Bromophenols as Candida albicans isocitrate lyase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.09.015

A new series of bromophenols was synthesized by reactions of corresponding phenol analogs with bromine. The synthesized compounds were tested for inhibitory activity against isocitrate lyase (ICL) of Candida albicans and antimicrobial activity against Gram-positive and, Gram-negative bacteria and fungi. Among the synthesized bromophenols, bis(3-bromo-4,5-dihydroxyphenyl) methanone (11) and (3-bromo-4,5-dihydroxyphenyl)(2,3-dibromo-4,5- dihydroxyphenyl)methanone (12) displayed potent inhibitory activities against ICL, showing a stronger inhibitory effects than were found with natural bromophenol 1. The preliminary structure-activity relationships were investigated in order to determine the essential structural requirements for the inhibitory activities of these compounds against ICL of C. albicans.

Full text of DOI:10.1016/j.bmcl.2010.09.015

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6644–6648
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Bromophenols as Candida albicans isocitrate lyase inhibitors
Ki-Bong Oh a, Heung Bae Jeon b, Yu-Ri Han c, Yeon-Ju Lee c, Jiyoung Park a, So-Hyoung Lee a, Dongsik Yang b,
Mihyun Kwon b, Jongheon Shin d, Hyi-Seung Lee c,
⇑
a Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea
b Department of Chemistry, Kwangwoon University, Seoul 139-701, Republic of Korea
c Marine Natural Products Laboratory, Korea Ocean Research & Development Institute, Ansan PO Box 29, Seoul 425-600, Republic of Korea
d Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2010
Revised 20 August 2010
Accepted 3 September 2010
Available online 21 September 2010
A new series of bromophenols was synthesized by reactions of corresponding phenol analogs with bro-
mine. The synthesized compounds were tested for inhibitory activity against isocitrate lyase (ICL) of
Candida albicans and antimicrobial activity against Gram-positive and, Gram-negative bacteria and fungi.
Among the synthesized bromophenols, bis(3-bromo-4,5-dihydroxyphenyl)methanone (11) and
(3-bromo-4,5-dihydroxyphenyl)(2,3-dibromo-4,5-dihydroxyphenyl)methanone (12) displayed potent
inhibitory activities against ICL, showing a stronger inhibitory effects than were found with natural bro-
mophenol 1. The preliminary structure–activity relationships were investigated in order to determine the
essential structural requirements for the inhibitory activities of these compounds against ICL of
C. albicans.
Keywords:
Bromophenols
Synthesis
Isocitrate lyase inhibition
Antibacterial activity
Antifungal activity
Ó 2010 Elsevier Ltd. All rights reserved.
The glyoxylate cycle serves to bypass the CO2-generating steps
of the tricarboxylic acid (TCA) cycle and facilitates the net assimi-
lation of carbon from C2 compounds, allowing microorganisms to
replenish the pool of TCA cycle intermediates necessary for gluco-
neogenesis and other biosynthetic processes.1 Enzymes unique to
this route include isocitrate lyase (ICL) and malate synthase (MS).
The glyoxylate cycle is widespread and well documented in ar-
chaea, bacteria, protists, plants, fungi, and nematodes. Genetic reg-
ulation of the glyoxylate cycle during bacterial growth on acetate
has been reviewed and in the last several years it has become evi-
dent that this pathway is important in fungal and bacterial
pathogenesis.2,3
The role of the glyoxylate cycle in microbial virulence has been
reported in many pathogens including Candida albicans. The genes
of the glyoxylate cycle are strongly induced when C. albicans is
phagocytosed by macrophage.3 The inside environment of phago-
lysosome, abundant in carbon sources such as fatty acids or their
breakdown products allows C. albicans to utilize the enzymes of
the glyoxylate cycle and permits the use of C2 carbon sources.
The C. albicans mutant strain lacking the glyoxylate cycle enzyme
ICL is markedly less virulent in a mouse model of systemic candi-
diasis and less persistent in internal organs than the wild-type
strain.3,4 Since expression of glyoxylate cycle genes is detected dur-
ing specific stages of the interaction between host and pathogen in
a variety of human-pathogenic bacteria and fungi, the develop-
ment of specific inhibitors against ICL and MS is an attractive
prospect.
