Synthesis of the calcilytic ligand NPS 2143

Article abstract of DOI:10.3762/bjoc.9.154

(R)-3 (NPS 2143) is a negative allosteric modulator of the human calcium-sensing receptor (CaSR) and as such represents an important pharmacological tool compound for studying the CaSR. Herein, we disclose for the first time a complete experimental description, detailed characterisation and assessment of enantiomeric purity for (R)-3. An efficient, reproducible and scalable synthesis of (R)-3 that requires a minimum of chromatographic purification steps is presented. (R)-3 was obtained in excellent optical purity (er > 99:1) as demonstrated by chiral HPLC and the pharmacological profile for (R)-3 is in full accordance with that reported in the literature.

Full text of DOI:10.3762/bjoc.9.154

Synthesis of the calcilytic ligand NPS 2143
Henrik Johansson1, Thomas Cailly1,2, Alex Rojas Bie Thomsen1,
Hans Bräuner-Osborne1 and Daniel Sejer Pedersen*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 1383–1387.
1Department of Drug Design and Pharmacology, University of
Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
and 2Université de Caen Basse-Normandie, CERMN (EA 4258 - FR
CNRS 3038 INC3M - SF 4206 ICORE) UFR des Sciences
Pharmaceutiques, Bd Becquerel, F-14032 Caen, France
doi:10.3762/bjoc.9.154
Received: 09 April 2013
Accepted: 11 June 2013
Published: 09 July 2013
Email:
Associate Editor: M. Rueping
Daniel Sejer Pedersen* - daniel.pedersen@sund.ku.dk
© 2013 Johansson et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
epoxides; GPCR; NPS 2143; nucleophilic aromatic substitution;
pyrylium chemistry
Abstract
(R)-3 (NPS 2143) is a negative allosteric modulator of the human calcium-sensing receptor (CaSR) and as such represents an
important pharmacological tool compound for studying the CaSR. Herein, we disclose for the first time a complete experimental
description, detailed characterisation and assessment of enantiomeric purity for (R)-3. An efficient, reproducible and scalable syn-
thesis of (R)-3 that requires a minimum of chromatographic purification steps is presented. (R)-3 was obtained in excellent optical
purity (er > 99:1) as demonstrated by chiral HPLC and the pharmacological profile for (R)-3 is in full accordance with that reported
in the literature.
Introduction
The first G-protein-coupled receptors (GPCRs) were identified acid type B (GABAB) receptors, three taste (TR) receptors, the
more than 30 years ago and have since grown to become the GPRC6A receptor, the calcium-sensing receptor (CaSR), and
largest class of membrane-bound receptors and the single most seven orphan receptors [4]. Class C GPCRs have proven viable
common target type in drug discovery and therapy [1,2]. as drug targets, exemplified by the marketed GABAB agonist
Excluding olfactory receptors there are 390 genes encoding baclofen, and by cinacalcet (1), a positive allosteric modulator
GPCRs in the human genome, divided into three major classes on the CaSR (1, Figure 1) [5].
(A, B and C) of which the rhodopsin-like class A is the largest
and most diverse, and which has been well-studied and subject The CaSR is the main regulator of serum calcium homeostasis
to drug targeting [3]. GPCR class C is much smaller and and plays a central role in several cellular processes such as
comprises only 22 known receptors including eight secretion and regulation of peptide hormones (most importantly
metabotropic glutamate (mGlu) receptors, two γ-aminobutyric parathyroid hormone), ion-channel activity and apoptosis in
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Scheme 1: Strategy for assembling (R)-3 from fragments 4, 5 and 6.
m-Ns = m-nitrobenzenesulfonyl.
Figure 1: Marketed calcium-sensing receptor agonist cinacalcet (1),
and CaSR antagonists Calhex 231 (2) and NPS 2143 ((R)-3).
