Structure characterization, biomimetic total synthesis, and optical purity of two new pyrrolidine alkaloids, pandamarilactonine-A and -B, isolated from Pandanus amaryllifolius Roxb

Article abstract of DOI:10.1021/ja0009929

Two new alkaloids, both possessing a pyrrolidinyl α,β-unsaturated γ-lactone residue and a γ-alkylidene α,β-unsaturated γ-lactone residue, were isolated from a tropical medicinal plant, Pandanus amaryllifolius Roxb. Their structures were deduced by spectroscopic analysis including the new NMR technique PFG J-HMBC 2D spectroscopy and then confirmed by biomimetic total synthesis. It was found that one diastereoisomer, pandamarilactonine-A (1), comprised a mixture enriched with (+)-enantiomer, while another diastereomeric isomer, pandamarilactonine-B (2), occurred as a racemate.

Full text of DOI:10.1021/ja0009929

J. Am. Chem. Soc. 2000, 122, 8635-8639
8635
Structure Characterization, Biomimetic Total Synthesis, and Optical
Purity of Two New Pyrrolidine Alkaloids, Pandamarilactonine-A and
-B, Isolated from Pandanus amaryllifolius Roxb.
Hiromitsu Takayama,*,† Tomotake Ichikawa,† Toshiyuki Kuwajima,† Mariko Kitajima,†
Hiroko Seki,‡ Norio Aimi,†,‡ and Maribel G. Nonato*,§
Contribution from the Faculty of Pharmaceutical Sciences, Chiba UniVersity, 1-33 Yayoi-cho, Inage-ku,
Chiba 263-8522, Japan, Chemical Analysis Center of Chiba UniVersity, 1-33, Yayoi-cho, Inage-ku,
Chiba 263-8522, Japan, and Research Center for the Natural Sciences, UniVersity of Santo Tomas,
Espana, Manila 1008, Philippines
ReceiVed March 20, 2000
Abstract: Two new alkaloids, both possessing a pyrrolidinyl R,â-unsaturated γ-lactone residue and a
γ-alkylidene R,â-unsaturated γ-lactone residue, were isolated from a tropical medicinal plant, Pandanus
amaryllifolius Roxb. Their structures were deduced by spectroscopic analysis including the new NMR technique
PFG J-HMBC 2D spectroscopy and then confirmed by biomimetic total synthesis. It was found that one
diastereoisomer, pandamarilactonine-A (1), comprised a mixture enriched with (+)-enantiomer, while another
diastereomeric isomer, pandamarilactonine-B (2), occurred as a racemate.
Introduction
The genus Pandanus belonging to the family Pandanaceae
comprises about 600 species which are distributed widely in
tropical and subtropical regions. Several Pandanus species are
recognized as medicinal plants used as a remedy for toothache
and rheumatism, and as a diuretic, cardiotonic, etc.1 Recently,
a new structural class of alkaloids, possessing a γ-alkylidene
R,â-unsaturated γ-lactone or γ-alkylidene R,â-unsaturated γ-lac-
tam moiety, has been isolated from Pandanus amaryllifolius.2-4
Since the Pandanus plants are potentially valuable as candidates
for new medicinal resources, we have started chemical studies
of Pandanus plants.5 Described herein is the isolation of two
new diastereomeric pyrrolidine alkaloids, pandamarilactonine-A
(1) and -B (2), from P. amaryllifolius Roxb. native to the
Philippines and Thailand, and their structure elucidation using
a new NMR technique, viz., PFG J-HMBC 2D analysis.
Biomimetic total synthesis of 1 and 2 and analysis of the optical
purity of the natural alkaloids are also reported.
Results and Discussion
Isolation and Structure Elucidation of Two New Alkaloids.
Two new alkaloids, 1 and 2, were isolated together with the
known compound pandamarilactone-1 (3)3 (Figure 1) by using
SiO2 column chromatography of the alkaloidal fraction obtained
Figure 1.
† Faculty of Pharmaceutical Sciences, Chiba University.
‡ Chemical Analysis Center of Chiba University.
by conventional methods from an EtOH extract of the fresh
leaves of P. amaryllifolius.
§ University of Santo Tomas.
(1) Tan, M. Philippine Medicinal Plants in Common Use: Their
Phytochemistry and Pharmacology; AKAP: Quezon City, Philipp 1980.
