Discovery of 1-(β-amino substituted-β-alanyl)-N,N- dimethylindoline-2-carboxamides as novel nonpeptide antagonists of nociceptin/orphanin FQ receptor: Efficient design, synthesis, and structure-activity relationship studies

Article abstract of DOI:10.1016/j.ejmech.2012.07.021

Since the discovery of endogenous nociceptin/orphanin FQ (N/OFQ) peptide and N/OFQ peptide (NOP) receptor [or opioid-receptor-like-1 (ORL1) receptor], the structures, distribution, and pharmacology have been reported in detail. N/OFQ and NOP receptor are

Full text of DOI:10.1016/j.ejmech.2012.07.021

European Journal of Medicinal Chemistry 55 (2012) 228e242
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of 1-(b-amino substituted-b-alanyl)-N,N-dimethylindoline-2-
carboxamides as novel nonpeptide antagonists of nociceptin/orphanin FQ
receptor: Efficient design, synthesis, and structureeactivity relationship studies
*
Shigeo Hayashi , Katsuyo Ohashi, Eriko Nakata, Chie Emoto
Pfizer Global Research & Development, Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo, Aichi 470-2393, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Since the discovery of endogenous nociceptin/orphanin FQ (N/OFQ) peptide and N/OFQ peptide (NOP)
receptor [or opioid-receptor-like-1 (ORL1) receptor], the structures, distribution, and pharmacology have
been reported in detail. N/OFQ and NOP receptor are located in the corticolimbic regions that are
involved in the integration of the emotional activity, and located in the spinal cord, the peripheral
nervous systems or other peripheral tissues that are related to pain as well as urinary signal trans-
missions, with a pattern distinct from that of classical opioid peptides and their receptors in rodents or
primates. Furthermore, N/OFQeNOP receptor system plays an important role in the regulation of
various human physiologies such as depression effect, hyperphasia effect, and blood pressure effect. In
Received 7 May 2012
Received in revised form
11 July 2012
Accepted 15 July 2012
Available online 25 July 2012
Keywords:
Nociceptin/Orphanin FQ (N/OFQ)
NOP (ORL1) receptor antagonist
NOP receptor selectivity
Half-life in HLM
this study, the structureeactivity relationship of novel NOP receptor antagonist for various 1-(
b-amino
substituted- -alanyl)-N,N-dimethylindoline-2-carboxamides was investigated in vitro to elucidate
b
structural requisites to identify and develop potent and selective NOP receptor antagonists, which
resulted in the discovery of 1-{3-[4-(substituted phenyl)piperidin-1-yl]propanoyl}-N,N-dimethylindo-
line-2-carboxamide analogues that display potent and selective human NOP (hNOP) receptor binding
affinity and potent hNOP receptor antagonist activity. The efficient design, synthesis, and structuree
activity relationship studies for potent and selective novel NOP receptor antagonists and significant
findings in vitro, that include insights for binding and functional mechanisms via receptoreligand
interactions, are reported herein.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
their sequence similarities, are widely distributed in the cortico-
limbic regions involved in the integration of the emotional activity,
An endogenous heptadecapeptide, nociception/orphanin FQ (N/
OFQa) [1a,b], and nociceptin/orphanin FQ peptide (NOP1) receptor
[opioid-receptor-like-1 (ORL1) receptor or the fourth opioid
peptide (OP4) receptor] [2a,b], that is a G-protein-coupled receptor
(GPCR) and distinct from classical opioid peptide receptors despite
and in the spinal cord, the peripheral nervous systems or other
peripheral tissues related to pain as well as urinary signal trans-
missions [3aei]. Significantly, N/OFQ has demonstrated the utilities
of its agonist activity in vivo in various animal models, such as
analgesic effect against neuropathic pain (chronic pain), acute pain
or inflammatory pain in the peripheral and spinal cord [4aem],
anxiolytic effect [5aei] as well as antianorectic effect [6a,b] in the
brain. In fact, we have been reported the discovery of novel, highly
selective and potent NOP receptor agonists, (i) HPCOM that
demonstrates systemically potent and peripherally- and/or spinal
cord-active/selective antiallodynic effect in animal peripheral
neuropathic pain model, and (ii) MCOPPB that demonstrates orally
potent and brain-active/selective anxiolytic effect in animal Vogel
conflict model, in the distinct two series of drug-design, synthesis,
and structureeactivity relationship (SAR) studies by Hayashi et al.,
respectively [7,8a,b]. As well, it has been reported that N/OFQ dis-
played inhibitory effect on micturition reflex in vivo [9a], and
attenuation effect on neurogenic detrusor overactivity without
Abbreviations: GPCR, G-protein-coupled receptor; N/OFQ, nociceptin/orphanin
FQ; NOP receptor, N/OFQ peptide receptor [or opioid-receptor-like-1 (ORL1)
receptor]; MOP receptor,
human NOP and human MOP receptors, respectively; [35S]GTP
50-(
-thiotriphosphate); HEK, human embryonic kidney; CHO, Chinese hamster
ovary; HLM, human liver microsome; WSCI, water soluble carbodiimide, that is, 1-
m
-opioid peptide receptor; hNOP and hMOP receptors,
g
S, [35S]-guanosine
g
ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride;
HOBT,
1-
hydroxybenzotriazole; SAR, structureeactivity
relationship;
SMR,
structureemetabolic stability relationship.
* Corresponding author.
E-mail address: Shigeo_Hayashi@nifty.com (S. Hayashi).
Nomenclature Committee of the International Union of Pharmacology (NC-
1
IUPHAR).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.021

S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
229
significant problems for the treatment of urinary incontinence in
clinical trial [9b], as well as no sensory effect on bladder function
and no anomalous vital signs in normal subjects [9c]. On the other
hand, depression effect [10a,b], hyperphasia effect [11], and blood
pressure effect [12] of N/OFQ in vivo by way of NOP receptoreN/
OFQ system have been studied, which might show the potential
of NOP receptor antagonists to modulate/attenuate N/OFQ activity
in pharmacological viewpoints. Therefore, potent and selective
NOP receptor antagonists per se are also needed for investigation
and recognition of N/OFQeNOP receptor system in detail and might
be useful for seeking potential pharmacological utility. Herein, the
design, synthesis, and structureeactivity relationship (SAR) studies
speed parallel synthesis, respectively, of which the various amines
were 4-(phenyl, heteroaryl, cycloalkyl, and other substituted)
piperidine derivatives 4a, 4b and 4jen, 4-(10-aza-cycloheptanyl)
phenyl derivative 4o, 1-(phenyl, heteroaryl, cycloalkyl, and other
substituted)piperazine derivatives 4cei and 4pev, and fused bi- or
tri-cycloamine derivatives 4wez, respectively (Scheme 2). As well,
(2S)-analogues 7aef were prepared from compound (S)-3 with
various 4-(phenyl or substituted phenyl)piperidine derivatives
6aef by manual synthesis, respectively (Scheme 3). In the struc-
tural analysis study for the isolated products 5aei and 7aef, their
1H NMR spectra showed the starting materials-derived amido- and
amino-portion signals with several signals attributed to their
amide-bond conformers, respectively, and had no olefinic-portion
signals of the Michael acceptor (rac)-3 or (S)-3, respectively (see
Sections 4.1.3e4.1.11 and 4.1.13e4.1.18 in the Experimental section,
respectively). Overall, over two hundreds analogues were produced
by this method for the study (further data not shown).
of
1-(
b-amino
substituted-b-alanyl)-N,N-dimethylindoline-2-
carboxamide derivatives for potent and selective novel NOP
receptor antagonists in vitro with efficient analogue-developing
strategy, and significant findings of the analogues are reported.
As a key amine for this study, 4-(4-fluoro-2-methylphenyl)
piperidine 6d was prepared by multistep manual synthesis similar
to the known synthetic conditions for phenylpiperidine derivatives
[15]. Thus, 1-benzyl-4-piperidone 8 was coupled with Grignard
reagent, 4-fluoro-2-methylphenylmagnesium bromide generated
from 1-bromo-4-fluoro-2-methylbenzene in situ, and then
dehydro-olefinylation for the resulting hydroxyl compound 9 was
performed by acidic condition to give 1-benzyl-4-(4-fluoro-2-
methylphenyl)-1,2,3,6-tetrahydropyridine 10. Finally, simulta-
neous hydrogenolysis of the N-benzyl portion and hydrogenation
of the olefinic portion for compound 10 with Pd(OH)2 were per-
formed to afford requisite 4-(4-fluoro-2-methylphenyl)piperidine
6d as a sole product (Scheme 4).
2. Results and discussion
2.1. Chemistry
For design and SAR studies of NOP receptor antagonists in vitro,
the synthesis of various 1-(b-amino substituted-b-alanyl)-N,N-
dimethylindoline-(2RS or 2S)-2-carboxamide derivatives were
efficiently performed as follows.
First, a racemic compound, tert-butyl (2RS)-2-(dimethylcarba-
moyl)indoline-1-carboxylate (rac)-1 that was prepared from 1-
(tert-butoxycarbonyl)indoline-(2RS)-2-carboxylic acid by amida-
tion with dimethylamine, water soluble carbodiimide [1-ethyl-3-
(3-dimethylaminopropylcarbodiimide) hydrochloride (WSCI)], and
1-hydroxybenzotriazole (HOBT) [13] was N-deprotected by acidic
condition to afford N,N-dimethylindoline-(2RS)-2-carboxamide
(rac)-2. The unprotected 1-amino portion of the indoline deriva-
tive (rac)-2 was then amidated with acryl chroride to afford 1-
The 1-b-alanyl-N,N-dimethylindoline-2-carboxamide deriva-
tives 5aei and 7aef prepared by manual synthesis were converted
into monocitrates using citric acid in CH2Cl2eMeOH, respectively
(see Sections 4.1.3e4.1.11 and 4.1.13e4.1.18 in the Experimental
section, respectively). Also, 1-b-alanyl-N,N-dimethylindoline-2-
acryloyl-N,N-dimethylindoline-(2RS)-2-carboxamide
(rac)-3.
carboxamide derivatives 5jez prepared by high-speed parallel
synthesis were obtained as salt-free compounds (5jey) or
a formate (5z), dependent on the purification condition, respec-
tively (see Section 4.1.20 in the Experimental section). These salts
and salt-free compounds were used for SAR study with pharma-
cological and/or pharmacokinetic evaluations, respectively.
Furthermore, in the same manner described above, 1-acryloyl-N,N-
dimethylindoline-(2S)-2-carboxamide (S)-3, that is the (S)-enan-
tiomer of compound (rac)-3, was prepared from the corresponding
tert-butyl (2S)-2-(dimethylcarbamoyl)indoline-1-carboxylate (S)-1
(Scheme 1).
Second, the requisite 1-(N-substituted b-alanyl)-N,N-dimethy-
lindoline-2-carboxamides were prepared for the designed SAR
study by Michel addition reaction [14] from the above 1-acryloyl-
N,N-dimethylindoline-2-carboxamide of racemate, (rac)-3 or of (S)-
form, (S)-3 with various types of amines that were obtained from
commercial suppliers or in-house library or by manual synthesis.
Also, the Michel addition reaction was applied to high-speed ana-
loging (HSA) study with library-amines selected for the SAR study.
Indeed, (2RS)-analogues 5aei were prepared from compound (rac)-
3 with amines 4aei by manual synthesis, and (2RS)-analogues 5jez
were prepared from compound (rac)-3 with amines 4jez by high-
2.2. Design, SAR, and mechanism studies in vitro for NOP receptor
antagonists
Actually, the design, synthesis, and SAR studies of 1-(
b-amino
substituted- -alanyl)-N,N-dimethylindoline-2-carboxamide
b
analogues were performed to explore and identify novel NOP
receptor antagonists that have potent and selective binding affinity
and potent functional antagonist activity in vitro. The biological
assays for the in vitro SAR study were performed as follows. The
Scheme 1. Synthesis of 1-acryloyl-N,N-dimethylindoline-2-carboxamides (rac)-3 and (S)-3. Reagents and conditions: (a) trifluoroacetic acid, THF, 0 ꢀC to room temp; (b) acryloyl
chloride, Et3N, CH2Cl2, 0 ꢀC.

