Original Paper
643
N-Oxide Alkaloids and Cytotoxic Constituents of Corydalis tashiroi
New Tetrahydroprotoberberine
1
2
3,
Jih-Jung Chen , Chang-Yih Duh , and Ih-Sheng Chen * 1 2 3
Department of Pharmacy, Tajen Junior College of Pharmacy, Pingtung, Taiwan, Republic of China
Institute of Marine Resources, National Sun Yat-sen University, Kaohsiung, Taiwan, Republic of China
Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical College, Kaohsiung, Taiwan, Republic of China Revision accepted: April 6, 2, 1998 Received: December 2, 1999; 1998; Received: Accepted:December April 6, 1999
Abstract:
Three new tetrahydroprotoberberine
loids, (±)-cis-corydalmine
2
N-oxide (1),
N-oxide
alka-
(±)-trans-corydalmine
oxide ( ), and (±)-trans-isocorypalmine
NN-oxide (3), along with
three known benzo[c]phenanthridine alkaloids, norsanguinar-
4
5
6 7 corydalmine (8), (±)-scoulerine (9), (±)-corynoxidine (10), (±)epicorynoxidine (11), palmatine (12), and protopine (13), have been isolated from the herb Corydalis tashiroi. The structures of
ine ( ), dihydrosanguinarine ( ), and oxysanguinarine ( ), six
known berberine alkaloids, (±)-tetrahydropalmatine ( ), (±)-
these compounds were elucidated by spectroscopic analysis. Three of the isolated compounds show significant cytotoxic activities (ED50 values < 4
mg/ml) against P-388, KB16, A549, and
HT-29 cell lines. Key words:
Corydalis tashiroi,
Fumariaceae, alkaloids, berber-
N-oxide (±)-cis-corydalmine NN-oxide, (±)-trans-isocorypalmine
ine, tetrahydroprotoberberine oxide, (±)-trans-corydalmine
N-oxide, cytotoxic activity.
Introduction
Makino (Fumariaceae) is a biennial herb without tubers (1), and distributed in southern Japan, southern China and Taiwan. Among the constituents of the Fumariaceae, the isoquinoline alkaloids are an important group of compounds. The plant is rich in isoquinoline alkaloids including tetrahydroprotoberberine, berberine, and protopine (2), (3). As a series of studies on the active constituents of cytotoxic and antiplatelet aggregation principles of Formosan plants, we have screened about 400 species till now and C. tashiroi was one of the active species in both screening programs.
Corydalis tashiroi
In this paper, we report on the isolation and structural elucidation of three new tetrahydroprotoberberine N-oxide alkaloids together with ten known compounds and on the cytotoxic activities of the isolates.
Planta Medica 65 (1999) 643±647 Georg Thieme Verlag Stuttgart New York ISSN: 0032-0943 ·
Materials and Methods
General experimental procedures
All melting points were determined on a Yanaco micro-melting point apparatus and were uncorrected. IR spectra were taken on a Hitachi 260-30 (KBr) spectrophotometer. UV spectra were obtained on a Shimadzu UV-160A spectrophotometer in EtOH. EI-mass spectra were recorded on a VG Biotech Quattro 5022 spectrometer. HR-mass spectra were recorded on a JEOL JMXHX 110 spectrometer. 1H-NMR (400 MHz), 13C-NMR (100 MHz) and NOESY spectra were measured on a Varian Unity 400 spectrometer and values are given in ppm (d). Silica gel (60±230, 230±400 mesh) (Merck) was used for CC and silica gel 60 F254 (Merck) for TLC and preparative TLC. Optical rotations were measured using a Jasco DIP-370 polarimeter in CHCl3. Plant material
