Ž .Immunopharmacology 43 1999 163–168www.elsevier.comrlocaterimmpharm

Discovery of orally active nonpeptide bradykinin B receptor2

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

Ž .Orally active nonpeptide bradykinin BK B receptor antagonists have been discovered by using directed random2w3 xscreening and chemical modification. These compounds displaced H BK binding to B receptors in guinea-pig ileum2

membranes, rat uterus membranes and human lung fibroblasts with nanomolar IC s. They did not inhibit different specific50

radio-ligand bindings to other receptor sites including B receptors. In isolated guinea-pig ileum preparations, these1

compounds had no agonistic effect on smooth muscle contraction at 10y6 M, and caused parallel rightward shifts of theconcentration–response curves to BK on contraction with higher p A values. They also blocked human B receptor-media-2 2

ted phosphatidylinositol hydrolysis without agonistic effect. In vivo, the oral administrations of these antagonists potentlyinhibited BK-induced bronchoconstriction in guinea-pigs. They also reduced carrageenin-induced paw edema and caerulein-induced pancreatitis in rats. Moreover, these compounds alleviated kaolin-induced pain in mice by oral administration. Theseresults show that our compounds are potent, selective, and orally active BK B receptor antagonists and that they may have2

therapeutic potential against inflammatory diseases and pain. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Orally active; Bradykinin; B2

1. Introduction

Ž .Bradykinin BK , an endogenous nonapeptideproduced by kallikrein, has various biological actionssuch as bronchoconstriction, plasma extravasation,release of prostaglandinsrleukotrienes, smooth mus-

Žcle contractionrrelaxation and nociception Burch 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: q81-6-6304-5367; e-mail: masayuki-asano@po.fujisawa.co.jp

potentially important roles in inflammatory diseasessuch as asthma, rhinitis, arthritis, and pancreatitis. Toinvestigate the pathophysiological role of BK and todevelop a drug for inflammatory diseases, a number

Žof BK antagonists have been synthesized Burch et.al., 1990; Stewart, 1995; Regoli et al., 1998 . Re-

cently, so-called ‘second-generation’ B antagonists,2

such as Icatibant and Bradycor, have been reportedŽHock et al., 1991; Wirth et al., 1991; Cheronis et

.al., 1992 . These compounds have higher affinity forB receptors and longer lifetimes than previous B2 2

antagonists. However, they are all peptide analogsand their therapeutic use is limited because of their

0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0162-3109 99 00131-9

( )M. Asano et al.r Immunopharmacology 43 1999 163–168164

poor oral bioavailability. Some nonpeptide B antag-2Žonists have already been discovered Sawutz et al.,

.1994 , but they are neither potent nor orally active.Therefore, we tried to find a potent and orally activenonpeptide B receptor antagonist by using directed2

random screening and chemical modification.

2. Finding a lead compound

We found the lead compound for nonpeptide B2Žreceptor antagonists by a unique process Abe et al.,

.1998 . In general, it is very difficult to get a goodlead compound. However, we took notice of therelationship between BK and angiotensin. An-giotensin II is a typical vasoconstrictor, whereas BKcauses vasodilation. They seem to play key roles inregulating cardiovascular homeostasis through theirspecific G-protein-coupled receptors.

Angiotensin-converting enzyme generates an-giotensin II from an inactive precursor, angiotensin I.This enzyme is identical with kininase II that is themajor enzyme for degrading BK. Moreover, an-giotensin AT receptors show the highest homology1

to BK B receptors, other than B receptors. This2 1

suggestive relationship between angiotensin and BK

prompted us to focus our initial screening efforts onnonpeptide angiotensin AT receptor antagonists and1

their related compounds in the Fujisawa chemicallibrary.

