Synthesis and antibiotic activity of novel acylated phloroglucinol compounds against methicillin-resistant Staphylococcus aureus

Article abstract of DOI:10.1038/s41429-019-0153-4

The rise in antibiotic resistance among pathogenic microorganisms has created an imbalance in the drugs available for treatment, in part due to the slow development of new antibiotics. Cystic fibrosis (CF) patients are highly susceptible to antibiotic-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Phloroglucinols and related polyketide natural products have demonstrated antimicrobial activity against a number of Gram-positive bacteria including S. aureus. In this study, we investigated a series of acylated phloroglucinol derivatives to determine their potential as lead compounds for the design of novel therapeutics. To assess the activity of these compounds, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively), the minimum biofilm inhibitory and biofilm eradication concentration (MBIC and MBEC, respectively), and evaluated hemolytic activity, as well as their interaction with clinically relevant antibiotics. Of the 12 compounds tested against MRSA and methicillin-susceptible strains, four showed MIC values ranging from 0.125 to 8 μg ml^?1 and all of them were bactericidal. However, none of the compounds were able to eradicate biofilms at the concentrations tested. Three of the four did not display hemolytic activity under the conditions tested. Further studies on the interactions of these compounds with clinically relevant antibiotics showed that phlorodipropanophenone displayed synergistic activity when paired with doxycycline. Our results suggest that these acylated phloroglucinols have potential for being further investigated as antibacterial leads.

Full text of DOI:10.1038/s41429-019-0153-4

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0153-4
ARTICLE
Synthesis and antibiotic activity of novel acylated phloroglucinol
compounds against methicillin-resistant Staphylococcus aureus
1
Navriti Mittal1 Haben H. Tesfu2 Andrew M. Hogan2 Silvia T. Cardona2,3 John L. Sorensen
●
●
●
●
Received: 14 September 2018 / Revised: 24 January 2019 / Accepted: 27 January 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
The rise in antibiotic resistance among pathogenic microorganisms has created an imbalance in the drugs available for
treatment, in part due to the slow development of new antibiotics. Cystic fibrosis (CF) patients are highly susceptible to
antibiotic-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Phloroglucinols and related
polyketide natural products have demonstrated antimicrobial activity against a number of Gram-positive bacteria including
S. aureus. In this study, we investigated a series of acylated phloroglucinol derivatives to determine their potential as lead
compounds for the design of novel therapeutics. To assess the activity of these compounds, we determined the minimum
inhibitory and bactericidal concentration (MIC and MBC, respectively), the minimum biofilm inhibitory and biofilm
eradication concentration (MBIC and MBEC, respectively), and evaluated hemolytic activity, as well as their interaction
with clinically relevant antibiotics. Of the 12 compounds tested against MRSA and methicillin-susceptible strains, four
showed MIC values ranging from 0.125 to 8 µg ml−1 and all of them were bactericidal. However, none of the compounds
were able to eradicate biofilms at the concentrations tested. Three of the four did not display hemolytic activity under the
conditions tested. Further studies on the interactions of these compounds with clinically relevant antibiotics showed that
phlorodipropanophenone displayed synergistic activity when paired with doxycycline. Our results suggest that these
acylated phloroglucinols have potential for being further investigated as antibacterial leads.
Introduction
the Gram-positive bacterium Staphylococcus aureus, a
ubiquitous commensal of the human skin. Naturally sus-
ceptible to most antibiotics, S. aureus is capable of causing
infections in predisposed individuals and can acquire for-
midable antibiotic resistance [3]. One of the most notorious
antibiotic-resistant members of the species is methicilin-
resistant S. aureus (MRSA) [4], which are grouped into two
categories referred as hospital-associated (HA-MRSA) and
comunity-associated (CA-MRSA) [5]. MRSA is also a
concerning pathogen for people with the genetic disease
cystic fibrosis (CF) [6]. Studies show that lower airway
inflamation in children with CF is associated with early
colonization with S. aureus [7] and that the prevalence of
MRSA in CF patients is high [8, 9].
