Molecular structures and antiviral activities of naturally occurring and modified cassane furanoditerpenoids and friedelane triterpenoids from Caesalpinia minax

Article abstract of DOI:10.1016/S0968-0896(02)00072-X

Further investigation of the active components of the chloroform fraction of the seeds of Caesalpinia minax led to the isolation of a new cassane furanoditerpenoid, caesalmin H (1), together with two known furanoditerpenoid lactones, caesalmin B (2) and bonducellpin D (3). Reduction of the naturally abundant caesalmin D (9), E (10) and F (11) resulted in three new furanoditerpenoid derivatives 4-6. Phytochemical study of the stem of the same plant and subsequent reduction afforded two friedelane triterpenoids (7-8), which were identified by spectroscopic methods. Compounds 1-2 and 4-8 were corroborated by single crystal X-ray analysis. The factors governing the reduction of cassane furanoditerpenoids and friedelane triterpenoids were investigated by correlating the crystallographic results with density functional theory. The inhibitory activities of 2-8 on the Para3 virus were evaluated by cytopathogenic effects (CPE) reduction assay.

Full text of DOI:10.1016/S0968-0896(02)00072-X

Bioorganic & Medicinal Chemistry 10 (2002) 2161–2170
Molecular Structures and Antiviral Activities of Naturally
Occurring and Modified Cassane Furanoditerpenoids and
Friedelane Triterpenoids from Caesalpinia minax
a
b
b
c
Ren-Wang Jiang, Shuang-Cheng Ma, Zhen-Dan He, Xue-Song Huang,
b
b
a
b
Paul Pui-Hay But, Hua Wang, Siu-Pang Chan, Vincent Eng-Choon Ooi,
b
a,
Hong-Xi Xu and Thomas C.W. Mak *
aDepartment of Chemistry & Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong SAR, PR China
bDepartment of Biology & Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong SAR, PR China
cCollege of Food Science and Engineering, Shandong Agricultural University, Taian, Shan Dong, PR China
Received 21 December 2001; accepted 9 February 2002
Abstract—Further investigation of the active components of the chloroform fraction of the seeds of Caesalpinia minax led to the isola-
tion of a new cassane furanoditerpenoid, caesalmin H (1), together with two known furanoditerpenoid lactones, caesalmin B (2) and
bonducellpin D (3). Reduction of the naturally abundant caesalmin D (9), E (10) and F (11) resulted in three new furanoditerpenoid
derivatives 4–6. Phytochemical study of the stem of the same plant and subsequent reduction afforded two friedelane triterpenoids
(7–8), which were identified by spectroscopic methods. Compounds 1–2 and 4–8 were corroborated by single crystal X-ray analysis.
The factors governing the reduction of cassane furanoditerpenoids and friedelane triterpenoids were investigated by correlating the
crystallographic results with density functional theory. The inhibitory activities of 2–8 on the Para3 virus were evaluated by
cytopathogenic effects (CPE) reduction assay. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
furanoditerpenoid derivatives, that is, 1a,5a,14a-trihy-
droxy-6a,7b-diacetoxyvouacapane (4), 1a,5a,6a,7b,14b-
pentahydroxyvouacapane (5) and 1a,5a-dihydroxy-14b-
methoxy-6a,7b-diacetoxyvouacapane (6). In addition,
investigation of the chemical components of the stem of
C. minax led to the isolation of a friedelane triterpenoid,
Natural products have proven to be a rich source of
biologically active materials and potentially useful lead
1
to drug development. Motivated by the severe health
hazards worldwide caused by respiratory infections, and
the pronounced resistance from some pathogens to
7
that is, friedelin (7), and a reduction experiment was
8
2
currently administered therapeutic agents, we started a
program to develop antiviral agents from natural sources.
Following a lead based on ethanomedical usage, we
investigated the bioactive components of the seeds of
Caesalpinia minax Hance. The results showed that the
tetracyclic furanoditerpenoids (caesalmin C, D, E and F)
from the brown precipitate after defatting with hexane
performed to afford epifriedelinol (8) (Fig. 1). We
report herein the structure elucidation of the above
compounds and their antiviral activities against Para3
3
9
virus, a major pathogen of some respiratory infections.
Detailed knowledge about the molecular geometry is
often necessary for relating biological activity to structure,
exploring potential clinical and chemical uses, and as a
starting point for further explorations using molecular
modeling. Thus X-ray crystallographic studies on com-
pounds 1–2 and 4–8 were performed for both molecular
structure determination and further optimization.
possessed potent anti-para3 (para-influenza virus type
4
3
) activity.
Further investigation of the chloroform fraction resulted
in the isolation of a new furanoditerpenoid, named cae-
5
salmin H (1), together with caesalmin B (2) and bon-
6
ducellpin D (3). Reduction of the naturally abundant
4
Results and Discussion
caesalmin D (9), E (10) and F (11) yielded three new
High-resolution liquid secondary ion mass spectrometry
(HRLSIMS) of (1) suggested the molecular formula
C H O which agrees well with the molecular ion m/z
*Corresponding author. Tel.: +852-2609-6279; fax: +852-2603-5057;
e-mail: tcwmak@cuhk.edu.hk
2
2
32
5
0
968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00072-X

2162
R.-W. Jiang et al. / Bioorg. Med. Chem. 10 (2002) 2161–2170
376 obtained from EIMS. A color change to reddish
purple with the Ehrlich reagent, in combination with a
+
fragment ion peak at m/z 108 [C H O] derivable from
7
8
characteristic reverse Diels–Alder cleavage of the furan
moiety, indicated a furanoid structure, which was also
substantiated by a pair of doublets at d 6.22 (1H, d,
J=1.0 Hz, H-15) and d 7.24 (1H, d, J=1.0 Hz, H-16)
2
and four sp carbon atoms at d 148.25 (C-12, s), 122.04
1
3
(C-13, s), 109.68 (C-15, d) and 140.60 (C-16, d). The
C
NMR resonance at d 168.82 (C-21, s) and the infrared
ꢀ
1
peak at 1740 cm revealed the presence of a carbonyl
group (Tables 1 and 2). Accordingly, four of the seven
degrees of unsaturation suggested by the molecular for-
mula can be ascribed to the furan ring and the carbonyl
group, and the three remaining degrees of unsaturation
account for the tricarbocyclic system. Thus 1 was inferred
to possess a cassane-type furanoditerpenoid skeleton.
