Structure-activity relationships of 6-Methyl-benzo[ b ]thiophene-2- carboxylic Acid (1-{(S)-1-Benzyl-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1- yl]butylcarbamoyl}cyclopentyl)amide, potent antagonist of the neurokinin-2 receptor

Article abstract of DOI:10.1021/jm100176s

As part of a project aimed at the identification of a series of small, orally available antagonists for the hNK₂ receptor, starting from one of our capped dipeptide libraries, we succeeded in the chemical optimization of the first identified leads, finally producing a class of molecules with significant activity in our animal model after iv administration. We herein report the results of further chemical modifications made to reduce the overall peptide character of this series and the consequent improvement of their in vivo antagonist activity. The present work identified 6-methylbenzo[b]thiophene-2- carboxylic acid (1-{(S)-1-benzyl-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1- yl]butylcarbamoyl}cyclopentyl)amide (10i), endowed with subnanomolar potency in all the in vitro tests and being highly potent and of long duration upon in vivo testing after both iv and id dosing.

Full text of DOI:10.1021/jm100176s

4148 J. Med. Chem. 2010, 53, 4148–4165
DOI: 10.1021/jm100176s
Structure-Activity Relationships of 6-Methyl-benzo[b]thiophene-2-carboxylic Acid
(1-{(S)-1-Benzyl-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}cyclopentyl)amide,
Potent Antagonist of the Neurokinin-2 Receptor
Daniela Fattori,*,† Marina Porcelloni,† Piero D’Andrea,† Rose-Marie Catalioto,‡ Alessandro Ettorre,† Sandro Giuliani,‡
Elena Marastoni,† Sandro Mauro,† Stefania Meini,‡ Cristina Rossi,† Maria Altamura,‡ and Carlo A. Maggi‡
†Menarini Ricerche, Via Tito Speri 10, 00040 Pomezia, Rome, Italy, and ‡Menarini Ricerche, Via Rismondo 12A, 50131 Florence, Italy
Received February 10, 2010
As part of a project aimed at the identification of a series of small, orally available antagonists for the
hNK2 receptor, starting from one of our capped dipeptide libraries, we succeeded in the chemical
optimization of the first identified leads, finally producing a class of molecules with significant activity in
our animal model after iv administration. We herein report the results of further chemical modifications
made to reduce the overall peptide character of this series and the consequent improvement of their in
vivo antagonist activity. The present work identified 6-methylbenzo[b]thiophene-2-carboxylic acid
(1-{(S)-1-benzyl-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}cyclopentyl)amide
(10i), endowed with subnanomolar potency in all the in vitro tests and being highly potent and of long
duration upon in vivo testing after both iv and id dosing.
Introduction
nanomolar potency on an in vitro functional test and sig-
nificant NK2 antagonist activity on guinea pig after iv
administration at a dose of 3 μmol/kg. One of the best
compounds reported was (R)-40-methyl-N-(1-(1-oxo-3-phenyl-
1-((1-((tetrahydro-2H-pyran-4-yl)methyl)piperidin-4-yl)methyl-
amino)propan-2-ylcarbamoyl)cyclopentyl)biphenyl-4-carbox-
amide (pKi=9.5, Figure 1).2
These molecules showed a general common topological
profile: a rigid and essentially hydrophobic portion (A), con-
taining the dipeptide R,R-cyclopentaneglycine-D-Phe capped
with a planar hydrophobic acyl group at the N-terminus, and a
flexible hydrophilic portion (B) as carboxy terminal capping
group.
Portion A seems to be strongly sensitive to even very small
chemical modifications, whereas portion B, which gives indeed
a large contribution to the binding energy, is very tolerant to
modifications. The two portions A and B are joined by an
amide bond. In addition to this, X-ray crystallography and in
solution NMR both are consistent with a bioactive where the
two aromatic groups of the portion A point toward opposite
directions in 3D space.2
Three mammalian neurokinin (tachykinin) receptors have
been cloned and expressed. They are classified as neurokinin-1
(NK1), neurokinin-2 (NK2), and neurokinin-3 (NK3) and are
activated by three endogenous peptides (tachykinins) widely
distributed in the mammalian peripheral and central nervous
systems. Although each of these tachykinins is the preferred
ligand for one of the receptors, NK1 for substance P (SPa),
NK2 for neurokinin A (NKA), and NK3 for neurokinin B
(NKB), all three are capable of full agonist activity at each of
the receptors, albeit with reduced affinity.
The human NK2 receptor (hNK2) has been identified and
supported as a suitable target for development of novel drugs
to be used for the treatment of a number of diseases in the
respiratory, gastrointestinal, and genitourinary tracts and in
the CNS. As part of a project aimed at the identification of a
series of small, orally available antagonists for the hNK2,
starting from one of our capped dipeptide libraries, we were
able to identify a number of molecules with subnanomolar
binding affinity for the hNK2 receptor.1 All the molecules
were characterized by a rigid core structure containing an R,R-
cyclopentaneglycine fragment. These first initial structures
were further elaborated and led to compounds with low
We herein report the results of the chemical modifications
made to further improve the in vivo antagonist activity of this
class of molecules.
We started with the idea to reduce the overall peptide
character through elimination of one amide linkage. From
previous work it was known that both the amide bonds
formed by the R,R-cyclopentaneglycine were essential for
the binding affinity. Every attempt to alkylate, eliminate, or
substitute them with classical isosteres resulted in a dra-
matic drop in pKi. As a consequence, our effort was con-
centrated on the elimination of the amide linkage between
portions A and B, with accompanying modifications of
portion B.
*To whom correspondence should be addressed. Phone: þ39 06
91184522. Fax: þ39 06 9106137. E-mail: Dfattori@menarini-ricerche.it.
a Abbreviations: AcOH, acetic acid; AcCN, acetonitrile; BSA, bis-
trimethylsilylacetamide; CNS, centralnervous system;DCC dicyclohexyl-
carbodiimide, DIPEA, diisopropylethylamine; DCM, dichloro-
methane; DME, 1,2-dimethoxyethane; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; EDCA, N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide; EtOAc, ethyl acetate; Et2O, diethyl ether; EtOH,
ethanol; GPC, guinea pig colon; HOAt, 1-hydroxy-7-azabenzotriazole;
HOBt, 1-hydroxybenzotriazole; HUB, human urinary bladder; IBS,
irritable bowel syndrome; MeOH, methanol; NK, neurokinin; SP,
susbtance P; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
r
pubs.acs.org/jmc
Published on Web 04/21/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4149
a selected panel of antagonists, an in vivo evaluation was
performed (iv dosing).
Chemistry
Wishing to remove the aforesaid amide bond, we decided
that the first modification to be realized was its substitution
with an ethylene group combined with the introduction of a
second nitrogen atom onto the piperidine ring (i.e., a piper-
azine) in order to avoid an excessive increase of local lipophilic
character, as shown in Figure 2.
A number of planar, relatively lipophilic aromatic systems
were used as N-terminal capping group R according to the
general synthetic sequence reported in Scheme 1. R,β-Unsatu-
rated ester 13 was saturated through catalytic hydrogenation
(2), submitted to basic hydrolysis (3), and finally coupled,
under standard conditions (EDCA, HOBt, DIPEA), with
amine 5 to afford amide 6. Removal of the Boc group (7)
followed by LiAlH4 reduction gave amine 9. The introduction
of portion A to produce the final molecules (10a-i) was
achieved in a single step by reaction of the free amine 9 with
oxazolones 15a-i in DMF at room temperature. These
oxazolones were obtained in a two-step synthetic sequence
from acyl chlorides of general formula 13 that were reacted
with 1-amino-1-cyclopentanecarboxylic acid in the presence
of BSA. BSA here has the double role of activating the amino
group on one side and preventing the free carboxylic acid to
form a mixed anhydride on the other, hence avoiding the need
to protect and deprotect the amino acid. The intramolecular
closure of the so obtained N-capped amino acid 14 with
EDCA completed the sequence.
Figure 1. General structure of our NK2 antagonists represented by
(R)-40-methyl-N-(1-(1-oxo-3-phenyl-1-((1-((tetrahydro-2H-pyran-
4-yl)methyl)piperidin-4-yl)methylamino)propan-2-ylcarbamoyl)-
cyclopentyl)biphenyl-4-carboxamide.2
Table 1. Binding Affinity for the hNK2 Receptor, in Vitro Functional
Activity, and Permeability of Compounds of General Formula 10
Through the application of the same synthetic pathway but
using the L isomer 11 as starting material, we prepared 12, the
enantiomer of 10i. The reaction of the intermediate 7 with
oxazolone 15i was repeated for the synthesis of 8, an analogue
of 10i where an amide group was reintroduced. However,
since in this case the amide is tertiary, the overall balance still
resulted in a net elimination of a hydrogen bond group donor
compared to the starting motif.
When possible, final compounds were simply crystallized;
otherwise, they were purified by preparative HPLC to obtain
the corresponding trifluoroacetate salts.
Pushed by the intention to explore further the motif of the
amide/ethylene substitution with slight modifications on the
flexible hydrophilic moiety, we coupled either acid 3 or its
N-succinimidyl activated form 4 (Scheme 2)4 to a series of
piperidine and piperazine derivatives. This led, after Boc
deprotection, to amine hydrochlorides 16-21. An exhaustive
reduction of the carbonyl groups with LiAlH4 in THF at
refluxing temperature, prior to the coupling with oxazolone
15i, allowed us to obtain the fully reduced compounds 22, 24,
26, 27, and 28 (Scheme 2). The direct reaction of the inter-
mediate amines 17, 18, and 21 with oxazolone 15i led to
compound 23, 25, and 29, giving the opportunity to further
explore the properties of the tertiary amides.
The increased flexibility of the C-terminal portion due to the
amide/ethylene replacement encouraged us to prepare and test
also the shorter homologues of 10i. The coupling reaction of
the commercially available acid 38 with amine 5, followed by
Boc deprotection, exhaustive reduction with LiAlH4, and final
reaction with oxazolone 15i gave 40, the one methylene unit
shorter homologue of 10i (Scheme 3). The two methylene units
shorter homologue 32 was prepared from reductive amina-
tion of the commercially available Boc-protected aldehyde of
a pKi for inhibition of specific binding of [125I]neurokinin A to
recombinant hNK2 receptor in a cell membrane preparation. b pKB
functional activity in guinea pig isolated proximal colon. For details
see the Experimental Section. c Caco-2 permeability. For details see the
Experimental Section.
Once prepared, the compounds were submitted to binding
affinity evaluation on the hNK2 receptor and the results are
reported in Tables 1-3. Since our in vivo test was going to be
performed on guinea pig, the most active molecules (pKi g
8.5) were evaluated for functional antagonist potency in
guinea pig isolated proximal colon (GPC pKB). Finally, for

4150 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Fattori et al.
Table 2. Binding Affinity for the hNK2 Receptor and in Vitro Functional Activitya
a pKi for inhibition of specific binding of [125I]neurokinin A to recombinant hNK2 receptor in a cell membrane preparation; for details see the
Experimental Section. pKB gunctional activity in guinea pig isolated proximal colon; for details see the Experimental Section.
D-phenylalanine (30) with amine 5 in presence of Na(AcO)3-
BH5 as reducing agent, followed by Boc deprotection in acidic
media and subsequent reaction with oxazolone 15i. We also
reduced the newly created flexibility by synthesizing the trans-
olefin as amide geometrical isoster. Wittig reaction of aldehyde
30 with (formylmethylene)triphenylphosphorane gave the un-
saturated aldehyde 33, which was submitted to reductive
amination with amine 5 in the Magid conditions to give olefin
36. Boc deprotection of the amine and then reaction with
oxazolone 15i led to the planned analogue 37. The reductive
amination of 33 with morpholine(piperidin-4-yl)methanone,
followed by the three-step sequence catalytic hydrogenation/
acidic Boc-deprotection/oxazolone reaction, afforded com-
pound 35, where the methylene joining the two C-terminal
six-membered rings is replaced by a carbonyl, with an overall
reduction of the system rotational freedom.
situ from phosphonium salt 50, which was in turn prepared
according to the sequence reported in Scheme 4, and afforded
42. Subsequent hydrogenation of 42 followed by selec-
tive removal of the trityl group in mild acidic media and
reaction with oxazolone 15i resulted in intermediate 45.
Removal of the Boc group in strong acidic medium and then
reductive amination with 4-tetrahydropyranylaldehyde gave
47 (Scheme 4).
The two compounds containing an ether junction between
portions A and B were both prepared from D-phenylalaninol
51. Alkylation with mesylate 57 or iodide 49 of the alcoholate
of 51 formed in situ gave ethers 52a and 52b, respectively.
Reaction with oxazolone 15i followed by Boc cleavage in
acidic media and reductive amination with 4-tetrahydropyr-
anylaldehyde yielded products 55a and 55b (Scheme 5).
The exploration was completed by the preparation of a
number of molecules differing from 10i only at the substituent
on the terminal piperazine nitrogen, which were prepared
according to two major routes, A and B, reported inScheme6.
Compound 47, where an additional nitrogen atom has been
eliminated, was obtained from aldehyde 41.6 This aldehyde
was submitted to a Wittig reaction with the ylide generated in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4151
Table 3. Binding Affinity for the hNK2 Receptor and in Vitro Functional Activity of Compounds of General Formula 67
a pKi for inhibition of specific binding of [125I]neurokinin A to recombinant hNK2 receptor in a cell membrane preparation. For details see the
Experimental Section. b pKB functional activity in guinea pig isolated proximal colon. For details see the Experimental Section.
Figure 2. Working strategy: removal of the amide bond linking portions A and B.
In the first one the cleavage of the Boc group of ester 2
afforded 58, which was reacted with oxazolone 15i to obtain
ester 59. Basic hydrolysis of the ester, followed by coupling
with N,O-dimethylhydroxylamine to prepare the Weinreb
amide and reduction with LiAlH4 afforded aldehyde 62. This
was reductively aminated in presence of Na(OAc)3BH with
variously functionalized piperidines 69 to produce the main
part of the derivatives 67.
Alternatively, reductive amination of aldehyde 33 with
Cbz-piperazine afforded 63, which was sequentially depro-
tected in acidic media, coupled with oxazolone 15i, and
submitted to hydrogenation in the presence of ammonium

4152 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Fattori et al.
Scheme 1a
a Reagents: (a) 10% Pd-C, H2, EtOH; (b) 1 M NaOH, MeOH/H2O, 1:1; (c) DCC, N-hydroxysuccinimide, THF; (d) EDCA, HOBt, DIPEA, DMF,
room temp; (e) 4 N HCl in dioxane, room temp; (f) 15i, DMF, room temp; (g) LiAlH4, THF, room temp; (h) 15a-i, DMF, room temp; (i) BSA, DCM,
room temp; (l) EDCA, DIPEA, DME, room temp.
formate. Through this latest step the double bond was satu-
rated and the Cbz group was removed to produce 66. Reduc-
tive amination with a variety of aldehydes gave the remaining
compounds 67. Formyl derivative 67p was obtained as a side
product of the homogeneous hydrogenation reaction and
isolated through preparative HPLC purification.
respect to the amide carbonyl (B, Figure 3), while for all the
benzothiophenes the situation is inverted (A, Figure 3).
