Discovery of novel motilin antagonists: Conversion of tetrapeptide leads to orally available peptidomimetics

Article abstract of DOI:10.1016/j.bmcl.2009.05.059

We successfully discovered peptidomimetic motilin antagonists (17c and 17d) through the improvement of physicochemical properties of a tetrapeptide antagonist (2). Furthermore, with oral administration and based on motilin antagonistic activity, both comp

Full text of DOI:10.1016/j.bmcl.2009.05.059

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3426–3429
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel motilin antagonists: Conversion of tetrapeptide leads
to orally available peptidomimetics
a
a
a
a
a
Naoki Taka a, , Hiroharu Matsuoka , Tsutomu Sato , Hitoshi Yoshino , Ikuhiro Imaoka , Haruhiko Sato ,
Ken-ichiro Kotake b, Yoshikazu Kumagai b, Kenshi Kamei c, Ken-ichi Ozaki d,
Atsuko Higashida e, Toshio Kuroki f
*
a Chemistry Research Dept. 1, Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd, 1-135 Komakado Gotemba City, Shizuoka 412-8513, Japan
b Pharmaceutical Technology Dept., Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd, 1-135 Komakado Gotemba City, Shizuoka 412-8513, Japan
c CMC Planning and Coordination Dept., Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd, 1-135 Komakado Gotemba City, Shizuoka 412-8513, Japan
d Pharmaceutical Research Dept. 1, Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd, 1-135 Komakado Gotemba City, Shizuoka 412-8513, Japan
e Discovery Platform Technology Dept., Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd, 1-135 Komakado Gotemba City, Shizuoka 412-8513, Japan
f Research Planning and Coordination Dept., Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd, 1-135 Komakado Gotemba City, Shizuoka 412-8513, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 November 2008
Revised 7 May 2009
Accepted 11 May 2009
Available online 20 May 2009
We successfully discovered peptidomimetic motilin antagonists (17c and 17d) through the improvement
of physicochemical properties of a tetrapeptide antagonist (2). Furthermore, with oral administration and
based on motilin antagonistic activity, both compounds suppressed motilin-induced colonic and gastric
motility in conscious dogs.
Ó 2009 Published by Elsevier Ltd.
Keywords:
Motilin
Motilin receptor antagonist
GM-109
Peptidomimetics
Irritable bowel syndrome (IBS) is a gastrointestinal (GI) disorder
characterized by altered bowel function and abdominal pain in the
absence of detectable structural abnormalities1 A variety of drugs
have been used to alleviate the major manifestations of IBS; how-
ever, no single drug has proven consistently effective in treating
IBS. Motilin, a 22-amino acid peptide intestinal hormone, has been
reported to stimulate contractile activity of the gastrointestinal
tract and to have some clinical relevance to some gastrointestinal
diseases, such as IBS2 and functional dyspepsia (FD).3 Taking
advantage of this finding, we started motilin derivatization. Previ-
ously, we reported a macrocyclic motilin agonist, GM-611 (1)
(Chart 1),4 which is currently under clinical study in the US as an
anti-diabetic gastroparesis. Our next focus is the discovery of mo-
tilin antagonists,5 with an anti-IBS or FD agent as the target. We
have developed an SAR on the peptidic motilin antagonist and
identified critical amino acid residues for the interaction with a
motilin receptor. From this research, a cyclic tetrapeptide, GM-
109 (2) has been identified as a potent motilin antagonist.6 How-
ever, 2 had a poor PK profile, an oral bioavailability of 0%, and
did not exhibit any pharmacological effects after oral administra-
Me
Me
OH
N
O
OH
HO
O
O
O
O
O
H
N
O
H2N
N
H
O
O
O
HN
O
H
N
O
OMe
O
GM-611, 1
GM-109, 2
Cl3C
O
NH
NH
N
O
O
N
O
RWJ-68023, 3
Chart 1. Structures of motilin agonist and antagonist.
tion. Recently, non-peptide motilin antagonist 3 was disclosed.7
However; there have been no reports of the oral activities of moti-
lin antagonists to date. Here we report on the chemical modifica-
* Corresponding author.
