Journal of Natural Products
Article
Preparation of MPA Esters of 1 and 2. The sample of cladosins
H and I (1 and 2) (0.5 mg each) was treated with (R)- or (S)-MPA
counting the living cells on the cell counting plate. Then, cell
suspensions, 90 μL, at a density of 8000 cells per well were plated in
96-well microtiter plates and exposed to 10 μL of test solution in each
well for 72 h. Then, 100 μL of each of the three linked dissolved
solutions was added to each well, which were incubated for 10 h.
Absorbance was then determined on a Spectra Max Plus plate reader
at 570 nm.
(
0.4 mg) with dicyclohexylcarbodiimide (DCC) (0.5 mg) and
dimethylaminopyridine (DMAP) (0.3 mg) in dry CH Cl (0.5 mL).
2
2
The reaction was stirred at room temperature (rt) under a nitrogen
atmosphere for 2 h. The organic phase was evaporated to dryness and
separated by semipreparative HPLC with MeCN−H O (45:55) to
2
obtain the (R)-MPA ester and (S)-MPA ester (1 and 2), respectively.
In the SRB assay, the cells were washed with 1 mL of phosphate-
buffered saline twice. Then 1 mL of pancreatin was added to 2 mL of
medium to digest parietal cells for 2 min, preparing the single-cell
suspensions. Cell viability was detected by the Trypan blue cell
counting assay. Then, 6000 cells per well, 90 μL, were seeded in 96-
well microtiter plates and exposed to 10 μL of test solution in each
well for 72 h. The cells were fixed with 12% trichloroacetic acid, and
the cell layer was strained with 0.4% SRB. The absorbance of SRB
solution was measured at 515 nm. Doxorubicin hydrochloride was
1
Cladosin H (1) (R)-MPA ester: H NMR (DMSO-d ) exoform A
6
(
4
(
exoform B) δ 12.82 (12.16), 9.40 (9.73), 7.36−7.49, 7.29−7.33,
.84−4.89, 4.73, 3.37−3.42, 3.28 (3.26), 3.09−3.20, 2.82 (2.85), 2.20
2.19), 1.81 (1.80), 1.55−1.65, 1.38−1.45, 0.96 (0.99); HRESIMS m/
+
z 507.2478 [M + H] (calcd for C H N O , 507.2490).
2
9
35
2
6
1
Cladosin H (1) (S)-MPA ester: H NMR (DMSO-d ) exoform A
6
(
exoform B) 12.78 (12.11), 9.40 (9.73), 7.42−7.46, 7.28−7.37, 7.21−
7
2
.24, 4.77 (4.79), 4.72−4.74, 3.27, 3.12−3.16, 2.98−3.06, 2.92−2.95,
18
.54 (2.55), 2.21 (2.24), 1.83 (1.82), 1.56−1.65, 1.31−1.39, 1.11
used as the positive control.
+
(
1.13); HRESIMS m/z 507.2490 [M + H] (calcd for C H N O ,
2
9
35
2
6
5
07.2490).
Cladosin I (2) (R)-MPA ester: H NMR (DMSO-d ) exoform A
ASSOCIATED CONTENT
Supporting Information
■
1
6
*
S
(
(
1
exoform B) 12.80 (12.16), 9.33 (9.69), 7.31−7.46, 4.81−4.85, 4.77
4.78), 3.28−3.40, 3.26 (3.24), 2.94 (2.99), 2.19 (2.15), 1.80 (1.78),
.62−1.68, 1.48−1.55, 0.92 (0.94); HRESIMS m/z 507.2491 [M +
+
H] (calcd for C H N O , 507.2490).
29
35
2
6
1
Cladosin I (2) (S)-MPA ester: H NMR (DMSO-d ) exoform A
6
Structures of cladosins A−K and cladodionen, MS, IR,
(
(
1
5
exoform B) 12.77 (12.11), 9.34 (9.68), 7.41−7.45, 7.31−7.36, 7.28
7.27), 4.80−4.88, 4.77, 3.25, 2.94−3.07, 2.85 (2.89), 2.19 (2.16),
.80 (1.78), 1.53−1.58, 1.42−1.49, 1.07 (1.09); HRESIMS m/z
+
07.2497 [M + H] (calcd for C H N O , 507.2490).
