Isolation, Synthesis, and Biological Activity of Chlorinated Alkylresorcinols from Dictyostelium Cellular Slime Molds

Article abstract of DOI:10.1021/acs.jnatprod.7b00456

Eight chlorinated alkylresorcinols, monochasiol A-H (1-8), were isolated from the fruiting bodies of Dictyostelium monochasioides. Compounds 1-8 were synthesized to confirm their structures and to obtain sufficient material for performing biological tests. Monochasiol A (1) selectively inhibited the concanavalin A-induced interleukin-2 production in Jurkat cells, a human T lymphocyte cell line. Monochasiols were biogenetically synthesized by the combination of biosynthetic enzymes relating to the principal polyketides, MPBD and DIF-1, produced by Dictyostelium discoideum.

Full text of DOI:10.1021/acs.jnatprod.7b00456

Article
pubs.acs.org/jnp
Isolation, Synthesis, and Biological Activity of Chlorinated
Alkylresorcinols from Dictyostelium Cellular Slime Molds
Haruhisa Kikuchi,*,† Ikuko Ito,† Katsunori Takahashi,‡ Hirotaka Ishigaki,‡ Kyoichi Iizumi,§
Yuzuru Kubohara,§ and Yoshiteru Oshima†
†Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aza-Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan
‡Department of Medical Technology, Faculty of Health Science, Gunma Paz University, 1-7-1, Tonyamachi, Takasaki 370-0006,
Japan
§Graduate School of Health and Sports Science, Juntendo University, 1-1 Hiraga-gakuendai, Inzai, Chiba 270-1695, Japan
S
* Supporting Information
ABSTRACT: Eight chlorinated alkylresorcinols, monochasiol
A−H (1−8), were isolated from the fruiting bodies of
Dictyostelium monochasioides. Compounds 1−8 were synthe-
sized to confirm their structures and to obtain sufficient
material for performing biological tests. Monochasiol A (1)
selectively inhibited the concanavalin A-induced interleukin-2
production in Jurkat cells, a human T lymphocyte cell line.
Monochasiols were biogenetically synthesized by the combination of biosynthetic enzymes relating to the principal polyketides,
MPBD and DIF-1, produced by Dictyostelium discoideum.
he cellular slime mold Dictyostelium discoideum is thought
to be an excellent model organism for studying cell and
of these compounds against the production of interleukin-2
(IL-2) in Jurkat cells, a human T lymphocyte cell line, are also
described.
T
developmental biology because of its simple developmental
pattern.1 Vegetative cells of D. discoideum grow as a single
amoeba by eating bacteria. When these cells are starved, they
initiate a developmental program of morphogenesis, forming a
slug-shaped multicellular aggregate. This aggregate differ-
entiates into two cell types, prespore and prestalk cells, which
are precursors to spores and stalk cells, respectively. At the end
of its development, the aggregate forms a fruiting body
consisting of spores and a multicellular stalk.2
In 2005, the genome sequencing of D. discoideum clearly
revealed the presence of approximately 45 polyketide synthase
(PKS) gene clusters.3 Another cellular slime mold species, D.
purpureum, has also been reported to contain 50 predicted PKS
genes.4 These numbers are even greater than those observed in
Streptomyces avermitilis, which is known to contain abundant
secondary metabolites. Thus, we have focused on the utility of
cellular slime molds as a source of natural compounds.
Recently, our group has isolated α-pyronoids,5a,d,g amino
sugar derivatives,5b and aromatics5c,e,f with unique structures
and various biological activities. Brefelamide exhibits an
inhibitory effect on osteopontin expression.5c,6 Ppc-1 is a
prenylated quinolone derivative isolated from Polysphondylium
pseudocandidum; it serves as a mitochondrial uncoupler and is a
candidate for new antiobesity drugs.5e,7 The above results
indicate that cellular slime molds are an important source of
lead compounds for drug discovery.
Figure 1. Structures of monochasiols A−H (1−8).
