Synthesis and antimicrobial activities of halogenated bis(hydroxyphenyl)methanes

Article abstract of DOI:10.1016/j.bmcl.2008.11.089

A series of halophenols was prepared by the reaction of bis(hydroxyphenyl)methanes with effective halogenating agents such as bromine and sulfuryl chloride. One of these compounds, a biologically active halophenol-2,2′,3,3′-tetrabromo-4,4′,5,5′-tetrahydroxydiphenylmethane (1)-frequently isolated from red algae, was synthesized for the first time. Other halophenols included several novel compounds, together with known derivatives that were synthesized from the phenolic intermediates, bis(3,4-dihydroxyphenyl)methane (5) and bis(2-hydroxyphenyl)methane (14). All of the synthesized compounds were tested for antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. The preliminary structure-activity relationship was investigated in order to determine the essential structural requirements for their antimicrobial activity. Of all these halophenols, 2,2′,3,3′,6-pentabromo-4,4′,5,5′-tetrahydroxydiphenylmethane (8) was found to be the most active against Candida albicans, Aspergillus fumigatus, Trichophyton rubrum, and Trichophyton mentagrophytes while 3,3′,5,5′-tetrachloro-2,2′-dihydroxydiphenylmethane (18) exerted a powerful antibacterial effect against Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Proteus vulgaris, and Salmonella typhimurium.

Full text of DOI:10.1016/j.bmcl.2008.11.089

Bioorganic & Medicinal Chemistry Letters 19 (2009) 945–948
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antimicrobial activities of halogenated
bis(hydroxyphenyl)methanes
Ki-Bong Oh a,b, Ji Hye Lee c, Jong Wook Lee c, Kyung-Mi Yoon a, Soon-Chun Chung a,
Heung Bae Jeon d, Jongheon Shin e, Hyi-Seung Lee c,
*
a Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea
b Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea
c Marine Natural Products Laboratory, Korea Ocean Research & Development Institute, Ansan PO Box 29, Seoul 425-600, Republic of Korea
d Department of Chemistry, Kwangwoon University, Seoul 139-701, Republic of Korea
e Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-921, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of halophenols was prepared by the reaction of bis(hydroxyphenyl)methanes with effective hal-
ogenating agents such as bromine and sulfuryl chloride. One of these compounds, a biologically active
halophenol—2,20,3,30-tetrabromo-4,40,5,50-tetrahydroxydiphenylmethane (1)—frequently isolated from
red algae, was synthesized for the first time. Other halophenols included several novel compounds,
together with known derivatives that were synthesized from the phenolic intermediates, bis(3,4-dihy-
droxyphenyl)methane (5) and bis(2-hydroxyphenyl)methane (14). All of the synthesized compounds
were tested for antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. The pre-
liminary structure–activity relationship was investigated in order to determine the essential structural
requirements for their antimicrobial activity. Of all these halophenols, 2,20,3,30,6-pentabromo-4,40,5,50-
tetrahydroxydiphenylmethane (8) was found to be the most active against Candida albicans, Aspergillus
fumigatus, Trichophyton rubrum, and Trichophyton mentagrophytes while 3,30,5,50-tetrachloro-2,20-
dihydroxydiphenylmethane (18) exerted a powerful antibacterial effect against Staphylococcus aureus,
Bacillus subtilis, Micrococcus luteus, Proteus vulgaris, and Salmonella typhimurium.
Received 15 October 2008
Revised 20 November 2008
Accepted 24 November 2008
Available online 27 November 2008
Keywords:
Halophenols
Antibacterial activity
Antifungal activity
Ó 2008 Elsevier Ltd. All rights reserved.
Bromophenol compounds frequently isolated from various
marine red algae,1–5 have been reported to exhibit a wide spectrum
of pharmacological activities including antibacterial4 and antimi-
crobial5 activities. We recently reported the isolation and synthesis
of some of these bromophenol derivatives and their antimicrobial
activities.3 Among natural bromophenols, 2,20,3,30-tetrabromo-
4,40,5,50-tetrahydroxydiphenylmethane (1) was found to be the
most active against various fungi, whereas a synthetic bromo-
phenol—3,30,5,50-tetrabromo-2,20-dihydroxydiphenylmethane
(2)—showed a powerful antibacterial effect against Gram-negative
and Gram-positive bacteria (Fig. 1).
