Monoterpenoid indole alkaloids from Gardneria ovata

Article abstract of DOI:10.1021/np2000254

Eight new monoterpenoid indole alkaloids, gardfloramine-9-O-β-d- glucopyranoside (1), 19(E)-18-demethoxygardfloramine-N(4)-oxide (2), gardfloramine-N(4)-oxide (3), 18-demethylgardfloramine (4), 19(E)-9,18- didemethoxygardneramine (5), 19(E)-11-methoxy-9,18-didemethoxygardneramine (6), 9-demethoxy-18-demethylgardneramine (7), and minfiensine-N(4)-oxide (8), along with six known alkaloids, were isolated from Gardneria ovata. The structures of the new alkaloids were established by means of spectroscopic methods. None of the compounds were cytotoxic to five human cancer cell lines. (Chemical Equation Presented).

Full text of DOI:10.1021/np2000254

ARTICLE
pubs.acs.org/jnp
Monoterpenoid Indole Alkaloids from Gardneria ovata
Xiao-Ning Li,†,‡ Xiang-Hai Cai,† Tao Feng,† Yan Li,† Ya-Ping Liu,†,‡ and Xiao-Dong Luo*,†
†State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy
of Sciences, Kunming 650204, People’s Republic of China
‡Graduate School of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
S Supporting Information
b
ABSTRACT: Eight new monoterpenoid indole alkaloids, gardfloramine-
9-O-β-D-glucopyranoside (1), 19(E)-18-demethoxygardfloramine-N(4)-
oxide (2), gardfloramine-N(4)-oxide (3), 18-demethylgardfloramine (4),
19(E)-9,18-didemethoxygardneramine (5), 19(E)-11-methoxy-9,18-dide-
methoxygardneramine (6), 9-demethoxy-18-demethylgardneramine (7),
and minfiensine-N(4)-oxide (8), along with six known alkaloids, were
isolated from Gardneria ovata. The structures of the new alkaloids were
established by means of spectroscopic methods. None of the compounds
were cytotoxic to five human cancer cell lines.
onoterpenoid indole alkaloids have attracted interest due
to their complicated structures and potent biological
M
activities.1ꢀ3 Our previous studies have reported the alkaloids
(19,20)-E/Z-alstoscholarine,4 melohenines A and B,5 melote-
nine A,6 scholarisines AꢀG,7,8 alstoyunines AꢀH,9 and melodi-
nines AꢀL.10,11 In addition, these alkaloids have anti-
inflammatory and cytotoxic activities.6,9 Plants of the genus
Gardneria in the Loganiaceae family are also sources of mono-
terpenoid indole alkaloids.12 As part of our search for novel and
bioactive alkaloids, phytochemical research on the alkaloids of
Gardneria ovata Wall. resulted in the isolation of eight new
alkaloids, gardfloramine-9-O-β-D-glucopyranoside (1), 19(E)-18-
demethoxygardfloramine-N(4)-oxide (2), gardfloramine-N(4)-
oxide (3), 18-demethylgardfloramine (4), 19(E)-9,18-didemethoxy-
gardneramine (5), 19(E)-11-methoxy-9,18-didemethoxygard-
neramine (6), 9-demethoxy-18-demethylgardneramine (7),
and minfiensine-N(4)-oxide (8), together with six known com-
pounds. The six known alkaloids were identified as gard-
floramine,13,14 19(E)-18-demethoxygardfloramine,13,14 18-demethyl-
gardneramine,15,16 gardneramine,15ꢀ18 19(E)-18-demethoxy-
gardneramine,18 and gardneramine-N(4)-oxide19 by comparison
with the literature. Compound 1 is the first example of Gardneria
alkaloids containing a glucose unit. None of the compounds were
cytotoxic to five human cancer cell lines.
