Non-peptide Bradykinin Research Articles

Synthesis of hexahydrofuro[3,2-c]quinoline, a martinelline type analogue and investigation of its biological activity.

BackgroundCandida susceptibility commonly occurs in breast cancer patients. Of which, Candida albicans is considered as a common pathogen causing candidiasis. Martinella iquitosensis (Bignoniaceae) is one of the species belonged to Martinella, distributed widely in Amazon basin. Its root extract yielded two complex substituted tetrahydroquinolines, Martinelline and Martinellic acid which were the first natural non-peptide bradykinin receptor antagonists identified.FindingsIn this study, a novel martinelline type analogue, named 2,3,3a,4,5,9b-hexahydro-8-phenoxy-4-(pyridin-2-yl)furo[3,2-c]quinoline, was synthesized and its preliminary anticancer activity and antifungal potential were investigated. This compound showed potential anticancer activity against MDAMB-231 breast cancer cells. Meanwhile it could enhance the fungistatic activity of miconazole against Candida albicans.ConclusionsThese findings provide an implication for the continue investigation and development of martinelline type analogues as therapeutic agents in the future.

Ocular Hypotensive Activity of a Non-Peptide Bradykinin B2-Receptor Antagonist (WIN-64338) In Dutch-Belt Rabbits- A Case of Poly-Pharmacology in Action

Ocular Hypotensive Activity of a Non-Peptide Bradykinin B2-Receptor Antagonist (WIN-64338) In Dutch-Belt Rabbits- A Case of Poly-Pharmacology in Action

Novel Potential Treatment Modalities for Ocular Hypertension: Focus on Angiotensin and Bradykinin System Axes

Despite the availability of modern surgical procedures, new drug delivery techniques, health authority-approved single topical ocular drugs, and combination products thereof, there continues to be an unmet medical need for novel treatment modalities for preserving vision. This is especially true for the treatment of glaucoma and the high risk factor often associated with this ocular disease, elevated intraocular pressure (IOP). Undesirable local or systemic side effects, frequency of dosing, lack of sustained IOP lowering, and lack of prevention of diurnal IOP spikes are among the greatest challenges. The very recent discovery, characterization, and publication of 2 novel IOP-lowering agents that pertain to the renin-angiotensin and kallikrein-kinin axes potentially offer novel means to treat and control ocular hypertension (OHT). Here, some contextual introductory information is provided first, followed by more detailed discussion of the properties and actions of diminazene aceturate (DIZE; a novel angiotensin-converting enzyme-2 activator) and FR-190997 (a nonpeptide bradykinin receptor-2 agonist) in relation to their anti-OHT activities in rodent and cynomolgus monkey eyes, respectively. It is anticipated that these compounds will pave the way for future discovery, development, and marketing of novel drugs to treat glaucoma and thus help save sight for millions of people afflicted with this slow progressive optic neuropathy.

Preclinical pharmacology, ocular tolerability and ocular hypotensive efficacy of a novel non-peptide bradykinin mimetic small molecule

We sought to characterize the ocular pharmacology, tolerability and intraocular pressure (IOP)-lowering efficacy of FR-190997, a non-peptidic bradykinin (BK) B2-receptor agonist. FR-190997 possessed a relatively high receptor binding affinity (Ki = 27 nM) and a high in vitro potency (EC50 = 18.3 ± 4.4 nM) for inositol-1-phosphate generation via human cloned B2-receptors expressed in host cells with mimimal activity at B1-receptors. It also mobilized intracellular Ca2+ in isolated human trabecular meshwork (h-TM), ciliary muscle (h-CM), and in immortalized non-pigmented ciliary epithelial (h-iNPE) cells (EC50s = 167–384 nM; Emax = 32–86% of BK-induced response). HOE-140, a selective B2-receptor antagonist, potently blocked the latter effects of FR-190997 (e.g. IC50 = 7.3 ± 0.6 nM in h-CM cells). FR-190997 also stimulated the release of prostaglandins (PGs) from h-TM and h-CM cells (EC50s = 60–84 nM; Emax = 29–44% relative to max. BK-induced effects). FR-190997 (0.3–300 μg t.o.) did not activate cat corneal polymodal nociceptors and did not cause ocular discomfort in Dutch-Belted rabbits, but it was not well tolerated in New Zealand albino rabbits and Hartley guinea pigs. A single topical ocular (t.o.) dose of 1% FR-190997 in Dutch-Belted rabbits and mixed breed cats did not lower IOP. However, FR-190997 efficaciously lowered IOP of conscious ocular hypertensive cynomolgus monkey eyes (e.g. 34.5 ± 7.5% decrease; 6 h post-dose of 30 μg t.o.; n = 8). Thus, FR-190997 is an unexampled efficacious ocular hypotensive B2-receptor non-peptide BK agonist that activates multiple signaling pathways to cause IOP reduction.

