Nitrogen-containing compounds from Salvia miltiorrhiza

Article abstract of DOI:10.1021/np0500934

Five new N-containing compounds, neosalvianen (1), salvianen (2), salvianan (3), salviadione (4), and 5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde (5), were isolated from Salvia miltiorrhiza. Their structures were mainly established by spectroscopic methods. Neosalvianen (1) and its analogues (6a, 6b) were synthesized for spectroscopic data comparison. Compounds 1,2,4, and 6a were evaluated for their cytotoxic activities against selected cancer cell lines. Among these components, salvianen (2) exhibited the most potent cytotoxicity with a CD₅₀ range of 30.4-39.5 μM against HeLa (cervical epitheloid carcinoma), HepG2 (hepatocellular carcinoma), and OVCAR-3 (ovarian adenocarcinoma) cell lines in a dose-dependent manner. The cytotoxicities of the tested compounds were not specific and showed similar activities to the selected cancer cell lines.

Full text of DOI:10.1021/np0500934

1066
J. Nat. Prod. 2005, 68, 1066-1070
Nitrogen-Containing Compounds from Salvia miltiorrhiza
Ming-Jaw Don,† Chien-Chang Shen,†,‡ Yun-Lian Lin,† Wan-Jr Syu,§ Yi-Huei Ding,† and Chang-Ming Sun*,†,‡
National Research Institute of Chinese Medicine, Taipei 112, Taiwan, Department of Biochemistry, National Yang-Ming
University, Taipei 112, Taiwan, and Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112,
Taiwan, Republic of China
Received March 18, 2005
Five new N-containing compounds, neosalvianen (1), salvianen (2), salvianan (3), salviadione (4), and
5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde (5), were isolated from Salvia miltiorrhiza. Their structures
were mainly established by spectroscopic methods. Neosalvianen (1) and its analogues (6a, 6b) were
synthesized for spectroscopic data comparison. Compounds 1, 2, 4, and 6a were evaluated for their cytotoxic
activities against selected cancer cell lines. Among these components, salvianen (2) exhibited the most
potent cytotoxicity with a CD50 range of 30.4-39.5 µM against HeLa (cervical epitheloid carcinoma),
HepG2 (hepatocellular carcinoma), and OVCAR-3 (ovarian adenocarcinoma) cell lines in a dose-dependent
manner. The cytotoxicities of the tested compounds were not specific and showed similar activities to the
selected cancer cell lines.
The traditional Chinese medicine “Danshen”, the dried
root of Salvia miltiorrhiza Bunge (Labiatae), has been used
for the treatment of menstrual disorders, menostasis,
menorrhalgia, insomnia, arthritis, and coronary heart
diseases, particularly angina pectoris and myocardial
infarction.1-3 Numerous diterpenoid tanshinones have been
isolated from S. miltiorrhiza, and several were shown to
possess various biological and pharmacological activities
including antitumor,3-7 antimicrobial,8,9 antioxidant,10-13
antiinflammatory,14,15 and antiplatelet aggregation16-18
activities.
This paper describes the isolation and identification of
five new N-containing compounds, neosalvianen (1), salvi-
anen (2), salvianan (3), salviadione (4), and 5-(methoxy-
methyl)-1H-pyrrole-2-carbaldehyde (5), from S. miltior-
rhiza. In addition, two known components, tanshinone IIA
and cryptotanshinone, previously reported from this plant,
were also isolated. Compound 1 and its analogues (6a, 6b)
d, J ) 1.0 Hz) (Table 1). The 13C and DEPT NMR spectra
were synthesized for spectroscopic data comparison. Fur-
of 1 showed 20 carbon signals (Table 2), which were
thermore, some selected compounds were assayed for
cytotoxic and antimicrobial properties.
