Inhibitory effects on HIV-1 protease of tri-p-coumaroylspermidine from Artemisia caruifolia and related amides

Article abstract of DOI:10.1248/cpb.49.915

From a methanol extract of Artemisia caruifolia, which showed a moderate inhibitory activity on HIV-1 protease in a preliminary screening, N¹,N⁵,N¹⁰-tri-p-coumaroylspermidine and three dicaffeoylquinic acids were isolated.

Full text of DOI:10.1248/cpb.49.915

July 2001
Notes
Chem. Pharm. Bull. 49(7) 915—917 (2001)
915
Inhibitory Effects on HIV-1 Protease of Tri-p-coumaroylspermidine from
Artemisia caruifolia and Related Amides
Chao-mei MA, Norio NAKAMURA, and Masao HATTORI*
Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930–0194, Japan.
Received January 11, 2001; accepted March 21, 2001
From a methanol extract of Artemisia caruifolia, which showed a moderate inhibitory activity on HIV-1 pro-
tease in a preliminary screening, N1,N5,N10-tri-p-coumaroylspermidine and three dicaffeoylquinic acids were iso-
lated. The former compound was found to appreciably inhibit HIV-1 protease. Of related amides which were
chemically synthesized, N1,N5,N10,N14-tetra-p-coumaroylspermine and N1,N4,N7,N10,N13-penta-p-coumaroylte-
traethylenepentamine inhibited HIV-1 protease more potently than N1,N5,N10-tri-p-coumaroylspermidine.
Key words Artemisia caruifolia; tri-p-coumaroylspermidine; tetra-p-coumaroylspermine; penta-p-coumaroyltetraethylenepenta-
mine; dicaffeoylquinic acid; human immunodeficiency virus type 1 protease inhibitor
During the replication of AIDS virus (HIV), the viral reported. The structure elucidation of compound 1 was per-
polyprotein must be cleaved by viral protease (PR) to gener- formed as follows.
ate essential viral enzymes, such as reverse transcriptase, in-
The molecular weight of 1 was determined to be 583 by
1
tegrase and PR itself, as well as the viral structural proteins. electrospray ionization (ESI)-MS. In its H-NMR spectrum,
HIV PR has been proved to be one of the therapeutic targets compound 1 displayed signals of large coupling constants
and some peptide mimetic compounds targeting the active (Jϭ15.5—16.0 Hz) at d 6.35—6.45 and 7.55—7.60, charac-
site of HIV PR have been synthesized as potent anti-HIV teristic of the spinning system of the olefinic part of E-p-
drugs able to be used clinically. However, high dosages of coumaroyl. The signals of aromatic protons were greatly
these drugs are usually needed, which has led to severe side overlapped. The 13C-NMR spectrum showed signals attribut-
effects and thus long term administration is difficult for able to E-p-coumaroyl moieties. The signals at the upper
AIDS patients to adhere to.1) This means that novel types of field could be assigned to a spermidine moiety by analysis of
anti-HIV agents are urgently needed.
its two dimensional (2D) NMR spectrum. The structure of 1
In the present paper, we report the isolation of N1,N5,N10- was then deduced to be N1,N5,N10-tri-p-coumaroylspermi-
tri-p-coumaroylspermidine as an HIV-1 PR inhibitor from dine. This compound had been reported from the flowers of
Artemisia caruifolia and the synthesis and inhibitory activity Crataegus species as a mixture of E and Z forms. The struc-
of some related amides.
ture had been established by spectral analysis of its perme-
thylated product.7) Most recently, this compound was isolated
Results and Discussion
from the species of the genera Aphelandra8) and Helianthus
In a preliminary screening, a methanol extract of annuus L.9) However, the H-NMR spectrum of this com-
Artemisia caruifolia was found to have a moderate inhibitory pound had not yet been assigned clearly. In the present exper-
activity on HIV-1 PR (38.5% at 100 mg/ml). This extract was iment, this compound was isolated as a pure E-form and its
divided into CHCl3-, AcOEt-, BuOH- and H2O-soluble frac- 500 MHz 1H-NMR spectrum could be interpreted by detailed
tions. Improved inhibitory activity was found in the CHCl3- analysis of its 2D NMR spectra. Though the spectrum was
and AcOEt-soluble fractions, which showed 44.6% and considerably complex due to the overlapping and splitting of
44.9% inhibition, respectively, at the same concentration. signals, it revealed that all of the three p-coumaroyl residues
1
The BuOH- and H2O-soluble fractions showed no activity.
