Discovery of orally active nonpeptide bradykinin B2 receptor antagonists

Immunopharmacology 43 Ž1999. 163–168 www.elsevier.comrlocaterimmpharm

Discovery of orally active nonpeptide bradykinin B 2 receptor antagonists Masayuki Asano ) , Noriaki Inamura, Chie Hatori, Hiroe Sawai, Tatsujiro Fujiwara, Yoshito Abe, Hiroshi Kayakiri, Shigeki Satoh, Teruo Oku, Kunio Nakahara Departments of Pharmacology and Chemistry, Exploratory Research Laboratories, Fujisawa Pharmaceutical, Tsukuba, Ibaraki 300-2698, Japan Accepted 3 May 1999

Abstract Orally active nonpeptide bradykinin ŽBK. B 2 receptor antagonists have been discovered by using directed random screening and chemical modification. These compounds displaced w3 HxBK binding to B 2 receptors in guinea-pig ileum membranes, rat uterus membranes and human lung fibroblasts with nanomolar IC 50 s. They did not inhibit different specific radio-ligand bindings to other receptor sites including B1 receptors. In isolated guinea-pig ileum preparations, these compounds had no agonistic effect on smooth muscle contraction at 10y6 M, and caused parallel rightward shifts of the concentration–response curves to BK on contraction with higher p A 2 values. They also blocked human B 2 receptor-mediated phosphatidylinositol hydrolysis without agonistic effect. In vivo, the oral administrations of these antagonists potently inhibited BK-induced bronchoconstriction in guinea-pigs. They also reduced carrageenin-induced paw edema and caeruleininduced pancreatitis in rats. Moreover, these compounds alleviated kaolin-induced pain in mice by oral administration. These results show that our compounds are potent, selective, and orally active BK B 2 receptor antagonists and that they may have therapeutic potential against inflammatory diseases and pain. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Orally active; Bradykinin; B 2

1. Introduction Bradykinin ŽBK., an endogenous nonapeptide produced by kallikrein, has various biological actions such as bronchoconstriction, plasma extravasation, release of prostaglandinsrleukotrienes, smooth muscle contractionrrelaxation and nociception ŽBurch et al., 1990; Bhoola et al., 1992.. Therefore, BK has )

Corresponding author. Medical Biology Research Laboratory, Fujisawa Pharmaceutical, 106 Kashima 2-chome, Yodogawa-ku, Osaka 532-8514, Japan. Tel.: q81-6-6390-1155; fax: q81-66304-5367; e-mail: [email protected]

potentially important roles in inflammatory diseases such as asthma, rhinitis, arthritis, and pancreatitis. To investigate the pathophysiological role of BK and to develop a drug for inflammatory diseases, a number of BK antagonists have been synthesized ŽBurch et al., 1990; Stewart, 1995; Regoli et al., 1998.. Recently, so-called ‘second-generation’ B 2 antagonists, such as Icatibant and Bradycor, have been reported ŽHock et al., 1991; Wirth et al., 1991; Cheronis et al., 1992.. These compounds have higher affinity for B 2 receptors and longer lifetimes than previous B 2 antagonists. However, they are all peptide analogs and their therapeutic use is limited because of their

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poor oral bioavailability. Some nonpeptide B 2 antagonists have already been discovered ŽSawutz et al., 1994., but they are neither potent nor orally active. Therefore, we tried to find a potent and orally active nonpeptide B 2 receptor antagonist by using directed random screening and chemical modification. 2. Finding a lead compound We found the lead compound for nonpeptide B 2 receptor antagonists by a unique process ŽAbe et al., 1998.. In general, it is very difficult to get a good lead compound. However, we took notice of the relationship between BK and angiotensin. Angiotensin II is a typical vasoconstrictor, whereas BK causes vasodilation. They seem to play key roles in regulating cardiovascular homeostasis through their specific G-protein-coupled receptors. Angiotensin-converting enzyme generates angiotensin II from an inactive precursor, angiotensin I. This enzyme is identical with kininase II that is the major enzyme for degrading BK. Moreover, angiotensin AT1 receptors show the highest homology to BK B 2 receptors, other than B 1 receptors. This suggestive relationship between angiotensin and BK