Several inhibitors of ICL have been identified, including 3-nitro-
propionate,5 3-bromopyruvate,6 3-phosphoglycerate,7 mycenon,8
oxalate,9 and itaconate.9 However, these inhibitor are not pharma-
cologically suitable for testing in vivo because of their toxicity and
low activity. The development of selective ICL inhibitors with suit-
able pharmacological properties would open the door to testing in
animal models to explore the role of the glyoxylate cycle in disease
and serve as proof of concept experiments to evaluate the thera-
peutic potential of ICL inhibition. As a result of our efforts to dis-
cover ICL inhibitors, we recently reported on the isolation and
evaluation of ICL inhibitory activities of sesterterpene sulfates10
from marine sponges.
In the course of our search for novel natural products with po-
tent antimicrobial activities from marine sources, we identified
bromophenols 1 from red alga Odonthalia corymbifera that showed
promising antibacterial activities.11 Further experiments demon-
strated that compound 1 exhibited potent isocitrate lyase (ICL)
inhibitory activity and protected rice plants from rice blast fungus
Magnaporthe grisea infection, whereas a synthetic bromophenol 2
showed antibacterial effects against Gram-negative and Gram-po-
sitive bacteria.12 Furthermore we previously reported preliminary
structure–activity relationships associated with halogenated deriv-
atives of bis(3,4-dihydroxydiphenyl)methane as a novel class of
antimicrobial agents (Fig. 1).12,13
⇑
Corresponding author. Tel.: +82 31 400 6179; fax: +82 31 400 6170.
E-mail address: hslee@kordi.re.kr (H.-S. Lee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.015

K.-B. Oh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6644–6648
6645
and bis(2-bromo-4,5-dihydroxyphenyl) ether (6) were also pre-
pared from the reaction15 of compound 4 with 2 equiv of bromine
in acetic acid: CH2Cl2 (1:1) yielding 20% and 56% yields,
respectively.
Br
Br
Br
Br
Br
Br
HO
OH
HO
OH
The second set of bromophenols, 11 and 12, bearing a carbonyl
group between two phenyl rings, was prepared following a se-
quence of reactions similar to the first one described. The treat-
ment of 4-bromo-1,2-(methylenedioxy)benzene with the mixture
of n-butyllithium and piperonal afforded a 64% yield of compound
8, which was reacted with PDC to form dibenzo[d][1,3]dioxol-5-
ylmethanone (9) with a 95% yield. Bis(3,4-dihydroxyphenyl)meth-
anone (10)16 was prepared from compound 9 by deprotection and
then treated with 2 equiv bromine furnished in a 50% of bis(3-bro-
mo-4,5-dihydroxyphenyl)methanone (11). And the reaction of
compound 10 with excess bromine yielded 47% of (3-bromo-4,5-
dihydroxyphenyl)(2,3-dibromo-4,5-dihydroxyphenyl)methanone
(12)16 (Scheme 2).
The synthesis of a third set, bromophenol 16 and 17, with an
ethylene between two phenyl rings, involved subjecting piperonal
to a mixture of zinc and TiCl4 in tetrahydrofuran to obtain coupling
compound 13 with a 53% yield.17 Compound 13 was reduced under
hydrogen in the presence of Pd/C to give compound 14 with an 81%
yield. When compound 14 was subjected to the same reaction se-
quence (deprotection and bromination) used for the synthesis of
compound 12, the corresponding compounds of 2,20-dibromo-
4,40,5,50-tetrahydroxybibenzyl (16) and 2,20,3-tribromo-4,40,5,50-
tetrahydroxybibenzyl (17) were prepared18 via compound 1519
(Scheme 3).
OH
OH
OH
OH
1
2
Figure 1. Bioactive bromophenols.
