Antibacterial acylphloroglucinols from hypericum olympicum

Article abstract of DOI:10.1021/np2003319

New antibacterial acylphloroglucinols (1-5) were isolated and characterized from the aerial parts of the plant Hypericum olympicum L. cf. uniflorum. The structures of these compounds were confirmed by extensive 1D- and 2D-NMR experiments to be 4,6-dihydroxy-2-O-(3″,7″-dimethyl-2″, 6″-octadienyl)-1-(2′-methylbutanoyl)benzene (1), 4,6-dihydroxy-2-O-(7″-hydroxy-3″,7″-dimethyl-2″, 5″-octadienyl)-1-(2′-methylbutanoyl)benzene (2), 4,6-dihydroxy-2-O-(6″-hydroxy-3″,7″-dimethyl-2″, 7″-octadienyl)-1-(2′-methylbutanoyl)benzene (3), 4,6-dihydroxy-2-O-(6″-hydroperoxy-3″,7″-dimethyl-2″, 7″-octadienyl)-1-(2′-methylbutanoyl)benzene (4), and 4,6-dihydroxy-2-O-(6″,7″-epoxy-3″,7″-dimethyloct- 2″-enyl)-1-(2′-methylbutanoyl)benzene (5). These new natural products have been given the trivial names olympicins A-E (1-5). All compounds were evaluated against a panel of methicillin-resistant Staph. aureus and multidrug-resistant strains of Staph. aureus. Compound 1 exhibited minimum inhibitory concentrations (MICs) of 0.5-1 mg/L against the tested Staph. aureus strains. Compounds 2 to 5 were also shown to be active, with MICs ranging from 64 to 128 mg/L. Compound 1 was synthesized using a simple four-step method that can be readily utilized to give a number of structural analogues of 1.

Full text of DOI:10.1021/np2003319

ARTICLE
pubs.acs.org/jnp
Antibacterial Acylphloroglucinols from Hypericum olympicum
Winnie K. P. Shiu, M. Mukhlesur Rahman, Jonathan Curry, Paul Stapleton, Mire Zloh, John P. Malkinson,
and Simon Gibbons*
Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square,
London WC1N 1AX, U.K.
S Supporting Information
b
ABSTRACT: New antibacterial acylphloroglucinols (1ꢀ5)
were isolated and characterized from the aerial parts of the
plant Hypericum olympicum L. cf. uniflorum. The structures
of these compounds were confirmed by extensive 1D- and
2D-NMR experiments to be 4,6-dihydroxy-2-O-(300,700-dimethyl-
200,600-octadienyl)-1-(20-methylbutanoyl)benzene (1), 4,6-dihy-
droxy-2-O-(700-hydroxy-300,700-dimethyl-200,500-octadienyl)-1-(20-
methylbutanoyl)benzene (2), 4,6-dihydroxy-2-O-(600-hydroxy-
300,700-dimethyl-200,700-octadienyl)-1-(20-methylbutanoyl)benzene
(3), 4,6-dihydroxy-2-O-(600-hydroperoxy-300,700-dimethyl-200,700-
octadienyl)-1-(20-methylbutanoyl)benzene (4), and 4,6-dihydroxy-2-O-(600,700-epoxy-300,700-dimethyloct-200-enyl)-1-(20-methylbu-
tanoyl)benzene (5). These new natural products have been given the trivial names olympicins AꢀE (1ꢀ5). All compounds were
evaluated against a panel of methicillin-resistant Staph. aureus and multidrug-resistant strains of Staph. aureus. Compound 1
exhibited minimum inhibitory concentrations (MICs) of 0.5ꢀ1 mg/L against the tested Staph. aureus strains. Compounds 2 to 5
were also shown to be active, with MICs ranging from 64 to 128 mg/L. Compound 1 was synthesized using a simple four-step
method that can be readily utilized to give a number of structural analogues of 1.
he widespread interest in the use of Hypericum perforatum
value of this class as antibacterial drug leads, which could be
further developed into antibiotics to treat resistant strains.
T
(St. John0s Wort) in mild to moderate depression has
attracted much attention in investigating the bioactive metabo-
lites from other species in the Hypericum genus. The acylphlor-
oglucinol hyperforin is the best characterized acylphloroglucinol
from this genus in terms of its bioactivity and has been shown to
be the major antibacterial constituent in H. perforatum1 and
active against Staph. aureus and MRSA strains at concentra-
tions as low as 0.1 mg/L.2 Other acylphloroglucinol deriva-
tives with antibacterial activities have been isolated and
characterized from this genus.3,4 Notable among these acylph-
loroglucinols are the antibacterial drummondins from Hyper-
icum drummondii, which are filicinic acid derivatives displaying
MIC values as low as 0.39 mg/L.5 This study is part of ongoing
research in our group to identify antibacterial metabolites from
various Hypericum species.
These levels of activity, coupled with interesting acylphlor-
oglucinol chemistry, which is often complex, prompted us
to screen a range of Hypericum species collected from the
UK National Collection at the Royal Botanic Gardens Kew
at Wakehurst Place.8 This led to the isolation and evaluation
of the antibacterial properties of a series of new acylphlor-
oglucinol natural products (1ꢀ5) from Hypericum olympicum.
This species is a small herb with typical yellow Hypericum
flowers.
