Novel agonists and antagonists for human protease activated receptor 2

Article abstract of DOI:10.1021/jm100984y

Human protease activated receptor 2 (PAR2) is a G protein-coupled receptor that is associated with inflammatory diseases and cancers. PAR2 is activated by serine proteases that cleave its N-terminus and by synthetic peptides corresponding to the new N-terminus. Peptide agonists are widely used to characterize physiological roles for PAR2 but typically have low potency (e.g., SLIGKV-NH₂, SLIGRL-NH₂), uncertain target selectivity, and poor bioavailability, limiting their usefulness for specifically interrogating PAR2 in vivo. Structure-activity relationships were used to derive new PAR2 agonists and antagonists containing nonpeptidic moieties. Agonist GB110 (19, EC₅₀ 0.28 μM) selectively induced PAR2-, but not PAR1-, mediated intracellular Ca²⁺ release in HT29 human colorectal carcinoma cells. Antagonist GB83 (36, IC₅₀ 2 μM) is the first compound at micromolar concentrations to reversibly inhibit PAR2 activation by both proteases and other PAR2 agonists (e.g., trypsin, 2f-furoyl-LIGRLO-NH ₂, 19). The new compounds are selective for PAR2 over PAR1, serum stable, and suitable for modulating PAR2 in disease models.

Full text of DOI:10.1021/jm100984y

7428 J. Med. Chem. 2010, 53, 7428–7440
DOI: 10.1021/jm100984y
Novel Agonists and Antagonists for Human Protease Activated Receptor 2
Grant D. Barry,† Jacky Y. Suen,† Giang T. Le, Adam Cotterell, Robert C. Reid, and David P. Fairlie*
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland,
Brisbane Qld 4072, Australia. †Joint first authors.
Received July 31, 2010
Human protease activated receptor 2 (PAR2) is a G protein-coupled receptor that is associated with
inflammatory diseases and cancers. PAR2 is activated by serine proteases that cleave its N-terminus and
by synthetic peptides corresponding to the new N-terminus. Peptide agonists are widely used to
characterize physiological roles for PAR2 but typically have low potency (e.g., SLIGKV-NH2,
SLIGRL-NH2), uncertain target selectivity, and poor bioavailability, limiting their usefulness for
specifically interrogating PAR2 in vivo. Structure-activity relationships were used to derive new PAR2
agonists and antagonists containing nonpeptidic moieties. Agonist GB110 (19, EC50 0.28 μM)
selectively induced PAR2-, but not PAR1-, mediated intracellular Ca2þ release in HT29 human
colorectal carcinoma cells. Antagonist GB83 (36, IC50 2 μM) is the first compound at micromolar
concentrations to reversibly inhibit PAR2 activation by both proteases and other PAR2 agonists (e.g.,
trypsin, 2f-furoyl-LIGRLO-NH2, 19). The new compounds are selective for PAR2 over PAR1, serum
stable, and suitable for modulating PAR2 in disease models.
Introduction
cathepsin G) and has been linked to inflammatory and prolif-
erative disorders.3-7
Among the most unusual of almost 1000 known human G
protein-coupled receptors (GPCRsa)1,2 are four protease acti-
vated receptors (PARs),3,4 which have no known endogenous
extracellular ligands. Instead, serine proteases indirectly activate
at least four PAR subtypes by cleaving their N-terminus,
exposing a new extracellular N-terminus that folds back onto
and intramolecularly self-activates PAR.5 Research on PARs
has focused on PAR1 because it is a receptor for the serine
protease thrombin, and thus a potential therapeutic target in
cardiovascular disease.3 PAR2 is not activated by thrombin, but
it is activated by other serine proteases (e.g., trypsin, tryptase,
Most PAR2 research has attempted to define physiological
and pathophysiological roles for this receptor. Distributed
widely throughout the body, PAR2 signals through intracellular
G proteins3,8 associated with release of intracellular Ca2þ via
phospholipase C activation and inositol triphosphate (GRq),8
p38 MAP kinase and ERK phosphorylation, as well as adenylyl
cyclase inhibition (GRi),9-11 and Rho-dependent phagocytosis
(GR12/13).12 PAR2 activation has been linked to proliferation,
metastasis, and angiogenesis in many cancers especially of the
stomach,13 colon,11,14 breast, and pancreas.11,15-17 PAR2 has
been implicated as a pro-inflammatory mediator in arthritis,18
inflammatory bowel disease,19,20 pancreatitis,21-27 and cardio-
vascular disease,28 while it has also been reported as anti-
inflammatory and protective in other conditions such as gastric
ulcer,29 colitis,30 asthma,27,31-33 and liver fibrosis.30,34-36
Clearly, these roles are not yet well understood.22,24-26,37
PAR2-/- deficient mice show delayed appearance and
decreased development of mammary adenocarcinoma38 and
abolished revascularization in hypoxia-induced angiogenesis.39
In inflammation, PAR2 knockouts had reduced dendritic cell
trafficking and T cell activation,40 and higher survival against
antiphospholipid syndrome by lowered neutrophil activa-
tion.41 PAR2 deficient mice also had impaired production
of IgE and IL-4,42 reduced contact sensitivity in airway
inflammation,43 resistance to adjuvant-induced arthritis,18
or delayed onset of inflammation.44 Roles for PAR2 in inflam-
mation were further supported by studies with small interfering
RNA (siRNA) for PAR2, which repeatedly showed suppres-
sion of innate immunity markers, such as IL8,40,45 CXCL5,
and CCL20.46 By contrast, PAR2 activation attenuated
pancreatitis-related hyperalgesia,47 and thus, the role of
PAR2 could be cell type-specific. This is supported by a
*To whom correspondence should be addressed. Telephone: þ61-
733462989. Fax: þ61 733462990. E-mail: d.fairlie@imb.uq.edu.au.
a Abbreviations: ACN, acetonitrile; Boc, tert-butoxycarbonyl; BOP,
(benzotriazol-1-yloxy)-tris(dimethylamino) phosphonium hexafluoro-
phosphate; Cha, cyclohexylalanine; DCM, dichloromethane; DIC, 1,3-
diisopropylcarbodiimide; DIPEA, N,N-diisopropylethylamine; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTPM, 1,3-dimethyl-
5-[(dimethylamino)methylene]-2,4,6-(1H,3H,5H)-trioxopyrimidine; EC50,
molar concentration that produces 50% of the maximum response of an
agonist; ERK, extracellular signal-regulated kinase; ESMS, electrospray
mass spectroscopy; FCS, fetal calf serum; Fmoc, 9H-fluoren-9-ylmethoxy-
carbonyl; GI, gastrointestinal, G protein, guanosine monophosphate-
protein; GPCR, G protein-coupled receptor; HBSS, Hank’s balanced
salt solution; HEK293, human embryonic kidney 293 cells; HBTU,
O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid); HOBt, 1-hydroxybenzotriazole; HT29, human colon adenocar-
cinoma grade II cells; HRMS, high resolution mass spectroscopy; IC50
,
molar concentration of an antagonist that inhibits 50% of a known
concentration of agonist activity; IgE, immunoglobulin E; IL, interleukin;
IMDM, Iscove’s modified Dulbucco’s medium; iCa2þ, intracellular Ca2þ
;
MAP kinase, mitogen-activated protein kinase; MBHA, methylbenzhy-
drylamine; PAR, protease activated receptor; rpHPLC, reversed phase high
performance liquid chromatography; SAR, structure-activity relation-
ships; siRNA, small interfering ribonucleic acid; SEM, standard error of
mean; SPPS, solid phase peptide synthesis; TFA, trifluoroacetic acid; TIPS,
triisopropylsilane; TCP, tritylchloride-polystyrene.
r
pubs.acs.org/jmc
Published on Web 09/28/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7429
Table 1. Intracellular Ca2þ Release by 1-7 in HT29 Cells
protective role for PAR2 on neurons, yet a pathogenic role on
glial cells in Alzheimer’s disease.48 PAR2 antibodies are avail-
able to sequences upstream of the PAR2 cleavage site, across
the cleavage site, at the newly exposed N-terminus, and at the
C-terminus, but they have not been extensively used in PAR2
research due to their lack of selectivity. Some studies have
shown that PAR2 interaction with tissue factors is important
for tumor growth.49
compd (50 μM)
X1
X2
% relative activitya
Trypsin is a potent activator of PAR2 in the GI tract where
pancreatic trypsin is found, and in colon, airway epithelium,
neuronal, and vascular endothelial cells and skin, intestine,
kidney, and pancreas where trypsinogen expression has been
demonstrated.50 However, like other serine proteases, trypsin
nonspecifically cleaves many proteins in addition to PAR2.
Thus, much of the evidence for PAR2 in disease has had to rely
upon the use of short synthetic hexapeptides, corresponding to
the new N-terminal sequence of PAR2 exposed after proteolyt-
ic cleavage. Hexapeptides SLIGKV-NH2 and SLIGRL-NH2,
corresponding to the tethered ligand human and murine
sequences, respectively, can activate human PAR2 at micro-
molar concentrations without the need for proteases which are
much more potent agonists (nM).3 Due to their low potency,
these short peptide agonists have been administered at high
concentrations to elicit responses51 and, although selective for
PAR2 over PAR1, at such high concentrations these peptides
also interact with other receptors.52,53 Similarly, the first known
antagonist of protease-activated PAR2 has only millimolar
affinity for PAR2 and selectivity is most unlikely.54 A second
antagonist reported for PAR2 is active at micromolar concen-
trations but is completely inactive against endogenous activa-
tors like the serine protease trypsin.55 Our understanding of
PAR2 physiology is thus still quite limited by the lack of truly
selective and potent PAR2 agonists and antagonists suitable for
in vivo studies.
More potent peptide agonists have been created for
PAR2.56-64 Hexapeptide analogues in which serine is
replaced by 2-furoyl (e.g., 2-furoyl-LIGRL-NH2),61,62 a residue
is added to the seventh position (e.g., SLIGRLI-NH2),63,64 the
sixth residue is changed to para-nitrophenylalanine (e.g.,
SLIGR(pNO2F)I-NH2),64 or combinations of these N- and
C-terminal changes have 5-30-fold higher agonist potency
than SLIGRL-NH2 and are selective for PAR2 over PAR1.
Other heterocyclic replacements for serine can also result in
equipotent PAR2 agonists,64 while large aromatic groups in
place of the C-terminal leucine impart a similar enhancement in
PAR2 agonist potency. Screening of 250,000 druglike com-
pounds produced two small molecule agonists of PAR2 with
similar potency to 2-furoyl-LIGRL-NH2, some selectivity for
PAR2, and metabolic stability in vivo.65,66 Although down-
sizing agonist peptides derived from SLIGRL-NH2 has been
found to reduce PAR2 agonist potency,58 this was not the case
for more potent agonist peptides with heterocyclic replacements
for serine at the N-terminus. The present study began with a
heterocyclic replacement for the N-terminal serine, then trunca-
tion and optimization of very short peptides with selective PAR2
agonist potency, before altering the C-terminus with nonpeptidic
fragments that dictate agonist or antagonist potency.
