New alkaloids from Daphniphyllum calycinum

Article abstract of DOI:10.1021/np040038f

Four new alkaloids, 17-hydroxyhomodaphniphyllic acid (1), daphcalycinosidine C (2, a new iridoid alkaloid), yuzurimine E (3), and yuzurimic acid B (4), were isolated from the seeds of Daphniphyllum calycinum. The structures of these Daphniphyllum alkaloids were determined by spectroscopic analysis including mass spectrometry and 2D NMR.

Full text of DOI:10.1021/np040038f

1094
J . Nat. Prod. 2004, 67, 1094-1099
New Alk a loid s fr om Da ph n iph yllu m ca lycin u m
Hoda El Bitar,† Van Hung Nguyen,‡ Anthony Gramain,§ Thierry Se´venet,§ and Bernard Bodo*,†
Laboratoire de Chimie et Biochimie des Substances Naturelles-UMR 5154 CNRS, Muse´um National d’Histoire Naturelle, 63
Rue Buffon, 75005 Paris, France, Institut de Chimie, Centre National des Sciences Naturelles et de la Technologie, Hanoi,
Vietnam, and Institut de Chimie des Substances Naturelles-CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
Received February 17, 2004
Four new alkaloids, 17-hydroxyhomodaphniphyllic acid (1), daphcalycinosidine C (2, a new iridoid alkaloid),
yuzurimine E (3), and yuzurimic acid B (4), were isolated from the seeds of Daphniphyllum calycinum.
The structures of these Daphniphyllum alkaloids were determined by spectroscopic analysis including
mass spectrometry and 2D NMR.
The Daphniphyllum genus, the only one of the Daph-
niphyllaceae family, is composed of 10 species and is
remarkable for its ability to biosynthesize various alkaloid
classes with highly complex and unique polycyclic struc-
tures.1,2 Recently, various novel types of Daphniphyllum
alkaloids were characterized, and more than 60 compounds
are now known.3-16 Heathcock and co-workers developed
biomimetic total syntheses of several of these alkaloids.17
Daphniphyllum species are used in folk medicines in
various Asian countries and especially D. calycinum Benth.,
a shrub native to North Vietnam and China, which is used
for wound healing and as an antiinflammatory remedy.18
Recent studies on different parts of D. calycinum reported
the isolation of new structural types of alkaloids such as
daphcalycine,3 daphcalycic acid, daphcalycinosidines A and
B,4 and calyciphyllines A and B.14 Further investigation
of the seeds of D. calycinum resulted in the isolation of four
new alkaloids: 17-hydroxyhomodaphniphyllic acid (1),
daphcalycinosidine C (2), yuzurimine E (3), and yuzurimic
acid B (4). This paper describes their isolation and struc-
tural elucidation.
Resu lts a n d Discu ssion
The seeds of D. calycinum Benth. collected in North
Vietnam were ground, defatted with cyclohexane, and then
extracted with MeOH. The methanolic extract was chro-
matographed over reversed-phase silica gel column to yield
fractions, which were further purified by combination of
column chromatography and TLC procedures to afford four
new compounds: 17-hydroxyhomodaphniphyllic acid (1,
0.0001%), daphcalycinosidine C (2, 0.0004%), yuzurimine
E (3, 0.0004%), and yuzurimic acid B (4, 0.001%).
Compound 1 is an optically active solid, [R]22 -17° (c
D
0.2, MeOH), the molecular formula of which was deter-
mined as C22H35NO3 by HRFABMS (m/z 362.2638, [M +
H]+, calc 362.2696), involving six sites of unsaturation. Its
IR absorption spectrum showed the presence of hydroxyl
1
(3302 cm-1) and carboxylic (1715 cm-1) functionalities. H
and 13C NMR spectra (Pyr-d5, Table 1) showed signals due
to four quaternary carbons (1 sp2, 3 sp3), six methines, nine
methylenes, and three methyls.
The 13C chemical shifts of the methylene at δC 45.4 (C-
7), the methine at δC 64.6 (C-1), and the quaternary carbon
at δC 79.4 (C-10) suggested they were linked to a nitrogen
* To whom correspondence should be addressed. Tel: 33(0)140793129.
Fax: 33(0)140793135. E-mail: bodo@mnhn.fr.
atom, whereas that of the methine at δC 78.0 (C-17)
† Museum of Natural History, Paris.
indicated it bore an oxygen atom. The 1H-1H COSY
‡ Institut of Chemistry of Hanoi.
