Inhibitors of the bacterial cell wall biosynthesis enzyme MurC

Article abstract of DOI:10.1016/S0960-894X(01)00251-7

A series of phosphinate transition-state analogues of the L-alanine adding enzyme (MurC) of bacterial peptidoglycan biosynthesis was prepared and tested as inhibitors of the Escherichia coli enzyme. Compound 4 was identified as a potent inhibitor of MurC

Full text of DOI:10.1016/S0960-894X(01)00251-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1451–1454
Inhibitors of the Bacterial Cell Wall Biosynthesis Enzyme MurC
Folkert Reck,a,* Stephen Marmor,b Stewart Fisherb and Mark A. Wuonolaa
aChemistry, Infection Discovery, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
bBiochemistry, Infection Discovery, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
Received 12 March 2001;accepted 6 April 2001
Abstract—A series of phosphinate transition-state analogues of the l-alanine adding enzyme (MurC) of bacterial peptidoglycan
biosynthesis was prepared and tested as inhibitors of the Escherichia coli enzyme. Compound 4 was identified as a potent inhibitor
of MurC from Escherichia coli with an IC50 of 49 nM. # 2001 Elsevier Science Ltd. All rights reserved.
Rapidly developing bacterial resistance to existing drugs
has become the major problem in antibacterial therapy
and necessitates continuous research for novel anti-
bacterials. Especially, agents directed against novel
bacterial targets are expected to have the potential for
low initial bacterial resistance. The biosynthesis of pep-
tidoglycan as a target in bacterial cell wall synthesis is
validated by several important classes of antibiotics.
One key enzyme, the ligase MurC, has been shown to be
essential in Escherichia coli.1 MurC catalyzes the con-
version of UDP-MurNAc to UDP-MurNAc-Ala in the
assembly of the disaccharide-peptide unit required for
peptidoglycan biosynthesis (Fig. 1).
We were interested in the design and synthesis of tran-
sition state analogues of MurC. The reaction pathway
catalyzed by MurC involves the acyl phosphate inter-
mediate 32,3 (Fig. 2).
Figure 2. MurC, acyl phosphate intermediate 3 and transition state
analogues 4 and 5.
For several ligases that involve acyl phosphate inter-
mediates, such as MurD,4,5 MurE,6 and d-Ala-d-Ala
ligase,7 potent phosphinate inhibitors have been devel-
oped. Phosphinates mimic the tetrahedral reaction cen-
ter of the transition state. In the case of d-Ala-d-Ala-
ligase, phosphinates have been shown to undergo phos-
Figure 1. The reaction catalyzed by MurC.
*Corresponding author. Fax: +1-781-839-4500;e-mail: folkert.reck@
astrazeneca.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00251-7

