Inhibition of Acinetobacter -Derived Cephalosporinase: Exploring the Carboxylate Recognition Site Using Novel β-Lactamase Inhibitors

Article abstract of DOI:10.1021/acsinfecdis.7b00153

Boronic acids are attracting a lot of attention as β-lactamase inhibitors, and in particular, compound S02030 (K_i = 44 nM) proved to be a good lead compound against ADC-7 (Acinetobacter-derived cephalosporinase), one of the most significant resistance determinants in A. baumannii. The atomic structure of the ADC-7/S02030 complex highlighted the importance of critical structural determinants for recognition of the boronic acids. Herein, to elucidate the role in recognition of the R2-carboxylate, which mimics the C₃/C₄ found in β-lactams, we designed, synthesized, and characterized six derivatives of S02030 (3a). Out of the six compounds, the best inhibitors proved to be those with an explicit negative charge (compounds 3a-c, 3h, and 3j, K_i = 44-115 nM), which is in contrast to the derivatives where the negative charge is omitted, such as the amide derivative 3d (K_i = 224 nM) and the hydroxyamide derivative 3e (K_i = 155 nM). To develop a structural characterization of inhibitor binding in the active site, the X-ray crystal structures of ADC-7 in a complex with compounds 3c, SM23, and EC04 were determined. All three compounds share the same structural features as in S02030 but only differ in the carboxy-R2 side chain, thereby providing the opportunity of exploring the distinct binding mode of the negatively charged R2 side chain. This cephalosporinase demonstrates a high degree of versatility in recognition, employing different residues to directly interact with the carboxylate, thus suggesting the existence of a carboxylate binding region rather than a binding site in ADC enzymes. Furthermore, this class of compounds was tested against resistant clinical strains of A. baumannii and are effective at inhibiting bacterial growth in conjunction with a β-lactam antibiotic.

Full text of DOI:10.1021/acsinfecdis.7b00153

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Article
Inhibition of Acinetobacter-Derived Cephalosporinase (ADC): Exploring
the Carboxylate Recognition Site Using Novel #-Lactamase Inhibitors
Emilia Caselli, CHIARA ROMAGNOLI, Rachel A. Powers, Magdalena A. Taracila, Alexandra A Bouza,
Hollister C Swanson, Kali A Smolen, Francesco Fini, Bradley J. Wallar, Robert A. Bonomo, and Fabio Prati
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00153 • Publication Date (Web): 16 Nov 2017
Downloaded from http://pubs.acs.org on November 17, 2017
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Inhibition of Acinetobacter-Derived Cephalosporinase (ADC): Exploring the
Carboxylate Recognition Site Using Novel β-Lactamase Inhibitors
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Emilia Caselli†, Chiara Romagnoli†, Rachel A. Powers#, Magdalena A. Taracila‡,
Alexandra A. Bouza#, Hollister C. Swanson#, Kali A. Smolen#, Francesco Fini†, Bradley J.
Wallar#, Robert A. Bonomo∞,‡* and Fabio Prati,†*
†University of Modena and Reggio Emilia, Department of Life Sciences, via Campi 101, 41125,
Modena, Italy
# Department of Chemistry, 1 Campus Drive, Grand Valley State University,
Allendale, MI, 49401
∞Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Research Service,
10701 East Blvd, Cleveland, OH, 44106
‡Case Western Reserve University, Departments of Medicine, Pharmacology, Biochemistry and
Molecular Biology and Microbiology, 10900 Euclid Avenue, Cleveland, OH, 44106
*Corresponding authors eꢀmail: fabio.prati@unimore.it; Robert.Bonomo@va.gov
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Boronic acids are attracting a lot of attention as βꢀlactamase inhibitors, and in particular
compound S02030 (Ki = 44 nM) proved to be a good lead compound against ADCꢀ7
(Acinetobacter Derived Cephalosporinase), one of the most significant resistance determinants in
A. baumannii. The atomic structure of the ADCꢀ7/S02030 complex highlighted the importance
of critical structural determinants for recognition of the boronic acids. Herein, to elucidate the
role in recognition of the R2ꢀcarboxylate, which mimics the C3/C4 found in βꢀlactams, we
designed, synthesized, and characterized six derivatives of S02030 (3a). Out of the six
compounds, the best inhibitors proved to be those with an explicit negative charge (compounds
3aꢀc and 3h,j, Ki = 44ꢀ115 nM), which is in contrast to the derivatives where the negative charge
is omitted, such as the amide derivative 3d (Ki = 224 nM) and the hydroxyamide derivative 3e
(Ki = 155 nM). To develop a structural characterization of inhibitor binding in the active site, the
Xꢀray crystal structures of ADCꢀ7 in a complex with compounds 3c, SM23, and EC04 were
determined. All three compounds share the same structural features as in S02030, but only differ
in the carboxyꢀR2 side chain, thereby providing the opportunity of exploring the distinct binding
mode of the negatively charged R2 side chain. This cephalosporinase demonstrates a high degree
of versatility in recognition, employing different residues to directly interact with the
carboxylate, thus suggesting the existence of a “carboxylate binding region” rather than binding
site in ADC enzymes. Furthermore, this class of compounds was tested against resistant clinical
strains of A. baumannii and are effective at inhibiting bacterial growth in conjunction with a βꢀ
lactam antibiotic.
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Keywords: βꢀlactamase, Boronic acid, Structure Activity Relationship, click chemistry,
carboxylate, structure activity relationship (SAR) study, enzyme plasticity, Acinetobacter
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Bacterial production of βꢀlactamases represents the most clinically challenging
mechanism of resistance to βꢀlactam antibiotics in Gramꢀnegative bacteria. Amongst these
multidrug resistant bacteria, Acinetobacter baumannii is one of the greatest clinical threats due to
its acquisition of numerous genetic determinants conferring resistance to all antibiotics.1 A.
baumannii harbors multiple antibiotic resistance determinants, such as class C, D and B βꢀ
lactamases, multidrug resistance efflux pumps, and a modified outer membrane that decreases
permeability to antimicrobials. Furthermore, A. baumannii possesses the ability to acquire new
resistance determinants by genetic exchange, natural transformation and outer membrane
vesicles. Preserving and restoring the efficacy of βꢀlactam antibiotics, which are still the major
class of drugs for the treatment of bacterial infections, is of critical importance as a strategy to
overcome and circumvent resistance mechanisms against this challenging pathogen.
