Design of selective peptidomimetic agonists for the human orphan receptor BRS-3

Article abstract of DOI:10.1021/jm0210921

New tool substances may help to unravel the physiological role of the human orphan receptor BRS-3 and its possible use as a drug target for the treatment of obesity and cancer. In continuation of our work on BRS-3, the solid- and solution-phase synthesis of a library of low molecular weight peptidomimetic agonists based on the recently developed short peptide agonist 4 is described. Functional potencies of the compounds were determined measuring calcium mobilization in a fluorometric imaging plate reader (FLIPR) assay. Focusing on the N-terminus, the D-Phe-Gln moiety of 4 was modified in a combinatorial SAR-oriented medicinal chemistry approach. With the incorporation of N-arylated glycine and alanine building blocks azaglycine, piperazine, or piperidine and the synthesis of semicarbazides and semicarbazones, a number of highly potent and selective compounds with a reduced number of peptide bonds were obtained, which also should have enhanced metabolic stability.

Full text of DOI:10.1021/jm0210921

1918
J . Med. Chem. 2003, 46, 1918-1930
Design of Selective P ep tid om im etic Agon ists for th e Hu m a n Or p h a n Recep tor
BRS-3#
Dirk Weber,† Claudia Berger,‡ Peter Eickelmann,‡,§ J ochen Antel,‡ and Horst Kessler*,†
Institut fu¨r Organische Chemie und Biochemie, Technische Universita¨t Mu¨nchen, Lichtenbergstrasse 4,
85747 Garching, Germany, and Solvay Pharmaceuticals GmbH, Hans-Bo¨ckler-Allee 20, 30173 Hannover, Germany
Received November 11, 2002
New tool substances may help to unravel the physiological role of the human orphan receptor
BRS-3 and its possible use as a drug target for the treatment of obesity and cancer. In
continuation of our work on BRS-3, the solid- and solution-phase synthesis of a library of low
molecular weight peptidomimetic agonists based on the recently developed short peptide agonist
4 is described. Functional potencies of the compounds were determined measuring calcium
mobilization in a fluorometric imaging plate reader (FLIPR) assay. Focusing on the N-terminus,
the D-Phe-Gln moiety of 4 was modified in a combinatorial SAR-oriented medicinal chemistry
approach. With the incorporation of N-arylated glycine and alanine building blocks azaglycine,
piperazine, or piperidine and the synthesis of semicarbazides and semicarbazones, a number
of highly potent and selective compounds with a reduced number of peptide bonds were obtained,
which also should have enhanced metabolic stability.
In tr od u ction
Data obtained from the knock-out mouse model impli-
cated BRS-3 with the regulation of energy balance,
body weight, and blood pressure because mice lacking
functional BRS-3 developed mild obesity, hypertension,
and diabetes.13 Second, it has been observed that
the BRS-3 receptor is expressed on several human
carcinoma cell lines.11,14 In general, bombesin-like
peptides (BLPs) are involved in the growth regulation
of various cancers, especially small-cell lung cancer
(SCLC) cell lines.15 Expression of BRS-3 in mammals,
as determined by distribution of BRS-3 mRNA, is
species-dependent and limited16,17 compared to the
widely expressed NMB-R and GRP-R.18 Very recently,
BRS-3 was connected with the treatment of neuro-
logical disorders.19 Despite the development of the high-
affinity ligand [D-Tyr6,â-Ala11,Phe13,Nle14]Bn(6-14) (1)
and its analogues,20 which allowed receptor pharma-
cology studies,20,21 more tool substances are needed
to find out about the possible usefulness of BRS-3 as a
drug target. Recently, we and others22,23 performed
structure-activity relationship studies of 1 mainly by
utilizing its ability to mobilize intracellular calcium in
BRS-3 transfected CHOGR-16 cells in a FLIPR assay.
With the obtained information, we applied a strategy
similar to the “peptoid” approach,24 which was used to
design peptidomimeticantagonists for severalGPCRs25-27
and subsequent C-terminal optimization to develop the
selective short peptide agonist 4 (Table 1 and Figure 1)
for the human BRS-3 receptor.22 In continuation of this
work, we describe the solid- and solution-phase synthe-
sis of a library of low molecular weight peptidomimetic
BRS-3 agonists based on the short peptide agonist 4,
which should have enhanced metabolic stability. Besides
the incorporation of peptoid building blocks,28-32 the
synthesized BRS-3 agonists include a number of aza-
peptides,33 a class of compounds that was successfully
developed into drugs,33-35 semicarbazides, semicarba-
zones, and compounds containing a piperazine/piperi-
dine scaffold (Figure 1).
G-protein-coupled receptors (GPCRs) represent the
largest and most important class of all drug targets.1
Currently, worldwide more than 50% of all drugs are
GPCR-based and their annual sales exceeded 30 billion
U.S. dollars in 2001.2 Owing to this proven track of
being excellent drug targets, it is commonly assumed
that orphan GPCRs, which emerged from genomic
research, will offer a similar aptitude in the future.3,4
About 1-3% of our genome encode approximately 1000
GPCRs, a number that was until recently predicted
to be much larger.5 Excluding sensory receptors, there
are presently only about 150 orphan GPCRs.4 Although
the orphan receptor strategy and reverse pharma-
cology approach6 have led to the identification of many
natural ligands in recent years, the proportion of
available orphan receptor sequences that have been
“deorphanized” in this manner remains relatively small.
New approaches such as the constitutively activating
receptor technology (CART) are gaining increasing
attention,7 also because it is now a generally accepted
concept that various ligands can bind to different
binding sites to have different effects on GPCRs.7-9 The
identification of a natural ligand is an important step
in the target validation of orphan GPCRs;10 however,
in the case of the orphan receptor bombesin receptor
subtype 3 (BRS-3), a naturally occurring high-affinity
ligand is still unknown. The BRS-3 receptor was as-
signed to the bombesin (Bn) receptor familiy because
of its high sequence homology with the two mammalian
bombesin receptors neuromedin B receptor (NMB-R)
and gastrin-releasing peptide receptor (GRP-R).11,12
#
Dedicated to Prof. George M. Sheldrick on the occasion of his 60th
birthday.
* To whom correspondence should be addressed. Phone: ++49+89/
289-13300. Fax: ++49+89/289-13210. E-mail: kessler@ch.tum.de.
† Technische Universita¨t Mu¨nchen.
‡ Solvay Pharmaceuticals GmbH.
§ Present address: Boehringer Ingelheim Pharma KG, Biberach an
der Riss, Germany.
10.1021/jm0210921 CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/15/2003

Design of Peptidomimetic Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1919
Ta ble 1. Functional Potencies from 4 to 30 Independent Concentration-Response Curves of [D-Phe6,â-Ala11,Phe13,Nle14]Bn(6-14) (1),
Endogenous Ligands NMB (2) and GRP (3), and the Synthetic Agonist 4 in CHO Cells Transfected with the Human Bombesin
Receptors NMB-R, GRP-R, and BRS-3a
EC50 [nM]
compd
peptide
NMB-R
GRP-R
BRS-3
1
2
3
4
[D-Phe6,â-Ala11,Phe13,Nle14]Bn(6-14)
310 (260-380)
120 (91-150)
3200 (2400-4400)
inactive
84 (69-100)
41 (36-47)
34 (24-49)
inactive
151 (134-170)
NMB
4500 (4100-4900)
6400 (6100-6700)
710 (590-860)
GRP
fQw-1-(2-phenylethyl)amide
a
For further experimental details see Table 2 or Supporting Information.
previously described.37 Chain extension including the
coupling of the N-terminal building blocks 7a -c,g38-41
was carried out using standard Fmoc protocols42 with
TBTU/HOBt/DIEA activation.43 Peptides with an addi-
tional substitution of Gln by Ala (16a -g) were prepared
similarly (Scheme 1).
In ser t ion of N-Su b st it u t ed Glycin e/Ala n in e
(Com p ou n d s 17a -d a n d 18a -c). In the second step,
the D-Phe-Gln unit was replaced with N-substituted
glycines and alanines. All Fmoc-protected peptoid
monomers, except 11c, were synthesized in two steps.
First, 2-(arylalkylamino)acetic acid ethyl esters (8a ,b,d )
and 2-(arylalkylamino)propionic acid ethyl esters
(10a -c) were prepared in a modified, previously de-
scribed procedure.44 Then, the esters were hydrolyzed
with aqueous NaOH and Fmoc was introduced with-
out intermediate purification to yield building blocks
11a ,b ,d and also 12a -c, which were obtained as
racemic mixtures. 2-{Pyridine-3-ylmethyl[(9H-fluorene-
9-ylmethoxy)carbonyl]amino}acetic acid (11c) was syn-
thesized in three steps45 via the tert-butyl ester 8c,
which was Fmoc-protected to give 9. Final cleavage of
the tert-butyl group was carried out with TFA/TIPS
(10:1) (v/v). Assembly of the peptoid-peptide hybrids
17a -d was carried out on solid support36 using TBTU/
HOBt/DIEA activation43 (Scheme 1). For synthesis of
compounds 18a -c, Fmoc-protected peptoid monomers
12a -c were coupled to 14 in solution with HATU/HOAt/
collidine activation46 (Scheme 1). Cleavage of the Boc-
protecting group from D-Trp turned out to be incomplete
after treatment with TFA/TIPS (10:1) (v/v) at 0 °C. ESI
mass spectral data indicated that the carbaminic acid
was stable under these highly acidolytic water-free
conditions.47 Destruction of the carbaminic acid was
achieved by treatment with DMSO/H2O/HOAc (8:1:1)
(v/v). Compounds 18a -c were tested as racemic mix-
tures.
F igu r e 1. Development of peptidomimetic BRS-3 agonists
from lead structure 4. The marked region of structure 4 shows
the previously optimized part of the molecule, which was left
unchanged in this study.
Ch em istr y
Gen er a l Str a tegy. Recently, we described the design
of the short peptide agonist H-D-Phe-Gln-D-Trp-1-(2-
phenylethyl)amide (4).22 Previous results suggested that
the side chain of the Gln residue is not essential for
functional activity; however, all three aromatic moieties
are essential.22 Furthermore, a stereochemical change
of the N-terminal D-Phe did not affect functional po-
tency.22 Consequently, our work was directed toward the
N-terminal part of the molecule while leaving the
C-terminal part D-Trp-1-(2-phenylethyl)amide, which
already has been optimized,22 unchanged. We tried to
modify the residues D-Phe-Gln step by step, to elaborate
the structural features of this part of the agonist
required for biological activity, to reduce it to a mini-
mum fragment, and moreover to replace it with pro-
teolytically more stable surrogates.
Rem ova l of N-Ter m in a l NH2 a n d Sid e Ch a in of
th e Gln (Com p ou n d s 15a-d , 16a -g). First, to remove
the N-terminal chirality and increase lipophilicity,
peptides with removed N-terminal amino function (15a-
d ) were prepared (Scheme 1).36 For synthesis, FMPE
resin was reductively aminated and loaded with Fmoc-
D-Trp-OH to yield 13 in a procedure similar to the one
Syn th esis of Aza p ep tid es 21a -e. In the next step,
Gln was replaced by azaglycine. Numerous routes have
been described for the synthesis of azapeptides in
solution,33 but besides the synthesis of azatides,48 only
a few were reported for the preparation on solid
support.49-51 Other methods suffer from major draw-
backs such as hydantoin formation and a slow reaction
rate50 or give rise to considerable amounts of byproducts
such as the in situ activation of Fmoc-hydrazine with
triphosgene.51,52a To overcome these drawbacks, we
incorporated azaglycine in the solid-phase synthesis of
azapeptides 21a -e using an excess of freshly prepared
5-(9H-fluoren-9-ylmethoxy)-1,3,4-oxadiazol-2(3H)-one
(19).52 Therefore, 13 was Fmoc-deprotected and reacted
with 19, which was obtained from the activation of
Fmoc-hydrazine with an excess of a solution of phosgene

1920 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Weber et al.
