Design and enantioselective synthesis of 3-(α-acrylic acid) benzoxaboroles to combat carbapenemase resistance

Article abstract of DOI:10.1039/d1cc03026d

Chiral 3-substituted benzoxaboroles were designed as carbapenemase inhibitors and efficiently synthesisedviaasymmetric Morita-Baylis-Hillman reaction. Some of the benzoxaboroles were potent inhibitors of clinically relevant carbapenemases and restored the activity of meropenem in bacteria harbouring these enzymes. Crystallographic analyses validate the proposed mechanism of binding to carbapenemases,i.e.in a manner relating to their antibiotic substrates. The results illustrate how combining a structure-based design approach with asymmetric catalysis can efficiently lead to potent β-lactamase inhibitors and provide a starting point to develop drugs combatting carbapenemases.

Full text of DOI:10.1039/d1cc03026d

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Design and enantioselective synthesis
of 3-(a-acrylic acid) benzoxaboroles to combat
carbapenemase resistance†
Cite this: Chem. Commun., 2021,
57, 7709
Received 8th June 2021,
Accepted 17th June 2021
a
You-Cai Xiao,
Xiao-Pan Chen,a Ji Deng,a Yu-Hang Yan,a Kai-Rong Zhu,a
Gen Li,a Jun-Lin Yu,a Jurgen Brem,
Fener Chen,a Christopher J. Schofield
*
b
b
¨
a
DOI: 10.1039/d1cc03026d
and Guo-Bo Li
*
rsc.li/chemcomm
Chiral 3-substituted benzoxaboroles were designed as carbapenemase little progress towards the asymmetric synthesis of 3-
inhibitors and efficiently synthesised via asymmetric Morita–Baylis–Hill- substituted benzoxaboroles suitable for biological testing, with
man reaction. Some of the benzoxaboroles were potent inhibitors of only a Wittig/oxa-Michael reaction cascade from 2-formyl aryl
clinically relevant carbapenemases and restored the activity of merope- boronic acid having been reported.4
nem in bacteria harbouring these enzymes. Crystallographic analyses
A major mechanism of carbapenem resistance involves produc-
validate the proposed mechanism of binding to carbapenemases, i.e. in a tion of carbapenemases, i.e. evolved b-lactamases that inactivate
manner relating to their antibiotic substrates. The results illustrate how carbapenem antibiotics, which can be divided into two categories:
combining a structure-based design approach with asymmetric catalysis serine-carbapenemases (e.g. Ambler class A KPC-2 and class D OXA-
can efficiently lead to potent b-lactamase inhibitors and provide a 48 types) and metallo-carbapenemases (e.g. NDM and VIM types).5
starting point to develop drugs combatting carbapenemases.
With the aim of helping enable agents to overcome carbapenem
resistance in Gram-negative pathogens,6 we analysed carbapenem-
The benzoxaborole ring system has emerged as a useful drug substrate derived complexes and identified benzoxaboroles with a
scaffold, due to its intrinsic reactivity, metabolic stability and C3 acrylic acid group as potential inhibitors (Fig. 1b). Here we
low toxicity.1 Benzoxaborole derivatives have been developed to
treat infectious, inflammatory and neoplastic diseases;2 Tava-
borole and Crisaborole are approved for treatment of onycho-
micosis and mild-to-moderate atopic dermatis, respectively and
other benzoxaboroles are in development (Fig. 1a).3 However,
most benzoxaborole containing drugs/drug candidates have
achiral substituents, hampering the development of selectivity.
The development of efficient new methods for the asymmetric
construction of chiral benzoxaboroles, including with C3 sub-
stituents is a current challenge in the development of boron
containing drugs.
Procedures have been recently described for synthesis of
C3-substituted benzoxaboroles via reaction of nucleophiles
with 2-formyl aryl boronic acid.1a Most, however, suffer from
low yields and limited substrate scope.1b Indeed, there has been
a Key Laboratory of Drug-Targeting and Drug Delivery System of the Ministry of
Education and Sichuan Research Center for Drug Precision Industrial Technology,
West China School of Pharmacy, Sichuan University, Chengdu 610041, China.
E-mail: liguobo@scu.edu.cn
b Department of Chemistry and the Ineos Oxford Institute for Antimicrobial
Research, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
E-mail: christopher.schofield@chem.ox.ac.uk
† Electronic supplementary information (ESI) available: Spectroscopy details
Fig. 1 Design of 3-substitued benzoxaboroles as b-lactamase Inhibitors.
