2-Phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives as new inhibitors of bacterial cell wall biosynthesis

Article abstract of DOI:10.1016/S0960-894X(03)00471-2

Twenty-five 2-phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives were synthesized for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good inhibitory activity against Staphylococcus aureus MurB, MurC and MurD enzymes in vitro and antimicrobial activity against gram-positive bacteria including MRSA, VRE and PRSP. However, when they were tested in the presence of 4% bovine serum albumin, the MIC values increased to greater than 128 μg/mL against PRSP. None of the compounds demonstrated activity against gram-negative bacteria at MIC <32 μg/mL.

Full text of DOI:10.1016/S0960-894X(03)00471-2

Bioorganic & Medicinal ChemistryLetters 13 (2003) 2591–2594
2-Phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol Derivatives as
New Inhibitors of Bacterial Cell Wall Biosynthesis
Zhong Li, Gerardo D. Francisco, William Hu, Pornpen Labthavikul, Peter J. Petersen,
AnatolySeverin, GuySingh, Youjun Yang, Beth A. Rasmussen, Yang-I Lin, *
Jerauld S. Skotnicki and Tarek S. Mansour
Chemical Sciences and Infectious Diseases, Wyeth Research, Pearl River, NY 10965, USA
Received 10 January2003; accepted 17 April 2003
Abstract—Twenty-five 2-phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives were synthesized for evaluation as new
inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good inhibitory activity against Staphylococcus aureus
MurB, MurC and MurD enzymes in vitro and antimicrobial activity against gram-positive bacteria including MRSA, VRE and
PRSP. However, when theywere tested in the presence of 4% bovine serum albumin, the MIC values increased to greater than 128
ꢀg/mL against PRSP. None of the compounds demonstrated activityagainst gram-negative bacteria at MIC <32 ꢀg/mL.
# 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 more than 10 synthetic transfor-
mations, each one of them requiring a specific enzyme.1
These enzymes include MurA, MurB, MurC, MurD,
MurE, MurF, MraY, MurG, and the transglycosylase
and transpeptidase families of enzymes. Inhibition of
anyof these essential enzymes leads to loss of cell shape
and integrityfollowed bybacterial death. 2,3 This applies
in both gram-positive and gram-negative organisms. Of
these enzymes, only MurA, the transglycosylases and
the transpeptidases have been the targets of commerical
antimicrobial agents. b-Lactam antibiotics inhibit
transpeptidases; vancomycin inhibits transglycosylases;
fosfomycin inhibits MurA.4 Despite the unprecedented
commercial success of b-lactam and glycopeptide anti-
biotics, their clinical use has recentlybeen compromised
bythe emergence of resistant bacterial strains. There-
fore, one of the attractive strategies to overcome resis-
tance to b-lactam antibiotics and vancomycin is to find
novel inhibitors of the cell wall biosynthetic enzymes
other than the transpeptidases and the transglycosy-
lases.5 The objective of our bacterial cell-wall program
was to identifynovel inhibitors of the first eight
enzymes of the peptidoglycan biosynthesis. In a pre-
vious publication,6 we reported the urea, hydantoin and
N-alkyl derivatives of muramycin C1 as novel inhibitors
of MraY. Here, we report the synthesis and anti-
microbial activity of 2-phenyl-5,6-dihydro-2H-thieno[3,2-
c]pyrazol-3-ol derivatives as new inhibitors of Staphylo-
coccus aureus MurB, MurC and MurD enzymes.
2-Phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol deri-
vatives 5 were synthesized in four steps in ꢀ60% overall
yields. Reaction of methyl acrylates 1 with methyl thio-
glycolate in the presence of a catalytic amount of
piperidine gave 3-methoxycarbonylmethylsulfanyl-
propionic acid methyl esters 2 in almost quantitative
yield. Cyclization of 2 with titanium tetrachloride and
triethylamine in dichloromethane gave 3-oxotetrahy-
drothiophene-2-carboxylates 3 in ꢀ60% yields.7 Con-
densation of 3 with substituted phenyl hydrazines,
followed bycyclization with sodium methoxide gave 2-
phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol deriva-
tives 5 in ꢀ95% yield (Scheme 1).8 Similarly, con-
densation of commerciallyavailable keto esters 6 with
substituted phenylhydrazines, followed by cyclization
with sodium methoxide gave 2-phenylbicyclopyrazol-3-
ol derivatives 8 (Scheme 2). Reaction of 2-(3-cyanophe-
nyl)-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol (5l) with
potassium hydroxyde in t-butanol gave 3-(3-hydroxy-
5,6-dihydro-thieno[3,2-c]pyrazol-2-yl)-benzamide (5o) in
good yield (Scheme 3).9 Oxidation of 5j with H2O2/
HCO2H in dichloromethane gave sulfoxide 5u in an
excellent yield (Scheme 4).10 However, further oxidation
*Corresponding author. Fax: +1-845-602-5561; e-mail: linf@wyeth.
com
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00471-2

