Discovery of N-(3-fluorophenyl)-1-[(4-([(35)-3-methyl-1-piperazinyl]methyl) phenyl)acetyl]-4-piperidinamine (GSK962040), the first small molecule motilin receptor agonist clinical candidate

Article abstract of DOI:10.1021/jm801332q

N-(3-Fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]methyl)phenyl) acetyl]-4-piperidinamine 12 (GSK962040) is a novel small molecule motilin receptor agonist. It possesses excellent activity at the recombinant human motilin receptor and also at the native rabbit motilin receptor where its agonist activity results in potentiation of the amplitude of neuronal-mediated contractions of isolated gastric antrum tissue. Compound 12 also possesses highly promising pharmacokinetic profiles in both rat and dog, and these results, in combination with further profiling in human native tissue and an in vivo model of gastrointestinal transit in the rabbit, have led to its selection as a candidate for further development.

Full text of DOI:10.1021/jm801332q

1180
J. Med. Chem. 2009, 52, 1180–1189
Discovery of N-(3-Fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]methyl)phenyl)acetyl]-4-
piperidinamine (GSK962040), the First Small Molecule Motilin Receptor Agonist Clinical Candidate
Susan M. Westaway,*,† Samantha L. Brown,† Stephen C. M. Fell,† Christopher N. Johnson,‡ David T. MacPherson,‡
Darren J. Mitchell,† James W. Myatt,‡ Steven J. Stanway,‡ Jon T. Seal,† Geoffrey Stemp,† Mervyn Thompson,‡ Kirk Lawless,§
Fiona McKay,§ Alison I. Muir,§ Jonathan M. Barford,‡ Chermaine Cluff,‡ Sadhia R. Mahmood,† Kim L. Matthews,‡
Shiyam Mohamed,‡ Beverley Smith,‡ Alexander J. Stevens,† Victoria J. Bolton,† Emma M. Jarvie,‡ and Gareth J. Sanger†
Immuno-Inflammation Centre of Excellence for Drug DiscoVery, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road,
SteVenage, Herts, SG1 2NY, U.K., Neurosciences Centre of Excellence for Drug DiscoVery, GlaxoSmithKline, New Frontiers Science Park,
Harlow, Essex, CM19 5AW, U.K., and Molecular DiscoVery Research, GlaxoSmithKline, New Frontiers Science Park, Harlow,
Essex, CM19 5AW, U.K.
ReceiVed October 22, 2008
N-(3-Fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]methyl)phenyl)acetyl]-4-piperidinamine 12 (GSK962040)
is a novel small molecule motilin receptor agonist. It possesses excellent activity at the recombinant human
motilin receptor and also at the native rabbit motilin receptor where its agonist activity results in potentiation
of the amplitude of neuronal-mediated contractions of isolated gastric antrum tissue. Compound 12 also
possesses highly promising pharmacokinetic profiles in both rat and dog, and these results, in combination
with further profiling in human native tissue and an in vivo model of gastrointestinal transit in the rabbit,
have led to its selection as a candidate for further development.
The treatment of gastric and upper intestinal stasis offers an
opportunity for the relief of symptoms associated with conditions
such as diabetic gastroparesis1 and functional dyspepsia2 and
also for improvement in recovery times of patients who are
subject to enteral feeding in a critical care setting.3 The antibiotic
erythromycin 1 (Figure 1) possesses gastroprokinetic activity
and is often used “off-label” in such circumstances.1 However,
there is a significant requirement for an alternative because of
the safety profile of erythromycin (potential for drug/drug
interactions and cardiac liabilities) as well as the need to avoid
the development of antibiotic resistance through chronic usage.
Erythromycin exerts its gastroprokinetic effects through agonism
of the motilin receptor4 (formerly known as GPR38a) for which
the endogenous ligand is the 22-amino acid peptide hormone
motilin.5 The motilin receptor (MTL-R) is located primarily in
the gastrointestinal tract on the enteric nerves, smooth muscle
and gastric vagal nerve terminals with the highest levels present
in the stomach and duodenum.5-7
Motilin is thought to act as a “housekeeper”, promoting the
Figure 1. Published motilin receptor agonists.
migrating motor complex that sweeps the GI tract in the fasted
state.8 However, it has also been demonstrated that motilin9 and
other motilin receptor agonists such as erythromycin10 also
promote gastric emptying and propulsion of GI tract contents
in an anal direction in the fed state.
Extensive progress in modifying erythromycin has been made
by a number of researchers giving rise to a class of motilin
receptor agonists known as the motilides.11 Despite demonstrat-
ing improved gastric emptying in healthy volunteers, some of
these motilides12,13 have suffered difficulties in the clinic.11,14
However, two members of the motilide class including mitem-
cinal (GM-611) 215 are currently in clinical trials.14d Non-
motilide agonists 316 and 417 (Figure 1) have also been disclosed,
but at present there is no further information on the progress of
these molecules that possess relatively high molecular weights
(MW 594 and 723 for 3 and 4, respectively).
At GlaxoSmithKline we embarked on a research program to
identify novel small molecule rather than motilide agonists of
the motilin receptor. We have previously reported on the results
of our high throughput screening approach and the subsequent
identification of an initial lead molecule 5.18 Further optimization
resulted in the discovery of amide 6, which possessed very
promising in vitro and in vivo profiles (Figure 2).19
* To whom correspondence should be addressed. Phone: +44 (0)1438
763469. Fax: +44 (0)1438 768232. E-mail: sue.m.westaway@gsk.com.
†
Immuno-Inflammation Centre of Excellence for Drug Discovery.
Neurosciences Centre of Excellence for Drug Discovery.
Molecular Discovery Research.
‡
§
a Abbreviations: CLi, intrinsic clearance; CYP, cytochrome P 450; DDI,
drug/drug interaction; EFS, electrical field stimulation; FLIPR, fluorometric
imaging plate reader; GI, gastrointestinal; GPR38, G-protein-coupled
receptor 38; MTL-R, motilin receptor; PK, pharmacokinetic; TDI, time-
dependent inhibition.
In this paper we report on further optimization of 6 that
resulted in the discovery of a series of 4-(piperazinylmeth-
yl)phenylacetamides of which compound 12 (GSK962040)20
possessed a profile suitable for further development.
10.1021/jm801332q CCC: $40.75
2009 American Chemical Society
Published on Web 02/03/2009

A 4-Piperidinamine Agonist
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1181
with diethyl malonate as previously described gave 29, which
was methylated, hydrolyzed, and decarboxylated to give acid
30. Coupling with the appropriate piperidinamine and depro-
tection then gave 14. Bromide 28 was also coupled with methyl
isobutyrate to give 31, which was converted to 15 using
conditions similar to those described above.
Results and Discussion
Replacement of the pyridyl B-ring of 6 with a two-carbon
linker 7 resulted in ∼12-fold drop in potency, and replacement
of the dimethylpiperazine warhead with the (2S)-methylpipera-
zine 8 offered no improvement. However, these changes in
combination with a terminal 3-fluoro substituent gave compound
9, which possessed sufficient potency for further evaluation and
we were pleased to observe a much improved TDI profile
compared to 6 (Table 3).
Further reduction in molecular weight in the dimethylpip-
erazine series was achieved by reducing the linker size to one
carbon as in 10. The TDI profile of 10 was very encouraging
(2.7-fold decrease in CYP3A4 (DEF) IC50), and we went on to
prepare the monomethyl analogues 11 and 12.
We were gratified to observe an increase in agonist potency
at the motilin receptor for both compounds as well as acceptable
TDI profiles (<2-fold decrease in CYP3A4 (DEF) IC50). Of
the two compounds, 12 was preferred because its initial IC50
values at CYP3A4 were significantly higher than our preferred
threshold of 10 µM. The corresponding (2R)-methyl analogue
13 of compound 12 was also synthesized but showed slightly
reduced agonist potency at the motilin receptor. We also
prepared analogues of 10 whereby the linker was substituted
with methyl groups in an attempt to improve potency in the
series 14 and 15; however, this was unsuccessful and the CYP
profiles were also unacceptable. Following further SAR explora-
tion around the terminal phenyl ring of 12, we found that
replacement of the 3-fluoro substituent with a 3-cyano group,
16, resulted in a further improvement in potency together with
acceptable CYP inhibition and TDI profiles.
Figure 2. Motilin receptor agonist lead compounds 5 and 6.
As previously described,19 the overall profile of 6 was very
promising as summarized in Table 1.21 However, on further
assessment of the compound as a potential candidate for
preclinical development we discovered that it showed an
increased potential for drug/drug interactions (DDI) in the clinic
due to time-dependent inhibition (TDI) of CYP3A4 in an in
vitro isolated enzyme assay as shown in Table 2. In our hands,
erythromycin showed similar levels of TDI although the initial
IC50 values were considerably lower than for 6. While these
data did not preclude further development, we rapidly undertook
a further assessment of the key features of 6 in order to identify
alternatives with a reduced potential for DDI.
Since inhibition of CYP3A4 is driven by size and lipophi-
licity, we targeted analogues of 6 with reduced molecular weight
and cLogP. We had previously shown that the benzylpiperazine
moiety of our lead series was crucial for motilin receptor agonist
activity.18 However, replacement of the B-ring of the biphenyl
motif present in the original lead series (see Figure 2) to give,
for example, the phenylpyridyl motif present in 6 was well
tolerated and resulted in a much improved CYP inhibition
profile.19 Therefore, we examined removal of the B-ring in
compound 6 and removal of one of the piperazine methyl groups
to give a series of 4-(piperazinylmethyl)phenylpropionamides
and -acetamides (Figure 3).
Chemistry
The ethylene linked analogues 7-9 were synthesized ac-
cording to Scheme 1. Hydrogenation of 4-formylcinnamic acid
gave a 1:1 mixture of 4-methyl- and 4-hydroxymethylphenyl-
propionic acids 18. The acid mixture was coupled with the
required piperidine, and oxidation with manganese dioxide
furnished the required aldehyde intermediates 20a and 20b.
Reductive amination with the appropriate protected piperazine
followed by deprotection gave 7-9. The first phenylacetamide
to be synthesized 10 was prepared according to Scheme 2.
4-Formylphenylacetic acid was coupled with the required
piperidine using standard conditions to give 21, which was
subject to reductive amination with Boc-protected cis-2,6-
dimethylpiperazine. A final acidic deprotection step then af-
forded 10.