assigned to three methyls, three saturated methylenes,
three aromatic methines, and 11 quaternary (one saturated
at δ 34.4 and 10 aromatic) carbons. The connectivities of
Results and Discussion
1H and 13C signals were determined by HMQC and
revealed that the signals at δH 1.41 and δC 31.8 were
attributed to two equivalent methyl groups. The COSY
spectrum showed the correlations of protons at δ 2.02
(H-2) with those at δ 3.51 (H-1) and 1.79 (H-3), indicating
three consecutive methylene groups. On the basis of the
HMBC spectrum, the selected correlations of protons to
carbons are shown in Figure 1. However, assignment of
the oxazole ring orientation of 1 was still ambiguous
because no 1H-13C correlation signal was observed at C-11
and C-12 in the HMBC spectrum. To study the orientation
of N and O atoms in the oxazole ring, compound 1 was
therefore synthesized.
The synthetic compound 1 was obtained by oxidation of
6a in the presence of DDQ using benzene as the solvent
(Scheme 1).19 Compound 6a was prepared by reaction of
cryptotanshinone in ethanol with aqueous ethylamine
solution.20 Ethylamine preferentially attacked the carbonyl
carbon at C-12 of cryptotanshinone and then cyclized with
C-11 to afford the oxazole ring.20,21 However, the three-bond
coupling of H-15 to nitrogen attached at C-12 (δC 135.5)
was not observed in HMBC experiments with coupling
The concentrated EtOH extract of S. miltiorrhiza was
chromatographed on silica gel. Repeated chromatography
of the fractions resulted in the isolation of five new
compounds (1-5). The structures of these new compounds
1
were elucidated from H and 13C NMR spectra with the
aid of COSY, HMQC, and HMBC experiments.
Compound 1 was obtained as colorless needles with a
molecular formula of C21H21NO2 determined by HREIMS
([M]+, m/z 319.1570). The IR spectrum of 1 indicated the
existence of an oxazole ring (1571 and 822 cm-1). The
1H NMR spectrum exhibited three aromatic protons at
δ 8.17 and 7.61 (1H each, d, J ) 9.0 Hz) and 7.57 (1H, q,
J ) 1.0 Hz), three aliphatic methylene protons at δ 3.51
(2H, t, J ) 6.0 Hz), 2.02, and 1.79, and three tertiary
methyl signals at δ 2.81 (3H, s), 1.41 (6H, s), and 2.60 (3H,
* To whom correspondence should be addressed. E-mail: cmsun@
nricm.edu.tw. Fax: +886-2-28264276.
† National Research Institute of Chinese Medicine.
‡ Department of Biochemistry, National Yang-Ming University.
§ Institute of Microbiology and Immunology, National Yang-Ming Uni-
versity.
10.1021/np0500934 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/29/2005

Nitrogen-Containing Compounds from Salvia
Journal of Natural Products, 2005, Vol. 68, No. 7 1067
Table 1. 1H NMR (500 MHz) Data of 1-4 and 6a in CDCl3
1
2
δ (J)
3
δ (J)
4
δ (J)
6a
δ (J)
position
δ (J)a
1
3.51 t (6.0)
2.02 m
3.75 m
3.68 m
1.96 m
1.76 m
7.49 d (9.0)
7.83 d (9.0)
3.42 t (6.0)
1.97 m
2
2.00 m
3
1.79 m
1.76 m
7.61 d (8.5)
8.16 d (8.5)
2.95 s
1.76 m
6
7.61 d (9.0)
8.17 d (9.0)
7.30 d (7.5)
7.66 d (7.5)
7.56 s
7.52 d (8.0)
7.85 d (8.0)
7
14
15
16
3.99 m
3.54 m
1.31 d (7.0)
4.08 m
7.57 q (1.0)
7.53 q (1.0)
a 4.36 dd (2.0, 9.0)
b 4.92 t (9.0)
1.49 d (7.0)
1.36 sb
a 4.40 dd (6.0, 8.5)
b 4.92 t (8.5)
1.56 d (6.5)
1.36 sb
17
18
19
21
2.60 d (1.0)
1.41 s
1.41 s
2.81 s
2.50 d (1.0)
1.39 s
1.39 s
2.81 s
1.31 d (7.0)
1.49 s
1.49 s
1.37 sb
2.74 s
1.37 sb
2.72 s
a J ) coupling constant in hertz. b Interchangeable.
