Synthesis of Avibactam Derivatives and Activity on β-Lactamases and Peptidoglycan Biosynthesis Enzymes of Mycobacteria

Article abstract of DOI:10.1002/chem.201800923

There is a renewed interest for β-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus because their β-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen–Sharpless cycloaddition reaction is reported. The amoxicillin–DBO combinations were active, indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 μg mL^?1 for both species). Mechanistically, β-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, the latter compounds were investigated as inhibitors of l,d-transpeptidases (Ldts), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of Ldts by S-carbamoylation indicating that optimization of DBOs for Ldt inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria.

Full text of DOI:10.1002/chem.201800923

A Journal of
Accepted Article
Title: Synthesis of avibactam derivatives and activity on β-lactamases
and peptidoglycan biosynthesis enzymes of mycobacteria
Authors: Zainab Edoo, Laura Iannazzo, Fabrice Compain, Inès Li de
la Sierra Gallay, Herman van Tilbeurgh, Matthieu Fonvielle,
Flavie Bouchet, Eva Le Run, Jean-Luc Mainardi, Michel
Arthur, Mélanie Ethève-Quelquejeu, and Jean-Emmanuel
Hugonnet
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
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To be cited as: Chem. Eur. J. 10.1002/chem.201800923
Link to VoR: http://dx.doi.org/10.1002/chem.201800923
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Synthesis of avibactam derivatives and activity on β-lactamases
and peptidoglycan biosynthesis enzymes of mycobacteria
Authors: Zainab Edoo,1 Laura Iannazzo,1 Fabrice Compain,[a,b] Inès Li de la Sierra Gallay,[d] Herman van
[a]
[c]
Tilbeurgh,[d] Matthieu Fonvielle,[a] Flavie Bouchet,[c] Eva Le Run,[a] Jean-Luc Mainardi,[a,b] Michel Arthur,*2 Mélanie
[a]
2
Ethève-Quelquejeu,2 and Jean-Emmanuel Hugonnet
[c]
[a]
1Equal contribution
2Equal contribution
*Corresponding author
[a] Ms Z Edoo, Dr. F Compain, Dr. M Fonvielle, Ms E Le Run, Dr. JL Mainardi, Dr. M Arthur, Dr. JE Hugonnet,
Sorbonne Université, Sorbonne Paris Cité, Université Paris Descartes, Université Paris Diderot, INSERM,
Centre de Recherche des Cordeliers, CRC, F-75006 Paris, France. E-mail: michel.arthur@crc.jussieu.fr; jean-
emmanuel.hugonnet@crc.jussieu.fr
[b] Dr. F Compain, Pr. JL Mainardi, Service de Microbiologie, Assistance Publique-Hôpitaux de Paris, Hôpital
Européen Georges Pompidou, Paris, France.
[c] Dr. L Iannazzo, Dr. M Ethève-Quelquejeu, Ms F Bouchet, Laboratoire de Chimie et de Biochimie
Pharmacologiques et Toxicologiques, Université Paris Descartes, UMR 8601, Paris, F-75005 France; CNRS UMR
8601, Paris, F-75006 France. E-mail: melanie.etheve-quelquejeu@parisdescartes.fr
[d] Dr. I Li de la Sierra Gallay, Dr. H van Tilbeurgh, Fonction et Architecture des Assemblages Macromoléculaires,
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198,
Gif-sur-Yvette cedex, France
Abstract: There is a renewed interest for β-lactams for treating infections due to Mycobacterium tuberculosis and
M. abscessus since their β-lactamases are inhibited by classical (clavulanate) or new generation (avibactam)
inhibitors, respectively. Here, we report access to an azido derivative of the diazabicyclooctane (DBO) scaffold of
avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction. The amoxicillin-DBO
combinations were active indicating that the triazole ring is compatible with drug penetration (minimal inhibitory
concentration of 16 µg/ml for both species). Mechanistically, β-lactamase inhibition was not sufficient to account
for the potentiation of amoxicillin by DBOs. Thus, we investigated the latter compounds as inhibitors of L,D-
transpeptidases (Ldts), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-
binding inhibitors of Ldts by S-carbamoylation indicating that optimization of DBOs for Ldt inhibition is an
attractive strategy to obtain drugs selectively active on mycobacteria.
