4-Alkyl and 4,4′-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5- dione derivatives as new inhibitors of bacterial cell wall biosynthesis

Article abstract of DOI:10.1016/j.bmcl.2005.03.058

Over 195 4-alkyl and 4,4-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5- dione derivatives were synthesized, utilizing microwave accelerated synthesis, for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good a

Full text of DOI:10.1016/j.bmcl.2005.03.058

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2527–2531
4-Alkyl and 4,40-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-
dione derivatives as new inhibitors of bacterial cell wall biosynthesis
Kristina M. K. Kutterer,a,* Jamie M. Davis,a Guy Singh,b Youjun Yang,b
William Hu,b Anatoly Severin,b Beth A. Rasmussen,b Girija Krishnamurthy,a
Amedeo Faillic and Alan H. Katzc
aWyeth Research, Chemical and Screening Sciences, 401 N. Middletown Rd, Pearl River, NY 10965, USA
bInfectious Disease Discovery Research, 401 N. Middletown Rd, Pearl River, NY 10965, USA
cWyeth Research, Chemical and Screening Sciences, 865 Ridge Road, Monmouth Junction, NJ 08852, USA
Received 29 December 2004; revised 14 March 2005; accepted 16 March 2005
Available online 12 April 2005
Abstract—Over 195 4-alkyl and 4,4-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-dione derivatives were synthesized, utilizing
microwave accelerated synthesis, for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated
good activity against MurB in vitro and low MIC values against Gram-positive bacteria, particularly penicillin-resistant Strepto-
coccus pneumoniae (PRSP). Derivative 7l demonstrated antibacterial activity against both Gram-positive and Gram-negative bacteria.
Derivatives 7f and 10a also demonstrated potent nanomolar Kd values in their binding to MurB.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Scientific effort for the design and synthesis of novel
antibacterials has focused mainly on modifications with-
in the same class of antibiotics already on the market.
Due to the ease with which bacteria can exchange genetic
material and develop resistance to the preexisting anti-
biotic classes, our focus has been to develop
antibacterials with a novel mode of action. As peptido-
glycan is an essential cell wall component of most bacte-
ria, cytosolic enzymes MurA-F at early stages of
peptidoglycan biosynthesis are unique and selective tar-
get for antibiotic action.1 Furthermore, the MurA-F
cascade is ubiquitous to both Gram-positive and
Gram-negative bacteria, but have no mammalian homo-
logue.1 Inhibition of any of these essential enzymes leads
to loss of cell shape and integrity followed by bacterial
death.2,3 From screening of a focused set of the corpo-
rate compound collection, the pyrazolidinedione scaf-
fold was identified as a potential inhibitor of MurB.
Here we report the synthesis and antimicrobial activity
of 4-alkyl and 4,4-bis-alkyl pyrazolidinedione deriva-
tives as new inhibitors of MurA and MurB.
Pyrazolidinediones are conventionally synthesized
through the condensation of diethylmalonates with
hydrazine using in situ generated sodium ethoxide as
the base and heating at elevated temperatures for ex-
tended time periods.4 In order to generate large numbers
of compounds, we developed a simple, fast, high
yielding, one step method for the synthesis of 4-alkyl-
pyrazolidinediones 3 via microwave heating of alkyl-
diethylmalonates
2 with diarylhydrazine 1 using
commercially available sodium ethoxide (Scheme 1).5
Diarylhydrazine derivative 1 was prepared in ꢀ50%
overall yield by oxidative dimerization of 2 equiv of 4-
chloroaniline (4) in the presence of manganese dioxide,
R
O
O
R
OEt OEt
O
O
a
2
N
N
HN NH
Keywords: Microwave-assisted synthesis; Pyrazolidinediones; MurB;
MurA; Antibacterial activity.
3
1
Cl
Cl
Cl
Cl
*
Corresponding author. Tel.: +1 845 602 2174; fax: +1 845 602
5561; e-mail: kutterk@wyeth.com
Scheme 1. Reagents and conditions: (a) NaOEt, microwave, 4 min.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.058

2528
K. M. K. Kutterer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2527–2531
followed by reduction in the presence of zinc dust and
ammonium chloride6 (Scheme 2).