The 13C and DEPTNMR spectra, indicating the pre-
3
sence of 17 sp carbon atoms (5 methyls, 4 methylenes, 5
2
methines and 3 quaternary carbons), four sp carbons (2
methines and 2 quaternary carbons) and a carbonyl
group, confirmed the molecular skeleton of 1. The pre-
sence of the carbonyl group and an oxygenated carbon
at d 75.82 (C-1, d) can be attributed to an acetate group,
which is supported by the methyl signal d 2.10 (3H, s,
H -22), and its position at C-1 was confirmed by the
3
HMBC spectrum which showed that H-1 (d 4.91, br. s)
is correlated to C-2, C-3, C-5, C-10, C-20 and the ace-
tate carbonyl attached to C-1. The other two C–O sig-
nals [d 78.59 (C-5, s) and 67.79 (C-7, d)] must be due to
hydroxyl groups which were supported by the absorp-
ꢀ
1
tion band (3586 cm ) in the IR spectrum. Normally, all
furanoditerpenoids isolated from Caesalpinia genus so
far possess an a-oriented hydroxyl group at C-5, and
thus the presence of 5-OH can be speculated from the
biogenetic view point. Another 7-OH group was con-
firmed by the HMBC spectrum which showed that H-7
Figure 1. Structural formulae of some cassane furanoditerpenes and
friedelane triterpenoids.
Table 1. 1H NMR data of compounds 1, 4, 5 and 6 (300 MHz, in CD
3
OD)
Position
1
4
5
6
1
2
4.91, 1H, br s
1.64, 1H, m, 2-H
1.92, 1H, m, 2-H
1.41, 1H, m, 3-H
3.77, 1H, br s
1.63, 1H, m, 2-H
1.89, 1H, m, 2-H
1.01, 1H, m, 3-H
1.65, 1H, m, 3-H
5.49, 1H, d, 8.4
3.66, 1H, br s
1.59, 1H, m, 2-H
1.92, 1H, m, 2-H
1.00, 1H, m, 3-H
1.61, 1H, m, 3-H
3.82, 1H, d, 9.0
3.66, 1H, br s
1.64, 1H, m, 2-Ha
a
b
a
b
a
a
b
a
b
b
a
b
b
1.93, 1H, m, 2-H
3
1.05, 1H, m, 3-Ha
1.68, 1H, m, 3-Hb
5.44, 1H, d, 8.1
1.96, 1H, m, 3-H
1.76, 2H, m
6
7
8
9
4.71, 1H, dt, 5.3, 12.9
2.13, 1H, ddd, 4.2, 10.4, 12.9
2.67, 1H, ddd, 5.1, 8.1, 10.4
2.28, 1H, dd, 5.1, 16.4, 11- H
2.44, 1H, dd, 8.1, 16.4, 11- H
3.11, 1H, m
5.86, 1H, dd, 8.4, 8.8
2.09, 1H, m
3.17, 1H, dt, 5.1, 11.5
2.78, 1H, dd, 5.1, 15.9, 11-H
2.42, 1H, dd, 11.5, 15.9, 11-H
4.17, 1H, dd, 9.0, 10.5
2.02, 1H, m
2.85, 1H, ddd, 4.5, 6.3, 10.2
2.64, 1H, dd, 6.3, 16.3, 11-H
2.45, 1H, dd, 10.8, 16.2, 11-H
5.64, 1H, dd, 8.1, 8.7
2.08, 1H, m
2.97, 1H, ddd, 5.7, 7.8, 11.1
2.74, 1H, dd, 5.7, 15.8, 11-Ha
2.46, 1H, dd, 11.1, 15.8, 11-Hb
a
a
a
1
1
b
b
b
1
1
1
1
1
1
2
1
6
7
1
4
5
6
7
8
9
0
6.22, 1H, d, 1.0
7.24, 1H, d, 1.0
1.11, 3H, d, 5.4
1.12, 3H, s
1.14, 3H, s
1.07, 3H, s
2.10, 3H, s
6.42, 1H, d, 2.0
7.29, 1H, d, 2.0
1.41, 3H, s
1.14, 3H, s
1.08, 3H, s
1.17, 3H, s
6.40, 1H, d, 2.0
7.29, 1H, d 2.0
1.47, 3H, s
1.24, 3H, s
1.08, 3H, s
1.28, 3H, s
6.23, 1H, d, 1.6
7.24, 1H, d 1.6
1.49, 3H, s
1.15, 3H, s
1.09, 3H, s
1.19, 3H, s
-OAc
-OAc
-OAc
4-OCH
1.96, 3H, s
2.03, 3H, s
2.00, 3H, s
2.05, 3H, s
3.04, 3H, s
3

R.-W. Jiang et al. / Bioorg. Med. Chem. 10 (2002) 2161–2170
2163
Table 2. 13C NMR data of compounds 1, 4, 5 and 6 (75 MHz, in
CD OD)
3
Position
1
4
5
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
1
75.82 d
21.56 t
30.03 t
38.50 s
78.59 s
22.55 t
67.79 d
42.56 d
35.37 d
43.47 s
22.07 t
148.25 s
122.04 s
31.93 d
109.68 d
140.60 d
28.18 q
27.42 q
25.28 q
17.17 q
168.82 s
68.60 d
22.40 t
32.45 t
38.89 s
80.14 s
72.52 d
72.52 d
33.93 d
33.93 d
44.32 s
25.49 t
150.94 s
123.58 s
77.10 s
107.98 d
150.94 d
30.56 q
27.80 q
24.62 q
16.44 q
72.68 d
22.04 t
32.47 t
39.14 s
80.69 s
74.38 d
75.57 d
35.12 d
35.12 d
44.70 s
25.59 t
149.35 s
124.28 s
73.28 s
107.52 d
141.75 d
31.23 q
25.58 q
24.77 q
16.40 q
73.18 d
23.19 t
32.56 t
38.94 s
80.91 s
73.77 d
76.23 d
38.22 d
35.69 d
45.12 s
23.76 t
151.80 s