Sch em e 1a
a
Reagents: (a) (only 13, 20% piperidine/NMP), Fmoc-Gln(Trt)-OH or Fmoc-Ala-OH, TBTU/HOBt/DIEA, NMP; (b) 20% piperidine/
NMP; (c) carboxylic acid building blocks 7a -g (compounds 7a -e were purchased from commercial sources), TBTU/HOBt/DIEA, NMP;
(d) TFA/TIPS/H2O (18:1:1); (e) {arylalkyl-[9H-fluoren-9-ylmethoxy)carbonyl]amino}acetic acid (11a -d ), TBTU/HOBt/DIEA, NMP; (f)
(1) 2-{arylalkyl-[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propionic acid (12a -c), HATU/HOAt/collidine, DMF, (2) TFA/CH2Cl2/TIPS
(10:10:1), (3) DMSO/HOAc/H2O (8:1:1), (4) 20% piperidine/DMF (65-87%).
52
in toluene as previously described
(Scheme 2). Dia-
to convert these building blocks into acylating agents
selectively at the unsubstituted nitrogen atom of the
hydrazine after Boc deprotection in the following steps.
Because the site of acylation is dependent on the nature
of the substituted hydrazine and the activation reagent
and therefore was difficult to predict,48,52a,54 Fmoc was
introduced at the arylalkylated nitrogen to give building
blocks 27a -c. Preliminary attempts to cleave the Boc
protecting group were carried out by careful treatment
with TFA/CH2Cl2/TIPS/H2O (4:14:1:1) (v/v) at 0 °C and
showed approximately 70% deprotection after 1 h as
monitored by TLC and HPLC-MS. However, we ob-
served unexpected degradation of the dried deprotected
species at room temperature within 24 h. Surprisingly,
attempts to remove the Boc with 4 N HCl in dioxane as
a solvent at room temperature35a failed because of
insufficiently low reaction rates. Therefore, it was
decided to cleave the Boc group of 27a -c under more
drastic conditions than aforementioned with TFA/CH2-
Cl2/TIPS (20:20:1) (v/v). Without further workup, acti-
vated carbazic acid esters were immediately generated
using bis(pentafluorophenyl)carbonate and without iso-
lation instantly reacted with the amine 14.48,56 This
method worked very well and was found to be superior
to the use of a solution of phosgene in toluene,52a which
seems to require more subtle adjustment of the reaction
conditions. Direct cleavage of Boc and Fmoc without
intermediate purification finally furnished the desired
semicarbazides 28a -c (Scheme 3).
stereomers 21d 1 and 21d 2 were separated by HPLC.
Syn th esis of Sem ica r ba zon es 24a -d . Analogues
of the azapeptides 21 lacking an N-terminal peptide
bond and a C-N double bond were prepared in solution
(Scheme 2). Acylation of amine 14 with freshly prepared
oxadiazolone 19 in DMF at room temperature resulted
in aza compound 20, which was then Fmoc-deprotected
and without intermediate purification reacted with
aldehydes 22a -d in THF at room temperature. Imine
bond formation was slow probably because of residual
basicity; however, since the desired compounds 23a -d
were obtained in acceptable yields, optimization of this
process was not pursued. Aldehydes 22a -c were pur-
chased from commercial sources, whereas 4-(2-thienyl)-
benzaldehyde (22d ) was synthesized from 4-bromo-
benzaldehyde via Suzuki coupling.53 Final Boc de-
protection under first strong and then mild acidic
conditions gave semicarbazones 24a -d .
Syn th esis of Sem ica r ba zid es 28a -c. The synthesis
of analogues of azapeptides 21 lacking an N-terminal
peptide bond afforded the preparation of suitably pro-
tected azaamino acid constituents (Scheme 3). There-
fore, Boc-hydrazine was reacted to the Boc-protected
hydrazones 25a -c with the appropriate aldehydes in
THF.54 Contrary to previous reports, no conversion was
observed when reduction of 25a was attempted through
hydrogenation over 5% Pd/C in THF as a solvent at
room temperature,54 both at atmospheric pressure or
at 50 atm. However, reduction with sodium cyanoboro-
hydride55 afforded the desired Boc-protected arylalkyl-
hydrazines 26a -c in good yields. It was further planned
Sca ffold s Con t a in in g P ip er id in e/P ip er a zin e
(Com p ou n d s 35 a n d 36). Building blocks (4-benzyl-
piperazin-1-yl)acetic acid (31) and its piperidine ana-

Design of Peptidomimetic Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1921
Sch em e 2a
a
Reagents: (a) 5-(9H-fluoren-9-ylmethoxy)-1,3,4-oxadiazol-2(3H)-one (19), CH2Cl2 (68%, for R1 ) H); (b) 20% piperidine/NMP; (c)
carboxylic acid building blocks 7a ,b,e-g (compounds 7a ,b,e were purchased from commercial sources), TBTU/ HOBt/DIEA, NMP; (d)
TFA, TIPS, H2O (18:1:1); (e) (1) 20% piperidine/DMF, (2) arylaldehydes 22a -d (compounds 22a -c were purchased from commercial
sources), THF (47-83%); (f) (1) TFA/CH2Cl2/TIPS (30:30:1), (2) DMSO/HOAc/H2O (8:1:1) (56-63%).
Sch em e 3a
for the synthesis of 18a -c yielded compounds 35
and 36.
Resu lts a n d Discu ssion
The synthesized library is derived from the structure
of the recently published BRS-3 agonist 4, which was
obtained from the C-terminal optimization of the lead
structure H-D-Phe-Gln-D-Trp-Phe-NH2.22 Functional ac-
tivity of the compounds was assessed in a FLIPR assay.
Therefore, their ability to increase the level of intra-
cellular calcium in CHO cells transfected with the
human bombesin receptors NMB-R, GRP-R, and BRS-3
was measured and referenced to the peptide agonist
[D-Phe6,â-Ala11,Phe13,Nle14]Bn(6-14) (1) for BRS-3, en-
dogenous ligands NMB (2) for NMB-R, and GRP (3) for
GRP-R. Further experimental details of this assay have
been published earlier22 and are given in the Supporting
Information.
One of the most reliable methods in medicinal chem-
istry to improve in vitro activity is to incorporate
properly positioned lipophilic groups or, as could be
suggested for our case, to remove “unnecessary” hydro-
philic groups. Although C-terminal optimization of the
aforementioned lead structure, which resulted in the
removal of the amide function, was sufficient to increase
functional potency about 15-fold to generate agonist 4
with an EC50 of 710 nM,22 we hoped that further
improvement of functional activity could be achieved by
exploiting the N-terminal SAR of this molecule. On the
other hand, we were aiming at oral availability, which
requires solubility. Therefore, regarding Lipinski’s “rule
of five”,57 lipophilicity, which can be estimated and
a
Reagents: (a) arylaldehydes 22a -c (compounds were pur-
chased from commercial sources), THF (82-96%); (b) (1) NaC-
NBH3, THF, AcOH, (2) NaHCO3, (3) 1 N NaOH, MeOH (71-93%);
(c) Fmoc-Cl, NaHCO3, dioxane (72-98%); (d) (1) TFA/CH2Cl2/TIPS
(20:20:1), (2) tert-butyl-3-{(2R)-2-amino-2-[1-(2-phenylethyl)car-
bamoyl]ethyl}-1H-1-indolecarboxylate (14), bis(pentafluorophenyl)-
carbonate, DMAP, CH2Cl2, (3) TFA/TIPS (40:1), CH2Cl2, (4) 20%
piperidine/DMF (9-21%).
logue 32 were prepared in two steps similar to that
described for 8a -d followed by saponification. After
coupling to amine 14 using TBTU/HOBt/DIEA activa-
tion, Boc deprotection carried out as described above

1922 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Weber et al.
Ta ble 2. Functional Potencies from 4 to 11 Independent Concentration-Response Curves of Modified Analogues of 4 with
N-Terminally Deleted Amino Function (15a -d )a
BRS-3
compd
R1
R2
EC50 [nM]
Emax
cLogP
15a
15b
15c
15d
16a
16b
16c
16d
16e
16f
16g
(CH2)2CONH2
(CH2)2CONH2
(CH2)2CONH2
(CH2)2CONH2
CH3
CH3
CH3
CH3
CH3
benzyl-
4-chlorobenzyl-
1-[2-(4-chlorophenyl)ethyl]-
1H-indole-2-ylmethyl-
benzyl-
1200 (680-2100)
1400 (990-2000)
230 (170-320)
57 (26-120)
38 ( 0.8
62 ( 3.8
68 ( 5.9
82 ( 4.7
71 ( 4.7
75 ( 8.5
30 ( 2.2
75 ( 7.0
74 ( 1.6
69 ( 8.1
69 ( 1.2
1.81
2.52
2.91
1.8
3.62
4.33
4.72
3.18
2.12
3.83
3.61
2.1 (1.2-3.9)
4-chlorobenzyl-
140 (75-270)
6400 (5800-7200)
7.8 (2.8-22)
32 (23-44)
310 (250-390)
79 (50-120)
1-[2-(4-chlorophenyl)ethyl]-
1,3-benzodioxol-5-ylmethyl-
pyridine-3-ylmethyl-
1,2,3,4-tetrahydroiso- quinoline-1-ylmethyl-
1H-indole-2-ylmethyl-
CH3
CH3
a
Additionally, in compounds 16a -g, Gln was replaced by Ala. Functional potencies of the peptides were determined in a previously
described FLIPR assay22 (see also Supporting Information) and are given as EC50 ( SEM.63 Emax represents the Ca2+ signal at 16 µM in
percent of maximal response of 1. All compounds tested were inactive on the NMB-R and GRP-R except for 15a , with EC50 ) 15.0 µM
(13.9-16.3) on NMB-R and EC50 ) 19.1 µM (15.5-23.5) on GRP-R, and for 15b, with EC50 ) 18.5 µM (16.6-20.7) on NMB-R and EC50
) 12.2 µM (11.1-13.3) on GRP-R. For further experimental details, see Supporting Information.
expressed by the n-octanol/water partition coefficient
cLogP value, should not exceed the magnitude of five.
First, we deleted the N-terminal aminofunction of the
agonist 4 (Scheme 1, Figure 1). As a result of this, the
cLogP value increased from 1.3 to 1.81 (Table 2),
compared to the augmentation of 0.1 of H-D-Phe-Gln-
D-Trp-Phe-NH2 to 1.3 of agonist 4. As shown in Table 2
(compounds 15a -d ), the effect of this increase of lipo-
philicity on functional potency at BRS-3 is low except
for compound 15d , with an increase of about 12-fold.
Compounds 15a and 15b showed functional potencies
on the NMR-R and GRP-R in the micromolar range.
In the second step, a series of compounds, where in
addition to the deleted amino function Gln was replaced
by Ala and the N-terminal aromatic moiety was differ-
ently substituted or replaced by heteroaromatic moi-
eties, were investigated (Table 2). Although the syn-
thetic effort to get from lead structure H-D-Phe-Gln-D-
Trp-Phe-NH2 to compounds 16 was relatively small, the
impact on improvement of functional potency was
enormous. With compounds 16a and 16d , potencies in
the nanomolar range were obtained. Furthermore, all
compounds showed excellent selectivity for BRS-3. As
seen in the preceding series, for compounds 15a -d ,
indole-2-yl/3-pyridyl was clearly favored as the N-
terminal aromatic moiety over 4-chlorophenyl. On the
other hand, these alterations probably do not represent
much progress concerning proteolytic stability, since
these compounds still comprised three peptide bonds.