2
9
35
2
6
Preparation of N-Phenyloctanamide. Aniline (50.0 mg) was
AUTHOR INFORMATION
■
*
dissolved in CH Cl (3.0 mL) containing DMAP (13.0 mg), followed
2
2
by adding Et N (111 μL). Octanoyl chloride (100 μL) was then
3
added, and after stirring 2 h at rt, the product was filtered through a
small plug of silica eluting with CH Cl (15 mL) prior to solvent
2
2
evaporation in vacuo. The product was purified by RP-HPLC eluting
with MeOH−H O (75:25) to obtain N-phenyloctanamide.
ORCID
2
1
N-Phenyloctanamide: white powder; H NMR (500 MHz,
Notes
DMSO-d ) δ 9.81 (s, 1H), 7.56 (d, J = 7.8 Hz, 2H), 7.25 (t, J =
6
7
.9 Hz, 2H), 6.99 (t, J = 7.4 Hz, 1H), 2.27 (t, J = 7.5 Hz, 2H), 1.56
(
dd, J = 13.4, 6.5 Hz, 2H), 1.31−1.19 (m, 8H), 0.84 (t, J = 6.9 Hz,
H); ESIMS m/z 220.55 [M + H] .
+
3
The authors declare no competing financial interest.
SAHA and Analogues Incubation Studies. Incubation studies
with SAHA, N-phenyloctanamide, and aniline (each at 300 μM) were
carried out in 500 mL flasks containing 150 mL of the potato dextrose
broth (PDB) medium (a separate flask containing PDB medium
without any additive served as a control). Cultures were each
inoculated with spores of C. sphaerospermum. After 6 days, cultures
were clarified via filter paper to separate the supernatant from mycelia.
The supernatant was extracted with EtOAc and evaporated under
reduced pressure to give an extract. Dried extracts were resuspended
in 2 mL of MeOH and analyzed by UPLC-MS.
ACKNOWLEDGMENTS
■
This work was financially supported by the Scientific and
Technological Innovation Project, Qingdao National Labo-
ratory for Marine Science and Technology (2015ASKJ02,
2016ASKJ08-02), NSFC-Shandong Joint Fund for Marine
Science Research Centers (U1606403), the National Natural
Science Foundation of China (21372208), and Global Change
and Air-Sea Interaction Project (Grant GASI-IPOVAI-01-03).
We also appreciate Professor B. A. Pfeifer (The State
University of New York) for reviewing the manuscript.
Enzyme Inactivation Experiment. The fungus C. sphaerosper-
mum was cultivated in an Erlenmeyer flask (500 mL) containing 150
mL of fermentation media for 5 days. The supernatant of the
fermentation broth was clarified by a 3 kDa filter, collected in a
separate Erlenmeyer flask (100 mL), and then heated to 110 °C in an
REFERENCES
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17
oil bath for 5 min to denature and/or remove enzymes. The
collected supernatant without enzymes was divided into three
portions (each contain 20 mL), with SAHA (300 μM) added to
one portion, aniline (300 μM) added to another portion, and nothing
added to the third portion (control), respectively. After stirring at 28
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°C for 24 h, the supernatant was extracted with EtOAc and
evaporated under reduced pressure to give an extract. Dried extracts
were dissolved in 1 mL of MeOH and analyzed by UPLC-MS.
Bioactivity Evaluation. Cytotoxic activities of 1−5 were
evaluated against PC-3, MGC-803, SH-SY5Y, and HCT-116 cell
14
lines by the SRB method; the K562 and HL-60 cell lines were
15
analyzed by the MTT method.
In the MTT assay, moderate cell suspensions were mixed with 0.8%
Trypan blue solution by equal volume. Cell viability was detected by
F
J. Nat. Prod. XXXX, XXX, XXX−XXX