Daphnicyclidins A-H, novel hexa- or pentacyclic alkaloids from two species of Daphniphyllum

Article abstract of DOI:10.1021/ja016955e

Eight highly modified Daphniphyllum alkaloids with unprecedented fused hexa- or pentacyclic skeletons, daphnicyclidins A-H (1-8), have been isolated from the stems of Daphniphyllum humile and D. teijsmanni, and their structures were elucidated on the basi

Full text of DOI:10.1021/ja016955e

11402
J. Am. Chem. Soc. 2001, 123, 11402-11408
Daphnicyclidins A-H, Novel Hexa- or Pentacyclic Alkaloids from
Two Species of Daphniphyllum
Jun’ichi Kobayashi,*,† Yasutada Inaba,† Motoo Shiro,‡ Naotoshi Yoshida,† and
Hiroshi Morita†
Contribution from the Graduate School of Pharmaceutical Sciences, Hokkaido UniVersity, Sapporo
060-0812, Japan, and X-ray Research Laboratory, Rigaku Corporation, Akishima, Tokyo 196-8666, Japan
ReceiVed August 28, 2001. ReVised Manuscript ReceiVed September 29, 2001
Abstract: Eight highly modified Daphniphyllum alkaloids with unprecedented fused hexa- or pentacyclic
skeletons, daphnicyclidins A-H (1-8), have been isolated from the stems of Daphniphyllum humile and D.
teijsmanni, and their structures were elucidated on the basis of spectroscopic data and chemical means. The
stereochemistry was elucidated by combination of NOESY correlations, X-ray crystallographic data, and CD
analyses.
Introduction
humile. In our continuing search for biogenetically interesting
Daphniphyllum alkaloids, daphnicyclidins A-H (1-8), eight
novel alkaloids with unprecedented fused hexa- and pentacyclic
skeletons, were isolated from the stems of D. humile and D.
teijsmanni.7 This paper describes the isolation and structural
elucidation of 1-8.
Daphniphyllum alkaloids are a structurally diverse group of
natural products, which are elaborated by trees of the genus
Daphniphyllum and have attracted great interest from biogenetic
and synthetic points of view.1,2 A number of Daphniphyllum
alkaloids have been isolated and classified into six different
types of backbone skeletons.1,2 Heathcock and co-workers have
demonstrated a marvelous biomimetic transformation of a
dialdehyde to a pentacyclic alkaloid due to formation of seven
new σ bonds.3 Recently, we have isolated two novel types of
Daphniphyllum alkaloids named as daphnezomines A and B4
with a unique aza-adamantane core, and daphnezomines F and
G5 with an 1-azabicyclo[5.2.2]undecane ring system as well as
some new related alkaloids6 from the leaves or stems of D.
Results and Discussion
Isolation of Daphnicyclidins A-H (1-8). The stems of D.
teijsmanni were extracted with MeOH, and the extract was
partitioned between EtOAc and 3% tartaric acid. Water-soluble
materials, which were adjusted at pH 9 with saturated Na2CO3,
were extracted with CHCl3. CHCl3-soluble materials were
subjected to an amino silica gel column (Hex/EtOAc, 9:1 f
1:1, and then CHCl3/MeOH, 1:0 f 0:1), in which a fraction
eluted with MeOH was purified by C18 HPLC (25% CH3CN/
0.1%TFA) and then amino silica gel HPLC (15% CH3CN) to
afford daphnicyclidins A (1, 0.003% yield), B (2, 0.0003%), C
(3, 0.001%), D (4, 0.002%), and F (6, 0.001%) as colorless
solids. Alkaloidal fractions prepared from the stems of D.
humile described in the previous paper4,5 were separated by the
same procedure as described above to give daphnicyclidins A
(1, 0.003% yield), B (2, 0.0005%), C (3, 0.003%), D (4,
0.001%), E (5, 0.001%), F (6, 0.003%), G (7, 0.001%), and H
(8, 0.004%).
Structural Elucidation of Daphnicyclidins A-C (1-3). The
FABMS spectrum of daphnicyclidin A (1) showed the pseudo-
molecular ion peak at m/z 368 (M + H)+, and the molecular
formula, C22H25NO4, was established by HRFABMS [m/z
368.1862, (M + H)+, ∆ +0.0 mmu]. IR absorptions implied
the presence of OH or NH (3440 cm-1) and conjugated carbonyl
(1680 cm-1) functionalities. 1H and 13C NMR data are presented
in Tables 1 and 2, respectively. The 13C NMR spectrum8
revealed 22 carbon signals due to one sp3 quaternary carbon,
* To whom corresondence should be addressed. J.K. Telephone: (011)-
706-4985. Fax: (011)706-4989. E-mail: jkobay@pharm.hokudai.ac.jp.
