N-[(R,R)-(E)-1-(4-chloro-benzyl)-3-(2-oxo-azepan-3-ylcarbamoyl)-allyl]-N- methyl-3,5-bis-trifluoromethyl-benzamide: An orally active neurokinin NK1/NK2 antagonist

Article abstract of DOI:10.1016/S0960-894X(00)00260-2

The stereoselective synthesis of N-[(R,R)-(E)-1-(4-chloro-benzyl)-3-(2-oxo-azepan-3-ylcarbamoyl)-allyl]-N-methyl-3 ,5-bis-trifluoromethyl-benzamide (4) and its NK₁ and NK₂ receptor binding properties are reported. In addition the potent inhibitory effects in vivo on sar⁹-SP- and β-Ala-NKA-induced airway bronchoconstriction in guinea pigs are demonstrated. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00260-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1467±1470
N-[(R,R)-(E)-1-(4-Chloro-benzyl)-3-(2-oxo-azepan-3-ylcarbamoyl)-
allyl]-N-methyl-3,5-bis-tri¯uoromethyl-benzamide: An Orally
Active Neurokinin NK1/NK2 Antagonist
Marc Gerspacher,a,* Andreas von Sprecher,b Robert Mah,a Gary P. Anderson,c
Claude Bertrand,d Natarajan Subramanian,a Kathleen Hausera and Howard A. Balla
aPharma Research, Novartis Pharma AG, CH-4002 Basel Switzerland
bActelion Ltd., CH-4123, Basel, Switzerland
cDepartment of Pharmacology, University of Melbourne, Parkville, 3052 VIC, Australia
dIn¯ammatory Disease Unit, Roche Biosciences, Palo Alto, CA, USA
Received 15 February 2000; accepted 2 May 2000
AbstractÐThe stereoselective synthesis of N-[(R,R)-(E)-1-(4-chloro-benzyl)-3-(2-oxo-azepan-3-ylcarbamoyl)-allyl]-N-methyl-3,5-
bis-tri¯uoromethyl-benzamide (4) and its NK1 and NK2 receptor binding properties are reported. In addition the potent inhibitory
e€ects in vivo on sar9-SP- and b-Ala-NKA-induced airway bronchoconstriction in guinea pigs are demonstrated. # 2000 Elsevier
Science Ltd. All rights reserved.
Neurokinins are proposed to be involved in a number of
pathological conditions including pain, arthritis, migraine,
emesis, cancer, anxiety, depression, schizophrenia, asthma
and airway diseases, and NK receptor antagonists have
been proposed to have potential clinical bene®ts.1 The
results obtained from animal and clinical studies in recent
years provide an emerging pharmacological evidence that
neurokinins play an important role in airway disease
induction and progression via the activation of NK1
and NK2 receptors.2 Furthermore, the studies suggest
that neurokinin receptor antagonists, especially dual NK1/
NK2 antagonists, may represent a new treatment option
for asthma and other airway diseases, particularly since
lung tissue from asthma patients has been shown to over-
express NK1 and NK2 receptors.3 As a consequence many
pharmaceutical companies have shifted their e€orts aim-
ing at selective NK-antagonists towards the discovery of
dual NK1/NK2 antagonists.
from the selective NK1 antagonist CGP49823 (Fig. 1).4,5
Formal elimination of a CH2-group from the piperidine
ring led to a new series of ``open chain'' neurokinin
receptor antagonists. In general, compounds from this
structural class exhibited highly potent anity to the NK1
3
receptor (inhibition of H-sar9-substance P binding to
bovine retinal membranes) and a number of compounds
exhibited an additional anity to the NK2 receptor (inhi-
bition of 125I-NKA binding to human NK2-CHO-cells).
In these binding assays, 1 and 2 exhibited IC50 values of
0.7 nM (NK1) and 55 nM (NK2), and 10 nM (NK1) and
49 nM (NK2) respectively.5
Compound 1 which exhibited highly potent anity to
the NK1 receptor and a promising anity to the NK2
receptor was prepared and tested as a mixture of 4 stereo-
isomers. In order to ®nd out which isomers (and to what
degree) are responsible for the anity of 1 to the neu-
rokinin receptors, all 4 isomers have been prepared as
outlined in Scheme 1. Coupling of the racemic acid 3
with either enantiomerically pure d(R)- or l(S)-a-amino-
Our e€orts aiming at the discovery of dual NK1/NK2
antagonists as potential anti-asthma agents led to the
discovery of a series of N-[(E)-3-carbamoyl-1-(4-chloro-
benzyl)-allyl]-N-methyl-3,5-bis-tri¯uoro-methyl-benza-
mides, as dual NK1/NK2 antagonists, derived originally
6
E-caprolactam followed by deprotection and acylation
led in both cases to a mixture of two diastereoisomers
which could easily be separated on a standard ¯ash-
chromatography column.
