4-Substituted trinems as broad spectrum β-lactamase inhibitors: Structure-based design, synthesis, and biological activity

Article abstract of DOI:10.1021/jm0703237

A wide variety of pathogens have acquired antimicrobial resistance as an inevitable evolutionary response to the extensive use of antibacterial agents. In particular, one of the most widely used antibiotic structural classes is the β-lactams, in which the

Full text of DOI:10.1021/jm0703237

J. Med. Chem. 2007, 50, 4113-4121
4113
4-Substituted Trinems as Broad Spectrum â-Lactamase Inhibitors: Structure-Based Design,
Synthesis, and Biological Activity
Ivan Plantan,† Lovro Selicˇ,† Tomazˇ Mesar,† Petra Sˇtefanicˇ Anderluh,† Marko Oblak,† Andrej Prezˇelj,† Lars Hesse,‡
Miha Andrejasˇicˇ,§ Mateja Vilar,† Dusˇan Turk,§ Andrej Kocijan,† Tadeja Prevec,† Gregor Vilfan,† Darko Kocjan,† Anton Cˇ opar,†
Urosˇ Urleb,† and Tom Solmajer*,†,#,
Drug DiscoVery, Lek Pharmaceuticals d.d., VeroVskoVa 57, SI-1526 Ljubljana, SloVenia, National Institute of Chemistry, POB 660, HajdrihoVa
19, 1001 Ljubljana, SloVenia, Antibiotic Research Institute, Sandoz, A-1235 Wien, Austria, and Department of Biochemistry and Molecular
Biology, Jozˇef Stefan Institute, JamoVa 39, SI-1000 Ljubljana, SloVenia
ReceiVed March 21, 2007
A wide variety of pathogens have acquired antimicrobial resistance as an inevitable evolutionary re-
sponse to the extensive use of antibacterial agents. In particular, one of the most widely used antibiotic
structural classes is the â-lactams, in which the most common and the most efficient mechanism of bac-
terial resistance is the synthesis of â-lactamases. Class C â-lactamase enzymes are primarily cephalosporinases,
mostly chromosomally encoded, and are inducible by exposure to some â-lactam agents and resistant to
inhibition by marketed â-lactamase inhibitors. In an ongoing effort to alleviate this problem a series
of novel 4-substituted trinems was designed and synthesized. Significant in Vitro inhibitory activity was
measured against the bacterial â-lactamases of class C and additionally against class A. The lead compound
LK-157 was shown to be a potent mechanism-based inactivator. Acylation of the active site Ser 64 of
the class C enzyme â-lactamase was observed in the solved crystal structures of two inhibitors complexes
to AmpC enzyme from E. cloacae. Structure-activity relationships in the series reveal the importance
of the trinem scaffold for inhibitory activity and the interesting potential of the series for further
development.
Introduction
Enzymes of classes A, C, and D are active site serine
â-lactamases, whereas class B enzymes are metallo-â-lactamases
that contain a zinc atom at the active site. Class A enzymes are
primarily penicillinases, mostly plasmid-mediated, usually
constitutively expressed and susceptible to inhibition by
the clinically available â-lactamase inhibitors. Class C en-
zymes, in contrast, are primarily cephalosporinases, mostly
chromosomally encoded, inducible by exposure to some â-lac-
tam agents, and resistant to inhibition by â-lactamase inhibi-
tors.6,7
Infectious diseases are among the leading causes of mortality.
A wide variety of pathogens have acquired antimicrobial
resistance as an inevitable evolutionary response to the extensive
use of antibacterial agents. Surveillance data indicate that
resistance has arisen to all established antibacterial classes,
including new agents with novel mechanisms of action, and has
spread between and within bacterial populations with great
efficiency. The dramatic increase in antibacterial resistance in
both community and hospital settings has resulted in therapeutic
failures with increased morbidity, mortality, and healthcare
costs.1-3
Although bacteria have developed several strategies for
escaping the lethal action of â-lactam antibiotics, the most
common and often the most efficient mechanism is the synthesis
of â-lactamases. These enzymes, which are usually secreted into
the external medium by Gram-positive species and into the
periplasm by their Gram-negative counterparts, catalyze the
irreversible hydrolysis of the amide bond of the â-lactam ring,
yielding biologically inactive products.4,5
Much effort has been devoted to the synthesis of â-lactam
agents which would bypass this defense mechanism, but the
changing epidemiology of â-lactamases has rendered these novel
agents inactive following their introduction to clinical use. A
highly effective approach for tackling â-lactamase-mediated
resistance to â-lactams is the use of â-lactam agent/â-lacta-
mase-inhibitor combinations, where the latter potentiates the
action of the former by protecting it from enzymatic hydrolysis.8
Commercially available inhibitors potassium clavulanate, sul-
bactam, and tazobactam, inhibit most class A and some class
D â-lactamases, but activity against class C types is poor.
Detailed knowledge of the binding mode of â-lactamase
inhibitors used in the clinic should facilitate the design of second
generation â-lactamase inhibitors that possess broad-spectrum
activity against clinically relevant producers of class A, class
D, and class C enzymes.9 The efficacy and safety of â-lactams
as a class has provided powerful motivation for the development
of novel molecules which would address the unmet medical
needs posed by bacterial resistance. The alkylidene penems and
2â-substituted penam sulfones, oxapenems, cephalosporin-
derived compounds, cyclic acyl phosphonates, and non-â-lactam
compounds are currently under investigation as potential â-lac-
tamase inhibitors, but their clinical applications are not yet
available.10-14
On the basis of amino acid sequence similarities, â-lactamases
have been broadly grouped into four molecular classes, A, B,
C, and D. The diversity of the â-lactamases is a most critical
aspect of antimicrobial therapy. The â-lactamase superfamily
currently has more than 450 members, many of which differ
only by a single amino acid (www.lahey.org).
This paper is dedicated to the late Professor J.-M. Ghuysen (University
of Liege) in honor of his pioneering work in the field.
* To whom correspondence should be addressed. Phone: +386-1-4760
277; Fax: +386-1-4760-300; E-mail: tom.solmajer@ki.si; tom.solmajer@
sandoz.com.
† Lek Pharmaceuticals d.d.
# National Institute of Chemistry.
‡ Sandoz.
§ Jozˇef Stefan Institute.
10.1021/jm0703237 CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/01/2007

4114 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Plantan et al.
ypropionitrile, respectively. Since the yields of the previously
described methods did not render realization of the six synthetic
steps for the preparation of 2-fluoroethoxy and 2-cyanoethoxy
final compounds 11g,h possible, it was necessary to prepare
adequate quantities of 2′-alkoxy-1-hydroxycyclohexylazetidi-
nones 6g,h.22
Figure 1. Chemical structure of lead compound (8S,9R)-10(E)-
ethylidene-4(S)-methoxy-11-oxoazatricyclo[7.2.0.03,8]undec-2-enecar-
boxylate, with trinem numbering included.