§ Institut of Chemistry of Natural Products, Gif-sur-Yvette.
spectrum revealed connectivities of partial structures a
10.1021/np040038f CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/22/2004

New Alkaloids from Daphniphyllum
J ournal of Natural Products, 2004, Vol. 67, No. 7 1095
Ta ble 1. 1H (400.13 MHz) and 13C (75.47 MHz) NMR Data for Compounds 1, 3, and 4
1 in pyridine-d5
δH J (Hz)
3 in CD3OD
δH J (Hz)
4 in CD3OD
δH J (Hz)
C no.
δC
δC
δC
1
2
3a
3b
4a
4b
3.50 d 3.5
64.6
37.5
26.5
101.5
48.1
21.9
3.30 d 4.2
2.52 m
1.81 m
1.76 m
1.98 ddd 12.5/12.5/6.0
1.62 ddd 12.5/5.0/5.0
70.0
37.9
22.2
1.46 m
1.68 m
1.41 m
1.93 m
1.34 m
2.00 ddd 8.7/7.7/5.1
1.54 m
1.48 m
1.66 m
1.64 m
35.9
34.4
33.1
5
6
7a
7b
37.1
39.2
45.4
43.8
40.0
56.3
39.1
41.4
58.6
1.50 m
3.52 brd 13.6
3.40 brd 13.6
2.29 m
3.52 dd 13.1/4.0
3.23 dd 13.1/9.6
2.25 brddd 9.5/6.5/3.0
3.59 dd 14.0/3.0
3.53 dd 14.0/9.5
8
9
10
47.2
49.3
79.4
22.6
51.6
144.6
137.0
26.0
45.0
145.8
135.5
25.8
2.48 dd 10.4/7.7
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
18
19a
19b
20
21a
21b
22
23
24
25
2.28 m
1.60 m
1.70 m
1.59 m
2.32 ddd 15.0/10.0/6.5
1.68 m
2.75 ddd 16.8/10.5/6.5
2.59 ddd 16.8/10.0/4.8
1.68 m
1.52 m
1.97 m
1.63 m
4.60 dd 11.4/6.0
2.45 m
2.08 m
1.90 m
1.45 m
2.62 dd 14.7/4.2
2.55 dd 14.7/9.3
2.99 ddd 13.4/9.3/4.2
2.32 ddd 15.0/7.5/7.5
2.16 ddd 15.0/4.0/4.0
1.48 m
20.4
26.8
32.2
22.7
31.2
78.0
28.1
39.8
44.5
57.5
29.1
44.0
28.3
40.6
47.8
55.7
29.2
44.1
2.28 m
3.02 dd 15.0/2.5
1.99 dd 15.0/8.9
2.82 ddd 11.6/8.9/2.5
3.48 m
3.52 m
1.83 dddd 11.5/7.0/7.0/1.0
1.42 dddd 11.5/11.5/11.5/11.5
2.54 m
2.34 brdd 14.2/7.6
2.46 m
3.34 m
2.50 m
1.02 d 6.8
4.32 d 11.6
1.93 ddd 12.5/7.0/7.0
1.63 dddd 12.5/11.0/11.0/9.2
2.68 brdd 14.5/11.0
2.37 brdd 14.5/7.0
2.65 m
3.81 dd 12.4/11.0
2.79 m
1.15 d 7.1
1.84 m
1.20 d 6.4
30.3
21.9
35.0
66.2
36.9
65.5
0.75 d 6.5
0.95 s
20.7
24.6
13.8
71.0
14.4
68.7
4.01 brd 11.7
3.53 d 11.7
4.30 d 11.6
175.8
177.2
51.7
173.0
20.9
182.8
3.67 s
2.04 s
(C-2 to C-1 and C-18, and C-18 to C-19 and C-20), b (C-3
to C-4), c (C-6 to C-7 and C-12, and C-12 to C-11), d (C-15
to C-9 and C-16, and C-16 to C-17), and e (C-13 to C-14)
(Figure 1). Connections between a and b units were
depicted by HMBC cross-peaks between H-1 and both C-2
(δC 37.5) and C-3 (δC 26.5) and between H-4 and C-2.
Connections among C-1, C-4, C-6, C-13, and C-21 via two
consecutive quaternary carbons C-5 (δC 37.1) and C-8 (δC
47.2) were deduced by HMBC cross-peaks for H-6 and H2-7
to C-5, H2-4 to C-6 (δC 39.2) and C-8, H2-13 to C-1 (δC 64.6),
C-5, and C-8, and H3-21 to C-4, C-5, C-6, and C-8,
indicating the linkage of the methyl group (δC 24.6) at C-5.