Therefore, efforts were directed toward a reduction of
the peptidic character of the compounds.
Ta ble 3. Functional Potencies from 7 to 11 Independent
Concentration-Response Curves of Modified Analogues of 4
Containing Peptoid Monomer Building Blocks (17a -d ) and
N-Arylated 2-Aminopropionic Acid Building Blocks (18a -c)a
BRS-3
compd R1
R2
EC50 [nM]
Emax
cLogP
17a
17b
17c
17d
18a
18b
18c
H
H
H
H
benzyl-
4-chlorobenzyl-
pyridine-3-ylmethyl- 21 (12-36)
1-(2-phenylethyl)-
21 (14-31)
87 ( 6.4
3.86
4.58
2.37
2.9 (1.2-6.9) 81 ( 1.7
82 ( 8.4
17 (8.9-31) 92 ( 16.0 4.08
46 (34-64) 86 ( 12.8 4.17
4.0 (2.3-7.3) 83 ( 12.5 4.89
25 (19-34) 82 ( 9.6 4.39
CH3 benzyl-
CH3 4-chlorobenzyl-
CH3 1-(2-phenylethyl)-
a
All compounds tested were inactive on the NMB-R and GRP-
R. For further experimental details, see Table 2 or Supporting
Information. Emax represents the Ca2+ signal at 16 µM in percent
of maximal response of 1.
Here, in contrast to the preceding series, the 4-chloro-
substituted compounds 17b and 18b showed the highest
functional potency with EC50 values around 3-4 nM.
The explanation for this shift of high functional potency
could probably be that a high lipophilicity is favorable
but only at the correct distance from the tryptophan.
This distance is obviously too large for compounds 15
and 16. Moreover, a basic functionality located more
closely to the tryptophan seems to enhance functional
activity. The overall series of compounds 17 and 18
demonstrated that the carbonyl function or the planar-
ity caused by the peptide bond was not contributing to
the binding mode of the compound to the receptor and
therefore could be omitted.
In compounds 17a -d the N-terminal peptide
bond was removed by incorporation of N-substituted
glycine, and in compounds 18a -c it was removed by
N-substituted alanine (Table 3). Thus, it is assumed
that these modifications introduce a greater flexibility
to the backbone accompanied by enhanced metabolic
stability.28-31 Deletion of the carbonylfunction caused
cLogP values to slightly increase by about 0.2-0.7.
Moreover, we attempted to exchange CRH by NR,
which leads to a loss of one chiral center and to a

Design of Peptidomimetic Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1923
Ta ble 4. Functional Potencies of Azapeptides 21a -e from 7 to
14 Independent Concentration-Response Curvesa
Ta ble 5. Functional Potencies of Semicarbazones 24a -d from
6 Independent Concentration-Response Curvesa
BRS-3
compd
R
EC50 [nM]
Emax
cLogP
compd
R
EC50 [nM]
Emax
cLogP
21a
21b
21c
benzyl-
4-chlorobenzyl-
pyridine-3-ylmethyl-
3.1 (2.1-4.6)
86 ( 9.5
3.61
4.33
0.19 (0.06-0.58) 89 ( 13.5
24a
24b
24c
24d
phenyl-
240 (110-490)
2700 (1500-5200)
1.5 (0.7-3.2)
89 ( 0.4 4.21
78 ( 0.1 4.92
94 ( 2.6 3.38
1.4 (0.7-2.5)
102 ( 26.1 2.12
4-chlorophenyl-
2-furyl-
4-(2-thienyl)phenyl- 7900 (3800-16200) 58 ( 7.3 5.98
21d 1 1,2,3,4-tetrahydroiso- 9.3 (7.1-12.3)
95 ( 15.0
91 ( 15.1
84 ( 4.3
3.83
3.83
3.6
21d 2 quinoline-1-ylmethyl- 19 (15-25)
21e
1H-indole-2-ylmethyl- 2.2 (0.8-5.9)
a
All compounds tested were inactive on the NMB-R and GRP-
R. For further experimental details see Table 2 or Supporting
Information. Emax represents the Ca2+ signal at 16 µM in percent
of maximal response of 1.
a
All compounds tested were inactive on the NMB-R and GRP-
R. For further experimental details, see Table 2 or Supporting
Information. Emax represents the Ca2+ signal at 16 µM in percent
of maximal response of 1.
Ta ble 6. Functional Potencies of Semicarbazides 28a -c from 6
Independent Concentration-Response Curvesa
conformational change.58 Nevertheless, there are many
examples where amino acids have been successfully
replaced by their corresponding azaamino acids with
retention of biological activity accompanied by an
increase of metabolic stability.35,49,52 Ab initio calcula-
tions showed that diacylhydrazine has relatively easy
access to different conformations; however, its flexibility
was substantially constrained by N-methylation.58a It
is generally believed that a critical amount of rigidity
is a prerequisite for high activity and selectivity.59 On
the other hand, scaffolds bearing azaglycine as their core
and structurally dominating unit demonstrated posses-
sion of these features.52 Obviously the same proves to
be true in this case. All azapeptides show excellent
activities and selectivities, especially compound 21b
with an EC50 value in the subnanomolar range (Table
4). Although the distance to the lipophilic 4-chloro-
substituted moiety seems to be larger than in com-
pounds 17 and 18 with the removed peptide bond, it is
feasible that the azaglycine induces a bentlike structure.
Therefore, the actual length of the compound in its
bioactive conformation could resemble that of the pep-
toid-peptide hybrids.
BRS-3
compd
R
EC50 [nM]
Emax
cLogP
28a
28b
28c
benzyl-
4-chlorobenzyl-
furyl-2-ylmethyl- 24 (18-33)
15 (8.5-25)
81 ( 0.4
3.9
4.62
3.08
6.0 (2.3-16) 83 ( 5.3
96 ( 14.3
a
All compounds tested were inactive on the NMB-R and GRP-
R. For further experimental details, see Table 2 or Supporting
Information. Emax represents the Ca2+ signal at 16 µM in percent
of maximal response of 1.
Ta ble 7. Functional Potencies of Compounds 35 and 36 from 6
Independent Concentration-Response Curvesa
In the next step, we investigated the necessity of the
N-terminal peptide bond in the aza compounds. Func-
tional potencies of the semicarbazides 28a -c were lower
than those of the corresponding azapeptides 21a -e but,
as expected, were still excellent (Table 6). As for the
peptoid-peptide hybrids, removal of the carbonyl group
caused the cLogP values to increase about 0.3 order of
magnitude, and the 4-chloro-substituted compound 28b
showed the highest activity. Insertion of a C-N double
bond (semicarbazones 24a -d , Table 5) caused another
increase of the cLogP value of about 0.3. This increase
was tolerated for 2-furyl as the aromatic moiety but not
for too lipophilic residues. In the latter, the drop of
functional activity correlated with increasing cLogP
value, and because of the high activity of 24c, it
probably was not conformationally related. Taken to-
gether, the results confirm those obtained from the
peptoid-peptide hybrids 17 and 18, namely, that the
N-terminal peptide bond is not involved in receptor
activation.
BRS-3
compd
Z
EC50 [nM]
Emax
cLogP
35
36
N
C
70 (57-85)
30 (4.9-190)
90 ( 1.2
84 ( 5.3
4.98
5.85
a
Compounds tested were inactive on the NMB-R and GRP-R.
For further experimental details, see Table 2 or Supporting
Information. Emax represents the Ca2+ signal at 16 µM in percent
of maximal response of 1.
high activities and selectivities were obtained too;
however, the cLogP value clearly exceeded the upper
limit of 5.
Additional FLIPR experiments were carried out
to assess the selectivity of the new agonists to the
BRS-3 receptor over the NMB and the GRP receptors.
The ability of compounds 15-18, 21, 24, 28, 35, and
36 to inhibit receptor activation of the agonists
NMB at NMB-R and GRP at GRP-R was investigated.
Finally compounds with incorporated piperazine (35)
and piperidine (36) have been tested (Table 7). Here,

1924 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Weber et al.
As a reference, the known antagonists [D-Phe12]Bn,
[D-Phe6,Leu13,p-chloro-Phe14]Bn(6-14), and [D-Phe6,Leu-
NHEt13,desMet14]Bn(6-14) were used.60-62 First, a
solution of the new agonists and reference antagonists
at eight different concentrations were added to CHO
cells transfected with the NMB or GRP receptor followed
by a solution of the agonist, and calcium mobilization
was permanently measured. In all measurements, no
reduction of the calcium emission induced by the pres-
ence of the aforementioned compounds could be ob-
served. Thus, we conclude that the investigated com-
pounds are not antagonists of the NMB-R and GRP-R.
At present, we are unable to explain the selectivity
of the compounds for the BRS-3 receptor.
Perseptive Biosystems (Hamburg, Germany), and TBTU and
HOBt from Quantum Appligene (Heidelberg, Germany). TFA
was obtained from Solvay (Hannover, Germany). Bis(penta-
fluorophenyl)carbonate was purchased from Senn Chemicals
(Dielsdorf, Switzerland) and 3-(4-chlorophenyl)propionic acid
from CPS Chemie + Service (Du¨ren, Germany). All other
chemicals were purchased from Aldrich (Deisenhofen, Ger-
many), Merck (Darmstadt, Germany), Lancaster (Mu¨hlheim,
Germany), or Fluka (Seelze, Germany). Solid-phase synthesis
was carried out in PE syringes (2, 5, or 20 mL) from Becton-
Dickinson (Fraga, Spain) or Braun (Melsungen, Germany)
equipped with PE frits from Roland Vetter Laborbedarf
(Ammerbuch, Germany). Air- or moisture-sensitive reactions
were carried out in dry glassware and under argon (99.996%)
atmosphere. There was no attempt to optimize yields. Thin-
layer chromatography was performed on TLC aluminum
sheets covered with silica gel 60 F254 from Merck (Darmstadt).
For flash chromatography, silica gel 60 (230-400 mesh ASTM,
Korngro¨sse 0.040-0.063 mm) from Merck (Darmstadt) was
used. Melting points were determined on a Bu¨chi 510 melting
point apparatus and are uncorrected. 1H and 13C NMR spectra
were recorded in CDCl3, DMSO-d6, or ACN-d3 as solvent on
Bruker AC250 and DMX500 instruments at 300 K. TMS or
the solvent peak was used as internal reference. Chemical
shifts (δ) are reported in parts per million (ppm), and coupling
constants (J values) are given in Hertz (Hz). Purification of
compounds 15-18, 21, 23d , 24, 28, 35, and 36, analyses, and
analytical purity determination were carried out on RP-HPLC
instruments from Amersham Pharmacia Biotech or Beckman
(System Gold), equipped with Omicron YMC columns (pre-
parative, ODS-A C18, 250 mm × 30 mm, 10 µm, flow rate of
25 mL/min; semipreparative, ODS-A C18, 250 mm × 20 mm,
Con clu sion s
In summary, we have performed detailed N-terminal
structure-activity studies on the known BRS-3 agonist
H-D-Phe-Gln-D-Trp-1-(2-phenylethyl)amide (4). On the
basis of previous results, which suggested that the side
chain of Gln is not essential for functional potency, we
developed a library of peptidomimetics with a conserved
C-terminal D-Trp-1-(2-phenylethyl)amide moiety and
structural variations on the H-D-Phe-Gln unit. It was
demonstrated that the N-terminal increase of lipophi-
licity by simple deletion of the amino function combined
with removal of the Gln side chain furnished selective
BRS-3 agonists in the nanomolar range. Furthermore,
substitution of the H-D-Phe-Gln unit by peptoid mono-
mers showed that the N-terminal peptide bond is not
required for receptor activation. Gln can further be
replaced by azaglycine, leading to compounds with
subnanomolar activities (21b : EC50 ) 0.19 nM). For
azapeptides, too, a removal of the N-terminal peptide
bond is possible, as realized in the semicarbazides 28a -
c. However, as shown by the semicarbazones 24a -d , a
too lipophilic N-terminus does not seem to be tolerated.
