Novel potent substance P and neurokinin A receptor antagonists. Conception, synthesis and biological evaluation of indolizine derivatives

Article abstract of DOI:10.1016/S0968-0896(02)00144-X

Exploration of SAR around dual NK₁/NK₂ antagonist Cbz-Gly-Leu-Trp-OBzl(CF₃)₂ and its derivatives disclosed the essential requirements for more potent dual NK₁/NK₂ binding. We report here the synthesis and the biological properties of a novel series of indolizine including pharmacophoric elements.

Full text of DOI:10.1016/S0968-0896(02)00144-X

Bioorganic & Medicinal Chemistry 10 (2002) 2905–2912
Novel Potent Substance P and Neurokinin A Receptor
Antagonists. Conception, Synthesis and Biological Evaluation of
Indolizine Derivatives
Regis Millet,a Juozas Domarkas,a Benoıt Rigo,b Laurence Goossens,a
a
Jean-Francois Goossens,a RaymondHoussin andJean-Pierre He nicharta,*
a
´
Institut de Chimie Pharmaceutique Albert Lespagnol, Universite de Lille 2, EA 2692,
3rue du Professeur Laguesse, B.P. 83, F-59006 Lille, France
b
´ ´
Laboratoire d’Ingenierie Moleculaire, Ecole des Hautes Etudes Industrielles,
13rue de Toul, F-59046 Lille, France
Received11 January 2002; accepted3 May 2002
Abstract—Exploration of SAR arounddual NK 1/NK2 antagonist Cbz-Gly-Leu-Trp-OBzl(CF3)2 and its derivatives disclosed the
essential requirements for more potent dual NK1/NK2 binding. We report here the synthesis and the biological properties of a novel
series of indolizine including pharmacophoric elements. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
from which they can be releasedby a variety of stimuli,
including allergen, ozone or inflammatory mediators.
Involvement of NK1 receptors seems to be more
important in inflammatory conditions including vaso-
dilatation, mucus secretion, plasma extravasation and
leukocyte adhesion-activation, while NK2 mediates the
bronchomotor tone. Taking into account these features,
it was hypothesizedthat dual NK 1/NK2 antagonism
may be useful in the treatment of asthma.11,12
Substance P (SP), Neurokinin A (NKA) andNeuro-
kinin B (NKB) are members of the tachykinin family
(Table 1) andshare a common C-terminal amino acid
sequence Phe-X-Gly-Leu-Met-NH2 (Table 1) often
1,2
referredto as the message moiety of the peptide.
The biological actions of tachykinins are mediated
through specific G-protein coupledreceptor. There are
three subtypes of receptor designated NK1, NK2 and
NK3. SP mediates its physiological effects3 mainly by
binding to a specific NK1 receptor whereas NKA and
We have already reported that the tripeptide Cbz-Gly-
Leu-Trp-OBzl(CF3)2 exhibitedudal NK
andNK
2
1
affinities.13ꢀ15 Studies of structure–activity relationships
revealedthat the C-terminal sequence [Trp-OBzl(CF ) ]
NKB exert their activities via the NK2 andNK recep-
3
3 2
tors, respectively.
was shown to be favourable for NK1 recognition and
indolylmethyl and Cbz carbamate groups for NK2
recognition. These results encouragedus to findnew
potent dual NK1/NK2 receptor antagonists by struc-
tural modifications of the tripeptide Cbz-Gly-Leu-Trp-
OBzl(CF3)2 (Table 2). Thus, we decided firstly to inves-
tigate chemical modifications of C-terminal sequence
since a new class of dual NK1/NK2 antagonists (Scheme
1) has recently been reportedas neurokinin receptor
antagonists including the N-methylbenzamide group in
their structure.17 For a better understanding of NK1/
NK2 profile binding and to improve metabolic stability,
we went on to replace the ester linkage of Cbz-Gly-Leu-
Trp-OBzl(CF3)2 by several amide groups (1a–c).
Neurokinin antagonists have been the subject of con-
siderable investigation since the association of their
central andperipheral actions with the treatment of
chronic diseases like asthma,4,5 pain,6 emesis7,8 and
psychiatric disorders.9,10
In the pathogenesis of asthma, SP andNKA have been
widely studied in animal and human airways. These
neuropeptides are localised in sensory airway nerves
*Corresponding author. Tel.: +33-3-2096-4374; fax: +33-3-2096-
4906; e-mail: henicha@phare.univ-lille2.fr
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00144-X

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R. Millet et al. / Bioorg. Med. Chem. 10 (2002) 2905–2912
Table 1. Chemical structure of three mammalian tachykinins
Tachykinin
Sequence
SP
NKA
NKB
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2
His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2
Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2
Amino acids common to carboxyl-terminal are italicized.
Scheme 1. Structure of dual NK1/NK2 antagonists.
Table 2. Binding affinity and calcium responses in hNK1- or hNK2-
receptors expressing CHO cells
b
b
CompoundhNK
Kia pA2 hNK2 Kia pA2
1
(nM)
(nM)
1a
1b
1c
2a
2b
2c
200ꢁ18 6.8ꢁ0.4 >3000
n.d.
n.d.
1580ꢁ82 n.d.c
>3000
100ꢁ7 6.7ꢁ0.3 79ꢁ5 7.1ꢁ0.5
79ꢁ8 7.0ꢁ0.2 >3000
n.d.