† Graduate School of Pharmaceutical Sciences, Hokkaido University.
‡ X-ray Research Laboratory, Rigaku Corporation. E-mail: jkobay@
pharm.hokudai.ac.jp.
(1) For reviews of Daphniphyllum alkaloids, see: (a) Yamamura, S.;
Hirata, Y. In The Alkaloids; Manske, R. H. F., Ed., Academic Press: New
York, 1975; Vol. 15, p 41. (b) Yamamura, S. In The Alkaloids; Brossi, A.,
Ed.; Academic Press: New York, 1986, Vol. 29, p 265.
(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. (d) Yamamura, S.; Lamberton, J. A.; Irikawa, H.; Okumura, Y.; Hirata,
Y. Chem. Lett. 1975, 923-926. (e) Yamamura, S.; Irikawa, H.; Okumura,
Y.; Hirata, Y. Bull. Chem. Soc. Jpn. 1975, 48, 2120-2123. (f) Yamamura,
S.; Hirata, Y. Tetrahedron Lett. 1974, 42, 3673-3676. (g) Yamamura, S.;
Sasaki, K.; Toka, M.; Hirata, Y. Tetrahedron Lett. 1974, 2023-2026 and
references therein.
(3) (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.; Joe, 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. Angew. Chem. 1992, 104, 675-691. (f)
Heathcock, C. H. Angew. Chem., Int. Ed. Engl. 1992, 31, 665-681 and
references therein.
(4) Morita, H.; Yoshida, N.; Kobayashi, J. J. Org. Chem. 1999, 64, 7208-
(7) In the previous study, daphnilactone B, daphniteijsmanine, daph-
nijsmine, desacetyldaphnijsmine, and zwitterionic alkaloid have been isolated
from the seeds of D. teijsmanni. (a) Niwa, H.; Toda, M.; Hirata, Y.;
Yamamura, S. Tetrahedron Lett. 1972, 2697-2700. (b) Yamamura, S.;
Hirata, Y. Tetrahedron Lett. 1974, 2849-2852. (c) Yamamura, S.; Hirata,
Y. Tetrahedron Lett. 1974, 3673-3676. (d) Yamamura, S.; Toda, M.; Hirata,
Y. Bull. Chem. Soc. Jpn. 1976, 49, 839.
7212.
(5) Morita, H.; Yoshida, N.; Kobayashi, J. J. Org. Chem. 2000, 65, 3558-
3562.
(6) (a) Morita, H.; Yoshida, N.; Kobayashi, J. Tetrahedron 1999, 55,
12549-12556. (b) Morita, H.; Yoshida, N.; Kobayashi, J. Tetrahedron 2000,
56, 2641-2646.
10.1021/ja016955e CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/27/2001

Isolation and Characterization of Daphnicyclidins
J. Am. Chem. Soc., Vol. 123, No. 46, 2001 11403
(δC 65.0) and C-19 (δC 52.1) and for H-4 of C-19 gave rise to
the connectivity of partial structures a and b through a nitrogen
atom (N-1). In addition, HMBC correlations for H-4 to C-5 and
C-21, H3-21 to C-5 and C-6, and Hb-7 and Hb-12 to C-5
suggested that partial structures a and b were also connected
through a quaternary carbon (C-5, δC 50.5) with a methyl group
(C-21). HMBC correlations for H-2 and H2-3 to C-1 (δC 187.0),
and H2-11 and H2-12 to C-10 (δC 202.8) indicative of adjacent
enolic carbon or ketone were observed.
The X-ray crystal structure (Figure 2) of daphnicyclidin A
(1) TFA salt revealed a unique fused-hexacyclic ring system
consisting of two of each five-, six-, and seven-membered rings
containing a nitrogen atom and two methyls at C-5 and C-18,
in which an intramolecular hydrogen bond was observed
between C-1 hydroxyl proton and C-22 carbonyl oxygen.12 The
relative configurations at C-4, C-5, C-6, and C-18 were deduced
from NOESY correlations of H3-21/H-6, H3-21/H-4, H-4/H-6,
Ha-3/H3-21, Ha-7/Ha-19, H-2/H3-20, and Hb-3/H3-20 together
with a stable chair conformation of ring B13 as depicted in the
computer-generated 3D drawing (Figure 3).
Figure 1. Selected 2D NMR Correlations for Daphnicyclidin A (1).
three tetra-substituted olefins, two carbonyls, four sp3 methines,
seven sp3 methylenes, and two methyls. Among them, one
methine (δC 65.0; δH 3.74) and two methylenes (δC 59.3; δH
2.52 and 4.07, δC 52.1; δH 3.05 and 3.24) were ascribed to those
bearing a nitrogen.