The finding that piperidine or piperazine can also be
incorporated suggests that a large number of different
spacers are able to mimic the function of the former Gln
at this position. From the analysis of the substitution
and distance pattern for the different series of com-
pounds, it seems very likely that a lipophilicity placed
at a certain distance from the tryptophan combined with
a basic functionality located between them is favorable
for functional potency. A further FLIPR experiment in
which the ability of the described peptidomimetics to
inhibit activation of the NMB-R and GRP-R by their
natural ligands was investigated, showed that these
compounds are not antagonists of the NMB-R and GRP-
R. This work therefore describes the development of
selective tool substances for BRS-3 with improved
pharmacokinetic properties, which may prove to be
helpful in the understanding of the physiological role
of this orphan receptor.
5 µm or 10 µm, flow rate of 8 mL/min; analytical, ODS-A C18
,
250 mm × 4.6 mm, 5 µm, flow rate of 1 mL/min) or a
Macherey-Nagel column (preparative, Nucleosil C18, 250 mm
× 40 mm, 7 µm, flow rate of 25 mL/min). Compounds were
eluted with linear gradients (30 min) of acetonitrile in water
and 0.1% (v/v) trifluoroacetic acid, detected at 220 nm, and
lyophilized after purification. ESI mass spectra were obtained
on a LCQ Finnigan mass spectrometer, and GC mass spectra
were obtained on a Finnigan MAT 8200 instrument. High-
resolution mass spectra were recorded on a Finnigan spec-
trometer using the electrospray ionization time-of-flight (ESI-
TOF) technique. cLogP values were calculated using Sybyl 6.8
from Tripos Inc.
Syn t h esis of N1-(2-P h en ylet h yl)-(2R)-2-[(9H-flu or en -
9-ylm eth oxy)ca r boxa m id o]-3-[1-(ter t-bu toxyca r bon yl)-3-
in d olyl]p r op a n a m id e on F MP E Resin (13).6 FMPE resin
(2.378 g, theoretical 0.5 mmol/g, 1.19 mmol) was preswollen
in DCE (24 mL) for 10 min. Then TMOF (12 mL), 1-(2-
phenylethyl)amine (11.89 mmol), and NaBH(OAc)3 (2.52 g,
11.89 mmol) were added and the mixture was submitted to
an ultrasonicator for 10 min and then shaken overnight at
room temperature. After the mixture was washed with CH2-
Cl2 (3 × 20 mL) and NMP (3 × 20 mL), Fmoc-D-Trp-OH (2.38
mmol, 2 equiv) was coupled to the resin using [HATU (2
equiv)]/[HOAt (2 equiv)]/[collidine (20 equiv)] activation in
NMP (20 mL) for 5 h. Washing with NMP (3 × 20 mL) was
followed by a double-coupling step overnight. Finally the resin
was washed with NMP (3 × 20 mL) and CH2Cl2 (3 × 20 mL)
and dried thoroughly in vacuo to give resin-bound N1-(2-
phenylethyl)-(2R)-2-[(9H-fluoren-9-ylmethoxy)carboxamido]-3-
[1-(tert-butoxycarbonyl)-3-indolyl]propanamide (13).
N1-{(1R)-2-(1H-3-In d olyl)-1-[1-(2-p h en yleth yl)ca r ba m -
oyl]et h yl}-(2S)-2-(b en zylca r b oxa m id o)p en t a n ed ia m id e
(15a ). Resin-bound 13 (124 mg, 0.315 mmol/g, 0.039 mmol)
was treated two times with 20% piperidine (v/v) in NMP (5
mL) for 15 min and washed after each time with NMP (5 × 5
mL). Then Fmoc-Gln(Trt)-OH (48 mg, 0.078 mmol) dissolved
in NMP (2 mL) was coupled two times to the resin for 45 min
using [TBTU (2 equiv)]/[HOBt (2 equiv)]/[DIEA (5.7 equiv)]
activation. Coupling steps were followed by a washing step
with NMP (5 × 5 mL). After repeated Fmoc removal and
coupling of 7a (10.6 mg, 0.078 mmol), the resin was washed
Exp er im en ta l Section
Gen er a l. Fmoc-protected amino acids and 2-(4-formyl-
3-methoxyphenoxy)ethylpolystyrene (FMPE) resin was
purchased from Novabiochem (Darmstadt, Germany). [D-
Phe6,â-Ala11,Phe13,Nle14]Bn(6-14) (1) was purchased from
Polypeptide Laboratories (Wolfenbu¨ttel, Germany). NMB (2),
GRP (3), [D-Phe12]Bn, [D-Phe6,Leu13,p-chloro-Phe14]Bn(6-14),
and [D-Phe6,Leu-NHEt,13desMet14]Bn(6-14) were obtained
from Bachem (Heidelberg, Germany), HATU and HOAt from

Design of Peptidomimetic Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1925
with CH2Cl2 (3 × 5 mL) for 30 min and the compound was
deprotected and cleaved from the resin with [TFA (18 equiv)]/
[TIPS (1 equiv)]/[H2O (1 equiv)] (3 × 2 mL, 30 min each). The
combined filtrates were reduced in vacuo. RP-HPLC purifica-
tion yielded 11.1 mg of 15a : colorless powder; mp 226-227
°C; 1H NMR (500 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.30 (d, 1H,
J ) 7.1 Hz), 8.18 (d, 1H, J ) 8.3 Hz), 7.95 (t, 1H, J ) 5.0 Hz),
7.55 (d, 1H, J ) 8.0 Hz), 7.31 (d, 1H, J ) 8.1 Hz), 7.23-7.27
(m, 6H), 7.16-7.19 (m, 2H), 7.11 (d, 2H, J ) 7.5 Hz), 7.07 (s,
1H), 7.05 (t, 1H, J ) 7.7 Hz), 6.97 (t, 1H, J ) 7.2 Hz), 6.72 (bs,
2H), 4.39-4.44 (m, 1H), 4.15-4.21 (m, 1H), 3.47 (d, 2H, J )
6.0 Hz), 3.16-3.21 (m, 2H), 3.11 (dd, 1H, J ) 4.9/14.5 Hz),
2.86 (dd, 1H, J ) 8.9/14.7 Hz), 2.57 (t, 2H, J ) 8.0 Hz), 1.92-
2.00 (m, 1H), 1.85-1.92 (m, 1H), 1.69-1.77 (m, 1H), 1.60-
1.69 (m, 1H); MS (ESI) m/z 554.2 [M + H]+.
9.6/14.6 Hz), 2.61 (t, 2H, J ) 7.6 Hz), 1.01 (d, 3H, J ) 7.0 Hz);
MS (ESI) m/z 497.2 (80) [M + H]+.
N1-(2-P h en ylet h yl)-(2R)-2-{[(1S)-1-((4-ch lor ob en zyl)-
ca r b oxa m id o)et h yl]ca r b oxa m id o}-3-(1H -3-in d olyl)p r o-
p a n a m id e (16b). Preparation as described for 16a , using 7b
(12.1 mg, 0.070 mmol), yielded 10.4 mg of 16b: colorless
powder; mp 198-200 °C; 1H NMR (500 MHz, DMSO-d6) δ
10.78 (s, 1H), 8.28 (d, 1H, J ) 6.8 Hz), 8.18 (d, 1H, J ) 8.4
Hz), 7.99 (t, 1H, J ) 5.5 Hz), 7.57 (d, 1H, J ) 7.9 Hz), 7.29-
7.32 (m, 3H), 7.24-7.27 (m, 4H), 7.18 (t, 1H, J ) 7.3 Hz), 7.13
(d, 2H, J ) 7.5 Hz), 7.07 (s, 1H), 7.05 (t, 1H, J ) 7.7 Hz), 6.97
(t, 1H, J ) 7.4 Hz), 4.37-4.44 (m, 1H), 4.20-4.26 (m, 1H),
3.45 (s, 2H), 3.16-3.26 (m, 2H), 3.11 (dd, 1H, J ) 4.5/14.7 Hz),
2.84 (dd, 1H, J ) 9.6/14.6 Hz), 2.60 (t, 2H, J ) 7.6 Hz), 1.00
(d, 3H, J ) 7.0 Hz); MS (ESI) m/z 531.1 [M + H]+.
N1-{(1R)-2-(1H-3-In d olyl)-1-[1-(2-p h en yleth yl)ca r ba m -
oyl]e t h yl}-(2S )-2-[(4-ch lor ob e n zyl)ca r b oxa m id o]p e n -
ta n ed ia m id e (15b). Preparation as described for 15a , using
128 mg of 13 (0.357 mmol/g, 0.046 mmol) and 7b (15.6 mg,
0.091 mmol), yielded 6.3 mg of 15b: colorless powder; mp 219-
N1-(2-P h en yleth yl)-(2R)-2-{[(1S)-1-(1-(2-(4-ch lor op h en -
yl)et h yl)ca r b oxa m id o)et h yl]ca r b oxa m id o}-3-(1H -3-in -
d olyl)p r op a n a m id e (16c). Preparation as described for 16a ,
using 7c (13.1 mg, 0.07 mmol), yielded 10.2 mg of 16c:
colorless powder; mp 214-217 °C; 1H NMR (500 MHz, DMSO-
d6) δ 10.78 (s, 1H), 8.14 (d, 1H, J ) 8.5 Hz), 8.02-8.04 (m,
2H), 7.57 (d, 1H, J ) 7.9 Hz), 7.24-7.32 (m, 5H), 7.17-7.21
(m, 5H), 7.07 (s, 1H), 7.05 (t, 1H, J ) 7.5 Hz), 6.97 (t, 1H, J )
7.4 Hz), 4.39-4.43 (m, 1H), 4.20-4.26 (m, 1H), 3.22-3.29 (m,
2H), 3.11 (dd, 1H, J ) 4.5/14.6 Hz), 2.86 (dd, 1H, J ) 9.5/14.6
Hz), 2.78 (t, 2H, J ) 7.7 Hz), 2.68 (t, 2H, J ) 7.5 Hz), 2.36-
2.40 (m, 2H), 0.97 (d, 3H, J ) 7.0 Hz); MS (ESI) m/z 545.1 [M
+ H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[(1S)-1-((1,3-ben zod ioxol-5-
ylm et h yl)ca r boxa m id o)et h yl]ca r b oxa m id o}-3-(1H-3-in -
d olyl)p r op a n a m id e (16d ). Preparation as described for 16a ,
using 7d (12.8 mg, 0.07 mmol), yielded 12.1 mg of 16d :
colorless powder; mp 211 °C; 1H NMR (500 MHz, DMSO-d6) δ
10.78 (s, 1H), 8.17-8.19 (m, 2H), 7.99 (t, 1H, J ) 5.5 Hz), 7.57
(d, 1H, J ) 7.9 Hz), 7.31 (d, 1H, J ) 8.1 Hz), 7.26 (t, 2H, J )
7.5 Hz), 7.18 (t, 1H, J ) 7.3 Hz), 7.13 (d, 2H, J ) 7.5 Hz), 7.07
(s, 1H), 7.05 (t, 1H, J ) 7.7 Hz), 6.97 (t, 1H, J ) 7.4 Hz), 6.81
(s, 1H), 6.78 (d, 1H, J ) 7.9 Hz), 6.68 (d, 1H, J ) 8.0 Hz), 5.91
(s, 2H), 4.38-4.43 (m, 1H), 4.19-4.24 (m, 1H), 3.35 (s, 2H),
3.18-3.27 (m, 2H), 3.11 (dd, 1H, J ) 4.5/14.6 Hz), 2.83 (dd,
1H, J ) 9.7/14.6 Hz), 2.61 (t, 2H, J ) 7.6 Hz), 0.99 (d, 3H, J
) 7.0 Hz); MS (ESI) m/z 541.2 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[(1S)-1-((3-p yr id yl)m eth yl-
ca r b oxa m id o)et h yl]ca r b oxa m id o}-3-(1H -3-in d olyl)p r o-
p a n a m id e (16e). Preparation as described for 16a , using 7e
(12.3 mg, 0.07 mmol), yielded 13.2 mg of 16e: colorless powder;
mp 95-97 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.78 (s, 1H),
8.57-8.59 (m, 2H), 8.40 (d, 1H, J ) 7.0 Hz), 8.20 (d, 1H, J )
8.4 Hz), 7.97-8.01 (m, 2H), 7.57 (d, 2H, J ) 7.8 Hz), 7.31 (d,
1H, J ) 8.1 Hz), 7.26 (t, 2H, J ) 7.5 Hz), 7.18 (t, 1H, J ) 7.2
Hz), 7.13 (d, 2H, J ) 7.5 Hz), 7.07 (s, 1H), 7.05 (t, 1H, J ) 7.7
Hz), 6.97 (t, 1H, J ) 7.4 Hz), 4.04-4.45 (m, 1H), 4.23-4.29
(m, 1H), 3.61 (s, 2H), 3.18-3.28 (m, 2H), 3.09 (dd, 1H, J )
4.7/14.6 Hz), 2.84 (dd, 1H, J ) 9.6/14.6 Hz), 2.60 (t, 2H, J )
7.5 Hz), 1.01 (d, 3H, J ) 7.1 Hz); MS (ESI) m/z 498.2 [M +
H]+.
