New coumarins and triterpenes from Calophyllum inophyllum

Article abstract of DOI:10.1002/hlca.200900469

Four new coumarins, 1-4, and two new triterpenes, 5 and 6, along with nine known compounds have been identified from the leaves of Calophyllum inophyllum on the basis of spectroscopic analyses and chemical methods. The structure of apetalolide (7), previously reported from C. apetalum, was revised by means of 2D-NMR, and the absolute configuration of the known compound inophyllum D (8) was established by a modified Mosher's method.

Full text of DOI:10.1002/hlca.200900469

1
812
Helvetica Chimica Acta – Vol. 93 (2010)
New Coumarins and Triterpenes from Calophyllum inophyllum
by Jian Zou, Jian Wu, Shuang-Zhu Liu, and Wei-Min Zhao*
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 201203, P. R. China
(
phone/fax: þ 86-21-50806052; e-mail: wmzhao@mail.shcnc.ac.cn)
Four new coumarins, 1 – 4, and two new triterpenes, 5 and 6, along with nine known compounds have
been identified from the leaves of Calophyllum inophyllum on the basis of spectroscopic analyses and
chemical methods. The structure of apetalolide (7), previously reported from C. apetalum, was revised by
means of 2D-NMR, and the absolute configuration of the known compound inophyllum D (8) was
established by a modified Mosherꢀs method.
Introduction. – Calophyllum inophyllum (Guttiferae) is an arbor widely distributed
in the southeast of Asia, and it has been used as herb medicine for the treatment of
rheumatism, arthritis, lumbago, and wounds in the south of China [1]. Previous
phytochemical investigations have revealed C. inophyllum to be a rich source of
secondary metabolites, including xanthones [2], coumarins [3 – 5], and triterpenes [6],
and some of them have been found to exhibit antimicrobial [2], cytotoxic [2], piscicidal
[
4], and anti-HIV activities [5]. To the best of our knowledge, no systematical
investigation has been undertaken on the chemical constituents of C. inophyllum
distributed in China. Since the same plant species grows in different regions, they may
contain different constituents. Phytochemicals of the leaves of C. inophyllum collected
in Hainan Island of China were investigated. We herein report the isolation and
structural characterization of six new compounds, named 12-O-butylinophyllum D (1),
12-O-ethylinophyllum D (2), inophyllum H (3), inophyllum I (4), 27-[(E)-p-
coumaroyloxy]friedelin-28-carboxylic acid (5), and 27-[(Z)-p-coumaroyloxy]friede-
lin-28-carboxylic acid (6), along with nine known compounds. The structure of the
known compound apetalolide (7), identified previously from C. apetalum, was revised
according to 2D-NMR spectral analysis, and the absolute configuration of inophyllum
D (8), a known compound isolated from C. inophyllum, was established by a modified
Mosherꢀs method.
Results and Discussion. – Compound 1 was obtained as a yellow amorphous powder
with the molecular formula C H O , determined by HR-EI-MS and NMR analyses.
2
9
32
5
ꢀ1
The IR spectrum of 1 revealed the existence of OH (3438 cm ) and a,b-unsaturated
ꢀ1
ꢀ1
lactone C¼O (1739 cm ) groups, and an olefinic C¼C bond (1637 cm ). Its UV
spectrum exhibited absorption maxima at 332, 286, 278, and 236 nm, which were similar
to those of inophyllum D (8), a known coumarin previously reported from the same
13
species [4]. The C-NMR spectrum of 1 (Table 1) showed 29 C-atom signals
ꢁ
2010 Verlag Helvetica Chimica Acta AG, Zꢂrich

Helvetica Chimica Acta – Vol. 93 (2010)
1813
3
3
2
3
corresponding to five Me, three sp CH , three sp CH, eight sp CH groups, and one sp
2
1
2
and nine sp quaternary C-atoms. The H-NMR spectrum of 1 (Table 2) exhibited two
Me singlets at d(H) 0.91 (Me(19)) and 0.94 (Me(20)), two Me doublets at d(H) 0.83
(
J ¼ 7.3, Me(21)), 1.44 (J ¼ 6.6 Hz, Me(22)), one Me triplet at d(H) 0.93 (J ¼ 7.4,
3
Me(26)), signals corresponding to three sp CH groups (d(H) 3.78 – 3.80 (m,
2
CH (23)), 1.58 – 1.62 (m, CH (24)), and 1.40 – 1.44 (m, CH (25))), three olefinic H-
2
2
2
atom signals (d(H) 5.36 (d, J ¼ 10.0, HꢀC(7)), 6.58 (d, J ¼ 10.0, HꢀC(8)), and 5.98 (s,
HꢀC(3))), signals due to a mono-substituted benzene ring (d(H) 7.38 – 7.40 (m,
3
HꢀC(14,16,18)), 7.20 – 7.24 (m, HꢀC(15,17))), and three sp CH signals (d(H) 4.59
(
dq, J ¼ 6.6, 2.0, HꢀC(10)), 4.51 (d, J ¼ 2.2, HꢀC(12)), and 2.05 (ddq, J ¼ 7.3, 2.0, 2.2,
1
HꢀC(11)). The H-NMR data of 1 were similar to those of 8 except three additional
3
sp CH signals and one Me triplet [4], and the doublet due to HꢀC(12) was shifted
2
upfield from d(H) 4.93 (J ¼ 2.1) to 4.51 (J ¼ 2.2), thus it was assumed that a BuO group
was at C(12) of 1. The planar structure of 1 was established by HMBC spectrum, in
13
1
which the following C, H long-range correlation signals were observed: C(4a)/
HꢀC(3), C(4b)/Me(19,20), C(6)/HꢀC(8), C(7)/Me(19,20), C(8a)/HꢀC(7), C(8b)/
HꢀC(10), C(10)/HꢀC(12) and Me(22), C(11)/Me(21), C(12)/Me(22) and CH (23),
2
C(13)/HꢀC(3,15,17), C(19,20)/HꢀC(7), C(22)/HꢀC(10,12), C(23)/HꢀC(12), and
CH (25), C(24)/Me(26), C(25)/CH (23), C(26)/CH (24). Hydrolysis of 1 yielded 8,
2
2
2
which was identified by co-TLC with authentic sample and optical-rotation data. Thus,
compound 1 was assigned as 12-O-butylinophyllum D.