Compounds 11-13 and 16 were synthesized as shown in
Scheme 3. The key protected 4-(piperazinylmethyl)phenylacetic
acid intermediates 25a/b and 27 could be synthesized from either
4-(bromomethyl)phenyl acetic acid or 4-bromobenzaldehyde
using standard methods as shown. Reaction with the appropriate
4-(substituted phenyl)piperidinamine right-hand sides under
carbodiimide coupling conditions, either in solution phase or
through use of polymer supported reagent, furnished the
protected final products. Standard Boc- and Cbz-deprotection
conditions gave the target compounds.
Compounds 12 and 16 were assessed for their prokinetic-
like activity in native rabbit gastric antrum tissue whereby their
ability to potentiate electrical-field-stimulated (EFS) cholinergic
contractions of the tissue was assessed24 (Table 4). Both
compounds were less potent than [Nle13]motilin (a more stable
analogue of motilin),24 as expected from their activity compared
with human motilin in the recombinant assay (Table 3), and
they also showed partial responses with respect to efficacy when
compared to [Nle13]motilin in this tissue.24 The cyano analogue
16 gave a response similar to that of erythromycin 1 but at a
3-fold lower concentration of 1 µM, and the fluoro analogue
12 gave a lower maximal response than 1 but at the same
concentration of 3 µM. With these promising yet different
profiles in hand, compounds 12 and 16 were assessed further
to determine their in vivo pharmacokinetic properties in the rat
(Table 4).
We found that the presence of the more polar cyano group
was detrimental to the oral pharmacokinetics of compound 16.
In the male Sprague-Dawley rat, 16 showed low and variable
oral exposure whereas the fluoro analogue 12 gave higher and
more consistent levels. Following determination of its intrave-
nous pharmacokinetics, the oral bioavailability (Fpo) of 12 was
found to be 48 ( 13%. This highly promising result led us to
determine the pharmacokinetic profile of 12 in the male beagle
dog, and we were pleased to find that its oral bioavailability
was 51 ( 16%.
R-Substituted analogues 14 and 15 were prepared according
to Scheme 4. Bromide 28 was prepared from 4-bromobenzal-
dehyde and 1-Boc-cis-2,6-dimethylpiperazine using standard
reductive amination conditions. Palladium mediated coupling
Compound 12 has also been assessed for its inhibition of the
other major human CYP isoforms, and it was found to possess

1182 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Table 1. Profile of Lead Amide 6
Westaway et al.
CYP IC50 CLi (mL·min-1 ·g-1
)
Fpo (%)
rat, dog tissue potentiation
rabbit native
hGhrelinR hERG (binding)
log D aqueous solubility
MW cLogP (pH 7.4)
hMTL-R pEC50
8.4
(µM)
human, rat, dog
pEC50
pIC50
(mg·mL-1
)
all >23
2.6, 2.8, <0.5
13, 58 343% at 3 µM
7.0
<4.5
501 4.1 0.7
g1 (HCl salt)
Table 2. hMTL-R FLIPR Potency22 and CYP3A4 (DEF) Time
Dependent Inhibition (TDI) Data23 for Lead 6 and Erythromycin 1
Scheme 2. Synthesis of Phenylacetamide 10a
CYP 3A4 (DEF)
initial IC50
(µM), 0-5 min
CYP 3A4 (DEF)
final IC50
(µM), 25-30 min decrease
hMTL-R
pEC50
fold
compd
1
6
7.3
8.4
1.9
20
0.19
1.4
10
14
Scheme 1. Synthesis of Phenylpropionamides 7-9a
a Reagents: (a) 4-(4-fluorophenyl)piperidinamine 17a, EDCI ·HCl, HOBt,
DMF, room temp; (b) (i) 1-Boc-cis-2,6-dimethylpiperazine, NaBH(OAc)3,
1,2-DCE, room temp, (ii) TFA/DCM, room temp.
dosed appropriately in vivo.24 General selectivity, efficacy in
human native stomach tissue, and prokinetic activity in a rabbit
model of whole gut transit have also been determined for 12,
and these data will also be reported in full elsewhere.25
Conclusion
a Reagents: (a) H2, 10% Pd/C, EtOH; (b) 4-(4-fluorophenyl)piperidi-
namine 17a or 4-(3-fluorophenyl)piperidinamine 17b, EDCI ·HCl, HOBt,
DMF, room temp; (c) MnO2, DCM, room temp; (d) 1-Boc-cis-2,6-
dimethylpiperazine or 1-Cbz-(2S)-methylpiperazine, NaBH(OAc)3, 1,2-DCE,
room temp; (e) TFA/DCM, room temp, or H2, 10% Pd/C, MeOH.
In summary, compound 12 demonstrated a highly favorable
overall profile and has been selected as a candidate for further
development. Thus, compound 12 represents a novel and
selective small molecule motilin receptor agonist (molecular
weight, 424) developed via an extensive SAR program based
on activity at the recombinant human motilin receptor and is
the first such compound to progress to phase I clinical trials for
potential use as a therapeutic agent for conditions associated
with delayed gastric emptying.
a favorable profile (1A2, 2C19, 2C9 IC50 > 100 µM, 2D6 IC50
) 34 µM). Furthermore, there were no TDI liabilities at any of
these isoforms or at CYP3A4 with 7BQ as the substrate.
Selectivity at the closely related human ghrelin acceptor was
high (pEC50 < 6.0), and there were no liabilities at the hERG
channel (binding assay pIC50 ) 4.8). In vitro plasma protein
binding levels were acceptable (human 83%, rat 63%), and
solubility in water and a range of simulated gastrointestinal fluids
was high (HCl salt, >1 mg/mL).
Additionally, the duration of action of 12 in the rabbit gastric
antrum native tissue assay has been determined. This will be
reported in full elsewhere,25 but in summary, 12 shows a long
lasting effect (T1/2 ) 46.9 ( 5.0 min at 3 µM) when compared
with the short-lived effect of [Nle13]motilin (T1/2 ) 11.4 ( 1.5
min at 0.3 µM). Its duration of action is also longer than that
of erythromycin 1 (T1/2 ) 24.0 ( 5.6 min at 3 µM), which is
used successfully in the clinic to improve gastric emptying when
dosed repeatedly at a low level. Therefore, these data may
indicate a low potential for 12 to cause tachyphylaxis when
Experimental Section
Chemistry. All anhydrous solvents were purchased from Romil
Ltd. Other solvents were purchased from Fisher Scientific. Com-
mercially available reagents were used as received. All reactions
were followed by TLC analysis (TLC plates GF254, Merck) or
LCMS (liquid chromatography-mass spectrometry). 1H NMR
spectra were recorded on Bruker AVANCE 400 or DPX250
spectrometers and are referenced to TMS (tetramethylsilane) at 0
ppm. Column chromatography was performed on prepacked silica
gel columns (Isolute SPE Flash Si II or Biotage Flash+) using
Flashmaster II or Biotage Horizon automated chromatography
systems. Electrospray mass spectra (ESI-MS) and purity were
analyzed using an LCMS system as described in further detail in
the Supporting Information. The UV detection was an averaged
signal from wavelength of 210 to 350 nm, and mass spectra were
recorded using alternate-scan positive and negative electrospray
ionization. Mass directed autoprep (MDAP) purification of samples
was carried out using the system and methods described in the
Supporting Information.
N-(4-Fluorophenyl)-4-piperidinamine 17a. A solution of 1,1-
dimethylethyl 4-oxo-1-piperidinecarboxylate (1 g, 5 mmol), 4-fluo-
roaniline (0.56 g, 5 mmol), and acetic acid (0.26 mL, 5 mmol) in
1,2-DCE (30 mL) was stirred at room temperature for 24 h. Sodium
tri(acetoxy)borohydride (1.48 g, 7 mmol) was then added, and
stirring continued for 24 h. The reaction mixture was washed with
water, dried (MgSO4), and then concentrated in vacuo to give 1,1-
dimethylethyl 4-[(4-fluorophenyl)amino]-1-piperidinecarboxylate as
Figure 3. 4-(Piperazinylmethyl)phenylpropionamide and -acetamide
targets.

A 4-Piperidinamine Agonist
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1183
Scheme 3. Synthesis of Phenylacetamides 11-13 and 16a
Scheme 4. Synthesis of R-Substituted Phenylacetamides 14 and
15a
a Reagents: (a) diethyl malonate, Pd(OAc)2, 2-(di-tert-butylphosphino)-
2′-methylbiphenyl, K3PO4, 1,4-dioxane, reflux; (b) (i) NaH, MeI, DMF, 0
°C to room temp, (ii) 2 M NaOH, 1,4-dioxane, 80 °C; (c) (i) 4-(4-
fluorophenyl)piperidinamine 17a, polymer supported carbodiimide, HOBt,
DMF/DCM, room temp, (ii) TFA/DCM, room temp; (d) iPrCO2Me,
Pd(dba)2, PtBu3, LiNCy2, PhMe, room temp; (e) (i) LiOH, 1,4-dioxane/
H2O, room temp, (ii) 4-(4-fluorophenyl)piperidinamine 17a, polymer
supported carbodiimide, HOBt, DMF/DCM, room temp, (iii) 4 M HCl/
1,4-dioxane, room temp.
overnight under an argon atmosphere. When the mixture was
cooled, the solvent was removed in vacuo and the residue
partitioned between water (100 mL) and EtOAc (100 mL). The
organic layer was separated, dried, and concentrated and the crude
product purified by column chromatography. Elution with a 0-50%
Et2O/petroleum ether gradient gave 1,1-dimethylethyl 4-[(3-cy-
anophenyl)amino]-1-piperidinecarboxylate as a white solid (1.49
g, 83%). δH (CDCl3, 250 MHz) 7.22 (1H, t, J 7.6 Hz), 6.95 (1H,
m), 6.77 (2H, m), 4.07 (2H, m), 3.77 (1H, m), 3.41 (1H, m), 2.93
(2H, m), 2.03 (2H, m), 1.47 (9H, s), 1.34 (2H, m). MS (ES+): 302
(MH+).
A solution of 1,1-dimethylethyl 4-[(3-cyanophenyl)amino]-1-
piperidinecarboxylate (750 mg, 2.43 mmol) in DCM (30 mL) was
cooled in an ice bath, and TFA (6 mL) was added. The reaction
mixture was then stirred at room temperature for 1 h. The solvent
was removed in vacuo and the residue loaded onto an Isolute SCX
cartridge. Elution with MeOH (100 mL) followed by 2 M NH3 in
MeOH (100 mL) followed by removal of solvent gave 17c as a
white solid (613 mg, 122% yield, 76% pure, contains residual DCM
and MeOH). δH (CDCl3, 250 MHz) 7.21 (1H, t, J 7.6 Hz)), 6.93
(1H, m), 6.77 (2H, m), 3.78 (1H, m), 3.35 (1H, m), 3.14 (2H, m),
2.73 (2H, m), 2.06 (2H, m), 1.34 (2H, m). MS (ES+): 202 (MH+).