(2) Byrne, L. T.; Guevara, B. Q.; Patalinghug, W. C.; Recio, B. V.; Ualat,
C. R.; White, A. H. Aust. J. Chem. 1992, 45, 1903-1098.
(3) Nonato, M. G.; Garson, M. J.; Truscott, R. J. W.; Carver, J. A.
Phytochemistry 1993, 34, 1159-1163.
(4) Sjaifullah, A.; Garson, M. J. ACGC Chem. Res. Commun. 1996, 5,
24-27.
(5) (a) Takayama, H.; Kuwajima, T.; Kitajima, M.; Nonato, M. G.; Aimi,
N. Heterocycles 1999, 50, 75-78. (b) Takayama, H.; Kuwajima, T.;
Kitajima, M.; Nonato, M. G.; Aimi, N. Nat. Med. (Tokyo) 1999, 53, 335.
The new compound 1, named pandamarilactonine-A, was
obtained as an amorphous powder, exhibiting [R]23 +35.0°
D
(c 4.37, CHCl3). High-resolution FABMS analysis gave m/z
318.1721 [M + H]+ and established the molecular formula as
C18H23NO4, which indicated 1 to be an isomer of the coexistent
alkaloid 3. The characteristic 1H and 13C NMR signals {δ 6.99
(1H, d-like, J ) 1.5 Hz, H-4), 5.18 (1H, dd, J ) 7.9, 7.9 Hz,
H-6), 1.99 (3H); δ 171.1 (C-2), 129.1 (C-3), 137.7 (C-4), 148.6
10.1021/ja0009929 CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/22/2000

8636 J. Am. Chem. Soc., Vol. 122, No. 36, 2000
Takayama et al.
(C-5), 114.1 (C-6), 10.5 (C-21)} and the UV absorption at 275
nm indicated the presence of a γ-alkylidene-R-methyl R,â-
unsaturated γ-lactone moiety. A three-carbon methylene chain
was connected with the terminal sp2 carbon (C-6) of the
γ-alkylidene-γ-lactone moiety, which was established by COSY,
HMQC, and HMBC spectra. Further, the presence of an
R-methyl R,â-unsaturated γ-lactone residue was shown by the
1
characteristic signals in the H and 13C NMR spectra {δ 7.09
(1H, dd, J ) 1.5, 1.8 Hz, H-16), 4.80 (1H, ddd, J ) 1.8, 1.8,
5.5 Hz, H-15), 1.93 (3H); δ 174.3 (C-18), 131.2 (C-17), 147.0
(C-16), 83.4 (C-15), 10.7 (C-20)}. Using the residual four
carbons (three methylenes and one methine) and one nitrogen
atom, a pyrrolidine ring could be constructed. In the HMBC
spectrum, the methine proton (δ 2.83, 1H, m, H-14) on the
pyrrolidine ring correlates with the methylene carbon at C-9 (δ
55.0) (the terminal of a three-carbon methylene chain) and the
sp2 carbon at C-16 (δ 147.0) in the R,â-unsaturated γ-lactone
ring. In addition, the methine proton (δ 4.80) at C-15 (δ 83.4)
in the γ-lactone ring has connectivity between the carbons at
C-14 and C-13 in the pyrrolidine ring. In the NOESY spectrum,
a clear cross-peak between H-4 and H-6 was observed,
demonstrating the (Z) configuration in the γ-alkylidene R,â-
unsaturated γ-lactone moiety. All the above findings enabled
us to compose the molecular structure of the new alkaloid to
be the formula 1, having a novel pyrrolidinyl R,â-unsaturated
γ-lactone skeleton, except for the stereochemistry of the vicinal
asymmetric center at the C-14 and C-15 positions.
The second new alkaloid (2), named pandamarilactonine-B,
was also obtained as an amorphous powder, exhibiting [R]23
D
0° (c 0.20, CHCl3). The UV and mass spectra, as well as the
molecular formula obtained by HR-FABMS (found 318.1704),
were completely the same as those of pandamarilactonine-A
(1). Further, the 1H and 13C NMR spectra were very similar to
those of 1, indicating that new alkaloid 2 is a stereoisomer of
compound 1. As the differential NOE experiment demonstrated
the (Z) configuration in the γ-alkylidene R,â-unsaturated γ-lac-
tone moiety in 2, a diastereomeric relation at the C-14 and C-15
positions between 1 and 2 is implied.
Figure 2.
