Pyrazolidine-3,5-diones and 5-hydroxy-1H-pyrazol-3(2H)-ones, inhibitors of UDP-N-acetylenolpyruvyl glucosamine reductases

Article abstract of DOI:10.1021/jm060499t

A series of pyrazolidine-3,5-dione and 5-hydroxy-1H-pyrazol-3(2H)-one inhibitors of Escherichia coli UDP-N-acetylenolpyruvyl glucosamine reductase (MurB) has been prepared. The 5-hydroxy-1H-pyrazol-3(2H)-ones show low micromolar IC₅₀ values versus E. coli MurB and submicromolar minimal inhibitory concentrations (MIC) against Staphylococcus aureus GC 1131, Enterococcus faecalis GC 2242, Streptococcus pneumoniae GC 1894, and E. coli GC 4560 imp, a strain with increased outer membrane permeability. None of these compounds show antimicrobial activity against Candida albicans, a marker of eukaryotic toxicity. Moreover, these compounds inhibit peptidoglycan biosynthesis, as assessed by measuring the amount of soluble peptidoglycan produced by Streptococcus epidermidis upon incubation with compounds. A partial least squares projection to latent structures analysis shows that improving MurB potency and MIC values correlate with increasing lipophilicity of the C-4 substituent of the 5-hydroxy-1H-pyrazol-3(2H)-one core. Docking studies using FLO and PharmDock produced several binding orientations for these molecules in the MurB active site.

Full text of DOI:10.1021/jm060499t

J. Med. Chem. 2006, 49, 6027-6036
6027
Pyrazolidine-3,5-diones and 5-Hydroxy-1H-pyrazol-3(2H)-ones, Inhibitors of
UDP-N-acetylenolpyruvyl Glucosamine Reductase
Adam M. Gilbert,*,§ Amedeo Failli,‡ Jay Shumsky,‡ Youjun Yang,†,§ Anatoly Severin,§ Guy Singh,§ William Hu,§
David Keeney,§ Peter J. Petersen,§ and Alan H. Katz‡
Chemical and Screening Sciences and Infectious Diseases Research, Wyeth, 401 North Middletown Road, Pearl RiVer, New York 10965, and
Chemical and Screening Sciences, Wyeth, CN 8000, Princeton, New Jersey 08543-8000
ReceiVed April 27, 2006
A series of pyrazolidine-3,5-dione and 5-hydroxy-1H-pyrazol-3(2H)-one inhibitors of Escherichia coli UDP-
N-acetylenolpyruvyl glucosamine reductase (MurB) has been prepared. The 5-hydroxy-1H-pyrazol-3(2H)-
ones show low micromolar IC50 values versus E. coli MurB and submicromolar minimal inhibitory
concentrations (MIC) against Staphylococcus aureus GC 1131, Enterococcus faecalis GC 2242, Streptococcus
pneumoniae GC 1894, and E. coli GC 4560 imp, a strain with increased outer membrane permeability.
None of these compounds show antimicrobial activity against Candida albicans, a marker of eukaryotic
toxicity. Moreover, these compounds inhibit peptidoglycan biosynthesis, as assessed by measuring the amount
of soluble peptidoglycan produced by Streptococcus epidermidis upon incubation with compounds. A partial
least squares projection to latent structures analysis shows that improving MurB potency and MIC values
correlate with increasing lipophilicity of the C-4 substituent of the 5-hydroxy-1H-pyrazol-3(2H)-one core.
Docking studies using FLO and PharmDock produced several binding orientations for these molecules in
the MurB active site.
Chart 1
Introduction
The majority of efforts to develop new antibacterial therapies
have focused on synthesizing new derivatives of known
compound classes (fluoroquinolones, tetracyclines, oxazolidi-
nones). While these new derivatives are clinically effective, new
strategies to treat bacterial infection are necessary due to the
rapid emergence of bacterial resistance. Therapeutic failure of
clinically used antibiotics against methycillin-resistant Staphy-
lococcus aureus (MRSA), vancomycin-resistant Enterococcus
faecalis (VRE), and penicillin-resistant Streptococcus pneumo-
niae (PRSP) have created an urgent need for development of
new structural classes of antibiotics with new mechanisms of
action.1
Since the intact cell wall is critical for bacterial survival, we
decided to target the cytoplasmic enzymes involved in the early
steps of bacterial cell wall biosynthesis. Specifically we focused
on targeting MurBa (UDP-N-acetylenolpyruvyl glucosamine
reductase),2 the enzyme that catalyzes the stereospecific reduc-
tion of enolpyruvyl uridine diphosphate N-acetylglucosamine
(UDP-EP-GluNAc) to uridine diphosphate N-acetylmuramic
acid (UDP-MurNAc). This enzyme is only found in prokary-
otes,3 has been shown to be essential for bacterial cell wall
biosynthesis, and has been the subject of biochemical4,5 and
structural studies in Escherichia coli and S. aureus.6 Thus MurB
represents an attractive target for the development of new
antibacterial therapeutics. To date, there have been few disclo-
sures of small molecule MurB inhibitors with antibacterial
properties.7-11
While doing a moderate-throughput screen of an in-house
collection of molecules with antibacterial activity against S.
aureus and no activity against a yeast strain, Candida albicans,
we identified examples of pyrazolidine-3,5-diones and 5-hy-
droxy-1H-pyrazol-3(2H)-ones (1 and 2, Chart 1) that inhibit E.
coli MurB and peptidoglycan biosynthesis and possess antibac-
terial activity against Gram-positive bacteria.12 The following
paper describes the synthesis and structure-activity relationship
development of these two classes of MurB inhibitors.
Chemistry
Preparations of the antibacterial pyrazolidine-3,5-diones and
5-hydroxy-1H-pyrazol-3(2H)-ones 1 and 2 are outlined in
Schemes 1 and 2, respectively. For pyrazolidine-3,5-diones 1,
anilines 3-6 were dimerized to the corresponding diazo
compounds 7-10 via oxidation with MnO2.13 Reduction to the
corresponding hydrazines 11-15 was accomplished via the
action of Zn dust and NH4Cl in aqueous acetone.14 Pyrazolidine-
3,5-diones 16-20 (Scheme 1) were prepared via heating with
diethyl malonate in EtOH and distilling the reaction solution to
dryness (oil bath temperature ∼200 °C).
* To whom correspondence should be addressed. Phone: (845) 602-
4865. FAX: (845) 602-5561. E-mail: gilbera@wyeth.com.
† Present address: Avon Products Inc., Suffern, NY 10901.
‡ Chemical and Screening Sciences, Wyeth, New Jersey.
§ Chemical and Screening Sciences and Infectious Diseases Research,
Wyeth, New York.
Pyrazolidine-3,5-diones 16-20 were treated with isocyanates
in the presence of Et3N in toluene15 to yield 4-amido compounds
21-39 and 41-47 (Scheme 2). The corresponding 4-keto
derivatives 48-57 were prepared from 17 using the above
conditions, except that acid chlorides were used in place of
isocyanates.16
a Abbreviations: MurB, UDP-N-acetylenolpyruvyl glucosamine reduc-
tase; UDP-EP-GluNAc, enolpyruvyl uridine diphosphate N-acetylglu-
cosamine; UDP-MurNAc, uridine diphosphate N-acetylmuramic acid; MIC,
minimal inhibitory concentrations; SPG, soluble peptidoglycan; PLS, partial
least squares projection to latent structures.
10.1021/jm060499t CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/13/2006

6028 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Scheme 1. Preparation of Pyrazolidine-3,5-diones 16-20a
Gilbert et al.
Biology
E. coli MurB was purified according to published proce-
dures.2,17 The inhibition of MurB activity was analyzed by
incubating an enzyme/inhibitor mixture with enolpyruvyl UDP-
N-acetyl glucosamine and NADPH. MurB activity was measured
by monitoring the amount of NADPH oxidation (reduction in
the absorbance at 340 nm using a SpectroMax 250-plate reader).