1
2
3
4
5
6
7
Leu
Leu
Leu
Leu
Cha
Phe
Chg
Ile
38 ( 5
43 ( 1
22 ( 2
19 ( 3
58 ( 10
67 ( 2
12 ( 1
Cha
Phe
Phe(p-F)
Ile
Ile
Ile
a Versus 100% iCa2þ release induced by 2f-LIGRLI-NH2 (50 μM).64
resin using Fmoc-protected amino acids. The intention was
to identify the minimum number of residues required for
PAR2 activity. Compounds were evaluated at a single con-
centration (50 μM) for induction of intracellular Ca2þ
(iCa2þ) release in HT29 colon cancer cells relative to the
calcium efflux produced by a PAR2 agonist, 2-furoyl-LIGR-
LI-NH2 (2f-LIGRLI-NH2),64 at the same concentration
(Table 1). Compounds were not active enough to determine
EC50, being too insoluble above 50 μM to test at higher
concentrations. Table 1 shows that bulky amino acids linked
to the 2-furoyl group have some agonist activity, with cyclic
aliphatic or aromatic side chains being preferred at position
X1 (5, 6 vs 1), while shortening the aliphatic side chain (7) was
detrimental.
Compounds 5 and 6 were next evaluated for PAR2 versus
PAR1 selectivity in order to choose between them for further
synthetic elaboration. In hexapeptide agonists based on
SLIGRL, substitution of leucine 2 with phenylalanine but
not cyclohexylalanine has been reported to favor binding to
PAR1 on HEK293 cells, thereby reducing PAR2 selecti-
vity.3,59 A receptor desensitization assay67 was therefore
performed (Figure 1) by treating HEK293 cells with PAR2
agonist64 2f-LIGRLI-NH2 (6.7 μM) followed by a second
treatment with the same agonist after 6 min (t370) to
re-establish a baseline (7.5 μM final concentration, Figure 1A),
consistent with desensitization of PAR2. Alternatively, if the
second treatment at t370 was with the PAR1-selective weak
agonist TFLLR-NH2 (80 μM), calcium efflux was observed
since PAR1 had not been densensitized by prior treatment
with PAR2 agonist (Figure 1B). In a parallel experiment,
cells were treated with 80 μM 5 or 6 at t370 following
desensitization of the PAR2 receptors with 2f-LIGRLI-
NH2. Lack of a second calcium efflux following treatment
with 5 (Figure 1C) indicated PAR2 selective activation,
whereas 6 induced a calcium response (Figure 1D), suggest-
ing no PAR2 selectivity and activation of another receptor(s).
The same experiment in which cells were first desensitized by
treatment with 100 μM PAR1 selective agonist TFLLR-NH2
(Figure 1E, F) showed that 6 was activating mainly through
PAR1, whereas 5 was capable of inducing a PAR1-independent
Ca2þ response consistent with selectivity for PAR2 over PAR1.
Our previous SAR studies64 with PAR2 activating hexa-
peptides on HEK293 cells indicated that serine could be
replaced by several heterocyclic alternatives to the 2-furoyl
group. We therefore compared Ca2þ efflux in HEK293 cells
in response to 5 (EC50 > 50 μM, n = 3) and analogues in which
the furoyl group was replaced with pyrazoyl (8, EC50 >100 μM,
Results and Discussion
Truncating PAR2 Activating Peptides. A series of short
peptide agonists 1-7 (Table 1), featuring a heterocyclic
replacement for serine at the N-terminus, was synthesized
by standard solid phase methodology on Rink amide MBHA

7430 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Barry et al.
Figure 2. Concentration dependent iCa2þ release in HEK293 cells by
compound 9, expressed as % response induced by ionophore A-23187.
Table 2. Effect of C-Terminal Substituent R in 11-18 on Agonist-
Induced Calcium Mobilization in HT29 Cells
Figure 1. PAR2 versus PAR1 selectivity via receptor desensitiza-
tion. (A) Treatment of HEK293 cells (t10) with PAR2 selective
agonist 2f-LIGRLI-NH2 (6.7 μM), followed by second treatment at
t370 (7.5 μM final conc) showing desensitization. (B) After initial
treatment at t10 with 2f-LIGRLI-NH2, cells were treated with PAR1
selective agonist TFLLR-NH2 (80 μM) at t370, showing Ca2þ
release. Cells desensitized with 2f-LIGRLI-NH2 (6.7 μM, t10) were
then treated at t370 with 80 μM PAR2 ligand 5 (C) or nonselective 6
(D). Cells desensitized at t10 with the PAR1 selective TFLLR-NH2
(100 μM) were then treated at t310 with 80 μM 5 (E), suggesting no
PAR1 activation, or 6 (F) that activates PAR1.
n=3), isoxazoyl (9, EC50 4 μM, n = 3), or methyloxazoyl
(10, EC50 >100 μM, n = 3). The most active compound 9 was
the only compound soluble enough at e100 μM concentrations
to permit determination of a reliable EC50 by varying concentra-
tions (Figure 2), and even 9 was not soluble above 100 μM,
limiting accurate determination of EC50. Thus, substituting Ser
by isoxazoyl and Ile by Cha had converted Ser-Leu-Ile-NH2 into
a small molecule agonist 9 (MW 378) of comparable potency to,
but half the size of, the hexapeptide SLIGRL-NH2 (MW 657).
1 At 1 μM relative to 1 μM SLIGRLI-NH2 (100% iCa2þ).
resin, and then isoleucine, cyclohexylalanine, and isoxazole-
5-carboxylic acid were sequentially coupled to each diamine
via standard Fmoc chemistry (Scheme S1, Supporting In-
formation). Similarly, 4-(aminomethyl)piperidine and 4-(2-
aminoethyl)aniline were separately coupled to TCP resin in
parallel, the resins were split two more times, and each func-
tional group was coupled to either the Fmoc-5-aminovaleric
acid or Fmoc-β-alanine linkers, followed by the sequential
coupling of the remaining amino acids (Scheme S2, Supporting
Information). Finally, the use of a pyridine moiety as a func-
tional group necessitated commencing the solid phase synthesis
by coupling the linkers to TCP resin (Scheme S3, Supporting
Information). Either Fmoc-protected 5-aminovaleric acid or
Fmoc-β-alanine was first coupled to TCP resin. Ile, Cha and
isoxazole were then sequentially coupled to the linkers via
Fmoc SPPS and then cleaved and purified by rpHPLC. The
two intermediates were then amidated with 3-aminomethyl
pyridine via the free acid to give compounds 11-18 (Table 2).
Derivatization of Agonist 9. Linking an amine to 9 in order
to mimic the arginine side-chain in SLIGRL-NH2 was
considered to be a worthwhile interim measure for both
enhancing agonist potency and increasing water solubility.
Compound 9 was linked to three different amine-containing
moieties using one of two aliphatic linkers of different length
(Schemes S1-S3, Supporting Information). Either 1,3-dia-
minopropane or 1,5-diaminopentane was coupled to TCP

Article
Compounds 11-18 were more water-soluble than 5 or 9.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7431
increasing the distance between the component analogous
to 9 and the amine terminus was advantageous, especially for
13 and 14. Whilst coupling the 4-(aminomethyl)piperidine
moiety to the lead with the shorter β-alanine (13) elicited a
97% response, extending the same ligand via 5-aminovaleric
acid (14) elicited an even stronger calcium response (181% of
that for SLIGRLI-NH2). A full concentration-response
curve for agonist 14 in HEK293 cells gave EC50 0.37 μM
(Figure 3).
To further improve agonist potency, a set of more con-
strained linkers was examined in place of the flexible ethy-
lenediamine linker in 14 while still enabling flexible orienting
and favorable positioning of the amine in the receptor.
Compounds 19-28 were obtained through parallel synthesis
by first coupling 4-(aminomethyl)piperidine to TCP resin
and then protecting the primary amine with 1,3-dimethyl-
5-[(dimethylamino)methylene]-2,4,6-(1H,3H,5H)-trioxo-
pyrimidine (DTPM) (Scheme S4, Supporting Information).
This allowed the analogues to be synthesized in parallel,
while reducing side-product formed when the 4-(amino-
methyl)piperidine was coupled to the linker via the second-
ary amine. The relatively low reactivity of the linker amine
resulted in some side products in which the adjoining iso-
leucine residue had racemized, as well as deletion products in
which the isoleucine failed to couple to the linker. These
impurities were readily separated from the product by
Compared with SLIGRLI-NH2,63,64 all were more effective
agonists at 1 μM in eliciting iCa2þ release in HT29 cells
(Table 2). Agonist potency improved over the more flexible
analogues (11, 12) when the amine was mounted on a more
constrained linker (e.g., 13-16) that restricts conformational
freedom, possibly directing the amine to a hydrogen bond
acceptor in PAR2. Alternatively, the cyclic group may
provide additional hydrophobic interactions with PAR2.
Terminating the ligand with a pyridine substituent (15, 16),
capable of aromatic, hydrophobic, or ionic interactions with
receptor, was also beneficial for agonist potency. In all cases,
Figure 3. Ca2þ release in HT29 cells for 14 (1) versus SLIGRLI-
NH2 (9), relative to 100% for A-23187 (mean ( SEM).
Table 3. Release of iCa2þ in HT29 Cells by Constrained PAR2 Agonists 19-28

7432 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Barry et al.
rpHPLC. Larger scale preparations of potent ligands were
made in solution using Boc-protected amino acids and
linkers, starting from DTPM-protected 4-aminomethylpi-
peridine with deprotection at the end of the synthesis using
2% hydrazine in DMF. Solution phase chemistry eliminated
racemization of the isoleucine residue. Agonist activity was
measured for compounds 19-28 by calcium efflux in HT29
human colon carcinoma cells (Table 3).
Restricting the conformational freedom of the linker with
a 4-(aminomethyl)piperidine- substituent in 19 was slightly more
effective than the aminobutyl- linker of 14 (EC50 0.28 vs 0.36
μM). However, coupling of the tightly constrained amino-
methyl piperidine moiety para to the isoleucine carbonyl via
an isonipecotic acid linker (20) proved to be detrimental for
agonist activity. Coupling the two functional groups para to
each other, but altering the length and conformation with a
phenyl linker (4-aminobenzoic acid, 21), further reduced
potency. Addition of a methylene group to the linker, adding
some flexibility to the scaffold, was beneficial for activity of
the para coupled linkers, more so when the methylene was
situated between the phenyl ring and the aminomethyl
piperidine group using 4-aminophenylacetic acid (22) than
when before the linker via 4-aminomethylbenzoic acid (23).
Similar observations were noted when meta-coupling linkers
were employed. While substitution with the aliphatic nipe-
cotic acid linker (24) decreased potency of the ligand
∼45-fold, substitution with 3-aminobenzoic acid (25) resulted
in agonist activity comparable to that of SLIGRL-NH2,
however still less potent than the valeric acid-linked agonist.
Extending the compound by adding a methylene with 3-amino-
methylbenzoic acid was beneficial, resulting in the most
potent ligand of this series (19, EC50 0.28 ( 0.01 μM).
Enforcing a rigid turn conformation, either an R-turn via
the pipecolinic acid linker (26) or a β-turn with a dipeptide
(27) or Freidinger’s lactam (28) linkers, was not conducive
for agonist activity. Thus, incorporating constraints in 14
improved agonist potency, provided that the linker was not
too rigid and that there was some degree of flexibility,
perhaps enabling the amine to make a key interaction with
receptor residues.
