Antimicrobial and KPC/AmpC inhibitory activity of functionalized benzosiloxaboroles

Article abstract of DOI:10.1016/j.ejmech.2019.03.028

A series of 22 benzosiloxaboroles, silicon analogues of strong antimicrobial agents - benzoxaboroles, have been synthesized and tested against β-lactamases KPC- and pAmpC-producing strains of Gram-negative rods. Comprehensive structural-property relationship studies supported by molecular modelling as well as biological studies reveal that 6-B(OH)₂-substituted derivative 27 strongly inhibits the activity of cephalosporinases (chromosomally encoded AmpC and plasmid encoded CMY-2) and KPC carbapenemases. It also shows strong ability to inhibit growth of the strains producing KPC-3 when combined with meropenem. In addition, halogen-substituted (mono-, di- or tetra-) benzosiloxaboroles demonstrate high antifungal activity (MIC 1.56–6.25 mg/L) against C. tropicalis, C. guilliermondii and S. cerevisiae. The highest activity against pathogenic yeasts (C. albicans, C. krusei and C. parapsilosis - MICs 12.5 mg/L) and against Gram-positive cocci (S. aureus and E. faecalis - 6.25 mg/L and 25 mg/L respectively) was displayed by 6,7-dichloro-substituted benzosiloxaborole. The studied systems exhibit low cytotoxity toward human lung fibroblasts.

Full text of DOI:10.1016/j.ejmech.2019.03.028

Accepted Manuscript
Antimicrobial and KPC/AmpC inhibitory activity of functionalized benzosiloxaboroles
Krzysztof Durka, Agnieszka E. Laudy, Łukasz Charzewski, Mateusz Urban, Karolina
Stępień, Stefan Tyski, Krystiana A. Krzyśko, Sergiusz Luliński
PII:
S0223-5234(19)30246-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.028
EJMECH 11198
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 January 2019
Revised Date: 11 March 2019
Accepted Date: 12 March 2019
Please cite this article as: K. Durka, A.E. Laudy, Ł. Charzewski, M. Urban, K. Stępień, S. Tyski,
K.A. Krzyśko, S. Luliński, Antimicrobial and KPC/AmpC inhibitory activity of functionalized
benzosiloxaboroles, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.03.028.
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ACCEPTED MANUSCRIPT
Antimicrobial and KPC/AmpC inhibitory activity of functionalized
benzosiloxaboroles
$
,[a]
$, [b]
$,[d]
[a]
Krzysztof Durka,
Agnieszka E. Laudy, ** Łukasz Charzewski,
Mateusz Urban,
[
b]
[b,c]
[d]
[a]
Karolina Stępień, Stefan Tyski,
Krystiana A. Krzyśko,*** Sergiusz Luliński*
[
a]
Physical Chemistry Department, Faculty of Chemistry, Warsaw University of Technology,
Noakowskiego 3, 00-664 Warsaw, Poland
[
b]
Department of Pharmaceutical Microbiology, Medical University of Warsaw, Oczki 3, 02-
07 Warsaw, Poland
0
[
c]
Department of Antibiotics and Microbiology, National Medicines Institute, Chełmska 30/34,
0-725 Warsaw, Poland
0
[
d]
Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland
*
Corresponding author responsible for chemical research. Email address: serek@ch.pw.edu.pl
(
S. Luliński).
*Corresponding author responsible for microbiological research. Email address:
alaudy@wp.pl (A.E. Laudy).
**Corresponding author responsible for molecular modelling. Email address:
*
*
krzysko@bioexploratorium.pl (K.A. Krzyśko).
$
These authors equally contributed to this work
Keywords: benzosiloxaboroles, antifungal activity, antibacterial activity, AmpC/KPC β-
lactamase inhibitory activity, molecular docking, efflux pump
Abstract
A series of 22 benzosiloxaboroles, silicon analogues of strong antimicrobial agents -
benzoxaboroles, have been synthesized and tested against β-lactamases KPC- and pAmpC-
producing strains of Gram-negative rods. Comprehensive structural-property relationship
studies supported by molecular modelling as well as biological studies reveal that 6-B(OH)2-
substituted derivative 27 strongly inhibits the activity of cephalosporinases (chromosomally
encoded AmpC and plasmid encoded CMY-2) and KPC carbapenemases. It also shows strong
1

ACCEPTED MANUSCRIPT
ability to inhibit the growth of the strains producing KPC-3 when combined with meropenem.
In addition, halogen-substituted (mono-, di- or tetra-) benzosiloxaboroles demonstrate high
antifungal activity (MIC 1.56-6.25 mg/L) against C. tropicalis, C. guilliermondii and S.
cerevisiae. The highest activity against pathogenic yeasts (C. albicans, C. krusei and C.
parapsilosis - MICs 12.5 mg/L) and against Gram-positive cocci (S. aureus and E. faecalis -
6
.25 mg/L and 25 mg/L respectively) was displayed by 6,7-dichloro-substituted
benzosiloxaborole. The studied systems exhibit low cytotoxity toward human lung fibroblasts.
Introduction
1
,3-Dihydro-1-hydroxy-2,1-benzoxaborole and its derivatives were synthesized and
characterized already in 1957 (Scheme 1) [1]. In the last decade, strong interest in these boron
heterocycles was stimulated to a major extent by discovery of their biological activity [2-8].
For instance, 5-fluoro- and 5-chloro-1,3-dihydro-2,1-benzoxaborole (referred to AN2690 as
tavaborole and AN2718, respectively) were identified as potent antifungal and antibacterial
agents [2]. Gratifyingly, these compounds exhibit low toxicity to humans and thus the former
compound has already been commercialized under trade name Kerydin, for treatment of
onychomycosis - a fungal infection of nail and nail bed [9-10]. Very recently, 5-(4-
cyanophenoxy)benzoxaborole (crisaborole, trade name Eucrisa) was approved for the
treatment of mild-to-moderate atopic dermatitis (eczema) [11]. Other benzoxaboroles are also
effective against important human diseases including malaria [12], human African
trypanosomiasis (HAT) [13], and selected liver disfunctions [14]. Recently we have presented
silicon-based analogues of benzoxaboroles – benzosiloxaboroles (Scheme 1). The electronic
properties of silicon are quite different from those of carbon. Specifically, the presence of
low-lying antibonding σ* orbitals at the silicon atom gives rise to significant interaction with
lone pair at the adjacent oxygen atom which, in turn, results in the increased Lewis acidity of
the boron atom in benzosiloxaborole compared to benzoxaborole (where the carbon atom is
present instead of the silicon). In addition, the increased atomic radius of silicon results in
slightly different geometry of the siloxaborole ring due to longer bonds formed by the silicon
atom. Our goal was to exploit specific electronic and steric (geometric) characteristics of
benzosiloxaboroles in order to obtain systems showing improved antimicrobial activity.
Indeed, our preliminary microbiological studies showed their promising activity against
selected yeast strains [15].
2

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Scheme 1. Benzoxa- and benzosiloxaboroles. Position numbering scheme is additionally
provided (note that it is different between benzoxa- and benzosiloxaboroles).
Herein, we report on comprehensive characterization of antimicrobial potential of 22
benzosiloxaboroles and the structure-properties relationships by varying the type, number and
positions of substituents. The studied compounds can be divided into 3 subgroups. The first
group comprises the parent benzosiloxaborole and its halogen (F, Cl, Br, CF )-substituted
3
derivatives including two structural analogues of tavaborole (Scheme 2, I). This is
rationalized by the fact that related benzoxaboroles containing halogen atoms located at the 4-
or 5-position show enhanced antimicrobial activity. The second group contains
benzosiloxaboroles bearing CHO or B(OH) group in different configurations (II). We
2
assumed that such systems might exhibit increased affinity to target biological sites due to the
auxiliary interactions between an enzyme and a polar functional group. Finally, three
bifunctional systems with two oxaborole rings (III) were studied in order to find any
synergistic effects resulting from such an extended structure.
Scheme 2. Studied groups of benzosiloxaboroles.
Results and Discussion
Synthesis
The synthesis of the parent benzosiloxaborole 1 and its simple fluorinated analogues 2-4
(
Scheme 3) was described in our seminal paper [15]. The synthesis of halogenated 10-14
Scheme 4) derivatives utilizes two step protocol. Thus, appropriate halogenated
(
bromobenzenes were subjected to deprotonative lithiation with LDA in THF at –78 °C
followed by addition of Me Si(H)Cl resulting in the formation of arylsilanes 5-8. In all cases
2
3