4,40-Differentially alkylated products, Scheme 4, were
obtained in a manner similar to Scheme 3. 4-Methyl
1,2-bis(4-chlorophenyl)pyrazolidine-3,5-dione 12 was
prepared in 85% yield by the microwave-assisted con-
densation of diarylhydrazine 1 with methyl diethylmalo-
nate 11. Next 12 was reacted with alkyl bromides or
alkyl chlorides and lithium carbonate under microwave
heating (900 W) to produce 40alkyl-4-methyl-pyrazoli-
dine-3,5-dione products (13). Similarly, 2-bromoaceto-
phenones were also added to yield a mixture of 40-
alkyl-4-methyl-pyrazolidine-3,5-dione (14) and 4-methyl-
5-alkyloxy-pyrazol-3-one (15) products.
Since limited numbers of diethyl alkylmalonates are
available, techniques for the alkylation of pyrazolidine-
dione itself were also developed utilizing microwave heat-
ing (Scheme 3). First unsubstituted pyrazolidinedione 6
was prepared in 95% yield by the microwave-assisted
condensation of 1,2-di(4-chlorophenyl)hydrazine 1 with
diethylmalonate 5. Next 6 was reacted with alkyl bro-
mides or alkyl chlorides and lithium carbonate under
microwave heating to produce a mixture of 4-alkyated
(7) and 4,40-dialkylated products (8).7 Of other bases at-
tempted, potassium carbonate produced somewhat
more dialkylated product and no alkylation resulted
when triethylamine, pyridine or N,N-diisopropylethyl-
amine were used. Higher boiling N,N-dimethylform-
amide was used as the solvent rather than acetone to
prevent evaporation and subsequent charring of reac-
tions in open vessels. For the addition of more reactive
2-bromoacetophenones to unsubstituted pyrazolidinedi-
one 6 N,N-diisopropylethylamine was utilized as a base
to yield a mixture of mono (9) and bis (10) products.
According to molecular modeling studies, the 4-position
of the pyrazolidinedione ring points into a long and
large lipophilic pocket. Hence, using Schemes 1, 3 and
4 over 195 4-alkyl or 4,40-alkyl derivatives were synthe-
sized to probe the lipophilic pocket and submitted for
evaluation8–10 as new inhibitors of bacterial cell wall
biosynthesis. SAR (structure activity relationship)
trends were established and some are highlighted in Ta-
bles 1–3. In terms of activity against MurB and Gram-
positive bacteria, particularly Streptococcus pneumoniae
(PRSP), lipophilic 4-alkyl groups were preferred over
somewhat more hydrophilic groups (3b vs 3f, 7i vs 3e).
Potency was improved against MurB by incorporating
an aromatic ring in the lipophilic chain (Table 1, 3a, b
compared to 7a–d). Longer chain length (Table 1, 7a–
d vs 3c, d) was also a factor in improving IC50 values
against MurB (n = 1 IC50 > 50 lM, n = 6 IC50 ꢀ 9 lM)
and MIC (minimum inhibitory concentration) values
for Streptococcus pneumoniae bacteria strains (n = 1,
200 lM and n = 6, 6.25 lM). Derivative 7d (chain length
Cl
Cl
NH2
a
b
NH
NH
N
N
1
4
Cl
Cl
Cl
Scheme 2. Reagents and conditions: (a) MnO2, benzene, reflux, 1 h;
(b) Zn dust, NH4Cl, acetone, 1 h.
O
O
O
O
O
O
OEt OEt
O
O
5
a
OEt OEt
N
N
HN NH
11
a
N
N
HN NH
6
Cl
Cl
1
Cl
Cl
12
Cl
Cl
1
Cl
Cl
O
R
R
R
N
R
N
O
O
O
O
O
b
N
N
R
b
N
N
7
8
Cl
c
Cl
Cl
Cl
13
Cl
O
Cl
O
6
R
R
12
O
O
O
R
O
O
O
O
O
O
O
O
c
R
N
N
N
N
N
N
N
N
15
10
14
9
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Scheme 3. Reagents and conditions: (a) NaOEt, microwave, 2 min; (b)
R-Br, Li2CO3, DMF, microwave, 2–4 min; (c) R-C(O)–CH2–Br,
DIPEA, toluene, microwave, 1–3 min.
