Effect of diphenyl diselenide, diphenyl ditelluride and ebselen on cerebral Na(+), K(+)-ATPase activity in rats

In the present study, we investigated the in vitro effect of diphenyl ditelluride, diphenyl diselenide and ebselen on Na(+), K(+)-ATPase activity of rat brain. The results demonstrated that all compounds significantly inhibited (in the muM range) Na(+), K(+)-ATPase activity. Diphenyl ditelluride, at low concentrations, provoked an increase in Na(+), K(+)-ATPase activity. Dithiothreitol (DTT), at 3mM, protected the inhibition caused by diphenyl ditelluride, diphenyl diselenide and ebselen in Na(+), K(+)-ATPase activity. Post-incubation of diphenyl diselenide-treated homogenate with DTT completely recovered enzyme activity. DTT was able to recover the enzyme inhibition induced by 20muM of diphenyl ditelluride, but was partially able to recover inhibition induced by high concentrations of organotellurium compound. Conversely, DTT did not recover ebselen-induced Na(+), K(+)-ATPase inhibition. The mechanism of inhibition by diphenyl diselenide, diphenyl ditelluride and ebselen in Na(+), K(+)-ATPase activity revealed: decreased maximal velocity and K(m). Cerebral Na(+), K(+)-ATPase is a potential molecular target for the toxic effect of organochalcogens and the inhibition may occur through a change in the crucial thiol groups of this enzyme.     

Synthesis and biological evaluation of alpha- and beta-6-amido derivatives of 17-cyclopropylmethyl-3, 14beta-dihydroxy-4, 5alpha-epoxymorphinan: potential alcohol-cessation agents

Substituted aryl and aliphatic amide analogues of 6-naltrexamine were synthesized and used to characterize the binding to and functional activity of human mu-, delta-, and kappa-opioid receptors. Competition binding assays showed 11-25 and 27-31 bound to the mu (K(i) = 0.05-1.2 nM) and kappa (K(i) = 0.06-2.4 nM) opioid receptors. Compounds 11-18 possessed significant binding affinity for the delta receptor (K(i) = 0.8-12.4 nM). Functional assays showed several compounds acted as partial or full agonists of delta or kappa receptors while retaining an antagonist profile at the mu receptor. Structure-activity relationship for aryl amides showed that potent compounds possessed lipophilic groups or substituents capable of hydrogen bonding. Metabolic stability studies showed that 11, 12, and 14 possessed considerable stability in the presence of rat, mouse, or human liver preparations. The ED 50 of inhibition of 10% ethanol self-administration in trained rats, using operant techniques for 11, was 0.5 mg/kg.     

A new class of highly potent and selective endomorphin-1 analogues containing α-methylene-β-aminopropanoic acids (map)

A new class of endomorphin-1 (EM-1) analogues were synthesized by introduction of novel unnatural α-methylene-β-amino acids (Map) at position 3 or/and position 4. Their binding and functional activity, metabolic stability, and antinociceptive activity were determined and compared. Most of these analogues showed high affinities for the μ-opioid receptor and an increased stability in mouse brain homogenates compared with EM-1. Examination of cAMP accumulation and ERK1/2 phosphorylation in HEK293 cells confirmed the agonist properties of these analogues. Among these new analogues, H-Tyr-Pro-Trp-(2-furyl)Map-NH(2) (analogue 12) exhibited the highest binding potency (K(i)(μ) = 0.221 nM) and efficacy (EC(50) = 0.0334 nM, E(max) = 97.14%). This analogue also displayed enhanced antinociceptive activity in vivo in comparison to EM-1. Molecular modeling approaches were then carried out to demonstrate the interaction pattern of these analogues with the opioid receptors. We found that, compared to EM-1, the incorporation of our synthesized Map at position 4 would bring the analogue to a closer binding mode with the μ-opioid receptor.     

