Pulvinones as bacterial cell wall biosynthesis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.09.021

Pulvinones were synthesized (>180) in arrays and evaluated as inhibitors of early stage cell wall biosynthesis enzymes MurA-MurD. Several pulvinones inhibited Mur enzymes with IC₅₀'s in the 1-10 μg/mL range and demonstrated antibacterial activity against Gram-positive bacteria including methicillin-resistant Staphyloccus aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

Full text of DOI:10.1016/j.bmcl.2005.09.021

Bioorganic & Medicinal Chemistry Letters 16 (2006) 176–180
Pulvinones as bacterial cell wall biosynthesis inhibitors
Schuyler Antane,a,* Craig E. Caufield,a William Hu,b David Keeney,b
Pornpen Labthavikul,b Koi Morris,a Shaughnessy M. Naughton,a Peter J. Petersen,b
Beth A. Rasmussen,b Guy Singhb and Youjun Yangb
aMedicinal Chemistry, Chemical and Screening Sciences, Wyeth Research, Princeton, NJ 08543, USA
bInfectious Diseases, Wyeth Research, Pearl River, NY 10965, USA
Received 21 July 2005; revised 26 August 2005; accepted 8 September 2005
Available online 10 October 2005
Abstract—Pulvinones were synthesized (>180) in arrays and evaluated as inhibitors of early stage cell wall biosynthesis enzymes
MurA–MurD. Several pulvinones inhibited Mur enzymes with IC50Õs in the 1–10 lg/mL range and demonstrated antibacterial activ-
ity against Gram-positive bacteria including methicillin-resistant Staphyloccus aureus, vancomycin-resistant Enterococcus faecalis,
and penicillin-resistant Streptococcus pneumoniae.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Enzymes of the murein pathway mediate bacterial cell
wall biosynthesis.1 The initial enzymes, MurA–MurB,
convert
uridinediphosphate
N-acetylglucosamine
(UDP-GlcNAc) into uridinediphosphate N-acetylmu-
ramic acid (UDP-MurNAc). A pentapeptide chain is
appended to the UDP-MurNAc by the amino acid
ligases MurC–MurF. The nucleotide of the UDP-
MurNAc-peptide unit is replaced with membrane bound
lipid phosphate. MurG couples a GlcNAc to the lipid
bound MurNAc and the resultant disaccharide is trans-
ferred across the membrane to the growing cell wall mol-
ecule. Although the early stage enzymes (MurA–MurD)
are well validated as antibacterial targets only fosfomy-
cin (a MurA inhibitor) has been used as a clinical
product.2
Figure 1. Structures of pulvinic acid 1 (R = H), vulpinic acid 1
(R = Me), pulvinamide derivative 2, and pulvinone 3.
resistant Enterococcus faecalis (VRE), and penicillin-
resistant Streptococcus pneumoniae (PRSP).6
Pulvinones (10) were prepared in five steps (Scheme 1)
starting from commercially available 4-methoxy-2(5H)-
furanone (4). Bromination with N-bromosuccinimide
yielded 5.7 Aldol reaction of various aldehydes with 5
deprotonated by lithium isopropyl cyclohexylamide
(LICA)8 afforded secondary alcohols (6). The resultant
alcohols were eliminated via in situ mesylate formation
to afford the halogenated Suzuki coupling partner 8 in
the more thermodynamically stable Z-form.9
Naturally occurring vulpinic acid (Fig. 1) has been
reported to possess in vitro activity against Gram-posi-
tive bacteria.3 In-house screening for Mur enzyme inhib-
itors identified pulvinamide 24 [MurC (IC50 = 8 lg/mL)].
Our discovery effort revealed analogues of the des-ami-
do pulvinone 35 also inhibit MurC with an IC50 of
68 lg/mL and demonstrated antibacterial activity
against Gram-positive bacteria including methicillin-
resistant Staphyloccus aureus (MRSA), vancomycin-
Alternatively, the aldol reaction may be performed first
using lithium hydroxide. Reaction of 4 with various
aldehydes under these conditions gave 7.10 Subsequent
bromination in acetonitrile afforded 6 that was eliminat-
ed as before to provide the bromo alkylidene furanone
Keywords: Pulvinones; Murein enzyme inhibitors; Antibacterial;
Microwave demethylation.
*
Corresponding author. E-mail: antanes@wyeth.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.021