Table 2. 13C NMR (125 MHz) Data of 1-4 and 6a in CDCl3
Scheme 1. Syntheses of 1, 6a, and 6ba
1
δ
2
δ
3
δ
4
δ
6a
δ
position
1
29.4
19.5
30.7
19.7
30.5
19.6
128.2
188.0
57.4
29.3
19.5
2
3
38.6
38.8
38.8
38.6
4
34.4
34.6
34.6
41.3
34.4
5
143.1
125.0
118.4
117.8
117.5
130.1
144.4
132.6
116.3
149.5
116.0
141.1
9.4
143.6
124.8
118.3
118.1
122.3
132.7
134.0
148.5
111.3
150.6
114.7
141.1
9.3
144.2
124.0
119.7
117.0
124.9
132.0
130.5
145.9
109.3
154.8
36.4
145.3
121.0
129.7
123.9
124.3
127.9
126.9
175.9
151.4
129.1
27.4
143.7
124.2
119.7
116.8
119.6
129.5
143.0
135.5
115.2
153.1
36.8
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
79.5
22.8
79.4
19.2
22.8
19.8
31.8
31.8
162.6
14.9
32.0
32.0
159.8
14.5
31.8a
31.9a
159.0
14.5
30.1
31.6a
31.7a
163.0
14.9
30.1
a Interchangeable.
a (i) 70% aqueous EtNH2, EtOH for 6a; Et15NH2, EtOH for 6b; (ii) DDQ,
benzene.
2 showed the presence of 20 carbon signals (Table 2) and
were similar to those of 1. The 1H NMR spectrum exhibited
three aromatic protons at δ 8.16 and 7.61 (1H each, d, J )
8.5 Hz) and 7.53 (1H, q, J ) 1.0 Hz), three methylene
protons at δ 3.75, 2.00, and 1.76, and three tertiary methyl
signals at δ 2.81 (3H, s), 1.39 (6H, s), and 2.50 (3H, d, J )
1.0 Hz) (Table 1). A set of equivalent methyl groups at δH
1.39 and δC 32.0 was observed in the HMQC spectrum. The
COSY spectrum of 2 indicated that the protons at δ 2.00
(H-2) correlated with those at δ 3.75 (H-1) and 1.76 (H-3).
Analysis of the COSY, HMQC, and HMBC data (Experi-
mental Section) revealed that 2 was similar to 1. The IR
spectrum exhibited absorption bands at 1591 and 821 cm-1
for the oxazole ring. The EIMS of 2 gave a molecular ion
peak at m/z 319 and major fragment peaks at m/z 304, 263,
and 235, suggesting [M - CH3]+, [M - CH3 - C2H3N]+,
and [M - CH3 - C2H3N - CO]+, respectively. By compari-
son with the spectra of 1, the above data suggested that
the N atom in the oxazole ring of 2 was alternatively
attached to C-11, resonating at δ 134.0. The structure of 2
was therefore assigned, and it was named salvianen.
Compound 3 was obtained as pale green plates with a
molecular formula of C21H23NO2 determined by HREIMS
([M]+, m/z 321.1728). The 1H NMR spectrum exhibited two
aromatic protons at δ 7.83 and 7.49 (1H each, d, J )
9.0 Hz), one methine signal at δ 3.99, three methylene
Figure 1. Selected HMBC correlations for compounds 1 and 4.
constants of 2.5, 4, 6, 8, and 10 Hz. To confirm the
orientation of N and O atoms in the oxazole ring of 6a,
compound 6b was prepared from 15N-enriched ethylamine.