Synthesis and pharmacological evaluation of novel octahydro-1H-pyrido[1,2-a]pyrazine as mu-opioid receptor antagonists

To better understand structural requirements for a mu ligand of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class to interact with the mu opioid receptor, we have described in the previous article (Le Bourdonnec, B. et al. J. Med. Chem. 2006, 25, 7278-7289) new, constrained analogues of the N-phenethyl derivative 3. One of the active constrained analogues, compound 4, exhibited subnanomolar mu-opioid receptor affinity (K(i) = 0.62 nM) and potent mu-opioid antagonist activity (IC(50) = 0.54 nM). On the basis of structure 4, a new series of mu-opioid receptor antagonists were designed. In these compounds the octahydroquinolizine template of 4 was replaced by an octahydro-1H-pyrido[1,2-a]pyrazine scaffold. The new derivatives were tested for their binding affinities and in vitro functional activity against the cloned human mu-, delta-, and kappa-opioid receptors. From this study, we identified compound 36, which displays high affinity toward the mu-opioid receptor (K(i) = 0.47 nM), potent mu in vitro antagonist activity (IC(50) = 1.8 nM) and improved binding selectivity profile mu/kappa and mu/delta, when compared to 4.     

New 2-arylpyrazolo[4,3-c]quinoline derivatives as potent and selective human A3 adenosine receptor antagonists

In this paper we report the synthesis and biological evaluation of a new class of 2-phenyl-2,5-dihydro-pyrazolo[4,3-c]quinolin-4-ones as A(3) adenosine receptor antagonists. We designed a new route based on the Kira-Vilsmeier reaction for the synthesis of this class of compounds. Some of the synthesized compounds showed A(3) adenosine receptor affinity in the nanomolar range and good selectivity as evaluated in radioligand binding assays at human (h) A(1), A(2A), A(2B), and A(3) adenosine receptor subtypes. We introduced several substituents on the 2-phenyl ring. In particular substitution at the 4-position by methyl, methoxy, and chlorine gave optimal activity and selectivity 6c (K(i)hA(1), A(2A)>1000 nM, EC(50)hA(2B)>1000 nM, K(i)hA(3) = 9 nM), 6d (K(i)hA(1), A(2A)>1000 nM, EC(50)hA(2B)>1000 nM, K(i)hA(3) = 16 nM), 6b (K(i)hA(1), A(2A) >1000 nM, EC(50)hA(2B)>1000 nM, K(i)hA(3) = 19 nM). In conclusion, the 2-phenyl-2,5-dihydro-pyrazolo[4,3-c]quinolin-4-one derivatives described herein represent a new family of in vitro selective antagonists for the adenosine A(3) receptor.     

Structure-activity relationship study of N⁶-(2-(4-(1H-Indol-5-yl)piperazin-1-yl)ethyl)-N⁶-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine analogues: development of highly selective D3 dopamine receptor agonists along with a highly potent D2/D3 agonist and their pharmacological characterization

In our effort to develop multifunctional drugs against Parkinson's disease, a structure-activity-relationship study was carried out based on our hybrid molecular template targeting D2/D3 receptors. Competitive binding with [(3)H]spiroperidol was used to evaluate affinity (K(i)) of test compounds. Functional activity of selected compounds in stimulating [(35)S]GTPγS binding was assessed in CHO cells expressing either human D2 or D3 receptors. Our results demonstrated development of highly selective compounds for D3 receptor (for (-)-40K(i), D3 = 1.84 nM, D2/D3 = 583.2; for (-)-45K(i), D3 = 1.09 nM, D2/D3 = 827.5). Functional data identified (-)-40 (EC(50), D2 = 114 nM, D3 = 0.26 nM, D2/D3 = 438) as one of the highest D3 selective agonists known to date. In addition, high affinity, nonselective D3 agonist (-)-19 (EC(50), D2 = 2.96 nM and D3 = 1.26 nM) was also developed. Lead compounds with antioxidant activity were evaluated using an in vivo PD animal model.     

Phenylguanidines as selective nonpeptide melanocortin-5 receptor antagonists

A series of phenylguanidine analogues represented by 10, 12, and 21 were synthesized and found to have high binding affinities for the human melanocortin-5 receptor. Their binding affinities for three other melanocortin receptor subtypes, MC1, MC3, and MC4, were low. Selected compounds were also tested for their functional activity and exhibited inhibition of alpha-MSH-stimulated cAMP production in cells expressing the human MC5 receptor. Compound 10 had a K(i) value of 2.1 nM in the binding assay and an IC(50) of 72 nM in the functional assay. Some analogues such as 13 from this series possessed weak agonist activity at the human MC4 receptor.     

