Conformationally-flexible benzamide analogues as dopamine D3 and sigma 2 receptor ligands

A series of conformationally-flexible analogues was prepared and their affinities for D2-like dopamine (D2, D3 and D4) were determined using in vitro radioligand binding assays. The results of this structure-activity relationship study identified one compound, 15, that bound with high affinity (K(i) value=2nM) and moderate selectivity (30-fold) for D3 compared to D2 receptors. In addition, this series of compounds were also tested for affinity at sigma1 and sigma2 receptors. We evaluated the affinity of these dopaminergic compounds at sigma receptors because (a) several antipsychotic drugs, which are high affinity antagonists at dopamine D2-like receptors, also bind to sigma receptors and (b) sigma receptors are expressed ubiquitously and at high levels (picomoles per mg proteins). It was observed that a number of analogues displayed high affinity and excellent selectivity for sigma2 versus sigma1 receptors. Consequently, these novel compounds may be useful for characterizing the functional role of sigma2 receptors and for imaging the sigma2 receptor status of tumors in vivo with PET.     

Synthesis and evaluation of optically pure [3H]-(+)-pentazocine, a highly potent and selective radioligand for sigma receptors

Tritium-labeled (+)-pentazocine ([3H]-1b) of specific activity 26.6 Ci/mmol was synthesized in 3 steps starting with (+)-normetazocine (2) of defined optical purity. [3H]-1b has been characterized as a highly selective ligand for labeling of sigma receptors. Competition data revealed that [3H]-1b could be displaced from guinea pig brain membrane preparations with a number of commonly used sigma receptor ligands. [3H]-1b exhibited saturable, enantioselective binding with a Kd of 5.13 +/- 0.97 nM and a Bmax of 1146 +/- 122 fmol/mg protein. Phencyclidine (PCP) displaced [3H]-1b with low affinity while MK-801 was inactive, thus indicating insignificant activity at the PCP-binding site; apomorphine failed to displace [3H]-1b indicating lack of dopamine receptor cross-reactivity. Since the affinity of [3H]-1b is about 6 times that of the two commonly employed sigma ligands ((+)-3-[3H]PPP and [3H]DTG) and since it is more selective for sigma receptors than the benzomorphan [3H]SKF-10,047, it represents the first example of a highly selective benzomorphan based sigma receptor ligand. [3H]-1b should prove useful for further study of the structure and function of sigma receptors.     

Tricyclic pyrazoles. Part 2: Synthesis and biological evaluation of novel 4,5-dihydro-1H-benzo[g]indazole-based ligands for cannabinoid receptors

A series of 4,5-dihydro-1H-benzo[g]indazole-3-carboxamides (2a-k) as analogues of the previously reported CB(2) ligands 6-chloro- and 6-methyl-1-(2',4'-dichlorophenyl)-N-piperidin-1-yl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamides (1a,b) was synthesized and their affinity and selectivity towards CB(1) and CB(2) receptors were evaluated. Several of the new compounds (2a,b,c,d and i) exhibited CB(1) affinity in the nanomolar range with moderate or negligible affinity towards CB(2) receptors. Compounds 2a and c increased intestinal propulsion in mouse. Their pro-kinetic effects were reversed by the reference CB agonist CP-55,940. Consequently, in vivo CB(1) antagonistic activity was highlighted for these compounds.     

Substituted 1-phenyl-2-cyclopropylmethylamines with high affinity and selectivity for sigma sites

A series of 1-phenyl-2-cyclopropylmethylamines structurally related to (+)- and (-)-MPCB were synthesized and their binding affinities for sigma1, sigma2, opioid and dopamine (D2) receptors were evaluated. Substitution of the cis-N-normetazocine with different aminic moieties provided compounds with high affinity and selectivity for sigma binding sites with respect to opioid and dopamine (D2) receptors. The observed increase in sigma2 affinity as compared to the parent (+)-MPCB, supports the idea that the particular stereochemistry of (+)-cis-N-normetazocine affects sigma1 selectivity but does not affect sigma1 affinity. The (+/-)-cis isomers of methyl 2-[(1-adamantylamino)methyl]-1-phenylcyclopropane-1-carboxyl ate (18) displayed a higher affinity and selectivity for the sigma1 and sigma2 receptor subtypes compared to the (+/-)-trans 19. Interestingly, the enantiomer (-)-cis 18 displayed a preference for sigma1 receptor subtype whereas the (+)-cis 18 did for sigma2. These results prompt us to synthesize compounds with modification of nitrogen and carboxyl groups. The compounds obtained showed high affinities and selectivity for sigma sites. Moreover, modifications of carboxyl groups provided compounds with the highest affinities in the series. In particular, compound 25 with reverse-type ester showed a Ki of 0.6 and 4.05 nM for sigma1 and sigma2 binding sites, respectively.     

