Monocyclic human tachykinin NK-2 receptor antagonists as evolution of a potent bicyclic antagonist: QSAR and site-directed mutagenesis studies

Article abstract of DOI:10.1021/jm011127h

A new series of monocyclic pseudopeptidic tachykinin NK-2 receptor antagonists has been derived from nepadutant with the help of site-directed mutagenesis studies and QSAR models. MEN11558 is the lead compound which is evaluated on a series of 13 new huma

Full text of DOI:10.1021/jm011127h

3418
J . Med. Chem. 2002, 45, 3418-3429
Mon ocyclic Hu m a n Ta ch yk in in NK-2 Recep tor An ta gon ists a s Evolu tion of a
P oten t Bicyclic An ta gon ist: QSAR a n d Site-Dir ected Mu ta gen esis Stu d ies
Alessandro Giolitti,*,† Maria Altamura,‡ Francesca Bellucci,§ Danilo Giannotti,‡ Stefania Meini,§
Riccardo Patacchini,§ Luigi Rotondaro,| Sabrina Zappitelli,| and Carlo A. Maggi§
Departments of Drug Design, Chemistry, Pharmacology, and Biotechnology, Menarini Ricerche S.p.A.,
Via Sette Santi 3, 50131 Firenze, Italy
Received December 18, 2001
A new series of monocyclic pseudopeptidic tachykinin NK-2 receptor antagonists has been
derived from nepadutant with the help of site-directed mutagenesis studies and QSAR models.
MEN11558 is the lead compound which is evaluated on a series of 13 new human tachykinin
NK-2 receptor mutants (Tyr107Ala, Gln109Ala, Asn110Ala, Phe112Ala, Ser164Phe, Cys167Gly,
Phe168Ala, Tyr169Ala, Ile202Phe, Trp263Ala, Tyr269Phe, Tyr269Ala, and Phe293Ala) and 8
mutants on which data from nepadutant were already available (Gln166Ala, Ser170Ala,
Thr171Ala, His198Ala, Tyr206Phe, Tyr266Phe, Tyr289Phe, and Tyr289Thr). The results show
that the two compounds share most of their binding sites, in agreement with their hypothesized
binding modes. This allows us to transfer the structural knowledge we already had for
nepadutant to the new series of compounds. At the same time, a sound QSAR model is developed
to assist the prioritization of new chemical syntheses. The result is the discovery of receptor
antagonists with a higher affinity than nepadutant for the hNK-2 receptor.
In tr od u ction
Neurokinin A, a member of the tachykinin peptide
family, is widely distributed in the mammalian periph-
eral nervous system and exerts its biological effects
mainly by activating the tachykinin NK-2 receptor.1
Human NK-2 receptor antagonists are considered po-
tential candidates for the treatment of asthma, bron-
chial hyperreactivity, irritable bowel syndrome, and
cystitis.2 Our studies identified MEN10627 as one of the
most potent antagonists for the tachykinin NK-2 recep-
tor, but its very low water solubility did not allow
clinical development. The glycosylated derivative ne-
padutant3 (cyclo{[Asn(â -D-GlcNAc)-Asp-Trp-Phe-Dap-
Leu]cyclo(2â-5â)}) (Figure 1) represents a great im-
provement in bioavailability, besides being metabolically
very stable, and is presently undergoing phase II clinical
trials. We have recently shown4 how a first step in
simplifying the structure of nepadutant was undertaken
with the discovery of a new series of monocyclic
pseudopeptide derivatives. This represented part of a
two-branched program: extensive site-directed NK-2
receptor mutagenesis and rationally driven chemical
syntheses. The first branch had the aim of identifying
and describing the binding site of our reference antago-
nist (nepadutant) and its chemical evolution derivatives
in addition to comparing this binding site with those of
other known antagonists; part of this work has already
been published.5 The second branch of the project had
the objective to reduce the structural complexity of
nepadutant and improve its receptor affinity through
the rational design of novel compounds.
F igu r e 1. Structure of nepadutant.
This paper reports the complete mutagenesis study,
the evolution of a new antagonist series, and the
development of a QSAR model with excellent predictive
ability.
Site-Dir ected Mu ta gen esis
Our previous study5 considered the residues Gln166,
Ser170, Thr171, His198, Tyr206, Tyr266, His267,
Phe270, and Tyr289 which are considered to be in the
seven transmembrane helices (TM1-7) of the human
tachykinin NK-2 receptor. We were able to describe part
of the binding site of the peptide NK-2 antagonist
nepadutant. The results indicated that Thr171, Tyr206,
Tyr266, and Phe270 were involved in the binding of
nepadutant, whose binding site was only partially
shared with the nonpeptide antagonist SR48968.6 The
two antagonists show significantly different binding
modes. The residues to be mutated were selected on the
basis of a three-dimensional receptor model which was
started from the available knowledge about the rel-
evance of some corresponding residues in the NK-1
receptor. Because of the high level of sequence homology
* To whom correspondence should be addressed. Tel: +39 055
5680240. Fax: +39 055 5680241. E-mail: agiolitti@menarini-ricerche.it.
† Department of Drug Design.
‡ Department of Chemistry.
§ Department of Pharmacology.
| Department of Biotechnology.
10.1021/jm011127h CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/03/2002