were in E-forms and only two conformational isomers were
The CHCl3-soluble fraction was further separated into sev- present. As represented by proton signals of 7, 8, 7Ј, 8Ј, 7Љ
eral sub-fractions, and from these, a guaianolide,2) a germa- and 8Љ, the nearer a proton to the center nitrogen, the larger
cranolide,2) eight guaiane dimers,2) two triterpenes,3)
a
was the split of its signal. This phenomenon indicated that
flavonoid,3) and eight lignans3) were isolated. These com- the splitting of signals was mainly due to the sterically hin-
pounds showed inhibitory activity on HIV-1 protease of 22— dered umbrella-like inversion at the center N atom. The
46% at 100 mg/ml.
structure of 1 was further confirmed by synthesis to be
The AcOEt soluble part was further divided into 3 frac- N1,N5,N10-tri-p-coumaroylspermidine.
tions by octadecylsilyl (ODS) column chromatography (40%
Compounds 1—4 were tested for their inhibitory activity
MeOH eluate, 65% MeOH eluate and MeOH eluate). In- on HIV-1 PR. In comparing their potent inhibitory activity on
creased inhibitory activity was observed in the second eluate HIV-1 integrase and moderate anti-HIV activity,10,11) 2—4
(53.9% at 100 mg/ml) while the other two eluates showed showed no inhibitory activity against HIV-1 PR. Appreciable
only weak activity (21—25% inhibition at 100 mg/ml). Re- HIV-1 PR inhibitory activity was observed only in 1. In addi-
peated chromatography of the 65% MeOH eluate on silica tion, the corresponding amine (spermidine) and acid (E-p-
gel with CHCl3–MeOH (8 : 2) and Sephadex LH-20 with coumaric acid) showed no activity against HIV-1 PR. To de-
20—100% MeOH afforded compounds 1—4.
velop more potent inhibitors, several similar amides were
Compounds 2—4 were identified as methyl 3,5-dicaf- synthesized and tested for their inhibitory activity against
feoylquinate,4) 1,3-dicaffeoylquinic acid5) and 3,5-dicaf- HIV-1 PR. Two of the synthesized amides (7, 8) with longer
feoylquinic acid6) by comparing their spectral data with those chains, more amide bonds and free hydroxy groups in their
To whom correspondence should be addressed. e-mail: saibo421@ms.toyama-mpu.ac.jp
© 2001 Pharmaceutical Society of Japan

916
Vol. 49, No. 7
GEMINI 300 (1H, 300 MHz; 13C, 75 MHz) or Varian UNITY 500 (1H,
500 MHz; 13C, 125 MHz) or JEOL JNM-LA 400WB-FT (1H, 400 MHz; 13C,
100 MHz) spectrometer, the chemical shifts being represented as ppm with
tetramethylsilane as an internal standard. ESI-MS spectra were measured
with a Perkin-Elmer SCIEX API-III biomolecular mass analyzer. FAB-MS
and high resolution (HR)-FAB-MS were measured with a JEOL JMS-700T
mass spectrometer, and m-nitrobenzyl alcohol was used as a matrix.
Plant Material The aerial part of Artemisia caruifolia BUCH.-HAM. ex
ROXB. was purchased from Yaocaigongyingzhan of Huhhot, Inner Mongolia
of the People’s Republic of China in September of 1998. The plant material
was identified by Dr. Katsuko Komatsu of the Analytical Research Center
for Ethnomedicines, Institute of Natural Medicine, Toyama Medical and
Pharmaceutical University. A voucher specimen (TMPW No. 19154) is
stored at the Museum of Materia Medica, Toyama Medical and Pharmaceu-
tical University, Japan.
