCC 25923)
MRSA (ATCC 43300)
PenG
5a
5b
0.025
16
64
16
8
8
64
5e
5g
64
0.25
0.25
N-thiolated lactams have the same activity against drug-
susceptible S. aureus and b-lactamase-producing
MRSA, in contrast to penicillin G (PenG) whose inhib-
itory activity is markedly diminished for MRSA.
We then extended these studies to N-thiolated lactams
having alicyclic or aromatic rings as branched residues
within the organothio chain. To this end, N-cyclohexyl-
thio, N-phenylthio, and N-benzylthio b-lactams 5i–k
were synthesized accordingly and tested against the
MRSAs. All three of these lactams were found to be
roughly equal to each other in activity, but showed low-
er effectiveness than the sec-butylthio (5g) and isopro-
pylthio (5f) analogues. This suggests that the presence
of bulkier cyclic side chains on the sulfur atom diminish-
es anti-MRSA activity to some degree.
2.2.2. Branched N-alkylthio b-lactams. Investigating the
effects of branching within the alkylthio chain should
provide some evidence for, or against, a steric effect on
bioactivity. We therefore conducted a study to ascertain
this by examining the anti-MRSA activity of branched
N-organothio lactams 5f–k (Fig. 5). Each of these lac-
tams was prepared analogously to that shown in Scheme
1. As the plot of bioactivities in Figure 6 shows, replace-
CH3
CH3
CH3
CH3
CH3
CH3
O
S
O
S
O
S
2.2.3. N-Sulfinyl and N-sulfonyl derivatives. While prior
studies in our laboratory1 suggested that the antibacteri-
al properties of N-thiolated b-lactams could be modulat-
ed by changing the sulfur oxidation state, we decided to
reinvestigate this in more detail using N-cyclohexylthio
lactam 5i as a point of reference. Thus, N-sulfinyl lactam
6i and N-sulfonyl analogue 7i were prepared as shown in
Scheme 2. Oxidation of 5i with 30% hydrogen peroxide
in glacial acetic acid gave the N-sulfinyl product 6i as a
1:1 diastereomeric mixture, whereas the use of mCPBA
in diethyl ether led to the N-protio lactam through
cleavage of the sulfur–nitrogen bond. Re-subjection of
N-sulfinyl compounds 6i to the hydrogen peroxide con-
ditions afforded N-sulfonyl product 7i in nearly quanti-
tative yield. Antimicrobial screening of 6i and 7i against
MRSA indicated that the N-sulfinyl lactam showed sim-
ilar but slightly lower anti-MRSA activities to that of
the N-sulfenyl precursor 5i, but the N-sulfonyl analogue
7i was largely devoid of activity (Fig. 7). This suggests
that the availability of an electron pair on sulfur may
be needed for bioactivity.
N
N
N
CH3
CH3O
CH3O
CH3O
Cl
Cl
Cl
5g
5h
5f
O
S
O
S
O
S
N
N
N
CH3O
CH3O
CH3O
Cl
Cl
Cl
5j
5i
5k
Figure 5. Branched N-alkylthio b-lactams 5f–k.
45
V 5mm
40
V 4mm
35
V 4mm
30
V 3mm
V 4mm
25
V 4mm
20
15
10
5
2.2.4. N-Sulfonate and N-sulfonic acid lactams. In fol-
low-up to the previous pair of S-oxidized lactams 6i
and 7i, we next investigated N-sulfonate salts 8a–c and
N-sulfonic acids 9a–c. These derivatives were formed
V 0mm
0
PenG 5b
5f
5g
5h
5i
5j
5k
O
O
O
O
S
O
S
Figure 6. Antimicrobial activities of N-ethylthio compound 5b and
lactams 5f–k against MRSA. Penicillin G (Pen G) is used as a control.
The vertical bars along the y axis indicate the average diameter in mm
of the growth inhibition zones produced on agar plates against nine
different MRSA strains (in triplicate). Margin of error for these
triplicate measurements is 1 mm. The number (V) above each vertical
bar indicates the range of variation observed among the nine MRSA
strains, taken from Table 1.
O
S
N
N
[O]
N
[O]
CH3O
CH3O
CH3O
Cl
5i
Scheme 2.