In order to assign the absolute stereochemistry of the
chiral center in the carbon chain of the four isomers, we
*Corresponding author. Tel.:+41-61-696-7636; fax: +41-61-696-
5966; e-mail: marc.gerspacher@pharma.novartis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00260-2

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M. Gerspacher et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1467±1470
chose to work out a stereoselective synthesis to prepare
speci®cally one isomer as an enantiomerically pure com-
pound. In the 4-amino-piperidine series of selective NK1
antagonists (e.g., CGP49823) it was shown that the com-
pounds possessing the S-con®guration at C-2 exhibited
higher anity to the NK1 receptor in comparison to the
corresponding R-enantiomers. Therefore, we focused our
e€orts on the stereoselective preparation of an isomer
that possessed the same stereochemistry at the corre-
sponding chiral center.
group and subsequent acylation of the nitrogen with 3,5-
bistri¯uoro-methylbenzoyl-chloride led to the ®nal pro-
duct in an overall yield of 20% after recrystallisation from
CH2Cl2/n-pentane (ee=95% before and ee=98.5% after
recryst.) (Scheme 2). During the synthesis starting with
d-4-chlorophenyl-alanin-methylester (8, ee>97%) no sig-
ni®cant epimerisation of the chiral center could be
observed.7 After checking all intermediates for optical
purity, we showed that the very minor loss of optical purity
had occurred during the N-methylation of N-BOC-d-4-
chlorophenyl-alanin-methylester in the presence of silver
oxide and DMF at the beginning of the synthesis
(Scheme 2).
The R,R-isomer was prepared starting from commer-
cially available d-4-chlorophenyl-alanin-methylester (8)
which, after BOC-protection followed by N-methyla-
tion, was reduced to the aldehyde. Chain elongation
using trimethylsilyl-P,P-diethyl-phosphonoacetate and
an acidic work up procedure led to the carboxylic acid
11. Introduction of d-a-amino-E-caprolactam (D-13) in
the presence of EDC, removal of the BOC protecting
On the basis of the chromatographical behaviour of 4
(R,R-isomer), the absolute con®guration of the stereo-
centers of all the isomers produced starting from 3 could
be assigned and all four compounds were tested for
their binding anities in the NK-receptor assays.8,9 As
can be seen from the NK1 and NK2-binding values in
Table 1, the R,R-isomer (4) clearly exhibits the highest
anity for both the NK1 and the NK2 receptor. Changing
the stereochemistry of the backbone chiral center from
R to S (resulting in the S,R-isomer, (5) leads to a decrease
in binding anities to the NK1 and NK2 receptors by a
factor of 30 and 5 respectively. Inversion of the stereo-
chemistry at the chiral center of the caprolactam moiety
(leading to the R,S-isomer, (7) results in reduced binding
Figure 1. The discovery of 5-aryl-4-benzoyl-amino-pent-2-ene-carbox-
amides.
Scheme 2. Stereospeci®c preparation of 4. (a) (BOC)2O, Et3N; b) MeI,
Ag2CO3, DMF; (c) DIBAH, toluene, 78 ꢀC; (d) n-BuLi, trimethyl-
silyl-P,P-diethylphosphonoacetate, then citric acid; (e) d-a-amino-E-
caprolactam, EDC, DMAP, CH2Cl2; (f) TFA, CH2Cl2; (g) 3,5-Bistri-
¯uoromethylbenzoylchloride; Et3N, CH2Cl2, chromatography, recrys-
tallisation.
Scheme 1. Preparation of diastereoisomers. (a) Reaction conditions:
see Scheme 2. (b) d-a-amino-E-caprolactam, EDC, DMAP, CH2Cl2;
(c) TFA, CH2Cl2; (d) 3,5-Bistri¯uoromethyl-benzoyl-chloride; Et3N,
CH2Cl2; (e) Chromatography on silica gel; (f) l-a-amino-E-capro-
lactam, EDC, DMAP, CH2Cl2.

M. Gerspacher et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1467±1470
1469
anities to the NK1 and NK2 receptors by a factor of 7
and 5 respectively. In comparison to the R,R-isomer (4),
it's enantiomer the S,S-isomer (6) exhibits a 170 times
lower binding anity to the NK1 and a 12 times lower
anity to the NK2 receptor.
preparation of the R,R-isomer (4) in an overall yield of
20% and with an ee>98%. 4 was also shown to exhibit a
protective e€ect against sar9-substance P- or b-Ala8-
NKA-induced bronchoconstriction in guinea pigs after
oral application.
