Calmodulin-binding peptide PEP-19 modulates activation of calmodulin kinase II In situ.

PEP-19 is a 6 kDa polypeptide that is highly expressed in select populations of neurons that sometimes demonstrate resistance to degeneration. These include the granule cells of the hippocampus and the Purkinje cells of the cerebellum. Its only identified activity to date is that of binding apo-calmodulin. As a consequence, it has been demonstrated to act as an inhibitor of calmodulin-dependent neuronal nitric oxide synthase in vitro, although PEP-19 regulation of calmodulin-dependent enzymes has never been characterized in intact cells. The activation of the calmodulin-dependent enzyme calmodulin kinase II (CaM kinase II) was studied in PC12 cells that had been transfected so as to express physiological levels of PEP-19. The expression of PEP-19 yielded a stable phenotype that failed to activate CaM kinase II upon depolarization in high K(+). However, CaM kinase II could be fully activated when calcium influx was achieved with ATP. The effect of PEP-19 on CaM kinase II activation was not attributable to changes in the cellular expression of calmodulin. The cellular permeability of the transfected cells to calcium ions also appeared essentially unchanged. The results of this study demonstrated that PEP-19 can regulate CaM kinase II in situ in a manner that was dependent on the stimulus used to mobilize calcium. The selective nature of the regulation by PEP-19 suggests that its function is not to globally suppress calmodulin activity but rather change the manner in which different stimuli can access this activity.

The signal transduction system of the leukotriene D4 receptor.

During the past several years, substantial progress in understanding the receptors and signal transduction processes for peptidyl leukotrienes has been reported. Receptors have been identified and characterized, the major steps in the signal transduction pathway have been described, and the genetic and epigenetic regulatory processes have been characterized. Very recent studies have defined the mechanisms by which LTE4 acts as a partial agonist at the LTD4 receptor. The cloning of the genes for the proteins involved in the major steps of the signalling process has also been initiated. Stanley Crooke and co-authors summarize this recent progress and present their current notions about the LTD4 receptor signalling process.

Inhibitors of protein kinase C selectively enhanced leukotriene D4-induced calcium mobilization in differentiated U-937 cells.

U-937 cells differentiated with dimethylsulphoxide for 3-4 days express receptors for leukotriene D4 (LTD4), which are coupled to Ca2+ mobilization and phosphatidylinositol (PI) metabolism. Treatment of U-937 cells with an inhibitor of protein kinase C (PKC) [staurosporine (100 nM)] augmented the Ca2+ mobilized by LTD4. The peak concentration of the LTD4-induced increase in [Ca2+]i was 1500 nM in untreated cells and 3000 nM in cells treated with staurosporine for 30 s. Maximal mobilization responses were observed at 1-10 microM LTD4 in both control and staurosporine-treated cells. The increased Ca2+ response to LTD4 after staurosporine treatment occurred within 30 s and was attributable to both intracellular and extracellular stores. Additionally, a second phase of Ca2+ mobilization occurred after stimulation with LTD4, which was elevated by pretreatment with staurosporine--this effect was maximal after 5-10 min of treatment. Staurosporine either had no effect or decreased the Ca2+ mobilization response of differentiated U-937 cells to other agonists, such as LTB4, platelet activating factor, ATP or the chemotactic peptide f-Met-Leu-Phe. Although staurosporine alone had no effect on basal PI metabolism it increased LTD4-induced PI metabolism. Staurosporine did not prevent the tachyphylaxis observed upon second challenge with LTD4, nor did it prevent LTD4-induced homologous densensitization. Other compounds which inhibit PKC (sphingosine and 1-O-hexadecyl-2-O-methylglycerol), also enhanced the Ca2+ response of U-937 cells to LTD4, but not to other agonists. These data show that inhibition of PKC enhanced responses of LTD4, suggesting that PKC plays a role in determining the responsiveness of LTD4 receptors.

Interleukin-8 receptor antagonists in pulmonary diseases.

