Insertion of an aspartic acid moiety into cyclic pseudopeptides: Synthesis and biological characterization of potent antagonists for the human tachykinin NK-2 receptor

Article abstract of DOI:10.1021/jm040832y

A new series of monocyclic pseudopeptide tachykinin NK-2 receptor antagonists has been derived from the lead compound MEN11558. A synthesis for these molecules sharing the same intermediate was designed and performed. The replacement of the succinic moiety with an aspartic acid and the functionalization of its amino group with a wide variety of substituents led to very potent and selective NK-2 antagonists. Best results were obtained through the insertion in position 12 of an amino group with R configuration, linked by a short spacer to a saturated nitrogen heterocycle (morpholine, piperidine, or piperazine). The study led to compounds 54 and 57, endowed with high in vivo potency at very low doses and long duration of action in animal models of bronchoconstriction. In particular 54 and 57 completely inhibited NK-2 agonist induced bronchoconstriction in guinea pig after intratracheal administration at subnanomolar doses (ED₅₀ = 0.27 nmol/kg and 0.15 nmol/kg, respectively).

Full text of DOI:10.1021/jm040832y

J. Med. Chem. 2004, 47, 6935-6947
6935
Insertion of an Aspartic Acid Moiety into Cyclic Pseudopeptides: Synthesis and
Biological Characterization of Potent Antagonists for the Human Tachykinin
NK-2 Receptor
Valentina Fedi,* Maria Altamura,* Giuseppe Balacco, Franca Canfarini, Marco Criscuoli, Danilo Giannotti,
Alessandro Giolitti, Sandro Giuliani, Antonio Guidi, Nicholas J. S. Harmat, Rossano Nannicini, Franco Pasqui,
Riccardo Patacchini, Enzo Perrotta, Manuela Tramontana, Antonio Triolo, and Carlo Alberto Maggi
Menarini Ricerche S.p.A., Via dei Sette Santi 3, I-50131 Florence, Italy
Received April 27, 2004
A new series of monocyclic pseudopeptide tachykinin NK-2 receptor antagonists has been
derived from the lead compound MEN11558. A synthesis for these molecules sharing the same
intermediate was designed and performed. The replacement of the succinic moiety with an
aspartic acid and the functionalization of its amino group with a wide variety of substituents
led to very potent and selective NK-2 antagonists. Best results were obtained through the
insertion in position 12 of an amino group with R configuration, linked by a short spacer to a
saturated nitrogen heterocycle (morpholine, piperidine, or piperazine). The study led to
compounds 54 and 57, endowed with high in vivo potency at very low doses and long duration
of action in animal models of bronchoconstriction. In particular 54 and 57 completely inhibited
NK-2 agonist induced bronchoconstriction in guinea pig after intratracheal administration at
subnanomolar doses (ED50 ) 0.27 nmol/kg and 0.15 nmol/kg, respectively).
Introduction
The tachykinins substance P (SP), neurokinin A
(NKA), and neurokinin B (NKB) form a family of peptide
neurotransmitters that are widely distributed in the
mammalian peripheral and central nervous system and
produce their biological actions by activating three
distinct receptor types, respectively termed NK-1, NK-
2, and NK-3.1 NKA exerts its biological effects mainly
by activation of the tachykinin NK-2 receptor. The
human NK-2 receptor has been identified and validated
as a suitable target for the development of novel drugs
to be used for the treatment of a number of diseases in
the respiratory, gastrointestinal, and genitourinary
tract and in the CNS.2 In particular, human NK-2
receptor antagonists are considered potential candidates
for the treatment of asthma, bronchial hyperreactivity,
irritable bowel syndrome, cystitis, or depression.3 In our
previous studies4 MEN11558, a monocyclic pseudopep-
tide, was identified as a potential new lead for a class
of antagonists with simpler structures with respect to
nepadutant (MEN11420, Figure 1),5 a bicyclic hexapep-
tide currently in phase II clinical trials. It was clear from
previous structural studies4 that MEN11558 maintains
its biological activity thanks to the fact that the Trp-
Phe fragment is adjusted in a type I â-turn by the
appropriate cyclic linker. The correct R configuration
of the carbon atom in position 8 (Figure 2), bearing the
benzyl substituent on the diamine moiety, was also
defined in previously reported investigations.4 In the
current contribution we present the results of the
replacement of the succinic portion (positions 12 and 13
of the structure of Figure 2) with an aspartic moiety,
Figure 1. The structure of nepadutant (MEN 11420).
Figure 2. Insertion and further modifications of an amino
moiety on the initial lead MEN11558.
* To whom correspondence should be addressed. Phone:
+3905556801. Fax: +390555680419. E-mail: vfedi@menarini-ricerche.it,
maltamura@menarini-ricerche.it.
which allowed us to obtain new potent antagonists with
enhanced physicochemical properties.
10.1021/jm040832y CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/07/2004

6936 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Fedi et al.
Scheme 1a
a
Reagents: (a) Benzyl chloroformate, NEt3, THF, 0 °C (75-80%); (b′) HCl in dioxane, MeOH, room temperature (95%); (b′′) CF3COOH,
CH2Cl2, 0 °C f room temperature (80%); (c) FmocOSu, THF (70-75%); (d) Boc-Phe-OH, HOBt, EDC‚HCl, NEt3, CH3CN (or DMF), 0 °C
f room temperature (85%); (e) HCl in dioxane, MeOH, 5 °C f room temperature (90-95%); (f) Boc-Trp-OH, HOBt, EDC‚HCl, NEt3,
DMF, 0 °C f room temperature (90%); (g) Et2NH, DMF, room temperature; (h) Boc-Asp(OH)-OBzl (D or L),b HOBt, EDC‚HCl, NEt3,
DMF, room temperature (70%); (i) Fmoc-Asp-(OtBu)-OH (D or L),b HOBt, EDC‚HCl, DMF (>95%); (j) Boc-Asp(OBzl)-OH (D or L),b HOBt,
EDC‚HCl, NEt3, DMF, room temperature (70%); (k) H2, Pd/C, DMF, H2O, room temperature (93%); (l) ethanedithiol, CF3COOH, CH2Cl2,
0 °C f room temperature (80%); (m) HOBt, EDC‚HCl, DMF, room temperature (80%, 62%). b The D or L amino acid derivative is used to
obtain compounds of the a or b series, respectively.
There were several advantages in selecting the as-
partic acid from the chiral pool for our purposes: it is
easily available in both enantiomers; with respect to
MEN11558, it guarantees the same number of terms
in the resulting cycle while introducing a new function-
alization; the use of differentially protected derivatives
(at the R- and â-carboxy groups) allows the insertion of
amino groups in both positions 12 and 13 of the original
molecule; primary or substituted amines were expected
to increase water solubility of the final products. Fur-
thermore, the QSAR model recently described6 by our
group suggested that modifications in that part of the
lead molecule should have been well tolerated by the
receptor. The R configuration of the amino acid was
initially chosen to minimize the risk of recognition by
hydrolytic enzymes (peptidases), but during our inves-
tigation we found that the derivatives of (R)-aspartic
acid were also more effective than the derivatives of (S)-
aspartic acid.
In the first part of the present work a small group of
unsubstituted or substituted derivatives was syntesized
to check the potential of the new class and to confirm
the validity of the new lead 7a in comparison to its
epimer 7b and its regioisomers 8a and 8b (Scheme 1).
It was immediately clear that introducing a hydrophilic
group in the succinic linker to enhance physicochemical

Antagonists for the Human Tachykinin NK-2 Receptor
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6937
Scheme 2a
properties did not negatively affect the binding affinity,
while some of the functionalizations led to a substantial
increment of the NK-2 receptor antagonist activity. In
a further development of the SAR study a larger space
of substituents was considered to improve the pharma-
cological and physicochemical profile of these com-
pounds.
This study eventually led to selection of 54 and 57,
whose in vitro and in vivo pharmacological profile is
discussed below.
Chemistry. The synthesis of the new lead compound
7a and that of its analogues bearing the amino group
in a different configuration (7b) or in a different position
(on C13, 8a,b), together with the preparation of dichlo-
robenzyl derivatives 14a,b, are depicted in Scheme 1.
The starting monoprotected diamine 17 was op-
portunely transformed to a derivative suitably protected
on the distal amino group, 2, and subsequently coupled
with Boc protected D-phenylalanine. Deprotection of Boc
under standard conditions gave amine 3. The corre-
sponding monoprotected diamine 9 was subjected to a
similar procedure, using a Fmoc protecting strategy.
During the synthesis all the intermediates containing
a free amino group in the presence of a Fmoc protected
amine were isolated or conveniently stored to avoid
partial deprotection. As presented in Scheme 1, 9 was
initially transformed to the diprotected derivative 10,
and then deprotection of Boc under standard conditions
and immediate coupling with Boc-L-phenylalanine were
performed to give 11, once again as a diprotected
intermediate. The second coupling step to introduce Boc-
L-tryptophan was followed in the first case (left) by the
Boc deprotection to obtain 4 while in the synthesis of
the dichloro derivatives (right) it was preceded by the
Boc-deprotection and followed by the Fmoc deprotection
to 12. The last synthetic strategy was chosen due to the
impossibility to perform any subsequent catalytic hy-
drogenation to remove benzyloxycarbonyl or benzyl
protective groups without the concomitant partial loss
of chloride from the aromatic ring of the diamine
portion. Following the synthetic pathway depicted in the
left part of Scheme 1, 4 was subsequently acylated with
diprotected aspartic acid, the regioselectivity being
defined by the position of the free carboxylic group. The
reaction of the two enantiomers of Boc-Asp-(OH)-OBzl
with 4 under standard activating conditions and the
double deprotection of the benzyl ester and the benzyl-
carbamate by hydrogenolysis gave 5a (from the D-Asp
derivative) and 5b (from the L-Asp derivative), respec-
tively. Under the same conditions the enantiomers of
Boc-Asp-(OBzl)-OH gave with comparable yields the
regioisomers 6a (from the D-Asp derivative) and 6b
(from the L-Asp derivative). Intermediate 12 (right) was
on the contrary acylated under standard conditions only
with the two enantiopure Fmoc-Asp-(OtBu)-OH and
subsequently doubly deprotected to 13a,b with TFA and
ethanedithiol. The final cyclization step of the obtained
zwitterions was performed under controlled high dilu-
tion conditions to minimize the parasite intermolecular
reaction to oligomeric compounds, the threshold for the
concentration of acyclic reactant being 0.01 M in DMF.
a
Reagents: (a) appropriate carbonyl compound, Na(CN)BH3,
AcOH, MeOH, room temperature.
Scheme 3a
a
Reagents: (a) appropriate carbonyl compound, Na(CN)BH3,
AcOH, MeOH, room temperature.
Scheme 2, using sodium cyanoborohydride and acetic
acid to obtain products 15a-18a.
To enlarge the structure-activity relationship study
about the substitution on the amino group, other alky-
lations employing aldehydes or ketones containing
optionally protected additional functional groups were
performed on the lead compound 7a (Scheme 3), giving
compounds listed in Table 2.
A wide range of carbohydrates (Scheme 4 and Table
3) was introduced in order to further increase the water
solubility of the final products and to evaluate, in this
series, the effect of the insertion of a sugar moiety
which, in the case of the previously described bicyclic
peptide nepadutant, gave a compound with excellent
pharmacological properties. Three general methods
were used for the polyhydroxylated substituents: method
A, to attach a linker to the amino group in position 12
of the lead 7a via an acylation, then create the linkage
with the new hydrophilic functional group through
another acylation step (see 34, 35); method B, to directly
attach the selected sugar to the amine in position 12
through its aldehydic function in a reductive medium
(see 36-40); method C, to directly acylate the amino
functionality of 7a with an oxidized sugar (41).
The last part of the present synthetic work regards
target compounds 48-57, obtained through the acyla-
tion of 7a with amino acids coming from a nucleophilic
substitution on bromoacetic acid. As described in Scheme
5, the amino nucleophile was added to the bromoacetic
The amino group of 7a,b, 14a, 8a,b was mono- or
dialkylated through reductive amination as shown in