E-mail address: takanok@chugai-pharm.co.jp (N. Taka).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.05.059

N. Taka et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3426–3429
3427
Table 1
tions of GM-109 resulting in orally active agents and their biolog-
Results of motilin receptor binding and motilin antagonistic action assay for 9
ical evaluations.
OH
In order to convert 2 to an oral agent, the physicochemical
drawbacks, such as high MW, too many hydrogen bonding accep-
tors/donors, and metabolically unstable peptide bonds, need to be
improved.8
We first focused on downsizing the molecular weight of 2. We
assumed that the macrocyclic backbone of 2 was unnecessary for
motilin receptor binding because the Phe and Thy(t-Bu) residues
are thought to play a critical role in motilin receptor binding and
that the backbone plays an important role in maintaining the ac-
tive conformation of the two resides. Therefore, we first prepared
tripeptide derivatives without the backbone.
As shown in Scheme 1, Fmoc-Thy(t-Bu) 6 was initially prepared
from Thy-OMe 4. The reaction of 4 with tert-butyl acetate in the
presence of HClO4 gave 5 in moderate yield. Hydrolysis of 5 and
subsequent Fmoc protection afforded Fmoc-Thy(t-Bu) 6 in 61%
yield in two steps. The tripeptide 9 series was synthesized using
a solid-phase reaction method. Fmoc-resin was de-protected by
piperidine, which was then coupled with Thy(t-Bu) 6, using BOP,
to afford 7. Next, 7 was de-protected and coupled with Fmoc-pro-
tected amino acid (AA) to give di-peptide 8. Coupling dipeptide 8
with N-terminal AA and deprotection gave tripeptide 9.
The results of motilin receptor binding activities and motilin
antagonistic activities of 9a–f are shown in Table 1.
Although 9a, of which central AA is Gly, did not exhibit motilin
receptor binding activity, 9b–f showed various extents of antago-
nistic activities. Importantly, 9c, 9d, 9e, and 9f exhibited motilin
antagonistic activity comparable to that of 2 (GM-109). The data
suggested that the relatively large R1 group kept the tripeptide in
active conformation and supports our hypothesis.
H
N
O
NH2
H2N
N
H
O
R1
O
R1
IC50 (nM)
pA2
a
b
Compound
9a
H
—
—
<6
9b
9c
9d
9e
CH3
CH(CH3)2
CH2CH(CH3)2
Ph
CH2Ph
185
46
27
12
6
6.9
7.3
7.8
7.2
7.4
9f
GM-109
12
a
IC50, expressing MTL receptor binding activity, was the concentration necessary
to displace 50% of the binding of 125I-motilin (rabbit duodenum homogenate).9
b
pA2, expressing MTL antagonist activity, is the negative logarithm of the molar
concentration of compound causing a twofold shift in the concentration–response
curve for motilin (rabbit duodenum specimen).10
by means of N-methylation at amide bond or replacement of ter-
minal tyrosine moiety with non-amino acid forms, 17a–d. Synthe-
ses of target molecules 17a–d are outlined in Scheme 2.
An amide intermediate 11 was prepared from tyrosine deriva-
tive 5. First, Z-protection of Thy(t-Bu) 5 gave 10, and amidation
of 10 using a mixed anhydride method and deprotection gave
the amide 11. Meanwhile, N-methyl tyrosine intermediate 13
was prepared from 10. Initially, the phenol moiety of 10 was pro-
tected by the benzyl group to give ether 12. Subsequent hydrolysis
of 12 afforded a carboxylic acid, which was then converted to the
corresponding N-methyl amide from treatment of MeI and NaH.
