Leukotriene (LT) receptor antagonists. Heterocycle-linked tetrazoles and carboxylic acids. LY203647

LY171883, 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl] ethanone, is an orally active antagonist of LTD4- and LTE4-induced responses in a variety of test systems. We prepared a new series of LT antagonists based on a proposed model of LY171883 binding to the LTE4 receptor in which the n-propyl and tetrazole moieties of LY171883 occupy those parts of the receptor to which the C1-C5 chain and the cysteinyl carboxyl of LTE4 bind, respectively. The new compounds have an acidic function corresponding to the glycine carboxyl of LTD4 linked through a heterocyclic group which is proposed to bind to the LTD4 receptor where the cysteinyl glycine amide bond of LTD4 binds. LY203647, 1-[2-hydroxy-3-propyl-4-[4-[2-[4-(1H-tetrazol-5-yl)butyl]-2H- tetrazol-5-yl]butoxy]phenyl] ethanone, showed good LTD4 antagonist activity with a suitable pharmacologic and toxicologic profile and has been chosen for clinical evaluation.     

(Phenylmethoxy)phenyl derivatives of omega-oxo- and omega-tetrazolylalkanoic acids and related tetrazoles. Synthesis and evaluation as leukotriene D4 receptor antagonists.

Two series of (phenylmethoxy)phenyl compounds derived from the structure of LY163443 were synthesized and evaluated as leukotriene D4 receptor antagonists. In the omega-[(phenylmethoxy)phenyl]-omega-oxoalkanoic acid series, 5-[4-[(4-acetyl-2-ethyl-3-hydroxyphenyl)methoxy]phenyl]-3,3-dimethyl-5- oxopentanoic acid (8) was the most potent antagonist of LTD4-induced contractions of guinea pig ileum (pKB of 7.60) and LTD4 pressor response in pithed rats (ED50 of 1.4 mg/kg iv). Replacing the carboxylic acid function with 5-tetrazole gave slightly more potent compounds. In the omega-[5-[[(phenylmethoxy)phenyl]alkyl] tetrazolyl]alkanoic acid series, replacing the carboxylic acid with 5-tetrazole gave compounds that were equally effective in the guinea pig ileum but more potent in vivo against the LTD4 pressor response in rat. The pKB value in the guinea pig ileum for 1-[2-hydroxy-3-propyl-4- [[4-[[2-[3-(1H-tetrazol-5-yl)propyl]-2H-tetrazol-5-yl]methyl ] phenoxy]methyl]phenyl]ethanone (25) was 7.87 and the ED50 for antagonism of the LTD4 pressor response was 4.0 mg/kg iv. The sodium salts of 8 (9) and 25 (26) given by the iv route of administration antagonized LTD4-induced cardiovascular alterations in anesthetized rat and LTD4-induced bronchoconstriction in guinea pig in a dose-dependent manner. Oral activity was also demonstrated against the LTD4-induced bronchoconstriction in guinea pig.     

1,3,6-trisubstituted indoles as peptidoleukotriene antagonists: benefits of a second, polar, pyrrole substituent.

1,6-Substituted and 3,5-substituted indoles and indazoles containing acylamino and N-arylsulfonyl amide appendages are potent antagonists of the peptidoleukotrienes LTD4 and LTE4. A compound from the 3,5-substituted indole series, N-[4-[[5-[[(cyclopentyloxy)carbonyl]amino]-1-methylindol- 3-yl]methyl]-3-methoxybenzoyl]-2-methyl-benzenesulfonamide (ICI 204,219), is undergoing clinical evaluation for asthma. Two new elements of structural diversity were introduced to this series of antagonists. An investigation of pyrrole substituents in the 1,6-substituted indoles demonstrated that substitution at C-2 was detrimental to biological activity, but the incorporation of hydrophilic groups at C-3 was beneficial. The introduction of a propionamide moiety at C-3 enhanced activity by 1 order of magnitude; N-[4-[[6-(cyclopentylacetamido)-3-[2-(N- methylcarbamoyl)ethyl]indol-1-yl]methyl]-3-methoxy- benzoyl]benzenesulfonamide (15c) has a pKB of 10.7 at the LTD4 receptor on guinea pig trachea. Modifications of the acylamino portion of the disubstituted antagonists demonstrated that a transposition of the amide CO and NH atoms was viable. N-Cyclopentylmethyl amides in both the 1,6- and 3,5-disubstituted indole series were 1 order of magnitude less potent than the corresponding cyclopentylacetamides. In both series this potency loss could be regained by the incorporation of a propionamide substituent at either C-3 or N-1, respectively. For example, N-[4-[[6-[N-(cyclopentylmethyl)carbamoyl]-3-[2-(pyrrolidin-1 - methylbenzenesulfonamide (39c) has a pKB of 9.5.     