RESULTS AND DISCUSSION
■
Isolation and Structural Elucidation. From the fruiting
bodies (85 g) of D. monochasioides, monochasiols A (1, 1.6
mg), B (2, 3.4 mg), C (3, 3.6 mg), D (4, 3.4 mg), E (5, 8.2
mg), F (6, 3.8 mg), G (7, 5.2 mg), and H (8, 2.9 mg) were
isolated. The aromatic regions of their H and 13C NMR data
1
showed signals at δH 6.45 (2H, s) and 5.29 (2H, br s) in the 1H
NMR data and δC 151.4, 143.8, 108.2, and 104.3 in the 13C
NMR data. These indicate that the compounds contain a
common aromatic skeleton.
This paper reports the isolation, structure elucidation, and
synthesis of new chlorinated alkylresorcinols, monochasiols A−
H (1−8), from the cellular slime mold Dictyostelium
monochasioides (Figure 1). The selective inhibitory activities
Received: May 27, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Two peaks observed in the EIMS of monochasiol A (1) at
m/z 326 and 328 for the molecular ion peaks at a ratio of 3:1
suggest that it contains one chlorine atom. Its HREIMS (m/z
326.2012 [M]+) indicated that its molecular formula is
C19H31O2Cl. Except for those in the common aromatic
skeleton, protons associated with 12 methylene groups (δ
2.49 (2H, t, J = 7.8 Hz), 1.56 (2H, quint, J = 7.8 Hz), and
1.22−1.32 (20H, m)) and one methyl group (δ 0.88 (3H, t, J =
Scheme 1. Determination of Position of the Double Bond of
Monochasiols D (4) and E (5)
1
6.9 Hz)) were observed in the H NMR data. These indicate
the presence of a tridecyl group (C13H27). Thus, the remaining
molecular formula for the common aromatic skeleton is
C6H4O2Cl, which corresponds to a symmetrical tetrasubsti-
tuted benzene ring (δC 151.4, 143.8, 108.2, and 104.3)
containing a chlorine atom and two phenolic hydroxy groups
(δH 5.29 (2H, br s)). In the HMBC spectrum of 1, the
correlation between two phenyl protons (δH 6.45 (2H, s)) and
a benzylic methylene carbon (δC 35.7, Figure 2) suggests that
data.10 The structure of monochasiol G (7), the HREIMS (m/z
366.2320 [M]+) of which showed the molecular formula
C22H35O2Cl, also contained a cis-cyclopropane ring in its C15
side chain. The opening of a cyclopropane ring into a methyl-
branched alkyl chain has been widely exploited for determining
the position of a cyclopropane ring in fatty acid methyl esters.11
This procedure was utilized for the hydrogenation of 6
catalyzed by platinum(IV) oxide; however, several products
were obtained by the partial hydrogenation of the benzene ring
of 6. Thus, we planned to synthesize plausible structures of 6
and 7 to elucidate these structures. Typically, a cyclopropane
ring in an alkyl chain is biogenetically synthesized by the
methylenylation of a double bond.12 Therefore, the structures
of 6 and 7 are hypothesized to contain a cyclopropane ring
between C-6′ and C-7′ and between C-8′ and C-9′,
respectively, which correspond to those of 4 and 5, respectively.
These plausible structures should be confirmed by the
comparison of the NMR and mass spectra of the natural and
synthesized compounds.
In addition, the absolute configurations of the cyclopropane
moieties of 6 and 7 were not determined because of the
extremely low optical activities observed for their quasisym-
metric structures ([α]D −1.4 (c 0.38, CHCl3) for 6; [α]D +0.9
(c 0.48, CHCl3) for 7). It is also possible that these compounds
are racemic. Although a method for forming α-oxocyclopro-
pane has been reported to determine such absolute
configurations,13 small amounts of isolated natural mono-
chasiols F (6) and G (7) restricted applying this method due to
very low yield of the oxidation. The ω-7 long alkyl chains of 6
and 7 suggest that the biogenetic origin of 6 and 7 is
lactobacillic acid (discussed later) (Figure 3), which is
widespread in several bacteria, e.g., Klebsiella aerogenes,14 fed
with D. monochasioides under the culture conditions employed
herein. Thus, the absolute configurations of 6 and 7 are
assumed to be 6′R, 7′S and 8′R, 9′S, respectively, which are the
same as those in lactobacillic acid.15
Figure 2. Structural elucidation of a common aromatic skeleton of
monochasiols.