Human Tachykinin NK-2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3419
F igu r e 2. Snake plot of the human NK-2 receptor, updated according to ref 7. Residues indicated with white letters on dark
gray circles are those mutated in this work.
and evidence for cross talking between these receptors,
some of the conserved residues represented a reasonable
starting point. In respect to the previously built model,
a new one has been obtained based on the now available
crystal structure of bovine rhodopsin.7 The relative
position of the residues in which we were interested was
not changed significantly, and we did not have to modify
our previous results. The new arrangement is shown
in Figure 2. However, a new series of point mutations
was necessary to gain a better understanding of the SAR
in our peptidic NK-2 antagonists and collect some hints
about structural modifications aimed at simplifying
nepadutant.
describe the development of a predictive QSAR model.
The complete synthetic work will be presented else-
where.
Str u ctu r e-Activity Rela tion sh ip s Mod el
Three-dimensional QSAR methods, such as CoMFA9
and related techniques, have been proven to be success-
ful tools for correlating structural features with a given
biological activity and for predicting the activity of
untested compounds.10 A limitation of these methods
is represented by the need to select a conformer (or more
than one) for each compound from a conformational
analysis which is assigned the label of “bioactive con-
formation”. It is then necessary to define an alignment
rule to properly superimpose the compounds to each
other and, finally, perform the SAR analysis to build
the model.
The new point mutations concern Tyr107, Gln109,
Asn110, and Phe112 on TM3; Ser164, Cys167, Phe168,
and Tyr169 on TM4; Ile202 on TM5; Trp263 and Tyr269
on TM6; and Phe293 on TM7. Data are reported in
Table 1.
In the past few years, a QSAR method has been
described, termed HQSAR (Hologram QSAR),11 which
is based on fragment fingerprints (a list of all the
fragments of a specific length that can be found in a
given structure) and their frequency (molecular holo-
grams). Methods such as HQSAR can also produce very
good models, as we show here, with a much faster
procedure than, for instance, CoMFA, and they are
suitable even for the “virtual screening” of large data-
bases. The trade-off is that at the end no real hints are
given by the model itself about the best structural
direction the synthetic work should take, because no
field contour plot is produced like in CoMFA, and the
statistical variables in the method have no immediate
physical meaning. Fingerprints represent the variables
for predicting the biological activity of the corresponding
compounds, without the need of selecting a suitable
conformer, and properly align each structure. This
Mon ocyclic An ta gon ists
In the preliminary work,4 we took a step back from
the bicyclic structure of MEN10627,8 the precursor of
nepadutant, to identify the smallest structural subset
retaining micromolar affinity for the human NK-2
receptor. This is represented in Figure 3.
The conformational equivalence of the common subset
was confirmed.4 An additional benzyl group was then
introduced in different positions of 5a . The result was
MEN11558 (compound 14c in ref 4) with a pKi value of
8.7.
MEN11558 (Figure 4) was chosen as the lead com-
pound of this new series of promising NK-2 receptor
antagonists. Here, only a small subset of this class of
antagonists will be presented, as it is instrumental to

3420 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Giolitti et al.

Human Tachykinin NK-2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3421
NK-1 receptor.12 This appears to support the hypothesis
that NKA binds differently to the NK-2 and NK-1
receptors. In agreement with the lack of binding by
[3H]nepadutant radioligand, competition binding ex-
periments, with respect to the nonpeptide antagonist
[3H]SR48968, confirmed the drastic loss of affinity by
both nepadutant and MEN11558.
Tyr 169Ala . While abolishing the binding of NKA,
this mutation does not impair the binding of nepadutant
and MEN11558. Therefore, it appears to be a residue
relevant to the binding of the natural agonist.
Th r 171Ala . No determination of a KD value was
F igu r e 3. Smallest structural subset in MEN10627 retaining
micromolar affinity (from ref 4).
possible by direct binding of [3H]nepadutant. Against
[
125I]NKA, both nepadutant and MEN11558 maintained
their affinity.
His198Ala . The mutation impairs the binding of
MEN11558 18-fold, even more than that of nepadutant,
in addition to drastically preventing the binding of the
natural agonist.
Ile202P h e. This residue was mutated to Phe as it is
in mouse, rat, and hamster NK-2 receptors. The muta-
tion increases the binding of nepadutant by about 1
order of magnitude but not that of NKA. This result is
in agreement with the functional data we previously
obtained from the human and mouse NK-2 receptors;
the apparent affinity of nepadutant, determined as pKB,
in blocking the contractile effect produced by the agonist
NKA was significantly higher at the mouse (pKB 9.83)
NK-2 receptors than at the human (pKB 8.513) NK-2
receptors, as expressed in urinary bladder preparations.
Tr p 263Ala . No binding could be detected for [3H]ne-
padutant and [125I]NKA; therefore, [3H]SR48968 was
evaluated. This nonpeptide ligand maintained an af-
finity value comparable to that observed with the wild
type, while both nepadutant and MEN11558 had their
binding strongly impaired (55- and 75-fold, respectively).
Tyr 266P h e. As in the case of Thr171Ala, [125I]NKA
had to be used as a radioligand, but with this mutant,
both nepadutant and MEN11558 had their affinity
decreased (6- and 13-fold, respectively).
Tyr 269Ala a n d -P h e. We have shown5 that Tyr266,
which is located about one helical turn above Tyr269,
is part of the binding site of nepadutant. Tyr269,
especially with its phenyl ring, affects the binding of
nepadutant and MEN11558 (5- and 2-fold, respectively).
As suggested previously,4 nepadutant and MEN11558
share most of their binding sites; here, we show different
binding behavior at Tyr107, Phe112, and Gln166 which
are located on TM3 and TM4. This is in agreement with
our proposed model4,5 and the structural differences
between the two compounds.
F igu r e 4. Structure of MEN11558.
allows a rapid obtainment of good QSAR models, even
from large sets of molecules; such models can be used
by the bench chemist to check proposed modifications
and to put them in rank order in terms of synthetic
priority.
Table 2 reports the compounds used in the training
set and their pKi values as produced by the HQSAR
models and experimental methods.
Resu lts
Site-Dir ected Mu ta gen esis. A total of 21 mutant
human NK-2 receptors were studied to define the
binding site of the reference compound MEN11558,
especially in respect to the binding site of nepadutant.
These results and those concerning the behavior of
MEN11558 on mutants previously tested against ne-
padutant5 are reported in Table 1. When the mutations
did not allow an evaluation of the binding of [3H]ne-
padutant, the affinities of nepadutant and MEN11558
were tested against [125I]NKA. Here, a comment is
outlined for each significant mutation.
Tyr 107Ala . It negatively affects the binding of
MEN11558 by 3-fold reduction but not that of nepadu-
tant.
Asn 110Ala . None of the radioligands we tested
exhibited binding affinity for this mutant; this suggests
a significant modification in either overall receptor
conformation (tertiary structure) or receptor expression.
P h e112Ala . The binding of nepadutant and NKA is
decreased by approximately 5- and 4-fold, respectively.
Gln 166Ala . This mutation was previously reported
not to affect the binding of nepadutant;4 on the other
hand, the affinity of MEN11558 is decreased 5-fold.
Cys167Gly. A relevant effect is produced by this
substitution which completely abolishes the binding of
nepadutant. Gly is the residue in the hNK-1 receptor
which corresponds to Cys167 in the hNK-2 receptor.
This mutation also abolishes the binding of NKA which,
on the other hand, binds, albeit with lower affinity, the
Mod elin g a n d QSAR. The first series of 25 com-
pounds, reported in rows 1-25 in Table 2, was examined
using the HQSAR module in Sybyl (Tripos Inc., St.
Louis, MO). The corresponding results are reported in
Table 2 as MOD1. The standard error in prediction (SE
of Q2) is quite high; with the evaluation of the absolute
differences between the experimental pKi and cross-
validated (predicted) values (CV), one of the compounds,
MEN11690, showed a gap of 2 log units (exp, 10.0; CV
predicted, 7.99). Compound competition curves of
[
125I]NKA displayed a shallow slope (significantly less
than unity) fitting a two-site model with the two Ki
values calculated as 3 and 1.2 nM, respectively. The