Inflammatory cells are thought to be instrumental in the pathophysiology of pulmonary diseases, and control of their recruitment and activation in the lung would appear to be an attractive strategy for therapeutic intervention. Interleukin-8 and related CXC chemokines are involved in the function of neutrophils and T cells, and have been implicated in several lung diseases. Small-molecule antagonists of the interleukin-8 receptors have been identified, which may help elucidate the role of interleukin-8 and related chemokines in the pathophysiology of lung diseases.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.

Cloning and characterization of a rabbit ortholog of human Galpha16 and mouse G(alpha)15.

A cDNA was cloned from a rabbit spleen cDNA library which encoded a G-protein alpha subunit peptide of 374 amino acids, that at the peptide level exhibited 86% and 79% identity with human Galpha16 and mouse G(alpha)15, respectively. The rabbit G(alpha)subunit cDNA was subcloned into a mammalian expression vector and transiently co-transfected into HEK-293 cells along with cDNAs encoding the human C3a, C5a, or nociceptin/orphanin FQ receptors. In all three cases the rabbit G alpha subunit behaved similarly to G(alpha)15 or G(alpha)16 and effectively coupled the transfected receptors to intracellular calcium mobilization pathways. By nucleotide sequence homology and functional activity the rabbit G(alpha) subunit appears to be the ortholog of human G(alpha)16 and mouse G(alpha)15.

Synthesis of structural analogs of leukotriene B4 and their receptor binding activity.

Structural analogs of leukotriene B4 (LTB4) were designed using a preferred conformation of LTB4 (1). Appending an aromatic ring scaffold between LTB4 carbons 7 and 11 led to quinoline analogs 3 and 15. A similar modification to the LTB4 structure between carbons 7 and 9 led to the pyridine analogs 41 and 46. The compounds of this study were evaluated in receptor binding assays using [3H]LTB4 and intact human DMSO differentiated U-937 cells. The first analog prepared, quinoline 3, displayed moderate potency in the LTB4 receptor binding assay (Ki = 0.9 microM). Modification of 3 by appending an aromatic ring between carbons 2 and 4 of the acid side chain produced a dramatic increase in receptor binding (15, Ki = 0.01 microM); a further improvement in receptor binding was achieved in the pyridine series (e.g., 41; Ki = 0.001 microM). The LTB4 receptor agonist/antagonist activity of the test compounds was determined using a functional assay that relies upon intracellular calcium mobilization induced by LTB4. Of the analogs prepared in this report only 47 demonstrated LTB4 receptor antagonist activity.

Trisubstituted pyridine leukotriene B4 receptor antagonists: synthesis and structure-activity relationships.

A series of trisubstituted pyridines have been prepared that exhibit in vitro leukotriene B4 (LTB4, 1) receptor antagonist activity. Previous disubstituted pyridines from these labs showed high affinity for the LTB4 receptor but demonstrated agonist activity in functional assays (e.g., 2, Ki = 1 nM). Compound 4, the initial lead compound of this new series, showed only modest affinity by comparison (Ki = 282 nM); however, 4 was a receptor antagonist with no demonstrable agonist activity up to 10 microM. Subsequent modifications of the lipid tail and aryl head group region led to the discovery of aniline 50 (SB 201146). This compound, also free of agonist activity, possesses high affinity for the LTB4 receptor (Ki = 4.7 nM).

Synthesis and LTB4 receptor antagonist activities of the naturally occurring LTB4 receptor antagonist Leucettamine A and related analogues.

The isolation and structure determination of the naturally occurring LTB4 receptor antagonist Leucettamine A (1) was recently reported. Herein we describe the synthesis of this natural product, the preparation of several analogues, and their effectiveness as antagonists of [3H]LTB4 binding to intact human U-937 cells. Total synthesis of Leucettamine A (1) is achieved by a convergent route which takes advantage of the elements of symmetry within the molecule. Syntheses of analogues of 1, which lacked the same degree of symmetry, are achieved by a different approach starting from alpha-amino acids. The natural product 1 inhibits [3H]LTB4 binding to its receptors on intact human U-937 cells with a Ki = 3.5 +/- 0.8 microM and is devoid of measurable agonist activity at the concentrations tested. 2-Amino imidazole analogues of 1 lacking the dioxolane groups were prepared. Generally these are significantly less potent than 1. However, one (26), designed on the basis of a putative structural overlay with LTB4, demonstrated potency comparable to that of the natural product (Ki = 2.4 +/- 0.2 microM).

