Synthesis and Penicillin-binding Protein Inhibitory Assessment of Dipeptidic 4-Phenyl-β-lactams from α-Amino Acid-derived Imines

Article abstract of DOI:10.1002/asia.201901470

Monocyclic β-lactams revive the research field on antibiotics, which are threatened by the emergence of resistant bacteria. A six-step synthetic route was developed, providing easy access to new 3-amino-1-carboxymethyl-4-phenyl-β-lactams, of which the penicillin-binding protein (PBP) inhibitory potency was demonstrated biochemically.

Full text of DOI:10.1002/asia.201901470

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Synthesis and penicillin-binding protein inhibitory assessment of
dipeptidic 4-phenyl-β-lactams from α-amino acid-derived imines
Authors: Lena Decuyper, Marko Jukic, Izidor Sosic, Ana Maria
Amoroso, Olivier Verlaine, Bernard Joris, Stanislav Gobec,
and Matthias D'hooghe
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To be cited as: Chem. Asian J. 10.1002/asia.201901470
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10.1002/asia.201901470
Chemistry - An Asian Journal
COMMUNICATION
Lena Decuyper, Marko Jukič, Izidor
Sosič, Ana Maria Amoroso, Olivier
Verlaine, Bernard Joris, Stanislav
Gobec and Matthias D'hooghe*
Page No. – Page No.
Synthesis
and
penicillin-binding
In light of the bacterial resistance problem, a new synthesis of C4-substituted
3-amino-1-carboxymethyl--lactams was developed and their biological potential
demonstrated.
protein inhibitory assessment of
dipeptidic 4-phenyl--lactams from
-amino acid-derived imines
1
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10.1002/asia.201901470
Chemistry - An Asian Journal
Synthesis and penicillin-binding protein inhibitory assessment of
dipeptidic 4-phenyl--lactams from -amino acid-derived imines
Lena Decuyper,[a] Marko Jukič,[b] Izidor Sosič,[b] Ana Maria Amoroso,[c] Olivier Verlaine,[c]
Bernard Joris,[c] Stanislav Gobec[b] and Matthias D'hooghe*[a]
Abstract: Monocyclic -lactams revive the research field on promoted to become our first hit, favoured with promising
antibiotics, which are threatened by the emergence of resistant
PBP inhibitory activity and high
to gain additional insights in the chemistry and structure-
activity relationships of these complex -lactams, an
explorative study on synthetic routes towards C4-substituted
analogues was now initiated, holding on to the same design
objectives (Figure 1). In light of synthetic feasibility, 4-phenyl-
-lactams were targeted first to provide a proof of concept.
-lactamase stability. In order
bacteria. A six-step synthetic route was developed, providing easy
access to new 3-amino-1-carboxymethyl-4-phenyl- -lactams, of


which the penicillin-binding protein (PBP) inhibitory potency was
demonstrated biochemically.
2
In light of the rapidly escalating bacterial resistance problem,

2
antibiotic discovery remains
discovery endeavour.[1] To break away from traditional
bicyclic -lactam scaffolds, the research on monocyclic
-lactam antibiotics gains in importance, as we previously
a highly important drug
Their synthesis was perceived to be achievable through
functionalisation, in a similar way as previously described,[3]

of 3-amino-1-carboxymethyl-
-lactams. As opposed to C4-

unsubstituted -lactams, C4-substituted variants can be

demonstrated through a three-decade literature overview.[2]
Taking into account the assembled state-of-the-art, and
combining the structural properties of aztreonam (the only
FDA-approved monobactam), the nocardicins (the first
easily prepared via Staudinger’s ketene-imine [2+2]-
cyclocondensation.[4] To date, this is one of the most
renowned and versatile methods for the construction of the
four-membered amide ring system and is often selected for
its simplicity in reaction procedures and predictability of the
stereochemical outcome.[5] For the synthesis of 3-amino-
described, natural monocyclic
cephalosporins, our group recently studied
pharmacophore to mimic D-alanyl-D-alanine, the natural