FR-190997, a nonpeptide bradykinin B2-receptor partial agonist, is a potent and efficacious intraocular pressure lowering agent in ocular hypertensive cynomolgus monkeys.

Preclinical Research FR-190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnaminoacetyl]-N-methylamino]benzyloxy]-2-methyl-4- (2-pyridylmethoxy) quinoline), a nonpeptide bradykinin (BK) B2-receptor-selective agonist, represents a novel class of ocular hypotensive agents. FR-190997 exhibited a high affinity for the human cloned B2-receptor (Ki = 9.8 nM) and a relatively high potency (EC50 = 155 nM) for mobilizing intracellular Ca(2+) ([Ca(2+)]i) in human ocular cells from nonpigmented ciliary epithelium; trabecular meshwork [h-TM]; ciliary muscle [h-CM] that are involved in regulating intraocular pressure (IOP). Unlike BK, FR-190997 behaved as a partial agonist (Emax = 38-80%) in these cells and its [Ca(2+)]i-mobilizing effects were blocked by the B2-receptor-selective antagonists (HOE-140, Ki = 0.8-7 nM; WIN-64338, Ki = 157-425 nM). FR-190997 stimulated the production of prostaglandins (PGs) in h-CM and h-TM cells (EC50 = 15-19 nM; Emax = 27-33%); an effect that was reduced by the cyclooxygenase-2 inhibitor bromfenac, and by HOE-140. FR-190997 also induced pro-matrix metalloproteinase (MMP)-1 and MMP-3 release from h-CM cells. FR-190997 significantly lowered IOP (37% [P < 0.001] with 30 μg, 24 h post-topical ocular dosing) in ocular hypertensive eyes of conscious Cynomolgus monkeys. This effect was reduced by bromfenac and completely blocked by a B2-antagonist. FR-190997 primarily stimulated uveoslceral outflow (UVSO) of aqueous humor (2.6 to 3.9-fold above baseline). In conclusion, FR-190997 is a B2-receptor selective partial agonist that activates phospholipase C, mobilizes [Ca(2+)]; induces PG and pro-MMP production, and that profoundly lowers IOP by promoting UVSO in ocular hypertensive Cynomolgus monkey eyes.

Strong cytotoxic effect of the bradykinin antagonist BKM‐570 in ovarian cancer cells – analysis of the molecular mechanisms of its antiproliferative action

