Bioorganic & Medicinal Chemistry Letters 23 (2013) 6569–6576
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
SAR-based optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as highly potent 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
a r t i c l e
i n f o
Article history: Received 14 September 2013 Revised 21 October 2013 Accepted 29 October 2013 Available online 6 November 2013
a b s t r a c t We describe a medicinal chemistry approach for generating a series of 2-(1H-pyrazol-1-yl)thiazoles as EP1 receptor antagonists. To improve the physicochemical properties of compound 1, we investigated its structure–activity relationships (SAR). Optimization of this lead compound provided small compound 25 which exhibited the best EP1 receptor antagonist activity and a good SAR profile. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: EP1 antagonist Pyrazole Thiazole Solubility Optimization
Overactive bladder (OAB) is a urological condition with symptoms of urgency, with or without urge incontinence, and is usually accompanied by frequency and nocturia. OAB is estimated to affect 16% of people in the United States and Europe,1 and 30% in Asia.2 Current OAB therapies consist primarily of antimuscarinic drugs, but their use is limited by unpleasant side effects such as dry mouth, constipation and blurred vision.3 Gradual bladder filling is associated with the activation of sensory nerves, and the intensity of the stimuli on these sensory nerves leads to a desire to void. A proposed etiology for OAB is an alteration of the excitability of these sensory nerves. Targeting of the hyperactive pathways represents an attractive therapeutic approach for OAB.4 Prostaglandin E2 (PGE2) has been suggested as a possible therapeutic agent because of its role in the excitement of afferent nerves via the EP1 receptor.5 This concept leads to EP1 receptor antagonists being one of the most promising candidates in developing OAB drugs.6 For example, evidence for a role of the EP1 receptor is provided by studies of EP1 receptor-selective antagonists. SC19220, an EP1 receptor antagonist, increased bladder capacity in normal rats7, and in a rat spinal cord injury model of overactive bladder, the EP1 receptor antagonist ONO-8711 produced a decrease in detrusor overactivity.8 We have previously reported hit-to-lead optimization of 2(1H-pyrazol-1-yl)-thiazole derivatives as novel EP1 receptor ⇑ Corresponding author. Tel.: +81 558 76 8493; fax: +81 558 76 5755. E-mail address: [email protected] (M. Atobe). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.065
antagonists.9 In this study we found that compound 1 appeared to give the best EP1 receptor antagonistic activity and good oral pharmacokinetics (Fig. 1). Unfortunately, 1 has low water solubility and high protein binding to plasma proteins, mainly albumin. To overcome these problems, we investigated the structure–activity relationships (SAR) of this lead compound and optimized it by characterizing its hydrophobic properties, such as the c LogP value. Our previous results showed that the pyrazole ring plays an important role in the expression of EP1 antagonistic activity. We therefore aimed to replace the 3-, 4- and 5-position on the pyrazole with various functional groups adjusting physicochemical property. Table 1 shows the results of SAR studies of the 5-position on the pyrazole. Replacement of the phenyl ring with 2-thienyl (2) or 3thienyl (3) resulted in moderate activity. We prepared compounds bearing 3-pyridyl (4), 5-pyrimidyl (5), 1-N-methyl-4-pyrazolyl (6) and 4-amino-phenyl (7) and found that these compounds had decreased EP1 activity, whereas the corresponding 3-fluorophenyl (8) and 4-fluorophenyl (9) showed moderate activity. These results suggested that the pocket at the 5 position should be hydrophobic and shallow, so substituting with a hydrophilic group, such as a heterocyclic group containing one or two nitrogen atoms, is incompatible with activity (Table 1). Table 2 shows SAR studies of the thiazole part. Replacement of the thiazole ring with 2-oxazolyl (10), 2-furyl (11), 2-pyridyl (12) or 3-pyridyl (13) resulted in decreased activity. Only the 2-thienyl substitution (14) was tolerated when the 5-methyl-thiazolyl (15) and carboxylethylene group (16) were retained. These data
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a. hEP1 rep.: ‘human EP1 reporter assay’ b. hCLint.: ‘human intrinsic clearance’ c. rCLint.: ‘rat intrinsic clearance’ d. Sol: ‘solubility’ e. MDCK: ‘Madin-Darby canine kidney’ Figure 1. Profile of 1.