Analogues of σ receptor ligand 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) with added polar functionality and reduced lipophilicity for potential use as positron emission tomography radiotracers

1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine 1 (PB28) represents an excellent lead candidate for therapeutic and/or diagnostic applications in oncology. However, because its utility is limited by its relatively high degree of lipophilicity, novel analogues of 1 with reduced lipophilic character were designed by substituting methylene groups with more polar functional groups in the propylene linker and at the tetralin C4 position. For the chiral analogues, separate enantiomers exhibited substantial and roughly equal affinities within a given receptor subtype, with the greatest difference observed for compound 9 at σ(1) (7.5-fold; (-)-(S)-9 K(i) = 94.6 nM, (+)-(R)-9 K(i) = 12.6 nM). Compound (-)-(S)-9 was also found to be the most σ(2)-selective agent (σ(2) K(i) = 5.92 nM), to possess a lipophilicity consistent with entry into tumor cells (log D(7.4) = 2.38), and to show minimal antiproliferative activity. However, (-)-(S)-9 exhibited moderate activity (EC(50) = 8.1 μM) at the P-gp efflux pump.     

Structure-activity relationship studies on a novel series of (S)-2beta-substituted 3alpha-[bis(4-fluoro- or 4-chlorophenyl)methoxy]tropane analogues for in vivo investigation

In general, 3alpha-(diphenylmethoxy)tropane (benztropine)-based dopamine uptake inhibitors do not demonstrate cocaine-like pharmacological activity in models of psychostimulant abuse and have been proposed as potential medications for the treatment of cocaine addiction. However, several (S)-2-carboalkoxy-substituted-3alpha-[bis(4-fluorophenyl)methoxy]tropane analogues were discovered to stimulate locomotor activity and substitute in subjects trained to discriminate cocaine, suggesting a role of the 2-position substituent in mediating these cocaine-like actions. Herein, we describe the synthesis of a series of novel N- and 2-substituted-3alpha-[bis(4-fluoro- or 4-chlorophenyl)methoxy]tropane analogues. Most of these analogues demonstrated high affinity binding to the dopamine transporter (DAT; K(i) = 1.8-40 nM), and selectivity over the other monoamine transporters and muscarinic M(1) receptors. When the (S)-2-carboalkoxy substituent was replaced with (S)-2-ethenyl, the resulting analogue 11 demonstrated the highest DAT binding affinity in the series (K(i) = 1.81 nM) with DAT selectivity over serotonin transporters (SERT; 989-fold), norepinephrine transporters (NET; 261-fold) and muscarinic receptors (90-fold). When the 4'-F groups of compounds 5 (K(i) = 2.94 nM) and 8 (K(i) = 6.87 nM) were replaced with 4'-Cl in the (S)-2-carboalkoxy series, DAT binding affinities were slightly reduced (K(i) = 12.6 and 14.6 nM for 6 and 7, respectively), yet inhibition of dopamine uptake potency remained comparably high (IC(50) range = 1.5-2.5 nM). Interestingly, the 4'-Cl analogue (+/-)-6 substituted less in rats trained to discriminate cocaine than the 4'-F analogue (+/-)-5. These studies demonstrate that manipulation of the 2-, N-, and 3-position substituents in the 3alpha-(diphenylmethoxy)tropane class of dopamine uptake inhibitors can result in ligands with high affinity and selectivity for the DAT, and distinctive in vivo pharmacological profiles that cannot be predicted by their effects in vitro.     

9-hydroxyazafluorenes and their use in thrombin inhibitors

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs). An effective strategy for reducing plasma protein binding of 2 and improving efficacy in an in vivo thrombosis model in rats was to replace the lipophilic fluorenyl group in P3 with an azafluorenyl group. Systematic investigation of all possible azafluorenyl P3 isomers and azafluorenyl-N-oxide analogues of 2 led to the identification of an optimal compound, 3-aza-9-hydroxyfluoren-9(R)-ylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (19b), with high potency (K(i) = 0.40 nM, 2x APTT = 0.18 microM), excellent pharmacokinetic properties (F = 55%, T(1/2) = 14 h in dogs), and complete efficacy in the in vivo thrombosis model in rats (inhibition of FeCl(3)-induced vessel occlusions in six of six rats receiving an intravenous infusion of 10 microg/kg/min of 19b). The stereochemistry of the azafluorenyl group in 19b was determined by X-ray crystallographic analysis of its N-oxide derivative (23b) bound in the active site of human thrombin.     