The standard chemotherapy for epithelial ovarian cancer (EOC) patients is currently a combination of taxane and platinum. However, most EOC patients still suffer relapses, and there is an immediate need for the development of novel and more effective therapeutic modalities against this deadly disease. Recently, the nonpeptide bradykinin (BK) antagonist 2,3,4,5,6-pentafluorocinnamoyl-(o-2,6-dichlorobenzyl)-l-tyrosine-N-(4-amino-2,2,6,6-tetramethyl-piperidyl) amide (BKM-570) was shown to cause impressive growth inhibition of lung and prostate tumors, displaying superior in vivo inhibitory effects than convential chemotherapeutic drugs. Here, we investigated BKM-570 cytotoxic effects in two EOC cell lines, derived from different EOC histopathologies: a clear cell carcinoma (TOV-21), and an endometrioid carcinoma (TOV-112). We showed that BKM-570 effectively inhibited the growth of ovarian cancer cells, as its cytotoxic effects were comparable to those of cisplatin, and were independent of the functional status of BK receptors. Moreover, BKM-570 synergized with cisplatin in inhibiting EOC cell growth. To better understand the molecular mechanisms of the antiproliferative action of this BK antagonist in EOC cells, we performed gene expression profiling in TOV-21 and TOV-112 cells following treatment with 10 μM BKM-570 for 24 h. BKM-570 displayed similar cytotoxic effects in the two cell lines analyzed, as genes with previously shown involvement in apoptosis/antiapoptosis and cell adhesion were proportionally upregulated and downregulated in both cell lines, whereas genes involved in basic cellular mechanisms, including cell growth and maintenance, metabolism, cell cycle control, inflammatory and immune response, signal transduction, protein biosynthesis, transcription regulation, and transport, were predominantly downregulated upon treatment. Our data are indicative of the therapeutic potential of BKM-570 and related compounds in EOC management.

The BRAIN TRIAL: a randomised, placebo controlled trial of a Bradykinin B2 receptor antagonist (Anatibant) in patients with traumatic brain injury

BackgroundCerebral oedema is associated with significant neurological damage in patients with traumatic brain injury. Bradykinin is an inflammatory mediator that may contribute to cerebral oedema by increasing the permeability of the blood-brain barrier. We evaluated the safety and effectiveness of the non-peptide bradykinin B2 receptor antagonist Anatibant in the treatment of patients with traumatic brain injury. During the course of the trial, funding was withdrawn by the sponsor.MethodsAdults with traumatic brain injury and a Glasgow Coma Scale score of 12 or less, who had a CT scan showing an intracranial abnormality consistent with trauma, and were within eight hours of their injury were randomly allocated to low, medium or high dose Anatibant or to placebo. Outcomes were Serious Adverse Events (SAE), mortality 15 days following injury and in-hospital morbidity assessed by the Glasgow Coma Scale (GCS), the Disability Rating Scale (DRS) and a modified version of the Oxford Handicap Scale (HIREOS).Results228 patients out of a planned sample size of 400 patients were randomised. The risk of experiencing one or more SAEs was 26.4% (43/163) in the combined Anatibant treated group, compared to 19.3% (11/57) in the placebo group (relative risk = 1.37; 95% CI 0·76 to 2·46). All cause mortality in the Anatibant treated group was 19% and in the placebo group 15.8% (relative risk 1.20, 95% CI 0.61 to 2.36). The mean GCS at discharge was 12.48 in the Anatibant treated group and 13.0 in the placebo group. Mean DRS was 11.18 Anatibant versus 9.73 placebo, and mean HIREOS was 3.94 Anatibant versus 3.54 placebo. The differences between the mean levels for GCS, DRS and HIREOS in the Anatibant and placebo groups, when adjusted for baseline GCS, showed a non-significant trend for worse outcomes in all three measures.ConclusionThis trial did not reach the planned sample size of 400 patients and consequently, the study power to detect an increase in the risk of serious adverse events was reduced. This trial provides no reliable evidence of benefit or harm and a larger trial would be needed to establish safety and effectiveness.Trial RegistrationThis study is registered as an International Standard Randomised Controlled Trial, number ISRCTN23625128.