S. Antane et al. / Bioorg. Med. Chem. Lett. 16 (2006) 176–180
177
Scheme 1. Synthesis of pulvinones. Reagents and conditions: (a) NBS, CCl4, reflux; (b) LICA, THF, R1CHO, ꢀ78 ꢁC or LiOH, CH3CN, R1CHO,
rt; (c) MsCl, TEA, CH2Cl2, 0 ꢁC, 90 min; (d) Pd(PPh3)4, K3PO4, R2B(OH)2 dioxane, Synthewaveꢂ 402 microwave (Prolabo), 15% pwr, 5 min or
100 ꢁC, 90 min; (e) LiBr, DMF, Synthewaveꢂ 402 microwave (Prolabo), 30% pwr, 4 min or heat 150 ꢁC, 15 min; (f) Br2, CH3CN, rt.
8. Suzuki coupling of 8 with various boronic acid deriv-
atives yielded the penultimate methoxy furanones (9),
which were demethylated with lithium bromide to afford
the final pulvinone products (10). The last two steps
were accelerated by microwaves.11
and 14c) appeared to show a slight preference for MurC
and were highly variable in MIC effectiveness. para-
Chloro substitution (12a,c) was slightly better than
para-methoxy substitution (12b,d) in MurB–MurC
inhibition and activity versus VRE.
Pulvinones (10) were initially prepared in a matrix of
five arrays (11–15). MurA–MurD inhibition was as-
sayed sequentially unlike the efficient combinatorial
enzymology approach utilized at Merck.13 The methoxy
precursors (9) were devoid of significant activity. Repre-
sentative final products from each array are shown in
Table 1. Overall these pulvinones appeared to
consistently inhibit MurC. In addition, the pulvinones
displayed varying MurB inhibition and inhibition of
MurA to a lesser extent.
The phenyl-m-trifluoromethylbenzylidene (12a) was
equipotent with naphthylidene derivatives (11a,b). The
overall antibacterial activity of the 1-,2-naphthyl-m-tri-
fluoromethylbenzylidenes (13a and 14a) was slightly
enhanced by replacement with biphenylmethylidene
(13b and 14b).
Biphenylmethylidene (14b) appeared to show a slight
preference for MurB. A double Suzuki14 coupling
reaction of dibromo o,m,p-isomers 16 (Scheme 2)
provided additional arrays of biphenylmethylidenes
18. Biphenyl directly bonded to the furanone core
15a demonstrated the best overall Gram-positive
The naphthylidene derivatives (11a,b) were nearly iden-
tical in activity. The cyclohexylidene derivatives (11c
Table 1. Minimal inhibitory concentration (MIC, lg/mL) and in vitro activity against MurA–MurD enzymes (IC50, lg/mL)12
11ad
11be
11cf
32
12ag,i 12bh,i 12cd,g 12dd,h 13ai
13bj 14ai 14bj 14cf
15ai
15bk
S. aureus GC 1131a
S. aureus GC 4543
8
8
8
8
16
8
1
128
128
64
8
8
32
64
32
64
8
4
8
8
16 >128
4
4
8
4
16
16
8
>128
64
32
4
8
4
2
16
32
E. faecalis GC 4555
E. faecalis GC 2242b
S. pneumoniae GC 1894c
16
8
16
4
16
8
16
8
>128
32
>128
32
32
8
32
8
8
4
0.5
0.25
60.12
1
2
0.5
8
8
4
8
60.12
0.5
1
MurA
MurB
MurC
MurD
>25
8
>25
11
>25
>25
10
14
9
>25
>25
14
>25
10
>25
>25
16
>25
16
20 >25
>25
>25
9
17
5
>25
—
>25 >25
13
10
6
2
10
6
>25
6
>25
6
>25
7
>25
9
8
>25
16
>25
>25
25
>25
>25 >25 >25 >25
>25
a MRSA.
b VRE.
c PRSP.
d R = 1-naphthyl.
e R = 2-naphthyl.
f R = cyclohexyl.
g X = Cl.
h X = OMe.
i R = 3-CF3Ph.
j R = biphenyl.
k R = 4-ClPh.