The absolute configuration of 8 was established by a modified Mosherꢀs method
[
7]. Both (R)- and (S)-MTPA (MTPA ¼ a-methoxy-a-(trifluoromethyl)phenylacetic

1
814
Helvetica Chimica Acta – Vol. 93 (2010)
Table 1. 13C-NMR Data (100 MHz, in CDCl ) of Compounds 1 – 4
3
C-Atom
1
2
3
4
Inophyllum D (8) [8]
C(2)
C(3)
C(4)
C(4a)
C(4b)
C(6)
160.8 (s)
112.3 (d)
156.2 (s)
103.7 (s)
151.4 (s)
77.2 (s)
160.5 (s)
111.9 (d)
155.9 (s)
103.4 (s)
151.1 (s)
76.8 (s)
160.8 (s)
111.4 (d)
154.9 (s)
98.9 (s)
158.6 (s)
72.7 (d)
12.9 (s)
160.9 (s)
111.8 (d)
154.6 (s)
98.8 (s)
158.3 (s)
73.0 (d)
13.8 (s)
160.5 (s)
111.9 (d)
156.3 (s)
103.4 (s)
151.1 (s)
77.1 (s)
C(7)
127.3 (d)
116.3 (d)
106.1 (s)
154.2 (s)
71.3 (d)
34.3 (d)
72.3 (d)
102.3 (s)
155.1 (s)
140.4 (s)
127.6 (d)
127.6 (d)
127.7 (d)
127.6 (d)
127.6 (d)
27.2 (q)
27.0 (q)
18.0 (q)
9.4 (q)
127.0 (d)
115.9 (d)
105.8 (s)
153.9 (s)
70.9 (d)
34.2 (d)
71.8 (d)
101.8 (s)
154.7 (s)
140.1 (s)
127.3 (d)
127.3 (d)
127.4 (d)
127.3 (d)
127.3 (d)
26.9 (q)
26.7 (q)
17.4 (q)
9.0 (q)
127.2 (d)
115.9 (d)
106.0 (s)
153.9 (s)
71.2 (d)
37.2 (d)
64.6 (d)
103.9 (s)
154.6 (s)
139.9 (s)
127.3 (d)
127.4 (d)
127.6 (d)
127.4 (d)
127.3 (d)
27.0 (q)
27.0 (q)
17.1 (q)
9.1 (q)
C(8)
29.2 (d)
111.5 (s)
155.1 (s)
70.9 (d)
37.4 (d)
64.8 (d)
104.3 (s)
153.7 (s)
137.5 (s)
127.7 (d)
127.7 (d)
128.8 (d)
127.7 (d)
127.7 (d)
22.6 (q)
12.9 (q)
17.7 (q)
9.2 (q)
30.9 (d)
113.0 (s)
152.6 (s)
112.0 (d)
132.1 (s)
76.1 (d)
103.9 (s)
152.4 (s)
137.8 (s)
127.6 (d)
127.9 (d)
128.8 (d)
127.9 (d)
127.6 (d)
22.7 (q)
12.8 (q)
19.6 (q)
19.4 (q)
C(8a)
C(8b)
C(10)
C(11)
C(12)
C(12a)
C(12b)
C(13)
C(14)
C(15)
C(16)
C(17)
C(18)
C(19)
C(20)
C(21)
C(22)
C(23)
C(24)
C(25)
C(26)
69.5 (t)
32.5 (t)
19.8 (t)
14.2 (q)
64.7 (t)
15.8 (q)
acid) esters of inophyllum D were prepared according to a usual method, and the Dd
1
values (Dd ¼ d ꢀ d ) of their H-NMR data were calculated (Fig.). In this way, the
S
R
absolute configuration at C(12) was determined to be (R). Since the relative
configuration of 8 had already been determined [5], 8 was thus identified to be
(10R,11S,12R)-12-hydroxy-11,12-dihydro-6,6,10,11-tetramethyl-4-phenyl-2H,6H,10H-
dipyrano[2,3-f :2’,3’-h]chromen-2-one, and, accordingly, compound 1 was established as
1
Figure. Dd Values (Dd ¼ d ꢀ d ) of H-NMR data (600 MHz, J in Hz)
S
R
of the MPTA esters of inophyllum D

Helvetica Chimica Acta – Vol. 93 (2010)
1815

1
816
Helvetica Chimica Acta – Vol. 93 (2010)
(10R,11S,12R)-12-butoxy-11,12-dihydro-6,6,10,11-tetramethyl-4-phenyl-2H,6H,10H-
dipyrano[2,3-f :2’,3’-h]chromen-2-one.
Compound 2 was obtained as a yellow amorphous powder with the molecular
1
3
formula C H O , determined by HR-EI-MS. The IR, UV, and C-NMR spectra of 2
27
28
5
3
13
were similar to those of 1, except with two sp CH signals less in its C-NMR spectrum.
2
Hydrolysis of 2 also yielded 8. Further analysis of 2D-NMR spectra allowed the
characterization of compound 2 as (10R,11S,12R)-12-ethoxy-11,12-dihydro-6,6,10,11-
tetramethyl-4-phenyl-2H,6H,10H-dipyrano[2,3-f :2’,3’-h]chromen-2-one.