158ꢁ16 6.9ꢁ0.3 398ꢁ35 6.2ꢁ0.2
100ꢁ9 6.7ꢁ0.4 100ꢁ12 6.7ꢁ0.4
Cbz-Gly-Leu-Trp-OBzl(CF3)2
40ꢁ3
n.d.
250ꢁ24
n.d.
aInhibition of [3H]SP or [3H]NKA binding to membranes of hNK1- or
hNK2-receptors expressing CHO cells. Each value represents the
meanꢁSD of three independent experiments (triplicate per experi-
ment) performedfrom a 10 ꢀ11–10ꢀ6 M range.
bInhibition of SP- or NKA-induced intracellular [Ca2+]i increase in
hNK1 or hNK2 receptors expressing CHO cells loaded with Fura-2.
Each value is the meanꢁSD derived from Schild plot using three
independent experiments (triplicate per experiment) in a 10ꢀ10–10ꢀ6 M
range for both agonist andantagonist compounds.
cn.d., not determined.
In addition, we focused our interest on a rational design
of peptidomimetics.18,19 In previous reports, we descri-
bed that introducing building units such as spirolactam
or lactam ledto restrictedCbz-Gly-Leu-Trp-
OBzl(CF3)2 conformations showing high binding affi-
Scheme 2. Drug design of tripeptides 1a–c andindolizines 2a–c.
nity andselectivity for NK receptors. To continue our
1
benzylamine, benzylamine, or N-methylbenzylamine
under EDCI/HOBt conditions (59–83% yield) to afford
the amides 3a–c. Removal of the Boc protective group
with methanolic HCl solution gave amines 4a–c which
were coupledwith commercially available Cbz-Gly-Leu-
OH by using PyBOP to produce tripeptides 1a–c at 58–
64% yields after purification.
investigation in the design of neurokinin antagonists, we
report here (Scheme 2) the synthesis andbinding profile
of new compounds which include an indolizine moiety
in their N-terminal structure (2a–c) mimicking the Gly-
Leu sequence of the tripeptide Cbz-Gly-Leu-Trp-
OBzl(CF3)2. Investigation of such a system shouldallow
an increasing neurokinin binding potency through a
decrease in the entropy system.
The synthesis of 5,6-fusedbicyclic skeleton ( 2a–c) is
outlinedin Scheme 3 andrepresents an extension of the
chemistry that we previously described.21,22 The strategy
Furthermore, these chemical modifications led us to
explore the importance of the amide function and Cbz
carbamate on NK2 recognition andto improve our
knowledge of the bioactive conformation of neurokinin
antagonists with a view to creating andoptimising dual
NK1/NK2 antagonists.
adopted uses enaminoesters 8a–b anddifferent N-sub-
23
stitutedaminoacrylic acisd
in a Michael conden-
sation24 followedby cyclisation promotedby
phosphorous trichloride (30–50% yield). Enaminoesters
8a–b were obtained according to a previously described
procedure25 which usedmethyl pyroglutamate as its
starting point. Conversion of pyroglutamate into
iminoether 5 was carriedout with dimethyl sulfate and
triethylamine anda follow-on condensation with Mel-
drum’s acid afforded enaminoester 6 at 83% yield.
Opening of Meldrum’s ring and saponification of ester
function were carried out with sodium methoxide in
methanol followed by acidification with hydrochloric
acidto give a Z/E mixture of carboxylic acid 7 at 70%
yield. Coupling reactions with tryptophan amides 4a or
4c (PyBOP) were finally completedto generate the
Results and Discussion
Chemistry
Cbz-Gly-Leu-Trp-N(R1)R2 peptides (1a–c) were
obtained (Scheme 3) according to classical peptidic
methods20 in solution using Boc as protective group for
Trp, andEDCI/HOBt or PyBOP as coupling agents.
Boc-Trp-OH was coupledwith 3,5-bis(trifluoromethyl)

R. Millet et al. / Bioorg. Med. Chem. 10 (2002) 2905–2912
2907
Scheme 3. Reagents andconditions: (a) EDCI, HOBt, amine, DIPEA, CH 2Cl2, 48 h, rt; (b) MeOH, HCl, 16 h, rt; (c) Cbz-Gly-Leu-OH, PyBOP,
DIPEA, CH2Cl2, 48 h, rt; (d) (1) (CH3)2SO4, 60 ꢂC, 12 h; (2) NEt3, 0 ꢂC; (3) Meldrum’s acid, 24 h, rt; (e) (1) MeONa, MeOH, reflux, 24 h; (2) H2O,
2 h, rt; (3) HCl, MeOH; (f) PyBOP, DIPEA, CH2Cl2, 48 h, rt; (g) PCl3, dioxane, toluene, reflux, 4 h.
desired enaminoesters 8a–b (70–71% yield) which were
directly used in the building of the indolizine ring (2a–c).
supposedandshows that -Trp-N(Me)Bzl is an interest-
ing pattern providing a good NK1/NK2 in binding affi-
nity ratio. Introduction of a primary amide as in
compounds 1a–b leads to a significant decrease in
binding affinity to NK2 receptors. Noting these results,
we decided to introduce a heterocyclic connector
between the previously identified -Trp-N(Me)-Bzl moi-
ety andthe Cbz ring of 1c in an attempt to induce a
structural constraint. The indolizine ring was chosen as
a scaffoldto mimic the Gly-Leu residue of 1c. Three
indolizine compounds (2a–c) were preparedandtheir
pharmacological results ledto two significant observa-
tions that enabledus to propose pharmacophoric ele-
ments (Scheme 4) of potential dual NK1/NK2 receptor
antagonists.
