Discovery of 2-(1H-indazol-1-yl)-thiazole derivatives as selective EP 1 receptor antagonists for treatment of overactive bladder by core structure replacement

Article abstract of DOI:10.1016/j.bmcl.2014.01.052

We have designed a series of potent EP₁ receptor antagonists. These antagonists are a series of 2-(1H-indazol-1-yl)-thiazoles in which the core structure was replaced with pyrazole-phenyl groups. In preliminary conscious rat cystometry experiments, two representative candidates, 2 and 22, increased bladder capacity. In particular, the increase using 22 was approximately 2-fold that of the baseline. More detailed profiling of this compound and further optimization of this series promises to provide a novel class of drug for treating overactive bladder (OAB).

Full text of DOI:10.1016/j.bmcl.2014.01.052

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1327–1333
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 2-(1H-indazol-1-yl)-thiazole derivatives as selective
EP1 receptor antagonists for treatment of overactive bladder by core
structure replacement
⇑
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
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed a series of potent EP1 receptor antagonists. These antagonists are a series of
2-(1H-indazol-1-yl)-thiazoles in which the core structure was replaced with pyrazole-phenyl groups.
In preliminary conscious rat cystometry experiments, two representative candidates, 2 and 22, increased
bladder capacity. In particular, the increase using 22 was approximately 2-fold that of the baseline. More
detailed profiling of this compound and further optimization of this series promises to provide a novel
class of drug for treating overactive bladder (OAB).
Received 5 November 2013
Revised 6 January 2014
Accepted 18 January 2014
Available online 28 January 2014
Keywords:
EP1 antagonist
Indazole
Ó 2014 Elsevier Ltd. All rights reserved.
Thiazole
Overactive bladder
Cystometry
Overactive bladder (OAB) is defined by The International Conti-
nence Society as a ‘symptom syndrome which includes urinary
urgency with or without urge incontinence, urinary frequency,
and nocturia’.1 In addition, urinary incontinence is generally
defined as an ‘involuntary loss of urine that is objectively demon-
strable and is a social or hygienic problem’, and urinary urgency is
generally understood as a ‘state at which strong and sudden desire
to urinate occurs and the urge cannot be controlled’.2
The causes of OAB may include a change in bladder function due
to aging, cerebral hemorrhage, cerebral infarction, Parkinson’s dis-
ease, a neuronal disorder such as spinal injury, lower urinary tract
obstruction due to prostatic hypertrophy, and a sensitive bladder
due to expression of an irritative voiding symptom caused by a
hypersensitive bladder resulting from chronic interstitial cystitis.
However, for most cases, the cause remains unknown.3
Pharmaceutical Co., Ltd and Kissei Pharmaceutical Co., Ltd are cur-
rently co-evaluating their candidate in Phase I clinical trials.8
We previously identified 2-(pyrazolyl-1-yl)-thiazole 1 as show-
ing good EP1 receptor antagonist activity and selectivity.9 We fur-
ther optimized 1 and identified 2 as having high activity and good
physicochemical properties.10 However, we thought it was needed
to improve the potency of this series for providing the clinical can-
didate. To identify new compounds with unique profiles including
high potent in vivo activity, we attempted synthetic approaches
different from previous strategies applied to 1. We modified the
core structure of 1 because our earlier studies suggested that a
smaller structure is more suitable for EP1 receptor affinity.
Prostaglandin E2 (PGE2) has been suggested as a putative stim-
ulant of afferent nerves of the bladder via the EP1 receptor. Conse-
quently, EP1 receptor antagonists are a promising strategy for
treating OAB.4 A number of companies are pursuing EP1 receptor
antagonists, but very limited information on their clinical develop-
ment is available, except for AstraZeneca’s ZD6416,5 and Ono Phar-
maceutical’s ONO-81306 and ONO-8539.7 Most recently, KYORIN
⇑
Corresponding author. Tel.: +81 558 76 8493; fax: +81 558 76 5755.
E-mail address: atobe.mb@om.asahi-kasei.co.jp (M. Atobe).
Figure 1. Replacement of core structure of 1. (a) hEP1 rep.: ‘human EP1 reporter
assay’.
http://dx.doi.org/10.1016/j.bmcl.2014.01.052
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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M. Atobe et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1327–1333
Therefore, we investigated replacing the pyrazole-phenyl with a