122.10 s
78.78 s
108.37 d
142.33 d
31.10 q
25.93 q
25.35 q
17.47 q
0
1
2
3
4
5
6
7
8
9
0
-OAc
17.84 q
6
-OAc
-OAc
171.78 s
170.74 s
21.97 q
171.68 s
22.37 q
2
1.05 q
171.78 s
1.05 q
7
2
13C multiplicities were determined using the DEPTpulse sequences.
(
d 4.71, dt, J=5.3, 12.9 Hz) is correlated to C-5, C-6, C-8,
C-9 and C-14. The 17-methyl appeared as a doublet at d
.11 (3H, d, J=5.4 Hz) which indicated that C-14 is a
non-oxygenated carbon as found in caesaldekarin A (12,
1
1
0
Fig. 1), which was isolated from Caesalpinia major.
Compound 1 is the first tetracyclic furanoditerpenoid
from C. minax that possesses a 17-methyl attached to a
non-oxygenated C-14.
Figure 2. (a) Molecular structure of 1 with atom labeling scheme. The
C and O atoms are drawn as 30% thermal ellipsoids. (b) Perspective
view showing the asymmetric unit of 2(1).H O, including hydrogen
2
atoms involved in hydrogen bonds, which are represented by dashed
lines. Two independent molecules of 1 (designated as I and II) forming
a hydrogen-bonded dimer.
The molecular structure and relative configuration of 1
were established unambiguously by X-ray crystallo-
graphic analysis as 5a,7b-dihydroxy-1a-acetoxyvouaca-
pane (Fig. 2a). The asymmetric unit consists of two
independent molecules of 1 (designated as I and II) and
a water molecule disordered over two sites (occupancy
Cassane furanoditerpenoids, also named ‘vouacapane’,
are characterized by the fusion of the tricarbocyclic ring
with a furan ring. Such a nucleus is mostly distributed in
1
2
13
0
.3 and 0.7). The two independent molecules are con-
the genera Caesalpinia and Pterodon of the same
1
4
nected by a pair of hydrogen bonds involving the hydroxyl
group at C-7 as both hydrogen-bond donor and acceptor,
family (Fabaceae), and differentiated by the presence
of a C-5 oxygenated group in the former genus. Exami-
nation of the previously reported cassane furanoditer-
penoids revealed three common structural features: (i) the
three six-membered rings A, B and C are fused as a
trans–anti–trans system; (ii) the substituents at C-1, C-5
and C-10 occupy axial positions and the substituents at
C-6 and C-7 are equatorial; (iii) the hydroxyl group at
C-5 forms an intramolecular hydrogen bond with the
oxygenated group at C-1. Thus cassane furanoditer-
penoids possess a highly stable and conserved skeleton.
0
2.920 A). The two water sites are linked to compound
˚
0
that is, O-4–Hꢁ ꢁ ꢁO-4 (2.920 A) and O-4 –Hꢁ ꢁ ꢁO-4
˚
(
(
0
˚
1) by hydrogen bonds O-1wꢁ ꢁ ꢁO-4 (2.889 A, x, 1+y, z)
0
˚
and O-2wꢁ ꢁ ꢁO-2 (3.045 A, 0.5+x, 1.5ꢀy,ꢀz) (Fig. 2b),
respectively. The two independent molecules take the
same conformation except that the acetate group exhi-
bits larger thermal motion in II than in I (the displace-
ments of O-2, C-21 and C-22 for I are 0.086, 0.051 and
0
respectively), indicating a significant degree of libra-
.071, respectively, and for II are 0.123, 0.103 and 0.120,
1
5
The absolute configurations of e-caesalpin and com-
pound 12, which share the same carbon skeleton with
1
1
10
tional disorder (Table S1, see supplementary mate-
rial). The thermal motion of the acetate group led to
slightly different orientations as indicated by the torsion
compound 1, were well established by X-ray analysis
using the anomalous dispersion method and comparison
of the NMR data of MTPA derivatives, respectively.
Accordingly, the absolute configuration of (1) can be
inferred by considering the biogenetic relationship in the
furanoditerpenoids, as shown in Figure 1.
