Stereoselective preparation of N-[(R,R)-(E)-1-(3,4-dichlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl- N-methyl-3,5-bis(trifluoromethyl)benzamide, a potent and orally active dual neurokinin NK1/NK2 receptor antagonist

Article abstract of DOI:10.1021/jm030786m

In a program aimed at the development of neurokinin antagonists, N-[(R,R)-(E)-1-(3,4-dichloro-benzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl- N-methyl-3,5-bis(trifluoromethyl)benzamide (1, DNK333) has been discovered as a potent and balanced neurokinin (tachykinin) NK₁/NK₂ receptor antagonist. Enantiomerically pure (>99.5% ee) 1 can be prepared in 6 + 1 synthetic steps starting from commercially available optically active BOC-D-3,4-dichlorophenylalanine in an overall yield of ca. 25-30%. 1 showed potent affinities to cloned human NK₁ (pK_i = 8.38) and NK₂ (pK_i = 8.02) receptors. When 1 was compared to the other possible three diastereoisomers, it could be demonstrated that only the R,R-isomer (1) exhibits potent and balanced affinity for the cloned human NK₁ and NK₂ receptors. 1 exhibited favorable pharmacokinetic properties in guinea pigs following oral administration and demonstrated in vivo activity in pharmacological models of substance P- and neurokinin A (NKA)-induced bronchoconstriction in guinea pigs after intravenous and in squirrel monkeys after oral application.

Full text of DOI:10.1021/jm030786m

3508
J . Med. Chem. 2003, 46, 3508-3513
Ster eoselective P r ep a r a tion of N-[(R,R)-(E)-1-(3,4-d ich lor oben zyl)-3-
(2-oxoa zep a n -3-yl)ca r ba m oyl]a llyl-N-m eth yl-3,5-bis(tr iflu or om eth yl)ben za m id e,
a P oten t a n d Or a lly Active Du a l Neu r ok in in NK1/NK2 Recep tor An ta gon ist
Marc Gerspacher,*,† Christine Lewis,‡ Howard A. Ball,† Colin Howes,‡ Natarajan Subramanian,†
Karin Ryffel,† and J ohn R. Fozard†
Pharma Research, Novartis Pharma AG, CH-4002 Basel, Switzerland, and Novartis Horsham Research Centre,
Horsham, West Sussex RH12 5AB, U.K.
Received J anuary 23, 2003
In a program aimed at the development of neurokinin antagonists, N-[(R,R)-(E)-1-(3,4-dichloro-
benzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (1,
DNK333) has been discovered as a potent and balanced neurokinin (tachykinin) NK1/NK2
receptor antagonist. Enantiomerically pure (>99.5% ee) 1 can be prepared in 6 + 1 synthetic
steps starting from commercially available optically active BOC-D-3,4-dichlorophenylalanine
in an overall yield of ca. 25-30%. 1 showed potent affinities to cloned human NK1 (pKi ) 8.38)
and NK2 (pKi ) 8.02) receptors. When 1 was compared to the other possible three diastereo-
isomers, it could be demonstrated that only the R,R-isomer (1) exhibits potent and balanced
affinity for the cloned human NK1 and NK2 receptors. 1 exhibited favorable pharmacokinetic
properties in guinea pigs following oral administration and demonstrated in vivo activity in
pharmacological models of substance P- and neurokinin A (NKA)-induced bronchoconstriction
in guinea pigs after intravenous and in squirrel monkeys after oral application.
In tr od u ction
Neurokinins are members of a family of small pep-
tides known as tachykinins that are widely distributed
throughout the central and peripheral nervous systems.1
The most prominent mammalian neurokinins are sub-
stance P (SP), neurokinin A (NKA), neurokinin B
(NKB), and the recently discovered hemokinin 1.2,3
Neurokinins exhibit their effects via specific G-protein-
coupled receptors (NK1, NK2, and NK3 receptors).