During this methylation, no methyl ester was obtained. Next, ami-
dation of the N-methyl amide followed by deprotection by Pd/C
gave N-methylated Thy(t-Bu)-NH2 13. Phenethyl amine 15 was
Next we focused on stabilization against metabolism through
the modification of 9c, which has the smallest molecular weight
among the potent antagonists 9c–f. Stabilization included design
and synthesize derivatives bearing cleavage-resistant amide bonds
OH
OH
OH
1) NaOH
2) Fmoc-Osu, NaHCO3
HClO4, AcOBut
40%
H2N
CO2Me
H2N
CO2Me
FmocHN
CO2H
61%
4
5
6
1) piperidine
2) Fmoc-AA-OH,
coupling reagent
or Fmoc-AA-oPfp
1) piperidine
2) 6,coupling reagent
OH
OH
O
H
N
H
N
FmocHN
H
N
N
H
FmocHN
Fmoc
R1
O
O
7
R1= H (AA=Gly)
R1= Me (AA=Ala)
R1= CH(CH
R1= CH 2CH(CH
R1= Ph (AA=Phg)
R1= CH 2Ph (AA=Phe)
8a
8b
8c
8d
8e
8f
1) piperidine
2) Boc-Phe-OH,
coupling reagent
3) TFA cleavage
)2 (AA=Val)
3
OH
)2 (AA=Leu)
3
O
H
N
NH2
N
H
H2N
R1
O
O
TFA
R1= H (AA=Gly)
R1= Me (AA=Ala)
R1= CH(CH
2 (AA=Val)
9a
MeO NHFmoc
9b
9c
9d
9e
9f
FmocHN
=
)
3
PS
MeO
O
R1= CH 2CH(CH
)2 (AA=Leu)
3
R1= Ph (AA=Phg)
R1= CH 2Ph (AA=Phe)
Scheme 1.

3428
N. Taka et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3426–3429
1) NaOH
OH
OH
OH
2) ClCO2Et, NEt3
3) NH3 aq.
4) H2, Pd-C
ZCl, Na2CO3
ZHN
CO2Me
H2N
CONH2
11
H2Z
CO2Me
dioxane-H2O
86%
61%, 4 steps
5
10
1) NaOH
2) MeI, NaH
3) ClCO2Et, NEt3, then NH3 aq.
4) H2, Pd-C
OBn
OH
BnBr, K2CO3
10
DMSO
80%
79%, 4 steps
ZHN
CO2Me
MeHN
CONH2
13
12
1) LiBH4
2) MsCl, NEt3
3) LiBEt3H
4) H2, Pd(OH)2
OBn
OH
BnBr, NaH
10
DMF
94%
59%, 4 steps
Z
N
Bn
CO2Me
MeHN
Me
15
14
1) Boc-Phe-OH
OH
OH
1) ZNR2Val-OH
DIC, HOBt
2) H2, Pd-C
DIC, HOBt
2) TFA
R2
N
O
R2
HN
O
11, 13, 15
R4
H2N
N
R4
N
R3
O
R3
16a R2= Me, R 3= H, R 4= CONH
16b R2= H, R 3=Me, R 4= CONH
17a R2= Me, R 3= H, R 4= CONH
17b R2= H, R 3= Me, R 4= CONH
2
2
2
2
16c R2= Me, R 3= Me, R 4= CONH
16d R2= Me, R 3= H, R 4= Me
17c R2= Me, R 3=Me, R 4= CONH
17d R2= Me, R 3= H, R 4= Me
2
2
Scheme 2.
prepared as follows. Double benzyl protection of 10 obtained 14 in
94% yield. Reduction of the ester group using lithium borohydride
gave the corresponding alcohol, which was methansulfonylated
and converted into a methyl group using Super-HydrideÒ. Depro-
tection of the benzyl group afforded 15 in 59% yield in four steps.
