The Journal of Organic Chemistry
Article
5% HCl aqueous solution and then partitioned with EtOAc three
times. The water layer was basified with KOH and successively
partitioned with EtOAc three times, resulting in 3.2 g of the alkaloid
fraction. The alkaloid fraction (3.2 g) was subjected to Sephadex LH-
20 open CC with an isocratic solvent system of 100% MeOH to
obtain 10 subfractions (F.1−F.10). F.5 (32 mg) was subjected to
semipreparative HPLC with CH3CN/H2O (v/v, 40/60) containing
0.05% formic acid (flow rate, 4 mL/min), resulting in the isolation of
compound 1 (5.3 mg, tR = 7.7 min). F.7 (25 mg) was subjected to
semipreparative HPLC with CH3CN/H2O (v/v, 40/60) containing
0.05% formic acid (flow rate, 4 mL/min), resulting in the isolation of
compound 2 (3.4 mg, tR = 10.8 min). F.9 (1.4 g) was subjected to
medium pressure liquid chromatography with a gradient solvent
system of MeOH/H2O (v/v, 10/90 → 100/0) containing 0.05%
formic acid to obtain subfractions (F.9.1−F.9.20). F.9.12 (130 mg)
was applied to Sephadex LH-20 open CC with an isocratic solvent
system of 100% MeOH to obtain nine subfractions (F.9.12.1−
F.9.12.9). F.9.12.9 (44.2 mg) was applied to semipreparative HPLC
with CH3CN/H2O (v/v, 29/71) containing 0.05% formic acid (flow
rate, 2 mL/min) to obtain compound 3 (4.2 mg, tR = 23.8 min),
compound 4 (2.4 mg, tR = 26.9 min), and compound 5 (3.5 mg, tR =
31.5 min).
negative mode) of the derivative of Thr in 1 was compared with that
of L-Thr (tR = 17.08), D-Thr (tR = 18.53), L-all-Thr (tR = 17.41), and
D-all-Thr (tR = 17.96), assigning its absolute configuration to be L-Thr.
In the same manner, Thr in 3 and 4 was determined to be L-Thr
CH3CN over 70 min) at 1.0 mL min−1 [0.05% TFA in H2O and
CH3CN] was used. The retention time (tR = 37.27) of Ile in 2 was
compared with that of L-Ile (tR = 37.29), L-all-Ile (tR = 36.86), D-Ile
(tR = 50.19), and D-all-Ile (tR = 50.19), assigning its absolute
configuration to be L-Ile (Figure S47). In the same manner, Ile in 5
was also determined to be L-Ile. For the rest of the amino acids, the
gradient system (20 → 65% CH3CN over 35 min) at 1.0 mL min−1
[H2O and 0.1% HCOOH in CH3CN] was used. The retention times
of the derivatives of amino acids in 1−5 were compared to those of
Cytopathic Effect Inhibition Assay.38,39 MDCK cells were
seeded on 96-well culture plates at a density of 1 × 105 cells per well.
After incubation for 24 h, the medium was removed, and the cells
were washed twice with phosphate-buffered saline (PBS). The
influenza H1N1 and H9N2 viruses at a multiplicity of infection of
0.01 were inoculated onto near-confluent MDCK cell monolayers
using Dulbecco’s modified Eagle’s medium (DMEM) containing 0.15
μg/mL trypsin and 5 μg/mL bovine serum albumin for 2 h. The
medium was removed, the cells were washed with PBS, and new
medium with several compounds at different concentrations was
added. After being incubated for 3 days at 37 °C under a 5% CO2
atmosphere, the cell medium was replaced with DMEM, 20 μL of 2
mg/mL MTT was added to each well, and the plate was incubated for
4 h at 37 °C. After that, the subsequent steps followed those of the
cytotoxicity assay procedures, and the 50% effective concentration
(EC50) was evaluated by regression analysis. A selective index (SI)
value was determined via the formula SI = CC50/EC50. The statistical
significance was calculated via comparison with the null group.
Melicopteline A (1). Yellow amorphous solid; [α]2D0 −39.5 (c 0.4,
MeOH); UV (MeOH) λmax (log ε) 200 (4.0), 269 (2.7) nm; CD
(MeOH) (Δε) 221 (−4.0); IR νmax: 3311, 2967, 1638, 1545, 1454,
1
1055 cm−1; H and 13C{1H} NMR data, see Tables 1 and 2; HRMS
(ESI) m/z: [M + H]+ calcd for C27H47N6O7, 567.3506, found,
567.3517.
