Minimum structural requirements for cell membrane leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s

Article abstract of DOI:10.1016/j.bmcl.2016.03.033

Macrocyclic bis(bibenzyl)-type phenolic natural products, found exclusively in bryophytes, exhibit potent antibacterial activity towards methicillin-resistant Staphylococcus aureus (anti-MRSA activity). Here, in order to identify the minimum essential structure for cell membrane leakage-mediated anti-MRSA activity of these compounds, we synthesized acyclic fragment structures and evaluated their anti-MRSA activity. The activities of all of the acyclic fragments tested exhibited similar characteristics to those of the macrocycles, i.e., anti-MRSA bactericidal activity, an enhancing effect on influx and efflux of ethidium bromide (EtBr: fluorescent DNA-binder) in Staphylococcus aureus cells, and bactericidal activity towards a Staphylococcus aureus strain resistant to 2-phenoxyphenol (4). The latter result suggests that they have a different mechanism of action from 4, which is a FabI inhibitor previously proposed to be the minimum active fragment of riccardin-type macrocycles. Thus, cyclic structure is not a necessary condition for cell membrane leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s.

Full text of DOI:10.1016/j.bmcl.2016.03.033

Bioorganic & Medicinal Chemistry Letters 26 (2016) 2324–2327
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Minimum structural requirements for cell membrane
leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s
⇑
Kana Fujii, Daichi Morita, Kenji Onoda, Teruo Kuroda, Hiroyuki Miyachi
Division of Pharmaceutical Sciences, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 1-1-1, Tsushima-Naka, Kita-ku, Okayama
700-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Macrocyclic bis(bibenzyl)-type phenolic natural products, found exclusively in bryophytes, exhibit
potent antibacterial activity towards methicillin-resistant Staphylococcus aureus (anti-MRSA activity).
Here, in order to identify the minimum essential structure for cell membrane leakage-mediated anti-
MRSA activity of these compounds, we synthesized acyclic fragment structures and evaluated their
anti-MRSA activity. The activities of all of the acyclic fragments tested exhibited similar characteristics
to those of the macrocycles, i.e., anti-MRSA bactericidal activity, an enhancing effect on influx and efflux
of ethidium bromide (EtBr: fluorescent DNA-binder) in Staphylococcus aureus cells, and bactericidal activ-
ity towards a Staphylococcus aureus strain resistant to 2-phenoxyphenol (4). The latter result suggests
that they have a different mechanism of action from 4, which is a FabI inhibitor previously proposed
to be the minimum active fragment of riccardin-type macrocycles. Thus, cyclic structure is not a neces-
sary condition for cell membrane leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 11 February 2016
Revised 7 March 2016
Accepted 10 March 2016
Available online 10 March 2016
Keywords:
Macrocyclic bis(bibenzyl) derivative
Methicillin resistance
Membrane
Cell membrane leakage
Structure–activity relationship
We have previously reported that macrocyclic bis(bibenzyl)-
type phenolic natural products, such as riccardin C (RC: 1) and iso-
plagiochin D (2), which are found exclusively in bryophytes,1,2
exhibit potent antibacterial activity towards methicillin-resistant
Staphylococcus aureus (anti-MRSA activity), comparable to that of
the clinically used drugs vancomycin and linezolid.3–5 Structural
development studies of these macrocyclic bis(bibenzyl)s,
prompted us to identify some synthetic bis(bibenzyl) derivatives
such as 3, which exhibited more potent anti-MRSA activity. In
studies to identify the minimum essential structure of these
macrocyclic bis(bibenzyl)s for anti-MRSA activity, we previously
prepared three fragments of RC, i.e., 4–6 (Fig. 1). Compound 6 is
identical with lunularin,6 which is a precursor in the biosynthetic
pathway of bis(bibenzyl)-type cyclic phenolic natural products.
Among these fragments, only the northern fragment, 2-phe-
noxyphenol (4) exhibited substantial anti-MRSA activity, with an
concentration of 4 (4 ꢀ MIC concentration) did not decrease the
survival of these strains.7 In other words, 4 exhibited bacteriostatic
activity, not bactericidal activity. 1 affected the inflow and outflow
of ethidium bromide (EtBr) through Staphylococcus aureus mem-
brane, whereas 4 did not.7 Therefore 4 is not a true minimum
essential structure for the activity of the macrocyclic bis(biben-
zyl)s.