Novel,potent ORL-1 receptor agonist peptides containing alpha-Helix-promoting conformational constraints

The ORL-1 receptor has recently been cloned and is implicated in a wide variety of physiological and pathophysiological processes. Toward the goal of elucidating important features of the receptor-bound conformation of the endogenous ligand, nociceptin (NC), several conformationally constrained analogues were prepared. Either alpha-aminoisobutyric acid (Aib) or N-methylalanine (MeAla) were inserted as replacement(s) for Ala7, Ala11, or Ala15 in the native NC sequence (FGGFTGARKSARKLANQ). In vitro assays measuring human ORL-1 receptor affinity (competition binding against [3H] NC), functional potency ([35S]GTP gamma S), and efficacy (as compared to NC) were performed for each new peptide. The receptor affinities of the Aib-containing peptides generally matched NC, showing K(i)'s in the range of 0.1-0.5 nM. By comparison, the receptor affinities of the MeAla-containing peptides were significantly diminished. Peptide 14 (FGGFTG[Aib]RKS[Aib]RKLANQ-NH2), which contains two constrained alanine residues (positions 7 and 11) and a C-terminal amide modification, was found to be a very potent agonist with K(i) = 0.05 nM and EC50 = 0.08 nM in the human ORL-1 assays. The data support a hypothesis that the receptor-bound form of NC might adopt an amphipathic helix in the "address" segment of the sequence.     

In vitro and in vivo pharmacology of synthetic olivetol- or resorcinol-derived cannabinoid receptor ligands

BACKGROUND AND PURPOSE: We have previously reported the development of CB-25 and CB-52, two ligands of CB1 and CB2 cannabinoid receptors. We assessed here their functional activity. EXPERIMENTAL APPROACH: The effect of the two compounds on forskolin-induced cAMP formation in intact cells or GTP-gamma-S binding to cell membranes, and their action on nociception in vivo was determined. KEY RESULTS: CB-25 enhanced forskolin-induced cAMP formation in N18TG2 cells (EC50 approximately 20 nM, max. stimulation = 48%), behaving as an inverse CB1 agonist, but it stimulated GTP-gamma-S binding to mouse brain membranes, behaving as a partial CB1 agonist (EC50 =100 nM, max. stimulation = 48%). At human CB1 receptors, CB-25 inhibited cAMP formation in hCB1-CHO cells (EC50 = 1600 nM, max. inhibition = 68% of CP-55,940 effect). CB-52 inhibited forskolin-induced cAMP formation by N18TG2 cells (IC50 = 450 nM, max. inhibition = 40%) and hCB1-CHO cells (EC50 = 2600 nM, max. inhibition = 62% of CP-55,940 effect), and stimulated GTP-gamma-S binding to mouse brain membranes (EC50 = 11 nM, max. stimulation approximately 16%). Both CB-25 and CB-52 showed no activity in all assays of CB2-coupled functional activity and antagonized CP55940-induced stimulation of GTP-gamma-S binding to hCB2-CHO cell membranes. In vivo, both compounds, administered i.p., produced dose-dependent nociception in the plantar test carried out in healthy rats, and antagonised the anti-nociceptive effect of i.p. WIN55,212-2. In the formalin test in mice, however, the compounds counteracted both phases of formalin-induced nociception. CONCLUSIONS AND IMPLICATIONS: CB-25 and CB-52 behave in vitro mostly as CB1 partial agonists and CB2 neutral antagonists, whereas their activity in vivo might depend on the tonic activity of cannabinoid receptors.     

