Design and synthesis of motilin antagonists derived from the [1-4] fragment of porcine motilin

Article abstract of DOI:10.1021/jm010332u

A series of cyclic peptides having the general structure H-Phe-c[-N^ε-Lys-X-NH-(CH₂)_n-CO-] were designed on the basis of structure-activity relationship studies of motilin. All were motilin antagonists. The cyclic peptides,

Full text of DOI:10.1021/jm010332u

670
J . Med. Chem. 2002, 45, 670-675
Design a n d Syn th esis of Motilin An ta gon ists Der ived fr om th e [1-4] F r a gm en t
of P or cin e Motilin
Masayuki Haramura,* Akira Okamachi, Kouichi Tsuzuki, Kenji Yogo, Makoto Ikuta, Toshiro Kozono,
Hisanori Takanashi, and Eigoro Murayama
Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd., 1-135 Komakado, Gotemba-shi,
Shizuoka 412-8513, J apan
Received J uly 18, 2001
A series of cyclic peptides having the general structure H-Phe-c[-Nꢀ-Lys-X-NH-(CH2)n-CO-] were
designed on the basis of structure-activity relationship studies of motilin. All were motilin
antagonists. The cyclic peptides, in which X is a 3-tert-butyl-substituted tyrosine residue
(H-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-âAla-] (3), H-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-Gly-] (6), H-Phe-c[-Nꢀ-Lys-
Tyr(3-tBu)-Abu-] (7), and H-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-Ahx-] (8)) showed potent motilin receptor
antagonistic activity in the rabbit smooth muscle (pA2 > 7). The 3-tert-butyl Tyr was found to
be the moiety responsible for enhanced binding to the motilin receptor, while the size of the
ring had little importance.
In tr od u ction
the motilin receptor (MTL-R) but is devoid of contractile
effects up to 100 µM (unpublished data). On the basis
of this result, the tetrapeptide was thought to be either
a metabolically unstable agonist, even in a contraction
assay, or an antagonist. However, metabolic inactivation
of the motilin tetrapeptide was not observed in the assay
system (unpublished data). On the basis of this result,
the compound was hypothesized to be a motilin receptor
antagonist. We have therefore focused on the design and
synthesis of more potent motilin antagonists via en-
hancement of the binding affinity of this tetrapeptide.
Motilin is a 22 amino acid gastrointestinal (GI)
peptide that stimulates contractile activity of gastro-
intestinal smooth muscle. In man and in dog motilin
appears to be involved in the regulation of the fasting
motility pattern.1 Motilin is also able to stimulate
gastric emptying, and the finding that erythromycin is
a motilin agonist2 has led to the development of potent
erythromycin derivatives, which have been named
motilides3 and which may be useful for the treatment
of hypomotility disorders such as gastroparesis.4
Recently, it was suggested that motilin may also be
involved in the production of some GI symptoms, such
as early satiety, abdominal distention, nausea, vomiting,
and anorexia,5 suggesting that motilin antagonists may
have clinical applications as well. Moreover, motilin
antagonists would be useful tools for studying the
physiological role of motilin and its mechanism of action.
The first motilin antagonist that was described was an
analogue of the 1-14 fragment of porcine motilin,
namely, [1-14] Phe3, Leu13 porcine motilin.6 Soon
thereafter, it was shown that the corresponding full
length molecule had higher affinity for the motilin
receptor and therefore was also a more potent antago-
nist.7 Unfortunately both these molecules also have
agonist properties. Moreover, they have a short half-
life, limiting their usefulness in vivo. Recently, a non-
peptide motilin receptor antagonist was identified
through electronic screening of a database against a 3D
pharmacophore for motilin.8 This molecule was effective
in inhibiting motilin-induced contractility in segments
of the rabbit duodenum.8 However, in vivo assay data
have not been reported.
It was anticipated that appropriate conformational
restriction of the peptide, through cyclization, would
result in the formation of improved motilin receptor
antagonists,9-11 and three cyclization modes for this
peptide were considered: (a) N-terminus to C-terminus,
(b) N-terminus to side chain, and (c) side chain to
C-terminus (Figure 1). Several 1H NMR studies of
motilin12-15 showed that Phe1 has considerable flex-
ibility. This residue is very important for binding
because its substitution by alanine in [1-14] Leu13
motilin causes the binding affinity to drop more than
100-fold.16 The next residue, Val2, does not seem to be
part of the motilin pharmacophore, which involves
residues 1, 4, and 7.16,17 Residue 7 is a Tyr, and our
fragment contained a tyrosine in position 3, the same
position that introduced antagonist properties to Leu13
porcine motilin when substituted with Phe.6,7 On the
basis of these considerations, we hypothesized that a
cyclic peptide, connecting the side chain of the second
residue with the C-terminus of the peptide (mode C),
would bind tightly to MTL-R. Also, a peptide, designed
with this mode, was thought to have almost the same
size ring as erythromycin. Therefore, we focused our
effort on the design and synthesis of cyclic peptides
bearing a modified Tyr with an aim of enhancing the
binding affinity.16-18 Herein, we report the syntheses
and biological activities of the cyclic motilin antagonists,
H-Phe-c[-Nꢀ-Lys-X-NH-(CH2)n-CO-] (Table 1).