The synthesis of amine 6 was accomplished from commercially
available 2-cyanonaphthalene (7) in four steps (Scheme 2).
certain cell types [6-8]. The CaSR is involved in several Reduction of 7 was easily achieved on multigram scale by using
calcium-regulation-related diseases including various types of LiAlH4 to provide amine 8 in excellent yield and purity after
hypo- and hyperparathyroidism, and its pronounced regulation work-up. According to the method of Katritzky et al. [16] amine
of parathyroid hormone secretion makes it an interesting target 8 was activated as the corresponding pyridinium salt 10 upon
for drug discovery [9]. Several potent CaSR modulators have treatment with triphenylpyrylium salt 9 in ethanol under reflux.
been reported over the past decade, including the negative Subsequently, the crude pyridinium salt 10 was exposed to the
allosteric modulators (calcilytics) 2 [10] and (R)-3 (Figure 1) sodium salt of 2-nitropropane (11) to give nitro compound 12.
[11,12].
Reduction of the nitro group in 12 by catalytic hydrogenation at
atmospheric pressure to produce amine 6 has previously been
To facilitate our pharmacological research program on the described by Kamal and Chouhan [17] but was unsuccessful in
CaSR we required calcilytic (R)-3 in quantities of several grams our hands, returning the starting material under a variety of
as a pharmacological tool compound. We quickly discovered conditions. Eventually, the reduction was realised using zinc
that the published [12-14] experimental details and characterisa- and HCl in ethanol to give amine 6 in good yield after
tion for ligand (R)-3 were unsatisfactory, and we were unable to chromatographic purification.
reproduce several of the reported synthetic procedures. More-
over, we were surprised to discover the lack of assessment of
optical purity for (R)-3. Considering that the R-enantiomer of
(R)-3 and related aminoalcohols display both significantly
higher potency and target selectivity than their S-enantiomers,
we believe that this is a critical shortcoming in the previously
reported syntheses [13,14].
Herein, we wish to report a detailed, reproducible and scalable
synthesis for racemic and enantiopure calcilytic agent (R)-3,
including detailed spectroscopic, chromatographic and pharma-
cological characterisation.
Results and Discussion
We decided to synthesise (R)-3 using the same overall strategy
published by others employing the three fragments depicted in
Scheme 1. Similar to a previously reported synthesis of (R)-3
[13], we decided to activate epoxide 5 as the m-nosyl derivative
Scheme 2: Synthesis of amine building block 6 by using Katritzky’s
pyrylium chemistry [16].
that has been shown to minimise racemisation during epoxide
ring opening [15].
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Next we turned our attention to the synthesis of phenol 4 formed by nucleophilic attack on the more sterically hindered
(Scheme 3). Previously, 4 has been synthesised by nucleophilic epoxide carbon. Fortunately, the side product could be removed
aromatic substitution on commercially available aryl fluoride 13 by recrystallization of the hydrochloric salt of 3 to produce
using crown ether and potassium acetate in acetonitrile [14]. 3·HCl of excellent purity and in good overall yield.
Unfortunately, we found it difficult to drive the reaction to
completion using the published method, obtaining only low
yields of acetate 14 even under forcing conditions. However,
performing the reaction in DMSO with potassium acetate,
conditions that we have previously applied successfully [18],
gave a clean reaction and high yield of acetate 14. Basic hydro-
lysis of crude acetate 14 produced phenol 4 of high purity and
in good yield after aqueous work-up.
Scheme 3: Synthesis of phenol 4 from commercially available aryl
fluoride 13.
Scheme 4: Synthesis of rac-3 and (R)-3 from commercially available
racemic- and (S)-glycidol 15, respectively. Only the optically active
compounds are depicted.
With compounds 4 and 6 in hand we turned our attention to the
synthesis of the remaining building block 5 (Scheme 4). In
order to accurately determine the optical purity of the target Analysis of optical purity by chiral HPLC showed that (R)-3
molecule (R)-3 by chiral HPLC, we performed the synthesis of was of excellent purity with an er > 99:1 (Figure 2).
racemic rac-3 and optically pure (R)-3 in parallel using the
method by Marquis et al. [13]. Commercially available glycidol
15 activated as the m-nitrobenzene sulfonyl (m-nosyl) ester 5
has previously been shown to be an ideal leaving group in reac-
tions with aryloxy nucleophiles (e.g., 4) as it promotes direct
SN2 over SN2’ attack thereby suppressing racemisation [15].