Hz (3JH-14/C-16) and 3.3 Hz (3JH-15/C-13), were indicative of
the gauche relationship of both H-14/C-16 and H-15/C-13.
Therefore, conformers I and II corresponded to compounds 1
and 2, respectively. This analysis was confirmed by differential
NOE experiments; specifically, NOEs were observed between
H-15 and H-13 and between H-16 and H-13 in compound 1
and between H-15 and H-13 and between H-16 and H-14 in
compound 2. From these data, we propose that pandamarilac-
tonine-A (1) has the erythro structure taking the conformation
I and pandamarilactonine-B (2) has the threo structure with
conformer II.
Biomimetic Total Synthesis of Pandamarilactonine-A and
-B. A biosynthetic route to the known Pandanus alkaloids, such
as pandamarine (4)2 and pandamarilactone-1 (3), has been
proposed by Garson2,4 (Figure 3). By condensation of two units
of 4-hydroxy-4-methylglutamic acid (5),10 which has been found
in a Pandanus species,11 and a C-4-N-C-4 dicarboxylic acid
(6) probably derived from glutamic acid, an intermediate (7)
possessing a basic backbone structure would be formed. Through
a decarboxylation, cyclization, reduction, and dehydration
process, plausible biogenetic intermediates 8 and 9 having a
Next, we attempted characterization of the relative stereo-
chemistry at C-14 and C-15 in the new alkaloids 1 and 2. Martin
et al. reported that the stereochemistry of the threo/erythro
diastereomers having the pyrrolidinyl R,â-unsaturated γ-lactone
skeleton could be assigned by comparison of the chemical shifts
of their 13C and 1H NMR spectra at particular positions.6
However, this approach was not adaptable to the case of
compounds 1 and 2, because of the indistinct difference in the
chemical shifts at the corresponding positions (C-15 and H-14).
Then, PFG J-HMBC 2D spectroscopy,7 which enables the
torsion angle between two heteroatoms to be determined, i.e.,
H-C-C-C,8 was applied with a combination of differential
NOE experiments to establish the relative stereochemistry in 1
and 2 (Figure 2). Each of the compounds 1 and 2 has the
possibility to take three conformations (I, I′, and I′′ and II, II′,
and II′′). From the small coupling constants between H-14 and
H-15 (1, J ) 4.9 Hz; 2, J ) 3.9 Hz), the possibility of
conformers I′′ and II′′ could be excluded. In pandamarilactonine-
A, a large coupling constant (5.6 Hz) between H-14 and C-16
and a small coupling constant (3.7 Hz) between H-15 and C-13
were obtained by PFG J-HMBC 2D spectroscopy, indicating
their anti and gauche orientations,9 respectively, while in the
case of pandamarilactonine-B, the small coupling constants, 2.7
(9) (a) Matsumori, N.; Murata, M.; Tachibana, K. Tetrahedron 1995,
51, 12229-12238. (b) Matsumori, N.; Nonomura, T.; Sasaki, M.; Murata,
M.; Tachibana, K.; Satake, M.; Yasumoto, T. Tetrahedron Lett. 1996, 37,
1269-1272. (c) Furihata, K.; Seto, H. Tetrahedron Lett. 1999, 40, 6271-
6275.
(10) Peterson, P. J.; Fowden, L. Phytochemistry 1972, 11, 663-673.
(11) Bell, E. A.; Meier, L. K.; Sorenson, H. Phytochemistry 1981, 20,
2213-2216.
(6) Martin, S. F.; Barr, K. J.; Smith, D. W.; Bur, S. K. J. Am. Chem.
Soc. 1999, 121, 6990-6997.
(7) Willker, W.; Leibfritz, D. Magn. Reson. Chem. 1995, 33, 632-638.
(8) Hansen, P. E. Prog. NMR Spectrosc. 1981, 14, 175-296.

Characterization of Pandamarilactonine-A and -B
J. Am. Chem. Soc., Vol. 122, No. 36, 2000 8637
Figure 3. Hypothetical biogenetic route of the Pandanus alkaloids.
symmetrical structure would be generated. Successive intra-
molecular cyclization of the secondary amine to one of the two
γ-alkylidene R,â-unsaturated γ-lactones or γ-alkylidene R,â-
unsaturated γ-lactams would produce 3 or 4. The newly isolated
alkaloid found in the present study could also be biosynthesized
in a divergent way via the common intermediate 9.
