A new nonpeptide tachykinin NK1 receptor antagonist isolated from the plants of compositae

Article abstract of DOI:10.1248/cpb.50.47

To find new tachykinin NK₁ receptor antagonists from natural sources, we examined the tachykinin antagonist activity in the extracts of approximately 200 species of plants by the use of isolated guinea pig ileum. As a result, we discovered a no

Full text of DOI:10.1248/cpb.50.47

January 2002
Chem. Pharm. Bull. 50(1) 47—52 (2002)
47
A New Nonpeptide Tachykinin NK1 Receptor Antagonist Isolated from the
Plants of Compositae
Atsushi YAMAMOTO, Ko NAKAMURA,* Kazuhito FURUKAWA, Yukari KONISHI, Takashi OGINO,
Kunihiko HIGASHIURA, Hisashi YAGO, Kaoru OKAMOTO, and Masanori OTSUKA
Institute of Bio-Active Science, Nippon Zoki Pharmaceutical Co., Ltd., Kinashi, Yashiro-cho, Kato-gun, Hyogo 673–1461,
Japan. Received July 9, 2001; accepted October 19, 2001
To find new tachykinin NK1 receptor antagonists from natural sources, we examined the tachykinin antago-
nist activity in the extracts of approximately 200 species of plants by the use of isolated guinea pig ileum. As a re-
sult, we discovered a novel and potent NK1 receptor antagonist in the extract of dried flowers of Matricaria
chamomilla L. (chamomile). The structure of the antagonist was established as N1,N5,N10,N14-tetrakis[3-(4-hy-
droxyphenyl)-2-propenoyl]-1,5,10,14-tetraazatetradecane (tetracoumaroyl spermine, 1a). The Ki values of 1a, es-
timated from the inhibitory action on the substance P (SP)-induced contraction of the guinea pig ileum and the
inhibition of the binding of [3H][Sar9, Met(O2)11]SP to human NK1 receptors, were 21.9 nM and 3.3 nM, respec-
tively. 1a is the first potent NK1 receptor antagonist from natural sources and it has a unique structure of a poly-
acylated spermine. 1a was concentrated in pollen of Matricaria chamomilla L. and was also found in the extracts
of flowers of other four species of Compositae. In addition, we found N1,N5,N10-tris[3-(4-hydroxyphenyl)-2-
propenoyl]-1,5,10,14-tetraazatetradecane (2) as a new compound in the extract of flowers of Matricaria
chamomilla L., which did not exhibit any tachykinin antagonist activity. A number of related compounds were
synthesized, and the structure–activity relationship was studied.
Key words tachykinin antagonist; NK1; chamomile; Matricaria chamomilla; Compositae
Since the report of the first nonpeptide tachykinin receptor
The residual major part of the MeOH extract, therefore,
antagonist, CP-96,345,1) a large number of nonpeptide was partitioned between n-hexane and H2O. The H2O layer
tachykinin antagonists have been reported. Among these, was further partitioned between n-BuOH and H2O. The n-
tachykinin NK1 receptor antagonists are most numerous and BuOH layer was subjected to a silica gel column chromato-
probably most important because the NK1 receptor is pre- graphy. The active compound (1a) was obtained in pure form
dominant in the human central nervous system,2) and because as a white powder by repeated reversed phase HPLC.
the selective endogenous ligand for NK1 receptor, substance
The molecular formula of 1a was determined to be
P (SP), is most abundant among tachykinin peptides in both C46H50N4O8 from the result of elemental analysis and the
central and peripheral nervous systems of various species. It electrospray ionization liquid chromatography mass spec-
was shown that many tachykinin NK1 receptor antagonists trometry (ESI-LC-MS) spectrum giving a pseudo-molecular
block the neurotransmitter action of SP mediated by NK1 re- ion peak at m/z 785.43 ([MϪH]Ϫ).
1
ceptors.3) Clinical application of NK1 antagonists for various
Because of the complicated signals in H-NMR spectrum
diseases has been a target of many recent studies, and indeed caused by the conformers of 1a, several modification and de-
a successful treatment of depression and anxiety by an NK1 rivatization reactions were needed for the structure analysis
antagonist, MK 806, was reported.4)
of 1a. The 1H-NMR spectrum of 1a showed 6 protons, which
Most of the NK1 antagonists so far reported are synthetic,
and a few of them are of microbial origin but of low poten-
cies.5,6) No potent tachykinin NK1 antagonist from natural
sources has been reported. In this paper we report a new po-
tent tachykinin NK1 receptor antagonist obtained from the
extracts of plants of Compositae. A preliminary report has
been published as a patent application No. P2000-256293A.7)
Results
Extraction, Isolation and Structure Determination
Dried flower heads of Matricaria chamomilla L. were ex-
tracted with MeOH at room temperature. A small portion of
the MeOH extract was subjected to octadecyl silica (ODS)
column chromatography. The obtained fractions were dried
and submitted to the bioassay for tachykinin NK1 receptor
antagonist activity using the isolated guinea pig ileum. Fig-
ure 1 shows the chromatogram monitored by UV (300 nm)
and the activities of the fractions. The major peak of active
fractions, exhibiting the NK1 antagonist activity, was eluted
with the retention time from 10.8 to 12.4 min. These frac-
tions, however, did not show any anticholinergic activity.
Fig. 1. Chromatogram of Methanol Extract of Flowers of Matricaria
Chamomilla L.
Relative inhibitory activities of the eluted fractions on the contractile responses to SP
(10 nM) and acetylcholine (ACh, 100 nM) of the guinea pig ileum are shown by black
bars.