Three partial structures a (from C-2 to C-4 and from C-18 to
C-19 and C-20), b (from C-6 to C-7 and C-12 and from C-11
to C-12), and c (from C-16 to C-17) were deduced from
extensive analyses of 2D NMR data of 1 including the 1H-1H
COSY, HOHAHA,9 HMQC, and HMBC10,11 spectra in CDCl3-
CD3OD (9:1) (Figure 1). HMBC correlations for Ha-7 of C-4
(10) (a) Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093-
2094. (b) Bax, A.; Aszalos, A.; Dinya, Z.; Sudo, K. J. Am. Chem. Soc.
1986, 108, 8056-8063.
(11) The HMBC correlations of 1: H-2/C-1 and C-13, H-3/C-5, H-4/
C-8, C-21, C-5, and C-19, H3-21/C-5, C-4, C-6, and C-8, H-7/C-5, C-4,
and C-19, H-19/C-7 and C-4, H-11, and H-12/C-10, H-12/C-5, H-16/C-15
and C-14, and H-17/C-15 and C-22.
(12) Distance and angle of the intramolecular hydrogen bond are
followed: O1-H‚‚‚O2, 1.377 Å, 166.322°.
(13) The piperidine ring (ring B) in the X-ray structure adopts a chairlike
conformation: internal torsion angles in the ring B are -46.0(4), -52.1-
(4), -55.1(4), 45.9(4), 55.4(4), and 51.7(5)°.
(8) The 13C NMR signals (C-1, C-8, C-9, C-13, C-14, C-15, and C-22)
around ring E in 1 were detected by setting delay time to 8 s. These NMR
anomalies seem to be due to long relaxation time around this ring.
(9) Edwards, M. W.; Bax, A. J. Am. Chem. Soc. 1986, 108, 918-923.

11404 J. Am. Chem. Soc., Vol. 123, No. 46, 2001
Kobayashi et al.
Table 1. 1H NMR Data (δH) of Daphnicyclidins A-H (1-8) at 300 K
proton
1a
2c
3c
4a
2
2.87 (1H, brs)
3.13 (1H, brs)
3.69 (1H, dd, 2.4, 13.8)
2.77 (1H, m)
2.58 (1H, brs)
3a
2.26 (1H, d, 15.8)
2.45 (1H, dt, 15.8, 6.0)
3.74 (1H, d, 6.7)
2.64 (1H, m)
2.45 (2H, m)
2.12 (1H, d, 15.4)
2.40 (1H, m)
3b
4
6
7a
7b
11a
11b
12a
12b
16a
16b
17a
17b
18
3.95 (1H, d, 7.2)
2.81 (1H, m)
2.81 (1H, m)
4.11 (1H, m)
2.66 (1H, dd, 3.3, 19.7)
2.74 (1H, dd, 13.0, 19.7)
1.79 (1H, m)
2.19 (1H, m)
2.81 (1H, m)
3.28 (1H, ddd, 5.7, 9.2, 18.1)
4.46 (1H, m)
4.59 (1H, m)
2.24 (1H, m)
3.36 (1H, dd, 2.9, 13.2)
3.47 (1H, brd, 13.2)
1.32 (3H, d, 7.3)
1.50 (3H, s)
3.77 (1H, m)
2.97 (1H, m)
2.58 (1H, m)
2.52 (1H, dd, 8.7, 12.2)
4.07 (1H, brt, 11.3)
2.61 (2H, m)
3.61 (1H, brt, 11.4)
4.43 (1H, dd, 8.7, 13.6)
2.57 (1H, dd, 6.6, 18.0)
2.79 (1H, m)
2.53 (1H, m)
3.96 (1H, m)
2.60 (1H, dd, 5.5, 18.6)
2.73 (1H, dt, 13.0, 18.6)
1.46 (1H, m)
1.67 (1H, m)
2.11 (1H, m)
2.69 (1H, dt, 18.8, 5.1)
3.27 (1H, m)
4.38 (1H, dt, 4.7, 9.8)
4.51 (1H, m)
2.24 (1H, m)
3.05 (1H, dd, 3.4, 12.8)
3.24 (1H, brd, 13.3)
1.32 (3H, d, 7.3)
1.42 (3H, s)
1.55 (1H, m)
2.18 (1H, m)
2.24 (1H, m)
2.81 (1H, m)
2.67 (1H, dd, 4.9, 17.0)
2.96 (1H, dd, 3.0, 17.0)
4.04 (1H, ddd, 3.0, 11.0, 14.5)
4.57 (1H, dd, 5.4, 11.0)
2.24 (1H, m)
2.89 (1H, ddd, 5.0, 13.8, 18.3)
4.20 (1H, ddd, 3.7, 10.5, 13.8)