Table 1 Optimization of the 5-position on the pyrazole
Table 2 Optimization of the thiazole part
Compd Compd
a
R
1
hEP1 rep. 1 lM inhibition
a
hEP1 rep. IC50 (nM)
c Log P
1
50
5.48
2
120
5.34
3
80
5.13
4
34%
3.98
5
24%
3.98
6
8%
3.60
7
10%
4.26
8
50
5.48
9
100
5.62
clogP value was calculated with ChemBioDraw 12.0.
suggested that the thiazole ring might fit into a small pocket, so the SAR studies were narrowed to focus on this moiety (Table 2). SAR of the 4-position on the pyrazole was investigated (Table 3). When R3 = Et (17), EP1 activity was decreased. When R3 = CF3 (18), EP1 activity was retained. We prepared halogen derivatives, such as fluorine (19), chlorine (20), bromine (21) and iodine (22), and found that 20 showed highly potent EP1 receptor antagonist activity. When R3 = OH (23), EP1 activity was retained. Substitution with the methoxy group (24) provided decreased EP1 activity. Finally,
R2
hEP1 rep. 1 lM inhibition
1
hEP1 rep. IC50 (nM)
c Log P
50
5.48
10
33%
4.63
11
24%
5.22
12
48%
5.23
13
26%
5.03
14
1000
5.99
15
330
5.68
16
500
5.11
the compound bearing an amine (25) appeared to give the best EP1 receptor antagonist activity (IC50: 20 nM) and the lowest LogP. These data suggested that this position is critical for controlling the physicochemical properties of the antagonist. The results in Tables 1–3 indicated that decreasing the size of the molecule increases potency. We then focused on optimizing the pyrazole ring by fusing this ring with both phenyl rings
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(Table 4). 2-(3-Phenylindeno[1,2-c]pyrazol-1(4H) (26) exhibited significantly increased EP1 receptor antagonist activity (IC50: 0.9 nM). 2-(3-Phenyl-1H-benzofuro[3,2-c]pyrazol-1-yl) (27) showed good EP1 activity and lower c LogP. 3-Phenyl fused (28), 2-(3-phenyl-4,5-dihydro-1H-benzo[g]indazol-1-yl), showed decreased EP1 receptor antagonist activity. However, 5-phenyl fused (29), 2-(3-phenyl-4,5-dihydro-2H-benzo[g]indazol-2-yl), exhibited significantly increased EP1 receptor antagonist activity (IC50: 2.0 nM). Table 5 shows the antagonist activity of highly potent compounds 1, 25, 26 and 29 as measured using an EP1 reporter-gene assay and a Ca assay. Stimulation of the EP1 receptor by PGE2 was evaluated by a reporter-gene assay and an intracellular Ca2+ release assay using human EP1 receptor-expressed recombinant cells. Table 5 also shows the intrinsic metabolic clearance (CLint) of 1, 25, 26 and 29 in rat and human hepatic microsomes. CLint was calculated from the elimination ratio (0/20 min at 0.5 lM). These results suggest that 1 and 25 would be stable in both species. The solubility of 1, 25, 26 and 29 in aqueous buffer at pH 1.2 and pH 6.8 showed that 25 exhibited the best solubility under these conditions. To evaluate the epithelial membrane permeability of compound 25, we used Madin–Darby canine kidney (MDCK) cells as an in vitro cell model Figure 2.10 The permeability of compound 25 through the MDCK cell monolayer was significantly higher than through the atenolol, showing that compound 25 exhibits good permeability. Next, the safety of compound 25 was evaluated by checking CYP inhibition and genetic toxicity using the Ames test. Compound 25 did not inhibit the major human CYP enzymes below 5 lM, as seen in Figure 2. The IC50 values obtained are above 5 lM for all human CYPs. A bacterial mutation test was conducted using an amino acid-requiring strain of Salmonella typhimurium (TA98) to detect the point mutations. No genetic toxicity was observed for compound 25. The pharmacokinetic parameters of 25 in rats are shown in Figure 2. A single oral administration of compound 25 to rats at 10 mg/kg (n = 3) provided a Cmax of 6.33 lM and an AUC(0–1) of 17.4 lM/L hr. The bioavailability (BA) in rats was calculated to be 26%. Compound 25 was tested against a battery of 57 receptors and 4 enzymes at a concentration of 10 lM (Cerep, Celle l’Evescault France). No significant activity (100
5.82
29
2
5.82
Table 5 Comparison of the profile of 1, 25, 26 and 29 Compd
hEP1 rep. IC50 (nM)
Ca assay IC50 (nM)
Human CLint (ml/min/kg)
Rat CLint (ml/min/kg)
Protein bound (%)
Sol. H2O (lg/ml)
Sol. pH 1.2 (lg/ml)
Sol. pH 6.8 (lg/ml)
1 25 26 29
50 20 0.9 2
500 60 40 20
35 19 425 149
15 29 927 236
99.71 99.00 99.68 99.88
129 865 4 20
0 867 0 0
401 1012 57 10
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a. The peak plasma concentration of a drug after administration. b. BA: ‘bioavailability’ Figure 2. Profile of 25.
Scheme 1. Reagents and conditions: (a) propionitrile, sodium ethoxide, EtOH, rt, (84%); (b) thiosemicarbazide, EtOH, 80 °C then ethyl bromopyrvate, 80 °C, (18%); (c) tBuONO, I2, MeCN, reflux, (66%); (d) Pd2dba3, (o-tol)3P, K3PO4, 34a–h, DMF, 80 °C, (35a: 65%, 35b: 70%, 35c: 68%, 35d: 62%, 35e: 53%, 35f: 67%, 35g: 65%, 35h: 68%); (e) 5 M NaOH, EtOH, rt, (2: 61%, 3: 72%, 4: 52%, 5: 48%, 6: 62%, 7: 74%, 8: 80%, 9: 78%); (f) TFA, CH2Cl2, (39%).