m-Nosylate 5 was synthesised by using nosyl chloride at low
temperature [19]. Upon cooling, the reaction mixture became a
viscous slurry, and it was important to maintain vigorous stir-
ring to ensure full conversion. In this manner, a spectroscopi-
cally pure product could be obtained in quantitative yield after
simple aqueous work-up (e.g., rac-5). Alternatively, any
residual alcohol 15 could be removed by chromatographic
purification (e.g., (R)-5). Next, the activated glycidols 5 were
treated with phenol 4 in acetone under reflux and basic condi-
tions to provide epoxides 16. Epoxides 16 were obtained in
good yield after purification by column chromatography and
subsequent recrystallization to remove residual starting ma-
terial 5. Finally the target molecules rac-3 and (R)-3 were
synthesised by ring-opening epoxides 16 with amine 6 in
ethanol under reflux. 1H NMR analysis of the crude product
Figure 2: Determination of the optical purity for (R)-3 by chiral HPLC
on a Daicel AD-H column. Top: Optically enriched product (er > 99:1).
Bottom: Racemic sample.
showed the presence of approximately 5–10% of a structurally Pharmacological testing of (R)-3 was performed as previously
similar side-product that proved extremely difficult to remove described [20]. (R)-3 showed inhibition of calcium-stimulated
by chromatography. The side product is likely the regioisomer D-myo-inositol monophosphate (IP1) accumulation in a concen-
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Beilstein J. Org. Chem. 2013, 9, 1383–1387.
tration-dependent manner, with a potency consistent with Pharma A/S, Aase og Ejnar Danielsens Fond, and Beckett-
that reported in the literature [21,22] (IC50 = 0.64 μM, Fonden.
pIC50 = 6.3 ± 0.2), thus confirming its biological activity
References
1. Lagerström, M. C.; Schiöth, H. B. Nat. Rev. Drug Discovery 2008, 7,
(Figure 3).
339–357. doi:10.1038/nrd2518
2. Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L.
Nat. Rev. Drug Discovery 2006, 5, 993–996. doi:10.1038/nrd2199
3. Congreve, M.; Langmead, C. J.; Mason, J. S.; Marshall, F. H.
J. Med. Chem. 2011, 54, 4283–4311. doi:10.1021/jm200371q
4. Wellendorph, P.; Bräuner-Osborne, H. Br. J. Pharmacol. 2009, 156,
869–884. doi:10.1111/j.1476-5381.2008.00078.x
5. Bräuner-Osborne, H.; Wellendorph, P.; Jensen, A. A.
Curr. Drug Targets 2007, 8, 169–184.
doi:10.2174/138945007779315614
6. Tfelt-Hansen, J.; Brown, E. M. Crit. Rev. Clin. Lab. Sci. 2005, 42,
35–70. doi:10.1080/10408360590886606
7. Brown, E. M.; MacLeod, R. J. Physiol. Rev. 2001, 81, 239–297.
8. Feng, J.; Petersen, C. D.; Coy, D. H.; Jiang, J.-K.; Thomas, C. J.;
Pollak, M. R.; Wank, S. A. Proc. Natl. Acad. Sci. U. S. A. 2010, 107,
17791–17796. doi:10.1073/pnas.1009078107
9. Chattopadhyay, N.; Brown, E. M. Mol. Genet. Metab. 2006, 89,
189–202. doi:10.1016/j.ymgme.2006.07.003
Figure 3: Characterisation of concentration-dependent (R)-3 inhibition
of 3.5 mM calcium-stimulated IP1 response in HEK293 cells stably
transfected with rat CaSR. The graph is representative of three inde-
pendent experiments.
10.Kessler, A.; Faure, H.; Petrel, C.; Rognan, D.; Césario, M.; Ruat, M.;
Dauban, P.; Dodd, R. H. J. Med. Chem. 2006, 49, 5119–5128.
doi:10.1021/jm051233+
11.Gowen, M.; Stroup, G. B.; Dodds, R. A.; James, I. E.; Votta, B. J.;
Smith, B. R.; Bhatnagar, P. K.; Lago, A. M.; Callahan, J. F.;
DelMar, E. G.; Miller, M. A.; Nemeth, E. F.; Fox, J. J. Clin. Invest. 2000,
105, 1595–1604. doi:10.1172/JCI9038
Conclusion
Herein, for the first time, we disclose a detailed, reproducible
and scalable synthetic procedure for the calcilytic ligand (R)-3
(NPS 2143) making this important pharmacological tool com-
pound readily available to the scientific community. All com-
pounds have been characterised carefully and the optical purity
of (R)-3 was deemed excellent by chiral HPLC (er > 99:1).