Special Issue: Special Issue in Honor of Gordon M. Cragg
The simplicity of compound 1, coupled with its level of activity
against a range of methicillin-resistant Staph. aureus (MRSA) and
multidrug-resistant (MDR) strains of Staph. aureus, shows the
Received: April 19, 2011
Published: September 07, 2011
r
Copyright
2011 American Chemical Society and
336
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American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data and 1Hꢀ C Long-Range Correlations for 1ꢀ3 Recorded
13
in CDCl3
1
2
3
position
1H (J, Hz)
13C
2J
3J
1H (J, Hz)
13C
2J
3J
1H (J, Hz)
13C
2J
3J
1
105.0
162.6
105.9
162.4
105.9
162.4
91.6
2
3
5.92 d (2.5)
5.98 d (2.5)
91.5 C-4
161.9
C-1, C-5
C-1, C-3
5.90 d (2)
5.99 d (2)
91.7 C-4
162.1
C-1, C-5
C-1, C-3
5.92 d (2)
5.98 d (2)
C-1
C-3
4
161.9
96.6
5
96.5 C-6
167.5
96.6 C-6
167.5
6
167.6
210.3
46.1
10
20
30
40
50
100
200
300
400
500
600
700
800
900
1000
210.4
210.3
3.66 m
46.1
3.63 m
46.1
3.64 m
1.37 m, 1.80 m 26.8
1.37 m, 1.80 m 26.9
1.37 m, 1.80 m 26.8
0.88 t (7.5)
11.9 C-30 C-20
C-10, C-30 1.12 d (6.5) 16.7 C-20 C-10, C-30
0.89 t (7.5)
1.12 d (6.5)
4.57 d (6.5)
5.51 m
11.8 C-30
C-20
C-10, C-30
C-3, C-300
0.88 t (7.5)
1.12 d (7)
4.58 d (6.5)
11.9 C-30
C-20
16.6 C-200
65.7 C-200
118.2
16.7 C-20
65.6 C-200
119.6
C-300
4.57 d (6.5)
5.53 dt
65.6
118.7
141.9
35.5
C-300
C-400, C-1000 5.30 m
142.3
140.5
2.13 m
2.10 m
5.10 m
39.5 C-300, C-500 C-200, C-600
26.3 C-400, C-600 C-300
2.81 d (6.5)
5.68 m
42.2 C-300, C-500 C-200, C-600 2.15 m
128.4
1.75
26.8 C-400 C-300
123.6
132.0
17.7 C-700
25.7 C-700
16.7 C-300
5.66 d (15)
135.9
4.08 t (6.5)
75.5
82.2
147.2
1.62 s
1.69 s
1.74 s
C-600, C-900
C-600, C-800
C-200, C-400
1.35 s
1.35 s
1.74 s
24.3 C-700
24.3 C-700
16.8 C-300
C-600, C-800 4.95 s, 4.87 t
C-600, C-800 1.76 s
C-200
111.4
C-600
16.7 C-700 C-600, C-800
17.6 C-300 C-200, C-400
1.74 s
4-OH 5.32 bs
6-OH 14.02 s
C-1
C-2, C-6
14.00 s
C-6
C-1, C-5
13.99 s
C-1, C5
’ RESULTS AND DISCUSSION
a deshielded carbon (δ 161.9, C-4). The second hydroxy group
was therefore placed between the aromatic protons at C-4.
The substituent at C-1 included a methine multiplet (δ 3.66,
H-20), a methylene multiplet (δ 1.37, 1.80, H2-30), a methyl
triplet (δ 0.89, H3-40), and a methyl doublet (δ 1.12, H3-50). In
the COSY spectrum, the methylene (H2-30) was coupled to the
methyl triplet (H3-40), and the methine multiplet (H-20) was
coupled to the methyl doublet (H3-50). The HMBC spectrum
revealed cross-peaks between this methyl doublet and C-20, C-30,
and a carbonyl carbon (δ 210.4, C-10), which could interact with
the hydroxy group at C-6 via a hydrogen bond and be responsible
for the downfield resonance of this hydroxy group. This con-
firmed the 2-methylbutanoyl side-chain at C-1.
Compound 1 was isolated as a pale yellow oil from the
n-hexane extract of H. olympicum L. cf. uniflorum. The HR-ESIMS
indicated an [M ꢀ H]ꢀ ion at m/z 345, suggesting a molecular
formula of C21H30O4. The 1H and 13C NMR data (Table 1)
were indicative of an acylphloroglucinol with a terpene sub-
stituent. The 1H NMR spectrum showed two signals for
hydroxy groups, one of which was highly deshielded and
hydrogen-bonded (δ 14.02) and the other of which appeared
as a broad singlet at δ 5.32. Other signals observed in the 1H
NMR spectrum included two meta-coupled aromatic protons
(δ 5.98 d, J = 2.5 Hz; 5.92 d, J = 2.5 Hz), two olefinic protons
(δ 5.51 m; 5.10 m), one methine (δ 3.66 m), four methylene
groups, three methyl singlets (δ 1.74, 1.69, 1.62), one methyl
doublet (δ 1.12, J = 6.5 Hz), and one methyl triplet (δ 0.89, J =
7.5 Hz). The 13C NMR spectrum displayed signals for six
aromatic carbons, three of which were highly deshielded,
implying that these carbons were attached to electron-with-
drawing groups. The pattern of these signals suggested a 1,3,5-
trihydroxybenzene (phloroglucinol) structure. In the HMBC
The final substituent at C-2 included an oxymethylene group
13
(δ 4.57 d, J = 6.5 Hz, C-100), which showed long-range 1Hꢀ C
correlations with a deshielded aromatic carbon (δ 162.6, C-2)
and two carbons associated with an olefinic group (δ 118.2, C-200;
δ 142.3, C-300). In the HMBC spectrum, an olefinic proton
(δ 5.51 m) at C-200 was coupled to the carbon of a methyl moiety
(δ 16.7, C-1000) and to a methylene carbon (δ 39.5, C-400) via
three bonds. This methylene group (δ 2.13 m, H-400) showed 2J
2
spectrum the hydrogen-bonded proton showed a J correla-
3
correlations with C-300 and C-500 (δ 26.3) and J correlations
tion with the carbon to which it was directly attached (δ 167.5,
with C-200 and C-600 (δ 123.6). Two methyl singlets (δ 1.62,
1.69) were coupled to C-600 and a quaternary carbon associated
with this olefinic carbon (δ 132.0, C-700), therefore completing
the substituent at C-2. This side-chain consisted of 10 carbons,
including two olefinic and three methyl groups, and was char-
acteristic of a geranyl group. The COSY and NOESY spectra also
3
C-6) and J correlations with an aromatic methine carbon
(δ 96.5, C-5) and a quaternary aromatic carbon (δ 105.0, C-1),
confirming the position of the hydroxy group. The other
aromatic hydrogen at δ 5.92 was then placed at C-3, as it
was meta-coupled to H-5. This was further confirmed by
HMBC correlations between this proton and C-1, C-5, and
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Journal of Natural Products
ARTICLE
Figure 1. Synthesis of compound S1.
provided evidence for the geranyl side-chain. Both double bonds
were assigned the trans-configuration on the basis of the nature of
the biosynthesis of a geranyl side-chain from two isoprene units.
