Modifications of the C6-substituent of penicillin sulfones with the goal of improving inhibitor recognition and efficacy

Article abstract of DOI:10.1016/j.bmcl.2010.10.134

In order to evaluate the importance of a hydrogen-bond donating substituent in the design of β-lactamase inhibitors, a series of C6-substituted penicillin sulfones, lacking a C2′ substituent, and having an sp ³ hybridized C6, was prepared and evaluated against a representative classes A and C β-lactamases. It was found that a C6 hydrogen-bond donor is necessary for good inhibitory activity, but that this feature alone is not sufficient in this series of C6β-substituted penicillin sulfones. Other factors which may impact the potency of the inhibitor include the steric bulk of the C6 substituent (e.g., methicillin sulfone) which may hinder recognition in the class A β-lactamases, and also high similarity to the natural substrates (e.g., penicillin G sulfone) which may render the prospective inhibitor a good substrate of both classes of enzyme. The best inhibitors had non-directional hydrogen-bonding substituents, such as hydroxymethyl, which may allow sufficient conformational flexibility of the acyl-enzyme for abstraction of the C6 proton by E166 (class A), thus promoting isomerization to the β-aminoacrylate as a stabilized acyl-enzyme.

Full text of DOI:10.1016/j.bmcl.2010.10.134

Bioorganic & Medicinal Chemistry Letters 21 (2011) 387–393
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Modifications of the C6-substituent of penicillin sulfones with the goal
of improving inhibitor recognition and efficacy
Micheal Nottingham a, Christopher R. Bethel b, Sundar Ram Reddy Pagadala a, Emily Harry a, Abishai Pinto a,
Zachary A. Lemons a, Sarah M. Drawz c, Focco van den Akker d, Paul R. Carey d, Robert A. Bonomo b,
John D. Buynak a,
⇑
a Department of Chemistry, Southern Methodist University, Dallas, TX 75275-0314, USA
b Research Service, Louis Stokes Veterans Affairs Medical Center, Cleveland, OH 44106, USA
c Department of Pathology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA
d Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to evaluate the importance of a hydrogen-bond donating substituent in the design of b-lactamase
inhibitors, a series of C6-substituted penicillin sulfones, lacking a C20 substituent, and having an sp3
hybridized C6, was prepared and evaluated against a representative classes A and C b-lactamases. It
was found that a C6 hydrogen-bond donor is necessary for good inhibitory activity, but that this feature
alone is not sufficient in this series of C6b-substituted penicillin sulfones. Other factors which may impact
the potency of the inhibitor include the steric bulk of the C6 substituent (e.g., methicillin sulfone) which
may hinder recognition in the class A b-lactamases, and also high similarity to the natural substrates (e.g.,
penicillin G sulfone) which may render the prospective inhibitor a good substrate of both classes of
enzyme. The best inhibitors had non-directional hydrogen-bonding substituents, such as hydroxymethyl,
which may allow sufficient conformational flexibility of the acyl-enzyme for abstraction of the C6 proton
by E166 (class A), thus promoting isomerization to the b-aminoacrylate as a stabilized acyl-enzyme.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 11 September 2010
Revised 26 October 2010
Accepted 27 October 2010
Available online 5 November 2010
Keywords:
b-Lactamase
Inhibitor
Antibiotic
Penicillin
The past two decades have witnessed an exponential growth in
the number of b-lactamases, now at more than 850. b-Lactamases
are grouped into classes A–D and many of the newly reported
enzymes have substantially broadened hydrolytic spectrum,
including the appearance and dissemination of extended-spectrum
b-lactamases (ESBLs)1–4 and classes A and D carbapenemases5–7
R'CONH
S
Antibiotic
On
S
O
HN
R
O
PBP 2X
O
R
OSer130
-
H
OSer130
SO2
CO2
R
Ser337
3
N
-
HOSer130
O
-
H2N+
O
Ser70
R
O
CO2
O
H
O
O2
H
R
6
R
1 Antibiotic: R = acylamido
-
-
S
Ser70
SO2
CO2
n = 0
Sulbactam
(R = H)
-
HN+
SO2
O
O
HN
2 Sulbactam: R = H
HN
O
n = 2
R
-
-
-
CO2
CO2
TEM-1
SO2
Ser70
Ser70
- H+
-
CO2
O
HN
O
4
5
O
Ser70
O
Ser70
-
CO2
7a
7b
Scheme 1. Interactions of a b-lactam antibiotic (1) or a b-lactamase inhibitor (2) with either a penicillin-binding protein (top) or a b-lactamase.