1
220 °C; H NMR (500 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.33
(d, 1H, J ) 7.5 Hz), 8.19 (d, 1H, J ) 9.0 Hz), 7.94 (t, 1H, J )
5.0 Hz), 7.55 (d, 1H, J ) 7.5 Hz), 7.29-7.32 (m, 3H), 7.23-
7.26 (m, 4H), 7.17 (t, 1H, J ) 7.3 Hz), 7.11 (d, 2H, J ) 7.6
Hz), 7.08 (s, 1H), 7.05 (t, 1H, J ) 7.7 Hz), 6.97 (t, 1H, J ) 8.0
Hz), 6.73 (bs, 2H), 4.39-4.44 (m, 1H), 4.16-4.20 (m, 1H), 3.47
(d, 2H, J ) 6.0 Hz), 3.16-3.21 (m, 2H), 3.10 (dd, 1H, J ) 5.0/
14.3 Hz), 2.86 (dd, 1H, J ) 8.5/14.8 Hz), 2.56 (t, 2H, J ) 7.0
Hz), 1.92-1.99 (m, 1H), 1.84-1.92 (m, 1H), 1.68-1.76 (m, 1H),
1.60-1.68 (m, 1H); MS (ESI) m/z 588.2 [M + H]+.
N1-{(1R)-2-(1H-3-In d olyl)-1-[1-(2-p h en yleth yl)ca r ba m -
oyl]eth yl}-(2S)-2-[1-(2-(4-ch lor op h en yl)eth yl)ca r boxa m i-
d o]p en ta n ed ia m id e (15c). Preparation as described for 15a ,
using 100 mg of 13 (0.354 mmol/g, 0.035 mmol) and 7c (13.1
mg, 0.07 mmol), yielded 16.8 mg of 15c: colorless powder; mp
213-215 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.78 (s, 1H),
8.14 (d, 1H, J ) 8.2 Hz), 8.08 (d, 1H, J ) 7.4 Hz), 7.99 (t, 1H,
J ) 5.8 Hz), 7.55 (d, 1H, J ) 8.5 Hz), 7.28-7.32 (m, 3H), 7.26
(t, 2H, J ) 7.4 Hz), 7.21 (d, 2H, J ) 8.0 Hz), 7.18 (t, 1H, J )
7.3 Hz), 7.15 (d, 2H, J ) 7.6 Hz), 7.08 (s, 1H), 7.05 (t, 1H, J )
7.7 Hz), 6.97 (t, 1H, J ) 7.7 Hz), 6.72 (bs, 2H), 4.39-4.45 (m,
1H), 4.15-4.21 (m, 1H), 3.18-3.29 (m, 2H), 3.11 (dd, 1H, J )
4.7/14.4 Hz), 2.88 (dd, 1H, J ) 8.8/14.8 Hz), 2.79 (t, 2H, J )
7.7 Hz), 2.64 (t, 2H, J ) 7.5 Hz), 2.34-2.46 (m, 2H), 1.82-
1.98 (m, 2H), 1.65-1.74 (m, 1H), 1.55-1.65 (m, 1H); MS (ESI)
m/z 602.2 [M + H]+.
N1-{(1R)-2-(1H-3-In d olyl)-1-[1-(2-p h en yleth yl)ca r ba m -
oyl]eth yl}-(2S)-2-[(1H-2-in dolylm eth yl)car boxam ido]pen -
ta n ed ia m id e (15d ). Preparation as described for 15a , using
100 mg of 13 (0.354 mmol/g, 0.035 mmol) and 7g (12.3 mg,
0.07 mmol), yielded 6.6 mg of 15d : colorless powder; mp 195-
1
201 °C; H NMR (500 MHz, DMSO-d6) δ 10.86 (s, 1H), 10.76
(s, 1H), 8.24 (d, 1H, J ) 7.5 Hz), 8.21 (d, 1H, J ) 8.2 Hz), 7.96
(t, 1H, J ) 5.6 Hz), 7.55 (d, 1H, J ) 7.8 Hz), 7.39 (d, 1H, J )
7.8 Hz), 7.29 (t, 2H, J ) 8.2 Hz), 7.22 (t, 2H, J ) 7.4 Hz), 7.14
(t, 1H, J ) 7.4 Hz), 7.06-7.08 (m, 3H), 7.04 (t, 1H, J ) 8.0
Hz), 6.95-6.99 (m, 2H), 6.90 (t, 1H, J ) 7.4 Hz), 6.72 (bs, 2H),
6.20 (s, 1H), 4.41-4.45 (m, 1H), 4.23-4.27 (m, 1H), 3.63 (d,
2H, J ) 3.4 Hz), 3.13-3.24 (m, 2H), 3.09 (dd, 1H, J ) 5.4/14.5
Hz), 2.86 (dd, 1H, J ) 8.7/14.5 Hz), 2.56 (t, 2H, J ) 7.6 Hz),
1.87-2.01 (m, 2H), 1.71-1.78 (m, 1H), 1.61-1.69 (m, 1H); MS
(ESI) m/z 593.2 (100) [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[(1S)-1-((1,2,3,4-tetr a h yd r o-
1-isoqu in olin yl)m eth ylcar boxam ido)eth yl]car boxam ido}-
3-(1H-3-in d olyl)p r op a n a m id e (16f). Preparation as de-
scribed for 16a , using 7f (29 mg, 0.07 mmol), yielded 4.7 mg
of 16f: colorless powder; cis/trans isomeric ratio of 1:1.08; mp
1
110-113 °C; H NMR (500 MHz, DMSO-d6) δ 10.78 (s, 1H),
8.80 and 9.21 (bs, 1H), 8.39 and 8.45 (d, 1H, J ) 7.5 Hz), 8.21
and 8.23 (d, 1H, J ) 8.5 Hz), 8.10 (s, 1H), 7.60 (d, 1H, J ) 7.8
Hz), 7.31 (d, 1H, J ) 8.1 Hz), 7.15-7.28 (m, 9H), 7.08 (s, 1H),
7.05 (t, 1H, J ) 7.6 Hz), 6.97 (t, 1H, J ) 7.5 Hz), 4.77-4.82
(m, 1H), 4.46-4.52 (m, 1H), 4.33-4.44 (m, 1H), 3.40-3.48 (m,
1H), 3.27-3.36 (m, 2H), 3.17-3.24 (m, 1H), 2.81-3.08 (m, 6H),
2.64 (t, 2H, J ) 7.5 Hz), 0.94 and 1.02 (d, 3H, J ) 7.0 Hz); MS
(ESI) m/z 552.3 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[(1S)-1-((1H-2-in dolyl)m eth -
ylca r boxa m id o)eth yl]ca r boxa m id o}-3-(1H-3-in d olyl)p r o-
p a n a m id e (16g). Preparation as described for 16a , using 7g
(12.3 mg, 0.07 mmol), yielded 5.8 mg of 16g: colorless powder;
N 1-(2-P h e n yle t h yl)-(2R )-2-{[(1S )-1-(b e n zylca r b oxa -
m id o)eth yl]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e
(16a ). Preparation as described for 15a , using 100 mg of 13
(0.354 mmol/g, 0.035 mmol) and Fmoc-Ala-OH (22 mg, 0.07
mmol), yielded 12.6 mg of 16a : colorless powder; mp 205-
1
207 °C; H NMR (500 MHz, DMSO-d6) δ 10.78 (s, 1H), 8.24
(d, 1H, J ) 6.8 Hz), 8.18 (d, 1H, J ) 8.4 Hz), 8.00 (t, 1H, J )
5.5 Hz), 7.57 (d, 1H, J ) 7.9 Hz), 7.31 (d, 1H, J ) 8.1 Hz),
7.23-7.27 (m, 6H), 7.16-7.19 (m, 2H), 7.14 (d, 2H, J ) 7.5
Hz), 7.06 (s, 1H), 7.05 (t, 1H, J ) 7.7 Hz), 6.97 (t, 1H, J ) 7.3
Hz), 4.38-4.43 (m, 1H), 4.21-4.25 (m, 1H), 3.45 (s, 2H), 3.19-
3.24 (m, 2H), 3.11 (dd, 1H, J ) 4.6/14.7 Hz), 2.84 (dd, 1H, J )

1926 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Weber et al.
mp 188-193 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.86 (s,
1H), 10.76 (s, 1H), 8.20 (d, 1H, J ) 8.5 Hz), 8.17 (d, 1H, J )
7.1 Hz), 8.01 (t, 1H, J ) 5.5 Hz), 7.56 (d, 1H, J ) 8.1 Hz), 7.38
(d, 1H, J ) 7.8 Hz), 7.29 (t, 2H, J ) 9.1 Hz), 7.22 (t, 2H, J )
7.4 Hz), 7.15 (t, 1H, J ) 7.4 Hz), 7.11 (d, 2H, J ) 7.6 Hz), 7.06
(s, 1H), 7.04 (t, 1H, J ) 7.6 Hz), 6.94-6.99 (m, 2H), 6.90 (t,
1H, J ) 7.0 Hz), 6.19 (s, 1H), 4.41-4.45 (m, 1H), 4.26-4.31
(m, 1H), 3.60 (d, 2H, J ) 3.5 Hz), 3.15-3.29 (m, 2H), 3.08 (dd,
1H, J ) 4.8/14.6 Hz), 2.84 (dd, 1H, J ) 9.5/14.4 Hz), 2.61 (t,
2H, J ) 7.6 Hz), 1.00 and 1.01 (s, 3H); MS (ESI) m/z 536.2 [M
+ H]+.
was added. After the mixture was cooled to 0 °C, TFA (2.5
mL) was added dropwise over 5 min and the solution was
stirred for 1 h. Then the solvent was removed in vacuo and
the residue was treated with a mixture of DMSO (20 mL),
water (2.5 mL), and acetic acid (2.5 mL) and stirred at room
temperature overnight. Evaporation to dryness was followed
by treatment with 20% (v/v) piperidine in DMF (10 mL) and
stirring for 30 min at room temperature. Finally the solvent
was removed in vacuo, and chromatography (ethyl acetate)
yielded 0.25 g (73%) of 18a : colorless powder; the ratio of the
two isomers was determined from the NMR integrals of the
CH3 group as 1:1.52; mp 100-105 °C; 1H NMR (500 MHz,
DMSO-d6) δ 10.83 and 10.85 (s, 1H), 9.1 (m, 1H), 8.74-8.78
(m, 1H), 8.27 and 8.34 (t, 1H, J ) 5.6 Hz), 7.63 and 7.68 (d,
1H, J ) 7.8 Hz), 7.17-7.21 and 7.24-7.33 and 7.33-7.37 and
7.40-7.43 (m, 11H), 7.09 and 7.14 (s, 1H), 6.96-7.07 (m, 2H),
4.62-4.68 (m, 1H), 3.98-4.01 (m, 1H), 3.76-3.78 (m, 1H),
3.61-3.67 and 3.41-3.43 (m, 1H), 3.34-3.39 (m, 1H), 3.26-
3.30 (m, 1H), 3.05-3.09 (m, 1H), 2.84-2.93 (m, 1H), 2.67-
2.72 (m, 2H), 1.11 and 1.34 (d, 3H, J ) 6.9 Hz); MS (ESI) m/z
469.2 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[1-((4-ch lor oben zyl)a m in o)-
eth yl]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e (18b).