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reactions occurred regioselectively at the position between bromine and another halogen atom
in agreement with the cumulated ortho-acidifying effect of those two substituents [16,17].
The silylated derivatives were successfully converted into respective benzosiloxaboroles by
bromine-lithium exchange performed using t-BuLi at low temperatures (–75 °C) followed by
trapping of generated aryllithium intermediates with an excess of B(OMe) . Final hydrolysis
3
with dilute aqueous H SO gave rise to targeted products with good yields. The synthesis of 6-
2
4
CF substituted derivative 14 utilized an analogous approach. In this case, the intermediate
3
arylsilane 9 was obtained by regioselective deprotonation of 4-bromobenzotrifluoride with
LTMP/THF at –80 °C [18].
Scheme 3. Parent benzosiloxaborole and its fluorinated analogues.
OH
1
. LDA or LTMP
Br
Br
o
o
1. t-BuLi (2 equiv), Et O, -95 C
B
THF, -78 C
2
O
o
X2
2. Me Si(H)Cl
3. H2O
X2
SiMe H
2. -85 C, 30 min
Si
2
2
X2
o
X1
X1
3. B(OEt) , -100 C
3
X1
4
. H3O+
5
-9
10-14
X1 = H, F, Cl, Br
X2 = H, F, Cl, CF3
5
6
7
8
9
: X , X = F
1
2
: X , X = Cl
1
2
: X ,= Cl, X = H
: X ,= Br, X = H
: X ,= H, X = CF
3
1
2
1
2
1
2
OH
OH
OH
OH
OH
B
B
B
B
B
O
O
O
O
O
Si
Si
Si
Si
Si
F
Cl
F3C
F
1
Cl
11
Cl
12
Br
13
0
14
Scheme 4. Synthesis of halogenated benzosiloxaboroles 10-14.
We were especially interested in the preparation of benzosiloxaboroles 23 and 24 as those
compounds are structural analogues of tavaborole due to the presence of halogen (X = F, Cl)
at the 6-position (para with respect to the boron atom). They were obtained in multistep
protocols starting with monobromination of respective 4-haloanilines (Scheme 5) [19,20].
Subsequent replacement of amino group in 15-16 with iodine gave rise to corresponding 1-
bromo-2-iodobenzenes 17-18 [21,22]. Then selective iodine-lithium exchange/in situ trapping
with B(OiPr) [23,24] was accomplished using n-BuLi in a mixed solvent (THF/Et O 1:1) at –
3
2
4

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9
0 °C. The resulting respective ate complexes were treated with Me SiCl to give halogenated
3
aryl boronates 19-20 which were further converted to dioxaazaborocans 21-22 by
transesterification with N-butyldiethanolamine (BDEA) in Et O. Finally, compounds 21-22
2
were subjected to bromine-lithium exchange with n-BuLi in THF and the generated
aryllithium intermediates were trapped with Me Si(H)Cl. Hydrolysis with dilute aqueous
2
H SO resulted in the formation of targeted products 23-24.
2
4
Scheme 5. Synthesis of 6-fluoro and 6-chloro substituted benzosiloxaboroles as direct
analogues of tavaborole.
In the next step we have turned our attention to functionalized benzosiloxaboroles
(
Scheme 2, II, III). For these studies we have selected compounds bearing B(OH) and CHO
2
functional groups. We have assumed that such functionalization may be beneficial for the
activity due to a stronger and specific binding to biological targets by means of hydrogen-
bond interaction or direct covalent/coordination bond. With boronic group, ester formation or
boron-nitrogen/oxygen coordination is expected, while in the case of formyl group hemiacetal
or acetal may be formed. The geometry of the molecule may be crucial for biological activity
and thus systems bearing B(OH) or CHO groups along with some fluorine atoms at various
2
positions have been obtained. Exceptionally 4-B(OH) and 7-CHO derivatives were not
2
obtained due to synthetic limitations. At this point it should be noted that the fluorine
substituents play a dual role, as it allows to regulate reaction selectivity and also, as will be
shown in the next paragraph, it may enhance biological activity of the system.
Synthesis of boronated derivatives 25, 27, and 29 (Scheme 6) was accomplished by
the treatment of respective silylated dibromobenzenes 8, 26, 28 with an excess of t-BuLi
which resulted in the generation of respective dilithio intermediates. Then boronation and
5

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subsequent workup were performed as described above. In a similar way, a symmetrical
species 31 composed of two siloxaborole rings fused with the central benzene ring was
readily accessible from disilylated halobenzene 30.
Scheme 6. Synthesis of boronic-functionalized benzosiloxaboroles.
The synthesis of formyl-functionalized compounds (32-35) along with some of their
reduction products (36-37) were described in our recent publication (Scheme 7) [25].
Synthesis of the newly obtained compound 40 started with 1,4-dibromo-2,5-difluorobenzene
and followed the general route developed for its close analogue 37. It involves the
introduction of acetal function resulting in compound 38 followed by silylation to give
intermediate 39. Final replacement of bromine atoms with boronic groups is accompanied by
net reduction of acetal group with hydride bound to silicon atom (Scheme 7) [25]. A special
attention should be paid to compounds 37 and 40 comprising benzoxaborole and siloxaborole
rings, which may influence biological activity due to competitive or synergistic effects. It is
also noticeable that the comparison between 37 and its bis(siloxaborole) analogue 31 should
give additional insight into the effect of methylene/dimethylsilyl group substitution.
6

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Scheme 7. Benzosiloxaboroles functionalized with CHO groups and their reduced derivatives.
1
13
11
19
All newly obtained compounds were characterized by multinuclear ( H, C, B, F)
NMR spectroscopy. The studied benzosiloxaborole are white, air- and moisture stable solids
readily soluble in polar organic solvents such as dichloromethane, acetone, MeOH and
DMSO. They are also sufficiently soluble in a mixed solvent H O/DMSO, which is relevant
2
to antimicrobial activity studies. Since acidity is an important parameter of a compound in
view of potential medicinal chemistry applications, we have measured the pK values for all
a
studied systems in water/methanol solution (1:1) by potentiometric titration with 0.1 M aq.
NaOH (Table 1). As we have found previously, the pK of the parent compound 1 is 7.9 [15].
a
This value is lower than that determined for the benzoxaborole analogue by 0.4 pK units [26]
a
reflecting the acidifying effect of the silicon centre. Acidity is strongly increased upon
substitution with halogens. The introduction of formyl groups has also a positive effect on
acidic properties. In the case of selected compounds possessing two boron atoms both pKa1
and pK were obtained. Overall, the obtained pK values indicate that the most of studied
a2
a
compounds tend to be converted into corresponding anionic forms under standard
physiological conditions (pH = 7.4), which should be beneficial for their solubility. In the
7

ACCEPTED MANUSCRIPT
case diboron derivatives monoanionic form is expected, with hydroxyl group probably located
on siloxaborole moiety.
Table 1. Acidic properties (pK values) of studied compounds.
a
1
2
3
4
10
11
12
13
14
23
24
25
[
a]
[a]
[a]
[a]
pKa
pKa
7.9
7.2
6.8
4.7
6.1
6.0
6.5
6.5
6.6
7.3
7.3
7.4
2
7
29
31
32 33 34 35 36 37
5.4 5.6 5.2 4.9 5.8 6.3
8.6
40
7.7 6.6
5.9
8.6
4.7
7.8
[
b]
[b]
[b]
[b]
1
0.2
[a]
[b]
ref. [15]; pKa2
Antifungal activity
We have investigated the antifungal activity of 19 benzosiloxaboroles 1, 10-14, 23-25, 27, 29,
1-38, 40 against 6 yeast strains. For comparison, we also included the data obtained for 2-4
15]. Five species from the Candida genus belong to the opportunistic fungi and cause
3
[
significant morbidity in people with decreased immunity. The highest mortality rate applies to
patients in intensive care units, where Candida albicans is the fourth leading cause of
nosocomial bloodstream infections [27]. Antifungal activity was determined according to
Clinical and Laboratory Standards Institute (CLSI) [28,29] and European Committee on
Antimicrobial Susceptibility Testing (EUCAST) [30] recommendations by disk-diffusion
method and by evaluation of the MIC values. Fluconazole was used as a reference compound
as it is the most commonly used antifungal compound in the case of suspected yeast
infections. However, among the Candida genus, Candida krusei is intrinsically resistant to
fluconazole (FL) [31,32]. The antifungal activity of the most active compounds is presented
in the Table 2 whereas complete set of data is given in the Supporting Information (Table
S1). Halogen-substituted benzosiloxaboroles from the group I (1-24) demonstrated the
highest antifungal activity (MIC 1.56-6.25 mg/L) against Saccharomyces cerevisiae ATCC
9
763, Candida tropicalis IBA 171 and Candida guilliermondii IBA 155. The presence of
chlorine at the 6-position of benzosiloxaboroles in 11 and 24 resulted in selectively higher
activity of these agents against C. albicans ATCC 90028 (MIC 12.5 mg/L). Comparable
activity in relation to fluconazole was observed for 3 and 4 against C. tropicalis as well as for
1
0 against C. guillermondii. Moreover, compound 23, the structural analogue of tavaborole,
demonstrated the highest activity against S. cerevisiae and promising activity against Candida
8

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krusei and C. tropicalis. We have also tried to find any dependence between the acidity and
activity of obtained compounds. However, the correlation is not monotonic as the highest
antifungal activity was found for dihalogenated compounds 3, 10, 11 having the pK of ca. 6-
a
7
, i.e., in the middle of the pK range of ca. 4-8.5 found for all characterized derivatives.
a
Introduction of additional electron-withdrawing formyl groups resulted in increased acidity
for derivatives 32-35, pKa = 4.9-5.6). However, the antifungal activity decreased
(
significantly which indicates that specific structural effects can be very important. It is also
plausible that an anionic form is much less effective in penetration through cell membrane
due to higher hydrophilicity although it is preferentially bound in the cell interior, e.g., in an
enzyme pocket. Thus, the possibility for equilibration between the neutral and anionic form
under physiological conditions would be a key factor responsible for high antimicrobial
activity.
Table 2. The MIC values of selected benzosiloxaboroles against yeasts.
Agent MIC [mg/L]
tested
C. albicans
C.
C. tropicalis C. guilliermondii C. krusei
S. cerevisiae
ATCC 90028 parapsilosis
IBA 171
IBA 155
ATCC 6258 ATCC 9763
ATCC 22019
1
2
3
4
1
1
2
2
100
50
25
25
25
25
3.125
6.25
3.125
25
a
a
a
100
100
50
50
12.5
6.25
50
50
50
0.78
1.56
6.25
3.125
12.5
6.25
0.38
50
200
100
12.5
25
200
100
12.5
12.5
25
0
1
3
4
12.5
12.5
25
3.125
25
12.5
6.25
1.563
6.25
25
12.5
1
25
25
b
c
d
FL
2
0.75
64
16
The highest activity indicated by the low MIC values (0.78–12.5 mg/L) is shown in bold.
a
The MIC values of compounds 2-4 against standard strains were previously described [15].
b
used as a reference antifungal agent.
c
The ellipse was visible at the MIC value 64 mg/L with macro-colonies up to ≥ 256 mg/L. The
MIC value of fluconazole (≥ 256 mg/L) against C. krusei was given in accordance with the
recommendations for Etest method [33,34].
9

