Synthesis and pharmacological characterization of 5-phenyl-2-[2-(1- piperidinylcarbonyl)phenyl]-2,3-dihydro-1H-pyrrolo[1,2-c]imidazol-1-ones: A new class of Neuropeptide S antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.10.067

A new class of selective NPS antagonist was developed starting from a commercially available product identified by screening activities. Experimental NMR observations and computational experiments allowed the discovery of a new class of derivatives. 5-Phe

Full text of DOI:10.1016/j.bmcl.2010.10.067

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7308–7311
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and pharmacological characterization of 5-phenyl-2-[2-(1-piperidi-
nylcarbonyl)phenyl]-2,3-dihydro-1H-pyrrolo[1,2-c]imidazol-1-ones: A new class
of Neuropeptide S antagonists
Fabrizio Micheli a, , Romano Di Fabio a, Angelo Giacometti b, Adelheid Roth a, Elisa Moro b, Giancarlo Merlo a,
⇑
Alfredo Paio a, Emilio Merlo-Pich a, Silvia Tomelleri a, Federica Tonelli a, Paola Zarantonello a, Laura Zonzini a,
Anna Maria Capelli b
a Neurosciences Centre of Excellence, GlaxoSmithKline Medicines Research Centre, Via Fleming 4, 37135 Verona, Italy
b Molecular Discovery Research, GlaxoSmithKline Medicines Research Centre, Via Fleming 4, 37135 Verona, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of selective NPS antagonist was developed starting from a commercially available product
identified by screening activities. Experimental NMR observations and computational experiments
allowed the discovery of a new class of derivatives. 5-Phenyl-2-[2-(1-piperidinylcarbonyl)phenyl]-2,3-
dihydro-1H-pyrrolo[1,2-c]imidazol-1-one represents a new lead compound in the NPS antagonist field.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 20 September 2010
Revised 13 October 2010
Accepted 14 October 2010
Available online 21 October 2010
Keywords:
Neuropeptide S
NPS
GPR154
Antagonist
Neuropeptide S (NPSa)1–4 was the last endogenous peptide
identified via the reverse pharmacology approach. Human NPS is
a 20 residue peptide showing the following primary sequence:
SFRNGVGTGMKKTSFQRAKS. This sequence is also well conserved
among species; in particular, the serine (S) N-terminal residue of
NPS is conserved among all species examined so far.2
After its pairing with NPS, the previously orphan G-protein cou-
pled receptor (GPCR) GPR154 was named the NPS receptor and
abbreviated as NPSR.
This receptor exhibits overall low homology to other members
of the GPCR family. However, phylogenetic tree analysis of 7TM
receptors has shown close proximity of NPSR to the vasopressin/
oxytocin receptor family.5
limited.11–13 Molecular modeling studies dealing with potential
binding sites in the NPS receptor model for non peptide antago-
nists were also recently reported.14
Considering that non peptide antagonists like SHA-66 and SHA-
68 (Fig. 1) show high MW and lipophilicity (MW = 427 and
C log P15 of 4.6 for SHA-66; MW = 445 and C log P = 4.7 for SHA-
68), it might be hypothesized that these structures are not ideal
starting points for a medicinal chemistry exploration considering
their potential developability issues (e.g., potential low solubility)
for CNS indications. Screening activities performed in house in re-
combinant system (hGPR154 HEK) using FLIPR assay16,17 allowed
for the identification of the competitive antagonist compound 1,
which shows a pIC50 value of 7.5, comparable to SHA-66, but lower
In situ hybridization studies1,6 showed that NPS mRNA is heav-
ily expressed in a few brain regions only. These include the locus
coeruleus area and a few nuclei of the brainstem. The medicinal
chemistry interest in this field was elicited by the high number
of potential applications for NPS ligands, in particular due to its po-
tential involvement in several biological processes such as arousal,
anxiety, and food intake.7–9
O
N
O
O
O
H
N
N
N
N
O
H
R
O
A series of studies were recently reported10 on the neuropeptide
modification, while nonpeptide NPRS antagonists are quite
1
R = H, SHA-66
R = F, SHA-68
⇑
Corresponding author. Tel.: +39 045 8218515; fax: +39 045 8218196.