230
S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
Scheme 2. Synthesis of N,N-dimethyl-1-{3-[(substituted)cycloamino]propanoyl}indoline-(2RS)-2-carboxamides 5aez with Michael addition reaction. Reagents and conditions: (a)
Et3N, THF, 60 ꢀC.
binding affinities (Ki values) of the synthetic analogues to human
NOP (hNOP) receptor were measured by the displacement of
tritium-labelled N/OFQ for the recombinant hNOP receptor
expressed in human embryonic kidney (HEK)-293 cells [7,8a,16]. To
Scheme 3. Synthesis of N,N-dimethyl-1-{3-[(substituted)cycloamino]propanoyl}
indoline-(2S)-2-carboxamides 7aef with Michael addition reaction. Reagents and
conditions: (a) Et3N, THF, 60 ꢀC.
Scheme 4. Synthesis of 4-(4-fluoro-2-methylphenyl)piperidine 6d. Reagents and
conditions: (a) 4-fluoro-2-methylphenylmagnesium bromide, THF, reflux; (b) p-tol-
uenesulfonic acid monohydrate, toluene, reflux; (c) Pd(OH)2, MeOH, H2 (1 atm).

S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
231
Table 1
evaluate in vitro binding selectivity of the analogues for NOP
receptor over
-opioid peptide (MOP1) receptor that is a classical
Structureeactivity relationships of in vitro binding affinities to human recombinant
NOP receptor and human recombinant MOP receptor, and in vitro antagonist
m
opioid peptide receptor, the binding affinities for the analogues to
human MOP (hMOP) receptor expressed in Chinese hamster ovary
(CHO)-K1 cells were measured by the displacement of corre-
sponding radiolabelled ligand, [3H]DAMGO [7,8a,16]. The hNOP
receptor antagonist activities of the analogues were determined as
activities against human recombinant NOP receptor for 1-
b
-alanyl-N,N-dimethy-
lindoline-(2RS)-2-carboxamide analogues 5aez.a
the inhibitory activities (IC50 values) against [35S]GTP
a
g
S binding to
-unit of G-protein stimulated by N/OFQ that bound to the
recombinant hNOP receptor in HEK-293 cells [7,8a].
First, synthetic 1-(N-substituted -alanyl)-N,N-dimethylindo-
b
line-(2RS)-2-carboxamide derivatives, especially, aryl or heteroaryl
group-substituted monocycloamine analogues, alicyclic group-
substituted or acyclic-alkyl group-substituted monocycloamine
analogues, fused bi- or tri-cycloamine analogues as the modifica-
Compounds
Binding
affinity
Antagonism
against N/OFQ
stimulated
Ki (nM)b
tion of b-amino group were explored. The results of the SAR study
[
35S]GTP
gS
were illustrated in Table 1. Among this study for hNOP receptor
binding affinity of the 2-racemic analogues, it was found that 40-
phenylpiperidine analogues 5a, 5j, 5k, and 5l were effective as the
binding
IC50 (nM)c
No
RR0Ne
hNOP
5.55
hMOP
138
b
-amino portion-modified analogues. In the SAR study of substit-
uent effect on phenyl moiety of the 40-phenylpiperidine analogues,
40-(ortho-substituted phenyl)piperidine analogues were significant
or tolerated for potent hNOP receptor binding affinity. And the rank
of orders of the binding affinities for the various 40-(ortho-
substituted/unsubstituted phenyl)piperidine analogues was Me
(5a) > H (5j) > F (5k) > CF3 (5l). In particular, 40-(ortho-tolyl)
piperidine analogue 5a exhibited the most potent NOP binding
affinity among them, that is, the Ki value of the binding affinity to
hNOP receptor was 5.55 nM, with less hMOP receptor binding
affinity, that is, the Ki value was 138 nM. Further, the ortho-meth-
ylation of the phenyl unit was favourable for the selectivity of hNOP
receptor binding over hMOP receptor binding, that is, the selec-
tivity of 40-(ortho-tolyl)piperidine analogue 5a was 25-fold and the
selectivity of 40-(unsubstituted phenyl)piperidine analogue 5j was
19-fold, respectively. In seeking study of other cycloamino-ring
species for the corresponding piperidinyl-ring portion of the 40-
phenylpiperidine analogues, several 40-(ortho-substituted phenyl)
piperazine analogues were also allowed for potent binding affini-
ties to hNOP receptor, and the rank of orders of the binding affin-
ities for the 40-(ortho-substituted/unsubstituted phenyl)piperazine
analogues was Me (5p) > OEt (5c) > H (5d). Interestingly, compared
to 40-(ortho-tolyl)piperazine analogue 5p, 40-(ortho-ethoxyphenyl)
piperazine analogue 5c showed reduced hNOP receptor binding
affinity (Ki ¼ 127 nM) and increased hMOP receptor binding affinity
(Ki ¼ 29.8 nM), thus rather hMOP receptor binding selectivity (4-
fold). As the SAR of cycloamino-ring portion, relative to the above
40-(ortho-tolyl)piperidine analogue 5a, 40-(ortho-tolyl)piperazine
analogue 5p showed a half less potent hNOP receptor binding
affinity (Ki ¼ 11.6 nM) with comparative hNOP receptor binding
selectivity over hMOP receptor binding (Ki ¼ 318 nM), that is, the
hNOP receptor selectivity for compound 5p was 27-fold.
5a
116
N
5b
>250 (inactive @1
m
M) >450
NTd
NT
N
N
OEt
5c
5d
5e
5f
128
29.8
N
N
N
>250 (43% @1
>250 (37% @1
>250 (21% @1
>250 (11% @1
m
m
m
m
M)
M)
M)
M)
397
48,000
NT
N
N
N
N
N
N
235
N
>450
>450
NT
5g
NT
N
N
5h
5i
101
>450
>450
NT
NT
N
N
>250 (40% @1
m
M)
N
N
5j
26.2
26.5
502
NT
2296
NT
N
F
In the functional study in vitro, the above 40-(ortho-tolyl)piper-
idine analogue 5a and 40-phenylpiperidine analogue 5j exhibited
hNOP receptor antagonist activities as inhibitory activities against
5k
N
F
F
35S]GTP
gS binding induced by N/OFQ, that is, the respective IC50
F
[
5l
31.5
NT
NT
values were 116 nM for compound 5a and 2296 nM for compound
5j, which were related to the degree of the respective hNOP
receptor binding affinities in vitro. Also 40-phenylpiperazine
analogue 5d that had very weak hNOP receptor binding affinity
exhibited much less hNOP receptor antagonist activity compared to
the above compounds 5a and 5j.
N
N
N
N
5m
5n
>250 (inactive @1
m
M) NT
NT
NT
286
NT
Besides, other mono- or di-methylphenyl analogues such as 40-
(meta-tolyl)piperazine analogue 5q, 40-(200,400-dimethylphenyl)
piperazine analogue 5r, and 40-(300,400-dimethylphenyl)piperazine
analogue (not shown) showed much less hNOP receptor binding
5o
>250 (15% @1
m
M)
NT
NT
(continued on next page)

232
S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
Table 1 (continued )
inactivity at 1
mM for the hNOP receptor binding, respectively,
showing electron-state (electron-density) effect for the aryl core to
exhibit hNOP receptor binding affinity.
Compounds
Binding
affinity
Antagonism
against N/OFQ
stimulated
Ki (nM)b
Further approaches around 1-(N-substituted
b-alanyl)-N,N-
[
35S]GTP
gS
dimethylindoline-2-carboxamide analogues as shown below to
explore chemical space for exhibiting hNOP receptor binding
affinity were not successful. Thus, (1) for 40-position modification
binding
IC50 (nM)c
No
RR0Ne
hNOP
11.6
hMOP
318
on piperidin-10-yl or piperazin-10-yl substituent as the
b-amino
portion, (i) alicyclic substituted-groups such as 40-piper-
idinopiperidine (5b), 40-cyclohexylpiperidine (5n), 40-cyclo-
5p
NT
NT
NT
N
N
N
N
pentylpiperazine
(5t),
40-cyclohexylpiperazine
(5g),
40-
cycloheptylpiperazine (5u), and 40-cyclooctylpiperazine (5v), as
well as (ii) acyclic-alkyl substituted-groups such as 40-(100-ethyl-
propyl)piperazine (5h) and 40-(100-methylbutyl)piperazine (5i)
groups gave much less activity or inactivity. (2) Ring enlargement
analogue of the piperidine moiety of 40-phenylpiperidine analogue,
that is, 40-phenyl-10-aza-cycloheptane analogue 5o was inactive.
(3) As a modification of 40-phenylpiperidine portion, insertion of
methylene bridge between the piperidinyl ring and phenyl ring
such as development to 40-benzylpiperidine analogues 5f and 5s, or
alkyl bridge insertion within the piperidinyl ring such as develop-
ment to bicycloamine analogues (not shown, also relative analogue
is discussed later) resulted in very weak activity or inactivity. (4) In
case benzo-fused bi- or tri-cyclic amines (5wez) were used as the
replacement of 40-phenylpiperidine, the affinities were much
reduced, respectively. (5) In case fused bi-alicyclic amine such as
decahydroisoquinoline or mono-alicyclic amine such as 10-aza-
5q
5r
>250 (13% @1
>250 (25% @1
m
m
M)
M)
NT
NT
N
N
5s
>250 (11% @1
m
M)
>450
NT
5t
>250 (3% @1
m
M)
NT
NT
NT
NT
N
N
N
5u
N
>250 (12% @1
m
M)
cyclooctane was used for b-amino portion of b-alanine analogues,
the affinities were very weak or inactive (Ki > 250 nM) (further data
not shown).
5v
>250 (12% @1
>250 (16% @1
m
m
M)
M)
NT
NT
NT
NT
N
N
Taken together, among the above various analogues in the SAR
study, 40-(ortho-methyl substituted-phenyl)piperidine analogues
were the most promising/encouraging leads for further design,
synthesis, and SAR development study, because of their appro-
priate structural features and chemical properties such as appro-
priate electron-state (electron-density) of the aryl (phenyl) ring
core, appropriate bulkiness and electron state of the ortho-position
substituent of phenyl ring, and preferable size/volume/bulkiness
and shape of the cycloamino (piperidinyl) ring portion which
contribute to their potent and selective hNOP receptor binding
affinities and potent hNOP receptor antagonist activities in vitro.
5w
N
N
5x
5y
>250 (11% @1
>250 (33% @1
m
m
M)
M)
NT
NT
NT
NT
N
Next, synthetic 1-(N-substituted
b-alanyl)-N,N-dimethylindo-
line-(2S)-2-carboxamide derivatives, especially around 40-phenyl-
piperidine analogues were designed and investigated to identify
potent and selective hNOP receptor antagonists in vitro. The results
were summarized as shown in Table 2.
5z
>250 (15% @1
m
M)
NT
NT
N
a
5aei: products by manual synthesis; 5jez: products by high-speed parallel
As comparison between (S)-configuration- and racemic-forms
of potent indoline amide analogues for hNOP receptor binding
affinity, (S)-form 40-ortho-tolylpiperidine analogue 7a and 40-phe-
nylpiperidine analogue 7b showed approximately twice potent
binding affinities than that of the corresponding racemic analogues
5a and 5j, respectively, that is, the Ki values of ortho-tolyl analogues
were 3.03 nM for (S)-form 7a and 5.55 nM for racemate 5a,
respectively, and the Ki values of phenyl analogues were 13.6 nM for
(S)-form 7b and 26.2 nM for racemate 5j, respectively. Furthermore,
the respective hNOP receptor antagonist activities for (S)-forms 7a
(IC50 ¼ 44 nM) and 7b (IC50 ¼ 138 nM) were more potent than the
corresponding racemates 5a and 5j, respectively. Hence, it was
clarified that (S)-configuration forms of these analogues were
apparently preferable to the corresponding racemates for both
hNOP receptor binding affinities and hNOP receptor antagonist
activities, respectively. Besides the selectivities of the binding
affinity to hNOP receptor over hMOP receptor for ortho-tolyl
analogues were comparative between (S)-form 7a (22-fold) and the
corresponding racemate 5a (25-fold), which were higher than that
synthesis.
b
Ki values for compounds were measured by displacement of [3H]N/OFQ binding
to hNOP receptor expressed in HEK-293 cells and of [3H]DAMGO binding to hMOP
receptor expressed in CHO-K1 cells, respectively. Ki ¼ IC50/(1þ[radioligand]/KD).
Radioligands: [3H]N/OFQ as hNOP receptor agonist, concentration 0.4 nM,
KD ¼ 0.135 nM; [3H]DAMGO as hMOP receptor agonist, concentration 1.0 nM,
KD ¼ 0.821 nM.
c
Antagonism IC50 values for compounds were measured as inhibitory activity
against [35S]GTP
gS binding to a-unit of G-protein due to binding of N/OFQ to hNOP
receptor expressed in HEK-293 cells.
d
NT, not tested.
affinity or inactivity at 1 mM. Hence, steric substituent on meta- or
para-position of the phenyl group was not allowed for hNOP
receptor binding, and the negative steric hindrance effect at the
position(s) was also supported by other analogues (not shown). As
well, heteroaryl group substitution instead of the phenyl group of
the
40-phenylpiperidine/40-phenylpiperazine
analogue,
for
example, 40-(pyridin-200-yl)piperidine analogue 5m and 40-(pyr-
azin-200-yl)piperazine analogue 5e gave very weak activity or