Scheme 4. Reagents and conditions: (a) NaOEt, microwave, 4 min; (b)
R-Br, Li2CO3, DMF, microwave, 2–4 min; (c) R-C(O)–CH2–Br,
DIPEA, toluene, microwave, 1–3 min.

K. M. K. Kutterer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2527–2531
2529
Table 1. Antimicrobial activities (lM) and IC50 values of selected 4-alkyl and 4,40-dialkyl-1,2-bis(4-chlorophenyl)pyrazolidine-3,5-diones
O
O
O
n
n
R
Cl
O
N
n
N
O
O
n
O
O
8a
3c-d, 7a-d
3a-b
13a
Cl
3a
3b
(CH2)5CH3
3c
1
3d
2
7a
3
7b
4
7c
5
7d
6
8a
6
13a
6
CH(CH2CH3)2
S. aureus GC 1131 (MRSA)
S. aureus GC 4543 (MSSA)
E. faecalis GC 4555 (ATCC)
E. faecalis GC 2242 (VRE)
S. pneumo GC1894 (PRSP)
S. pneumo GC1894 (PRSP)a
MSCNS GC 646
100
100
200
100
25
100
100
200
100
6.25
>200
200
>200
>200
>50
42
200
200
200
200
>200
200
50
200
200
>200
200
25
200
200
200
100
25
200
100
100
200
25
25
25
>200
200
>200
>200
200
>200
>200
200
50
50
>200
200
100
100
6.25
>200
100
>200
200
34.9
8.9
0.78
>200
>200
>200
200
>200
200
>200
200
>50
50
>200
>200
>200
>200
>50
>50
—
>200
200
>200
200
>50
35
>200
>200
>200
200
44.3
10
>200
200
>200
>200
23.8
5.6
>200
>200
>200
>200
42.3
10
>200
>200
>200
>200
9.9
E. coli GC 4559
E. coli GC 4560
MurA, E. coli (IC50, lM)
MurB, E. coli (IC50, lM)
MurB, S. aureus (IC50, lM)
>50
>50
7.2
8.2
—
—
—
—
6.8
32
12
>50
R = CH(CH2CH3)2 or (CH2)5CH3; n = 1–6.
a Tested in the presence of 4% bovine serum albumin.
Table 2. Antimicrobial activities (lM) and IC50 values of selected 4-alkyl and 4,40-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-diones
O
O
O
X
X
O
O
X
R
O
O
O
X
7e-g
7h-l
8b
13b
7e
NEt
7f
7g
O
8b
O
13b
O
7h
2-Cl
7i
3-CF3
7j
2,4-DiCl
7k
3,5-DiCF3
7l
4-(1-CN-Phenyl)
S
S. aureus GC 1131 (MRSA)
S. aureus GC 4543 (MSSA)
E. faecalis GC 4555 (ATCC)
E. faecalis GC 2242 (VRE)
S. pneumo GC1894 (PRSP)
S. pneumo GC1894 (PRSP)a
MSCNS GC 646
200
25
25
100
200
200
100
25
100
100
25
200
>200
>200
>200
>200
12.5
>200
>200
>200
>200
50
50
50
25
25
12.5
6.25
25
>200
>200
200
>200
>200
>200
100
100
50
100
50
50
25
50
12.5
3.12
>200
6.25
>200
25
100
6.25
>200
50
12.5
>200
200
>200
200
20.1
7.2
12.5
>200
50
6.25
>200
50
>200
>200
>200
>200
>50
>200
200
>200
200
33.4
11
>200
>200
>200
>200
>50
20
>200
>200
>200
>200
>50
>200
>200
>200
>200
>50
E. coli GC 4559
>200
200
9.8
>200
100
>50
11
50
E. coli GC 4560
>200
>50
12
MurA, E. coli (IC50, lM)
MurB, E. coli (IC50, lM)
MurB, S. aureus (IC50, lM)
>50
11
>50
>50
5.2
5.1
>50
>50
18
—
11
7.5
—
29
—
12
X = NEt, S or O; R = 2-Cl, 3-CF3, 2,4-diCl, 3,5-diCF3, or 4-(1-CN-Phenyl).