A multireceptorial binding reinvestigation on an extended class of sigma ligands: N-[omega-(indan-1-yl and tetralin-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine reveal high affinities towards sigma1 and EBP sites

New 1-[omega-(2,3-dihydro-1H-inden-1-yl)- and (2,3-dihydro-5-methoxy-1H-inden-1-yl)alkyl]- and 1-[omega-(1,2,3,4-tetrahydronaphthalen-1-yl)- and (6-methoxy- or 6-fluoro-1,2,3,4-tetrahydronaphthalen-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine were synthesized, as homologous compounds of an existing series of sigma ligands, in order to carry out sigma receptor subtypes structure-affinity relationships. The new compounds and some of their related analogues, already reported, were tested in new multireceptorial radioligand binding assays. As reference compounds, the known sigma(1) ligands SA 4503, BD 1008 and NE 100 were also prepared and tested. All reported compounds showed high sigma(1) affinity assayed by (+)-[(3)H]-pentazocine on guinea-pig brain (apparent K(i)=1.75-72.2 nM) and moderate or low sigma(2) affinity by [(3)H]-DTG on rat liver, in contrast with previous results. One tertiary amine function spaced by a five-membered chain from a phenyl group is the structural feature shared by the most active compounds 26 and 43 and some reference sigma(1) ligands. The reported sigma(1) ligands, including reference compounds, also demonstrated a high affinity towards EBP (Delta(8)-Delta(7) sterol isomerase) site (apparent K(i)=0.48-14.8 nM) and some of them (37 and 44) were good ligands at L-type Ca(++) channel. 1-[4-(2,3-Dihydro-1H-inden-1-yl)butyl]-3,3-dimethylpiperidine (26) was the best mixed sigma(1) and EBP ligand (apparent K(i)=1.75 and 1.54 nM, respectively) with a good selectivity versus sigma(2) receptor (138- and 157-fold, respectively).     

2,3,7-Trisubstituted pyrazolo[1,5-d][1,2,4]triazines: functionally selective GABAA alpha3-subtype agonists

Novel synthetic routes have been devised for the preparation of previously inaccessible 2,3,7-trisubstituted pyrazolo[1,5-d][1,2,4]triazines 2. These compounds are high affinity ligands for the GABA(A) benzodiazepine binding site and some analogues show functional selectivity for agonism at alpha3-containing receptors over alpha1-containing receptors with the lead compound being 32.     

Synthesis of N-substituted 9-azabicyclo[3.3.1]nonan-3alpha-yl carbamate analogs as sigma2 receptor ligands

A series of N-substituted 9-azabicyclo[3.3.1]nonan-3alpha-yl phenylcarbamate analogs was prepared and their affinities for sigma (sigma(1) and sigma(2)) receptors were measured in vitro. The results of their structure-activity relationship study identified two new compounds, N-(9-(4-aminobutyl)-9-azabicyclo[3.3.1]nonan-3alpha-yl)-N'-(2-methoxy-5-methylphenyl)carbamate and N-(9-(6-aminohexyl)-9-azabicyclo[3.3.1]nonan-3alpha-yl)-N'-(2-methoxy-5-methylphenyl)carbamate, having a high affinity and selectivity for sigma(2) versus sigma(1) receptors. These compounds were also used in the preparation of biotinylated and fluorescent probes of the sigma(2) receptor.     