Next, the selectivity for PAR2 over PAR1 of the most
potent compounds from the constrained agonist library was
evaluated via a desensitization assay (Figure 4). As described
above, HT29 cells were administered with 2f-LIGRLI-NH2
(6.7 μM) at t0 and then re-exposed at t300 to a second
treatment of the same compound (7.5 μM). This second
addition of PAR2 agonist failed to generate calcium efflux,
indicating that cells had been desensitized to PAR2 activa-
tion (Figure 4A). Following desensitization via exposure to
2f-LIGRLI-NH2, cells were treated at t300 with 100 μM 19
(Figure 4B). This also failed to generate a calcium response,
suggesting that 19 is acting via the PAR2 receptor. Treating cells
with 22 at t300 after first desensitizing with 2f-LIGRLI-NH2
(Figure 4C) did generate a calcium response, indicating that
agonist 22 was not selective for PAR2 and thus it is likely
activating other receptors.
Figure 4. Desensitization assays for constrained PAR2 agonists.
(A) Control using 2-furoyl-LIGRLI-NH2 at t0 to desensitize PAR2
on HT29 cells, then treated at t300 with 2-furoyl-LIGRLI-NH2
(6.7 μM) to demonstrate PAR2 desensitization. (B) Cells treated
with 2-furoyl-LIGRLI-NH2 at t0 and then at t300 with compound 19
(100 μM). (C) After desensitizing cells with 2-furoyl-LIGRLI-NH2,
compound 22 (100 μM) was administered at t300. Thus, agonist 19 is
PAR2 selective, and 22 is not PAR2 selective.
in ref 64) and is one of the most potent and serum-stable
peptide agonists reported to date. Each compound was
added to filtered fetal calf serum (5 mL) so that the final
concentration of agonist was 1 mM. At regular intervals,
500 μL aliquots were removed and added to 1.5 mL MeCN.
The solution was centrifuged (2500 rpm, 5 min) before
removing supernatant (1 mL) and analyzing via ESMS and
rpHPLC. Data from these experiments are expressed as
percent of peak area recorded from the HPLC trace at t0
(Figure 5). Over 95% 19 was present in FCS after 4 h,
compared with ∼78% 29 and 57% SLIGRL-NH2. In addi-
tion to serum stability, compound 19 has cLogP 3.1,
4 H-bond donors, and 7 H-bond acceptors, and it conforms
to Lipinski’s rule of five with the exception of molecular
weight (608.8). These properties have enabled ongoing stud-
ies in experimental pharmacology with 19 in animal models
of inflammatory diseases. The potency, selectivity, serum
stability, and druglike composition of 19 makes it more
Serum Stability of PAR2 Agonists 19 and 29. To assess
whether reducing the peptidic content of the PAR2 agonist
improved serum stability, resistance to proteolysis was com-
pared for (a) SLIGRL-NH2, (b) 4-(2-methyloxazoyl)-
LIGR-(4-I)Phe-NH2 (29) containing a heterocyclic moiety
in place of serine and the unnatural amino acid 4-iodophe-
nylalanine in place of leucine, and (c) compound 19. Com-
pound 29 has previously been reported by us (compound 52

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7433
Figure 5. Comparative serum stability of PAR2 agonists SLIGRL-
NH2 (1), 19 (2), and 29 ([) in FCS at 37 °C over 4 h. Data expressed
as percent of peak area from HPLC trace at t = 0.
effective than known PAR2 agonists and should enable its
use as an effective probe of the biological functions of PAR2
in vivo.
Figure 6. Comparison of iCa2þ release in HT29 cells. (A) PAR2
agonist 19 (EC50 0.34 ( 0.05 μM). (B) PAR2 antagonist 30 alone (2)
or its inhibition of 2.5 μM agonist SLIGRLI-NH2 (9, IC50 57 μM).
Data = mean ( SEM (n = 3).
40-80%, and at 50 μM compound 36 completely inhibited
the intracellular calcium efflux. To determine if the observed
inhibition could be attributed to PAR2 desensitization, the
compounds were also evaluated alone for agonist activity at
50 μM concentrations (Figure 7B). Compounds 31, 33, and
34 were clearly weak agonists, eliciting a calcium efflux
greater than that observed for 2.5 μM SLIGRLI-NH2.
Compound 32 appeared to have little activity toward
PAR2, as either an agonist or antagonist. Compounds 35
and 36 showed negligible agonist activity as defined by Ca2þ
release in HT29 cells, and thus, they can be confidently
assigned as antagonists of PAR2 on these cells. Compound
36 was the more potent antagonist in full concentration-
response curves (2 μM ( 1.0 μM, Figure 8).
Conversion to PAR2 Antagonists 30, 35, and 36. Based on
the PAR2 agonist activity of compound 22, we decided to
probe the importance of the primary amine substituent
appended to the piperidine group, without substantially
altering the space-filling characteristics of 22. Replacing
the aminomethyl piperidine moiety with morpholine gave
compound 30. In contrast to the agonist profile shown by 19
on HT29 cells (Figure 6A), compound 30 showed no agonist
activity but did inhibit intracellular Ca2þ release induced by
2.5 μM SLIGRLI-NH2 (Figure 6B). The inhibition assay
was performed by preincubating HT29 cells with increasing
concentrations of 30 15 min prior to administration of
SLIGRLI-NH2 at 2.5 μM (EC80). Although not a potent
antagonist (IC50 57 μM), this was a clue for the design and
development of PAR2 antagonists.
The steric effect on antagonist potency was examined by
appending bulky uncharged moieties directly to the agonist
template 9, amidating with different amines bearing bi and
tricyclic fused rings that could interrogate different pharma-
cophore space. These included tetrahydroisoquinoline (31),
1-naphthylamine (32), N-phenylpiperizine (33, 34), 2-ami-
nobiphenyl (35), and spiroindanepiperidine (36) (Figure 7).
Antagonist activity was assessed in single point inhibition
assays at 5 and 50 μM concentrations against 2.5 μM
SLIGRLI-NH2 (Figure 7A). At 5 μM, these compounds
attenuated the agonist response of SLIGRLI-NH2 by
Figure 8A compares intracellular Ca2þ release induced in
HT29 colon carcinoma cells by three different PAR2 ago-
nists, demonstrating with concentration response curves that
agonist 19 (9, pEC50 6.6 ( 0.05) is an equipotent agonist with
hexapeptide 2f-LIGRLO-NH2 (O, pEC50 6.7 ( 0.05), but it
is not as potent as trypsin (pEC50 8.2 ( 0.08). Compound 19
was also selective (up to 100 μM) for PAR2 over PAR1, as
indicated by lack of response after PAR2 was desensitized by
5 μM 2f-LIGLRO-NH2 (Figure 4B).
Figure 8B shows that antagonist 36 selectively inhibited
iCa2þ release induced in HT29 cells by two different PAR2
agonists at their EC80 concentrations: trypsin (pIC50 5.7 (
0.1) and 2f-LIGRLO-NH2 (pIC50 5.1 ( 0.2). It also antag-
onizes 19 (pIC50 5.1 ( 0.2; data not shown). Moreover, 36
had no effect against PAR1 agonist thrombin at 10 U/mL.
Compound 36 is the only PAR2 antagonist reported to date
that is able to inhibit at low micromolar concentrations both
trypsin- and peptide-agonist-induced iCa2þ in PAR2 expres-
sing native cells, with others being either several orders of
magnitude less potent54 or unable to block endogenous
protease agonists of PAR2.55 The present data support the
idea that the nonpeptidic PAR2-selective agonist 19 and

7434 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Barry et al.
antagonist 36 could be extremely valuable tools for probing
PAR2 activation and blockade in vivo.
Conclusions. The G protein coupled receptor known as
protease activated receptor 2 (PAR2) is upregulated in many
inflammatory diseases and multiple cancers. It is activated in
vivo by mainly serine proteases (e.g. trypsin, tryptase), which
cleave the N-terminus of the receptor, but there are no known
nonproteolytic endogenous agonists. Although short syn-
thetic peptides, corresponding to the new PAR2 N-terminus
created through cleavage, are weak PAR2 agonists, they are
mainly of low potency, questionable selectivity, and poor
bioavailability that limit their usefulness and reliability as
PAR2 investigative tools in vivo. Here we have derived short
agonist peptides, converted them to more potent PAR2
agonists by replacing amino acids with certain nonpeptidic
fragments often present in other GPCR ligands,1 and derived
nonpeptidic PAR2 antagonists through structure-activity
relationships. Nonpeptidic agonist 19 (EC50 280 nM) selec-
tively induced intracellular Ca2þ release in human colorectal
carcinoma cells with comparable potency to the most potent
known peptide agonist 2f-LIGRLO-NH2. Nonpeptidic antag-
onist 36 (IC50 2 μM) selectively and reversibly inhibits intra-
cellular Ca2þ release induced in HT29 cells by three differ-
ent PAR2 agonists (trypsin, 2f-LIGRLO-NH2, and 19), and it
does not inhibit the PAR1 agonist thrombin. Compound 36 is
the most potent and only knownPAR2antagonist reported to
inhibit both trypsin- and peptide-induced activation of PAR2
at low micromolar concentrations. PAR2 agonist 19 will be
designated GB110 and PAR2 antagonist 36 will be GB83 in
future reports by our group on in vivo properties of these
compounds. Compounds 19 and 36, which were selective for
PAR2 over PAR1 with no effect on intracellular Ca2þ release
in cells desensitized to PAR2 activation, are serum stable and
suitable for oral administration, thus enabling pharmacolo-
gical investigation of the effects of PAR2 regulation in animal
models of human diseases.
Figure 7. Prospective PAR2 antagonists (31-36). (A) Compounds
31-36 tested for antagonism at 5 μM (clear) and 50 μM (black) by
inhibition of calcium efflux elicited in HT29 cells by SLIGRLI-NH2
(2.5 μM). Each column represents percent inhibition versus 100%
for complete inhibition. (B) Compounds 31-36 tested at 50 μM for
agonist activity (% calcium efflux relative to 100% for SLIGRLI-
NH2 at 2.5 μM). Compounds 35 and 36 are antagonists.
Experimental Methods
Compounds 1-7. Portions (7 ꢀ 200 mg) of rink amide MBHA
resin (0.14 mmol scale) were sequentially coupled with two
Fmoc-protected amino acids (4 equiv amino acid, 4 equiv
HBTU, 4 equiv DIPEA, in DMF), followed by furan-2-car-
boxylic acid (4 equiv HBTU, 4 equiv DIPEA, in DMF),
following standard Fmoc SPPS. Ligands were then cleaved
from resin using 95:2.5:2.5 TFA/TIPS/H2O stirred for 1.5 h.