3422 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Giolitti et al.
Ta ble 2. Structures of the 29 Compounds Used To Develop the Initial HQSAR Models MODna

Human Tachykinin NK-2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3423
Ta ble 2 (Continued)

3424 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Ta ble 2 (Continued)
Giolitti et al.

Human Tachykinin NK-2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3425
Ta ble 2 (Continued)
a
Numbers in each MODn column are predicted pKi values in cross-validated mode, except for the “MOD4 pred” column. Numbers in
the MOD5 column refer to the values predicted (cross-validated mode) by the final model for these compounds. Values in brackets in
column “MOD2” for the last four compounds are those predicted by model 2 before the compounds were included in model 3.
resulting mean pKi value is higher than that corre-
sponding to the common binding site only. Therefore,
this compound was excluded from the training set, as
its value could not properly be explained in a simple
way by the model.
MEN11987 ) 8.4 (exp, 8.4), MEN11990 ) 8.7 (exp, 9.5),
and MEN11995 ) 9.2 (exp, 9.8).
A new model was calculated including these 28
compounds, with the results reported as MOD3 in Table
2. This model was also tested for chance correlation
using a randomization routine which confirmed the
quality of the result. A parameter was then modified to
take chirality into account. With the option “chirality
ON” in HQSAR, the model resulted in MOD4 in Table
2. Since then, the HQSAR model has progressed to the
present MOD5 (Tables 2 and 3) which includes 313
The new model is MOD2 with 24 rows in Table 3.
While Q2 is slightly worse, R2 and the standard error
improved, even if the corresponding standard error is
lower. This model was used to predict the binding values
of four new compounds (rows 26-29 in Table 2) with
the following results: MEN11986 ) 8.3 (exp, 8.4),

3426 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Giolitti et al.
Ta ble 3
interactions discriminating between the two. However,
since the binding site appears to be proximal to the
extracellular site of the receptor, we can suppose that
the glycosidic moiety of nepadutant could interact with
more polar loop residues. This is shown in Figures 3
and 4. Figure 5 shows a proposed binding mode for
MEN11558 with the relevant receptor residues evident.
It should be mentioned again that the models are still
speculative, because there is no crystal structure avail-
able for these complexes. However, we could maintain
the starting hypotheses about the main structural
features of the monocyclic compounds, as derived from
our knowledge of the nepadutant series. Different
binding behavior is found, as expected, for mutations
in the residues belonging to TM3 and TM4 which, in
our model, correspond to the nonconserved part of the
ligand structure. Since the mutant receptors are stably
transfected, they are usefully employed to check struc-
tural modifications in the monocyclic antagonist series.
no.
model compd
R2 (SE)
Q2 (SE)
compd
3
optionsa
A,B,C: ON
H,Ch,DA: OFF
Atm in Fr.: 4-7
A,B,C: ON
H,Ch,DA: OFF
Atm in Fr.: 4-7
A,B,C: ON
H,Ch,DA: OFF
Atm in Fr.: 4-7
A,B,C,Ch: ON
H,DA: OFF
Atm in Fr.: 4-7
A,B,C,Ch: ON
H,DA: OFF
1
2
3
4
5
25
24
0.95 (0.29) 0.77 (0.64)
0.98 (0.19) 0.75 (0.62)
0.98 (0.19) 0.90 (0.42)
0.99 (0.12) 0.91 (0.40)
0.88 (0.44) 0.86 (0.49)
4
5
6
6
28
28
313
Atm in Fr.: 4-7
a
Options in HQSAR model building: A, atoms; B, bonds; C,
connections; H, hydrogens; Ch, chirality; DA, donor & acceptor.
compounds. While statistics for this model are clearly
weaker than those for the previous MOD4, MOD5 is
able to accommodate a greater variability in functional
groups and shows excellent stability by being able to
include and predict different classes of compounds (data
not shown).
As we said before, we cannot deduce the nature of the
molecular interactions, that is, which part of the ligand
interacts with which part of the receptor, from HQSAR
models directly. However, the binding modes suggested
even by the manual docking of the ligands in receptor
models14 on the basis of mutagenesis data can be a very
useful guide for selecting new ligand moieties and
substituents. The HQSAR model, on the other hand, is
an efficient tool for helping assign priorities to the
proposed chemical syntheses, as it is very fast in
checking new structures, because it does not require the
assignment of a “bioactive conformation” or an align-
ment rule. It is not efficient, however, in proposing
structural modifications favorable for activity, which
rely on the knowledge and intuition of the medicinal
chemists. For instance, it is clear that a proper amino
group and a hydrogen bond acceptor close to it have a
favorable effect on the binding to the receptor (see, for
instance, the amino side chain in MEN11995). On the
Discu ssion
Results from site-directed mutagenesis studies al-
lowed us to hypothesize a binding site for the repre-
sentative of the new series of antagonists, namely,
MEN11558, which are largely superimposable to that
of nepadutant. The residues significantly affecting the
binding of nepadutant and MEN11558 are Cys167 in
TM4, His198 in TM5, and Trp263, Tyr266, and Tyr269
in TM6. Gln166, on the other hand, seems to interact
with MEN11558 but not with nepadutant. These resi-
dues are proposed to interact with the common struc-
tural features of the two antagonists, although there is
no clear evidence, among the residues we mutated, of
F igu r e 5. CR trace of the three-dimensional receptor model (stereoview, TM only), as viewed from the extracellular side, reporting
the hypothesis for nepadutant binding mode on the human NK-2 receptor as reported in ref 5.

