Cremastrine, a pyrrolizidine alkaloid from Cremastra appendiculata

Article abstract of DOI:10.1021/np049650x

A new pyrrolizidine alkaloid, cremastrine (1), was isolated from the bulbs of Cremastra appendiculata. Its configuration was determined by spectroscopic and chemical analyses. Compound 1 inhibited the binding of tritium-labeled N-methylscopolamine to the muscarinic M3 receptor with a K_i value of 126 nM.

Full text of DOI:10.1021/np049650x

572
J. Nat. Prod. 2005, 68, 572-573
Cremastrine, a Pyrrolizidine Alkaloid from Cremastra appendiculata
Yoshitaka Ikeda,*,† Hikaru Nonaka,† Tamotsu Furumai,‡ and Yasuhiro Igarashi‡
Pharmaceuticals Research Unit, Mitsubishi Pharma Corporation, 2-25-1, Shodai-Ohtani, Hirakata, Osaka 573-1153, Japan,
and Biotechnology Research Center, Toyama Prefectural University, 5180, Kurokawa, Kosugi, Toyama 939-0398, Japan
Received November 4, 2004
A new pyrrolizidine alkaloid, cremastrine (1), was isolated from the bulbs of Cremastra appendiculata.
Its configuration was determined by spectroscopic and chemical analyses. Compound 1 inhibited the
binding of tritium-labeled N-methylscopolamine to the muscarinic M3 receptor with a Ki value of 126
nM.
Anticholinergic alkaloids such as atropine have been
used for a century for the treatment of obstructive airway
diseases, although they have unfavorable peripheral and
central nervous system effects such as tachycardia, my-
driasis, and dementia.1 To date, five muscarinic acetylcho-
line receptor subtypes, m1-m5, have been identified and
cloned.2 Of these, M3 receptors, localized in smooth muscle
and mucosal glands, mediate contraction and mucus secre-
tion, respectively. Subtype-selective surrogate ligands that
recognize the different localizations and functions of these
receptor subtypes provide the possibility of developing
drugs since they may avoid the occurrence of adverse
effects. It has been hypothesized that the selective blockade
of muscarinic M3 receptors may be therapeutically useful
in the treatment of respiratory disorders such as chronic
obstructive pulmonary disease, gastrointestinal disorders
such as irritable bowel syndrome, and urinary tract
disorders such as urinary incontinence.3 Plants and mi-
croorganisms have been reliable sources of new drugs, and
much emphasis has been placed on finding novel drug
candidates from these sources. Despite extensive screening
studies, however, there is still no compound that can bind
selectively to M3 receptors.
In the course of our screening for muscarinic M3 receptor
binding inhibitors from natural sources, the extract of
Santsigu Tuber, a dried bulb of Cremastra appendiculata
(D.Don) Nakai (Orchidaceae), was found to be highly active.
Santsigu Tuber is used for the treatment of burns, chaps,
and frostbite in Japan and used as an antidote for snake
and insect bites in China. This plant is widely distributed
in Japan and China, but its chemical constituents and their
bioactivities have not been studied. Bioassay-guided chro-
matography of the extract of Santsigu Tuber provided a
new pyrrolizidine alkaloid, cremastrine (1), as an active
principle. This paper describes the isolation, structure
determination, and its inhibition of the muscarinic M3
receptor.
pound 1 was also obtained from the n-BuOH extract in a
similar manner.
Compound 1 was isolated as colorless syrup. It is alkaline
but was negative in the ninhydrin color reaction test,
indicating the presence of a tertiary amine group. The
molecular formula of 1 was determined as C14H25NO3 on
the basis of the high-resolution LC/MS ([M + H]+, m/z
256.1900) and 13C NMR spectroscopic data.
1
The H NMR spectrum of 1 showed the presence of one
hydroxyl group (δ 6.90) and two methyl groups (δ 1.08 and
0.95, H-5′ and -6′). The 13C NMR and DEPT spectra
revealed the presence of 14 carbon signals comprising one
ester carbonyl carbon (δ 174.0, C-1′), one oxymethine
carbon (δ 72.8, C-2′), one oxymethylene carbon (δ 63.0, C-9),
one nitrogen-bearing methine carbon (δ 66.5, C-8), and two
nitrogen-bearing methylene carbons (δ 54.3 and 52.4, C-3
and -5). The remaining carbon signals were assigned to two
methyls, four methylenes, and two methines. Further
analyses of COSY, HMQC, and HMBC spectra suggested
that 1 consisted of a pyrrolizidine (C-1-C-8) moiety and a
2-hydroxy-3-methylpentanoic acid (Hmp, C-1′-C-6′) moiety
connected through hydroxymethyl (C-9) at C-1. In addition,
HMBC correlations from the hydroxymethylene protons (H-
9) to the carbonyl carbon (C-1′) and to C-1, -2, and -8
indicated the position of the ester attachment at C-9. Thus,
1 was characterized as the Hmp ester of 1-hydroxymeth-
ylpyrrolizidine.
