Journal of Natural Products
Note
All air- and moisture-sensitive reactions were carried out in a flame-
dried, argon-flushed, two-necked flask sealed with rubber septa, and
the dry solvents and reagents were introduced using a syringe.
Tetrahydrofuran (THF) was freshly distilled under an argon
atmosphere from sodium benzophenone ketyl. Dichloromethane
(CH2Cl2) was freshly distilled from phosphoric pentaoxide (P2O5).
Flash column chromatography was carried out on a Kanto Chemical
silica gel 60N (spherical, neutral, 40−50 μm), and precoated Merck
silica gel plates (Art5715 Kieselgel 60F254, 0.25 mm) were used for
thin-layer chromatography (TLC). TLC visualization was accom-
panied using a UV lamp (254 nm) or a charring solution (ethanolic p-
anisaldehyde, ethanolic phosphomolybdic acid).
Analytes. Commercially available antimycin A (Sigma-Aldrich, a
mixture of A1−A4) was submitted to the evaluation. UK-2A was a
generous gift from Meiji Seika Pharma Co., Ltd. Splenocin B was
prepared as we have recently reported.12 Compounds 4−6 were
synthesized by the methods described in our previous report9 as
Isovaleric acid (3S,4R,7R,8R,9S)-[3-(3-hydroxy-4-methoxy-
piridine-2-carbonyl)-amino-7-benzyl-9-methyl-2,6-dioxo-
[1,5]dioxonan-8-yl] ester (4): 1H NMR (600 MHz, CDCl3) δ
11.78 (s, 1H), 8.59 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 5.2 Hz, 1H), 7.25
(t, J = 7.0 Hz, 2H), 7.20 (t, J = 7.3 Hz, 1H), 7.13 (d, J = 7.3 Hz, 2H),
6.87 (d, J = 5.2 Hz, 1H), 5.35 (m, 1H), 5.23 (t, J = 9.8 Hz, 1H), 5.17
(m, 1H), 4.97 (m, 1H), 3.94 (s, 3H), 3.62 (m, 1H), 3.00 (t, J = 12.6
Hz, 1H), 2.93 (td, J = 2.9, 11.4 Hz, 1H), 2.74 (d, J = 13.0 Hz, 1H),
2.26 (d, J = 7.1 Hz, 2H), 2.16 (sept, J = 6.8 Hz, 1H), 1.34 (d, J = 6.4
Hz, 3H), 1.00 (d, J = 6.6 Hz, 6H); 13C NMR (150 MHz, CDCl3) δ
171.79, 171.72, 169.73, 168.95, 155.42, 148.83, 140.70, 137.81,
129.94, 128.75, 128.59, 126.68, 109.70, 75.20, 74.77, 65.25, 56.10,
51.85, 49.98, 43.16, 34.63, 25.46, 22.45, 17.90; HRMS (ESI+) m/z
calcd for C27H32N2NaO9 [M + Na]+ 551.20055, found 551.20308; IR
(KBr) 3369, 2966, 1749, 1369, 1241, 849, 760 cm−1; [α]D +83.2 (c
0.223, CHCl3).
1H), 3.41 (m, 1H), 2.51 (td, J = 2.9, 11.2 Hz, 1H); 13C NMR (150
MHz, CDCl3) δ 75.24, 74.20, 66.99, 49.85.
Cell Culture. RBL-2H3 cells (Health Science Resources Bank,
Osaka, Japan) were cultured in Dulbecco’s modified Eagle’s medium
(Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine
serum (Life Technologies, CA, USA), 100 U/mL penicillin, and 100
μg/mL streptomycin (Sigma) as a growth medium at 37 °C under 5%
CO2.
Measurement of IL-4 Released from Cells. RBL-2H3 cells
were inoculated into a 96-well plate (2.5 × 104 cells/well) and
cultured overnight. The cells were further incubated in a growth
medium containing 50 ng/mL of DNP-IgE (Sigma) for 2 h. After
washing with the growth medium, each drug in N,N-dimethylforma-
mide (0.05% final concentration) was added to the culture. After
incubation for 10 min, DNP-labeled human serum albumin (50 ng/
mL final concentration, Sigma) was added, and the culture was
incubated for 1 h. The release levels of IL-4 in the supernatant were
measured using rat IL-4 platinum ELISA (Invitrogen BMS628) and
the multiplex immunoassay Luminex 200 system (Luminex Co., TX,
USA).
Cytotoxicity Assay. RBL-2H3 cells were seeded on 96-well plates
at an initial density of 105 cells per well and incubated under 5% CO2
for 12 h at 37 °C. After incubation, RBL-2H3 cells were further
incubated in Dulbecco’s modified Eagle’s medium with each drug for
24 h. The medium was then replaced with 100 μL of the culture
medium containing serum. For the detection of relative cell viability, a
Cell Counting Kit-8 (Dojindo Molecular Technologies, 10 μL)
solution was added into each well of the 96-well plate containing a
completely water-soluble tetrazolium salt, WST-8 [2-(2-methoxy-4-
nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,
monosodium salt], which is reduced by dehydrogenase in living cells
to give an orange-colored outcome, WST-8 formazan, which was also
soluble in the cell medium. The quantity of the formazan was directly
proportional to the amount of living cells after 1 h of incubation, and
results were obtained by a microplate reader at 450 nm.
