Discovery of orally active nonpeptide bradykinin B2 receptor antagonists.

Orally active nonpeptide bradykinin (BK) B2 receptor antagonists have been discovered by using directed random screening and chemical modification. These compounds displaced [3H]BK binding to B2 receptors in guinea-pig ileum membranes, rat uterus membranes and human lung fibroblasts with nanomolar IC50s. They did not inhibit different specific radio-ligand bindings to other receptor sites including B2 receptors. In isolated guinea-pig ileum preparations, these compounds had no agonistic effect on smooth muscle contraction at 10(-6) M, and caused parallel rightward shifts of the concentration-response curves to BK on contraction with higher p A2 values. They also blocked human B2 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 caerulein-induced 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 B2 receptor antagonists and that they may have therapeutic potential against inflammatory diseases and pain.

Nonpeptide mimic of bradykinin with long-acting properties.

Kinins, members of a family of peptides released from kininogens by the action of kallikreins, have been implicated in a variety of biological activities including vasodilation, increased vascular permeability, contraction of smooth muscle cells and activation of sensory neurons. However, investigation of the physiological actions of kinins have been greatly hampered because its effects are curtailed by rapid proteolytic degradation. We examined the pharmacological characteristics of the first nonpeptide bradykinin receptor agonist 8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl+ ++]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinolin e (FR190997). FR190997, whose structure is quite different from the natural peptide ligand, but is similar to the nonpeptide antagonists FR165649, FR167344 and FR173657, potently and selectively interacts with the human B2 receptor and markedly stimulates inositol phosphate formation in transfected Chinese hamster ovary (CHO) cells. FR190997 induces concentration-dependent contraction of isolated guinea pig ileum. In vivo, FR190997 mimics the biological action of bradykinin and induces hypotensive responses in rats with prolonged duration, presumably as a consequence of its resistance to proteolytic degradation. Therefore, FR190997 is a highly potent and subtype-selective nonpeptide agonist which displays high intrinsic activity at the bradykinin B2 receptor. This compound represents a powerful tool for further investigation of the physiology and pathophysiology of bradykinin receptors.

Probes for narcotic receptor mediated phenomena. 26. Synthesis and biological evaluation of diarylmethylpiperazines and diarylmethylpiperidines as novel, nonpeptidic delta opioid receptor ligands.

We recently reported (+)-4-¿(alphaR)-alpha-¿(2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl¿-3-methoxybenzyl-N,N-diethylbenzamide (1b, SNC80) as a novel nonpeptidic delta receptor agonist and explored the structure-activity relationships (SAR) of a series of related derivatives. We have found that delta binding activities and selectivity showed little change when the 3-methoxy group in 1b was removed or replaced by the other substituents, whereas the N, N-diethylbenzamide group is important for interaction with the delta receptor. Extensive modification of the piperazine nucleus led to the synthesis of a new series of N, N-diethyl(alpha-piperazinylbenzyl)benzamides (2, 3a-e), N, N-diethyl(alpha-piperidinyl or piperidinylidenebenzyl)benzamides (4a, 5a-c, 6a-b), and related derivatives (4b, 7a-c). Several compounds (2, 3a, 3e, 6a) strongly bound to the delta receptor with K(i) values in the low nanomolar range. On the other hand, the binding affinities of these compounds for the mu and kappa receptors were negligible, indicating excellent delta opioid receptor subtype selectivity. The two nitrogen atoms on the piperazine nucleus showed different SAR in the interaction of this series of compounds at the delta receptor. Nitrogen N(4) appears to be an important structural element and is essential for electrostatic interaction, while N(1) seems to be unnecessary for recognition at the delta receptor.

Studies on 5-lipoxygenase inhibitors. II. Discovery, optical resolution and enantioselective synthesis of FR110302, a highly potent non-redox type 5-lipoxygenase inhibitor.

A novel series of 2,2-dialkyl-5-(2-quinolylmethoxy)-1,2,3, 4-tetrahydro-1-naphthols was synthesized and evaluated as 5-lipoxygenase (5-LO) inhibitors. Systematic optimization led to identification of several highly potent non-redox type 5-LO inhibitors with nanomolar IC50s as racemic mixtures. Optical resolution of racemate 50 indicated that its 5-LO inhibitory activity was enantiospecific and due to the (+)-enantiomer. An efficient synthetic route to the (+)-enantiomers via asymmetric reduction of tetralone intermediates was established. The best compound, (+)-2,2-dibutyl-5-(2-quinolylmethoxy)-1,2,3,4-tetrahydro-1-naphtho l (FR110302, (+)-50), showed potent inhibitory activity against leukotriene (LT) biosynthesis by intact neutrophiles in rats (IC50 4.9 nM) and in humans (IC50 40 nM). Furthermore oral administration of FR110302 significantly inhibited neutrophil migration in the rat air pouch model at 1 mg/kg.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 4. Discovery of novel frameworks mimicking the active conformation.

