Polybrominated Diphenyl Ethers: Structure Determination and Trends in Antibacterial Activity

Article abstract of DOI:10.1021/acs.jnatprod.6b00229

Antibacterial-guided fractionation of the Dictyoceratid sponges Lamellodysidea sp. and two samples of Dysidea granulosa yielded 14 polybrominated, diphenyl ethers including one new methoxy-containing compound (8). Their structures were elucidated by inter

Full text of DOI:10.1021/acs.jnatprod.6b00229

Note
pubs.acs.org/jnp
Polybrominated Diphenyl Ethers: Structure Determination and
Trends in Antibacterial Activity
Hongbing Liu,† Katheryn Lohith,† Margaret Rosario,† Thomas H. Pulliam,† Robert D. O’Connor,†
Lori J. Bell,‡ and Carole A. Bewley*,†
†Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, Maryland 20892-0820, United States
‡Coral Reef Research Foundation, Koror, PW 96940, Palau
S
* Supporting Information
ABSTRACT: Antibacterial-guided fractionation of the Dictyo-
ceratid sponges Lamellodysidea sp. and two samples of Dysidea
granulosa yielded 14 polybrominated, diphenyl ethers including
one new methoxy-containing compound (8). Their structures
were elucidated by interpretation of spectroscopic data of the
natural product and their methoxy derivatives. Most of the
compounds showed strong antimicrobial activity with low- to
sub-microgram mL−1 minimum inhibitory concentrations
against drug-susceptible and drug-resistant strains of Staph-
ylococcus aureus and Enterococcus faecium, and two compounds
inhibited Escherichia coli in a structure-dependent manner.
he occurrence of bacterial infections by antibiotic-resistant
organisms in hospital as well as community settings
widespread marine bacterium.21 In addition to isolating known
PBDEs 1−7 from D. granulosa and Lamellodysidea sp., we
describe the structural assignment of a new PBDE, compound 8
(Figure 1), along with an approach to assign bromination and
hydroxylation patterns in substituted diphenyl ethers. Un-
expected trends in antibacterial activities were observed for
these compounds.
The extracts of Lamellodysidea sp. and two collections of D.
granulosa inhibited the growth of both Gram-positive and
Gram-negative bacteria. The Gram-positive bacteria included
Bacillus subtilis and drug-susceptible and drug-resistant strains
of Staphylococcus aureus and Enterococcus faecium, with E. coli
representing Gram-negative bacteria. With an emphasis on
identifying the compounds responsible for the Gram-negative
inhibitory activity, we fractionated these extracts on HP20SS
eluting with H2O to MeOH followed by acetone. LC-MS and
NMR analyses of the E. coli-active fractions identified a number
of known PBDEs, along with a new compound, 8. Compound
8 was isolated as a white solid, and the low-resolution ESIMS
spectrum of 8 showed a distribution of negatively charged ions
at m/z 620.6, 622.6, 624.6, 626.6, 628.6, and 630.6 having
relative intensities of 1:4:6:6:4:1, which indicated the presence
of five bromine atoms in the molecule. The molecular formula
of C13H7Br5O4 was determined by HRESIMS, indicating eight
degrees of unsaturation.
T
continues to be a major burden in public health. Knowing that
our current arsenal of antibiotics comes almost exclusively from
bacterial- and fungal-derived natural products1 and that high-
throughput screening of synthetic small-molecule libraries for
the discovery of new antibiotics has met with limited success,
some experts have proposed continued investigations into
natural products libraries for discovery of new antibiotics.2−4
While screening organic extracts from the NCI Open
Repository for antibiotic activity against the model Gram-
negative bacterium Escherichia coli, we identified three
antibacterial extracts that originated from related marine
sponges of the family Dysideidae. These included two separate
collections of Dysidea granulosa and one collection of
Lamellodysidea sp.
