Novel prenylated and geranylated aromatic compounds isolated from Polysphondylium cellular slime molds

Article abstract of DOI:10.1016/j.tet.2010.06.029

We have studied the diversity of secondary metabolites of cellular slime molds to utilize them as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium tenuissimum, we obtained five prenyla

Full text of DOI:10.1016/j.tet.2010.06.029

Tetrahedron 66 (2010) 6000e6007
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Novel prenylated and geranylated aromatic compounds isolated from
Polysphondylium cellular slime molds
,
a
a
b
a,
*
Haruhisa Kikuchi a , Shinya Ishiko , Koji Nakamura , Yuzuru Kubohara , Yoshiteru Oshima
*
a Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai 980-8578, Japan
b Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have studied the diversity of secondary metabolites of cellular slime molds to utilize them as new
biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Pol-
ysphondylium tenuissimum, we obtained five prenylated and geranylated aromatic compounds, Pt-1e5
(1e5). An additional aromatic compound, Ppc-1 (6), was isolated from Polysphondylium pseudo-candi-
dum. The structures of these compounds were determined by spectral analysis, and synthetic routes to 4,
5, and 6 were developed. Compound 5 showed the glucose consumption-promotive activity on 3T3-L1
cells.
Received 9 April 2010
Received in revised form
8 June 2010
Accepted 9 June 2010
Available online 16 June 2010
Keywords:
Cellular slime molds
Ó 2010 Elsevier Ltd. All rights reserved.
Polysphondylium tenuissimum
Prenylated aromatic compounds
Geranylated aromatic compounds
Glucose consumption
1. Introduction
although no other reports have, to date, described the secondary
metabolites of cellular slime molds.
The cellular slime mold Dictyostelium discoideum is thought to
be an excellent model organism for the study of cell and de-
velopmental biology because of its simple pattern of development.1
Vegetative cells of D. discoideum grow as single amoebae by eating
bacteria. When starved, they initiate a developmental program of
morphogenesis and gather to form a slug-shaped multicellular
aggregate. This aggregate then differentiates into two distinct cell
types, prespore, and prestalk cells, which are precursors of spores
and stalk cells, respectively. At the end of its development, the
aggregate forms a fruiting body consisting of spores and a multi-
cellular stalk.
We have focused on the utility of cellular slime molds as a re-
source for novel drug development, and have studied the diversity
of secondary metabolites of cellular slime molds.12 Cellular slime
molds are classified into three genera, Dictyostelium, Poly-
sphondylium, and Acytostelium, according to their morphology.13
We have recently isolated
a
-pyronoids,12a,f amino sugar deriva-
tives,12c,d and aromatics12b,e with unique structures from several
species of Dictyostelium. This paper reports the isolation, structure
elucidation and synthesis of new prenylated and geranylated aro-
matic compounds Pt-1e5 (1e5) and Ppc-1 (6) from Poly-
sphondylium cellular slime molds (Fig. 1). The antiproliferative and
glucose consumptionepromotive activities of these compounds in
mammalian cells are also described.
Several small molecules including DIFs (Differentiation-In-
ducing Factors),2 discadenine,3 and cAMP4 have been reported as
development-regulating substances of cellular slime molds. DIFs
and their derivatives also exhibit many biological effects in mam-
malian cells, such as suppression of cell growth,5e7 induction/
promotion of cell differentiation,5a,d promotion of glucose con-
2. Results and discussion
2.1. Isolation and structure elucidation
sumption7,8 and regulation of IL-2 production.9
A chlorine-
substituted dibenzofuran derivative AB0022A10 and a resorcinol
derivative MPBD11 were also isolated from cellular slime molds,
Fruiting bodies (dry weight 73.6 g) of the cellular slime mold,
Polysphondylium tenuissimum, were cultured on plates and extrac-
ted three times with methanol at room temperature to yield an
extract (18.2 g) that was subsequently partitioned between ethyl
acetate and water. The ethyl acetate solubles (5.49 g) were sepa-
rated by repeated column chromatography over SiO2 and ODS to
* Corresponding authors. Tel.: þ81 22 795 6822; fax: þ81 22 795 6821; e-mail
addresses: hal@mail.pharm.tohoku.ac.jp (H. Kikuchi), oshima@mail.pharm.tohoku.
ac.jp (Y. Oshima).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.06.029

H. Kikuchi et al. / Tetrahedron 66 (2010) 6000e6007
6001
with C-5 and C-8; and H2-10 with C-4 and C-8, in the HMBC spec-
trum, indicated that two methyl groups (C-11 and C-12) and a ger-
anyl group were attached to N-3, N-7, and N-9, yielding the
complete structure of 1 (Fig. 2).
Figure 2. Structural elucidation of Pt-1 (1).
HREIMS of 2 (m/z 318.1688 [M]þ) indicated a molecular formula,
C16H22N4O3, which differed from that of 1 by a methylene unit. The
1H and 13C NMR spectra of 2 were nearly identical to those of 1
(Table 1), suggesting that the methyl group and geranyl group were
attached to the uric acid moiety in the structure of 2. In the HMBC
spectrum of 2, the two sets of correlation peaks: H3-11 to the sig-
nals at dc 137.6 (C-4) and 151.5; and H2-10 to the signals at dc 137.6
(C-4) and 153.2, indicated that these groups were bonded to N-3
and N-9 (Fig. 3). However, the positions of each substituent could
not be determined because the chemical shifts of the signals at dc
151.5 and 153.2 were too close to assign them to C-2 and C-8, re-
spectively. To resolve this issue, compound 2 was methylated. The
HMBC spectrum of the product 7 showed two sets of correlation
peaks: between H3-12 and the signals at dc 99.5 and 151.9; and
between H2-10 and the signals at dc 135.4 and 151.9. Because the
signals at dc 99.5 and 135.4 could be readily assigned to C-5 and C-4,
respectively, the position of the geranyl group was assigned to N-9.
Figure 1. Structures of Pt-1e5 (1e5) and Ppc-1 (6).
yield Pt-1 (1) (1.8 mg), Pt-2 (2) (9.7 mg), Pt-3 (3) (0.8 mg), Pt-4 (4)
(2.6 mg), and Pt-5 (5) (4.7 mg). In the same manner, Ppc-1 (6)
(1.8 mg) was obtained from the fruiting bodies (dry weight 57.3 g)
of Polysphondylium pseudo-candidum.