S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
233
Table 2
selectivity for hNOP receptor over hMOP receptor was 37-fold.
Furthermore, 40-(200,600-dimethylphenyl)piperidine analogue 7e was
themostpotentanalogueamongthisseriesofanalogues,thatis, theKi
value of the hNOP receptor binding affinity was 0.6 nM with 86-fold
hNOP receptor selectivity over hMOP receptor. As for a bicycloa-
minoanalogue, thatis, 80-phenyl-30-azabicyclo[3.2.1]octaneanalogue
7f showed very weak hNOP receptor binding affinity and rather
potent hMOP receptor binding affinity, thereby supporting profit-
Structureeactivity relationships of in vitro binding affinities to human recombinant
NOP receptor and human recombinant MOP receptor, and in vitro antagonist
activities against human recombinant NOP receptor for 1-
b
-alanyl-N,N-dimethy-
lindoline-(2S)-2-carboxamide analogues 7aef.a
O
N
N
O
S
R
N
R'
ability of the piperidine as cycloamino ring for
b-portion modification
of the -alanine analogue as described already.
b
Significantly, the above 40-phenylpiperidine analogues 7aee
that possessed potent hNOP receptor binding affinities exhibited
potent hNOP receptor antagonist activities, respectively, that is,
their IC50 values were 12e138 nM. The potencies of the antagonist
activities are depended on that of the respective hNOP receptor
binding affinities for the analogues, and the relationships between
them are clearer than that of the mentioned cases of the racemic 40-
phenyl(piperidine or piperazine) analogues. Thus, for hNOP
receptor antagonists 7aee, there are highly significant positive
correlations of their IC50 values against N/OFQ-stimulated [35S]
Compounds
Binding affinity
Ki (nM)b
Antagonism
against
N/OFQ
stimulated
[
35S]GTP
gS
binding
IC50 (nM)c
No
RR0Ne
hNOP
3.03
hMOP
67.2
GTPg
S binding to the respective Ki values for inhibition of [3H]N/
OFQ binding as shown in Fig. 1 (r2 ¼ 0.9911, p < 0.0005). In the
present study, it is indicated that the binding of hNOP receptor
antagonists to NOP receptor is the primary mechanism of the
inhibition of G-protein activation response that is stimulated by N/
OFQ. More specifically, the present data demonstrated that the
appropriately modified 40-phenylpiperidine portion, that plays an
essential role for the relationships in the study, and/or the other
part or whole entity of the analogue that is assisted by the 40-
phenylpiperidine portion, effectively blocks or interferes on an
indispensable site or region for both the receptor binding and
biological activity in the hNOP receptor protein. By contrast, as
previously reported study by Hayashi et al. [8a], MCOPPB analogues
{2-substituted 1-[1-(1-methylcyclooctyl)piperidin-4-yl]-1H-benz-
imidazole derivatives} as hNOP receptor agonists exhibited highly
significant positive correlations of their in vitro EC50 (potency)
values, that were the concentrations producing a half-maximal
7a
44
N
7b
13.6
9.84
3.79
80.7
238
138
107
42
N
7c
F
F
N
N
7d
139
7e
0.96
82.1
28.4
12
N
response of them evaluated by the
[ gS binding in
35S]GTP
response to the agonistereceptor binding, to their respective
in vitro Ki values, that were evaluated by the inhibition of [3H]N/
OFQ binding, indicating that the binding of hNOP receptor agonist
to hNOP receptor is the primary mechanism of the G-protein
activation response and the potency of the functional activity
depends on the potency of the binding affinity for each analogue.
And the results also demonstrated that the 2-substituent portion of
the benzimidazole analogue that plays an essential role for the
relationships in the study, and/or the other part or whole entity of
the analogue that is assisted by the 2-substituent portion, effects on
an indispensable site or region for both the receptor binding and
biological activity in the hNOP receptor protein. On the other hand,
7f
>250
NTd
N
(23% @1 mM)
a
7aef: products by manual synthesis.
b
Ki values for these compounds were measured by displacement of [3H]N/OFQ
binding to hNOP receptor expressed in HEK-293 cells and of [3H]DAMGO binding to
hMOP receptor expressed in CHO-K1 cells, respectively (see also footnotes of
Table 1).
c
Antagonism IC50 for compounds were measured against N/OFQ-stimulated [35S]
GTP
g
S binding.
d
NT, not tested.
of (S)-form 40-phenylpiperidine analogue 7b (5.9-fold) and the
corresponding racemate 5j (19-fold).
Therefore, further design and synthetic studies were performed
around (S)-forms of 40-phenylpiperidine derivatives for achieving
potent and selective in vitro activity as hNOP receptor antagonist, as
well as metabolic stability (mentioned later). Indeed, (S)-form
40-(400-fluorophenyl)piperidine analogue 7c also showed greater
hNOP receptor binding affinity (Ki ¼ 9.84 nM) than that of (S)-form
40-(unsubstituted phenyl)piperidine analogue 7b, and further, on the
basis of multi-viewpoint results (described later) of 40-(ortho-tolyl)
piperidine analogue 7a and 40-(400-fluorophenyl)piperidine analogue
7c, (S)-40-(400-fluoro-200-methylphenyl)piperidine analogue 7d was
designed, which resulted in retained potency with improved selec-
tivity: the Ki value of hNOP receptor binding affinity was 3.79 nM, and
the Ki value of hMOP receptor binding affinity was 139 nM, thus the
Fig. 1. Correlation between Ki and IC50 for human nociceptin/orphanin FQ peptide
(hNOP) receptor antagonists 7aee. Ki values for binding affinity to hNOP receptor; IC50
values for hNOP receptor antagonist activity against nociceptin/orphanin FQ (N/OFQ)-
stimulated [35S]-guanosine 50-(
g-thiotriphosphate) binding.

234
S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
supported or complementally results have been reported for bio-
logical function of NOP receptor. Thus, (i) several cavities in its
transmembrane regions work for binding to N/OFQ and following
biological activity as a GPCR, where are able to be affected by
mutation of amino acid residues around the binding site [17]. For
example, one cavity in a transmembrane region works for binding
to N/OFQ with biological activity, but loses the biological function
after the binding if Gln286 of the binding site is point mutated,
namely, the transduction of N/OFQ signal is not conveyed after the
binding, thereby the functional switch remains off [17]. Similarly,
(ii) Asn133 mutation of NOP receptor causes no biological response
after N/OFQ binding [18]. In other words, these residues of NOP
receptor in the reported studies such as (i) Gln286 and (ii) Asn133
were estimated as functionally important, respectively. Therefore,
the results of the present SAR study for hNOP receptor antagonists
and of the previous SAR study for hNOP receptor agonists (MCOPPB
analogues as well as HPCOM analogues) [7,8], and these analogues
per se might be quite useful to elucidate functional mechanisms of
NOP receptoreligand interactions, that includes further exploring/
identifying the dynamics of intercepting (by antagonists) or causing
(by N/OFQ or other agonists) effect via receptoreligand binding on
biological signal-transduction mechanisms/pathways of GPCR
systems with the receptor, as well as specifying/focusing further or
detailed intrinsic portion, character or pharmacophore of the
receptor or the ligands in terms of biological function after the
binding.
clearly shows that the stereochemistry of the antagonists/agonists
is closely related to the degrees of molecular-recognition and
functional-action (intercepting or causing signal transduction) of
the antagonists/agonists for NOP receptor, respectively.
In conclusion, it was revealed that the respective structural
features and chemical properties of the b-amino substituent of b-
alanine part for the present series of analogues, such as appropriate
total and partial size/volume/bulkiness, shape, and electron state
(electron-density degree or electron-density distribution), the
position/orientation pattern of substituent(s)/pharmacophore(s),
and the stereochemical character of the molecule would deeply
contribute to the underlying dynamic interaction mechanisms of
hNOP receptor binding and hNOP receptor antagonism for the
analogues in the biological field, which would be also related to the
underlying interaction mechanisms of hNOP receptor binding and
hNOP receptor function for N/OFQ or hNOP receptor agonist(s).
2.3. Design and SMR studies in vitro with multi-viewpoint
integrated strategy for NOP receptor antagonists
During the course of design, synthesis, and in vitro SAR studies
of the above (S)-form N,N-dimethylindoline-2-carboxamide
analogues to identify potent and selective NOP receptor antago-
nists as described, simultaneously, design and in vitro
structureemetabolic stability relationship (SMR) for the analogues
were investigated to identify metabolically stable analogues as
well, for an integrated drug-design strategy with multi-viewpoint
evaluations as described already in the discovery study of orally
potent anxiolytic by Hayashi et al. [8b]. For the present study, the
half-lives of NOP receptor antagonists in human liver microsomes
(HLMs), that is related to long duration of drug efficacy with good
bioavailability [7,8b], were investigated as pharmacokinetic evalu-
ation in vitro. Also, physicochemical properties for the analogues
were estimated, for example, the lipophilicity value for the whole
structure of the respective molecule at pH 7.4 was calculated as ACD
log D calculated by ACD software (ACD log D7.4). The results of the
relationships of structureemetabolic stabilityephysicochemical
properties for NOP receptor agonists were summarized in Table 3.
As described, regarding with the in vitro SAR study of 40-phe-
nylpiperidine portion of (S)-form N,N-dimethylindoline-2-
carboxamide analogues, 40-(ortho-tolyl)piperidine analogue 7a
was more potent and selective hNOP receptor antagonist compared
to 40-phenylpiperidine analogue 7b; however, 40-(ortho-tolyl)
piperidine analogue 7a showed less half-life (24 min) than that of
40-phenylpiperidine analogue 7b (55 min) in HLM as a result of SMR
study. Besides, as a significant finding, further designed 40-(400-
fluorophenyl)piperidine analogue 7c prolonged half-life in HLM
(82 min) compared to 40-phenylpiperidine analogue 7b, which
might be due to blocking effect by introduction of fluorine atom
against the potential metabolic site at para-position of the phenyl
nucleus (also discussed later), with the improved in vitro characters
as hNOP receptor antagonist relative to 40-phenylpiperidine
analogue 7b, and comparable hNOP receptor selectivity to that of
40-(ortho-tolyl)piperidine analogue 7a. Therefore, 40-(400-fluoro-200-
methylphenyl)piperidine analogue 7d was designed for the
compatibility of potent and selective hNOP receptor binding
affinity and potent hNOP receptor antagonist activity in vitro, and
long half-life in vitro. Actually, the novel analogue 7d achieved
longer lasting half-life in vitro (43 min) than that of 400-position
unblocked 40-(ortho-tolyl)piperidine analogue 7a, as well as the
potent and selective binding affinity, and the potent antagonist
activity in vitro as described already.
Indeed, for example, in a viewpoint of the structural feature of
the b-portion substituent for the present series of b-alanine amide
analogues regarding the molecular-recognition mechanisms and
functional mechanisms as receptoreligand interactions, the rela-
tionship for the position/orientation between phenyl ring and
piperidinyl ring is significant, thus restriction of torsion angle
(dihedral angle) of phenyl-ring plain and cycloamino-ring portion
would influence on the potency of hNOP receptor binding affinity
and hNOP receptor antagonist activity. Actually, when the torsion
angle between two rings was restricted to (or fixed for) non-
coplanar or was ranged around perpendicular because of the
steric hindrance by ortho-substituent on phenyl ring, for example,
in the cases of 40-(ortho-tolyl)piperidine analogue 7a and 40-(200,600-
dimethylphenyl)piperidine analogue 7e, their respective hNOP
receptor binding affinities and hNOP receptor antagonist activities
that were closely correlated to the binding affinities were greater
than that of 40-phenylpiperidine analogue 7b. On the contrary,
when the phenyl ring was united/fixed with the cycloamino ring as
nearly coplanar position as a whole, thus in the cases of 10,20,30,40-
tetrahydroisoquinoline analogue 5x and 10,20,30,40,40a,50,60,100b-
octahydrobenzo[f]isoquinoline analogue 5y, their hNOP receptor
binding affinities were low or inactive. By contrast, in the case of 40-
(unsubstituted phenyl)piperidine analogue 5j, the phenyl moiety is
placed at 40-piperidine moiety via a single bond without no
significant steric hindrance, the angle between phenyl ring and
cycloamino ring is freely variable, thereby showing much greater
hNOP receptor binding affinity than that of 10,20,30,40,40a,50,60,100b-
octahydrobenzo[f]isoquinoline analogue 5y and less than that of 40-
(ortho-tolyl)piperidine analogue 5a. Hence it would be estimated
that the single-bond rotation between phenyl- and piperidinyl-
rings for compound 5j would allow its active conformation in the
compoundehNOP receptor complex at binding mode, whereas the
coplanar-like moiety consisted of phenyl- and piperidinyl-rings for
compound 5y would avoid/inhibit the active conformation.
In other structural viewpoints, it is noteworthy that the
stereochemistry of the present (S)-configuration of hNOP receptor
antagonists and previous (R)-configuration of hNOP receptor
agonists [8a] is also important for their in vitro potent antagonist
activities and in vitro potent agonist activities, respectively, which
As another side of SMR viewpoints, ortho-methyl substituent
effect in terms of in vitro metabolic stability for compound 7a
relative to compound 7b as 40-(400-unsubstituted phenyl)piperidine