Fig. 1 shows our screening process for finding thelead compound. From this initial random screeningof AT receptor antagonists and their related com-1

pounds, we found that a synthetic intermediate forw3 xAT antagonists inhibited the binding of H BK to1

B receptors in guinea-pig ileum membrane with2

IC of 31 mM. As a next step, we carried out a50

second screening of more structurally diverse com-pounds that incorporated a benzyloxy-heteroaromaticsubstructure. And then, we identified dichloro-ben-zyloxy-imidazopyridine as a better screening leadcompound with an IC of 7.6 mM. With this unique50

lead compound in hand, we started extensive chemi-cal modification to obtain more potent and orallyactive compounds.

3. Potent and orally active nonpeptide B antago-2

nists

We obtained potent and orally active B receptor2Ž .antagonists by chemical modifications Fig. 2

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

( )M. Asano et al.r Immunopharmacology 43 1999 163–168 165

Ž .Fig. 2. Chemical structure of nonpeptide B antagonists: a2Ž . Ž .FR167344, b FR173657, c FR165649.

Ž .Asano et al., 1997b, 1998; Inamura et al., 1997 .Ž .First, we found FR167344 Fig. 2a from the

dichloro-benzyloxy-imidazopyridine structure. Amethyl group at the 2-position is very important forthe binding to B receptors, and the incorporation of2

halogen at the 3-position remarkably increases B2

Table 1Effects of nonpeptide antagonists on B and B binding in1 2

guinea-pig, rat and human tissues

Ž .Receptor IC nM50

binding FR167344 FR173657 FR165649 Hoe140

Guinea-pig 0.66 0.56 0.47 0.09Ž .ileum B2

Rat uterus 1.2 1.5 1.5 0.16Ž .B2

Human 13 2.9 1.6 2.7Ž .fibroblast B2

Human )10000 )10000 )10 000 )10000Ž .fibroblast B1

Table 2Inhibitory activities of FR167344 and FR173657 against differentradio-ligand bindingsŽ . Ž . Ž .y Negative, " slightly positive, qq very potent.

Receptor binding FR167344 FR173657

Ž .Adenosine nonselective y yŽ .Adrenergic a y y1Ž .Muscarinic nonselective " yŽ .Histamine H y y1Ž .Endotheline ET y yAŽ .Neurokinin NK y y1

Ž .Leukotriene D4 CysLT y y1Ž .Angiotensin AT y y1Ž .Bradykinin B qq qq2

binding activity. As a next step, we tried to find thereplacement of the imidazopyridine skeleton. Andthen, the methylquinoline skeleton was discovered. Itwas almost equivalent to the bromo-methyl-im-idazopyridine. Moreover, this structure has higheraffinity to human B receptors. Finally, we have2

obtained very potent and orally active antagonists,Ž . Ž .FR173657 Fig. 2b and 165649 Fig. 2c from this

structure.Table 1 shows the antagonistic activity of our

three nonpeptide antagonists against B and B bind-1 2

ing in guinea-pig, rat and human tissues. Theseantagonists are very potent against B binding with2

nanomolar IC , but they have no effect on B50 1

binding. FR173657 and 165649 are more potent inhuman B receptors than FR167344, although they2

have the same potency in guinea-pig and rat B2

receptors. Scatchard analysis demonstrated that FRcompounds caused reduction of ligand–receptoraffinity without change of maximal binding of ligand

Table 3Effects of nonpeptide antagonists on BK-induced responses invitro and in vivo

FR167344 FR173657 FR165649

pA2

BK-induced contraction 9.3 9.2 9.2in guinea-pig ileum

( )ED mgrkg, p.o.50

BK-induced 0.28 0.08 0.14bronchoconstrictionin guinea-pigs

( )M. Asano et al.r Immunopharmacology 43 1999 163–168166

Žin human fibroblasts Asano et al., 1997b; Inamura.et al., 1997 . It suggests that these compounds may

act in a competitive manner in human cells. Theantagonists also displaced BK binding to humanrecombinant B receptors, and inhibited human B2 2

receptors-mediated phosphatidylinositol hydrolysisŽ .Aramori et al., 1997 .

