Synthesis and biological evaluation of an antibacterial azaborine retinoid isostere

Article abstract of DOI:10.1016/j.tetlet.2020.152667

Our continued synthetic interest in this class of retinoids, CD437 and its analogs, against methicillin-resistant Staphylococcus aureus (MRSA) has brought us to explore further isosteric substitutions within the scaffold. Although our previous findings ha

Full text of DOI:10.1016/j.tetlet.2020.152667

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Synthesis and biological evaluation of an antibacterial azaborine retinoid iso‐
stere
Brittney A. Haney, Cassandra L. Schrank, William M. Wuest
PII:
S0040-4039(20)31178-3
DOI:
Reference:
https://doi.org/10.1016/j.tetlet.2020.152667
TETL 152667
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Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
6 October 2020
5 November 2020
12 November 2020
Please cite this article as: Haney, B.A., Schrank, C.L., Wuest, W.M., Synthesis and biological evaluation of an
antibacterial azaborine retinoid isostere, Tetrahedron Letters (2020), doi: https://doi.org/10.1016/j.tetlet.
2
020.152667
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Synthesis and biological evaluation of an
antibacterial azaborine retinoid isostere
Leave this area blank for abstract info.
Brittney A. Haney, Cassandra L. Schrank,
and William M. Wuest
X
X
HO
HO
B
N
H
OH
OH
CD437 (X = O)
Analog 2 (X = H,H)
Azaborine Analogs
(X = O or H,H)

1
Tetrahedron Letters
journal homepage: www.elsevier.com
Synthesis and biological evaluation of an antibacterial azaborine retinoid isostere#
Brittney A. Haney, Cassandra L. Schrank, and William M. Wuest
Department of Chemistry, Emory University, 30322, United States
#
Dedicated to Prof. Dale Boger for his scholarship in the field of medicinal chemistry and continued support for the next generation of scientists
—
——
ARTICLE INFO
ABSTRACT

Corresponding author. Tel.: E-mail: wwuest@emory.edu
Article history:
Received
Received in revised form
Accepted
Our continued synthetic interest in this class of retinoids, CD437 and its analogs, against
methicillin-resistant Staphylococcus aureus (MRSA) has brought us to explore further isosteric
substitutions within the scaffold. Although our previous findings have shown promising activity
against gram-positive pathogens, their therapeutic viability remained an issue. Specifically,
through preliminary analysis, our best performing compound, analog 2, displayed low solubility
within serum as well as high affinity for retinoid binding proteins with a concentration
dependent relationship. To circumvent this issue, we proposed a class of analogs containing an
azaborine substitution in place of the naphthalene moiety. Azaborines have a nitrogen-boron
bond substituting a carbon-carbon double bond that alters the electronics of the parent scaffold.
This motif has been explored successfully in cancer research but to the best of our knowledge
has yet to be applied to antibiotics. Herein, we describe the synthesis of the desired analogs,
antimicrobial activity, and surprising physiochemical properties.
Available online
Keywords:
CD437
Retinoids
Azaborine
Methicillin-resistant Staphylococcus aureus
2
009 Elsevier Ltd. All rights reserved.
Introduction
According to the Centers for Disease Control and Prevention,
more than 2.8 million antibiotic resistant infections occur
1
annually in the United States alone. One of the most prevalent
O
antibiotic
resistant
bacteria
is
methicillin-resistant
Staphylococcus aureus (MRSA), which is also known to develop
persistent populations. Persister cells are dormant bacterial cells
that are genetically identical, but phenotypically different than
HO
HO
OH
OH
2
the wild type. These cells have slowed growth rates, diminished
CD437 (1)
Analog 2 (2)
MIC = 2 g/mL
3
metabolism, and are found in many S. aureus populations. As
MIC = 1 g/mL
most antibiotics target metabolic and growth processes, persister
cells are often difficult to eradicate.4
X
Previously, our laboratory, in collaboration with the
Mylonakis laboratory at Brown University, disclosed the potent
activity of the synthetic retinoid CD437 (1) at eradicating both
wild type and persistent MRSA infections via membrane
perturbation with a minimum inhibitory concentration (MIC) of 1
HO
B
N
H
Azaborine Analogs
X = H,H (3)
OH
5
X = O (4)
μg/mL (Fig. 1). Since this finding in 2018, we have disclosed
three generations of analogs to further explore the structure
activity relationship of this class of retinoids.6-7 In our
investigations, our lead compound was found to be the primary
alcohol derivative we deemed analog 2 (2). This compound
exhibited an MIC of 2 μg/mL while also reducing the toxicity of
the parent compound.
Molander and co-workers'
Azaborine Method
K
F3B
O
MeO
OH
Figure 1. The structure and antimicrobial activity of CD437
+
NH2
(
1) and Analog 2 (2) are shown at the top followed by the
proposed azaborine analogs (3, 4) and a retrosynthetic plan
invoking the method developed by Molander and co-
workers.
5
6