Marine sponges of the Dysideidae family are known for
containing a rich array of diverse natural products. Examples
include unusual sterols,5−7 glycolipids,8 sesterterpenes,9 and
sesquiterpene quinones;10 the peptidic natural products
dysinosins,11,12 dysideaprolines, and barbaleucamides;13 and
numerous polybrominated and hydroxylated diphenyl ethers
(PBDEs).14−17 Halogenated diphenyl ethers in general have
garnered attention from different fields. Revealed by natural
products chemists, they represent some of the earliest
discoveries of polyhalogenated compounds coming from
nature.18 The distribution of synthetic polychlorinated and
polybrominated biphenyls continues to be studied by environ-
mental toxicologists.19,20 Most recently Agarwal et al. described
a biosynthetic scheme that can lead to formation of PBDEs in a
Received: March 13, 2016
This article not subject to U.S. Copyright.
Published XXXX by the American Chemical
Society
A
DOI: 10.1021/acs.jnatprod.6b00229
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Figure 1. Compounds isolated from Lamellodysidea sp. (1−8), two collections of Dysidea granulosa (9−14), synthetic methylates 3a−6a and 8a, and
a commercial 2,2′-dihydroxydiphenyl ether (15).
a
1
Table 1. H (500 MHz) and 13C NMR (126 MHz) Data for 8 and 8a
8
8a
position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
ΔδC(δ8a−δ8)
1
144.9, C
142.7, C
111.3, C
116.3, CH
111.8, C
129.5, CH
145.1, C
138.8, C
119.2, C
149.0, C
113.4, C
114.2, C
150.8, C
145.5, C
119.2, C
116.9, CH
116.8, C
129.4, CH
148.5, C
145.0, C
119.4, C
152.6, C
113.5, C
121.7, C
5.9
2.8
7.9
0.6
5.0
−0.1
3.4
6.2
0.1
3.6
0.2
7.5
2
3
4
7.37, d (2.2)
6.54, d (2.2)
7.40, d (2.2)
6.49, d (2.2)
5
6
1′
2′
3′
4′
5′
6′
−OH
−OH
1′-OCH3
4′-OCH3
2-OCH3
7.08, br s
7.56, br s
61.6, CH3
61.0, CH3
61.3, CH3
3.79, s
3.91, s
4.00, s
61.1, CH3
3.87, s
a
Chemical shifts (δ) are referenced to residual CDCl3 (1H: 7.26/13C: 77.16 ppm).
1
The H NMR spectroscopic data of compound 8 (Table 1)
exhibited a pair of meta-coupled (2.2 Hz) signals at δH 6.54 and
7.37 and one methoxy group at δH 3.87 (s, 3H). Two broad
1
singlets (δH 7.08 and 7.56) in the H NMR spectrum were
tentatively assigned as hydroxy groups. The presence of one
methoxy carbon, two sp2 methines, and 10 sp2 nonprotonated
carbons was apparent from the 13C NMR and HSQC spectra,
consistent with 8 being a polybrominated diphenyl ether.22
Methylation of 8 to give 8a confirmed the presence of two
hydroxy groups (Table 1).
The locations of the bromines and hydroxy groups in ring A
of 8 and 8a were clear from the chemical shifts, meta-coupling,
and HMBC correlations. The HMBC spectra (Figure 2)
showed couplings from H-4 to C-2, C-3, C-5, and C-6 and from
H-6 to C-1, C-2, C-4, and C-5 in 8 and 8a. Additionally, the
coupling from the methoxy group at δH 4.00 to C-2 in
derivative 8a established the positions of the ether and hydroxy
groups in ring A and the chemical shifts of C-1 and C-2. These
data indicated that the meta-coupled protons were para to the
Figure 2. HMBC correlations used in structure determination of
compounds 8 and 8a.
ether and to a brominated carbon, leaving only the H-4/H-6
arrangement possible.