Compounds 1 and 2 were confirmed to be natural compounds by re-extraction of
the plant material and preparative TLC purification without using BuOH and EtOH,
and further LC/MS analyses.
Compound 3 was obtained as a yellow amorphous powder with the molecular
formula C H O , determined by HR-EI-MS and NMR analyses. The IR spectrum of
2
5
24
5
ꢀ
1
ꢀ1
3
revealed presence of OH (3426 cm ) and a,b-unsaturated lactone C¼O (1708 cm )
ꢀ1 13
functionalities, and a monosubstituted aromatic ring (700, 748 cm ). The C-NMR
3
spectrum of 3 (Table 1) showed 25 C-atom signals including those for four Me, five sp
2
3
2
1
CH, six sp CH groups, and one sp and nine sp quaternary C-atoms. The H-NMR
spectrum of 3 (Table 2) exhibited two Me singlets (d(H) 0.75 (Me(19)), 1.02
(
Me(20))), two Me doublets (d(H) 1.46 (d, J ¼ 6.5, Me(21)), 0.81 (J ¼ 7.2, Me(22))),
2
one sp H-atom singlet (d(H) 6.01 (HꢀC(3))), signals due to a monosubstituted
aromatic ring (d(H) 7.40 – 7.42 (m, HꢀC(14,16,18)), 7.34 – 7.38 (m, HꢀC(15,17))), and
3
five sp CH signals (d(H) 4.21 (d, J ¼ 5.7, HꢀC(6)), 2.42 (d, J ¼ 5.7, HꢀC(8)), 4.39 (dq,
J ¼ 6.6, 2.0, HꢀC(10)), 4.95 (d, J ¼ 2.3, HꢀC(12)), and 2.04 (ddq, J ¼ 7.2, 2.0, 2.2,
1
HꢀC(11))). The H-NMR spectrum of 3 was thus similar to that of 8 [4]; the
difference between the two spectra consists in an upfield shift of HꢀC(7) and HꢀC(8),
the cis-coupled olefinic doublet from d(H) 5.35 (d, J ¼ 10.0, HꢀC(7)), 6.52 (d, J ¼ 10.0,
HꢀC(8)) in 8 to 4.21 (d, J ¼ 5.7, HꢀC(6)), 2.42 (d, J ¼ 5.7, HꢀC(8)) in 3, indicating the
loss of the C(7)¼C(8) bond and presence of a dimethylcyclopropane ring in ring A of 3
as in inophyllum G and G [5]. The observed upfield shift of the geminal Me singlet
1
2
resonance (8: d(H) 0.95 (s, Me(19,20)); 3: d(H) 0.75 (s, Me(19)), 1.02 (s, Me(20))) also
supported the presence of a cyclopropane ring in ring A of 3. The constitutional
formula of 3 was established via the HMBC spectra, in which the following long-range
1
13
H, C-correlation signals were observed: C(4a)/HꢀC(3), C(4b)/HꢀC(6,8), C(6)/
Me(19,20), C(8)/Me(19,20), C(8a)/HꢀC(6), C(8b)/HꢀC(8), C(10)/HꢀC(12), C(11)/
Me(21), C(12)/HꢀC(10) and Me(22), C(13)/HꢀC(3,15,17), C(19)/HꢀC(6,8), C(20)/
HꢀC(6,8), C(21)/HꢀC(11), C(22)/HꢀC(10,12). Since the chemical shifts and the
coupling constants of HꢀC(10), HꢀC(11), and HꢀC(12) are almost the same as those
of 8 [5], it can be assumed that the relative configuration of the chromanol ring is the
same as that of 8. To the best of our knowledge, compound 3 is a new compound, and
has been assigned the name inophyllum H. It was difficult to completely specify the
relative configuration of 3, since it possesses two additional stereogenic centers, and
the H-atoms in the cyclopropane ring are too far from the chromanol ring H-atoms to
display a NOE-based relationship between the two spin systems, thus it was not pos-
sible to assign the configuration of the cyclopropane ring in 3 without an X-ray analysis.
Compound 4 was obtained as a yellow amorphous powder with the molecular
formula of C H O , determined by HR-EI-MS and NMR analysis. The IR spectrum
25
22
4

Helvetica Chimica Acta – Vol. 93 (2010)
1817
13
and UV spectra were similar to those of 3. The C-NMR spectrum of 4 showed 25 C-
3
2
atom signals including those for four Me, three sp CH, and seven sp CH groups, and
3
2
1
one sp and ten sp quaternary C-atoms. The H-NMR spectrum of 4 exhibited four Me
signals (d(H) 0.70 (s, Me(19)), 1.04 (s, Me(20)), 1.90 (s, Me(22)), and 1.40 (d, J ¼ 6.5,
2
Me(21))), two sp H-atom signals (d(H) 5.98 (s, HꢀC(3)) and 6.68 (s, HꢀC(12))),
signals due to a monosubstituted aromatic ring (d(H) 7.39 – 7.40 (m, HꢀC(14,16,18)),
3
7
.24 – 7.26 (m, HꢀC(15,17))), and three sp CH signals (d(H) 4.25 (d, J ¼ 5.7, HꢀC(6)),
2
.41 (d, J ¼ 5.7, HꢀC(8)), and 4.93 (q, J ¼ 6.5, HꢀC(10))). Compared with the
1
3
H-NMR spectra of 11,12-dehydroxyinophyllum P [4], the presence of two sp CH
signals at d(H) 4.20 (d, J ¼ 5.7, HꢀC(6)), 2.41 (d, J ¼ 5.7, HꢀC(8)) and the
disappearance of the cis-coupled olefinic doublet indicated the loss of the
C(7)¼C(8) bond, and the presence of a dimethylcyclopropane ring in ring A of 4 as
in 3. The planar structure of 4 was established on the HMBC spectra, in which the
1
13
following long-range H, C-correlation signals were observed: C(4)/HꢀC(14,18),
C(4a)/HꢀC(3), C(4b)/HꢀC(6,8), C(6)/Me(19,20), C(8)/Me(19,20), C(8a)/HꢀC(6),
C(8b)/HꢀC(12), C(10)/HꢀC(12) and Me(22), C(11)/Me(21), C(12)/Me(22), C(13)/
HꢀC(3), C(19)/HꢀC(6,8), C(20)/HꢀC(6,8), C(22)/HꢀC(10). Compound 4 is a new
compound, and has been named inophyllum I.