We planned a total synthesis of 1 and 2 by a route which
mimics the final stage of the biosynthesis (from 9 to 1/2)
proposed above. The key secondary amine derivative 9, which
corresponds to the hypothetical biogenetic intermediate, was
prepared as follows (Figure 4). N-Dialkylation of benzylamine
(10) with O-tetrahydropyranyl-4-chlorobutanol (11) was carried
out using potassium carbonate in the presence of a catalytic
amount of sodium iodide to yield the tertiary amine 12 in 61%
yield. Then, the benzyl group was switched to a â,â,â-
trichloroethoxycarbonyl group12 to give carbamate 13. After
removal of the THP ether in 13, the free alcohols in 14 were
converted to aldehyde 15 in 72% yield by Swern oxidation.
The aldol reaction of aldehyde 15 with siloxyfuran using boron
Figure 4.
trifluoride etherate (BF3‚Et2O)13 gave a mixture of stereoiso-
meric adducts 16 in quantitative yield. Next, installation of the
exo double bond was performed by treatment of the adducts 16
with a combination of TMS-Cl and DBU to give γ-alkyli-
denebutenolide (17) in 41% yield together with the (Z,E) isomer
in 25% yield. Finally, the protecting group on nitrogen in 17
was removed with Zn in AcOH. Because of the lability of amine
9 toward SiO2 column chromatography, the crude residue
obtained by usual workup was directly treated with a catalytic
amount of trifluoroacetic acid in CH3CN. By careful separation
of the crude products, pandamarilactonine-A (1) and -B (2) were
obtained in 9% and 9% yields, respectively. They were
respectively identical with the natural products by direct
comparison of the chromatographic behavior and high-resolution
MS and 1H and 13C NMR spectra. Although the chemical yield
(12) Takayama, H.; Tominaga, Y.; Kitajima, M.; Aimi, N.; Sakai, S. J.
Org. Chem. 1994, 59, 4381-4385.
(13) Ohe, F.; Brueckner, R. Tetrahedron Lett. 1998, 39, 1901-1904.

8638 J. Am. Chem. Soc., Vol. 122, No. 36, 2000
Takayama et al.
CHCl3. The organic layer was dried over magnesium sulfate and
evaporated to give a crude alkaloidal fraction (2.56 g). The portion of
the crude base (1.10 g) was roughly separated by silica gel flash column
chromatography eluted with a CHCl3 to MeOH/CHCl3 gradient to give
the 10 fractions. The 2-5% MeOH/CHCl3 eluate was subjected to SiO2
medium-pressure liquid chromatography using 2% EtOH/CHCl3 to give
95 mg of pandamarilactonine-A (1), 10 mg of pandamarilactonine-B
(2), and 9 mg of pandamarilactone-1 (3).
of the final stage in the present synthesis was not satisfactory,
we succeeded in the first and biomimetic total synthesis of the
new alkaloids, which provided chemical support for the sug-
gested biogenetic route of 1 and 2 as well as proof for the
chemical structures deduced by spectroscopic analysis.
Optical Purity of Natural Pandamarilactonine-A and -B.
As described in the isolation section above, pandamarilacto-
nine-A (1) exhibited [R]23D +35.0° (c 4.37, CHCl3); in contrast,
the specific rotation of pandamarilactonine-B (2) was almost
zero. We were interested in the optical purity of these natural
products.14 With the synthetic racemates in hand, we first
examined the resolution of the enantiomers using chiral column
chromatography. Synthetic pandamarilactonine-A (1) exhibited
two peaks at 43.2 and 51.7 min when Chiralcel OB was used,
and synthetic pandamarilactonine-B (2) showed two peaks at
18.7 and 20.4 min by using Chiralcel OD. Then, natural
alkaloids were subjected to chiral HPLC analysis, which
established that the natural pandamarilactonine-A (1) contained
dominantly the (+)-enantiomer over the (-)-enantiomer in a
ratio of 63:37, while natural 2 existed as a racemate. On the
basis of the biosynthetic proposal and the chemical conversion
of 9 to 1 and 2, it is conceivable that pandamarilactonine-A
and pandamarilactonine-B undergo acid-catalyzed interconver-
sion during the isolation process and that pandamarilactonine-B
(2) is an artifact from pandamarilactonine-A (1). To test this
hypothesis, the natural alkaloids 1 and 2 were respectively
treated with 5% H2SO4 under the conditions used in the
partitioning of alkaloids and the recovered alkaloids were
carefully analyzed with 500 MHz NMR spectroscopy. In the
recovered 1 and 2, the presence of the stereoisomers and/or
pandamarilactonine-1 could not be observed. Further, recovered
pandamarilactonine-A exhibited same optical purity (63:37) by
chiral HPLC analysis, revealing that no racemization has
occurred. These experiments demonstrate that pandamarilacto-
nine-A and -B are not interconvertible under the isolation
conditions and that pandamarilactonine-B is not an artifact
produced during the isolation process.