In view of these complexities the ultrasonically aided opening
of epoxide 5, with triflate catalysts, was studied.23 According
to those findings, 6g was prepared from 2-fluoroethanol with a
52% yield, and 6h from 3-hydroxypropionitrile with a 55%
yield. Subsequent Swern oxidation24 of 6g,h yielded 4g,h.
The pure diastereomers 4a-h (at least 96% de) obtained by
synthetic routes I and II were further acylated with allyl oxallyl
chloride and cyclized with triethyl phosphite in refluxing xylene
in the presence of hydroquinone to give the trinem intermediates
8a-h.25 Subsequent deprotection of the tert-butyldimethylsilyl
group by tetrabutylammonium fluoride provided intermediates
9a-h.26 In order to prepare the designed ethylidene moiety on
the trinem scaffold, various synthetic approaches were studied,
among which only water elimination under the conditions of
the Mitsunobu reaction27 enabled the preparation of isomers
10a-h possessing the exclusively E configuration of the
ethylidene group.28 The byproducts of the Mitsunobu reaction,
triphenylphosphine oxide and diethyl hydrazine-1,2-dicarboxy-
late, were removed by selective precipitation with diethyl ether
and the crude products further purified with column chroma-
tography.
Recently, following a rational structure-based drug design
approach, novel tricyclic carbapenem (trinema) compounds with
tetrahydro-2H-thiopyran and/or adjacent fused cyclohexane rings
were synthesized by our research group (Figure 1).15,16 The
fusion of the hydrophobic cyclohexane or 6-thia ring onto the
carbapenem moiety, which was hypothesized to arrest the
deacylation step of the tetrahedral acyl-enzyme intermediate15
by blocking the access of water, was found to augment inhibitory
activity toward class C and class A â-lactamases (unpublished
work from this laboratory). However, by introducing the
4-methoxy substituent16,17 to the trinem scaffold, the inhibitory
activity was enhanced by 2 orders of magnitude in Vitro to both
enzymes AmpC â-lactamase (class C) and TEM 1 â-lactamase
(class A) from E. coli. On the basis of these preliminary findings,
we did not pursue the development of the unsubstituted trinem
compounds any further.
In the present paper we report on the design, improved
synthesis procedure, and inhibitory activity of novel 4-substi-
tuted trinems as broad-spectrum â-lactamase inhibitors of both
class A and class C enzymes. In addition, the solved crystal
structure of our lead compound 11a (Figure 2), selected from a
series of ethylidene derivatives of tricyclic carbapenems in
complex with AmpC â-lactamase from Enterobacter cloacae,
offers a possible explanation for its in Vitro efficacy, which was
determined to be superior to clinically available â-lactamase
inhibitors. In addition, the solved crystal structure of the
4-butyloxy derivative offers a possible explanation to the
structure-activity relationship of 4-substituted derivatives with
variable lipophilicity.
In the last step, allyl group deprotection with sodium
6-ethylhexanoate, Pd[(PPh)3]4 and PPh3 in tetrahydrofuran
afforded target compounds 11a-h.29 In general, the final
compounds precipitated from the reaction mixture spontaneously
or after addition of diethyl ether or petroleum ether; otherwise
extraction to water medium and subsequent lyophilization was
necessary.
Results and Discussion
Determination of the Acyl-enzyme Complex Structure.
Several structures of â-lactamases from both classes A and C
have been available for some time now.30-41 Their use for the
design, using computer-assisted molecular modeling, of novel
molecules is necessitated and based on the knowledge of the
complex enzyme-inhibitor structure.
Chemistry. The designed 4-alkoxy-10-ethylidene trinem
analogues were prepared employing previously described syn-
thetic procedures18 for trinem scaffold synthesis, followed by
ethylidene moiety formation and allyl ester deprotection. These
procedures were significantly optimized to give intermediates
with considerably higher yields.
However, the principal reason for a possible uncertainty in
this procedure is that the reactions of the inhibitors with
â-lactamases frequently involve branched pathways with a
variety of putative intermediates. A manifested example of such
a process was the solved structure of the complex clavulanate-
â-lactamase TEM-1 from E. coli, in which an unexpected
breakup of the clavulanate ligand was observed.42 Thus, we
deemed it necessary to substantiate our mechanistic hypothesis
and attempt to explain the inhibitory activity of the trinem series
toward â-lactamases of class C by solving the structure of the
complex AmpC â-lactamase from Enterobacter cloacae with
two compounds from the trinem series. 11a was chosen due to
its inhibitory potency in the nanomolar range toward both class
A and class C â-lactamases.17 A second structure of the complex
AmpC â-lactamase with 4-butyloxy trinem derivative (vide
infra) was solved to provide clues to structure-activity relation-
ship of inhibitors series designed to optimize ADME properties
of the lead compound 11a.
Two different synthetic procedures were used (Scheme 1) to
prepare the key intermediates, (3R)-[(1R)-(tert-butyldimethyl-
silyloxy)-ethyl]-(4R)-((1S/R)-(3R/ S)-alkoxy-2-oxo-cyclohexyl)-
azetidin-2-ones (4a-h). According to synthetic route I, the
starting 2-alkoxycyclohexanones 2c,d were synthesized, using
known synthetic procedures, from cyclohexene oxide,19 except
for the 2-methoxy- and 2-ethoxycyclohexanones (2a, 2b) which
were commercially available. Coupling of the corresponding
2-alkoxycyclohexanones 2a-d with ((3R,4R)-3-((1R)-1-{[tert-
butyl(dimethyl)silyl]oxy}ethyl)-4-acetoxy-2-azetidinone 3, in the
presence of SnCl4 as a Lewis acid, afforded the mixtures of
(1′R,3′S) and (1′S, 3′R) diastereomers 4a-f, which were
separated by column chromatography or by crystallization.20 The
2-fluoroethoxy and 2-cyanoethoxy intermediates 4g and 4h were
prepared Via synthetic route II, which included the stereoselec-
tive opening of epoxide21 5 with 2-fluoroethanol and 3-hydrox-
Interactions of the Acyl-enzyme Intermediate in the
AmpC Binding Site. The crystal structure of the complex
revealed the presence of inhibitor 11a covalently linked to the
Oγ atom of the Ser 64 side chain in the AmpC active site (Figure
a Abbreviations: trinem, tricyclic carbapenem; EDIPA, N,N-diisopro-
pylethylamine; TBAF, tetra-n-butylammonium fluoride; THF, tetrahydro-
furan; DEAD, diethyl azodicarboxylate; PEG, polyethylene glycol; PMSF,
phenylmethanesulfonyl fluoride.