ꢂ
ꢂ
angle of C-1–O-1–C-21–O-2 (ꢀ4.7 for I and 2.0 for
II). Cyclohexane rings A and B adopt a chair con-
formation, while ring C exists as a twisted half-chair due
to fusion to the planar furan ring D.

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R.-W. Jiang et al. / Bioorg. Med. Chem. 10 (2002) 2161–2170
Caesalmin B (2) was reported previously as its (2/1)
solvate with acetone (triclinic, space group P1) crystal-
lized from a mixture of hexane/acetone (8:1). Interest-
ingly, it was also obtained here as a (3/1) hexane solvate
the acetyl group at C-1 (9: d : 2.03, 3H, s and d :
H
C
169.88, s) and the high-field nature of H-1 (4: d 3.77; 9:
H
5
dH 4.88). This information indicates that the acetyl
group at C-1 had been reduced. The molecular structure
of 4 was confirmed by X-ray analysis as 1a,5a,14a-tri-
hydroxy-6a,7b-diacetoxyvouacapane (Fig. 4a). In the
solid state, the intermolecular hydrogen bond O-1–
(
orthorhombic, space group P2 2 2 ) from a different
1 1 1
mixing ratio of the same solvents (hexane/acetone 20:1).
The asymmetric unit consists of three independent
molecules of 2 and a hexane molecule (Fig. 3). The
conformations of the three molecules closely resemble
one another and that in the triclinic form, that is, the A,
B, C, D and E rings adopt chair, chair, twist-chair, planar
and envelope conformations, respectively (Table S2, see
supplementary material). The hexane molecules are
arranged in an infinite chain along the a-axis. The
˚
Hꢁ ꢁ ꢁO-4 (x, yꢀ1, z, 2.873 A) links the molecules into a
chain along the b-axis. Adjacent chains are cross-linked
by another intermolecular hydrogen bond O-7—Hꢁ ꢁ ꢁ
˚
O-6 (1ꢀx, yꢀ0.5,ꢀz, 2.920 A) (Fig. 4b).
0
shortest intermolecular contact with (2) being C-6 ꢁ ꢁ ꢁ
˚
O-3 (3.71 A).
Pseudopolymorphism occurs when a compound co-crys-
tallizes with different solvents or the same solvent but in
different stoichiometric ratios.16 Such phenomenon is
1
7
common in host–guest systems and also observed
among natural products; for example, compound 9 has
been found to crystallize as a methanol solvate and a
hydrate, and stigmasterol forms both a monohydrate
4
and a hemihydrate. Compound 2 represents a rare
example of pseudopolymorphism whereby the host
selectively encloses different guests when the molar ratio
of the two-component mixed solvent system is varied.
The phenomenon of peseudopolymorphism highlights
the role of solvent control in the organic molecular
packing arrangement.
Bonducellpin D (3) was obtained as colorless needle-like
crystals and identified as a cassane furanoditerpenoid
lactone by comparing its physical and NMR data with
6
the literature values. Compound (3) had already been
1
2b
isolated from Caesalpinia bonduc;
however, it was
isolated from C. minax for the first time and it is the
only furanoditerpenoid isolated from both plants.
Attempts to obtain diffraction quality crystals of 3 were
unsuccessful, although the crystal structures of similar
5
lactones had been reported.
Compound 4 was obtained by reduction of 9 with
1
13
LiAlH in THF. The H and C NMR spectra are
4
similar to those of 9 except for the absence of signals for
Figure 4. (a) Molecular structure of 4. The C and O atoms are drawn
as 30% thermal ellipsoids. (b) Hydrogen-bonding network of 4 viewed
roughly down the a-axis. Selected hydrogen atoms highlight the
scheme of hydrogen bonding.
.
6 14
Figure 3. Perspective view showing the asymmetric unit of 3(2) C H .
Only one of the two independent molecules of (2) and the hexane
molecule are labeled.

R.-W. Jiang et al. / Bioorg. Med. Chem. 10 (2002) 2161–2170
2165
Compound 5 was obtained by treatment of 10 with the
1
Compound 7 was isolated from the stem of C. minax
and identified as the pentacyclic friedelane triterpene
friedelin by comparison of its spectral and crystal data
3
same method as 9. T he C NMR spectra show only
twenty diterpenoid skeleton carbon atoms and no signal
1
19
for acetate carbonyl was observed. In the H NMR
with the reported values. Although friedelin (7) had
7
spectrum, signals for H-1, H-6 and H-7 were shifted to
high-field (5: dH 3.66, 3.82 and 4.17; 10: dH 4.84, 5.51
and 5.64). These data indicate that all three acetyl
groups had been reduced. The molecular structure of 5
was confirmed by X-ray analysis as 1a,5a,6a,7b,14b-
pentahydroxyvouacapane. The asymmetric unit consists
of two same molecules of 5 and a water molecule which
is located on the crystallographic 2-axis (Table S3, see
supplementary material). The two molecules in the
asymmetric unit are linked through three intermolecular
been isolated from numerous plants, it was isolated
from the present source for the first time. Reduction of
1
7 with LiAlH resulted in compound 8. In the H NMR
4
spectrum, the oxygenated methine resonates at d 3.73
(H-3, m). The width of the entire signal (8 Hz) indicates
that the hydroxyl group should be b-oriented because
the a-oriented proton H-3, adopting the equatorial
position, is split by two axial protons (H-2a and H-4a)
and an equatorial proton (H-2b). As all three dihedral
ꢂ
angles are close to 60 , the J values should be small (2–
0
˚
0
hydrogen bonds, that is O-3–Hꢁ ꢁ ꢁO-4 (2.708A), O-4 –
3 Hz). Accordingly, 8 is identified as epifriedelinol. The
molecular structure of 8 was confirmed by X-ray ana-
lysis (Fig. 7). The crystal structure of 8 was reported
˚
0
molecule is connected to 5 through an intermolecular
˚
Hꢁ ꢁ ꢁO-3 (2.708 A) and O-3 –Hꢁ ꢁ ꢁO-4 (2.665A). The water
0
˚
hydrogen bond O1w—Hꢁ ꢁ ꢁO-5 (2.856A) (Fig. 5).