Design of potent dicyclic (4-10/5-8) gonadotropin releasing hormone (GnRH) antagonists

With the ultimate goal of identifying a consensus bioactive conformation of GnRH antagonists, the compatibility of a number of side chain to side chain bridges in bioactive analogues was systematically explored. In an earlier publication, cyclo[Asp(4)-Dpr(10)]GnRH antagonists with high potencies in vitro and in vivo had been identified.(1) Independently from Dutta et al. (2) and based on structural considerations, the cyclic [Glu(5)-Lys(8)] constraint was also found to be tolerated in GnRH antagonists. We describe here a large number of cyclic (4-10) and (5-8) and dicyclic (4-10/5-8) GnRH antagonists optimized for affinity to the rat GnRH receptor and in vivo antiovulatory potency. The most potent monocyclic analogues were cyclo(4-10)[Ac-DNal(1), DFpa(2),DTrp(3),Asp(4),DArg(6),Xaa(10)]GnRH with Xaa = D/LAgl (1, K(i) = 1.3 nM) or Dpr (2, K(i) = 0.36 nM), which completely blocked ovulation in cycling rats after sc administration of 2.5 microgram at noon of proestrus. Much less potent were the closely related analogues with Xaa = Dbu (3, K(i) = 10 nM) or cyclo(4-10)[Ac-DNal(1), DFpa(2),DTrp(3),Glu(4),DArg(6),D/LAgl(10)]GnRH (4, K(i) = 1.3 nM). Cyclo(5-8)[Ac-DNal(1),DCpa(2),DTrp(3),Glu(5),DArg++ +(6),Lys(8), DAla(10)]GnRH (13), although at least 20 times less potent in the AOA than 1 or 2 with similar GnRHR affinity (K(i) = 0.84 nM), was found to be one of the most potent in a series of closely related cyclo(5-8) analogues with different bridge lengths and bridgehead chirality. The very high affinity of cyclo(5,5'-8)[Ac-DNal(1), DCpa(2),DPal(3),Glu(5)(betaAla),DArg(6),(D or L)Agl,(8)DAla(10)]GnRH 14 (K(i) = 0.15 nM) correlates well with its high potency in vivo (full inhibition of ovulation at 25 microgram/rat). Dicyclo(4-10/5-8)[Ac-DNal(1),DCpa(2),DTrp(3),Asp (4),Glu(5),DArg(6), Lys(8),Dpr(10)]GnRH (24, K(i) = 0.32 nM) is one-fourth as potent as 1 or 2, in the AOA; this suggests that the introduction of the (4-10) bridge in 13, while having little effect on affinity, restores functional/conformational features favorable for stability and distribution. To further increase potency of dicyclic antagonists, the size and composition of the (5-8) bridge was varied. For example, the substitution of Xbb(5') by Gly (30, K(i) = 0.16 nM), Sar (31, K(i) = 0.20 nM), Phe (32, K(i) = 0.23 nM), DPhe (33, K(i) = 120 nM), Arg (36, K(i) = 0.20 nM), Nal (37, K(i) = 4.2 nM), His (38, K(i) = 0.10 nM), and Cpa (39, K(i) = 0.23 nM) in cyclo(4-10/5,5'-8)[Ac-DNal(1),DCpa(2),DPal(3),Asp(4),G lu(5)(Xbb(5')), DArg(6),Dbu,(8)Dpr(10)]GnRH yielded several very high affinity analogues that were 10, ca. 10, 4, >200, 1, ca. 4, >2, and 2 times less potent than 1 or 2, respectively. Other scaffolds constrained by disulfide (7, K(i) = 2.4 nM; and 8, K(i) = 450 nM), cyclo[Glu(5)-Aph(8)] (16, K(i) = 20 nM; and 17, K(i) = 0.28 nM), or cyclo[Asp(5)-/Glu(5)-/Asp(5)(Gly(5'))-Amp(8)] (19, K(i) = 1.3 nM; 22, K(i) = 3.3 nM; and 23, K(i) = 3.6 nM) bridges yielded analogues that were less potent in vivo and had a wide range of affinities. The effects on biological activity of substituting DCpa or DFpa at position 2, DPal or DTrp at position 3, and DArg, DNal, or DCit at position 6 are also discussed. Interestingly, monocyclo(5-8)[Glu(5), DNal(6),Lys(8)]GnRH (18, K(i) = 1. (ABSTRACT TRUNCATED)     

