Synthesis, enantiomeric separation and docking studies of spiropiperidine analogues as ligands of the nociceptin/orphanin FQ receptor

Article abstract of DOI:10.1039/c4md00082j

A series of triazospirodecanone derivatives were synthesized as potential NOP ligands. 8-(Chroman-4-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (4) and its 5-fluoro analogue (18) proved to be active as agonists with EC₅₀ values in the submicromolar range. Single enantiomers of compound 4 were separated and tested as NOP agonists; the eutomer R-(+)-4 showed a pEC ₅₀ of 7.34. Finally docking studies were performed on the NOP receptor to identify the most significant stereospecific interactions.

Full text of DOI:10.1039/c4md00082j

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Synthesis, enantiomeric separation and docking
studies of spiropiperidine analogues as ligands of
the nociceptin/orphanin FQ receptor†
Cite this: DOI: 10.1039/c4md00082j
Umberto M. Battisti,a Sandra Corrado,a Claudia Sorbi,a Andrea Cornia,b Annalisa Tait,a
c
c
c
a
*
Davide Malfacini, Maria Camilla Cerlesi, Girolamo Calo and Livio Brasili
`
A series of triazospirodecanone derivatives were synthesized as potential NOP ligands. 8-(Chroman-4-yl)-
1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (4) and its 5-fluoro analogue (18) proved to be active as
agonists with EC50 values in the submicromolar range. Single enantiomers of compound 4 were
separated and tested as NOP agonists; the eutomer R-(+)-4 showed a pEC50 of 7.34. Finally docking
studies were performed on the NOP receptor to identify the most significant stereospecific interactions.
Received 24th February 2014
Accepted 22nd April 2014
DOI: 10.1039/c4md00082j
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active as central nervous system drugs showed good affinity for
the NOP receptor.13
Introduction
Identication of the essential chemical features responsible
for NOP affinity may lead to the development of new small-
molecule NOP ligands. NOP agonists are being investigated as
potential therapeutics for anxiety, cough, and drug abuse. NOP
antagonists have been studied for their utility in treating Par-
kinson's, depression, obesity and learning decits.14,15
Among the neuroleptics tested, spiroxatrine (1) (Fig. 1), an a2
adrenergic and 5-HT1A antagonist, displayed a notable affinity
for NOP.13,14,16
Since their discovery, many advances have been made regarding
the biological signicance and functions of the neuropeptide
nociceptin/orphanin FQ (N/OFQ) and its receptor, namely the
N/OFQ peptide (NOP) receptor.1–6 Indeed several studies sug-
gested that the N/OFQ–NOP receptor system could be involved
in the control of several functions and processes. Although its
role and properties are still under active investigation, it is clear
that the N/OFQ–NOP receptor system regulates a wide range of
biological functions including cough, urinary incontinence,
anxiety and mood, pain, stress, feeding, learning and memory,
locomotor activity, substance abuse, cardiovascular functions,
and Parkinson's disease.7–12
On the basis of these results, spiroxatrine was selected as the
lead compound for the design of new potential NOP ligands by
several research groups.17,18
In particular, the 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one
portion of 1, due to its chemical features, was found to be
responsible for the binding affinity of this class of
compounds.17 The resulting spiropiperidine analogues exhibi-
ted high affinity for the NOP receptor. In particular, the spi-
ropiperidine series developed by Roche and Pzer
demonstrated high affinity associated with moderate selectivity
for the NOP receptor.15
The complex pharmacological effects of NOP are distinct
from the classical opioid receptors. N/OFQ showed sequence
similarity with classical opioid peptides but interestingly it does
not interact with classical opioid receptors (d, k, and m). More-
over the opioid peptides have no or very low affinity for the NOP
receptor. Similarly, the NOP receptor shared a high structural
homology (47% overall homology, 65% homology in the trans-
membrane domains) with the three classical opioid receptors;
but NOP is insensitive to most of the morphine-like ligands
such as naloxone. However, several known small molecules
Herein we report the results of the SAR study aimed at
identifying a series of triazaspirodecanone derivatives as NOP
receptor ligands. In particular, the SAR development plan was to
aDepartment of Life Sciences, University of Modena & Reggio Emilia, Via G. Campi 183,
41125 Modena, Italy. E-mail: livio.brasili@unimore.it; Fax: +39 0592055131; Tel: +39
0592055139
bDepartment of Chemical and Geological Sciences, University of Modena & Reggio
Emilia, Via G. Campi 183, 41125 Modena, Italy
cDepartment of Medical Sciences, Section of Pharmacology and National Institute of
Neuroscience, University of Ferrara, Via Fossato di Mortara, 64/B, 44121 Ferrara, Italy
† Electronic supplementary information (ESI) available: Characterization of all
compounds, crystallographic data, and computational methods. See DOI:
10.1039/c4md00082j
Fig. 1 Spiroxatrine (1).
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investigate possible substitutions of the chromane core of 8-
(chroman-4-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one. This
compound appeared previously in a Hoffmann-La Roche
patent; however no data regarding its binding affinity and
activity were reported.19 Moreover, we developed a useful
method to obtain single enantiomers for this class of
compounds. The method was applied to 8-(chroman-4-yl)-1-
phenyl-1,3,8-triazaspiro[4.5]decan-4-one and single enantio-
mers were separated and tested, highlighting a good enantio-
selective interaction with the NOP receptor. Docking studies
were also performed on the NOP receptor in order to identify
possible stereoselective interactions of these ligands.
Scheme 2 Synthesis of 4-chromanone derivatives. Reagents and
conditions: (a) pyrrolidine, EtOH, reflux.
Results and discussion
Chemistry
A synthetic approach different from the reductive amination,
previously reported for these compounds, was employed in an
attempt to increase the reaction yield and to avoid highly toxic
reagents (e.g. cyanoborohydride).19 As described in Scheme 1, the
commercially available 1-phenyl-1,3,8-triazaspiro[4,5]decan-4-
one, 2, was alkylated in the presence of various benzyl halides to
produce 3, where the benzyl halides consist of chromanyl
analogues. The halides were generated via treatment of the
corresponding alcohols with SOCl2 as described in Scheme 1.
Similarly, the alcohols were generated by NaBH4 reduction
from the corresponding chromane analogues (Scheme 1). The
required chromane derivatives were prepared as described in
Scheme 2 by condensation of 20-hydroxyacetophenone with an
appropriate ketone or aldehyde.20
Scheme 3 Synthesis of 17a. Reagents and conditions: (a) CH3I, KOtBu,
THF, ꢁ70 ꢀC.
Scheme 4 Synthesis of 18a. Reagents and conditions: (a) acrylonitrile,
Triton B, 70 ꢀC; (b) HCl conc., 100 ꢀC; (c) SOCl2, toluene, 0 ꢀC to r.t.; (d)
triflic acid, CH2Cl2, ꢁ70 ꢀC to r.t.