was collected from Wutai, Pingtung Hsien, Taiwan in December 1994. A voucher sample (Chen 2439) was depos-
C. tashiroi
644
Planta Med. 65 (1999)
ited in the Herbarium of the School of Pharmacy, Kaohsiung Medical College, Kaohsiung, Taiwan, Republic of China. Extraction and isolation
Fresh herb (9.32 kg) was extracted with cold MeOH and the extract concentrated under reduced pressure. The MeOH extract was partitioned between CHCl3-H2O (1:1) to afford a CHCl3-soluble fraction (fr. A, 70 g). Fr. A (70 g) was chromatographed on silica gel (1450 g) eluting with CHCl3, gradually increasing the polarity with MeOH to obtain 24 frs: fr. A1 ~ A3 (each 500 ml, CHCl3), fr. A4 ~ A5 (each 1000 ml, CHCl3), fr. A6 (500 ml, CHCl3), fr. A7 ~ A12 (each 3000 ml, CHCl3), fr. A13 ~ A16 (each 3000 ml, CHCl3-MeOH, 99:1), fr. A17 (2500 ml, CHCl3-MeOH, 98:2), fr. A18 ~ A19 (each 3000 ml, CHCl3-MeOH, 95:5), fr. A20 (3000 ml, CHCl3-MeOH, 90:10), fr. A21 ~ A22 (each 3000 ml, CHCl3-MeOH, 80:20), fr. A23 (3000 ml, CHCl3-MeOH, 50:50), fr. A24 (5000 ml, MeOH). Fr. A5 (387 mg) was rechromatographed with silica gel (11.6 g) by using CHCl3 as eluent to give frs A5-1 ~ A5-5 (each 250 ml). Fr. A5-2 (79 mg) was separated by preparative TLC (n-hexaneEtOAc, 4:1) to give 4 (7.3 mg) (Rf 0.55) and 5 (9.5 mg) (Rf 0.68). Fr. A5-4 (71 mg) was separated by preparative TLC (benzene-MeOH, 15:1) to give 6 (6.4 mg) (Rf 0.46). Fr. A15 (7.64 g) was rechromatographed on silica gel (210 g) eluting with CHCl3-Me2CO (10:1) to obtain frs A15-1 ~ A15-7 (each 400 ml). Part (200 mg) of fr. A15-4 (1.4 g) was further purified by preparative TLC (CHCl3-EtOAc, 7:1) to give 7 (149 mg) (Rf 0.35) after recrystallization from CHCl3-MeOH. Part (104 mg) of fr. A15-7 (453 mg) was further purified by preparative TLC (CHCl3-MeOH, 3:1) to give 12 (16 mg) (Rf 0.89). Fr. A17 (566 mg) was rechromatographed on silica gel (15.8 g) eluting with CH2Cl2-MeOH (10:1) to obtain 8 frs (each 200 ml, fr. A17-1 ~ fr. A17-8). Fr. A17-7 (67 mg) was separated by preparative TLC (Me2CO-MeOH, 3:1) to give 3 (8.1 mg) (Rf 0.31). Fr. A18 (5.925 g) was rechromatographed with silica gel (180 g) by using CH2Cl2-MeOH as eluent to give frs A181 ~ A18-8 (each 200 ml). Part (102 mg) of fr. A18-4 (794 mg) was separated by preparative TLC (CHCl3-Me2CO, 4:1) to give 8 (11.4 mg) (Rf 0.58) and 9 (9.8 mg) (Rf 0.46). Fr. A18-6 (89 mg) was separated by preparative TLC (CHCl3-EtOH, 5:2) to give 1 (5.1 mg) (Rf 0.53) and 2 (4.8 mg) (Rf 0.47). Fr. A21 (6.266 g) was rechromatographed on silica gel (185 g) eluting with CHCl3-MeOH (10:1) to obtain 6 frs (each 400 ml, fr. A211 ~ fr. A21-6). Fr. A21-1 (473 mg) was rechromatographed with silica gel (14 g) by using EtOAc-MeOH (10:1) as eluent to give frs A21-1-1 ~ A21-1-3 (each 250 ml). Fr. A21-1-2 (29 mg) was separated by preparative TLC (CHCl3-MeOH, 10:1) to give 13 (6.4 mg) (Rf 0.49). Fr. A21-1-3 (35 mg) was separated by preparative TLC (Me2CO-MeOH, 6:1) to give 10 (4.9 mg) (Rf 0.28) and 11 (5.6 mg) (Rf 0.11). Isolates
