Discovery of novel 2′,3′,4′-trihydroxy-2- phenylacetophenone derivatives as anti-gram-positive antibacterial agents

Article abstract of DOI:10.1271/bbb.80532

A number of 2′,3′,4′-trihydroxy-2-phenylacetophenone derivatives were synthesized and examined for growth inhibition of several kinds of bacteria. 2′,3′,4′-Trihydroxy-2-phenylacetophenone itself exhibited no antibacterial activity, but some of its derivat

Full text of DOI:10.1271/bbb.80532

Biosci. Biotechnol. Biochem., 73 (1), 124–128, 2009
Discovery of Novel 20,30,40-Trihydroxy-2-phenylacetophenone Derivatives
as Anti-Gram-Positive Antibacterial Agents
y
2;
Hideyuki GOTO,1 Yuji KUMADA,2 Hitoshi ASHIDA,2 and Ken-ichi YOSHIDA
1Institute for Environmental Sciences, University of Shizuoka, Suruga, Shizuoka 422-8526, Japan
2Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-5801, Japan
Received August 1, 2008; Accepted September 26, 2008; Online Publication, January 7, 2009
[doi:10.1271/bbb.80532]
A number of 20,30,40-trihydroxy-2-phenylacetophe-
none derivatives were synthesized and examined for
growth inhibition of several kinds of bacteria. 20,30,40-
Trihydroxy-2-phenylacetophenone itself exhibited no
antibacterial activity, but some of its derivatives showed
various antibacterial activities depending on functional
groups introduced on the 2-phenyl ring. Eighteen out of
24 compounds synthesized in this study appeared to
possess antibacterial activities against at least two
Gram-positive strains of Bacillus subtilis and Staph-
ylococcus aureus, 2-(biphenyl-4-yl)-20,30,40-trihydroxy-
acetophenone being the most active with LC50 of
5.8 ꢀM and 5.6 ꢀM respectively. However, none of the
synthesized compounds exhibited inhibitory effects on
Gram-negative strains, such as Escherichia coli, Proteus
mirabilis, Pseudomonas aeruginosa, and Salmonella
enterica, suggesting that anti-Gram-positive specificity
of the antibacterial compounds.
activity, yielding 18 candidates for new antibacterial
agents specific for Gram-positive bacteria.
Materials and Methods
General procedure for synthesis of 20,30,40-trihydroxy-2-phenyl-
acetophenones. Pyrogallol (y in Fig. 1, 3.9 mmol) and one of the
phenylacetic acid derivatives (a–x in Fig. 1, 3.7 mmol) were added to
BF3 OEt2 (8.8 mmol). The mixture was heated once at 120 ꢀC for
.
10 min, cooled to room temperature, and diluted with cold water. The
resulting mixture was extracted with Et2O. The organic layer was
washed with brine and sat. aq. NaHCO3, and dried over anhydrous
MgSO4. After complete evaporation of the diethyl ether, the residual
solid containing the product was subjected to reverse-liquid chroma-
tography using an ODS column eluted with AcCN–H2O (70:30, v/v).
The elluent was evaporated, and the product was crystallized in EtOH.
After filtration, the product crystals were dried under reduced pressure
to obtain purified compounds 1–24 (Fig. 1). The FAB-MS spectrum of
the synthesized compounds was recorded on a JEOL JMS-700
spectrometer. 1H (500 MHz)- and 13C (125 MHz)-NMR were per-
formed in DMSO-d6 (containing < 0.003% water) with TMS as
internal standard at 25 ꢀC on a JEOL ECA-500 spectrometer.
Compound 1, 20,30,40-trihydroxy-2-phenylacetophenone, was ob-
tained as pale violet crystals. M.p. 121–122 ꢀC. Yield: 56%. 1H-NMR
(DMSO-d6) ꢁ: 4.29 (2H, s, CH2), 6.42 (1H, d, J = 8.6 Hz, 50-H), 7.23
(2H, t, J = 7.4 Hz, 4-H), 7.29 (2H, d, J = 7.4 Hz, 2,6-H), 7.31 (2H, t,
J = 7.4 Hz, 3,5-H), 7.51 (1H, d, J = 8.6 Hz, 60-H), 12.45 (1H, s,
20-OH). 13C-NMR (DMSO-d6) ꢁ: 44.00 (CH2), 107.80 (50-C), 112.47
(10-C), 123.03 (60-C), 126.57 (4-C), 128.36 (3,5-C), 129.54 (2,6-C),
132.35 (30-C), 135.29 (1-C), 152.54 (20-C), 152.63 (40-C), 202.87
(C=O). FAB-HRMS m=z (M + H)þ calcd. for C14H13O4 245.0814,
found 245.0805.
Key words: antibacterial agent; 2-phenylacetophenone;
Gram-positive; Staphylococcus aureus
Over the past few decades, antibiotics have a history of
success in controlling morbidity due to infectious
diseases. However, as a consequence of frequent and
excessive use of antibiotics, antibiotic-resistant bacteria
have emerged and now cause severe clinical problems.1,2)
One of the most serious problems is provoked by
methicillin-resistant Staphylococcus aureus (MRSA), a
Gram-positive pathogen responsible for difficult-to-treat
infections in humans, since it is resistant to a large group
of ꢀ-lactam antibiotics. Accordingly, there is urgent and
increasing need to discover and develop novel classes of
antibacterial agents to make possible control of such
antibiotic-resistant and infectious bacteria as MRSA. To
date, numbers of natural and synthetic compounds have
been investigated for their possible antibacterial activ-
ities.3,4) 2-Phenylacetophenone (1,2-diphenylethanone,
or deoxybenzoin) derivatives are a class of potential
candidates for novel antibacterial agents, because pre-
vious reports suggest that they might have certain
chemical, medical, and biological activities, includ-
ing estrogenicity, protein tyrosine phosphatase inhib-
itory activity, and antimicrobial activity.5–7) As this
class of compounds came to receive increasing attention,
we were prompted to synthesize various derivatives to
test for the antibacterial activity. Here we report the
synthesis of 24 novel 20,30,40-trihydroxy-2-phenylaceto-
phenone derivatives with a series of substitutions on the
2-phenyl ring and the evaluation of their antibacterial
Compound 2, 20,30,40-trihydroxy-2-(2-methylphenyl) acetophenone,
was obtained as beige crystals. M.p. 120–121 ꢀC. Yield: 61%. 1H-NMR
(DMSO-d6) ꢁ: 2.18 (3H, s, 2-Me), 4.36 (2H, s, CH2), 6.45
(1H, d, J = 9.2 Hz, 50-H), 7.14 (2H, t, J = 8.0 Hz, 4,5-H), 7.15
(1H, d, J = 8.0 Hz, 6-H), 7.16 (1H, d, J = 8.0 Hz, 3-H), 7.52
(1H, d, J = 9.2 Hz, 60-H), 8.64 (1H, s, 40-OH), 10.11 (1H, s, 30-OH),
12.44 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 19.30 (2-Me), 42.06
(CH2), 107.76 (50-C), 112.78 (10-C), 122.55 (60-C), 125.68 (5-C),
126.72 (4-C), 129.80 (3-C), 130.48 (6-C), 132.34 (30-C), 134.12
(1-C), 136.80 (2-C), 152.27 (20-C), 152.52 (40-C), 202.76 (C=O). FAB-
HRMS m=z (M + H)þ calcd. for C15H15O4 259.0970, found 259.0956.