⇑
Corresponding author.
E-mail address: jbuynak@smu.edu (J.D. Buynak).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.134

388
M. Nottingham et al. / Bioorg. Med. Chem. Lett. 21 (2011) 387–393
O
R
R
N
S
S
S
O
OH
H
O
SER70
N
N
N
N
H
H
O
O
O
O
PhCH2
N
O
-
+
+
O
O
NH3
NH3
O
-
-
O
H
H
H
N
O
O
H
SER403
O
O
N H
N
O
+
+
H
H2N
H2N
NH2
NH2
O
H
N
O
O
O
ALA237
ALA237
LYS234
LYS234
NH
NH
SER598
N
H
N
THR600
O
ARG244
ARG244
A
B
Figure 1. (A) Computer-assisted docking of penam substrate in TEM-1 b-lactamase (L shows H-bonding in oxyanion hole and R shows H-bonding of side C6 acylamido side
chain); (B) Acyl-enzyme of S. aureus PBP 2a with Penicillin G (code 1MWT).
YH
administration of antibiotic/b-lactamase inhibitor combinations
remains a highly effective mechanism for overcoming b-lacta-
mase-mediated resistance,8 understanding the factors leading to
optimal inhibitory efficacy is of utmost importance.
On
S
N
O
CO2Na
The b-lactamases are postulated to have evolved from the
penicillin-binding proteins (PBPs), the transpeptidase targets of
b-lactam antibiotics,9 with the mechanistic difference that, while
PBPs are inactivated by the antibiotics through formation of a
stable acyl-enzyme, b-lactamases have evolved the ability to
hydrolyze the corresponding covalent intermediate10 and have
also lost the ability to recognize peptidoglycan.11 In general,
b-lactamase inhibitors are designed to resemble the antibiotics,
with minor structural modifications, such as the oxidation of the
(penam) thiazolidine sulfide to the sulfone, and the omission of
8
Y = O, n = 0
Y = O, n = 2
Y = S, n = 2
9
10
Figure 2. Inhibitors of b-lactamase possessing either a C6-hydroxymethyl or a
C6-mercaptomethyl group.
Since b-lactam antibiotics continue to play a dominant role as first-
line agents in the treatment of bacterial infections, and since the
H
O2
O2
N
H2N
S
F3CO2S
S
c
N
N
O
O
CO2CHPh2
CO2R'
13
14
= R' = CHPh2
= R' = Na
a,b
12
d,e
16 = R = CH2Ph
17 = R =
H2N
O
O2
S
H
N
H3N+
O
R
O2
S
g
f
S
N
OMe
O
N
N
O
11 CO2H
-
CO2
CO2Na
15
OMe
h
O
O
Ph
N
N
H
H
N
O2
S
N
O
O
N
S
i
Ph
Ph
O
O
Cl
N
N
18
O
O
19
CO2H
2Na
20
O
O
BnO
NaO
O2
O2
S
H2N
O
HN
O
O2
S
S
HN
j
k
l
15
N
N
N
O
CO2Bn
CO2Bn
CO2Na
23
22
21
Scheme 2. Reagents and conditions:: (a) Ph2CN2, acetone, 0 °C to rt, 20 h; (b) 3 equiv mCPBA, DCM, rt, 1 h; (c) Tf2O, TEA, 0 °C, 30 min; (d) anisole, CF3CO2H, ꢀ10 °C, 15 min; (e)
NaHCO3, EtOAc/H2O, rt, 15 min; (f) KMnO4, H2SO4, H2O/MeCN, ꢀ10 °C, pH 3.2, 2 h; (g) (i) RCOCl, TEA, DCM, rt, 30 min; (ii) HCl; (iii) NaHCO3; (h) (i) TEA, DCM, rt, 30 min; (ii) aq
HCl; (i) (i) KMnO4, H3PO4, H2O, rt, 2 h; (ii) NaHCO3; (j) PhCHN2, acetone, rt, 30 min; (k) benzyl bromoacetate, DIPEA, DMF, 50 °C, 5 h; (l) (i) Pd/C, H2, EtOH/EtOAc (4:1), 4 h, 40
psi; (ii) EtOAc/H2O, NaHCO3.

