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Vol. 58, No. 11
Table 1. 1H- (500 MHz) and 13C-NMR (125 MHz) Spectral Data of 1 and 2 in CD3OD (d in ppm)
Position
1
2
1, 2
HMBC (H→C)
dH
dC
dH
dC
COSY (H→H)
1
2/6
3/5
4
7
8
127.9
131.1
116.9
160.9
143.9
116.0
168.4
53.1
128.2
131.4
116.4
159.5
136.1
120.9
171.0
52.6
7.48 (d, 8.5)
6.80 (d, 8.5)
7.28 (d, 8.5)
6.73 (d, 8.5)
C-4, 7
C-1
H-3, 5
H-2, 6
7.53 (d, 15.8)
6.72 (d, 15.8)
6.62 (d, 12.5)
5.91 (d, 12.5)
C-1, 2, 6, 8, 9
C-7, 9
H-8
H-7
9
1Ј
3.71 (dd, 12.0, 5.5)
3.59 (dd, 12.0, 2.5)
4.11 (m)
4.18 (m)
3.91 (dd, 12.0, 5.5)
3.67 (dd, 12.0, 2.5)
3.66 (dd, 12.0, 5.5)
3.51 (dd, 12.0, 2.5)
4.10 (m)
4.01 (m)
3.53 (dd, 12.0, 5.5)
3.35 (dd, 12.0, 2.5)
C-9, 2Ј, 3Ј
H-2Ј
2Ј
3Ј
4Ј
74.9
76.5
53.7
75.0
76.1
54.1
C-1Ј, 3Ј, 4Ј
C-1Ј, 2Ј, 4Ј
C-2Ј, 3Ј
H-1Ј, 3Ј
H-2Ј, 4Ј
H-3Ј
Assignments were based on 2D-NMR including COSY, HMQC and HMBC. Well-resolved couplings are expressed with coupling patterns and coupling constants in Hz in
parentheses.
Ϫ6.4 (cϭ0.12, MeOH); UV (MeOH) lmax (log e) 230 (3.9) 315 (4.7) nm; IR
found that compounds 1 and 2 showed moderate cytotoxicity
against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines
(IC50 (1): 24.3, 37.0, 19.7, and 25.8 mM, and IC50 (2): 25.5,
45.4, 13.9, and 25.1 mM, respectively).
The anti-neuroinflammatory effects of 1 and 2 were also
evaluated using lipopolysaccharide (LPS)-activated BV-2
cells, a microglia cell line. However, none of the compounds
inhibited nitric oxide (NO) production in LPS-activated mi-
croglia cells.
(KBr) nmax 3356, 2945, 2834, 1666, 1650, 1452, 1028, 676 cmϪ1 1H-
;
(500 MHz) and 13C- (125 MHz) NMR data, see Table 1; FAB-MS (positive
mode) m/z: 289 [MϩNa]ϩ; HR-FAB-MS m/z: 289.1157 [MϩNa]ϩ (Calcd
for C13H18N2NaO4, 289.1164).
Pharnilatin B (2): White amorphous powder; mp 217—220 °C; [a]D25
Ϫ7.7 (cϭ0.23, MeOH); UV (MeOH) lmax (log e) 229 (3.9) 315 (4.6) nm; IR
(KBr) nmax 3355, 2945, 2833, 1667, 1651, 1452, 1028, 676 cmϪ1 1H-
;
(500 MHz) and 13C- (125 MHz) NMR data, see Table 1; FAB-MS (positive
mode) m/z: 289 [MϩNa]ϩ; HR-FAB-MS m/z: 289.1167 [MϩNa]ϩ (Calcd
for C13H18N2NaO4, 289.1164).
Preparation of cis-1,5-Dioxa-3-aza-7-cis-p-coumaroyl-decalin (1a)
Formaldehyde (paraformaldehyde, 2 mM) was added to an aqueous solution
of 1 (1 mM) and the solution was heated in an ultrasonic bath at 60 °C for
2 h. The reaction solution was extracted with CHCl3 to give 1a (1.0 mg).
Experimental
General Experimental Procedures All melting points were determined
on a Gallenkamp melting point apparatus and are uncorrected. Optical rota-
tions were measured on a Jasco P-1020 polarimeter. IR spectra were
recorded on a Bruker IFS-66/S FT-IR spectrometer. UV spectra were
recorded with a Shimadzu UV-1601 UV–Visible spectrophotometer. FAB
and HR-FAB mass spectra were obtained on a JEOL JMS700 mass spec-
trometer. NMR spectra, including 1H–1H COSY, HMQC, and HMBC exper-
iments, were recorded on a Varian UNITY INOVA 500 NMR spectrometer
operating at 500 MHz (1H) and 125 MHz (13C), with chemical shifts given in
ppm (d). Preparative high-performance liquid chromatography (HPLC) was
performed using a Gilson 306 pump with a Shodex refractive index detector.
Silica gel 60 (Merck, 230—400 mesh) and RP-C18 silica gel (Merck, 230—
400 mesh) were used for column chromatography. Merck precoated Silica
gel F254 plates and RP-18 F254s plates were used for thin layer chromatogra-
phy (TLC). Spots were detected on TLC under UV light or by heating after
spraying with 10% H2SO4 in EtOH (v/v). The packing material for molecu-
lar sieve column chromatography was Sephadex LH-20 (Pharmacia Co.,
Sweden).
