Discovery of a Potent and Novel Motilin Agonist

Article abstract of DOI:10.1021/jm0304865

A novel series of dihydro- and tetrahydrotriazolopyridazine-1, 3-dione-based amino acid derivatives were identified as very potent motilin receptor agonists. Incorporating one additional phenylethyl glycinamide subunit to 1 (EC₅₀ = 660 nM) was

Full text of DOI:10.1021/jm0304865

1704
J . Med. Chem. 2004, 47, 1704-1708
Discover y of a P oten t a n d Novel Motilin Agon ist
J ames J . Li,*,† Hann-Guang Chao,† Haixia Wang,† J oseph A. Tino,† R. Michael Lawrence,† William R. Ewing,†
Zhengping Ma,‡ Mujing Yan,‡ Dorothy Slusarchyk,‡ Ramakrishna Seethala,‡ Huabin Sun,‡ Danshi Li,‡
Neil T. Burford,| Robert H. Stoffel,| Mary Ellen Salyan,§ Cindy Y. Li,§ Michael Witkus,§ Ning Zhao,§
Adam Rich,*,‡ and David A. Gordon*,‡
Discovery Chemistry, Metabolic Disease Research, and Discovery Analytical Science, Bristol-Myers Squibb Pharmaceutical
Research Institute, P.O. Box 5400, Princeton, New J ersey 08643-5400, and Lead Discovery, Bristol-Myers Squibb
Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, Connecticut 06492
Received October 1, 2003
A novel series of dihydro- and tetrahydrotriazolopyridazine-1,3-dione-based amino acid
derivatives were identified as very potent motilin receptor agonists. Incorporating one additional
phenylethyl glycinamide subunit to 1 (EC50 ) 660 nM) was found to improve in vitro potency
approximately 3000-fold, resulting in compound 10 (EC50 ) 0.22 nM). The more potent
enantiomer 11A has an EC50 of 0.047 nM in the motilin receptor functional assay and a Ki of
0.7 nM in the binding assay. In addition, compound 11A was shown to have a significantly
reduced tendency to cause receptor desensitization as compared with the motilin receptor
agonist ABT-229.
In tr od u ction
The motilin receptor is a G-protein-coupled receptor
(GPCR) expressed on smooth muscle and enteric neu-
rons in the gastrointestinal (GI) tract.1 The endogenous
ligand, motilin, is a 22 amino acid peptide that is
secreted by enterochromaffin cells in the small intestine
and is involved in the normal regulation of coordinated
motility of the GI tract.2 It has been shown that
intravenous infusion of motilin peptide stimulates con-
tractile activity of GI smooth muscle and accelerates
gastric emptying.3-6 Therefore, a motilin agonist could
be utilized therapeutically for the treatment of GI
F igu r e 1. Motilin agonist ABT-229 and HTS lead 1.
hypomotility disorders such as diabetic gastroparesis
and constipation type irritable bowel syndrome.7-8
Although erythromycin is primarily used as an anti-
biotic, it can also be used clinically to stimulate GI
motility.9 The prokinetic effects of erythromycin are
believed to be mediated by stimulation of the motilin
receptor.10 Long-term use of erythromycin, however, is
limited due to its antibiotic activity. Efforts to find
erythromycin analogues with functional activity at the
motilin receptor and without antibiotic activity have led
to the discovery of ABT-229.11,12 ABT-229, however,
failed in several clinical trials due to lack of long-term
efficacy.13,14 Clinical failure might be attributed to
tachyphylaxis,15 i.e., a diminished response upon re-
peated treatment. Preliminary studies in our laboratory
using cellular expression of the cloned human motilin
receptor revealed that ABT-229 caused a profound
tachyphylaxis after the first treatment, and a long
washout period was needed for the receptor to recover.
The motilin peptide desensitized the receptor to a much
lesser extent, and receptor recovery was faster than that
observed for ABT-229. We hypothesize that compounds
with a similar agonism profile to that of the natural
ligand motilin would be less likely to induce tachyphy-
laxis and thus offer a more desirable profile as a motilin
agonist.