Structure and function of eritadenine and its 3-deaza analogues: potent inhibitors of S-adenosylhomocysteine hydrolase and hypocholesterolemic agents

d-Eritadenine (DEA) is a potent inhibitor of S-adenosyl-l-homocysteine hydrolase (SAHH) and has hypocholesterolemic activity. We have hypothesized that 3-deaza-DEA (C3-DEA) and its analogues retain high level of SAHH inhibitory activity and have resistance to deamination and glycosidic bond hydrolysis in vivo. Such C3-DEA analogues would have much higher hypocholesterolemic activity. C3-DEA, and its methyl ester (C3-OMeDEA) and its methyl amido (C3-NMeDEA) were synthesized to examine their SAHH inhibitory and hypocholesterolemic activities. A crystal structure of SAHH containing C3-DEA was determined and confirmed that DEA and C3-DEA bound to the same site of SAHH with the same binding mode. The SAHH inhibitory activities of C3-DEA (K(I)=1.5 microM) and C3-OMeDEA (K(I)=1.5 microM) are significantly lower than that of DEA (K(I)=30 nM), while rats fed by C3-DEA and C3-OMeDEA decrease the total plasma cholesterol and phospholipids by 36-40% and 23%, respectively, which is similar to the level of reductions (42% and 27%) by DEA. C3-NMeDEA lost most of the SAHH inhibitory activity (K(I)=30 microM) and dietary C3-NMeDEA does not decrease cholesterol and phospholipid in plasma but decreases the triacylglycerol level by 16%. DEA and C3-DEA analogues are neither substrates nor inhibitors of adenosine deaminase.     

2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors

The affinity and efficacy at four subtypes (A(1), A(2A), A(2B) and A(3)) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N(6)-position, several 2-substituents were found to be critical structural determinants for the A(3)AR activation. The following adenosine 2-ethers were moderately potent partial agonists (K(i), nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A(3)AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)adenosine as an A(3)AR antagonist right-shifted the concentration-response curve for the inhibition by NECA of cyclic AMP accumulation with a K(B) value of 212 nM, which is similar to its binding affinity (K(i) = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A(1)AR in comparison to the A(3)AR, but fully efficacious, with binding K(i) values over 100 nM. The 2-phenylethyl moiety resulted in higher A(3)AR affinity (K(i) in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (K(i) = 3.8 nM) was found to be the most potent and selective (>50-fold) A(2A) agonist in this series. Mixed A(2A)/A(3)AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A(2B)AR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC(50) = 1.4 microM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC(50) = 1.8 microM) were found to be relatively potent A(2B) agonists, although less potent than NECA (EC(50) = 140 nM).     

Design of 2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7-dipropylaminopyrazolo[1,5-a]pyrimidine (NBI 30775/R121919) and structure--activity relationships of a series of potent and orally active corticotropin-releasing factor receptor antagonists

We have previously shown that 3-phenylpyrazolo[1,5-a]pyrimidines exemplified by 8 were potent antagonists of the human corticotropin-releasing factor-1 receptor. A series of 3-pyridylpyrazolo[1,5-a]pyrimidines 15, 25-30, 34, and 35 containing a weakly basic pyridine ring at the 3-position of the bicyclic nucleus was designed to reduce lipophilicity from the initial leads such as 7. Here, we showed that these 3-pyridyl compounds exhibited potent antagonists at the human CRF(1) receptor. Moreover, the hydrophilic and weakly basic pyridine moiety increased the water solubility of some analogues. Compound 26 h exhibited good binding affinity at the human CRF(1) receptor with a K(i) value of 3.5 nM. As a functional antagonist, it dose-dependently inhibited CRF-stimulated cAMP production in cells expressing the CRF(1) receptor (IC(50) = 50 nM), and CRF-stimulated ACTH release from cultured rat pituitary cells (IC(50) = 20 nM). 26 h had a log P value of 4.9 and water solubility of greater than 10 mg/mL. Pharmacokinetic studies in rats showed that 26 h was orally bioavailable and able to penetrate into the brain. 26 h has been demonstrated in vivo efficacy in animal behavioral models that measure anxiolytic activity. These results suggest that analogues from this series were potent CRF(1) receptor antagonists with proper physicochemical properties and good pharmacokinetic profiles. 26 h was developed into a clinical compound and exhibited efficacy in patients with major depression.     

Structure-activity relationships at the monoamine transporters and sigma receptors for a novel series of 9-[3-(cis-3, 5-dimethyl-1-piperazinyl)propyl]carbazole (rimcazole) analogues.