which were consistent with indole alkaloids.14 The 1H and 13
C
NMR and DEPT spectra displayed a trisubstituted indolenine
ring [δC 181.8 (s, C-2), 62.1 (s, C-7), 123.0 (s, C-8), 135.8 (s,
C-9), 134.7 (s, C-10), 149.5 (s, C-11), 96.0 (d, C-12), 145.1 (s,
C-13); δH 6.53 (s, H-12)].13 Besides the signals of the indolenine
ring, the 13C NMR and DEPT spectra displayed 19 additional
carbon signals, including a quaternary carbon (δC 146.2), 10
’ RESULTS AND DISCUSSION
The alkaloid fraction of the aerial parts from G. ovata was separated
as described in the Experimental Section to yield a total of 14
monoterpenoid indole alkaloids, including eight new ones (1ꢀ8).
The molecular formula C27H32N2O10 of compound 1 was
determined by the molecular ion peak at m/z 544.2079 [M]þ in
the HREIMS in combination with 1H and 13C NMR and DEPT
spectra. Its UV and IR spectra showed absorption bands at 295
and 233 nm and bands at 3415 and 1583 cmꢀ1, respectively,
Received: January 9, 2011
Published: March 22, 2011
r
Copyright
2011 American Chemical Society and
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American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
1
Table 1. H NMR Data of Compounds 1ꢀ4 (methanol-d4, δ in ppm and J in Hz)
position
1
2
3
4
3
3.98, d (8.2)
3.64, m
3.98, overlap
4.03, overlap
3.82, dd (13.2, 3.6)
2.60, d (13.2)
6.45, s
4.03, overlap
4.07, m
3.70, d (8.0)
3.65, overlap
2.53, overlap
2.36, overlap
6.41, s
5
6a
2.58, overlap
2.37, overlap
6.53, s
3.79, dd (13.2, 3.5)
2.61, d (13.2)
6.45, s
6b
12
14a
2.04, dd (14.4, 5.3)
1.84, dd (14.4, 8.2)
2.61, m
2.30, dd (15.0, 8.0)
2.24, dd (15.0, 5.9)
3.16, m
2.37, dd (15.0, 7.9)
2.25, dd (15.0, 5.8)
2.79, m
2.01, dd (14.5, 5.4)
1.84, dd (14.5, 8.0)
2.56, m
14b
15
16
2.39, m
2.74, m
2.83, m
2.38, m
17a
4.58, d (12.5)
4.53, dd (12.5, 3.4)
3.93, overlap
3.89, overlap
5.41, t (6.2)
3.90, overlap
3.70, d (17.0)
5.91, 5.80
4.67, d (13.0)
4.55, overlap
1.70, d (6.7)
4.68, d (13.0)
4.53, dd (13.0, 4.1)
3.94, d (5.8)
4.58, d (12.8)
4.50, dd (12.8, 3.8)
4.07, dd (12.5, 7.5)
4.01, dd (12.5, 6.2)
5.41, t (6.7)
3.90, d (17.5)
3.65, overlap
5.87, s
17b
18a
18b
19
5.31, q (6.7)
4.58, overlap
4.18, d (16.3)
5.91, 5.89
5.51, t (5.8)
4.61, d (17.6)
4.49, d (17.6)
5.92, 5.90
4.00, s
21a
21b
ꢀOCH2Oꢀ
9-OMe
4.01, s
4.05, s
18-OMe
3.33, s
3.34, s
10
20
30
40
5.02, d (7.2)
3.48, overlap
3.45, overlap
3.37, m
50
60a
60b
3.34, overlap
3.84, d (12.8)
3.67, overlap
methine carbons (δC 36.2, 41.4, 59.8, 59.8, 70.2, 73.7, 76.1, 76.5,
102.8, 114.9), seven methylene carbons (δC 30.0, 31.8, 45.0,
61.2, 67.3, 72.2, 100.9), and a methoxy group (δC 56.6). These
data indicated that 1 was similar to gardfloramine with an
additional hexose unit. The connection between the hexose unit
and C-9 was established by the HMBC correlation of the
anomeric proton H-10 at δH 5.02 with C-9 (δC 135.8). After
acidic hydrolysis of 1, D-glucose was identified by comparison
with an authentic sample on TLC and a specific rotation of
[R]24D þ53.2 (c 0.24, H2O). Analysis of the 2D NMR spectra
established the structure of 1 as gardfloramine-9-O-glucopyrano-
side. The coupling constant of the anomeric proton at δH 5.02
(J = 7.2 Hz) suggested that the glucose was the β-glycoside.
