Potent macrocyclic antagonists to the motilin receptor presenting novel unnatural amino acids

Article abstract of DOI:10.1016/j.bmcl.2007.05.043

Novel, potent small molecule motilin receptor antagonists are described. These peptidomimetic macrocycles are composed of a tripeptide cyclized backbone-to-backbone with a nonpeptidic tether and bear new unnatural amino acids containing basic side chains.

Full text of DOI:10.1016/j.bmcl.2007.05.043

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4187–4190
Potent macrocyclic antagonists to the motilin receptor
presenting novel unnatural amino acids
Eric Marsault,* Kamel Benakli, Sylvie Beaubien, Carl Saint-Louis,
´
Robert Deziel and Graeme Fraser
Tranzyme Pharma Inc. 3001, 12e av Nord, Sherbrooke, PQ, Canada J1H5N4
Received 27 March 2007; revised 13 May 2007; accepted 14 May 2007
Available online 18 May 2007
Abstract—Novel, potent small molecule motilin receptor antagonists are described. These peptidomimetic macrocycles are com-
posed of a tripeptide cyclized backbone-to-backbone with a nonpeptidic tether and bear new unnatural amino acids containing basic
side chains.
Ó 2007 Elsevier Ltd. All rights reserved.
Motilin is a peptide hormone released from entero-chro-
maffin cells in the gut wall, that acts locally on the mo-
tilin receptor (hMOT-R) to stimulate contraction of the
gut smooth muscle.1,2 Activation of hMOT-R provokes
peristaltic contractions of the gut in a pattern analogous
to phase III of the migrating motor complex (MMC).3
hMOT-R is a G-protein coupled receptor (GPCR) pre-
dominantly found in the gut wall of the stomach and
upper intestine. Molecules interacting with hMOT-R
have potential for treatment of GI disorders associated
with altered gut motility, particularly irritable bowel
syndrome or functional dyspepsia.4,5
led to the synthesis of analogous macrocycles bearing
polar residues at the AA3 position, including 2 bearing
an Asp residue, clearly indicating that a carboxylate
was unsuitable in this position (Ki 2.5 lM). On the other
hand, macrocycles 3 and 4, bearing Lys and Orn
residues, were associated with 5- to 7-fold decrease in
potency (Ki 664 and 889 nM). Despite lower potency,
the different polarity profile of these molecules opened
an avenue to broaden the class and create compounds
with modified physicochemical and pharmaceutical
properties. This led to the synthesis of several potent
hMOT-R antagonists bearing basic or heterocyclic resi-
dues, as described below.
To expand on a previous report of a new class of mac-
rocyclic peptidomimetics antagonists to hMOT-R,6 we
herein describe a subclass of macrocycles with increased
potency as hMOT-R antagonists. These macrocycles
contain new unnatural amino acids bearing basic side
chains. SAR as well as the synthesis of the unnatural
amino acids and the corresponding macrocycles are
presented.
As an initial effort, the length of the exocyclic arm bear-
ing the polar residue at AA3 was shortened (Fig. 2).7
Compared to macrocycles 3 and 4, macrocycles bearing
an L-diaminobutyrate (Dab, 5) or diamino-propionate
residue (Dap, a basic isostere of Nva, see 6) were signif-
icantly more potent, the latter essentially equipotent to
initial lead 1 (6 vs 1, Ki 171 vs 137 nM). Alkylation of
the Dap residue with an isopropyl group led to a mod-
erate decrease in potency (7, Ki 376 nM), whereas acety-
lation had a more dramatic impact (8, Ki 2 lM). While
these two analogues are isosteric, they are not isoelec-
tronic and results clearly indicate the importance of
basicity at that position.
High throughput screening of ca 10,000 macrocyclic
peptidomimetics led to the identification of macrocycle
1 (Ki 137 nM, Fig. 1, functional IC50 74 nM, Fig. 4)
bearing a Nva residue as the AA3 amino acid as the first
hMOT-R antagonist of this class.6 Initial SAR studies
Within the newly discovered Dap series, SAR was nar-
rowed by finer modifications to the aromatic ring substi-
tution pattern (Fig. 2). Replacement of the 4-OH residue
of (D)Tyr by less polar substituents such as 3-Cl, 4-Cl and
Keywords: Motilin; Antagonist; Macrocycles; Peptidomimetics.