(±)-cis-Corydalmine N-oxide (1): Colorless prisms from CHCl3-MeOH, m.p. 205±207 8C. UV: lmax nm (log e) = 206 (4.71), 227 (sh, 4.10), 284 (3.71); (KOH) 213 (5.09), 252 (4.04), 286 (3.85). IR: nmax (cm±1) = 3350 (OH), 1600, 1505, 1458 (aromatic ring C=C stretch). EI-MS: m/z (rel. int.) = 341 ([M ± 16]+, 31), 340 (42), 339 (78), 338 (83), 324 (17), 323 (57), 308 (12), 307 (15), 192 (100), 190 (42), 150 (25), 149 (30), 139 (32), 135 (37), 121 (35); FAB-MS: m/z (rel. int.) = 358 ([M + H]+, 42); HR/ FAB-MS: C20H24O5N, found: 358.1664 [M + H]+, calcd:
Jih-Jung Chen, Chang-Yih Duh, and Ih-Sheng Chen
358.1654. 1H-NMR (CDCl3, 400 MHz): see Table 1. 13C-NMR (CDCl3, 100 MHz): d = 24.9 (C-5), 36.5 (C-13), 56.0 (OMe-2), 56.1 (OMe-3), 56.9 (C-6), 60.2 (OMe-9), 65.8 (C-8), 70.5 (C14), 109.9 (C-1), 111.7 (C-4), 117.9 (C-11), 119.5, 121.8, 122.6, 123.3 (C-12), 126.5, 144.0, 147.9, 148.6, 149.5. [a]26D: ±28.28 (c 0.31, CHCl3). (±)-trans-Corydalmine N-oxide (2): Colorless prisms from CHCl3-MeOH, m.p. 176±178 8C. UV: lmax nm (log e) = 209 (4.58), 228 (sh, 4.17), 284 (3.74); (KOH) 213 (4.68), 240 (sh, 4.11), 286 (3.78). IR: nmax (cm±1) = 3375 (OH), 1610, 1515, 1458 (aromatic ring C=C stretch). EI-MS: m/z (rel. int.) = 341 ([M ± 16]+, 21), 340 (35), 339 (97), 338 (100), 324 (21), 323 (81), 308 (16), 307 (22), 192 (56), 190 (29), 149 (26), 140 (20), 135 (32), 107 (21), 77 (21); FAB-MS: m/z (rel. int.) = 358 ([M + H]+, 51), 342 ([M + H ± O]+, 9); HR/FAB-MS: C20H24O5N, found: 358.1645 [M + H]+, calcd: 358.1654. 1H-NMR (CDCl3, 400 MHz): see Table 1. 13C-NMR (CDCl3, 100 MHz): d = 24.4 (C5), 29.8 (C-13), 56.0 (OMe-2), 56.3 (OMe-3), 59.8 (OMe-9), 64.5 (C-6), 66.5 (C-8), 68.3 (C-14), 108.8 (C-11), 111.5 (C-12), 118.0 (C-4), 120.7, 121.8, 123.1 (C-1), 123.8, 124.3, 144.8, 148.3, 148.5, 148.6. [a]24D: ±75.38 (c 0.22, CHCl3). (±)-trans-Isocorypalmine N-oxide (3): Colorless needles from CHCl3, m.p. 224±226 8C. UV: lmax nm (log e) = 210 (4.53), 225 (sh, 4.18), 284 (3.73); (KOH) 214 (4.62), 250 (3.84), 286 (sh, 3.62), 302 (3.71). IR: nmax (cm±1) = 3430 (OH), 1610, 1500, 1460 (aromatic ring C=C stretch). EI-MS: m/z (rel. int.) = 341 ([M ± 16]+, 14), 340 (15), 339 (27), 338 (22), 324 (14), 280 (15), 265 (15), 221 (18), 177 (17), 176 (21), 164 (27), 162 (24), 149 (55), 147 (43), 121 (34), 91 (35), 77 (47), 73 (100); FAB-MS: m/z (rel. int.) = 358 ([M + H]+, 100), 342 ([M + H ± O]+, 14); HR/FAB-MS: C20H24O5N, found: 358.1664 [M + H]+, calcd: 358.1654. 1HNMR (CDCl3, 400 MHz): see Table 1. 13C-NMR (CD3OD, 100 MHz): d = 25.0 (C-5), 30.4 (C-13), 56.4 (OMe-3), 56.4 (OMe-10). 60.6 (OMe-9), 65.3 (C-6), 67.5 (C-8), 68.8 (C-14), 112.4 (C-1), 113.4 (C-4), 113.5 (C-11), 123.9, 124.3, 124.9 (C-12), 125.5, 126.7, 146.8, 146.9, 148.6, 152.1. [a]25D: ±38.68 (c 0.23, CHCl3). Cytotoxicity test
KB16 and P-388 cells were kindly provided by Prof. J. M. Pezzuto, University of Illinois at Chicago; A549 (human lung adenocarcinoma) and HT-29 (human colon carcinoma) were purchased from American Type Culture Collection. P-388 cells were cultured in Fisher©s medium supplemented with 10% heat-inactivated (56 8C for 30 min) fetal calf serum (FCS). The KB cells were maintained in Basal Medium Eagle (BME) containing 10% heat-inactivated FCS. A549 cells were cultured in Eagle Minimum Essential Medium (EMEM) containing Earle©s salts and supplemented with 0.1 mM of nonessential amino acids and 10% heat-inactivated FCS. HT-29 cells were maintained in Rosewell Park Memorial Institute (RPMI) 1640 Medium containing 10% heat-inactivated FCS. All cell lines were maintained in an incubator at 37 8C in humidified air containing 5% CO2. The cytotoxic activities of compounds against P-388, KB16, A549, and HT-29 were assayed by a modification of the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method (4). For P-388 cells, 200 ml cultures
