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Mitochondrial fragmentation from defective fusion or unopposed fission contributes to many neurodegenerative diseases. Small molecule mitofusin activators reverse mitochondrial fragmentation in vitro , promising a novel therapeutic approach. The first-in-class mitofusin activator, 2 , has a short plasma t 1/2 and limited neurological system bioavailability, conferring "burst activation". Here, pharmacophore-based rational redesign generated analogues of 2 incorporating cycloalkyl linker groups. A cyclopropyl-containing linker, 5 , improved plasma and brain t 1/2 , increased nervous system bioavailability, and prolonged neuron pharmacodynamic effects. Functional and single-crystal X-ray diffraction studies of stereoisomeric analogues of 5 containing sulfur as a "heavy atom", 14A and 14B , showed that 5 biological activity resides in the trans- R/R configuration, 5B . Structural analysis revealed stereoselective interactions of 5 associated with its mimicry of MFN2 Val372, Met376, and His380 side chains. Modification of murine ALS phenotypes in vitro and in vivo supports advancement of 5B for neurological conditions that may benefit from sustained mitofusin activation.

The exploitation of GLU988 and LYS903 residues in PARP1 as targets to design isoquinolinone (I & II) and naphthyridinone (III) analogues is described. Compounds of structure I have good biochemical and cellular potency but suffered from inferior PK. Constraining the linear propylene linker of structure I into a cyclopentene ring (II) offered improved PK parameters, while maintaining potency for PARP1. Finally, to avoid potential issues that may arise from the presence of an anilinic moiety, the nitrogen substituent on the isoquinolinone ring was incorporated as part of the bicyclic ring. This afforded a naphthyridinone scaffold, as shown in structure III. Further optimization of naphthyridinone series led to identification of a novel and highly potent PARP1 inhibitor 34, which was further characterized as preclinical candidate molecule. Compound 34 is orally bioavailable and displayed favorable pharmacokinetic (PK) properties. Compound 34 demonstrated remarkable antitumor efficacy both as a single-agent as well as in combination with chemotherapeutic agents in the BRCA1 mutant MDA-MB-436 breast cancer xenograft model. Additionally, compound 34 also potentiated the effect of agents such as temozolomide in breast cancer, pancreatic cancer and Ewing's sarcoma models., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

PI3Kδ inhibitors have been approved for B-cell malignancies like CLL, small lymphocytic lymphoma, and so forth. However, currently available PI3Kδ inhibitors are nonoptimal, showing weakness against at least one of the several important properties: potency, isoform selectivity, and/or pharmacokinetic profile. To come up with a PI3Kδ inhibitor that overcomes all these deficiencies, a pharmacophoric expansion strategy was employed. Herein, we describe a systematic transformation of a "three-blade propeller" shaped lead, 2,3-disubstituted quinolizinone 11 , through a 1,2-disubstituted quinolizinone 20 to a novel "four-blade propeller" shaped 1,2,3-trisubstituted quinolizinone 34 . Compound 34 has excellent potency, isoform selectivity, metabolic stability across species, and exhibited a favorable pharmacokinetic profile. Compound 34 also demonstrated a differentiated efficacy profile in human germinal center B and activated B cell-DLBCL cell lines and xenograft models. Compound 34 qualifies for further evaluation as a candidate for monotherapy or in combination with other targeted agents in DLBCLs and other forms of iNHL.

Mutations in the mitochondrial fusion protein mitofusin (MFN) 2 cause the chronic neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A), for which there is currently no treatment. Small-molecule activators of MFN1 and MFN2 enhance mitochondrial fusion and offer promise as therapy for this condition, but prototype compounds have poor pharmacokinetic properties. Herein, we describe a rational design of a series of 6-phenylhexanamide derivatives whose pharmacokinetic optimization yielded a 4-hydroxycyclohexyl analogue, 13 , with the potency, selectivity, and oral bioavailability of a preclinical candidate. Studies of 13 cis - and trans -4-hydroxycyclohexyl isostereomers unexpectedly revealed functionality and protein engagement exclusively for the trans form, 13B . Preclinical absorption, distribution, metabolism, and excretion (ADME) and in vivo target engagement studies of 13B support further development of 6-phenylhexanamide derivatives as therapeutic agents for human CMT2A.

A new series of potent and selective histamine-3 receptor (H3R) antagonists was identified on the basis of an azaspiro[2.5]octane carboxamide scaffold. Many scaffold modifications were largely tolerated, resulting in nanomolar-potent compounds in the H3R functional assay. Exemplar compound 6s demonstrated a selective profile against a panel of 144 secondary pharmacological receptors, with activity at only σ2 (62% at 10 μM). Compound 6s demonstrated free-plasma exposures above the IC50 (∼50×) with a brain-to-plasma ratio of ∼3 following intravenous dosing in mice. At three doses tested in the mouse novel object recognition model (1, 3, and 10 mg/kg s.c.), 6s demonstrated a statistically significant response compared with the control group. This series represents a new scaffold of H3 receptor antagonists that demonstrates in vivo exposure and efficacy in an animal model of cognition.

Herein, we describe the discovery of inhibitors of norepinephrine (NET) and dopamine (DAT) transporters with reduced activity relative to serotonin transporters (SERT). Two compounds, 8b and 21a, along with nomifensine were tested in a rodent receptor occupancy study and demonstrated dose-dependent displacement of radiolabeled NET and DAT ligands. These compounds were efficacious in a rat forced swim assay (model of depression) and also had activity in rat spontaneous locomotion assay.