In recent articles we reported the identification of a series of 8-[[2, 6-dichloro-3-[N-methyl-N-[(E)-(substituted)acryloylglycyl]amino]++ +benzy l]oxy]-2-methylimidazo[1,2-a]pyridines as the first orally active non-peptide bradykinin (BK) B2 receptor antagonists. Optimization of the terminal glycine part and the imidazo[1,2-a]pyridine moiety led to the discovery of a clinical candidate (5, FR173657). With the aim of completion of the structure-activity relationship (SAR) study, we next investigated the roles of the substituents on the central phenyl ring. The results suggested that the 2,6-dichloro or 2, 6-dimethyl groups may play important roles in regulating the conformations of the 1- and 3-substituents and also may interact with hydrophobic pockets of the B2 receptors. Furthermore, according to the results of a molecular modeling study reported in part 1 of this series, we designed and synthesized a series of sterically constrained analogues by replacing the N-methylamide group with cis-amide-like rigid moieties. We discovered several bioisosteres and chemically proved that the N-methylamide moiety adopts the cis-amide form in the active conformation. Extensive chemical modification led to the identification of a novel class of highly potent and orally active non-peptide B2 antagonists represented by a pyrrole derivative (52a, FR193517). Compound 52a inhibited the specific binding of [3H]BK to recombinant human B2 receptors expressed in Chinese hamster ovary (CHO) cells and guinea pig ileum membrane preparations expressing B2 receptors with IC50s of 0.37 and 0.56 nM, respectively. This compound also displayed excellent in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs at 1 mg/kg by oral administration.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 2. Overcoming the species difference between guinea pig and man.

Recently we reported the identification of a series of 8-[[3-(N-acylglycyl-N-methylamino)-2, 6-dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridines as the first orally active non-peptide bradykinin (BK) B2 receptor antagonists (1-3). These compounds inhibited the specific binding of [3H]BK to guinea pig ileum membrane preparations expressing B2 receptors with nanomolar IC50's and also displayed in vivo functional antagonistic activities against BK-induced bronchoconstriction in guinea pigs at 1 mg/kg by oral administration. However, it was found that their affinities for the B2 receptors in human A-431 cells (human epidermoid carcinoma) were much lower. Intensive modifications of the terminal substituents at the glycine moiety elucidated the structure-activity relationships (SAR) for human B2 receptors, leading to an extended basic framework which incorporated a novel key pharmacophore. Thus, we overcame the species difference and identified the first clinical candidate 18c (FR167344) with IC50's of 0.66 and 1.4 nM for guinea pig ileum and human A-431 cells, respectively. This compound displayed in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs with an ED50 value of 0.17 mg/kg by oral administration. This novel non-peptide B2 antagonist is extremely potent both in vitro and in vivo by oral administration and is expected to be the first member of a new class of drug for the treatment of various inflammatory diseases.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 3. Discovering bioisosteres of the imidazo[1,2-a] pyridine moiety.

Recently we reported on overcoming the species difference of our first orally active non-peptide bradykinin (BK) B2 receptor antagonists, incorporating an 8-[[3-(N-acylglycyl-N-methylamino)-2, 6-dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridine skeleton, leading to identification of the first clinical candidate 4a (FR167344). With this potent new lead compound in hand, we then investigated further refinement of the basic framework by replacement of the imidazo[1,2-a]pyridine moiety and discovered several bioisosteric heterocycles. Extensive optimization of these new heteroaromatic derivatives revealed the detailed structure-activity relationships (SAR) around the imidazo[1, 2-a]pyridine ring and the 2,6-dichlorobenzyl moiety, leading to the discovery of our second clinical candidate 87b (FR173657) which inhibited the specific binding of [3H]BK to recombinant human B2 receptors expressed in Chinese hamster ovary (CHO) cells and guinea pig ileum membrane preparations expressing B2 receptors with IC50's of 1.4 and 0.46 nM, respectively. This compound also displayed excellent in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs with an ED50 value of 0.075 mg/kg by oral administration. Further modifications of the terminal substituents on the pyridine moiety led to a novel pharmacophore and resulted in the identification of 99 (FR184280), whose IC50 value for human B2 receptors (0.51 nM) was comparable to that of the second-generation peptide B2 antagonist Icatibant.