1-[3-(4-([(3R,5S)-3,5-Dimethyl-1-piperazinyl]methyl)phenyl)pro-
panoyl]-N-(4-fluorophenyl)-4-piperidinamine 7. (2E)-3-(4-Formylphe-
nyl)-2-propenoic acid (2.55 g) was dissolved in EtOH (250 mL)
and hydrogenated at atmospheric pressure with 10% Pd/C (0.8 g)
as catalyst. After 5 h the reaction mixture was filtered and
concentrated to give an ∼1:1 mixture of 3-[4-(hydroxymethyl)phe-
nyl]propanoic acid and 3-(4-methylphenyl)propanoic acid 18 (2.43
g, 98%). LCMS: hydroxymethyl product, (ES+) 163 (MH+ - H2O),
(ES-) 179 (M - H+), retention time 1.55 min; methyl product,
(ES+) 147 (MH+ - H2O), (ES-) 163 (M - H+), retention time
2.43 min.
Acid mixture 18 (500 mg), N-[3-(dimethylamino)propyl]-N′-
ethylcarbodiimide hydrochloride (723 mg, 3.78 mmol), and 1-hy-
droxybenzotriazole (578 mg, 3.78 mmol) in DMF (10 mL) were
treated with N-(4-fluorophenyl)-4-piperidinamine 17a (561 mg, 2.9
mmol), and the mixture was stirred at room temperature for 2 h.
The DMF was removed in vacuo, and EtOAc and water were added.
The aqueous layer was extracted with EtOAc, and the combined
organics were washed with saturated aqueous NaHCO3 solution
a Reagents: (a) 1-Cbz-(2S)-methylpiperazine, NaBH(OAc)3, 1,2-DCE;
(b) diethyl malonate, Pd(OAc)2, 2-(di-tert-butylphosphino)-2′-methylbiphe-
nyl, K3PO4, 1,4-dioxane, reflux; (c) (i) 2 M NaOH, 1,4-dioxane, room temp,
then 2 M HCl, (ii) PhMe, reflux; (d) (i) TMS-Cl, MeOH, room temp, (ii)
1-Boc-(2S)-methylpiperazine or 1-Cbz-(2R)-methylpiperazine, iPr2NEt,
DMF, 0 °C to room temp; (e) 2 M NaOH, THF, room temp. (f) For 12, 13,
and 16: 4-(3-fluorophenyl)piperidinamine 17b or 4-(3-cyanophenyl)pip-
eridinamine 17c, EDCI ·HCl, HOBt, Et3N, DMF, room temp. For 11: 4-(4-
fluorophenyl)piperidinamine 17a, polymer supported carbodiimide, HOBt,
DMF/DCM, room temp. (g) P ) Boc: TFA/DCM, 0 °C to room temp, or
2 M HCl, dioxane 50 °C. P ) Cbz: H2, 10% Pd/C or Pd black, MeOH.
a crude solid (1.6 g). δH (CDCl3, 250 MHz) 6.88 (2H, t, J 8.8 Hz),
6.54 (2H, dd, J 8.8, 4.4 Hz), 4.04 (2H, m), 3.35 (1H, m), 2.91 (2H,
m), 2.02 (2H, m), 1.46 (9H, s), 1.30 (2H, m).
A solution of 1,1-dimethylethyl 4-[(4-fluorophenyl)amino]-1-
piperidinecarboxylate (1.6 g) in 2 M HCl (5 mL) and 1,4-dioxane
(20 mL) was heated at 60 °C for 24 h. On cooling, the solution
was diluted with water, basified with 2 M NaOH solution, and
extracted with EtOAc (×3). The combined organics were dried
(MgSO4) and concentrated in vacuo to give the title compound as
a yellow oil (0.71 g, 73% over two steps). δH (CDCl3, 250 MHz)
6.88 (2H, t, J 8.8 Hz), 6.54 (2H, dd, J 8.8, 4.4 Hz), 3.30 (1H, m),
3.20 (2H, m), 2.70 (2H, m), 2.05 (2H, m), 1.62 (2H, br), 1.29 (2H,
m).
N-(3-Fluorophenyl)-4-piperidinamine 17b. 17b was prepared
as for 17a. δH (CDCl3, 400 MHz) 7.07 (1H, dt, J 6.8, 8.0 Hz),
6.33 (3H, m), 3.83 (1H, br s), 3.33 (1H, br s), 3.12 (2H, m), 2.71
(2H, m), 2.04 (2H, m), 1.30 (2H, m).
N-(3-Cyanophenyl)-4-piperidinamine 17c. A mixture of BI-
NAP (560 mg, 0.9 mmol), palladium acetate (135 mg, 0.6 mmol),
and cesium carbonate (2.932 g, 9 mmol) in 1,4-dioxane (10 mL)
was sonicated for 50 min. 1,1-Dimethylethyl 4-amino-1-piperidi-
necarboxylate (1.2 g, 6 mmol) and 3-bromobenzonitrile (1.638 g,
9 mmol) were added, and the mixture was heated to 105 °C

1184 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Westaway et al.
Table 3. hMTL-R FLIPR Potency22 for Compounds 7-16, Human Motilin, and Erythromycin 1 and CYP3A4 and Time Dependent Inhibition (TDI)
Data23 for Compounds 7-16
CYP 3A4 IC50 (µM)b
CYP 3A4 (DEF) TDI
compd
R1
R2
n
X
Y
hMTL-R pEC50 hMTL-R IAa MW cLogP
DEF
7BQ
fold decrease
7
8
9
10
11
12
13
14
15
(R)-Me (S)-Me
1
1
1
0
0
0
0
0
0
0
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CHMe 4-F
CMe2
CH2
4-F
4-F
3-F
4-F
4-F
3-F
3-F
7.3
7.2
7.6
7.4
8.0
7.9
7.7
7.5
7.5
8.4
10.4
7.3
0.9
1.2
1.0
0.9
0.8
0.9
0.7
1.1
0.7
1.1
1.0
1.3
452
438
438
438
424
424
424
452
466
431
2699
733
3.6
3.1
3.1
3.8
3.3
3.3
3.3
4.1
4.5
3.0
nd
nd
10
41
4.9
26
29
8.7
4.7
36
nd
nd
18
59
14
69
69
nd
nd
58
nd
nd
H
H
(S)-Me
(S)-Me
3.2
2.7
0.4
1.4
nd
7.6
6.1
2.0
(R)-Me (S)-Me
H
H
H
(S)-Me
(S)-Me
(R)-Me
(R)-Me (S)-Me
(R)-Me (S)-Me
4-F
3-CN
16
h-motilin
1
H
(S)-Me
a IA: intrinsic activity relative to motilin (1.0). b nd: not determined.
Table 4. Prokinetic-like Activity in Isolated Rabbit Gastric Antrum of 12, 16, Erythromycin 1, and [Nle13]Motilin24 and Rat Pharmacokinetic Profiles of
12 and 16
% potentiation of EFS-evoked contractions (n ) 4-8)a
rat PK 5 mg ·kg-1 po (n ) 3)
concn
3 µM
concn
1 µM
concn
0.3 µM
concn
0.1 µM
CLi (mL·min-1 ·g-1),
Cmax
(µM)
Tmax
(h)
AUC(0-t)/dose
compd
human, rat
(min·kg·L-1
)
12
16
1
248 ( 47
nd
490 ( 17
nd
108 ( 21
462 ( 99
189 ( 71
nd
53 ( 30
nd
0.6, e1.0
0.5, <0.5
0.4 ( 0.1
0.2 ( 0.2
2-3
4-6
12 ( 3
2.8 ( 2.6
218 ( 86
69 ( 32
98 ( 16
14 ( 6
[Nle13]motilin
740 ( 151
721 ( 185
a nd: not determined.
and brine, then dried (Na2SO4) and concentrated to give a crude
mixture of [4-(3-(4-[(4-fluorophenyl)amino]-1-piperidinyl)-3-oxo-
propyl)phenyl]methanol and N-(4-fluorophenyl)-1-[3-(4-methylphe-
nyl)propanoyl]-4-piperidinamine 19a (1.1 g). LCMS: hydroxym-
ethyl product, (ES+) 357 (MH+), retention time 2.08 min; methyl
product, (ES+) 341 (MH+), retention time 2.94 min.
Mixture 19a (1.1 g) was dissolved in DCM (20 mL) and treated
with manganese dioxide (2 g). After the mixture was stirred
overnight, further manganese dioxide was added (5 g), and stirring
continued for 30 min. The mixture was filtered, concentrated, and
purified by column chromatography (10-90% EtOAc/pentane) to
give 20a as a yellow gum (282 mg, 28% over two steps). δH
(CDCl3, 400 MHz) 9.96 (1H, s), 7.80 (2H, d, J 8.0 Hz), 7.39 (2H,
d, J 8.0 Hz), 6.86 (2H, t, J 9.0 Hz), 6.53 (2H, dd, J 9.2, 4.4 Hz),
4.49 (1H, br d), 3.80 (1H, br d), 3.41 (2H, m), 3.11 (1H, m), 3.06
(2H, t, J 7.8 Hz), 2.84 (1H, m), 2.68 (2H, t, J 7.8 Hz), 2.05 (2H,
m), 1.25 (2H, m). LCMS: (ES+) 355 (MH+), retention time 2.43
min, >98% pure.
mixture was stirred for 15 min and then extracted with EtOAc.
The combined extracts were dried (Na2SO4) and concentrated in
vacuo to give the crude product which was purified by column
chromatography, eluting with 0-100% EtOAc/pentane to give 1,1-
dimethylethyl (2R,6S)-4-([4-(3-(4-[(4-fluorophenyl)amino]-1-pip-
eridinyl)-3-oxopropyl)phenyl]methyl)-2,6-dimethyl-1-piperazine-
carboxylate. δH (CDCl3, 400 MHz) 7.29 (2H, d, J 7.8 Hz), 7.17
(2H, d, J 7.8 Hz), 6.87 (2H, t, J 8.8 Hz), 6.53 (2H, dd, J 9.2, 4.4
Hz), 4.52 (1H, br d), 4.05 (2H, m), 3.80 (1H, br d), 3.45 (2H, s),
3.39 (2H, m), 3.10 (1H, m), 2.96 (2H, m), 2.84 (1H, m), 2.60 (4H,
m), 2.08 (4H, m), 1.46 (9H, s), 1.29 (6H, d, J 6.8 Hz), 1.22 (2H,
m). LCMS: (ES+) 553 (MH+), retention time 2.39 min, >98% pure.