Human Tachykinin NK-2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3427
ethylcarbamoyl]-2-(S)-(1H-indol-3-yl)-ethyl]-succinamic acid.
The compound was suspended in anhydrous ethanol (150 mL),
and 10% Pd/C (450 mg) was added; the mixture was main-
tained under a hydrogen flow for 4 h. After being filtered and
washed with ethanol (2 × 30 mL), the solution was evaporated
giving N-[1-[1-(2-amino-1-(S)-carbamoyl-ethylcarbamoyl)-2-(S)-
phenyl-ethylcarbamoyl]-2-(S)-(1H-indol-3-yl)-ethyl]-succin-
amic acid (499 mg, 0.93 mmol, 82% yield). The compound was
other hand, there are no good candidates among the
residues in the receptor model for such an interaction
once the ligand has been docked. We suspect, therefore,
that the basic moiety of the ligands interacts with the
residues belonging to the extracellular loops of the
receptor which lie on top of the transmembrane helices,
as suggested by the crystal structure of bovine rhodop-
sin.7 Since these are usually not conserved among
GPCRs and depend strongly on the specific receptor,
they are not properly modeled and, therefore, are
generally left out from the receptor model itself, as in
this case.
In conclusion, the aim of this program was two-fold:
(1) to describe in molecular terms the binding site of
our pseudopeptide hNK-2 receptor antagonists, cor-
relate ligand structural features to specific residues in
the receptor, and define how much of the available
knowledge gained from the bicyclic antagonists, such
as nepadutant, could be transferred to the new simpli-
fied series; and (2) to develop a QSAR working tool to
support the synthetic effort toward smaller and more
active antagonists.
dissolved in anhydrous DMF (75 mL) under
a nitrogen
atmosphere; PyBOP (600 mg, 1.11 mmol) and DIPEA (0.58
mL, 3.34 mmol) were added, and the solution was stirred for
2 h at room temperature. After evaporation in vacuo, the
residue was purified by reverse phase flash chromatography
(Matrex C18, 60 Å pore diameter, 50 mm particle size; mobile
phase: acetonitrile/water, 50:50 v/v) giving 154 mg (0.30 mmol,
32% yield) of 1: 1H-NMR (DMSO-d6, ∂) 2.15-2.35 (2H, m),
2.55-2.85 (8H, m), 3.60-3.72 (1H, m), 3.95-4.18 (1H, m),
4.25-4.42 (1H, m), 6.71 (1H, d, J ) 9 Hz), 6.90-7.40 (9H, m),
7.43 (1H, d, J ) 8 Hz), 8.09 (1H, m), 8.50 (1H, d, J ) 6 Hz),
10.82 (1H, br s); MS (ES+) m/z 519 (MH+).
5(S )-B e n z y l-8(R )-(3,4-d i c h lo r o -b e n z y l)-2(S )-(1H -
in dol-3-ylm eth yl)-3,6,9,14-tetr aoxo-1,4,7,10-tetr aaza-cyclo-
tetr a d eca n e-11-ca r boxylic Acid Allyl Ester (13). H-Trp-
Phe-OtBu (580 mg, 1.42 mmol) and Fmoc-Asp(OH)-OAll (570
mg, 1.42 mmol) were dissolved in DMF (30 mL). EDC (280
mg, 1.46 mmol) and HOBT (767 mg, 5.67 mmol) were added,
and the solution was stirred overnight at room temperature.
After evaporation of the solvent, water (150 mL) was added
to the residue, giving a white solid that was filtered and
dissolved in ethyl acetate (50 mL). The organic phase was
washed with 10% aqueous (aq) citric acid (2 × 30 mL); it was
then washed with 5% aq NaHCO3 (2 × 30 mL), and then with
water (30 mL). After the solution was dried over Na2SO4 and
evaporated under reduced pressure, the residue was purified
by flash chromatography (ethyl acetate/cyclohexane, 50:50 v/v),
giving 800 mg (73% yield) of 4-[1-(1-tert-butoxycarbonyl-2(S)-
phenyl-ethylcarbamoyl)-2(S)-(1H-indol-3-yl)-ethylcarbamoyl]-
2-(2-9H-fluoren-9-yl-oxycarbonylamino)-butyric acid allyl ester.
The residue was dissolved in anhydrous THF (25 mL), and
the Fmoc group was removed by stirring the solution for 90
min with excess diethylamine (2.5 mL, 24 mmol). After the
solvent was evaporated, the residue was purified by flash
chromatography (ethyl acetate/methanol, 95:5 v/v), giving 430
mg (82% yield) of 2-amino-4-[1-(1-tert-butoxycarbonyl-2(S)-
phenyl-ethylcarbamoyl)-2(S)-(1H-indol-3-yl)-ethylcarbamoyl]-
butyric acid allyl ester. The compound was dissolved in
anhydrous DMF (20 mL) together with 256 mg (0.77 mmol) of
N-Boc-3,4-dichlorophenylalanine. EDC (154 mg, 0.80 mmol)
and HOBt (311 mg, 2.3 mmol) were added in sequence, and
the mixture was stirred for 20 h at room temperature. After
the solvent was concentrated to a small volume, the residue