Compound 3, 20,30,40-trihydroxy-2-(3-methylphenyl) acetophenone,
was obtained as violet crystals. M.p. 127–128 ꢀC. Yield: 56%. 1H-
NMR (DMSO-d6) ꢁ: 2.27 (3H, s, 3-Me), 4.23 (2H, s, CH2), 6.42 (1H,
d, J = 9.2 Hz, 50-H), 7.04–7.09 (3H, m, 2,4,6-H), 7.19 (1H, t,
J = 7.7 Hz, 5-H), 7.50 (1H, d, J = 9.2 Hz, 60-H), 8.63 (1H, s,
40-OH), 10.12 (1H, s, 30-OH), 12.53 (1H, s, 20-OH). 13C-NMR
(DMSO-d6) ꢁ: 20.95 (3-Me), 43.92 (CH2), 107.78 (50-C), 112.46
(10-C), 123.03 (60-C), 126.54 (6-C), 127.19 (4-C), 128.25 (5-C), 130.01
(2-C), 132.33 (30-C), 135.16 (1-C), 137.40 (3-C), 152.56 (20-C), 152.61
(40-C), 202.90 (C=O). FAB-HRMS m=z (M + H)þ calcd. for
C15H15O4 259.0970, found 259.0954.
y
To whom correspondence should be addressed. Tel: +81-78-803-5862; Fax: +81-78-803-5815; E-mail: kenyoshi@kobe-u.ac.jp

2-Phenylacetophenone Derivatives as Antibacterial Agents
125
OH
OH
HO
OH
2
5
HO
a
HO
OH
3
4
2
5
+
3
4
6
O
R
6
O
R
Product
Phenylacetic acid derivatives
Pyrogallol
R
Compound
C2
H
C3
C4
C5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C6
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
a
b
c
phenylacetic acid
o -tolylacetic acid
m -tolylacetic acid
p -tolylacetic acid
mesitylacetic acid
1
2
CH3
H
H
CH3
H
H
3
CH3
H
d
e
4
CH3
H
CH3
H
5
CH(CH3)2
H
f
4-isopropylphenylacetic acid
2-hydroxyphenylacetic acid
3-hydroxyphenylacetic acid
4-methoxyphenylacetic acid
4-ethoxyphenylacetic acid
2-fluorophenylacetic acid
3-fluorophenylacetic acid
4-fluorophenylacetic acid
2-chlorophenylacetic acid
3-chlorophenylacetic acid
4-chlorophenylacetic acid
2,4-dichlorophenylacetic acid
2-bromophenylacetic acid
3-bromophenylacetic acid
4-bromophenylacetic acid
3-iodophenylacetic acid
1-naphthaleneacetic acid
2-naphthaleneacetic acid
4-biphenylacetic acid
6
OH
H
H
H
g
h
i
7
OH
H
H
8
OCH3
H
9
OCH2CH3
H
H
j
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
F
H
H
H
F
k
l
H
F
y
pyrogallol
H
H
m
n
o
p
q
r
Cl
H
H
H
H
Cl
Cl
H
H
Br
H
H
Cl
H
H
Cl
Br
H
H
H
Br
H
s
H
t
H
I
u
v
w
x
CH=CH-CH=CH
CH=CH-CH=CH
Ph
H
H
H
Fig. 1. Molecular Structure of the 20,30,40-Trihydroxy-2-phenylacetophenone Derivatives Synthesized in This Study.
.
Schematic protocol for the synthesis of compounds 1–24 is shown at the top. Reagent, (a) BF3 OEt2. Compounds 1–24 with indicated
functional groups on the 2-phenyl ring were produced from the respective phenylacetic acid derivatives (a–x) and pyrogallol (y).
Compound 4, 20,30,40-trihydroxy-2-(4-methylphenyl) acetophenone,
was obtained as pale violet crystals. M.p. 125–126 ꢀC. Yield: 55%. 1H-
NMR (DMSO-d6) ꢁ: 2.26 (3H, s, 4-Me), 4.22 (2H, s, CH2), 6.41 (1H,
d, J = 9.2 Hz, 50-H), 7.11 (2H, d, J = 8.0 Hz, 3,5-H), 7.17 (2H, d,
J = 8.0 Hz, 2,6-H), 7.49 (1H, d, J = 9.2 Hz, 60-H), 8.61 (1H, s, 40-OH),
10.10 (1H, s, 30-OH), 12.52 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ:
20.59 (4-Me), 43.62 (CH2), 107.74 (50-C), 112.39 (10-C), 123.00
(60-C), 128.92 (3,5-C), 129.29 (2,6-C), 132.16 (1-C), 132.31 (30-C),
135.58 (4-C), 152.56 (20,40-C), 203.02 (C=O). FAB-HRMS m=z
(M + H)þ calcd. for C15H15O4 259.0970, found 259.0970.