Lea d Id en tifica tion a n d Ch em istr y. A number of
potent motilin agonists reported in the literature are
erythromycin derivatives16 as exemplified by ABT-229
(EC50 ) 4.2 nM; Ki ) 3.4 nM).17 Because of the
tachyphylaxis displayed by ABT-229 and the potential
for other potent erythromycin analogues to share this
property, we decided to search for a nonmacrolide lead
that might offer a different profile with respect to
motilin receptor desensitization. A high throughput
screen (HTS) of the BMS compound collection identified
a novel (L)-phenylethyl glycinamide derivative 1 (Figure
1) as a modest motilin receptor agonist with an EC50 of
660 nM.
The lead compound 1 was originally prepared in a
chemical library and was obtained as a mixture of two
diastereomers with 90% purity.18 As part of our HTS
validation process, compound 1 was resynthesized as
shown in Scheme 1. (L)-Phenylethyl glycinamide 3 was
prepared by converting N-Boc-(L)-phenylethylglycine 2
to corresponding N-Boc-(L)-phenylethylglycine carboxa-
mide, followed by removal of the Boc group with
hydrogen chloride in a mixture of dioxane and methanol.
* Corresponding authors: (J ames Li) tel 609-818-7124, fax 609-818-
6810, e-mail james.li@bms.com; (David Gordon) tel 609-818-4882, fax
609-818-7877, e-mail david.gordon@bms.com; (Adam Rich) tel 585-395-
5740, e-mail: Arich@brockport.edu, current address: Department of
Biological Sciences, SUNY at Brockport, Brockport, NY 14420.
† Discovery Chemistry.
‡ Metabolic Disease Research.
§ Discovery Analytical Science.
| Lead Discovery.
10.1021/jm0304865 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/02/2004

Potent and Novel Motilin Agonist
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1705
Sch em e 1a
a
Reagents and conditions: (a) EDAC, HOAT, NH4OH; (b) ∼ 3 N HCl in dioxane/methanol; (c) saturated HCl in methanol; (d) EDAC,
HOAT, 3; (e) ∼15% TFA in CH2Cl2; (f) PhI(CO2CF3)2; (g) 1 N NaOH; (h) EDAC, HOAT, 3 and/or 4; (i) EDAC, HOAT, 5; (j) H2/Pd-C; (k)
TMSiCHN2; (l) chiral separation.
quently used to react with carboxylic acid 8, resulting
in the formation of the corresponding amides 1A and
1B, along with methyl esters 9A and 9B as side
products (∼7% yield). Interestingly, the second eluting
methyl ester 9B was found to be a potent, functionally
active motilin receptor agonist, with an EC50 of 7 nM.
Additional amounts of compound 9B was therefore
needed for further evaluation. Pure amino methyl ester
4 was prepared directly from N-Boc-phenethylglycine
2 by treatment with a saturated solution of hydrogen
chloride in methanol. Subsequent amide formation from
F igu r e 2. LC/MS/MS fragment pattern for 10.
amine 4 and acid 8, followed by Boc deprotection and
separation, provided compound 9B. To our surprise, the
second batch of 9B was inactive in our in vitro motilin
assay when tested at concentrations up to 10 µM. A
detailed structural analyses between the two batches
of 9B was carried out to determine if there were any
differences. Comparison of 1H, 13C and various 2D NMR
spectra (COSY, HMQC, and HMBC) as well as HPLC
coinjections confirmed that these two lots had the same
Two key intermediates 6 and 7 were prepared according
to a modification of literature procedures.19,20 The spiro
bicyclic dihydrotriazolopyridazine-1,3-dione scaffold was
then constructed via a [4 + 2] cycloaddition reaction
between 6 and 7 followed by basic hydrolysis of the ester
to provide the late-stage intermediate acid 8.21,22 Amino
acid coupling between acid 8 and amine 3, followed by
Boc deprotection, gave compound 1 as a mixture of two
diastereomers. The two isomers were subsequently
major component, compound 9B, in approximately 95%
separated to 1A and 1B.23 Motilin activity resulted
primarily from the second eluting isomer 1B (Table 1).