The connectivities between c and d through C-10 (δC 79.4)
were deduced from HMBC correlations of H-9 to C-11 (δC
22.6), and of H2-11, H2-16, and H-17 to C-10, with a
hydroxyl group at C-17 (δC 78.0). Long-range couplings for
H2-13 to C-9 (δC 49.3) indicated the connection between d
and e units via C-8. The HMBC correlations of H2-14 and
H2-13 to C-22 (δC 175.8) revealed that the carboxyl group
was attached to C-14. Thus, 1 has the daphnane skeleton
and its structure is related to that of methyl homodaph-
niphyllate19 and was named 17-hydroxyhomodaphniphyllic
acid.
R*. A chair conformation for the cyclohexane bearing (C-
1-C-5 and C-8) was suggested by NOESY cross-peaks of
H-3b with H-7a and of H-4a with H-13a.
A plausible pathway for the biosynthesis of 1 from
methyl homosecodaphniphyllate is proposed in Figure 2:
an elimination-fragmentation process takes place through
an oxidation of the nitrogen atom of methyl homosecodaph-
niphyllate(I), providing the intermediates II and III, and
the reduction of the latter yields IV.17c Then, the C10-C17
double bond of IV is oxidized to form the intermediate
epoxide V. Finally, 17-hydroxyhomodaphniphyllic acid is
suggested to result from a rearrangement due to the
nucleophilic attack of the nitrogen atom at C-10.
Daphcalycinosidine C (2) was isolated as an optically
active solid, [R]22 -15° (c 0.6, MeOH). Its HRFABMS
D
showed the quasimolecular ion [M + H]+ at m/z 712.3349
(calc 712.3334), corresponding to the molecular formula
C
38H49NO12, which indicated 15 degrees of unsaturation.
The IR absorption spectrum showed the presence of hy-
droxyls (3307 cm-1) and carbonyls (1735, 1720, and 1705
cm-1).
The 13C NMR spectrum (DMSO-d6, Table 2) had 38
carbon signals due to two methyls, 11 methylenes, 16
methines (2 sp2, 14 sp3), and nine quaternary carbons (7
sp2, 2 sp3).
The carbonyl signal at δC 215.5 was assigned to a ketone
and those at 173.8 and 169.3 to carboxyls, with the latter
being conjugated. The 13C NMR data (Table 2) suggested
that the methine at 65.8 (C-4) and the methylenes at 55.6
(C-7) and 48.8 (C-19) were linked to the nitrogen atom. The
chemical shifts of the methine carbons at δC 98.5 (C-1′) and
NOESY correlations for 1 indicated the same relative
configuration as that of methyl homodaphniphyllate (Fig-
ure 1). The protons of the methyl H3-21 showed NOE
correlations with H-6, H-9, and H-12a. The hydroxyl group
at C-17 was located on the same side of the cyclopentane
ring as H2-11, because a strong NOE cross-peak was
observed between its geminal proton (H-17) and H-1 and
not with H2-11; the relative configuration at C-17 is thus

1096 J ournal of Natural Products, 2004, Vol. 67, No. 7
El Bitar et al.
Ta ble 2. 1H (400.13 MHz) and 13C (75.47 MHz) NMR Data for
Compound 2 (in DMSO-d6)
C no.
δ
H J (Hz)
δC
1
2
3a
3b
215.5
43.3
19.8
2.09 m
1.97 brdd 14.3/6.0
1.82 brdd 14.3/4.6
3.15 brd 4.6
4a
65.8
4b
5
6
7a
7b
51.3
50.6
55.6
2.12 m
2.79 dd 11.8/6.9
2.49 dd 11.8/8.2
8
9
10
61.0
141.8
136.8
25.1
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
18
19a
19b
20
21a
21b
22
1′
2′
3′
4′
1.97 m
1.87 m
1.82 m
1.52 m
2.63 dd 13.5/7.5
2.22 dd 13.5/10.0
2.65 m
26.3
39.2
41.6
53.2
27.1
41.5
3.19 m
1.71 m
1.19 m
2.53 m
2.28 m
2.64 m
2.63 m
2.35 m
0.87 d 6.3
1.20 s
33.1
48.8
18.2
23.9
173.8
98.5
73.1
76.3
70.0
74.0
63.6
4.52 d 8.0
2.99 dd 8.8/8.0
3.19 dd 9.0/8.8
3.04 dd 9.5/9.0
3.30 ddd 9.5/6.9/1.9
4.34 dd 11.9/1.9
3.92 dd 11.9/6.9
4.87 d 7.3
5′
6′a
6′b
1′′
3′′
4′′
95.8
148.5
114.4
35.6
7.20 d 1.3
F igu r e 1. Selected 2D NMR correlations for 17-hydroxyhomodaph-
niphyllic acid (1).