We might suppose that the proximity of the heterocyclic
oxygen helps somewhat the reduction of the hydrogen bond
potential of the amide bond. This would facilitate the desolva-
tion of the solute, necessary to enter the hydrophobic, non-
hydrogen bonding interior of the membrane. In fact, desolva-
tion is considered the rate limiting step in transport across the
cell membrane.7 These conformational preferences appeared
to further confirm our general observation, formulated for
very close chemical series, that the SARs relative to com-
pounds bearing 6-substituted benzothiophenes as acyl capping
groups paralleled well with those of compounds capped with
the corresponding 5-substituted benzofuranes and vice versa.
Considering the data reported in Table 1, we decided that the
best balance between binding affinity to the human receptor
and functional activity was that given by the 6-methylbenzo-
thiophene capping group, and therefore, we used this moiety
for the study of further modifications in the flexible hydro-
phobic portion. Our previous work had shown a clear pre-
ference for the phenyl alanine in the D configuration over the
L one. However, we wondered if the increase in flexibility due to
the elimination of the amide could reverse the situation.
Compound 12, an enantiomer of 10i, demonstrated that
this was not the case. In fact both the binding affinity and
the functional activity of 12 were almost 1 log unit lower
Results and Discussion
In the first set of compounds (Table 1) of general formula
10, portion B was maintained constant while varying the
N-terminal capping group. Binding affinities were always in
the subnanomolar range, reaching the low picomolar value
with the methylbenzothiophene derivative 10i. The results
from the functional activity test on guinea pig isolated proxi-
mal colon were also quite good, with values always in the low
nanomolar range. The permeability evaluated in the Caco-2
cell system was also generally good (Table 1). We found the
high values of permeability for the benzofurane derivatives
10d(20.2) and 10g (30.3)intriguingwhencomparedwiththose
of the corresponding benzothiophenes 10h (11.0) and 10i
(11.1). Further experimental work with the Caco-2 cells
excluded that the observed difference was due to active
transport. A search in the Cambridge Crystallographic Data-
base for benzothiophene and benzofurane-1-carboxylic acid
amides showed an interesting feature: all the benzofurans
seemed to have the ring oxygen in the anti configuration with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4153
Scheme 2a
the role of an anchoring section for the receptor according to
the Verkhivker definition.8 The results concerning the in vitro
functional activity reported in Tables 1-3 show that the pKB
isgenerally 1 logunitor morelowerthanthe pKi and this is not
dependent on the hydrophylicity/lipophlicity balance. The
fact that the antagonist potency at the guinea pig receptor is
within 1 log unit lower than the affinity for the human
receptor is a general trend for this series of compounds,
already observed in the past, and seems not to be dependent
on the species but rather on the different experimental condi-
tions used to estimate the affinity (radioligand binding) and
the antagonist potency (smooth muscle contractility assay in
the GPC). In addition, because of the different incubation
times of the experiments and considering the high values of
binding affinities, we cannot exclude effects due to differences
in residence time.9 Very likely, when such differences are
higher than 1 log unit, in addition to the different testing
conditions, they are also related to the differences between
human and GP receptors. Indeed this was the case for the few
compounds within this project that were tested for functional
activity on isolated urinary bladder (HUB), where the differ-
ence remained within 1 log unit.
In Vivo Pharmacology. As anticipated, selected compounds
were tested in our animal model.10 The potency in inhibiting the
colonic contractions induced by the selective tachykinin NK2
receptor agonists [βAla8]neurokinin A(4-10) ([βAla8]neuro-
kinin A(4-10), 3 nmol kg-1 iv) in guinea pig was evaluated
after iv administration at a cutoff dose of 3 μmol kg-1. The
results, obtained after iv administration of the antagonist, are
expressed both as maximal inhibitory effect reached (%imax)
and as i%max (Table 4). The latter value is expressed as the
P
sum of the percent inhibition vs the basal response of the colon
contractions induced by the selective agonist at nine time-point
observations (5, 30, 60, 90, 120, 150, 180, 210, and 240 min)
after iv administration of the antagonist and further calculated
a Reagents: (a) EDCA, HOBt, DIPEA, amine, DMF, room temp; (b)
DIPEA, amine, THF; (c) 4 N HCl in dioxane; (d) LiAlH4, THF, reflux;
(e) 15i, DMF, room temp.
as a percentage of the theoretical maximal inhibitory response
P
(
(%imax-th)), which is a constant and equal to 900. The mean
percent inhibition over the entire experiment is expressed as
(9.2 vs 10.3 for pKi and 7.7 vs 8.6 for pKB, respectively) than
those of 10i.
P
X
ð%iÞ
Analogous results were obtained by introducing a trans
double bond as a geometrical mimic of the anti amide linkage
(compound 37). Shortening the aliphatic chain connecting the
hydrophobic portion by one (40) or two (32) methylenes was
also deleterious for binding affinity, with a drop of 1 and 2 log
units, respectively (Table 2).
P
i%max ¼
ꢀ 100
ð%imax-th
Þ
This parameter gives a measure of the activity during
the entire experimental period therefore allowing the evalua-
tion of both the intensity and the duration of the anta-
gonist effect. The maximal inhibition corresponds to Σi%
max =100, while the absence of effect results in Σi%max =0.
In this case, as expected, activities were very different for
the different compounds, reflecting the complexity of the
multiparametric nature of the interaction of a small molecule
with a living organism. The interplay between tissues dis-
tribution, metabolic degradation, protein binding, etc. resul-
ted in almost no in vivo activity for 22 to the full antagonist
potency of 10i, in spite of the two compounds differing only
for the position of one nitrogen atom. In fact, while in 22 the
nitrogen atom is in the second ring of the flexible portion B,
in 10i it is in the first heterocyclic ring of the same molecular
portion.
The reintroduction of a carbonyl group near the piperazine
ring (7), the displacement of the nitrogen atom from the first
ring to the second one (22), the deletion of a nitrogen atom on
the first ring through substitution with one carbon unit (47),
and the introduction of an oxygen atom on the flexible
C-terminal chain (55a and 55b) seemed to have no major
effect on the binding but a considerable impact on functional
activity, which drops below 8. The same was true for many
analogues withthe exception of the derivativewhere the pyran
ring was opened to form an alcohol (27). In this case a
significant fall in binding affinity was also observed.
In the subsequent panel of derivatives, the C-terminal
piperazine was maintained whereas the molecular properties
were modulated through the piperazine N substituent
(Table 3). Also in this case, in spite of the different chemical
nature of the groups introduced, like basic aromatic hetero-
cycles, hydrophilic heterocycles, sulfonamides, sulfamides,
and guanidines, the binding affinities were all subnanomolar,
confirming that very likely the rigid lipophilic portion plays
Because of its very good activity, 10i was further profiled.
It was tested for functional activity on human bladder
(HUB) and found to have pA2=9.7. Administration via id
at a dose of 10 μmol/kg showed a significant systemic activity
(Figure 4).
In conclusion a work that started with libraries of capped
dipeptides and then optimized separately at the various

4154 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Fattori et al.
Scheme 3a
a Reagents: (a) 5 or morpholine(piperidin-4-yl)methanone, Na(AcO)3BH, THF, 0 °C; (b) 4 N HCl in 1,4-dioxane, room temp; (c) 15i, DMF, room
temp; (d) (formylmethylene)triphenylphosphorane, DCM, room temp; (e) 10% Pd/C, EtOH; (f) 5, EDCA, HOAt, DIPEA, DMF, room temp; (g)
LiAlH4, THF.
Scheme 4a
a Reagents: (a) NaHMDS 1M, THF, 50; (b) 10% Pd-C, H2, EtOH; (c) 1% TFA, DCM; (d) 15i, DIPEA, DMF; (e) 4 N HCl in 1,4-dioxane;
(f) tetrahydropyranecarboxaldehyde, Na(OAc)3BH, DCM; (g) PPh3, imidazole, I2, DCM; (h) PPh3, CH3CN.
components while continuing to reduce the peptidic char-
acter led to the identification of compound 10i endowed with
subnanomolar potency in all the in vitro tests and that was
highly potent and of long duration upon in vivo testing, after
both iv and id dosing.
Experimental Section
(A) Chemistry. Commercial chemicals and solvents were of
reagent grade and used without further purification. Merck silica
gel (Kieselgel 60) was used for analytical thin-layer chromatogra-
phy (TLC, F254 plates) and flash chromatography (230-400 mesh).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4155
Figure 3. Possible planar conformations for benzothiophene- and benzofurane-2-carboxamides. Conformations A and B are represented in
the Cambridge Crystallographic Database, while conformations C and D are not represented.
Scheme 5a
a Reagents: (a) KH, THF, 57 or 49; (b) 15i, DMF, room temp; (c) 4 N HCl in dioxane, room temp; (d) 4-tetrahydropyranylaldehyde, Na(AcO)3BH,
DCM, room temp; (e) MsCl, DCM, DMAP.
Scheme 6a
a Reagents: (a) 4 N HCl in 1,4-dioxane; (b) 15i, DIPEA, DMF, room temp; (c) 1 M NaOH, H2O/MeOH; (d) EDAC, HOBt, N,O-dimethylhydroxyl-
amine HCl, DIPEA, DMF; (e) LiAlH4, THF; (f) A, Na(OAc)3BH, DCM; (g) Cbz-piperazine, Na(OAc)3BH, DCM; (h) ammonium formate, 10% Pd/C,
EtOH; (i) RCHO, Na(OAc)3BH, DCM.
Purity was evaluated by analytical HPLC using a 600 E Waters
pump coupled to a Jasco 875 UV detector and a Merck-Hitachi
D-2500 integrator or a system comprising a Jasco PU-980 pump,
LG-980-02 gradient unit, a UV-975 UV/vis detector, and a
Merck-Hitachi D-2500 integrator or a Beckman System Gold
apparatus or an Agilent 1100 analytic HPLC system. The solvents
were (A) water þ 0.1% TFA and (B) AcCN þ 0.1% TFA with
flow rate of 1 mL/min. System A consisted of the following:
Symmetry RP-C18 column, 3.5 μm, 4.6 mm ꢀ 100 mm; λ=214
nm; 1 min 10% solvent B, then from 10% B to 80% B in 10 min;
flow of 1.0 mL/min. System B consisted of the following: Vydac
pept and prot C18 column, 5 μm, 4.6 mm ꢀ 250 mm; λ=223 nm;
gradient from 20% B to 100% B in 20 min. All the tested
compounds had purities of g95%.
Preparative reverse phase HPLC was performed on a Waters
600E apparatus with a Jasco 874 UV detector or on a Waters

4156 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Fattori et al.
4-tert-Butoxycarbonylamino-5-phenylpentanoic Acid (3). A
solution of 1 (9.39 g, 29.4 mmol) in EtOH (180 mL) was stirred
with 10% Pd/C (939 mg) under hydrogen atmosphere for 5 h.
The catalyst was filtered off and the solvent was removed in
vacuo affording 2 as a white solid (9.00 g, 95%). Ester 2 (2.00 g,
6.22 mmol) was dissolved in 1 M NaOH and 1:1 MeOH/H2O.
The hydrolysis was immediate. Then 1 M HCl was added
dropwise until complete precipitation of the crude acid, which
was collected by filtration, washed with water, and finally dried
in vacuo to obtain crude 3 (1.66 g, 91%), used as such without
further purification. 1H NMR (δ, DMSO-d6, 300 MHz): 11.99
(bs, 1H), 7.29-7.16 (m, 5H), 6.72 (d, 1H, J=6.7 Hz), 3.60 (m,
1H), 2.65 (m, 2H), 2.20 (m, 2H), 1.70-1.62 (m, 1H), 1.54-1.46
(m, 1H), 1.32 (s, 9H).
{1-Benzyl-4-oxo-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-
1-yl]butyl}carbamic Acid tert-Butyl Ester (6). Acid 3 (1.00 g, 3.40
mmol) was dissolved in dry DMF (10 mL). Amine 5 (690 mg,
4.08 mmol), HOBt (551 mg, 4.08 mmol), EDCA (783 mg, 4.08
mmol), and DIPEA (0.59 mL, 3.40 mmol) were added. The
mixture was stirred at room temperature. At the end of the
reaction (HPLC control), NaHCO3 aqueous saturated solution
(30 mL) was added and stirring was continued for an additional
30 min. Then EtOAc was added (50 mL). The two phases were
separated, and the organic one was washed with NaHCO3
saturated aqueous solution (ꢀ3), 1 N NaOH, and brine until
neutral pH was obtained. Then it was dried over Na2SO4 and
concentrated under reduced pressure to obtain crude 6 (1.29 g,
82.3%). 1H NMR (δ, DMSO-d6, 300 MHz): 7.35-7.10 (m, 5H),
6.70 (d, 1H), 3.95 (m, 1H), 3.90-3.75 (m, 2H), 3.65-3.50 (m,
2H), 3.30-3.20 (m, 4H), 2.70-2.60 (m, 2H), 2.40-2.20 (m, 6H),
2.20-2.05 (m, 2H), 1.85-1.45 (m, 5H), 1.45-1.20 (m, 9H),
1.20-1.0 (m, 2H). MS m/z calcd for C26H41N3O4: 459.62.
Found 460.3 [M þ 1]þ. HPLC purity: system A, tR=7.265 min.
4-Amino-5-phenyl-1-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-
yl]pentan-1-one Dihydrochloride (7). HCl, 4 M in dioxane
(20 mL), was added to a solution of 6 (1.29 g, 2.80 mmol) in
dioxane (5 mL), and the resulting mixture was stirred at room
temperature for 1 h. The solvent was removed in vacuo. Et2O
was added and removed (ꢀ3) in order to totally eliminate the
residue HCl. The resulting solid was finally triturated with Et2O
and collected by filtration to afford 7 as a yellow solid (1.20 g,
quantitative). MS m/z calcd for C21H33N3O2: 359.51. Found
360.1 [M þ 1]þ. HPLC purity: system A, tR=4.138 min.
1-Benzyl-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]-
butylamine Hydrochloride (9). The hydrochloride salt of amine 7
(1.10 g, 2.08 mmol) was suspended in dry THF (30 mL). The
suspension was cooled in an ice bath under nitrogen atmosphere,
and a 1 M THF solution of LiAlH4 (20.8 mmol) was added. The
mixture was then stirred at room temperature for 3 h. LC-MS
control showed the total consumption of the starting material and
the formation of the product. The reaction mixture was cooled to
3-4 °C. Then the excess of LiAlH4 was quenched by slow and
cautious addition of ice. When the hydride was completely
quenched (no more H2 evolution was observed), NaOH pellets
was added and the resulting suspension was stirred at room
temperature for 1 h. The inorganic salts were filtered off through-
out Celite washing with DCM. The biphasic system obtained was
saturated with NaOH. Then it was transferred into a separation
funnel and the phases were separated. The organic phase was dried
over Na2SO4, filtered, and the solvent was removed under reduced
pressure. The desired product was obtained as a pale-yellow oil
used in the next reaction without any further purification. MS m/z
calcd for C21H35N3O: 345.52. Found 346.2 [M þ 1]þ.