Development of a novel series of styrylquinoline compounds as high-affinity leukotriene D4 receptor antagonists: synthetic and structure-activity studies leading to the discovery of (+-)-3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3- (dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid.

Based on LTD4 receptor antagonist activity of 3-(2-quinolinyl-(E)-ethenyl)pyridine (2) found in broad screening, structure-activity studies were carried out which led to the identification of 3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3- (dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid (1, MK-571) as a potent and orally active LTD4 receptor antagonist. These studies demonstrated that a phenyl ring could replace the pyridine in 2 without loss of activity, that 7-halogen substitution in the quinoline group was optimal for binding, that the (E)-ethenyl linkage was optimal, that binding was enhanced by incorporation of a polar acidic group or groups in the 3-position of the aryl ring, and that two acidic groups could be incorporated via a dithioacetal formed from thiopropionic acid and the corresponding styrylquinoline 3-aldehyde to yield compounds such as 20 (IC50 = 3 nM vs [3H]LTD4 binding to the guinea pig lung membrane). It was found that one of the acidic groups could be transformed into a variety of the amides without loss of potency and that the dimethylamide 1 embodied the optimal properties of intrinsic potency (IC50 = 0.8 nM on guinea pig lung LTD4 receptor) and oral in vivo potency in the guinea pig, hyperreactive rat, and squirrel monkey. The evolution of 2 to 1 involves the increase of > 6000-fold in competition for [3H]LTD4 binding to guinea pig lung membrane and a > 40-fold increase in oral activity as measured by inhibition of antigen-induced dyspnea in hyperreactive rats.     

Evolution of a series of peptidoleukotriene antagonists: synthesis and structure/activity relationships of 1,3,5-substituted indoles and indazoles

1,3,5-Substituted indoles and indazoles have been studied as receptor antagonists of the peptidoleukotrienes. The best of these compounds generally had a methyl group at the N1 position, a [(cyclopentyloxy)carbonyl]amino or 2-cyclopentylacetamido or N'-cyclopentylureido group at the C-5 position, and an arylsulfonyl amide group as part of the acidic chain at the C-3 position of the ring. Such compounds had in vitro dissociation constants (KB) in the range 10(-9) - 10(-11) M on guinea pig trachea against LTE4 as agonist and inhibition constants (Ki) less than or equal to 10(-9) M on guinea pig parenchymal membranes against [3H]LTD4. A number of compounds were orally effective at doses less than or equal to 1 mg/kg in blocking LTD4-induced "dyspnea" in guinea pigs. Compound 45 [N-[4-[[5-[[(cyclopentyloxy)carbonyl]-amino]-1-methylindol-3- yl]methyl]-3-methoxybenzoyl]-2-methylbenzenesulfonamide, ICI 204,219; pKB = 9.67 +/- 0.13, Ki = 0.3 +/- 0.03 nM, po ED50 = 0.3 mg/kg] is currently under clinical investigation for asthma. In the indole series, certain alkylsulfonyl amides possessing a 3-cyanobenzyl substituent at the N-1 position (60, 61) were produced that had KB less than or equal to 10(-9) M on guinea pig trachea.     

Development of a series of phenyltetrazole leukotriene D4 (LTD4) receptor antagonists.

