Structure-activity studies on the nociceptin/orphanin FQ receptor antagonist 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide

Article abstract of DOI:10.1016/j.bmc.2009.05.068

Twelve derivatives of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) antagonist 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide (Comp 24) were synthesized and tested in binding experiments performed on CHO

Full text of DOI:10.1016/j.bmc.2009.05.068

Bioorganic & Medicinal Chemistry 17 (2009) 5080–5095
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity studies on the nociceptin/orphanin FQ receptor
antagonist 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),40-piperidin-1-yl]propyl}
pyrrolidine-2-carboxamide
b
Claudio Trapella a, , Carmela Fischetti b,c, , Michela Pela’ a, Ilaria Lazzari a, Remo Guerrini a, , Girolamo Calo’ ,
*
Anna Rizzi b, Valeria Camarda b, David G. Lambert c, John McDonald c, Domenico Regoli b, Severo Salvadori a
a Department of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, via Fossato di Mortara 19, 44100 Ferrara, Italy
b Department of Experimental and Clinical Medicine, Section of Pharmacology and National Institute of Neuroscience,
University of Ferrara, via Fossato di Mortara 19, 44100 Ferrara, Italy
c Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management,
University of Leicester, Leicester Royal Infirmary, Leicester, LE1 5WW, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Twelve derivatives of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) antagonist 1-benzyl-N-{3-[spi-
roisobenzofuran-1(3H),40-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide (Comp 24) were synthesised
and tested in binding experiments performed on CHOhNOP cell membranes. Among them, a novel inter-
esting NOP receptor antagonist (compound 35) was identified by blending chemical moieties taken from
different NOP receptor ligands. In vitro in various assays, Compound 35 consistently behaved as a pure,
highly potent (pA2 in the range 8.0–9.9), competitive and NOP selective antagonist. However compound
35 was found inactive when challenged against N/OFQ in vivo in the mouse tail withdrawal assay. Thus
the usefulness of the novel NOP ligand compound 35 is limited to in vitro investigations.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 27 March 2009
Revised 20 May 2009
Accepted 23 May 2009
Available online 2 June 2009
Keywords:
SAR studies
NOP receptor antagonists
Receptor and [35S]GTP
Mouse vas deferens
Calcium mobilization
Tail withdrawal assay
cS binding
1. Introduction
Recently, a novel non-peptide NOP antagonist, 1-benzyl-N-{3-
[spiroisobenzofuran-1(3H),40-piperidin-1-yl]propyl} pyrrolidine-
Nociceptin/orphanin FQ (N/OFQ)1,2 modulates different biologi-
cal functions via activation of the N/OFQ peptide receptor (NOP).3
Few non-peptide molecules have been reported to selectively
interact with the NOP receptor: these include the NOP agonist Ro
64-6198,4 and the antagonists J-1133975 and SB-6121116 (see
Chart 1). However, the chemical synthesis of such molecules,
which are characterized by two chiral centres, is extremely com-
plex, time consuming and of low yield, making the availability of
these tools to the scientific community very limited.
2-carboxamide, has been reported by Banyu researchers (as Comp
24).7 Comp 24 binds with high affinity (pIC50 9.57) to the human
recombinant NOP receptor showing an impressive selectivity
(>3000-fold) over classical opioid receptors. Comp 24 behaves as
a pure NOP antagonist in the [35S]GTP
cS assay with very high po-
tency (pIC50 9.82). Moreover, in vivo in mice Comp 24 at 10 mg/kg
sc completely reverses the locomotor inhibitory effect elicited by a
NOP receptor agonist.7 Recently this molecule was synthesized and
characterized in our laboratories confirming its excellent in vitro
pharmacological profile in terms of high antagonist potency and
selectivity of action over classical opioid receptors.8 Moreover, in
the mouse tail withdrawal assay, Comp 24 at 10 mg/kg ip did not
modify per se tail withdrawal latencies but prevented the pronoci-
ceptive and antinociceptive effects of 1 nmol N/OFQ given suprasp-
inally and spinally, respectively.8 Collectively, these studies
demonstrate that Comp 24 behaves as a pure, highly potent, selec-
tive and competitive NOP antagonist.
Abbreviations: EtOAc, ethyl acetate; Bn-Cl, benzyl chloride; Boc2O, di-tert-butyl-
dicarbonate; BSA, bovine serum albumin; n-BuLi, normal butyllithyum; DCC, N,
N0-dicicloexyl-carbodiimide; DCM, methylen chloride; DMF, N,N0-dimetilformam-
mide; DPN, diprenorphine; Et3N, triethylamine; Et2O, diethylether; EtPt, light
petroleum boiling fraction 40–60°; HOBt, 1-hydroxy-benzotriazole; i-PrOH,
isopropanol; MeOH, methanol; rt, room temperature; NSB, non-specific binding;
TBAF, tetrabutylamonium fluoride; TBDMS-Cl, tert-butyldimethylsilylchloride; TFA,
trifluoro acetic acid; THF, tetrahydrofurane; TLC, thin layer chromatography; WSC,
1-ethyl-(3-dimethyl-amino-propyl)-carbodiimmide hydrochloride.
Interestingly, Comp 24 displays some chemical characteristics
typical of N/OFQ related peptides (see Chart 1), these include: (i)
a spacer of 12 atoms between the two phenyl rings which matches
* Corresponding author. Tel.: +39 0532 455 988; fax: +39 0532 455953.
E-mail address: r.guerrini@unife.it (R. Guerrini).
Both authors equally contributed to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.068

C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
5081
O
O
O
H
N
H
N
O
N
H2N
N
H
N
H
N
O
O
N/OFQ message domain
Comp 24
Cl
O
O
NH
N
N
N
N
N
N
Cl
HO
J-113397
OH
SB-612111
Ro 64-6198
Chart 1. Chemical formulae of nociceptin/orphanin FQ receptor ligands.
the Phe-Gly-Gly-Phe sequence of the N/OFQ message domain; (ii)
an amide bond which is quite uncommon in non-peptide NOP li-
gands; (iii) a N-benzyl amino acid, a chemical moiety also present
in the N-terminal of the NOP receptor peptide antagonists
cule with the highest affinity, that is, compound 35 has been fur-
ther characterized in vitro at the recombinant human NOP in
[
35S]GTP
cS binding and calcium mobilization assays and at native
NOP receptors expressed in isolated animal (mouse, rat, guinea-
pig) tissues. Finally compound 35 was assayed in vivo against the
effects elicited by N/OFQ in the mouse tail withdrawal assay.
9
[Nphe1]N/OFQ(1-13)-NH2 and UFP-101.10 Based on these consid-
erations, in the present study, the importance of the N-benzyl
D-
Pro of Comp 24 was assessed by replacement with - or -Phe,
L
D
and Nphe. In addition, the amide bond of Comp 24 was substituted
with other amide bond isosters. Moreover, the spiroisobenzofurane
nucleus of Comp 24 was replaced with chemical moieties derived
from other non-peptide NOP ligands that is, Ro 64-6198,4
SB-6121116 and J-1133975 (Chart 1). The novel molecules were
evaluated for their ability to bind the human recombinant NOP
receptor expressed in CHO cell (CHOhNOP) membranes. The mole-
2. Chemistry
Compound 24 was synthesized following procedures reported
by Goto et al.7 The key intermediate 5 (Scheme 1) was obtained
starting from commercially available benzanilide 1 that reacts with
n-butyllithium at ꢀ78 °C and subsequentially with the N-benzyl-
O
O
O
O
O
b
a
c
d
N
H
H2N
N
N
N
N
H
O
2
3
1
5
4
O
O
f
BocHN
H2N
H2N
N
H
N
N
H
N
O
O
9
6
O
O
e
f
BocHN
N
N
N
N
H
O
H
O
e
10
7
e
H2N
N
O
O
O
f
H
N
N
Boc
N
N
N
H
N
e
H
5
O
O
11
12
8
O
O
g
N
N
N
N
H
N
N
N
N
O
O
Comp 24
h
N
H
O
13
*
Scheme 1. Reagents and conditions: (a) n-BuLi 2.5 M, ꢀ78 °C, N-benzyl-piperidine-4-one, 0 °C, THF; (b) (CH3)2S BH3, THF, 0 °C to reflux; (c) H2, C/Pd 10%, EtOH, overnight;
(d) N-Boc-3-bromo-propyl amine DMF, 60 °C, 3 h, then DCM TFA 1 h; (e)
(CH3)2S BH3, THF, 0 °C to reflux O.N.; (h) CH3I, NaH, DMF, 0 °C to rt, O.N.
L-Phe,
D-Phe, Nphe or N-Bn-D-Pro, DMF, HOBt. WSC, 0 °C to rt, 24 h; (f) TFA, 0 °C, 1 h; (g)
*

5082
C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
piperidone to give the spiroisobenzofuran-2-one 2. Compound 2
was reduced with borane dimethyl sulfide complex in THF to give
the corresponding isobenzofurane 3. The deprotection of the piper-
idine nitrogen using H2 in presence of palladium on charcoal 10%
afforded the desired spiroisobenzofurane piperidine 4 in good
yield.
All the attempt to prepare the tosylate of 16 using tosyl chloride
and Et3N, generated the corresponding chloride 18, which is em-
ployed in the esterification reaction12 with Bn-
D-prolinol 19 using
tetrabutyl ammonium iodide as an exchanging agent to obtain the
ether 20.
The replacement of the amide bond present in Comp 24 with a
olefin moiety was the most difficult procedure, in particular, all
the attempts to prepare the desired olefin via Wittig13 or Horner–
Wadsworth–Emmons14 reaction on the N-Bn-Prolinale15 using the
(3-carboxy-propyl)-triphenylphosphonium bromide 21, failed. In
contrast, using the dimethylsulfynil anion16,17 to generate the corre-
sponding ylide allowed us to obtain the corresponding olefin 22 in
moderate yield.
Compound 22 was reacted with 4 in the presence of WSC/HOBt
and the amide bond of 23 reduced using borane dimethylsulfide
complex to obtain the desired olefin derivative 25 as a inseparable
mixture of E/Z isomers (Scheme 3).
The synthesis of compound 28 (Scheme 4) started from the
commercially available 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-
one 26 that was alkylated using the conditions previously reported
for 5 with N-Boc-3-bromo-propyl amine to obtain in a good yield
the derivative 27. Deprotection of tert-butoxycarbonil moiety and
Alkylation with N-Boc-3-bromo-propyl amine followed by
TFA treatment gave the free amine 5. The substitution in com-
pound 24 of the
D-Pro amino acid with the D-Phe, L-Phe and
Nphe was achieved using compound 5 and the corresponding
protected aminoacid for the coupling reaction, removing the
Boc protected group from the amino function with TFA at
0 °C. This allowed us to obtain compounds 9, 10 and 11
(Scheme 1).
The N-methyl alkylation or the reduction of the amide bond of
compound 24 produced the final compounds 12 and 13.
The substitution of the amide bond in Comp 24 with ester or
ether bond was achieved following Scheme 2. 3-Bromo-1-propa-
nol, was protected as TBDMS derivative (14) and reacted with 4
in DMF at 60 °C in the presence of potassium carbonate to obtain
compound 15. Treatment of 15 with TBAF in THF allows us to ob-
tain the corresponding alcohol 16 which was condensed with the
N-Bn-
D
-Pro using the Steglich esterification11 to obtain the desired
the coupling with N-Bn-D-Pro allowed us to obtain the chimeric
ester 17
compound 28 as depicted in Scheme 4.
a
b
Si
Br
OH
TBDMSO
N
N
Br
O
O
O
14
15
c
d
O
N
HO
O
N
O
16
17
e
Bn
N
f
OH
N
Cl
N
O
N
O
O
19
20
18
Scheme 2. Reagents and conditions: TNDMS-Cl, imidazole, THF, rt, 24 h; (b) (4), K2CO3, DMF, 60 °C, O.N.; (c) TBAF, THF, 24 h; (d) N-Bn-
tosyl chloride, Et3N, CH2Cl2, 0 °C to rt, O.N.; (f) (15) TBAI, NaH, DMF, 0 °C to rt, O.N.
D-Pro, HOBt, WSC, DMF, rt, O.N.; (e)
Br-
O
a
O
Ph
Ph
N
b
P+
Ph
COOH
N
+
COOH
H
N
N
O
21
22
23
O
c
N
N
N
N
O
O
23
25
*
Scheme 3. Reagents and conditions: (a) NaH, DMSO, 77 °C 40 min, than N-Bn-
D
-Pro at 0 °C to rt, O.N.; (b) (4) DMF, HOBt, WSC, rt, O.N.; (c) THF, (CH3)2S BH3, 0 °C to reflux
O.N.