4.39 (1H, dd, 5.0, 10.5)
2.56 (1H, m)
19a
19b
20
4.10 (1H, dd, 7.7, 14.2)
3.32 (1H, dd, 9.1, 14.2)
1.33 (3H, d, 6.9)
3.13 (2H, m)
1.17 (3H, d, 6.7)
1.34 (3H, s)
3.66 (3H, s)
8b
2.54 (1H, m)
2.35 (2H, m)
21
1.82 (3H, s)
22-OMe
proton
2
5c
6a
7c
3.17 (1H, brs)
3.72 (1H, m)
2.90 (1H, m)
2.36 (1H, d, 15.5)
2.43 (1H, dd, 7.1, 15.5)
3.93 (1H, d, 7.0)
2.77 (1H, m)
2.88 (1H, dd, 9.2, 12.6)
4.00 (1H, dd, 10.1, 12.6)
2.71 (1H, dd, 5.5, 18.5)
2.94 (1H, dd, 12.2, 18.5)
1.65 (1H, m)
2.35 (1H, m)
6.51 (1H, br s)
2.81 (1H, m)
2.83 (1H, m)
4.16 (1H, ddd, 5.9, 10.8, 11.2)
4.68 (1H, ddd, 1.8, 4.8, 10.8)
2.07 (1H, m)
3.44 (1H, br d, 11.4)
3.37 (1H, dd, 4.5, 13.3)
1.21 (3H, d, 7.1)
1.49 (3H, s)
3a
3b
4
6
7a
7b
11a
11b
12a
12b
16a
16b
17a
17b
18
19a
19b
20
21
22-OMe
2.06 (1H, d, 15.7)
2.50 (1H, brd, 15.7)
4.05 (1H, m)
3.58 (1H, t, 3.8)
2.61 (1H, m)
2.77 (1H, m)
3.08 (1H, m)
3.76 (1H, m)
2.73 (1H, m)
2.57 (1H, m)
4.52 (1H, dd, 8.8, 13.7)
2.77 (1H, dd, 6.4, 18.9)
3.12 (1H, m)
4.01 (1H, m)
3.97 (1H, dd, 10.7, 12.3)
2.58 (1H, m)
2.68 (1H, m)
2.85 (1H, m)
2.63 (1H, m)
1.49 (1H, m)
1.51 (1H, m)
1.84 (1H, m)
2.42 (1H, m)
2.35 (1H, m)
2.10 (1H, m)
2.91 (1H, m)
2.76 (1H, m)
2.76 (1H, m)
3.00 (1H, dd, 4.0, 18.7)
4.27 (1H, ddd, 4.0, 10.9, 15.0)
4.80 (1H, dd, 5.3, 10.9)
2.49 (1H, m)
3.19 (1H, dd, 3.0, 17.6)
4.15 (1H, ddd, 3.0, 11.0, 14.9)
4.67 (1H, dd, 5.3, 11.0)
2.29 (1H, m)
2.84 (1H, m)
3.70 (2H, m)
2.26 (1H, m)
4.11 (1H, dd, 7.1, 14.0)
3.41 (1H, dd, 9.5, 14.0)
1.33 (3H, d, 6.8)
1.84 (3H, s)
3.28 (1H, m)
3.36 (1H, m)
1.15 (3H, d, 6.7)
1.41 (3H, s)
3.76 (3H, s)
3.20 (1H, dd, 1.6, 13.0)
3.62 (1H, dd, 4.1, 13.0)
1.29 (3H, d, 7.4)
1.45 (3H, s)
3.73 (3H, s)
3.72 (3H, s)
a CDCl3/CD3OD (9:1). b CDCl3/CD3OD (1:9). c CD3OD
Table 2. 13C NMR Data (δC) of Daphnicyclidins A-H (1-8) at
300 K
carbon
1a
2c
3c
4a
5c
6a
7c
8b
1
2
3
4
5
6
7
8
9
187.0 199.1 190.9 197.4 197.3 196.7 199.0 196.9
43.3 50.4 73.6 47.3
16.8 27.2 25.6 16.8
54.0 72.9 74.3 48.1
26.6 26.2 25.8 18.0
65.0 200.8 69.5 65.3 197.5 66.7 69.7 68.8
50.5 60.9 50.8 51.2
47.9 46.3 49.5 47.4
59.3 65.2 60.5 59.1
61.1 51.0 52.2 51.3
45.6 47.6 49.0 49.5
65.0 58.5 60.1 61.2
146.7 135.6 145.9 137.3 134.4 138.9 140.7 132.7
132.6 123.1 131.8 120.8 120.3 120.9 124.5 126.9
202.8 203.2 204.9 180.6 184.9 181.5 180.9 204.5
10
11
39.0 40.8 40.2 31.0
27.1 27.5 28.2 29.7
32.1 31.0 32.0 40.5
29.3 29.1 31.2 29.5
12
13
117.4 122.8 118.8 134.4 133.8 131.1 134.0 123.4
113.1 113.9 114.0 122.9 122.5 123.1 118.4 123.1
149.3 145.0 149.9 130.2 133.5 131.7 127.6 131.6
14
15
16
22.8 26.2 24.6 22.4
68.7 69.6 70.0 69.1
29.8 36.9 36.3 27.7
52.1 54.2 56.9 52.4
16.1 19.3 11.8 16.9
34.8 28.9 34.9 33.3
24.4 22.6 24.5 30.8
71.7 69.3 71.2 65.3
36.4 32.9 35.6 30.4