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The Acinetobacterꢀderived cephalosporinases (ADCs) are chromosomallyꢀencoded class
C βꢀlactamases found in A. baumannii and other Acinetobacter spp. and are responsible for
resistance to penicillins, cephalosporins, and βꢀlactam/βꢀlactamase inhibitor combinations. Since
clavulanic acid, sulbactam and tazobactam, the commercially available βꢀlactamase inhibitors
bearing a βꢀlactam ring as their core structure, are not effective against ADC,2 the urgency of
discovering new active inhibitors is pressing. The bridged diazabicyclo [3.2.1]ꢀoctanone
avibactam entered the market in combination with ceftazidime and demonstrated potent
inhibitory activity against most Enterobacteriaceae that produce class A and class C βꢀ
lactamases, with some inhibitory capabilities against organisms producing class D enzymes.
Notably, this combination is not targeted for A. baumannii3 and other solutions are urgently
needed to treat infections caused by A. baumannii.
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A class of nonꢀβꢀlactam βꢀlactamase inhibitors that is recently attracting considerable
attention for their activity and pharmacological profile are boronic acid transitionꢀstate inhibitors
(BATSIs).4ꢀ7 Mechanistically, the electrophilic boron reacts with the nucleophilic catalytic serine
leading to the formation of a tetrahedral adduct that mimics the highꢀenergy intermediate of the
deacylation reaction with βꢀlactams (Scheme 1). Previous studies showed that the insertion of
different chemical groups on the carbon bearing the boronic moiety results in specific
interactions with different βꢀlactamase enzymes, allowing for a gain in inhibitory function and
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selectivity tuned for a specific class of βꢀlactamases. Boronic acids have been widely used for
inhibition of class C βꢀlactamases, such as AmpC, and recently they have been studied primarily
for inhibition of the cephalosporinase of Acinetobacter spp. (ADC).8 Of the reported BATSIs,
S02030 (compound 3a) is comprised with a set of features that makes it a promising lead
compound against ADC, both for its structure and activity (Ki = 44 nM), as well as its efficient
synthetic pathway.7
The chemical structure of S02030 was specifically designed to mimic the structure of the
natural substrate cephalothin in the deacylation high energy intermediate with the βꢀlactamase
(Figure 1). The structural features important for enzyme recognition and inhibition are depicted
in Figure 1: an R1 amide side chain bearing a 2ꢀthienylmethyl group, an oxyanion group
represented by one boronic hydroxyl, and a R2 cyclic side chain bearing a carboxylate meant to
mimic the C3/C4 carboxylate found in βꢀlactams. All these groups are spatially oriented as in the
natural substrate, in order to gain the proper interactions with the enzyme.
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R2 side chain
R1 amide side chain
R2 side chain
R1 amide side chain
H
N
N
S
S
N
N
H
O-
N
S
O
HO
N
B
OH
O-
O
O
OH
O
O-Ser-BL
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O
carboxylate
oxyanion
hole
O-Ser-BL
carboxylate
oxyanion
hole
a.
b.
Scheme 1. a. cephalothinꢀβꢀlactamase (BL) deacylation high energy intermediate. b. S02030 in
complex with a serine βꢀlactamase.
The crystal structure of ADCꢀ7 in complex with S02030 demonstrated that the inhibitor is
interacting with highly conserved residues in the catalytic pocket.7 In particular, the R1 amide
side chain is positioned in a region analogous to where βꢀlactam substrates bind in other class C
βꢀlactamases, interacting with highly conserved Asn152 and Gln120. Also, one hydroxyl group
of the boronic acid is positioned in the oxyanion hole and made hydrogen bonds with the main
chain nitrogens of Ser64 and Ser315 as well as the main chain carbonyl oxygen of Ser315.
Interestingly, the position and role of the R2 side chain, and specifically the carboxylate, is not
similar to other class C structures. In general, the C3/C4 carboxylate recognition site in class C
enzymes is reported to be comprised of Asn346 and Arg349 (AmpC numbering), but while these
residues were conserved in ADCꢀ7 (Asn343 and Arg346), the carboxylate of S02030 interacted
with Arg340, a residue that distinguishes ADCꢀ7 from other AmpCs. In class A βꢀlactamases,
the carboxylate binding pocket and the role of the carboxylate itself is well established;9ꢀ11 in
class C βꢀlactamases, we confirmed that neither the residues involved in recognition nor the role
of the carboxylate are sufficiently known.
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To elucidate the residues that take part in carboxylate recognition in ADCꢀ7 and its
importance for βꢀlactamase inhibition, we report the synthesis and characterization of six S02030
derivatives (3bꢀe, 3h, 3j) specifically designed to insert unique modifications localized on the R2
triazole substituent. Specific localized modifications were easily introduced using the flexible
and efficient synthesis previously described which relies on “click chemistry”, specifically, the
wellꢀknown copperꢀcatalyzed azideꢀalkyne cycloaddition (CuAAC).12 According to this
procedure, the substituted triazole moiety is formed by reaction of the chiral boronic scaffold 1,
bearing both the R1 thienylacetamido side chain and an azido group, with the proper
monosubstituted alkyne (Scheme 2). These alkynes are specifically chosen to introduce rational
variation of the carboxylate.
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Scheme 2.
3e = -CONHOH
3a = -COOH
H
N
H
N
S
S
N
3b = -CH2COOH
3c = -m-C6H5COOH
3d = -CONH2
1)
2) deprotection
3h =
Cu(I)
NH
N
R
N3
N
N
R
N
O
B
N
O
B
O
O
HO
OH
H
N
3a-j
3j =
1
N
N
N
All synthesized BATSIs were tested against ADCꢀ7 and confirmed to be potent
inhibitors. Since the best inhibition values were obtained for compounds with an explicit
negative charge, two additional boronic acids (SM2313 and EC01414) were tested and confirmed
to be amongst the best inhibitors. To elucidate the specific residues that interact with the
carboxylate, we determined the crystal structures of ADCꢀ7 in complex with the boronic acids
3c, SM23 and EC04. Comparison of these structures reveals critical differences in binding of the
carboxylate. The variability in the active site interactions with the R2ꢀcarboxylate suggests that
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class C βꢀlactamases display a high degree of plasticity and flexibility for recognition, pointing
to the existence of a carboxylate “binding region” rather than a “binding site” in ADCꢀ7. The
synergistic effect of these inhibitors paired with βꢀlactams to reverse antibiotic resistance in
whole cell assays against Escherichia coli and Acinetobacter baumannii strains expressing ADCꢀ
7 was also explored.
