Design and Synthesis of novel tetra-peptide motilin agonists

Article abstract of DOI:10.1016/S0968-0896(02)00027-5

A series of novel tetra-peptide motilin agonists, having the general structure H-Phe-Val-X-Ile-NH₂, were designed, on the basis of structure-activity relationship studies of motilin. Peptides, in which X is a side chain substituted tryptophan residue, have agonistic activity. H-Phe-Val-Trp(2′-CH₂CH₂OH)-Ile-NH₂(7), H-Phe-Val-Trp(2′-SCH₃)-Ile-NH₂(8), and H-Phe-Val-Trp(2′-SCH₂CH₂CH₃)-Ile-NH ₂ (9), showed an EC₅₀ for contractile activity in the rabbit smooth muscle of 14.1±3.2, 12.9±4.1, and 4.6±1.6 μM, respectively. Interaction of the tryptophan aliphatic side chain with motilin receptor appears to influence the signal transduction via motilin receptor.

Full text of DOI:10.1016/S0968-0896(02)00027-5

Bioorganic & Medicinal Chemistry 10 (2002) 1805–1811
Design and Synthesis of Novel Tetra-Peptide Motilin Agonists
Masayuki Haramura,* Kouichi Tsuzuki, Akira Okamachi, Kenji Yogo, Makoto Ikuta,
Toshiro Kozono, Hisanori Takanashi and Eigoro Murayama
Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
Received 24 October 2001; accepted 8 January 2002
Abstract—A series of novel tetra-peptide motilin agonists, having the general structure H-Phe-Val-X-Ile-NH2, were designed, on
the basis of structure–activity relationship studies of motilin. Peptides, in which X is a side chain substituted tryptophan residue,
have agonistic activity. H-Phe-Val-Trp(20-CH2CH2OH)-Ile-NH2 (7), H-Phe-Val-Trp(20-SCH3)-Ile-NH2 (8), and H-Phe-Val-Trp(20-
SCH2CH2CH3)-Ile-NH2 (9), showed an EC50 for contractile activity in the rabbit smooth muscle of 14.1ꢀ3.2, 12.9ꢀ4.1, and
4.6ꢀ1.6 mM, respectively. Interaction of the tryptophan aliphatic side chain with motilin receptor appears to influence the signal
transduction via motilin receptor. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
late new studies on the biological and physiological
mechanism of the motilin and perhaps lead to the dis-
covery of new drugs for the treatment of patients with
hypomotility syndromes.
Motilin, a single-chain peptide of 22 amino acid resi-
dues, was isolated from endocrine cells in gastro-
intestinal (GI) mucosa of various species.1 Although the
biological function of motilin has not been fully eluci-
dated, motilin is known to stimulate GI motility in
many species.1
The previous studies10,11 on the SAR of pMTL suggest
that Phe1, Ile4, and Tyr7 play an important role in
agonism of the motilin receptor. Several 1H NMR
studies of motilin indicate that residues 1–6 adopt a
wide nonclassical turn conformation, which brings Pro3
and Tyr7 into close proximity with one another.12ꢁ14
Therefore, the Tyr7 binding site of MTL-R was expec-
ted to be occupied by the 3rd residue side chain. In
addition, Pro3 was reported not to contribute to the
agonistic effect of the pMTL partial peptide.10,11 From
these considerations, we hypothesized that if the third
amino acid (Pro) was replaced with Tyr, even the
N-terminal tetra-peptide would have the agonistic effect
without Tyr7. With this idea in mind, we synthesized
and evaluated tetra-peptide 1 (H-Phe-Val-Tyr-Ile-NH2).
Peptide 1 was found to bind to MTL-R with an IC50 of
11 mM. However, the contractile activity of 1 was found
to be extremely weak (EC50:>100 mM). The low ago-
nistic activity of this peptide could be due to the inad-
equate interaction with the Tyr7 binding site of MTL-R.
The importance of such an interaction was reported in a
previous study of a motilin analogue peptide.15 In other
words, the third amino acid of the N-terminal tetra-
peptide may play a critical role for its agonism. There-
fore, we have focused our efforts on modifications of 1,
with the aim of conducting an extensive SAR analysis of
the N-terminal moiety of motilin, in order to enhance
The amino acid sequence of porcine motilin (pMTL)
was first determined in 1973 (FVPIF TYGEL QRMQE
KERNK GQ).2,3 Human motilin, isolated in 1995,4 was
found to be identical with porcine motilin, but different
from canine motilin by five residues at the 7th, 8th,
12th, 13th, and 14th amino acid residues.