New Tetrahydroprotoberberine
N-Oxide Alkaloids and
Cytotoxic Constituents of
were established at 1500 cells/well in 96-well tissue culture plates (Falcon). Compounds were dispensed to established cultures at eight concentrations in triplicate. After three days of incubation, P-388 cells were enumerated with MTT.
Results and Discussion
(±)-cis-Corydalmine N-oxide (1) was isolated as colorless prisms. The FAB-MS afforded the positive ion at m/z 358 [M + H]+, implying a molecular formula of C20H24O5N, which was confirmed by the HRFAB-MS ([M + H]+ found 358.1664, calcd 358.1654). The fragment peak in the EI-MS at m/z 341 was due to the loss of an oxygen atom and suggested the presence of an N-oxide function. The UV absorptions at 206, 227 sh, 284 nm were similar to those of epicorynoxidine (11) and were characteristic of the 2,3,9,10-oxygenated tetrahydroprotoberberine skeleton (5). The presence of a phenolic hydroxy group was indicated by the IR absorption at 3350 cm±1 and a 1
Planta Med. 65 (1999)
bathochromic shift of UV absorption in alkaline solution. The 1H-NMR spectrum of 1 showed four mutually coupling aliphatic protons at d 2.90 (1H, m, H-5a), d 3.45 (1H, m, H-6b), d 3.66 (1H, m, H-5b) and d 3.71 (1H, m, H-6a) in ring B, and other three mutually coupling aliphatic protons at d 2.97 (1H, dd, J = 18.0, 10.0 Hz, H-13b), d 3.55 (1H, dd, J = 18.0, 6.4 Hz, H13a), and d 4.58 (1H, dd, J = 10.0, 6.4 Hz, H-14) in ring C. In addition, the chemical shifts of aliphatic protons of ring B and C, including the signals at d 4.75 and 4.84 (each 1H, d, J = 16.0 Hz, H-8a and H-8b) were similar to those of epicorynoxidine (11) and different from those of corynoxidine (10) (5) (Table 1), and suggested that the N-oxide function occupied the a orientation. The 1H-NMR spectrum of 1 also revealed the presence of three methoxy signals at d 3.78, 3.83 and 3.86 (each 3H, s) due to OMe-9; OMe-2 and OMe-3. The aromatic region of the spectrum showed four protons: two at d 6.58, 6.70 (each 1H, s) were assigned to H-1 and H-4, and the other two ortho-coupled protons at d 6.70 and 6.83 (each 1H, d, J = 8.4 Hz) due to H-12 and H-11. Furthermore, the chemical shifts of C-6 (d 56.9), C-13 (d 36.5) and C-14 (d 70.5) of 1 were similar to those of epicorynoxidine (11) and cis-xylopinine Noxide, and different from those of corynoxidine (10) and trans-xylopinine N-oxide (6) (Table 2). Therefore, a cis-stereochemistry at the B/C ring junction was determined for 1. The above assignments were further confirmed by the 1H-1H COSY, HETCOR and NOESY experiments (Fig. 1). The new compound has been named (±)-cis-corydalmine N-oxide, and its specific rotation is negative, so 1 also possesses an S configuration at C-14 as in the cases of epicorynoxidine (11) (5) and cis-(±)-caseamine N-oxide (7).