Dopamine receptor agonists: 3-allyl-6-chloro-2,3,4,5-tetrahydro- 1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-diol and a series of related 3-benzazepines.

The N-allyl derivative (SK&F 85174) of 6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-dio l (SK&F 82526) not only retains the exceptional D-1 agonist potency of its parent but also displays reasonably potent D-2 agonist activity, as measured by a dopamine-sensitive adenylate cyclase test and a rabbit ear artery assay, respectively. Several additional N-substituted compounds were prepared to explore the D-2/D-1 agonist relationship. The N-methyl analogue retained good D-2 agonist potency, but this substitution converted D-1 agonist activity into antagonist activity. Most other N-substituents sharply decreased D-2 agonist potency including the N-n-propyl group. This observation was surprising since the introduction of mono- or di-N-n-propyl substituent(s) is commonly linked with retention or enhancement of D-2 agonist potency in other series of dopamine agonists. The N-(2-hydroxyethyl) analogue retains about one-fourth the D-2 potency of SK&F 85174. Several synthetic methods were used to prepare these compounds. N-Allylation of a trimethoxybenzazepine followed by cleavage of the methyl ethers with boron tribromide was the preferred method. Other methods used were direct alkylation of the trihydroxy secondary amine, i.e., SK&F 82526, and an acylation-amide reduction-cleavage method.

Absolute stereochemistry and dopaminergic activity of enantiomers of 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine.

Resolution of the unique dopamine receptor agonist 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (1) was achieved by a stereospecific multistep conversion of the readily separated enantiomers of its O,O,N-trimethylated precursor 2. The absolute stereochemistry of the antipodes of 2-MeI was determined by single-crystal X-ray diffractometric analysis, thus permitting assignment of the configuration of stereospecifically related 1, as well as that of the synthetic intermediates. High-performance liquid chromatography of diastereoisomeric derivatives was utilized to determine the enantiomeric excess of the R (greater than 97%) and S (greater than 89%) isomers of 1. Examination of the isomers in several in vitro and in vivo tests for both central and peripheral dopaminergic activity revealed that activity resided almost exclusively in the R isomer. The results suggest that the properly oriented 1-phenyl substituent of 1 is important for dopamine-like activity; it may contribute to receptor binding by interaction with a chirally defined accessory site. Configurational and conformational requirements for receptor binding of 1 are considered in relationship to previously described dopaminergic agents. These studies, in accord with previous suggestions, indicate that (R)-1 interacts with dopamine receptors in a conformation in which the catecholic hydroxyls and basic nitrogen are at least nearly maximally separated.

Dopamine agonists related to 3-allyl-6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzaz epi ne-7, 8-diol. 6-Position modifications.

The N-allyl derivative (SK&F 85174) of 6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-diol (SK&F 82526) retains the DA-1 agonist potency of the latter compound but unlike the parent also shows substantial DA-2 agonist activity. In a previous study of N-substituted benzazepines these combined agonist effects were shown to be uniquely associated with the N-allyl group. A continuation of this research has examined dependency of combined DA-2/DA-1 agonist activities on 6-position modification with the specific objective of developing an agonist with maximum effectiveness and potency at the DA-2 receptor subtype. DA-2 agonist activity was measured in a rabbit ear artery assay, and DA-1 agonist activity was determined in an adenylate cyclase assay. Replacing chloro with bromo retains the activity pattern and the potency of the chloro compound; replacement with a hydrogen causes a decrease of both DA-1 and DA-2 receptor activating potency. Introduction of a 6-methyl group causes loss of DA-2 agonist activity and reduction in DA-1 agonist potency. Substitution with a 6-fluoro provides the best balance of DA-2 and DA-1 agonist activities; this compound was moderately potent in both assays.