The corresponding 3,3-dimethylchromane derivative (17a)
was obtained by alkylation of compound 4a (Scheme 3).21
Differently the 5-uoro analogue of 4a was synthesized as
outlined in Scheme 4.22
Compound 16 was obtained by basic hydrolysis of the acetyl
group of the triazaspirodecanone derivative 14 (Scheme 5).
All compounds were fully characterized and the nal spi-
ropiperidine derivatives were converted into the corresponding
salts with oxalic acid.
Scheme 5 Synthesis of 16. Reagents and conditions: (a) NaOH 10%,
MeOH, 80 ꢀC.
In vitro activity at human recombinant NOP receptors
calcium signaling. This assay has been previously validated by
The compounds described were evaluated by measuring studying the effects of a large series of standard NOP receptor
calcium mobilization in CHONOP cells, stably expressing the ligands encompassing full and partial agonist as well as
Gaqi5 chimeric protein that forces the receptor to couple with antagonist activities and of novel NOP ligands including the
Scheme 1 Synthesis of triazaspirodecanone derivatives. Reagents and conditions: (a) NaBH4, MeOH, 0 ꢀC; (b) SOCl2, CH2Cl2, 0 ꢀC; (c) K2CO3, KI,
CH3CN, reflux.
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Table 1 Effects of standard and novel ligands in calcium mobilization
experiments performed in CHO cells coexpressing the human NOP
receptor and the Gaqi5 chimeric proteina
antagonists C-24, C-35 and the mixed NOP/opioid receptor
agonist [Dmt1]N/OFQ(1–13)-NH2.23–26
The novel compounds were evaluated as NOP receptor
agonists using N/OFQ as the standard (Fig. 2, le panel). Under
the present experimental conditions, N/OFQ produced a
concentration-dependent stimulation of calcium mobilization
with a pEC50 of 9.68 and maximal effect of 254 ꢂ 16% over the
basal value. These results are close to those previously pub-
lished.23,24 Results obtained with the novel compounds are
summarized in Table 1. Spiroxatrine (1), taken as the lead
compound for this study, behaved as a low potency NOP full
agonist with a pEC50 of 6.49. Compounds 4 and 18 produced a
concentration-dependent calcium mobilization with pEC50
values of 6.80 and 6.37, respectively.
Antagonist
pKB (CL95%
n.d.
Agonist
Compound
pEC50 (CL95%
)
a ꢂ SEM
)
9.68 (9.59–9.78) 1.00
N/OFQ
The maximal effects elicited by the compounds were not
signicantly different from that of N/OFQ (Table 1). However,
the potency of these compounds was signicantly lower than
that produced by the standard agonist N/OFQ. The same
compounds were found to be inactive up to 10 mM when eval-
uated in CHO cells expressing the Gaqi5 protein, but not the
NOP receptor, indicating that the observed stimulation of
calcium mobilization is exclusively due to their ability to bind
and activate the NOP receptor.
Compounds 5–10, 13–15 and 17 produced a weak stimula-
tion of the NOP receptor, generating incomplete concentration
response curves. Thus these molecules behaved as very low
potency NOP agonists. Differently, compounds 11, 12 and 16
did not stimulate calcium mobilization up to 10 mM.
SB-612111
Inactive
8.10 (7.95–8.24)
1
4
6.49 (5.95–7.03) 0.81 ꢂ 0.03 n.d.
6.80 (5.79–7.63) 1.02 ꢂ 0.04 n.d.
5
6
Crc incomplete
Crc incomplete
n.d.
n.d.
These compounds were further evaluated in antagonist-type
experiments. In these studies SB-612111 was used as a positive
control. Inhibition response curves were performed against a
xed concentration of N/OFQ, approximately corresponding to
its EC80. As shown in Fig. 2, right panel, under the present
experimental conditions SB-612111 elicited a complete and
concentration-dependent inhibition of the N/OFQ stimulatory
effect, with a pKB value of 8.10. This value is in line with
previously reported ndings.23,27,28 Compounds 11, 12, and 16
up to 10 mM did not modify the stimulatory effect of N/OFQ. In
addition, compounds 7 and 13, which displayed a weak stim-
ulatory effect only at 10 mM, were also evaluated as NOP
antagonists up to 1 mM. These compounds produced a weak
inhibitory effect only at the highest concentration tested.
7
8
Crc incomplete
Crc incomplete
<6
n.d.
9
Crc incomplete
n.d.
10
11
12
Crc incomplete
Inactive
n.d.
Inactive
Inactive
Inactive
13
14
Crc incomplete
Crc incomplete
<6
Fig. 2 Calcium mobilization experiments performed in CHONOP cells
stably expressing the Gaqi5 protein. Concentration response curve to
N/OFQ (left panel) and inhibition response curve to SB-612111 vs. 1 nM
N/OFQ (right panel). Data are the mean ꢂ sem of at least 4 separate
experiments performed in duplicate.
n.d.
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Table 1 (Contd.)
introduction of a third cycle could lead to an increase in activity
through additional interactions. However, this strategy was not
successful and compounds 6 and 15 did not show promising
activity, suggesting very low steric tolerance on the chromane
ring. Interestingly, homologation of the spiro structure of 6, to
give 7, seems to convert the compound into a weak antagonist.
However, the very low potency of this compound does not allow
a detailed characterization of its pharmacological activity.
Further homologation of the spiro alkyl substituents (8) led to a
decrease in activity both as agonist and antagonist. The same
behaviour was observed for the isosters of 7, compounds 9, 10
and 16. Indeed the latter was completely inactive as its methyl
and ethyl derivatives (11 and 12, respectively), probably because
at physiological pH the nitrogen atom is protonated and the
positive charge may negatively interfere with the binding
process. Compound 13, with a non-protonable sulfonamidic
nitrogen which also carries the hindered substituent such as the
methanesulfonyl group, showed a weak antagonist activity
similar to compound 7. However, this is not observed with
compound 14 which has a similar nitrogen atom (acetamidic),
indicating the relevant and discriminating role of steric inter-
actions in the antagonist activity.
Antagonist
pKB (CL95%
n.d.
Agonist
Compound
pEC50 (CL95%
)
a ꢂ SEM
)
9.68 (9.59–9.78) 1.00
N/OFQ
SB-612111
Inactive
8.10 (7.95–8.24)
15
16
Crc incomplete
n.d.
Inactive
Inactive
n.d.
17
18
S-4
Crc incomplete
Synthesis and biological evaluation of (S)-4 and (R)-4
Since previous studies suggested that chiral spiropiperidine
derivatives display a stereoselective pharmacological action on
NOP receptors, it was important to evaluate the activity of single
stereoisomers of 4, the most active compound of this series.29
Initial attempts at fractional crystallization of the racemic
(ꢂ)-4 in the presence of (L)(+)-tartaric or (D)(ꢁ)-mandelic acids as
resolving agents were performed with no success. Moreover the
use of semi-preparative chiral HPLC gave no results since a
baseline resolution was not achieved. For these reasons we
were prompt to develop a new synthetic pathway to obtain
single enantiomers. Resolution of (ꢂ)-4 was achieved by
conversion of the two enantiomers into their diastereomeric
amide derivatives (S,S)-19 and (S,R)-19 using N-tosyl-(S)-prolyl
chloride as the optically active resolving agent (Scheme 6).30
N-Tosyl-(S)-prolyl chloride was previously obtained by N-tosyla-
tion of (S)-proline, followed by the formation of the acyl chloride
derivative (Scheme 6).30 The epimers (S,S)-19 and (S,R)-19 were
6.37 (6.31–6.43) 1.00 ꢂ 0.09 n.d.