Figure 4. In vivo activity of compound 10i after id administration
at a dose of 10 μmol/kg in inhibiting the colonic contractions
induced by the NK2 receptor agonist [βAla8]neurokinin A(4-10).
The vehicle is DMSO.
Table 4. In Vivo Evaluation of Selected Compoundsa
P
P
compd
i%max b %imax ( SEM c compd
i%max b %imax ( SEM c
7
65
44
66
55
88
80
91
67
94
8
99 ( 1
60 ( 7
86 ( 6
65 ( 5
100 ( 0
98 ( 1
100 ( 0
82 ( 9
100 ( 0
62 ( 9
90 ( 6
47 ( 11
99 ( 1
81 ( 8
72 ( 14
67 ( 11
46 ( 3
100 ( 0
76 ( 7
67c
67d
67e
67f
67g
67h
67i
45
63
72
71
55
50
83
47
22
35
79
71
39
62
48
43
19
29
47
63 ( 5
66 ( 4
90 ( 4
84 ( 9
86 ( 7
83 ( 8
88 ( 9
67 ( 13
83 ( 8
72 ( 9
97 ( 2
100 ( 0
85 ( 5
97 ( 2
80 ( 9
62 ( 5
85 ( 7
77 ( 5
100 ( 0
10a
10b
10d
10e
10f
10g
10h
10i
22
67j
67k
67l
25
27
60
11
59
26
42
41
23
75
60
67n
67o
67q
67r
67s
67t
67u
67v
67y
29
35
37
47
55a
67a
67b
a Inhibition of colonic contractions induced by [βAla8]NKA(4-10) in
P
P
guinea pig after iv administration at 3 μmol/kg. b i%max = (%i)/
P
(%imax-th) ꢀ 100 = mean % inhibition over the entire experiment,
that is, the sum of the % inhibition, vs the basal response of the colon
contraction to [βAla8]neurokinin A(4-10) at the nine times of observa-
tion (5, 30, 60, 90, 120, 150, 180, 210, and 240 min) after iv administration
of the antagonist and further calculated as the percentage of the
theoretical maximal inhibitory response [ (%imax-th) = 900%]. c Maxi-
mal inhibitory effect as % inhibition of the basal response.
P
Delta-Prep 3000 apparatus. The mobile phases were the same as
for the analytical systems. Gradient elution was employed. The
columns used were a SymmetryPrep C18, 7 μm, 19 mm ꢀ 300 mm,
a Hibar Lichrosorb RP-18, 7 μm, 25 mm ꢀ 250 mm, a Vydac C18,
10 μm, 22 mm ꢀ 250 mm, or Jupiter, 15 μm, 250 mm ꢀ 21.2 mm.
Peak detection was at 220 and 254 nm. Chemical yields are not
optimized. NMR experiments were recorded on a Varian Gemini
200 model J200 HC, a Varian 300 MHz spectrometer (equipped
with a 5 mm inverse probe), a Bruker Avance 400 MHz, or a
Bruker Avance 600 MHz machine and are referenced to residual
solvent signals: CDCl3 (δ 7.26) or DMSO-d6 (δ 2.49). Chemical
shifts are reported in δ units (parts per million) and are assigned as
singlet (s), doublet (d), doublet of doublets (dd), triplet (t), multi-
plet (m), broad signal (br), or very broad signal (vbr). Coupling
constants (J) are reported in hertz (Hz).
General Procedure for the Synthesis of Oxazolones 15a-i. To
a suspension of the 1-amino-1-cyclopentanecarboxylic acid
(5.70 g, 97%, 44 mmol) in dry DCM (200 mL), BSA (22 mL,
90 mmol) was rapidly added under inert atmosphere. The
mixture was kept under magnetic stirring at room temperature
until the solution became completely clear. A solution of the
selected acyl chloride 13 (37 mmol) in DMC (100 mL) was added
Mass spectra were recorded using a Waters Alliance 2795
HPLC system fitted with a UV-PDS 996 diode array detector, a
ZMD mass spectrometer, and a GL Science Inertsil ODS-3
column (50 mm ꢀ 3 mm, 3 μm) or a ThermoFinnigan LCQ
equipped with APCI or ESI source.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4157
dropwise. After one night at room temperature the solvent was
removed under reduced pressure and the residue treated with
5% aqueous K2CO3 (300 mL) and extracted with EtOAc (ꢀ3).
The aqueous phase was acidified with 37% HCl to the complete
precipitation of intermediate 14 as a solid which was collected by
filtration and dried under vacuum. Intermediate 14 (9.9 mmol)
was dissolved in DME (48 mL). Then EDCA (2.08 g, 10.8 mmol)
and DIPEA (1.69 mL, 9.9 mmol) were added. If necessary,
DMF was added in order to obtain a clear solution. The reaction
mixture was stirred at room temperature overnight and then
concentrated under reduce pressure. The residue was parti-
tioned between EtOAc and 5% NaHSO4. The phases were
separated and the organic one was washed with 5% NaHCO3
and brine, dried over Na2SO4, filtered, and concentrated to give
oxazolones 15a-i in quantitative yield.
General Procedure for the Reaction of Oxazolones 15 with
Amines 7 and 9. To a stirred solution of the amine (0.87 mmol) in
DMF (5 mL), the selected oxazolone 15 (1.21 mmol) was added.
The resulting mixture was stirred at room temperature for 15 h.
HPLC showed total consumption of the amine. EtOAc (20 mL)
and 10% NaHCO3 (20 mL) were added, and the two phases
were separated. The organic layer was washed with brine
(10 mL), dried over Na2SO4, filtered, and concentrated under
reduced pressure. The resulting crude product was purified by
flash chromatography (silica, CHCl3/MeOH various propor-
tions) or by preparative HPLC.
(m, 2H), 3.30 (m, 2H), 3.20-2.60 (m, 10H), 2.40 (m, 2H), 2.30 (m,
2H), 2.00 (m, 1H), 1.90 (m, 1H), 1.80-1.35 (m, 13H), 1.10 (m, 2H).
MS m/z calcd for C38H50N4O3: 610.3. Found 611.5 [M þ 1]þ.
HPLC purity: system B, tR=8.53 min.
Benzofuran-2-carboxylic Acid (1-{(S)-1-Benzyl-4-[4-(tetrahydro-
pyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}cyclopentyl)-
amide Trifluoroacetate Salt (10d). Amine 9 was reacted with oxa-
zolone 15d according to the general procedure. The crude product
was purified by preparative HPLC. 1H NMR (δ, DMSO-d6, 400
MHz): 9.25 (brs, 1H), 8.50 (s, 1H), 7.80 (d, 1H), 7.70 (d, 1H), 7.65
(s, 1H), 7.50 (m, 2H), 7.35 (dd, 1H), 7.20 (m, 5H), 4.05 (m, 1H),
3.85 (m, 2H), 3.35 (m, 2H), 3.20-2.80 (m, 8H), 2.70 (m, 2H), 2.40
(m, 2H), 2.20 (m, 2H), 2.00 (m, 1H), 1.85 (m, 1H), 1.80-1.35 (m,
13H), 1.15 (m, 2H). MS m/z calcd for C36H48N4O4: 600.4. Found
601.5 [M þ 1]þ. HPLC purity: system B, tR=8.25 min.
5-Chlorobenzofuran-2-carboxylic Acid (1-{(S)-1-Benzyl-
4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}-
cyclopentyl)amide Trifluoroacetate Salt (10e). Amine 9 was reacted
with oxazolone 15e according to the general procedure. The crude
product was purified by preparative HPLC. 1H NMR (δ, DMSO-
d6, 400 MHz): 8.60 (s, 1H), 7.93 (d, 1H), 7.74 (d, 1H), 7.63 (s, 1H),
7.50 (m, 2H), 7.20 (m, 5H), 4.10-2.90 (m, 17H), 2.72 (m, 2H),
2.20 (m, 1H), 2.00-1.05 (m, 16H). MS m/z calcd for C36-
H47ClN4O4: 634.3. Found 635.4 [M þ 1]þ. HPLC purity: system
B, tR=8.84 min.
5-Methoxybenzofuran-2-carboxylic Acid (1-{(S)-1-Benzyl-
4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}-
cyclopentyl)amide Trifluoroacetate Salt (10f). Amine 9 was
reacted with oxazolone 15f according to the general procedure.
The crude product was purified by preparative HPLC. 1H NMR
(δ, DMSO-d6, 400 MHz): 8.50 (s, 1H), 7.60 (d, 1H), 7.58 (s, 1H),
7.50 (d, 1H), 7.30-7.07 (m, 7H), 4.11-2.50 (m, 18H), 3.85 (s,
3H), 2.20 (m, 1H), 2.00-1.05 (m, 17H). MS m/z calcd for
C37H50N4O5: 630.4. Found 631.4 [M þ 1]þ. HPLC purity:
system B, tR=8.84 min.
5-Methylbenzofuran-2-carboxylic Acid (1-{(S)-1-Benzyl-
4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}-
cyclopentyl)amide Trifluoroacetate Salt (10g). Amine 9 was
reacted with oxazolone 15g according to the general procedure.
The crude product was purified by preparative HPLC. 1H NMR
(δ, DMSO-d6, 400 MHz): 8.45 (s, 1H), 7.55 (3H, m), 7.45 (m,
1H), 7.30 (d, 1H), 7.20 (m, 5H), 4.00 (m, 1H), 3.90-2.80
(m, 16H), 2.70 (m, 2H), 2.40 (s, 3H), 2.20 (m, 1H), 2.00
(m, 1H), 1.90-1.05 (m, 15H). MS m/z calcd for C37H50-
N4O4: 614.8. Found 615.4 [M þ 1]þ. HPLC purity: system A,
tR=7.43 min.
Benzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Benzyl-
4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}-
cyclopentyl)amide Trifluoroacetate Salt (10h). Amine 9 was
reacted with oxazolone 15h according to the general procedure.
The crude product was purified by preparative HPLC. 1H NMR
(δ, DMSO-d6, 400 MHz): 9.25 (brs, 1H), 8.60 (s, 1H), 8.25 (s,
1H), 8.00 (dd, 2H), 7.45 (m, 3H), 7.20 (m, 5H), 4.00 (m, 1H),
3.95-2.80 (m, 16H), 2.73 (m, 2H), 2.20 (m, 1H), 2.00 (m, 1H),
1.95-1.25 (m, 13H), 1.15 (m, 2H). MS m/z calcd for
C36H48N4O3S: 616.3. Found 617.5 [M þ 1]þ. HPLC purity:
system B, tR=8.40 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-oxo-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]-
butylcarbamoyl}cyclopentyl)amide Trifluoroacetate Salt (8).
Amine 7 was reacted with oxazolone 15i according to the general
procedure. The crude product was purified by preparative
HPLC. 1H NMR (δ, DMSO-d6, 400 MHz): 9.11 (brs, 1H), 8.3
(s, 1H), 8.05 (s, 1H), 8.03 (d, 1H), 7.85 (s, 1H), 7.69-7.38
(m, 3H), 7.32-7.00 (m, 4H), 4.02-3.98 (m, 1H), 3.72-3.50
(m, 4H), 3.44-3.36 (m, 3H), 2.80-2.65 (m, 3H), 2.43 (s, 3H),
2.52-2.33 (m, 4H), 2.09-1.81 (m, 7H), 1.69-1.51 (m, 6H),
1.45-1.28 (m, 6H). MS m/z calcd for C37H48N4O4S: 644.3.
Found 645.3 [M þ 1]þ.
40-Methylbiphenyl-4-carboxylic Acid (1-{(S)-1-Benzyl-4-[4-
(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}-
cyclopentyl)amide Trifluoroacetate Salt (10a). Amine 9 was
reacted with oxazolone 15a according to the general proce-
dure. The crude product was purified by preparative HPLC.
1H NMR (δ, DMSO-d6, 400 MHz): 9.15 (brs, 1H), 8.40
(s, 1H), 7.96 (d, 2H, J = 8.4 Hz), 7.75 (d, 2H, J = 8.4 Hz),
7.62 (d, 2H, J=8.4 Hz), 7.40 (d, 1H), 7.30 (d, 2H, J=8.4 Hz),
7.20 (m, 5H), 4.00 (m, 1H), 3.80 (m, 2H), 3.25 (m, 2H),
3.15-2.70 (m, 10H), 2.40 (m, 2H), 2.35 (s, 3H), 2.25 (m,
2H), 2.00 (m, 1H), 1.85 (m, 1H), 1.80-1.35 (m, 13H). MS
m/z calcd for C41H54N4O3: 650.4 Found 651.5 [M þ 1]þ.
HPLC purity: system A, tR=8.265 min.
Biphenyl-4-carboxylic Acid (1-{(S)-1-Benzyl-4-[4-(tetrahydro-
pyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}cyclopentyl)-
amide Trifluoroacetate Salt (10b). Amine 9 was reacted with
oxazolone 15b according to the general procedure. The crude
product was purified by preparative HPLC. 1H NMR (δ,
DMSO-d6, 400 MHz): 9.15 (brs, 1H), 8.40 (s, 1H), 8.00 (dd,
2H), 7.75 (m, 4H), 7.50 (m, 2H), 7.30 (m, 1H), 7.25 (m, 1H), 7.20
(m, 5H), 4.00 (m, 1H), 3.80 (m, 2H), 3.25 (m, 2H), 3.15-2.70 (m,
10H), 2.40 (m, 2H), 2.25 (m, 2H), 2.00 (m, 1H), 1.85 (m, 1H),
1.80-1.35 (m, 13H), 1.15 (m, 2H). MS m/z calcd for
C40H52N4O3: 636.9. Found 637.5 [M þ 1]þ. HPLC purity:
system B, tR=9.46 min.
Naphthalene-2-carboxylic Acid (1-{(S)-1-Benzyl-4-[4-(tetra-
hydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}cyclo-
pentyl)amide Trifluoroacetate Salt (10c). Amine 9 was reacted with
oxazolone 15c according to the general procedure. The crude
product was purified by preparative HPLC. 1H NMR (δ,
DMSO-d6, 400 MHz): 9.15 (brs, 1H), 8.50 (m, 2H), 8.00 (m,
4H), 7.60 (m, 2H), 7.45 (m, 1H), 7.20 (m, 5H), 4.00 (m, 1H), 3.80
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarba-
moyl}cyclopentyl)amide (10i). Amine 9 was reacted with oxazolone
15i according to the general procedure. The crude product was
purified by flash chromatography (silica, CHCl3/MeOH 95:5). 1H
NMR (δ, DMSO-d6, 400 MHz): 8.34 (s, 1H), 8.15 (s, 1H),
7.83-7.80 (m, 2H), 7.28 (d, 1H, J=8.4 Hz), 7.23-7.12 (m, 6H),
4.00-3.94 (m, 1H), 3.83-3.79 (m, 2H), 3.29-3.16 (m, 3H),
2.73-2.61 (m, 2H), 2.45 (s, 3H), 2.25-2.08 (m, 10H), 2.08-1.83
(m, 6H), 1.67-1.51 (m, 6H), 1.46-1.22 (m, 5H), 1.21-1.00 (m,
2H). MS m/z calcd for C37H50N4O3S: 630.3. Found 631.2 [M þ
1]þ. HPLC purity: system A, tR=7.488 min.