To confirm the difference in mode of antibacterial action, we
selected an MRSA mutant resistant to 32 mg/L 4 from Staphylococcus
aureus strain N315, for which the genome has been fully
sequenced.7 We found that the macrocyclic bis(bibenzyl) 1 still
exhibited potent activity towards this strain, whereas the activity
of 4 was greatly reduced, as expected.7 Based on these experiments,
we concluded that the northern fragment 4 is not the minimum
essential structure of 1 for potent anti-MRSA activity. In addition,
our genetic studies indicated that the mutation in the 4-resistant
strain was located in the enoyl-acyl carrier protein reductase gene
fabI, i.e., the molecular target of 4 is FabI.7 The molecular target of
the parent macrocyclic bis(bibenzyl) derivative 1 remains to be
identified, but is clearly different from FabI.
MIC (minimum inhibitory concentration) of 8 lg/mL, while 5 and
6 were inactive (Table 1).
However, it turned out that the antibiotic activity of 4 is distinct
from that of the macrocyclic bis(bibenzyl)s. For example, survival
assay indicated that an excess concentration of 1 (4 ꢀ MIC concen-
tration) shows potent bactericidal activity towards MRSA strains
(survival rate is less than 1/10,000).7 On the other hand, an excess
In continuing studies to identify the minimum essential struc-
ture of the macrocyclic bis(bibenzyl) derivatives, aimed at develop-
ing
a new lead structure for anti-MRSA agents, we further
synthesized two larger acyclic fragment structures, 7 and 8 and
evaluated their anti-MRSA activities. These results are presented
here.
⇑
Corresponding author. Tel.: +81 086 251 7930.
E-mail address: miyachi@pharm.okayama-u.ac.jp (H. Miyachi).
http://dx.doi.org/10.1016/j.bmcl.2016.03.033
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

K. Fujii et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2324–2327
2325
OH
OH
OH
O
HO
HO
HO
HO
1
HO
3
HO
2
OH
OH
O
4
OH
HO
HO
5
6
HO
Me
HO
HO
HO
7
8
Figure 1. Chemical structures of the macrocyclic bis(bibenzyl)s (1–3) and their fragments (4–8), including those synthesized here (7, 8).
l
g/mL).9 The MRSA strains used for the
Table 1
concentration (MIC,
Anti-MRSA activities of macrocyclic bis(bibenzyl) derivatives and their fragments
evaluation were OM481 and N315. OM481 is a clinical isolate
from Okayama University Hospital Japan, and N315 is a MRSA
strain isolated in 1982 in Japan as a prototype of Japanese
hospital-associated MRSA strains.10 The activity of these
compounds towards a compound 4-resistant MRSA strain was also
measured. The results are summarized in Table 1.
The inflow of EtBr to energized S. aureus N315 cells was evalu-
ated as the increase in fluorescence intensity after addition of the
test compounds (excitation 530 nm and emission 600 nm).7 The
outflow of EtBr from ethidium-loaded S. aureus N315 cells was
evaluated in terms of decrease in fluorescence intensity (excitation
530 nm and emission 600 nm).7
Compd
MIC (
l
g/mL) S. aureus
N-315
OM481
Compd 4-resistant strain
1
3
4
5
6
7
8
2
0.5
8
>128
>128
8
2
2
0.5
8
>128
>128
8
0.5
>128
>128
>128
8
16
16
16
Vancomycin
1
1
N.T.
Measurement of intracellular ion concentrations were per-
formed with the inductively coupled plasma optical emission spec-
trometry (ICP-OES) analysis with a VISTA-PRO (Seiko Instruments
Inc.).7
Compounds 7 and 8 were prepared basically by Suzuki coupling
to construct the biphenyl framework, and subsequent Wittig
reaction to attach the side chain phenethyl group.8 Wittig reaction
of (20,4-dimethoxy-40-(methoxycarbonyl)[1,10-biphenyl]-2-yl)
methyl)triphenyl-phosphonium bromide (9) with benzaldehyde
in the presence of K2CO3 and 18-crown-6 ether afforded the stil-
bene derivative (10) as a mixture of geometrical isomers. This mix-
ture was hydrogenated to afford the phenethyl derivative (11). The
methoxycarbonyl group of 11 was reduced with lithium aluminum
hydride to obtain the hydroxymethyl derivative (12). The hydrox-
ymethyl derivative was treated with triphenylphosphine HBr, fol-
lowed by Wittig reaction, and finally hydrogenation to afford the
bis(phenethyl) derivative (15). Demethylation with BBr3 gave the
target compound 7.