We examined the effect of our antagonists onother ligand-binding assay. As shown in Table 2, ourcompounds have good specificity. They have no

Ž . Ž .Fig. 3. Effects of FR167344 on animal models of inflammation. a Carrageenin-induced paw edema in rats. b Kaolin-induced writhingŽ . Ž . Ž . Ž . Ž .response in mice ns19 or 20 . c Pancreatic edema caerulein-treated rats . d Amylase and lipase in blood caerulein-treated rats . Data

U UU Ž .are expressed as mean"standard error. P-0.05, P-0.01 vs. control Dunnett’s test .

( )M. Asano et al.r Immunopharmacology 43 1999 163–168 167

inhibitory effect on these bindings, except a weakactivity of FR167344 against muscarinic binding.These compounds do not have a significant effect onangiotensin receptor binding, although they camefrom an intermediate for AT antagonists.1

Table 3 shows p A values and ED values of2 50

three antagonists in BK-induced in vitro and in vivoresponses. In guinea-pig isolated ileum preparations,FR compounds had no agonistic effect on smoothmuscle contraction at 10y6 M, and caused parallelrightward shifts of the concentration–response curvesto BK on contraction. The oral administrations ofthese antagonists potently inhibited BK-inducedbronchoconstriction in guinea-pigs. All of their p A2

values are higher than 9, and all of their ED values50

are much lower than 1 mgrkg. These results indicatethat our antagonists are very potent and orally active.

4. Effects of nonpeptide B antagonists on animal2

models of inflammation

We have examined effects of nonpeptide B an-2

tagonists on animal models of inflammation to studytheir therapeutic potential against inflammatory dis-

Ž .eases Asano et al., 1997a . Our data reveal thatFR167344 and other nonpeptide antagonists have theinhibitory effects on paw edema, pain reaction, andpancreatitis models.

Fig. 3a shows the effect of FR167344 on thecarrageenin-induced rat paw edema. The carrageenininjection provoked edema time-dependently, and theoral administration of the compound reduced thecarrageenin-induced paw edema dose-dependently.Its ED was 2.7 mgrkg at the 2-h time point after50

the carrageenin injection. FR173657 and FR165649Ž .also diminished the paw edema Asano et al., 1997b .

FR167344 inhibited kaolin-induced pain reactionin mice as demonstrated in Fig. 3b. Open columnsshow counts of writhing, i.e., pain reaction in 10-minperiod after kaolin injection. Solid columns showthose in the 15-min period. FR compound blockedthe early phase of this pain reaction more effectively.

In addition, we investigated the effect ofFR167344 on the caerulein-induced pancreatitismodel in rats. Caerulein caused the increase in wetweight of pancreas that indicated pancreatic edema.

As shown in Fig. 3c, the oral administration of theantagonist reduced this pancreatic edema. In thismodel, both amylase and lipase in blood weremarkedly increased, and the antagonist suppressedthese increases with ED values of 10.3 and 7.450

Ž .mgrkg, respectively Fig. 3d .

5. Conclusions

We have discovered orally active nonpeptide BKB receptor antagonists by a directed random screen-2

ing process and chemical modification. The antago-nists specifically inhibited BK-induced responsesboth in vitro and in vivo. They also had inhibitoryeffects on several in vivo animal models of inflam-mation. These results indicate that our nonpeptideBK B receptor antagonists may have therapeutic2

potentials against inflammatory diseases. We hopethat our nonpeptide antagonists will not only be goodtools for studying the role of kinins but will also auseful medicine for inflammatory diseases.

References

Abe, Y., Kayakiri, H., Satoh, S., Inoue, T., Sawada, Y., Imai, K.,Inamura, N., Asano, M., Hatori, C., Katayama, A., Oku, T.,Tanaka, H., 1998. A novel class of orally active nonpeptidebradykinin B receptor antagonists: 1. Construction of the2

basic framework. J. Med. Chem. 41, 564–578.Aramori, I., Zenkoh, J., Morikawa, N., O’Donnell, N., Asano, M.,

Nakamura, K., Iwami, M., Kojo, H., Notsu, Y., 1997. Novelsubtype-selective nonpeptide bradykinin receptor antagonistsFR167344 and FR173657. Mol. Pharmacol. 51, 171–176.

