Identification of small molecule agonists of the motilin receptor

Article abstract of DOI:10.1016/j.bmcl.2008.10.071

High-throughput screening resulted in the identification of a series of novel motilin receptor agonists with relatively low molecular weights. The series originated from an array of biphenyl derivatives designed to target 7-transmembrane (7-TM) receptors. Further investigation of the structure-activity relationship within the series resulted in the identification of compound (22) as a potent and selective agonist at the motilin receptor.

Full text of DOI:10.1016/j.bmcl.2008.10.071

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6423–6428
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of small molecule agonists of the motilin receptor
b
b
a
c
Tom D. Heightman a, , Elizabeth Conway , David F. Corbett , Gregor J. Macdonald , Geoffrey Stemp ,
Susan M. Westaway c, Paolo Celestini d, Stefania Gagliardi d, Mauro Riccaboni d, Silvano Ronzoni d,
Kalindi Vaidya b, Sharon Butler b, Fiona McKay b, Alison Muir b, Ben Powney b, Kim Winborn b,
Alan Wise b, Emma M. Jarvie a, Gareth J. Sanger c
*
a Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
b Molecular Discovery Research, GlaxoSmithKline R&D, Third Avenue, Harlow, Essex CM19 5AW, UK
c Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK
d NiKem Research Srl, Via Zambeletti, 25, 20021 Baranzate (MI), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
High-throughput screening resulted in the identification of a series of novel motilin receptor agonists
with relatively low molecular weights. The series originated from an array of biphenyl derivatives
designed to target 7-transmembrane (7-TM) receptors. Further investigation of the structure–activity
relationship within the series resulted in the identification of compound (22) as a potent and selective
agonist at the motilin receptor.
Received 11 September 2008
Revised 15 October 2008
Accepted 16 October 2008
Available online 19 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Motilin
Receptor
Agonist
GPCR
Motilin, a 22-amino acid peptide found in endocrine cells with-
in the gut mucosa, can enhance gastrointestinal motility and pro-
mote gastric emptying in both animals and humans.1 The
macrolide antibiotic erythromycin mimics the effects of motilin,
and has been used clinically for the treatment of motility disorders
including post-operative ileus.2 These effects have been shown to
result from agonist activity at the motilin receptor (previously
known as GPR38), providing attractive clinical validation of the
receptor as a target for small molecule therapeutic intervention,3
and prompting the development of a family of non-antibiotic mac-
rolides known as motilides, exemplified by KC11458, ABT229 and
mitemcinal (GM-611).4 Until recently the motilides were the only
non-peptide agonists of the motilin receptor, and despite success
in demonstrating enhanced gastric emptying in humans, some
have encountered difficulties during clinical development.5 How-
ever two molecules of this class, mitemcinal from Roche/Chugai
and PF-0454803/KOS2197 from Pfizer/Kosan, are currently pro-
gressing in clinical trials.6 In 2004 a series of non-motilide motilin
receptor agonists was disclosed (Fig. 1), represented by compounds
(1) and (2), possessing molecular weights in the range 560–720,
multiple chiral centres and a relatively complex synthesis.7
At GSK we initiated a programme towards the discovery of
small molecule rather than motilide agonists of the human motilin
receptor. A FLIPR (Fluorescence Imaging Plate Reader) based high-
throughput screen8 of the in-house compound collection using
cells recombinantly expressing the human motilin receptor identi-
fied a series of biphenylmethyl piperazine analogues with promis-
ing agonist activity. These screening hits originated from a large
array of biphenyl containing compounds which had been prospec-
tively designed to contain the 3D display of pharmacophoric ele-
ments known to be important in small molecule interactions
with 7-TM receptors. We have described previously a subset of
compounds from this array which led to the discovery of a series
of selective 5HT5a receptor antagonists.9 The identification of a
Ph
O
O
O
O
O
O
H
N
H
N
NH2
O
O
NH2
N
H
O
O
O
N
N
N
N
N
N
O
O
N
H
N
H
1
2
* Corresponding author. Tel.: +44 1865617574.
E-mail address: tom.heightman@sgc.ox.ac.uk (T.D. Heightman).
Figure 1. Non-macrolide motilin receptor agonists.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.071

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T. D. Heightman et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6423–6428
Table 2
Motilin receptor agonist potency of piperazine analogues (12)–(18). FLIPR data8 on
compounds from solid.