Table 1. Inhibitory Activity of Compounds Isolated from the EtOAc Ex-
tract of A. caruifolia and Related Amides on HIV PR
Compound
% Inhibition (100 mg/ml)
IC50 (mg/ml)
1
2
3
4
5
6
7
8
70.2Ϯ1.4
30.1Ϯ7.8
0.0Ϯ3.2
2.5Ϯ1.4
2.7Ϯ0.6
4.0Ϯ4.3
99.5Ϯ0.8
97.3Ϯ1.7
53
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
27
30
Extraction and Isolation The aerial part of A. caruifolia (3.0 kg) was
extracted with MeOH under reflux (20 lϫ3, each 2 h). The combined MeOH
solutions were evaporated to give a residue (190 g). The residue was sus-
pended in water and extracted with CHCl3, AcOEt, and BuOH to give the
respective fractions in yields of 96, 11 and 21 g, together with the residual
water-soluble fraction (62 g). The AcOEt-soluble fraction was chro-
matographed on an ODS column eluted with increasing amounts of MeOH
in H2O (40% MeOH eluate, 3.4 g; 65% MeOH eluate, 5.1 g; MeOH eluate,
1.1 g). Further repeated chromatography of the second eluate on silica gel
with CHCl3–MeOH (8 : 2) and Sephadex LH-20 with 20—100% MeOH af-
forded compounds 1—4 (30, 40, 30 and 50 mg, respectively).
N1,N5,N10-Tri-p-coumaroylspermidine (1): White powder, IR (KBr): nmax
3300 (OH), 1660 (CONH–), 1610, 1580, 1520, 1450 (benzene ring), 1220,
1160, 980, 830, 520 cmϪ1. Negative ESI-MS: m/z 582 ([MϪ1]Ϫ, 70), 462
(50), 342 (45), 205 (30), 115 (100). Positive ESI-MS: m/z 607 ([MϩNa]ϩ,
30), 584 ([MϩH]ϩ, 35), 438 (70), 204 (70), 147 (100). 1H-NMR (500 MHz,
CD3OD): d: 1.60 (2H, m, H-8), 1.67 (2H, m, H-7), 1.85 (quin.
Jϭ7.0 Hz)/1.92 (quin. Jϭ7.0 Hz) (2H, H-3), 3.33 (4H, m, H-2, 9), 3.52 (4H,
m, H-4, 6), 6.37 (d, Jϭ15.0 Hz)/6.40 (d, Jϭ15.0 Hz) (H-8Ј), 6.42 (d,
Jϭ15.5 Hz) (H-8ٞ), 6.71 (d, Jϭ9.0 Hz)/6.76 (overlapped) (H-3Љ, 5Љ), 6.77
(overlapped)/6.78 (overlapped) (H-3Ј, 5Ј), 6.79 (overlapped) (H-3ٞ, 5ٞ),
6.81 (d, Jϭ15.5 Hz)/6.86 (d, Jϭ15.5 Hz) (H-8Љ), 7.35 (d, Jϭ9.0 Hz)/7.39 (d,
Jϭ9.0 Hz) (H-2Ј, 6Ј), 7.39 (d, Jϭ9.0 Hz) (H-2ٞ, 6ٞ), 7.43 (overlapped)/7.44
(overlapped) (H-7Ј), 7.43 (overlapped)/7.47 (d, Jϭ9.0 Hz) (H-2Љ, 6Љ), 7.47
(d, Jϭ15.5 Hz)/7.48 (d, Jϭ15.5 Hz) (H-7ٞ), 7.50 (d, Jϭ15.5 Hz)/7.53 (d,
Jϭ15.5 Hz) (H-7Љ). 13C-NMR (100 MHz, CD3OD): d: 26.3/27.9 (C-7),
27.8/27.9 (C-8), 28.9/30.6 (C-3), 37.9/38.2 (C-2), 39.9/40.1 (C-9), 45.7/47.0
(C-4), 47.6/49.0 (C-6), 114.9 (C-8Љ), 116.8 (C-3Ј, 3Љ, 3ٞ, 5Ј, 5Љ, 5ٞ),
118.2/118.3 (C-8Ј), 118.5 (C-8ٞ), 127.7 (C-1Ј, 1ٞ), 127.9 (C-1Љ), 130.6 (C-
2Ј, 6Ј, 2ٞ, 6ٞ), 130.9 (C-2Љ, 6Љ), 141.8/141.9/142.2 (C-7Ј, 7ٞ), 144.4 (C-7Љ),
160.5/160.7 (C-4Ј, 4Љ, 4ٞ), 169.3 (C-9Ј), 169.2/169.4 (C-9Љ), 169.3 (C-9ٞ).