Following con®rmation of activity in in vitro functional
tests (Ca2+ in¯ux measurements and IL6 release in cul-
tured cell lines), the ability of 4 to antagonise either
sar9-substance P (selective and stable NK1 agonist)- or
b-Ala8-NKA (selective and stable NK2 agonist)-induced
bronchoconstriction (airway pressure measurement) in
anaesthetised guinea pigs was also tested.10 As can be seen
from the results shown in Table 2, after oral application
2 h prior to the challenge with the agonists, 4 is able to
inhibit sar9-substance P-induced bronchoconstriction
(NK1) with an ED50 of 0.036 mg/kg and the b-Ala8-
NKA-induced bronchoconstriction (NK2) with an ED50
of 0.9 mg/kg. An inhibition of 85% after a 1 mg/kg oral
dose 12 h prior to the challenge with the NK1 agonist
could also be observed indicating a long duration of
action (Table 2).
Acknowledgements
We would like to thank Ms. K. Ry€el, Ms. V. Pawelzik
and Mr. D. Wyss for their excellent technical assistance.
We also thank Dr. E. Francotte for the determination
of ee-values.
References and Notes
1. (a) Longmore, J.; Swain, C. J.; Hill, R. G. Drug News Per-
spec. 1995, 8, 5. (b) Kucharczyk, N. Exp. Opin. Invest. Drugs
1995, 4, 299. (c) Elliott, J.; Seward, E. M. Exp. Opin. Ther.
Patents 1997, 7, 43-54. (d) Longmore, J.; Hill, R. G.; Har-
greaves, R. J. Can. J. Physiol. Pharmacol. 1997, 75, 612. (e)
von Sprecher, A.; Gerspacher, M.; Anderson, G. P. Drugs
1998, 1, 73.
2. (a) Ford-Hutchinson, A. W.; Rodger, I. W.; Jones, T.R.
Drug. News Perspec. 1992, 5, 542. (b) Geppetti, P.; Bertrand,
C.; Ricciardolo, F. M. L; Nadel, J. A. Can. J. Physiol. Phar-
macol. 1995, 7, 843. (c) Advenier, C.; Lagente, V.; Boichot, E.
Eur. Respir. J. 1997, 10, 1892. (d) Chapman, R. W.; Hey, J.
A.; McLeod, R.; Minnicozzi, M.; Rizzo, Ch. Drug News Per-
spect. 1998, 11, 480.
From a series of N-[(E)-3-carbamoyl-1-(4-chloro-benzyl)-
allyl]-N-methyl-3,5-bis-tri¯uoro-methyl-benzamides, 1 (a
mixture of 4 isomers) has been identi®ed as a dual NK1/
NK2 antagonist exhibiting highly potent anity to the
NK1 receptor and good anity to the NK2 receptor.
Consequently, all four isomers were prepared and tested
as enantiomerically pure compounds in NK1 and NK2
binding assays. In addition, a stereoselective synthesis (8
steps) starting from a commercially available d-amino
acid derivative has been worked out, which allows the
3. (a) Murai, M.; Morimoto, H.; Maeda, Y.; Kiyotoh, S.;
Nishikawa, M.; Fujii, T. J. Pharmacol. Exp. Ther. 1992, 262,
403. (b) Joos, G. F.; Van Schoor, J.; Kips, J. C.; Pauwels, R.
A. Am. J. Respir. Crit. Care Med. 1996, 153,1781.
4. Ofner, S.; Hauser, K.; Schilling, W.; Vassout, A.; Veenstra,
S. J. Bioorg. Med. Chem. Lett. 1996, 6, 1623.
Table 1. In vitro binding anities of compounds 1 and 4±7 to NK1-
and NK2-receptors
5. Gerspacher, M.; von Sprecher, A.; Mah, R.; Roggo, S.;
Ofner, S.; Auberson, Y.; Betschart, C.; Schilling, W.; Ander-
son, G. P.; Ball, H.; Bertrand, C.; Subramanian, N.; Hauser,
K. 5-Aryl-4-benzoyl-amino-pent-2-ene-carboxamides: a new
class of NK1 and dual NK1/NK2 antagonists. 212th ACS
National Meeting (Aug. 23±27, Boston, USA) 1998, MEDI 52.
6. Rezler, E. M.; Fenton, R. P.; Esdale, W. J.; McKeage, M.
J.; Russell, P. J.; Hambley, T. W. J. Med. Chem. 1997, 40, 3508.
l-a-amino-E-caprolactam is commercially available from
Fluka Chemie AG, CH-9471 Buchs: Switzerland.