5′′
3.00 brddd 8.0/7.3/7.0
2.70 dd 17.0/7.0
1.95 dd 17.0/8.0
5.63 s
6′′a
6′′b
7′′
8′′
9′′
10′′a
10′′b
11′′
38.7
95.8 (C-1′′) revealed they were hemiketal carbons. The
methylenes at δC 63.6 (C-6′) and 59.4 (C-10′′) were linked
to oxygen atoms. Of the 15 degrees of unsaturation, six
were assigned on the basis of this spectrum to the three
carbonyls and three carbon-carbon double bonds (Table
125.6
144.6
45.8
2.52 dd 8.0/7.3
4.04 brd 14.1
3.96 dd 14.1/2.0
59.4
1
2), and thus 2 has nine rings. Analysis of the H-1H COSY
169.3
spectrum showed spin systems of the six partial struc-
tures: a (C-2 to C-4), b (C-18 to C-20), c (C-6 to C-7 and
C-12, and C-12 to C-11), d (C-13 to C-17), e (C-1′ to C-6′),
and f (C-6′′ to C-5′′ and C-7′′, and C-9′′ to C-1′′ and C-5′′)
(Figure 3). The carbon signals at δC 98.5, 76.3, 74.0, 73.1,
70.0, and 63.6 (substructure e) together with 2D NMR data
indicated the presence of a glucopyranose moiety. The large
coupling (J ) 8.0 Hz) of the anomeric proton at δH 4.52
indicated a â-glucose. HMBC correlations characterized
substructure f as a de-O-methylgenipin unit (Figure 3). The
HMBC connectivities between H-1′′ and C-1′ (δC 98.5) and
between H-1′ and C-1′′ (δC 95.8) supported the C-1′ to C-1′′
link through an oxygen, and this linkage formed a geni-
posidic acid substructure. HMBC correlations were ob-
served for H-4 and H2-19 to C-7, and for H2-7 and H2-19 to
C-4, suggesting that C-4, C-7, and C-19 were connected to
each other through the nitrogen atom. Connections among
the a and b units were depicted by HMBC cross-peaks
between H2-19 and H3-20 and C-2 (δC 43.3) and between
H-18 and C-3 (δC 19.8). The long-range correlations of both
H-2 and H2-3 to C-1 (δC 215.5) linked the ketone carbonyl
C-1 to C-2. HMBC cross-peaks for H2-13 to C-1, C-5 (δC
51.3), and C-8 (δC 61.0) and for H-4 to C-8 indicated
connectivities of C-1 to C-13 through C-8 and also of C-4
to C-8 through C-5. The linkage of C-21 (δC 23.9) to C-5
was suggested by HMBC correlations for H3-21 to C-4, C-5,
C-6 (δC 50.6), and C-8. Connections among c and d units
and the tetrasubstituted olefin C-9 (δC 141.8) and C-10 (δC
136.8) were provided by HMBC correlations of H2-13, H-14,
H-15, H2-16, and H2-17 to C-9 and of H2-11, H2-12, and
H2-17 to C-10. Furthermore, HMBC correlations of H2-13,
H-14, and H2-6′ to C-22 (δC 173.8) showed that C-22 was
linked to C-14 and attached to C-6′ by an ester bond.
Saponification of 2 with KOH (2%) afforded, after acidifica-
tion and purification by TLC, de-N-oxide-calyciphylline A14
and geniposidic acid; spectral data and [R]D of the latter
were identical with those of the natural product, [R]22D +3°

New Alkaloids from Daphniphyllum
J ournal of Natural Products, 2004, Vol. 67, No. 7 1097
F igu r e 2. Biogenetic pathway proposed for 17-hydroxyhomodaphniphyllic acid (1) formation from methylhomosecodaphniphyllate.
(c 0.1, MeOH).20 Thus, 2 is a new Daphniphyllum iridoid
alkaloid, for which we propose the name daphcalycinosidine
C.
