Synthesis and Structure-Activity Relationships of Triazaspirodecanone Derivatives as Nociceptin/Orphanin FQ Receptor Ligands

Article abstract of DOI:10.1111/cbdd.12505

Several spiroxatrine derivatives were synthesized and evaluated as potential NOP receptor ligands. Structural modifications of the 1,4-benzodioxane moiety of spiroxatrine have been the focus of this research project. The structure-activity relationships that emerged indicate that the presence of an H-bond donor group (hydroxyl group) is more favorable for NOP activity when it is positioned α with respect to the CH₂ linked to the 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one portion. Moreover, cis diastereoisomers of the hydroxyl derivatives4 and 22 show a moderately higher degree of stereoselectivity than trans isomers. In particular, the spiropiperidine derivative cis-4 has submicromolar agonistic activity, and it will be the reference compound for the design and synthesis of new NOP agonists.

Full text of DOI:10.1111/cbdd.12505

Chem Biol Drug Des 2015; 86: 447–458
Research Article
Synthesis and Structure–Activity Relationships of
Triazaspirodecanone Derivatives as Nociceptin/
Orphanin FQ Receptor Ligands
1
1
Sandra Corrado , Umberto M. Battisti , Claudia
several biological functions such as learning and memory,
attention, emotions, homeostasis, neuroendocrine secre-
tions, anxiety, drug abuse, cardiovascular control, immu-
nity, and motor activity (5–11). The endogenous ligand of
ORL-1 receptor is a 17-amino acid neuropeptide which
was isolated from the brain and named nociceptin/orphanin
FQ (N/OFQ) (1,2). The ORL-1 receptor was then renamed
nociceptin/orphanin FQ peptide (NOP) receptor. The
endogenous peptide ligand inhibits synaptic transmission in
the CNS and thereby contributes to a reduction in respon-
siveness to stress and evokes anxiolytic effects (12). There-
1
1
2
Sorbi , Annalisa Tait , Davide Malfacini , Valeria
Camarda , Girolamo Cal oꢀ and Livio Brasili *
2
2
1,
1
Department of Life Sciences, University of Modena &
Reggio Emilia, Via G. Campi 183, 41125 Modena, Italy
Department of Medical Sciences, Section of
Pharmacology and National Institute of Neuroscience,
University of Ferrara, 44121 Ferrara, Italy
2
*
Corresponding author: Livio Brasili, livio.brasili@unimore.it
Several spiroxatrine derivatives were synthesized and
evaluated as potential NOP receptor ligands. Structural
modifications of the 1,4-benzodioxane moiety of spir-
oxatrine have been the focus of this research project.
The structure–activity relationships that emerged indi-
cate that the presence of an H-bond donor group
fore, NOP selective ligands may represent
a novel
therapeutic approach for the treatment of neuronal dys-
function, anxiety, and several other CNS pathologies (13).
In addition, NOP ligands may be new promising tools for
the treatment of acute and chronic pain, possibly by gener-
ating drugs with a lower side-effect profile (3–5). In the last
decade, several classes of NOP agonists have been
reported; they can be divided into peptide and non-peptide
ligands. Different chemical classes of non-peptide ligands
have been discovered; among them, spiropiperidine ana-
logues have shown relatively higher NOP-binding affinities
(
hydroxyl group) is more favorable for NOP activity
when it is positioned a with respect to the CH linked
2
to the 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one por-
tion. Moreover, cis diastereoisomers of the hydroxyl
derivatives 4 and 22 show a moderately higher degree
of stereoselectivity than trans isomers. In particular,
the spiropiperidine derivative cis-4 has submicromolar
agonistic activity, and it will be the reference com-
pound for the design and synthesis of new NOP agon-
ists.
(14). Spiroxatrine (1, Figure 1), an a2 adrenergic and 5-
HT1A antagonist, due to its good affinity for NOP receptor,
has been extensively studied to develop ORL-1 selective
ligands (15,16). These studies led to the development of
the most extensively studied agonist Ro 64-6198 and to
other several triazaspirodecanone derivatives (17–19).
Key words: agonists, NOP ligands, ORL-1, spiroxatrine, tri-
azaspirodecanone
Herein, SAR studies on triazaspirodecanone derivatives as
potential NOP agonists are reported. In particular, we
explored possible substitution of the lipophilic moiety
attached to the piperidine nitrogen by a ‘1-carbon’ linker.
This kind of studies could represent a useful tool for the
development of agonists as the presence of a 1-carbon
linker could influence the intrinsic activity of NOP receptor
Received 17 July 2014, revised 17 October 2014 and
accepted for publication 15 December 2014
The superfamily of G protein-coupled receptors (GPCRs)
still represents the majority of current drug targets. Beside
the three classical opioid receptors, namely l,d and j, a
new rhodopsin-type GPCR, belonging to the opioid family,
has been discovered in 1994 and named opioid receptor-
like 1 (ORL-1) (1,2). The similarity of postreceptor transduc-
tion and the high degree of structural homology (~60%)
with the classical opioid receptors suggested that this
receptor could be involved in pain modulation (3,4). More-
over, due to its wide distribution, it has been suggested
that the ORL-1 receptor may play an important role in many
other central nervous system (CNS) processes and in
(19). These investigations were focused on the structural
variations of the 1,4-benzodioxane moiety of spiroxatrine.
