Isolation and Total Synthesis of Bromoiesol sulfates, Antitrypanosomal arylethers from a Salileptolyngbya sp. Marine Cyanobacterium

Article abstract of DOI:10.1021/acs.joc.1c01214

Bromoiesol sulfates A (1) and B (2), new polyhalogenated aryl sulfates, were isolated from a Salileptolyngbya sp. marine cyanobacterium along with their hydrolyzed compounds, bromoiesols A (3) and B (4). To pick up the candidates of their structures, we u

Full text of DOI:10.1021/acs.joc.1c01214

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Isolation and Total Synthesis of Bromoiesol sulfates,
Antitrypanosomal arylethers from a Salileptolyngbya sp. Marine
Cyanobacterium
Akira Ebihara, Arihiro Iwasaki, Youhei Miura, Ghulam Jeelani, Tomoyoshi Nozaki,
and Kiyotake Suenaga*
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Supporting Information
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ABSTRACT: Bromoiesol sulfates A (1) and B (2), new
polyhalogenated aryl sulfates, were isolated from a Salileptolyngbya
sp. marine cyanobacterium along with their hydrolyzed com-
pounds, bromoiesols A (3) and B (4). To pick up the candidates
of their structures, we used Small Molecule Accurate Recognition
Technology (SMART), an artificial intelligence-based structure-
prediction tool, and their structures were elucidated on the basis of
single-crystal X-ray diffraction analysis of bromoiesols (3 and 4). In
addition, to verify the structures, the total synthesis of bromoiesol
A sulfate (1) and bromoiesol A (3) was achieved. The bromoiesol
family, especially bromoiesols (3 and 4), selectively inhibited the
growth of the bloodstream form of Trypanosoma brucei rhodesiense,
the causative agent of human African sleeping sickness.
bromoiesols (3 and 4), from a Salileptolyngbya sp. marine
cyanobacterium. Structurally, 1 and 2 are aryl ethers containing
three polyhalogenated benzene rings and a sulfate group,
which is rare in natural products. In addition, we clarified that
the bromoiesol family (1−4), especially bromoiesols (3 and
4), exhibited antitrypanosomal activity. Here, we report the
isolation and structure elucidation of the bromoiesol family
(1−4), and the first total synthesis of 1 and 3.
INTRODUCTION
■
Tropical diseases caused by Trypanosoma sp., such as African
sleeping sickness and Chagas disease, are some of the most
serious infectious diseases worldwide. They are endemic in
developing countries, where they threaten the health of over
400 million people and slow economic development.1
Although some drugs for these diseases are already available
for use, they have limitations such as serious side effects and
the emergence of drug resistance.1 Therefore, new anti-
protozoal drugs are needed.
Meanwhile, marine natural products are considered to be
good sources for drug leads.2 In particular, secondary
metabolites produced by marine cyanobacteria have unique
structures and versatile biological activities3 including anti-
protozoal activity. For example, gallinamide A4 shows very
potent antimalarial activity by inhibiting plasmodial cysteine
proteases,5 and almiramides6 perturb the function of
glycosome, that is, an essential organelle for survival of
kinetoplastid parasites.7 As for other activities, a number of
important bioactive natural products have been discovered
from marine cyanobacteria such as largazole,8 carmaphycins,9
and antillatoxin B.10 Against this background, we have
investigated the secondary metabolites of Japanese marine
cyanobacteria and have discovered several novel natural
products possessing antiparasitic activities such as ikoamide,11
iheyamides,12 and hoshinoamides.13
Received: May 25, 2021
Recently, we found new antitrypanosomal aryl ethers,
bromoiesol sulfates (1 and 2), and their hydrolysates,
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A

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1
Table 1. H and 13C{1H} NMR Data for Bromoiesol Family (1−4) in CD3OD
Bromoiesol A (3)
Bromoiesol B (4)
Bromoiesol A sulfate (1)
Bromoiesol B sulfate (2)
a
b
a
b
a
b
a
b
position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
c
1
2
3
4
5
6
7
8
153.0, C
139.3, C
123.0, C
126.9, CH
120.5, C
120.8, CH
142.1, C
151.0, C
119.8, CH
123.8, C
121.6, C
118.9, C
153.9, C
117.9, C
132.4, CH
116.1, C
136.7, CH
113.4, C
154.5, C
139.0, C
118.6, C
125.4, CH
119.9, C
121.5, CH
142.4, C
151.3, C
119.9, CH
120.6, C
121.1, C
123.0, C
156.5, C
116.8, C
133.2, CH
116.3, C
142.5, CH
147.2, C
140.8, C
119.4, C
130.6, CH
119.0, C
124.2, CH
140.8, C
149.9, C
119.1, CH
122.5, C
121.0, C
122.1, C
152.7, C
117.3, CH
132.1, CH
115.0, C
135.4, CH
111.9, C
-
141.0, C
c
-
7.27, d (2.3)
7.07, d (2.3)
7.16, d (2.3)
7.02, d (2.3)
7.62, d (2.3)
7.88, d (2.3)
130.0, CH
c
7.62, d (2.3)
7.89, d (2.3)
-
123.9, CH
141.0, C
149.6, C
118.9, CH
c
9
6.96, s
6.96, s
7.00, s
7.02, s
10
11
12
13
14
15
16
17
18
-
-
-
c
c
155.1, C
115.9, C
132.4, CH
115.1, C
140.7, CH
84.8, C
6.66, d (9.1)
7.36, dd (2.3, 9.1)
6.59, d (9.0)
7.38, dd (2.4, 9.0)
6.83, d (9.1)
7.43, dd (2.3, 9.1)
6.73, d (9.0)
7.44, dd (2.4, 9.0)
7.89, d (2.3)
7.97, d (2.4)
7.74, d (2.3)
7.93, d (2.4)
86.7, C
a
b
c
Measured at 100 MHz. Measured at 400 MHz. Not observed.