Additionally, compound 1 exhibited potent isocitrate lyase (ICL)
inhibitory activity and protected rice plants from Magnaporthe gri-
sea infection.6 Due to these interesting bioactivities, we decided to
develop a synthetic strategy which would allow us to produce not
only the natural bromophenol, but also its analogues. In this paper,
we describe the synthesis and antimicrobial activity of some hal-
ophenols and their structure–activity relationship.
Br
Br
OH
Br
OH
Br
Br
Br
Br
Br
HO
OH
OH
OH
1
2
Figure 1. Bioactive bromophenols.
In our previous study,3 we reported the synthesis of bis(3,4-
dihydroxydiphenyl)methane (5) from bromophenol 1 by a cata-
lytic hydrodehalogenation reaction, in which 5 could be utilized
as a common building block in the synthesis of 1 and its analogues.
We thus undertook the synthesis of bioactive bromophenol 1
via compound 5 as shown in Scheme 1. Compound 3 was obtained
by the sequential treatment of 4-bromo-1,2-(methylenedioxy)ben-
zene with n-butyllithium and 3,4-(methylenedioxy)benzaldehyde
with a 79% yield. Subsequently, the reductive removal of the
benzylic alcohol under standard hydrogenation conditions in the
presence of a catalytic amount of Pd(OH)2 afforded bis(3,4-methyl-
enedioxyphenyl)methane (4)7 with a 51% yield. Deprotection of
* 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 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.089

946
K.-B. Oh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 945–948
O
R
HO
OH
OH
Br
O
O
O
O
O
O
O
O
H
a
c
d
1
+
HO
3 R=OH
4 R=H
5
b
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, ꢀ78 °C to rt; (b) H2, Pd(OH)2/C, MeOH/THF, rt; (c) BBr3, CH2Cl2, rt; (d) excess Br2, AcOH, rt.
the two methylene groups in 4 with BBr3 yielded 64% of the phe-
nolic compound 5 and the treatment of compound 5 with excess
bromine in acetic acid resulted in a 57% conversion to bioactive
bromophenol 1.
In the course of our research to enhance the spectrum and po-
tency of bromophenols, we embarked on the synthesis and bioac-
tivity test of the relatively less explored halophenol derivatives of
compound 5.
Br
Br
O
a
4
O
O
O
9
Scheme 3. Reagent: (a) excess Br2, AcOH.
With this purpose, we directed our efforts to investigate the
changes of antibacterial activity resulting from the number and po-
sition of bromine atoms attached to the phenol ring. Therefore the
analogues 2-bromo-30,4,40,5-tetrahydroxydiphenylmethane (6),
2,20-dibromo-4,40,5,50-tetrahydroxydiphenylmethane (7) and
2,20,3,30,6-pentabromo-4,40,5,50-tetrahydroxydiphenylmethane (8)
were synthesized by the reaction of phenolic compound 5 with
bromine as illustrated in Scheme 2.8
In order to examine the relationship between biological activity
and free hydroxyl groups in aromatic rings, bis(2-bromo-4,5-meth-
ylenedioxyphenyl)methane (9), a form of 7 in which hydroxyl
groups are protected, was also prepared from compound 4 with
an 87% yield (Scheme 3).9
The in vitro antimicrobial activities of the new bromophenols
6–9 together with bioactive compounds 1 and 2 were assessed
against three representative Gram-positive bacteria viz. Staphylo-
coccus aureus (ATCC 6538p), Bacillus subtilis (ATCC 6633), and
Micrococcus luteus (IFC 12708), three Gram-negative bacteria viz.