bicyclic indazole system and generated compound 3. Compound
3 exhibited significantly increased EP1 receptor antagonist activity
(IC50: 2.4 nM), confirming that we had identified a new structure
with high potency (Fig. 1).
bicyclic system is unsuitable for polar groups, leading us to identify
the indazole ring as the best core structure. We thus further substi-
tuted this position (Table 1).
To identify the candidate position for increasing activity, we
scanned using fluorine. 4-F (13) showed significantly decreased
We next investigated the replacement of the pyrazole-phenyl
activity (IC50: 500 nM) whereas 5-F (14) was tolerated (IC50:
with other bicyclic systems. Indole 4 showed high potency (IC50
:
80 nM). Since 6-F (15) was found to retain EP1 receptor antagonist
activity (IC50: 2 nM), we focused on the 6-position on the indazole
to further optimize the compound. 6-Me (16) exhibited increased
potency (IC50: 1.0 nM), and the 6-OH substitution (17) was toler-
ated (IC50: 140 nM). These data were similar to that obtained dur-
ing optimization of the core structure. Conversion of 6-OH (17) to
6-OMe (18) improved potency (IC50: 1.1 nM) whereas conversion
to 6-OEt (19) decreased potency (IC50: 100 nM). 6-NH2 (20) re-
tained potency (IC50: 19 nM), whereas there was an increase in
the potency of 6-NHMe (21) (IC50: 50 nM). Finally, 6-CF3 (22)
was identified as exhibiting the best EP1 activity (IC50: 0.6 nM)
(Table 2).
4.0 nM), but was intrinsically unstable (rat CLint: 2396 mL/min/
kg). Tetrahydro six-membered derivative 5 retained potency,
whereas five, seven and eight membered ring compounds
exhibited decreased potency. These data suggested that the
six-membered moieties fit into a hydrophobic pocket. Dihydropyran
9 showed decreased potency, together with pyrazolopyridine 10, 11
and 12. These data suggested that the pocket accommodating the
Table 1
Optimization of core structure R1
We next optimized the 3-position phenyl-group on the indazole
ring. Comparison of the activity of 4-F (23) and 3-F (24) showed
that the para position was preferred to the meta position. 4-Cl
(25) showed slightly decreased activity whereas 4-MeO (26) re-
tained potency. However, 4-CF3 (27) exhibited significantly de-
creased activity, suggesting that this bulky group could not be
accommodated by the ring structure of the compound. Although
we speculated that a smaller group will be preferred in this part
of the compound, cyclopentenyl (28) showed decreased activity
Compd R1
hEP1 rep. 1
inhibition
l
M
hEP1 rep.
IC50 (nM)
rat CLint
(mL/kg/min)
(IC50: 80 nM) and cyclohexyl (29) showed high potency (IC50
:
4 nM). These data suggested that a six-membered ring was a suit-
able moiety. We also evaluated other heterocyclic systems. 3-Thi-
enyl (30) was tolerated, whereas 5-pyrimidyl (31) showed
significantly decreased activity, indicating that an external position
for the hydrophobic structure was preferred. Compounds contain-
ing the saturated cyclic system, 1-N-piperidyl (32) or 4-methyl-1-
N-piperidyl (33), retained potency. However, both these com-
pounds showed positive reactions in the Ames test and so were
considered unsuitable due to safety concerns. Consequently, the
phenyl group was chosen as the best structure for the 3-position
on the indazole ring (Table 3).
3
2.4
120
16
194
4
2396
5
6
290
ND
Figure 2 compares the profiles of compounds 22 and 2, which
we previously reported as having good profiles.10 Both compounds
show good selectivity for the EP1 receptor and a good profile,
except for low water solubility between pH 1.2 and pH 6.8. The
0%
7
8
9
1000
ND
ND
ND
Table 2
Discovery of substituted group R2
6%
8%
Compd
R2
hEP1 rep. IC50 (nM)
rat CLint (mL/kg/min)
10
11
12
200
39
65
69
ND
3
–H
4-F
5-F
6-F
6-Me
6-OH
6-OMe
6-OEt
6-NH2
6-NHMe
6-CF3
2.4
500
80
2
1
140
1.1
100
19
50
0.6
194
280
345
337
ND
522
196
ND
13
14
15
16
17
18
19
20
21
22
13%
208
ND
186

M. Atobe et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1327–1333
1329
Table 3
Discovery of substituted group R3
Compd
R3
hEP1 rep. IC50 (nM)
2.4
rat CLint (mL/kg/min)
194
3
23
24
25
26
27
4.5
20
12
4
278
385
753
618
809
140
28
29
30
80
4
196
490
190
30
31
32
33
>100
16
ND
Figure 2. Comparison of the profiles of 2 and 22. (a) hCLint.: ‘human intrinsic
clearance’. (b) rCLint.: ‘rat intrinsic clearance’. (c) Sol: ‘solubility’. (d) MDCK.:
‘Madin-Darby canine kidney’. (e) The peak plasma concentration of a drug after
administration. (f) BA: ‘bioavailability’.
139
260
32
Figure 3. Effect of2and 22 onurinationreflectionparametersin aratmodel.Eachvaluerepresentsthe mean SE of 3 animals.2 and 22 wereintravenously administrated (1 mg/kg).