Figure 1. X-ray ORTEP drawings of bromoiesols 3 and 4.
respectively). Therefore, we first determined the structures of
3 and 4 and then elucidated the structures of the original
compounds, 1 and 2, based on the structures of bromoiesols (3
and 4).
RESULTS AND DISCUSSION
■
A Salileptolyngbya sp. marine cyanobacterium was collected at
Ie Island, Okinawa, Japan, in July 2020. The collected
cyanobacterium (650 g, wet weight) was extracted with
EtOH, and the extract was filtered and concentrated. The
residue was partitioned between EtOAc and H2O. The
material obtained from the organic layer was partitioned
between 90% aqueous MeOH and hexane. The aqueous
MeOH fraction was separated by reversed-phase column
chromatography (ODS silica gel, MeOH-H2O). Reversed-
phase HPLC analysis of the fraction eluted with 80% aqueous
MeOH showed the presence of two compounds with strong
UV absorption (bromoiesol sulfates 1 and 2). Since 1 and 2
were eluted as sharp peaks only with the use of an acidified
mobile phase, we used ODS HPLC with 91% aqueous MeOH
with 0.1% TFA as a solvent for the isolation of bromoiesol
sulfates (1 and 2). However, after evaporation of the solvent,
large percentages of 1 and 2 were converted to other
compounds, bromoiesols (3, 8.6 mg, and 4, 3.6 mg,
Compared to 1 and 2, 3 and 4 are much less polar (see
isolation conditions in the Supporting Information) and 80
units smaller m/z in the mass spectrum. In addition,
bromoiesols (3 and 4) showed a sharp peak in HPLC without
the use of an acidified solvent. Therefore, 3 and 4 were easily
purified by HPLC (Cosmosil 5PE-MS, MeCN-H2O, 8.6 mg of
3 and 3.6 mg of 4). The molecular formula of bromoiesol A
(3) was determined on the basis of negative HRESIMS data
(m/z 822.4606, calcd for C18H6Br7O3 [M-H]− 822.4606). The
presence of seven bromine atoms was supported by octet
isotopic [M-H]− peaks with a ratio of 1:7:21:35:35:21:7:1.
1
The NMR data for 3 are summarized in Table 1. In the H
NMR spectrum in CD3OD, 3 showed only six aromatic signals
with a simple coupling pattern (δH 6.66, 6.96, 7.07, 7.27, 7.36,
and 7.78), and the analyses of the coupling constants and the
B
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decoupling experiments clarified the presence of three aromatic
rings as follows: 1, 2, 4-trisubstituted, 1, 3, 4, 5-tetrasubstituted,
and 1, 2, 3, 4, 5-pentasubstituted benzene rings. Meanwhile,
the 13C{1H} NMR spectrum revealed the existence of 18 sp2
Scheme 1. Outline of Synthesis of Bromoiesol A sulfate (1)
1
carbons (δC 113.4−153.9), supporting the result of the H
NMR analysis. To obtain clues about the structure, we
analyzed HMQC spectral data of 3 using Small Molecule
Accurate Recognition Technology (SMART), an artificial
intelligence-based structure-prediction tool (see Supporting
Information).14 As a result, several oxygenated arylethers, such
as spongiadioxin A15 (Cosine score: 0.9803) and ambigol C16
(cosine score: 0.9602), were proposed as natural products that
have a similar structure to bromoiesol A (3), suggesting that
the three aromatic rings in 3 are connected by ether bonds.
However, the scarcity of protons in the structure prevented us
from clarifying the connectivity of the three aromatic rings
using 2D NMR data. Therefore, we tried to crystallize 3 for
single-crystal X-ray diffraction analysis. Recrystallization was
attempted using hexane, acetone, acetonitrile, and methanol,
but only methanol gave a single crystal. The structure of
bromoiesol A (3) was determined by single-crystal X-ray
diffraction analysis, as shown in Figure 1.
The molecular formula of bromoiesol B (4) was determined
by HRESIMS (m/z 870.4491, calcd for C18H6Br6IO3 [M-H]−
870.4467). The NMR data for 4 are similar to those for
bromoiesol A (3) except for a chemical shift at C-18, which is
the typical value for an iodine-attached aromatic carbon (Table
1). Bromoiesol B (4) was crystallized in the same way, and the
structure was determined by single-crystal X-ray diffraction
analysis as shown in Figure 1.
Next, we deduced the structures of the original compounds,
bromoiesol sulfates (1 and 2), based on the structures of
bromoiesols (3 and 4). As we mentioned above, 1 and 2 have
much higher polarity and 80 units greater m/z. In addition,
HRESIMS analysis clarified that the difference in the molecular
formulas between 1 and 3, and 2 and 4 was one sulfur and
three oxygens. On the basis of these experimental findings, we
deduced that 1 and 2 were sulfates of bromoiesols (3 and 4). If
so, 1 and 2 seem unstable under strong acidic conditions such
as with TFA. Therefore, we modified the solvent for the HPLC
purification of bromoiesol sulfates (1 and 2). As a result, we
found that a solvent containing 20 mM AcOH/AcONa buffer
at pH 4 was suitable for the isolation of 1 and 2 without
hydrolysis of the sulfate group. We recollected the same
cyanobacterium at Ie Island in September 2020 and
successfully isolated bromoiesol sulfates (1, 1.8 mg, and 2,
1.3 mg) without decomposition by using the modified
condition. In this second isolation experiment, we obtained
neither 3 nor 4. The NMR data of 1 and 2 were summarized in
Table 1.