The preclinical pharmacology and pharmacokinetic properties of (2R)-6-methoxy-8-(4-methylpiperazin-1-yl)-N-(4-morpholin-4-ylphenyl)chromane-2-carboxamide (AZD3783), a potent 5-hydroxytryptamine 1B (5-HT(1B)) receptor antagonist, were characterized as part of translational pharmacokinetic/pharmacodynamic hypothesis testing in human clinical trials. The affinity of AZD3783 to the 5-HT(1B) receptor was measured in vitro by using membrane preparations containing recombinant human or guinea pig 5-HT(1B) receptors and in native guinea pig brain tissue. In vivo antagonist potency of AZD3783 for the 5HT(1B) receptor was investigated by measuring the blockade of 5-HT(1B) agonist-induced guinea pig hypothermia. The anxiolytic-like potency was assessed using the suppression of separation-induced vocalization in guinea pig pups. The affinity of AZD3783 for human and guinea pig 5-HT(1B) receptor (K(i), 12.5 and 11.1 nM, respectively) was similar to unbound plasma EC(50) values for guinea pig receptor occupancy (11 nM) and reduction of agonist-induced hypothermia (18 nM) in guinea pig. Active doses of AZD3783 in the hypothermia assay were similar to doses that reduced separation-induced vocalization in guinea pig pups. AZD3783 demonstrated favorable pharmacokinetic properties. The predicted pharmacokinetic parameters (total plasma clearance, 6.5 ml/min/kg; steady-state volume of distribution, 6.4 l/kg) were within 2-fold of the values observed in healthy male volunteers after a single 20-mg oral dose. This investigation presents a direct link between AZD3783 in vitro affinity and in vivo receptor occupancy to preclinical disease model efficacy. Together with predicted human pharmacokinetic properties, we have provided a model for the quantitative translational pharmacology of AZD3783 that increases confidence in the optimal human receptor occupancy required for antidepressant and anxiolytic effects in patients.

We present a technique for computing activity discriminants of in vitro (pharmacological, DMPK, and safety) assays and the application to the prediction of in vitro activities of proposed synthetic targets during the lead optimization phase of drug discovery projects. This technique emulates how medicinal chemists perform SAR analysis and activity prediction. The activity discriminants that are functions of 6 commonly used medicinal chemistry descriptors can be interpreted easily by medicinal chemists. Further, visualization with Spotfire allows medicinal chemists to analyze how the query molecule is related to compounds tested previously, and to evaluate easily the relevance of the activity discriminants to the activities of the query molecule. Validation with all compounds synthesized and tested in AstraZeneca Wilmington since 2006 demonstrates that this approach is useful for prioritizing new synthetic targets for synthesis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We describe herein the discovery of novel, de novo designed, 5-HT(1B) receptor antagonists that lack a basic moiety and that provide improved hERG and in vitro phospholipidosis profiles. We used a known 5-HT(1B) antagonist template as our starting point and focused on replacing the piperazine moiety. Pyrazole-based ideas were designed and synthesized among a small library of piperazine replacements. To our knowledge, these are the first potent, nonbasic, functionally active antagonists of the 5-HT(1B) receptor.

Previously we reported on the synthesis and properties of a series of highly potent piperidinyl 2-subsituted-3-cyano-1-naphthamide NK1 antagonists that includes 3 and 4. Here we report our efforts to alleviate a troublesome atropisomeric property of those derivatives by introduction of a tethering bridge that, in addition, could be used to lock the resulting cyclic derivatives in a purported NK1 pharmacophore conformation. Using 3 as a starting point, the naphtho[2,1-b][1,5]oxazocine, 17, was found to contain the optimal ring tether size (8) for retaining NK1 activity, was more NK1 versus NK2 selective, and reduced the number of atropisomers from four to two. Cyclic derivatives 29 and 32, which exist as essentially single atropisomers in the purported pharmacophore conformation, were prepared in the closely related naphtho[1,2-f][1,4]oxazocine series as part of an effort to use mono methyl substitution of the tethering bridge as a conformation stabilizing factor. Both 29 and 32 were found to be less active as NK1 antagonists than the non-methylated parent 28 possibly due to methyl group destabilization of receptor interaction. We discuss the above findings in the context of a previously proposed NK1 pharmacophore model and present a further refinement of that model.

We have previously described a series of antagonists that showed high potency and selectivity for the NK(1) receptor. However, these compounds also had the undesirable property of existing as a mixture of interconverting rotational isomers. Here we show that alteration of the 2-naphthyl substituent can modulate the rate of isomer exchange. Comparisons of the NK(1) receptor affinity for the various conformational forms has facilitated the development of a detailed NK(1) pharmacophore model.

Through optimization of compounds based on the dual NK(1)/NK(2) antagonist ZD6021, it was found that alteration of two key regions could modulate the balance of NK(1) and NK(2) potency. Substitution of the 2-naphthalene position in analogues of ZD6021 resulted in increased NK(1) potency and thus afforded NK(1) preferential antagonists. Alterations of the piperidine region could then increase NK(2) potency to restore dual NK(1)/NK(2) selectivity. Through these efforts, three novel receptor antagonists from a single chemically related series were identified; two are dual NK(1)/NK(2) antagonists, and the third is an NK(1) preferential antagonist. In this paper, the factors affecting the balance of NK(1) and NK(2) selectivity in this series are discussed and the in vitro and in vivo properties of the novel antagonists are described.