The solvent was filtered in a sintered glass funnel and then
removed in vacuo, and products were purified via rpHPLC
fitted with a tunable absorbance detector (λ 214 nm), using a
˚
Phenomenex C18 column (300 A, 22 ꢀ 250 mm) with a gradient
of 0% B to 100% B over 30 min (solvent A 0.1% TFA in H2O,
solvent B 0.1% TFA, 10% acetonitrile, 90% H2O). Compounds
were characterized by HRMS, compounds were characterized by
HRMS and 1H NMR using DMSO-d6 as solvent, and purity of
compounds was assessed via analytical rpHPLC under different
conditions (0% B to 100% B over 30 min gradient, Phenomenex
˚
C18 column, 300 A, 4.6 ꢀ 250 mm, 214 nm λ). Compounds were
Figure 8. Characterization of nonpeptide agonist GB110 (19) and
antagonist GB83 (36) of PAR2. (A) Comparison of PAR2 agonist-
induced intracellular Ca2þ release in HT29 colon carcinoma cells by
hexapeptide SLIGRL-NH2 (0, pEC50 5.6 ( 0.15), hexapeptide
2f-LIGRLO-NH2 (O, pEC50 6.7 ( 0.05), and 19 (9, pEC50 6.6 (
0.05). 19 is equipotent with one of the most potent known PAR2
activating peptides, 2f-LIGRLO-NH2. (B) 36 selectively inhibits
intracellular Ca2þ release induced in HT29 cells by two different
PAR2 agonists at their EC80 concentrations: trypsin (b, pIC50 5.7 (
0.1) and 2f-LIGRLO-NH2 (O, pIC50 5.1 ( 0.2). But it has no effect
against PAR1 agonist thrombin (0) at 10U/mL.
repurified if found to be below 95%, with all final compounds
being >95% pure by analytical HPLC trace analysis.
2-Furoyl-Leu-Ile-NH2 (1). Yield 15 mg (28% isolated); Rt
22.24 min; HRMS 338.2074 (calc), 338.2068 (found). 1H NMR
(400 MHz), δ 0.81(t, 5H, J = 6.90Hz); 0.82 (d, 6H, J = 6.90 Hz);
0.85 (d, 3H, J = 6.43 Hz); 0.89 (d, 3H, J = 6.37 Hz); 1.01-1.09
(m, 1H); 1.39-1.53 (m, 2H); 1.59-1.72 (m, 3H); 4.13 (dd, 1H, J
7.30, 8.98 Hz); 4.46-4.52 (m 1H); 6.63 (q, 1H); 7.00 (s 1H); 7.18
(dd, 1H, J = 0.82, 3.48 Hz); 7.38(s, 1H); 7.72 (d, 1H, J=9.02 Hz);
7.86 (dd, 1H, J = 0.81, 1.75 Hz); 8.33 (d, 1H, J = 8.48 Hz).

Article
2-Furoyl-Leu-Cha-NH2 (2). Yield 26 mg (49% isolated); Rt
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7435
MHz), δ 0.79-0.83 (m, 7H); 0.889 (d, 7H, J = 5.74 Hz);
1.02-1.10 (m, 1H); 1.37-1.45 (m, 1H); 1.52-1.61 (m, 2H);
1.67-1.72 (m, 2H); 4.152 (dd, 1H, J = 7.46, 8.94 Hz); 4.62-4.68
(m, 1H); 6.997 (s, 1H); 7.405 (s, 1H); 7.984 (d, 1H, J = 8.97 Hz);
8.770 (dd, 1H, J = 1.53, 2.52 Hz); 8.807 (d, 1H, J = 8.98 Hz);
8.903 (d, 1H, J = 2.39 Hz); 9.190 (d, 1H, J = 1.43 Hz).
2-Pyrazoyl-Cha-Ile-NH2 (8). Yield 30 mg (55% isolated); Rt
24.46 min; HRMS 390.2500 (calc), 390.2514 (found). 1H NMR
(400 MHz), δ 0.79-0.83 (m, 8H,); 0.85-0.94 (m, 2H); 1.02-1.17
(m, 5H); 1.23-1.30 (m, 1H); 1.37-1.46 (m, 1H); 1.55-1.70 (m,
8H); 4.15 (dd, 1H, J = 7.47, 8.90 Hz); 4.64-4.70 (m, 1H); 7.00
(s, 1H); 7.40 (s, 1H); 7.99 (d, 1H, J = 12.74 Hz); 8.77 (dd, 1H,
J=1.48, 2.50 Hz); 8.90 (d, 1H, J = 2.42 Hz); 9.19 (d, 1H,
J = 1.55 Hz).
5-Isoxazoyl-Leu-Ile-NH2. Yield 8 mg (17% isolated); Rt 21.17
min; HRMS 339.2027 (calc), 339.2037 (found). 1H NMR (400
MHz), δ 0.81 (t, 5H, J = 7.62 Hz); 0.82 (d, 4H, J = 7.49 Hz);
0.86 (d, 3H, J = 6.42 Hz); 0.90 (d, 3H, J = 6.38 Hz); 1.00-1.10
(m, 1H); 1.40-1.54 (m, 2H); 1.58-1.73 (m, 3H); 4.13 (dd, 1H,
J 7.40, 8.98 Hz); 4.50-4.56 (m 1H); 7.00 (s, 1H); 7.15 (d, 1H, J =
1.88 Hz); 7.83 (d, 1H, J = 8.98 Hz); 8.75 (d, 1H, J = 1.92 Hz);
8.96 (d, 1H, J = 8.47 Hz).
25.38 min; HRMS 378.2387 (calc), 378.2394 (found). 1H NMR
(400 MHz), δ 0.86 (d, 4H, J = 6.32 Hz); 0.90 (d, 4H, J = 6.24
Hz); 1.11-1.20 (m, 3H); 1.29-1.43 (m, 1H); 1.43-1.68 (m 10H);
4.25-4.28 (m, 1H); 4.41-4.47 (m, 1H); 6.63 (q, 1H); 6.95 (s 1H);
7.18 (dd, 1H, J = 0.78, 3.49 Hz); 7.24 (s, 1H); 7.84-7.86 (m,
2H); 8.30 (d, 1H, J = 8.30 Hz).
2-Furoyl-Leu-Phe-NH2 (3). Yield 10 mg (19% isolated); Rt
23.34 min; HRMS 372.1918 (calc), 372.1924 (found). H NMR
1
(400 MHz), δ 0.81 (d, 3H, J = 6.43 Hz); 0.85 (d, 3H, J = 6.38 Hz);
1.37-1.42 (m, 1H); 1.48-1.59 (m, 2H); 2.81 (dd, 1H, J= 9.03, 13.84
Hz); 3.00 (dd, 1H, J = 4.95, 13.78 Hz); 4.36-4.46 (m, 2H); 6.64 (q,
1H); 7.07 (s, 1H); 7.17-7.19 (m, 5H); 7.36 (s, 1H); 7.86 (dd, 1H, J =
0.81, 1.75 Hz); 7.92 (d, 1H, J = 8.41 Hz); 8.25 (d, 1H, J = 8.26 Hz).
2-Furoyl-Leu-(p-F)Phe-NH2 (4). Yield 15 mg (27% isolated);
Rt 23.87 min; HRMS 390.1824 (calc), 390.1812 (found). 1H
NMR (400 MHz), δ 0.81 (d, 3H, J = 6.43 Hz); 0.86 (d, 3H, J =
6.37 Hz); 1.35-1.41 (m, 1H); 1.45-1.59 (m, 2H); 2.79 (dd, 1H,
J=9.24, 13.65 Hz); 2.99 (dd, 1H, J = 5.07, 13.98 Hz); 4.35-
4.45 (m, 2H); 6.64 (q, 1H); 6.96-7.00 (m, 2H); 7.09 (s, 1H); 7.18
(dd, 1H, J = 0.81, 3.49 Hz); 7.21 (dd, 2H, J = 5.63 8.72 Hz); 7.39
(s, 1H); 7.86 (dd, 1H, J = 0.82 1.75 Hz); 7.92 (d, 1H, J = 8.45
Hz); 8.24 (d, 1H, J = 8.20 Hz).
2-Furoyl-Cha-Ile-NH2 (5). Yield 26 mg (49% isolated); Rt
25.11 min; HRMS 378.2387 (calc), 378.2375 (found). 1H NMR
(400 MHz), δ 0.80 (t, 7H, 6.90 Hz); 0.81 (d, 5H, 6.90 Hz);
1.00-1.15 (m, 3H); 1.24-1.34 (m, 1H); 1.37-1.45 (m, 1H);
1.50-1.72 (m, 8H); 4.12 (dd, 1H, J = 7.39, 9.02 Hz); 4.48-4.54
(m, 1H); 6.63 (q, 1H); 7.01 (s, 1H); 7.18 (dd, 1H, J = 0.84, 3.46
Hz); 7.37 (s, 1H); 7.71 (d, 1H, J = 8.91 Hz); 7.86 (dd, 1H, J =
0.83, 1.75 Hz); 8.31 (d, 1H J = 8.43 Hz).
2-Furoyl-Phe-Ile-NH2 (6). Yield 13 mg (25% isolated); Rt
22.61 min; HRMS 372.1918, (calc) 372.1927 (found). 1H NMR
(400 MHz), δ 0.80-0.85 (m, 7H); 1.02-1.11 (m, 1H); 1.39-1.48
(m, 1H); 1.68-1.75 (m, 1H); 2.96 (dd, 1H, J = 11.11, 10.36 Hz);
3.08 (dd, 1H, J = 4.18 14.39 Hz); 4.17 (dd, 1H, J = 7.14 9.05
Hz); 4.70-4.76 (m, 1H); 7.04 (s, 1H); 7.10 (d, 1H, J = 4.04 Hz);
7.15 (t, 1H, J = 7.23 Hz); 7.23 (t, 2H, J = 7.31 Hz); 7.30 (d, 1H,
J = 8.41 Hz); 7.38 (s, 1H); 7.82 (d, 1H, J = 1.66 Hz); 7.93 (d, 1H,
J = 9.04 Hz); 8.34 (d, 1H, J = 8.67 Hz).
5-Isoxazoyl-Cha-Ile-NH2 (9). Yield 18 mg (34% isolated); Rt
24.09 min; HRMS 379.2340 (calc), 379.2351 (found). 1H NMR
(400 MHz), δ 0.79-0.83 (m, 7H,); 0.86-0.95 (m, 2H); 1.00-1.19
(m, 4H); 1.26-1.35 (m, 1H); 1.38-1.46 (m, 1H); 1.51-1.72 (m,
8H); 4.12 (dd, 1H, J = 7.36, 8.97 Hz); 4.52-4.57 (m, 1H); 7.01
(s, 1H); 7.15 (d, 1H, J = 1.91 Hz); 7.37 (s, 1H); 7.82 (d, 1H, J =
11.81 Hz); 8.75 (d, 1H, J = 1.88 Hz); 8.97 (d, 1H, J = 8.18 Hz).
4-(2-Methyloxazoyl)-Leu-Ile-NH2. Yield 16 mg (32% iso-
lated); Rt 21.67 min; HRMS 353.2183 (calc), 353.2196
1
(found). H NMR (400 MHz), δ 0.811 (t, 7H, J = 7.50 Hz);
0.841 (d, 3H, J = 9.68 Hz); 0.877 (d, 4H, J = 6.50 Hz);
1.02-1.09 (m, 1H); 1.38-1.71 (m, 5H); 4.146 (dd, 1H, J =
7.34, 9.01 Hz); 4.51-4.57 (m 1H); 6.994 (s, 1H); 7.405 (s, 1H);
7.881 (d, 1H, J = 9.02 Hz); 8.112 (d, 1H, J = 8.90 Hz); 8.487
(s, 1H).