To verify the structure of bromoiesol A sulfate (1), we
achieved the total synthesis of 1 along with bromoiesol A (3).
Our synthetic strategy is shown in Scheme 1. Bromoiesol A
sulfate (1) could be synthesized from bromoiesol A (3) by
condensation with sulfur trioxide. Bromoiesol A (3) could be
obtained by sequential coupling of known fragments 5, 6, and
7.17−19 In these key coupling reactions, we used aryliodonium
salts,20,21 derived from 5 and 7 as an electrophile, respectively.
Our total synthesis commenced with the preparation of aryl
iodonium salts 9 and 11 (Scheme 2). Known amine 517 was
converted to known iodide 8 by the Sandmeyer reaction.22
The oxidation of 8 using mCPBA followed by the addition of
trimethoxybenzene (TMB) afforded iodonium salt 9.20
Meanwhile, known amine 719 was converted to iodonium
salt 11 in the same way as 9.
The first coupling reaction between iodonium salt 9 and
known phenol 618 under basic condition gave aryl ether 12
(Scheme 3).21 After the demethylation of 12 by boron
tribromide,19 the second coupling reaction with 11 afforded
arylether 14. Demethylation of 14 provided bromoiesol A (3),
and 3 was successfully converted to bromoiesol A sulfate (1)
by sulfur trioxide. The spectral data of synthetic bromoiesol A
sulfate (1) and bromoiesol A (3) were consistent with those of
natural 1 and 3. Therefore, we verified the correctness of our
structure determination.
As for biological activity, the bromoiesol family (1−4)
showed antitrypanosomal activity against the bloodstream
form of Trypanosoma brucei rhodesience IL-1501 strain, the
causative agent of African sleeping sickness, without affecting
the growth of HeLa cells (Table 2) at 10 μM. In addition,
although iodine substitution did not change the degree of
activity of the bromoiesol family (1−4), hydrolysis of the
sulfate group drastically increased the antiparasitic activity.
Considering the instability of the sulfate group and the
significant change in biological activities between the sulfates
(1 and 2) and the hydrolysates (3 and 4), bromoiesol sulfates
(1 and 2) may act as prodrug-like compounds in the natural
environment.
In conclusion, bromoiesol sulfates (1 and 2), polyhalo-
genated aryl ethers, and their hydrolysates (3 and 4) were
isolated from the marine cyanobacterium Salileptolyngbya sp.
We determined their structures based on SMART analysis of
the HMQC data of 3 and single-crystal X-ray diffraction
analyses of 3 and 4. In addition, to verify the structures, the
total synthesis of bromoiesol A sulfate (1) and bromoiesol A
(3) was achieved. The bromoiesol family, especially
bromoiesols (3 and 4), showed selective growth-inhibitory
activity against the causative agent of Human African
trypanosomiasis. To date, several structurally related natural
products, such as crossbyanols A−D,23 have been reported.
They also showed significant differences in biological activities
between sulfates and the corresponding hydrolysates. The
biosynthetic pathways of these polyhalogenated aromatic
compounds are revealed by Moore’s group.24 Each aromatic
ring is derived from 4-hydroxybenzoic acid with bromination
by flavin-dependent halogenase Bmp5, and the 4-hydrox-
ybenzoic acid is synthesized from chorismic acid by chorismate
lyase Bmp6. Meanwhile, the aryl ether bonds are constructed
by cytochrome P450 enzyme Bmp7. In fact, a recent study
C
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Scheme 2. Synthesis of Aryliodonium Salts 9 and 11
Scheme 3. Synthesis of Bromoiesol A (3) and Bromoiesol A sulfate (1)
EXPERIMENTAL SECTION
Table 2. Growth-Inhibitory Activities of the Bromoiesol
Family (1−4)
■
General Experimental Procedures. Optical rotations were
measured with a JASCO DIP-1000 polarimeter. IR spectra were
recorded on a JASCO RT/IR-4200 instrument. All NMR spectral
IC50 (μM)
1
compounds
HeLa cells
T. b. rhodesiense
data were recorded on a JEOL JNM-ECS400 spectrometer for H
(400 MHz) and 13C{1H} (100 MHz). 1H NMR chemical shifts
(referenced to residual CHCl3 and CHD2OD observed at δH 7.26 and
3.31, respectively) were assigned using a combination of data from 1H
decoupling and HMQC experiments. Similarly, 13C{1H} NMR
chemical shifts (referenced to CDCl3 and CD3OD observed at δC
77.16 and 49.0) were assigned based on HMBC and HMQC
experiments. HRESIMS and HRAPCIMS spectra were obtained on
an LCT Premier XE time-of-flight (TOF) mass spectrometer.