Isobutyric acid (3S,4R,7R,8R,9S)-7-benzyl-3-(3-formylami-
no-2-hydroxybenzoylamino)-9-methyl-2,6-dioxo-[1,5]-
dioxonan-8-yl ester (5). 5 was prepared as a 6:1 mixture of
rotamers. Major: 1H NMR (600 MHz, CDCl3) δ 12.57 (s, 1H), 8.65
(d, J = 8.1 Hz, 1H), 8.53 (dd, J = 1.1, 8.0 Hz, 1H), 8.49 (d, J = 1.1 Hz,
1H), 7.92 (s, 1H), 7.26 (t, J = 6.9 Hz, 2H), 7.21 (t, J = 7.8 Hz, 1H),
7.18 (dd, J = 1.1, 8.1 Hz, 1H), 7.12 (d, J = 6.3 Hz, 2H), 6.89 (t, J =
8.1 Hz, 1H), 5.46 (m, 1H), 5.24 (t, J = 9.7 Hz, 1H), 5.16 (m, 1H),
5.02 (m, 1H), 3.60 (m, 1H), 3.03−2.90 (m, 2H), 2.72 (d, J = 12.1 Hz,
1H), 2.63 (sept, J = 7.0 Hz, 1H), 1.34 (d, J = 6.4 Hz, 3H), 1.252 (d, J
= 6.9 Hz, 3H), 1.246 (d, J = 6.9 Hz, 3H); 13C NMR (150 MHz,
CDCl3) δ 175.62, 171.89, 170.06, 169.47, 158.97, 150.58, 137.73,
128.74, 128.63, 127.37, 126.76, 124.76, 120.11, 118.93, 112.45, 75.13,
74.91, 65.88, 52.03, 50.90, 34.53, 34.10, 18.97, 18.96, 17.82; HRMS
(ESI+) m/z calcd for C27H30N2NaO9 [M + Na]+ 549.18490, found
549.18644; IR (KBr) 3369, 2978, 2937, 1752, 1364, 1242, 740 cm−1;
[α]D +79.6 (c 6.54 × 10−3, CHCl3). Minor (diagnostic peaks only):
1H NMR (600 MHz, CDCl3) δ 12.41 (s, 1H), 8.77 (d, J = 11.5 Hz,
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
■
S
Synthetic route, 1D NMR spectra (1H and 13C) of 4−6,
effects of UK-2A (1) and compounds 4−6 on anti-
inflammatory cytokine IL-4 release, and effects of
compounds 4−6 on RBL-2H3 cell viability (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
1H), 7.74 (d, J = 11.2 Hz, 1H), 6.87 (t, J = 8.0 Hz, 1H), 5.23 (t, J =
9.0 Hz, 1H); 13C NMR (150 MHz, CDCl3) δ 160.84, 118.78.
Isobutyric acid (3S,4R,7R,8R,9S)-[3-(3-hydroxy-4-methoxy-
piridine-2-carbonyl)-amino-7-butyl-9-methyl-2,6-dioxo-[1,5]-
dioxonan-8-yl] ester (6). 6 was prepared as a 4:1 mixture of
rotamers. Major: 1H NMR (600 MHz, CDCl3) δ 11.09 (s, 1H), 8.65
(d, J = 8.1 Hz, 1H), 8.01 (d, J = 5.2 Hz, 1H), 6.89 (d, J = 5.2 Hz, 1H),
5.48 (br s, 1H), 5.19 (m, 1H), 5.07 (t, J = 9.9 Hz, 1H), 4.94 (m, 1H),
3.95 (s, 3H), 3.76 (m, 1H), 2.61 (sept, J = 7.0 Hz, 1H), 2.58 (td, J =
2.9, 13.0 Hz, 1H), 1.72 (m, 1H), 1.28 (d, J = 8.3 Hz, 3H), 1.38−1.09
(m, 5H), 1.217 (d, J = 6.9 Hz, 3H), 1.211 (d, J = 6.9 Hz, 3H), 0.86 (t,
J = 7.0 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 175.55, 172.67,
169.73, 168.98, 155.43, 148.84, 140.71, 129.97, 109.71, 75.17, 74.90,
65.12, 56.10, 52.06, 50.03, 34.09, 29.17, 28.06, 22.33, 18.93, 18.91,
17.77, 13.73; HRMS (ESI−) m/z calcd for C23H31N2O9 [M − H]−
479.20295, found 479.20270; IR (KBr) 3100, 2937, 1751, 1284, 1240,
810 cm−1; [α]D +71.1 (c 2.73 × 10−1, CHCl3). Minor (diagnostic
peaks only): 1H NMR (600 MHz, CDCl3) δ 5.14 (br s, 1H), 4.80 (m,
ORCID
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This paper is dedicated to the memory of the late emeritus
Professor Makoto Taniguchi, Osaka City University. We also
thank Dr. Matsumi Doe, Analytical Division, Osaka City
University, for 2D NMR measurements. The present research
was supported in part by the Osaka City University (OCU)
Strategic Research Grant 2017 for top priority research, which
is greatly appreciated.
D
J. Nat. Prod. XXXX, XXX, XXX−XXX