This was also supported by an NOE correlation between H-200
and H2-400. The 1H and 13C NMR data on the geranyloxy side-
chain showed good agreement with reported data.7
J = 15 Hz, H-600), two methylene doublets including one shifted
downfield at δH 4.58 (J = 6.5 Hz, H2-100) and one at δ 2.81 (J =
6.5 Hz, H2-400), two equivalent methyl groups integrating to six
protons (δ 1.35 s, H3-800, H3-900), and an additional methyl
singlet (δ 1.74, H3-1000). The remaining signals in the 13C NMR
spectra included one quaternary olefinic carbon (δ 140.5, C-300),
three olefinic methines (δ 119.6, C-200; 128.4, C-500; 135.9,
C-600), an oxygenated quaternary carbon (δ 82.2, C-700), two
methylenes (δ 65.6, C-100; 42.2, C-400), two equivalent methyl
carbons at δ 24.3 (C-800, C-900), and a further methyl carbon at δ
16.8 (C-100). An oxymethylene (δ 4.58 d, H2-100) was coupled to
the olefinic proton at δ 5.30 (H-200) in the COSY spectrum. It
The position of the O-geranyl substituent was assigned as
ortho to the 2-methylbutanoyl group on the basis of the HMBC
correlations as described and also the observation that the meta-
coupled aromatic protons were nonequivalent. The configura-
tion at the C-20 stereogenic center can be identified by comparing
the specific rotation of this compound with that of the synthetic
compound S1, which was synthesized from (+)-(S)-2-methyl-
butanoic acid (Figure 1). The specific rotations for 1 and S1
were found to be [α]D22 +6.0 (c 0.25, CHCl3) and [α]2D2 +6.0
(c 0.30, CHCl3), respectively. The data implied that the stereo-
genic center in the natural product 1 was of the S-configuration.
Compound 1 was therefore identified as the new natural product
(S)-4,6-dihydroxy-2-O-(300,700-dimethyl-200,600-octadienyl)-1-(20-
methylbutanoyl)benzene and given the trivial name olympicin A.
A patent was filed for this compound for its application as a
potential antibacterial agent.8
13
also displayed 1Hꢀ C 2J correlation to the olefinic carbon at δ
119.6 (C-200) and 3J correlation to a quaternary olefinic carbon
(δ 140.5, C-300) to which a methyl group (δ 1.74 s, H3-1000) was
directly attached. The C-1000 methyl group was correlated to a
methylene carbon at δC 42.2 (C-400) in the HMBC spectrum,
thus placing the methylene group (δ 2.81 d, H2-400) next to C-300.
13
This methylene doublet in turn displayed 1Hꢀ C correlations
to four olefinic carbons, confirming its position between the two
olefinic bonds. An olefinic multiplet at δH 5.68 (H-500) was
placed between the methylene group (H2-400) and the olefinic
doublet (δ 5.66, H-600), as they showed correlations in the COSY
spectrum. The splitting of the olefinic multiplet (δ 5.68, H-500)
was due to the fact that the signal was split by both the
neighboring methylene (H2-400) and the C-600 olefinic proton.
The olefinic proton at H-600 appeared as a clear doublet because
the signal was only split by the neighboring H-500. The large
coupling constant between H-500 and H-600 (J = 15 Hz) indicated
that these two protons were in a trans-configuration. The
equivalent methyl groups (δ 1.35 s, 6 H, H3-800, H3-900) at the
terminus of the side-chain were directly attached to a quaternary
carbon (δ 82.2, C-700), which was shifted downfield by a hydroxy
group directly attached to this carbon. This connection was
supported by HMBC correlations between the methyl singlet
and C-700 and the olefinic carbon at C-600. This completed the
structural elucidation of compound 2. The specific rotation of
this compound was found to be [α]2D2 +5.8 (c 0.22, CHCl3),
which might again imply that C-20 was of an S-configuration
(synthetic compound S1, [α]2D2 +6.0 (c 0.30, CHCl3)). Com-
pound 2 was therefore identified as 4,6-dihydroxy-2-O-(700-
hydroxy-300,700-dimethyl-200,500-octadienyl)-1-(20-methylbutanoyl)-
benzene, assigned as olympicin B, and is described here for the
first time.
Compound 2 was isolated as a pale yellow oil from the CH2Cl2
extract of H. olympicum L. cf. uniflorum. High-resolution ESIMS
gave an [M + H]+ ion at m/z 363, indicating a molecular formula
of C21H30O5. The 1H and 13C NMR spectroscopic data (Table 1)
1
were similar to those of compound 1. The H NMR signals
indicated the presence of a 2-methylbutanoyl phloroglucinol
nucleus with one highly deshielded hydrogen-bonded hydroxy
group (δ 14.00), two meta-coupled aromatic protons (δ 5.90 d,
J = 2 Hz, H-3; 5.99 d, J = 2 Hz, H-5), one methine multiplet
(δ 3.63, H-20), one methylene (δ 1.37 m, 1.80 m, H2-30), one
methyl triplet (δ 0.88, J = 7.5 Hz, H3-40), and one methyl doublet
(δ 1.12, J = 7.5 Hz, H3-50). The 13C NMR signals were again
indicative of a phloroglucinol moiety with six aromatic carbons,
comprising three deshielded quaternary carbons (δ 167.5, C-6;
162.4, C-2; 162.1, C-4), one quaternary carbon at δ 105.9 (C-1),
and two methines at δ 96.6 (C-5) and 91.7 (C-3). The presence
of a carbonyl carbon (δ 210.3, C-10), one methine (δ 46.1, C-20),
one methylene (δ 26.9, C-30), and two methyl carbon signals
(δ 11.9, C-40; 16.7, C-50) again suggested a 2-methylbutanoyl side-
chain in 2. The structure of the 2-methylbutanoyl-phloroglucinol
nucleus was further confirmed by HMBC correlations, which
were described earlier in the structural elucidation of compound 1.
1
The remaining signals in the H NMR spectrum included
Compound 3 was isolated as a pale yellow oil from the CH2Cl2
three olefinic protons (δ 5.30 m, H-200; 5.68 m, H-500; 5.66 d,
extract of H. olympicum. The HR-ESIMS gave an [M ꢀ H]ꢀ ion
338
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Journal of Natural Products
ARTICLE
Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data and 1Hꢀ C Long-Range Correlations of 4 and 5 Recorded
13
in CDCl3
4
5
position
1H (J, Hz)
13C
2J
3J
position
1H (J, Hz)
13C
2J
3J
1
105.9
162.4
91.7
1
105.5
162.3
92.7
162.8
96.8
167.6
210.0
46.1
26.5
12.0
16.6
66.4
121.9
138.5
37.0
26.5
65.1
59.1
18.9
24.6
16.0
2
2
3
5.91 d (2.5)
5.98 d (2.5)
C-1, C-5
C-1, C-3
3
6.02 d (2.5)
5.98 d (2.5)
C-1, C-5
C-3
4
162.0
96.6
4
5
C-6
5
6
10
20
30
40
50
100
200
300
400
500
600
700
800
900
1000
4-OH
6-OH
167.5
210.3
46.1
6
10
20
30
40
50
100
200
300
400
500
600
700
800
900
1000
1-OH
5-OH
3.63 m
3.63 m
1.35 m, 1.80 m
0.88 t (7.5)
1.11 d (6)
4.58 d (6.5)
5.52 m
26.8
1.38 m, 1.81 m
0.90 t (7.5)
1.1 d (7)
4.67 m
11.9
C-30
C-20
C-200
C-20
C-10, C-30
C-300
C-30
C-20
C-20
C-10, C-30
16.6
65.6
119.2
141.2
35.2
5.50 m
2.21 m
1.75 m
4.32 t
2.30 m
26.8
1.65 m, 1.90 m
2.77 dt
89.0
143.3
114.6
17.2
5.02 s, 5.05 t
1.75 s
C-600, C-900
C-600, C-800
C-200, C-400
1.31 s
1.34 s
1.76 s
13.95 s
6.90 bs
C-700
C-700
C-300
C-600, C-900
C-600, C-800
C-200, C-400
C-1, C-5
C-700
C-300
1.75 s
16.6
5.32 bs
13.99 s
C-6
C-1, C-5
at m/z 361, suggesting a molecular formula of C21H30O5. The 1H
C-800), two quaternary olefinic carbons (δ 141.0, C-300; 147.2,
C-700), and two methyl carbons (δ 16.7, C-900; 17.6, C-1000).