*
Corresponding author. Tel.: +1 819 820 6839; fax: +1 819 820
6841; e-mail: emarsault@tranzyme.com
Present address: MethylGene, Montreal, QC, Canada.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.043

4188
E. Marsault et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4187–4190
AA2 = (D)Val
O
O
R
O
AA3
O
O
R
O
NH HN
AA1 = (D)Tyr
NH HN
Ki (nM)
137
Entry
R
Et
AA3
Nva
Asp
HN
NH
X
1
2
HN
NH
CO2H
2553
O
3
4
(CH2)3NH2
(CH2)2NH2
Lys
Orn
664
889
O
HO
1-4
19-33
X
Tether
Ki
(nM)
Ki
(nM)
Entry
19
X
R
Entry
27
R
Figure 1. Initial hit from HTS and early analogues.
N
CF3
CF3
20
4.6
17
HN
Entry
R
X
Ki (nM)
N
N
CH2NH2
NH2
4-OH
4-OH
4-OH
4-OH
342
171
5
6
7
8
N
N
N
CF3
CF3
CF3
Cl
20
Cl
Cl
Cl
Cl
41
51
28
HN
N
S
NH-CH(CH3)2
NH-COCH3
376
N
N
N
N
1946
AA3
O
HN
21
22
23
29
30
31
23
21
18
O
R
O
3-Cl
4-Cl
64
60
15
31
6
9
N
NH HN
H
NH2
10
11
12
13
N
N
N
N
3-CF3
4-Cl
N
HN
NH
HN
87
HN
NHCH3
N(CH3)2
X
3-CF3
O
5-18
H2N
CN
N
3-Cl
4-Cl
4-Cl
27
16
6
14
15
16
N
100
N
NH
NH(C=NH)NH2
NH(C=O)NH2
N
3-CF3
4-Cl
6
17
18
NH2
NH
24
25
26
Cl
Cl
Cl
57
27
33
32
33
Cl
F
62
N
543
N
H2N
HN
S
Figure 2. Macrocycles bearing shorter AA3 exocyclic substituents.
116
NH
NH
NH2
3-CF3 led to significant increase in potency (respectively,
9, 10 and 11, Ki 64, 60 and 15 nM). Monomethylation and
dimethylation of the amine proved beneficial for potency
(12, 13, Ki 31 and 6 nM and 14, 15, Ki 27 and 16 nM,
respectively). To be noted, the Dap(Me2) residue consti-
tutes a polar isosteric replacement for Leu. Replacement
of Dap with an Ala(guanidinyl) residue was found to also
generate very potent macrocycles (16, 17, Ki 6 nM). Com-
pounds 11 and 13 proved to be robust antagonists to
hMOT-R functionally (IC50 8 and 3 nM, Fig. 4). It is
interesting to note that replacement of the guanidine
functionality by the neutral yet isosteric urea dropped po-
tency by two orders of magnitude (18, Ki 543 nM), in
agreement with the result obtained with Dap(Ac) 8. Alto-
gether these results confirm that basicity in this subclass is
a key element to potency.
Figure 3. Guanidine mimetics and heterocycles at AA3 position.
Ki 33 nM). Altogether, even though the preferred group
was the guanidine, isosteric analogues gave interesting
results in terms of potency.
Other heteroaromatic groups were also studied, such as
pyrrolidine (27, Ki 4.6 nM). The latter proved to be the
most potent antagonist in this class, with an IC50 of
2 nM (Fig. 4). It was found that both triazole, tetrazole
and pyrazole were reasonably well tolerated at this posi-
tion (Ki ꢀ 20 nM) although functional IC50 suffered
compared to the pyrrolidine moiety (Fig. 4). Finally,
imidazole and thiazole analogues both showed a de-
crease in potency.
The next part of the effort investigated the impact of de-
creased basicity of the guanidine moiety on the one
hand, and evaluating heterocyclic appendages on the
other hand. A variety of approaches to maintain the
guanidine’s unique hydrogen bonding capability while
decreasing its basicity have been reported,8 several of
which were applied to this system in the form of guani-
dine mimetics (Fig. 3). Heteroaryl-substituted Dap ana-
logues gave interesting results, albeit with a moderate
decrease in potency (19 and 20, Ki 20 and 41 nM). Cycli-
zation of the guanidine further reduced potency (21 and
22, Ki 51 and 87 nM), as did modification to a cyano-
guanidine (23, Ki 100 nM), the latter result being in
agreement with results of 8 and 18. Replacement of
the guanidine by an amidine also had a negative impact
(24 and 25, Ki 57 and 27 nM). Finally, lengthening of the
distance between backbone and guanidine chain by one
carbon atom also resulted in a decrease in potency (26,
Essentially, these results confirmed that the presence of a
basic appendage on the macrocycle is beneficial for com-
pound affinity to hMOT-R, with a preference for alkyl-
ated or cyclic variants of the Dap moiety (13, 16, 17, 27),
and Ala(guanidinyl) or guanidine mimetics (16, 17, 19).