54.0 52.8 55.6 55.2
18.7 12.0 12.2 17.1
27.0 32.9 34.4 36.1
17
18
19
20
21
22
169.7 168.9 171.1 167.3 167.9 166.7
51.5 52.1 51.8
174.2
52.1
22-OMe
a CDCl3/CD3OD (9:1). b CDCl3/CD3OD (1:9). c CD3OD
Figure 2. Molecular structure of daphnicyclidin A (1) TFA salt
obtained by X-ray analysis (ORTEP drawing; ellipsoids are drawn at
the 30% probability level).
The FABMS spectrum of daphnicyclidin B (2) showed the
molecular ion peak at m/z 366 (M+), and the molecular formula
was inferred as C22H24NO4 by HRFABMS [m/z 366.1702 (M+),
∆ -0.3 mmu]. The IR spectrum was indicative of the presence
of OH (3440 cm-1), conjugated carbonyl and imine (1680 and
1630 cm-1) functionalities. 13C NMR data (Table 2) including

Isolation and Characterization of Daphnicyclidins
J. Am. Chem. Soc., Vol. 123, No. 46, 2001 11405
Figure 5. Selected 2D NMR correlations for daphnicyclidin D (4).
was indicated to be C22H25NO5 by HRFABMS [m/z 384.1821,
(M + H)+, ∆ +1.0 mmu], which was larger than that of
daphnicyclidin A (1) by one oxygen atom. The 1H and 13C NMR
(Tables 1 and 2) spectra of daphnicyclidin C (3) were almost
the same as those of daphnicyclidin A (1), except for the
downfield shift of the C-2 carbon (δC 73.6) and the lack of a
methine signal assignable to H-2. HMBC correlations for H2-
2, H-4, H3-20, and Hb-19 to C-2, and extensive 2D NMR data
indicated that daphnicyclidin C (3) was the 2-hydroxy form of
daphnicyclidin A (1).
Figure 3. Selected NOESY correlations (dotted arrows) and relative
configurations for daphnicyclidin A (1).
Structural Elucidation of Daphnicyclidins D-G (4-7).
HRFABMS data [m/z 382.2029, (M + H)+, ∆ +1.1 mmu] of
daphnicyclidin D (4) established the molecular formula to be
C23H27NO4, which was larger than that of daphnicyclidin A (1)
1
by a CH2 unit. H and 13C NMR data (Tables 1 and 2) of 4
were analogous to those of 1 in the left-hand part, consisting
of rings A to C with a nitrogen atom, except that a methoxy
signal (δH 3.76) lacking for 1 was observed for 4. The structure
of 4 was elucidated by 2D NMR (1H-1H COSY, HOHAHA,
HMQC, and HMBC) data. HMBC cross-peaks of H2-11 and
Hb-17 to C-10 (δC 180.6) indicated that C-10 and C-17 (δC 69.1)
were connected to each other through an oxygen atom to form
a dihydropyran ring (Figure 5). The presence of a methoxy group
at C-22 was suggested by the HMBC correlation for H3-OCH3
to C-22 (δC 167.3). In addition, the presence of a conjugated
cyclopentadiene moiety (C-8∼C-9 and C-13∼C-15) like 1 was
suggested by HMBC correlations for H2-16 to C-9, C-14, and
C-15, Hb-17 to C-15, and Ha-11 to C-9. Treatment of 1 with
methanolic p-TsOH gave daphnicyclidin D (4) (Scheme 1).
Thus, the structure of daphnicyclidin D was assigned as 4.