Exploration of the SAR around selective NK2 antagonists, SR48968 and ZD7944, led to the discovery that naphth-1-amide analogues provide potent dual NK1 and NK2 antagonists. ZD6021 inhibited binding of [3H]-NKA or [3H]-SP to human NK1 and NK2 receptors, with high-affinity (K(i)=0.12 and 0.62nM, respectively). In functional assays ZD6021 had, at 10(-7)M, in human pulmonary artery pK(B)=8.9 and in human bronchus pK(B)=7.3, for NK1 and NK2, respectively. Oral administration of ZD6021 to guinea pigs dose-dependently attenuated ASMSP induced extravasation of plasma proteins, ED(50)=0.5mg/kg, and NK2 mediated bronchoconstriction, ED(50)=13mg/kg.

The tachykinins, substance P, neurokinin A, and neurokinin B, have been implicated in many diseases. The present study evaluated the pharmacological properties of a novel tachykinin antagonist ZD6021 [3-cyano-N-((2S)-2-(3,4-dichlorophenyl)-4-[4-[2-(methyl-(S)-sulfinyl)-phenyl]piperidino]butyl)-N-methyl-]-napthamide]. The affinity (K(i)) of ZD6021 for the cloned human neurokinin (NK)1, NK2, and NK3 receptors was 0.12 +/- 0.01, 0.64 +/- 0.08, and 74 +/- 13 nM, respectively. Mucin secretion by Chinese hamster ovary cells transfected with the human NK1 receptor was dose dependently inhibited by ZD6021: pIC(50) = 7.6 +/- 0.1. For NK1 and NK2 receptors, the agonist concentration-response curves using isolated tissues were displaced rightward in the presence of ZD6021: rabbit pulmonary artery, pA2 = 8.7 and 8.5; human pulmonary artery and bronchus, pKB = 8.9 +/- 0.4 and 7.5 +/- 0.2, at 10(-7) M, respectively. Senktide-induced contractions of isolated guinea pig ileum were also blocked by low concentrations of ZD6021. Oral administration of ZD6021 to guinea pigs dose dependently attenuated tracheal extravasation of plasma proteins induced by the NK1 receptor agonist Ac-[Arg6,Sar9,Met(O2)11]-SP(6-11), ED50 = 0.8 micromol/kg, and bronchoconstriction, elicited by the NK2 receptor agonist [beta-Ala8]-NKA(4-10), ED50 = 20 micromol/kg. Potency was unaffected by feeding. After oral administration of ZD6021, the time to peak activity was 150 min for the NK1 receptor and 60 min for the NK2 receptor with pharmacodynamic half-lives of 280 and 458 min, respectively. These data indicate that ZD6021 is a potent, orally active antagonist of all three tachykinin receptors. This compound may be useful for future studies of tachykinin-related pathology such as asthma.

Neutrophils release elastase, which is known secondarily to cause tissue damage. However, it is rapidly inactivated by the endogenous alpha1-proteinase inhibitor (alpha1Pi). Nevertheless, under pathological conditions, alpha1i is inactivated by oxidants released from neutrophils, resulting in an excess of elastase at the site of inflammation. This elastase/alpha1Pi imbalance has been implicated as a pathogenic factor in cystic fibrosis, acute respiratory distress syndrome, and emphysema. Elastase inhibitors, which do not interfere with the microbicidal activity of neutrophils and are resistant to neutrophil-released oxidants, would undoubtedly represent an important advance in the management of neutrophil-mediated tissue injury. We report that a new family of elastase inhibitors ICI200355 and ZD0892 was found to be resistant toward superoxide, hypochlorous acid, hydrogen peroxide, hydroxyl radical, and peroxynitrite mediated degradation as well as having no effect on the formation of these oxidants by activated neutrophils. More importantly, we found that these inhibitors did not interfere with the ability of human neutrophils to phagocytose and to kill Staphylococcus aureus. In conclusion, a new potent class of elastase inhibitors, while blocking the effects of neutrophil elastase, was found not to impede various physiological functions of human neutrophils, in particular the ability of these phagocytic cells to phagocytose and kill bacteria.

Several strategies have been employed by medicinal chemists in the design of potent and selective leukotriene receptor antagonists-leukotriene structural analogs, FPL 55712 analogs, and random screening of corporate compound banks. Lead compounds were optimized, often through the exchange of ideas with groups working on other chemical series of leukotriene antagonists. Pranlukast can likely be traced to a lead compound identified by random screening that was initially modified by incorporating structural components present in FPL 55712. Montelukast originated from an early quinoline lead, which was modified with leukotriene structural elements. Zafirlukast is based on a lead compound that incorporated structural components from both FPL 55712 and the leukotrienes. Therefore, each medicinal chemistry strategy that was originally employed has successfully identified clinically effective leukotriene receptor antagonists. Bernstein PR. Chemistry and structure-activity relationships of leukotriene receptor antagonists.