M. Nottingham et al. / Bioorg. Med. Chem. Lett. 21 (2011) 387–393
389
the acylamido side chains found at C6 in the antibiotics. Inhibitors
are recognized in a similar fashion to the substrate b-lactam anti-
biotics themselves, and such structural similarity may advanta-
geously render it difficult for the microorganism to evolve
inhibitor resistance while maintaining substrate recognition.
Mechanistically, b-lactam antibiotics and the b-lactamase inhibi-
tors react with their respective enzymes to generate stabilized
acyl-enzymes of differing structure. The penicillin sulfones, such
as the commercial b-lactamase inhibitors, sulbactam and tazobac-
tam, inactivate serine b-lactamases by the mechanism shown in
Scheme 1 (bottom), leading to either the ‘transiently inhibited’
b-aminoacrylates 7a and 7b, or to the doubly covalently linked
intermediates 6, with both 6 and 7 having been observed crystallo-
graphically on the (soluble) b-lactamases. While crystallographic
data on the membrane PBP transpeptidases has accumulated more
slowly,12 it is now established that the b-lactam antibiotics them-
selves, having sulfur in the sulfide oxidation state, maintain an
unfragmented thiazolidine (or dihydrothiazole, in case of cephems)
in the stabilized acyl-enzyme (3), as shown in Scheme 1 (top).13
The respective active sites of the PBPs and the serine
b-lactamases have similar recognition elements, as shown in Fig-
ure 1, including a positively charged pocket to recognize the C3
carboxylate, an oxyanion hole (electrophilic center) to facilitate
addition of serine to the b-lactam, and a suitably situated backbone
carbonyl to serve as a hydrogen-bond acceptor for the C6 acylam-
ido N–H (circled). While commercial inhibitors lack a hydrogen-
bond donating C6 substituent, it is known that compounds which
have an H-bond donor suitably positioned, such as the 6-hydrox-
ymethylpenicillin sulfones (9, Fig. 2),14–16 and the corresponding
mercaptomethylpenicillin sulfones (10),17 are recognized as potent
b-lactamase inactivators. The analogous 6-(hydroxymethyl)peni-
cillin sulfides (8), while less potent b-lactamase inhibitors,15,17
are valuable mechanistic probes which can define the direction
of approach of the active site water to the acyl-enzyme.18,19 While
covalent complexes of these 6-(hydroxymethyl)penicillin sulfides
with representative b-lactamases are available,20,21 corresponding
structural data is unfortunately not available on the more potent
6-(hydroxymethyl)penicillin sulfones, which will undoubtedly
exhibit a mechanistically dissimilar course of transformations
subsequent to acylation. Although progress was recently made in
understanding the recognition of the antibiotic substrates by the
PBPs,22 it is also not clear why, given their propensity to form
stable acyl-enzymes of the b-lactamases, most b-lactamase inhibi-
tors exhibit little or no binding to essential PBP transpeptidases,23
with the notable exception of the binding of PBP 2 of Acinetobacter
baumannii by sulbactam.24,25
In view of these factors, especially including the evolution of
new enzymes and the lack of data of C6-substituted penicillin
O
OH
O
OH
O
N
O
O
a
N
b
O
N
OH
24
O
OH
O
26
25
O
S
O
O
OH
Ar
Ph
O
e
c
d
O
O
N
H
O
N
H
O
Ph
=
Ar
H
Ar
Ar
Ar
29
30
27
28
Br
Br
O2
S
Br
Br
Br
S
Br
f
g
h
N
N
11
N
O
O
O
CO2H
CO2CHPh2
CO2CHPh2
O
33
32
31
N
O
a
b
R =
R =
HO
Br
HO
O2
S
HO
O2
S
O2
S
H3N
i
k
j
R
O
R
33
O N
N
N
N
(for 33b)
H
O
O
CO2CHPh2
CO2R'
35a,c R' = CHPh2
CO2
34
37
c
R = H
l
k