Chem. Commun., 2021, 57, 7709–7712 | 7709
have been deposited. See DOI: 10.1039/d1cc03026d
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report use of an asymmetric Morita–Baylis–Hillman (MBH) cascade owing to their low reactivity.7 However, electron-donating groups,
reaction7 to construct the desired chiral C3-substituted benzoxa- e.g. methyl and alkoxy groups at C4 of the 2-formylphenylboronic
boroles. Some of the compounds manifested inhibition of clinically ring are well tolerated in our conditions (Table 1, entries 1–5); it is
relevant carbapenemases, with the proposed mode of action being possible that the aldehyde is activated by an intramolecular
validated by crystallography and potentiation of carbapenem activ- hydrogen bond between its carbonyl oxygen and the boronic acid,
ity in cells.
so promoting MBH reaction.9 By contrast, electron-withdrawing
Initially, we utilized 2-formyl-phenylboronic acids as the elec- substituents at the same position in the phenyl ring delivered
trophiles and hexafluoroisopropyl acrylate (HFIPA) as the acti- relatively low enantioselectivities (entries 6 and 7). Similarly,
vated alkene in model reactions. We envisaged challenges relating substituents at C5 of the 2-formylphenylboronic ring were amen-
to the limited substrate scope of the MBH reaction, the desire for able to reaction regardless of the electronic properties of the
asymmetric induction,7c and the presence of a boronic acid group tested substituents (entries 8–12). However, substituents at either
at the benzaldehyde ortho-position. After optimization of the C3- or C6- of the 2-formylphenylboronic ring were not amenable
model reaction, we identified reaction conditions using cinchona to reaction, with only traces of the desired products being
alkaloid derived chiral tertiary amines C6 and C7 as catalysts (see observed, revealing a limitation of the methodology. We applied
ESI† for optimization details).
catalyst C7 for the asymmetric MBH synthesis of (S)-enantiomers;
We explored the scope of the asymmetric reaction with the desired products were observed in good yield, albeit with
various substituted 2-formylphenylboronic acids using C6 moderate ee (entries 13–16); pleasingly, excellent ee’s were
(Table 1). The products 3 were hydrolyzed to the corresponding obtained after recrystallization.
carboxylic acids 4 for biological evaluation. The chiral MBH
adducts made using C6 were assigned the R-configuration, e.g. aryloxy and benzyloxy groups, at C4 and C5- of 2-for-
based on X-ray analysis of a single crystal of 4a.8
mylphenylboronic acids, since structural analyses suggested these
We next examined the reaction scope with larger substituents,
The asymmetric MBH reaction with aromatic aldehydes derivatives are of interest as b-lactamase inhibitors (representatives
bearing electron-donating groups is reported to be challenging, are given in Scheme 1).10 Aryloxy substituents, (p-tolyloxy, m-tolyloxy,
p-trifluoromethyl-phenoxy) at C4 of 2-formylphenylboronic acids
gave products (4m–4o) in high yields and good enantioselectivities.
A benzyloxy group at C4 was also amenable to reaction, affording 4p
in 81% yield and 87% ee. By contrast, aryloxyl- and benzyloxy
substituents at C5 of 2-formylphenylboronic acids gave relatively
lower ee‘s compared to the C4 derivatives (4q–4t). Density functional
Table 1 Substrate scope with small substituents on the phenyl ring of 2-
formylphenylboronic acidsa
Entry
R1
R2
Timeb (h)
ID, Yieldc (%)
eede (%)
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
Me
OMe
F
Cl
OMe
H
H
H
F
29
29
43
10
24
46
22
7
17
36
36
48
96
90
76
48
(R)-4a, 80
(R)-4b, 80
(R)-4c, 85
(R)-4d, 82
(R)-4e, 71
(R)-4f, 86
(R)-4g, 70
(R)-4h, 84
(R)-4i, 72
(R)-4j, 91
(R)-4k, 81
(R)-4l, 84
(S)-4a, 88
(S)-4c, 80
(S)-4f, 79
(S)-4j, 65
90(96)
89
90(99)
86
82
82(90)
75
88
84
81(96)
78
82
Me
OMe
OEt
OiPr
F
Cl
H
H
H
H
OMe
H
9
10
11
12
13f
14f
15f
16f
60(99.5)
52(96)
48(90)
57(95)
OMe
F
H
Scheme 1 Scope of C3 substituted benzoxaborole synthesis employing
aryloxy and benzyloxy substituted 2-formylphenylboronic acids.a,b,c
aUnless otherwise stated, reactions were performed with 1 (0.1 mmol), 2
(0.5 mmol), C6 or C7 (0.01 mmol), Al2O3 (0.4 mmol), 1,4-dioxane (4.0 mL),
r.t. 12–48 h. bIsolated yields over two steps. cee’s determined by chiral
HPLC. dee after recrystallization.