Moreover, the pharmacological profile of (R)-3 was consistent
with that reported in the literature.
12.Nemeth, E. F.; Delmar, E. G.; Heaton, W. L.; Miller, M. A.;
Lambert, L. D.; Conklin, R. L.; Gowen, M.; Gleason, J. G.;
Bhatnagar, P. K.; Fox, J. J. Pharmacol. Exp. Ther. 2001, 299,
323–331.
13.Marquis, R. W.; Lago, A. M.; Callahan, J. F.; Trout, R. E. L.;
Gowen, M.; DelMar, E. G.; Van Wagenen, B. C.; Logan, S.;
Shimizu, S.; Fox, J.; Nemeth, E. F.; Yang, Z.; Roethke, T.; Smith, B. R.;
Ward, K. W.; Lee, J.; Keenan, R. M.; Bhatnagar, P. J. Med. Chem.
2009, 52, 3982–3993. doi:10.1021/jm900364m
14.Del Mar, E. G.; Barmore, R. M.; Sheehan, D.; Van Wagenen, B. C.;
Callahan, J. F.; Keenan, R. M.; Kotecha, N. R.; Lago, M. A.;
Southall, L. S.; Thompson, M. Method of using calcilytic compounds.
U.S. Patent US6,022,894, Feb 8, 2000.
Supporting Information
1H and 13C NMR spectra and HPLC chromatograms for
compounds 3, 4, 5, 6, 8, and 16. Chromatograms from
chiral HPLC analysis of rac-3 and (R)-3 and experimental
details for the pharmacological characterisation for (R)-3.
15.Klunder, J. M.; Onami, T.; Sharpless, K. B. J. Org. Chem. 1989, 54,
1295–1304. doi:10.1021/jo00267a014
16.Katritzky, A. R.; De Ville, G.; Patel, R. C. Tetrahedron 1981, 37, 25–30.
doi:10.1016/0040-4020(81)85037-5
Supporting Information File 1
Experimental procedures and full characterisation of the
calcilytic ligand NPS 2143.
17.Kamal, A.; Chouhan, G. Tetrahedron: Asymmetry 2005, 16,
2784–2789. doi:10.1016/j.tetasy.2005.07.022
18.Johansson, H.; Jørgensen, T. B.; Gloriam, D. E.; Bräuner-Osborne, H.;
Sejer Pedersen, D. RSC Adv. 2013, 3, 945–960.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-154-S1.pdf]
doi:10.1039/c2ra21902f
19.Fagerström, A.; Nilsson, M.; Berg, U.; Isaksson, R. Org. Biomol. Chem.
2006, 4, 3067–3076. doi:10.1039/b605603b
20.Thomsen, A. R. B.; Hvidtfeldt, M.; Bräuner-Osborne, H. Cell Calcium
2012, 51, 107–116. doi:10.1016/j.ceca.2011.11.009
21.Petrel, C.; Kessler, A.; Dauban, P.; Dodd, R. H.; Rognan, D.; Ruat, M.
J. Biol. Chem. 2004, 279, 18990–18997. doi:10.1074/jbc.M400724200
Acknowledgements
This work was supported by the Danish Council for Inde-
pendent Research – Medical Sciences, the Lundbeck Founda-
tion, the Carlsberg Foundation, Drug Research Academy, LEO
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Beilstein J. Org. Chem. 2013, 9, 1383–1387.
22.Faure, H.; Gorojankina, T.; Rice, N.; Dauban, P.; Dodd, R. H.;
Bräuner-Osborne, H.; Rognan, D.; Ruat, M. Cell Calcium 2009, 46,
323–332. doi:10.1016/j.ceca.2009.09.004
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