Pyrrolidinoindoline alkaloids from selaginella moellendorfii

Article abstract of DOI:10.1021/np9001515

Eight new pyrrolidinoindoline alkaloids (1-8) were isolated from the whole plant of Selaginella moellendorfii. Their structures were determined by mass spectrometry, 1D and 2D NMR spectroscopy, and chemical interconversions. These alkaloids have a 3-carboxybut-2-enyl group at C-3a and two methyl groups at N-8. The possible biogenetic route from selaginellic acid (1) to neoselaginellic acid (6) was postulated and chemically mimicked. Tautomerization between 6 and 6a was observed. Selected compounds were evaluated for antibacterial, cytotoxic, and acetylcholinesterase inhibitory activities.

Full text of DOI:10.1021/np9001515

J. Nat. Prod. 2009, 72, 1151–1154
1151
Pyrrolidinoindoline Alkaloids from Selaginella moellendorfii
Yue-Hu Wang,† Chun-Lin Long,*,†,‡ Fu-Mei Yang,§ Xi Wang,§ Qian-Yun Sun,§ Hong-Sheng Wang,†,⊥,| Ya-Na Shi,†,⊥ and
Gui-Hua Tang†
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China, College of Life and EnVironmental
Sciences, Minzu UniVersity of China, Beijing 100081, People’s Republic of China, Key Laboratory of Chemistry for Natural Products, Guizhou
ProVince and Chinese Academy of Sciences, Guiyang 550002, People’s Republic of China, Yunnan Agricultural UniVersity, Kunming 650201,
People’s Republic of China, and Henan Institute of Science and Technology, Xinxiang 453003, People’s Republic of China
ReceiVed March 5, 2009
Eight new pyrrolidinoindoline alkaloids (1-8) were isolated from the whole plant of Selaginella moellendorfii. Their
structures were determined by mass spectrometry, 1D and 2D NMR spectroscopy, and chemical interconversions. These
alkaloids have a 3-carboxybut-2-enyl group at C-3a and two methyl groups at N-8. The possible biogenetic route from
selaginellic acid (1) to neoselaginellic acid (6) was postulated and chemically mimicked. Tautomerization between 6
and 6a was observed. Selected compounds were evaluated for antibacterial, cytotoxic, and acetylcholinesterase inhibitory
activities.
Pyrrolidinoindoline alkaloids bearing a C-3a-substituted hexahy-
dropyrrolo[2,3-b]indole ring system possess a variety of biological
activities.1 For example, (-)-physostigmine isolated from the seeds
of Physostigma Venenosum and (-)-debromoflustramine B occur-
ring in the cheilostome bryozoan of Flustra foliacea serve as
inhibitors of cholinesterase (ChE),2 while hodgkinsine identified
from Psychotria colorata exhibits antinociceptive activity,3 and (-)-
pseudophrynaminol synthesized from tryptamine has antibacterial
activity.4
Selaginella moellendorfii Hieron. (Selaginellaceae) is a perennial
herb mainly distributed in the southern area of the Changjiang River
in mainland China. It has a history of use in treating bleeding,
gonorrhea, jaundice, and idiopathic thrombocytopenic purpura (ITP)
in traditional Chinese folk medicine.5 A coumarin,5a a lignanoside,5b
flavone glycosides,5c and cytotoxic5d and antioxidative5e biflavones
have been found in this plant. A positive reaction of the MeOH
extract of S. moellendorfii with Dragendorff’s reagent led us to
further investigate this species. This study resulted in the isolation
of a series of new pyrrolidinoindoline alkaloids (1-8) from the
whole plant. These alkaloids are characterized by possessing a
3-carboxybut-2-enyl side chain at the 3a position and two N8-methyl
groups. Compound 6 might be biogenetically derived from 1 and
was biomimetically synthesized. Further, selected compounds
(1, 4, and 6) were evaluated for activities against Escherichia coli,
Staphylococcus aureus, human leukemia K562, and lung adeno-
carcinoma A549 cell lines, and acetylcholinesterase (AChE). This
paper reports the structure elucidation of these new compounds as
well as the results of chemical transformation and bioassays.