Methods and Materials
Chemistry
All reagents, solvents, and other chemicals were used as
purchased from Sigma-Aldrich without further purification
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12505
447

Corrado et al.
Biology
O
O
O
N
N/OFQ and dermorphin used in this study were prepared
and purified as previously described (20,21). All cell culture
media and supplements were from Invitrogen (Paisley,
UK). Dynorphin A, DPDPE, and J-113397 were from To-
cris Bioscience (Bristol, UK), while other reagents were
from Sigma Chemical Co. (Poole, UK) or E. Merck (Darms-
tadt, Germany) and were of the highest purity available. N/
OFQ, dermorphin, dynorphin A, and DPDPE were solubi-
lized in bidistilled water at a final concentration of 1 mM.
All the tested compounds were solubilized in dimethyl sulf-
oxide at a final concentration of 10 mM. Stock solutions of
ligands were stored at ꢀ20 °C. The successive dilutions
were made in HBSS/HEPES (20 mM) buffer (containing
0.005% BSA fraction V).
NH
N
1
Figure 1: Spiroxatrine (1).
unless otherwise specified. Air- or moisture-sensitive reac-
tants and solvents were employed in reactions carried out
under nitrogen atmosphere unless otherwise noted. Flash
column chromatography purifications (medium pressure
liquid chromatography) were carried out using Merck silica
gel 60 (230–400 mesh; ASTM). The purity of compounds
was determined by elemental analysis (C, H, N) that was per-
formed on a Carlo Erba 1106 Analyzer in the Microanalysis
Laboratory of the Life Sciences Department of Universit aꢀ de-
gli Studi di Modena e Reggio Emilia. Melting points were
determined with a Stuart SMP3, and they are uncorrected.
The structures of all isolated compounds were established
by nuclear magnetic resonance (NMR) and mass spectrom-
Calcium mobilization assay
Chinese hamster ovary (CHO) cells stably co-expressing
human recombinant NOP, MOP, or KOP receptors and
the C-terminally modified Gaqi5 and CHO cells stably co-
expressing the human recombinant DOP receptor and the
GaqG66Di5 chimeric protein were generated as previously
described (22).
1
13
etry. H and C NMR (1D and 2D experiments) spectra
were recorded on a DPX-200 Avance (Bruker, Faellanden,
Switzerland) spectrometer at 200 MHz ( H-NMR) and on a
DPX-400 Avance (Bruker) spectrometer at 400 MHz ( H-
NMR) and 100 MHz ( C NMR). Chemical shifts are
The cells were cultured in culture medium consisting of
Dulbecco’s MEM/HAMS F12 (50/50) supplemented with
10% fetal calf serum, penicillin (100 IU/mL), streptomycin
(100 mg/mL), geneticin (G418; 200 lg/mL), and hygro-
mycin B (100 lg/mL). Cell cultures were kept at 37 °C in
1
1
13
1
expressed in d (ppm). H NMR chemical shifts are relative to
2
5% CO /humidified air. When confluence was reached
1
3
tetramethylsilane (TMS) as internal standard.
chemical shifts are relative to TMS at d 0.0 or to the C sig-
nal of the solvent: CDCl d 77.04, CD OD d 49.8, DMSO-d
d 39.5. H NMR data are reported as follows: chemical shift,
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, mul-
tiplet; and br, broadened), number of protons, coupling con-
stants (Hz) and assignment (chr = chromane, chroman-4-
C NMR
(3–4 days), cells were subcultured as required using tryp-
sin/EDTA and used for experimentation. Cells were
seeded at a density of 50 000 cells/well into 96-well
black, clear-bottom plates. After 24-h incubation, the
cells were loaded with medium supplemented with
2.5 mM probenecid, 3 lM of the calcium-sensitive fluores-
cent dye Fluo-4 AM, and 0.01% pluronic acid, for 30 min
at 37 °C. Afterward, the loading solution was aspirated,
and 100 lL/well of assay buffer: Hank’s balanced salt
solution (HBSS) supplemented with 20 mM HEPES,
2.5 mM probenecid, and 500 lM brilliant black was
added. After placing both plates (cell culture and com-
pound plate) into the FlexStation II, fluorescence changes
were measured.