Substance P and hemokinin A display the highest
affinity for the NK1 receptor, NKA displays the highest
affinity for the NK2 receptor, and NKB displays the
highest affinity for the NK3 receptor.4 Neurokinins and
the neurokinin receptors have been proposed to be
involved in a number of pathological conditions includ-
ing pain, arthritis, migraine, emesis, cancer, anxiety,
depression, schizophrenia, asthma and airways dis-
F igu r e 1. N-[1-(Arylmethyl)-3-carbamoyl]allyl-N-methylbenz-
amides and the structure of 1.
eases, inflammatory bowel disease, and incontinence,
and NK receptor antagonists have been proposed to
have potential clinical benefits.5
NK1/NK2 receptor antagonists.9 From this series 1,
Over the past few years, many pharmaceutical com-
(DNK333, (N-[(R,R)-(E)-1-(3,4-dichlorobenzyl)-3-(2-oxo-
panies have successfully shifted research efforts aimed
azepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoro-
at selective NK1 or NK2 receptor antagonists toward the
methyl)benzamide) was found to exhibit potent and
identification of non-peptide dual NK1/NK2 receptor
balanced affinity at NK1 and NK2 receptors9 (Figure 1).
antagonists,6 since increasing evidence was found for
We here describe the stereospecific synthesis of 1 and
the contribution of both the NK1 and the NK2 receptors
the preparation of its stereoisomers, together with NK1
on a variety of diseases.7,8
and NK2 affinity data. We also provide results of in vivo
We have recently described N-[1-(arylmethyl)-3-car-
studies with 1 in bronchoconstriction studies as well as
bamoyl]allyl-N-methylbenzamides as a series of com-
the results from pharmacokinetic studies.
pounds capable of delivering potent, orally active dual
Ch em istr y
* To whom correspondence should be addressed. Phone:
+41 61 696 7636. Fax: +41 61 696 6071. E-mail: marc.gerspacher@
1 (R,R-isomer) was prepared starting from com-
mercially available BOC-D-3,4-dichlorophenylalanine
(2), which, after N-methylation and esterification with
pharma.novartis.com.
† Novartis Pharma AG.
‡ Novartis Horsham Research Centre.
10.1021/jm030786m CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/27/2003

Preparation of a Benzamide Antagonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3509
Sch em e 2. Preparation of Diastereoisomersa
Sch em e 1. Preparation of 1 (R,R-Isomer)a
a
Reagents and conditions: (a) reaction conditions, see Scheme
a
Reagents and conditions: (a) MeI, Ag2CO3, DMF; (b) DIBAH,
1; (b) D-R-amino-ꢀ-caprolactam, EDC, DMAP, CH2Cl2; (c) TFA,
CH2Cl2; (d) 3,5-bis(trifluoromethyl)benzoyl chloride, Et3N, CH2Cl2;
(e) chromatography on silica gel; (f) L-R-amino-ꢀ-caprolactam, EDC,
DMAP, CH2Cl2.
toluene, -78 °C; (c) n-BuLi, trimethylsilyl P,P-diethylphosphono-
acetate, then dilute HCl; (d) D-R-amino-ꢀ-caprolactam, EDC,
DMAP, CH2Cl2; (e) TFA, CH2Cl2; (f) 3,5-bis(trifluoromethyl)benzoyl
chloride, Et3N, CH2Cl2, crystallization from CH2Cl2/n-pentane.
that could easily be separated by standard flash chro-
matography on silica gel to afford the diastereoisomers
8-10 as pure compounds (Scheme 2).
excess methyl iodide in the presence of silver oxide in
DMF, was converted to the ester 3. The ester group of
this compound was reduced with diisobutylaluminum
hydride (DIBAH) in toluene at -78 °C to afford the cor-
responding aldehyde 4. Chain elongation under Wittig-
Horner conditions with trimethylsilyl P,P-diethylphos-
phonoacetate10 in THF followed by hydrolysis of the
trimethylsilyl ester group with diluted hydrochloric acid
during the workup procedure yielded R-4-(tert-butoxy-
carbonylmethylamino)-5-(3,4-dichlorophenyl)pent-2-eno-
ic acid 5. Condensation of 5 with D-R-amino-ꢀ-caprolac-
tam11 in the presence of N-ethyl-N′,N′-dimethylamino-
propylcarbodiimide-HCl (EDC) and 4-(dimethylamino)-
pyridine (DMAP) afforded N-[(R,R)-(E)-1-(3,4-dichloro-
benzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-
carbamic acid tert-butyl ester (6). Removal of the BOC
protecting group with trifluoroacetic acid in dichloro-
methane, subsequent acylation of the nitrogen with 3,5-
bistrifluoromethylbenzoyl chloride in the presence of
triethylamine in dichloromethane, and purification on
silica gel led to the final product in an overall yield of
ca. 25-30%. Crystallization of the product from CH2Cl2/
n-pentane yielded 1 as a crystalline compound with a
melting point of 127-129 °C. An enantiomeric excess
(ee) value of >99.5% could be determined by HPLC
analysis using a Chiralcel OJ column (Scheme 1).