36a,c R' = Na
O
HO
O2
O2
O2
S
O2
Br
O
S
S
S
j
n
e
N
N
O
N
N
O
O
CO2CHPh2
CO2CHPh2
CO2CHPh2
k
CO2CHPh2
38
42
44
39
m,k
39 R' = CHPh2
40 R' = Na
k
k
HO
HO
HO
O2
S
O2
S
O2
S
HO
N
O
N
N
O
O
CO2Na
CO2Na
CO2Na
41
43
45
Scheme 3. Reagents and conditions: (a) pyrrolidine, 70 °C, 18 h; (b) HIO4, Et2O, 0 °C, 4 h; (c) PhOCOCl, pyr, DCM, 0 °C, 1 h; (d) allylamine, CHCl3, 50 °C, 12 h; (e) (i) O3, DCM,
ꢀ78 °C, 30 min; (ii) Me2S, DCM, ꢀ78 °C to rt, 2 h; (f) Ref. 27; (g) Ph2CN2, acetone, 0 °C to rt; (h) mCPBA, DCM, rt, 1 h; (i) (i) MeMgBr, THF, ꢀ78 °C, 30 min; (ii) RCHO (26, 30, or
CH2O), THF, ꢀ78 °C to rt; 2 h; (j) n-Bu3SnH, cat. AIBN, tol, reflux, 1 h; (k) (i) anisole, CF3CO2H, ꢀ10 °C, 15 min; (ii) NaHCO3, EtOAc/H2O, rt, 15 min; (l) allyltributyltin, cat. AIBN,
tol, reflux, 1 h; (m) (i) 9-BBN, THF, rt, 1 h; (ii) H2O2, NaOAc, MeOH; (n) NaBH3CN, HOAc–THF, rt, 30 min.

390
M. Nottingham et al. / Bioorg. Med. Chem. Lett. 21 (2011) 387–393
sulfones on these new serine b-lactamases, we proceeded to sys-
tematically study the inhibitory activity of a series of structurally
modified penicillin sulfones (i.e., penicillin sulfones possessing an
sp3-hybridized carbon at C6 and lacking a C20 substituent) with
representative classes A and C b-lactamases, and to compare
them with the prototypical C20-unsubstituted penicillin sulfone,
sulbactam. All compounds possess C6b geometry, analogous to the
natural penicillin series.
As shown in Scheme 2, commercial 6-aminopenicillanic acid
(6-APA, 11) was esterified and oxidized to produce sulfone 12,
which was sulfonylated and deprotected to produce inhibitor 14.
Depending on the bulkiness of the acylamido side chain the 6-acy-
lamido inhibitors were prepared either by an oxidation-acylation
or an acylation-oxidation sequence. Thus 11 was oxidized to the
corresponding sulfone with potassium permanganate,26 and sub-
sequently acylated to produce inhibitors 16 and 17. The more
bulky 20, however, was prepared in the converse fashion as shown.
Inhibitor 23 was generated from the corresponding benzyl ester as
shown.
6b-(Hydroxyalkyl)penicillanic acids were prepared as shown in
Scheme 3. In preparation for linking to the penicillin, 6-APA was
converted to 6,6-dibromopenicillanic acid (31) by the method of
Volkmann,27 esterified, and oxidized to sulfone 33 as shown. 33
was metallated and reacted with aldehydes to produce bromoalco-
hols 34, which were reduced and deprotected to produce inhibitors
36a, 36c, and 37. Dibromide 33 was reacted with allyltributylst-
annane in the presence of a catalytic amount of AIBN to produce
38 as a mixture of epimers. These were reduced with tributyltin
O
O
O
O
a
b
c
O
O
O
O
HN
HN
NH2
Cl
OH
OAc
46
47
48
+
NH2
NH3
NHAlloc
NHAlloc
O2
S
O2
O2
O2
S
S
f
g
S
e
d
Br
33
N
N
N
N
O
O
O
O
-
CO2CHPh2
CO2CHPh2
CO2
CO2R
52
53
49
50 R = CHPh2
51 R = Na
g
O
O2
S
h
54
55
R = CHPh2
R = Na
N
H
H
g
g
N
O
CO2R
O2
O
H2N
S
O2
S
N
R'
56a,b
57a,b
N
R = CHPh2
R = Na
i
j
H
O
CO2CHPh2
N
O
CO2R
a
b
R' = (CH3)C
R' = PhCH2
52
O2
S
CF3SO2NH
58
59
R = CHPh2
R = Na
g
N
O
CO2R
Scheme 4. Reagents and conditions: (a) aq NH4OH, THF, 0 °C, 1 h; (b) Na2CO3, paraformaldehyde, H2O, 24 h; (c) pyr, Ac2O, 0 °C, 18 h; (d) (i) MeMgBr, THF, ꢀ78 °C, 30 min; (ii)
48, THF, ꢀ78 °C, 2 h; (e) nBu3SnH, cat AIBN, tol, reflux; 1 h; (f) n-Bu3SnH, PPh3, cat. Pd(PPh3)4, DCM/H2O, rt, 90 min; (g) (i) TFA, anisole, ꢀ10 °C, 1 h; (ii) NaHCO3, EtOAc/H2O, rt,
15 min; (h) AcOCHO, pyr, DCM, 0 °C, 15 min; (i) RCOCl, pyr, DCM, 0 °C, 15 min; (j) Tf2O, pyr, THF, ꢀ78 °C, 20 min.