These results are summarized in Figures. 2 and 3.
All of the new acyclic fragments exhibited substantial anti-
MRSA activity. Although these compounds were less potent than
macrocycles such as 1, their activity was comparable to that of 4.
However, it is very important to note that 4 exhibited decreased
activity towards the 4-resistant mutant strain compared to the
parent MRSA strain, as expected, whereas 7 and 8 exhibited com-
parable activities towards both MRSA strains. This result suggested
that the mechanism of action of 7 and 8 might be distinct from that
of 4, i.e., the activities of 7 and 8 might not be mediated through
inhibition of FabI.
The hydroxyl group of methyl 4-hydroxy-3-methoxybenzoate
(16) was treated with trifluoromethanesulfonic anhydride, and
subsequent Suzuki coupling with (4-methoxy-2-methylphenyl)
boronic acid afforded methyl biphenylcarboxylate derivative 18.
18 was reduced with lithium aluminum hydride to obtain the
hydroxymethyl derivative (19). 19 was treated with triph-
enylphosphine HBr, followed by Wittig reaction, and finally hydro-
genation to afford the phenethyl derivative (22). Demethylation
with BBr3 gave the target compound 8 (Scheme 1).
Encouraged by this result, we evaluated the effects of these
fragments on influx and efflux of EtBr in S. aureus N315 cells. The
results are summarized in Figure 2. When the fragments were
added to energized S. aureus N315 cells, the fluorescence of EtBr
immediately increased (Fig. 2A and C), indicating increased intra-
cellular binding of EtBr to N315 DNA. We also investigated the
effect of the acyclic fragments on outflow of EtBr from EtBr-loaded
S. aureus cells. Addition of the fragments caused a rapid decrease in
EtBr-mediated fluorescence (Fig. 2B and D). These results indicate
that all the acyclic fragments tested, increase the permeability of
the cytoplasmic membrane of S. aureus.
Anti-MRSA activities were determined by the liquid microdilu-
tion method, and are expressed in terms of minimum inhibitory

2326
K. Fujii et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2324–2327
MeO
MeO
MeO
MeO
MeO
PPh3Br
c
g
a
e
b
d
MeO
COOMe
MeO
CH2OH
MeO
MeO
COOMe
MeO
MeO
COOMe
9
10
14
11
15
12
MeO
HO
MeO
f
HO
PPh3Br
COOMe
MeO
13
7
MeO
Me
MeO
Me
HO
TfO
h
l
j
i
k
MeO
MeO
COOMe
OH
MeO
COOMe
MeO
19
16
17
18
MeO
Me
MeO
Me
HO
Me
MeO
Me
m
n
MeO
MeO
HO
PPh3Br
MeO
20
21
22
8
Scheme 1. Synthetic routes to compounds 7 and 8. Reagents and conditions: (a) benzaldehyde, K2CO3, 18-crown-6-ether, CH2Cl2, reflux, 11.5 h (90%); (b) 10%-Pd/C, 0.35 MPa
H2, EtOAc, rt, 0.5 h (90%); (c) LAH, THF, 0 °C to rt, 14 h (95%); (d) P(Ph)3HBr, CH3CN, reflux, 13 h (94%); (e) benzaldehyde, K2CO3, 18-crown-6-ether, CH2Cl2, reflux, 14 h (90%);
(f) 10%-Pd/C, 0.35 MPa H2, EtOAc, MeOH, rt, 2 h (78%); (g) BBr3, CH2Cl2, ꢁ78 °C to rt, 3 days (quant); (h) Tf2O, Py, CH2Cl2, 0 °C to rt, 1.5 h (94%); (i) 4-methoxy-2-methylphenyl
boronic acid, Pd(PPh3), K3PO4, DMF, 100 °C, 15.5 h (92%); (j) LAH, THF, 0 °C to rt, 14 h (95%); (k) PPh3HBr, CH3CN, reflux, 4 h (quant); (l) benzaldehyde, K2CO3, 18-crown-6-
ether, CH2Cl2, reflux, 21.5 h (99%); (m) 10%-Pd/C, EtOAc, 0.31 MPa H2, rt, 1 h (quant); (n) BBr3, CH2Cl2, ꢁ78 °C to rt, 6 h (69%).