9-[3-(cis-3,5-Dimethyl-1-piperazinyl)propyl]carbazole (rimcazole) has been characterized as a sigma receptor antagonist that binds to the dopamine transporter with moderate affinity (K(i) = 224 nM). Although the binding affinities at the dopamine transporter of rimcazole and cocaine are comparable, rimcazole only depressed locomotor activity in mice and antagonized the stimulant effects produced by cocaine. The neurochemical mechanisms underlying the attenuation of cocaine's effects are not understood, although interaction at a low affinity site/state of the dopamine transporter has been suggested. To explore further this class of compounds, a series of rimcazole analogues was designed and synthesized. Displacement of [(3)H]WIN 35,428 binding at the dopamine transporter in rat caudate-putamen revealed that aromatic substitutions on rimcazole were not well tolerated, generally, with significant reductions in affinity for the 3,6-dibromo (5; K(i) = 3890 nM), 1,3, 6-tribromo (6; K(i) = 30300 nM), 3-amino (8; K(i) = 2400 nM), and 3, 6-dinitro (9; K(i) = 174000 nM) analogues. The N-phenylpropyl group was the only terminal piperazine nitrogen substituent that retained moderate affinity at the dopamine transporter (11; K(i) = 263 nM). Analogues in which the carbazole ring was replaced with a freely rotating diphenylamine moiety were also prepared. Although the diphenylamino analogue in which the terminal piperazine nitrogen was unsubstituted, as in rimcazole, demonstrated relatively low binding affinity at the dopamine transporter (24; K(i) = 813 nM), the N-phenylpropyl analogue was found to have the highest affinity for the dopamine transporter within the series (25; K(i) = 61.0 nM). All of the analogues that had affinity for the dopamine transporter inhibited [(3)H]dopamine uptake in synaptosomes, and potencies for these two effects showed a positive correlation (r(2) = 0.7731, p = 0.0018). Several of the analogues displaced [(3)H]paroxetine from serotonin transporters with moderate to high affinity, with the N-phenylpropyl derivative (11) having the highest affinity (K(i) = 44.5 nM). In contrast, none of the analogues recognized the norepinephrine transporter with an affinity of <1.3 microM. Binding affinities for sigma(1) and sigma(2) receptors were also determined, and several of the compounds were more potent than rimcazole with affinities ranging from 97 nM to >6 microM at sigma(1) sites and 145 to 1990 nM at sigma(2) sites. The compound with the highest affinity (25) at sigma(1) sites was also the compound with highest affinity at the dopamine transporter. These novel rimcazole analogues may provide important tools with which to characterize the relationship between the low affinity site or state of the dopamine transporter, sigma receptors, and their potential roles in modulating cocaine's psychostimulant actions.     

Synthesis and evaluation of 17-aliphatic heterocycle-substituted steroidal inhibitors of 17alpha-hydroxylase/C17-20-lyase (P450 17)

In the search for potent inhibitors of P450 17, the key enzyme in androgen biosynthesis, a series of steroidal inhibitors were synthesized and tested toward rat and human P450 17. Small aliphatic heterocycles (aziridine, oxirane, thiirane, diaziridine, diazirine, azetidine) were introduced into the 17beta-position of anstrost-5-en-3beta-ol. After identifying that aziridine is the most suitable functional group to coordinate with the heme iron, modifications of the steroidal skeleton were performed for further optimization. A wide range of inhibitory potencies toward P450 17 were found for the 21 test compounds. The most potent inhibitors toward the human and rat enzyme were aziridine compounds 3 (IC(50) rat: 0.21 microM, K(i) = 3 nM; IC(50) human: 0.54 microM, K(i) = 8 nM), 5 (IC(50) rat: 0.43 microM, K(i) = 7 nM; IC(50) human: 0.29 microM, K(i) = 4 nM), and 8 (21R:21S = 1:1; IC(50) rat: 0.53 microM, K(i) = 9 nM; IC(50) human: 0.40 microM, K(i) = 6 nM) which were more potent than the reference ketoconazole (IC(50) rat: 67 microM; IC(50) human: 0.74 microM). The inhibitory potency depends markedly on the stereochemistry at C20 of the inhibitors. This effect is more pronounced for the rat enzyme. Tested for selectivity, the highly potent inhibitors show poor inhibitory activity toward P450 arom, P450 scc, P450 TxA(2), and 5alpha-reductase. Tested for in vivo activity, 3 and 8 (0.019 mmol/kg) decreased the plasma testosterone concentration in rats by 81% and 84% after 2 h.     