Methy 3, 5-Dicaffeoylquinate (2)4): White powder, positive ESI-MS m/z
553 ([MϩNa]ϩ, 100), 385 (40). Negative ESI-MS m/z: 529 ([MϪ1]Ϫ, 100),
367 (80), 179 (77). 1H-NMR (500 MHz, CD3OD): d: 2.14 (dd, Jϭ13.5,
8.5 Hz, Ha-2), 2.18 (dd, Jϭ13.5, 3.5 Hz, Ha-6), 2.29 (dd, Jϭ13.5, 6.5 Hz,
Hb-6), 2.32 (dd, Jϭ13.5, 4.0 Hz, Hb-2), 3.68 (3H, –OCH3), 3.98 (dd, Jϭ6.5,
3.0 Hz, H-4), 5.31 (m, H-5), 5.39 (dt, Jϭ8.5, 4.0 Hz, H-3), 6.21 (d,
Jϭ16.0 Hz, H-8Љ), 6.34 (d, Jϭ16.0 Hz, H-8Ј), 6.77* (d, Jϭ8.5 Hz, H-5Ј),
6.78* (d, Jϭ8.5 Hz, H-5Љ), 6.95** (dd, Jϭ8.5, 2.5 Hz, H-6Љ), 6.96** (dd,
Jϭ8.5, 2.5 Hz, H-6Ј), 7.05 (d, Jϭ2.5 Hz, H-2Ј), 7.06 (d, Jϭ2.5 Hz, H-2Љ),
7.54 (d, Jϭ16.0 Hz, H-7Ј), 7.61 (d, Jϭ16.0 Hz, H-7Љ). (*, **: Assignments
may be exchangeable.)
1,3-Dicaffeoylquinic Acid (3)5): White powder, positive ESI-MS m/z 539
([MϩNa]ϩ, 100), 413 (80), 385 (50). 1H-NMR (400 MHz, CD3OD): d: 2.02
(dd, Jϭ13.9, 8.9 Hz, Ha-2), 2.38 (2H, m, H-6), 2.52 (dd, Jϭ13.9, 3.8 Hz,
Hb-2), 3.74 (dd, Jϭ8.3, 3.4 Hz, H-4), 4.25 (q, Jϭ4.3 Hz, H-5), 5.34 (td,
Jϭ8.3, 3.6 Hz, H-3), 6.25* (d, Jϭ16.3 Hz, H-8Ј), 6.26* (d, Jϭ16.0 Hz, H-
8Љ), 6.74 (2H, d, Jϭ8.3 Hz, H-5Ј, 5Љ), 6.92 (2H, dd, Jϭ8.3, 2.0 Hz, H-6Ј, 6Љ),
7.01 (2H, d, Jϭ2.0 Hz, H-2Ј, 2Љ), 7.54 (2H, d, Jϭ16.3 Hz, H-7Ј, 7Љ). (*: As-
signments may be exchangeable.)
p-coumaroyl moieties were found to be more potent HIV-1
PR inhibitors than 1. However, tris(2-p-coumaroy-
laminoethyl)amine (5), which possesses the same number of
amide bonds as 1, showed no inhibitory activity on HIV-1
PR. On acetylation of hydroxyl groups of the p-coumaroyl
residues as in 1a, 7a and 8a, the activity disappeared com-
pletely. These polyamides are a new type of HIV-1 PR in-
hibitors and are easy to synthesize from inexpensive starting
materials, which may enable investigaton of their interaction
with the enzyme at the molecular level, and conduct of the
rational design and synthesis of more preferable inhibitors
against HIV-1 PR.
3,5-Dicaffeoylquinic Acid (4)6): White powder, positive ESI-MS m/z 539
1
([MϩNa]ϩ, 100), 413 (70). H-NMR (300 MHz, CD3OD): d: 2.26 (4H, m,
H-2, 6), 4.01 (dd, Jϭ7.5, 3.3 Hz, H-4), 5.46 (2H, m, H-3, 5), 6.30* (d,
Jϭ16.0 Hz, H-8Ј), 6.39* (d, Jϭ16.0 Hz, H-8Љ), 6.82 (2H, d, Jϭ8.1 Hz, H-5Ј,
5Љ), 7.01 (2H, dd, Jϭ8.1, 1.5 Hz, H-6Ј, 6Љ), 7.10 (2H, d, Jϭ1.5 Hz, H-2Ј, 2Љ),
7.62** (d, Jϭ16.0 Hz, H-7Ј), 7.66** (d, Jϭ16.0 Hz, H-7Љ). (*, **: Assign-
ments may be exchangeable.)