Because methanol was used as a cosolvent in the Boc
removal reaction during the preparation of glycinamide
3 (step b in Scheme 1), a small amount (∼8% by HPLC)
of phenylethylglycine methyl ester 4 was formed. The
mixtures of amide 3 and methyl ester 4 were subse-
purity. Chiral HPLC also showed that both batches of
9B were the same enantiomer. Further HPLC analysis
revealed that even though both lots had a similar
impurity profile, the first lot of methyl ester 9B con-
tained a small impurity (0.2% by HPLC) that was not
observed in the second inactive lot. Extensive LC/MS/
MS analysis indicated that the fragmentation pattern

1706 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Ta ble 1. Summary of in Vitro Potency
Li et al.
F igu r e 3. Measurement of receptor desensitization in FLIPR
Assay.
they were hydrolyzed to corresponding acids 12A and
12B and coupled with carboxamide 5 to yield the
dipeptide analogues 11A and 11B, both as single
isomers (Scheme 1).
Resu lts a n d Discu ssion
In Vitr o SAR Stu d ies. Initial SAR studies were
directed toward simple amino acid analogues of HTS
lead 1 (Table 1). To probe the preferred stereochemistry
with respect to the amino acid side chain, the corre-
sponding (D)-phenylethyl analogues were prepared and
separated as 13A and 13B, but both isomers led to a
substantial loss of functional activity. Two amino acid
derivatives with smaller side chains, (L)-phenylalanine
in 14 and (L)-alanine in 15, were also prepared to probe
the optimal size. Similarly this modification resulted in
a loss of motilin activity. To our surprise, approximately
3000-fold improvement of in vitro potency (10 vs 1) was
obtained by adding one additional phenylethyl glycina-
mide moiety to 1. The corresponding reduced analogue
of dipeptide 10, tetrahydrotriazolopyridazine-1,3-dione
11, was also evaluated and showed a comparable in vitro
potency (EC50 of 0.35 vs 0.22 nM). The more potent
isomer 11A gave an EC50 of 0.047 nM in the motilin
functional assay and was 3- and 80-fold more potent
than natural ligand motilin and macrolide analogue
ABT-229 (Table 1). To the best of our knowledge, 11A
is the most potent motilin agonist reported in the
literature.
Ta ch yp h yla xis. Tachyphylaxis, or receptor desen-
sitization, is a common phenomenon with G-protein-
coupled receptors when treated with an agonist.25 We
suspect the lack of long-term efficacy observed for ABT-
229 in humans may be due to motilin receptor desen-
sitization after multiple treatments. Therefore, finding
a compound with a lower tendency to cause tachyphy-
laxis was identified as a key issue. Toward that end, a
functional-based fluorescence imaging plate reader
(FLIPR) assay was developed to measure the receptor
desensitization in a HeLa cell line stably expressing the
human motilin receptor. In this assay, the receptor cells
were first treated with compounds at a concentration
of 1- to 50-fold of their EC50, followed by a washout and
recovery period. The maximum functional response for
these treated cells was then measured after a challenge
using a high dose (i.e., 100-fold EC50) of the testing
compound. Treatment with the motilin peptide or
erythromycin even at a relatively high concentration
(50-fold EC50) showed a complete recovery of receptor
to the second stimulation as shown in Figure 3. ABT-
a
Compounds 1, 10, and 11 are diastereomeric mixtures; all
others are single enantiomers.
for the impurity was consistent with the dipeptide
derivative 10, with a molecular weight of 721 and a loss
of 44 mass units for the terminal carboxamide group
and 177 for the phenylethylglycinamide moiety (Figure
2). The proposed dipeptide analogue 10 was then
synthesized by coupling acid 8 with the dipeptidyl
carboxamide 5, followed by removal of the Boc group
(Scheme 1). Compound 10 was subsequently confirmed
as a very potent motilin agonist with an EC50 of 0.22
nM. It is evident from the result that a small amount
of dipeptide 10 present in the first batch of 9B was
responsible for the motilin activity observed.
Reduction of the double bond in 10 provided tetrahy-
drotriazolopyridazine-1,3-dione analogue 11. Unfortu-
nately neither 10 nor 11, both as diastereomeric mix-
tures, could be separated under a variety of conditions
attempted. The problem was circumvented by repeating
the last amide formation with enantiomeric pure acid
12A and 12B. Reduction of 8 proceeded smoothly. The
resultant acid was subsequently converted to methyl
ester for ease of prep-scale chiral separation.24 After
both enantiomerically pure methyl esters were obtained,

Potent and Novel Motilin Agonist
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1707
measure the motilin receptor binding constant. To a 96-well
OptiPlate (Packard) were added 40 µL of 2X SPA binding
buffer (1X: 50 mM Tris-HCl, 5 mM MgCl2, 50 mM NaCl, 5
mM KCl, 1 mM PMSF, 2 mM CHAPS, and 0.1% BSA, pH 7.6),
10 µL of 1 mg/mL cell membranes (10 µg), 15 µL of 2 nM
229, on the other hand, gave 73% of recovery when the
cells were first treated at a concentration at its EC50.