The β-lactams remain the first-line treatment for numerous bacterial infections despite the emergence of
multiple resistance mechanisms resulting from an intensive use of the drugs in the last 70 years. This reflects the
covalent mechanism-based mode of action of β-lactams and the unique properties of their targets, the
peptidoglycan D,D
-transpeptidases belonging to the penicillin-binding protein (PBP) family.[1] These properties
include the presence of multiple essential paralogues in nearly all bacteria and a periplasmic location. To combat
resistance, the β-lactam scaffold has been successfully engineered to obtain drugs that are active against low-
affinity PBPs, are poorly hydrolyzed by β-lactamases, or act as inhibitors of these enzymes.[2] However, this
strategy is presently not sufficient to counteract the rapidly evolving resistance mechanisms involving the
production of extended-spectrum and inhibitor-resistant β-lactamases and of low-affinity PBPs.
Avibactam, a recently approved β-lactamase inhibitor, is original in its structure since it is based on a
diazabicyclooctane (DBO) scaffold containing a cyclic urea rather than a β-lactam ring found in classical inhibitors
such as clavulanate (Figure 1a).[3] Avibactam is also original in its mode of action since it reversibly inactivates β-
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lactamases containing an active-site serine by formation of a carbamoyl-enzyme, which is not, or poorly, prone
to hydrolysis.[2b, 4]
Although avibactam has been developed for inhibiting β-lactamases from multi-drug resistant Gram-negative
bacteria, we have recently reported that avibactam is also active against the β-lactamase BlaMab from
Mycobacterium abscessus.[5] Avibactam potentiates the activity of imipenem, a β-lactam of the carbapenem class
used in the reference treatment of lung infections due to M. abscessus in cystic fibrosis patients.[6] Of note,
avibactam is a poor inhibitor of BlaC from Mycobacterium tuberculosis. Clavulanate, a classical β-lactam-
containing β-lactamase inhibitor, is hydrolyzed by BlaMab.
[7] In contrast, clavulanate irreversibly inhibits BlaC[8] and
this molecule is currently evaluated for the treatment of tuberculosis in combination with the carbapenem
meropenem.[9] The opposite behavior of BlaMab and BlaC with avibactam and clavulanate is almost exclusively
determined by a single amino acid polymorphism, i.e. the presence of motif SDG in BlaC instead of the nearly
invariant motif SDN present in BlaMab and in most class A β-lactamases.[10]
The potential of avibactam for improving the efficacy of β-lactams against mycobacterial infections prompted
us to further investigate DBOs and to develop a synthetic route to easily obtain functionalized DBOs. Synthesis of
L L-pyroglutamic
avibactam was previously described using chiral piperidine derivatives,[11] -glutamic acid,[12] or
acid[12] as starting material. Recently, the process has been optimized starting from commercially available Boc-
benzylglutamate.[13] Synthetic efforts devoted to the discovery of new DBOs have also been based on the olefin
metathesis reaction[14] or on a lipase catalytic resolution.[15] Here, we report a versatile route for the
functionalization of DBOs via the copper(I)-catalyzed Huisgen-Sharpless cycloaddition reaction (Figure 1c). We
show that the resulting triazole-containing DBOs retain the capacity to reach the periplasm of mycobacteria and
to inhibit their β-lactamases. We also unexpectedly show that DBOs act as slow-binding inhibitors of
transpeptidases, opening the way to the development of antibiotics specific to mycobacteria.
L,D-
CH2OH
H2N
O
H2N
O
O
a)
H
CH2OH
H+
O
H+
N
N
N
N
CO2H
O
O
O
O
HN
N
CO2H
O
O
O
O
O
O
O
O
Fragmentation and
hydrolysis
OH
S
S
OH
O
Clavulanate
O
Ser
Ser
Ser
EI*
EI
Ser
EI
EI*
BnO
NaO3SO
c)
Avibactam
NaO3SO
b)
Click chemistry
R
N
N
CO2Me
N
N
N
N
Boc
O
N3
N
N
N
O
O
NH2
New DBO
N
O
O
Figure 1. a) Mechanism of action of β-lactamase inhibitors. EI, non-covalent β-lactamase:inhibitor complex; EI*,
covalent adduct resulting from the carbamoylation (avibactam) or acylation (clavulanate) of the active-site serine
of the β-lactamase. b) Structure of avibactam. c) Retrosynthetic scheme for the synthesis of DBO analogues.