Cl
6i
Cl
7i

B. Heldreth et al. / Bioorg. Med. Chem. 14 (2006) 3775–3784
3779
25
20
15
10
5
the sulfur side chain, we thought it would be informative
to examine the effect of absolute stereochemistry within
the organothio side chain. We therefore focused on our
most potent compound, N-sec-butylthio b-lactam 5g,
which was initially tested above as a 1:1 mixture of dia-
stereomeric racemates (four stereoisomers). For com-
parison, we decided to independently examine each of
the four stereoisomeric forms of 5g, compounds 15a–d,
to look for possible differences in anti-MRSA activity.
The syntheses of these compounds are shown in Scheme
4. To prepare these four lactams in enantiomerically
pure form, we were able to take advantage of a method
we reported earlier for the enzymatic resolution of race-
mic 3-acetoxy lactams 11 using lipase PS-30.8 This led to
the production of 3-hydroxy lactam 12 and unreacted
3-acetoxy lactam 11b, each in better than 95% ee. These
intermediates could then be taken on to 15a, b using
enantiomeric sec-butylthio phthalimide transfer re-
agents, (R)-14 and (S)-14 (Fig. 9).9 The four lactam
stereoisomers were independently obtained with better
V 4mm
V 3mm
V 3mm
0
5i
6i
7i
Figure 7. Antimicrobial activities of lactams 5i, 6i, and 7i. Penicillin G
(Pen G) is used as a control. The vertical bars along the y axis indicate
the average diameter in mm of the growth inhibition zones produced
against nine different MRSA strains (in triplicate). Margin of error for
these triplicate measurements is 1 mm. The number (V) above each
vertical bar indicates the range of variation observed among the nine
MRSA strains, taken from Table 1.
from the respective N-protio lactams 4a–c using sulfur
trioxide/pyridine/DMF conditions (Scheme 3).7 This
provides the N-sulfonate potassium salts which can be
converted by ion exchange to the tetrabutylammonium
salts 8. Eluting the tetrabutylammonium salt through
OCH3
OCH3
OCH3
a
silica gel chromatography column gave the
O
O
O
N
N
N
N-sulfonic acid derivatives 9a–c in good overall yield.
Testing of lactams 8 and 9 revealed that both forms of
the N-sulfonic acid were devoid of anti-MRSA activity.
Neither compound produced growth inhibition zones
against MRSA on agar plates, nor inhibited bacterial
growth in broth at 128 lg/mL concentration levels.
Lipase PS-30
+
acetone
phosphate buffer
pH 7.2
AcO
HO
AcO
(-)-12
(3S,4R)
(+)-11b
(3R,4S)
(+/-)-11
a) Ac2O/CH2Cl2
b) CAN/CH3CN
CAN/CH3CN
2.2.5. Effect of chirality in the organothio chain on anti-
MRSA activity. Recently, we reported that the absolute
stereochemistry of 3,4-disubstituted N-methylthio b-lac-
tams has no discernible effect on anti-MRSA activity.
This was demonstrated with the enantiomeric lactams
10a and b (Fig. 8).3 Both antipodes displayed the same
potencies, suggesting that absolute chirality of the lac-
tam has no influence on bioactivity. Since biological
activity of the lactams seems to be more dependent on
O
O
NH
NH
AcO
AcO
(-)-13
(+)-13
(S)-14
TEA
(R)-14
(R)-14
(S)-14
TEA
TEA
TEA
O
X
SO3+-NBu4
O
X
H
O
X
SO3H
SiO2
N
N
1)SO3 . DMF
N
Column
O
S
O
O
S
S
O
S
N
N
N
N
2) Ion Exchange
with TBAB
+
+
Cl
Cl
Cl
AcO
AcO
AcO
AcO
4
a X=OMe
b X=Cl
c X=I
8
9
15a
Scheme 4.
15b
15c
15d
Scheme 3.
O
N
O
N
O
O
N
O
O
SCH3
SCH3
N
S
S
AcO
AcO
(R)-14
(S)-14
10a
10b
Figure 9. Enantiomeric sec-butylthio transfer reagents (R)-14 and
(S)-14.
Figure 8. Enantiomeric b-lactams 10a and b.

3780
B. Heldreth et al. / Bioorg. Med. Chem. 14 (2006) 3775–3784
tions, penicillin G is completely destroyed within
20 min.
