Hit-to-lead optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as a novel class of EP1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2013.09.032

We describe a medicinal chemistry approach to generate a series of 2-(1H-pyrazol-1-yl)thiazole compounds that act as selective EP₁ receptor antagonists. The obtained results suggest that compound 12 provides the best EP₁ receptor ant

Full text of DOI:10.1016/j.bmcl.2013.09.032

Accepted Manuscript
Hit-to-lead optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as a novel
class of EP receptor antagonists
1
Masakazu Atobe, Kenji Naganuma, Masashi Kawanishi, Akifumi Morimoto,
Ken-ichi Kasahara, Shigeki Ohashi, Hiroko Suzuki, Takahiko Hayashi, Shiro
Miyoshi
PII:
S0960-894X(13)01106-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.09.032
BMCL 20884
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 August 2013
2 September 2013
10 September 2013
Please cite this article as: Atobe, M., Naganuma, K., Kawanishi, M., Morimoto, A., Kasahara, K-i., Ohashi, S.,
Suzuki, H., Hayashi, T., Miyoshi, S., Hit-to-lead optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as a novel
class of EP receptor antagonists, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/
1
j.bmcl.2013.09.032
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Hit-to-lead optimization of 2-(1H-pyrazol-1-
yl)-thiazole derivatives as a novel class of EP1
receptor antagonists
Leave this area blank for abstract info.
Masakazu Atobe*, Kenji Naganuma, Masashi Kawanishi, Akifumi Morimoto, Ken-ichi Kasahara, Shigeki
Ohashi, Hiroko Suzuki, Takahiko Hayashi, Shiro Miyoshi

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Hit-to-lead optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as a novel
class of EP1 receptor antagonists
Masakazu Atobe*, Kenji Naganuma, Masashi Kawanishi, Akifumi Morimoto, Ken-ichi Kasahara, Shigeki
Ohashi, Hiroko Suzuki, Takahiko Hayashi, Shiro Miyoshi
Pharmaceutical Research Center, Asahi Kasei Pharma Corporation, 632-1 Mifuku, Izunokuni-shi, Shizuoka 410-2321, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
We describe a medicinal chemistry approach to generate a series of 2-(1H-pyrazol-1-yl)thiazole
compounds that act as selective EP1 receptor antagonists. The obtained results suggest that
compound 12 provides the best EP1 receptor antagonist activity and demonstrates good oral
pharmacokinetics.
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
EP1 receptor antagonist
pyrazole
thiazole
overactive bladder
HTS
Prostaglandin E2 (PGE2) is a metabolite of arachidonic acid
and is involved in various physiological actions such as cell
protection, oxytocic activity, pain generation, peristaltic
movement in the digestive tract, anti-hypertensive activity, and
diuretic activity. PGE2 is released from the urothelium or smooth
muscle of the bladder, and intravesicular PGE2 contributes to the
and showed a 3–8 fold EP1 selectivity relative to EP2, EP3 and
EP4.
pathophysiology of bladder overactivity1, 2
.
It is believed that PGE2 acts on bladder sensation by
contractile action of the detrusor smooth muscle and by
stimulating the afferent nerves of the bladder3, 4. According to
recent studies, there are four receptor subtypes of PGE2: EP1,
EP2, EP3 and EP4 receptor5, 6. Of these, the EP1 receptor is the
most abundant on the C fiber of the bladder7. It was found that
antagonizing this receptor inhibits the voiding reflex7. It was also
reported that the symptoms of overactive bladder accompanying
prostatic hyperplasia were improved by inhibiting afferent neural
activities. EP1 receptor antagonists inhibit afferent activity and
are used to treat overactive bladder symptoms8.
Figure 1. HTS hit compound
To obtain more potent compounds with higher EP1 activity,
we introduced different functional groups into compound 1
(Table 1). Regioisomer 2 showed improved EP1 receptor
antagonist activity. Compound 3 (R1 = Ph) also showed increased
activity, whereas 4 (R1 = 4-Me-Ph) showed a loss of activity.
These results suggested that the 4-methoxy group and 4-methyl
group are not important for EP1 receptor activity. However, the
4-phenol analog 5 and 3-phenol analog 6 showed moderate
potency, whereas the 2-phenol 7 showed increased potency; these
results indicated that ortho substitution is favored over meta or
para substitution.
A
number of companies are pursuing EP1 receptor
antagonists, but very limited information on their clinical
development is available. The few exceptions to this are
AstraZeneca’s ZD64169, Ono Pharmaceutical’s ONO-813010 and
Ono Pharmaceutical’s ONO-853911.
The in vitro ADME profile of 1, 3 and 5 was determined (Table
2). Compound 5 was found to be intrinsically unstable in human
plasma (human CLint: 500 ml/min/kg; rat CLint: 130
ml/min/kg). We checked the metabolites of 5 by LC/MS/MS and
found that the major metabolite arises from glucoronidation. In
contrast, compound 3 was stable in human and rat plasma, so we
investigated modifications of compound 3.
Our starting compound was identified by screening our in-
house compound library. 2-(1H-pyrazol-1-yl)thiazole 1 was
identified as having potent EP1 receptor antagonist activity by
hEP1 reporter gene assay (Fig 1). Compound 1 was then tested
for activity towards the other PGE2 receptors (EP2, EP3 and EP4)