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RESULTS
Design. In previous studies, we demonstrated that boronic acids of general structure 3
were excellent inhibitors against class C βꢀlactamases,12 and in particular 3a was a 44 nM
inhibitor of ADCꢀ7 cephalosporinase.7 In this study, our goal was to elucidate the role of the
carboxylate in enzymeꢀinhibitor recognition. To achieve this end, we introduced specific
modifications near this R2ꢀcarboxylate group. Specifically, the carboxylate of 3a is elongated
with addition of a methylene bridge (3b), inserted on the metaꢀposition of a phenyl ring (3c),
modified to an amide group (3d) and to an hydroxamate (3e) and finally replaced by the
bioisostere tetrazole, either directly bonded to the triazole (3h) or as a metaꢀsubstituent on the
phenyl ring (3j). Compounds 3b, 3c, 3h and 3j are explicitly designed to introduce distance
modifications between the negative charge of the carboxylate and the electrophilic boron atom,
whereas the amide 3d and the hydroxamate 3e change the protonation profile of the substituent
while still maintaining heteroatoms able to form hydrogen bonds with the enzyme residues.
Synthesis of α-2-Thienylacetamido-β-Triazolylethaneboronic Acids.
The synthesis of compounds 3bꢀj is depicted in Scheme 3.
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H
N
H
N
H
N
S
N
S
S
N
R
Deprotection
81-96%
N3
N
N
N
R
N
R
O
B
B
O
O
B
72-99%
O
O
O
O
OH
HO
7
8
9
2f R= -CN
3e R= -CONHOH
1
3a R= -COOH
2b R= -CH2COOH
2c R= -C6H5COOH
2d R= -CONH2
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NH
2h R= N
3b R= -CH2COOH
3c R= -C6H5COOH
3d R= -CONH2
3h R=
N
NH
N
N
N
N
2g R= -C6H5CN
H
3j R=
N
N
N
2e R= -CONHOH
H
N
2j R=
N
N
N
N
Scheme 3. General Synthesis of αꢀ2ꢀThienylacetamidoꢀβꢀTriazolylethaneboronic Acids 3.
(+)ꢀPinanediol (1R)ꢀ2ꢀAzidoꢀ1ꢀ[(2ꢀthienylacetyl)amino]ꢀethaneboronate 1 was obtained
as previously described.12 Compound 1 was then reacted with six terminal alkynes in the copper
catalyzed AzideꢀAlkyne Cycloaddition (CuAAC). Amongst the six chosen alkynes, 3ꢀbutynoic
acid (R= –CH2COOH, for the synthesis of compound 2b) and 3ꢀcarboxyphenylacetylene (R= –
mꢀC6H5COOH, for compound 2c) are commercially available, whereas the other four alkynes,
propiolamide15 (R= –CONH2, for compound 2d), Nꢀhydroxypropiolamide (R= –CONHOH, for
compound 2e),16 propiolonitrile (2f)17 and 3ꢀethynylbenzonitrile (2g)18 were prepared following
established procedures.
As already described for compound 3a, formation of the triazole was conducted in very
mild conditions, in water/tertꢀbutyl alcohol as solvent, generating the Cu(I) catalyst in situ from
CuSO4 in the presence of an excess of ascorbate as reducing agent (5% catalyst, 20% ascorbate)
and at 60°C for approximately two hours. The expected triazole 2bꢀg were recovered in goodꢀ
excellent yields (72ꢀ99%) by extraction with ethyl acetate and crystallization of the crude
(Scheme 3). The formation of the triazole was confirmed by the presence in the 1HNMR spectra
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of a singlet downfield at 8.0ꢀ8.7 ppm which correlates (HSQC experiments) with a signal at 118ꢀ
131 ppm of the 13C NMR spectra, as expected for a triazolic CH. Moreover, the 1,4 substitution
was confirmed by the longꢀrange correlation (HMBC experiment). Triazoles 2hꢀj were obtained
by treatment of the cyano precursors with tetrabutylammonium fluoride trihydrate in
trimethylsililazide as solvent. Final deprotection of (+)ꢀpinanediolesters was accomplished by
transesterification with phenylboronic acid (isobutylboronic acid in case of 3h and 3j) in a
biphasic system of acetonitrile/nꢀhexane, to afford the final BATSIs 3bꢀe, 3h and 3j in 81ꢀ96%
yield.
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Inhibition Kinetics. The binding affinities for each of the BATSIs with ADCꢀ7 were
determined using competition kinetics. Utilizing nitrocefin, NCF, as a colorimetric substrate of
ADCꢀ7, boronic acids 3a-e, 3h and 3j were tested as inhibitors of ADCꢀ7 βꢀlactamase.
Additionally, three reference BATSIs were tested: compound s08127,12 which lacks the
substituent on the triazole ring, compound EC04,14 which replaces the triazole with a phenyl
group, and compound SM2313 where the phenyl aromatic moiety is directly connected to the
boron bearing carbon atom. The Ki values were calculated for all ten BATSIs and reported in
Table 1.
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Table 1: Ki values of Compound 3 derivatives with ADCꢀ7
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Cmp
Structure
R
Ki [nM]
COOH
44 ± 2
3a
3b
CH2-COOH
COOH
115 ± 2
46 ± 3
3c
CONH2
HN OH
O
224 ± 2
155 ± 3
3d
3e
H
N
S
N
N
N
R
O
HO
B
OH
76 ± 2
99 ± 2
3h
3j
N
N
N
N
N
N
NH
N
658 ± 6
53±4
s08127
EC04
H
N
S
–
–
O
B
HO OH
COOH
COOH
H
N
S
22±2
SM23
O
B
HO OH
All BATSIs were shown to be very active ADCꢀ7 inhibitors with Ki values in the
nanomolar range. We observed that affinity is diminished when the inhibitor lacks the
carboxylate group, such as for s08127 (Ki = 658 nM), and is less significant when the negative
charge is not present, as for the amide and hydroxyamate derivatives 3d and 3e (Ki = 224 and
155 nM respectively). Compound 3a proved to be a potent inhibitor of ADCꢀ7, with a Ki of 44
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nM. The boronic acid 3b, which exhibits an additional methylene linker between the carboxylate
and the triazole, demonstrates less activity by threeꢀfold (Ki = 115 nM), whereas 3c (Ki = 46
nM), bearing a phenyl linker, exhibits an equivalent activity to 3a. Inhibition is diminished when
the carboxylate negative charge is replaced by an amide derivative 3d (Ki = 224 nM) or a
hydroxyamide group 3e (Ki = 155 nM). We synthesized two additional boronates where the
negatively charged carboxylate is substituted with its bioisostere tetrazole, a group with a similar
pKa value, but a wider distribution of the negative charge. Indeed, the presence of a net negative
charge as for compound 3h (Ki = 76 nM) and for the phenyltetrazole 3j (Ki = 99 nM) improves
activity with respect to inhibitors lacking this negative charge.