13
3
3
6
1
one,
chroman-4-ol,
chromene,
chromen-4-one;
Ph = phenyl; ox. ac. = oxalic acid; tasd = 1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one; tetr = tetralone, tetralinol;
thn = tetrahydronaphthalene; and Ts = tosyl). H- H corre-
lation spectroscopy (COSY), H- C heteronuclear single-
quantum coherence (HSQC) and heteronuclear multiple-
bond connectivity (HMBC) NMR 2D experiments were
1
1
1
13
1
1
1
13
recorded for determination of H- H and H- C correlations,
respectively. Mass spectra were obtained on a hybrid Q-TOF
mass spectrometer (PE SCIEX-QSTAR) using electrospray
ionization mode (HRMS (ESI), ion voltage of 4800 V). The
HPLC experimental conditions of the HPLC–MS system
were as follows: flow rate 5 mL/min, sample solution (100–
Data analysis and terminology
All data are expressed as means ꢁ standard error of the
mean (SEM) of at least three experiments performed in
duplicate. For potency values, 95% confidence limits were
indicated. Calcium mobilization data are expressed as fluo-
rescence intensity units (FIU) in percent over the baseline.
Agonist potencies are given as pEC50 corresponding to
the negative logarithm of the concentration of the agonist
that produces 50% of the maximal effect. Concentration–
response curve to agonists was fitted with the following
equation:
1
000 pmol/mL) of the selected compound with 0.1% acetic
acid, mobile phase consisting of methanol (50%) and water
50%). All compounds were synthesized as reported in
(
Schemes 1–5. Full characterization as well as detailed
experimental procedures for all compounds is reported in
Appendix S1. The oxalate salts of all tested compounds
were used for the pharmacological evaluations.
4
48
Chem Biol Drug Des 2015; 86: 447–458

Nociceptin/Orphanin FQ Receptor Ligands
O
O
N+
(b)
–
I
2
a
(
c)
82%
95%
O
O
O
O
O
O
NH
N
(
a)
N
(b)
0%
8
5%
O
1
N
3
2
8
0%
(d)
OH
O
O
O
NH
N
N
O
N
(
f)
(e)
4%
O
NH
O
N
12%
N
NH
2
N
6
5
cis-4
trans-4
Scheme 1: Synthesis of 3 and its derivatives 4, 5 and 6. Reagents and conditions: (a) (HCHO)
n
, NH(CH
3
)
2
ꢂHCl, HCl, EtOH, reflux; (b) 1-
4
phenyl-1,3,8-triaza-spiro[4.5]decan-4-one, Na CO , acetonitrile, reflux; (c) CH I, diethyl ether, 0 °C to r.t.; (d) NaBH
2
3
3
, MeOH, 0 °C to r.t.;
3 4 2
(e) H PO , 60 °C; (f) H , Pd/C, MeOH, r.t.
O
O
OH
O
NH
(
a)
(b)
37%
OH
(c)
OTs
(d)
N
7
0%
O
52%
43%
O
O
O
O
N
6
a
6b
6c
6
Scheme 2: Alternative synthesis of 6. Reagents and conditions: (a) HCOOC
2
H
5
, NaOtBu, diethyl ether, 0 °C to r.t.; (b) BF
3
ꢂetherate,
2 3
BH
3
3 2
ꢂN(t-Bu) , CH Cl
2
, ꢀ78 °C to r.t.; (c) TsCl, N(Et)
3
, CH
2
2
Cl , 0 °C to r.t.; (d) 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one, Na
CO ,
acetonitrile, reflux.
O
O
O
O
O
O
O
O
(
a)
(b)
OH
(c)
(d)
N
Cl
9
9%
64%
99%
OH
98%
NH
O
OH
O
N
7
8
9
10
Scheme 3: Synthesis of 10. Reagents and conditions: (a) HCOOC
H
2 5
, NaOMe, THF, 0 °C to r.t.; (b) CH
2
O 37%, NaOAc, HCl, acetone,
r.t.; (c) SOCl , CH Cl , 0 °C to r.t.; (d) 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one, K
2
2
2
2
CO , KI, acetonitrile, reflux.
3
Emaxꢀbaseline
Agonist efficacy is expressed as intrinsic activity (a) using
the maximal effect elicited by N/OFQ as internal standard
Effect ¼ baseline þ
ðlogEC50ꢀXÞꢂn
1
þ 10
(
a = 1.00).
Antagonist potencies are given as pKB values. These were
where X is the agonist concentration and n is the Hill
coefficient.
derived from inhibition–response curves and calculated,
Chem Biol Drug Des 2015; 86: 447–458
449

Corrado et al.