To understand more about the activities of bromophenols,
derivatives of compound 1 were prepared and their antimicrobial
activities were examined. The promising results of experiments
with certain derivatives prompted us to further investigate these
compounds. A variety of bis-bromophenols with different linkers
such as oxygen, a carbonyl group, ethylene, or oxybis(methylene)
were prepared. Most derivatives were synthesized in the lab by a
simple halogenation process as a mixture of multiple products
with different degrees of substitutions, which were then separated
using HPLC techniques.
The synthesis of bis(2,3-dibromo-4,5-dihydroxyphenyl) ether
(7) via compound 4, which has an electron-donating ether linkage
between two phenols, is illustrated in Scheme 1. Compound 3 was
obtained by the sequential treatment of sesamol and 4-bromo-1,2-
(methylenedioxy)benzene in the presence of cesium carbonate,
copper bromide, and 2-oxocyclohexane carboxylate for a 62%
yield.14 Subsequently, deprotection of the two methylene groups
in 3 with BBr3 yielded 90% 4,40-oxydibenzene-1,2-diol (4).15 The
treatment of phenolic compound 4 with excess bromine in acetic
acid resulted in a 67% conversion to bromophenol 7.15 Meanwhile,
(2-bromo-4,5-dihydroxyphenyl)(3,4-dihydroxyphenyl) ether (5)
The final set of bromophenols, 20 and 21, was prepared follow-
ing a reaction sequence similar to the first one. Compound 18 was
prepared by the treatment of 5-(bromomethyl)benzo[d][1,3]diox-
OH
Br
O
O
a
b
+
O
HO
OH
O
O
O
O
O
O
O
OH
OH
4
3
c
d
Br
Br
Br
Br
Br
O
O
Br
O
Br
+
HO
OH HO
OH
HO
OH
OH
OH
OH
OH
OH
OH
5
6
7
Scheme 1. Reagents and conditions: (a) CuBr, Cs2CO3, oxocyclohexane carboxylate, DMSO; (b) BBr3, CH2Cl2, rt; (c) Br2 (2 equiv), CH2Cl2 + AcOH, rt; (d) excess Br2, AcOH, 50 °C.
O
O
OH
O
Br
H
a
b
c
+
HO
OH
O
O
O
O
O
d
O
O
O
O
O
O
O
OH
Br
OH
OH
10
8
9
e
O
O
Br
Br
OH
Br
Br
HO
HO
OH
OH
OH
OH
11
12
Scheme 2. Reagents and conditions: (a) n-BuLi, THF, rt; (b) PDC, CH2Cl2, rt; (c) BBr3, CH2Cl2, rt; (d) excess Br2, AcOH, rt; (e) excess Br2, AcOH, 50 °C.

6646
K.-B. Oh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6644–6648
O
O
O
O
O
H
a
b
c
O
O
O
O
O
O
+
14
13
OH
Br
OH
Br
OH
OH
OH
OH
Br
Br
Br
d
HO
HO
HO
OH
OH
OH
16
17
15
Scheme 3. Reagents and conditions: (a) TiCl4, Zn, THF, reflux; (b) H2, Pd/C, EtOAc/EtOH; (c) BBr3, CH2Cl2, rt; (d) excess Br2, CH2Cl2 + AcOH, rt.
OH Br
+
O
a
b
O
O
O
O
O
O
O
O
18
Br
Br
Br
OH
Br
Br
HO
Br
Br
O
c
O
O
+
HO
OH
OH HO
OH
OH
OH
OH
OH
OH
19
20
21
Scheme 4. Reagents and conditions: (a) NaH, THF, rt; (b) BBr3, CH2Cl2, rt; (c) excess Br2, AcOH, rt.
Table 1
ole in the presence of piperonyl alcohol and NaH in THF. The next
deprotection and bromination reactions, as used previously in
Scheme 1, gave the corresponding bromophenols, (2-bromo-4,5-
dihydroxybenzyl)(2,3-dibromo-4,5-dihydroxybenzyl) ether (20)
and bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (21) via com-
pound 1920 (Scheme 4). Compound 21, which had been reported
as a natural product from red alga O. corymbifera,21 was synthe-
sized for the first time.