It was found also that several heteroaromatic substitu-
tions could be tolerated, albeit less preferred.
Ki (nM) IC50 (nM)
Entry
1
137
15
31
6
27
6
74
8
33
3
95
28
2
11
12
13
14
17
27
31
4.6
18
56
Figure 4. Functional assay results for best hits.

E. Marsault et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4187–4190
4189
Binding studies were performed in duplicate on CHO
X
cells transfected with hMOT-R using [125I]-motilin (test
concentration, 0.36 nM) as the radioligand. Antagonist
activity was measured on selected compounds using
the AequoScreenTM (Euroscreen, Belgium) cell line in
an aequorin assay measuring the inhibition of motilin-
induced (EC80 0.14 nM) calcium release (Fig. 4).
N3
O
O
O
H
N
b-e
BocNH
Bts
N
N
O
N
H
N
H
O
40
Y = OMs
O
a
Y
42
41 Y = N3
f-j
X
O
O
N3
From a synthetic point of view, macrocycles 1–6, 9–11,
20, 31–33, 48, 49, 51 and 52 were made using solid phase
parallel synthesis as described previously,6 other macro-
cycles were synthesized in solution (Scheme 2). All mac-
rocycles were purified by reverse phase HPLC, and
testing was performed on pure materials exclusively.9
H
NH HN
O
N
O
k
N
H
HN
NH
O
O
NH
X
O
HO2C
44 (X=3Cl)
45 (X=4Cl)
(X=3CF3)
NH2 x TFA
N3
43
46
Scheme 2. Reagents and conditions: (a) NaN3, DMF, 50 °C (70%); (b)
TFA, Et3SiH, DCM (quant.); (c) Boc–(D)Val, EDCI, (6Cl)HOBt,
DIPEA, DCM:THF (98%); (d) TFA, Et3SiH, DCM (quant.); (e) Bts–
Unnatural amino acids b-(2-aminothiazolyl) alanine 36,
b-(imidazol-1-yl)alanine 38 and b-(pyrazol-1-yl)alanine
39 (Scheme 1) were obtained by Mitsunobu reaction
on serine amide 3410 or using Vederas’ b-lactone
(Scheme 1)11 and used as such for macrocycle synthesis.6
(D)Phe(X)–OH,
HATU,
DIPEA,
DCM–THF (95%);
(f)
HO(CH2)2OAC6H4A(CH2)3ANHBoc, DIAD, PPh3, THF (90%); (g)
HS(CH2)2CO2H, Na2CO3, DMF (90%); (h) LiOH, THF:H2O (98%);
(j) TFA, Et3SiH, DCM (quant.); (k) DEPBT, DIPEA, THF (80%).
Azidoalanine-containing macrocycles 44–47 were pre-
pared in solution (Scheme 2). b-Azidoalanine amide 41
was synthesized and the peptide assembled using Boc
chemistry through the attachment of betsylated (Bts)12
aromatic AA1 to give 42. The tether moiety was intro-
duced by Fukuyama–Mitsunobu reaction.13 Subsequent
deprotections and macrolactamization in the presence of
DEPBT14 gave macrocycles 44–46.
X = NHiPr
Y
a,b
a,c-e
7 (Y = OH)
O
O
X
X=N3
X = NHCH3
NH HN
44
45
46
47
(Y=3-Cl)
(Y=4-Cl)
(Y=3-CF3)
(Y=4-OH)
12
(Y = 4-Cl)
O
13 (Y = 3-CF3)
HN
NH
X = N(CH3)2
O
a,f-h
14
(Y = 3-Cl)
15 (Y = 4-Cl)
N-monosubstituted amines (Scheme 3) were obtained
from macrocycles 44–47 by Staudinger azide reduction
followed by reductive alkylation with acetone to give 7
or by Bts protection followed by Mitsunobu alkylation
and Bts removal to give 12 and 13.
a,j,k
X=NH(C=NH)NH2
16
(Y = 4-Cl)
17 (Y = 3-CF3)
Scheme 3. Reagents: (a) PPh3, H2O; (b) acetone, NaBH3CN, AcOH,
HC(OMe)3:MeCN (75%); (c) Bts–Cl, DIPEA, DCM (95%); (d)
MeOH, DIAD–PPh3, THF (90%); (e) HS(CH2)2CO2H, K2CO3,
DMF (90%); (f) Boc2O, Na2CO3, THF–water (85%); (g) H2CO,
NaBH3CN, AcOH, HC(OMe)3, MeCN (75%); (h) HCl–dioxane
(98%); (j) BocNHAC(@NTf)ANHBoc, Et3N, THF; (k) TFA, Et3SiH,
DCM (70%, 2 steps).