prompted us to focus our initial screening efforts on nonpeptide angiotensin AT1 receptor antagonists and their related compounds in the Fujisawa chemical library. Fig. 1 shows our screening process for finding the lead compound. From this initial random screening of AT1 receptor antagonists and their related compounds, we found that a synthetic intermediate for AT1 antagonists inhibited the binding of w3 HxBK to B 2 receptors in guinea-pig ileum membrane with IC 50 of 31 mM. As a next step, we carried out a second screening of more structurally diverse compounds that incorporated a benzyloxy-heteroaromatic substructure. And then, we identified dichloro-benzyloxy-imidazopyridine as a better screening lead compound with an IC 50 of 7.6 mM. With this unique lead compound in hand, we started extensive chemical modification to obtain more potent and orally active compounds. 3. Potent and orally active nonpeptide B 2 antagonists We obtained potent and orally active B 2 receptor antagonists by chemical modifications ŽFig. 2.

Fig. 1. Finding of the lead compound by a two-step directed random screening.

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Table 2 Inhibitory activities of FR167344 and FR173657 against different radio-ligand bindings Žy. Negative, Ž". slightly positive, Žqq. very potent.

Fig. 2. Chemical structure of nonpeptide B 2 antagonists: Ža. FR167344, Žb. FR173657, Žc. FR165649.

ŽAsano et al., 1997b, 1998; Inamura et al., 1997.. First, we found FR167344 ŽFig. 2a. from the dichloro-benzyloxy-imidazopyridine structure. A methyl group at the 2-position is very important for the binding to B 2 receptors, and the incorporation of halogen at the 3-position remarkably increases B 2 Table 1 Effects of nonpeptide antagonists on B1 and B 2 binding in guinea-pig, rat and human tissues Receptor binding

IC 50 ŽnM. FR167344 FR173657 FR165649 Hoe140

Guinea-pig ileum ŽB 2 . Rat uterus ŽB 2 . Human fibroblast ŽB 2 . Human fibroblast ŽB1 .

0.66

0.56

0.47

0.09

1.2

1.5

1.5

0.16

13

2.9

1.6

2.7

)10 000

)10 000

)10 000

)10 000

Receptor binding

FR167344

FR173657

Adenosine Žnonselective. Adrenergic Ž a 1 . Muscarinic Žnonselective. Histamine ŽH 1 . Endotheline ŽETA . Neurokinin ŽNK 1 . Leukotriene D4 ŽCysLT1 . Angiotensin ŽAT1 . Bradykinin ŽB 2 .

y y " y y y y y qq

y y y y y y y y qq

binding activity. As a next step, we tried to find the replacement of the imidazopyridine skeleton. And then, the methylquinoline skeleton was discovered. It was almost equivalent to the bromo-methyl-imidazopyridine. Moreover, this structure has higher affinity to human B 2 receptors. Finally, we have obtained very potent and orally active antagonists, FR173657 ŽFig. 2b. and 165649 ŽFig. 2c. from this structure. Table 1 shows the antagonistic activity of our three nonpeptide antagonists against B 1 and B 2 binding in guinea-pig, rat and human tissues. These antagonists are very potent against B 2 binding with nanomolar IC 50 , but they have no effect on B 1 binding. FR173657 and 165649 are more potent in human B 2 receptors than FR167344, although they have the same potency in guinea-pig and rat B 2 receptors. Scatchard analysis demonstrated that FR compounds caused reduction of ligand–receptor affinity without change of maximal binding of ligand Table 3 Effects of nonpeptide antagonists on BK-induced responses in vitro and in vivo

pA 2 BK-induced contraction in guinea-pig ileum ED50 (mg r kg, p.o.) BK-induced bronchoconstriction in guinea-pigs