A hypothetical model for receptor binding of leukotriene D4 (LTD4) was deduced from conformational analysis of LTD4 and from the structure-activity relationships (SAR) of known LTD4 receptor antagonists. A new structural series of LTD4 receptor antagonists exemplified by 5-[4-(4-phenylbutoxy)phenyl]-2-[4-(tetrazol-5-yl)butyl]-2H-t etrazole was designed in which a phenyltetrazole moiety was incorporated as a receptor binding equivalent of the triene unit of LTD4. A number of these phenyltetrazoles were prepared and found to possess LTD4 receptor antagonist activity. The structure-activity relationship (SAR) of this series is described.     

N-[(arylmethoxy)phenyl] and N-[(arylmethoxy)naphthyl] sulfonamides: potent orally active leukotriene D4 antagonists of novel structure

Two series of compounds, N-[(arylmethoxy)phenyl] sulfonamides and N-[(arylmethoxy)naphthyl] sulfonamides, were prepared as leukotriene D4 (LTD4) antagonists. In the phenyl series, N-[3-(2-quinolinylmethoxy)phenyl]-trifluoromethanesulfonamide (Wy-48,252, 16) was the most potent inhibitor of LTD4-induced bronchoconstriction in the guinea pig. With an intragastric ID50 of 0.1 mg/kg (2-h pretreatment), 16 was 300 times more potent than LY-171,883. Compound 16 also intragastrically inhibited ovalbumin-induced bronchoconstriction in the guinea pig with an ID50 of 0.6 mg/kg. In vitro against LTD4-induced contraction of isolated guinea pig trachea pretreated with indomethacin and L-cysteine, 16 produced a pKB value of 7.7. In the rat PMN assay 16 inhibited both 5-lipoxygenase and cyclooxygenase (IC50's = 4.6 and 3.3 microM). In the naphthyl series, N-[7-(2-quinolinylmethoxy)-2-naphthyl]trifluoromethanesulfonamide (Wy-48,090, 47) in addition to potent LTD4 antagonist activity (on isolated guinea pig trachea 47 had a pKB value of 7.04) also had antiinflammatory activity (63% inhibition at 50 mg/kg in the rat carrageenan paw edema assay and 34% inhibition of TPA-induced inflammation at 1 mg/ear in the mouse ear edema model). Perhaps the antiinflammatory activity of 47 was due to its additional activity of inhibiting both 5-lipoxygenase and cyclooxygenase enzymes (IC50's = 0.23 and 11.9 microM, respectively, in rat PMN).     

The development of a novel series of (quinolin-2-ylmethoxy)phenyl-containing compounds as high-affinity leukotriene receptor antagonists. 3. Structural variation of the acidic side chain to give antagonists of enhanced potency

This paper is the third in a series outlining the development of orally active sulfido peptide leukotriene antagonists containing a (quinolin-2-ylmethoxy)phenyl moiety. In this work the systematic variation of the acid side chain substituents led to dramatic and reproducible changes in the oral activity of these compounds, presumably due to alterations in their pharmacokinetic properties. The most potent compound identified, 5-[4-[4-(quinolin-2-yl-methoxy)phenyl]-3-methylbutyl]tetrazole (32), represents a convergence of good in vitro antagonist activity and a 3-10-fold improvement in oral potency over the current clinical candidate 2. The new findings from these optimization studies are as follows: oxygen substitution in the acid side chain was not necessary for antagonist activity, in vitro and in vivo activity was enhanced by alkyl or phenyl substitution on the gamma-carbon of the acid side chain of para-substituted (quinolin-2-ylmethoxy)phenyl derivatives, and free rotation about the side chain carbon atom adjacent to the (quinolin-2-ylmethoxy)phenyl ring was required for activity. The lead compound of this report (32) is a competitive inhibitor of [3H]LTD4 binding to receptor membrane purified from guinea pig lung (Ki = 12 +/- 3 nM) and of the spasmogenic activity of LTC4, LTD4, and LTE4 in guinea pig lung strip. Dosed orally in guinea pigs, this compound blocks LTD4-induced bronchoconstriction (ED50 0.8 mg/kg) and antigen-induced systemic anaphylaxis (ED50 = 1.2 mg/kg).     