HRFABMS data [m/z 380.1853, (M)+, ∆ -0.9 mmu] of
daphnicyclidin E (5) established the molecular formula, C23H25-
NO4. The IR spectrum was indicative of the presence of
conjugated carbonyl and/or imine (1680 cm-1) functionalities.
1H and 13C NMR data (Tables 1 and 2) of 5 were analogous to
those of daphnicyclidin D (4). The presence of an iminium
carbon [C-4, δC 197.5 (s)] was elucidated by HMBC correlations
for H3-21 and Hb-3 to C-4, and H2-7 and Ha-19 to C-4 through
a nitrogen atom. Treatment of 5 with NaBH4 afforded daphni-
cyclidin D (4) (Scheme 1). Thus, daphnicyclidin E (5) was
concluded to be the imine form at C-4 of daphnicyclidin D (4).
Daphnicyclidin F (6) had the molecular formula of C23H27-
NO5 as revealed by HRFABMS data [m/z 398.1950, (M + H)+,
∆ -1.7 mmu]. IR absorptions at 3430 and 1680 cm-1 indicated
Figure 4. Key HMBC (arrows) and NOESY correlations (dotted
arrows) for daphnicyclidin B (2).
DEPT experiments revealed that the chemical shift [δC 200.8
(s)] of C-4 was remarkably shifted to lower field as compared
with that [δC 65.0 (d)] of 1. The presence of an iminium carbon14
(C-4) was elucidated by HMBC correlations for H3-21 and Hb-3
to C-4, and H2-7 and Ha-19 to C-4 though a nitrogen atom
(Figure 4). In addition, the 1H and 13C signals at 3-, 5-, 7-, and
19-positions around the imine functionality were observed at
lower field due to deshielding effects (Tables 1 and 2). 2D NMR
data of 2 including the 1H-1H COSY, HOHAHA, HMQC, and
HMBC15 spectra corroborated well with those of the imine (C-4
and N-1) form of 1. The relative stereochemistry and ring
conformation16 of 2 elucidated by NOESY correlations (Figure
4) were almost same as those of 1. Thus, daphnicyclidin B (2)
was concluded to be the imine (C-4 and N-1) form of
daphnicyclidin A (1).
The FABMS spectrum of daphnicyclidin C (3) showed the
pseudomolecular ion peak at m/z 384, and the molecular formula
(14) Iminium carbons have been observed at ca. 200 ppm. Some
examples; Uemura, D.; Chou, T.; Haino, T.; Nagatsu, A.; Fukuzawa, S.;
Zheng, S.-z.; Chem, H.-s. J. Am. Chem. Soc. 1995, 117, 1155-1156: Chou,
T.; Haino, T.; Kuramoto, M.; Uemura, D. Tetrahedron Lett. 1996, 37, 4027-
4030.
(15) The HMBC correlations of 2: H-2 and H-3/C-1, H-3, H-19, H-7,
and H3-21/C-4, H-7, H3-21, and H-12/C-5, H3-21/C-6, H-6 and H3-21/C-8,
H-11 and H-12/C-10, H-16 and H-17/C-15, and H-17/C-22.
(16) Conformational search and molecular mechanics calculations were
conducted by the Macromodel program: Mohamadi, F.; Richards, N. G.
J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield, C.; Chang, G.;
Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440-467. All
conformers took a bort form in the ring A.

11406 J. Am. Chem. Soc., Vol. 123, No. 46, 2001
Kobayashi et al.
Scheme 1. Chemical Correlations for Daphnicyclidins A (1) and D-H (4-8)
Figure 8. Two representative stable conformers (A and B) of
daphnicyclidin B (2) analyzed by Monte Carlo simulation followed by
minimization and clustering analysis. Conformer A indicated by a
NOESY correlation of Hâ-11/H3-21 in solution is energetically stabler
than conformer B (more than 17 kJ/mol). Bold lines denote the electric
transition dipole of the chromophore. The alternative conformation
would provide the same positive chirality.
Figure 6. Stereostructure of p-bromo benzoate (10) of daphnicyclidin
F (6). Arrows denote the electric transition dipole of the chromophore.
Figure 9. Two representative stable conformers (C and D) of
daphnicyclidin D (4) analyzed by Monte Carlo simulation followed
by minimization and clustering analysis. Conformer C is energetically
stabler than conformer D (more than 4 kJ/mol). Bold lines denote the
electric transition dipole of the chromophore.