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The binding affinity of all the other BATSIs spans from 22 nM (for SM23) to 115 nM
(3b), thus confirming the importance of a negative charge in the R2 side chain for recognition,
and simultaneously suggesting the presence of a “binding region” for this carboxylate rather than
of a “binding site” (Table 1).
X-ray Crystal Structures of ADC-7 in Complex with SM23, EC04 and 3c BATSIs.
To further define the role of the carboxylate group for ADCꢀ7 inhibition, the complexes of ADCꢀ
7 with SM23, EC04 and 3c were determined by Xꢀray crystallography to resolutions ranging
from 1.88ꢀ1.95 Å (Table 2, Figure 1). Each of the three inhibitors bears an unmasked carboxylate
at varying distances from the electrophilic boron. Complexes were obtained by soaking the
inhibitors into preꢀformed ADCꢀ7 crystals. The complexes crystallized in the same space group
(P21) with similar cell parameters. Inspection of the initial Fo – Fc maps countered at 3σ showed
unambiguous electron density for the inhibitors bound in the active sites of each structure. The
density was contiguous with the Oγ atom of Ser64, suggesting that the inhibitors were covalently
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attached to the catalytic serine residue, Ser64. Refinement of the inhibitors modeled into the
density revealed a tetrahedral geometry around the boron atom as expected. For compounds
EC04 and 3c, the position of the carboxylate was unambiguous, but electron density for the
SM23 carboxylate suggests two distinct conformations for the negative charge.
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Table 2. Crystallographic summary for the ADCꢀ7/BATSI complexes.
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ADC-7/3c
ADC-7/EC04
ADC-7/SM23
a=89.01
a=89.53
a=88.71
b=81.30
c=106.44;
β=112.70
b=81.30
c=106.69;
β=112.89
b=81.65
c=106.42;
β=112.84
Cell constants (Å; °)
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Space group
P21
P21
P21
Resolution (Å)
Unique reflections
Total observations
Rmerge (%)
1.88 (1.89ꢀ1.88)a
113,977
1.88 (1.89ꢀ1.88)a
113,662
1.95 (1.96ꢀ1.95)a
101,377
475,114
477,304
418,250
6.7 (62.4)
99.8 (99.8)
13.5 (2.2)
0.998 (0.732)
50.00ꢀ1.88
1,424
5.9 (65.3)
99.0 (98.1)
15.7 (2.2)
0.996 (0.734)
98.28ꢀ1.88
1,423
9.0 (70.6)
99.6 (99.6)
11.9 (2.3)
0.997 (0.719)
98.07ꢀ1.95
1,426
Completeness (%)b
<I/σI>
Resolution range for
refinement (Å)
Number of protein
residues
Number of water
molecules
RMSD bond lengths
(Å)
RMSD bond angles
(°)
589
516
628
0.018
0.018
0.018
Rꢀfactor (%)
1.896
20.2
1.865
20.1
1.915
18.7
R
free (%)c
Average
Bꢀfactor,
protein atoms (Å2)
25.6
25.5
24.6
Average
Bꢀfactor,
water molecules (Å2) 37.0
40.5
38.4
37.0
37.1
Average
Bꢀfactor,
36.7
inhibitor atoms (Å2)
a. Values in parentheses are for the highest resolution shell.
b. Fraction of theoretically possible reflections observed.
c. Rfree was calculated with 5% of reflections set aside randomly.
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SM23
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EC04
3c
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Figure 1: Stereoview of the SM23, EC04, 3c BATSIs bound to the catalytic residue, Ser64 of
ADCꢀ7. FoꢀFc omit maps are contoured at 2.5 σ. Atom coloring is as follows: oxygen atoms are
red, nitrogens blue, sulfurs yellow, borons pink, carbon atoms of SM23 are white, for EC04
carbons are cyan, and for 3c carbons are green. This figure was generated with PyMOL
[Schrodinger].
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The structures of the three BATSIs bound in the active site reconfirm the features essential for
enzymeꢀinhibitor recognition of both the R1 and R2 groups (Figure 2), initially described in the
ADCꢀ7/S02030 complex. Briefly, the inhibitor R1 side chain is bound in the highlyꢀconserved
amide binding site, where the amide oxygen hydrogen bonds with the side chain nitrogen atoms
of Gln120 and Asn152 and the main chain amide nitrogen and oxygen atoms of Ser315,
reminiscent of βꢀlactamase recognition of the R1 amide group of βꢀlactam substrates. The
inhibitor O1 hydroxyl groups are positioned in the oxyanion hole, hydrogen bonding with the
main chain nitrogens of Ser315 and Ser64 and also with the main chain oxygen of Ser315. The
inhibitor O2 hydroxyl groups make hydrogen bonds with the side chain hydroxyl of Tyr150, as
well as water molecule observed in most class C βꢀlactamase complexes. The distal thiophene
ring common to the inhibitors is positioned near the lip of the active site in all three structures,
extending over Tyr222 (Tyr221 in AmpC numbering). The thiophene ring of EC04 and SM23 is
observed to adopt two distinct conformations that exhibit an approximate 180° rotation of the
thiophene ring; in 3c, only one conformer is observed.
The R2 side chains display more variability in their binding modes than the R1 groups.