1
1a: White amorphous powder; H-NMR (CDCl3, 500 MHz) d: 7.30 (1H,
d, Jϭ15.5 Hz, H-7Ј), 7.10 (2H, d, Jϭ8.5 Hz, H-2Ј/6Ј), 6.80 (2H, d, Jϭ8.5
2
4
Hz, H-3Ј/5Ј), 6.68 (1H, d, Jϭ15.5 Hz, H-8Ј), 4.22 (1H, dd, Jϭ9.5, Jϭ2.0
Hz, H-2eq), 4.17 (1H, dd, 2Jϭ9.5, 4Jϭ2.0 Hz, H-6eq), 4.09 (1H, d, 2Jϭ9.5 Hz,
2
H-2ax), 3.94 (1H, d, Jϭ9.5 Hz, H-6ax), 3.54 (1H, m, H-9), 3.51 (1H, m, H-
10), 3.40 (1H, dd, 2Jϭ13.5, 3Jϭ2.5 Hz, H-4eq), 3.35 (1H, m, 2Jϭ13.5 Hz, H-
2
3
2
3
8eq), 2.76 (1H, dd, Jϭ13.5, Jϭ2.5 Hz, H-4ax) 2.70 (1H, dd, Jϭ13.5, Jϭ
2.5 Hz, H-8ax); FAB-MS (positive mode) m/z: 291 [MϩH]ϩ.
Acidic Hydrolysis of Compounds 1 and 2 Compounds 1 (2 mg) and 2
(1.5 mg) were individually hydrolyzed with 6 N HCl under reflux for 24 h.
After cooling, each reaction mixture was diluted with H2O and extracted
with CHCl3. The aqueous layer was neutralized by passage through an Am-
berlite IRA-67 column and was repeatedly evaporated to give each fraction.
The fraction was individually chromatographed by silica gel Waters Sep-Pak
Vac 6 cc (CHCl3–MeOH, 10 : 1) to give the side chain, 1b (1 mg) from 1 and
2a (0.7 mg) from 2.
1b: White powder; [a]D25 Ϫ13.5 (cϭ0.05, H2O); 1H-NMR (D2O,
500 MHz) d: 3.90 (4H, dd, Jϭ7.0, 4.0 Hz, H-1/4), 3.12 (2H, m, H-2/3);
FAB-MS (positive mode) m/z: 143 [MϩNa]ϩ. Compound 2a was identical
Plant Materials The seeds of P. nil were purchased at Kyungdong
herbal market, Seoul, Korea, in July 2006, and were identified by one of the
authors (K.R.L.). A voucher specimen (SKKU 2006-7) was deposited in the
herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon,
Korea.
1
to 1b in terms of H-NMR and TLC and optical rotation of 2a was [a]D25
Ϫ14.4 (cϭ0.03, H2O).
Extraction and Isolation The dried seeds of P. nil (10 kg) were extracted
with 50% EtOH (3ϫ4 l, each 3 d) at room temperature and filtered. The
filtrate was evaporated in vacuo to obtain EtOH extract (1.4 kg), which was
suspended in distilled H2O (7.2 l) and then successively partitioned with
n-hexane, CHCl3, EtOAc, and n-BuOH, yielding 10, 7, 10, and 550 g,
respectively. The EtOAc-soluble fraction (10 g) was chromatographed on a
silica gel (230—400 mesh, 300 g) column and eluted with CHCl3–MeOH
(10 : 1→1 : 1, gradient system) to yield five fractions (A—E). Fraction E
(3.0 g) was chromatographed further on an RP-C18 silica gel (230—400
mesh, 150 g) column and eluted with MeOH–H2O (1 : 1→1 : 0, gradient sys-
tem) to give 11 subfractions (E1—E11). Fraction E1 (250 mg) was subjected
to Sephadex LH-20 column chromatography (MeOH–H2O, 4 : 1) and further
purified by preparative reversed-phase HPLC (MeCN–H2O, 1 : 4) to yield
two new compounds 1 (5 mg) and 2 (5 mg).
Synthesis of 1c Compound 1b (1 mg) was dissolved in 2,2-
dimethoxypropane (1 ml) and methanol (0.3 ml), and p-toluenesulfonic acid
(2 mg) was added. The reaction was allowed to proceed for 12 h at room
temperature and then quenched with 5% aqueous NaHCO3, extracted 3
times with CH2Cl2. The CH2Cl2 solution was dried (anhydrous MgSO4), the
solvent was removed under reduced pressure, and the residue was purified
by silica gel Waters Sep-Pak Vac 6 cc (CHCl3–MeOH, 30 : 1) to provide
compound 1c (0.5 mg).
1c: Oil; [a]D25 Ϫ73.1 (cϭ0.02, acetone); H-NMR (CDCl3, 500 MHz) d:
1
3.75 (2H, m, –OCHCH2–), 2.88 (2H, dd, Jϭ13.5, 4.0 Hz, –CH2NH2), 2.80
(2H, dd, Jϭ13.5, 6.0 Hz, –CH2NH2), 1.40 (6H, s, –CCH3); FAB-MS (posi-
tive mode) m/z: 161 [MϩH]ϩ.
Cytotoxicity Assay A sulforhodamine B (SRB) bioassay was used to
determine the cytotoxicity of each compound isolated against four cultured
human tumor cell lines.24) The assays were performed at the Korea Research
Pharnilatin A (1): White amorphous powder; mp 221—223 °C; [a]D25