The 13C NMR spectrum showed a two-bond coupling of C-13
(δC 115.2, d, 2JC-N ) 5.6 Hz) with 15N, indicating that the
nitrogen atom was located at C-12.22 Thus, the structure
of 6a was established, and it was named neosalvianan.
Furthermore, compound 1 was also prepared by treatment
of tanshinone IIA in EtOH with aqueous ethylamine
solution. The 1H and 13C NMR spectra of synthetic 1 were
identical to those of naturally occurring material (1). The
structure of compound 1 was therefore confirmed, and 1
was given the trivial name neosalvianen.
Compound 2 was obtained as brown needles with a
molecular formula of C21H21NO2 determined by HREIMS
([M]+, m/z 319.1581). The 13C and DEPT NMR spectra of

1068 Journal of Natural Products, 2005, Vol. 68, No. 7
Don et al.
Table 3. Cytotoxicities of Selected Compounds against Human
protons at δ 3.68, 1.96, and 1.76, an oxygenated methylene
at δ 4.92 (1H, t, J ) 9.0 Hz) and 4.36 (1H, dd, J ) 2.0 and
9.0 Hz), and four methyl groups at δ 2.74, 1.37, and 1.36
(3H each, s), and 1.49 (3H, d, J ) 7.0 Hz) (Table 1). The
13C and DEPT NMR spectra displayed 21 carbons (Table
2), which were assigned to four methyls (three tertiary and
one secondary), four aliphatic methylenes (one oxygenated
at δ 79.5), three methines (one aliphatic at δ 36.4 and two
aromatic), and 10 quaternary (one aliphatic at δ 34.6 and
nine aromatic) carbons. In the COSY spectrum, it showed
correlations of the methylene protons at δ 1.96 (H-2) with
those at δ 3.68 (H-1) and 1.76 (H-3). In addition, the
correlations of the methine signal at δ 3.99 (H-15) with the
oxygenated methylene protons at δ 4.36 (H-16a) and 4.92
(H-16b) and the methyl group at δ 1.49 (H-17) were also
observed. Analysis of the COSY, HMQC, and HMBC data
(Experimental Section) indicated that 3 was partially
similar to 2. The long-range correlations between H-16 and
C-13, C-14, and C-17 in the HMBC spectrum revealed the
presence of a 3-methyldihydrofuran ring. Further compari-
sons of the COSY, HMQC, and HMBC data with those of
6a showed that 3 was similar to synthetic 6a. In contrast
to 6a, the N atom in the oxazole ring of 3 was attached to
C-11, resonating at δ 130.5. The structure of 3 was
therefore determined, and it was given the trivial name
salvianan.
Cancer Cell Lines
CD50 (µM)
compound
HeLa
HepG2
OVCAR-3
1
63.9
32.3
178
59.2
30.4
137
74.6
39.5
169
2
4
6a
108
121
126
crytotanshinone
tanshinone IIA
cisplatin
17.2
8.50
18.3
29.7
26.5
27.7
9.12
9.52
31.7
presence of a pyrrole NH stretch band (3263 cm-1) and a
conjugated aldehyde (1652 cm-1). The EIMS of 5 gave a
molecular ion at m/z 139 and major fragment ions at m/z
110 and 108, indicating [M - CHO]+ and [M - OCH3]+,
respectively. The 1H NMR spectrum exhibited one aldehyde
proton at δ 9.45, two aromatic protons at δ 6.89 and 6.20,
one oxygenated methylene at δ 4.17, one methoxy group
at δ 3.37, and one N-H proton at δ 9.62. The 13C NMR
and DEPT spectra showed one methoxy (δ 58.4), one
oxygenated methylene (δ 67.0), one aldehyde methine
(δ 178.8), and four aromatic carbons. Of the four aromatic
carbons, two quaternary carbons were deshielded (δ 137.6
and 132.7) and two protonated carbons were upfield
(δ 121.5 and 109.8), which suggested that the pyrrole ring
was 2,5-disubstituted. The HMBC spectrum showed that
the aldehyde proton and the oxygenated methylene protons
correlated with C-3 and C-4, respectively. The 1H and
13C NMR data were comparable with those of 5-(hydroxy-
methyl)-1H-pyrrole-2-carbaldehyde.23 Thus, the structure
of this new compound was deduced as 5-(methoxymethyl)-
1H-pyrrole-2-carbaldehyde.