-lactams) and late-generation
a
new
1

-lactams specifically, phthalimido- (FtN) and azido-
substrate of the penicillin-binding proteins, playing a crucial
role in the bacterial cell wall biosynthesis (Figure 1). Using a
virtual screening approach as an onset, a ten-step synthetic
containing ketenes are typically employed.[6] As a means to
shorten the synthetic pathway as much as possible, the
variable side chain ‘R’ could be introduced directly in the
pathway was developed towards
4-unsubstituted -lactams , characterised by diverse side
chains ‘R’. -Benzylidene-substituted analogue 1A was
a model library of
amine part of the imine. Indeed, when protected
-amino

1
acids would be used as cheap and easily available substrates

* [a] Dr. L. Decuyper, Prof. Dr. M. D’hooghe
SynBioC Research Group
Department of Green Chemistry and Technology
Faculty of Bioscience Engineering
Ghent University, Coupure Links 653, 9000 Ghent (Belgium)
E-mail: Matthias.Dhooghe@UGent.be
[b] Dr. M. Jukič, Dr. I. Sosič, Prof. Dr. S. Gobec
Department of Pharmaceutical Chemistry
Faculty of Pharmacy
University of Ljubljana, Aškerčeva 7, 1000 Ljubljana (Slovenia)
[c] Dr. A. M. Amoroso, O. Verlaine, Prof. Dr. B. Joris
Centre for Protein Engineering
Faculty of Sciences
University of Liège, Quartier Agora, Allée du 6 Août 13, Bât B6a, 4000 Liège Sart-Tilman (Belgium)
Supporting information for this article is given via a link at the end of the document.
2
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10.1002/asia.201901470
Chemistry - An Asian Journal
synthetic procedures
(
L-
/
DL-Phe); ii. introduce terminal
and condensed with benzaldehyde, the
-functionalised
hydrogen bond donors or acceptors (L-Glu, L-Trp and L-/D-
carboxymethyl moiety could be installed quite easily onto the
resulting -lactam N1-position. Based on the in silico design
criteria regarding the
-side chain ‘R’ (Figure 1[c]),[3] several
divergent -amino acids were selected to i. optimise the
Met, for oxidation to sulphones); and iii. change the linker
type to aromatic nocardicin-like motifs (D-Phg).[2]