Compound 5 was obtained as a white amorphous powder with the molecular
formula of C H O , determined by HR-EI-MS and NMR analysis. The IR spectrum
3
9
54
6
ꢀ1
ꢀ1
of 5 revealed the existence of a OH (3415 cm ) and a C¼O group (1701 cm ), and of
ꢀ1
an aromatic ring (1604, 1514, 1450 cm ). The presence of the (E)-p-coumaroyl group
1
13
was established by characteristic H- and C-NMR data (Table 3) [7], and character-
istic peaks at m/z 164.0470 (C H O ), 163.0401 (C H O ), 147.0441 (C H O ), and
9
8
3
9
7
3
9
7
2
1
4
19.0498 (C H O) in the HR-EI-MS. In the HR-EI-MS spectra of 5, the peak at m/z
8 7
þ
09.3447 (C H O, [M ꢀ coumaroyloxy group ꢀ COOH] ) implied the presence of a
29
45
free COOH group in the triterpene part of 5. In the HMBC spectra of 5, the following
long-range C, H correlation signals were observed: C(3)/CH (1) and Me(23), C(5)/
13
1
2
Me(23), C(6)/HꢀC(8) and Me(24), C(8)/Me(25,26), C(9)/CH (12), C(10)/CH (2)
2
2
and Me(24,25), C(11)/HꢀC(8) and Me(25), C(12)/CH (27), C(13)/Me(26), C(14)/
2
CH (27), C(15)/Me(26), C(17)/CH (19), C(18)/CH (16,27), C(19)/Me(29,30), C(20)/
2
2
2
CH (22), C(21)/CH (19) and Me(29,30), C(22)/HꢀC(18), C(24)/HꢀC(4), C(25)/
2
2
HꢀC(8,10), C(26)/CH (15), C(27)/CH (12) and HꢀC(18), C(28)/CH (22), C(29,30)/
2
2
2
CH (19,21), C(1’)/HꢀC(3’,5’,8’), C(9’)/CH (27) and HꢀC(7’). Thus, a friedelin
2
2
skeleton was suggested for 5, with the free COOH group at C(28) and the (E)-p-
coumaroyloxy group at C(27), which was confirmed by NOE correlation signals
observed between Me(23), and CH (2,6) and Me(24); Me(24) and CH (1,7) and
2
2
Me(25); Me(25), and CH (1,11,12) and Me(26); Me(26), and CH (7) and HꢀC(18);
2
2
H ꢀC(27), and CH (15,12) and Me(29); H ꢀC(27) and CH (19,22); Me(29), and
b
2
a
2
CH (22) and Me(30); and Me(30) and CH (19,22). Compound 5 was finally
2
2
characterized as 27-[(E)-p-coumaroyloxy]friedelin-28-carboxylic acid.
Compound 6 was obtained as a white amorphous powder with the molecular
formula of C H O determined by HR-EI-MS and NMR analysis. The IR spectrum of
3
9
54
6
ꢀ
1
ꢀ1
6
revealed the presence of a OH (3392 cm ) and a C¼O group (1701 cm ), and an
ꢀ1 13
aromatic ring (1604, 1514, 1451 cm ). The C-NMR spectrum showed 39 C-atom
signals including those for six Me groups, and ten aromatic and conjugated C¼C bond

1
818
Helvetica Chimica Acta – Vol. 93 (2010)
Table 3. 1H- (400 MHz) and 13C-NMR (100 MHz) Data of 5 and 6 (C D N)
5
5
5
6
d(H)
d(C)
d(H)
d(C)
CH (1)
1.56 – 1.80 (m)
2.18 – 2.32 (m)
22.3 (t)
41.4 (t)
211.7 (s)
57.7 (d)
41.8 (s)
40.7 (t)
18.7 (t)
53.3 (t)
37.8 (s)
59.0 (d)
36.7 (t)
25.5 (t)
43.4 (s)
38.4 (s)
32.1 (t)
30.3 (t)
45.1 (s)
38.7 (d)
36.4 (t)
28.8 (s)
33.1 (t)
36.5 (t)
7.2 (q)
1.54 – 1.78 (m)
2.08 – 2.30 (m)
22.4 (t)
41.5 (t)
211.7 (s)
57.7 (d)
41.9 (s)
40.8 (t)
18.5 (t)
53.3 (t)
37.7 (s)
59.0 (d)
36.7 (t)
25.5 (t)
43.2 (s)
38.5 (s)
32.1 (t)
30.3 (t)
45.1 (s)
38.7 (d)
36.0 (t)
28.8 (s)
33.2 (t)
36.5 (t)
7.2 (q)
2
CH (2)
2
C(3)
HꢀC(4)
2.12 (q, J ¼ 6.6)
2.16 (q, J ¼ 6.5)
C(5)
HꢀC(26)
1.58 – 1.62 (m)
1.57 – 1.59 (m)
1.59 – 1.62 (m)
1.60 – 1.64 (m)
1.57 – 1.63 (m)
1.62 – 1.66 (m)
CH (7)
2
HꢀC(8)
C(9)
HꢀC(10)
1.54 – 1.56 (m)
1.60 – 1.64 (m)
2.30 – 2.36 (m)
1.56 – 1.60 (m)
1.63 – 1.70 (m)
2.35 – 2.39 (m)
CH (11)
2
CH (12)
2
C(13)
C(14)
CH (15)
1.23 – 1.27 (m)