Biological evaluation and structure–activity relationships
The NK1/NK2 receptor binding affinity and antagonist
activity data are summarized in Table 2. Our peptidic
approaches in the design of a dual NK1/NK2 antagonist
consistedfirstly in modifying the ester function of the tri-
peptide Cbz-Gly-Leu-Trp-OBzl(CF3)2 by different amide
groups to optimize NK1 andNK
affinities andto
2
enhance metabolic stability. Three tripeptides (1a–c) were
preparedandtheir affinities towardneurokinin receptors
were measured. Analysis of the neurokinin binding
profile revealedthat it was possible to obtain dual NK
/
1
NK2 antagonists by substitution of the 3,5-bis(tri-
fluoromethyl)benzyl ester function with N-methyl(benzyl)-
amide moiety. This observation confirms what we
Firstly, as it was observedfor 1c-Trp-N(Me)Bzl residues
in 2b,c were well toleratedboth by NK
andNK
2
1

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R. Millet et al. / Bioorg. Med. Chem. 10 (2002) 2905–2912
Conclusion
For this paper, we prepareddual NK 1/NK2 antagonists
using several pharmacophoric elements proposedin
Scheme 4. The development of small molecules -antago-
nists of SP andNKA—offers a goodpossibility for the
treatment of asthma. Few dual NK1/NK2 antagonists
have been reported29ꢀ36 andthe pharmacophoric ele-
ments proposedhere open the way for the development of
such molecules. Moreover, we have demonstrated that the
indolizine ring seems to be an interesting template in the
construction of dual NK1/NK2 antagonists andeasy to
use in other drug design peptidic strategies. In our strat-
egy, the introduction of an indolizine scaffold disposed
pharmacophoric elements favorably to interact with both
the NK1 andthe NK 2 receptors.
Scheme 4. Pharmacophoric elements of potential dual NK1/NK2
receptor antagonists.
receptors suggesting that these residues are optimal for
NK1/NK2 recognition. The -Trp-NHBzl(CF3)2 residue
in 2a saw a significant reduction in NK2 affinity. Sec-
ondly, replacement of the acetamido group in 2b by Cbz
(2c) ledto an increase in NK 2 affinity. This information
is in total agreement with that observed13 before andled
to the conclusion that Cbz carbamate moiety hada
direct influence on NK2 recognition.
Experimental
Chemistry
All information reportedherein andin previous
papers13ꢀ16 correlate andresult in establishing the
essential requirements (Scheme 4) for more potent dual
NK1/NK2 binding affinity. The C-terminal sequence of
tripeptides 1a–c seems to be responsible for NK1 recog-
nition—l-tryptophan benzyl esters have already been
Melting points were determined on a Buchi 535 capil-
lary melting point apparatus andare uncorrected. Ana-
lytical tlc were performedon precoatedKieselgel 60F
plates (Merck). The spots were locatedby uv (254 and
366 nm). Column chromatographies were performedon
silica gel 60 230–400 mesh (Merck). IR spectra were
determined in potassium bromide with a Bruker Vector
254
reportedto be selective NK
antagonists—whereas
1
indolylmethyl and Cbz moieties proved particularly sig-
nificant for NK2 recognition. In addition, the tertiary
benzyl amide function of l-Trp has been shown to be an
essential requirement for a goodNK 1/NK2 binding
ratio.
22 spectrophotometer; absorbances are reportedin
n
(cmꢀ1). H NMR spectra were recorded on a Bruker
AC 300 spectrometer (300 MHz) using tetramethylsilane
as an internal standard. Chemical shifts were expressed
in d units (ppm) andthe splitting patterns were desig-
natedas follows: s singlet, bs broadsinglet, t triplet, d
doublet, dd doublet of doublets, m multiplet, bm broad
multiplet. Mass spectra were recorded on a quadripolar
Finnigan Mat SSQ 710 instrument in the electron
impact (EI) mode. Elemental analyses for C, H, N, were
performedby the ‘Service Central d’Analyses’ (CNRS,
Vernaison, France), andare within 0.4% of theory.
1
Furthermore, the in vitro antagonist activities of high
affinity selective ligands have been evaluated on all
hNK1- or hNK2-transfectedCHO cells. The ability of
compounds to antagonize the calcium mobilization
causedby activation of hNK 1- or hNK2-receptors
expressedin CHO cells was studiedusing Fura-2 fluor-
escence, as described for hNK1- or hNK2-transfected
cells.26ꢀ28 SP andNKA stimulate the phosphoinositide-
turnover signaling system following binding to its NK1
General procedure for preparation of N-protected amides
(3a–c). DIPEA (12.7 mL, 72.9 mmol), EDCI (5.90 g,
30.8 mmol) andHOBt (4.16 g, 30.8 mmol) were addedto
a solution of Boc-Trp-OH (6.23 g, 20.5 mmol) in
CH2Cl2 (40 mL) (ice bath). The mixture was stirredfor
1 h, then amine (20.5 mmol) was added. The mixture
was stirredfor 48 h, then the solution was washedwith
HCl (0.25 N) then NaHCO3 (10%). The organic phase
was dried (MgSO4) andevaporatedto yieldan oil.