2
2
(A)
(B)
Cpd. 7
7
1.5
1
7
1.5
1
EtBr
0.5
0
0.5
0
0
100
200
300
0
100
200
300
Time (sec)
Time (sec)
8
2
1.5
1
2
(C)
(D)
Cpd. 8
1.5
8
1
0.5
0
EtBr
0.5
0
0
100
200
300
0
100
200
300
Time (sec)
Time (sec)
Figure 2. Effects of 7, 8 on the inflow (left) and outflow (right) of EtBr in S. aureus N315 cells.
We have previously reported that macrocyclic bis(bibenzyl)s,
such as 1 and 3 exhibited rapid EtBr inflow and outflow. Two pos-
sibilities were considered for the increase of fluorescence intensity
observed in intracellular EtBr. One was the increase of membrane
permeability, and the other was the inhibition of multidrug efflux
pumps that extrude EtBr. However we would be able to omit latter
possibility, for, 1 and 3 caused a rapid decrease in EtBr fluorescence
from EtBr-loaded S. aureus N315 cells. If 1 and 3 inhibited efflux
pumps, we would not be able to detect any changes in fluorescence
in the EtBr outflow assay. These results strongly suggested that 1
and 3 increased the permeability of the cytoplasmic membrane
of S. aureus.
Thus, both the inflow and outflow of EtBr suggest that 7 and 8
may not be an inhibitor of efflux pumps.

K. Fujii et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2324–2327
2327
(A)
9
(B)
0.8
(C)
K+
Na+
10
9
8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Control
7
8
6
7
5
6
4
1
MIC
5
3
4
2
3
1
4
MIC
2
0
pre
pre
DMSO
1 (4×MIC)
7
DMSO
1 (4×MIC)
7
0
3
6
9
12
Time (hr)
15
18
21
24
Figure 3. Effect of the representative fragment 7 on intracellular Na+ and K+ concentrations (A, B), and survival of S. aureus N315 (C).
indirectly through the formation of mesosomes, intracellular struc-
tures formed by invagination of the cell membrane (Fig. 4).7
OH
Me
HO
O
HO
HO
Acknowledgments
HO
HO
HO
This work was supported in part by a grant for the Platform for
Drug Discovery, Informatics, and Structural Life Science from the
Ministry of Education, Culture, Sports, Science, and Technology,
Japan, and a Grant-in-aid for Scientific Research <KAKENHI> (B)
(grant number 26293027 to H.M.) from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT). We also thank the
SC-NMR Laboratory of Okayama University for NMR
measurements.
OH
O
Na+, K+ etc
inflow/outflow
Cell wall/membrane
damage/leakage !
FabI inhibition
cell wall
MRSA
cell membrane
Mesosome
formaꢀon
References and notes
FabI
1. Asakawa, Y.; Toyota, M.; Tori, M.; Hashimoto, T. Spectroscopy 2000, 14, 149.
2. Asakawa, Y. In Progress in the Chemistry of Organic Natural Products; Herz, W.,
Kirby, G. W., Moore, R. E., Steglich, W., Tamm, C., Eds.; Springer: Vienna,
Austria, 1995; Vol. 65, p 618.
Figure 4. Schematic illustration of the putative molecular targets and target sites of
bis(bibenzyl) derivatives and their fragments for anti-MRSA activity.
3. Sawada, H.; Onoda, K.; Morita, D.; Ishitsubo, E.; Matsuno, K.; Tokiwa, H.;
Kuroda, T.; Miyachi, H. Bioorg. Med. Chem. Lett. 2013, 23, 6563.
4. Sawada, H.; Okazaki, M.; Morita, D.; Kuroda, T.; Matsuno, K.; Hashimoto, Y.;
Miyachi, H. Bioorg. Med. Chem. Lett. 2012, 22, 7444.
5. Onoda, K.; Sawada, H.; Morita, D.; Fujii, K.; Tokiwa, H.; Kuroda, T.; Miyachi, H.
Bioorg. Med. Chem. 2015, 23, 3309.