4158 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Fattori et al.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-morpholin-4-ylmethylpiperidin-1-yl)butylcarbamoyl]-
cyclopentyl}amide (22). A solution of acid 3 (250 mg, 0.85
mmol), EDCA (180 mg, 0.94 mmol), and HOBt (128 mg, 0.93
mmol) in DMF (7 mL) was stirred at room temperature for 1 h.
A solution of 4-(morpholine-4-carbonyl)piperidinium chloride
(200 mg, 0.85 mmol) and DIPEA (242 mg, 1.87 mmol) in DMF
(2 mL) was added, and the resulting mixture was stirred over-
night. It was then partitioned between 2 N NaOH and EtOAc,
and the resulting phases were separated. The organic phase was
washed with water (ꢀ3), dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was dissolved
in 4 N HCl in dioxane and stirred for 1 h. The solvent was
removed in vacuo to afford 16 (280 mg, 80%) that was used as
such without further purification. Compound 16 (250 mg, 0.61
mmol) was treated with a 1 M LiAlH4 solution in THF (10 mL)
at refluxing temperature for 3 h. The LC-MS control showed
the total consumption of the starting material and the formation
of the product. The reaction mixture was cooled to 3-4 °C.
Then the excess of LiAlH4 was quenched by slow and cautious
addition of ice. When the hydride was completely quenched (no
more H2 evolution was observed), NaOH pellets were added and
the resulting suspension was stirred at room temperature for 1 h.
The inorganic salts were filtered off throughout Celite washing
with DCM. The biphasic system obtained was saturated with
NaOH. Then it was transferred into a separatory funnel and the
phases were separated. The organic phase was dried over
Na2SO4 and filtered, and the solvent was removed under
reduced pressure. The desired product was obtained as a pale-
yellow oil (110 mg, 52%) and used in the next reaction without
any further purification. The crude amine was reacted with
oxazolone 15i according to the general procedure. The resulting
product was purified by flash chromatography (silica, from
CHCl3/MeOH, 99:1 to 95:5, with final elution with EtOH) to
obtain 70 mg (37%) of 22. 1H NMR (δ, DMSO-d6, 400 MHz):
8.40 (s, 1H), 8.18 (s, 1H), 7.80 (m, 2H), 7.28 (m, 2H), 7.20 (m,
5H), 4.00 (m, 1H), 3.58 (m, 4H), 2.70 (m, 4H), 2.45 (s, 3H), 2.30
(m, 4H), 2.20 (m, 2H), 2.10 (m, 2H), 2.00 (m, 1H), 1.90 (m, 3H),
1.75-1.20 (m, 13H), 0.95 (m, 2H). MS m/z calcd for
C37H50N4O3S: 630.8. Found 631.3 [M þ 1]þ. HPLC purity:
system A, tR=7.482 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-morpholin-4-ylpiperidin-1-yl)-4-oxobutylcarbamoyl]-
cyclopentyl}amide (23). A solution of acid 3 (203 mg, 0.69 mmol),
EDCA (146 mg, 0.76 mmol), and HOBt (104 mg, 0.76 mmol) in 1:1
THF/DMF (6 mL) was stirred at room temperature for 1 h.
4-Morpholinopiperidine (129 mg, 0.76 mmol) was added, and the
mixture was stirred for an additional 10 h. The mixture was then
partitioned between EtOAc and 10% NaHCO3. The two phases
were separated and the organic one was washed with water, dried
over Na2SO4, filtered, and concentrated under reduced pressure.
The resulting solid was triturated with Et2O and collected by
filtration. It was then treated with 4 N HCl in dioxane for 1 h.
The solvent was removed under reduced pressure to obtain 17
(218.9 mg, 83%), which was used as such without further purifica-
tion. Crude amine 17 was reacted with oxazolone 15i according to
the general procedure. The resulting crude was purified by flash
chromatography (CHCl3/MeOH, 95:5, 70 mg). 1H NMR (δ,
DMSO-d6, 400 MHz): 8.40 (s, 1H), 8.18 (s, 1H), 7.81 (m, 2H),
7.37 (m, 1H), 7.28 (d, 1H), 7.20 (m, 5H), 4.28 (m, 1H), 3.95 (m,
1H), 3.75 (m, 1H), 3.55 (m, 4H), 2.9-2.68 (m, 3H), 2.50-2.00 (m,
8H), 2.45 (s, 3H), 2.00-1.45 (m, 12H), 1.15 (m, 2H). MS m/z calcd
for C36H46N4O4S: 630.8. Found 631.3 [M þ 1]þ. HPLC purity:
system A, tR=8.144 min.
was stirred for an additional hour. Then the inorganic salts were
filtered off through Celite washing with DCM. The biphasic
resulting solution was separated into its components and the
organic one was dried over Na2SO4, filtered, and concentrated
under reduced pressure to give a colorless oil (110 mg) that was
reacted with oxazolone 15i (94 mg) according to the general
procedure. The resulting crude was purified by preparative HPLC.
1H NMR (δ, DMSO-d6, 400 MHz): 10.3 (brs, 1H), 9.37 (brs, 1H),
8.58 (s, 1H), 8.25 (s, 1H), 7.90 (d, 1H), 7.80 (s, 1H), 7.45 (m, 1H),
7.30 (d, 1H), 7.20 (m, 5H), 4.20-2.85 (m, 16H), 2.75 (m, 2H),
2.45 (s, 3H), 2.40-1.30 (m, 16H). MS m/z calcd for C36H48N4-
O3S: 616.8. Found 631.3 [M þ 1]þ. HPLC purity: system A,
tR=7.299 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid [1-((S)-1-Ben-
zyl-4-{4-[methyl(tetrahydropyran-4-yl)amino]piperidin-1-yl}-4-
oxobutylcarbamoyl)cyclopentyl]amide (25). A solution of acid 3
(400 mg, 1.36 mmol), EDCA (287 mg, 1.49 mmol), and HOBt (203
mg, 1.49 mmol) in THF (10 mL) was stirred at room tempera-
ture for 1 h. N-Methyl-N-(tetrahydro-2H-pyran-4-yl)piperidin-4-
amine dihydrochloride salt (370 mg, 1.36 mmol) and DIPEA (579
mg, 4.49 mmol) were then added, and the resulting mixture was
stirred at room temperature until the total consumption of the
starting materials. EtOAc and 0.1 N HCl were added, and the
phases were separated. The organic phase was washed with 10%
NaHCO3, water, and brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The so obtained crude
product was dissolved in 4 N HCl in dioxane (10 mL) and stirred
at room temperature. At the end of the reaction (HPLC control)
the solvent was distilled off and the resulting solid was triturated
with Et2O to afford crude 18 (346 mg, 0.77 mmol) which was used
as such without further purification. Crude amine 18 was reacted
with oxazolone 15i according to the general procedure. The crude
mixture was purified by flash chromatography (from 99:1 to 95:5
CHCl3/MeOH). 1H NMR (δ, DMSO-d6, 400 MHz): 8.45 (s, 1H),
8.20 (s, 1H), 7.90 (d, 1H), 8.80 (dd, 1H), 7.32 (m, 2H), 7.20 (m, 5H),
4.35 (m, 1H), 4.00 (m, 1H), 3.85 (m, 3H), 3.30 (m, 4H), 2.90-2.60
(m, 5H), 2.48 (s, 3H), 2.48-2.05 (m, 2H), 2.10 (s, 3H), 2.00-1.15
(m, 17H). MS m/z calcd for C38H50N4O4S: 658.9. Found 659.3 [M
þ 1]þ. HPLC purity: system A, tR=8.173 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid [1-((S)-1-Ben-
zyl-4-{4-[methyl(tetrahydropyran-4-yl)amino]piperidin-1-yl}-
butylcarbamoyl)cyclopentyl]amide Trifluoroacetate Salt (26). Crude
18 (226 mg, 0.506 mmol) was dissolved in dry THF (10 mL) under
nitrogen atmosphere, and LiAlH4 (115 mg, 3.03 mmol) was added.
The resulting mixture was refluxed overnight. It was then cooled in
an ice bath and solid ice followed by careful addition of 1 N NaOH.
The resulting system was stirred for an additional 1 h. The white
precipitate formed was removed by filtration through a Celite pad,
washing with Et2O, DCM, and water. NaOH pellets were added to
the biphasic filtrate. The phases were separated, and the organic one
was washed with brine, dried over Na2SO4, filtered, and concen-
trated under reduced pressure to obtain the intermediate amine as a
solid, which was reacted as such with oxazolone 15i according to the
general procedure. Crude 24 was purified by preparative HPLC. 1H
NMR (δ, DMSO-d6, 400 MHz): 9.70 (brs, 1H), 9.30 (brs, 1H), 8.55
(s, 1H), 8.25 (s, 1H), 7.90 (d, 1H), 7.80 (s, 1H), 7.50 (d, 1H), 7.30 (d,
1H), 7.20 (m, 5H), 4.10 (m, 1H), 3.7-3.2 (m, 4H), 3.20-2.90 (m,
4H), 2.73 (m, 5H), 2.50 (s, 3H), 2.25 (m, 3H), 2.00 (s, 3H),
2.00-1.30 (m, 17H). MS m/zcalcd for C38H52N4O3S: 644.9. Found
645.1 [M þ 1]þ. HPLC purity: system A, tR=7.357 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(2-hydroxyethyl)piperidin-1-yl]butylcarbamoyl}cyclo-
pentyl)amide (27). A solution of 4 (500 mg, 1.28 mmol) and
4-ethanolpiperidine (165 mg, 1.28 mmol) in THF (10 mL) was
stirred at room temperature. At the end of the reaction the mixture
was partitioned between EtOAc and 10% NaHCO3 and the two
resulting phases were separated. The organic layer was washed
with water and brine, dried over Na2SO4, filtered, and concen-
trated under reduced pressure to give crude Boc-protected 19 as an
oil. This oil was dissolved in 4 N HCl in dioxane and stirred for 1 h
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-morpholin-4-ylpiperidin-1-yl)butylcarbamoyl]cyclopentyl}-
amide Trifluoroacetate Salt (24). Crude amine 17 (110 mg) was
treated with a 1 M solution of LiAlH4 in THF at reflux for 4 h. The
solution was cooled to room temperature and quenched by
cautious addition of ice and NaOH pellets. The resulting mixture

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4159
at room temperature. The solvent was removed under reduced
pressure, and the resulting 19 yellow oil was used in the next
reaction without any further purification. Crude 19 was dissolved
in dry THF (10 mL) and treated with LiAlH4 (5 mL) at refluxing
temperature. After 20 h MeOH (1 mL) and then water (10 mL)
were sequentially added. The resulting system was stirred at room
temperature for an additional hour. The inorganic salts were
filtered off through a pad of Celite, washing with water and
Et2O. The solvents were removed under reduced pressure, and
the residue was treated with 4 N HCl. The solvents were eliminated
under reduced pressure and the solid obtained was dried to obtain
the intermediate amine as the hydrochloride salt (127 mg, 0.35
mmol). This was reacted with oxazolone 15i (100 mg, 0.35 mmol)
according to the general procedure. 1H NMR (δ, DMSO-d6, 400
MHz): 8.30 (s, 1H), 8.05 (s, 1H), 8.02 (d, 1H), 7.93 (m, 1H),
7.66-7.40 (m, 4H), 7.32-7.11(m, 3H), 4.01-3.97 (m, 1H), 3.65 (s,
1H), 3.47 (m, 2H), 2.81-2.51 (m, 4H), 2.43 (s, 3H), 2.47-2.40 (m,
4H), 2.10-1.81 (m, 4H), 1.60-1.40 (m, 11H), 1.39-1.29 (m, 4H).
MS m/z calcd for C34H45N3O3S: 575.8. Found 576.2 [M þ 1]þ.
HPLC purity: system A, tR=8.342 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(3-ethoxypropyl)piperazin-1-yl]butylcarbamoyl}cyclo-
pentyl)amide Trifluoroacetate Salt (28). A solution of acid 3 (190
mg, 0.782 mmol), EDCA (165 mg, 0.86 mmol), and HOBt (117
mg, 0.86 mmol) in DMF/THF, 1:1 (6 mL), was stirred at room
temperature for 1 h. 1-(2-Ethoxyethyl)piperazine-1,4-diium
chloride (190 mg, 0.78 mmol) and DIPEA (252 mg, 1.955 mmol)
were added, and stirring was continued for an additional 4 h.
The solution was then partitioned between EtOAc and 0.1 N
HCl. The two phases were separated, and the organic one was
washed with 10% NaHCO3 and brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The crude
residue was dissolved in 4 N HCl in dioxane and stirred at room
temperature for 2 h. The solvent was distilled off under reduced
pressure and the residue was triturated with Et2O, collected by
filtration, and dried to obtain crude 20 (210 mg, 0.56 mmol) that
was used as such without further purification. The amide 20 was
treated with a 1 M LiAlH4 solution in THF (10 mL) at refluxing
temperature for 3 h. The LC-MS control showed the total
consumption of the starting material and the formation of the
product. The reaction mixture was cooled to 3-4 °C. Then the
excess of LiAlH4 was quenched by slow and cautious addition of
ice. When the hydride was completely quenched (no more H2
evolution was observed), NaOH pellets was added and the
resulting suspension was stirred at room temperature for 1 h.
The inorganic salts were filtered off throughout Celite, washing
with DCM. The biphasic system obtained was saturated
with NaOH. Then it was transferred into a separatory funnel
and the phases were separated. The organic phase was dried over
Na2SO4 and filtered, and the solvent was removed under reduced
pressure. The desired product was obtained as a pale -yellow oil
(115 mg, 64%) and used in the next reaction without any further
purification. A solution of oxazolone (81 mg, 0.28 mmol) and the
so obtained amine (100 mg, 0.31 mmol) in DMF (5 mL) was stirred
at room temperature for 3 h. The HPLC showed the total
consumption of the oxazolone and the formation of the product.
NaOH, 2 N (7 mL), and EtOAc (10 mL) were then added and the
phases separated. The organic phase was washed with water, brine,
dried over Na2SO4, and filtered, and the solvent was removed
under reduced pressure. The crude reaction was purified by
preparative HPLC to give a white solid (62 mg, 36%). 1H NMR
(δ, DMSO-d6, 400 MHz): 8.50 (s, 1H), 8.20 (s, 1H), 7.88 (d, 1H),
7.80 (s, 1H), 7.40 (brs, 1H), 7.30 (d, 1H), 7.20 (m, 5H), 4.00
(m, 1H), 3.40 (m, 6H), 2.80 (m, 8H), 2.75 (m, 3H), 2.45 (s, 3H), 2.20
(m, 1H), 2.00-1.35 (m, 14H), 1.15 (t, 3H). MS m/z calcd for
C36H50N4O3S: 618.9. Found 619.3 [M þ 1]þ. HPLC purity: system
A, tR=7.844 min.