andNK receptor, leading to an elevation in intra-
2
cellular cytosolic Ca2+ concentration. Our interest was
to examine the effects of blocking the NK1- or NK2-
receptor with selectedcompounds ( Ki<400 nM) on SP-
or NKA-mediated increase in cytosolic Ca2+ con-
centration in CHO cells. SP andNKA induceda max-
imal elevation of intracellular cytosolic Ca2+
concentration from 25 (ꢁ3) to 312 (ꢁ16) nM andfrom
18 (ꢁ4) to 215 (ꢁ12) nM (meanꢁSEM of 15 separate
NK1 andNK -CHO cells. All selectedcompounds 1a–c
N-ꢀ-tert-Butoxycarbonyl-N-3,5-bis(trifluoromethyl) ben-
zyltryptophan amide (3a). The product 3a was recrys-
tallizedfrom CH Cl2 to give a white solid(6.40 g) at
2
and 2a–c (10ꢀ6 M) do not increased intracellular cal-
cium concentration in both cell lines. Inhibition increase
in SP- or NKA-induced intracellular calcium con-
centration was calculatedfrom Schildplot representa-
tions, giving pA2 values indicated in Table 2.
Compounds 1a–c and 2a–c antagonize the SP- and
NKA-induced calcium pathway. Furthermore, for all
selectedligands, potent functional antagonistic proper-
ties correlate well with the respective specific binding
affinities.
2
59% yield: mp 180–182 ꢂC; TLC Rf [hexane/ethyl ace-
tate (5:5)]=0.55; IR (KBr) n: 1651 cmꢀ1 (CO),
1
1680 cmꢀ1 (CO), 3260 cmꢀ1 (NH); H NMR (300 MHz,
DMSO-d6) d: 1.32 (s, 9H), 2.90–3.10 (m, 2H), 4.20–4.59
(m, 3H), 7.00 (t, 1H, J=7.5 Hz), 7.07 (t, 1H, J=7.0 Hz),
7.18 (s, 1H), 7.34 (d, 1H, J=8.0 Hz), 7.63 (d, 1H,
J=8.0 Hz), 7.98 (s, 3H), 8.71 (s, 1H), 10.83 (s, 1H); MS
(EI) m/z 529 (M+).

R. Millet et al. / Bioorg. Med. Chem. 10 (2002) 2905–2912
2909
N-ꢀ-tert-Butoxycarbonyl-N-benzyltryptophan amide (3b).
The product 3b was flash chromatographedon a
5ꢃ40 cm column using cyclohexane/ethyl acetate (5:5)
as the eluting solvent. The product thus obtained crys-
tallizedfrom CH 2Cl2/hexane (8:2) as a white solid
(7.10 g) in 88% yield: mp 136–139 ꢂC; TLC Rf [cyclo-
hexane/ethyl acetate (5:5)]=0.3; IR (KBr) n: 1651 cmꢀ1
(CO), 1680 cmꢀ1 (CO), 3260 cmꢀ1 (NH); 1H NMR
(300 MHz, DMSO-d6) d: 1.32 (s, 9H), 2.89–3.13 (m,
2H), 4.20–4.30 (m, 3H), 6.86 (d, 1H, J=8.1 Hz), 7.00 (t,
1H, J=7.2 Hz), 7.09 (t, 1H, J=7.2 Hz), 7.13 (s, 1H),
7.16–7.29 (m, 5H), 7.34 (d, 1H, J=8.0 Hz), 7.63 (d, 1H,
J=8.1 Hz), 8.43 (t, 1H, J=7.5 Hz), 10.83 (s, 1H); MS
(EI) m/z 393 (M+).
TLC Rf [CH2Cl2/MeOH saturatedwith NH
(9:1)]=0.6; IR (KBr) n: 1643 cmꢀ1 (CO), 3415 cmꢀ13
(NH); H NMR (300 MHz, DMSO-d6) d: 2.57 (s, 2H),
2.65 (s, 1H), 3.14–3.33 (m, 2H), 4.30–4.70 (m, 3H),
6.97–7.45 (m, 9H), 7.61 (d, 1H, J=7.7 Hz), 8.51 (s, 3H),
11.18 (s, 1H); MS (EI) m/z 307 (M+).
1
General procedure for preparation of tripeptides 1a–c.
Amide (4a–c) (1.00 g, 2.15 mmol), PyBOP (1.23 g,
2.36 mmol) and DIPEA (1.1 mL, 6.44 mmol) were added
to a solution of Cbz-Gly-Leu-OH (693 mg, 2.15 mmol)
in 40 mL of CH2Cl2. The reaction mixture was cooledin
an ice bath andstirredfor 48 h; then the solvent was
removedin vacuum.
N-ꢀ-tert-Butoxycarbonyl-N-benzyl-N-methyltryptophan
amide (3c). The product 3c was flash chromatographed
on a 5ꢃ40 cm column using cyclohexane/ethyl acetate
(7:3) as the eluting solvent. The product thus obtained
crystallizedfrom idisopropyl ether as a white solid
(6.27 g) in 75% yield: mp 144–145 ꢂC; TLC Rf [cyclo-
hexane/ethyl acetate (5:5)]=0.5; IR (KBr) n: 1645 cmꢀ1
(CO), 1686 cmꢀ1 (CO), 3226 cmꢀ1 (NH); 1H NMR
(300 MHz, DMSO-d6) d: 1.30 (s, 3H), 1.33 (s, 6H), 2.72
(s, 1H), 2.78 (s, 2H), 2.89–3.13 (m, 2H), 4.30–4.75 (m,
3H), 7.16–7.29 (m, 8H), 7.34 (d, 1H, J=8.3 Hz), 7.56 (d,
1H, J=7.8 Hz), 10.82 (s, 0.33H), 10.88 (s, 0.66H); MS
(EI) m/z 407 (M+).