Anatibant ®, a selective non-peptide bradykinin B 2 receptor antagonist, reduces intracranial hypertension and histopathological damage after experimental traumatic brain injury

Bradykinin, the main metabolite of the kallikrein-kinin system and one of the first mediators released during inflammation, is well known to increase the permeability of the blood brain barrier (BBB) by activation of kinin B 2 receptors and hence promote brain edema formation following traumatic brain injury (TBI). Anatibant ® (LF 16-0687), a selective non-peptide bradykinin B 2 receptor antagonist, reduces brain edema after experimental TBI, however, so far no data are available if Anatibant ® reduces also the sequels of brain edema formation, i.e. morphological brain damage. Therefore, we investigated the effect of Anatibant (3.0 mg/kg b.w.) on intracranial pressure (ICP) and contusion volume after experimental TBI. Male C57/Bl6 mice (25–28 g) were subjected to Controlled Cortical Impact trauma (CCI). Anatibant ® was administrated as a subcutaneous bolus 15 min and 8 h after TBI. ICP was measured 3, 6, and 10 h after injury and contusion volume was quantified 24 h after trauma. Our data demonstrate a significant reduction of ICP (16.6 ± 1.67 mmHg vs. 24.40 ± 3.58 mmHg; n = 6; p = 0.002) and of contusion volume 24 h after trauma (28.28 ± 5.18 mm 3 vs. 35.0 ± 3.32 mm 3 n = 7; p = 0.003) in treated mice. Therefore we conclude, that inhibition of bradykinin B 2 receptors seems to be a promising treatment option, and might therefore be investigated in clinical trails for the treatment of TBI.

Design and Synthesis of Novel Sulfonamide-Containing Bradykinin hB2 Receptor Antagonists. 2. Synthesis and Structure−Activity Relationships of α,α-Cycloalkylglycine Sulfonamides

Recently we reported on the design and synthesis of a novel class of selective nonpeptide bradykinin (BK) B2 receptor antagonists (J. Med. Chem. 2006, 3602-3613). This work led to the discovery of MEN 15442, an antagonist with subnanomolar affinity for the human B2 receptor (hB2R), which also displayed significant and prolonged activity in vivo (for up to 210 min) against BK-induced bronchoconstriction in the guinea-pig at a dose of 300 nmol/kg (it), while demonstrating only a slight effect on BK-induced hypotension. Here we describe the further optimization of this series of compounds aimed at maximizing the effect on bronchoconstriction and minimizing the effect on hypotension, with a view to developing topically delivered drugs for airway diseases. The work led to the discovery of MEN 16132, a compound which, after intratracheal or aerosol administration, inhibited, in a dose-dependent manner, BK-induced bronchoconstricton in the airways, while showing minimal systemic activity. This compound was selected as a preclinical candidate for the topical treatment of airway diseases involving kinin B2 receptor stimulation.

Bradykinin B2 Receptor Antagonism With LF 18-1505T Reduces Brain Edema and Improves Neurological Outcome After Closed Head Trauma in Rats

We evaluated the effect of LF 18-1505T, a novel nonpeptide bradykinin type-2 receptor antagonist, on brain edema and neurologic severity score (NSS) after closed head trauma (CHT). There were 132 rats anesthetized and assigned for sham or CHT; infusion of saline or LF 18-1505T (0.3, 1, 3, 10, or 30 microg x kg x min); and determination of neurologic outcome (brain water content and NSS) or physiologic variables (blood pressure, glucose concentration, etc.). Post-CHT brain water content was less with LF 18-1505T doses of 3 and 10 microg x kg x min (80.1 +/- 3.8 through 81.6 +/- 2.6%, mean +/- SD) than in the untreated group (84.6 +/- 1.9%, p < 0.01). Post-CHT NSS improved with doses of 3, 10, and 30 microg x kg x min (median, 7; range, 0-12 through median, 10; range, 8-18) as compared with that in the untreated group (median, 17; range, 14-23; p < 0.05). LF 18-1505T with or without CHT did not significantly alter physiologic variables. LF 18-1505T decreased brain edema and improved neurologic status after CTH in rats without significantly altering physiologic values.

Discovery of Non‐Peptide Bradykinin B2 Receptor Antagonists and Agonists

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access the actual ChemInform Abstract, please click on HTML or PDF.