2.90 (dd, J ¼ 14.7, 11.6)
1,27 – 1.31 (m)
2.88 (dd, J ¼ 14.5, 11.2)
2
CH (16)
2
C(17)
HꢀC(18)
3.00 (d, J ¼ 13.9)
1.60 – 1.66 (m)
2.98 (d, J ¼ 13.6)
1.58 – 1.62 (m)
CH (19)
2
C(20)
CH (21)
1.30 – 1.34 (m)
1.70 – 1.80 (m)
0.91 (d, J ¼ 6.6)
0.61 (s)
0.80 (s)
1.18 (s)
1.27 – 1.33 (m)
1.70 – 1.80 (m)
0.90 (d, J ¼ 6.6)
0.60 (s)
0.78 (s)
1.15 (s)
2
CH (22)
2
Me(23)
Me(24)
Me(25)
Me(26)
14.7 (q)
17.5 (q)
21.8 (q)
25.5 (t)
14.6 (q)
17.5 (q)
21.8 (q)
25.5 (t)
CH (27)
4.90 (d, J ¼ 12.3),
4.90 (d, J ¼ 12.5),
2
5.08 (d, J ¼ 12.3)
5.08 (d, J ¼ 12.5)
C(28)
Me(29)
Me(30)
181.2 (s)
30.0 (q)
34.9 (q)
181.2 (s)
30.0 (q)
34.9 (q)
1.38 (s)
1.02 (s)
1.36 (s)
1.00 (s)
(
E)-p-Coumaroyl:
C(1’)
126.1 (s)
130.8 (d)
116.9 (d)
161.6 (s)
145.3 (d)
115.6 (d)
167.8 (s)
126.6 (s)
133.7 (d)
116.0 (d)
160.7 (s)
144.1 (d)
116.5 (d)
167.2 (s)
HꢀC(2’,6’)
HꢀC(3’,5’)
C(4’)
HꢀC(7’)
HꢀC(8’)
C(9’)
7.71 (d, J ¼ 8.7)
7.20 (d, J ¼ 8.7)
8.15 (d, J ¼ 8.6)
7.20 (d, J ¼ 8.6)
8.10 (d, J ¼ 15.8)
6.85 (d, J ¼ 15.8)
7.08 (d, J ¼ 12.8)
6.18 (d, J ¼ 12.8)
1
C-atoms. In the H-NMR spectra of 6 (Table 3), the presence of a (Z)-coumaroyl
13
group was revealed supported by the C-NMR signals (Table 3) [9] and characteristic
peaks at m/z 164.0473 (C H O ), 147.0446 (C H O ), and 119.0497 (C H O) in the HR-
9
1
8
3
9
7
2
8
7
13
EI-MS of 6. Analysis of H, C, DEPT, and EI-MS data indicated that 6 possesses also
13
a triterpene skeleton and a (Z)-p-coumaroyl group. Since the C-NMR spectral data
Table 3) of the triterpene skeleton in 6 are resembled closely to those of 5, it can be
(

Helvetica Chimica Acta – Vol. 93 (2010)
1819
assumed that compound 6 is an isomer of 5 with a (Z)-p-coumaroyloxy group at C(27).
As a result, the structure of 6 was elucidated as 27-[(Z)-coumaroyloxy]friedelin-28-
carboxylic acid.
Compound 7 was obtained as a white amorphous powder with the molecular
formula of C H O , determined by HR-EI-MS. Its NMR data were identical to those
26
24
5
of 7a previously identified by Nigam et al. under the name apetalolide [10]. Palmer
1
et al. reported the total synthesis of 7a in 1995; however, H-NMR data of the synthetic
and the natural product were different, and the structure 7 was proposed for apetalolide
[
8]. To confirm the structure of apetalolide, a series of 2D-NMR analyses were
performed. In the ROESY spectrum, two NOE correlation were observed between the
MeO signal (Me(22)) and that of HꢀC(6), and also between the MeO signal and the
H-atom signal in the benzene ring at C(4), which excluded the possibility of the
13
1
structure 7a. Furthermore, the following long-range C, H correlation signals were
observed in the HMBC spectrum: C(4)/HꢀC(17,21), C(4a)/HꢀC(3), C(5)/HꢀC(6)
and Me(22)O, C(6a)/HꢀC(7), C(6b)/HꢀC(6), C(7)/Me(23,24), C(8)/HꢀC(6), C(11)/
HꢀC(13) and Me(15), C(12)/Me(14), C(13)/Me(15), and C(16)/HꢀC(3,18,20). Thus,
the structure of apetalolide had to be revised to 5-methoxy-10-[(E)-2-methylcroto-
noyl]-8,8-dimethyl-4-phenyl-8H-pyrano[3,2-g]chromen-2-one (7).
Besides compounds 1 – 7, inophyllum A [4], 12-methoxyinophyllum D [11],
inophyllum C [4], inophyllum D (8) [4], inophyllum E [4], calophyllolide [5],
calophyllic acid [5], and astragalin [12] were also isolated and characterized by
comparison of their spectroscopic data with those in the literature.