The dried bulbs of C. appendiculata were extracted with
70% aqueous EtOH, and the extract was concentrated in
vacuo. The residual aqueous solution was acidified with
HOAc and extracted with EtOAc. The aqueous layer was
then basified with aqueous ammonia solution and extracted
successively with EtOAc and n-BuOH. The alkaline EtOAc
extract was fractionated by preparative HPLC using an
ODS column. The active fractions were further purified to
afford 1 by preparative HPLC using a C30 column. Com-
Alkaline hydrolysis of 1 gave Hmp and 1-hydroxymeth-
ylpyrrolizidine. The absolute configuration of the Hmp
moiety was determined to be (2R, 3R) by comparison with
four synthetic stereoisomers of Hmp in a chiral HPLC
analysis.4 The 1-hydroxymethylpyrrolizidine moiety was
estimated as (-)-isoretronecanole (1S, 8S-form) or its
antipode based on the NOESY spectrum of 1 that showed
a positive NOE between the protons at H-1 and H-8,
indicating the cis-configuration for these protons. As the
optical rotation of the 1-hydroxymethylpyrrolizidine, [R]20
D
* To whom correspondence should be addressed. Tel: +81-72-856-9205.
Fax: +81-72-868-9597. E-mail: Ikeda.Yoshitaka@mc.m-pharma.co.jp.
† Mitsubishi Pharma Corporation.
-75.9 (c 0.027, EtOH), was in good agreement with that
of (-)-isoretronecanole, [R]20 -77.0 (c 1.0, EtOH),5 the
D
‡ Toyama Prefectural University.
absolute configuration of the 1-hydroxymethylpyrrolizidine
10.1021/np049650x CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/03/2005

Notes
Journal of Natural Products, 2005, Vol. 68, No. 4 573
Table 1. Ki Values of 1 to Five Subtypes, M1-M5
Ki (nM)
1.75-1.70 (2H, m, H-2b, H-4′a), 1.59 (1H, m, H-7b), 1.43 (1H,
m, H-4′b), 1.08 (3H, d, J ) 7.5 Hz, H-6′), 0.95 (3H, t, J ) 6.8
Hz, H-5′); 13C NMR (pyridine-d5, 125 MHz) δ 174.0 (C, C-1′),
72.8 (CH, C-2′), 66.5 (CH, C-8), 63.0 (CH2, C-9), 54.3 (CH2, C-5),
52.4 (CH2, C-3), 39.1 (CH, C-1), 38.1 (CH, C-3′), 25.9, 25.4, 25.3,
25.0 (CH2, C-2, -6, -7, -4′), 13.3 (CH3, C-6′), 10.9 (CH3, C-5′);
HRMS (positive mode) m/z 256.1900 [M + H]+ (calcd for C14H26-
NO3, 256.1913).
Ki (nM)
M1
M2
M3
505
>5517
126
M4
M5
498
1220
was determined as (1S, 8S). There are many reports on
the esters of 1-hydroxymethylpyrrolizidine6 such as the (-)-
trachelanthic acid ester (heliocurassavicine) and the (-)-
curassavic acid ester (heliocurassavine) of (-)-isoretrone-
canole isolated from Heliotropium curassavicum L.7 Crema-
strine (1) is the first example of the Hmp ester of 1-hy-
droxymethylpyrrolizidine including (-)-isoretronecanole.8
The inhibition activities of cremastrine (1) to muscarinic
receptor subtypes are summarized in Table 1. Compound
1 showed selective inhibition of the muscarinic M3 receptor.
It inhibits the binding of tritium-labeled N-methylscopol-
amine ([3H]-NMS) to the muscarinic M3 receptor with an
IC50 of 594 nM (Ki ) 126 nM). Some pyrrolizidine alkaloids
such as cynaustraline [viridifloric acid ester of (+)-iso-
retronecanole] showed antagonistic activity against the
muscarinic receptor in the guinea-pig ileum, but their
selectivity for M1-M5 receptors is unknown.9 Further
studies on the pharmacological functions of cremastrine (1)
are in progress.