S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
235
Table 3
SAR of metabolic stabilities (half-lives in human liver microsomes) versus physicochemical properties for (S)-1-
b-alanyl-N,N-dimethylindoline-2-carboxamide analogues 7aef.
O
N
N
O
S
R
N
R'
Compounds
Half-life in HLM
min
Physicochemical properties
Lipophilicity
Size
HBAb
TPSAc
Å2
a
No
RR0Ne
ACD log D7.4
MW
Number
7a
24
55
2.41
1.95
419.56
405.53
3
3
43.86
43.86
N
7b
7c
N
82
2.03
423.52
3
43.86
F
F
N
N
7d
7e
43
19
2.49
2.87
437.55
433.59
3
3
43.86
43.86
N
7f
10
2.11
431.57
3
43.86
N
a
Predicted by ACD/laboratories 9.0.
HBA, hydrogen bond acceptor.
TPSA, topological polar surface area.
b
c
analogues, and the effect for compound 7c relative to compound 7d
as 40-(400-fluorophenyl)piperidine analogues were almost same,
thus the ortho-methyl substituent, that is, more lipophilic and
bulkier than hydrogen, influenced to shorten half-life in each case.
As well, in comparison between 40-phenylpiperidine analogue 7b
and 80-phenyl-30-azabicyclo[3.2.1]octane analogue 7f, the latter
one showed shorter half-life than that of the former one, owing to
its bicycloamino moiety that is more lipophilic and bulkier than
monocycloamino moiety of the former one. Besides, the major
metabolic site of a 200,600-dimethylphenyl analogue was 400-position
of the phenyl core, which was identified as 400-hydroxylated
metabolite by HPLCeMS/MS analysis in our metabolite study with
liver microsome (further data not shown). Therefore, the lip-
ophilicity and/or steric effect, and metabolic-site protection would
be estimated as key factors for the present analogues to vary
metabolic stability in vitro. Furthermore, it is significant to consider
the SMR study with a viewpoint of physicochemical property for
the present series of analogues (Table 3). Thus, except 40-(400-fluo-
rophenyl)piperidine analogues (7c and 7d), the relationship
between half-life evaluated in HLM and lipophilicity estimated as
ACD log D7.4 value for 40-(substituted or unsubstituted phenyl)
piperidine analogues (7a, 7b, and 7e), that have similarity of other
structural features such as molecular weight (Mw), hydrogen-
bonding functionality (for example, number of hydrogen bond
acceptor), and topological polar surface area (TPSA), indicates that
the degree of metabolic stability is inversely related to the degree of
lipophilicity. As well, for the potential metabolic site-blocked
analogues such as 7c and 7d, the relationship of metabolic
stabilityelipophilicity was the same, as mentioned above for the
ortho-methyl substituent effect in the SMR study. Hence, the
tendency of the relationship for the present series of analogues,
that is, lower the lipophilicity, the longer the half-life, is in line with
the previously reported results for HPCOM analogues or MCOPPB
analogues by Hayashi et al. [7,8b] on condition that the other
structural features or chemical properties are almost same or
similar, and that the potential metabolic-site issue is removable or
negligible or similar, for example, in case the major determinant-
moiety of lipophilicty-difference and the major metabolic-site are
located at different portions each other for the series of analogues.
In summary, as the results of the present design and SMR study,
it was clarified that the important factors to achieve long-lasting
metabolic stability in vitro for
b-amino substituent of the present
series of -alanine analogues were protection of potential meta-
b
bolic-reaction, low lipophilicity, and/or low steric-effect, with
appropriate other structural/chemical properties. Furthermore,
through the present drug-design, synthesis, SAR, and SMR studies
with efficient, logical, and integrated strategy in vitro, 1-{3-[4-
(substituted phenyl)piperidin-1-yl]propanoyl}-N,N-dimethylindo-
line-(2S)-2-carboxamide analogues were discovered as potent,
selective, and long-lasting metabolically stable novel hNOP

236
S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
receptor antagonists. These analogues and their SAR/SMR
outcomes might be quite useful to elucidate functional mechanisms
of N/OFQeNOP receptor system in vitro as well as in vivo for further
studies.
multiplicities (s, singlet; d, doublet; t, triplet; q, quartet; dd, double
doublet; ddd, double double doublet; m, multiplet; and br, broad-
ened), and coupling constants. Infrared spectraweremeasured byan
IR-470 (Shimadzu) infrared spectrometer. Low-resolution mass
spectral data (EI) were obtained on an Automass 120 (JEOL) mass
spectrometer. Low-resolution mass spectral data (ESI) were ob-
tained on a Quattro II (Micromass) mass spectrometereAgilent 1100
HPLC system. General reagents and solvents were purchased from
commercial chemical suppliers. Some amines were in-house library
compounds in Pfizer Global Research & Development (PGRD) or
prepared by us at PGRD, Nagoya Laboratories. The compounds 5aei
and 7aef prepared by manual synthesis were used for pharmaco-
logical and/or pharmacokinetic evaluations after citrate formation
using one equivalent of citric acid in MeOHeCH2Cl2, respectively,
and the purities of the citrate salts of the compounds 5aei, 7aed,
and 7f were confirmed by elementary analysis to be within ꢁ0.4%
of calculated values before the biological evaluations, respectively.
As well, the compounds 5jez prepared by high-speed parallel
synthesis were used for the evaluation study as salt-free form or
formate salt dependent on the purification condition of the corre-
sponding compound, respectively.
3. Conclusions
In the present drug-discovery study, 1-(
b
-amino substituted-
b-
alanyl)-N,N-dimethylindoline-(2RS)-2-carboxamide
derivatives
were explored to elucidate structural requisites for hNOP receptor
antagonist activity with efficient synthesis and well-designed SAR
investigation in vitro, which resulting in the discovery of 1-{3-[4-
(substituted phenyl)piperidin-1-yl]propanoyl}-N,N-dimethylindo-
line-(2RS)-2-carboxamide analogues as novel hNOP receptor
antagonists. Furthermore, simultaneous investigation of SAR and
SMR in vitro for 1-{3-[4-(substituted phenyl)piperidin-1-yl]prop-
anoyl}-N,N-dimethylindoline-(2S)-2-carboxamide analogues were
very useful and effective to design and identify potent, selective,
and metabolically stable hNOP receptor antagonists in vitro, and the
significant findings are as follows: (i) The (2S)-form analogues were
superior to the corresponding racemic analogues for hNOP receptor
binding affinities and antagonist activities. (ii) There are highly
significant positive correlations of the IC50 values to the respective
Ki values for hNOP receptor antagonists 7aee, and the structural
features and chemical properties of the hNOP receptor antagonists
would influence on (or contribute to) their inhibition mechanisms
against dynamic signal-transduction of N/OFQeNOP receptor
system. (iii) As a representative, 1-{3-[4-(4-fluoro-2-methylphenyl)
piperidin-1-yl]propanoyl}-N,N-dimethylindoline-(2S)-2-
4.1.2. 1-Acryloyl-N,N-dimethylindoline-(2RS)-2-carboxamide
[(rac)-3]
4.1.2.1. tert-Butyl 2-(dimethylcarbamoyl)indoline-1-carboxylate [(rac)-
1]. To
a
stirred suspension of indoline-2-carboxylic acid
(SigmaeAldrich, 9.790 g, 60.00 mmol) in dioxane (130 mL)eH2O
(98.0 mL) was added 2 N NaOH (32.6 mL, 65.2 mmol) and di-tert-
butyl dicarbonate (Boc2O) (16.5 mL, 71.8 mmol) at 0 ꢀC under N2. The
mixture was allowed to warm to room temperature and stirred
under N2 for 1 day. The mixture was concentrated in vacuo. The
aqueous residue was dissolved in H2O (total amount 130 mL), acid-
ified by adding 10% aqueous citric acid, and extracted with AcOEt
(150 mL ꢂ 2). The combined organic extracts were dried over
anhydrous MgSO4, concentrated in vacuo to afford 15.66 g of 1-(tert-
carboxamide 7d, that was rationally designed through the inte-
grated SAR and SMR studies, demonstrates potent and selective
hNOP receptor binding affinity, potent hNOP receptor antagonist
activity, and long-lasting half-life in HLM owing to the introduction
of the methyl group to restrict position and orientation (torsion
angle) between the phenyl portion and the piperidinyl portion of
the
b
-amino moiety, and to the introduction of the fluorine atom at
butoxycarbonyl)indoline-(2RS)-2-carboxylic
acid
(crude)
as
potential metabolic-reaction site on the phenyl nucleus. Overall,
the SAR/SMR outcomes for the present series of analogues and the
analogues per se might be quite useful to elucidate further under-
lying functional mechanisms of N/OFQeNOP receptor system in
detail for further hNOP receptor antagonist studies as well as NOP
receptor studies.
a brownish-white solid. In the spectrum of 1H NMR (270 MHz, CDCl3)
for the product, a broad signal of 9H proton at 1.53e1.43 ppm
attributed to tert-butoxycarbonyl (Boc) group was confirmed, with
several broad signals for other parts of the product at other positions
of the chart (further data not shown; see also an NMR signal data for
Boc group of compound (rac)-1 as shown below).
To a stirred mixture of 1-(tert-butoxycarbonyl)indoline-(2RS)-2-
carboxylic acid (15.66 g, crude) and dimethylamine hydrochloride
(14.68 g, 180 mmol) in dry CH2Cl2 (150 mL) was added water
soluble carbodiimide, that is, 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (WSCI) (23.00 g, 120 mmol), 1-
hydroxybenzotriazole (HOBT) (16.33 g, 120 mmol), and dry Et3N
(42.0 mL, 300 mmol) at 0 ꢀC under N2. The mixture was allowed to
room temperature and stirred under N2 for 1 day. The mixture was
poured into ice-cooled saturated aqueous NaHCO3 (100 mL). The
resulting mixture was shaken, and the organic layer was separated.
The aqueous layer was extracted with CH2Cl2 (100 mL). The organic
layers were combined, dried over anhydrous MgSO4, and concen-
trated in vacuo. The residue was purified by flash column chro-
matography (silica gel, hexane/AcOEt ¼ 1:2) to afford 11.03 g of the
title product (rac)-1 in 63% yield (2 steps) as a slight brownish-
4. Experimental section
4.1. Chemistry
4.1.1. General procedures
In general, reagents, solvents, and other chemicals were used as
purchased without further purification. All reactions with air- or
moisture-sensitive reactants and solvents were carried out under
nitrogen atmosphere. Flash column chromatography (medium
pressure liquid chromatography) purifications were carried out
using Merck silica gel 60 (230e400 mesh ASTM). Preparative thin-
layer chromatography (PTLC) purifications were carried out on
Merck silica gel 60 F254 precoated glass plates at a thickness of 0.5 or
1.0 mm. The structures of all isolated compounds were assured by
the following techniques, such as NMR, IR, MS or elementary anal-
ysis. 1H nuclear magnetic resonance (1H NMR) data were deter-
mined at 270 MHz on a JNM-LA 270 (JEOL) spectrometer and at
300 MHz on a JNM-LA300 (JEOL) spectrometer. Chemical shifts are
white solid. 1H NMR (270 MHz, CDCl3)
d 7.92e6.88 (4H, m, Ar H),
5.25e5.08 (1H, m, CH), 3.47 (1H, dd, J ¼ 16.0 Hz, J ¼ 11.2 Hz, CH2),
3.18e2.93 (7H, m, CH2 and N(CH3)2), 1.59e1.48 (9H, m, OeC(CH3)3).
MS (EI direct) m/z: Mþ 290.
expressed in
d
(ppm). 1H NMR chemical shifts including several
signals for major amide-bond conformers of compounds were
determined relative totetramethylsilane (TMS) as internal standard.
NMR data are reported as follows: chemical shift, number of atoms,
4.1.2.2. N,N-Dimethylindoline-(2RS)-2-carboxamide
a stirred solution of tert-butyl (2RS)-2-(dimethylcarbamoyl)indoline-
1-carboxylate (rac)-1 (11.03 g, 37.99 mmol) in anhydrous THF
[(rac)-2]. To