Compound 5, 20,30,40-trihydroxy-2-(2,4,6-trimethylphenyl) aceto-
phenone, was obtained as gray crystals. M.p. 166–167 ꢀC. Yield: 73%.
1H-NMR (DMSO-d6) ꢁ: 2.11 (6H, s, 2,6-Me), 2.22 (3H, s, 4-Me), 4.35
(2H, s, CH2), 6.46 (1H, d, J = 8.6 Hz, 50-H), 6.83 (2H, s, 3,5-H), 7.62
(1H, d, J = 8.6 Hz, 60-H), 8.63 (1H, s, 40-OH), 10.09 (1H, s, 30-OH),
12.41 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 19.87 (2,6-Me), 20.50
(4-Me), 38.07 (CH2), 107.73 (50-C), 112.94 (10-C), 122.22 (60-C),
128.19 (3,5-C), 129.73 (1-C), 132.34 (30-C), 135.08 (4-C), 136.71
(2,6-C), 152.06 (20-C), 152.45 (40-C), 202.65 (C=O). FAB-HRMS m=z
(M + H)þ calcd. for C17H18O4 287.1283, found 287.1289.
1H-NMR (DMSO-d6) ꢁ: 1.17 (6H, d, J = 6.9 Hz, 4-isopropy-diMe),
2.84 (1H, tt, J = 13.7, 6.9 Hz, 4-isopropy-H), 4.22 (2H, s, CH2), 6.42
(1H, d, J = 8.6 Hz, 50-H), 7.17 (1H, d, J = 8.0 Hz, 3,5-H), 7.20 (1H, d,
J = 8.0 Hz, 2,6-H), 7.50 (1H, d, J = 8.6 Hz, 60-H), 8.62 (1H, s, 40-OH),
10.11 (1H, s, 30-OH), 12.53 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ:
23.86 (4-isopropy-diMe), 33.02 (4-isopropy-C), 43.58 (CH2), 107.77
(50-C), 112.41 (10-C), 123.01 (60-C), 126.26 (3,5-C), 129.34 (2,6-C),
132.34 (30-C), 132.53 (1-C), 146.60 (4-C), 152.56 (40-C), 152.59
(20-C), 203.02 (C=O). FAB-HRMS m=z (M + H)þ calcd. for
C17H18O4 287.1283, found 287.1283.
Compound 7, 20,30,40-trihydroxy-2-(2-hydroxyphenyl) acetophe-
none, was obtained as reddish-brown crystals. M.p. 159–161 ꢀC.
Yield: 8%. 1H-NMR (DMSO-d6) ꢁ: 4.19 (2H, s, CH2), 6.41 (1H, d,
J = 9.2 Hz, 50-H), 6.74 (1H, t, J = 8.0 Hz, 5-H), 6.80 (1H, d,
J = 8.0 Hz, 3-H), 7.07 (1H, t, J = 8.0 Hz, 4-H), 7.08 (1H, d,
J = 8.0 Hz, 6-H), 7.49 (1H, d, J = 9.2 Hz, 60-H), 8.60 (1H, s,
40-OH), 9.45 (1H, s, 2-OH), 10.03 (1H, s, 30-OH), 12.54 (1H, s,
20-OH). 13C-NMR (DMSO-d6) ꢁ: 38.80 (CH2), 107.61 (50-C), 112.66
(10-C), 114.88 (3-C), 118.82 (5-C), 121.85 (1-C), 122.47 (60-C), 127.82
(4-C), 131.09 (6-C), 132.28 (30-C), 152.30 (20-C), 152.33 (40-C),
155.04 (2-C), 203.12 (C=O). FAB-HRMS m=z (M + H)þ calcd. for
C14H13O5 261.0763, found 261.0758.
Compound 6, 20,30,40-trihydroxy-2-(4-isopropylphenyl) acetophe-
none, was obtained as beige crystals. M.p. 130–131 ꢀC. Yield: 67%.

126
H. GOTO et al.
Compound 8, 20,30,40-trihydroxy-2-(3-hydroxyphenyl) acetophe-
107.86 (50-C), 112.66 (10-C), 122.30 (60-C), 127.04 (4-C), 128.69
(5-C), 128.93 (6-C), 132.37 (30-C), 132.58 (3-C), 133.56 (2-C), 133.83
(1-C), 152.08 (20-C), 152.60 (40-C), 201.02 (C=O). FAB-HRMS m=z
(Mþ) calcd. for C14H11ClO4 278.0346, found 278.0360.
none, was obtained as violet crystals. M.p. 147–148 ꢀC. Yield: 51%.
1H-NMR (DMSO-d6) ꢁ: 4.16 (2H, s, CH2), 6.41 (1H, d, J = 8.6 Hz,
50-H), 6.62 (1H, dd, J = 8.0, 1.7 Hz, 4-H), 6.68 (1H, d, J = 1.7 Hz,
2-H), 6.70 (1H, d, J = 8.0 Hz, 6-H), 7.09 (1H, t, J = 8.0 Hz, 5-H), 7.47
(1H, d, J = 8.6 Hz, 60-H), 8.62 (1H, s, 40-OH), 9.33 (1H, s, 3-OH),
10.12 (1H, s, 30-OH). 13C-NMR (DMSO-d6) ꢁ: 44.04 (CH2), 107.76
(50-C), 112.45 (10-C), 113.58 (4-C), 116.16 (2-C), 120.00 (6-C), 123.12
(60-C), 129.32 (5-C), 132.33 (30-C), 136.56 (1-C), 152.62 (20,40-C),
157.30 (3-C), 202.94 (C=O). FAB-HRMS m=z (M + H)þ calcd. for
C14H13O5 261.0763, found 261.0738.