Alkaline Hydrolysis of 1. A solution of 1 (2.3 mg) in 0.5
N NaOH (0.4 mL) was refluxed for 1 h. After cooling, the
reaction mixture was neutralized with HOAc and further
purified to afford Hmp (1.1 mg) and 1-hydroxymethylpyrrolizi-
dine (0.27 mg) by preparative HPLC under the following
conditions: column, XTerra Prep RP18 column (10 mm i.d. ×
150 mm, Waters, MA); eluent, CH3CN-aqueous ammonia
solution (pH 10.9, a linear gradient from 2.5% to 50% of CH3-
CN over 20 min); flow rate, 4.0 mL/min; oven temperature,
40 °C; detection, UV at 220 nm.
Synthesis of Hmp Stereoisomers. A solution of NaNO2
(103 mg) in water (1.0 mL) was added to a stirred and ice-
cooled solution of L-isoleucine (L-Ile, 131 mg) in 1 N H2SO4
(2.0 mL). The mixture was stirred for 13 h and then extracted
with Et2O (6.0 mL). The Et2O solution was washed with brine,
dried over Na2SO4 (anhydrous), and evaporated in vacuo to
give 2S,3S-Hmp (52.5 mg, 40%): 1H NMR (pyridine-d5, 500
MHz) δ 10.56 (2H, br s, -OH), 4.48 (1H, d, J ) 4.4 Hz, H-2),
2.21 (1H, dddq, J ) 4.4, 4.5, 7.0, 9.0 Hz, H-3), 1.86 (1H, ddq,
J ) 4.5, 7.5, 13.5 Hz, H-4a), 1.54 (1H, ddq, J ) 7.5, 9.0, 13.5
Hz, H-4b), 1.21 (1H, d, J ) 7.0 Hz, H-6), 0.96 (3H, t, J ) 7.5
Hz, H-5); 13C NMR (pyridine-d5, 125 MHz) δ 176.4 (C, C-1),
74.6 (CH, C-2), 38.4 (CH, C-3), 23.5 (CH2, C-4), 15.0 (CH3, C-6),
11.0 (CH3, C-5). In the same manner as described above, d-Ile,
allo-L-Ile, and allo-D-Ile afforded 2R,3R-Hmp, 2S,3R-Hmp, and
2R,3S-Hmp, respectively.
Experimental Section
General Experimental Procedures. Optical rotations
were measured on a JASCO P-1020 digital polarimeter using
a 5 cm cell. NMR spectra were measured on a Bruker AMX-
500 spectrometer using standard Bruker pulse programs.
Chemical shifts are given in δ values with reference to
tetramethylsilane as an internal standard. IR specra were
recorded on a Perkin Elmer 1725X FT-TR spectrophotometer.
The MS spectra were measured on an Agilent MSD spectrom-
eter, and high-resolution MS spectra were measured on a
JEOL JMS-700 spectrometer.
Chiral HPLC Analysis of Hmp. Chiral HPLC analysis
was achieved using SUMICHIRAL OA-5000 [4 mm i.d. × 150
mm (Osaka, Japan); flow rate, 0.7 mL/min; eluent, i-PrOH-
H2O containing 2.0 mM of CuSO4 (15:85); oven temperature,
40 °C; detection, UV at 238 nm]. The retention times of the
synthetic Hmp stereoisomers were as follows; 2S,3R-Hmp (37.0
min), 2S,3S-Hmp (42.4 min), 2R,3R-Hmp (58.9 min), and
2R,3S-Hmp (67.8 min). The retention time of the Hmp derived
from 1 was 58.9 min.
Plant Material. The plant material was purchased from
Alps Pharmaceutical Ind. Co. Ltd. (Gifu, Japan). A small
amount of the sample is preserved in our laboratory.
