Journal of Natural Products
Article
2800SPC UV/vis spectrometer (Shanghai, China). ECD spectra were
obtained using a DSM 1000 spectrometer (OLIS, USA) using a 1 mm
cell. NMR spectra were measured on an INOVA-600 (Varian, USA)
and an AVANCE III-400 (Bruker, Switzerland) spectrometer. HR-
ESI-MS spectra were acquired using an Orbitrap Elite spectrometer
equipped with an electrospray ionization (ESI) interface (Thermo,
USA). Column chromatography (CC) was performed using silica gel
(200−300 mesh) (Qingdao Haiyang Chemical Group Corporation,
Qingdao, China) and Sephadex LH-20 (Mitsubishi, Japan). Precoated
silica gel plates (GF254, 10−40 μm, Qingdao, China) were used for
thin-layer chromatography (TLC). Semipreparative HPLC was
performed on a system equipped with a 1525 liquid chromatograph
(Waters, USA) and a 2489 UV/visible (254 nm) (Waters, USA) peak
detector, using a Synergi C18 column (250 × 10 mm, 4 μm,
Phenomenex, USA). All solvents were of analytical grade for CC and
chromatographic grade for HPLC.
dropwise over 20 min. After stirring the mixture for 1 h at room
temperature, the reaction mixture was kept under reflux (80 °C) for 4
h, and then MeOH was removed under reduced pressure and H2O
(25 mL) added. The pH of the aqueous solution was adjusted to 9−
10 with NaOH, the solution was extracted with EtOAc, the organic
layer was washed with brine, dried (Na2SO4), and filtered, and solvent
was evaporated. The residue was purified by column chromatography
(CH2Cl2−MeOH, 50:1) to produce 11i or 11ii (41.1% yield) as a
yellow solid. To a solution of 11i or 11ii (1.0 mmol) in CH2Cl2 were
added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC) (1.2 mmol), triethylamine (Et3N) (1.2 mmol), 1-hydrox-
ybenzotriazole (HOBT) (1.2 mmol), and N-Boc-threonine (12i or
12ii) (1.2 mmol) at room temperature. The solution was stirred at
room temperature for 6 h. After completion of the reaction, the
solution was washed with brine (20 mL × 3). The organic layer was
dried over anhydrous Na2SO4 and the solvent evaporated. The
products 13i−13iv were obtained by purification with silica gel
column chromatography (petroleum ether−acetone, 5:2) in 52.6−
59.7% yields. 13i−13iv (1.0 mmol) were dissolved in 6.0 mL of 1,4-
dioxane−H2O (1:3) in a sealed tube. The solution was stirred at 150
°C for 7 h. After completion of the reaction, the crude products were
obtained by evaporation of solvent under reduced pressure. The crude
product was washed with water to give 3i−3iv in 36.0−49.7% yields.
ECD, NMR, and Specific Rotation Calculations. The analysis
of the absolute configuration of 1 and 2 was supported by the
Supercomputing Center of Lanzhou University, China. Conforma-
tional searches were performed with molecular dynamics using the
xtb-200702 and the ORCA-4.2.1 software. The initial conformers
were batch optimized with GFN0-xTB and GFN2-xTB using xtb-
200702 in turn and filtered by an energy threshold of 0.5 kcal/mol
and geometry threshold of 0.25 Å. The DFT calculations of the
resulting conformers of ECD and NMR of 1 were subjected to
geometry optimizations and frequency calculations at the B3LYP/6-
31G(d) level, while the specific rotations of 1 and 2 were obtained at
the PBEPBE/6-31G(d) level using the Gaussian 09 program. The
single-point-energy calculations were both performed at the RI-
PWPB95/DEF2-QZVPP level using ORCA-4.2.1 with SMD in
methanol. All of these programs were used through Molclus-1.9.6
software.26 The dominant conformations of ECD spectra of 1 were
calculated by the TDDFT methodology at the B3LYP/6-311G(d)
level with SMD in methanol. ECD spectra were simulated using
SpecDis 1.71.27 The NMR shielding tensors of 1 were computed with
the GIAO methodology at the B972/pcSseg-1 level of theory with
SMD in acetone, and the Boltzmann-weighted chemical shift data
were acquired with Multiwfn-3.8 software.28 The specific rotation data
were calculated by the DFT methodology at the B3LYP/6-31G(d)
level for 1 and the B3LYP/ma-def2TZVPP level for 2 with SMD in
methanol, and the Boltzmann-weighted data were acquired with
Cell Culture and Cell Viability Assay. HeLa and HepG2 tumor
cells were purchased from the Shanghai Institute of Biochemistry and
Cell Biology, Chinese Academy of Sciences (September 2019). The
cells were maintained in RPMI 1640 medium (containing 100 units/
mL of penicillin/streptomycin and 10% FBS). Cell viability was
assessed by the Cell Counting Kit-8 assay (CCK-8) (TopScience,
China). Cells (8000 cells/well) were seeded into 96-well plates. After
culturing for 24 h, cells were incubated with 20 μM compounds 1−9
for an additional 48 h. Subsequently, 10% CCK-8 solution was added
to each well and incubated for 1.5 h. Absorbance at 450 nm was then
measured using a microplate reader (BioTek, USA). Cell viability was
calculated as a percentage relative to the DMSO group.