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c
The ellipse was visible pointing the MIC value 64 mg/L, however, with macro-colonies up to
concentration ≥256 mg/L. In accordance with the recommendations for Etest method, the
MIC value of FL against C. krusei can be also interpreted as ≥256 mg/L [31,32]. C. krusei is
intrinsically resistant to fluconazole.
d
The ellipse was visible pointing the MIC value 16 mg/L, with colonies up to concentration
≥
256 mg/L. There are no recommendations for Etest method interpretation of the MIC value
of FL against S. cerevisiae. The obtained MIC 16 mg/L is in line with the published results
32].
[
Direct antibacterial activity
Inspired by reported high activity of benzoxaborole derivative- epetraborole
(GSK2251052), [35-37] we have investigated the activity of benzosiloxaboroles against group
of 5 Gram-positive and 10 Gram-negative strains. All data are deposited in the Supporting
Information, Tables S2-S3 and compared with those found for nitrofurantoin in accordance
with EUCAST and CLSI recommendations [38,39]. All tested compounds showed a higher
activity against Staphylococcus sp. than Enterococcus sp. strains (MICs 6.25-200 mg/L and
2
5->400 mg/L, respectively). The highest activity was observed for compound 11 and 40,
with the MICs of 6.25-25 mg/L. Moreover, Gram-positive cocci are more susceptible to
chloro-substituted compounds than to their fluoro counterparts. It is also worth to pay
attention to diboronic compounds 37 and 40, for which low MICs (3.12-6.25 mg/L) for S.
aureus and S. epidermidis were obtained. These values are comparable to those reported for
nitrofurantoin and epetraborole [35,40]. Finally, it is noticeable that the 6-CF -substituted
3
benzosiloxaborole 14 showed the highest bactericidal activity (MBC) in the range of 50-100
mg/L.
1
4 of 22 tested benzosiloxaboroles displayed only weak activity against Gram-negative
bacilli (Table S3). The highest activity was found for 40 against Stenotrophomonas
maltophilia ATCC 13637 and Burkholderia cepacia ATCC 25416 (MICs 50 mg/L). It should
be stressed that MDR efflux pumps, especially those from the RND family, play a significant
role in the resistance of Gram-negative bacteria to antibiotics and chemotherapeutics [41].
Therefore we undertaken the study on the influence of the efflux pump inhibitor PAβN
[
42,43] on the activity of our compounds. It is known that PAβN potently inhibits the efflux
systems from the Mex family in P. aeruginosa (especially MexAB-OprM) [44,45] as well as
it inhibits the AcrAB-TolC efflux system, described in the strains of the Enterobacteriaceae
family species, e.g., E. coli [43], K. pneumoniae [46], Proteus mirabilis [47], Enterobacter
aerogenes [48], and Salmonella enterica serovar Typhimurium [49]. We found that MIC
1
0

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values for all benzosiloxaboroles significantly (at least 4-fold) decreased in the presence of
PAβN for the majority of the standard strains (Table 3). Large decrease (from 32-fold to 64-
fold) in the MIC values in the presence of PAβN was noticed in the case of chloro-substituted
derivatives 11 (for 6 strains) and 24 (for 4 strains). The highest increase in bacterial
susceptibility to benzosiloxaboroles in the presence of PAβN was observed also in the case 11
and 24 as MIC values decreased from 400 mg/L to 6.25 mg/L for E. coli and from 100–200
mg/L to 6.25 mg/L for S. maltophilia. Obtained results indicate that benzosiloxaboroles are
effectively extruded through bacterial walls by MDR efflux pumps, and thus further studies
are necessary to develop more effective systems.
β-Lactamase inhibitory activity
The β-lactamase inhibitory activity of boronic acids has been known since the late 1970’s
[
50,51]. According to EUCAST recommendation, phenylboronic acid (PBA) is commonly
used in phenotypic detection methods of KPC-producing strains among Gram-negative rods
52]. PBA inhibits also AmpC cephalosporinases [53]. A report on the ability of
[
phenoxybenzoxaboroles to inhibit AmpC enzyme has been published [54]. Furthermore, in
August 2017 U.S. FDA approved Vabomere containing vaborbactam in a combination with β-
lactam antibiotic (meropenem) for treatment of complicated urinary tract infections (cUTIs)
and acute pyelonephritis (AP), caused by carbapenem-resistant Gram-negative bacteria from
Enterobacteriaceae family (CRE) [55,56]. Vaborbactam is based on a cyclic boronic acid
pharmacophore and shows activity against Ambler classes A and C enzymes [55, 57-59].
Taking this into account, we have investigated the β-lactamase inhibitory activity of all 22
presented organoboron compounds using strains expressing the various classes of β-
lactamases. In the first stage, we looked for compounds, which possess the significant β-
lactamase (KPC, AmpC and ESBL) inhibitory activity at high concentrations. The screening
was performed by three combination disc tests (CDTs) and PBA was used as the reference
inhibitor of KPC [52] and AmpC enzymes [53]. The results are presented in Table 3.
Interestingly for 5 out of 22 tested compounds very promising BLI activity was observed at
the concentration of 0.3 mg per disc. It should be noted here that these agents (compounds 4,
1
0, 27, 37, 40) are essentially inactive (MICs > 400 mg/L) against the studied Gram-negative
strains when used alone (for details see Table S3 in the Supporting Information). Compound
7 bearing the 6-B(OH) group exhibited the highest activity. This agent inhibited the activity
2
2
of KPC-2 carbapenemase as well as cephalosporinases (chromosomally encoded AmpC and
plasmid encoded CMY-2). In the CDT tests, compound 27 significantly altered the diameter
1
1

ACCEPTED MANUSCRIPT
of bacterial growth inhibition zone of KPC- and AmpC-producing strains around the disc with
β-lactams, whereas no activity against ESBL-positive K. pneumoniae was detected. The BLI
activity was also observed at the lower concentration 16 mg/L, however, only in the case of
2
7 (Table 4 and Supporting Information, Table S4 and S5). It is worth noting that
vaborbactam, a new BLI at the concentration of 8 mg/L, was approved in combination with
meropenem for clinical use for treatment of infections caused by CRE [55,60]. In the presence
of 27 at least a four-fold decrease in the MIC values of ceftazidime and meropenem were
observed for AmpC-positive and KPC-positive strains, respectively. However, our studies
have shown that 27 inhibits more significantly the growth of strains producing KPC-3 rather
than KPC-2 enzymes. The relationship between structure and activity was observed very
clearly in the case of boronated derivatives as structural analogues of 27 with the B(OH)2
group at the 5 (25) or 7 (29) positions, were not active. Moreover, in the CDT tests the BLI
activity against CMY-2-producing strain was observed for derivatives 4, 10, 37 and 40 (Table
3
) at the concentration of 16 mg/L (Supporting Information, Table S4 and S5). SAR analysis
indicates that the substitution pattern plays a major role in the inhibitory activity. Unlike
compounds 4 and 10 bearing two fluorine atoms at positions 6 and 7, the monofluorinated
derivatives 2 and 23 as well as 5,6-difluorosubstituted derivative 3 were inactive. Also,
introduction of chlorine atoms did not result in any significant activity. Finally, the AmpC
inhibitory activity of compounds 37 and 40 probably results from their unique structure
created through a fusion of oxaborole and siloxaborole heterocycles with a central benzene
ring. Remarkably, the most promising compounds (10, 27, 37) are not cytotoxic toward
human lung fibroblasts (MRC-5) in a concentration range of 1-16 µg/ml. For compounds 10
and 37, the cell viability was decreased to 74.0±7.3% and 54.3±3.6%, respectively at the
higher concentration (32 µg/ml) indicating their weak cytotoxicity (Supporting Information,
Table S6).
Table 3. β-Lactamase inhibitory activity of selected benzosiloxaboroles.
b
Diameter of inhibition zone (mm)
Disc with
K. pneumoniae P. aeruginosa
K. pneumoniae
ATCC 700603
ESBL(+)
E. coli 77
antibiotic +/-
ATCC BAA
1705 KPC(+)
MUW 700
AmpC(+)
CMY-2(+)
a
studied agent
b
b
b
b
MEM-10
CAZ-30
CAZ-30
CAZ-30/CTX-30
without agent
12
11
13
13/19
1
2

