Successful bridging from a peptide to a non peptide antagonist at the human tachykinin NK-2 receptor

Article abstract of DOI:10.1016/S0960-894X(02)00539-5

Non peptide products have been found to show nanomolar binding and functional affinities at the human tachykinin NK-2 receptor. The new antagonists do not possess stereogenic centers and their thermal behaviour in solution is featured by a peculiar set of conformational stereoisomers. A macroscopic viewpoint is preferentially adopted to rationalize the obtained results.

Full text of DOI:10.1016/S0960-894X(02)00539-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2945–2948
Successful Bridging from a Peptide to a Non Peptide Antagonist
at the Human Tachykinin NK-2 Receptor
Maria Altamura, Franca Canfarini, Rose-Marie Catalioto, Antonio Guidi,*
Franco Pasqui, Anna R. Renzetti, Antonio Triolo and Carlo A. Maggi
Menarini Ricerche S.p.A., Via dei Sette Santi 3, 50131 Florence, Italy
Received 30 April 2002; revised 20 June 2002; accepted 4 July 2002
Abstract—Non peptide products have been found to show nanomolar binding and functional affinities at the human tachykinin
NK-2 receptor. The new antagonists do not possess stereogenic centers and their thermal behaviour in solution is featured by a
peculiar set of conformational stereoisomers. A macroscopic viewpoint is preferentially adopted to rationalize the obtained results.
# 2002 Elsevier Science Ltd. All rights reserved.
The tachykinins form a family of peptide neuro-
trasmitters which share a common carboxy terminal
sequence, Phe-Xxx-Gly-Leu-Met-NH2, and Neurokinin
A (NKA, Xxx=Val) is the tachykinin preferentially
acting at the NK-2 receptor whose activation in mam-
mals is thought to play a role in the regulation of
airways, gut and urinary tract motility.1,2
antagonist could be essential for the bioactivity and is
characterized by the pseudosymmetric juxtaposition of
a second benzyl group in the place of the Leu-Met
containing fragment.
Results and Discussion
Figure 1. Molecular structure of two potent and selective antagonists
at the hNK-2 receptor.
Figure 2. Early rationale to bridge from a peptide to a non peptide
antagonist at the hNK-2 receptor.
Amongst the known most potent and selective antago-
nists3 of NKA at the human NK-2 (hNK-2) receptor,
MEN10627, and MEN11558 have been disclosed by our
research team (Fig. 1). Particularly, MEN10627 was
rationally derived from the structure of the natural
agonist,3a while MEN115583d stemmed from the insight
that only one of the two cycles present in the bicyclic
During our study on MEN 11558 analogues,4 it was
noted that 1 (Fig. 2) retained the potency of the parent
compound (Table 1) and it was proposed to use the
exocyclic diamide framework to access to a fully non
peptide antagonist; on the other hand, a product of
general structure x could furnish an entry to the use of
the benzodiazepine scaffold, the most famous amongst
the so-called privileged platforms,5 because of the
possibility of an intramolecular hydrogen bond (Fig. 2).
*Corresponding author. Tel.: +39-055-568-0757; fax: +39-055-568-
0419; e-mail: aguidi@menarini-ricerche.it
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00539-5

2946
M. Altamura et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2945–2948
Early biological results, however, showed that the
arrangement with two tertiary amides was preferred
over that concerning one secondary and one tertiary
amide, and eventually the double tertiary amide strategy
resulted successful (Table 1), allowing us to obtain the
antagonists 5 and 6, endowed with nanomolar potency
at the hNK-2 receptor and featured by a molecular frame-
work inconsistent with those of the other non peptide
NK-2 antagonists.3fꢀi,6
The secondary amine 7, necessary to synthesize 5 and 6,
was obtained as reported in Scheme 1. The final cou-
pling was accomplished by making o-phthaloyl dichlor-
ide to react with a mixture of N-methyl tryptamine and
7 in the presence of triethylamine, this technique being
possible because the polarity difference of the three
resulting amides permitted a facile chromatographic
separation.
Scheme 1. (a) Thiomorpholine, PyBrop, TEA, DCM; (b) HCl, ethyl
acetate, Et2O 29%, as the hydrochloride, two steps; (c) 3-indolylacetic
acid, PyBop, TEA, DCM; (d) LAH, THF reflux. 83%, two steps; (e)
N-methyltryptamine, TEA, DCM, FCC, followed by preparative
HPLC for 6.