Correlations of δH 5.41 (1H, t, J = 6.2 Hz, H-19) and δH 2.61
(1H, m, H-15), δH 3.89 (1H, H-18b), and δH 3.70 (1H, d, J =
17.0 Hz, H-21b) in the ROESY spectrum indicated that the
C-19ꢀC-20 double bond had the Z-configuration. The relative
configuration was the same as gardfloramine, from the 13C NMR
and the specific rotation data;13,14 therefore, the structure of 1
was established as 19-Z-gardfloramine-9-O-β-D-glucopyrano-
side, which is a rare glucoside linked to an aromatic ring in
monoterpene indole alkaloids.
was 19(E)-18-demethoxygardfloramine-N(4)-oxide. The ROESY
spectrum showed correlations of H-19 to H-21b and of H-18 to
H-15, which indicated that the configuration of the C-19ꢀC-20
double bond was E.
Compound
3 was assigned the molecular formula
C22H24N2O6, by HREIMS. The 13C NMR and DEPT data were
similar to those of gardfloramine,13,14 except for three downfield
signals of C-3 (δC 80.7), C-5 (δC 76.3), and C-21 (δC 64.9) due
to the N(4)-oxide functional group, supported by the HMBC
correlations of δH 4.49 (H-21b), 2.79 (H-15), 2.61 (H-6b), and
2.37 (H-14a) with δC 80.7 (C-3). The ROESY correlations of
H-19 to H-15 and of H-18 to H-21 indicated that the configura-
tion of the C-19ꢀC-20 double bond was Z. Thus, compound 3
was named gardfloramine-N(4)-oxide.
The molecular formula of compound 4 was determined to be
C21H22N2O5 by HREIMS. The 13C NMR and DEPT data were
related to those of gardfloramine,13,14 except for one missing
methoxy group and one upfield methylene carbon signal of δC
58.4 (C-18) in 4. Instead, a C-18 hydroxy group in compound 4
was supported by the HMBC correlations of δH 4.07 (H-18a)
and 4.01 (H-18b) with both δC 145.8 (C-20) and 119.1 (C-19).
The ROESY correlations of H-19 with H-15 and of H-18a
with H-21b indicated that the configuration of the C-19ꢀC-20
double bond was Z. Thus, compound 4 was identified as 18-
demethylgardfloramine.
The molecular formula of compound 2 was determined as
C21H22N2O5 by HREIMS. The 13C NMR and DEPT data were
related to those of 19(E)-18-demethoxygardfloramine13,14 with
the exception of three downfield chemical shifts at δC 81.0 (C-3),
76.0 (C-5), and 67.1 (C-21) in compound 2, indicating that it was an
N(4)-oxide derivative of 19(E)-18-demethoxygardfloramine.