30
29
28
27
26
25
24
23
22
21
V 3mm
V 3mm
While work is in progress to identify the biological tar-
get of the lactams and to determine the effects these lac-
tams have on the primary cellular processes in bacteria,
one of the more immediate issues we are examining is
the effect of cellular thiols on bioactivity and microbial
selectivity of the lactams. Among the more ubiquitous
of these organothiols in bacteria is glutathione, a natural
reductant which helps maintain proper redox conditions
in cells. We therefore decided to examine the sensitivity
of the lactams to glutathione by bioassaying N-methyl-
thio lactam 5a, ethylthio lactam 5b, and N-sec-butylthio
lactam 5g against MRSA in the presence of different
amounts of glutathione. In these experiments, shown
in Table 3, the growth inhibition zones of the three lac-
tams produced after 24 h of incubation with MRSA
were compared first in the absence of additional gluta-
thione versus when 20 lg of glutathione was added to
the wells along with the drug, and then when 50 lg of
glutathione was added. Using 20 lg of glutathione,
N-methylthio lactam 5a loses all of it activity, while
N-ethylthio lactam 5b and N-sec-butylthio compound
5g retain most of their activity. Similarly, using 50 lg
of glutathione, the activity of 5b is completely sacrificed,
while 5g is still quite active. Thus, these experiments
indicate an order in relative stabilities of the different
N-alkylthio lactams to glutathione, with N-methyl-
thio < N-ethylthio < N-sec-butylthio, which coincides
with the observed trend in anti-MRSA activity. MIC
testing of the most active of these three compounds, lac-
tam 5g, in broth media gives an MIC value of 0.25 lg/
mL against both MSSA and MRSA, which increases
to 2 lg/mL in the presence of 20 lg of glutathione. To
study the interaction of glutathione with lactams 5a, b,
and g, we followed the reaction of glutathione with equi-
molar amounts of the individual lactams in buffer solu-
tion, finding that all three lactams reacted rapidly with
glutathione to produce the N-protio lactam and the S-
alkylthio glutathione disulfide adduct. Thus, transfer
of the N-alkylthio substituent from the lactam to the
thiol occurs spontaneously. To further substantiate the
neutralizing effect of glutathione, a Kirby–Bauer plate
was prepared with a 1 mg reservoir of glutathione locat-
ed at the center and three surrounding wells containing
20 lg of different N-methylthio b-lactams (Fig. 11). The
ability of glutathione to consume the lactams was again
clearly evident from the presence of concaved regions
within the growth inhibition zones, between the wells
containing glutathione and the lactam, where the con-
centration of glutathione was sufficient to deactivate
the lactams.
V 2mm
V 3mm
15a
15b
15c
15d
Figure 10. Comparison of antimicrobial activities of the four stereo-
isomers 15a–d. The vertical bars along the y axis indicate the average
diameter in mm of the growth inhibition zones produced against nine
different MRSA strains (in triplicate). Margin of error for these
triplicate measurements is 1 mm. The number (V) above each vertical
bar indicates the range of variation observed among the nine MRSA
strains, taken from Table 1.
than 95% de and % ee, and tested individually for anti-
bacterial activity (Fig. 10).
To our surprise, there was a clear difference in bioactiv-
ity between these four stereoisomers. In particular, we
noted that for each of the nine clinical strains of MRSA,
two of the compounds, lactams 15a and b, were consis-
tently equal in activity but also considerably less potent
than their enantiomers, compounds 15c and d. This was
unexpected, since it appears in this case that activity is
not dependent on the chirality of the organothio moiety,
but instead is related to the stereochemistry at the C3
and C4 centers of the lactam. This contrasts with our
previous observations for enantiomeric N-methylthio
lactams 10a and b, which carry a non-stereogenic thio
side chain. It is rather curious that absolute stereochem-
istry of the lactam ring could have an effect on bioactiv-
ity when the N-organothio group is chiral, but not when
the N-moiety is achiral (N-methylthio). We do not have
a reasonable explanation for this yet.
Previously, we described experiments which showed that
the N-thiolated lactams are stable to penicillinases, thus
accounting for their ability to retain full antibacterial
activity against b-lactamase-producing strains of S.
aureus. Our studies indicate that compounds 5a, b, and
g are likewise impervious to the hydrolytic destruction
of the b-lactam ring by pencillinases, as evidenced by
the fact that they can be recovered completely un-
changed after 24 h of standing in pH 7.4 buffer solution
containing penicillinase protein. Under identical condi-
Table 3. Effect of glutathione on the growth inhibition zones of lactams 5a, b, and g against MRSA (ATCC 43300) by Kirby–Bauer diffusion
Compound
No glutathione (mm)
20 lg glutathione (mm)
100 lg glutathione (mm)
5a
5b
5g
29
33
40
0
0
0
27
38
25
In each case, 20 lg of the lactam (applied as a 1 mg/mL solution in DMSO) was added to the well, along with the indicated amount of glutathione.