The oxymethylene group showed HMBC correlations with an
olefinic carbon at δ 118.7 (C-200) and COSY correlations to the
olefinic proton associated with this carbon (δ 5.53). A methyl
singlet at δ 1.74 (H3-1000) showed HMBC correlations with
C-200, an olefinic carbon to which it was directly attached
(δ 141.9, C-300), and to a methylene carbon at δ 35.5 (C-400).
The methylene protons at C-400 were coupled to another
methylene group (δ 1.75 m), which was coupled to a methine
triplet (δ 4.08, J = 6.5 Hz, H-600), as shown in the COSY
spectrum. This triplet was shifted downfield and was attached
to a carbon at δ 75.5 (C-600), indicating that a hydroxy group
should be attached here, and this was supported by the mass
spectrum. The remaining methyl group (δ 1.76) was placed at
and 13C NMR spectroscopic data (Table 3) were again similar to
1
those of compound 1. The common signals in the H NMR
spectrum included one highly deshielded hydrogen-bonded
hydroxy group (δ 13.99, 6-OH), two meta-coupled aromatic
protons (δ 5.92 d, J = 2 Hz, H-3; 5.98 d, J = 2 Hz, H-5), one
methine (δ 3.64 m, H-20), one methylene (δ 1.37 m, 1.80 m,
H2-30), one methyl triplet (δ 0.88 t, J = 7.5 Hz, H3-40), and one
methyl doublet (δ 1.12 d, J = 6.5 Hz, H3-50). In the 13C NMR
spectrum, signals suggesting a (2-methylbutanoyl)phlorogluci-
nol nucleus were again seen, including three deshielded qua-
ternary aromatic carbons (δ 167.6, C-6; 162.4, C-2; 161.9, C-4),
one quaternary aromatic carbon (δ 105.9, C-1), two methine
aromatic carbons (δ 91.6 C-3; 96.6, C-5), one carbonyl carbon
(δ 210.3, C-10), one methine (δ 46.1, C-20), one methylene
(δ 26.8, C-30), and two methyl carbons (δ 11.9, C-40; 16.7,
C-60). HMBC correlations further confirmed the structure
of the (2-methylbutanoyl)phloroglucinol nucleus and were
similar to those described in the structural elucidation of
compound 1.
2
C-700, as it exhibited a J HMBC correlation to a quaternary
carbon at δ 143.3 (C-700) and 3J correlations to the oxygenated
methine carbon (C-600) and to an olefinic carbon at δ 111.4
(C-800) bearing two exo-methylene protons. The specific rotation
of 3 was [α]2D2 +2.5 (c 0.41, CHCl3). Although it showed the
same direction of rotation as S1, the configuration of 3 could not
be determined, as two stereogenic centers were present in this
compound. Compound 3 was therefore identified as the new
natural product 4,6-dihydroxy-2-O-(600-hydroxy-300,700-dimeth-
yl-200,700-octadienyl)-1-(20-methylbutanoyl)benzene and was
given the trivial name olympicin C.
1
The H NMR signals for the side-chain at the 2-O position
consisted of four methylenes, including one shifted downfield
(δ 4.57 d, J = 6.5 Hz, H2-100) and an exo-methylene(δ 4.87 t, 4.95 s,
H2-800), one olefinic proton (δ 5.53 dt, H-200), one deshielded
methine triplet (δ 4.08, J = 6.5 Hz, H-600), and two methyl
singlets (δ 1.76, H3-900; 1.74, H3-1000). Ten carbons were
accountable for this side-chain, again indicative of a geranyl
moiety. These included two methines (δ 118.7, C-200; 75.5, C-600),
four methylenes (δ 65.6, C-100; 35.5, C-400; 26.8, C-500; 111.4,
Compound 4 was isolated as a pale yellow oil from the CH2Cl2
extract of H. olympicum L. cf. uniflorum. High-resolution ESIMS
gave an [M ꢀ H]ꢀ ion at m/z 377, indicating a molecular formula
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Journal of Natural Products
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Table 3. MIC (μM) Values of Compound 1 and Control
Antibiotics against Staph. aureus, Mycobacterium species,
P. aeruginosa, and Salmonella enterica serovar Typhimurium
(C-300) and COSY correlation with the olefinic proton at C-200.
2
The methyl singlet at δ 1.74 (H-1000) showed a J HMBC
correlation to the olefinic carbon to which it was directly attached
3
(C-300) and J HMBC correlations to C-200 and a methylene
control compound 1
carbon at δ 35.2 (C-400). The methylene protons at C-400 (2.21
m) were coupled to another methylene group (δ 1.75 m, H-500),
which was coupled to a methine triplet (δ 4.32, H-600), as shown
in the COSY spectrum. This triplet was shifted downfield and
was attached to a carbon at δ 89.0 (C-600), indicating that an
electron-withdrawing group with a stronger deshielding effect
than a hydroxy group was present at this carbon (δC 75.5 for
hydroxy-bearing C-600 in compound 3). This chemical shift was
characteristic of a hydroperoxy-bearing methine. The molecular
formula for this compound (C21H30O6) indicated that it had
an extra oxygen when compared to that of compound 3
(C21H30O5). The remaining methyl group (δH 1.75, H-900)
was placed at C-700 (δC 143.3), as the methyl singlet showed
HMBC correlations to this carbon, the hydroperoxy-bearing
carbon (C-600), and the exo-methylene carbon at δC 114.6
(C-800). This was further confirmed by HMBC correlations
between the exo-methylene group and C-600 and C-900. This
completed the structural elucidation of compound 4. Like S1 and
other acylphloroglucinols isolated from H. olympicum L. cf.