M. Atobe et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6569–6576
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Scheme 2. Reagents and conditions: (a) Pd(OAc)2, cBRIDP, K3PO4, 38a, mesitylene, 180 °C (30%) or CuI, K3PO4, 37, 38b–f, mesitylene, 185 °C, (39b: 10%, 39c: 42%, 39d: 63%, 39e: 32%, 39f: 40%); (d) 5 M NaOH, EtOH, rt, (10: 67%, 11: 72%, 12: 32%, 13: 62%, 14: 53%, 15: 54%).
Scheme 3. Reagents and conditions: (a) LiAlH4, THF, rt, (97%); (b) Dess–Martin periodinane, CH2Cl2, rt, (58%); (c) ethyldiethylphosphonic acid, KHMDS, THF, 0 °C, (52%); (c) 5 M NaOH, EtOH, (78%).
to give 2–6, 8 or 9. In the case of 35f, after deprotection of the Boc group with TFA, hydrolysis of 35i afforded the corresponding carboxylic acid 7 (Scheme 1). Compounds 10, 11 and 13–16 listed in Table 2 were synthesized according to Scheme 2. For compound 10, N-arylation of 36 with chloride 38a in the presence of cBRIDP, manufactured by Takasago11, was used to introduce the oxazole-ring moiety, followed by hydrolysis to give 10. When bromide 38b–f was used, Buchwald
copper mediated N-arylation was used to give the thiazole or heterocyclic moiety, followed by hydrolysis to provide 11–15 (Scheme 2). For compound 16, the synthetic route started from previously reported compound 40 (Scheme 3), which was first reduced to the corresponding primary alcohol 41 with LiAlH4. Alcohol 41 was oxidized with Dess–Martin periodinane to give aldehyde 42, and subsequent reaction under the Horner–Wadsworth–Emmons
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Scheme 4. Reagents and conditions: (a) propionitrile, sodium ethoxide, EtOH, rt, (74%); (b) (NH2)2-H2O, EtOH, rt, (98%); (c) CuI, K3PO4, 37, ethyl 2-bromothiazolecarboxylate, mesitylene, 185 °C, (16%); (d) 5 M NaOH, EtOH, rt, (68%).
Scheme 5. Reagents and conditions: (a) sodium nitrite, urea, DMSO, 0 °C, (53%); (b) thiosemicarbazide, EtOH, 80 °C then ethyl bromopyruvate, 80 °C, (14%); (c) 5 M NaOH, EtOH, rt, (25: 52%, 20: 74%, 21: 52%, 22: 83%, 18: 74%); (d) tBuONO, CuCl2 (R2 = Cl) or CuBr2 (R2 = Br) or I2 (R2 = I), MeCN, 80 °C, (51a: 46%, 51b: 32%, 51c: 40%); (e) 2,2-difluoro2-(fluorosulfonyl)-acetic acid, CuI, DMF, 100 °C, (43%).
condition resulted in the formation of acrylate 43. Finally, hydrolysis of 43 afforded the corresponding carboxylic acid 16. For compound 17, the synthetic route started from the commercially available 1-phenylbutan-1-one 44 (Scheme 4), which was first a-acylated to the corresponding dione 45 with LHMDS and PhCOCl at room temperature and then condensed with (NH2)2-H2O to give 3,5-diphenyl-4-methyl pyrazole 46. Buchwald N-arylation of compound 47 was used to give the thiazole-ring moiety, followed by hydrolysis to give 17. Compounds 18, 20–22 and 25 listed in Table 3 were synthesized according to Scheme 5. The synthetic route started from compound 48 reported by Quyen et al.12, which was first converted to the corresponding a-nitrosodiketone 49 with NaNO2 and urea. Compound 49 was reacted with thiosemicarbazide and ethyl bromopyruvate in ethanol under reflux conditions in a one-pot pyrazole-thiazole cyclization to form 2-(1H-pyrazol-1-yl)thiazole, which was reacted
with a reducing nitroso to give 2-(1H-4-amino-pyrazole-1-yl) thiazole 50. Finally, hydrolysis of 50 afforded the corresponding carboxylic acid 25 (Scheme 5). Chlorination, bromination or iodation of 4-amino-pyrazole 50 under typical Sandmeyer conditions gave the corresponding 4-Cl (51a), 4-Br (51b) and 4-I (51c), followed by hydrolysis to give 20–22. Trifluoromethylation of 51c under copper-mediated conditions gave 4-CF3 (52), followed by hydrolysis to give 18. Compounds 19 and 24 listed in Table 3 were synthesized according to Scheme 6. 2-Fluoro-1,3-diphenylpropane-1,3-dione (53), reported by Stavber et al.13, was reacted with thiosemicarbazide and ethyl bromopyravate under one-pot pyrazole-thiazole cyclization conditions to give 54, followed by hydrolysis to provide 19. In the same manner 2-hydroxy-1,3-diphenylpropane-1,3dione14 (55) was converted to 56, then hydrolysis of 56 gave the corresponding carboxylic acid 23. Methylether formation of 56
M. Atobe et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6569–6576
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Scheme 6. Reagents and conditions: (a) thiosemicarbazide, EtOH, 80 °C then ethyl bromopyruvate, 80 °C, (54: 21%, 56: 31%); (b) 5 M NaOH, EtOH, rt, (19: 42%, 23: 60%); (c) MeI, NaH, DMF, 0 °C, (62%).