To measure the cytotoxic activities of purified compounds against KB16, A549, and HT-29 cells, each cell line was initiated at 1000 cells/well in 96-well microtiter plates. Eight concentrations of test compounds encompassing a 128-fold range were added to each cell line. KB16, A549, and HT-29 cells were enumerated using MTT after exposure to test compounds for 3, 6, and 6 days, respectively. Fifty ml of 1 mg/ml MTT were added to each well, and plates were incubated at 37 8C for a further 5 h. Formazan crystals were redissolved in DMSO (E. Merck) for 10 min with shaking, and the plate was read immediately on a microtiter plate reader (Dynatech) at a wavelength of 540 nm. The ED50 was defined as the concentration of the test compound resulting in a 50% reduction of absorbance compared to untreated cells in the MTT assay.
Table 1
Corydalis tashiroi
(±)-trans-Corydalmine N-oxide (2) was isolated as colorless prisms. The molecular formula was established as C20H24O5N
H-NMR data of compounds 1, 2, 3, 10, and 11. Compound*
H
1
2
3
10
11
1
6.58 s
6.71 s
6.77 s
6.71 s
6.60 s
4
6.70 s
6.71 s
6.64 s
6.71 s
6.70 s
5
2.90 m
2.76 m
2.69 m
2.73 dd (16.2, 3.4)
2.93 m
3.66 m
3.93 m
3.91 m
3.88 m
3.63 m
3.71 m
3.58 m
3.52 m
3.57 m
3.72 m
3.45 m
3.93 m
3.91 m
3.96 m
3.43 m
a 5b 6a 6b 8a 8b
4.75 d (16.0)
4.40 d (15.0)
4.45 d (15.4)
4.52 d (15.0)
4.68 d (16.4)
4.84 d (16.0)
4.83 d (15.0)
4.72 d (15.4)
4.72 d (15.0)
4.85 d (16.4)
11
6.83 d (8.4)
6.47 s
6.78 d (8.4)
6.89 d (8.4)
6.86 s
12
6.70 d (8.4)
6.47 s
6.88 d (8.4)
7.02 d (8.4)
6.86 s
13
3.55 dd
3.27 dd
3.10 dd
3.31 dd
3.66 dd
(18.0, 6.4)
(15.8, 4.2)
(16.4, 4.0)
(16.2, 4.0)
(18.0, 6.2)
a b
13
14
2.97 dd
3.48 dd
3.52 dd
3.64 dd
3.02 dd
(18.0, 10.0)
(15.8, 11.8)
(16.4, 12.0)
(16.2, 12.0)
(18.0, 9.4)
4.58 dd
4.51 dd
4.34 dd
4.52 dd
4.55 dd
(10.0, 6.4)
(11.8, 4.2)
(12.0, 4.0)
(12.0, 4.0)
(9.4, 6.2)
OMe-2
3.83 s
3.88 s
±
3.89 s
3.85 s
OMe-3
3.86 s
3.90 s
3.86 s
3.90 s
3.86 s
OMe-9
3.78 s
3.84 s
3.79 s
3.87 s
3.84 s
OMe-10
±
±
3.81 s
3.88 s
3.84 s
* Recorded at 400 MHz in CDCl3; chemical shifts in ppm from TMS; coupling constants (Hz) in parentheses.