6938 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Fedi et al.
Table 1. Structures of Compounds 7, 8, and 14-18 and Affinities for the Tachykinin NK-2 Receptor Evaluated in Binding
Experiments (pKi Values) and Functional in Vitro Experiments (pKB Values)
NK-2 activitye
b
c
NR1R2
position
pKia
(hNK-2)
pKB
pKB
entry
R1
R2
X
(RPA)
(RUB)
MEN11558d
H
Cl
H
H
H
H
Cl
Cl
H
H
H
H
H
H
Cl
8.7 ( 0.1
10.0 ( 0.1
8.7 ( 0.1
8.5 ( 0.2
7.8 ( 0.1
8.5 ( 0.2
9.8 ( 0.2
9.7 ( 0.1
10.2 ( 0.1
9.0 ( 0.2
8.1 ( 0.1
8.2 ( 0.2
9.8 ( 0.2
9.1 ( 0.2
9.6 ( 0.1
7.6 ( 0.1
8.1 ( 0.1
7.9 ( 0.2
7.5 ( 0.2
7.2 ( 0.2
7.7 ( 0.2
8.1 ( 0.2
8.6 ( 0.2
9.2 ( 0.2
8.6 ( 0.2
8.3 ( 0.1
8.1 ( 0.1
MEN11690d
7a
12-(R)
12-(S)
13-(R)
13-(S)
12-(R)
12-(S)
12-(R)
12-(S)
13-(R)
13-(S)
12-(R)
12-(S)
12-(R)
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
7b
8a
8b
14a
14b
15a
15b
16a
16b
17a
17b
18a
8.9 ( 0.2
9.1 ( 0.2
4-tetrahydropyranyl
4-tetrahydropyranyl
4-tetrahydropyranyl
4-tetrahydropyranyl
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
7.4 ( 0.2
7.8 ( 0.2
9.2 ( 0.2
8.9 ( 0.2
8.0 ( 0.2
8.9 ( 0.2
8.5 ( 0.3
8.4 ( 0.2
a pKi ) -log Ki. Affinities for the human NK-2 receptor estimated against [125I]neurokinin A in radioligand binding experiments. b pKB
) -log KB. Antagonist affinities of compounds for rabbit NK-2 receptor estimated against neurokinin A in the rabbit isolated pulmonary
artery (RPA). c Antagonist affinities of compounds for rat NK-2 receptor estimated against [â-Ala8]NKA(4-10) in the rat isolated urinary
bladder (RUB). d Data from ref 4. e Each value is the mean ( SEM of 4-8 determinations.
Table 2. In Vitro Activity of Compounds 19-33 Evaluated in Binding Experiments (pKi Values) and Functional Experiments (pKB
Values)
NK-2 activitye
b
pKia
(hNK-2)
pKB
entry
R1
R2
(RUB)
MEN11558c
8.7 ( 0.1
8.7 ( 0.1
9.8 ( 0.2
8.8 ( 0.1
8.3 ( 0.1
8.8 ( 0.1
9.4 ( 0.1
9.9 ( 0.1
8.5 ( 0.1
9.7 ( 0.2
9.7 ( 0.1
10.2 ( 0.1
10.0 ( 0.1
10.6 ( 0.1
9.8 ( 0.1
9.8 ( 0.2
10.2 ( 0.1
10.3 ( 0.1
9.7 ( 0.1
8.3 ( 0.1
7.9 ( 0.2d
9.2 ( 0.2
8.2d ( 0.2
8.5 ( 0.1
7.7d ( 0.2
9.2 ( 0.1
9.6 ( 0.2
9.3 ( 0.2
8.9 ( 0.2
8.9 ( 0.1
9.1 ( 0.2
7.8 ( 0.1
9.1 ( 0.2
9.3 ( 0.2
8.8 ( 0.2
9.5 ( 0.2
9.5 ( 0.2
9.0 ( 0.2
7ac
17ac
19
-CH2CH2OCH2CH2-
-(CH2)5-
20
-CH2CH2CH(NH2)CH2CH2-
-CH2CH2NHCH2CH2-
21
22
-CH2CH2N(CH3)CH2CH2-
-CH2CH2N(COCH3)CH2CH2-
-CH2CH2N(SO2CH3)CH2CH2-
-CH2CH2CH(1-piperidin)CH2CH2-
-CH2CH2CH(4-morpholin)CH2CH2-
4-tetrahydropyranyl
23
24
25
26
15ac
27
H
H
4-tetrahydrothiopyranyl
28
H
H
CH3
H
H
H
4-tetrahydrothiopyranyl-1-oxide
4-tetrahydrothiopyranyl-1,1-dioxide
4-tetrahydropyranyl
29
30
31
4-piperidinyl-1-methanesulfonyl
4-piperidinyl-1-sulfonic acid amide
4-piperidinyl-1-methyl
32
33
a See corresponding footnote in Table 1. b See footnote c in Table 1. c Data from Table 1. d pKB measured on RPA, see footnote b in
Table 1. e Each value is the mean ( SEM of 4-8 determinations.
tert-butyl ester in the presence of triethylamine in THF,
and the resulting amino ester was hydrolyzed under
acid catalysis to give the corresponding amino acid. In
the particular case of 54 the aminosulfonylpiperazine
nucleophile was obtained by reaction of the freshly
prepared sulfamoyl chloride8 with N-benzyl protected
piperazine in triethylamine and THF and further
hydrogenation catalyzed by Pd/C in methanol. Finally
the coupling reactions with 7a were performed through
activation of the carboxylic function with HOBt, EDC‚
HCl, usually in DMF as a solvent.
Results and Discussions
The binding affinity at the human tachykinin NK-2
receptor together with the functional activity on isolated
tissues was measured for all the derivatives according

Antagonists for the Human Tachykinin NK-2 Receptor
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6939
Scheme 4a
a
Reagents: (a) succinic anhydride, CH3CN, DMF; (b) starting sugar moiety (see Table 3), HOBt, EDC‚HCl, DMF; (c) Ac-Asp-(OtBu)-
OH, HOBt, EDC.HCl, DMF; (d) TFA, CH2Cl2; (e) starting sugar moiety (see Table 3), CH3COOH, NaCNBH3, MeOH; (f) lactobionic acid,
PyBOP, diisopropylethylamine, DMF.
Table 3. In Vitro Activity of Compounds 34-41 Evaluated in Binding Experiments (pKi Values) and Functional Experiments (pKB
Values)
NK-2 activityg
b
pKia
(hNK-2)
pKB
entry
spacer
starting sugar moiety
method
(RUB)
34
35
36
37
38
39
40
41
succinic acid
N-acetyl-aspartic acid
2-acetamido-2-deoxy-â-D-glucopyranosyl amine
2-acetamido-2-deoxy-â-D-glucopyranosyl amine
2-acetamido-2-deoxy-â-D-glucose
D-(+)-glucose
A
8.8 ( 0.1
7.6 ( 0.2
8.7 ( 0.1
9.4 ( 0.1
8.9 ( 0.1
9.2 ( 0.1
9.3 ( 0.1
8.4 ( 0.2
7.6 ( 0.2
7.7 ( 0.2
8.1 ( 0.2
8.8 ( 0.1
8.2 ( 0.2
8.5 ( 0.2
8.5 ( 0.2
8.7 ( 0.1
A
B
B
D-(+)-lactosec
B
D-(+)-maltosed
B
maltotriosee
B
lactobionic acidf
C (A)
a See corresponding footnote in Table 1. b See footnote c in Table 1. c 4-O-â-D-galactopyranosyl-D-glucose. d 4-O-R-D-glucopyranosyl-D-
glucose. e O-R-D-glucopyranosyl-(1f4)-O-R-D-glucopyranosyl-(1f4)-D-glucose. f 4-O-â-D-galactopyranosyl-D-gluconic acid. g Each value is
the mean ( SEM of 4-8 determinations.
to experimental methodologies reported in the Experi-
mental Section. Results are listed in Tables 1-4.
Regarding functional assays, after initial testing of
the early compounds in the rabbit pulmonary artery
(RPA) test, the more reliable and convenient RUB (rat
urinary bladder) test was chosen as a functional model
for the NK-2 receptor, once results had demonstrated
the equivalence between data obtained in the RPA vs
data obtained in the RUB (see Table 1).
In Table 1 the in vitro pharmacological profiles of the
two reference compounds MEN11558 and MEN116904
are compared with the amino substituted derivatives.
The introduction of the amino moiety in the R config-
uration in position 12 of MEN11558 gave product 7a,
which luckily showed comparable activity with respect
to the starting lead molecule while bearing an amino
group suitable to further functionalization. To confirm
the opportunity of the selection of 7a as a new lead, the