The 13C NMR spectrum depicted signals for two meth-
ylenes at δC 56.3 (C-7) and 66.2 (C-19), bearing a nitrogen,
and one quaternary carbon δC 101.5 (C-1) linked to both a
nitrogen and an oxygen atom. The chemical shift of the
methylene at δC 71.0 (C-21) suggested it is linked to an
oxygen atom. The presence of four partial structures
(Figure 4), a (C-2 to C-4, and C-18 to C-2, C-19, and C-20),
b (C-6 to C-7 and C-12, and C-12 to C-11), c (C-13 to C-14),
and d (C-15 to C-17), was demonstrated by detailed
analyses of 1H-1H COSY and HSQC data. The a -d units
were connected to one another on the basis of the following
HMBC correlations: H-2 and H2-3 to C-1 (δC 101.5), H2-7
to C-1 and C-19 (δC 66.2), H2-21 to C-4 (δC 34.4), C-6 (δC
40.0), and C-8 (δC 51.6), H2-13 to C-1, C-5, C-8, C-9 (δC
144.6), and C-15 (δC 57.5), and H2-11, H2-16, and H2-17 to
C-9 and C-10 (δC 137.0). The linkage of the methoxycar-
bonyl at C-14 was established by HMBC correlations of H2-
13, H-14, and H3-23 to C-22 (δC 177.2). HMBC cross-peaks
of H2-21 and H3-25 with carbonyl at C-24 (δC 173.0)
indicated an acetoxyl group to be at C-21 (δC 71.0).
Compound 3 was thus assigned as the C-4 deacetoxy form
of yuzurimine21 and named yuzurimine E. The NOESY
correlations in 3 indicated the same relative configuration
as yuzurimine (Figure 4). The conformations of the cyclo-
hexane (C-1-C-5 and C-8) and piperidine (N, C-1, C-8, and
C-5-C-8) rings were assigned as chairs from NOE correla-
tions of H-3b with H-7a, of H-4a with H-13a, and of H-2
with H-13b.
Analysis of the fragmentation pattern of the quasi-
molecular ion [M + H]+ of daphcalycinosidine C (m/z 712)
by positive ion ESI-TOF MS/MS showed fragment ions
supporting the proposed structure: the ions at m/z 518 and
500 corresponded to the cleavage of the ether bond between
the de-O-methylgenipin and the glucose moieties at the
O/C-1′′ and O/C-1′ level, respectively. Other major frag-
ments at m/z 356 and 338 corresponded to the cleavage of
the ester bond between the glucose and the alkaloidal
moieties at the O/C-6′ and O/C-22 level, respectively.
The relative configuration of daphcalycinosidine C was
deduced from NOESY correlations (Figure 3). Strong NOEs
for the alkaloidal moiety were observed between H-3a and
the two protons of the methylene H2-13, suggesting a boat
conformation for the cyclohexanone ring bearing C-3.
Furthermore, the NOESY correlations between H-3b and
H-19b on one hand, and between H-3b and H3-20 on the
other hand, suggested an equilibrium between the boat and
chair conformations for the piperidine ring involving C-18
and C-19. The strong NOE correlations of H-5′′ with H-9′′
and the absence of cross-peaks between H-1′′ and both H-5′′
and H-9′′ indicated the same relative configuration as
geniposidic acid.20 In addition, H-1′′ (δH 4.87) gave a large
coupling with H-9′′ (J ) 7.3 Hz), in agreement with a trans
relative disposition. Another strong NOE correlation was
depicted between H-1′′ and H-16a, suggesting a preferred
conformation of the two substituents at C-1′ and C-6′ of
the glucose moiety.
Yuzurimic acid B (4), [R]22 +11° (c 0.3, MeOH), was
D
shown to have the molecular formula C22H31NO3 by HR-
FABMS (m/z 358.2379, for [M + H]+, calc 358.2383). In its
13C NMR spectrum (CD3OD, Table 1), 22 carbon signals
including five quaternary carbons (3 sp2, 2 sp3), six me-
thines, 10 methylenes, and one methyl were observed. The
IR spectrum showed the presence of hydroxyl (3429 cm-1),
carboxyl (1720 cm-1), and olefinic (1646 cm-1) functional-
ities. The 1H and 13C NMR data suggested that 4 was
structurally related to yuzurimine E (3) (Table 1), but
differed from the latter as it lacked the hydroxyl group at
C-1, the acetyl group on the C-21 oxygen, and the methyl
group at C-22. In fact, detailed analyses of the 2D NMR
data indicated that 4 was the acid form of yuzurimine B,22
and thus named yuzurimic acid B.
The molecular formula of yuzurimine E (3), [R]22 -33°
D
(c 0.3, MeOH), was determined as C25H35NO5 by HR-
FABMS (m/z 430.2590, [M + H]+, calc 430.2594), corre-
sponding to nine degrees of unsaturation. Its 13C NMR
spectrum (CD3OD, Table 1) showed signals due to seven
quaternary carbons (four sp2 and three sp3), five methines,
10 methylenes, and three methyls including one methoxyl
and one acetoxyl group. The IR absorption spectrum
suggested the presence of hydroxyl (3425 cm-1), ester
carbonyl (1734 cm-1), and olefinic (1639 cm-1) functional-
ities. Of the nine levels of unsaturation, three corresponded
to one tetrasubstituted double bond (Table 1) and two ester
carbonyls (δC 173.0 and 177.2), and thus six rings were
assigned to 3.