On the other hand, N,N-dimethylation required Boc
protection of the secondary amine, followed by reduc-
tive alkylation of the exocyclic primary amine and Boc
removal (14 and 15).
Guanidino alanine macrocycles 16 and 17 were obtained
by reaction with Goodman’s guanidinylation reagent15
followed by Boc acidolysis.
X
O
R'
NH
O
48 (R=Bts, X=4-OH, R'=H)
8 (R=H,X=4-OH, R'=COCH3)
NH HN
a,b
c,b
O
N-Ac and N-urea analogues (8 and 18) were made from
Bts-protected macrocycles 48 and 49 (Scheme 4).
HN
N
R
49 (R=Bts, X=4-Cl, R'=H)
O
18 (R=H, X=4-Cl, R'=CONH2)
O
O
a,b
BocNH
BocNH
X
N
Scheme 4. Reagents: (a) Ac2O, DIPEA, DCM; (b) PS–thiophenoxide,
THF:EtOH (60–70%, 2 steps); (c) TMS–NCO, Et3N, DMAP, DCM.
N
S
O
N
Cbz
Y
34 (Y=OH)
35 X=N(Me)OMe
36 X=OH
Amidine analogues 24 and 25 were obtained from
nitriles 51 and 52 by Pinner synthesis (Scheme 5).16
O
O
O
BocNH
BocNH
d
OH
OH
N
c
BocNH
O
37
N
N
Guanidine analogues 21–23 were synthesized by known
methods,17,18 and pyrimidine analogue 19 was obtained
by SNAr reaction on 2-bromopyrimidine (Scheme 6).
38
39
N
Scheme 1. Reagents and conditions: (a) N-Cbz-2-aminothiazole,
DIAD, PPh3, THF (50%); (b) LiOH, THF–water, rt (98%); (c)
TMS–imidazole, MeCN, rt (55%); (d) TMS–pyrazole, MeCN:THF,
50 °C (60%).
Finally, heterocyclic AA3 analogues were obtained by
reductive alkylation of the primary amine (27), or by

4190
E. Marsault et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4187–4190
Janssens, J. Gastroenterology 1980, 79, 716; (c) Fraser, G.;
O
Cl
Marsault, E.; Coulie, B.; ver Donck, L. Abstracts of the
Digestive Disease Week Meeting, Los Angeles, May 2006.
4. (a) Itoh, Z. Peptides 1997, 18, 593; (b) Peeters, T.
Neurogastroenterol. Motil. 2006, 18, 1; For a review on
motilin agonists and their applications, please refer to (c)
Peeters, T. Neurogastroenterol. Motil. 2006, 18, 1, and
references cited therein; for precedents on motilin antag-
onists, see (d) Haramura, M.; Okamachi, A.; Tsuzuki, K.;
Yogo, K.; Ikuta, M.; Kozono, T.; Takanashi, H.;
Murayama, E. J. Med. Chem. 2002, 45, 670; (e) 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. T. Drug Des.
Disc. 2001, 17, 243; (f) 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.
O
nCN
O
NH HN
NH2
NH
a,b
51 (n=1)
52 (n=2)
HN
n
NH
O
24 (n=1)
25 (n=2)
Scheme 5. Reagents: (a) HCl, EtOH; (b) NH3, EtOH (35%, 2 steps).
N
19
O
X = NH
F3C
O
X
a
N
NH HN
H
N
b,c
O
21
22
(n=1)
(n=2)
50
(X=NH2)
n
X = NH
HN
NH
N
d,e
O
X = NH CN
N
23
5. (a) Besterman, H. S.; Mallinson, C. N.; Modigliani,
R.; Christofides, N. D.; Pera, A.; Ponti, V.; Sarson,
D. L.; Bloom, S. R. Scand. J. Gastroenterol. 1983, 18,
NH2
46
f-h
N
(X = N3)
´
845; (b) Simren, M.; Bjo¨rnsson, E. S.; Abrahamsson,
Y
N
27
X =
N
j
i
k
H. Neurogastroenterol. Motil. 2005, 17, 51; (c) Cuomo,
R.; Vandaele, P.; Coulie, B.; Peeters, T.; Depoortere,
I.; Janssens, J.; Tack, J. Am. J. Gastroenterol. 2005,
100, 1.