Compound 6 was obtained by reduction of 11 under the
+
same conditions as 9 and 10. Compound 6 gave a [M]
peak at m/z 464 corresponding for the molecular for-
mula C H O . The absence of signals for the acetyl
2
5
36
8
group at C-1 (11: d : 2.06, 3H, s and d : 170.93) and the
H
C
high field nature of H-1 (6: dH 3.66; 11: dH 4.86) indi-
cated that the acetyl group at C-1 had been reduced to a
hydroxyl group. The molecular structure of (6) was
confirmed by X-ray analysis as 1a,5a-dihydroxy-14b-
methoxy-6a,7b-diacetoxyvouacapane.
view of the molecular structure is presented in Figure
a. In the crystalline state, the intermolecular hydrogen
A
perspective
6
˚
bond O-1—Hꢁ ꢁ ꢁO-4 (ꢀx+1, y+1/2,ꢀz+1/2, 2.817 A)
links the molecules into a chain along the b direction.
Adjacent chains are connected by a weak C–Hꢁ ꢁ ꢁO inter-
18
action (C-24—Hꢁ ꢁ ꢁO-4, symmetry, ꢀ0.5+x, 0.5ꢀy, ꢀz,
˚
˚
ꢂ
D=3.56 A, d=2.72 A and y=145.5 ). Effectively, O-4
behaves as a bifurcated acceptor (Fig. 6b).
Figure 5. An ORTEP drawing (30% thermal ellipsoids) of
.
(5) 0.5H O showing the asymmetric unit. Only atoms in one of the
two independent molecules is labeled. The intermolecular hydrogen
bonds in the asymmetric unit are represented by dashed lines.
Figure 6. (a) Molecular structure of 6. The C and O atoms are drawn
as 30% thermal ellipsoids. (b) Hydrogen-bonding network of 6 viewed
roughly down the a-axis. Selected hydrogen atoms highlight the
scheme of hydrogen bonding.
2
2

2166
R.-W. Jiang et al. / Bioorg. Med. Chem. 10 (2002) 2161–2170
Figure 7. Molecular structure of 8. The C and O atoms are drawn as
0% thermal ellipsoids.
3
previously, but with low precision (R=0.17 for 2651
2
0
8
reflections). Compound 8 also occurs in many plants;
Figure 8. DFT-based structural optimization of 4, 4a (6-OH deriva-
tive) and 4b (7-OH derivative). Molecule 4b is more stable with a
ground state energy 7.23 kJ/mol lower than 4 and 12.49 kJ/mol lower
than (4a).
however, it is a reduction derivative of 7 in this context.
The wide distribution of 7 and 8 in combination with their
low biological activity prompted a presumption that such
triterpenoids might serve as a hydrophobic coating dur-
ing plant evolution and as the precursors of the highly
2
1
oxygenated and biologically active triterpenoids.
seen that the reduction products are not the ones bearing
the lowest energy. Thus the reduction of cassane fur-
anoditerpenoids and friedelane triterpenoids by LiAlH4
are dictated by steric control rather than energetic
preference.
It is interesting to investigate the factors governing the
reduction of cassane furanoditerpenoids and friedelane
triterpenoids. The reduction of polyacetyl cassane fur-
anoditerpenoid is influenced by the steric hindrance
induced by the substituent patterns at C-14. When there
is a large substituent, for example, CH of 9 or OCH of
Anti-para3activity
3
3
1
1, at the quasi-equatorial position of ring C (the same
Compounds 2–8 were evaluated for their inhibitory
effects on the para3 virus in vitro by CPE reduction
orientation as the 7b-OAc), only the 1-OAc group will
be reduced, whereas when the substituent is small, for
example, OH of compound 10, all three acetyl groups
will be reduced. It can also be seen that the acetyl group
at C-1, occupying the axial position and possessing the
smallest steric hindrance, is more easily reduced than
those adopting the equatorial positions at C-6 and C-7.
2
5
assay according to an established procedure. (Table 3)
The fuanoditerpenoid lactones 2–3 showed moderate
activity against para3 virus and exhibited high toxicity
with TI values of 2.8 and 2.7, respectively. Reduction of
the acetoxyl group significantly decreased the activity by
comparing the IC50 values of compounds 4–6 with the
4
Normally, LiAlH reduction of unhindered cyclohexa-
4
corresponding parent furanoditerpenoids 9–11. It is
nones occurs mainly by axial attack, producing the
2
2
equatorial alcohol, whereas in the case of 7 possessing
a C-5 axial methyl group, the result is altered to produce
the axial alcohol with an equatorial attack.