To whom correspondence should be addressed. e-mail: k-nakamura@nippon-zoki.co.jp
© 2002 Pharmaceutical Society of Japan

48
Vol. 50, No. 1
Fig. 2. Chemical Structures of Coumaroyl Spermines, 1a and 2
were exchangeable with D2O, at d 9.69—9.92 (4H, m), and d
7.94—7.98 and 8.05—8.11 (total 2H, m). Acetylation of 1a
with acetic anhydride in pyridine gave tetra-acetate (4a). In
Table 1. Contents of 1a and 2 in Flowers of the Plants of Compositae
Contents
(mg/g wet weight)
1
Genus, species
Part
Sourcea)
the H-NMR spectrum of 4a, signals at d 9.69—9.92 (4H,
m) of 1a disappeared, whereas new signals at d 2.24 (3H, s),
2.25 (3H, s), and 2.27 (6H, br s) were observed. The residual
two protons of 4a were at about d 8, suggesting the presence
of the amide protons.
1a
2
Matricaria chamomilla L.
Anthemis nobilis L.
Pollen
Tubular
Ligulate
Pollen
Tubular
Ligulate
Tubular
Ligulate
Tubular
A
A
A
A
A
A
A
A
B
B
C
C
8.92
0.49
0.00
8.60
0.55
0.00
On the other hand, catalytic hydrogenation of 1a gave a
single product (6) which showed pseudo-molecular ion at m/z
795 ([MϩH]ϩ) in the secondary ion (SI)-MS spectrum.
Since, as compared with 6, 1a possessed 8 more protons at d
16.31 28.15
0.48
0.00
1.48
0.00
0.85
0.00
0.00
0.00
Cosmos bipinnatus Cav.
Erigeron annus L.
1
6.36—7.56 and 16 less protons at d 2.24—3.02 in the H-
0.20 n.d.b)
0.00 n.d.b)
0.86c) 0.00
1.05c) 0.00
NMR spectrum, the presence of four olefins in 1a was sug-
gested. After acid hydrolysis of 6, the hydrolysate was parti-
tioned between H2O and n-BuOH. The n-BuOH extract con-
tained 3-(4-hydroxyphenyl)propanoic acid as a single com-
pound, whose 1H-NMR spectrum and retention time in
HPLC analysis were identical with those of the authentic
compound, whereas the H2O extract contained spermine as a
Ligulate
Inula britanica L.
Tussilago farfara L.
Flower heads
Flower buds
a) A: cultivated at the herb garden of Nippon Zoki Pharmaceutical Co., Ltd.; B: col-
lected locally in Hyogo; C: purchased from Shigatensanbutsu Laboratory Co., Ltd. b)
Not determined. c) mg/g dry weight.
sole compound, which was identical with the authentic com- caria chamomilla L. and Anthemis nobilis L.), 1a was specif-
pound in 1H-NMR spectra5) and in HPLC analysis.7)
ically concentrated in pollen (Table 1). In addition, we found
The structure of 6 was, therefore, presumed to be 2 in pollen and tubular flowers of both German and Roman
N1,N5,N10,N14-tetrakis[3-(4-hydroxyphenyl)propanoyl]- chamomiles.
1,5,10,14-tetraazatetradecane and confirmed by direct com-
Structure–Activity Relationship A number of com-
parison of its Rf value in TLC (silica gel, MeOH–EtOAc, pounds with structures related to that of 1a were synthesized
1
1 : 9), retention time in HPLC, and H-NMR chemical shifts by the methods shown in Charts 1—3. Thus, the acid chlo-
with those of the synthetic material (see Experimental sec- rides 3a—g8—12) and 513) prepared from the corresponding
tion).
carboxylic acids were condensed with spermine or its
Since 1a possessed four olefins, the structure of 1a was analogs to give the amides 4a—g, whose protecting groups
easily deduced to be N1,N5,N10,N14-tetrakis[3-(4-hydroxy- were saponified with 1 N NaOH to afford 1a—d, g, 6, 10—
phenyl)-2-propenoyl]-1,5,10,14-tetraazatetradecane (Fig. 2), 12. The activities of these amides as tachykinin NK1 re-
which was confirmed by direct comparison with the synthetic ceptor antagonists were estimated by their inhibitory actions
sample in a manner similar to that for 6 (see Experimental on SP-induced contraction of the guinea pig ileum and by
section).
their competitive inhibition of the binding of [3H][Sar9,
A ninhydrin positive analog of 1a, designated as 2, was Met(O2)11]SP to human tachykinin NK1 receptors expressed
also isolated from CHCl3–MeOH–H2O (10 : 5 : 1) eluates in on Chinese hamster ovary (CHO) cells. The results for the
silica gel column chromatography of the MeOH extract. The selected 13 compounds are shown in Table 2. Most of the
structure of 2 was deduced to be N1,N5,N10-tris[3-(4-hy- other derivatives were inactive. Modification of the coumaroyl
droxyphenyl)-2-propenoyl]-1,5,10,14-tetraazatetradecane group (1b—d, g, 4a, e, f, 6) as well as the spermine skeleton
(Fig. 2) by instrumental analyses of trifluoroacetic acid (10—12) resulted in reduction of activity. The results suggest
(TFA) salt of 2, its reduction product, and its acid-hy- that the structure of 1a is considerably optimized for the an-
drolysates (see Experimental section).
tagonist activity on the guinea pig and human NK1 receptors.
Distribution of Coumaroyl Spermines The extracts of In contrast with the inactivity of 2, the activity of 10 was
about 200 species of plants were screened for NK1 antagonist noteworthy indicating that the lack of one coumaroylamino-
activity by the use of guinea pig ileum, and when the active propyl moiety of 1a does not abolish the NK1 antagonist ac-
extracts were found, the presence of 1a was examined in the tivity completely. This difference may be due to the differ-
extracts by HPLC (see Experimental section). As a result, 1a ence between their stable conformations. A hypothesis was
was found in flowers of 6 species of Compositae. In four postulated that two phenyl rings with specific relative spatial
species examined, 1a was found in tubular but not in ligulate orientation may be an important factor for the NK1 antago-
flowers. In both German and Roman chamomiles (Matri- nist activity.14) 1a and 10, however, were too flexible to pre-

January 2002
49
Chart 1
Chart 2
Chart 3

50
Vol. 50, No. 1
Table 2. Tachykinin NK1 Antagonist Activities of the Compounds
Compositae. Compound 1a is the first alkaloid with a potent
NK1 receptor antagonist activity from natural sources and
has a unique structure, which is entirely different from those
of the previously reported synthetic NK1 antagonists.