was diluted with water (30 mL), giving a white solid that was
dissolved in ethyl acetate (30 mL). The organic phase was
separated and washed with 10% aq citric acid (2 × 20 mL); it
was then washed with 5% aq NaHCO3 (2 × 20 mL), and then
with water (20 mL). After being dried over Na2SO4, the
separated organic phase was evaporated under reduced pres-
sure to obtain 2-[2-tert-butoxycarbonylamino-3(R)-(3,4-dichloro-
phenyl)-propionylamino]-4-[1-(1-tert-butoxycarbonyl-2(S)-phenyl-
et h ylca r ba m oyl)-2(S )-(1H -in dol-3-yl)-et h ylca r ba m oyl]-
butyric acid allyl ester as a yellowish solid (657 mg, 97% yield).
The N-Boc protecting group and the tert-butyl ester were
removed by treatment with TFA (10 mL, 0.13 mol) and methyl
sulfide (70 µL, 1.6 mmol) for 30 min at room temperature. After
the evaporation of TFA, the crude residue (white solid, 500
mg, 96% yield) was dissolved in anhydrous DMF. TEA (96 µL,
0.69 mmol) was added followed by EDC (135 mg, 0.7 mmol)
and HOBt (281 mg, 2.07 mmol). The solution was stirred
overnight at room temperature and evaporated to give a yellow
oil. The addition of water gave a yellowish solid that was
filtered out and dissolved in ethyl acetate (30 mL). The organic
phase was washed with 5% aq KHSO4 (20 mL); it was then
washed with 10% aq NaHCO3 (20 mL), and then with water
(2 × 20 mL). After the solution was dried over Na2SO4 and
Exp er im en ta l Section
(A) Com p ou n d Syn th eses. Compounds 2-7, 11, 12, 16,
and 24 in Table 2 were synthesized as in ref 4. The compounds
8-10, 17-22, and 29 were synthesized as in Patent WO
9834949, and compounds 26-28 were synthesized as in Patent
WO 0008046. The other compounds were obtained as follows.
1
Gen er a l Meth od s. H-NMR spectra were acquired at 500
MHz on a Bruker Advance spectrometer. Mass spectra were
acquired by positive-ion electrospray ionization (ES) on a
Finnigan LCQ ion trap mass spectrometer, introducing the
sample by infusion via a built-in syringe pump. Melting points
were determined using a Bu¨chi melting point apparatus and
were not corrected. Preparative HPLC was performed on a
Shimadzu LC-10 apparatus. The following abbreviations are
used for the reagents and solvents: All, allyl; Boc, tert-
butyloxycarbonyl; dba, dibenzylideneacetone; DIPEA, N,N-
diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO,
dimethyl sulfoxide; Dpr, (S)-2,3-diaminopropionic acid; EDC,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;
Fmoc, (9-fluorenyl-methoxy)carbonyl; HOBt, 1-hydroxybenzo-
triazole; Phe, (S)-phenylalanine; PyBOP, (benzotriazol-1-yloxy)-
tripyrrolidinophosphonium hexafluorophosphate; TEA, tri-
ethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
Trp, (S)-tryptophan; and Z, benzyloxycarbonyl.
6-(S)-Ben zyl-9-(S)-(1H-in dol-3-ylm eth yl)-5,8,11,14-tetr a-
oxo-1,4,7,10-tetr a a za -cyclotetr a d eca n e-3-(S)-ca r boxylic
Acid Am id e (1). To a solution of Boc-Trp-Phe-OH (1.20 g, 2.66
mmol) in dichloromethane (10 mL) were added H-Dpr(Z)-NH2‚
TFA (0.95 g, 2.7 mmol) and PyBOP (1.70 g, 3.27 mmol) in
sequence under a nitrogen atmosphere. After the mixture was
stirred for 10 min, TEA (1.48 mL, 10.6 mmol) was added, and
the resulting solution was stirred overnight. Evaporation of
the solvent gave a residue that was purified by flash chroma-
tography (silica gel, ethyl acetate) giving 0.95 g (1.42 mmol,
53% yield) of Boc-Trp-Phe-Dpr(Z)-NH2 as a white solid. The
compound was suspended in dichloromethane (45 mL), and
TFA (5 mL, 64.9 mol) was added; the solution was stirred for
90 min and evaporated. After the mixutre was dried in vacuo
for 24 h, 787 mg (1.38 mmol, 97% yield) of H-Trp-Phe-Dpr(Z)-
NH2 was obtained. The compound was dissolved in anhydrous
DMF (20 mL) under a nitrogen atmosphere. Succinic anhy-
dride (166 mg, 1.66 mmol) and DIPEA (0.29 mL, 1.66 mmol)
were added, and the solution was stirred for 2 h. After
evaporation under reduced pressure, the residue was treated
with water (8 mL), and the obtained white solid was filtered
off, washed on the filter with water (2 × 8 mL), and dried
giving 760 mg (1.13 mmol, 82% yield) of N-[1-[1-(2-benzyloxy-
carbonylamino-1-(S)-carbamoyl-ethylcarbamoyl)-2-(S)-phenyl-