HN
N
HN
N
O
O
H
N
O
N
NH
N
N
MeO
R
4 pEC50 5.8
3 pEC50 7.0
Figure 2. Motilin receptor agonist potency of regioisomeric 4- and 3-piperazinyl
Compound
R
Motilin R pEC50 (IA)a
6.1 (0.7)
derivatives. HTS FLIPR data8 on array compounds from DMSO solution.
NH
N
12
second subset of compounds from the same array which has moti-
lin receptor agonist activity provides clear validation of this pro-
spective design approach.
The significant number of close analogues in the array allowed
rapid generation of SAR without the need for further synthesis.
Thus, the data for regioisomeric analogues (3) and (4) indicated a
clear preference for the piperazinylmethyl group to be linked in a
1,4-orientation with respect to the first phenyl ring of the biphenyl
core (Fig. 2).
N Me
N
N
13
14
<4.9
<4.9
O
N
Me
O
N
N
15
16
17
<4.9
The SAR around the most potent hit from the array, urea (3), is
shown in Table 1. Removal of the cyclohexyl urea (5) effectively
abolished activity, whilst replacement of the methoxyphenethyl
<4.9
Me
N
5.5 (0.4)
<5.5
NMe2
NH
Table 1
Motilin receptor agonist potency of analogues of HTS hit (3). HTS FLIPR data8 on array
O
compounds from DMSO solution.
18
N
R1
H
R2
N
a
N
All compounds n P 3 except (12), (13) and (17) where n = 2. IA = intrinsic
activity.8
N
group with the smaller, less lipophilic methoxypropyl group (6)
gave a fivefold reduction in activity. Replacement of the urea with
amides (7)–(10) or a sulfonamide (11) was tolerated.
R1
OMe
R2
Motilin R pEC50
7.0
a
Compound
O
3
Table 3
N
H
Motilin receptor agonist potency of piperazine analogues (3) and (19)–(22). FLIPR
data8 on compounds from solid.
OMe
H
5
5.3
O
N
H
N
O
O
6
7
6.3
6.0
MeO
N
H
MeO
R
OMe
Compound
R
Motilin R pEC50 (IA)a
7.3 (0.9)
3
NH
N
OMe
O
O
O
O
8
6.6
6.4
6.2
6.2
19
20
21
5.3 (0.9)
<4.9
NH
N
9
MeO
NH
N
6.4 (0.6)
NH
O
10
MeO
MeO
Me
F
Me
NH
Me
O
O
22
8.0 (1.0)
N
S
11
a
All compounds n P 3 with SEM 6 0.1 except (21) where n = 2. IA = intrinsic
a
activity.8
All compounds n = 2.

T. D. Heightman et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6423–6428
6425
Table 4
The dimethylpiperazine (22) was selected for further profiling
and >100-fold selectivity was demonstrated against a panel of over
100 receptor and enzyme targets. When tested against the closely
related ghrelin receptor (previously known as the growth hormone
secretagogue receptor) in a similar human recombinant FLIPR as-
say (22) had a pEC50 of 6.9, representing ꢀ10-fold selectivity.10
In experiments to assess the potential for gastric prokinetic-like
activity (measuring an ability to enhance neuronally mediated
contractions evoked by electrical field stimulation of isolated rab-
bit gastric antrum11), (22) significantly enhanced the contractile
response. The enhancement is partial in magnitude compared to
motilin and erythromycin, and interestingly shows a longer dura-
tion (Table 4). This profile is encouraging in the light of speculation
that the lack of clinical efficacy of motilides such as ABT-229 in
functional dyspepsia and diabetic gastroparesis may arise from
their potential to evoke tachyphylaxis.5
The synthesis of the hit generation array, including analogues
(3)–(11), was carried out using solid phase chemistry (Scheme 1).
Wang resin was activated with the 2-pyridylcarbonate group
(23), then nucleophilic displacement with the regioisomeric bro-
mobenzyl piperazines gave the carbamates (24). Suzuki coupling
with 2-, 3- or 4-formylbenzeneboronic acid was followed by reduc-
tive amination to give the regioisomeric biphenylmethylamines
(26). Treatment with the appropriate acid chloride or sulfonyl chlo-
ride in the presence of triethylamine, or with the appropriate iso-
cyanate gave the resin bound final products (27). TFA-mediated
cleavage from the resin and purification then gave the library of
piperazinylmethyl biphenyl methyl amides, sulfonamides and ur-
eas respectively, represented by (28). Array compounds for which
data are shown in Table 1 exceeded 80% purity (LC/MS and 1H
NMR) and were screened without further purification.