The cloning, expression, and purification of ICL from the geno-
mic DNA of C. albicans (ATCC 10231) were carried out as described
previously.9 The eleven bromophenols 1, 2, 5–7, 11, 12, 16, 17, 20,
21 and four bisphenols 4, 10, 15, 19 were evaluated for their inhib-
itory activities against ICL of C. albicans according to a previously
documented procedure.9,22,23 The inhibitory potencies of the tested
compounds, expressed as IC50 values, are shown in Table 1 and are
compared to that of a known ICL inhibitor, 3-nitropropinate.24 As
expected, the synthesized bromophenols did significantly affect
C. albicans ICL. The most active compound was compound 12
Inhibitory effect of synthetic compounds on the activity of ICL
enzyme
Compound
ICL IC50 (lM) (lg/ml)
1
2
4
5
6
7
10
11
12
15
16
17
19
20
21
18.44 (10.1)
46.15 (18.0)
>100 (61.23)
68.19 (21.35)
30.88 (12.10)
11.63 (6.39)
22.58 (5.56)
8.89 (3.57)
2.65 (1.27)
58.47 (14.40)
21.21 (8.57)
14.77 (7.13)
(>100)
15.61 (7.74)
28.06 (16.1)
50.70 (6.0)
NDb
3-Nitropropionatea
Amphotericin Bb
(IC50: 2.65
than 3-nitropropinate (IC50: 50.7
tion of a carbonyl group between two phenols resulted in com-
l
M) possessing an activity nearly 20 times stronger
a
3-Nitropropinate and amphotericin B inhibitors of ICL and
l
M). Interestingly, the introduc-
fungus, respectively, were used as positive controls.
b
ND, not determined.
pounds 11 and 12 which displayed nearly 2–7 times higher
activity than the natural compound 1 (IC50: 18.44 lM). The reason
for this increased activity is not clear at this moment. It might be
due to the changes in electron density of the phenyl ring caused
by the electron-withdrawing properties of carbonyl groups, or it
could be due to formation of an additional binding site between
the carbonyl group of the compounds and enzyme. In contrast,
compounds 5–7, bearing an ether linkage between two phenols,
an oxybis(methylene) group instead of methylene in bromophe-
nols were also investigated. However, the lengths of linkers had lit-
tle effect on C. albicans ICL inhibition activities.
In the current series, an increase of the number of Br atom in
bromphenols resulted in an increase in activity. Concerning the
number of bromines, compounds 1, 7, 11, 12, 17, 20, and 21, bear-
ing three or four bromines, were more active than the others. The
reason for this increase in activity remains unclear. To our surprise,
were found to be similar or less active (IC50: 11.63–68.19
lM) as
compared to 1. The effects of the substitution of an ethylene or

K.-B. Oh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6644–6648
6647
Table 2
Antibacterial activity
Compound
MIC (lg/ml)
S. aureusa
B. subtilisa
M. luteusa
P. vulgarisa
S. typhimuriuma
1b
25
25
50
100
100
50
25
25
>100
>100
100
25
25
50
25
>100
>100
100
25
50
25
100
100
100
50
2c
12.5
100
100
50
4
5
6
7
50
10
11
12
15
>100
>100
100
50
>100
100
50
100
50
>100
100
50
100
50
>100
>100
100
100
50
>100
>100
100
100
50
16
50
17
19
20
21
100
>100
>100
>100
1.56
50
100
>100
>100
>100
1.56
100
>100
>100
>100
3.12
100
>100
>100
>100
3.12
>100
>100
>100
1.56
Ampicillin
a
Microorganisms: Staphylococcus aureus ATCC 6538p; Bacillus subtilis ATCC 6633; Micrococcus luteus IFO 12708; Proteus vulgaris ATCC 3851; Salmonella typhimurium ATCC
14028.