Muscarinic M3 Receptor Binding Assay. The binding
affinities (Ki) to five subtypes were determined by inhibition
of specific binding of [3H]-NMS using the human receptor
membranes at MDS Pharma Services (Taiwan). In competitive
experiments, membranes from insect Sf9 cells stably express-
ing cloned human m1-m5 were incubated with 0.29 nM [3H]-
NMS in the medium consisting of a buffer containing 50 mM
Tris-HCl, 10 mM MgCl2, and 1 mM EDTA at pH 7.4 and 25
°C for 1 h. Nonspecific binding was determined in the presence
of 1 µM atropine. IC50 values were determined from competi-
tion binding curves and converted to apparent Ki values using
the Cheng-Prusoff equation.10
Extraction and Isolation. The plant material (150 g) was
extracted with 70% aqueous EtOH (1.5 L) overnight at room
temperature, and the EtOH solution was concentrated in
vacuo. The residual aqueous solution (ca. 400 mL) was
adjusted to pH 2.8 with HOAc and extracted with EtOAc (250
mL, ×2), and the EtOAc layer was discarded. Then, the
aqueous layer was adjusted to pH 9.4 with aqueous ammonia
solution and extracted with EtOAc (250 mL ×3) and n-BuOH
(250 mL ×3). The EtOAc and n-BuOH layers were separately
evaporated in vacuo. The alkaline EtOAc extract (274.3 mg)
was fractionated by preparative HPLC using an ODS column
(Delta-Pak C18, 40 mm i.d. × 100 mm, Waters, MA) with the
eluent of CH3CN-0.05% TFA (a linear gradient from 5% to
50% of CH3CN over 20 min) at a flow rate of 80 mL/min. On
the basis of results of a muscarinic M3 receptor binding assay,
the active fractions were combined and evaporated in vacuo.
The active fraction was further purified to afford 1 (15.4 mg)
by preparative HPLC using a C30 column (Develosil C30-UG,
20 mm i.d. × 150 mm, Nomura Chemical Co. Ltd., Japan) with
CH3CN-0.2% HOAc (a linear gradient from 0% to 50% of CH3-
CN over 30 min) at a flow rate of 20 mL/min. Compound 1
(10.1 mg) was also obtained from the n-BuOH extract (998.0
mg) in a manner similar to that described above.
Acknowledgment. The authors thank Mr. T. Ogawa for
high-resolution LC/MS measurements.
References and Notes
(1) Goodman & Gilman’s The Pharmacologocal Basis of Therapeutics;
McGraw-Hill Companies, Inc.: New York, 1985; pp 130, and refer-
ences therein.
(2) Kubo, T.; Fukuda, K.; Mikami, A.; Maeda, A.; Takahashi, H.; Mishina,
M.; Haga, T.; Haga, K.; Ichiyama, A.; Kangawa, K.; Kojima, M.;
Matsuo, H.; Hirose, T.; Numa, S. Nature 1986, 323, 411-416.
(3) Wallis, R. M. Life Sci. 1995, 56, 861-868.
(4) Kobayashi, J.; Itagaki, F.; Shigemori, H.; Takao, T.; Shimonishi, Y.
Tetrahedron 1995, 51, 2525-2532.
(5) Southon, I. W.; Bukingham, J. Dictionary of Alkaloids; Chapman &
Hall Ltd.: London, 1989; p 543, and references therein.
(6) Roeder, E. Pharmazie 2000, 55, 711-726, and references therein.
(7) Mohanraj, S.; Subramanian, P. S.; Herz, W. Phytochemistry 1982,
21, 1775-1779.
(8) Dictionary of Natural Products on CD-ROM; Chapman & Hall Ltd.:
London, 2004; Version 13.2.
(9) Pomeroy, A. R.; Raper, C. Br. J. Pharmacol. 1971, 41, 683-690.
(10) Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099-3108.
Cremastrine (1): colorless syrup; [R]25 -26.8 (c 1.0,
D
EtOH); IR (film) νmax 3412, 2967, 2879, 1732, 1681, 1463, 1203,
1
1141 cm-1; H NMR (pyridine-d5, 500 MHz) δ 6.90 (1H, br s,
-OH), 4.50 (1H, d, J ) 3.9 Hz, H-2′), 4.33 (1H, dd, J ) 6.7,
11.1 Hz, H-9a), 4.24 (1H, dd, J ) 7.9, 11.1 Hz, H-9b), 4.17 (1H,
m, H-8), 3.64 (1H, br t, J ) 7.5 Hz, H-5a), 3.45 (1H, dt, J )
6.4, 11.1 Hz, H-3a), 2.93 (1H, br t, J ) 8.3 Hz, H-3b), 2.76
(1H, m, H-1), 2.66 (1H, dt, J ) 6.4, 10.3 Hz, H-5b), 2.03 (1H,
m, H-3′), 1.89 (1H, m, H-2a), 1.85-1.75 (3H, m, H-6, H-7a),
NP049650X