Figure 1. Design strategy. [a] 3-Amino-1-carboxymethyl--lactam, mimicking the natural PBP substrate, D-alanyl-D-alanine, and enabling crucial molecular
interactions with the enzyme’s active site; [b] Optimised 2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido substituent;[2] [c] Variable side chain embedding a
lipophilic linker and terminal hydrogen bond-donating or -accepting functionalities, enabling additional non-covalent interactions with the key active site residues.
Taking all the above into account, L-phenylalanine 3A (R = imines 5A-A’ to yield 4-phenyl-3-phthalimidoazetidin-2-ones
Bn) as a model substrate was converted to the corresponding 8A-A’
. Profound variation of the Staudinger reaction
methyl ester 4A by means of 2,2-dimethoxypropane and conditions was necessary to enhance the conversion rate
hydrochloric acid (Method A, Scheme 1, Table 1). The and yield. The optimised set of reaction parameters and
resulting amine HCl salt was converted to the free base and corresponding reaction outcomes are displayed in Table 1.
subsequently condensed with benzaldehyde, affording As expected, comparison of the results of Methods E and F,
aldimine 5A in quantitative yield (Method C). Also the racemic showed that a higher amount of trans-isomer was obtained
starting material 3A’
view of chiral HPLC analysis at a later stage. Meanwhile, N- The accessibility of both cis- and trans-isomers was
phthaloylglycine was converted to the corresponding glycyl considered an asset with respect to future evaluation of the
chloride . After dehydrochlorination of acid chloride using biological potency of the corresponding target products
(
DL-Phe) was converted in this way in when 2,6-lutidine was used as a base in boiling toluene.[5]
6
7
7
2.
triethylamine, the in situ generated ketene was coupled with
Scheme 1. Staudinger synthesis of Ft-protected 3-amino-1-methoxycarbonylmethyl-4-phenyl--lactams 8.
3
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10.1002/asia.201901470
Chemistry - An Asian Journal
Table 1. Optimised synthesis of -substituted 3-amino-1-methoxycarbonylmethyl-4-phenylazetidin-2-ones 8.
Compound
Esterification
Imination
Staudinger synthesis
Method
Method
4 (%) (ee)[a]
Method
5 (%) (ee)[a]
dr before purif.
8 (%) (dr)
(Amino acid)
(Eq. base; Eq. 7)
E (1.5; 0.8)
3A (L-Phe)
A
99 (99)
C
D
C
D
D
D
D
D
46-99 (6)
99 (86)
64 (2)
31
56/44/0/0
8Acis: 5 (58/42)[b]
3A’ (DL-Phe)
3B (L-Glu)
3C (L-Trp)
3D (L-Met)
3D’ (D-Met)
3E (D-Phg)
A
B
99 (0)
79
F (4; 1.5)
F (2; 1.5)
F (2; 1.5)
F (2; 1.5)
F (2; 1.5)
F (3; 2.25)
3/2/53/42
2/1/51/46
0/0/63/37
4/4/53/39
4/4/53/39
8A’cis: 6 (59/41); 8A’trans: 64 (55/45)[b]
8Btrans: 89 (52/48)[c]
-
-
77
8Ctrans: 85 (59/41)[c]
8Dtrans: 64 (56/44)[c]
[d]
[d]
A
96
99
-
-
87
8D’trans: 60 (57/43)[c]
[d]
[d]
[b],[e]
[b],[e]
A
99
69
-
-
[a] Chromatographic conditions: see Supporting information. [b] Purified via automated column chromatography (C18). [c] Purified via automated column
chromatography (SiO2). [d] Commercially available. [e] Complex mixture, containing 29% of coupling product of 4E and 7, 4% 8Ecis (dr = 62/38) and 11% 8Etrans
(dr = 51/49).
Next, phthaloyl deprotection of compounds 8A-A’ using chemical transformations (Scheme 1, Scheme 2, Table 1,
hydrazine hydrate furnished 3-aminoazetidin-2-ones 9A-A’ Table 2). Despite the (presumed) use of chiral imines, a more
(Table 2, Scheme 2).[7] In the next step, the obtained free or less equimolar amount of the two trans-isomers 8B-E was
amines were coupled with 2-(2-aminothiazol-4-yl)-2-(methoxy- obtained in each case, which is in good agreement with the
imino)acetic acid (2-ATMO, predominantly syn) in alkaline statement that a chiral imine derived from a chiral amine and
environment
using
O-(benzotriazol-1-yl)-N,N,N',N'- an achiral aldehyde is not an appropriate chiral inductor in the
tetramethyluronium tetrafluoroborate (TBTU) as a coupling Staudinger synthesis.[10] Both L- and D-methionine 3D-D’ were
reagent. The resulting cyclic amides 10A-A’ were obtained in employed individually to be able to use the enriched mixtures
variable yields of 35-69% after conventional purification of compounds 10D-D’ for chiral HPLC method optimisation
techniques. Additional separation of the obtained and determine if the configuration of the chiral centre in
diastereomeric mixtures via preparative HPLC permitted to
isolate the individual diastereomers 10A1 10A2 and 10A’1
10A’2 (all trans). The isolated isomers were used to assess the
optical purity of the compounds with respect to the -position