The remaining three stereoisomers (and 1) can be
prepared as outlined in Scheme 2 starting from racemic
312 under exactly the same reaction sequence used for
the stereospecific preparation of 1 (see Scheme 1).
Coupling of the racemic carboxylic acid derivative rac-5
with either enantiomerically pure D(R)- or L(S)-R-amino-
ꢀ-caprolactam11 in the presence of ′′EDC and DMAP,
followed by cleavage of the BOC group and acylation of
the nitrogen with 3,5-bistrifluoromethylbenzoyl chloride,
led in both cases to a mixture of two diastereoisomers
Resu lts a n d Discu ssion
By comparison of the chromatographical behavior of
compounds 8-10 with 1 (R,R-isomer), the absolute
configuration of the stereocenters of the three remaining
isomers could be assigned, and all four compounds were
tested for their binding affinities to NK1 and NK2
receptors. In comparison to the other three possible
isomers, 1 (R,R-isomer) clearly exhibits the most potent
NK2 binding affinity showing at least 10-fold higher
affinity for this receptor in comparison to the other
isomers. With respect to affinity for human NK1 recep-
tors however, the S,R-isomer (8) is some 5 times more
potent than 1 but exhibits the lowest affinity to the NK2
receptor. Of the four stereoisomers, the S,S-isomer 10
(i.e., enantiomer of 1) exhibits the lowest affinity to the
NK1 receptor. Only the R,R-isomer (1) exhibits potent
and balanced affinity for the cloned human NK1 and
NK2 receptors (Table 1).
Following confirmation of activity in in vitro func-
tional tests, where the compound demonstrated block-
ade of bronchoconstrictor responses induced by selective
NK1 (pA2 ) 7.93) and selective NK2 (pA2 ) 7.27)
agonists in the guinea pig isolated trachea model,13 in
in vivo experiments the ability of 1 to antagonize either
Sar9 substance P (a selective and stable NK1 receptor
agonist) or (Ala5,â-Ala8)-R-NKA (fragments 4-10) (a
selective and stable NK2 receptor agonist) induced
bronchoconstriction in anesthetised guinea pigs was
tested. The results are presented in Figure 2. 1, 1 mg/
kg, dosed iv at 5 min or 1 h prior to administration of
the NK1 agonist Sar9-SP induced 15.8- or 15.7-fold shifts
in the dose-response curves to the right, respectively.
1, 10 mg/kg, dosed iv at 5 min or 1 h prior to adminis-

3510 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Gerspacher et al.
Ta ble 1. In Vitro Binding Affinities (pKi Values) of 1 and
Compounds 8-10 to Human NK1 and NK2 receptorsa
pKi
compd
(configuration of *1, *2)
NK1 binding
NK2 binding
1 (R,R-isomer)
8 (S,R-isomer)
9 (R,S-isomer)
10 (S,S-isomer)
8.38 ( 0.12
9.08 ( 0.09
8.06 ( 0.06
7.74 ( 0.08
8.02 ( 0.02
6.34 ( 0.10
6.99 ( 0.09
6.74 ( 0.06
a
n ) 3 for all compounds.
tration of the NK2 agonist (Ala5,â-Ala8)-R-NKA (frag-
ments 4-10) demonstrated 2.3- or 2.6-fold shifts in the
dose-response curves to the right, respectively.
There is no loss of activity up to 1 h after iv
administration of 1, and the relative potency was ca.
6-fold in favor of NK1 blockade, a finding that is broadly
consistent with the in vitro relative antagonist potency
at guinea pig NK1 (pA2 ) 7.93) and NK2 (pA2 ) 7.27)
receptors (guinea pig isolated trachea13).
1 was tested in a second in vivo study, the broncho-
constrictor response to the selective NK2 receptor ago-
nist â-Ala8-NKA, in the anesthetised squirrel monkey.
When administered orally 2 h prior to the agonist, 1
inhibited â-Ala8-NKA induced bronchoconstriction dose-
dependently with ED50 values of 1 mg/kg (airways
resistance) and 2.8 mg/kg (dynamic compliance) (Figure
3).