uniflorum, the specific rotation of compound 4 showed a positive
value ([α]2D2 +2.7 (c 0.32, CHCl3)). However, two stereogenic
centers (C-20 and C-600) were present in 4, and thus the
configuration at each stereogenic center could not be determined
by direct comparison with the specific rotation of S1. A further
experiment using a starch-iodide test strip (Fisher) was con-
ducted to confirm the presence of the hydroperoxide group in
this compound. A starch-iodide test strip was dipped into a
CHCl3 solution of compound 4 (approximately 2 mg/mL) and
showed a positive color reaction (light brown). CHCl3 was used
as a negative control and did not result in any color change. A
CHCl3 solution of mCPBA (20 mg/mL) was used as a positive
control, and it turned the test strip dark blue. When a lower
concentration of mCPBA was used (approximately 2 mg/mL),
the test strip turned light brown, implying that the color change
was dependent on the concentration of the hydroperoxide
present. Compound 4 was therefore identified as 4,6-dihy-
droxy-2-O-(600-hydroperoxy-300,700-dimethyl-200,700-octadienyl)-
1-(20-methylbutanoyl)benzene (olympicin D) and is reported
here for the first time.
Compound 5 was isolated as a pale yellow oil from the CH2Cl2
extract of H. olympicum L. cf. uniflorum. The molecular formula of
compound 5 was C21H30O5, indicated by an [M + H]+ ion at
m/z 363 in the high-resolution ESIMS. The 1H NMR spectrum
(Table 2) revealed signals typical of a (2-methylbutanoyl)phlor-
oglucinol, including a broad signal accounting for a hydroxy
group (δ 6.90, 4-OH), two meta-coupled aromatic protons
(δ 6.02 d, J = 2.5, H-3; 5.98 d, J = 2.5, H-5), a methine multiplet
(δ3.63, 1H, H-20), andamethylenegroup(δ1.38 m, 1.81 m, H2-30).
More interestingly, duplication of the following signals was
observed: a highly deshielded hydrogen-bonded singlet (δ 13.95,
6-OH), a methyl triplet (δ 0.90, J = 7.5, H3-40), and a methyl
doublet (δ 1.13, J = 7, H3-50). The carbon signals corresponding
to the phloroglucinol nucleus included three deshielded aromatic
carbons (δ 167.6, C-6; 162.3, C-2; 162.8, C-4), a quaternary
aromatic carbon (δ 105.5, C-1), and two aromatic methines
(δ 92.7, C-3; 96.8, C-5). Duplication of the signals in the 13C
NMR spectrum was also observed for the 2-methylbutanoyl side-
chain: a carbonyl carbon (δ 210.1 and 210.0, C-10), a methine
strain
control
(μM)
(μM)
S. aureus
SA-1199B
XU212
norfloxacin
100
2.9
tetracycline
erythromycin
norfloxacin
oxacillin
288
349
3.1
2.9
2.9
2.9
1.45
2.9
RN4220
ATCC25923
EMRSA-15
79
EMRSA-16
oxacillin
1276
Mycobacterium
M. smegmatis ATCC 14468 isoniazid; ethambutol 14.6; 2.4
M. fortuitum ATCC 6841 isoniazid; ethambutol 29.2; 2.4
M. smegmatis MC2 2700
M. phlei ATCC 11758
P. aeruginosa
K 1119
11.6
23.2
11.6
11.6
isoniazid; ethambutol 14.6; 2.4
isoniazid; ethambutol 14.6; 2.4
norfloxacin
norfloxacin
6.2
6.2
>1 mM
>1 mM
K 767
S. enterica serovar
Typhimurium
L 354
tetracycline
tetracycline
2.3
2.3
>1 mM
>1 mM
L 10
of C21H30O6. The 1H and 13C NMR data (Table 2) for compound
4 were similar to those of compound 3. The characteristic H
1
NMR features of a (2-methylbutanoyl)phloroglucinol were again
seen in this compound, including one highly deshielded hydro-
gen-bonded hydroxy group (δ 13.99, 6-OH), two meta-coupled
aromatic protons (δ 5.91 d, J = 2.5 Hz, H-3; 5.98 d, J = 2.5 Hz,
H-5), one methine (δ 3.63 m, H-20), one methylene (δ 1.35 m,
1.80 m, H2-30), one methyl triplet (δ 0.88, J = 7.5 Hz, H3-40), and
one methyl doublet (δ 1.11, J = 6 Hz, H3-50). Signals in the 13C
NMR spectrum accounting for a (2-methylbutanoyl)phloroglu-
cinol nucleus consisted of three deshielded quaternary aromatic
carbons (δ 167.5, C-6; 162.3, C-3; 162.0, C-4), one quaternary
aromatic carbon (δ 105.9, C-1), two aromatic methine carbons
(δ 91.7, C-3; 96.6, C-5), one carbonyl carbon (δ 210.3, C-10),
one methine (δ 46.1, C-20), one methylene (δ 26.8, C-30), and
two methyl carbons (δ 11.9, C-40; 16.6, C-50). The connection
of these protons and carbons was made through HMBC studies
and was identical to those seen in the structural elucidation of
compound 1.
1
The H NMR signals for the side-chain at the 2-O position
consisted of four methylenes, including an oxymethylene (δ 4.58
d, J = 6.5 Hz, H2-100) and an exo-methylene (δ 5.02 s, 5.05 t,
H2-800), one olefinic proton (δ 5.52 dt, H-200), one deshielded
methine triplet (δ 4.32, J = 6.5 Hz, H-600), and two overlapping
methyl singlets (δ 1.75, 6H, H3-900, H3-1000). Ten carbon signals
were observed in the 13C NMR spectrum for this side-chain,
including two methines (δ 89.0, C-600; 119.2, C-200), four
methylenes (δ 65.6, C-100; 35.2, C-400; 26.8, C-500; 114.6, C-800),
two quaternary olefinic carbons (δ 141.2, C-300; 143.3, C-700),
and two methyl carbons (δ 17.2, C-900; 16.6 C-1000). These
signals were indicative of a geranyl derivative and were again
similar to those of compound 3.