Minimal inhibitory concentrations (MIC) against S. aureus GC
1131; E. faecalis GC 2242; S. pneumoniae GC 1894; the E.
coli GC 4560 imp strain, a strain with increased outer membrane
permeability;12 and a yeast strain, C. albicans, were measured
using the microbroth dilution method. None of the 5-hydroxy-
1H-pyrazol-3(2H)-ones showed activity against C. albicans. The
inhibition of peptidoglycan biosynthesis was determined by
measuring the amount of soluble peptidoglycan (SPG) produced
by a Streptococcus epidermidis upon incubation with compounds
under study.
Results and Discussion
a Reagents: (a) MnO2, PhMe, 110 °C; (b) Zn(dust), NH4Cl, acetone, H2O,
23 °C; (c) NaOEt, diethyl malonate, EtOH, 50-200 °C (dryness).
Four out of the five pyrazolidine-3,5-diones prepared, 16, 17,
19, 20, display weak MurB inhibition (IC50 > 68 µM) (Table
1). However, the bis((3,4-Cl)Ph) analogue 18 shows moderately
potent MurB inhibition with an IC50 ) 25 µM. Moreover, 18
has moderate antimicrobial activity against S. aureus GC 1131,
E. faecalis GC 2242, S. pneumoniae GC 1894, and the E. coli
GC 4560 imp strain (respective MICs: 8, 8, 8, and 32 µg/mL).
Both 18 and 19 were tested for SPG inhibitory activity, and
both show weak inhibition (18, SPG IC50 ) 41 µM; 19, SPG
IC50 ) 56 µM).
Scheme 2. Preparation of 5-Hydroxy-1H-pyrazol-3(2H)-ones
21-39, 41-47, and 48-57a
The 4-substitued 5-hydroxy-1H-pyrazol-3(2H)-ones exhibited
more potent MurB inhibition and improved MIC values (Table
2). The phenyl carboxamide 21 possesses a MurB IC50 of 9.8
µM and an excellent MIC profile. Of special note is that 21
shows MIC activity against the E. coli imp strain (MIC ) 4
µg/mL) and submicromolar SPG inhibition (IC50 ) 0.78 µM).
Substitution of the carboxamide phenyl ring affects MurB
inhibition, MICs, and SPG inhibition. Electron-withdrawing/
lipophilic moieties such as chlorine (22, 25, 26) give compounds
with comparable MurB IC50 values to that of 21 or compounds
that are more potent. MIC values for these analogues are
generally comparable to that of 21 except that only 22 shows
similar MIC activity in the E. coli imp strain (MIC ) 2 µg/
mL). The high value for 22 in the SPG assay is not understood
at this time.
Other electron-withdrawing substituents (CF3, 23, 24; CN,
28, CO2Et, 30) exhibit similar MurB IC50s compared to 21. The
MIC profile of these compounds is also similar, but they do
not show comparable antimicrobial activity against the E. coli
imp strain. All of these analogues possess SPG inhibitory
activity with IC50 values in the micromolar range. Polar electron-
withdrawing substituents (CO2H, 31; C(O)NH(CH2)3NMe2, 33)
give inactive compounds. Electron-donating substituents (OMe,
27; OH, 32) provide compounds that are slightly less active
against MurB and a panel of microorganisms than 21. Extension
of the phenyl carboxamide 21 to a phenethyl carboxamide 37
generates a compound with similar MurB inhibition but
comparatively weaker antibacterial activity against S. aureus
and E. coli imp. The corresponding benzyl carboxamides (34-
36) have similar profiles in that they show good MurB inhibition
but comparatively weaker MICs against S. aureus and E. coli
imp. Compounds 34 and 35 also show comparable SPG
inhibition activity to the corresponding phenyl analogues. Methyl
substitution of the benzyl carbon (38 and 39) is tolerated, but
these compounds do not offer improvements in MurB potency
a Reagents: (a) R3NCO, Et3N, PhMe, 0-100 °C; (b) R3COCl, Et3N,
PhMe, 0-100 0 °C.
Methyl amide 40 was prepared by using N-methyl-N-
phenylcarbamoyl chloride in place of an isocyanate, as shown
in eq 1.

Cell Wall Biosynthesis Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6029
Table 1. E. coli MurB Inhibition, Minimal Inhibition Concentrations (MIC), and Soluble Peptidoglycan Biosynthesis Inhibition of
Pyrazolidine-3,5-diones
MIC (µg/mL)b
MurB
inhibn of
SPG IC50
(µM)c
IC50
S. aureus
GC 1131
E. faecalis
S. pneumo
E. coli
imp
compd
X
Y
(µM)a
GC 2242
GC 1894
16
17
18
19
20
4-H
4-H
>100
71.6
25.1
68.5
>87.0
-
8
8
16
128
-
8
8
16
128
-
8
4
8
64
-
-
4-Cl
3,4-Cl
3-Cl
4-F
4-Cl
3,4-Cl
3-Cl
4-F
16
32
200
64
41.0
56.0
a Average of three runs. Standard deviation (5%. b Average of two runs. Standard deviation (5%. c Average of three runs. Standard deviation (6%.
Table 2. E. coli MurB Inhibition, Minimal Inhibition Concentrations (MIC), and Soluble Peptidoglycan Biosynthesis Inhibition of
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazole-4-carboxamides
MIC (µg/mL)b
MurB
IC50
inhibn of
SPG IC50
(µM)c
S. aureus
GC 1131
E. faecalis
S. pneumo
E. coli
imp
compd
R3
(µM)a
GC 2242
GC 1894
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Ph
9.8
13.7
5.5
5.9
5.9
12.2
16
13.1
8.1
17.6
>52
21.9
>44
<6.2
<6.2
<6.2
9.6
1
1
1
16
0.5
1
8
2
1
8
64
8
8
26.1
8
6.1
11.7
3.4
26
<0.12
0.50
0.25
0.25
<0.12
0.25
1
0.25
0.25
0.5
128
8
8
0.41
0.25
0.76
0.37
0.21
0.4
<0.12
<0.12
<0.12
>128
<0.12
<0.12
0.5
<0.12
0.25
0.25
64
4
4
0.2
0.25
<0.10
<0.10
0.21
0.2
4
2
16
16
32
8
>128
>128
8
>128
64
4
4
>105
>128
>100
>100
>109
>103
0.78
42.2
4.7
8.3
7.0
(4-Cl)Ph
(3-CF3)Ph
(4-CF3)Ph
(3-Cl)Ph
(2-Cl)Ph
(4-OMe)Ph
(4-CN)Ph
cyclohexyl
(4-CO2Et)Ph
(4-CO2H)Ph
(4-OH)Ph
(4-CONH(CH2)3NMe2)Ph
(3,4-Cl)PhCH2
(2,4-Cl)PhCH2
(2-Cl)PhCH2
PhCH2CH2
(4-Br)PhCH(Me)
naphthylCH(Me)
6.9
<0.87
11.1
2.6
>44.0
7.5
8.8
5.6
<6.2
a Average of three runs. Standard deviation (5%. b Average of two runs. Standard deviation (5%. c Average of three runs. Standard deviation (6%.
or antimicrobial activity when compared to corresponding
phenyl analogues.
analogues show MurB IC50s and antibacterial activity against
the Gram-positive strains that are similar to the corresponding
compounds where R1 is (4-Cl)Ph (see 22 and 24). The R1 )
(3,4-Cl) analogues 46 and 47 show similar MurB inhibition to
44 and 45 but weaker S. aureus antibacterial activity.
That the internal hydrogen bond between the amide N-H
and the 5-hydroxy-1H-pyrazol-3(2H)-one carbonyl may be
important for activity is shown in N-Me amide analogue 40
(eq 1). This compound is devoid of MurB inhibitory activity
(MurB IC50 > 55 µM) as well as antibacterial activity (MIC >
128 µg/mL). The rigidification of the pyrazolidinedione-4-
carboxamide structure provided by this internal hydrogen bond
could lock these amides into a bioactive conformation and is
the probable cause of this effect.
The effects of varying the N(1) and N(2) substituents of the
5-hydroxy-1H-pyrazol-3(2H)-ones can be seen in Table 3.