Synthesis and in vitro LTD4 antagonist activity of bicyclic and monocyclic cyclopentylurethane and cyclopentylacetamide N-arylsulfonyl amides

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.     

N-[(arylmethoxy)phenyl] carboxylic acids, hydroxamic acids, tetrazoles, and sulfonyl carboxamides. Potent orally active leukotriene D4 antagonists of novel structure

Four series of N-[(arylmethoxy)phenyl] compounds were prepared as leukotriene D4 (LTD4) antagonists. In the hydroxamic acid series, methyl 3-(2-quinolinylmethoxy)benzeneacetohydroxamate (Wy-48,422, 20) was the most potent inhibitor of LTD4-induced bronchoconstriction with an oral ED50 of 7.9 mg/kg. Compound 20 also orally inhibited ovalbumin-induced bronchoconstriction in the guinea pig with an ED50 of 3.6 mg/kg. In vitro, against LTD4-induced contraction of isolated guinea pig trachea pretreated with indomethacin and 1-cysteine, 20 produced a pKB value of 6.08. In the sulfonyl carboxamide series, N-[(4-methylphenyl)sulfonyl]-3-(2-quinolinylmethoxy)-benzamide (Wy-49,353, 30) was the most potent antagonist. Compound 30 orally inhibited both LTD4- and ovalbumin-induced bronchoconstriction with ED50s of 0.4 and 20.2 mg/kg, respectively. In vitro, against LTD4-induced contraction of isolated guinea pig trachea, 30 produced a pKB value of 7.78. In the carboxylic acid series, which served as intermediates for the above two series, 3-(2-quinolinylmethoxy)benzeneacetic acid (Wy-46,016, 5) was the most potent inhibitor of LTD4-induced bronchoconstriction (99% at 25 mg/kg, intraduodenally); however, the pKB for this compound was disappointing (5.79). In the tetrazole series, the most potent inhibitor was 2-[[3-(1H-tetrazol-5-ylmethyl)phenoxy]methyl]quinoline (Wy-49,451, 41). The respective inhibitory ED50s were 3.0 mg/kg versus LTD4 and 17.5 mg/kg versus ovalbumin. In the isolated guinea pig trachea, 41 produced a pKB value of 6.70.     

N-(3-acyloxy-2-benzylpropyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues: novel potent and high affinity antagonists and partial antagonists of the vanilloid receptor

Isosteric replacement of the phenolic hydroxyl group in potent vanilloid receptor (VR1) agonists with the alkylsulfonamido group provides a series of compounds which are effective antagonists to the action of the capsaicin on rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells. In particular, compound 61, N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N'-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea was a full antagonist against capsaicin, displayed a K(i) value of 7.8 nM (compared to 520 nM for capsazepine and 4 nM for 5-iodoRTX), and showed excellent analgesic activity in mice. Structure-activity analysis of the influence of modifications in the A- and C-regions of 4-methylsulfonamide ligands on VR1 agonism/antagonism indicated that 3-fluoro substitution in the A-region and a 4-tert-butylbenzyl moiety in the C-region favored antagonism, whereas a 3-methoxy group in the A-region and 3-acyloxy-2-benzylpropyl moieties in the C-region favored agonism.     

Synthesis of carbon-11 labeled iodinated cocaine derivatives and their distribution in baboon brain measured using positron emission tomography.