The oxymethylene group (δ 4.58, H2-100) exhibited HMBC
correlations with two carbons at δC 119.2 (C-200) and 141.2
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Journal of Natural Products
ARTICLE
(δ 46.09 and 46.12, C-20), a methylene (δ 26.93 and 26.96, C-30),
and two methyl carbons (δ 11.96 and 12.00, C-40; 16.52 and
16.55, C-50). The HMBC correlations observed for the acylph-
loroglucinol nucleus of this compound were identical to those of
compound 1 and were as described previously. The duplication
of NMR signals was indicative of the presence of two rotamers,
and this phenomenon has been observed in some natural pro-
ducts, including acylphloroglucinols.9 In the case of acylphlor-
oglucinols, two isomers in rotameric forms were possible: one
with the acyl chain above the plane of the phloroglucinol and the
other below the plane. Molecular modeling calculation was
carried out for this compound. Since two relatively stable con-
formations were possible, two different energy minima were
expected. However, there was only one energy minimum observed
in the calculation.
25923). The activity of olympicin A seemed to be unaffected by
the MDR mechanisms, as shown by the fairly consistent MIC
values against the different effluxing strains. It was also active
against four Mycobacterium strains at MIC values ranging from 4
to 8 mg/L, but was not as active as the control antibiotics.
Olympicin A was not active against any of the Gram-negative
species at 512 mg/L. This may result from the impermeability of
the outer membrane of the Gram-negative bacteria, which pre-
vents the influx of chemicals from the surrounding environment
into the cells, or may be due to the different efflux pumps these
bacteria express.
Compounds 2ꢀ5 (olympicins BꢀE) were moderately active
against the Staph. aureus strains, with MICs ranging from 64 to
128 mg/L. The simple nature of olympicin A and the ease of its
synthesis, coupled with its activity toward Gram-positive strains
of Staph. aureus, some of which are effluxing and methicillin-
resistant, make this chemotype worthy of further evaluation, and
synthetic efforts are underway to optimize its antibacterial action.
The need for new topical antibacterials, particularly those used in
the decolonization of MRSA from patients, offers a potential use
for these simple acylphloroglucinols, particularly given the resis-
tance occurring to conventional agents such as mupirocin and
fusidic acid.
1
The H NMR signals for the side-chain at the 2-O position
included three methylenes (δ 4.67 m, H2-100; 2.30 m, H2-400; 1.65
m, 1.90 m, H2-500), one olefinic proton (δ 5.50 m, H-200), one
deshielded oxymethine (δ 2.77 dt, H-600), and three methyl
singlets (δ 1.34 s, H3-800; 1.31 s, H3-900; 1.76 s, H3-1000). Ten
carbon signals were observed in the 13C NMR spectrum for this
side-chain, including three methylenes (δ 66.4, C-100; 37.0, C-400;
26.5, C-500), an olefinic carbon (δ 121.9, C-200), an oxymethine
(δ 65.1, C-600), and three methyl carbons (δ 24.6, C-800; 18.9,
C-900; 16.0, C1000).
’ EXPERIMENTAL SECTION
Due to the small quantity of the compound, signals in the
HMBC spectrum were very weak. The COSY spectrum and the
structures of the other related derivatives isolated from this plant
played an important role in the structural elucidation of this
compound. As with the other acylphloroglucinols, the oxymethy-
lene group (δ 4.67 m, H2-100) was placed at C-2 of the
acylphloroglucinol. The oxymethylene protons showed a COSY
correlation with the olefinic proton (δ 5.50 m, H-200), thus
placing the olefinic proton at C-200. The methyl group shifted
downfield at δH 1.76 (H3-1000) showed a 2J HMBC correlation to
General Experimental Procedures. Optical rotations were
measured on a Bellingham and Stanley ADP 200 polarimeter. UV
spectra were recorded on a Thermo Electron Corporation Helios
spectrophotometer, and IR spectra were recorded on a Nicolet 360
FT-IR spectrophotometer. NMR spectra were recorded on a Bruker
AVANCE 500 MHz spectrometer. Chemical shift values (δ) are
reported in ppm, relative to appropriate internal solvent standard, and
J values are given in Hz. Mass spectra were recorded on a Finnigan MAT
95 high-resolution, double-focusing, magnetic sector mass spectrometer.
Accurate mass measurement was achieved using voltage scanning of the
accelerating voltage. This was nominally 5 kV, and an internal reference
of heptacosa was used. Resolution was set between 5000 and 10 000.
Plant Material. The aerial parts of H. olympicum L. cf. uniflorum N.
Robson (accession number 1969-31184) were collected from the Royal
Botanic Garden at Wakehurst Place, Surrey, which forms part of the
National Hypericum Collection.
Extraction and Isolation. Dried, powdered plant material (937 g)
was sequentially extracted with 3.5 L of n-hexane, CH2Cl2, and MeOH
using a Soxhlet apparatus. The n-hexane and CH2Cl2 extracts were active
at a concentration of 32 and 16 mg/L, respectively, whereas the MeOH
extract was active at 512 mg/L. The n-hexane extract (15.2 g) was
fractionated by VLC (silica gel PF254+366; Merck) using a step-gradient
solvent system from 100% n-hexane to 100% EtOAc with a 10%
increment and a final MeOH wash. VLC fractions 6 to 8 were active
against SA-1199B with an MIC value of 64 mg/L. They displayed
similar TLC profiles and were combined (total of 842.0 mg). The
combined fraction was separated by Sephadex LH-20 (Amersham
Biosciences) chromatography, giving five fractions eluted with
CHCl3/MeOH (1:1) and one fraction eluted with MeOH. The
fraction eluted with MeOH (80.9 mg) was active at an MIC of 1 mg/L.
Compound 1 (29.1 mg) was isolated from this fraction using pre-
parative TLC (silica; toluene/EtOAc/HOAc, 80:18:2, Rf = 0.62, yield
0.0031%).
3
a quaternary olefinic carbon at δ 138.5 (C-300) and J HMBC
correlations to C-200 and a methylene carbon at δ 37.0 (C-400).
The methylene protons at C-400 (δ 2.30 m) showed a COSY
correlation to a further methylene group (δ 1.65 m, 1.90 m,
C-500), which was coupled to an oxymethine, as revealed by the
COSY spectrum (δ 2.77 dt, H-600). The remaining methyl groups
at δ 1.34 (H3-800) and 1.31 (H3-900) showed HMBC correlations
to δ 18.9 (C-900) and 24.6 (C-800), respectively, indicating that
the methyl groups were geminal to each other. These methyl
groups also correlated to a quaternary carbon at δ 59.1 (C-700)
and the oxymethine carbon (C-600). The 13C NMR chemical
shifts of C-600 and C-700 were indicative of an epoxy moiety,10
1
and the H and 13C NMR data of this side-chain showed
close agreement with the corresponding substituent of 60,70-
epoxygeranyloxypsoralen.10 Again, this compound showed a
positive specific rotation ([α]2D2 +2.6 (c 0.19, CHCl3)). However,
due to the presence of two stereogenic centers (C-20 and C-600) in
the molecule, the configuration at the stereocenter could not be
determined by direct comparison with that of S1. Compound 5
was therefore identified as the new 4,6-dihydroxy-2-O-(600,700-
epoxy-300,700-dimethyloct-200-enyl)-1-(20-methylbutanoyl)benzene
and was given the trivial name olympicin E.