C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
5083
O
N
O
N
O
H
N
NH
NH
a
b
N
HN
NH
BocHN
N
N
N
O
26
28
-Pro, DMF, HOBt, WSC, 0 °C to rt, O.N.
27
Scheme 4. Reagents and conditions: (a) DMF, BocNH-(CH2)3-Br, K2CO3, 60 °C, 1 h; (b) (i) TFA, 0 °C, 1 h; (ii) N-Bn-
D
O
H
Cl
EtOOC
Cl
COOEt
O
Cl
Cl
Cl
Cl
c
Cl
Cl
O
d
b
a
Cl
Cl
+
COOEt
HOOC
COOH
O
N
H
O
N
H
OH
29
31
30
32
33
Cl
Cl
H
N
Cl
Cl
N
e
N
f
HN
H2N
N
O
Cl
33
Cl
34
g
35
Cl
H
N
N
N
O
Cl
36
*
Scheme 5. Reagents and conditions: (a) EtOH, Pip, rt, O.N.; (b) NaOH 35%, EtOH, reflux, O.N.; (c) NH4OH, 200 °C, 6 h; (d) (CH3)2S BH3, THF, 0 °C to reflux O.N.; (e) DMF K2CO3,
BocNH-(CH2)2-Br, 60 °C, O.N., then TFA; (f) DMF, HOBt, WSC, N-Bn- -Pro, 0 °C to rt, O.N.; (g) DMF, HOBt, WSC, N-Bn- -Pro, 0 °C to rt O.N.
D
L
The synthesis of the 4-(2,6-dichlorophenyl)-piperidine 33 starts
from the reaction of 2,6-dichloro-benzaldehyde 29 with ethyl ace-
toacetate to give the diester 30. Hydrolysis under basic conditions
of 30 allowed us to obtain the corresponding diacid 31 that was
treated with ammonia at 200 °C for 6 h to gave the immide 32.
Reduction using borane dimethyl sulfide complex of 32 gave the
desired 4-(2,6-dichloro-phenyl)-piperidine 33 in moderate yield.
The final compounds 35 and 36 were prepared using 33 following
procedures reported in Scheme 2d (Scheme 5).
The compound 41 was prepared starting from the tert-butyloxy-
carbonyl protection of the commercially available 1-piperidin-4-yl-
1,3-dihydro-benzoimidazol-2-one37. N3ethylationof 38withethyl
a
b
c
NH
N
HN
N
NH
N
H2N
N
N
BocN
N
HN
N
O
O
O
O
37
38
39
40
d
H
N
N
H2N
N
N
N
N
N
N
O
O
O
40
41
Scheme 6. Reagents and conditions: (a) CH2Cl2, (Boc)2O, DMAP; (b) (i) DMF, EtBr, NaH, 0 °C to rt, 5 h; (ii) TFA, 1 h,rt; (c) (i) DMF, K2CO3, BocNH-(CH2)3-Br, 60 °C, 1 h; (ii) TFA,
1 h; (d) DMF, HOBt, N-Bn- -Pro, 0 °C to rt, O.N.
D

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C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
bromide in presence of sodium hydride followed by TFA treatment
gave compound 39. The reaction of 39 with N-Boc-3-bromo-propyl
receptor ligands to generate novel biologically active molecules.
In particular, the chemical groups benzoisofurane and 2,6-dichlo-
rophenyl seem to contribute to NOP receptor binding in a very
similar manner. This is corroborated by the finding that the substi-
amine followed by deprotection and acylation with N-Bn-
allowed us to obtain the final compound 41 (Scheme 6).
D-Pro
Compound 35 was selected as the most interesting molecule of
this series. It was obtained with an overall yield of 21%. This value
is similar to the overall yield of Comp 24 (25% in our laboratories8,
31% in Banju laboratories7). On the contrary the overall yield of
other NOP antagonists such as J-113397 and SB-612111 is much
lower (approx. 1–2% in our laboratories). Moreover, the synthesis
of the 2,6-dichlorophenyl-piperidine moiety of SB-612111 used
for generating compound 35 has been performed avoiding the te-
dious, money and time consuming chromatographic purifications.
In addition, the complete synthesis of compound 35 has been ob-
tained in six steps while those of Comp 24 and of SB-612111 re-
quire 8 and 12 steps, respectively.
tution of D-Pro with L-Pro in the chimeric molecule compound 36
produced a 100-fold reduction in receptor affinity. This result is
in agreement with compounds 22 and 23 of the Goto series.7 Thus,
D chirality of the Pro residue represents a crucial requirement for
high affinity NOP receptor recognition for both Comp 24 and com-
pound 35.
Based on its high affinity for the NOP receptor, compound 35
was selected for further pharmacological characterization in vitro
and in vivo. As shown in Figure 1 (top left panel) in CHOhNOP cell
membranes N/OFQ concentration dependently stimulated
[
35S]GTP
cS binding with pEC50 and Emax values of 8.91 and
10.93 0.18 (stimulation factor), respectively. Over the concentra-
tion range of 1–100 nM, compound 35 was inactive per se but pro-
duced a rightward shift of the concentration response curve to N/
OFQ in a parallel manner and without modifying the agonist max-
imal effect. Schild analysis of these data (Fig. 1 top right panel) is
compatible with a competitive type of antagonism and a pA2 value
of 9.91 was derived. This potency value is close to that previously
reported for Comp 24 by Goto et al. (pIC50 9.82)7 and by us (pA2
9.98).8 These results suggest that the benzoisofurane and 2,6-
dichlorophenyl moieties of Comp 24 and compound 35 play a sim-
ilar role not only in receptor binding but also in determining phar-
macological activity that is, pure and competitive antagonism. This
may be derived from the common ability of the spiro junction
(Comp 24) and of the 2,6-dichloro substitution (compound 35) to
favour an orthogonal spatial disposition between their piperidine
and phenyl nuclei. Therefore, this conformational feature may
likely be crucial for both NOP receptor binding and antagonist
activity.
3. Results and discussion
The ability of the reference ligand, Comp 24, and the novel mol-
ecules to bind to the NOP receptor was evaluated using CHOhNOP
cell membranes (Table 1). Comp 24 produced a concentration
dependent inhibition of [3H]N/OFQ binding with a pKi value of
9.62. This result perfectly matches with that previously reported
by Goto et al.7 (pIC50 9.57). The substitution of the N-benzyl
D-
Pro with
11) produced a profound (>100-fold) loss of NOP affinity suggest-
ing a pivotal role of the N-benzyl -Pro moiety for Comp 24 bioac-
D or L-Phe (compounds 9 and 10) or Nphe (compound
D
tivity. This result is not surprising since the simple inversion of Pro
chirality was reported to generate an analogue 200-fold less potent
than Comp 24.7
Modifications of the amide bond obtained by N-methylation
(compound 12) or by replacement with a methyleneamino (com-
pound 13), an ester (compound 17), a methyleneoxy (compound
20), or an alkene bond (compound 25), produced a drastic reduc-
tion in NOP receptor binding or, in the case of compound 25, a
complete loss of affinity. These results indicated that the amide
bond represents a chemical feature crucial for the bioactivity of
Comp 24. It is worthy of note, that an amide bond is a relatively
uncommon chemical feature in non-peptide NOP ligands. How-
ever, this chemical bond is also present in the NOP receptor antag-
onist JTC-801 and its chemical modification (i.e., N-methylation
and retro-inverso bond) was reported to be detrimental for binding
affinity.18 Thus, the amide bond might play a similar important
role in both Comp 24 and JTC-801. In particular, according to the
non-peptide NOP ligand pharmacophoric model proposed by
Zaveri,19 the amide bond may be part of the B-moiety that links
and contributes to the maintainance of the correct spatial arrange-
ment of the A-moiety (important for ligand affinity and selectivity)
and the C-moiety (important for ligand efficacy).
Next, we considered replacement of the benzoisofurane group
in position 4 of the Comp 24 piperidine scaffold with chemical
moieties taken from the some position of other high affinity NOP
receptor ligands. The substitution with 1-phenylimidazolidin-4-
one, the chemical group of the NOP receptor agonist Ro 64-6198
(compound 28), or with 1-ethylbenzoimidazol-2-one, the chemical
group of the NOP receptor antagonist J-113397 (compound 41), in
position 4 generated inactive molecules. In contrast, the insertion
of the piperidine scaffold of the 2,6-dichlorophenyl moiety (com-
pound 35), the pharmacophore of the NOP receptor antagonist
SB-612111, in position 4 generated a molecule with high affinity
for the NOP receptor (pKi 9.14). In particular compound 35 is only
threefold less potent than Comp 24. The results obtained with this
limited series of chimeric compounds suggest that it is possible to
combine pharmacophoric moieties taken from different NOP
The pharmacological actions of compound 35 were further as-
sessed at the hNOP receptor coupled to calcium signalling via the
chimeric protein Gaqi5; this assay has been previously validated
with a large panel of NOP receptor full and partial agonists and
antagonists.20 In CHO cells stably expressing the hNOP receptor
and the Gaqi5 protein, N/OFQ produced a concentration dependent
stimulation of intracellular calcium with a pEC50 of 9.24 and Emax
of 198 12% over the basal values. Compound 35 was inactive
per se up to 10 lM while inhibiting the stimulatory effect of
10 nM N/OFQ in a concentration dependent manner. A pKb value
of 8.47 was derived from these experiments (Table 2). This potency
value is approx threefold lower than that obtained with Comp 24
(pKb 9.03).8
The competitive antagonist behaviour of compound 35 was
confirmed at the native NOP receptor expressed in the mouse vas
deferens. In this preparation, N/OFQ inhibited electrically evoked
twitches in a concentration dependent manner with pEC50 and
Emax values of 7.49 and ꢀ80 2%, respectively (Fig. 1, right top
panel). Compound 35 was inactive per se but caused a concentra-
tion dependent (10–1000 nM) and a parallel rightward shift of the
concentration response curve to N/OFQ without modifying the
agonist maximal effects. The relative Schild plot, depicted in Figure
1 right bottom panel, demonstrated a competitive type of antago-
nism with a pA2 value of 8.00. This value of potency is close to that
previously reported for Comp 24 that is, 8.24.8 Similar results were
obtained in other N/OFQ sensitive preparations such as the rat vas
deferens and guinea pig ileum where compound 35 at 100 nM was
inactive per se while antagonizing N/OFQ inhibitory actions with
pKb values of 8.06 and 8.84, respectively (Table 2).
Finally the selectivity of action of compound 35 over classical
opioid receptors was assessed in animal tissues expressing native
receptors and at recombinant human proteins. Compound 35 at
1 lM was inactive per se and did not modify the inhibitory effects

C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
5085
Table 1
Binding affinities at CHOhNOP cell membranes of Comp 24 and related compounds
Compound
Structure
pKi
O
Comp 24
9.62 0.07
N
N
H
N
O
9
7.23 0.19
7.23 0.05
7.22 0.08
H
N
N
N
O
O
H2N
O
10
11
H
N
H2N
O
H
N
N
O
N
H
O
O
12
5.94 0.28
N
N
N
N
O
13
17
20
25
7.31 0.06
6.97 0.02
6.80 0.80
N
H
N
O
O
N
O
N
O
O
O
N
N
O
N
N
<5
(continued on next page)