178
S. Antane et al. / Bioorg. Med. Chem. Lett. 16 (2006) 176–180
(Scheme 3). For comparative purposes we prepared 20
from the commercially available 2-fluoro-4-biphenyl
boronic acid.
Polychlorinated pulvinone derivatives 18 evoked toxici-
ty comparisons to the persistent environmental pollu-
tant PCBs.16 Prudently we reduced the number of
halogens in a follow-up array (Scheme 4). Suzuki
reaction of ortho-substituted boronic acids with p-bro-
mo or p-triflate aldehydes represented by 23 yielded
2,20-disubstituted biphenyl aldehydes 24. These alde-
hydes were reacted with the bromofuranone 5 as before
(Scheme 1, steps b–e) to produce the less halogenated
pulvinones (25).
Scheme 2. Double Suzuki. Reagents: (a) Pd(PPh3)4, K3PO4, dioxane;
(b) LiBr, DMF.
antibacterial activity of the first matrix with MICÕs in
the range 0.5–8 lg/mL.
Inspired by the oxazolidinone antibiotic linezolid15 and
its suggestive 6-6-5-framework similarity to 15, we re-
placed the biphenyl of 15 with an ortho-fluor-
ophenylmorpholine to generate 19 (Fig. 2). The
preparation of 19 necessitated the synthesis of the
boronate derivative 22 from 4-bromo-2-fluoroaniline
Potent antibacterial activities were observed for these
biphenyl arrays versus Gram-positive organisms: S.
aureus including MRSA, E. faecalis including VRE,
and penicillin-resistant S. pneumoniae (Table 2). Clearly
the most potent were the isomeric tetrachlo-
robiphenylmethylidenes 18a–c. The dichloro analogue
18d inhibited MurA–Mur D but exhibited less antibacte-
rial activity.
The morpholine-substituted pulvinone 19 was ineffec-
tive, despite showing modest MurB inhibition.
Replacement of the morpholine with a phenyl ring
(20c) increased inhibition of MurA–MurD. The wider
spectrum and enhanced potency of Mur enzyme inhibi-
tion exhibited by these pulvinones were difficult to ex-
plain. It seemed less probable for the pulvinones to
mimic the common phosphate motifs in the natural
substrates and co-factors than it was to occupy an
allosteric inhibitory site, perhaps located in a solvent
exposed region.17
Figure 2. Substructure graft of linezolid onto pulvinones.
The 2,20-disubstituted biphenylmethylidenes (25a,b,d,
and e) displayed primarily MurA and MurB inhibition,
except for a dimethoxy analogue (25c) that also inhibit-
ed MurC. For the most part, the Gram-positive (S. aure-
us) enzyme data seemed to be in agreement with the
Gram-negative (Escherichia coli) enzyme data (Table
2). However, concerning MurD enzyme data, it has been
noted that Gram-negative is more restrictive than
Gram-positive.18
Scheme 3. Synthesis of 3-fluoro-4-morpholinophenylboronic acid
pinacol ester. Reagents: (a) (ClCH2CH2)2O, NaI, K2CO3, DMF;
(b) pinacolborane, PdCl2(PPh3)2, dioxane, TEA.
Pulvinones may be considered decarboxylated ana-
logues of pulvinic acids. To summarize, we have
found that the carboxyl group of pulvinic acid deriv-
atives is not essential for murein enzyme inhibition.
Therefore, pulvinones are novel inhibitors of bacterial
cell wall biosynthesis enzymes. Matrix production of
pulvinone arrays, some expedited by microwave-assist-
ed Suzuki and demethylation reactions, hastened the
identification of several derivatives that possessed
antibacterial activity against Gram-positive organisms:
S. aureus including MRSA, E. faecalis including
VRE, and penicillin-resistant S. pneumoniae. Finally,
the ability of selected pulvinones to block multiple
Mur enzymes may be preferred since bacterial resis-
tance might require genetic mutations at multiple
targets.19
Scheme 4. Synthesis of 2,20-disubstituted biphenylmethylidene pulvi-
nones. Reagents and conditions: (a) 2-R0PhB(OH)2, Pd(PPh3)4,
K3PO4, dioxane, 100 ꢁC, overnight.