The relative configuration was elucidated to be the same
as that yuzurimine E (3) from NOESY data. The meth-

1098 J ournal of Natural Products, 2004, Vol. 67, No. 7
El Bitar et al.
F igu r e 4. Selected 2D NMR correlations for yuzurimine E (3).
obtained either on a ZAB2-SEQ (VG analytical) for the FABMS
or on an API Q-STAR (Applied Biosystem) for the ESI-TOFMS.
13C NMR spectra were recorded on a Bruker AC 300 spec-
1
trometer (13C: 75.47 MHz) and H NMR spectra on an Avance
400 Bruker (1H: 400.13 MHz) with internal reference (1H:
CHD2OD at δH 3.31, CHCl3 at δH 7.27, pyridine at δH 7.19,
DMSO at δH 2.49; 13C: CD3OD at δC 49.0, CDCl3 at δC 77.0,
Pyr-d5 at δC 123.5, and DMSO-d6 at δC 39.5).
F igu r e 3. Selected 2D NMR correlations for daphcalycinosidine C (2).
ylation of 4 with diazomethane afforded the corresponding
methyl ester (5), spectral data of which were identical with
those of yuzurimine B.22 Thus, the structures of yuzurimine
E and yuzurimic acid B were assigned as 3 and 4,
respectively.
As compounds 1, 3, 4, yuzurimine B, and yuzurimine
have a plausible common precursor, such as methyl homo-
secodaphniphyllate, for which the absolute configuration
has been determined by X-ray crystallography, the absolute
configuration of the new compounds could be related to that
of yuzurimine and methyl homosecodaphniphyllate.22b
P la n t Ma ter ia l. The seeds of D. calycinum Benth. (Daph-
niphyllaceae) were harvested in North Vietnam in October
1999. A voucher specimen has been deposited in the herbarium
(VN 579) of CNSNT, Hanoi.
Extr a ction a n d Isola tion . The seeds of D. calycinum (1.2
kg) were ground, defatted with cyclohexane, and extracted at
room temperature with MeOH for 3 days, and the extract was
concentrated under reduced pressure to yield 80 g of crude
methanolic extract. The extract was chromatographed on a
RP2 silica gel column eluted with a H2O/MeOH (1:0 to 0:1)
gradient and afforded 20 fractions (F1-F20). The less polar
fraction F20 (50 mg) was purified by TLC on SiO2 gel (CH2-
Cl2/MeOH, 90:10) to yield 1 (1.5 mg). Fractions F7-F12 were
combined (4 g) and further separated on a RP2 silica gel
column eluting with a H2O/MeOH (1:0 to 0:1) gradient followed
by Sephadex LH20 CC (MeOH) to yield 2 (5 mg). Fractions
F16-F19 were combined (5 g) and purified on silica gel CC
using a CH2Cl2/MeOH (1:0 to 0:1) gradient to give 3 (5 mg).
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured in MeOH at 28 °C on a Perkin-Elmer 241
polarimeter. IR spectra were recorded in KBr disks on a
Nicolet Impact 400 D spectrophotometer. Mass spectra were

New Alkaloids from Daphniphyllum
J ournal of Natural Products, 2004, Vol. 67, No. 7 1099
Refer en ces a n d Notes
Then, fractions F4-F6 (0.9 g) were chromatographed on a RP2
silica gel column eluted with a H2O/MeOH (1:0 to 0:1) gradient
to yield compound 4 (15 mg).
(1) (a) Yamamura, S.; Hirata, Y. In The Alkaloids; Manske, R. H. F.,
Ed.; Academic Press: New York, 1975; Vol. 15, pp 41-81. (b)
Yamamura, S. In The Alkaloids; Brossi, A., Ed.; Academic Press: New
York, 1986; Vol. 29, pp 265-286. (c) Kobayashi, J .; Morita, H. In The
Alkaloids; Cordell, G. A., Ed.; Academic Press: New York, 2003; Vol.
60, pp 165-205.
17-Hyd r oxyh om od a p h n ip h yllic a cid (1): C22H35NO3 (M
) 361), microcrystals (MeOH), mp 97-99 °C, [R]D -17° (c 0.2,
MeOH); HRFABMS, m/z 362.2638, [M + H]+, calc 362.2696;
IR (KBr) νmax 3302, 2935, 1715, and 1568 cm-1
NMR data (Pyr-d5, Table 1).