This whole sample was dissolved in 2:1 DCM/TFA (3 mL) and
stirred for 1.5 h. The mixture was concentrated and eluted through
an Isolute SCX cartridge (2 M NH3 in MeOH) to give 7 (100 mg,
78% over two steps). δH (CDCl3, 400 MHz) 7.23 (2H, d, J 8.0
Hz), 7.17 (2H, d, J 8.0 Hz), 6.88 (2H, t, J 8.8 Hz), 6.53 (2H, m, J
8.8, 4.4 Hz), 4.52 (1H, m), 3.79 (1H, m), 3.42 (4H, m), 3.09 (1H,
m), 2.94 (4H, m), 2.83 (1H, m), 2.75 (2H, m), 2.63 (2H, m), 2.03
(2H, m), 1.59 (2H, t, J 10.6 Hz), 1.29 (1H, m), 1.17 (1H, m), 1.02
(6H, d, J 6.4 Hz). LCMS: (ES+) 453 (MH+), retention time 1.50
min, >98% pure. Free base 7 (100 mg) was dissolved in DCM
To a mixture of 20a (100 mg, 0.282 mmol) and 1,1-dimethylethyl
(2R,6S)-2,6-dimethyl-1-piperazinecarboxylate (61 mg, 0.283 mmol)
in 1,2-DCE (5 mL) was added sodium tri(acetoxy)borohydride (90
mg, 0.423 mmol), and the mixture was stirred at room temperature
overnight. Saturated aqueous NaHCO3 solution was added, and the

A 4-Piperidinamine Agonist
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1185
and treated with 1 M HCl in Et2O (243 µL) to give the
hydrochloride salt (98 mg).
(Na2SO4) and concentrated in vacuo to give the crude product,
which was purified by column chromatography. Elution with
20-90% EtOAc/pentane gave 1,1-dimethylethyl (2R,6S)-4-([4-(2-
(4-[(4-fluorophenyl)amino]-1-piperidinyl)-2-oxoethyl)phenyl]methyl)-
2,6-dimethyl-1-piperazine carboxylate (143 mg, 61%). δH (CDCl3,
400 MHz) 1.06 (1H, m), 1.27 (6H, d, J 6.4 Hz), 1.30 (1H, m),
1.46 (9H, s), 1.93 (1H, m), 2.05 (1H, m), 2.12 (2H, dd, J 11.2, 4.4
Hz), 2.59 (2H, d, J 11.2 Hz), 2.88 (1H, m), 3.13 (1H, m), 3.29
(1H, br s), 3.38 (1H, m), 3.45 (2H, s), 3.74 (2H, s), 3.86 (1H, m),
4.07 (2H, m), 4.50 (1H, m), 6.50 (2H, m), 6.87 (2H, t, J 8.8 Hz),
7.20 (2H, d, J 8.0 Hz), 7.31 (2H, d, J 8.0 Hz). LCMS: (ES+) 539
(MH+), retention time 1.85 min, >98% pure.
This whole sample was dissolved in 2:1 DCM/TFA and stirred
for 1 h. The mixture was concentrated and eluted through an Isolute
SCX cartridge (2 M NH3 in MeOH) to give free base 10. δH (CDCl3,
400 MHz) 1.01 (6H, d, J 6.4 Hz), 1.06 (1H, m), 1.29 (1H, m),
1.62 (2H, t, J 10.4 Hz), 1.93 (1H, m), 2.04 (1H, m), 2.74 (2H, d,
J 10.4 Hz), 2.83-2.94 (3H, m), 3.12 (1H, m), 3.36 (2H, m), 3.46
(2H, s), 3.73 (2H, s), 3.86 (1H, m), 4.51 (1H, m), 6.55 (2H, m),
6.86 (2H, t, J 8.8 Hz), 7.20 (2H, d, J 8.0 Hz), 7.26 (2H, d, J 8.0
Hz). LCMS: (ES+) 439 (MH+), retention time 1.20 min, >98%
pure. Free base 10 was converted to the dihydrochloride salt (104
mg, 77% from Boc-protected material).
N-(4-Fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]meth-
yl)phenyl)acetyl]-4-piperidinamine 11. A mixture of 4-bromoben-
zaldehyde (1.19 g, 6.42 mmol), phenylmethyl (2S)-2-methyl-1-
piperazinecarboxylate (1.505 g, 6.42 mmol), and sodium triacetoxy
borohydride (2.04 g, 9.63 mmol) in 1,2-DCE (15 mL) was stirred
at room temperature overnight. Saturated aqueous NaHCO3 solution
was added and the mixture stirred for 30 min and then extracted
with EtOAc. The extracts were dried (Na2SO4) and concentrated.
Column chromatography (0-30% EtOAc in pentane) gave 22 (2.18
g, 84%). δH (CDCl3, 400 MHz) 7.43 (2H, d, J 8.4 Hz), 7.34 (5H,
m), 7.21 (2H, d, J 8.4 Hz), 5.13 (2H, AB 2 × d), 4.28 (1H, br s),
3.90 (1H, m), 3.47 (1H, d, J 13.6 Hz), 3.34 (1H, d, J 13.6 Hz),
3.18 (1H, td, J 12.8, 3.2 Hz), 2.74 (1H, m), 2.57 (1H, dd, J 11.2,
3.6 Hz), 2.03 (1H, m), 1.26 (3H, m). LCMS: (ES+) 403/405 (MH+),
retention time 2.39 min, >98% pure.
A mixture of 22 (1.62 g, 4 mmol), diethyl malonate (0.73 mL,
4.8 mmol), palladium(II) acetate (27 mg, 0.12 mmol), potassium
phosphate (1.95 g, 2.3 mmol), and 2-(di-tert-butylphosphino)-2′-
methylbiphenyl (83 mg, 0.264 mmol) in 1,4-dioxane (20 mL) was
refluxed under argon for ∼20 h. The mixture was filtered through
Celite and concentrated. Column chromatography (0-40% EtOAc/
hexane) gave 23 as a clear oil (1.165 g, 60%). δH (CDCl3, 400
MHz) 7.35 (9H, m), 5.12 (2H, AB 2 × d), 4.60 (1H, s), 4.10-4.31
(5H, m), 3.89 (1H, m), 3.51 (1H, d, J 13.4 Hz), 3.41 (1H, d, J 13.4
Hz), 3.19 (1H, td, J 12.8, 3.2 Hz), 2.76 (1H, m), 2.61 (1H, m),
2.15 (1H, dd, J 11.2, 4.0 Hz), 2.02 (1H, td, J 11.6, 3.2 Hz), 1.26
(9H, m). MS: (ES+) 483 (MH+).
A mixture of 23 (761 mg, 1.58 mmol), 2 M NaOH solution (6
mL), and 1,4-dioxane (6 mL) was stirred at room temperature for
2 h. The solvents were removed, and the residue was dissolved in
water and the pH adjusted to 4 with 2 M HCl. The product was
extracted with EtOAc, and the combined extracts were dried and
concentrated. The product was refluxed in toluene (∼20 mL) for
2 h and the solvent was evaporated to give 25a as a yellow foam
(505 mg, 84%). δH (CDCl3, 400 MHz) 7.34 (5H, m), 7.28 (2H, d,
J 8.0 Hz), 7.26 (2H, d, J 8.0 Hz), 5.13 (2H, AB 2 × d), 4.28 (1H,
m), 3.90 (1H, m), 3.63 (2H, s), 3.52 (1H, d, J 13.0 Hz), 3.41 (1H,
d, J 13.0 Hz), 3.19 (1H, td, J 12.8, 3.2 Hz), 2.80 (1H, m), 2.63
(1H, m), 2.16 (1H, dd, J 11.6, 4.0 Hz), 2.03 (1H, td, J 11.6, 3.2
Hz), 1.27 (3H, d, J 6.8 Hz). MS: (ES+) 383 (MH+), (ES-) 381 (M
- H+).
A mixture of 25a (115 mg, 0.3 mmol), polymer-supported
carbodiimide (270 mg, 1.7 mmol/g, 0.45 mmol), and 1-hydroxy-
benzotriazole (55 mg, 0.36 mmol) in 2:1 DMF/DCM (3 mL) was
treated with N-(4-fluorophenyl)-4-piperidinamine 17a (58 mg, 0.3
mmol) and stirred overnight. Scavenger resins (PS-trisamine, PS-
isocyanate, and Si-carbonate) together with DCM (∼3 mL) were
added. The mixture was stirred for ∼2 h and then filtered and
N-(4-Fluorophenyl)-1-[3-(4-([(3S)-3-methyl-1-piperazinyl]meth-
yl)phenyl)propanoyl]-4-piperidinamine 8. To a mixture of 20a (100
mg, 0.282 mmol) and phenylmethyl (2S)-2-methyl-1-piperazin-
ecarboxylate (66 mg, 0.281 mmol) in 1,2-DCE (5 mL) was added
sodium tri(acetoxy)borohydride (90 mg, 0.423 mmol), and the
mixture was stirred at room temperature overnight. Saturated
aqueous NaHCO3 solution was added, and the mixture was stirred
for 15 min and then extracted with EtOAc. The combined extracts
were dried (Na2SO4) and concentrated in vacuo to give the crude
product which was purified by column chromatography, eluting with
0-100% EtOAc/pentane to give phenylmethyl (2S)-4-([4-(3-(4-
[(4-fluorophenyl)amino]-1-piperidinyl)-3-oxopropyl)phenyl]methyl)-
2-methyl-1-piperazinecarboxylate. δH (CDCl3, 400 MHz) 7.27-7.35
(5H, m), 7.24 (2H, d, J 8.0 Hz), 7.16 (2H, d, J 8.0 Hz), 6.87 (2H,
t, J 8.8 Hz), 6.52 (2H, m), 5.12 (2H, 2 × AB d), 4.51 (1H, br d),
4.27 (1H, br s), 3.88 (1H, br d), 3.79 (1H, br d), 3.48 (1H, d, J
13.2 Hz), 3.39 (2H, m), 3.18 (1H, m), 3.09 (1H, m), 2.96 (2H, m),
2.83 (1H, m), 2.75 (1H, br d), 2.65-2.58 (3H, m), 2.13 (1H, m),
2.05-1.97 (4H, m), 1.27 (3H, d, J 6.8 Hz), 1.23 (2H, m). LCMS:
(ES+) 573 (MH+), retention time 2.21 min, 95% pure.
