Phenyl thiazolyl urea and carbamate derivatives as new inhibitors of bacterial cell-wall biosynthesis

Article abstract of DOI:10.1016/j.bmcl.2003.09.082

Over 50 phenyl thiazolyl urea and carbamate derivatives were synthesized for evaluation as new inhibitors of bacterial cell-wall biosynthesis. Many of them demonstrated good activity against MurA and MurB and gram-positive bacteria including MRSA, VRE and

Full text of DOI:10.1016/j.bmcl.2003.09.082

Bioorganic & Medicinal Chemistry Letters 14 (2004) 235–238
Phenyl thiazolyl urea and carbamate derivatives as new inhibitors
of bacterial cell-wall biosynthesis
Gerardo D. Francisco, Zhong Li, J. Donald Albright, Nancy H. Eudy, Alan H. Katz,
Peter J. Petersen, Pornpen Labthavikul, Guy Singh, Youjun Yang, Beth A. Rasmussen,
Yang-I Lin* and Tarek S. Mansour
Chemical Sciences and Infectious Diseases, Wyeth Research, Pearl River, NY 10965, USA
Received 30 June 2003; accepted 10 September 2003
Abstract—Over 50 phenyl thiazolyl urea and carbamate derivatives were synthesized for evaluation as new inhibitors of bacterial
cell-wall biosynthesis. Many of them demonstrated good activity against MurA and MurB and gram-positive bacteria including
MRSA, VRE and PRSP. 3,4-Difluorophenyl 5-cyanothiazolylurea (3p) with clog P of 2.64 demonstrated antibacterial activity
against both gram-positive and gram-negative bacteria.
# 2003 Elsevier Ltd. All rights reserved.
Since peptidoglycan is an essential bacterial cell-wall
polymer, peptidoglycan biosynthesis provides a unique
and selective target for antibiotic action.1 Peptidoglycan
biosynthesis requires 10 synthetic transformations, each
one of them requiring a specific enzyme.1 These enzymes
include MurA, MurB, MurC, MurD, MurE, MurF,
MurG, MraY, and the transglycosylase and transpepti-
dase families of enzymes. Inhibition of any of these
essential proteins leads to loss of cell shape and integrity
followed by bacterial death.2ꢀ4 This applies in both
gram-positive and gram-negative organisms. Our bac-
terial cell-wall program is committed to identify the
inhibitors of the first eight enzymes of the peptidoglycan
biosynthesis. In a previous publication,5 we reported the
synthesis and antimicrobial activity of 2-phenyl-5,6-
dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives as new
inhibitors of MurB, MurC, and MurD. Here we report
the synthesis and antimicrobial activity of phenyl thia-
zolyl urea and carbamate derivatives as new inhibitors
of MurA and MurB.
dichlorobenzyl alcohol (4) with p-nitrophenyl-
chloroformate, followed by reaction with aminothia-
zoles in the presence of Hunig’s base (Scheme 2). Since
Scheme 1.
Thiazolyl urea derivatives 3 were prepared in one step in
ꢁ80% yield from reaction of substituted aminothiazole
2 with isocyanate 1 in the presence of Hunig’s base in
DMF (Scheme 1). Carbamate derivatives 5 were pre-
pared in ꢁ40% overall yields by reaction of 3,4-
* Corresponding author. Fax: +1-845-602-5561; e-mail: linf@wyeth.
com
Scheme 2.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.082

