Studies on the synthesis and opioid agonistic activities of mitragynine-related indole alkaloids: Discovery of opioid agonists structurally different from other opioid ligands

Article abstract of DOI:10.1021/jm010576e

Mitragynine (1) is a major alkaloidal component in the Thai traditional medicinal herb, Mitragyna speciosa, and has been proven to exhibit analgesic activity mediated by opioid receptors. By utilizing this natural product as a lead compound, synthesis of some derivatives, evaluations of the structure-activity relationship, and surveys of the intrinsic activities and potencies on opioid receptors were performed with guinea pig ileum. The affinities of some compounds for μ-, δ-, and κ-receptors were determined in a receptor binding assay. The essential structural moieties in the Corynanthe type indole alkaloids for inducing the opioid agonistic activity were also clarified. The oxidative derivatives of mitragynine, i.e., mitragynine pseudoindoxyl (2) and 7-hydroxymitragynine (12), were found as opioid agonists with higher potency than morphine in the experiment with guinea pig ileum. In addition, 2 induced an analgesic activity in the tail flick test in mice.

Full text of DOI:10.1021/jm010576e

J . Med. Chem. 2002, 45, 1949-1956
1949
Stu d ies on th e Syn th esis a n d Op ioid Agon istic Activities of
Mitr a gyn in e-Rela ted In d ole Alk a loid s: Discover y of Op ioid Agon ists
Str u ctu r a lly Differ en t fr om Oth er Op ioid Liga n d s
,
†
†
†
†
†
‡
Hiromitsu Takayama,* Hayato Ishikawa, Mika Kurihara, Mariko Kitajima, Norio Aimi, Dhavadee Ponglux,
§
§
§
§
§
Fumi Koyama, Kenjiro Matsumoto, Tomoyuki Moriyama, Leonard T. Yamamoto, Kazuo Watanabe,
Toshihiko Murayama, and Syunji Horie*
§
,§
Laboratory of Molecular Structure and Biological Function, Graduate School of Pharmaceutical Sciences, Chiba University,
Yayoi-cho, 1-33, Inage-ku, Chiba 263-8522, J apan, Faculty of Pharmaceutical Sciences, Chulalongkorn University,
Bangkok 10330, Thailand, Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences,
Chiba University, Yayoi-cho, 1-33, Inage-ku, Chiba 263-8522, J apan
Received December 21, 2001
Mitragynine (1) is a major alkaloidal component in the Thai traditional medicinal herb,
Mitragyna speciosa, and has been proven to exhibit analgesic activity mediated by opioid
receptors. By utilizing this natural product as a lead compound, synthesis of some derivatives,
evaluations of the structure-activity relationship, and surveys of the intrinsic activities and
potencies on opioid receptors were performed with guinea pig ileum. The affinities of some
compounds for µ-, δ-, and κ-receptors were determined in a receptor binding assay. The essential
structural moieties in the Corynanthe type indole alkaloids for inducing the opioid agonistic
activity were also clarified. The oxidative derivatives of mitragynine, i.e., mitragynine
pseudoindoxyl (2) and 7-hydroxymitragynine (12), were found as opioid agonists with higher
potency than morphine in the experiment with guinea pig ileum. In addition, 2 induced an
analgesic activity in the tail flick test in mice.
In tr od u ction
In the recent chemical1-5 and pharmacological
studies on the Rubiaceous plant, Mitragyna speciosa,
derivatives in the guinea pig ileum test will be de-
scribed. In addition, an analgesic activity of one of the
mitragynine derivatives, i.e., 2, will be reported.
6-10
1-13
1
which has been traditionally used in tropical areas as
a substitute for opium,14 we have found that mitragy-
nine (1), a major Corynanthe type indole alkaloid of this
plant, elicited potent analgesic activity mainly via
1
5
µ-opioid receptors. In addition, mitragynine pseudo-
indoxyl (2),16 an oxidative derivative of 1, was found to
show potent opioid agonistic activity in the guinea pig
ileum and in mouse vas deferens. The potency of 2 on
µ-opioid receptors in the guinea pig ileum was about
1
00- and 20-fold higher than that of 1 and morphine
(4), respectively. Additionally, its potency on δ-opioid
receptors in mouse vas deferens was about 700- and 35-
fold higher than that of 1 and 4, respectively. These
Mitragyna alkaloids from Thai herbal medicine have
different chemical structures as compared with the
morphine skeleton. These findings prompted us to
embark on the creation of novel lead compounds based
on the mitragynine skeleton, which may be utilized for
development of practical analgesics and as a new type
of probe for the study of opioid receptor systems. In the
present paper, our findings on a survey of the structure-
activity relationship using natural and semisynthetic
derivatives of 1 as well as potent and unique opioid
agonistic and antagonistic activities of the mitragynine
F igu r e 1.
Ch em istr y
*
To whom correspondence should be addressed. H.T.: Tel. and
Fax: (81)43-290-2902. E-mail: htakayam@p.chiba-u.ac.jp. S.H.: Tel.:
In investigating the structure-activity relationship,
we initially directed our attention to the presence of a
methoxyl group at the C9 position on the indole ring in
1, because it was a structural characteristic of Mi-
tragyna alkaloids, as compared with common Corynan-
(
81)43-290-2924. E-mail: syunji@p.chiba-u.ac.jp.
†
Laboratory of Molecular Structure and Biological Function, Gradu-
ate School of Pharmaceutical Sciences, Chiba University.
‡
Chulalongkorn University.
Laboratory of Chemical Pharmacology, Graduate School of Phar-
§
maceutical Sciences, Chiba University.
1
0.1021/jm010576e CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/20/2002

1
950 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Takayama et al.
Sch em e 1a
methyl formamide (DMF) under air gave the 4-qui-
nolone derivative (15) in 49% yield.
Biologica l Resu lts a n d Discu ssion
P h a r m a cologica l E va lu a t ion of P ot en cies a n d
Affin ities of Com p ou n d s for Op ioid Recep tor s
Usin g Electr ica lly Stim u la ted Con tr a ction in Iso-
la ted Gu in ea P ig Ileu m a n d Recep tor Bin d in g
Assa y. The opioid agonistic activities of the natural
analogue of mitragynine and semisynthetic compounds
derived from 1 were evaluated using twitch contraction
induced by electrical stimulation in guinea pig ileum
preparation. Opioid agonistic activities are defined as
the inhibition of the twitch contraction, which is re-
versed by the opioid antagonist naloxone. The affinities
for µ-, δ-, and κ-receptors were determined by displace-
3
3
ment upon the binding of [ H]DAMGO, [ H]DPDPE, and
[
3
H]U69593, respectively, to guinea pig brain mem-
branes.