3428 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Giolitti et al.
the solvent evaporated under reduced pressure, 13 was
solution was stirred overnight at room temperature, and then
evaporated; the residue was dissolved in ethyl acetate. The
organic phase was washed with 5% aq KHSO4, 5% aq NaHCO3,
and brine. After the mixture was dried over MgSO4, evapora-
tion of the solvent under reduced pressure gave a yellowish
solid. Purification by reverse phase flash chromatography
(Matrex C18, 60 Å pore diameter, 50 µm particle size; mobile
phase: acetonitrile/water, 50:50 v/v) gave 275 mg (0.486 mmol,
45% yield) of 15 as a white lyophilized solid: 1H-NMR (DMSO-
d6, ∂) 1.45-1.53 (1H, m), 1.87-1.97 (1H, m), 2.21-2.37 (2H,
m), 2.56-2.90 (5H, m), 3.22-3.26 (1H, m), 3.28-3.35 (m,
partially overlapped with the water signal), 3.58-3.65 (1H,
m), 4.07-4.12 and 4.15-4.18 (2H, 2 m), 4.46-4.52 (1H, m),
6.95-7.25 (13, m), 7.33 (1H, d, J ) 8 Hz), 7.45 (1H, d, J ) 8
Hz), 8.32 (1H, d, J ) 5 Hz), 8.51 (1H, d, J ) 8 Hz), 10.8 (1H,
d, J ) 3 Hz); MS (ES+) m/z 566 (MH+).
2-[1,4′]Bipiper idin yl-1′-yl-N-[(5S,8R)-diben zyl-2-(S)-(1H-
in d ol-3-ylm eth yl)-3,6,11,14-tetr a oxo-1,4,7,10-tetr a a za -cy-
clotetr a d ec-12-(R)-yl]-a ceta m id e (23). To a suspension of
500 mg (2.2 mmol) of [1,4′-bipiperidine]-1′-acetic acid15 were
added, in 30 mL of DMF, HOBt (770 mg, 5.7 mmol) and EDC
(360 mg, 1.88 mmol) in sequence. After the mixture was stirred
at room temperature for 10 min, 1.11 g (1.91 mmol) of (12R)-
amino-(5S,8R)-dibenzyl-2-(S)-(1H-indol-3-ylmethyl)-1,4,7,10-
tetraaza-cyclotetradecane-3,6,11,14-tetraone16 was added, and
the solution was stirred for 2 h at the same temperature.
Evaporation of the solvent gave a crude residue that was
treated with ethyl acetate. The solid precipitate was filtered
off and dried, giving a crude product. Purification by reverse
phase flash chromatography (Matrex C18, 60 Å pore diameter,
50 mm particle size; mobile phase: acetonitrile/water, 50:50
v/v) gave 23 as a white solid (655 mg, 0.83 mmol, 43% yield):
1H-NMR (DMSO-d6, ∂) 1.45-1.65 (2H, m), 1.65-1.85 (6H, m),
1.91-2.03 (2H, m), 2.33-2.50 (3H, m), 2.78-2.82 (5H, m),
2.85-3.70 (m, overlapped with the water signal), 4.04-4.09
(1H, m), 4.10-4.14 (1H, m), 4.23-4.35 (1H, m), 4.74-4.80 (1H,
m), 6.81-6.69 (1H, d, J ) 9 Hz), 6.95-7.40 (12H, m), 7.46 (1H,
d, J ) 8 Hz), 7.52-7.61 (1H, m), 8.30 (1H, d, J ) 9 Hz), 8.50
(1H, d, J ) 5 Hz), 10.62 (1H, br s); MS (ES+) m/z 789 (MH+).
obtained as a white solid (335 mg, 0.48 mmol, 34% overall
1
yield): H-NMR (DMSO-d6, ∂) 2.53 (2H, m), 2.63 (2H, ABq, J
) 5, 14 Hz), 4.30 (1H, m), 4.31 (1H, m), 4.37 (1H, m), 4.57
(2H, m), 4.71 (1H, m), 5.19 (1H, m), 5.32 (1H, m), 5.89 (1H,
m), 6.97 (1H, m), 7.67 (1H, d, J ) 8.6 Hz), 7.94 (1H, d, J ) 8.3
Hz), 8.77 (1H, d, J ) 8.5 Hz), 10.87 (1H, d, J ) 2.2 Hz); MS
(ES+) m/z 704 (MH+).
5(S)-Ben zyl-8(R)-(3,4-d ich lor o-ben zyl)-2(S)-(1H-in d ol-
3-ylm et h yl)-3,6,9,14-t et r a oxo-1,4,7,10-t et r a a za -cyclot et -
r a d eca n e-11-ca r boxylic Acid Allyl Ester (14). Compound
13 (150 mg, 0.21 mmol) was dissolved under a nitrogen
atmosphere in anhydrous THF (30 mL). Pd(dba)2 (126 mg, 0.22
mmol) was added, followed by triphenylphosphine (82 mg, 0.31
mol) and acetic acid (18 µL, 0.31 mmol). After the mixture was
stirred for 1 h at room temperature, the solvent was evapo-
rated under reduced pressure giving a greenish residue.
Diethyl ether (10 mL) was added, and the solid residue was
filtered under a nitrogen atmosphere, washed several times
on the filter with diethyl ether, and dried. Purification by
reverse phase flash chromatography (Matrex C18, 60 Å pore
diameter, 50 µm particle size; mobile phase: acetonitrile/water,
50:50 v/v) gave 70 mg (0.105 mmol, 50% yield) of 14 as a white
1
solid: mp 290-305 °C dec; H-NMR (DMSO-d6, ∂) 2.53 (2H,
m), 2.64 (2H, ABq, J ) 5, 14 Hz), 4.29 (1H, m), 4.36 (1H, m),
4.52 (1H, m), 7.64 (1H, m), 7.94 (1H, m), 8.76 (1H, d, J ) 8.5
Hz), 10.86 (1H, d, J ) 2.2 Hz); MS (ES+) m/z 664 (MH+).
(3S,6R)-Diben zyl-9-(S)-(1H-in d ol-3-ylm eth yl)-1,4,7,10-
tetr a a za -cyclotetr a d eca n e-2,5,8,11-tetr a on e (15). N-Boc-
4-aminobutyric acid (500 mg, 2.46 mmol) was dissolved in 25
mL of anhydrous DMF. EDC (480 mg, 2.50 mmol), HOBt (1.0
g, 6.5 mmol), and H-Trp-Phe-OtBu (1.0 g, 2.45 mmol) were
added in sequence, and the resulting solution was stirred
overnight at room temperature. After the solution was con-