Preparation as described for 18a , using 12b (0.32 g, 0.734
mmol), yielded 320 mg (87%) of 18b: colorless powder; the
ratio of the two isomers was determined from the NMR
integrals of the CH3 group as 1:1.24; mp 107-110 °C; 1H NMR
(500 MHz, DMSO-d6) δ 10.85 and 10.83 (s, 1H), 9.2 (m, 1H),
8.72 and 8.77 (d, 1H, J ) 8.5 Hz), 8.28 and 8.34 (t, 1H, J )
5.5 Hz), 7.68 and 7.63 (d, 1H, J ) 7.7 Hz), 7.17-7.20 and 7.24-
7.33 and 7.40-7.50 (m, 10H), 7.09 and 7.15 (s, 1H), 6.96-7.07
(m, 2H), 4.61-4.69 (m, 1H), 3.98-4.02 (m, 1H), 3.75 (m, 1H),
3.60-3.67 and 3.39-3.43 (m, 1H), 3.34-3.38 (m, 1H), 3.24-
3.29 (m, 1H), 3.05-3.08 (m, 1H), 2.85-2.92 (m, 1H), 2.67-
2.71 (m, 2H), 1.33 and 1.10 (d, 3H, J ) 6.9 Hz); MS (ESI) m/z
503.2 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[1-(1-(2-ph en yleth yl)am in o)-
eth yl]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e (18c).
Preparation as described for 18a , using 12c (0.305 g, 0.737
mmol), yielded 230 mg (65%) of 18c: colorless powder; the ratio
of the two isomers was determined from the NMR integrals
of the CH3 group as 1:1.59; mp 89-91 °C; 1H NMR (500 MHz,
DMSO-d6) δ 10.83 and 10.79 (s, 1H), 8.88 (m, 1H), 8.77 (m,
1H), 8.24 and 8.32 (t, 1H, J ) 5.4 Hz), 7.63 (d, 1H, J ) 7.8
Hz), 7.15-7.18 and 7.21-7.28 and 7.29-7.33 (m, 11H), 7.04-
7.12 (m, 2H), 6.94-7.00 (m, 1H), 4.59-4.64 (m, 1H), 3.82-
3.86 (m, 1H), 3.29-3.35 (m, 1H), 3.22-3.30 (m, 1H), 2.75-
3.09 (m, 6H), 2.66 (t, 2H, J ) 7.2 Hz), 1.35 and 1.12 (d, 3H, J
) 6.9 Hz); MS (ESI) m/z 483.3 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[N′-(ben zoyl)h ydr azin o]car -
b oxa m id o}-3-(1H -3-in d olyl)p r op a n a m id e (21a ). Resin-
bound 13 (100 mg, 0.354 mmol/g, 0.035 mmol) was treated two
times with 20% piperidine (v/v) in NMP (5 mL) for 15 min,
washed after each time with NMP (5 × 5 mL), finally washed
with dry CH2Cl2 (5 × 5 mL), and subsequently left in dry CH2-
Cl2 (5 mL) for 30 min. Then a solution of 19 (30.5 mg, 0.108
mmol) in dry CH2Cl2 (1 mL) was added and the resin was
shaken for 90 min at room temperature and washed with CH2-
Cl2 (5 × 5 mL) and NMP (5 × 5 mL). After repeated Fmoc
cleavage, 7a (9.5 mg, 0.07 mmol) was coupled two times to
the resin for 45 min using [TBTU (2 equiv)]/[HOBt (2 equiv)]/
[DIEA (5.7 equiv)] activation. Coupling steps were followed
by a washing step with NMP (5 × 5 mL). Finally the resin
was washed with CH2Cl2 (3 × 5 mL) for 30 min and the
compound was deprotected and cleaved from the resin with
[TFA (18 equiv)]/[TIPS (1 equiv)]/[H2O (1 equiv)] (3 × 2 mL,
30 min each). The combined filtrates were reduced in vacuo.
RP-HPLC purification yielded 2.6 mg of 21a : colorless powder;
mp 109-114 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.79 (s,
1H), 9.80 (s, 1H), 7.95-8.00 (m, 2H), 7.51 (d, 1H, J ) 7.9 Hz),
7.30 (d, 1H, J ) 8.2 Hz), 7.23-7.28 (m, 6H), 7.19-7.22 (m,
1H), 7.16 (t, 1H, J ) 7.1 Hz), 7.13 (d, 2H, J ) 7.5 Hz), 7.02-
7.04 (m, 2H), 6.95 (t, 1H, J ) 7.5 Hz), 6.36 (d, 1H, J ) 8.0
Hz), 4.36 (q, 1H, J ) 6.6 Hz), 3.41 (s, 2H), 3.14-3.29 (m, 2H),
N1-(2-P h en yleth yl)-(2R)-2-{[(ben zyl)a m in o]m eth ylca r -
boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e (17a ). Prepara-
tion as described for 15a , using 100 mg of 13 (0.354 mmol/g,
0.035 mmol) and 11a (27 mg, 0.07 mmol), yielded 9.8 mg of
1
17a : colorless powder; mp 107-109 °C; H NMR (500 MHz,
DMSO-d6) δ 10.82 (s, 1H), 9.11 (bs, 1H), 8.69 (d, 1H, J ) 8.1
Hz), 8.24 (m, 1H), 7.62 (d, 1H, J ) 7.9 Hz), 7.38-7.43 (m, 5H),
7.32 (d, 1H, J ) 8.1 Hz), 7.26 (t, 2H, J ) 7.4 Hz), 7.18 (t, 1H,
J ) 7.2 Hz), 7.16 (d, 2H, J ) 7.4 Hz), 7.10 (s, 1H), 7.06 (t, 1H,
J ) 7.4 Hz), 6.98 (t, 1H, J ) 7.5 Hz), 4.57 (q, 1H, J ) 5.7 Hz),
4.02 (m, 2H), 3.63-3.66 (m, 1H), 3.53-3.56 (m, 1H), 3.27-
3.34 (m, 1H), 3.20-3.26 (m, 1H), 3.04 (dd, 1H, J ) 5.1/14.5
Hz), 2.86 (dd, 1H, J ) 9.0/14.5 Hz), 2.64 (t, 2H, J ) 7.5 Hz);
MS (ESI) m/z 455.2 [M + H]+.
N1-(2-P h en ylet h yl)-(2R)-2-{[(4-ch lor ob en zyl)a m in o]-
m eth ylcar boxam ido}-3-(1H-3-in dolyl)pr opan am ide (17b).
Preparation as described for 17a , using 11b (29.8 mg, 0.07
mmol), yielded 11 mg of 17b: colorless powder; mp 138-140
1
°C; H NMR (500 MHz, DMSO-d6) δ 10.82 (s, 1H), 9.13 (bs,
1H), 8.68 (d, 1H, J ) 8.2 Hz), 8.25 (m, 1H), 7.61 (d, 1H, J )
7.9 Hz), 7.49 (d, 2H, J ) 8.2 Hz), 7.41 (d, 2H, J ) 8.1 Hz),
7.32 (d, 1H, J ) 8.1 Hz), 7.26 (t, 2H, J ) 7.4 Hz), 7.18 (t, 1H,
J ) 7.3 Hz), 7.16 (d, 2H, J ) 7.5 Hz), 7.10 (s, 1H), 7.06 (t, 1H,
J ) 7.3 Hz), 6.97 (t, 1H, J ) 7.5 Hz), 4.57 (q, 1H, J ) 5.8 Hz),
4.03 (m, 2H), 3.62-3.67 (m, 1H), 3.51-3.56 (m, 1H), 3.26-
3.34 (m, 1H), 3.19-3.26 (m, 1H), 3.04 (dd, 1H, J ) 5.1/14.5
Hz), 2.86 (dd, 1H, J ) 9.0/14.5 Hz), 2.64 (t, 2H, J ) 7.5 Hz);
MS (ESI) m/z 489.2 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[(3-p yr id yl)m eth yla m in o]-
m eth ylcar boxam ido}-3-(1H-3-in dolyl)pr opan am ide (17c).
Preparation as described for 17a , using the TFA salt of 11c
(35.4 mg, 0.07 mmol), yielded 6.6 mg of 17c: colorless powder;
mp 89-93 °C; 1H NMR (500 MHz, DMSO-d6) δ 10.82 (s, 1H),
9.20 (bs, 1H), 8.71 (d, 1H, J ) 8.0 Hz), 8.59-8.62 (m, 2H),
8.26 (t, 1H, J ) 5.5 Hz), 7.83 (d, 1H, J ) 8.0 Hz), 7.62 (d, 1H,
J ) 8.0 Hz), 7.46 (dd, 1H, J ) 4.7/7.9 Hz), 7.32 (d, 1H, J ) 8.0
Hz), 7.26 (d, 2H, J ) 7.4 Hz), 7.18 (t, 1H, J ) 7.2 Hz), 7.16 (d,
2H, J ) 7.5 Hz), 7.11 (s, 1H), 7.06 (t, 1H, J ) 7.0 Hz), 6.98 (t,
1H, J ) 7.0 Hz), 4.57 (q, 1H, J ) 6.0 Hz), 4.09 (m, 2H), 3.69-
3.75 (m, 1H), 3.57-3.64 (m, 1H), 3.27-3.35 (m, 1H), 3.19-
3.27 (m, 1H), 3.05 (dd, 1H, J ) 5.2/14.9 Hz), 2.87 (dd, 1H, J )
8.8/14.6 Hz), 2.64 (t, 2H, J ) 7.1 Hz); MS (ESI) m/z 456.2 [M
+ H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[1-(2-p h en yleth yl)a m in o]-
m eth ylcar boxam ido}-3-(1H-3-in dolyl)pr opan am ide (17d).