Trinems as Broad Spectrum â-Lactamase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4115
Figure 2. Structures of the synthesized compounds illustrating stereoisomerism at substituents 4 and 8.
Scheme 1a
a Reagents and conditions: (i) 1. ROH, FeCl3, r.t.; 2. Pyridinium chlorochromate, CH2Cl2, r.t.; (ii) 1. SnCl4, CH2Cl2, 3, 0 °C, 2. EDIPA, 0 °C; (iii) for
6g: FCH2CH2OH, Yb(OTf)3, ultrasound; for 6h: NCCH2CH2OH, Sn(OTf)2, ultrasound; (iv) 1. (COCl)2, DMSO, CH2Cl2, 2. Et3N; (v) for 7a-f: allyl
oxallyl chloride, Et3N, CH2Cl2, 0 °C; for 7g, 7h: allyl oxallyl chloride, Et3N, K2CO3, CH2Cl2, 0 °C; (vi) (EtO)3P, hydroquinone, xylene, reflux; (vii) 1 M
TBAF, AcOH, THF, r.t.; (viii) PPh3, DEAD, CH2Cl2, 0 °C; (ix) PPh3, Pd[(PPh)3]4, NaEH, THF, r.t.
3). The â-lactam carbonyl oxygen of 11a is oriented in the
oxyanion hole, forming two hydrogen bonds with the backbone
amide groups of Ser 64 (2.9 Å) and Ser 318 (3.0 Å). In turn,
the acyl intermediate appears to be well positioned in the active
site, forming numerous hydrophobic interactions with the
nonpolar side chains of residues Leu 119 and Leu 293.
Surprisingly, the carboxylic group of 11a is directed out of
the active site and has no direct interactions with the enzyme.
In the structure of the noncovalent Michaelis complex43 of a
cephem sulfone which is a step in the mechanism prior to the
acyl-enzyme formation, such interactions are clearly observed.
It appears that the ethylidene group, which is present at position
C-10 in 11a, is not bulky enough to provide sufficient steric
hindrance for such a dramatic conformational change. However,
the acyl intermediate of 11a is stabilized against hydrolysis
through favorable conjugation of the acyl carbonyl with the
ethylidene group, which is pointed toward residues Leu 119
and Gln 120.15 Similar resonance stabilization effects have also
been described for cephem sulfone43 and penem inhibitors44 of
â-lactamases.
Proposed Inhibition Mechanism in Class C â-Lactamases.
The catalytic water molecule, which is responsible for deacy-
lation of enzyme-substrate covalent intermediates and is
observed in several class C â-lactamase complexes,39-41 is not
present in the AmpC/11a complex structure. Since the bicyclic
ring of the intermediate 11a itself is not able to completely block
the approach of a water molecule to the acyl ester bond, rotation
of the opened â-lactam ring of 11a about the C9-C10 and/or
C10-C11 bonds (Figure 1) upon acylation appears to be
responsible for displacement of the putative deacylating water
molecule. The crystal structure of acyl-enzyme intermediate for
imipenem bound to TEM-1 â-lactamase from E. coli indicates
a conformational change for the complex which supposedly

4116 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Plantan et al.
Figure 3. P99 â-lactamase from the E. cloacae active-site with bound inhibitor 11a in stereoview (a, left; b, right). The â-lactamase is presented
in cyan, and main chain bonds are thicker. The inhibitor is drawn in orange. The nitrogen atoms are colored blue, and oxygen atoms red. The
hydrogen bond between the inhibitor and the enzyme is shown in gray. 2Fo - Fc electron density map (inhibitor atoms only) at 2.05 Å resolution
is contoured at 0.8σ. The image was generated by programs Raster3d52 and MAIN.54 PDB ID entry code of the structure is 2Q9M.
The results of the inhibitory activities evaluations against class
A (TEM-1 and SHV-1 from Escherichia coli) and class C
(AmpC from E. cloacae) â-lactamases for compounds 11a-h
are presented in Table 2 and are compared to clavulanate48 and
tazobactam.49 The in Vitro activities of these compounds against
class A enzymes are comparable to clavulanate and tazobactam.
In contrast, significant improvement of potency by 2-30-fold
compared to tazobactam and 100-2000-fold compared to
clavulanate was achieved against the representative class C
enzyme AmpC with 11a being the most potent inhibitor.
Surprisingly, and contrary to our hypothesis, the IC50 values
of compounds 11b-h against class C â-lactamase AmpC were
3-16-fold higher compared to the lead compound 11a. On the
other hand, introduction of bulkier substituents at position 4
roughly preserved the in Vitro activity of compounds 11b-h
against class A â-lactamases. Similarly, our initial hypothesis
based on the structure of the complex AmpC/11a that a
structural change at position 4 such as introduction of fluorine
(11g) or cyano group (11h) on an ethoxy spacer would enable
additional favorable electrostatic contacts with the enzyme
surface did not yield any beneficial effect to binding of these
derivatives.
Furthermore, four compounds, 11b, 11e, 11g, and 11h, exhibit
a 3-fold increase in potency against TEM-1 enzyme compared
to 11a. In the case of TEM-1 enzyme the (4R,8R)-stereoisomer
of the ethoxy and butoxy derivatives is twice as potent as the
(4S,8S)-stereoisomer. This pattern of stereoselectivity, which
is not completely preserved for SHV-1 and AmpC enzymes,
might be useful for further improvement of trinem inhibitory
activity.
Two possibilities to explain the somewhat unexpected
diminished AmpC inhibitory activities in this series could be
suggested: (i) the preferred conformation in compounds 11b-h
in the acyl-AmpC complex is different from the observed
conformation of 11a. Alternatively, (ii) the acyl intermediate
conformations are similar to the determined crystal structure
of the complex, but the bulkier substituents at position C-4 might
not be favorably positioned in the previous step of the catalytic
process, i.e., when the noncovalent Michaelis complex is formed
and after subsequent reorganization in the active site upon
acylation. Preliminary docking studies (unpublished results from
this laboratory)50 of the noncovalently bound compounds from
this series in the AmpC active site indicate that the lipophilic
substituents at position C-4 of the trinem structure are directed
toward the segment of residues 280-295. This part of the
binding site has previously been observed to be conformationally
disordered in some crystal structures of AmpC â-lactamases.43,44
Figure 4. The structure of the intermediate C after deacylation by Ser
64 of â-lactamase.17
accounts for the ability of the antibiotic to resist hydrolytic
deactivation by â-lactamase.45
In our previous study,17 we showed that the product from
the â-lactamase-catalyzed and basic hydrolysis of compound
11a is intermediate C (Figure 4), where tautomerization of the
cleaved â-lactam ring and elimination of the 4-methoxy group
has occurred. This behavior was previously documented for
other â-lactams, such as cephalosporins43 and oxacephems,44
with the leaving group at position C3.