Effects of YM218, a nonpeptide vasopressin V1A receptor-selective antagonist, on human vasopressin and oxytocin receptors.

The binding and signal transduction characteristics of YM218 ((Z)-4'-{4,4-difluoro-5-[2-oxo-2-(4-piperidinopiperidino)ethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl}-2-methyl-3-furanilide hemifumarate), a newly synthesized, potent arginine vasopressin (AVP) V(1A) receptor-selective antagonist, were examined using cloned human AVP receptors (V(1A), V(1B) and V(2)) stably expressed in Chinese hamster ovary (CHO) cells and human uterine smooth muscle cells (USMCs) expressing oxytocin receptors. YM218 potently inhibited specific binding of [(3)H] AVP to V(1A) receptors, exhibiting a K(i) value of 0.30 nM. In contrast, YM218 exhibited much lower affinity for V(1B), V(2) and oxytocin receptors, exhibiting K(i) values of 25,500 nM, 381 nM and 71.0 nM, respectively. In CHO cells expressing V(1A) receptors, YM218 potently inhibited the AVP-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), exhibiting an IC(50) value of 0.25 nM. However, in human USMCs expressing oxytocin receptors, YM218 exhibited a much lower potency in inhibiting the oxytocin-induced [Ca(2+)](i) increase, showing an IC(50) value of 607 nM, and had no effect on the AVP-induced [Ca(2+)](i) increase in CHO cells expressing V(1B) receptors. Furthermore, in CHO cells expressing V(2) receptors, YM218 did not potently inhibit the production of cAMP stimulated by AVP, showing an IC(50) value of 62.2 nM. In all assays used, YM218 did not exhibit any agonistic activity. These results demonstrate that YM218 is a potent, nonpeptide human V(1A) receptor-selective antagonist, and that YM218 will be a valuable new tool to gain further insight into the physiologic and pharmacologic actions of AVP.     

PD 158771, a potential antipsychotic agent with D(2)/D(3) partial agonist and 5-HT(1A) agonist actions. I. Neurochemical effects

The neurochemical effects of a novel dopamine (DA) D(2)-like and serotonin (5-HT) 5-HT(1A) agonist, PD 158771, are described. PD 158771 exhibited affinities for human D(2L), D(3) and D(4.2) receptors expressed in Chinese hamster ovary (CHO)-K1 cells with K(i) (nM) values of 5.2, 13.7 and 34.8 respectively. PD 158771 showed high affinity for cloned human 5-HT(1A) (K(i) = 2.6 nM) and rat hippocampal 5-HT(1A) receptors (K(i) = 3.5 nM). Weaker affinities were observed at alpha 1-adrenergic (K(i) = 43 nM), histamine H(1) (IC(50) = 30 nM), 5-HT(2A) (K(i) = 24.5 nM) and sigma (sigma) -1 binding sites (K(i) = 24.5 nM). In measures of in vitro functional activity, PD 158771 stimulated [(3)H]thymidine uptake in CHO p-5 cells transfected with hD(3) receptors with a maximal effect of 23% relative to quinpirole. In hD(2)L, the corresponding value was 60% with an EC(50) of 29 nM, again indicating partial DA agonist action of PD 158771. In vivo, PD 158771 produced a dose-related decrease in DA synthesis in the striatum and mesolimbic regions of rat brain treated with gamma-butyrolactone (GBL), indicating a DA autoreceptor agonist action. In animals not treated with GBL, PD 158771 produced a dose-related decrease in DA synthesis and extracellular DA. A decrease in 5-HT synthesis in several brain areas was observed consistent with an agonist response. Further support for DA autoreceptor agonist action is that PD 158771 produced a partial inhibition of the firing of substantia nigra zona compacta DA neurons, an effect reversed by haloperidol. In conclusion, PD 158771 exhibited affinities for DA and 5-HT receptors, appears to possess DA and 5-HT agonist actions; and it could provide improved antipsychotic profile with minimal side effects.