O
O
O
O
O
O
O
NH
(
a)
(b)
(c)
(
86
d)
%
N
O
99%
O
99%
OH
60%
Cl
N
O
O
O
O
OH
O
1
1
12
13
14
(e)
2
7%
O
O
O
O
O
OH
NH
O
NH
(
h) or (i)
N
(g)
(f)
N
N
N
OTs
OH
O
99% or 28%
O
36%
78%
O
cis-18
17
1
6
15
Scheme 4: Synthesis of 14, 17 and cis-18. Reagents and conditions: (a) MeOH, H
SOCl , CH Cl , 0 °C to r.t.; (d) 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one, K CO , KI, acetonitrile, reflux; (e) H
TsCl, N(Et) , CH Cl , 0 °C to r.t.; (g) 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one, Na CO , acetonitrile, reflux; (h) NaBH
4
2
SO
4
, reflux; (b) NaBH
4
, MeOH, 0 °C to r.t.; (c)
, Pd/C, CH Cl , r.t.; (f)
, MeOH, 0 °C to
2
2
2
2
3
2
2
2
3
2
2
2
3
r.t.; (i) L-Selectride, THF, ꢀ78 °C to r.t.
OH
O
N
NH
N
trans-22
(
d)
O
O
87%
20%
O
O
O
N
(
a)
4%
OH
(b)
Cl
(c)
4
99%
77%
NH
N
1
9
20
21
OH
(
e)
O
N
NH
N
cis-22
Scheme 5: Synthesis of trans-22 and cis-22. Reagents and conditions: (a) HCHO 37%, Na
2 3 2 2 2
CO , acetonitrile, 40 °C (b) SOCl , CH Cl ,
0
ꢀ
°C to r.t.; (c) 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one, K
78 °C to r.t.
2 3 4
CO , KI, acetonitrile, reflux; (d) NaBH , MeOH, r.t.; (e) L-Selectride, THF,
assuming a competitive type of antagonism, using the
following equation:
substituted with the commercially available 1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one to provide compound 3 (23).
Under these conditions, 3 was obtained in poor yield (10%
yield). Accordingly, 2 was converted into the correspond-
ing trimethylammonium salt 2a to increase the efficiency of
the leaving group and to enhance the yield of the subse-
quent substitution reaction (78% yield two steps). From
compound 3, several derivatives were then synthesized.
IC50
pK ¼ ꢀlog ꢂ
ꢃ
B
h
ꢀ
ꢁ i
n
1=n
½
Aꢃ
2
þ
ꢀ 1
EC50
where IC50 is the concentration of antagonist that pro-
duces 50% inhibition of the agonist response, [A] is the
concentration of agonist, EC50 is the concentration of ago-
nist that produces a 50% of the maximal response, and n
is the Hill coefficient of the concentration–response curve
to the agonist.
The reduction of the ketone carbonyl group by NaBH
4
gave 4 (23) in good yield as cis/trans diastereoisomeric
mixture (40/60 cis/trans ratio). The two diastereoisomers
were separated by flash chromatography. The cis/trans
configuration, relative to 1-phenyl-1,3,8-triaza-spiro[4.5]
decan-4-one and OH substituents respectively on 3 and 4
position of chromane ring, was unambiguously assigned
through NMR 2D experiments. Positive NOE effect was
detected between H-3 and H-4 of the chromane moiety of
cis isomer (Figure 2). The same signal was not observed
in the same NMR 2D experiment performed on trans iso-
mer. Stereochemical assignments were confirmed also by
Results and Discussion
Synthesis
Synthesis of 8-(4-oxo-chroman-3-ylmethyl)-1-phenyl-1,3,8-
triaza-spiro[4.5]decan-4-one (3) and its derivatives is out-
lined in Scheme 1. Compound 2 was prepared by Man-
nich reaction from dimethylamine hydrochloride and
chroman-4-one. Then, the dimethylamino portion of 2 was
1
the H-NMR correlations. The relative chemical shifts of
the H-4 protons were particularly informative, as were the
H-3-H-4 coupling constants. H-4 signal of the trans
4
50
Chem Biol Drug Des 2015; 86: 447–458

Nociceptin/Orphanin FQ Receptor Ligands
1
Figure 2: Upper panels: NOESY spectra (DMSO, 400 MHz) of cis-4 (left) and trans-4 (right). Lower panels: enlarged H-NMR spectra
(DMSO, 400 MHz) of cis-4 (left) and trans-4 (right): 3.9–4.8 ppm region.
isomer occurred at higher field with respect to the H-4
signal of the cis isomer (Δd: 0.28 ppm; Figure 2). The
increased deshielding effect produced by the aromatic ring
of chromane indicated an equatorial orientation of H-4
proton. Moreover, the H-4 signal of the cis isomer
appeared as broadened single peak. This small magnitude
coupling constant clearly indicated an axial orientation of
the H-3 (Figure 2), and these equatorial/axial positions can
be observed only in a cis configuration. Spectrum of the
trans isomer showed the H-4 proton as a doublet
Furthermore, 8-(4-oxo-4H-chromen-3-ylmethyl)-1-phenyl-
1,3,8-triaza-spiro[4.5]decan-4-one (10), the 2,3-dehydro
analogue of 3, was synthesized as displayed in Scheme 3.