This material was hydrogenated in MeOH (5 mL) with 10% Pd/C
catalyst (20 mg) for 2.5 h. The mixture was filtered, concentrated,
and purified by MDAP to give 8 (69 mg, 56% over two steps). δH
(CDCl3, 400 MHz) 7.24 (2H, d, J 8.0 Hz), 7.17 (2H, d, J 8.0 Hz),
6.88 (2H, t, J 8.8 Hz), 6.53 (2H, m), 4.51 (1H, m), 3.79 (1H, m),
3.43 (4H, m), 3.09 (1H, m), 2.73-2.98 (8H, m), 2.63 (2H, t, J 7.8
Hz), 1.96-2.06 (4H, m), 1.66 (1H, t, J 10.4 Hz), 1.27 (1H, m),
1.15 (1H, m), 1.00 (3H, d, J 6.4 Hz). LCMS: (ES+) 439 (MH+),
retention time 1.46 min, >98% pure. Free base 8 (69 mg) was
treated with 1 M HCl in Et2O (174 µL) to give the hydrochloride
salt (69 mg).
N-(3-Fluorophenyl)-1-[3-(4-([(3S)-3-methyl-1-piperazinyl]meth-
yl)phenyl)propanoyl]-4-piperidinamine 9. Compound 9 was pre-
pared from 3-[4-(hydroxymethyl)phenyl]propanoic acid and 3-(4-
methylphenyl)propanoic acid 18, N-(3-fluorophenyl)-4-piperi-
dinamine 17b, and phenylmethyl (2S)-2-methyl-1-piperazinecar-
boxylate using the same procedure as that described for
compound 8. δH (CDCl3, 400 MHz) 7.24 (2H, d, J 8.0 Hz), 7.17
(2H, d, J 8.0 Hz), 7.08 (1H, m), 6.36 (2H, m), 6.27 (1H, m),
4.52 (1H, br d), 3.79 (1H, br d), 3.46 (2H, s), 3.43 (1H, m),
3.09 (1H, m), 2.73-2.98 (8H, m), 2.63 (2H, m), 2.01 (3H, m),
1.83 (1H, br s), 1.65 (1H, t, J 10.6 Hz), 1.30 (1H, m), 1.16 (1H,
m), 1.00 (3H, d, J 6.4 Hz). LCMS: (ES+) 439 (MH+), retention
time 1.76 min, >98% pure.
1-[(4-([(3R,5S)-3,5-Dimethyl-1-piperazinyl]methyl)phenyl)ace-
tyl]-N-(4-fluorophenyl)-4-piperidinamine 10. A mixture of (4-
formylphenyl)acetic acid (87 mg, 0.53 mmol), N-(4-fluorophenyl)-
4-piperidinamine 17a (102 mg, 0.53 mmol), 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (123 mg, 0.64 mmol),
and 1-hydroxybenzotriazole (98 mg, 0.64 mmol) in DMF (2 mL)
was stirred at room temperature overnight. The DMF was removed
in vacuo, and then EtOAc and water were added. The product was
extracted into EtOAc, and the combined organic layers were washed
with saturated aqueous NaHCO3 solution (×2) and brine and then
dried (Na2SO4). The solvent was removed in vacuo and the resulting
residue was purified by column chromatography, eluting with an
EtOAc/petroleum ether gradient to afford 21 (149 mg, 83%). δH
(CDCl3, 400 MHz) 10.0 (1H, s), 7.86 (2H, d, J 8.0 Hz), 7.43 (2H,
d, J 8.0 Hz), 6.86 (2H, m), 6.57 (2H, m), 4.51 (1H, m), 3.85 (1H,
m), 3.83 (2H, s), 3.37 (2H, m), 3.16 (1H, m), 2.90 (1H, m), 2.03
(2H, m), 1.29 (1H, m), 1.12 (1H, m). LCMS: (ES+) 341 (MH+),
(ES-) 339 (M - H+), retention time 2.17 min, >98% pure.
A mixture of 21 (149 mg, 0.438 mmol) and 1,1-dimethylethyl
(2R,6S)-2,6-dimethyl-1-piperazinecarboxylate (94 mg, 0.438 mmol)
in 1,2-DCE (3 mL) was stirred for 5 min at room temperature.
Sodium tri(acetoxy)borohydride (139 mg, 0.66 mmol) was added,
and the mixture was stirred for 3 h. Then saturated aqueous
NaHCO3 solution was added. The mixture was stirred for 15 min
and then extracted with EtOAc. The combined extracts were dried

1186 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Westaway et al.
concentrated. Column chromatography (0-60% EtOAc/pentane)
gave phenylmethyl (2S)-4-([4-(2-(4-[(4-fluorophenyl)amino]-1-pi-
peridinyl)-2-oxoethyl)phenyl]methyl)-2-methyl-1-piperazinecar-
boxylate (148 mg, 88%). δH (CDCl3, 400 MHz) 7.34 (5H, m), 7.27
(2H, d, J 8.0 Hz), 7.19 (2H, d, J 8.0 Hz), 6.85 (2H, t, J 8.8 Hz),
6.50 (2H, dd, J 9.2, 4.4 Hz), 5.13 (2H, AB 2 × d), 4.50 (1H, m),
4.27 (1H, br s), 3.90 (2H, m), 3.73 (2H, s), 3.49 (1H, d, J 13.2
Hz), 3.38 (1H, d, J 13.2 Hz), 3.37 (1H, m), 3.14 (2H, m), 2.87
(1H, m), 2.75 (1H, m), 2.58 (1H, m), 2.14 (1H, dd, J 11.2, 3.6
Hz), 2.01 (2H, m), 1.93 (1H, m), 1.30 (1H, m), 1.25 (3H, m), 1.07
(1H, m). LCMS: (ES+) 559 (MH+), retention time 2.12 min, >95%
pure.
This whole sample was hydrogenated in MeOH (∼5 mL) with
10% Pd/C catalyst (∼20 mg) for 2 h. Column chromatography
(0-20% MeOH/DCM) gave 11 (19 mg, 17%). δH (CDCl3, 400
MHz) 7.27 (2H, d, J 8.0 Hz), 7.20 (2H, d, J 8.0 Hz), 6.85 (2H, t,
J 9.2 Hz), 6.50 (2H, dd, J 9.2, 4.4 Hz), 4.51 (1H, m), 3.86 (1H,
m), 3.73 (2H, s), 3.47 (2H, s), 3.38 (1H, m), 3.12 (1H, m),
2.83-3.00 (4H, m), 2.75 (2H, m), 2.27 (1H, br s), 2.03 (2H, m),
1.94 (1H, m), 1.70 (1H, m), 1.30 (2H, m), 1.06 (1H, m), 1.02 (3H,
d, J 6.4 Hz). LCMS: (ES+) 425 (MH+), retention time 1.42 min,
>95% pure. Free base 11 (19 mg) was treated with 1.1 equiv of 1
M HCl in Et2O to give the hydrochloride salt (25 mg).
A mixture of 25b (27.8 g, 79.8 mmol), N-[3-(dimethylamino)-
propyl]-N′-ethylcarbodiimide hydrochloride (22.9 g, 119.7 mmol),
1-hydroxybenzotriazole hydrate (16.2 g, 119.7 mmol), triethylamine
(45 mL, 319.1 mmol), and N-(3-fluorophenyl)-4-piperidinamine 17b
hydrochloride salt (18.4 g, 79.8 mmol) in dry DMF (400 mL) was
stirred at room temperature overnight. The solvent was removed
in vacuo, and the residue was redissolved in DCM (300 mL) and
washed with 2 M NaOH (2 × 200 mL), water (200 mL), and brine
(200 mL). All aqueous washings were combined and back-extracted
with DCM (2 × 100 mL). The combined organics were dried and
concentrated to give a solid which was purified by column
chromatography (silica prewashed with 50% EtOAc/hexane).
Elution with 70% EtOAc/hexane gave 1,1-dimethylethyl (2S)-4-
([4-(2-(4-[(3-fluorophenyl)amino]-1-piperidinyl)-2-oxoethyl)phenyl-
]methyl)-2-methyl-1-piperazinecarboxylate as a white solid (34.95
g, 84%). δH (CDCl3, 400 MHz) 7.28 (2H, d, J 8.0 Hz), 7.19 (2H,
d, J 8.0 Hz), 7.07 (1H, dt, J 8.0, 6.4 Hz), 6.23-6.40 (3H, m), 4.52
(1H, m), 4.17 (1H, m), 3.78-3.88 (2H, m), 3.74 (2H, s), 3.60 (1H,
d, J 7.6 Hz), 3.40 (1H, d, J 13.4 Hz), 3.43 (1H, m), 3.38 (1H, d, J
13.4 Hz), 3.06-3.17 (2H, m), 2.89 (1H, m), 2.74 (1H, m), 2.57
(1H, m), 1.94-2.13 (4H, m), 1.45 (9H, s), 1.32 (1H, m), 1.21 (3H,
d, J 6.4 Hz), 1.09 (1H, m). LCMS: (ES+) 525 (MH+), retention
time 2.18 min, 95% pure.
A solution of 1,1-dimethylethyl (2S)-4-([4-(2-(4-[(3-fluorophe-
nyl)amino]-1-piperidinyl)-2-oxoethyl)phenyl]methyl)-2-methyl-1-
piperazinecarboxylate (83.01 g, 0.158 mol) in DCM (900 mL) was
split into two portions and each cooled to 0 °C and treated with
TFA (100 mL). After being stirred for 0.5 h at 0 °C, the mixtures
were warmed to room temperature and stirred for 3.25 h. The
solvent was removed in vacuo, and the residues were combined
and partitioned between DCM and 2 M NaOH solution. The
aqueous phase was re-extracted with DCM (×2), and the combined
organics were then washed with 2 M NaOH and brine. The organics
were dried and concentrated in vacuo to give an off-white solid.