Experimental Part
General. HPTLC Plates (Yantai Chemical Industrial Institute, Yantai, P. R. China). Column
chromatography (CC): silica gel H60 (SiO ; Qingdao Haiyang Chemical Group Corporation, Qingdao,
2
P. R. China), MCI-gel CHP-20P (Mitsubishi Chemical, Japan), and Sephadex LH-20 (Pharmacia Biotech
AB, S-Uppsala) as packing materials. Prep. HPLC: Varian SD-1 instrument, equipped with a Merck
NW25 C18 column (10 m, 20 mm ꢁ 250 mm), and ProStar 320 UV/VIS detector. Optical rotations:
Horiba Sepa-300 polarimeter. UV Spectra: Beckman DU-7 spectrometer; lmax in nm. IR Spectra: Perkin-
ꢀ
1
Elmer 577 spectrometer; ˜n in cm . NMR Spectra: Bruker AM-300, AM-400, and AM-600 spectrometers
in CDCl or C D N; d in ppm rel. to Me Si as internal standard, J in Hz. EI- and HR-EI-MS: Finnigan
3
5
5
4
MAT 95 instrument; in m/z (rel. %). LC/MS: Bruker esquir3000plus instrument equipped with an Varian
Dynmax C18 column (5 mm, 4.6 mm i.d. ꢁ 25 cm), and ionization mode, ESI; column flow, 0.5 ml/min;
eluting program, solvent A/solvent B 100%:0 ! 0 :100%; solvent A: H O þ 0.05% HCOOH, solvent B:
2
MeCN þ 0.05% HCOOH; stop time, 15 min.
Plant Material. The leaves of C. inophyllum were collected in Hainan Island, P. R. China, in June
2
003, and identified by Prof. Ding Fang of Guangxi Institute of Chinese Traditional Medicine. A voucher
specimen (No. SIMM20001201) was deposited with the Herbarium of Shanghai Institute of Materia
Medica, Chinese Academy of Sciences.
Extraction and Isolation. Powdered air-dried leaves of C. inophyllum (1.2 kg) were extracted three
times (3 ꢁ 3 l) with MeOH/CHCl 1:1 at 708, and then concentrated in vacuo to give a crude extract
3
(
5
28.5 g). The extract was subjected to an MCI gel column (7 ꢁ 15 cm) and eluted with a mixture of 10, 30,
0, 75, 90, and 100% MeOH and H O (each 500 ml) successively to give Fractions A (4.75 g), B (3.80 g),
2
C (2.05 g), D (1.25 g), E (2.10 g), F (2.35 g), and G (10.3 g). Fr. F (2.35 g) was subjected to CC (SiO2;
petroleum ether (PE)/AcOEt gradient 15 :1 ! 4 :1) to give 1 (84.0 mg), 2 (72.3 mg), 3 (11.0 mg), and
inophyllum A (1.05 g). Fr. E (2.10 g) was subjected to CC (SiO ; PE/AcOEt gradient 8 :1 ! 2 :1) to give
2
inophyllum A (350.5 mg), 12-methoxyinophyllum D (145.0 mg), and 8 (12.8 mg). Fr. D (1.25 g) was

1
820
Helvetica Chimica Acta – Vol. 93 (2010)
subjected to CC (SiO ; PE/AcOEt gradient 10 :1 ! 3 :1) to give inophyllum A (250.6 mg), calophyllolide
2
(
564.0 mg), inophyllum C (80.3 mg), 8 (123.0 mg), and Fr. D1 (13.3 mg). Fr. D1 was subjected to
HPTLC (hexane/AcOEt 3 :1) to give 4 (7.2 mg) and 5 (1.2 mg). Fr. C (1.05 g) was subjected to prep.
HPLC (MeOH/H O gradient 3 :1 ! 20 :1) to give isocalophyllic acid (132.5 mg), calophyllic acid
2
(
(
120.8 mg), inophyllum E (110.6 mg), and Fr. C1 (80.3 mg). Fr. C1 was subjected to prep. HPLC
MeCN/H O 19 :1) to give 6 (32.1 mg) and 7 (34.8 mg). Fr. B (3.80 g) was passed through a Sephadex
2
LH-20 column with 95% EtOH to give astragalin (3.06 g).
1
2-O-Butylinophyllum D (¼(10R,11S,12R)-12-Butoxy-11,12-dihydro-6,6,10,11-tetramethyl-4-phe-
2
0
nyl-2H,6H,10H-dipyrano[2,3-f :2’,3’-h]chromen-2-one; 1). Yellow amorphous powder. [a] ¼ þ23.0
D
(
1
c ¼ 0.07, CHCl ). UV (MeOH): 236, 278, 286, 332 nm. IR (KBr): 3438, 2931, 1739 (C¼O), 1637, 1587,
3
1
13
463, 1373, 1315, 1181, 1128,1085, 865, 748, 702. H- and C-NMR: Tables 1 and 2, resp. EI-MS: 460 (24,
þ
þ
M ), 445 (100), 417 (4), 387 (57, [M ꢀ C H OH] ), 371 (25), 333 (20), 149 (25). HR-EI-MS: 460.2254
4
9
þ
þ
5
(
M , C H O ; calc. 460.2250).
29 32
1
2-O-Ethylinophyllum D (¼(10R,11S,12R)-12-Ethoxy-11,12-dihydro-6,6,10,11-tetramethyl-4-phe-
2
0
nyl-2H,6H,10H-dipyrano[2,3-f :2’,3’-h]chromen-2-one; 2). Yellow amorphous powder. [a] ¼ ꢀ4.0
D
(
1
c ¼ 0.075, CHCl ). UV (MeOH): 236, 279, 285, 335. IR (KBr): 3435, 2971, 1736 (C¼O), 1637, 1587,
3
1
13
444, 1373, 1315, 1181, 1141, 1128, 1078, 1012, 858, 748, 702. H- and C-NMR: Tables 1 and 2, resp. EI-
þ
þ
þ
MS: 432 (20, M ), 417 (100), 403 (65, [M ꢀ C H ] ), 387 (21, [M ꢀ C H OH] ), 371 (25), 333 (16), 178
2
5
2
5
þ
þ
5
(
6). HR-EI-MS: 432.1944 (M , C H O ; calc. 432.1937).