The pharmacokinetic properties of 1 were investigated
in guinea pigs (3 mg/kg, iv; 10 mg/kg, po) following
administration of 1 as a cremophor formulation (iv) and
as microemulsion or cremophor oral formulations. A
terminal elimination half-life of ca. 4.2 h after intrave-
nous administration and apparent termination half-
lives of 3.8 h (microemulsion formulation) and 5.4 h
(cremophor formulation) after oral administration of the
compound could be observed. After oral administration
of 10 mg/kg 1, an AUC0-24h of 6378 ng h/mL and a
bioavailability of approximately 41% can be observed
when the compound was applied as a microemulsion.
A prolonged half-life but lower AUC of 2712 ng h/ml
and a bioavailability of just 17.4% were observed after
oral application of 1 as a cremophor formulation (Table
2).
F igu r e 2. Inhibition of NK1 (A) and NK2 (B) agonist induced
bronchoconstriction in guinea pigs. Inhibitory effects on airway
resistance of 1 mg/kg (upper graph) or 10 mg/kg (lower graph)
1 dosed iv: 5 min (2, n ) 9 or 5) or 1 h (9, n ) 4 or 5) prior to
establishing a dose-response curve to Sar9SP, a selective NK1
receptor agonist (A), or to (Ala5,â-Ala8)-R-NKA (fragments
4-10), a selective NK2 receptor agonist (B) compared with
vehicle dosed iv (b, n ) 4, both).
F igu r e 3. Inhibition of changes in airway resistance (0) and
dynamic compliance (9) induced by â-Ala8-NKA (1 mM aerosol)
by 1. Dose-response curves for 1 in the anesthetized squirrel
monkey, n ) 12.
Con clu sion s
(>99.5% ee) from commercially available optically active
starting material using a short synthesis comprising 6
+ 1 chemical steps in an overall yield of ca. 30%. We
have also shown that 1 exhibits potent and balanced
affinity to cloned human NK1 and NK2 receptors. NK1
and NK2 receptor blocking activity could be demon-
strated in bronchoconstriction studies in guinea pigs
(after intravenous administration) and in squirrel mon-
keys (after oral administration). Favorable pharmaco-
kinetic properties after oral administration of the
compound formulated as a microemulsion to guinea pigs
have been observed.
With the availability of potent and orally bioavailable
dual NK1/NK2 neurokinin receptor antagonists, it is now
possible to further elucidate the role of NK1 and NK2
receptors in contributing to pathophysiological proc-
esses, in particular in respiratory diseases, and to de-
fine their utility as drugs. N-[(R,R)-(E)-1-(3,4-dichloro-
benzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-
3,5-bis(trifluoromethyl)benzamide (1) has been discov-
ered as a potent, balanced (human receptors), orally
active dual NK1/NK2 receptor antagonist. The compound
can be prepared as an enantiomerically pure product

Preparation of a Benzamide Antagonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3511
Ta ble 2. Pharmacokinetic Parameters of 1 in Guinea Pigs
route of administration
t1/2 (h)
Cmax (ng/mL)
AUC0-24h (ng h/mL)
bioavailability (%)
guinea pig, iv, n ) 6, 3 mg/kg
guinea pig, po, n ) 7, 10 mg/kg
4.2
3.8
5.4
4687
600
415
6378 (microemulsion)
2712 (cremophor)
41
17.4
cooling bath was removed and the reaction mixture was stirred
for 1 h. To the reaction mixture, 0.1 N HCl (100 mL) was
added. After being stirred for 20 min, the reaction mixture
was extracted three times with EtOAc. The combined organic
layers were washed with water and brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by flash
chromatography (EtOAc) to give 5 (1.83 g, 76%) as a colorless
oil. 1H NMR (400 MHz, DMSO, +150 °C): δ 7.49 (d, 1H), 7.48
(s, 1H), 6.78 (dd, 1H), 5.85 (d, 1H), 3.00 (m, 2H), 2.68 (s, 3H),
1.36 (s, 9H). Anal. (C17H21Cl2NO4) H, N. C: calcd, 54.56; found,
54.15.