Synthesis, resolution, absolute stereochemistry, and enantioselectivity of 3',4'-dihydroxynomifensine.

3',4'-Dihydroxynomifensine, 8-amino-1,2,3,4-tetrahydro-4-(3,4-dihydroxyphenyl)-2-methylisoquinoli ne (1a), is an agonist of dopamine receptors in central and peripheral systems. Since this dopamine receptor agonist bears an asymmetric center at position 4, its synthesis and resolution were undertaken as part of a study directed toward determining the mode of interaction of these agents with the receptor(s). The enantiomers of 3',4'-dihydroxynomifensine are of particular interest, as they provide additional probes of present conceptual models of the dopamine receptor(s). Initial attempts to prepare 1a were inefficient or unsuccessful; instead, an isomeric compound, 1,2,4,5-tetra-hydro-2-(3,4-dihydroxyphenyl)-4- methyl-3H-1,4-benzodiazepine (9), was obtained. For this reason, a new route to 3',4'-dihydroxynomifensine was employed. The racemic dimethoxy intermediate 1d, thus obtained, was resolved. Methoxyl cleavage of the isomers of 1d afforded the enantiomers of 1a. Enantiomeric excess of these antipodes or appropriate derivatives was examined by NMR, CD, and HPLC methods. CD analysis suggests an enantiomeric excess greater than 99%. Determination of the absolute configuration of the enantiomers of 1a was determined by single-crystal X-ray diffractometric analysis. Examination of the isomers in several pharmacological test systems revealed a high degree of enantioselectivity. D-1 dopaminergic activity resides almost exclusively in the S enantiomer. The findings of the study have been employed to suggest an accessory binding site on the dopamine receptor(s) that differs from that advanced earlier. This accessory binding site may be specific for the D-1 subpopulation of dopamine receptors.

Dopaminergic activity of substituted 6-chloro-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepines.

6-Chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepines were synthesized and evaluated as agonists of central and peripheral dopamine receptors. These benzazepines were prepared by cyclization of certain amino alcohols followed by demethylation of the 7,8-dimethoxy groups of the precursors to the 7,8-catecholic moiety. Preliminary evidence of dopaminergic activity was determined in anesthetized dogs by measuring the effects on renal blood flow and calculating the accompanying changes in renal vascular resistance. The most potent compounds contained an hydroxyl group on the 1-phenyl group or were substituted at the 3' position with a chloro, methyl, or trifluoromethyl group. Evidence for central dopaminergic activity was obtained by measuring rotational effects in rats lesioned in the substantia nigra and also in an in vitro assay which measured stimulation of rat striatal adenylate cyclase. The compounds with the best central dopaminergic activity were generally the benzazepines which were the most lipophilic, were substituted on the 3' position of th 1-phenyl group, and contained either a 3-N-methyl or 3-N-allyl group.

Discovery of a novel class of selective non-peptide antagonists for the human neurokinin-3 receptor. 1. Identification of the 4-quinolinecarboxamide framework.

A novel class of potent and selective non-peptide neurokinin-3 (NK-3) receptor antagonists, featuring the 4-quinolinecarboxamide framework, has been designed based upon chemically diverse NK-1 receptor antagonists. The novel compounds 33-76, prompted by chemical modifications of the prototype 4, have been characterized by binding analysis using a membrane preparation of chinese hamster ovary (CHO) cells expressing the human neurokinin-3 receptors (hNK-3-CHO), and clear structure-activity relationships (SARs) have been established. From SARs, (R)-N-[alpha-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (65, SB 218795, hNK-3-CHO binding Ki = 13 nM) emerged as one of the most potent compounds of this novel class. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 65 is about 90-fold selective for hNK-3 versus hNK-2 receptors (hNK-2-CHO binding Ki = 1221 nM) and over 7000-fold selective versus hNK-1 receptors (hNK-1-CHO binding Ki = > 100 microM). In vitro functional studies in rabbit isolated iris sphincter muscle preparation demonstrated that 65 is a competitive antagonist of the contractile response induced by the potent and selective NK-3 receptor agonist senktide with a Kb = 43 nM. Overall, the data indicate that 65 is a potent and selective hNK-3 receptor antagonist and a useful lead for further chemical optimization.