Proteus vulgaris (ATCC 3851), Salmonella typhimurium (ATCC
14028), and Escherichia coli (ATCC 25922), and four fungi viz. Can-
dida albicans (ATCC 10231), Aspergillus fumigatus (HIC 6094), Trich-
ophyton rubrum (IFO 9185), and Trichophyton mentagrophytes (IFO
40996).10,11 The minimum inhibitory concentrations (MICs) of
the compounds are displayed in Tables 1 and 2. Among the three
bromophenols, the multi-brominated phenolic compounds 7 and
8 exhibited moderate inhibition activity against all tested Gram-
positive and Gram-negative organisms except E. coli, whereas com-
pounds 6 and 9 showed no antibacterial activity whatsoever (Table
1). However, the activity of compound 8 was especially interesting,
Table 1
Antibacterial activity
Compound Antibacterial activity (MIC,
lg/ml)
S. aureus B. subtilis M. luteus P. vulgaris S. typhimurium E. coli
1
25
25
25
25
25
50
2
6
7
1.56
>100
25
1.56
>100
25
1.56
100
12.5
25
>100
100
25
12.5
25
50
1.56
>100
25
12.5
>100
>100
50
3.12
>100
25
>100
>100
>100
50
>100
>100
>100
100
50
100
50
100
>100
12.5
8
25
25
25
9
>100
>100
50
12.5
25
50
25
12.5
0.78
>100
>100
50
25
25
100
25
12.5
1.56
1.56
>100
>100
50
25
25
100
25
6.25
3.12
3.12
10
11
12
13
15
16
17
18
25
25
100
25
12.5
0.78
3.12
25
12.5
0.78
1.56
Ampicillin 1.56
Microorganisms: Staphylococcus aureus ATCC 6538p; Bacillus subtilis ATCC 6633;
Micrococcus luteus IFC 12708; Proteus vulgaris ATCC 3851; Salmonella typhimurium
ATCC 14028; Escherichia coli ATCC 25922.
Table 2
Antifungal activity
Compound
Antifungal activity (MIC,
lg/ml)
C. albicans
A. fumigatus
T. rubrum
T. mentagrophytes
1
25
12.5
>100
>100
>100
3.12
>100
>100
>100
>100
50
100
50
50
>100
3.12
12.5
>100
>100
>100
1.56
>100
>100
>100
>100
50
100
50
50
>100
3.12
1.56
>100
>100
50
1.56
>100
>100
50
2
6
7
8
9
10
11
12
>100
>100
>100
6.25
>100
>100
>100
100
50
Br
Br
Br
Br
b
a
5
1
+
HO
Br
OH
25
OH
Br
OH
Br
13
3.12
12.5
6.25
12.5
12.5
3.12
8
15
50
16
50
17
25
Br
18
100
6.25
Amphotericin B
+
Microorganisms: Candida albicans ATCC 10231; Aspergillus fumigatus HIC 6094;
Trichophyton rubrum IFO 9185; Trichophyton mentagrophytes IFO 40996.
HO
OH
HO
OH
OH
OH
OH
OH
6
7
exhibiting MIC values against C. albicans, A. fumigatus, T. rubrum,
and T. mentagrophytes some twofold times lower than the refer-
ence compound, amphotericin B (Table 2). These results revealed
Scheme 2. Reagents and conditions: (a) Br2, CH2Cl2/AcOH, rt; (b) excess Br2, AcOH,
50 °C.

K.-B. Oh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 945–948
947
Antibacterial and antifungal activity in vitro testing was per-
formed on the eight chlorophenols 10–13 and 15–18 prepared as
described above. Results expressed as minimum inhibitory concen-
trations are listed in Tables 1 and 2. As can be appreciated, mono-
chlorinated compound 10 resulted to be almost inactive against all
Gram-negative, Gram-positive bacteria and fungi tested. These re-
sults were similar to those obtained with the mono-brominated
compound 6.