;
1H and 13C
(2) (a) Hao, X.; Zhou, J .; Node, M.; Fuji, K. Yunnan Zhiwu Yanjiu 1993,
15, 205-207. (b) Arbain, D.; Byrne, L. T.; Cannon, J . R.; Patrick, V.
A.; White, A. H. Aust. J . Chem. 1990, 43, 185-190. (c) Yamamura,
S.; Lamberton, J . A.; Niwa, M.; Endo, K.; Hirata, Y. Chem. Lett. 1980,
393-396, and references therein.
Da p h ca lycin osid in e C (2): C38H49NO12 (M ) 711), color-
less amorphous solid, [R]22 -15° (c 0.6, MeOH); HRFABMS
D
m/z 712.3349, [M + H]+, calc 712.3334; IR (KBr) νmax 3307,
2922, 1735, 1720, 1705, and 1588 cm-1; UV (MeOH) λmax 222
(3) J ossang, A.; El Bitar, H.; Pham, V. C.; Sevenet, T. J . Org. Chem. 2003,
68, 300-304.
1
nm (ꢀ 10.000); H and 13C NMR (DMSO-d6, Table 2).
(4) El Bitar, H.; Nguyen, V. H.; Gramain, A.; Se´venet, T.; Bodo, B.
Tetrahedron Lett. 2004, 45, 515-518.
Yu zu r im in e E (3): C25H35NO5 (M ) 429), colorless amor-
phous solid, [R]22 -33° (c 0.3, MeOH); HRFABMS m/z
(5) Morita, H.; Yoshida, N.; Kobayashi, J . J . Org. Chem. 1999, 64, 7208-
D
430.2590, [M + H]+, calc 430.2594; IR (KBr) νmax 3425, 2930,
7212.
1
(6) Morita, H.; Yoshida, N.; Kobayashi, J . J . Org. Chem. 2000, 65, 3558-
3562.
1734, and 1639 cm-1; H and 13C NMR data (CD3OD, Table
1).
(7) Morita, H.; Yoshida, N.; Kobayashi, J . Tetrahedron 1999, 55, 12549-
Yu zu r im ic a cid B (4): C22H31NO3 (M ) 357), microcrystals
12556.
(MeOH), mp 253-255 °C, [R]22 +11° (c 0.3, MeOH); HR-
(8) Morita, H.; Yoshida, N.; Kobayashi, J . Tetrahedron 2000, 56, 2641-
2646.
D
FABMS m/z 358.2379, [M + H]+, calc 358.2383; IR (KBr) νmax
3429, 2930, 1720, and 1646 cm-1; 1H and 13C NMR data (CD3-
OD, Table 1).
(9) Kobayashi, J .; Inaba, Y.; Shiro, M.; Yoshida, N.; Morita, H. J . Am.
Chem. Soc. 2001, 123, 11402-11408.
(10) Morita, H.; Yoshida, N.; Kobayashi, J . J . Org. Chem. 2002, 67, 2278-
Meth yla tion of Yu zu r im ic Acid B (4). A solution of 4 (2
mg) in methanol (0.5 mL) was treated with diazomethane at
5 °C. The mixture was stirred at room temperature for 30 min
and concentrated in vacuo. The residue was purified by TLC
(100% MeOH) to give the corresponding methyl derivative (5),
whose spectral data were identical with those of yuzurimine
B. C23H33NO3 (M ) 371), microcrystals (MeOH), mp 280-282
°C, [R]22D -22° (c 0.2, MeOH); HRFABMS m/z 372.2531, [M +
H]+, calc 372.2540; IR (KBr) νmax 3435, 2928, 1732, and 1646
2282.
(11) Kobayashi, J .; Ueno, S.; Morita, H. J . Org. Chem. 2002, 67, 6546-
6549.
(12) Morita, H.; Takatsu, H.; Kobayashi, J . Tetrahedron 2003, 59, 3575-
3579.
(13) Kobayashi, J .; Takatsu, H.; Shen, Y. C.; Morita, H. Org. Lett. 2003,
5, 1733-1736.
(14) Morita, H.; Kobayashi, J . Org. Lett. 2003, 5, 2895-2898.
(15) Yang, S. P.; Yue, J . M. J . Org. Chem. 2003, 68, 7961-7966.
(16) Morita, H.; Takatsu, H; Shen, Y. C.; Kobayashi, J . Tetrahedron Lett.
2004, 45, 901-904.