The cytotoxicities of N-containing compounds 1, 2, 4, and
6a against the HeLa (cervical epitheloid carcinoma), HepG2
(hepatocellular carcinoma), and OVCAR-3 (ovarian adeno-
carcinoma) cell lines have not been previously reported. The
cytotoxic evaluation of these compounds showed that 2 was
the most potent, with a CD50 range of 30.4-39.5 µM against
the selected cell lines (Table 3), which is comparable with
the positive control, cisplatin. Compounds 1, 2, 4, and 6a
were further subjected to evaluation of antibacterial activ-
ity against Gram-positive Staphylococcus aureus and En-
terococcus faecalis and Gram-negative Escherichia coli. No
significant activity was observed for the dosage up to
100 µg/disk of the tested compounds.
Compound 4 was obtained as an orange solid with a
molecular formula of C19H19NO2 determined by HREIMS
([M]+, m/z 293.1419). The 1H NMR spectrum exhibited
three aromatic protons at δ 7.66 and 7.30 (1H each, d, J )
7.5 Hz) and 7.56 (1H, s), one aliphatic methine at δ 3.54,
one aliphatic methylene at δ 2.95 (2H, s), and two methyl
signals at δ 1.49 (6H, s) and 1.31 (6H, d, J ) 7.0 Hz) (Table
1). The 13C NMR spectrum of 4 showed 17 carbon signals
(Table 2). The DEPT NMR spectrum displayed two meth-
yls, one aliphatic methylene, four methines (one aliphatic
and three aromatic), and 10 quaternary (one aliphatic,
seven aromatic, and two carbonyl) carbons. The connec-
1
tivities of H-13C signals were determined by the HMQC
spectrum, which revealed two sets of two equivalent methyl
groups (δΗ 1.49, δC 30.1; δΗ 1.31, δC 22.8). The structure
elucidation was further facilitated through analyses of the
COSY and HMBC spectra. The COSY spectrum showed
correlation between the methine proton at δ 3.54 (H-15)
and the two equivalent methyl protons at δ 1.31 (H-16 and
H-17), which indicated the presence of an isopropyl group.
Some selected HMBC correlations are shown in Figure 1.
However, it was difficult to assign the C-11 carbon on the
basis of the HMBC spectrum by standard pulse sequence
(JH-C ) 8 Hz). When the 1H-13C coupling constant of
2.5 Hz was applied, the four-bond correlations from H-7
and H-14 to C-11 were observed in the HMBC spectrum,
hence confirming the assignment of C-11. The 1H NMR
spectrum of 4 in DMSO-d6 exhibited a broad signal at
δ 14.23, which indicated the existence of the intramolecular
hydrogen bonding between the pyrrole N-H and the
carbonyl groups. The EIMS of 4 gave a molecular ion at
m/z 293 and major fragment ions at m/z 278, 265, 250, and
237, suggesting [M - CH3]+, [M - CO]+, [M - CH3 - CO]+,
and [M - 2CO]+, respectively. The IR spectrum of 4 showed
the presence of a pyrrole NH stretch (3429 cm-1) and
conjugated ketones (1679 and 1582 cm-1). The structure
of 4 was therefore assigned; 4 was given the trivial name
salviadione.