Chromatographic analyses were performed using an HPLC system
consisting of a pump (model PU-2080, JASCO) and a UV detector
(model UV-2075, JASCO). All chemicals and solvents used in this
study were the best grade and available from a commercial source
(Nacalai Tesque). Reactions were monitored by thin-layer chroma-
tography (TLC), and TLC plates were visualized by both UV
detection and phosphomolybdic acid solution. Silica Gel 60N
(Irregular, 63-212 μm) were used for open column chromatography
unless otherwise noted. Automated Flash Chromatography System
(AFCS, EPCLC AI-580, YAMAZEN) was also used for purification of
synthesized compounds. X-ray crystal structure analysis was
bromoiesol A (3)
bromoiesol B (4)
bromoiesol A sulfate (1)
bromoiesol B sulfate (2)
21
21
>10
>10
6
5
1.2 0.1
0.70 0.23
8.8 1.3
7.9 1.8
a
a
a
Because of the scarcity of the sample, the exact IC50 value was not
determined.
clarified that ambigols A−C, polyhalogenated triphenyls
produced by the terrestrial cyanobacterium Fischerella ambigua
108b, are synthesized by the similar pathway as described
above.25 Therefore, it is likely that bromoiesols are constructed
in the same way.
Now that we have developed a synthetic route for the
bromoiesol family, a detailed study on the structure−activity
relationships of synthetic analogs may contribute to the
development of new antitrypanosomal drugs.
D
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performed using a Bruker D8 VENTURE instrument and APEX3. All
moisture-sensitive reactions were performed under an atmosphere of
nitrogen, and the starting materials were azeotropically dried with
toluene before use.
detection UV 215 nm] to give bromoiesol A (3) (8.6 mg, tR = 45
min) and bromoiesol B (4) (3.6 mg, tR = 48 min).
Bromoiesol A (3). A white solid; IR (neat) 3094, 1576, 1460, 1427,
1348, 1283, 1213, 1036, 924, 892, 804 cm−1; UV (MeCN) λmax (log
ε) 289 nm (3.54); melting point of crystal 128.0−129.5 °C; 1HNMR,
13C{1H} NMR, and HMBC data, Table S1 in the Supporting
Identification of Marine Cyanobacterium. A cyanobacterial
filament was isolated under a microscope and crushed with freezing
and thawing. The 16S rDNA genes were PCR-amplified from the
isolated DNA using the primer set CYA106F (a cyanobacterial-
specific primer) and 16S1541R (a universal primer). The PCR
reaction contained DNA derived from a cyanobacterial filament, 0.5
μL of KOD-Multi and Epi- (Toyobo), 0.25 μL of each primer, 12.5
μL of 2 × PCR Buffer for KOD-Multi and Epi-, and H2O for a total
volume of 24 μL. The PCR reaction was performed as follows: initial
denaturation for 2 min at 94 °C, and amplification by 40 cycles of 10 s
at 98 °C and 10 s at 58 °C, and 1 min at 66 °C. PCR products were
analyzed on agarose gel (1%) in TBE buffer and visualized by
ethidium bromide staining. The obtained DNA was sequenced with
CYA106F and 16S1541R primers.
This sequence is available in the DDBJ/EMBL/Genbank databases
under accession number LC603015. The obtained nucleotide
sequence of 16S rRNA gene was used for phylogenetic analysis
with the sequences of related cyanobacterial 16S rRNA genes. All
sequences were aligned by the SINA web service (version 1.2.11)26
with default settings. The poorly aligned positions and divergent
regions were removed by Gblocks Server (version 0.91b),27
implementing the options for a less stringent selection, including
the ‘Allow smaller final blocks’, ‘Allow gap positions within the final
blocks’, and ‘Allow less strict flanking positions’ options. The obtained
904 nucleotide positions were used for phylogenetic analysis.
JModeltest (version 2.1.7)28,29 with default settings was used to
select the best model of DNA substitution for the maximum
likelihood (ML) analysis and Bayesian analysis according to the
Akaike information criterion (AIC). The ML analysis was conducted
by PhyML (version 20131016) using the GTR+I+G model with a
gamma shape parameter of 0.4850, a proportion of invariant sites of
0.5220, and nucleotide frequencies of F(A) = 0.2490, F (C) = 0.2307,
F (G) = 0.3118, F(T) = 0.2084. Bootstrap resampling was performed
on 1000 replicates. The ML tree was visualized with Njplot (version
2.3).30 The Bayesian analysis was conducted by MrBayes (version
3.2.5)31 using the GTR+I+G model. The Markov chain Monte Carlo
process was set at 2 chains, and 1 000 000 generations were
conducted. Sampling frequency was assigned at every 500 generations.
After analysis, the first 100 000 trees were deleted as burn-in, and the
consensus tree was constructed. The Bayesian tree was visualized with
FigTree (version 1.4.0, http://tree.bio.ed.ac.uk/software/figtree). As
a result, the cyanobacterium (accession no. LC603015) formed a
clade with Salileptolyngbya sp. Therefore, the cyanobacterium was
classified into Salileptolyngbya sp. The phylogenetic tree is shown in
Figure S1 in the Supporting Information.