Zone diameters were then measured after 24 h of incubation at 37 °C.

B. Heldreth et al. / Bioorg. Med. Chem. 14 (2006) 3775–3784
3781
gel (40 mm). NMR spectra were recorded in CDCl3un-
less otherwise noted. 13C NMR spectra were proton
broad-band decoupled.
Lactam
5a
4.1. Procedure for the preparation of N-alkylthio-phthal-
imide reagents
In dry benzene, chlorine gas was bubbled until the in-
crease in weight equaled 0.93 g (13 mmol). This solution
was then added to a round-bottomed flask containing
1.00 g (13 mmol) of 2-propanethiol in a minimal quanti-
ty of benzene. The mixture was then sealed at 0 °C with
stirring for 1 h, then added dropwise to a second round-
bottomed flask containing a slurry of 1.93 g (13 mmol)
of phthalimide and 2.89 g (22 mmol) of Hunig’s base.
The mixture was allowed to reach room temperature
over 2 h with stirring. The solution was then poured into
water, extracted three times with benzene, dried over
magnesium sulfate and concentrated via rotary evapora-
tion. Any unreacted phthalimide was removed by
trituration with chloroform. Further purification was
done by recrystallization with methanol or column
chromatography.
glutathione
Figure 11. Effect of glutathione on the anti-MSSA activity of
N-thiolated b-lactams. The center well was loaded with 1 mg of
glutathione in aqueous solution, and the three peripheral wells each
contain 20 lg of test compound. The dotted circles indicate the regions
where the lactam (yellow) and glutathione (white) are present. Where
the circles meet is where bacterial growth occurs, indicating that
glutathione blocks the anti-MRSA activity of lactam 5a in this region.
4.2. 2-Propylthioisoindoline-1,3-dione
It is likely, then, that the in vitro anti-MRSA activity,
and the selectivity of the lactams to S. aureus over many
other microbes, could be directly attributed to cellular
glutathione levels. Additional studies are underway in
our laboratory to investigate this in more detail and will
be reported in due course.
White solid, mp 77–78 °C; 1H NMR (250 MHz, CDCl3)
d 7.87 (4H, m), 2.86 (2H, t, J = 7.3 Hz), 1.62 (2H, m)
1.04 (3H, t, J = 7.3 Hz).
4.3. 2-Methylthioisoindoline-1,3-dione
White solid; mp 178–180 °C; 1H NMR (250 MHz,
CDCl3) d 7.82 (4H, m), 3.77 (3H, s).
3. Conclusions
The results of this study, in concert with our previously
acquired data, support the empirical model presented in
Figure 1 for the mode of antibacterial activity of N-thi-
olated b-lactams. While the lipophilic nature of the C3/
C4 ring substituents may help the molecule transverse
the lipid bilayer of the bacterial cell membrane, having
too much lipophilicity in the organothio side chain ap-
pears to have a detrimental affect on antibacterial prop-
erties. This suggests that the organothio moiety must be
available for presentation to the thiol target in the cell,
rather than sequestered within the membrane. There-
fore, the distribution of lipophilicity on the periphery
of the molecule must be balanced among the substitu-
ents. Ongoing work in our laboratories is attempting
to delineate the full mechanistic basis for the bioactivity
of these compounds and to develop suitable candidates
for clinical development.
4.4. 2-Ethylthioisoindoline-1,3-dione
White solid; mp 158–160 °C; 1H NMR (250 MHz,
CDCl3) d 7.86 (4H, m), 2.92 (2H, q, J = 7.3 Hz), 1.56
(3H, t, J = 8.1 Hz).
4.5. 2-Butylthioisoindoline-1,3-dione
White solid; mp 189–190 °C; 1H NMR (250 MHz,
CDCl3) d 7.8 (4H, m), 2.85 (2H, t, J = 7.4 Hz), 1.61
(2H, m), 1.42 (2H, m), 0.91 (3H, t, J = 5.0 Hz).
4.6. 2-Isopropylthioisoindoline-1,3-dione
White solid, mp 61–62 °C; 1H NMR (250 MHz,
CDCl3) d 7.8–7.5 (4H, m), 4.9 (1H, m), 2.17 (6H, d,
J = 6.9 Hz).