Preparation as described for 17a , using 11d (28.3 mg, 0.07
mmol), yielded 2.7 mg of 17d : colorless powder; mp 197-199
1
°C; H NMR (500 MHz, DMSO-d6) δ 10.82 (s, 1H), 8.79 (bs,
1H), 8.74 (d, 1H, J ) 8.5 Hz), 8.26 (t, 1H, J ) 5.5 Hz), 7.62 (d,
1H, J ) 8.0 Hz), 7.31-7.34 (m, 3H), 7.25-7.28 (m, 3H), 7.19-
7.21 (m, 3H), 7.16 (d, 2H, J ) 7.6 Hz), 7.12 (s, 1H), 7.05 (t,
1H, J ) 7.5 Hz), 6.98 (t, 1H, J ) 7.5 Hz), 4.58 (q, 1H, J ) 5.5
Hz), 3.73-3.79 (m, 1H), 3.62-3.67 (m, 1H), 3.29-3.34 (m, 1H),
3.20-3.25 (m, 1H), 3.04-3.08 (m, 3H), 2.84-2.92 (m, 3H), 2.64
(t, 2H, J ) 7.4 Hz); MS (ESI) m/z 469.2 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[1-(ben zyla m in o)eth yl]ca r -
boxam ido}-3-(1H-3-in dolyl)pr opan am ide (18a). Compound
14 (0.30 g, 0.736 mmol) and 12a (0.295 g, 0.735 mmol) were
coupled in DMF (7 mL) at room temperature within 1 h using
[HATU (1.5 equiv)]/[HOAt (1.5 equiv)]/[collidine (15 equiv)]
activation. The solvent was removed in vacuo, the residue was
chromatographed (ethyl acetate/hexane, 1:1) and subsequently
dissolved in CH2Cl2 (2.5 mL), and triisopropylsilane (0.25 mL)

Design of Peptidomimetic Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1927
3.01 (dd, 1H, J ) 5.7/14.6 Hz), 2.91 (dd, 1H, J ) 7.5/14.5 Hz),
2.58 (t, 2H, J ) 7.1 Hz); MS (ESI) m/z 484.3 [M + H]+.
J ) 7.5 Hz), 7.14-7.19 (m, 3H), 7.11 (s, 1H), 7.05 (t, 1H, J )
7.4 Hz), 6.94 (t, 1H, J ) 7.5 Hz), 6.72 (d, 1H, J ) 8.0 Hz), 4.44
(q, 1H, J ) 7.4 Hz), 3.27-3.35 (m, 1H), 3.19-3.27 (m, 1H),
3.11 (d, 2H, J ) 6.4 Hz), 2.66 (t, 2H, J ) 7.4 Hz); MS (ESI)
m/z 454.2 [M + H]+.
N 1-(2-P h e n y le t h y l)-(2R )-2-{[N ′-(4-c h lo r o b e n zo y l)-
h yd r a zin o]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e
(21b). Preparation as described for 21a , using 7b (12 mg, 0.07
mmol), yielded 3.0 mg of 21b: colorless powder; mp 195/196
°C; 1H NMR (500 MHz, DMSO-d6) δ 10.79 (s, 1H), 9.81 (s, 1H),
8.00 (s, 1H), 7.94 (bs, 1H), 7.51 (d, 1H, J ) 7.9 Hz), 7.23-7.34
(m, 7H), 7.17 (t, 1H, J ) 7.3 Hz), 7.13 (d, 2H, J ) 7.5 Hz),
7.02-7.05 (m, 2H), 6.95 (t, 1H, J ) 7.5 Hz), 6.38 (d, 1H, J )
8.1 Hz), 4.36 (q, 1H, J ) 6.7 Hz), 3.42 (s, 2H), 3.16-3.24 (m,
2H), 3.01 (dd, 1H, J ) 5.6/14.9 Hz), 2.91 (dd, 1H, J ) 7.5/14.5
Hz), 2.58 (t, 2H, J ) 7.5 Hz); MS (ESI) m/z 518.2 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[N′-(2-(3-p yr id yl)eth a n oyl)-
h yd r a zin o]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e
(21c). Preparation as described for 21a , using 7e (12 mg, 0.07
mmol), yielded 4.5 mg of 21c: colorless powder; mp 116-120
°C; 1H NMR (500 MHz, DMSO-d6) δ 10.79 (s, 1H), 9.89 (s, 1H),
8.63 (s, 1H), 8.61 (d, 1H, J ) 5.0 Hz), 8.02-8.04 (m, 2H), 7.96
(bs, 1H), 7.63 (dd, 1H, J ) 5.8/7.3 Hz), 7.51 (d, 1H, J ) 7.9
Hz), 7.30 (d, 1H, J ) 8.2 Hz), 7.25 (t, 2H, J ) 7.6 Hz), 7.17 (t,
1H, J ) 7.4 Hz), 7.13 (d, 2H, J ) 7.5 Hz), 7.02-7.04 (m, 2H),
6.95 (t, 1H, J ) 8.0 Hz), 6.44 (d, 1H, J ) 8.1 Hz), 4.32-4.36
(m, 1H), 3.59 (s, 2H), 3.22-3.26 (m, 1H), 3.15-3.19 (m, 1H),
3.01 (dd, 1H, J ) 5.6/14.4 Hz), 2.91 (dd, 1H, J ) 7.4/14.6 Hz),
2.58 (t, 2H, J ) 7.5 Hz); MS (ESI) m/z 485.3 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[N′-(4-ch lor op h en ylm eth yl-
en e)h yd r a zin o]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a -
m id e (24b). Preparation from 23b (120 mg, 0.204 mmol) as
described for 24a yielded 56 mg (56%) of 24b: colorless
powder; mp 173-175 °C; 1H NMR (500 MHz, DMSO-d6) δ
10.85 (s, 1H), 10.51 (s, 1H), 8.05 (t, 1H, J ) 5.5 Hz), 7.79 (s,
1H), 7.59 (d, 1H, J ) 8.0 Hz), 7.56 (d, 2H, J ) 8.4 Hz), 7.46 (d,
2H, J ) 8.3 Hz), 7.33 (d, 1H, J ) 8.1 Hz), 7.25 (t, 2H, J ) 7.5
Hz), 7.15-7.18 (m, 3H), 7.11 (s, 1H), 7.05 (t, 1H, J ) 7.4 Hz),
6.94 (t, 1H, J ) 7.5 Hz), 6.73 (d, 1H, J ) 8.0 Hz), 4.43 (q, 1H,
J ) 7.5 Hz), 3.27-3.35 (m, 1H), 3.19-3.27 (m, 1H), 3.11 (d,
2H, J ) 6.2 Hz), 2.66 (t, 2H J ) 7.5 Hz); MS (ESI) m/z 488.2
[M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[N′-(fu r a n -2-ylm eth ylen e)-
h yd r a zin o]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e
(24c). Preparation from 23c (100 mg, 0.184 mmol) as described
for 24a yielded 46 mg (57%) of 24c: colorless powder; mp 100/
1
101 °C; H NMR (500 MHz, DMSO-d6) δ 10.82 (s, 1H), 10.41
(s, 1H), 8.09 (t, 1H, J ) 5.5 Hz), 7.76 (s, 1H), 7.73 (s, 1H), 7.55
(d, 1H, J ) 7.9 Hz), 7.30 (d, 1H, J ) 8.1 Hz), 7.25 (t, 2H, J )
7.4 Hz), 7.14-7.17 (m, 3H), 7.07 (s, 1H), 7.03 (t, 1H, J ) 7.4
Hz), 6.93 (t, 1H, J ) 7.5 Hz), 6.74 (d, 1H, J ) 3.2 Hz), 6.58-
6.60 (m, 2H), 4.45 (q, 1H, J ) 6.8 Hz), 3.24-3.31 (m, 1H), 3.17-
3.24 (m, 1H), 3.02-3.10 (m, 2H), 2.62 (t, 2H, J ) 7.4 Hz); MS
(ESI) m/z 444.2 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[N′-(2-(1,2,3,4-tetr a h yd r o-1-
isoqu in olin yl)eth a n oyl)h yd r a zin o]ca r boxa m id o}-3-(1H-
3-in d olyl)p r op a n a m id e (21d ). Prepared as described for
21a , using 7f (29 mg, 0.07 mmol). The two isomers were
separated by RP-HPLC and obtained as colorless powders.
N1-(2-P h en ylet h yl)-(2R)-2-{[N′-(4-(2-t h ien yl)p h en yl-
m et h ylen e)h yd r a zin o]ca r b oxa m id o}-3-(1H -3-in d olyl)-
p r op a n a m id e (24d ). Preparation from 23d (40 mg, 0.063
mmol) as described for 24a yielded 25 mg (74%) of 24d :
colorless powder; mp 219-221 °C; 1H NMR (500 MHz, DMSO-
d6) δ 10.87 (s, 1H), 10.48 (s, 1H), 8.06 (t, 1H, J ) 5.5 Hz), 7.81
(s, 1H), 7.69 (d, 2H, J ) 8.2 Hz), 7.57-7.61 (m, 5H), 7.33 (d,
1H, J ) 8.1 Hz), 7.25 (t, 2H, J ) 7.5 Hz), 7.16-7.18 (m, 4H),
7.13 (s, 1H), 7.06 (t, 1H, J ) 7.7 Hz), 6.95 (t, 1H, J ) 7.5 Hz),
6.74 (d, 1H, J ) 7.9 Hz), 4.43 (q, 1H, J ) 7.7 Hz), 3.27-3.34
(m, 1H), 3.20-3.27 (m, 1H), 3.12 (d, 2H, J ) 6.4 Hz), 2.66 (t,
2H, J ) 7.4 Hz); MS (ESI) m/z 536.1 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[N′-(ben zyl)h yd r a zin o]ca r -
boxam ido}-3-(1H-3-in dolyl)pr opan am ide (28a). Compound
27a (0.24 g, 0.54 mmol) was dissolved in CH2Cl2 (2 mL),
triisopropylsilane (0.1 mL) was added, and the solution was
cooled to 0 °C. Then TFA (2 mL) was added dropwise over 5
min and the solution was stirred for 30 min. The solvent was
removed in vacuo, and the residue was redissolved in a solution
of dry CH2Cl2 (8 mL) and DMAP (66 mg, 0.54 mmol). This
solution was added dropwise and under stirring to a solution
of bis(pentafluorophenyl)carbonate (213 mg, 0.54 mmol) in dry
CH2Cl2 (20 mL) over a period of 20 min. Upon completion of
the addition, a solution of 14 (0.22 g, 0.54 mmol), DMAP (66
mg, 0.54 mmol), and dry CH2Cl2 (8 mL) was added. The
resulting mixture was stirred for 30 min at room temperature.
Then the solvent was removed in vacuo, the residue was
redissolved in CH2Cl2 (4 mL) and triisopropylsilane (0.1 mL),
and the solution was cooled to 0 °C. TFA (4 mL) was added
dropwise over 5 min, and the solution was stirred for 30 min.
The solution was evaporated to dryness and then treated with
a solution of 20% (v/v) piperidine in DMF (10 mL) for 30 min
at room temperature. Finally the solvent was removed in vacuo
and RP-HPLC purification yielded 34.2 mg (14%) of 28a :
colorless powder; mp 81-84 °C; 1H NMR (500 MHz, CDCl3) δ
8.13 (s, 1H), 7.56 (d, 1H, J ) 7.9 Hz), 7.34 (d, 1H, J ) 8.1 Hz),
7.16-7.29 (m, 9H), 7.11 (t, 1H, J ) 7.1 Hz), 6.94-6.95 (m, 3H),
6.88 (bs, 1H), 5.99 (bs, 1H), 4.43 (q, 1H, J ) 6.5 Hz), 3.91 (bs,
2H), 3.34-3.42 (m, 1H), 3.16-3.24 (m, 1H), 3.14 (dd, 1H, J )
5.5/14.0 Hz), 3.07 (dd, 1H, J ) 7.4/14.3 Hz), 2.45-2.59 (m, 2H);
MS (ESI) m/z 456.1 [M + H]+.