6. Keseru, G. M.; Nogradi, M. Phytochemistry 1992, 31, 1573.
7. Morita, D.; Sawada, H.; Ogawa, W.; Miyachi, H.; Kuroda, T. Biochim. Biophys.
Acta. 2015, 1848, 2057.
We further confirmed the effect of 7 on membrane permeability
by analyzing changes in the intracellular concentrations of Na+ and
K+ (Fig. 3A and B). When S. aureus cells were treated with 7, intra-
cellular Na+ concentration was increased (Fig. 3A), while K+ con-
centration was decreased (Fig. 3B).
Figure 3C shows the results of survival assay of S. aureus N315.
When 7 was added at the concentration of the MIC, the number of
viable cells was reduced by at least three orders of magnitude,
while a concentration of 4 times the MIC rapidly killed essentially
all the cells. These results are consistent with the idea that 7 and 8
exhibit bactericidal (not bacteriostatic) activity by disrupting the
membrane of S. aureus.
In conclusion, we synthesized acyclic fragment structures of
macrocyclic bis(bibenzyl) derivatives, and evaluated their antibac-
terial activities towards methicillin-resistant Staphylococcus aureus
in order to identify the minimum essential structure for cell mem-
brane leakage-mediated anti-MRSA activity. The results showed
that cyclic structure is not necessary for anti-MRSA activity. In con-
trast to the previously reported fragment 2-phenoxyphenol (4),
which exhibited bacteriostatic anti-MRSA activity by inhibiting
the function of FabI, the present compounds 7 and 8 appear to
directly permeabilize the cell membrane of methicillin-resistant
Staphylococcus aureus in a similar manner to the macrocyclic bis
(bibenzyl)s, which cause membrane leakage mediated directly or
8. Physico-chemical properties of compounds 7 and 8:
Compound 7: 1H NMR (400 MHz, CDCl3) d = 7.30–7.26 (m, 2H), 7.23–7.18 (m,
5H), 7.15–7.13 (m, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.96–6.92 (m, 3H), 6.84–6.81
(m, 2H), 6.79–6.76 (m, 2H), 5.00–4.72 (m, 2H), 3.00–2.90 (m, 4H), 2.74–2.69
(m, 4H). 13C NMR (100 MHz, CDCl3): d = 155.94, 152.88, 143.35, 143.32, 141.91,
141.66, 132.37, 130.75, 128.60, 128.50, 128.47, 128.42, 127.73, 126.09, 126.03,
124.68, 120.71, 116.77, 115.24, 113.88, 37.94, 37.87, 37.35, 35.68. MS (FAB)
395 (M+H)+. HPLC purity was estimated to be 92.0% by means of reversed
phase-HPLC, using a Pegasil ODS sp100 column (4.6 mm ꢀ 250 mm, Senshu
Chemical, Japan) fitted on a JASCO HPLC system, with CH3CN/H2O = 2:1 v/v as
the eluent and detection at 254 nm.
Compound 8: 1H NMR (400 MHz, CDCl3) d = 7.32–7.28 (m, 2H), 7.23–7.19 (m,
3H), 7.10 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), 6.84–6.79 (m, 3H), 6.75 (dd,
J = 8.0 Hz, 2.4 Hz, 1H), 4.96 (s, 1H), 4.77 (s, 1H), 2.99–2.89 (m, 4H), 2.12 (s, 3H).
13C NMR (100 MHz, CDCl3): d = 155.50, 152.61, 143.09, 141.81, 139.42, 131.93,
130.39, 128.43, 128.36, 127.98, 125.95, 124.86, 120.60, 117.33, 115.00, 113.35,
37.69, 37.67, 19.91. MS (FAB) 305 (M+H)+. HPLC purity was estimated to be
98.0% by means of reversed phase-HPLC, using a Pegasil ODS sp100 column
(4.6 mm ꢀ 250 mm, Senshu Chemical, Japan) fitted on a JASCO HPLC system,
with CH3CN/H2O = 2:1 v/v as the eluent and detection at 254 nm.
9. Andrews, J. M. J. Antimicrob. Chemother. 2001, 48, 5.
10. Ito, T.; Katayama, Y.; Hiramatsu, K. Antimicrob. Agents. Chemother. 1999, 43,
1449.