H
TBSOHN
O2
S
HO
O2
S
N
O2
S
HO
c
b
O
a
O
O
42
N
N
N
O
O
O
CO2CHPh2
CO2CHPh2
CO2Na
60
61
62
c
O2
S
NaO2C
N
O
CO2Na
63
Scheme 5. Reagents and conditions: (a) NaClO2, NaH2PO4, 2-methyl-2-butene, tBuOH/THF/H2O, 0 °C, 1 h; (b) (i) EtOCOCl, TEA, THF, 0 °C, 1 h; (ii) TBSONH2, DCM, 0 °C, 30 min;
(c) (i) TFA, anisole, ꢀ10 °C, 1 h; (ii) NaHCO3, EtOAc/H2O, rt, 15 min.

M. Nottingham et al. / Bioorg. Med. Chem. Lett. 21 (2011) 387–393
391
Table 1
hydride to stereoselectively generate the 6b-allylpenicillin sulfone
39 by addition of the hydrogen atom to the intermediate radical
from the less-hindered -face. Compound 39 was deprotected to
produce inhibitor 40 and also reacted with 9-BBN, followed by
hydrogen peroxide, and deprotected to produce 41, or alterna-
tively, subjected to ozonolysis and deprotected to produce the
aldehyde hydrate 43, as shown. Intermediate aldehyde 42 was re-
duced to the corresponding alcohol 44 and deprotected to produce
the 6b-(hydroxyethyl)penicillin sulfone 45.
The 6b-(aminomethyl)penicillanic acid sulfones were prepared
as shown in Scheme 4. Dibromide 33 was aminomethylated using
the alloc-protected N,O-acetal 48 (prepared from allycarbamate
46, as shown) and stereoselectively reduced to produce 50. This
acylamidomethylpenicillinate was deprotected to produce inhibi-
tor 51. The allyloxycarbonyl protecting group was removed to pro-
duce the parent 6b-aminomethylpenicillin sulfone 52, which was
further deprotected to produce inhibitor 53 as shown. Then 52
was reacted with formic acetic anhydride, pivaloyl chloride, phen-
ylacetyl chloride, and triflic anhydride, to produce, after subse-
quent depotection, inhibitors 55, 57a, 57b, and 59, respectively.
Lastly, the hydroxamate 62 was prepared, through oxidation of
aldehyde 42 to generate acid 60, (which was also deprotected to
produce diacid 63) followed by coupling with O-TBS hydroxyl-
amine and deprotection, as shown in Scheme 5.