1
21d 1: 1.6 mg; mp 105-110 °C; H NMR (500 MHz, DMSO-
d6) δ 10.79 (s, 1H), 9.94 (s, 1H), 8.75 and 9.25 (bs, 1H), 8.12 (s,
1H), 8.04 (t, 1H, J ) 5.5 Hz), 7.54 (d, 1H, J ) 7.5 Hz), 7.31 (d,
1H, J ) 8.0 Hz), 7.17-7.26 (m, 7H), 7.14 (d, 2H, J ) 7.4 Hz),
7.05 (s, 1H), 7.04 (t, 1H, J ) 7.7 Hz), 6.96 (t, 1H, J ) 7.0 Hz),
6.49 (d, 1H, J ) 8.0 Hz), 4.75-4.84 (m, 1H), 4.35-4.39 (m,
1H), 3.31-3.48 (m, 2H), 3.24-3.28 (m, 1H), 3.16-3.20 (m, 1H),
2.87-3.01 (m, 6H), 2.59 (t, 2H, J ) 7.5 Hz); MS (ESI) m/z 539.3
[M + H]+. 21d 2: 0.9 mg; mp 120-123 °C; 1H NMR (500 MHz,
DMSO-d6) δ 10.79 (s, 1H), 9.95 (s, 1H), 8.78 and 9.26 (bs, 1H),
8.12 (s, 1H), 8.03 (t, 1H, J ) 5.8 Hz), 7.54 (d, 1H, J ) 7.7 Hz),
7.31 (d, 1H, J ) 8.1 Hz), 7.17-7.26 (m, 7H), 7.14 (d, 2H, J )
7.3 Hz), 7.03-7.05 (m, 2H), 6.95 (t, 1H, J ) 7.0 Hz), 6.49 (d,
1H, J ) 8.2 Hz), 4.80-4.86 (m, 1H), 4.37 (q, 1H, J ) 3.6 Hz),
3.31-3.48 (m, 2H), 3.22-3.28 (m, 1H), 3.15-3.20 (m, 1H),
2.87-3.05 (m, 6H), 2.59 (t, 2H, J ) 7.5 Hz); MS (ESI) m/z 539.3
[M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[N′-(2-(1H-2-in d olyl)eth a n -
oyl)h yd r a zin o]ca r b oxa m id o}-3-(1H -3-in d olyl)p r op a n a -
m id e (21e). Preparation as described for 21a , using 7g (12.3
mg, 0.07 mmol), yielded 5.7 mg of 21e: colorless powder; mp
1
109-115 °C; H NMR (500 MHz, DMSO-d6) δ 10.92 (s, 1H),
10.79 (s, 1H), 9.80 (s, 1H), 8.04 (s, 1H), 7.97 (bs, 1H), 7.52 (d,
1H, J ) 7.9 Hz), 7.40 (d, 1H, J ) 7.7 Hz), 7.30 (d, 2H, J ) 8.0
Hz), 7.24 (t, 2H, J ) 7.4 Hz), 7.16 (t, 1H, J ) 7.3 Hz), 7.12 (d,
2H, J ) 7.5 Hz), 6.90-7.05 (m, 5H), 6.45 (d, 1H, J ) 8.0 Hz),
6.23 (s, 1H), 4.36-4.40 (m, 1H), 3.60 (s, 2H), 3.15-3.26 (m,
2H), 3.02 (dd, 1H, J ) 5.7/14.5 Hz), 2.91 (dd, 1H, J ) 7.4/14.6
Hz), 2.58 (t, 2H, J ) 7.4 Hz); MS (ESI) m/z 523.2 [M + H]+.
N 1-(2-P h e n yle t h yl)-(2R )-2-{[N ′-(p h e n ylm e t h yle n e )-
h yd r a zin o]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e
(24a ). Compound 23a (120 mg, 0.217 mmol) was dissolved in
CH2Cl2 (3 mL), triisopropylsilane (0.1 mL) was added, and the
solution was cooled to 0 °C. Then TFA (3 mL) was added
dropwise over 5 min and the solution was stirred for 30 min.
The solvent was removed in vacuo and the residue was treated
with DMSO (8 mL), water (1 mL), and acetic acid (1 mL) and
stirred overnight at room temperature. Then the solution was
evaporated to dryness. RP-HPLC purification yielded 62 mg
(63%) of 24a : colorless powder; mp 183 °C; 1H NMR (500 MHz,
DMSO-d6) δ 10.85 (s, 1H), 10.43 (s, 1H), 8.06 (t, 1H, J ) 5.5
Hz), 7.81 (s, 1H), 7.60 (d, 1H, J ) 7.9 Hz), 7.54 (d, 2H, J ) 7.4
Hz), 7.36-7.41 (m, 3H), 7.32 (d, 1H, J ) 8.1 Hz), 7.25 (t, 2H,
N1-(2-P h en yleth yl)-(2R)-2-{[N′-(4-ch lor oben zyl)h yd r a -
zin o]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e (28b).
Preparation from 27b (0.30 g, 0.63 mmol) and 14 (0.26 g, 0.63

1928 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Weber et al.
mmol) as described for 28a yielded 26.8 mg (9%) of 28b:
colorless powder; mp 75-80 °C; 1H NMR (500 MHz, ACN-d3)
δ 9.74 (bs, 1H), 7.91 (d, 1H, J ) 7.7 Hz), 7.98 (d, 1H, J ) 8.1
Hz), 7.74-7.83 (m, 5H), 7.71 (t, 1H, J ) 7.4 Hz), 7.66-7.69
(m, 4H), 7.62 (t, 1H, J ) 7.5 Hz), 7.58 (s, 1H), 6.99 (bs, 1H),
6.89 (bs, 1H), 4.87 (q, 1H, J ) 6.9 Hz), 4.34 (bs, 2H), 3.87-
3.94 (m, 1H), 3.76-3.82 (m, 1H), 3.66 (d, 2H, J ) 6.2 Hz), 3.17
(t, 2H, J ) 7.3 Hz); MS (ESI) m/z 490.1 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[N′-(2-fu r ylm eth yl)h yd r a -
zin o]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e (28c).
Preparation from 27c (0.30 g, 0.69 mmol) and 14 (0.28 g, 0.69
mmol) as described for 28a yielded 65.1 mg (21%) of 28c:
colorless powder; mp 59-62 °C; 1H NMR (500 MHz, ACN-d3)
δ 9.72 (s, 1H), 8.14 (d, 1H, J ) 7.9 Hz), 7.97 (d, 1H, J ) 8.3
Hz), 7.95 (s, 1H), 7.82 (t, 2H, J ) 7.5 Hz), 7.76 (t, 1H, J ) 7.2
Hz), 7.70 (t, 1H, J ) 7.4 Hz), 7.67 (d, 2H, J ) 7.5 Hz), 7.62 (t,
1H, J ) 7.6 Hz), 7.60 (s, 1H), 7.05 (bs, 2H), 6.89 (s, 1H), 6.80
(s, 1H), 4.89 (q, 1H, J ) 6.8 Hz), 4.41 (bs, 2H), 3.82-3.92 (m,
2H), 3.68 (dd, 1H, J ) 5.9/14.5 Hz), 3.64 (dd, 1H, J ) 7.7/14.0
Hz), 3.17 (t, 2H, J ) 7.0 Hz); MS (ESI) m/z 446.2 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[(4-ben zylp ip er a zin o)m eth -
yl]ca r boxa m id o}-3-(1H-3-in d olyl)p r op a n a m id e (35). Com-
pound 33 (170 mg, 0.273 mmol) was dissolved in CH2Cl2 (3
mL), and triisopropylsilane (0.1 mL) was added. After the
mixture was cooled to 0 °C, TFA (3 mL) was added dropwise
over 5 min and the solution was stirred for 1 h. Then the
solvent was removed in vacuo and the residue was treated with
DMSO (8 mL), water (1 mL), and acetic acid (1 mL). The
mixture was stirred overnight at room temperature. Evapora-
tion to dryness and RP-HPLC purification yielded 97 mg (68%)
of 35: colorless powder; mp 67-70 °C; 1H NMR (500 MHz,
CDCl3) δ 8.40 (s, 1H), 7.60 (d, 1H, J ) 7.9 Hz), 7.55 (d, 1H, J
) 7.5 Hz), 7.43-7.49 (m, 3H), 7.34-7.37 (m, 3H), 7.23-7.27
(m, 2H), 7.19 (t, 2H, J ) 7.2 Hz), 7.13 (t, 1H, J ) 7.5 Hz), 7.05
(d, 2H, J ) 7.4 Hz), 6.96 (s, 1H), 6.26 (s, 1H), 4.68 (q, 1H, J )
7.5 Hz), 4.03 (dd, 2H, J ) 12.9/23.8 Hz), 3.48-3.55 (m, 1H),
3.38-3.45 (m, 1H), 3.23 (dd, 1H, J ) 7.4/14.8 Hz), 3.14 (dd,
1H, J ) 7.2/14.8 Hz), 3.08 (m, 2H), 2.70 (t, 2H, J ) 6.9 Hz),
2.61-2.75 (m, 8H); MS (ESI) m/z 524.4 [M + H]+.
N1-(2-P h en yleth yl)-(2R)-2-{[(4-ben zylp ip er id in o)m eth -
yl]car boxam ido}-3-(1H-3-in dolyl)pr opan am ide (36). Prepa-
ration from 34 (180 mg, 0.289 mmol) as described for 35
yielded 109 mg (72%) of 36: colorless powder; mp 73-75 °C;
1H NMR (500 MHz, DMSO-d6) δ 10.84 (s, 1H), 8.81 (d, 1H, J
) 8.3 Hz), 8.25 (t, 1H, J ) 5.2 Hz), 7.61 (d, 1H, J ) 7.7 Hz),
7.14-7.31 (m, 11H), 7.09 (s, 1H), 7.02 (t, 1H, J ) 7.3 Hz), 6.96
(t, 1H, J ) 6.9 Hz), 4.60 (q, 1H, J ) 6.9 Hz), 3.81 (d, 1H, J )
15.2 Hz), 3.67 (d, 1H, J ) 13.1 Hz), 3.21-3.34 (m, 3H), 3.06
(dd, 1H, J ) 4.9/14.4 Hz), 2.97-2.93 (m, 3H), 2.59-2.67 (m,
3H), 2.46-2.48 (m, 2H), 1.57-1.70 (m, 3H), 1.32-1.46 (m, 2H);
MS (ESI) m/z 523.3 [M + H]+.
diisopropylethylamine; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; EC, effect concentration;
ESI, electrospray ionization; FLIPR, fluorometric imag-
ing plate reader; Fmoc, 9-fluorenylmethoxycarbonyl;
FMPE, 2-(4-formyl-3-methoxyphenoxy)ethyl; GPCR, G-
protein-coupled receptor; GRP, gastrin-releasing pep-
tide; GRP-R, gastrin-releasing peptide receptor; HATU,
2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate; HOAc, acetic acid; HOAt,
N-hydroxy-9-azabenzotriazole; HOBt, N-hydroxybenzo-
triazole; mRNA, messenger ribonucleic acid; MS, mass
spectrometry; NMB, neuromedin B; NMB-R, neurome-
din B receptor; NMP, N-methylpyrrolidinone; PE, poly-
ethylene; propylamide is n-propylamide; RP-HPLC,
reverse-phase high-pressure performance chromatog-
raphy; SAR, structure-activity relationship; SCLC,
small-cell lung cancer; TBTU, 2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium tetrafluoroborate; TFA, tri-
fluoroacetic acid; THF, tetrahydrofuran, TIPS, triiso-
propylsilane; TLC, thin-layer chromatography; TMS,
trimethylsilane; TMOF, trimethyl orthoformate; TOF,
time of flight.
Su p p or tin g In for m a tion Ava ila ble: Detailed descrip-
tions of molecular cloning, transfection, cell culture, the FLIPR
assay, and the FLIPR antagonist experiment, synthesis pro-
cedures of compounds 7f, 7g, 8-12, 14, 20, 22d , 23, 25-27,
29-34, and tables of analytical data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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