Compounds 41-43 all possess unsubstituted Ph groups at R1
and all of these compounds exhibit weak MurB activity
(IC50 ) 42-54 µM) and only moderate MIC values. An
improvement in MurB inhibition and MICs is seen in com-
pounds 44 and 45, which have (3-Cl)Ph groups at R1. These
The corresponding ketone analogues of several compounds
in Tables 2 and 3 show good MurB inhibition and MICs (Table
4). Analogues with electron-withdrawing groups on the phenyl
ketones [48, (4-Cl)Ph; 49, (3,4-Cl)Ph; 52, (4-OCF3)Ph; and 55,
(4-CF3)Ph] possess good MurB inhibition (IC50 ) 5.4-15.0 µM)
and good antibacterial activity against the Gram-positive bacteria
tested (MIC ) 0.39-12.7 µg/mL). These analogues also have
moderate MICs against the E. coli imp strain (MIC ) 16.0-49
µg/mL). Analogues with electron-donating groups on the phenyl
ketones [50, (4-OMe)Ph; 51, (4-O-n-Bu)Ph] exhibit similar
MurB inhibition and MICs [IC50 ) 4.5-10.0 µM; MIC ) 0.39-
11.3 µg/mL). The thiophene ketone 54 (IC50 ) 5.8 µM) shows

6030 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Gilbert et al.
Table 3. E. coli MurB Inhibition, Minimal Inhibition Concentrations (MIC), and Soluble Peptidoglycan Biosynthesis Inhibition of
5-Hydroxy-3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazole-4-carboxamides
MIC (µg/ mL)b
MurB
inhibn of
SPG IC50
(µM)c
IC50
S. aureus
GC 1131
E. faecalis
S. pneumo
E. coli
imp
compd
R1
R3
(µM)a
GC 2242
GC 1894
41
42
43
44
45
46
47
Ph
Ph
Ph
(3-Cl)Ph
(3-Cl)Ph
(4-Cl)Ph
(4-Br)Ph
Ph
(4-Cl)Ph
(4-CF3)Ph
(4-Me)Ph
(3,4-Cl)Ph
54
42
54
8.6
8.7
4.8
4.8
32
16
64
0.5
0.5
16
64
1.0
1.0
8.0
0.25
<0.12
0.25
0.5
0.5
1.0
>128
>128
>128
>128
64
>128
>128
16.0
<0.12
<0.12
<0.12
0.5
3
2.2
4
(3,4-Cl)Ph
(3,4-Cl)Ph
a Average of three runs. Standard deviation ( 5%. b Average of two runs. Standard deviation ( 5%. c Average of three runs. Standard deviation ( 6%.
Table 4. E. coli MurB Inhibition, Minimal Inhibition Concentrations (MIC), and Soluble Peptidoglycan Biosynthesis Inhibition of
4-Acetyl-1,2-bis(4-chlorophenyl)-5-hydroxy-1H-pyrazol-3(2H)-ones
MIC (µg/mL)b
MurB
IC50
inhibn of
SPG IC50
(µM)c
S. aureus
GC 1131
E. faecalis
S. pneumo
E. coli
imp
compd
R3
(µM)a
GC 2242
GC 1894
48
49
50
51
52
53
54
55
56
57
(4-Cl)Ph
11.0
13.0
10.0
4.5
5.4
6.1
5.7
6.2
11.3
1.6
12.7
3.1
5.4
8.0
11.7
1.3
1.40
0.77
5.7
0.39
0.79
0.39
1.3
2.0
1.4
0.34
0.72
0.39
2.8
0.39
0.79
0.3
0.67
0.50
0.73
0.17
23.0
49.0
23.0
>100
25.4
>100
21.6
16.0
>94
5.4
(3,4-Cl)Ph
(4-OMe)Ph
(4-On-Bu)Ph
(4-OCF3)Ph
(4-Ph)Ph
7.3
2.0
4.9
1.0
thiophene
5.8
(4-CF3)Ph
(4-OMe)PhCH2
cyclohexyl
15.0
40.0
24.0
a Average of three runs. Standard deviation (5%. b Average of two runs. Standard deviation (5%. c Average of three runs. Standard deviation (6%.
good MurB inhibitory activity while the cyclohexyl ketone 57
(IC50 ) 24.0 µM) is less potent.
this increasing activity are increasing R5_clogP, decreasing
R5_HBA (hydrogen-bond-acceptor count), and decreasing
R5_PSA. E. coli imp MICs improve less strongly than MICs
against Gram-positive bacteria, but the activity is modulated
by the same calculated properties. Increasing SPG inhibition
(moving from the origin into the SE quadrant of the figure) is
not as closely correlated to the MurB inhibition and/MIC activity
as SPG inhibition is located in a different quadrant that the other
biological activities. Decreasing R5_HBD (hydrogen-bond-
donor count), R5_MW, and R5_cMR appear to be the variables
that correlate best with increasing SPG inhibition.
In line with what we have previously reported,12 all of the
test compounds 21-57 showed no antimicrobial activity against
S. pneumoniae in the presence of 4% bovine serum albumin
(BSA) (MIC > 128 µg/mL). This effect of serum albumin
reducing MIC values is presumably due to the high protein-
binding properties of these compounds, leaving them unavailable
to interact with the bacterial target.18 This BSA effect prevents
us from testing the 5-hydroxy-1H-pyrazol-3(2H)-ones for ef-
ficacy in animal models.
While not normally expected for antibacterial agents, cor-
relation of the 5-hydroxy-1H-pyrazol-3(2H)-one physical prop-
erties, MurB inhibition, MIC activity, as well as SPG inhibitory
activity is possible using partial least squares projection to latent
structures (PLS) analysis19,20 using Simca-P21 (Figure 1). A four-
component PLS model is generated where 75% of the calculated
physical properties data (R2X ) 0.75) and 50% of the MurB,
MIC, and SPG data (R2Y ) 0.5) is used in the model. As MurB
inhibition increases (moving from the origin into the NE
quadrant of the figure), the MICs against the Gram-positive
bacteria, S. aureus, E. faecalis, and S. pneumoniae, also improve.
The calculated physicochemical properties that correlate with
A computational chemistry effort was undertaken to ascertain
the binding mode of 5-hydroxy-1H-pyrazol-3(2H)-ones in the
MurB active site. All attempts to obtain well-defined density
for a member of the 5-hydroxy-1H-pyrazol-3(2H)-one series
were unsuccessful.22 Diffuse density is identified that radiates
from above the flavine ring of the FAD cofactor. Docking
studies were carried out using FLO23 and PharmDock24 against
the available structure of the S229A MurB mutant/substrate
complex.25 Each docking algorithm produces three families of
orientations (Figure 2). The first coincides with the substrate-
binding site and places the 4-substituent above the flavine ring,
and the 1,3-dicarbonyl group overlays the diphosphate of the

Cell Wall Biosynthesis Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6031
Figure 1. PLS loadings plot (w*c[1] vs w*c[2]) of 5-hydroxy-1H-pyrazol-3(2H)-ones 16-38 and 47-56: calculated physiochemical properties
(X-matrix), negative log (E. coli MurB IC50, MIC Data and SPG Inhibitory Data) (Y-Matrix) (A ) 4; R2X ) 0.75; R2Y ) 0.5; Q2(cum) ) 0.35).
Figure 2. The top panel shows the orientation of the MurB substrate in the crystal structure and a proposed orientation of NADP in the perpendicular
site. The bottom panel shows how 5-hydroxy-1H-pyrazol-3(2H)-one docks into the two sites. A third orientation was found in which the NADP-
overlapped orientation is rotated 180°.
substrate. The second and third families of orientations extend
the molecule into the perpendicular site. Preliminary studies,
to be published elsewhere, suggest that NADPH, which is
required to recycle FADH after reduction of the substrate, may
occupy the site. In the crystal structure of the substrate, the enol
ether is situated directly above and parallel to the flavine ring.
The orientation of NADP described in this study places the
nicotinamide ring in the same position, from which the reduced
ring can transfer a hydrogen to FAD. One orientation places
the 4-substituent overlapping the first orientation above the
flavine ring. The other related orientation places the compound
in the same position, but rotated 180° so that the diaryl pyrazole
ring system lies above FAD. All three orientations dock with
similar scores, and we believe the poor resolution obtained by
X-ray may be due to the presence of a mixture of these
orientations.