6.68 (6.57–6.80) 0.95 ꢂ 0.04 n.d.
R-4
7.34 (7.29–7.39) 0.98 ꢂ 0.04 n.d.
a
Data are mean of at least 3 separate experiments performed in
duplicate. n.d.: not determined. Inactive: inactive up to 10 mM. Crc
incomplete: the concentration response curve could not be completed
because of the low potency of the agonist. <6: the pKB could not be
precisely determined because the ligand produced a weak inhibitory
effect only at the highest concentration tested, i.e. 1 mM.
Structure–activity relationships of the NOP receptor ligands
Introduction of the chromane core on the piperidine nitrogen of separated by ash chromatography and then individually
1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, as in compound 4, deprotected to yield (S)-4 and (R)-4 by hydrolysis with sodium
moderately increases the agonist activity with respect to 1. methoxide. The optical rotatory power of the single stereo-
Introduction of a halogen atom at position 5 of compound 4, to isomer was recorded, conrming their stereochemical purity
give 18, leads to a slight decrease in potency. The presence of a (R[a]2D0 ¼ +42.6ꢀ; S[a]D20 ¼ ꢁ41.6ꢀ).
steric hindrance at position 3 of the chromane moiety as in
The absolute conguration of the enantiomers was deter-
compound 17 decreases the ligand potency. A similar trend was mined for the corresponding hydrogenoxalate hemihydrate
observed for the introduction of alkyl and aromatic substituents salts 4$H2C2O4$1/2H2O. X-ray diffraction assigned to the
on position 2 of the chromane as in compounds 5 and 15. dextrorotatory enantiomer the R conguration (Fig. 3).
Anyway since recent studies suggested that the complementary
Subsequently, the single enantiomers were evaluated as
lipophilic binding pocket in the NOP receptor should be able to hydrogenoxalate as NOP agonists as described above. (+)-(R)-4
accommodate even bigger substituents, we decided to increase and (ꢁ)-(S)-4 produced a concentration-dependent calcium
the steric hindrance at position 2 of the chromane core. In fact mobilization with pEC50 values of 7.34 and 6.68, respectively
several most active agonists or antagonists of the NOP receptor (Table 1 and Fig. 4). Thus the results identied the (R)-enan-
bear a tricyclic fused ring system. Thus we hypothesized that the tiomer as the most active component possessing an activity
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Scheme 6 Synthesis of R-4 and S-4. Reagents and conditions: (a) TsCl, Na2CO3, H2O, r.t., (b) SOCl2, toluene, reflux; (c) Et3N, CH2Cl2, reflux; (d)
NaOMe, MeOH, r.t.
Fig. 3 X-ray structure of one of the crystallographically independent
(+)-(R)-4 molecules (protonated on the piperidine nitrogen atom) in
(+)-R-4$H2C2O4$1/2H2O. Thermal ellipsoids are drawn at 50% prob-
ability. Cutout ellipsoids are shown for nitrogen and oxygen atoms.
Hydrogen atoms are represented as spheres of arbitrary radius.
5-fold higher than the (S)-enantiomer. Moreover, in order to
demonstrate the involvement of the NOP receptor in the
Fig. 4 Calcium mobilization experiments were performed in CHONOP
cells stably expressing the Gaqi5 protein. Top panels display the
concentration response curves to N/OFQ and (S)-4 (left) or (R)-4
(right). Bottom panels show the inhibition response curves to SB-
biological action of (+)-(R)-4 and (ꢁ)-(S)-4, inhibition
response experiments were performed by testing increasing
concentrations of SB-612111 against xed concentrations of
(+)-(R)-4 and (ꢁ)-(S)-4 (1 mM), approximately corresponding to
612111 vs. 1 mM (S)-4 (left) and 1 mM (R)-4 (right). Data are mean ꢂ sem
of at least 4 separate experiments performed in duplicate.
their EC80
.
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The pKB values obtained for SB-612111 vs. (+)-(R)-4 and should be exercised while using such structures for studying
(ꢁ)-(S)-4 were 8.67 and 8.58, respectively (Fig. 4B), indicating the docking of a receptor agonist. However, recent ndings
that the latter compounds behaved as full agonists interacting obtained by comparing the few examples of the structure of the
with the NOP receptor at the orthosteric binding site.
same GPCR in complex with an agonist or with an antagonist
demonstrated that the agonist bound form of the receptor
showed relatively minor backbone deviations compared to the
antagonist bound form, allowing the initial docking of agonists
into such model.32 Substantial conformational changes,
particularly in the cytoplasmic ends of helices V and VI, are
observed only when the agonist bound receptor is crystallized
together with the G protein.33
The soware MVD was initially evaluated on the peptide
mimetic antagonist C-24 to verify whether the docking method
can reproduce the crystal binding model. The average root-
mean-square distance (rmsd) of the best ranking pose of the
compound as compared to its binding pose in the respective
Docking studies
In order to understand possible stereospecic interactions of
(+)-(R)-4 and (ꢁ)-(S)-4 docking studies were performed. Taking
advantage of the crystal structure of the NOP receptor (PDB code
4EA3), recently published by Thompson et al., Molegro Virtual
Docker (MVD) soware was applied to dock (+)-(R)-4 and (ꢁ)-(S)-
4 within the binding pocket of the NOP receptor.31 It should be
emphasized that the crystal structure of the NOP receptor has
been solved in complex with an antagonist (C-24) and thus
represents the inactive form of the receptor. Thus caution
˚
crystal structure was found to be 0.68 A, proving that MVD is
able to accurately dock this type of compound (see the ESI†).
The crystal structure obtained for the R stereoisomer was
employed as an input le to build the ligand with Spartan'08.
The docking output clearly indicates a similar binding mode for
(+)-(R)-4 and (ꢁ)-(S)-4 (Fig. 5 and 6). In particular, two hydro-
philic interactions were observed: the salt bridge between the
protonated piperidine nitrogen and Asp-130, an H-bond
between the amide nitrogen of the agonist and the oxygen of
Thr-305 (Fig. 6).
These polar interactions are probably essential for the
activity, playing a crucial role in anchoring the ligand to the
binding site. Moreover, the benzopyran moiety is accommo-
dated within the lipophilic cavity lined by Ile-127, Tyr-131, Phe-
135, Ile-204, Phe-215, Ile-219, Phe-220, Phe-224, Phe-272, Trp-
276, Val-279, and Val-283, further stabilizing the compound in
the binding pocket by multiple interactions. The main differ-
ence observed in the binding mode of the two stereoisomers is
Fig. 5 Cartoon representation of the NOP crystal structure with its
ligand-binding pocket shown as a green transparent surface. The best
results obtained from docking output for (S)-4 (orange) and (R)-4
(blue) are reported within the binding pocket.