The central ring of compound 12 was also replaced with two
types of imidazole rings (Table 4) to provide imidazole 13, which
exhibited decreased activity, and imidazole 14, in which the
replacement was tolerated. These results show that the pyrazole
ring plays an important role in the expression of EP1 antagonistic
activity.
Table 4. Optimization of the central ring
EP1 signals primarily through Gq, producing a transient rise
in intracellular calcium. Therefore, compound 12 was tested
using an intracellular Ca2+ release assay. In addition, compound
12 was tested for its ability to bind EP2, EP3 and EP4 receptors.
As shown in Table 4, 12 demonstrated excellent EP1 receptor
selectivity over other EP family receptors. The in vitro ADME
profile of 12 was also evaluated. Compound 12 was found to be
intrinsically stable in plasma (human CLint: 35 ml/min/kg;
mouse CLint: 15 ml/min/kg). We delineated the metabolites of
compound 12 by LC/MS/MS and observed several oxidized
products. Compound 12 displayed excellent apparent
permeability compared with atenolol as measured by flux
through MDCK cells in transwell cultures (A/BPapp = 51.64 x
10-6 cm/s). Compounds 1, 7 and 12 were tested using the Ames
test and were found to be negative against both TA98 and
TA100.
Table 1. Optimization of hit compound
Table 2. Profile of selected compounds
Starting from compound 3, modification of the 4- or 5-
position moiety was investigated (Table 3). The analogues with
t
R2 = Me (8) and R2 = Bu (9) showed a loss of EP1 antagonistic
activity, whereas when R2 was a phenyl group (10), EP1 activity
decreased. When we prepared compound 11 (R2 = CF3, R3 = Me)
and 12 (R2 = Ph, R3 = Me), it was found that 12 showed the most
potent EP1 receptor antagonist activity.
Table 5. EP family profile of 1, 7 and 12
Table 6. In vitro ADMET profile of 1, 7 and 12
The pharmacokinetics of compounds 7 and 12 were examined
via intravenous and oral administration to male Sprague–Dawley
rats. The animals received a single administration of 3 mg/kg, i.v.
or 10 mg/kg, p.o. Compound 12 exhibited good oral
pharmacokinetics and oral bioavailability was 32%. This
prolonged pharmacokinetic half-life suggests that the metabolic
stability of 12 is improved (cf., Fig 2 and Fig 3).
The synthetic route for the synthesis of the 2-(1H-pyrazol-1-
yl)thiazole derivatives 8–10 is highlighted in Schemes 1–3.
Table 3. Optimization of 3