Structural optimization using density functional theory
2
3
(
DFT) was used to compare the energies of 4 (reduction
product from 9), and its 6-OH (4a) and 7-OH (4b) deri-
vatives (4a and 4b were modeled from 9). The calculations
starting with X-ray coordinates were performed using the
2
4
Gaussian 98 software package (B3LYP based DFT
with 6–31G(d) basis set and double-zeta plus polariza-
tion function on a heavy atom). The results showed that
4
b is more stable with a ground state energy 7.23 kJ/mol
lower than that of 4 and 12.49 kJ/mol lower than 4a
Fig. 8). The same computation on epifriedelinol (8) and
(
friedelinol (8a, friedelin-3a-ol, modeled from 7) showed
that 8a is more stable by 2.95 kJ/mol (Fig. 9). It can be
Figure 9. DFT-based structural optimization of 8 and 8a (friedelin-3a-
ol). Molecule 8a is more stable than 8 by 2.95 kJ/mol.

R.-W. Jiang et al. / Bioorg. Med. Chem. 10 (2002) 2161–2170
2167
Table 3. Inhibitory effects of compounds 2–8 on the para3 virus
induced cytopathogenicity
Experimental
General
I
Compounds
IC50 (Mꢃ10ꢀ5)
T
50 (MꢃC10ꢀ5
)
T
Melting points were determined using a Fisher Scientific
instrument and uncorrected. The UV spectra were
obtained on a Beckman DU650 spectrophotometer in
MeOH. IR spectra were recorded on a Nicolet Impact 420
FT-IR spectrometer. EIMS were recorded on a Finnigan
MAT TS Q 7000 instrument. HRLSIMS measurements
were made on an APEX 47e FTMS spectrometer. NMR
spectra were obtained with a Bruker spectrometer oper-
2
3
4
5
6
7
5.5
4.8
5.2
6.1
15.6
12.9
37.9
48.9
120.0
42.3
41.0
24.0
2.8
2.7
7.3
8.0
18.8
3.0
3.3
24.0
6.4
14.0
12.5
1.0
8
Ribavirin
1
13
ating at 300 MHz for H and 75MHz for C, respec-
tively. Chemical shifts are reported in ppm with TMS as
the internal standard, and coupling constants are in Hz.
Column chromatographies were performed with silica gel
(Qingdao Haiyang Chemical Group Co. Ltd., China);
TLC was performed on precoated Si gel 60 F254 plates
(0.2 mm thick, Merck) and spots were detected by UV
illumination and by spraying with Ehrlich reagent.
noteworthy that the toxicity of compound 6 is sig-
nificantly smaller than other compounds with a TI value
comparable to its parent furanoditerpenoid 11. The
high hydrophobic friedelane triterpenoids 7–8 were
inactive for para3 virus.
4
Acute respiratory viral infections have long been recog-
nized as important contributors to morbidity and mor-
Plant material. The seed and stem of C. minax Hance
were collected in Guangxi Province, PR China in Sep-
tember, 1999. The materials were identified at the Institute
of Chinese Medicine, The Chinese University of Hong
Kong, where the voucher specimen (No. cm-99 for the
seed and No. cm-99s for the stem) is preserved.
2
6
tality in young children and older adults. The search
of natural products as antiviral agents against respira-
2
7
tory viruses, for example respiratory syncytial virus
2
8
and influenza virus, has attracted recent attention;
however, natural products against para3 virus have
been less studied. Although the mechanisms of action of
furanoditerpenoids are still under investigation, the
results achieved so far showed useful clue to develop
new antiviral agents against para3 virus from this plant.
Extraction and isolation. The 95% ethanol extract of
seeds of C. minax (10 kg) was divided into six fractions
4
as described previously. The chloroform fraction (50 g)
was subjected to column chromatography and eluted with
hexane/acetone (10:1). Fractions of 150 mL each were
taken and 32 fractions collected. Fractions 9, 10 and 11
were combined to afford 2 (50 mg), and recrystallized
from hexane/acetone (20:1) at room temperature. Frac-
tions 17 afford 1 (6 mg), and fractions 19 and 20 afford 3
(30 mg). The stem of the same plant (5 kg) were refluxed
with methanol to afford a crude extract (67 g), which
was then separated between hexane and water. The
hexane fraction (23 g) was subjected to column chro-
matography and eluted with hexane/acetone (15:1).
Compound 7 was obtained from fractions 5–7 and
recrystallized from acetone/hexane mixture (1.0 g).
Conclusions
A number of conclusions may be drawn from this work:
(
i) Three cassane furanoditerpenoids were isolated from
the seed of C. minax: compound 1 represents the first
tetracyclic furanoditerpenoid possessing a 17-methyl
attached to a non-oxygenated C-14 from this plant,
compound 2 provides a new example of pseudopoly-
morphism through crystallization from variable mixing
ratios of a two-component solvent system, and com-
pound 3 is the only furanoditerpenoid isolated from two
different plants in the same genus. (ii) Isolation of frie-
delane triterpenoid 7 in good yield (0.02%) provides a
new rich source and an easy methodology. Notwith-
standing its poor bioactivity, its can be used as a pre-
cursor to synthesize other physiologically active
Reduction of compounds 7 and 9–11. Compound 7
(21.3 mg) was dissolved in anhydrous THF, then 6 mg
LiAlH was added to the solution. The solution was
4
stirred for 30 min. After the reaction a few drops of
water was added to hydrolyze the excess LiAlH and
4
2
0
triterpenoids. (iii) In order to investigate the influence
of molecular polarity on antiviral activity, four deriva-
tives 4–6 and 8 were obtained by reduction of 9–11 and 7,
respectively, and the factors governing the reduction of
cassane furanoditerpenoids and friedelane triterpenoids
were investigated using X-ray structural data in combi-
nation with density functional theory. (iv) The antiviral
activities of 2–8 against para3 were assessed by CPE
reduction assay and compared with previously known
compounds. The results of this study shed light on the
direction of developing antiviral agents by showing that
tetracyclic furanoditerpenoids are superior to pentacylic
furanoditerpenoid lactones, and an increase in polarity
results in a decrease of activity.
filtered. The filtrate was condensed in reduced pressure
and the residue was recrystallized from acetone to
afford 19.3 mg compound 8 (yield 90%). Compounds 9–
11 was reduced with the same method as 7; however, the
resulting products of compounds 4 (yield 76%), 5 (yield
85%) and 6 (yield 81%) were recrystallized from
methanol solution.