Assignment of the substitution pattern in ring B was more
challenging due to the lack of HMBC correlations. In addition,
the 13C chemical shifts did not match any known PBDEs. This
was likely due to the presence of a methoxy group that affects
13C chemical shifts in PBDEs.23 We tentatively assigned the
position of the naturally occurring −OCH3 group in ring B
using a combination of NOEs and distance comparisons from
molecular modeling. In selective 1D-NOE spectra of 8 an NOE
B
DOI: 10.1021/acs.jnatprod.6b00229
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
between the OCH3 and H-6 was the only one observed (Figure
S13, Supporting Information). As shown in Figure 3, this
Table 2. Occurrence of Compounds in Three Dysideae
Sponges
NPID
a
sponge
number
collection site
Papua New Guinea 1−8
Palau 9−13
Papua New Guinea 9, 10, and 14
compounds
Lamellodysidea sp.
Dysidea granulosa
Dysidea granulosa
C024121
C031381
C024075
a
Corresponds to the NCI Open Repository Natural Products ID
number.
We tested compounds 1−14 for antibacterial activities
against the panel of drug-susceptible and drug-resistant bacteria
and fungi shown in Table 3. Compounds 2−13 showed strong
antibacterial effects toward S. aureus and E. faecium strains,
exceeding the potency of control antibiotics oxacillin and
vancomycin. Surprisingly and unlike the other compounds, 9
and 11 also inhibited the growth of E. coli with MICs of 3.1 and
12.5 μg/mL, respectively. Though previously reported to have
broad-spectrum antibacterial activity24 in our antimicrobial
assays, compound 10 did not inhibit the growth of E. coli. None
of the compounds were active against P. aeruginosa or C.
albicans.
Compounds 1−14, 3a−6a, and 8a were tested for
cytotoxicity against a monkey kidney cell line (BSC-1) and a
human colorectal tumor cell line (HCT-116).25 Compounds
1−8, 3a−6a, and 8a were nontoxic at the maximum tested
concentration (50 μg/mL). Compounds 9−14 showed some
toxicity against the kidney cell line BSC-1 with IC50’s between 7
and 35 μg/mL. Although their ring B structures are similar,
rings A of compounds 9−14 lack a hydroxy and contain
bromine atoms ortho and para to the ether compared to
compounds 2−8. This difference in conjunction with the cell-
based screens suggests that the lack of the hydroxy group on
ring A and/or the bromine substitution pattern leads to
increased cytotoxicity. To explore whether halogens are
required for these activities, we tested the commercially
available compound 2,2′-dihydroxydiphenyl ether (15) for
cytotoxity and antibacterial activity; it was inactive in all assays,
as were the permethylated derivatives 3a−6a and 8a. The
observed structure−activity relationship suggests that ring B
needs two bromine atoms and a C-1′ hydroxy group for
antibacterial activity. Further, the presence of two phenolic
hydroxy groups at C-1′ and C-2 in PBDEs decreases cytotoxity,
but also corresponds to a loss in activity against the Gram-
negative bacterium E. coli.
In summary, we isolated 14 polybrominated and hydroxy-
lated diphenyl ethers from related Dysideidae sponges and
demonstrated a simple method employing methylation and
associated changes in 13C chemical shifts to assign complex
substitution patterns in proton-deficient structures. Because it
can be used on compounds of this class that contain both
hydroxy and methoxy substitutents, this method augments
previous NMR approaches that have been used in PBDE
analyses.23 Although superficially compounds 1−14 appear to
be highly similar to one another, their antibacterial spectrum
and potencies, as well as cellular cytotoxicities, differed greatly
as a function of the presence and pattern of hydroxy and
bromine substituents. Given the difficulty of identifying small
molecules that inhibit the growth of Gram-negative organisms,
these results may provide insights into features that lead to this
activity.