In all three orientations, there is an intramolecular bond
formed between the amide N-H and the 5-hydroxy-1H-pyrazol-
3(2H)-one’s carbonyl, which is consistent with the loss of
activity of the N-methyl analogue. The orientation that mimics
the substrate shares some of its key interactions near the
diphosphate group with the pyrazolidinedione carbonyls forming

6032 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Gilbert et al.
through Celite. The cake was washed with several portions of
acetone until a clear filtrate was obtained. The combined filtrates
were concentrated to about half the original volume, and the residue
was diluted with 200 mL of ice/water to precipitate the product.
The precipitate was stirred under nitrogen for 1 h and then
concentrated to remove residual acetone. The tan solid was
collected, washed with water, and dried in vacuo over P2O5 to give
5.49 g (21.9 mmol, a 97% yield) of 12 as a pale yellow solid: mp
122-124 °C; MS (EI) m/z 252 (M + H)+. Anal. (C12H10Cl2N2) C,
H, N.
N,N′-Bis(3,4-dichlorophenyl)hydrazine (13). The title com-
pound was prepared according to the procedure of compound 12
using bis(3,4-dichlorophenyl)diazene 8 in place of bis(4-chloro-
phenyl)diazene: yield 94%. Anal. (C12H8Cl4N2) C, H, N.
N,N′-Bis(3-chlorophenyl)hydrazine (14). The title compound
was prepared according to the procedure of compound 12 using
bis(3-chlorophenyl)diazene 9 in place of bis(4-chlorophenyl)dia-
zene: yield 85%; MS (EI) m/z 252 (M + H)+.
hydrogen bonds with TYR190 and GLN288. In both of the
orientations that lie in the putative NADPH binding pocket,
hydrogen bonds exist between the carbonyl groups and TYR190
and ASN233. It is interesting to note that TYR190 bisects and
is therefore available for hydrogen bonds with both general
orientations. In all three orientations, hydrogen-bond-donating
residues exist that can interact with small substituents at the
4-position of the bis-aryl rings of the series.
Conclusion
In summary, we have identified a series of 5-hydroxy-1H-
pyrazol-3(2H)-ones as novel potent inhibitors of E. coli MurB
with antibacterial activity against S. aureus GC 1131, E. faecalis
GC 2242, S. pneumonia GC 1894, and the E. coli GC 4560
imp strain. Inhibition of the cell wall synthetic enzyme MurB
by these compounds affects peptidoglycan biosynthesis and leads
to the inhibition of cell growth.
N,N′-Bis(4-fluorophenyl)hydrazine (15). The title compound
was prepared according to the procedure of compound 12 using
bis(4-fluorophenyl)diazene 10 in place of bis(4-chlorophenyl)-
diazene: yield 95%; MS (APCI+) m/z 221 (M + H)+. Anal.
(C12H10F2N2) C, H, N.
Experimental Section
Chemistry. Melting points were determined on a Thomas-
Hoover Mel-temp apparatus and are uncorrected. The proton nuclear
magnetic resonance (1H NMR) spectra were recorded at 300 MHz
on a Bruker DPX-300 spectrometer using tetramethylsilane (δ 0.0)
as an internal standard. Combustion analyses were obtained using
a Perkin-Elmer Series II 2400 CHNS/O analyzer. Mass spectra were
obtained using a Micromass platform electrospray ionization
quadrapole mass spectrometer. Flash chromatography was per-
formed using EM Science 230-400 mess silica gel. Thin-layer
chromatography (TLC) was performed in Analtech silica gel GHLF
250 µm prescored plates. Analytical LC/MS was performed using
an HP Agilent 1100 LC/MS with an Agilent 1100 diode array
detector (Agilent MS Column, Waters Xterra MS C18 30 mm ×
2.1 mm i.d., 3.5 µm; flow rate, 1.00 mL/min; run time, 5.00 min;
gradient elution, 0 min 90% water, 10% acetonitrile; 3 min 10%
water, 90% acetonitrile; column temperature, 50.0 °C; UV signals,
215, 254 nm. MS parameters: mass range, 100-1000, Fragmentor
140, Gain EMV 1.0). Preparative RP-HPLC was performed using
a Gilson HPLC [Phenomenex LUNA C18 column, 60 mm × 21.20
mm i.d., 5 µm particle size, eluted with an acetonitrile/water gradient
(containing 0.2% Et3N)].
Bis(4-chlorophenyl)diazene (7). To a mechanically stirred
suspension of 4-chloroaniline (25.51 g, 200 mmol) 3 in toluene
(700 mL) was added manganese(IV) dioxide (86.9 g, 1000 mmol).
The mixture was refluxed for 8 h with water removal (Dean-Stark)
and then filtered through Celite. The filter cake was washed with
several volumes of toluene, and the filtrate was evaporated to give
7 as a dark orange solid (9.6 g, 38 mmol). An additional quantity
of 7 was obtained by exhaustive extraction of the Celite cake with
toluene and CH2Cl2 (2.7 g, 10.8 mmol). The total yield of 7 was
12.3 g (49.0 mmol, a 49% yield). The material was used in the
next step without further purification: mp 180-183 °C; MS
(APCI+) m/z 251 (M + H)+. Anal. (C12H8Cl2N2) C, H, N.
Bis(3,4-dichlorophenyl)diazene (8). The title compound was
prepared according to the procedure of compound 7 using 3,4-
dichloroaniline 4 in place of 4-chloroaniline: yield 89%; MS
(APCI+) m/z 321 (M + H)+.
1,2-Diphenylpyrazolidine-3,5-dione (16). To a solution of
sodium metal (574 mg, 23.9 mmol) in anhydrous ethanol (25 mL)
under nitrogen was added 4.2 g (22.8 mmol) of 1,2-bisphenylhy-
drazine 11. The suspension was heated at 50 °C to achieve complete
solution. Diethyl malonate (3.5 mL, 3.67 g, 23.1 mmol) was added,
and after stirring for 15 min, the mixture was placed in an oil bath
preheated at 50 °C. The flask was fitted with a takeoff adapter and
the temperature was slowly raised to 150 °C (over approximately
90 min) to distill off the volatiles. The dry residue was heated at
200 °C for an additional 3 h, cooled, and partitioned between water
(200 mL) and diethyl ether (100 mL). The aqueous layer was again
extracted with diethyl ether and then acidified in the cold with 2 N
HCl. The precipitate was extracted with CH2Cl2. The extracts were
washed with brine, dried over anhydrous MgSO4, and evaporated
to dryness to provide a tan-colored solid. Recrystallization from
ethanol provided 16 (3.0 g, 11.9 mmol, a 52% yield) as a yellow
solid: mp 178-180 °C; MS (ES-) m/z 251 (M - H)-. Anal.
(C15H12N2O2) C, H, N.
1,2-Bis(4-chlorophenyl)pyrazolidine-3,5-dione (17). The title
compound was prepared according to the procedure of compound
16 using N,N′-bis(4-chlorophenyl)hydrazine 12 in place of 1,2-
bisphenylhydrazine: yield 47%; mp 194-197 °C; 1H NMR
(DMSO-d6) δ 7.42 (m, 4H), 7.32 (m, 4H), 3.78 (brs, 2H); MS
(ES-) m/z 319.1 (M - H)-. Anal. (C15H10Cl2N2O2·0.2H2O) C, H,
N.
1,2-Bis(3,4-dichlorophenyl)pyrazolidine-3,5-dione (18). The
title compound was prepared according to the procedure of
compound 16 using N,N′-bis(3,4-dichlorophenyl)hydrazine 13 in
place of 1,2-bisphenylhydrazine: yield 25%; mp 206 °C dec; MS
(ES-) m/z 387 (M - H)-. Anal. (C15H8Cl4N2O2) C, H, N.
1,2-Bis(3-chlorophenyl)pyrazolidine-3,5-dione (19). The title
compound was prepared according to the procedure of compound
16 using N,N′-bis(3,4-dichlorophenyl)hydrazine 14 in place of 1,2-
bisphenylhydrazine: yield 30%; mp 145-147 °C; MS (APCI+)
m/z 321 (M + H)+. Anal. (C15H10Cl2N2O2) C, H, N.
