Synthesis and biological evaluation of penem inhibitors of bacterial signal peptidase

Article abstract of DOI:10.1016/j.bmcl.2009.04.034

We report the first synthesis of a 5S penem, known to bind bacterial type I signal peptidase, from the commercially available and inexpensive 6-aminopenicillanic acid. We report the first in vivo activity of the compound and use structure-activity relationship studies to begin to define the determinants of signal peptidase binding and also to begin to optimize the penem as an antibiotic.

Full text of DOI:10.1016/j.bmcl.2009.04.034

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3787–3790
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of penem inhibitors of bacterial
signal peptidase
*
David A. Harris, Michael E. Powers, Floyd E. Romesberg
Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the first synthesis of a 5S penem, known to bind bacterial type I signal peptidase, from the
commercially available and inexpensive 6-aminopenicillanic acid. We report the first in vivo activity of
the compound and use structure–activity relationship studies to begin to define the determinants of sig-
nal peptidase binding and also to begin to optimize the penem as an antibiotic.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 2 March 2009
Revised 10 April 2009
Accepted 10 April 2009
Available online 18 April 2009
Keywords:
Antibiotic
SAR
Staphylococcus epidermidis
MRSA
The emergence of drug resistant bacteria poses a serious threat
to human health, and the preservation of the ‘antibiotic era’ will
likely require the discovery of novel antibiotics that act via novel
mechanisms. One potential novel mechanism is the inhibition of
bacterial type I signal peptidase (SPase). SPase is conserved across
both Gram-positive and Gram-negative bacteria and is required to
process cell surface bound preproteins during export from the
cytoplasm.1,2 Its location in the outer leaflet of the cytoplasmic
membrane should make it accessible to small molecule drugs. In
addition, its unique catalytic mechanism, which relies on an atyp-
ical Ser-Lys catalytic dyad and attack of the peptide substrate from
the si-face, rather than the re-face characteristic of the common
Ser-His-Asp catalytic triad serine proteases,3 should limit off-target
toxicity. Moreover, SPase is not only required for viability, but is
also required to process and secrete proteins involved in a number
of bacterial processes including adhesion, antibiotic resistance, vir-
ulence, lateral gene transfer, and toxin production.4,5 SPase inhibi-
tors should thus attenuate virulence, potentially increasing their
utility as therapeutics.
Consistent with its unconventional proteolysis mechanism,
standard serine protease inhibitors do not inhibit SPase.6–9
However, researchers at SmithKline Beecham Pharmaceuticals
identified a racemic penem with moderate in vitro activity from
a screen against the Escherichia coli protein.10–13 Interestingly, all
inhibitor activity was found in the 5S diastereomer of the penem,
which is the stereochemistry opposite to that of other known
b-lactam drugs.14
Generally, b-lactam antibiotics are effective irreversible cova-
lent inhibitors of serine proteases and hydrolases, such as elastase
and b-lactamase.16,17 These antibiotics act by forming long lived
acyl enzyme intermediates via the electrophilicity of their cyclic
amide carbonyl.
A medicinal chemistry effort at SmithKline
Beecham directed at the optimization of the initially identified
penem culminated in the identification of the (5S,6S)-6-[(R)-
hydroxyethyl]-penem-3-carboxylate (Fig. 1A, R1,R2 = H) which
inhibits E. coli SPase with an IC50 of 180 nM.10–13 In addition, highly
strained 5S tricyclic penems, in which a third heterocyclic ring is
fused to the C2 and C3 positions of the (5S,6S)-6-[(R)-hydroxy-
ethyl]-penem core, inhibit E. coli and MRSA SPase in vitro with
IC50 values of 0.2 lM and 5 l
M, respectively.18 Thus, potent
in vitro SPase inhibition has been demonstrated by derivatization
of the 5S,6S-penem, however; antibacterial activity in vivo has
not been reported for any of these compounds.
Since the efforts of SmithKline Beecham, Paetzel et al. reported
the crystal structure of the E. coli SPase complexed with the allyl
(5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate (Fig. 1A, R1 =
acetyl, R2 = allyl).15 In addition to confirming the unique mecha-
nism of SPase catalysis, the structure offered insight into the bind-
ing of both the inhibitor and the natural leader peptide substrates.
In general, an E. coli signal peptide consists of a C-terminal cleavage
recognition sequence containing small uncharged residues at the
P1 and P3 sites, with the former almost always Ala and the latter
Ala, Val, Leu, or Ile.19,20 The P1 and P3 side chains are thought to
bind in the corresponding substrate binding pockets S1 and S3 in
SPase. The crystal structure reveals that the penem does not access
the S3 pocket, but its C10 methyl group is oriented towards the S1
pocket. The structure also reveals that substituents attached to the
* Corresponding author. Tel.: +1 858 784 7290; fax: +1 858 784 7472.
E-mail address: floyd@scripps.edu (F.E. Romesberg).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.034