S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
237
(44.0 mL) was added trifluoroacetic acid (TFA) (133 mL) at 0 ꢀC under
N2. The mixture was stirred at 0 ꢀC under N2 for 30 min, allowed to
room temperature, stirred for 2 h, then concentrated in vacuo. The
residue was dissolved in CH2Cl2 (150 mL)eH2O (150 mL). The organic
layer was separated, dried over anhydrous MgSO4, and concentrated
in vacuo to afford 7.15 g of the title product (rac)-2 in 99% yield as
25% aqueous ammonia ¼ 10:1:0.5) was performed to afford 71.9 mg
of the title product 5b in 87% yield. 1H NMR (300 MHz, CDCl3)
8.28
d
(0.4H, d, 8.25 Hz, Ar H that is a peak for an amide-bond conformer),
7.26e7.08 (2.6H, m, Ar H including a peak for an amide-bond
conformer), 6.99 (1H, ddd, J ¼ 7.32 Hz, J ¼ 7.14 Hz, J ¼ 1.11 Hz, Ar
H), 5.47e5.44 (0.6H, CHeCO that is a peak for an amide-bond
conformer), 5.21 (0.4H, d, 8.61 Hz, CHeCO that is a peak for an
amide-bond conformer), 3.72e1.76 (23H, m, NCH(CH2)2, CH2, and
N(CH3)2), 1.69e1.38 (8H, m, CH2). Monocitrate of compound 5b: IR
(KBr): 3396, 2943, 1653, 1597, 1483, 1406, 1271, 1123, 1061, 989, 760,
a slight brownish-white solid. 1H NMR (270 MHz, CDCl3)
d 7.08e7.03
(2H, m, Ar H), 6.80e6.70 (2H, m, Ar H), 4.57 (1H, dd, J ¼ 10.7 Hz,
J¼ 5.62Hz,CH), 3.50(1H, dd, J¼ 15.7 Hz, J¼ 10.7 Hz, CH2), 3.16(1H, dd,
J ¼ 15.7 Hz, J ¼ 5.62 Hz, CH2), 3.07 (3H, s, NCH3), 2.99 (3H, s, NCH3).
598 cmꢃ1
(C24H36N4O2$C6H8O7) C, H, N.
.
MS (ESI positive) m/z: [M
þ
H]þ 413.28. Anal.
4.1.2.3. 1-Acryloyl-N,N-dimethylindoline-(2RS)-2-carboxamide
[(rac)-3]. To a stirred solution of N,N-dimethylindoline-(2RS)-2-
carboxamide (rac)-2 (7.150 g, 37.58 mmol) in dry CH2Cl2 (140 mL)
was added dry Et3N (13.1 mL, 94.0 mmol) at room temperature
under N2. The mixture was cooled to 0 ꢀC, then acryloyl chloride
(3.70 mL, 45.1 mmol) was added. The reaction mixture was stirred
at 0 ꢀC under N2 for 2 h, then poured into saturated aqueous
NaHCO3 (150 mL) at 0 ꢀC. The mixture was stirred at 0 ꢀC for 15 min,
then the organic layer was separated. The aqueous layer was
extracted with CH2Cl2 (100 mL ꢂ 2). The organic layers were
combined, dried over anhydrous MgSO4, and concentrated in vacuo.
The residue was purified by flash column chromatography (silica
gel, hexane/acetone ¼ 1:1) followed by recrystallization from
AcOEtehexane to afford 5.335 g of the title product (rac)-3 in 58%
yield as a yellowish-white solid. 1H NMR (300 MHz, DMSO-d6)
4.1.5. 1-{3-[4-(2-Ethoxyphenyl)piperazin-1-yl]propanoyl}-N,N-
dimethylindoline-(2RS)-2-carboxamide (5c)
A solution of 1-(2-ethoxyphenyl)piperazine monohydrochloride
(SigmaeAldrich, 48.6 mg, 0.200 mmol), compound (rac)-3
(48.9 mg, 0.200 mmol), and dry Et3N (72.5
mL, 0.52 mmol) in
anhydrous THF (3.0 mL) was stirred at 60 ꢀC under N2 for 36 h,
cooled to room temperature, then concentrated in vacuo. PTLC
purification (silica gel, CH2Cl2/MeOH ¼ 20:1) was performed to
afford 61.1 mg of the title product 5c in 68% yield. 1H NMR
(300 MHz, CDCl3)
d
8.61 (0.4H, d, J ¼ 7.50 Hz, Ar(indoline) H that is
a peak for an amide-bond conformer), 7.29e6.83 (7.6H, m, Ar H
including Ar(indoline) H that is a peak for an amide-bond
conformer), 5.49e5.46 (0.6H, m, CHeCO that is a peak for an
amide-bond conformer), 5.26e5.23 (0.4H, m, CHeCO that is a peak
for an amide-bond conformer), 4.07 (2H, q, 6.96 Hz, OCH2CH3),
3.75e1.70 (20H, m, CH2 and N(CH3)2), 1.46 (3H, t, J ¼ 6.96 Hz,
OCH2CH3). Monocitrate of compound 5c: IR (KBr): 3410, 2932,1728,
1655, 1501, 1485, 1404, 1248, 1121, 1042, 984, 935, 754, 598,
d
8.20 (1H, d, J ¼ 8.40 Hz, Ar H), 7.21e7.16 (2H, m, Ar H), 7.03e6.98
(1H, m, Ar H), 6.37e6.21 (2H, m, CH2]CH(CO) and CH2]CH(CO)),
5.76e5.68 (2H, m, CH2]CH(CO) and CHeCON(CH3)2), 3.68 (1H, dd,
J ¼ 17.1 Hz, J ¼ 11.1 Hz, CH2), 3.11 (3H, s, NCH3), 3.03 (1H, d,
J ¼ 18.3 Hz, CH2), 2.84 (3H, s, NCH3). MS (EI direct) m/z: Mþ 244.
536 cmꢃ1
(C26H34N4O3$C6H8O7) C, H, N.
.
MS (ESI positive) m/z: [M
þ
H]þ 451.26. Anal.
4.1.3. N,N-Dimethyl-1-[3-(4-o-tolylpiperidin-1-yl)propanoyl]
indoline-(2RS)-2-carboxamide (5a)
A solution of 4-o-tolylpiperidine hydrochloride (Arch, 52.9 mg,
0.250 mmol), compound (rac)-3 (61.7 mg, 0.252mmol), and dry Et3N
4.1.6. N,N-Dimethyl-1-[3-(4-phenylpiperazin-1-yl)propanoyl]
indoline-(2RS)-2-carboxamide (5d)
A solution of 1-phenylpiperazine (Tokyo Chemical Industry,
32.4 mg, 0.200 mmol), compound (rac)-3 (48.9 mg, 0.200 mmol),
(91
m
L, 0.65 mmol) in anhydrous THF (3.0 mL) was stirred at 60 ꢀ
C
under N2 for 36 h, cooled to room temperature, then concentrated in
vacuo. PTLC purification (silica gel, CH2Cl2/MeOH ¼ 10:1) was per-
formed to afford70.9 mgof the title product 5a in 68% yield as a solid.
and dry Et3N (45 mL, 0.32 mmol) in anhydrous THF (3.0 mL) was
stirred at 60 ꢀC under N2 for 36 h, cooled to room temperature, then
concentrated in vacuo. PTLC purification (silica gel, CH2Cl2/
MeOH ¼ 20:1) was performed to afford 41.2 mg of the title product
1H NMR (270 MHz, CDCl3)
d
8.31 (0.4H, d, J ¼ 7.72 Hz, Ar(indoline) H
that is a peak for an amide-bond conformer), 7.30e7.06 (6.6H, m, Ar
H including Ar(indoline) H that is a peak for an amide-bond
conformer), 7.03e6.97 (1H, m, Ar(indoline) H), 5.50e5.45 (0.6H,
m, CHeCO that is a peak for an amide-bond conformer), 5.26e5.21
(0.4H, m, CHeCO that is a peak for an amide-bond conformer),
3.75e2.47 (15H, m, AreCH, CH2, and N(CH3)2), 2.29e2.11 (2H, m,
CH2), 2.34 (3H, s, AreCH3),1.87e1.74 (4H, m, CH2). General procedure
of monocitrate formation. A solution of compound 5a (70.9 mg,
0.169 mmol) and one equivalent of citric acid (32.5 mg, 0.169 mmol)
in dry MeOH (15.0 mL)edry CH2Cl2 (12.0 mL) was stirred at room
temperature under N2 for 2 h, and then concentrated in vacuo. The
residue was solidified from dry CH2Cl2edry hexane. After removing
solvent in vacuo, the solid was collected and dried under vacuum at
60 ꢀC for 2 h to afford 93.1 mg of monocitrate of compound 5a as
awhite solid: IR (KBr): 3398, 2932,1724,1655,1485,1406,1261,1123,
756, 598 cmꢃ1. Anal. (C26H33N3O2$C6H8O7) C, H, N.
5d in 51% yield. 1H NMR (300 MHz, CDCl3)
d 8.31e8.28 (0.4H, m,
Ar(indoline) H that is a peak for an amide-bond conformer),
7.29e6.83 (8.6H, m, Ar H including Ar(indoline) H that is a peak for
an amide-bond conformer), 5.49e5.46 (0.6H, m, CHeCO that is
a peak for an amide-bond conformer), 5.24e5.21 (0.4H, m, CHeCO
that is a peak for an amide-bond conformer), 3.73e1.80 (20H, m,
CH2 and N(CH3)2). Monocitrate of compound 5d: IR (KBr): 3418,
2932, 1728, 1655, 1599, 1485, 1402, 1250, 1124, 982, 928, 758, 696,
596, 581 cmꢃ1. MS (ESI positive) m/z: [M þ H]þ 407.22. Anal.
(C24H30N4O2$C6H8O7) C, H, N.
4.1.7. N,N-Dimethyl-1-[3-(4-pyrazin-2-ylpiperazin-1-yl)propanoyl]
indoline-(2RS)-2-carboxamide (5e)
A
solution of 1-(2-pyrazinyl)piperazine (Chess, 32.8 mg,
0.200 mmol), compound (rac)-3 (48.9 mg, 0.200 mmol), and dry
Et3N (45 mL, 0.32 mmol) in anhydrous THF (3.0 mL) was stirred at
4.1.4. 1-[3-(1,40-Bipiperidin-10-yl)propanoyl]-N,N-dimethylindoline-
60 ꢀC under N2 for 36 h, cooled to room temperature, then
concentrated in vacuo. PTLC purification (silica gel, CH2Cl2/
MeOH ¼ 20/1/) was performed to afford 70.1 mg of the title product
(2RS)-2-carboxamide (5b)
A
solution of 1,40-bipiperidine (SigmaeAldrich, 33.7 mg,
0.200 mmol), compound (rac)-3 (48.9 mg, 0.200 mmol), and dry
5e in 86% yield. 1H NMR (300 MHz, CDCl3)
d 8.30 (0.4H, d,
Et3N (45
m
L, 0.32 mmol) in anhydrous THF (3.0 mL) was stirred at
J ¼ 8.40 Hz, Ar(indoline) H that is a peak for an amide-bond
conformer), 8.13 (1H, m, pyrazine H), 8.06 (1H, dd, J ¼ 2.55 Hz,
J ¼ 1.47 Hz, pyrazine H), 7.85 (1H, d, J ¼ 2.58 Hz, pyrazine H),
60 ꢀC under N2 for 36 h, cooled to room temperature, then
concentrated in vacuo. PTLC purification (silica gel, CH2Cl2/MeOH/

