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Discovery of Diphenyloxazole and Nδ-Z-Ornithine Derivatives as Highly Potent and Selective Human Prostaglandin EP4 Receptor Antagonists Kouji Hattori,*,† Akira Tanaka,† Naoaki Fujii,† Hisashi Takasugi,† Yoshiyuki Tenda,‡ Masayuki Tomita,§ Shoko Nakazato,§ Keiko Nakano,§ Yasuko Kato,§ Yutaka Kono,§ Hidetsugu Murai,| and Kazuo Sakane† Medicinal Chemistry Research Laboratories, Exploratory Research Laboratories, Medicinal Biology Research Laboratories, and Biopharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan Received January 29, 2005 Abstract: Two novel classes of diphenyloxazole and Nδ-Zornithine derivatives as highly potent and selective EP4 antagonists have been discovered. The optimized diphenyloxzole 8 and Nδ-Z-ornithine 11 effectively competed with [3H]PGE2 binding to human recombinant EP4, with Ki values of 0.30 nM and 0.91 nM, respectively, and were selective for all members of the human prostanoid receptor family. 8 was shown to exhibit good pharmacokinetic properties in rats and dogs and potent inhibitory activity toward in vitro PGE2promoted IgE synthesis.
The development of agents active at prostaglandin (PG) E2 receptors has been an area of interest to the pharmaceutical industry for the past three decades, with both agonists and antagonists anticipated to have therapeutic utility in treating diverse conditions including asthma, inflammation, pain, ulcers, cancer, and osteoporosis.1 However, success has been limited by the problems of identifying suitably selective ligands. Many of the problems encountered have been due to the existence of multiple prostanoid receptor subtypes and the lack of the selectivity of prostaglandin analogues. PGE2 has been shown to be the preferred prostanoid ligand for four of these receptor subtypes, the EP1, EP2, EP3, and EP4 receptors: PGD2 for the DP receptor, PGF2R for the FR receptor, PGI2 for the IP receptor, and TXA2 for the TP receptor.2 The molecular characterization of these receptors has resulted in renewed interest in the field because the availability of cloned receptors facilitates the identification of subtype-selective ligands and in delineating the pathophysiological roles of each of these receptor subtypes.3 EP2 and EP4 receptors coupled to an increase in intracellular cAMP are smooth muscle relaxants.4 Fedyk and Phipps have reported that EP2 and/or EP4 antagonists play an important role in diminishing allergic and IgE-mediated asthmatic responses since EP2 and EP4 subtypes regulate activation and differentiation of mouse B lymphocytes to IgE-secreting cells.5 In a mixed * To whom correspondence should be addressed. Phone: 81-6-63901220. Fax: 81-6-6304-5435. E-mail address: kouji_hattori@ po.fujisawa.co.jp. † Medicinal Chemistry Research Laboratories. ‡ Exploratory Research Laboratories. § Medicinal Biology Research Laboratories. | Fujisawa Pharmaceutical Co.
Figure 1. Lead generation.