In guinea pig ileum, the main constituent 1 inhibited
the twitch contraction induced by electrical stimulation,
which was reversed by the addition of naloxone.1
Figure 2 shows that the inhibitory effect of 1 was
concentration-dependent. Its potency is one-fourth of
that of 4 (Table 1). The first noteworthy result is that a
0,15
9
-demethoxy analogue of mitragynine, i.e., 3, did not
a
Reagents: (a) EtSH, AlCl3, CH2Cl2; (b) for 6, EtI, 15% aqueous
show any opioid agonistic activity at all (Figure 2) but
reversed the morphine-inhibited twitch contraction in
guinea pig ileum (Figure 3). Its antagonistic effect was
in a concentration-dependent manner (Table 2). Com-
pound 3 did not affect the muscarinic receptor antago-
nist atropine or the Ca channel blocker verapamil-
inhibited twitch contraction (Table 2). These results
suggest that 3 inhibits the effect of morphine via
functional antagonism of opioid receptors. The receptor
binding assay substantiated that 3 selectively binds to
µ-receptors (Table 3). Taken together, 3 was found to
have an opioid antagonistic property on µ-opioid recep-
tors.
NaOH, n-Bu4NHSO4, benzene; (c) for 7, i-PrI, 15% aqueous NaOH,
n-Bu4NHSO4, benzene; (d) for 8, CH3OCH2Cl, 15% NaOH, meth-
yltrialkyl(C8-C10)ammonium chloride, CH2Cl2; (e) for 9, Ac2O,
pyridine; (f) m-chloroperbenzoic acid, CH2Cl2.
2
+
the type indole alkaloids isolated from other plant
genus. As described in the Biological Results and
Discussion in detail, corynantheidine (3), which corre-
sponds to 9-demethoxy-mitragynine, was devoid of
agonistic activity in guinea pig ileum preparation. This
fact indicated that the methoxy group on C9 in 1 is
essential for eliciting the analgesic activity. On the basis
of this result, we initially carried out chemical modifica-
tion of the C9 function in 1. As shown in Scheme 1, the
methyl ether at C9 was selectively cleaved with EtSH
and AlCl3 in CH2Cl2 to give 9-hydroxycorynantheidine
(5) in 90% yield. The thus-obtained phenolic function
was converted to ethyl, i-propyl, and methoxymethyl
ether derivatives (6-8). In addition, acetate (9) was also
prepared with a view to its relation to morphine and
heroin. The Nb oxide derivative (10) was also prepared
by treatment of 1 with one equivalent of m-chloroper-
benzoic acid.
As described in the Introduction, we have already
found that 2, an oxidative derivative of 1, has high
agonist potency in in vitro experiments.16 On the basis
of this knowledge, we prepared some oxidative deriva-
tives of the indole nucleus in 1, as shown in Scheme 2.
Treatment of 1 with lead tetraacetate gave the 7-ac-
etoxyindolenine derivative (11) in 50% yield. By alkaline
hydrolysis of 11, 7-hydroxy-7H-mitragynine (12), which
has been found as a minor constituent in the leaves of
M. speciosa,17 was obtained in 95% yield. Treatment of
F igu r e 2. Effects of 1, 3, and 9 on twitch contraction induced
by electrical stimulation in guinea pig ileum. Each point
represents means ( SEM of five animals.
The 9-demethyl analogue of mitragynine, 5, also
inhibited electrically induced twitch contraction in
guinea pig ileum, but its maximum inhibition percent
is less potent than that of 1 (Table 1). On the other
hand, the receptor binding assay clarified that 5 binds
to µ-receptors (Table 3). Taken together, it is suggested
1
2 with sodium methoxide in methanol gave the pseudo-
indoxy derivative (2). Compound 1 afforded 7-methoxy
or 7-ethoxy indolenine derivatives (13 and 14) by
treatment with idosobenzene diacetate in MeOH or
EtOH, respectively. Treatment of 1 with NaH in di-

Mitragynine-Related Indole Alkaloids
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1951
Sch em e 2a
a
(
a) Pb(OAc)4, CH2Cl2; (b) 15% aqueous NaOH, MeOH; (c) NaOMe, MeOH; (d) for 13, idosobenzene diacetate, MeOH; (e) for 14,
idosobenzene diacetate, EtOH; (f) NaH, air, DMF.
Ta ble 1. Opioid Agonistic Activities of Mitragynine-Related Compounds and Morphine in Electrically Stimulated Guinea Pig Ileum
a
Preparation
compds
n
pD2 value
relative potency (%)
maximum inhibition (%)
relative inhibitory activity (%)
4
1
2
3
5
6
7
8
9
0
1
2
3
4
5
6
5
5
6
5
5
5
5
5
5
5
6
5
5
5
5
5
7.17 ( 0.05
100
26
3467
87.2 ( 1.8
83.1 ( 3.7
83.5 ( 3.3
-18.1 ( 8.6***
49.4 ( 3.1***
NS
100
95
96
-21
57
NS
NS
NE
38
6.59 ( 0.13**
8.71 ( 0.07***
6.78 ( 0.23
NS
NS
NE
5.39 ( 0.12
41
NS
NS
NE
2
b
b
NS
NE
c
33.2 ( 8.8***
-123.5 ( 39.2***
13.4 ( 12.7***
86.3 ( 4.8
60.9 ( 7.2**
22.9 ( 1.1***
74.1 ( 5.6
85.9 ( 2.7
1
1
1
1
1
1
1
-142
6.50 ( 0.16**
8.20 ( 0.14***
6.45 ( 0.04***
5.29 ( 0.12***
6.70 ( 0.04***
5.40 ( 0.07***
21
1071
19
1
34
15
99
70
26
85
101
2
a
Opioid agonistic activities of the compounds are evaluated by their ability to inhibit the electrically induced twitch contraction, which
is reversed by naloxone (300 nM). Relative potency is expressed as a percentage of the pD2 value of the compound against that of morphine.
The pD2 values of 2 and 12 are significantly high as compared with that of morphine, while the pD2 values of 1, 11, and 13-16 are
significantly low as compared with that of morphine. Maximum inhibition (%), which is elicited by the compound when the response
reached a plateau, was calculated by regarding electrically induced contraction as 100%. The concentration range of tested compounds
was from 100 pM to 30 µM. Relative inhibitory activity, which means intrinsic activity on opioid receptors, is expressed as a percentage
of the maximum inhibition by compounds against that by morphine. Each value represents the mean ( SEM of the results obtained from
b
five to six animals. *, P < 0.05; **, P < 0.01; ***, P < 0.001, significantly different from the morphine group. In the case of the naloxone
insensitive inhibition, the effect was regarded as “nonspecific (NS)”. c In the case that significant inhibition was not obtained at 30 µM of
the compound, the effect was regarded as “no effect (NE)”.