3788
D. A. Harris et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3787–3790
Scheme 1. Reagents and conditions: (i) Br2 (3.0 equiv), NaNO2 (2.0 equiv), 2.5 N
H2SO4 (2.0 equiv), CH2Cl2, 5 °C; (ii) K2CO3 (0.9 equiv), p-nitrobenzyl bromide
(1.0 equiv), DMF, 40 °C, (70% for i–ii); (iii) MeMgBr (3.0 M in Et2O) (1.0 equiv),
MeCHO (6.5 equiv), THF, À78 °C, (35%); (iv) Bu3P (1.6 equiv), MeOH, 0 °C, (95%); (v)
t-BuMe2SiCl (1.5 equiv), imidazole (3.0 equiv), DMF, rt, (98%); (vi) MCPBA
(0.9 equiv), CH2Cl2, 0 °C, (80%); (vii) mercaptobenzothiazole (0.99 equiv), toluene,
reflux; (viii) Et3N (0.1 equiv), toluene, 0 °C; (ix) HCO2H (4.4 equiv), Ac2O (4.4 equiv),
pyridine (1.03 equiv), PPh3 (1.01 equiv), toluene, À15 °C, (65% for vii–ix); (x) O3,
Figure 1. (A) Structure of (5S,6S)-6-[(R)-hydroxyethyl] (R1, R2 = H) and 3-allyl ester
(R1 = acetyl and R2 = allyl) penems identified by SmithKline Beecham.12,13 (B)
Structure of the 3-allyl ester penem bound to E. coli SPase. The S1 and S3 binding
pockets as well as the catalytically essential serine (Ser90) and lysine (Lys145) are
highlighted.15 The C6 hydroxyethyl group is indicated with an arrow.
EtOAc, À78 °C, (98%); (xi) P(OMe)3 (5.0 equiv), toluene, 70 °C, (40%); (xii) h
t, EtOAc,
rt, (60%); (xiii) Bu4NF (1.0 M in THF) (4.0 equiv), HOAc (12.0 equiv), rt, (90%).
penem at its C2 and C3 positions roughly correspond to substrate
side chains P2 and P4, and that they offer little to no opportunity
for optimization of inhibitor binding because they are oriented into
solvent. This structural data is consistent with results from
SmithKline Beecham12,13 as well the natural substrate sequence
diversity at P2 and P4.19
hydroxyethyl group, oxidation with m-chloroperbenzoic acid
(MCPBA) afforded the sulfoxide. Heating of the sulfoxide to reflux
in toluene induced a sigmatropic rearrangement that resulted in
the formation of the sulfenic acid, which was intercepted by mer-
captobenzothiazole to yield the disulfide product. Base-catalyzed
double-bond isomerization produced the more stable conjugated
ester disulfide, and the C4 formylthio substituent was introduced
via reductive formylation with pyridine, acetic formic anhydride,
(in situ from formic acid and acetic anhydride), and triphenylphos-
phine, yielding the aldehyde 4.27 Ozonolysis of this material fol-
lowed by trialkyl phosphite-mediated cyclization of the
oxalamide by reductive carbonyl-carbonyl coupling yielded the
hydroxyethyl penem ester.28 Photochemical isomerization at C5
converted the core 5R,6S-trans-penem to the 5S,6S-cis stereochem-
istry.29 Tetrabutylamonium fluoride (TBAF)-mediated desilylation
gave the desired hydroxyethyl penem.
Before commencing our structure–activity studies we first
determined the susceptibility of several important human patho-
gens to the parent penem 1 (Table 1). Specifically, we determined
the sensitivity of E. coli (strain ATCC25922 treated with permeabi-
lizing agent polymyxin b (PMBn) to eliminate penetrance issues),
two Staphylococcus aureus strains (8325 and the methicillin resis-
tant clinical isolate USA300, which for convenience will be referred
to as MRSA), and Staphylococcus epidermidis (strain RP62A). In each
case, the minimal inhibitory concentration (MIC) was determined
using the liquid dilution method. As reported previously, the
parent 5S-OH penem 1 showed no significant activity against per-
This structural data suggest that the optimization of binding
within S1 and/or accessing the S3 pocket of the enzyme offers
the best opportunity to increase the affinity of the penem for SPase
and that both of these sites should be accessible from the C6 posi-
tion of the inhibitor core (Fig. 1). Toward exploring this possibility,
we report the first synthesis of a 5S penem from the commercially
available and inexpensive 6-aminopenicillanic acid, which has pro-
ven to be a useful starting material for the synthesis of numerous
5R b-lactam drugs.21,22 With access to the compound, we explored
its activity against several bacteria and demonstrated the first
in vivo activity. Finally, we report an initial structure–activity rela-
tionship (SAR) study designed to probe the accessibility of the S1
and/or S3 pockets of SPases from both Gram-positive and Gram-
negative bacteria from the C6 position of the 5S penem core.
For our initial SAR studies we examined modifications of the
5S,6S-penem core with a C3 p-nitrobenzyl protected carboxylic
acid and a C6 hydroxyethyl moiety (1). Preliminary experiments
showed little differences in activity with a free acid at position
C3 or with p-nitrobenzyl-, allyl-, or methyl-esters. The C6 hydroxy-
ethyl substituent is known to be important for activity12,13 and we
also found that it facilitated stereoinversion during synthesis. Syn-
thesis of this penem core (Scheme 1) commenced with preparation
of p-nitrobenzyl 6,6-dibromopenicillanate which is readily
obtained from aminopenicillanic acid 2 by diazotization-bromina-
tion, followed by esterification of the crude di-bromo acid.23
Metal–halogen exchange with methylmagnesium bromide in THF
at À78 °C, gave an enolate intermediate, which upon quenching
with an excess of acetaldehyde, afforded the hydroxyethyl prod-
uct.24,25 Diastereoselective reduction of the 6-bromo-6-substituted
penicillanate was achieved by treatment with tributylphosphine
meabilized E. coli or S. aureus (MIC P 200
observe activity against S. epidermidis (MIC = 50
l
g/mL). However, we did
g/mL), which is
l
the first demonstration of antibacterial activity for a penem SPase
inhibitor on whole cells. In contrast, the 5R diastereomer had no
activity, suggesting that the antibacterial activity was indeed asso-
ciated with SPase inhibition.
Our initial SAR studies focused on attaching simple ethers to the
C6 hydroxyethyl moiety. Synthetic challenges limited our ability to
introduce branched ethers, but we were able to introduce the
methoxy methyl ether (5), ethoxy methyl ether (6), and methoxy
yielding the 6-substituted penicillanate ester
3 under mild
conditions.26 After tert-butyldimethylsilyl protection of the