Scheme 7. Reagents and conditions: (a) LHMDS, PhCOCl, toluene, rt, 10 min; (b) (NH2)2-H2O, AcOH, rt, 3 h, (2 steps, 60a: 72%, 60b: 53%, 64: 85%); (c) CuI, K3PO4, ethyl 2bromo-thiazolecarboxylate, 37, mesitylene, 185 °C, (61a: 10%, 61b: 11%, 65a: 6%, 65b: 5%); (d) 5 M NaOH, EtOH, rt, on, (26: 73%, 27: 75%, 28: 72%, 29: 75%).
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under typical alkylation conditions provided 57, and finally hydrolysis of 57 afforded 24 (Scheme 6). The synthetic route of compounds 26–29 listed in Table 4 is shown in Scheme 7. In the case of phenylindeno[1,2-c]pyrazol compounds 26 and 27, the synthetic route started from the commercially available 2,3-dihydro-1H-inden-1-one (58a) and benzofuran-3(2H)-one (58b) (Scheme 7). These compounds were acylated with LHMDS and PhCOCl at room temperature to give the corresponding diones 59a and 59b, and then condensed with (NH2)2-H2O to give tricyclic-pyrazole 60a and 60b. Buchwald N-arylation of compound 60a and 60b was used to give the thiazole-ring moiety as a single isomer, followed by hydrolysis to give 26 and 27. NOESY correlations observed between 8-H and the ethylester proton confirmed the structure of 61a. In a similar sequence, the a-acylation of a -tetlarone (62) afforded 63, which underwent (NH2 )2-H2O pyrazole cyclization to provide the 3-phenyl-4,5-dihydro-1H-benzo[g]indazol (64). Buchwald N-arylation of compound 64 was used to give the thiazole-ring moiety as a mixture of regioisomers, followed by separation by HPLC to provide regioisomers 65a and 65b. The configuration of 65a and 65b was determined from NOE difference data. Hydrolysis of the ethyl esters 65a and 65b with 5 M NaOH provided 28 and 29, respectively. In conclusion, we have designed and synthesized a series of 2(1H-pyrazol-1-yl)thiazoles as EP1 antagonists. The results suggest that small compound 25 gives the best EP1 antagonistic activity and exhibits a good pharmacokinetic profile. Compound 25 may therefore serve as a lead compound for further development of
OAB drugs. The continuation of this study will be reported elsewhere. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 10.065. References and notes 1. Abrams, P.; Cardozo, L.; Fall, M.; Griffiths, D.; Rosier, P.; Ulmsten, U.; van Kerrebroeck, P.; Victor, A.; Wein, A. Neurourol. Urodyn. 2002, 21, 167. 2. Yamaguchi, O. Clinics 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. Ikeda, M.; Kawatani, M.; Maruyama, T.; Ishihama, H. Biomed. Res. 2006, 27, 49. 6. Palea, S.; Toson, G.; Pietra, C.; Trist, D. G.; Artibani, W.; Romano, O.; Corsi, M. Br. J. Pharmacol. 1998, 124, 865. 7. Maggi, C. A.; Giuliani, S.; Patacchini, R.; Conte, B.; Furio, M.; Santicioli, P. Eur. J. Pharmacol. 1988, 152, 273. 8. Yoshida, M.; Inadome, A.; Takahashi, W.; Yono, M.; Miyamoto, Y.; Murakami, S., et al J. Urol. 2000, 163, 44. abstract 191. 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. Avdeef, A.; Tam, K. Y. J. Med. Chem. 2010, 53, 3566. 11. Suzuki, K.; Hori, Y.; Kobayashi, T. Adv. Synth. Catal. 2008, 350, 652. 12. Quyen, T. H. Le; Umetani, S.; Suzuki, M.; Matsui, M. J. Chem. Soc., Dalton Trans. 1997, 643. 13. Stavber, S.; Šket, B.; Zajc, B.; Zupan, M. Tetrahedron 1989, 45, 6003. 14. Blatt, A. H.; Hawkins, W. L. J. Am. Chem. Soc. 1936, 58, 81.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
SAR-based optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as highly potent 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
a r t i c l e
i n f o
Article history: Received 14 September 2013 Revised 21 October 2013 Accepted 29 October 2013 Available online 6 November 2013
a b s t r a c t We describe a medicinal chemistry approach for generating a series of 2-(1H-pyrazol-1-yl)thiazoles as EP1 receptor antagonists. To improve the physicochemical properties of compound 1, we investigated its structure–activity relationships (SAR). Optimization of this lead compound provided small compound 25 which exhibited the best EP1 receptor antagonist activity and a good SAR profile. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: EP1 antagonist Pyrazole Thiazole Solubility Optimization