We recently found that [1-4] Tyr3 porcine motilin (H-
Phe-Val-Tyr-Ile-NH2) has an affinity of 11 µM (IC50) for
* To whom correspondence should be addressed. Address: Chugai
Biopharmaceuticals, Inc., 6275 Nancy Ridge Drive, San Diego, CA
92121. Phone: +1-858-626-2532. Fax: +1-858-535-5994. E-mail:
mharamura@chugaibio.com.
10.1021/jm010332u CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/08/2002

Motilin Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 671
F igu r e 1. Various modes of cyclization.
Ta ble 1. Structures, Contractile Potency, and Binding Affinity of Cyclic Peptides
contractile potencya
binding affinity
b
compound
IC50 (nM)
EC50 (nM)
pA2
1
2
3
4
5
6
7
8
9
H-Phe-c[-Nꢀ-Lys-Tyr-âAla-]
25 000 and 42 000c
480d
>100 000
4.05 ( 0.07 (n ) 6)
H-Phe-c[-Nꢀ-Lys-Tyr(O-secBu)-âAla-]
H-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-âAla-]
H-Phe-c[-Nꢀ-Lys-Tyr(O-tBu)-âAla-]
H-Phe-c[-Nꢀ-Lys-Tyr(3,5-di-tBu)-âAla-]
H-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-Gly-]
H-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-Abuf-]
H-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-Ahxg-]
H-D-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-âAla-]
H-Phe-c[-Nꢀ- D-Lys-Tyr(3-tBu)-âAla-]
H-Phe-c[-Nꢀ-Lys-D-Tyr(3-tBu)-âAla-]
10 ( 4 (n ) 3)e
7500d
>100 000
>100 000
>10 000
>100 000
>100 000
>100 000
>100 000
>100 000
>100 000
7.37 ( 0.24 (n ) 10)
3.92 ( 0.38 (n ) 6)
460d
7 and 33c
33 and 59c
17 and 65c
1800d
7.33 ( 0.06 (n ) 6)
7.46 ( 0.17 (n ) 6)
7.08 ( 0.08 (n ) 6)
5.70 ( 0.11 (n ) 6)
5.66 ( 0.08 (n ) 6)
5.27 ( 0.15 (n ) 6)
10
11
2100d
3100d
a
Contractile potency data are presented as the mean ( SEM with the number of repetitions from independent assays (n) listed in
parentheses. b pA2 is the negative logarithm of the molar concentration of antagonist causing a 2-fold shift to the right of the concentration-
response curve for Motilin. c Binding affinity data are based on two determinations. Binding affinity data are based on one determination.
d
e Binding affinity data are presented as the mean ( SEM with the number of repetitions from independent assays (n) listed in parentheses.
f Abu: 4-aminobutyric acid. Ahx: 6-aminohexanoic acid.
g
Sch em e 1. Synthesis of Cyclic Peptide
Ch em istr y
The peptides were synthesized by a solid-phase
peptide synthesis (SPPS) methodology (Scheme 1),19,20
according to an NR-Fmoc (NR-9-fluorenylmethyloxycar-
bonyl) group strategy21 on a HMPA resin (p-hydroxy-
methylphenylacetic acid substituted resin). The depro-
tection of the NR-Fmoc group was conducted with 20%
piperidine in N,N-dimethylformamide (DMF) solution
prior to coupling with the next protected amino acid.
All amino acids were coupled as a pentafluorophenyl
(Pfp) esters in the presence of N-hydroxybenzotriazole
(HOBt) or as symmetrical anhydrides in the presence
of diisopropylcarbodiimide (DIC) and HOBt. The comple-
tion of the couplings was verified by the Kaiser test.22
The peptides were simultaneously deprotected and
cleaved from the resin by treatment with trifluoroacetic
acid (TFA)/anisole/ethanedithiol (94:5:1; v/v) for 1-3 h
at 0 °C. Crude Fmoc peptides were cyclized with BOP
reagent, followed by deprotection of the Fmoc moiety.
Alkylation of Tyr was carried out with isobutene or
1-butene in the presence of TFA and trimethylsilyl
triflate (TMSOTf).
The cyclic peptides were purified by preparative
reversed-phase (RP) HPLC. The purified peptides were
analyzed by analytical RP-HPLC and characterized by
Fourier transform (FT) NMR and high-resolution FAB-
MS (see Experimental Section and Supporting Informa-
tion).

672 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3
Haramura et al.