Design and synthesis of a piperazinylalkylisoxazole library for subtype selective dopamine receptor ligands

A piperazinylbutylisoxazole libary was designed, synthesized and screened for the binding affinities to dopamine D2, D3, and D4 receptors. Several ligands were identified to possess high binding affinity and selectivity for the D3 and D4 receptors over the D2 receptor. Compounds 6s and 6t showed K(i) values of 2.6 nM and 3.9 nM for the D3 receptor with 46- and 50-fold selectivity over the D2 receptor, respectively.     

Synthesis and pharmacological evaluation of substituted naphth[1,2,3-de]isoquinolines (dinapsoline analogues) as D1 and D2 dopamine receptor ligands

Dinapsoline ((2); (+/-)-dihydroxy-2,3,7,11b-tetrahydro-1H-naphth[1,2,3-de]isoquinoline) is a full D(1) dopamine agonist that also has significant D(2) receptor affinity. Based on a similar pharmacophore, dinapsoline has pharmacological similarities to dihydrexidine ((1); (+/-)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine), the first high affinity full D(1) agonist. Small alkyl substitutions on the dihydrexidine backbone are known to alter markedly the D(1):D(2) selectivity of dihydrexidine, and it was of interest to determine whether similar SAR exists within the dinapsoline series. This report describes the synthesis and pharmacological evaluation of six analogues of dinapsoline: N-allyl-(3);N-n-propyl- (4); 6-methyl- (5); 4-methyl- (6); 4-methyl-N-allyl- (7); and 4-methyl-N-n-propyl-dinapsoline (8). As expected from earlier studies with the dihydrexidine backbone, N-allyl (3) or N-n-propyl (4) analogues had markedly decreased D(1) affinity. Unexpectedly, and unlike the dihydrexidine series, these same substituents did not markedly increase D(2) affinity. The addition of a methyl group to position 6 (5) increased D(1):D(2) selectivity, but less markedly than did the analogous 2-methyl substituent added to 1. Unlike the analogous 4-methyl substituent of 1, the addition of a 4-methyl-group (6) actually decreased D(1) affinity without affecting D(2) affinity. These data demonstrate that the dinapsoline (2) backbone can be modified to produce dopamine agonists with novel properties. Moreover, as rigid ligands in which small substituents can cause significant changes in selectivity, they are important tools for deriving 'differential' SARs of the dopamine receptor isoforms.     

High-affinity carbamate analogues of morphinan at opioid receptors

A series of carbamate analogues were synthesized from levorphanol (1a), cyclorphan (2a) or butorphan (3a) and evaluated in vitro for their binding affinity at mu, delta, and kappa opioid receptors. Functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. Phenyl carbamate derivatives 2d and 3d showed the highest binding affinity for kappa receptor (K(i)=0.046 and 0.051 nM) and for mu receptor (K(i)=0.11 and 0.12 nM). Compound 1c showed the highest mu selectivity. The preliminary assay for agonist and antagonist properties of these ligands in stimulating [(35)S]GTPgammaS binding mediated by the kappa opioid receptor illustrated that all of these ligands were kappa agonists. At the mu receptor, compounds 1b, 1c, 2b, and 3b were agonists, while compounds 2c-e and 3c-e were mu agonists/antagonists.     

Phenolic derivatives from Wigandia urens with weak activity against the chemokine receptor CCR5

Three compounds, 2,3-dihydroxy-4-methoxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (1), 8-methoxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-6-ol (2) and 4-methoxy-3-(3-methyl-2-butenyl)-benzoic acid (3), have been isolated from Wigandia urens. The structures of compounds 1, 2 and 3 were determined from spectroscopic data and showed activity in a CCR5 assay with IC(50) values of 33, 46 and 26 muM respectively.     

Synthesis and structure-affinity relationships of novel spirocyclic sigma receptor ligands with furopyrazole structure

The synthesis of novel spirocyclic sigma receptor ligands with high affinity is described. The cyclization of the hydroxy acetal 8, which represents a key step in the synthesis of the spirocyclic compounds 3, was supported by theoretical considerations. The affinity of the spirocyclic furopyrazoles 3a-c to the sigma receptors was determined in receptor binding studies with radioligands. The N-benzyl (3b) and N-butyl (3c) derivatives display very high sigma(1) receptor affinity (3b, K(i)=0.50 nM; 3c, K(i)=1.28 nM) and high selectivity toward the sigma(2) receptor and some other receptor systems. Calculation of crucial distances of the spirocyclic furopyrazole derivatives 3b and 3c shows good correlation with the pharmacophore model of Glennon.     