Analysis of HMBC and HSQC data confirmed compound 2
The molecular formula of 5, C21H24N2O3, was established by
HREIMS, indicating 11 degrees of unsaturation. The UV absorp-
tion bands at 314 and 272 nm and an IR absorption band at
1579 cmꢀ1 supported an indolenine fragment.15 The 13C NMR
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1
Table 2. H NMR Data of Compounds 5ꢀ8 (methanol-d4, δ in ppm and J in Hz)
position
5
6
7
8
2
6.33, s
3
3.56, d (7.8)
3.70, overlap
3.59, d (7.9)
3.74, overlap
3.57, d (7.8)
3.72, overlap
5a
3.86, dd (20.2, 11.0)
3.46, br, t
5b
6a
2.57, d (12.6)
2.60, d (12.8)
2.18, dd (12.8, 3.5)
7.03, s
2.56, d (12.6)
2.29, overlap
6b
2.16, dd (12.6, 3.4)
6.77, d (2.2)
2.17, dd (12.6, 3.6)
6.78, d (2.1)
2.13, dd (13.9, 8.8)
7.16, d (7.4)
9
10
6.76, t (7.4)
11
6.45, d (2.2)
6.45, d (2.1)
7.05, t (7.7)
12
6.68, d (7.7)
14a
14b
15
2.06, dd (14.4, 5.6)
1.81, dd (14.4, 7.8)
3.02, m
2.08, dd (14.4, 5.6)
1.84, dd (14.4, 7.9)
3.04, m
2.09, dd (14.3, 5.5)
1.84, dd (14.3, 7.8)
2.60, m
2.34, dd (13.4, 2.5)
1.83, dd (13.4, 3.6)
3.57, s
16
2.32, m
2.34, m
2.42, m
17a
17b
18a
18b
19
4.59, d (12.9)
4.52, dd (12.9, 3.7)
1.65, d (6.8)
4.62, d (12.7)
4.56, dd (12.7, 3.7)
1.65, d (6.6)
4.59, d (12.7)
4.50, dd (12.7, 3.7)
4.09, dd (12.7, 7.3)
4.00, dd (12.7, 6.3)
5.44, t (6.3)
3.99, overlap
1.79, d (6.4)
5.23, q (6.8)
3.68, overlap
5.25, q (6.6)
3.70, overlap
5.67, q (6.4)
4.34, d (13.9)
3.72, d (13.9)
21a
21b
10-OMe
11-OMe
12-OMe
3.97, overlap
3.70, overlap
3.77, s
3.83, s
3.83, s
3.78, s
3.93, s
3.78, s
3.82, s
spectrum showed similar patterns to those of 19(E)-18-
demethoxygardneramine,18 the difference being a missing meth-
oxy group at C-9 (δC 99.9) in 5. The connectivity between H-9
and C-7 was established by a HMBC correlation at the proton of
δH 6.77 (H-9) with C-7 (δC 63.7). The ROESY correlations of
H-19 with H-21 and of H-18 with H-15 indicated that the
configuration of the C-19ꢀC-20 double bond was E. Analysis of
the 2D NMR spectra established the structure of 5 as 19(E)-9,18-
didemethoxygardneramine.
Compound 6 had the molecular formula C22H26N2O4, as
suggested by HREIMS. The 13C NMR spectrum showed similar
patterns to those of 5 with an additional methoxy group (δH 3.78;
δC 61.5) in 6. The connectivity between the methoxy group and
C-11 was established by the HMBC correlation of the protons of
methoxy group with δC 142.9 (s, C-11). The ROESY correlations of
H-19 to H-21 and of H-15 to H-18 indicated that the configuration
of the C-19ꢀC-20 double bond was E. Thus, the structure of 6 was
established as 19(E)-11-methoxy-9,18-didemethoxygardneramine.
Compound 7 had the molecular formula C21H24N2O4 on the
basis of the HREIMS. The 13C NMR spectrum was similar to that
of 18-demethylgardneramine,15,16 except for a missing methoxy
group at C-9 (δC 99.7) in 7, as established by a HMBC correla-
tion at the proton of δH 6.78 (H-9) with C-7 (δC 63.8). The
correlations of H-19 to H-15 and of H-18 to H-21b indicated
that the configuration of the C-19ꢀC-20 double bond was Z.