2
Tetrahedron
O
B
K
OH
Br
F3B
i) NaH, MEMCl
THF, 0 °C to rt
OH
O
AcOH, H2SO4
5:1)
(
4.5 M KHF2
(94%)
OH
OMEM
OMEM
+
Br
ii) B2pin2, Na2CO3,
Pd(dppf)Cl2 (3 mol%)
Toluene, 80 °C
CH2Cl2, rt
61%)
MeOH, 0 °C
(62%)
(
7
8
(66%)
9
10
5
O
O
Tributylvinyltin,
Pd(PPh3)4 (10 mol%)
O
SiCl4, NEt3
MeCN:THF
Br
NH2
SOCl2, MeOH
Br
NH2
HO
MeO
MeO
(
1:1)
50 °C
25%)
0
°C to reflux
92%)
Toluene, 100 °C
(80%)
NH2
(
(
11
12
6
O
O
conditions
HO
HO
LiAlH4, THF
0 °C to rt
MeO
B
B
N
B
N
N
H
H
H
(97%)
OH
OH
OH
4
3
13
conditions
Scheme 1. Synthesis of the trifluoroborate fragment (5), aminostyrene fragment (6), and primary alcohol azaborine analog (3).
Attempted conditions to achieve the carboxylic acid analog (4) are described in the text.
Though this compound showed promise, it displayed high
the focus on T-cell lysozyme targets.14 Based on this background
we proposed to explore this isosteric model within the
antibacterial space.
levels of binding to plasma binding proteins, namely retinoid
binding proteins (unpublished data), thereby reducing its
effectiveness in a therapeutic setting. To combat this issue, our
To achieve this target, we employed a process developed by
1
5-16
laboratory proposed to investigate an isosteric substitution of the
naphthalene scaffold known as an azaborine (3, 4). It is well-
known that N-B bonds are isosteric to carbon-carbon double
bonds. The N-B bond, although isoelectronic, introduces a local
dipole moment with the lone pair of the nitrogen donating into
the empty p-orbital of boron.8-10 This polarity sharply alters the
molecular and solid states of the compound, namely widening the
HOMO-LUMO gap due to the lowered energy of the HOMO in
comparison to parent C=C bonds.
Molander and co-workers at the University of Pennsylvania.
This method proved to be amenable to our previously disclosed
syntheses of the parent compound and allowed us to prepare the
azaborine derivative quickly. Herein, we report on the synthesis
of the azaborine analog as well as its biological and
physiochemical properties in comparison to the parent
compounds.
We began by synthesizing the trifluoroborate salt fragment (5)
utilizing a previously described route for the synthesis of CD437
5
-7
With physical and molecular changes, N-B isosteres have
garnered interest within the medicinal community to expand the
structural diversity of therapeutic agents. Beginning in the early
(Scheme 1). Upon achieving the borylated compound, 10 was
then subjected to aqueous potassium bifluoride in methanol to
1
7-18
afford 5 in 62% yield.
With the trifluoroborate fragment in
1
960s, this isostere was mainly explored in diazaborines (B-N-
hand, we then began synthesis of the aminostyrene fragment (6,
Scheme 1). Beginning with commercially available 4-amino-3-
bromobenzoic acid (11), a methyl protection in the presence of
thionyl chloride and methyl iodide afforded 12. This was then
N), which exhibit antibacterial properties against Gram-negative
strains.
1
1-13
Their specific mode of action has been extensively
researched and hypothesized to target enoyl reductase; an
enzyme involved in fatty acid synthesis.12 Although diazaborines
have been studied in bacterial systems, their sister structure,
azaborine, to the best of our knowledge has not. To date,
azaborines have been applied in various anti-cancer studies with
19
subjected to a tin-mediated vinylation to afford 6 in 80% yield.
With both fragments in hand, we then employed the method
developed by Molander and co-workers utilizing silicon
Table 1. Antimicrobial activity and toxicity data for analog 2 (2) and azaborine analog (3).
Lysis20
M)
Compound
Minimum Inhibitory Concentration (M)
(
MSSA
HA-MRSA
CA-MRSA
E. faecalis
125
P. aeruginosa
125
E. coli
32
BAC
2
4
4
32
2
3
2
2
2
>250
>250
>250
>250
125
250
125
64
250
>250
>250
All MIC and Lysis20 data were acquired through an average of the highest value of three independent trials; all trials were
within one dilution.