Collection, Extraction, and Isolation of Bromoiesols A and B
(3 and 4). The Salileptolyngbya sp. cyanobacterium (650 g, wet
weight) was collected at Ie-Island, Okinawa, Japan in July 2020. The
collected cyanobacterium was extracted with EtOH (2 L) for 1 week
at room temperature. The extract was filtered, and the residue was re-
extracted with EtOH (2 L) at room temperature for 2 days. The
extract was filtered, and the combined filtrates were concentrated. The
residue was partitioned between EtOAc (3 × 300 mL) and water (300
mL). The combined organic layers were concentrated, and the residue
was partitioned between 90% aqueous MeOH (300 mL) and hexane
(3 × 300 mL). The aqueous MeOH layer was concentrated, and the
obtained residue (387 mg) was separated by column chromatography
on ODS (5 g) eluted with 25 mL of 40%, 60%, and 80% aqueous
MeOH, followed by 25 mL of MeOH. The fraction eluted with 80%
MeOH (96.3 mg) was subjected to HPLC [Cosmosil 5C18-MS-II (ϕ
20 mm × 250 mm); solvent 91% aqueous MeOH with 0.1% TFA;
flow rate 5 mL/min; detection UV 215 nm] in three batches to give a
fraction that contained 3 and 4 (18 mg, tR = 35−45 min). This
fraction was further separated by HPLC [Cosmosil 5PE-MS (ϕ 20
mm × 250 mm); solvent 78% aqueous MeCN; flow rate 5 mL/min;
Information; HRESIMS m/z 822.4606 [M-H]− (calcd for
C18H679Br7O3 822.4606).
Bromoiesol B (4). A white solid; IR (neat) 1572, 1461, 1429, 1351,
1277, 1213, 1029, 768 cm−1; UV (MeCN) λmax (log ε) 288 nm
(3.75);melting point of crystal 140.0−147.5 °C;1HNMR, 13C{1H}
NMR, and HMBC data, Table S2 in the Supporting Information;
HRESIMS m/z 870.4491 [M-H]− (calcd for C18H679Br6IO3
870.4467).
Collection, Extraction, and Isolation of Bromoiesol sulfates
A and B (1 and 2). The Salileptolyngbya sp. cyanobacterium (300 g,
wet weight) was collected at Ie-Island, Okinawa, Japan in September
2020. The experiments before the ODS column chromatography step
were conducted in the same way as the previous ones. The residue
(108 mg) obtained by concentration of the 90% aqueous MeOH layer
was separated by column chromatography on ODS (3 g) eluted with
15 mL of 40%, 60%, and 80% aqueous MeOH, followed by 25 mL of
MeOH. Among them, the fraction eluted with 80% MeOH (24.9 mg)
contained bromoiesol sulfates (1 and 2). We examined several
conditions for the purification of bromoiesol sulfates (1 and 2), and
the most successful procedure is described below.
One-third of the fraction eluted with 80% MeOH (8.3 mg) was
separated by HPLC [Cosmosil 5C18-MS-II (ϕ 20 mm × 250 mm);
solvent 92% MeOH, 8% pH4 20 mM AcOH/AcONa buffer; flow rate
5 mL/min; detection UV 215 nm] to give a fraction that contained 1
and 2 (4.3 mg, tR = 25−35 min). This fraction was further purified by
HPLC [Cosmosil 5PE-MS (ϕ 20 mm × 250 mm); solvent 65%
MeCN, 35% pH4 20 mM AcOH/AcONa buffer; flow rate 5 mL/min;
detection UV 215 nm] to give two fractions that contained almost
pure 1 (2.3 mg, tR = 29−33 min) and almost pure 2 (1.1 mg, tR = 33−
40 min), respectively.
These fractions were combined with other fractions containing 1 or
2 that were purified by other methods to give two fractions containing
1 (3.0 mg) and 2 (2.6 mg), respectively. The fraction containing 1
(3.0 mg) was finally purified by column chromatography on ODS (0.2
g) with 80% aqueous MeOH to give 1.8 mg of bromoiesol A sulfate
(1). The fraction containing 2 (2.6 mg) was purified by HPLC
[Cosmosil 5PE-MS (ϕ 20 mm × 250 mm); solvent 65% MeCN, 35%
pH 4 20 mM AcOH/AcONa buffer; flow rate 5 mL/min; detection
UV 215 nm], and the resulting crude 2 (tR = 33−40 min) was finally
purified by column chromatography on ODS (0.2 g) with 80%
aqueous MeOH to give 1.3 mg of bromoiesol B sulfate (2).
Bromoiesol A sulfate (1). A pale white solid; IR (neat) 1464, 1427,
1284, 1260, 1055, 927 cm−1; UV (MeCN) λmax (log ε) 283 nm
1
(3.62); H NMR, 13C{1H} NMR, and HMBC data, Table S3 in the
Supporting Information; HRESIMS m/z 902.4178 [M-H]− (calcd for
C18H679Br7O6S 902.4174).
Bromoiesol B sulfate (2). A pale white solid; IR (neat) 1460, 1420,
1280, 1260, 1055, 920 cm−1; UV (MeCN) λmax (log ε) 285 nm
1
(3.68); H NMR, 13C{1H} NMR, and HMBC data, Table S4 in the
Supporting Information; HRESIMS m/z 950.4043 [M-H]− (calcd for
C18H679Br6IO6S 950.4036).
X-ray Crystallographic Analysis of Bromoiesols A (3) and B
(4). Bromoiesol A (3, 1.0 mg) MeOH solution (0.1 mL) was allowed
to stand at −20 °C for 2 days. Single crystal of 3 was precipitated and
was collected by a spatula. Single crystal of bromoiesol B (4) was also
prepared in the same procedure. The crystals were kept at 300 K
during data collection. The structures were solved with the ShelXT
structure solution program using intrinsic phasing and refined with
the ShelXL refinement package using least squares minimization. The
data for 3 and 4 were summarized in Table S5 in the Supporting
Information. Crystallographic data for 3 and 4 have been deposited at
the Cambridge Crystallographic Data Center (CCDC 2082191 and
CCDC 2082192).