centrated to a small volume, the residue was diluted with ethyl
acetate. The organic phase was washed with 10% aq citric acid;
it was then washed with 5% aq sodium bicarbonate, and finally
with water. After the mixture was dried over Na2SO4, evapo-
ration under reduced pressure gave (2R)-{2-(S)-[2-(4-tert-
butoxycarbonylamino-butyrylamino)-3-(S)-(1H-indol-3-yl)-pro-
pionylamino]-3-phenyl-propionylamino}-3-phenyl-propionic acid
tert-butyl ester as a yellowish solid (1.27 g, 2.40 mmol, 98%
yield). The crude product was dissolved in dichloromethane
(20 mL), and the solution was cooled to 0 °C. TFA (4.25 mL,
55 mmol) was added dropwise. The resulting solution was
allowed to warm to room temperature and was stirred for 1
h. After evaporation of the solvent, the addition of water and
sodium carbonate gave a white precipitate that was filtered
out and dissolved in ethyl acetate. The organic phase was
washed with water to a neutral pH. After the mixture was
dried over MgSO4 and the solvent evaporated under reduced
pressure, (2R)-{2-(S)-[2-(4-amino-butyrylamino)-3-(S)-(1H-in-
dol-3-yl)-propionylamino]-3-phenyl-propionylamino}-3-phenyl-
propionic acid tert-butyl ester was obtained as a porous white
solid (662 mg, 1.34 mmol, 56% yield). The crude product was
added to a solution of N-Boc-(R)-phenylalanine (353 mg, 1.34
mmol), EDC (274 mg, 1.42 mmol), and HOBt (542 mg, 4.0
mmol) in anhydrous DMF (40 mL). The mixture was stirred
overnight at room temperature, and then evaporated; the
residue was dissolved in ethyl acetate and washed with
5% aq KHSO4, 5% aq NaHCO3, and water. After the mixture
was dried over MgSO4 and the solvent evaporated under
reduced pressure, (2R)-{2-(S)-[2-[4-(2-tert-butoxycarbonylamino-
3-phenyl-propionylamino)-butyrylamino]-3-(S)-(1H-indol-3-yl)-
propionylamino]-3-phenyl-propionylamino}-3-phenyl-propion-
ic acid tert-butyl ester was obtained as a white solid (803 mg,
1.08 mmol, 81% yield). The crude product was added to a
solution of methyl sulfide (0.26 mL, 3.5 mmol) in 18 mL of
TFA, and the mixture was stirred for 1 h at room temperature.
After the evaporation of TFA, the crude residue was repeatedly
treated with diethyl ether to give a white solid; the white solid
was filtered off, dried, and dissolved in 90 mL of anhydrous
DMF. After the addition of EDC (210 mg, 1.09 mmol), HOBt
(440 mg, 3.25 mmol), and TEA (0.175 mL, 1.25 mmol), the
(B) Biology. Ra d ioliga n d Bin d in g Exp er im en ts. Stable
transfection of wild type and mutant receptors and membrane
preparation were carried out as previously described.5 The
buffer used for binding experiments was Tris-HCl (50 mM, pH
7.4) which contained bacitracin (100 mg mL-1), chymostatin
(10 mg mL-1), leupeptin (5 mg mL-1), thiorphan (10 mM), NaCl
(150 mM), MnCl2 (5 mM), and bovine serum albumin (1 g L-1).
The binding assay was performed in a final volume of 0.5 mL
for 30 min ([125I]NKA and [3H]SR48968) or 60 min ([3H]ne-
padutant) at 20 °C. [3H]Nepadutant (specific activity, 62 Ci
mmol-1) was synthesized by SibTech Inc. (Elmsford, NY).
[
125I]NKA (specific activity, 2000 Ci mmol-1) and [3H]SR48968
(specific activity, 25 Ci mmol-1) were purchased from Amer-
sham (Buckinghamshire, U.K.) and NEN Life Sciences Prod-
ucts (Boston, MA), respectively. Competition binding experi-
ments were carried out at the agonist and antagonist
radioligand concentration below its affinity constant, which
bound less than 10% of the total added radioligand. Prelimi-
nary experiments were performed to determine membrane
protein concentrations (50-300 µg mL-1) of each receptor in
order to obtain a signal of 1500-4000 dpm/assay of specific
binding. Nonspecific binding was defined as the amount of
labeled radioligand bound in the presence of 1 mM appropriate
unlabeled ligand, and it represented less than 5% of total
bound radioligand. All of the compound competition curves
were tested in a wide range of concentrations (0.3 pM to 10
µM). The assay was started by the addition of 0.4 mL of
membrane suspension, and it was terminated by the rapid
filtration through UniFilter-96 plates (Packard) that had been
presoaked for at least 2 h in polyethylenimine (PEI) 0.3%,
using a MicroMate 96 Cell Harvester (Packard Instrument
Company). The tubes and filters were then washed 5 times
with 0.5 mL aliquots of Tris buffer (50 mM, pH 7.4, 4 °C).
Filters were dried and soaked in Microscint 40 (Packard
Instrument Company), and bound radioactivity was counted