4.77mmol) inTHF(25mL) wascooledinanicebath. N-Hydroxy-
succinimide (0.55 g, 4.77 mmol) and DCC (1.08 g, 5.24 mmol) were
added in three portions during 10 min. The resulting mixture was
stirred at room temperature for 12 h. Then it was filtered and
concentrated under reduced pressure to obtain 4 (1.53 g, 88%) as a
white solid. A solution of compound 4 (350 mg, 0.90 mmol),
4-(tetrahydro-2H-pyran-4-yl)piperazin-1-ium chloride (218 mg,
0.90 mmol), and DIPEA (255 mg, 1.98 mmol) in THF (10 mL)
was stirred at room temperature. At the end of the reaction (HPLC
control) the mixture was partitioned between EtOAc and 10%
NaHCO3. The two phases were separated, and the organic one was
further washed with water, dried over Na2SO4, and concentrated
under reduced pressure to give crude N-Boc derivative of 21 (401
mg, 75%) which was used as such without further purification.
This crude was dissolved in 4 N HCl in dioxane and stirred at room
temperature. At the end of the reaction the solvent was distilled off
under reduced pressure and the resulting solid was collected and
triturated with Et2O to afford crude 21 (258 mg, 94%). This
product was reacted with oxazolone 15i according to the general
procedure to obtain 29 as a white solid after flash chromatographic
purification (silica, MeOH in CHCl3 from 1% to 5%, 90 mg, 35%
yield). 1H NMR (δ, DMSO-d6, 400 MHz): 8.45 (s, 1H), 8.13 (s,
1H), 7.82 (dd, 2H), 7.40 (d, 1H), 7.32 (d, 1H), 7.18 (m, 5H), 3.95
(m, 1H), 3.82 (m, 2H), 3.43-3.14 (m, 6H), 2.73 (m, 2H), 2.43 (s,
3H), 2.39-2.09 (m, 7H), 2.04 (m, 1H), 1.93 (m, 1H), 1.88-1.41 (m,
10H), 1.27 (m, 2H). MS m/z calcd for C36H46N4O4S: 630.8. Found
631.2 [M þ 1]þ. HPLC purity: system A, tR=8.141 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(R)-2-
Phenyl-1-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-ylmethyl]-
ethylcarbamoyl}cyclopentyl)amide Trifluoroacetate Salt (32). A
mixture of 30 (72 mg, 0.28 mmol), 1-[(tetrahydro-2H-pyran-4-
yl)methyl]piperazine dihydrochloride salt (75 mg, 0.29 mmol) and
Na(AcO)3BH (42 mg, 0.28 mmol) in DCM (5 mL) was stirred at
room temperature. At the end of the reaction 10% NaHCO3
aqueous solution was added and the resulting phases were sepa-
rated. The aqueous one was back-extracted with DCM (ꢀ2). The
combined organic phase was washed with water, brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure to
afford crude 31. This crude was dissolved in 4 N HCl in dioxane
(4 mL) and stirred at room temperature for 1 h. At the end of the
reaction the solvent was distilled off under reduced pressure. The
resulting solid was triturated with Et2O and then collected by
filtration to give the intermediate amine (75 mg, 75% yield of two
steps), which was reacted without further purification with oxazo-
lone 15i (70 mg, 0.24 mmol) according to the general procedure.
The crude product was purified by preparative HPLC to obtain 32
as the trifluoroacetate salt (95 mg, 63%). 1H NMR (δ, DMSO-d6,
400 MHz): 8.90 (brs, 1H), 8.30 (brs, 1H), 7.85 (m, 2H), 7.70 (brs,
1H), 7.25 (m, 6H), 4.10 (m, 1H), 3.85 (m, 4H), 3.50-2.60 (m, 13H),
2.45 (s, 3H), 2.30 (m, 1H), 2.00-1.45 (m, 11H), 1.20 (m, 2H). MS
m/z calcd for C35H46N4O3S: 602.8. Found 603.3 [M þ 1]þ. HPLC
purity: system A, tR=13.63 min.
(R,E)-tert-Butyl 5-Oxo-1-phenylpent-3-en-2-ylcarbamate (33).
A mixture of 30 (600 mg, 2.41 mmol) and (formylmethylene)-
triphenylphosphorane (805 mg, 2.64 mmol) in DCM (40 mL) was
stirred at room temperature overnight. At the end of the reaction
(TLC control, silica, CHCl3/MeOH, 9:1) the solvent was distilled
off in vacuo at low temperature and the residue, because of
stability concerns, was used as such, calculating the title aldehyde
1
1
33 from the H NMR spectrum. H NMR (δ, DMSO-d6, 400
MHz): 9.51 (d, 1H, J=7.8 Hz), 7.32-7.20 (m, 5H), 7.00 (dd,, 1H,
J=5.4 and 15.6 Hz), 7.00 (dd, 1H, J=5.4 and 13.5 Hz), 4.45 (m,
1H), 2.92 (dd, 1H, J=5.3, 13.6 Hz), 2.77 (dd, 1H, J=9.3, 13.6 Hz),
1.31 (s, 9H). HPLC purity: system A, tR=10.26 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(morpholine-4-carbonyl)piperidin-1-yl]butylcarbamoyl}-
cyclopentyl)amide Trifluoroacetate Salt (35). A mixture of 33
(379 mg, 1.36 mmol), morpholino(piperidin-4-yl)methanone
(213 mg, 0.906 mmol), and Na(AcO)3BH (576 mg, 2.72 mmol)
in DCM (5 mL) was stirred at room temperature. At the end of
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-oxo-4-[4-(tetrahydropyran-4-yl)piperazin-1-yl]butylcarba-
moyl}cyclopentyl)amide (29). A solution of acid 3 (1.40 g,

4160 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Fattori et al.
the reaction 2 M NaOH was added, the resulting phases were
separated, and the aqueous one was back-extracted with DCM
(ꢀ3). The combined organic phase was washed with water,
brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. This crude mixture was purified by flash
chromatography (silica, CHCl3/MeOH, 99:1 and then 95:5) to
obtain the allylic amine in 40% yield. A portion of this amine
was submitted to catalytic hydrogenation (EtOH, 5% Pd/C).
The catalyst was filtered off over a pad of Celite, and the solvent
was removed under reduced pressure to give crude 34. This was
treated with 4 N HCl in dioxane (5 mL) and EtOAc (5 mL) for
1 h. The solvents were distilled off under reduced pressure, and
the so obtained amine hydrochloride (120 mg, 0.277 mmol) was
reacted with oxazolone 15i according to standard conditions.
The crude product was purified by preparative HPLC to obtain
35 as the trifluoroacetate salt. 1H NMR (δ, DMSO-d6, 400
MHz): 8.95 (brs, 1H), 8.50 (d, 1H), 8.20 (d, 1H), 7.80 (m, 2H),
7.45 (m, 1H), 7.25 (m, 6H), 4.10 (m, 1H), 3.50 (m, 10H), 3.10 (m,
1H), 2.90 (m, 4H), 2.75 (m, 2H), 2.45 (s, 3H), 2.15 (m, 1H),
2.00-1.30 (m, 15H). MS m/z calcd for C37H48N4O4S: 644.8.
Found 645.3 [M þ 1]þ. HPLC purity: system A, tR=8.337 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(E)-(R)-1-
Benzyl-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]but-
2-enylcarbamoyl}cyclopentyl)amide Trifluoroacetate Salt (37). A
solution of 33 (100 mg, 0.36 mmol), amine 5 (66 mg, 0.36 mmol),
and Na(OAc)3BH (228 mg, 1.07 mmol) in THF (2 mL) was stirred
at 0 °C for 1 h. At the end of the reaction (HPLC control) the
mixture was partitioned between EtOAc and 5% NaHCO3 aqu-
eous solution and the phases were separated. The organic one was
washed with water, brine, dried over Na2SO4, filtered, and con-
centrated to give crude 36 which was used as such without further
purification. This crude was treated with 4 N HCl in dioxane for
1 h. Then the solvent was distilled off in vacuo and the residue was
reacted as such with oxazolone 15i according to the general
procedure. Crude 37 was purified by preparative HPLC. 1H
NMR (δ, DMSO-d6, 400 MHz): 9.40 (brs, 1H), 8.50 (s, 1H),
8.20 (s, 1H), 7.90 (d, 1H), 7.80 (s, 1H), 7.70 (m, 1H), 7.30 (d, 1H),
7.20 (m, 5H), 5.90 (m, 1H), 5.60 (m, 1H), 4.60 (m, 1H), 3.80 (brm,
6H), 3.30 (m, 4H), 3.20 (brm, 6H), 2.80 (m, 2H), 2.45 (s, 3H), 2.20
(m, 1H), 2.00-1.45 (m, 10H), 1.15 (m, 2H). MS m/z calcd for
C37H48N4O3S: 628.9. Found 629.1 [M þ 1]þ. HPLC purity: system
A, tR=7.624 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(R)-1-Ben-
zyl-3-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]propylcarb-
amoyl}cyclopentyl)amide Trifluoroacetate Salt (40). A solution of
Boc-β-Homophe-OH (85 mg, 0.3 mmol), EDCA (60 mg, 0.3
mmol), and HOAt (40 mg, 0.3 mmol) in DMF (5 mL) was stirred
at room temperature for 1 h. N-Methyl-N-(tetrahydro-2H-pyran-
4-yl)piperidin-4-amine dihydrochloride salt (75 mg, 0.3 mmol) and
DIPEA (113 mg, 0.90 mmol) were then added, and the resulting
mixture was stirred at room temperature. At the end of the
reaction, DMF was distilled off under reduced pressure and the
oily residue was partitioned between EtOAc and 0.1 N HCl. The
phases were separated and the organic one was washed with 10%
NaHCO3 aqueous solution, water, and brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The so obtained
crude product was dissolved in 4 N HCl in dioxane (4 mL) and
stirred at room temperature. At the end of the reaction (HPLC
control) the solvent was distilled off and the resulting solid was
triturated with Et2O and collected by filtration to afford the
intermediate amine hydrochloride (91 mg, 80% yield of two steps).
The amine was dissolved in dry THF (10 mL) and treated with 1 M
LiAlH4 in THF (1 mL) at room temperature. After 1 h MeOH
(1 mL) and water (10 mL) were sequentially added and the mixture
was left stirring at room temperature for an additional hour.
Inorganic salts were removed by filtration through a pad of Celite,
washing with water and Et2O. The solvents were removed under
reduced pressure and the residue was treated with 4 N HCl for 1 h.
The solvent was removed under reduced pressure to obtain the
intermediate amine hydrochloride (80 mg, 87%). This was reacted
with oxazolone 15i (63 mg, 0.22 mmol) according to the general
procedure. The crude product was purified by preparative HPLC
to obtain 40 as trifluoroacetate salt (110 mg, 71%). 1H NMR (δ,
DMSO-d6, 400 MHz): 8.70 (s, 1H), 8.25 (s, 1H), 7.85 (d, 1H), 7.75
(s, 1H), 7.60 (d, 1H), 7.25 (m, 6H), 4.05 (m, 1H), 3.85 (m, 4H),
3.60-3.00 (m, 10H), 2.80 (m, 2H), 2.70 (m, 2H), 2.45 (s, 3H), 2.20
(m, 1H), 2.00-1.45 (m, 12H), 1.20 (m, 2H). MS m/z calcd for
C36H48N4O3S: 616.8. Found 617.4 [M þ 1]þ. HPLC purity: system
A, tR=13.57 min.
4-[5-Phenyl-4-(tritylamino)pent-2-enyl]piperidine-1-carboxylic
Acid tert-Butyl Ester (42). A suspension of 50 (0.614 g, 1.02
mmol) in dry THF (3.0 mL) was stirred under nitrogen at room
temperature. A 1 M solution of NaHMDS in THF (1.0 mL, 1.02
mmol) was added dropwise. After about 10 min the salt was
completely dissolved and the solution was bright-orange. The
mixture was then cooled to -40 °C, and a solution of 41 (0.600 g,
1.53 mmol) in dry THF (3 mL) precooled at -40 °C was added
dropwise. The resulting mixture was stirred under nitrogen while
allowing it to warm to room temperature. Et2O (10 mL) and
brine (10 mL) were added, and the resulting phases were
separated. The organic layer was further washed with brine (10
mL), dried on Na2SO4, filtered, and concentrated under reduced
pressure. The so obtained crude product was purified by flash
chromatography (silica, petroleum ether/EtOAc from 99:1 to
95:5) to obtain pure 42 (0.200 g, 0.34 mmol) as a colorless oil. 1H
NMR (δ, DMSO-d6, 300 MHz): 7.53-7.42 (m, 9H), 7.31-7.22
(m, 9H), 7.22-7.03 (m, 6H), 6.78 (d, 2H, J=7.2 Hz), 5.29 (t, 1H,
J=10.4 Hz), 4.97 (m, 1H), 3.72 (m, 2H), 3.30 (m, 1H), 2.41 (m,
2H), 2.18-2.04 (m, 2H), 1.38 (s, 9H), 1.30-0.36 (m, 2H).
Benzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Benzyl-4-[1-
(tetrahydropyran-4-ylmethyl)piperidin-4-yl]butylcarbamoyl}cyclo-
pentyl) Trifluoroacetate Salt (47). Compound 42 (200 mg, 0.34
mmol) was dissoled in EtOH (10 mL) and hydrogenated in the
presence of 5% Pd/C. At the end of the reaction the catalyst was
filtered off throughout Celite and the solvents were concentrated
under reduced pressure to give crude 43 (0.140 g, 0.23 mmol) that
was treated for 15 min with a 1% solution of TFA in DCM
(5 mL). Two drops of water were added, and stirring was
continued for additional 10 min. Then 2 M NaOH (5 mL) was
added, and the resulting phases were separated. The aqueous one
was extracted with DCM (2 ꢀ 10 mL), and the combined organic
phase was dried over Na2SO4, filtered, and concentrated under
reduced pressure to obtain crude 44 which was used as such in the
next reaction. A solution of amine 44 (100 mg, 0.288 mmol) and
oxazolone 15i (82 mg, 0.288 mmol) in DMF (4 mL) was stirred at
room temperature overnight. It was then partitioned between
EtOAc and 5% aqueous Na2HCO3, and the phases were sepa-
rated. The organic one was washed with water, brine, dried over
Na2SO4, filtered, and concentrated to afford crude 45. This crude
was treated with 4 N HCl in dioxane for 20 min. The solvent was
removed under reduced pressure. Et2O was added and distilled off
severaltimestoobtain46 asasolidresidue (70 mg) whichwasused
as such without further purification. Na(AcO)3BH (78 mg, 0.37
mmol) was added to a stirred solution of 46 (70 mg, 0.123 mmol)
and tetrahydro-2H-pyran-4-carbaldehyde (42 mg, 0.37 mmol) in
DCM (5 mL), and the resulting mixture was stirred at room
temperature overnight. A few drops of 1 N NaOH were added,
and the crude mixture was concentrated and finally purified by
preparative HPLC. 1H NMR (δ, DMSO-d6, 400 MHz): 8.65 (brs,
1H), 8.40 (s, 1H), 8.15 (s, 1H), 7.80 (m, 2H), 7.30 (d, 1H), 7.15 (m,
6H), 4.00 (m, 1H), 3.75 (m, 2H), 3.40 (m, 4H), 2.90 (m, 2H), 2.75
(m, 4H), 2.50 (s, 3H), 2.20 (m, 1H), 2.10-1.00 (m, 23H). MS m/z
calcd for C38H51N3O3S: 629.9. Found 630.3 [M þ 1]þ. HPLC
purity: system A, tR=9.069 min.