Resu lts a n d Discu ssion
Exp er im en ta l Section
Ma ter ia ls. NR-Fmoc amino acids and Pfp esters were
purchased from MilliGen/Biosearch (Burlington, MA) and
Watanabe Chemical (J apan). HMPA resin (PEPSYN KA, 0.2
mmol/g) was purchased from MilliGen/Biosearch (Burlington,
MA). TFA, TMSOTf, 1,3-diisopropylcarbodiimide (DIC), and
HOBt were purchased from Watanabe Chemical. 4-(Dimeth-
ylamino)pyridine (DMAP), piperidine, triethylamine, 1-butene,
isobutene, ethanedithiol, and anisole were purchased from
Tokyo Chemical Industry (J apan). Methanol (MeOH), acetic
acid (AcOH), diethyl ether, and NaHCO3 were from J unsei
Chemical (J apan). Pd/C was purchased from Kawaken Fine
Chemicals (Japan). DMF was purchased from Kokusan Chemi-
cal (J apan). HPLC grade acetonitrile and water were from
Kanto Chemical (J apan) and J unsei Chemical (J apan).
Solid -P h a se P ep tid e Syn th esis of H-P h e-c[-NE-Lys-Tyr -
âAla -] (1). HMPA resin (PEPSYN KA, 0.2 mmol/g, 50 g) was
placed in DMF and allowed to swell overnight. NR-Fmoc-âAla-
OH (8 equiv) was dissolved in DMF, and DIC (4 equiv) was
added to the solution. DMAP (1 equiv) was dissolved in DMF.
The amino acid/DIC solution and DMAP solution were added
to the resin in DMF, and this was shaken slowly and left
overnight at room temperature.
The resin was washed twice with DMF, twice with MeOH,
twice with AcOH, twice with MeOH, and three times with
DMF. The following NR-Fmoc amino acid Pfp esters were
sequentially coupled to a growing peptide chain: NR-Fmoc-
Tyr(tBu)-OPfp, NR-Fmoc-Lys(Boc)-OPfp, NR-Fmoc-Phe-OPfp.
Each coupling cycle consisted of the following: (1) treating
three times with 20% piperidine in DMF, (2) washing six times
with DMF, (3) coupling the amino acid active ester, and (4)
washing six times with DMF. All the NR-Fmoc amino acid Pfp
esters (2.5 equiv) were coupled to a growing peptide chain
using HOBt (2.5 equiv) in DMF. All the coupling reactions
were shaken on a manual shaker for 3-12 h at room temper-
ature. After coupling of the last NR-Fmoc amino acid, the
peptide resin was washed with MeOH and dried in vacuo to
yield the protected Fmoc-Phe-Lys(Boc)-Tyr(tBu)-âAla-HMPA
resin (58.6 g).
The synthesized cyclic tetrapeptides were tested for
binding affinity to the motilin receptor (MTL-R) and for
smooth muscle contractile activity in the rabbit smooth
muscle, and the data are summarized in Table 1.
All peptides showed binding affinity, but those with
an alkyl-substituted Tyr residue (compounds 2-11)
were more potent than the one compound with the
nonsubstituted Tyr (compound 1). This result suggested
that the alkyl substituent on Tyr3 contributes to a
hydrophobic interaction with the MTL-R. The 3-tert-
butyl-substituted Tyr derivatives (compounds 3 and
6-8) especially showed good binding affinity, and all
were at least 400 times more potent than 1. The most
potent compound, 3, bound to MTL-R with an IC50 of
10 ( 4 nM.
The table also shows that cyclic peptides with varying
ring sizes (compounds 3 and 6-8) had similar affinity
with an IC50 in the range 7-65 nM. The data indicate
that the size of the ring is not important in determining
the affinity for the MTL-R, although it was important
in the macrolides.4 The molecule was initially designed
so that the peptide backbone, via cyclization, would
mimic the macrolide ring of erythromycin. The results,
however, suggest that the peptide ring may play a role
different from that of the macrolide ring.
In contrast, replacement in compound 3 of any of the
first three residues with the D isomer (compounds 9-11)
strongly reduced binding affinity, suggesting that the
stereochemistry of the compound was important. In
motilin, changing Phe1 to its D isomer did not reduce
the affinity drastically.16 The stereoconformation of
these compounds seemed to be more important than in
motilin itself.