N-ꢀ-Benzyloxycarbonyl-Gly-Leu-N-3,5-bis-(trifluoro-
methyl)benzyltryptophan amide (1a). The product 1a
was recrystallizedfrom ethyl acetate as a white solid
(960 mg) at 61% yield: mp 213–215 ꢂC; TLC Rf [hexane/
ethyl acetate (3:7)]=0.3; IR (KBr) n: 1640 cmꢀ1 (CO),
1718 cmꢀ1 (CO), 1723 cmꢀ1 (CO), 3250–3400 cmꢀ1
1
(NH); H NMR (300 MHz, DMSO-d6) d: 0.79–0.83 (m,
6H), 1.30–1.40 (m, 2H), 1.47–1.55 (m, 1H), 3.01–3.17
(m, 2H), 3.65 (d, 2H, J=5.8 Hz), 4.30–4.53 (m, 4H),
5.03 (s, 2H), 6.97 (t, 1H, J=7.1 Hz), 7.06 (t, 1H,
J=6.6 Hz), 7.13 (s, 1H), 7.31–7.39 (s, 5H), 7.45–7.49 (m,
1H), 7.55 (d, 1H, J=7.5 Hz), 7.91 (s, 2H), 7.99 (s, 1H),
8.17 (d, 1H, J=7.8 Hz), 8.59 (t, 1H, J=8.0 Hz), 10.82 (s,
1H); MS (CI) m/z 734 (MH+), 227 CH2Ph(CF3)2, 130
(Ind-CH+2 ), 91 Ph-CH2+. Anal. calcdfor C 36H37F6N5O5:
C, 58.94; H, 5.08; N, 9.55. Found: C, 59.01; H, 5.37; N,
9.42.
General procedure for preparation of amides 4a–c.
N-Protectedamide ( 3a–c) (10.4 mmol) was dissolved in
a mixture of dry tetrahydrofuran (20 mL) and of
methanol saturatedwith hydrochloric acid(30 mL), and
the solution was left to standfor 16 h. The solvent was
removedin vacuum andthe residue was recrystallized.
N-ꢀ-Benzyloxycarbonyl-Gly-Leu-N-benzyltryptophan
amide (1b). The product 1b crystallizedfrom CH Cl2 as
2
a white solid(822 mg) at 64% yield: mp 211–212 ꢂC;
TLC Rf [hexane/ethyl acetate (3:7)]=0.3; IR (KBr) n:
1631 cmꢀ1 (CO), 1763 cmꢀ1 (CO), 1718 cmꢀ1 (CO),
N-3,5-Bis(trifluoromethyl)benzyltryptophanamide hydro-
chloride (4a). The product 4a crystallizedfrom MeOH/
CH2Cl2 (1:1) as a white solid(4.61 g) in 95% yield: mp
3282 cmꢀ1 (NH), 3351 cmꢀ1
(NH); 1H NMR
245–247 ꢂC; TLC Rf [CH2Cl2/MeOH saturatedin NH
(300 MHz, DMSO-d6) d: 0.78–0.85 (m, 6H), 1.30–1.40
(m, 2H), 1.47–1.55 (m, 1H), 3.01–3.17 (m, 2H), 3.66 (d,
2H, J=5.4 Hz), 4.30–4.53 (m, 4H), 5.03 (s, 2H), 6.97 (t,
1H, J=7.0 Hz), 7.06 (t, 1H, J=6.8 Hz), 7.13 (s, 1H),
7.20–7.40 (m, 10H), 7.45–7.49 (m, 1H), 7.55 (d, 1H,
J=7.5 Hz), 8.12 (d, 1H, J=7.8 Hz), 8.41 (t, 1H,
J=8.0 Hz), 10.76 (s, 1H); MS (CI) m/z 598 (MH+), 130
(Ind-CH+2 ). Anal. calcdfor C 34H39N5O5: C, 70.09; H,
6.56; N, 12.57. Found: C, 70.00; H, 6.71; N, 12.44.
3
(9:1)]=0.6; IR (KBr) n: 1661 cmꢀ1 (CO), 3200–
3350 cmꢀ1 (NH), 3423 cmꢀ1 (NH); 1H NMR (300 MHz,
DMSO-d6) d: 3.14–3.33 (m, 2H), 4.08 (bs, 1H), 4.41 (dd,
1H, J=6.5 Hz, J0=13.0 Hz), 4.56 (dd, 1H, J=6.4 Hz,
J0=13.1 Hz), 6.97 (t, 1H, J=7.5 Hz), 7.08 (t, 1H,
J=7.0 Hz), 7.21 (s, 1H), 7.35 (d, 1H, J=8.0 Hz), 7.68
(d, 1H, J=7.5 Hz), 7.97 (s, 2H), 8.00 (s, 1H), 8.38 (s,
3H), 9.40 (s, 3H), 11.07 (s, 1H); MS (EI) m/z 429 (M+).