238
S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
7.28e7.10 (2.6H, m, Ar H including Ar(indoline) H that is a peak for
an amide-bond conformer), 7.00 (1H, dd, J ¼ 7.32 Hz, J ¼ 6.96 Hz,
Ar(indoline) H), 5.50e5.46 (0.6H, m, CHeCO that is a peak for an
amide-bond conformer), 5.23e5.19 (0.4H, m, CHeCO that is a peak
for an amide-bond conformer), 3.75e1.65 (20H, m, CH2 and
N(CH3)2). Monocitrate of compound 5e: IR (KBr): 3390, 1717, 1653,
1485, 1427, 1259, 1205, 1078, 831, 758 cmꢃ1. MS (ESI positive) m/z:
[M þ H]þ 409.21. Anal. (C22H28N6O2$C6H8O7) C, H, N.
amide-bond conformer), 5.23e5.19 (0.4H, m, CHeCO that is a peak
for an amide-bond conformer), 3.72e2.37 (20H, m, CH2 and
N(CH3)2), 2.13 (1H, quintet, 6.42 Hz, NCH(CH2CH3)2), 1.56e1.24 (4H,
m, NCH(CH2CH3)2), 0.89 (6H, t, 7.50 Hz, NCH(CH2CH3)2). Mono-
citrate of compound 5h: IR (KBr): 3414, 2966, 2936, 1720, 1655,
1597, 1483, 1406, 1271, 1123, 758, 598 cmꢃ1. MS (ESI positive) m/z:
[M þ H]þ 401.24. Anal. (C23H36N4O2$C6H8O7) C, H, N.
4.1.11. N,N-Dimethyl-1-{3-[4-(1-methylbutyl)piperazin-1-yl]
propanoyl}indoline-(2RS)-2-carboxamide (5i)
4.1.8. N,N-Dimethyl-1-{3-[4-(1-phenylethyl)piperazin-1-yl]
propanoyl}indoline-(2RS)-2-carboxamide (5f)
A solutionof1-(2-pentyl)piperazine(Chess, 31.3mg, 0.200 mmol),
A solution of 1-(1-phenylethyl)piperazine (Chess, 38.1 mg,
0.200 mmol), compound (rac)-3 (48.9 mg, 0.200 mmol), and dry
compound (rac)-3 (48.9 mg, 0.200 mmol), and dry Et3N (45 mL,
0.32 mmol) in anhydrous THF (3.0 mL) was stirred at 60 ꢀC under N2
for 48 h, then cooled to room temperature. The reaction solution was
poured into H2O, extracted with CH2Cl2 twice. The combined extracts
were dried over anhydrous MgSO4, and then, concentrated in vacuo.
PTLC purification (silica gel, CH2Cl2/MeOH/25% aqueous
ammonia ¼ 10:1:0.05) was performed to afford 62.0 mg of the title
product 5i in 77% yield as a white solid. 1H NMR (300 MHz, CDCl3)
Et3N (45 mL, 0.32 mmol) in anhydrous THF (3.0 mL) was stirred at
60 ꢀC under N2 for 36 h, cooled to room temperature, then
concentrated in vacuo. PTLC purification (silica gel, CH2Cl2/
MeOH ¼ 20/1/) was performed to afford 75.4 mg of the title product
5f in 87% yield. 1H NMR (300 MHz, CDCl3)
d 8.27 (0.4H, d,
J ¼ 7.68 Hz, Ar(indoline) H that is a peak for an amide-bond
conformer), 7.33e7.07 (7.6H, m, Ar H including Ar(indoline) H
that is a peak for an amide-bond conformer), 7.01e6.95 (1H, m,
Ar(indoline) H), 5.46e5.43 (0.6H, m, CHeCO that is a peak for an
amide-bond conformer), 5.24e5.19 (0.4H, m, CHeCO that is a peak
for an amide-bond conformer), 3.70e1.73 (21H, m, CHCH3, CH2, and
N(CH3)2), 1.37 (3H, d, 6.60 Hz, CHCH3). Monocitrate of compound
5f: MS (ESI positive) m/z: [M þ H]þ 435.26. IR (KBr): 3400, 2934,
1717, 1655, 1483, 1406, 1269, 1123, 760, 706, 600 cmꢃ1. Anal.
(C26H34N4O2$C6H8O7) C, H, N.
d
8.28 (0.5H, d, J ¼ 8.07 Hz, Ar H that is a peak for an amide-bond
conformer), 7.25e7.09 (2.5H, m, Ar H including a peak for an amide-
bond conformer), 6.98 (1H, ddd, J ¼ 7.53 Hz, J ¼ 7.14 Hz, J ¼ 1.08 Hz,
Ar H), 5.45 (0.5H, dd, J ¼ 10.8 Hz, J ¼ 3.51 Hz, CHeCO that is a peak for
an amide-bond conformer), 5.22 (0.5H, d, J ¼ 10.8 Hz, CHeCO that is
a peak for an amide-bond conformer), 3.71e0.84 (31H, m, NCH(CH3)
CH2, CH2, CH3, and N(CH3)2). Monocitrate of compound 5i: IR (KBr):
3437, 2961,1720,1655,1483,1271, 758, 600 cmꢃ1. MS (ESI positive) m/
z: [M þ H]þ 401.27. Anal. (C23H36N4O2$C6H8O7) C, H, N.
4.1.9. 1-[3-(4-Cyclohexylpiperazin-1-yl)propanoyl]-N,N-
dimethylindoline-(2RS)-2-carboxamide (5g)
4.1.12. 1-Acryloyl-N,N-dimethyl-2,3-dihydro-1H-indole-(2S)-2-
carboxamide [(S)-3]
A
solution of 1-cyclohexylpiperazine (Acros, 33.7 mg,
1-(tert-Butoxycarbonyl)indoline-(2S)-2-carboxylic acid, that was
prepared from indoline-(2S)-2-carboxylic acid (Tokyo Chemical
Industry) by tert-butoxycarbonyl (Boc) protection as the above
described manner, was amidated into tert-butyl (2S)-2-(dime-
thylcarbamoyl)indoline-1-carboxylate (S)-1 in the same manner for
compound (rac)-1 except the temperature for the amidation
reagents-adding condition was ꢃ20 ꢀC instead of 0 ꢀC at the corre-
sponding synthetic protocol for compound (rac)-1 (see Section
4.1.2.1.). Then compound (S)-1 was converted into N,N-dimethyl-2,3-
dihydro-1H-indole-(2S)-2-carboxamide (S)-2 in the same manner for
compound (rac)-2 (see Section 4.1.2.2.). To a stirred solution of
compound (S)-2 (11.07 g, 58.19 mmol) and dry Et3N (17.8 mL,
128 mmol) in dry CH2C12 (200 mL) was added acryloyl chloride
(4.98 mL, 61.3 mmol) at 0 ꢀC under N2. The reaction mixture was
stirred at 0 ꢀC under N2 for 2 h, then poured into ice-cooled saturated
aqueous NaHCO3. The resulting mixture was extracted with CH2C12,
and the combined extracts were dried over anhydrous Na2SO4,
filtered, and concentrated. The residue was purified by column
chromatography(silicagel, hexane/acetone¼ 2:1to1:1)togive8.00g
of title product (S)-3 in 56% yield as a white solid. This compound
showed a broadened spectrum in 1H NMR. MS (EI direct) m/z: Mþ 244
(see also Section 4.1.2.1. for the above procedure of (rac)-3).
0.200 mmol), compound (rac)-3 (48.9 mg, 0.200 mmol), and dry
Et3N (45 mL, 0.32 mmol) in anhydrous THF (3.0 mL) was stirred at
60 ꢀC under N2 for 48 h, cooled to room temperature, then
concentrated in vacuo. PTLC purification (silica gel, CH2Cl2/MeOH/
25% aqueous ammonia ¼ 10:1:0.5) was performed to afford 67.5 mg
of the title product 5g in 82% yield as a white solid. 1H NMR
(300 MHz, CDCl3)
d
8.24 (0.4H, d, J ¼ 8.04 Hz, Ar H that is a peak for
an amide-bond conformer), 7.22e7.04 (2.6H, m, Ar H including
a peak for an amide-bond conformer), 6.94 (1H, ddd, J ¼ 7.50 Hz,
J ¼ 7.14 Hz, J ¼ 1.26 Hz, Ar H), 5.43e5.39 (0.6H, m, CHeCO that is
a peak for an amide-bond conformer), 5.18e5.14 (0.4H, m, CHeCO
that is a peak for an amide-bond conformer), 3.68e0.99 (31H, m,
NCH(CH2)2, CH2, and N(CH3)2). Monocitrate of compound 5g: IR
(KBr): 3410, 2937, 2860,1719, 1649,1483, 1406, 1121, 1080, 943, 758,
598 cmꢃ1
(C24H36N4O2$C6H8O7) C, H, N.
.
MS (ESI positive) m/z: [M
þ
H]þ 413.28. Anal.
4.1.10. 1-{3-[4-(1-Ethylpropyl)piperazin-1-yl]propanoyl}-N,N-
dimethylindoline-(2RS)-2-carboxamide (5h)
A
solution of 1-(3-pentyl)piperazine (Chess, 31.3 mg,
0.200 mmol), compound (rac)-3 (48.9 mg, 0.200 mmol), and dry
Et3N (45 mL, 0.32 mmol) in anhydrous THF (3.0 mL) was stirred at
60 ꢀC under N2 for 48 h, then cooled to room temperature. The
reaction solution was poured into H2O, and the mixture was
extracted with CH2Cl2 twice. The combined extracts were dried
over anhydrous MgSO4, and then concentrated in vacuo. PTLC
4.1.13. N,N-Dimethyl-1-[3-(4-o-tolylpiperidin-1-yl)propanoyl]
indoline-(2S)-2-carboxamide (7a)
A solution of 4-o-tolylpiperidine hydrochloride (Arch, 76.2 mg,
0.360 mmol), compound (S)-3 (87.9 mg, 0.360 mmol), and dry Et3N
purification
(silica
gel,
CH2Cl2/MeOH/25%
aqueous
(131 m
L, 0.94 mmol) in anhydrous THF (4.4 mL) was stirred at 60 ꢀC
ammonia ¼ 10:1:0.05) was performed to afford 67.8 mg of the title
under N2 for 36 h, cooled to room temperature, then concentrated
in vacuo. PTLC purification (silica gel, CH2Cl2/MeOH ¼ 10:1) was
performed to afford 121.0 mg of the title product 7a in 80% yield as
a solid. 1H NMR data of this compound 7a was the same as that of
the corresponding (RS)-form 5a. Monocitrate of compound 7a: IR
data of this salt was the same as that of the corresponding
product 5h in 85% yield as a white solid. 1H NMR (300 MHz, CDCl3)
d
8.28 (0.4H, d, J ¼ 8.25 Hz, Ar H that is a peak for an amide-bond
conformer), 7.26e7.08 (2.6H, m, Ar H including a peak for an
amide-bond conformer), 6.98 (1H, ddd, J ¼ 7.35 Hz, J ¼ 7.32 Hz,
J ¼ 1.11 Hz, Ar H), 5.47e5.43 (0.6H, m, CHeCO that is a peak for an