Importantly, the antagonistic activity of these com-
pounds in the contractile assay correlated with their
binding affinity, and no contractile activity was observed
even at high concentration (>100 µM). Thus, 3-tert-
butyl-substituted Tyr derivatives (compounds 3 and
6-8) showed good antagonistic activities, with pA2
values of 7.37 ( 0.24, 7.33 ( 0.06, 7.46 ( 0.17, and
7.08 ( 0.08, respectively in the rabbit duodenum
contractile assay. Compound 3 (GM-109), which has the
highest binding affinity (pKi ) 7.99 ( 0.04),23 showed
antagonism in an in vivo assay in dog.24 It has also been
reported that compound 3 (GM-109) antagonizes moti-
lin-induced calcium signaling in TE671 cells (a cell line
of human cerebellar origin)25 and in cultured myocytes
from the human colon.26 In these systems, compound 3
(GM-109) did not show any agonist activity. Also,
compound 3 (GM-109) had no effect on the contractions
induced by substance P, acetylcoline ACh, KCl, and
prostaglandin F2R in the rabbit duodenum contractile
assay.23
In conclusion, we designed and synthesized potent
cyclic peptide motilin antagonists based on the structure
H-Phe-c[-Nꢀ-Lys-Tyr-âAla-]. Substitution of Tyr with
3-tert-butyl-Tyr strongly increases potency. A change of
cycle length by replacing âAla4 with Gly, Abu, or Ahx
has little effect. Replacement of residues 1-3 with their
D isomers reduced the potency. These antagonists
should be useful in the investigation of the physiological
role of motilin and may find application in diseases
associated with increased motilin secretion.
The protected peptide resin was treated with TFA/anisole/
ethanedithiol (94:5:1; v/v) for 3 h at 0 °C. The solution was
filtered, concentrated under the reduced pressure, and added
dropwise to excess cold diethyl ether. The precipitated peptide
was filtered, washed with diethyl ether, and dried. The peptide
was dissolved in DMF (3 L) and pyridine (3 L), BOP reagent
(28 g) was added, and the mixture was left for 24 h at room
temperature. The solvent was removed under reduced pres-
sure, and water was added to the residue. The precipitate was
collected by filtration, washed with water, and dried. The Fmoc
cyclic peptide was treated with 20% piperidine in DMF for 0.5
h at room temperature, and the solvent was removed under
reduced pressure.
The crude peptide was purified by preparative HPLC with
a linear gradient of 0-60% of 0.1% TFA in acetonitrile against
0.1% aqueous TFA over 60 min at 10 mL/min. The lyophilized
product was a white amorphous solid (6.7 g). Overall yield:
86%. 1H NMR (DMSO-d6): δ 1.00-1.66 (6H, m, 3 × CH2), 2.21
(2H, m, CH2), 2.71-2.97 (4H, m, 2 × CH2), 3.10-3.65 (4H, m,
2 × CH2), 3.93 (1H, m, CH), 4.27 (1H, m, CH), 4.44 (1H, m,
CH), 6.62 (2H, d, J ) 7.6 Hz, aromatic-H), 6.95 (2H, d, J )
7.9 Hz, aromatic-H), 7.11-7.30 (5H, m, aromatic-H), 7.39 (1H,
m, NH), 7.64 (1H, m, NH), 8.39 (1H, m, NH), 8.51 (1H, m,
NH). FAB-MS m/z: calcd 510.2716, found 510.2722 (MH)+.
H-P h e-c[-NE-Lys-Tyr (O-secBu )-âAla -] (2). H-Phe-c[-Nꢀ-
Lys-Tyr-âAla-] (1) (500 mg) was dissolved in TFA (10 mL), and
TMSOTf (0.1 mL) was added. 1-Butene gas was added for 20
min at -10 °C, and the mixture was left for 18 h at room
temperature. The reaction solution was added dropwise to
excess cold diethyl ether, and the precipitated peptide was
collected by centrifugation and dried. The crude peptide was
purified by preparative HPLC with a linear gradient of 0-60%
of 0.1% TFA in acetonitrile against 0.1% aqueous TFA over
60 min at 10 mL/min. The lyophilized product was a white

Motilin Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 673
amorphous solid. H-Phe-c[-Nꢀ-Lys-Tyr(O-secBu)-âAla-] (2) (21.7
mg, yield 4%). 1H NMR (DMSO-d6): δ 0.81 (3H, m, CH3), 1.07
(3H, t, J ) 5.6 Hz, CH3), 1.12-1.36 (4H, m, 2 × CH2), 1.37-
1.67 (4H, m, 2 × CH2), 2.23 (2H, m, CH2), 2.66-2.95 (4H, m,
2 × CH2), 3.20-3.55 (4H, m, 2 × CH2), 3.96 (1H, m, CH), 4.14
(1H, m, CH), 4.30 (1H, m, CH), 4.46 (1H, m, CH), 6.74 (2H, d,
J ) 8.3 Hz, aromatic-H), 7.06 (2H, d, J ) 8.3 Hz, aromatic-
H), 7.16-7.26 (5H, m, aromatic-H), 7.40 (1H, brt, J ) 5.4 Hz,
NH), 7.67 (1H, m, NH), 8.49 (1H, d, J ) 9.2 Hz, NH), 8.61
(1H, d, J ) 17.5 Hz, NH). FAB-MS m/z: calcd 566.3342, found
566.3352 (MH)+.