6940 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Fedi et al.
Scheme 5a
the first, consisting of the introduction of a 4-tetrahy-
dropyranyl pendant on the primary amine of the four
isomers, gave rise to products 15a,b and 16a,b; the
second, consisting of the transformation of the free
amine pendant in a morpholine, was performed on the
precursors 7a,b and on the chlorinated analogue 14a
to give the corresponding 17a,b and 18a. In the first
four-component library the regiochemistry and absolute
configuration of 7a were confirmed as the best (compare
15a with 15b and 16a,b), in the second three-component
group the R isomer was once again the best (17a vs 17b)
and, interestingly, the dihalogenated product 18a seemed
to lose the advantage over the nonhalogenated com-
pounds shown by the unsubstituted precursor (14a). In
conclusion, considering that the dichlorinated series
would have been more expensive, heavier in terms of
molecular weight, and consequently less favorable for
some of the physicochemical properties (e.g. solubility),
and also that the in vitro activity shown by the first
derivative 18a was not better than that of the corre-
sponding unsubstituted product 17a, it was reasonable
to elect compound 7a as the new lead for further
derivatization.
Results on an enlarged group of both tertiary and
secondary amino derivatives are presented in Table 2.
Starting from the consideration that 17a showed good
activity, simple modifications on the terminal cyclic
moiety were performed: the substitution of the oxygen
atom of the morpholine with CH2 (19), CH-NH2 (20),
NH (21), and NSO2CH3 (24) turned out in evident loss
of affinity, while the introduction in the same position
of N-(CH3) (22) and N-(COCH3) (23) gave rise to
products with comparable activity with respect to 17a.
The insertion of an extra amino functionality in a distal
heterocycle, as in 25 and 26, was well tolerated by the
receptor and is potentially advantageous for the hydro-
philicity of the resulting compounds. A brief structure-
activity study was undertaken also on the tetrahydro-
pyranyl derivative 15a presented in the first part of this
discussion. The attempt to substitute the oxygen atom
of the heterocycle with sulfur (27) seemed to emphasize
the differences in terms of activity between the two
considered species giving rise to a product with high
affinity for the human receptor but with a pKB of more
than 100-fold smaller in rat tissues. This gap was easily
recovered passing from the sulfur derivative to the
sulfoxide (28) and sulfone (29) functionalities in the
same position. The substitution of the remaining hy-
drogen on the amino functionality of 15a with a methyl
group does not seem to improve further the biological
results.
a
Reagents: (a) amine (see corresponding X group in Table 4),
NEt3, THF, 65 °C (>98%); (b) TFA, CH2Cl2, room temperature
(>98%); (c) sulfamoyl chloride, THF, 0 °C f room temperature
(26%); (d) H2, Pd/C 10%, MeOH, H2O (76%); (e) 44a-j, HOBt,
ECD‚HCl, DMF, room temperature (20-50%).
Table 4. In Vitro Activity of Compounds 48-57 Evaluated in
Binding Experiments (pKi Values) and Functional Experiments
(pKB Values)
a See corresponding footnote in Table 1. b See footnote c in Table
1. c Each value is the mean ( SEM of 4-8 determinations.
Thanks to the information obtained with this first
sample set of molecules, N-substituted piperidines were
used in alternative to the tetrahydropyran of 15a and
tested. The two sulfonyl derivatives (31 and 32) turned
out to be among the most active compounds. These
levels of affinity result extremely interesting even if
compared with the other known selective antagonists
for this receptor.9
Another set of products was prepared and tested, on
the same target, to verify the opportunity of the
introduction of a sugar moiety for receptor affinity and,
in the case of a positive result, in terms of ADME
features. This approach was dictated by our previous
regio- and stereoisomers 7b and 8a,b were synthesized
and tested: these all showed a worse or at most
comparable value of pKi for the binding at the human
NK-2 receptor and pKB in the rabbit pulmonary artery
(RPA) functional test. The insertion of two chlorine
atoms on the benzyl substituent in position 8 was
suggested by the encouraging results shown by the
reference compound MEN11690.4 As expected the cor-
responding dichloro derivative 14a and also its epimer
14b showed interesting values in both binding and
functional tests. To further check the potential of 7a as
new lead in comparison with the other candidates, two
types of substitution on the amino group were realized:

Antagonists for the Human Tachykinin NK-2 Receptor
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6941
Table 5. In Vitro Activity of Selected Compounds for the
Tachykinin NK-1, NK-2, and NK-3 Receptors Evaluated in
Functional Experiments (pKB Values) in Comparison with the
Reference Compound MEN11558
investigations around one of the bicyclic lead structures
(MEN 10627),10 onto which the introduction of an
N-acetyl-glucosamine pendant in place of the residue
of a cysteine gave rise to nepadutant (MEN11420), a
compound with improved pharmacokinetic and physi-
cochemical properties, presently under clinical trials.5,11
In general sugar scaffolds are quite interesting hydro-
philic groups because of the abundance of hydroxylic
functions that insist on a small structure in a precise,
naturally defined stereochemistry. Results on this sub-
set are reported in Table 3. Even at a first glance it is
clear that the mean value of binding and functional
activity for this series is not so exciting with respect to
the other investigated manipulations, although some of
these derivatives maintain an interesting pharmacologi-
cal pattern. Our first attempt (see 34 and 35) was to
insert an ω-carboxylated linker able to support the
aminosugar (2-acetamido-2-deoxy-â-D-glucopyranosyl
amine)12 characteristic of our reference compound (ne-
padutant), and mimicking at the same time the distance
between the peptide skeleton and the sugar moiety in
the above-mentioned molecule. With this purpose in
mind, succinic acid and N-acetyl-aspartic acid were
introduced and further functionalized with the afore-
mentioned aminosugar as described in Scheme 3. Both
34 and 35 not being satisfactory for their pharmacologi-
cal profile, the elongation chain was suppressed and
other derivatives were obtained by direct reductive
amination on the aldehydic function of diverse monosac-
charides as easily as with di-and trisaccharides: 36 with
N-acetyl-D-glucose, 37 with D-glucose, 38 with D-lactose,
39 with D-maltose, 40 with D-maltotriose. This unusual
synthetic methodology allowed the amino functionality
to be maintained in conjunction with the insertion of
highly hydrophilic moieties giving rise to compounds
with subnanomolar affinity at the human NK-2 receptor
and favorable activity in functional tests. On the other
hand the loss of the basic functionality resulting from
the introduction of the D-lactobionic fragment (see 41)
provoked a sensible decrease in the binding ability.
NK-1
NK-2
NK-3
entry
pKB (GPI)a,e
pKB (RUB)b,e
pKB (GPI)c,e
MEN11558d
15a
17a
54
55
57
5.7 ( 0.1
6.1 ( 0.1
6.2 ( 0.1
<5.5
6.8 ( 0.1
7.5 ( 0.2
7.6 ( 0.1
9.1 ( 0.2
9.2 ( 0.2
9.5 ( 0.1
9.2 ( 0.2
9.7 ( 0.1
6.1 ( 0.1
6.4 ( 0.1
6.1 ( 0.1
5.9 ( 0.2
5.8 ( 0.1
6.4 ( 0.2
a pKB ) -log KB. Antagonist affinities of compounds for guinea
pig NK-1 receptor estimated against substance P methyl ester
(SPOMe) in the isolated guinea pig ileum (GPI). b See footnote c
in Table 1. c Antagonist affinities of compounds for guinea pig
NK-3 receptor estimated against senktide in the isolated guinea
pig ileum (GPI). d Data from ref 4. e Each value is the mean ( SEM
of 4-8 determinations.
subnanomolar affinity for the human receptor together
with a good activity in the functional test on the isolated
rat tissue. The synthesis of the thiomorpholine sulfoxide
53 took its origin from the three results previously
obtained on the differently oxidized thio derivatives
depicted in Table 2 and suggesting an advantage for the
monooxidized 28. However, even maintaining a good in
vitro profile, 53 resulted less active than its above-
mentioned precursor.
Finally, the insertion of differently substituted amino
groups in position 12 on our reference compound
MEN11558 does not seem to alter the selectivity profile
respect to the other tachykinin receptors, shown by the
latter compound. Table 5 compares the results of
functional experiments at the NK-1, NK-2, and NK-3
receptors for a number of “key” compounds: 15a and
17a, the lead compounds of the two groups based on
the insertion of a 4-tetrahydropyranyl pendant (15a) or
a morpholine (17a), respectively; compound 55, a more
advanced compound bearing the 4-piperidylpiperidine
moiety; and two of the most active compounds of the
series, 54 and 57. All compounds reported in Table 5
show an improved selectivity when compared to that of
MEN11558, with at least 100-fold higher affinity at the
NK-2 receptor versus the other two tachykinin recep-
tors.
Compound 54, bearing an aminosulfonyl piperazine,
and compound 57, with a 4-morpholine-piperidine pen-
dant, were among the most active, in in vitro tests, we
have ever encountered during our work, and these
compounds were chosen for deeper pharmacological
investigations. In addition to the affinity shown at the
human tachykinin NK-2 receptor at subnanomolar
concentrations (pKi ) 9.8 for 54, pKi ) 10.0 for 57), they
showed very high selectivity in binding tests at the
human tachykinin NK-1 and NK-3 receptors, with
affinity at least 1000-fold lower for NK-1 (pKi ) 6.7 for
54, pKi ) 6.9 for 57) and 10000-fold lower for NK-3 (pKi
) 5.1 for 54, pKi ) 5.9 for 57) than for the NK-2
receptor.
Both compounds 54 and 57 showed high in vivo
potency at very low doses, and long duration of action
in animal models of bronchoconstriction. In particular,
after intratracheal (i.t.) administration in anesthetized
guinea pigs the two compounds were active at sub-
nanomolar concentrations. At a dose of 10 nmol/kg i.t.,
54 and 57 strongly inhibited (about 80%) bronchocon-
Some deductions arise by the analysis of these
results: (a) the presence of an amino group in this part
of the molecule does seem to increase the potency
against the NK-2 receptor; (b) among the considered
nitrogen-containing heterocycles, morpholine, piperi-
dine, and acylated piperazine emerged as the best.
Further work has been done with these thoughts in
mind to probe the better position for the basic nitrogen,
and a new small subset was obtained as depicted in
Scheme 5. The binding and functional properties of the
new compounds are listed in Table 4. The common
feature of these derivatives is the presence of the amide
functionality in place of the original amine in the
aspartic portion and the concomitant reinsertion of a
basic nitrogen in a farther position with respect to the
other compounds, usually spaced out by a single meth-
ylene. As evident from Table 4, all compounds, except
the N-methyl-piperazine derivative 51, showed subna-
nomolar affinity to the receptor, with pKi > 9.0, indicat-
ing that this strategy is as easy as it is satisfactory.
Many basic moieties selected among the most active of
the previous series were inserted in the pendant giving
rise to very active products. In particular derivatives
with 4-substituted piperidines (49 and 52) showed