236
G. D. Francisco et al. / Bioorg. Med. Chem. Lett. 14 (2004) 235–238
isocyanates and benzyl alcohols are commercially avail-
able, one of the major efforts has been the synthesis of
substituted aminothiazoles.
Enterococus (VRE) and penicillin-resistant Strepto-
coccus pneumoniae (PRSP) with MICs (minimum inhi-
bitory concentrations) in the range of 0.5–4 mg/mL.
Using 3a as the lead, over 50 urea and carbamate deri-
vatives were synthesized and submitted for evaluation
as new inhibitors of bacterial cell-wall biosynthesis. The
introduction of the 4-cyano group( 3b) to 3a slightly
improved the activity against VRE and PRSP but
slightly decreased the activity against MRSA. However,
the introduction of the 5-cyano group( 3c) to 3a
improved the activity against MRSA, VRE and PRSR
by a factor of 2->4-fold and considerably improved
IC50 values (4.5–12.6 mg/mL) against MurA and MurB.
The introduction of a 4-t-butyl groupto 3c further
improved both MIC and IC50 values. Of 19 derivatives
tested, the derivative 3d demonstrated the best anti-
bacterial activity against gram-positive bacteria with
MICs in the range of <0.12–0.25 mg/mL and IC50
values in the range of 2.8–6.2 mg/mL. Attempts to fur-
ther improve the MIC and IC50 values of 3d by the
replacement of the 4-t-butyl groupwith 4-iso-butoxyl,
4-ethoxyl, 4-methoxyl, 4-chloro or 4-morpholino groups
in the thiazolyl ring failed. The 5-nitrothiazoyl deriva-
tive 3o was as active as the 5-cyanothiazoyl derivative
3c. Antibacterial activity was diminished, when the 3,4-
dichlorophenyl group of 3c and 3d was replaced by 3,4-
dichlorobenzyl group( 3q and 3n).
2-Amino-5-cyanothiazole 10 was prepared in four steps
in 37% yield (Scheme 3).6
Three 2-amino-4-alkoxy-5-cyanothiazoles were prepared
in two steps in about 40% overall yields (Scheme 4).7
A very effective new synthesis of 4-substituted 2-amino-
5-cyanothiazoles was developed. 2-Amino-4-chloro-5-
cyanothiazole 18 was prepared in four steps in 27%
overall yield by the literature process.8,9 Reaction of 18
with morpholine gave 2-amino-5-cyanothiazole 19 in
about 85% yield (Scheme 5). This process was applied
to the synthesis of other 4-substituted 2-amino-5-
cyanothiazoles.10
As shown in Table 1, 3,4-dichlorophenyl thiazolyl deri-
vative 3a demonstrated an IC50 value of 20 mg/mL
against MurA and good antimicrobial activity against
gram-positive bacteria including methicillin resistant
Staphlococcus aureus (MRSA), vancomycin resistant
The MIC values of 3c and 3d against PRSP were
ꢂ0.12 mg/mL. However, when 3c and 3d were tested in
the presence of 4% bovine serum albumin, their MIC
values increased to 128 mg/mL. Since this increase in
MIC might be due to serum protein binding, attempts
were made to minimize the serum protein binding
problem by increasing the polarity of compounds. The
3,4-dichlorophenyl group of 3d was replaced by 3,4-
dimethylphenyl, 3,4-difluorophenyl, 3-cyanophenyl, 3-
pyridinyl, or 3,4-dichlorobenzyl group. These new deri-
vatives (3j–n) failed to improve the MIC against PRSP
when tested in the presence of 4% bovine serum albu-
min. In general, the antimicrobial activity decreased as
the polarity increased. However, when the 3,4-dichlor-
ophenyl group of 3c with clog P of 3.44 was replaced by
a 3,4-difluorophenyl group, the resulting compound
Scheme 3.
Scheme 4.
Figure 1. Preferred orientation of 3d in the MurB crystal structure
based on the Dock and Flo programs.
Scheme 5.