Table 1. hNK-2 receptor binding affinity (pKi) and in vitro func-
tional activity (pA2) for the reference peptides and the new ligands
9
Compd7
Structure
pKi
pA2
In order to make the synthetic route more expeditious,
we had also thought to perform a solvent-free double
amidation10 of dimethyl phthalate. Unfortunately, this
idea was frustrated by the unexpected results obtained
in the simplified experiment (Scheme 2) involving only
one secondary amine. Briefly, the disappearance of
dimethyl phthalate corresponded to the formation of
three new products, identified as phthalamic acid 8,
N,N0-dimethyltryptamine, and [2-(1H-Indol-3-yl)-ethyl]-
trimethyl-ammonium salt.11
MEN 11558
See Figure 1
8.7
na
1
2
See Figure 2
8.7
6.3
nt
nt
The structural features of the non peptides ligands
reported in Table 1 deserve consideration. If at a first
glance, compounds 2–6 can be imagined as highly flex-
ible molecules, that is not completely true. o-Phthaloyl
tertiary diamides are known for the discrete pattern of
their conformational isomers,12 a characteristic which
has been claimed to be potentially useful for the con-
struction of molecular switches.13 This peculiarity arises
from the 2-fold combination of cis/trans amide iso-
merization with the rotation around the CAr-CONR1R2
bond. Indeed, the 500 MHz PMR spectra of compounds
3–6 are coherent14 with those expected on the base of
literature findings.12,13 In addition, we report that four
isomers are observed in the PMR spectrum of 5, and
that 4 isomers resulted physically isolable to some
extent in the case of compound 9 (Fig. 3),7 an analogue
with sub-micromolar binding affinity.8 The chromato-
gram of 9, obtained under our standard, non chiroptical
HPLC conditions,15 consists of four peaks in relative
amounts similar to those measured in the 500 MHz
3
4
5
6
6.9
nt
7.5
nt
8.08.0
8.4
8.4
Scheme 2. (a) N-methyltryptamine, two molar equivalents, 90–100 ꢁC
without solvent. R=(1H-Indol-3-yl)-ethyl residue. Seemingly,
X=CF3COO under the conditions of HPLC analysis and chlorine
after extraction of the crude with aqueous hydrochloric acid.

M. Altamura et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2945–2948
2947
PMR by integration of the four N-methyl singlets, and a
clear separation afforded also by TLC on silica (10%
methanol in ethyl acetate as eluent); an observation that
allowed us to accomplish a partial separation by FCC
and to observe the re-equilibration of the solutes in the
collected fractions.16
progress aiming at their complete pharmacological eval-
uation, with particular attention to their selectivity pro-
file. The results of this investigation will be reported in
due time.
Acknowledgements
The isomeric nature of the four chromatographic peaks
was then proven by LC–MS analysis which showed the
same protonated quasi-molecular ions, featured by iso-
topic patterns in agreement with the presence of four
chlorine atoms and superimposable product ion tandem
mass spectra.
Thanks are due to Dr. Giuseppe Balacco and Mr.
Marco Guelfi for their technical assistance.
References and Notes
1. Maggi, C. A. Gen. Pharmac. 1995, 26, 911.
2. For the therapeutic relevance of tachykinin NK-2 receptor
selective antagonists in humans, see: (a) Van Schoor, J.; Joos,
G. F.; Chasson, B. L.; Brouard, R. J.; Pauwels, R. A. Eur.
Respir. J. 1998, 12, 17. (b) Lordal, M.; Navalesi, G.; Theo-
dorsson, E.; Maggi, C. A.; Hellstrom, P. M. Br. J. Pharmacol.
2001, 134, 215.
3. (a) Pavone, V.; Lombardi, A.; Nastri, F.; Saviano, M.;
Maglio, O.; D’Auria, G.; Quartara, L.; Maggi, C. A.; Pedone,
C. J. Chem. Soc., Perkin Trans. 2 1995, 987. (b) Maggi, C. A.;
Astolfi, M.; Giuliani, S.; Goso, C.; Manzini, S.; Meini, S.;
Patacchini, R.; Pavone, V.; Pedone, C.; Quartara, L.; Renzetti,
A. R.; Giachetti, A. J. Pharmacol. Exp. Ther. 1994, 271, 1489.
(c) Catalioto, R.-M.; Criscuoli, M.; Cucchi, P.; Giachetti, A.;
Giuliani, S.; Lecci, A.; Lippi, A.; Patacchini, R.; Quartara, L.;
Renzetti, A. R.; Tramontana, M.; Arcamone, F.; Maggi, C. A.