Compound
Ki (nM)a)
Guinea pig ileum Human NK1 receptors on CHO cells
Experimental
1a
1b
1c
1d
1g
2
4a
4e
4f
6
21.9
Ͼ1000.0
Ͼ1000.0
Ͼ1000.0
367.1
Ͼ1000.0
74.0
Ͼ1000.0
Ͼ1000.0
Ͼ1000.0
304.0
3.3
Ͼ1000.0
Ͼ1000.0
Ͼ1000.0
53.3
Ͼ1000.0
196.4
Ͼ1000.0
Ͼ1000.0
Ͼ1000.0
357.9
General Melting points were determined on a Yamato MP-21 melting
point apparatus and are uncorrected. UV spectra were recorded on a Beck-
man DU 650 spectrophotometer and IR spectra on a Horiba FT-200 Fourier
transform (FT) IR spectrophotometer. 1H-NMR spectra were taken on a
Bruker ARX-500 (500 MHz) spectrometer. Chemical shifts are reported in d
units (parts per million downfield from internal tetramethylsilane). MS were
measured on a Micromass Q-Tof or a Hitachi M-80B mass spectrometer.
High resolution (HR)-ESI-MS was measured with Bruker Daltonics Matrix-
Assisted Laser Desorption Ionization (MALDI)-Time-of-Flight (TOF)-MS:
reflex III. The elemental analyses (C, H, N) were performed on a Yanaco
CHN Corder MT-5 elemental analyzer. TLC was performed by using Silica
gel 60F254 (Merck). Column chromatography was performed with Silica gel
60 (70—230 mesh, Merck). HPLC was performed on a Jasco system (pump,
PU-920; detectors, UV-970 and FP-970; auto-sampler, 851-AS; controller,
802-SC; column oven, 860-CO; solvent mixing module, HG-980-31). Evap-
oration of the solvent was carried out under reduced pressure. Anhydrous
Na2SO4 was employed as the drying agent.
Plant Materials Dried plants, Inula britanica L. and Tussilago farfara
L., were obtained from commercial sources (Shigatensanbutsu Laboratory
Co., Ltd., Shiga, Japan; Kishida Chemical Co., Ltd.). On the other hand, the
fresh plants were obtained as follows; Erigeron annus L. was collected in
Hyogo in June, Matricaria chamomilla L. and Anthemis nobilis L. were
grown in the garden of our institute. They were seeded in September 1999
and were harvested between June and July 2000. Cosmos bipinnatus Cav.
was also grown in the garden of our institute. It was seeded in April 2000
and was harvested in September 2000.
Extraction and Isolation The dried flower heads of Matricaria
chamomilla L. (400 g) were extracted with MeOH (3 lϫ3 times) at room
temperature. After filtration, the MeOH solution was evaporated to give the
residue (78.8 g).
A portion of the residue (20 mg) was subjected to preparative HPLC [col-
umn, STR PREP-ODS, 20ϫ250 mm; eluent, 0.1% TFA–MeOH (30 : 70)
(0—6 min), 0.1% TFA–MeOH (30 : 70)→MeOH (6—16 min, linear gradi-
ent), MeOH (16—30 min); flow rate, 8 ml/min; column temperature, ambi-
ent; detection, 300 nm] and fractionated. 2% aliquot of each fraction was
dried and dissolved in 10 ml dimethylsulfoxide (DMSO), and added to 5 ml
Tyrode solution followed by application to the ileum (Fig. 1).
A residual part of the MeOH extract (78 g) was suspended in water (1 l)
and extracted with n-hexane (0.5 l). The obtained aqueous layer was further
extracted with n-BuOH (0.5 l). The n-BuOH extract (20.5 g) was subjected
to silica gel column chromatography (5ϫ40 cm), and elution was carried out
in steps with a mobile phase of CHCl3–MeOH (10 : 1, yield 5.8 g),
CHCl3–MeOH (4 : 1, yield 13.1 g), and CHCl3–MeOH–H2O (10 : 5 : 1, yield
1.8 g).
The CHCl3–MeOH (4 : 1) eluate (13.0 g) was further separated repeatedly
by HPLC [STR PREP-ODS, 20ϫ250 mm, Shimadzu Techno Research Inc.,
MeOH–0.1% TFA (6 : 4)] and gave an active fraction (299 mg). The active
fraction was concentrated and then crystallized from MeOH–H2O to give 1a
(270 mg) as white powder: mp 139—141 °C (dec.). 1H-NMR (DMSO-d6) d:
1.45—1.80 (8H, m), 3.12—3.55 (12H, m), 6.36—6.44 (2H, m), 6.68—6.94
(10H, m), 7.29—7.56 (12H, m), 7.94—7.98, 8.05—8.11 (total 2H, m),
9.69—9.92 (4H, m). UV lmax (MeOH) nm (log e): 227 (4.70), 299 (4.93),
311 (4.94). IR (KBr) cmϪ1: 3300, 1650, 1606, 1514, 1440, 1234, 1169. Neg-
ative ion ESI-MS: m/z 785 (MϪH)Ϫ, 665 (MϪHϪ120)Ϫ, 639 (MϪ
HϪ146)Ϫ. Positive ion ESI-MS: m/z 787 (MϩH)ϩ, 642, 623. Anal. Calcd
for C46H50N4O8·H2O: C, 68.64; H, 6.51; N, 6.96. Found: C, 68.56; H, 6.47;
N, 6.97.