4.7. ( )-2-sec-Butylthioisoindoline-1,3-dione
4. Experimental
White solid, mp 43–45 °C; 1H NMR (250 MHz, CDCl3)
d 7.83 (4H, m), 3.21 (1H, m), 1.55 (2H, m), 1.24 (3H, d,
J = 6.8 Hz), 1.04 (3H, t, J = 7.3 Hz).
All reagents were purchased from Sigma–Aldrich Chem-
ical Company and used without further purification.
Solvents were obtained from Fisher Scientific Company.
Thin layer chromatography (TLC) was carried out using
EM Reagent plates with a fluorescence indicator (SiO2-
60, F-254). Products were purified by flash chromato-
graphy using J.T. Baker flash chromatography silica
4.8. 2-Cyclohexylthioisoindoline-1,3-dione
White solid, mp 92–94 °C; 1H NMR (250 MHz, CDCl3)
d 7.80 (4H, m), 3.05 (1H, m), 1.90–1.25 (1H, m).

3782
B. Heldreth et al. / Bioorg. Med. Chem. 14 (2006) 3775–3784
4.9. 2-Phenylthioisoindoline-1,3-dione
0.82 (3H, t, J = 6.9 Hz); 13C NMR (63 MHz, CDCl3) d
171.0, 133.6, 128.8, 128.3, 86.3, 66.9, 58.3, 38.2, 30.8,
21.5, 13.6.
White solid, mp 149–155 °C; 1H NMR (250 MHz,
CDCl3) d 8.0–6.8 (9H, m).
4.16. ( )-(3S,4R)-N-Butylthio-4-(2-chlorophenyl)-3-
methoxyazetidin-2-one (5d)
4.10. 2-Benzylthioisoindoline-1,3-dione
White solid, mp 165–168 °C; 1H NMR (250 MHz,
CDCl3) d 7.9–7.5 (4H, m), 7.31 (5H, m), 1.75 (2H, s).
Light yellow viscous oil; 1H NMR (250 MHz, CDCl3) d
7.30 (4H, m), 5.35 (1H, d, J = 4.9 Hz), 4.89 (1H, d,
J = 4.9 Hz), 3.23 (3H, s), 2.85 (2H, t, J = 7.0), 1.60
(4H, m), 0.91 (3H, t, J = 7.3 Hz); 13C NMR (63 MHz,
CDCl3) d 176.5, 134.0, 132.1, 129.0, 126.9, 86.8, 64.8,
58.1, 48.0, 12.3.
4.11. Procedure for the preparation of N-thiolated
azetidin-2-ones
In
a
round-bottomed flask were added 5.0 mg
(0.24 mmol) of N-protio b-lactam 4, 6.7 mg (0.24 mmol)
of N-phenylthiophthalimide, and 11.9 mg (0.12 mmol)
of triethylamine, in a minimal quantity of dichlorometh-
ane. The solution was refluxed and monitored by TLC
until all of the starting lactam was consumed. Reaction
was generally complete within 12 h. After cooling, the
solution was poured into an equal volume of water,
washed with aqueous solution of 5% sodium bicarbon-
ate, 1% sodium bisulfate, and saturated sodium chlo-
ride. The extracts were then dried over magnesium
sulfate and concentrated via rotary evaporation. Phthal-
imide was removed via trituration with chloroform and
the remaining impurities were removed by column chro-
matography, eluting with dichloromethane.
4.17. ( )-(3S,4R)-4-(2-Chlorophenyl)-3-methoxy-N-
octylthio azetidin-2-one (5e)
White amorphous powder; 1H NMR (250 MHz, CDCl3)
d 7.35 (4H, m), 5.35 (1H, d, J = 4.9 Hz), 4.89 (1H, d,
J = 4.9 Hz), 3.23 (3H, s), 2.77 (2H, m), 1.64–1.26
(12H, m), 0.88 (t, 3H, J = 6.2 Hz); 13C NMR
(63 MHz, CDCl3) d 167.8, 132.5, 131.1, 127.5, 127.0,
80.3, 52.7, 43.7, 28.0, 26.0, 24.5, 22.3, 18.9.