In the case of our covalently bound trinem complex the
electron density of the 4-methoxy group, which is clearly
observed in the acylated complex, indicates that the acylation
process cannot be accompanied by the simultaneous elimination
of the 4-methoxy group, at least for 11a under these particular
crystallization conditions. Our crystallographic data corroborates,
that in the case of 11a, elimination of the leaving methoxy group
proceeds only after the deacylation step and release of the
product from the enzyme is completed.
Structure-Activity Relationship of Novel Trinem Inhibi-
tors. Recently, the paradigm of rational drug design has shifted
its focus from optimization of in Vitro enzymatic inhibitory
activity toward accounting for ADME properties of the inhibi-
tors.46 Thus, in order to vary the lipophilic character of the lead
compound 11a we exploited the details of its crystal structure
in the complex with the enzyme. Since the oxygen atom of the
4-methoxy group of compound 11a forms no interactions with
the active site and at the same time the methyl moiety is exposed
to the solvent, we hypothesized that position 4 would be suitable
for balancing the lipophilicity while maintaining inhibitory
activity (Table 1). Compounds 11b-h were designed to test
this hypothesis, and their lipophilic character was predicted by
calculating the octanol/water partition coefficient.47 Rather
conservative substitutions at position 4 offered a spread of 2
log units in log P values. With the exception of the fluoroethoxy
derivative 11g (log Pcalc ) 0.97; log Dcalc ) -1.82) all the
compounds possess higher calculated log P and log D (pH )
7.4) values compared to the parent compound 11a (log Pcalc
1.28; log Dcalc ) -1.48), whereas compounds 11e and 11f are
the most lipophilic in the series.
)

Trinems as Broad Spectrum â-Lactamase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4117
Figure 5. P99 â-lactamase from E. cloacae active site with bound inhibitor 11e in stereoview (a, left; b, right). 2Fo - Fc electron density map
(inhibitor atoms only) at 2.20 Å resolution is contoured at 0.8σ. Details same as in Figure 3. PDB ID entry code of the structure is 2Q9N.
Table 1. Stereoisomerism of the Intermediates 2a-9h and Final Compounds 11a-ha
compound
R
compound
R
compound
R
2a
2b
R ) Me
R ) Et
4a, 7a
4b, 7b
4c, 7c
4d, 7d
4e, 7e
4f, 7f
(1R,3S)
(1S,3R)
(1R,3S)
(1R,3S)
(1S,3R)
(1R,3S)
(1R,3S)
(1R,3S)
R ) Me
8a, 9a, 10a, 11a
8b, 9b, 10b, 11b
8c, 9c, 10c, 11c
8d, 9d, 10d, 11d
8e, 9e, 10e, 11e
8f, 9f, 10f, 11f
(4S,8S)
(4R,8R)
(4S,8S)
(4S,8S)
(4R,8R)
(4S,8S)
(4S,8S)
(4S,8S)
R ) Me
R ) Et
R ) Et
R ) Et
R ) Et
2c
2d
R ) iPr
R ) Bu
R ) iPr
R ) iPr
R ) Bu
R ) Bu
R ) Bu
R ) Bu
6g, 4g, 7g
6h, 4h, 7h
R ) (CH2)2F
R ) (CH2)2CN
8g, 9g, 10g, 11g
8h, 9h, 10h, 11h
R ) (CH2)2F
R ) (CH2)2CN
a (4R,8R) isomer of 4-OiPr analogue was not prepared with sufficient purity and is therefore not included.
Table 2. In Vitro Activity and Calculated Partition Coefficients of
Compounds 11a and 11b-h against Class A (TEM-1 and SHV-1) and
Class C (AmpC) â-Lactamases
increased lipophilicity compared to parent compound 11a (as
estimated by log P value of 2.88) with AmpC â-lactamase. It
was found that the 4-butyloxy substituent is exposed toward
solvent in a similar way to 4-OMe substituent (Figure 5). Thus,
on one hand, the notion of the unfavorable energetic influence
of a more lipophilic substituent exposed to solvent for an
inhibitory constant of compound 11e which is weakened by a
factor of 7 (0.430 nmol) compared to 62 nmol for compound
11a appears to be correct. On the other hand, this crystal
structure enables answer to the question: is the effect of lipo-
philic functionality at position 4 of the trinem negligible? By
comparing both solved crystal structures, we are in position to
describe the variation in the interaction pattern in compounds
11e and 11a. The interactions of the covalently bound â-lactam
fragment of the trinem scaffold with residues Ser 64, Lys 67,
and Ser 318 of the active site are very similar. The carboxylate
substituents are oriented toward lipophilic environment of Leu
119 and Gln 120 in both compounds, and the bulky trinem rings
B and C are positioned in the enzymatic cleft. Most interestingly,
both C4 substituents are exposed to solvent. This similarity leads
us to the conclusion that the introduction of a bulkier C4
substituent in compound 11e probably interacts unfavorably with
the loop 280-295 in the noncovalent Michaelis complex formed
before reorganization in the active site upon acylation step. This
appears to be responsible for the unfavorable change in the
binding and weakening the effect of an additional hydrogen bond
between carboxylate group of compound 11e with backbone
amide of Gln 120 as well.
calculated partition
IC50 [µM]a
coefficientb
log Dcalc
compound
TEM-1
SHV-1
AmpC
log Pcalc
(pH 7.4)
clavulanate
tazobactam
11ac
11b
11c
11d
11e
11f
11g
0.030
0.017
0.055
0.022
0.057
0.039
0.012
0.032
0.021
0.018
0.028
0.222
0.151
0.284
0.385
0.340
0.219
0.206
0.148
0.152
136.2
1.808
0.062
0.295
0.229
0.341
0.430
0.720
1.020
0.209
-1.98
-1.70
1.28
1.81
1.81
2.16
2.88
2.88
0.97
1.42
-5.68
-5.43
-1.48
-0.94
-0.94
-0.58
0.12
0.12
-1.82
-1.36
11h
a IC50 values are presented as an average of at least two measurements
with variability of 0.1 units. b log P and log D (at pH 7.4) were calculated
using ACD/Labs7.0 software. c Lead compound 11a.