Coupling of each intermediates 11, 13, and 15 with Z-protected va-
line derivative in the presence of HOBT and DIC afforded dipeptide
derivatives, then Z group of the dipeptides were removed by Pd/C–
H2 to give 16a-d. Finally, 16a–d were coupled with Boc-Phe and
the following deprotection yielded the target compounds 17a–d.
The results of the motilin receptor binding activities and motilin
antagonistic activities are shown in Table 2.
Although 17a was the most potent binder, its antagonistic activity
was relatively weaker, compared with that of 17d. This reduced
activity may have been due to insufficient metabolic stability in
the ex vivo assay.
Compounds 17a–d were further evaluated on metabolic stabil-
ity and permeability, using rat liver S-9 fraction and Caco-2 cells,
respectively (Table 3). Compound 17a, which had an N-methylated
amide bond at the valine moiety, showed considerable metabolic
stability, but it did not show permeability. Interestingly, 17b, a
mono-methylated derivative like 17a but showed neither the sta-
bility nor permeability, suggesting a methylation site is critical
Compounds 17a–d showed potent motilin binding activity and
motilin antagonistic activity. In particular, 17c and 17d, bearing a
phenetyl moiety, were the potent antagonists, with more potent
motilin antagonistic activity in pA2 value than that of GM-109.
Table 3
Metabolic stability and permeability of 17a–d
OH
Table 2
R2
N
O
Results of motilin receptor binding and motilin antagonistic action assay for 9c and 17
OH
H2N
N
R4
O
R3
O
H
N
NH2
N
H
H2N
Compd.
R2
R3
R4
Metabolic stabilitya
Permeabilityb
BA (%)c
R1
O
O
17a
17b
17c
17d
Me
H
Me
Me
H
CONH2
CONH2
CONH2
Me
69
7
80
100
0
0
2.31
12.1
6
Me
Me
H
n.t.
30
65
Compound
R2
R3
R4
IC50 (nM)
pA2
9c
H
H
CONH2
CONH2
CONH2
CONH2
Me
46
1.1
24
4.3
1.9
12
6.9
7.9
7.2
8.6
8.4
7.4
17a
17b
17c
17d
Me
H
Me
Me
H
a
Metabolic stability was expressed as residual % value using rat liver S-9 after
Me
Me
H
incubation for 30 min at 37 °C.
Permeability was expressed as apparent permeability value (Papp: 10À6 cm/s.)
using Caco-2 cells (apical pH 6.8).
b
GM-109
c
BA was expressed as bioavailability in rat at 10 mg/kg.

N. Taka et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3426–3429
3429
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Coulie, B.; Peeters, T. Am. J. Gastroenterol. 2005, 100, 1.
4. (a) Koga, H.; Takanashi, H.; Itoh, Z.; Omura, S. Drug Future 2002, 27, 255; (b)
Haramura, M.; Okamachi, A.; Tsuzuki, K.; Yogo, K.; Ikuta, M.; Kozono, T.;
Takanashi, H.; Murayama, E. Chem. Pharm. Bull. 2001, 49, 40; (c) Peeters, T. L.
Curr. Opin. Investig. Drugs 2001, 2, 555.
5. (a) Takanashi, H.; Yogo, K.; Ozaki, K.; Ikuta, M.; Akima, M.; Koga, H.; Nabata, H.
J. Pharmacol. Exp. Ther. 1995, 273, 624; (b) Marsault, E.; Hoveyda, H. R.;
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500
400
300
200
100
0
Gastric antrum
Colon 1
*** **
*** **
vehicle
17c
17d
5mg/kg
5mg/kg
7. (a) Kamerling, L. M. C.; van Haarst, A. D.; burggraaf, J.; Schoemaker, R. C.; De
Kam, M. L.; Heinzerling, H.; Cohen, A. F.; Masclee, A. A. Br. J. Clin. Pharmacol.