Experimental Section
General Experimental Procedures. Melting points were
measured using a Yanaco MP-S9 micro-melting point ap-
paratus and are uncorrected. UV spectra were measured with
a Hitachi U-3310 spectrophotometer. IR spectra were recorded
on a Nicolet Avatar 320 FT-IR spectrometer. 1H and 13C NMR
spectra were recorded on a Varian Unity Inova 500 spectrom-
eter in CDCl3 with TMS as an internal standard. 1H, 13C,
COSY, HMQC, HMBC, and DEPT spectra were obtained using
standard Varian pulse sequences. EIMS spectra were mea-
sured with a direct insertion probe on a Finnigan GCQ
spectrometer at 30 eV. HREIMS data were taken on a
Finnigan MAT 95S mass spectrometer. Silica gel (Kieselgel
60, 70-230 mesh, Macherey-Nagel) was used for column
chromatography. TLC was carried out on aluminum sheets
precoated with silica gel 60 F254 (layer thickness 0.2 mm,
Merck). The chromatograms were visualized under UV light
(254 or 365 nm) or by spraying with 5% phosphomolybdic acid
in 5% H2SO4 containing a trace of ceric sulfate, followed by
heating on a hot plate (120 °C). Ethylamine-15N hydrochloride
was purchased from Aldrich Chemical Co.
Compound 5 was obtained as a pale brown oil with a
molecular formula of C7H9NO2 determined by HREIMS
([M]+, m/z 139.0637). The IR spectrum indicated the

Nitrogen-Containing Compounds from Salvia
Journal of Natural Products, 2005, Vol. 68, No. 7 1069
Plant Material. The dried root of S. miltiorrhiza was
purchased from the Cherng-Chi Chinese herbal shop in Taipei
in January 2002. A voucher specimen (NRICM 02006) was
deposited in the herbarium of the National Research Institute
of Chinese Medicine, Taipei.
58.4 (q, OCH3); HREIMS m/z 139.0637 (calcd for C7H9NO2
139.0633); EIMS m/z 139 [M]+ (92), 110 (100), 108 (83), 80 (52).
Synthesis of Neosalvianan (6a).20 To a solution of crypto-
tanshinone (100 mg, 0.34 mmol) in EtOH (20 mL) was added
EtNH2 in H2O (70%, 15 mL). After stirring at room temper-
ature for 24 h, the mixture was concentrated and subjected to
column chromatography with hexane/EtOAc (95:5) as the
solvent to give 6a (25 mg, 23%) as a colorless solid: mp 230-
231 °C; UV (CH3OH) λmax (log ꢀ) 230 (4.52), 252 (4.46) nm; IR
(KBr) νmax 2953, 2910, 1568, 1459, 1398, 1240, 1121, 988, 957,
Extraction and Isolation. The dried root of S. miltiorrhiza
(20 kg) was crushed and extracted with EtOH (3 × 100 L) at
60 °C for 24 h. The EtOH extracts were combined and
concentrated in vacuo to 3 L. The concentrated extract was
suspended in H2O (15 L) and partitioned successively with
EtOAc. After concentration of the EtOAc extract, the concen-
trate was mixed with 1.5 kg of silica gel (230-400 mesh). The
air-dried mixture was divided into three equal parts, and each
part was subjected to column chromatography (10 cm i.d. ×
100 cm). Each column was then eluted with a stepwise
gradient eluent of hexane/EtOAc (95:5, 90:10, 80:20, 70:30,
60:40, 50:50, 25:75, 0:100) (15 L each). Fractions (1 L each)
were collected, and similar fractions were combined to give
16 fractions (A-P). Fraction E was separated by column
chromatography (2 cm i.d. × 100 cm) and eluted with hexane/
EtOAc (95:5) to afford 2 (5 mg). Fraction F was rechro-
matograped on a column (1 cm i.d. × 100 cm) with CHCl3/
hexane (20:80) as the eluent to yield 3 (3 mg). Fraction G was
separated by column chromatography (2 cm i.d. × 100 cm).