Concentration-dependent potentiation of the Electrical Field Stimulated (EFS)
contractile response in rabbit isolated gastric antrum by compound 22.
Compound
Max response % increase
Conc (uM)a
T
(min)
n
½
Rabbit motilin
Erythromycin
22
506 112
490 117
182 56
0.3
3
10
9
4
5
3
23
39
a
Concentration of agonist at which maximum response was observed.
Since the piperazinyl ring had been the point of attachment to
the solid phase in the original array, there were no existing ana-
logues of this group, and hence the early chemistry program fo-
cused on generating diverse SAR in this region of the molecule.
Amide (12) was chosen as the starting point for these modifica-
tions, as although not the most potent analogue, its relatively
low molecular weight would allow incorporation of larger pipera-
zine replacements without overly compromising physicochemical
properties. A 96-member solution phase array was prepared using
reductive alkylation chemistry, and screened in the FLIPR assay.8
The results indicated that the NH of the piperazine was essential
for agonist activity (Table 2). Methylation (13), or acetylation
(14) of the terminal piperazine nitrogen abolished activity, as did
replacement by oxygen (15) or methylene (16). The only analogue
showing measurable but weak activity was the trimethyl ethylene-
diamine (17). Replacement of the methylene linker between the
phenyl ring and the piperazine with a carbonyl group (18), also
abolished activity, suggesting that both of the piperazine nitrogens
need to be basic.
Following these results, a series of more conservative modifica-
tions were made in this region of the molecule, using the side
chains of the most potent urea (3) as the starting point (Table 3).
The narrow SAR observed for the diversity array was confirmed.
Shortening the methylene linker to a bond (19) or lengthening to
ethylene (20) effectively abolished activity and replacement of
the proximal piperazine nitrogen with carbon (21) reduced activity
by ꢀ10-fold. However, addition of methyl groups flanking the ter-
minal piperazine nitrogen (22) led to a significant increase in
potency.
The solution phase piperazine analogue array, including com-
pounds (12)–(17), was prepared according to Scheme 2. Hence,
2-bromobenzaldehyde was reacted with 3-methoxypropylamine
under reductive amination conditions, and the resulting secondary
amine was acylated with phenyl–acetyl chloride to give amide
(29). Suzuki coupling of (29) with 4-formylbenzeneboronic acid
gave the biphenylaldehyde (30). Reductive amination using
sodium triacetoxyborohydride with 80 diverse amines followed
N
i)
ii)
O
O
N
Br
OH
POL
N
O
POL
POL
O
O
23
24
iii)
CHO
H
iv)
N
N
N
R2
N
O
N
O
POL
POL
O
O
26
25
v)
2'
3'
X
3
4
X
N
vi)
R3
N
4'
R3
N
N
R2
HN
N
O
R2
POL
O
28
3,2'-, 3,3'-, 3,4'-, 4,2'-, 4,3'-, 4,4'-isomers
27
X = CO, SO2, CONH
Scheme 1. Reagents and conditions: (i) Wang resin (1.7 mmol/g), (2-PyO)2C@O, Et3N, DCM; (ii) 3- or 4-bromobenzylpiperazine, DCM; (iii) 2-, 3- or 4-formylbenzeneboronic
acid, Pd(Ph3P)4, Na2CO3, 1,2-dimethoxyethane-water (9:1), 80 °C under argon; (iv) R2NH2, NaBH(OAc)3, AcOH, Na2SO4, 1,2-dichloroethane; (v) R3COCl, Et3N; R3SO2Cl, Et3N; or
R3NCO in DCM; (vi) 1:4 TFA/CH2Cl2.

6426
T. D. Heightman et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6423–6428
by solid phase cation exchange purification and HLPC gave the tar-
get biphenylmethylamines represented by (31). The amide (18)
was prepared by oxidation of aldehyde (30) followed by standard
amide coupling with N-Boc-piperazine and deprotection.
Synthesis of the phenylpiperazine analogue (19) is shown in
Scheme 3. Protection of 4-bromophenyl piperazine and subsequent
Suzuki coupling with 2-formylboronic acid yielded the aldehyde
(33) which was treated with (3-methoxy)phenethylamine under
reductive amination conditions to give amine (34). Reaction of
(34) with cyclohexyl isocyanate and final deprotection yielded tar-
get compound (19) (Scheme 3).