N-methyl groups [δH 3.34 (3H, s), 3.09 (3H, s), and 2.99 (3H, s)],
and one methyl group [δH 1.90 (3H, br s)]. Comparison of NMR
data of 1 with those of pseudophrynamine A and pseudophrynami-
nol6 showed that 1 is a prenylated pyrrolodinoindoline alkaloid.
The prenylated moiety was elucidated as a 3-carboxybut-2-enyl
group and located at the C-3a position from the HMBC correlations
of H3-12 to C-10, C-11, and C-13; H-10 to C-3a; and H2-9 to C-3,
C-4a, C-8a, and C-11. The E-configuration of the double bond in
this group, the same as that in tiglic acid, was confirmed by the
ROESY correlation between H3-12 and H2-9. The three N-methyl
groups were located at N-1, N-8, and N-8, from the HMBC
correlations of N1-CH3 to C-2 and C-8a and of N8-CH3 to C-7a
and C-8a, respectively. Thus, the planar structure of 1 was
determined as 3a-[(2E)-3-carboxybut-2-enyl]-N1,N8,N8-trimethyl-
1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole. The presence of a
carboxyl group and a quaternary nitrogen atom (N-8) in 1 led to
the inference that this compound is an inner salt.
The relative configuration of 1 was deduced as cis by the ROESY
correlation of H-8a to H2-9. The absolute configuration of C-3a
was determined as S by transformation of 1 to noselaginellic acid
(6, Scheme 2), for which the configuration was suggested by
comparing its CD data with those of known compounds (see the
structure elucidation paragraph for 6 below).
Compound 2 was assigned the molecular formula C18H24N2O3
by HRESIMS. By comparing the MS and NMR (Tables 1 and 3)
data of 2 with those of 1, compound 2 was inferred as being a
derivative of selaginellic acid (1) substituted by a hydroxy group,
which was located at C-5 by the presence of an aromatic ABX
coupling system [δH 7.45 (d, J ) 8.8 Hz, H-7), 6.89 (dd, J ) 8.8
Results and Discussion
Selaginellic acid (1) was obtained as an optically active ([R]23
D
-61.8), colorless, amorphous solid having the molecular formula
C18H24N2O2, as determined by HRESIMS (obsd [M + H]+ at m/z
1
301.1912, calcd 301.1916). The H NMR spectra of 1 (Table 1)
revealed the signals of four aryl protons [δH 7.68 (1H, d, J ) 7.5
Hz) and 7.56-7.59 (3H, m)], one olefin proton [δH 6.33 (1H, br t,
J ) 7.5 Hz)], one aminoacetal proton [δH 5.75 (1H, s)], three
* To whom correspondence should be addressed. Tel: +86-871-5223233.
Fax: +86-871-5223233. E-mail: long@mail.kib.ac.cn or chunlinlong@
hotmail.com.
† Kunming Institute of Botany.
‡ Minzu University of China.
§ Key Laboratory of Chemistry for Natural Products.
⊥ Yunnan Agricultural University.
| Henan Institute of Science and Technology.
10.1021/np9001515 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/07/2009

1152 Journal of Natural Products, 2009, Vol. 72, No. 6
Wang et al.