b
c
Published results.13
Published results.12
compounds with no halogen substituents (10, and 15) also showed
potent ICL inhibition activities. In particular, compound 10, bearing
carbonyl groups, was more effective than 3-nitropropionate, with
activities against Gram-positive and Gram-negative bacteria with
minimum inhibitory concentration (MIC) values in the range of
25–50 lg/ml, as shown in comparison to ampicillin. In an anti-
an IC50 value of 5.56
l
g/mL. Activities obtained with compound
fungal activity assay using medically important pathogenic fungi,
10 can be regarded as more interesting results considering that
the brominated derivative 12 might cause possible concerns
regarding toxicities.
most of the synthesized bromophenols, except compound 7 and
16, were inactive at 100 l
g/ml. In our previous Letters,12,13 natural
bromophenol 1 exhibited inhibitory activity against A. fumigatus, T.
The in vitro antimicrobial activities of the nine bromophenols
5–7, 11, 12, 16, 17, 20, 21 and four bisphenols 4, 10, 15, 19 were
assessed against three representative Gram-positive bacteria
including Staphylococcus aureus (ATCC 6538p), Bacillus subtilis
(ATCC 6633), and Micrococcus luteus (IFO 12708), two Gram-nega-
tive bacteria, Proteus vulgaris (ATCC 3851) and Salmonella typhimu-
rium (ATCC 14028), and four fungi, C. albicans (ATCC 10231),
Aspergillus fumigatus (HIC6094), Trichophyton rubrum (IFO 9185),
and T. mentagrophytes (IFO 40996).25,26
rubrum, and T. mentagrophytes with MIC values in the range of
1.56–12.5 lg/ml. These results revealed that the methylene group
in bromophenol was important for antimicrobial activity.
In conclusion, a new series of bromophenols was synthesized and
their inhibitory activities against isocitrate lyase of C. albicans were
investigated. Among the synthesized bromophenols, (3-bromo-
4,5-dihydroxyphenyl)(2,3-dibromo-4,5-dihydroxyphenyl)metha-
none (12) displayed a potent inhibitory activity against isocitrate
lyase (ICL) of C. albicans, which showed a much stronger inhibitory
effect than did natural bromophenol 1. During the investigation of
bromophenols, bis(3,4-dihydroxyphenyl)methanone (10), which
showed very high activity despite its lack of halide substituents,
was discovered as a new and promising lead compound for the
development of potent ICL inhibitors. Investigations into this com-
pound are already underway. Since the enzymes of the glyoxylate
cycle are not foundin mammals, the synthesized bromophenol com-
pounds tested in this study are good starting candidates for ICL
inhibitor design.
The minimum inhibitory concentrations (MIC) of the com-
pounds are displayed in Tables 2 and 3. Among the bromophenols
tested, compounds 7 and 16 exhibited only weak inhibitory
Table 3
Antifungal activity
Compound
MIC (lg/ml)
C. albicansa A. fumigatusa T. rubruma T. mentagrophytesa
1b
2c
4
5
6
1.56
>100
>100
>100
>100
25
>100
>100
100
>100
50
100
>100
>100
>100
0.78
>100
>100
>100
>100
25
>100
>100
>100
>100
50
>100
>100
>100
>100
3.12
1.56
>100
>100
>100
>100
12.5
>100
>100
>100
>100
50
>100
>100
>100
>100
3.12
1.56
50
>100
>100
>100
25
>100
>100
>100
100
Acknowledgments
This work was supported by the grant of Ministry of Land,
Transport and Maritime Affairs, Republic of Korea (PM55420) and
a grant of the Korea Healthcare Technology R&D Project, Ministry
of Health and Welfare, Republic of Korea (A080051). H.B.J. also
thanks to the Research Grant of Kwangwoon University in 2010.
7
10
11
12
15
16
17
19
20
21
100
References and notes
>100
>100
>100
>100
3.12
1. Dunn, M. F.; Ramirez-Trujillo, J. A.; Hernandez-Lucas, I. Microbiology 2009, 155,
3166.
2. Vereecke, D.; Cornelis, K.; Temmerman, W.; Jaziri, M.; Van Montagu, M.;
Holsters, M.; Goethals, K. J. Bacteriol. 2002, 184, 1112.