10
11
12
94.6
52.1
25.6
63.7
a) The concentration of the antagonist which displaces the log concentration re-
sponse curve by log 2.
dict the active conformations by conformational analysis and
to assign the roles of the coumaroyl groups.
Effects of 1a on the Receptors Other Than NK1 In
order to examine the specificity of the action of 1a as an NK1
receptor antagonist, its effects on other types of receptors
were studied. 1a at a concentration of 300 nM did not dis-
place the concentration–contractile response curves on the
guinea pig ileum to ACh, histamine and bradykinin. More-
over, 1a altered neither the concentration–contractile re-
sponse curves to neurokinin A (NK2 receptor agonist) on the
rat vas deferens, an NK2 monoreceptor system,15) nor the
contraction of the rat portal vein, an NK3 monoreceptor sys-
tem,15) evoked by senktide (NK3 receptor agonist, 10 nM).
Discussion
The original purpose of the present study was to find in the
plant kingdom clinically efficacious antagonists for
tachykinin NK1 receptors. In this respect, although 1a is a
potent NK1 receptor antagonist, this compound appears un-
likely to serve as a clinically useful drug for the following
reasons. Firstly, the solubility of 1a in water and human
plasma is extremely low. The estimated maximal concentra-
tion of 1a in the human plasma after deproteination was
around 10 nM, and when 1a was dissolved in non-depro-
teinated human plasma, it did not display any inhibitory ac-
tion on the binding of [3H][Sar9, Met(O2)11]SP to human NK1
receptors expressed on CHO cells. Secondly, when 1a, dis-
solved in 0.5% carboxymethyl cellulose sodium salt solution,
was orally administered in rats, 1a was not detected in the
blood. It is noteworthy that several commercially available
preparations of chamomile were found to contain significant
amounts of 1a, which, if it were absorbed efficiently from
gastrointestinal tract, could afford a significant concentration
in the blood. However, when these preparations were orally
administered in rats, again 1a was not detected in the blood
(unpublished observation). Therefore, it seems unlikely that
The CHCl3–MeOH–H2O (10 : 5 : 1) eluate was chromatographed by
HPLC [STR PREP-ODS, 20ϫ250 mm, Shimadzu Techno Research Inc.,
MeOH–0.1% TFA (55 : 45)]. The fraction containing the main UV peak was
the effects of the chamomile preparations administered orally concentrated and then precipitated with ether to give TFA salt of 2 (230 mg)
as an yellow powder: mp 109—111 °C (dec.). 1H-NMR (DMSO-d6) d:
in humans, such as anti-inflammatory and anti-asthmatic ef-
fects, involve the inhibition of tachykinin NK1 receptors in-
duced by 1a.
In conclusion, we have discovered a new nonpeptide
tachykinin NK1 antagonist, 1a, in the extracts of the plants of
1.43—1.86 (8H, m), 2.71—2.92 (2H, m), 3.13—3.56 (10H, m), 6.37—6.46
(1H, m), 6.70—6.97 (8H, m), 7.23—7.60 (9H, m), 7.68—7.76 (3H, br s),
8.00—8.13 (1H, m), 9.83—9.96 (3H, m). UV lmax (MeOH) nm (log e): 209
(4.41), 304 (4.55), 312 (4.58). IR (KBr) cmϪ1: 3409, 1641, 1606, 1514,
1447, 1202, 1171. Positive ion SI-MS: (m/z) 641 (MϩH)ϩ. Positive ion HR-

January 2002
51
ESI-MS: (m/z) 641.3355 (MϩH)ϩ, Calcd for C37H44N4O6ϩH, 641.3334.
Modification and Decomposition Reaction of 1a and 2. Acetylation
of 1a A solution of 1a (40 mg) was stirred with Ac2O (5 ml) in pyridine
(5 ml) at room temperature for 2 h. The reaction solution was concentrated
and crystallized from MeOH–pyridine to give 4a (26 mg) as a white powder:
1H-NMR (DMSO-d6) d: 1.49—1.62 (4H, m), 1.70—1.81 (4H, m) , 2.24
(3H, s), 2.25 (3H, s), 2.27 (6H, br s), 3.14—3.54 (12H, m), 6.57—6.63 (2H,
m), 7.00—7.06 (4H, m), 7.09—7.18 (6H, m), 7.40—7.53 (4H, m), 7.57—
7.62 (4H, m), 7.64—7.79 (4H, m), 8.09—8.14 (1H, m), 8.21—8.26 (1H, m).
UV lmax (MeOH) nm: 218, 280. IR (KBr) cmϪ1: 3400, 2900, 1760, 1639,
1623. Negative ion ESI-MS: m/z 953 (MϪH)Ϫ.
100 °C for 25 h. The reaction mixture was poured into 2 N HCl and extracted
with EtOAc. The extract was washed with brine, dried, and condensed to
give a solid, which was recrystallized from EtOAc to give (E)-3-(4-hydroxy-
phenyl)-2-methyl-2-propenoic acid (4.2 g, 27%) as white crystals: mp 210—
1
213 °C. H-NMR (DMSO-d6) d: 2.02 (3H, s), 6.83 (2H, d, Jϭ8.6 Hz), 7.35
(2H, d, Jϭ8.6 Hz), 7.51 (1H, s), 9.82 (1H, s), 12.12 (1H, s). The acid ob-
tained was acetylated with acetic anhydride (10 ml) in pyridine (50 ml) at
room temperature for 5 h. The mixture was condensed and partitioned be-
tween EtOAc and water. The organic layer was washed with brine, dried, and
subjected to silica gel column chromatography (CHCl3–MeOH, 9 : 1) to give
(E)-3-(4-acetoxyphenyl)-2-methyl-2-propenoic acid (3.6 g, 70%) as crystals:
1
mp 142—144 °C. H-NMR (DMSO-d6) d: 2.03 (3H, s), 2.28 (3H, s), 7.20
Catalytic Hydrogenation of 1a A solution of 1a (80 mg) in dimethyl-
formamide (DMF) (40 ml) was stirred in the presence of 10% Pd/C (5 mg) at
room temperature for 4 h under an H2 atmosphere (1 atm). The reaction mix-
ture was filtered, and the filtrate was concentrated. The residue was purified
by HPLC (Chemcosorb I-7C18, Chemco, 20ϫ250 mm, 60% MeOH) to give
(2H, d, Jϭ8.2 Hz), 7.52 (2H, d, Jϭ8.2 Hz), 7.59 (1H, s), 12.52 (1H, s). A
mixture of the propenoic acid (3.4 g, 15.6 mmol) and thionyl chloride (5 ml)
in CH2Cl2 was stirred at room temperature for 24 h. The reaction mixture
was condensed to give quantitatively the acid chloride 3g as a pale yellow
oil, which was applied to the next step without further purification.