Compound 15, 2-(3-chlorophenyl)-20,30,40-trihydroxyacetophenone,
was obtained as violet crystals. M.p. 138–139 ꢀC. Yield: 51%. 1H-
NMR (DMSO-d6) ꢁ: 4.35 (2H, s, CH2), 6.44 (1H, d, J = 9.2 Hz, 50-H),
7.25 (1H, d, J = 7.4 Hz, 6-H), 7.31 (1H, dt, J = 7.4, 1.7 Hz, 4-H), 7.35
(1H, t, J = 7.4 Hz, 5-H), 7.37 (1H, t, J = 1.7 Hz, 2-H), 7.50 (1H, d,
J = 9.2 Hz, 60-H), 8.66 (1H, s, 40-OH), 10.15 (1H, s, 30-OH), 12.36
(1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 43.37 (CH2), 107.85 (50-C),
112.50 (10-C), 122.80 (60-C), 126.53 (5-C), 128.47 (6-C), 129.59 (2-C),
130.03 (4-C), 132.37 (30-C), 132.78 (3-C), 137.68 (1-C), 152.38 (20-C),
152.69 (40-C), 202.03 (C=O). FAB-HRMS m=z (Mþ) calcd. for
C14H11ClO4 278.0346, found 278.0345.
Compound 16, 2-(4-chlorophenyl)-20,30,40-trihydroxyacetophenone,
was obtained as reddish-brown crystals. M.p. 147–148 ꢀC. Yield: 60%.
1H-NMR (DMSO-d6) ꢁ: 4.33 (2H, s, CH2), 6.44 (1H, d, J = 8.6 Hz,
50-H), 7.31 (2H, d, J = 8.6 Hz, 2,6-H), 7.38 (2H, d, J = 8.6 Hz, 3,5-H),
7.49 (1H, d, J = 8.6 Hz, 60-H), 8.65 (1H, s, 40-OH), 10.14 (1H, s,
30-OH), 12.40 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 43.18 (CH2),
107.83 (50-C), 112.47 (10-C), 122.84 (60-C), 128.19 (3,5-C), 131.30
(4-C), 131.53 (2,6-C), 132.35 (30-C), 134.24 (1-C), 152.41 (20-C),
152.66 (40-C), 202.30 (C=O). FAB-HRMS m=z (Mþ) calcd. for
Compound 9, 20,30,40-trihydroxy-2-(4-methoxyphenyl) acetophe-
none, was obtained as pale violet crystals. M.p. 131–132 ꢀC. Yield:
42%. 1H-NMR (DMSO-d6) ꢁ: 3.72 (3H, s, 4-OMe), 4.20 (2H, s, CH2),
6.41 (1H, d, J = 9.2 Hz, 50-H), 6.87 (2H, d, J = 8.6 Hz, 3,5-H), 7.20
(2H, d, J = 8.6 Hz, 2,6-H), 7.50 (1H, d, J = 9.2 Hz, 60-H), 8.64 (1H, s,
40-OH), 10.12 (1H, s, 30-OH), 12.54 (1H, s, 20-OH). 13C-NMR
(DMSO-d6) ꢁ: 43.12 (CH2), 55.01 (4-OMe), 107.76 (50-C), 112.36
(10-C), 113.81 (3,5-C), 123.01 (60-C), 127.07 (1-C), 130.51 (2,6-C),
132.35 (30-C), 152.57 (20-C), 152.58 (40-C), 158.00 (4-C), 203.23
(C=O). FAB-HRMS m=z (M + H)þ calcd. for C15H15O5 275.0919,
found 275.0945.
Compound 10, 2-(4-ethoxyphenyl)-20,30,40-trihydroxyacetophe-
none, was obtained as violet crystals. M.p. 152–153 ꢀC. Yield: 71%.
1H-NMR (DMSO-d6) ꢁ: 1.30 (3H, t, J = 6.9 Hz, 4-ethoxy-CH3), 3.98
(2H, q, J = 6.9 Hz, 4-ethoxy-CH2), 4.19 (2H, s, CH2), 6.41 (1H, d,
J = 8.6 Hz, 50-H), 6.85 (2H, d, J = 8.6 Hz, 3,5-H), 7.18 (2H, d,
J = 8.6 Hz, 2,6-H), 7.49 (1H, d, J = 8.6 Hz, 60-H), 8.61 (1H, s, 40-OH),
10.10 (1H, s, 30-OH), 12.54 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ:
14.62 (4-ethoxy-CH3), 43.09 (CH2), 62.87 (4-ethoxy-CH2), 107.73
(50-C), 112.35 (10-C), 114.27 (3,5-C), 122.97 (60-C), 126.91 (1-C),
130.44 (2,6-C), 132.32 (30-C), 152.53 (20-C), 152.56 (40-C), 157.24
(4-C), 203.19 (C=O). FAB-HRMS m=z (M + H)þ calcd. for
C16H17O5 289.1076, found 289.1079.
Compound 11, 2-(2-fluorophenyl)-20,30,40-trihydroxyacetophenone,
was obtained as violet crystals. M.p. 119–120 ꢀC. Yield: 39%. 1H-NMR
(DMSO-d6) ꢁ: 4.41 (2H, s, CH2), 6.46 (1H, d, J = 9.2 Hz, 50-H), 7.15–
7.20 (2H, m, 3,5-H), 7.31–7.35 (2H, m, 4,6-H), 7.50 (1H, d, J = 9.2 Hz,
60-H), 8.66 (1H, s, 40-OH), 10.14 (1H, s, 30-OH), 12.25 (1H, s, 20-OH).
13C-NMR (DMSO-d6) ꢁ: 38.05 (CH2), 107.85 (50-C), 112.50 (10-C),
114.95 (d, J = 21.5 Hz, 3-C), 122.33 (d, J = 15.5 Hz, 1-C), 122.46
(60-C), 124.23 (d, J = 3.6 Hz, 5-C), 128.92 (d, J = 8.3 Hz, 4-C), 132.34
(d, J = 3.5 Hz, 6-C), 132.36 (30-C), 152.15 (20-C), 152.63 (40-C),
160.76 (d, J = 243.2 Hz, 2-C), 201.10 (C=O). FAB-HRMS m=z
(M + H)þ calcd. for C14H12FO4 263.0720, found 263.0701.