E-mail address: fabrizio.micheli@aptuit.com (F. Micheli).
Figure 1. NPS antagonists.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.067

F. Micheli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7308–7311
7309
MW and lipophilicity (MW = 374 and C log P = 4). Further selectiv-
ity studies confirmed its selectivity for this target and a detailed
chemical exploration around this lead was performed.
In this Letter, we report how an experimental observation led to
the discovery of an alternative lead series.
The high value of the amide proton chemical shift in the NMR
spectra of compound 1 clearly suggested the existence of a hydro-
gen bond interaction between such proton and an appropriate
acceptor.18 The value of the chemical shift remained unchanged
upon dilution of the sample and upon titration of DMSO to the
sample dissolved in chloroform (data not shown). This demon-
strated the intra-molecular character of this interaction.19 In order
to test whether or not the receptor affinity was linked to the afore-
mentioned folded conformation, a series of aromatic/heteroaro-
matic rings with different H-bond capabilities either as donor or
acceptor were prepared. The results of this exploration are re-
ported in Table 1.
Figure 2. Conformational studies performed on compound 1. In the left panel, the
lowest energy conformation is reported. Conf #1
DE = 0. s1 = À1.0 (O–C–C–N),
s2 = 22.8 (H–N–C@C),
s
3 = À112.6 (C@C–C@O). Boltzmann factor21 (Bf = 2.7 Â 10À1).
In the right panel, the first ‘trans’ conformation is reported. Conf #2
DE = 0.56 kcal/
mol (2.35 kJ/mol).
s
1 = À175.9,
s
2 = À26.7,
s
3 = 112.2. Bf = 1.0 Â 10À1
.
In agreement with the working hypothesis, the pyridyl scaffold
(2) demonstrated the best potency at the receptor, while the phe-
nyl (4) and thienyl (3) derivatives showed a substantial drop in the
NPSR potency. Finally, the pyrrolyl derivative (5) resulted com-
pletely inactive; this might be due to a potential clash between
the two acidic hydrogens.
To further refine this working hypothesis and to provide
rational basis for the exploitation of such results, in silico confor-
mational analyses20 were performed with low-mode conforma-
tional sampling in implicit water model.
Results of these studies demonstrated the capability of the
acidic hydrogen to interact with the furane oxygen rather then
with the carbonyl group of the piperidine amide because of the
particular geometry adopted by the template in the space as re-
ported in Figure 2.
Within a 3 kcal/mol energy window, the 37% of the conforma-
tions sampled for compound 1 are characterized by the presence
of such interaction.
The same computational analysis was performed for the pyridyl
derivative 2 and results are reported in Figure 3.
In this case, within a 3 kcal/mol energy window, 100% of the
conformations sampled had the possibility to show H-bonding
capabilities. As shown in the left panel of Figure 3, only the confor-
mation with the amide NH and the pyridyl N close in space were
populated. The same analysis demonstrate that, for the phenyl
derivative 4, an almost equal distribution of cis- and trans-confor-
mations were obtained with in silico calculations.
Based on these evidences, conformationally locked analogues of
derivative 1 were designed.
Three main structures were identified as ‘ideal’ candidates for
this task supported by conformational analyses. Their structures
Figure 3. Conformational studies performed on compound 2. In the left panel, the
lowest energy conformation is reported. Conf #1 E = 0.
1 = À2.9 (N–C–C–N),
(C@C–C@O). Boltzmann
factor21
D
s
s2 = 17.4
(H–N–C@C),
s3 = À111.7
(Bf = 4.3 Â 10À1). In the right panel, the first ‘trans’ conformation is reported. Conf
#17
D
E = 3.24 kcal/mol (13.53 kJ/mol).
s
1 = 137.6,
s2 = À24.9,
s3 = 112.6.
Bf = 3.4 Â 10À2
.
are reported in Figure 4 in order of synthetic feasibility from left
to right.
As it can be clearly appreciated from Figure 5 (left panel), the
compound 8 can be nicely overlapped on derivative 2, while for
structure 7 (Fig. 5, right panel) and structure 6 (data not shown),
the steric bulk of the newly formed ring might cause a clash within
the receptor pocket.
In agreement with the synthetic feasibility, the first compound
to be prepared was derivative 6.22 Results for this compound are
reported in Table 2.