Microorganisms such as bacteria and fungi produce
chemical compounds that have been sucessfuly used as
therapeutic antibiotics [10]. Lichenized fungi are a rich
source of mostly under-explored natural products, many of
which have chemical structures that suggest an origin from
acetate via the polyketide pathway. One of the most widely
occuring lichen natural products is the dibenzofuran usnic
acid (UA), and the biological activity of this polyketide has
The emergence of multidrug resistant bacterial infections is
a major health threat [1]. Agencies like the World Health
Organization and the Public Health Agency of Canada [2]
have highlighted the need for investment in research and
development of new antibiotics. A remarkable example is
These authors contributed equally: Navriti Mittal, Haben H. Tesfu
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0153-4) contains supplementary
material, which is available to authorized users.
* John L. Sorensen
John.Sorensen@umanitoba.ca
1
Department of Chemistry, University of Manitoba,
Winnipeg, Canada
2
Department of Microbiology, University of Manitoba,
Winnipeg, Canada
3
Department of Medical Microbiology & Infectious Diseases,
University of Manitoba, Winnipeg, Canada

N. Mittal et al.
been extensivly studied. The biosynthesis of UA has been
demonstrated to proceed via the intermediate methylphlor-
acetophenone (MPA [11], Fig. 1) which is produced from
acetyl-CoA, malonyl-CoA, and S-adenosylmethionine
(SAM) by a polyketide synthase (PKS). The intermediate
MPA is in turn dimerized by an oxidative P450-type enzyme
to produce UA. The biological activity of MPA vs. UA has
recently been examined [12] and it was demonstrated that
UA is more bioactive than MPA in a series of antibacterial
and biofilm disruption assays. However, a systematic
examination of the biological activity of other related
phloracetophenone analogs has not yet been reported. We
chose to explore this chemical space as a potential source of
novel bioactivity. Althougth MPA is a known natural pro-
duct none of the compounds reported here appear to be of
natural origin.
In this study, we synthesized 12 novel phlor-
acetophenone analogs (Compounds 1–12, Schemes 1–3,
Fig. 2) and explored their biological activity against S.
aureus ATCC 29213 and MRSA strains isolated from CF
patients. Our results show that 4 of the 12 novel compounds
displayed promising activity as a monotherapy and one
strongly synergized with doxycycline. These results suggest
that phloracetophenone analogs may serve as promising
lead compounds for the design of novel antibiotics.
sequentially longer hydrocarbon chain of 3–6 carbon atoms
in length. The synthesis of compounds 1–4 is summarized
in Scheme 1. Commercially available trihydroxytoluene
was reacted with 0.5 equivalent of the appropriate anhy-
dride in the presence of 1 equivalent of Lewis acid
(BF3•OEt2) using dioxane as the solvent. Using the anhy-
dride as the limiting reagent resulted in a correspondingly
low yield (18–40%), but prevented over acylation of the
substrate. We also undertook the synthesis of a series of
acylated phloroglucinols, compounds 5–8, as described in
Scheme 2. Phloroglucinol was dissolved in carbon disulfide