D. A. Harris et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3787–3790
3789
Table 1
glycine, alanine, valine, or leucine was coupled with 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and
catalytic dimethyl amino pyridine (DMAP). In each case, we found
that the modifications did not bestow the penem with activity
against E. coli or S. aureus and actually eliminated activity against
S. epidermidis (MICs > 200 lg/mL) (Table 1). Thus, at least when
linked via an ester, these amino acids do not appear to mimic the
constituents of a natural leader sequence substrate.
We reasoned that poor recognition may be the result of the
penem scaffold mispositioning the peptidic side chain or hetero-
atom functionality of 8–11 in the SPase binding site, relative to
the natural substrates. To begin to explore this possibility, we
examined a series of simple alkyl esters (12–16, Scheme 2). The
esters were synthesized by triethylamine treatment of the penem
followed by the appropriate acetic anhydride or acid chloride and
a catalytic quantity of DMAP.11 Again, none of the ester substitu-
ents conferred activity against E. coli or S. aureus. However, they
all restored activity against S. epidermidis, and the isopropyl ester
15 was as active as the parent penem (Table 1). This data suggests
that while simple ester derivatization reduces SPase binding,
appropriate derivatization of the alkyl group can restore affinity,
presumably by accessing the S1 and/or S3 pockets.
MICs of penem inhibitors 1–18a
Compound E. coli+PMBn (
#
l
g/ S. aureus (
l
g/
MRSA (
mL)
lg/
S. epidermidis
(lg/mL)
mL)
mL)
1
5
6
7
8
9
10
11
12
13
14
15
16
17
18
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
100
50
50
50
>200
>200
200
100
>200
>200
>200
>200
100
100
100
50
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
100
100
100
100
a
MICs were determined using compounds that were serially diluted 2-fold in
cation-adjusted Mueller–Hinton broth to a total volume of 100 L in 96-well plates.
Bacteria were grown to 108 cfu/mL, and 5
L of a 10-fold dilution was used to
l
l
inoculate 100 lL of penem-containing media to a final concentration of
5 Â 105 cells/mL. Microplates were incubated at 37 °C for 18 h with MICs defined as
the lowest concentration necessary to inhibit visible growth.
To examine the reintroduction of peptide-like functionality into
the derivatives, we synthesized and examined two carbamates (17
and 18, Scheme 2),11 which restore a nitrogen atom, but in a posi-
tion shifted relative to the amino acid derivatives 8–11. Neither the
ethyl nor the isopropyl carbamate derivatization resulted in
detectable activity against E. coli or S. aureus 8325, and both
slightly reduced activity against S. epidermidis, but surprisingly
both conferred the penem with modest activity against MRSA
ethoxy ether (7) substituents via diisopropylethylamine (DIEA)
treatment followed by the corresponding chloride (Scheme 2).11
These derivatizations did not confer activity against E. coli or S. aur-
eus, and had little to no effect on the sensitivity of S. epidermidis
(Table 1).
We next explored the attachment of different amino acids via
their C-terminus to the hydroxyethyl moiety of the penem core
(8–11, Scheme 2). We hypothesized that these compounds might
mimic a natural leader peptidase substrate. Acetyl protected
(MIC = 100 lg/mL) (Table 1).
We have demonstrated that while the penem derivatives
synthesized have no activity against the Gram-negative pathogen
Scheme 2. Reagents and conditions: (i) R-Cl (4.0 equiv), DIEA (4.0 equiv), CH2Cl2, rt, (80%); (ii) COCH3NH-RCH-COOH (4.0 equiv), DMAP (1.0 equiv), EDC (8.0 equiv), CH2Cl2,
À15 °C, (40%); (iii) for compound 12: Et3N (2.0 equiv), Ac2O (2.5 equiv), DMAP (1.0 equiv), CH2Cl2, 0 °C, (90%); for compounds 13–16: Et3N (5.0 equiv), R-Cl (5.0 equiv), DMAP
(1.0 equiv), rt, (75%); (iv) R-isocyanate (50.0 equiv), DMAP (1.0 equiv), rt (50%).