[(E)-(R)-1-(3,4-Dich lor ob en zyl)-3-((R)-2-oxoa zep a n -3-
ylca r ba m oyl)a llyl]m eth ylca r ba m ic Acid ter t-Bu tyl Ester
(6). A solution of 5 (0.5 g, 1.34 mmol), D-R-amino-ꢀ-caprolactam
(0.172 g, 1.34 mmol), N-ethyl-N′-(3-dimethylaminopropyl)-
carbodiimid hydrochloride (0.405 g, 2.01 mmol), and 4-(di-
methylamino)pyridine (0.16 g, 1.34 mmol) in methylene chlo-
ride (15 mL) was stirred at room temperature for 20 h. The
reaction mixture was concentrated in vacuo and the residue
was purified by flash chromatography to yield a colorless oil
(0.46 g, 76%). The oil was recrystallized from Et2O to give 6
as white crystals. Mp 153-154 °C. [R]20D +1.6° (c 2.50, EtOH).
1H NMR (400 MHz, DMSO, +120 °C): δ 7.62 (bs, 1H), 7.49
(d, 1H), 7.47 (s, 1H), 7.37 (bs, 1H), 7.22 (d, 1H), 6.62 (dd, 1H),
6.18 (d, 1H), 4.80 (bs, 1H), 4.51 (m, 1H), 3.25-3.12 (m, 2H),
2.95 (m, 2H), 2.68 (s, 3H), 1.90 (m, 2H), 1.80-1.73 (m, 2H),
1.46 (m, 1H), 1.30 (s, 9H), 1.29 (m, 1H). Anal. (C23H31N3O4-
Cl2) C, H, N, Cl.
Exp er im en ta l Section
Gen er a l. Melting points were determined on a Reichert
melting point apparatus and are uncorrected. IR spectra were
recorded on a Perkin-Elmer 781 spectrometer. Proton NMR
(1H NMR) were recorded at 300 MHz on a Varian Mercury
spectrometer and at 400 MHz on Bruker AMX 400 and Varian
VXR-400 S spectrometers. Deutero-DMSO or CDCl3 were used
as solvents. In some cases NMR spectra were recorded at 120
or 150 °C to ease NMR spectral interpretation. Chemical shifts
are given in δ units (ppm) and relative to deuterated solvent.
NMR multiplicity are denoted by s (singlet), d (doublet), m
(multiplet), and bs (broad singlet). HPLC experiments were
carried out on Chiralcel OJ or AD columns (25 cm × 0.46 cm),
with hexane/2-propanol ) 85:15 + 0.1% TFA gradients at a
flow rate of 1 mL/min and detection at 210 nM. Optical
rotations were measured in absolute ethanol on a Perkin-
Elmer 241 polarimeter at 20 °C. Elemental analyses were
performed in the labs of Solvias AG Elemente-Analytik, Basel
(CH), Switzerland, and are within (0.4% of theoretical values
unless otherwise noted. Reagents and starting materials were
obtained from commercial sources unless otherwise stated.
Reactions were in general carried out under an argon atmo-
sphere. Solvents used were distilled or dried by standard
techniques prior to use. After aqueous workup, magnesium
sulfate was used as drying agent for the organic phase.
Chromatography on silica gel was carried out on Merck silica
gel 60 (230-400 mesh) under flash conditions.
(R )-2-(t er t -Bu t oxyca r b on ylm e t h yla m in o)-3-(3,4-d i-
ch lor op h en yl)p r op ion ic Acid Meth yl Ester (3). To a
solution of (R)-2-tert-butoxycarbonylamino-3-(3,4-dichloro-
phenyl)propionic acid (2.0 g, 5.7 mmol) in DMF (30 mL) was
added silver oxide (8 g), methyl iodide (20 g), and acetic acid
(0.8 mL). This mixture was stirred at room temperature for
20 h. Then Et2O (200 mL) was added and the resulting
suspension was filtered. The filtrate was washed with water
(three times) and brine, dried over MgSO4, and concentrated
in vacuo. The residue was purified by flash chromatography
(hexane/EtOAc ) 5:1) to give 3 (2.0 g, 93%) as a colorless oil.
1H NMR (300 MHz, CDCl3, room temp): δ 7.36 (d, 1H), 7.25
(s, 1H), 7.02 (m, 1H), 4.86 and 4.52 (2m, 1H), 3.76 (bs, 3H),
3.28 (m, 1H), 3.00 (m, 1H), 2.74 and 2.70 (2s, 3H), 1.40 and
1.35 (2s, 9H). Colorless oil. Anal. (C16H21NO4Cl2) C, H, N, Cl.