Fig. 6 Binding mode of (R)-4 (blue, left panel) and (S)-4 (orange, right panel). Polar interactions are indicated with green dashes.
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Fig. 7 Aromatic ring of (R)-4 (blue, left panel) strongly interacts with Tyr-131 and aromatic ring of (S)-4 (orange, right panel) lined opposite to the
Tyr-131 residue.
the interaction with Tyr-131 (Fig. 7). As described by Thompson ¼ spirocycloheptane, pipd. ¼ spiropiperidine, phen. ¼
et al., point mutation of this residue with alanine residue had a phenyl, tasd ¼ 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one,
deleterious effect on agonist potency suggesting that Tyr-131 arom. tasd ¼ aromatic portion of 1-phenyl-1,3,8-triazaspiro
participates in crucial p-stacking interactions.31,34
[4.5]decan-4-one, Tos-pro ¼ tosylproline, arom. Tos-pro ¼
aromatic portion of tosylproline). 1H–1H correlation spec-
troscopy (COSY), 1H–13C heteronuclear multiple quantum
coherence (HMQC) and heteronuclear multiple bond
connectivity (HMBC) experiments were performed for the
determination of 1H–1H and 1H–13C correlations, respec-
tively. Mass spectra were obtained on a hybrid QTOF mass
spectrometer (PE SCIEX-QSTAR) using electrospray ioniza-
tion mode (HR-ESI-MS, ion voltage of 4800 V). The HPLC
experimental conditions of the HPLC-MS system are as
Experimental section
Chemistry
Materials and methods. All reagents, solvents and other
chemicals were used as purchased from Sigma-Aldrich
without further purication unless otherwise specied. Air-
or moisture-sensitive reactants and solvents were employed
in reactions carried out under a nitrogen atmosphere unless
otherwise noted. Flash column chromatography purica-
tions (medium pressure liquid chromatography) were carried
out using Merck silica gel 60 (230–400 mesh, ASTM). The
purity of the compounds was determined by elemental
analysis (C, H, N) that was performed on a Carlo Erba 1106
Analyzer in the Microanalysis Laboratory of the Life Sciences
follows: ow rate 5 ml minꢁ1, sample solution (10 pmol mlꢁ1
)
of the selected compound with 0.1% acetic acid, mobile
phase consisting of methanol (50%) and water (50%).
The oxalate salts of all tested compounds were used for
pharmacological evaluations.
`
Department of Universita degli Studi di Modena e Reggio
General procedure A
Emilia and the results reported here are within ꢂ0.4% of the
theoretical values. Melting points were determined with a A solution of 20-hydroxyacetophenone (1.0 mmol) and the
Stuart SMP3 and they are uncorrected. The structures of all selected ketone/aldehyde (1.5 mmol) in EtOH was treated with
isolated compounds were ensured by nuclear magnetic pyrrolidine (1.5 mmol) and then heated at reux for 24 h. Aer
resonance (NMR) and mass spectrometry. 1H and 13C NMR completion (monitored by TLC), the reaction mixture was
(1D and 2D experiments) spectra were recorded on a DPX-200 concentrated in vacuo. The residue was dissolved in EtOAc
Avance (Bruker) spectrometer at 200 MHz and on a DPX-400 and washed with HCl 1 N and brine. The organic layer was dried
Avance (Bruker) spectrometer at 400 MHz. Chemical shis on anhydrous Na2SO4, ltered, and evaporated in vacuo. Puri-
are expressed in d (ppm). 1H NMR chemical shis are relative cation by ash column chromatography gave the desired
to tetramethylsilane (TMS) as internal standard. 13C NMR compound.
chemical shis are relative to TMS at d 0.0 or to the 13C signal
2,3-Dihydro-4H-chromen-4-one (4a). The compound was
of the solvent: CDCl3 d 77.04, CD3OD d 49.8, DMSO-d6 d 39.5. obtained from paraformaldehyde following the general proce-
NMR data are reported as follows: chemical shi, number of dure A as a white solid (95% yield).
protons/carbons, multiplicity (s, singlet; d, doublet; t, triplet;
Mp: 41–42 ꢀC; 1H NMR (200 MHz, CDCl3): d ¼ 2.82 (t, J ¼ 6.5
q, quartet; m, multiplet; br, broadened), coupling constants Hz, 2H, CH2-3 chrom.), 4.54 (t, J ¼ 6.5 Hz, 2H, CH2-2 chrom.),
(Hz) and assignment (chrom. ¼ chromane, cyclopen. ¼ spi- 6.96–7.05 (m, 2H, CH-6, CH-8 chrom.), 7.45–7.48 (m, 1H, CH-7
rocyclopentane, cyclohex. ¼ spirocyclohexane, cyclohep. chrom.), 7.90 ppm (dd, J ¼ 8.1, 1.5 Hz, 1H, CH-5 chrom.)
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2.88 (m, 3H, CHb-6/CHb-10 tasd, CHa-7/CHa-9 tasd, CHb-
7/CHb-9 tasd), 3.29–3.36 (m, 1H, CHb-7/CHb-9 tasd), 3.95 (dd, J
¼ 5.5, 9.0 Hz, 1H, CH-4 chrom.), 4.11–4.16 (m, 1H, CHa-2
chrom.), 4.34–4.39 (m, 1H, CHb-2 chrom.), 4.72 (q, J ¼ 4.2 Hz,
2H, CH2-2 tasd), 6.77 (dd, J ¼ 0.8, 8.2 Hz, 1H, CH-8 chrom.), 6.85
(t, J ¼ 7.3 Hz, 1H, CH-4 arom. tasd), 6.90 (ddd, J ¼ 0.8, 7.7, 8.2
Hz, 1H, CH-6 chrom.), 6.94 (d, J ¼ 8.1 Hz, 2H, CH-2, CH-6 arom.
tasd), 7.11 (ddd, J ¼ 1.2, 7.7, 8.2 Hz, 1H, CH-7 chrom.), 7.27
(s, 1H, NH), 7.33 (dd, J ¼ 7.5, 8.5 Hz, 2H, CH-3, CH-5 arom.
tasd), 7.59 ppm (d, J ¼ 7.7 Hz, 1H, CH-5 chrom.); 13C NMR
(400 MHz, CDCl3): d ¼ 21.3 (C-3 chrom.), 29.1 (C-6/C-10 tasd),
29.5 (C-6/C-10 tasd), 42.3 (C-7/C-9 tasd), 47.9 (C-7/C-9 tasd), 58.6
(C-4 chrom.), 59.0 (C-2 tasd), 59.1 (C-5 tasd), 65.0 (C-2 chrom.),
114.4 (C-2, C-6 arom. tasd), 116.4 (C-8 chrom.), 118.2 (C-4 arom.
tasd), 120.0 (C-6 chrom.), 123.7 (C-4a chrom.), 127.9 (C-7
chrom.), 128.3 (C-5 chrom.), 129.0 (C-3, C-5 arom. tasd), 142.9
(C-1 arom. tasd), 155.5 (C-8a chrom.), 178.3 ppm (CO tasd).