4.18. ( )-(3S,4R)-4-(2-Chlorophenyl)-N-isopropylthio-3-
methoxyazetidin-2-one (5f)
1
Light yellow solid, mp 30–38 °C; H NMR (250 MHz,
CDCl3) d 7.34 (4H, m), 5.39 (1H, d, J = 4.9 Hz), 4.95
(1H, d, J = 4.9 Hz), 3.27 (1H, m), 3.25 (3H, s), 1.27
(6H, d, J = 5.7 Hz); 13C NMR (63 MHz, CDCl3) d
171.3, 133.2, 128.9, 127.0, 87.1, 65.2, 59.7, 49.4, 34.0,
30.1, 28.5, 25.5, 21.5.
4.12. ( )-(3S,4R)-4-(2-Chlorophenyl)-3-methoxy-N-
phenylthioazetidin-2-one (5j)
1
Light yellow solid, mp 60–62 °C; H NMR (250 MHz,
CDCl3) d 7.34 (9H, m), 5.35 (1H, d, J = 5.0 Hz), 4.91
(1H, d, J = 5.0 Hz), 3.25 (3H, s); 13C NMR (63 MHz,
CDCl3) d 172.0, 137.0, 135.0, 134.0, 132.0, 130.0,
129.0, 128.0, 127.0, 125.0, 82.0, 59.0, 48.0.
4.19. ( )-(3S,4R)-N-sec-Butylthio-4-(2-chlorophenyl)-3-
methoxyazetidin-2-one (5g)
Light yellow viscous oil; 1H NMR (250 MHz, CDCl3) d
7.4 (1H, d, J = 7.4 Hz), 7.3 (3H, m), 5.3 (1H, d,
J = 4.7 Hz), 4.9 (1H, d, J = 4.8 Hz), 3.2 (3H, s), 3.0
(1H, m), 1.48 (1H, m), 1.2 (3H, dd, J = 6.8, 4.9 Hz),
0.94 (3H, q, J = 6.0 Hz); 13C NMR (63 MHz, CDCl3)
d 171.0, 133.8, 131.4, 129.5, 128.9, 126.8, 86.3, 64.1,
58.8, 48.1, 28.1, 19.0, 18.6, 11.1.
4.13. ( )-(3S,4R)-4-(2-Chlorophenyl)-3-methoxy-N-
(methylthio)azetidin-2-one (5a)
White crystal, mp 71–73 °C; 1H NMR (250 MHz,
CDCl3) d 7.35 (1H, d, J = 7.4 Hz), 7.24 (3H, m), 5.29
(1H, d, J = 4.9 Hz), 4.80 (1H, d, J = 4.9 Hz), 3.16 (3H,
s), 2.40 (3H, s); 13C NMR (63 MHz, CDCl3) d 170.4,
133.8, 131.4, 129.6, 128.9, 126.8, 86.7, 62.7, 58.9, 21.8.
4.20. ( )-(3S,4R)-N-tert-Butylthio-4-(2-chlorophenyl)-3-
methoxyazetidin-2-one (5h)
4.14. ( )-(3S,4R)-4-(2-Chlorophenyl)-N-ethylthio-3-
methoxyazetidin-2-one (5b)
White amorphous powder; 1H NMR (250 MHz, CDCl3)
d 7.34 (4H, m), 5.50 (1H, d, J = 4.8 Hz), 4.99 (1H, d,
J = 4.8 Hz), 3.25 (3H, s), 1.35 (9H, s); 13C NMR
(63 MHz, CDCl3) d 166.0, 133.0, 129.0, 88.0, 65.0,
60.0, 44.0, 34.0, 31.0, 27.0, 19.0.
White solid, mp 68–70 °C; 1H NMR (250 MHz, CDCl3)
d 7.34 (4H, m), 5.33 (1H, d, J = 5.0 Hz), 4.88 (1H, d,
J = 5.0 Hz), 3.20 (3H, s), 1.35 (2H, q, J = 10.0 Hz),
0.92 (3H, t, J = 6.8 Hz); 13C NMR (63 MHz, CDCl3) d
172.5, 142.1, 137.5, 130.8, 128.8, 128.4, 87.5, 70.5,
46.7, 34.4, 22.0.