H-P h e-c[-NE-Lys-Tyr (3-tBu )-âAla -] (3) a n d H-P h e-c[-NE-
Lys-Tyr (3,5-d i-tBu )-âAla -] (5). H-Phe-c[-Nꢀ-Lys-Tyr-âAla-]
(1) (170 mg) was dissolved in TFA (10 mL), and TMSOTf (0.1
mL) was added. Isobutene gas was added for 20 min at -10
°C, and the mixture was left for 12 h at room temperature.
The reaction solution was added dropwise to excess cold diethyl
ether, and the precipitated peptide was collected by centrifu-
gation and dried. The crude peptide was purified by prepara-
tive HPLC with a linear gradient of 0-60% of 0.1% TFA in
acetonitrile against 0.1% aqueous TFA over 60 min at 10 mL/
min. The lyophilized products were white amorphous solids.
H-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-âAla-] (3) (85.5 mg, yield 46%). 1H
NMR (DMSO-d6): δ 1.14 (2H, m, CH2), 1.29 (9H, s, 3 × CH3),
1.30, 1.40 (2H, 2m, CH2), 1.54 (2H, m, CH2), 2.19 (2H, m, CH2),
2.67 (1H, dd, J ) 7.9, 13.9 Hz, CH2), 2.84 (2H, m, CH2), 2.94
(1H, dd, J ) 5.5, 14.0 Hz, CH2), 3.26-3.44 (4H, m, 2 × CH2),
4.00 (1H, brt, J ) 5.6 Hz, CH), 4.29 (1H, dt, J ) 5.0, 7.6 Hz,
CH), 4.42 (1H, dt, J ) 7.1, 7.6 Hz, CH), 6.63 (1H, d, J ) 7.9
Hz, aromatic-H), 6.78 (1H, dd, J ) 2.0, 8.0 Hz, aromatic-H),
6.94 (1H, d, J ) 2.0 Hz, aromatic-H), 7.16 (2H, m, aromatic-
H), 7.23 (3H, m, aromatic-H), 7.38 (1H, t, J ) 6.0 Hz, NH),
7.63 (1H, dd, J ) 4.5, 7.5 Hz, NH), 8.38 (1H, d, J ) 8.9 Hz,
NH), 8.57 (1H, d, J ) 7.6 Hz, NH). FAB-MS m/z: calcd
566.3342, found 566.3328 (MH)+.
J ) 6.9, 6.9 Hz, CH), 4.63 (1H, dd, J ) 6.6, 14.5 Hz, CH), 6.89
(2H, d, J ) 8.3 Hz aromatic-H), 7.17 (2H, d, J ) 8.3 Hz,
aromatic-H), 7.20-7.31 (5H, m, aromatic-H). FAB-MS m/z:
calcd 566.3342, found 566.3343 (MH)+
.
H-P h e-c[-NE-Lys-Tyr (3-t Bu )-Gly-] (6). H-Phe-c[-Nꢀ-Lys-
Tyr-Gly-] was synthesized by the same procedure as that for
1 but using NR-Fmoc-Gly-OH, NR-Fmoc-Tyr(tBu)-OPfp, NR-
Fmoc-Lys(Boc)-OPfp, and NR-Fmoc-Phe-OPfp. H-Phe-c[-Nꢀ-
Lys-Tyr(3-tBu)-Gly-] was synthesized by the same procedure
as that for 3 from H-Phe-c[-Nꢀ-Lys-Tyr-Gly-] (878 mg). H-Phe-
c[-Nꢀ-Lys-Tyr(3-tBu)-Gly-] (6) (288 mg, yield 29%). 1H NMR
(DMSO-d6): δ 0.99-1.65 (6H, m, 3 × CH2), 1.32 (9H, s, 3 ×
CH3), 2.67-2.98 (6H, m, 3 × CH2), 3.18-3.34 (2H, m, CH2),
3.82 (1H, dd, J ) 6.1, 15.4 Hz, CH), 4.43 (2H, m, 2 × CH),
6.67 (1H, d, J ) 8.3 Hz, aromatic-H), 6.86 (1H, d, J ) 7.6 Hz,
aromatic-H), 7.02 (1H, s, aromatic-H), 7.20 (5H, s, aromatic-
H), 8.27 (1H, m, NH), 8.55 (1H, d, J ) 7.3 Hz, NH), 8.67 (1H,
m, NH), 8.90 (1H, d, J ) 7.9 Hz, NH). FAB-MS m/z: calcd
552.3186, found 552.3180 (MH)+.