Compounds 1–7 are commercially available. As shown in
Scheme 1, a SNAr reaction of the known 3,5-disubstituted
pyrazole derivatives 15a–c and ethyl 2-bromothiazolecarboxylate
using NaH in DMF at 150oC afforded the thiazoleacetates 16a–
m. Saponification of the resultant esters with NaOH provided 8–
10 (Scheme 1).
Scheme 2. Reagents and conditions: (a) thiosemicarbazide, EtOH, 80oC, 1 hour; (b)
ethyl 2-bromopyruvate, EtOH, 80oC, (2 steps, 17%); 5M NaOH, EtOH, rt, o.n., 99%.
The synthetic route to compound 12 started from
commercially available propiophenone 20 (Scheme 3), which
was first α-acylated to provide the corresponding 1,3-diketone
21 (LiHMDS, BzCl). Subsequent condensation with
NH2NH2•H2O afforded 3,5-diphenyl-4-methyl pyrazole 2212.
Buchwald N-arylation13 of compound 22 was employed to install
the thiazole-ring moiety, followed by hydrolysis to provide 1214.
Figure 2. Change in plasma concentration of 7 and 12.
Each compound was dosed at 10 mg/kg, p.o. in rats.
Scheme 3. Reagents and conditions: (a) LiHMDS, PhCOCl, toluene, rt, 1 min; (b)
NH2NH2•H2O, AcOH, rt, 30 min, (2 steps, 99%); (c) CuI, K3PO4, ethyl 2-bromo-
thiazolecarboxylate, 23, mesitylene, 185oC, (15%); (d) 5M NaOH, EtOH, rt, o.n., (82%).
The synthetic route providing compound 13 is illustrated in
Scheme 4. Ethyl 2-formylthiazole-4-carboxylate was first
condensed with 1-phenylpropane-1,2-dione and NH4OAc to give
the imidazole 26. Buchwald N-arylation of compound 26 was
used to install the N-phenyl moiety in 27, followed by hydrolysis
to give 13. The NOESY correlation of the imidazole confirmed
that the structure of 27 is as shown below.
Figure 3. Change in plasma concentration of 7 and 12.
Each compound was dosed at 10 mg/kg, p.o. in rats.
Scheme 4. Reagents and conditions: (a) NH4OAc, phenylpropane-dione, MeOH, rt, on,
(74%); (b) CuI, K3PO4, iodobenzene, 23, mesitylene, 185oC, (7%); (c) 5M NaOH, EtOH,
rt, o.n., 71%.
Scheme 1. Reagents and conditions: (a) ethyl 2-bromothiazole-carboxylate, NaH, DMF,
150oC, o.n. (16a: 44%, 16b: 20%, 16c: 18%); (b) 5M NaOH, EtOH, rt, o.n., (8: 55%, 9:
63%, 10: 60%).
Compound 14 was prepared using the route shown in Scheme
5. Suzuki-Miyaura coupling of compound 29 installed the vinyl
moiety, and the styrenyl disubstituted olefin was converted by a
two-step sequence (OsO4-catalyzed dihydroxylation followed by
oxidation with Dess-Martin periodinane) to the dione 32. Dione
32 was condensed with benzaldehyde and NH4OAc to give the
imidazole 33. N-alkylation of compound 33 effected the
installation of the N-methyl moiety in 34, followed by hydrolysis
to afford 13. The NOESY correlation of the imidazole confirmed
the structure of 34 to be as shown below.
As shown in Scheme 2, reaction of 4,4,4-trifluoro-2-methyl-1-
phenyl butane-1,3-dione 17 and thiosemicarbazide in ethanol
under reflux produced the pyrazolylthiourea 18, which was then
alkylated in situ with ethyl 2-bromopyruvate (Hanzsch reaction)
to give 2-(1H-pyrazol-1-yl)thiazole 19 in 37% yield in a two-
step, one-pot reaction. Finally, basic hydrolysis of 19 provided 11
(Scheme 2).