ACCEPTED MANUSCRIPT
PBA
22
13
14
18
12
12
22
11
14
18
16
11
24
22
22
22
20
20
14/19
13/19
13/19
13/19
13/19
13/19
4
10
27
37
40
MEM-10: A disc with meropenem 10 µg per disc. CAZ-30: A disc with ceftazidime 30 µg per
disc. CTX-30: A disc with cefotaxime 30 µg per disc. The diameter of all used antibiotic discs
was 6 mm. The amount of reference compound PBA and other agents added onto the
antibiotic discs was 0.3 mg per disc.
a
The addition of compounds 1-3, 11-14, 23-25, 29, 31-36 onto antibiotic discs did not
significantly alter the diameter of the bacterial growth inhibition zones in comparison to the
results obtained with the antibiotic discs alone.
b
A significant difference in the inhibition zones around the antibiotic disc with an agent
versus the same antibiotic disc without agent is presented in boldface. In the case of
meropenem (MEM) a significant difference is at least 4 mm, while for ceftazidime (CAZ) and
cefotaxime (CTX) it is at least 5 mm [52,53].
Table 4. The MICs for β-lactam antibiotics alone and in combination with 27 against standard
and clinical strains of Gram-negative rods producing various classes of β-lactamases (the
results for other agents are given in Tables S4-S5 in the Supporting Information).
MICs (mg/L) of antibiotic in the absence or in the presence of
a
additional agent
Strain
β-lactamase
Ambler
CAZ
CAZ
MEM
+PBA
MEM
CAZ
64
+ PBA
+27
MEM
64
+27
class [65]
K. pneumoniae
ATCC BAA-1705
K. pneumoniae 75
E. coli 76
KPC-2
A-CARB
32
64
16
32
KPC-2
KPC-2
KPC-3
KPC-3
KPC-3
A-CARB
A-CARB
A-CARB
A-CARB
A-CARB
>64
>64
64
16
>64
16
8
64
>64
64
32
>64
16
2
16
32
2
16
>64
4
b
K. pneumoniae 81
c
E. coli 82 TR(pl 81)
32
32
0.25
8
0.25
8
b
K. pneumoniae 83
>64
16
64
32
1
3

ACCEPTED MANUSCRIPT
CTX-M-3
A-ESBL
B-MBL
D-CARB
C
P. aeruginosa 698
VIM
64
>64
>64
32
64
>64
8
64
>64
32
8
16
>64
16
>64
0.25
nd
16
>64
0.25
nd
d
A. baumannii 181
P. aeruginosa 700
CHDL
AmpC
CMY-2
CMY-2
0.5
b
E. coli 77
C-pAmpC
C-pAmpC
8
<0.125
<0.125
e
K. pneumoniae 78
K. pneumoniae
ATCC 700603
E. coli 557
>64
32
64
nd
nd
SHV-12
A-ESBL
64
64
64
<0.125
nd
nd
CTX-M-2
OXA-15
A-ESBL
D-ESBL
16
16
16
<0.125
32
nd
16
nd
32
P. aeruginosa 852
>64
>64
>64
CAZ: ceftazidime. CAZ+PBA: ceftazidime with phenylboronic acid. CAZ+27: ceftazidime
with new agent no 27. MEM: meropenem. MEM+PBA: meropenem with phenylboronic acid.
MEM+27: meropenem with new agent no 27. A-CARB: class A carbapenemases. A-ESBL:
class A extended-spectrum β-lactamases. B-MBL: class B metalo-β-lactamases. D-CARB:
class D carbapenemases. C: class C cephalosporinases. C-pAmpC: class C plasmid-acquired
AmpC cephalosporinases. D-ESBL: class D extended-spectrum β-lactamases. nd: not
determined.
Significant decrease (at least 4-fold) of MIC values of β-lactam antibiotic in the presence of a
tested compound are presented in boldface.
a
All new agents and the reference compound PBA were used at the concentration of 16 mg/L.
b
In addition, these strains produce the narrow spectrum β-lactamase TEM-1-like.
c
The transformant of E. coli DH5α with plasmid from clinical strain of K. pneumoniae 81.
d
This strain produces two enzymes from CHDL family: OXA-24-like and OXA-51-like.
e
In addition, this strain produces the narrow spectrum β-lactamases TEM-1-like and OXA-1.
Docking and molecular dynamics with KPC-2 enzyme
In order to get deeper understanding of the β-lactamase inhibition mechanism we have
performed the docking simulations complemented by the molecular dynamic studies on the
group of the most active benzosiloxaboroles (10, 27, 37) with the KPC enzyme. To date, at
least 39 variants of KPC carbapenemases have been reported [61] although KPC-2 appears to
be the most widespread in the world among various types of bacteria in the
Enterobacteriaceae family [62,63]. The RCSB Protein Data Base (PDB) contains the crystal
structure of 3-nitrophenyl boronic acid (3-NPBA) bound to the KPC-2 enzyme, and this
1
4

ACCEPTED MANUSCRIPT
system was used as a starting point for our calculations (Scheme 7) [64]. In the crystal
structure, 3-NPBA interacts with Ser70, Lys73, Asn132, Asn170 and Thr237 of KPC-2
enzyme mainly through hydrogen bonds. Molecular dynamics revealed two more hydrogen
bonds formed with Cys69 and Ser130. Assuming that considered compounds bind to the same
site as the reference one, they should form similar interactions in order to trigger the same
biological effect. As the obtained pK values span below 7 for most of studied systems, it is
a
expected that the anionic forms are strongly preferred over the neutral ones. Accordingly,
crystallographic data show that 3-NPBA is present in its anionic form and binds to the KPC-2
enzyme through the B-O-Ser70 covalent bond. In the case of diboron systems, the
monoanionic form seems to be the most abundant under biological conditions. Nevertheless,
the compound may be further ionized in a binding pocket due to a local environment. For that
reason, all possible ionization states were generated. Compound 10 was considered in only
one ionization state (10a). Compounds 27 and 37 were investigated in all three possible
ionization states – with additional OH group bound to the boron atom either in
benzosiloxaborole (27a and 37a) or benzoxaborole ring (27b and 37b, respectively), or
dianionic form with two hydroxyl groups bonded to both oxaborole rings (27ab and 37ab, see
Scheme 8). For comparison we have also performed docking simulations for 4-
fluorobenzoxaborole (4-FBOB), (Scheme 7). It was investigated in its neutral and anionic
forms (Scheme 8).
Scheme 7. Referential compounds for docking and MD simulations.
1
5

ACCEPTED MANUSCRIPT
Scheme 8. Discussed anionic forms of 4FBOB, 10, 27 and 37.
Molecular docking results show that compounds 27 and 37 in their corresponding
dianionic forms are strongly bound to the enzyme pocket. In general, all obtained modes
scored between -5.5 and -5.0 kcal/mol. In the case of 27 the binding of monoanionic form 27a
with hydroxyl group attached on the oxaborole site is energetically favorable over 27b.
Conversely, the binding of 37b is slightly more preferable than 37a, indicating that stronger
interactions are formed with ionized oxaborole ring than with siloxaborole one. This can be
rationalized by the higher steric hindrance of SiMe groups compared to the CH bridge.
2
2
In the next step all anionic forms were subjected to molecular dynamics (MD)
simulations. Furthermore, for each system two molecule-enzyme binding modes have been
considered. The first one is based on intermolecular (non-covalent) forces such as hydrogen
bonds, C-H⋯π interactions, etc., while the second involves the formation of the covalent B-O
bond with the Ser70 moiety. The most stable modes were determined based on binding
energy values in MD simulations. Their stability as well as ligand interactions were similar to
those found for 3-NPBA. In every MD simulation with non-covalent binding mode, we have
found that at least 3 out of 7 intermolecular interactions that are present in reference system
(with Ser70, Lys73 and Thr237) have been preserved. In turn, in simulations where B-O-
Ser70 covalent bond was introduced, two or three intermolecular interactions were preserved
from the 3-NPBA – KPC-2 reference system (Figure 1). The covalent bond with Ser70
locates all compounds in the active site in such a manner that their aromatic rings are
similarly oriented during the MD simulations. This indicates that studied compounds are well
fitted to the active site shape.
1
6

ACCEPTED MANUSCRIPT
Analysis of all forms for each compound revealed that the most stable, hence the most
probable, are 10a, 27ab, 37a, presenting similar interaction energies with KPC-2. For non-
covalently bonded systems mean net interaction energies during the simulations are in range
from –57.48 to –105.45 kcal/mol while for covalently bonded systems they are in range from
–
107.69 to –124.34 kcal/mol (Table 5). Our results indicate that in cell-free systems
compound 27ab exhibits the strongest inhibitory activity. In turn, 37a is less effective (and
comparable to 3-NPBA), whereas 10a exhibits weaker, yet still significant binding to the
KPC-2, which corresponds to results of β-lactamase inhibitor activity test (Table 5).
The ionization aspects of 27 seem to be particularly interesting. Since the existence of a
dianionic form strongly depends on the environment, it is crucial to be determined if they can
occur in this state in a bacterial cell or not. If acquiring of a dianionic form were possible, this
would result in the strongest biological effect of this compound among the others yet binding
to Ser70 with side a is still more probable than with side b due to its significantly lower
interaction energy with KPC-2. If ionization is possible only on one side of the compound the
interaction with KPC is weaker and remains on the same level as for 10a regardless of which
boron atom is directed to the interior of a KPC-2 pocket. These results should also be
applicable to KPC-3 containing systems. The sequence alignment of KPC-2 and -3 revealed
that the only difference between these proteins in a region close to binding pocket is a single
amino acid substitution. Instead of His274 residue, the KPC-3 contains Tyr272 structurally
located in the same position. However, this particular amino acid does not take part in ligand
binding directly and it is separated from the binding site by Thr237 (KPC-2 numbering).
The obtained results have been compared to referential system 4-FBOB. We observed
that its docking scores are significantly lower than in benzosiloxaboroles, ranging from -4.2 to
-3.9 kcal/mol. Similarly to all other investigated compounds, the ionized form is energetically
favorable over the neutral one. In MD simulations, the interactions with Ser70, Thr237 are
preserved, however the interaction with Lys73 is replaced with Lys234 residue. This behavior
is a consequence of lack of two Si-bound Me groups in 4-FBOB, which limits its mobility in
the binding site. 4-FBOB in its non-covalently bounded form is displaced by about 1 Å as
compared to the location of 10a. After formation of the covalent bond it rearranges to the
same position as in the other compounds. Summing up, the performed calculations show that
the benzosiloxaboroles interact less effectively with KPC-2 in their non-covalently bounded
forms than benzoxaboroles. Conversely, it seems that the covalent binding of
benzosiloxaboroles is more advantageous than for benzoxaboroles represented by 4-FBOB.
1
7