5086
C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
Table 1 (continued)
Compound
Structure
pKi
O
O
NH
N
N
H
N
28
35
36
<5
N
Cl
O
O
9.14 0.05
N
N
N
H
N
N
Cl
Cl
7.08 0.02
N
H
Cl
N
O
41
<5
N
N
N
N
H
O
Data are means sem of four separate experiments performed in duplicate.
elicited by the DOP selective agonist DPDPE in the mouse vas def-
zoisofurane with the 2,6-dichlorophenyl moiety not only allows
maintenance of high affinity and antagonist potency but also of
high selectivity over classical opioid receptors.
erens (control pEC50 8.38 (CL95% 8.20–8.56), Emax ꢀ98 1%; 1
lM
compound 35 pEC50 8.44 (CL95% 8.26–8.62), Emax ꢀ98 1%) or those
produced by the MOP agonist dermorphin in the guinea pig ileum
Finally the pharmacological activity of compound 35 was as-
sessed in vivo using the mouse tail withdrawal assay. In this test
N/OFQ was reported to elicit opposite effects depending on the
route of administration: the peptide induced pronociceptive ac-
tions when injected intracerebroventricularly (icv) while antinoci-
ceptive effects were reported after intrathecal (i.t.) injection (see
for reviews3,21 and for results obtained in our laboratories22,23).
As shown in Figure 2 left panel, mice injected with saline dis-
played similar tail withdrawal latencies (ꢁ6 s) over the time
course of the experiment. N/OFQ 1 nmol given icv produced a
clear but short lasting pronociceptive effect. Compound 35 at
10 mg/kg given intraperitoneally 30 min before icv injection, did
not modify tail withdrawal latencies per se and did not affect
the pronociceptive action of the peptide. When 1 nmol N/OFQ
was given i.t., the peptide elicited a robust antinociceptive effect
(Fig. 2 right panel). Again compound 35 at 10 mg/kg did not mod-
ify the effect of N/OFQ. Thus, in the mouse tail withdrawal assay
compound 35 at 10 mg/kg was found inactive against the prono-
ciceptive and antinociceptive effects of N/OFQ injected supraspi-
nally and spinally, respectively. Under these same experimental
conditions Comp 24 at 10 mg/kg significantly counteracted the
actions of N/OFQ8, while SB-612111 already at 1 mg/kg fully pre-
vented the effects of the peptide.24 Thus, despite a very similar
in vitro pharmacological profile, the three NOP antagonists dis-
played very different in vivo potency that is, SB-612111 > Comp
24 ꢂ compound 35. Although the reasons for such variable
in vivo potency are at present unknown, they might be due to
(control pEC50 8.52 (CL95% 8.35–8.69), Emax ꢀ90 3%;
1 lM
compound 35 pEC50 8.44 (CL95% 8.19–8.69), Emax ꢀ85 5%). Results
obtained with compound 35 and the universal opioid receptor
antagonist naloxone in selectivity studies performed with receptor
binding and calcium mobilization assays are summarized in Tables
3 and 4, respectively. Compound 35 up to 10 lM did not displace
[3H]DPN from DOP sites and showed very low affinity (less than
300-fold compared to NOP) at MOP and KOP sites (Table 3). In con-
trast, naloxone did not bind to the NOP receptor up to 10 lM, while
it displaced [3H]DPN at classical opioid receptors showing very
high affinity for MOP and lower affinities for KOP and DOP (Table
3). Similar results were found in functional studies measuring cal-
cium mobilization in CHO cells stably expressing the hNOP recep-
tor or classical opioid receptors and the Gaqi5 protein (Table 4).
Dermorphin, DPDPE and dynorphin A were used in these experi-
ments as agonists for MOP, DOP and KOP receptors, respectively;
they produced a concentration dependent stimulation of intracel-
lular calcium with the following pEC50 values: 7.93 (CL95% 7.67–
8.19), 8.82 (CL95% 8.43–9.21) and 8.47 (CL95% 8.16–8.78). Naloxone
inhibited the effects of these agonists with higher potency at MOP
than KOP and DOP while being inactive against N/OFQ (Table 4).
Thus, compound 35 was at least 300-fold less potent at classical
opioid receptors than at the NOP receptor (Table 4).
These in vitro results demonstrated that compound 35 behaves
as a pure, potent and competitive NOP receptor antagonist. More-
over selectivity studies indicated that the substitution of the ben-

C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
5087
control
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
12
10
8
compound 35 1nM
compound 35 10nM
compound 35 100nM
6
4
pA2= 9.91
r2 = 0.91
slope = 1.05 0.10
2
0
-12 -11 -10
-9
-8
-7
-6
-5
-4
-9
-8
Log[compound 35]
-7
Log[N/OFQ]
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
100
80
60
40
20
0
control
pA2 = 8.00
compound 35 10nM
compound 35 100nM
compound 35 1µM
r2 = 0.99
slope = 1.06 0.09
-10
-9
-8
-7
-6
-5
-8
-7
Log[compound 35]
Figure 1. Concentration–response curve to N/OFQ in the absence and presence of increasing concentrations of compound 35 for [35S]GTP
-6
Log[N/OFQ]
c
S binding to CHOhNOP cell
membranes (top left panel) and in electrically stimulated mouse vas deferens (bottom left panel). The corresponding Schild plots are shown in the right panels. Data are
means SEM of four separate experiments.
Table 2
Compound 35 affinity/antagonist potency in various pharmacological assays
CHOhNOP cell membranes
CHOhNOP/G
cells
qi5
Electrically stimulated tissues
Rat vas deferens
a
Receptor binding
9.14 (9.04–9.24)
[
35S]GTP
9.91 (9.23–10.59)
c
S binding
Ca2+ mobilization
Mouse vas deferens
8.00 (7.32–8.68)
Guinea pig ileum
8.84 (8.64–9.04)
Compound 35
8.47 (8.31–8.63)
8.06 (7.58–8.54)
Data are expressed as means (CL95%) of at least four separate experiments.
Table 3
Affinities of compound 35 and naloxone at NOP and classical opioid receptors expressed in CHO cell membranes
Receptor radioligand
NOP [3H]N/OFQ
MOP [3H]DPN
DOP [3H]DPN
KOP [3H]DPN
Naloxone
Compound 35
<6
9.25 (9.04–9.46)
6.72 (6.47–6.97)
7.67 (7.59–7.75)
<6
8.35 (8.20–8.50)
6.50 (6.37–6.63)
9.14 (9.04–9.24)
Data are expressed as mean (CL95%) of four separate experiments. Naloxone data at classical opioid receptors are obtained from Vergura et al.33
Table 4
Antagonist potencies of compound 35 and naloxone evaluated in calcium mobilization experiments performed in CHO cells expressing NOP or classical opioid receptors and the
aqi5 protein
G
Receptor agonist
NOP N/OFQ 10 nM
MOP dermorphin 100 nM
DOP DPDPE 100 nM
KOP dynorphin A 100 nM
Naloxone
Compound 35
<6
9.09 (8.73–9.45)
6.11 (5.92–6.30)
7.32 (6.80–7.84)
<6
7.14 (6.60–7.68)
<6
8.47 (8.31–8.63)
Data are expressed as mean (CL95%) of four separate experiments performed in duplicate.
marked differences in pharmacokinetics between the three mole-
cules. Therefore the blending of Comp 24 and SB-612111
chemical moieties to generate compound 35 did not affect phar-
macodynamics (i.e., receptor affinity, antagonist potency and
selectivity of action) while it seems to have a detrimental effect
on pharmacokinetics.

5088
C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
20
8
6
4
2
0
saline
N/OFQ 1 nmol
compound 35 10 mg/kg
compound 35 vs N/OFQ
15
10
5
saline
N/OFQ 1 nmol
compound 35 10 mg/kg
compound 35 vs N/OFQ
0
0
15
30
45
60
0
15
30
45
60
time (min)
time (min)
Figure 2. Mouse tail withdrawal assay. Effects of compound 35 (10 mg/kg ip, 30 min pre-treatment) on the pronociceptive or antinociceptive effects induced by 1 nmol N/
OFQ injected icv (left panel) or i.t. (right panel). Data are mean SEM of four separate experiments.
HCl 3 N and the aqueous layer partitioned in chloroform and ex-
tracted three times with chloroform (30 mL each). The organic
layer was dried and concentrated in vacuo to afford a yellow so-
lid after crystallization with Et2O (6.54 g, 22.345 mmol, yield
88%). mp = 210–215 °C. MS (ESI): [MH]+ = 294. 1H NMR (CDCl3):
d 7.872 (td, 1H, J = 8, 0.9 Hz); 7.660 (dt, 1H, J = 7.4, 1.2 Hz);
7.51 (dt, 1H, J = 7.4, 0.8 Hz); 7.42 (td, 1H, J = 7.6, 0.7 Hz); 7.38–
7.31 (m, 4H); 7.29–7.25 (m, 1H); 3.62 (s, 2H); 2.92 (dd, 2H,
J = 9, 2.2 Hz); 2.56 (dt, 2H, J = 12, 2.4 Hz); 2.23 (dt, 2H, J = 13.2,
4.4 Hz); 1.71 (dd, 2H, J = 14.4, 2.4 Hz). 13C NMR (CDCl3):
168.80; 151.37; 135.09; 131.33 (2C); 130.36; 130.21; 129.53
(2C); 126.14 (2C); 124.80; 121.79; 81.56; 61.31; 48.92 (2C);
33.02 (2C).
4. Conclusions
The present study demonstrated that the N-benzyl D-Pro moi-
ety as well as the amide bond of Comp 24 is essential for biological
activity. In contrast the spirobenzoisofurane can be replaced with
2,6-dichlorophenyl moiety without significant loss of ligand po-
tency, antagonist activity and selectivity of action. Thus by blend-
ing chemical moieties taken from known molecules (Comp 24 and
SB-612111) we were able to identify compound 35 as a novel non-
peptide selective NOP antagonist. While the in vitro pharmacolog-
ical profile of compound 35 is similar to that of Comp 24 and SB-
612111, the in vivo activities of these compounds are substantially
different with the following order of antagonist potency SB-
612111 > Comp 24 ꢂ compound 35. This limits the usefulness of
compound 35 to in vitro investigations.
5.2.2. 10-Benzyl-spiro[isobenzofuran-1(3H),40-piperidine] (3)
Under argon atmosphere 6.54 g (22.34 mmol) of compound 2
were suspended in anhydrous THF, the reaction mixture cooled
5. Experimental
5.1. Materials
*
at 0 °C and under vigorously stirring (CH3)2S BH3 (4.30 mL,
44.68 mmol) was added drop wise. The reaction mixture was
heated at reflux overnight. After this time the reaction was acidi-
fied (HCl 10%) until pH 2 and heated again at reflux for 4 h. The
reaction was quenched with NaOH 2 N until pH 12, the THF was re-
moved in vacuo and the aqueous layer extracted twice with EtOAc
(30 mL each). The organic layer was dried, concentrated in vacuo
and purified by flash chromatography (EtOAc/light petroleum,
1:1) to afford compound 3 in 55% yield. MS (ESI): [MH]+ = 280.
1H NMR (CDCl3): d 7.39–7.15 (m, 9H); 5.07 (s, 2H,); 3.60 (s, 2H);
2.85 (dd, 2H, J = 10, 2 Hz); 2.44 (dt, 2H, J = 12, 2.4 Hz); 2.01 (dt,
2H, J = 13.2, 4.4 Hz); 1.76 (dd, 2H, J = 14.4, 2.4 Hz). 13C NMR
(CDCl3): 145.78; 138.98 (2C); 129.43 (2C); 128.28 (2C); 127.59;
127.38; 127.10; 121.10; 120.89; 84.81; 70.78; 63.54; 50.21 (2C);
36.64 (2C).
Melting points (uncorrected) were measured with a Buchi-Tot-
toli apparatus, and 1H, 13C, and NMR spectra were recorded on a
VARIAN400 MHz instrument unless otherwise noted. Chemical
shifts are given in ppm (d) relative to TMS and coupling constants
are in hertz. MS analyses were performed on a ESI-Micromass ZMD
2000. Infrared spectra were recorded on a Perkin–Elmer FT-IR
Spectum 100 spectrometer. Flash chromatography was carried
out on a silica gel (Merck, 230–400 Mesh). Elemental analyses
were performed at the analytical laboratories of the Department
of Chemistry, University of Ferrara.
5.2. Synthetic procedure
5.2.1. 10-Benzyl-spiro[isobenzofuran-1(3H),40-piperidin]-3-one
(2)
5.2.3. Spiro[isobenzofuran-1(3H),40-piperidine] (4)
A solution of 3 (3.43 g, 12.29 mmol) in 200 mL of ethanol was
hydrogenated in the Parr apparatus (50 psi) in the presence of
10% palladium on charcoal (0.1 g) for 48 h. Filtration of the catalyst
through a Celite pad and solvent evaporation gave (4) (2.20 g,
11.64 mmol, yield 70%) as a colorless oil. MS (ESI): [MH]+ = 190.
1H NMR (CDCl3): d 7.31–7.28 (m, 2H); 7.23–7.21 (m, 2H); 5.79
(br, 1H); 5.06 (s, 2H); 3.40–3.87 (m, 2H); 2.75–2.65 (m, 2H);
2.37–2.28 (m, 2H); 1.83–1.81 (m, 2H).
To a stirred solution of 1 (5 g, 25.38 mmol) in anhydrous THF
at ꢀ78 °C and in argon atmosphere, n-BuLi (25.98 mL,
64.97 mmol) was added drop wise. The reaction was warmed
at 0 °C for 1 h until a red-orange color appeared. At this time,
N-benzyl-piperidone (10.68 mL, 57.61 mmol) was added and
the reaction stirred at room temperature overnight. The reaction
was checked by TLC (EtOAc/light petroleum, 3:2), quenched with