H-P h e-c[-NE-Lys-Tyr (3-tBu )-Abu -] (7). H-Phe-c[-Nꢀ-Lys-
Tyr-Abu-] was synthesized by the same procedure as that for
1 but using NR-Fmoc-Abu-OH, NR-Fmoc-Tyr(tBu)-OPfp, NR-
Fmoc-Lys(Boc)-OPfp, and NR-Fmoc-Phe-OPfp. H-Phe-c[-Nꢀ-
Lys-Tyr(3-tBu)-Abu-] was synthesized by the same procedure
as that for 3 from H-Phe-c[-Nꢀ-Lys-Tyr-Abu-] (747 mg). H-Phe-
1
c[-Nꢀ-Lys-Tyr(3-tBu)-Abu-] (7) (300 mg, yield 25%). H NMR
(DMSO-d6): δ 1.07 (2H, m, CH2), 1.24-1.38 (2H, m, CH2), 1.31
(9H, s, 3 × CH3), 1.54 (2H, m, CH2), 1.87 (2H, m, CH2), 2.22
(2H, m, CH2), 2.64-2.94 (6H, m, 3 × CH2), 3.31 (2H, m, CH2),
4.07 (1H, m, CH), 4.29 (1H, brd, J ) 5.6 Hz, CH), 4.43 (1H, m,
CH), 6.66 (1H, d, J ) 6.6 Hz, aromatic-H), 6.86 (1H, brd, J )
6.3 Hz, aromatic-H), 7.03 (1H, s, aromatic-H), 7.19 (5H, s,
aromatic-H), 7.58 (2H, m, 2 × NH), 8.45 (2H, m, 2 × NH).
FAB-MS m/z: calcd 580.3499, found 580.3495 (MH)+.
H-P h e-c[-NE-Lys-Tyr (3-tBu )-Ah x-] (8). H-Phe-c[-Nꢀ-Lys-
Tyr-Ahx-] was synthesized by the same procedure as that for
1 but using NR-Fmoc-Ahx-OH, NR-Fmoc-Tyr(tBu)-OPfp, NR-
Fmoc-Lys(Boc)-OPfp, and NR-Fmoc-Phe-OPfp. H-Phe-c[-Nꢀ-
Lys-Tyr(3-tBu)-Ahx-] was synthesized by the same procedure
as that for 3 from H-Phe-c[-Nꢀ-Lys-Tyr-Ahx-] (712 mg). H-Phe-
c[-Nꢀ-Lys-Tyr(3-tBu)-Ahx-] (8) (428 mg, yield 34%). 1H NMR
(DMSO-d6): δ 1.02-1.69 (12H, m, 6 × CH2), 1.30 (9H, s, 3 ×
CH3), 2.00 (2H, m, CH2), 2.60-3.23 (8H, m, 4 × CH2), 4.09
(1H, m, CH), 4.39 (2H, m, 2 × CH), 6.64 (1H, d, J ) 8.3 Hz,
aromatic-H), 6.82 (1H, d, J ) 7.9 Hz, aromatic-H), 6.97 (1H,
brs, aromatic-H), 7.20 (5H, brs, aromatic-H), 7.54 (1H, m, NH),
7.72 (1H, m, NH), 8.28 (1H, m, NH), 8.50 (1H, brd, J ) 6.9
Hz, NH). FAB-MS m/z: calcd 608.3812, found 608.3806 (MH)+.
H-Phe-c[-Nꢀ-Lys-Tyr(3,5-di-tBu)-âAla-] (5) (41 mg, yield
20%). 1H NMR (DMSO-d6): δ 1.10-1.67 (6H, m, 3 × CH2),
1.34 (18H, s, 6 × CH3), 2.24 (2H, m, CH2), 2.42-2.58 (2H, m,
CH2), 2.63-3.02 (6H, m, 3 × CH2), 4.03 (1H, m, CH), 4.34 (1H,
m, CH), 4.43 (1H, m, CH), 6.92 (2H, s, aromatic-H), 7.20 (5H,
m, aromatic-H), 7.43 (1H, m, NH), 7.61 (1H, m, NH), 8.65 (1H,
d, J ) 9.1 Hz, NH), 8.58 (1H, d, J ) 8.0 Hz, NH). FAB-MS
m/z: calcd 622.3968, found 622.3961 (MH)+.