Several strategies have been employed by medicinal chemists in the design of potent and selective leukotriene receptor antagonists-leukotriene structural analogs, FPL 55712 analogs, and random screening of corporate compound banks. Lead compounds were optimized, often through the exchange of ideas with groups working on other chemical series of leukotriene antagonists. Pranlukast can likely be traced to a lead compound identified by random screening that was initially modified by incorporating structural components present in FPL 55712. Montelukast originated from an early quinoline lead, which was modified with leukotriene structural elements. Zafirlukast is based on a lead compound that incorporated structural components from both FPL 55712 and the leukotrienes. Therefore, each medicinal chemistry strategy that was originally employed has successfully identified clinically effective leukotriene receptor antagonists.

This paper describes the development a series of peptidyl trifluoromethyl ketone inhibitors of human leukocyte elastase which are found to have excellent pharmacological profiles. Methods have been developed that allow for the synthesis of these inhibitors in stereochemically pure form. Two of these compounds, 1k and 1l, have high levels of oral bioavailability in several species. Compound 1l has entered development as ZD8321 and is presently undergoing clinical evaluation. These compounds demonstrate that peptidyl trifluoromethyl ketone inhibitors can achieve high levels of oral activity and bioavailability, and therefore they may prove useful as therapeutic agents in the treatment of diseases in which elastase is implicated.

The effects of changes in substitution in a series of 5-amino-2-pyrimidin-6-ones on both in vitro activity and oral activity in an acute hemorrhagic assay have been explored. These compounds contained either a trifluoromethyl ketone or a boronic acid moiety to bind covalently to the Ser-195 hydroxyl of human leukocyte elastase (HLE). Boronic acid-containing inhibitors were found to be more potent than the corresponding trifluoromethyl ketones in vitro but were less active upon oral administration. Compound 13b was found to offer the best combination of oral potency, duration of action, and enzyme selectivity and, as such, was selected for further biological testing. X-ray crystallography of a cocrystallized complex of compound 19m and porcine pancreatic elastase demonstrated that the inhibitor is bound to the enzyme in a manner similar to that found previously for a closely related series of pyridone-containing inhibitors of HLE.

Further modification of the 3-amino substituent in a trifluoromethyl ketone-based series of 3-amino-6-phenylpyridin-2-ones that had been optimized for oral activity led to analogs that were potent intratracheal inhibitors in a model of HLE-induced lung damage in the hamster. The best 3-amino substituent for intratracheal activity is [4-[N-[(4-chlorophenyl)sulfonyl]-carbamoyl]phenyl]sulfonyl. At a 30 min prechallenge interval, compound 9, which incorporates this substituent, had an ED50 of approximately 2 nmol/animal and, qualitatively, afforded a very similar dose-response relationship to that found with a peptidic trifluoromethyl ketone inhibitor, ICI 200,355.

A series of nonpeptidic inhibitors of human leukocyte elastase (HLE) is reported. These trifluoromethyl ketone-based inhibitors contain a 3-amino-6-phenylpyridone group as a central template. The effect of varying the N-3 substituent in these inhibitors on in vitro potency, physical properties, and oral activity in a hamster based, HLE-induced lung damage model is described. The variety of substituents at this position that have little effect on in vitro potency supports the idea that this region of the molecule does not interact strongly with the enzyme. One exception to this generality is 13k, which is substituted with a (4-acetamidophenyl)sulfonyl group. This compound has a K(i) of 0.7 nM and is, in vitro, the most potent inhibitor in the series. In contrast, variation of the N-3 substituent was found to have a dramatic effect on activity after oral administration. Several analogs, including the parent amine, 7, formamide, 2u, benzyl sulfamide, 13e, and benzyl sulfonamide, 13f, show significant activity when administered at an oral dose of 2.5 mg/kg. Support for the modeling-based design concepts was obtained through in vitro SAR results and X-ray crystallographic analysis of the complex between 13d and porcine pancreatic elastase (PPE), a closely related enzyme.

The continued exploration of a series of 3-(arylmethyl)-1H-indole-5-carboxamides by the introduction of fluorinated amide substituents has resulted in the discovery of 4-[[5-[((2R)-2-methyl-4,4,4-trifluorobutyl)carbamoyl]-1-methyli ndol- 3-yl]methyl]-3-methoxy-N-[(2-methyl-phenyl)sulfonyl]benzamide (38p, ZENECA ZD3523), which has been chosen for clinical evaluation. This compound exhibited a Ki of 0.42 nM for displacement of [3H]LTD4 on guinea pig lung membranes, a pKB of 10.13 +/- 0.14 versus LTE4 on guinea pig trachea, and an oral ED50 of 1.14 mumol/kg opposite LTD4-induced bronchoconstriction in guinea pigs. The R enantiomer was found to be modestly more potent than the S enantiomer 38o. Modification of the amide substituent to afford achiral compounds was unsuccessful in achieving comparable levels of activity. Profiling of 38p opposite a variety of functional assays has demonstrated the selectivity of this compound as a leukotriene receptor antagonist. The enantioselective synthesis of 38p, which employed a diastereoselective alkylation of (4R,5S)-3-(1-oxo-4,4,4-trifluorobutyl)-4-methyl-5-phenyl-2-oxazoli dinone (27) as the key step to establish the chirality of the amide substituent, provided an efficient route for generating 38p in > 99% enantiomeric purity.