Thus, compound 7 was determined to be 9-demethoxy-18-
demethylgardneramine.
bands at 292, 236, and 215 nm were consistent with an indoline
moiety.20 The 1H and 13C NMR and DEPT spectra displayed a
substituted indoline ring [δC 99.6 (C-3), 52.9 (C-7), 135.6 (C-
8), 123.0 (C-9), 121.0 (C-10), 129.5 (C-11), 111.4 (C-12), 147.6
(C-13); δH 7.16 (H-9), 6.76 (H-10), 7.05 (H-11), 6.68 (H-
12)].13 Besides the signals of the indoline ring, the 13C NMR and
DEPT spectra displayed 11 additional carbon signals, includ-
ing two quaternary carbons (δC 140.0, 129.9), three methine
carbons (δC 125.2, 33.1, 126.7), five methylene carbons (δC
66.1, 39.0, 31.1, 64.7, 69.9), and one methyl group (δC 13.2).
The carbons at δC 125.2 and 140.0 were ascribed to two olefinic
carbons located at C-2 and C-16, respectively, according to the
HMBC correlations of δH 3.99 (H-17) and δH 2.29 (H-6a)
with δC 125.2 (C-2) and of δH 3.99 (H-17), δH 3.57 (H-15),
and δH 2.34 (H-14a) with δC 140.0 (C-16). These data
suggested that 8 possessed a similar carbon skeleton to that
of minfiensine,20 except for three downfield signals of δC 99.6
(C-3), δC 66.1 (C-5), and δC 69.9 (C-21) due to the N(4)-
oxide functionality. Compound 8 displayed [R]25 þ183.4
D
(c 0.10, MeOH), whereas an [R]25D þ134 (c 0.82, CHCl3) was
reported for minfiensine,20 which suggested the same config-
uration for both compounds. The ROESY correlations of H-19
with H-21 and of H-18 with H-15 indicated that the config-
uration of the C-19ꢀC-20 double bond was E; therefore, the
structure of compound 8 was established as minfiensine-N(4)-
oxide.
The NMR data of compounds 1ꢀ8, as shown in Tables 1,2,
and 3, were assigned on the basis of 2D NMR spectra (HSQC,
HMBC, ROESY).
All compounds were evaluated for their cytotoxicity against
five human cancer cell lines using the MTT method as reported
The molecular formula of compound 8 was determined to be
C19H22N2O2 on the basis of its HREIMS, requiring 10 degrees of
unsaturation. The IR absorption bands at 3407 and 1609 cmꢀ1
suggested hydroxy and olefinic groups, and the UV absorption
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ARTICLE
Table 3. 13C NMR Data of Compounds 1ꢀ8 (methanol-d4, δ in ppm)
position
1
2
3
4
5
6
7
8
2
3
5
6
7
8
9
181.8, qC
59.8, CH
59.8, CH
31.8, CH2
62.1, qC
181.1, qC
81.0, CH
76.0, CH
28.6, CH2
63.6, qC
180.9, qC
80.7, CH
76.3, CH
28.4, CH2
63.6, qC
183.4, qC
62.9, CH
61.9, CH
32.0, CH2
64.1, qC
180.9, qC
65.6, CH
61.5, CH
35.5, CH2
63.7, qC
181.8, qC
65.6, CH
62.0, CH
35.4, CH2
63.4, qC
180.7, qC
65.0, CH
61.9, CH
35.4, CH2
63.8, qC
125.2, CH
99.6, qC
66.1, CH2
39.0, CH2
52.9, qC
123.0, qC
135.8, qC
134.7, qC
149.5, qC
96.0, CH
145.1, qC
30.0, CH2
36.2, CH
41.4, CH
72.2, CH2
67.3, CH2
114.9, CH
146.2, qC
45.0, CH2
100.9, CH2
120.3, qC
142.5, qC
136.8, qC
151.5, qC
96.1, CH
147.1, qC