Inhibitory activity against TEM-1 (class A) and PDC-3 (extended spectrum class C28
)
b-lactamase (IC50
,
l
M, 10%)29
a
Inhibitor
-R
TEM-1
PDC-3
Sulbactam
14
15
16
17
20
23
36a
36c
37
40
41
43
45
51
53
55
–H
–NHSO2CF3
—NH3
–NHCOCH2Ph
–NHCO(2,6-C6H3(OCH3)2)
–NHCO(5-Me-3-Ph-(1,2-C3NO)
–NHCH2CO2Na
–CH(OH)CO(NC4H8)
–CH2OH
1.3
>50
8.4
>50
>50
>50
>50
0.18
0.012
1.2
4.3
7.0
þ
>50
>50
1.1
1.7
13.8
0.16
0.18
1.2
1.2
30
>50
43
45
>50
10
þ
—CHðOHÞCH2NH3
–CH2CH@CH2
35
–CH2CH2CH2OH
–CH2CH(OH)2
–CH2CH2OH
–CH2NHCO2CH2CH@CH2
—CH2NH3
>50
>50
>50
>50
0.45
9.9
>50
>50
6.1
þ
–CH2NHCHO
57a
57b
59
62
63
–CH2COBut
1.7
4.2
1.8
>50
12.7
–CH2COCH2Ph
–CH2NHSO2CF3
–CH2CONHOH
–CH2CO2Na
>50
>50
As seen in Table 1 and Figure 3, the most active inhibitors in this
series have an hydroxymethyl group at C6, including inhibitors 36a
and 36c, which result in improvements in inhibitory activity
against both the representative class A and the class C b-lactamase,
relative to sulbactam. Related compounds 37 and 53, which also
have hydrogen bonding potential at the C60 position, are also
capable of maintaining inhibitory activity of the class A TEM-1 en-
zyme. Surprisingly, the inhibitors most similar to the natural anti-
biotics and possessing C6 substituents able to mimic their
hydrogen bond-donating capability through a directly attached
C6 acylamino group (e.g., 16, 17, 20, and 14) have little activity
as inhibitors of the class A enzyme.
O2
R
S
N
O
CO2Na
Figure 3. General inhibitor structure for Table 1.
k1
k2
k3
EC
E +
P
E +
C
EC*
k-1
k4
Like other serine hydrolases (albeit lacking the usual catalytic
triad), classes A and C b-lactamases function through a three-step
process involving formation of the Michaelis complex (EC) of the
enzyme (E) with the antibiotic/inhibitor (C), acylation of the active
k5
E + P'
EC**
Scheme 6. Minimal kinetic scheme for interaction of a b-lactamase (E) with a
substrate and/or inhibitor (C).
*
site serine to form the acyl-enzyme (EC ), and deacylation, as
thus leading to poor recognition (high Km) for branched
inhibitors (Fig. 4). This same bulkiness possibly leads to a
longer lifetime of the acyl-enzyme in the class C b-lactamase
(e.g., low activity of 17 and 20 against the class A enzyme,
but improved inhibition of class C).
shown in Scheme 6. After acylating the active site serine, inhibitors
partition, via subsequent fragmentation of the dioxothiazolidinone
(Scheme 1 and 4?5) to produce a more stabilized acyl-enzyme
**
(Scheme 6, EC ; Scheme 1, 6 or 7) with a dramatically reduced rate
of hydrolysis (k5). Classes A and C b-lactamase differ kinetically.
Class A enzymes are less able to recognize (higher Km) sterically
bulky b-lactams (such as third generation cephalosporins and
methicillin30), but possess enhanced deacylation capability due to
the presence of Glu166, which activates the hydrolytic water.31
Class C b-lactamases, while having a broader recognition of sub-
strates (lower Km) and rapid acylation (k2), have reduced deacyla-
tion rates (k3),32,33 and thus the kcat for many class C enzymes is
equal to the rate of this final step of the hydrolytic process.10
As a result of these arguments, we advance the following
hypotheses:
(3) Inhibitors which closely resemble good substrates (e.g., 16)
have little potency. This is probably due to their functioning
as substrates of both enzymes, and thus having a large par-
tition ratio (k3/k4, i.e., kcat/kinact).34
(4) Inhibitors having a non-directional C60 H-bond donating
group (OH) (e.g., 36a, 36c, and 37) are the best overall inhib-
itors. In these inhibitors, the side chain CH2O–H group may
be improving recognition (lowering Km), and also lowering
the partition ration (k3/k4) by providing sufficient conforma-
tional freedom in the acyl-enzyme to allow the catalytic
base (E166, in the case of Class A) to abstract a proton
directly from C6, leading to formation of the b-aminoacry-
(1) In this series of inhibitors, an H-bond donor, at either C6, or
at the adjacent carbon (C60) is needed for activity at either
the class A or the class C b-lactamase (e.g., low activity of
41, 43, 45, 62, and 63 against both enzymes).