a
Standard reaction conditions: 1 (0.1 mmol), 2 (0.5 mmol), C6 or C7
b
(0.01 mmol), Al2O3 (0.4 mmol), 1,4-dioxane (4.0 mL), r.t. Reaction
time for the first step. Isolated yields over two steps. ee’s were
determined by chiral HPLC. ee after recrystallization from ethyl
acetate. Reaction at 0 1C.
c
d
e
f
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Table 3 In vitro cell-based screening of 4a, 4c, and 4qa
theory (DFT) calculations inform on the mechanism of reaction and
rationalise its stereoselectivity (see E. 1 Chemistry in ESI†).
MEM (mg mlÀ1) in presence or absence of inhibitor
We then tested the activity of the benzoxaborole derivatives
against purified recombinant forms of 7 types of clinically
relevant b-lactamases, i.e. class A SBL KPC-2, class C SBL AmpC,
class D SBL OXA-48, class B1 MBLs NDM-1, IMP-1, VIM-1, and
VIM-2; the SBL inhibitor avibactam was used as a positive
control (Table 2 and Table S1, ESI†).
The results (Table 2) show both enantiomers of some of the
chiral benzoxaborole derivatives manifest moderate, or potent,
inhibition of the carbapenemase KPC-2, demonstrating their
potential for inhibition of clinically relevant SBLs. In all cases,
the (S)-configured compounds (4a, 4c, 4f, 4j, 4q) were more
potent than the corresponding (R)-enantiomers, illustrating
how chirality can confer selectivity in boron based SBL inhibi-
tors. This selectivity was mostly, but not exclusively observed
for the other b-lactamases, substantial inhibition of which was
more sporadic than for KPC-2 (Table 2). The KPC-2 stereo-
K. pneumoniae
Conc. C692 (blaAmpC, E. coli BAA-2340 E. coli 11119
Compound (mM)
blaTEM-1)
(blaKPC-2)
(blaKPC-2)
—
(R)-4a
—
100
10
4128
16
128
8
16
64
0.5
4
0.5
2
2
16
1
4
2
16
2
16
0.25
0.5
0.25
0.5
0.5
2
0.25
4
1
(S)-4a
(R)-4c
(S)-4c
(R)-4q
(S)-4q
100
10
100
10
100
10
100
10
64
128
4128
32
128
4128
4128
64
2
0.25
1
100
10
4128
a
Clinically isolated strains producing KPC-2, AmpC, and/or TEM-1.
selectivity for the benzoxaborole derivatives is interesting since susceptible to MEM alone (MIC Z 16 mg mlÀ1); for the control
the penicillin substrates of some of the b-lactamases have a (2S) strain ATCC25922, the MEM MIC is 0.06 mg mlÀ1
C-3 carbon adjacent to their C-2 carboxylate. The difference in
Addition of 4a, 4c or 4q at 100 mM substantially reduced the
.
results for the (R)- and (S)-benzoxaboroles may reflect different MEM MICs, i.e. by 16-128 fold for the KPC-2 producing E. coli
distances between their carboxylate and electrophilic boron; strains; notably, the presence of 10 mM (R)-4a and (S)-4a
the analogous carboxylate to b-lactam distance is proposed to decreased the MEM MICs to below 2 mg mlÀ1 for E. coli
be important with respect to antibacterial activity.11
BAA-2340 and E. coli 11119 (Table 3). Consistent with the
Overall, these results reveal that when combined with an biochemical results, reduced (but still clear) activity was
appropriate C-5 or C-6 substituent, the C-3 acrylate substituted observed for AmpC and TEM-1 (both class A SBLs) producing
benzoxaborole derivatives are potent KPC-2 inhibitors. They K. pneumoniae C692, i.e. the addition of 100 mM (S)-4a resulted
were in general much less potent inhibitors for the other tested in a 16-fold decrease in the MEM MICs. Overall, consistent with
SBLs/MBLs, but in some cases potent inhibition was observed, the biochemical results, the (S)-compounds show better cellular
suggesting that with optimisation the scaffold could be activity than the corresponding (R)-enantiomers, illustrating
developed into potent broad spectrum b-lactamase inhibitors. the importance of stereochemistry. Interestingly, although 4q
The abilities of selected Inhibitors to potentiate the anti- was a better inhibitor than 4a versus against the isolated
bacterial activity of meropenem (MEM), a clinically important b-lactmases (Table 2), it was less active in restoring MEM
carbapenem, was then investigated for AmpC, TEM-1, or KPC-2 activity in cells (Table 3), possibly reflecting different permea-
producing clinically derived strains, including Klebsiella Pneu- tion or efflux properties.
