J. L. Ralbovsky et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2661–2663
2663
When the benzyl group (R1) of the original HTS hit (1) was
substituted in the para position with an electron donating group
such as methoxy (20), a significant increase in potency was
achieved. Similar results were obtained when halogens were
substituted in the para position (16, 17, 27). Methoxy substitutions
in the meta or ortho positions (18, 19) provided compounds with
much less potency. When electron withdrawing functional groups,
such as cyano (23), were substituted in the para position of the
benzyl group, we observed similar activity to the HTS lead. Adding
an extra carbon between the phenyl group and the triazinedione
provided a less potent compound (22). Replacing the benzyl group
with groups such as n-butyl (14) or furanyl (15) yielded less potent
compounds.
mucosal exposure to PK1 peptide added cumulatively at 10 and
100 nM, respectively. Pre-treatment of isolated rat ileum mucosa
with compound 16 (1 lM) added to the serosa 20 min prior to
addition of PK1 peptide significantly suppressed the sustained in-
crease in baseline short-circuit current (Isc) over time evoked by
PK1 peptide by approximately 79.2% at 10 nM, and 60.7% at
100 nM (Fig. 3). These data suggest the potential for the efficacious
use of PK1 receptor antagonists from this chemical class in gut dis-
ease states that have a significant secretory diarrhea component.
In summary, through the discovery of an HTS hit, a series of
Prokineticin 1 receptor antagonists were synthesized with excel-
lent cellular functional activity. The SAR of the triazinedione scaf-
fold was explored and novel compounds with eighteen times
greater potency were identified. Benzyl groups with methoxy or
halogens substituted in the para position were optimal for R1,
and the p-methoxy, p-hydroxyl or p-difluoromethoxy groups were
the best benzyl substituents for R2. These are the first reported
small molecule inhibitors of the PK1 receptor. We further demon-
strated the anti-secretory property of these inhibitors ex vivo by
inhibiting the PK1 invoked prosecretory response in rat ileum tis-
sue. Further publications will discuss SAR of the guanidine moiety.
When the p-methoxybenzyl group (R2) of 27 was replaced with
just a benzyl group (26), a significant loss of potency was observed.
Interestingly, the methoxy group could be replaced with difluoro-
methoxy (35) but not propyloxy (29). Substituents such as para ni-
tro (28) provided less potent compounds. The p-methoxy benzyl
group was successfully replaced with the methoxy substituted pyr-
idine (34) but not with n-hexyl (30) or 3-furyl (33). As was seen for
R1, adding an extra carbon between the triazinedione and the phe-
nyl group (25) of R2 did not yield greater potency. Unlike R1, para
substituted halogens like fluoro (36) substituted on the benzyl
group R2 were less potent. Also, para hydroxyl benzyl derivative
worked well for R2 but not for R1 (32 compared to 21). The p-meth-
oxy, p-hydroxyl or p-difluoromethoxy groups were the best benzyl
substituents for R2.
When an extra carbon was added to the linker between the het-
erocyclic ring and the guanidine (24 compared to 17), there was a
significant loss in potency. The ethylenediamine linker was
optimal.
To investigate the potential anti-secretory efficacy of selective
small molecule PK1 receptor antagonists, we established a model
of secretory diarrhea ex vivo in the Ussing-type flux chambers with
Acknowledgments
We would like to thank Jack Kauffman and Hong Xin for run-
ning the High Throughput Screening Assays. Also, we would like
to thank Diane Gauthier for spectroscopy support and Dr. Jian Li
for molecular modeling.
References and notes
1. (a) Kaser, A.; Winklmayer, M.; Lepperdinger, G.; Kreil, G. EMBO Reports 2003, 4,
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S. L.; Chevalier, K. M.; Hornby, P. J. Gastroenterology 2007, 132, A223.
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Control (DMSO),N = 7
50
16, 1 μM
N = 6
40
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30
7. For recent reviews see: (a) Wood, J. D. World J. Gastroentrol 2007, 13, 1313; (b)
Zisman, T. L.; Cohen, R. D. Curr. Treat. Opt. Gastroentrol 2007, 10, 185; (c) Nyrop,
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8. Recently, a paper was published describing some of our compounds: Balboni,
G.; Lazzari, I.; Trapella, C.; Negri, L.; Lattanzi, R.; Giannini, E.; Nicotra, A.;
Melchiorri, P.; Visentin, S.; De Nuccio, C.; Salvadori, S. J. Med. Chem. 2008, 51,
7635.
20
Means + SE
*P < 0.05
10
0
*
10 nM
100 nM
9. See: (a) Gopalsamy, A.; Yang, H. J. Comb. Chem. 2001, 3, 278; (b) Yu, Y.; Ostresh,
J. M.; Houghten, RA. J. Comb. Chem. 2004, 6, 83.
10. The reaction could also be done in the microwave in ethanol at 160 °C.
Prokineticin-1
Figure 3. Effect of PK1 peptide tested at two concentrations, 10 and 100 nM,
applied in a cumulative fashion to the basolateral side of mucosal tissues mounted
in Ussing-type flux chambers on basal short-circuit current (Isc) in rat ileal mucosa
in the absence (filled bars; N = 7 tissues) and the presence (hatched bars; N = 6
11. GPR73 expressing HEK 293 cells are grown on 96 well poly-
D-lysine coated
plate (Costar), in selective media at 37 °C and 5% CO2. On day of experiment
media is removed and replaced with Calcium plus dye solution (Molecular
Devices). After equilibrations in dark 1st at 37 °C followed by rt, compound is
added to the cells followed by addition of hPK1 ligand (at
a previously
tissues) of 16 (1 lM). Compound 16 was added to the basolateral surface of the
determined EC50). Fluorescence is measured on a Fuorometric Imaging Plate
Reader (FLIPR), and IC50 determined using GraphPad Prism.
tissues 20 min before addition of the test concentrations of PK1 peptide used to
evoke prosecretory response to these tissues. DMSO which was used to dissolve 16
served as a vehicle control in non-treated tissues.