[(R)-1-(3,4-Dich lor oben zyl)-2-oxoeth yl]m eth ylca r ba m -
ic Acid ter t-Bu tyl Ester (4). A solution of (R)-2-(tert-
butoxycarbonylmethylamino)-3-(3,4-dichlorophenyl)propion-
ic acid methyl ester (3) (2.0 g, 5.52 mmol) in toluene (50 mL)
was cooled to -78 °C. Then diisobutylaluminum hydride (11.5
mL of 20% solution in toluene) was added dropwise over a
period of 0.5 h. After an additional stirring period of 1 h,
methanol (2 mL) was added and the reaction mixture warmed
to 0 °C and poured into 50 mL of an ice-cold citric acid solution
(18 g/50 mL). This mixture was stirred at 0 °C for 2 h. Then
the layers were separated and the aqueous layer was extracted
twice with Et2O. The combined organic layers were washed
with water and brine, dried, and concentrated under vacuum
to give 4 (1.76 g, 96%) as a pale-yellow oil. This material was
used without further purification in the next step. 1H NMR
(300 MHz, CDCl3, room temp): δ 9.60 (s, 1H), 7.36 (d, 1H),
7.25 (s, 1H), 7.02 (m, 1H), 4.14 and 4.02 (2m, 1H), 3.24 (m,
1H), 3.02-2.80 (m, 1H), 2.76 and 2.68 (2s, 3H), 1.44 and 1.37
(2s, 9H).
(E)-(R)-5-(3,4-Dich lor op h en yl)-4-m et h yla m in op en t -2-
en oic Acid (R)-2-Oxoa zep a n -3-yl)a m id e (7). A solution of
6 (0.230 g, 0.478 mmol) and trifluoroacetic acid (1.5 mL) in
methylene chloride (10 mL) was stirred for 4 h at room
temperature. The reaction mixture was concentrated in vacuo,
and the residue was disolved in 50 mL of EtOAc. This solution
was washed twice with 0.1 N NaOH, water, and brine, dried,
and concentrated in vacuo to give 7 as a crude product that
was directly used in the next step.
N-[(E)-(R)-1-(3,4-Dich lor oben zyl)-3-(R)-2-oxoa zep a n -3-
ylca r b a m oyl)a llyl]-N-m et h yl-3,5-b is(t r iflu or om et h yl)-
ben za m id e (1). To a solution of 7 (ca. 0.144 g, ca. 0.375 mmol),
triethylamine (0.27 mL), and 4-(dimethylamino)pyridine (0.01
g) in dichloromethane (3.5 mL), 3,5-bis(trifluoromethyl)benzoyl
chloride (0.081 mL dissolved in 1 mL of methylenchloride) was
added dropwise at 0 °C. The reaction mixture was stirred for
0.5 h at 0 °C and 2 h at room temperature. Then EtOAc (30
mL) was added, and the resulting mixture was washed with
0.1 N HCl, water, and brine. The organic layer was dried and
concentrated in vacuo. The residue was purified by flash
chromatography (EtOAc) followed by crystallization from
methylenchloride/n-pentane to yield 1 as white crystals (0.142
g, 61%). Mp 127-129 °C. [R]20 +39.9° (c 0.94, EtOH). ee g
D
99.5 (tR ) 21.64 min, Chiralcel OJ , hexane/2-propanol ) 85:
15 + 0.1% TFA, flow rate 1 mL/min). 1H NMR (400 MHz,
DMSO, +150 °C): δ 8.02 (s, 1H), 7.65 (s, 2H), 7.55 (bs, 1H),
7.44 (m, 2H), 7.28 (bs, 1H), 7.21 (bs, 1H), 6.71 (dd, 1H), 6.32
(dd, 1H), 5.03 (bs, 1H), 4.51 (m, 1H), 3.18 (m, 2H), 3.05 (m,
2H), 2.82 (s, 3H), 1.96 (m, 2H), 1.75 (m, 2H), 1.45 (m, 1H), 1.3
(m, 1H). Anal. (C27H25Cl2F6N3O3) C, H, N, Cl, F. IR (CH2Cl2,
cm-1): 1668 (-CHdCH-CdO), 1643 (Ph-CdO), 1286 (Ph-
CF3), 975 (C-H, trans disubstituted CdC).