HREIMS (m/z 332.1835 [M]þ), 1H and 13C NMR spectra indicated
that the molecular formula of 1 was C17H24N4O3. The 13C NMR
spectrum of 1 showed the presence of seven quaternary sp2 car-
bons, two tertiary sp2 carbons, three methylene carbons and five
methyl carbons (Table 1). 1He1H COSY revealed that C-10eC-20 and
C-40eC-50eC-60 were connected. The correlations of H3-90eC-20, C-
30, and C-40; and H3-100eC-60, C-70, and C-80 in the HMBC spectrum
indicated a geranyl group. The molecular formula of the structure
with neither the geranyl group (C10H17) nor the two methyl groups
was C5HN4O3, suggesting the presence of a trisubstituted uric acid
moiety. This was supported by the observation that the chemical
shifts of the five remaining sp2 carbons (
149.7 (C-2), 137.1 (C-4), and 99.7 (C-5)) were consistent with those
of uric acid14
153.4, 152.6, 150.1, 136.6, and 97.1). The correlations
d 152.6 (C-6), 151.8 (C-8),
(d
between the three sets of peaks: H3-11 with C-2 and C-4; H3-12
Table 1
13C and 1H NMR spectral data of Pt-1 (1), Pt-2 (2) and 7
Pt-1 (1)a
Pt-2 (2)b
7a
13C
1H
13C
1H
13C
2
4
5
149.7
137.1
99.7
151.5
137.6
99.8
150.8
135.4
99.5
Figure 3. Structural elucidation of Pt-2 (2).
6
8
152.6
151.8
154.4
153.2
153.5
151.9
28.2
30.5
29.3
10
11
12
10
Compound 3 showed a molecular ion peak at m/z 241.1091 in
HREIMS, suggesting that its molecular formula was C15H15NO2. The
13C NMR spectrum of 3 showed the presence of one carbonyl, five
sp2 quaternary, six sp2 tertiary, one methylene, and two methyl
carbons (Table 2). The 1H NMR spectrum showed the presence of
30.5 3.61 (3H, s)
29.3 3.56 (3H, s)
41.8 4.66 (2H, d, J¼6.0 Hz)
119.4 5.08e5.12 (1H, m)
140.7
39.2 2.03 (2H, t, J¼7.2 Hz)
26.1 2.07 (2H, q, J¼7.2 Hz)
123.3 4.98e5.03 (1H, m)
132.3
25.7 1.64 (3H, d, J¼1.0 Hz)
16.7 1.72 (3H, d, J¼1.0 Hz)
17.7 1.56 (3H, s)
30.0 3.68 (3H, s)
41.2 4.82 (2H, d, J¼5.7 Hz)
121.4 5.36e5.40 (1H, m)
139.5
39.4 2.05 (2H, t, J¼7.0 Hz)
26.4 2.11 (2H, q, J¼7.0 Hz)
124.3 5.07e5.12 (1H, m)
131.8
25.7 1.63 (3H, d, J¼0.8 Hz)
16.4 1.71 (3H, d, J¼1.0 Hz)
17.7 1.52 (3H, s)
41.8
20
119.6
140.5
39.2
30
40
a 1,2,3-trisubstituted benzene ring (
5), 7.62 (1H, dd, J¼8.1, 7.1 Hz, H-6), and 7.65 (1H, dd, J¼7.1, 1.2 Hz, H-
7)). Other low field-shifted proton signals at
8.26 (1H, d, J¼8.3 Hz,
d
7.78 (1H, dd, J¼8.1, 1.2 Hz, H-
50
26.1
60
123.3
132.3
25.7
16.7
17.7
70
d
80
H-3) and 8.39 (1H, d, J¼8.3 Hz, H-4) implied the presence of a ni-
trogen-containing heterocycle, and the HMBC correlations shown
in Figure 4 gave 2,8-disubstituted quinoline moiety. Moreover, the
presence of a prenyl group at C-8 was revealed by the correlation of
H2-10eH-20 in the 1He1H COSY and the correlations of H3-50eC-20,
C-30, and C-40; and H2-10eC-7, C-8, and C-8a in the HMBC spectrum.
Therefore, the remaining moiety, CHO2, was assigned as a carboxyl
90
100
1-NH
9-NH
8.08 (1H, br s)
13.40 (1H, br s)c
13.29 (1H, br s)c
1H (600 MHz) and 150 MHz for 13C in CDCl3.
1H (600 MHz) and 150 MHz for 13C in pyridine-d5.
These signals were indistinguishable.
a
b
c

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H. Kikuchi et al. / Tetrahedron 66 (2010) 6000e6007
group, and the structure of 3 was determined to be 8-pre-
nylquinoline-2-carboxylic acid. This structure was supported by the
fact that 13C NMR signals of the quinoline ring in 3 corresponded
with those from 8-ethylquinoline-2-carboxylic acid.15
tetrasubstituted benzene ring (
d
6.05 (1H, d, J¼2.4 Hz) and 5.92 (1H,
d, J¼2.4 Hz)). The 1H NMR signal at
d 14.09 (1H, s) was assigned to
a phenolic proton hydrogen-bonded to a carbonyl group. A buta-
noyl group was suggested by the correlation of H2-20eH2-30eH3-40
in the 1He1H COSY and the correlations of H2-20eC-10 in the HMBC
spectrum. A prenyl group was also deduced by the correlation of
H2-100eH-200 in the 1He1H COSYand the correlations of H3-500eC-200,
C-300, and C-400 in the HMBC spectrum. The hydrogen bond between
a phenolic proton and a carbonyl group indicated that the butanoyl
group was located on ortho position of the phenolic hydroxyl group.
Thus, the HMBC correlations of a phenolic proton to C-1, C-2, and C-
3 revealed that the hydroxyl group and the butanoyl group were
attached to C-2 and C-1, respectively. The HMBC correlations of H-3,
H-5, and H2-100eC-4 showed that the prenyloxy group was bonded
to C-4. Finally, the remaining methoxyl group was attached to C-6,
and the structure of 4 was elucidated (Fig. 5).
Table 2
13C and 1H NMR spectral data of Pt-3 (3)a
13C
1H
2
3
4
4a
5
6
7
8
8a
9
10
20
30
40
50
144.7
118.8
139.4
130.3
126.0
129.2
130.2
140.5
144.4
164.3
30.1
8.26 (1H, d, J¼8.3 Hz)
8.39 (1H, d, J¼8.3 Hz)
7.78 (1H, dd, J¼8.1, 1.2 Hz)
7.62 (1H, dd, J¼8.1, 7.1 Hz)
7.65 (1H, dd, J¼7.1, 1.2 Hz)
3.97 (2H, d, J¼7.2 Hz)
5.38e5.44 (1H, m)
122.2
133.4
25.7
1.76 (3H, br s)
1.80 (3H, br s)
17.9
a
600 MHz for 1H and 150 MHz for 13C in CDCl3.
Figure 5. Structural elucidation of Pt-4 (4).