In summary, two new alkaloids having a pyrrolidinyl R,â-
unsaturated γ-lactone moiety and a γ-alkylidene R,â-unsaturated
γ-lactone residue were isolated from a tropical medicinal plant,
P. amaryllifolius. Their structures were first deduced by
spectroscopic analysis including the new NMR technique PFG
J-HMBC 2D spectroscopy and then confirmed by biomimetic
total synthesis. Interestingly, it was found that one of the
diastereoisomers, pandamarilactonine-A (1) having the erythro
structure, was comprised of a mixture enriched with the (+)-
enantiomer, while another diastereomeric threo isomer, pan-
damarilactonine-B (2), occurred as a racemate in nature.
Further investigation of the minor constituents in this plant,
development of an efficient synthetic pathway of 1 and 2,
determination of the absolute configuration of (+)-pandama-
rilactonine-A, and biological evaluation of these alkaloids are
in progress in our laboratories.
Pandamarilactonine-A (1). An amorphous powder. Rf value 0.5
[SiO2, solvent system 5% MeOH in CHCl3]. [R]23 +35.0° (c 4.37,
D
CHCl3). UV (MeOH): λmax (nm) (log ꢀ) 275 (2.43), 232 (sh), 207
(1.82). IR (neat): νmax (cm-1) 1750 (lactone). FABMS (NBA): m/z
318 [M + H]+. HR-FABMS (NBA): calcd for C18H24NO4 318.1704,
1
found 318.1721. H NMR (500 MHz, CDCl3): δ 7.09 (1H, dd, J )
1.5 and 1.8 Hz, H-16), 6.99 (1H, d-like, J ) 1.5 Hz, H-4), 5.18 (1H,
dd, J ) 7.9 and 7.9 Hz, H-6), 4.80 (1H, ddd, J ) 1.8, 1.8 and 5.5 Hz,
H-15), 3.12 (1H, dd, J ) 6.7 and 7.6 Hz, H-11), 2.88 (1H, ddd, J )
4.0, 7.9 and 12.9 Hz, H-9), 2.83 (1H, m, H-14), 2.45 (1H, m, H-9),
2.43 (2H, dd, J ) 7.3 and 15.0 Hz, H2-7), 2.21 (1H, m, H-11), 1.99
(3H, d-like, J ) 0.9 Hz, H3-21), 1.93 (3H, dd, J ) 1.5 and 1.8 Hz,
H3-20), 1.70-1.80 (2H, m, H-12 and H-13), 1.59-1.70 (3H, m, H2-8
1
and H-13), 1.42 (1H, m, H-12). H NMR (400 MHz, acetone-d6): δ
7.27 (1H, dd, J ) 1.5 and 2.9 Hz, H-16), 7.26 (1H, ddd, J ) 1.7, 1.7
and 3.2 Hz, H-4), 5.33 (1H, dd, J ) 7.8 and 8.1 Hz, H-6), 4.86 (1H,
ddq, J ) 4.9, 2.9 and 2.0 Hz, H-15), 3.10 (1H, ddd-like, J ) 2.0, 7.1
and 8.5 Hz, H-11), 2.90 (1H, ddd, J ) 8.1, 8.1 and 11.7 Hz, H-9),
2.85 (1H, ddd, J ) 4.9, 4.9 and 9.3 Hz, H-14), 2.44 (1H, ddd, J ) 4.9,
6.8 and 11.7 Hz, H-9), 2.38 (2H, ddd, J1 ) J2 ) J3 ) 7.6 Hz, H-7),
2.19 (1H, ddd, J ) 6.1, 8.8 and 10.2 Hz, H-11), 1.90 (3H, dd-like, J
) 0.5 and 1.5 Hz, H-21), 1.83 (3H, dd, J ) 1.5 and 2.0 Hz, H-20),
1.57-1.77 (5H, m, 2 × H-8, H-12, and 2 × H-13), 1.42 (1H, m, H-12).