The recovery was reduced to 33-36% when the first
dose was at a higher concentration of 5 or 10-fold EC50.
It desensitized the receptor further with only 17%
recovery after an initial concentration of 50-fold EC50,
essentially resulting in a profound down-regulation for
the motilin receptor. When 11A was tested at concen-
trations of 1- to 10-fold EC50, the receptor showed a
complete recovery after 5 h. Desensitization (67%
recovery) occurred only at the highest concentration
tested (50-fold EC50). These results suggested that ABT-
229 would cause a more profound tachyphylaxis whereas
compound 11A will act more like the endogenous ligand
motilin, therefore offering a more desirable profile as a
motilin agonist.
[
125I]-motilin (NEN NEX 378, 2200 Ci/mmol; or Amersham
Pharmacia IM337, 2000 Ci/mmol), 15 µL of 10X compound or
unlabeled 2 µM motilin, and 0.5 mg of PVT-WGA SPA beads
in 70 µL of 1X SPA binding buffer. Contents in 150 µL of total
volume were incubated at room temperature for 2 h. The plate
was counted in a TopCount (Packard) for 1 min/well to
determine radioactive ligand bound to the receptor.
Gen er a l Ch em istr y Meth od s. Proton NMR spectra were
recorded on a J EOL 400 or 500 MHz instrument. Analytical
HPLC was performed on a Shimadzu instrument using YMC
C18 5 micron 4.6 × 50 mm column with a 4-min gradient of
0-100% solvent A (90%MeOH/10%H2O/0.2% H3PO4) and
100-0% solvent B (10%MeOH/90%H2O/0.2% H3PO4) with a
1-min hold. LC-MS spectra were obtained on a Shimadzu
HPLC and Micromass Platform using electrospray ionization.
HRMS were obtained on a Micromass LCT in lockspray with
electrospray ionization. The preparative HPLC was performed
on an automated Shimadzu system using the YMC ODS C18
5 µm preparative columns with mixtures of solvent C (10%
MeOH/90%H2O/0.2% TFA) and solvent D (90% MeOH/10%
H2O/ 0.2% TFA) or mixtures of solvent E (10% CH3CN/
90%H2O/0.2% TFA) and solvent F (90% CH3CN/10% H2O/ 0.2%
TFA). Preparation of intermediate compounds 3-8 and 12A
are described in the Supporting Information. All other reagents
and solvent were obtained from commercial sources and were
used without further purification.
Com p ou n d s 1A, 1B, 9A, a n d 9B. To a stirred solution of
compound 8 (140 mg, 0.28 mmol) in 2 mL of dichloromethane
were added EDAC (107 mg, 0.56 mmol), HOAT (76 mg, 0.56
mmol), Et3N (78 µL, 0.56 mmol), and mixtures of 3 and 4 (120
mg), and the reaction was stirred at RT overnight. The
mixtures were diluted with EtOAc, washed with water (2×),
1 N NaOH (2×), and brine, dried over Na2SO4, and concen-
trated. The Boc-intermediate was deprotected with 3 mL of
15% TFA/CH2Cl2 solution at RT for 3 h. The final compounds
were separated on an automated Shimadzu HPLC system to
give compounds 1A, 1B, 9A, and 9B, all as TFA salts. The
first eluting isomer was assigned as A, and the second eluting
isomer was assigned as B.