Figure 3. Interproton distances of three possible isomers of 8 and
NOEs observed for 8a. Coordinates for the three possible structures of
compound 8 were energy minimized using Chem3D, and interproton
distances between H-4 and H-6 of ring A and the −OCH3 group in
ring B were measured in each and compared to the experimental NOE
data (A−C). The isomer shown in B (1′-hydroxy-3′-methyoxy)
predicts NOEs that are not observed, and the isomer in C (1′-hydroxy-
5′-methyoxy) shows a distance greater than 5.5 Å for an NOE that is
present. Panel D shows the NOEs observed for methylated 8a, in
agreement with experimental data. 1D NOE spectra are shown in
Figure S13.
suggests that the −OCH3 group is located meta to the ether
and attached at C-4′ or C-6′. In the NOE spectra of methylated
derivative 8a, NOEs between the new methoxy group in ring B
and H-6 and −OCH3 in ring A were present (Figure 3D);
however no NOEs were observed between the two −OCH3
groups in ring B (Supporting Information). This suggested that
the hydroxy group in 8 was para to the −OCH3 group and thus
located at C-1′.
To provide additional support for the location of substituents
in ring B of compound 8, we prepared the methoxy derivatives
of known compounds 3−6 by treatment with iodomethane to
give compounds 3a−6a, with 3a and 5a being new compounds
(Supporting Information), and compared their 13C chemical
shifts. Compared to the parent compounds 3−6, chemical shifts
for C-1, C-3, and C-5 in ring A and C-2′, C-4′, and C-6′ in ring
B were deshielded by an average of 6.4, 8.3, 4.6, 4.7, 3.2, and 4.0
ppm, respectively. Similarly, the corresponding carbons in 8
and 8a were deshielded by 5.9, 7.9, 5.0, 6.2, 3.6, and 7.5 ppm
(Table 1). These trends in chemical shifts are consistent with
the substitution pattern suggested above and confirm the
locations of the bromine, hydroxy, and methoxy substituents.
We note that these chemical shift changes at positions ortho
and para to a methoxy group are seen at both protonated and
halogenated carbons, making this a useful approach for
assigning these complex substitution patterns. Interestingly,
compounds originating from Lamellodysidea all contained a 2-
hydroxy-3,5-dibromophenyl moiety for ring A, while com-
pounds from D. granulosa uniformly contained a 2,4-
dibromophenyl moiety, making this chemistry specific for
taxonomically distinct marine sponge samples (Table 2).
C
DOI: 10.1021/acs.jnatprod.6b00229
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
a
Table 3. Minimum Inhibitory Concentrations and Cytotoxicity of Compounds 1−14, 3a−6a, 8a, and 15
S. aureus ATCC S. aureus ATCC E. faecium ATCC E. faecium ATCC E. coli ATCC
P. aeruginosa
ATCC 27853
C. albicans ATCC
29213
43300
29212
51299
25922
28517
Bsc-1
1
100
1.6
>50
12.5
3.1
3.1
1.6
1.6
0.39
0.78
3.1
50
25
1.6
>100
50
>50
>50
>50
>50
50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
7.0
2
1.25
0.31
0.16
0.16
0.078
0.16
0.39
3
1.6
3.1
100
50
4
0.78
0.31
0.31
0.39
0.78
>50
>50
>50
>50
3.1
0.78
0.78
0.39
0.39
1.56
>50
50
5
50
6
25
50
7
50
50
8
>50
>50
>50
>50
>50
>50
3.1
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
3a
>50
4a
>50
>50
>50
50
5a
>50
>50
13
>50
>50
13
6a
8a
3.1
9
0.042
0.78
0.14
3.7
0.08
0.19
0.015
0.4
1.2
0.8
0.4
1.2
33
1.2
10
0.8
>100
12.5
>100
>100
>100
32
11
0.4
8.8
12
1.2
15
13
1.6
0.8
33
29
14
3.7
0.4
11
11
35
15
50
50
>50
>50
32
oxacillin
gentamicin
vancomycin
amphotericin B
0.13
>32
1
0.5
1
2
<7.8
a
MICs and cytotoxity data are reported in μg/mL.