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M. Atobe et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1327–1333
Scheme 1. Reagents and conditions: (a) 2-bromo-thiazolecarboxylate, (1S,2S)-N,N0-dimethylcyclohexane-1,2-diamine, CuI, K3PO4, mesitylene, 185 °C, (35a: 45%, 35b: 47%,
35c: 48%, 39a: 16%, 39b: 5%, 39c: 6%, 39d: 15%, 39e: 19%); (b) 5 M NaOH, EtOH, rt, (3: 41%, 4: 90%, 5: 89%, 6: 44%, 7: 32%, 8: 36%, 9: 59%, 10: 58%); (c) PhCOCl, LHMDS, toluene,
0 °C, 1 h; (d) (NH2)2–H2O, AcOH, rt, 3 h (38a: 35%, 38b: 29%, 38c: 22%, 38d: 39%, 38e: 11%).
pharmacokinetic parameters of these compounds in rats are shown.
A single oral administration of compound 22 to rats at 10 mg/kg
micturition volume and the micturition interval, but had no effect
on basal pressure, micturition pressure or residual volume (Fig. 3).
Both the intercontraction interval and the micturition volume were
increased: approximately 1.3 and 2-fold that of the baseline for
compounds 2 and 22, respectively. These data support the hypoth-
esis that the hyperactivity of afferent fiber C of the sensory nerves
in the bladder was reduced, indicating that these EP1 receptor
antagonists hold promise for the treatment of OAB.
(n = 3) provided a Cmax of 5.34
The bioavailability (BA) in rats was calculated to be 26%.
Compound 22 was tested against a battery of 57 receptors and 4
enzymes at a concentration of 10 M (Cerep, Celle l’Evescault
France). No significant activity (<25%) was seen with the exception
of the A1 and A3 receptors (IC50 = >1.0 M) (data not shown). Thus,
lM and AUC(0–1) of 9.27 lM/L hr.
l
l
compound 22 shows excellent selectivity. These data suggested
that the selectivity of compound 22 was improved compared to
compound 2.
In vivo cystometry studies in conscious rats using compounds 2
and 22 were then performed. The effects of compound 2 and 22
(1 mg/kg, iv) on cystometric parameters such as basal pressure,
micturition pressure, intercontraction interval, micturition volume
and residual volume were evaluated. Both compounds increased
The synthesis of the 2-(1H-indazol-1-yl)-thiazole derivatives 3–
10 listed in Table 1 is shown in Scheme 1. Buchwald N-arylation11
of starting material 34a–c12–14 was used to give the thiazole-ring
moiety, followed by hydrolysis to give 3, 4 and 10. In the case of
compounds 5–9, the synthetic route started from the commercially
available ketone 36a–e, which was first
a-acylated to the
corresponding dione 37a–e with LHMDS and PhCOCl at
room temperature and then condensed with (NH2)2–H2O to give
Scheme 2. Reagents and conditions: (a) PhMgBr, THF, 0 °C to rt, on, (quant); (b) Dess–Martin periodinane, CH2Cl2, rt, on, (97%); (c) (NH2)2–H2O, EtOH, reflux, on, (51%); (d) 2-
bromo-thiazolecarboxylate, (1S,2S)-N,N0-dimethylcyclohexane-1,2-diamine, CuI, K3PO4, mesitylene, 185 °C, (44: 63%, 48: 19%); (e) 5 M NaOH, EtOH, rt, (11: 20%, 12: 50%); (f)
N-methoxy-N-methyl-benzamide, LDA, THF, ꢀ78 °C to rt; (g) (NH2)2–H2O, EtOH, rt, 1 h, (2 steps 63%).