Olympicin A (1) displayed exceptional activity against all of
the Staph. aureus strains tested, with MIC values ranging from
0.5 to 1 mg/L (Table 3). It was more active than the control
antibiotics against the MDR and epidemic strains and as active as
norfloxacin with the standard susceptibility testing strain (ATCC
A portion (2.5 g) of the CH2Cl2 extract was applied to a Sephadex
LH-20 column, giving five fractions eluted with CHCl3/MeOH (1:1)
and one fraction eluted with MeOH. Fractions 4 to 6 exhibited excellent
antibacterial activity at 0.5 mg/L. They showed similar TLC profiles and
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Journal of Natural Products
ARTICLE
were combined, giving 62.0 mg. The combined fraction was directly
purified by p-TLC (silica; toluene/EtOAc/HOAc, 75:23:2), yielding
compounds 2 (4.2 mg, Rf = 0.48, yield 0.00045%), 3 (3.8 mg, Rf = 0.44,
yield 0.00041%), and 4 (1.8 mg, Rf = 0.34, yield 0.00019%). Sephadex
fraction 3 (140.8 mg) was active at 512 mg/L. It was further separated by
SPE on a silica gel column using a step-gradient system from 100%
n-hexane to 100% EtOAc. SPE fraction 8 (17.8 mg), which was eluted
with 70% EtOAc in n-hexane, was purified by p-TLC (silica; toluene/
EtOAc/HOAc, 75:23:2), yielding compound 5 (1.3 mg, Rf = 0.57, yield
0.00013%). All compounds gave an orange color reaction with vanillin-
H2SO4 spray on a TLC plate.
Information; HR-ESIMS (m/z) 209.0813 [M ꢀ H]ꢀ (calcd for C11H14O4,
209.0814).
(S)-1-(2,4-Bis[(tert-butyldimethylsilyl)oxy]-6-hydroxyphenyl)-2-
methylbutan-1-one (10) (ref 13). Acylphloroglucinol 9 (9.71 g, 46.19
mmol) was dissolved in 150 mL of dry acetone. Imidazole (3.43 g, 138.6
mmol, 3 equiv) was added to the solution, and the reaction mixture
stirred for 5 min before the addition of TBDMS-Cl (14.61 g, 97.0 mmol,
2.1 equiv). The reaction mixture was stirred for 2 h at room temperature.
The reaction mixture was diluted with CHCl3 and washed with 1 M HCl
(150 mL). The solvent was removed under reduced pressure, and the
crude product purified by VLC over silica gel to afford TBDMS-
protected phloroglucinol 10 in the fraction eluted with 9:1 n-hexane/
EtOAc. The title compound was isolated as a pale yellow oil (16.4 g,
37.26 mmol, 81%): [α]2D2 +4.9 (c 0.39, CHCl3); UV (CHCl3) λmax
(log ε) 239 (4.26), 290 (4.23) nm; IR νmax (thin film) cmꢀ1 3276, 2973,
4,6-Dihydroxy-2-O-(300,700-dimethyl-200,600-octadienyl)-1-(20-methyl-
butanoyl)benzene (1), olympicin A: pale yellow oil; [α]2D2 +6.0 (c 0.25,
CHCl3); UV (CHCl3) λmax (log ε) 240 (4.05), 289 (4.35) nm; IR νmax
(thin film) cmꢀ1 3348, 2968, 2931, 1624, 1593, 1448, 1377, 1216, 1162,
1099, 826; 1H NMR and 13C NMR (CDCl3) see Table 1; HR-ESIMS
(m/z) 345.2056 [M ꢀ H]ꢀ (calcd for C21H30O4, 345.2071).
1
1688, 1572, 1531, 1256, 1131, 1072, 850; H NMR and 13C NMR
(CDCl3), see Supporting Information; HR-ESIMS (m/z) 437.2554
4,6-Dihydroxy-2-O-(700-hydroxy-300,700-dimethyl-200,500-octadienyl)-
1-(20-methylbutanoyl)benzene (2), olympicin B: pale yellow oil; [α]2D2
+5.8 (c 0.12, CHCl3); UV (CHCl3) λmax (log ε) 240 (4.26), 290
(3.62) nm; IR νmax (thin film) cmꢀ1 3338, 2967, 2934, 2873, 1620,
[M ꢀ H]ꢀ (calcd for C23H42O4Si2, 437.2549).
(S)-4,6-Dihydroxy-2-O-(300,700-dimethyl-200,600-octadienyl)-1-(20-
methylbutanoyl)benzene (S1), Olympicin A (ref 13). TBDMS-pro-
tected acylphloroglucinol 10 (6.6 g, 15.0 mmol) was dissolved in 100 mL
of dry DMF, to which anhydrous K2CO3 was added (3.1 g, 22.5 mmol,
1.5 equiv). The mixture was stirred for approximately 5 min before the
addition of geranyl bromide (3.43 mL, 18 mmol, 1.2 equiv). The mixture
was heated at 80 °C for 3 h with stirring. The reaction mixture was
poured over H2O and extracted with CHCl3. The solvent in the organic
layer was removed under reduced pressure. The crude product was
purified by chromatography over silica gel by VLC. Compound S1 was
eluted with 9:1 n-hexane/EtOAc, and removal of the solvents under
reduced pressure yielded the title compound as a pale yellow oil. All
spectral data were identical to those of the natural product 1, and the
overall yield was 1.6%.
1
1594, 1448, 1217; H NMR and 13C NMR (CDCl3) see Table 1;
HR-ESIMS (m/z) 363.2168 [M + H]+ (calcd for C21H30O5, 363.2172).
4,6-Dihydroxy-2-O-(600-hydroxy-300,700-dimethyl-200,700-octadienyl)-
1-(20-methylbutanoyl)benzene (3), olympicin C: pale yellow oil; [α]2D2
+2.5 (c 0.41, CHCl3); UV (CHCl3) λmax (log ε) 240 (4.44), 288
(4.90) nm; IR νmax (thin film) cmꢀ1 3357, 1734, 1653, 1558, 1540,
1506, 1457; 1H NMR and 13C NMR (CDCl3) see Table 1; HR-ESIMS
(m/z) 363.2163 [M + H]+ (calcd for C21H30O5, 363.2172).