Pharmacological characterization of a nonpeptide bradykinin B2 receptor antagonist, FR165649, and agonist, FR190997.

1. The nonpeptide bradykinin (BK) B2 receptor antagonist, FR165649 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl ]-N-methylamino]benzyloxy]-2-methylquinoline), and agonist, FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridyl methoxy)quinoline) have been identified. These compounds have a common chemical structure, and the 2-pyridylmethoxy group is the only structural difference between them. 2. Both FR165649 and FR190997 displaced [3H]-BK binding to B2 receptors in guinea-pig ileum membranes, with an IC50 of 4.7 x 10(-10) M and 1.5 x 10(-9) M, respectively. They also displaced [3H]-BK binding to B2 receptors in human lung fibroblast IMR-90 cells, with an IC50 of 1.6 x 10(-9) M and 9.8 x 10(-10) M, respectively. 3. In guinea-pig isolated ileum-preparations, FR165649 had no agonistic effect on contraction and caused parallel rightward shifts of the concentration-response curves to BK on contraction. Analysis of the data produced a nominal pA2 value of 9.2+/-0.1 (n=5) and a slope of 1.4+/-0.1 (n=5). On the other hand, FR190997 induced concentration-dependent contraction of guinea-pig ilea with a pD2 of 7.9+/-0.2 and the contraction was inhibited by a specific peptide bradykinin B2 receptor antagonist, Hoe 140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]BK) in a non-competitive manner. 4. In IMR-90 cells, FR165649 had no agonistic effect on phosphatidyl inositol (PI) hydrolysis and caused parallel rightward shifts (approximately 200 fold shift at 10(-7) M) of the concentration-response curves to BK on PI hydrolysis. FR190997 induced concentration-dependent PI hydrolysis in IMR-90 cells with a pD2 of 8.4+/-0.1, and this effect was inhibited by Hoe 140. 5. These results indicate that FR165649 and FR190997 are, respectively, a potent bradykinin B2 receptor antagonist and agonist, and that the agonistic activity depends on the small part of the nonpeptide ligand. FR165649 and FR190997 may be useful tools for studying the relationship between ligands and receptors.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 1. Construction of the basic framework.

A novel class of potent, selective, and orally active non-peptide bradykinin (BK) B2 receptor antagonists were designed and synthesized starting from 8-benzyloxyimidazo[1,2-a]pyridine derivative 2. The unique screening lead (2) was discovered by a two-step intentional random screening process, involving recognition of the relationship between BK and angiotensin II (Ang II) and the common structural features. Systematic chemical modification of 2 elucidated the structural requirements essential for B2 binding affinity leading to the identification of 8-[[3-(N-acylglycyl-N-methylamino)-2,6-dichlorobenzyl]oxy]-3-halo- 2- methylimidazo[1,2-a]pyridine skeleton as 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-dichlorobenzene ring to allow these compounds to adopt the characteristic active conformation. The representative lead compounds inhibited the specific binding of [3H]BK to guinea pig ileum membrane preparations expressing B2 receptors, with nanomolar IC50S and also displayed in vivo functional antagonistic activities against BK-induced bronchoconstriction in guinea pigs at an oral dose of 1 mg/kg. Pharmacokinetic studies of compounds 47c and 50b in rats highlighted their excellent oral bioavailabilities, indicating that they represent the first orally active non-peptide B2 antagonists reported to date.

Pharmacological characterization of a novel, orally active, nonpeptide bradykinin B2 receptor antagonist, FR167344.

To investigate the pathophysiological role of bradykinin and to develop a drug for inflammatory diseases, we discovered an orally active, nonpeptide bradykinin B2 receptor antagonist, FR167344, N-[N-[3-[(3-bromo-2-methylimidazo[1,2-a]pyridin-8-yl)oxymethyl]-2, 4dichlorophenyl]-N-methylaminocarbonylmethyl]-4-(dimethyl aminocarbonyl) cinnamylamide hydrochloride. This compound competitively displaced [3H]bradykinin binding to bradykinin B2 receptors present in guinea-pig ileum membrane with an IC50 value of 6.6 X 10(-10) M. In isolated guinea-pig ileum preparations, it also antagonized bradykinin-induced contraction with a pA2 value of 9.3. In human lung fibroblast IMR-90 cells, FR167344 displaced [3H]bradykinin binding to human bradykinin B2 receptors with an IC50 value of 1.3 X 10(-8) M, but not [3H]des-Arg10-kallidin binding to human bradykinin B1 receptors. In vivo, oral administration of FR167344 inhibited bradykinin-induced bronchoconstriction in guinea pigs and the bradykinin-induced hypotensive response for 6 h in rats. These results show that FR167344 is a potent, selective, orally active and long acting bradykinin B2 receptor antagonist.