N-Benzyltryptophan amide hydrochloride (4b). The pro-
duct 4b recrystallizedfrom MeOH/acetone (2:8) as a
white solid(3.26 g) in 95% yield: mp 228–230 ꢂC; TLC
N-ꢀ-Benzyloxycarbonyl-Gly-Leu-N-Benzyl-N-methyl-
tryptophan amide (1c). The product was flash chromato-
graphedon a 5 ꢃ40 cm column using cyclohexane/ethyl
acetate (3:7) as the eluting solvent andrecrystallized
from hexane/CH2Cl2 (3:7) to give 1c as a white solid
(763 mg) at 58% yield: mp 92–94 ꢂC; TLC Rf [cyclohex-
ane/ethyl acetate (3:7)]=0.3; IR (KBr) n: 1632 cmꢀ1
(CO), 1711 cmꢀ1 (CO), 3293 cmꢀ1 (NH); 1H NMR
(300 MHz, DMSO-d6) d: 0.79–0.83 (m, 6H), 1.30–1.40
(m, 2H), 1.47–1.55 (m, 1H), 2.66 (s, 1H), 2.68 (s, 2H),
2.92–3.05 (m, 1H), 3.62–3.68 (m, 2H), 4.30–4.53 (m,
4H), 5.05 (s, 2H), 6.90–7.09 (m, 1H), 7.15 (s, 1H), 7.19–
7.39 (m, 10H), 7.45–7.49 (m, 1H), 7.57 (d, 1H,
J=8.0 Hz), 7.92 (m, 1H), 8.46 (d, 0.33H, J=8.0 Hz),
Rf [CH2Cl2/MeOH saturatedwith NH (9:1)]=0.6; IR
3
(KBr) n: 1663 cmꢀ1 (CO), 3261 cmꢀ1 (NH), 3315 cmꢀ1
1
(NH); H NMR (300 MHz, DMSO-d6) d: 3.14–3.33 (m,
2H), 4.02–4.12 (m, 1H), 4.20–4.34 (m, 2H), 6.99 (t, 1H,
J=7.5 Hz), 7.14–7.30 (m, 7H), 7.38 (d, 1H, J=8.3 Hz),
7.70 (d, 1H, J=7.7 Hz), 8.45 (s, 3H), 9.17 (t, 1H,
J=5.8 Hz), 11.16 (s, 1H)); MS (EI) m/z 293 (M+).
N-Benzyl-N-methyltryptophan amide hydrochloride (4c).
The product 4c crystallizedfrom MeOH/ethyl acetate
(1:5) as a white solid(3.50 g) at 98% yield: mp 76–77 ꢂC;

2910
R. Millet et al. / Bioorg. Med. Chem. 10 (2002) 2905–2912
8.53 (d, 0.66H, J=8.0 Hz), 10.81 (s, 0.33H), 10.87 (s,
0.66H); MS (CI) m/z 612 (MH+), 130 (Ind-CH2+), 91
(Ph-CH+2 ). Anal. calcdfor C 35H41N5O5: C, 68.72; H,
6.75; N, 11.45. Found: C, 68.73; H, 7.01; N, 11.70.
1.70–1.80 (m, 0.66H), 1.95–2.10 (m, 1H), 2.40–2.50 (m,
2H), 2.95–3.20 (m, 2H), 3.48 (s, 1H), 3.50 (s, 2H), 4.20–
4.60 (m, 5H), 6.97 (t, 1H, J=7.5 Hz), 7.05 (t, 1H,
J=7.4 Hz), 7.12 (bs, 1H), 7.32 (d, 1H, J=8.3 Hz), 7.57
(d, 1H, J=7.8 Hz), 7.92–7.97 (m, 3H), 8.10 (s, 0.33H),
8.14 (s, 0.66H), 8.23 (d, 0.66H, J=7.8 Hz), 8.30 (d,
0.33H, J=7.8 Hz), 8.70–8.80 (m, 1H), 10.82 (s, 1H); MS
(EI) m/z 596 (M+), 565 (M-OCH3), 130 (CH2-Ind+).
Anal. calcdfor C 28H26F6N4O4: C, 56.38; H, 4.39; N,
9.39. Found: C, 56.38; H, 4.43; N, 9.55.
Isopropylidene(methoxycarbonylpyrrolidinylidene)malonate
(6). A mixture of dimethyl sulfate (100 mL, 1.06 mol)
andmethyl pyroglutamate (120 g, 0.85 mol) was stirred
at 60 ꢂC for 12 h. The iminoether salt was slowly drop-
pedinto an ice well stirredcooledsolution of NEt
3
(150 mL, 1.05 mol). Meldrum’s acid (123 g, 0.85 mol)
was added and the mixture was stirred at room tem-
perature for 24 h. The b-enaminoester 6 was filteredand
recrystallizedfrom water as a white solid(189 g) at 83%
yield: mp 140–141 ꢂC; TLC Rf [hexane/ethyl acetate
(1:1)]=0.35; IR (KBr) n: 1720 cmꢀ1 (CO), 1745 cmꢀ1
(E)- and (Z)-5-Methoxycarbonylmethine-(RS)-prolyl-N-
benzyl-N-methyltryptophan amide (8b). The product 8b
was flash chromatographedon a 5 ꢃ40 cm column using
ethyl acetate as the eluting solvent. The product was
isolatedas an unstable oil (1.07 g) at 70% yieldandwas
not purifiedbut utilizedidrectly to synthesize com-
pounds 2a and 2c. TLC Rf (ethyl acetate)=0.2; IR
(KBr) n: 1630 cmꢀ1 (CO), 1650 cmꢀ1 (CO), 3210 cmꢀ1
(NH), 3320 cmꢀ1 (NH).
1
(CO), 3300 cmꢀ1 (NH); H NMR (300 MHz, CDCl3) d:
1.70 (s, 6H), 2.20–2.70 (m, 2H), 3.20–3.70 (m, 2H), 3.70
(s, 3H), 4.40–4.90 (m, 1H), 10.10 (s, 1H); MS (EI) m/z
269 (M+). Anal. calcdfor C 12H15NO6: C, 53.53; H,
5.61; N, 5.20. Found: C, 53.46; H, 5.59; N, 5.39.