Compound 1. Colorless needle-like crystals; IR (KBr)
MeOH
ꢀ
1
nmax 3586, 1740 cm ; UV lmax 220 nm (log e 3.72);
EIMS m/z (relative intensity,%) 376 [M] (24), 316
+
+
+
[MꢀHOAc] (100), 298 [MꢀHOAc–H O] (27), 283
2
+
[MꢀHOAc–H O–CH ] (27), 265 [MꢀHOAc–2H O–
2
3
2
+
+
CH ]
3
(21), 108[C H O]
8
(93), 209 [MꢀC H O–
7
7
8

2168
R.-W. Jiang et al. / Bioorg. Med. Chem. 10 (2002) 2161–2170
OAc](20), HR-LSIMS m/z [MH]+ 377.2328, calculated
for C H O , requires 377.2319.
HR-LSIMS m/z [MH]+ 367.2104, C H O , requires
20 30 6
367.2112.
2
2
32
5
ꢂ
ꢂ
Compound 4. Mp 192–194 C, EIMS m/z (relative inten-
+
Compound 6. Mp 248–250 C, EIMS m/z (relative
+
+
+
sity, %): 450 [M] (20), 435 [MꢀCH ] (15), 317 [Mꢀ
intensity, %): 464 [M] (17), 432 [MꢀCH OH] (3),
3
3
+
+
+
CH –2OAc] (22). HR-LSIMS m/z [MH] 451.2300,
3
373 [MꢀCH OH–OAc]
(18), 312 [MꢀCH OH–
3
3
+
+
C H O requires 451.2314.
8
2OAc] (18), 138 [C H O+OCH ] (100). HR-LSIMS
7 7 3
2
4
34
+
m/z [MH] 465.2472, C H O requires 465.2478.
2
5
36
8
ꢂ
Compound 5. Mp 224–226 C, EIMS m/z (relative
+
+
intensity, %): 366 [M] (2), 351 [MꢀCH ] (12), 333
Single crystal X-ray analysis. The X-ray intensities of
. . .
2(2) H O, 3(2) C H , 4, 2(5) 0.5H O, 6, 7 and 8 were
2 6 14 2
measured over a hemisphere of reciprocal space, by a
combination of three sets of exposures on a Bruker
3
+
+
[
[
[
MꢀCH –H O] (18), 315 [MꢀCH –2H O] (4), 297
3
2
3
2
+
+
MꢀCH –3H O] (3), 279 [MꢀCH –4H O] (8), 236
3
2
3
2
+
+
Mꢀ2CH –4H O–CO]
(100), 109[C H OH]
8
(27).
3
2
7
Table 4. Crystal data and structure refinement parameters
.
2(1) H
.
6
Compound
2
O
3(2) C
H
14
(4)
CCDC deposit no.
Color/shape
Crystal dimensions (mm )
Chemical formula
Formula weight
Temperature (K)
Crystal system
Space group
175941
Colorless/needle
175942
Colorless/prism
175943
Colorless/prism
0.61ꢃ0.25ꢃ0.17
3
0.67ꢃ0.12ꢃ0.09
0.65ꢃ0.26ꢃ0.18
.
.
C
6
2C22H
32
O
5
H
2
O
3C22H
28
O
6
H
14
24 34 8
C H O
770.96
293(2)
1251.57
293(2)
450.51
293(2)
Orthorhombic
P2 (No. 19)
Orthorhombic
P2 (No. 19)
Monoclinic
P2 (No. 4)
a=10.0824(8) A
1
2
1
2
a=8.817(1) A
1
1
2
1
2
a=8.7592(7) A
1
1
˚
˚
˚
Unit cell dimensions
˚
b=10.112(2) A
˚
c=46.686(9) A
˚
b=19.732 (1) A
˚
c=37.815(3) A
˚
b=9.6405(8) A
˚
c=11.897(1) A
b=100.285(2)
1137.8(1)
2
˚
3
Volume (A )
Z
Density (calculated) (mg/m )
4162.6(14)
4
6535.9(9)
4
1.258
0.090
3
1.227
0.087
Bruker SMARTBruker SMARTBruker SMART
1.315
0.098
Absorption coefficient (mm 1)
Diffractometer/scan
ꢀ
1
000 CCD/$
1.74–25.20
22582
1000 CCD/$
1.92–25.00
36615
1000 CCD/$
1.74–25.03
6175
ꢂ
y range ( )
Reflections measured
Independent reflections (Rint
)
7416 (0.1818)
3014
7416/0/491
0.0178(17)
0.909
11516 (0.0522)
6963
11516/0/782
0.0007(3)
0.995
3889(0.0696)
3226
3889/1/289
0.000(2)
0.990
Observed reflections
Data/restrains/parameters
Extinction coefficient
2
Goodness of fit on F
Final R indices [I>2s(I)]
R indices (all data)
0.0794
0.1964
0.0611
0.1098
0.0588
0.0694
.