Nonpeptide mimic of bradykinin with long-acting properties at the bradykinin B2 receptor.

Kinins, members of a family of peptides released from kininogens by the action of kallikreins, exhibit a variety of biological activities including vasodilation, increased vascular permeability, contraction of smooth muscle cells, and activation of sensory neurons. However, investigation of the physiological actions of kinins has been greatly hampered because its effects are curtailed by rapid proteolysis in blood, lung, and liver. We describe the pharmacological characteristics of a novel nonpeptide bradykinin receptor agonist FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl ]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoli ne). FR190997 markedly stimulated phosphatidylinositol hydrolysis in Chinese hamster ovary cells permanently expressing the human bradykinin B2 receptor. The response of phosphatidylinositol hydrolysis was antagonized by the B2 receptor selective antagonist Hoe 140 (D-Arg-[hydroxyproline3,beta-thienylalanine4,D-Tic7,++ +Oic8]bradykinin). In competitive experiments using membranes prepared from Chinese hamster ovary cells expressing the human bradykinin receptor subtypes, FR190997 showed a high affinity binding to the B2 receptor with IC50 value of 5.3 nM and no binding affinity for the B1 receptor. In vivo, FR190997 mimics the biological action of bradykinin and induces hypotensive responses in rats with prolonged duration. Therefore, FR190997 is a highly potent and subtype-selective nonpeptide agonist which displays high intrinsic activity. This compound should represent a powerful tool for further investigation of the physiology and pathophysiology of bradykinin receptors.

Characterization of FR173657, a novel nonpeptide B2 antagonist: in vitro and in vivo studies.

Bradykinin (BK) is involved in different pathophysiological conditions, including allergic and (or) inflammatory reactions. Thus, BK antagonists are considered as a potential drug in allergic and (or) inflammatory diseases. Orally active BK antagonist would be desirable for this purpose. Here, we describe the pharmacological characterization of FR173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8- quinolinyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acr ylamide) obtained from our screening for nonpeptide, orally active B2 antagonists. (i) FR173657 antagonized [3H]BK binding with IC50 values of 4.6 x 10(-10) and 8.6 x 10(-9) M in membrane preparations of guinea pig ileum and lung, respectively. FR173657 inhibited [3H]BK binding to A431, W138, and IMR90 cell lines of human origin with IC50 values of 2.0 x 10(-9), 2.3 x 10(-9), and 1.7 x 10(-9) M, respectively. FR173657 did not affect [3H]des-Arg10-kallidin (B1 ligand) binding onto IMR90 cells. (ii) FR173657 inhibited guinea pig ileum contractions by BK (6 x 10(-8) M) with an IC50 value of 6.1 x 10(-9) M. Acetylcholine- and histamine-induced contraction of guinea pig ileum was unaffected by FR173657. (iii) Oral administration of FR173657 dose-dependently inhibited BK (5 micrograms/kg) and dextran sulfate (activator of kinin-kallikrein cascade) induced bronchoconstriction with ED50 values of 0.075 and 0.057 mg/kg, respectively. In conclusion, FR173657 is a selective potent, orally active B2 receptor antagonist that can be used to investigate the role of BK in allergic and inflammatory diseases.

The identification of an orally active, nonpeptide bradykinin B2 receptor antagonist, FR173657.