Elution with hexane/EtOAc (100:0, 95:5, 90:10) (3 L each) gave
1 (15 mg). Fraction I was similarly rechromatograped on a
column (2 cm i.d. × 100 cm) and eluted with hexane/EtOAc
(95:5, 90:10, 85:15, 80:20, 75:25) (2 L each), affording 4
(10 mg) and 5 (35 mg).
932, 820 cm-1 1H and 13C NMR, see Tables 1 and 2,
;
respectively; HMBC correlations H-1/C-2, C-3, C-5, C-9, C-10;
H-2/C-1, C-3, C-4, C-10; H-3/C-1, C-2, C-4, C-5, C-18; H-6/
C-4, C-8, C-10; H-7/C-5, C-9, C-14; H-16a/C-13, C-14, C-15,
C-17; H-16b/C-13, C-14, C-17; H-17/C-13, C-15, C-16; H-18/
C-3, C-4, C-5; H-21/C-20; EIMS m/z 321 [M]+ (100), 306 (74),
277 (3), 265 (40), 236 (13), 221 (12).
Synthesis of 15N-Enriched Neosalvianan (6b). To a
solution of cryptotanshinone (100 mg, 0.34 mmol) in EtOH
(15 mL) was added Et15NH2‚HCl (260 mg, 3.3 mmol) in 2 N
NaOH solution (1.6 mL). After stirring at room temperature
for 22 h, the mixture was concentrated and subjected to column
chromatography eluting with hexane/EtOAc (95:5) to give 6b
1
(15 mg, 15%) as a colorless solid. The H and 13C NMR data
were the same as those of 6a except H-21 (d, JH-N ) 2.0 Hz),
C-13 (d, JC-N ) 5.6 Hz), C-20 (d, JC-N ) 2.2 Hz), and C-21 (d,
JC-N ) 5.6 Hz), which coupled with 15N.
Synthesis of Neosalvianen (1) from Neosalvianan (6a).
To a solution of 6a (18 mg, 0.056 mmol) in dry benzene (5 mL)
was added DDQ (32 mg, 0.14 mmol).19 After stirring at room
temperature for 48 h, the mixture was filtered through Celite
and concentrated. The crude residue was subjected to column
chromatography eluting with hexane/EtOAc (95:5) to give 1
Neosalvianen (1): colorless needles (EtOAc/hexane);
15 mg; mp 213-215 °C; TLC Rf 0.44 (15% EtOAc/hexane); UV
(CH3OH) λmax (log ꢀ) 201 (4.90), 255 (5.34), 263 (5.43) nm; IR
(KBr) νmax 2956, 2924, 2851, 1729, 1571, 1466, 1418, 1375,
1
1237, 1133, 989, 941, 822 cm-1; H and 13C NMR, see Tables
1
(10 mg, 56%) as a colorless solid. The H, 13C NMR, mp, and
1 and 2, respectively; COSY correlations H-2/H-1, H-3; H-6/
H-7; H-16/H-17; HREIMS m/z 319.1570 (calcd for C21H21NO2
319.1567); EIMS m/z 319 [M]+ (93), 304 (100), 276 (5), 263 (34),
235 (5).
EIMS data were identical with those of natural product 1.
Synthesis of Neosalvianen (1) from Tanshinone IIA.
A solution of EtNH2 in H2O (70%, 10 mL) was added to a
solution of tanshinone IIA (50 mg, 0.17 mmol) in EtOH
(10 mL). The reaction mixture was stirred at room temperature
for 48 h and then concentrated. The residue was subjected to
column chromatography and eluted with hexane/EtOAc
(95:5) to afford 1 (8 mg, 15%).