33.3, CH2
29.5, CH
42.7, CH
71.5, CH2
12.5, CH3
116.2, CH
134.7, qC
67.1, CH2
102.5,CH2
60.2, CH3
120.1, qC
142.1, qC
136.1, qC
151.5, qC
96.1, CH
147.0, qC
33.9, CH2
35.9, CH
42.7, CH
71.3, CH2
69.0, CH2
117.8, CH
138.4, qC
64.9, CH2
102.5, CH2
60.2, CH3
122.3, qC
140.9, qC
134.5, qC
151.1, qC
95.6, CH
146.7, qC
32.1, CH2
37.7, CH
43.0, CH
73.4, CH2
58.4, CH2
119.1,CH
145.8, qC
46.7, CH2
102.2,CH2
59.9, CH3
144.0, qC
99.9, CH
160.7, qC
100.0, CH
151.5, qC
133.1, qC
30.6, CH2
30.5, CH
42.8, CH
73.6, CH2
12.7, CH3
114.4, CH
141.3, qC
49.7, CH2
137.8, qC
102.5, CH
153.0, qC
142.9, qC
145.1, qC
137.0, qC
30.5, CH2
30.4, CH
42.7, CH
73.7, CH2
12.7, CH3
114.6, CH
140.8, qC
49.4, CH2
143.8, qC
99.7, CH
160.7, qC
99.9, CH
151.5, qC
133.1, qC
31.3, CH2
37.5, CH
42.9, CH
73.4, CH2
58.4, CH2
119.8, CH
144.7, qC
46.5, CH2
135.6, qC
123.0, CH
121.0, CH
129.5, CH
111.4, CH
147.6, qC
31.1, CH2
33.1, CH
140.0, qC
64.7, CH2
13.2, CH3
126.7, CH
129.9, qC
69.9, CH2
10
11
12
13
14
15
16
17
18
19
20
21
ꢀOCH2Oꢀ
9-OMe
10-OMe
56.2, CH3
56.4, CH3
57.0, CH3
61.5, CH3
61.6, CH3
56.2, CH3
56.3, CH3
11-OMe
12-OMe
18-OMe
56.6, CH3
102.8, CH
73.7, CH
76.1, CH
70.2, CH
76.5, CH
61.2, CH2
58.6, CH3
10
20
30
40
50
60
previously;21 however, all were inactive and showed IC50 values
of >40 μM.
Plant Material. The aerial parts of Gardneria ovata were collected
from Mengna County, Yunnan Province, P. R. China, and authenticated
by Mr. Jing-Yun Cui, Xishuangbanna Tropical Plant Garden. A voucher
specimen (No. Cui20081129) has been deposited at Kunming Institute
of Botany, Chinese Academy of Sciences.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were ob-
tained on an X-4 micro melting point apparatus. Optical rotations were
measured with a Horiba SEPA-300 polarimeter. UV spectra were
obtained using a Shimadzu UV-2401A spectrometer. IR spectra were
recorded on a Bruker FT-IR Tensor 27 spectrometer using KBr pellets.
1D and 2D NMR spectra were recorded on Bruker Avance III 600 MHz,
Bruker DRX-500 MHz, or AV-400 MHz spectrometers with TMS as the
internal standard. Chemical shifts (δ) are expressed in ppm relative to
TMS. HREIMS was recorded on a Waters Auto Premier P776 spectro-
meter. Column chromatography (CC) was performed on silica gel
(200ꢀ300 mesh, Qingdao Marine Chemical Ltd., Qingdao, People’s
Republic of China), RP-18 gel (20ꢀ45 μm, Fuji Silysia Chemical Ltd.,
Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd.,
Sweden). Fractions were monitored by TLC (GF254, Qingdao Haiyang
Chemical Co., Ltd. Qingdao), and spots were visualized with Dragen-
dorff’s reagent. HPLC was performed using Waters 600 pumps coupled
with analytical and semipreparative Sunfire C18 columns (150 ꢁ 4.6 and
150 ꢁ 10 mm, respectively). The HPLC system employed a Waters
2996 photodiode array detector and a Waters fraction collector II.