a Tested in the presence of 4% bovine serum albumin.
n = 6) also demonstrated good activity against other
Gram-positive bacteria including methicillin-resistant
Staphylococcus aureus (MRSA), methicillin-susceptible
S. aureus (MSSA), vancomycin-susceptible Enterococcus
faecalis (ATCC) and vancomycin-resistant E. faecalis
(VRE) with MICs in the range of 25–50 lM. The 4,40
bis-alkyl derivative of 7d, 8a, showed no change in
MurB activity but almost complete loss of antibacterial
activity against PRSP. Contrarily, the 40-methyl deriva-
tive 13a showed complete loss of MurB activity and a
significant improvement of PRSP MIC to 0.78 lM, indi-
cating that for this derivative bacterial inhibition was
not operating through inhibition of MurB.
In general, the addition of more polar heteroatoms into
the chains spanning between the pyrazolidinedione and
phenyl ring resulted in a decrease in MurB IC50 (7b vs
7e and 7g) and also a 4-fold decrease in MIC for 7e.
On the other hand, addition of a sulfur atom in the
chain (7f) lead to a maintenance of MurB IC50, a

2530
K. M. K. Kutterer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2527–2531
Table 3. Antimicrobial activities (lM) and IC50 values of selected 4-alkyl and 4,40-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-diones
F
F
O
F
F
O
F
R
F
O
O
O
n
F
O
O
n
O
n
3e
F
O
O
O
R
n
F
O
O
O
O
3f
O
14a
15a
7m-n, 9a
10a
O
7m
3
7n
2
9a
1
10a
1
14a
1
15a
1
3e
3-OMe
3f
CH2N(CH2CH3)2
S. aureus GC 1131 (MRSA)
S. aureus GC 4543 (MSSA)
E. faecalis GC 4555 (ATCC)
E. faecalis GC 2242 (VRE)
S. pneumo GC1894 (PRSP)
S. pneumo GC1894 (PRSP)a
MSCNS GC 646
50
50
100
100
100
100
50
25
25
12.5
12.5
12.5
12.5
3.12
>200
>200
200
>200
>200
>200
200
>200
>200
>200
>200
200
100
>200
200
200
50
50
50
50
100
12.5
25
>200
>200
>200
>200
>200
>50
>200
>200
>200
>200
>200
>50
100
>200
200
>200
100
>200
200
32.4
10
>200
200
>200
200
46.9
13
>200
>200
>200
200
8.4
>200
25
>200
200
E. coli GC 4559
>200
100
9.6
>200
>200
>50
50
>200
>200
>50
>50
—
E. coli GC 4560
MurA, E. coli (IC50, lM)
MurB, E. coli (IC50, lM)
MurB, S. aureus (IC50, lM)
5.4
5.2
5.8
5.2
49
>50
>50
>50
11
18
—
R = 3-OMe, CH2N(CH2CH3)2; n = 1–3.
a Tested in the presence of 4% bovine serum albumin.
significant increase in MurA activity, and a 4-fold
improvement in PRSP MIC. In contrast to the previous
4,40-dialkyl case (7d vs 8a) the 4,40-dialkyl with a hetero-
atom in the chains causes a increase in both MurB and
MIC activity (8b). The 40-methyl derivative 13b, how-
ever, resulted in a loss of MurB activity and antimicro-
bial activity.
bovine serum albumin, their MIC values increased to
greater than 200 lM against PRSP. Activity against
Gram-negative bacteria was not detected with the excep-
tion of derivative 7l with a MIC of 25 lM. Derivatives
7f (Kd = 400 nM) and 10a (Kd = 710 nM) were also
shown to display potent nanomolar Kd values in their
binding to MurB. Since many derivatives, 7d, 7f, 7k, 7l
and 10a show good selectivity against MurB as well as
good activity against Gram-positive penicillin resistant
bacteria, they are good leads for further investigation.