Compound 7 (2-hydroxy-chroman-4-one) was obtained by
0
Claisen condensation of 2 -hydroxyacetophenone with
ethyl formate. Treatment of 7 with formaldehyde provided
the corresponding unstable 3-hydroxymethyl derivative,
which was then dehydrated in situ to give 3-(hydroxymeth-
yl)-4H-chromen-4-one (8) (26). Compound 8 was then
2
converted into 3-chloromethyl intermediate 9, using SOCl ,
(
J = 5.0 Hz) confirming the axial orientation of both H-3
which was subsequently employed to alkylate 1-phenyl-
1,3,8-triaza-spiro[4.5]decan-4-one providing the final com-
pound 10.
and H-4 of the chromane ring and thus the trans configu-
ration.
Subsequently, compound 5 was obtained by dehydration
of chroman-4-ol portion of 4, in acidic medium. To avoid
product degradation due to the harsh conditions of this
type of reaction, phosphoric acid was employed. Then,
compound 6 was produced by catalytic reduction (Pd/C)
of double bond of the chromene moiety of 5. As we iso-
lated 6 with poor yield (12% yield), we developed a differ-
ent synthesis which is described in Scheme 2 (23,24).
Chroman-4-one was treated in basic conditions with ethyl
formate to give 6a (24). This intermediate was then
reduced by the synergistic action of boron trifluoride
diethyl etherate and borane tert-butylamine complex giv-
ing the second intermediate 6b (25). The latter was then
converted into the corresponding tosylated product 6c,
which was then reacted with 1-phenyl-1,3,8-triaza-spiro
Moreover, the 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one
portion was inserted at position 2 of the chromane nucleus
(Scheme 4). The 4-oxo-4H-chromene-2-carboxylic acid
was converted into the corresponding methyl ester 11,
and the latter was reduced with NaBH to give 2-(hydrox-
4
ymethyl)-4H-chromen-4-one (12) (27,28). Compound 12
reacted with SOCl2 to give the chloro-intermediate 13
which furnished 14 in good yields by nucleophilic substitu-
tion, under basic conditions, with 1-phenyl-1,3,8-triaza-
spiro[4.5]decan-4-one. On the other hand, the reduction
of the intermediate 12 provided the 2,3-dihydro derivative
15, which was subsequently tosylated (16) and then
reacted with 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one
to give final compound 17. The reduction of the latter with
NaBH
4
or L-Selectride yielded in both cases the 4-
[4.5]decan-4-one to give 6.
hydroxy-chromane derivative 18 exclusively as cis isomer
Chem Biol Drug Des 2015; 86: 447–458
451

Corrado et al.
configuration assignment was performed as previously
(
hydroxymethylation of tetralone in the a position with
respect to the carbonyl function was obtained (19) using
aqueous formaldehyde with base catalysis (29). Subse-
quently, the corresponding halide 20 was generated by
described for 4).
For the preparation of the other 1-phenyl-1,3,8-triaza-spiro
[4.5]decan-4-one analogues, containing the tetralin
2
treating the alcohol with SOCl . Then, alkylation of 1-phe-
nucleus, the same synthetic pathway shown in Scheme 1
was initially followed (5–10% yield). However, to obtain
these derivatives in superior yields, we developed a new
synthetic strategy (Scheme 5), as the Mannich reaction
was proved to be the bottleneck of the previous one,
probably due to the instability of the first intermediate. The
nyl-1,3,8-triaza-spiro[4.5]decan-4-one furnished compound
21 (34% yield three steps). Stereoselective reduction of
the latter with NaBH
4
or L-Selectride gave respectively
trans-22 and cis-22 diastereoisomers in good yields (con-
figuration assignment was performed as previously
described for 4) (23). Differently, compound 23 was
synthesized starting from a-tetralone as described for
compound 6 and as depicted in Scheme 2.
1
13
a
Table 1: H and C NMR data in CDCl
3
for compound 3
1
Substructures
of compound 3
d
H (multiplicity,
b
c
13 c
C
Position
J in Hz)
d
Purity of the final products
d
Full assignments of protons and carbons for all the final 1-
phenyl-1,3,8-triaza-spiro[4.5]decan-4-one derivatives were
tasd
2
3
4
5
4.79 (s)
7.73 (br s)
–
–
1.77 (m), 2.85 (m)
2.85 (m)
–
6.94 (d, 8.0)
7.34 (dd, 7.5, 8.3)
6.91 (t, 7.5)
59.2
–
178.4
59.5
29.1, 29.4
51.3, 54.4
143.2
115.2
129.32
118.9
1
13
performed by H and C NMR analysis, through related
1D spectra acquisition. Also the evaluation of the key
e
1
1
1
13
6
7
1
2
3
4
/10
cross-peaks detected in the H- H COSY, H- C HSQC
and HMBC 2D NMR experiments was performed. Particu-
e
/9
0
1
13
larly, the NMR H and C assignments of compound 3
are reported in Table 1. All the tested compounds were
converted into their oxalate salts using oxalic acid in ace-
tone or methanol. The purities of all salts were checked by
elemental analysis. The exact mass of all of them was
obtained with HPLC–QTOF analysis.