6942 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
striction induced by the selective NK-2 agonist [â-Ala8]-
Fedi et al.
Methods. Binding Experiments. Radioligand binding
experiments were performed at human NK-1, NK-2, and NK-3
tachykinin receptors stably transfected on membranes of
U-373MG (NK-1) or CHO-K1 (NK-2 or NK-3) cells, as reported
previously.5,14,15 All compounds were tested for their ability to
displace the following radioligands from the three tachykinin
receptors (in brackets): [3H][Sar9]SP sulfone, 1.2 nM (NK-1);
NKA(4-10) (1 nmol/kg iv) for at least 4 h, with ED50
)
0.27 nmol/kg for 54 and ED50 ) 0.15 nmol/kg for 57. In
comparison, previous leads 15a (bearing a 4-tetrahy-
dropyranyl pendant, see Table 1) and 17a (with a
morpholine substituent, see Tables 1 and 2) produced
similar effects, as duration of action and maximal
inhibition of bronchocostriction, only after i.t. adminis-
tration of 100 nmol/kg, a 10 times higher dose compared
to 54 and 57.
[
125I]neurokinin A, 0.15 nM (NK-2); [125I][MePhe7]NKB, 50 pM
(NK-3). All radioligands were from Amersham Biosciences
(Buckinghamshire, U.K.). Nonspecific binding was determined
in the presence of unlabeled [Sar9]SP sulfone 1 µM (at NK-1
receptor), or of unlabeled neurokinin A 1 µM (at NK-2
receptor), or of unlabeled [MePhe7]NKB 1 µM (at NK-3
receptor), and ranged between 5% and 10% of total binding at
each receptor. Affinity of test compounds for the tachykinin
receptors determined in these competition experiments was
expressed in terms of pKi (-log Ki).
Conclusions
Starting from the previously described lead
MEN11558, we have designed and realized a new series
of potent and selective NK-2 antagonists. Results pre-
sented in this work show that the identification of a
versatile position in the cyclic moiety of the reference
compound (C-12) and the juxtaposition there of a
functionalizable exocyclic amine in that precise position
led to a wide range of compounds with high affinity. A
careful examination of structure-activity relationships
and an optimization process were conducted through the
investigation on many types of substitution on the
primary amine of the newly identified lead molecule 7a.
This iterative process finally gave rise to two interesting
candidates, 54 and 57, which showed the ability to act
as potential drugs for the therapy of respiratory dis-
eases, such as asthma, with excellent performances
after intratracheal administration.
Organ Bath Experiments. The experiments were per-
formed on the following isolated smooth muscle preparations:
rabbit pulmonary artery circular muscle strips deprived of the
endothelium (RPA), guinea pig ileum longitudinal muscle
myenteric plexus strips (GPI), and strips of detrusor muscle
of rat urinary bladder (RUB). All of the experiments were
performed in oxygenated (96% O2 and 4% CO2) Krebs-
Henseleit solution having the following composition: NaCl, 119
mM; NaHCO3, 25 mM; KH2PO4, 1.2 mM; MgSO4, 1.5 mM;
CaCl2, 2.5 mM; KCl, 4.7 mM; and glucose 11 mM.
RPA, GPI, and RUB preparations were set up according to
the methods previously described:16 briefly, RPA strips were
connected to isometric force transducers (load 10 mN), whereas
GPI (load 3 mN) and RUB (load 0.5 g) strips were prepared
for isotonic recording of mechanical activity. Activity of test
compounds at tachykinin NK-1 receptors was evaluated in the
GPI (in the presence of atropine and chlorpheniramine 1 µM,
indomethacin 3 µM) by using substance P methyl ester
(SPOMe) as tachykinin NK-1 receptor-selective agonist. Activ-
ity of test compounds at tachykinin NK-2 receptors was
assessed against neurokinin A (in the RPA) or [â-Ala8]NKA-
(4-10) (in the RUB) as the agonists. The activity of test
compounds at tachykinin NK-3 receptors was evaluated in the
GPI (in the absence of premedication) by using senktide as
the tachykinin NK-3 selective agonist. Cumulative concentra-
tion-response curves to the agonists were obtained in all
preparations, each concentration being added when the effect
of the preceding one had reached a steady state. The antago-
nist affinity of all test compounds (incubation period of 15 min)
was expressed as pKB (negative logarithm of KB, the antagonist
dissociation constant), which was estimated as the mean of
the individual values obtained with the equation
Experimental Section
General. All reagents and solvents were used without
further purification or drying. Amino acids and coupling
reagents were purchased from Bachem AG (Bubendorf, Swit-
zerland) and Novabiochem (La¨ufelingen, Switzerland). All
other reagents were purchased from Sigma-Aldrich (Milan,
Italy) unless otherwise specified. Commercial grade anhydrous
solvents were purchased from J. T. Baker (Deventer, Holland).
All reactions were performed under an atmosphere of nitrogen,
unless otherwise specified. 1H NMR spectra were acquired on
a Varian 200 MHz or a Bruker 500 MHz instrument and
recorded in parts per million (ppm) δ values, relative to CHCl3
(δ 7.27) or DMSO (δ 2.50) as the internal standards. The data
were transferred to an Apple Macintosh computer and pro-
cessed using the program SwaN-MR.13 Mass spectra were
obtained with a Finnigan LCQ ion trap mass spectrometer,
operated in positive-ion electrospray ionization. The samples
(about 10 mg/mL) were dissolved in acetonitrile/ammonium
acetate (10 mM) 1:1 v/v and introduced by direct infusion at 5
mL/min through the built-in syringe pump. The samples were
analyzed by full-scan MS and product ion MS/MS of the
protonated quasi-molecular ions, at 30% relative collision
energy, using helium as the collision gas. Exact mass mea-
surements were performed using a microTOF orthogonal
acceleration time-of-flight mass spectrometer (Bruker Dalton-
ics, Billerica, MA), using positive-ion electrospray ionization.
The samples, dissolved in 50:50:0.1 water/acetonitrile/formic
acid, were directly infused into the instrument at 4 µL/min
via a syringe pump. Mass calibration was performed internally
using Agilent APCI tuning solution. HPLC were recorded on
a HP-1100 instrument using the following methods. Method
A: Symmetry column RP-8 (3.6 × 150 mm) H2O + 0.1% TFA/
MeCN + 0.1% TFA 80/20 f 20/80 in 20′, flow rate 1 mL/min,
λ ) 215, 254 nm. Method B: Luna column RP-8(2) (4.6 × 250
mm) H2O + 1% TFA/MeCN + 0.1% TFA 80/20 f 20/80 in 20′
+ 6′ isocratic, flow rate 1 mL/min, λ ) 215, 254 nm. Method
C: Zorbax column SB-18, 3.5 mm, 100 Å (4.6 × 50 mm), H2O
+ 0.1% TFA/MeCN + 0.1% TFA, 95/5 f 5/95 in 6.5′ + 1′
isocratic, flow rate 3 mL/min, λ ) 220, 270 nm.
pKB ) log[dose ratio -1] - log[antagonist concentration]
In Vivo Experiments. The activity of test compounds, after
intratracheal administration, was checked on bronchoconstric-
tion produced by intravenous (iv) administration of the selec-
tive tachykinin NK-2 receptor agonist, [â-Ala8]NKA(4-10), in
anesthetized guinea pigs as previously described.17
Male Dunkin Hartley guinea pigs (Charles River, Italy),
weighing 350-400 g, were anesthetized with urethane (1.5
g/kg sc), and the body temperature was kept constant at 36
°C. The left jugular vein was cannulated for drug administra-
tion, and the animals were mechanically ventilated at a rate
of 60 strokes/min. The basal insufflation pressure and bron-
choconstriction were measured by connecting a pressure
transducer to a sidearm of tracheal cannula and recorded by
a MacLab (ADInstruments, Australia) data acquisition system.
A polyethylene catheter (PE10, Clay Adams) was stably
inserted into the tracheal cannula and its tip positioned closely
to the bronchi bifurcation to allow intratracheal administration
of compounds.
After two reproducible control responses to the agonist (1
nmol/kg iv) the antagonist was administered intratracheally
5 and 30 min before the challenges with the agonist that was
repeated every 30 min up to 4 h from antagonist administra-
tion.