N
30
(Y = N)
29
(Y = C-CH3)
28
(Y = CH)
Scheme 6. Reagents and conditions: (a) 2-bromopyrimidine, K2CO3,
DMF 50 °C, (96%); (b) guanidinylation reagent, Et3N, THF 50 °C; (c)
TFA, Et3SiH, DCM (35%, 2 steps); (d) (MeS)2C@NACN, EtOH,
50 °C; (e) NH3, EtOH, 50 °C (30%, 2 steps); (f) PPh3, THF–water,
40 °C (90%); (g) OHCCH2CH2CHO, HC(OMe)3, acetic acid, THF
(65%); (h) H2, PtO2, AcOH, EtOH (98%); (i) Propyne, CuI, DIPEA,
MeCN, rt (76%); (j) 2-butyne, DMSO, 140 °C (65%); (k) CH3CN,
ZnBr2, DMSO, 140 °C (35%).
6. Marsault, E.; Hoveyda, H. R.; Peterson, M. L.; Saint-
´
Louis, C.; Landry, A.; Vezina, M.; Ouellet, L. G.; Wang,
Z.; Ramaseshan, M.; Beaubien, S.; Benakli, K.; Beauche-
´
min, S.; Deziel, R.; Peeters, T.; Fraser, G. J. Med. Chem.
2006, 49, 7190.
7. During the course of this research (see Ref. 6), it was
realized that replacement of the alkene moiety on the
tether arm was slightly beneficial for potency.
8. For a review of guanidine mimetics, see Peterlin-Masic, L.;
Kikelj, D. Tetrahedron 2001, 57, 7073, and references cited
therein.
azide [2+3] cycloaddition with a nitrile or an acetylene
(28–30) (Scheme 6).19
9. Purity criteria: P95% (ELSD detector) and P90%
(CLND detector) and P85% (UV detector).
10. Panda, G.; Rao, N. V. Synlett 2004, 714.
11. Arnold, L. D.; Kalantar, T. H.; Vederas, J. C. J. Am.
Chem. Soc. 1985, 107, 7105.
12. (a) Vedejs, A.; Lin, S.; Klapars, A.; Wang, J. J. Am. Chem.
Soc. 1996, 118, 9796; (b) Wuts, P. G. M.; Gu, R. L.;
Northuis, J. M.; Thomas, C. L. Tetrahedron Lett. 1998,
39, 9155.
In summary, we have identified a new series of macrocy-
clic antagonists to the human motilin receptor (hMOT-
R). These compounds are characterized by the presence
of a basic or heteroaromatic residue at the AA3 position.
In combination with halogenated residues at the AA1
position, the resulting macrocycles displayed very potent
antagonist activity at hMOT-R.
13. Fukuyama, T.; Cheung, M.; Jow, C. K.; Hidai, Y.; Kan,
T. Tetrahedron Lett. 1997, 38, 5831.
14. Li, H.; Jiang, X.; Ye, Y.; Fan, C.; Romoff, T.; Goodman,
M. Org. Lett. 1999, 1, 91.
Acknowledgments
15. Fechtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J.
Org. Chem. 1998, 63, 3804.
´
Dr. Rene Gagnon and Mrs. Annie Doucet are acknowl-
16. Schaefer, F. C.; Peters, G. A. J. Org. Chem. 1961, 26, 412.
17. St Laurent, D. R.; Balasubramanian, N.; Han, W. T.;
Trehan, A.; Federici, M. E.; Meanwell, N. A.; Wright,
J. J.; Seiler, S. M. Bioorg. Med. Chem. 1995, 3, 1145.
18. (a) Tagmose, T. M.; Schou, S. C.; Mogensen, J. P.;
Nielsen, F. E.; Arkhammar, P. O. G.; Wahl, P.; Hansen,
B. S.; Worsaae, A.; Boonen, H. C. M.; Antoine, M. H.;
Lebrun, P.; Hansen, J. B. J. Med. Chem. 2004, 47, 3202;
(b) Penning, T. D.; Russell, M. A.; Chen, B. B.; Chen, H.
Y.; Desai, B. N.; Docter, S. H.; Edwards, D. J.; Gesicki,
G. J.; Liang, C. D.; Malecha, J. W.; Yu, S. S.; Engleman,
V. W.; Freeman, S. K.; Hanneke, M. L.; Shannon, K. E.;
Westlin, M. M.; Nickols, G. A. Bioorg. Med. Chem. Lett.
2004, 14, 1474.
edged for analytical support, as well as Dr. Daniel Veber
for helpful discussions.
References and notes
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Abramovitz, M.; Stocco, R.; Kargman, S.; O’Neill, G.;
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B. Angew. Chem. Int. Ed. Engl. 2002, 41, 2596, and
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references cited therein; (b) Peeters, T. L.; Vantrappen, G.;