Overactive bladder (OAB) is a urological condition with symptoms of urgency, with or without urge incontinence, and is usually accompanied by frequency and nocturia. OAB is estimated to affect 16% of people in the United States and Europe,1 and 30% in Asia.2 Current OAB therapies consist primarily of antimuscarinic drugs, but their use is limited by unpleasant side effects such as dry mouth, constipation and blurred vision.3 Gradual bladder filling is associated with the activation of sensory nerves, and the intensity of the stimuli on these sensory nerves leads to a desire to void. A proposed etiology for OAB is an alteration of the excitability of these sensory nerves. Targeting of the hyperactive pathways represents an attractive therapeutic approach for OAB.4 Prostaglandin E2 (PGE2) has been suggested as a possible therapeutic agent because of its role in the excitement of afferent nerves via the EP1 receptor.5 This concept leads to EP1 receptor antagonists being one of the most promising candidates in developing OAB drugs.6 For example, evidence for a role of the EP1 receptor is provided by studies of EP1 receptor-selective antagonists. SC19220, an EP1 receptor antagonist, increased bladder capacity in normal rats7, and in a rat spinal cord injury model of overactive bladder, the EP1 receptor antagonist ONO-8711 produced a decrease in detrusor overactivity.8 We have previously reported hit-to-lead optimization of 2(1H-pyrazol-1-yl)-thiazole derivatives as novel EP1 receptor ⇑ Corresponding author. Tel.: +81 558 76 8493; fax: +81 558 76 5755. E-mail address: [email protected] (M. Atobe). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.065
antagonists.9 In this study we found that compound 1 appeared to give the best EP1 receptor antagonistic activity and good oral pharmacokinetics (Fig. 1). Unfortunately, 1 has low water solubility and high protein binding to plasma proteins, mainly albumin. To overcome these problems, we investigated the structure–activity relationships (SAR) of this lead compound and optimized it by characterizing its hydrophobic properties, such as the c LogP value. Our previous results showed that the pyrazole ring plays an important role in the expression of EP1 antagonistic activity. We therefore aimed to replace the 3-, 4- and 5-position on the pyrazole with various functional groups adjusting physicochemical property. Table 1 shows the results of SAR studies of the 5-position on the pyrazole. Replacement of the phenyl ring with 2-thienyl (2) or 3thienyl (3) resulted in moderate activity. We prepared compounds bearing 3-pyridyl (4), 5-pyrimidyl (5), 1-N-methyl-4-pyrazolyl (6) and 4-amino-phenyl (7) and found that these compounds had decreased EP1 activity, whereas the corresponding 3-fluorophenyl (8) and 4-fluorophenyl (9) showed moderate activity. These results suggested that the pocket at the 5 position should be hydrophobic and shallow, so substituting with a hydrophilic group, such as a heterocyclic group containing one or two nitrogen atoms, is incompatible with activity (Table 1). Table 2 shows SAR studies of the thiazole part. Replacement of the thiazole ring with 2-oxazolyl (10), 2-furyl (11), 2-pyridyl (12) or 3-pyridyl (13) resulted in decreased activity. Only the 2-thienyl substitution (14) was tolerated when the 5-methyl-thiazolyl (15) and carboxylethylene group (16) were retained. These data
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a. hEP1 rep.: ‘human EP1 reporter assay’ b. hCLint.: ‘human intrinsic clearance’ c. rCLint.: ‘rat intrinsic clearance’ d. Sol: ‘solubility’ e. MDCK: ‘Madin-Darby canine kidney’ Figure 1. Profile of 1.