and nitrobenzene and treated with 10 equivalents of the
appropriate acid chloride using AlCl3 (10 equivalents) as
catalyst. Heating for one hour followed by work-up affor-
ded compounds 5–8 in a range of 41–53% yield. A series of
diacylated phloroglucinol derivatives were synthesized as
described in Scheme 3. Phloroglucinol was dissolved in
dioxane and reacted with an excess of the appropriate
anhydride (10 equivalents) and an excess (10 equivalents)
of Lewis acid. Heating for 30 min followed by work up
produced diacylphloroglucinols 9–12 in a range of 17 to
55% yield. The complete characterization data for each
compound is provided in the supplementary material
Evaluation of antibiotic properties
The minimum inhibitory concentration (MIC) of UA against
S. aureus ranges between 6 and 32 μg ml−1 depending on the
strain [12]. To evaluate the antibiotic activity of the
synthesized compounds, we performed MIC assays using a
laboratory strain of S. aureus (ATCC 29213) and MRSA
strains isolated from the sputum of CF patients [13]. Table 1
shows MICs of the synthesized compounds. Group 3 had the
Results and discussion
Synthesis of phloroglucinol compounds
We first set out to synthesize a series of analogs of MPA
where the methyl ketone had been replaced with a
CH3
H3C
HO
OH
H3C
O
O
HO
OH
P450
Acetyl-CoA
Malonyl-CoA
SAM
PKS
CH3
OH
O
O
O
OH CH3
(MPA)
CH3
(UA)
Fig. 1 The biosynthesis of usnic acid (UA) from acetyl-CoA, malonyl-CoA, and S-adenosylmethionine (SAM) has been demonstrated to proceed
via methylphloracetophenone (MPA)
CH3
CH3
OH
O
R =
CH2 CH3 (1)
HO
OH
HO
OH
CH2 CH3 (2)
R
O
2
(0.5 eq.)
OEt2
30 min, Δ
dioxane
2
CH2 CH3 (3)
R
1 eq. BF3
•
3
CH2 CH3 (4)
4
OH
O
Scheme 1 The synthesis of compounds 1–4 is shown

Synthesis and antibiotic activity of novel acylated phloroglucinol compounds against. . .
CH3
HO
OH
O
HO
O
OH
O
HO
OH
O
OH
5
OH
9
OH
1
CH3
HO
OH
HO
OH
O
HO
OH
O
OH
O
OH
6
OH
10
O
2
CH3
HO
HO
OH
HO
OH
HO
HO
OH
OH
3
CH3
O
O
OH
11
O
OH
7
O
OH
OH
HO
OH
OH
4
O
OH
8
O
O
OH
12
O
Fig. 2 Chemical structure of the synthetized compounds. Compounds were grouped according to the synthesis scheme. Group 3 contains the most
bioactive compounds
O
R =
CH2 CH3 (5)
HO
OH
HO
OH
CH2 CH3 (6)
2
Cl (10 eq.)
R
CH2 CH3 (7)
R
10 eq. AlCl3
3
1 hr, Δ
CH2 CH3 (8)
4
OH
OH
O
CS2, Ph–NO2
Scheme 2 The synthesis of compounds 5–8 is shown
O
R =
CH2 CH3 (9)
CH2 CH3 (10)
HO
OH
HO
OH
R
O
CH2 2CH3 (11)
(10 eq.)
OEt2
2
R
R
10 eq. BF3
•
3
30 min, Δ
CH2 CH3 (12)
4
OH
O
OH O
dioxane
Scheme 3 The synthesis of compounds 9–12 is shown
lowest MICs, ranging between 0.125 and 8 μg ml−1. Among
them, compound 11 had the lowest MIC against all the
mutual ortho phenolic group. The compounds differ only in
the chain length of the alkyl group on the anhydride that was
used in the acylation step. We then tested the ability of
compounds 9–12 to effectively kill S. aureus by performing
minimum bactericidal concentration (MBC) assays. All
compounds displayed a MBC to MIC ratio lower than 4 for
strains of S. aureus tested ranging from 0.125 to 0.5 μg ml−1
,
while compounds 9 and 10 had MIC values ranging from 2
to 8 μg ml−1. The common structural feature for the four
most active compounds (9–12) is a diketo moiety with a