EIMS of 5 showed two peaks, m/z 366 and 368, for molecular ion
peaks in a ratio of 3:1, suggesting that compound 5 contained one
chlorine atom. HREIMS (m/z 366.1597 [M]þ), 1H and 13C NMR
spectra indicated the molecular formula of 5 as C20H27ClO4. The 13
C
NMR spectrum of 5 showed the presence of a keto carbonyl, seven
sp2 quaternary, three sp2 tertiary, one oxymethylene, four methy-
lene, and four methyl carbons (Table 3). The presence of a ger-
anyloxy group and a butanoyl group in 5 was deduced by the
1He1H COSY and HMBC spectra as well as in compound 1 and 4,
Figure 4. Structural elucidation of Pt-3 (3).
The molecular formula for 4, C16H22O4, was proposed based
on the observation of a molecular ion peak at m/z 278.1498 in
HREIMS. The 13C NMR spectrum of 4 showed the presence of a keto
carbonyl, five sp2 quaternary, three sp2 tertiary, one oxymethylene,
one methoxyl, two methylene, and three methyl carbons (Table 3).
respectively. The 1H NMR signal at
d 14.83 (1H, s) was assigned to
a phenolic proton hydrogen-bonded to a carbonyl group. Five
quaternary sp2 carbons (dC 162.0, 160.9, 157.5, 106.3, 99.9) and
a tertiary sp2 carbon (dC 91.0 and dH 6.13) revealed a benzene ring
substituted by two phenolic hydroxy groups, a geranyloxy group,
a butanoyl group and a chlorine atom. The HMBC correlation peaks
for H-2eOH (dH 14.83 (1H, s)) to C-1, C-2, and C-3 showed that the
butanoyl group and the chlorine atom were connected to either of
C-1 (dC 106.3) and C-3 (dC 99.9). The HMBC correlation peaks for H-5
to C-1, C-3, C-4, and C-6 showed that the geranyloxy group and the
hydroxy group were connected to either of C-4 (dC 157.5) and C-6
The 1H NMR spectrum revealed the presence of
a 1,2,3,5-
Table 3
13C and 1H NMR spectral data of Pt-4 (4) and Pt-5 (5)a
Pt-4 (4)
Pt-5 (5)
13C
1H
13C
1H
1
2
3
4
105.7
167.6
106.3
162.0
(dC 160.9). Thus, there are two possible structures of 5 (Fig. 6). To
94.2 6.05 (1H, d, J¼2.4 Hz)
99.9
157.5
resolve this problem, the methylation and catalytic hydrogenation
165.0
5
91.3 5.92 (1H, d, J¼2.4 Hz)
91.0 6.13 (1H, s)
160.9
206.3
46.4 2.99 (2H, t, J¼7.4 Hz)
6
162.7
10
205.8
20
46.1 2.94 (2H, t, J¼7.4 Hz)
30
18.1 1.67 (2H, sextet, J¼7.4 Hz) 18.3 1.68 (2H, sextet, J¼7.4 Hz)
40
14.0 0.96 (3H, t, J¼7.4 Hz)
65.0 4.50 (2H, d, J¼6.8 Hz)
118.7 5.42e5.48 (1H, m)
139.3
13.9 0.97 (3H, t, J¼7.4 Hz)
65.9 4.58 (2H, d, J¼6.7 Hz)
117.9 5.48e5.51 (1H, m)
142.7
39.5 2.08e2.16 (2H, m)
26.2 2.08e2.16 (2H, m)
123.5 5.07e5.11 (1H, m)
132.1
25.6 1.68 (3H, br s)
16.7 1.75 (3H, br s)
17.7 1.61 (3H, br s)
14.83 (1H, s)
100
200
300
400
500
600
700
800
900
1000
2-OH
4-OH
25.8 1.78 (3H, br s)
18.2 1.72 (3H, br s)
14.09 (1H, s)
6.14 (1H, br s)
6-OCH3 55.5 3.82 (3H, s)
a
600 MHz for 1H and 150 MHz for 13C in CDCl3.
Figure 6. Structural elucidation of Pt-5 (5).

H. Kikuchi et al. / Tetrahedron 66 (2010) 6000e6007
6003
of 5 were carried out. The 1H NMR spectrum of the resulting
compound showed two aromatic and two methoxyl proton signals,
suggesting that the product was the non-symmetrical compound 8,
instead of the symmetric 80. Therefore, the geranyloxy group in 5
was shown to be located on C-6.
hydroxyl group of 13 was prenylated to give 14. Finally, removal of
the SEM group on C-2 by using the TBAFeHMPA system again
allowed us to synthesize 4.
Compound 6 showed a molecular ion peak at m/z 355.1754 in
HREIMS, suggesting that its molecular formula was C21H25NO4. The
13C NMR spectrum of 6 showed the presence of one carboxyl, seven
sp2 quaternary, six sp2 tertiary, two oxymethylene, one methoxyl, and
four methyl carbons (Table 4). The 1He1H COSY, and HMBC spectra
showedthe presenceoftwoprenyl groups in 6aswellasincompound
4. The 1H NMR spectrumshowed thepresenceofa1,2,3-trisubstituted
benzene ring (
6) and 7.08 (1H, dd, J¼8.1,1.1 Hz, H-7)). Other low field-shifted proton
signal at 7.60 (1H, s, H-3) implied the presence of a nitrogen-con-
d
7.76 (1H, dd, J¼8.1,1.1 Hz, H-5), 7.48 (1H, t,J¼8.1 Hz, H-
d
taining heterocycle, and the HMBC correlationpeaks for H-3 to C-2, C-
4, C-4a, and C-9; H-5 to C-4 and C-8a; H-6 to C-4a and C-8; and H-7 to
C-8 and C-8a depicted in Figure 7 gave 2,4,8-trisubstituted quinoline
moiety. Finally, the correlationpeaks foramethoxylprotontoC-4; H2-
10 to C-9; and H2-100 to C-8 in HMBC spectrum revealed the methoxyl
group, the two prenyloxy groups were attached to C-8, C-4, and C-9,
respectively, the structure of 6 was elucidated (Fig. 7).
Scheme 1. Synthesis of Pt-4 (4). Reagents and conditions: (a) butanoyl chloride, AlCl3,
CH2Cl2, 0 ꢀC (81%); (b) BBr3, CH2Cl2, ꢁ78 ꢀC/rt (95%); (c) SEMCl, DIPEA, CH2Cl2, rt (86%);
(d) MeI, K2CO3, acetone, rt (90%); (e) TBAF, HMPAeTHF (8:1), 40 ꢀC (23%); (f) 1-bromo-3-
methyl-2-butene, K2CO3, DMF, rt (60%); (g) TBAF, HMPAeTHF (8:1), 40 ꢀC (45%).