27 28
Inophyllum H (¼(2R*,3R*,4R*)-3,4,10,10a-Tetrahydro-4-hydroxy-2,3,10,10-tetramethyl-8-phenyl-
H,6H,9aH-cyclopropa[4,5]furo[2,3-f]pyrano[2,3-h]chromen-6-one; 3). White amorphous powder.
2
[
1
4
20
a] ¼ þ136.6 (c ¼ 0.20, CHCl ). UV (CHCl ): 226, 256, 337. IR (KBr): 3426 (OH), 2971, 1708, 1604,
D
3
3
1
13
568, 1421, 1384, 1324, 1228, 1139, 1099, 1031, 748, 700. H- and C-NMR: Tables 1 and 2, resp. EI-MS:
þ
þ
04 (15, M ), 389 (100), 371 (65), 334 (44), 317 (19), 178 (8), 164 (4), 84 (12). HR-EI-MS: 404.1630 (M ,
þ
C H O ; calc. 404.1624).
25
24
5
Inophyllum
I
(¼10,10a-Dihydro-2,3,10,10-tetramethyl-8-phenyl-2H,6H,9aH-cyclopropa[4,5]-
20
furo[2,3-f]pyrano[2,3-h]chromen-6-one; 4). White amorphous powder. [a] ¼ ꢀ4.0 (c ¼ 0.005, CHCl ).
D
3
UV (MeOH): 228, 255, 338. IR (KBr): 3404 (OH), 2924, 1701, 1631, 1572, 1458, 1419, 1363, 1198, 1074,
þ
1
1
020, 903, 764, 698. EI-MS: 386 (21, M ), 371 (100), 333 (41), 317 (3), 178 (5), 149 (6). H- and
C-NMR: Tables 1 and 2, resp. HR-EI-MS: 386.1502 (M , C H O ; calc. 386.1518).
1
3
þ
þ
25
22
4
2
7-[(E)-Coumaroyloxy]friedelin-28-carboxylic Acid (¼(4aS,6aR,6bS,8aS,9R,12aS,12bS,14aR,
1
4bS)-14a-({[(2E)-3-(4-Hydroxyphenyl)prop-2-enoyl]oxy}methyl)-2,2,6a,8a,9,12b-hexamethyl-10-oxo-
2
0
icosahydropicene-4a(2H)-carboxylic Acid; 5). White amorphous powder. [a] ¼ þ49.4 (c ¼ 0.37,
D
CHCl ). UV (MeOH): 315, 231, 210. IR (KBr): 3415, 2948, 1701, 1604, 1514, 1451, 1392, 1267, 1169,
3
1
13
þ
þ
8
4
31, 733. H- and C-NMR: Tables 3. ESI-MS: 619 ([M þ H] ). EI-MS: 618 (8, M ), 455 (4), 454 (10),
þ þ
41 (12), 409 (6), 395 (45), 164 (40), 163 (9), 147 (100), 119 (31). HR-EI-MS: 618.3925 (M , C H O ;
39 54 6
calc. 618.3920).
2
7-[(Z)-Coumaroyloxy]friedelin-28-carboxylic Acid (¼(4aS,6aR,6bS,8aS,9R,12aS,12bS,14aR,
1
4bS)-14a-({[(2Z)-3-(4-Hydroxyphenyl)prop-2-enoyl]oxy}methyl)-2,2,6a,8a,9,12b-hexamethyl-10-oxo-
2
0
icosahydropicene-4a(2H)-carboxylic Acid; 6). White amorphous powder. [a] ¼ ꢀ17.0 (c ¼ 0.05,
D
CHCl ). UV (MeOH): 310, 228, 212. IR (KBr): 3392, 2949, 2870, 1701, 1604, 1514, 1450, 1390, 1168,
3
þ
1
13
þ
9
(
6
83, 841, 733. ESI-MS: 619 ([M þ H] ). H- and C-NMR: Tables 3. EI-MS: 618 (14, M ), 454 (9), 441
20), 409 (17), 396 (15), 395 (48), 189 (17), 177 (25), 164 (12), 163 (17), 147 (100), 119 (19). HR-EI-MS:
þ
þ
6
18.3928 (M , C H O ; calc. 618.3920).
39 54
Apetalolide (¼ 5-Methoxy-8,8-dimethyl-10-[(2E)-2-methylbut-2-enoyl]-4-phenyl-2H,8H-pyrano[3,2-
g]chromen-2-one; 7). White amorphous powder. UV (MeOH): 352, 272, 237. IR (KBr): 3435, 2927, 2852,
þ
1
3
737, 1641, 1577, 1463, 1344, 1135, 1016, 864, 749, 700. EI-MS: 416 (19, M ), 402 (24), 401 (100), 389 (49),
þ
þ
71 (74), 333 (27), 317 (5), 178 (4), 77 (5). HR-EI-MS: 416.1628 (M , C H O ; calc. 416.1623).
26
24
5
Preparation of the (R)- and (S)-MTPA Esters of Inophyllum D (8). A soln. of (R)-a-methoxy-a-
trifluoromethyl)phenylacetic acid ((R)-MTPA) chloride (4.0 mg in 80 ml of benzene) was added to a
(
soln. of 8 (4 mg) in dry benzene (3 ml). A 0.05-mg aliquot of 4-(dimethylamino)pyridine (DMAP) and
Et N (15 ml) were added, and the mixture was refluxed. After 3 h, a second portion of (R)-MTPA
3
chloride (4.0 mg) was added, and the mixture was refluxed for an additional 2 h. When the mixture was

Helvetica Chimica Acta – Vol. 93 (2010)
1821
cooled, benzene (10 ml) was added, and the org. phase was succesively washed with 10% HCl (5 ml),
n NaHCO (10 ml), and H O (20 ml). The soln. was evaporated to dryness, and subjected to HPTLC
1
3
2
(
(
PE/AcOEt 4 :1) to give (R)-MTPA ester of inophyllum D (2.2 mg). (S)-MTPA Ester of inophyllum D
2.3 mg) was produced in the same way.
