Triazinediones as prokineticin 1 receptor antagonists. Part 1: SAR, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2009.03.157

A series of guanidine triazinediones were identified as potent PK1 receptor antagonists. A compound in this series inhibited the PK1 invoked prosecretory response in rat ileum tissue.

Full text of DOI:10.1016/j.bmcl.2009.03.157

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2661–2663
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Triazinediones as prokineticin 1 receptor antagonists. Part 1: SAR, synthesis
and biological evaluation
*
Janet L. Ralbovsky , Joseph G. Lisko, Jeffrey M. Palmer, John Mabus, Kristen M. Chevalier, Mark J. Schulz,
Alexey B. Dyatkin, Tamara A. Miskowski, Steven J. Coats, Pamela Hornby, Wei He
Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, L. L. C., Welsh and McKean Rds., Spring House, PA 19477, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of guanidine triazinediones were identified as potent PK1 receptor antagonists. A compound in
this series inhibited the PK1 invoked prosecretory response in rat ileum tissue.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 22 January 2009
Revised 25 March 2009
Accepted 30 March 2009
Available online 5 April 2009
Keywords:
PK1
Prokineticin
Triazinediones
The cysteine rich peptide prokineticin 1 (PK1), a member of the
AVIT protein family, is the human homolog of a peptide originally
isolated from snake venom (mamba intestinal toxin-1).1 The pep-
tide was named ‘prokineticin’ due to its ability to potently and
selectively evoke contractions in mammalian intestinal smooth
muscle.2
Immunostaining has demonstrated expression of PKR1 in
approximately 25% of the myenteric neurons in mouse proximal co-
lon.3 PK1 applied directly to the isolated intact mouse colon inhibits
spontaneous generation of large amplitude propulsive contrac-
tions.3 We recently reported fluid enteropooling and transit stimu-
lation effects of PK1 in the rat small intestine in vivo.4 We have also
found PK1 peptide to act as a novel potent intestinal secretagogue
in vitro.4 The distribution of PKR1 in the rat and mouse gastrointes-
tinal (GI) tracts characterized via RT-PCR taken together with PK1-
mediated functional effects suggest that it plays a significant role in
modulating GI motility and secretomotor function.2,5,6
Dysmotility and altered secretomotor activity are functional
hallmarks of GI diseases like irritable bowel syndrome (IBS) and
inflammatory bowel disease (IBD).7 Due to the pivotal role that
PK1 and its receptor appear to play in the modulation of GI motil-
ity, novel agents that are designed to modulate the activation of
PKR1 as antagonists could be highly efficacious in the treatment
of functional bowel disorders that accompany IBS and IBD. There-
fore, a program was initiated to discover a novel PK1 receptor
antagonist.
A review of the literature revealed no reports of a small mole-
cule inhibitor of the PK1 receptor.8 A high throughput screening
of our compound collection identified guanidine triazinedione 1
as a moderate PK1 receptor antagonist. (Fig. 1) Efforts were under-
taken to increase potency and to explore structure–activity rela-
tionships (SAR) by optimizing R1 and R2 of scaffold 2. (Fig. 2)
New analogs were synthesized via two different routes.9 The
first method was developed to explore changes to R2, keeping R1
constant (Scheme 1). Initially, the commercially available sulfonic
acid salt of 2-methyl-isothiourea 3 was free-based with 3 N NaOH
and the resulting compound reacted with commercially available
isocyanates to provide intermediate 4. Acylation of the isothiourea
4 with methylchloroformate under basic conditions yielded cycli-
zation precursor 5. Subsequently, 5 was cyclized under basic con-
ditions to yield triazinedione 6. Intermediate 6 was alkylated with
alkyl halides under basic conditions or substituted with commer-
cially available alcohols using Mitsunobu conditions to provide
key intermediate 7. Displacement of the thiomethyl group with
O
N
N
H
N
NH2
NH
O
N
N
H
MeO
PK1 IC50 = 0.36 μM
1
* Corresponding author.
E-mail address: jralb@msn.com (J.L. Ralbovsky).
Figure 1. Compound identified via HTS.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.157