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6. Ko, Y. H.; McFadden, B. A. Arch. Biochem. Biophys. 1990, 278, 373.
Amphotericin B 6.25
a
Microorganisms: Candida albicans ATCC 10231; Aspergillus fumigatus HIC6094;
Trichophyton rubrum IFO 9185; Trichophyton mentagrophytes IFO 40996.
Published results.13
b
Published results.12
c

6648
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7. Ko, Y. H.; Vanni, P.; McFadden, B. A. Arch. Biochem. Biophys. 1989, 274, 155.
8. Hautzel, R.; Anke, H.; Sheldrik, W. S. J. Antibiot. 1990, 43, 1240.
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Chem. Lett. 2008, 18, 5377.
11. Lee, H.-S.; Lee, T.-H.; Lee, J. H.; Chae, C.-S.; Chung, S.-C.; Shin, D.-S.; Shin, J.; Oh,
K.-B. J. Argic. Food Chem. 2007, 55, 6923.
12. Oh, K.-B.; Lee, J. H.; Lee, J. W.; Yoon, K.-M.; Chung, S.-C.; Jeon, H. B.; Shin, J.; Lee,
H.-S. Bioorg. Med. Chem. Lett. 2009, 19, 945.
(3-Bromo-4,5-dihydroxyphenyl)(2,3-dibromo-4,5-dihydroxyphenyl)methanone
(12): 1H NMR (CD3OD, 500 MHz) d 6.99 (1H, s, H-60), 7.17 (1H, s, H-6), 7.36 (1H,
s, H-2); 13C NMR (CD3OD, 125 MHz) d 109.7, 110.4, 114.7, 116.5, 124.1, 128.1,
130.0, 134.2, 145.8, 146.9, 147.1, 150.4, 194.3; ESI (ꢁ) m/z 480.8 (MꢁH)ꢁ.
17. Bringmann, G.; Pabst, T.; Henschel, P.; Michel, M. Tetrahedron 2001, 57, 1269.
18. The reaction of compound 1519 (21 mg, 85
lmol) with bromine (40 ll) yielded
5 mg (15%) and 9 mg (22%) of bromophenols 16 and 17, respectively.
2,20-Dibromo-4,40,5,50-tetrahydroxybibenzyl (16): mp 205–206 °C (dec.); 1H
NMR (CD3OD, 500 MHz) d 2.86 (4H, m, H-7, H-70), 6.61 (2H, s, H-6, H-60),
6.91 (2H, s, H-3, H-30); 13C NMR (CD3OD, 125 MHz) d 146.0, 145.8, 132.8, 120.0,
118.1, 113.3, 37.2; ESI (ꢁ) m/z 402.9 (MꢁH)ꢁ.
13. Oh, K.-B.; Lee, J. H.; Chung, S.-C.; Shin, J.; Shin, H. J.; Kim, H.-K.; Lee, H.-S. Bioorg.
Med. Chem. Lett. 2008, 18, 104.
14. Lv, X.; Bao, W. J. Org. Chem. 2007, 72, 3863.
2,20,3-Tribromo-4,40,5,50-tetrahydroxybibenzyl (17): mp 220–225 °C (dec.); 1H
NMR (CD3OD, 500 MHz) d 2.86 (4H, m, H-7-70), 6.73 (1H, s, H-6), 6.91 (1H, s, H-
60), 6.98 (1H, s, H-30); 13C NMR (CD3OD, 125 MHz) d 35.3, 38.2, 113.4, 113.8,
117.9, 118.9, 120.0, 131.7, 132.8, 145.8, 146.1; ESI (ꢁ) m/z 480.9 (MꢁH)ꢁ.
19. Nishimura, N.; Kobayashi, K. Angew. Chem., Int. Ed. 2008, 47, 6255.
15. Experimental: The 1D and 2D NMR spectra were obtained at 500 and 125 MHz
for 1H and 13C, respectively, on
a Varian UNITY 500 spectrometer in
chloroform-d, methanol-d4 and acetone-d6 with solvent peaks as references.