Melicopteline B (2). Pale yellow amorphous solid; [α]2D0 −24.7 (c
0.4, MeOH); UV (MeOH) λmax (log ε) 200 (4.3), 225 (3.9), 275
(3.4) nm; CD (MeOH) (Δε) 222 (−3.7); IR νmax: 3303, 2966, 1655,
1517, 1455, 1369 cm−1; 1H and 13C{1H} NMR data, see Tables 1 and
2; HRMS (ESI) m/z: [M + H]+ calcd for C28H42N5O6, 544.3135,
found, 544.3134.
Melicopteline C (3). White amorphous solid; [α]2D0 +2.6 (c 0.37,
MeOH); UV (MeOH) λmax (log ε) 201 (4.0), 235 (3.1), 289 (2.7)
nm; CD (MeOH) (Δε) 214 (−1.1), 227 (−1.1); IR νmax: 3435, 2969,
1734, 1653, 1560, 1033 cm−1; 1H and 13C{1H} NMR data, see Tables
1 and 2; HRMS (ESI) m/z: [M + H]+ calcd for C33H42N7O8,
664.3095, found 664.3099.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
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sı
Melicopteline D (4). White amorphous solid; [α]2D0 +30.8 (c 0.4,
MeOH); UV (MeOH) λmax (log ε) 201 (4.1), 233 (3.3), 288 (2.8)
nm; CD (MeOH) (Δε) 212 (−0.9), 231 (−1.7); IR νmax: 3317, 2966,
1654, 1560, 1032 cm−1; 1H and 13C{1H} NMR data, see Tables 1 and
2; HRMS (ESI) m/z: [M + H]+ calcd for C33H42N7O8, 664.3095,
found, 664.3123.
1D and 2D NMR, HRESIMS, ECD spectra, and
chromatograms after advanced Marfey’s analysis of
compounds 1−5; comparison of retention times of
derivatized amino acids in 1−5 to those of authentic
amino acids; and processes of logical deduction to
determine the absolute configuration of the HPI moiety
using extensive ROESY analysis (PDF)
Melicopteline E (5). White amorphous solid; [α]2D0 −10.5 (c 0.4,
MeOH); UV (MeOH) λmax (log ε) 201 (4.7), 226 (4.1), 284 (3.6)
nm; CD (MeOH) (Δε) 226 (4.1), 284 (3.6); IR νmax: 3318, 2966,
1644, 1518, 1438, 1282, 1033 cm−1; 1H and 13C{1H} NMR data, see
Tables 1 and 2; HRMS (ESI) m/z: [M + H]+ calcd for C35H46N7O8,
692.3408, found, 692.3415.
AUTHOR INFORMATION
Corresponding Author
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Advanced Marfey’s Analysis for Compounds 1−5.25,36,37
Compounds 1−5 (0.5 mg each) were hydrolyzed with 6 N HCl (200
μL) at 115 °C for 1.5 h. The residual HCl was removed using a rotary
evaporator. The dried hydrolysates of 1−5 were suspended in H2O
(20 μL) and treated with 1 M NaHCO3 (20 μL) and 25 mM L-FDLA
in acetone (100 μL). The solutions were heated at 37 °C for 1 h. The
reaction was quenched by adding 20 μL of 1 N HCl and diluted with
CH3CN. Aliquots of the solutions were used for the subsequent
HPLC analysis. For comparison with the derivatives of authentic
standard amino acids, pairs of L- and D-Val, Tyr, Leu, Pro, Thr, allo-
Thr, Ile, allo-Ile, and Phe were also derivatized successfully with L-
FDLA. The retention times of amino acids derivatives in 1−5 were
compared with those of authentic ones. Especially, Thr was analyzed
by HPLC−ESI-MS for clarity, and a 5 μm Kinetex C-18 column (250
× 4.6 mm, 100 Å) was eluted at 0.4 mL min−1 [0.1% HCOOH in
H2O and CH3CN] with the gradient system (20 → 80% CH3CN over
15 min, isocratic 80% CH3CN over 5 min). The retention time (tR =
17.09) with mass (412 m/z from extraction ion chromatogram of the
Won Keun Oh − Korea Bioactive Natural Material Bank,
Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 08826, Republic
Authors
Ba Wool Lee − Korea Bioactive Natural Material Bank,
Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 08826, Republic
Thi Kim Quy Ha − Korea Bioactive Natural Material Bank,
Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 08826, Republic
of Korea; Cantho University, Cantho City 900000, Vietnam
Eun Jin Park − Korea Bioactive Natural Material Bank,
Research Institute of Pharmaceutical Sciences, College of
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J. Org. Chem. 2021, 86, 1437−1447