Investigation of non-polar substituents on the terminal
phenyl ring (Table 2, 7h–l) indicated that increased lipo-
philic bulk improves MurB activity as well as antimicro-
bial activity against Gram-positive bacteria. Derivative
7l was the most promising compound with activity
against Gram-positive bacteria in the range between
3.12 and 12.5 lM and Gram-negative MIC for Escher-
ichia coli GC 4560 being 25 lM.
Acknowledgements
We would like to thank Dr. David Shlaes for his support
of the program.
When a carbonyl (Table 3, derivatives 7m,n and 9a) is
placed in the chain, the chain length effect reverses with
the shorter chain, 9a, showing the best potency. The
dialkyl derivative, 10a, and methylalkyl derivative,
14a, however, show the same trends of increased po-
tency for the dialkyl and loss of potency for the
methyl-alkyl, as was noted previously for other deriva-
tives. The interesting O-alkyl side product 15a obtained
in this case showed absolutely no activity.
References and notes
1. (a) Wong, K. K.; Pompiano, D. L. In Resolving the
Antibiotic Paradox: Progress in Understanding Drug
Resistance and Development of New Antibiotics; Rosen,
B. P., Mobashery, S., Eds.; Plenum: NY, 1998, pp 197–
217; (b) Goldman, R. C.; Branson, A. Curr. Pharm.
Design 1999, 5, 473.
2. Isono, K.; Uramoto, M.; Kusakabe, H.; Kimura, K.;
Izaki, K.; Nelson, C. C.; McCloskey, J. A. J. Antibiot.
1985, 38, 1617.
3. Kimura, K.; Ikeda, Y.; Kagami, S.; Yoshihara, M. J.
Antibiot. 1998, 51, 1099.
4. El-Rayyes, N. R.; Al-Awadi, N. A. Synthesis 1985, 11,
1028.
5. Representative procedure for microwave assisted pyrazo-
lidine-3,5-dione synthesis: Reactions were carried out in
open glass vials using a CEM Corporation Mars 5
commercial multimode microwave at a setting of 900 W
In summary, microwave heating has proven to be effi-
cient in many steps in the synthesis of 4-alkyl and 4,40-
dialkyl pyrazolidinediones. Alkyl pyrazolidinedione
derivatives were identified as a new class of inhibitors
in bacterial cell wall biosynthesis. In general they dem-
onstrated good activity against MurB and to a lesser de-
gree against MurA and in some cases good activity
against Gram-positive bacteria, particularly PRSP.
However, when they were tested in the presence of 4%

K. M. K. Kutterer et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2527–2531
2531
(note: temperature setting was not used since these
reactions were run to dryness given that the driving force
of the reaction is the elimination of ethanol). To 1,2-bis(4-
chlorophenyl)hydrazine 1 (2.53 g, 10 mmol) was added
10 mL of sodium ethoxide (21 wt% in ethanol) and diethyl
malonate (5 mL) and subjected to microwave heating for
2 min. The residue was dissolved in water (50 mL),
acidified with 1 N hydrochloric acid, and extracted with
dichloromethane (100 mL). The organic extracts were
concentrated to give 3.05 g of 1,2-bis(4-chlorophenyl)pyr-
5748) and consisted of 150 nM MurA, 200 lM PEP, and
200 lM UDP-N-acetylglucosamine (UDP-GlcNAc) in
25 lL of 25 mM Tris, pH 8. The reaction was incubated
for 30 min at 37 ꢁC and was terminated by the addition of
210 lL of malachite green reagent (0.03% malachite green,
1.4% ammonium molybdate, and 1.3 N HCl). Inhibition
of MurA was performed by preincubating of the enzyme
and compound for 10 min at 37 ꢁC before addition of the
substrates. The amount of phosphate released during the
reaction was detected spectrometrically at 660 nm using a
Molecular Devices Spectra Max 250 with SoftMax soft-
ware. The results were compared to the inhibition of the
enzyme by fosfomycin (commercially available MurA
inhibitor). The results are presented as % inhibition at a
compound concentration of 25 lg/mL. The calculation
follows the formula: % inhibition = 100 ꢁ (OD for sam-
ple)/(OD mean of control) · 100. IC50s were then esti-
mated by linear regression analysis using the percent
inhibition data bracketing 50% inhibition.