Mass spectra were recorded on a ThermoFinnigan Surveyor MSQ spectrometer.
Column chromatography was performed with RP-18 reversed-phase silica gel
20. The reaction of 26 mg (0.10 mmol) of 19 with bromine (40 ll) yielded 8 mg
(43–60
General procedure:
0.11 mmol) of and bromine (12
l
m).
(16%) and 26 mg (45%) of bromophenols 20 and 21,21 respectively.
Bis(3,4-dihydroxybenzyl) ether (19): 1H NMR (CD3OD, 500 MHz) d 6.77–6.53 (m,
6H), 4.28 (s, 4H); 13C NMR (CD3OD, 125 MHz) d 146.2, 146.1, 130.7, 121.1,
116.5, 116.1, 75.7; ESI (ꢁ) m/z 261.1 (MꢁH)ꢁ.
A
mixture of 4,40-oxydibenzene-1,2-diol (4) (26 mg,
l, 0.23 mmol) in a mixture of acetic acid
l
and dichloromethane (2 ml, 1:1) was stirred at room temperature for 1 h. The
excess of bromine was removed by blowing with N2, and the solvent was
evaporated under reduced pressure. The crude products were purified by
reversed-phase HPLC (YMC ODS-A column, 1 cm ꢀ 25 cm, 45% aqueous
(2-Bromo-4,5-dihydroxybenzyl)(2,3-dibromo-4,5-dihydroxybenzyl) ether (20):
1
mp 155–157 °C (dec.); H NMR (CD3OD, 500 MHz) d 4.40 (4H, s, H-7, 70),
6.95 (1H, s, H-6), 7.00 (1H, s, H-60), 7.15 (1H, s, H-30); ESI (ꢁ) m/z 495.8
(MꢁH)ꢁ.
CH3OH) to give 6.9 mg (20%) and 24 mg (56%) of compounds
respectively.
5 and 6,
21. Kurihara, H.; Mitani, T.; Kawabata, J.; Takahashi, K. J. Nat. Prod. 1999, 62, 882.
22. Dixon, G. H.; Kornberg, H. L. J. Biochem. 1959, 72, 3P.
4,40-Oxydibenzene-1,2-diol (4): mp 124–126 °C; 1H NMR (CD3OD, 500 MHz) d
6.60 (2H, d, J = 8.4 Hz), 6.33 (2H, d, J = 2.4 Hz), 6.19 (2H, dd, J = 8.4, 2.4 Hz); 13
C
23. Hautzel, R.; Anke, H.; Sheldrick, W. S. J. Antibiot. 1990, 43, 1240.
24. Sharma, V.; Sharma, S.; Hoener zu Bentrup, K.; McKinney, J. D.; Russell, D. G.;
Jacobs, W. R., Jr.; Sacchettini, J. C. Nat. Struct. Biol. 2000, 7, 663.
25. Three Gram-positive bacteria (Staphylococcus aureus ATCC 6538p, Bacillus
subtilis ATCC 6633 and Micrococcus luteus IFO 12708) and two Gram-negative
bacteria (Proteus vulgaris ATCC 3851 and Salmonella typhimurium ATCC) were
used for antimicrobial activity tests. Bacteria were grown overnight in Luria
Bertani (LB) broth at 37 °C, harvested by centrifugation, and then washed twice
with sterile distilled water. Stock solutions of the series compound were
prepared in DMSO. Each stock solution was diluted with Standard method
NMR (CD3OD, 125 MHz) d 152.4, 146.9, 141.8, 116.5, 110.4, 107.5; ESI (ꢁ) m/z
233.1 (MꢁH)ꢁ.
(2-Bromo-4,5-dihydroxyphenyl)(3,4-dihydroxyphenyl) ether (5): mp 93–95 °C
(dec.); 1H NMR (CD3OD, 500 MHz) d 6.21 (1H, dd, J = 8.0, 2.6 Hz, H-6), 6.35
(1H, d, J = 2.6 Hz, H-60), 6.43 (1H, s, H-30), 6.67 (1H, d, J = 8.0 Hz, H-20), 6.95 (1H,
s, H-3); 13C NMR (CD3OD, 125 MHz) d 103.5, 106.6, 109.2, 116.4, 119.9, 120.1,
141.9, 143.6, 146.9, 147.1, 148.2, 152.4; ESI (ꢁ) m/z 311.1 (MꢁH)ꢁ.