Under our conditions for crystallization of AmpC from E.
cloacae with 11a only one monomer is located in an asymmetric
unit. However, in other crystal structures of AmpC from E. coli
in complex with various ligands two monomers were observed
in each asymmetric unit.51 While monomer 2 always adopts a
similar conformation to that observed in our complex (rmsd
for all backbone atom pairs is ∼0.5 Å) the main difference is
located in monomer 1 in which residues 280-295 are disordered
and its structure could not be determined. Similarly, Lobkovsky
et al.51 have observed positional disorder of the tetrahedral
phosphonate derivative intermediate in the complex with E.
cloacae P99 cephalosporinase. Their seminal observation in this
respect was that the difference in active site volume available
for specific ligand binding between class A and class C
â-lactamase enzymes might be accounted for by the unexpected
position of the â-lactam carbonyl in the observed acyl tetrahedral
intermediate in class C â-lactamase enzyme.
Conclusions
The compound 11a was previously identified as a promising
candidate for a â-lactamase inhibitor in bacterial infections
which show resistance to currently available antibiotics. A series
of analogues of our lead compound 11a with a variety of
substituents at position C4 was synthesized and evaluated.
Potency of the derivatives compared to 11a decreased,
however, some of the compounds still maintain interesting
inhibitory activity to both class A and class C â-lactamase
To resolve these issues we have determined the crystal
structure of the 4-OBu derivative (compound 11e) which has

4118 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Plantan et al.
Interestingly, the space group and unit cell dimensions are not
similar to P99 â-lactamase’s crystals determined previously (PDB
code 1XX256) but are closer to the values of E. cloacae GC1
â-lactamase crystals (PDB ID code 1GCE,.57 Although the crystal
dimensions are similar, there is no density space between Asp 205
and Lys 207 for insertion of three additional amino acids present
in the GC1 â-lactamase sequence. Results are presented in Table
S2 of the Supporting Information.
(d) â-Lactamase-11e Complex. The protein (AmpC â-lacta-
mase) was cloned with the purification His-tag attached. It was
linked via a thrombin cleavage site linker consisting of six residues
(LVPRvGS) which was removed prior to crystallization. PBS was
used as the cleavage buffer (140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM KH2PO4, pH ) 7.3). Thrombin was prepared
as a stock solution by dissolving 250 U of thrombin into 250 µL
of PBS buffer. Cleavage was performed at 19 ×bcC for 24 h. Further
cleavages were blocked with addition of 0.5 µL of PMSF, a
thrombin irreversible inhibitor. After 30 min, the excess PMSF and
cleaved His-Tag were removed by dialysis on MICROCON using
a 3000 Da membrane.
The crystals were grown by the hanging drop vapor diffusion
method with a solution of 22% PEG4000, 0.1 M Na acetate at pH
4.8, 0.2 M ammonium acetate. Crystals of the size 0.1 × 0.2 × 0.5
mm appeared within 14 to 21 days and were suitable for cryodata
collection. They belonged to P21212 space group with unit cell
dimensions a ) 77.33A, b ) 68.98, c ) 62.02, R ) â ) γ ) 90
and contained a single molecule per asymmetric unit.
Solution of (∼50 mM) inhibitor was prepared in INH buffer
(22% PEG4000, 10% glycerol, 0.1 M Na acetate pH ) 7.0, 0.2 M
ammonium acetate). Soaking was performed just before the
measurement in 1 µL drops on glass. During a 4 h soak, the solution
was exchanged three times. The crystals were transferred into a
liquid nitrogen stream and exposed to X-rays immediately after
the soaking.
X-ray diffraction data were collected in-house at a RIGAKU RU-
200 rotating anode (Cu, λ ) 1.5418 Å) at 100 K using a MAR345
IP detector. Frames were counted over 1×bc oscillation images for
600 s. There were a total of 100 images over a 100×bc range. The
reflections were indexed, integrated, and scaled using the HKL2000
software suite.53 Results are presented in Table S2 of the Supporting
Information.
(e) Structure Determination of the Enzyme-11e Complex.
The previously determined structure of the AmpC â-lactamase
molecule was used for the starting model. The final R factor was
0.252 using diffraction data in the resolution range 10-2.2 Å. The
structure of inhibitor 11e was clearly visible in the final electron
density map. The orientation of the 11e is different from the 11a
structure. The difference in orientation is presumably a consequence
of the larger nonpolar side chain present in 11e.
General Chemical Methods. Analytical TLC was performed
on Merck silica gel 60 F254 plates (0.25 mm), and the components
detected using specific spray reagents. Column chromatography was
carried out on silica gel 60 (particle size 240-400 mesh). Melting
points were determined on a Reichert hot stage microscope and
are uncorrected or obtained from DSC spectra, which were recorded
on a Mettler Toledo DSC822e. IR spectra were obtained with a
Nicolet FT-IR Nexus spectrometer and optical rotation was
measured on a Perkin-Elmer 1241 MC polarimeter. 1H NMR spectra
were recorded on a Varian Inova300 spectrometer in CDCl3 or
DMSO-d6 solution, with TMS as the internal standard. Mass spectra
were obtained using a VG-Analytical Autospec Q mass spectrom-
eter. HPLC analyses were performed using a Waters 2695 Separa-
tion Module with a Waters 2996 PDA detector and an XTerra RP
C18 (150 × 4.6 mm I.D., 3.5 µm particle size) analytical column
and a gradient elution method combining mobile phase A with 25
mM ammonium acetate (pH ) 6)/acetonitrile (95/ 5 v/v) and mobile
phase B with 25 mM ammonium acetate (pH ) 6)/acetonitrile (10/
90 v/v). LC-MS analyses were performed on an Alliance HT Waters
2795 separations module with a Waters 2996 (photodiode array)
UV-detector and a Micromass Quatro Micro mass spectrometer.
HRMS analyses were performed on Micromass QTOF Ultima
enzymes. The variations in lipophilicity of the derivatives in
the series offers possibilities for optimization of pharmacokinetic
properties. Although we cannot confirm this hypothesis as yet,
it is reasonable to expect that changing log P and log D in the
series would enable optimization of properties such as clearance.
The solved crystal structures of the inhibitors 11a and 11e
in complexes with enzyme AmpC from E. cloacae P99 provide
a viable rationale for further cycles of rational structure-based
design of the first tricyclic carbapenem inhibitor of â-lactamases.