Motilin has been suggested to have physiological rele-
vance to a number of gastrointestinal symptoms,
including early satiety, abdominal distention, nausea,
vomiting, and anorexia.1 Motilin agonists, such as
motilin analogues5 and erythromycin derivatives,6ꢁ8 and
their three-dimensional structure–activity relationship
(SAR) study9 have been reported. Although they have
been suggested to be effective in the treatment of such
symptoms, the detailed mechanisms of motilin agonistic
action, after binding to the motilin receptor (MTL-R),
have not yet been explored. The discovery of small
molecule motilin agonists would be expected to stimu-
*Corresponding author. Chugai Biopharmaceuticals, Inc, 6275 Nancy
Ridge Drive, San Diego, CA 92121, USA. Tel.: +1-858-626-2532; fax:
+1-858-535-5994; e-mail: mharamura@chugaibio.com
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00027-5

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M. Haramura et al. / Bioorg. Med. Chem. 10 (2002) 1805–1811
Scheme 1. Modification of Trp on tetra-peptide.
the contractile effect of this type of molecule. Herein, we
would like to report the syntheses and biological activ-
ities of novel tetra-peptide motilin agonists, H-Phe-Val-
X-Ile-NH2.
sole (100:28.6:7.47:16.7; v/v) or TFA/TMSOTf/m-cre-
sol/thioanisole/ethanedithiol (100:12:6.5:15:5; v/v) for
1–4 h at 0 ꢂC or room temperature depending on the
side-chain protecting groups. Modification of the indole
ring of Trp was performed with the corresponding dis-
ulfide in the presence of silver triflate (Scheme 1).
Chemistry
Crude peptides were then purified to homogeneity by
preparative reversed-phase high-performance liquid
chromatography (RP-HPLC) eluting with a linear gra-
dient of 0.1% aqueous TFA in acetonitrile. Peptide
fractions found to be homogeneous by analytical RP-
HPLC were combined, concentrated, and lyophilized.
The peptides were characterized by Fourier transform
(FT) NMR and high-resolution fast atom bombard-
ment mass spectrometry (FABMS) (Table 3).
The peptides were synthesized by a solid phase method-
ology,16,17 according to an Na-Boc (Na-t-butyloxy-
carbonyl) protecting group strategy or Na-Fmoc (Na-9-
Fluorenylmethyloxycarbonyl) protecting group strategy
a MBHA-resin (p-methylbenzhydrylamine sub-
stituted polystyrene resin).18,19
on
In the case of the Na-Boc group strategy, the deprotec-
tion of Na-Boc group was conducted with 33–50% tri-
fluoroacetic acid (TFA) in dichloromethane (DCM)
solution prior to coupling with the next protected amino
acid. A preformed Na-Boc amino acid symmetric anhy-
dride was utilized as the acylating species. The pro-
cedure for generating an amino acid symmetric
anhydride consists of reacting 2.0 equivalents of
dicyclohexylcarbodiimide (DCC) with 4.0 equivalents of
Na-Boc amino acid in DCM.
Results and Discussion
The synthesized peptides (Table 1), H-Phe-Val-X-Ile-
NH2 (X=Tyr, Pro, Lys, Glu, Gln, Trp), were tested for
binding activity to MTL-R and for the rabbit smooth
muscle contractile activity.
As shown in Table 1, 1–3 and 6 showed the binding
activity to the MTL-R, whereas 4 and 5 did not. Tryp-
tophan derivative 6 was 30 times more potent than tyr-
osine derivative 1. The binding activities of the proline
and lysine derivatives 2 and 3 were comparable to that
of 1. However, 1, 2 and 6 showed extremely weak ago-
nistic activity in the rabbit duodenal smooth muscle
contractile assay. On the basis of these results, we con-
cluded that 1, 2 and 6 bound to MTL-R, but the signal
transduction via MTL-R was incomplete. In order to
achieve more complete signal transduction, a series of
extensively modified peptides were prepared (Table 2).
In the case of the Na-Fmoc group strategy, Na-Fmoc
group was removed with 20% piperidine in N,N-di-
methylformamide (DMF) prior to coupling with the
next protected amino acid. All Na-Fmoc amino acids
were coupled as their pentafluorophenyl (Pfp) esters, in
presence of N-hydroxybenzotriazole (HOBt).
The completion of the couplings was verified by the
Kaiser test.20 The peptides were simultaneously depro-
tected and cleaved from the resin by treatment with
TFA/trimethylsilyltriflate (TMSOTf)/m-cresol/thioani-

M. Haramura et al. / Bioorg. Med. Chem. 10 (2002) 1805–1811
1807
Based on the binding activity of the tryptophan con-
taining peptide 6, we focused our attention on the
preparation of peptides with a substituted tryptophan at
residue 3.
A structural feature, distinguishing the contractile-
active peptides, 7, 8, and 9 from the contractile-inactive
derivative 6, is the aliphatic side chain on the tryptophan
ring. Although the Trp 20-substituents do not contribute
MTL-R binding, they appear to play an important role
in inducing receptor mediated signal transduction. In
the model proposed by Gouda et al.,9 ethyl group of an
erythromycin derivative was presumed to correspond to
Tyr7 of Motilin. Aliphatic side chain in 7, 8, and 9
seemed to play the same role as this ethyl group.
The 20-substituted tryptophan derivatives, H-Phe-Val-
X-Ile-NH2 (X=20-substituted tryptophan) were tested
for binding and contractile activity. The binding activ-
ities of 7, 8, and 9 were comparable to that of the tryp-
tophan derivative 6, whereas, those of 10 and 11 were
less potent (Table 2). Importantly, 7, 8, and 9 were all
effective in the contractile assay (EC50: 14.1ꢀ3.2,
12.9ꢀ4.1, and 4.6ꢀ1.6 mM, respectively), demonstrat-
ing that the N-terminal tetra-peptide can act as motilin
agonist in vitro. As shown in Figure 1, 7 induced con-
tractile activity in the rabbit duodenal smooth muscle in
a concentration-dependent manner.