MEN16132, a novel potent and selective nonpeptide antagonist for the human bradykinin B 2 receptor. In vitro pharmacology and molecular characterization

The pharmacological characterization of the novel nonpeptide antagonist for the B 2 receptor, namely MEN16132 (4-( S)-Amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2 H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride) is presented. The affinity of MEN16132 for the bradykinin B 2 receptor has been investigated by means of competition studies at [ 3H]bradykinin binding to membranes prepared from Chinese Hamster Ovary (CHO) cells expressing the human bradykinin B 2 receptor (pK i 10.5), human lung fibroblasts (pK i 10.5), guinea pig airways (pK i 10.0), guinea pig ileum longitudinal smooth muscle (pK i 10.2), or guinea pig cultured colonic myocytes (pK i 10.3). In all assays MEN16132 was as potent as the peptide antagonist Icatibant, and from 3- to 100-fold more potent than the reference nonpeptide antagonists FR173657 or LF16-0687. The selectivity for the bradykinin B 2 receptor was checked at the human bradykinin B 1 receptor (pK i < 5), and at a panel of 26 different receptors and channels. The antagonist potency was measured in functional assays, i.e., in blocking the bradykinin induced inositolphosphates (IP) accumulation at the human (CHO: pK B 10.3) and guinea pig (colonic myocytes: pK B 10.3) B 2 receptor, or in antagonizing the bradykinin induced contractile responses in human (detrusor smooth muscle: pK B 9.9) and guinea pig (ileum longitudinal smooth muscle: pK B 10.1) tissues. In both functional assay types MEN16132 exerted a different antagonist pattern, i.e., surmountable at the human and insurmountable at the guinea pig bradykinin B 2 receptors. Moreover, the receptor determinants important for the high affinity interaction of MEN16132 with the human bradykinin B 2 receptor were investigated by means of radioligand binding studies performed at 24 point-mutated receptors. The results obtained revealed that residues in transmembrane segment 2 (W86A), 3 (I110A), 6 (W256A), and 7 (Y295A, Y295F but not much Y295W), were crucial for the high affinity of MEN16132. In conclusion, MEN16132 is a new, potent, and selective nonpeptide bradykinin B 2 receptor antagonist.

Antagonistic effects of novel non-peptide chlorobenzhydryl piperazine compounds on contractile response to bradykinin in the guinea-pig ileum

Two novel compounds, N-phenylacetyl- N′-(4-methoxybenzyl)- N″-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid triamide (compound I) and N-phenylacetyl- N′-(4-methylbenzyl)- N″-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid triamide (compound II), designed and synthesized as novel non-peptide bradykinin B 2 receptor antagonists, were studied for their functional activities in isolated guinea-pig ileum smooth muscle. These compounds were compared with the conventional peptide bradykinin B 2 receptor antagonist, icatibant (H– dArg-Arg-Pro-Hyp-Gly-Thi-Ser- dTic-Oic-Arg–OH) for their in vitro functional activities. Compounds I and II showed highly potent, time-dependent insurmountable antagonism against contractile responses to bradykinin (p K B 8.80 and 8.57, respectively) with progressive reduction of maximum effect maintaining the concentration producing half maximal-response unchanged. Otherwise, icatibant, known as a non-competitive antagonist, showed a rightward displacement of cumulative concentration–response curves to bradykinin with decrease of its maximum effect (p K B 8.73). The IC 50 values of compounds I and II were 3.56 × 10 − 8 and 6.30 × 10 − 8 M, respectively, while that of icatibant was 5.02 × 10 − 8 M. The profile of action of compounds I and II varied when contact time was prolonged from 5 to 60 min, whereas that of icatibant did not. The inhibitory effects of the newly synthesized compounds and icatibant on the contractile response to bradykinin were differently reverted by washout (icatibant < 100 min, compounds I and II > 100 min). This class of compounds containing the chlorobenzhydryl piperazine moiety is expected to be a novel non-peptide bradykinin B 2 receptor antagonists.