ACCEPTED MANUSCRIPT
Table 5. Interactions with KPC-2 for compounds 3-NPBA, 4-FBOB, 10a, 27ab and 37a.
Compound
Net interaction energy
Amino acids contacts
(mean ± SEM kcal/mol)
Non-covalently
bound
Covalently
bound
Cys69, Ser70, Lys73, Ser130,
Asn132, Asn170, Thr237
Ser70, Lys73/Lys234, Ser130,
Asn132, Asn170, Thr237
Ser70, Lys73, Asn132,
Asn170, Thr237
3
4
1
2
-NPBA
-FBOB
0a
- 97.28 ± 0.11
-98.36 ± 0.23
- 57.48 ± 0.14
- 75.11 ± 0.12
-116.95 ± 0.12
-95.70 ± 0.07
-107.69 ± 0.07
-124.34 ± 0.34
Cys69, Ser70, Lys73, Ser130,
Asn132, Asn170, Arg220,
Thr237
7ab
Ser70, Lys73, Asn170, Thr237
3
7a
- 105.45 ± 0.11
-113.37 ± 0.07
1
8

ACCEPTED MANUSCRIPT
Figure 1. KPC-2 (PDB ID: 3RXX [62]) contacts formed with compounds 10a (A), 37a (B),
2
7ab (C) and 4-FBOB (D). Visible amino acids form interactions with compounds. Blue and
green dashed lines depict hydrogen bonding and C-H⋯π interactions, respectively.
Conclusions
In conclusion, a series of benzosiloxaboroles with varying substitution patterns were
obtained and their biological properties were comprehensively studied in order to elucidate
the structural factors that affect their bioactivities. Most of compounds were prepared from
simple starting materials based on two general protocols elaborating introduction of silicon-
containing group in the first step followed by the Br/Li exchange and boronation, or the
reversed one. In selected cases (especially, for compounds 23 and 24) multistep reaction
sequences were developed. We would like to stress that the synthesis of benzosiloxaboroles is
relatively simple as compared to many biologically active systems currently used in medicine.
This indicates that they can be easily accessed on a significant scale that can allow their
further use toward desired application.
The most significant result of this work is demonstration of the KPC/AmpC enzyme
inhibitory activity by some benzosiloxaboroles (compounds 4, 10, 27, 37 and 40). The highest
activity was observed for 6-B(OH) substituted derivative 27. Biological activity of selected
2
compounds has been confirmed with molecular modelling methods. Docking results with
KPC-2 enzyme showed the strongest binding with compounds 27 and 37. Importantly, they
also exhibit low cytotoxity as indicated from the tests performed for human lung fibroblast.
At this point it should be stressed, that only one organoboron compound, namely
vaborbactam, was approved for treatment as a novel β-lactamase inhibitor with KPC
inhibitory activity, so far. Thus our results may initiate new progress in this area.
Moreover, obtained series of benzosiloxaboroles showed direct antifungal activity,
especially against C. tropicalis, C. guilliermondii and S. cerevisiae. The presence of chlorine
at the 6-position of benzosiloxaboroles in compounds 11 and 24 resulted in selectively higher
activity of these agents against C. albicans. It is interesting that 11 showed also the highest
activity against Gram-positive cocci from Staphylococcus and Enterococcus genera (MIC
values 6.25 mg/L and 25 mg/L, respectively). The clinical strains of S. aureus MRSA and E.
faecalis VRE are still a severe health public problem. Regarding the weak activity against
Gram-negative rods, the MIC values for a group of 22 studied benzosiloxaboroles in the
presence of PAβN were significantly (a minimum 4-fold) reduced against majority of the
1
9

ACCEPTED MANUSCRIPT
studied strains. Presumably, a limited activity (MIC 100->400 mg/L) of analyzed compounds
resulted from the presence of specific Gram-negative rods efflux pumps.
To summarize, our study highlights a new group of organoboron heterocycles as a source
of potential KPC inhibitors as well as direct antifungal agents. Further work in the field is
currently in progress in our group and the results will be given in due course. The search for
organoboron compounds that inhibit β-lactamases, particularly carbapenemases, is one of the
latest ways to look for new therapeutic options. The rapid progress in this field is highly
demanded as the mortality of patients infected with KPC-producing strains reaches 70%,
constituting one of the major worldwide health threats [65,66].
Experimental Section
Synthesis of benzosiloxaboroles.
Solvents used for reactions were dried by heating to reflux with sodium/benzophenone and
distilled under argon. n-BuLi (10 M in hexane), t-BuLi (1.7 M in pentane), trialkyl borates
B(OR) , Me Si(H)Cl and starting haloarenes were used as received without further
3
2
purification. Detailed procedures for the synthesis of benzosiloxaborole precursors are given
in the Supporting Information. Syntheses of benzosiloxaboroles 1-4 [15] and 32-37 [25] were
13
reported by us recently. In the C NMR spectra the resonances of boron-bound carbon atoms
1
were not observed in most cases as a result of broadening by a quadrupolar boron nucleus. H
1
3
and C NMR chemical shifts are given relative to TMS using residual solvent resonances.
1
1
19
B and F NMR chemical shifts are given relative to BF ·Et O and CFCl , respectively. The
3
2
3
purity of all new compounds (> 95%) was established by elemental analysis and HRMS.
6
,7-Difluoro-1,3-dihydro-3-hydroxy-1,1-dimethyl-1,2,3-benzosiloxaborole
(10):
A
solution of 5 (5.67 g, 0.023 mol) in Et O (10 mL) was added dropwise to a solution of tBuLi
2
(
1.7 M in pentane, 28.2 mL, 0.048 mol) in Et O (100 mL) and cooled to –95 °C. The solution
2
o
was stirred for 30 min, warmed to –85 C and then again cooled to –100 °C followed by the
addition of B(OEt) (3.9 mL, 0.023 mol). The obtained white slurry was warmed to –30 °C,
3
quenched with 1.5 M aq. H SO to reach the pH = 2-3 and stirred at room temperature until
2
4
evolution of H ceased. The aqueous phase was separated followed by the extraction with
2
Et O (2 × 20 mL). The extracts were added to the organic phase, which was concentrated
2
under reduced pressure. Water (5 mL) acidified with a few drops of conc. aqueous HCl was
added and the white solid precipitated during the evaporation. It was filtered, and
2
0