Compound 12, 2-(3-fluorophenyl)-20,30,40-trihydroxyacetophenone,
was obtained as pale violet crystals. M.p. 137–138 ꢀC. Yield: 47%. 1H-
NMR (DMSO-d6) ꢁ: 4.16 (2H, s, CH2), 6.41 (1H, d, J = 8.6 Hz, 50-H),
7.06–7.09 (1H, m, 4-H), 7.11–7.15 (2H, m, 2,6-H), 7.33–7.37 (1H, m,
5-H), 7.50 (1H, d, J = 8.6 Hz, 60-H), 8.65 (1H, s, 40-OH), 10.14 (1H, s,
30-OH), 12.37 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 43.53 (CH2),
107.84 (50-C), 112.50 (10-C), 113.35 (d, J = 21.5 Hz, 4-C), 116.50
(d, J = 20.3 Hz, 2-C), 122.86 (60-C), 125.83 (d, J = 3.6 Hz, 6-C),
130.05 (d, J = 8.4 Hz, 5-C), 132.36 (30-C), 137.97 (d, J = 7.2 Hz,
1-C), 152.41 (20-C), 152.67 (40-C), 162.02 (d, J = 242.0 Hz, 3-C),
202.09 (C=O). FAB-HRMS m=z (M + H)þ calcd. for C14H12FO4
263.0720, found 263.0720.
C
14H11ClO4 278.0346, found 278.0331.
Compound 17, 2-(2,4-dichlorophenyl)-20,30,40-trihydroxyacetophe-
none, was obtained as beige crystals. M.p. 182 ꢀC decompose. Yield:
20%. 1H-NMR (DMSO-d6) ꢁ: 4.52 (2H, s, CH2), 6.46 (1H, d,
J = 8.6 Hz, 50-H), 7.44–7.42 (2H, m, 5,6-H), 7.51 (1H, d, J = 8.6 Hz,
60-H), 7.62 (1H, d, J = 1.7 Hz, 3-H), 8.67 (1H, s, 40-OH), 10.16 (1H, s,
30-OH), 12.13 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 41.90 (CH2),
107.90 (50-C), 112.62 (10-C), 122.28 (60-C), 127.17 (5-C), 128.39
(3-C), 132.31 (4-C), 132.36 (30-C), 132.82 (2-C), 133.83 (6-C), 134.86
(1-C), 152.01 (20-C), 152.66 (40-C), 200.46 (C=O). FAB-HRMS m=z
(Mþ) calcd. for C14H10Cl2O4 311.9956, found 311.9978.
Compound 18, 2-(2-bromophenyl)-20,30,40-trihydroxyacetophenone,
was obtained as violet crystals. M.p. 131–132 ꢀC. Yield: 40%. 1H-
NMR (DMSO-d6) ꢁ: 4.52 (2H, s, CH2), 6.46 (1H, d, J = 8.6 Hz, 50-H),
7.23 (1H, t, J = 7.6 Hz, 5-H), 7.34–7.40 (2H, m, 3,4-H), 7.53 (1H, d,
J = 8.6 Hz, 60-H), 7.62 (1H, d, J = 7.6 Hz, 6-H), 8.65 (1H, s, 40-OH),
10.13 (1H, s, 30-OH), 12.22 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ:
44.79 (CH2), 107.85 (50-C), 112.72 (10-C), 122.24 (60-C), 124.81 (1-C),
127.57 (4-C), 128.86 (5-C), 132.16 (6-C), 132.36 (30-C), 132.62 (3-C),
135.38 (2-C), 152.05 (20-C), 152.58 (40-C), 200.95 (C=O). FAB-
HRMS m=z (Mþ) calcd. for C14H11BrO4 321.9841, found 321.9828.
Compound 19, 2-(3-bromophenyl)-20,30,40-trihydroxyacetophenone,
was obtained as beige crystals. M.p. 134–135 ꢀC. Yield: 42%. 1H-
NMR (DMSO-d6) ꢁ: 4.35 (2H, s, CH2), 6.44 (1H, d, J = 9.2 Hz, 50-H),
7.28–7.29 (2H, m, 4,6-H), 7.43–7.46 (1H, m, 5-H), 7.49 (1H, d,
J = 9.2 Hz, 60-H), 7.51 (1H, s, 2-H), 8.65 (1H, s, 40-OH), 10.15 (1H, s,
30-OH), 12.36 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 43.32 (CH2),
107.85 (50-C), 112.50 (10-C), 121.42 (1-C), 122.80 (60-C), 128.86
(6-C), 129.41 (4-C), 130.33 (5-C), 132.37 (30-C), 132.44 (2-C), 137.96
(3-C), 152.38 (20-C), 152.69 (40-C), 202.04 (C=O). FAB-HRMS m=z
(Mþ) calcd. for C14H11BrO4 321.9841, found 321.9833.
Compound 20, 2-(4-bromophenyl)-20,30,40-trihydroxyacetophenone,
was obtained as reddish-brown crystals. M.p. 140–141 ꢀC. Yield: 64%.
1H-NMR (DMSO-d6) ꢁ: 4.31 (2H, s, CH2), 6.43 (1H, d, J = 9.2 Hz,
50-H), 7.25 (2H, d, J = 8.6 Hz, 2,6-H), 7.49 (1H, d, J = 9.2 Hz, 60-H),
7.51 (2H, d, J = 8.6 Hz, 3,5-H), 8.65 (1H, s, 40-OH), 10.14 (1H, s,
30-OH), 12.39 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 43.26 (CH2),
107.82 (50-C), 112.47 (10-C), 119.79 (1-C), 122.84 (60-C), 131.11
(3,5-C), 131.93 (2,6-C), 132.35 (30-C), 134.68 (4-C), 152.41 (20-C),
152.66 (40-C), 202.23 (C=O). FAB-HRMS m=z (Mþ) calcd. for
C14H11BrO4 321.9841, found 321.9817.