Synthesis and dopaminergic activity of some halogenated mono- and dihydroxylated 2-aminotetralins.

In a series of 7,8-dihydroxy-1-phenyltetrahydro-3-benzazepine dopamine receptor agonists introduction of a chloro or fluoro substituent into the 6-position increases dopaminergic potency. Also, in this series replacement of the 7-hydroxyl group with a halogen results in inversion of activity from dopamine receptor agonist to antagonist. The present study was aimed at exploring the possibility that the structure-activity observations in the 3-benzazepine series of dopaminergic agents might be extrapolated to another class of dopamine receptor agonists, the 2-aminotetralins. Thus, a series of chloro- and fluoro-substituted mono- and dihydroxylated 2-aminotetralins was prepared and evaluated for dopaminergic properties in D-1 and D-2 receptor-related tests. Introduction of a chloro substituent into the 8-position of the prototype of this series, i.e. 2-amino-6,7-dihydroxytetralin (ADTN), resulted in a compound with a high degree of selectivity for the D-1 subpopulation of dopamine receptors; it was equally or more potent than ADTN in the D-1 receptor-related tests with greatly decreased effectiveness in the tests involving D-2 receptors. A similar effect was observed with 8-fluoro-ADTN; however, the N-(4-hydroxyphenethyl)-N-propyl derivative 4g of the 8-chloro-substituted ADTN showed marked D-2 binding affinity. Conversely, introduction of a chloro substituent into the 5-position of ADTN markedly decreased D-1 receptor affinity and efficacy. This effect was not seen with the related 5-fluoro derivative, suggesting D-1 receptors are more sensitive to bulk in the 5-position of ADTN than are the D-2 receptors. Replacement of either the 6- or 7-hydroxyl groups of ADTN with a chloro or fluoro substituent, in contrast, did not parallel the response seen in the benzazepine series (i.e., the compounds uniformly demonstrated less receptor affinity and did not have dopamine receptor antagonist activity); however, the decrease in agonist potency was less marked in the case of 2-amino-6-fluoro-7-hydroxytetralins than in the chlorinated monohydroxyaminotetralins. Thus, a parallelism in structure-activity relationships in the benzazepine and aminotetralin series of dopamine receptor agonists was not observed. The differences may reflect altered modes of receptor binding in the two series.

Synthesis and renal vasodilator activity of some dopamine agonist 1-aryl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diols: halogen and methyl analogues of fenoldopam.

Certain 6-halo-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepines were found to be potent D-1 dopamine agonists. The 1-(4-hydroxyphenyl) analogues did not have central nervous system activity because their high polarity inhibited entry into the brain. However, these compounds were potent renal vasodilators. Fenoldopam, the 6-chloro analogue, is an especially significant member of the series, and its synthesis, pharmacology, and clinical properties have been studied extensively. The 6-methyl and 6-iodo congeners were potent renal vasodilators, but nonpotent partial D-1 agonists as measured by stimulation of rat caudate adenylate cyclase. A possible rationalization suggests different receptor reserves for these activities. The 9-substituted benzazepines were either inactive or of low potency as dopamine agonists, while the N-methyl analogues had significant antagonist potency as measured by inhibition of dopamine stimulation of rat caudate adenylate cyclase.

(E)-3-[6-[[(2,6-dichlorophenyl)thio]methyl]-3-(2-phenylethoxy)-2- pyridinyl]-2-propenoic acid: a high-affinity leukotriene B4 receptor antagonist with oral antiinflammatory activity.