Table 4
In the course of synthesis of 5 (Scheme 2), C-3 in compound 10 was
selectively chlorinated by using 1 equiv of sulfuryl chloride to give
15. The two phenolic hydroxyl groups on C-2 and C-4 were sub-
sequently protected by SEM groups to produce 16. During ger-
anylation of 16, the SEM groups were simultaneously eliminated to
afford compounds 17 and Pt-5 (5). By using TBAFeHMPA system,
13C and 1H NMR spectral data of Ppc-1 (6).a
13C
1H
2
3
4
4a
5
6
7
8
148.2
100.7
163.2
123.5
113.1
127.9
110.0
155.3
140.7
166.0
63.2
118.8
138.8
25.8
18.4
66.2
7.60 (1H, s)
7.76 (1H, dd, J¼8.1, 1.1 Hz)
7.48 (1H, t, J¼8.1 Hz)
7.08 (1H, dd, J¼8.1, 1.1 Hz)
8a
9
10
4.95 (2H, d, J¼7.3 Hz)
5.55e5.61 (1H, m)
20
30
40
1.78 (3H, br s)
1.79 (3H, br s)
50
100
4.81 (2H, d, J¼6.4 Hz)
200
120.1
136.7
25.8
18.2
56.1
5.67e5.73 (1H, m)
300
400
1.80 (3H, br s)
1.81 (3H, br s)
4.11 (3H, s)
500
4-OCH3
Scheme 2. Synthesis of Pt-5 (5). Reagents and conditions: (a) SO2Cl2, CHCl3eEtOH
(100:1), rt (95%); (b) SEMCl, DIPEA, CH2Cl2, rt (53%); (c) geranyl bromide, K2CO3, DMF,
rt (25% (for 17) and 19% (for 5)); (d) TBAF, HMPA, 50 ꢀC (66% borsm).
a
600 MHz for 1H and 150 MHz for 13C in CDCl3.
compound 17 was also converted into 5.
Synthesis of 6 proceeded via the synthetic method described for
quinolobactin (19),17 a siderophore from Pseudomonas fluorescens
Figure 7. Structural elucidation of Ppc-1 (6).
2.2. Syntheses of 4e6
Compounds 4e6 were synthesized to confirm the structures
and obtain a sufficient quantity of sample for biological assays. The
synthesis of 4 was shown in Scheme 1. FriedeleCrafts acylation of
commercially available 5-methoxyresorcinol with butanoyl chlo-
ride gave compound 9.6 After demethylation of the 4-methoxyl
group, both of 2- and 4-hydroxyl groups were protected by SEM
groups, and the 6-hydroxyl group was methylated to produce 12.
The TBAFeHMPA system16 removed only one SEM group to afford
4-hydroxyl compound 13 and the 2-hydroxyl isomer. The phenolic
Scheme 3. Synthesis of Ppc-1 (6). Reagents and conditions: (a) 5% NaOHaq, MeOH,
reflux; (b) 1-bromo-3-methyl-2-butene, K2CO3, DMF, rt (87% for two steps).

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H. Kikuchi et al. / Tetrahedron 66 (2010) 6000e6007
(Scheme 3). Hydrolysis of 18, prepared from xanthurenic acid, gave
quinolobactin (19), which was prenylated to produce 6.
2.3. Biological evaluation
The antiproliferative activities of 1e6 were evaluated on K562
human leukemia, Hela cervical carcinoma, and 3T3-L1 mouse
embryonic fibroblast cells (Fig. 8A). Ppc-1 (6) showed about 50%
inhibition at 15
mM in all cell lines. Pt-1 (1) and Pt-5 (5) decreased
the cell number into less than 30% of control on K562 cells. The
Figure 10. Effects of 1e6 on glucose consumption in 3T3-L1 cells. Confluent 3T3-L1
cells were incubated for several hours in DMEM-MG containing 0.2% DMSO or 20
m
M
of compounds. The glucose concentration was measured, and the rate of glucose
consumption was calculated. The mean values and SD (bars) of the triplicate (n¼3) are
presented.
3. Conclusions
The prenylatedand geranylated compounds, such asPt-1e5 (1-5)
and Ppc-1 (6) have been scarcely reported, although their structural
skeletons are composed of familiar aromatic rings. Isolation of this
type of compounds shows that cellular slime molds are promising
resources for natural product chemistry. Compounds 4 and 5, which
bear butanoyl groups, may be biosynthesized via an analog of DIF-1
(20), which contains a hexanoyl group. The difference in length of
the acyl groups may be a result of the chemotaxonomic differences
between the genera Dictyostelium and Polysphondylium.
4. Experimental section
4.1. General methods
Analytical TLC was performed on silica gel 60 F254. Column
chromatography was carried out on silica gel 60 (70e230 mesh). 1H
NMR spectra were recorded on at 600 MHz, 500 MHz or 400 MHz.
13C NMR spectra were recorded on at 150 MHz, 125 MHz or
100 MHz. Chemical shifts for 1H and 13C NMR are given in parts per
million (d) relative to tetramethylsilane (dH 0.00) and residual sol-
vent signals (dC 77.0, 29.8, and 123.5 for CDCl3, acetone-d6 and
pyridine-d5, respectively) as internal standards.
Figure 8. A. Antiproliferative activities of 15 mM of 1e6 on K562, Hela and 3T3-L1 cells.
B. Detailed analysis of antiproliferative activities of 1, 5, and 6 on K562 cells. Cells were
incubated in vitro with the indicated concentrations of compounds for several days,
and the relative cell number was assessed. The mean values and SD (bars) of the
triplicate (n¼3) are presented.
4.2. Organism and culture conditions
concentration response curves for K562 cells exposed to 1, 5 and 6
gave the EC50 values of 4.4
(Fig. 8B).
mM, 10 mM, and 13 mM, respectively
The cellular slime molds, P. tenuissimum and P. pseudo-candi-
dum, were kindly supplied by Kyorin Pharmaceutical Co. Ltd.,
Tokyo, Japan and Dr. Hagiwara, National Science Museum, Tokyo,
Japan. Spores were cultured at 22 ꢀC with Escherichia coli Br on A-
medium consisting of 0.5% glucose, 0.5% polypeptone, 0.05% yeast
extract, 0.225% KH2PO4, 0.137% Na2HPO4$12H2O, 0.05%
MgSO4$7H2O, and 1.5% agar. When fruiting bodies had formed after
four days, they were harvested for extraction.
Compound 5 is a chlorine-containing acylphloroglucinol de-
rivative, and is related to DIF-1 (20) (Fig. 9),2 which induces dif-
ferentiation in Dictyostelium cellular slime molds. As mentioned, 20
promoted glucose consumption in 3T3-L1 cells.8 Therefore, we
examined the activities of 1e6 toward glucose consumption. As
shown in Figure 10, only 5 showed activity at 20
1 (20).
mM, similar to DIF-
4.3. Isolation of Pt-1e5 (1e5)
The cultured fruiting bodies (dry weight 73.6 g) of P. tenuissimum
were extracted three times with MeOH at room temperature to give
an extract (18.2 g), which was then partitioned with EtOAc and H2O
to yield EtOAc solubles (5.49 g). The EtOAc solubles were chroma-
tographed over SiO2 and the column eluted with hexaneeEtOAc and
EtOAceMeOH solutions with increasing polarity to afford hex-
aneeEtOAc (19:1) eluent (fraction A, 718 mg), hexaneeEtOAc (4:1)
Figure 9. Structure of DIF-1 (20).