For more than two decades investigators around the world, in both academic and industrial institutions, have been developing inhibitors of human neutrophil elastase. A number of very elegant and insightful strategies have been reported. In the case of reversible peptidic inhibitors, this has resulted in the identification of some extremely potent compounds with dissociation constants in the 10(-11) M range. This is quite an accomplishment considering that these low molecular-weight inhibitors are only tri- and tetrapeptides. In the case of the heterocyclic-based inhibitors, the challenge of balancing the heterocycle's inherent reactivity and aqueous stability with the stability of the enzyme-inhibitor adduct has been meet by either using a latent, reactive functionality which is only activated within the enzyme, or by incorporating features which selectively obstruct deacylation but have little effect on the enzyme acylation step. The underlying goal of this research has been the identification of agents to treat diseases associated with HNE. Several animal models have been developed for evaluating the in vivo activity of elastase inhibitors, and compounds have been shown to be effective in all of these models by the intravenous, intratrachael or oral routes of administration. However, only a very small percentage of compounds have possessed all the necessary properties, including lack of toxicity, for progression into the clinic. The peptidyl TFMK ICI 200,880 (25-12) has many of the desired characteristics of a drug to treat the diseases associated with HNE: chemical stability, in vitro and in vivo activity, a long duration of action, and adequate metabolic stability. Currently ICI 200,880 is the only low molecular-weight HNE inhibitor known to be undergoing clinical trials, and may be the compound which finally demonstrates the clinical utility of a synthetic HNE inhibitor.

A systematic structure-activity exploration of the carboxylic acid region in a series of indole- or indazole-derived leukotriene antagonists 1 led to several discoveries. Use of the 3-methoxy-p-tolyl fragment (illustrated in acid 1) for connecting the indole and the acidic site provides the most potent carboxylic acids 1, tetrazoles 20, and aryl sulfonimides 21. The aryl sulfonimides are 5-500 times more potent (in vitro and/or in vivo) than the corresponding carboxylic acids 1. The o-tolyl sulfonimides such as 114 show greater oral potency than the phenyl sulfonimides at a given level of in vitro activity. Acidic keto sulfone derivatives 10 (Nu = CH-(CO2CH3)SO2Ph) mimic the activity of the sulfonimides.

The dissociation constants (KB) at the LTD4 receptor on guinea pig trachea of a series of monocyclic and bicyclic cyclopentylurethane and cyclopentylacetamide N-arylsulfonyl amides have been measured. The KB was found to be remarkably tolerant of changes in the electronic constitution and lipophilicity of the bicyclic ring system (template). Thus, N-[4[[6-[[(cyclopentyloxy)carbonyl]amino]benzimidazol-1- yl]methyl]-3-methoxybenzoyl]benzenesulfonamide (11a) and N-[4-[[5-[[(cyclopentyloxy)carbonyl]amino]benzo[b]thien-3- yl]methyl]-3-methoxybenzoyl]benzene-sulfonamide (25a) had closely similar affinities (pKB, 9.20 and 9.31, respectively; LTE4 as agonist). It has been shown that the hetero-ring of the template need not be aromatic in order to achieve high affinity, since indoline 31 and 2,3-dihydrobenz-1,4-oxazines 37a-c had pKBs greater than 9. Further, it has been shown that an o-aminophenone (see 42 and Figure 3) can function as a template; the template in 42 [see iii] is bicyclic by virtue of the presence of an intramolecular hydrogen bond. In contrast, when the template is a phenyl ring (48), receptor affinity is markedly reduced. These findings support the notion that central bicyclic ring system in this family of peptidoleukotriene antagonists is a molecular feature which helps to preorganize the acylamino and acidic chains and thereby facilitate the molecular recognition event.

ICI 204,219 (4-(5-cyclopentyloxycarbonylamino-1-methylindol-3-ylmethy l)-3- methoxy-N-o-tolylsulfonylbenzamide) was designed as a peptide leukotriene (LT) antagonist. The compound is a competitive antagonist of LTD4- and LTE4-induced contraction of guinea pig lung tracheal and parenchymal strips with an apparent negative log molar dissociation constant (KB) of approximately 9.6. ICI 204,219 did not antagonize LTC4-induced contractions of guinea pig trachea when the metabolism of LTC4 to LTD4 and, subsequently, to LTE4 was inhibited. The compound inhibited the binding of [3H]LTD4, [3H]LTE4, and [3H]ICI 198,615 (a potent LT antagonist from a different heterocyclic series) to guinea pig lung parenchymal membranes in a competitive manner, and also inhibited [3H]ICI 198,615 binding to human lung parenchymal membranes. ICI 204,219 did not bind to a variety of other receptors when evaluated at concentrations 1,000- to 10,000-fold higher than the apparent KB value for peptide LT receptors. When administered orally, intravenously, or by aerosol, the compound provided dose-related antagonism of the airway effects of aerosol LTD4 in conscious guinea pigs. ED50 values and pharmacodynamic t1/2 (min) for oral, intravenous and aerosol routes of administration were, respectively: 0.52 mumol/kg, greater than 816 min; 0.046 mumol/kg, 85 min; 5.1 x 10(-6) M, 109 min. ICI 204,219 also produced dose-related inhibition of the effects of LTC4 (aerosol or intravenous administration) on pulmonary mechanics in anesthetized guinea pigs when administered orally, intraduodenally, intravenously, or by aerosol. The compound also reversed bronchospasm produced by LTs. Aerosol ovalbumin antigen-induced bronchospasm in guinea pigs was both inhibited and reversed by ICI 204,219. Lastly, the compound inhibited LTD4-induced increases in cutaneous vascular permeability in guinea pigs, being 1,006- and 679-fold more potent than the first generation LT antagonists LY 171,883 and FPL 55712, respectively. ICI 204,219 is a potent, selective, orally active LT antagonist currently undergoing clinical trials.