The free amine was then converted to the corresponding
hydrogenoxalate from acetone.
Mp: 178–182 C; H NMR (200 MHz, DMSO): d ¼ 1.80–1.86
(m, 2H, CHa-6/CHa-10 tasd), 2.21–2.32 (m, 2H, CH2-3 chrom.),
2.55–2.91 (m, 2H, CHa-6/CHa-10 tasd), 3.05–3.15 (m, 1H, CHa-
7/CHa-9 tasd), 3.26–2.31 (m, 1H, CHa-7/CHa-9 tasd), 3.43–3.51
(m, 1H, CHb-7/CHb-9 tasd), 3.69–3.82 (m, 1H, CHb-7/CHb-9
tasd), 4.18–4.23 (m, 1H, CHa-2 chrom.), 4.35–4.43 (m, 1H, CHb-
2 chrom.), 4.56–4.62 (m, 3H, CH2-2 tasd, CH-4 chrom.), 6.75–
7.00 (m, 5H, CH-2, CH-4, CH-6 arom. tasd; C-6, C-8 chrom.), 7.25
(m, 3H, CH-3, CH-5 arom. tasd; CH-7 chrom.), 7.55 (d, J ¼ 7.5
Hz, 1H, CH-5 chrom.), 8.86 ppm (s, 1H, oxalic acid).
General procedure B
The selected ketone (1.0 mmol) was suspended in methanol
(10 ml) and treated with an excess of NaBH4 (1.5 mmol) at 0 ꢀC.
The resulting mixture was stirred for 30 minutes at room
temperature, then concentrated in vacuo. The residue was par-
titioned between CH2Cl2 and H2O. The organic layer was sepa-
rated, and the aqueous layer was extracted with CH2Cl2. The
organic layers were then combined, washed with H2O, dried
over anhydrous Na2SO4, ltered, and concentrated in vacuo to
yield the desired compound.
3,4-Dihydro-2H-chromen-4-ol (4b). The compound was
obtained from 4a following the general procedure B as an oil
(94% yield).
1H NMR (200 MHz, CDCl3): d ¼ 1.87 (dd, J ¼ 4.7 Hz, 1H,
–OH), 1.95–2.26 (m, 2H, CH2-3 chrom.), 4.18–4.38 (m, 2H, CH2-2
chrom.), 4.79 (dd, J ¼ 4.1, 8.3 Hz, 1H, CH-4 chrom.), 6.79–7.00
(m, 2H, CH-6, CH-8 chrom.), 7.13–7.37 ppm (m, 2H, CH-5, CH-7
chrom.)
1
ꢀ
General procedure C
To an ice-bath cooled solution of the selected alcohol derivative
(1.0 mmol) in CH2Cl2 (10 ml), thionyl chloride (1.5 mmol) was
added dropwise. The reaction mixture was stirred at room
temperature for 1 h then water was added. The aqueous layer
was decanted, separated and then extracted two times with
CH2Cl2. The organic layers were combined, washed with H2O,
dried over anhydrous Na2SO4, ltered, and concentrated in
vacuo to yield the desired compound.
HRMS-ESI m/z [M + H]+ calc. for C22H26N3O2: 364.2026;
found: 364.2029; anal. calcd for C24H27N3O6 : C, 63.56; H, 6.00;
N, 9.27; found C, 63.71; H, 6.15; N, 9.41.
N-Tosyl-(S)-prolyl chloride. To a solution of (S)-proline (1 g,
8.7 mmol) in 7.5 ml of water was added Na2CO3 (1.70 g, 16.0
mmol) and then p-toluenesulfonyl chloride (2.00 g, 10.5 mmol)
was added slowly at 0 ꢀC. The reaction mixture was stirred at
room temperature overnight. HCl 1 N was added until the solu-
tion was acidic and the resulting mixture was taken up in ethyl
acetate (4 ꢃ 15 ml). The organic phase was dried over Na2SO4
4-Chloro-3,4-dihydro-2H-chromene (4c). The compound was
obtained from 4b following the general procedure C as a black
oil (70% yield).
1H NMR (200 MHz, CDCl3): d ¼ 2.15–2.34 (m, 1H, CHa-3
chrom.), 2.39–2.63 (m, 1H, CHb-3 chrom.), 4.20–4.59 (m, 2H,
CH2-2 chrom.), 5.24 (m, 1H, CH-4 chrom.), 6.70–7.03 (m, 2H,
CH-6, CH-8 chrom.), 7.07–7.40 ppm (m, 2H, H-5, CH-7 chrom.)
General procedure D
To a solution of 1-phenyl-1,3,8-triazaspiro[4.5]deacan-4-one (1.5 and ltered, and the solvent was evaporated. The product
mmol), K2CO3 (3.0 mmol), KI (1.0 mmol) in anhydrous aceto- (N-tosyl-(S)-proline) was obtained in 78% yield and used for
nitrile (10 ml) was added the selected benzyl chloride (1.0 the next step. N-Tosyl-(S)-proline (1 g, 3.5 mmol) was dissolved in
mmol) portionwise. The resulting mixture was stirred under 6 ml of toluene; to this solution was carefully added thionyl
reux for 4 h, then cooled to room temperature and concen- chloride (1.32 g, 11.1 mmol). The reaction mixture was reuxed
trated in vacuo. The residue was partitioned between EtOAc and for 30 min. Aer removal of the solvent, 0.98 g (3.4 mmol, 97%
H2O. The organic layer was separated, and the aqueous layer yield) of N-tosyl-(S)-prolyl chloride were obtained and it was
was extracted with EtOAc. The organic layers were combined, immediately used for the next step without further purication.
washed with H2O, dried over anhydrous Na2SO4, ltered, and
concentrated in vacuo. The residue was puried by ash chro- 1H, CHa-4 Tos-pro), 1.92–2.05 (m, CHb-4, CHa-3 Tos-pro), 2.15–
Mp: 56–57 ꢀC; 1H NMR (400 MHz, CDCl3): d ¼ 1.84–1.90 (m,
matography to yield the desired compound.
2.23 (m, 1H, CHb-3 Tos-pro), 2.48 (s, 3H, CH3 Tos-pro), 3.35–
8-(Chroman-4-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one 3.45 (m, 1H, CHa-5 Tos-pro), 3.50–3.56 (m, 1H, CHb-5 Tos-pro),
(4). The compound was obtained from 4c following the general 4.65 (dd, J ¼ 6.1, 7.9 Hz, 1H, CH-2 Tos-pro), 7.37 (d, J ¼ 8.0 Hz,
procedure D as a white solid (48% yield).