Ta ble 2. Effects of 3 on Twitch Contraction Inhibited by 4,
Atropine, and Verapamil in Electrically Stimulated Guinea Pig
Ileum Preparationa
contraction (%)
contraction (%)
reversed by 3
inhibited by
drugs (concn)
drugs (control)
1 µM
10 µM
4
(1 µM)
27.4 ( 3.5
3.9 ( 1.3
6.8 ( 1.8
43.0 ( 4.2* 109.2 ( 6.2***
atropine (0.1 µM)
verapamil (3 µM)
5.7 ( 1.6
7.0 ( 1.9
6.3 ( 1.4
13.2 ( 3.8
a
Each value represents the mean ( SEM of the results obtained
from five animals. *, P < 0.05; ***, P < 0.001, significantly
different from the control group.
F igu r e 3. Typical recording of effect of 3 on twitch contraction
inhibited by morphine in guinea pig ileum.
that 5 is a partial agonist of opioid receptors. It is an
interesting finding that a fine transformation of the
substituent on C9, i.e., from OMe to OH or to H, led to
a shift of activity from that of a full agonist via a partial
agonist to that of an antagonist on opioid receptors.
Thus, it is found that the functional group on C9 of

1
952 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Takayama et al.
Ta ble 3. Opioid Receptor Binding Affinities of
Mitragynine-Related Compounds at µ-, δ-, and κ-Receptora
relative
affinity (%)
pKi
compds [3H]DAMGO [3H]DPDPE [3H]U69593
µ
δ
κ
4
1
2
3
5
2
8.46 ( 0.29 6.38 ( 0.13 6.33 ( 0.22 98.5
0.8 0.7
8.14 ( 0.28 7.22 ( 0.21 5.96 ( 0.22 88.7 10.7 0.6
10.06 ( 0.39 8.52 ( 0.22 7.10 ( 0.32 97.1
7.14 ( 0.18 5.54 ( 0.44 5.59 ( 0.04 95.8
7.92 ( 0.05 4.51 ( 0.15 5.53 ( 0.07 99.6 < 0.1 0.4
7.87 ( 0.16 6.81 ( 0.19 6.91 ( 0.07 83.3 6.6 10.1
2.8 0.1
1.0 3.2
1
a
Receptor binding assays were carried out using membranes
prepared from guinea pig brain. Specific binding of each radioli-
gand for opioid receptor subtype was saturated, and Scatchard
3
3
plots were linear. The KD values of [ H]DAMGO, [ H]DPDPE, and
[
3
H]U69593 were 0.44 ( 0.04, 0.83( 0.05, and 0.69 ( 0.10 nM,
respectively. Radioligand displacement curves were generated
from binding data. The binding constants (pKi) were calculated
using the IC50 values obtained from the displacement curves.
Values are the means ( SEM of 3-5 separate displacement curves,
each assayed in triplicate. Relative affinities (%) of compounds
were calculated according to the following equations: relative
affinity ) Kax/(Kaµ + Kaδ + Kaκ), (Ka ) 1/Ki).
mitragynine-related compounds managed the relative
inhibitory activity, which means the intrinsic activity
on opioid receptors. The affinities of 1, 3, and 5 on µ-,
δ-, and κ-receptors were determined by displacement of
the binding of specific radioligands at each receptor in
guinea pig brain membranes. Table 3 shows a close
similarity of the pKi values of 3 and 5 on each receptor.
The relative affinities for µ-, δ-, and κ-receptors are also
shown in Table 3. The binding assay demonstrated that
these compounds have relatively high selectivity for
µ-receptors.
F igu r e 4. Stereostructure of mitragynine and speciociliatine.
agonistic property on opioid receptors. The introduction
of a hydroxy group at the C7 position led to higher
potency as compared with that of 1. As shown in Table
3
, 12 tends to show selectivity at µ-receptors. The
relative affinity for κ-receptors of 12 is higher than that
of 1.
In turn, 11 has lower intrinsic activity than 1,
although its potency is nearly equal to that of 1 (Table
1
). The introduction of a methoxy or an ethoxy group
The introduction of the acetoxy group at C9 on the
indole ring (compound 9) led to marked reduction in
both intrinsic activity and potency as compared with
those of 1 (Figure 2). The compounds 6 and 7 with the
elongation of the methyl group of 9-methoxy ether
induced naloxone insensitive inhibition on twitch con-
traction (Table 1), suggesting a inhibitory effect via
mechanisms distinct from the stimulation of opioid
receptors. The compound 8 did not show any opioid
agonistic activities. These results demonstrate that the
intrinsic activities of compounds on opioid receptors are
determined by their functional groups at the C9 position
and that a methoxy group at the C9 position is the most
suitable functional group for pharmacophore binding to
opioid receptors.
Speciociliatine (16), a minor constituent of this plant,
is the C3 stereoisomer of mitragynine, which takes a
folded cis-quinolizidine conformation in the C/D-ring
junction as depicted in Figure 4. The potency of this
compound to opioid receptors was 14-fold weaker than
that of 1 in ileal preparation (Table 1), indicating that
the flat trans-quinolizidine form was a more efficient
conformation for exhibiting the activity than the folded
cis form. The Nb oxide derivative (10) showed no opioid
agonistic activity in guinea pig ileum. The Nb lone
electron pair was also found to be essential for opioid
agonistic activity. These results suggest that the Nb lone
electron pair of this series of compounds constitutes
pharmacophore binding to opioid receptors.
at the C7 position (compounds 13 and 14) led to a
dramatic reduction in both intrinsic activity and potency
for opioid receptors (Table 1). It is conceivable that the
hydroxyl group at the C7 position in the mitragynine
skeleton is a necessary functional group for the in-
creased potency to opioid receptors. 4-Quinolone deriva-
tive (15) retained almost the same opioid agonistic
activity as that of 1.
P h a r m a cologica l Eva lu a tion of An a lgesic Ac-
tivities of Com p ou n d s in Mice. Among the mitragy-
nine derivatives described above, 2 emerged as com-
pounds of potential analgesics in the guinea pig ileum
test. Thereupon, the antinociceptive activities of com-
pound 2 having a different structural skeleton from
morphine were evaluated using mice tail flick test.
Compound 2, administered by intracerebroventricular
(icv) injections, showed antinociceptive effects in the tail
flick test in mice. Its effect reached a maximum at about
15-45 min after the injection (Figure 6). The effect of
2 is less potent than that of morphine (Figure 5). In
addition, the effect of 1 is less potent than that of 2
(Figure 5). The analgesic effects of these compounds are
dose-dependent. The ED50 value estimated for 2 was
6.51 nmol/mouse (95% confidence limits 3.78-11.20
nmol/mouse), while that of 4 was 3.20 nmol/mouse (95%
confidence limits 2.11-4.88 nmol/mouse). The ED50
value estimated for 1 was 60.22 nmol/mouse (95%
confidence limits 39.10-92.75 nmol/mouse). The anti-
nociceptive effects of 1, 2, and 4 were completely
inhibited by naloxone at 2 mg/kg, s.c. (Figure 6).