13C NMR (125 MHz, CDCl3): δ 174.3 (C-18), 171.1 (C-2), 148.6 (C-
5), 147.0 (C-16), 137.7 (C-4), 131.2 (C-17), 129.1 (C-3), 114.1 (C-6),
83.4 (C-15), 65.3 (C-14), 55.0 (C-9), 54.2 (C-11), 28.3 (C-8), 25.7
(C-12), 24.0 (C-7), 23.8 (C-13), 10.7 (C-20), 10.5 (C-21).
Pandamarilactonine-B (2). An amorphous powder. Rf value 0.5
[SiO2, solvent system 5% MeOH in CHCl3]. [R]23D 0° (c 0.20, CHCl3).
UV (MeOH): λmax (nm) (log ꢀ): 275 (2.49), 232 (sh), 207 (1.72). IR
(neat): νmax (cm-1) 1758 (lactone). FABMS (NBA): m/z 318 [M +
H]+. HR-FABMS (NBA): calcd for C18H24NO4 318.1704, found
1
318.1704. H NMR (500 MHz, CDCl3): δ 7.05 (1H, dd, J ) 1.5 and
1.7 Hz, H-16), 7.00 (1H, d-like, J ) 1.5 Hz, H-4), 5.18 (1H, dd, J )
7.8 and 8.0 Hz, H-6), 4.71 (1H, ddd, J ) 1.7, 2.0 and 5.9 Hz, H-15),
3.12 (1H, m, H-11), 2.73 (1H, m, H-9), 2.70 (1H, m, H-14), 2.42-
2.48 (2H, m, H-7 and H-9), 2.36 (1H, m, H-7), 2.25 (1H, m, H-11),
1.99 (3H, d-like, J ) 0.7 Hz, H3-21), 1.93 (3H, dd, J ) 1.7 and 1.7
Hz, H3-20), 1.73-1.87 (4H, m, H2-12 and H2-13), 1.59-1.67 (2H, m,
H2-8). 1H NMR (400 MHz, acetone-d6): δ 7.28 (1H, dd-like, J ) 1.5
and 2.9 Hz, H-16), 7.26 (1H, ddd, J ) 1.5, 1.7 and 3.2 Hz, H-4), 5.32
(1H, dd-like, J ) 7.8 and 8.1 Hz, H-6), 4.81 (1H, ddq, J ) 3.9, 2.9
and 2.0 Hz, H-15), 3.08 (1H, m, H-11), 2.80 (1H, ddd, J ) 8.1, 8.1
and 12.0 Hz, H-9), 2.77 (1H, ddd, J ) 3.9, 5.1 and 8.8 Hz, H-14),
2.45 (1H, m, H-9), 2.33 (2H, ddd, J ) 7.8, 7.8 and 15.6 Hz, H-7),
2.24 (1H, m, H-11), 1.92 (3H, dd-like, J ) 0.7 and 1.5 Hz, H-21),
1.83 (3H, dd-like, J ) 1.5 and 2.0 Hz, H-20), 1.59-1.77 (6H, m, 2 ×
H-8, 2 × H-12, and 2 × H-13). 13C NMR (125 MHz, CDCl3): δ 174.3
(C-18), 171.1 (C-2), 148.5 (C-5), 147.5 (C-16), 137.7 (C-4), 130.8 (C-
17), 129.1 (C-3), 114.1 (C-6), 83.4 (C-15), 66.3 (C-14), 55.8 (C-9),
54.2 (C-11), 28.4 (C-8), 27.1 (C-12), 24.0 (C-7), 24.0 (C-13), 10.8
(C-20), 10.5 (C-21).
Experimental Section15
Extraction and Isolation of Alkaloids. Fresh young leaves (1.2
kg) of P. amaryllifolius purchased at flower market in Bangkok
(Thailand) were macerated with ethyl alcohol (6 L) three times, and
filtered. The combined filtrate was concentrated under reduced pressure
to give a crude extract (105 g), which was then partitioned between
Et2O and 5% aqueous H2SO4. The water-soluble fraction was alkalinized
with concentrated NH4OH until pH 10 and exhaustively extracted with
Experimental Conditions for PFG J-HMBC 2D Spectroscopy.
All NMR experiments were performed at 303 K for solution of ca. 10
mg of alkaloid dissolved in 0.5 mL of acetone-d6 on a JEOL LA600
spectrometer equipped with a 5 mm NALORAC HX inverse probe.