Table 1 Optimization of the 5-position on the pyrazole
Table 2 Optimization of the thiazole part
Compd Compd
a
R
1
hEP1 rep. 1 lM inhibition
a
hEP1 rep. IC50 (nM)
c Log P
1
50
5.48
2
120
5.34
3
80
5.13
4
34%
3.98
5
24%
3.98
6
8%
3.60
7
10%
4.26
8
50
5.48
9
100
5.62
clogP value was calculated with ChemBioDraw 12.0.
suggested that the thiazole ring might fit into a small pocket, so the SAR studies were narrowed to focus on this moiety (Table 2). SAR of the 4-position on the pyrazole was investigated (Table 3). When R3 = Et (17), EP1 activity was decreased. When R3 = CF3 (18), EP1 activity was retained. We prepared halogen derivatives, such as fluorine (19), chlorine (20), bromine (21) and iodine (22), and found that 20 showed highly potent EP1 receptor antagonist activity. When R3 = OH (23), EP1 activity was retained. Substitution with the methoxy group (24) provided decreased EP1 activity. Finally,
R2
hEP1 rep. 1 lM inhibition
1
hEP1 rep. IC50 (nM)
c Log P
50
5.48
10
33%
4.63
11
24%
5.22
12
48%
5.23
13
26%
5.03
14
1000
5.99
15
330
5.68
16
500
5.11
the compound bearing an amine (25) appeared to give the best EP1 receptor antagonist activity (IC50: 20 nM) and the lowest LogP. These data suggested that this position is critical for controlling the physicochemical properties of the antagonist. The results in Tables 1–3 indicated that decreasing the size of the molecule increases potency. We then focused on optimizing the pyrazole ring by fusing this ring with both phenyl rings
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(Table 4). 2-(3-Phenylindeno[1,2-c]pyrazol-1(4H) (26) exhibited significantly increased EP1 receptor antagonist activity (IC50: 0.9 nM). 2-(3-Phenyl-1H-benzofuro[3,2-c]pyrazol-1-yl) (27) showed good EP1 activity and lower c LogP. 3-Phenyl fused (28), 2-(3-phenyl-4,5-dihydro-1H-benzo[g]indazol-1-yl), showed decreased EP1 receptor antagonist activity. However, 5-phenyl fused (29), 2-(3-phenyl-4,5-dihydro-2H-benzo[g]indazol-2-yl), exhibited significantly increased EP1 receptor antagonist activity (IC50: 2.0 nM). Table 5 shows the antagonist activity of highly potent compounds 1, 25, 26 and 29 as measured using an EP1 reporter-gene assay and a Ca assay. Stimulation of the EP1 receptor by PGE2 was evaluated by a reporter-gene assay and an intracellular Ca2+ release assay using human EP1 receptor-expressed recombinant cells. Table 5 also shows the intrinsic metabolic clearance (CLint) of 1, 25, 26 and 29 in rat and human hepatic microsomes. CLint was calculated from the elimination ratio (0/20 min at 0.5 lM). These results suggest that 1 and 25 would be stable in both species. The solubility of 1, 25, 26 and 29 in aqueous buffer at pH 1.2 and pH 6.8 showed that 25 exhibited the best solubility under these conditions. To evaluate the epithelial membrane permeability of compound 25, we used Madin–Darby canine kidney (MDCK) cells as an in vitro cell model Figure 2.10 The permeability of compound 25 through the MDCK cell monolayer was significantly higher than through the atenolol, showing that compound 25 exhibits good permeability. Next, the safety of compound 25 was evaluated by checking CYP inhibition and genetic toxicity using the Ames test. Compound 25 did not inhibit the major human CYP enzymes below 5 lM, as seen in Figure 2. The IC50 values obtained are above 5 lM for all human CYPs. A bacterial mutation test was conducted using an amino acid-requiring strain of Salmonella typhimurium (TA98) to detect the point mutations. No genetic toxicity was observed for compound 25. The pharmacokinetic parameters of 25 in rats are shown in Figure 2. A single oral administration of compound 25 to rats at 10 mg/kg (n = 3) provided a Cmax of 6.33 lM and an AUC(0–1) of 17.4 lM/L hr. The bioavailability (BA) in rats was calculated to be 26%. Compound 25 was tested against a battery of 57 receptors and 4 enzymes at a concentration of 10 lM (Cerep, Celle l’Evescault France). No significant activity (100
5.82
29
2
5.82
Table 5 Comparison of the profile of 1, 25, 26 and 29 Compd
hEP1 rep. IC50 (nM)
Ca assay IC50 (nM)
Human CLint (ml/min/kg)
Rat CLint (ml/min/kg)
Protein bound (%)
Sol. H2O (lg/ml)
Sol. pH 1.2 (lg/ml)
Sol. pH 6.8 (lg/ml)
1 25 26 29
50 20 0.9 2
500 60 40 20
35 19 425 149
15 29 927 236
99.71 99.00 99.68 99.88
129 865 4 20
0 867 0 0
401 1012 57 10
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a. The peak plasma concentration of a drug after administration. b. BA: ‘bioavailability’ Figure 2. Profile of 25.
Scheme 1. Reagents and conditions: (a) propionitrile, sodium ethoxide, EtOH, rt, (84%); (b) thiosemicarbazide, EtOH, 80 °C then ethyl bromopyrvate, 80 °C, (18%); (c) tBuONO, I2, MeCN, reflux, (66%); (d) Pd2dba3, (o-tol)3P, K3PO4, 34a–h, DMF, 80 °C, (35a: 65%, 35b: 70%, 35c: 68%, 35d: 62%, 35e: 53%, 35f: 67%, 35g: 65%, 35h: 68%); (e) 5 M NaOH, EtOH, rt, (2: 61%, 3: 72%, 4: 52%, 5: 48%, 6: 62%, 7: 74%, 8: 80%, 9: 78%); (f) TFA, CH2Cl2, (39%).