Synthesis of the ethylene-linked analogue (20) started from N-
Boc-piperazine, which was coupled with 4-bromophenylacetic
acid to give amide (35) (Scheme 4). Reduction with borane/THF
complex followed by basic work up to avoid hydrolysis of the
Boc group yielded compound (36), which was converted to (20)
following the same synthetic pathway described in Scheme 3.
Synthesis of piperidine analogue (21) was carried out accord-
ing to Scheme 5. Reductive amination of 2-bromobenzaldehyde
with 3-methoxyphenylethylamine followed by electrophilic trap-
ping with cyclohexylisocyanate gave the bromobenzyl urea (38).
Suzuki coupling with 4-formylbenzeneboronic acid gave the
biphenylaldehyde (39), which was reduced with sodium borohy-
dride, converted to the bromide with phosphorus tribromide,
and then to the triphenylphosphonium bromide Wittig precursor
(40). Deprotonation of (40) with butyllithium followed by reac-
tion with N-Boc-4-oxopiperidine, then hydrogenation of the
resulting alkene and acidic deprotection gave the target com-
pound (21).
Compound (22) was prepared in 67% yield from the biphenylal-
dehyde (39) (Scheme 5), via reductive amination with cis-2,6-
dimethylpiperazine using the same conditions as in the preparation
of the array compounds (31).
In summary, following the discovery of a series of biphenyl-
methyl piperazine analogues from a high-throughput screen, sub-
sequent optimisation has identified compound (22) as a potent and
selective motilin receptor agonist with prokinetic-like activity in a
native tissue assay. Compound (22)12 represents a lead which has
OMe
OMe
OMe
R2 R1
N
O
O
N
O
O
N
H
O
N
H
i), ii)
iii)
iv)
Br
Br
Ph
Ph
Ph
29
30
31
v), vi), vii)
H
N
OMe
N
N
O
O
Ph
18
Scheme 2. Reagents and conditions: (i) 3-methoxypropyl-1-amine, NaBH(OAc)3, THF, rt, 37%; (ii) phenyl–acetyl chloride, Et3N, DCM, 0 °C, 64%; (iii) 4-formylbenzeneboronic
acid, Pd(PPh3)4, Na2CO3, DME, H2O, reflux, 82%; (iv) R1R2NH, NaBH(OAc)3, DCM, rt, 5–75%; (v) H2NSO3H, NaClO2, THF, H2O, rt, 54%; (vi) 1-Boc-piperazine, EDCI.HCl, HOBt, Et3N,
DCM, rt; (vii) TFA, DCM, rt, 59% (2 steps).
Boc
Boc
HN
N
N
i)
ii)
N
N
N
CHO
Br
Br
32
33
iii)
OMe
MeO
Boc
HN
N
iv), v)
N
N
O
N
NH
HN
19
34
Scheme 3. Reagents and conditions: (i) (Boc)2O, NaOH, t-BuOH/H2O (2:1), rt, quant.; (ii) 2-formyl-benzeneboronic acid, Pd(PPh3)4, 2M Na2CO3, DME, 80 °C, 65%; (iii)
3-MeOPh(CH2)2NH2, DCM, 4 Å mol. sieves, rt, 6 h then NaBH(OAc)3, rt, 42%; (iv) CyNCO, DCM, rt, quant.; (v) 2.0 M HCl in Et2O, DCM, rt, quant.

T. D. Heightman et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6423–6428
6427
Boc
Boc
N
N
HO
O
N
O
N
i)
ii)
Boc
N
+
NH
Br
Br
Br
35
36
iii)
OMe
Boc
N
HN
N
N
iv) - vi)
N
O
CHO
HN
20
37
Scheme 4. Reagents and conditions: (i) HOBt, NMM, EDCIÁHCl, DMF, rt, 98%; (ii) BH3.THF, THF, 50 °C then rt, 83%; (iii) 2-formyl-benzeneboronic acid, Pd(PPh3)4, 2M Na2CO3,
DME, 80 °C, 74%; (iv) 3-MeOPh(CH2)2NH2, DCM, 4 Å mol. sieves, rt, 6 h then NaBH(OAc)3, rt, 90%; (v) CyNCO, DCM, rt, 60%; (vi) 2.0 M HCl in Et2O, DCM, rt, quant.