Cl
Cl
Cl
a
5
+
HO
OH
HO
OH
OH
OH
OH
OH
b
10
11
In terms of antibacterial activity, chlorophenol 18 exhibited
roughly twofold lower MIC values against S. aureus, B. subtilis, M. lu-
teus, P. vulgaris, and S. typhimurium as compared to the reference
compound, ampicillin. It was interesting that E. coli was resistant
towards compound 18. Conversely, the other multi-chlorinated
chlorophenols 11–13 and 17 revealed only moderate to weak activ-
ity against various bacteria with the exception of E. coli (Table 1).
In summary, a number of halophenols were prepared and
their antimicrobial activity tested against various Gram-positive,
Gram-negative bacteria and fungi. Among these, one of them—
2,20,3,30,6-pentabromo-4,40,5,50-tetrahydroxydiphenylmethane
(8)—displayed the highest activity against C. albicans, A. fumigatus,
T. rubrum, and T. mentagrophytes while another chlorophenol—
3,30,5,50-tetrachloro-2,20-dihydroxydiphenylmethane (18)—showed
a potent antibacterial effect against S. aureus, B. subtilis, M. luteus,
P. vulgaris, and S. typhimurium. By modifying the naturally occur-
ring product, bromophenol, we were thus able to obtain several
compounds which showed promising potency profiles in antimi-
crobial assays.
Cl
Cl
Cl
Cl
Cl
OH
Cl
Cl
+
HO
HO
OH
OH
OH
OH
OH
12
13
Scheme 4. Reagents: (a) SO2Cl2, AcOH/CH2Cl2; (b) excess SO2Cl2, AcOH.
OH
OH
OH
OH
OH
OH
Cl
Cl
a
+
14
15
16
b
OH
OH
OH
Cl
OH
Cl
Cl
+
Acknowledgments
Cl
Cl
Cl
This work was supported by the Korea Ocean Research & Devel-
opment Institute (PE98202), Korea Research Foundation Grant
(KRF-2004-005-F00055) and the Ministry of Land, Transport and
Maritime Affairs, Republic of Korea.
17
18
Scheme 5. Reagents: (a) SO2Cl2, AcOH/CH2Cl2; (b) excess SO2Cl2, AcOH.
References and notes
that the presence of free hydroxyl groups and two or more bromine
atoms attached to the phenol ring are important for antimicrobial
activity.
The study continued, focused now on establishing whether the
type of substituting halogen, specifically chlorine, could alter the
potency of the antimicrobial activity of the halophenols. These hal-
ophenol analogues have been synthesized as shown in Schemes 4
and 5.
1. Li, K.; Li, X.-M.; Ji, N.-Y.; Gloer, J. B.; Wang, B.-G. Org. Lett. 2008, 10, 1429.
2. Li, K.; Li, X.; Ji, N.; Wang, B. J. Nat. Prod. 2008, 71, 28.
3. 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.
4. Xu, N.; Fan, X.; Yan, X.; Li, X.; Niu, R.; Tseng, C. K. Phytochemistry 2003, 62,
1221.
5. Kurata, K.; Amiya, T. Phytochemistry 1980, 19, 141.
6. Lee, H.-S.; Lee, T.-H.; Lee, J. H.; Chae, C.-S.; Chung, S.-C.; Shin, D.-S.; Shin, J.; Oh,
K.-B. J. Agric. Food Chem. 2007, 55, 6923.
7. Berryman, K. A.; Cheng, X.-M.; Doherty, A. M.; Edmunds, J. J.; Klutchko, S. U.S.
Patent 5,658,943, 1997.
To investigate the effect on the antimicrobial activity resulting
from the different positions and number of chlorine atoms attached
to the phenol ring, several chlorinated analogues were synthesized
by reaction of 5 with sulfuryl chloride (Scheme 4). The resulting
compounds were identified as 2-chloro-30,4,40,5-tetrahydroxydi-
phenylmethane (10), 2,20-dichloro-4,40,5,50-tetrahydroxydiphenyl
methane (11), 2,20,3-trichloro-4,40,5,50-tetrahydroxydiphenylme-
thane (12), and 2,20,3,30-tetrachloro-4,40,5,50-tetrahydroxydiphenyl-
methane (13).12
In order to examine the bioactivities of analogues of the potent
antibacterial bromophenol 2, similar chlorophenols were prepared
from commercially available bis(2-hydroxyphenyl)methane (14).