Compound 21, 20,30,40-trihydroxy-2-(3-iodophenyl) acetophenone,
was obtained as violet crystals. M.p. 147–148 ꢀC. Yield: 51%. 1H-
NMR (DMSO-d6) ꢁ: 4.29 (2H, s, CH2), 6.41 (1H, d, J = 8.6 Hz, 50-H),
7.12 (1H, t, J = 7.4 Hz, 5-H), 7.30 (1H, d, J = 7.4 Hz, 6-H), 7.47 (1H,
d, J = 8.6 Hz, 60-H), 7.61 (1H, d, J = 7.4 Hz, 4-H), 7.68 (1H, s, 2-H).
13C-NMR (DMSO-d6) ꢁ: 43.15 (CH2), 94.62 (3-C), 107.94 (50-C),
112.20 (10-C), 122.90 (60-C), 129.18 (6-C), 130.38 (5-C), 132.40
(30-C), 135.22 (4-C), 137.98 (1-C), 138.18 (2-C), 152.21 (20-C), 153.37
Compound 13, 2-(4-fluorophenyl)-20,30,40-trihydroxyacetophenone,
was obtained as violet crystals. M.p. 145–146 ꢀC. Yield: 64%. 1H-NMR
(DMSO-d6) ꢁ: 4.31 (2H, s, CH2), 6.43 (1H, d, J = 8.6 Hz, 50-H), 7.12–
7.16 (2H, m, 3,5-H), 7.30–7.33 (2H, m, 2,6-H), 7.50 (1H, d, J = 8.6 Hz,
60-H), 8.64 (1H, s, 40-OH), 10.13 (1H, s, 30-OH), 12.44 (1H, s, 20-OH).
13C-NMR (DMSO-d6) ꢁ: 42.99 (CH2), 107.79 (50-C), 112.44 (10-C),
114.98 (d, J = 20.3 Hz, 3,5-C), 122.82 (60-C), 131.32 (1-C), 131.50
(d, J = 7.2 Hz, 2,6-C), 132.34 (30-C), 152.43 (20-C), 152.61 (40-C),
161.07 (d, J = 240.9 Hz, 4-C), 202.60 (C=O). FAB-HRMS m=z
(M + H)þ calcd. for C14H12FO4 263.0720, found 263.0704.
Compound 14, 2-(2-chlorophenyl)-20,30,40-trihydroxyacetophenone,
was obtained as pale violet crystals. M.p. 133–134 ꢀC. Yield: 50%. 1H-
NMR (DMSO-d6) ꢁ: 4.51 (2H, s, CH2), 6.46 (1H, d, J = 8.6 Hz, 50-H),
7.30–7.32 (2H, m, 4,5-H), 7.38–7.40 (1H, m, 3-H), 7.44–7.46 (1H, m,
6-H), 7.52 (1H, d, J = 8.6 Hz, 60-H), 8.66 (1H, s, 40-OH), 10.14 (1H, s,
30-OH), 12.23 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 42.36 (CH2),

2-Phenylacetophenone Derivatives as Antibacterial Agents
127
(40-C), 201.83 (C=O). FAB-HRMS m=z (M + H)þ calcd. for
C14H12IO4 370.9780, found 370.9769.
above in ‘‘Materials and Methods.’’ The spectral data for
all the newly synthesized compounds were in full
agreement with the proposed structures.
Compound 22, 20,30,40-trihydroxy-2-(1-naphthyl) acetophenone,
was obtained as violet crystals. M.p. 178–179 ꢀC. Yield: 46%. 1H-
NMR (DMSO-d6) ꢁ: 4.83 (2H, s, CH2), 6.49 (1H, d, J = 9.2 Hz, 50-H),
7.43 (1H, d, J = 8.0 Hz, 2-H), 7.47 (1H, t, J = 8.0 Hz, 3-H), 7.49–7.52
(2H, m, 6,7-H), 7.67 (1H, d, J = 9.2 Hz, 60-H), 7.85 (1H, d,
J = 8.0 Hz, 4-H), 7.88 (1H, d, J = 8.6 Hz, 8-H), 7.94 (1H, d,
J = 8.6 Hz, 5-H), 8.65 (1H, s, 40-OH), 10.14 (1H, s, 30-OH), 12.37
(1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 41.59 (CH2), 107.85 (50-C),
112.72 (10-C), 122.68 (60-C), 124.24 (8-C), 125.47 (2-C), 125.61 (6-C),
126.08 (7-C), 127.25 (4-C), 128.31 (3,5-C), 128.40 (1-C), 132.10
(8a-C), 132.38 (30-C), 133.31 (4a-C), 152.30 (40-C), 152.62 (20-C),
202.78 (C=O). FAB-HRMS m=z (M + H)þ calcd. for C18H15O4
295.0970, found 295.0971.
Compound 23, 20,30,40-trihydroxy-2-(2-naphthyl) acetophenone,
was obtained as pale violet crystals. M.p. 144–145 ꢀC. Yield: 69%.
1H-NMR (DMSO-d6) ꢁ: 4.48 (2H, s, CH2), 6.44 (1H, d, J = 8.6 Hz,
50-H), 7.44 (1H, d, J = 8.0 Hz, 3-H), 7.47–7.50 (2H, m, 6,7-H), 7.57
(1H, d, J = 8.6 Hz, 60-H), 7.81 (1H, s, 1-H), 7.88 (2H, m, 5,8-H), 7.86
(1H, d, J = 8.0 Hz, 4-H), 8.64 (1H, s, 40-OH), 10.14 (1H, s, 30-OH),
12.50 (1H, s, 20-OH). 13C-NMR (DMSO-d6) ꢁ: 44.14 (CH2), 107.82
(50-C), 112.54 (10-C), 122.99 (60-C), 125.65 (6-C), 126.10 (7-C),
127.38 (5-C), 127.46 (8-C), 127.68 (4-C), 127.88 (1-C), 128.00 (3-C),
131.78 (4a-C), 132.35 (8a-C), 132.98 (2-C), 133.01 (30-C), 152.52
(40-C), 152.64 (20-C), 202.78 (C=O). FAB-HRMS m=z (M + H)þ
calcd. for C18H15O4 295.0970, found 295.0971.