The NaOH phase was re-extracted with DCM, which was dried
and concentrated to give a further batch of white foam. The two
batches were combined to give 12 as an off-white solid (66.94 g,
100%). δH (CDCl3, 400 MHz) 7.26 (2H, d, J 8.0 Hz), 7.20 (2H, d,
J 8.0 Hz), 7.06 (1H, q, J 8.0 Hz), 6.36 (1H, m), 6.31 (1H, m), 6.25
(1H, m), 4.52 (1H, m), 3.86 (1H, m), 3.73 (2H, s), 3.66 (1H, m),
3.48 (2H, s), 3.41 (1H, m), 2.84-3.16 (6H, m), 2.76 (2H, d, J 11.2
Hz), 2.03-2.13 (2H, m), 1.94 (1H, m), 1.78 (1H, t, J 10.8 Hz),
1.34 (1H, m), 1.07 (4H, m). LCMS: (ES+) 425 (MH+), retention
time 1.64 min, 98% pure, mp 144 °C (onset by DSC).
A batch of free base 12 (66.22 g, 0.156 mol) was dissolved in
EtOAc (1.7 L) at 45-50 °C to give a homogeneous pale-yellow
solution which was then cooled to room temperature and flushed
with argon. Then 1 M HCl in Et2O (156 mL, 0.156 mol) was added
with vigorous stirring, and after 15 min, the resultant creamy-white
precipitate was collected by filtration under a blanket of argon. This
was washed with further EtOAc (0.8 L) and partially dried on the
filter under a blanket of argon for 15 min The solid was further
dried at 80 °C in vacuo to give the hydrochloride salt of 12 (58.95
g, 82%). δH (CDCl3, 400 MHz) 9.70 (1H, br), 7.32 (2H, m), 7.24
(2H, d, J 8.0 Hz), 7.08 (1H, q, J 8.0 Hz), 6.38 (2H, m), 6.31 (1H,
m), 4.52 (1H, m), 3.86 (1H, m), 3.74 (4H, m), 3.51 (1H, br s),
3.41 (2H, m), 3.24 (1H, br s), 3.16 (1H, m), 2.99 (2H, m), 2.87
(2H, m), 2.64 (1H, br s), 2.02 (2H, m), 1.45 (3H, d, J 6.8 Hz),
1.34 (1H, m), 1.12 (1H, m). LCMS: (ES+) 425 (MH+), retention
time 1.61 min, >98% pure.
N-(3-Fluorophenyl)-1-[(4-([(3S)-3-methyl-1-piperazinyl]meth-
yl)phenyl)acetyl]-4-piperidinamine 12. Compound 12 was initially
prepared as for compound 11 described above using N-(3-
fluorophenyl)-4-piperidinamine 17b instead of N-(4-fluorophenyl)-
4-piperidinamine 17a. Alternatively, a larger scale preparation was
used as follows:
To a solution of 4-(bromomethyl)phenylacetic acid (20 g, 87.3
mmol) in MeOH (200 mL) was added trimethylsilyl chloride (2
mL), and the mixture was stirred for 2 h. The solvent was removed
in vacuo and the residue was twice redissolved in MeOH (200 mL)
and reconcentrated to give methyl 4-(bromomethyl)phenylacetate
(21.08 g, 99%). δH (CDCl3, 250 MHz) 7.36 (2H, d, J 8.0 Hz), 7.26
(2H, d, J 8.0 Hz), 4.49 (2H, s), 3.69 (3H, s), 3.62 (2H, s). LCMS:
(ES+) 243/245 (MH+), 183/185 (M - CO2Me)+ retention time 2.84
min, >98% pure.
To a solution of methyl 4-(bromomethyl)phenylacetate (20.8 g,
85.6 mmol) and diisopropylethylamine (16.4 mL, 94.1 mmol) in
dry DMF (100 mL) was added a solution of 1,1-dimethylethyl (2S)-
2-methyl-1-piperazinecarboxylate (18.8 g, 94.1 mmol) in dry DMF
(75 mL) with cooling in an ice bath. The mixture was warmed to
room temperature and stirred for 15 min. The solvent was removed
in vacuo and the residue partitioned between EtOAc and 2 M NaOH
solution (400 mL, 1:1). The organic phase was washed with water
(200 mL) and brine (200 mL), and the combined aqueous washings
were back-extracted with EtOAc (200 mL). The EtOAc extracts
were combined, dried (MgSO4), and concentrated in vacuo to give
the crude product which was purified by column chromatography.
Elution with 20-25% EtOAc/hexane gave 24 as a colorless oil
(29.3 g, 95%). δH (CDCl3, 250 MHz) 7.29 (2H, d, J 8.0 Hz), 7.22
(2H, d, J 8.0 Hz), 4.18 (1H, m), 3.79 (1H, m), 3.69 (3H, s), 3.62
(2H, s), 3.51 (1H, d, J 13.2 Hz), 3.39 (1H, d, J 13.2 Hz), 3.10 (1H,
td, J 12.8, 3.5 Hz), 2.75 (1H, m), 2.59 (1H, m), 2.12 (1H, dd, J
11.1, 3.9 Hz), 1.99 (1H, m), 1.45 (9H, s), 1.24 (3H, d, J 6.8 Hz).
LCMS: (ES+) 363 (MH+), retention time 1.78 min, 90% pure.
To a solution of 24 (29.3 g, 80.9 mmol) in THF (200 mL) was
added 2 M NaOH solution (100 mL), and the two-phase reaction
mixture was stirred at room temperature for 3 h. The mixture was
concentrated in vacuo to remove the THF, and the aqueous solution
was extracted with EtOAc (2 × 100 mL). The aqueous phase was
acidified to pH 6 with concentrated HCl and extracted with DCM
(3 × 300 mL). The combined organics were washed with brine
(×2), dried and concentrated in vacuo to give 25b as a colorless
foam (27.8 g, 99%). δH (CDCl3, 400 MHz) 7.27 (2H, d, J), 7.23
(2H, d), 4.20 (1H, m), 3.81 (1H, m), 3.62 (2H, s), 3.59 (1H, d, J
8.3 Hz), 3.49 (1H, d, J 8.3 Hz), 3.15 (1H, m), 2.88 (1H, m), 2.69
(1H, m), 2.20 (1H, dd, J 11.2, 4.4 Hz), 2.05 (1H, m), 1.45 (9H, s),
1.25 (3H, d, J 6.8 Hz). LCMS: (ES+) 349 (MH+), (ES-) 347 (M
- H+), retention time 1.53 min, 94% pure.
N-(3-Fluorophenyl)-1-[(4-([(3R)-3-methyl-1-piperazinyl]meth-
yl)phenyl)acetyl]-4-piperidinamine 13. A mixture of methyl 4-(bro-
momethyl)phenylacetate (243 mg, 1 mmol) (see compound 12 for
synthesis), diisopropylethylamine (174 µL, 1 mmol), and 1,1-
dimethylethyl (2R)-2-methyl-1-piperazinecarboxylate (234 mg, 1
mmol) in DMF was stirred at room temperature for 1 h and then
allowed to stand overnight. The solvent was removed in vacuo.
Then the residue was diluted with water (20 mL) and extracted
with EtOAc (2 × 20 mL). The combined organic extracts were
dried (MgSO4) and concentrated in vacuo to give 26 as a yellow
oil (383 mg, 97%). δH (CDCl3, 250 MHz) 7.21-7.40 (9H, m), 5.13

A 4-Piperidinamine Agonist
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1187
(2H, AB 2 × d) 4.29 (1H, m), 3.91 (1H, m), 3.70 (3H, s), 3.62
(2H, s), 3.43 (2H, m), 3.20 (1H, m), 2.78 (1H, m), 2.62 (1H, m),
2.05-2.18 (2H, m), 1.29 (3H, d, J 6.3 Hz). MS: (ES+) 397 (MH+).
A solution of 26 (380 mg, 0.96 mmol) in THF (4 mL) and 2 M
NaOH (1 mL) was stirred at room temperature overnight. Water
(10 mL) was added and the solution washed with EtOAc (20 mL).
The aqueous phase was adjusted to pH 6 and extracted with EtOAc
(2 × 20 mL). The combined extracts were washed with brine, dried
(MgSO4), and concentrated in vacuo to give 27 as a colorless gum
(255 mg, 69%). δH (CDCl3, 250 MHz) 7.20-7.40 (9H, m), 5.12
(2H, AB 2 × d) 4.29 (1H, m), 3.91 (1H, d, J 13.5 Hz), 3.61 (2H,
s), 3.51 (2H, AB, 2 × d), 3.20 (1H, m), 2.85 (1H, d, J 10.8 Hz),
2.69 (1H, d, J 11.5 Hz), 2.20 (1H, m), 2.10 (1H, m), 1.29 (3H, m).
A mixture of 27 (100 mg, 0.261 mmol), N-[3-(dimethylamino)-
propyl]-N′-ethylcarbodiimide hydrochloride (75 mg, 0.392 mmol),
1-hydroxybenzotriazole (53 mg, 0.392 mmol), triethylamine (110
µL, 0.784 mmol), and N-(3-fluorophenyl)-4-piperidinamine 17b
hydrochloride salt (60 mg, 0.261 mmol) in DMF (2 mL) was stirred
at room temperature for 3 days. The solvent was removed in vacuo,
and the residue was purified by column chromatography. Elution
with 0-10% MeOH/DCM gave phenylmethyl (2R)-4-([4-(2-(4-[(3-
fluorophenyl)amino]-1-piperidinyl)-2-oxoethyl)phenyl]methyl)-2-
methyl-1-piperazine carboxylate as a colorless oil (136 mg, 93%).
δH (CDCl3, 400 MHz) 7.31-7.38 (5H, m), 7.28 (2H, d, J 8.0 Hz),
7.19 (2H, d, J 8.0 Hz), 7.07 (1H, m), 6.23-6.39 (3H, m), 5.13
(2H, AB 2 × d), 4.52 (1H, m), 4.27 (1H, br s), 3.88 (2H, m), 3.74
(2H, s), 3.62 (1H, br s), 3.50 (1H, d, J 13.2 Hz), 3.42 (1H, m),
3.38 (1H, d, J 13.2 Hz), 3.16 (2H, m), 2.88 (1H, m), 2.76 (1H, d,
J 10.8 Hz), 2.59 (1H, d, J 10.8 Hz), 1.94-2.15 (4H, m), 1.32 (1H,
m), 1.26 (3H, m), 1.08 (1H, m). LCMS: (ES+) 559 (MH+), retention
time 2.26 min, 91% pure.
A solution of phenylmethyl (2R)-4-([4-(2-(4-[(3-fluorophenyl)-
amino]-1-piperidinyl)-2-oxoethyl) phenyl]methyl)-2-methyl-1-pip-
erazinecarboxylate (136 mg, 0.24 mmol) was hydrogenated in
MeOH (5 mL) with palladium black catalyst (68 mg) for 0.75 h.