Experimental Section
Enzymatic Assays. Assays were performed with â-lactamases
expressed in the pET system (Novagen, San Diego, CA) without
signal peptides. They contained an N-terminal hexahistidine tag
that was used for purification on Ni-NTA resin (Qiagen, Hilden,
Germany). Compounds were prepared as 50 mM stocks in DMSO
and diluted with buffer P1 (50 mM phosphate, pH 7) to a final
concentration of 10% DMSO. All further dilutions were done using
P2 (P1 with 10% DMSO). Enzyme and compound dilutions were
preincubated for 10 min at 37 °C, and the reaction started with the
addition of prewarmed (37 °C) nitrocefin at a final concentration
of 50 mM. The change in absorption at 490 nm was followed at
37 °C for 10 min at 30 s intervals in a Spectramax 384 Plus
microplate reader (Molecular Devices, Sunnyvale, CA) using 96-
well plates. The initial rate was determined, and IC50 values were
calculated using nonlinear regression and sigmoidal dose-response
analysis with the PRISM 4.0 software (Graphpad Software Inc.,
San Diego, CA). IC50 data is expressed as µM within the 95%
confidence interval (95% CI) and was calculated from at least two
independent experiments.
Crystal Growth and Structure Determination. (a) â-Lacta-
mase-11a Complex. The crystals of AmpC â-lactamase were
grown by vapor diffusion method by hanging 2 µL drops of
precipitant and protein solution over a solution of 22% PEG 4000,
0.1 M Na acetate at pH 4.8, and 0.2 M ammonium acetate. Crystals
of the size 0.1 × 0.1 × 0.5 mm grew in 3-4 weeks at 19 °C and
were suitable for cryodata collection. A solution of 50 mM inhibitor
was prepared using INH buffer (22% Peg4000, 10% glycerol, 0.1
M Na acetate pH 7.0, 0.2 M ammonium acetate). Soaking was
performed just before the measurement in 1 µL drops on glass.
Crystals were soaked at 21 ×bcC for 2.5 h. Subsequently, the
solution containing inhibitor was replaced and soaking continued
for another 2.5 h. Crystals prepared as described were frozen in
liquid nitrogen.
(b) Diffraction Data Collection. The crystal diffracted to 2.0
Å resolution in the space group P21212 with unit cell parameters a
) 76.65 Å, b ) 69.77 Å, and c ) 62.82 Å, R ) â ) γ ) 90. The
AmpC structure of the apoenzyme was used as a starting structure,35
and the inhibitor was modeled in the active site. The data set of
20733 reflections (96.8% complete) were collected at XRD-1 beam
line at Elettra Synchrotron Facility in Trieste at 100 K using 162
mm MAR CCD detector. Frames were counted over 0.75×bc
oscillation images for 40 s. There were total of 112 images over
84×bc range. The reflections were indexed, integrated, and scaled
using the HKL2000 software suite.53
(c) Structure Determination of the Enzyme-11a Complex.
Molecular replacement was solved using the AMoRe program with
one molecule in units and an R factor of 25.4 and correlation
coefficient of 60.0. The model of P99 â-lactamase with PDB ID
1XX2 was loaded into program suite MAIN.54 Several cycles of
model fitting and refinement were carried out using the Engh and
Huber parameter set.55 The resulting electron density difference
maps clearly revealed the presence of inhibitor covalently bound
to the reactive site Ser 64. Water molecules were added, and
resolution of the data was gradually extended to maximum 2.05
Å. The final crystallographic R factor is 0.209 and Rsymm is 0.121.
The final model of the enzyme-inhibitor complex was composed
of 3282 proteins, 19 atoms of inhibitor 11a, and 367 crystal water
molecules.

Trinems as Broad Spectrum â-Lactamase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4119
Global using ESI (negative and positive mode). All reported yields
are yields of purified products. Alternative additional HPLC
analyses were performed (denoted as method 1 below) using Agilent
1100 Separation Module and Luna C18 (250 × 4.6 mm I.D., 5
mm particle size) analytical column by an isocratic elution method
combining 25 mM ammonium phosphate buffer (pH ) 7.57)/
acetonitrile (60/40 v/v).
General Procedure F. Synthesis of Sodium (8S/ 8R,9R)-10-
(E)-Ethylidene-4(S/ R)-alkoxy-11-oxo-1-azatricyclo[7.2.0.03,8]-
undec-2-enecarboxylate (11a-h). Compounds 10a-h were pre-
pared according to general procedures A-E described in the
Supporting Information. PPh3 (42.6 mg, 1.63 mmol, 0.1 equiv),
Pd[(PPh)3]4 (43.1 mg, 0.041 mmol, 0.025 equiv), and 0.21 M
sodium 6-ethylhexanoate solution in THF (NaEH) (7.8 mL, 1.63
mmol, 1 equiv) were added during stirring into a solution of 10a-h
(1.63 mmol, 1 equiv) in THF (10 mL). The mixture was left stirring
at room temperature until complete conversion was detected. If the
product precipitated from the reaction mixture spontaneously or
after the addition of diethyl ether or petroleum ether, it was filtered
off and dried. Otherwise, water (30 mL) was added, washed (3 ×
10 mL) with ethyl acetate, and lyophilized.
170.9 (CdO). HRMS ([M - Na]-): Anal. calcd for C15H18NO4:
276.1236; found: 276.1246. HPLC purity: 98.00 area%. HPLC
purity (method 1): 99.9%.
Sodium (8S,9R)-10(E)-Ethylidene-4(S)-isopropoxy-11-oxo-1-
azatricyclo[7.2.0.03,8]undec-2-enecarboxylate (11d). Compound
11d was prepared from 10d according to General Procedure F. The
obtained product was precipitated from the reaction mixture after
the addition of diethyl ether to give 11d; yield: 21.7%, yellow
amorphous powder; mp decomp above 160 °C; [R]20D (H2O, 1.055
mg/mL) ) +40.5. IR (KBr) ν [cm-1] 3439, 2931, 1752, 1634, 1395,
1018. 1H NMR (DMSO-d6) δ [ppm] 1.04 (t, 3H, J ) 5.3 Hz, CH3-
CH2O), 1.05 (t, 3H, J ) 5.3 Hz, CH3CH2O), 1.75 (d, 3H, J ) 7.2
Hz, CH3CHd), 1.22-1.32 (m, 1H), 1.39-1.44 (m, 1H), 1.60-
1.74 (m, 4H), 2.96-3.06 (m, 1H, H-8), 3.46-3.55 (m, 2H,
CH3CH2O), 4.55 (d, 1H, J ) 10.1 Hz, H-9), 5.32 (t, 1H, J ) 2.9
Hz, H-4), 6.24 (dq, 1H, J1 ) 7.1 Hz, J2 ) 1.6 Hz, CH3CHd). 13
C
NMR (DMSO-d6) δ [ppm] 14.7 (CH3CHd), 20.1 (CH3CH2O), 21.8
(CH3CH2O), 23.6 (C-6), 30.3 (C-5), 32.6 (C-7), 43.5 (C-8), 59.7
(CH3CH2O), 66.9 (C-9), 76.5 (C-4), 126.5 (CH3CHd), 135.2 (C-
3), 135.6 (C-2), 140.2 (C-10), 164.4 (COONa), 170.8 (CdO).