C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
5089
5.2.4. 3-[Spiro[isobenzofuran-1(3H),40-piperidin-1-yl]]
propylamine (5)
9.5:0.5:0.3) to give compound
8
in 22% yield. MS (ESI):
[MH]+ = 494. 1H NMR (CDCl3): d 7.31–7.18 (m, 9H); 5.05 (s, 2H);
4.58–4.43 (m, 6H); 3.82–3.76 (m, 4H); 3.46–3.32 (m, 4H); 2.98–
2.65 (m, 4H); 2.38 (br, 1H); 1.45 (s, 9H). 13C NMR (CDCl3):
169.96; 156.34; 143.97; 138.51; 137.65; 128.68; 128.13; 128.00;
127.76; 127.46; 127.08; 121.14 (2C); 84.25; 80.79; 79.92; 71.13;
54.86; 51.67; 50.78; 50.28; 49.56; 36.96; 34.53; 28.40 (3C);
24.46; 14.25.
To a stirred solution of 4 (2 g, 10.58 mmol) in DMF a solution of
N-Boc-3-bromo-propyl-amine (2.99 g, 12.59 mmol) in 10 mL of
DMF was added followed by addition of K2CO3 (2.92 g,
21.16 mmol); the reaction was warmed at 60 °C for 1 h. The reac-
tion was concentrated and the residue was diluted with EtOAc
and washed twice with NH4Cl saturated solution (30 mL each)
and brine (30 mL). The organic layer was dried and evaporated un-
der reduced pressure. The crude material (3.45 g, 9.99 mmol) was
treated with TFA at 0 °C for 1 h. NaOH 2 N was added until pH
12–14, the aqueous phase was extracted several times with EtOAc
and the organic phases were combined, dried and evaporated un-
der vacuum to give compound 5 (2.32 g, 9.417 mmol, 95% yield).
MS (ESI): [MH]+ = 247. 1H NMR (CDCl3): d 7.27–7.24 (m, 2H);
7.20–7.14 (m, 2H); 5.06 (s, 2H); 2.89 (dd, 2H, J = 9.4, 2 Hz); 2.81
(t, 2H, J = 6.8 Hz); 2.50 (t, 2H, J = 7.2 Hz); 2.38 (t, 2H, J = 12.4 Hz);
2.23 (br, 2H); 1.98 (dt, 2H, J = 12.4, 4.4 Hz); 1.79–1.70 (m, 4H).
5.2.8. (D)-2-Amino-N-{3-[spiro[isobenzofuran-1(3H),40-piperidin-
1-yl]]-propyl}-3-phenyl-propyl-amide (9)
Compound 6 (160 mg, 0.324 mmol) was dissolved in TFA (5 mL)
at room temperature for 1 h, after cooling at 0 °C the reaction was
basified with NaOH 2 N until pH 13 and the aqueous layer was ex-
tracted three times with EtOAc (20 mL each). The solvent was
evaporated under reduced pressure and the crude material was
purified by flash chromatography (EtOAc/MeOH/NH4OH, 9.5/0.5/
0.3) to give compound 9 as oil in quantitative yield. MS (ESI):
[MH]+ = 394. 1H NMR (CDCl3): d 7.27–7.19 (m, 9H); 5.06 (s, 2H);
3.61–3.20 (m, 6H); 2.98–2.65 (m, 4H); 2.54–2.38 (m, 5H); 1.80–
1.73 (m, 5H). 13C NMR (CDCl3): 174.36; 145.72; 140.72; 138.88;
138.082; 129.40; 128.73; 127.73; 127.48; 126.83; 121.18;
120.80; 84.43; 70.89; 60.48; 57.11; 56.92; 50.371; 41.42; 38.48;
5.2.5. (D)-1-{3-[Spiro[isobenzofuran-1(3H),40-piperidin-1-yl]-pro-
pylcarbamoyl}-2-phenyl-ethyl)-carbamic acid-tert-butyl estere
(6)
To a stirred solution of Boc-D-Phe-OH (539 mg, 2.032 mmol) in
DMF at 0 °C, HOBt (373 mg, 2.44 mmol) and WSC (467 mg,
2.44 mmol) were added and the reaction stirred for 10 min. After
this time compound 5 (500 mg, 2.032 mmol) dissolved in DMF
(20 mL) was added and the reaction mixture stirred for 24 h. The
DMF was removed in vacuo and the product partitioned between
EtOAc and NaHCO3 5%. The organic phase was dried, concentrated
in vacuum and the crude product purified by flash chromatography
(EtOAc/NH4OH, 10:0.3) to give compound 6 in 30% yield. MS (ESI):
[MH]+ = 494. 1H NMR (CDCl3): d 7.42 (br, 1H); 7.28–7.15 (m, 9H);
5.83 (br, 1H); 5.03 (s, 2H); 3.43 (s, 1H); 3.55–3.16 (m, 3H); 3.08–
2.95 (m, 2H); 2.90–2.80 (m, 3H); 2.50–2.44 (m, 4H); 1.76–1.68
(m, 4H); 1.36 (s, 9H). 13C NMR (CDCl3): 173.02; 171.38; 144.70;
138.62; 136.946; 129.35 (2C); 128.63 (2C); 128.54; 127.83;
127.56; 126.82; 121.05 (2C); 83.92; 70.90; 60.41; 56.31; 50.498;
36.34; 25.89; 21.15; 14.27. ½a D20
ꢃ
= +15 (c = 0.2 g/100 mL, chloro-
form). Anal. (C24H31N3O2) C, H, N.
5.2.9. (L)-2-Amino-N-{3-[spiro[isobenzofuran-1(3H),40-piperidin-
1-yl]]-propyl}-3-phenyl-propyl-amide (10)
Compound 7 was treated as for obtaining 9 to give compound
10 in quantitative yield. MS (ESI): [MH]+ = 394. 1H NMR (CDCl3):
d 7.27–7.19 (m, 9H); 5.06 (s, 2H); 3.61–3.20 (m, 6H); 2.98–2.65
(m, 4H); 2.54–2.38 (m, 5H); 1.80–1.73 (m, 5H). 13C NMR (CDCl3):
174.36; 145.72; 140.72; 138.88; 138.08; 129.40; 128.73; 127.77;
127.48; 126.83; 121.18; 120.80; 84.43; 70.89; 60.48; 57.11;
56.92; 50.37; 41.42; 38.48; 36.34; 25.89; 21.15; 14.27.
½
a 2D0
H, N.
ꢃ
= ꢀ15 (c = 0.2 g/100 mL, chloroform). Anal. (C24H31N3O2) C,
49.88; 39.06; 38.38; 35.82; 35.72; 28.29 (3C); 24.67. ½a D20
= ꢀ8
ꢃ
(c = 0.1 g/100 mL, chloroform).
5.2.10. 2-Benzylamino-N-{3-[spiro[isobenzofuran-1(3H),4’-pipe-
ridin-1-yl]]-propyl}-acetamide (11)
5.2.6. (L)-1-{3-[Spiro[isobenzofuran-1(3H),40-piperidin-1-yl]-pro-
pylcarbamoyl}-2-phenyl-ethyl)-carbamic acid-tert-butyl estere
(7)
This product was obtained as reported for 6 using Boc-Phe-OH
instead of Boc-D-Phe-OH. The crude product was purified by flash
Compound 8 was treated as for obtaining 9 to give 11 in quan-
titative yield. MS (ESI): [MH]+ = 394. 1H NMR (CDCl3): d 7.35–7.19
(m, 9H); 7.20–7.19 (m, 1H); 5.05 (s, 2H); 3.80 (s, 2H); 3.32–3.29 (m,
5H); 3.25–3.21 (m, 2H); 2.94–2.82 (m, 4H); 2.19–2.10 (m, 2H);
1.91–1.82 (m, 4H). 13C NMR (CDCl3): 173.72; 145.30; 140.09;
139.72; 129.68 (2C); 129.61 (2C); 129.24; 128.67; 128.55;
122.34; 121.68; 84.28; 71.90; 56.31; 54.06; 51.59; 50.92 (2C);
37.77; 35.85 (2C); 26.44. Anal. (C24H31N3O2) C, H, N.
chromatography (EtOAc/NH4OH, 10:0.3) to give compound 7 in
30% yield. MS (ESI): [MH]+ = 494. 1H NMR (CDCl3): d 7.42 (br,
1H); 7.28–7.15 (m, 9H); 5.83 (br, 1H); 5.03 (s, 2H); 3.43 (s, 1H);
3.55–3.16 (m, 3H); 3.08–2.95 (m, 2H); 2.90–2.80 (m, 3H); 2.50–
2.44 (m, 4H); 1.76–1.68 (m, 4H); 1.36 (s, 9H). 13C NMR (CDCl3):
173.02; 171.38; 144.70; 138.62; 136.946; 129.35 (2C); 128.63
(2C); 128.54; 127.83; 127.56; 126.82; 121.05 (2C); 83.92; 70.90;
60.41; 56.31; 50.498; 49.88; 39.06; 38.38; 35.82; 35.72; 28.29
5.2.11. (D)-1-Benzyl-pirrolidin-2-carboxilic acid {3-[spiro[iso-
benzofuran-1(3H ),40-piperidin-1-yl]]-propyl}-methyl-amide
(12)
A solution of the Comp 24 (200 mg, 0.460 mmol) in DMF
(10 mL) was added was added to a stirred suspension of sodium
hydride 60% (20.276 mg, 0,5069 mmol) in DMF (5 mL) dropwise
at 0 °C. The reaction mixture was left in the same conditions for
(3C); 24.67. ½a 2D0
ꢃ
= +8 (c = 0.1 g/100 mL, chloroform).
5.2.7. Benzyl-({3-[spiro[isobenzofuran-1(3H),40-piperidin-1-yl]-
propylcarbamoil}-methyl)-carbamic acid tert-butyl estere (8)
To stirred solution of (benzyl-tert-butoxycarbonyl-amino)acetic
acid (200 mg, 0.754 mmol) in DMF, at 0 °C, HOBt (138 mg,
0.905 mmol) and WSC (173 mg, 0.905 mmol) were added and the
reaction stirred for 10 min. After this time compound 5 (188 mg,
0.754 mmol) dissolved in DMF (20 mL) was added and the reaction
mixture stirred for 24 h. The DMF was removed in vacuo and the
product partitioned between EtOAc and NaHCO3 5%. The organic
phase was dried, concentrated in vacuum and the crude product
purified by flash chromatography (EtOAc/MeOH/NH4OH,
half an hour, then a solution of methyl iodide (38 ll, 0.599 mmol)
in DMF (1 mL) was added. After 24 h at room temperature, the
reaction was monitored by TLC (EtOAc/MeOH/NH3 9.5:0.5:0.3).
The DMF was removed under vacuum and the residue was diluted
with Brine (5 mL) and extracted thrice with EtOAc. The organic lay-
ers were combined, dried and evaporated under reduced pressure
to give a crude product as a yellow oil. This was purified by flash
chromatography to obtain compound 12 (53 mg, 0.118 mmol, yield
26%). MS (ESI): [MH]+ = 448. 1H NMR (CDCl3): d 7.80–7.25 (m, 9H);
5.07 (s, 2H); 3.78–3.29 (m, 10H); 2.95–2.87 (m, 4H); 2.56–2.12 (m,