The reaction mixture was concentrated in vacuo to give a pale-
yellow crude oil which was purified by column chromatography.
Elution with 0-10% (2 M NH3 in MeOH)/DCM gave 13 as a
colorless oil (38 mg, 37%). δH (CDCl3, 400 MHz) 7.27 (2H, d, J
8.0 Hz), 7.20 (2H, d, J 8.0 Hz), 7.07 (1H, q, J 8.0 Hz), 6.37 (1H,
m), 6.31 (1H, m), 6.25 (1H, m), 4.52 (1H, m), 3.86 (1H, m), 3.74
(2H, s), 3.62 (1H, m), 3.47 (2H, s), 3.42 (1H, m), 3.13 (1H, m),
2.90 (4H, m), 2.75 (2H, m), 2.00 (4H, m), 1.69 (1H, t, J 10.4 Hz),
1.30 (1H, m), 1.06 (1H, m), 1.02 (3H, d, J 6.4 Hz). LCMS: (ES+)
425 (MH+), retention time 1.64 min, >98% pure. Free base 13
(38 mg) was treated with 1.1 equiv of 1 M HCl in Et2O to give the
hydrochloride salt (41 mg).
1-[2-(4-([(3R,5S)-3,5-Dimethyl-1-piperazinyl]methyl)phenyl)pro-
panoyl]-N-(4-fluorophenyl)-4-piperidinamine 14. Malonate 29 was
prepared in the same manner as 23 using 1,1-dimethylethyl (2R,6S)-
2,6-dimethyl-1-piperazinecarboxylate instead of phenylmethyl (2S)-
2-methyl-1-piperazinecarboxylate in the first step (see compound
11 for details). LCMS: (ES+) 463 (MH+), retention time 2.62 min,
87% pure.
A solution of 29 (538 mg, 1.16 mmol) in DMF (10 mL) was
added dropwise to sodium hydride (61 mg, 60% w/w in oil, 1.51
mmol) in DMF (2 mL) at 0 °C under argon. After the mixture was
stirred for 10 min, methyl iodide (0.144 mL, 2.32 mmol) was added
and the reaction mixture allowed to warm to room temperature over
1 h. Ammonium chloride solution was added and the mixture
extracted with EtOAc. The extracts were washed with saturated
aqueous NaHCO3 solution and water, then dried (Na2SO4) and
concentrated in vacuo. Column chromatography, eluting with
0-10% EtOAc/hexane, gave diethyl (4-[((3S)-4-([(1,1-dimethyl-
ethyl)oxy]carbonyl)-3-methyl-1-piperazinyl)methyl]phenyl)pro-
panedioate as a clear gum (356 mg, 65%). LCMS: (ES+) 477
(MH+), retention time 2.72 min, >95% pure.
for 3 h. The solvents were removed in vacuo, water was added,
and the mixture was adjusted to pH 4 with 2 M HCl. The product
was extracted into EtOAc and the extracts were washed with brine
and then dried (Na2SO4) and concentrated to give 30 (256 mg, 91%).
δH (CDCl3, 400 MHz) 7.32 (2H, d, J 8.0 Hz), 7.27 (2H, d, J 8.0
Hz), 4.10 (2H, m), 3.74 (1H, q, J 7.2 Hz), 3.49 (2H, br s), 2.64
(2H, m), 2.14 (2H, m), 1.52 (3H, d, J 7.2 Hz), 1.46 (9H, s), 1.30
(6H, d, J 6.8 Hz). LCMS: (ES+) 377, (ES-) 375 (M - H+),
retention time 1.93 min, >95% pure.
A mixture of 30 (100 mg, 0.27 mmol), polymer-supported
carbodiimide (310 mg, 1.3 mmol/g, 0.40 mmol), and 1-hydroxy-
benzotriazole (50 mg, 0.324 mmol) in 2:1 DMF/DCM (3 mL) was
treated with N-(4-fluorophenyl)-4-piperidinamine 17a (51 mg, 0.26
mmol) and the mixture stirred overnight. Scavenger resins (PS-
trisamine, PS-isocyanate, and Si-carbonate) together with DCM (∼3
mL) were added. The mixture was stirred for ∼3 h and then filtered
and concentrated to give 1,1-dimethylethyl (2R,6S)-4-([4-(2-(4-[(4-
fluorophenyl)amino]-1-piperidinyl)-1-methyl-2-oxoethyl)phenyl]m-
ethyl)-2,6-dimethyl-1-piperazinecarboxylate. LCMS: (ES+) 553
(MH+), retention time 2.47 min, >98% pure.
This whole sample was dissolved in DCM (2 mL) and TFA (1
mL) and then stirred for 1.5 h. The solvents were removed in vacuo
and the residue re-evaporated from toluene and ether. Purification
using an Isolute SCX cartridge, eluting with MeOH followed by 2
M NH3 in MeOH, gave 14 as ∼1:1 mixture of diastereoisomers.
δH (CDCl3, 400 MHz) 7.26 (2H, m), 7.18 (2H, m), 6.83 (2H, m),
6.50 (0.5H, m), 6.44 (0.5H, m), 4.61 (0.5H, br d), 4.43 (0.5H, m),
3.87 (2H, m), 3.46 (2H, s), 3.31 (2H, m), 3.05 (1H, m), 2.91 (3H,
m), 2.73 (3H, m), 2.00 (1H, m), 1.84 (0.5H, br d), 1.72 (0.5H, br
d), 1.59 (2H, m), 1.44 (3H, d, J 8.8 Hz), 1.31 (1H, m), 1.14 (0.5H,
m), 0.97-1.03 (6H, m), 0.45 (0.5H, m). LCMS: (ES+) 453 (MH+),
retention time 1.53 min, >98% pure. Free base 14 was dissolved
in DCM and treated with 1.1 equiv of 1 M HCl in ether to give the
hydrochloride salt of the title compound (122 mg, 94% from acid
30). LCMS: (ES+) 453 (MH+), retention time 1.55 min, >98%
pure.
1-[2-(4-([(3R,5S)-3,5-Dimethyl-1-piperazinyl]methyl)phenyl)-2-
methylpropanoyl]-N-(4-fluorophenyl)-4-piperidinamine 15. Bro-
mobenzene 28 was prepared in the same manner as 22 using 1,1-
dimethylethyl (2R,6S)-2,6-dimethyl-1-piperazinecarboxylate instead
of phenylmethyl (2S)-2-methyl-1-piperazinecarboxylate (see com-
pound 11 for details). δH (CDCl3, 250 MHz) 7.44 (2H, d, J 8.0
Hz), 7.25 (2H, d, J 8.0 Hz), 4.09 (2H, m), 3.42 (2H, s), 2.57 (2H,
d, J 11.0 Hz), 2.12 (2H, dd, J 11.3, 4.5 Hz), 1.46 (9H, s), 1.28
(6H, d, 7.8 Hz). LCMS: (ES+) 383/385 (MH+), retention time 3.10
min, >95% pure.
A solution of methyl 2-methylpropanoate (188 µL, 1.64 mmol)
in toluene (3 mL) was added to lithium dicyclohexylamide (362
mg, 1.93 mmol) under glovebox conditions. The suspension was
stirred for 10 min and then added to a mixture of 30 (570 mg, 1.49
mmol) and bis(dibenzylideneacetone) palladium(0) (43 mg, 0.074
mmol). Tri-(tert-butyl)phosphine (18 µL, 0.074 mmol) was added
and the reaction mixture stirred at room temperature overnight. The
solvent was removed in vacuo, and column chromatography, eluting
with 0-90% EtOAc/petroleum ether, gave 31 as a yellow oil (395
mg, 66%). δH (CDCl3, 250 MHz) 7.29 (4H, m), 4.07 (2H, m), 3.66
(3H, s), 3.46 (2H, s), 2.61 (2H, m), 2.12 (2H, dd, J 11.5, 4.3 Hz),
1.58 (6H, s), 1.46 (9H, s), 1.29 (6H, d, J 6.5 Hz). MS: (ES+) 405
(MH+).
A mixture of 31 (395 mg, 0.978 mmol) and lithium hydroxide
monohydrate (82 mg, 1.95 mmol) in water (5 mL) and 1,4-dioxane
(10 mL) was stirred at room temperature for 3 days. The solvents were
removed in vacuo, and the residue was dissolved in water. The solution
was washed with ether, acidified to pH 4 with 1 M HCl and then
extracted with DCM (×2). The combined organics were dried
(Na2SO4) and concentrated in vacuo to give 2-(4-[((3R,5S)-4-([(1,1-
dimethylethyl)oxy]carbonyl)-3,5-dimethyl-1-piperazinyl)methyl]-phen-
yl)-2-methylpropanoic acid as a yellow foam (316 mg, 83%). δH
(CDCl3, 250 MHz) 7.35 (4H, m), 4.11 (2H, br), 3.71 (2H, br), 2.67
(2H, br), 2.17 (2H, br), 1.61 (6H, s), 1.46 (9H, s), 1.33 (6H, br d).
MS: (ES+) 391 (MH+), (ES-) 389 (M - H+).
A solution of diethyl (4-[((3S)-4-([(1,1-dimethylethyl)oxy]car-
bonyl)-3-methyl-1-piperazinyl)methyl]-phenyl)propanedioate (356
mg, 0.748 mmol) in 2 M NaOH solution (3 mL) and 1,4-dioxane
(3 mL) was stirred at room temperature for 1 h and then at 80 °C

1188 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Westaway et al.
Table 5
A mixture of 2-(4-[((3R,5S)-4-([(1,1-dimethylethyl)oxy]-carbo-
nyl)-3,5-dimethyl-1-piperazinyl)methyl]phenyl)-2-methylpropano-
ic acid (100 mg, 0.256 mmol), polymer-supported carbodiimide
(296 mg, 1.3 mmol/g, 0.385 mmol), and 1-hydroxybenzotriazole
(18 mg, 0.128 mmol) in 1:4 DMF/DCM (5 mL) was treated with
N-(4-fluorophenyl)-4-piperidinamine 17a (50 mg, 0.256 mmol), and
the mixture was stirred overnight. Scavenger resins (PS-trisamine,
PS-isocyanate, and Si-carbonate) were added, and the mixture was
shaken for 2 h and then filtered and concentrated to give crude
1,1-dimethylethyl (2R,6S)-4-([4-(2-(4-[(4-fluorophenyl)-amino]-1-
piperidinyl)-1,1-dimethyl-2-oxoethyl)phenyl]methyl)-2,6-dimethyl-
1-piperazinecarboxylate (contains DMF, ∼12% by mass by 1H
NMR). δH (CDCl3, 400 MHz) 7.32 (2H, d, J 8.4 Hz), 7.18 (2H, d,
J 8.4 Hz), 6.83 (2H, t, J 8.8 Hz), 6.45 (2H, dd, J 9.2, 4.4 Hz), 4.51
(1H, br), 4.06 (2H, m), 3.45 (2H, s), 3.26 (2H, m), 2.75 (2H, br),
2.58 (2H, d, J 11.2 Hz), 2.12 (2H, dd, J 11.2, 4.4 Hz), 1.69 (1H,
br), 1.54 (6H, s), 1.46 (9H, s), 1.26 (6H, d, J 6.8 Hz). Some
piperidine signals were very broad and merged into baseline. MS:
(ES) 567 (MH+). This whole sample was treated with 4 M HCl in
1,4-dioxane (2 mL) for 1 h. The solvents were removed in vacuo
to give a colorless solid which was dried under vacuum to give the
dihydrochloride salt of 15 (115 mg, 83% from acid). LCMS: (ES+)
467 (MH+), retention time 1.66 min, >98% pure.