The carboxylateꢀbearing R2 side chain is structurally different in the three BATSIs, and the
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negatively charged group is observed to bind in two regions of the ADCꢀ7 active site. The
chemical structures of SM23 and EC04 are relatively similar, differing from each other by a
single carbon spacer between the boronꢀbearing carbon atom and the aromatic ring of EC04. The
carboxylate groups are bound in essentially the same region, a site comprised of residues Ser315,
Arg340, and Asn343. In the EC04 complex, the inhibitor carboxylate hydrogen bonds with the
amide nitrogen of Asn343 (3.2Å) and interacts with Arg340 via a waterꢀmediated bridge
(Wat412). This water molecule is not observed in the ADCꢀ7/SM23 complex, where the
carboxylate of SM23 occupies that space and adopts two conformations. In the first
conformation, the carboxylate hydrogen bonds directly to Arg340 (2.6Å) and Asn343Nδ2 (3.2Å)
and in the second, with the side chain hydroxyl of Ser315 (3.0Å) and a water molecule (Wat227;
2.9Å). Compound 3c varies more from the chemical structures of SM23 and EC04 in that 3c is
more extended due to the addition of a triazole linker between the boron and the benzoic acid
functionality. This linker allows the R2 group of 3c to extend over the R1 group and places the
carboxylate group toward an area near the edge of the active site. In this location, the carboxylate
is observed in a hydrogen bond with the side chain of Ser317 and near the bulk solvent.
Additionally, in this extended conformation, the aryl ring of 3c is positioned near Arg340 (Cζꢀ
centroid of aryl ring = 4.0 Å), making a potential cationꢀπ interaction with this conserved residue
in the ADC enzymes.
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SM23
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EC04
3c
Figure 2: SM23 (white), EC04 (cyan), 3c (lime green). Stereoview of the ADCꢀ7 active site in
complex with three, R2ꢀcontaining BATSIs. Hydrogen bonds observed are shown as yellow,
dashed lines. Cationꢀπ interactions are shown as magenta dashed lines. Water molecules are
shown as red spheres. Oxygens are colored red, nitrogens blue, boron pink and sulfur yellow.
The carbon atoms of the active site residues and BATSIs are colored by the various inhibitors in
complex with ADCꢀ7.
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Antimicrobial Activity. Disk susceptibility assays (DSAs) were performed using an
E.coli carrying the phagemid pBC SK expressing blaADCꢀ7 construct and in an A. baumannii
clinical strain. In both cases, significant inhibition of ADCꢀ7 βꢀlactamase by the BATSIs is
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observed at a concentration of 10µg/mL when partnered with the βꢀlactam ceftazidime (CAZ)
for the E. coli strain, and sulbactam (SULB) for the A. baumannii isolate tested (Table 3). The
DSA zone disk increased from 12 mm for CAZ alone to up to 26 mm for the best inhibitor 3a.
The results were very encouraging as the boronate compounds show cell penetration against
clinical isolates of A. baumannii when paired with sulbactam with the best ones, 3a and 3c
increasing the disk size from 6 mm to 16 and 15 mm, respectively. The broth MICs results show
that the addition of the BAI decreases the CAZ MIC from 32µg/ml to 1ꢀ2µg/ml for 3a, SM23,
3b, 3c, 3e, 3h, EC04. The results from DSA and broth MIC reveal that most of the compounds
perform similarly under the agar or liquid condition. The slight difference is for S08127, which
perform relatively better in liquid that in agar media, which can be attributed to lower diffusion
properties in agar. The highest MIC (8 µg/ml) for compound 3j (despite a very good affinity
Ki=99nM) can be due to cell penetration.
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Table 3. MIC were determined using the broth microdilution method with 10µg/ml compound,
and varying amounts of CAZ (from 0 to 128µg/ml) The DSAs were performed against E. coli
DH10B using ceftazidime as the BATSI antibiotic partner, and sulbactam for the AB0057
clinical strain (10µg BAI and 10 µg antibiotic partner).
9
E. coli carrying blaADC-7
AB0057
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encoded in pBC SK (-)
A. baumannii spp.
clinical strain
Cmpd.
MIC broth dilution
[µg/ml]
Disk assay
Disk assay
[mm]
[mm]
none
3a
32
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2
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8
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2
1
CAZ 12 mm
SUL 6 mm
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3b
3c
3d
3e
10
6
3h
3j
6
s08127
EC04
SM23
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DISCUSSION AND CONCLUSIONS
Although boronic acids are a class of compounds known since the 1980s to act as βꢀ
lactamase inhibitors,19,20 a significant amount of effort is presently focused on the discovery of
potent and selective boronic inhibitors to be administered together with a βꢀlactam. Currently, a
boronic acid, RPX7009 (vaborbactam), is commercially available in combination with
meropenem to treat carbapenem resistant Enterobacteriaceae infections.21 Unfortunately, this
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combination displays only a little enhancement of meropenem activity when tested against
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carbapenemꢀresistant A. baumannii or P. aeruginosa.22
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Thus far, several different classes of boronic acid inhibitors have been discovered. These
compounds display different profiles of activity, both in terms of microbiological and enzymatic
potency. In this study, we report our attempt to identify specific molecular determinants for the
recognition of BATSIs by a representative class C βꢀlactamase expressed in A. baumannii, ADCꢀ
7. We conducted a SAR study on the BATSI’s R2ꢀcarboxylate which is designed to mimic the
C3/C4 carboxylate found in βꢀlactams, starting from the lead compound S02030 (compound 3a).
The high degree of flexibility of the synthesis developed for this lead compound that bears a
simple carboxylate and the triazole substituent, allows easy access of a focused small library of
compounds bearing different substituents that aim either to: 1) distance the carboxylate from the
electrophilic center, 2) substitute the carboxylate with the bioisostere tetrazole, or 3) replace the
negative charge with an amide or a hydroxamide.
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The data reported herein suggest that ADCꢀ7 βꢀlactamase recognizes a negative charge in
this region, probably employing different residues in recognition. Indeed, compound s08127,
which lacks the substituent on the triazole ring, shows a significantly less potent Ki of 658 nM,
whereas the other two BATSIs bearing a mꢀcarboxyphenyl (SM23, Ki = 22 nM) or a mꢀ
carboxybenzyl (EC04, Ki = 56 nM) side chain, restore a comparable activity to the βꢀtriazole
derivatives.
To better understand the role of the carboxylate in this class of inhibitors, we determined
crystallographic complexes of ADCꢀ7 in a complex with three BATSIs that all have a
carboxylate in the R2 side chain: SM23, EC04 and 3c. The carboxylate is positioned on a phenyl
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ring in both SM23 and EC04, with the only difference being that EC04 contains an additional
methylene bridge between the aromatic moiety and the αꢀcarbon. Compound 3c is structurally
different, since it keeps the additional methylene linker, but replaces the phenyl with a triazole
that bears a metaꢀcarboxyphenyl substituent at position 4. The presence of two aromatic rings
between the boronic group and the carboxylate not only alters the distance of the negative charge
to the electrophilic center, but also adds hydrophobic character to the inhibitor.