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Z. Li et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2591–2594
Scheme 4.
Scheme 1.
Scheme 5.
para-substituted derivatives were slightlymore active or
equallyas active as meta-substituted derivatives. ortho-
Substituted derivative 5p was devoid of enzyme activity
at IC50 ꢁ25 mg/ml, but demonstrated slight anti-
bacteria activity. Di-substituted derivative 5q was less
active than mono-substituted derivatives 5k and 5e. As
lipophilicityof compounds decreased ( 5c: clog P=3.06
to 5f: clog P=2.53 to 5o: clog P=1.41 and 5j: clog
P=3.62 to 5u: clog P=2.01), activityagainst the Mur
enzymes and gram-positive bacteria decreased. The
rates of passive diffusion of uncharged molecules across
lipid membranes correlate reasonablywell with their
clog P. That is, the more polar a compound is, the less
readilyit enters and diffuses across the ctyoplasmic
membrane.12 Suprisingly, p-isopropyl derivative 5h and
t-butyl derivative 5i were devoid of enzyme inhibitory
activityat ꢁ25 mg/mL and gave MIC >64 mg/mL. 5-
Methyl derivative 5r and 5-phenyl derivative 5s were
less active than the lead 5c. The carbocyclic derivative
10b is devoid of inhibitoryactivityagainst enzymes at
ꢁ25 mg/mL and bacteria at <64 mg/mL. When the sul-
fur or oxygen was placed in the middle of the ring such
as in 8a, 8b, 8c, and 10a, the compounds were also devoid
of enzyme inhibitoryactivityat ꢁ25 mg/mL and gave MIC
ꢁ32 mg/mL. (Table 2). None of twentyfive 2-phenyl-
bicyclopyrazol-3-ol derivatives tested demonstrated activ-
ityagainst gram-negative bacteria at <32 mg/mL.
Scheme 2.
Scheme 3.
of 5u with m-chloroperbenzoic acid failed to yield the
corresponding sulfone derivative. Condensation of keto
esters 9 with substituted phenylhydrazines in acetic acid
gave 2-phenylbicyclopyrazol-3-ol derivatives 10 in good
yield (Scheme 5).
The activityof 5c against S. aureus MurB-MurD and
gram-positive bacteria was discovered through biologi-
cal screens. Using 5c as the lead, 25 derivatives were
synthesized and submitted for evaluation as new inhibi-
tors of bacterial cell wall biosynthesis. Among the
twenty five 2-phenyl-bicyclopyrazol-3-ol derivatives tes-
ted, five of them11 (5b, 5c, 5d, 5e, and 5k with MIC
values in the range of 0.25–32 mg/mL) demonstrated the
best antibacterial activityagainst gam-positive bacteria
including methicillin resistant S. aureus (MRSA), van-
comycin resistant Enterococus (VRE) and penicillin
resistant Staphylococcus pneumoniae (PRSP). However,
when tested in the presence of 4% bovine serum albu-
min (BSA), the MIC values increased to >128 mg/mL
against PRSP, suggesting that these compounds are
highlyprotein bonded byBSA. These five derivatives
also demonstrated good activityagainst at least one of
S. aureus MurB-MurD enzymes (Table 1). In general,
In summary, several 2-phenyl-5,6-dihydro-2H-thieno-
[3,2-c]pyrazol-3-ol derivatives were identified as a new
class of bacterial cell wall biosynthesis inhibitors. They
demonstrated good activityagainst S. aureus MurB,
MurC and MurD and gram-positive bacteria including
MRSA, VRE and PRSP. However, when theywere
tested in the presence of 4% bovine serum albumin,
their MIC values increased to greater than 128 mg/mL
against PRSP. Although compounds 5b, 5c, 5d, 5e and
5k are less active than vancomycin against gram-posi-
tive bacteria, theyare good leads for further investiga-
tion. None of them demonstrated activityagainst gram-
negative bacteria at MIC <32 mg/mL.