Treatment of compound 14 with two equivalents of sulfuryl chlo-
ride in acetic acid:CH2Cl2 (1:1) yielded a mixture of chlorophenols,
consisting of 38% of 3-chloro-2,20-dihydroxydiphenylmethane
(15)14 and 51% of 3-chloro-20,6-dihydroxydiphenylmethane
(16).15 Similarly, two well known chlorophenols—3,30-dichloro-
6,60-dihydroxydiphenylmethane (17)16 and 3,30,5,50-tetra-
chloro-2,20-dihydroxydiphenylmethane (18)17—were prepared
from the reaction of compound 14 with excess sulfuryl chloride
in the acetic acid yielding 9% and 58%, respectively (Scheme 5).
8. 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 methanol-
d4 with solvent peaks as references. Mass spectra were recorded on
a
ThermoFinnigan Surveyor MSQ spectrometer. Column chromatography was
performed with silica gel (230–400 mesh), RP-18 reversed-phase silica gel (43–
60 lm).General procedure: A mixture of bis(3,4-dihydroxydiphenyl)methane
(5) (24 mg, 0.10 mmol) of and bromine (31 mg, 0.24 mmol) in a mixture of
acetic acid and dichloromethane (2 ml, 1:1) was stirred at room temperature
for 2 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, 40% aq
CH3OH) to give 6.5 mg (21%) and 18 mg (46%) of bromophenols 6 and 7,
respectively.
2-Bromo-30,4,40,5-tetrahydroxydiphenylmethane (6): 1H NMR (CD3OD, 500 MHz)
d 6.92 (1H, s, H-3), 6.66 (1H, d, J = 7.80 Hz, H-50), 6.57 (1H, d, J = 1.95 Hz, H-20),
6.56 (1H, s, H-6), 6.48 (1H, dd, J = 7.80, 1.95 Hz, H-60), 3.76 (2H, s, CH2); 13C
NMR (CD3OD, 125 MHz) d 146.1, 146.0, 145.6, 144.4, 133.3, 133.2, 121.2, 119.9,
118.5, 117.0, 116.2, 113.5, 41.1; APCI(ꢀ) m/z 309 (MꢀH).
2,20-Dibromo-4,40,5,50-tetrahydroxydiphenylmethane (7): 1H NMR (CD3OD,
500 MHz) d 6.97 (2H, s, H-3, H-30), 6.45 (2H, s, H-6, H-60), 3.83 (2H, s, CH2);
13C NMR (CD3OD, 125 MHz) d 146.0 (2ꢁ), 145.8 (2ꢁ), 131.6 (2ꢁ), 120.0 (2ꢁ),
118.2 (2ꢁ), 113.8 (2ꢁ), 41.3; APCI(ꢀ) m/z 387 (MꢀH).
Reacting 21 mg of 5 with 124 mg of bromine at 50 °C for 6 h yielded 22 mg
(45%) and 11 mg (19%) of bromophenols 1 and 8, respectively.
2,20,3,30,6-Pentabromo-4,40,5,50-tetrahydroxydiphenylmethane (8): 1H NMR
(CD3OD, 500 MHz) d 6.04 (1H, s, H-60), 4.37 (2H, s, CH2); 13C NMR (CD3OD,

948
K.-B. Oh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 945–948
125 MHz) d 146.3, 145.5, 145.2, 144.0, 131.9, 131.0, 118.4, 116.3, 114.6, 114.5,
114.35, 114.32, 46.6; APCI(ꢀ) m/z 626 (MꢀH).