Our synthetic approach to obtain avibactam analogues leads to azido DBO 13 from a commercially available
oxopyrrolidine in 12 steps followed by a Huisgen-Sharpless cycloaddition as a key step (Figure 1c). In the first step
(Scheme 1), a premixed slurry of trimethylsulfoxonium iodide and potassium tert-butoxide are added to the
oxopyrrolidine to afford the β-keto sulfoxonium ylide 1.[16] The next step, catalyzed by an iridium complex, leads
to the formation of the piperidinone 2. The latter compound is selectively reduced to alcohol 3 and converted
into compound 5 by a Mitsunobu reaction with N-protected O-benzyl hydroxylamine 4.[17] Compounds 6 and 7
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are obtained by deprotection of the N-O-benzyl hydroxylamine and of the N-Boc, respectively. Reduction of the
methyl ester affords alcohol 8, which is then protected by a silyl group to obtain compound 9. The bicyclic urea
ring of 10 is formed in the presence of triphosgene.[18] Final modification steps of the side-chain lead to the
formation of precursor 13 by deprotection and activation of the alcohol, and addition of sodium azide.
Scheme 1: a) S(O)(CH3)3I, tBuOK, DMSO, rt, 2 h (53%), b) [Ir(COD)Cl]2, DCE, 80°C, overnight (72%), c) NaBH4, MeOH, 2 h, 0°C
(91%), d) DEAD, PPh3, THF, rt, overnight (83%), e) PhSH, K2CO3, MeCN, rt, overnight (98%), f) TFA, DCM, rt, overnight (97%),
g) LiAlH4, THF, 0°C, 2 h (51%), h) TBDMSCl, imidazole, DCM, rt, overnight (79%), i) triphosgene, DIPEA, MeCN, 0°C, 2 h (61%),
j) TBAF, THF, 0°C, 1 h (98%), k) MsCl, NEt3, DMAP, DCM, 0°C, 1 h (73%), l) NaN3, DMF, 80°C, overnight (78%).
Functionalization of azido 13 (Scheme 2) was performed by copper(I)-catalyzed 1,3-cycloaddition in the
presence of commercially available alkynes affording compounds 14a and 14b in good yield (Scheme 2). Access
to the final DBOs, 15a and 15b, included debenzylation of 14a and 14b by hydrogenolysis followed by sulfation
with pyridinium sulfate.
Scheme 2: a) CuSO4 30 mol%, sodium ascorbate 60 mol%, THF/H2O (2/1), rt overnight (14a 77%, 14b 99%), b) H2, Pd/C,
MeOH, rt, 48 h, c) SO3-Pyr, Pyr, rt, overnight, d) DOWEX Na+ (15a 10%, 15b 9%).
The biological activity of avibactam and 15a was evaluated by determining their ability to potentiate the
activity of amoxicillin, a β-lactam of the penicillin class, against mycobacteria (Table 1). Avibactam inhibited β-
lactamases BlaC and BlaMab in the periplasm of M. tuberculosis and M. abscessus since the minimal inhibitory
concentrations (MICs) of amoxicillin were reduced at least 16 fold (from 128 to 8 µg/ml and from >256 to 16
µg/ml, respectively). Inhibition of BlaC and BlaMab was apparently partial since the MICs of amoxicillin remained
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higher than those observed for isogenic strains obtained by deletion (Δ) of the blaC and blaMab genes. 15a was
similarly active in reducing the MICs of amoxicillin indicating that functionalization of the DBO scaffold by the
cycloaddition reaction affords biologically active compounds. Since the antibacterial activity of 15a and 15b
against M. abscessus was similar (Supplementary Table S1) biological evaluation was only pursued with 15a.
Table 1. Potentiation of the activity of amoxicillin by β-lactamase inhibitorsa
MIC of amoxicillin (µg/ml) in the
presence or absence of DBOs
Strain
β-lactamase
BlaC
None
None
128
1
>256
4
Avibactam
15a
16
1
16
4
M. tuberculosis H37Rv
M. tuberculosis H37Rv ΔblaC
M. abscessus
8
0.5
16
4
CIP104536
BlaMab
M. abscessus
a
CIP104536 ΔblaMab None
Minimal inhibitory concentrations (MICs) are defined as the lowest drug concentration that
prevents visible growth. Avibactam and 15a were used at a concentration of 15 µM (4 and 6
µg/ml, respectively). DBOs had no antibacterial activity when tested alone (MIC > 64 µg/ml).