Assay of Antimicrobial Activity. The filter paper diffusion
method was used. A 200 μg amount of compounds 1, 2, and 3i−3iv
was dissolved in 50 μL of DMSO and 950 μL of sterile water,
respectively, for the solutions of 200 μg/mL. Five concentration
gradients were prepared according to the double-dilution method.
Filter papers were impregnated with different concentrations. The
filter paper was placed on agar plates inoculated with bacterial and
fungal pathogens, including Bacillus cereus, Bacillus subtilis, Erwinia
Fungi Material. The isolation and identification of the used
fungus were described previously.13
Fermentation, Extraction, and Isolation. Erlenmeyer flasks
(1000 mL) containing rice (100 g) and distilled water (150 mL) were
inoculated with spores after sterilization. The flasks were cultured
under static conditions at 26 °C for 26 days.
The culture was macerated and extracted three times with
Me2CO−CH2Cl2−MeOH (4:3:1), and the extract was first
partitioned by liquid−liquid extraction with petroleum ether (15 L),
ethyl acetate (15 L), and n-butanol (15 L). The EtOAc fraction (53.9
g) was subjected to silica gel open CC (200−300 mesh) eluting with a
mixture of CH2Cl2−MeOH (50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1, 5.0 L
each) to yield 12 fractions (A−G).
Fr.A2 (6.0 g) was further fractionated by silica gel CC eluting with
a mixture of petroleum ether−Me2CO (10:1−0:1) to yield nine
fractions (A21−A29). Fr.A25 (556.8 mg) was subjected to silica gel
chromatography eluting with a mixture of CH2Cl2−Me2CO (20:1−
0:1) and CH2Cl2−EtOAc (5:1) to yield 7 (2.1 mg), 8 (13.0 mg), and
9 (4.0 mg). Fr.A28 (411.2 mg) was subjected to a Sephadex LH-20
column eluting with MeOH and silica gel chromatography eluting
with a mixture of petroleum ether−Me2CO (2:1) to yield 6 (1.5 mg).
Fr.B2 (1.7 g) was further fractionated by silica gel chromatography
eluting with a mixture of petroleum ether−EtOAc (5:1−1:1) to yield
10 fractions (B21−B210). Fr.B27 (333.4 mg) was subjected to silica
gel CC eluting with a mixture of petroleum ether−Me2CO (4:1−2:1)
and a Sephadex LH-20 CC eluting with MeOH to yield 1 (2.4 mg)
and 2 (1.0 mg). Fr.B29 (26.5 mg) was repeatedly eluted with
methanol to yield insoluble compound 5 (7.1 mg).
Fr.C1 (3.4 g) was further fractionated by Sephadex LH-20 CC
eluting with MeOH, semipreparative HPLC (MeOH−H2O, 40:60,
250 × 10 mm, 4 μm, Synergi 4u Hydro-RP 80A), and Sephadex LH-
20 CC eluting with petroleum ether−CH2Cl2−MeOH (2:1:1) to
yield 4 (3.5 mg). Fr.C2 (2.4 g) was further fractionated by Sephadex
LH-20 CC eluting with MeOH, semipreparative HPLC (MeOH−
H2O, 25:75, 250 × 10 mm, 4 μm, Synergi 4u Hydro-RP 80A), and
Sephadex LH-20 CC eluting with petroleum ether−CH2Cl2−MeOH
(2:1:1) to yield 3 (7.0 mg).
Clonorosin A (1): pale yellow powder; [α]2D0 −10.00 (c 0.30,
CH3OH); IR (KBr) νmax 3333, 2926, 2855, 1729, 1657, 1612, 1444,
1380, 1265, 1194, 1121, 1094, 1023, 866, 792, 739, 703, 664, 607
cm−1; UV (MeOH) λmax (log ε) 220 (3.93), 274 (3.27) nm; ECD
(MeOH) λmax (mdeg, 0.24 mg/mL) 200 (+22.50), 214 (−5.36), 231
(+18.22), 274 (−29.68), and 309 (+0.10) nm; HR-ESI-MS [M + H]+
m/z 355.1648 (calcd 355.1652); for 1H NMR and 13C NMR data, see
Table 1.
Clonorosin B (2): white powder; [α]2D0 +80.00 (c 0.30, CH3OH);
IR (KBr) νmax 3344, 2922, 2852, 1735, 1720, 1638, 1595, 1460, 1385,
1301, 1261, 1095, 854, 794, 738, 660, 615 cm−1; UV (MeOH) λmax
(log ε) 206 (3.37), 246 (3.10). 286 (2.91), 341 (3.17) nm; HR-ESI-
MS [M + H]+ m/z 216.0650 (calcd 216.0655); for 1H NMR and 13
C
Synthesis of 2,5-Diketopiperazine Derivatives. To a stirred
solution of tryptophan (10i or 10ii) (10 mmol) in 50 mL of dry
MeOH, under ice-cooling, was added SOCl2 (2.18 mL, 30 mmol)
E
J. Nat. Prod. XXXX, XXX, XXX−XXX