HRFABMS data [m/z 340.1920, (M + H)+, ∆ +0.7 mmu]
of daphnicyclidin G (7) revealed the molecular formula, C21H25-
NO3, which was smaller than that of daphnicyclidin F (6) by a
1
C2H2O2 unit. Though H and 13C NMR data (Tables 1 and 2)
were close to those of daphnicyclidin F (6), differences were
observed for the methoxy carbonyl moiety. Daphnicyclidin F
1
(6) possessed a methoxy carbonyl moiety at C-14, while H
and 13C NMR data of 7 showed signals due to an sp2 methine
[δH 6.51 (1H, s), δC 118.4 (d)]. Treatment of 6 with p-TsOH at
70 °C for 2 days gave daphnicyclidin G (7) (Scheme 1).
Therefore, daphnicyclidin G (7) was elucidated to be the
demethoxycarbonyl form at C-14 of daphnicyclidin F (6).
Structure of Daphnicyclidin H (8). Daphnicyclidin H (8)
had the molecular formula of C23H29NO5 as revealed by
Figure 7. CD and UV spectra of daphnicyclidins A (1), B (2), C (3),
and H (8).
the presence of hydroxyls and conjugated carbonyl groups,
respectively. 1H and 13C NMR data (Tables 1 and 2) disclosed
that 6 possessed a hydroxyl at C-2 (δC 72.9) as compared with
those of daphnicyclidin D (4). HMBC and NOESY correla-
tions17 indicated the proposed structure for 6. Treatment of 4
with iodosobenzene acetate18 for 2 days afforded daphnicyclidin
F (6) (Scheme 1). Thus, daphnicyclidin F (6) was assigned as
the 2-hydroxy form of daphnicyclidin D (4).
(17) Key HMBC and NOESY correlations of 5 are followed: HMBC
(H-3/C-1, H-3 and H3-20/C-2, H3-21/C-4, C-5, and C-6, H-11/C-6, H3-21/
C-8, H-16/C-9, H-11/C-9, C-10, and C-12, H-17/C-10, H-16/C-14, C-15,
and C-17, H-17/C-15, H-3 and H3-20/C-18, H3-20/C-19, and H-OMe/C-
22); NOESY (H-3/H3-20, H-4 and H-6/H3-21, and H-4/H-6).
(18) Moriarty, R. M.; Hu, H. Tetrahedron Lett. 1981, 22, 2747-2750.

Isolation and Characterization of Daphnicyclidins
J. Am. Chem. Soc., Vol. 123, No. 46, 2001 11407
Scheme 2. Plausible Biogenetic Path of Daphnicyclidins A-H (1-8)
observed equivalently as compared with those of 1-7. Treat-
ment of 8 with acetic anhydride afforded the monoacetate (9),
in which the hydroxyl group at C-17 was acetylated. On the
other hand, the presence of a methoxy carbonyl group at C-14
and rings A∼E with a ketone at C-10 (δC 204.5) was deduced
from 2D NMR analysis.19,20 The 2D NMR data indicated that
the conjugated keto-enol moiety of 8 was the same as that of
daphnicyclidin A (1). Treatment of daphnicyclidin H (8) with
p-TsOH gave daphnicyclidin D (4) (Scheme 1).21 Thus, the
structure of 8 was assigned as shown.
Absolute Stereochemistry of Daphnicyclidins A-H (1-
8). Absolute stereochemistry of daphnicyclidin F (6) was
analyzed by applying exciton chirality method22 after introduc-
tion of p-bromobenzoyl chromophore into the hydroxyl group
at C-2. As the sign of the first Cotton effect [λmax 280 (θ
+20000) and 225 (-16000) nm] was positive, the chirality
between the cyclopentene moiety and the benzoate group of
the p-bromobenzoyl derivative (10) of 6 was assigned as shown
(19) The HMBC correlations of 6: H-2 and H-3/C-1, H-3, H-4, and
H-19/C-2, H-3 and H-7/C-5, H-7, and H-11/C-6, H-4/C-8, H-16/C-9, H-11,
and H-12/C-10, H-6, and H-7/C-12, H-16/C-14 and C-15, H-17/C-15, H-3,
H-19, and H3-20/C-18, H-2, and H-7/C-19, H-19/C-20, and H-OMe/C-
Figure 10. CD and UV spectra of daphnicyclidins D (4), F (6), and
G (7).
HRFABMS data [m/z 400.2140, (M + H)+, ∆ +1.6 mmu]. IR
absorptions at 3435 and 1680 cm-1 indicated the presence of
hydroxyls and conjugated carbonyl groups, respectively. The
1H NMR (Table 1) signals assignable to H2-17 (δH 3.70) were
22.
(20) Acid-catalyzed chemical conversion of daphnicyclidin H (8) under
this condition afforded daphnicyclidin D (4) but not daphnicyclidin A (1).
(21) The NOESY correlations of 8: H-3 and H-2/H3-20, H-7/H-19, H-3,
H-4, and H-6/H3-21 and H-4/H-6.