In the earlier study derived for a large series of motilin
analogues, Peeters et al. mentioned the correlation
between binding affinity and agonistic effect
(pEC50=1.13+0.74 pIC50),10 which led to a difference
between pEC50 and pIC50 of about one log unit. This
correlation was also true for motilin partial peptides,21
and erythromycin derivatives.22,23 However, 7 and 8
showed the almost two log units difference between
pEC50 and pIC50 in this study. The fact suggested that
these peptides bound to MTL-R, but receptor mediate
signal transductions by these peptides were weaker than
those of other Tyr7 containing motilin analogues and
erythromycin derivatives.
Figure 1. Contractile activity of compound 7 in the rabbit duodenal
smooth muscle. aContraction % of 100 mM ACh max. Data are pre-
sented as the meanꢀSEM with the number of repetitions from inde-
pendent assays (n=6).
Table 1. Structures, binding activity and contractile activity of tetra-peptides
No.
Structures
Binding activity (IC50: mM)
Contractile activitya
EC50 (mM)
Maximum contraction (% of 100 mM ACh max)
n
1
2H-Phe-Val-Pro-Ile-NH
H-Phe-Val-Tyr-Ile-NH2
11
33
6.4
>100
>300
—
—
—
8.7b
17.5ꢀ6.0c
—
2
6
—
—
—
6
2
3
4
5
6
H-Phe-Val-Lys-Ile-NH2
H-Phe-Val-Glu-Ile-NH2
H-Phe-Val-Gln-Ile-NH2
H-Phe-Val-Trp-Ile-NH2
>100
>100
0.37
—
—
>100
14.2ꢀ5.1b
aContractile activity data are presented as the meanꢀSEM with the number of repetitions from independent assays (n).
bMaximum contraction (%) at 100 mM.
cMaximum contraction (%) at 300 mM.
Table 2. Structures, binding activity and contractile activity of Trp modificated peptides
No.
Structures
Binding activity (IC50: mM)
Contractile activitya
EC50 (mM)
Maximum contraction
(% of 100 mM ACh max)
n
6
7
8
9
10
11
H-Phe-Val-Trp-Ile-NH2
H-Phe-Val-Trp(20-SCH2CH2OH)-Ile-NH2
H-Phe-Val-Trp(20-SCH3)-Ile-NH2
H-Phe-Val-Trp(20-SCH2CH2CH3)-Ile-NH2
H-Phe-Val-Trp(20-SCH2CH2NMe2)-Ile-NH2
H-Phe-Val-Trp(20-SCH2COOH)-Ile-NH2
0.37
0.57
0.4212
0.23
1.0
>100
14.1ꢀ3.285.7
ꢀ4.1
14.2ꢀ5.1b
ꢀ6.8b
6
6
5
3
3
3
.9
58.2ꢀ3.6c
67.7ꢀ5.0c
2.9ꢀ2.9d
1.5ꢀ1.5c
4.6ꢀ1.6
>300
>30
5.6
aContractile activity data are presented as the meanꢀSEM with the number of repetitions from independent assays (n).
bMaximum contraction (%) at 100 mM.
cMaximum contraction (%) at 30 mM.
dMaximum contraction (%) at 300 mM.

1808
M. Haramura et al. / Bioorg. Med. Chem. 10 (2002) 1805–1811
Table 3. Analytical properties of peptides
Solid-phase peptide synthesis
No.
HPLC tR (min), (purity, %)
FAB-MS (MH+) (calcd)
The peptides were synthesized by solid phase peptide
chemistry.16,17 The Boc strategy syntheses were carried
out on an ABI Model 430A peptide synthesizer. The
Fmoc strategy syntheses were performed using a man-
ual shaker.
a
b
tR
tR
1
13.13 (100)
15.91 (100)
10.85 (99.1)
17.71 (100)
540.3175 (540.3181)
4(7447.43.0380280)
211.98 (100)
3
4
5
6
7
8
9
15.14 (100)
16.37 (100)c
15.72(100)
21.07 (100)
21.15 (100)
22.24 (100)
24.45 (100)
19.86 (100)
21.14 (97.9)
505.3504 (505.3502)
506.2980 (506.2978)
505.3141 (505.3138)
563.3350 (563.3345)
639.3328 (639.3328)
609.3230 (609.3223)
637.3529 (637.3536)
666.3802(666.3801)
653.3125 (653.3121)
11.85 (99.3)
11.56 (98.5)
15.38 (99.6)
15.04 (100)
16.25 (96.2)
18.01 (99.0)
13.62(99.4)
15.20 (95.2)
H-Phe-Val-Tyr-Ile-NH2 (1) (Boc method). MBHA resin
(0.42mmol/g; 1.2094 g) was used to prepare the peptide:
Boc-Phe-Val-Tyr(Br-Z)-Ile-MBHA-resin, by stepwise
coupling of the amino acids Boc-Ile-OH, Boc-Tyr(Br-
Z)-OH, Boc-Val-OH, Boc-Phe-OH. All the Boc amino
acids (4 equiv) were coupled to the growing peptide
chain as a symmetric anhydride by using DCC (2
equiv). Boc group was removed with 33–50% TFA in
DCM solution prior to coupling with the next protected
amino acid. After coupling of the last Boc amino acid,
the peptide resin was washed with methanol and dried
in vacuo to yield the Boc-Phe-Val-Tyr(Br-Z)-Ile-
MBHA-resin (1.5447 g).