Non-peptide ligands for bradykinin receptors 1995 – 2004

It has been a decade since the area of bradykinin (BK) research was last reviewed in this journal. Within this period of time, selective, high-affinity non-peptide BK ligands have been identified by different research groups for both the B1 and B2 receptors (B1R and B2R). The efficacies of these agents in animal models of human diseases suggest that it may be possible to develop such compounds into novel classes of therapeutic drugs. The emerging pharmacological evidence from these studies suggests that B1R and B2R antagonists may be clinically useful for the treatment of pain, inflammation, cancer, hypotension, asthma, colitis, rhinitis, pancreatitis, sepsis and rheumatoid arthritis. B2R agonists, on the other hand, may alleviate cardiovascular disorders, such as congestive heart failure, hypertension and ischaemic heart disease, and are potentially useful for the treatment of diabetic disorders by mimicking the beneficial effects of BK on glucose uptake and insulin sensitivity. A major challenge in targeting the B2R is maintaining a balance between the opposing beneficial (cardioprotective, anti-inflammatory) and debilitating (pro-inflammatory, cardiocompromising) effects upon activation or inhibition of this receptor subtype. For this reason, the last few years have seen a significant shift in research emphasis to the B1R subtype due to its disease-dependent expression. This review will focus on the advances in BK medicinal chemistry reported between 1995 and the beginning of December 2004, with particular emphasis on small-molecule patent disclosures made during this period.

Antihyperalgesic activity of a novel nonpeptide bradykinin B1 receptor antagonist in transgenic mice expressing the human B1 receptor

We describe the properties of a novel nonpeptide kinin B1 receptor antagonist, NVP-SAA164, and demonstrate its in vivo activity in models of inflammatory pain in transgenic mice expressing the human B1 receptor. NVP-SAA164 showed high affinity for the human B1 receptor expressed in HEK293 cells (K(i) 8 nM), and inhibited increases in intracellular calcium induced by desArg10kallidin (desArg10KD) (IC50 33 nM). While a similar high affinity was observed in monkey fibroblasts (K(i) 7.7 nM), NVP-SAA164 showed no affinity for the rat B1 receptor expressed in Cos-7 cells. In transgenic mice in which the native B1 receptor was deleted and the gene encoding the human B1 receptor was inserted (hB1 knockin, hB1-KI), hB1 receptor mRNA was induced in tissues following LPS treatment. No mRNA encoding the mouse or human B1 receptor was detected in mouse B1 receptor knockout (mB1-KO) mice following LPS treatment. Freund's complete adjuvant-induced mechanical hyperalgesia was similar in wild-type and hB(1)-KI mice, but was significantly reduced in mB1-KO animals. Mechanical hyperalgesia induced by injection of the B1 agonist desArg10KD into the contralateral paw 24 h following FCA injection was similar in wild-type and hB1-KI mice, but was absent in mB1-KO animals. Oral administration of NVP-SAA164 produced a dose-related reversal of FCA-induced mechanical hyperalgesia and desArg10KD-induced hyperalgesia in hB1-KI mice, but was inactive against inflammatory pain in wild-type mice. These data demonstrate the use of transgenic technology to investigate the in vivo efficacy of species selective agents and show that NVP-SAA164 is a novel orally active B1 receptor antagonist, providing further support for the utility of B1 receptor antagonists in inflammatory pain conditions in man.

非ペプチド性ブラジキニンＢ２受容体アンタゴニストおよびアゴニストの創製

A novel class of potent and selective non-peptide bradykinin (BK) B2 receptor antagonists and agonists was designed and synthesized. The unique screening lead (9) was discovered by a two-step directed random screening process. Systematic chemical modification of 9 elucidated the structural requirements essential for the B2 binding affinity, leading to the identification of the basic framework of this new series of B2 antagonists. A molecular modeling study suggested the key role of the N-methylanilide moiety at the 3-position of the 2, 6-dichlorophenyl ring to allow these compounds to adopt the characteristic active conformation. It was further revealed that the 4-substituent of the quinoline moiety is the key pharmacophore to determine the agonist/antagonist profiles. During these studies, we discovered the orally active non-peptide B2 antagonist, FK 3657 (2), which is undergoing clinical development, and potent B2 agonists represented by FR 190997 (5) and FR 198100 (6).