Antagonists for the Human Tachykinin NK-2 Receptor
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6943
The bronchoconstriction was calculated as amplitude (mmHg)
of the response over the basal value of insufflation pressure.
The effect of the antagonist at various times after treatment
was expressed as % inhibition of the control response. The ED50
were calculated (Prism 3 software, Graphpad, San Diego, CA)
considering the maximum value of inhibition reached at each
dose during the experimental time.
Synthetic Method for the Obtainment of Compounds
7a,b, 8a,b, and 14a,b. The synthesis of (2-amino-1-(R)-benzyl-
ethyl)-carbamic acid tert-butyl ester 1 was conducted as
previously described,7 and that of [2-amino-1-(R)-(3,4-dichloro-
benzyl)-ethyl]-carbamic acid tert-butyl ester 9 was conducted
in a similar way with the simple substitution of the catalytic
hydrogenation step with a reduction with LiAlH4 according
to a known procedure.4
(2-(R)-Amino-3-phenyl-propyl)-carbamic Acid Benzyl
Ester Hydrochloride (2). Benzyl chloroformate (0.7 mL, 3.7
mmol) was dropped slowly into a solution of 1 (0.925 g, 3.7
mmol) and triethylamine (1.25 mL, 8.8 mmol) in anhydrous
THF (30 mL) and the mixture stirred at 0 °C for 1 h. The
resulting suspension was filtered and the residue washed with
THF. The collected filtrates were evaporated to dryness, then
treated with ethyl acetate, and extracted with NaHCO3 and
brine. The organic phase was evaporated to a solid, which was
treated with diethyl ether. The suspended solid was filtrated
and dried under vacuum to give the diprotected intermediate
(1.09 g, 77% yield). TLC (ethyl acetate/petroleum ether 40-
60° 1/3): Rf ) 0.40. Mp: 147-148 °C. MS (m/z): 385 (MH+),
402 (MNH4+), 346, 329, 285, 177. 1H NMR (200 MHz, CDCl3):
δ 7.4-7.1 (m, 10H), 5.3-5.1 (m, 1H), 5.11 (s, 2H), 4.9-4.6 (m,
1H), 4.05-3.75 (m, 1H), 3.43-3.05 (m, 2H), 2.95-2.62 (m, 2H),
1.37 (s, 9H).
Three grams (7.8 mmol) of that solid were subsequently
suspended in methanol (55 mL) and treated with a 4 M
solution of HCl in dioxane (22.5 mL, 90 mmol). After 1 h the
solvent was evaporated under reduced pressure and the solid
residue treated with diethyl ether in an ice bath for 10 min
and then filtered off by suction to give 2.49 g (7.8 mmol, 99%
yield) of 2 as a white powder (overall yield from 1 76%). HPLC
(A): 6.76′, 98% purity. 1H NMR (500 MHz, DMSO-d6): δ 7.99
(br s, 3H), 7.62-7.42 (br m, 1H), 7.41-7.14 (m, 10H), 5.02 (s,
2H), 3.46-3.33 (m, 1H), 3.27-3.08 (m, 2H), 3.05-2.72 (m, 2H).
(2-(R)-{2-(S)-[2-(S)-Amino-3-(1H-indol-3-yl)-propionyl-
amino]-3-phenyl-propionylamino}-3-phenyl-propyl)-car-
bamic Acid Benzyl Ester Hydrochloride (4). A solution
of Boc-L-tryptophan (19.5 g, 64 mmol), HOBt (27 g, 199 mmol),
and EDC‚HCl (15 g, 78.2 mmol) in 300 mL of DMF was
prepared and stirred for 15 min. After that time a suspension
of 3 (30 g, 64 mmol) and triethylamine (10 mL, 72 mmol) in
150 mL of DMF was added and the resulting mixture stirred
for 2 h at room temperature. The solvent was then evaporated
under reduced pressure and the crude residue treated with
NaHSO4 (5% aqueous solution, 500 mL) and filtered. This
procedure was repeated four times, and the resulting solid was
further washed with water (3 × 500 mL). After stripping with
toluene and with absolute ethanol the solid was dried under
vacuum for 4 h. The obtained Boc-protected derivative (42 g,
58.5 mmol, 91% yield) was used without further purification
for the next deprotecting step. HPLC: (B) 22.53′, (A) 19.23′.
MS (m/z) (ES+): 718.0 (MH+). 1H NMR (200 MHz, DMSO-
d6): δ 10.77 (s, 1H), 7.99 (d, J ) 7.77 Hz, 1H), 7.18 (d, J )
7.77 Hz, 1H), 7.65-6.69 (m, 22H), 5.01 (br s, 2H), 4.60-4.32
(m, 1H), 4.24-3.78 (m, 2H), 3.00-2.35 (m, 8H), 1.25 (s, 9H).
A suspension of that solid (58.5 mmol) in methanol (700 mL)
was cooled to 5 °C and HCl 7 N in dioxane (55 mL, 385 mmol)
and subsequently 100 mL (400 mmol) of HCl 4 N in dioxane
were dropped slowly, the internal temperature being kept
under 10 °C during the addition. The reaction mixture was
then warmed to room temperature, stirred for 5.5 h, and then
evaporated down under reduced pressure. The resulting solid
was treated with tert-butyl methyl ether (200 mL), filtered by
suction, and dried under vacuum to give 4 (35.8 g, 55 mmol,
92% yield). Overall yield from 3: 84%. HPLC: (B) 15.7′, >99%
purity. MS (m/z) (ES+): 618.2 (MH+). 1H NMR (200 MHz,
DMSO-d6): δ 11.02 (m, 1H), 8.88 (d, J ) 8.04 Hz, 1H), 8.24
(d, J ) 8.5 Hz, 1H), 8.04 (br s, 3H), 7.73 (d, J ) 7.3 Hz, 1H),
7.50-6.89 (m, 20H), 5.03 (s, 2H), 4.58-4.39 (m, 1H), 4.15-
3.87 (m, 2H), 3.35-2.55 (m, 8H).
General Procedure for the Coupling of 4 with Mono-
protected Aspartic Acid. N-[1-(S)-[1-(S)-(2-Amino-1-(R)-
benzyl-ethylcarbamoyl)-2-phenyl-ethylcarbamoyl]-2-
(1H-indol-3-yl)-ethyl]-2-(R)-tert-butoxycarbonylamino-
succinamic Acid (5a). Boc-(D)-Asp-OBzl (18 g, 55.6 mmol)
was dissolved in 1 L of DMF with magnetic stirring. HOBt
(27 g, 200 mmol) and subsequently 15 g (78.2 mmol) of EDC‚
HCl were added to that solution. After 15 min a suspension
of 4 (35.8 g, 54.8 mmol) and triethylamine (12.7 mL, 91.2
mmol) in DMF (150 mL) was added dropwise. After the
addition of 15 mL (108 mmol) of triethylamine, the reaction
mixture was left at room temperature for 4 h, and then the
solvent was evaporated under reduced pressure. The residue
was treated with portions of aqueous KHSO4 (5%, 2 × 500 mL),
water (2 × 500 mL), NaHCO3 (5%, 2 × 500 mL), and water (2
× 500 mL). The resulting solid was washed with hot ethanol
(96%) until the alcoholic solution was approximately colorless,
filtered, and dried under high vacuum for 4 h obtaining 35 g
(38 mmol, 69% yield) of the benzyl ester as a solid, directly
used for the next synthetic step. HPLC: (B) 23.80′; (A) 19.94′,
>99% purity. MS (m/z) (ES+): 923.0 (MH+). 1H NMR (200
MHz, DMSO-d6): δ 10.77 (br s, 1H), 8.12 (d, J ) 7.6 Hz, 1H),
7.94 (d, J ) 8.1 Hz, 1H), 7.78 (d, J ) 8.4 Hz, 1H), 7.80 (d, J )
7.6 Hz, 1H), 7.40-6.80 (m, 26H), 5.05-4.94 (m, 4H), 4.57-
4.21 (m, 3H), 4.12-3.86 (m, 1H), 3.18-2.3 (m, 10H), 1.34 (s,
9H).
The product (17.5 g, 18.9 mmol) was subsequently treated
with hydrogen in the presence of Pd/C (2 g, 10% Pd) in DMF
(550 mL) and water (50 mL) during 6 h. The solution was
filtered and evaporated under reduced pressure. The obtained
solid was washed repeatedly with tert-butyl methyl ether and
dried under vacuum to give 5a as a whitish solid (12.3 g, 93%
yield). HPLC: (B) 14.2′, >98% purity. MS (m/z) (ES+): 699.3
(MH+). 1H NMR (200 MHz, DMSO-d6): δ 10.79 (d, J ) 1.9
Hz, 1H), 8.75 (d, J ) 9.2 Hz, 1H), 8.53 (d, J ) 6.0 Hz, 1H),
8.61-7.91 (br s, 3H), 7.55-6.82 (m, 16H), 6.24 (d, J ) 6.0 Hz,
1H), 4.50-3.99 (m, 5H), 3.40-2.60 (m, 8H), 2.03 (dd, J ) 12.4,
11.6 Hz, 1H), 1.40 (s, 9H).
[2-(R)-(2-(S)-Amino-3-phenyl-propionylamino)-3-phen-
yl-propyl]-carbamic Acid Benzyl Ester Hydrochloride
(3). Boc-L-phenylalanine (20.6 g, 77.6 mmol) was combined
with HOBt (15.8 g, 117 mmol) in acetonitrile (280 mL) with
mechanical stirring. The solution was cooled to 0 °C, and EDC‚
HCl (17.9 g, 93.3 mmol) was added. After 30 min a solution of
2 (25 g, 77.9 mmol) and triethylamine (11 mL, 79 mmol) in
acetonitrile (70 mL) was added dropwise, and the mixture was
warmed to room temperature and stirred for a further 3 h.
The formed precipitate was filtered, washed with acetonitrile
(2 × 75 mL) and tert-butyl methyl ether (3 × 100 mL), and
dried to give the crude diprotected dipeptide (35.3 g, 66.8
mmol, 85% yield). MS (m/z): 532 (MH+), 476, 432. HPLC: (A)
1
18.06′, >95% purity; (B) 21.80′, >95% purity. H NMR (200
MHz, DMSO-d6): δ 7.94 (d, J ) 8.6 Hz, 1H), 7.54-6.96 (m,
16H), 6.72 (d, J ) 8.21 Hz, 1H), 5.05 (s, 2H), 4.18-3.85 (m,
2H), 3.28-2.93 (m, 2H), 2.90-2.40 (m, 4H), 1.30 (s, 9H).
The diprotected derivative (35.3 g, 66.4 mmol) was sus-
pended in methanol (350 mL) and cooled to 5 °C with an ice
bath. HCl 7 M in dioxane (150 mL, 1.05 mol) was added
dropwise through a dropping funnel. The reaction mixture was
left to warm to room temperature. After 8 h a mixture of
methanol and dioxane (7:3 v/v, 50 mL) was added to make the
solution completely clear, and after 1 h of stirring the solvent
was evaporated under reduced pressure and the resulting
viscous oil was crystallized from 100 mL of tert-butyl methyl
ether. The obtained solid 3 was filtered by suction and dried
under high vacuum (30 g, 64 mmol, 96% yield). Overall yield
from 2: 82%. HPLC: (B) 13.49′. MS (m/z) (ES+): 432.2 (MH+).
1H NMR (200 MHz, DMSO-d6): δ 8.57 (d, J ) 7.9 Hz, 1H), 8.1
(br s, 3H), 7.48-6.86 (m, 16H), 5.00 (br s, 2H), 4.12-3.78 (m,
2H), 3.24-2.4 (m, 6H).