Experimental
Synthesis of Polycoumaroylamines A mixture of E-p-coumaric acid
Apparatus 1H- and 13C-NMR spectra were measured with a Varian (1.0 g) and Ac2O (10 ml) in 10 ml of pyridine was stirred over night at room

July 2001
917
temperature and worked up as usual to afford 1.1 g of (E)-4-O-acetyl- (12H, s, CH3CO–), 3.42 (4H, overlapped with solvent, H-2, 13), 3.51 (8H,
coumaric acid. One gram of (E)-4-O-acetylcoumaric acid in 7 ml of SOCl2 m, H-4, 6, 9, 11), 6.48 (2H, m), 6.81 (2H, m), 7.08 (8H, m), 7.55 (12H, m)
was refluxed at 80 °C for 16 h, then the mixture was evaporated to dryness to (H-ac-cou).
give (E)-4-O-acetylcoumaroyl chloride.
N1,N5,N10,N14-Tetra-p-coumaroylspermine (7)13)
: White powder, 91%
To (E)-4-O-acetylcoumaroyl chloride (0.5 mmol) in 15 ml tetrahydrofuran yield, negative ESI-MS: m/z 785 ([MϪH]Ϫ, 7). 1H-NMR (300 MHz,
(THF) was added 15 ml THF solution of 0.17 mmol of spermidine [or CD3OD): d: 1.69 (4H, m, H-7, 8), 1.86 (4H, m, H-3, 12), 3.31 (4H, over-
0.17 mmol of tris (2-aminoethyl)amine, or 0.13 mmol of spermine, or lapped with solvent, H-2, 13), 3.54 (8H, m, H-4, 6, 9, 11), 6.40 (2H, m),
0.25 mmol of putrescine, or 0.10 mmol of tetraethylenepentamine], and 6.79 (10H, m), 7.40 (12H, m) (H-cou).
100 ml triethylamine. The mixture was stirred at room temperature overnight
N1,N4,N7,N10,N13-Penta(4-O-acetylcoumaroyl)tetraethylenepentamine
and then water (20 ml) was added. After being concentrated, the residue was (8a): White powder, 51% yield, positive FAB-MS: m/z 1130 ([Mϩ1]ϩ, 25),
purified with ODS column chromatography eluted with H2O–MeOH (6 : 4— 307 (13). 1H-NMR (300 MHz, CDCl3): d: 2.30 (15H, s, CH3CO–), 3.5—3.8
0 : 10) to afford the corresponding 4-O-acetylcoumaroylamides (1a, 5a—
(16H, m, H-2, 3, 5, 6, 8, 9, 11, 12), 6.3—6.5 (2H, m), 6.9—7.7 (28H, m) (H-
8a). To a solution of 4-O-acetylcoumaroylamide (50 mg) in CH3OH–THF ac-cou). HR-FAB-MS m/z 1130.4347 (Calcd for C63H64N5O15 [MϩH]ϩ,
(1 : 1) was added 0.1 N KOH/MeOH 12 ml. After stirring for 1.5 h at room 1130.4400).
temperature, the solution was neutralized with 0.1 N HCl and purified by
N1,N4,N7,N10,N13-Penta-p-coumaroyltetraethylenepentamine (8): White
ODS column chromatography eluted with H2O–MeOH (8 : 2—0 : 10) to give powder, 87% yield, positive FAB-MS: m/z 920 ([Mϩ1]ϩ, 7), 307 (15), 289
the corresponding coumaroylamides (1, 5—8).
(10). 1H-NMR (300 MHz, CD3OD): d: 3.4—3.9 (16H, m, H-2, 3, 5, 6, 8, 9,
N1,N5,N10-Tri(4-O-acetylcoumaroyl)spermidine (1a): White powder, 69% 11, 12), 6.2—6.5 (2H, m), 6.6—7.0 (13H, m), 7.2—7.6 (15H, m) (H-ac-
yield, positive FAB-MS: m/z 710 ([Mϩ1]ϩ, 15). 1H-NMR (300 MHz, cou). HR-FAB-MS m/z 920.3834 (Calcd for C53H54N5O10 [MϩH]ϩ,
CD3OD): d: 1.68 (4H, m, H-7, 8), 1.89 (2H, m, H-3), 2.25 (9H, s, CH3CO–), 920.3872).