3
tetrachloride and triethylamine (Scheme 1).15-16 This method
proved difficult with our scaffold as the MEM-protecting group
was subsequently removed during the reaction progress due to
the production of hydrochloric acid in situ. We hypothesized that
the deprotected free phenol subsequently formed a stable bond
with a neighboring boron, eliminating a fluoride anion and
thereby limiting the availability of the trifluoroborate in solution.
Although triethylamine was added to the reaction to neutralize
the hydrochloric acid production, the removal of MEM still
occurred regardless of the concentration of triethylamine used. In
future use, the addition of a proton sponge would be a potential
route to avoid these issues. However, the desired protected
azaborine scaffold (13) could still be obtained in 25% yield.
Although a change in protecting group strategy could potentially
circumvent these issues, we sought to pursue a direct path to the
desired analog for biological studies.
4, we decided to focus on the primary alcohol analog (3) as it
did mimic our best in class analog, analog 2 (2).
The antimicrobial activity of 3 was analyzed along with 2
against a panel of bacterial strains including three different
strains of Staphylococcus aureus (SH1000, ATCC 33591, USA
3
00-0114), Pseudomonas aeruginosa (PAO1), Enterococcus
2
0
faecalis (OG1RF), and Escherichia coli (MC4100). We chose
to use benzalkonium chloride (BAC) as a positive control since it
is a common, commercially available quaternary ammonium
compound that also induces bacterial killing via membrane
perturbation. In addition to antimicrobial activity, the toxicity
was analyzed using a red blood cell (RBC) lysis assay utilizing
mechanically defibrillated sheep’s blood. These values are
indicated as Lysis20 and are measured as the concentration that
lyse 20% or less of RBC. Surprisingly, the azaborine analog (3)
possessed a higher MIC than its isosteric partner (2, Table 1)
across all strains. However, the toxicity was comparable to the
parent compound (Table 1).
In achieving the formation of the desired scaffold, we
proceeded with reduction and deprotection pathways to afford
our planned primary alcohol and carboxylic acid analogs,
respectively. Firstly, protected 13 was subjected to lithium
aluminum hydride to afford the desired primary alcohol
azaborine (3) in 97% yield. Upon obtaining 3, we then turned to
deprotection of the methyl ester 13. Several hydrolysis methods
were attempted to achieve the desired carboxylic acid analog (4)
including base mediated hydrolysis (NaOH, KOH), acid-
To further understand this decrease in biological activity,
partition coefficients (P) for both the analog 2 (2) and azaborine
analog (3) were theoretically calculated via ChemDraw-3D and
experimentally determined through extraction protocols with n-
2
1-22
octanol and deionized water.
This experiment was performed
in triplicate, and the average P was determined. From this data,
log(base 10) of P was calculated. Somewhat surprisingly, we
found that 2 had a logP of 0.93 and 3 had a logP of 0.12 (Table
). These results indicate that the isosteric substitution in 3
switches the compound to become more hydrophilic in
2 4
mediated hydrolysis (HCl, H SO ), as well as milder approaches
with pig liver esterase (PLE). However, each method proved
unsuccessful. The base mediated hydrolysis showed degradation
in which the proposed degradative mechanism involved the
hydroxide anion attacking the empty p-orbital of the boron,
forming a stable O-B bond and eliminating the nitrogen, thereby
breaking apart the scaffold. This degraded product was confirmed
by NMR spectroscopy (data not shown). Additionally, the acid
mediated hydrolysis and the PLE-mediated hydrolysis reactions
yielded only recovered starting material.
2
comparison to 2.
HO
OH
Analog 2 (2)
Table 2. Experimentally determined P and logP for analog 2
HO
HO
(2) and azarborine analog (3) as well as quantum mechanical
B
B
calculated dipole expressed as debye (D).
N
N
H
H
OH
OH
2
3
Azaborine Analog (3)
P
logP
9.16
0.93 ± 0.20
7.687
1.39
0.12 ± 0.19
6.792
Figure 2. The structures of Analog 2 and the azaborine analog,
displaying contributing resonance structures. Highlighted in red
are hydrogen-bond donors and acceptors.
cPa
To further explore this surprising finding, we performed a
quantum mechanical analysis with Schrodinger 2020-3 suite of
modelling software to analyze the dipole moment of both
compounds. Through these calculations, we found that analog 2
had a higher dipole moment of 2.42 D vs 1.34 D for azaborine
clogPa
0.89
0.83
QM Dipole (D)
2.42
1.34
(
Table 2). As was previously disclosed by our group, the
proposed mechanism of action of this class of molecules is
membrane perturbation via amphiphilic attachment to the
phospholipids. We propose that the presence of the N-B bond
greatly reduces the molecular dipole moment of the primary
alcohol, thereby limiting its amphiphilicity. Additionally, within
the dipole analysis it was shown that the phenol dipole moment
was greatly reduced in comparison to the parent molecule. This is
most likely due to the resonance induction into the boron atom
Partition coefficients shown as an average over 3 independent trials.
logP data shown with + / – standard deviation. “c” indicates
calculated data for the partition coefficient utilizing the
ChemDraw3D program.
a
To avoid degradation of the scaffold, we attempted utilizing
oxidation pathways from the primary alcohol 3. First, we
implemented a Swern-Pinnick oxidation sequence that yielded
starting material as well as minor degradative products.
Additionally, we attempted a TEMPO oxidation that yielded only
recovered starting material. After these failed attempts to access
(not shown). Also, as highlighted in Figure 2, the azaborine
analog has more hydrophilic regions, explaining the observed
differences between clogP and logP (Fig. 2). Therefore, with a