645
646
Planta Med. 65 (1999)
Table 2
13
Jih-Jung Chen, Chang-Yih Duh, and Ih-Sheng Chen
Fig. 1
C-NMR data of compounds 1, 2, 3, 10, and 11.
NOESY
interac-
tions observed for 1. Compound* C
1
2
3
10
11
5
24.9
24.4
25.0
25.1
25.7
6
56.9
64.5
65.3
65.4
58.7
8
65.8
66.5
67.5
67.8
66.0
13
36.5
29.8
30.4
30.4
36.3
14
70.5
68.3
68.8
68.9
71.7
* 1, 2 , 10 , and 11 were recorded in CDCl 3 and 3 was recorded in CD 3 OD at 100 MHz; chemical shifts in ppm from TMS.
Fig. 2
NOESY
interac-
tions observed for 2.
by FAB ([M + H]+, m/z 358) and HR/FAB-MS. The existence of an N-oxide function was also evidenced by the fragment peak (m/z 341) in the EI-MS due to the loss of an oxygen atom. The UV absorptions at 209, 228 sh, 284 nm were similar to those of 1, and were typical of a 2,3,9,10-oxygenated tetrahydroprotoberberine skeleton. The presence of a phenolic hydroxy group in the molecule was indicated by the IR absorption at 3375 cm±1 and a bathochromic shift of UV absorption in alkaline solution. The 1H-NMR spectrum of 2 showed four protons [d 2.76 (1H, m, H-5a), d 3.58 (1H, m, H-6a), d 3.93 (2H, m, H5b and H-6b)] assignable to the two methylene units at C-5 and C-6 of ring B, and three mutually coupling aliphatic protons at d 3.27 (1H, dd, J = 15.8, 4.2 Hz, H-13a), d 3.48 (1H, dd, J = 15.8, 11.8 Hz, H-13b), and d 4.51 (1H, dd, J = 11.8, 4.2 Hz, H14) in ring C. In addition, the chemical shifts at d 4.40 and 4.83 (each 1H, d, J = 15.0 Hz, H-8a and H-8b) were similar to those of corynoxidine (10), and different from those of 1 and epicorynoxidine (11) (5) (Table 1), and suggested that the Noxide function occupied the b orientation. The 1H-NMR spectrum of 2 also included three methoxy signals at d 3.84, 3.88 and 3.90 (each 3H, s) that were characteristic of OMe-9, OMe2 and OMe-3, respectively. The aromatic region of the spectrum showed two singlet signals, one at d 6.47 (2H, s) was due to H-11 and H-12, and the other at d 6.71 (2H, s) was assigned to H-1 and H-4. Furthermore, the chemical shifts of aliphatic protons of ring B and C were similar to those of corynoxidine (10), and different from those of (±)-cis-corydalmine N-oxide (1) and epicorynoxidine (11) (Table 1). Therefore, a trans-stereochemistry at the B/C ring junction was determined for 2, and this was also supported by 13C-NMR data of 2, which showed the chemical shifts of C-6 (d 64.5), C-13 (d 29.8) and C-14 (d 68.3) were similar to those of corynoxidine (10), and different from those of 1 and epicorynoxidine (11) (Table 2). The structure of 2 was further confirmed by the 1H-1H COSY, HETCOR and NOESY experiments (Fig. 2). The new compound has been named (±)-trans-corydalmine N-oxide, and it possesses an S configuration at C-14 due to laevorotation as in the case of corynoxidine (5). (±)-trans-Isocorypalmine N-oxide (3) was isolated as colorless needles. The FAB-MS afforded the positive ion at m/z 358 [M + H]+, implying a molecular formula of C20H24O5N, which was confirmed by the HRFAB-MS ([M + H]+ found 358.1664, calcd 358.1654). The fragment peak in the EI-MS at m/z 341 was due to the loss of an oxygen atom and suggested the presence of an N-oxide function. The presence of a 2,3,9,10-oxygenated tetrahydroprotoberberine skeleton was characterized by the UV spectrum showing absorptions at 210, 225 sh, 284 nm (5).