H-P h e-c[-NE-Lys-Tyr (O-tBu )-âAla -] (4). Z-Lys-Tyr(tBu)-
âAla-HMPA resin was synthesized by the same procedure as
that for 1 but using NR-Fmoc-âAla-OH, NR-Fmoc-Tyr(O-tBu)-
OH, and Z-Lys(Fmoc)-OH. The peptide resin was treated with
MeOH (270 mL) and triethylamine (30 mL) for 18 h at room
temperature. After filtration, the solution was evaporated to
dryness and treated with MeOH/H2O/Et3N (2:2:1; v/v) for 24
h at room temperature. After evaporation, the residue was
dissolved in DMF (200 mL) and pyridine (200 mL). BOP
reagent (2 g) was added to the solution, and the mixture was
left for 18 h at room temperature. After evaporation, water
was added and the precipitate was filtered, washed once with
5% NaHCO3 (aq) and twice with water. The dried solid was
dissolved in DMF (20 mL) and hydrogenated with Pd/C (200
mg) for 3 h. The solution was filtered and evaporated to
dryness. The residue was dissolved in DMF (5 mL) and coupled
with NR-Fmoc-Phe-OPfp (1.11 g) using HOBt (306 mg) for 18
h at room temperature. After evaporation, water was added
and the precipitate was filtered, washed once with water, and
washed twice with dry ether. The dried solid was dissolved in
DMF (8 mL), treated with piperidine (2 mL) for 1 h, and
evaporated to dryness. The crude peptide was purified by
preparative HPLC with a linear gradient of 0-60% of 0.1%
TFA in acetonitrile against 0.1% aqueous TFA over 60 min at
10 mL/min. The lyophilized product was a white amorphous
solid. H-Phe-c[-Nꢀ-Lys-Tyr(O-tBu)-âAla-] (4) (43 mg). 1H NMR
(CD3OD): δ 1.15-1.28 (2H, m, CH2), 1.25 (9H, s, 3 × CH3),
1.40, 1.53 (2H, 2m, CH2), 1.68 (2H, m, CH2), 2.37 (2H, m, CH2),
2.84 (2H, m, CH2), 3.06 (4H, m, 2 × CH2), 3.41-3.72 (2H, m,
CH2), 3.97 (1H, dd, J ) 5.6, 8.6 Hz, CH), 4.34 (1H, dd,
H-D-P h e-c[-NE-Lys-Tyr (3-tBu )-âAla -] (9). H-D-Phe-c[-Nꢀ-
Lys-Tyr-âAla-] was synthesized by the same procedure as that
for 1 but using NR-Fmoc-âAla-OH, NR-Fmoc-Tyr(tBu)-OPfp,
NR-Fmoc-Lys(Boc)-OPfp, and NR-Fmoc-D-Phe-OPfp (697 mg,
yield 74%). H-D-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-âAla-] was synthe-
sized by the same procedure as that for 3 from H-D-Phe-c[-
Nꢀ-Lys-Tyr-âAla-] (878 mg). H-D-Phe-c[-Nꢀ-Lys-Tyr(3-tBu)-
1
âAla-] (9) (111 mg, yield 34%). H NMR (DMSO-d6): δ 0.74-
1.07 (2H, m, CH2), 1.12-1.40 (4H, m, 2 × CH2), 1.31 (9H, s,
3 × CH3), 2.20 (2H, m, CH2), 2.60-2.85 (4H, m, 2 × CH2), 2.95
(2H, d, J ) 6.9 Hz, CH2), 3.26-3.44 (2H, m, CH2), 4.15 (1H,
m, CH), 4.27 (1H, m, CH), 4.38 (1H, m, CH), 6.63 (1H, d, J )
7.9 Hz, aromatic-H), 6.76 (1H, brd, J ) 7.6 Hz, aromatic-H),
6.92 (1H, brs, aromatic-H), 7.20-7.40 (6H, m, NH + aromatic-
H), 7.57 (1H, m, NH), 8.38 (2H, d, J ) 8.3 Hz, 2 × NH). FAB-
MS m/z: calcd 566.3342, found 566.3341 (MH)+.
H-P h e-c[-NE-D-Lys-Tyr (3-t Bu )-âAla -] (10). H-Phe-c[-Nꢀ-
D-Lys-Tyr-âAla-] was synthesized by the same procedure as
that for 1 but using NR-Fmoc-âAla-OH, NR-Fmoc-Tyr(tBu)-
OPfp, NR-Fmoc-D-Lys(Boc)-OPfp, and NR-Fmoc-Phe-OPfp (796
mg, yield 85%). H-Phe-c[-Nꢀ-D-Lys-Tyr(3-tBu)-âAla-] was syn-
thesized by the same procedure as that for 3 from H-Phe-c[-
Nꢀ-D-Lys-Tyr-âAla-]. H-Phe-c[-Nꢀ-D-Lys-Tyr(3-tBu)-âAla-] (10)
(100 mg, yield 31%). 1H NMR (DMSO-d6): δ 0.92 (2H, m, CH2),
1.10-1.53 (4H, m, 2 × CH2), 1.30 (9H, s, 3 × CH3), 2.16 (2H,

674 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3
Haramura et al.
m, CH2), 2.55-3.14 (6H, m, 3 × CH2), 3.28-3.58 (2H, m, CH2),
4.02 (1H, m, CH), 4.12 (1H, m, CH), 4.23 (1H, dt, J ) 7.3, 7.3
Hz, CH), 6.62 (1H, d, J ) 7.9 Hz, aromatic-H), 6.76 (1H, brd,
J ) 7.6 Hz, aromatic-H), 6.89 (1H, brs, aromatic-H), 7.22-
7.40 (6H, m, NH + aromatic-H), 7.49 (1H, brd, J ) 7.3 Hz,
NH), 7.75 (1H, d, J ) 7.8 Hz, NH), 8.27 (3H, m, NH2 + NH).