Asano, M., Hatori, C., Inamura, N., Sawai, H., Hirosumi, J.,Fujiwara, T., Nakhara, K., 1997a. Effects of a nonpeptidebradykinin B receptor antagonist, FR167344, on different in2

vivo animal models of inflammation. Br. J. Pharmacol. 122,1436–1440.

Asano, M., Inamura, N., Hatori, C., Sawai, H., Fujiwara, T.,Katayama, A., Kayakiri, K., Satoh, S., Abe, Y., Inoue, T.,Sawada, Y., Nakahara, K., Oku, T., Okuhara, M., 1997b. Theidentification of an orally active, nonpeptide bradykinin, B2

receptor antagonist, FR173657. Br. J. Pharmacol. 120, 617–624.

Asano, M., Hatori, C., Sawai, H., Johki, S., Inamura, N., Kayakiri,H., Satoh, S., Abe, Y., Inoue, T., Sawada, Y., Mizutani, T.,Oku, T., Nakahara, K., 1998. Pharmacological characterizationof a nonpeptide bradykinin B receptor antagonist, FR165649,2

and agonist, FR190997. Br. J. Pharmacol. 124, 441–446.

( )M. Asano et al.r Immunopharmacology 43 1999 163–168168

Bhoola, K.D., Figueroa, C.D., Worthy, K., 1992. Bioregulation ofkinins: kallikreins, kininogens, and kininases. Pharmacol. Rev.44, 1–80.

Burch, R.M., Farmer, S.G., Steranka, L.R., 1990. Bradykininreceptor antagonists. Med. Res. Rev. 10, 237–269.

Cheronis, J.C., Whalley, E.T., Nguyen, K.T., Eubanks, S.R.,Allen, L.G., Duggan, M.J., Loy, S.D., Bonham, K.A., Blod-gett, J.K., 1992. A new class of bradykinin antagonists: syn-thesis and in vitro activity of bissuccinimidoalkane peptidedimers. J. Med. Chem. 35, 1563–1572.

Hock, F.J., Wirth, K., Albus, U., Linz, W., Gerhards, H.J.,Wiemer, G., Henke, S., Breipohl, G., Konig, W., Knolle, J.,Scholkens, B.A., 1991. Hoe140 a new potent and long-actingbradykinin-antagonist: in vitro studies. Br. J. Pharmacol. 102,769–773.

Inamura, N., Asano, M., Hatori, C., Sawai, H., Hirosumi, J.,Fujiwara, T., Kayakiri, H., Satoh, S., Abe, Y., Inoue, T.,Sawada, Y., Oku, T., Nakahara, K., 1997. Pharmacological

characterization of a novel, orally active, nonpeptidebradykinin B receptor antagonist, FR167344. Eur. J. Pharma-2

col. 333, 79–86.Regoli, D., Nsa Allogho, S., Rizzi, A., Gobeil, F.J., 1998.

Bradykinin receptors and their antagonists. Eur. J. Pharmacol.348, 1–10.

Sawutz, D.G., Salvino, J.M., Dolle, R.E., Casiano, F., Ward, S.J.,Houck, W.T., Faunce, D.M., Douty, B.D., Baizman, E., Awad,M.M., Marceau, F., Seoane, P.R., 1994. The nonpeptide WIN64338 is a bradykinin B receptor antagonist. Proc. Natl.2

Acad. Sci. U.S.A. 91, 4693–4697.Stewart, J.M., 1995. Bradykinin antagonists: development and

applications. Biopolymers 37, 143–155.Wirth, K., Hock, F.J., Albus, U., Linz, W., Alpermann, H.G.,

Anagnostopoulos, H., Henk, S., Breipohl, G., Konig, W.,Knolle, J., Scholkens, B.A., 1991. Hoe140, a new potent andlong-acting bradykinin antagonist: in vivo studies. Br. J. Phar-macol. 102, 774–777.