10
11
a100:0 to 30:70 (0.1% aqueous TFA/0.1% TFA in acetonitrile) over 25
min, 1.0 mL/min.
b100:0 to 30:70 (0.1% aqueous TFA/MeOH) over 25 min, 1.0 mL/min,
unless otherwise noted.
c100:0 to 2:98 (0.1% aqueous TFA/MeOH) over 35 min, 1.0 mL/min.
In conclusion, we designed motilin N-terminal tetra-
peptide derivatives (H-Phe-Val-X-Ile-NH2), and explored
extensive SAR analyses of the N-terminal moiety of
motilin. Peptides 7, 8, and 9, possessing an aliphatic side
chain on the tryptophan ring, showed contractile activ-
ity in the rabbit smooth muscle. The interaction of the
aliphatic side chain with MTL-R appears to exert an
influence on the signal transduction via MTL-R.
The protected peptide resin was treated with TFA/
TMSOTf/m-cresol/thioanisole (100:28.6:7.47:16.7; v/v)
for 1 h at 0 ꢂC and for 2h at room temperature. The
solution was filtered and added drowse to excess cold
diethyl ether (600 mL) and n-hexane (300 mL). The
precipitated peptide was collected by centrifugation,
washed with diethyl ether and n-hexane (1:1), and dried.
The residue was treated with 2-mercaptoethanol (0.4
mL) and 1 N NH4F (0.4 mL) in MeOH (8 mL) and H2O
(8 mL) at 0 ꢂC. The solution was adjusted to pH 8.0
with triethylamine and then acidified to pH 4.0 with
AcOH, and applied to gel chromatography with
Sephadex G-10 (Amersham Pharmacia Biotech, Swe-
den) in 1 N AcOH. The lyophilized crude peptide was
purified by RP-HPLC using a Waters semi-prep system
with C18 YMC-Pack S-343-15 (YMC, Japan), 15 mm,
120 A, 2 0ꢃ250 mm, eluting with a linear acetonitrile
gradient (0–60%) in water containing a constant con-
centration of TFA (0.1%, v/v) over 60 min at a flow
rate of 10 mL/min. The peptide fractions, which were
verified by analytical HPLC, were lyophilized. Yield:
0.0758 g (28%).1H NMR (DMSO-d6) d 0.82(12H, m,
Information from this report will hopefully stimulate
studies on the biological and physiological mechanism
of motilin, and assist in developing a remedies for
motilin associated diseases.
Experimental
Materials
Fmoc amino acids, and Pfp esters were purchased from
MilliGen/Biosearch (Burlington, MA, USA) and Wata-
nabe Chemical (Japan). Boc amino acids were purchased
from Applied Biosystems (ABI; Foster City, CA, USA)
and Watanabe Chemical (Japan). MBHA-resin was pur-
chased from the Peptide Institute (Japan) and Watanabe
Chemical (Japan). All reagents used in ABI 430A peptide
synthesizer were purchased from ABI. TFA, TMSOTf,
1,3-diisopropylcarbodiimide (DIC), and HOBt were
purchased from Watanabe Chemical (Japan). Di-n-
propyl disulfide, dithiodiglycolic acid, silver tri-
fluoromethanesulfonate, triethylamine, dithiothreitol,
NH4F, 2-mercaptoethanol, m-cresol, ethanedithiol and
thioanisole were purchased from Tokyo Chemical
Industry (Japan). Dimethyl disulfide was purchased
from Nakarai Tesque (Japan). DMF, DCM, and N-
methyl-2-pyrrolidinone (NMP) were purchased from
Kokusan Chemical (Japan). Methanol (MeOH), diethyl
ether, n-hexane, NaOH, and acetic acid (AcOH) were
purchased from Junsei Chemical (Japan). HPLC grade
acetonitrile, and water were purchased from Kanto
Chemical (Japan) and Junsei Chemical (Japan).
4ꢃCH3), 1.06 (1H, m, CH2), 1.42(1H, m, CH ), 1.70
2
(1H, m, CH), 1.99 (1H, m, CH), 2.65–3.03 (4H, m,
2ꢃCH2), 4.12(2H, m, 2 ꢃCH), 4.27 (1H, dd, J=6.3, 8.6
Hz, CH), 4.52(1H, dd, J=4.6, 7.9 Hz, CH), 6.63 (2H,
d, J=8.6 Hz, aromatic–H), 7.06 (2H, d, J=7.1 Hz,
aromatic–H), 7.21 (5H, m, aromatic–H), 7.77 (1H, d,
J=8.9 Hz, NH), 8.18 (1H, d, J=7.9 Hz, NH), 8.44 (1H,
d, J=8.9 Hz, NH).