ACCEPTED MANUSCRIPT
recrystallized from hexane. Drying in vacuo afforded 10 as white powder, m.p. 81–82 °C.
1
Yield 3.69 g (75%). H NMR (400 MHz, CDCl ): δ 7.53 (dd, J = 7.8, 3.8 Hz, 1H, Ar), 7.25
3
1
3
1
(
ddd, J = 10.8, 7.8, 7.4 Hz, 1H, Ar), 5.20 (br, 1H, B(OH)), 0.51 (s, 6H, SiMe ) ppm. C{ H}
2
NMR (100.6 MHz, CDCl ): δ 152.09 (dd, J = 254.5, 15.3 Hz), 151.51 (dd, J = 247.0, 11.6
3
Hz), 137.96 (d, J = 26.7 Hz), 128.84 (dd, J = 6.1, 3.5 Hz), 120.00 (d, J = 17.9 Hz), –0.76
19
ppm. F NMR (282.44 MHz, CDCl ): δ –129.53 (dd, J = 24.0, 7.3 Hz), –135.13 (ddd, J =
3
11
2
4.0, 10.8, 3.7 Hz) ppm. B NMR (96.32 MHz, CDCl ): δ 29.7 ppm. HRMS (EI): calcd. for
3
+
C H BF O Si [M] 214.0433; found 214.0431. Anal. Calcd for C H BF O Si: C, 44.89; H,
8
9
2
2
8
9
2
2
4
.24. Found: C, 44.71; H, 3.97.
,7-Dichloro-1,3-dihydro-3-hydroxy-1,1-dimethyl-1,2,3-benzosiloxaborole (11). This
6
compound was obtained from 6 (0.01 mol) using the procedure described for 10. The product
1
was obtained as a white powder, m.p. 107-109 °C. Yield 1.74 g (68%). H NMR (400 MHz,
CDCl ): δ 7.62 (dd, J = 7.7, 2.5 Hz, 1H, Ar), 7.53 (d, J = 7.7 Hz, 1H, Ar), 0.53 (s, 6H, SiMe )
3
2
13
1
ppm. C{ H} NMR (101 MHz, CDCl ): δ 151.80, 135.33, 135.16, 132.54, 130.79, –1.59
3
+
ppm. HRMS (EI): calcd. for C H BCl O Si [M] 245.9842; found 245.9844. Anal. Calcd for
8
9
2
2
C H BCl O Si: C, 38.91; H, 3.67. Found: C, 38.69; H, 3.59.
8
9
2
2
7
-Chloro-1,3-dihydro-3-hydroxy-1,1-dimethyl-1,2,3-benzosiloxaborole
(12).
This
compound was obtained from 7 (0.01 mol) using the procedure described for 10. The product
1
was obtained as a white powder, m.p. 133-134 °C. Yield 1.74 g (68%). H NMR (300 MHz,
CDCl ): δ 7.67 (dd, J = 4.7, 3.4 Hz, 1H, Ar), 7.45 – 7.38 (m, 2H, Ar), 4.95 (s, 1H, B(OH)),
3
1
3
1
0
1
.50 (s, 6H, SiMe ) ppm. C{ H} NMR (100.6 MHz, CDCl ): δ 149.38, 137.68, 131.76,
2 3
1
1
30.54, 129.45, –1.45 ppm. B NMR (96.3 MHz, CDCl ): δ 29.6 ppm. HRMS (EI): calcd.
3
+
for C H BClO Si [M] 212.0232; found 212.0234. Anal. Calcd for C H BClO Si: C, 45.22;
8
10
2
8
10
2
H, 4.74. Found: C, 44.76; H, 3.91.
-Bromo-1,3-dihydro-3-hydroxy-1,1-dimethyl-1,2,3-benzosiloxaborole
7
(13).
This
compound was obtained from 8 (0.01 mol) using the procedure described for 10. However in
this case nBuLi (10 M, 1.05 mL, 10.5 mmol) was used instead of tBuLi. The product was
1
obtained as a white powder, m.p. 120-122 °C. Yield 1.12 g (43%). H NMR (300 MHz,
CDCl ): δ 7.76 (dd, J = 7.2, 0.9 Hz, 1H, Ar), 7.58 (dd, J = 7.9, 0.9 Hz, 1H, Ar), 7.33 (dd, J =
3
1
3
1
8
.0, 7.2 Hz, 1H, Ar), 6.22 (br, 1H, B(OH)), 0.54 (d, J = 0.8 Hz, 6H SiMe ) ppm. C{ H}
2
11
NMR (100.57 MHz, CDCl ): δ 152.33, 133.62, 131.93, 130.03, 126.55, –1.64 ppm. B NMR
3
+
(
96.3 MHz, CDCl ): δ 29.8 ppm. HRMS (EI): calcd. for C H BBrO Si [M] 255.9726; found
3
8
10
2
2
55.9729. Anal. Calcd for C H BBrO Si: C, 37.39; H, 3.92. Found: C, 37.58; H, 3.63.
8 10 2
2
1

ACCEPTED MANUSCRIPT
6
-Trifluoromethyl-1,3-dihydro-3-hydroxy-1,1-dimethyl-1,2,3-benzosiloxaborole
(14):
This compound was obtained from 9 (0.035 mol) using the procedure described for 10. The
1
product was obtained as a white powder, m.p. 101-103 °C. Yield 4.90 g (71%). H NMR (400
MHz, CDCl ): δ 7.94 (dd, J = 7.7, 0.8 Hz, 1H, Ar), 7.86 (m, 1H, Ar), 7.71 (dd, J = 7.7, 0.9
3
1
3
1
Hz, 1H, Ar), 5.87 (br, 1H, B(OH)), 0.48 (s, 6H, SiMe ) ppm. C{ H} NMR (101 MHz,
2
CDCl ): δ 151.00, 132.43 (q, J = 31.7 Hz, CF ), 131.58, 126.89 (q, J = 3.8 Hz), 126.34 (q, J =
3
3
1
9
11
3
.8 Hz), 125.66, 122.95, –0.74 ppm. F NMR (282.44 MHz, CDCl ): δ –62.74 ppm.
B
3
+
NMR (96.3 MHz, CDCl ): δ 30.2 ppm. HRMS (EI): calcd. for C H BO F Si [M] 246.0495;
3
9
10
2 3
found 246.0497. Anal. Calcd for C H BO F Si: C, 44.93; H, 4.10. Found: C, 44.17; H, 4.12.
9
10
2 3
6
-Fluoro-1,3-dihydro-3-hydroxy-1,1-dimethyl-1,2,3-benzosiloxaborole (23). A solution of
1 (5.16 g, 0.015 mol) in THF (15 mL) was added to the previously prepared solution of
2
nBuLi (10 M solution in hexanes, 1.5 mL, 0.015 mol) in THF (30 mL) at –95 °C. After 30
min of stirring Me (H)SiCl (1.66 mL, 0.015 mol) was slowly added. Then it was warmed
2
gradually to –40 °C, which resulted in a white, thick slurry. It was quenched with H SO (1.5
2
4
o
M) to pH = 4 and stirred at 40 C until hydrogen evolution ceased. The water phase was
separated followed by the extraction with Et O (2 × 20 mL). The extracts were added to the
2
organic phase, which was concentrated under reduced pressure, to give a white residue. It was
crystallized from hexane (20 mL) to give 23 as a white powder, m.p. 63-64 °C. Yield: 1.76 g
1
(
60%). H NMR (300 MHz, CDCl ): δ 7.82 (ddd, J = 8.2, 5.4, 0.6 Hz, 1H, Ar), 7.27 (ddd, J =
3
8
.2, 2.4, 0.6 Hz, 1H, Ar), 7.14 (ddd, J = 9.7, 8.1, 2.4 Hz, 1H, Ar), 6.02 (br, 1H, OH), 0.45 (s,
13
1
6
H, SiMe ) ppm. C{ H} NMR (101 MHz, CDCl ): δ = 165.02 (d, J = 252.6 Hz), 153.41 (d,
2
3
J = 5.8 Hz), 133.62 (d, J = 7.8 Hz), 117.05 (d, J = 21.5 Hz), 116.89 (d, J = 19.0 Hz), –0.74
19
11
ppm. F NMR (376 MHz, CDCl ): δ –109.33 (ddd, J = 9.7, 8.1, 5.2 Hz) ppm. B NMR (96
3
+
MHz, CDCl ): δ 30.0 ppm. HRMS (EI): calcd. for C H BO FSi [M] 196.0527; found
3
8
10
2
1
96.0524. Anal. Calcd for C H BO FSi: C, 49.01; H, 5.14. Found: C, 48.82; H, 5.10.
8 10 2
6
-Chloro-1,3-dihydro-3-hydroxy-1,1-dimethyl-1,2,3-benzosiloxaborole
(24).
This
compound was obtained from 22 (2.38 g, 5.8 mol) using the procedure described for 23.
1
Yield: 0.67 g (55%), m.p. 105-108 °C H NMR (400 MHz, CDCl ): δ 7.75 (dd, J = 7.9, 0.7
3
Hz, 1H, Ar), 7.57 (d, J = 1.9 Hz, 1H, Ar), 7.43 (ddd, J = 7.9, 1.9, 0.5 Hz, 1H, Ar), 6.06 (s, 1H,
13
1
OH), 0.45 (s, 6H, SiMe ) ppm. C{ H} NMR (75 MHz, CDCl ): δ 152.43, 137.76, 132.80,
2
3
1
1
1
30.41, 129.90, -0.68 ppm. B NMR (96 MHz, CDCl ): δ 30.0 ppm. HRMS (EI): calcd. for
3
+
C H BO SiCl [M] 212.0232; found 212.0225. Anal. Calcd for C H BO SiCl: C, 45.22; H,
8
10
2
8
10
2
4
.74. Found: C, 44.85; H, 4.39.
2
2