2H, CH-3,CH-5 arom. Tos-pro), 7.78 ppm (d, J ¼ 8.0 Hz, 2H, CH-
Mp: 210–211 ꢀC; 1H NMR (400 MHz, CDCl3): d ¼ 1.63 (m, 1H, 2, CH-6 arom. Tos-pro).
CHa-6/CHa-10 tasd), 1.78 (m, 1H, CHa-6/CHa-10 tasd), 1.97–
8-(Chroman-4-yl)-1-phenyl-3-((S)-1-tosylpyrrolidine-2-carbonyl)-
2.02 (m, 1H, CHa-3 chrom.), 2.08–2.17 (m, 1H, CHb-3 chrom.), 1,3,8-triazaspiro[4.5]decan-4-one (S,S)-(19) (S,R)-19. To a solution
2.56–2.64 (m, 2H, CHb-6/CHb-10 tasd, CHa-7/CHa-9 tasd), 2.80– of (ꢂ)-4 (0.544 g, 1,5 mmol) in CH2Cl2 was added triethylamine
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(1.67 ml, 12 mmol) and a solution of N-tosyl-(S)-prolyl chloride residue was dissolved in H2O and extracted three times with
(1.72 g, 6 mmol) in CH2Cl2 at 0 ꢀC. The reaction was warmed to RT CH2Cl2. The organic phase was washed with brine and dried
and reuxed for 24 min. NaHCO3 was added and the organic layer over Na2SO4, and the solvent was removed under vacuum. The
was separated and washed with H2O, dried over Na2SO4 and products were isolated without further purication (0.222 g,
concentrated under reduced pressure. The residue was puried by 0.612 mmol, 95% yield).
ash chromatography (cyclohexane–EtOAc 70 : 30) to yield single
diasteromers.
8-((S)-Chroman-4-yl)-1-phenyl-3-((S)-1-tosylpyrrolidine-2- (m, 1H, CHa-6/CHa-10 tasd), 1.97–2.02 (m, 1H, CHa-3 chrom.),
carbonyl)-1,3,8-triazaspiro[4.5]decan-4-one (less polar isomer) 2.08–2.17 (m, 1H, CHb-3 chrom.), 2.56–2.64 (m, 2H, CHb-6/
(S,S)-19. The compound was obtained as a yellow solid (0.267 g, CHb-10 tasd, CHa-7/CHa-9 tasd), 2.80–2.88 (m, 3H, CHb-6/CHb-
Mp: 210–211 ꢀC; [a]D20 ¼ +42.6ꢀ (15 mg mlꢁ1 CH2Cl2); 1H NMR
(400 MHz, CDCl3): d ¼ 1.63 (m, 1H, CHa-6/CHa-10 tasd), 1.78
0.435 mmol, 29% yield).
10 tasd, CHa-7/CHa-9 tasd, CHb-7/CHb-9 tasd), 3.29–3.36
1
ꢀ
Mp: 141–142 C; H NMR (400 MHz, CDCl3): d ¼ 1.87–1.93 (m, 1H, CHb-7/CHb-9 tasd), 3.95 (dd, J ¼ 5.5, 9.0 Hz, 1H, CH-4
(m, 2H, CHa-6/CHa-10 tasd, CHa-4 Tos-pro), 1.92–2.08 (m, 4H, chrom.), 4.11–4.16 (m, 1H, CHa-2 chrom.), 4.34–4.39 (m, 1H,
CHa-3 chrom., CHa-3 Tos-pro, CHb-4 Tos-pro, CHa-6/CHa-10 CHb-2 chrom.), 4.72 (q, J ¼ 4.2 Hz, 2H, CH2-2 tasd), 6.77 (dd, J ¼
tasd), 2.17–2.25 (m, 1H, CHb-3 chrom.), 2.26–2.32 (m, 1H, CHb- 0.8, 8.2 Hz, 1H, CH-8 chrom.), 6.85 (t, J ¼ 7.3 Hz, 1H, CH-4 arom.
3 Tos-pro), 2.37–2.43 (m, 1H, CHb-6/CHb-10 tasd), 2.48 (s, 3H, tasd), 6.90 (ddd, J ¼ 0.8, 7.7, 8.2 Hz, 1H, CH-6 chrom.), 6.94 (d, J
CH3 Tos-pro), 2.52–2.57 (m, 1H, CHb-6/CHb-10 tasd), 2.66–2.73 ¼ 8.1 Hz; 2H, CH-2, CH-6 arom. tasd), 7.11 (ddd, J ¼ 1.2, 7.7, 8.2
(m, 1H, CHa-7/CHa-9 tasd), 2.83–2.88 (m, 2H, CHa-7/CHa-9 Hz, 1H, CH-7 chrom.), 7.27 (bs, 1H, NH), 7.33 (dd, J ¼ 7.5, 8.5
tasd, CHb-7/CHb-9 tasd), 3.17–3.20 (m, 1H, CHb-7/CHb-9 tasd), Hz, 2H, CH-3, CH-5 arom. tasd), 7.59 ppm (d, J ¼ 7.7 Hz, 1H,
3.30–3.38 (m, 1H, CHa-5 Tos-pro), 3.55–3.60 (m, 1H, CHb-5 Tos- CH-5 chrom.); 13C NMR (400 MHz, CDCl3): d ¼ 21.3 (C-3
pro), 4.01 (dd, J ¼ 5.6, 8.1 Hz, 1H, CH-4 chrom.), 4.15–4.22 chrom.), 29.0 (C-6/C-10 tasd), 29.5 (C-6/C-10 tasd), 42.2 (C-7/C-9
(m, 1H, CHa-2 chrom.), 4.39–4.44 (m, 1H, CHb-2 chrom.), 5.03 tasd), 47.9 (C-7/C-9 tasd), 58.5 (C-4 chrom.), 59.0 (C-2 tasd), 59.1
(d, J ¼ 7.0 Hz, 1H, CHa-2 tasd), 5.13 (d, J ¼ 7.0 Hz, 1H, CHb-2 (C-5 tasd), 65.0 (C-2 chrom.), 114.4 (C-2, C-6 arom. tasd), 116.4
tasd), 5.61 (dd, J ¼ 3.8, 9.0 Hz, 1H, CH-2 Tos-pro), 6.82 (d, J ¼ 8.0 (C-8 chrom.), 118.2 (C-4 arom. tasd), 120.0 (C-6 chrom.), 123.7
Hz, 1H, CH-8 chrom.), 6.94 (dd, J ¼ 7.3, 7.4 Hz, 1H, CH-6 (C-4a chrom.), 127.8 (C-7 chrom.), 128.3 (C-5 chrom.), 129.0
chrom.), 7.08 (t, J ¼ 7.3 Hz, 1H, CH-4 arom. tasd), 7.12–7.18 (C-3, C-5 arom. tasd), 142.9 (C-1 arom. tasd), 155.5 (C-8a
(m, 3H, CH-7 chrom., CH-2, CH-6 arom. tasd), 7.36–7.42 (m, 4H, chrom.),178.3 ppm (C]O tasd).