S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
239
monocitrate of compound 5a. Anal. (C26H33N3O2$C6H8O7) C, H, N
(17.4 m
L, 0.125 mmol) in anhydrous THF (1.5 mL) was stirred at 60 ꢀC
(see also Section 4.1.3. for the above 1H NMR and IR data).
under N2 for 3 days, cooled to room temperature, then concentrated in
vacuo. The residue was diluted with CH2Cl2 (40 mL), washed with ice-
cooled saturated aqueous NaHCO3 (30 mL). The organic layer was
separated and the aqueous layer was extracted with CH2Cl2
(30 mL ꢂ 2). The organic layers were combined, dried over anhydrous
MgSO4, and concentrated in vacuo. PTLC purification was performed
twice (first, silica gel, CH2Cl2/MeOH ¼ 16:1; second, silica gel, CH2Cl2/
MeOH/25% aqueous ammonia ¼ 10:1:0.05) to afford 9.4 mg of the title
4.1.14. N,N-Dimethyl-1-[3-(4-phenylpiperidin-1-yl)propanoyl]
indoline-(2S)-2-carboxamide (7b)
A
solution of 1-phenylpiperidine hydrochloride (71.2 mg,
0.360 mmol), compound (S)-3 (87.9 mg, 0.360 mmol), and dry Et3N
(131
L, 0.936 mmol) in anhydrous THF (4.4 mL) was stirred at 60 ꢀC
m
under N2 for 36 h, then concentrated in vacuo. PTLC purification
(silica gel, CH2Cl2/MeOH ¼ 10:1) was performed to afford 110.2 mg
of the title product 7b in 75% yield as a white solid. 1H NMR
product 7e in 28% yield. 1H NMR (300 MHz, CDCl3)
d 8.31 (0.4H, d,
J¼ 8.61 Hz, Ar(indoline) H that is a peak for an amide-bond conformer),
7.30e6.98 (6.6H, m, Ar H including Ar(indoline) H that is a peak for an
amide-bond conformer), 5.50e5.42 (0.6H, m, CHeCO that is a peak for
an amide-bond conformer), 5.30e5.21 (0.4H, m, CHeCO that is a peak
for an amide-bond conformer), 3.75e2.09 (23H, m, AreCH, CH2,
N(CH3)2, and AreCH3), 1.69e1.60 (4H, m, CH2). MS (ESI positive) m/z:
[M þ H]þ 434.29 for salt-free compound 7e. Compound 7e was con-
verted into the corresponding monocitrate as usual, and the salt was
used for pharmacological and pharmacokinetic evaluations without
elementary analysis because of the requisite amount for the analysis;
the purity of the salt-free compound 7e was assured by the above 1H
NMR analysis that had no significant impurity signal except small
amounts of solvent signal.
(300 MHz, CDCl3) d 8.31e8.29 (0.4H, m, Ar(indoline) H that is a peak
for an amide-bond conformer), 7.33e7.09 (7.6H, m, Ar H including
Ar(indoline) H that is a peak for an amide-bond conformer),
7.02e6.97 (1H, m, Ar(indoline) H), 5.49e5.44 (0.6H, m, CHeCO that
is a peak for an amide-bond conformer), 5.26e5.21 (0.4H, m,
CHeCO that is a peak for an amide-bond conformer), 3.73e2.75
(13H, m, AreCH, CH2, and N(CH3)2), 2.58e2.46 (2H, m, CH2),
2.25e2.10 (2H, m, CH2), 1.90e1.71 (4H, m, CH2). Monocitrate of
compound 7b: IR (KBr): 3422, 2932, 1732, 1653, 1485, 1406, 1271,
1217, 1123, 758, 702, 598 cmꢃ1. Anal. (C25H31N3O2$C6H8O7) C, H, N.
4.1.15. 1-{3-[4-(4-Fluorophenyl)piperidin-1-yl]propanoyl}-N,N-
dimethylindoline-(2S)-2-carboxamide (7c)
A
solution of 4-(4-fluorophenyl)piperidine hydrochloride
4.1.18. N,N-Dimethyl-1-[3-(8-phenyl-3-azabicyclo[3.2.1]oct-3-yl)
(41.4 mg, 0.192 mmol), compound (S)-3 (46.4 mg, 0.190 mmol), and
propanoyl]indoline-(2S)-2-carboxamide (7f)
dry Et3N (69
m
L, 0.50 mmol) in anhydrous THF (2.3 mL) was stirred
A
solution of 8-phenyl-3-azabicyclo[3.2.1]octane [36.6 mg,
at 60 ꢀC under N2 for 36 h, cooled to room temperature, then
concentrated in vacuo. PTLC purification (silica gel, CH2Cl2/
MeOH ¼ 10:1) was performed to afford 58.9 mg of the title product
0.195 mmol; 1H NMR (270 MHz, CDCl3)
d
7.38e7.15 (5H, m), 3.12e3.07
(3H, m), 2.60e2.55 (2H, m), 2.44 (2H, dd, J ¼ 12.4 Hz, J ¼ 3.13 Hz),
2.02e1.80 (4H, m), 1.64 (1H, br s)], compound (S)-3 (47.6 mg,
7c in 73% yield as a solid. 1H NMR (300 MHz, CDCl3)
d
8.30 (0.4H, d,
0.195 mmol), and dry Et3N (43 mL, 0.31 mmol) in anhydrous THF
J ¼ 8.07 Hz, Ar(indoline) H that is a peak for an amide-bond
conformer), 7.29e6.94 (7.6H, m, Ar H including Ar(indoline) H
that is a peak for an amide-bond conformer), 5.49e5.44 (0.6H, m,
CHeCO that is a peak for an amide-bond conformer), 5.25e5.20
(0.4H, m, CHeCO that is a peak for an amide-bond conformer),
3.75e2.75 (13H, m, AreCH, CH2, N(CH3)2), 2.55e2.46 (2H, m, CH2),
2.23e2.11 (2H, m, CH2), 1.88e1.69 (4H, m, CH2). Monocitrate of
compound 7c: IR (KBr): 3400, 2932, 1719, 1655, 1485, 1406, 1221,
1123, 835, 758, 592, 540 cmꢃ1. Anal. (C25H30N3O2F$C6H8O7) C, H, N.
(2.4 mL) was stirred at 60 ꢀC under N2 for 36 h, cooled to room
temperature, then concentrated in vacuo. PTLC purification (silica gel,
CH2Cl2/MeOH ¼ 10:1) was performed to afford 60.2 mg of the title
product 7g in 72% yield as a solid. 1H NMR (300 MHz, CDCl3)
d 8.25
(0.4H, d, J ¼ 7.32 Hz, Ar(indoline) H that is a peak for an amide-bond
conformer), 7.34e7.08 (7.6H, m, Ar H including Ar(indoline)H that is
a peak for an amide-bond conformer), 7.00e6.95 (1H, m, Ar H),
5.43e5.38 (0.6H, m, CHeCO that is a peak for an amide-bond
conformer), 5.10e5.04 (0.4H, m, CHeCO that is a peak for an amide-
bond conformer), 3.65e1.68 (23H, m, CH, CH2, and N(CH3)2). Mono-
citrate of compound 7f: IR (KBr): 2955, 1719, 1655, 1485, 1406, 1123,
993, 754 cmꢃ1. Anal. (C27H33N3O2$C6H8O7) C, H, N.
4.1.16. 1-{3-[4-(4-Fluoro-2-methylphenyl)piperidin-1-yl]
propanoyl}-N,N-dimethylindoline-(2S)-2-carboxamide (7d)
A solution of 4-(4-fluoro-2-methylphenyl)piperidine (compound
6d, see Section 4.1.19; 42.5 mg, 0.220 mmol), compound (S)-3
4.1.19. Preparation of 4-(4-fluoro-2-methylphenyl)piperidine
4.1.19.1. 1-Benzyl-4-(4-fluoro-2-methylphenyl)-1,2,3,6-
tetrahydropyridine (10). A mixture of Mg (turnings, 48.6 mg,
2.00 mmol), 1-bromo-4-fluoro-2-methylbenzene (Tokyo Chemical
(53.7 mg, 0.220 mmol), and dry Et3N (56 mL, 0.40 mmol) in anhydrous
THF (4.0 mL) was stirred at 60 ꢀC under N2 for 2 days, then cooled to
room temperature, then concentrated in vacuo. PTLC purification
(silica gel, CH2Cl2/MeOH ¼ 10:1) was performed to afford 58.6 mg of
Industry, 253
mL, 2.00 mmol), iodine (25.4 mg, 0.100 mmol), and
the title product 7d in 61% yield. 1H NMR (300 MHz, CDCl3)
d
8.30
MgBr2 (7.0 L, 0.10 mmol) in anhydrous THF (5.0 mL) was stirred
m
(0.4H, d, J ¼ 8.04 Hz, Ar(indoline) H that is a peak for an amide-bond
conformer), 7.29e7.10 (3.6H, m, Ar H including Ar(indoline) H that is
a peak for an amide-bond conformer), 7.00 (1H, dd, J ¼ 7.32 Hz,
J¼ 7.32Hz, ArH),6.88e6.82(2H, m, ArH),5.49e5.44(0.6H, m, CHeCO
that is a peak for an amide-bond conformer), 5.26e5.21 (0.4H, m,
CHeCO that is a peak for an amide-bond conformer), 3.75e2.45 (15H,
m, AreCH, CH2, and N(CH3)2), 2.32 (3H, s, AreCH3), 2.27e2.11 (2H, m,
CH2), 1.83e1.73 (4H, m, CH2). Monocitrate of compound 7d: IR (KBr):
3414, 2930,1728,1655,1485,1410,1246,1126, 955, 758, 594 cmꢃ1. MS
(ESI positive) m/z: [M þ H]þ 438. Anal. (C26H32N3O2F$C6H8O7) C, H, N.
under reflux conditions under N2 for 1 h, and cooled to room
temperature. To the resulting solution of Grignard reagent (4-
fluoro-2-methylphenylmagnesium bromide) was added a solution
of 1-benzyl-4-piperidone (Tokyo Chemical Industry, 371
mL,
2.00 mmol) in anhydrous THF (7.0 mL) at room temperature under
N2 within 5 min. The mixture was stirred at room temperature
under N2 for 10 min, then stirred under reflux conditions for 6 h.
The reaction solution was cooled to 0 ꢀC then poured into ice-
cooled aqueous NH4Cl (40 mL). The mixture was extracted with
CH2Cl2 (40 ꢂ 4), then the combined extracts were dried over
anhydrous MgSO4 and concentrated in vacuo to afford 658.5 mg of
4.1.17. 1-{3-[4-(2,6-Dimethylphenyl)piperidin-1-yl]propanoyl}-
N,N-dimethylindoline-(2S)-2-carboxamide (7e)
1-benzyl-4-(4-fluoro-2-methylphenyl)piperidin-4-ol
TLC: Rf ¼ 0.4, AcOEt/hexane ¼ 1:8.
9
(crude).
A
solution of 4-(2,6-dimethylphenyl)piperidine (16.6 mg,
To a stirred solution of 1-benzyl-4-(4-fluoro-2-methylphenyl)
piperidin-4-ol 9 (658.5 mg, crude) in toluene (50.0 mL) was added
0.0880 mmol), compound (S)-3 (19.1 mg, 0.0782 mmol), and dry Et3N