0
0
0
0
0
/6
/5
chroman-4-one 2″
4.53 (dd, 8.3, 11.4),
69.7
4
.71 (dd, 4.2, 11.3)
e
3
4
4
5
6
7
″
″
″a
″
″
2.85 (m)
–
–
7.94 (dd, 1.7, 7.9)
7.07 (dd, 7.1, 7.9)
7.52 (ddd, 1.7,
44.4
193.8
161.7
127.34
121.4
135.9
In vitro biological activity at NOP receptors
The activities at NOP receptors of the 1-phenyl-1,3,8-tri-
aza-spiro[4.5]decan-4-one analogues were evaluated mea-
suring calcium mobilization in CHOhNOP cells stably
expressing the Gaqi5 chimeric protein that forces the NOP
receptor to couple to calcium signaling. This assay has
been previously validated by studying the effects of a large
series of NOP receptor ligands encompassing full and par-
tial agonist as well as antagonist activities (30). The novel
compounds have been evaluated as NOP receptor agon-
ists using as standard N/OFQ (Figure 3, left panel). Under
″
7
.1, 8.3)
8
8
Z
″
″a
7.03 (d, 8.3)
117.8
120.9
48.9
e
2.85 (m)
a
See Methods and Materials.
Atom position in the respective portion of 3.
Measured at 400 MHz.
tasd: 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one.
Overlapped with each other.
b
c
d
e
N/OFQ 10 nM
2
2
1
1
50
00
50
00
250
200
150
100
50
Figure 3: Calcium mobilization experiments
performed in CHO cells coexpressing the
human NOP receptor and the Gaqi5 protein.
Left panel: concentration response curve to
N/OFQ. Right panel: inhibition response
curve obtained by challenging 10 nM N/OFQ
with increasing concentrations of SB-
5
0
0
0
6
12111. Data are the mean ꢁ SEM of at
1
2
11 10
9
8
7
6
5
12 11 10
9
8
7
6
5
least five separate experiments performed in
duplicate.
–
Log [N/OFQ]
–Log [SB-612111]
4
52
Chem Biol Drug Des 2015; 86: 447–458

Nociceptin/Orphanin FQ Receptor Ligands
Table 2: Effects of standard and novel NOP ligands in calcium mobilization assays, performed in CHO cells co-expressing the human
NOP receptor and the Gaqi5 protein
Agonist
Antagonist
Compound
pEC50 (CL95%
)
a ꢁ SEM
B
pK (CL95%)
N/OFQ
SB-612111
1
9.55 (9.07–10.04)
Inactive
6.49 (5.95–7.03)
1.00
–
8.50 (8.03–8.97)
–
O
O
0.81 ꢁ 0.03
1.09 ꢁ 0.05
6
5
5.97 (5.78–6.17)
–
–
–
O
O
crc incomplete
2
3
3
crc incomplete
Inactive
O
Inactive
O
O
1
2
7
1
crc incomplete
–
O
O
Inactive
<6
cis-4
OH
6.67 (6.43–6.91)
0.83 ꢁ 0.04
0.70 ꢁ 0.02*
1.01 ꢁ 0.41
–
O
trans-4
cis-22
OH
6.45 (5.77–7.13)
6.38 (5.51–7.25)
–
–
O
OH
Chem Biol Drug Des 2015; 86: 447–458
453

Corrado et al.
Table 2: continued
Agonist
Antagonist
Compound
pEC50 (CL95%
)
a ꢁ SEM
B
pK (CL95%)
trans-22
OH
O
crc incomplete
–
cis-18
6.57 (6.06–7.08)
1.08 ꢁ 0.32
–
OH
O
1
0
4
crc incomplete
–
O
O
1
Inactive
<6
O
Agonist efficacy is expressed as intrinsic activity (a) using the maximal effect elicited by N/OFQ as internal standard (a = 1.00).
Inactive: inactive up to 10 lM.
crc incomplete: the concentration–response curve could not be completed because of the low potency of the agonist.
<
6: the pK
tested, that is, 10 lM.
p < 0.05 according to ANOVA followed by the Dunnett test for multiple comparisons.
B
could not be precisely determined because the ligand produced a weak inhibitory effect only at the highest concentration
*
the present experimental conditions, N/OFQ produced a
concentration-dependent stimulation of calcium mobiliza-
tion with a pEC50 of 9.55 and maximal effects of
bind and activate the NOP receptor. trans-22 produced
an incomplete concentration–response curve at 10 lM,
stimulating calcium mobilization to 39 ꢁ 14% over the
basal value. Similar results were obtained testing com-
pounds 5, 10, 17, and 23. Thus, these molecules behaved
as low-potency NOP agonists. On the contrary, com-
pounds 3, 14, and 21 did not modify calcium levels up to
10 lM. Therefore, these compounds were further evalu-
ated in antagonist-type experiments. In these studies, SB-
612111 was used as positive control (30). Inhibition–
response curves were performed against a fixed concen-
1
98 ꢁ 29% over the basal values. These results are close
to those previously published (30,31). Results obtained
with the novel compounds are summarized in Table 2.