(22) Harada, N.; Nakanishi, K.; Tatsuoka, S. J. Am. Chem. Soc. 1969,
91, 5896-5898.

11408 J. Am. Chem. Soc., Vol. 123, No. 46, 2001
Kobayashi et al.
in Figure 6 (right-handed screw), indicating the absolute
stereochemistry at C-2 was S.
alkaloids such as yuzurimine A24 and macrodaphniphyllamine25
with an appropriate leaving group at C-4 and a methyl group at
C-21. Rings B and C might be constructed by loss of the leaving
group at C-4 followed by N-1-C-4 bond formation. Subse-
quently, cleavage of C-1-C-8 bond followed by formation of
C-1-C-13 bond would result in enlargement of ring A, and
aromatization of ring E to generate an intermediate A. Further-
more, oxidative cleavage of C-10-C-17 bond could lead to
daphnicyclidin H (8), followed by cyclization and dehydration
to produce daphnicyclidin D (4), which may be oxidized to give
daphnicyclidins E (5) and F (6). On the other hand, cyclization
of 17-OH to C-22 in 8 to form ring F would generate
daphnicyclidins A (1), B (2), and C (3).
Cytotoxicity of Daphnicyclidins A-H (1-8). Daphnicy-
clidins A-H (1-8) exhibited cytotoxicity against murine
lymphoma L1210 cells (IC50, 0.8, 0.1, 3.0, 1.7, 0.4, 4.3, 4.2,
and 0.5 µg/mL, respectively) and human epidermoid carcinoma
KB cells (IC50, 6.0, 2.6, 7.2, 4.6, 5.2, 7.6, >10, and 0.9 µg/mL,
respectively) in vitro.
The absolute stereochemistry of dapnicyclidins A (1), B (2),
C (3), D (4), G (7), and H (8) was examined by CD
spectroscopy.23 Daphnicyclidins A (1), B (2), C (3), and H (8)
with a ketone at C-10 exhibited Cotton effects centered at 290
nm corresponding to the UV maximum of a cyclopentene group
conjugated with a ketone at C-22. This transition was coupled
with the ketone transition at C-10, and consequently the CD
spectra had two Cotton effects at 320 and 285 nm (Figure 7).
The positive sign of the first Cotton effect was coincident with
the chirality between the long axis of the conjugate cyclopentene
moiety and the ketone group at C-10. This helicity was
essentially independent of their conformations as described
(Figure 8) in the case of daphnicyclidin B (2). On the other
hand, in daphnicyclidins D (4), F (6), and G (7), the two long
axes of the fulvene moiety conjugated with the ketones at C-1
and C-22 were coupled to each other. The conformational space
for 4 was searched using the MMFF force field implemented
in the Macromodel program.16 The lowest-energy conformers
belonging to two separate clusters were represented as conform-
ers C and D (Figure 9). Conformer C was energetically more
stable than conformer D (>4 kJ/mol). As the sign of the first
Cotton effect at 345 nm was negative (Figure 10), the long axis
transitions of the two chromophores coupled as depicted in
Figure 9 (negative chirality), and consequently the absolute
configurations of 4, 6, and 7 were elucidated.
Acknowledgment. We thank Mrs. S. Oka and M. Kiuchi,
Center for Instrumental Analysis, Hokkaido University, for
measurements of FABMS. This work was partly supported by
a Grant-in-Aid from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan, and a Grant from the Takeda
Science Foundation.
Supporting Information Available: Experimental proce-
dures, 1D and 2D NMR spectra for compounds 1-8, and X-ray
crystallographic data of 1 (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
Plausible Biogenesis of Daphnicyclidins A-H (1-8).
Daphnicyclidins A-G (1-7) and H (8) are novel types of
natural products consisting of fused hexa- or pentacyclic ring
system, respectively. A plausible biogenetic pathway for daph-
nicyclidins A-H (1-8) is proposed as shown in Scheme 2.
The biogenetic origin of them seems to be yuzurimine-type
JA016955E
(24) Sakurai, H.; Irikawa, H.; Yamamura, S.; Hirata, Y. Tetrahedron
Lett. 1967, 2883-2888; Irikawa, H.; Yamamura, S.; Hirata, Y. Tetrahedron
1972, 28, 3727-3738.
(23) The CD spectra of daphnicyclidins E (5) showed different Cotton
curves (5, ∆ꢀ320 -2.5 and ∆ꢀ265 +2.0) from those of daphnicyclidins D
(4), F (6), and G (7).
(25) Nakano, T.; Saeki, Y. Tetrahedron Lett. 1967, 4791-4797.