4-(2-Iodoethyl)piperidine-1-carboxylic Acid tert-Butyl Ester (49).
A solution of PPh3 (1.20 g, 4.60 mmol) and imidazole (0.32 g, 4.60
mmol) in DCM (30 mL) was treated with I2 (1.17 g, 4.60 mmol) for
30 min at room temperature. A solution of 48 (0.81 g, 3.54 mmol)
in DCM (5 mL) was added, and the resulting mixture was stirred at
room temperature overnight. Et2O (100 mL) and water (50 mL)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4161
were added. The biphasic mixture was transferred into a sepa-
ratory funnel, and the phases were separated. The organic phase
was washed with a saturated solution of NaHCO3 (100 mL), a 5%
aqueous solution of Na2SO3 (100 mL), and brine (100 mL), dried
over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was purified by flash chromatography (silica,
petroleum ether/EtOAc, 9:1) to obtain 49 (1.08 g, 90%) as a
was stirred at room temperature for an additional 12 h. It was
then diluted with EtOAc, washed with 10% NaHCO3 and brine,
dried over Na2SO4, filtered, and the solvent was removed under
reduced pressure. The crude product was purified by flash
chromatography (silica, CHCl2/MeOH, 98:2) to obtain 53b
1
(0.40 g, 0.62 mmol). H NMR (δ, DMSO-d6, 400 MHz): 8.45
(s, 1H), 8.20 (s, 1H), 7.85 (d, 1H), 7.80 (s, 1H), 7.30 (d, 1H), 7.25
(dd, 1H), 7.20-7.10 (d, 1H), 4.05 (m, 1H), 3.85 (m, 2H),
3.40-3.20 (m, 4H), 2.80-2.60 (m, 4H), 2.45 (s, 3H), 2.15 (m,
1H), 2.00-1.20 (m, 21H), 0.90 (m, 2H). A solution of 53b (0.40 g,
0.62 mmol) in 4 N HCl in dioxane (5 mL) was stirred at room
temperature for 30 min. The solvent was distilled off in vacuo,
and to the gummy residue Et2O was added and removed until
the formation of a solid occurred. The solid was triturated with
Et2O and collected by filtration to obtain 54b (0.33 g, 91%) as a
hydrochloride salt. A solution of 54b (100 mg, 0.17 mmol) and
4-tetrahydropyranylaldehyde (58 mg, 0.51 mmol) in dichloro-
methane (20 mL) was stirred at room temperature under nitro-
gen. Sodium triacetoxyborohydride (110 mg, 0.51 mmol) was
added once, and stirring was continued for 2 h. The solvent was
distilled off under reduced pressure, and the residue was dis-
solved in EtOAc. The resulting solution was washed with 10%
NaHCO3, brine, dried over Na2SO4, filtered, and the solvent
was removed under reduced pressure. The resulting crude
product was purified by flash chromatography (silica, CHCl3/
MeOH, 95:5) to obtain 55b (70 mg, 64%). 1H NMR (δ, DMSO-
d6, 400 MHz): 8.45 (s, 1H), 8.20 (s, 1H), 7.85 (d, 1H), 7.80 (s, 1H),
7.30 (d, 1H), 7.25 (dd, 1H), 7.20-7.10 (m, 5H), 4.05 (m, 1H),
3.85 (m, 2H), 3.40-3.20 (m, 6H), 2.80-2.65 (m, 4H), 2.45 (s,
3H), 2.15 (m, 1H), 2.00-1.85 (m, 5H), 1.70-1.00 (m, 18H). MS
m/z calcd for C38H51N3O4S: 645.9. Found 646.3 [M þ 1]þ.
HPLC purity: system A, tR=8.947 min.
(S)-5-Ethoxy-5-oxo-1-phenylpentan-2-aminium Chloride (58).
Compound 2 (4.50 g, 14.0 mmol) was dissolved in dry dioxane
(17 mL) and cooled to 0 °C. A 4 M solution of HCl in dioxane
(70 mL) was added, and stirring was continued for 20 min,
letting the mixture reach room temperature. At the end of the
reaction (HPLC control) the solvent was distilled off in vacuo to
obtain 58 as a white solid (3.50 g, 97%). 1H NMR (δ, DMSO-d6,
300 MHz): 8.15 (s, 3H), 7.40-7.20 (m, 5H), 4.00 (q, 2H), 3.40
(m, 1H), 3.00 (dd, 1H), 2.80 (dd, 1H), 2.60-2.30 (m, 2H), 1.75
(m, 2H), 1.15 (t, 3H). MS m/z calcd for C13H19NO2: 221.3.
Found 222.1 [M þ 1]þ. HPLC purity: system B, tR=5.29 min.
(S)-4-(1-(6-Methylbenzo[b]thiophene-2-carboxamido)cyclo-
pentanecarboxamido)-5-phenylpentanoic Acid (60). Amine 58
(1.99 g, 7.74 mmol) was dissolved in dry DMF (32 mL).
Oxazolone 15i (2.00 g, 7.74 mmol) and DIPEA (501 mg, 7.74
mmol) were sequentially added, and the mixture was stirred at
room temperature. At the end of the reaction the solution
was diluted with EtOAc and washed with 2% NaHSO4 (ꢀ2)
and brine, dried over Na2SO4, filtered, and concentrated.
The residue was triturated with Et2O to obtain 59 as a white
solid (2.50 g, 4.90 mmol, 64%) used as such without further
purification.
1
colorless oil. H NMR (δ, DMSO-d6, 300 MHz): 3.91 (m, 2H),
3.30 (m, 2H), 2.68 (m, 2H), 1.72 (q, 2H, J=6.9 Hz), 1.62 (d, 2H,
J=14.1 Hz), 1.46-1.56 (m, 1H), 1.39 (s, 9H), 1.04 (dq, 2H, J=4.3,
11.5 Hz).
[2-(1-tert-Butoxycarbonylpiperdin-4-yl)ethyl]triphenylphospho-
nium Iodide (50). A solution of iodide 49 (0.696 g, 2.05 mmol) and
PPh3 (0.592 g, 2.25 mmol) in acetonitrile (5 mL) was refluxed
under stirring for 2 days. The solvent was then distilled off under
reduced pressure to obtain a white solid mixture of 50 and excess
PPh3. Et2O (10 mL) was added and the resulting suspension was
stirred for 10 min in order to dissolve the PPh3. Crude 50 was then
collected by filtration as a white solid (0.94 g, 74%). 1H NMR (δ,
DMSO-d6, 300 MHz): 7.91-7.84 (m, 3H), 7.83-7.78 (m, 12H),
3.94 (m, 2H), 3.50-3.65 (m, 2H), 2.66 (m, 2H), 1.73 (m, 3H),
1.60-1.45 (m, 2H), 1.38 (s, 9H), 1.10-0.96 (m, 2H).
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(R)-2-Phe-
nyl-1-[1-(tetrahydropyran-4-ylmethyl)piperidin-4-ylmethoxymethyl]-
ethylcarbamoyl}cyclopentyl)amide (55a). KH (102 mg, 2.56 mmol)
was added to a solution of D-phenylalaninol (386 mg, 2.56 mmol) in
dry THF (24 mL). The resulting suspension was stirred under
nitrogen for 2 h at room temperature. Then a solution of tert-butyl
4-((methylsulfonyloxy)methyl)piperidine-1-carboxylate (750 mg,
2.56 mmol) in dry THF was added dropwise and the resulting
mixture was stirred for further 12 h. The solvent was partially
distilled off in vacuo. The residual solution was diluted with EtOAc,
then washed with 10% NaHCO3 and brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The crude
product was purified by flash chromatography (silica, CHCl3/
MeOH, 95:5) to obtain 52a (125 mg). Amine 52a (125 mg, 0.36
mmol) was reacted with oxazolone 15i (102 mg, 0.36 mmol)
according to the standard procedure, and the crude product was
treated with 4 N HCl in dioxane to produce crude 54a. Crude 54a
(89 mg, 0.16 mmol) and 4-tetrahydropyranylaldehyde (54 mg, 0.47
mmol) were dissolved in DCM (3 mL) and treated with Na-
(AcO)3BH (100 mg, 0.47 mmol) at room temperature. At the end
of the reaction 2 N NaOH was added and the resulting phases were
separated. The aqueous one was back-extracted with DCM (ꢀ2).
The combined organic phase was washed with water, brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure.
This crude mixture was purified by flash chromatography (DCM/
1
MeOH, 95:5) to afford 55a (50.4 mg, 51% yield). H NMR (δ,
DMSO-d6, 400 MHz): 8.50 (s, 1H), 8.20 (s, 1H), 7.85 (m, 2H), 7.25
(m, 1H), 7.20 (m, 6H), 4.05 (m, 3H), 3.80 (m, 2H), 3.20 (m, 10H),
2.75 (m, 2H), 2.45 (s, 3H), 2.20-1.00 (m, 18H). MS m/z calcd for
C37H49N3O4S: 631.9. Found 632.4 [M þ 1]þ. HPLC purity: system
A, tR=8.710 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid [1-((R)-2-Phe-
nyl-1-{2-[1-(tetrahydropyran-4-ylmethyl)piperidin-4-yl]ethoxy-
methyl}ethylcarbamoyl)cyclopentyl]amide (55b). KH (530 mg,
13.2 mmol) was added to a solution of D-phenylalaninol (2.00 g,
13.24 mmol) in dry THF (100 mL). The resulting suspension was
stirred under nitrogen for 2 h at room temperature. Then a
solution of 4-(2-iodoethyl)piperidine-1-carbamic acid tert-butyl
ester (4.50 g, 13.24 mmol) in dry THF (50 mL) was added
dropwise and the resulting mixture was stirred for a further 12 h.
The solvent was partially distilled off in vacuo. The residual
solution was diluted with EtOAc, then washed with 2 N NaOH,
brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The crude product was purified by flash
chromatography (silica, CHCl3/MeOH, 95:5) to obtain 52b
(420 mg). A solution of amine 52b (0.25 g, 0.69 mmol) in
DMF (5 mL) was added to a stirred solution of oxazolone 15i
(0.197 g, 0.69 mmol) in DMF (10 mL). The resulting solution
This crude was dissolved in MeOH (20 mL) and THF
(15 mL). Then 4 N NaOH (20 mL) was added under stirring.
At the end of the reaction (HPLC control) 6 N HCl was added
dropwise until complete precipitation of the product. The pre-
cipitate was collected by filtration and dried to afford crude 60
(2.34 g, quantitative yield) which was used as such without
further purification. 1H NMR (δ, DMSO-d6, 400 MHz): 8.40 (s,
1H), 8.20 (s, 1H), 7.83 (m, 2H), 7.30 (m, 2H), 7.15 (m, 5H), 4.00
(m, 1H), 2.70 (m, 2H), 2.45 (s, 3H), 2.38-1.40 (m, 12H). MS m/z
calcd for C27H30N2O4S: 478.2. Found 479.3 [M þ 1]þ.
(S)-N-(1-(5-(Methoxy(methyl)amino)-5-oxo-1-phenylpentan-
2-ylcarbamoyl)cyclopentyl)-6-methylbenzo[b]thiophene-2-carbox-
amide (61). Acid 60 (2.24 g, 4.68 mmol) was dissolved in dry
DMF (22 mL) and cooled down to 0 °C. EDCA (1.07 g, 5.60
mmol), HOBt (756 mg, 5.60 mmol), N,O-dimethylhydroxylamine
(546.3 mg, 5.60 mmol), and DIPEA (2.40 mL, 14.0 mmol) were

4162 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Fattori et al.
sequentially added, and stirring was continued at room tempera-
ture. At the end of the reaction (HPLC control) the mixture was
diluted with EtOAc and saturated aqueous solution of NaHCO3
while stirring. The two phases were separated, and the organic one
was washed with saturated aqueous solution of NaHCO3 (ꢀ3),
5% NaHSO4 (ꢀ2), and brine, dried over Na2SO4, filtered, and
concentrated to obtain crude 61 (2.39 g, 98%). 1H NMR (δ,
DMSO-d6, 400 MHz): 8.40 (s, 1H), 8.20 (s, 1H), 7.90-7.80 (m,
2H), 7.40-7.25 (m, 2H), 7.25-7.10 (m, 5H), 4.10-3.90 (m, 1H),
3.60 (s, 3H), 3.05 (s, 3H), 2.80-2.65 (m, 2H), 2.45 (s, 3H),
2.40-1.40 (m, 12H). MS m/z calcd for C29H35N3O4S: 521.7.
Found 522.3 [M þ 1]þ. HPLC purity: system B, tR=12.98 min.
(S)-6-Methyl-N-(1-(5-oxo-1-phenylpentan-2-ylcarbamoyl)cyclo-
pentyl)benzo[b]thiophene-2-carboxamide (62). Compound 61 (1.19
g, 2.29 mmol) was dissolved in dry THF (20 mL) and cooled to
-30 °C under nitrogen atmosphere. A 1 M solution of LiAlH4 in
THF (2.98 mL, 2.98 mmol) was added dropwise while stirring. At
the end of the reaction (TLC control, silica, petroleum ether/
EtOAc, 1:1) 0.1 N HCl was added while stirring at -30 °C until
complete elimination of residual LiAlH4. The mixture was parti-
tioned between water and EtOAc. The organic phase was then
separated, washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure to obtain crude 62 (1.08 g,
and hydrogenated in the presence of 10% Pd/C (1.20 g) and
ammonium formate (938 mg, 14.9 mmol). At the end of the reaction
(HPLC control) the catalyst was filtered off on a Celite pad and
partitioned between DCM and 0.1 N NaOH. The phases were
separated, and then the organic one was washed with brine, dried
over Na2SO4, filtered, and concentrated to obtain crude 66 (780mg,
79%), which was used as such without further purification. It
contained around 9% of the N-formyl derivative 67p. MS m/z
calcd for C31H40N4O2S: 532.7. Found 533.2 [M þ 1]þ.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(tetrahydropyran-4-yl)piperazin-1-yl]butylcarbamoyl}cyclo-
pentyl)amide Trifluoroacetate Salt (67a). 1H NMR (δ, DMSO-d6,
400 MHz): 9.10 (brs, 1H), 8.55 (brs, 1H), 8.22 (s, 1H), 7.82 (m, 2H),
7.41 (m, 1H), 7.30 (m, 1H), 7.22 (m, 5H), 4.00 (m, 1H), 3.90 (m,
2H), 3.50-2.65 (m, 13H), 2.70 (m, 4H), 2.20 (m, 1H), 2.00-1.20
(m, 16H). MS m/z calcd for C36H48N4O3S: 616.9. Found 617.3 [M
þ 1]þ. HPLC purity: system A, tR=7.422 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-pyridin-2-ylpiperazin-1-yl)butylcarbamoyl]cyclopentyl}-
1
amide Trifluoroacetate Salt (67b). H NMR (δ, DMSO-d6, 400
MHz): 9.75 (brs, 1H), 8,60 (s, 1H), 8.30 (s, 1H), 8.20 (d, 1H), 7.90
(d, 1H), 7.80 (s, 1H), 7.65 (m, 1H), 7.50 (d, 1H), 7.35 (d, 1H), 7.20
(m, 5H), 6.90 (d, 1H), 6.80 (d, 1H), 4.30 (m, 2H), 4.10 (m, 1H), 3.50
(m, 2H), 3.20 (m, 3H), 3.15 (m, 3H), 2.75 (m, 2H), 2.45 (s, 3H), 2.20
(m, 1H), 1.95 (m, 1H), 1.90-1.20 (m, 10H). MS m/z calcd for
C36H43N5O2S: 609.8. Found 610.3 [M þ 1]þ. HPLC purity: system
A, tR=7.605 min.