Condensation of Acid Chlorides with Spermine. N1,N5,N10,N14-
Tetrakis[3-(4-acetoxyphenyl)-2-propenoyl]-1,5,10,14-tetraazatetradecane
(4a) The acid chloride 3a (4.7 g, 20.9 mmol) was added to a solution of
spermine (1.0 g, 5.0 mmol) and Et3N (2.1 g, 21.0 mmol) in dry tetrahydrofu-
ran (THF) (80 ml) at 0 °C under an argon atmosphere. The mixture was
stirred at an ambient temperature overnight and diluted with 10-fold volume
of water. The precipitated solid was filtered, washed with water, and dried to
give 4a as a white solid (4.0 g, 83% yield): mp 171—173 °C. 1H-NMR
(DMSO-d6) d: 1.49—1.62 (4H, m), 1.70—1.81 (4H, m), 2.24 (3H, s), 2.25
(3H, s), 2.27 (6H, 2s), 3.14—3.54 (12H, m), 6.57—6.63 (2H, m), 7.00—
7.06 (4H, m), 7.09—7.18 (6H, m), 7.40—7.53 (4H, m), 7.57—7.62 (4H, m),
7.64—7.79 (4H, m), 8.09—8.14 (1H, m), 8.21—8.26 (1H, m). Anal. Calcd
for C54H58N4O12: C, 66.35; H, 6.24; N, 5.73. Found: C, 66.42; H, 6.18; N,
5.75.
1
6 (65 mg) as a white amorphous solid: H-NMR (DMSO-d6) d: 1.29—1.37
(4H, m), 1.48—1.59 (4H, m), 2.24—2.31 (4H, m), 2.43—2.51 (4H, m),
2.64—2.72 (8H, m), 2.94—3.08 (4H, m), 3.11—3.21 (8H, m), 6.61—6.66
(8H, m), 6.93—7.01 (8H, m), 7.71—7.75 (1H, m), 7.78—7.82 (1H, m), 9.14
(4H, br s). Positive ion SI-MS: m/z 795 (MϩH)ϩ.
Acid Hydrolysis of 6 The compound 6 (25 mg) in 6 N HCl (1 ml) was
heated at 110 °C for 24 h. The reaction mixture was concentrated and parti-
tioned between H2O (5 ml) and n-BuOH (5 ml). The H2O layer and the n-
BuOH layer were concentrated to yield an H2O extract (21 mg) and n-BuOH
extract (11 mg), respectively.
The H2O Extract: 1H-NMR (D2O) d: 1.79—1.86 (4H, m), 2.09—2.17
(4H, m), 3.12—3.22 (12H, m).
The n-BuOH Extract: 1H-NMR (CD3OD) d: 2.53 (2H, t, Jϭ7.5 Hz), 2.80
(2H, t, Jϭ7.5 Hz), 6.69 (2H, dd, Jϭ2.0, 8.0 Hz), 7.02 (2H, dd, Jϭ2.0,
8.0 Hz).
N1,N5,N10,N14-Tetrakis[3-(3-acetoxyphenyl)-2-propenoyl]-1,5,10,14-
tetraazatetradecane (4b): mp 85—90 °C. Yield 58%. 1H-NMR (DMSO-d6)
d: 1.44—1.62 (4H, m), 1.64—1.80 (4H, m), 2.26 (6H, s), 2.28 (6H, s),
3.16—3.25 (4H, m), 3.39—3.45 (4H, m), 3.47—3.55 (4H, m), 6.60—6.66
(2H, m), 7.05—7.21 (6H, m), 7.29—7.33 (3H, m), 7.39—7.61 (13H, m),
8.11—8.20 (2H, m). Anal. Calcd for C54H58N4O12·H2O: C, 66.65; H, 6.22;
N, 5.76. Found: C, 66.45; H, 6.22; N, 5.77.
N1,N5,N10,N14-Tetrakis[3-(2-acetoxyphenyl)-2-propenoyl]-1,5,10,14-
tetraazatetradecane (4c): mp 94—96 °C. Yield 31%. 1H-NMR (DMSO-d6)
d: 1.46—1.64 (m, 4H), 1.67—1.82 (m, 4H), 2.29 (s, 3H), 2.30—2.38 (m,
9H), 3.13—3.27 (m, 4H), 3.34—3.44 (m, 4H), 3.46—3.58 (m, 4H), 6.61—
6.68 (m, 2H), 7.06—7.56 (m, 18H), 7.61—7.69 (m, 2H), 7.83—8.02 (m,
2H), 8.17—8.31 (m, 2H). Anal. Calcd for C54H58N4O12·H2O: C, 66.65; H,
6.22; N, 5.76. Found : C, 66.67; H, 6.24; N, 5.94.