Compound 24, 2-(biphenyl-4-yl)-20,30,40-trihydroxyacetophenone,
was obtained as violet crystals. M.p. 203–204 ꢀC. Yield: 61%. 1H-
NMR (DMSO-d6) ꢁ: 4.34 (2H, s, CH2), 6.45 (1H, d, J = 8.6 Hz, 50-H),
7.35 (1H, t, J = 7.4 Hz, 4-phenyl-4-H), 7.38 (2H, d, J = 8.0 Hz, 2,6-
H), 7.45 (2H, t, J = 7.4 Hz, 4-phenyl-3,5-H), 7.54 (1H, d, J = 8.6 Hz,
60-H), 7.61 (2H, d, J = 8.0 Hz, 3,5-H), 7.64 (2H, d, J = 7.4 Hz, 4-
phenyl-2,6-H), 8.69 (1H, s, 40-OH), 10.13 (1H, s, 30-OH), 12.51 (1H, s,
20-OH). 13C-NMR (DMSO-d6) ꢁ: 43.63 (CH2), 107.83 (50-C), 112.48
(10-C), 123.00 (60-C), 126.54 (4-phenyl-2,6-C), 126.64 (3,5-C), 127.29
(4-phenyl-4-C), 128.87 (4-Phenyl-3,5-C), 130.08 (2,6-C), 132.36
(30-C), 134.52 (1-C), 138.48 (4-C), 139.89 (4-phenyl-1-C), 152.54
(40-C), 152.66 (20-C), 202.74 (C=O). FAB-HRMS m=z (M + H)þ
calcd. for C20H17O4 321.1127, found 321.1138.
The 24 compounds synthesized in this study were
screened for the antibacterial activity against various
strains of bacteria, including Bacillus subtilis 168,
Staphylococcus aureus KSA-1, Escherichia coli
ATCC43888, Proteus mirabilis Pm1, Pseudomonas
aeruginosa T8, and Salmonella enterica KK1; the first
two were Gram-positive and the last four Gram-negative.
The activities were expressed as LC50 of the compounds
against the six bacterial strains listed in Table 1. Also
listed are the activities of reference compounds, includ-
ing penicillin G, chloramphenicol, rifampicin, and
mitomycin C. Eighteen of the 24 compounds were found
to be active against both Gram-positive strains of
B. subtilis and S. aureus, while they had no inhibitory
effect on the four Gram-negative strains even at the
highest concentration used in this study (250 mM),
suggesting possible selective toxicity and specific inhib-
itory effects against Gram-positive bacteria.
20,30,40-Trihydroxy-2-phenylacetophenone (compound
1) itself did not show any antibacterial activity, but some
functional groups introduced onto its 2-phenyl ring
appeared to be able to enhance the activity. The
introduction of 3-methyl, 4-methyl, 2,4,6-trimethyl
(compounds 3–5) and of 4-isopropyl (compound 6) was
able to confer the antibacterial activities, although 2-
methyl (compound 2) was not effective. On the other
hand, 2-hydroxy, 3-hydroxy, and 4-methoxy abolished
the activity completely (compounds 7–9), and 4-ethoxy
(compound 10) resulted in moderate activity, suggesting
that oxygen atoms attached on the 2-phenyl ring cause
negative effects on the antibacterial activity. The
introduction of halogens, including chloro, bromo, and
iodo (compounds 14–21), generally increased the anti-
bacterial activity. Especially, 2,4-dichloro, 2-bromo, and
3-bromo (compounds 17–19) were effective at increasing
the activity against S. aureus. However, 3- and 4-fluoro
(compounds 12 and 13) were not as effective as the other
halogen groups, and even 2-fluoro (compound 11)
abolished the activity. Replacing the entire 2-phenyl
group with the naphthyl group (compounds 22 and 23)
also enhanced activity. Finally, 2-(biphenyl-4-yl)-
20,30,40-trihydroxyacetophenone (compound 24) exhibit-
ed the highest activities against both B. subtilis and
S. aureus, with LC50 calculated at 5.8 mM and 5.6 mM
respectively, almost comparable to the positive control
chloramphenicol.
Bacterial and yeast strains, cell-line, and bioassay. Bacterial strains
were grown in LB medium with shaking at 37 ꢀC. The yeast strain
Saccharomyces cerevisiae Hansen BY4742 (Invitrogen, Carlsbad, CA)
was grown in YPD medium with shaking at 30 ꢀC. LC50 values were
determined basically according to the microtiter plate method
previously described.8) Briefly, cells of the bacteria were inoculated
into LB medium in the wells of a 96-well microtiter plate, each of
which contained one of the serially diluted 20,30,40-trihydroxy-2-
phenylacetophenone derivatives (ranging from 0 to 250 mM), and were
allowed to grow. After 3 h, the optical density for the cells at 630 nm
was measured, and the LC50 of each compound was calculated from a
plot of the optical density values against the concentrations of the
compound. In the case of yeast, the same procedure as for the bacterial
assay was employed, except that YPD was used as the culture medium.
Mouse hepatoma Hepa-1c1c7 cells9) obtained from the American Type
Culture Collection were maintained in Eagle’s medium (Nissui
Pharmaceuticals, Tokyo) supplemented with 5% (v/v) fetal bovine
serum, 4 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml strep-
tomycin at 37 ꢀC in an atmosphere containing 5% CO2. The cells were
treated with diluted 20,30,40-trihydroxy-2-phenylacetophenone deriva-
tives for 24 h, and then cell survival was monitored by counting after
trypan blue exclusion.
Our results suggest that S. aureus is more susceptible
than B. subtilis to these antibacterial 20,30,40-trihydroxy-
2-phenylacetophenone derivatives, implying that these
compounds deserve further investigation as potential
antimicrobial agents to control clinical problems with
S. aureus. To clarify this possibility, however, we still
need to test other strains, especially those including
MRSA. Only recently, it was found that B. subtilis cells
treated with compound 19 became more susceptible to
osmotic stress (data not shown), suggesting that this
compound disturbs the integrity of the cell surface.
Further study is required to determine the mode of action
of the active compounds, and would be worthwhile in the
effort to design more effective antibacterial agents.