lymphocyte model of the cellular immune response, Nataraj and colleagues have reported that EP2 and EP4 receptors can regulate the function of antigen-presenting cells.6 These finding may also be related to the observation that homozygous deletion of the EP4 receptor decreased the incidence and severity of collagen antibody-induced arthritis by McCoy and co-workers.7 Gene knockout study has also been used to study the potential involvement of the EP2 and EP4 receptors in colon cancer. For example, Mutoh and co-workers have reported homozygous deletion of the EP4 receptor decreased the formation of aberrant crypt foci in animals treated with azoxymethane, a known colon carcinogen.8 On the other hand, there is little known about selective ligands as EP2 and EP4 receptor antagonists. Several PG congeners have been used as probes for the EP2 and EP4 receptor antagonist ligands, although subtype selectivity was not satisfaction.9 We disclose herein two different structural scaffolds as high potent and selective EP4 antagonists.10 Figure 1 shows two structural different series as a seed/lead compound. One approach to find the lead compound is to modify the previously reported diphenyloxazole series which was a potent and orally active prostacyclin mimetic.11 1 was found to have affinity to not only the IP receptor but also the EP4 receptor, with Ki ) 54 nM for IP and Ki ) 1020 nM for EP4; therefore, we modified 1 to generate a more selective EP4 ligand. Initial SAR work on replacement of the acid moiety of 1 rapidly afforded a dramatic change in affinity and selectivity. Phenylacetic acid type 2 was approximately 20-fold improved affinity for the EP4 receptor (Ki for EP4 ) 50 nM and Ki for IP ) 170 nM, data not shown), and the benzoic acid type 3, which we identified as a promising lead, was improved 200-fold (Ki for EP4 ) 5.5 nM and Ki for IP ) 610 nM) and also showed excellent bioavailability. The second approach to find a different scaffold was to explore the Fujisawa sample collection. Random screening identified the promising seed compound 4 with Ki for EP4 ) 80 nM, which had a unique dipeptide structure containing N-R-indoylcarbonyl-N-δ-benzyloxycarbonyl ornithine and phenylalanine benzylmethylamide. Transformation of the terminal benzylmethyla-
10.1021/jm050085k CCC: $30.25 © 2005 American Chemical Society Published on Web 04/13/2005
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Scheme 1a
a Reagents and conditions: (a) Tf O, 2,6-lutidine, dichloromethane, 91%; (b) Pd(OAc) , 1,3-bis(diphenylphosphino)propane, CO, Et N, 2 2 3 DMF-MeOH, 83%; (c) 1 N NaOH, THF, MeOH, 92%; (d) BnONH2, WSC-HCl, HOBT, DMF, 95% for 7, BnSO2NH2, WSC-HCl, DMAPy, DMF, 89% for 8.
Table 1. Profile of Eight Prostanoid Binding human Ki (nM) 1 3 7 8 5 11
rat Ki (nm)
IP
DP
FP
TP
EP1
EP2
EP3
EP4
R-EP4
54 610 >3000 >3000 >1000 >10000
>1000 >3000 >1000 >3000 >1000 >10000
>1000 >3000 >1000 >3000 >1000 >10000
>1000 >3000 >1000 >3000 >1000 >10000
>1000 2500 >3000 >3000 >1000 >10000
>1000 >3000 >3000 >3000 >1000 >10000
>1000 >3000 >3000 >3000 >1000 >10000
1020 5.5 ( 0.23 0.68 0.30 ( 0.09 43 0.91 ( 0.10
0.74 ( 0.21 5.1 ( 0.2
The results were presented as the mean ( SE of three independent experience for 3, 8, and 11 and as the average of two experiment for 1, 7, and 5. Competitive binding assay based on the displacement of [3H]-Iloprost for human IP receptor, [3H]-PGD2 for human DP receptors, [3H]-PGF2 for human FP receptors, [3H]-SQ29548 for human TP receptor, and [3H]-PGE2 for human EP1-4 receptors. Assays were performed by the reported method; see in refs 2 and 4. a
Table 2. Profile of Pharmacokinetica rat: iv (n ) 2-3) t1/2 (h) 1 6.6 ( 0.33 3 4.0 8 4.4 ( 1.6 a
po (n ) 3)
dog: iv (n ) 2-3)
Cl (mL/min/kg) dose (mg/kg) Cmax (ng/mL) F(%)b 17.7 ( 0.33 11.2 10.2 ( 0.7
0.32 10 10
16.4 ( 0.88 9100 ( 1000 2100 ( 300
50 99 51
t1/2 (h) 1 3.0 ( 1.4 3 5.5 8 5.5 ( 0.8
po (n ) 3)
Cl (mL/min/kg) dose (mg/kg) Cmax (ng/mL) F(%) 1.59 ( 0.29 0.52 0.52 ( 0.18
0.32 0.10 3.2
592 ( 196 57.3 ( 12.0 8200 ( 1400
72 25 97
Results were shown as the mean ( SE. b F ) Bioavailability.