1. An orally active, nonpeptide bradykinin (BK) B2 receptor antagonist, FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2-4-dichloro-3-[(2-methyl-8-quinolin yl) oxymethyl]phenyl]-N-methylaminocarbonyl-methyl] acrylamide) has been identified. 2. This compound displaced [3H]-BK binding to B2 receptors present in guinea-pig ileum membranes with an IC50 of 5.6 x 10(-10) M and in rat uterus with an IC50 of 1.5 x 10(-9) M. It did not inhibit different specific radio-ligand binding to other receptor sites. 3. In human lung fibroblast IMR-90 cells, FR173657 displaced [3H]-BK binding to B2 receptors with an IC50 of 2.9 x 10(-9) M and a Ki of 3.6 x 10(-10) M, but did not reduce [3H]-des]Arg10-kallidin binding to B1 receptors. 4. In guinea-pig isolated preparations, FR173657 antagonized BK-induced contractions with an IC50 of 7.9 x 10(-9) M, but did not antagonize acetylcholine or histamine-induced contractions even at a concentration of 10(-6) M. FR173657 caused parallel rightward shifts of the concentration-response curves to BK at concentrations of 10(-9) M and 3.2 x 10(-9) M, and a little depression of the maximal response in addition to the parallel rightward shift of the concentration-response curve at a concentration of 10(-8) M. Analysis of the data yield a pA2 of 9.2 +/- 0.2 (n = 5) and a slope of 1.5 +/- 0.2 (n = 5). 5. In vivo, the oral administration of FR173657 inhibited BK-induced bronchoconstriction dose-dependently in guinea-pigs with an ED50 of 0.075 mg kg-1, but did not inhibit histamine-induced bronchoconstriction even at 1 mg kg-1. FR173657 also inhibited carrageenin-induced paw oedema with an ED50 of 6.8 mg kg-1 2 h after the carrageenin injection in rats. 6. These results show that FR173657 is a potent, selective, and orally active bradykinin B2 receptor antagonist.

Probes for narcotic receptor mediated phenomena. 23. Synthesis, opioid receptor binding, and bioassay of the highly selective delta agonist (+)-4-[(alpha R)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]- N,N-diethylbenzamide (SNC 80) and related novel nonpeptide delta opioid receptor ligands.

The highly selective delta (delta) opioid receptor agonist SNC 80 [(+)-4- [(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N ,N- diethylbenzamide, (+)-21] and novel optically pure derivatives were synthesized from the enantiomers of 1-allyl-trans-2,5-dimethylpiperazine (2). The piperazine (+/-)-2 was synthesized, and its enantiomers were obtained on a multigram scale in > 99% optical purity by optical resolution of the racemate with the camphoric acids. The absolute configuration of (+)-2 was determined to be 2S,5R by X-ray analysis of the salt with (+)-camphoric acid. Since the chirality of the starting material was known, and the relative configuration of compounds (-)-21, (-)-22, and (+)-23 were obtained by single-crystal X-ray analysis, the assignment of the absolute stereochemistry of the entire series could be made. Radioreceptor binding studies in rat brain preparations showed that methyl ethers (+)-21 (SNC 80) and (-)-25 exhibited strong selectivity for rat delta receptors with low nanomolar affinity to delta receptors and only micromolar affinity for rat mu (mu) opioid receptors. Compounds (-)-21, (-)-22, and (-)-23 showed micromolar affinities for delta opioid receptors. The unsubstituted derivative (+)-22 and the fluorinated derivative (-)-27 showed > 2659- and > 2105-fold delta/mu binding selectivity, respectively. The latter derivatives are the most selective ligands described in the new series. Studies with some of the compounds described in the isolated mouse vas deferens and guinea pig ileum bioassays revealed that all were agonists with different degrees of selectivity for the delta opioid receptor. These data show that (+)-21 and (+)-22 are potent delta receptor agonists and suggest that these compounds will be valuable tools for further study of the delta opioid receptor at the molecular level, including its function and role in analgesia and drug abuse.

Ligand selectivity of cloned human and rat opioid mu receptors.

Opiate receptors play major roles in analgesic and euphoric effects of opiate drugs. Recent cloning of cDNAs encoding the rodent and human mu receptor revealed high homology between the predicted receptors but also some sequence differences. To determine if these sequence differences produced significant changes in ligand-selectivity profiles, we assessed these profiles in expressing COS and CHO cell lines using the agonist ligand [125I]IOXY-AGO (6 beta-[125Iodo]-3,14-dihydroxy-17-methyl-4,5 alpha- epoxymorphinan). This ligand's high specific activity (2,200 Ci/mmol) and high affinity for mu opioid receptors generated high signal-to-noise ratio binding. The resulting ligand-selectivity profiles of the human and rat mu receptors reveal modest differences in affinities for morphine and naloxone in COS cells but not CHO cells. Ligand-selectivity profiles of the rat and human mu receptors were otherwise similar. Interesting differences between these data and data previously obtained with the peptide agonist [3H]DAMGO suggest that the peptide and alkaloid agonists may label different domains of the mu receptor.

6 beta-[125iodo]-3, 14-dihydroxy-17-methyl-4, 5 alpha-epoxymorphinan ([125I]IOXY-AGO): a potent and selective radioligand for opioid mu receptors.