3-((1-[(4-([(3S)-3-Methyl-1-piperazinyl]methyl)phenyl)acetyl]-4-
piperidinyl)amino)benzonitrile 16. Compound 16 was prepared by
methods similar to those used for the synthesis of compounds 11
and 12 using N-(3-cyanophenyl)-4-piperidinamine 17c as required.
δH (CDCl3, 400 MHz), 7.28 (2H, m), 7.20 (3H, m), 6.94 (1H, d, J
7.6 Hz), 6.74 (2H, m), 4.54 (1H, m), 3.88 (1H, m), 3.80 (1H, m),
3.74 (2H, s), 3.47 (2H, s), 3.14 (1H, m), 2.89 (4H, m), 2.74 (2H,
br d), 1.92-2.06 (3H, m), 1.66 (1H, m), 1.33 (1H, m), 1.06 (1H,
m), 1.00 (3H, d, J 6.4 Hz). LCMS: (ES+) 432 (MH+), retention
time 1.59 min, >98% pure. The free base of 16 was converted to
the hydrochloride salt as for previous compounds. LCMS: (ES+)
432 (MH+), retention time 1.59 min, >98% pure.
enzyme
CYP3A4
incubation/assay temperature (°C)
substrate
37
DEF
1
final substrate concentration
(equivalent to Km) (µM)
final enzyme concentration (mg/mL)
final volume of incubation mix (µL)
volume of test compound (µL)
volume of cofactor (µL)
buffer
0.1
220
5
25
50 mM K3PO4 at pH 7
excitation wavelength (nm)
emission wavelength (nm)
485
530
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 concen-
tration-response curve.
CYP 3A4 Time Dependent Inhibition (TDI) Assay. Inhibition
was determined by quantifying the production of fluorescent
metabolite following incubation of CYP3A4 specific profluorescent
probe substrate diethoxyfluorescein (DEF), with heterologously
expressed CYP3A4 in E. coli (Cypex) and the test compound or
positive control (troleandomycin). A NADPH-regenerating system
(cofactor) was prepared as follows: 7.8 mg/mL glucose 6-phosphate
(27.65 mM), 1.7 mg/mL NADP (2.22 mM), and glucose 6-phos-
phate dehydrogenase at 6 enzyme units/mL were made up in 2%
w/v sodium bicarbonate solution. Incubation mixtures containing
enzyme, probe substrate, and 50 mM potassium phosphate buffer
(at pH 7.4) were prepared, and 220 µL was added to each well of
a 96-well plate. An amount of 5 µL of the serially diluted test
compounds was added, and the plate was incubated at 37 °C for
10 min. To start the reaction, 25 µL of cofactor was added. The
production of fluorescence was then measured every minute over
a 30 min time frame at 37 °C. A summary of the assay conditions
is given in Table 5.
Motilin Receptor Agonist FLIPR Assay. The potency and
efficacy of target compounds at the human motilin receptor were
studied using a fluorometric imaging plate reader (FLIPR) and
Chinese hamster ovary (CHO-K1) cells which stably express the
human motilin receptor. Briefly, the human motilin receptor was
cloned (PCR from human genomic DNA) into pCDNA3.1 (Invit-
rogen) in the vector, subcloned into pENTR/D-TOPO (using the
pENTR D-TOPO directional cloning kit) and then recombined into
pCIN1GW vector (LR clonase gateway reaction kit) to give the
pCIN1 motilin receptor. CHO-K1 cells (ATCC No. CCl-61) were
then stably transfected with the pCIN1 motilin receptor plasmid.
These cells were grown as a monolayer in DMEM/HamF-12
supplemented with 10% v/v of FBS, 2 mM GlutaMAX, and 1 mg/
mL Geneticin. Stimulation of this cell line with motilin causes
intracellular signaling leading to an increase in intracellular calcium
which was measured using calcium sensitive fluorescent dyes and
quantified using a FLIPR. Briefly, cells were seeded onto 384-well
black-walled, clear-bottom microtiter plates (Greiner) (10 000 cells/
well) and incubated for 24 h. On the day of assay, media were
aspirated from cell plates using a cell washer (leaving 10 µL of
media). Cells 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 + 2.5 µM Fluo 4 dye) and incubated at 37.5 °C for 1 h.
Master compound plates were prepared in 100% DMSO. A top
concentration of 3 mM was used (giving 12 µM final concentration
in assay), and this was serially diluted 1 in 4. Then 1 µL from the
master plate was transferred to a daughter plate, to which 50 µL of
compound dilution buffer (Tyrodes + 1 mg/mL BSA + 1.5 mM
CaCl2) was added. An amount of 10 µL from the compound plates
was then added immediately to cell plates using a FLIPR 3 calcium
imaging instrument, and changes in fluorescence were measured
over a 1 min time frame. Maximum change in fluorescence over
baseline was used to determine agonist response, and concentration
response curves were constructed using a four-parameter logistic
A seven point compound concentration curve was used to
determine IC50 values in addition to no-compound controls and the
positive control. Thus, the control rate of fluorescent metabolite
production was established from no-compound control incubations
(uninhibited). The extent of inhibition at each test compound
concentration (0.1, 0.33, 1.0, 3.3, 10, 33, and 100 µM) was
calculated as a percentage of the uninhibited control rate (assigned
as 100%), and the IC50 value was determined from these results.
An IC50 was determined for each 5 min interval (0-5, 5-10,
10-15, 15-20, 20-25, and 25-30 min). The ratio of the first and
last IC50 gave a fold change (IC50(initial)/IC50(final)), and if this
was >2, the compound was denoted as displaying TDI.
Rat Pharmacokinetic Studies. The pharmacokinetics and oral
bioavailability of the HCl salt of compound 12 were investigated in
the male Sprague-Dawley rat (n ) 3). The study was carried out on
2 study days with a period of 2 days between each study day. On
study day 1, compound 12 was dissolved in 0.9% (w/v) saline at a
target concentration of 0.2 mg of free base/mL. Compound 12 was
administered as a 1 h intravenous infusion at 5 (mL/kg)/h to three rats
to achieve a target dose of 1 mg of free base/kg. Serial blood samples
were taken from each rat up to 12 h after the start of the infusion. On
study day 2, compound 12 was suspended in 1% (w/v) methylcellulose
at a target concentration of 1 mg of free base/mL. Three rats received
an oral gavage dose of compound 12 administered at 5 mL/kg to
achieve a target dose of 5 mg of free base/kg. Serial blood samples
were taken from each rat up to 12 h after dosing. Diluted blood samples
were analyzed for compound 12 by LC/MS/MS (LLQ was 5 ng/mL,
0.012 µM).
The systemic exposure of the HCl salt of compound 16 following
oral suspension administration was investigated the male Sprague-
Dawley rat. Compound 16 was dosed to three rats orally by gavage
at a target dose of 5 mg of free base/kg. Compound 16 was prepared
on the day of dosing in 1% (w/v) aqueous methylcellulose at a
concentration of 1 mg of free base/mL and administered at 5 mL/
kg. Serial blood samples were taken from each rat up to 8 h after

A 4-Piperidinamine Agonist
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1189
agonist KC 11458 on gastric emptying in diabetic gastroparesis.
Aliment. Pharmacol. Ther. 2004, 20, 333–338.
dose administration. Diluted blood samples were analyzed for parent
compound by LC/MS/MS.
(13) (a) Verhagen, M. A. M. T.; Samson, M.; Maes, B.; Geypens, B. J.;
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229, on gastric emptying and postprandial antroduodenal motility in
healthy volunteers. Aliment. Pharmacol. Ther. 1997, 11, 1077. (b)
Talley, N. J.; Verlinden, M.; Snape, W.; Beker, J. A.; Ducrotte, P.;
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Dog Pharmacokinetic Studies. The pharmacokinetics and oral
bioavailability of the HCl salt of compound 12 were investigated
in the male beagle dog (n ) 3). The study was carried out on 2
study days with a period of 7 days between each study day. On
study day 1, compound 12 was dissolved in 0.9% (w/v) saline at a
concentration of 0.4 mg of free base/mL. Compound 12 was
administered as a 1 h intravenous infusion at 5 (mL/kg)/h to three
dogs to achieve a target dose of 2 mg of free base/kg. Serial blood
samples were taken from each dog up to 30 h after the start of the
infusion. On study day 2, compound 12 was suspended in 1% (w/
v) methylcellulose at a concentration of 1 mg of free base/mL. The
same three dogs each received an oral gavage dose of compound
12 administered at 5 mL/kg to achieve a target dose of 5 mg of
free base/kg. Serial blood samples were taken from each dog up to
30 h after dosing. Diluted blood samples were analyzed for
compound 12 by LC/MS/MS (LLQ was 5 ng/mL, 0.012 µM).
Supporting Information Available: Details of the liquid
chromatography-mass spectrometry (LCMS) procedure and hardware
used for the determination of purity; LCMS chromatograms and spectra
for key compounds 12 and 16; details of the procedure and hardware
for mass directed autoprep (MDAP) purification. This material is
available free of charge via the Internet at http://pubs.acs.org.
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on gastrointestinal symptoms in patients with symptoms suggesting
diabetic gastropathy: a randomized, multi-center, placebo-controlled
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(16) Jasserand, D.; Antel, J.; Preuschoff, U.; Brueckner, R.; Sann, H.; Wurl,
M.; Eickelmann, P. PCT Int. Appl. WO 2002092592, CAN 137:
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(17) 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,
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