E
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Total Synthesis Procedures. 2,4-Dibromo-1-iodobenzene (8).
This compound is a known compound, and we synthesized it in the
same manner as in the previous report.22
H2O (5 mL) at 0 °C. THF was removed in vacuo, and the residual
aqueous mixture was extracted with Et2O (2 × 5 mL). The combined
organic layers were washed with brine, dried over Na2SO4, and
concentrated. The obtained residue was purified by AFCS (gradient
condition; Hexane/EtOAc, 100/0 → 82/18) to give aryl ether 12
(119 mg, 0.20 mmol, 35%) as a white solid; IR (neat) 1571, 1464,
(2,4-Dibromophenyl)(2,4,6-trimethoxyphenyl)iodonium tosylate
(9). To a stirred solution of aryl iodide 8 (274 mg, 0.757 mmol) in dry
acetonitrile (2.3 mL) were added p-toluenesulfonic acid monohydrate
(158 mg, 0.83 mmol) and mCPBA (70% active oxidant, 205 mg, 0.83
mmol). The reaction mixture was stirred at 55 °C in an oil bath for 50
min before being treated with 1,3,5-trimethoxybenzene (140 mg, 0.83
mmol). The reaction mixture was stirred at 55 °C in an oil bath for
another 15 min and concentrated under reduced pressure. The
obtained crude residue was washed with Et2O (4 × 1 mL) and dried
in vacuo to give diaryliodonium salt 9 (397 mg, 0.57 mmol, 75%) as
an orange solid; IR (neat)1696, 1694, 1592, 1457, 1346, 1203, 1125,
1051, 1009, 695, 681, 566 cm−1; 1H NMR (400 MHz, CD3OD) δ8.10
(d, J = 2.4 Hz, 1H), 8.00 (d, J = 8.9 Hz, 1H), 7.70 (d, J = 7.9 Hz, 2H),
7.59 (dd, J = 2.4, 8.9 Hz, 1H), 7.23 (d, J = 7.9 Hz, 2H), 6.42 (s, 2H),
3.97 (s, 6H), 3.90 (s, 3H), 2.37 (s, 3H); 13C{1H} NMR (100 MHz,
CD3OD) δ 168.0, 160.5 (2C), 140.7, 140.0, 136.1, 133.4, 128.8 (2C),
128.0, 127.7, 126.0 (2C), 119.2, 91.9 (2C), 85.6, 56.6 (2C), 55.7,
20.3; HRESIMS m/z 526.8359 [M]+ (calcd for C15H1479Br2IO3
526.8349).
1
1427, 1360, 1290, 1250, 1223, 1177, 1041, 864, 772, 726 cm−1; H
NMR (400 MHz, CDCl3) δ 7.75 (d, J = 2.4 Hz, 1H), 7.29 (s, 1H),
7.23 (dd, J = 2.4, 8.7 Hz, 1H), 7.23 (dd, J = 2.4, 8.7 Hz, 1H), 3.77 (s,
3H); 13C{1H} NMR (100 MHz, CDCl3) δ 152.7, 152.3, 141.1, 136.0,
131.2, 122.6, 122.4, 119.2, 116.7, 115.3, 115.1, 112.4, 56.8;
HRAPCIMS m/z 589.6363 [M]+ (calcd for C13H779Br5O2 589.6369).
3,4,5-Tribromo-2-(2,4-dibromophenoxy)phenol (13). This com-
pound is a known compound, and we synthesized it in the same
manner as in the previous report.19
1,2,3-Tribromo-5-(2,4-dibromo-6-methoxyphenoxy)-4-(2,4-
dibromophenoxy)benzene (14). To a stirred solution of t-BuOK (24
mg, 0.20 mmol) in dry THF (1 mL) was added phenol 13 (104 mg,
0.18 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15
min. After addition of iodonium salt 11 (131 mg, 0.18 mmol), the
reaction mixture was stirred at 40 °C in an oil bath for 44 h and was
diluted with H2O (5 mL) at 0 °C. THF was removed in vacuo, and
then the residue was extracted with Et2O (2 × 5 mL). The combined
organic layers were washed with brine, dried over Na2SO4, and
concentrated. The obtained residue was purified by AFCS (gradient
condition, Hexane/EtOAc, 100/0 → 83/17) to give aryl ether 14
(110 mg, 0.13 mmol, 73%) as a white solid; IR (neat) 1579, 1468,
1,5-Dibromo-2-iodo-3-methoxybenzene (10). To aryl amine 7
(1.00 g, 3.56 mmol) were added H2O (7.1 mL) and 37% aq. HCl (7.1
mL), and the mixture was heated to 80 °C in an oil bath while stirring.
The reaction mixture was stirred for 30 min followed by cooling to 0
°C on an iced water bath. To the reaction mixture was added NaNO2
(270 mg, 3.92 mmol, 1.1 eq) maintaining the internal temperature
below 10 °C. The mixture was stirred at 0 °C for 30 min followed by
addition of KI (650 mg, 3.92 mmol, 1.1 equiv). Next, the reaction
mixture was warmed to room temperature and was stirred overnight.