Pharmacological characterization and radioligand binding properties of a high-affinity, nonpeptide, bradykinin B 1 receptor antagonist

Compound A ( N-{2-[4-(4,5-dihydro-1 H-imidazol-2-yl)phenyl]ethyl}-2-[(2 R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide) is a member of a new class of aryl sulfonamide dihydroquinoxalinone bradykinin B 1 receptor antagonists that should be useful pharmacological tools. Here we report on some of the pharmacological properties of compound A as well as the characterization of [ 35S]compound A as the first nonpeptide bradykinin B 1 receptor radioligand. Compound A inhibited tritiated peptide ligand binding to the cloned human, rabbit, dog, and rat bradykinin B 1 receptors expressed in CHO cells with K i values of 0.016, 0.050, 0.56, and 29 nM, respectively. It was inactive at 10 μM in binding assays with the cloned human bradykinin B 2 receptor. In functional antagonist assays with the cloned bradykinin B 1 receptors, compound A inhibited agonist-induced signaling with activities consistent with the competition binding results, but had no antagonist activity at the bradykinin B 2 receptor. Compound A was also found to be a potent antagonist in a rabbit aorta tissue bath preparation and to effectively block des-Arg 9 bradykinin depressor responses in lipopolysaccharide-treated rabbit following intravenous administration. The binding of [ 35S]compound A was evaluated with the cloned bradykinin B 1 receptors. In assays with human, rabbit, and dog receptors, [ 35S]compound A labeled a single site with K d values of 0.012, 0.064, and 0.37 nM, respectively, and with binding site densities equivalent to those obtained using the conventional tritiated peptide ligands. Binding assays with the cloned rat bradykinin B 1 receptor were not successful, presumably due to the low affinity of the ligand for this species receptor. There was no specific binding of the ligand detected in CHO cells expressing the human bradykinin B 2 receptor. In assays with the cloned human bradykinin B 1 receptor, the pharmacologies of the binding of [ 35S]compound A and [ 3H][Leu 9]des-Arg 10-kallidin were the same. The high signal-to-noise ratio obtained with [ 35S]compound A will allow this ligand to be a very useful tool for future investigations of the bradykinin B 1 receptor.

The N-terminal of Icatibant and bradykinin interact with the same Asp residues in the human B 2 receptor

The pharmacology of peptide and non-peptide bradykinin B 2 receptor ligands was evaluated in the inositol phosphate (IP) production assay in CHO cells expressing the human bradykinin B 2 receptor. The effect of single and double alanine mutation of D266 and D284 residues at the human bradykinin B 2 receptor was evaluated on the agonist profile of bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) and the synthetic agonist FR190997 (8-[2,6-dichloro-3-[ N-methylcarbamoyl)cinnamidoacetyl]- N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline). Bradykinin potency (EC 50 0.5 nM at the wild-type receptor) was reduced by 16-fold at D266A and D284A mutants and by 2300-fold at the D266A/D284A double mutant. None of the mutants affected the potency or the efficacy of FR190997. Peptide antagonists, Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH) and MEN11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7γ-10α)) (100 nM) similarly antagonized the concentration–response curve to bradykinin or FR190997 (pA 2 values 8.5 and 8.4 versus bradykinin and 8.2 and 8.4 versus FR190997) at the wild-type receptor. Non-peptide antagonists FR173657 ((E)-3-(6-acetamido-3-pyridyl)- N-[ N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl]phenyl]- N-methylaminocarbonyl methyl]acrylamide) and LF16-0687 (1-[[2,4-dichloro-3-[(2,4-dimethylquinolin-8-yl)oxy] methyl]-phenyl]sulfonyl]- N-[3-[[4-(aminoiminomethyl)-phenyl]carbonylamino]propyl]-(S)-pyrrolidine carboxamide) (100 nM) showed an equivalent potency values in blocking the IP production induced by bradykinin or FR190997 (pA 2 values 8.7 and 8.8 versus bradykinin and 8.8 and 8.6 versus FR190997). Whilst the antagonist potency of FR173657 and LF16-0687 was not affected by D266A/D284A double mutation (IP production induced by the synthetic agonist), that of Icatibant and MEN11270 was reduced by 50- and 200-fold. The antagonist potency of [Ala 1]-Icatibant and [Ala 2]-Icatibant (pA 2 values at wild-type 7.7 and 6.4) was significantly less reduced (20-fold and 13-fold, respectively) by the D266A/D284A double mutation. Our results highlight a crucial role for two aspartic residues, D266 and D284, located at the top of transmembrane segments 6 and 7, in the high-affinity interaction of peptide antagonists with the human bradykinin B 2 receptor. An interaction of these receptor residues with the N-terminal basic residues of Icatibant is hypothesized.