H. Kikuchi et al. / Tetrahedron 66 (2010) 6000e6007
6005
eluent (fraction B, 450 mg), EtOAc eluent (fraction C, 36 mg) and
EtOAceMeOH (1:1) eluent (fraction D, 510 mg). Fraction A was
chromatographed over ODS using H2Oeacetonitrile (3:7) to give Pt-
4 (4) (2.6 mg). Fraction B was separated by ODS column (elutant:
H2Oeacetonitrile (1:1)) and silica gel column (elutant: hex-
aneeCHCl3 (1:1)) to give Pt-5 (5) (4.7 mg). Fraction C was chroma-
tographed over silica gel using CHCl3eMeOH (99:1) and then ODS
using H2OeMeOH (1:4) to afford Pt-2 (2) (9.7 mg). Fraction D was
further separated by silica gel column using CHCl3eMeOH solvent
system to give CHCl3eMeOH (99:1) elutant (fraction D1, 25 mg) and
CHCl3-MeOH (49:1) elutant (fraction D2, 8 mg). Fraction D1 was
purified by ODS column using H2Oeacetonitrile (1:2) to give Pt-3 (3)
(0.8 mg). Fraction D2 was also purified by ODS column using
H2OeMeOH (1:1) to give Pt-1 (1) (1.8 mg).
46 mmol). After being stirred for 3 h at room temperature, the re-
action mixture was poured into 0.3 M HCl, and extracted with EtOAc
three times. The combined organic layer was washed with brine,
dried over sodium sulfate, and evaporated. The residue was chro-
matographed over silica gel and eluted by CHCl3eMeOH (99:1) to
give 7 (1.9 mg, 5.5 mmol, 87%).
Data for 7 ((E)-9-(3,7-dimethylocta-2,6-dienyl)-1,3,7-trimethyl-
1H-purine-2,6,8(3H,7H,9H)-trione). Colorless amorphous solid; 1H
NMR (600 MHz, CDCl3)
d 5.08e5.12 (1H, m), 4.97e5.02 (1H, m),
4.65 (2H, d, J¼6.0 Hz), 3.61 (3H, s), 3.56 (3H, s), 3.36 (3H, s), 2.07
(2H, q, J¼7.2 Hz), 2.03 (2H, t, J¼7.2 Hz), 1.72 (3H, d, J¼1.0 Hz), 1.64
(3H, d, J¼1.0 Hz), 1.56 (3H, s); 13C NMR datum is shown in Table 1;
EIMS m/z 346 [M]þ, 210 (100%), 153, 69; HREIMS m/z 346.2012 [M]þ
(346.2005 calcd for C18H26N4O3).
Data for 1 ((E)-9-(3,7-dimethylocta-2,6-dienyl)-3,7-dimethyl-
1H-purine-2,6,8(3H,7H,9H)-trione). Colorless amorphous solid; UV
4.6. Conversion of Pt-5 (5) into 8
(EtOH) lmax, nm (log 3) 204 (4.34), 236 (3.83), 295 (4.01); IR (KBr)
nmax (cmꢁ1) 1693; 1H NMR and 13C NMR data are shown in Table 1;
EIMS m/z 332 [M]þ, 196 (100%), 153, 139, 81, 69; HREIMS m/z
332.1835 [M]þ (332.1848 calcd for C17H24N4O3).
To a solution of 5 (2.1 mg, 5.7
methyl iodide (10 L, 161 mol) and potassium carbonate (6.1 mg,
44 mol). After being stirred for 1 h at room temperature, the re-
mmol) in DMF (1.0 mL) were added
m
m
m
Data for 2 ((E)-9-(3,7-dimethylocta-2,6-dienyl)-3-methyl-1H-
purine-2,6,8(3H,7H,9H)-trione). Colorless amorphous solid; UV
action mixture was poured into 0.3 M HCl, and extracted with
EtOAc three times. The combined organic layer was washed with
brine, dried over sodium sulfate, and evaporated. The residue and
20% Pd(OH)2 on carbon (5.5 mg) in MeOH (1.0 mL) were stirred at
room temperature for 24 h under hydrogen atmosphere. After fil-
tration, the filtrate was evaporated. The residue was chromato-
graphed over silica gel and eluted by hexaneeEtOAc (49:1) to give 8
(EtOH) lmax, nm (log 3) 204 (4.31), 234 (3.81), 294 (3.99); IR (KBr)
nmax (cmꢁ1) 1700; 1H NMR and 13C NMR data are shown in Table 1;
EIMS m/z 318 [M]þ, 182 (100%), 137, 81, 69; HREIMS m/z 318.1688
[M]þ (318.1692 calcd for C16H22N4O3).
Data for 3 (8-(3-methylbut-2-enyl)quinoline-2-carboxylic acid).
Brownishamorphoussolid;UV(EtOH) lmax, nm(log
3
)206(4.09), 239
(1.0 mg, 2.9
dimethyloctyloxy)-3,5-dimethoxybenzene): colorless oil; 1H NMR
(400 MHz, CDCl3) 6.09e6.14 (2H, m), 3.92e3.98 (2H, m), 3.79 (3H,
s), 3.78 (3H, s), 2.55 (2H, t, J¼7.5 Hz), 1.77e1.84 (1H, m), 1.68e1.73
(1H, m), 1.50e1.57 (2H, m), 1.40e1.45 (2H, m), 1.28e1.35 (6H, m),
1.13e1.18 (2H, m), 0.90e0.94 (6H, m), 0.86e0.88 (6H, m); 13C NMR
mmol, 50% (two steps)). Data for 8 (2-butyl-1-(3,7-
(4.06), 314 (3.29); IR (KBr) nmax (cmꢁ1) 2924, 2852,1723; 1H NMR and
13C NMR data are shown in Table 2; EIMS m/z 241 [M]þ, 198 (100%),
180, 154; HREIMS m/z 241.1091 [M]þ (241.1103 calcd for C15H15NO2).
d
Data for
4 (1-(2-hydroxy-6-methoxy-4-(3-methylbut-2-eny-
loxy)phenyl)butan-1-one). Yellowish amorphous solid; UV (EtOH)
lmax, nm (log
3
) 206 (4.26), 225 (sh, 4.11), 288 (4.19); IR (KBr) nmax
(100 MHz, CDCl3) d 158.9, 158.8, 158.3, 112.4, 91.4, 90.5, 66.4, 55.7,
(cmꢁ1) 2959, 2932, 2872,1620, 1594; 1H NMR and 13C NMR data are
shown in Table 3; EIMS m/z 278 [M]þ, 210, 167 (100%), 69; HREIMS
m/z 278.1498 [M]þ (278.1518 calcd for C16H22O4).