Bis(3-chlorophenyl)diazene (9). The title compound was pre-
pared according to the procedure of compound 5 using 3-chloro-
aniline 5 in place of 4-chloroaniline: yield 88%; MS (APCI+) m/z
321 (M + H)+.
Bis(4-fluorophenyl)diazene (10). The title compound was
prepared according to the procedure of compound 7 using 3-chloro-
aniline 6 in place of 4-chloroaniline: yield 79%; MS (APCI+) m/z
219 (M + H)+.
N,N′-Bis(4-chlorophenyl)hydrazine (12). A suspension of 5.64
g (22.5 mmol) of bis(4-chlorophenyl)diazene 7 (5.64 g, 22.46 mmol)
and 115 mL of acetone was treated with saturated aqueous
ammonium chloride. Zinc dust (11.3 g, 172.3 mmol) was added,
the suspension was stirred at room temperature for 5 h and filtered
1,2-Bis(4-fluorophenyl)pyrazolidine-3,5-dione (20). The title
compound was prepared according to the procedure of compound
16 using N,N′-bis(4-fluorophenyl)hydrazine 15 in place of 1,2-
bisphenylhydrazine: mp 150-152 °C; 1H NMR (DMSO-d6) δ 7.37
(m, 4H), 7.21 (m, 4H), 3.35 (brs, 2H); MS (APCI+) m/z 289
(M + H)+. Anal. (C15H10F2N2O2) C, H, N.
1,2-Bis(4-chlorophenyl)-3,5-dioxo-N-phenylpyrazolidine-4-
carboxamide (21). To a 0 °C suspension of 200 mg (0.62 mmol)
of 1,2-bis(4-chlorophenyl)-5-hydroxy-1,2-dihydropyrazol-3-one 17
and 20 mL of toluene under nitrogen was added 958 µL (6.88
mmol) of Et3N followed by 819 mg (6.88 mmol) of phenyl
isocyanate. After stirring at room temperature for 15 min, the
mixture was heated at 100 °C for 12 h. After cooling to room

Cell Wall Biosynthesis Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6033
temperature, the reaction mixture was diluted with ethyl acetate,
washed with 1 N hydrochloric acid and brine/water (1:1, v/v), dried
over anhydrous Na2SO4, and evaporated to dryness. The residue
was redissolved in dichloromethane and evaporated to provide a
brown foam. Trituration with hexane containing a small amount
of Et2O precipitated a solid which was collected and dried in vacuo
to provide 170 mg (0.38 mmol, 61% yield) of 21 as a light brown
solid: mp 200-203 °C; MS (ES+) m/z 440 (M + H)+. Anal.
(C22H15Cl2N3O3) C, H, N.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid (4-Chlorophenyl)amide (22). The title
compound was prepared according to the procedure of compound
21 using 4-chlorophenyl isocyanate in place of phenyl isocyanate:
yield 65%; mp 215-218 °C; 1H NMR (DMSO-d6) δ 10.55 (s, 1H),
7.57 (d, J ) 6.3 Hz, 2H), 7.32 (m, 8H), 7.27 (d, J ) 6.3 Hz, 2H),
OH proton not visible; MS (APCI+) m/z 474 (M + H)+. Anal.
(C22H14Cl3N3O3) C, H, N.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid (3-Trifluoromethylphenyl)amide (23).
The title compound was prepared according to the procedure of
compound 21 using 3-trifluoromethylphenyl isocyanate in place of
phenyl isocyanate: yield 51%; mp 148.5-151.5 °C; MS (APCI+)
m/z 508 (M + H)+.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid (4-Trifluoromethylphenyl)amide (24).
The title compound was prepared according to the procedure of
compound 21 using 4-trifluoromethylphenyl isocyanate in place of
phenyl isocyanate: yield 66%; mp 208-210 °C; 1H NMR (DMSO-
d6) δ 10.80 (s, 1H), 7.74 (d, J ) 6.3 Hz, 2H), 7.58 (d, J ) 6.3 Hz,
2H), 7.33 (m, 7H), OH proton not visible; MS (EI+) m/z 508
(M + H)+. Anal. (C23H14Cl2F3N3O3) C, H, N.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid (3-Chlorophenyl)amide (25). The title
compound was prepared according to the procedure of compound
21 using 3-chlorophenyl isocyanate in place of phenyl isocyanate:
yield 85%; mp 143-145 °C; 1H NMR (DMSO-d6) δ 10.62 (s, 1H),
7.38-7.30 (m, 9H), 7.27-7.18 (m, 2H), 6.96 (m, 1H), OH proton
not visible; MS (ESI+) m/z 474 (M + H)+. Anal. (C22H14Cl3N3O3)
C, H, N.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid (2-Chlorophenyl)amide (26). The title
compound was prepared according to the procedure of compound
21 using 2-chlorophenyl isocyanate in place of phenyl isocyanate:
yield 33%; mp 226-230 °C; MS (ESI+) m/z 474 (M + H)+. Anal.
(C22H14Cl3N3O3) C, H, N.
4-[1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-
pyrazole-4-carbonyl]aminobenzoic Acid (31). To a suspension
of 170 mg (0.33 mmol) of 4-[1,2-bis(4-chlorophenyl)-5-hydroxy-
3-oxo-2,3-dihydro-1H-pyrazole-4-carbonyl]aminobenzoic acid ethyl
ester 30 and 10 mL of EtOH under nitrogen was added 660 µL
(0.66 mmol) of 1 N NaOH, and the mixture was refluxed for 12 h.
The mixture was acidified to pH 4 with 1 N HCl, and the EtOH
removed in vacuo. The residue was diluted with H2O, and the
precipitate was collected, washed with H2O, and dried in vacuo at
40 °C to provide 120 mg (0.25 mmol, a 76% yield) of 31 as a
white solid: mp >260 °C; MS (APCI-) m/z 482 (M - H)-. Anal.
(C23H15Cl2N3O5·0.20H2O) C, H, N.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid (4-Hydroxyphenyl)amide (32). To a -78
°C solution of 200 mg (0.43 mmol) of 1,2-bis(4-chlorophenyl)-5-
hydroxy-3-oxo-2,3-dihydro-1H-pyrazole-4-carboxylic acid (4-meth-
oxyphenyl)amide 27 in 4 mL of CH2Cl2 was added 1.3 mL (1.3
mmol) of a 1 M solution of BBr3/CH2Cl2, and the mixture was
allowed to warm to 0 °C. After stirring for 3 h at 0 °C, the reaction
was quenched by the addition of aqueous NH4OH, and the resulting
mixture was stirred at room temperature overnight. After extraction
with EtOAc and drying over MgSO4, the solvent was evaporated
in vacuo. Flash chromatography on SiO2, eluting with 5% to 10%
MeOH in CH2Cl2, gave 31 as an amorphous pale brown solid: MS
(APCI-) m/z 455 (M - H)-.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid [4-(3-(Dimethylamino)propylcarbamoyl)-
phenyl]amide (33). To a 23 °C solution of 250 mg (0.52 mmol)
of 4-[1,2-bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-
pyrazole-4-carbonyl]aminobenzoic acid 31 and 3 mL of DMF
(0.097 g, 0.20 mmol) was added 230 µL (1.32 mmol) of i-Pr2NEt,
followed by 85 mg (0.63 mmol) of 1-hydroxybenzotriazole hydrate
and 120 mg (0.63 mmol) of 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride. After stirring at 23 °C for 30 min,
70 mg (0.67 mmol) of diethanolamine was added. After stirring at
23 °C for an additional 26 h, the solvent was removed in vacuo,
and the residue diluted with EtOAc and 2 N HCl. The organic layer
was washed with brine/H2O (1:1, v/v), dried over MgSO4, and
evaporated to dryness. Flash chromatography on SiO2, eluting with
1% to 5% MeOH in CH2Cl2, gave 33 as an amorphous solid: mp
212-220 °C; MS (ESI-) m/z 569 (M - H)-.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid 3,4-Dichlorobenzylamide (34). The title
compound was prepared according to the procedure of compound
21 using 3,4-dichlorobenzyl isocyanate in place of phenyl isocyanate
with the following differences: (1) parallel synthesis format was
used to prepare this compound, (2) analytical an LC/MS as
described above (Chemistry) was used for reaction monitoring, and
(3) compound purification was performed using preparative RP-
HPLC as described above (Chemistry): HPLC 100.0%; MS (ESI-)
m/z 520 (M - H)-.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid (4-Methoxyphenyl)amide (27). The title
compound was prepared according to the procedure of compound
21 using 4-methoxyphenyl isocyanate in place of phenyl isocyan-
1
ate: yield 66%; mp 187-190 °C; H NMR (DMSO-d6) δ 10.30
(s, 1H), 7.44 (d, J ) 6.6 Hz, 2H), 7.33 (m, 8H), 6.84 (d, J ) 6.6
Hz, 2H), 3.71 (s, 3H), OH proton not visible; MS (APCI+) m/z
470 (M + H)+. Anal. (C23H17Cl2N3O4) C, H, N.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid 2,4-Dichlorobenzylamide (35). The title
compound was prepared according to the procedure of compound
34 using 2,4-dichlorobenzyl isocyanate in place of 3,4-dichloroben-
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid (4-Cyanophenyl)amide (28). The title
compound was prepared according to the procedure of compound
21 using 4-cyanophenyl isocyanate in place of phenyl isocyanate:
yield 66%; mp 250-253 °C; MS (APCI+) m/z 465 (M + H)+.