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F. Reck et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1451–1454
phorylation in the active site by the co-substrate ATP.
Therefore, we expected that phosphinate analogues of
the product of the MurC reaction, such as compounds 4
and 5 might produce tight binding inhibitors of MurC.
In the design of 4 we did not include the methyl group
of the lactic acid moiety, and chose not to define the
stereochemistry at the l-alanyl-derived moiety, in order
to simplify the synthesis considerably. For additional
simplification, in 5 the hexose is substituted by a 1,3-
propanediol spacer.
give 9 in 97% yield. Arbuzov reaction with methyl bro-
momethyl acrylate under silylating conditions10 affor-
ded 10 in 83% yield. Concomitant hydrogenation of the
double bond and benzylether in 10 proceeded smoothly
to the alcohol 11 which was phosphorylated with diphe-
nyl chlorophosphate/DMAP to give 12 in 94% yield.
Deprotection of 12 by hydrogenation over PtO2 to 13,
followed by saponification gave phosphate 14 in nearly
quantitative yield. Coupling of 13 with UMP-morpholi-
date/tetrazole in pyridine afforded the UDP-containing
derivative 15 in 27% yield. Finally, saponification of 15
gave the diastereomeric mixture of 5 in 88% yield.
Here, we report on the synthesis and biological evalua-
tion of compounds 4 and 5 along with compounds
derived from their synthetic precursors.
For the synthesis of 4 (Scheme 2) the major problem
was again formation of the ether linkage alpha to the
phosphinate. Commercially available 16 was chosen as
a suitable and convenient starting material. Alkylation
of 16 with 1.6 equiv of mesylate 17 (the latter was pre-
pared from the corresponding alcohol11 with
mesylchloride/DIPA in 73% yield) and sodium hydride
as base gave 18 as a mixture of two diastereomers in
10% yield. The low yield is the result of decomposition
of the mesylate under reaction conditions;most of 16
could be recovered during workup. Removal of the
ketal protecting group in 18 was effected with CHCl3/
TMSCl. In our experience, deprotection of the ketal of
the Baylis protecting group requires at least a trace of
water, and as a consequence the benzylidene protecting
group in 18 was partially intermittently lost during ketal
removal in 18. However, after ketal removal the ben-
zylidene protection could be reestablished in good yield
by azeotropic distillation with toluene. The resulting
crude intermediate was then subjected to Arbuzov
reaction with methyl bromomethyl acrylate under sily-
lating conditions10 to afford 19 in 49% overall yield. In
19, saturation of the double bond and removal of the
benzylidene and benzyl groups was effected by hydro-
genation over Pd/C. The crude product was then per-
acetylated with acetic anhydride/pyridine. Treatment
with aminoethanol in THF selectively removed the
anomeric acetate to give the free anomeric intermediate
Compounds 4 and 5 include an ether linkage in a-posi-
tion to the phosphinate moiety, a structural feature that
is difficult to establish, and has little literature precedent
for complex molecules. Other known phosphinate tran-
sition state analogues of ligases have at this position a
nitrogen instead of an oxygen. Formation of this ether
linkage was perceived as the major challenge in the
synthesis of 4 and 5.
Synthesis of 5 is outlined in Scheme 1. The starting
material 6 was prepared from phosphinic acid according
to Baylis.8 Compound 6 has the advantage that it allows
selective deprotection under mild conditions for step-
wise assembly of complex phosphinates. The chloro-
methyl ether 7 was obtained in quantitative yield by
treating commercially available monobenzylated 1,3-
propanediol with formaldehyde/HCl. Arbuzov reaction
of 6 and 7 proceeded to 8 under mild silylating condi-
tions9 in 29% yield. Side reactions arose from partial
loss of the ketal and ester protection groups on the
phosphinate. An alternative route via alkylation of the
corresponding primary alcohol with mesylate 17 was
explored, but proved to be less compatible with the
protecting groups on the phosphinic acid moiety. Selec-
tive removal of the ketal protecting group in 8 was
achieved under mild conditions with CHCl3/TMSCl8 to
Scheme 1. Reagents: (a) 2,6-lutidine (4 equiv), BSA (1 equiv)/CH3CN, 90 ꢀC, 1 h, 29%;(b) TMSCl (1.1 equiv), H 2O (0.5 equiv)/CHCl3, rt, 5 h, 97%;
(c) TEA (2.2 equiv), TMSCl (2 equiv), then methyl (2-bromomethyl) acrylate (1.1 equiv), 0 ꢀC–rt, 2 h, 83%;(d) Pd/C, H 2/MeOH, 15 min, 96%;(e)
PClO(OPh)2 (1.4 equiv), DMAP (1.6 equiv)/CH2Cl2, 0 ꢀC, 15 min, 94%;(f) PtO , H2/MeOH/AcOH, 2 h, 99%;(g) LiOH (2.3 M, aq), rt, 2 h, 99%;
(h) UMP-morpholidate (2 equiv), 1H-tetrazole (4 equiv)/pyridine, rt, 27%;(i) LiOH (2.3 M, aq), rt, 2 h, 88%.
2

F. Reck et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1451–1454
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Scheme 2. Reagents: (a) NaH/DMF, 0 ꢀC–rt, 18 h, 10%;(b) TMSCl (1.1 equiv), H 2O (0.5 equiv)/CHCl3, rt, 1 h;(c) TMSCl (4 equiv), TEA (4.5
equiv), 0 ꢀC, 30 min, then methyl (2-bromomethyl) acrylate (1.2 equiv), 5 min, 49% (b-c);(d) Pd/C, H 2/MeOH, HOAc (10:1), rt, 1 day;(e) Ac O/
2
pyridine, rt, 16 h;(f) aminoethanol (2 equiv)/THF, rt, 4 h, 50% (d-f);(g) n-BuLi (1 equiv)/THF, À78 ꢀC, 2 min, then PClO(OPh)2 (1.3 equiv), 1 h;(h)
PtO2, H2/THF, À78 ꢀC–rt, 16 h, 96% (g-h);(i) NaOH (0.5 M, aq), rt, 1.5 h, 80%;(j) 20, UMP-morpholidate (2.5 equiv)/pyridine, rt, 9 days, 37%;(k)
NaOH (0.5 M, aq), 1.5 h, 79%.
yield to 22. Finally, saponification of 22 gave 4 in 79%
yield as a 1:1 mixture of the two diastereomers.
Compounds 4, 5, 14, and 21 were tested for inhibition of
MurC from E. coli using 37 nM enzyme, 200 mM ATP,
120 mM l-alanine and 75 mM UDP-MurNAc. The read-
out was detection of the product phosphate by the
malachite green assay.13 Results are summarized in
Figure 3. The phosphinate 4 exhibited an IC50 of 49 nM
and is thus the most potent inhibitor of MurC reported.
The results indicate that substitution of the des-methyl-
muramic acid in 4 with a flexible 1,3-propanediol
spacer decreases potency by over three orders of mag-
nitude. Also, the UDP moiety is necessary for potent
inhibition, since the terminal phosphates 14 and 21 are
inactive.
References and Notes
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Figure 3. Inhibition of MurC from E. coli by compounds 4, 5, 14, and
21.
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