-position of the carboxylic acid was now conserved
throughout the pathway (Table 2). When compared with L-
Phe-derived -lactams 10A (synthesised using imination
Method C, Table 1, ee = 6%), the epimerisation issue of most
other derivatives was far more limited (e.g., ee >76% for 10D’
Method D). As expected, only D-Phg-derived compounds
8-10E were completely racemised as a result of the
formation of an extensive conjugated system after
optically pure L-phenylalanine. Retrospective analysis of all deprotonation at the benzylic position.[11] In previous work,[3] it
,
,


after the different reaction steps via chiral HPLC. This
,
technique, however, demonstrated a low enantiomeric excess
(6%) for compounds 10A1 and 10A2
, which implied
5
,
epimerisation of the -position of the methyl ester derived from

intermediates revealed that epimerisation took place already
during imination (Scheme 1, Table 1, Method C), which is not
in accordance with preceding literature reports, stating
conservation of the chirality of similar amino acid-derived
imines using comparable or even harsher reaction conditions
(alkaline environment, elevated temperatures).[8] For this
reason, a new imination procedure was tested (Method D),
which did give rise to imine 5A with a considerably higher ee
of 86%.[9] Consequently, this method was applied for the
was shown that epimerisation at the -position can also take
place during subsequent hydrolysis, depending on the reagent
used. The deprotection reaction of diastereomerically pure
trans ester 10A1 using LiOH.H2O indeed caused slight
epimerisation (Method A, Table 2). SN2-type dealkylation with
LiI resulted in high stereoselectivity but lower conversion
(Method B). Similar results were obtained for the racemic
analogues 10A’. In either way, the loss of enantiopurity at the

-position was not a major issue as long as the biological
synthesis of the other derivatives
our target library was enlarged by converting the above-
mentioned -amino acids 3B-E to -lactams 10B-E via similar
5,8-10B-E as well. As such,
potency of these compounds had not been proven. Analogues
10B-E were therefore hydrolysed using the LiOH-mediated
procedure.


4
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10.1002/asia.201901470
Chemistry - An Asian Journal
Scheme 2. Functionalisation of key intermediates 8 towards target products 11.
Table 2. Yields and diastereomeric ratios of intermediate and target products 9-11.
Compound
AA
Deprotection
9 (%) (dr)
61 (53/47)
90 (59/41)
90 (58/42)
70 (53/47)
89 (51/49)
67 (59/41)
Acylation
Oxidation
Hydrolysis
10A-E (%) (dr)
35 (53/47)[b],[c]
36 (60/40)[b]
69 (54/46)[b],[c]
16 (51/49)[b]
22 (56/44)[e]
35 (55/45)[f]
10F-F’ (%) (dr)
Method
11 (%) (dr)
[a]
8Atrans
L-Phe
-
A (on 10A1); B (on 10A1)
99 (83/17); 50 (96/4)
99 (67/33)
8A’cis
DL-Phe
-
A
8A’trans
8Btrans
8Ctrans
8Dtrans
-
A (on 10A’1-2); A (on 10A’2)
99 (69/31); 99 (58/42)
99 (62/38)
L-Glu
L-Trp
L-Met
-
A[d]
-
A
99 (54/46)
-
A (on 10D)
A (on 10F)
A (on 10D’)
A (on 10F’)
A
99 (66/34)
10F: 30 (62/38)[g],[h]
99 (61/39)
8D’trans
D-Met
D-Phg
84 (55/45)
69 (54/46)[f],[h]
-
99 (58/42)
10F’: 35 (53/47)[f]
99 (57/43)
8Ecis
38 (66/34)
79 (62/38)
99 (60/40)[g],[h]
38 (52/48)[g],[h]
-
-
99 (74/26)
8Etrans
A
99 (70/30)
[a] dr = 68/32. [b] Purified via automated column chromatography (C18). [c] Additional purification via prep. HPLC permitted to separate the two diastereomers
10Atrans-1 and 10Atrans-2 (6% ee each) and 10A’trans-1 and 10A’trans-2 (0% ee each). [d] 2 eq. LiOH.H2O were used. [e] Purified via prep. TLC. [f] Purified via automated
column chromatography (SiO2). [g] Purified via prep. TLC. [h] Chiral HPLC analysis was performed on compounds 10D’trans (ee >76%), 10Ecis,trans (ee = 0%) and
10Ftrans (ee = 85%).
As mentioned before, in case of methionine-derived In order to provide some preliminary data on the biological
analogues 10D-D’, sulphide oxidation was performed prior to potential of this series of functionalised 3-amino-1-
hydrolysis using meta-chloroperbenzoic acid to bring along
the insertion of two additional hydrogen bond acceptors in a
terminal position, which is one of the premised design
requirements (Figure 1).[3],[12] Sulphones 10F-F’ were obtained
in this way without significant loss of enantiopurity at the
carboxymethyl--lactams 2 (after neutralisation of 11 with HCl
to provide the corresponding carboxylic acids), the
compounds were subjected to a PBP binding and competition
assay. The purified PBP3 of E. coli K12, PBP5fm of E. faecium
D63r and R39 DD-carboxypeptidase of Actinomadura spp.[13]