H-Phe-Val-Pro-Ile-NH2 (2). The title compound was
synthesized using the same procedure as that for 6, but
using Fmoc-Ile-OPfp, Fmoc-Pro-OPfp, Fmoc-Val-
OPfp, Fmoc-Phe-OPfp. Yield: 0.0553 g (24%). 1H
NMR (CD3OD) d 0.96 (12H, m, 4ꢃCH3), 1.23 (1H, m,
CH2), 1.60 (1H, m, CH2), 1.80 (1H, m, CH), 2.00–2.16
(5H, m, CH, 2ꢃCH2), 3.01, 3.21 (2H, 2dd, J=5.6, 8.1,
14.2Hz, CH ), 3.66 (1H, m, CH2), 3.83 (1H, m, CH2),
2
4.19 (2H, m, 2ꢃCH), 4.49 (2H, m, 2ꢃCH), 7.29 (6H, m,
NH, aromatic–H), 7.96 (1H, d, J=8.6 Hz, NH)

M. Haramura et al. / Bioorg. Med. Chem. 10 (2002) 1805–1811
1809
H-Phe-Val-Lys-Ile-NH2 (3). The title compound was
synthesized using the same procedure as that for 1, but
using Boc-Ile-OH, Boc-Lys(Cl-Z)-OH, Boc-Val-OH,
for 0.5 h, and then acidified to pH 4.0 with AcOH. The
crude peptide was purified by preparative HPLC with a
linear gradient of 0–60% 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.
Yield: 0.134 g (9%).1H NMR (DMSO-d6) d 0.83 (12H,
m, 3ꢃCH2), 1.06 (1H, m, CH2), 1.37 (1H, m, CH2), 1.65
(1H, m, CH), 1.98 (1H, m, CH), 2.72–3.24 (4H, m,
2ꢃCH2), 4.13 (2H, m, 2ꢃCH), 4.29 (1H, m, CH), 4.67
(1H, m, CH), 6.94–7.30 (10H, m, aromatic–H), 7.62
(1H, d, J=6.9 Hz, NH), 8.25 (2H, m, 2ꢃNH) 8.35 (1H,
d, J=9.2Hz, NH).
1
Boc-Phe-OH. Yield: 0.10 g (38%). H NMR (DMSO-
d6) d 0.84 (12H, m, 4ꢃCH3), 1.02–1.70 (9H, m, 4ꢃCH2,
CH), 1.96 (1H, m, CH), 2.75 (2H, t, J=7.1 Hz, CH2),
2.90, 3.07 (2H, 2dd, J=4.3, 7.6, 13.9 Hz, CH2), 4.17
(2H, m, 2ꢃCH), 4.28 (2H, m, 2ꢃCH), 7.27 (5H, m,
aromatic–H), 7.70 (1H, d, J=8.9 Hz, NH), 8.18 (1H, d,
J=7.6 Hz, NH), 8.54 (1H, d, J=8.9 Hz, NH).
H-Phe-Val-Glu-Ile-NH2 (4). The title compound was
synthesized in the same way as that for 1, but using Boc-
Ile-OH, Boc-Glu(OBzl)-OH, Boc-Val-OH, Boc-Phe-
OH. Yield: 0.069 g (33%).1H NMR (DMSO-d6) d 0.86
(12H, m, 4ꢃCH3), 1.10 (1H, m, CH2), 1.39 (1H, m,
CH2), 1.63–2.11 (4H, m, 2ꢃCH, CH2), 2.25 (2H, m,
CH2), 2.92, 3.08 (2H, 2dd, J=4.8, 7.9, 13.9 Hz, CH2),
4.09–4.44 (4H, m, 4ꢃCH), 7.30 (5H, m, aromatic–H),
8.21–8.40 (2H, m, 2ꢃNH), 8.52(1H, t, J=8.4 Hz, NH).
H-Phe-Val-Trp(20-SCH2CH2OH)-Ile-NH2 (7). 6 (0.191
g, 0.339 mmol) was added to a solution of Bis-(2-acet-
oxyethyl)disulfide (0.82g, 3.44 mmol) in TFA (20 mL)
under ice-water cooling, followed by trifluoro-
methanesulfonic acid silver salt (0.8449 g, 3.29 mmol).
The solution was stirred for 8 h under ice water cooling,
and for 120 h at room temperature. The solvent was
removed under reduced pressure, and the residue was
dissolved in MeOH (10 mL). The solution was adjusted
to pH 9 with 1 N NaOHaq for 0.5 h, filtered, and then
acidified to pH 4 with AcOH. The solvent was removed
under reduced pressure to give a residue that was pur-
ified by preparative RP-HPLC. The lyophilized product
H-Phe-Val-Gln-Ile-NH2 (5). The title compound was
synthesized in the same way as that for 1, but using Boc-
Ile-OH, Boc-Gln-OH, Boc-Val-OH, Boc-Phe-OH. Boc-
Gln-OH was activated as the HOBt ester formed using
4.0 equivalents of DCC in DMF and DCM. Yield:
0.123 g (66%).1H NMR (DMSO-d6) d 0.84 (12H, m,
4ꢃCH3), 1.07 (1H, m, CH2), 1.38 (1H, m, CH2), 1.56–
2.03 (4H, m, 2ꢃCH, CH2), 2.12 (2H, t, J=7.9 Hz,
CH2), 4.14 (2H, m, 2ꢃCH), 4.28 (2H, m, 2ꢃCH), 7.29
(5H, m, aromatic–H), 7.69 (1H, d, J=8.3 Hz, NH), 8.25
(2H, m, 2ꢃNH) 8.53 (1H, d, J=8.9 Hz, NH).