N1,N5,N10,N14-Tetrakis[3-(3,4-diacetoxyphenyl)-2-propenoyl]-
1,5,10,14-tetraazatetradecane (4d): mp 89—94 °C. Yield 87%. 1H-NMR
(DMSO-d6) d: 1.48—1.60 (4H, m), 1.66—1.79 (4H, m), 2.26—2.31 (24H,
m), 3.16—3.54 (12H, m), 6.56—6.63 (2H, m), 7.07—7.66 (18H, m), 8.08—
8.13 (1H, m), 8.16—8.22 (1H, m). Anal. Calcd for C62H66N4O20·0.65H2O:
C, 62.11; H, 5.66; N, 4.67. Found: C, 62.14; H, 5.69; N, 4.65.
N1,N5,N10,N14-Tetrakis[3-(4-methoxyphenyl)-2-propenoyl]-1,5,10,14-
tetraazatetradecane (4e): mp 145—149 °C. Yield 66%. 1H-NMR (DMSO-d6)
d: 1.36—1.76 (8H, m), 3.17—3.51 (12H, m), 3.65 (3H, s), 3.75—3.82 (9H,
m), 6.49—6.63 (2H, m), 6.79—7.03 (10H, m), 7.38—7.64 (12H, m), 8.01—
8.04 (1H, m), 8.16—8.18 (1H, m).
HPLC Analysis of Hydrolysate of 6 The analysis of spermine was per-
formed with the on-column method of Saito et al.16) with minor modifica-
tion. Briefly, a 1 ml aliquot of the H2O layer was injected into a reverse phase
column (YMC-Pack Polymer C18, YMC, 4.6ϫ250 mm). The mobile phase
was a mixture of 50 mM sodium borate buffer (pH 9.9)–acetonitrile (77 : 23,
v/v) containing 2 mM o-phthalaldehyde and 2 mM N-acetyl-L-cysteine. The
flow rate was adjusted to 0.8 ml/min and the peaks were detected at excita-
tion and emission wavelengths of 330 and 430 nm, respectively. The separa-
tion was performed at a temperature of 40 °C. The water-soluble derivative
thus obtained showed a single peak at 10.5 min, identical to that of authentic
spermine.
The analysis of 3-(4-hydroxyphenyl)propanoic acid was carried out on
YMC-Pack Polymer C18 (CH3CN–10 mM phosphate buffer pH 2.0, 22 : 78)
at a temperature of 30 °C. The mobile phase was delivered isocratically at a
flow rate of 1.0 ml/min. The detection wavelength was set to 280 nm and a
4 ml aliquot of the n-BuOH layer was injected. A single peak at a retention
time of 13.8 min was identical to that of authentic 3-(4-hydroxyphenyl)-
propanoic acid.
Catalytic Hydrogenation of 2 and Acid Hydrolysis of Its Reduction
Product A solution of 2 (17 mg) in MeOH (15 ml) was stirred with 10%
Pd/C (5 mg) at room temperature for 4 h under an H2 atmosphere. The reac-
tion mixture was filtered, and the filtrate was concentrated. The residue gave
a pseudo-molecular ion at m/z 647 (MϩH)ϩ in the SI-MS spectrum. A por-
tion of the material was hydrolyzed in 6 N HCl (1 ml) at 110 °C for 24 h. The
hydrolysate was concentrated and partitioned between H2O (4 ml) and n-
BuOH (4 ml). The analysis of each layer was performed by HPLC with the
same conditions as for 6. The H2O layer was found to contain spermine, and
the n-BuOH layer contained 3-(4-hydroxyphenyl)propanoic acid.
HPLC Analysis of Coumaroyl Spermines (1a, 2) Plant materials were
powdered and extracted by percolation with 10 volume of MeOH at room
temperature in the dark. The MeOH solution was concentrated, and the
residue was dissolved in DMSO. The quantification of 1a and 2 in the ex-
tracts was performed by HPLC using 1a and 2 isolated from Matricaria
chamomilla L. as standards (Develosil ODS-HG-5, 4.6ϫ250 mm, Nomura
Chemical, 50 mM phosphate buffer (pH 2.5)–MeOH (48 : 52), 1 ml/min,
40 °C, UV at 300 nm, tR of 1a and 2: 10.4 and 4.1 min, respectively).
Synthesis. Preparation of the Acid Chlorides The acid chlorides
3a,8) 3b,9) 3c,10) 3d,11) 3e,12) and 513) were prepared from the corresponding
carboxylic acids by the reported procedures. 3-Phenyl-2-propenoyl chloride
(3f) was obtained from Aldrich.
N1,N5,N10,N14-Tetrakis(3-phenyl-2-propenoyl)-1,5,10,14-tetraazate-
tradecane (4f): mp 173—182 °C. Yield 86%. 1H-NMR (DMSO-d6) d:
1.51—1.61 (4H, m), 1.71—1.79 (4H, m), 3.18—3.54 (12H, m), 6.60—6.63
(2H, m), 7.08—7.19 (2H, m), 7.29—7.72 (24H, m), 8.14—8.16 (1H, m),
8.22—8.24 (1H, m). Anal. Calcd for C46H50N4O4·0.5H2O: C, 75.60; H,
7.05; N, 7.45. Found: C, 75.49; H, 7.02; N, 7.65.
N1,N5,N10,N14-Tetrakis[3-(4-acetoxyphenyl)-2-methyl-2-propenoyl]-
1,5,10,14-tetraazatetradecane (4g): An amorphous solid. Yield 98%. 1H-
NMR (DMSO-d6) d: 1.48—2.10 (8H, m), 2.26 (6H, s), 3.08—3.42 (12H,
m), 6.41 (2H, br s), 7.02—7.45 (18H, m), 8.06 (2H, br s).