Unfortunately, in agreement with the computational sugges-
tions, the compound was completely inactive when tested versus
the hNPSR.
The second compound to be prepared was derivative 7.23 Once
again, in agreement with what reported in the right part of
Figure 5, a negative result was observed, potentially suggesting
that either the steric bulk of the core ring or the change in the
spatial orientation of the pendant phenyl group prevented their
interaction in the receptor binding site.
Table 1
Finally, derivative 824 was prepared, in agreement to Scheme 1.
In this case, the compound showed the expected potency at the
hNPS receptor, confirming the working hypothesis and the compu-
tational predictions.
Affinity results for derivatives 1–516
O
N
H
X
O
N
O
O
O
N
N
N
N
Entry
X
hNPS
pIC50
N
N
O
O
O
SHA-66
NA
8.7 0.1
7.5 0.2
7.7 0.1
6.1 0.1
6.6 0.2
<5.3
N
N
N
1
2
3
4
5
2,5-Furanyl
2,6-Pyridyl
2,5-Thienyl
2,6-Phenyl
2,5-Pyrrolyl
6
7
8
Figure 4. The proposed conformationally locked templates.

7310
F. Micheli et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7308–7311
Acknowledgments
We would like to thank Dr. S. Braggio and Dr. S. Fontana and
their groups for the DMPK support received. Dr. Jens Klein and
Dr. E. Chiarparin are acknowledge for the NMR support. Finally,
Drs. G. Bonanomi, M. Biagetti, and L. Tarsi are acknowledged for
their synthetic support.
Figure 5. Left: Atom-based superimposition of the lowest energy conformation of
References and notes
compound
8 (atom type colored) on the 4th energy ranked conformation of
derivative 2 (green). Right: Atom-based superimposition of the lowest energy
conformation of compound 2 (green) on the 3rd energy ranked conformation of
derivative 7 (purple).
1. Xu, Y. L.; Reinscheid, R. K.; Huitron-Resendiz, S.; Clark, S. D.; Wang, Z.; Lin, S. H.;
Brucher, F. A.; Zeng, J.; Ly, N. K.; Henriksen, S. J.; de Lecea, L.; Civelli, O. Neuron
2004, 43, 487.
2. Reinscheid, R. K. Peptides 2007, 28, 830.
3. Civelli, O. Trends Pharmacol. Sci. 2005, 26, 15.
4. Reinscheid, R. K.; Xu, Y. L.; Okamura, N.; Zeng, J.; Chung, S.; Pai, R.; Wang, Z.;
Civelli, O. J. Pharmacol. Exp. Ther. 2005, 315, 1338.
5. Surgand, J.-S.; Rodrigo, J.; Kellenberger, E.; Rognan, D. Proteins: Struct. Funct.
Bioinformatics 2006, 62, 509.
6. Xu, Y. L.; Gall, C. M.; Jackson, V. R.; Civelli, O.; Reinscheid, R. K. J. Comp. Neurol.
2007, 500, 84.
7. Okamura, N.; Reinscheid, R. K. Stress 2007, 10, 221.
8. Smith, K. L.; Patterson, M.; Dhillo, W. S.; Patel, S. R.; Semjonous, N. M.; Gardiner,
J. V.; Ghatei, M. A.; Bloom, S. R. Endocrinology 2006, 147, 3510.
9. Gloriam, D. E.; Schioth, H. B.; Fredriksson, R. Biochim. Biophys. Acta Gen. Subj.
2005, 1722, 235.
Table 2
Affinity results for derivatives 6–816
Entry
hNPS
pIC50
6
7
8
<5.3
<5.3
8.0 0.1
10. Roth, A.; Marzola, E.; Rizzi, A.; Arduin, M.; Trapella, C.; Corti, C.; Vergura, R.;
Martinelli, P.; Salvadori, S.; Regoli, D.; Corsi, M.; Cavanni, P.; Calo, G.; Guerrini,
R. J. Biol. Chem. 2006, 281, 20809.
11. Fukatsu, K.; Nakayama, Y.; Tarui, N.; Mori, M.; Matsumoto, H.; Kurasawa, O.;
Banno, H. PCT Int. Appl.: WO 2005021555, 2006.