The resulting mixture was extracted with CH2Cl2, and the organic
layer was washed with 10% sodium hydroxide, 1 M sodium
thiosulfate, 10% hydrochloric acid, water, and brine. After the
removal of organic solvents, the obtained crude compound was
purified by AFCS (gradient condition, Hexane/EtOAc, 100/0 → 81/
19) to afford 493 mg (1.26 mmol, 35%) of aryl iodide 10 asa pale
yellow solid; IR (neat) 1558, 1460, 1384, 1262, 1220, 1038, 1007,
1
1433, 1394, 1350, 1259, 1218, 1181, 1035, 871, 834, 757 cm−1; H
NMR (400 MHz, CD3OD) δ 7.73 (d, J = 2.5 Hz, 1H), 7.36 (d, J = 2.2
Hz, 1H), 7.27 (dd, J = 2.5, 8.8 Hz, 1H), 7.04 (d, J = 2.2 Hz, 1H), 6.81
(s, 1H), 6.58 (d, J = 8.8 Hz, 1H), 3.73 (s, 3H); 13C{1H} NMR(100
MHz, CD3OD) δ 153.1, 152.6, 149.6, 140.8, 138.7, 135.9, 130.8,
127.6, 123.1, 122.1, 121.2, 119.9, 118.5, 118.0, 116.4, 115.8, 115.3,
112.8, 56.6; HRMS data were not obtained probably due to the lack
of the polarity. Therefore, we fully characterized a compound in the
next step.
Bromoiesol A (3). To a stirred solution of aryl ether 14 (60 mg,
0.071 mmol) in CH2Cl2 (1 mL) was added dropwise a 1 M solution
of boron tribromide (1.07 mL, 1.07 mmol) at 0 °C. The reaction
mixture was stirred at 0 °C for 2 h, followed by at room temperature
for 28 h. Next, the reaction mixture was quenched with water at 0 °C.
The organic layer was separated, and then the aqueous layer was
extracted with CH2Cl2 (2 × 5 mL). The combined organic layers
were washed with brine, dried over Na2SO4, and concentrated. The
obtained residue was purified by AFCS (gradient condition, Hexane/
EtOAc, 73/27 → 52/48) to give bromoiesol A (3, 45 mg, 0.55 mmol,
77%) as a white solid; IR (neat) 3094, 1576, 1460, 1427, 1348, 1283,
1
833, 773 cm−1; H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 2.1 Hz,
1H), 6.84 (d, J = 2.1 Hz, 1H), 3.88 (s, 3H); 13C{1H} NMR (100
MHz, CD3OD) δ 160.4, 131.1, 127.5, 123.3, 112.7, 93.1, 57.2;
HRESIMS m/z 374.7503 [M-CH3]− (calcd for C6H279Br2IO
374.7517).
(2,4-Dibromo-6-methoxyphenyl)(2,4,6-trimethoxyphenyl)-
iodonium tosylate (11). To a stirred solution of aryl iodide 10 (246
mg, 0.63 mmol) in acetonitrile (2.0 mL) were added p-toluene-
sulfonic acid monohydrate (132 mg, 0.69 mmol) and mCPBA (70%
active oxidant, 171 mg, 0.69 mmol). The reaction mixture was stirred
at 55 °C in an oil bath for 50 min, and then 1,3,5-trimethoxybenzene
(117 mg, 0.69 mmol) was added. The reaction mixture was stirred at
55 °C in an oil bath for another 15 min and was concentrated under
reduced pressure. The obtained residue was washed with Et2O (4 × 1
mL) and dried in vacuo to give a crude compound. The crude
compound was purified by column chromatography (SiO2, CH2Cl2/
MeOH, 20/1→ 10/1) to afford 185 mg (0.25 mmol, 40%) of
iodonium salt 11 as a pale purple solid; IR (neat) 1583, 1457, 1413,
1388, 1346, 1262, 1208, 1162, 1126, 1032, 1009, 868, 814, 755, 680,
566 cm−1; 1H NMR (400 MHz, CD3OD) δ 7.68 (d, J = 8.3 Hz, 2H),
7.62 (d, J = 1.8 Hz, 1H), 7.33 (d, J = 1.8 Hz, 1H), 7.21 (d, J = 8.3 Hz,
2H), 6.36 (s, 2H), 3.92 (s, 6H), 3.90 (s, 3H), 3.87 (s, 3H), 2.36 (s,
3H); 13C{1H} NMR (100 MHz, CD3OD) δ 167.4, 160.7, 160.2,
142.4, 140.6, 129.7, 128.9, 128.8 (2C), 127.9, 125.9 (2C), 114.7,
110.4, 91.6 (2C), 84.6, 57.4, 56.5 (2C), 55.6, 20.4; HRESIMS m/z
556.8458 [M]+ (calcd for C16H1679Br2IO4 556.8455).
1
1213, 1036, 924, 892, 804 cm−1; H NMR (400 MHz, CD3OD) δ
7.78 (d, J = 2.3 Hz, 1H), 7.36 (dd, J = 2.3, 9.1 Hz, 1H), 7.27 (d, J =
2.3 Hz, 1H), 7.07 (d, J = 2.3 Hz, 1H), 6.96 (s, 1H), 6.66 (d, J = 9.1
Hz, 1H); 13C{1H} NMR (100 MHz, CD3OD) δ 153.9, 153.0, 151.0,
142.1, 139.3, 136.7, 132.4, 126.9, 123.8, 123.0, 121.6, 120.8, 120.5,
119.8, 118.9, 117.9, 116.1, 113.4; HRESIMS m/z 822.4626 [M-H]−
(calcd for C18H679Br7O3 822.4606).