HRMS ([M - Na]-): Anal. calcd for C16H20NO4: 290.1392;
found: 290.1384. HPLC purity: 98.86 area%. HPLC purity
(method 1): 98.45%.
Sodium (8S,9R)-10(E)-Ethylidene-4(S)-methoxy-11-oxo-1-
azatricyclo[7.2.0.03,8]undec -2-enecarboxylate (11a). Compound
11a was prepared from 10a according to General Procedure F. The
obtained product was precipitated from the reaction mixture; yield
90.0%, white amorphous powder; mp 151-152 °C; [R]20D (H2O,
10.0 mg/mL) ) +100.0. IR (KBr) ν [cm-1] 2934, 1763, 1609, 1397,
Sodium (8R,9R)-10(S)-[1(R)-Hydroxyethyl]-4(R)-butoxy-11-
oxo-1-azatricyclo[7.2.0 .03,8]undec-2-enecarboxylate (11e). Com-
pound 11e was prepared from 10e according to General Procedure
F. The obtained product was precipitated from the reaction mixture;
yield: 66.0%, white crystals; mp 181 °C, [R]20D (H2O, 1.045 mg/
mL) ) +59.1. IR (KBr) ν [cm-1] 1102, 1219, 1390, 1595, 1757,
1
1189, 1088, 959, 779. H NMR (DMSO-d6) δ [ppm] 1.00-1.64
(m, 6H), 1.80 (d, 3H, J ) 7.2 Hz, CH3CHd), 3.00-3.09 (m, 1H),
3.11 (s, 3H, OCH3), 4.60 (d, 1H, J ) 10.4 Hz, H-9), 5.12 (t, 1H,
J ) 2.7 Hz, H-4), 6.29 (dq, 1H, J1 ) 7.0 Hz, J2 ) 1.5 Hz, CH3CHd
). 13C NMR (DMSO-d6) δ [ppm] 14.7, 20.1, 30.1, 31.8, 43.4, 54.8,
59.8, 72.0, 126.7, 134.0, 136.0, 140.2, 164.4, 170.9. HRMS ([M -
Na]-): Anal. calcd for C14H16NO4: 262.1079; found: 262.1086.
HPLC purity: 99.55 area%. HPLC purity (method 1): 100%.
1
2861, 2939, 3439. H NMR (DMSO-d6): δ 0.80 (t, 3H, J ) 7.3,
CH3-(CH2)3O), 1.18-1.83 (m, 10H), 1.73 (dd, 3H, J ) 7.1, 0.8,
CH3CHd), 2.01-2.12 (m, 1H), 2.80-2.92 (m, 1H, H-9), 3.24-
3.41 (m, 2H, OCH2(CH2)2CH3), 4.07 (d, 1H, J ) 7.0 Hz, H-9),
5.10 (deg. dd, 1H, J ) 2.6 Hz, H-4), 6.16 (dq, 1H, J ) 7.0, 1.5
Hz, CH3CHd). 13C NMR (75 MHz, DMSO-d6): δ 14.5, 14.6, 18.9,
19.4, 31.6, 31.8, 34.2, 43.9, 63.3, 66.0, 67.0, 68.8, 125.4, 133.3,
136.8, 142.9, 164.35, 170.90. MS (M + H)+ ) 328 m/z. HRMS
([M - Na]-): Anal. calcd for C17H22NO4: 304.1549; found:
304.1563. HPLC purity: 98.69 area%. HPLC purity (method 1):
98.76%.
Sodium
(8R,9R)-10(E)-Ethylidene-4(R)-ethoxy-11-oxo-1-
azatricyclo[7.2.0.03,8]undec-2 -enecarboxylate (11b). Compound
11b was prepared from 10b according to General Procedure F. The
obtained product was precipitated from the reaction mixture to give
11b; yield: 63.4%, yellow amorphous solid; mp decomp above
160 °C; [R]20d (H2O, 1.065 mg/mL) ) +10.2. IR (KBr) ν [cm-1
]
Sodium (8S,9R)-10(S)-[1(R)-Hydroxyethyl]-4(S)-butoxy-11-oxo-
1-azatricyclo[7.2.0. 03,8]undec-2-enecarboxylate (11f). Compound
11f was prepared from 10f according to General Procedure F. The
reaction mixture was dissolved in 50 mL of H2O and washed with
2 × 25 mL CH2Cl2. The product was obtained after lyophilization;
yield 36.9%, white-yellow crystals; mp 195 °C, [R]20D (H2O, 1.045
mg/mL) ) +47.7. IR (KBr) ν [cm-1] 1092, 1396, 1599, 1751, 2863,
2932, 3439. 1H NMR (DMSO-d6): δ 0.86 (t, 3H, J ) 7.3 Hz, CH3-
(CH2)3O), 0.98-1.85 (m, 10H), 1.76 (d, 3H, J ) 7.1 Hz, CH3-
CHd), 2.95-3.08 (m, 1H, H-9), 3.20-3.40 (m, 2H, OCH2(CH2)2-
CH3), 4.59 (d, 1H, J ) 10.1 Hz, H-9), 5.20 (t, 1H, J ) 2.8 Hz),
6.28 (dq, 1H, J1 ) 7.1, J2 ) 1.6 Hz, CH3CHd). 13C NMR (75
MHz, DMSO-d6): δ 13.8, 14.7, 19.0, 20.1, 30.2, 31.8, 32.1, 43.5,
59.7, 66.4, 70.2, 126.6, 134.9, 135.4, 140.1, 164.4, 170.9. HRMS
([M - Na]-): Anal. calcd for C17H22NO4: 304.1549; found:
304.1558. HPLC purity: 97.03 area%. HPLC purity (method 1):
96.81%.