5090
C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
6H); 2.06–1.69 (m, 6H). 13C NMR (CDCl3): 175.37; 171.47; 162.61;
141.82; 138.36; 138.25; 132.65; 129.390; 129.22; 128.70; 128.06;
127.54; 121.42; 81.22; 81.05; 71.47; 67.08; 66.69; 59.985; 58.04;
54.63; 45.77; 36.56; 36.25; 36.12; 31.45; 30.97; 24.18. Anal.
(C28H37N3O2) C, H, N.
luted with EtOAc and washed twice with water. The organic layer
was dried and concentrated under reduced pressure to give the
crude product, which was purified by flash chromatography to ob-
tain compound 16 (1.239 g, 5.015 mmol, yield 94%). MS (ESI):
[MH]+ = 248. 1H NMR (CDCl3): d 7.29–7.13 (m, 4H); 5.06 (s, 2H);
3.84 (t, 1H, J = 5.2 Hz); 3.61–3.60 (m, 1H); 3.08–3.01 (m, 2H);
2.72 (t, 1H, J = 5.6 Hz); 2.51–2.44 (m, 3H); 2.05–1.95 (m, 3H);
1.82–1.72 (m, 4H). 13C NMR (CDCl3): 145.20; 138.87; 127.78;
127.52; 121.14; 120.89; 84.46; 70.88; 64.64; 62.85; 59.16; 50.42;
36.55; 32.83; 27.16.
5.2.12. (1-Benzyl-pirrolidin-2-yl-methyl)-{3-[spiro[isobenzofu-
ran-1(3H),40-piperidin-1-yl]]-propyl}-amine (13)
In a two neck round bottom flask, under argon atmosphere,
Comp 24 (150 mg, 0.346 mmol) was suspended in anhydrous
*
THF (50 mL) and (CH3)2S BH3 (100 ll, 1.038 mmol) was added
drop wise. The reaction was heated at reflux overnight and moni-
tored by TLC (EtOAc/light petroleum/NH3 9:1:0.3). The mixture
was cooled at 0 °C and acidified with HCl 10%. Then it was heated
again at reflux and, after 4 h, it was cooled at 0 °C and basified with
NaOH 2 N. THF was evaporated under reduced pressure and the
remaining aqueous layer was extracted three times with EtOAc.
The organic layers were combined, dried and evaporated under
vacuum to obtain the crude product, which was purified by flash
chromatography to give compound 13 (20 mg, 0.239 mmol, yield
69%). MS (ESI): [MH]+ = 420. 1H NMR (CDCl3): d 7.34–7.23 (m,
9H); 5.05 (s, 2H); 3.84–3.59 (m, 1H); 3.70–3.65 (m, 2H); 3.59 (m,
1H); 2.99–2.87 (m, 4H); 2.61–2.35 (m, 8H); 2.06–1.92 (m, 2H);
1.83–1.79 (m, 4H); 1.71–1.65 (m, 4H). 13C NMR (CDCl3): 145.18;
139.28; 138.99; 128.92; 128.59; 127.81; 127.49; 127.43; 121.23;
120.69; 84.31; 70.83; 62.87; 61.88; 59.69; 57.89; 55.14; 51.69;
50.99; 49.85; 49.74; 36.28; 36.11; 29.96; 29.06; 24.14; 23.29. Anal.
(C27H37N3O) C, H, N.
5.2.16. (D)-1-Benzyl-pirrolidin-2-carboxilic acid {3-[spiro[iso-
benzofuran-1(3H),40-piperidin-1-yl]-propyl}estere (17)
To a solution of 1-benzyl-pirrolidin-2-carbossilic acid (264 mg,
1.288 mmol) in DMF, cooled at 0 °C, HOBt (236 mg, 1.546 mmol)
and DCC (319 mg, 1.546 mmol) were added. After 10 min com-
pound 16 (350 mg, 1.417 mmol) dissolved in DMF was added.
The reaction mixture was stirred at room temperature for 24 h
and monitored by TLC (EtOAc/light petroleum/NH3 4:1:0.3). The
solvent was evaporated under vacuum and the residue was basi-
fied with NaHCO3 5% and extracted with EtOAc. The organic layers
were combined, dried and concentrated under reduced pressure to
obtain the crude product, which was purified by flash chromatog-
raphy to obtain compound 17 (170 mg, 0.392 mmol, yield 30%). MS
(ESI): [MH]+ = 435. 1H NMR (CDCl3): d 7.31–7.23 (m, 9H); 5.04 (s,
2H); 4.12 (dt, 2H, J = 6.6, 2.4 Hz); 3.91 (d, 1H, J = 12.6 Hz); 3.57
(d, 1H, J = 12.8 Hz); 3.27–3.20 (m, 2H); 3.03–2.95 (m, 2H); 2.86–
2.81 (m, 2H); 2.51–2.33 (m, 5H); 1.90–1.72 (m, 8H). 13C NMR
(CDCl3): 174.21; 145.52; 138.85; 138.41; 129.177 (2C); 128.19
(2C); 127.61; 127.39; 127.09; 121.06; 120.83; 84.62; 70.76;
65.27; 63.10; 58.65; 55.29; 53.20; 50.16 (2C); 36.51 (2C); 29.38;
5.2.13. (3-Bromo-propyloxi)-tert-butyl-dimethyl-silane (14)
A mixture of 3-bromo-propanol (5 mL, 57.190 mmol), imidazole
(11.68 g, 171.57 mmol) and TBDMS-Cl (11.206 g, 74.347 mmol) in
anhydrous THF (100 mL) was stirred at room temperature for
24 h. The reaction was monitored by TLC (EtOAc/light petroleum
1:3). The solvent was removed under reduced pressure and the res-
idue was diluted with water and extracted three times with DCM.
The organic layers were combined, washed with Brine, dried and
evaporated to obtain compound 14 in quantitative yield. 1H NMR
(CDCl3): d 3.73 (t, 2H, J = 2.8 Hz); 3.51 (t, 2H, J = 3.2 Hz); 2.07–
1.99 (m, 2H); 0.89 (s, 9H); 0.06 (s, 6H).
26.26; 23.03.
(C27H34N2O3) C, H, N.
½
a 2D0
= +23 (c = 0.1 g/100 mL, Chloroform). Anal.
ꢃ
5.2.17. 1-(3-Chloro-propyl)-4-spiro[isobenzofuran-1(3H),
40-piperidine] (18)
In a two neck round bottom flask compound 16 (200 mg,
0.81 mmol) was dissolved in dry DCM (10 mL) and cooled at 0 °C,
tosylchloride (170 mg, 0.89 mmol) and Et3N (90 mg, 0.89 mmol)
were added drop wise. The reaction was stirred at room tempera-
ture for 24 h and after evaporation, the crude material was dis-
solved in 20 mL of EtOAc. The organic phase was washed twice
with NaHCO3 saturated solution (20 mL each), the combined or-
ganic phases were concentrated to dryness and the product puri-
fied by flash chromatography (EtOAc/MeOH/NH4OH, 9:1:0.3) to
obtain compound 18 (85 mg, 0.320 mmol, yield 40%). MS (ESI):
[MH]+ = 266. 1H NMR (CDCl3): d 7.27–7.14 (m, 4H); 5.05 (s, 2H);
3.61 (t, 2H, J = 6.6 Hz); 2.85–2.80 (m, 2H); 2.55 (t, 2H, J = 7.2 Hz);
2.40 (dt, 2H); 2.04–1.95 (m, 4H); 1.79–1.78 (m, 2H).
5.2.14. 1-[3-(tert-Butyl-dimethyl-silan-yl-oxi)-propyl]-spiro
[isobenzofuran-1(3H),40-piperidine] (15)
To a stirred solution of 4 (1 g, 5.71 mmol) in DMF (50 mL) com-
pound 14 (1.72 g, 6.8 mmol) and K2CO3 (1.58 g, 11.43 mmol) dis-
solved in DMF (10 mL) were added. The reaction was stirred and
warmed at 60 °C for an hour and it was monitored by TLC
(EtOAc/light petroleum/NH3 5:1:0.3). The solvent was removed
under vacuum and the residue was dissolved in EtOAc and washed
twice with saturated NH4Cl and once with Brine. The organic layer
was dried and evaporated under reduced pressure to give the crude
product as a red-orange oil. This was purified by flash chromatog-
raphy to obtain compound 15 (1.93 g, 5.35 mmol, yield 94%). MS
(ESI): [MH]+ = 362. 1H NMR (CDCl3): d 7.28–7.13 (m, 4H); 5.06 (s,
2H); 3.67 (t, 2H, J = 6 Hz); 2.91–2.83 (m, 2H); 2.53–2.36 (m, 4H);
1.99 (dt, 2H, J = 6, 4 Hz); 1.81–1.73 (m, 4H); 0.89 (s, 9H); 0.05 (s,
6H). 13C NMR (CDCl3): 145.77; 138.97; 127.60; 127.40; 121.08;
120.88; 84.82; 70.79 (2C); 61.68; 55.73; 50.30 (2C); 36.69 (2C);
30.32; 26.06 (3C); ꢀ5.29 (2C).
5.2.18. 1-[3-(1-Benzyl-pirrolidin-2-ylmetoxy)-propyl]-4-spiro-
[isobenzofuran-1(3H),40piperidine] (20)
To a stirred suspension of sodium hydride (14 mg, 0.34 mmol)
in anhydrous THF at 0 °C, a solution of 19 (60 mg, 0.31 mmol) in
THF was added. After 30 minutes, a catalytic amount of TBAI was
added followed by drop wise addiction of compound 18 (85 mg,
0.31 mmol) dissolved in THF. After 24 h at room temperature, the
reaction was heated at reflux for 12 h, the solvent was removed
in vacuo, diluted with Et2O and the resulting precipitate filtrated
on Celite pad. The solvent was concentrated to dryness and the
crude material purified by flash chromatography (EtOAc/MeOH/
NH4OH, 9.5:0.5:0.3) to give compound 20 in 56% yield. MS (ESI):
[MH]+ = 421. 1H NMR (CDCl3): d 7.32–7.21 (m, 9H); 5.03 (s, 2H);
4.12 (dt, 2H, J = 6.6, 2.4 Hz); 3.91 (d, 1H, J = 12.6 Hz); 3.57 (d, 1H,
J = 12.8 Hz); 3.45–3.21 (m, 2H); 3.27–3.20 (m, 2H); 3.03–2.95 (m,
1H); 2.86–2.81 (m, 2H); 2.51–2.33 (m, 5H); 1.90–1.72 (m, 8H).
5.2.15. 3-{Spiro[isobenzofuran-1(3H),40-piperidin-1-yl]}-pro-
pan-1-ol (16)
To a solution of compound 15 (1.930 g, 5.346 mmol) in anhy-
drous THF (35 mL), cooled at 0 °C, TBAF (5.06 g, 16.038 mmol)
was added. The reaction was stirred at room temperature for
24 h and the solvent removed under vacuum. The residue was di-