Anal. (C23H14Cl2N4O3·0.25H2O) C, H, N.
1
zyl isocyanate: HPLC 100.0%; H NMR (DMSO-d6) δ 8.80 (s,
1H), 7.59 (d, J ) 2.0 Hz, 1H), 7.41 (dd, J ) 2.0, 6.3 Hz, 1H),
7.35-7.31 (m, 9H), 4.45 (s, 2H), OH proton not visible; MS (ESI-)
m/z 520 (M - H)-.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid 2-Chlorobenzylamide (36). The title
compound was prepared according to the procedure of compound
34 using 2-chlorobenzyl isocyanate in place of 3,4-dichlorobenzyl
isocyanate: HPLC 100.0%; MS (ESI+) m/z 488 (M + H)+.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid Phenethylamide (37). The title compound
was prepared according to the procedure of compound 34 using
phenethyl isocyanate in place of 3,4-dichlorobenzyl isocyanate.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid [1-(4-Bromophenyl)ethyl]amide (38). The
title compound was prepared according to the procedure of
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid Cyclohexylamide (29). The title compound
was prepared according to the procedure of compound 21 using
cyclohexyl isocyanate in place of phenyl isocyanate: yield 32%;
mp 144-149 °C; MS (APCI+) m/z 465 (M + H)+. Anal. (C22H21-
Cl2N3O3) C, H, N.
4-[1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-
pyrazole-4-carbonyl]aminobenzoic Acid Ethyl Ester (30). The
title compound was prepared according to the procedure of
compound 21 using ethyl 4-isocyanatobenzoate in place of phenyl
isocyanate: yield 85%; mp 214-215 °C; MS (APCI-) m/z 511
(M - H)-. Anal. (C25H19Cl2N3O5) C, H, N.

6034 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Gilbert et al.
compound 34 using 1-(4-bromophenyl)ethyl isocyanate in place of
3,4-dichlorobenzyl isocyanate.
4-(4-Chlorobenzoyl)-1,2-bis(4-chlorophenyl)-5-hydroxy-1,2-
dihydropyrazol-3-one (48). The title compound was prepared
according to the procedure of compound 34 using 4-chlorobenzoyl
chloride in place of 3,4-dichlorobenzyl isocyanate: HPLC 100.0%;
MS (ESI-) m/z 457 (M - H)-.
1,2-Bis(4-chlorophenyl)-4-(3,4-dichlorobenzoyl)-5-hydroxy-
1,2-dihydropyrazol-3-one (49). The title compound was prepared
according to the procedure of compound 34 using 3,4-dichloroben-
zoyl chloride in place of 3,4-dichlorobenzyl isocyanate: HPLC
100.0%; MS (ESI-) m/z 491 (M - H)-.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid (1-Naphthalen-2-ylethyl)amide (39). The
title compound was prepared according to the procedure of
compound 34 using 2-(1-isocyanatoethyl)naphthalene in place of
3,4-dichlorobenzyl isocyanate.
1,2-Bis(4-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid Methylphenylamide (40). The title com-
pound was prepared according to the procedure of compound 21
using N-methyl-N-phenylcarbamoyl chloride in place of phenyl
1,2-Bis(4-chlorophenyl)-5-hydroxy-4-(4-methoxybenzoyl)-1,2-
dihydropyrazol-3-one (50). The title compound was prepared
according to the procedure of compound 34 using 4-methoxyben-
zoyl chloride in place of 3,4-dichlorobenzyl isocyanate.
1
isocyanate: mp 181-183 °C; H NMR (DMSO-d6) δ 7.50-6.90
(m, 13H), 3.21 (s, 3H); MS (ESI+) m/z 454 (M + H)+. Anal.
(C23H17Cl2N3O3) C, H, N.
5-Hydroxy-3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazole-4-
carboxylic Acid (4-Chloro-phenyl)amide (41). The title compound
was prepared according to the procedure of compound 21 using
5-hydroxy-1,2-diphenyl-1,2-dihydropyrazol-3-one 16 in place of
1,2-bis(4-chlorophenyl)-5-hydroxy-1,2-dihydro-pyrazol-3-one 17
and 4-chlorophenyl isocyanate in place of phenyl isocyanate: mp
166.5-168 °C; HPLC 100.0%; MS (ESI-) m/z 404 (M - H)-.
5-Hydroxy-3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazole-4-
carboxylic Acid (4-Bromophenyl)amide (42). The title compound
was prepared according to the procedure of compound 21 using
5-hydroxy-1,2-diphenyl-1,2-dihydropyrazol-3-one 16 in place of
1,2-bis(4-chlorophenyl)-5-hydroxy-1,2-dihydropyrazol-3-one 17 and
4-bromophenyl isocyanate in place of phenyl isocyanate: mp 156-
160 °C; yield 53%; MS (APCI+) m/z 450 (M + H)+. Anal. (C22H16-
BrN3O3) C, H, N.
5-Hydroxy-3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazole-4-
carboxylic Acid Phenylamide (43). The title compound was
prepared according to the procedure of compound 21 using
5-hydroxy-1,2-diphenyl-1,2-dihydropyrazol-3-one 16 in place of
1,2-bis(4-chlorophenyl)-5-hydroxy-1,2-dihydropyrazol-3-one 17: mp
171-175 °C; yield 47%; MS (APCI+) m/z 389 (M + H)+. Anal.
(C22H17N3O3) C, H, N.
1,2-Bis(3-chlorophenyl)-3,5-dioxopyrazolidine-4-carboxylic Acid
(4-Chlorophenyl)amide (44). The title compound was prepared
according to the procedure of compound 21 using 1,2-bis(3-
chlorophenyl)-5-hydroxy-1,2-dihydropyrazol-3-one 17 in place of
1,2-bis(4-chlorophenyl)-5-hydroxy-1,2-dihydropyrazol-3-one 17: mp
189-192 °C; yield 70%; MS (APCI+) m/z 474 (M + H)+. Anal.
(C22H14Cl3N3O3) C, H, N.
1,2-Bis(3-chlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-pyra-
zole-4-carboxylic Acid (4-Trifluoromethylphenyl)amide (45).
The title compound was prepared according to the procedure of
compound 21 using 1,2-bis(3-chlorophenyl)-5-hydroxy-1,2-dihy-
dropyrazol-3-one 19 in place of 1,2-bis(4-chlorophenyl)-5-hydroxy-
1,2-dihydropyrazol-3-one 17 and 4-trifluoromethylphenyl isocyanate
in place of phenyl isocyanate: mp 151-156 °C; yield 37%; MS
(APCI+) m/z 508 (M + H)+. Anal. (C23H14Cl2F3N3O3·0.2H2O) C,
H, N.