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A superposition of the ADCꢀ7 active site in complex with the three R2ꢀcontaining
BATSIs highlights that the R1 side chain of all three structures maintain a similar conformation
and therefore the same interactions with active site residues. However, the R2ꢀcarboxylate is
located in distinct binding regions (Figure 3).
Figure 3. Stereoview superposition of the ADCꢀ7 active site in complex with the R2ꢀcontaining
BATSIs; 3c (green), EC04 (cyan), SM23 (white).
In compounds SM23 and EC04, the carboxylate is pointing towards a binding region
previously identified in the crystal structure of ADCꢀ7 in complex with the lead compound
S02030. This region is positively charged due to the presence of Arg340, which is either
interacting directly with the carboxylate (SM23) or through a water molecule(EC04) making it
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an attractive binding site for a negatively charged carboxylate group in ADCꢀ7 and possibly
other ADCs that contain this conserved residue. However, Arg340 is not ubiquitous among all
class C βꢀlactamases. For example, the crystal structure of E. coli AmpC in complex with
SM2313 shows a direct hydrogen bond between the inhibitor carboxylate and Asn289; a residue
that is not conserved in related class C lactamases, such as E. cloacae P99, where this residue is
a serine. Interestingly in ADCꢀ7, this interaction is not present due to an Asp at the analogous
position, as this negatively charged residue is flipped away from the inhibitor R2ꢀcarboxylate
group. The presence of Arg340 seems crucial for inhibitor recognition in ADCꢀ7: compound 3c
has an extended R2 side chain due to the presence of two aromatic rings, therefore its
carboxylate is placed in a different region of the enzyme, interacting with the side chain of
Ser317, whereas Arg340 is in the position for a cationꢀπ interaction with the inhibitor aryl ring.
Together, these two interactions seem to contribute to reach the same level of inhibition as for
compounds SM23 and EC04 (Figure 2 and Table 1). Cationꢀπ interactions have been shown to
contribute to proteinꢀprotein and proteinꢀligand interactions across a broad range of the
biological realm23 and arginine residues have been proposed to play a role in the structural
stability of numerous βꢀlactamases.24 The Arg340 residue therefore seems to attract a negative
charge in this region, but also demonstrates a high degree of versatility in recognition.
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On the other hand, data previously obtained on compounds SM23 and EC04 against E.
coli AmpC (Ki values of 1 nM)13, and Pseudomonas PDCꢀ3 (Ki values of 4 and 220 nM,
respectively)14, two class C enzymes lacking Arg340, point to the high plasticity of this class of
enzymes. This plasticity allows the class C βꢀlactamases to take advantage of different functional
groups that are not conserved with respect to position in primary sequence, but which can
mediate the same role in recognition of inhibitors and possibly βꢀlactam substrates.
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Microbiological data suggest the importance of the carboxylate, not only for recognition,
but also for cell activity. Our data suggest that cell penetration is enhanced by compounds
possessing a carboxylate functional group, followed by ones with hydroxamide functionality.
These data also show that cell penetration correlates well with affinity. The only compound with
good binding affinity but poor microbiological activity is compound 3j. For this compound, the
R2 chain comprises three aromatic rings with the triazole and the phenyl ring that are likely to
conserve the binding interaction observed with 3c (cationꢀπ) with Arg340, and the third tetrazole
ring placing the negative charge toward an area near the distal site (Ser317ꢀAsn213) and the bulk
solvent. However, the dispersion of the negative charge on an aromatic moiety is not beneficial
for the cell penetration.
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In conclusion, this work describes a SAR study on the role of the carboxylate R2 group
of a previously identified βꢀlactamase BATSI, compound S02030 (3a), which proved to be an
excellent inhibitor of ADCꢀ7. The synthetic approach to 3a and its derivatives, which relies on
the robust CuAAC click reaction, gives easy access to specifically designed modifications of the
carboxylate position. This study demonstrates that insertion of a negative charge in this position
improves binding affinity and cell penetration; however, due to the high degree of plasticity of
ADC, a wider choice of R2 negatively charged groups could be explored in future inhibitor
design.
METHODS
Protein Preparation and Purification
A version of ADCꢀ7 beginning at amino acid D24 and ending at K383 was cloned into a
pET28a(+) plasmid (no Hisꢀtag), expressed in the competent E. coli strain, BL21 (DE3), and
affinity purified by a mꢀaminophenyl boronic acid column as previously described.7 Using the
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A280 with an extinction coefficient of 46,300 Mꢀ1, the concentration of ADCꢀ7 was determined.
The extinction coefficient was calculated for the expressed residues in the ADCꢀ7 construct by
the ProtParam tool on the ExPASy bioinformatics portal.
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ADC-7 X-ray structure determination
The ADCꢀ7 crystals were grown by hanging drop vapor diffusion at room temperature.
The crystallization conditions consisted of 3 mg/mL ADCꢀ7 in 25% w/v polyethylene glycol
1500, 0.1 M succinate/phosphate/glycine buffer at pH 5.0 (SPG buffer, Molecular Dimensions).
The crystals were harvested from the drop using a nylon loop and soaked in crystallization buffer
containing 1 mM BATSI for 5ꢀ10 min and subsequently cryocooled in liquid nitrogen. Data were
measured for each ADCꢀ7/BATSI complex from an individual crystal at LSꢀCAT sector (21ꢀ1Dꢀ
G Beamline) at the Advanced Photon Source at Argonne National Laboratory (Argonne, IL).
The images were indexed, integrated and scaled with XDS.25 Molecular replacement was carried
out with Phaser26 using the ADCꢀ7 apo structure (PDB 4U0T).7 Refmac5 in the CCP4 program
suite was utilized to perform refinements.27,28 Repeated rounds of model building optimization
were completed using Coot.29 The coordinates and structure parameters for the ADCꢀ7/BATSI
complexes are stored with the Protein Data Bank as 5W12 (EC04), 5W13 (SM23), and 5W14
(3c).