Spiroxatrine (1), taken as lead compound of this study,
behaved as a low-potency NOP agonist with a pEC50 of
6
.49. Compounds cis-4, cis-18, and cis-22 produced a
concentration-dependent calcium mobilization with pEC50
values of 6.67, 6.57, and 6.38, respectively. The maximal
effects elicited by these compounds were not significantly
different from that of N/OFQ (Table 2). Compound 6 also
behaved as a full agonist showing however lower potency
tration of N/OFQ, approximately corresponding to its EC80
.
As shown in Figure 3 right panel, under the present exper-
imental conditions, SB-612111 elicited a complete and
concentration-dependent inhibition of the N/OFQ stimula-
tory effect. A pKB value of 8.50 was derived from these
experiments. This value is in line with previously reported
findings (21,30). In parallel experiments, compound 3 did
not modify the N/OFQ stimulatory effect up to 10 lM.
Compound 14 and 21 displayed weak antagonist activity
only at the higher concentration tested, that is, 10 lM.
(pEC50 = 5.97). Compound trans-4 stimulated calcium
mobilization in a concentration-dependent manner with a
potency value of 6.45. However, the maximal effect
induced by this compound was significantly lower than
those produced by the standard agonist N/OFQ (Table 2
and Figure 4 panel A). Thus, trans-4 behaved as a partial
NOP agonist (Figure 4 panel A). These compounds were
inactive up to 10 lM when assayed in CHO cells express-
ing the Gaqi5 protein but not the NOP receptor. Thus, the
stimulatory effects of compounds 6, trans-4, cis-4, cis-
Finally, the selectivity of compounds trans-4 and cis-4
was evaluated measuring calcium mobilization in
CHOhNOP cells stably expressing the Gaqi5 chimeric
1
8, and cis-22 are due to the ability of these molecules to
4
54
Chem Biol Drug Des 2015; 86: 447–458

Nociceptin/Orphanin FQ Receptor Ligands
protein that forces MOP, DOP, and KOP receptors to
couple to calcium signaling. This assay has been previ-
ously reported and validated (20). The results obtained
are summarized in Table 3 and Figure 4. Compounds
trans-4 and cis-4 did not modify calcium levels up to
10 lM in agonist-type experiment for KOP and DOP
A
B
C
D
Figure 4: Calcium mobilization experiments
performed in CHO cells coexpressing the
chimeric G proteins and the human NOP,
MOP, KOP, DOP receptors (panels A, B, C,
and D, respectively). Concentration response
curves to standard agonists, cis-4 and
trans-4. Data are the mean ꢁ SEM of at
least three separate experiments performed
in duplicate.
Table 3: Effects of trans-4, cis-4, and standard NOP, MOP, KOP, and DOP ligands in calcium mobilization experiments performed in
CHO cells co-expressing the chimeric G proteins and the human NOP, MOP, KOP, or DOP receptors
NOP
MOP
KOP
DOP
pEC50
pEC50
pEC50
pEC50
(CL95%)
a ꢁ SEM
(CL95%)
a ꢁ SEM
(CL95%)
a ꢁ SEM (CL95%)
a ꢁ SEM
N/OFQ
9.35
1.00
<5
<6
<5
<5
(8.86–9.83)
Dermorphin
<5
8.43
1.00
<6
(
7.98–8.87)
a
a
Dynorphin-A <6
6.67
(
0.99 ꢁ 0.05
8.29
(7.45–9.12)
Inactive
1.00
7.16
(7.07–7.25)
8.73
1.10 ꢁ 0.20
6.17–7.17)
DPDPE
trans-4
cis-4
Inactive
Inactive
1.00
(8.39–9.07)
<6
6.21
(
0.87 ꢁ 0.06
0.73 ꢁ 0.05
Inactive
Inactive
Inactive
6.00–6.42)
6.68 (5.96–7.41) 0.85 ꢁ 0.08 6.17
5.88–6.45)
Inactive
(
Agonist efficacy is expressed as intrinsic activity (a) using the maximal effect elicited by N/OFQ as internal standard (a = 1.00).
Inactive: inactive up to 10 lM.
<5 and <6: the concentration–response curve could not be completed because of the low potency of the agonist; the estimated pEC50
values obtained by constraining the maximal effects were <5 or 6.
a
These data were taken from [31].