Table 1. 1H NMR Data of Compounds 1-3 and 6a (δ in ppm, J in Hz)
position
2
1a
2b
3c
6ab
3.65, 2H, m
3.58, ddd (9.0, 9.0, 4.0)
3.40, ddd (9.0, 9.0, 9.0)
2.40, ddd (13.5, 9.0, 4.0)
2.15, ddd (13.5, 9.0, 9.0)
7.57, md
3.55, ddd (8.6, 8.6, 3.6)
3.31, overlapped
2.34, ddd (13.5, 8.6, 3.6)
2.13, ddd (13.5, 8.6, 8.6)
6.81, d (2.4)
3.41, m
3.21, ddd (8.5, 8.5, 8.5)
3
2.18, m
2.43, 2H, m
1.91, ddd (13.5, 8.5, 8.5)
6.75, d (2.5)
4
7.24, m
7.54, m
7.57, m
8.02, m
5
6
7.59, m
7.56, md
6.89, dd (8.8, 2.4)
7.45, d (8.8)
5.62, s
6.79, dd (8.5, 2.5)
7.31, d (8.5)
5.46, s
7
8a
9
7.68, d (7.5)
5.75, s
2.89, 2H, m
2.87, 2H, m
2.56, 2H, m
2.71, dd (16.0, 13.0)
2.34, d (16.0)
4.04, d (13.0)
3.68, m
10
11
12
6.33, br t (7.5)
1.90, 3H, br s
6.62, br t (7.6)
1.90, 3H, br s
5.15, br t (7.6)
1.55, 3H, br s
3.79, 2H, s
2.88, 3H, s
3.09, 3H, s
2.77. 3H, s
1.47, 3H, d (7.2)
13
N1-CH3
N8-CH3
3.09, 3H, s
3.34, 3H, s
2.99. 3H, s
3.02, 3H, s
3.25, 3H, s
2.92. 3H, s
3.01, 3H, s
3.80, 3H, s
3.58. 3H, s
a Measured in CD3OD at 500 MHz. b In CD3OD at 400 MHz. c In acetone-d6 at 500 MHz. d Assignments with the same superscript are
interchangeable.
Scheme 1. Biogenetic Pathway for Neoselaginellic Acid (6)
compound
5 was elucidated as N-(5-hydroxyselaginelloyl)-
L-phenylalanine.
Neoselaginellic acid (6) was assigned the molecular formula
C18H24N2O3, as determined by HRESIMS. Similar to selaginellic
acid (1), 6 has a disubstituted phenyl ring, a 3-carboxybut-2-enyl
group, and three N-methyl groups. The difference between 1 and 6
is that the signals of the aminoacetal proton and carbon disappeared
in the NMR data of 6 and were replaced by the signal for a lactam
carbonyl group [δC 178.5 (qC, C-8a)]. This difference signifies that
C-8a is oxidized and the N-8-C-8a bond is broken in the structure
of 6. This was supported by the HMBC correlations of N1-CH3 to
C-8a and N8-CH3 to C-7a. Compound 6 is also structurally similar
to a known compound, chimonamidine,7 except that the latter has
a hydroxy group at C-3a rather than a 3-carboxybut-2-enyl group
and has only one methyl at N-8 rather than having two. The C-3a
substituent of 6 was suggested as having an S-configuration by
comparing its CD data [∆ε -2.00 (218), -1.86 (198)] with those
of (-)- and (+)-chimonamidine. (-)-Chimonamidine exhibited
negative Cotton effects [∆ε -2.9 (224) and -27 (201)] at 224 and
201 nm, while (+)-chimonamidine has positive values [∆ε +3.1
(226) and +24.4 (202)] at 226 and 202 nm.7
Compounds 7 and 8 were assigned the molecular formulas of
C27H33N3O4 and C27H33N3O5, respectively, as determined by HRES-
IMS. By comparing the MS and NMR data of 7 and 8 with those
of 4-6, both 7 and 8 were assigned with two units, a neoselaginel-
loyl or 5-hydroxyneoselaginelloyl group and a phenylalanine resi-
due. On the basis of their 2D NMR correlations, the two compounds
were elucidated as N-neoselaginelloyl-L-phenylalanine and N-(5-
hydroxyneoselaginelloyl)-L-phenylalanine, respectively.
Scheme 2. Chemical Transformation from 1 to 6 and
Tautomerization between 6 and 6a
and 2.4 Hz, H-6), and 6.81 (d, J ) 2.4 Hz, H-4)] in 2 and the
HMBC correlations of H-7 to C-5 and H-4 to C-3a. Therefore,
compound 2 was elucidated as 5-hydroxyselaginellic acid.
The molecular formula of 3 was determined as C18H27N2O2 by
HRESIMS. The NMR spectra of 3 were very similar to those of
pseudophrynaminol. Comparison of their MS and NMR data
revealed that 3 has one more hydroxy group and two more N-methyl
groups than the latter.6 As in 2, the hydroxy group was located at
C-5 and the two N-methyl groups at N-8. On the basis of these
findings, 3 was elucidated as 5-hydroxy-N8,N8-dimethylpseudo-
phrynaminol.