N1,N5,N10,N14-Tetrakis[3-(4-hyroxyphenyl)-2-propenoyl]-1,5,10,14-
tetraazatetradecane (1a) The compound 4a (954 mg, 1.0 mmol) was hy-
drolyzed with 1 N NaOH (4.8 ml) at room temperature for 1 h. The reaction
mixture was neutralized with 1 N HCl (4.8 ml) and the solid precipitated was
filtered, washed with water, and dried to give 1a (676 mg, 86%) as a white
1
solid: mp 138—140 °C (dec). H-NMR (DMSO-d6) d: 1.45—1.80 (8H, m),
(E)-3-(4-Acetoxyphenyl)-2-methyl-2-propenoyl Chloride (3g) A mix-
ture of 4-hydroxybenzaldehyde (9.3 g, 76.2 mmol), methylmalonic acid
(18.0 g, 150 mmol), and piperidine (15 ml) in pyridine (10 ml) was stirred at
3.12—3.55 (12H, m), 6.36—6.44 (2H, m), 6.68—6.94 (10H, m), 7.29—7.56
(12H, m), 7.94—7.98 (1H, m), 8.05—8.11 (1H, m), 9.69—9.92 (4H, m).

52
Vol. 50, No. 1
Anal. Calcd for C46H50N4O8·H2O: C, 68.64; H, 6.51; N, 6.96. Found: C,
68.83; H, 6.47; N, 6.90.
in the absence of atropine, that produced by histamine in the absence of
mepyramine and that produced by bradykinin in normal Tyrode solution
were evaluated in the guinea pig ileum.
N1,N5,N10,N14-Tetrakis[3-(3-hyroxyphenyl)-2-propenoyl]-1,5,10,14-
tetraazatetradecane (1b): mp 106 °C (dec.). Yield 88%. 1H-NMR (DMSO-
The rat vas deferens and the rat portal vein were used to examine the re-
d6) d: 1.50—1.77 (8H, m), 3.17—3.52 (12H, m), 6.54 (2H, d, Jϭ15.8 Hz), ceptor subtype selectivity of 1a.15) The methods used for experiments are the
6.74—6.78 (4H, m), 6.96—7.20 (14H, m), 7.32—7.42 (4H, m), 8.11—8.21 same as previously described.17)
(2H, m), 9.51—9.55 (4H, m). Anal. Calcd for C46H50N4O8·H2O: C, 68.64;
H, 6.51; N, 6.96. Found: C, 68.61; H, 6.60; N, 6.89.
Binding Assay with Human Tachykinin NK1 Receptors A CHO cell
line expressing human tachykinin NK1 receptors was established according
to the procedures described previously18) and was a generous gift from
N1,N5,N10,N14-Tetrakis[3-(2-hyroxyphenyl)-2-propenoyl]-1,5,10,14-
tetraazatetradecane (1c): mp 106 °C (dec.). Yield 82%. 1H-NMR (DMSO-d6) Amersham Pharmacia Biotech (Tokyo). The CHO cells were cultured in
d: 1.56—1.77 (8H, m), 3.17—3.57 (12H, m), 6.20 (2H, br s), 6.64—7.41
(22H, m), 7.67—7.81 (2H, m), 10.65 (4H, br s). Anal. Calcd for C46H50N4O8·
alpha minimum essential medium (a-MEM) supplemented with 100 IU/ml
penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum. Cultures
were kept at 37 °C in a humidified atmosphere of 5% CO2. Binding assays
were carried out on whole cells. Cells grown to confluence in culture flask
were detached with trypsin–EDTA (0.25% trypsin, 1 mM EDTA·Na) solu-
tion (GIBCO). Collected cells were washed twice with 50 mM Tris–Cl buffer
(pH 7.5) containing 150 mM NaCl and 0.02% bovin serum albumin (BSA).
The pellet was then resuspended in 50 mM Tris–Cl buffer containing 150 mM
NaCl, 0.02% BSA, 40 mg/ml bacitracin (Sigma), 4 mg/ml leupeptin (Sigma),
4 mg/ml chymostatin (Sigma) and 4 mg/ml phosphoramidon (Sigma). CHO
5H2O: C, 63.00; H, 6.90; N, 6.39. Found: C, 62.79; H, 6.74; N, 6.08.
N1,N5,N10,N14-Tetrakis[3-(3,4-dihyroxyphenyl)-2-propenoyl]-1,5,10,14-
tetraazatetradecane (1d): mp 159—163 °C (dec.). Yield 74%. 1H-NMR
(DMSO-d6) d: 1.49—1.76 (8H, m) , 3.16—3.53 (12H, m), 6.24—6.46 (2H,
m), 6.70—7.43 (18H, m), 7.66—7.98 (1H, m), 7.99—8.02 (1H, m), 9.08
(4H, br s), 9.47 (4H, br s). Anal. Calcd for C46H50N4O12·1.25H2O: C, 63.26;
H, 6.06; N, 6.41. Found: C, 63.26; H, 6.14; N, 6.32.
N1,N5,N10,N14-Tetrakis[3-(4-hyroxyphenyl)-2-methyl-2-propenoyl]-
1,5,10,14-tetraazatetradecane (1g): mp 158—162 °C. Yield 99%. 1H-NMR cells (106 cells) were incubated in 500 ml of buffer containing [3H]-
(DMSO-d6) d: 1.51 (4H, br s), 1.73 (4H, br s), 1.93 (12H, br s), 3.14 (4H,
br s), 3.34 (8H, br s), 6.30 (4H, br s), 6.75—6.81 (8H, m), 7.05—7.19 (12H,
m), 7.93 (2H, br s). Anal. Calcd for C50H58N4O8: C, 71.24; H, 6.93; N, 6.65.
Found: C, 70.45; H, 7.18; N, 5.64.