The synthesis and biological characterization of a series of novel leukotriene antagonists and agonists are reported. All of these compounds are derivatives of (5S,6R,7Z)-5-hydroxy-6-mercapto-9-phenyl-7-nonenoic acid. One of the more potent compounds is (5S,6R,7Z)-6-[[(4-carboxy-2-methoxyphenyl)methyl]thio]-5-hydroxy-9 -(4- heptylphenyl)-7-nonenoic acid (3f). In vitro evaluation of this compound on guinea pig trachea revealed that it is a competitive antagonist of LTD4 and LTE4 with pKB values of 6.4 and 5.8, respectively. On guinea pig ileum, the pKB values obtained for it with LTD4 and E4 were both 7.2. The selectivity of 3f was shown by its lack of effect on carbachol, histamine, and barium chloride concentration-response curves in guinea pig trachea.

We designed studies to determine if Leukotriene D4 (LTD4) could alter airway reactivity such that rhesus monkeys with positive skin reactivity and consistently negative airway responses would respond to ascaris airway challenge. The experiments were complicated by the observation that aerosolized LTD4 would occasionally increase airway hyperreactivity in some monkeys used as controls such that an airway response occurred to saline, the diluent for ascaris antigen. In spite of this, we were able to demonstrate induction of airway responsiveness to ascaris antigen. These results demonstrate that LTD4 will induce airway hyperreactivity to a nonspecific stimulus such as aerosolized saline or to an allergen.

Rhesus monkeys with IgE-mediated asthmatic-type responses to Ascaris suum antigen were pretreated with a combination of 2 receptor antagonists to leukotriene D4 (LTD4) and platelet-activating factor (PAF). This combination of anti-LTD4 and anti-PAF was shown to inhibit airway responses to either LTD4 or PAF in separate experiments in these Ascaris airway-reactive animals. When the same combination of LTD4 and PAF receptor antagonists was used to pretreat the same animal prior to aerosol challenge with Ascaris antigen we could not demonstrate inhibition of the antigen-induced airway response. Thus, combined receptor blockade for LTD4 and PAF did not alter IgE-mediated acute airway responses to antigen in this species. We review the current status of rhesus asthma and LTD4 and PAF receptor antagonist activity in this model.

Leukotriene (LT) D4 is a potent constrictor of human airways and a putative mediator of asthma. However, its mechanism of action in man has not been established. In the present study we sought to determine the role of upper airway reflexes, cholinergic pathways, and cyclooxygenase products of arachidonic acid metabolism in mediating LTD4-induced bronchoconstriction in man. Six normal subjects underwent bronchoprovocation testing with LTD4 after pretreatment with either aerosolized phosphate-buffered saline, aerosolized atropine (1.5 mg), aerosolized lidocaine (80 to 160 mg), or oral indomethacin (50 mg three times daily for 10 doses). Specific airway conductance (SGaw), the flow rate at 30% of vital capacity from a partial forced expiratory maneuver (V30P), and the FEV1 were measured at each concentration of LTD4. We calculated the provocative concentration of LTD4 required to produce a 35% fall in SGaw (PC35SGaw) or a 30% fall in V30P (PC30V30P) and the slope of the LTD4 dose-response curve. Atropine increased baseline SGaw 49% (p less than 0.01) and V30P 43% (p less than 0.005). Atropine also increased the PC35SGaw and PC30V30P and increased the slope of the LTD4 dose-response curve. However, neither of these opposing effects was significant. Lidocaine had no effect on either baseline function or the airway response to LTD4. Indomethacin produced small decreases in baseline V30P (12%) and FEV1 (3%) (p less than 0.05 for both), but it too had no effect on the airway response to LTD4. These results indicate that the bronchoconstriction produced by aerosolized LTD4 in normal subjects is not mediated by cyclooxygenase products of arachidonic acid metabolism or irritant receptors in the upper airways. Although cholinergic pathways may play some role, the data suggest that bronchoconstriction results, at least in part, from a direct effect of LTD4 on airway smooth muscle.

Contractile responses to leukotriene (LT)C4, LTD4 and LTE4 were examined in intralobar airways from human lung obtained after surgical resection. In addition, the ability of the LT receptor antagonist FPL55712 to antagonize responses to LTC4 and LTD4 was quantified (by calculating -log molar KB values) under experimental conditions designed to minimize metabolic transformation of the LTs. In the absence of drug pretreatment, the three peptide LTs were approximately equipotent and produced similar maximum degrees of contraction. L-Serine borate complex, 45 mM, used as an inhibitor of the degradation of LTC4 to LTD4 by the enzyme gamma-glutamyl transpeptidase, in paired airway segments (adjacent segments from the same branch), produced a small degree (about 3-fold) of shift to the right of the dose-response curve and reduction of the maximum response to LTC4. L-Cysteine, 3 mM, used as an inhibitor of the degradation of LTD4 to LTE4 by the enzyme aminopeptidase, in paired segments, did not alter the dose-response effects of LTD4 or appear to further alter the dose-response effects of LTC4 when applied together with L-serine borate complex in unpaired (nonadjacent) segments. The -log molar KB value for FPL55712 (about 6) was similar for antagonism of responses to both LTC4 and LTD4 in the absence or presence of treatment with the metabolic inhibitors L-serine borate complex, L-cysteine or a combination of the two treatments. The results suggest that inhibition of the enzymes involved in the pathway from LTC4 to LTE4 has little consequence in human airways because the three peptide LTs are approximately equipotent.(ABSTRACT TRUNCATED AT 250 WORDS)