4.21. ( )-(3S,4R)-4-(2-Chlorophenyl)-N-cyclohexylthio-
3-methoxyazetidin-2-one (5i)
4.15. ( )-(3S,4R)-4-(2-Chlorophenyl)-3-methoxy-N-(pro-
pylthio)azetidin-2-one (5c)
Brown solid; mp 78–79 °C; 1H NMR (250 MHz, CDCl3)
d 7.34 (4H, m), 5.35 (1H, d, J = 4.9 Hz), 4.93 (1H, d,
J = 4.9 Hz), 3.24 (3H, s), 3.05 (1H, m), 2.01–1.41
(10H, m); 13C NMR (63 MHz, CDCl3) d 171.3, 133.5,
128.9, 128.7, 128.3, 85.2, 67.6, 58.3, 49.5, 32.2, 30.9,
25.6, 25.4.
Colorless oil; 1H NMR (250 MHz, CDCl3) d 7.6–7.2
(4H, m), 5.45 (1H, d, J = 4.9 Hz), 5.09 (1H, d,
J = 4.9 Hz), 3.21 (3H, s), 2.49 (2H, m), 1.55 (2H, m),

B. Heldreth et al. / Bioorg. Med. Chem. 14 (2006) 3775–3784
3783
4.22. ( )-(3S,4R)-N-Benzylthio-4-(2-chlorophenyl)-3-
methoxyazetidin-2-one (5k)
J = 4.9 Hz), 3.32 (2H, m), 0.89 (1H, s); 13C NMR
(63 MHz, CDCl3) d 179.9, 145.2, 137.7, 131.0, 122.7,
83.4, 55.2, 43.3.
1
Light yellow solid; mp 68–70 °C; H NMR (250 MHz,
CDCl3) d 7.22 (9H, m), 5.30 (1H, d, J = 4.7 Hz), 4.55
(1H, d, J = 4.8 Hz), 3.16 (3H, s), 1.25 (3H, s); 13C NMR
(63 MHz, CDCl3) d 179.0, 142.0, 138.0, 136.0, 135.0,
134.0, 132.0, 130.0, 128.0, 126.0, 88.0, 59.0, 48.0, 40.0.
4.28. Testing of antimicrobial susceptibilities (Kirby–
Bauer well diffusion)
Staphylococcus aureus (ATCC 25923) and MRSA
(ATCC 43300) were purchased from ATCC sources.
Eight additional strains of MRSA were obtained
from Lakeland Regional Medical Center (Lakeland,
FL).
4.23. Procedure for the preparation of N-sulfoxylated
b-lactams 6i and 7i
In
a
round-bottomed flask was added 135 mg
(0.41 mmol) of N-cyclohexylthio b-lactam (5i) in a mini-
mal amount of glacial acetic acid at 0 °C with stirring. To
this, a solution of 80 mg (0.41 mmol) of 30% hydrogen
peroxide in several drops of glacial acetic acid was added
dropwise. The solution was allowed to come to room
temperature over 3 h with stirring. The reaction mixture
was then poured into an equivalent portion of water,
extracted with three equivalent volumes of benzene,
washed with water until the washings were at neutral
pH, as observed via pH paper, dried over magnesium sul-
fate, and concentrated by rotary evaporation.
4.29. Culture preparation
From a freezer stock in tryptic soy broth (Difco
Laboratories, Detroit, MI) and 20% glycerol, a cul-
ture of each microorganism was transferred with a
sterile Dacron swab to TrypticaseÒ Soy Agar
(TSA) plates (Becton-Dickinson Laboratories, Coc-
keysville, MD), streaked for isolation, and incubated
at 37 °C for 24 h. A 108 standardized cell count sus-
pension was then made in sterile phosphate-buffered
saline (pH 7.2) and swabbed across fresh TSA
plates.
4.24. ( )-(3S,4R)-N-Cyclohexylsulfinyl-4-(2-chlorophen-
yl)-3-methoxyazetidin-2-one (6i)
4.30. Antimicrobial testing
(1:1 mixture of diastereomers); clear oil; 1H NMR
(250 MHz, CDCl3) d 7.41 (m, 8H), 5.85 (1H, d,
J = 6.0 Hz), 5.71 (1H, d, J = 5.6 Hz), 4.94 (1H, d,
J = 6.0 Hz), 4.91 (1H, d, J = 6.0 Hz), 3.75 (2H, m), 3.29
(3H, s), 3.23 (3H, s), 2.72 (2H, m), 2.2–0.80 (20H, m);
13C NMR (63 MHz, CDCl3) d 171.4, 133.6, 130.9,
130.8, 129.3, 123.5, 83.4, 61.6, 53.2, 50.5, 37.4, 29.9, 25.6.