(4.2 mg, tR 29.5 min), and 7 (114 mg, tR 31.0 min). The mixture
eluting at 27 min was resubjected to semipreparative HPLC (5 mL/
min, 30 min, 50−70% MeCN−H2O gradient elution) to give
compounds 6 (6.4 mg, tR 23.5 min) and 3 (15 mg, tR 25.0 min).
The organic extract (2.19 g) of D. granulosa (NPID no. C031381)
was dissolved in MeOH and chromatographed on a Diaion HP20SS-
gel column eluting with water to MeOH, followed by acetone, to give
seven fractions (A−G), which were combined on the basis of their LC-
MS profiles. The acetone fraction G (1.44 g) was subjected to
reversed-phase HPLC (XBridge prep, Waters C18, 5 μm) eluting with
a linear gradient of 0.1% TFA in H2O and 0.05% TFA in MeCN (60−
100% in 40 min, flow rate 5 mL/min) to yield compounds 9 (38 mg,
tR 14.5 min), 10 (1.2 mg, tR 17.3 min), 11 (38.6 mg, tR 18.0 min), 12
(13.4 mg, tR 19.0 min), and 13 (1.7 mg, tR 22.7 min).
The organic extract (2 g) of D. granulosa (NPID no. C024075) was
dissolved in MeOH and chromatographed on a Diaion HP20SS-gel
column as described above. The acetone fraction G (0.35 g) was
subjected to reversed-phase HPLC (5 mL/min, 60−80% H2O−MeCN
with 0.01% TFA, gradient elution in 40 min) to yield compounds 9
(20.2 mg, tR 18.5 min), 10 (6.0 mg, tR 18.5 min), and 14 (2.1 mg, tR
37.5 min).
EXPERIMENTAL SECTION
■
General Experimental Procedures. UV−vis spectra were
recorded on an Agilent 8453 spectrophotometer, and IR spectra on
a PerkinElmer Frontier FT-IR spectrophotometer (KBr). NMR
spectra were recorded at 298 K on a Bruker Avance500 spectrometer
equipped with a triple resonance cryoprobe and z gradients. Transient
NOE spectra were acquired with a 1 s mixing time using a selective 1D
double pulsed field gradient spin echo pulse program incorporating
perfect-echo excitation, alternating gradient polarity, and zero
quantum suppression.26−29 LRESIMS was carried out on an Agilent
1100 LC system and a 6310 MSD. HRESIMS was carried out on a
Waters time-of-flight mass spectrometer model LCT Premier.
Semipreparative HPLC (Agilent 1100) was performed using a Waters
XBridge preparative C18 column (10 × 250 mm, S-5 μm, 12 nm). All
solvents (HPLC grade) and Diaion HP20SS gel were obtained from
Sigma-Aldrich.
Animal Material. Extracts from the three Dysideidae sponge
samples used in this study were obtained from the NCI Open
Repository. Lamellodysidea sp. (NPID no. C024121) was collected at a
depth of 43 m in Papua New Guinea in June 2003. Dysidea granulosa
(NPID no. C024075) was collected in Papua New Guinea at a depth
of 10 m, and a separate collection of D. granulosa (NPID no.
C031381) was collected in June 2010 in the Palau Islands. Taxonomic
identifications were made by Michelle Kelly or one of the authors,
L.J.B. Voucher specimens are available at the NCI, Frederick, MD,
USA, and at the Smithsonian Museum, Washington, DC.