To better understand the role of leukotriene D4 (LTD4) in the pathogenesis of airway hyperreactivity, we administered aerosolized LTD4 on multiple occasions to 6 normal subjects and measured specific airway conductance (SGaw) by body plethysmography and the flow rate at 30% of vital capacity from partial forced expiratory maneuvers (V30P). Dose-response curves generated from standard bronchoprovocation tests revealed that LTD4 was 280 to 590 times more potent as a bronchoconstrictor than was methacholine. The airway response to single bronchoconstricting doses of LTD4 administered on separate days was reproducible for SGaw (r = 0.86, p less than 0.05) and V30P (r = 0.95, p less than 0.005). The response to 3 consecutive single-dose LTD4 challenges performed on the same day, allowing SGaw and V30P to return to greater than 90% of the control value between challenges, demonstrated tachyphylaxis: the response to the third dose was significantly less than the response to the first dose for SGaw (p less than 0.001) and for V30P (p less than 0.01). A single bronchoconstricting dose of LTD4, given before a standard methacholine dose-response challenge, lowered the concentration of methacholine needed to decrease V30P 30% (p less than 0.05) and SGaw 35% (p = 0.09). A single bronchoconstricting dose of methacholine preceding a standard methacholine challenge did not have this effect. LTD4 and methacholine demonstrated a cumulative dose-effect when progressively higher concentrations were inhaled. In summary, the airway response to LTD4 is reproducible, exhibits tachyphylaxis, and a cumulative dose-effect occurs. As well, LTD4 increases the responsiveness of normal airways to methacholine.

The synthesis and biological characterization of a series of stable leukotriene analogues (2) are reported. They are derivatives of (5S,6R,7Z)-6-peptidyl-5-hydroxy-9-phenyl-7-nonenoic acid, in which the phenyl group is variously substituted with a heptanyl, 2-heptenyl, or hexanyloxy chain (R1) and the peptide is either glutathionyl, cysteinylglycinyl, or cysteinyl. The most potent agonist is (5S,6R,7Z)-6-S-glutathionyl-5-hydroxy-9-(4-heptanylphenyl)-7 -nonenoic acid. This analogue has an EC50 value of 74.5 nM, in the presence of 1-serine borate (45 mM), on guinea pig tracheal spirals. The agonist activity of the cysteinylglycinyl- and the cysteinyl-substituted analogues was inhibited by FPL-55712. Three of the analogues were weak leukotriene antagonists in vitro on guinea pig tracheal spirals. The most potent of these was (5S,6R,7Z)-6-S-cysteinyl-5-hydroxy-9-(2-heptanylphenyl)-7-++ +nonenoic acid. At 10 microM, this analogue inhibited by 28% the contraction induced by 8 nM LTE4.

A sensitive radioimmunoassay for leukotrienes (LTs) has been developed. Rabbits were immunized with a conjugate of LTD4 and bovine serum albumin, prepared by using 1,5-difluoro-2,4-dinitrobenzene as the coupling agent. The assay can detect 0.045 pmol LTD4 at a final plasma dilution of 1:72. 50% displacement of bound 3H-LTD4 was obtained with 0.43 +/- 0.03 pmol LTD4. LTC4, LTE4 and LTF4 cross-react 159%, 57% and 85%, respectively, whereas LTB4, 5-HETE and prostaglandins did not. The assay was validated by measuring the antigen-induced release of LTs from sensitized guinea-pig chopped lungs. High correlation (0.9434, p less than 0.05) was found when LTs were simultaneously determined by this assay and a bioassay on guinea pig ileum.

Peptide leukotrienes are potent constrictors of airway smooth muscle but lack chemical stability. Replacement of the natural triene backbone of leukotrienes with 9-(x-heptylphenyl)-7-nonenoic acid (x = 2, 3, or 4) renders these analogs chemically stable and pharmacologically active. The para, meta and ortho substitutions of the heptyl (C7H15) moiety were combined with the different leukotriene peptide substitutions of glutathionyl, C-peptide of leukotriene C4, cysteinylglycinyl, D-peptide of leukotriene D4, and cysteinyl, E-peptide of leukotriene E4 rendering nine active analogs. The pharmacology of these analogs was evaluated in isolated guinea-pig tracheal strips. The para-substituted C-peptide analog was the most potent; however, it was 100-fold less potent than leukotriene C4. The contractile activities of the para- and meta-substituted C-peptide analogs were enhanced by L-serine borate (45 mM), indicating they were substrates for gamma-glutamyl transpeptidase. FPL55712 (10 microM) failed to antagonize the substituted C-peptide analogs independent of the presence of L-serine borate. The contractile activities of the three substituted D-peptide analogs were enhanced by L-cysteine, indicating they are all substrates for aminopeptidase. The para-substituted D-peptide analog was the most potent D-peptide analog, but it was 10- and 1,000-fold less active than the para-substituted C-peptide analog and leukotriene D4, respectively. The para- and meta-substituted E-peptide analogs were approximately 1,000-fold less potent than leukotriene E4, and like the substituted D-peptide analogs, they were antagonized by FPL55712. In contrast, the ortho-substituted E-peptide analog was devoid of intrinsic activity but antagonized leukotriene E4-induced contractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Considerations of the possible similarities between leukotriene D4 and its prototypical antagonist, FPL 55712, led to the development of a new series of leukotriene antagonists incorporating a hydroxyacetophenone group (e.g., the toluic acids 16 and 18). Although considerable attention has focused on FPL 55712-derived analogues, only limited investigations into alternatives for the standard 4-acetyl-3-hydroxy-2-propylphenoxy moiety have been reported. Therefore, an extensive study of modifications to the hydroxyacetophenone portion of toluic acid 18 was undertaken. Although no viable alternative to the 3-hydroxy moiety was discovered, replacements for the 2-propyl group (34, 37) and the 4-acetyl functionality (56, 59) yielded potent antagonists. A number of compounds exhibited longer duration of action in vivo than FPL 55712.