O
Ph
N
H
O
NH
8
Ph
COOH
N
H
12. Okamura, N.; Habay, S. A.; Zeng, J.; Chamberlin, A. R.; Reinscheid, R. K. J.
Pharmacol. Exp. Ther. 2008, 325, 893.
N
i
ii
13. Zhang, Y.; Gilmour, B. P.; Navarro, H. A.; Runyon, S. P. Bioorg. Med. Chem. Lett.
2008, 18, 4064.
9
10
14. Dal Ben, D.; Antonini, I.; Buccioni, M.; Lambertucci, C.; Marucci, G.; Vittori, S.;
Volpini, R.; Cristalli, G. Chem. Med. Chem. 2010, 5, 371.
15. Daylight 4.81, http://www.daylight.com.
Scheme 1. Preparation of compound 8. (i) SOCl2, 2-(1-piperidinylcarbonyl)aniline;
(ii) ICH2Cl, DMF.
16. The compounds functional activity was tested on recombinant human GPR154
receptors, transiently expressed in HEK293 cells. Briefly: Cells were seeded at a
density of 18.000 cells/well in 384-well plates and incubated at 37 °C, 5% CO2
for 24 h before the experiment.
Considering the results reported in Table 2, it might be hypoth-
esized that, within the NPS receptor, very stringent conformational
requirements have to be met to achieve the desired interactions
and that minimal variations to the structure might lead to poten-
tial steric clashes with the amino-acid residues in the binding site.
The identification of a small, rigid, non peptidic hNPSR antago-
nist elicited the interest to derivative 8.
Accordingly, the molecule was better characterized in the pro-
gramme screening cascade; the CYPEX bactosome P450 inhibition
and rat and human in vitro clearance in liver microsomes were
performed to further evaluate its developability potential.
IC50 values for all major P450 isoforms tested (CYP1A2, CYP2C9,
On the day of the experiment, cells were loaded with the fluorescent calcium
indicator dye FLUO-4-AM for 1 h at 37 °C, after which the dye was removed by
washing cells with HBSS. Compound modulation of intracellular calcium levels,
induced by an EC80 concentration of Neuropeptide S, was assessed using
FLIPR.17 Concentration response data are expressed in terms of percentage
response relative to a maximum test concentration of NPS (30 nM final concn)
calculated from a maximum minus minimum of the relative fluorescence units,
and pIC50 values were determined by nonlinear regression analysis.
17. Sullivan, E.; Tucker, E. M.; Dale, I. L. In Calcium Signaling Protocols; Lambert, D.
G., Ed.; Humana: Totowa, 1999; p p 125.
18. Wagner, G.; Pardi, A.; Wuethrich, K. J. Am. Chem. Soc. 1983, 105, 5948.
19. Yang, D.; Qu, J.; Li, B.; Ng, F.-F.; Wang, X.-C.; Cheung, K.-K.; Wang, D.-P.; Wu, Y.-
D. J. Am. Chem. Soc. 1999, 121, 589.
20. Conformational analyses were carried out with BatchMin V9.5 and V9.6
(Schrodinger, http://www.schrodinger.com). Conformational sampling was
done with Low-frequency-Mode Conformational Search (LMCS keyword),
OPLS_2005 force field, implicit water model. 1000 Monte Carlo steps per
rotatable bond, 10,000 minimization steps and 50 kJ/mol energy window were
set. A root mean square deviation cutoff of 0.5 Å was utilized to discard
duplicated conformations. Conformations were visually inspected within
Maestro (Schrodinger). Atom-based superimpositions were performed with
the use of the routines available within Maestro.
CYP2C19, CYP2D6, and CYP3A4) were greater than 5 lM. Intrinsic
clearance (Cli) values both in human and in rat resulted relatively
high (6.9 and 26.3) ml/min/g of protein. This result was quite ex-
pected considering that the template was completely not substi-
tuted and therefore potentially prone to metabolic degradation.
In vitro results found confirmation in in vivo studies in rat.25
Derivative 8 (1 mg/kg, po, 5%DMSO + 0.5% HPMC in water) actually
showed high blood clearance (69 ml/min/kg), a relatively low half-
life (0.7 h), a moderate distribution volume (2.4 l/kg) and a low
bioavailability (F = 1%). Brain/blood ratio was 0.3.