ACCEPTED MANUSCRIPT
1
,3-Dihydro-3-hydroxy-7-(dihydroxyboryl)-1,1-dimethyl-1,2,3-benzosiloxaborole (25).
This compound was obtained from 8 (5.88 g, 0.02 mol) in a similar procedure as 10. 4 equiv
of tBuLi (1.7 M pentane, 47 mL, 0.08 mol) and 3 equiv of B(OMe) (6.4 mL, 0.06 mol) were
3
1
used. The product was obtained as a white powder, m.p. 340-344 °C. Yield 3.30 g (74 %). H
NMR (400 MHz, acetone-d + D O): δ 7.97 (dd, J = 7.4, 1.2 Hz, 1H, Ar), 7.87 (dd, J = 7.3,
6
2
1
3
1
.2 Hz, 1H, Ar), 7.39 (t, J = 7.4 Hz, 1H, Ar), 3.93 (br, 3H, OH), 0.34 (s, 6H) ppm. C NMR
1
1
(
101 MHz, acetone-d + D O): δ 158.16, 135.99, 132.87, 128.45, -1.15 ppm. B NMR (96
6 2
+
MHz, acetone-d + D O) δ 28.8 ppm. HRMS (EI): calcd. for C H B O Si [M] 222.0691;
6
2
8
12
2
4
found 222.0690. Anal. Calcd for C H B O Si: C, 43.31; H, 5.45. Found: C, 43.40; H, 5.79.
8
12
2
4
1
,3-Dihydro-3-hydroxy-6-(dihydroxyboryl)-1,1-dimethyl-1,2,3-benzosiloxaborole (27).
This compound was obtained from 26 (2.95 g, 0.01 mol) using the procedure described for
o
1
2
5. Yield 1.57 g (71%). m.p. 340-346 C. H NMR (300 MHz, acetone-d + D O): δ 8.16 (s,
6 2
1
H, Ar), 7.92 (d, J = 7.5 Hz, 1H, Ar), 7.82 (d, J = 7.5 Hz, 1H, Ar), 3.61 (br, 3H, OH), 0.36 (s-
13
br, 6H, SiMe ) ppm. C NMR (101 MHz, acetone-d + D O): δ 149.14, 136.33, 134.98,
2
6
2
11
1
30.15, –1.36 ppm. B NMR (96 MHz, acetone-d + D O): δ 28.8 ppm. HRMS (EI): calcd.
6 2
+
for C H B O Si [M] 222.0691; found 222.0689. Anal. Calcd for C H B O Si: C, 43.31; H,
8
12
2
4
8
12
2
4
5
.45. Found: C, 42.94; H, 5.80.
-Fluoro-1,3-dihydro-3-hydroxy-5-(dihydroxyboryl)-1,1-dimethyl-1,2,3-
7
benzosiloxaborole (29). A solution of 28 (3.12 g, 0.01 mol) in Et O (15 mL) was added
2
dropwise to a solution of t-BuLi (1.7 M in pentane, 24 mL, 0.04 mol) in THF (70 mL) at –100
°
C. After 30 min of stirring at –100 °C, B(OMe) (4.4 mL, 0.04 mol) was added and the
3
reaction mixture was warmed to –30 °C, quenched with 1.5 M aq. H SO to reach the pH = 2-
2
4
3
and stirred at room temperature until evolution of H ceased. The aqueous phase was
2
separated followed by the extraction with Et O (2 × 20 mL). The extracts were added to the
2
organic phase, which was concentrated under reduced pressure. The white solid precipitated
during the evaporation. It was filtered and washed several times with water. Then it was
suspended in CH Cl (10 mL) and stirred for 30 min. Then hexane (10 mL) was added and the
2
2
resulting slurry was filtered. The product was washed with hexane (2 × 5 mL) and dried in
1
vacuo to give 29, m.p. 310–315 °C. Yield 2.1 g (87%). H NMR (400 MHz, acetone-d ): δ
6
8
.19 (s, 1H, BOH), 8.12 (d, J = 3.0 Hz, 1H, Ph), 7.57 (d, J = 8.2 Hz, 1H, Ph), 7.43 (s, 2H,
1
3
B(OH) ), 0.43 (s, 6H, SiMe ) ppm. C NMR (101 MHz, acetone-d ): δ 164.63 (d, J = 243.5
2
2
6
Hz), 143.8 (broad, C ), 139.6 (broad, C ), 136.85 (d, J = 30 Hz), 133.30 (d, J = 2.4 Hz),
B
B
1
1
19
1
21.48 (d, J = 22.2 Hz), –1.51 (s) ppm. B NMR (96 MHz, acetone-d ): δ 28.5 ppm.
F
6
NMR (376 MHz, acetone-d ): δ –107.14 (m) ppm. HRMS (EI): calcd. for C H B O Si F
6
8
11
2
4
2
2
3

ACCEPTED MANUSCRIPT
+
[
M] 240.0597; found 240.0593. Anal. Calcd for C H B O Si F: C, 40.06; H, 4.62. Found:
8 11 2 4 2
C, 39.98; H, 4.58.
8
-Fluoro-1,3,5,7-tetrahydro-3,5-dihydroxy-1,1,7,7-
tetramethylbenzo[1,2,3,7,6,5]bis(siloxaborole) (31). This compound was prepared as
described for 29 starting with 30. It was isolated as a white solid, m.p. 136–140 °C. Yield 2.2
1
g (74%). H NMR (300 MHz, acetone-d ): δ 8.20 (broad, 2H, BOH), 8.09 (d, J = 5.1 Hz, 1H,
6
13
Ph), 0.46 (s, 12H, SiMe ) ppm. C NMR (101 MHz, acetone-d ): δ 166.89 (d, J = 247.0 Hz),
2
6
1
1
1
48.87 (broad, C ), 136.70 (d, J = 34.3 Hz), 130.63 (d, J = 2.8 Hz), –1.45 (s) ppm. B NMR
B
1
9
(
96 MHz, acetone-d ): δ 29.5 ppm. F NMR (282 MHz, acetone-d ) δ = –96.62 (s) ppm.
6
6
+
HRMS (EI): calcd. for C H B O Si F [M] 296.0679; found 296.0675. Anal. Calcd for
10
15
2
4
2
C H B O Si F: C, 40.58; H, 5.11. Found: C, 40.33; H, 5.25.
10
15
2
4
2
4
,8-difluoro-3,3-dimethyl-3,7-dihydro-1H,5H-[1,2]oxaborolo[4',3':4,5]benzo[1,2-
c][1,2,5]oxasilaborole-1,5-diol (40). A solution of 39 (4.04 g, 0.01 mol) in THF (15 mL) was
added dropwise to a solution of t-BuLi (1.7 M in pentane, 24 mL, 0.04 mol) in THF (80 mL)
and cooled to –100 °C. After 30 min of stirring at –100 °C, B(OMe) (4.5 mL, 0.04 mol) was
3
added and the reaction mixture was warmed to –30 °C, quenched with 1.5 M aq. H SO to
2
4
reach the pH = 2-3 and stirred at room temperature. The aqueous phase was separated
followed by the extraction with Et O (2 × 20 mL). The extracts were added to the organic
2
phase, which was concentrated under reduced pressure. A solid residue was washed with
water, CH Cl (2 × 5 mL), hexane (2 × 5 mL), and dried in vacuo to give the final product 40,
2
2
1
m.p. 195–198 °C. Yield 0.75 g (28%). H NMR (400 MHz, acetone-d ): δ 5.08 (s, 2H), 2.95
6
1
3
(
s, 1H), 0.45 (s, 6H) ppm. C NMR (101 MHz, acetone-d ): δ 161.3 (d, J = 244.6 Hz), 156.0
6
(dd, J = 251.0, 3.0 Hz), 144.8 (dd, J = 20.7, 8.0 Hz), 136.1 (d, J = 39.6 Hz), 120.65, 67.0, –
1
9
1
.67 ppm. F NMR (376 MHz, acetone-d ): δ –104.43 (dd, J = 33.5, 9.8 Hz), –121.66 (d, J =
6
1
1
3
3.5 Hz) ppm. B NMR (96 MHz, acetone-d ): δ 30.5 ppm. HRMS (EI): calcd. for
6
+
C H B F O Si [M] 270.0503; found 270.0492. Anal. Calcd for C H B F O Si: C, 40.05; H,
9
10
2
2
4
9
10
2
2
4
3
.74. Found: C, 40.28; H, 3.58.
Antimicrobial activity
Bacterial and fungal strains and their growth conditions. The following standard strains to
determine the direct antimicrobial activity were used in the study: (1) Gram-positive cocci:
Staphylococcus aureus ATCC 6538P, S. epidermidis ATCC 12228, Enterococcus faecalis
ATCC 29212, E. faecium ATCC 6057, Bacillus subtilis ATCC 6633; (2) Gram-negative
bacteria from Enterobacteriaceae family: Escherichia coli ATCC 25922, Klebsiella
2
4

ACCEPTED MANUSCRIPT
pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453, Enterobacter cloacae DSM 6234,
Serratia marcescens ATCC 13880; (3) Gram-negative non-fermentative rods: Pseudomonas
aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Stenotrophomonas
maltophilia ATCC 13637, Burkholderia cepacia ATCC 25416, Bordetella bronchiseptica
ATCC 4617; (4) yeasts: Candida albicans ATCC 90028, C. parapsilosis ATCC 22019, C.
tropicalis IBA 171, C. guilliermondii IBA 155, C. krusei ATCC 6258 and Saccharomyces
cerevisiae ATCC 9763.
The following strains (see Table 4) were used to examine β-lactamase inhibitory activity
of studied agents: (1) two standard strains - K. pneumoniae ATCC BAA-1705 (with
carbapenemase KPC-2) and K. pneumoniae ATCC 700603 (with extended-spectrum β-
lactamase, ESBL, SHV-12); (2) eleven clinical isolates producing various classes of β-
lactamases – carbapenemases KPC-2 (E. coli 76, K. pneumoniae 75) and KPC-3 (K.
pneumoniae 81 and 83), metallo-β-lactamase from VIM family (P. aeruginosa 698),
carbapenem-hydrolyzing class D β-lactamase (CHDL) from OXA-24 family (A. baumannii
1
81), ESBL-type enzymes CTX-M-2 (E. coli 557) and OXA-15 (P. aeruginosa 852),
plasmid-acquired AmpC cephalosporinase CMY-2 (E. coli 77, K. pneumoniae 78), and with
overexpression of chromosomally encoded cephalosporinase AmpC (P. aeruginosa MUW
7
00); (3) the transformant of E. coli DH5α with plasmid from clinical strain of K.
pneumoniae 81 carrying the gene blaKPC-3. All strains were stored at −80 °C. Prior to testing,
each bacterial strain was subcultured twice on tryptic soy agar TSA (bioMérieux) medium and
yeast strains on Sabouraud dextrose agar (bioMérieux) for 24–48 h at 30 °C to ensure
viability.
Determination of antimicrobial activity. Direct antimicrobial activity against yeast, Gram-
positive and Gram-negative bacterial strains was examined by the disc-diffusion test and the
MIC determination assays according to the EUCAST [30,67] and CLSI [28,29,66]
recommendations. Additionally, in the study of antimicrobial activity of new
benzosiloxaboroles two following reference agents were used: fluconazole (in the case of
fungi) and nitrofurantoin (for bacteria). The MIC value of fluconazole was tested by Etest
method [31,32]. Determination of MIC/MBC values of nitrofurantoin was done using the
CLSI methods [68,69], however, its concentration range was compatible with Etest. The
solutions of all tested compounds were prepared in DMSO (Sigma). The disc-diffusion test
was determined on Mueller-Hinton II agar medium (MHA) (Becton Dickinson) for bacteria
and on MHA supplemented with 2% glucose and 0.5 mg/L methylene blue dye (Sigma)
(MHA+GMB medium) for yeasts. The MIC determination was performed in Mueller-Hinton
2
5