Fifteen J-HMBC 2D spectra were acquired with 16 scans for a 1024
(F2) × 128 (F1) data matrix and with 300 ms of constant time and
varying evolution time (∆) from 10 to 290 ms in 20 ms steps. The
HMBC signal intensity is given by a sine function of the evolution
time (∆) and coupling constant (JHX). Therefore, the J value (coupling
constant) can be obtained from a least-squares approximation by fitting
(14) Takayama, H.; Kurihara, M.; Kitajima, M.; Said, I. M.; Aimi, N. J.
Org. Chem. 1999, 64, 1772-1773.
(15) See the Supporting Information for general procedures.

Characterization of Pandamarilactonine-A and -B
J. Am. Chem. Soc., Vol. 122, No. 36, 2000 8639
a sine curve to the signal amplitude of the HMBC correlation peak
depending on the characteristic sin(πJHX) with increasing evolution time
(∆).
Chiral HPLC Analysis of Synthetic and Natural Pandamarilac-
tonine-A and B. Pandamarilactonines were respectively analyzed by
HPLC using chiral column chromatography (pandamarilactonine-A,
Chiralcel OB, Daicel Chemical Industries, Ltd., solvent 40% i-PrOH/
n-hexane, flow rate 0.3 mL/min, column temperature 30 °C; pandama-
rilactonine-B, Chiralcel OD, Daicel Chemical Industries, Ltd., solvent
20% EtOH/n-hexane, flow rate 0.5 mL/min, column temperature 30
°C). The synthetic and natural 1 exhibited two peaks at 43.2 and 51.9
min in ratios of 50:50 and 63:37, respectively. Both the synthetic and
natural 2 exhibited two peaks at 18.8 and 20.5 min in a ratio of 50:50,
respectively.
Synthesis of Pandamarilactonine-A (1) and -B (2). Zinc dust (50
mg, 0.076 mmol) was added to a solution of γ-alkylidenebutenolides
[17, (Z,Z)-form] (25.2 mg, 0.051 mmol) in acetic acid (2 mL), and the
mixture was stirred at room temperature under argon atmosphere for 4
h. After the reaction mixture was filtered, the filtrate was concentrated
under reduced pressure to give a residue. A portion of the residue was
roughly purified by SiO2 flash column chromatography (10%MeOH/
CHCl3) to characterize the secondary amine 9. [1H NMR (400 MHz,
CDCl3): δ 7.02 (2H, d, J ) 1.5 Hz, H-4), 5.14 (2H, dd, J ) 7.8 and
8.1 Hz, H-6), 2.94 (4H, dd, J ) 8.1 and 8.1 Hz), 2.47 (4H, dd, J )
15.3 and 7.3 Hz), 2.08 (4H, m), 2.00 (6H, s, -CH3). 13C NMR (125
MHz, CDCl3): δ 170.74 (C-2), 149.38 (C-5), 137.61 (C-4), 130.00
(C-3), 110.99 (C-6), 47.32 (C-9), 25.26 (C-8), 23.34 (C-7), 10.56
(-CH3). EIMS: m/z (rel intens) 317 (M+, 1), 220 (29), 151 (3), 55
(100).] The crude residue obtained by the reaction above was dissolved
in CH3CN (1 mL). After addition of trifluoroacetic acid (2 µL), the
reaction mixture was stirred at 80 °C under argon atmosphere for 5 h.
The reaction mixture was concentrated under reduced pressure, and
dissolved in CHCl3. The CHCl3 solution was poured into saturated
aqueous NaHCO3, washed with brine, dried over MgSO4, and evapo-
rated. The residue was separated by SiO2 MPLC (2% EtOH/CHCl3) to
give pandamarilactonine-A (1) (1.4 mg, 9%), and -B (2) (1.4 mg, 9%).
They were respectively identical with the natural products by direct
comparison of the chromatographic behavior and high-resolution MS
Acknowledgment. This work was supported in part by
Grants-in-Aid (Nos. 10877349 and 10044325) for Scientific
Research from the Ministry of Education, Science, Sports, and
Culture, Japan.
Supporting Information Available: Detailed experimental
1
procedures for the syntheses of 12-17 and copies of the H
and 13C NMR spectra of natural and synthetic pandamarilac-
tonine-A and -B and those of synthetic intermediates 12-17
and 9 (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
1
and H and 13C NMR spectra.
JA0009929