Therefore, these compounds induce analgesic effect via
opioid receptors. Compound 2 exhibited less potent
Compound 12, a minor constituent of M. speciosa, was
found to exhibit high potency to opioid receptors: about
1
3- and 46-fold higher than those of 4 and 1, respec-
tively. The intrinsic activity of 12 suggested its full

Mitragynine-Related Indole Alkaloids
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1953
C9 position, i.e., OMe f OH f H, of mitragynine, the
activities of compounds dramatically shifted from that
of full agonists through partial agonists to that of
antagonists on opioid receptors. Among the mitragynine
derivatives prepared in the present study, 2, which is
an oxidized derivative of 1, and 12, which was also
isolated as a minor constituent of the Thai medicinal
herb, M. speciosa, were found as opioid agonists with a
higher potency than 4 in the experiment with guinea
pig ileum. In addition, 2 induced an analgesic activity
in the tail flick test in mice. Detailed studies on their
analgesic effects are under way to investigate the
potential abilities of mitragynine derivatives for clinical
use.
Exp er im en ta l Section
F igu r e 5. Dose-response curves for analgesic activities
induced by intracerebroventricular administration of 1, 2, and
Gen er a l Meth od s. UV spectra were recorded in MeOH.
Hitachi U 3400. H and C nuclear magnetic resonance (NMR)
spectra were recorded at 500 and 125.65 MHz, respectively
1
13
4
in tail flick test in mice. Tail flick latencies are presented as
%
MPE. Values are mean ( SEM of values obtained from 9 to
(
ppm, J in Hz with tetramethylsilane (TMS) as internal
1
2 animals. Each point represents % MPE at 30 min after the
standard). J EOL J NM A-500. Electron ionization mass spec-
trometry (EI-MS): direct probe insertion at 70 eV. J EOL J MS-
AM20. Fast atom bombardment (FAB)-MS: J EOL J MS-
HX110. Thin-layer chromatography (TLC): precoated Kieselgel
60 F254 plates (Merck, 0.25 mm thick). Column chromatogra-
phy: Kieselgel 60 [Merck, 70-230 (for open chromatography)
and 230-400 mesh (for flash chromatography)], Sephadex LH-
0 [Pharmacia Biotech]. Performance liquid chromatography
MPLC): silica gel prepacked column Kusano CPS-HS-221-
5. Preparative TLC: silica gel 60 GF254 (Merck 7730, 0.5 mm
administration of each drug. *, P < 0.05; **, P < 0.01; ***, P
<
0.001, significantly different from the control (vehicle) group.
analgesic activity than 4, despite very high opioid
activity in isolated guinea pig ileum test. We speculate
that the low analgesic activity of 2 results from the
instability of the compound in the brain.
2
(
0
Com p u ta tion a l Su p er p osition of Mor p h in e a n d
Mitr a gyn in es. Complementarily, we tried to look for
any structural similarity between morphine and mi-
tragynine or its derivative using molecular modeling
techniques. A theory that spatial arrangement of the
three functional groups, i.e., a nitrogen atom, a benzene
residue, and a phenol function, in the structure of
morphine plays a significant role in exhibiting analgesic
thick). Male albino Dunkin-Hartley guinea pigs (300-400 g)
and male ddY mice (30-35 g) were purchased from Takasugi
Laboratory Animals Co. (Saitama, J apan) and J apan SLC Inc.
(
Shizuoka, J apan), respectively. They were housed in 12 h/12
h light/dark cycles, at 25 °C ( 1 for at least 1 week before the
experiments with free access to food and water. Each animal
was used only once. The experiments were carried out in strict
accordance with “Guiding Principles for the Care and Use of
Laboratory Animals” approved by The J apanese Pharmaco-
logical Society and the guideline approved by the Ethical
Committee on Animal Care and Animal Experiment of our
Faculty. Chemicals for pharmacological assay were obtained
from the following sources: acetylcholine chloride (Dai-ichi
Seiyaku Co., Ltd., Tokyo, J apan); morphine hydrochloride
1
8
activity has been proposed by Beckett and Casy. After
that, Portoghese has suggested a new concept “different
1
9
binding modes for different opioid skeletons”. These
models have since been refined by researchers and now
include the receptor-based models to interpret the
interactions between the opioid receptors and the ago-
nists including the nonmorphinan skeletons so far
3
2
4
(Takeda Chemical Industries, J apan); [ H][D-Ala , MePhe ,
5
3
3
2,5
Glyol ] enkephalin ([ H]DAMGO), [ H][D-Pen ] enkephalin
3
3
2
0
([ H]DPDPE), and [ H]U-69593 (Du Pont NEN Life Science
developed. At the outset, we examined the respective
superposition between the nitrogen atom, benzene ring,
and oxygen function on the benzene ring in 4 and those
in 1 or 2. However, as shown in Figure 7, we have not
been able to superimpose all three functions of the two
molecules. Therefore, we concluded that Mitragyna
alkaloids are structurally different from the morphine
skeleton.
2
4
5
Products, Boston, MA); [D-Ala , Met-Phe , Glyol ] enkephalin
DAMGO), naloxone hydrochloride, U-69593 (Sigma Chemical
(
2
,5
Co., St. Louis, MO); [D-Pen ] enkephalin (DPDPE) (BACHEM
Feinchemikalien, Switzerland).
Ch em istr y. Con ver sion of 1 to 5. To a stirred solution of
1 (300 mg, 0.75 mmol) in dry CH Cl (6 mL) was successively
2
2
3
added AlCl (302 mg, 2.26 mmol) and then EtSH (1.0 mL, 13.5
mmol) at 0 °C under argon atmosphere. After the reaction
mixture was stirred for 3 h at room temperature, the volatile
material was thoroughly removed under reduced pressure. The
Con clu sion
residue was separated by Al
hexane/AcOEt 1:1) to give (-)-5 (258 mg, 90%) as an amor-
phous powder. UV (MeOH) λmax: 294, 226 nm. IR νmax (CHCl ):
): δ 7.78 (1H, br.s, Na-
H), 7.43 (1H, s, H-17), 6.89 (1H, dd, J ) 7.8, 7.8, H-11), 6.84
2 3
O column chromatography (n-
A series of novel Corynanthe type ligands derived
from a natural indole alkaloid, 1, was prepared and
evaluated for pharmacological activities on opioid recep-
tors. Consequently, we found that some mitragynine
derivatives, whose basic skeleton is completely different
from that of 4, exhibit potent agonistic properties on
opioid receptors in guinea pig ileum. It was also found
that the methoxy group on the indole ring at the C9
position as well as the Nb lone electron pair in the
fundamental structure of Corynanthe type indole alka-
loid are essential structural functions for opioid ago-
nistic activity. With altering the functional group at the
3
-1 1
3
213, 1695, 1636 cm . H NMR (CDCl
3
(
1H, d, J ) 7.3, H-12), 6.37 (1H, d, J ) 7.3, H-10), 3.71 (6H, s,
17-OCH3 and 22-OCH ), 3.19 (1H, m, H-6), 3.13 (1H, br.d, J
3
) 11.6, H-3), 3.04 (2H, m, H-15 and H-21), 2.94 (2H, m, H-5
and H-6), 2.56 (1H, m, H-5), 2.08 (1H, m, H-14), 2.44 (1H, m,
H-21), 1.76 (2H, m, H-14 and H-19), 1.63 (1H, m, H-20), 1.22
1
3
(
(
(
1H, m, H-19), 0.86 (3H, dd, J ) 7.3, 7.3, H-18). C NMR
CDCl ): δ 169.3 (C-22), 160.6(C-17), 150.0 (C-9), 137.9
C-13), 134.0 (C-2), 121.9 (C-11), 116.6 (C-7), 111.4 (C-16),
3
106.7 (C-8), 104.3 (C-10), 103.8 (C-12), 61.5 (17-OCH ), 61.2
3
3
(C-3), 57.6 (C-21), 53.5 (C-5), 51.4 (22-OCH ), 40.6 (C-20), 39.8

1
954 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Takayama et al.