6944 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Fedi et al.
With the same methodology and with comparable yields, the
following products were obtained: 5b (using Boc-(L)-Asp-OBzl),
6a (using Boc-(D)-Asp(OH)-OBzl), and 6b (using Boc-(D)-Asp-
(OH)-OBzl).
and treated with CF3COOH (13 mL), then warmed to room
temperature, and stirred for further 30 min. After the evapo-
ration of the solvent the crude product was treated three times
with diethyl ether, to give after settling 1.33 g (2.4 mmol, 80%
yield) of the deprotected derivative, which was immediately
used for the coupling reaction. HPLC: (A) 13.1′, 95% purity.
MS (m/z) (ES+): 441 (MH+, isotopic pattern for dichloro
compound). Boc-L-phenylalanine (0.49 g, 1.85 mmol) was
combined with HOBt (0.66 g, 4.9 mmol) and EDC‚HCl (0.46
g, 2.4 mmol) in DMF (9 mL). The solution was cooled to 0 °C.
After 30 min a solution of the aforementioned intermediate
(1.03 g, 1.85 mmol) in DMF (8 mL) and subsequently triethyl-
amine (0.6 mL, 4.8 mmol) was added dropwise, and the
mixture was warmed to room temperature and stirred for a
further 4 h. The solvent was removed under reduced pressure,
and the residue was dissolved in ethyl acetate (400 mL) and
washed with brine, 5% aqueous NaHCO3, and brine. The
organic phase gave, after drying and evaporation, a solid
compound (1.13 g, 1.64 mmol, 82% yield), which was directly
subjected to the deprotection conditions. HPLC: (A) 23.34′,
>99% purity. MS (m/z) (ES+): 687 (MH+, isotopic pattern for
General Procedure for the Obtainment of the Cy-
clized Pseudopeptides Bearing the Free Amino Group
(7ab, 8ab). 12-(R)-Amino-5-(S),8-(R)-dibenzyl-2-(S)-(1H-
indol-3-ylmethyl)-1,4,7,10-tetraaza-cyclotetradecane-
3,6,11,14-tetraone (7a). Product 5a (12.2 g, 17.45 mmol) was
dissolved in DMF (1000 mL) and maintained at room temper-
ature with stirring. HOBt (9.3 g, 68.8 mmol) and, after 10 min,
EDC‚HCl (5.6 g, 29.2 mmol) were added in portions. After 2.5
h the mixture was evaporated down under reduced pressure
and the remaining residue was dissolved in ethyl acetate (1250
mL) and extracted with KHSO4 (5%, 2 × 200 mL), NaHCO3
(5%, 2 × 200 mL), and finally water (2 × 250 mL). The organic
phase, dried over anhydrous sodium sulfate (40 g) with
magnetic stirring, was then filtered and evaporated to dryness.
The resulting solid was washed twice with tert-butyl methyl
ether (60 mL). After recrystallization from ethyl acetate (155
mL) the obtained solid (7.3 g, 62% yield) was directly depro-
tected.
1
dichloro compound). H NMR (200 MHz, DMSO-d6): δ 8.05-
7.82 (m, 3H), 7.78-7.59 (m, 2H), 7.55-7.00 (m, 13H), 6.66 (d,
J ) 8.1 Hz, 1H), 4.42-4.17 (m, 3H), 4.15-3.87 (m, 2H), 3.27-
2.95 (m, 2H), 2.90-2.37 (m, 4H), 1.28 (s, 9H).
Seven grams of the solid (10.28 mmol) were suspended in
200 mL of methanol, and the temperature of the solution was
maintained around 5°C during the addition of 25 mL of HCl
4 M in dioxane. After that operation the mixture was left to
reach room temperature, and after 20 h the solvent was
removed under reduced pressure and the residue, after treat-
ments with ethyl acetate (50 mL) and tert-butyl methyl ether
(50 mL), was filtered and maintained under vacuum for 2 h,
giving the hydrochloride of 7a as a white solid (5.8 g, 9.4 mmol,
92% yield). HPLC: (B) 12.5′, 97.7% purity. MS (ES+): 581
(MH+).
The corresponding free amine was extracted with ethyl
acetate from a saturated solution of NaHCO3, washed with
brine, and purified by flash column chromatography using
solutions of methanol in ethyl acetate with increasing polarity.
HPLC: (C) 3.42′, 99% purity. MS (m/z) (ES+): 581 (MH+),
603 (MNa+), 619 (MK+), 564, 378, 281, 231. 1H NMR (500 MHz,
DMSO-d6): δ 10.80 (d, J ) 1.35 Hz, 1H), 8.63 (d, J ) 5.2 Hz,
1H), 7.82 (bs, 1H), 7.68 (bs, 1H), 7.43 (d, J ) 7.8 Hz, 1H), 7.33
(d, J ) 8.1 Hz, 1H), 7.29-7.22 (m, 6H), 7.21-7.16 (m, 4H),
7.07 (t, J ) 7.03 Hz, 2H), 6.98 (t, J ) 7.6 Hz, 1H), 6.73 (d, J
) 9.1 Hz, 1H), 4.24 (m, 1H), 4.14 (m, 1H), 4.04 (m, 1H), 3.53
(bs, 1H), 3.51 (t, J ) 5.2 Hz, 1H), 3.38 (dd, J ) 14.3, 3.8 Hz,
1H) 3.09 (d, J ) 12.0 Hz, 1H), 2.95 (dd, J ) 14.4, 4.5 Hz, 1H),
2.81-2.69 (m, 5H), 2.20 (dd, J ) 14.3, 6.1 Hz, 1H).
[1-(S)-{1-(S)-[2-Amino-1-(R)-(3,4-dichloro-benzyl)-eth-
ylcarbamoyl]-2-phenyl-ethylcarbamoyl}-2-(1H-indol-3-
yl)-ethyl]-carbamic Acid tert-Butyl Ester (12). 11 (1.13 g,
1.64 mmol) was suspended in dichloromethane (32 mL), cooled
to 0 °C, treated with trifluoroacetic acid (7.5 mL), then warmed
to room temperature, and stirred for further 30 min. After the
evaporation of the solvent the crude residue was treated three
times with diethyl ether, to give, after filtration, the depro-
tected intermediate (1.5 mmol, 91% yield), readily used for the
next coupling step.
A solution of the aforemetioned intermediate (1.5 mmol) in
10 mL of DMF was added at room temperature to a solution
of Boc-L-tryptophan (0.457 g, 1.5 mmol), HOBt (0.625 g, 4.6
mmol), and EDC‚HCl (0.385 g, 2.0 mmol) in DMF (10 mL).
Then DIPEA (0.33 mL, 1.84 mmol) was dropped slowly into
the resulting mixture. After 2 h the reaction was complete and
the solvent was evaporated under reduced pressure. The
residue, dissolved in ethyl acetate, was treated with citric acid
(10%), NaHCO3 solution (5%), and brine and then was dried
over anhydrous sodium sulfate and evaporated. The resulting
solid was subsequently washed, with stirring, with ethyl
acetate (20 mL), collected by filtration, and dried to obtain the
diprotected derivative (1.14 g, 1.3 mmol, 87% yield). This solid
was dissolved in DMF (30 mL), treated with Et2NH (3 mL),
and stirred for 2 h. After evaporation of the solvent under high
vacuum the residue was pulverized in diethyl ether and
filtered to give 12 as a crystalline white solid (0.666 g, 1.02
mmol, 68% yield). HPLC: (A) 13.75′, 96.4% purity. MS (m/z)
(ES+): 652 (MH+, isotopic pattern for dichloro compound). 1H
NMR (500 MHz, DMSO-d6): δ 10.80 (s, 1H), 7.91 (d, J ) 8.6
Hz, 1H), 7.80 (d, J ) 8.1 Hz, 1H), 7.55-7.46 (m, 3H), 7.31 (d,
J ) 8.1 Hz, 1H), 7.22-7.15 (m, 4H), 7.11-6.92 (m, 5H), 6.75
(d, J ) 8.2 Hz, 1H), 4.56-4.41 (m, 1H), 4.17-4.04 (m, 1H),
3.90-3.80 (m, 1H), 2.97-2.51 (m, 8H), 1.28 (s, 9H).
General Procedure for the Coupling of 12 with the
Appropriate Diprotected Aspartic Acid for the Obtain-
ment of 13a,b and the Obtainment of the Cyclized
Pseudopeptides (14a,b) Bearing the Free Amino Group.
12-(R)-Amino-5-(S)-benzyl-8-(R)-(3,4-dichloro-benzyl)-2-
(S)-(1H-indol-3-ylmethyl)-1,4,7,10-tetraaza-cyclotetrade-
cane-3,6,11,14-tetraone (14a). Fmoc-(D)-Asp-(OtBu)-OH (0.13
g, 0.316 mmol) was combined with HOBt (0.128 g, 0.95 mmol)
and EDC‚HCl (0.073 g, 0.38 mmol) in DMF (3 mL). After 30
min was slowly added, with stirring, a solution of 12 (0.841 g,
1.29 mmol) in DMF (6 mL). The reaction mixture was stirred
at room temperature for 2 h, then the solution was concen-
trated to dryness, and the residue was treated with citric acid
(5%), then filtered, and washed with water. The solid was
suspended in NaHCO3 (5%), then newly filtered, and washed
In a similar way the following products were obtained
starting from the appropriate intermediates: 7b (from 5b),
8a (from 6a), 8b (from 6b). Purifications in these cases were
conducted through preparative HPLC.
[1-(R)-(3,4-Dichloro-benzyl)-2-(9H-fluoren-9-ylmeth-
oxycarbonylamino)-ethyl]-carbamic Acid tert-Butyl Es-
ter (10). The starting amine 9 (2.79 g, 8.75 mmol) was
dissolved in anhydrous THF (75 mL). Addition of triethylamine
(1.25 mL, 8.8 mmol) and of N-(9-fluorenylmethoxycarbonylox-
y)succinimide (2.95 g, 8.75 mmol) were conducted slowly at
room temperature, and the resulting mixture was stirred for
1 h. The solvent was evaporated under reduced pressure, and
the residue, solubilized in ethyl acetate (1 L), was treated with
10% citric acid, brine, 5% aqueous NaHCO3, and brine. The
organic layer was evaporated to dryness and the solid treated
with diethyl ether, obtaining 3.29 g (6.55 mmol, 70% yield) of
the diprotected intermediate. HPLC: (A) 23.31′, 91% purity.
MS (m/z) (TS+): 541 (MH+, isotopic pattern for dichloro
compound). 1H NMR (200 MHz, DMSO-d6): δ 7.88 (d, J ) 7.0
Hz, 2H), 7.69 (d, J ) 7.1 Hz, 2H), 7.56-7.23 (m, 7H), 7.16 (dd,
J ) 1.7, 8.2 Hz, 1H), 6.7 (d, J ) 8.6 Hz, 1H), 4.42-4.11 (m,
3H), 3.78-3.52 (m, 1H), 3.14-2.90 (m, 2H), 2.81-2.36 (m, 2H),
1.27 (s, 9H).
[2-(R)-(2-(S)-tert-Butoxycarbonylamino-3-phenyl-pro-
pionylamino)-3-(3,4-dichlorophenyl)-propyl]-carbamic
Acid 9H-Fluoren-9-ylmethyl Ester (11). 10 (1.62 g, 3 mmol)
was suspended in dichloromethane (50 mL), cooled to 0 °C,