1
was a white amorphous solid. Yield: 0.108 g (50%). H
NMR (DMSO-d6) d 0.80 (12H, m, 4ꢃCH3), 1.03 (1H,
m, CH2), 1.38 (1H, m, CH2), 1.66 (1H, m, CH), 1.92
(1H, m, CH), 2.77 (2H, m, CH2), 2.87 (2H, m, CH2),
3.04, 3.20 (2H, 2dd, J=5.9, 8.3, 14.2Hz, CH ), 3.47
2
(2H, m, CH2), 4.12(1H, dd, J=7.3, 8.6 Hz, CH), 4.26
(1H, dd, J=6.9, 8.2Hz, CH), 4.68 (1H, dd, J=6.6, 7.9
Hz, CH), 4.91 (1H, t, J=5.6 Hz, CH), 6.95 (2H, m,
aromatic–H), 7.03 (1H, t, J=7.4 Hz, aromatic–H), 7.12
(5H, s, aromatic–H), 7.20 (1H, d, J=7.6 Hz, aromatic–
H), 7.66 (1H, d, J=8.9 Hz, NH), 7.69 (1H, d, J=7.9
Hz, NH), 8.19 (1H, d, J=7.9 Hz, NH) 8.40 (1H, d,
J=8.9 Hz, NH).
H-Phe-Val-Trp-Ile-NH2 (6) (Fmoc method). MBHA
resin (0.79 mmol/g; 3.418 g) was treated twice with 20%
piperidine in DMF and washed eight times with DMF.
Fmoc-Ile-OPfp (3.5 equiv) was then added followed by
HOBt (3.5 equiv). The coupling reaction mixture was
shaken on a manual shaker for 3 h at room tempera-
ture. The resin was then washed six times with DMF.
The following Fmoc amino acid Pfp esters were
sequentially coupled to the growing peptide chain:
Fmoc-Trp-OPfp, Fmoc-Val-OPfp, Fmoc-Phe-OPfp. All
the Fmoc amino acid Pfp esters (3 equiv) were coupled
to a growing peptide chain using HOBt (3 equiv) in
DMF. All the coupling reactions were shaken on a
manual shaker for 3–30 h at room temperature. Fmoc
group was removed with 20% piperidine in DMF prior
to coupling with the next protected amino acid. After
deprotection of the last Fmoc group, the peptide resin
was washed with MeOH and dried in vacuo to yield the
H-Phe-Val-Trp-Ile-MBHA resin (4.725 g).
H-Phe-Val-Trp(20-SCH3)-Ile-NH2 (8). 6 (0.589 g, 1.05
mmol) was added to a solution of dimethyl disulfide
(1.028 g, 10.9 mmol) in TFA (20 mL) under ice-water
cooling, followed by trifluoromethanesulfonic acid sil-
ver salt (1.308 g, 5.09 mmol). The solution was stirred
for 0.5 h under ice water cooling, and for 48 h at room
temperature. The solution was added dropwise to cold
diethyl ether (400 mL) and n-hexane (100 mL). The
precipitated peptide was collected by centrifugation,
and washed with diethyl ether. The residue was treated
with dithiothreitol (0.8 g) in MeOH (30 mL), AcOH (30
mL), and H2O (20 mL) for 1 h. The precipitate was
removed by centrifugation. The solvent was removed
under reduced pressure to give a residue that was puri-
fied by preparative RP-HPLC. The lyophilized product
The peptide resin was treated with TFA/TMSOTf/m-
cresol/thioanisole/1,2-ethandithiol (100:12:6.5:15:5; v/v)
for 2h at 0 ꢂC and additional 2h at room temperature.
The solution was filtered and added dropwise to cold
diethyl ether (500 mL) and n-hexane (1500 mL). The
precipitated peptide was collected by centrifugation,
and washed with diethyl ether and n-hexane (1:3), and
dried. The residue was treated with 2-mercaptoethanol
1
was a white amorphous solid. Yield: 0.322 g (43%). H
NMR (DMSO-d6) d 0.81 (12H, m, 4ꢃCH3), 1.03 (1H,
m, CH2), 1.34 (1H, m, CH2), 1.64 (1H, m, CH), 1.93
(1H, m, CH), 2.40 (3H, s, CH3), 2.87 (2H, m, CH2),
3.03, 3.24 (2H, 2dd, J=6.3, 7.6, 13.9 Hz, CH2), 4.12
(2H, m, 2ꢃCH), 4.23 (1H, dd, J=6.9, 8.3 Hz, CH), 4.69
(1H, dd, J=7.1, 14.4 Hz, CH), 6.90–7.06 (4H, m, aro-
matic–H), 7.17 (5H, m, aromatic–H), 7.60 (1H, d,
(0.2mL) in MeOH (10 mL) and H O (6 mL) at 0 ꢂC.
2
The solution was adjusted to pH 8.0 with triethylamine

1810
M. Haramura et al. / Bioorg. Med. Chem. 10 (2002) 1805–1811
J=8.9 Hz, NH), 7.67 (1H, d, J=7.9 Hz, NH), 8.21 (1H,
d, J=7.9 Hz, NH), 8.44 (1H, d, J=8.9 Hz, NH).
TFA/0.1% TFA in acetonitrile) over 25 min at 1 mL/
min using YMC-Pack A-302analytical column
(4.6ꢃ150 mm, 120 A, 5 mm particle size, YMC, Japan).