Bromoiesol sulfate (1). To a stirred solution of sulfur trioxide
pyridine complex (57 mg, 0.36 mmol) in pyridine (500 μL) was
added bromoiesol A (3, 10 mg, 0.012 mmol) at room temperature.
The reaction mixture was stirred at 45 °C in an oil bath for 1 h, and
then 5 mL of 1 M KOH aq. was added at 0 °C. The resulting mixture
was applied on a ODS plug (5.0 g) and washed with H2O and 40%
aqueous MeOH to remove potassium sulfate and unreacted reagents.
The elution with 80% aqueous MeOH afforded bromoiesol A sulfate
(1, 13 mg, 0.014 mmol, quant.) as a pale white solid; IR (neat) 1464,
1
1427, 1284, 1260, 1055, 927 cm−1; H NMR (400 MHz, CD3OD)
δ7.88 (d, J = 2.3 Hz, 1H), 7.74 (d, J = 2.3 Hz, 1H), 7.62 (d, J = 2.3
Hz, 1H), 7.43 (dd, J = 2.3, 9.1 Hz, 1H), 7.00 (s, 1H), 6.83 (d, J = 9.1
Hz, 1H); 13C{1H} NMR (100 MHz, CD3OD) δ 152.7, 149.9, 147.2,
140.8, 135.4, 132.1, 130.6, 124.2, 122.5, 122.1, 121.0, 119.4, 119.4,
119.1, 119.0, 117.3, 115.0, 111.9; HRESIMS m/z 902.4178 [M-H]−
(calcd for C18H679Br7O6S 902.4174).
1,2,3-Tribromo-4-(2,4-dibromophenoxy)-5-methoxybenzene
(12). To a stirred solution of t-BuOK (70 mg, 0.63 mmol) in dry THF
(3 mL) at 0 °C was added phenol 6 (206 mg, 0.57 mmol). The
reaction solution was stirred at 0 °C for 15 min. After addition of the
iodonium salt 9 (397 mg, 0.57 mmol), the reaction mixture was
further stirred at 40 °C in an oil bath for 47 h and was diluted with
F
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Cell Growth Analysis. HeLa cells were cultured at 37 °C with 5%
CO2 in DMEM (Nissui, Japan) supplemented with 10% heat-
inactivated FBS, 100 units/mL penicillin, 100 μg/mL streptomycin,
0.25 μg/mL amphotericin, 300 μg/mL L-glutamine, and 2.25 mg/mL
NaHCO3. HeLa cells were seeded at 5 × 103 cells/well in 96-well
plates (Iwaki, Japan) and cultured overnight. Various concentrations
of compounds were then added, and cells were incubated for 72 h.
Cell proliferation was measured by the MTT assay. The assay was
performed in triplicate.
Tomoyoshi Nozaki − Department of Biomedical Chemistry,
Graduate School of Medicine, The University of Tokyo,
Tokyo 113-0033, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.1c01214
Notes
The authors declare no competing financial interest.
In Vitro Antitrypansomal Assay. The Trypanosoma brucei
rhodesiense strain IL-150132 was cultured at 37 °C under a humidified
5% CO2 atmosphere in HMI-9 medium33 supplemented with 10%
heat-inactivated fetal bovine serum (FBS). For in vitro studies,
compounds were dissolved in DMSO and diluted in culture medium
prior to being assayed. The maximum DMSO concentration in the in
vitro assays was 1%. The compounds were tested in an AlamarBlue
serial drug dilution assay34 to determine the 50% inhibitory
concentrations (IC50). Serial drug dilutions were prepared in 96-
well microtiter plates containing the culture medium, and wells were
inoculated with 2.0 × 104 cells/mL T. b. rhodesiense IL-1501 parasites.
Cultures were incubated for 69 h at 37 °C under a humidified 5%
CO2 atmosphere. After this time, 10 μL of resazurin (12.5 mg
resazurin [Sigma] dissolved in 100 mL of phosphate-buffered saline)
was added to each well. The plates were incubated for an additional 3
h. The plates were read in a SpectraMax Gemini XS microplate
fluorescence scanner (Molecular Devices) using an excitation
wavelength of 536 nm and an emission wavelength of 588 nm. The
assay was performed in duplicate.
ACKNOWLEDGMENTS
■
This work was supported by JSPS KAKENHI Grant Nos.
18K14346 and 20H02870, and Keio University Academic
Development Funds.
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
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AUTHOR INFORMATION
Corresponding Author
Kiyotake Suenaga − Department of Chemistry, Keio
University, Yokohama, Kanagawa 223-8522, Japan;
orcid.org/0000-0001-5343-5890; Email: suenaga@
chem.keio.ac.jp
■
Authors
Akira Ebihara − Department of Chemistry, Keio University,
Yokohama, Kanagawa 223-8522, Japan
Arihiro Iwasaki − Department of Chemistry, Keio University,
Yokohama, Kanagawa 223-8522, Japan; orcid.org/0000-
0002-3775-5066
Youhei Miura − Department of Applied Chemistry, Keio
University, Yokohama, Kanagawa 223-8522, Japan;
orcid.org/0000-0001-5003-0687
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Ghulam Jeelani − Department of Biomedical Chemistry,
Graduate School of Medicine, The University of Tokyo,
Tokyo 113-0033, Japan
G
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