moniae C692, Escherichia coli BAA-2340, and Escherichia coli
To investigate the structural basis of inhibition by the chiral
11119 (for details, see ESI†). None of these isolates were benzoxaboroles, we obtained KPC-2:(S)-4a and OXA-48:(R)-4a
complex structures by co-crystallisation (Table S2, ESI†). The
KPC-2: (S)-4a structure reveals a covalent bond between boron
and Ser70, with hydrogen bonds between the inhibitor and b3
Table 2 Comparison of the inhibitory activities (IC50, mM) of (R)- and (S)-
configuration of selected benzoxaborolesa
Thr237, a8 Ser130, b3 Thr235, and b3 Thr237 (Fig. 2a and
Fig. S4, ESI†). The OXA-48:(R)-4a structure similarly reveals a
tetrahedral boron with hydrogen bonds between the inhibitor
a3 Ser70, b5 Tyr211, a5 Ser118, and a10 Arg250; Lys73 of OXA-
48 was refined in its catalytically active carbamylated form
(Fig. 2b and Fig. S5, ESI†). Superimposition of structures of
KPC-2:(S)-4a with KPC-2:reacted imipenem (PDB: 6XJ8),12 and
OXA-48:(R)-4a with OXA-48:reacted meropenem (PDB: 6P98)13
reveals that (S)-4a and (R)-4a have binding modes closely
related with those of ring-opened products of carbapenems
(for more details, see Fig. S7–S9, ESI†). Based on these struc-
tures and computational results, it is proposed that the C-6
4-methoxyphenoxyl group of (S)-4q, which occupies a hydro-
phobic subpocket adjacent to OXA-48 active site, forms an
additional hydrogen bond with Asn170 of KPC-2 (Fig. S6, ESI†).
Class
C
Class
Class D B1
Class Class
B1 B1
Class
Class
Cpd ID
A KPC-2 AmpC OXA-48 NDM-1 B1 IMP-1 VIM-1 VIM-2
(R)-4a
(S)-4a
(R)-4c
(S)-4c
(R)-4f
(S)-4f
(R)-4j
(S)-4j
(R)-4q
(S)-4q
3.08
0.48
7.62
0.54
3.87
0.34
0.97
0.49
0.07
0.01
134.10 15.84 549.80 4600
185.7 14.06 4600 142.5
178.20 42.52 4600 4600
4600 225.40
308.6 12.05
4600 232.60
74.14 34.15 169.7
140.90 10.13 4600 65.65
149.8 14.68 325.3 46.5
140.50 5.97 4600 4600
108.7 12.03 719.1 142.5
18.38 0.43 260.00 497.36
22.52 0.74 34.06 4600
0.24 0.17 4600 425
4600
466.3
513.8
22.40
82.80
4600 120.5
4600 55.87
308.6 105.7
388.3
4600
4600
57.37
58.49
19.08
Avibactam 0.004
a
All tested compounds were recrystallised. IC50 curves are shown in ESI
Fig. S1.
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Conflicts of interest
There are no conflicts to declare.
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This putative interaction may explain why 4q is more potent
than 4a and suggests that further optimization of substituents
particularly at C-6 should improve the potency of the series for
carbapenemase inhibition.
In summary, an enantioselective MBH cascade reaction,
employing a bifunctional tertiary amine-carbamate catalyst, was
developed for efficient synthesis of 3-substituted benzoxaboroles.
The reaction enabled synthesis of C3 acrylate substituted benzox-
aboroles in high yield and enantiomeric excess. Screening against
clinically relevant SBLs and MBLs, revealed that some of the
benzoxaborole derivatives are potent inhibitors of SBLs/MBLs.
Crystallographic analyses revealed that active site of (S)-4a to KPC-
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serine. (R)-4a and (S)-4a potentiate carbapenem activity in cells.
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7712 | Chem. Commun., 2021, 57, 7709–7712
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