CH-3, CH-5 arom. tasd, CH-3, CH-5 arom. Tos-pro), 7.57 (d, J ¼
The free amine was then converted to the corresponding
8.2 Hz, 1H, CH-5 chrom.), 7.84 ppm (d, J ¼ 8.2 Hz, 2H, CH-2, hydrogenoxalate hemihydrate salt (+)-R-4$H2C2O4$1/2H2O from
CH-6 arom. Tos-pro).
acetone.
8-((R)-Chroman-4-yl)-1-phenyl-3-((S)-1-tosylpyrrolidine-2-
carbonyl)-1,3,8-triazaspiro[4.5]decan-4-one (more polar isomer) CHa-6/CHa-10 tasd), 2.21 (m, 2H, CH2-3 chrom.), 2.55–2.91
(R,S)-19. The compound was obtained as a yellow solid (0.396 g, (m, 2H, CHa-6/CHa-10 tasd), 3.05–3.10 (m, 1H, CHa-7/CHa-9
Mp: 178–182 ꢀC; 1H-NMR (400 MHz, DMSO): d ¼ 1.80 (m, 2H,
0.645 mmol, 43% yield).
tasd), 3.26 (m, 1H, CHa-7/CHa-9 tasd), 3.43–3.50 (m, 1H, CHb-7/
Mp: 146–148 ꢀC; 1H NMR (400 MHz, CDCl3): d 1.77–1.83 CHb-9 tasd), 3.69–3.74 (m, 1H, CHb-7/CHb-9 tasd), 4.18–4.22
(m, 2H, CHa-6/CHa-10 tasd, CHa-4 Tos-pro), 1.96–2.08 (m, 4H, (m, 1H, CHa-2 chrom.), 4.35–4.39 (m, 1H, CHb-2 chrom.), 4.56–
CHa-3 chrom., CHa-3 Tos-pro, CHb-4 Tos-pro, CHa-6/CHa-10 4.66 (m, 3H, CH2-2 tasd, CH-4 chrom.), 6.75–7.00 (m, 5H, CH-2,
tasd), 2.17–2.25 (m, 1H, CHb-3 chrom.), 2.26–2.30 (m, 1H, CHb- CH-4, CH-6 arom. tasd; CH-6, CH-8 chrom.), 7.25 (m, 3H, CH-3,
3 Tos-pro), 2.31–2.34 (m, 1H, CHb-6/CHb-10 tasd), 2.47 (s, 3H, CH-5 arom. tasd; CH-7 chrom.), 7.55 ppm (d, J ¼ 7.5 Hz, 1H,
CH3 Tos-pro), 2.52–2.58 (m, 1H, CHb-6/CHb-10 tasd), 2.72–2.80 CH-5 chrom.)
(m, 2H, CHa-7/CHa-9 tasd, CHa-7/CHa-9 tasd), 2.82–2.85
HRMS-ESI m/z [M + H]+ calc. for C22H26N3O2: 364.2019;
(m, 1H, CHb-7/CHb-9 tasd), 3.22–3.27 (m, 1H, CHb-7/CHb-9 found: 364.2018; anal. calcd for C24H27N3O6: C, 63.56; H, 6.00;
tasd), 3.32–3.38 (m, 1H, CHa-5 Tos-pro), 3.56–3.61 (m, 1H, CHb- N, 9.27; found C, 63.44; H, 5.91; N, 9.01.
5 Tos-pro), 3.99 (m, 1H, CH-4 chrom.), 4.15–4.22 (m, 1H, CHa-2
(S)-8-(Chroman-4-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-
chrom.), 4.40–4.45 (m, 1H, CHb-2 chrom.), 5.07 (d, J ¼ 7.1 Hz, one (ꢁ)-(S)-4. (S,S)-19 was added to a 30% (p/v) solution of
1H, CHa-2 tasd), 5.11 (d, J ¼ 7.1 Hz, 1H, CHb-2 tasd), 5.58 (dd, sodium methoxide in methanol. The mixture was stirred at
J ¼ 3.7, 8.9 Hz, 1H, CH-2 Tos-pro), 6.83 (d, J ¼ 7.5 Hz, 1H, CH-8 room temperature for 1 h. The solvent was evaporated and the
chrom.), 6.93 (dd, J ¼ 7.2, 7.4 Hz, 1H, CH-6 chrom.), 7.08 (t, residue was dissolved in H2O and extracted three times with
J ¼ 7.3 Hz, 1H, CH-4 arom. tasd), 7.12–7.18 (m, 3H, CH-7 CH2Cl2. The organic phase was washed with brine and dried
chrom., CH-2, CH-6 arom. tasd), 7.36–7.42 (m, 4H, CH-3, CH-5 over Na2SO4, and the solvent was removed under vacuum. The
arom. tasd, CH-3, CH-5 arom. Tos-pro), 7.55 (d, J ¼ 7.1 Hz; 1H, products were isolated without further purication (0.151 g,
CH-5 chrom.), 7.82 ppm (d, J ¼ 8.2 Hz, 2H, CH-2, CH-6 arom. 0.417 mmol, 96% yield).
Tos-pro).
Mp: 210–211 ꢀC; [a]D20 ¼ ꢁ41.6ꢀ (16 mg mlꢁ1 CH2Cl2); 1H
(R)-8-(Chroman-4-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4- NMR (400 MHz, CDCl3): d ¼ 1.63 (m, 1H, CHa-6/CHa-10 tasd),
one (+)-(R)-4. (R,S)-19 was added to a 30% (p/v) solution of 1.78 (m, 1H, CHa-6/CHa-10 tasd), 1.97–2.02 (m, 1H, CHa-3
sodium methoxide in methanol. The mixture was stirred at chrom.), 2.08–2.17 (m, 1H, CHb-3 chrom.), 2.56–2.64 (m, 2H,
room temperature for 1 h. The solvent was evaporated and the CHb-6/CHb-10 tasd, CHa-7/CHa-9 tasd), 2.80–2.88 (m, 3H,
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CHb-6/CHb-10 tasd, CHa-7/CHa-9 tasd, CHb-7/CHb-9 tasd), humidied air. When conuence was reached (3–4 days), cells
3.29–3.36 (m, 1H, CHb-7/CHb-9 tasd), 3.95 (dd, J ¼ 5.5, 9.0 Hz, were sub-cultured as required using trypsin/EDTA and used for
1H, CH-4 chrom.), 4.11–4.16 (m, 1H, CHa-2 chrom.), 4.34–4.39 experimentation. CHONOP stably expressing the Gaqi5 protein
(m, 1H, CHb-2 chrom.), 4.72 (q, J ¼ 4.2 Hz, 2H, CH2-2 tasd), 6.77 was seeded at a density of 50 000 cells per well into 96-well
(dd, J ¼ 0.8, 8.2 Hz, 1H, CH-8 chrom.), 6.85 (t, J ¼ 7.3 Hz, 1H, black, clear-bottom plates. Aer 24 hours incubation, the cells
CH-4 arom. tasd), 6.90 (ddd, J ¼ 0.8, 7.7, 8.2 Hz, 1H, CH-6 were loaded with a medium supplemented with 2.5 mM
chrom.), 6.94 (d, J ¼ 8.1 Hz; 2H, CH-2, CH-6 arom. tasd), 7.11 probenecid, 3 mM of the calcium sensitive uorescent dye Fluo-4
ꢀ
(ddd, J ¼ 1.2, 7.7, 8.2 Hz, 1H, CH-7 chrom.), 7.27 (bs, 1H, NH), AM and with 0.01% pluronic acid, for 30 min at 37 C. Aer-
7.33 (dd, J ¼ 7.5, 8.5 Hz, 2H, CH-3, CH-5 arom. tasd), 7.59 ppm wards the loading solution was aspirated and 100 ml per well of
(d, J ¼ 7.7 Hz, 1H, CH-5 chrom.); 13C NMR (400 MHz, CDCl3): d assay buffer, Hank's Balanced Salt Solution (HBSS) supple-
¼ 21.3 (C-3 chrom.), 29.0 (C-6/C-10 tasd), 29.5 (C-6/C-10 tasd), mented with 20 mM HEPES, 2.5 mM probenecid and 500 mM
42.2 (C-7/C-9 tasd), 47.9 (C-7/C-9 tasd), 58.5 (C-4 chrom.), 59.0 Brilliant Black, was added. Aer placing both plates (cell culture
(C-2 tasd), 59.1 (C-5 tasd), 65.0 (C-2 chrom.), 114.4 (C-2, C-6 and compound plate) into the FlexStation II, uorescence
arom. tasd), 116.4 (C-8 chrom.), 118.2 (C-4 arom. tasd), 120.0 changes were measured.