ACCEPTED MANUSCRIPT
II broth medium (MHB) (Becton Dickinson) for bacteria and in RPMI 1640 broth medium
(Sigma) with 2% glucose (Sigma) for yeasts. Results of antimicrobial activity were evaluated
o
after incubation at 35 C for 18 h (bacteria) and 24 h (yeasts). Determination of bactericidal
(MBC) activity was performed according to the CLSI recommendations [69].
Determination of the MICs of agents in the presence of PAβN. To determine the ability of
the strains to remove benzosiloxaboroles by MDR efflux pumps, the MIC values of studied
agents, with or without the pump inhibitor, PAβN (50 mg/L) (Sigma) were evaluated [41].
The MIC determination was performed in MHB with 1 mM MgSO (Sigma), using 2-fold
4
serial dilutions, according to the CLSI guidelines [68] in order to compare these two assays.
The presence of 1 mM MgSO stabilizes the outer membrane [70]. At least a 4-fold decrease
4
in the MIC value after the addition of PAβN was considered significant [40,41].
Study of BLI activity of tested agents
The ability of new studied agents to serve as BLIs was examined into two steps, initially by
using combination disc tests (CDTs) for screening all compounds and then by microdilution
test for selected agents.
Combination disc tests for detection of BLI activity. The three following CDTs were
performed on Müller-Hinton agar (MHA) plates (Becton Dickinson) according to the general
EUCAST recommendations [52] and methodology described by Yagi et al. [53]:
(1) CDT-KPC is the EUCAST phenotypic test using discs with meropenem (MEM-10)
(Becton Dickinson) alone and discs with this β-lactam supplemented with PBA (Sigma) at the
concentration of 0.3 mg per disc. Additionally, in this research the discs with meropenem
supplemented with one of the studied compounds (at the concentration of 0.3 mg per disc)
were also used. The CDT-KPC test was performed using the recommended by EUCAST
reference strain K. pneumoniae ATCC BAA-1705. In the case of PBA, the standard KPC
inhibitor, a difference of at least 4 mm in the inhibition zone of MEM-PBA versus MEM-10
for K. pneumoniae ATCC BAA-1705 should be obtained [52]. The KPC carbapenemases
inhibitor activity of new tested agents was evaluated by comparison of the diameter of
inhibition zone around the disc with meropenem supplemented with a new agent to the
diameter of inhibition zone around the disc with meropenem alone. In this study, we assumed
that if the new compound has BLI activity then the difference in these diameters should also
be at least 4 mm;
(
2) CDT-AmpC is the modified phenotypic test previously described by Yagi et al. [51]
for detection class β-lactamase-producing Enterobacteriaceae by using 3-
aminophenylboronic acid (APB). This test has been specially modified for the purpose of this
C
2
6

ACCEPTED MANUSCRIPT
work in order to meet general procedures for detection of resistance mechanisms
recommended by EUCAST [52]. The CDT-AmpC test was performed using two AmpC-
producing clinical isolates: P. aeruginosa MUW 700 with overexpression of chromosomally
encoded cephalosporinase AmpC and E. coli 77 with plasmid-acquired AmpC
cephalosporinase CMY-2. The AmpC cephalosporinases inhibitor activity of new tested
agents was evaluated by comparison of the diameter of inhibition zone around the disc with
ceftazidime (CAZ-30) supplemented with a new agent (0.3 mg per disc) to the diameter of
inhibition zone around the disc with ceftazidime alone. PBA at the concentration of 0.3 mg
per disc was used as the reference BLI which is known to inhibit the activity of both KPC
[
52] and AmpC enzymes [53]. The results of BLI activity were evaluated after incubation at
o
3
5 C for 18 hours. It was assumed in this study that, in the presence of reference compound
PBA and new agents with BLI activity, a difference of at least 5 mm in the inhibition zones
around the disc with CAZ-PBA or CAZ+new agent versus CAZ-30 in the case of P.
aeruginosa MUW 700 as well as E. coli 77 should be observed. Ceftazidime was chosen for
this study also following Xia et al. [54] who observed increased susceptibility of AmpC-
producing strains to this cephalosporin in the presence of some new benzoxaboroles as β-
lactamase inhibitors;
(
3) CDT-ESBL is the EUCAST phenotypic test using disc with ceftazidime (CAZ-30) or
cefotaxime (CTX-30) alone and disc with this β-lactam supplemented with clavulanic acid
CAZ-CL and CTX-CL) (Becton Dickinson) in concentration 0.01 mg per disc. The ESBL
(
inhibitor activity of new tested agents was evaluated by comparison of the diameter of
inhibition zone around the disc with ceftazidime and cefotaxime supplemented with a new
agent (0.3 mg per disc) to the diameter of inhibition zone around the disc with β-lactam alone.
In the case of clavulanic acid, the standard ESBL inhibitor, a difference of at least 5 mm in
the inhibition zone of CAZ-CL versus CAZ-30 and CTX-CL versus CTX-30 for K.
pneumoniae ATCC 700603 should be obtained [52]. Using the new compounds as BLI, we
assumed that the difference in these diameters should also be at least 5 mm.
Microdilution test for detection of BLI activity. The ability of new studied agents to inhibit
the activity of various classes of carbapenemases, AmpC-type and ESBL enzymes was
examined by the MIC values of β-lactam determination assays, with or without
benzosiloxaboroles, according to the CLSI [68] recommendations. Ceftazidime was used to
determine the BLI activity against various classes of β-lactamases, While meropenem allowed
the analyzes of new agent activity only against carbapenemases. All studied
benzosiloxaboroles and PBA were added at a the concentration of 0.016 mg/L. The MIC
2
7

ACCEPTED MANUSCRIPT
determination was performed in Mueller-Hinton II broth medium (MHB) (Becton Dickinson).
o
Results of BLI activity were evaluated after incubation at 35 C for 18 hours. It was assumed
that at least a 4-fold decrease in the MIC value of β-lactams after the addition of PBA and
new agent with BLI activity should be observed.
Cytotoxic studies. (1) Culture method. MRC-5 pd30 human fibroblasts were cultured in
MEM (Gibco) supplemented with 10% fetal bovine serum (EuroClone), 2mM L-glutamine,
antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, Sigma-Aldrich) and 1% non-
2
essential amino-acids (Sigma-Aldrich). Cells were grown in 75 cm cell culture flasks
(
Sarstedt), in a humidified atmosphere of CO /air (5/95%) at 37˚C.
2
MTT-based viability assay
2) MTT-based viability assay. Stock solutions of the tested compounds were prepared in
DMSO, so the final concentration of vehicle was 0.5% in each case. For cytotoxicity studies
-fold serial dilutions were prepared in the proper medium containing 0.5% DMSO. All the
(
2
experiments were performed in exponentially growing cultures. Before treatment MRC-5
cells were trypsinized in 0.25% trypsin-EDTA solution (Sigma-Aldrich) and seeded into 96-
3
well microplates (Sarstedt) at a density of 8×10 cells/well. Cells were treated with the tested
compounds or DMSO (0.5%) at the appropriate concentrations 18 h after plating. After 72h
incubation with the compounds, the supernatants were discarded, and subsequently MTT
stock solution (Sigma-Aldrich) was added to each well to the final concentration of 1 mg/ml.
After 1 h incubation at 37˚C, water-insoluble dark blue formazan crystals were dissolved in
DMSO (100 µl) (37˚C/10 min incubation). Optical densities were measured at 570 nm using
BioTek microplate reader. All measurements were carried out in four replicates and the
results expressed as a percent of viable cells versus control cells.
Docking and molecular dynamics
Three most promising compounds (10, 27, 37 and 4-FBOB) were modeled with semi-flexible
molecular docking approach. The search of PDB database for boron-compoundcontaining β-
lactamase structures revealed that all systems are covalently bonded to the serine residue in a
pocket. Furthermore, organoboron molecules are present in their anionic form with
tetracoordinate boron atom. In the current studies all possible ionized forms of the compounds
were thus generated and docked using MOE2016.08 [71] to the KPC-2 β-lactamase structure
derived from PDB (ID: 3RXX) [64]. The geometry binding pocket was minimized in the
absence of a compound. Scoring function GBVI/WSA dG was applied. Results, where boron
atom was placed closer than 4 Å to Ser70 sidechain oxygen atom, were qualified as potent to
form a covalent bond. Only those results were retained and B-O-Ser70 bod was introduced
2
8