(17) (a) Wallace, G. A.; Heathcock, C. H. J . Org. Chem. 2001, 66, 450-
454. (b) Heathcock, C. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
14323-14327. (c) Heathcock, C. H.; J oe, D. J . Org. Chem. 1995, 60,
1131-1142. (d) Heathcock, C. H.; Kath, J . C.; Ruggeri, R. B. J . Org.
Chem. 1995, 60, 1120-1130. (e) Heathcock, C. H.; Ruggeri, R. B.;
Mcclure, K. F. J . Org. Chem. 1992, 31, 665-681. (f) Heathcock, C.
H. Angew. Chem. 1992, 104, 675-691. (g) Heathcock, C. H. Angew.
Chem., Int. Ed. Engl. 1992, 31, 665-681.
(18) (a) Arthur, H. R.; Han, R. P. K.; Loo, S. N. Phytochemistry 1965, 4,
627-629. (b) Gamez, E. J . C.; Luyengi, L.; Lee, S. K.; Zhou, B.-N.;
Fong, H. H. S.; Pezzuto, J . M.; Kinghorn, A. D. J . Nat. Prod. 1998,
61, 706-708. (c) Gray, D. E.; Leung, W. N. Asian J . Med. 1971, 7,
409-412. (d) Fang, S. D.; Hou, W.; Hen, Y.; Hu, J .-H. Acta Chem.
Sin. 1964, 30, 271-274.
(19) (a) Irikawa, H.; Toda, M.; Yamamura, S.; Hirata, S. Tetrahedron Lett.
1969, 1821-1824. (b) Toda, M.; Yamamura, S.; Hirata, S. Tetrahedron
Lett. 1969, 2585-2586.
(20) Takeda, Y.; Nishimura, H.; Inoue, H. Phytochemistry 1975, 14, 2647-
2650.
(21) (a) Sakurai, H.; Sakabe, N.; Hirata, Y. Tetrahedron Lett. 1966, 6309-
6314. (b) Irikawa, H.; Yamamura, S.; Hirata, Y. Tetrahedron 1972,
28, 3727-3738.
(22) (a) Sakurai, H.; Irikawa, H.; Yamamura, S.; Hirata, Y. Tetrahedron
Lett. 1967, 2883-2888. (b) Sasaki, B. K.; Hirata, Y. J . Chem. Soc.
(B) 1971, 1565-1568.
cm-1 13C NMR data (75 MHz, CD3OD) δ 69.0 (1), 39.2 (2), 23.1
;
(3), 33.8 (4), 40.1 (5), 39.5 (6), 59.7 (7), 45.5 (8), 145.0 (9), 136.1
(10), 26.2 (11), 29.5 (12), 40.1 (13), 43.9 (14), 55.7 (15), 29.2
(16), 44.1 (17), 38.6 (18), 66.1 (19), 15.2 (20), 67.6 (21), 177.5
1
(22), 51.6 (23); H NMR data (400 MHz, CD3OD) δ 2.89 (m,
H-1), 2.30 (m, H-2), 1.66 (m, H-3a), 1.66 (m, H-3b), 2.00 (m,
H-4a), 1.63 (m, H-4b), 2.34 (m, H-6), 3.31 (m, H-7a), 3.30 (m,
H-7b), 2.34 (m, H-11a), 2.11 (m, H-11b), 2.18 (m, H-12a), 1.50
(m, H-12b), 2.66 (dd 14.9/3.1, H-13a), 2.08 (dd 14.9/9.2, H-13b),
2.94 (ddd 12.4/9.2/3.1, H-14), 3.53 (m, H-15), 1.93 (m, H-16a),
1.63 (m, H-16b), 2.68 (m, H-17a), 2.37 (m, H-17b), 2.45 (m,
H-18), 3.50 (m, H-19a), 2.48 (m, H-19b), 1.12 (d 6.9, H-20),
3.99 (d 11.6, H-21a), 3.73 (d 11.6, H-21b), 3.65 (s, H-23).
Hyd r olysis of Da p h ca lycin osid in e C (2). A solution of
the daphcalycinosidine C (2, 4 mg) in 2% KOH (1 mL) was
stirred at room temperature for 2 h. The residue was dissolved
in methanol (0.3 mL) and neutralized with HCl (2 N). After
evaporation, the residue was purified by TLC (50 MeOH, 50
CH2Cl2) to afford de-N-oxide-calyciphylline A (1.5 mg) and
geniposidic acid (1.3 mg).
Ack n ow led gm en t. We thank Dr. J . P. Brouard and Mr.
L. Dubost for the mass spectra, Miss C. Caux for the NMR
spectra, and Dr. R. J oyeau for helpful discussions.
NP040038F