The recent cloning and expression of an opioid mu receptor has opened up new opportunities for research in opioid pharmacology. The relatively low level of transient receptor expression in COS cells emphasizes the need for radioligands with high specific activity and low nonspecific binding with which to label receptors. In addition, recent data indicating that agonists and antagonists bind to different domains on the same receptor protein indicate the utility of having both agonist and antagonist radioligands available for the study of opioid receptor mechanisms. Previous studies characterized the binding of the opioid antagonist 6 beta-[125iodo]-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([125I]IOXY) and showed that this naltrexone analog labels mu and kappa 2 receptors in rat and guinea pig brain with high affinity and low nonspecific binding. In the present study, we synthesized the agonist congener of IOXY, 6 beta-iodo-3,14-dihydroxy-17-methyl-4,5 alpha-epoxymorphinan. We named this novel agent IOXY-AGO for IOXY-agonist. Competition binding studies showed that IOXY-AGO has high affinity for mu receptors (Ki = 0.28 nM) and lower affinity for delta (Ki = 18.7 nM) and kappa 1 (Ki = 33.9 nM), kappa 2a (Ki = 38.4 nM) and kappa 2b (Ki = 58.2 nM) binding sites. IOXY-AGO was radioiodinated to a specific activity of 2,200 Ci/mmol. [125I]IOXY-AGO binding was rapid, readily reversible, and characterized by low nonspecific binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Opioid peptide receptor studies. 3. Interaction of opioid peptides and other drugs with four subtypes of the kappa 2 receptor in guinea pig brain.

Using guinea pig, rat, and human brain membranes depleted of mu and delta receptors by pretreatment with the site-directed acylating agents BIT (mu selective) and FIT (delta selective), previous studies from our laboratory resolved two subtypes of the kappa 2 binding site, termed kappa 2a and kappa 2b. In more recent studies, we used 6 beta-[125Iodo]-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([125I]IOXY) to characterize multiple kappa 2 binding sites in rat brain. The results indicated that [125I]IOXY, like [3H]bremazocine, selectively labels kappa 2 binding sites in rat brain membranes pretreated with BIT and FIT. In the rat brain, using 100 nM [D-Ala2-MePhe4,Gly-ol5]enkephalin to block [125I]IOXY binding to the kappa 2b site, we resolved two subtypes of the kappa 2a binding site. In the present study we examined the binding of [125I]IOXY to the kappa 2 receptors of guinea pig brain. As observed in rat brain, [125I]IOXY, under appropriate assay conditions, selectively labels kappa 2 binding sites. Quantitative binding studies readily demonstrated the presence of kappa 2a and kappa 2b binding sites. The kappa 2a binding sites were selectively assayed using 5 microM [Leu5]enkephalin to block [125I]IOXY binding to the kappa 2b sites, and kappa 2b sites were selectively assayed using 5 microM (-)-(1S,2S)-U50,488 to block [125I]IOXY binding to the kappa 2a sites. Under these conditions, two subtypes of the kappa 2a site were resolved with high (kappa 2a-1) and low (kappa 2a-2) affinity for nor-BNI (Ki values = 0.88 and 476 nM) and CI977 (Ki values = 17.5 and 95,098 nM). Similarly, two subtypes of the kappa 2b site were observed with high (kappa 2b-1) and low (kappa 2b-2) affinity for [D-Ala2-MePhe4,Gly-ol5]enkephalin (DAMGO) (Ki values = 97 and 12,321 nM) and alpha-neoendorphin (Ki values = 33 and 5308 nM). Two-site models were also resolved in the presence of 100 microM 5'-guanylyimidodiphosphate (GppNHp). We carried out detailed ligand selectivity analysis of the multiple kappa 2 binding sites. Most test agents were either nonselective or selective for the kappa 2a-1 site. Nalbuphine was moderately selective for the kappa 2a-2 site. Similarly, although most test agents were either nonselective or selective for the kappa 2b-1 site, butorphanol, and the delta antagonists naltrindole, naltriben, and 7-benzylidene-7-dehydronaltrexone were moderately selective for the kappa 2b-2 site.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure and synthesis of nectrisine, a new immunomodulator isolated from a fungus.

The structure of a novel immunomodulator, nectrisine (1), has been elucidated on the basis of chemical and spectroscopic evidence. Its absolute stereochemistry was predicted on the basis of the dibenzoate chirality rule and finally confirmed by a synthesis from D-glucose.