4-(2-Methyloxazoyl)-Cha-Ile-NH2 (10). Yield 23 mg (42%
isolated); Rt 24.60 min; HRMS 393.2496 (calc), 393.2509
(found). 1H NMR (400 MHz), δ 0.79-0.90 (m, 8H,); 1.02-
1.14 (m, 4H); 1.19-1.27 (m, 1H); 1.36-1.44 (m, 1H); 1.48-1.75
(m, 9H); 4.14 (dd, 1H, J = 7.42, 8.95 Hz); 4.53-4.59 (m, 1H);
6.99 (s, 1H); 7.39 (s, 1H); 7.881 (d, 1H, J = 9.08 Hz); 8.08 (d, 1H,
J=8.92 Hz); 8.49 (s, 1H).
2-Furoyl-Chg-Ile-NH2 (7). Yield 3 mg (6% isolated); Rt 23.15
min; HRMS 364.2231 (calc), 364.2243 (found). H NMR (400
1
MHz), δ 0.79-0.83 (m, 8H); 1.00-1.09 (m, 1H); 1.37-1.45 (m,
1H); 1.65-1.70 (m, 1H); 2.96 (dd, 1H, J = 11.11, 10.36 Hz); 4.12
(dd, 1H, J = 8.14 9.04 Hz); 4.332 (t, 1H, J = 8.43 Hz); 6.63 (q,
1H); 6.70 (s, 1H); 7.19 (dd, 1H, J = 0.80, 3.47 Hz); 7.33 (s, 1H);
7.85 (dd, 1H, J = 0.80, 1.75 Hz); 7.88 (d,1H, J = 8.88 Hz); 8.01
(d, 1H, J=8.90 Hz).
Compounds 11 and 12. 1,5-Diaminopentane 2 HCl (800 mg,
3
4.6 mmol) was dissolved in a solution of KOH (516 mg, 2 equiv)
in MeOH (4 mL) and allowed to stir at rt for 10 min, at which
time the KCl salt had precipitated out. The solution was then
filtered in a sintered glass funnel and washed with DCM before
the solvent was removed in vacuo. Two portions of TCP resin
Compounds 8-10. Portions (6 ꢀ 200 mg) of rink amide
MBHA resin (0.14 mmol scale) were sequentially coupled with
Fmoc-Ile-OH (4 equiv) and then either Fmoc-Leu-OH or Fmoc-
Cha-OH in a solution consisting of HBTU (4 equiv) and DIPEA
(4 equiv) in DMF (2 mL). Upon completion, compounds
were coupled with either pyrazine-2-carboxylic acid, isoxazole-
5-carboxylic acid, or 2-methyloxazole-4-carboxylic acid (all
4 equiv) in the presence of HBTU (4 equiv), and of DIPEA
(4 equiv) in DMF (2 mL). Ligands were then cleaved from resin
using 95:2.5:2.5 TFA/TIPS/H2O, solvent was removed in vacuo,
and products were purified via rpHPLC fitted with a tunable
absorbance detector (214 nm λ), using a Phenomenex C18
(200 mg, substitution 0.9 mmol g-1) were reacted with either
3
1,5-diaminopentane (74 mg, 4 equiv) or 1,3-diaminopropane
(53 mg, 4 equiv) dissolved in anhydrous DCM and allowed to
shake overnight. Compounds were sequentially coupled with
Fmoc-Ile-OH (2 equiv), Fmoc-Cha-OH (2 equiv), and isoxa-
zole-5-carboxylic acid (2 equiv) in the presence of HOBt (4
equiv), DIC (2 equiv), and DIPEA (2 equiv) in DMF (2 mL).
The products were cleaved from the resin by treatment with a
solution of 20% TFA and 2.5% TIPS in DCM 5 (mL) for 1.5 h
and then filtered in a sintered glass funnel before the filtrate was
removed in vacuo. Products were isolated by rpHPLC fitted
with a tunable absorbance detector (λ 214 nm), using a Phenom-
˚
column (300 A, 22 ꢀ 250 mm) with a gradient of 0% B to
100% B over 30 min (solvent A 0.1% TFA in H2O, solvent B
0.1% TFA, 10% acetonitrile, 90% H2O). Compounds were
characterized by HRMS and 1H NMR using DMSO-d6 as
solvent, and purity of compounds was assessed via analytical
rpHPLC (0% B to 100% B over 30 min gradient, Phenomenex
˚
enex C18 column (300 A, 22 ꢀ 250 mm) with a gradient of 0% B
to 100% B over 30 min (solvent A 0.1% TFA in H2O, solvent B
0.1% TFA, 10% acetonitrile, 90% H2O). Compounds were
characterized by HRMS and 1H NMR using DMSO-d6 as
solvent, and purity of compounds was assessed via analytical
rpHPLC (0% B to 100% B over 30 min gradient, Phenomenex
˚
C18 column, 300 A, 4.6 ꢀ 250 mm, (λ 214 nm). Compounds were
repurified if found to be below 95%.
2-Pyrazoyl-Leu-Ile-NH2. Yield 5 mg (10% isolated); Rt 21.23
˚
C18 column, 300 A, 4.6 ꢀ 250 mm, 214 nm λ). Compounds were
1
min; HRMS 350.2187 (calc), 350.2199 (found). H NMR (400
repurified if found to be below 95%.

7436 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
5-Isoxazoyl-Cha-Ile-amino-3-aminopropane (11). Rt 21.16
min; HRMS 436.2918 (calc), 436.2929 (found). H NMR (400
MHz), δ 0.79-0.83 (m, 7H,); 0.85-0.95 (m, 2H); 1.03-1.16 (m,
5H); 1.27-1.34 (m, 1H); 1.38-1.46 (m, 1H); 1.51-1.70 (m,
12H); 2.77 (t, 2H, J = 7.73 Hz); 3.00-3.09 (m, 1H); 3.13-3.22
(m, 1H); 4.09 (t, 1H, J = 8.28 Hz); 4.51-4.57 (m, 1H); 7.16 (d,
1H, J = 1.88 Hz); 7.70 (s, 3H); 7.93 (d, 1H, J = 13.85 Hz); 8.13
(t, 1H, J = 5.68 Hz); 8.76 (d, 1H, J = 1.90 Hz); 8.95 (d, 1H, J =
8.24 Hz).
Barry et al.
(2 mL). Products were then cleaved from the resin by treatment
with a solution of 20% TFA and 2.5% TIPS in DCM (5 mL) for
1.5 h. The solution was then filtered in a sintered glass funnel,
and the filtrate was removed in vacuo. Crude products were
isolated by rpHPLC fitted with a tunable absorbance detector
1
˚
(λ 214 nm), using a Phenomenex C18 column (300 A, 22 ꢀ 250
mm) with a gradient of 0% B to 100% B over 30 min (solvent A
0.1% TFA in H2O, solvent B 0.1% TFA, 10% acetonitrile, 90%
H2O). Compounds were characterized by HRMS and 1H NMR
using DMSO-d6 as solvent, and purity of compounds was
assessed via analytical rpHPLC (0% B to 100% B over 30 min
5-Isoxazoyl-Cha-Ile-amino-5-aminopentane (12). Rt 21.74 min;
HRMS 464.3231 (calc), 464.3245 (found). 1H NMR (400 MHz),
δ 0.79-0.82 (m, 8H,); 0.85-0.95 (m, 2H); 1.02-1.16 (m, 5H);
1.23-1.31 (m, 4H); 1.35-1.44 (m, 4H); 1.47-1.69 (m, 12H); 2.75
(t, 2H, J = 7.85 Hz); 2.93-3.01 (m, 1H); 3.05-3.13 (m, 1H); 4.09
(t, 1H, J = 8.55 Hz); 4.51-4.57 (m, 1H); 7.15 (d, 1H, J = 1.89
Hz); 7.64 (s, 3H); 7.88 (d, 1H, J = 8.90 Hz); 7.96 (t, 1H, J = 5.41
Hz); 8.76 (d, 1H, J = 1.91 Hz); 8.95 (d, 1H, J = 8.18 Hz).
Compounds 13 and 14. 4-Aminomethyl piperidine (82 mg,
4 equiv) was dissolved in anhydrous DCM (2 mL) and then
˚
gradient, Phenomenex C18 column, 300 A, 4.6 ꢀ 250 mm, 214
nm λ). Compounds were repurified if found to be below 95%.
5-Isoxazoyl-Cha-Ile-amino-(3-(4-aminomethyl)piperidin-1-yl)-
propan-3-one (15). Rt 21.64 min; HRMS 569.3446 (calc), 569.3458
1
(found). H NMR (400 MHz), δ 0.75-0.81 (m, 7H,); 0.85-0.95
(m, 2H); 1.01-1.15 (m, 4H); 1.26-1.34 (m, 1H); 1.38-1.44 (m,
1H); 1.51-1.71 (m, 9H); 2.21 (t, 2H, J = 7.41 Hz); 2.57 (t, 1H, J =
7.65 Hz); 3.13-3.20 (m, 1H); 3.24-3.31 (m, 1H); 4.11 (dd, 1H, J =
7.85, 16.44 Hz); 4.52-4.57 (m, 1H); 7.00 (d, 1H, J = 8.76 Hz);
7.15-7.17 (m, 2H); 7.87-7.92 (m, 2H); 7.98 (t, 1H, J = 5.70 Hz);
8.75 (d, 1H, J = 1.86 Hz); 8.95 (d, 1H, J = 8.32 Hz).
added to TCP resin (2 ꢀ 200 mg, substitution 0.9 mmol g-1
)
3
and shaken overnight. The free amine was then reacted with
either Fmoc-β-Ala-OH (2 equiv) or Fmoc-5-aminoveleric acid
(2 equiv) and then dissolved in a solution of DCM (2 mL)
and DIC (1 equiv). Fmoc-Ile-OH (2 equiv), Fmoc-Cha-OH
(2 equiv), and isoxazole-5-carboxylic acid (2 equiv) were then
sequentially coupled following standard Fmoc-SPPS, using
HOBt (4 equiv) and DIC (2 equiv) to activate the acid and
DIPEA (2 equiv) as base dissolved in DMF (2 mL). Upon
completion, products were cleaved from the resin by treatment
with a solution of 20% TFA and 2.5% TIPS in DCM (5 mL) for
1.5 h. The solution was then filtered in a sintered glass funnel,
and the filtrate was removed in vacuo. Crude products were
purified by rpHPLC fitted with a tunable absorbance detector
5-Isoxazoyl-Cha-Ile-amino-[5-(4-(aminomethyl)piperidin-1-yl]-
pentan-5-one (16). Rt 22.05 min; HRMS 597.3756(calc), 597.3761
(found). 1H NMR (400 MHz), δ 0.75-0.81 (m, 7H,); 0.84-0.94
(m, 2H); 1.01-1.15 (m, 4H); 1.29-1.36 (m, 2H); 1.40-1.46 (m,
3H); 1.51-1.71 (m, 9H); 2.02 (t, 2H, J = 7.31 Hz); 2.63 (t, 1H,
J = 7.10 Hz); 2.91-3.00 (m, 1H); 3.03-3.11 (m, 1H); 3.21 (dd,
1H, J = 6.69, 14.43 Hz); 4.10 (t, 3H, J = 8.52 Hz); 4,51-4.57 (m,
1H); 6.98 (d, 1H, J = 8.29 Hz); 7.13-7.17 (m, 3H); 7.82 (t, 1H,
J = 5.57 Hz); 7.88 (d, 1H, J = 8.98 Hz); 7.93-7.99 (m, 1H); 8.75
(d, 1H, J = 1.87 Hz); 8.95 (d, 1H, J = 8.28 Hz).