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
9. Bis(2-bromo-4,5-methylenedioxyphenyl)methane (9): 1H NMR (CD3OD, 500 MHz)
d 7.06 (2H, s, H-3, H-30), 6.49 (2H, s, H-6, H-60), 5.95 (4H, s, 2CH2), 3.97 (2H, s,
CH2); 13C NMR (CD3OD, 125 MHz) d 149.1 (2ꢁ), 148.7 (2ꢁ), 133.3 (2ꢁ), 115.7
(2ꢁ), 113.5 (2ꢁ), 111.1 (2ꢁ), 103.2 (2ꢁ), 42.5; APCI(ꢀ) m/z 411 (MꢀH).
10. Three Gram-positive bacteria (Staphylococcus aureus ATCC 6538p, Bacillus
subtilis ATCC 6633, and Micrococcus luteus IFO 12708) and three Gram-negative
bacteria (Proteus vulgaris ATCC 3851, Salmonella typhimurium ATCC 14028, and
Escherichia coli ATCC 35218) 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 broth (Difco) to prepare serial twofold
a 96-well microtiter plate. Culture plates were incubated for 48–72 h at 28 °C.
12. The reaction of 28 mg (0.12 mmol) of
5 with 29 mg (0.21 mmol) of
sulfuryl chloride in an ice bath for 2 h yielded 15 mg (47%) and 6 mg
(17%) of chlorophenols 10 and 11,13 respectively.
2-Chloro-30,4,40,5-tetrahydroxydiphenylmethane (10): 1H NMR (CD3OD,
500 MHz) d 6.75 (1H, s, H-3), 6.66 (1H, d, J = 7.80 Hz, H-50), 6.57 (1H, d,
J = 1.95 Hz, H-20), 6.54 (1H, s, H-6), 6.48 (1H, dd, J = 7.80, 1.95 Hz, H-60), 3.75
(2H, s, CH2); 13C NMR (CD3OD, 125 MHz) d 146.1, 145.5, 145.4, 144.4, 133.3,
131.4, 124.2, 121.2, 118.4, 117.0, 116.8, 116.2, 38.6; ESI(ꢀ) 265 m/z (MꢀH)
The reaction of 33 mg (0.14 mmol) of 5 with 130 mg (0.96 mmol) of sulfuryl
chloride in an ice bath for 4 h gave 27 mg (58%) and 10 mg (19%) of
chlorophenols 12 and 13, respectively.
dilutions in the range of 100–0.78
l
g/ml. Ten microliters of the broth
2,20,3-Trichloro-4,40,5,50-tetrahydroxydiphenylmethane (12): 1H NMR (CD3OD,
500 MHz) d 6.79 (1H, s, H-30), 6.46 (1H, s, H-60), 6.42 (1H, s, H-6), 3.89 (2H, s,
CH2); 13C NMR (CD3OD, 125 MHz) d 146.0, 145.9, 145.6, 142.9, 130.7, 129.1,
124.6, 123.2, 120.9, 118.1, 117.0, 115.9, 37.1; ESI(ꢀ) m/z 334 (MꢀH).
2,20,3,30-Tetrachloro-4,40,5,50-tetrahydroxydiphenylmethane (13): 1H NMR
(CD3OD, 500 MHz) d 6.42 (2H, s, H-6, H-60), 3.95 (2H, s, CH2); 13C NMR
(CD3OD, 125 MHz) d 146.1 (2ꢁ), 143.2 (2ꢁ), 130.0 (2ꢁ), 123.3 (2ꢁ), 121.0 (2ꢁ),
115.8 (2ꢁ), 38.1; ESI(ꢀ) m/z 369 (MꢀH).
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.
11. 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
13. Inatomi, S.; Mori, S.; Nohara, T. Jpn. Kokai Tokyo Koho JP 07089888, 1995.
14. Renga, J. M.; Riley, B. K.; Ray, P. G.; Smith, M. G. U.S. Patent 4979978,
1990.
15. Katritzky, A. R.; Zhang, Z.; Lang, H.; Lan, X. J. Org. Chem. 1994, 59, 7209.
16. Commercially available 17 can be purchased from Aldrich, TCI, and so forth.
17. Commercially available 18 can be purchased from Pfaltz & Bauer, Inc.