A plausible biogenetic pathway for compound 6 is proposed as
shown in Scheme 1. To substantiate the proposal, 1 was oxidized
with I2 in the presence of NaOAc in refluxing EtOH followed by
basification with NaOH in H2O to give 6 (Scheme 2).8 On the basis
of the biogenetic relationship between selagenellic acid (1) and
neoselagenellic acid (6), we suggested that the latter and its
derivatives (7 and 8) as well as the chimonamidines should be
regarded as pyrrolidinoindoline alkaloids.
Compound 4 was found to have a molecular formula of
C27H33N3O3, according to HRESIMS. Detailed analysis of the MS
and NMR data of 4 showed that this compound is comprised of
two moieties, a selaginellic acid (1) unit and a phenylalanine unit,
which are joined through an amide bond formed by the carboxyl
group of the selaginellic acid and the amino group of the
phenylalanine, and supported by the HMBC correlation of H-8′
[δH 4.68 (dd, J ) 9.5 and 4.5 Hz)] to C-13 [δC 171.3 (qC)]. The
configuration of phenylalanine was determined as L by acidic
hydrolysis of 4 to give a product having a negative specific rotation.
Thus, the structure of 4 was determined as N-selaginelloyl-L-
phenylalanine.
We also noted that compound 6 is unstable. Signals for an
impurity were observed in its NMR spectra (see the Supporting
Information), although it was purified repeatedly. The possible
reason is that a nucleophilic addition reaction may occur between
the nucleophilic atom of N-8 and the partially positive atom of
C-10, especially under acidic conditions. It was found that 6 and
its tautomer 6a interconverted easily. As shown in Scheme 2,
compound 6 could be transformed to this major product, 6a, under
acidic conditions, and in turn, 6a was converted to 6 under basic
Compound 5 gave the molecular formula C27H33N3O4 according
to HRESIMS. By comparing the MS and NMR data of 5 with those
of 4, it was found that the former has an additional hydroxy group.
As in 2 and 3, the hydroxy group was also located at C-5. Accordingly,

Pyrrolidinoindoline Alkaloids from Selaginella moellendorfii
Journal of Natural Products, 2009, Vol. 72, No. 6 1153
Table 2. 1H NMR Data of Compounds 4-8 (δ in ppm, J in Hz)
position
2
4a
5a
6a
3.68, m
3.60, m
2.72, 2H, m
7a
8a
3.53, ddd (9.0, 9.0, 3.5)
3.26, overlapped
2.34, ddd (13.5, 9.0, 3.5)
2.10, ddd (13.5, 9.0, 9.0)
7.49, dd (7.5, 1.5)
7.56, m
3.52, ddd (9.0, 9.0, 3.0)
3.23, m
3.58, 2H, m
3.55, 2H, m
3
2.28, m
2.68, 2H, m
2.64, m
2.56, m
6.81, br s
2.08, ddd (13.9, 9.0, 9.0)
6.78, d (2.0)
4
5
7.59, mb
7.54, mb
7.60, mb
7.83, m
7.54, m
7.50, m
7.57, m
6
7
8a
9
7.54, m
7.65, dd (7.5, 1.5)
5.67, s
2.85, 2H, d (8.0)
6.02, br t (8.0)
1.84, 3H, br s
7.17, 2H, m
7.21, 2H, m
7.18, m
3.26, overlapped
3.00, overlapped
4.68, dd (9.5, 4.5)
2.99, 3H, s
6.89, dd (9.0, 2.0)
7.44, d (9.0)
5.51, s
2.78, 2H, d (7.5)
6.02, br t (7.5)
1.84, 3H, br s
7.17, 2H, m
7.21, 2H, m
7.18, m
3.25, m
3.00, m
4.67, dd (9.5, 4.5)
2.95, 3H, s
3.20, 3H, s
2.89, 3H, s
6.94, br d (9.0)
7.57, d (9.0)
7.77, br d (7.5)
2.84, 2H, m
6.49, br t (7.5)
1.68, 3H, br s
2.77, 2H, m
2.71, 2H, m
10
12
2′, 6′
3′, 5′
4′
6.03, br t (7.5)
1.53, 3H, br s
7.22, 2H, d (7.2)
7.29, 2H, t (7.2)
7.23, t (7.2)
3.25, dd (14.5, 5.0)
2.99, overlapped
4.65, dd (10.0, 5.0)
2.98, 3H, s
6.05, br t (7.5)
1.59, 3H, br s
7.20, 2H, d (7.5)
7.28, 2H, t (7.5)
7.21, m
3.23, dd (14.0, 4.3)
2.97, overlapped
4.65, dd (10.0, 4.3)
2.96, 3H, s
7′
8′
N1-CH3
N8-CH3
3.00, 3H, s
3.29, 3H, s
3.25, 3H, s
3.25, 3H, s
2.94, 3H, s
3.17, 3H, s
3.05, 3H, s
3.11, 3H, s
3.01, 3H, s
a Measured in CD3OD at 500 MHz. b Assignments with the same superscript are interchangeable.