Compound 1A (38 mg), HPLC: 98% purity; LCMS m/z 561
(M + H); HRMS calcd for (M + H) 561.2462, found 561.2466;
1H NMR (MeOH-d4) δ 1.87-2.04 (m, 3H), 2.09-2.19 (m, 1H),
2.49 (dt, J ) 5, 14 Hz, 1H), 2.55-2.71 (m, 2H), 3.21 (dt, J ) 3,
14 Hz, 1H), 3.29-3.52 (m, 4H), 4.26-4.33 (m, 1H), 4.61 (dd, J
) 15, 39 Hz, 2H), 5.12 (dd, J ) 2, 5 Hz, 1H), 5.90 (s, 2H), 6.24
(dd, J ) 5, 11 Hz, 1H), 6.61 (dd, J ) 2, 10 Hz, 1H), 6.73 (d, J
) 8 Hz, 1H), 6.84 (dd, J ) 2, 8 Hz, 1H), 6.88 (d, J ) 1 Hz, 1H),
7.12-7.27 (m, 5H), 8.58 (d, J ) 8 Hz, 1H).
Compound 1B (34 mg), HPLC: 98% purity; LCMS m/z 561
(M + H); HRMS calcd for (M + H) 561.2462, found 561.2465;
1H NMR (MeOH-d4) δ 1.90-2.12 (m, 4H), 2.47 (dt, J ) 5, 14
Hz, 1H), 2.58-2.77 (m, 2H), 3.16-3.52 (m, 5H), 4.32-4.39 (m,
1H), 4.63 (dd, J ) 15, 39 Hz, 2H), 5.13 (dd, J ) 2, 5 Hz, 1H),
5.90 (s, 2H), 6.25 (dd, J ) 5, 11 Hz, 1H), 6.57 (dd, J ) 2, 10
Hz, 1H), 6.71 (d, J ) 8 Hz, 1H), 6.85 (dd, J ) 2, 8 Hz, 1H),
6.89 (d, J ) 1 Hz, 1H), 7.12-7.26 (m, 5H), 8.56 (d, J ) 8 Hz,
1H).
Con clu sion
We have discovered a novel series of dihydro- and
tetrahydrotriazolopyridazine-1,3-dione-based amino acid
derivatives as potent motilin agonists. Addition of one
phenylethyl glycinamide moiety to 1 was found to
markedly improve the in vitro potency 3000-fold (i.e.,
10 vs 1). The most potent motilin agonist 11A has an
EC50 of 0.047 nM in the functional assay and a Ki of 0.7
nM in the binding assay. In addition, 11A was shown
to have a lower tendency to cause tachyphylaxis of the
motilin receptor as compared with ABT-229, thus
exhibiting a potentially more desirable profile as a
motilin receptor agonist. Motilin agonists with a profile
similar to that of 11A may be able to maintain long-
term efficacy for the treatment of GI hypomotility
disorders.
Exp er im en ta l Section
Motilin Recep tor F u n ction a l Assa y (EC50). HeLa-MR9
cell line stably expressing the human motilin receptor was
used. The cells were grown in T-150 flask in Dulbecco’s
modified eagle medium (Gibco, #12320-032) containing 10%
fetal bovine serum (Gibco, #16140-071), 1X MEM nonessential
amino acids (Gibco, #11140-050), and 3 mg/mL G418 (Gibco,
#10131-035). Hela-MR9 cells were plated into a black/clear
flat bottom 96-well plate (BD Falcon #353948) at a density of
20 000 per well, and cultured for 20-24 h at 37 °C with 5%
CO2 to make them about 90% to 95% confluent. Compounds
were diluted to 5× of final concentration with assay buffer
(Hanks balanced salt solution without Ca2+ and Mg2+, 10 mM
HEPES, pH 7.5, 0.1% BSA), and the final DMSO concentration
was kept below 0.5%. When cells were confluent, the medium
was aspirated from the cells. To each well was added 100 µL
of dye solution made in the assay buffer, consisting of 5 µg/
mL fluo-3 (Molecular Probes, #F-1242), 2.5 mM probenecid
(Sigma, #P-8761), and 0.5% pluronic acid (Molecular Probe
#P-3000) and incubated at 37 °C. After incubation for 60 min,
the dye solution was aspirated, and 120 µL of assay buffer was
added to each well. The cell and compound plates were loaded
in a fluorescence imaging plate reader (FLIPR), and 30 µL of
compound was added to the cell plate. The intracellular Ca2+
signal was measured at a 1 s interval for first 90 s and 3 s
interval for the next 3 min in the FLIPR.