F igu r e 6. Effects of naloxone on analgesic activities of 1 (75 nmol/mouse, icv), 2 (12 nmol/mouse, icv), and 4 (9 nmol/mouse, icv)
in tail flick test in mice. Naloxone (2 mg/kg, s.c.) was administered 15 min before injection of each drug. Tail flick latencies are
presented as a percentage of MPE. Values are mean ( SEM of values obtained from 7 to 12 animals. *, P < 0.05; **, P < 0.01;
*
**, P < 0.001, significant difference in comparison of each nontreated group. #, P < 0.05; ##, P < 0.01; ###, P < 0.001, significant
difference in comparison of each drug group.
F igu r e 7. Overlays of the low-energy conformation of morphine (gray)/mitragynine (black) (A) and morphine (gray)/mitragynine
pseudoindoxyl (black) (B). Hydrogen atoms are omitted.
(
C-15), 29.8 (C-14), 23.6 (C-6), 19.1 (C-19), 12.8 (C-18). EI-MS
(C-5), 51.5 (22-OCH
3
), 40.7 (C-20), 40.0 (C-15), 29.9 (C-14), 22.9
), 19.1 (C-19), 12.9 (C-18). EI-MS m/z
(%): 426 (M , 85), 411 (35), 297 (35), 242 (100). HR-FABMS:
+
m/z (%): 384 (M , 100), 369 (35), 255 (30), 200 (76). HR-
FABMS: calcd for C22
P r ep a r a tion of 9. A mixture of 5 (11 mg, 0.028 mmol) and
acetic anhydride (250 µL, 0.265 mmol) in dry pyridine (0.5 mL)
was stirred at room temperature under argon atmosphere for
(C-6), 21.1 (9-OCOCH
3
+
+
29 4 2
H O N [MH ], 385.2127; found, 385.2123.
+
calcd for C24
P r ep a r a tion of 10. To a stirred solution of 1 (52 mg, 0.13
mmol) in dry CH Cl (1 mL) was added a solution of m-
chloroperbenzoic acid (25 mg, 0.16 mmol) in dry CH Cl (0.3
31 5 2
H O N [MH ], 427.2233; found, 427.2233.
2
2
1
h. After the volatile material was removed under reduced
pressure, the residue was diluted with 5% aqueous NaHCO
solution. The whole mixture was extracted with CHCl three
times. The combined organic layer was washed with brine,
dried over MgSO , and evaporated. The residue was separated
by SiO column chromatography (3% MeOH-CHCl ) to give
(9 mg, 76%) as an amorphous powder. UV (MeOH) λmax: 279
2
2
3
mL) at -50 °C under argon atmosphere. After the reaction
mixture was stirred for 4 h, the reaction mixture was poured
3
into the chilled 5% NaHCO
CHCl . The combined organic layer was washed with brine,
dried over MgSO , and evaporated. The residue was separated
by SiO column chromatography (5% MeOH-CHCl ) to give
10 (19 mg, 36%) as an amorphous powder. UV (MeOH) λmax:
3
solution and was extracted with
4
3
2
3
4
9
(
(
2
3
1
sh), 227 nm. H NMR (CDCl
3
): δ 7.95 (1H, br.s, Na-H), 7.43
1
1H, s, H-17), 7.15 (1H, dd, J ) 8.1, 0.8, H-12), 7.05 (1H, dd,
291, 283 (sh), 247 (sh), 224 nm. H NMR (CDCl ): δ 7.87 (1H,
3
J ) 7.8, 7.8, H-11), 6.75 (1H, dd, J ) 7.7, 0.8, H-10), 3.72 (3H,
s, 17-OCH ), 3.71 (3H, s, 22-OCH ), 3.15 (1H, br.d, J ) 11.3,
H-3), 3.04 (3H, m, H-6 and H-15 and H-21), 2.92 (1H, m, H-5),
2
2
1
br.s, Na-H), 7.52 (1H, s, H-17), 7.00 (1H, dd, J ) 7.9, 7.9,
H-11), 6.87 (1H, d, J ) 8.1, H-12), 6.45 (1H, d, J ) 7.8, H-10),
3
3
4.31 (1H, br.d, J ) 11.9, H-3), 3.86 (3H, s, 9-OCH
s, 17-OCH ), 3.74 (3H, s, 22-OCH
3
), 3.79 (3H,
.71 (1H, br.d, J ) 10.4, H-6), 2.55 (2H, m, H-5 and H-14),
.46 (1H, dd, J ) 11.5, 2.5, H-21), 2.35 (3H, s, 9-OCOCH ),
.80 (1H, br.d, J ) 12.6, H-14) 1.75 (1H, m, H-19), 1.63 (1H,
3
3
), 3.61∼3.86 (4H, m, H-5 and
3
H-21), 3.38∼3.48 (1H, m, H-14), 3.31 (1H, dd, J ) 12.2, 4.6,
H-6), 3.19 (1H, br.d, J ) 13.4, H-15), 3.06 (1H, dd, J ) 16.1,
3.9, H-6), 2.65∼2.80 (1H, m, H-19), 1.80∼1.95 (2H, m, H-14
and H-20), 1.30∼1.45 (1H, m, H-19), 0.99 (3H, dd, J ) 7.4,
br.d, J ) 10.9, H-20), 1.21 (1H, m, H-19), 0.86 (3H, dd, J )
1
3
7
(
1
(
.4, 7.4, H-18). C NMR (CDCl
C-22), 160.7 (C-17), 143.7 (C-9), 137.9 (C-13), 136.2 (C-2),
21.3 (C-11), 120.4 (C-7), 112.0 (C-10), 111.4 (C-16), 108.9
C-12), 106.3 (C-8), 61.6 (17-OCH ), 61.1 (C-3), 57.7 (C-21), 53.4
3
): δ 170.1 (9-OCOCH
3
), 169.3
7.4, H-18). 13C NMR (CDCl
): δ 168.86 (C-22), 161.27 (C-17),
3
154.68 (C-9), 137.65 (C-13), 127.20 (C-2), 122.57 (C-11), 117.45
(C-7), 109.92 (C-16), 107.17 (C-8), 104.34 (C-12), 100.08
3

Mitragynine-Related Indole Alkaloids
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1955
H-19), 1.10∼1.14 (1H, m, H-14R), 0.85 (3H, dd, J ) 7.4, 7.4,
(
5
2
C-10), 77.21 (C-5), 71.47 (C-3), 66.85 (C-21), 61.79 (17-OCH
3
),
), 40.27 (C-20), 39.42 (C-15),
9.95 (C-6), 24.15 (C-14), 20.82 (C-19), 13.31 (C-18). EI-MS
5.41 (9-OCH
3
), 51.50 (22-OCH
3
H-18). 13C NMR (CDCl
): δ 199.46 (C-7), 168.85 (C-22), 162.12
3
(C-9), 160.16 (C-17), 158.61 (C-13), 138.62 (C-11), 111.76
(C-16), 109.83 (C-8), 103.76 (C-12), 99.07 (C-10), 75.16 (C-2),
+
m/z (%): 414 (M , 1.7), 398 (16), 397 (18), 383 (10), 200 (100).