(C-6 chrom.), 123.7 (C-4a chrom.), 127.8 (C-7 chrom.), 128.3 (C-5
chrom.), 129.0 (C-3, C-5 arom. tasd), 142.9 (C-1 arom. tasd), mean ꢂ standard error of the mean (sem) of at least 3 experi-
155.5 (C-8a chrom.),178.3 ppm (C]O tasd). ments performed in duplicate. For potency values 95% con-
Data analysis and terminology. All data are expressed as
The free amine was then converted to the corresponding dence limits were indicated. Calcium mobilization data are
hydrogenoxalate hemihydrate salt (ꢁ)-S-4$H2C2O4$1/2H2O from expressed as uorescence intensity units (FIU) in percent over
acetone.
the baseline. Agonist potencies are given as pEC50 correspond-
Mp: 178–182 ꢀC; 1H-NMR (400 MHz, DMSO): d ¼ 1.80 (m, 2H, ing to the negative logarithm of the concentration of the agonist
CHa-6/CHa-10 tasd), 2.21 (m, 2H, CH2-3 chrom.), 2.55–2.91 that produces 50% of the maximal effect. Concentration
(m, 2H, CHa-6/CHa-10 tasd), 3.05–3.10 (m, 1H, CHa-7/CHa-9 response curves to agonists were tted by using the following
tasd), 3.26 (m, 1H, CHa-7/CHa-9 tasd), 3.43–3.50 (m, 1H, CHb-7/ equation:
CHb-9 tasd), 3.69–3.74 (m, 1H, CHb-7/CHb-9 tasd), 4.18–4.22
Emax ꢁ baseline
Effect ¼ baseline þ
(m, 1H, CHa-2 chrom.), 4.35–4.39 (m, 1H, CHb-2 chrom.), 4.56–
4.66 (m, 3H, CH2-2 tasd, CH-4 chrom.), 6.75–7.00 (m, 5H, CH-2,
CH-4, CH-6 arom. tasd; CH-6, CH-8 chrom.), 7.25 (m, 3H, CH-3,
CH-5 arom. tasd; CH-7 chrom.), 7.55 ppm (d, J ¼ 7.5 Hz, 1H,
CH-5 chrom.)
50ꢁXÞ$n
1 þ 10ðlog EC
where X is the agonist concentration and n is the Hill
coefficient.
Agonist efficacy is expressed as intrinsic activity (a) using
the maximal effect elicited by N/OFQ as an internal standard
(a ¼ 1.00).
HRMS-ESI m/z [M + H]+ calc. for C22H26N3O2: 364.2019;
found: 364.2016; anal. calcd for C24H27N3O6: C, 63.56; H, 6.00;
N, 9.27; found C, 63.31; H, 5.88; N, 9.03.
Antagonist potencies are given as pKB values. These were
derived from inhibition response curves and calculated,
assuming a competitive type of antagonism, using the following
equation:
Biology
General. N/OFQ used in this study was prepared and puried
as previously described.35 All cell culture media and supple-
ments were purchased from Invitrogen (Paisley, UK), SB-612111
was from Tocris Bioscience (Bristol, UK) and other reagents
were purchased from Sigma Chemical Co. (Poole, UK) or from
E. Merck (Darmstadt, Germany) and were of the highest purity
available. N/OFQ was solubilized in bidistilled water at a nal
concentration of 1 mM. All the tested compounds were solubi-
lized in dimethyl sulfoxide at a nal concentration of 10 mM.
Stock solutions of ligands were stored at ꢁ20 ꢀC. The successive
dilutions were made in HBSS–HEPES buffer (20 mM, containing
0.005% BSA fraction V).
IC50
ꢂ
!
pKB ¼ ꢁlog
ꢀ
ꢃ
1=n
ꢁ
n
½Aꢄ
2 þ
ꢁ 1
EC50
where IC50 is the concentration of the antagonist that produces
50% inhibition of the agonist response, [A] is the concentration
of the agonist, EC50 is the concentration of the agonist that
produces 50% of the maximal response and n is the Hill coef-
cient of the concentration response curve to the agonist.
Conclusions
Calcium mobilization assay. Chinese Hamster Ovary (CHO)
cells, stably co-expressing human recombinant NOP receptors In summary, we have developed several small molecule ligands
and the C-terminally modied Gaqi5 protein, were generated as for the NOP receptor, among them compound 4 demonstrated a
described by Camarda et al. (2009).23
good activity as a NOP receptor agonist. Then an enantiose-
The cells were cultured in culture medium consisting of parative method has been developed to obtain single enantio-
Dulbecco's MEM/HAMS F12 (50/50) supplemented with 10% mers of 4. Further biological studies have shown (R)-4 as the
foetal calfꢁ1serum, penicillin (100 IU mlꢁ1), streptomycin eutomer, indicating a clear stereospecic interaction. Docking
(100 mg ml ), geneticin (G418, 200 mg mlꢁ1) and hygromycin B studies were performed on the NOP receptor crystal structure in
(100 mg mlꢁ1). Cell cultures were kept at 37 ꢀC in 5% CO2/ order to understand the binding mode of the two stereoisomers
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Acknowledgements
We thank Dr Samuele Ciattini (Centro di Cristallograa Strut-
`
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collection.
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