55.3, 39.3, 37.3, 36.5, 31.9, 29.8, 28.0, 24.8, 22.8, 22.7, 22.6, 22.4,19.6,
14.1; EIMS m/z 350 [M]þ, 307 (100%), 169, 167; HREIMS m/z
350.2812 [M]þ (350.2821 calcd for C22H38O3).
Data for 5 ((E)-1-(3-chloro-6-(3,7-dimethylocta-2,6-dienyloxy)-
2,4-dihydroxyphenyl)butan-1-one). Yellowish amorphous solid;
4.7. 1-[2-Hydroxy-4,6-bis[(2-trimethylsilylethoxy)methoxy]
phenyl]butan-1-one (11)
UV (EtOH) lmax, nm (log 3) 205 (4.36), 229 (sh, 4.01), 293 (3.97), 329
(3.91); IR (KBr) nmax (cmꢁ1) 2964, 2924, 2852, 1613, 1556; 1H NMR
and 13C NMR data are shown in Table 3; EIMS m/z 368[Mþ2]þ, 366
[M]þ, 232, 230, 189, 187, 137, 93, 81, 69 (100%); HREIMS m/z
366.1597 [M]þ (366.1598 calcd for C20H2735ClO4).
To a solution of 96 (793 mg, 3.77 mmol) in dichloromethane
(20 mL) was added 1.0 M boron tribromide solution in dichloro-
methane (20 mL) at ꢁ78 ꢀC. After being stirred for 2 h at room tem-
perature, the reaction mixture was poured into saturated sodium
bicarbonate solution and extracted with EtOAc three times. The or-
ganic layer was washed with water and brine, dried over
anhydrous sodium sulfate, and evaporated. The residue was chro-
matographed over silica gel eluted by hexaneeEtOAc (2:1) to give
1 -(2,4,6-Trihydroxyphenyl)butan-1-one (10) (701 mg, 3.57 mmol,
95%).
4.4. Isolation of Ppc-1 (6)
In a similar manner to the isolation of 1e5, the cultured fruiting
bodies of P. pseudo-candidum (dry weight 85.1 g) yielded a MeOH
extract (16.9 g), which was then partitioned with EtOAc and H2O to
yield EtOAc solubles (3.60 g). The EtOAc solubles were chromato-
graphed over SiO2, and the column eluted with hexaneeEtOAc with
increasing polarity. HexaneeEtOAc elutant (55 mg) was subjected
To a solution of 10 (205 mg, 1.05 mmol) in dichloromethane
(4.0 mL) were added 2-(trimethylsilyl)ethoxymethyl chloride
(0.4 mL, 2.26 mmol) and DIPEA (0.4 mL, 2.30 mmol). After being
stirred for 2.5 h at room temperature, the reaction mixture was
poured into saturated ammonium chloride solution, and extracted
with EtOAc three times. The combined organic layer was washed
with brine, dried over sodium sulfate, and evaporated. The residue
was chromatographed over silica gel eluted by hexaneeEtOAc
(19:1) to give 11 (411 mg, 0.90 mmol, 86%). Data for 11: yellowish
to recycle preparative HPLC (column, JAIGEL-GS310
(4
20.0 mmꢂ500 mm); elutant, CHCl3) to give Ppc-1 (6) (1.8 mg). Data
for 6 (3-methylbut-2-enyl 4-methoxy-8-(3-methylbut-2-enyloxy)
quinoline-2-carboxylate): colorless oil; UV (EtOH) lmax, nm (log 3)
204 (4.33), 212 (sh, 4.32), 248 (4.36), 301 (3.43), 343 (3.45); IR (KBr)
nmax (cmꢁ1) 2925, 2855, 1715; 1H NMR and 13C NMR data are shown
inTable 4; EIMS m/z 355 [M]þ, 286, 219 (100%), 201,173; HREIMS m/z
355.1754 [M]þ (355.1784 calcd for C21H25NO4).
oil; 1H NMR (400 MHz, CDCl3)
d
13.79 (1H, s), 6.26 (1H, d, J¼2.4 Hz),
6.25 (1H, d, J¼2.4 Hz), 5.28 (2H, s), 5.20 (2H, s), 3.71e3.80 (4H, m),
4.5. Conversion of Pt-2 (2) into 7
2.99 (2H, t, J¼7.4 Hz), 1.70 (2H, sextet, J¼7.4 Hz), 0.93e1.01 (7H, m),
0.01 (9H, s), 0.00 (9H, s); 13C NMR (100 MHz, CDCl3)
d 205.9, 166.8,
To a solution of 2 (2.0 mg, 6.3
m
mol) in DMF (1.0 mL) were added
163.4, 160.4, 106.7, 97.2, 94.1, 92.9, 92.5, 67.1, 66.7, 46.2, 18.2, 18.01,
methyl iodide (10 L, 161 mol) and potassium carbonate (6.3 mg,
m
m
18.00, 14.0, ꢁ1.44 (3C), ꢁ1.45 (3C); EIMS m/z 456 [M]þ, 398, 355,

6006
H. Kikuchi et al. / Tetrahedron 66 (2010) 6000e6007
340, 325, 297, 281, 103, 73 (100%); HREIMS m/z 456.2361 [M]þ
4.11. Synthesis of Pt-4 (4)
(456.2363 calcd for C22H40O6Si2).
To a solution of 14 (10.8 mg, 26 mmol) in HMPA (1.0 mL) was added
1.0 M TBAF in THF (50 mL). After being stirred for 4 h at room tem-
4.8. 1-[2-Methoxy-4,6-bis[(2-trimethylsilylethoxy)methoxy]
phenyl]butan-1-one (12)
perature, the reaction mixture was poured into water, and extracted
with EtOAc threetimes. The combined organic layer waswashed with
water and brine, dried over sodium sulfate, and evaporated. The
residuewaschromatographed over silicageleluted byhexaneeEtOAc
To a solution of 11 (188 mg, 0.41 mmol) in acetoneeDMF (1:1)
(4.0 mL)were addedmethyliodide(75 mL,1.20 mmol)andpotassium
(19:1) to give Pt-4 (4) (3.3 mg, 12 mmol, 45%). All spectral data of the
carbonate (249 mg, 2.30 mmol). After being stirred for 6 h at room
temperature, the reaction mixture was poured into water, and
extracted with EtOAc three times. The combined organic layer was
washed with water and brine, dried over sodium sulfate, and evap-
orated. The residue was chromatographed over silica gel eluted by
hexaneeEtOAc (39:1) to give 12 (473 mg, 0.37 mmol, 90%). Data for
synthetic product were identical with those of the natural product.