Extraction and Isolation. An air-dried, powdered sample (7 kg)
was extracted with 90% MeOH (25 L ꢁ 4). The extract was partitioned
between EtOAc and 0.5% HCl solution. The acidic water-soluble
materials, adjusted to pH 9ꢀ10 with 10% ammonia solution, were
extracted with EtOAc to give an alkaloidal extract (11 g). The extract was
subjected to silica gel column chromatography (CHCl3/MeOH, 1:0,
30:1, 20:1, 10:1, 5:1, 0:1) to afford fractions AꢀF. Fraction A (2.4 g)
was separated by RP-18 CC (MeOH/H2O, 1:1ꢀ1:0) to afford gard-
floramine (712 mg), gardneramine (760 mg), 19(E)-18-demethoxy-
gardfloramine (110 mg), and subfraction A1. Subfraction A1 was
chromatographed over silica gel CC (CHCl3/MeOH, 50:1ꢀ10:1)
and then chromatographed over Sephadex LH-20 CC (MeOH) to
afford 19(E)-18-demethoxygardneramine (35 mg), 5 (13 mg), and 6 (5
mg). Fraction C (745 mg) was subjected to RP-18 CC (MeOH/H2O,
4:6ꢀ4:1) to yield 18-demethylgardneramine (76 mg) and 7 (5 mg).
Fraction D (590 mg) was separated by RP-18 CC (MeOH/H2O,
3:7ꢀ8:2) and purified by silica gel CC (CHCl3/MeOH, 40:1ꢀ10:1)
to give 8 (11 mg) and 4 (40 mg). Fraction E (700 mg) was
chromatographed over RP-18 (MeOH/H2O, 6:4ꢀ8:2) and further
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Journal of Natural Products
ARTICLE
resolved by HPLC to yield 2 (3 mg) and 3 (9 mg). Fraction F (1.2 g) was
separated by RP-18 CC (MeOH/H2O, 3:7ꢀ9:1) and further purified
by silica gel CC (CHCl3/MeOH, 40:1ꢀ5:1) to give 1 (31 mg) and
gardneramine-N(4)-oxide (46 mg).
Cytotoxicity Assay. The cytotoxicity assay was performed accord-
ing to the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide] method,21 by use of the following five human cancer
cell lines: breast cancer MCF-7, hepatocellular carcinoma SMMC-
7721, human myeloid leukemia HL-60, colon cancer SW480, and lung
cancer A-549 cells, and IC50 values were calculated by Reed and
Muench’s method.22
Gardfloramine-9-O-β-D-glucopyranoside (1): white needles
(MeOH); mp 207ꢀ208 °C; [R]24 ꢀ252.8 (c 0.13, MeOH); UV
D
(MeOH) λmax (log ε) 295 (0.91), 233 (3.43) nm; IR (KBr) νmax 3415,
2897, 1583, 1459, 1346, 1073 cmꢀ1 1H (500 MHz) and 13C
;
NMR (100 MHz) data (MeOH-d4), see Tables 1 and 3, respec-
tively; HREIMS m/z 544.2079 (calcd for C27H32N2O10 [M]þ,
544.2057).
’ ASSOCIATED CONTENT
S
Supporting Information. 1D and 2D NMR and HREIMS
b
19(E)-18-Demethoxygardfloramine-N(4)-oxide (2): yellow
oil; [R]24D ꢀ125.9 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 296
(0.07), 240 (0.42), 203 (0.43), 196 (0.31) nm; IR (KBr) νmax 3424,
spectra of compounds 1ꢀ8. These materials are available free of
charge via the Internet at http://pubs.acs.org.
1
2920, 1622, 1587, 1449, 1349, 1050 cmꢀ1; H (500 MHz) and 13C
NMR (125 MHz) data (MeOH-d4), see Tables 1 and 3, respec-
tively; HREIMS m/z 382.1530 (calcd for C21H22N2O5 [M]þ,
382.1529).
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ86-871-5223177. Fax: þ86-871-5150227. E-mail: xdluo@
Gardfloramine-N(4)-oxide (3): yellow oil; [R]24D ꢀ232.3 (c 0.11,
MeOH); UV (MeOH) λmax (logε) 307 (0.88), 298 (0.87), 229 (3.97) nm;
mail.kib.ac.cn.