1,2-Bis(3,4-dichlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-
pyrazole-4-carboxylic Acid p-Tolylamide (46). The title com-
pound was prepared according to the procedure of compound 21
using 1,2-bis(3,4-dichlorophenyl)-5-hydroxy-1,2-dihydropyrazol-
3-one 18 in place of 1,2-bis(4-chlorophenyl)-5-hydroxy-1,2-dihy-
dropyrazol-3-one 17 and 4-methylphenyl isocyanate in place of
phenyl isocyanate: mp 185-187 °C; yield 70%; 1H NMR (DMSO-
d6) δ 10.24 (s, 1H), 7.69 (d, J ) 2.1 Hz, 2H), 7.52 (d, J ) 6.6 Hz,
2H), 7.42 (d, J ) 6.3 Hz, 2H), 7.20 (dd, J ) 2.1, 6.6 Hz, 2H), 7.04
(d, J ) 6.3 Hz, 2H), 2.23 (s, 3H), OH proton not visible; MS
(APCI+) m/z 522 (M + H)+. Anal. (C23H15Cl4N3O3) C, H, N.
1,2-Bis(3,4-dichlorophenyl)-5-hydroxy-3-oxo-2,3-dihydro-1H-
pyrazole-4-carboxylic Acid (3,4-Dichlorophenyl)amide (47). The
title compound was prepared according to the procedure of
compound 21 using 1,2-bis(3,4-dichlorophenyl)-5-hydroxy-1,2-
dihydropyrazol-3-one 18 in place of 1,2-bis(4-chlorophenyl)-5-
hydroxy-1,2-dihydropyrazol-3-one 17 and 3,4-dichlorophenyl iso-
cyanate in place of phenyl isocyanate: mp 160-163 °C; yield 35%;
MS (APCI+) m/z 576 (M + H)+. Anal. (C22H11Cl6N3O3) C, H, N.
4-(4-Butoxybenzoyl)-1,2-bis(4-chlorophenyl)-5-hydroxy-1,2-
dihydropyrazol-3-one (51). The title compound was prepared
according to the procedure of compound 34 using 4-n-butoxyben-
zoyl chloride in place of 3,4-dichlorobenzyl isocyanate: HPLC
98.0%; MS (ESI-) m/z 497 (M + H)+.
1,2-Bis(4-chlorophenyl)-5-hydroxy-4-(4-trifluoromethoxyben-
zoyl)-1,2-dihydropyrazol-3-one (52). The title compound was
prepared according to the procedure of compound 34 using
4-trifluoromethyoxybenzoyl chloride in place of 3,4-dichlorobenzyl
isocyanate: HPLC 100.0%; MS (ESI-) m/z 507 (M - H)-.
4-(Biphenylyl-4-carbonyl)-1,2-bis(4-chlorophenyl)-5-hydroxy-
1,2-dihydropyrazol-3-one (53). The title compound was prepared
according to the procedure of compound 34 using 4-biphenylcar-
bonyl chloride in place of 3,4-dichlorobenzyl isocyanate: HPLC
80.0%; MS (ESI-) m/z 499 (M - H)-.
1,2-Bis(4-chlorophenyl)-5-hydroxy-4-(thiophene-2-carbonyl)-
1,2-dihydropyrazol-3-one (54). The title compound was prepared
according to the procedure of compound 34 using 2-thiophenecar-
bonyl chloride in place of 3,4-dichlorobenzyl isocyanate.
1,2-Bis(4-chlorophenyl)-5-hydroxy-4-(4-trifluoromethylben-
zoyl)-1,2-dihydropyrazol-3-one (55). The title compound was
prepared according to the procedure of compound 34 using
4-trifluoromethylbenzoyl chloride in place of 3,4-dichlorobenzyl
isocyanate: HPLC 100.0%; MS (ESI-) m/z 491 (M - H)-.
1,2-Bis(4-chlorophenyl)-5-hydroxy-4-[2-(4-methoxyphenyl)-
acetyl]-1,2-dihydropyrazol-3-one (56). The title compound was
prepared according to the procedure of compound 34 using
4-methoxyphenylacetyl chloride in place of 3,4-dichlorobenzyl
isocyanate: HPLC 88.0%; MS (ESI+) m/z 469 (M + H)+.
1,2-Bis(4-chlorophenyl)-4-cyclohexanecarbonyl-5-hydroxy-
1,2-dihydropyrazol-3-one (57). The title compound was prepared
according to the procedure of compound 34 using cyclohexanecar-
bonyl chloride in place of 3,4-dichlorobenzyl isocyanate: HPLC
100.0%; MS (ESI-) m/z 429 (M - H)-.
Biology.12 MurB Purification. The purification of E. coli MurB
followed the method of Benson2 and Dhalla17 with some modifica-
tions. E. coli BL21 (λDE3) cells harboring the expression plasmid
were grown in Luria-Bertani broth containing kanamycin (50 µg/
mL) at 37 °C. The expression of E. coli MurB was induced by the
addition of IPTG to a final concentration of 1 mM. After incubation
at 37 °C for 2 h, the cells were harvested; resuspended in 25 mM
Tris-HCl, pH 8.0, 5 mM DTT (buffer A); and lysed by two passages
through a French press, at approximately 15 000 psi. The lysate
was centrifuged at 100 000g for 30 min at 4 °C. Ammonium sulfate
was added to the supernatant to a final concentration of 40%.
Precipitated material was removed by centrifugation and the
supernatant was loaded onto a Phenyl-Sepharose 6 Fast Flow
column (General Electric, Piscataway, NJ) equilibrated with buffer
B (1.6 M (NH4)2SO4, 25 mM Tris-HCl, pH 8.0, and 5 mM DTT).
MurB was eluted with a linear gradient from buffer B to buffer A.
The eluted fractions containing MurB were pooled and concentrated.
MurB was further purified using ion-exchange column chroma-
tography on MonoQ column (General Electric, Piscataway, NJ).
MurB was eluted from the column by a gradient of KCl from 0 to
1 M in buffer A. The purity of E. coli MurB was analyzed using
SDS-PAGE.

Cell Wall Biosynthesis Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6035
(2) Benson, T. E.; Marquardt, J. L.; Marquardt, A. C.; Etzkorn, F. A.;
Walsh, C. T. Overexpression, purification, and mechanistic study of
UDP-N-acetylenolpyruvylglucosamine reductase. Biochemistry 1993,
32 (8), 2024-30.
(3) Benson, T. E.; Filman, D. J.; Walsh, C. T.; Hogle, J. M. An enzyme-
substrate complex involved in bacterial cell wall biosynthesis. Nat.
Struct. Biol. 1995, 2 (8), 644-53.
(4) Benson, T. E.; Walsh, C. T.; Massey, V. Kinetic characterization of
wild-type and S229A mutant MurB: Evidence for the role of Ser
229 as a general acid. Biochemistry 1997, 36 (4), 796-805.
(5) Lees, W. J.; Benson, T. E.; Hogle, J. M.; Walsh, C. T. (E)-
enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of
UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Biochem-
istry 1996, 35 (5), 1342-51.
(6) Benson, T. E.; Harris, M. S.; Choi, G. H.; Cialdella, J. I.; Herberg,
J. T.; Martin, J. P., Jr.; Baldwin, E. T. A structural variation for
MurB: X-ray crystal structure of Staphylococcus aureus UDP-N-
acetylenolpyruvylglucosamine reductase (MurB). Biochemistry 2001,
40 (8), 2340-50.
(7) Francisco, G. D.; Li, Z.; Albright, J. D.; Eudy, N. H.; Katz, A. H.;
Petersen, P. J.; Labthavikul, P.; Singh, G.; Yang, Y.; Rasmussen, B.
A.; Lin, Y. I.; Mansour, T. S. Phenyl thiazolyl urea and carbamate
derivatives as new inhibitors of bacterial cell-wall biosynthesis.
Bioorg. Med. Chem. Lett. 2004, 14 (1), 235-8.