1
quantitative yield). H NMR (δ, DMSO-d6, 400 MHz): 9.60 (s,
1H), 8.40 (s, 1H), 8.20 (s, 1H), 7.85-7.75 (m, 2H), 7.35-7.20 (m,
2H), 7.20-7.05 (m, 5H), 3.95 (m, 1H), 2.75-2.60 (m, 2H), 2.45 (s,
3H), 2.25-1.40 (m, 12H). MS m/z calcd for C27H30N2O3S: 462.6.
Found 463.2 [M þ 1]þ. HPLC purity: system B, tR=12.99 min.
Route A. General Procedure To Obtain Compounds 35 through
Reductive Amination on Aldehyde 62. Aldehyde 62 (90 mg, 0.19
mmol) was dissolved in dry THF (5 mL). Compound 63 (0.21
mmol) was added, followed by NaB(OAc)3H (62 mg, 0.29
mmol) and some drops of acetic acid to reach a pH of around
5. Stirring was continued at room temperature until the end of
the reaction. The mixture was then diluted with a saturated
aqueous solution of NaHCO3 and extracted with EtOAc. The
organic phase was washed with a saturated aqueous solution of
NaHCO3 and brine, dried over Na2SO4, filtered, and concen-
trated under reduced pressure to obtain crude 35 that was
purified by preparative HPLC.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-pyrimidin-2-ylpiperazin-1-yl)butylcarbamoyl]cyclopentyl}-
amide Trifluoroacetate Salt (67c). 1H NMR (δ, DMSO-d6, 400
MHz): 9.55 (brs, 1H), 8.55 (s, 1H), 8.45 (m, 2H), 8.22 (s, 1H),
7.85 (d, 1H), 7.75 (s, 1H), 7.45 (d, 1H), 7.30 (d, 1H), 7.20 (m, 5H),
6.80 (m, 1H), 4.70 (m, 1H), 4.15 (m, 1H), 3.60-2.90 (m, 8H),
2.80 (2H, m), 2.45 (s, 3H), 2.20 (s, 1H), 1.95 (s, 1H), 1.90-1.30
(m, 10H). MS m/z calcd for C35H42N6O2S: 610.8. Found 611.3
[M þ 1]þ. HPLC purity: system A, tR=8.997 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(4-fluorobenzyl)piperazin-1-yl]butylcarbamoyl}cyclo-
pentyl)amide (67d). 1H NMR (δ, DMSO-d6, 400 MHz): 9.10 (brs,
1H), 8.50 (s, 1H), 8.20 (s, 1H), 7.85 (d, 1H), 7.70 (s, 1H), 7.40 (m,
4H), 7.30 (d, 1H), 7.30 (m, 5H), 4.05 (m, 1H), 3.65-2.30 (m, 12H),
2.45 (s, 3H), 2.20 (m, 1H), 1.95 (m, 1H), 1.85-1.30 (m, 12H). MS
m/z calcd for C38H45FN4O2S: 640.8. Found 641.4 [M þ 1]þ.
HPLC purity: system A, tR=8.406 min.
(R,E)-Benzyl 4-(4-(tert-butoxycarbonylamino)-5-phenylpent-
2-enyl)piperazine 1-Carboxylate (63). To a solution of aldehyde
33 (4.534 g, 16.48 mmol) in THF (160 mL), Cbz-piperazine (3.63
g, 16.5 mmol), NaB(OAc)3H (4.887 g, 23.06 mmol), and AcOH
(1.046 mL, 18.29 mmol) were added. At the end of the reaction,
the mixture was concentrated, diluted with EtOAc, and washed
with 5% NaHCO3 aqueous solution and H2O. The organic
phase was dried over Na2SO4, filtered, and concentrated to
obtain a crude containing product 63, some byproduct, and
triphenylphosphine oxide (which contaminated the starting
material 33). This crude material was treated with hexane,
affording a supernatant solution and an oily residue enriched
with triphenylphosphine oxide. The solution was separated, and
the oily residue was further treated with hexane. The combined
supernatant solution was dried over MgSO4, filtered, and
evaporated under vacuum to give 63 (5.60 g). Purification of
this crude by flash chromatography (silica, hexane/EtOAc/Et3N
from 85:13:2 to 65:33:2) afforded compound 63 of sufficient
purity to be used as such in the subsequent reactions. MS m/z
calcd for C28H37N3O3: 463.2. Found 464.2 [M þ 1]þ.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-pyridin-2-ylmethylpiperazin-1-yl)butylcarbamoyl]cyclo-
pentyl}amide Trifluoroacetate Salt (67e). 1H NMR (δ, DMSO-
d6, 400 MHz): 9.21 (brs, 1H), 8.50 (d, 1H), 8.27 (s, 1H), 8.07 (s,
1H), 8.03 (d, 1H), 7.85 (s, 1H), 7.71-7.37 (m, 4H), 7.33-7.06 (m,
6H), 4.02-3.96 (m, 1H), 3.94 (s, 2H), 2.80-2.65 (m, 2H), 2.45 (s,
3H), 2.51-2.30 (m, 10H), 2.10-1.84 (m, 4H), 1.56-1.51 (m,
4H), 1.46-1.30 (m, 4H). MS m/z calcd for C37H45N5O2S: 623.8.
Found 624.3 [M þ 1]þ. HPLC purity: system A, tR=7.606 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-pyridin-3-ylmethylpiperazin-1-yl)butylcarbamoyl]cyclo-
pentyl}amide Trifluoroacetate Salt (67f). 1H NMR (δ, DMSO-
d6, 400 MHz): 9.15 (brs, 1H), 8.65 (m, 2H), 8.52 (s, 1H), 8.20 (s,
1H), 7.95 (m, 1H), 7.85 (d, 1H), 7.77 (s, 1H), 7.58 (m, 1H), 7.40
(m, 1H), 7.22 (m, 6H), 4.05 (m, 1H), 4.70 (m, 2H), 3.42 (m, 2H),
3.15-2.85 (m, 6H), 2.73 (m, 2H), 2.45 (s, 3H), 2.40 (m, 2H), 2.20
(m, 1H), 1.90 (m, 1H), 1.85-1.03 (m, 10H). MS m/z calcd for
C37H45N5O2S: 623.8. Found 624.3 [M þ 1]þ. HPLC purity:
system A, tR=8.406 min.
(S)-6-Methyl-N-(1-(1-phenyl-5-(piperazin-1-yl)pentan-2-yl-
carbamoyl)cyclopentyl)benzo[b]thiophene-2-carboxamide (66). Boc-
protected amine 63 (350 mg, 0.73 mmol) was dissolved in 4 N HCl
in dioxane (8 mL) while cooling at 0 °C. At the end of the reaction
(HPLC control) the solvent was distilled off under reduced pres-
sure. The residue was triturated with Et2O, collected by filtration,
and dried to obtain crude 64 (200 mg, 0.42 mmol) which was
directly used in the reaction with oxazolone for the preparation
of 65 following the general procedure. Crude compound 65
(1.23 g, 1.86 mmol) was dissolved in absolute EtOH (100 mL)
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-pyridin-4-ylmethylpiperazin-1-yl)butylcarbamoyl]cyclo-
pentyl}amide Trifluoroacetate Salt (67g). 1H NMR (δ, DMSO-
d6, 400 MHz): 9.30 (brs, 1H), 8.75 (m, 2H), 8.54 (s, 1H), 8.20 (s,
1H), 7.83 (d, 1H), 7.70 (m, 3H), 7.42 (d, 1H), 7.20 (m, 6H),
4.05 (m, 1H), 3.80 (m, 2H), 3.45 (m, 2H), 3.20-2.80 (m,
6H), 2.70 (m, 2H), 2.45 (s, 3H), 2.40 (m, 2H), 2.20 (m, 1H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4163
1.95 (m, 1H), 1.80-1.30 (m, 10H). MS m/z calcd for C37H45-
N5O2S: 623.8. Found 624.3 [M þ 1]þ. HPLC purity: system A,
tR=8.406 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-bBen-
zyl-4-[4-(1-methyl-1H-pyrrole-2-carbonyl)piperazin-1-yl]butyl-
carbamoyl}cyclopentyl)amide Trifluoroacetate Salt (67o). 1H NMR
(δ, DMSO-d6, 400 MHz): 9.60 (brs, 1H), 8.55 (s, 1H), 8.20 (s, 1H),
8.50 (m, 2H), 8.72 (s, 1H), 7.45 (d, 1H), 7.20 (m, 6H), 7.00 (s, 1H),
6. 45 (s, 1H), 6.10 (s, 1H), 4.40 (m, 1H), 4.00 (m, 1H), 3.70 (s,
3H), 3.10 (m, 6H), 2.75 (m, 2H), 2.45 (s, 3H), 2.15 (m, 1H), 1.95
(m, 1H), 1.90-1.30 (m, 11H). MS m/z calcd for C37H45N5-
O3S: 639.8. Found 640.3 [M þ 1]þ. HPLC purity: system A,
tR=9.056 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-formylpiperazin-1-yl)butylcarbamoyl]cyclopentyl}amide
(67p). 1H NMR (δ, DMSO-d6, 400 MHz): 8.40 (s, 1H), 8.15 (s,
1H), 7.90 (s, 1H), 7.80 (m, 2H), 7.30 (m, 2H), 7.15 (m, 5H), 4.00
(m, 1H), 3.25 (m, 2H), 3.20 (m, 2H), 2.70 (m, 2H), 2.42 (s, 3H),
2.20 (m, 6H), 1.90 (m, 3H), 1.75-1.30 (m, 9H). MS m/z calcd for
C32H40N4O3S: 560.75. Found 561.5 [M þ 1]þ. HPLC purity:
system B, tR=9.62 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(morpholine-4-carbonyl)piperazin-1-yl]butylcarbamoyl}-
cyclopentyl)amide Trifluoroacetate Salt (67q). 1H NMR (δ,
DMSO-d6, 400 MHz): 9.40 (brs, 1H), 8.58 (brs, 1H), 8.25 (s,
1H), 7.88 (d, 1H), 7.80 (s, 1H), 7.45 (d, 1H), 7.20 (m, 6H), 4.25
(m, 1H), 3.50 (m, 12H), 3.10 (m, 6H), 2.75 (m, 2H), 2.45 (s, 3H),
2.15 (m, 1H), 2.00-1.30 (m, 11H). MS m/z calcd for
C36H47N5O4S: 645.85. Found 646.3 [M þ 1]þ. HPLC purity:
system A, tR=8.37 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(morpholine-4-sulfonyl)piperazin-1-yl]butylcarbamoyl}-
cyclopentyl)amide Trifluoroacetate Salt (67r). 1H NMR (δ, DMSO-
d6, 400 MHz): 9.50 (brs, 1H), 8.55 (s, 1H), 8.25 (s, 1H), 7.85 (m,
1H), 7.80(s, 1H), 7.45 (m, 1H), 7.20(m, 6H), 4.05(m, 1H), 3.60(M,
8H), 3.15 (m, 10H), 2.75 (m, 2H), 2.45 (s, 3H), 2.20 (m, 1H),
1.90-1.30 (m, 11H). MS m/z calcd for C35H47N5O4S2: 681.9.
Found 682.1 [M þ 1]þ. HPLC purity: >98%, system B, tR =
11.14 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-dimethylsulfamoylpiperazin-1-yl)butylcarbamoyl]cyclo-
pentyl}amide Trifluoroacetate Salt (35s). 1H NMR (δ, DMSO-
d6, 400 MHz): 9.50 (brs, 1H), 8.55 (s, 1H), 8.25 (s, 1H), 7.90 (m,
1H), 7.80 (s, 1H), 7.45 (m, 1H), 7.25 (m, 6H), 4.05 (m, 1H), 3.60
(m, 4H), 3.15 (m, 6H), 2.80 (s, 6H), 2.75 (m, 2H), 2.45 (s, 3H),
2.20 (m, 1H), 1.95 (m, 1H), 1.90-1.30 (m, 10H). MS m/z calcd
for C33H45N5O4S2: 639.9. Found 641.0 [M þ 1]þ. HPLC purity:
system B, tR=9.046 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-methylsulfamoylpiperazin-1-yl)butylcarbamoyl]cyclo-
pentyl}amide Trifluoroacetate Salt (67t). 1H NMR (δ, DMSO-d6,
400 MHz): 9.40 (brs, 1H), 8.50 (s, 1H), 8.20 (s, 1H), 7.85 (d, 1H),
7.75 (s, 1H), 7.45 (d, 2H), 7.20 (m, 5H), 4.00 (m, 1H), 3.70 (m, 4H),
3.10 (m, 6H), 2.75 (m, 2H), 2.60 (s, 3H), 2.45 (s, 3H), 2.15 (m, 1H),
1.95 (m, 1H), 1.90-1.30 (m, 11H). MS m/z calcd for
C32H43N5O4S2: 625.8. Found 626.4 [M þ 1]þ. HPLC purity:
system B, tR=10.63 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(propane-2-sulfonyl)piperazin-1-yl]butylcarbamoyl}cyclo-
pentyl)amide Trifluoroacetate Salt (67u). A solution of 66 (100 mg,
0.188 mmol) in dry DCM (5 mL) was stirred at room temperature.
i-PrSO2Cl (32.1 mg, 0.22 mmol) and DIPEA (33 μL, 0.188 mmol)
were added. At the end of the reaction (HPLC control) the mixture
was diluted with DMC and washed with 0.1 N NaOH and brine.
The resulting organic phase was dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was
purified by preparative HPLC to obtain pure 35u (22 mg, 18%).