M. Atobe et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1327–1333
1331
Scheme 3. Reagents and conditions: (a) (NH2)2–H2O, CuO, K2CO3, xylenes, reflux, on, (50a: 16%, 50b: 21%, 50c: quant, 50d: 99%, 50e: quant, 50f: 50%, 55: 24%); (b) 2-bromo-
thiazolecarboxylate, (1S,2S)-N,N0-dimethylcyclohexane-1,2-diamine, CuI, K3PO4, mesitylene, 185 °C, (51a: 14%, 51b: 93%, 51c: 52%, 51d: 64%, 51e: 23%, 51f: 27%, 56: 62%); (c)
5 M NaOH, EtOH, rt, (13: 65%, 14: 95%, 15: 67%, 16: 82%, 22: 99%, 23: 39%, 24: 95%); (d) PhMgBr, THF, 0 °C to rt, on, (73%); (e) Dess–Martin periodinane, CH2Cl2, rt, on, (quant).
3,5-diphenyl-4-methyl tetrahydro-indazole derivative 38a–e. The
thiazole-ring moiety was synthesized in a similar manner, fol-
lowed by hydrolysis to give 5–9.
49a–f was reacted using the same conditions described by Olmo
et al.15 to give indazole 50a–f. The thiazole-ring moiety was syn-
thesized in a similar manner, followed by hydrolysis to give 13–
15 and 22–24. For compound 16, the synthetic route started from
2-fluoro-4-methylbenzaldehyde (52), which was alkylated to the
corresponding alcohol 53 with PhMgBr, and then oxidized using
Dess–Martin periodinane to give benzoyl 54. Benzoyl 54 was
condensed with (NH2)2–H2O under copper-mediated conditions16
to give indazole 55. A similar procedure was used to synthesize
compound 16.
Compounds 17–19 listed in Table 2 were synthesized according
to Scheme 4. The phenol of 5717 was protected with TBDMS-Cl,
then regioselective iodation in the presence of potassium
t-butylate gave intermediate 58. Protection of the 1-nitrogen on
the indazole and Suzuki–Miyaura coupling was used to prepare
The synthetic route for compounds 11 and 12 is shown in
Scheme 2. Commercially available 3-fluoroisonicotinaldehyde
(40) was first alkylated to alcohol 41 with phenylMgBr and then
oxidized with Dess–Martin periodinane to give benzoyl 42. Ben-
zoyl 42 was condensed with (NH2)2–H2O to give 1H-pyrazol-
o[3,4-c]pyridine 43, then the thiazole-ring moiety was
synthesized and hydrolysis was conducted in a manner similar to
11. Lithiation of the 3-position of 4-chloropyridine (45) and subse-
quent quenching with Weinreb amide resulted in the formation of
benzoyl 46. In a similar manner, compound (12) was synthesized.
Compounds 13–16 and 22–24 were synthesized according to
Scheme 3. Commercially available 1-benzoyl-2-fluorobenzene
Scheme 4. Reagents and conditions: (a) TBDMS-Cl, imidazole, DMF, rt, 3 h, (88%); (b) tBuOK, I2, THF, 0 °C, on, (quant); (c) Boc2O, Et3N, DMAP, CH2Cl2, rt, 1 h, (quant); (d)
PhB(OH)2, Pd2dba3, (o-tol)3P, K3PO4, DMF, 80 °C, on, (31%); (e) TFA, CH2Cl2, rt, 3 h, (96%); (f) 2-bromo-thiazolecarboxylate, (1S,2S)-N,N0-dimethylcyclohexane-1,2-diamine, CuI,
K3PO4, mesitylene, 185 °C, (54%); (g) 5 M NaOH, EtOH, rt, (17: 89%, 18: 97%, 19: 90%); (h) MeI, NaH, DMF, rt, 1 h, (18%); (i) EtI, NaH, DMF, rt, 1 h, (30%).