Leukotriene D, (LTD4) is a potent bronchoconstrictor that may play an important role in asthma. To better characterize the effect of LTD4 on human airways, normal, rhinitic, and asthmatic subjects underwent inhalation challenge tests with LTD4 and methacholine. In all subjects increasing concentrations of LTD4 produced a parallel decrease in specific airway conductance and flow at 30% of vital capacity measured from a partial forced expiratory maneuver (V30P). These changes occurred independent of any decrease in FEV1 or increase in functional residual capacity. The airway response to LTD4 was maximal within 2 to 3 min, persisted for approximately 30 min, and resolved over 1 to 3 h. Late effects were not seen. Rhinitic subjects were 3 to 5 times (p less than 0.01) and asthmatic subjects were 25 to 100 times (p less than 0.001) more sensitive than were normal subjects to LTD4. All 3 groups of subjects were 250 to 850 times more sensitive to LTD4 than to methacholine. Significant correlations were found between the concentration of LTD4 and the concentration of methacholine that produced a 35% decrease in specific airway conductance (r = 0.874; p less than 0.006) and a 30% decrease in V30P (r = 0.751; p less than 0.04). These results confirm that LTD4 is a potent bronchoconstrictor in humans, with a rapid onset of action and a prolonged effect. In contrast to previous reports, we found that LTD4 has an equal effect on both large and small airways and that both asthmatic and normal subjects have the same degree of increased sensitivity to LTD4 as to methacholine.

This study examined both TMJ and normal groups for any changes in the masseteric silent period with variation of the subjects' occlusal force. An analog meter was used to give each subject feedback on how hard the teeth were clenched together. Five silent periods were elicited at each of the four levels of occlusal force from 40% to 100% of maximum force. Results showed a small decrease in silent period duration for both groups when occlusal force was increased.

Chemically stable analogs of peptide leukotrienes (LT) have been developed in our laboratories by replacement of the natural triene backbone with a C7H15 substituted aromatic moiety (1). These analogs are potent agonists of airway smooth muscle. Substitution in the peptide region resulted in U19052, an LT receptor antagonist. U19052 antagonized LT-induced contractions of guinea-pig tracheal spirals in a concentration-related manner. The pA2 values versus LTD4 and LTE4 were 6.0 and 5.7, respectively, with slopes which were not significantly different from unity. LTC4-induced contractions were antagonized by U19052 with a pKB of 5.6 obtained either in the absence or presence of L-serine borate. In contrast, carbachol and histamine concentration-response curves were not altered by U19052. LTD4 or LTE4 contractions of isolated guinea-pig ileum were antagonized by U19052 with pKB values of 7.2. The results indicate that potent selective LT antagonists can be developed from stable analogs of leukotrienes. U19052, an example of this series, appears to be as effective in antagonizing LTC4- as well as LD4- and LTE4-induced contractions in guinea-pig tracheal spirals.

We studied the effect of leukotriene D4(LTD4) given by aerosol challenge on asthmatic and normal monkeys. The response to LTD4 occurred within 2 min., was prolonged at higher concentrations, was reversed by epinephrine, and more closely resembled an antigen response than a histamine response. Since LTD4 concentrations of 30-100 micrograms/ml produced a response similar to 2-20 mg/ml of histamine, the LTD4 was 300-900 times as potent as histamine on a molar basis. Thus, LTD4, which appears to reproduce an antigen-induced response in rhesus monkeys, may be a mediator of allergic asthma in these animals.

Leukotriene D4 and synthetic platelet-activating factor have been shown to qualitatively reproduce an antigen-induced airway response in rhesus monkeys at concentrations several hundred times less than histamine. In normal rhesus monkeys, challenges with concentrations of leukotriene D4 or platelet-activating factor, established as subthreshold doses (not producing a response), result in abnormal pulmonary function parameters when followed by a second challenge of a subthreshold dose. Results were similar with any sequence of leukotriene D4 and platelet-activating factor combination. This was not a result of additive effects of agonists, because previous experiments have shown that log or 0.5 log increases over the subthreshold dose are necessary for a response. Our results may suggest that these mediators that possess remarkable biologic activity might sensitize the lower airway for response to stimulation with the same or other mediators.