mide group of 4 to simple carboxylic acid led to the identification of the 2-fold more potent compound 5 as a second lead. We further optimize these two series of diphenyloxazole-based lead and peptide-based lead toward high EP4 subtype selectivity. The diphenyloxazole derivatives in this study were prepared as shown in Scheme 1. Key optically active intermediate 6 was prepared by a simple and stereoselectve synthetic method.11 Optically active 6 was treated with Tf2O, followed by carbonization with Pd(OAc)2, 1,3bis(diphenylphosphino)propane, and CO gas in a mixture of DMF and MeOH and hydrolysis with 1 N NaOH to give the desired compound 3 69% yield and >99% ee from 6. We focused our attention on further modification of the benzoic acid part, which was transformed into amide derivatives using various commercial available amines to find a new pharmacophore for the EP4 receptor. We discovered that aryl-substituted amide compounds formed from the amines with two and three carbon atoms between the nitrogen atom and the phenyl, such as 7, had 10-fold improved potency toward the EP4. Phenoxymethylamide derivative 7 had high affinity for the EP4 receptor with Ki ) 0.68 nM and good selectivity over other EP receptors (Table 1). However, despite amide derivatives showing high affinity and good selectivity, compound 7 exhibited poor PK properties, hindering further pharmacological evaluation. In consideration of the PK profile of 1 and 3, acidic character of the
molecule would be essential for good bioavailability. Combining both compounds character for potency and bioavailability, we attempted to utilize acylsulfonamide linked with benzyl substitution. The acyl sulfonamide group is probably the most widely used replacement for carboxylic acid and also recently has shown up in some prostanoid directed ligands12 The new designed acylsulfonamide analogue 8 showed good affinity for the EP4 receptor and excellent selectivity over other human prostanoid receptors (each receptor selectivity >3000 nM). The acidic character of 8 was also displayed in the good oral availability in rats and dogs, 50% and 90%, respectively, in Table 2.13 Our next efforts on modifying another lead compound 5 started by exploring possible replacements for the three fragments, as shown in Scheme 2. To optimize rapidly and efficiently, we applied solid-phase synthesis involving three major steps of three units: (i) coupling of 2-chlorotrityl resin-bound to the first amino acid unit; (ii) coupling of the second amino acid unit; (iii) capping of the terminal amine with a substituted carboxylic acid. This method produced the desired dipeptide-products on a 1-10 mg scale in a total yield 80-90%. We first determined the optimal side chain length of the terminal carboxylic acid using building block X as a carboxylic acid mimetic of the R-side chain of natural PGE2. The homologue 9a was found to be same potency while 9b was 50-fold more potent than lead 5 with 0.83 nM for the EP4 receptor. From this study, five-carbon
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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3105
Scheme 2a
a Reagents and condition: (a) HO-AAx-NHFmoc, diisopropylethylamine, dichloromethane; (b) piperazine, DMF; (c) HO-AAy-NHFmoc, TBTU, HOBT, diisopropylethyamine, DMF; (d) R-OH, 1,3-carbodiimide, diisopropylethyamine, dichloromethane; (e) TFA, dichloromethane.
chains were selected as the optimum length for further optimization. Replacement of the Z-ornithine unit with building block Y resulted in completely loss of affinity for the EP4 receptor (Ki > 1000 nM) except Z-lysine. L-Lysine 10a and D-lysine 10b showed different discrimination for the EP4 receptor, 8.4 nM and 240 nM, respectively. In the terminal capping unit R, 2-indoyl, 2-furanyl, 2-thiophenyl, and 2-naphthyl were the best groups. Preliminary SAR work for the dipeptide series indicated that substituted Z-ornithines 9b and 11 provide the best compound for potency and selectivity; however, 9b and 11 showed problems in bioavailability. The preliminary SAR results led to the generation of 11 are reported as Supporting Information and the further optimization focused on improving bioavailability will be published soon. Next, we examined the in vitro antagonistic action of 8 and 11. It has already been confirmed that PGE2 is a powerful immunomodulator that shifts the balance of the cellular immune response away from Th1 and toward Th2 and drives the humoral response toward IgE.14 Figure 2 shows EP4 antagonists have an inhibitory effect on PGE2-promoted IgE synthesis using BDF1 mice B cells.15 PGE2 were preincubated with small dense B lymphocytes stimulated with IL-4 and LPS. IgE were assayed in supernatants 6 days after addition of IL-4 and LPS. PGE2 promotes IL-4, and LPS induced IgE synthesis, which was significantly attenuated by pretreatment with 8 at concentrations from 10-8 M to 10-6 M and 11 at concentrations from 10-7 M to 10-6 M. In this model, pretreatment of antagonists 8 and 11 without PGE2 had no effect on IgE level (data not shown).