FAB-MS m/z: calcd 566.3342, found 566.3332 (MH)+.
H-P h e-c[-NE-Lys-D-Tyr (3-t Bu )-âAla -] (11). H-Phe-c[-Nꢀ-
Lys-D-Tyr-âAla-] was synthesized by the same procedure as
that for 1 but using NR-Fmoc-âAla-OH, NR-Fmoc-D-Tyr(tBu)-
OPfp, NR-Fmoc- Lys(Boc)-OPfp, and NR-Fmoc-Phe-OPfp (472
mg, yield 50%). H-Phe-c[-Nꢀ-Lys-D-Tyr(3-tBu)-âAla-] was syn-
thesized by the same procedure as that for 3 from H-Phe-
c[-Nꢀ-Lys-D-Tyr-âAla-]. H-Phe-c[-Nꢀ-Lys-D-Tyr(3-tBu)-âAla-] (11)
(78 mg, yield 24%). 1H NMR (DMSO-d6): δ 1.17 (2H, m, CH2),
1.28 (9H, s, 3 × CH3), 1.37 (2H, m, CH2), 1.61 (2H, m, CH2),
2.22 (2H, m, CH2), 2.83 (4H, m, 2 × CH2), 3.22 (2H, brdd, J )
3.6, 14.5 Hz, CH2), 3.50 (2H, m, CH2), 4.18 (3H, m, 3 × CH),
6.58 (1H, d, J ) 7.9 Hz, aromatic-H), 6.70 (1H, brd, J ) 8.9
Hz, aromatic-H), 6.87 (1H, brs, aromatic-H), 7.33 (5H, m,
aromatic-H), 7.42 (1H, m, NH), 7.66 (1H, m, NH), 7.89 (1H, d,
J ) 8.6 Hz, NH), 8.62 (1H, d, J ) 7.3 Hz, NH). FAB-MS m/z:
calcd 566.3342, found 566.3343 (MH)+.
attachment, and cut along the longitudinal axis to obtain
muscle strips approximately 10 mm long and 3 mm wide. The
preparation was mounted in an organ bath containing 10 mL
of modified Krebs’ solution kept at 28 °C to prevent excessive
spontaneous contractions.28 The solution was gassed with a
mixture of 95% O2 and 5% CO2. The longitudinal strips were
initially loaded with a 1.0 g weight, and contractile activity
was measured by means of isotonic transducers (ME-4012,
Medical Electronics Co., Tokyo) and recorded on an ink-writing
recorder (type 3066, Yanagisawa-Denki, Tokyo). Before the
experiments, each strip was subjected to repeated stimulation
with 100 µM ACh until a reproducible response was obtained.
The contractile potency of each compound was expressed as a
percentage of that induced by 100 µM ACh. The dose giving
50% of the response (EC50) was determined by interpolation.
A commutative concentration-dependent contractile curve
for pMTL was established by adding increasing concentrations
of pMTL. The effect of each antagonist on the contractile
activity of pMTL was further studied by incubating the strip
for 15 min with different concentrations of each antagonist
and then recording a cumulative concentration-response
curve in the presence of each antagonist. The pA2 value was
obtained by regression analysis of Schild plots, as has been
described previously.29
P ep tid e P u r ifica tion . The crude peptides were purified
by RP-HPLC using a Waters semiprep system with C18 YMC-
Pack S-343-15 column (YMC, J apan, 15 µm, 120 Å, 20 mm ×
250 mm). The peptide fractions that were purified by prepara-
tive HPLC were lyophilized, and the powder was kept at -20
°C until biological assay.
Ack n ow led gm en t. We thank Dr. Theo L. Peeters,
Dr. Hiroharu Matsuoka, and Dr. Thomas Arrhenius for
their useful advice.
P ep tid e An a lysis. The purity of the final products was
confirmed by RP-HPLC using a Hitachi D-7000 HPLC system
with an analytical column (YMC-Pack A-302, 4.6 mm × 150
mm, 120 Å, 5 µm particle size, YMC, J apan). The gradient for
analytical RP-HPLC was as follows: (A) 100:0 to 30:70 (0.1%
aqueous TFA/0.1% TFA in acetonitrile) over 25 min at 1 mL/
min; (B) 100:0 to 30:70 (0.1% aqueous TFA/MeOH) over 25
min at 1 mL/min (see Supporting Information).
Su p p or tin g In for m a tion Ava ila ble: Table of HPLC
analytical data. This material is available free of charge via
the Internet at http://pubs.acs.org.
Refer en ces
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The molecular weight of the compounds was determined by
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expressed for MH+.
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