The presence of a phenolic hydroxy group in the molecule was indicated by the IR absorption at 3430 cm±1 and a bathochromic shift of UV absorption in alkaline solution. The 1HNMR spectrum of 3 showed four protons [d 2.69 (1H, m, H5a), d 3.52 (1H, m, H-6a), and d 3.91 (2H, m, H-5b and 6b)] assignable to the two methylene units at C-5 and C-6 of ring B, and three mutually coupling aliphatic protons at d 3.10 (1H, dd, J = 16.4, 4.0 Hz, H-13a), d 3.52 (1H, dd, J = 16.4, 12.0 Hz, H13b), and d 4.34 (1H, dd, J = 12.0, 4.0 Hz, H-14) in ring C. In addition, the chemical shifts of d 4.45 and 4.72 (each 1H, d, J = 15.4 Hz, H-8a and H-8b) were similar to those of 2 and corynoxidine (10) and different from those of 1 and epicorynoxidine (11) (5) (Table 1), thus suggesting that the N-oxide function occupied the b orientation. The 1H-NMR spectrum of 3 also contained three methoxyl signals at d 3.79, 3.81 and 3.86 (each 3H, s), which were characteristic of OMe-9, OMe-10 and OMe-3, respectively. The aromatic region of the spectrum showed four protons: two at d 6.64 and 6.77 (each 1H, s) assignable to H-4 and H-1, and the other two ortho-coupled protons at d 6.78 and 6.88 (each 1H, d, J = 8.4 Hz) due to H-11 and H-12. Furthermore, the chemical shifts of aliphatic protons of ring B and C were similar to those of 2 and corynoxidine (10), and different from those of 1 and epicorynoxidine (11) (Table 1). Therefore, a trans-stereochemistry at the B/C ring junction was determined for 3, and this was also supported by 13CNMR data of 3, which showed the chemical shifts of C-6 (d 65.3), C-13 (d 30.4) and C-14 (d 68.8) similar to those of 2 and corynoxidine (10), and different from those of 1 and epicorynoxidine (11). On the basis of the above data, the structure of 3 was elucidated as (±)-trans-isocorypalmine N-oxide, which was further confirmed by the 1H-1H COSY, HETCOR and NOESY experiments (Fig. 3). Its specific rotation is negative, so 3 possesses an S configuration at C-14 as in the case of 2. The known compounds including three benzo[c]phenanthridine alkaloids, norsanguinarine (4) (8), dihydrosanguinarine (5) (8), and oxysanguinarine (6) (8), six berberine alkaloids, (±)-tetrahydropalmatine (7) ([a]24D: ±288.48, c 0.3, CHCl3) (2), (9), (±)-corydalmine (8) ([a]24D: ±289.58, c 0.22, CHCl3) (10),
New Tetrahydroprotoberberine
N-Oxide Alkaloids and
Cytotoxic Constituents of
(11), (±)-scoulerine (9) ([a]24D: ±292.28, c 0.25, MeOH) (10), (12), (±)-corynoxidine (10) ([a]24D: ±59.28, c 0.2, CHCl3), (5), (± )-epicorynoxidine (11) ([a]24D: ±4.78, c 0.21, CHCl3) (5), and palmatine (12) (2), and one protopine alkaloid, protopine (13) (2) were readily identified by comparison of physical and spectroscopic data (UV, IR, 1H-NMR, [a]D, and mass spectrometry data) with the literature values. The cytotoxic effects of the isolates were tested in vitro against P-388, KB16, A549, and HT-29 cell lines. The cytotoxicity data are shown in Table 3, and the clinically applied anticancer agent mithramycin was used as reference compound. By comparison, the 2,3,7,8-tetraoxygenated benzo[c]phenanthridine alkaloids (4 and 5) exhibited more potent