C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
5091
13C NMR (CDCl3): 145.52; 138.85; 138.41; 129.17 (2C); 128.19
(2C); 127.61; 127.39; 127.09; 121.06; 120.83; 84.62; 74.34;
70.76; 65.27; 63.10; 58.65; 55.29; 53.20; 50.16 (2C); 36.51 (2C);
give Comp 24 in 37% yield. MS (ESI): [MH]+ = 434. 1H NMR (CDCl3):
d 7.60 (br, 1H); 7.30–7.20 (m, 9H); 5.05 (s, 2H); 3.87 (d, 1H,
J = 12.8 Hz); 3.48 (d, 1H, J = 12.8 Hz); 3.42–3.16 (m, 4H); 3.09–
3.00 (m, 1H); 2.87–2.80 (m, 2H); 2.48–2.29 (m, 5H); 2.23–2.13
(m, 3H); 2.03–1.87 (m, 2H); 1.77–1.65 (m, 4H). 13C NMR (CDCl3):
174.62; 145.61; 138.94; 138.64; 128.81 (2C); 128.56 (2C);
127.62; 127.38 (2C); 121.10; 120.79; 84.69; 70.79; 67.61; 60.02;
56.76; 54.07; 50.54; 50.21; 37.67; 36.60 (2C); 30.78; 26.99;
29.38; 26.26; 23.03. ½a D20
ꢃ
= +21 (c = 0.2 g/100 mL, chloroform).
Anal. (C27H36N2O2) C, H, N.
5.2.19. 5-(1-Benzyl-pirrolidin-2-yl)-pent-4-enoic acid (22)
In a two neck round bottom flask, under argon atmosphere, was
placed NaH 60% (12.57 g, 64.31 mmol) and washed five times with
anhydrous pentane (20 mL each). The resulting white solid was
dried flushing argon and anhydrous DMSO (50 mL) was added;
the mixture was heated at 77 °C for 40 min. At this time, the reac-
tion was cooled at 0 °C and the (3-carboxy-propyl)-triphenyl-phos-
phonium bromide 21 (12.55 g, 29.23 mmol) dissolved in DMSO
was added drop wise. When the reaction becomes a brown-red col-
or, 1.105 g (5.85 mmol) of 1-benzyl-pirrolidine-2-carbaldheyde
were added drop wise. The reaction was checked by TLC (EtOAc/
MeOH/NH4OH, 9:1:0.3), the DMSO was removed under vacuum
and the crude material dissolved in EtOAc (60 mL) and washed
with a 10% solution of citric acid (30 mL). The organic phases were
dried and evaporated to dryness, the compound was purified by
flash chromatography (EtOAc/MeOH/NH4OH, 9:1:0.3) to give com-
pound 22 (350 mg, 1.135 mmol, yield 23%). MS (ESI): [MH]+ = 260.
1H NMR (CD3OD): d 7.76–7.32 (m, 5H); 5.95–5.86 (m, 1H); 5.60–
5.50 (m, 1H); 4.45–4.39 (m, 2H); 4.16–4.10 (m, 1H); 3.34–3.29
(m, 6H); 2.40–2.39 (m, 4H). 13C NMR (CD3OD): 177.93; 139.63;
132.54; 131.91; 131.02; 130.47; 124.63; 66.22; 57.71; 53.82;
24.09.
(C27H35N3O2) C, H, N.
½
a 2D0
ꢃ
= +43
(c = 0.1 g/100 mL,
chloroform).
Anal.
5.2.22. 1-[5-(1-Benzyl-pirrolidin-2-yl)-pent-4-enyl]-spiro[iso-
benzofuran-1(3H),40-piperidine] (25)
In a round-bottomed flask under argon atmosphere, to a stirred
solution of 23 (140 mg, 0.325 mmol) in anhydrous THF (10 mL)
cooled at 0 °C, was added drop wise to a solution of borane dimeth-
ylsulfide complex (0.094 mL, 0.977 mmol) dropwise. The solution
was heated at reflux overnight, after this time, cooled at 0 °C and
HCl 10% was added until pH 2. The resulting solution was stirred at
reflux for 4 h, basified with NaOH 2 N and the solvent was removed
under vacuum. The crude product was purified by flash chromatog-
raphy (EtOAc/light petroleum/NH4OH, 2:1:0.3) to give 25 (35 mg,
0.084 mmol, yield 26%). 1H NMR (CDCl3): d 7.326–7.216 (m, 9H);
5.42–5.37 (m, 2H); 5.06 (s, 2H); 4.09 (t, 1H, J = 6.4 Hz); 3.77 (s, 1H);
3.67 (t, 1H, J = 6.4 Hz); 2.91–2.86 (m, 2H); 2.68–2.34 (m, 4H);
2.09–1.96 (m, 4H); 1.80–1.50 (m, 8H); 1.24–1.15 (m, 2H). 13C NMR
(CDCl3): 145.70; 141.23; 138.93; 130.53; 130.06; 128.44; 128.19;
127.63; 127.43; 126.95; 121.09; 120.92; 84.77; 70.83; 64.34;
62.38; 59.00; 54.08; 50.27; 49.80; 36.60; 32.51; 30.40; 29.87;
50.47; 40.62; 35.48; 31.32; 25.01; 22.35. ½a D20
= ꢀ7.14 (c = 0.21 g/
ꢃ
100 mL, ethanol). IR: 3398.72; 2993.34; 1658.97; 1560.22;
1435.77; 1405.21; 1315.37; 1211.62; 1159.59; 1124.14; 1013.97;
952.12; 752.60; 701.61.
27.88; 27.71; 26.48; 25.143. ½a D20
ꢃ
= +1.67 (c = 0.31 g/100 mL, chloro-
form). Anal. (C28H36N2O) C, H, N.
5.2.20. 5-(1-Benzyl-pirrolidin-2-yl)-1-[spiro[isobenzofuran-1(3H),
40-piperidin-1-yl]]-pent-4-en-1-one (23)
5.2.23. [3-(4-Oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-8-yl)-pro-
pyl]-carbamic acid tert-butyl estere (27)
Compound 22 (590 mg, 2.278 mmol) was dissolved in DMF
(50 mL) and at 0 °C HOAt (372 mg, 2.773 mmol), WSC (524 mg,
2.733 mmol) and compound 4 (430 mg, 2.278 mmol) were added.
After 12 h at room temperature, the solvent was removed by evap-
oration under reduced pressure and the residue diluted with EtOAc
(50 mL); the organic layer washed with NaHCO3 5% (10 mL) and
the aqueous phase extracted three times with EtOAc (30 mL each).
The combined organic phases were dried and concentrated in va-
cuo. The crude product was purified by flash chromatography
(EtOAc/light petroleum/NH4OH, 1:1:0.3) to obtain compound 23
(783 mg, 1.822 mmol, yield 80%). MS (ESI): [MH]+ = 431. 1H NMR
(CDCl3): d 7.2967.22 (m, 9H); 5.67–5.58 (m, 1H); 5.51–5.44 (m,
1H); 5.08 (s, 2H); 4.67–4.61 (m, 1H); 4.04–3.98 (m, 1H); 3.86–
3.80 (m, 1H); 3.67 (t, 1H, J = 6.4 Hz); 3.57 (t, 1H, J = 6.4 Hz); 3.22–
2.89 (m, 4H); 2.55–2.44 (m, 4H); 1.95–1.55 (m, 8H). 13C NMR
(CDCl3): 170.73; 144.70; 139.43; 138.86; 133.47; 130.75; 129.11;
128.19; 127.97; 127.58; 126.85; 121.30; 120.69; 84.76; 71.03;
61.94; 58.52; 53.28; 44.99; 42.61; 38.73; 37.09; 36.19; 33.39;
To a stirred solution of 26 (500 mg, 2.62 mmol) in DMF, was
added N-Boc-3-bromo-propyl-amine (612 mg, 2.57 mmol) in
10 mL of DMF and K2CO3 (596 mg, 4.32 mmol), the reaction was
warmed at 60 °C for 1 h. Most of the solvent was removed and
the residue was diluted with EtOAc and washed twice with NH4Cl
saturated solution (30 mL each) and brine (30 mL). The organic
layer was dried and evaporated under reduced pressure. The crude
product was crystallized from Et2O to give the final compound
(535 mg, 1.38 mmol, yield 64%). mp = 163–165 °C. MS (ESI):
[MH]+ = 389. 1H NMR (CDCl3): d 7.31–7.23 (m, 3H); 6.90–6.84 (m,
2H); 5.38 (br, 2H); 4.73 (s, 2H); 3.23–3.19 (m, 2H); 2.82–2.45 (m,
8H); 1.75–1.66 (m, 4H); 1.41 (s, 9H). 13C NMR (CDCl3): 178.14;
156.20; 143.21; 129.37 (2C); 119.02 (2C); 115.50 (2C); 79.00;
59.50; 59.38; 56.98; 49.84 (2C); 40.16; 29.31; 28.55 (3C); 26.91.
5.2.24. Acid-(D)-1-benzyl-pirrolidin-2-carboxilic-[3-(4-oxo-1-
fenyl-1,3,8-triaza-spiro[4.5]dec-8-yl)-propyl]-amide (28)
Compound 27 (535 mg, 1.37 mmol) was treated with TFA
(5 mL) at 0 °C for 1 h. NaOH 2 N was added until pH 12–14, the
aqueous phase was extracted several times with EtOAc and the or-
ganic phases were combined, dried and evaporated under vacuum
to give the free amine (395 mg, 1.37 mmol) in 100% yield. This
31.40; 29.97; 23.84; 22.10.
chloroform).
½
a 2D0
= +18 (c = 0.1 g/100 mL,
ꢃ
5.2.21. (D)-1-Benzyl-pirrolidin-2-carboxilic acid {3-[spiro[iso-
benzofuran-1(3H),40-piperidin-1-yl]]-propyl}-amide (Comp 24)
product was condensed with N-Bn-D-Pro in a similar manner as re-
To a stirred solution of N-Bn-
D
-Pro (860 mg, 3.496 mmol) in
ported for Comp 24. The crude product was purified by flash chro-
matography (EtOAc/MeOH/NH4OH, 9.5:0.5:0.3) to give 28 in 56%
yield. MS (ESI): [MH]+ = 476. 1H NMR (CDCl3): d 7.94–7.83 (m,
2H); 7.35–6.86 (m, 10H); 4.72 (s, 2H); 3.84 (d, 1H, J = 13 Hz);
3.51 (d, 1H, J = 13 Hz); 3.27–3.19 (m, 3H); 3.07–2.81 (m, 6H);
2.76–2.47 (m, 2H); 2.36–1.66 (m, 10H). 13C NMR (CDCl3): 178.00;
175.06; 143.32; 138.80; 129.52 (2C); 128.91 (2C); 128.74 (2C);
127.54; 125.82; 119.61; 116.19 (2C); 115.30; 67.58; 60.17;
59.59; 56.04; 54.33; 49.86; 37.55; 30.98; 29.90; 29.34; 27.22;
DMF (20 mL) cooled at 0 °C, HOBt (642 mg, 4.195 mmol), WSC
(804 mg, 4.195 mmol) were added and the reaction stirred for
10 min. After this time compound 5 (716 mg, 3.496 mmol) dis-
solved in DMF (20 mL) was added and the reaction mixture was
stirred for 24 h. The DMF was removed in vacuo and the product
partitioned between EtOAc and NaHCO3 5%. The organic phase
was dried, concentrated in vacuum and the crude product purified
by flash chromatography (EtOAc/MeOH/NH4OH, 9.5:0.5:0.3) to

5092
C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
24.37.
N5O2) C, H, N.
½
a 2D0
ꢃ
= +24 (c = 0.1 g/100 mL, chloroform). Anal. (C28H37
5.2.29. 3-[4-(2,6-Dichloro-phenyl)-piperidin-1-yl]-propylamine
(34)
To a stirred solution of 33 (284 mg, 1.236 mmol) in DMF (5 mL), N-
Boc-3-bromo-propyl-amine (350 mg, 1.471 mmol) and K2CO3
(341.77 mg, 2.473 mmol) were added. The reaction mixture was stir-
red at 60 °C for 1 h and than evaporated under vacuum, the residue
was dissolved in EtOAc and washed twice with a saturated solution
of NH4ClandoncewithBrine. Theorganiclayerwasdriedandconcen-
trated under reduced pressure to obtain the product as a red-orange
oil. This crude product was then purified by flash chromatography
(EtOAc/light petroleum/NH4OH 5:1:0.3) (yield 94%). At the product
so obtained (1.163 mmol), cooled at 0 °C, was added drop wise 1 mL
of TFA. The reaction was monitored by TLC (EtOAc/light petroleum
5:1) and after about 1 h was cooled at 0 °C and basified with NaOH
2 N. The aqueous layer was extracted three times with EtOAc and
the organic layers were combined, dried and evaporated under vac-
uum to give compound 34 with a quantitative yield. MS (ESI):
[MH]+ = 288. 1H NMR (CDCl3): d 7.25–7.00 (m, 3H); 3.53–3.46 (m,
1H); 3.36 (br, 2H); 3.116 (dd, 2H, J = 9.6, 4 Hz); 2.90 (t, 2H,
J = 6.8 Hz); 2.67–2.56 (m, 2H); 2.50 (t, 2H, J = 6.8 Hz); 2.05 (dt, 2H,
J = 10, 2.4 Hz); 1.75 (t, 2H, J = 6.4 Hz); 1.58 (d, 2H, J = 12.4 Hz).
5.2.25. 2-(2,6-Dichloro-phenyl)-4-hydroxy-4-methyl-6-oxo-cy-
clohexane-1,3-dicarboxylic acid diethyl ester (30)
Compound 29 (5 g, 28.57 mmol) and ethyl aceto-acetate
(7.22 mL, 57.14 mmol) were dissolved in 20 mL of ethanol 96%.
At this stirred solution was added piperidine (0.5 mL) and the reac-
tion mixture was stirred at room temperature overnight.
The solvent was removed under reduced pressure and the prod-
uct was crystallized from Et2O to give compound 30 with a quan-
titative yield. mp = 112–115 °C. MS (ESI): [MH]+ = 418. 1H NMR
(CDCl3): d 12.50 (s, 1H); 7.28–7.18 (m, 2H); 7.05 (t, 1H, 8 Hz);
5.01 (d, 1H, J = 11 Hz); 4.05–3.83 (m, 5H); 3.10 (d, 1H,
J = 11.4 Hz); 2.48 (s, 2H); 1.32 (s, 3H); 0.98 (t, 3H, 7.4 Hz); 0.85 (t,
3H, 7 Hz). 13C NMR (CDCl3): 174.77; 171.11; 170.52; 138.03;
137.03; 134.28; 130.16; 128.04; 127.90; 98.09; 69.35; 61.36;
60.29; 50.85; 41.72; 38.28; 28.05; 13.83; 13.39.
5.2.26. 3-(2,6-Dichloro-phenyl)-pentane-dioic acid (31)
Compound 30 (7 g, 16.79 mmol) dissolved in ethanol 96%
(55 mL), NaOH 35% (40.75 mL) and of water (16.3 mL) were added.
The reaction mixture was stirred at reflux for 3 h. The solvent was
removed under reduced pressure, the resulting aqueous layer was
acidified at 0 °C with HCl 20% and extracted with ethyl acetate. The
organic layers were combined, dried and evaporated under vac-
uum to obtain the crude product, which was crystallized from
ethyl ether to give 31 (4.026 g, 14.53 mmol, yield 87%).
5.2.30. (D)-1-Benzyl-pirrolidin-2-carbossilic acid {3-[4-(2,
6-dichloro-phenyl)-piperidin-1-yl]-propyl}-amide (35)
N-Bn-D-Pro and compound 34 were condensed in the same
experimental condition as for Comp 24 yielding 35 (48 mg,
0.101 mmol, yield 65%). MS (ESI): [MH]+ = 475. 1H NMR (CDCl3):
d 7.56–7.52 (br, 1H); 7.35–7.24 (m, 7H); 7.03 (t, 1H, J = 8 Hz);
3.86 (d, 1H, J = 13.2 Hz); 3.50 (d, 1H, J = 13.2 Hz); 3.28–3.17 (m,
2H); 3.07–3.03 (m, 3H); 2.69–2.64 (m, 2H); 2.47–2.34 (m, 3H);
2.27–2.19 (m, 2H); 2.06–2.01 (m, 2H); 1.92–1.90 (m, 2H); 1.75–
1.72 (m, 4H); 1.56 (d, J = 12.8 Hz). 13C NMR (CDCl3): 174.71;
138.67; 130.41; 128.76 (2C); 128.57 (2C); 127.78 (2C); 127.35
(2C); 67.52; 59.95 (2C); 56.41; 54.64; 54.10; 40.57; 37.43 (2C);
mp = 153–155 °C. MS (ESI): [MH]+ = 278. 1H NMR (CDCl3):
d
12.20 (s, 2H); 7.41 (t, 2H, J = 8 Hz); 7.23 (t, 1H, J = 8 Hz); 3.43–
3.32 (m, 1H); 2.92–2.70 (m, 4H). IR: 2918.07; 1713.09; 1560.58;
1438.02; 1310.02; 1226.35; 1202.58; 1085.05; 903.48; 779.13.
5.2.27. 4-(2,6-Dichloro-phenyl)-piperidin-2,6-dione (32)
Compound 31 (4.026 g, 15.60 mmol) was dissolved in NH4OH
(40 mL). The solvent was evaporated under vacuum and the solid
residue was heated at 200 °C for 6 h. At the crude product was
added DCM (150 mL) and was washed twice with Na2CO3
0.1 M. The organic layer was dried and concentrated in vacuum
to obtain a crude product, which was crystallized from ethyl
ether to give 32 with a quantitative yield. mp = 155–157 °C. MS
(ESI): [MH]+ = 259. 1H NMR (CDCl3): d 8.20 (br, 1H); 7.36–7.13
(m); 4.43–4.30 (m, 1H); 3.60 (dd, 2H, J = 18, 13.5 Hz); 2.69 (dd,
2H, J = 17.7, 4.6 Hz). IR: 3208.19; 3078.49; 2921.50; 2385.98;
2342.76; 1696.60; 1560.07; 1437.89; 1361.16; 1271.72;
1150.48; 1079.12; 770.20; 731.23.
30.78 (2C); 27.64; 26.80; 24.14 (2C). ½a D20
ꢃ
= +27 (c = 0.1 g/100 mL,
chloroform). Anal. (C26H33Cl2N3O) C, H, N, Cl.
5.2.31. (L)-1-Benzyl-pirrolidin-2-carboxilic acid {3-[4-(2,
6-dichloro-phenyl)-piperidin-1-yl]-propyl}-amide (36)
N-Bn-Pro and compound 34 were condensed in the same experi-
mental condition as for Comp 24. yielding 36 (48 mg, 0.101 mmol,
yield 35%). MS (ESI): [MH]+ = 475. 1H NMR (CDCl3): d 7.53 (br, 1H);
7.33–7.25 (m, 7H); 7.03 (t, 1H, J = 8 Hz); 3.86 (d, 1H, J = 13.2 Hz);
3.50 (d, 1H, J = 13.2 Hz); 3.30–3.17 (m, 3H); 3.07–3.03 (m, 2H);
2.67–2.64 (m, 2H); 2.42–2.32 (m, 3H); 2.24–2.19 (m, 2H); 2.07–2.01
(m, 3H); 1.88–1.85 (m, 2H); 1.78–1.67 (m, 3H); 1.57–1.54 (m, 2H).
13C NMR (CDCl3): 174.69; 138.69; 130.40; 128.73 (2C); 128.57 (2C);
127.74 (2C); 127.35 (2C); 67.53; 59.95 (2C); 56.51; 54.68; 54.09;
40.64; 37.47 (2C); 30.78 (2C); 27.74; 26.86; 24.14 (2C). ½a D20
= ꢀ27
ꢃ
5.2.28. 4-(2,6-Dichloro-phenyl)-piperidine (33)
Compound 32 (4 g, 15.5 mmol) was suspended, under argon
(c = 0.1 g/100 mL, chloroform). Anal. (C26H33Cl2N3O) C, H, N, Cl.
*
atmosphere, in anhydrous THF (150 mL) and (CH3)2S BH3
(14.9 mL, 155 mmol) added at 0 °C under stirring. The reaction
was heated at reflux overnight. The solution was cooled at 0 °C,
HCl 10% (66.6 mL) was added until pH of the solution was strongly
acidic and the solution was left at reflux for 4 h. The reaction mix-
ture was cooled again at 0 °C, basified with NaOH 2 N and moni-
tored by TLC (EtOAc/light petroleum 3:2). The THF was
evaporated under vacuum and the aqueous layer was extracted
with ethyl acetate. The organic layers were combined, dried and
concentrated under vacuum to obtain the crude product, which
was crystallized with ethyl ether to give 33 (1.23 g, 5.348 mmol,
yield 35%). mp = 210–215 °C. MS (ESI): [MH]+ = 231. 1H NMR
5.2.32. 4-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-
1-carboxylic acid tert-butyl ester (38)
To a stirred solution of compound 37 (1 g, 4.60 mmol) in DCM
(50 mL) at 0 °C, a catalytical amount of DMAP and (Boc)2O
(1.57 g, 6.9 mmol) were added. After 6 h at room temperature,
water (100 mL) was added and the organic layer washed with citric
acid 10% (10 mL ꢄ 3), NaHCO3 saturated solution (10 mL ꢄ 3) and
brine (10 mL ꢄ 3). The organic phase was dried and concentrated
in vacuo to give compound 38 in quantitative yield pure enough
to be used in the next step without further purification.
(CDCl3):
d 9.77 (br, 1H); 7.28–7.25 (m, 2H); 7.089 (t, 1H,
5.2.33. 1-Ethyl-3-piperidin-4-yl-1,3-dihydro-benzoimidazol-2-
one (39)
To a suspension of sodium hydride (92 mg, 3.79 mmol) in anhy-
drous DMF (5 mL), compound 38 was added dropwise at 0 °C. After
J = 8 Hz); 3.78–3.72 (m, 1H); 3.67–3.64 (m, 2H); 3.13–3.02 (m,
4H); 1.82–1.79 (m, 2H). 13C NMR (CDCl3): 137.08; 135.21;
130.67; 128.65 (2C); 44.73 (2C); 38.30 (2C); 24.80 (2C).