M. Atobe et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6569–6576
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Scheme 2. Reagents and conditions: (a) Pd(OAc)2, cBRIDP, K3PO4, 38a, mesitylene, 180 °C (30%) or CuI, K3PO4, 37, 38b–f, mesitylene, 185 °C, (39b: 10%, 39c: 42%, 39d: 63%, 39e: 32%, 39f: 40%); (d) 5 M NaOH, EtOH, rt, (10: 67%, 11: 72%, 12: 32%, 13: 62%, 14: 53%, 15: 54%).
Scheme 3. Reagents and conditions: (a) LiAlH4, THF, rt, (97%); (b) Dess–Martin periodinane, CH2Cl2, rt, (58%); (c) ethyldiethylphosphonic acid, KHMDS, THF, 0 °C, (52%); (c) 5 M NaOH, EtOH, (78%).
to give 2–6, 8 or 9. In the case of 35f, after deprotection of the Boc group with TFA, hydrolysis of 35i afforded the corresponding carboxylic acid 7 (Scheme 1). Compounds 10, 11 and 13–16 listed in Table 2 were synthesized according to Scheme 2. For compound 10, N-arylation of 36 with chloride 38a in the presence of cBRIDP, manufactured by Takasago11, was used to introduce the oxazole-ring moiety, followed by hydrolysis to give 10. When bromide 38b–f was used, Buchwald
copper mediated N-arylation was used to give the thiazole or heterocyclic moiety, followed by hydrolysis to provide 11–15 (Scheme 2). For compound 16, the synthetic route started from previously reported compound 40 (Scheme 3), which was first reduced to the corresponding primary alcohol 41 with LiAlH4. Alcohol 41 was oxidized with Dess–Martin periodinane to give aldehyde 42, and subsequent reaction under the Horner–Wadsworth–Emmons
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Scheme 4. Reagents and conditions: (a) propionitrile, sodium ethoxide, EtOH, rt, (74%); (b) (NH2)2-H2O, EtOH, rt, (98%); (c) CuI, K3PO4, 37, ethyl 2-bromothiazolecarboxylate, mesitylene, 185 °C, (16%); (d) 5 M NaOH, EtOH, rt, (68%).
Scheme 5. Reagents and conditions: (a) sodium nitrite, urea, DMSO, 0 °C, (53%); (b) thiosemicarbazide, EtOH, 80 °C then ethyl bromopyruvate, 80 °C, (14%); (c) 5 M NaOH, EtOH, rt, (25: 52%, 20: 74%, 21: 52%, 22: 83%, 18: 74%); (d) tBuONO, CuCl2 (R2 = Cl) or CuBr2 (R2 = Br) or I2 (R2 = I), MeCN, 80 °C, (51a: 46%, 51b: 32%, 51c: 40%); (e) 2,2-difluoro2-(fluorosulfonyl)-acetic acid, CuI, DMF, 100 °C, (43%).
condition resulted in the formation of acrylate 43. Finally, hydrolysis of 43 afforded the corresponding carboxylic acid 16. For compound 17, the synthetic route started from the commercially available 1-phenylbutan-1-one 44 (Scheme 4), which was first a-acylated to the corresponding dione 45 with LHMDS and PhCOCl at room temperature and then condensed with (NH2)2-H2O to give 3,5-diphenyl-4-methyl pyrazole 46. Buchwald N-arylation of compound 47 was used to give the thiazole-ring moiety, followed by hydrolysis to give 17. Compounds 18, 20–22 and 25 listed in Table 3 were synthesized according to Scheme 5. The synthetic route started from compound 48 reported by Quyen et al.12, which was first converted to the corresponding a-nitrosodiketone 49 with NaNO2 and urea. Compound 49 was reacted with thiosemicarbazide and ethyl bromopyruvate in ethanol under reflux conditions in a one-pot pyrazole-thiazole cyclization to form 2-(1H-pyrazol-1-yl)thiazole, which was reacted
with a reducing nitroso to give 2-(1H-4-amino-pyrazole-1-yl) thiazole 50. Finally, hydrolysis of 50 afforded the corresponding carboxylic acid 25 (Scheme 5). Chlorination, bromination or iodation of 4-amino-pyrazole 50 under typical Sandmeyer conditions gave the corresponding 4-Cl (51a), 4-Br (51b) and 4-I (51c), followed by hydrolysis to give 20–22. Trifluoromethylation of 51c under copper-mediated conditions gave 4-CF3 (52), followed by hydrolysis to give 18. Compounds 19 and 24 listed in Table 3 were synthesized according to Scheme 6. 2-Fluoro-1,3-diphenylpropane-1,3-dione (53), reported by Stavber et al.13, was reacted with thiosemicarbazide and ethyl bromopyravate under one-pot pyrazole-thiazole cyclization conditions to give 54, followed by hydrolysis to provide 19. In the same manner 2-hydroxy-1,3-diphenylpropane-1,3dione14 (55) was converted to 56, then hydrolysis of 56 gave the corresponding carboxylic acid 23. Methylether formation of 56
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Scheme 6. Reagents and conditions: (a) thiosemicarbazide, EtOH, 80 °C then ethyl bromopyruvate, 80 °C, (54: 21%, 56: 31%); (b) 5 M NaOH, EtOH, rt, (19: 42%, 23: 60%); (c) MeI, NaH, DMF, 0 °C, (62%).