Chem Biol Drug Des 2015; 86: 447–458
455

Corrado et al.
receptors highlighting a fine NOP/DOP and NOP/KOP
selectivity (Figure 4 panel C and D, respectively). Differ-
ently, trans-4 and cis-4 produced a concentration-
dependent calcium mobilization for the MOP receptor
with pEC50 values of 6.21 and 6.17, respectively, with a
low NOP/MOP selectivity ratio (<0.6 and 3.2, respec-
tively; Table 3). Notably, a reversed behavior in selectiv-
ity was observed in comparison with NOP agonist
activity, and trans-4 proved to be the most active iso-
mer suggesting complete different stereointeractions
with the two NOP/MOP receptors.
Finally, we wanted to investigate the effect of the introduc-
tion of a double bond in the chromanone structure of regio-
nal isomers 3 and 17 with the synthesized chromones 10
and 14. Proceeding from 3 to 10, a small and barely detect-
able agonist efficacy was seen, while the contrary seems to
be true going from 17 to 14, as in this case the already weak
activity disappears. Certainly, such consideration is based
on a very small variation of activity and might be speculative.
However, it is suggested that the more constrained struc-
ture of these chromones, that makes therefore planar this
part of the molecule, does not favor agonist activity. This
indirectly seems to confirm the importance first of a certain
distortion of the lateral chain and secondly of the oxygen-
ated function in position 4, indicating that the interaction at
NOP receptor site is highly stereoselective.
Structure–activity relationships of NOP receptor
ligands
Isosteric substitution O-1/CH2 in the 1,4-benzodioxane
nucleus of spiroxatrine (1), as represented by compound
6
, results in a threefold decrease of agonist potency. A fur-
ther decrease in potency was observed with the unsatu-
rated benzopyran derivative and with the tetralin
Conclusions
5
We have synthesized and evaluated several spiroxatrine
derivatives as possible NOP receptor ligands. They repre-
sent novel tools for better understanding the structural
requirements necessary for NOP receptor binding and
agonist activity. Moreover, we demonstrated that by intro-
ducing specific group on the chromane core of this type
of molecules, it is possible to modulate the agonist activity
notwithstanding the lipophilic portion is attached to the
piperidine nitrogen by a 1-carbon linker. Because the
majority of the agonists reported in literature are con-
nected directly to the piperidine nitrogen, this evidence
could be useful for the development of new NOP ligands.
derivative 23 with respect to 6. These results indicate that
both oxygen atoms are necessary for spiroxatrine activity
at NOP receptors, and the one at position 1 is probably
more sensitive to isosteric replacement.
Alternatively, dislocating out of the ring, the oxygen at posi-
tion 1 and 4 of the 1,4-benzodioxane core of 1 produced
the chroman-4-one derivatives 3 and 17, and it was possi-
ble to determine that the introduction of a carbonyl group
led to a severe loss of activity. These findings suggest that
the introduction of an H-bond acceptor is not well tolerated.
In addition, the replacement of oxygen at position 1 of the
chroman-4-one derivative 3 provided the inactive a-tetr-
alone derivative 21, confirming the role of this oxygen. Com-
pounds 3, 17, and 21 proved to be inactive also in
antagonist-type experiments; these data suggest that these
compounds do not bind NOP receptor.
Some considerations on structure–activity relationships
have led to these conclusions:
1
. The presence of an H-bond donator group (hydroxyl
group) is more favorable than an H-bond acceptor one
carbonyl group).
(
The reduction of carbonyl group of chromanone moiety
of 3, which gave chroman-4-ols cis- and trans-4,
allowed the recovery of agonist activity, especially in the
case of cis isomer that showed a pEC50 value of 6.67,
higher than that of spiroxatrine (6.49). The favorable effect
of carbonyl reduction was also observed for compound
2
. The H-bond donor group is preferred in a position with
respect to the CH
dine core.
2
linker interposed before the spiropiperi-
3
. The activities shown by the hydroxyl derivatives display
2
2 obtained from the other chromanone derivative 21.
a certain degree of stereoselectivity, as cis isomers are
moderately more active than trans isomers.
Also in this case, the cis isomer was more potent than
the trans isomer, with a pEC50 value of 6.38. Further,
confirmation of the positive role of the hydroxyl group in
this position came from the comparison of the activities
of chromanone 17, regional isomer of chromanone 3,
and its carbonyl-reduced derivative cis-18 which showed
a pEC50 value of 6.57. Unfortunately, in the case of this
4
. Compound cis-4 shows a promising submicromolar
agonistic activity at NOP receptor, and it will be employed
as reference compound for the further design and synthe-
sis of other new spiropiperidine-based agonists.
4
-hydroxyl derivative, only the cis isomer was obtained.
We did not made any further effort to obtain the trans
isomer as the same isomer of the alcohols 4 and 22 was
less active than the cis one. For this reason, most likely
also in the case of trans-18, it would have been obtained
the same result.
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3
Appendix S1. Full characterization of compounds 2–23,
along with detailed experimental procedures, is given in
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4
58
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