2662
J. L. Ralbovsky et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2661–2663
O
H
S
N
R1
b
a
N
N
HN
NH2
H
N
HN
R2
SMe
R2
NH2
NH
O
N
N
H
R2
9
10
2
O
O
R1
O
Figure 2. Scaffold 2.
HN
N
N
N
c
2
O
SMe
SMe
N
N
NH
SMe
R2
R2
O
NH
SMe
c
a,b
H2N
R1
7
11
O
S
O
N
H
N
H
HO
3
OH
Scheme 2. Reagents: (a) MeI, MeOH; (b) ClCONCO, DIEA,CH2Cl2; (c) R1–OH, PPh3,
4
DEAD, THF.
O
O
O
R1
R1
O
N
OMe
d
e
acted with pyrazole-1-carboxamidine hydrochloride and diisopro-
pylethylamine in acetonitrile at room temperature.
N
N
R1
N
N
N
H
N
H
SMe
O
N
R2
7
SMe
O
N
H
6
SMe
In order to synthesize target compounds varying R1 and keep-
ing R2 constant, we utilized the reaction sequence shown in
Scheme 2. Commercially available thiourea 9 was methylated
with methyl iodide in methanol to provide isothiourea 10. Upon
addition of N-chlorocarbonyl isocyanate, isothiourea 10 was cy-
clized to give triazinedione 11. Alkylation of the ring was accom-
plished either by utilizing Mitsunobu conditions as shown or alkyl
halides under basic conditions to provide key intermediate 7.
Using the same procedures outlined in Scheme 1, 7 was converted
to guanidine 2. All final compounds were purified using reverse
phase chromatography and isolated as trifluoroacetic acid (TFA)
salts.
5
O
O
R1
R1
f
N
N
g
N
N
H
N
NH2
NH2
O
N
N
H
O
N
N
H
R2
R2
NH
8
2
Scheme 1. Reagents and conditions: (a) 3 N NaOH; (b) R1NCO, H2O/MeOH/THF, 0 °C
to rt; (c) methylchloroformate, NEt3, CH2Cl2, À10 °C to rt; (d) NaOMe,MeOH; (e) R2-
OH, PPh3, DEAD, THF or R2–Cl, NaOMe, CH3CN, heat; (f) ethylenediamine, toluene,
90 °C; (g) pyrazole-1-carboxamidine hydrochloride, DIEA, CH3CN.
Compounds were tested by measuring the compounds ability to
inhibit hPK1 induced calcium mobilization in GPR73 (hPKR1)
expressing HEK 293 cells.11 The results are shown in Table 1.
ethylenediamine in toluene at 90 °C yielded compound 8.10 To
form the desired guanidine derivative 2, the amine of 8 was re-
Table 1
The inhibition of prokineticin 1 by triazinediones with R1 and R2 modifications
O
R1
N
N
H
X
N
NH2
NH
O
N
N
H
R2
12
Compd #
R1
R2
X
PK1 IC50 (lM)
1
Benzyl
Phenethyl
n-Butyl
2-Furyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Methoxyphenethyl
Benzyl
4-Methoxybenzyl
4-Nitrobenzyl
4-Propoxybenzyl
n-Hexyl
4-Methoxy-methylcyclohexyl
4-Hydroxybenzyl
3-Furyl
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
0.336
2.50
2.18
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
2.59
4-Fluorobenzyl
3,4-Dichlorobenzyl
3-Methoxybenzyl
2-Methoxybenzyl
4-Methoxybenzyl
4-Hydroxybenzyl
4-Methoxyphenethyl
4-Cyanobenzyl
3,4-Dichlorobenzyl
4-Fluorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Fluorobenzyl
3,4-Dichlorobenzyl
3,4-Dichlorobenzyl
4-Methoxybenzyl
4-Methoxybenzyl
4-Chlorobenzyl
4-Methoxybenzyl
3,4-Dichlorobenzyl
Benzyl
0.033
0.022
3.69
>10
0.021
1.16
0.461
0.318
1.21
2.86
4.78
0.019
>10
0.515
6.51
3.26
0.027
1.87
0.051
0.047
>10
2-Methoxy-5-methylpyridyl
4-Difluoromethoxybenzyl
4-Fluorobenzyl

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,
469; (b) Schweitz, H.; Pacaud, P.; Diochot, S.; Moinier, D.; Lazdunski, M. FEBS
Lett. 1999, 461, 183.
2. Li, M.; Bullock, C. M.; Knauer, D. J.; Ehlert, F. J.; Zhou, Q. Y. Mol. Pharmacol. 2001,
59, 692.
3. Hoogerwerf, W. A. Neurogastroenterol. Motil. 2006, 186, 455.
4. Wade, P. R.; Mabus, J. R.; Palmer, J. M.; Prouty, S. M.; Saunders, P. R.; McKenney,
S. L.; Chevalier, K. M.; Hornby, P. J. Gastroenterology 2007, 132, A223.
5. Masuda, Y.; Takatsu, Y.; Terao, Y.; Kumano, S.; Ishibashi, Y.; Suenaga, M.; Abe,
M.; Fukusumi, S.; Watanabe, T.; Shintani, Y.; Yamada, T.; Hinuma, S.; Inatomi,
N.; Ohtaki, T.; Onda, H.; Fujino, M. Biochem. Biophys. Res. Commun. 2002, 293,
396.
Control (DMSO),N = 7
50
16, 1 μM
N = 6
40
6. Lin, D. C.; Bullock, C. M.; Ehlert, F. J.; Chen, J. L.; Tian, H.; Zhou, Q. Y. J. Biol. Chem.
2002, 277, 19276.
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,
K. A.; Palsson, O. S.; Levy, R. L.; Korff, M. B.; Feld, A. D.; Turner, M. J.; Whitehead,
W. E. Aliment Pharmacol. Ther. 2007, 26, 237.
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.