Three iodine-substituted derivatives of cocaine, methyl esters of 3-[(2'-, 3'-, and 4'-iodobenzoyl)oxy]-8-methyl-[1R-(exo,exo)]-8- azabicyclo[3.2.1]octane-2 carboxylic acid (2a-c), were synthesized and subjected to N-demethylation to give the corresponding noriodococaines 3-[(2'-,3'-, and 4'-iodobenzoyl)oxy]-[1R-(exo,exo)]-8- azabicyclo[3.2.1]-octane-2-carboxylic acid (3a-c). These were remethylated with [11C]CH3I to give the [N-11C-methyl]iodococaines 4a-c which were examined in baboon brain in vivo using positron emission tomography (PET). Compared to [N-11C]cocaine itself the regional distributions were changed from a highly specific localization in the corpus striatum to more diffuse patterns which included the cerebellum and cortex. Peak brain uptakes and clearance kinetics were also changed. [N-11C]-o-Iodococaine (4a) had a peak uptake in the striatum at 4-5 min after injection of only 17% that of cocaine in the same animal. The peak uptake of [N-11C]-p-iodococaine (4c) was 60% of that of [N-11C]cocaine and a clearance half-time of approximately 55 min, twice that of [N-11C]cocaine. [N-11C]-m-Iodococaine (4b) displayed half the uptake of [N-11C]cocaine, buts its clearance was similar to that of the parent molecule. The fractions of unmetabolized tracer in blood plasma at 1-30 min were higher for 4a-c than for [N-11C]cocaine. Plasma protein binding experiments showed 10%, 0.3%, 1.6%, and 6% as the free fraction for cocaine and o-, m-, and p-iodococaines respectively, consistent with the low brain uptake observed for the ortho isomer, and implicated alpha 1-acid glycoprotein as responsible for the low free fraction of o-iodococaine. The potencies of 2a-c to displace tritiated cocaine from striatal membranes were p-iodo approximately cocaine greater than m-iodo approximately o-iodo.     

Nonpeptide angiotensin II receptor antagonists: the discovery of a series of N-(biphenylylmethyl)imidazoles as potent, orally active antihypertensives

A new series of nonpeptide angiotensin II (AII) receptor antagonists has been prepared. These N-(biphenylyl-methyl)imidazoles, e.g. 2-butyl-1-[(2'-carboxybiphenyl-4-yl)methyl]-4-chloro-5- (hydroxymethyl)imidazole, differ from the previously reported N-(benzamidobenzyl)imidazoles and related compounds in that they produce a potent antihypertensive effect upon oral administration; the earlier series generally were active only when administered intravenously. It has been found that the acidic group at the 2'-position of the biphenyl is essential. Only ortho-substituted acids possess both high affinity for the AII receptor and good oral antihypertensive potency. The carboxylic acid group has been replaced with a variety of acidic isosteres, and the tetrazole ring has been found to be the most effective. The tetrazole derivative, DuP 753, is currently in development for the treatment of hypertension.     

The effect of RG 12525 on leukotriene D4-mediated pulmonary responses in guinea pigs

RG 12525 (5-(2-[4-quinolin-2-yl)methoxyl] phenoxymethyl)benzyl tetrazole) is under investigation as a specific inhibitor of leukotriene D4 (LTD4). The present studies examine the effect of orally administered RG 12525 on LTD4 mediated pulmonary responses in three separate guinea pig models. The compound inhibited antigen-induced mortality in the systemic anaphylaxis model with an ED50 (95% confidence interval) = 2.2 (0.8-6.4) mg/kg. In this model, the activity half-life of RG 12525 was shown to be 6.5 hours and the compound offered significant protection within 15 minutes of administration. RG 12525 also protected against LTD4-induced bronchoconstriction in a model measuring changes in pulmonary function with an ED50 = 0.6 (0.4-1.0) mg/kg. The same level of activity was observed in a similar model which monitored changes in pulmonary function in response to exogenous antigen in actively-sensitized guinea pigs. Together, these data indicate that RG 12525 is a potent, orally active LTD4 antagonist which possesses the requisite profile for potential clinical development.     

Design and synthesis of potent non-polyglutamatable quinazoline antifolate thymidylate synthase inhibitors.

The synthesis is described of a series of analogues of the potent thymidylate synthase (TS) inhibitor, N-[4-[N-[(3,4-dihydro-2, 7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-f luorob enzoyl]-L-glutamic acid (4, ZM214888), in which the glutamic acid moiety is replaced by homologous amino acids and alpha-amino acids where the omega-carboxylate is replaced by acylsulfonamides and acidic heterocycles. In general these modifications when compared to 4 gave compounds with increased potency as inhibitors of isolated TS and as cytotoxic agents against murine tumor cell lines. The new compounds require transport by the reduced folate carrier for entry into cells but are not converted intracellularly into polyglutamated species. Agents with this profile are expected to show activity against tumors that are resistant to classical antifolates due to low expression of folylpolyglutamate synthetase. The analogue (S)-2-[4-[N-[(3,4-dihydro-2, 7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-f luorob enzamido]-4-(1H-1,2,3,4-tetrazol-5-yl)butyric acid (35, ZD9331) has been selected as a clinical development candidate and is currently undergoing phase I studies.     