Inhibition of the β-lactamase activity of BlaC and BlaMab was determined by using the chromogenic
cephalosporin nitrocefin as the substrate (Table 2 and supplementary Figure S1). As previously described, BlaMab
was effectively inhibited by avibactam,[10a, 19] whereas an 8-fold lower carbamoylation efficacy was obtained for
15a. As previously described, BlaC was poorly inhibited by avibactam[8a] and marginally inhibited by 15a
preventing quantitative assessment of the carbamoylation constant for the latter compound. This is in marked
contrast with potentiation of the antibacterial activity of amoxicillin by avibactam and 15a against M. tuberculosis
(Table 1). This observation suggests that the DBOs might not only inhibit the β-lactamases, but additionally act
on peptidoglycan polymerases.
Table 2. Efficacy of β-lactamase inhibition by DBOs
k2/Ki (M-1 s-1)
β-lactamase
BlaMab
BlaC
Avibactam
15a
(2.2 ± 0.1) x 104
< 5 x 100
(1.7 ± 0.1) x 105
(2.4 ± 0.8) x 101
The target of β-lactams is unusual in M. abscessus and M. tuberculosis since the last cross-linking step of
peptidoglycan synthesis is mostly performed by
-transpeptidases, which replace classical PBPs.[20] This
prompted us to investigate inhibition of the model enzyme of the -transpeptidase family, Ldtfm, by avibactam.
L,D
L,D
Incubation of Ldtfm with avibactam for various times followed by titration of the free active site by nitrocefin
indicated time-dependent and complete inactivation of the enzyme (Figure 2a and b). Further analyses showed
that the rate of inactivation of Ldtfm by avibactam linearly increases with the inhibitor concentration (Figure 2c).
Pre-incubation
with avibactam
0 min
100
80
60
40
20
0
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
25
20
15
10
5
a)
b)
c)
20 min
30 min
40 min
60 min
90 min
0
00
0.02
0.04
0.06
0.08
0.1
0
200 400 600 800 1,000 1,200 1,400
0
20
40
60
80
100
120
Time (min)
Pre-incubation time with avibactam (min)
Avibactam (µM)
Figure 2. Slow-binding inhibition of Ldtfm by avibactam assayed by titration of the free active site with nitrocefin. a) Ldtfm (40
µM) was incubated with avibactam (1,000 µM) for 0 to 170 min and the free active site was titrated by nitrocefin. b) Time-
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k
t
dependent inactivation of Ldtfm. Data were fitted to exponential decay (E/E° = e- obs , in which E/E° is the proportion of free
enzyme). c) kobs linearly increased with the concentration of avibactam revealing an S-carbamoylation efficacy of 0.7 M-1 s-1.
Mass spectrometry revealed formation of a covalent adduct between Ldtfm and avibactam (Supplementary Table
S2). The S-carbamoyl-enzyme was stable upon prolonged incubation at 22°C (up to 8 days) although minor forms
resulting from desulfation or hydrolysis of the thioester bond were detected (Supplementary Table S3 and figure
S2). The adduct was not observed for a derivative of Ldtfm in which the catalytic Cys was replaced by Ala.
Accordingly, co-crystallization of Ldtfm with avibactam established that the inhibitor was covalently linked to the
catalytic Cys residue (Figure 3 and Supplementary figure S3). The position of the sulfate of avibactam suggests
that it might be in hydrogen interaction with two His (421 and 440) and two Ser (405 and 439) residues. Since
desulfation of the S-carbamoyl-enzyme may have occurred during crystallogenesis, we assessed the stability of
the adduct in nitrate buffer, which was also used for crystallogenesis. No free form of Ldtfm was detected after
four weeks of incubation indicating that the S-carbamoyl-enzyme was stable (Supplementary Table S4). However,
this does not formally exclude the possibility that desulfation occurred in the crystal and that the electron density
attributed to the avibactam sulfate could in fact correspond to a phosphate originally present in the Ldtfm
purification buffer.
Figure 3. a) Structure of avibactam in the Ldtfm active site (PDB accession code 6FJ1) (Supplementary Table S5 for data-
collection and refinement statistics). The sulfate of avibactam might be stabilized by hydrogen interactions with S405, H421
S439, and H440 (dashed lines). b) Electron density map showing covalent linkage of avibactam to the catalytic Cys.
,
Our next objective was to determine whether DBOs could inhibit the peptidoglycan cross-linking reaction in
mycobacteria. There are five
-transpeptidase paralogues in mycobacteria,[20a] including LdtMt2, which has been
L,D
identified as the main cross-linking enzyme during exponential growth of M. tuberculosis.[21] DBO 15a and
avibactam were both found to inhibit LdtMt2 (Supplementary Figure S4).