Compound 9A (15 mg), HPLC: 95% purity; LCMS m/z 576
(M + H); HRMS calcd for (M + H) 576.2458, found 576.2460;
1H NMR (MeOH-d4) δ 1.90-2. 05 (m, 3H), 2.06-2.18 (m, 1H),
2.42 (dt, J ) 5, 14 Hz, 1H), 2.54-2.72 (m, 2H), 3.20 (dt, J ) 3,
14 Hz, 1H), 3.33-3.52 (m, 4H), 3.68 (s, 3H), 4.28-4.36 (m, 1H),
4.61 (dd, J ) 15, 42 Hz, 2H), 5.12 (dd, J ) 2, 5 Hz, 1H), 5.90
(s, 2H), 6.25 (dd, J ) 5, 11 Hz, 1H), 6.60 (dd, J ) 2, 11 Hz,
1H), 6.73 (d, J ) 8 Hz, 1H), 6.85 (dd, J ) 1, 8 Hz, 1H), 6.88 (d,
J ) 1 Hz, 1H), 7.13-7.27 (m, 6H), 8.66 (d, J ) 8 Hz, 1H).
Mea su r em en t of Ta ch yp h yla xis for Motilin Recep tor .
Effect of compounds on HeLa-MR9 cell FLIPR functional
response was used to measure the receptor desensitization
(tachyphylaxis). The HeLa-MR9 cells were first treated with
compounds at a concentration of 1- to 50-fold of their EC50
followed by a 5-h washout and recovery period. The cells were
stimulated with second addition of the compound at maximum
effective dose (100× EC50) and Ca2+ signal was then measured
in FLIPR.
Compound 9B (15 mg), HPLC: 95% purity; LCMS m/z 576
(M + H); HRMS calcd for (M + H) 576.2458, found 576.2482;
1H NMR (MeOH-d4) δ 1.90-2.20 (m, 4H), 2.44 (dt, J ) 5, 14
Motilin Bin d in g Assa y (Ki). A homogeneous scintillation
proximity assay (SPA) in HeLa-MR9 cell line was used to

1708 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Li et al.
(5) Peeters, T. L.; Muls, E.; J anssens, J .; Urbain, J . L.; Bex, M.;
Van Cutsem, E.; Depoortere, I.; DeRoo, M.; Vantrappen, G.;
Bouillon, R. Effect of Motilin on Gastric Emptying in Patients
with Diabetic Gastroparesis. Gastroenterology 1992, 102, 97-101.
(6) Schmid, R.; Schusdziarra, V.; Allescher, H. D.; Bofilias, I.;
Buttermann, G.; Classen, M. Effect of Motilin on Gastric
Emptying in Patients with Diabetic Gastroparesis. Diabetes Care
1991, 14, 65-68.
Hz, 1H), 2.58-2.76 (m, 2H), 3.21 (dt, J ) 3, 14 Hz, 1H),
3.34-3.52 (m, 4H), 3.67 (s, 3H), 4.33-4.40 (m, 1H), 4.63 (dd,
J ) 15, 42 Hz, 2H), 5.13 (dd, J ) 2, 5 Hz, 1H), 5.90 (s, 2H),
6.25 (dd, J ) 5, 11 Hz, 1H), 6.60 (dd, J ) 2, 11 Hz, 1H), 6.72
(d, J ) 8 Hz, 1H), 6.85 (dd, J ) 1, 8 Hz, 1H), 6.88 (d, J ) 1 Hz,
1H), 7.12-7.28 (m, 6H), 8.80 (d, J ) 8 Hz, 1H).
Com p ou n d 10. To a stirred solution of compound 8 (50 mg,
0.1 mmol) in 0.8 mL of DMF were added EDAC (19.2 mg, 0.1
mmol), HOAT (14 mg, 0.1 mmol), DIEA (18 µL, 0.1 mmol),
and 5 (34 mg, 0.09 mmol), and the mixtures were stirred at
RT overnight. The reaction was diluted with EtOAc, washed
with water (2×), 1 N NaOH (2×), and brine, dried over Na2SO4
and concentrated. The intermediate was purified on HPLC,
and then the Boc group was deprotected with 1 mL of 50%
TFA/CH2Cl2 solution at RT for 30 min. It was concentrated to
give 31 mg (40%) of compound 10 as foam solid. HPLC: 97%
purity; LCMS m/z 722 (M + H); HRMS calcd for (M + H)
722.3302, found 722.3328; 1H NMR (MeOH-d4) δ 1.85-2.20
(m, 6H), 2.37-2.80 (m, 5H), 3.15-3.50 (m, 5H), 4.25-4.70 (m,
4H), 5.10-5.16 (m, 1H), 5.83 and 5.85 (s, total 2H), 6.20-6.28
(m, 1H), 6.48-6.90 (m, 4H), 7.10-7.26 (m, 10H), 8.12 and 8.23
(d, J ) 7 Hz, total 1H), 8.67 and 8.81 (d, J ) 7 Hz, total 1H).