N-[(E)-(S)-1-(3,4-Dich lor oben zyl)-3-(R)-2-oxoa zep a n -3-
ylca r b a m oyl)a llyl]-N-m et h yl-3,5-b is(t r iflu or om et h yl)-
ben za m id e (8). 8 is a white crystalline powder (from meth-
(E)-(R)-4-(ter t-Bu t oxyca r b on ylm et h yla m in o)-5-(3,4-
d ich lor op h en yl)p en t-2-en oic Acid (5). To a solution of
trimethyl P,P-diethylphosphonoacetate (2.74 g, 10.2 mmol) in
THF (27 mL), n-BuLi (6.6 mL, 1.6 M in hexane) was added at
0 °C. After the mixture was strirred for 1 h, a solution of 4
(2.15 g, 6.48 mmol in 8 mL of THF) was added. Then the
ylene chloride/n-pentane). Mp 114-122 °C. [R]20 -59.6° (c
D
1
1
0.55, EtOH). H NMR (400 MHz, DMSO, +150 °C). H NMR
(400 MHz, DMSO, +120 °C): δ 8.02 (s, 1H), 7.65 (s, 2H), 7.55
(bs, 1H), 7.44 (m, 2H), 7.28 (bs, 1H), 7.23 (bs, 1H), 6.70 (dd,

3512 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Gerspacher et al.
1H), 6.315 (dd, 1H), 5.03 (bs, 1H), 4.50 (m, 1H), 3.18 (m, 2H),
3.05 (m, 2H), 2.82 (s, 3H), 1.96 (m, 2H), 1.75 (m, 2H), 1.45 (m,
1H), 1.3 (m, 1H). Anal. (C27H25Cl2F6N3O3) C, H, N.
animals were anesthetized and airway parameters were
monitored. The monkeys were exposed 2 h after administration
of 1 to an aerosol of a 1 mM solution of â-Ala8-NKA for 5 min.
P h a r m a cok in etic Testin g. Male Dunkin-Hartley guinea
pigs (350 g) were anesthetized with 4% halothane (Rhone
Merieux, U.K.) and 1:1 oxygen/nitrous oxide (2 L each per min)
and maintained at a reduced level for surgery (1.5% halo-
thane). Analgesia was initiated using 0.06 mg/kg intra-
muscular buprenorphine (Vestergesic, Reckitt and Coleman,
U.K.). The carotid artery was cannulated with pp50 polythene
tubing (Portex, U.K.) attached to a 1 mL syringe containing
500 IU/mL heparin (Multiparin, CP Pharmaceuticals Ltd,
U.K.) in 0.9% w/v saline. For animals receiving an intravenous
dose, the jugular vein was also cannulated. All cannulae were
exteriorized between the shoulders and tied in place. Animals
were allowed to recover, and up to 6 h postcannulation, each
cannula was flushed with 50 µL of 500 IU/mL heparinized
0.9% w/v saline. Animals to be dosed orally had food with-
drawn up to 18 h predose. The following morning, cannula
patency was checked using 101 IU/mL heparinized 0.9% w/v
saline. 1 was administered to guinea pigs at 3 mg/kg via the
jugular vein (iv, n ) 6) or at 10 mg/kg orally (po, n ) 7) as a
microemulsion (2% DMSO/20% Sandimmun placebo/78% sa-
line) or cremophor/2% DMSO formulation. Blood samples were
taken at 5, 30, 60, 120, 240, 360, 720, and 1440 min (iv) and
at 60, 120, 240, 360, 480, 720, and 1440 min (oral). Blood
samples were analyzed for 1 by HPLC mass spectrometry
(LC-MS).
N-[(E)-(R)-1-(3,4-Dich lor oben zyl)-3-(S)-2-oxoa zep a n -3-
ylca r b a m oyl)a llyl]-N-m et h yl-3,5-b is(t r iflu or om et h yl)-
ben za m id e (9). 9 is a white crystalline powder (from meth-
ylenchloride/n-pentane). Mp 115-120 °C. [R]20D +58.7° (c 0.65,
1
EtOH). H NMR (400 MHz, DMSO, +120 °C): δ 8.03 (s, 1H),
7.67 (d, 1H), 7.60 (s, 2H), 7.44 (m, 2H), 7.40 (bs, 1H), 7.20 (bs,
1H), 6.68 (dd, 1H), 6.37 (dd, 1H), 5.06 (bs, 1H), 4.52 (m, 1H),
3.15 (m, 2H), 3.07 (m, 2H), 2.81 (s, 3H), 1.90 (m, 2H), 1.80-
1.63 (m, 2H), 1.45 (m, 1H), 1.3 (m, 1H). Anal. (C27H25
Cl2F6N3O3) C, H, N, Cl.