4
Tetrahedron
more hydrophilic structure and additional points for hydrogen
9.
Bonifazi, D.; Fasano, F.; Lorenzo-Garcia, M. M.; Marinelli, D.;
Oubaha, H.; and Tasseroul, J. Boron–nitrogen doped carbon
scaffolding: organic chemistry, self-assembly and materials
applications of borazine and its derivatives. Chem. Comm., 2015,
bonding with water, 3 is likely unable to associate strongly with
the lipid bilayer thereby reducing its biological activity.
5
1(83), 15222–15236.
To confirm that the dipole moment, and not chemical stability,
was the reason for differential activity we characterized the
stability of 3 in water. Toward this end, we suspended the
compound in deionized water and heated it to 37 ⁰C for 23 h to
mimic the in vitro conditions employed in our biological assays.
The resulting mixture was then evacuated and analyzed by NMR
spectroscopy, which proved the compound to be stable in neutral
water. We recognize that there are limitations in this evaluation
as we chose deionized water over the growth media used.
However, we wanted a simple system to quantify degradation
with minimal background noise in the NMR spectroscopy
analysis and did not anticipate the additional nutrients found in
growth media to cause further degradation. Overall, we found
that the azaborine isosteric substitution is not a viable option for
improving the biological activity of our retinoid class of
antibacterial agents.
1
0. Knack, D.H.; Marshall, J.L.; Harlow, G.P.; Dudzik, A.; et al.
BN/CC isosteric compounds as enzyme inhibitors: N- and B-
ethyl-1,2-azaborine inhibit ethylbenzene hydroxylation as
nonconvertible substrate analogues. Angew Chem Int Ed Engl.
2
013, 52(9), 2599–2601.
1
1
1. Högenauer, G. and Woisetschläger, M. A diazaborine derivative
inhibits lipopolysaccharide biosynthesis. Nature, 1981, 293(5834),
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62–664.
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Stuitje, A. R.; Slabas, A. R.; Hawkes, T. R.; and Rice, D. W. A
Mechanism of Drug Action Revealed by Structural Studies of
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G.A. A Convergent, Modular Approach to Functionalized 2,1-
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In summary, we successfully completed the synthesis of the
first azaborine isostere of a retinoid derivative. Although, the
biological activity was inferior to our lead compound we did
uncover some surprising physiochemical properties. Namely that
the naphthalene to azaborine isosteric substitution increased
water solubility, which could find promising applications to other
drug candidates. This isosteric substitution warrants further
analysis in other antimicrobial natural products to fully
understand its availability for drug discovery.
5
49−555.
7. Santos-Filho, E.F.; Sousa, J.C.; Bezerra, N.M.M.; Menezes, P.H.;
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media. Tet Lett, 2011, 52, 5288–5291.
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R.; and Schrimpf, M. R. Asymmetric Memory at Labile,
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Am. Chem. Soc. 1999, 121, 2460.
This work was funded by the National Institute of
General Medical Sciences (GM119426) and the Georgia
Research Alliance based in Atlanta, Georgia to W.M.W.
C.L.S. was supported by the National Institute of Allergy and
Infectious Diseases of the National Institutes of Health under
Award Number T32AI106699. The content is solely the
responsibility of the authors and does not necessarily
represent the official views of the National Institutes of
Health. The NMR instruments used in this work were
1
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Design, Synthesis, and Biological Evaluation of Novel
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Graphical Abstract

5
To create your abstract, type over the instructions in the
template box below.
Synthesis and biological evaluation of an
antibacterial azaborine retinoid isostere
Leave this area blank for abstract info.
Brittney A. Haney, Cassandra L. Schrank,
and William M. Wuest
X
X
HO
HO
B
N
H
OH
OH
CD437 (X = O)
Analog 2 (X = H,H)
Azaborine Analogs
(X = O or H,H)
Fonts or abstract dimensions should not be changed or altered.