Compounds 17 and 18. TCP resin (2 ꢀ 200 mg portions, 0.18
mmol scale) was first reacted with either Fmoc-β-Ala-OH (112
mg, 2 equiv) or Fmoc-5-aminovaleric acid (122 mg, 2 equiv) and
DIPEA (62 μL, 2 equiv) dissolved in anhydrous DCM (2 mL)
and then allowed to shake overnight. Compounds were then
sequentially coupled with Fmoc-Ile-OH (2 equiv), Fmoc-Cha-
OH (2 equiv), and isoxazole-5-carboxylic acid (2 equiv) in the
presence of HOBt (4 equiv), DIC (2 equiv), and DIPEA (2 equiv)
in 2 mL of DMF. Intermediates were then cleaved from resin
using 5 mL of 20% TFA and 2.5% TIPS in DCM, solvent was
removed under vacuum, and products were isolated via
rpHPLC (25 mm OD, C18 column). Each was then dissolved
in DMF (2 mL) and 3-(aminomethyl)pyridine (4 equiv), and
BOP (2 equiv) was added and allowed to stir overnight. The
solvent was removed in vacuo, and the crude products were
˚
(λ 214 nm), using a Phenomenex C18 column (300 A, 22 ꢀ 250
mm) with a gradient of 0% B to 100% B over 30 min (solvent A
0.1% TFA in H2O, solvent B 0.1% TFA, 10% acetonitrile, 90%
H2O). Compounds were characterized by HRMS and 1H NMR
using DMSO-d6 as solvent, and purity of compounds was
assessed via analytical rpHPLC (0% B to 100% B over 30 min
˚
gradient, Phenomenex C18 column, 300 A, 4.6 ꢀ 250 mm, 214
nm λ). Compounds were repurified if found to be below 95%.
5-Isoxazoyl-Cha-Ile-amino-(3-(4-aminomethyl)piperidin-1-yl)-
propan-3-one (13). Rt 21.15 min; HRMS 547.3602 (calc),
547.3617 (found). 1H NMR (400 MHz), δ 0.77-0.81 (m,
8H,); 0.83-0.95 (m, 2H); 1.02-1.13 (m, 5H); 1.34-1.47 (m,
5H); 1.55-1.71 (m, 12H); 1.96-2.01 (m, 1H); 2.11-2.17 (m,
1H); 2.65 (s, 1H); 2.67-2.72 (m, 2H); 3.00-3.08 (m, 1H); 4.06
(t, 1H, J = 8.09 Hz); 4.46-4.51 (m, 1H); 7.12 (d, 1H, J = 1.87
Hz); 7.719 (s, 2H); 7.85-7.89 (m, 1H); 7.95 (t, 1H, J = 5.84
Hz); 8.754 (d, 1H, J = 1.91 Hz); 8.960 (d, 1H, J = 15.84 Hz).
5-Isoxazoyl-Cha-Ile-amino-[5-(4-(aminomethyl)piperidin-1-yl]-
pentan-5-one (14). Rt 21.49min; HRMS 575.3915(calc), 575.3929
(found). 1H NMR (400 MHz), δ 0.78-0.82 (m, 7H,); 0.85-0.91
(m, 2H); 1.03-1.12 (m, 5H); 1.37-1.43 (m, 1H); 1.54-1.75 (m,
12H); 2.04-2.07 (m, 1H); 2.25-2.29 (m, 1H); 2.71 (s, 1H);
2.92-2.98 (m, 2H); 3.06-3.11 (m, 1H); 4.10 (t, 1H, J = 7.97
Hz); 4.51-4.57 (m, 1H); 7.15 (d, 1H, J = 1.87 Hz); 7.76 (s, 2H);
7.84-7.89 (m, 1H); 7.96 (t, 1H, J = 5.79 Hz); 8.75 (d, 1H, J =
1.91 Hz); 8.95 (d, 1H, J = 8.26 Hz).
˚
purified by rpHPLC (Phenomenex C18 column, 300 A, 22 ꢀ 250
mm, with a gradient of 0% B to 100% B over 30 min, solvent A
0.1% TFA in H2O, solvent B 0.1% TFA, 10% acetonitrile, 90%
H2O). Compounds were characterized by HRMS and 1H NMR
using DMSO-d6 as solvent, and purity of compounds was
assessed via analytical rpHPLC (0% B to 100% B over 30 min
˚
gradient, Phenomenex C18 column, 300 A, 4.6 ꢀ 250 mm, (λ 214
nm). Compounds were repurified if found to be below 95%.
5-Isoxazoyl-Cha-Ile-amino-3-[(pyridin-3-yl)methylamino]propan-
3-one (17). Rt 21.29 min; HRMS 541.3133 (calc), 541.3153 (found).
1H NMR (400 MHz), δ 0.75-0.79 (m, 8H,); 0.84-0.86 (m, 2H);
1.00-1.15 (m, 4H); 1.25-1.32 (m, 1H); 1.38-1.44 (m, 1H); 1.50-
1.70 (m, 9H); 2.33 (t, 2H, J = 7.32 Hz); 3.17-3.25 (m, 1H); 3.29-
3.38 (m, 1H); 4.10 (t, 1H, J = 8.68 Hz); 4.34 (d, 1H, J = 5.82 Hz);
4,51-4.57 (m, 1H); 7.15 (d, 1H, J = 1.91 Hz); 7.62 (dd, 1H,
J = 5.27, 7.89 Hz); 7.88 (d, 1H, J = 8.87 Hz); 7.97 (d, 1H, J=
8.17 Hz); 8.04 (t, 1H, J = 11.17, 5.66 Hz); 8.49 (t, 1H, J =
11.90, 5.99 Hz); 8.60 (s, 2H); 8.75 (d, 1H, J = 1.90 Hz); 8.96 (d,
1H, J = 8.31 Hz).
Compounds 15 and 16. 4-(2-Aminoethyl) aniline (98 mg,
0.72 mmol,) dissolved in anhydrous DCM (2 mL) was added
to two portions of TCP resin (200 mg, substitution 0.9
mmol g-1) and allowed to shake overnight. The solution was
3
then removed from the resin, and each was coupled with either
Fmoc-β-Ala-OH (112 mg, 2 equiv) or Fmoc-5-aminovaleric acid
(122 mg, 2 equiv) using DIC (28 μL) in DCM (2 mL) to form an
acid anhydride intermediate. Next, Fmoc-Ile-OH (2 equiv),
Fmoc-Cha-OH (2 equiv), and isoxazole-5-carboxylic acid
(2 equiv) were sequentially coupled following Fmoc SPPS, with
HOBt (4 equiv), DIC (2 equiv), and DIPEA (2 equiv) in DMF
5-Isoxazoyl-Cha-Ile-amino-5-[(pyridin-3-yl)methylamino]pentan-
5-one (18). Rt 21.68 min; HRMS 569.3446 (calc), 569.3462 (found).
1H NMR (400 MHz), δ 0.78-0.81 (m, 8H,); 0.84-0.94 (m, 2H);
1.01-1.15 (m, 4H); 1.29-1.42 (m, 4H); 1.47-1.67 (m, 12H); 2.147

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7437
(t, 2H, J = 7.39 Hz); 2.92-3.00 (m, 1H); 3.05-3.13 (m, 1H); 4.10
(t, 1H, J = 8.62 Hz); 4.36 (d, 3H, J = 5.85 Hz); 4,51-4.57 (m, 1H);
7.15 (d, 1H, J = 1.87 Hz); 7.70 (dd, 1H, J = 5.27, 7.93 Hz); 7.88 (d,
1H, J = 8.94 Hz); 7.96 (t, 1H, J = 11.18, 5.68 Hz); 8.04 (d, 1H, J=
11.44 Hz); 8.45 (t, 1H, J = 5.87 Hz); 8.64 (s, 2H); 8.75 (d, 1H, J =
1.89 Hz); 8.95 (d, 1H, J = 8.29 Hz).
J= 7.71 Hz); 7.69 (s, 2H); 8.04-8.11 (m, 2H); 8.75 (d, 1H, J=2.40
Hz); 8.94 (d, 1H, J = 8.18 Hz).
5-Isoxazoyl-Cha-Ile-(4-[aminomethyl]phenyl)-(4-[aminomethyl]-
piperidin-1-yl)methanone (23). Rt 22.15 min; HRMS 609.3759
1
(calc), 609.3779 (found). H NMR (400 MHz), δ 0.78-0.82
(m, 9H); 0.85-0.94 (m, 2H); 1.03-1.18 (m, 7H); 1.27-1.35
(m, 1H); 1.40-1.47 (m, 1H); 1.50-1.74 (m, 10H); 2.74 (t, 1H,
J = 6.31 Hz); 4.19 (t, 1H, J = 8.66 Hz); 4.30 (dd, 1H, J = 3.47,
6.73 Hz); 4.54-4.60 (m, 1H); 7.15 (d, 1H, J = 1.88 Hz); 7.29-7.31
(m, 5H); 7.69 (s, 2H); 7.99 (d, 1H, J = 12.27 Hz); 8.55 (t, 1H, J =
6.09 Hz); = 8.75 (d, 1H, J = 1.85 Hz); 8.96 (d, 1H, J = 9.65 Hz).
5-Isoxazoyl-Cha-Ile-(piperidin-4-yl)-(4-[aminomethyl]piperidin-
1-yl)methanone (24). Rt 21.98 min; HRMS 587.3915 (calc), 587.3903
(found). 1H NMR (400 MHz), δ 0.77-0.80 (m, 7H); 0.82-0.90 (m,
2H); 1.00-1.12 (m, 8H); 1.23-1.31 (m, 1H); 1.49-1.66 (m, 8H);
2.70 (t, 1H, J = 5.87 Hz); 4.07 (t, 1H, J = 8.67 Hz); 4.32 (dd, 1H,
J = 5.41, 7.42 Hz); 4.54-4.60 (m, 1H); 7.137 (d, 1H, J = 5.95 Hz);
7.708 (s, 1H); 8.748 (d, 1H, J = 10.69 Hz).
Compounds 19-28. TCP resin (3 g, substitution 0.9 mmol g-1
)
was reacted with 4-(aminomethyl)piperidine (617 mg, 2 equiv) in
3
anhydrous DCE (10 mL) and allowed to shake at rt overnight.
Solution was removed, and resin was washed with DMF, treated
with DTPM (1.14 g, 2 equiv) in DMF (12 mL), and then shaken for
2 h, after which the resin was washed with DMF and dried in vacuo.