In conclusion, we have developed access to an azido-DBO that can be easily functionalized by the Huisgen-
Sharpless cycloaddition reaction. Using this click chemistry reaction, we synthesized two new DBOs and showed
that the resulting triazole linker is compatible with biological activity and penetration into the periplasm of M.
tuberculosis and M. abscessus. In these mycobacteria, the potential targets involved in peptidoglycan cross-
linking are complex, as they include the
L,
D-transpeptidases (Ldts) and the
D,
D-transpeptidases (PBPs), responsible
for formation of 80% and 20% of the cross-links, respectively, as well as the
D,D-carboxypeptidases that generate
the tetrapeptide-containing substrate of the Ldts.[22] The potentiation of the antibacterial activity of amoxicillin
by avibactam and 15a (Table 1) is likely to involve inhibition of these biosynthetic enzymes by the DBOs since the
antibacterial activity of the amoxicillin-DBO combinations poorly correlates with the inhibition efficacy of the β-
lactamases BlaMab and BlaC (Table 2). In agreement, we showed that the DBOs act as slow-binding inhibitors of
the model
observations suggest that inhibition of the Ldts may contribute to antibacterial activity, although the inhibition of
the -carboxypeptidases and of the -transpeptidases, that were not explored in this study, may also be
L,D-transpeptidase Ldtfm (Figure 2) by S-carbamoylation of the active-site Cys residue (Figure 3). These
D,
D
D,D
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important. In agreement DBOs active on PBPs have been reported.[23] Since transpeptidases of the
L,D and D,D
specificities are structurally unrelated and proceed via distinct catalytic mechanisms involving Cys and Ser
nucleophiles,[24] development of DBOs specifically active on Ldts is an attractive strategy to obtain drugs
selectively active on mycobacteria. Specificity of such compounds would rely on the fact that the L,D-
transpeptidation pathway is absent or plays a minor unessential role in peptidoglycan polymerization in most
bacteria.[25] Development of such Ldt-specific drugs would prevent side effects of current β-lactams, such as the
carbapenems, which lead to opportunistic fungal infections and the selection of resistance to these last resort
antibiotics. It is therefore worth noting that the carbapenemase KPC-2, one of the key targets in the development
of avibactam, was carbamoylated 50-fold less effectively by 15a than by avibactam (Supplementary Figure S5).
This observation indicates that selectivity could potentially be achieved by the functionalization of the DBO
scaffold. The route of synthesis of DBOs and the various bacteriologic and enzymatic assays described in this study
will pave the way to the development of such specific inhibitors.
Acknowledgements
We thank L. Dubost and A. Marie for technical assistance in the collection of mass spectra. This work was
supported by the French National Research Agency (grant MycWall, ANR-17-CE18-0010), the Fondation pour la
Recherche Médicale (grant ECO20160736080 to ZE), and Vaincre la Mucoviscidose (grant RIF2017050244 to JLM).
Keywords: Avibactam • β-lactamase • L,D-transpeptidase • Mycobacterium abscessus • Mycobacterium
tuberculosis
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Chemistry - A European Journal
COMMUNICATION
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10.1002/chem.201800923
Chemistry - A European Journal
COMMUNICATION
COMMUNICATION
Zainab Edoo,1[a] Laura Iannazzo,1[c]
[a,b]
A synthetic route to functionalized
diazabicyclooctanes (DBOs) was
developed based on incorporation
of an azido group for CuI-catalyzed
Huisgen-Sharpless cycloaddition.
Fabrice Compain,
Gallay,[d]
Inès Li de la
Herman
Sierra
van
Tilbeurgh,[d]
Matthieu
Fonvielle,[a]
Flavie Bouchet,[c] Eva Le Run,[a] Jean-
Luc Mainardi,[a,b] Michel Arthur,2[a]
Mélanie Ethève-Quelquejeu,2[c] and
DBOs
inhibited
the
L,D-
Jean-Emmanuel Hugonnet2[a]
1Equal contribution
transpeptidases
involved
in
2Equal contribution and corresponding authors
peptidoglycan polymerization in
addition to the expected activity
on β-lactamases. Optimization of
Page No. – Page No.
DBOs
for
inhibition
of
peptidoglycan polymerases is an
attractive strategy to obtain drugs
Synthesis of avibactam derivatives
and activity on β-lactamases and
peptidoglycan biosynthesis
selectively
active
on
mycobacteria.
enzymes of mycobacteria.
This article is protected by copyright. All rights reserved.