-
N-[(E)-(S)-1-(3,4-Dich lor ob en zyl)-3-(S)-2-oxoa zep a n -
3-ylca r b a m oyl)a llyl]-N-m et h yl-3,5-b is(t r iflu or om et h yl-
ben za m id e (10). 10 is a white crystalline powder (from
methylene chloride/n-pentane). Mp 122-127 °C. [R]D20 -39.25°
1
(c 0.53, EtOH). H NMR (400 MHz, DMSO, +120 °C): δ 8.03
(s, 1H), 7.67 (d, 1H), 7.60 (s, 2H), 7.44 (m, 2H), 7.40 (bs, 1H),
7.20 (bs, 1H), 6.70 (dd, 1H), 6.35 (dd, 1H), 5.06 (bs, 1H), 4.51
(m, 1H), 3.15 (m, 2H), 3.07 (m, 2H), 2.81 (s, 3H), 1.90 (m, 2H),
1.80-1.63 (m, 2H), 1.45 (m, 1H), 1.3 (m, 1H). Anal. (C27H25
Cl2F6N3O3) C, H, N, Cl.
-
NK1, NK2 Recep tor Bin d in g Assa ys. Cloned human
receptors, expressed in CHO cells, were purchased from NEN
Life Sciences. Ninety-six-deep-well plates were used, with each
well containing 375 µL of a membrane receptor suspension
(1:50 dilution of the original stock), 125 µL of 3H-[Sar9Met-
(O2)11]SP (final concentration of 0.5 nM) for NK1 or 125I-
neurokinin A (final concentration of 1.4 nM) for NK2 recetors,
25 µL of 1, 8, 9, or 10, all prepared and diluted in HEPES
buffer composition: 20 mM HEPES, 1 mM MnCl2, pH 7.4.
Nonspecific binding was determined in the presence of 0.3 µL
of Sar9SP or 10 µL of NKA for the NK1 receptor or the NK2
receptor, respectively. The mixture was incubated for 60 min
at 28 °C, after which the unbound ligand was removed by
inverse flash filtration into 96-well filter plates followed by
three washes with ice-cold Tris buffer (50 mM, pH 7.4). The
filter plates were dried for 15 min at 56 °C, followed by the
addition of 50 µL of scintillation, and sealed on top with an
adhesive sheet. The radioactivity was counted in a Packard
96-well plate counter.
Neu r okin in -In du ced Br on ch ocon str iction in th e Gu in ea
P ig. Male Dunkin-Hartley guinea pigs (400-550 g) were
anesthetized and mechanically ventilated. The jugular vein
and carotid artery were cannulated for injection of drug and
blood pressure measurements, respectively. All signals were
recorded using a computer data acquisition system (Lfr,
Mumed Systems). After establishment of a stable baseline,
dose-response curves were constructed for Sar9 substance P
(0.6-10 µg/kg) or (Ala5,â-Ala8)-R-neurokinin (fragments 4-10)
(0.06-3 µg/kg). The agonists were dissolved in 0.9% NaCl and
injected iv every 15 min as increasing quarter-log increments.
Increases in lung resistance were determined. The timing of
anesthesia and animal preparation were such that the dose-
response curves to Sar9 substance P or (Ala5,â-Ala8)-R-neuro-
kinin (fragments 4-10) were obtained 5 min and 1 h after the
iv injection of vehicle or 1. 1 was given at doses of 1 or 10
mg/kg in a vehicle of 2% DMSO/17% cremophor EL/ 81% NaCl.
Results were analyzed by calculating the percent change from
baseline of each agonist response. Best-fit dose-response
graphs were constructed, and values corresponding to a 300%
increase in resistance to agonist were calculated to quantify
the inhibitory effects of the antagonists. Student’s t-test (for
normally distributed data) and Mann-Whitney rank sum test
(for nonparametric data) were used to evaluate the significance
of the changes in resistance values at 300%. Results are quoted
as the mean ( SEM. Significance was determined as P < 0.05.
Ack n ow led gm en t. We thank Ms. E. Schuler, Ms.
V. Pawelzik, Mr. H. Bammerlin, Mr. R. Ernst, and Mr.
D. Wyss for their excellent technical assistance. We
thank Mr. P. Hug for recording NMR spectra and for
helpful discussions. We also thank Dr. E. Francotte for
the determination of ee values and HPLC analyses.
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