Table 3. 13C NMR Data of Compounds 1-8 and 6a (δ in ppm)
Qingdao, People’s Republic of China), and Sephadex LH-20 (40-70
µm; Amersham Pharmacia Biotech AB, Uppsala, Sweden). TLC was
conducted on precoated silica gel plates GF254 (Qingdao). HPLC
separations were performed using an Agilent 1200 series pump equipped
with a diode array detector and a semipreparative Zorbax SB-C18 (5
µm, φ 9.4 × 250 mm) column. L-Phenylalanine (L-Phe) was purchased
from Shanghai Xinxing Chemical Reagent Institute (Shanghai, People’s
Republic of China), and trifluoroacetic acid (TFA) from Sinopharm
Chemical Reagent Co. Ltd. (Shanghai, People’s Republic of China).
Plant Material. Selaginella moellendorfii was collected from Jingxi
County of Guangxi Zhuang Autonomous Region, People’s Republic
of China, in June 2008. The plant was identified by Dr. Guang-Wan
Hu (Kunming Institute of Botany, Chinese Academy of Sciences), and
a voucher specimen (No. JX0801) was deposited at the Laboratory of
Ethnobotany, Kunming Institute of Botany.
position 1a
2a
3b
4a
5a
6a
7c
8a
6ac
2
3
3a
56.0 56.0 55.0 56.1 56.0
38.9 38.6 37.6 38.6 38.6
57.7 57.5 56.7 57.5 57.4
48.3
33.9
57.4
48.3 48.2 47.2
34.2 34.1 35.7
57.6 57.3 50.0
4
4a
5
6
7
7a
8a
126.3 111.5 111.0 126.2 111.5 132.0d 132.0 117.8 131.2
140.4 141.5 141.2 140.2 141.4 133.9 135.7 137.1 134.7
132.4 161.4 158.6 132.5 161.4 132.4d 132.4 160.3 132.1
131.2 118.2 117.1 131.3 118.2 131.5d 131.4 118.0 131.2
120.6 121.4 120.8 120.7 121.4 123.5 123.3 124.6 123.1
146.2 137.3 137.0 146.0 137.3 142.8 142.8 134.3 146.4
124.0 122.5 123.1 124.1 122.4 178.5 178.3 178.2 177.1
9
36.2 36.4 34.2 35.9 35.6
40.4
40.5 40.5 29.0
10
11
131.7 136.7 119.4 131.2 131.3, 135.2 129.9 130.1 74.2
137.5 133.0 139.4 135.7 135.7 135.5 136.8 136.7 39.2
12
14.0 13.0 13.7 13.2 13.3
12.7
12.8 12.8 17.9
Extraction and Isolation. The air-dried whole plant of S. moellen-
dorfii (6 kg) was exhaustively extracted with MeOH. The solvent was
evaporated under reduced pressure, and the remaining residue (780 g)
was partitioned into three fractions, CHCl3 (A, 32 g), Me2CO (B, 154 g),
and MeOH (C, 350 g), by silica gel column chromatography. Fraction
C was separated into two further fractions, H2O (C1, 270 g) and MeOH
(C2, 71 g), by D101 resin column chromatography. Fraction C2, which
was alkaloid positive by Dragendorff’s reagent, was fractionated by
C18 column chromatography (MeOH-H2O, 5:95 to 95:5) into three
major alkaloid positive fractions, 20% MeOH (C21, 2.0 g), 30% MeOH
(C22, 0.5 g), and 50% MeOH (C23, 1.5 g).