General procedure for preparation of indolizines (2a–c).
Acrylic acid(4.21 mmol) andPCl 3 (0.41 mL, 4.63 mmol)
were added to a solution of b-enaminoester (8a,b)
(4.21 mmol) in dry dioxane (30 mL) and dry toluene
(30 mL). The solution was heatedat reflux in an inert
atmosphere andstirredfor 4 h. The solvents were
removed under reduced pressure.
(E) and (Z)-(RS) 5-Methoxycarbonylmethineproline (7).
A solution of sodium methoxide (276 mmol) in 140 mL
of MeOH was added to a solution of compound 6 (24 g,
88.9 mmol) in MeOH (60 mL). The mixture was refluxed
for 24 h then water (1.6 mL, 88.9 mmol) was added.
After 2 h, the solution was neutralizedwith con-
centratedHCl (23.1 mL, 276 mmol). The inorganic salts
were filteredandMeOH was removedunder vacuum.
The residue obtained was flash chromatographed on a
6ꢃ60 cm column using CH2Cl2/MeOH (1:1) as the elut-
ing solvent to give 7 as a white solid(11.5 g) at 70%
yield: mp 93–95 ꢂC; TLC Rf [CH2Cl2/MeOH (1:1)]=0.5;
IR (KBr) n: 1669 cmꢀ1 (CO), 1716 cmꢀ1 (CO),
3255 cmꢀ1 (NH), 3443 cmꢀ1 (OH); 1H NMR (300 MHz,
DMSO-d6) isomers E and Z were observedin 1/9 pro-
portion d: 1.85–1.90 (m, 1H), 2.09–2.16 (m, 1H), 2.49–
2.51 (m, 2H), 3.42 (s, 0.3H), 3.48 (s, 2.7H), 3.98 (t, 0.1H,
J=6.9 Hz), 4.14 (t, 0.9H, J=6.8 Hz), 4.34 (s, 0.9H), 4.66
(s, 0.1H), 7.85 (sꢂ, 0.1H), 8.05 (s, 0.9H); MS (EI) m/z 185
(M+). [a]2D5=0 (MeOH, c=1). Anal. calcdfor
C8H11NO4: C, 51.89; H, 5.99; N, 7.56. Found: C, 51.74;
H, 6.09; N, 7.69.
6-Acetamido-5-oxo-8-methoxycarbonyl-1,2,3,5,6,7-hexa-
hydroindolizine-3-carbonyl-3,5-bis(trifluoromethyl)benzyl-
tryptophan amide (2a). The product 2a was dissolved in
boiling acetone. The salts were filteredandthe acetone
solution was evaporated under reduced pressure to give
an oil which was flash chromatographedon a 4 ꢃ40 cm
column using CH2Cl2/acetone (1:1) as the eluting sol-
vent. The product obtained was isolated as a white solid
(1.49 g) at 50% yield: mp 208–212 ꢂC; TLC Rf [CH2Cl2/
acetone (1:1)]=0.4; IR (KBr) n: 1645 cmꢀ1 (CO),
1
1690 cmꢀ1 (CO), 3280 cmꢀ1 (NH); H NMR (300 MHz,
DMSO-d6) d: 1.80–1.90 (m, 4H), 2.12–2.40 (m, 2H),
2.65–2.87 (m, 2H), 3.05–3.20 (m, 3H), 3.63 and3.65 (2s,
3H), 4.25–4.75 (m, 5H), 6.98 (t, 1H, J=7.5 Hz), 7.05 (t,
1H, J=7.4 Hz), 7.15 and7.18 (2s, 1H), 7.30–7.35 (m,
1H), 7.55–7.60 (m, 1H), 7.91–7.96 (m, 3H), 8.14–8.27
(m, 1H), 8.50–8.60 (m, 1H), 8.63–8.71 (m, 1H), 10.80–
10.85 (m, 1H); MS (EI) m/z 707 (M+), 648
General procedure for preparation of ꢁ-enaminoesters
(8a,b). PyBOP (2.68 g, 3.22 mmol), carboxylic acid 7
(895 mg, 4.83 mmol) andDIPEA (1.7 mL, 9.66 mmol)
were added to a solution of tryptophan amide hydro-
chloride (4a or 4c) (3.22 mmol) in CH2Cl2 (50 mL) (ice
bath). The solution was stirredfor 48 h, then the solvent
was evaporated. The residue obtained was dissolved in
ethyl acetate andwashedsuccessively with saturated
NaHCO3 solution, HCl (1 N) andH 2O. The organic
phases were dried over MgSO4, filteredthen evaporated.
.
(MꢀCO2CH3). Anal. calcdfor C 33H31F6N5O6 1/2H2O:
C, 55.31; H, 4.50; N, 9.77. Found: C, 55.25; H, 4.60; N,
9.90.
6-Acetamido-5-oxo-8-methoxycarbonyl-1,2,3,5,6,7-hexa-
hydroindolizine-3-carbonyl-N-benzyl-N-methyltryptophan
amide (2b). The product 2b was dissolved in boiling
acetone. The salts were filteredandthe acetone solution
was evaporated under reduced pressure to give an oil
which was flash chromatographedon a 4 ꢃ40 cm col-
umn using CH2Cl2/acetone (7:3) as the eluting solvent.