Br. J. Pharmacol. 1998, 123, 81. (d) Giannotti, D.; Perrotta,
E.; Di Bugno, C.; Nannicini, R.; Harmat, N. J. S.; Giolitti, A.;
Patacchini, R.; Renzetti, A. R.; Rotondaro, L.; Giuliani, S.;
Altamura, M.; Maggi, C. A. J. Med. Chem. 2000, 43, 4041. (e)
Quartara, L.; Rovero, P.; Maggi, C. M. Med. Res. Rev. 1995,
15, 139. (f) Emonds-Alt, X.; Proietto, V.; Van Broeck, D.;
Vilain, P.; Advenier, C.; Neliat, G.; Le Fur, G.; Breliere, J. C.
Bioorg. Med. Chem. Lett. 1993, 3, 925. (g) Emonds-Alt, X.;
Advenier, C.; Cognon, C.; Croci, T.; Daoui, S.; Ducoux, J. P.;
Landi, M.; Naline, E.; Neliat, G.; Poncelet, M.; Proietto, V.;
Van Broeck, D.; Vilain, P.; Soubrie, P.; Le Fur, G.; Maffrand,
J. P.; Breliere, J. C. Neuropeptides 1997, 31, 449. (h) Cooper,
A. W. J.; Adams, H. S.; Bell, R.; Gore, P. M.; McElroy, A. B.;
Pritchard, J. M.; Smith, P. W.; Ward, P. Bioorg. Med. Chem.
Lett. 1994, 4, 1951. (i) Smith, P. W.; Cooper, A. W. J.; Bell,
R.; Beresford, I. J. M.; Gore, P. M.; McElroy, A. B.; Pritch-
ard, J. M.; Saez, V.; Taylor, N. R.; Sheldrick, R. L. G.; Ward,
P. J. Med. Chem. 1995, 38, 3772.
Figure 3. Structure of compound 9, reproduction of its HPLC chro-
matogram at 280nm and schematic representation of the 4 putative
conformational isomers observed in the chromatogram. The four syn
arrangements have not been taken into consideration because
they have been found much less stable in the congeners described in
literature.12,13
Apparently, the structural features of the known
o-phthaloyl tertiary diamides, which also compounds 3–
6 and 9 show, are an obstacle both to infer guesses on
the binding mode and to put into practice an investiga-
tion strategy based, as often occurs, on the imaginary
active conformer. Thus, since the number of conjectur-
able active conformers17 is at least 16 for compound 5,
and at least 10for compound 6,14 it is likely that here,
the desire to uncover the conformation preferred by
hNK-2 receptor, by the investigation of restricted ana-
logues, could result a synthetic sisyphean toil. On the
other hand, if speculations on ligand–receptor interac-
tions, as well as those on the receptor-bound conforma-
tion are neglected at all, as we did when we decided to
overlook the cyclopeptide moiety in 1, to concentrate on
the redundant pyrazine (redundant, in the sense of bind-
ing affinity), it is easy to observe that the pseudosymmetry
introduced into MEN11558 by the added benzyl group,
has been conserved in the structures of 5 and 6.18
4. (a) Altamura, M.; Criscuoli, M.; Guidi, A.; Perrotta, E.;
Maggi, C. A. WO00/08046, 2000; Chem. Abstr. 2000, 136,
166521. (b) Harmat, N. J. S.; Giannotti, D.; Nannicini, R.;
Perrotta, E.; Criscuoli, M.; Patacchini, R.; Renzetti, A. R.;
Giuliani, S.; Altamura, M.; Maggi, C. A. Bioorg. Med. Chem.
Lett. 2002, 12, 693. (c) Valenza, S.; Cordero, F. M.; Brandi,
A.; Guidi, A.; Altamura, M.; Giolitti, A.; Giuntini, F.; Pasqui,
F.; Renzetti, A. R.; Maggi, C. A. J. Org. Chem. 2000, 65,
4003.
5. Patchett, A. A.; Nargund, R. P. Annu. Rep. Med. Chem.
2000, 35, 289.
6. Curiously, N,N,N0,N0-tetraethylphthalamide, the com-
pound which can be considered the structural progenitor of
products 3–6, is a known drug. See: The Merck Index, 13th
ed., ref 9278; p 1642. Merck & Co.: Whitehouse Station, 2001.