13
1′
174.9 170.8 67.1 171.3 171.4 170.4 171.1 171.1 174.8
138.7 138.6
130.2 130.1
138.8 138.9
130.3 130.3
2′, 6′
3′, 5′
4′
129.4 129.4
129.6 129.6
127.8 127.8
127.8 127.8
7′
37.8 37.8
37.9 37.9
8′
55.1 55.2
55.2 55.3
9′
174.6 174.7
174.6 174.6
N1-CH3
N8-CH3
39.2 39.1 38.6 39.0 39.1
53.0 53.5 52.7 53.0 53.5
48.6 49.0 48.1 48.4 49.1
31.2
47.1
48.3
31.2 31.2 30.6
48.3 48.4 55.6
47.0 47.2 53.1
a Measured in CD3OD at 100 MHz. b In acetone-d6 at 125 MHz. c In
CD3OD at 125 MHz. d Assignments with the same superscript are
interchangeable.
Fraction C21 was submitted to chromatography on a silica gel column
(CHCl3-Et2NH, 1:1; CHCl3-MeOH-Et2NH, 90:30:1) to afford 6 (100
mg), which was purified by LH-20 column chromatography (MeOH)
and semipreparative HPLC [MeOH-H2O (containing 0.05% TFA), 35:
65], and 1 (200 mg), which was purified by LH-20 column chroma-
tography (MeOH). Fraction C22 was separated by silica gel column
chromatography (Me2CO-H2O, 10:1 and 5:1) to yield 3 (13.0 mg),
which was purified by HPLC [MeCN-H2O (0.05% TFA), 20:80], and
2 (8.3 mg), which was purified by HPLC [MeOH-H2O (0.05% TFA),
conditions. The structure of 6a was confirmed by 1D and 2D NMR
spectra, except for the configuration of C-11.
Compounds 1, 4, and 6 were tested for biological activity against
two bacteria, Escherichia coli and Staphylococcus aureus, two
cancer cell lines, K562 and A549, and acetylcholinesterase (AChE).
However, none of these compounds showed inhibitory effects up
to the 200 µg/mL dose used.
35:65]. Fraction C23 was chromatographed over
a silica gel
(CHCl3-MeOH-Et2NH, 90:30:1) and a Sephadex LH-20 column
(MeOH) and by HPLC [MeOH-H2O (0.05% TFA), 45:55] to give 8
(2.5 mg), 7 (14.0 mg), 5 (11.0 mg), and 4 (23.8 mg).
Experimental Section
Selaginellic acid (1): colorless, amorphous solid (MeOH); [R]23
D
General Experimental Procedures. Optical rotations were deter-
mined on a JASCO DIP-370 automatic digital polarimeter. UV spectra
were recorded on a Shimadzu double-beam 210A spectrometer. CD
spectra were recorded on a JASCO J-715 spectropolarimeter. IR spectra
were recorded on a Bio-Rad FTS-135 infrared spectrophotometer. 1D
and 2D NMR spectra were recorded on Bruker AM-400 and DRX-
500 spectrometers with TMS as internal standard. MS were measured
on a VG Auto Spec-3000 mass spectrometer. Column chromatography
was performed over silica gel G (80-100 and 300-400 mesh), silica
gel H (10-40 µm), D101 resin (Qingdao Marine Chemical Ltd.,
-61.8 (c 0.51, H2O); UV (H2O) λmax (log ε) 260 (3.40), 205 (4.11)
nm; CD (c 0.060, MeOH) ∆ε -2.91 (205), -6.19 (194); IR (KBr)
ν
max 3428, 1718, 1695, 1677, 1460 cm-1; 1H and 13C NMR, see Tables
1 and 3; ESIMS m/z 301 [M + H]+; HRESIMS m/z 301.1912 [M +
H]+ (calcd for C18H25N2O2, 301.1916).