two phenyl protons are located on C-2 and C-6. Thus, two
phenolic hydroxy groups are located on C-3 and C-5, and a
chlorine atom is located on C-4 because of the symmetry of
substitutions on a benzene ring, confirming the structure of 1.
The molecular formulas of monochasiols B (2) and C (3)
were C20H33O2Cl and C21H35O2Cl, as revealed by the HREIMS
data (m/z 340.2162 [M]+ and 354.2333 [M]+ for 2 and 3,
respectively). The formulas differ from that of 1 by one and two
methylene units, respectively. In addition, the signals of
methylene protons (δ 1.20−1.34 (22H, m) and 1.21−1.36
1
(24H, m)) were observed in the H NMR data of 2 and 3,
respectively, instead of the signal of δ 1.22−1.32 (20H, m) in 1.
Thus, the structures of 2 and 3 contain a tetradecyl and
pentadecyl side chain, respectively.
From the HREIMS data (m/z 324.1873 [M]+) of
monochasiol D (4), its molecular formula was determined to
be C19H29O2Cl, which differs from that of 1 by two hydrogen
atoms. Olefinic protons (δ 5.34 (2H, m)) and allylic protons (δ
2.01 (4H, m)) in the 1H NMR data indicate that 4 contains one
double bond in its C13 side chain. The double-bond positions
were determined through the dimethyl disulfide (DMDS)
adduct method.8 In the EIMS of the DMDS adduct of 4, we
observed two strong peaks at m/z 319 and 145, respectively,
which indicate that the double bond is located between C-6′
and C-7′ (Scheme 1A). In the 13C NMR data of 4, the signals
corresponding to allylic carbons (C-5′ and C-8′) at 27.2 and
27.1 ppm, respectively, are indicative of the cis double-bond
configuration.9 Similarly, monochasiol E (5) contained a C15
alkenyl chain with a cis double bond between C-8′ and C-9′
(Scheme 1B).
The molecular formula of monochasiol H (8) as suggested
from its HREIMS (m/z 350.2000 [M]+) was C21H31O2Cl. This
formula differs from that of 3 by four hydrogen atoms. Olefinic
protons (δ 5.38 (4H, m)) and allylic protons (δ 2.05 (8H, m))
in the 1H NMR data indicate that 8 contains two double bonds
in its C15 side chain. The DMDS adduct method was applied;
however, no products affording mass fragments that reveal the
The molecular formula of monochasiol F (6) as revealed by
its HREIMS (m/z 338.2035 [M]+) was C20H31O2Cl. This
formula differs from that of 1 by one carbon atom. The
structure of 6 contains a cis-cyclopropane ring in its C13 side
chain, due to the higher-field signals (δ 0.64 (2H, m), 0.56 (1H,
dt, J = 8.3, 4.9 Hz), −0.34 (1H, q, J = 4.9 Hz)) in its 1H NMR
B
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sorcinol was directly chlorinated, a mixture of the favored 2-
chloro compound and the disfavored 4-chloro compound was
obtained. Thus, after the selective chlorination of 3,5-
dimethoxyaniline, the Sandmeyer iodination of 8 provided 9,
the methoxy groups of which were converted into the
methoxymethyl (MOM) groups to give 10 (Scheme 4).