Synthesis of new 1,2,3-benzotriazin-4-one-arylpiperazine derivatives as 5-HT1A serotonin receptor ligands

A series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radioligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5-HT1A receptor, some of which were selective with respect 5-HT2A and 5-HT2C receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected and further evaluated for their binding affinities on D1, D2 dopaminergic and alpha1-, alpha2-adrenergic receptors. A o-OCH3 derivative (2e) bound at 5-HT1A sites with subnanomolar affinity (IC50 = 0.059 nM) and shows high selectivity over all considered receptors and may offer a new lead for the development of therapeutically efficacious agents.     

ERbeta ligands. Part 6: 6H-Chromeno[4,3-b]quinolines as a new series of estrogen receptor beta-selective ligands

A new class of estrogen receptor beta (ERbeta) ligands based on the 6H-chromeno[4,3-b]quinoline scaffold has been prepared. Several C7-substituted analogues displayed high affinity and modest selectivity for ERbeta.     

Chiral, nonracemic (piperazin-2-yl)methanol derivatives with sigma-receptor affinity

Starting with the proteinogenic amino acid (S)-serine a series of chiral nonracemic (piperazin-2-yl)methanols 3 with various N-4 substituents is described. The key step in the synthesis of 3 is the reaction of the chloroacetamide 5 with various primary amines to yield the diastereomeric bicyclic piperazinediones cis-6 and trans-6. The scope and limitation of this transformation is thoroughly investigated. The alpha1- and sigma2-receptor affinities of the piperazines 3 are determined in receptor binding studies with guinea pig brain and rat liver membrane preparations using [3H]-labeled (+)-pentazocine and ditolylguanidine, respectively. It was found, that an additional phenyl residue in the N-4 substituent is favorable to high sigma1-receptor affinity. In this series the p-methoxybenzyl substituted piperazine 3d reveals the highest sigma1-receptor affinity (Ki=12.4 nM) with selectivity toward sigma2-, NMDA-, kappa-opioid, and mu-opioid receptors.     

8,9-dihydroxy-1,2,3,11b-tetrahydrochromeno[4,3,2,-de]isoquinoline (dinoxyline), a high affinity and potent agonist at all dopamine receptor isoforms

The synthesis and preliminary pharmacological evaluation of 8,9-dihydroxy-1,2,3,11b-tetrahydrochromeno[4,3,2,-de]isoquinoline (5, now named dinoxyline) is described. This molecule was designed as a potential bioisostere that would conserve the essential elements of our beta-phenyldopamine D(1) pharmacophore (i.e., position and orientation of the nitrogen, hydroxyls, and phenyl rings). Previously, we have rigidified these elements using alkyl bridges, as exemplified in the dopamine D(1) full agonist molecules dihydrexidine (1) and dinapsoline (2). This approach has been modified and we now show that it is possible to tether these elements using an ether linkage. Preliminary pharmacology has revealed that 5 is a potent full D(1) agonist (K(0.5) <10 nM; EC(50)=30 nM), but also has high affinity for brain D(2)-like and cloned D(2) and D(3) receptors. Interestingly, whereas 1 and 2 and their analogues have only moderate affinity for the human D(4) receptor, 5 also has high affinity for this isoform. Moreover, although N-alkylation of 1 and 2 increases D(2) affinity, the N-allyl (15) and N-n-propyl (17) derivatives of 5 had decreased D(2) affinity. Therefore, 5 may be engaging different amino acid residues than do 1 and 2 when they bind to the D(2) receptor. This is the first example of a ligand with high affinity at all dopamine receptors, yet with functional characteristics similar to dopamine. These rigid ligands also will be useful tools to determine specific residues of the receptor transmembrane domains that are critical for agonist ligand selectivity for the D(4) receptor.     

Design of high affinity cyclic pentapeptide ligands for kappa-opioid receptors.