Com p ou n d 11A. To a stirred solution of 12A (80 mg, 0.2
mmol) in 2 mL of DMF were added PyAOP (115 mg, 0.22
mmol), 5 (97 mg, 0.22 mmol), and DIEA (80 µL, 0.44 mmol) at
RT, and the reaction was stirred at RT overnight. The reaction
was diluted with EtOAc, washed with water (2×), dried over
Na2SO4, and concentrated. The Boc intermediate was depro-
tected with 1 mL of 30% TFA in dichloromethane (v/v) at RT
for 2 h. It was purified on HPLC to give a TFA salt which was
washed with NaHCO3 solution to give 87 mg (60%) of com-
pound 11A as a white powder. HPLC: 95% purity; LCMS m/z
724 (M + H); HRMS calcd for (M + H) 724.3459, found
(7) Haramura, M.; Tsuzuki, K.; Okamachi, A. Yogo, K. Ikuta, M.;
Kozono, T.; Takanashi, H.; Murayama, E. Design and Synthesis
of Novel Tetra-Peptide Motilin Agonists Bioorg. Med. Chem. Lett.
2002, 10, 1805-1811.
(8) Itoh, Z. Motilin and Clinical Application. Peptides 1997, 18, 593-
608.
(9) J anssens, J .; Peeters, T. L.; Vantrappen, G.; Tack, J .; Urbain,
J . L.; DeRoo, M.; Muls, E.; Bouillon, R. Improvement of Gastric
Emptying in Diabetic Gastroparesis by Erythromycin. N. Engl.
J . Med. 1990, 322, 1028-1031.
(10) Peeters, T.; Matthijs, G.; Depoortere, I.; Cachet, T.; Hoogmartens,
J .; Vantrappen, G. Erythromycin is A Motilin Agonist. Am. J .
Physiol. Gastrointest Liver Physiol. 1989, 257, G470-G474.
(11) Lartey, P. A.; Nellans, H. N.; Faghih, R.; et al. Synthesis of 4′-
Deoxy Motilides: Identification of a Potent and Orally Active
Prokinetic Drug Candidate. J . Med. Chem. 1995, 38, 1793-1798.
(12) Faghih, R.; Nellans, H. N.; Plattner, J . J . Motilides and Moti-
lactides: Design and Development of Motilin Receptor Agonists
As A New Class of Gastrointestinal Prokinetic Drugs. Drugs
Future 1998, 23, 861-872.
(13) Talley, N. J .; Verlinden, M.; Snape, W.; Beker, J . A.; Ducrotte,
P.; Dettmer, A.; Brinkhoff, H.; Eaker, E.; Ohning, G.; Miner, P.
B.; Mathias, J . R.; Fumagalli, I.; Staessen, D.; Mack, R. J . Failure
of a Motilin Receptor Agonist (ABT-229) To Relieve the Symp-
toms of Functional Dyspepsia in Patients With and Without
Delayed Gastric Emptying: A Randomized Double-blind Placebo-
controlled Trial. Aliment. Pharmacol. Ther. 2000, 14, 1653-1661.
(14) Netzer, P.; Schmitt, B.; Inauen, W. Effects of ABT-229, A Motilin
Agonist, on Acid Reflux, Oesophageal Motility and Gastric
Emptying in Patients with Gastro-oesophageal Reflux Disease.
Aliment. Pharmacol. Ther. 2002, 16, 1481-1490.