Antagonists for the Human Tachykinin NK-2 Receptor
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6945
with water. The dried solid (0.278 g, 0.3 mmol, quantitative
yield) was used without further purification for the double
deprotecting step. This intermediate (0.3 mmol) was suspended
in dichloromethane (4 mL), cooled to 0 °C, and treated with
ethanedithiol (0.025 mL, 0.3 mmol) and trifluoroacetic acid (2.5
mL). The reaction mixture was then warmed to room temper-
ature and maintained under magnetic stirring for 1 h. The
residue obtained after evaporation of the solvent was washed
repeatedly with diethyl ether to give 13a as a solid (0.233 g).
HPLC: (A) 15.07′, 78.9% purity. MS (m/z) (ES+): 889 (MH+,
(d, J ) 8.5 Hz, 1H), 4.33 (m, 1H), 4.25 (m, 1H), 4.06 (m, 1H),
4.00 (m, 1H), 3.90 (m, 2H), 3.73 (m, 1H), 3.29-3.13 (m, 1H),
2.95-2.72 (m, 5H), 2.42 (t, J ) 11.6 Hz, 1H), 1.97 (m, 2H),
1.57 (m, 2H).
In a similar way, products 15b-18a were obtained using
the appropriate carbonyl compound and the desired starting
cyclic amine. Target compounds 19-33 were obtained starting
from the same amine 7a.
N-(3-(R)-Acetylamino-4-(R),5-(R)-dihydroxy-6-(R)-hy-
droxymethyl-tetrahydro-pyran-2-(R)-yl)-N′-[5-(S),8-(R)-
dibenzyl-2-(S)-(1H-indol-3-ylmethyl)-3,6,11,14-tetraoxo-
1,4,7,10-tetraaza-cyclotetradec-12-(R)-yl]-succinamide (34).
To a solution of 7a (0.2 g, 0.34 mmol) as free base in CH3CN
(3 mL) was added succinic anhydride (0.06 g, 0.6 mmol). DMF
was subsequently added till the solution was completely clear
(0.5 mL), and the reaction mixture was stirred for 2 h at room
temperature. The solvent was evaporated under reduced
pressure, and the residue was treated with diethyl ether to
obtain, after filtration, a white solid (200 mg), which was used
directly for further transformations. The obtained solid (0.2
g, 0.29 mmol calcd) was dissolved in DMF (7 mL), and HOBt
(0.13 g, 0.99 mmol), EDC‚HCl (0.067 g, 0.35 mmol), and, after
10 min, 2-acetamido-2-deoxy-â-D-glucopyranosyl amine (0.067
g, 0.29 mmol) were added at room temperature. After 2 h of
stirring, the solution was evaporated to dryness and the
residue was treated with 5% KHSO4, filtrated, and finally
washed with water, 5% NaHCO3, and water. The solid was
extracted with ethyl acetate, and the organic phase was
evaporated and purified by preparative HPLC (Vydac column
(19 × 250 mm), flow rate 20 mL/min, H2O + 0.1% TFA/CH3-
CN + 0.1% TFA, gradient 77/23 f 47/53 in 60′) giving 34 (72
1
isotopic pattern for dichloro compound). H NMR (200 MHz,
DMSO-d6): δ 10.97 (s, 1H), 8.80 (d, J ) 7.8 Hz, 1H), 8.35-
6.85 (m, 24H), 4.60-3.85 (m, 7H), 3.28-2.35 (m, 10H).
In the same way 13b was obtained with comparable yield.
Both compounds were used for the following synthesis of 14a
and 14b without further purification.
Compound 13a (0.23 g, 0.2 mmol) was solubilized in DMF
(20 mL). To this solution HOBt (99.5 mg, 1 mmol) and EDC.
HCl (89 mg, 0.46 mmol) were added and the mixture was
stirred for 2 h. After that time the reaction was complete and
the solvent was evaporated. The residue was treated with citric
acid (5%) then filtered and washed with water, NaHCO3 (5%)
and water. The obtained solid (0.210 g, 0.188 mmol, calcd. yield
93%) showed an HPLC purity of about 78% and was used for
the deprotecting step without purification.
The cyclic compound (0.188 mol) was solubilized in DMF
(10 mL), treated with Et2NH (1 mL), and maintained under
stirring for 2 h. After evaporation of the solvent under high
vacuum, the residue was triturated in diethyl ether and
filtered to give a white solid, which was further purified via a
preparative HPLC (Deltapak RP18 column (300 × 19 mm),
flow rate 15 mL/min, H2O + 0.1% TFA/CH3CN + 0.1% TFA,
gradient 75/25 > 15/85 in 60′) obtaining 47 mg (0.06 mmol,
35% yield) of lyophilized 14a (total yield 32%). HPLC: (A)
11.67′, >99% purity. MS (m/z) (ES+): 649 (MH+, isotopic
mg, 0.072 mmol, 24% yield) as a white lyophile. HPLC: (B)
1
11.3′, >99% purity. MS (m/z) (ES+): 883.2 (MH+
). H NMR
(500 MHz, DMSO-d6): δ 10.81 (d, J ) 1.7 Hz, 1H), 8.78 (d, J
) 4.8 Hz, 1H), 8.11 (m, 2H), 8.01 (bs, 1H), 7.79 (d, J ) 8.7 Hz,
1H), 7.48 (bs, 1H), 7.43 (d, J ) 7.9 Hz, 1H), 7.33 (d, J ) 8.1
Hz, 1H), 7.28-7.14 (m, 8H), 7.09-7.04 (m, 2H), 6.97 (t, J )
7.6 Hz, 1H), 6.81 (d, J ) 9.0 Hz, 1H), 4.95 (bs, 2H), 4.80 (t,
1H, J ) 9.3 Hz, 1H), 4.55 (m, 1H), 4.48 (bs, 1H), 4.19-4.14
(m, 2H), 4.00 (bs, 1H), 3.59-3.49 (m, 3H), 3.42-3.23 (m, 5H),
3.07 (m, 2H), 2.92 (dd, J ) 13.8, 4.4 Hz, 1H), 2.85-2.71 (m,
5H), 2.55 (t, J ) 5.3 Hz, 1H), 2.38-2.23 (m, 5H), 1.81 (s, 3H).
1
pattern for dichloro compound). H NMR (500 MHz, DMSO-
d6): δ 10.88 (m, J ) 1.8 Hz, 1H), 8.86 (d, J ) 5.1 Hz, 1H), 8.59
(d, J ) 5.9 Hz, 1H), 8.17 (s, 3H), 7.54 (d, J ) 1.9 Hz, 1H), 7.50
(d, J ) 8.2 Hz, 1H), 7.46 (d, J ) 7.9 Hz, 1H), 7.39 (m, 1H),
7.35 (d, J ) 8.1 Hz, 1H), 7.26 (t, J ) 7.2 Hz, 1H), 7.22 (dd, J
) 8.3, 1.9 Hz, 1H), 7.18 (tt, J ) 7.4, 1.33 Hz, 1H), 7.13 (d, J )
7.1 Hz, 1H), 7.08 (ddd, J ) 8.1, 7.0, 1.2 Hz, 1H), 7.06 (d, J )
2.4 Hz, 1H), 6.99 (ddd, J ) 8.0, 7.0, 1.0 Hz, 1H), 6.78 (d, J )
9.0 Hz, 1H), 4.28 (m, 1H), 4.16-4.11 (m, 2H), 4.05 (m, 1H),
3.70 (dd, J ) 12.6, 8.9 Hz, 1H), 3.11 (dd, J ) 15.0, 3.8 Hz,
1H), 3.06 (dt, J ) 13.6, 4.0 Hz, 1H), 2.89-2.72 (m, 5H).
2-(S)-Acetylamino-N4-(3-(R)-acetylamino-4-(R),5-(R)-di-
hydroxy-6-(R)-hydroxymethyl-tetrahydro-pyran-2-(R)-
yl)-N1-[5-(S),8-(R)-dibenzyl-2-(S)-(1H-indol-3-ylmethyl)-
3,6,11,14-tetraoxo-1,4,7,10-tetraaza-cyclotetradec-12-(R)-
yl]-succinamide (35). To a solution of Ac-Asp(OtBu)-OH (0.04
g, 0.17 mmol) in DMF (5 mL) were added, at room tempera-
ture, HOBt (0.07 g, 0.52 mmol), EDC‚HCl (0.033 g, 0.17 mmol),
and, after 10 min, 7a (0.1 g, 0.17 mmol). Stirring of the solution
was maintained overnight, then the solvent was evaporated
under reduced pressure, and the residue, dissolved in CH2-
Cl2, was washed subsequently with 5% KHSO4, 5% NaHCO3,
and water. The organic layer was anhydrified over MgSO4, the
solvent was completely removed under reduced pressure, and
the crude ester (100 mg) was used without any purification
for the deprotecting step. It was directly dissolved in 20 mL
of CH2Cl2 and treated with TFA (10 mL) for 90 min. The
solvent was then removed and the residue treated three times
with ethyl acetate and finally with diethyl ether to obtain a
white solid (70 mg, approximately 0.09 mmol), which showed
a mass spectrum consistent with the deprotected carboxylic
acid. The crude product was dissolved in DMF (5 mL), and
HOBt (0.039 g, 0.29 mmol), EDC‚HCl (0.018 g, 0.09 mmol),
and, after 10 min, 2-acetamido-2-deoxy-â-D-glucopyranosyl
amine (0.021 g, 0.09 mmol) were added at room temperature.
After 12 h at room temperature the mixture was directly
purified by preparative HPLC (Symmetry RP-18 column (19
× 300 mm), flow rate 15 mL/min, H2O + 0.1% TFA/CH3CN +
0.1% TFA, gradient 70/30 f 30/70 in 60′) to obtain 35 as a
lyophile (0.05 g, 0.05 mmol, 30% yield). HPLC: (B) 11.10′,
>99% purity. MS (m/z) (ES+): 962.4 (MH+). 1H NMR (500
MHz, DMSO-d6): δ 10.79 (d, J ) 1.9 Hz, 1H), 8.86 (d, J ) 5.1
Hz, 1H), 8.22 (d, J ) 9.0 Hz, 1H), 8.17 (d, J ) 7.0 Hz, 1H),
With the same method the product 14b was obtained from
13b.
General Procedure for the Obtainment of Products
15-33: Reductive Amination on the Primary Amino
Group of Compounds 7a,b, 8a,b, 14a. 5-(S),8-(R)-Diben-
zyl-2-(S)-(1H-indol-3-ylmethyl)-12-(R)-(tetrahydro-pyran-
4-ylamino)-1,4,7,10-tetraaza-cyclotetradecane-3,6,11,14-
tetraone Trifluoroacetate (15a). To a solution of 7a (0.044
g, 0.076 mmol) in methanol (5 mL), with magnetic stirring,
were added at room temperature acetic acid (0.1 mL, 1.6
mmol), a solution of tetrahydro-4H-pyran-4-one (0.018 g, 0.18
mmol) in methanol (1 mL), and finally sodium cyanoborohy-
dride (0.012 g, 0.2 mmol). After a further 30 min of stirring
and 8 h at rest the solution was acidified to pH 1-2 with HCl
(1 N in water). After dilution with water (20 mL), methanol
was evaporated under reduced pressure and the resulting
solution treated with NaHCO3 to alkaline pH and then
extracted with ethyl acetate. The organic phase was washed
with brine, anhydrified with Na2SO4, then filtered, and
evaporated to dryness. Purification by preparative HPLC
(Deltapak RP18 column (300 × 19 mm), flow rate 15 mL/min,
H2O + 0.1% TFA/CH3CN + 0.1% TFA, gradient 75/25 f 15/
85 in 120′) gave 15a as trifluoroacetate (0.025 g, 0.032 mmol,
37% yield). HPLC: (B) 10.40′, >99% purity. MS (m/z) (ES+):
1
665.4 (MH+). H NMR (500 MHz, DMSO-d6): δ 10.89 (d, J )
2.3 Hz, 1H), 8.99-8.92 (m, 4H), 7.48 (d, J ) 7.9 Hz, 1H), 7.45
(bs, 1H), 7.35 (d, J ) 8.1 Hz, 1H), 7.27-7.23 (m, 6H), 7.20-
7.15 (m, 2H), 7.12-7.07 (m, 4H), 7.00 (t, J ) 8 Hz, 1H), 6.86