Steady State Kinetics
Using an Agilent 8453 diode array spectrophotometer at room temperature, competition
kinetics were conducted in 10 mM phosphateꢀbuffered saline, pH 7.4. The βꢀlactam substrate and
colorimetric indicator, nitrocefin (NCF, ꢁε482 = 17400 Mꢀ1 cmꢀ1), was utilized. In each assay, the
measurements were taken after a 5 minutes preꢀincubation of enzyme with BATSI. The average
velocities (v0) were then fitted to equation 1,
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ꢃ [ꢅ]
ꢄ
ꢀꢁ = ꢀꢂ − ꢆ
(1)
ꢏ[ꢅ]
ꢇ(ꢈꢉꢊꢋꢌꢍꢋꢎ)
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where vu represents the uninhibited turnover of NCF, Ki(observed) is the concentration of inhibitor
that results in a 50 percent reduction of vu, and [I] is the concentration of inhibitor in the
experiment. Ki values were also corrected for NCF affinity (Km = 21.2 ꢂM) according to equation
2,
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ꢆ
ꢇ(ꢈꢉꢊꢋꢌꢍꢋꢎ)
[ꢙꢚꢛ]
ꢐ
=
.
(2)
ꢑ(ꢒꢓꢔꢔꢕꢒꢖꢕꢗ)
ꢘꢏ
ꢜ
ꢝꢙꢚꢛ
Disk Susceptibility Assays (DSAs) and Minimum Inhibitory Concentrations (MICs)
DSAs and MICs broth microdilutions were performed as previously described7 and according to
Clinical and Laboratory Standards Institute (CLSI) guidelines and methods30. Bacterial cultures
were grown overnight in MuellerꢀHinton (MH) broth supplemented with 20 ꢂg/mL of
chloramphenicol to ensure maintenance of the βꢀlactamase plasmid in pBC SK (−) containing
Escherichia coli DH10B strains. Liquid cultures were then diluted using MH broth to a
McFarland Standard (OD600 = 0.224). For DSA assay, bacteria were plated on MH agar and a
disk containing 10 ꢂg of boronic acid compounds and 10 ꢂg of ceftazidime or sulbactam were
added. After an overnight incubation at 37 °C, zone sizes were measured and reported. For the
MIC broth microdilution determinations, 200µl MH broth containing 10µg/ml BAI and
increasing concentration of CAZ (from 0 to 128µg/ml) was inoculated with the overnight
culture. After overnight incubation, MICs were determined.
Synthesis
All reactions were performed under argon using ovenꢀdried glassware and dry solvents. Dry
tetrahydrofuran (THF) and diethyl ether were obtained by standard methods and freshly distilled
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under argon from sodium benzophenone ketyl prior to use. The –100 °C bath was prepared by
addition of liquid nitrogen to a preꢀcooled (–78 °C) mixture of ethanol/methanol (1:1). Preloaded
(0.25 mm) glass supported silica gel plates (Kieselgel 60, Merck) were used for TLC analysis,
and compounds were visualized by exposure to UV light and by dipping the plates in 1 %
Ce(SO4)·4H2O, 2.5% (NH4)6Mo7O24·4H2O in 10% sulfuric acid followed by heating on a hot
plate. Chromatographic purification of the compounds was performed on silica gel (particle size
0.05ꢀ0.20 mm). Melting points were measured in open capillary tubes on a Stuart SMP30
Melting Point apparatus and are uncorrected. Optical rotations were determined at +20 °C on a
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1
13
PerkinꢀElmer 241 polarimeter and are expressed in 10ꢀ1 deg cm2 gꢀ1. H and C NMR spectra
were recorded on a Bruker Avanceꢀ400 MHz spectrometer. Chemical shifts (δ) are reported in
ppm and were calibrated to the residual signals of the deuterated solvent.27,28 Multiplicity is given
as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad signal; coupling
constants (J) are given in Hz. Twoꢀdimensional NMR techniques (COSY, HMBC, HSQC) were
used to aid in the assignment of signals in 1H and 13C spectra. Particularly, in the 13C spectra, the
signal of the boronꢀbearing carbon atom tends to be broadened, often beyond the detection limit;
however, its resonance was unambiguously determined by HSQC. Mass spectra were determined
on an Agilent Technologies LCꢀMS (n) Ion Trap 6310A (ESI, 70 eV). Highꢀresolution mass
spectra were recorded on an Agilent Technologies 6520 AccurateꢀMass QꢀTOF LC/MS.
The purity of all tested compounds was above 95%, determined by elemental analysis
performed on a Carlo Erba elemental analyzer 1110; results from elemental analyses for the
compounds were within 0.3% of the theoretical values. Combustion analysis and mass
spectrometry of free boronic acids provided inaccurate results because of the formation of
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dehydration products.31 Nevertheless, these boronic acids could be converted into analytically
pure pinanediol esters by exposure to an equimolar amount of pinanediol in anhydrous THF.
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(+)-Pinanediol (1R)-2-azido-1-(2-thienylacetylamino)-ethaneboronate (1), (1R)-2-[4-
carboxy-[1,2,3]triazol-1-yl]-1-(2-thienylacetylamino)ethaneboronic acid (3a) and (1R)-2-[4-
(3-carboxyphenyl)-[1,2,3]triazol-1-yl]-1-(2-thienylacetylamino)ethaneboronic acid (3c) were
synthesized according to the literature.12
General Procedure for Cu/C-catalyzed “click” reaction:
In a glass vial (+)ꢀPinanediol (1R)ꢀ2ꢀazidoꢀ1ꢀ(2ꢀthienylacetylamino)ꢀethaneboronate 1 (0.4
mmol) and the proper alkyne (0.4 mmol) were dissolved in tertꢀBuOH (3 mL) and CuSO4 (0.04
mmol), sodium ascorbate (0.08 mmol) and water (3 mL) were added. The vessel was sealed and
temperature raised at 60 °C. The reaction was monitored through TLC until disappearance of the
azidoboronate. After the proper reaction time, that spanned from 0.5 hour for more activated
alkynes to 2 hours for less activated alkynes, the reaction mixture was partitioned between
EtOAc (30 mL), H2O (12 mL) and saturated NaCl (7 mL). The aqueous phase was extracted with
EtOAc (2 x 20 mL) and the combined organic phases were washed with saturated NaCl, dried
over Na2SO4, and concentrated under vacuum to afford the crude product which was thereafter
purified as described.