240
S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
p-toluenesulfonic acid monohydrate (760.1 mg, 4.00 mmol) under
N2 at room temperature. The resulting solution was heated to reflux
and stirred under N2 for 10 h. The solution was cooled to 0 ꢀC,
basified by adding 2 N NaOH (30 mL), then the mixture was
extracted with chloroform (50 mL ꢂ 2). The combined extracts was
dried over anhydrous MgSO4, and concentrated in vacuo. The
residue was purified by flash column chromatography (silica gel,
CH2Cl2/MeOH ¼ 36:1) to afford 343.3 mg of the title product 10 in
61% yield (2 steps) as a yellow oil. 1H NMR (270 MHz, CDCl3)
N,N-Dimethyl-1-[3-(4-pyridin-2-ylpiperidin-1-yl)propanoyl]
indoline-(2RS)-2-carboxamide (5m) (salt free) from 2-(piperidin-
4-yl)pyridine (4m);
1-[3-(4-Cyclohexylpiperidin-1-yl)propanoyl]-N,N-dimethylindo-
line-(2RS)-2-carboxamide (5n) (salt free) from 4-cyclohexylpiperidine
(4n);
N,N-Dimethyl-1-[3-(4-phenylazepan-1-yl)propanoyl]indoline-
(2RS)-2-carboxamide (5o) (salt free) from 4-phenylazepane (4o);
N,N-Dimethyl-1-[3-(4-o-tolylpiperazin-1-yl)propanoyl]indo-
line-(2RS)-2-carboxamide (5p) (salt free) from 1-o-tolylpiperazine
(4p);
N,N-Dimethyl-1-[3-(4-m-tolylpiperazin-1-yl)propanoyl]indo-
line-(2RS)-2-carboxamide (5q) (salt free) from 1-m-tolylpiperazine
(4q);
d
7.40e7.24 (5H, m, Ar H), 7.05 (1H, dd, J ¼ 8.24 Hz, J ¼ 6.10 Hz, Ar
H), 6.88e6.77 (2H, m, Ar H) 5.53e5.49 (1H, m, CH), 3.65 (2H, s,
PheCH2eN), 3.13 (2H, dd, 5.91 Hz, 2.81 Hz, NeCH2), 2.68 (2H, t,
5.62 Hz, NeCH2), 2.36e2.29 (2H, m, CH2), 2.28 (3H, s, AreCH3).
1-{3-[4-(2,4-Dimethylphenyl)piperazin-1-yl]propanoyl}-N,N-
dimethylindoline-(2RS)-2-carboxamide (5r) (salt free) from 1-(2,4-
dimethylphenyl)piperazine (4r);
1-[3-(4-Benzylpiperazin-1-yl)propanoyl]-N,N-dimethylindo-
line-(2RS)-2-carboxamide (5s) (salt free) from 1-benzylpiperazine
(4s);
1-[3-(4-Cyclopentylpiperazin-1-yl)propanoyl]-N,N-dimethylindo-
line-(2RS)-2-carboxamide (5t) (salt free) from 1-cyclopentylpiperazine
(4t);
4.1.19.2. 4-(4-Fluoro-2-methylphenyl)piperidine (6d). A mixture of
1-benzyl-4-(4-fluoro-2-methylphenyl)-1,2,3,6-tetrahydropyridine
10 (343.3 mg, 1.220 mmol) and Pd(OH)2 (177.0 mg) in MeOH
(13.0 mL) was stirred at room temperature under H2 at 1 atm for 2
days. The catalyst was filtered through a Celite pad with MeOH. The
filtrate was concentrated in vacuo to afford 167.5 mg of the title
product 6d in 71% yield as a white solid. 1H NMR (300 MHz, CDCl3)
d
7.21e7.16 (1H, m, Ar H), 6.90e6.84 (2H, m, Ar H), 3.59 (1H, br s,
NH), 3.33 (2H, d, J ¼ 12.3 Hz, CH2), 2.88e2.77 (3H, m, CH, CH2), 2.33
1-[3-(4-Cycloheptylpiperazin-1-yl)propanoyl]-N,N-dimethy-
(3H, s, Ar-CH3), 1.82e1.69 (4H, m, CH2).
lindoline-(2RS)-2-carboxamide
1-cycloheptylpiperazine (4u);
(5u)
(salt
free)
from
4.1.20. High-speed parallel synthesis using Michael addition
reaction
(1) Reaction. To a half-dram vial, in which reagent amine or its
hydrochloride (0.050 mmol) was pre-weighed, were added 0.65 mL
1-[3-(4-Cyclooctylpiperazin-1-yl)propanoyl]-N,N-dimethy-
lindoline-(2RS)-2-carboxamide (5v) (salt free) from 1-
cyclooctylpiperazine (4v);
1-[3-(Isoindolin-2-yl)propanoyl]-N,N-dimethylindoline-(2RS)-
2-carboxamide (5w) (salt free) from isoindoline (4w);
1-{3-[3,4-Dihydroisoquinolin-2(1H)-yl]propanoyl}-N,N-dime-
thylindoline-(2RS)-2-carboxamide (5x) (salt free) from 1,2,3,4-
tetrahydroisoquinoline (4x);
1-(3-{1,2,4,4a,5,6-cis-Hexahydrobenzo[f]isoquinolin-3(10bH)-yl}
propanoyl)-N,N-dimethylindoline-(2RS)-2-carboxamide (5y) (salt
free) from 1,2,3,4,4a,5,6,10b-cis-octahydrobenzo[f]isoquinoline (4y);
1-(3-{1H-Benzo[de]isoquinolin-2(3H)-yl}propanoyl)-N,N-dime-
of
a 0.1 M solution of 1-acryloyl-N,N-dimethylindoline-2-
carboxamide (rac)-3 in 3.75% dry Et3Neanhydrous THF and
0.25 mL of 3.75% dry Et3N/dry dimethylacetamide and the resulting
mixture was shaken at 60 ꢀC for 16 h. After cooling to room
temperature, the reaction mixture was loaded onto an Oasis MCX
cartridge (1 g/6 mL) preconditioned 8 mL of MeOH. The solid-phase
matrix was washed with 10 mL of MeOH and then eluted with 5 mL
of 1 M ammonia/MeOH. The elute was concentrated to dryness by
vacuum centrifuge, providing crude product. In case it is needed,
the crude material was purified with preparative LS/MS to give the
desired product as salt-free form or formic acid salt dependent on
the purification condition. (2) HPLC/LCeMS method. (i) Analytical
condition: equipment, Waters 2795; column: Waters XTerra C18,
thylindoline-(2RS)-2-carboxamide
(5z)
(formate)
from
2,3-dihydro-1H-benzo[de]isoquinoline (4z).
4.2. Biology
5
m
m, 4.6 ꢂ 50 mm; column temperature, 40 ꢀC; detector, photo-
4.2.1. In vitro characterization of NOP receptor antagonists
diodearray (210e400 nm); flow, 1.0 mL/min; solvent (a) as acidic
condition, A: 0.1% HCO2H; B: MeOH, gradient, solvent (b) as basic
condition, A: 0.1% aqueous ammonia; B: MeOH, gradient; MS
condition (ionization method), ESI (positive). (ii) Preparative
conditions: equipment, Waters prep LC/MS system; column,
In vitro studies of synthetic compounds for hNOP receptor
binding affinities and hMOP receptors binding affinities, and for
antagonist activities against N/OFQ stimulated [35S]GTP
were conducted.
gS binding
Waters XTerra C18, 5
m
m, 20 ꢂ 50 mm; column temperature,
4.2.1.1. Materials. The hNOP receptor transfected human embry-
onic kidney (HEK)-293 cell membranes and the hMOP receptor
transfected Chinese hamster ovary (CHO)-K1 cell membranes were
purchased from Receptor Biology Inc., respectively. [3H]N/OFQ
ambient temperature; flow, 20.0 mL/min; solvent (a) as acidic
condition, A: 0.1% HCO2H; B: MeOH, gradient, solvent (b) as basic
condition, A: 0.1% aqueous ammonia; B: MeOH, gradient.
Utilizing the above protocol (1) and (2), parallel synthesis was
performedfortheanaloguesincluding5jez(assalt-freeformsof5jey
oraformatesaltof5z)thatwerepreparedfromcompound(rac)-3and
the corresponding amines 4jez, respectively, as listed below:
N,N-Dimethyl-1-[3-(4-phenylpiperidin-1-yl)propanoyl]indoline-
(2RS)-2-carboxamide (5j) (salt free) from 4-phenylpiperidine (4j);
1-{3-[4-(2-Fluorophenyl)piperidin-1-yl]propanoyl}-N,N-dime-
thylindoline-(2RS)-2-carboxamide (5k) (salt free) from 4-(2-
fluorophenyl)piperidine (4k);
(150 Ci/mmol), [35S]GTP
gS (1060e1150 Ci/mmol) and wheatgerm
agglutinin (WGA)-scintillation proximity assay (SPA) beads were
obtained from Amersham Pharmacia Biotech K.K., and [3H]DAMGO
(54.0 Ci/mmol) was provided from NENÔ Life Science Products Inc.,
respectively. N/OFQ was from Peptide Institute Inc. DAMGO was
from Sigma Chemical, respectively.
4.2.1.2. Evaluation of receptor binding affinities to hNOP receptor and
hMOP receptor. All competitive displacement analyses (IC50 and Ki)
for the hNOP receptor and hMOP receptor were performed in
duplicate in a 96-well plate using a scintillation proximity assay
(SPA), respectively. After the reaction, the assay plate was
N,N-Dimethyl-1-(3-{4-[2-(trifluoromethyl)phenyl]piperidin-1-yl}
propanoyl)indoline-(2RS)-2-carboxamide (5l) (salt free) from
4-[2-(trifluoromethyl)phenyl]piperidine (4l);

S. Hayashi et al. / European Journal of Medicinal Chemistry 55 (2012) 228e242
241
centrifuged at 1000 rpm for 1 min, and then the radioactivity was
measured by a 1450 MicroBetaÔ (Wallac) liquid scintillation
counter. IC50 values were calculated by nonlinear regression with
the software GraphPad Prism version 4.0 (GraphPad Software, Inc.,
San Diego, USA), respectively. Ki values were calculated by the
following equation, Ki ¼ IC50/(1 þ [L]/KD), where [L] is the radio-
labelled ligand concentration and KD is the dissociation constant
[7,8a,16].
with CH3CN. The extracted samples were measured by HPLC/MS/
MS system [7,8b].
4.4. Physicochemistry
The lipophilicity values of NOP receptor antagonists were esti-
mated as values of ACD log D7.4, the octanolewater distribution
coefficient for ionizable compounds at pH 7.4, calculated by ACD
software, ACD/Laboratories 9.0 (Advanced Chemistry Development,
Inc., Ontario, Canada).
4.2.1.2.1. hNOP receptor binding assay. The hNOP receptor
membranes (8.3 m
g) were incubated at 25 ꢀC for 45 min with 0.4 nM
[3H]N/OFQ, 1.0 mg of WGA-SPA beads, and six different concen-
trations of compounds (10ꢃ11e10ꢃ5 M, 10-fold) in a final volume of
0.2 mL of 50 mM HEPES buffer, pH 7.4, containing 10 mM MgCl2 and
1 mM EDTA. Non-specific binding was determined by the addition
Acknowledgement
We thank chemical technology group in PGRD for technical
support to our HSA study.
of 1 mM unlabelled N/OFQ. Approximately 900 cpm of total binding
were obtained, of which 3.3% was the non-specific binding.
4.2.1.2.2. hMOP receptor binding assay. The hMOP receptor
Appendix A. Supplementary data
membranes (18 m
g) were incubated at 25 ꢀC for 45 min with 1.0 nM
[3H]DAMGO, 1.0 mg of WGA-SPA beads, and six different concen-
trations of compounds (10-fold) in a final volume of 0.2 mL of
50 mM TriseHCl buffer, pH 7.4, containing 5 mM MgCl2. Non-
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.07.
021. These data include MOL files and InChiKeys of the most
important compounds described in this article.
specific binding was determined by the addition of 1 mM of unla-
belled DAMGO. Approximately 240 cpm of total binding were ob-
tained, of which 9.6% was the non-specific binding.
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