1522, 1499; HRMS (ESI) calcd for C20H15N3O2S [M+H]+ 362.0958,
found 362.0960.
Scheme 5. Reagents and conditions: (a) trans-2-phenylvinyl-boronic acid, Pd2(dba)3, tri-
o-tolylphosphine, K3PO4, DMF, 120oC, o.n., (62%); (b) OsO4, N-methylmorpholine,
MeCN-H2O(4:1), rt, (38%); (c) Dess-Martin periodinane, CH2Cl2, rt, (48%); (d)
NH4OAc, benzaldehyde, MeOH, rt, o.n., (41%); (e) MeI, NaH, DMF, rt, 1 hour, (91%);
(f) 5M NaOH, EtOH, rt, o.n., (82%).
In conclusion, we present a 2-(1H-pyrazol-1-yl)thiazole as a
novel class of EP1 receptor antagonists. Compound 12 appeared
to give the best EP1 receptor antagonist activity and good
selectivity over the other PGE2 receptors. Our research effort
focused on substituting the 2-phenol ring and identified
compound 12 as having good oral pharmacokinetics. Compound
12 may serve as a lead compound for further development of
overactive bladder drugs. Ongoing studies will be reported
elsewhere.
References and notes
1. Andersson, K. E., Pharmacol. Rev. 1993, 45, 253.
2. Khan, M. A.; Thompson C. S.; Mumtaz F. H.; Jeremy J. Y.; Morgan R.
J.; Mikhailidis D. P., Prostaglandins Leukot. Essent. Fatty Acids, 1998,
59, 415.
3. Palea, S.; Toson G.; Pietra C.; Trist D. G.; Artibani W.; Romano O.;
Corsi M., Br. J. Pharmacol., 1998, 124, 865.
4. Maggi, C. A., Pharmacol. Res. 1992, 25, 13.
5. Negishi, M.; Sugimoto Y.; Ichikawa A., J. Lipid Mediators Cell
signaling, 1995, 12, 379.
6. Narumiya S.; Sugimoto Y.; Ushikubi F., Pharmacol. Rev. 1999, 79, 1193.
7. Ikeda M.; Kawatani M.; Maruyama T.; Ishihama H., Biomed. Res. 2006,
27, 49.
8. Yamaguchi, O., Folia Pharmacologica, 2003, 121, 331.
9. Palea, S.; Toson G.; Pietra C.; Trist D. G.; Artibani W.; Romano O.;
Corsi M., Br. J. Pharmacol., 1998, 124, 865.
10. Miki, T.; Matsunami, M.; Nakamura, S.; Okada, H.; Matsuya, H.;
Kawabata, A., PAIN, 2011, 152, 1373.
11. Okada, H.; Konemura, T.; Maruyama, T., European Urology
Supplements, 2010, 9, 72
12. Stephen T. H.; Swaminathan R. N., Org. Lett., 2006, 8, 2675.
13. Antilla J. C.;, Baskin J. M.; Barder T. B.; Buchwald S. L., J. Org. Chem.,
2004, 69 5578.
14. Characterization data for compound 12: 2-(4-methyl-3,5-diphenyl-1H-
1
pyrazol-1-yl)thiazole-4-carboxylic acid ; H NMR (400 MHz, DMSO-
d6) ppm: 2.13 (3H, s), 7.46-7.56 (m, 8H), 7.78 (1H, s), 7.80 (1H, s),
8.20 (1H, s), 13C NMR (100 MHz, DMSO-d6) ppm:
δ
9.69 (CH3),
116.4 (C), 126.4 (CH), 127.6 (C and 2 carbons of CH), 128.1 (2 carbons
of CH), 128.54 (CH), 128.6 (C), 128.69 (3 carbons of CH), 128.9 (CH),
130.3 (2 carbons of CH), 132.0 (C), 142.1 (C), 152.44 (C), 159.89 (C),
161.52 (C), FT-IR (KBr, cm-1) 3071-2540 broad, 1686 (C=O), 1671,