N. Mittal et al.
Table 1 Minimum inhibitory
concentration (MIC) of the 12
novel phloroglucinol derivatives
against S. aureus strains
Compound no. MIC (μg ml−1
)
CF66 ATTC
(MSSA) 29213
CF225
(MRSA) (MRSA) (MRSA) (MRSA) (SCV
MRSA)
CF188
CF224
CF4
CF250
CF3
(MRSA)
1
>32
>32
>32
>32
>32
>32
>32
>32
8
>32
>32
>32
>32
>32
>32
>32
>32
2
>32
>32
>32
>32
>32
>32
>32
>32
2
>32
>32
>32
>32
>32
>32
>32
>32
2
>32
>32
>32
>32
>32
>32
>32
>32
2
>32
>32
>32
>32
>32
>32
>32
>32
4
>32
>32
>32
>32
>32
>32
>32
>32
>32
8
2
>32
>32
>32
>32
>32
>32
>32
4
3
4
5
6
7
8
9
10
11
12
8
2
4
8
8
8
4
8
0.25
0.5
0.25
0.25
0.5
0.5
0.125
0.5
0.125
0.25
0.25
0.5
0.5
1
0.125
0.5
Values presented are the median of three biological replicates
SCV small colony variant
Table 2 MBC and ratio of MBC
to MIC for the four novel
phloroglucinol derivatives
against S. aureus strains
Compound no. S. aureus CF3
S. aureus CF4
S. aureus CF66
S. aureus CF188
MBC
MBC/
MBC
MBC/
MBC
MBC/
MBC
MBC/
MIC
ratio
(μg ml−1
)
MIC
ratio
(μg ml−1
)
MIC
ratio
(μg ml−1
)
MIC
ratio
(μg ml−1
)
9
16
32
0.5
1
2
4
4
2
16
32
0.5
1
4
4
2
2
16
32
0.5
1
2
4
2
2
8
4
4
8
2
10
11
12
32
1
1
Table 3 In vitro hemolysis of ovine red blood cells by novel
phloroglucinol derivatives
all the strains tested with exception of compound 11 and
strain CF188 (Table 2), indicating a bactericidal action [14].
We also evaluated the ability of the synthesized phlor-
oglucinol compounds to eradicate biofilms by performing
the minimum biofilm eradication concentration (MBEC)
assay (Suppl. Table 1). None of the four compounds from
group 3 that had antibacterial activity possessed biofilm
eradication activity (Suppl. Table 2).
Compound no.
Percentage hemolysisa
32 μg ml−1
16 μg ml−1
8 μg ml−1
9
0.60 1.14
0.17 1.08
8.10 0.89
2.86 1.88
1.07 1.48
0.07 0.56
0.49 0.55
2.27 2.14
0.74 1.18
0.28 1.0
10
11
12
−0.52 0.93
1.44 1.49
To further investigate the therapeutic potential of the four
active compounds, we evaluated their hemolytic properties
against red blood cells (RBC) (Table 3). Only 11 displayed
minor hemolytic activity of 8% at the highest concentration
tested (32 μg ml−1), suggesting it would be unsuitable for
therapeutic purposes. Furthermore, the lack of hemolytic
activity for compounds 9, 10, and 12 suggests that this class
of compounds may have promising selectivity against
bacterial cells. Hence, more research on understanding the
mechanism of action and the apparent selectivity is merited.
We also investigated the interaction of the compounds
with clinically relevant antibiotics that have various
mechanisms of action. We selected compound 9 as repre-
sentative for checkerboard assays as it displayed strong
aValues presented are the mean of three biological replicates SD
activity, but without hemolysis. As shown in Table 4,
Compound 9 interacted additively with oxacillin, cepha-
lexin, rifampicin, and trimethoprim, and acted synergisti-
cally with doxycycline. This trend was seen in ATCC
29213 and the MRSA strains. Studies have shown that
synergies often occur between drugs that target the same
cellular process [15] and also can happen due to modulation
of intracellular drug concentrations. Doxycycline targets the
small ribosome subunit (30S subunit) interrupting protein
synthesis. In a study conducted by Sahuquillo-Arce et al. it

Synthesis and antibiotic activity of novel acylated phloroglucinol compounds against. . .