FR167344

FR173657

FR165649

9.3

9.2

9.2

0.28

0.08

0.14

166

M. Asano et al.r Immunopharmacology 43 (1999) 163–168

in human fibroblasts ŽAsano et al., 1997b; Inamura et al., 1997.. It suggests that these compounds may act in a competitive manner in human cells. The antagonists also displaced BK binding to human recombinant B 2 receptors, and inhibited human B 2

receptors-mediated phosphatidylinositol hydrolysis ŽAramori et al., 1997.. We examined the effect of our antagonists on other ligand-binding assay. As shown in Table 2, our compounds have good specificity. They have no

Fig. 3. Effects of FR167344 on animal models of inflammation. Ža. Carrageenin-induced paw edema in rats. Žb. Kaolin-induced writhing response in mice Ž n s 19 or 20.. Žc. Pancreatic edema Žcaerulein-treated rats.. Žd. Amylase and lipase in blood Žcaerulein-treated rats.. Data are expressed as mean " standard error. U P - 0.05, UU P - 0.01 vs. control ŽDunnett’s test..

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inhibitory effect on these bindings, except a weak activity of FR167344 against muscarinic binding. These compounds do not have a significant effect on angiotensin receptor binding, although they came from an intermediate for AT1 antagonists. Table 3 shows p A 2 values and ED50 values of three antagonists in BK-induced in vitro and in vivo responses. In guinea-pig isolated ileum preparations, FR compounds had no agonistic effect on smooth muscle contraction at 10y6 M, and caused parallel rightward shifts of the concentration–response curves to BK on contraction. The oral administrations of these antagonists potently inhibited BK-induced bronchoconstriction in guinea-pigs. All of their p A 2 values are higher than 9, and all of their ED50 values are much lower than 1 mgrkg. These results indicate that our antagonists are very potent and orally active.

4. Effects of nonpeptide B 2 antagonists on animal models of inflammation We have examined effects of nonpeptide B 2 antagonists on animal models of inflammation to study their therapeutic potential against inflammatory diseases ŽAsano et al., 1997a.. Our data reveal that FR167344 and other nonpeptide antagonists have the inhibitory effects on paw edema, pain reaction, and pancreatitis models. Fig. 3a shows the effect of FR167344 on the carrageenin-induced rat paw edema. The carrageenin injection provoked edema time-dependently, and the oral administration of the compound reduced the carrageenin-induced paw edema dose-dependently. Its ED50 was 2.7 mgrkg at the 2-h time point after the carrageenin injection. FR173657 and FR165649 also diminished the paw edema ŽAsano et al., 1997b.. FR167344 inhibited kaolin-induced pain reaction in mice as demonstrated in Fig. 3b. Open columns show counts of writhing, i.e., pain reaction in 10-min period after kaolin injection. Solid columns show those in the 15-min period. FR compound blocked the early phase of this pain reaction more effectively. In addition, we investigated the effect of FR167344 on the caerulein-induced pancreatitis model in rats. Caerulein caused the increase in wet weight of pancreas that indicated pancreatic edema.

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As shown in Fig. 3c, the oral administration of the antagonist reduced this pancreatic edema. In this model, both amylase and lipase in blood were markedly increased, and the antagonist suppressed these increases with ED50 values of 10.3 and 7.4 mgrkg, respectively ŽFig. 3d..

5. Conclusions We have discovered orally active nonpeptide BK B 2 receptor antagonists by a directed random screening process and chemical modification. The antagonists specifically inhibited BK-induced responses both in vitro and in vivo. They also had inhibitory effects on several in vivo animal models of inflammation. These results indicate that our nonpeptide BK B 2 receptor antagonists may have therapeutic potentials against inflammatory diseases. We hope that our nonpeptide antagonists will not only be good tools for studying the role of kinins but will also a useful medicine for inflammatory diseases.

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