S. Antane et al. / Bioorg. Med. Chem. Lett. 16 (2006) 176–180
179
Table 2. Minimal inhibitory concentration (MIC, lg/mL) and in vitro activity against MurA–MurD enzymes (IC50, lg/mL) of select pulvinones12
18ae,n 18bf,o
18ce,p 18dg,n 19h
20ai
20bj 20ch 20dk 25ae,l 25be,m 25ce,q 25de,r
25eg,r
S. aureus GC 1131a
S. aureus GC 4543
0.25
0.25
1
0.5
0.25
1
4
>128
64
128
128
128
16
2
2
1
2
2
2
2
1
1
4
1
1
0.5
0.5
2
0.5
0.5
2
2
0.5
0.5
2
2
1
4
2
0.5
1
1
8
4
1
1
2
E. faecalis GC 4555
E. faecalis GC 2242b
S. pneumoniae GC 1894c
2
8
0.5
0.5
0.5
0.25
0.5
0.25
1
0.5
8
0.5
1
0.25 60.12 60.12
0.25 0.5 0.25
0.5 60.12
0.5 60.12
MurAd
MurB
MurCd
MurDd
2
3
3
2
1
3
5
4
5
6
>25
10
10
12
8
1
1
2
6
3
1
5
9
1
1
4
2
2
1
17
(2)
4
3
6
6
3
(>25)
(24)
11
(6)
(18)
(5)
17
6
(>25)
10
16
(20)
(17)
(23)
(9)
(15)
(12)
(>25) >25
>25
a MRSA.
b VRE.
c PRSP.
d Values in parentheses are S. aureus enzymes, all others are for E. coli.
e X,Y = 3,5-diCl.
f X,Y = 3,4-diCl.
g X = 3-Cl, Y = H.
h R = 2-Cl,5-CF3-Ph.
i R = 3-CF3Ph.
j R = 2-F,5-CF3-Ph.
k R = 2-Cl,3-CF3-Ph.
l R = OMe, R0 = CF3.
m R = R0 = CH3.
n meta-biPh.
o ortho-biPh.
p para-biPh.
q R = R0 = OMe.
r R = CH3, R0 = CF3.
1.1 mmol lithium bromide. The mixture is diluted with
1.5 mL DMF and irradiated in a Synthewaveꢂ 402 micro-
wave (Prolabo) at 30% power for 4 min. Upon cooling,
crude material was poured over 2 M sulfuric acid and
extracted with ethylacetate. The crude lithium tetronates
were purified by parallel chromatography (Biotage Quad3)
and re-acidified to the tetronic acids 10 by washing with 2 N
hydrochloric acid.
References and notes
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No 2, NCCLS, Wayne, Pa.MurA–MurD enzyme bio-
chemical assays: novel compounds were tested for inhibi-
tion of murein enzymes in buffered solutions. Optical
densities (OD) due to the release of phosphate during
MurA, MurC or MurD enzymatic reactions were mea-
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a
SPECTRAmaxꢂ250 (Molecular Devices)
660 nm. The OD measurement in the Mur B assay,
however, directly detected the appearance of nicotin-
amide-adenine dinucleotide phosphate (NADP) at
340 nm. The difference in readings between control con-
ditions (buffered solutions of natural substrate(s), enzyme,
and co-factors) and in the presence of novel compounds
determines the percent inhibition. IC50Õs are then calcu-
lated by linear regression analysis using SoftMaxꢂ soft-
ware (Molecular Devices). The MurA assay detected the
quantity of inorganic phosphate cleaved from phospho-
enolpyruvate (PEP) and consisted of 25 mM Tris–HCl
buffer, pH 8, 150 nM MurA, 200 lM PEP, and 200 lM
UDP-GlcNAc in final volume 25 lL. The procedure is
11. Typical microwave Suzuki procedure: in a 2.5 mL Wheaton
Reacti-Vial was placed 0.2 mmol bromofuranone
8
(Scheme 1), 0.25 mmol aryl boronic acid, 0.7 mmol potas-
sium phosphate, and 5 mol% tetrakis(triphenyl-phos-
phine)-palladium. The mixture was diluted with dioxane
to 0.1 M and irradiated at 15% power for 5 min in a
Synthewaveꢂ 402 microwave (Prolabo). The vials were
centrifuged and decanted onto silica gel in order to purify by
parallel chromatography (Biotage Quad 3). Typical micro-
wave demethylation procedure: a 20 · 150 mm test tube was
charged with 1 mmol of methoxy furanone 9 (Scheme 1) and

180
S. Antane et al. / Bioorg. Med. Chem. Lett. 16 (2006) 176–180
similar to that described by Marquardt, Walsh, and others
1.3 N HCl). In order to minimize the acid hydrolysis of
ATP in MurC and MurD reactions, 50 lL of 34% (v/w)
sodium citrate was added to the reaction after the
malachite green reagent. This procedure is similar to that
described by Lanzetta and others (Anal. Biochem. 1979,
100, 95).
(Biochemistry 1994, 33, 10646). The second enzyme assay,
MurB, followed the appearance of NADP upon reduction
of enolpyruvate (EP) via direct readings taken at 340 nm
and contained 50 mM Tris–HCl, pH 8.0, 10 mM KCl,
100 lM NADPH, and 50 lM EP-UDP-GlcNAc, in a total
volume of 200 lL. This procedure was similar to that
described by Dhalla and others (Biochemistry 1995, 34,
5390). The amino acid ligase MurC assay detected the
quantity of inorganic phosphate released (OD at 660 nm)
from the adenosinetriphosphate (ATP) hydrolysis and
contained 83 mM Tris–HCl buffer, pH 8.5, 200 nM MurC,
50 mM MgCl2, 50 mM (NH4)2SO4, 2 mM ATP,
1.275 mM UDP-MurNAc, and 2 mM L-alanine in a final
volume of 25 lL. The amino acid ligase MurD assay
detected the quantity of inorganic phosphate released (OD
at 660 nm) from ATP hydrolysis and consisted of 83 mM
Tris–HCl buffer, pH 8.5, 68 nM MurD, 50 mM MgCl2,
50 mM (NH4)2SO4, 2 mM ATP, 0.375 mM UDP-
MurNAc-L-Ala, and 2 mM D-glutamate (final vol-
ume = 25 lL). These reactions were initiated by the
addition of appropriate UDP-linked substrate and incu-
bated at 37ꢁC for 30 min. The reactions were terminated
by the addition of 210 lL of malachite green reagent
(0.03% malachite green, 1.4% ammonium molybdate, and
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