Prior to swabbing with the culture solution, 20 lL of
a 1 mg/mL stock solution of the test lactam com-
pound in dimethylsulfoxide (DMSO) was added to a
6-mm diameter well bored into the agar. The plates
were swabbed uniformly with the test microbe com-
pound above and then incubated for 24 h at 37 °C.
The antimicrobial susceptibilities were determined by
measuring the zones of growth inhibition around each
well.
4.25. ( )-(3S,4R)-N-Cyclohexylsulfonyl-4-(2-chloro-
phenyl)-3-methoxyazetidin-2-one (7i)
4.31. Determination of minimum inhibitory
concentrations
White, waxy solid; mp 152–158 °C; 1H NMR (250 MHz,
CDCl3) d 7.47 (4H, m), 5.79 (1H, d, J = 5.0 Hz), 4.97
(1H, d, J = 4.6 Hz), 3.29 (3H, s), 3.12 (1H, m), 2.20–
0.86 (10H, m); 13C NMR (63 MHz, CDCl3) d 174.9,
143.8, 127.9, 125.8, 120.1, 118.5, 84.6, 61.9, 52.0, 45.2,
40.1, 34.9, 33.6.
The minimum inhibitory concentration (MIC) values
of the lactams were determined for Staphylococcus
aureus and MRSA by serial dilution according to
NCCLS protocols.10 The test medium was prepared
in 24-well plates (Costar 3524, Cambridge, MA) by
adding the test drug in DMSO to Mueller–Hinton II
agar (Becton-Dickinson Laboratories, Cockeysville,
MD) to bring the total volume in each well to
1.0 mL. Starting with an initial well concentration of
256 lg of drug/mL, each sequential dilution contained
half the concentration of drug. The medium was al-
lowed to solidify at room temperature for 24 h before
inoculation with the bacteria. Using a sterilized inocu-
lating loop, a small amount of each standardized
Staphylococcus strain cultured on TSA plates for 24 h
was transferred into sterile test tubes containing 5 mL
of TSA broth and incubated at 37 °C for 24 h. One
microliter of each culture was then applied to the
appropriate well of Mueller–Hinton agar and incubat-
ed at 37 °C overnight. After 24 h, the MICs were deter-
mined as being the lowest concentration of drug where
bacterial growth was visibly inhibited.
4.26. Preparation of a b-lactam sulfonic acid
In a round-bottomed flask, a solution of 100 mg
(0.47 mmol) of N-protio b-lactam (4) in 2 mL of freshly
distilled dichloromethane and 2 mL of dry DMF was
stirred with 140 mg (0.94 mmol) of 50% sulfur triox-
ide-pyridine under nitrogen at room temperature for
2 h. The solution was concentrated via rotary evapora-
tor and converted to the potassium salt by passing
through an ion exchange column. Column chromatogra-
phy afforded the sulfonic acid in pure form.
4.27. ( )-(3S,4R)-2-(2-Chlorophenyl)-3-methoxy-4-oxo-
azetidine-1-sulfonic acid (9a)
1
Dark brown oil; H NMR (250 MHz, CDCl3) d 7.32
(4H, m), 5.69 (1H, d, J = 4.9 Hz), 4.98 (1H, d,

3784
B. Heldreth et al. / Bioorg. Med. Chem. 14 (2006) 3775–3784
6. (a) Long, T. E.; Turos, E.; Konaklieva, M. I.; Blum, A.
Acknowledgment
L.; Amry, A.; Baker, E. A.; Suwandi, L. S.; McCain, M.
D.; Rahman, M. F.; Dickey, S.; Lim, D. V. Bioorg.
Med. Chem. 2003, 11, 1859; (b) Long, T. E., Ph.D.
Dissertation, University of South Florida, 2003.; (c)
Heldreth, B., Ph.D. Dissertation, University of South
Florida, 2004.
Funding for this research was generously provided by
the National Institutes of Health (R01 AI51351).
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Thioacetic acid
SO2Cl2
HO
Cl
Et3N, Benzene
O
1) SO2Cl2/CCl4
O
N
S
S
2) Phthalimide
O
Hunig's base
Benzene
(R)-14
10. NCCLS (National Committee for Clinical Laboratory
Standards) Methods for Dilution of Antimicrobial Suscep-
tibility Tests for Bacteria that Grow Aerobically. NCCLS
Document M7-A4, Vol. 17, No. 2, 1997.
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Lett. 1985, 26, 3891.