Table 4 Interaction of Compound 9 with several clinically relevant
antibiotics against S. aureus
statically at 37 °C for 24 h. To determine the MIC the plates
were visually inspected for growth and by reading the
optical density (OD600) with a BioTek plate reader. To
determine the MBC, 10−1–10−3 dilutions of cell suspen-
sions exposed to the antibiotic dilution gradients were pla-
ted on LB plates and incubated at 37 °C for 24 h. The
number of colony forming units (CFU) per dilution was
recorded. As a positive control, cells suspensions treated
similarly but without exposure to the compounds were used.
The MBC was calculated using the formula:
Antibiotic
S. aureus ATCC 29213
S. aureus CF225
FIC Index Interpretation FIC Index Interpretation
Oxacillin
0.625
1
Additive
Additive
Additive
Synergistic
Additive
0.75
1
Additive
Additive
Additive
Synergistic
Additive
Cephalexin
Rifampicin
0.75
1
Doxycycline 0.325
Trimethoprim 1.5
0.325
1.5
Values presented are the median of three biological replicates
No: of CFU in a pspecific concentration
MBC ¼
 100
No: of CFU in the postive control
was shown that linezolid interacted synergistically with
doxycycline [16]. Linezolid binds to the 50S ribosomal
subunit inhibiting protein synthesis. Therefore, the syner-
gistic interaction between doxycycline and linezolid occurs
because the two compounds act on interacting targets [16].
It is tantalizing to speculate that the mechanism of action of
compound 9 is related to the inhibition of protein synthesis,
a hypothesis worth to further be tested.
where number of CFU in a specific concentration is the
average CFU of each dilution at each concentration tested.
A concentration was considered bactericidal if it killed
99.9% of the bacterial population [14].
Minimum biofilm inhibitory concentration (MBIC)
assay
Assays were performed as previously described [18].
Briefly, an overnight culture was diluted 100-fold and
incubated at 37 °C for 3 h to reach mid-log phase. The
subculture of S. aureus was prepared by diluting the over-
night culture in 1:1000 (∼105 CFU ml−1) into TSB. Wells
of a 96-well plate were used to serially diluted the desired
compound in TSB and then inoculated with 5 × 106 cells
each and grown statically at 37 °C for 24 h. The wells were
then rinsed twice with PBS (pH 7.2, 0.8% NaCl, 0.02%
KCl, 0.17% Na2HPO4, 0.8% KH2PO4) and then followed
with the resazurin cell variability assay below.
Conclusion
In the present study, we have synthesized 12 phloroglucinol
derivatives and characterized their antibiotic properties
against S. aureus, an important CF pathogen. Diacylated
derivatives displayed the strongest bactericidal activity
against MRSA clinical isolates. Furthermore, these com-
pounds displayed apparent selectivity in their action, as they
were not hemolytic. Of interest, compound 9 interacted
synergistically with doxycycline. Hence, it could potentially
be used as a drug combination to treat highly resistant
MRSA strains.
MBEC assay
Assays were performed as previously described [18].
Briefly, an overnight culture was diluted 100-fold and
incubated at 37 °C for 3 h to reach mid-log phase. The
subculture of S. aureus was prepared by diluting the over-
night culture in 1:1000 (∼105 CFU ml−1) into TSB. Wells
of a 96-well plate were inoculated with 5 × 106 cells each
and grown statically at 37 °C for 24 h. The media was then
removed from each well. Then serial dilutions of the desired
compound in TSB were added to the wells and grown
statically at 37 °C for 24 h. The wells were then rinsed twice
with PBS (pH 7.2, 0.8% NaCl, 0.02% KCl, 0.17%
Na2HPO4, 0.8% KH2PO4) and then followed with the
resazurin cell variability assay.