OMe
OMe
O
N
O
H
N
O
CHO
i), ii)
HN
iii)
OMe
H2N
Br
HN
Br
+
38
39
iv) - vi)
OMe
OMe
H
N
Ph3P+Br-
vii), viii), ix)
N
O
N
O
HN
HN
21
40
Scheme 5. Reagents and conditions: (i) 2-BrPhCHO, MeOH, rt, 16 h, then NaBH4, 1 h, rt, 85%; (ii) CyNCO, THF, rt, quant. (crude); (iii) 4-formylbenzeneboronic acid, Pd(PPh3)4,
3 M Na2CO3, DME, 80 °C, 86%; (iv) NaBH4, EtOH, 0 °C, quant. (crude); (v) PBr3, DCM, 0 °C–rt, 98%; (vi) PPh3, toluene, reflux, 90%; (vii) nBuLi, THF, 0 °C–rt, then 4-oxo-N-Boc-
piperidine, rt, 77%; (viii) H2/Pd/C, MeOH, 20 psi, rt, 96%; (ix) 2.0 M HCl in Et2O, DCM, rt, 88%.
5. (a) Russo, A.; Stevens, J. E.; Giles, N.; Krause, G.; O’Donovan, D. G.; Horowitz, M.;
the potential for further optimization to provide orally active small
molecule motilin receptor agonists for the treatment of gastric
Jones, K. L. Aliment Pharmacol. Ther. 2004, 203, 333; (b) Talley, N. J.; Verlinden,
M.; Snape, W.; Beker, J. A.; Ducrotte, P.; Dettmer, A.; Brinkhoff, H.; Eaker, E.;
motility disorders such as gastroparesis, functional dyspepsia,
post-operative ileus, and others. Further development of struc-
ture–activity relationships and DMPK properties of the series are
described in the succeeding paper.13
Ohning, G.; Miner, P. B.; Mathias, J. R.; Fumagalli, I.; Staessen, D.; Mack, R. J.
Aliment Pharmacol. Ther. 2000, 1412, 1653; (c) Talley, N. J.; Verlinden, M.;
Geenen, D. J.; Hogan, R. B.; Riff, D.; McCallum, R. W.; Mack, R. J. Gut 2001, 49,
395; (d) Peeters, T. L. Neurogastroenterol. Motil. 2006, 18, 1; (e) Poitras, P.;
Peeters, T. L. Curr. Opin. Endocrinol. Diabetes Obes. 2008, 15, 54; (f) Sanger, G. J.;
Alpers, D. H. Neurogastroenterol. Motil. 2008, 30, 177.
6. Mccallum, R. W.; Cynshi, O. Aliment Pharmacol. Ther. 2007, 26(1), 107; See
company websites: www.roche.com and www.pfizer.com.
References and notes
7. Li, J. J.; Chao, H.-G.; Wang, H.; Tino, J. A.; Lawrence, R. M.; Ewing, W. R.; Ma, Z.;
Yan, M.; Slusarchyk, D.; Seethala, R.; Sun, H.; Li, D.; Burford, N. T.; Stoffel, R. H.;
Salyan, M. E.; Li, C. Y.; Witkus, M.; Zhao, N.; Rich, A.; Gordon, D. A. J. Med. Chem.
2004, 477, 1704.
8. High-throughput screening was carried out using recombinant human motilin
receptor stably expressed in a HEK293 cell line. Subsequent SAR analysis was
carried out using recombinant human motilin receptor stably expressed in a
CHO cell line. Assay conditions for the latter are as follows. Briefly, cells were
seeded onto 384-well black-walled, clear bottom microtitre plates (Greiner)
(10,000 cells/well) and incubated for 24 h. On the day of assay, media was
1. Christofides, N. D.; Modlin, I. M.; Fitzpatrick, M. L.; Bloom, S. R. Gastroenterology
1979, 765, 903; Peeters, T. L. Old Herborn University Seminar Monograph 1997, 9,
77.
2. Dhir, R.; Richter, J. E. J. Clin. Gastroenterol. 2004, 383, 237.
3. Carreras, C. W.; Siani, M. A.; Santi, D. V.; Dillon, S. B. Analyt. Biochem. 2002,
3002, 146.
4. (a) Sann, H. In Functional Disorders of the Gastrointestinal Tract; IOS Press: G.