7. Analytical data, 4: mp 110±112 ꢀC. [a]d20=+42ꢀ (c=0.895,
EtOH). 1H NMR (400 MHz, d, DMSO, +120 ꢀC) 8.02 (s,
1H); 7.67 (b, 1H); 7.55 (s,b, 2H); 7.41 (b, 1H); 7.31 (d, 2H);
7.23 (b, 2H); 6.69 (dd, 1H); 6.34 (dd, 1H); 4.98 (b, 1H); 4.51
(m, 1H); 3.17 (m, 1H); 3.06 (d, 2H); 2.82 (s,b, 3H); 1.96±1.22
(m, 6H). Anal. calcd for C27H26ClF6N3O3: C: 54.97, H:4.44,
N:7.12. Found: C: 54.94, H:4.73, N:7.13. Raman (pulv., cm 1):
1643 (-CHˆCH-CˆO), 1095 (Ph-Cl), 1003 (1,3,5-trisubst.
phenyl). ee-values determined using HPLC on Chiracel OJ
(hexane:ethanol=9:1+0.1% TFA).
Compound
NK1 binding
IC50, nMa
NK2 binding
IC50, nMa
1 (Mixture of 4 stereoisomers)
4 (R,R-isomer)
5 (S,R-isomer)
6 (S,S-isomer)
7 (R,S-isomer)
0.76
0.5
16
86
3.6
55
24
123
300
120
aValues are means of three experiments.
8. For experimental details see: Bittiger H., Heid J. In Sub-
stance P, Skrabanek, P.; Powell, D. Boole Eds.; Press Ltd:
Dublin 1983; pp 198±199.
9. Inhibition of [125I]-NKA binding to transfected Chinese
hamster ovary cells (CHO cells) expressing recombinant
human neurokinin 2 receptors: The assay was performed in 96
well plates (Nunclon) containing 200 mL 20 mM HEPES bu€er,
pH 7.4 containing 2 mM MnSO4 and 6 mM MgCl2, 3Â105 h
NK2 CHO cells, 0.05 nM [125I]-NKA (2200 Ci mmol 1) and
various drug concentrations. Nonspeci®c binding was estimated
Table 2. In vivo activity of 4 against NK1- and NK2-agonist-induced
bronchoconstriction in guinea pigs
Inhibition of sar9SP-induced
bronchoconstriction
ED50, mg/kg
Inhibition of b-Ala8-NKA-induced
bronchoconstriction
ED50, mg/kg
0.036 ( 2 h)
0.03 ( 4 h)
85% (1 mg/kg, 12 h)
0.9 ( 2 h)
0.73 ( 4 h)
n.d. ( 12 h)

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M. Gerspacher et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1467±1470
in the presence of 50 nM NKA. The mixture was incubated for
20 min at room temperature after which the unbound ligand
was removed by rapid ®ltration and washed 4 times with ice
cold Tricine bu€er. Filter bound radioactivity was counted in
Microscint 20 in a scintillation counter. All samples were
measured in triplicate. Culture conditions and cell isolation for
h NK2 CHO cells: Subramanian, N.; Ruesch, C.; Bertrand, C.
Biochem. Biophys. Res. Comm. 1994, 200, 1512.
Northridge, CA). Polyethylene catheters (250 I.U. ml 1 heparin
in 0.9% NaCl) were inserted into the right jugular vein for drug
injection and the left carotid for blood pressure measurements
(Statham transducer P23XL). All signals were recorded using a
computer data-acquisition system (Mi2 Bio Report software,
Modular Instruments). The timing of anaesthesia and animal
preparation were such that after a baseline period was obtained,
Sar9-SP (3 mg kg 1; ED80 dose for increase in intratracheal
1
10. Dunkin±Hartley guinea-pigs (500±700g) were anaesthetised
with ip urethane (1.5 g kg 1), tracheotomised and ventilated
with a constant-volume ventilator (Model 683; Harvard
pressure) or b-Ala8-NKA (0.8 mg kg ED80 dose) was injec-
ted, corresponding to a time of 2, 4 or 12 h since the oral
dosing of vehicle or drug. The antagonists were given in doses
ranging from 0.01 to 1 mg kg 1 in a vehicle consisting of 0.0067±
0.67% DMSO in 0.5% methylcellulose, in a volume of 10 mL
kg 1. Five to six animals per dose were studied. The percent
inhibition for each animal was calculated by dividing the elicited
change in intratracheal pressure for the antagonist-treated ani-
mals by the mean value obtained for the vehicle-treated group.
A linear regression analysis was then performed of the logarith-
mically transformed dose data and the ED50 value interpolated.
apparatus Co., S. Natick, MA) at a frequency of 60 breaths
min 1. Pavulon (pancuronium bromide, Organon, 1 mg kg
1
)
and atropine (Fluka, 1mg kg 1) were administered (iv) to pre-
vent spontaneous breathing and cholinergic re¯exes, respec-
1
tively. The tidal volume was adjusted to about 1 mL 100 g
body weight so as to maintain normal arterial blood gases.
Intratracheal pressure was measured with a di€erential pressure
transducer (Model DP 45-28, Validyne Engineering Corp.,