Scheme 4. Synthesis of the Aromatic Part of Monochasiols F
(6) and G (7)
Figure 3. Structural comparison between plausible strucutures of
monochasiols F (6) and G (7) and lactobacillic acid.
location of double bonds were obtained. Instead, the molecular
masses of the product obtained from the cross-metathesis16 of
8 with styrene were 234, indicating the presence of two
methylene units between two double bonds. In addition,
1H−1H COSY showed the linkage of benzylic methylene (δ
2.51 (2H, t, J = 7.8 Hz), H-1′)−homoallylic methylene (1.64
(2H, quint, J = 7.8 Hz), H-2′)−allylic methylene (2.04−2.11
(2H, m), H-3′). Therefore, the double bonds are located
between C-4′ and C-5′ and between C-8′ and C-9′, respectively
(Scheme 2).
Scheme 2. Determination of Position of the Double Bonds
of Monochasiol H (8)
Sonogashira coupling of 10 with 3-butyn-1-ol afforded
phenylalkyne 11f. Its triple bond was hydrogenated using a
palladium-activated carbon ethylenediamine complex [Pd/
C(en)]19 instead of ordinary Pd/C to avoid dechlorination to
give 12f; next, 12f was subjected to thioetherification and
subsequent oxidation to form phenyltetrazole sulfone 13f,
which corresponds to the aromatic part of 6.
((1R,2S)-2-Hexylcyclopropyl)methanol, the precursor of the
chiral cyclopropane part of 6 and 7, has been already
synthesized.20 Oxidation into an aldehyde and the subsequent
Julia−Kocienski olefination with 13f afforded 14f as a mixture
of cis/trans isomers (E/Z = 7:1) (Scheme 5). Then, the diimide
Synthesis of Monochasiols. To confirm the structures
and to obtain a sufficient amount of material for performing
several biological tests, monochasiols were synthesized. In
particular, the structures of 6 and 7 had to be reconfirmed.
Although the synthesis of alkylresorcinols has been reported in
some studies,17 cyclopropane-containing and halogenated
alkylresorcinols have not been synthesized thus far. Our
strategy to synthesize plausible structures of 6 and 7 involved
the connection of an aromatic part and a cyclopropane part via
the Julia−Kocienski olefination18 (Scheme 3).
Scheme 5. Synthesis of Monochasiols F (6) and G (7)
A tetrasubstituted benzene ring of the aromatic part had to
be constructed regioselectively. Nevertheless, when 5-alkylre-
Scheme 3. Retrosynthetic Pathway of Monochasiols F (6)
and G (7)
reduction of 14f and deprotection of the MOM groups
produced monochasiol F (6). Similarly, monochasiol G (7) was
also synthesized by the connection of ((1R,2S)-2-
hexylcyclopropyl)methanol and 13g. The spectral data of
synthetic 6 and 7 were identical to those of natural 6 and 7,
respectively. Particularly, all fragment ion peaks from the EIMS
of synthetic and natural compounds were completely consistent
(Supporting Information). Thus, the proposed planar struc-
tures of 6 and 7 are confirmed, although their absolute
C
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configurations are not elucidated because of low optical
activities ([α]D +0.2 (c 0.54, CHCl3) for synthetic 6; [α]D
−0.2 (c 0.90, CHCl3) for synthetic 7).
Monochasiols A−C (1−3) were synthesized by the
Sonogashira coupling of 10 with terminal alkynes and
subsequent saturation of triple bonds catalyzed by Pd/C(en)
(Scheme 6).
Scheme 6. Synthesis of Monochasiols A−C (1−3)
For the synthesis of monochasiol D (4), acetylenic alcohol
16d was obtained by a similar synthetic pathway to that for 11f
and 11g (Scheme 7). After the hydrogenation and bromination
Scheme 7. Synthesis of Monochasiols D (4), E (5), and H
(8)
Figure 4. (A) Antiproliferative activities of monochasiols A−H (1−8)
on HeLa cells. Cells were incubated with 20 μM of each compound for
4 days, and the relative cell number was estimated. (B) Effects of
monochasiol A−H (1−8) on ConA-induced IL-2 production in Jurkat
cells. (C) Selective immunosuppressive effect of monochasiol A (1).
Cells were preincubated for 30 min with 0.1% DMSO (control) or
indicated concentrations of each compound. After addition of ConA,
cells were further incubated for 12 h and assayed for IL-2 protein
production and cell viability. Results are presented as mean values of
three independent experiments (n = 3). The bars indicate the standard
errors.
of 16d, the extension of the chain with 1-octyne and
subsequent deprotection of the MOM groups provided 18d.