HR-FABMS: calcd for C23 , 415.2233; found, 415.2243.
P r ep a r a tion of 11. To a stirred solution of 1 (105 mg, 0.26
mmol) in dry CH Cl (14 mL) was added Pb(OAc) (259 mg,
1% purity, 0.53 mmol) at 0 °C under argon atmosphere. After
H
31
O
5
N
2
73.20 (C-3), 61.38 (17-OCH
3.15 (C-5), 51.13 (22-OCH3), 40.12 (C-20), 38.41 (C-15), 35.06
C-6), 23.79 (C-14), 19.31 (C-19), 12.90 (C-18). EI-MS m/z (%):
3 3
), 55.65 (9-OCH ), 54.82 (C-21),
5
(
4
2
2
4
+
14 (M , 24), 239 (43), 238 (100). HR-FABMS: calcd for
, 415.2233; found, 415.2207.
Molecu la r Mod elin g. Compounds 4, 1, and 2 were sub-
9
23 31 5 2
C H O N
the reaction mixture was stirred for 1.5 h, the reaction mix-
ture was poured onto the chilled water and was extracted with
CH
with brine, dried over MgSO
was separated by Al column chromatography (Woelm N,
grade III, CH Cl ) to give 11 (60 mg, 50%) as a yellowish
amorphous powder. UV (MeOH) λmax: 222, 246 (sh), 310 nm.
2
Cl
2
five times. The combined organic layer was washed
jected to an energy minimization using the semiempirical
quantum mechanics method AM1 as implemented in the
MOPAC 5.0 programs. The superimposed ensembles of the
compounds, 4/1 and 4/2, were subjected to the overlay program
implemented in Chem 3D 6.0.
4
, and evaporated. The residue
2
O
3
2
2
1
H NMR (CDCl
.4, 8.4, H-11), 7.25 (1H, dd, J ) 7.6, 0.9, H-12), 6.72 (1H, d,
J ) 7.6, H-10), 3.83 (3H, s, 9-OCH ), 3.81 (3H, s, 17-OCH ),
.70 (3H, s, 22-OCH ), 3.03 (1H, dd, J ) 11.4, 2.5, H-3), 2.05
3H, s, 7-OAc), 0.82 (3H, dd, J ) 7.4, 7.4, H-18), 2.40∼3.00
3
): δ 7.44 (1H, br.s, H-17), 7.28 (1H, dd, J )
Ma gn u s Assa ys Usin g Gu in ea P ig Ileu m P r ep a r a tion s.
8
Male, albino guinea pigs (Dunkin-Hartley) weighing 300-
3
3
4
00 g purchased from Takasugi Laboratory Animals Co. Ltd.
3
3
were stunned by a blow on the head and exsanguinated. The
(
(
(
(
(
(
(
ileum was removed and placed in Krebs-Henseleit solution
1
3
7H), 1.23∼1.92 (5H). C NMR (CDCl
C-22), 168.54 (7-OCOCH ), 160.78 (C-17), 155.72 (C-9), 155.46
C-13), 130.78 (C-11), 122.87 (C-8), 114.49 (C-12), 111.09
C-16), 108.88 (C-10), 84.76 (C-7), 61.95 (17-OCH ), 61.79
C-3), 58.09 (C-21), 55.41 (9-OCH ), 51.29 (22-OCH ), 50.03
C-5), 40.53 (C-20), 39.38 (C-15), 35.24 (C-6), 25.79 (C-14),
3
): δ 180.92 (C-2), 169.29
2 2 2
(mM): NaCl, 112.1; KCl, 5.9; CaCl , 2.0; MgCl , 1.2; NaH -
3
4 3
PO , 1.2; NaHCO , 25.0; and glucose, 11.5. The solution was
21
prepared as reported by Cox and Weinstock. The ileum was
set up under 1 g of tension in a 5 mL organ bath containing
the nutrient solution. The bath was maintained at 37 °C and
3
3
3
continuously bubbled with a gas mixture of 95% O
2
and 5%
2
4
3
0.77 (7-OCOCH ), 18.97 (C-19), 12.82 (C-18). EI-MS m/z (%):
CO . At the start of each experiment, a maximum response to
2
+
+
56 (M , 0.26), 397 ([M - OAc] , 87), 396 (100). HR-FABMS:
acetylcholine (3 µM) was obtained in each tissue to check its
suitability. Tissues were stimulated through platinum needle-
ring (a ring was placed 20 mm above the base of the needle 5
mm in length) electrodes using square wave pulses of supra-
maximal voltage. The ileum was transmurally stimulated with
monophasic pulses (0.2 Hz) and 0.3 ms duration by a stimula-
tor (SEN-7203, Nihon Kohden, Tokyo, J apan). Contractions
were isotonically recorded by using a displacement transducer
(NEC, San-ei Instruments Ltd., Type 45347), DC strain
amplifier (San-ei 6M92), and a DC recorder (Hitachi, Mod 056,
Tokyo, J apan). All concentration-response curves were con-
structed in a cumulative manner. The height of the twitch
response to transmural stimulation was measured before and
after drug challenge. The percentage of the inhibition response
remaining after the agonist was determined by dividing the
contractile height after each agonist addition by the twitch
height before agonist administration multiplied by 100. To
obtain the percentage inhibition of the twitch height, this value
was subtracted from 100. Agonist activity was expressed as a
calcd for C25
33 6 2
H O N
, 457.2339; found, 457.2324.