Extraction and Isolation. Organic extracts from the above three
sponges were prepared by extraction with MeOH−CH2Cl2 and stored
at −50 °C until shipment. The organic extract of Lamellodysidea sp. (5
g) was chromatographed on a Diaion HP20SS-gel column equilibrated
in H2O and was eluted with a 10% stepwise gradient from H2O to
MeOH. Fractions were combined into three groups, A−C, on the basis
of antimicrobial activity. Fraction C (4.5 g) was purified by
semipreparative HPLC (5 mL/min, 0−15 min, 50−85%; 15−40 min
85−95% MeCN−H2O gradient elution) to yield compounds 1 (0.4
mg, tR 14.5 min), 2 (1.1 mg, tR 24.0 min), 8 (3.3 mg, tR 24.5 min), a
mixture of compounds with a tR of 27 min, 4 (10 mg, tR 27.5 min), 5
1-(3′,5′,6′-Tribromo-4′-methoxy-1′-hydroxyphenoxy)-3,5-dibro-
mo-2-phenol (8): white solid; UV (MeOH) λmax (log ε) 213 (5.64),
297 (4.57) nm; IR (film) νmax 2918, 2865, 1711, 1415, 1234 cm−1; 1H
and 13C NMR data, Table 1; LRESIMS m/z 620.6 [M − H]−;
HRESIMS m/z 620.6187 [M − H]− (calcd for C13H6Br5O4,
620.6183).
1-(3′,4′,6′-Tribromo-1′-methoxyphenoxy)-3,5-dibromo-2-
methoxybenzene (3a): white solid; UV (MeOH) λmax (log ε) 214
(5.12) nm; IR (film) νmax 2917, 2864, 1701, 1413, 1232 cm−1; 1H and
13C NMR data, Supporting Information; HRESIMS m/z 619.6458 [M
+ H]+ (calcd for C14H9Br5O3, 619.6468).
1-(4′,5′,6′-Tribromo-1′-methoxyphenoxy)-3,5-dibromo-2-
methoxybenzene (5a): white solid; UV (MeOH) λmax (log ε) 216
(5.34) nm; IR (film) νmax 2917, 2865, 1701, 1510, 1493, 1482 cm−1;
1H and 13C NMR data, Supporting Information; HRESIMS m/z
619.6473 [M + H]+ (calcd for C14H9Br5O3, 619.6468).
D
DOI: 10.1021/acs.jnatprod.6b00229
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Journal of Natural Products
Note
1-(3′,5′,6′-Tribromo-1′,4′-dimethoxy-1′-phenoxy)-3,5-dibromo-
2-methoxybenzene (8a): UV (MeOH) λmax (log ε) 214 (5.68), 295
(4.44) nm; IR (film) νmax 2917, 2865, 1711, 1415, 1234 cm−1; 1H and
13C NMR data, Table 1; HRESIMS m/z 649.6567 [M + H]+ (calcd for
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Methylation of Compounds 3−6 and 8. Compound 3 (2.0 mg,
3 μmol) was added to a solution of acetone (500 μL) in the presence
of K2CO3 (0.1 mg) and MeI (400 μL, 6 μM) at rt. The solution was
stirred for 1 h, dried under a stream of N2, and extracted with CHCl3
to give 3a as a white solid (2.3 mg, 100%). By the same method
compounds 4a (1.7 mg, 95%), 5a (2.0 mg, 84%), 6a (3.3 mg, 95%),
and 8a (0.6 mg, 100%) were obtained from 4 (1.5 mg), 5 (2.0 mg), 6
(3.0 mg), and 8 (0.5 mg), respectively.
MIC and Cytotoxicity Determination. MICs were determined
by testing all compounds in duplicate in at least two independent
experiments for their antimicrobial activity against laboratory strains
listed in Table 3 and as described in the Clinical and Laboratory
Standards Institute (CLSI) guidelines.30 All laboratory bacterial strains
were obtained from the American Type Culture Collection.
Experimental details for MIC and cytotoxicity data are provided in
the Supporting Information.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00229.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1-(301)-594-5187. E-mail: caroleb@mail.nih.gov.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We thank J. Lloyd for HRMS data and the NCI Open
Repository for organic extracts. This work was supported by the
NIH Intramural Research Program (NIDDK). T.P. acknowl-
edges a Park Scholarship.
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