Figure 2. Effects of EP4 antagonists on in vitro PGE2promoted IgE synthesis in BDF1 mice B cells. Values were presented as the mean ( SE, N ) 6. ##p < 0.01 vs base. *p < 0.05 vs control. **p < 0.01 vs control.
We have discovered potent and selective EP4 antagonists according to two discovery approaches, from a PGI2 agonist and from random screening. Newly designed EP4 antagonists 8 and 11 exhibited highly potent and selective EP4 receptor activity. 8 has good pharmacokinetic properties, and the inhibitory effect against PGE2 promoted B lymphocyte Ig isotype switching to IgE in vitro. These findings suggest 8 could be an
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attractive therapeutic candidate for diminishing allergic, asthmatic, and atopic disorders mediated by IgE.
E receptor subtype EP4 in colon carcinogenesis. Cancer Res. 2002, 62, 28-32. Ono-AE2-227 and EP4A have been reported as EP4 antagonists. Ono-AE2-227: Ki values were found to be 2.7 nM for the mouse EP4 receptor and 21 nM for mouse EP3 receptor, see in ref 8. EP4A: Ki values were found to be 24 nM for the human EP4 receptor and 710 nM for human TP receptor, see in Machwate, M.; Harada, S.; Leu, C. T.; Seedor, G.; Labelle, M.; Gallant, M.; Hutchins, S.; Lachance, N.; Sawyer, N.; Slipetz, D.; Metters, K. M.; Rodon, S. B.; Young, R.; Rodan, G. A. Prostaglandin receptor EP4 mediates the bone anabolic effects of PGE2. Mol. Pharamacol. 2001, 60, 36-41. Saturation analysis was performed to determine the equilibrium dissociation constants (Kd) and maximum number of binding sites (Bmax) of radioligand [3H]PGE2 for EP4 receptors expressed in COS cells: HumanEP4, Kd ) 0.28 ( 0.03 (nM), Bmax ) 3.1 ( 0.21 (pmol/mg protein). RatEP4, Kd ) 1.3 + 0.7 (nM), Bmax ) 3.8 ( 0.9 (pmol/mg protein). Competition binding assays were conduced to determine the inhibition constants (Ki) for ligands in the previously reported method; see in refs 2 and 4. Hattori, K.; Tabuchi, S.; Okitsu, O.; Taniguchi, K. A simple stereoselective synthesis and biological evaluation of FR181157: orally active prostacyclin mimetic. Bioorg. Med. Chem. Lett. 2003, 13, 4277-4279. Gallant, M.; Carriere, M. C.; Chateauneuf, A.; Denis, D.; Gareau, Y.; Godbout, C.; Greig, G.; Juteau, H.; Lachance, N.; Lacombe, P.; Lamontagne, S.; Metters, K. M.; Rochette, C.; Ruel, R.; Slipetz, D.; Sawyer, N.; Tremblay, N.; Labelle, M. Structureactivity relationship of biaryl acylsulfonamide analogues on the human EP3 prostanoid receptor. Bioorg. Med. Chem. Lett. 2002, 12, 2583-2586. Ruel, R.; Lacombe, P.; Abramovitz, M.; Godbout, C.; Lamontagne, S.; Rochette, C.; Sawyer, N.; Stocco, R.; Tremblay, N. M.; Metters, K. M.; Labelle, M. New class of biphenylene dibenzazocinones as potent ligands for the human EP1 prostanoid receptor. Bioorg. Med. Chem. Lett. 1999, 9, 2699-2704. Yee, Y. K.; Bernstein, P. R.; Adams, E. J.; Brown, F. J.; Cronk, L. A.; Hebbel, K. C.; Vacek, E. P.; Krell, R. D.; Snyder, D. W. A new series of selective leukotriene antagonists: exploration and optimization of the acidic region in 1,6-disubstituted indoles and indazoles. J. Med. Chem. 1990, 33, 2437-2451. Measurements of the concentration of 1, 3, and 8 in plasma were measured using HPLC or LC/MS. Roper, R. L.; Brown, D. M.; Phipps, R. P. Prostaglandin E2 promotes B lymphocyte Ig isotype switching to IgE. J. Immunol. 1995, 154, 162-170. Ropper, R. L.; Conrad, D. H.; Brown, D. M.; Warner G. L.; Phipps, R. P. Prostaglandin E2 promotes IL4-induced IgE and IgG1 synthesis. J. Immunol. 1990, 145, 2644-2651. Assay was performed by the previously described method in refs 14 and 16. Fedric, E. R.; Phipps, R. P. Reactive oxygen species and not lipoxygenase products are required for mouse B-lymphocyte activation and differentiation. Int. J. Immunopharmacol. 1994, 16, 533-546.