The resin was split into 10-300 mg portions, and each was reacted
with either Fmoc-4-aminophenylactetic acid (202 mg), Fmoc-4-
aminomethylbenzoic acid (202 mg), Fmoc-3-aminomethylbenzoic
acid (202 mg), Fmoc-DL-pipecolinic acid (190 mg), Fmoc-3-ami-
nobenzoic acid (194 mg), Fmoc-4-aminobenzoic acid (194 mg),
Fmoc-β-turn-dipeptide (Fmoc-Btd-OH, 237 mg), Fmoc-isonipe-
cotic acid (190 mg), Fmoc-DL-nipecotic acid (190 mg), or Fmoc-
Freidinger’s lactam (236 mg) in a solution consisting of HOBt
(4 equiv), DIC (2 equiv), and DIPEA (2 equiv) dissolved in DMF
(2 mL). Solutions were left to shake overnight before the Fmoc
protecting groups were removed by washing the resin twice with 1:1
piperidine/DMF for 3 min. Fmoc-Ile-OH (1.91 g, 10 ꢀ 2 equiv),
HOBt (4 equiv), DIC (2 equiv), and DIPEA (2 equiv) were
dissolved in DMF (20 mL), and 2 mL was added to each reaction.
Fmoc-Cha-OH and isoxazole-5-carboxylic acid were then sequen-
tially coupled following Fmoc SPPS. Compounds were cleaved
from the resin by the addition of 20% TFA and 2.5% TIPS in
DCM (5 mL) and stirred for 1 h. The solvent was removed under
vacuum, and the crude products were purified by rpHPLC
5-isoxazoyl-Cha-Ile-(3-aminophenyl)-(4-[aminomethyl]piperidin-
1-yl)methanone (25). Rt 22.46 min; HRMS 595.3602 (calc),
1
595.3620 (found). H NMR (400 MHz), δ 0.82-0.88 (m, 8H);
1.05-1.19 (m, 6H); 1.44-1.70 (m, 10H); 1.76-1.85 (m, 2H); 2.74
(d, 1H, J = 7.04 Hz); 4.30 (t, 1H, J 8.47 Hz); 4.56-4.62 (m, 1H);
7.04 (d, 1H, J = 7.59 Hz); 7.16 (d, 1H, J = 1.91 Hz); 7.37 (t, 1H,
J = 7.97 Hz); 7.57 (d, 1H, J = 8.61); 7.71 (s, 4H); 8.14 (d, 1H, J =
8.73 Hz); 8.75 (d, 1H, J = 1.91 Hz); 10.21 (s, 1H).
5-Isoxazoyl-Cha-Ile-(piperidin-2-yl)-(4-[aminomethyl]piperidin-
1-yl)methanone (26). Rt 22.65 min (2 peaks for R- and S-en-
antomers); HRMS 587.3915 (calc), 587.3929 (found). 1H NMR
(400 MHz), δ 0.78-0.82 (m, 7H); 0.84-0.92 (m, 2H); 1.01-1.15
(m, 8H); 1.26-1.36 (m, 1H); 1.51-1.84 (m, 8H); 2.72 (t, 1H, J =
5.87 Hz); 4.07 (t, 1H, J = 8.67 Hz); 4.32 (dd, 1H, J = 6.38, 8.24
Hz); 4.55-4.59 (m, 1H); 7.153 (t, 1H, J = 1.76 Hz); 8.755 (d, 1H,
J=10.36 Hz).
˚
(Phenomenex C18 column, 300 A, 22 ꢀ 250 mm, with a gradient
of 0% B to 100% B over 30 min, solvent A 0.1% TFA in H2O,
solvent B 0.1% TFA, 10% acetonitrile, 90% H2O). Compounds
were characterized by HRMS and H NMR using DMSO-d6 as
1
5-Isoxazoyl-Cha-Ile-(3R,6S)-6-amino-3-(4-[aminomethyl]piperi-
dine-1-carbonyl)tetrahydro-2H-thiazolo(3,2-a)pyridin-5(3H)-one
(27). Rt 22.05 min; HRMS 674.3694 (calc), 674.3680 (found).
5-Isoxazoyl-Cha-Ile-(R)-3-amino-1-([S]-1-[4-(aminomethyl)-
piperidin-1-yl]-4-methyl-1-oxopentan-2-yl)pyrrolidin-2-one (28).
Rt 23.60 min; HRMS 672.4443 (calc), 672.4461 (found).
Compounds 30-36 (General Synthetic overview). Compounds
were synthesized in solution phase using Boc-protected amino
acids. For the initial coupling, the amino acid (1.2 equiv), HBTU
(1.2 equiv), HOBT (3 equiv), and DIPEA (1.2 equiv) were
dissolved in a volume of DMF. The solution was then added
to an amino bearing C-terminal moiety and left to stir overnight
at rt. The solutions were then diluted with ethyl acetate and
washed twice with saturated NaHCO3. The organic phase was
dried with MgSO4, and the solvent removed in vacuo. Boc
protecting groups were removed from amino acids with a
solution of 20% TFA in DCM stirred for 1 h. The solution
was neutralized by washing with saturated NaHCO3 (2ꢀ), dried
with MgSO4, and then removed in vacuo. Subsequent amino
acids and N-terminal carboxylic acids were then sequentially
coupled under the same condition. Coupling was determined by
ESI MS, with most reaching completion overnight. Crude
products were purified by rpHPLC fitted with a tunable absor-
bance detector (λ 214 nm), using a Phenomenex C18 column
solvent, and purity of compounds was assessed via analytical
rpHPLC (0% B to 100% B over 30 min gradient, Phenomenex
˚
C18 column, 300 A, 4.6 ꢀ 250 mm, (λ 214 nm). Compounds were
repurified if found to be below 95%.
5-Isoxazoyl-Cha-Ile-(3-[aminomethyl]phenyl)-(4-[aminomethyl]-
piperidin-1-yl)methanone (19). Rt 22.81 min; HRMS 609.3759
1
(calc), 609.3763 (found). H NMR (400 MHz), δ 0.78-0.81 (m,
8H); 0.85-0.91 (m, 2H); 1.03-1.19 (m, 7H); 1.27-1.36 (m, 1H);
1.38-1.49 (m, 1H); 1.51-1.84 (m, 10H); 2.74 (t, 1H, J = 6.07 Hz);
4.19 (t, 1H, J = 8.53 Hz); 4.30 (dd, 1H, J = 6.05, 9.03 Hz);
4.53-4.59 (m, 1H); 7.16 (d, 1H, J = 1.92 Hz); 7.21-7.23 (m, 2H);
7.29 (d, 1H, J = 7.87 Hz); 7.36 (t, 1H, J = 7.79 Hz); 7.74 (s, 3H);
7.97 (d, 1H, J = 11.95 Hz); 8.54 (t, 1H, J = 6.11 Hz); 8.75 (d, 1H,
J = 1.89 Hz); 8.95 (d, 1H, J = 10.62 Hz).
5-Isoxazoyl-Cha-Ile-(piperidin-3-yl)-(4-[aminomethyl]piperidin-
1-yl)methanone (20). Rt 21.74 min; HRMS 587.3915 (calc),
1
587.3902 (found). H NMR (400 MHz), δ 0.78-0.81 (m, 7H);
0.84-0.92 (m,2H);1.01-1.15 (m, 8H); 1.26 -1.36(m,1H);1.51-
1.84 (m, 8H); 2.72 (t, 1H, J = 5.87 Hz); 4.20 (t, 1H, J = 8.65 Hz);
4.35 (dd, 1H, J = 6.01, 8.88 Hz); 4.54-4.60 (m, 1H); 8.14 (d, 1H,
J=1.88 Hz); 8.91 (d, 1H, J = 9.94 Hz).
5-Isoxazoyl-Cha-Ile-(4-aminophenyl)-(4-[aminomethyl]piperidin-
1-yl)methanone (21). Rt 24.33 min; HRMS 595.3602 (calc),
1
˚
(300 A, 22 ꢀ 250 mm) with a gradient of 0% B to 100% B over
595.3616 (found). H NMR (400 MHz), δ 0.79-0.85 (m, 8H);
30 min (solvent A 0.1% TFA in H2O, solvent B 0.1% TFA, 10%
acetonitrile, 90% H2O). Compounds were characterized by
HRMS and H NMR using DMSO-d6 as solvent, and purity
1.01-1.18 (m, 6H); 1.42-1.63 (m, 10H); 1.75-1.82 (m, 2H); 2.72
(d, 1H, J = 8.27 Hz); 4.27 (t, 1H, J 7.86 Hz); 4.56-4.62 (m, 1H);
7.03 (d, 1H, J = 8.02 Hz); 7.12 (d, 1H, J = 2.04 Hz); 7.35 (t, 1H,
J = 8.22 Hz); 7.54 (d, 1H, J = 8.66); 7.71 (s, 4H); 8.02 (d, 1H, J =
9.02 Hz); 8.84 (d, 1H, J = 1.87 Hz).
1
of compounds was assessed via analytical rpHPLC under vary-
ing conditions (0% B to 100% B over 30 min gradient, Phenom-
˚
5-Isoxazoyl-Cha-Ile-4-aminophenyl-(4-[aminomethyl]piperidin-1-
yl)ethan-2-one (22). Rt 22.87 min; HRMS 609.3759 (calc), 609.3782
enex C18 column, 300 A, 4.6 ꢀ 250 mm, (λ 214 nm). Compounds
were repurified by further gradient-varied rpHPLC if necessary
until >95% pure, as assessed by analytical HPLC.
5-Isoxazoyl-Cha-Ile-4-aminophenyl-(4morpholin-1-yl)ethan-2-
1
(found). H NMR (400 MHz), δ 0.81-0.87 (m, 9H,); 1.06-1.21
(m, 4H); 1.24-1.34 (m, 1H); 1.48-1.80 (m, 12H); 2.65-2.71 (m,
1H); 2.92-3.00 (m, 1H); 3.64 (s, 1H); 4.30 (t, 1H, J = 8.62 Hz);
4.55-4.61 (m, 1H); 7.16 (dd, 1H, J = 8.10, 9.97 Hz); 7.50 (t, 1H,
one (30). TCP resin (200 mg, substitution 0.9 mmol g-1) was first
3
reacted with Fmoc-4-aminophenylacetic acid (269 mg, 4 equiv)

7438 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Barry et al.
in the presence of DIPEA (124 μL, 4 equiv) in DMF (2 mL) and
allowed to shake overnight. The Fmoc protecting group was
removed with 2 ꢀ 3 min washes with excess 1:1 piperidine/DMF,
and the resin was washed with DMF. Fmoc-Ile-OH (127 mg, 2
equiv), Fmoc-Cha-OH (142 mg, 2 equiv), and isoxazole-5-car-
boxylic acid (41 mg, 2 equiv) were then sequentially coupled
following standard Fmoc-SPPS, using HOBT (49 mg, 4 equiv),
DIC (56 μL, 2 equiv), and DIPEA (62 μL, 2 equiv) in DMF (2
mL). The intermediate was cleaved from resin in a solution of
20% TFA and 2.5% TIPS in DCM (5 mL) stirred for 1.5 h and
then filtered in a sintered glass funnel. The filtrate was removed
under vacuum, and the product was isolated via rpHPLC (25
mm OD, C18 column). HOBT (2 equiv), DIC (2 equiv), and
DIPEA (2 equiv) were added to the intermediate (30 mg) and
dissolved in DMF (2 mL), followed by the addition of morpho-
line (14 μL, 4 equiv), and the solution was left to stir overnight at
rt. Ethyl acetate (20 mL) wasadded to the solution, it waswashed
with 3 ꢀ 20 mL aliquots of saturated NaHCO3, and the organic
phases were combined, dried with MgSO4, and evaporated in
vacuo. The crude product was isolated via rpHPLC fitted with a
tunable absorbance detector (λ 214 nm), using a Phenomenex
2H); 7.15 (d, 1H, J = 2.95); 7.26-7.38 (m, 8H); 7.51 (d, 1H, J =
8.52 Hz); 8.75 (d, 1H, J =1.91 Hz); 8.95 (d, 1H, J = 8.12 Hz);
9.35 (s, 1H).