Table 1. Antimicrobial activities MIC (mg/mL) and IC50 values (mg/mL) of 2-phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives
5a
H
5b
4-F
5c5d
4-Cl
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
4-Br 4-CF3 4-CN 4-NO2 4-iPr 4-tBu 3,4-Cl2 3-CF3 3-CN 3-NO2 3-Cl 3-CONH2 2-CF3 3,5-CF3
5r
5s
Vancomycin
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 >128 >128
MSCNS GC 646
E. coli GC 4559
E. coli GC 4560
16
2
4
2
8
8
2
16
2
2
16
16
1
16
4
2
16
32
1
8
1
2
16
8
0.5
32
16
8
8
8
64
2
4
128 >128
128 128
128 >128
32
8
8
128
64
4
4
1
2
16
8
0.5
32
16
8
8
8
32
32
16
64
32
4
32
32
8
32
8
128
64
128
64
32
8
>128
>128
>128
128
32
32
32
32
16
8
32
32
32
32
16
2
16
16
32
128
128
64
2
0.5
0.5
2
32
16
32
4
32 >128 >128
64 >128 >128
4
32 >128
2
4
64
64
>128
<0.12
—
4
4
32
64
4
2
>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128
128 >128 16 32 32
>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128
>128 >128 >128 >128
64 >128 16 128
>128 >128 >128 >128
16
>128 >128
>128
0.25
4
1
4
4
1
2
2
>128
32
128
128
128
64 >128 >128 >128
128
128
64 >128 >128
128 >128 >128
128
0.25
IC50 (mg/mL):
S. aureus MurB
S. aureus MurC
S. aureus MurD
>25
>25
20.7 >25
12
>25
7.1
10.1
11.5
3.6
11
>25
>25
12.0
5.6 >25 >25
19.1 >25 >25
9.5 >25 >25
11
7
14
21
>25
19.2
14
13.3
11.4
6.2 >25
23.1 15.8
9.1 8.3
>25
>25
12.5
>25
>25
>25
>25 >25
>25 >25
11
>25
24.5
11.4 >25
9.0 12.3
>25
17.1
aTested in the presence of 4% bovine serum albumin.

2594
Z. Li et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2591–2594
3. Kimura, K.; Ikeda, Y.; Kagami, S.; Yoshihara, M. J.
Antibiot. 1998, 51, 1099.
Table 2. Activity of other 2-phenyl-bicyclopyrazol-3-ol derivatives
4. Russell, A. D.; Chopra, I. In Understanding Antibacterial
Action and Resistance; Ellis Horwood: UK 1990; Chapter 2, p
39.
5. Green, D. W. Expert Opin. Ther. Targets 2002, 6, 1.
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Davis, R. A.; Singh, G.; Yang, Y.; Mansour, T. S. Bioorg.
Med. Chem. Lett. 2002, 12, 2341.
7. Deshmukh, M. N.; Gangakhedkar, K. K.; Kumar, U. S.
Synth. Commun. 1996, 26, 1657.
8. Bauer, V. J.; Williams, R. P.; Safir, S. R. J. Med. Chem.
1971, 14, 454.
9. Hall, J. H.; Gisler, M. J. Org. Chem. 1976, 41, 3769.
10. Tanaka, M.; Konoike, T.; Yoshioka, M. Synthesis 1989,
197.
11. Spectra data of the five most active compounds are sum-
marized as follows: 5b, calcd for C11H9FN2OS: 236.3, Elec-
trospray-MS m/z 237.0 (M+H)+, 1H NMR (DMSO-d6) d
11.56 (1H, br), 7.66 (2H, m), 7.25 (2H, m), 3.58 (2H, t, J=5.7
Hz), 2.89 (2H, t, J=5.7 Hz). 5c, calcd for C11H9ClN2OS:
252.7, Electrospray-MS m/z 253.1 (M+H)+, 1H NMR
(DMSO-d6) d 11.79 (1H, br), 7.69 (2H, d, J=6 Hz), 7.48 (2H,
t, J=6 Hz), 3.58 (2H, t, J=5.7 Hz), 2.89 (2H, t, J=5.7 Hz).
5d, calcd for C11H9BrN2OS: 297.2, Electrospray-MS m/z 297.0
Acknowledgements
1
(M+H)+, H NMR (DMSO-d6) d 11.74 (1H, br), 7.64 (4H,
We would like to thank Dr. David Shlaes for his sup-
port of the program.
m), 3.59 (2H, t, J=5.7 Hz), 2.89 (2H, t, J=5.7 Hz). 5e, calcd
for C12H9F3N2OS: 286.3, Electrospray-MS m/z 284.9
(MÀH)À, 1H NMR (DMSO-d6) d 11.97 (1H, br), 7.94 (2H, d,
J=6 Hz), 7.77 (2H, t, J=6 Hz), 3.60 (2H, t, J=5.7 Hz), 2.92
(2H, t, J=5.7 Hz). 5k, calcd for C12H9F3N2OS: 286.3, Elec-
trospray-MS m/z 284.9 (MÀH)À, 1H NMR (DMSO-d6) d
11.94 (1H, br), 8.02 (2H, m), 7.64 (2H, m), 3.60 (2H, t, J=5.7
Hz), 2.91 (2H, t, J=5.7 Hz).
References and Notes
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1617.
12. Franklin, T. J.; Snow, G. A. In Biochemistry of Anti-
microbial Action, 4th ed., Chapman and Hall: London, 1989;
Chapter 7, p 161.