Finally, partial hydrogenation of triple bonds catalyzed by
palladium-polyethylenimine (Pd/PEI)21 afforded monochasiol
D (4). Similarly, the chain extension of bromides 17e,h with 1-
octyne and dodec-5-en-1-yne (19) afforded monochasiols E (5)
and H (8), respectively.
Biological Evaluation. The biological activity of mono-
chasiols A−H (1−8) was investigated. Some monochasiols (20
μM) only exhibited weak antiproliferative activity against HeLa
cells (Figure 4A). On the other hand, monochasiols exhibited
inhibitory activities against concanavalin A (ConA)-induced IL-
2 production22 in Jurkat cells. Monochasiols (20 μM) inhibited
the production of IL-2 to less than 50% of the control (Figure
4B). In particular, monochasiol A (1) exhibited inhibitory
activity (IC50 16 μM) in a concentration-dependent manner
without suppressing cell proliferation (Figure 4C). Thus, this
effect of monochasiol A (1) is selective for IL-2 production,
suggesting that compound 1 is a promising candidate for novel
immunosuppressive drugs.
Plausible Biosynthesis. Although some studies have
reported chlorinated alkylresorcinols from nature,23 mono-
chasiols are the first examples of chlorinated resorcinols bearing
a long alkyl chain (particularly, those greater than 10 carbons).
Monochasiols are assumed to be biogenetically synthesized by
analogous enzymes to essential biosynthetic enzymes for
principal polyketides, 4-methyl-5-pentylbenzene-1,3-diol
(MPBD),24 and differentiation-inducing factor-1 (DIF-1)25
(Scheme 8) produced by D. discoideum. The plausible
biosynthetic pathways for 6 and 7 could involve one or two
β-oxidations of lactobacillic acid followed by elongation of the
polyketide chain three times by an analogous enzyme of Steely
A,26 an essential polyketide synthase for MPBD, affording
tetraketides 23f and 23g. They were cyclized into resorcinols
24f and 24g, respectively. Then, chlorination of 24f and 24g
was done using an analogous enzyme of flavin-dependent
halogenase,27 an essential biosynthetic enzyme for DIF-1, to
afford 6 and 7. These plausible biosynthetic pathways support
D
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with increasing polarity to afford hexane−EtOAc (9:1) eluent (fraction
A, 163 mg). Fraction A was further separated by ODS column using a
water−acetonitrile solvent system to give a water−acetonitrile (1:4)
elutant (fraction B, 86.1 mg). Fraction B was subjected to recycle
preparative HPLC (column, JAIGEL-GS310 (ϕ 20 mm × 500 mm,
Japan Analytical Industry Co., Ltd.); solvent, ethyl acetate) to give
three fractions, C-1 (20.9 mg), C-2 (36.1 mg), and C-3 (6.5 mg).
Fraction C-1 was subjected to preparative HPLC (column,
Mightysil RP-18GP (ϕ 20 mm × 250 mm, Kanto Chemical Co.,
Inc.); solvent, water−acetonitrile (1:9)) to give monochasiol D (4)
(3.4 mg), monochasiol F (6) (3.8 mg), and monochasiol H (8) (2.9
mg).
Scheme 8. (A) Biosynthetic Pathway of MPBD and DIF-1;
(B) Plausible Biosynthetic Pathway of Monochasiols F (6)
and G (7)
Fraction C-2 was subjected to preparative HPLC (column,
Mightysil RP-18GP (ϕ 20 mm × 250 mm, Kanto Chemical Co.,
Inc.); solvent, water−acetonitrile (2:23)) to give monochasiol B (2)
(3.4 mg), monochasiol C (3) (3.6 mg), and monochasiol G (7) (5.2
mg).