5-Hydroxyselaginellic acid (2): colorless, amorphous solid (MeOH);
[R]23 -25.0 (c 0.36, H2O); UV (H2O) λmax (log ε) 274 (3.07), 257
D
(3.02) nm; CD (c 0.060, MeOH) ∆ε +1.87 (211), 0 (204), -6.93 (194);
IR (KBr) νmax 3432, 1687, 1605, 1476, 1460 cm-1; 1H and 13C NMR,

1154 Journal of Natural Products, 2009, Vol. 72, No. 6
Wang et al.
see Tables 1 and 3; ESIMS m/z 317 [M + H]+; HRESIMS m/z 317.1860
[M + H]+ (calcd for C18H25N2O3, 317.1865).
5-Hydroxy-N8,N8-dimethylpseudophrynaminol (3): colorless, amor-
phous solid (MeOH); [R]24D -17.0 (c 0.24, H2O); UV (H2O) λmax (log
ε) 274.0 (3.06), 195.4 (4.14) nm; CD (c 0.055, MeOH) ∆ε -1.25 (224),
0 (209), +1.52 (199); IR (KBr) νmax 3424, 1679, 1606, 1460 cm-1; 1H
and 13C NMR, see Tables 1 and 3; ESIMS m/z 303 [M]+; HRESIMS
m/z 303.2071 [M]+ (calcd for C18H27N2O2, 303.2072).
Tautomerization of 6a to 6. Compound 6a (20 mg, 0.063 mmol)
was dissolved in 2 mL of 10% NaOH (5 mmol) and stirred for 1 h at
60 °C. Then, 2 N HCl (2.5 mL, 5 mmol) was added to the reaction
mixture and concentrated and separated by HPLC [MeOH-H2O (0.05%
TFA), 35:65] to give 6 (17.2 mg, 0.0544 mmol, 86%). Analytical data:
HPLC retention time identical to that of natural neoselagenellic acid
isolated from the plant.
Biological Testing. The activity of compounds 1, 4, and 6, at the
concentrations of 2, 20, and 200 µg/mL, against E. coli and Staphy-
lococcus aureus were measured by the microdilution assay with
gentamicin as positive control (MIC ) 0.10 µM),9 K562 cells by the
MTT method with adriamycin as positive control (IC50 ) 0.32 µM),10
A549 cells by the SRB method with 5-fluorouracil as positive control
(IC50 ) 20.8 µM),11 and AChE by Ellman’s method with tacrine as
positive control (IC50 ) 0.20 µM),12 respectively.
N-Selaginelloyl-L-phenylalanine (4): colorless, amorphous solid
(MeOH); [R]24D -37.8 (c 0.48, H2O); UV (H2O) λmax (log ε) 205 (4.08)
nm; CD (c 0.070, MeOH) ∆ε +0.22 (221), 0 (217), -1.16 (205), -3.34
1
(193); IR (KBr) νmax 3428, 1684, 1631, 1532, 1457 cm-1; H and 13C
NMR, see Tables 2 and 3; ESIMS m/z 448 [M + H]+; HRESIMS m/z
448.2585 [M + H]+ (calcd for C27H34N3O3, 448.2600).
N-(5-Hydroxyselaginelloyl)-L-phenylalanine (5): colorless, amor-
phous solid (MeOH); [R]23D -67.5 (c 0.20, H2O); UV (H2O) λmax (log
ε) 268 (3.46) nm; CD (c 0.057, MeOH) ∆ε +1.81 (214), 0 (203), -2.22
Acknowledgment. This work was financially supported by the
Ministry of Education of China through its 111 & 985 projects (B08044
& MUC 985-3-3), the Ministry of Science and Technology of China
(2005DKA21006), and the Knowledge Innovation Program of the
Chinese Academy of Sciences. Selena Ahmed, a Ph.D. candidate in
Biology at the City University of New York, helped edit the English
of this paper.