Two of the most effective compounds, 19 and 24,
were applied to yeast strain BY4742 and mouse
hepatoma Hepa-1c1c7 cells, but neither of them ex-
hibited detectable toxicity at 10 mM (data not shown),
Results and Discussion
The 20,30,40-trihydroxy-2-phenylacetophenone deriva-
tives were synthesized by condensation of pyrogallol
(y in Fig. 1) with the corresponding phenylacetic acid
derivatives (a–x in Fig. 1).10,11) Compounds 1–24
(Fig. 1) were synthesized successfully, and their chemi-
cal identity and purity were confirmed by spectral
data (1H- and 13C-NMR, and mass), as summarized

128
H. GOTO et al.
Table 1. Antibacterial Activities of the Synthesized Compounds and Antibiotics Expressed as LC50 (mM)
B. subtilis
168a
S. aureus
KSA-1b
E. coli
ATCC43888c
P. mirabilis
Pm1b
P. aeruginosa
T8b
S. enterica
KK1b
Compound
d
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
230
15
20
110
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
170
40
19
2.6
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
57
14
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
210
13
51
25
2
—
120
98
3
18
14
4
5
28
11
28
6
5.8
—
7
—
—
8
—
—
9
10
—
110
—
33
—
11
12
100
94
43
32
13
14
55
14
24
11
15
16
12
10
11
17
18
5.8
7.7
6.1
11
42
18
19
20
15
23
21
22
12
12
12
14
23
24
11
5.8
190
8.4
NDe
1.3
5.6
180
9.8
ND
9.4
Penicillin G
Chloramphenicol
Rifampicin
Mitomycin C
Values are means from three independent measurements.
a
´
´
Obtained from Genetique Microbienne, Institut National de la Recherche Agronomique, Jouy-en-Josas, France
bObtained from the Bacterial Culture Collection of the Research Center for Food Safety and Security, Kobe University, Kobe, Japan
cObtained from American Type Culture Collection, Georgetown University, Washington, DC
d—, no antibacterial activity was observed (higher than 250 mM)
eND, not determined (lower than 0.98 mM)
4) Emami, S., Foroumadi, A., Faramarzi, M. A., and Samadi, N.,
Synthesis and antibacterial activity of quinolone-based
compounds containing a coumarin moiety. Arch. Pharm., 341,
42–48 (2008).
suggesting that both compounds might not be suffi-
ciently toxic to eukaryotic cells, at least at the lower
concentrations to exert antibacterial activity. In addition,
both chemicals can be regarded as harmless to yeast,
because no growth inhibition was seen even at 250 mM,
the highest concentration used under the assay con-
ditions (data not shown). However, to Hepa-1c1c7 cells,
both compounds appeared to be toxic at concentrations
higher than 100 mM (data not shown). The possible dose-
dependent toxicity to mammalian cells will be also
investigated in the future.
5) Fokialakis, N., Lambrinidis, G., Mitsiou, D. J., Aligiannis, N.,
Mitakou, S., Skaltsounis, A. L., Pratsinis, H., Mikros, E., and
Alexis, M. N., A new class of phytoestrogens; evaluation of the
estrogenic activity of deoxybenzoins. Chem. Biol., 11, 397–406
(2004).
6) Dufresne, C., Roy, P., Wang, Z., Asante-Appiah, E., Cromlish,
W., Boie, Y., Forghani, F., Desmarais, S., Wang, Q., Skorey, K.,
Waddleton, D., Ramachandran, C., Kennedy, B. P., Xu, L.,
Gordon, R., Chan, C. C., and Leblanc, Y., The development of
potent non-peptidic PTP-1B inhibitors. Bioorg. Med. Chem.
Lett., 14, 1039–1042 (2004).
Acknowledgments
7) Li, H. Q., Xue, J. Y., Shi, L., Gui, S. Y., and Zhu, H. L.,
Synthesis, crystal structure and antimicrobial activity of
deoxybenzoin derivatives from genistein. Eur. J. Med. Chem.,
43, 662–667 (2008).
The authors thank Dr. Hirosato Takikawa for critical
reading of the manuscript, Dr. Hiroaki Matsuzaki for
providing the yeast strain, and Ms. Emi Kurimoto and
Mr. Daisuke Kashiwada for the help with experiments.
8) Almeida, A. A., Farah, A., Silva, D. A., Nunan, E. A., and
´
Gloria, M. B., Antibacterial activity of coffee extracts and
selected coffee chemical compounds against enterobacteria.
J. Agric. Food Chem., 54, 8738–8743 (2006).
References
1) Bozdogan, B., Ednie, L., Credito, K., Kosowska, K., and
Appelbaum, P. C., Derivatives of a vancomycin-resistant
9) Bernhard, H. P., Darlington, G. J., and Ruddle, F. H.,
Expression of liver phenotypes in cultured mouse hepatoma
cells: synthesis and secretion of serum albumin. Dev. Biol., 35,
83–96 (1973).
Staphylococcus aureus strain isolated at Hershey Medical
Center. Antimicrob. Agents Chemother., 48, 4762–4765 (2004).
2) Harthug, S., Digranes, A., Hope, O., Kristiansen, B. E., Allum,
A. G., and Langeland, N., Vancomycin resistance emerging in a
clonal outbreak caused by ampicillin-resistant Enterococcus
faecium. Clin. Microbiol. Infect., 6, 19–28 (2000).
10) Chang, Y. C., Nair, M. G., Santell, R. C., and Helferich, W. G.,
Microwave-mediated synthesis of anticarcinogenic isoflavones
from soybeans. J. Agric. Food Chem., 42, 1869–1871 (1994).
11) Lang’at-Thoruwa, C., Kirby, G. C., Phillipson, J. D., Warhurst,
D. C., Watt, R. A., and Wright, C. W., Enhancement of the
antiplasmodial activity of quassin by transformation into a
gamma-lactone. J. Nat. Prod., 66, 1486–1489 (2003).
3) Friedman, M., Overview of antibacterial, antitoxin, antiviral,
and antifungal activities of tea flavonoids and teas. Mol. Nutr.
Food Res., 51, 116–134 (2007).