1H NMR (δ, DMSO-d6, 400 MHz): 9.45 (brs, 1H), 8.55 (brs, 1H),
8.20 (s, 1H), 7.90 (d, 1H), 7.80 (s, 1H), 7.45 (m, 1H), 7.20 (m, 6H),
4.00 (m, 1H), 3.70 (m, 1H), 3.60-3.00 (m, 10H), 2.70 (m, 2H), 2.45
(s, 3H), 2.20 (m, 1H), 2.00-1.30 (m, 11H), 1.20 (d, 6H). MS m/z
calcd for C34H46N4O4S2: 638.9. Found 639.4 [M þ 1]þ. HPLC
purity: system B, tR=11.44 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-thiophen-2-ylmethylpiperazin-1-yl)butylcarbamoyl]cyclo-
pentyl}amide Trifluoroacetate Salt (67h). 1H NMR (δ, DMSO-d6,
400 MHz): 9.20 (brs, 1H), 8.52 (s, 1H), 8.20 (s, 1H), 7.85 (d, 1H),
7.75 (s, 1H), 7.55 (d, 1H), 7.40 (dd, 1H), 7.30 (d, 1H), 7.20 (m, 5H),
7.10 (m, 2H), 4.10 (m, 1H), 3.95 (m, 2H), 3.45 (m, 2H), 3.00
(m, 6H), 2.75 (m, 2H), 2.45 (s, 3H), 2.40 (m, 2H), 2.20 (m, 1H), 1.95
(m, 1H), 1.90-1.30 (m, 10H). MS m/z calcd for C36H44N4-
O2S2: 628.9. Found 629.3 [M þ 1]þ. HPLC purity: system A,
tR=8.455 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-thiophen-3-ylmethylpiperazin-1-yl)butylcarbamoyl]cyclo-
pentyl}amide Trifluoroacetate Salt (67i). 1H NMR (δ, DMSO-
d6, 400 MHz): 9.45 (brs, 1H), 8.55 (s, 1H), 8.20 (s, 1H), 7.80 (d,
1H), 7.75 (s, 1H), 7.60 (d, 1H), 7.50 (s, 1H), 7.40 (d, 1H), 7.30 (d,
1H), 7.20 (m, 6H), 4.00 (m, 1H), 3.90 (m, 2H), 3.50 (m, 2H), 3.00
(m, 6H), 2.70 (m, 2H), 2.65 (m, 2H), 2.45 (s, 3H), 0.20 (m, 1H),
1.95 (m, 1H), 1.85-1.30 (m, 10H). MS m/z calcd for
C36H44N4O2S2: 628.9. Found 629.3 [M þ 1]þ. HPLC purity:
system A, tR=8.119 min.
6-Methyl-benzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-furan-3-ylmethylpiperazin-1-yl)butylcarbamoyl]cyclo-
pentyl}amide Trifluoroacetate Salt (67j). 1H NMR (δ, DMSO-d6,
400 MHz): 9.15 (brs, 1H), 8.32 (s, 1H), 8.05 (s, 1H), 8.03 (d, 1H),
7.88 (s, 1H), 7.70-7.40 (m, 3H), 7.30-7.05 (m, 6H), 6.10 (d, 1H),
4.00-3.95 (m, 1H), 3.60 (s, 2H), 2.80-2.65 (m, 2H), 2.43 (s, 3H),
2.50-2.35 (m, 10H), 2.09-1.85 (m, 4H), 1.56-1.51 (m, 4H),
1.46-1.30 (m, 4H). MS m/z calcd for C36H44N4O3S: 612.8. Found
613.3 [M þ 1]þ. HPLC purity: system A, tR=7.922 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(3H-imidazol-4-ylmethyl)piperazin-1-yl]butylcarbamoyl}-
cyclopentyl)amide Trifluoroacetate Salt (67k). 1H NMR (δ,
DMSO-d6, 400 MHz): 9.20 (brs, 1H), 9.10 (s, 1H), 8.55 (s, 1H),
8.25 (s, 1H), 7.85 (dd, 1H), 7.75 (s, 1H), 7.60 (s, 1H), 7.45 (m, 1H),
7.30 (m, 1H), 7.20 (m, 5H), 4.05 (m, 1H), 3.70 (brs, 2H), 3.48 (m,
2H), 3.10 (m, 1H), 2.95 (m, 5H), 2.70 (m, 2H), 2.45 (s, 3H), 2.45 (m,
2H), 2.25 (m, 1H), 1.95 (m, 1H), 2.85-1.30 (m, 10H). MS m/z
calcd for C35H44N6O2S: 612.8. Found 613.3 [M þ 1]þ. HPLC
purity: system A, tR=7.922 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(2-methyl-2H-tetrazol-5-ylmethyl)piperazin-1-yl]butyl-
carbamoyl}cyclopentyl)amide Trifluoroacetate Salt (67l). 1H NMR
(δ, DMSO-d6, 400 MHz): 9.10 (brs, 1H), 8.55 (s, 1H), 8.20 (s, 1H),
7.85 (m, 2H), 7.45 (d, 1H), 7.30 (d, 1H), 7.20 (m, 5H), 7.35 (s, 3H),
4.00 (m, 1H), 3.85 (s, 2H), 3.70 (m, 2H), 3.45 (m, 2H), 3.00 (m, 6H),
2.70 (m, 2H), 2.45 (s, 3H), 2.20 (m, 1H), 1.95 (m, 1H), 1.85-1.30
(m, 10H). MS m/z calcd for C34H44N8O2S: 628.8. Found 629.4 [M
þ 1]þ. HPLC purity: system A, tR=8.329 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(3-ethoxypropyl)piperazin-1-yl]butylcarbamoyl}cyclo-
pentyl)amide Trifluoroacetate Salt (67m). 1H NMR (δ, DMSO-d6,
400 MHz): 8.50 (brs, 1H), 8.20 (s, 1H), 7.85 (d, 1H), 7.80 (s, 1H),
7.40 (brs, 1H), 7.30 (d, 1H), 7.20 (M, 5H), 4.05 (m, 1H), 3.40 (m,
6H), 3,00 (m, 10H), 2.75 (m, 2H), 2.50 (s, 3H), 2.20 (m, 1H),
2.00-1.30 (m, 13H), 1.10 (t, 3H). MS m/z calcd for C36H50N4-
O3S: 618.9. Found 619.3 [M þ 1]þ. HPLC purity: system A,
tR=7.844 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid (1-{(S)-1-Ben-
zyl-4-[4-(tetrahydropyran-4-carbonyl)piperazin-1-yl]butylcarba-
moyl}cyclopentyl)amide Trifluoroacetate Salt (67n). 1H NMR (δ,
DMSO-d6, 400 MHz): 9.55 (brs, 1H), 8.55 (s, 1H), 8.25 (s, 1H),
7.85 (d, 1H), 7.80 (s, 1H), 7.45 (d, 1H), 7.30 (d, 1H), 7.20 (m, 5H),
4.40-4.00 (m, 3H), 2.85 (m, 2H), 3.45 (m, 5H), 3.00 (m, 6H), 2.75
(m, 2H), 2.45 (s, 3H), 2.15 (m, 1H), 2.00-1.30 (m, 15H). MS m/z
calcd for C37H48N4O4S: 644.8. Found 645.2 [M þ 1]þ. HPLC
purity: system A, tR=8.302 min.

4164 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Fattori et al.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-ethanesulfonylpiperazin-1-yl)butylcarbamoyl]cyclo-
pentyl}amide (67v). A solution of 66 (90 mg, 0.17 mmol) in dry
DCM (5 mL) was stirred at room temperature. EtSO2Cl (24
mg, 0.186 mmol) and DIPEA (until pH 7-8) were added. At
the end of the reaction (HPLC control) the solution was
partitioned between DMC and 0.1 M NaOH, and the organic
phase was separated, washed with brine, and concentrated.
The crude product was purified by flash chromato-
graphy (silica, CHCl3/MeOH, 99:1 to 97:3) to obtain 35v
(56 mg, 53%). 1H NMR (δ, DMSO-d6, 400 MHz): 8.40 (s,
1H), 8.15 (s, 1H), 7.85 (m, 2H), 7.25 (m, 2H), 7.15 (m, 5H), 3.95
(m, 1H), 2.95 (m, 6H), 2.70 (m, 2H), 2.45 (s, 3H), 2.25
(m, 2H), 2.20 (m, 4H), 1.90 (m, 3H), 1.75-1.20 (m,
(-log Ki), derived from the following equation:13
IC50
Ki ¼
½radioligandꢁ
1 þ
Kd
(D) Organ Bath Experiments. The experiments were per-
formed on guinea pig (Dunkin Hartley, Charles River, Italy)
isolated proximal colon circular smooth muscle preparation
(GPC). All experiments were performed in oxygenated (96%
O2 and 4% CO2) Krebs-Henseleit solution. The preparations
were set up according to methods previously described.12 The
activity of test compounds at tachykinin NK2 receptors in GPC
was assessed against selective NK2 receptor agonist [βAla8]-
NKA(4-10) in the presence of the NK1 receptor selective
antagonist SR 140333 (1 μM). The antagonist affinity of all test
compounds (15 min of incubation period) was expressed as pKB
(-log KB, the antagonist dissociation constant), which was
estimated as the mean of the individual values obtained with
the following equation:
9H), 1.15 (t, 3H). MS m/z calcd for
C33H44N4O4S2:
624.8. Found 625.5 [M þ 1]þ. HPLC purity: system B, tR =
11.06 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid [1-((S)-1-Ben-
zyl-4-{4-[[(E)-ethylimino](tetrahydropyran-4-yl)methyl]piper-
azin-1-yl}butylcarbamoyl)cyclopentyl]amide Trifluoroacetate Salt
1
pKB ¼ log½ðCRÞ - 1ꢁ - logðantagonist concentrationÞ
(67w). H NMR (δ, DMSO-d6, 400 MHz): 8.60 (brs, 1H), 8.30
(s, 1H), 8.15 (s, 1H), 7.85 (d, 1H), 7.80 (s, 1H), 7.20 (m, 7H), 4.05
(m, 1H), 3.09 (m, 2H), 3.80 (m, 4H), 3.60-2.90 (m, 8H), 2.70 (m,
2H), 2.45 (s, 3H), 2.20 (m, 1H), 2.00-1.35 (m, 18H), 1.25 (t, 3H).
MS m/z calcd for C39H53N5O3S: 671.9. Found 672.3 [M þ 1]þ.
HPLC purity: system A, tR=7.687 min.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-{[(E)-isopropylimino]morpholin-4-ylmethyl}piperazin-1-
yl)butylcarbamoyl]cyclopentyl}amide Trifluoroacetate Salt
(67x). 1H NMR (δ, DMSO-d6, 400 MHz): 10.00 (brs, 1H),
8.60 (s, 1H), 8.20 (s, 1H), 7.95 (d, 1H), 7.85 (d, 1H), 7.75 (s,
1H), 7.45 (m, 1H), 7.30 (d, 1H), 7.20 (m, 5H), 4.05 (m, 1H),
3.90-3.00 (m, 17H), 2.75 (m, 2H), 2.45 (s, 3H), 2.15 (m, 1H),
1.95 (m, 1H), 1.90-1.35 (m, 11H), 1.20 (d, 6H). MS m/z calcd for
C39H54N6O3S: 686.9. Found 687.4 [M þ 1]þ. HPLC purity:
system A, tR=7.97 min.
where CR is the ratio of equieffective concentrations (EC50) of
agonist in the presence and in absence of the antagonist.
(E) Functional Experiments on Human Urinary Bladder.
Mucosa-free strips of detrusor muscle were excised from the
urinary bladder dome of patients undergoing cystectomy because
of carcinoma of the bladder base, as described previously.14 The
strips were placed in 5 mL organ baths filled with oxygenated
Krebs-Henseleit solution at 37 °C, under a resting tension of 10
mN. Mechanical activity developed by the preparations was
recorded isometrically. The test compounds were tested for their
ability to block neurokinin A-induced contractions. Antagonist
affinity was expressed as pKB; pKB = log(CR - 1) - log-
(antagonist concentration).
(F) In vivo Experiments. All the experiments were performed
in accordance with the Declaration of Helsinki, with the prin-
ciples and the guidelines of the European Union regulations and
the local ethical committee. Briefly, male Dunkin-Hartley
guinea pigs (Charles River, Italy) weighing 350-400 g were
anesthetized with urethane (1.5 g/kg sc) and a polyethylene
catheter was inserted into the left jugular vein for intravenous
(iv) administration of drugs. Guinea pigs were mechanically
ventilated with a ventilation pump at a rate of 50 strokes/min
and a respiration volume of 10 mL/kg. The body temperature
was kept constant at 36 °C by a thermoregulated lamp. The
abdomen was opened, and a latex balloon, which was obtained
by a condom head, was connected to a PE90 polyethylene
catheter, inserted into the proximal colon at about 2-3 cm from
the cecum, and filled with 0.5 mL of saline. The intracolonic
balloon was connected to a pressure transducer (Transpac IV,
Abbott, Italy) for intraluminal pressure recording by means of a
MacLab/8S ML 780 data acquisition system (ADInstruments,
U.K.). Five minutes before starting the experiments, guinea pigs
were treated with the ganglionic blocker hexamethonium bro-
mide (13.8 μmol/kg iv) followed by continuous infusion of the
same solution at a rate of 300 μL/h to prevent reflex cholinergic
responses. The compounds of their vehicle (DMSO) were
administered iv (0.3-3 μmol/kg) in a volume of 100 μL/kg.
The selective NK2 receptor agonist [βAla8]NKA(4-10) (3
nmol/kg, iv) was administered two or three times before the
antagonist of the vehicle in order to stabilize the colon contrac-
tile responses, and the challenge was repeated at 5, 30, and then
every 30 min until 4 h after antagonist administration.
6-Methylbenzo[b]thiophene-2-carboxylic Acid {1-[(S)-1-Ben-
zyl-4-(4-{[(Z)-methoxyimino]morpholin-4-ylmethyl}piperazin-1-
yl)butylcarbamoyl]cyclopentyl}amide Trifluoroacetate Salt
1
(67y). H NMR (δ, DMSO-d6, 400 MHz): 9.80 (brs, 1H), 8.60
(s, 1H), 8.25 (s, 1H), 7.85 (d, 1H), 7.80 (s, 1H), 7.50 (d, 1H), 7.30 (d,
1H), 7.20 (m, 5H), 4.05 (m, 1H), 3.65 (s, 3H), 3.50 (m, 8H),
3.40-3.00 (m, 10H), 2.75 (m, 2H), 2.45 (s, 3H), 2.20 (m, 1H), 2.00
(m, 1H), 1.90-1.40 (m, 10H). MS m/z calcd for C37H50N6-
O4S: 674.9. Found 675.3 [M þ 1]þ. HPLC purity: system A,
tR=7.79 min.
(B) In Vitro Intestinal Permeability. The intestinal absorption
potential of the synthesized compounds was evaluated in vitro
using the human adenocarcinoma cell line Caco-2, as previously
described.11
Compounds were first dissolved in DMSO at 0.1 M, then in
Hank’s balanced salt solution, 25 mM HEPES, pH 7.4, at 100
μM and applied on the apical side of differentiated epithelial
monolayers. The amount of compound transported from the
apical to the basolateral compartment was quantified by
HPLC-MS in order to determine the apparent permeability
coefficient (Papp) in the absorption direction. The Papp was
obtained from the linear regression analysis of the appearance
rate of the compound in the receiver compartment and is
expressed as 10-6 cm/s. All the experiments were carried out
in the presence of markers of both passive and active transport
systems, and the integrity of the monolayer before and after
compound addition was also verified.
(C) Binding Experiments. All compounds were tested for their
ability to displace the [125I]neurokinin A specifically bound to
the recombinant hNK2 receptor in a cell membrane preparation
as reported previously.12 The radioligand was from Amersham
Biosciences (Buckinghamshire, U.K.). Nonspecific binding was
determined in the presence of unlabeled neurokinin A (1 μM).
The affinity of test compounds was expressed in terms of pKi
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