**
late, (EC , 7) rather than abstracting a proton from the
hydrolytic water (to form E and P) as shown in Scheme 7.35
(2) Branched, bulky side chains, combined with a C6 H-bond
donor, lower activity against the class A b-lactamase, but
improve activity against class C. This is probably due to
the smaller, more well-defined active site of the class A
enzyme as a consequence of the presence of the omega loop
In conclusion, it is clear that inhibitor design in the penicillin
sulfone series involves achieving a balance between inhibitor rec-
ognition and the hydrolytic stability of the resultant acyl-enzyme.
One needs sufficient similarity to the natural substrate to ensure
that the inhibitor is recognized and can efficiently acylate the

392
M. Nottingham et al. / Bioorg. Med. Chem. Lett. 21 (2011) 387–393
Figure 4. Computer-assisted docking of methicillin sulfone (17) in the active sites of a representative class A (TEM-1, 1ZG4, left) and class C (P99, 1XX2) b-lactamase.
Electrostatic potential is mapped onto the surface of the enzyme, with blue being most positive and red most negative.
E166
E166
O
O
H
O
O
H
H
O
H
O2
S
H
-
O
O
SO2
k4
E +
P
k3
EC**, 7
H
HN+
O
HN
(hydrolysis and
(inhibition)
O
reactivation of enzyme)
O
O
O
O
-
O
O
O
H
N
H
N
SER70
-
O
O
SER70
O
ALA237
4
EC*,
5
ALA237
Scheme 7. Proposed interactions of the acyl-enzyme derived from inhibitor 36c with the class A, TEM-1 b-lactamase, leading either to turnover (left) or inhibition.
6. Poirel, L.; Naas, T.; Nordmann, P. Antimicrob. Agents Chemother. 2010, 54, 24.
7. Queenan, A. M.; Bush, K. Clin. Microbiol. Rev. 2007, 20, 440.
8. Perez-Llarena, F. J.; Bou, G. Curr. Med. Chem. 2009, 16, 3740.
enzyme, but excessive resemblance to natural substrates may re-
sult in poor activity due to turnover (i.e., high partition ratio). Sub-
stituents which, through their placement and interactions with the
enzyme, facilitate the formation of the b-aminoacrylate and/or en-
hance its stability, improve inhibitory potency. The evolution of the
b-lactamase active site necessitates the redesign of both b-lactam
antibiotics and b-lactamase inhibitors to counter new modifica-
tions of the enzymes. This work, which systematically explores
inhibitory activity of a series of penicillin sulfones having an sp3
hybridized C6 substituent, demonstrates that recognition may be
an important factor, with both the conformational freedom and
the hydrogen-bond donating capability of the side chain at C6
being essential factors in attaining optimal activity.
9. Massova, I.; Mobashery, S. Antimicrob. Agents Chemother. 1998, 42, 1.
10. Pratt, R. F. J. Chem. Soc., Perkin Trans. 2 2002, 851.
11. Meroueh, S. O.; Minasov, G.; Lee, W.; Shoichet, B. K.; Mobashery, S. J. Am. Chem.
Soc. 2003, 125, 9612.
12. Sauvage, E.; Kerff, F.; Terrak, M.; Ayala, J. A.; Charlier, P. FEMS Microbiol. Rev.
2008, 32, 234.
13. Gordon, E.; Mouz, N.; Duee, E.; Dideberg, O. J. Mol. Biol. 2000, 299, 477.
14. Kellogg, M. S. Ger. Pat. DE 3039504 19810430, Chem. Abstr. 1981, 95, 480946.
15. Bitha, P.; Li, Z.; Francisco, G. D.; Rasmussen, B. A.; Lin, Y.-I. Bioorg. Med. Chem.
Lett. 1999, 9, 991.
16. Bitha, P.; Li, Z.; Francisco, G. D.; Yang, Y.; Petersen, P. J.; Lenoy, E.; Lin, Y.-I.
Bioorg. Med. Chem. Lett. 1999, 9, 997.
17. Buynak, J. D.; Chen, H.; Vogeti, L.; Gadhachanda, V. R.; Buchanan, C. A.; Palzkill,
T.; Shaw, R. W.; Spencer, J.; Walsh, T. R. Bioorg. Med. Chem. Lett. 2004, 14, 1299.