4.12. 1-(3-Chloro-2,4,6-trihydroxyphenyl)butan-1-one (15)
To a solution of 10 (106 mg, 0.54 mmol) in CHCl3 (5.0 mL) were
added ethanol (50 mL) and sulfuryl chloride (72.5 mg, 0.54 mmol) at
12: colorless oil; 1H NMR (400 MHz, CDCl3)
d
6.48 (1H, d, J¼1.9 Hz),
0 ꢀC. After being stirred for 30 min, the reaction mixture was
evaporated. The residue was chromatographed over silica gel
eluted by CHCl3eMeOH (19:1) to give 15 (119 mg, 0.51 mmol, 95%).
Data for 15: yellowish amorphous solid; 1H NMR (400 MHz, ace-
6.30 (1H, d, J¼1.9 Hz),, 5.20 (2H, s), 5.16 (2H, s), 3.69e3.77 (4H, m),
3.76 (3H, s), 2.71 (2H, t, J¼7.4 Hz), 1.68 (2H, sextet, J¼7.4 Hz),
0.92e0.98 (7H, m), 0.01 (9H, s), 0.00 (9H, s); 13C NMR (100 MHz,
CDCl3) d 204.5, 159.7, 157.6, 155.4, 115.5, 95.9, 93.6, 93.0, 92.9, 66.38,
tone-d6)
d
6.13 (1H, s), 3.06 (2H, t, J¼7.3 Hz), 1.65 (2H, sextet,
J¼7.3 Hz), 0.94 (3H, t, J¼7.3 Hz); 13C NMR (100 MHz, acetone-d6)
66.33, 55.7, 46.9,18.0,17.9,17.3,13.7, ꢁ1.44 (3C), ꢁ1.48 (3C); EIMS m/z
470 [M]þ, 397, 369, 354, 339, 311 (100%), 295, 103, 73; HREIMS m/z
470.2519 [M]þ (470.2520 calcd for C23H42O6Si2).
d
206.8, 161.8, 161.7, 160.2, 105.5, 100.1, 95.7, 46.4, 18.6, 14.1; EIMS m/
z 232 [Mþ2]þ, 230 [M]þ, 189, 187 (100%); HREIMS m/z 230.0335
[M]þ (230.0246 calcd for C10H1135ClO4).
4.9. 1-[4-Hydroxy-2-methoxy-6-[(2-trimethylsilylethoxy)
methoxy]phenyl]butan-1-one (13)
4.13. 1-[3-Chloro-2-hydroxy-4,6-bis[(2-trimethylsilylethoxy)
methoxy]phenyl]butan-1-one (16)
To a solution of 12 (160 mg, 0.34 mmol) in HMPA (8.0 mL) was
added 1.0 M TBAF in THF (1.0 mL). After being stirred for 4 h at
50 ꢀC, the reaction mixture was poured into water, and extracted
with EtOAc three times. The combined organic layer was washed
with water and brine, dried over sodium sulfate, and evaporated.
The residue was chromatographed over silica gel eluted by hex-
aneeEtOAc (17:3) and hexaneeEtOAc (4:1) to give 2-hydroxyl iso-
mer (42.1 mg, 0.124 mmol, 36%) and 13 (26.4 mg, 0.076 mmol, 23%),
respectively. Data for 13: colorless oil; 1H NMR (400 MHz, CDCl3)
To a solution of 15 (80.3 mg, 0.35 mmol) in dichloromethane
(2.0 mL) were added 2-(trimethylsilyl)ethoxymethyl chloride
(0.13 mL, 0.73 mmol) and DIPEA (0.13 mL, 0.75 mmol). After being
stirred for 30 min at room temperature, the reaction mixture was
poured into saturated ammonium chloride solution, and extracted
with EtOAc three times. The combined organic layer was washed
with water and brine, dried over sodium sulfate, and evaporated. The
residue was chromatographed over silica gel eluted by hex-
aneeEtOAc (99:1) and hexaneeEtOAc (49:1) to give 16 (91.0 mg,
0.19 mmol, 53%) and tri-SEM compound (62.9 mg, 0.10 mmol, 29%),
respectively. Data for 16: colorless oil; 1H NMR (400 MHz, CDCl3)
d
6.26 (1H, d, J¼2.0 Hz), 6.07 (1H, d, J¼2.0 Hz), 5.66 (1H, br s), 5.13
(2H, s), 3.69e3.73 (2H, m), 3.71 (3H, s), 2.71 (2H, t, J¼7.3 Hz), 1.69
(2H, sextet, J¼7.3 Hz), 0.92e0.96 (5H, m), 0.00 (9H, s); 13C NMR
d
12.99 (1H, s), 6.59 (1H, s), 5.30 (2H, s), 5.20 (2H, s), 3.75e3.81 (4H,
m), 3.14 (2H, t, J¼7.3 Hz), 1.70 (2H, sextet, J¼7.3 Hz), 0.92e0.98 (7H,
m), 0.01 (9H, s), 0.00 (9H, s); 13C NMR (100 MHz, CDCl3)
206.6,163.2,
(100 MHz, CDCl3)
d 205.1, 158.23, 158.16, 155.8, 114.2, 95.0, 92.95,
92.94, 66.5, 55.7, 47.0, 18.0, 17.5, 13.8, ꢁ1.47 (3C); EIMS m/z 340
[M]þ, 311, 282, 267, 239 (100%), 210, 167, 73; HREIMS m/z 340.1714
[M]þ (340.1706 calcd for C17H28O5Si).
d
158.7, 155.7, 111.8, 108.5, 100.3, 99.0, 93.4, 68.9, 67.2, 45.2, 18.2, 17.98,
17.97,13.8, ꢁ1.4 (3C), ꢁ1.5 (3C); EIMS m/z 492 [Mþ2]þ, 490 [M]þ, 361,
359 (100%), 333, 331, 103, 73; HREIMS m/z 490.2012 [M]þ (490.2012
calcd for C22H3935ClO6Si2).