3.31 (4H, overlapped, H-2, 9), 3.58 (4H, m, H-4, 6), 6.52 (d,
HIV PR Assay HIV PR assay kit (3700 Horizon Drive, King of Prussia,
Jϭ16.0 Hz)/6.57 (d, Jϭ16.0 Hz), 6.57 (1H, d, Jϭ16.0 Hz), 6.97 (2H, m), PA 19406, Kit Lot No 1) was used. Assay was performed and inhibitory ac-
7.11 (5H, m), 7.58 (9H, m) (aromatic and olefinic protons). HR-FAB-MS tivity was calculated as described previously.14) Acetyl pepstatin was used as
m/z 710.3086 (Calcd for C40H44N3O9 [MϩH]ϩ, 710.3078).
a positive control and showed an IC50 of 0.07 mM.
Tris[2-(4-O-acetylcoumaroyl)aminoethyl]amine (5a): White powder, 53%
yield, positive FAB-MS: m/z 711 ([Mϩ1]ϩ, 30), 492 (10). 1H-NMR (300 References
MHz, CD3OD): d: 2.29 (9H, s, CH3CO–), 2.70 (6H, m, H-1, 1Ј, 1Љ), 3.43
(6H, m, H-2, 2Ј, 2Љ), 6.63 (3H, d, Jϭ15.8 Hz, H-ac-cou-8), 6.86 (6H, d,
Jϭ8.4 Hz, H-ac-cou-3, 5), 7.25 (6H, d, Jϭ8.4 Hz, H-ac-cou-2, 6), 7.39 (3H,
d, Jϭ15.8 Hz, H-ac-cou-7). HR-FAB-MS m/z 711.3002 (Calcd for
C39H43N4O9 [MϩH]ϩ, 711.3031).
Tris(2-p-coumaroylaminoethyl)amine (5): White powder, 89% yield, posi-
tive FAB-MS: m/z 585 ([Mϩ1]ϩ, 25), 307 (15). 1H-NMR (300 MHz,
CD3OD): d: 2.67 (6H, t, Jϭ5.1 Hz, H-1, 1Ј, 1Љ), 3.39 (6H, t, Jϭ5.1 Hz, H-2,
2Ј, 2Љ), 6.49 (3H, d, Jϭ15.7 Hz, H-cou-8), 6.59 (6H, d, Jϭ8.5 Hz, H-cou-3,
6), 7.19 (6H, d, Jϭ8.5 Hz, H-cou-2, 6), 7.39 (3H, d, Jϭ15.7 Hz, H-cou-7).
HR-FAB-MS m/z 585.2714 (Calcd for C33H37N4O6 [MϩH]ϩ, 585.2714).
N1,N6-Di(4-O-acetylcoumaroyl)putrescine (6a): White powder, 64%
yield, positive FAB-MS: m/z 465 ([Mϩ1]ϩ, 15), 307 (10), 289 (8). 1H-NMR
(300 MHz, CDCl3–CD3OD 1 : 2): d: 1.64 (4H, m, H-3, 4), 2.32 (6H, s,
CH3CO–), 3.36 (4H, overlapped with solvent, H-2, 5), 6.49 (2H, d,
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N1,N6-Di-p-coumaroylputrescine (6)12): White powder, 90% yield, no
1
peak appeared in ESI-MS, H-NMR (300 MHz, CD3OD): d: 1.64 (4H, m,
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A1 12 Jul 2000, 11 pp.
N1,N5,N10,N14-Tetra(4-O-acetylcoumaroyl)spermine (7a)13): White pow-
der, 70% yield, positive ESI-MS: m/z 978 ([MϩNa]ϩ, 100), 955 ([MϩH]ϩ, 14) Ma C-M., Nakamura N., Miyashiro H., Hattori M., Shimotohono K.,
75). negative ESI-MS: m/z 953 ([MϪH]Ϫ, 7). 1H-NMR (300 MHz,
CDCl3–CD3OD 2 : 1): d: 1.68 (4H, m, H-7, 8), 1.84 (4H, m, H-3, 12), 2.31
Chem. Pharm. Bull., 47, 141—145 (1999).