Experimental procedures
Bacterial strains and growth conditions
Strains (Suppl. Table 1) were grown in Tryptic soy broth
(TSB) (Difco) at 37 °C and 230 rpm shaking for routine
overnight culturing unless otherwise stated.
MIC and MBC assays
The CLSI protocol for determining the MIC and MBC of
antibiotic activities in S.aureus was followed [17]. Briefly,
an overnight culture was diluted equivalent to a 0.5
McFarland standard and then further diluted to inoculate
wells of a 96-well plate, containing an antibiotic dilution
gradient, with 5 × 105 cells each. The plate was grown
Resazurin cell viability assay
Viable cells with active metabolism reduce resazurin into
fluorescent resorufin [19]. To the washed wells, 20 μl of

N. Mittal et al.
CellTitre-Blue (Promega) was added with 100 μl of PBS
and incubated at 37 °C for 1 h. Fluorescence was mea-
sured (530/25 nm excitation and 590/35 nm emission)
using a BioTek Synergy plate reader. Wells that had the
same fluorescence as a resazurin-only control were con-
sidered to have no cell viability.
Synthesis and characterization of compounds 1–12
Reagents and solvents were used as purchased without
further purification. Thin layer chromatography (TLC) was
conducted with 0.25 mm F254 silica gel on aluminum plates
and visualization was conducted with 254 nm UV light. The
purification of all compounds was carried out over flash
grade-silica gel and fractions combined according to TLC.
Solvent was removed under reduced pressure. The 1H (300
MHz) and 13C NMR (125 MHz) spectra were collected on a
Bruker Avance-300 console and were referenced to solvent
(CDCl3 = 7.24 ppm). The complete synthetic procedures
and characterization data for compounds 1–12 are included
in the electronic supplementary material.
Checkerboard assay
Checkerboard assays in 96-well format were performed
as previously described [20]. Briefly, the inoculum
was prepared as for the MIC testing above and added to
a
two-dimensional gradient of the compound of
interest. Plates were grown statically at 37 °C for 24 h
then visually inspected for growth. The fractional
inhibition concentration (FIC) index is calculated using
the formula
Acknowledgements This study was supported by a Discovery Grant
from the Natural Sciences and Engineering Research Council
(NSERC) to John Sorensen and a Mid-Career Award from Research
Manitoba to Silvia Cardona. The assistance of Harmanpreet Tatla in
the initial synthesis of compounds 9–12 is also gratefully
acknowledged.
C1
C2
FIC Index ¼
þ
MIC1 MIC2
where MIC1 and MIC2 are the MICs of antibiotics 1 and 2
alone, respectively, and C1 and C2 are the concentrations of
antibiotics 1 and 2, respectively, when at the combined
MIC. When the FIC index was ≤0.5 the antibiotic
combination was considered to be synergistic; when the
FIC index was >0.5 and ≤4.0 the effect of the two
antibiotics was considered to be additive. Finally, when the
FIC index was ≥4.0, the antibiotic combination was defined
as antagonistic [21].
Author contributions NM designed and performed synthesis of the
compounds, HHT tested the activity of the compounds. NM and HHT
interpreted the data and wrote the manuscript. AMH supervised and
edited the final version of the manuscript. STC and JLS conceived the
research approach, contributed to writing and edited the final version
of the manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Hemolysis assay
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Hemolytic activity on ovine erythrocytes was assessed as
previously described [22, 23]. Briefly, ovine erythrocytes
were gently pelleted by centrifugation and washed thrice
with PBS then resuspended in PBS at a 1:5 dilution. In 96-
well format, 100 µl erythrocyte suspension was added to a
dilution gradient of the desired compound and incubated
statically at 37 °C for 1 h. Triton X-100 at 0.1% was
included as a positive control and a dimethyl sulfoxide
(DMSO) concentration gradient as a negative control.
Erythrocytes were pelleted and the absorbance of the
supernatant was measured at 540 nm. The percentage of
lysis was calculated as follows:
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