New bronchodilators. Synthesis and bronchodilating activity of some 3-(alkoxymethyl)-alpha-(N-substituted aminomethyl)-4-hydroxybenzyl alcohols.

A series of 3-(alkoxymethyl)-alpha-(N-substituted aminomethyl)-4-hydroxybenzyl alcohols was synthesized as potential bronchodilators. The ability to prevent effects against histamine-induced bronchoconstriction in guinea pigs was studied to determine their bronchodilating activity. Introduction of a methoxymethyl group in place of the m-hydroxyl group of beta-adrenergic catecholamines afforded compounds especially effective in delaying histamine-induced bronchoconstriction in guinea pigs. Appropriate N-substitution also enhanced the potency of these catecholamine analogues. 4-Hydroxy-3-(methoxymethyl)-alpha-[N-[4-(methoxymethyl)-alpha-methylphenyl]aminoethyl]benzyl alcohol hemifumarate (3r) was the most potent compound in this series.     

Synthesis and LTD4 antagonist activity of 2-norleukotriene analogues

A series of structural analogues of 4(R)-hydroxy-5(S)-cysteinylglycyl-6(Z)-nonadecenoic acid [4R,5S,6Z)-2-nor-LTD1 (10b), SK&F 101132) has been synthesized and pharmacologically characterized. (4R,5S,6Z)-2-nor-LTD1 significantly antagonized LTD4-induced contractile responses on isolated guinea pig trachea. The cis double-bond geometry appears to be critical for antagonist activity, whereas the trans isomer 17 exhibited weak contractile activity. Replacement of the cysteinylglycyl moiety with cysteine afforded 20, which retained significant antagonist activity, while lengthening or shortening the lipid tail by five methylene groups resulted in complete loss of activity. The eicosanoid amide 15, glycinamide 14, and C-1 carbinol 18 analogues all possessed antagonist activity, whereas the diol derivative 19 exhibited increased intrinsic agonist activity.     

Molecular interaction of a potent nonpeptide agonist with the chemokine receptor CCR8

Most nonpeptide antagonists for CC-chemokine receptors share a common pharmacophore with a centrally located, positively charged amine that interacts with the highly conserved glutamic acid (Glu) located in position 6 of transmembrane helix VII (VII:06). We present a novel CCR8 nonpeptide agonist, 8-[3-(2-methoxyphenoxy)benzyl]-1-phenethyl-1,3,8-triaza-spiro[4.5]decan-4-one (LMD-009), that also contains a centrally located, positively charged amine. LMD-009 selectively stimulated CCR8 among the 20 identified human chemokine receptors. It mediated chemotaxis, inositol phosphate accumulation, and calcium release with high potencies (EC50 from 11 to 87 nM) and with efficacies similar to that of the endogenous agonist CCL1, and it competed for 125I-CCL1 binding with an affinity of 66 nM. A series of 29 mutations targeting 25 amino acids broadly distributed in the minor and major ligand-binding pockets of CCR8 uncovered that the binding of LMD-009 and of four analogs [2-(1-(3-(2-methoxyphenoxy)benzyl)-4-hydroxypiperidin-4-yl)benzoic acid (LMD-584), N-ethyl-2-4-methoxybenzenesulfonamide (LMD-902), N-(1-(3-(2-methoxyphenoxy)benzyl)piperidin-4-yl)-2-phenyl-4-(pyrrolidin-1yl)butanamide (LMD-268), and N-(1-(3-(2-methoxyphenoxy)benzyl)piperidin-4-yl)-1,2,3,4-tetrahydro-2-oxoquinoline-4-carboxamide (LMD-174)] included several key-residues for nonpeptide antagonists targeting CCR1, -2, and -5. It is noteworthy that a decrease in potency of nearly 1000-fold was observed for all five compounds for the Ala substitution of the anchor-point GluVII:06 (Glu(286)) and a gain-of-function of 19-fold was observed for LMD-009 (but not the four other analogs) for the Ala substitution of PheVI:16 (Phe(254)). These structural hallmarks were particularly important in the generation of a model of the molecular mechanism of action for LMD-009. In conclusion, we present the first molecular mapping of the interaction of a nonpeptide agonist with a chemokine receptor and show that the binding pocket of LMD-009 and of analogs overlaps considerably with the binding pockets of CC-chemokine receptor nonpeptide antagonists in general.     