1
3439, 2971, 2932, 2863, 1754, 1594, 1391, 1228, 1194, 1070. H
NMR (DMSO-d6) δ [ppm] 1.02 (t, 3H, J ) 7.0 Hz, CH3CH2O),
1.74 (d, 3H, J ) 7.0 Hz, CH3CHd), 1.22-1.33 (m, 2H), 1.42-
1.46 (m, 1H), 1.56-1.65 (m, 1H), 1.78-1.83 (m, 1H), 2.05-2.11
(m, 1H), 2.83-2.92 (m, 1H, H-8), 3.27-3.45 (m, 2H, CH3CH2O),
4.01 (d, 1H, J ) 7.2 Hz, H-9), 5.15 (brs, 1H, H-4), 6.18 (dq, 1H,
J ) 7.1 Hz, 1.5 Hz, CH3CHd). 13C NMR (DMSO-d6) δ [ppm] δ
14.7 (CH3CHd), 15.6 (CH3CH2O), 19.4 (C-6), 31.8 (C-5), 34.2
(C-7), 43.9 (C-8), 61.9 (CH3CH2O), 63.4 (C-9), 68.7 (C-4), 125.6
(CH3CHd), 133.1 (C-3), 137.0 (C-2), 142.9 (C-10), 164.3 (COOK),
170.8 (CdO). HRMS ([M - Na]-): Anal. calcd for C15H18NO4:
276.1236; found: 276.1227. HPLC purity: 98.02 area%. HPLC
purity (method 1): 99.9%.
Sodium
(8S,9R)-10(E)-Ethylidene-4(S)-ethoxy-11-oxo-1-
azatricyclo[7.2.0.03,8]undec-2 -enecarboxylate (11c). Compound
11c was prepared from 10c according to General Procedure F. The
obtained product was precipitated from the reaction mixture after
the addition of diethyl ether and petroleum ether to give 11c;
yield: 19.2%, yellow amorphous powder; mp decomp above
Sodium (8S,9R)-10(E)-Ethylidene-4(S)-(2′-fluoroethoxy)-11-
oxo-1-azatricyclo[7.2.0.03, 8]undec-2-enecarboxylate (11g). Com-
pound 11g was prepared from 10g according to General Procedure
F. The product was precipitated from the reaction mixture; 37%
180 °C, [R]20D (H2O, 1.04 mg/mL) ) +75.5. IR (KBr) ν [cm-1
]
3456, 2931, 1753, 1606, 1396, 1236, 1090, 1071. 1H NMR (DMSO-
d6) δ [ppm] 1.07 (t, 3H, J ) 7.0 Hz, CH3CH2O), 0.99-1.13 (m,
1H), 1.22-1.32 (m, 1H), 1.41-1.45 (m, 1H), 1.58-1.81 (m, 3H),
1.76 (dd, 3H, J1 ) 7.0 Hz, J2 ) 0.6 Hz, CH3CHd), 2.96-3.06 (m,
1H, H-8), 3.24-3.37 (m, 2H, CH3CH2O), 4.58 (d, 1H, J ) 10.1
Hz, H-9), 5.22 (t, 1H, J ) 2.7 Hz, H-4), 6,28 (dq, 1H, J1 ) 7.1
Hz, J2 ) 1.6 Hz, CH3CHd). 13C NMR (DMSO-d6) δ [ppm] 14.7
(CH3CHd), 15.5 (CH3CH2O), 20.2 (C-6), 30.2 (C-5), 32.1 (C-7),
43.5 (C-8), 59.7 (CH3CH2O), 62.1 (C-9), 70.0 (C-4), 126.6
(CH3CHd), 134.7 (C-3), 135.6 (C-2), 140.2 (C-10), 164.4 (COONa),
yield; white amorphous powder; mp decomp above 194 °C; [R]20
D
(H2O, 1.055 mg/mL) ) +89.0. 1H NMR (DMSO-d6) δ [ppm] 1.08
(m, 1H), 1.30 (m, 1H), 1.47 (m, 1H), 1.60-1.86 (m, 3H), 1.77 (d,
J ) 7.2 Hz, 3H, CH3CHd), 3.03 (m, 1H), 3.47 (m, 1H), 3.57 (m,
1H), 4.41 (m, 1H), 4.58 (m, 1H), 4.60 (d, J ) 10.5 Hz, 1H, H-9),
5.30 (t, J ) 2.60 Hz; 1H, H-4), 6.31 (dd, J1 ) 7.2, J2 ) 1.7 Hz,
1H, CH3CHd). 13C NMR (DMSO-d6) δ [ppm] 14.7, 20.0, 30.0,
31.9, 43.3, 59.8, 66.3 (J ) 19 Hz), 70.5, 82.8 (J ) 165 Hz), 127.0,
134.1, 135.6, 140.1, 164.4, 171.0. HRMS ([M - Na]-): Anal. calcd

4120 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
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489,318, Dec 2002.
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Gaviraghi, G.; Carlesso, R.; Bismara, C. EP 0416953B1; Chem. Abstr.
1991, 116:235337. (b) Andreotti, D.; Rossi, T.; Gaviraghi, G.; Donati,
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Lett. 1996, 6, 1683-1688.
for C15H17NO4F: 294.1136; found: 294.1142. HPLC purity: 98.2
area%. HPLC purity (method 1): 99.9%.
Sodium (8S,9R)-10(E)-Ethylidene-4(S)-(2′-cyanoethoxy)-11-
oxo-1-azatricyclo[7.2.0.03, 8]undec-2-enecarboxylate (11h). Com-
pound 11h was prepared from 10h according to General Procedure
F. The product was precipitated from the reaction mixture; 79%
yield; white amorphous powder; mp decomp above 188 °C. [R]20
D
(H2O, 1.075 mg/mL) ) +58.6. 1H NMR (DMSO-d6) δ [ppm]
1.04-1.85 (m, 6H), 1.78 (d, J ) 7.0 Hz, 3H, CH3CHd), 2.80 (m,
2H), 3.08 (m, 1H), 3.48 (m, 2H), 4.63 (d, J ) 10.2 Hz, 1H, H-9),
5.34 (t, J ) 2.6 Hz, 1H, H-4), 6.32 (qd, J1 ) 1.6, J2 ) 7.0 Hz, 1H,
CH3CHd). 13C NMR (DMSO-d6) δ [ppm] 14.7, 17.9, 20.0, 29.9,
31.9, 43.3, 59.7, 61.9, 70.1, 119.4, 127.1, 133.7, 136.2, 140.0, 164.2,
171.0. HRMS ([M - Na]-): Anal. calcd for C16H17N2O4: 301.1188;
found: 301.1181. HPLC purity: 97.0 area%. HPLC purity (method
1): 97.92%.
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Acknowledgment. We gratefully acknowledge the technical
expertise and contributions of Dr. Ursˇka Bratusˇek-Zavadlal and
Mr. B. Anzˇicˇ. The authors thank the members of the Analytical
group of Lek Pharmaceuticals d.d. for analytical and spectro-
scopic determinations. We are deeply indebted to Professor J.
M. Frere and members of the Centre for Protein Engineering
of the University Liege for their encouragement and continued
help.
Supporting Information Available: Purity of compounds
characterization data, details of data collection and structure
refinement statistics, and general synthetic procedures A-E for
preparation of intermediates 10a-h. This material is available free
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