1
azolidine-3,5-dione 6. H NMR (DMSO-d6, 400 MHz): d
3.78 (s, 2H), 7.32 (m, 4H), 7.42 (m, 4H), MS [(ꢁ)ESI, m/z]:
319.1 [MꢁH].
6. (a) Banthorpe, D. V.; Cooper, A. J. Chem. Soc. B 1968, 6,
608; (b) Baldwin, C. M.; Song, P. S.; Shine, H. J.
J. Am. Chem. Soc. 1970, 92, 3284; (c) Oae, S.; Tsujimoto,
N.; Nakanishi, A. Bull. Chem. Soc. Jpn. 1973, 46,
535.
7. Representative procedure for microwave assisted alkyl-
ation of pyrazolidine-3,5-diones: Reactions were carried
out in open glass vials using a CEM Corporation Mars 5
commercial multimode microwave at a setting of 200 ꢁC
and a maximum of 900 W or in closed glass vessels using a
Personal Chemistry Smith Workstation single mode
microwave at 200 ꢁC. To a solution of 1,2-bis(4-chloro-
phenyl)pyrazolidine-3,5-dione (0.064 g, 0.2 mmol) in
N,N-dimethylformamide (1 mL) was added lithium car-
bonate (0.044 g, 0.6 mmol) and benzyl 2-bromoethyl ether
(0.043 g, 0.2 mmol). The reaction was subjected to micro-
wave heating for 3.5 min, and then concentrated. The
residue was dissolved in water (1.0 mL), acidified with 1 N
hydrochloric acid, and extracted with dichloromethane
(4.0 mL). The organic extract was concentrated, taken up
in acetonitrile and purified by semi-preparative RP-HPLC
(Gilson Semi-Preparative HPLC system with Unipoint
Software v. 1.71, Phenomenex C18 Luna column,
21.6 · 100 mm, 5 l particle size, water–acetonitrile solvent
system with added 0.02% TFA buffer, at 22.5 mL/min) to
give 7g (0.012 g) and 8b (0.004 g) [LC/MS (Hewlett
Packard 1100 MSD with ChemStation Software, xterra
C18 2.1 mm · 30 mm column, 3.5 l particle size, at 50 ꢁC,
water–acetonitrile solvent system with added 0.02% TFA
buffer at 1.0 mL/min flow rate, 254 nm DAD detection;
API-ES scanning mode, fragmentor 140 mV): 7g m/z 455.0
(M+H); retention time 3.279 min, 8b m/z 589.1 (M+H);
retention time 3.622 min].
9. MurB inhibition studies: The inhibition of MurB
enzymatic activity by pyrazolidinedione derivatives was
determined using substrates NADPH (Sigma), and bio-
chemically synthesized, HPLC purified sub EP-UNAG
following the method of Dhalla et al. (Biochemistry 1995,
34, 5390). The reaction was determined following an initial
20 min preincubation of 20 nM of the enzyme with the
inhibitor. The substrate mixture was then added to the
enzyme-inhibitor mixture, to a final concentration of
50 lM EP-UNAG and 100 lM NADPH. MurB activity
was monitored using Molecular Devices Spectra Max 250
with SoftMax software. At least six concentrations of
inhibitor, between 1.5 and 50 lM, were used for each
compound tested. The IC50 values were derived using data
analysis function of Microsoft Excel program (Sigmoid
Curve Hill analysis 0–100).
10. MICs: The in vitro determination of the MICs against
aerobic bacteria was performed by the microdilution broth
method as recommended by the National Committee for
Clinical Laboratory Standards (Approved Standards M7 -
A3, 1997). Mueller–Hinton Broth was used for the
Staphylococci, and Enterococci. Streptococci were tested
in Mueller–Hinton Broth supplemented with 5% sheep
blood. Microtiter plates containing 50 lL sheep blood per
well of 2-fold serial dilutions of the antibacterial agents in
the appropriate broth were inoculated with 50 lL of
inoculum to yield a final density of 1–5 · 105 CFU/mL.
The MICs were determined after 18–22 h of incubation at
35 ꢁC in ambient air.
8. MurA inhibition studies: The MurA reaction followed the
method of Marquardt et al. (J. Bacteriol. 1992, 174(17),