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M. Atobe et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1327–1333
Scheme 5. Reagents and conditions: (a) KOH, I2, DMF, rt, 1 h, (85%); (b) PhB(OH)2, Pd2dba3, (o-tol)3P, K3PO4, DMF, 80 °C, on, (39%); (c) 2-bromo-thiazolecarboxylate, (1S,2S)-
N,N0-dimethylcyclohexane-1,2-diamine, CuI, K3PO4, mesitylene, 185 °C, (48%); (d) H2 gas, Pd-C, MeOH, rt, on, (55%); (e) 5 M NaOH, EtOH, rt, (20: 99%, 21: 87%); (f) 36% HCHO
aq, NaBH(OAc)3, AcOH, CH2Cl2, rt, on, (7%).
the phenyl moiety after deprotection of the TBDMS and Boc group
with TFA. The thiazole-ring moiety was synthesized in a similar
manner, followed by hydrolysis to give 17. Methylation and ethy-
lation of 60 provided 61 and 62, followed by hydrolysis to give 18
and 19, respectively.
For compounds 20 and 21, the synthetic route started from
commercially available 6-nitro-indazole 63, which was iodized
with iodine under basic conditions to give 64. Suzuki–Miyaura
coupling of 64 was used to construct the phenyl ring, followed
by N-arylation and hydrolysis to give 20. Methylation of 66 using
reductive N-alkylation provided 67, followed by hydrolysis to give
21 (Scheme 5).
Compounds 25–33 listed in Table 3 were synthesized according
to Scheme 6. Suzuki–Miyaura coupling of compound 6817 or 7018
were used to prepare the aryl moiety, cyclopentyl and cyclohexyl
moiety. Buchwald N-arylation of compounds 68 was used to pre-
pare the piperidine moiety. After deprotection of Boc or THP with
cHCl, the thiazole-ring moiety was synthesized in a similar man-
ner, followed by hydrolysis to give 25–33.
In conclusion, we have designed several EP1 receptor antago-
nists from a series of 2-(1H-indazol-1-yl)-thiazoles. In preliminary
conscious rat cystometry experiments, two representative candi-
dates, 2 and 22, increased bladder capacity (intercontraction inter-
val). Compound 22 in particular holds excellent promise. This
compound may serve as a candidate for further development of
an OAB drug.
References and notes
1. Wein, A. J. Urology 2002, 60, 7.
2. Yamaguchi, O. Clin. Drug Ther. 2002, 21, 2.
3. Andersson, K. E. Pharmacol. Rev. 1993, 45, 253.
4. 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.
5. Palea, S.; Toson, G.; Pietra, C.; Trist, D. G.; Artibani, W.; Romano, O.; Corsi, M. Br.
J. Pharmacol. 1998, 124, 865.
6. Miki, T.; Matsunami, M.; Nakamura, S.; Okada, H.; Matsuya, H.; Kawabata, A.
Pain 2011, 152, 1373.
7. Okada, H.; Konemura, T.; Maruyama T. EAU Congress 2010, Poster Session 125.
8. Cima Science Journal, inc. New Current, 2013, 24, 40.
9. Atobe, M.; Naganuma, K.; Kawanishi, M.; Morimoto, A.; Kasahara, K.; Ohashi, S.;
Suzuki, H.; Hayashi, T.; Miyoshi, S. Bioorg. Med. Chem. Lett. 2013, 23, 6064.
10. Atobe, M.; Naganuma, K.; Kawanishi, M.; Morimoto, A.; Kasahara, K.; Ohashi,
S.; Suzuki, H.; Hayashi, T.; Miyoshi, S. Bioorg. Med. Chem. Lett. 2013, 24, 6569.
11. Antilla, J. C.; Baskin, J. M.; Barder, T. B.; Buchwald, S. L. J. Org. Chem. 2004, 69,
Scheme 6. Reagents and conditions: (a) Pd2dba3, (o-tol)3P, K3PO4, DMF, 80 °C, 12 h;
(b) cHCl, MeOH, rt, 3 h, (69a: 81%, 69b: 76%, 69c: 97%, 69d: 72%, 69e: 75%, 69f: 80%,
69g: 83%); (c) Pd(OAc)2, xantPhos, Cs2CO3, dioxane, 90 °C, 12 hours; (d) trifluoro-
acetic acid, CH2Cl2, rt, 3 h, (69h: 40%, 69i: 56%); (e) 2-bromo-thiazolecarboxylate,
(1S,2S)-N,N0-dimethylcyclohexane-1,2-diamine, CuI, K3PO4, mesitylene, 185 °C,
(71a: 52%, 71b: 48%, 71c: 53%, 71d: 45%, 71e: 55%, 71f: 41%, 71g: 45%, 71h: 48%,
71i: 25%); (f) 5 M NaOH, EtOH, rt, (25: 83%, 26: 94%, 27: 87%, 28: 91%, 29: 84%, 30:
88%, 31: 83%, 32: 61%, 33: 24%).
5578.

M. Atobe et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1327–1333
1333
12. Taylor, E. C.; Sobieray, D. M. Tetrahedron 1991, 47, 9599.
13. Rodriguez, J. G.; Lafuente, A.; Garcïa-Almaraz, P. J. Heterocycl. Chem. 2000, 37,
1281.
15. Vina, D.; Olmo, E.; López-Pérez, J. L.; Feliciano, A. Org. Lett. 2007, 9, 525.
16. Stephen, T. H.; Swaminathan, R. N. Org. Lett. 2006, 8, 2675.
17. Valerie, C.; Didier, V.; Sylvain, R. Tetrahedron Lett. 2000, 41, 4363.
18. Elodie, L.; Valerie, C.; Silvia, S.; Sylvain, R. Synthesis 2011, 16, 2651.
14. Lynch, B. M.; Khan, M. A.; Teo, H. C.; Pedrotti, F. Can. J. Chem. 1988, 66, 420.