cytotoxic activities than the berberine-type alkaloids (7, 8, 10, 11, and 12) against P-388, KB16, A549, and HT-29 cell lines. Among them, norsanguinarine (4), dihydrosanguinarine (5), and (±)-scoulerine (9) exhibited effective cytotoxicities (ED50 value < 4 mg/ml) against P-388, KB16, A549, and HT-29 cell lines, and palmatine (12) showed selective cytotoxicity (ED50 value < 4 mg/ml) only against the P-388 cell line. In addition, the tetrahydroprotoberberine N-oxides (1, 2, 3, 10, and 11) were inactive as ()-tetrahydroberberine N-oxide, ()-tetrahydrojatrorrhizine N-oxide and ()-tetrahydropalmatine N-oxide (13). Furthermore, norsanguinarine (4) is the most cytotoxic isolate, and exhibited a more potent cytotoxicity (ED50 value = 0.051 mg/ml) against the P388 cell line than mithramycin (ED50 value = 0.056 mg/ml). Fig. 3
NOESY
interac-
Corydalis tashiroi
Planta Med. 65 (1999)
Acknowledgements
This work was kindly supported by a grant (NSC 88-2314-B127-001) from the National Science Council of the Republic of China. We are very grateful to Prof. Tsutomu Ishikawa, Faculty of Pharmaceutical Sciences, Chiba University, Japan, for the HR/FAB-MS measurements. References
1 Liu
TS, Yang KC. Fumariaceae in Flora of Taiwan, Second Edition, Editorial Committee of the Flora of Taiwan, Taipei, Taiwan, R.O.C., 1996; Vol. 2: p. 726 2 Lu ST, Lin CN, Wu TS. J. Chinese Chem. 1972; 19: 41±44 3 Tani C, Nagakura N, Saeki S, Kao MT. Planta Med.1981; 41: 403±405 4 Mosmann T. J. Immunol. Methods 1993; 65: 55±63 5 Tani C, Nagakura N, Hattori S. Chem. Lett. 1975: 1081±1084 6 Chinnasamy P, Minard RD, Shamma M. Tetrahedron 1980; 36: 1515±1519 7 Suau R, Silva MV, Valpuesta M. Phytochemistry 1993; 34: 559±561 8 Krane BD, Fagbule MO, Shamma M, Gozler B. J. Nat. Prod. 1984; 47: 1±43 9 Ruangrungsi N, Lange GL, Lee M. J. Nat. Prod. 1986; 49: 253±258 10 Ohiri FC, Verpoorte R, Baerheim Svendsen A. Planta Med. 1983; 49: 162±164 11 Kametani T, Ihara M, Honda T. J. Chem. Soc. (D) 1969;n :1310 12 Brochmann-Hanssen E, Nielsen B. Tetrahedron Lett. 1966;n :2261± 2263 13 Wu YC, Liou YF, Lu ST, Chen CH., Chang JJ, Lee KH. Planta Med. 1989; 55: 163±165
tions observed for 3.
Prof. Dr. I. S. Chen
Graduate Institute of Pharmaceutical Sciences Kaohsiung Medical College Kaohsiung, Taiwan Republic of China E-mail:
[email protected] Fax: +886-7-3210683 Table 3
Cytotoxic effects of compounds isolated from
Corydalis tashiroi against A549, HT-29, KB16 and P-388 cell lines.
m
ED50 ( g/ml) Compound
Mithramycin*
cis-Corydalmine N-oxide (1) (±)-trans-Corydalmine N-oxide (2) (±)-trans-Isocorypalmine N-oxide (3)
(±)-
A549
0.072
HT-29
0.080
KB
0.081
0.056
>50
>50
>50
>50
12.76
23.67
>50
>50
>50
>50
>50
>50
Norsanquinarine (4)
1.840
1.600
0.340
Dihydrosanquinarine (5)
2.310
3.110
3.060
Oxysanquinarine (6)
>50
>50
>50
(±)-Tetrahydropalmatine (7)
10.75
24.37
>50
(±)-Corydalmine (8)
>50
14.52
>50
(±)-Scoulerine (9)
P-388
2.250
1.920
2.840
0.051 0.083 >50 6.200 6.050 0.860
(±)-Corynoxidine (10)
>50
>50
>50
>50
(±)-Epicorynoxidine (11)
>50
>50
>50
25.53
Palmatine (12)
4.75
Protopine (13)
33.81
* Mithramycin was used as a positive control.
4.16 8.850
7.11 >50
3.72 21.41
647