1
IR (KBr) νmax 3425, 2927, 1624, 1588, 1451, 1348, 1045 cmꢀ1; H
(400 MHz) and 13C NMR (100 MHz) data (MeOH-d4), see Tables 1 and
3, respectively; HREIMS m/z 412.1642 (calcd for C22H24N2O6 [M]þ,
412.1634).
’ ACKNOWLEDGMENT
The authors are grateful to the National Basic Research Program
of China (973 Program 2009CB522300), Chinese Academy of
Sciences (KSCX2-YW-R-202 and KSCX2-EW-R-15), and the
National Natural Science Foundation of China for partial financial
support.
18-Demethylgardfloramine (4): yellow oil; [R]25 ꢀ270.4
D
(c 0.12, MeOH); UV (MeOH) λmax (log ε) 295 (0.81), 237
(3.64) nm; IR (KBr) νmax 3422, 2926, 1622, 1575, 1449, 1344,
1052, 751 cmꢀ1; 1H (500 MHz) and 13C NMR (125 MHz) data (MeOH-
d4), see Tables 1 and 3, respectively; HREIMS m/z 382.1535 (calcd for
C21H22N2O5 [M]þ, 382.1529).
19(E)-9,18-Didemethoxygardneramine (5): yellow oil; [R]25
’ REFERENCES
D
ꢀ333.2 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 375 (0.01), 314
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(0.72), 272 (1.32), 228 (3.44) nm; IR (KBr) νmax 3432, 2933, 1579, 1474,
1
1280, 1080, 822 cmꢀ1; H (400 MHz) and 13C NMR (125 MHz) data
(MeOH-d4), see Tables 2 and 3, respectively; HREIMS m/z 352.1797 (calcd
for C21H24N2O3 [M]þ, 352.1787).
19(E)-11-Methoxy-9,18-didemethoxygardneramine (6):
colorless needles (MeOH); mp 102ꢀ104 °C; [R]24D ꢀ135.7 (c 0.20,
MeOH); UV (MeOH) λmax (log ε) 279 (0.82), 233 (3.50), 206
(2.25) nm; IR (KBr) νmax 3426, 2942, 1576, 1472, 1344, 1119,
1049 cmꢀ1; 1H (500 MHz) and 13C NMR (100 MHz) data (MeOH-
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C22H26N2O4 [M]þ, 382.1893).
9-Demethoxy-18-demethylgardneramine (7): yellow oil;
[R]24 ꢀ349.1 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 312
D
(0.41), 272 (0.81) 227 (2.40), 207 (2.20) nm; IR (KBr) νmax 3424,
2928, 1577, 1467, 1296, 1074 cmꢀ1; 1H (400 MHz) and 13C NMR (100
MHz) data (MeOH-d4), see Tables 2 and 3, respectively; HREIMS m/z
368.1737 (calcd for C21H24N2O4 [M]þ, 368.1736).
Minfiensine-N(4)-oxide (8): yellow oil; [R]25 þ183.4 (c 0.10,
D
MeOH); UV (MeOH) λmax (log ε) 360 (0.04), 292 (1.02), 236 (2.09),
215 (2.99), 193 (0.87) nm; IR (KBr) νmax 3407, 2920, 1609, 1468, 1127,
750 cmꢀ1; 1H (400 MHz) and 13C NMR (100 MHz) data (MeOH-d4),
see Tables 2 and 3, respectively; HREIMS m/z 310.1675 (calcd for
C19H22N2O2 [M]þ, 310.1681).
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of the aqueous layer was performed by Sephadex LH-20 CC (MeOH)
followed by silica gel CC (CHCl3/MeOH/H2O, 3:1:0.1) to afford
D-glucose with a specific rotation [R]24D þ53.2 (c 0.24, H2O).
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Journal of Natural Products
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