4,6-Dihydroxy-2-O-(600-hydroperoxy-300,700-dimethyl-200,700-octadienyl)-
1-(20-methylbutanoyl)benzene (4), olympicin D: pale yellow oil; [α]2D2
+2.7 (c 0.32, CHCl3); UV (CHCl3) λmax (log ε) 240 (4.05), 289
(4.35) nm; IR νmax (thin film) cmꢀ1 3397, 2970, 2928, 1622, 1594,
1456, 1217; 1H NMR and 13C NMR (CDCl3) see Table 2; HR-ESIMS
(m/z) 379.2120 [M + H]+ (calcd for C21H30O6, 379.2121).
Antibacterial Assay with Staphylococcus aureus. Unless
otherwise stated, all chemicals were obtained from Sigma-Aldrich
Company Ltd., UK. Cation-adjusted Mueller-Hinton broth was ob-
tained from Oxoid and was adjusted to contain 20 and 10 mg/L of Ca2+
and Mg2+, respectively. The Staph. aureus strains used in this study
included ATCC 25923, SA-1199B, RN4220, XU212, EMRSA-15, and
EMRSA-16. ATCC 25923 is a standard laboratory strain sensitive to
antibiotics.14 SA-1199B overexpresses the NorA MDR efflux pump.15
RN4220 possesses the MsrA macrolide efflux protein.16 XU212 is a
Kuwaiti hospital isolate that is a MRSA strain possessing the TetK
tetracycline efflux pump.14 EMRSA-1517 and EMRSA-1618 are epidemic
strains in the U.K. Mycobacterium species used in this study included the
fast-growing species M. smegmatis ATCC14468, M. fortuitum ATCC6841,
and M. phlei ATCC11758. All were obtained from the National
Collection of Type Cultures (NCTC). M. smegmatis MC22700, which
possesses the M. tuberculosis fatty acid synthase I gene (Fas 1), was
obtained from Zimhony et al.19 Two wild-type Gram-negative bacteria
were used in this study, namely, Pseudomonas aeruginosa K767 and
Salmonella typhimurium L354, and both were obtained from the NCTC.
Staph. aureus, P. aeruginosa, and S. typhimurium strains were cultured
on nutrient agar (Oxoid) and incubated for 24 h at 37 °C prior to MIC
determination. Mycobacterium species were cultured on Columbia agar
(Oxoid) supplemented with 7% defibrinated horse blood (Oxoid) and
were subcultured and incubated for 72 h at 37 °C prior to the assay. An
inoculum density of 5 ꢁ 105 colony forming units of each bacterial strain
was prepared in normal saline (9 g/L) by comparison with a 0.5
MacFarland turbidity standard. The inoculum (125 μL) was added to
all wells, and the microtiter plate was incubated at 37 °C for the
corresponding incubation time. For MIC determination, 20 μL of a 5
mg/mL methanolic solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-
nyltetrazolium bromide (MTT) was added to each of the wells and
incubated for 20 min. Bacterial growth was indicated by a color change
4,6-Dihydroxy-2-O-(600,700-epoxy-300,700-dimethyloct-200-enyl)-1-(20-
methylbutanoyl)benzene (5), olympicin E: pale yellow oil; [α]2D2 +2.6
(c 0.19, CHCl3); UV (CHCl3) λmax (log ε) 240 (4.05), 289 (3.68) nm;
IR νmax (thin film) cmꢀ1 3376, 2970, 2934, 1622, 1593, 1447, 1217; 1H
NMR and 13C NMR (CDCl3) see Table 2; HR-ESIMS (m/z) 363.2181
[M + H]+ (calcd for C21H30O5, 363.2166).
Synthesis of Compound 1. (S)-2-Methylbutanoyl Chloride (7)
(ref 11). (S)-2-Methylbutanoic acid (6; 10 g, 97.91 mmol) and SOCl3
(10.71 mL, 146.9 mmol, 1.5 equiv) were heated together at 80 °C under
reflux for 2 h. Distillation of the reaction mixture afforded (S)-2-
methylbutanoyl chloride (10.63 g, 88.21 mmol, 90%): colorless liquid;
[α]2D2 +10.1 (c 0.54, CHCl3); bp 119ꢀ120 °C; 1H NMR and 13C NMR
(CDCl3) see Supporting Information.
(S)-2-Methyl-1-(2,4,6-trihydroxyphenyl)butan-1-one (9) (ref 12). AlCl3
(46.43 g, 351.8 mmol, 4.1 equiv) was added to a stirred suspension
of phloroglucinol (8; 10.81 g, 85.8 mmol, 1 equiv) in CS2 (50 mL).
Nitrobenzene (40 mL) was added to the solution over 30 min. The solution
was heated under reflux at 55 °C for 30 min. A solution of 7 (10.34 g,
85.8 mmol) dissolved in 5 mL of nitrobenzene was added to the reaction
mixture over 30 min, followed by heating for another 30 min. The reaction
mixturewasallowedtocoolwithstirringandthenpouredintoaniceꢀwater
bath (400 mL). Then 100 mL of 3 M HCl was added. The organic solvents
were removed under reduced pressure, and the oily residue containing the
acylphloroglucinol was extracted into Et2O. After the removal of the Et2O,
the crude product was purified by VLC using silica. The fraction eluting with
6:4 n-hexane/EtOAc was identified to be the title compound, isolated as a
pale yellow oil (9.71 g, 46.19 mmol, 54%): [α]2D2 +8.5 (c 0.35, CHCl3); UV
(CHCl3) λmax (log ε) 240 (4.17), 290 (3.97) nm; IR νmax (thin film) cmꢀ1
3297, 1628, 1602, 1222; 1H NMR and 13C NMR (CDCl3), see Supporting
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Journal of Natural Products
ARTICLE
from yellow to dark blue. The MIC was recorded as the lowest concentra-
tion at which no growth was observed.14
’ ASSOCIATED CONTENT
S
Supporting Information. This material is available free
b
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +44 207 753 5913. Fax: +44 207 753 5964. E-mail: simon.
gibbons@pharmacy.uk.
’ ACKNOWLEDGMENT
We thank the School of Pharmacy for the award of an EPSRC
DTA to W.K.P.S.
’DEDICATION
Dedicated to Dr. Gordon M. Cragg, formerly Chief, Natural
Products Branch, National Cancer Institute, Frederick,
Maryland, for his pioneering work on the development of
natural product anticancer agents.
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