Fraction C-3 was subjected to preparative HPLC (column,
Wakopak Navi C30-5 (ϕ 20 mm × 250 mm, Wako Pure Chemical
Industries, Ltd.); solvent, water−acetonitrile (13:87)) to give
monochasiol A (1) (1.6 mg) and monochasiol E (5) (8.2 mg).
Monochasiol A (1): colorless, amorphous solid; 1H NMR (400
MHz, CDCl3) δ 6.45 (2H, s), 5.29 (2H, br s), 2.49 (2H, t, J = 7.8 Hz),
1.50−1.60 (2H, m), 1.22−1.34 (20H, m), 0.88 (3H, t, J = 6.9 Hz); 13C
NMR (100 MHz, CDCl3) δ 151.4 (2C), 143.8, 108.2 (2C), 104.3,
35.7, 32.0, 31.0, 29.74, 29.72, 29.71, 29.70, 29.6, 29.5, 29.4, 29.2, 22.8,
14.2; EIMS m/z 328 [M + 2]+ (11%), 326 [M]+ (31%), 160 (34%),
158 (100%); HREIMS m/z 326.2012 [M]+ (326.2013 calcd for
C19H31O235Cl).
Monochasiol B (2): colorless, amorphous solid; 1H NMR (400
MHz, CDCl3) δ 6.45 (2H, s), 5.28 (2H, br s), 2.49 (2H, t, J = 7.8 Hz),
1.52−1.61 (2H, m), 1.20−1.34 (22H, m), 0.88 (3H, t, J = 7.0 Hz); 13C
NMR (100 MHz, CDCl3) δ 151.5 (2C), 143.9, 108.2 (2C), 104.3,
35.7, 31.9, 31.0, 29.71, 29.69, 29.68, 29.67 (2C), 29.57, 29.5, 29.4, 29.2,
22.7, 14.1; EIMS m/z 342 [M + 2]+ (8%), 340 [M]+ (22%), 160
(35%), 158 (100%); HREIMS m/z 340.2162 [M]+ (340.2169 calcd
for C20H33O235Cl).
our assumption of the absolute configurations of 6 and 7, which
are described earlier. Other monochasiols can also be
biosynthesized from their corresponding fatty acids. The
biosynthesis of monochasiols via the combination of the
biosynthetic enzymes for principal polyketides, MPBD and
DIF-1, suggests that cellular slime molds produce considerably
diverse secondary metabolites; thus, cellular slime molds are a
promising source for novel metabolites.
Monochasiol C (3): colorless, amorphous solid; 1H NMR (400
MHz, CDCl3) δ 6.45 (2H, s), 5.28 (2H, br s), 2.49 (2H, t, J = 7.8 Hz),
1.51−1.61 (2H, m), 1.21−1.36 (24H, m), 0.88 (3H, t, J = 6.9 Hz); 13C
NMR (100 MHz, CDCl3) δ 151.5 (2C), 143.9, 108.3 (2C), 104.3,
35.7, 31.9, 31.0, 29.71, 29.70, 29.69, 29.68, 29.67 (2C), 29.57, 29.48,
29.37, 29.2, 22.7, 14.1; EIMS m/z 356 [M + 2]+ (8%), 354 [M]+
(23%), 160 (34%), 158 (100%); HREIMS m/z 354.2333 [M]+
(354.2326 calcd for C21H35O235Cl).
Monochasiol D (4): colorless, amorphous solid; 1H NMR (400
MHz, CDCl3) δ 6.45 (2H, s), 5.30−5.40 (4H, m), 2.49 (2H, t, J = 7.4
Hz), 1.97−2.09 (4H, m), 1.57 (2H, quint, J = 7.2 Hz), 1.21−1.39
(12H, m), 0.88 (3H, t, J = 6.7 Hz); 13C NMR (100 MHz, CDCl3) δ
151.5 (2C), 143.8, 130.1, 129.6, 108.2 (2C), 104.3, 35.7, 31.8, 30.9,
29.7, 29.6, 29.0, 28.8, 27.2, 27.1, 22.7, 14.1; EIMS m/z 326 [M + 2]+
(7%), 324 [M]+ (20%), 230 (6%), 228 (20%), 160 (38%), 158
(100%); HREIMS m/z 324.1873 [M]+ (324.1856 calcd for
C19H29O235Cl).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Analytical TLC was
performed on silica gel 60 F254 (Merck). Column chromatography
was carried out on silica gel 60 (70−230 mesh, Merck). NMR spectra
were recorded on JEOL ECA-600 and AL-400. Chemical shifts for 1H
and 13C NMR are given in parts per million (δ) relative to
tetramethylsilane (δ H 0.00) and residual solvent signals (δC 77.0)
as internal standards. Mass spectra were measured on JEOL JMS-700
and JMS-DX303.