Salvianen (2): brown needles (EtOAc/hexane); 5 mg; mp
90-92 °C; TLC Rf 0.60 (10% EtOAc/hexane); IR (KBr) νmax
2957, 2923, 2851, 1813, 1591, 1456, 1376, 1125, 1073, 941,
1
821 cm-1; H and 13C NMR, see Tables 1 and 2, respectively;
Cytotoxicity Assay. The cell line culture conditions24 and
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide] colorimetric assay for CD50 were carried out according
to the procedures previously described.25
Antibacterial Activity. Bacteria, culture conditions, and
the antibacterial activity evaluation for compounds 1, 2, 4, and
6a and the positive control were the same as previously
outlined.25
COSY correlations H-2/H-1, H-3; H-6/H-7; H-16/H-17; HMBC
correlations H-1/C-2, C-3, C-5, C-9, C-10; H-2/C-1, C-3, C-4,
C-10; H-3/C-1, C-2, C-4, C-5, C-18; H-6/C-4, C-8, C-10; H-7/
C-5, C-9, C-14; H-16/C-13, C-14, C-15; H-17/C-13, C-15, C-16;
H-18/C-3, C-4, C-5; H-21/C-20; HREIMS m/z 319.1581 (calcd
for C21H21NO2 319.1567); EIMS m/z 319 [M]+ (93), 304 (100),
290 (25), 263 (39), 235 (34).
Salvianan (3): pale green plates (EtOAc/hexane); 3 mg;
TLC Rf 0.55 (15% EtOAc/hexane); 1H and 13C NMR, see Tables
1 and 2, respectively; COSY correlations H-2/H-1, H-3; H-6/
H-7; H-15/H-16a, H-16b, H-17; HMBC correlations H-1/C-2,
C-3, C-5, C-9, C-10; H-2/C-1, C-3, C-4, C-10; H-3/C-1, C-2, C-4,
C-5, C-18, C-19; H-6/C-4, C-8, C-10; H-7/C-5, C-9, C-14; H-15/
C-13, C-14, C-17; H-16a/C-13, C-14, C-15, C-17; H-16b/C-13,
C-14, C-15, C-17; H-17/C-13, C-15, C-16; H-18/C-3, C-4, C-5,
C-19; H-19/ C-3, C-4, C-5, C-18; H-21/C-20; HREIMS m/z
321.1728 (calcd for C21H23NO2 321.1723); EIMS m/z 321 [M]+
(100), 306 (96), 292 (40), 265 (15), 263 (14), 237 (71).
Acknowledgment. We gratefully acknowledge the finan-
cial support through the National Science Council (NSC
93-2113-M-077-004 to C.M.S.) and NRICM for this work.
References and Notes
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Salviadione (4): orange solid (EtOAc/hexane); 10 mg; mp
212-214 °C; TLC Rf 0.51 (40% EtOAc/hexane); IR (KBr) νmax
1
3429, 2954, 2925, 2853, 1679, 1650, 1582, 1266 cm-1; H and
13C NMR, see Tables 1 and 2, respectively; COSY correlations
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C19H19NO2 293.1410); EIMS m/z 293 [M]+ (100), 278 (50), 265
(41), 250 (32), 237 (25), 209 (15).
5-(Methoxymethyl)-1H-pyrrole-2-carbaldehyde (5): pale
brown oil; 35 mg; TLC Rf 0.53 (40% EtOAc/hexane); IR (neat)
νmax 3263, 3017, 2927, 2854, 1652, 1496, 1422, 1373, 1216,
1
1186, 1093, 1041, 759, 667 cm-1; H NMR (CDCl3, 500 MHz)
δ 3.37 (3H, s, OCH3), 4.17 (2H, s, OCH2), 6.20 (1H, m, H-4),
6.89 (1H, m, H-3), 9.45 (1H, s, CHO), 9.62 (1H, br, NH);
13C NMR (CDCl3, 125 MHz) δ 178.8 (d, CHO), 137.6 (s, C-5),
132.7 (s, C-2), 121.5 (d, C-3), 109.8 (d, C-4), 67.0 (t, OCH2),

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