P r ep a r a tion of 12. A mixture of 11 (70 mg, 0.16 mmol)
and aqueous 15% NaOH (0.3 mL) in MeOH (2 mL) was stirred
at 0 °C under argon atmosphere for 2 h. The reaction mixture
was poured onto the chilled water and was extracted with
CHCl
brine, dried over MgSO
separated by Al column chromatography (n-hexane/AcOEt
:4) to give 12 (62 mg, 95%) as an amorphous powder. UV
EtOH) λmax(logꢀ): 305 (sh, 3.43), 245 (sh, 4.06), 221 (4.36) nm.
IR νmax (CHCl ): 3590, 2850, 2820, 2750, 1700, 1645, 1630,
600, 1490, 1465, 1440 cm . H NMR (CDCl
H-17), 7.27 (1H, dd, J ) 8.0, 8.0, H-11), 7.19 (1H, d, J ) 7.6,
H-12), 6.71 (1H, d, J ) 8.0, H-10), 3.85 (3H, s, 9-OCH ), 3.80
3H, s, 17-OCH ), 3.68 (3H, s, 22-OCH ), 3.12 (1H, dd, J )
1.0, 2.5, H-3), 3.03 (1H, dd, J ) 11.4, 2.2, H-21), 3.00 (1H,
3
five times. The combined organic layer was washed with
4
, and evaporated. The residue was
2
O
3
6
(
3
-
1 1
1
3
): δ 7.43 (1H, s,
3
(
3
3
1
ddd, J ) 13.8, 3.6, 3.6, H-15), 2.75∼2.84 (1H, m, H-14),
2
1
1
.75∼2.84 and 2.61∼2.66 (2H, each m, H-5), 2.61∼2.66 and
.60∼1.73 (2H, each m, H-6), 2.48 (1H, dd, J ) 11.2, 2.7, H-21),
.87 (1H, br.d, J ) 13.7, H-14), 1.60∼1.73 and 1.23 (2H, each
2
pD value, which is the negative logarithm of the molar con-
centration required to produce 50% of the maximum responses
to the drug (EC50). To investigate the involvement of opioid
receptors in the inhibitory effect of the samples, antagonistic
effect of naloxone on the inhibited contraction was examined.
The inhibitory effects of samples on electrically stimulated
contraction can be regarded as opioid activities when the
antagonistic effect of naloxone is observed.
m, H-19), 1.57∼1.61 (1H, m, H-20), 0.82 (3H, dd, J ) 7.3, 7.3,
1
3
H-18). C NMR (CDCl
3
): δ 184.33 (C-2), 169.22 (C-22), 160.65
C-17), 155.85 (C-9), 154.98 (C-13), 130.55 (C-11), 126.51
C-8), 114.10 (C-12), 111.18 (C-16), 108.79 (C-10), 80.88 (C-7),
(
(
6
5
1.69 (17-OCH3), 61.41 (C-3), 58.10 (C-21), 55.37 (9-OCH
3
),
1.19 (22-OCH ), 49.98 (C-5), 40.44 (C-20), 39.25 (C-15), 35.64
3
(
4
C-6), 25.99 (C-14), 18.87 (C-19), 12.75 (C-18). EI-MS m/z (%):
14 (M , 91), 397 (100), 383 (38), 367 (39). HR-FABMS: calcd
Ra d ioliga n d Bin d in g Assa ys for µ-, δ-, a n d K-Recep -
tor s. Male Dunkin-Hartley guinea pigs were killed by cervical
dislocation and exsanguinated. The whole brain (excluding
cerebellum) was quickly removed and placed in ice-cold 0.05
M Tris-HCl buffer, pH 7.4, at 25 °C, weighed, and immediately
frozen in dry ice containing acetone. For each experiment,
frozen brains from two animals were thawed and homogenized
with a homogenizer (Kinematica GmbH LITTAU, Polytron, PT
10-35, Switzerland) for 60 s in 40 volumes of 50 mM Tris HCl
(pH 7.4) and centrifuged at 49 000g for 10 min. The pellet was
rehomogenized and centrifuged again. For the binding assays,
membrane fractions were suspended in assay buffer at a
protein concentration of 50 mg/mL. Saturation binding iso-
therms were produced by incubating each labeled compound
at nine or ten different concentrations (10-2000 pM) with 2
mg of membrane protein. To start the reaction, 0.1 mL aliquots
of protein were added to 0.9 mL of 50 mM Tris-HCl (pH 7.4)
+
for C23 , 415.2233; found, 415.2235.
31 5 2
H O N
P r ep a r a tion of 2 fr om 12. A solution of 12 (66 mg, 0.16
mmol) and NaOMe (19 mg, 0.35 mmol) in dry MeOH (6 mL)
was heated under reflux for 12 h under argon atmosphere.
The reaction mixture was cooled, poured onto the chilled water,
and then extracted with CHCl
organic layer was washed with brine, dried over MgSO
evaporated. The residue was separated by SiO column chro-
matography (n-hexane/AcOEt 1:2) to give 2 (32 mg, 48%) as
an amorphous powder. UV (MeOH) λmax: 395, 289, 238, 216,
2
7
3
three times. The combined
4
, and
2
1
02 nm. H NMR (CDCl
3
): δ 7.32 (1H, dd, J ) 8.2, 8.2, H-11),
.28 (1H, s, H-17), 6.40 (1H, d, J ) 7.8, H-12), 6.14 (1H, d, J
7.8, H-10), 5.17 (1H, br.s, Na-H), 3.90 (3H, s, 9-OCH ), 3.66
3H, s, 17-OCH ), 3.62 (3H, s, 22-OCH
)
(
3
3
3
), 3.09∼3.15 (2H, m,
H-5 and H-21), 2.77 (1H, m, H-15), 2.30∼2.36 (2H, m, H-5 and
3
3
H-6R), 2.15∼2.26 (2H, m, H-14â and H-3), 2.14 (1H, br.dd, J
assay buffer containing 1 nM [ H]DAMGO, [ H]DPDPE, or
3
)
11.4, 3.1, H-21), 1.87∼1.94 (1H, m, H-6â), 1.60∼1.68 (1H,
[ H]U-69593 and appropriate concentrations of competing
m, H-19), 1.50 (1H, br.d, J ) 11.2, H-20), 1.14∼1.24 (1H, m,
unlabeled ligands in a total volume of 1 mL. The incubation

1
956 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Takayama et al.