3790
D. A. Harris et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3787–3790
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E. coli, even with a permeabilized outer membrane, they do gener-
ally have antibiotic activity against the important human pathogen
S. epidermidis. S. epidermidis is a very common nocosomial patho-
gen and the major cause of indwelling device infection.30,31 Inter-
estingly, the penem’s activity is only ꢀ50-fold less than that of
antibacterials currently used to treat S. epidermidis infections.32
Importantly, the carbamate derivatives also resulted in activity
against MRSA. One interpretation of the structure–activity rela-
tionship data is that the ester linkage employed mispositions the
amino acid side chains or H-bonding functionality relative to the
SPase binding site, and that the carbamate nitrogen, which is
shifted by one bond, is better positioned to favorably engage the
enzyme. This suggests that amino acids attached via different link-
ers might be better mimics of the natural substrate and might
increase penem activity.
The structure–activity relationship data also reveal interesting
differences among the SPases from the different organisms. The
unique activity against S. epidermidis might result from unique
aspects of the pathogen’s SPase or from a unique aspect of its biol-
ogy, perhaps being more sensitive to SPase inhibition. Also, not
only is there variation in the SPases from Gram-negative and
Gram-positive pathogens, but the cell wall changes associated with
methicilin resistance33,34 appear to confer S. aureus with sensitiv-
ity, at least to the carbamate-derivatized penems. This latter effect
may result from increased or altered protein secretion in MRSA
relative to methicillin sensitive S. aureus. Thus, the characterization
of additional penem derivatives should help further define both
SPase biochemistry and possibly protein secretion in general.
Increasingly, there is a pressing need for antibiotics with novel
mechanisms of action, and in this regard penem inhibitors of SPase
appear attractive. The demonstration that a derivative of the
penem possesses at least modest in vivo antibacterial activity
against two important human pathogens suggests that with fur-
ther optimization more potent and broad spectrum activity may
be achievable.
Acknowledgments
30. Catheter-Related Infections; Seifert, H., Jansen, B., Farr, B. M., Eds.; Marcel
Dekker: New York, 1997.
31. Infections Associated with Indwelling Medical Devices; Waldvogel, F. A., Bisno, A.
L., Eds.; American Society for Microbiology: Washington, DC, 2000.
32. John, M. A.; Pletch, C.; Hussain, Z. J. Antimicrob. Chemother. 2002, 50, 933.
33. McAleese, F.; Wu, S. W.; Sieradzki, K.; Dunman, P.; Murphy, E.; Projan, S.;
Tomasz, A. J. Bacteriol. 2006, 188, 1120.
We acknowledge the Office of Naval Research (N00014-08-1-
0478 and N00014-03-1-0126) and Achaogen Inc. for funding.
References and notes
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