An extensive structure-activity study based around the high-affinity leukotriene B4 (LTB4) receptor antagonist SB 201146 (1) led to the identification of (E)-3-[6-[[(2,6-dichlorophenyl)-thio]methyl]-3-(2-phenylethoxy)-2- pyridinyl]-2-propenoic acid (3). This compound displays high affinity for the human neutrophil LTB4 receptor (Ki = 0.78 nM), blocks LTB4-induced Ca2+ mobilization with an IC50 of 6.6 +/- 1.5 nM, and demonstrates potent oral and topical antiinflammatory activity in a murine model of dermal inflammation.

Discovery of a novel class of selective non-peptide antagonists for the human neurokinin-3 receptor. 2. Identification of (S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (SB 223412).

Optimization of the previously reported 2-phenyl-4-quinolinecarboxamide NK-3 receptor antagonist 14, with regard to potential metabolic instability of the ester moiety and affinity and selectivity for the human neurokinin-3 (hNK-3) receptor, is described. The ester functionality could be successfully replaced by the ketone (31) or by lower alkyl groups (Et, 21, or n-Pr, 24). Investigation of the substitution pattern of the quinoline ring resulted in the identification of position 3 as a key position to enhance hNK-3 binding affinity and selectivity for the hNK-3 versus the hNK-2 receptor. All of the chemical groups introduced at this position, with the exception of halogens, increased the hNK-3 binding affinity, and compounds 53 (3-OH, SB 223412, hNK-3-CHO binding Ki = 1.4 nM) and 55 (3-NH2, hNK-3-CHO binding Ki = 1.2 nM) were the most potent compounds of this series. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 53 is about 100-fold selective for the hNK-3 versus hNK-2 receptor, with no affinity for the hNK-1 at concentrations up to 100 microM. In vitro studies demonstrated that 53 is a potent functional antagonist of the hNK-3 receptor (reversal of senktide-induced contractions in rabbit isolated iris sphincter muscles and reversal of NKB-induced Ca2+ mobilization in CHO cells stably expressing the hNK-3 receptor), while in vivo this compound showed oral and intravenous activity in NK-3 receptor-driven models (senktide-induced behavioral responses in mice and senktide-induced miosis in rabbits). Overall, the biological data indicate that (S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (53, SB 223412) may serve as a pharmacological tool in animal models of disease to assess the functional and pathophysiological role of the NK-3 receptor and to establish therapeutic indications for non-peptide NK-3 receptor antagonists.

Stepwise modulation of neurokinin-3 and neurokinin-2 receptor affinity and selectivity in quinoline tachykinin receptor antagonists.

A stepwise chemical modification from human neurokinin-3 receptor (hNK-3R)-selective antagonists to potent and combined hNK-3R and hNK-2R antagonists using the same 2-phenylquinoline template is described. Docking studies with 3-D models of the hNK-3 and hNK-2 receptors were used to drive the chemical design and speed up the identification of potent and combined antagonsits at both receptors. (S)-(+)-N-(1-Cyclohexylethyl)-3-[(4-morpholin-4-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 25, SB-400238: hNK-3R binding affinity, K(i) = 0.8 nM; hNK-2R binding affinity, K(i) = 0.8 nM) emerged as the best example in this approach. Further studies led to the identification of (S)-(+)-N-(1,2,2-trimethylpropyl)-3-[(4-piperidin-1-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 28, SB-414240: hNK-3R binding affinity, K(i) = 193 nM; hNK-2R binding affinity, K(i) = 1.0 nM) as the first hNK-2R-selective antagonist belonging to the 2-phenylquinoline chemical class. Since some members of this chemical series showed a significant binding affinity for the human mu-opioid receptor (hMOR), docking studies were also conducted on a 3-D model of the hMOR, resulting in the identification of a viable chemical strategy to avoid any significant micro-opioid component. Compounds 25 and 28 are therefore suitable pharmacological tools in the tachykinin area to elucidate further the pathophysiological role of NK-3 and NK-2 receptors and the therapeutic potential of selective NK-2 (28) or combined NK-3 and NK-2 (25) receptor antagonists.