3
3
times were 1.5, 2, and 1 h for [ H]DAMGO, [ H]DPDPE, and
[
(4) Takayama, H.; Aimi, N.; Sakai, S. Chemical studies on the
analgesic indole alkaloids from the traditional medicine (Mi-
tragyna speciosa) used for opium substitute. Yakugaku Zasshi
3
H]U-69593, respectively, at 25 °C. The reaction was termi-
nated by rapid filtration under reduced pressure through glass
microfiber filters (Whatman GF/B, presoaked in Tris-HCl
buffer) followed by the addition of 4 mL of ice-cold Tris-HCl
buffer. Filters were further washed with 4 mL of ice cold buffer
and left to dry for 12 h. Radioactivity bound to the filters was
quantitated by liquid scintillation spectrometry (ALOKA LSC-
100, J apan). Nonspecific binding for [ H]DAMGO, [ H]DP-
DPE, and [ H]U-69593 was determined in the presence of 1
µM unlabeled DAMGO, naltrindole, and U-69593, respectively.
The apparent dissociation constant (K ) and maximum binding
D
2
000, 120, 959-967 and references therein.
(
5) Takayama, H.; Ishikawa, H.; Kurihara, M.; Kitajima, M.; Sakai,
S.; Aimi, N.; Seki, H.; Yamaguchi, K.; Said, I. M.; Houghton, P.
J . Structure revision of mitragynaline, an indole alkaloid in
Mitragyna speciosa. Tetrahedron Lett. 2001, 42, 1741-1743.
6) Horie, S.; Yamamoto, L. H.; Futagami, Y.; Yano, S.; Takayama,
H.; Sakai, S.; Aimi, N.; Ponglux, D.; Shan, J .; Pang, P. K. T.;
(
3
3
5
2
+
Watanabe, K. Analgesic, neuronal Ca channel-blocking and
smooth muscle relaxant activities of mitragynine, an indole
alkaloid, from the Thai folk medicine ‘Kratom’. J . Traditional
Med. 1995, 12, 366-367.
3
site density (Bmax) for radioligands were estimated by Scat-
chard analysis of the saturation data over a concentration
range of 0.01-2 nM. The ability of unlabeled drugs to inhibit
specific radioligand binding was expressed as the IC50 value,
which was the molar concentration of unlabeled drug neces-
sary to displace 50% of the specific binding. Competitive
inhibition studies were carried out in the presence of 6-9
different inhibitor concentrations. Inhibition constant values
(7) Matsumoto, K.; Mizowaki, M.; Thongpradichote, S.; Murakami,
Y.; Takayama, H.; Sakai, S.; Aimi, N.; Watanabe, H. Central
antinociceptive effects of mitragynine in mice: Contribution of
descending noradrenergic and serotonergic systems. Eur. J .
Pharmacol. 1996, 317, 75-81.
8) Tohda, M.; Thongpraditchote, S.; Matsumoto, K.; Murakami, Y.;
Sakai, S.; Aimi, N.; Takayama, H.; Tongroach, P.; Watanabe,
H. Effects of mitragynine on cAMP formation mediated by
δ-opiate receptors in NG108-15 cells. Biol. Pharm. Bull. 1997,
(
2
0, 338-340.
i
(K ) of unlabeled compounds were calculated as described by
(
9) Matsumoto, K.; Mizowaki, M.; Takayama, H.; Sakai, S.; Aimi,
N.; Watanabe, H. Suppressive effect of mitragynine on the
2
2
Cheng and Prusoff. Their negative logarithm values were
shown as pK values.
An tin ocicep tive Assa ys Usin g Mou se Ta il F lick Test.
i
5
-methoxy-N, N-dimethyltryptamine-induced head-twitch re-
sponse in mice. Pharmacol. Biochem. Behav. 1997, 57, 319-323.
2
3
(10) Watanabe, K.; Yano, S.; Horie, S.; Yamamoto, L. T. Inhibitory
effect of mitragynine, an indole alkaloid with analgesic effect
from Thai medicinal plant, Mitragyna speciosa, on electrically
stimulated contraction of isolated guinea-pig ileum through the
opioid receptor. Life Sci. 1997, 60, 933-942.
The tail flick test described by D’Amour and Smith employing
a tail flick apparatus (Ugo Basile, Verese, Italy) was used.
Before injections, three drug-free trials were used to establish
basal reaction times. The ventral surface of the tail (2-5 cm
from the tip) was exposed to radiant heat, and the latency of
the tail movement was recorded. The radiant heat source was
set such that basal latencies were 2-4 s. Only mice with a
control reaction between the basal latencies were used. The
test latency after drug treatment was assessed at appropriate
times, and a 10 s maximum cutoff time was used to prevent
damage to the tail. Antinociception was quantified as the
maximum possible effect (% MPE) using the following for-
mula: % MPE ) 100 × [(test - control)/(10 - control)]. The
(11) Shellard, E. J .; Houghton, P. J .; Resha, M. The Mitragyna
speciosa of Asia. Planta Med. 1978, 33, 223-227.
12) Houghton, P. J .; Said, I. M. 3-Dehydromitragynine: An alkaloid
from Mitragyna speciosa. Phytochemistry 1986, 25, 2910-2912.
13) Houghton, P. J .; Latiff, A.; Said, I. M. Alkaloids from Mitragyna
speciosa. Phytochemistry 1991, 30, 347-350.
(14) J ansen, K. L. R.; Prast, C. J . Ethnopharmacology of Kratom and
the Mitragyna alkaloids. J . Ethnopharmacol. 1988, 23, 115-
119 and references therein.
15) Watanabe, K.; Yano, S.; Horie, S.; Yamamoto, L. T.; Takayama,
H.; Aimi, N.; Sakai, S.; Ponglux, D.; Tongroach, P.; Shan, J .;
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(
(
(
%
MPE was calculated for each mouse with the use of at least
five mice per dose. Intracerebroventricular injection of test
related indole alkaloids contained in the Asian herbal medicines,
2
4
drugs was performed as described previously. The test drugs
were injected in total volumes of 5 µL. The median antinoci-
ceptive dose (ED50) values and 95% confidence limits were
calculated by log-probit analysis according to the method of
hirsutine and mitragynine, with special reference to their Ca2+
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2
5
Litchfield and Wilcoxon. ED50 was defined as the dose of the
compound required to induce 50% of analgesia.
(
16) Yamamoto, L. T.; Horie, S.; Takayama, H.; Aimi, N.; Sakai, S.;
Yano, S.; Shan, J .; Pang, P. K. T.; Ponglux, D.; Watanabe, K.
Opioid receptor agonistic characteristics of mitragynine pseudo-
indoxyl in comparison with mitragynine derived from Thai
medicinal plant Mitragyna speciosa. Gen. Pharmacol. 1999, 33,
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between two values was considered significant when P was
less than 0.05.
7
3-81.
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17) Ponglux, D.; Wongseripipatana, S.; Takayama, H.; Kikuchi, M.;
Kurihara, M.; Kitajima, M.; Aimi, N.; Sakai, S. A New Indole
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Ack n ow led gm en t. This work was supported in part
by Grant-in-Aid (No. 10557229) for Scientific Research
from the Ministry of Education, Science, Sports, and
Culture, J apan.
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i
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