5-Isoxazoyl-Cha-Ile-spiroindane-1,40-piperidine (36). Rt = 35.00
min; HRMS [MHþ] 549.3435 (calc), 549.3451 (found). 1H NMR
(400 MHz), δ 0.79-0.82 (m, 6H); 1.03-1.14 (m, 4H); 1.42-1.60
(m, 10H); 2.02-2.08 (m, 2H); 2.87 (t, 1H, J = 7.40 Hz); 4.05 (t, 2H,
J = 10.46 Hz); 4.35 (d, 2H, J = 13.49 Hz); 4.54-4.66 (m, 2H); 7.03
(d, 1H, J= 6.53 Hz); 7.12 (d, 1H, J= 5.10 Hz); 7.14-7.16 (m, 4H);
7.20 (d, 1H, J = 5.42 Hz); 8.12 (d, 1H, J = 9.74 Hz); 8.27 (d, 1H,
J = 9.64 Hz); 8.75 (d, 1H, J = 1.89 Hz); 8.95 (d, 1H, J = 8.38 Hz).
Intracellular Calcium Efflux. Assay and Dye buffer. To
Hank’s balanced salt solution (HBSS, 1 L) diluted 1 in 10 (1ꢀ)
with distilled water was added 1 M HEPES (20 mL) and
probenecid (710 mg) disolved in 1 N NaOH (5 mL); pH was
then adjusted to 7.4. Aliquots of the fluorescent calcium in-
dicator Fluo-3 a.m. (2 mM) dissolved in DMSO (25 μL), FCS
(120 μL), and Pluronic acid (F-127, 25 μL) were dissolved in
assay buffer (12 mL) prior to use.
Sample Preparation. Stock solutions of agonists were pre-
pared by weighing compounds in Eppendorf vials and dissol-
ving them in a volume of DMSO to give a final concentration of
10 mM. Two more stock solutions were prepared by serially
diluting the 10 mM sample with DMSO to give 500 and 10 μM.
These solutions were then diluted to 4 mL by dissolving 80 μL of
the 10 mM, 500 μM, and 10 μM standards in 3.92 mL of assay
buffer to give three working solutions of 200, 10, and 0.2 μM.
Similarly, blanks were prepared by diluting DMSO (80 μL) to
4 mL with assay buffer. Using two pumps during the assay to
alter the volume of working stock to volume of blank delivered
(so that the final volume is always 100 μL), final concentrations
in the range of 10 nM to 100 μM are achieved and the concen-
tration of DMSO is consistently 2%.
Assay Protocol. To adhere intact human embryonic kidney
cells (HEK293) or colon carcinoma cells (HT29) to the base of a
96-well clear-bottomed black-walled assay plate (Corning), they
were incubated at 37 °C overnight in either Dulbecco’s modified
essential serum (Gibco) or RPMI 1640 medium, supplemented
with 2 mM L-glutamine (Gibco), 10% FCS, 0.1 mM MEM
nonessential amino acids (Invitrogen), penicillin (100 U/mL),
and streptomycin (100 U/mL). At 1 h before assaying, the
medium was removed and cells were incubated with 100 μL
per well of dye loading buffer; this was removed prior to the
assay, and wells were washed two times with assay buffer before
a final volume of assay buffer (100 μL) was added to each well.
Fluorescence was measured (excitation 495 nm, emission 520
nm) from the bottom of the plate for 60 s using a Polarstar
(BMG LABTECH) fluorescent plate reader, with blank deliv-
ered at 10 s and agonist delivered after 12 s to a final volume of
100 μL.63 For dose dependent concentration curves, agonists
were delivered to cells with final concentrations of 0.01, 0.03,
0.1, 0.3, 1, 3, 10, 30, and 100 μM, using three replicates per
concentration. Ca2þ efflux is reported as percent response of the
Ca2þ ionophore A-23187 (calcimycin) and plotted as mean (
SEM.
˚
C18 column (300 A, 22 ꢀ 250 mm) with a gradient of 0% B to
100% B over 30 min (solvent A 0.1% TFA in H2O, solvent B
0.1% TFA, 10% acetonitrile, 90% H2O) to yield 30 as a white
solid (8.7 mg, 0.015 mmol, 8% yield). Rt 21.89 min; HRMS
[MHþ] 582.3286 (calc), 582.3298 (found). 1H NMR (400 MHz),
δ 0.81-0.88 (m, 8H,); 1.06-1.16 (m, 4H); 1.46-1.67 (m, 10H);
1.74-1.83 (m, 2H); 2.65-2.71 (m, 1H); 2.92-3.00 (m, 1H); 3.64
(s, 1H); 4.30 (t, 1H, J = 8.69 Hz); 4.47 (t, 1H, J = 8.42 Hz); 7.74
(d, 1H, J = 6.00 Hz); 8.11 (d, 1H, 5.76 Hz); 8.19 (d, 1H, J = 8.72
Hz); 10.06 (s, 1H).
5-Isoxazoyl-Cha-Ile-3,4-dihydroisoquinoline (31). Rt = 31.79
min; HRMS [MHþ] 495.2966 (calc), 495.2984 (found). 1H NMR
(400 MHz), δ 0.77-0.84 (m, 7H); 1.02-1.13 (m, 4H); 1.30-1.36
(m, 1H); 1.46-1.65 (m, 7H); 1.77-1.85 (m, 1H); 2.74 (t, 1H, J =
5.91 Hz); 2.80-2.91 (m, 1H); 3.56-3.63 (m, 1H); 3.71-3.80 (m,
1H); 3.86-3.92 (m, 1H); 4.47-4.57 (m, 2H); 4.63-4.73 (m, 2H);
7.14-7.17 (m, 4H); 8.21 (t, 1H, J = 8.45 Hz); 8.74 (d, 1H, J =
1.88 Hz); 8.86-8.90 (m, 1H).
5-Isoxazoyl-Cha-Ile-1-amino-naphthalene (32). Rt = 23.27
min; HRMS [MHþ] 505.2809 (calc), 505.2821 (found). 1H
NMR (400 MHz), δ 0.90 (t, 2H, J = 7.37 Hz); 1.00 (d, 2H,
J = 6.75 Hz); 1.20-1.25 (m, 2H); 1.53-1.64 (m, 4H); 1.70-1.73
(m, 2H); 4.51 (t, 1H, J = 8.70 Hz); 4.60-4.66 (m, 2H); 7.17 (d,
1H, J = 1.90 Hz); 7.47-7.54 (m, 2H); 7.60 (d, 1H, J = 7.34 Hz);
7.78 (t, 1H, J = 8.19 Hz); 7.94 (d, 1H, J = 9.43 Hz); 8.02 (d, 1H,
J = 6.73 Hz); 8.23 (d, 1H, J = 8.21 Hz); 8.75 (d, 1H, J = 1.86
Hz); 9.00 (d, 1H, J = 8.33 Hz).
5-Isoxazoyl-Cha-Ile-(4-phenyl)piperazine) (33). Rt = 28.61
min; HRMS [MHþ] 424.3231 (calc), 524.3243 (found). 1H
NMR (400 MHz), δ 0.78-0.72 (m, 6H); 0.84-0.89 (m, 2H);
1.02-1.11 (m, 4H); 1.22-1.30 (m, 1H); 1.45-1.64 (m, 8H);
1.77-1.83 (m, 1H); 2.94-3.13 (m, 5H); 3.49-3.77 (m, 8H);
4.49-4.55 (m, 1H); 4.59 (t, 1H, J = 8.64 Hz); 6.79 (t, 1H, J =
7.27 Hz); 6.92 (d, 2H, J = 8.84); 7.10 (d, 1H, J = 4.00 Hz); 7.20
(dd, 1H, J = 7.28, 8.74 Hz); 8.19 (d, 1H, J = 8.75 Hz); 8.88 (d,
1H, J = 8.26 Hz).
5-Isoxazoyl-Cha-Ile-4-(o-tolyl)piperazine (34). Rt = 32.85
min; HRMS [MHþ] 538.3388 (calc), 538.3399 (found). 1H
NMR (400 MHz), δ 0.81-0.85 (m, 6H); 0.87-0.95 (m, 2H);
1.04-1.12 (m, 4H); 1.28-1.36 (m, 1H); 1.47-1.53 (m, 1H);
1.55-1.70 (m, 7H); 1.80-1.86 (m, 1H); 2.26 (s, 2H); 2.65-2.70
(m, 1H); 2.75-2.83 (m, 4H); 3.48-3.53 (m, 1H); 3.62-3.68 (m,
1H); 3.72-3.78 (m, 2H); 6.94-6.99 (m, 2H); 7.14 (d, 1H, J =
1.91); 8.22 (d, 1H, J = 8.83 Hz); 8.74 (d, 1H, J = 1.90 Hz); 8.93
(d, 1H, J = 8.29 Hz).
Statistical Analysis. For agonist and antagonist assays, non-
linear regression was performed using Prism 5 (GraphPad
Software, San Diego, CA).
Serum Stability Assay. A stock solution of compound 19
(20 mM) was prepared in water with 2% DMSO. An aliquot
(500 μL) was then added to filtered FCS (4.5 mL) at 37 °C.
At intervals of 0, 5, 15, 30, 60, 120, 180, and 240 min, 500 μL was
removed and added to 1.5 mL of MeCN and then centrifuged at
2500 rpm for 5 min. A total of 1 mL of the supernatant was
removed and analyzed via LCMS (20% B to 100% B in 20 min).
Data were plotted as a percentage of the peak area calculated
from t = 0 s samples.
5-Isoxazoyl-Cha-Ile-1-aminobiphenyl (35). Rt = 33.45 min;
HRMS [MHþ] 531.2966 (calc), 531.2977 (found). 1H NMR
(400 MHz), δ 0.77-0.93 (m, 2H); 1.07-1.18 (m, 3H); 1.24-1.33
(m, 1H); 1.46-1.52 (m, 1H); 1.56-1.68 (m, 7H); 4.51-4.57 (m,
Acknowledgment. We thank the Australian Research
Council (ARC) for a Federation Fellowship to D.P.F. and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7439
both ARC (Grants FF668733 and DP1093245) and the
National Health and Medical Research Council of Australia
(Grants 569595 and APP1000745) for research support.
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Supporting Information Available: Schemes S1-4 for syn-
thesis of 14 and 15 (Scheme S1), 16-19 (Scheme S2), 20 and
21 (Scheme S3), and 22 (Scheme S4) and analytical rp-HPLC
traces for agonist 19 (GB110) and antagonist 36 (GB83). This
material is available free of charge via the Internet at http://
pubs.acs.org.
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