(+)-Pinanediol
(1R)-2-(4-carboxymethyl-[1,2,3]-triazol-1-yl)-1-(2-
thienylacetylamino)-ethaneboronate (2b). The reaction was terminated after 1 hour. The crude
residue was triturated from diethylether to afford 2b as a white solid (117 mg, 80% yield), mp
1
180ꢀ182°C. [α]D – 94.5 (c 0.9, MeOH). H NMR (400 MHz, DMSO):
δ 0.81 (3H, s, pinanyl
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CH3), 1.22 (3H, s, pinanyl CH3), 1.24 (3H, s, pinanyl CH3), 1.36 (1H, d, J = 10.0, pinanyl
5
6
Hendo), 1.64ꢀ2.33 (5H, m, pinanyl protons), 2.98ꢀ3.02 (1H, m, BCHCH2), 3.66 (2H, s,
7
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9
CH2COOH), 3.90 (2H, s, CH2CONH), 4.05 (1H, d, J = 6.7, CHOB), 4.22ꢀ4.39 (2H, m,
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BCHCH2), 6.95ꢀ6.99 (2H, m, Har), 7.42ꢀ7.44 (1H, m, Har), 8.00 (1H, s, CHtriazole), 9.75 (1H, s,
13
CH2COOH). C NMR (100 MHz, DMSO):
δ 24.4, 26.7, 27.7, 29.6, 32.1, 37.0, 38.1, 43.1 (br,
CB), 52.4, 76.0, 83.0, 124.2 (CHtriazole), 126.2, 127.3, 127.4, 135.2, 140.6, 171.9, 175.4. MS (ESI,
Ion Trap): 473 [M + H]+, MS/MS 473, m/z (%): 346 (50), 321 (100), 168 (8). Anal. Calcd for
C22H29BN4O5S: C, 55,94; H, 6,19; N, 11,86; S, 6, 79. Found: C, 55.69; H, 6.21; N, 11.70; S, 6.93
(+)-Pinanediol
(1R)-2-(4-carboxamido-[1,2,3]triazol-1-yl)-1-(2-
thienylacetylamino)ethaneboronate (2d). Propiolamide was prepared from ethylpropiolate
according to literature procedure.15 The reaction was terminated after 30 min. The crude residue
was crystallized from EtOAc and nꢀhexane to afford 2d as a beige solid (162 mg, 89% yield), mp
1
144ꢀ146 °C. [α]D – 50.9 (c 0.9, MeOH). H NMR (400 MHz, MeOD):
δ 0.91 (3H, s, pinanyl
CH3), 1.33 (3H, s, pinanyl CH3), 1.38 (3H, s, pinanyl CH3), 1.43 (1H, d, J = 9.4, pinanyl Hendo),
1.80ꢀ2.40 (5H, m, pinanyl protons), 3.20 (1H, dd, J = 3.9, 10.6, BCHCH2), 3.98 (2H, s,
CH2CONH), 4.22 (1H, d, J = 6.9, CHOB), 4.43 (1H, dd, J = 10.6, 14.5, BCHCH2), 4.60 (1H, dd,
J = 4.0, 14.5, BCHCH2), 6.97ꢀ7.00 (2H, m, Har), 7.34 (1H, dd, J = 1.5, 5.0, Har), 8.45 (1H, s,
CHtriazole). 13C NMR (100 MHz, MeOD):
δ 23.2, 26.2, 26.4, 28.3, 31.0, 36.3, 37.7, 40.0, 44.4 (br,
CB), 52.2, 52.3, 76.1, 83.2, 125.4, 126.7 (CHtriazole), 126.8, 127.3, 133.2, 142.3, 163.4, 177.8. MS
(ESI, Ion Trap): 458 [M + H]+, MS/MS 458, m/z (%): 387 (3), 346 (22), 306 (100), 168 (4).
Anal. Calcd for C21H28BN5O4S: C, 55.15; H, 6.17; N, 15.31, S, 7.01. Found: C, 54.88; H, 6.01;
N, 15.25; S, 6.88.
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(+)-Pinanediol
(1R)-2-(4-idroxycarbamoyl-[1,2,3]triazol-1-yl)-1-(2-
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thienylacetylamino)ethaneboronate (2e). Nꢀhydroxypropiolamide was prepared from propiolic
acid according to literature procedure.16 The reaction was terminated after 30 min. The crude
residue was crystallized from EtOAc and nꢀhexane to afford 2e as a beige solid (155 mg, 82%
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yield), mp 105°C (dec). [α]D – 64.3 (c 0.6, CHCl3). 1H NMR (400 MHz, MeOD):
pinanyl CH3), 1.31 (3H, s, pinanyl CH3), 1.38 (3H, s, pinanyl CH3), 1.43 (1H, d, J = 9.4, pinanyl
endo), 1.79ꢀ2.40 (5H, m, pinanyl protons), 3.20 (1H, dd, J = 3.9, 10.3, BCHCH2), 3.98 (2H, s,
δ 0.90 (3H, s,
H
CH2CONH), 4.22 (1H, d, J = 8.5, CHOB), 4.40ꢀ4.62 (2H, m, BCHCH2), 6.95ꢀ7.00 (2H, m, Har),
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7.34 (1H, dd, J = 1.5, 5.0, Har), 8.44 (1H, s, CHtriazole). C NMR (100 MHz, MeOD):
δ 24.6,
27.6, 27.8, 29.7, 32.4, 37.7, 39.2, 41.4, 45.7 (br, CB), 53.6, 53.7, 77.5, 84.6, 126.8, 127.8
(CHtriazole), 128.1, 128.7, 134.6, 139.8, 160.6, 178.6. MS (ESI, Ion Trap): 474 [M + H]+, MS/MS
474, m/z (%): 346 (18), 322 (100), 168 (4). Anal. Calcd for C21H28BN5O5S: C, 53,28; H, 5,96; N,
14,80, S, 6,77. Found: C, 53.01; H, 6.10; N, 14.66; S, 6.55.
(+)-Pinanediol (1R)-2-[4-cyano-[1,2,3]triazol-1-yl]-1-(2-thienylacetylamino)ethaneboronate
(2f). Propiolonitrile was prepared according to literature procedure.17 The reaction was
terminated after 2 hrs. The crude residue triturated from Et2O to afford 2f as a beige solid (174
1
mg, 99% yield), mp 73 °C (dec). [α]D – 60.0 (c 1.0, MeOH). H NMR (400 MHz, MeOD):
δ
0.90 (3H, s, pinanyl CH3), 1.31 (3H, s, pinanyl CH3), 1.38 (3H, s, pinanyl CH3), 1.43 (1H, d, J =
10.4, pinanyl Hendo), 1.79ꢀ2.40 (5H, m, pinanyl protons), 3.19 (1H, dd, J = 3.9, 10.2, BCHCH2),
3.97 (2H, s, CH2CONH), 4.22 (1H, d, J = 6.8, CHOB), 4.47 (1H, dd, J = 10.2, 14.6, BCHCH2),
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