4.10. 1-[2-Methoxy-4-(3-methylbut-2-enyloxy)-6-[(2-
trimethylsilylethoxy)methoxy]phenyl] butan-1-one (14)
4.14. (E)-1-[3-Chloro-6-(3,7-dimethylocta-2,6-dienyloxy)-2-
hydroxy-4-[(2-trimethylsilylethoxy)-methoxy]phenyl]butan-1-
one (17) and Pt-5 (5)
To a solution of 13 (14.9 mg, 44
added 1-bromo-3-methyl-2-butene (20
m
mol) in DMF (1.0 mL) were
mL, 0.17 mmol) and potas-
sium carbonate (18.1 mg, 0.13 mmol). After being stirred for 3 h at
room temperature, the reaction mixture was poured into saturated
ammonium chloride solution, and extracted with EtOAc three
times. The combined organic layer was washed with water and
brine, dried over sodium sulfate, and evaporated. The residue was
chromatographed over silica gel eluted by hexaneeEtOAc (19:1) to
To a solution of 16 (70.3 mg, 0.14 mmol) in DMF (1.0 mL) were
added geranyl bromide (30 mL, 0.14 mmol) and potassium carbonate
(39.8 mg, 0.29 mmol). After being stirred for 1.5 h at room temper-
ature, the reaction mixture was poured into saturated ammonium
chloride solution, and extracted with EtOAc three times. The com-
bined organic layer was washed with water and brine, dried over
sodium sulfate, and evaporated. The residue was chromatographed
over silica gel eluted by hexaneeEtOAc (99:1) and hexaneeEtOAc
give 14 (10.8 mg, 26
(400 MHz, CDCl3)
5.44e5.48 (1H, m), 5.16 (2H, s), 4.49 (2H, d, J¼6.9 Hz), 3.74 (3H, s),
3.70e3.73 (2H, m), 2.71 (2H, t, J¼7.4 Hz), 1.79 (3H, d, J¼0.9 Hz), 1.75
(3H, d, J¼1.0 Hz), 1.67 (2H, sextet, J¼7.4 Hz), 0.91e0.96 (5H, m), 0.00
m
mol, 60%). Data for 14: colorless oil; 1H NMR
d
6.36 (1H, d, J¼2.0 Hz), 6.15 (1H, d, J¼2.0 Hz),
(19:1) to give 17 (17.5 mg, 35
28
mol, 19%), respectively. Data for 17: colorless oil; 1H NMR
(400 MHz, CDCl3) 14.51 (1H, s), 6.33 (1H, s), 5.46e5.50 (1H, m), 5.33
mmol, 25%) and Pt-5 (5) (10.1 mg,
m
(9H, s); 13C NMR (100 MHz, CDCl3)
d
204.6, 161.1, 157.8, 155.7, 138.6,
d
119.2, 114.7, 93.9, 93.2, 92.9, 66.4, 64.9, 55.7, 47.0, 25.8, 18.2, 18.0,
17.4, 13.8, ꢁ1.4 (3C); EIMS m/z 408 [M]þ, 378, 350, 335, 307, 267, 239
(100%), 223, 167, 73; HREIMS m/z 408.2346 [M]þ (408.2332 calcd
for C22H36O5Si).
(2H, s), 5.05e5.08 (1H, m), 4.58 (2H, d, J¼6.8 Hz), 3.78e3.82 (2H, m),
2.98 (2H, t, J¼7.5 Hz), 2.07e2.15 (4H, m), 1.75 (3H, s), 1.62e1.70 (5H,
m), 1.59 (3H, s), 0.93e0.97 (5H, m), 0.00 (9H, s); 13C NMR (100 MHz,
CDCl3)
d 206.5, 161.8, 160.5, 158.9, 142.7, 132.0, 123.5, 118.0, 107.0,

H. Kikuchi et al. / Tetrahedron 66 (2010) 6000e6007
6007
103.5, 93.2, 90.7, 67.1, 65.8, 46.6, 39.5, 26.3, 25.6, 18.3, 18.1, 17.7, 16.7,
13.9, ꢁ1.4 (3C); EIMS m/z 498 [Mþ2]þ, 496 [M]þ, 362, 360, 304, 302
(100%), 261, 259, 137, 81, 73; HREIMS m/z 496.2397 [M]þ (496.2412
calcd for C26H4135ClO5Si). All spectral data of synthetic Pt-5 (5) were
identical with those of the natural product.
1 ml of DMEM-MG containing DMSO (vehicle) or sample com-
pounds. Aliquots of the incubation media were then assayed for
their glucose concentration using a blood glucose test meter and
appropriate sensor chips, and the rate of glucose consumption
was calculated as previously described.8
4.15. Conversion of 17 into Pt-5 (5)
Acknowledgements
To a solution of 17 (10.0 mg, 20
mmol) in HMPA (1.0 mL) was
This work was supported in part by Chugai Pharmaceutical Co.,
Ltd. and a Grant-in-Aid for Scientific Research (No. 21590090) from
the Ministry of Education, Science, Sports and Culture of Japan (Y.
K.). The authors thank Ms. K. Nakata for her technical assistance.
added 1.0 M TBAF in THF (30
m
L). After being stirred for 4 h at 50 ꢀC,
the reaction mixture was poured into water, and extracted with
EtOAc three times. The combined organic layer was washed with
water and brine, dried over sodium sulfate, and evaporated. The
residue was chromatographed over silica gel eluted by hex-
aneeEtOAc (19:1) and hexaneeEtOAc (9:1) to give recovered 17
Supplementary data
(1.8 mg, 4 mmol, 18%) and 5 (4.0 mg, 11 mmol, 54%), respectively.
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2010.06.029.
4.16. Synthesis of Ppc-1 (6)
To a solution of 1817 (11.6 mg, 50
mmol) in MeOHewater (9:1)
References and notes
(2.0 mL) was added sodium hydroxide (50 mg). After being refluxed
for 30 min, the reaction mixture was acidified by 3 M HCl, and
extracted with EtOAc three times. The combined organic layer was
washed with brine, dried over sodium sulfate, and evaporated to
give crude quinolobactin (19). This crude was dissolved in DMF
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(1.0 mL), and 1-bromo-3-methyl-2-butene (20 mL, 0.17 mmol) and
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K562 cells were maintained at 37 ꢀC (5% CO2) in tissue culture
dishes filled with RPMI growth medium (RPMI1640 medium with
10% [v/v] fetal bovine serum, 25 mg/ml penicillin, and 50 mg/ml
streptomycin). HeLa and 3T3-L1 cells were maintained at 37 ꢀC (5%
CO2) in DMEM-HG (Dulbecco’s modified Eagle’s medium contain-
ing a high concentration (4,500 mg/l) of glucose supplemented
with 25 mg/ml penicillin, 50 mg/ml streptomycin and 10% [v/v] fetal
bovine serum). K562 cells (2ꢂ104 cells/well), HeLa and 3T3-L1 cells
(3e10ꢂ103 cells/well) were allowed to grow for 3 days in 12-well
plates; each well contained 1 ml of RPMI (for K562 cells) or DMEM-
HG (for HeLa and 3T3-L1 cells) with DMSO or sample compounds.
The relative cell number was assessed using Alamar blue (cell
number indicator) as described previously.6,8b
4.18. Assay for glucose consumption in confluent 3T3-L1 cells
3T3-L1 cells were transferred into 12-well plates, each con-
taining 1 ml of DMEM-HG, and incubated for 5e6 days until they
reached the confluent state with exchanging DMEM-HG every
2e3 days. The cells were then pre-incubated for 2e3 days with
1 ml of DMEM-MG [DMEM containing a medium concentration
(2,000 mg/l) of glucose supplemented with the antibiotics, 10%
[v/v] fetal bovine serum, and 10 mM HEPESeNaOH (pH 7.4)] with
exchanging the media every day. The confluent cells thus
obtained were treated for the appropriate number of hours with