[Sar9,Met(O2)11]SP (NEN Life Science Products, Inc.; final conc. 0.3 nM,
39.5 Ci/mmol) and one of the test compounds at room temperature for
60 min and then filtered over GF/B filters (Whatman) that had been pre-
soaked in 0.1% polyethylenimine (Wako). The radioactivities in the filters
Preparation of 6, 10, 11, and 12. N1,N5,N10,N14-Tetrakis[3-(4-hy- were determined after addition of scintillator (AL-1, Dojindo).
droxyphenyl)propanoyl]-1,5,10,14-tetraazatetradecane (6) The acid
chloride (5) (3.4 g, 15.0 mmol) was added to a solution of spermine (606 mg,
Acknowledgements We thank Prof. Shigetada Nakanishi (Department
3.0 mmol) and Et3N (1.5 g, 15.0 mmol) in THF (20 ml) . The mixture was of Molecular and System Biology, Kyoto University), Dr. Ichiro Aramori
stirred at room temperature overnight and filtered to remove insoluble solid. (Fujisawa Pharmaceutical Co., LTD.) and Dr. Satoshi Ono (Amersham Phar-
The filtrate was condensed to give a residue, which was purified by silica gel macia Biotech) for generous gift of the cell line expressing human NK1 re-
column chromatography (CHCl3–MeOH, 24 : 1) to give N1,N5,N10,N14- ceptors. We are also grateful to Professor Emeritus Shoji Shibata of Univer-
tetrakis[3-(4-acetoxyphenyl)propanoyl]-1,5,10,14-tetraazatetradecane as
colorless oil in quantitative yield.
a
sity of Tokyo and Dr. Robert W. Baughman of NINDS for much helpful ad-
vice.
The oil obtained above was stirred with 1 N NaOH (14.4 ml) in MeOH
(20 ml) at room temperature for 1 h. The reaction mixture was neutralized References
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with 1 N HCl and concentrated. After addition of MeOH to the residue, in-
soluble NaCl was filtered and the filtrate was purified by HPLC (Chem-
cosorb I-7C18, 20ϫ250 mm, 60% MeOH) to give 6 (1.45 g, 61%) as a white
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1
amorphous solid. H-NMR (DMSO-d6) d: 1.29—1.37 (4H, m), 1.48—1.59
(4H, m), 2.24—2.31 (4H, m), 2.43—2.51 (4H, m), 2.64—2.72 (8H, m),
2.94—2.30 (4H, m), 3.11—3.21 (8H, m), 6.61—6.66 (8H, m), 6.93—7.01
(8H, m), 7.71—7.75 (1H, m), 7.78—7.82 (1H, m), 9.14 (4H, br s).
N1,N5,N10-Tris[3-(4-hydroxyphenyl)-2-propenoyl]-1,5,10-triazadecane
(10): mp 138—140 °C. Yield 95%. 1H-NMR (DMSO-d6) d: 1.39—1.79
(6H, m), 3.14—3.50 (8H, m), 6.36—6.44 (2H, m), 6.71 (1H, d, Jϭ8.3 Hz),
6.75—6.91 (7H, m), 7.29—7.40 (6H, m), 7.49 (1H, d, Jϭ8.3 Hz), 7.54 (1H,
d, Jϭ8.3 Hz), 7.96—8.11 (2H, m), 8.79—9.83 (3H, m). Anal. Calcd for
C34H37N3O6·0.8H2O: C, 68.25; H, 6.42; N, 6.99. Found: C, 68.28; H, 6.51;
N, 7.03.
N1,N5,N9,N13-Tetrakis[3-(4-hydroxyphenyl)-2-propenoyl]-1,5,9,13-
1
tetraazatridecane (11): mp 123 °C (dec.). Yield 78%. H-NMR (DMSO-d6)
d: 1.69—1.89 (6H, m), 3.15—3.51 (12H, m), 6.38—6.43 (2H, m), 6.71—
6.86 (10H, m), 7.30—7.50 (12H, m), 7.97—8.10 (2H, m), 9.83 (4H, br s).
Anal. Calcd for C45H48N4O8·1.5H2O: C, 67.57; H, 6.43; N, 7.00. Found: C,
67. 67; H, 6.43; N, 7.01.
N1,N5,N10,N14-Tetrakis[3-(4-hydroxyphenyl)-2-propenoyl]-1,5,10,14-
tetraaza-7-tetradecene (12): mp ca. 126 °C (dec.). Yield 99%. 1H-NMR
(DMSO-d6) d: 1.67—1.74 (4H, m), 3.15—3.39 (8H, m), 3.99—4.13 (4H,
m), 5.56—5.70 (2H, m), 6.38—6.44 (2H, m), 6.70—6.85 (10H, m), 7.30—
7.49 (12H, m), 7.95—8.07 (2H, m), 9.84 (4H, m). Anal. Calcd for
C46H48N4O8·2H2O: C, 67.30; H, 6.38; N, 6.82. Found: C, 67.56; H, 6.23; N,
6.89.
Bioassays The antagonist activities for tachykinin NK1 receptor of chro-
matographic fractions of plant extracts and synthetic compounds were deter-
mined using the isolated ileum of the guinea pig as described previously.17)
Samples to be tested were added to the bath as a DMSO solution 5 min be-
fore the application of SP. Contractile responses were recorded isotonically
and were expressed as percentages of the maximal response to 10 nM SP. In
order to eliminate the anticholinergic and anti-histamine activities as well as
the interference of prostaglandins, the Tyrode solution used for the experi-
ments contained atropine 4 mM, mepyramine 4 mM, and indomethacin 3.6 mM.
To examine the specificity of the NK1 antagonist action of 1a, its effects
on the concentration–contractile response curve produced by acetylcholine
15) Regoli D., Drapeau G., Dion S., Couture R., Trends Pharmacol. Sci.,
9, 290—295 (1988).
16) Saito K., Horie M., Nose N., Nakagomi K., Nakazawa H., Anal. Sci.,
8, 675—680 (1992).
17) Hosoki R., Yanagisawa M., Onishi Y., Yoshioka K., Otsuka M., Eur. J.
Phramacol., 341, 235—241 (1998).
18) Nakajima Y., Tsuchida K., Negishi M., Ito S., Nakanishi S., J. Biol.
Chem., 267, 2437—2442 (1992).