The other (B) was 100:0 to 30:70, or 100:0 to 2:98 (0.1%
aqueous TFA/MeOH) over 25 min, 1.0 mL/min using m
Bondasphere 5m C18 300 A (3.9ꢃ150 mm, Waters).
H-Phe-Val-Trp(20-SCH2CH2CH3)-Ile-NH2 (9). The title
compound was synthesized from 6 (0.592g, 1.05 mmol)
and di-n-propyl disulfide following the procedure
describe for 8. Yield: 0.369 g (47%). 1H NMR (DMSO-
d6) d 0.76 (12H, m, 4ꢃCH3), 0.93 (3H, t, J=7.3 Hz,
CH3), 1.05 (1H, m, CH2), 1.41 (1H, m, CH2), 1.48 (2H,
six, J=7.3 Hz, CH2), 1.64 (1H, m, CH), 1.95 (1H, m,
CH), 2.79–2.95 (4H, m, 2ꢃCH2), 3.04 3.26, (2H, 2dd,
FT-NMR was performed on a JEOL JNM-A500, Var-
ian Mercury 300, and JEOL JNM-EX270. Spectra was
taken in DMSO-d6 or MeOH-d4. The molecular weight
of the compounds was determined by FABMS (VG70-
250SEQ, VG Analytical, UK). The values are expressed
as MH+ (Table 3).
J=6.3, 8.0, 14.2Hz, CH ), 4.12(2H, m, 2 ꢃCH), 4.33
2
(1H, dd, J=7.3, 8.2Hz, CH), 4.66 (1H, dd, J=7.3, 14.9
Hz, CH), 6.90–7.06 (4H, m, aromatic–H), 7.17 (5H, m,
aromatic–H), 7.55 (1H, d, J=8.9 Hz, NH), 7.66 (1H, d,
J=7.6 Hz, NH), 8.19 (1H, d, J=8.2Hz, NH), 8.44 (1H,
d, J=8.9 Hz, NH).
Receptor binding assay
Binding assay for MTL-R was performed according to
the procedure introduced by Bormans et al.25 with a
slight modification. After exsanguination, the upper
part of intestine (about 50 cm) of the rabbit was rapidly
removed and rinsed with ice cold 0.9% saline. The
smooth muscle tissue was dissected free from connective
tissue and mucosa, finely minced and homogenized in
50 mM Tris–HCl buffer (pH=7.4) at 0 ꢂC, using
tapered tissue grinders (Wheaten, Milliville, NJ, USA)
at 2000 rpm for 30 s. The homogenate was centrifuged
at 1500ꢃg for 5 min and was washed twice with a fresh
buffer. The final pellet was resuspended in 50 mL of 50
mM Tris–HCl buffer (pH=8.0, containing 10 mM
MgCl2, 1.5% bovine serum albumin) for binding
studies.
H-Phe-Val-Trp(20-SCH2CH2NMe2)-Ile-NH2 (10). The
title compound was synthesized from 6 (0.592g, 1.05
mmol) and tetramethyl cystamine24 following the pro-
cedure describe for 8. Yield: 0.589 g (63%).1H NMR
(DMSO-d6) d 0.82(12H, m, 4 ꢃCH3), 1.02(2H, m, CH2),
1.38 (2H, m, CH2), 1.65 (1H, m, CH), 1.94 (1H, m, CH),
2.77 (6H, s, 2ꢃCH3), 2.87 (2H, m, CH2), 3.00–3.33 (6H,
m, 3ꢃCH2), 4.11 (2H, m, 2ꢃCH), 4.27 (1H, dd, J=6.8,
8.4 Hz, CH), 4.73 (1H, dd, J=6.8, 7.6 Hz, CH), 6.91–
7.27 (9H, m, aromatic–H), 7.69 (1H, d, J=9.2Hz, NH),
7.74 (1H, d, J=8.2Hz, NH), 8.28 (1H, m, NH), 8.45
(1H, d, J=8.9 Hz, NH).
H-Phe-Val-Trp(20-SCH2COOH)-Ile-NH2 (11). The title
compound was synthesized from 6 (0.584 g, 1.04 mmol)
and dithiodiglycolic acid following the procedure
The homogenate (about 1.0 mg protein/assay) was
incubated at 25 ꢂC for 120 min with 25 pM 125I-pMTL
(specific activity, 33–66 kBq/pmol); the final volume was
1 mL. After incubation, the reaction was stopped by
adding 2mL of cold buffer. Bound and free reagents
were separated by centrifugation at 1500ꢃg for 5 min.
The pellet was washed with a cold buffer, and its radio-
activity was determined with a gamma counter (ARC-
300, Aroka, Tokyo, Japan). The concentration, displa-
cing 50% of the label, is expressed (IC50).