724.3466; 1H NMR (MeOH-d4)
δ 1.75-2.33 (m, 12H),
2.55-2.80 (m, 4H), 3.00-3.24 (m, 3H), 3.26-3.30 (m, 1H),
4.31-4.40 (m, 2H), 4.57 (dd, J ) 15, 49 Hz, 2H), 4.75 (t, J )
6 Hz, 1H), 5.85 and 5.86 (s, total 2H), 6.70 (d, J ) 8 Hz, 1H),
6.84 (d, J ) 8 Hz, 1H), 6.88 (s, 1H), 7.10-7.30 (m, 10H),
8.27 (d, J ) 7 Hz, 1H), 8.43 (d, J ) 7 Hz, 1H). Anal.
(C39H45N7O7‚0.39H2O) C, H, N.
(15) Tack, J .; Peeters, T. What Comes After Macrolides and Other
Motilin Stimulants? Gut 2001, 49, 317-318.
(16) Koga, H.; Takanashi, H.; Itoh, Z.; Omura, S. Design, SAR and
Pharmacology of GM-611, the First Acid-stable Nonpeptide
Motilin Receptor Agonist. Drugs Future 2002, 27, 255-272.
(17) A research sample of ABT-229 was obtained from Abbott
Laboratories, so the in vitro data reported herein were obtained
in our laboratory. For detailed protocol of biological assays, see
Experimental Section and Supporting Information.
Ack n ow led gm en t. The authors would like to thank
Abbott Laboratories for a research sample of ABT-229.
Su p p or tin g In for m a tion Ava ila ble: Preparation of in-
termediate compounds 3-8 and 12A as well as detailed
biological assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
(18) Compound 1 was obtained from Molecumetics Ltd.
(19) Cookson, R. C.; Gupte, S. S.; Stevens, I. D. R.; Watts, C. T.
Organic Syntheses; Wiley: New York, 1988; Collect. Vol. VI, p
pp 936-939.
(20) Takacs, J . M.; J aber, M. R.; Clement, F.; Walters, C. J . Org.
Chem. 1998, 63, 6757-6760.
(21) Boldi, A. M.; J ohnson, C. R.; Eissa, H. O. Solid-Phase Library
Synthesis of Triazolopyridazine via [4+2] Cycloadditions. Tet-
rahedron Lett. 1999, 40, 619-622.
(22) Hansen, S. U.; Bols, M. Synthesis of Labeled 1-Azafagomine J .
Chem. Soc., Perkin Trans. 1 1999, 3323-3325.
(23) The first eluting isomer was assigned as A, and the second
eluting isomer was assigned as B.
(24) The racemic methyl ester was separated on a ChiralPak AS
column (5 × 50 cm) using 10% ethanol/90% hexanes as the
solvents. The first eluting enantiomer was assigned as A, and
the second eluting enantiomer was assigned as B.
(25) Ferguson, S. S. Evolving Concepts in G Protein-Coupled Receptor
Endocytosis: The Role in Receptor Desensitization and Signal-
ing. Pharmacol. Rev. 2001, 53, 1-24.
Refer en ces
(1) Feighner, S. D.; Tan, C. P.; McKee, K. K.; Palyha, O. C.; Hreniuk,
D. L.; Pong, S.-S.; Austin, C. P.; Figueroa, D.; MacNeil, D.;
Cascieri, M. A.; Nargund, R.; Bakshi, R.; Abramovitz, M.; Stocco,
R.; Kargman, S.; O′Neil, G.; Van Der Ploeg, L. H. T.; Evans, J .;
Patchett, A. A.; Smith, R. G.; Howard, A. D. Receptor for Motilin
Identified in the Human Gastrointestinal System. Science 1999,
284, 2184-2188.
(2) Brown, J . C.; Cook, M. A.; Dryburgh, J . R. Motilin, a Gastric
Motor Activity Stimulating Polypeptide: The Complete Amino
Acid Sequence. Can. J . Biochem. 1973, 51, 533-537.
(3) Vantrappen, G.; J anssens, J .; Peeters, T. L.; et al. Motilin and
the Interdigestive Migrating Motor Complex in Man. Dig. Dis.
Science 1979, 24, 497-500.
(4) Christofides, N. D.; Long, R. G.; Fitzpatrick, M. L.; McGregor,
G. P.; Bloom, S. R. Effect of Motilin on the Gastric Emptying of
Glucose and Fat in Humans. Gastroenterology 1981, 80, 456-460.
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