Using results from our previously reported cyclic opioid peptide series and reliable models for mu-, delta-, and kappa-opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr-C[D-Cys-Phe-Phe-X]-NH2, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = D- or L-Cys; or D- or L-penicillamine (Pen; beta,beta-dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = D- or L-Cys display KOR affinities in the low nanomolar range, demonstrating that a 'DPDPE-like' tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8, Tyr-C[D-Cys-Phe-Phe-Cys]-NH2-cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm) with more than 100-fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Synthesis and characterization of selective dopamine D2 receptor antagonists

A series of indole compounds have been prepared and evaluated for affinity at D2-like dopamine receptors using stably transfected HEK cells expressing human D2, D3, or D4 dopamine receptors. These compounds share structural elements with the classical D2-like dopamine receptor antagonists, haloperidol, N-methylspiperone, and benperidol. The compounds that share structural elements with N-methylspiperone and benperidol bind non-selectively to the D2 and D3 dopamine receptor subtypes. However, several of the compounds structurally similar to haloperidol were found to (a) bind to the human D2 receptor subtype with nanomolar affinity, (b) be 10- to 100-fold selective for the human D2 receptor compared to the human D3 receptor, and (c) bind with low affinity to the human D4 dopamine receptor subtype. Binding at sigma (sigma) receptor subtypes, sigma1 and sigma2, were also examined and it was found that the position of the methoxy group on the indole was pivotal in both (a) D2 versus D3 receptor selectivity and (b) affinity at sigma1 receptors. Adenylyl cyclase studies indicate that our indole compounds with the greatest D2 receptor selectivity are neutral antagonists at human D2 dopamine receptor subtypes. With stably transfected HEK cells expressing human D2 (hD2-HEK), these compounds (a) have no intrinsic activity and (b) attenuated quinpirole inhibition of adenylyl cyclase. The D2 receptor selective compounds that have been identified represent unique pharmacological tools that have potential for use in studies on the relative contribution of the D2 dopamine receptor subtypes in physiological and behavioral situations where D2-like dopaminergic receptor involvement is indicated.     

Characterization of Novel Fluorescent Ligands with High Affinity for D1 and D2 Dopaminergic Receptors

We have synthesized and characterized a series of novel fluorescently labeled ligands with high affinity and specificity for D1 and D2 dopamine receptors. D1-selective probes were synthesized using (R,S)-5-(4'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl- [1H]-3-benzazepin-7-ol, the 4'-amino derivative of the high-affinity, D1-selective antagonist SCH-23390, whereas D2-selective probes were synthesized using the high-affinity, D2-selective antagonist N-(p-aminophenethyl)spiperone (NAPS). These ligands were coupled via spacer arms of various lengths to the fluorophores fluorescein and bodipy, which fluoresce in the yellow-green region, and to tetramethylrhodamine, which is a red fluorophore. The interaction of these fluorescent ligands with dopamine receptors was evaluated by examining their ability to compete for the binding of the radiolabeled antagonists [3H]SCH-23390 or [3H]methylspiperone to rat striatal D1 or D2 dopamine receptors, respectively. We report here that these novel fluorescent ligands exhibit very high affinity and specificity for either D1 or D2 dopamine receptors. The availability of various fluorescent ligands with different emission maxima and with high affinity and specificity for D1 and D2 dopamine receptors will now permit investigations involving the visualization and localization of these receptor subtypes at the single cell and intracellular levels in the CNS and on intact cells in culture.     

2-(N-acyl) and 2-N-acyl-N(6)-substituted analogues of adenosine and their affinity at the human adenosine receptors

A series of 2-(N-acyl) and 2-(N-acyl)-N(6)-alkyladenosine analogues have been synthesized from the intermediate 2-amino-6-chloroadenosine derivatives (2b and 7) and evaluated for their affinity at the human A(1), A(2A), and A(3) receptors. We found that 2-(N-acyl) derivatives of adenosine showed relatively low affinity at A(2A) and A(3) receptors, while the N(6)-cyclopentyl substituent in 4h and 4i induced high potency [A(1) (K(i))=20.7 and 31.8 nM respectively] at the A(1) receptor and resulted therefore in increased selectivity for this subtype. The general synthetic methods and their binding studies are presented herein.