The product obtained was recrystallized from acetone
as a white solid(740 mg) at 30% yield: mp >240 ꢂC;
TLC Rf [CH2Cl2/acetone (1:1)]=0.25; IR (KBr) n:
1634 cmꢀ1 (CO), 1697 cmꢀ1 (CO), 3303 cmꢀ1 (NH),
3373 cmꢀ1 (NH); 1H NMR (300 MHz, DMSO-d6) d:
(E)- and (Z)-5-Methoxycarbonylmethine-(RS)-prolyl-
N-3,5-bis(trifluoromethyl)benzyltryptophan amide (8a).
The product 8a was recrystallizedfrom CH 2Cl2 as a
white solid(1.36 g) at 71% yield: mp 190–194 ꢂC; TLC
Rf (ethyl acetate)=0.5; IR (KBr) n: 1630 cmꢀ1 (CO),
1
1650 cmꢀ1 (CO), 3210 cmꢀ1 (NH), 3320 cmꢀ1 (NH); H
NMR (300 MHz, DMSO-d6) d: 1.45–1.60 (m, 0.33H),

R. Millet et al. / Bioorg. Med. Chem. 10 (2002) 2905–2912
2911
1.40–1.60 (m, 1H), 1.87 (s, 3H), 2.02–2.15 (m, 1H),
2.30–2.73 (m, 2H), 2.76–2.80 (m, 4H), 3.00–3.20 (m,
3H), 3.62 (s, 1H), 3.65 (s, 2H), 4.38–5.14 (m, 5H), 6.84–
7.37 (m, 9H), 7.59 (d, 1H, J=7.6 Hz), 8.23 (d, 1H,
J=7.6 Hz), 8.72 (bs, 1H), 10.81 (s, 0.33H), 10.87 (s,
0.66H); MS (EI) m/z 585 (M+), 130 (Ind-CH2+). Anal.
(C32H35N5O6) C, H, N. Anal. calcdfor C 32H35N5O6: C,
65.63; H, 6.02; N, 11.96. Found: C, 65.37; H, 5.83; N,
11.84.
the cells were resuspended at 1 million/mL in a thermo-
statedquartz cuvette (3 mL). Cells were maintainedin
suspension by a magnetic stirrer. A spectrofluorometer
equippedfor dual excitation andmono emission wave-
length (Fluorolog, HORIBA group) was usedto record
the ratio of fluorescence emission intensity at 510 nm
resulting for alternative excitation of cells at 340 and
380 nM. The recorded ratios (6 per second) were then
2+
automatically convertedto cytosolic Ca
concentration
40
using the establishedequation describedby Grynkiewicz.
Schildplots were establishedfrom six activity curves
with concentrations of selectedcompounds 1a–c and 2a–c
(10ꢀ10–10ꢀ6 M) in three independent experiments.
6-Benzyloxycarbonylamino-5-oxo-8-methoxycarbonyl-
1,2,3,5,6,7-hexahydroindolizine-3-carbonyl-N-benzyl-N-
methyltryptophan amide (2c). The product 2c was dis-
solvedin boiling ethyl acetate. The salts were filtered
andthe ethyl acetate solution was evaporatedunder
reduced pressure to give an oil which was flash chroma-
tographedon a 4 ꢃ40 cm column using CH2Cl2/acetone
(75:25) as the eluting solvent. The product obtained was
isolatedas a white solid(285 mg) at 30% yield: mp 125–
135 ꢂC; TLC Rf [CH2Cl2/acetone (75:25)]=0.25; IR
(KBr) n: 1641 cmꢀ1 (CO), 1707 cmꢀ1 (CO), 3320 cmꢀ1
References and Notes
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1
(NH); H NMR (300 MHz, DMSO-d6) d: 1.20–2.45 (m,
5H), 2.50–2.82 (m, 4H), 2.85–3.20 (m, 2H), 3.60–3.79
(m, 3H), 4.20–5.20 (m, 7H), 6.80–7.37 (m, 14H), 7.50–
7.58 (m, 1H), 8.14–8.17 (m, 1H), 8.79–8.86 (bs, 1H),
10.85–10.93 (4s, 1H); MS (EI) m/z 677 (M+), 130 (Ind-
CH+2 ). Anal. calcdfor C 38H39N5O7: C, 67.34; H, 5.80;
N, 10.93. Found: C, 66.97; H, 5.92; N, 10.76.
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Binding assays
Binding experiments were performed according to stan-
dard techniques37 using clones of Chinese Hamster
Ovary (CHO) as the receptor source for both NK1 and
NK2 subtypes. Crude membranes were prepared and
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specific activities of 170 Ci/mmol (Amersham). Incu-
bation conditions were the following: Tris 50 mM, Mg
2 mM (final concentrations), pH 7.4 and additional
Bacitracine 160 mg/mL at 25 ꢂC for 1 h. The reaction was
terminatedby rapidvacuum filtration onto glass fiber
filters (GF/C Wathman presoaked2 h in PEI 0.1%):
after four 2 mL washes with Tris 50 mM at 4 ꢂC, pH 7.4,
the radioactivity trapped onto the filters was counted
and binding was calculated. Non-specific binding was
determined with additional non-radioactive Substance P
1 mM. Competition curves were fittedaccording to the
Cheng andPrussof equation (Kaleidagraph software,
Microsoft for Macintosh).38
Intracellular [Ca++]i measurements
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trifugedat 700 rpm during 10 min. After three washes,

2912
R. Millet et al. / Bioorg. Med. Chem. 10 (2002) 2905–2912
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