C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
5093
30 minutes, ethyl bromide (413 mg, 3.79 mmol) was added, and
the reaction mixture was allowed to stir at room temperature
overnight. The DMF was removed in vacuo and the salts were dis-
solved in water, the aqueous layer was extracted with EtOAc
(25 mL ꢄ 3), dried and concentrated in vacuo. The crude material
was dissolved in 5 mL of TFA and stirred for 1 h; after this time,
the TFA was evaporated and the solution was basified with NaOH
20% until pH 12. The aqueous layer was extracted twice with EtOAc
(30 mL each), concentrated to dryness and purified by flash chro-
matography using EtOAc/light petroleum/NH4OH: 2:1:0.3 as elu-
ent to give 0.84 g of desired compound (yield 75%). MS (ESI):
[MH]+ = 246.3 1H NMR (CDCl3): d 7.25 (m, 1H); 7.07–7.00 (m,
3H); 4.43 (m, 1H); 3.98–3.87 (q, 2H, J = 6 Hz); 3.22 (br s, 2H);
2.79 (dt, 2H, J = 15.6 Hz, J = 2.4 Hz); 2.37–2.30 (dq, 2H, J = 8.4 Hz,
J = 4.2 Hz); 1.85 (bs, 2H); 1.32 (t, 3H, J = 7.4 Hz).
via homogenisation and centrifugation at 13,500 rpm for 10 min at
4 °C. The final protein concentration was determined according to
Lowry.27
5.4. Receptor binding
[3H]N/OFQ binding experiments. 5
protein was incubated in 0.5 mL volumes of Tris-HCl (50 mM) buf-
fer supplemented with 10 M peptidase inhibitors (amastatin,
lg of CHOhNOP homogenate
l
bestatin, captopril and phosphoramidon), 0.5% bovine serum albu-
min (BSA), increasing concentrations of compounds under study
and approximately 200 pM [3H]-N/OFQ. Total radiolabel bound
was ꢅ10%. Non-specific binding was determined in the presence
of 1 lM unlabeled N/OFQ. In all experiments Comp 24 was in-
cluded as a reference ligand. Reactions were incubated for 1 h at
room temperature and terminated by vacuum filtration (Brandel
Harvester) through Whatman GF/B filters soaked in 0.5% polyethy-
lenimine (PEI). Radioactivity was determined after 8 h extraction
in scintillation cocktail. [3H]-Diprenorphine binding experiments.
5.2.34. 1-[1-(3-Amino-propyl)-piperidin-4-yl]-3-ethyl-1,3-di-
hydro-benzoimidazol-2-one (40)
To a stirred solution of 39 (92 mg, 0.375 mmol) in DMF (5 mL),
N-Boc-3-bromo-propyl-amine (106 mg, 0.447 mmol) and K2CO3
(103 mg, 0.750 mmol) were added. After 1 h at 60 °C, most of the
solvent was removed and the residue diluted with EtOAc and
washed twice with NH4Cl saturated solution (30 mL each) and
brine (30 mL). The organic layer was dried and evaporated under
reduced pressure. The crude material was treated at 0 °C with
TFA (5 mL) for 1 h and then treated with NaOH 2 N until pH 12–
14. The aqueous phase was extracted several times with EtOAc
and the organic phases were combined, dried and evaporated un-
der vacuum to give compound 40 in 80% yield. The product was en-
ough pure to be used in the next step without further purification.
50 lg (CHOhMOP), 25 lg (CHOhDOP) and 40 lg (CHOhKOP) membrane
protein were incubated in 0.5 ml buffer containing Tris-HCl
(50 mM) pH 7.4, BSA (0.5%), ꢆ0.7 nM [3H]Diprenorphine and
increasing concentrations of naloxone and compound 35. Non-spe-
cific binding was determined in the presence of 10 lM naloxone.
Reactions were incubated at room temperature for 1 h, harvesting
and determination of radioactivity were as for [3H]N/OFQ binding.
5.5. [35S]GTP
cS binding
20 g of CHOhNOP membranes were incubated in 0.5 mL buffer
l
containing Tris–HCl (50 mM), EGTA (0.2 mM) MgCl2 (1 mM), NaCl
5.2.35. 1-Benzyl-pyrrolidine-2-carboxylic acid {3-[4-(3-ethyl-2-
oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidin-1-yl]-propyl}-
amide (41)
(100 mM), bacitracin (0.15 mM) peptidase inhibitors (as above),
GDP (100
35 was pre-incubated for 15 min at 30 °C. Non-specific binding
was determined in the presence of unlabeled 10 M GTP S. The
l cS. Compound
M) and approximately 150 pM [35S]GTP
N-Bn-D-Pro and compound 40 were condensed in the same
l
c
experimental condition as for Comp 24 yielding 41 in 24% yield
after purification by flash chromatography (EtOAc/MeOH/NH4OH,
9.5:0.5:0.3). MS (ESI): [MH]+ = 490. 1H NMR (CDCl3): d 7.58–7.52
(m, 1H); 7.29–7.25 (m, 5H); 7.04–7.01 (m, 4H); 4.58–4.42 (m,
1H); 3.92 (q, 2H, J = 7.2 Hz); 3.866–3.560 (dd, a–b sistem, 2H,
J = 13 Hz); 3.24–3.09 (m, 5H); 2.59–2.19 (m, 9H); 1.87–1.72 (m,
7H); 1.315 (t, 3H, J = 7.2 Hz). 13C NMR (CDCl3): 175.07; 153.40;
138.58; 129.14; 128.71; 128.586; 127.87; 127.39; 121.06;
120.98; 109.80; 107.67; 67.41; 59.95; 55.44; 54.17; 53.00; 52.92;
49.83; 36.93; 35.95; 30.77; 28.11; 26.36; 24.13; 13.62. Anal.
(C29H39N5O2) C, H, N.
reaction was incubated for 1 h with increasing concentration of
N/OFQ at 30 °C with gentle shaking and terminated by filtration
through Whatman GF/B filters using a Brandel Harvester. PEI was
not used.
5.6. Calcium mobilization experiments
CHOhMOP, CHOhDOP, CHOhKOP and CHOhNOP stably expressing the
Gaqi5 protein were seeded at a density of 40,000 cells/well into 96-
well black, clear-bottom plates. After 24 h incubation the cells
were loaded with medium supplemented with 2.5 mM of proben-
ecid, 3
0.01% pluronic acid, for 30 min at 37 °C. Afterwards, the loading
solution was aspirated and 100 L/well of assay buffer: Hank’s Bal-
anced Salt Solution (HBSS) supplemented with 20 mM HEPES,
2.5 mM probenecid and 500 M Brilliant Black (Aldrich) was
lM of the calcium sensitive fluorescent dye Fluo-4 AM and
5.3. Cell culture and cell membrane preparation
l
CHOhNOP cells were cultured in DMEM and Hams F-12 (1:1)
supplemented with 5% foetal calf serum, penicillin (100 IU/mL),
l
Streptomycin (100
tures were further supplemented with geneticin (G418, 200
mL) and Hygromycin B (200
g/mL) as described previously.25
l
g/mL) and Fungizone (2.5
l
g/mL). Stock cul-
added. Stock solutions (1 mM) of ligands were prepared in distilled
water and stored at ꢀ20 °C. Serial dilutions of ligands for experi-
mental use were made in HBSS/HEPES (20 mM) buffer (containing
0.02% BSA fraction V). After placing both plates (cell culture and
compound plate) into the FlexStation II (Molecular Device, Union
City, CA), fluorescence changes were measured at room tempera-
lg/
l
CHO cell lines stably expressing the human MOP, DOP, KOP and
26
NOP receptors and the C-terminally modified Gaqi5 were gener-
ated as described previously8 and maintained in Dulbecco Mini-
mum Essential Medium (DMEM) and Hams F-12 (1:1)
ture. On-line additions were carried out in a volume of 50 lL/well.
supplemented with 10% fetal bovine serum, 2 mM
L-glutamine,
200 g/mL Geneticin and 100 g/mL Hygromicin B. Cells were cul-
l
l
5.7. Electrically stimulated isolated tissues
tured at 37 °C in 5% carbon dioxide humidified air, and used when
confluent.
Tissues were taken from male Swiss mice (30–35 g), albino gui-
nea pigs (300–350 g) and Sprague-Dawley rats (300–350 g). The
mouse and the rat vas deferens and the guinea pig ileum were pre-
pared as previously described.28 Tissues were continuously stimu-
lated through two platinum ring electrodes with supramaximal
For binding experiments membranes were prepared from
freshly harvested cell suspensions in Tris–HCl (50 mM), Mg++
(5 mM) pH 7.4 ([3H]N/OFQ binding experiments) or in Tris–HCl
(50 mM), EGTA (0.2 mM) pH 7.4 ([35S]GTP
cS binding experiments)