(8) Li, Z.; Francisco, G. D.; Hu, W.; Labthavikul, P.; Petersen, P. J.;
Severin, A.; Singh, G.; Yang, Y.; Rasmussen, B. A.; Lin, Y. I.;
Skotnicki, J. S.; Mansour, T. S. 2-Phenyl-5,6-dihydro-2H-thieno[3,2-
c]pyrazol-3-ol derivatives as new inhibitors of bacterial cell wall
biosynthesis. Bioorg. Med. Chem. Lett. 2003, 13 (15), 2591-4.
(9) Bronson, J. J.; DenBleyker, K. L.; Falk, P. J.; Mate, R. A.; Ho, H.
T.; Pucci, M. J.; Snyder, L. B. Discovery of the first antibacterial
small molecule inhibitors of MurB. Bioorg. Med. Chem. Lett. 2003,
13 (5), 873-5.
(10) Antane, S.; Caufield, C. E.; Hu, W.; Keeney, D.; Labthavikul, P.;
Morris, K.; Naughton, S. M.; Petersen, P. J.; Rasmussen, B. A.; Singh,
G.; Yang, Y. Pulvinones as bacterial cell wall biosynthesis inhibitors.
Bioorg. Med. Chem. Lett. 2006, 16 (1), 176-80.
(11) Kutterer, K. M.; Davis, J. M.; Singh, G.; Yang, Y.; Hu, W.; Severin,
A.; Rasmussen, B. A.; Krishnamurthy, G.; Failli, A.; Katz, A. H.
4-Alkyl and 4,4′-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-
dione derivatives as new inhibitors of bacterial cell wall biosynthesis.
Bioorg. Med. Chem. Lett. 2005, 15 (10), 2527-31.
(12) Yang, Y.; Severin, A.; Chopra, R.; Krishnamurthy, G.; Singh, G.;
Hu, W.; Keeney, D.; Svenson, K.; Petersen, P. J.; Labthavikul, P.;
Shlaes, D. M.; Rasmussen, B. A.; Failli, A. A.; Shumsky, J. S.;
Kutterer, K. M.; Gilbert, A.; Mansour, T. S. 3,5-dioxopyrazolidines,
novel inhibitors of UDP-N- acetylenolpyruvylglucosamine reductase
(MurB) with activity against gram-positive bacteria. Antimicrob.
Agents Chemother. 2006, 50 (2), 556-64.
(13) Shine, H. J.; Rhee, E. S. The preparation of 4,4′-dichloroazobenzene-
15N,15N′ and -4,4′-13C2 and 4-chloroazobenzene-15N,15N′. J.
Labelled Compd. Radiopharm. 1984, 21 (6), 569-73.
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compounds. III. The kinetics and mechanism of the rearrangement
of 2,2′-hydrazonaphthalene in polar solvents. J. Am. Chem. Soc. 1960,
82 (15), 4054-8.
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1963.
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reactions with 3,5-dioxo-1,2-diphenylpyrazolidine. Chem. Ber. 1955,
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J. J.; Robertson, J. G. Steady-state kinetic mechanism of Escherichia
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MurB Assay. MurB activity was determined using a continuous
assay4 monitoring the oxidation of NADPH as measured by
reduction in the absorbance at 340 nm. The reaction mixture
contained 50 mM Tris-HCl, pH 8.0, 10 mM KCl, 100 µM NADPH,
and 50 µM EP-UNAC, in a total volume of 200 µL. The reaction
was initiated by the addition of enzyme. The OD340 was monitored
over 5 min. One unit of enzyme activity was defined as the amount
of enzyme that catalyzed the oxidation of 1 µmol of NADPH/min.
A molar absorption coefficient of 6220 cm-1 M-1 for NADPH
absorption at 340 nm was used.
MurB inhibition Studies. The inhibition of MurB enzymatic
activity was determined following an initial 20-minute 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 µM enolpyruvyl UDP-N-acetylglycosamine and
100 µM NADPH. Six concentrations of inhibitor, 1.6, 3.2, 6.4, 12.8,
25, and 50 µM, were used for each compound tested. The IC50
values were derived using the data analysis function of Microsoft
Excel (Sigmoid Curve Hill analysis 0-100). Phosphomycin, a
MurA inhibitor, was included as a negative control.
Determination of in Vitro Antibacterial Activity. The micro-
organisms used for antimicrobial activity testing comprised a
spectrum of Gram-positive bacteria including species of Staphy-
lococci, Streptococci, and Enterococci. The organisms included
recent clinical isolates that are resistant to methicillin, penicillin,
and vancomycin. Gram-negative bacteria, a modified E. coli with
altered outer membrane permeability (imp),26 and a yeast strain,
C. albicans, were also included in the test panel. The minimal
inhibitory concentration (MIC) was determined by the broth dilution
method using Muller-Hinton II media (Baltimore Biological
Laboratories) following the recommendations of the National
Committee for Clinical Laboratory Standards.27 An inoculum of
5 × 105 cfu/mL and a range of compound concentrations (0.06-
128 µg/mL) were used. The MICs were determined after incubation
for 18 h at 35 °C in an ambient air incubator.
Effect of Pyrazolidine-3,5-diones and 5-Hydroxy-1H-pyrazol-
3(2H)-ones on Peptidoglycan Biosynthesis. Peptidoglycan bio-
synthesis was measured by determining the amount of soluble
peptidoglycan (SPG) produced by a S. epidermidis strain. The
method was originally reported by Boothby28 and was modified to
fit a 96-well microplate. Briefly, cells were grown in BHI to an
OD600nm of 0.6, harvested, washed twice with cold H2O, and
resuspended in cell wall enriched media28 at 10% of the original
volume. The reaction mixture contained 200 µL of cell suspension,
50 µg/mL chloramphenicol, 100 µg/mL penicillin G, 2.9 µM/40nCi
[14C]-N-acethylglucosamine, and test compound, at concentrations
of 0.0, 3.1, 6.2, 12.5, and 25 µg/mL, in a total volume of 250 µL.
After incubation with agitation at 37 °C for 1 h, the reaction mixture
was centrifuged at 1500g for 20 min. A quantity of 200 µL of the
supernatant was transferred into a Millipore MultiScreen 1.2 µm
glass fiber filter plate. BSA (bovine serum albumin) and TCA
(trichloroacetic acid) were added to each well, giving final
concentrations of 0.4% and 5%, respectively. After a 30-min
incubation at 4 °C, the plates were filtered, washed with 5% TCA,
and counted on a Packard TopCount using MicroScint scintillation
fluid (Perkin-Elmer Life Sciences, Boston, MA).
(18) Merrikin, D. J.; Briant, J.; Rolinson, G. N. Effect of protein binding
on antibiotic activity in vivo. J. Antimicrob. Chemother. 1983, 11
(3), 233-8.
(19) Staahle, L.; Wold, S. Partial least-squares analysis with cross-
validation for the two-class problem: A Monte Carlo study. J.
Chemometr. 1987, 1 (3), 185-96.
(20) Eriksson, L.; Johnasson, E.; Kettaneh-Wold, N.; Wold, S. Introduction
to Multi- and MegaVariate Data Analysis using Projection Methods
(PCA & PLS); Umetrics AB: Umea, Sweden, 1999.
Acknowledgment. The authors would like to acknowledge
the members of the Chemical and Screening Sciences pharma-
ceutical profiling group who determined the physicochemical
properties (aqueous solubility, PAMPA permeability, CYP
inhibition profiles and microsomal stability) for a portion of
the compounds described in this paper.
(21) Umetrics. SIMCA-P, 11.0; 2005.
(22) Xu, W. Unpublished results.
Supporting Information Available: Elemental analysis and
HPLC results for compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
(23) Joseph-McCarthy, D.; Thomas, B. E. t.; Belmarsh, M.; Moustakas,
D.; Alvarez, J. C. Pharmacophore-based molecular docking to account
for ligand flexibility. Proteins 2003, 51 (2), 172-88.
(24) McMartin, C.; Bohacek, R. S. QXP: Powerful, rapid computer
algorithms for structure-based drug design. J. Comput. Aided Mol.
Des. 1997, 11 (4), 333-44.
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