Monochasiol E (5): colorless, amorphous solid; 1H NMR (400
MHz, CDCl3) δ 6.45 (2H, s), 5.32−5.37 (2H, m), 5.29 (2H, br s),
2.49 (2H, t, J = 7.8 Hz), 1.96−2.08 (4H, m), 1.52−1.62 (2H, m),
1.22−1.39 (16H, m), 0.88 (3H, t, J = 7.0 Hz); 13C NMR (100 MHz,
CDCl3) δ 151.5 (2C), 143.8, 130.0, 129.8, 108.2 (2C), 104.3, 35.7,
31.8, 30.9, 29.7 (2C), 29.3, 29.2, 29.1, 29.0, 27.2, 27.1, 22.7, 14.1;
EIMS m/z 354 [M + 2]+ (6%), 352 [M]+ (16%), 258 (5%), 256
(15%), 160 (35%), 158 (100%); HREIMS m/z 352.2153 [M]+
(352.2169 calcd for C21H33O235Cl).
Monochasiol F (6): colorless, amorphous solid; [α]D −1.4 (c 0.38,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.45 (2H, s), 5.20−5.40 (2H,
br s), 2.50 (2H, t, J = 7.6 Hz), 1.58 (2H, quint., J = 7.6 Hz), 1.20−1.42
(14H, m), 1.08−1.17 (2H, m), 0.88 (3H, t, J = 7.1 Hz), 0.60−0.69
(2H, m), 0.56 (1H, dt, J = 8.3, 4.9 Hz), −0.34 (1H, q, J = 4.9 Hz); 13C
NMR (100 MHz, CDCl3) δ 151.5 (2C), 143.9, 108.3 (2C), 104.3,
35.7, 32.0, 31.0, 30.2, 30.0, 29.4, 29.2, 28.7, 28.6, 22.7, 15.8, 15.7, 14.1,
Organism and Culture Conditions. Dictyostelium monochasioides
JKS728 was kindly supplied by Dr. Hagiwara, National Science
Museum, Tokyo, Japan. Spores were cultured at 22 °C with Klebsiella
aerogenes 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 4 days, they were harvested and stored in a refrigerator at −18
°C. Collected fruiting bodies were lyophilized for extraction.
Isolation of Monochasiols. The lyophilized fruiting bodies (84.6
g) of D. monochasioides were extracted three times with methanol at
room temperature to give an extract (19.4 g), which was then
partitioned with ethyl acetate and water to yield ethyl acetate solubles
(3.04 g). The ethyl acetate solubles were chromatographed over silica
gel, and the column was eluted with hexane−ethyl acetate solutions
E
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10.9; EIMS m/z 340 [M + 2]+ (4%), 338 [M]+ (13%), 227 (19%), 225
(55%), 160 (35%), 158 (100%); HREIMS m/z 338.2035 [M]+
(338.2013 calcd for C20H31O235Cl).
determine cell viability, cells were incubated under the same
conditions, and the percentage of viable cells compared with controls
was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazo-
lium bromide (MTT) assay according to standard procedures.