Anyway, it does not bind to the hNK-2 receptor up to micro-
molar concentrations.7,8
Conclusions
In this report we have disclosed two novel antagonists
of NKA at the human NK-2 receptor. Work is in

2948
M. Altamura et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2945–2948
1
7. All final compounds, 1 included, were characterized by H
NMR, mass spectrometry and had a purity by analytical
HPLC of ꢂ95%.
temperature NMR timescale: 2ꢃ2 dissymmetric isomers and
2ꢃ1 asymmetric anti isomer. Observed isomers in the PMR
spectra, without the addition of external chiral substances: 3:
Noteworthy, in one of compound 3 isomers, the two pyrazinic
protons resonate as an AB system (d=8.76 and 8.79 ppm,
J=2.6 Hz).
8. Binding affinity for the hNK-2 receptor transfected in CHO
cells were determined in competition experiments using
[
125I]NKA as the radioligand. The pKi values were calculated
according to the procedure described by: Munson, P. J. Anal.
Biochem. 1980, 107, 220.
Compound 5: 16 different conformational isomers com-
puted in a chiral environment by combining the two kinds of
thermal motion. That is: 2ꢃ4 anti asymmetric isomers and
2ꢃ4 syn asymmetric isomers. Expected isomers12,13 on the
room temperature NMR timescale: 2ꢃ4 anti isomers.
Observed isomers in the PMR spectrum, without the addition
of external chiral substances: 4. The spectroscopic behaviour
in the presence of a chiral shift reagent will be reported else-
where. For an investigation on the stereomutation processes in
compounds of very similar geometry, see: (a) Dell’ Erba, C.;
Gasparrini, F.; Grilli, S.; Lunazzi, L.; Mazzanti, A.; Novi, M.;
Pierini, M. Tavani, C.; Villani, C. J. Org. Chem. 2002, 67,
1663.
15. Luna C8(2) 5 mm, 25 cm length, 4.6 mm internal diameter
column. Mobile phase: A water, B acetonitrile, both contain-
ing 0.1% trifluoroacetic acid. Gradient conditions: from 20 to
80% B in 20 min, at a flow rate of 1 mL/min.
16. So far, we have not attempted to exploit this finding to
know, by means of focussed experiments, which racemic mix-
ture is most potent at the hNK-2 receptor.
9. Functional affinity was evaluated by measuring the intra-
cellular calcium concentration in CHO cells transfected with
the human NK-2 receptor using Fura-2, according to the
procedure described by: Iridale, A. I.; Dickenson, J. M. In
Methods in Molecular Biology; Kendall, D. A., Hill, S. J. Eds.;
Humana: Totowa, NJ, 1995; Vol. 41, p 203. pA2 values were
calculated from the parallel rightward shift of the NKA con-
centration response curve in the presence of the antagonists,
according to: Tallarida, R. J.; Cowan, A.; Adler, M. W. Life
Sci. 1979, 25, 637.
10. Chou, W.-C.; Tan, C.-W.; Chen, S.-F.; Ku, H. J. Org.
Chem. 1998, 63, 10015.
11. The three products were identified by the comparison of
HPLC analyses and PMR spectra at 200 MHz, accomplished
before and after the extraction of the crude reaction mixture
with aqueous hydrochloric acid. Both UV spectroscopy and
MS spectrometry were used as chromatographic revelators.
For examples of carboxylic acids methyl esters acting as alky-
lating agents of amines see: Zaugg, H. E.; Helgren, P. F.;
Schaefer, A. D. J. Org. Chem. 1963, 28, 2617.
17. Janssen, L. H. M. Bioorg. Med. Chem. 1998, 6, 785. In this
paper, the author argues that theoretical reasons could exist to
survey the most potent active conformers amongst the less
stable conformers of the isolated ligand candidate..
12. Clayden, J.; Pink, J. H.; Yasin, S. A. Tetrahedron Lett.
1998, 39, 105.
13. Schneider, H.-J.; Kasper, C.; Palyulin, V.; Samoshin, V. V.
Supramol. Chem. 1997, 8, 225.
18. Liu, J.; Underwood, D. J.; Cascieri, M. A.; Rohrer, S. P.;
Cantin, L.-D.; Chicchi, G.; Smith, A. B., III; Hirschmann, R.
J. Med. Chem. 2000, 43, 3827. For a more general discussion
concerning the conservation of symmetry in natural phenom-
ena, see: Hargittai, I.; Hargittai, M. In Symmetry through the
Eyes of a Chemist, 2nd ed., Plenum: New York and London,
1995; p 70, and references cited.
14. Compounds 3, 4 and 6. 10different conformational iso-
mers computed in a chiral environment by combining the two
kinds of thermal motion. That is two achiral syn isomers, 2ꢃ2
dissymmetric (C2) isomers, 2ꢃ2 asymmetric isomers (C1), one
of which syn and one anti. Expected isomers12,13 on the room