Human Tachykinin NK-2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3429
(5) Giolitti, A.; Cucchi, P.; Renzetti, A. R.; Rotondaro, L.; Zappitelli,
S.; Maggi, C. A. Molecular determinants of peptide and nonpep-
tide NK-2 receptor antagonists binding sites of the human
tachykinin NK-2 receptor by site-directed mutagenesis. Neuro-
pharmacology 2000, 39, 1422-1429.
(6) Emonds-Alt, X.; Vilain, P.; Goulaouic, P.; Proietto, V.; Van
Broeck, D.; Advenier, C.; Naline, E.; Neliat, G.; Brelie`re, J .-C.;
Le Fur, G. Life Sci. 1992, 50, PL101-PL106.
(7) Palczewski, K.; Takashi, K.; Tetsuya, H.; Behnke, C. A.; Mo-
toshima, H.; Fox, B. A.; et al. Crystal Structure of Rhodopsin:
A G Protein-Coupled Receptor. Science 2000, 489, 739-745.
(8) Pavone, V.; Lombardi, A.; Maggi, C. A.; Quartara, L.; Pedone,
C. Conformational Rigidity versus Flexibility in a Novel Peptidic
Neurokinin A Receptor Antagonists. J . Pept. Sci. 1995, 41, 236-
240.
(9) Cramer, R. D., III; Patterson, D. E.; Bunce, J . D. Comparative
Molecular Field Analysis (CoMFA). 1. Effect of Shape on Binding
of Steroids to Carrier Proteins. J . Am. Chem. Soc. 1988, 110,
5959-5967.
(10) Waller, C. L.; Oprea, T. I.; Giolitti, A.; Marshall, G. R. Three-
Dimensional QSAR of Human Immunodeficiency Virus (I)
Protease Inhibitors. 1. A CoMFA Study Employing Experimen-
tally-Determined Alignment Rules. J . Med. Chem. 1993, 36,
4152-4160.
(11) Heritage, T. W.; Lowis, D. R. Molecular Hologram QSAR. In
Rational Drug Design; Parrill, A. L., Reddy, M. R., Eds.; ACS
Symposium Series 719; American Chemical Society: Washing-
ton, DC, 2000; pp 212-225.
(12) Bellucci, F.; Carini, F.; Catalani, C.; Cucchi, P.; Lecci, A.; Meini,
S.; Patacchini, R.; Quartara, L.; Ricci, R.; Tramontana, M.;
Giuliani, S.; Maggi, C. A. Pharmacological profile of the novel
mammalian tachykinin hemokinin 1. Br. J . Pharmacol. 2002,
135, 266-274.
(13) Patacchini, R.; Giuliani, S.; Turini, A.; Navarra, G.; Maggi, C.
A. Effect of nepadutant at tachykinin NK2 receptors in human
intestine and urinary bladder. Eur. J . Pharmacol. 2000, 398,
389-397.
by a TopCount Microplate Scintillation Counter (Packard
Instrument Company).
Radioligand binding experiments were analyzed by fitting
the data with the GraphPad Prism program (San Diego, CA)
for Apple Macintosh in order to determine the -log of the
inhibitory affinity constant (pKi) for heterologous competition
experiments and the -log of the radioligand affinity constant
(-log KD) for homologous competition experiments, and were
expressed as the mean ( standard error of the mean from the
stated number of experiments, each performed in duplicate.
(C) Mod elin g a n d QSAR. In the case of our NK-2
antagonists, the model has been built initially from a small
set of 24 compounds (Table 2). Newly synthesized compounds
have been progressively tested against the available model,
and then included in a new recomputed one.
HQSAR parameters were essentially set at their default
values, as shown in Table 3, and are reported here. Atom count
in fragments: min 4; max 7 (default 3-6). Atoms, bonds,
connections: ON. Hydrogen atoms, chirality, donor & accep-
tor: OFF. Hologram lengths: 53-353. Select best model based
on: least standard error. In the last model the “Chirality” flag
was ON. Table 3 reports the statistics for these five models.
The structures were built in Sybyl 6.4 (Tripos, Inc.) with a
careful check of atom types but no conformational search. The
parameters in HQSAR were initially set at their default
values, and were later adjusted to include chirality and the
optimal fragment length of 4-7 atoms (no hydrogens). Q is
the cross-validated correlation coefficient derived from the
predictive residual sum of squares.
Refer en ces
(1) Regoli, D.; Boudon, A.; Fauchere, J . Receptors and antagonists
for substance P and related peptides. Pharmacol. Rev. 1994, 46
(4), 551-599.
(2) Maggi, C. A.; Patacchini, R.; Rovero, P.; Giachetti, A. Tachykinin
receptors and tachykinin receptor antagonists. J . Auton. Phar-
macol. 1993, 13, 23-93.
(3) Catalioto, R.-M.; Criscuoli, M.; Cucchi, P.; Giachetti, A.; Gian-
notti, D.; Giuliani, S.; Lecci, A.; Lippi, A.; Patacchini, R.;
Quartara, L.; Renzetti, A. R.; Tramontana, M.; Arcamone, F.;
Maggi, C. A. MEN 11420 (Nepadutant), a novel glycosylated
bicyclic peptide tachykinin NK-2 receptor antagonist. Br. J .
Pharmacol. 1998, 123, 81-91.
(4) Giannotti, D.; Perrotta, E.; Di Bugno, C.; Nannicini, R.; Harmat,
N. J . S.; Giolitti, A.; Patacchini, R.; Renzetti, A. R.; Rotondaro,
L.; Giuliani, S.; Altamura, M.; Maggi, C. A. Discovery of Potent
Cyclic Pseudopeptide Human Tachykinin NK-2 Receptor An-
tagonists. J . Med. Chem. 2000, 43 (22), 4041-4044.
(14) Gershengorn, M. C.; Osman, R. Minireview: Insights into G
Protein-Coupled Receptor Function Using Molecular Models.
Endo 2001, 142 (1), 2-10.
(15) Hipskind, P. A.; Howbert, J . J .; Cho, S.; Cronin, J . S.; Fort, S.
L.; Ginah, F. O.; Hansen, G. J .; Huff, B. E.; Lobb, K. L.; et al.
Practical and Enantiospecific Synthesis of LY303870, a Novel
NK-1 Antagonist. J . Org. Chem. 1995, 60, (21), 7033-7036.
(16) Giorgi, R.; Di Bugno, C.; Giannotti, D.; Maggi, C. A. Monocyclic
compounds with four bifunctional residues having NK-2 antago-
nist action. PCT Int. Appl. WO 98 34949, Aug 13, 1998.
J M011127H