G. D. Francisco et al. / Bioorg. Med. Chem. Lett. 14 (2004) 235–238
237
3p with clog P of 2.64 demonstrated antibacterial
activity against both gram-positive and gram-negative
bacteria with MIC values in the range of 0.5–8 mg/
mL. It also demonstrated improved MIC (64 mg/mL)
against PRSP when tested in the presence of 4% serum
albumin.
suggest that 3d prefers to bind to the substrate-binding
region of the structure. The carbamate occupies the
bisphosphate binding region of the MurB substrate, and
mimics a number of its interactions, in particular,
strong hydrogen bonds between the urea carbonyl and
Lys217. There are also strong interactions Gln288
Tyr125 and Lys275. There are several alternative bind-
ing orientations available to the diaryl urea structure
with slightly lower interaction energies. In some of these
orientations the urea remains in the same place, but the
aromatic ring and the heterocycle ring switch positions.
In these orientations, the carbamate derivatives form
stronger interactions with the enzyme than the corre-
sponding urea derivatives due to the introduction of a
hydrogen bond between the carbamate oxygen and
Gln288. Therefore, carbamate derivatives 5a and 5b
Molecular modeling studies were carried to explore
the potential binding preferences of this series within the
MurB structure. An expanded set of conformations of
t-butyl analogue 3d were generated using an in-house
script described previously.11 The resulting conformer
database was docked into the MurB crystal structure12
with the substrate removed, using Dock 3.5.13 The same
structures were docked together using Flo.14 The lowest
energy complex obtained by both methods (Fig. 1)
Table 1. Antimicrobial activities (mg/mL) and IC50 values (mg/mL) of thiazolyl urea and carbamate derivatives
3a
H
3b
CN
3c
H
3d
t-Bu
3e
Oi-Bu
3f
OEt
3g
Cl
3h
3i
OMe morpholino 3,4-diMePh
3j
R, R0 or R00=
MIC (mg/mL)
S. aureus GC 1131 (MRSA)
S. aureus GC 4543 (MSSA)
S. aureus GC 2216 (ATCC)
E. faecalis GC 4555 (ATCC)
E. faecalis GC 2242 (VRE)
S. pneumo GC1894 (PRSP)
S. pneumo GC1894 (PRSP)a >128
MSCNS GC 646
E. coli GC 4559
E. coli GC 4560
2
2
2
4
4
8
4
4
8
2
1
<0.12
<0.12
<0.12
0.25
<0.12
<0.12
128
0.5
1
0.5
0.5
0.5
0.25
0.25
0.5
0.25
<0.12
>128
0.5
>128
0.5
>128
>128
>128
32
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
0.5
0.25
0.25
0.5
0.5
0.5
1
0.25
0.5
0.25 >128
0.25
<0.12
128
0.25
>128
>128
0.5
0.5
4
0.5
0.5
<0.12 <0.12
128
4
>128
8
<0.12
128
0.5
>128
0.5
>128
>128
>128
>128
>128
<0.12
128
0.5
>128
2
128
2
>128
1
>128
4
<0.12
>128
0.50
20
0.5
MurA, E. coli: IC50=mg/mL
MurB, E. coli: IC50=mg/mL >25
MurB, Staph: IC50=mg/mL
clog P
>25
>25
14
12.6
4.5
5.6
3.44
3.3
6.2
2.8
5.57
>25
5.8
—
>25
23
—
>25
>25
>25
21.6
9.8
—
>25
>25
13
>25
6.2
6.3
5.42
>25
3.35
3.81
5.25
4.37
4.34
4.03
4.04
3k
3l
3m
3n
3,4-diFPh 3-CNPh 3-Py 3,4-diClPh CH2
3o
—
3p 3q
3,4-diFPh 3,4-diClPh CH2
5a
H
5b
t-Bu
Vancomycin
R00, R000 or R=
MIC (mg/mL)
S. aureus GC 1131 (MRSA)
S. aureus GC 4543 (MSSA)
S. aureus GC 2216 (ATCC)
E. faecalis GC 4555 (ATCC)
E. faecalis GC 2242 (VRE)
S. pneumo GC1894 (PRSP)
S. pneumo GC1894 (PRSP)a
MSCNS GC 646
<0.12 >128
<0.12 >128
<0.12 >128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
1
0.5
0.25
1
0.5
<0.12
128
1
1
0.5
0.25
1
1
0.25
4
2
4
4
4
0.5
64
4
8
4
>128
>128
>128
>128
64
64
128
>128
>128
>128
>128
>128
>128
64
64
128
>128
>128
>128
>128
2
1
8
2
1
2
0.5
0.5
2
1
0.5
16
8
8
>128
<0.12
–
<0.12
128
0.25
>128
0.5 >128
128
0.5
>128
>128
>128
>128
>128
–
E. coli GC 4559
E. coli GC 4560
>128
1
>128
>128
>128
2
>128
0.25
2
MurA, E. coli: IC50=mg/mL >25
MurB, E. coli: IC50=mg/mL >25
>25
>25
>25
>25
>25
>25
20.8
4.6
3.8
3.0
>25
19
—
>25
>25
24
>25
>25
24
25
18
—
>25
11
5.64
MurB, Staph: IC50=mg/mL
clog P
—
4.77
4.48
3.11
—
2.64
3.51
4.19
6.31
a Tested in the presence of 4% bovine serum albumin.

238
G. D. Francisco et al. / Bioorg. Med. Chem. Lett. 14 (2004) 235–238
Table 2. Activity of other phenyl heteroaryl urea derivatives
were prepared. There was no significant difference in
activity between the carbamate derviatives (5a and 5b)
and the urea derivatives (3q and 3n).
Acknowledgements
We would like to thank Dr. Jerauld Skotnicki for help-
ful discussion. We would like to thank Dr. David Shlaes
for his support of the program.
As is evident from Table 2, other phenyl heteroaryl
urea derivatives investigated (20–24) were not as active
as many of the phenyl thiazoyl derivatives listed in
Table 1.
References and notes
In summary, many substituted phenyl thiazolyl urea
and carbamate derivatives were identified as a new
class of bacterial cell-wall biosynthesis inhibitors.
They demonstrated good activity against MurA and
MurB and gram-positive bacteria including MRSA,
VRE and PRSP. However, when tested in the pre-
sence of 4% bovine serum albumin, their MIC values
increased to greater than 128 mg/mL against PRSP.
The derivative 3p with clog P of 2.64 demonstrated
antibacterial activity against both gram-positive and
gram-negative bacteria with MIC values in the range
of 0.5–8.0 mg/mL. It also demonstrated improved
MIC value (64 mg/mL) against PRSP when tested in
the presence of 4% serum albumin. Since many com-
pounds 3c–e, 3j, 3k, 3n, and 3o are more active than
vancomycin against gram-positive bacteria, they are
good leads for further investigation. The compounds 3c
and 3d demonstrated IC50 of 32.1 and 4.2 mg/mL in the
soluble peptidoglycan screen using a strain of S. epi-
dermides.15 Thus, the cell wall biosynthesis inhibition
(MurA and MurB) of 3c and 3d is responsible for at
least part of their antimicrobial activity. Recently, anti-
microbial activity of some related diaryl ureas16,17 have
been shown to act by inhibition of bacterial respira-
tion.16 It is likely that the antimicrobial activity of the
phenyl thiazolyl urea and carbamate derivatives are due
to inhibition of both bacterial cell-wall biosynthesis and
bacterial respiration.
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