5094
C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
rectangular pulses of 1 ms duration and 0.05 Hz frequency. The
electrically evoked contractions (twitches) were measured isoton-
ically with a strain gauge transducer (Basile 7006, UgoBasile s.r.l.,
Varese, Italy) and recorded with the PC based acquisition system
Power Lab (ADInstrument, USA).
of the maximal possible effect. Antagonist potencies have been
evaluated (i) using pA2 derived from the classical Schild protocol
in [35S]GTP
cS binding and mouse vas deferens experiments (Fig. 1
and Table 2), (ii) using pKb derived from the Gaddum Schild
equation:
Following an equilibration period of 60 min, the contractions
induced by electrical field stimulation were stable. At this time,
cumulative concentration–response curves to N/OFQ were per-
formed (0.5 log unit steps) in the absence or presence of compound
35 (15 min pre-incubation time). For selectivity studies, in some
experiments the DOP selective agonist DPDPE was used in the
mouse vas deferens and the MOP selective agonist dermorphin
was used in the guinea pig ileum.
Kb ¼ ððCR ꢀ 1Þ=½antagonistꢃÞ
assuming a slope value equal to unity, where CR indicate the ratio
between agonist potency in the presence and in the absence of
the antagonist, in rat vas deferens and guinea pig ileum experi-
ments (Table 2). Finally, (iii) using pKb values derived from the fol-
lowing equation:32
n
1=n
Kb ¼ IC50=ð½2 þ ð½Aꢃ=EC50Þ ꢃ ꢀ 1
5.8. Mouse tail withdrawal assay
where IC50 is the concentration of antagonist that produces 50%
inhibition of the agonist response, [A] is the concentration of the
agonist, EC50 is the concentration of agonist producing a 50% max-
imal response and n is the Hill coefficient of the concentration re-
sponse curve to the agonist in inhibition response experiments
measuring calcium mobilization (Tables 2 and 4).
Male Swiss albino mice weighing 25–30 g were used. Animals
were handled according to guidelines published in the European
Communities Council directives (86/609/EEC) and Italian national
regulations (D.L. 116/92). They were housed in 425 ꢄ 266 ꢄ 155-
mm cages (Techniplast, Milan, Italy), fifteen animals/cage, under
standard conditions (22 °C, 55% humidity, 12-h light/dark cycle,
light on at 7:00 am) with food (MIL, standard diet; Morini, Reggio
Emilia, Italy) and water ad libitum for at least 5 days before exper-
Acknowledgments
This work was supported by funds from the University of Ferr-
ara (FAR grants to SS and GC) and the Italian Ministry of the Uni-
versity (PRIN and FIRB grants to RG).
iments began. Each mouse was used only once. Icv (2 ll/mouse) or
i.t (5 ll/mouse) injections were given according to the procedure
described by Laursen and Belknap29 and Hylden and Wilcox30
respectively.
,
Supplementary data
All experiments were started at 10:00 am and performed
according to the procedure described previously in detail.22 Briefly,
the mice were placed in a holder and the distal half of the tail was
immersed in water at 48 °C. Withdrawal latency time was mea-
sured by an experienced observer blind to drug treatment. A cut-
off time of 20 s was chosen to avoid tissue damage. For each exper-
iment sixteen mice were used by randomly assigning four animals
to each treatment group. The experiment was repeated four times;
therefore, each experimental point shown in Figure 2 is the mean
of the results obtained in 16 mice. Tail-withdrawal latency was
determined immediately before and 5, 15, 30, and 60 min after
icv or i.t. injection of vehicle (saline) or N/OFQ (1 nmol). Compound
35 (10 mg/kg) or its vehicle (2% DMSO and 10% encapsin) were gi-
ven i.p. 30 min before icv injection. Increased and decreased tail
withdrawal latencies compared with baseline indicated antinoci-
ceptive and pronociceptive effects, respectively.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.05.068.
References and notes
1. Meunier, J. C.; Mollereau, C.; Toll, L.; Suaudeau, C.; Moisand, C.; Alvinerie, P.;
Butour, J. L.; Guillemot, J. C.; Ferrara, P.; Monserrat, B.; Mazarguil, H.; Vassart,
G.; Parmentier, M.; Costentin, J. Nature 1995, 377, 532.
2. Reinscheid, R. K.; Nothacker, H. P.; Bourson, A.; Ardati, A.; Henningsen, R. A.;
Bunzow, J. R.; Grandy, D. K.; Langen, H.; Monsma, F. J., Jr.; Civelli, O. Science
1995, 270, 792.
3. Lambert, D. G. Nat. Rev. Drug Disc. 2008, 7, 694.
4. Jenck, F.; Wichmann, J.; Dautzenberg, F. M.; Moreau, J. L.; Ouagazzal, A. M.;
Martin, J. R.; Lundstrom, K.; Cesura, A. M.; Poli, S. M.; Roever, S.; Kolczewski, S.;
Adam, G.; Kilpatrick, G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 4938.
5. Ozaki, S.; Kawamoto, H.; Itoh, Y.; Miyaji, M.; Azuma, T.; Ichikawa, D.; Nambu,
H.; Iguchi, T.; Iwasawa, Y.; Ohta, H. Eur. J. Pharmacol. 2000, 402, 45.
6. Zaratin, P. F.; Petrone, G.; Sbacchi, M.; Garnier, M.; Fossati, C.; Petrillo, P.;
Ronzoni, S.; Giardina, G. A.; Scheideler, M. A. J. Pharmacol. Exp. Ther. 2004, 308,
454.
5.9. Data analysis and terminology
7. Goto, Y.; Arai-Otsuki, S.; Tachibana, Y.; Ichikawa, D.; Ozaki, S.; Takahashi, H.;
Iwasawa, Y.; Okamoto, O.; Okuda, S.; Ohta, H.; Sagara, T. J. Med. Chem. 2006, 49,
847.
All data are expressed as means standard error of the mean
(sem) of n experiments. For potency values confidence limits 95%
were indicated. Data have been analyzed statistically using one-
way ANOVA followed by Dunnett’s test for multiple comparisons
or the Student’s t test. In competition binding studies (Tables 2
and 3), the log concentration of competitor producing 50% inhibi-
tion of specific binding (pIC50) was corrected for the competing
mass of radiolabel according to Cheng and Prusoff31 to yield pKi
values. KD values for [3H]N/OFQ in CHOhNOP was 83 pM and for
[3H]diprenorphine in MOP, DOP and KOP were 125, 323, and
134 pM, respectively.
8. Fischetti, C.; Camarda, V.; Rizzi, A.; Pela’, M.; Trapella, C.; Guerrini, R.;
McDonald, J.; Lambert, D. G.; Salvadori, S.; Regoli, D.; Calo, G. Eur. J.
Pharmacol. 2009, 614, 50.
9. Guerrini, R.; Calo, G.; Bigoni, R.; Rizzi, A.; Varani, K.; Toth, G.; Gessi, S.; Hashiba,
E.; Hashimoto, Y.; Lambert, D. G.; Borea, P. A.; Tomatis, R.; Salvadori, S.; Regoli,
D. J. Med. Chem. 2000, 43, 2805.
10. Guerrini, R.; Calo, G.; Lambert, D. G.; Carra, G.; Arduin, M.; Barnes, T. A.;
McDonald, J.; Rizzi, D.; Trapella, C.; Marzola, E.; Rowbotham, D. J.; Regoli, D.;
Salvadori, S. J. Med. Chem. 2005, 48, 1421.
11. Neises, B.; Steglich, W. Angew. Chem., Int. Ed. Engl. 1978, 17, 522.
12. Sudau, A.; Muench, W.; Bats, J. W.; Nubbemeyer, U. Eur. J. Org. Chem. 2002,
2002, 3304.
13. Blackburn, L.; Kanno, H.; Taylor, R. J. K. Tetrahedron Lett. 2003, 44, 115.
14. Wadsworth, W. S.; Emmons, W. D. J. Am. Chem. Soc. 1961, 83, 1733.
15. Cremonesi, G.; Dalla Croce, P.; Fontana, F.; Forni, A.; La Rosa, C. Tetrahedron:
Asymmetry 2007, 18, 1667.
16. Corey, E. J.; Chaykovsky, M. J. Org. Chem. 1962, 84, 866.
17. Corey, H. S.; McCormick, J. R. D.; Swensen, W. E. J. Org. Chem. 1964, 86, 1884.
18. Shinkai, H.; Ito, T.; Iida, T.; Kitao, Y.; Yamada, H.; Uchida, I. J. Med. Chem. 2000,
43, 4667.
19. Zaveri, N.; Jiang, F.; Olsen, C.; Polgar, W.; Toll, L. Aaps J. 2005, 7, E345.
20. Camarda, V.; Fischetti, C.; Anzellotti, N.; Molinari, P.; Ambrosio, C.; Kostenis, E.;
Regoli, D.; Trapella, C.; Guerrini, R.; Salvadori, S.; Calo, G. Naunyn-
Schmiedebergs Arch. Pharmacol. 2009, 379, 599.
[
35S]GTP
ratio between agonist-stimulated [35S]GTP
cS data are expressed as a stimulation factor that is, the
cS specific (minus NSB)
binding and basal specific binding. Calcium mobilization data are
expressed as fluorescence intensity units (FIU) in percent over
the baseline. Isolated tissue data are expressed as percent of the
twitch response to electrical field stimulation.
Agonist potencies are given as pEC50 = the negative logarithm to
base 10 of the molar concentration of an agonist that produces 50%

C. Trapella et al. / Bioorg. Med. Chem. 17 (2009) 5080–5095
5095
21. Zeilhofer, H. U.; Calo, G. J. Pharmacol. Exp. Ther. 2003, 306, 423.
22. Calo, G.; Rizzi, A.; Marzola, G.; Guerrini, R.; Salvadori, S.; Beani, L.; Regoli, D.;
Bianchi, C. Br. J. Pharmacol. 1998, 125, 373.
23. Nazzaro, C.; Rizzi, A.; Salvadori, S.; Guerrini, R.; Regoli, D.; Zeilhofer, H. U.; Calo,
G. Peptides 2007, 28, 663.
24. Rizzi, A.; Gavioli, E. C.; Marzola, G.; Spagnolo, B.; Zucchini, S.; Ciccocioppo, R.;
Trapella, C.; Regoli, D.; Calo, G. J. Pharmacol. Exp. Ther. 2007, 321, 968.
25. McDonald, J.; Calo, G.; Guerrini, R.; Lambert, D. G. Naunyn-Schmiedeberg’s Arch.
Pharmacol. 2003, 367, 183.
26. Coward, P.; Chan, S. D.; Wada, H. G.; Humphries, G. M.; Conklin, B. R. Anal.
Biochem. 1999, 270, 242.
27. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193,
265.
28. Bigoni, R.; Giuliani, S.; Calo, G.; Rizzi, A.; Guerrini, R.; Salvadori, S.; Regoli, D.;
Maggi, C. A. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1999, 359, 160.
29. Laursen, S. E.; Belknap, J. K. J. Pharmacol. Methods 1986, 16, 355.
30. Hylden, J. L.; Wilcox, G. L. Eur. J. Pharmacol. 1980, 17, 313.
31. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
32. Kenakin, T. A Pharmacology Primer; Elsevier Academic Press: San Diego, 2004.
33. Vergura, R.; Valenti, E.; Hebbes, C. P.; Gavioli, E. C.; Spagnolo, B.; McDonald, J.;
Lambert, D. G.; Balboni, G.; Salvadori, S.; Regoli, D.; Calo, G. Peptides 2006, 27,
3322.