In summary, the exploitation of the experimental observations
relative to a HTS hit allowed the identification of a promising
new scaffold. The new compound is a low molecular weight, non
peptidic hNPSR antagonist.
21. The Boltzmann factor (Bf) of each conformation j was calculated as the ratio
*
*
between p(j)/totp where p(j) = exp(À1 ((energy(j) À energy(lowest)/(R T)))
and totp = sum(p(j)) over all the conformations sampled within 50 kJ/mol
energy window, energy(j) is the energy of the conformation j, energy(lowest) is
the energy of the lowest energy conformation, R = 8.31434/1000 and T = 300 K.
22. 7-Phenyl-2-[2-(1-piperidinylcarbonyl)phenyl]-2,3,4,5-tetrahydro-1H-
pyrrolo[1,2-a][1,4]diazepin-1-one 6. To
a solution of the commercially
available methyl 5-phenyl-1H-pyrrole-2-carboxylate, in dry DMF at 0 °C
under inert atmosphere, NaH was added. Subsequently, 1-bromo-3-
chloropropane was added and the resulting solution was stirred at 60 °C for
3 h. After quenching and work-up, the intermediate compound was dissolved
in ammonia (7 N in MeOH) and stirred at 65 °C for 20 h. The reaction
intermediate was cyclized with EtONa in EtOH (2 h, 60 °C) and the resulting 7-
phenyl-2,3,4,5-tetrahydro-1H-pyrrolo[1,2-a][1,4]diazepin-1-one was coupled
to 1-[(2-bromophenyl)carbonyl]piperidine in a mixture of dry 1,4-dioxane/
DMSO at 120 °C for 4 h using CuI, K3PO4 and dimethylethane diamine to give
the desired product.
The use of computational chemistry was critical to design the
new scaffold and a good agreement between experimental findings
and calculations was observed.
Further refinements have to be performed to transform the new
scaffold in a potential lead series for in vivo experimental activities.

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7311
23. 6-Phenyl-2-[2-(1-piperidinylcarbonyl)phenyl]-3,4-dihydropyrrolo[1,2-
a]pyrazin-1(2H)-one 7. To a solution of the commercially available methyl 5-
phenyl-1H-pyrrole-2-carboxylate, in dry DMF at 0 °C under inert atmpsphere,
NaH was added. Subsequently, bromoacetonitrile was added and the resulting
solution was stirred at rt °C for 3 h. After quenching and work up, the resulting
nitrile was reduced using NaBH4 and CoCl2 in MeOH. The resulting amino
derivative was cyclized with EtONa and EtOH to give 6-phenyl-3,4-
dihydropyrrolo[1,2-a]pyrazin-1(2H)-one. This intermediate was coupled with
1-[(2-bromophenyl)carbonyl]piperidine as described in Ref. 14 to give the title
material.
and the solution was added drop-wise to a previously prepared solution of [2-
(1-piperidinylcarbonyl)phenyl]amine (commercially available) and Et3N in dry
THF at 0 °C. After quenching and work-up, the resulting -phenyl-N-[2-(1-
piperidinylcarbonyl)phenyl]-1H-pyrrole-2-carboxamide was suspended in
DMF at 0 °C under a N2 blanket. NaH was added and the mixture stirred for
0.3 h; iodochlorometane was added drop-wise and the reaction was stirred for
24 h at 60 °C to give, after quenching and work-up, the title compound.
25. All the works involving animals were carried out in accordance with European
directive 86/609/EEC governing animal welfare and protection, which is
acknowledged by Italian Legislative Decree no. 116, 27 January 1992, and
according to internal review performed by the GlaxoSmithKline Committee on
Animal Research & Ethics (CARE) and to the company Policy on the Care and
Use of Laboratory Animals.
24. 5-Phenyl-2-[2-(1-piperidinylcarbonyl)phenyl]-2,3-dihydro-1H-pyrrolo[1,2-
c]imidazol-1-one 8. Thionyl chloride was added to the commercially available
5-phenyl-1H-pyrrole-2-carboxylic acid and the resulting mixture was stirred
at rt for 2 h. After work-up, the resulting acyl chloride was dissolved in dry THF