2(5) 0.5H
175944
2
O
6
175945
7
175946
8
175847
Colorless/block
Colorless/block
0.63ꢃ0.24ꢃ0.17
Colorless/needle
0.62ꢃ0.12ꢃ0.09
Colorless/block
0.53ꢃ0.34ꢃ0.27
0
2
7
2
.58ꢃ0.33ꢃ0.24
C
20
H
30
O
6
.0.5H
2
O
C
25
H
36
O
8
C
30
H
50
O
30 52
C H O
41.89
93(2)
464.54
293(2)
426.70
293(2)
Orthorhombic
428.72
293(2)
Tetragonal
P4 (No. 77)
a=16.764(2) A
Orthorhombic
P2 (No. 19)
Monoclinic
C2 (No. 5)
a=13.440(1) A
2
1
2
1
2
a=6.9582(8) A
1
1 1 1
P2 2 2 (No. 19)
˚
˚
˚
˚
˚
a=6.3794(6) A
˚
b=16.492(1) A
˚
c=20.840(2) A
˚
b=13.936(1) A
b=6.4089(5) A
˚
c=13.267(3) A
˚
c=28.425(3) A
˚
c=29.594(2) A
b=92.290(2)
2547.1(3)
4
3
4
1
0
728.5(11)
2391.4(5)
4
1.290
0.095
2527.1(4)
4
1.122
0.065
.322
.097
1.118
0.064
Bruker SMARTBr uBk r euBr krSeu Mr k SeArMRS AMT R AT RT
1
1
2
6
4
6
0
0
0
0
000 CCD/$
.21–25.04
0348
561 (0.0542)
828
561/1/475
.0013(4)
.958
.0445
.0656
1000 CCD/$
1.57–25.04
12954
4232 (0.0582)
2872
4232/0/300
0.008(9)
0.907
0.0399
0.0655
1000 CCD/$
1.43–24.99
13762
4457(0.0815)
2194
4457/0/281
0.0032(6)
0.908
0.0501
0.1343
1000 CCD/$
1.38–25.03
6912
4066 (0.0277)
3629
4066/1/280
0.000(3)
1.039
0.0442
0.0495

R.-W. Jiang et al. / Bioorg. Med. Chem. 10 (2002) 2161–2170
2169
SMART1000 CCD diffractometer. The intensities
were corrected for Lorentz and polarization effects but
not for absorption. The structures were solved by
direct methods and refined by full-matrix least-squares
Fund AF/281/97 from the Hong Kong SAR Govern-
ment. We thank Dr. Zhi-Feng Liu for helpful discus-
sions regarding the structural optimizations.
2
29
on F using SHELXTL-97 software package. The
crystallographic data of the above compounds are
shown in Table 4. Among these crystal structures,
References and Notes
.
(1) H O has one very long unit-cell axis and the crys-
2
2
1
. (a) Newman, D. J.; Cragg, G. M.; Snader, K. M. Nat.
Prod. Rep. 2000, 17, 215. (b) Matthee, G.; Wright, A. D.;
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122, 2116. (d) Henkel, T.; Brumne, R. M.; Muller, H.; Reichel,
F. Angew, Chem. Int. Ed. 1999, 38, 643.
tals occur as very slim needles, so that the diffraction
patterns are weak. Furthermore, the water molecule is
disordered over two positions in the asymmetric unit,
being represented by O-1w with a site occupancy factor
(sof)=0.7 and O-2w with sof=0.3; thus the R indices of
the hemihydrate of (1) are higher than usual. In addi-
´
¨
¨
2. Treanor, J.; Falsey, A. Antiviral Res. 1999, 44, 79.
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tion, the solvent molecule (hexane) in 3(2) C H exhib-
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6
14
4
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Antiviral assay
5
Para3 virus and HEp-2 cells were obtained from Amer-
ican Type Culture Collection. Dulbecco’s modified
eagle’s medium was purchased from Sigma Company,
USA. Fetal bovine serum was obtained from Biofluids
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M. J. Nat. Prod. 1997, 60, 1219.
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microtiter plates using the procedures described pre-
2
5
viously. Ribavirin was used as a positive control, and an
infection control was made in the absence of samples. A
fixed quantity of para3 virus in suspension was added to
the monolayers of HEp-2 cells, and the cytopathogenic
effects (CPE, loss of monolayer, rounding, shrinking of
the cells, granulation, and vacuolization in the cyto-
plasm) were observed under an inverted microscope.
The concentration that reduced CPE by 50% with
respect to virus control was defined as IC . The con-
9
. Galasso, G. J.; Merigan, T. C.; Buchanan, R. A. Antiviral
Agents and Viral Diseases of Man, 2nd ed.; Raven Press, New
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10. Kitagawa, I.; Simanjuntak, P.; Watano, T.; Shibuya, H.;
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1
994, 42, 1798.
1. Collings, J. C.; Roscoe, K. P.; Thomas, R. L. I.; Batsanov,
5
0
centration that showed 50% cytotoxic effect was defined
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5
0
50
L). The selectivity against Para3 virus was characterized
by the therapeutic index (TI)=TC /IC . The antiviral
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12. (a) For examples from Caesalpinia genus, see: Ashok, D. P.;
5
0
50
Alan, J. F.; Lee, W.; Gary, Z.; Rex, R.; Mark, F. B.; Leo, F.;
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(
1
3. (a) For examples from Pterodon genus, see: Mahajan,
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3
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.
independent molecules in 2(1) H O, three independent
2
.
molecules in 3(2) C H and two independent molecules
6
14
.
in 2(5) 0.5H O are listed in Tables S1, S2 and S3,
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2
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