M.-K. Yau et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
The pharmacokinetic profile and anti-inflammatory activity of
compounds and 10 were comparatively evaluated in rats
4
(Fig. 4). Compounds 4 and 10 reached a maximum plasma concen-
tration at 2.5–3 h after oral administration at 10 mg/kg, with a
maximum plasma concentration of 2.0 and 0.5 lM, respectively.
Compound 10 gave a lower plasma concentration after 6 h than
4. These compounds were compared (10 mg/kg, p.o.) for their
capacity to inhibit rat paw swelling induced 2 h later by intraplan-
tar (i.pl.) administration of 2f-LIGRLO-NH2 (Fig. 4). Compound 4
has been reported as an orally active anti-inflammatory agent in
inhibiting PAR2-induced paw inflammation.20,21 Similarly com-
pound 10 is orally active in reducing the severity of paw oedema
following oral administration at 10 mg/kg, but had lower potency
than 4 which was attributed to its lower oral bioavailability
(Fig. 4a). However, when both compounds were compared after
subcutaneous (sc) administration at 5 mg/kg (in saline), 10 was
more potent than 4 in suppressing paw swelling (Fig. 4b).
All compounds reported in this study were synthesized in par-
allel using standard solution phase peptide synthesis protocols and
Boc-protected amino acids (Scheme 1).22 All compounds were
characterized by HRMS, 1H NMR spectroscopy and rpHPLC (SI).
The spectral data for the most potent compound in this study
(10) is reported under reference and notes section.23
In conclusion, several benzylamine derivatives are reported as
novel PAR2 antagonists with superior or equal potency to 4. Com-
pound 10 was able to inhibit intracellular calcium release induced
by PAR2 agonists, the hexapeptide 2f-LIGRLO-NH2 (1) and trypsin,
but not by the PAR1 agonist thrombin. Compound 10 showed anti-
inflammatory activity in a rat paw model of acute inflammation, as
well as inhibition of the secretion of pro-inflammatory cytokines.
Compound 10 was a more effective anti-inflammatory agent than
4 when given sc to rats. These findings offer new clues for develop-
ing PAR2 antagonists for cellular and animal models of disease.
Figure 4. Anti-inflammatory activity of
Compound 10 (Tmax 158 45 min; Cmax 0.51 0.25
not as effective as 4 (Tmax 167 18 min; Cmax 1.97 0.38
in reducing paw oedema following oral administration at 10 mg/kg. Paw oedema in
rats were induced through intraplantar (i.pl.) administration of 2f-LIGRLO-NH2
4
versus 10 in rat paw oedema. (a)
l
M; AUC6h 444 ngꢁh/mL) was
l
M; AUC6h 3420 ngꢁh/mL)
(350 lg per paw in 100 lL saline control). Compounds were given orally in olive oil
120 min prior to 2f-LIGRLO-NH2. (b) Compound 10, at 5 mg/kg subcutaneous
injection, was more effective in reducing paw oedema compared to 4. Each data
point represents mean SEM (n = 3–5). Samples were compared to 2f-LIGRLO-NH2
treated control, **p <0.01, ***p <0.001; significant differences as indicated.
Compound 10 was able to inhibit iCa2+ release induced by two
PAR2 agonists, the PAR2 selective peptide agonist 1 (Fig. 2) and tryp-
sin (Fig. 3a) in HT29 cells. This compound was also tested in a com-
petitive binding assay on human PAR2-transfected CHO-cells (SI,
Acknowledgments
We thank the National Health and Medical Research Council of
Australia (NHMRC) for a Senior Principal Research Fellowship
(APP1027369) to DPF and grants from the NHMRC (APP1084083,
APP1083131, APP1047759), the Australian Research Council
(DP130100629, CE140100011) and the Queensland Government
(CIF grant), and acknowledge the Centre for Inflammation and Dis-
ease Research, University of Queensland for supporting the research.
Fig. S2) where it showed binding affinity (IC50 4.5 lM) for PAR2 by
competing with Europium-labeled 2f-LIGRLO-NH2 (1). To evaluate
the selectivity of 10 for PAR2 over PAR1, a human prostate cancer
cell line (PC3) was used as it has higher PAR1 expression than
HT29. Compound 10 was found to be selective for PAR2 over PAR1,
since it had no effect in blocking the release of calcium in PC3 cells
stimulated with PAR1 agonists (a-thrombin or TFLLR; Fig. 3b).
Previously our group reported that antagonist 4 (GB88) inhib-
ited PAR2-induced pro-inflammatory cytokines released in pri-
mary human kidney tubule epithelial cells (HTEC).19 ELISA
Supplementary data
Supplementary data (synthetic procedures, compound charac-
terization data (1H NMR, HRMS, HPLC) and assay results of all final
compounds) associated with this article can be found, in the online
results here indicate that 10 (5 and 10 lM) also showed inhibitory
effects in attenuating secretion of inflammatory cytokines TNF-
a
and IL6 induced by PAR2 agonist 1 in HTEC (Fig. 3c and d).
Scheme 1. General synthesis of benzylamine/alkylamine derivatives.