18. Golemi, D.; Maveyraud, L.; Ishiwata, A.; Tranier, S.; Miyashita, K.; Nagase, T.;
Massova, I.; Mourey, L.; Samama, J. P.; Mobashery, S. J. Antibiot. 2000, 53, 1022.
19. Testero, S. A.; O’Daniel, P. I.; Shi, Q.; Lee, M.; Hesek, D.; Ishiwata, A.; Noll, B. C.;
Mobashery, S. Org. Lett. 2009, 11, 2515.
Acknowledgments
20. Maveyraud, L.; Massova, I.; Birck, C.; Miyashita, K.; Samama, J.-P.; Mobashery,
S. J. Am. Chem. Soc. 1996, 118, 7435.
21. Mourey, L.; Miyashita, K.; Swaren, P.; Bulychev, A.; Samama, J.-P.; Mobashery,
S. J. Am. Chem. Soc. 1998, 120, 9382.
22. Pratt, R. F. Cell. Mol. Life Sci. 2008, 65, 2138.
23. Moosdeen, F.; Williams, J. D.; Yamabe, S. Antimicrob. Agents Chemother. 1988,
32, 925.
J.D.B. was supported by the Robert A. Welch Foundation (Grant
N-0871). R.A.B. was supported by the Veterans Affairs Merit Re-
view Program, GRECC, and the NIH (R01 AI063517-01). F.v.d.A.
was supported by NIH (R01 AI062968). P.R.C. was supported by
NIH (R01 GM54072).
24. Urban, C.; Go, E.; Mariano, N.; Berger, B. J.; Avraham, I.; Rubin, D.; Rahal, J. J. J.
Infect. Dis. 1993, 167, 448.
25. Urban, C.; Go, E.; Mariano, N.; Rahal, J. J. FEMS Microbiol. Lett. 1995, 125, 193.
26. Akkerboom, P. J.; Oldenhof, C. US patent 4,422,971. Chem. Abstr. 1981, 95,
587244.
27. Volkmann, R. A.; Carroll, R. D.; Drolet, R. B.; Elliott, M. L.; Moore, B. S. J. Org.
Chem. 1982, 47, 3344.
28. Rodriguez-Martinez, J.-M.; Poirel, L.; Nordmann, P. Antimicrob. Agents
Chemother. 2009, 53, 1766.
References and notes
1. Perez, F.; Endimiani, A.; Hujer, K. M.; Bonomo, R. A. Curr. Opin. Pharmacol. 2007,
7, 459.
2. Rossolini, G. M.; D’Andrea, M. M.; Mugnaioli, C. Clin. Microbiol. Infect. 2008(14),
33.
3. Bush, K. Clin. Microbiol. Infect. 2008(14), 134.
4. Rodriguez-Bano, J.; Pascual, A. Expert Rev. Anti Infect. Ther. 2008, 6, 671.
5. Poirel, L.; Pitout, J. D.; Nordmann, P. Future Microbiol. 2007, 2, 501.
29. IC50 assays were performed in 10 mM Tris (pH 7.7), 90 mM NaCl for TEM-1 and
10 mM phosphate-buffered saline (pH 7.4) for PDC-3. Enzymes ([E] = 4 nM)

M. Nottingham et al. / Bioorg. Med. Chem. Lett. 21 (2011) 387–393
393
were pre-incubated with inhibitor for 5 min prior to the addition of nitrocefin
[NCF] = 100 M. The error range in each measured IC50 value is 10%.
32. Galleni, M.; Frère, J.-M. Biochem. J. 1988, 255, 119.
33. Galleni, M.; Amicosante, G.; Frère, J.-M. Biochem. J. 1988, 255, 123.
l
30. Batchelor, F. R.; Cameron-Wood, J.; Chain, E. B.; Rolinson, G. N. Proc. R. Soc.
London 1963, 158, 311.
34. Fisher, J.; Charnas, R. L.; Bradley, S. M.; Knowles, J. R. Biochemistry 1981, 20,
2726.
31. Lobkovsky, E.; Moews, P. C.; Liu, H.; Zhag, H.; Frère, J.-M.; Knox, J. R. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 11257.
35. Kalp, M.; Buynak, J. D.; Carey, P. R. Biochemistry 2009, 48, 10196.