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Acknowledgment. We express our thanks to Dr. David Barrett for his critical reading of the manuscript. Supporting Information Available: The experimental procedures and compound characterization data. The preliminary SAR results of the dipeptide derivatives. This material is available free of charge via the Internet at http://pubs. acs.org.
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References (1) For recent reviews on prostaglandin receptors see: Regan, J. W. EP2 and EP4 prostanoid receptor signaling. Life Sci. 2003, 74, 143-153. Benoit, P.; De Leval, X.; Pirotte, B.; Dogne, J.-M. Latest discoveries in prostaglandin receptor modulators. Expert Opin. 2002, 12, 1225-1235. DeLong, M. Prostaglandin receptor ligands: Recent patent activity. IDrugs 2000, 3, 1039-1052. (2) Abramovitz, A. M.; Adam, M.; Boie, Y. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim. Biophys. Acta 2000, 1483, 285-293. Negishi, M.; Sugimoto, Y.; Ichikawa, A. Molecular mechanisms of diverse action of prostanoid receptors Biochim. Biophys. Acta 1995, 1259, 109-120. (3) Coleman, R. A.; Smith, W. L.; Narumiya, S. International union of pharmacology classification of prostanoid receptors, distribution and structure of the receptors and their subtypes. Pharmacol. Rev. 1994, 46, 205-229. (4) EP2: Regan, J. W.; Bailey, T. J.; Pepperl, D. J.; Pierce, K. L.; Bogardus, A. M.; Donello, J. E.; Fairbairn, C. E.; Kedzie, K. M.; Woodward, D. F.; Gil, D. W. Cloning of a novel human prostaglandin receptor with characteristics of the pharmacologically defined EP2 subtype. Mol. Pharmacol. 1994, 46, 213. EP4: Bastien, L.; Sawyer, N.; Grygorczyk, R.; Metters, K. M.; Adam, M. Cloning, function expression, and characterization of the human prostaglandin E2 receptor EP2 subtype. J. Biol. Chem. 1994, 269, 11873-11877. Marshall, F. H.; Patel, K.; Lundstrom, K.; Camacho, J., Foord, S. M.; Lee, M. G. Characterization of [3H]-prostaglandin E2 binding to prostaglandin EP4 receptors expressed with semliki forest virus. Br. J. Pharamcol. 1997, 121, 1673-1678. (5) Fedyk, E. R.; Phipps, R. P. Prostaglandin E2 receptors of the EP2 and EP4 subtypes regulate activation and differentiation of mouse B lymphocytes to IgE-secreting cells. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10978-10983. (6) Nataraj, C.; Thomas, D. W.; Tilley, S. L.; Nguyen, M.; Mannon, R.; Koller, B. H.; Coffman, T. M. Receptors for prostaglandin E2 that regulate cellular immune responses in the mouse. J. Clin. Invest. 2001, 108, 1229-1235. (7) McCoy, J. M.; Wicks, J. R.; Audoly, L. P. The role of prostaglandin E2 receptors in the pathogenesis of rheumatoid arthritis. J. Clin. Invest. 2002, 110, 651-658. (8) Mutoh, M.; Watanabe, K.; Kitamura, T.; Shoji, Y.; Takahashi, M.; Kawamori, T.; Tani, K.; Kobayashi, M.; Maruyama, T.; Kobayashi, K.; Ohuchida, S.; Sugimoto, Y.; Narumiya, S.; Sugimura, T.; Wakabayashi, K. Involvement of prostaglandin
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JM050085K