Scheme 7. Reagents and conditions: (a) LHMDS, PhCOCl, toluene, rt, 10 min; (b) (NH2)2-H2O, AcOH, rt, 3 h, (2 steps, 60a: 72%, 60b: 53%, 64: 85%); (c) CuI, K3PO4, ethyl 2bromo-thiazolecarboxylate, 37, mesitylene, 185 °C, (61a: 10%, 61b: 11%, 65a: 6%, 65b: 5%); (d) 5 M NaOH, EtOH, rt, on, (26: 73%, 27: 75%, 28: 72%, 29: 75%).
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under typical alkylation conditions provided 57, and finally hydrolysis of 57 afforded 24 (Scheme 6). The synthetic route of compounds 26–29 listed in Table 4 is shown in Scheme 7. In the case of phenylindeno[1,2-c]pyrazol compounds 26 and 27, the synthetic route started from the commercially available 2,3-dihydro-1H-inden-1-one (58a) and benzofuran-3(2H)-one (58b) (Scheme 7). These compounds were acylated with LHMDS and PhCOCl at room temperature to give the corresponding diones 59a and 59b, and then condensed with (NH2)2-H2O to give tricyclic-pyrazole 60a and 60b. Buchwald N-arylation of compound 60a and 60b was used to give the thiazole-ring moiety as a single isomer, followed by hydrolysis to give 26 and 27. NOESY correlations observed between 8-H and the ethylester proton confirmed the structure of 61a. In a similar sequence, the a-acylation of a -tetlarone (62) afforded 63, which underwent (NH2 )2-H2O pyrazole cyclization to provide the 3-phenyl-4,5-dihydro-1H-benzo[g]indazol (64). Buchwald N-arylation of compound 64 was used to give the thiazole-ring moiety as a mixture of regioisomers, followed by separation by HPLC to provide regioisomers 65a and 65b. The configuration of 65a and 65b was determined from NOE difference data. Hydrolysis of the ethyl esters 65a and 65b with 5 M NaOH provided 28 and 29, respectively. In conclusion, we have designed and synthesized a series of 2(1H-pyrazol-1-yl)thiazoles as EP1 antagonists. The results suggest that small compound 25 gives the best EP1 antagonistic activity and exhibits a good pharmacokinetic profile. Compound 25 may therefore serve as a lead compound for further development of
OAB drugs. The continuation of this study will be reported elsewhere. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 10.065. References and notes 1. Abrams, P.; Cardozo, L.; Fall, M.; Griffiths, D.; Rosier, P.; Ulmsten, U.; van Kerrebroeck, P.; Victor, A.; Wein, A. Neurourol. Urodyn. 2002, 21, 167. 2. Yamaguchi, O. Clinics 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. Ikeda, M.; Kawatani, M.; Maruyama, T.; Ishihama, H. Biomed. Res. 2006, 27, 49. 6. Palea, S.; Toson, G.; Pietra, C.; Trist, D. G.; Artibani, W.; Romano, O.; Corsi, M. Br. J. Pharmacol. 1998, 124, 865. 7. Maggi, C. A.; Giuliani, S.; Patacchini, R.; Conte, B.; Furio, M.; Santicioli, P. Eur. J. Pharmacol. 1988, 152, 273. 8. Yoshida, M.; Inadome, A.; Takahashi, W.; Yono, M.; Miyamoto, Y.; Murakami, S., et al J. Urol. 2000, 163, 44. abstract 191. 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. Avdeef, A.; Tam, K. Y. J. Med. Chem. 2010, 53, 3566. 11. Suzuki, K.; Hori, Y.; Kobayashi, T. Adv. Synth. Catal. 2008, 350, 652. 12. Quyen, T. H. Le; Umetani, S.; Suzuki, M.; Matsui, M. J. Chem. Soc., Dalton Trans. 1997, 643. 13. Stavber, S.; Šket, B.; Zajc, B.; Zupan, M. Tetrahedron 1989, 45, 6003. 14. Blatt, A. H.; Hawkins, W. L. J. Am. Chem. Soc. 1936, 58, 81.