6946 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Fedi et al.
7.99 (d, J ) 8.7 Hz, 1H), 7.78 (d, J ) 8.8 Hz, 1H), 7.63 (bs,
1H), 7.57 (bs, 1H), 7.42 (d, J ) 8.1 Hz, 1H), 7.33 (d, J ) 8.1
Hz, 1H), 7.29-7.15 (m, 10H), 7.06 (t, J ) 7.5 Hz, 1H), 7.01-
6.96 (m, 2H), 6.69 (d, J ) 9.1 Hz, 1H), 4.80 (t, J ) 9.4 Hz,
1H), 4.54 (m, 1H), 4.41 (q, J ) 6.5 Hz, 1H), 4.23 (m, 1H), 4.15
(m, 1H), 4.02 (m, 1H), 3.64 (d, J ) 11.3 Hz, 1H), 3.55 (m, 1H),
3.35 (m, 1H), 3.12-2.99 (m, 4H), 2.79-2.71 (m, 5H), 2.56 (dd,
J ) 15.7, 5.7 Hz, 1H), 2.43 (dd, J ) 15.7, 6.9 Hz, 1H), 2.29
(dd, J ) 14.9, 6.1 Hz, 1H), 1.83 (s, 3H).
a gentle reflux of the solvent. At the end of the addition of the
amine, the reaction mixture was refluxed for 2 h. After this
time the mixture was cooled to room temperature and then to
0 °C. The suspension was then filtered off by suction and the
filtrate evaporated down to dryness. The semisolid residue was
then taken up in ethyl acetate (100 mL) and the extract
washed with water (100 mL) and then dried over sodium
sulfate. Removal of the solvent under reduced pressure gave
43a (12.11 g, 57 mmol, >98% yield) as a clear yellow brown
liquid. This material was used for the next synthetic step
without further purification. TLC (ethyl acetate/MeOH/Et3N
94/5/1): Rf ) 0.71. MS (m/z) (ES+): 202.1 (MH+). 1H NMR
(200 MHz, CDCl3): δ 3.66-3.42 (m, 4H), 2.96 (2, 2H), 2.50-
2.25 (m, 4H), 1.32 (s, 9H).
Morpholin-4-yl-acetic Acid (44a). The morpholine ester
43a (2.5 g, 12 mmol) was dissolved in dichloromethane (35
mL), the solution treated with TFA (15 mL), and the mixture
stirred for 5 h at room temperature. After this time the
solution was evaporated down to dryness and the product
crystallized out by treatment of the residue with diisopropyl
ether. The off-white crystalline solid 44a (2.47 g, 12 mmol, 99%
yield), filtered off by suction and dried over CaCl2 overnight,
was obtained as a nonstoichiometric complex with trifluoro-
acetic acid and used for the next synthetic step. 1H NMR (200
MHz, DMSO-d6): δ 12.65-11.66 (br s, 1H), 4.06 (s, 2H), 3.84-
3.73 (m, 4H), 3.30-3.27 (m, 4H).
N-[5-(S),8-(R)-Dibenzyl-2-(S)-(1H-indol-3-ylmethyl)-3,6,-
11,14-tetraoxo-1,4,7,10-tetraaza-cyclotetradec-12-(R)-yl]-
2-morpholin-4-yl-acetamide Trifluoroacetate (48). The
amino acid 44a (0.021 g, 0.1 mmol) was dissolved in DMF (5
mL), and the solution was treated with HOBt (0.06 g, 0.45
mmol) and EDC‚HCl (0.02 g, 0.1 mmol). After 10 min of
stirring the solution was completely clear, 7a (0.06 g, 0.1 mmol)
was added, and the resulting mixture was stirred for 4 h. After
that time the solvent was removed under reduced pressure
and the residue was purified by preparative HPLC (Symmetry
RP-18 column (19 × 300 mm), flow rate: 15 mL/min, H2O +
0.1% TFA/CH3CN + 0.1% TFA, gradient 70/30 f 30/70 in 60′)
to give 48 (0.015 g, 0.02 mmol, 20%) as lyophile. HPLC: (B)
12.2′, 97.7 purity. MS (m/z) (ES+): 708.5 (MH+). 1H NMR (500
MHz, DMSO-d6): δ 10.71 (bs, 1H), 8.62 (bs, 1H), 8.60 (d, J )
5.2 Hz, 1H), 8.04 (bs, 1H), 7.46 (d, J ) 7.8 Hz, 1H), 7.34 (d, J
) 8.1 Hz, 1H), 7.28-7.24 (m, 7H), 7.19-7.16 (m, 4H), 7.08 (t,
J ) 7.2 Hz, 1H), 7.07 (m, 1H), 6.99 (t, J ) 7.1 Hz, 1H), 6.79
(d, J ) 8.8 Hz, 1H), 4.64 (m, 1H), 4.23 (m, 1H), 4.18 (m, 1H),
4.05 (m, 1H), 3.79 (bs, 4H), 3.36 (dd, J ) 14.2, 4.1 Hz, 1H),
3.12 (bs, 3H), 2.97 (dd, J ) 14.2, 4.1 Hz, 1H), 2.87-2.74 (m,
5H), 2.34 (dd, J ) 14.1, 8.3 Hz, 1H).
General Procedure To Obtain Products 36-40: Re-
ductive Amination on the Carbonylic Function of the
Starting Sugar Moiety. N-(1-(S)-{[5-(S),8-(R)-Dibenzyl-2-
(R)-(1H-indol-3-ylmethyl)-3,6,11,14-tetraoxo-1,4,7,10-tet-
raaza-cyclotetradec-12-(R)-ylamino]-methyl}-2-(R),3-(S),4-
(R),5-tetrahydroxy-pentyl)-acetamide (36). To a solution
of 7a (0.041 g, 0.07 mmol) in anhydrous methanol (5 mL) were
added acetic acid (0.08 mL, 1.3 mmol), 2-acetamido-2-deoxy-
â-D-glucopyranosyl amine (0.018 g, 0.08 mmol), and Na(CN)-
BH3 (0.02 g, 0.3 mmol). After 48 h, the conversion being not
more than 25%, further 2-acetamido-2-deoxy-â-D-glucopyra-
nosyl amine (0.018 g, 0.08 mmol) and Na(CN)BH3 (0.02 g, 0.3
mmol) were added and the reaction mixture was maintained
under magnetic stirring at room temperature totally for 7 days.
The solution was then acidified to pH 1-2 with HCl 1 N,
diluted with water, and concentrated to eliminate methanol,
then NaHCO3 was added, and extractions with ethyl acetate
were repeated three times. The organic phase, after treatments
with brine and MgSO4, was concentrated to dryness, and the
residue was purified by preparative HPLC (Symmetry RP-18
column (19 × 300 mm), flow rate 15 mL/min, H2O + 0.1% TFA/
CH3CN + 0.1% TFA, gradient 90/10 f 60/30 in 60′) to give 36
(0.018, 0.018 mmol, 27% yield) as trifluoroacetate. HPLC: (B)
11.70′, 98% purity. MS (m/z) (ES+): 786.3 (MH+). 1H NMR
(500 MHz, DMSO-d6): δ 10.87 (bs, 1H), 8.91 (bs, 2H), 8.78 (bs,
1H), 7.76 (bs, 1H), 7.47 (d, J ) 7.6 Hz, 1H), 7.44 (bs, 1H), 7.35
(d, J ) 8.1 Hz, 1H), 7.26-6.97 (m, 17H), 6.85 (bs, 1H), 5.02
(bs, 1H), 4.56 (bs, 1H), 4.45 (bs, 1H), 4.30 (bs, 1H), 4.20 (bs,
1H), 4.11 (bs, 1H), 4.04 (bs, 2H), 3.80 (bs, 1H), 3.72 (bs, 1H),
3.57 (d, J ) 8.3 Hz, 1H), 3.41 (m, 3H), 3.20-3.11 (m, 3H),
2.94-2.72 (m, 6H), 1.86 (s, 3H).
In a similar way starting from 7a and the correct starting
sugar (Table 3) products 37-40 were obtained (25-40%
yields).
2-(S),3-(R),5-(R),6-Tetrahydroxy-4-(R)-(O-r-D-glucopy-
ranosyl)-hexanoic Acid [5-(S),8-(R)-Dibenzyl-2-(S)-(1-H-
indol-3-ylmethyl)-3,6,11,14-tetraoxo-1,4,7,10-tetraaza-cy-
clotetradec-12-(R)-yl]-amide (41). 7a (0.049 g, 0.084 mmol),
PyBOP (0.133 g, 0.25 mmol), 4-O-â-D-galactopyranosyl-D-
gluconic acid (0.09 g, 0.25 mmol), and diisopropyl ethyl amine
(0.13 mL, 0.07 mmol) were dissolved in DMF (1 mL) and
stirred for 4 days at room temperature. The crude mixture was
then directly passed through preparative HPLC (Vydac C-18
column (19 × 250 mm), flow rate 24 mL/min, H2O + 0.1% TFA/
CH3CN + 0.1% TFA, gradient 90/10 f 60/40 in 120′) for
purification of 41, which was obtained as a lyophile (0.026 g,
0.032 mmol, 40% yield). HPLC: (C) 3.06′, >99% purity. MS
In a similar way products 49-57 were obtained.
Procedure for the Obtainment of the Amine Portion
47 (Precursor of 54). 4-Benzyl-piperazine-1-sulfonic Acid
Amide (46). To a solution of benzyl piperazine (14.1 g, 80
mmol) in THF (100 mL) cooled at 0 °C was added dropwise a
solution of sulfamoyl chloride (9.25 g, 80 mmol) in THF (250
mL) during a period of 1 h. The reaction mixture was then
stirred at room temperature for 4 h and left without stirring
overnight. The insoluble solid was then filtered by suction,
washed with THF, and treated with a saturated solution of
NaHCO3. Extracts with ethyl acetate were collected, washed
with brine, and dried over sodium sulfate. The organic phase
was then evaporated to dryness under reduced pressure and
the resulting solid treated with diethyl ether, filtered, washed,
and dried to give 5.24 g (20.8 mmol, 26% yield) of 46, which
was directly used for the deprotecting treatment. TLC (ethyl
acetate): Rf ) 0.54. HPLC: (A) 3.2′. MS (m/z) (ES+): 256.1
1
(m/z) (ES+): 921.4 (MH+). H NMR (500 MHz, DMSO-d6): δ
10.79 (m, J ) 2.1 Hz, 1H), 8.81 (d, J ) 4.8 Hz, 1H), 7.96 (d, J
) 8.9 Hz, 1H), 7.68 (d, J ) 6.5 Hz, 1H), 7.55 (t, J ) 5.6 Hz,
1H), 7.42 (d, J ) 7.9 Hz, 1H), 7.32 (d, J ) 8.1 Hz, 1H), 7.29-
7.15 (m, 10H), 7.06 (t, J ) 7.9 Hz, 1H), 6.99-6.96 (m, 2H),
6.75 (d, J ) 9.1 Hz, 1H), 5.32 (bs, 1H), 5.09 (bs, 1H), 4.78 (bs,
1H), 4.67 (m, 1H), 4.55 (bs, 1H), 4.29-4.22 (m, 3H), 4.14 (m,
1H), 4.05-4.00 (m, 2H), 3.71 (m, 2H), 3.60-3.58 (m, 2H), 3.53-
3.50 (m, 5H), 3.15 (dd, J ) 12.3, 6.3 Hz, 1H), 3.04 (dd, J )
14.5, 4.7 Hz, 1H), 2.80-2.70 (m, 5H), 2.29 (dd, J ) 14.5, 7.0
Hz).
1
(MH+). H NMR (200 MHz, DMSO-d6): δ 7.40-7.17 (m, 5H),
6.74 (br s, 2H), 3.48 (s, 2H), 3.04-2.87 (m, 4H), 2.48-2.37 (m,
4H).
General Procedure for the Obtainment of Products
49-57 via Acylation of 7a with Carboxylic Acids 44a-j.
Morpholin-4-yl-acetic Acid tert-Butyl Ester (43a). tert-
Butyl bromoacetate (11.19 g, 57 mmol) was dissolved in THF
(70 mL) and the solution treated by dropwise addition with
an equimolar mixture of morpholine (5 g, 57 mmol) and
triethylamine (8 mL, 57 mmol) at such a rate as to maintain
Piperazine-1-sulfonic Acid Amide (47). 4-Benzyl-pip-
erazine-1-sulfonic acid amide 46 (1.35 g, 5.3 mmol) was
dissolved in methanol (25 mL), and the solution was treated
with a suspension of Pd/C 10% (0.15 g) in methanol/H2O 1/1
v/v (5 mL). The resulting suspension was shaken under H2
atmosphere at normal pressure for 3 h. During that time 120

Antagonists for the Human Tachykinin NK-2 Receptor
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6947
Directed Mutagenesis Studies. J. Med. Chem. 2002, 45, 3418-
3429.
mL of H2 was absorbed. The solution was then filtered off by
suction and evaporated to dryness, obtaining a white solid,
which was treated with chloroform (20 mL). The unsoluble
solid was filtered off, washed again with chloroform (10 mL),
and dried to give 47 (0.67 g, 4.05 mmol, 76% yield). TLC
(chloroform/MeOH 1:1): Rf ) 0.18. MS (m/z) (ES+): 166.0
(MH+). 1H NMR (200 MHz, DMSO-d6): δ 6.72 (br s, 2H), 2.20
(br s, 2H), 2.86-2.6 (m, 8H).
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Acknowledgment. The authors thank Dr. Andrea
Raffaelli for performing exact mass measurements.
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Supporting Information Available: Table with HPLC
purity of all products according to two different methods
(retention time, purity). 1H NMR of the remaining final
products. LRMS of all final products (MH+ and significative
fragments). HRMS for key compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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JM040832Y