Bis(2-bromo-4,5-dihydroxyphenyl) ether (6): mp 146–149 °C (dec.); 1H NMR
(CD3OD, 500 MHz) d 6.31 (1H, s, H-6, 60), 6.96 (1H, s, H-3, 30); 13C NMR (CD3OD,
125 MHz) d 102.5, 107.9, 120.1, 143.5, 146.9, 148.1; ESI (ꢁ) m/z 390.9 (MꢁH)ꢁ.
broth (Difco) to prepare serial twofold dilutions in the range of 100–0.78 lg/
Reacting 14 mg of 4 with 40
of compound 7.
l
l of bromine at 50 °C for 6 h yielded 22 mg (67%)
ml. Ten microliters of the broth containing approximately 105 colony-forming
units (cfu)/ml of test bacteria were added to each well of a 96-well microtiter
plate. Culture plates were incubated for 24 h at 37 °C.
Bis(2,3-dibromo-4,5-dihydroxyphenyl) ether (7): mp 144–146 °C (dec.); 1H NMR
(CD3OD, 500 MHz) d 6.37 (1H, s, H-6, 60); 13C NMR (CD3OD, 125 MHz) d 106.5,
106.8, 114.6, 142.3, 147.0, 148.5; ESI (ꢁ) m/z 548.9 (MꢁH)ꢁ.
26. Candida albicans ATCC 10231, Aspergillus fumigatus HIC 6094, Trichophyton
rubrum IFO 9185 and Trichophyton mentagrophytes IFO 40996 were used for
antifungal activity tests. C. albicans was grown for 48 h at 28 °C in YPD broth
(1% yeast extract, 2% peptone and 2% dextrose), harvested by centrifugation,
and then washed twice with sterile distilled water. A. fumigatus, T. rubrum and
T. mentagrophytes were plated in potato dextrose agar (PDA) (Difco), incubated
at 28 °C for 2 weeks. Spores were washed three times with sterile distilled
water and resuspended in distilled water to obtain an initial inoculum size of
105 spores/ml. Each test compound was dissolved in DMSO and diluted with
potato dextrose broth (Difco) to prepare serial twofold dilutions in the range of
16. The reaction of compound 10 (25 mg, 0.10 mmol) with bromine (12
0.23 mmol) yielded compound 11 (20 mg, 50%).
ll,
Bis(3,4-dihydroxyphenyl)methanone (10): mp 220–222 °C; 1H NMR (CD3OD,
500 MHz) d 7.28 (2H, d, J = 1.6), 7.20 (2H, dd, J = 8.4, 1.6 Hz) 6.88 (2H, d, J = 8.4);
13C NMR (CD3OD, 125 MHz) d 197.4, 151.3, 146.1, 131.2, 124.8, 118.1, 115.6;
ESI (ꢁ) m/z 245.1 (MꢁH)ꢁ.
Bis(3-bromo-4,5-dihydroxyphenyl)methanone (11): mp 183–185 °C (dec.); 1H
NMR (CD3OD, 500 MHz) d 7.18 (2H, s, H-6, 60), 7.38 (2H, s, H-2, 20); 13C NMR
(CD3OD, 125 MHz) d 110.1, 116.6, 127.4, 130.9, 146.9, 149.1, 194.4; ESI (ꢁ) m/z
401.8 (MꢁH)ꢁ.
100–0.8 l
g/ml. Ten microliters of the broth containing about 103 (for yeast)
and 104 (for filamentous fungi) cells/ml of test fungi was added to each well of
Reacting 10 (12 mg) with bromine (40
ll) yielded compound 12 (11 mg, 47%).
a 96-well microtiter plate. Culture plates were incubated for 48–72 h at 28 °C.