Monochasiol G (7): colorless, amorphous solid; [α]D +0.9 (c 0.48,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.45 (2H, s), 5.30 (2H, br s),
2.49 (2H, t, J = 7.8 Hz), 1.57 (2H, quint., J = 7.8 Hz), 1.21−1.42
(18H, m), 1.08−1.17 (2H, m), 0.88 (3H, t, J = 7.0 Hz), 0.60−0.69
(2H, m), 0.56 (1H, dt, J = 8.3, 4.9 Hz), −0.34 (1H, q, J = 4.9 Hz); 13C
NMR (100 MHz, CDCl3) δ 151.5 (2C), 143.9, 108.3 (2C), 104.3,
35.7, 32.0, 31.0, 30.2 (2C), 29.55, 29.52, 29.4, 29.2, 28.73, 28.69, 22.7,
15.8, 15.7, 14.1, 10.9; EIMS m/z 368 [M + 2]+ (5%), 366 [M]+ (14%),
330 (6%), 270 (8%), 197 (10%), 160 (35%), 158 (100%); HREIMS
m/z 366.2320 [M]+ (366.2326 calcd for C22H35O235Cl).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00456.
Experimental methods for synthesis of monochasiols and
NMR spectra of the new compounds (PDF)
Monochasiol H (8): colorless, amorphous solid; 1H NMR (600
MHz, CDCl3) δ 6.45 (2H, s), 5.33−5.39 (4H, m), 5.31 (2H, br s),
2.51 (2H, t, J = 7.8 Hz), 2.04−2.11 (6H, m), 2.00 (2H, q, J = 6.9 Hz),
1.64 (2H, quint, J = 7.8 Hz), 1.22−1.35 (8H, m), 0.88 (3H, t, J = 7.0
Hz); 13C NMR (150 MHz, CDCl3) δ 151.5 (2C), 143.5, 130.5, 130.0,
129.4, 129.1, 108.3 (2C), 104.4, 35.2, 31.8, 30.9, 29.7, 29.0, 27.5, 27.34,
27.29, 26.7, 22.7, 14.1; EIMS m/z 352 [M + 2]+ (9%), 350 [M]+
(27%), 314 (10%), 253 (4%), 251 (11%), 225 (20%), 199 (10%), 197
(29%), 160 (35%), 158 (100%); HREIMS m/z 350.2000 [M]+
(350.2013 calcd for C21H31O235Cl).
AUTHOR INFORMATION
Corresponding Author
*E-mail (H. Kikuchi): hal@mail.pharm.tohoku.ac.jp. Phone:
+81-22-795-6824. Fax: +81-22-795-6821.
■
ORCID
Haruhisa Kikuchi: 0000-0001-6938-0185
Notes
Dimethyl Disulfide Adduction of Monochasiols D and E. To
a solution of monochasiol D (4) (0.23 mg) in diethyl ether (0.1 mL)
were added dimethyl disulfide (20 μL) and iodine (1.2 mg). After
being stirred for 5 h at 50 °C, the reaction mixture was cooled to room
temperature, poured into 5% sodium thiosulfate solution (0.5 mL),
and extracted with diethyl ether (0.3 mL) three times. The combined
organic layer was concentrated in vacuo to give a crude DMDS adduct
of 4 for MS analysis. EIMS m/z 466 (25%), 464 (52%), 420 (24%),
418 (54%), 370 (28%), 321 (37%), 319 (79%), 275 (25%), 273
(69%), 227 (14%), 225 (40%), 145 (100%).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by the Grants-in-Aid for
Scientific Research (no. 16H03279) from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT),
Japan; the Platform Project for Supporting Drug Discovery and
Life Science Research funded by Japan Agency for Medical
Research and Development (AMED); the Shorai Foundation
for Science and Technology; Kobayashi International Scholar-
ship Foundation; and the Takeda Science Foundation.
In a similar way, monochasiol E (5) was converted into a DMDS
adduct for MS analysis. EIMS m/z 494 (25%), 492 (50%), 448 (14%),
446 (35%), 398 (23%), 349 (48%), 347 (100%), 303 (19%), 301
(54%), 145 (78%).
REFERENCES
■
Cross-Metathesis of Monochasiol H (8) with Styrene. To a
solution of monochasiol H (8) (0.85 mg) in dichloromethane (1 mL)
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