1
describe for 8. Yield: 0.35 g (42%). H NMR (DMSO-
d6) d 0.79 (12H, m, 4ꢃCH3), 1.00 (1H, m, CH2), 1.39
(1H, m, CH2), 1.66 (1H, m, CH), 1.91 (1H, m, CH),
2.81, 2.93 (2H, 2dd, J=4.6, 8.3, 14.2Hz, CH ), 3.09,
2
3.20 (2H, 2dd, J=5.9, 8.3, 14.2Hz, CH ), 3.63 (2H, d,
2
J=1.7 Hz, CH2), 4.11 (2H, m, 2ꢃCH), 4.25 (1H, dd,
J=6.9, 8.2Hz, CH), 4.71 (1H, dd, J=6.9, 7.4 Hz, CH),
6.90–7.23 (9H, m, aromatic–H), 7.65 (1H, d, J=10.2
Hz, NH), 7.68 (1H, d, J=8.6 Hz, NH), 8.12(1H, d,
J=8.2Hz, NH), 8.51 (1H, br, NH).
Contraction assay
Male Japanese-white rabbits (about 3 kg) were used.
The animals were anesthetized with thiopental sodium
(30 mg/kg, iv) and exsanguinated. The upper part of the
small intestine was rapidly removed after laparotomy
and placed in an ice-cold modified Krebs’ solution
composed of (in mM): NaCl 120.0, KCl 4.7, CaCl2 2.4,
KH2PO4 1.0, MgSO4 1.2, NaHCO3 24.5 and glucose 5.6
(pH=7.4). The duodenum was washed, freed from
mesenteric 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’ solu-
tion kept at 28 ꢂC to prevent excessive spontaneous
contractions.26 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 con-
tractile activity was measured by means of isotonic
transducers (ME-4012, Medical Electronics Co., Tokyo,
Peptide purification
The crude peptides were purified by RP-HPLC using a
Waters semi-prep system with C18 YMC-Pack S-343-15
(YMC, Japan), 15 mm, 120 A, 2 0ꢃ250 mm, eluting with
a linear acetonitrile gradient (0–60%) in water contain-
ing a constant concentration of TFA (0.1%, v/v) over
60 min at a flow rate of 10 mL/min. The peptide frac-
tions, which were verified by analytical HPLC, were
lyophilized, and the powder was kept atꢁ20 ꢂC until
their biological assay.
Peptide analysis
The purity of the final products was confirmed by RP-
HPLC using a Hitachi D-7000 HPLC system in two
different columns and linear gradient solvent systems. One
solvent system (A) was 100:0 to 30:70 (0.1% aqueous

M. Haramura et al. / Bioorg. Med. Chem. 10 (2002) 1805–1811
1811
Japan) and recorded on an ink-writing recorder (Type
3066, Yanagisawa-Denki, Tokyo, Japan). Before the
experiments, each strip was subjected to repeated stim-
ulation with 100 mM acetylcholine (Ach) until a repro-
ducible response was obtained. The contractile potency
of each compound was expressed as a percentage of that
induced by 100 mM ACh. The dose giving 50% of the
response is expressed (EC50).
9. Gouda, H.; Sunazuka, T.; Omura, S.; Hirano, S. Chem.
Pharm. Bull. 2000, 48, 1835.
10. Peeters, T. L.; Macielag, M. J.; Depoortere, I.; Konteatis,
Z. D.; Florance, J. R.; Lessor, R. A.; Galdes, A. Peptides 1992,
13, 1103.
11. Haramura, M.; Tsuzuki, K.; Okamachi, A.; Yogo, K.;
Ikuta, M.; Kozono, T.; Takanashi, H.; Murayama, E. Chem.
Pharm. Bull. 1999, 47, 1555.
12. Khan, N.; Graslund, A.; Ehrenberg, A.; Shriver, J. Bio-
chemistry 1990, 29, 5743.
13. Edmondson, S.; Khan, N.; Shriver, J. Biochemistry 1991,
30, 11271.
Acknowledgements
14. Backlund, B.-M.; Wikander, G.; Peeters, T. L.; Graslund,
A. Biochim. Biophys. Acta 1994, 1190, 337.
15. Haramura, M.; Okamachi, A.; Tsuzuki, K.; Yogo, K.;
Ikuta, M.; Kozono, T.; Takanashi, H.; Murayama, E. Chem.
Pharm. Bull. 2001, 49, 40.
16. Bodansky, M.; Bodansky, A. The Practice of Peptide
Synthesis; Springer: Berlin, Heidelberg, 1984.
17. Stewart, J. M.; Young, J. D. Solid Phase Peptide Synth-
esis, 2nd ed; Pierce Chemical Co: Rockford, IL, 1984.
18. Matsueda, G. R.; Stewart, J. M. Peptides 1981, 2, 45.
19. Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404.
20. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I.
Anal. Biochem. 1970, 34, 595.
21. Macielag, M. J.; Peeters, T. L.; Konteatis, Z. D.; Flor-
ance, J. R.; Depoortere, I.; Lessor, R. A.; Bare, L. A.; Cheng,
Y-S.; Galdes, A. Peptides 1992, 13, 565.
22. Depoortere, I.; Peeters, T. L.; Matthijs, G.; Cachet, T.;
Hoogmartens, J.; Vantrappen, G. J. Gastrointest. Motility
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25. Bormans, V.; Peeters, T. L.; Vantrappen, G. Regul. Pept.
1986, 15, 143.
The authors wish to thank Dr. Theo L. Peeters, Dr.
Hiroharu Matsuoka and Dr. Thomas Arrhenius for
their help in the preparation of this paper and for many
useful suggestions for its improvement, and Mr. Kenichi
Ozaki for the biological data analysis.
References and Notes
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