Krause, 2005; (b) Verhagen, M. A. M. T.; Samson, M.; Maes, B.; Geypens, B. J.;
Ghoos, Y. F.; Smout, A. J. P. M. Aliment Pharmacol. Ther. 1997, 11, 1077; (c) Koga,
H.; Takanashi, H.; Itoh, Z.; Omura, S. Drugs Fut. 2002, 273, 255.
aspirated from cell plates using a cell washer (leaving 10
ll of media). Cells

6428
T. D. Heightman et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6423–6428
were immediately loaded with loading buffer (Tyrodes (Elga water +145 mM
NaCl +5 mM KCl +20 mM HEPES +10 mM glucose +1 mM MgCl2) +1.5 mM CaCl2
+0.714 mg/mL Probenicid (predissolved in 1 M NaOH) +0.5 mM brilliant black
receptor: other analogues in the series with no activity at the ghrelin
receptor showed comparable or greater activity in the antrum assay
(unpublished data); and ghrelin itself does not show enhancement of EFS
mediated contractions in rabbit gastric antrum, see Dass, N. B.; Hill, J.; Muir,
A.; Testa, T.; Wise, A.; Sanger, G. J. Brit. J. Pharmacol. 2003, 140, 948;
Depoortere, I.; Thijs, T.; Theelemans, L.; Robberecht, P.; Peters, T. L. J.
Pharmacol. Exp. Ther. 2003, 305, 660.
+2.5
were prepared in 100% DMSO. A top concentration of 3 mM was used (giving
12 M final concentration in assay) and this was serially diluted 1 in 4. One
microliter from the master plate was transferred to a daughter plate, to which
50 l of compound dilution buffer (Tyrodes + 1 mg/mL BSA + 1.5 mM CaCl2)
was added. 10 l from the compound plates was then added immediately to
cell plates using FLIPR calcium imaging instrument and changes in
lM Fluo 4 dye, and incubated at 37.5 C for 1 h. Master compound plates
l
l
11. Jarvie, E. M.; North Laidler, V. J.; Corcoran, S.; Bassil, A.; Sanger, G. J. Brit. J.
Pharmacol. 2007, 150, 455.
l
a
3
12. Characterising data for (22): 1H NMR (400 MHz, CDCl3) d (ppm): 7.29–7.38 (5H,
m), 7.23 (1H, m), 7.21 (2H, d, J 8.0 Hz), 7.15 (1H, t, J 7.8 Hz), 6.73 (1H, m), 6.66
(1H, d, J 7.5 Hz), 6.62 (1H, m), 4.35 (2H, s), 3.86 (1H, d, J 7.7 Hz), 3.76 (3H, s),
3.56 (2H, s), 3.52 (1H, m), 3.35 (2H, m), 3.03 (2H, m), 2.83 (2H, m), 2.65 (2H, m),
1.76 (4H, m), 1.56 (3H, m), 1.28 (2H, m), 1.10 (6H, d, J 6.4 Hz), 1.07 (1H, m), 1.86
(2H, m). MS: (ES+) 569 (MH+).
fluorescence were measured over a 1 min timeframe. Maximum change in
fluorescence over baseline was used to determine agonist response and
concentration-response curves were constructed, using a 4-parameter logistic
equation. The intrinsic activity of target compounds was calculated by using
the maximum asymptote of its concentration-response curve relative to the
maximum asymptote of the motilin concentration-response curve.
9. Corbett, D. F.; Heightman, T. D.; Moss, S. F.; Bromidge, S. M.; Coggon, S. A.;
Longley, M. J.; Roa, A. M.; Williams, J. A.; Thomas, D. R. Bioorg. Med. Chem. Lett.
2005, 1518, 4014.
13. Westaway, S. M.; Brown, S. L.; Conway, E.; Heightman, T. D.; Johnson, C. N.;
Lapsley, K.; Macdonald, G. J.; MacPherson, D. T.; Mitchell, D. J.; Myatt, J. W.;
Seal, J. T.; Stanway, S. J.; Stemp, G.; Thompson, M.; Colombo, A.; Consonni, A.;
Gagliardi, S.; Roccaboni, M.; Ronzoni, S.; Briggs, M. A.; Matthews, K. L.; Stevens,
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10. It is unlikely that the activity observed for Compound 22 in the rabbit
gastric antrum assay is partly or wholly due to action on the ghrelin