1
(197); IR (KBr) νmax 3379, 1674, 1628, 1604, 1500, 1457 cm-1; H
and 13C NMR, see Tables 2 and 3; ESIMS m/z 464 [M + H]+;
HRESIMS m/z 464.2553 [M + H]+ (calcd for C27H34N3O4, 464.2549).
Neoselaginellic acid (6): colorless, amorphous solid (MeOH); [R]17
D
-17.8 (c 2.58, MeOH); UV (MeOH) λmax (log ε) 206 (3.93) nm; CD
(c 0.060, MeOH) ∆ε -2.00 (218), -1.86 (198); IR (KBr) νmax 3435,
1
1686, 1650, 1619, 1471 cm-1; H and 13C NMR, see Tables 2 and 3;
ESIMS m/z 317 [M + H]+; HRESIMS m/z 317.1870 [M + H]+ (calcd
for C18H25N2O3, 317.1865).
Supporting Information Available: 1D and 2D NMR spectra for
compounds 1-8 and 6a. This material is available free of charge via
the Internet at http://pubs.acs.org.
N-Neoselaginelloyl-L-phenylalanine (7): colorless, amorphous solid
(MeOH); [R]22D -19.3 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 207
(4.10) nm; CD (c 0.060, MeOH) ∆ε -0.70 (216), 0 (209), +5.29 (193);
IR (KBr) νmax 3411, 3399, 1733, 1646, 1617, 1528, 1497, 1454 cm-1
;
References and Notes
1H and 13C NMR, see Tables 2 and 3; ESIMS m/z 464 [M + H]+;
HRESIMS m/z 464.2536 [M + H]+ (calcd for C27H34N3O4, 464.2549).
N-(5-Hydroxyneoselaginelloyl)-L-phenylalanine (8): colorless, amor-
phous solid (MeOH); UV (MeOH) λmax (log ε) 281 (3.22), 204 (4.39)
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1
nm; IR (KBr) νmax 3430, 1676, 1501, 1454 cm-1; H and 13C NMR,
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[R]17 -22.2 (c 0.06, H2O); HPLC retention time was identical with
D
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Transformation of 1 to 6. I2 (11 mg, 0.043 mmol) and NaOAc
(3.9 mg, 0.048 mmol, 1.1 equiv) were added to a stirred solution of
compound 1 (13 mg, 0.043 mmol) in EtOH (1 mL) at 75 °C. After
3 h, the reaction mixture was concentrated. Then, 25% NaOH (0.5 mL,
3.12 mmol) was added and stirred for 1 h at room temperature. This
was followed by the addition of 2 N HCl (1.6 mL, 3.20 mmol) into
the reaction mixture, which was then concentrated and separated by
HPLC [MeOH-H2O (0.05% TFA), 35:65] to yield 6 (2.4 mg, 0.0076
mmol, 18%). Analytical data: [R]18D -45.8 (c 0.12, MeOH); 1H NMR
spectrum (see the Supporting Information) and HPLC retention time
were identical to those of natural neoselagenellic acid isolated from
the plant.
Tautomerization of 6 to 6a. Compound 6 (80 mg, 0.253 mmol)
was dissolved in 2 mL of 2 N HCl and stirred for 4 h at 90 °C.
Following this procedure, the acidic solution was concentrated and
separated by HPLC [MeCN-H2O (0.05% TFA), 10:90] to result in
the purification of 6a (30 mg, 0.095 mmol, 38%). Analytical data:
colorless, amorphous solid (MeOH); [R]18D +5.4 (c 0.37, MeOH); UV
(H2O) λmax (log ε) 262 (2.58), 205 (3.75) nm; CD (c 0.057, MeOH)
∆ε +0.53 (229), 0 (224), -1.93 (215), -7.34 (197); 1H and 13C NMR,
see Tables 1 and 3; ESIMS m/z 317 [M + H]+.
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