Synthesis and anticonvulsant properties of a series of N-substituted 2-aza-spiro[4.5]decane-1,3-diones and 8-phenyl-2-aza-spiro[4.5]decane-1,3-diones

A series of N-phenyl-2-aza-spiro[4.5]decane-1,3-diones [III-VIIII] structurally related to the previously described N-phenyl-3-arylpyrrolidine-2,5-dione (11) was synthesized and tested for their anticonvulsant activity in the maximum electroshock seizure (MES) and metrazole seizure threshold (sc. MET) tests. The most potent of the series were N-(2-methylphenyl)-2-aza-spiro[4.5]decane-1,3-dione [III] and N-(3-methylphenyl)-2-aza-spiro [4.5]decane-1,3-dione [IV], which inhibited seizures in the MES and sc.MET tests. On the other hand, as a preliminary assay we synthesized and tested for the anticonvulsant activity a new N-substituted 8-phenyl-2-aza-spiro[4.5]decane-1,3-dione, containing either a benzyl or a cyclohexyl moiety [IX-XII] at the nitrogen atom. The obtained results showed that the presence and position of the methyl group in the aryl ring [III, IV], as well as an cyclohexane moiety [XI, XIII connected with the imide nitrogen atom, played the essential role for anticonvulsant activity.     

Rational design, synthesis, and structure-activity relationships of aryltriazoles as novel corticotropin-releasing factor-1 receptor antagonists

Following the discovery of the very high binding affinity of 4-anilinopyrimidines against corticotropin-releasing factor receptor-1 (CRF(1)) (e.g., 1, K(i) = 2 nM), a new series of triazoles bearing different groups has been synthesized and evaluated. The compounds were prepared by cyclizations of N-acyl-S-methylisothioureas with alkylhydrazines or by cyclizations with hydrazine followed by alkylation. While members of this series showed potent binding affinity against CRF(1) receptor, there were important differences between the different regio- (7 and 12) and stereoisomeric aryltriazoles where the R(1) or R(2) side chain in 7 has an asymmetric center. In terms of overall potency, aryltriazole analogues such as 7r bearing an N-(alpha-branched benzyl)-N-propylamino side chain were the most potent, followed by analogues such as 7a, with an N-bis(cyclopropyl)methyl-N-propylamino side chain, and analogues such as 7m, with an N-(alpha-branched aliphatic)-N-propylamino side chain. While the N-propyl group was crucial for high potency, we hypothesized that the terminal methyl mimicked the 5-methyl of pyrazolo[1,5-a]pyrimidines 3 and 4. Correlation of the low-energy conformers of compounds of type 3 and 7 generated by computational analyses was very good. The size and shape of the N-alkyl group dramatically changed the potency of the triazoles, which is in contrast to the SAR seen for bicyclic CRF(1) antagonists. In general, the S-enantiomer was much more potent than the corresponding R-isomer. Furthermore, to a limited extent in the aryltriazole series the substituent on the 5-phenyl ring changed the potency up to 9-fold. (S)-1-Methyl-3-[N-(4-fluorophenylpentyl)-N-propyl]amino-5-(2-methoxy-4-dichlorophenyl)-1H-[1,2,4]triazole [(S)-7r] showed very potent binding affinity (K(i) = 2.7 nM) to CRF(1) receptors with an IC(50) of 49 nM in a cAMP inhibition assay.