-position (ee(10Ftrans) = 85%, Table 2), and could
were incubated with acids 2, after which their residual activity
subsequently be hydrolysed in quantitative yields relying on (%RA) was determined by labelling the free enzymes with
LiOH.H2O. As such, this pathway enabled the preparation of a Bocillin FL, a fluorescent reporter molecule and penicillin V
set of novel cis- and/or trans-3-acetamido-1-carboxymethyl-4- analogue (Table 3).[14] The results revealed the remarkably
phenylazetidin-2-ones 11 in a limited amount of six (or seven low residual activity of PBP3 (10% RA), a lethal target of
for 11F-F’) steps in up to 30% overall yield, remarkably more E. coli, provoked by compound 2Etrans. The inhibitory potency
efficient compared with the synthesis of their C4-unsubstituted is higher than that of our initial hit 1A (28% RA),[3] confirming
analogues (ten steps, up to 2% overall yield).[3]
the potential of C4-substituted,

-aromatic – and therefore
-lactam scaffolds
The reactant scope of the synthetic pathway can be expanded
towards other aromatic or aliphatic aldehydes, combined with
nocardicin-like – 3-amino-1-carboxymethyl

for further PBP inhibitor design. The unprecedented and highly
straightforward six-step synthetic route described in this work,
readily available (un)natural -amino acids, if desired.
5
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10.1002/asia.201901470
Chemistry - An Asian Journal
converting simple substrates into highly functionalised C4-
(phenyl-)substituted nocardicin analogues, might therefore be
used to further guide expansion studies in the future starting
Acknowledgements
The authors gratefully acknowledge the Research Foundation
Flanders (FWO), the Slovenian Research Agency (research
program P1-0208) and Belspo IAP-Project P44-iPros for
financial support.
from 1-carboxymethyl--lactam 2Etrans as a promising new hit
structure.
Keywords: Amino acids • antibiotics • biological activity •
-lactams • Staudinger synthesis
Table 3. Residual enzymatic activities after incubation of PBP3, PBP5fm and R39
(2.5 µM) with compounds 2 (1 mM, pH 7, 30 °C, 3 h) and IC50 values.
Compound
%RA
%RA
%RA
PBP3 (IC50 (µM))
PBP5fm
R39 (IC50 (µM))
[b]
Aztreonam
Blanc
0 (0.004-0.022)[a]
-
0 (4)[c]
100
100
100
2A’cis; 2A’trans
2Btrans
87; 77
-
-
88
-
-
2Ctrans
79
-
-
2D’trans
89
-
-
2Ecis; 2Etrans
2Ftrans
73; 10 (720)[d]
-; 100
78
-; 100
99
83
]
[a] Computed from literature data.[15 [b] Aztreonam is not active against Gram-
positive bacteria. [c] Computed from literature data.[16] [d] Indicatory value due to
low solubility at >0.8 mM.
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6
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