2004, 57, 393; (b) Beavers, M. P.; Gunnet, J. W.; Hageman, W.; Miller, W.;
Moore, J. B.; Zhou, L.; Chen, R. H. K.; Xiang, A.; Urbanski, M.; Combs, D. W.;
Mayo, K. H.; Demarest, K. Ts. Drug Des. Discovery 2001, 17, 243; (c) Johnson, S.
G.; Gunnet, J. W.; Moore, J. B.; Miller, W.; Wines, P.; Rivero, R. A.; Combs, D.;
Demarest, K. T. Bioorg. Med. Chem. Lett. 2006, 16, 3362.
Figure 1. In vivo MTL antagonistic activities of 17c and 17d.10 Each column
represents the mean SE from 6 dogs, **P < 0.01 and ***P < 0.001 compared with the
vehicle group by student’s t-test.
8. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3.
for stability. Importantly, 17c and 17d showed satisfactory stability
and permeability. The drastic improvements were thought to be
due to the conversion of the peptide bond to the usual amide bond
and a reduction in hydrogen bonding donors/acceptors. In a rat PK
study, oral bioavailability results for 17c and 17d were 30% and
65%, respectively. These findings encouraged us to conduct an
in vivo pharmacological study with oral administration. Figure 1
shows the results of the pharmacological evaluation of 17c and
17d in dog.
Compounds 17c and 17d orally suppressed motilin-induced co-
lonic and gastric motility in conscious dogs. The data strongly sug-
gest that 17c and 17d exhibit motilin receptor antagonist behavior
and have the desired profile of a candidate anti-IBS or anti-FD drug.
In summary, we successfully modified the peptidic antagonist
GM109 and generated peptidomimetic antagonists 17c and 17d.
We confirmed that N-methylation at the peptide bond and replace-
ment of the amino acid with a non-amino acid, such as a phenetyl
group, effectively improved the ADME properties. Compounds 17c
and 17d exhibited good bioavailability, and thus potent antago-
nists even in vivo.
9. Motilin receptor-binding assay was performed according to the procedure
introduced by Depoortere et al. with a slight modification. A portion of the
colonic smooth muscle homogenate was incubated at 25 °C with 25 pM [125I]
motilin. After incubation for 120 min, the reaction was stopped by adding 2 mL
of ice-cold buffer. Bound and free ligands were separated by centrifugation at
1500g for 5 min. The pellet was then washed with ice-cold buffer, and
radioactivity determined using a gamma counter. IC50, determined as the
concentration to displace 50% of the binding of 125I-motilin, expressed the MTL
receptor binding activity. Rabbit upper small intestine longitudinal muscle
strips were mounted in an organ bath containing 10 mL of modified Krebs’
solution kept at 28 °C to prevent excessive spontaneous contraction, the
solution gassed with a mixture of 95% O2 and 5% CO2, and each strip loaded
with
a
1.0 g weight. Before each experiment, the strips were repeatedly
acetylcholine until reproducible response was
stimulated with 100
lM
a
obtained. Contractile activity was measured using an isotonic transducer and
recorded using a pen recorder while increasing the concentration of motilin
(0.1–1000 nM) in the bath solution. Next, the same experiment was repeated
with compounds added in the bath solution 15 min before addition of the
motilin was started. EC50 was defined as the molar concentration of compound
causing a twofold shift to the right of the motilin concentration–response
curve.
10. In the interdigestive state, 17c or 17d (3 or 10 mg/kg) or vehicle (3% gum
arabic) was administered into the stomach via the chronically implanted
silicon tube approximately 15 min after the end of the phase III contractions of
the MMC in the gastric antrum. Thirty minutes later, motilin (3 lg/kg) was
intravenously administered into the vena cava via the silicon tube.
Quantitative analysis of gastric and colonic contractile activities was
performed by calculating the absolute motility indices from the area
between the contractile wave and the baseline. The motility index induced
by intravenous motilin injection (3 lg/kg) was designated as 100% (control) in
each animal, and values obtained in the presence of 17c, 17d, or vehicle were
References and notes
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calculated as a percentage of the control.