Discovery of the first non-peptide full agonists for the human bradykinin B(2) receptor incorporating 4-(2-picolyloxy)quinoline and 1-(2-picolyl)benzimidazole frameworks.

In the course of our studies on non-peptide bradykinin (BK) B(2) receptor ligands, it was suggested that the 4-substituent of the quinoline ring may play a critical role in determining binding affinities for human and guinea pig B(2) receptors, as well as agonist/antagonist properties. We carried out an extensive investigation to elucidate the structure-activity relationships (SAR) for this key pharmacophore. Introduction of lower alkoxy groups to the 4-position of the quinoline ring of 3 led to the identification of 4-ethoxy derivative 22b as a unique partial agonist. This compound significantly stimulated inositol phosphates (IPs) formation in Chinese hamster ovary cells expressing the cloned human B(2) receptor at concentrations greater than 10 nM and displayed one-tenth of the intrinsic activity of BK. The agonist activity of 22b was selective for the B(2) receptor and was inhibited by selective peptide and non-peptide B(2) antagonists. On the other hand, 22b strongly suppressed BK-induced IPs formation through the cloned human B(2) receptor. Further studies on the key pharmacophore led to identification of a 2-picolyloxy moiety as a powerful agonist switch, leading to the discovery of a potent and efficacious non-peptide B(2) agonist, 19a. Successive optimization of the acyl side chain afforded 38, which exhibited full agonist activity on stimulation of IPs formation. Furthermore, this strategy could be applied successfully to the benzimidazole series. The representative 1-(2-picolyl)benzimidazole derivative 47c increased PGE(2) production at a 1 microM concentration to the same level as the maximum effect of BK. Thus, we have established the medicinal chemistry modifications required to convert our highly potent non-peptide B(2) antagonists to agonists with potent efficacy.

A new class of nonpeptide bradykinin B(2) receptor ligand, incorporating a 4-aminoquinoline framework. Identification of a key pharmacophore to determine species difference and agonist/antagonist profile.

Introduction of various aliphatic amino groups at the 4-position of the quinoline moiety of our nonpeptide bradykinin (BK) B(2) receptor antagonists afforded highly potent ligands for human B(2) receptor with various affinities for guinea pig B(2) receptor, indicating remarkable species difference. A representative 4-dimethyamino derivative 40a exhibited subnanomolar and nanomolar binding affinities for human and guinea pig B(2) receptors, respectively, and significantly inhibited BK-induced bronchoconstriction in guinea pigs at 10 microg/kg by intravenous administration. Further chemical modification led us to discover unique partial agonists for the human B(2) receptor that increase inositol phosphates (IPs) production by themselves in Chinese hamster ovary (CHO) cells expressing the cloned human B(2) receptor. Although their potency and efficacy were much lower than those of BK, we identified them as screening leads for nonpeptide B(2) agonists. In these studies it was revealed the 4-substituent of the quinoline moiety is the key pharmacophore to determine species difference and agonist/antagonist profiles.