1
(
S)-MTPA Ester of Inophyllum D. H-NMR (600 MHz, CDCl ): 7.62 (m, 2 arom. H of MPTA); 7.40
3
(
m, 3 arom. H of MPTA); 7.22 – 7.39 (m, HꢀC(14,15,16,17,18)); 6.47 (d, J ¼ 9.8, HꢀC(8)); 5.97 (s,
HꢀC(3)); 5.34 (d, J ¼ 9.8, HꢀC(7)); 4.06 (dq, J ¼ 2.1, 6.0, HꢀC(10)); 3.55 (s, MeO of MPTA); 2.21 (ddq,
J ¼ 2.1, 1.9, 6.5, HꢀC(11)); 6.14 (d, J ¼ 1.9, HꢀC(12)); 0.93 (s, Me(19,20)); 1.33 (d, J ¼ 6.0, Me(21)); 0.93
þ
(d, J ¼ 6.5, Me(22)). ESI-MS: 621.2 ([M þ H] ).
1
(
R)-MTPA Ester of Inophyllum D. H-NMR (600 MHz, CDCl ): 7.63 (m, 2 arom. H of MPTA); 7.39
3
(
m, 3 arom. H of MPTA); 7.20 – 7.36, (m, HꢀC(14,15,16,17,18)); 6.51 (d, J ¼ 10.0, HꢀC(8)); 5.93 (s,
HꢀC(3)); 5.35 (d, J ¼ 9.8, HꢀC(7)); 4.46 (dq, J ¼ 2.0, 6.6, HꢀC(10)); 3.55 (s, MeO of MPTA); 2.22 (ddq,
J ¼ 2.0, 1.9, 6.9, HꢀC(11)); 6.25 (d, J ¼ 1.9, HꢀC(12)); 0.93 (s, Me(19,20)); 1.44 (d, J ¼ 6.6, Me(21)); 0.96
þ
(d, J ¼ 6.9, Me(22)). ESI-MS: 621.2 ([M þ H] ).
Hydrolysis of 1, 2, and 3. A soln. of 1 (5.0 mg) in THF/H O 2 :1 (2 ml) was treated with 6n HCl
2
(
40 ml). The mixture was stirred at r.t. for 72 h, and then CHCl (20 ml) was added. The org. phase was
3
washed with 1n NaHCO and evaporated to dryness to give 8 (2.5 mg), which was confirmed by co-TLC.
3
2
0
20
[
a] ¼ þ40.0 (c ¼ 0.10, CHCl ; [a] ¼ þ35.0 (c ¼ 1.86, CHCl ) [4]). The same procedure was repeated
D
3
D
3
2
0
2
0
with 2 and 3 to give 8 with [a] ¼ þ29.0 (c ¼ 0.06, CHCl ) and [a] ¼ þ33.0 (c ¼ 0.08, CHCl ), resp.
D
3
D
3
This work was financially supported by the National Science & Technology Major Project ꢀKey New
Drug Creation and Manufacturing Programꢁ, P. R. China (No. 2009ZX09301-001). The authors also
thank Prof. Ding Fang at the Guangxi Institute of Chinese Traditional Medicine for identification of the
plant material.
REFERENCES
[
1] Chinese Traditional Medicine Corporation, ꢃResource of Chinese Traditional Medicineꢀ, Science
Press, Beijing, 1994, p. 419.
[
2] M. C. Yimdjo, A. G. Azebaze, A. E. Nkengfack, A. M. Meyer, B. Bodo, Z. T. Fomum, Phytochem-
istry 2004, 65, 2789.
[3] K. Kawazu, H. Ohigashi, T. Mitsui, Tetrahedron Lett. 1968, 9, 2383.
[4] K. Kawazu, H. Ohigashi, N. Takahashi, T. Mitsui, Bull. Inst. Chem. Res. 1972, 50, 160.
[5] A. D. Patil, A. J. Freyer, D. S. Eggleston, R. C. Haltiwanger, M. F. Bean, P. B. Taylor, M. J. Caranfa,
A. L. Breen, H. R. Bartus, R. K. Johnson, R. P. Hertzberg, J. W. Westley, J. Med. Chem. 1993, 36,
4
131.
6] T. R. Govindachari, N. Viswanathan, B. R. Pai, U. Ramadas Rao, M. Srinivasan, Tetrahedron 1967,
3, 1901.
[
[
2
7] Y. Kashman, K. R. Gustafson, R. W. Fuller, J. H. Cardellina II, J. B. McMahon, M. J. Currens, R. W.
Buckheit Jr., S. H. Hughes, G. M. Cragg, M. R. Boyd, J. Med. Chem. 1992, 35, 2735.
8] C. J. Palmer, J. L. Josephs, J. Chem. Soc., Perkin Trans. 1 1995, 3135.
[
[
9] C.-I. Chang, Y.-H. Kuo, Chem. Pharm. Bull. 1998, 46, 1627.
[10] S. K. Nigam, C. R. Mitra, G. Kunesch, B. C. Das, J. Polonsky, Tetrahedron Lett. 1967, 18, 2633.
[11] S. P. Joshi, V. B. Deodhar, U. D. Phalgune, Indian J. Chem., Sect. B 2000, 39, 560.
[12] K. R. Markham, B. Ternai, R. Stanley, H. Geiger, T. J. Mabry, Tetrahedron 1978, 34, 1389.
Received December 28, 2009