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In searching for putative new therapeutic strategies to treat neurodegenerative diseases, the mitochondrial 18 kDa translocator protein (TSPO) and cerebral isoforms of carbonic anhydrase (CA) were exploited as potential targets. Based on the structures of a class of highly affine and selective TSPO ligands and a class of CA activators, both developed by us in recent years, a small library of 2-phenylindole-based dual TSPO/CA modulators was developed, able to bind TSPO and activate CA VII in the low micromolar/submicromolar range. The interaction with the two targets was corroborated by computational studies. Biological investigation on human microglia C20 cells identified derivative 3 as a promising lead compound worthy of future optimization due to its (i) lack of cytotoxicity, (ii) ability to stimulate TSPO steroidogenic function and activate CA VII, and (iii) ability to effectively upregulate gene expression of the brain-derived neurotrophic factor.

Saturated N -heterocycles constitute a vital scaffold for pharmaceutical chemistry but are challenging to access synthetically, particularly asymmetrically. Here, we demonstrate how imine reductases can achieve annulation through tandem inter- and intramolecular reductive amination processes. Imine reductases were used in combination with further enzymes to access unsubstituted, α-substituted, and α,α'-disubstituted N -heterocycles from simple starting materials in one pot and under benign conditions. This work shows the remarkable flexibility of these enzymes to have broad activity against numerous substrates derived from singlular starting materials., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Mitochondria dysfunctions are typical hallmarks of cardiac disorders (CDs). The multiple tasks of this energy-producing organelle are well documented, but its pathophysiologic involvement in several manifestations of heart diseases, such as altered electromechanical coupling, excitability, and arrhythmias, is still under investigation. The human 18 kDa translocator protein (TSPO) is a protein located on the outer mitochondrial membrane whose expression is altered in different pathological conditions, including CDs, making it an attractive therapeutic and diagnostic target. Currently, only a few TSPO ligands are employed in CDs and cardiac imaging. In this Perspective, we report an overview of the emerging role of TSPO at the heart level, focusing on the recent literature concerning the development of TSPO ligands used for fighting and imaging heart-related disease conditions. Accordingly, targeting TSPO might represent a successful strategy to achieve novel therapeutic and diagnostic strategies to unravel the fundamental mechanisms and to provide solutions to still unanswered questions in CDs.

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N -hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N -hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5'-acylation using Novozym 435.

Hydrogels are useful platforms as three-dimensional (3D) scaffolds for cell culture, drug-release systems, and regenerative medicine applications. Here, we propose a novel chemical cross-linking approach by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione or diethyl squarate for the preparation of 5 and 10% w/v gelatin-based hydrogels. Hydrogels showed good swelling properties, and the 5% gelatin-based hydrogel proved suitable as a 3D cell culture scaffold for the chondrocyte cell line C28/I2. In addition, diffusion properties of different sized molecules inside the hydrogel were determined.

Translocator protein 18 kDa (TSPO) is a biomarker of neuroinflammation. [ 11 C]ER176 robustly quantifies TSPO in the human brain with positron emission tomography (PET), irrespective of subject genotype. We aimed to develop an ER176 analog with potential for labeling with longer-lived fluorine-18 ( t 1/2 = 109.8 min). New fluoro and trifluoromethyl analogs of ER176 were prepared through a concise synthetic strategy. These ligands showed high TSPO affinity and low human genotype sensitivity. Each ligand was initially labeled by a generic 11 C-methylation procedure, thereby enabling speedy screening in mice. Each radioligand was rapidly taken up and well retained in the mouse brain at baseline after intravenous injection. Preblocking of TSPO showed that high proportions of brain uptake were specifically bound to TSPO at baseline. Overall, the 3-fluoro analog of [ 11 C]ER176 ([ 11 C] 3b ) displayed the most promising imaging properties. Therefore, a method was developed to label 3b with [ 18 F]fluoride ion. [ 18 F] 3b gave similarly promising PET imaging results and deserves evaluation in higher species.

Translocator protein 18 kDa (TSPO) is a validated pharmacological target for the development of new treatments for neurological disorders. N , N -Dialkyl-2-phenylindol-3-ylglyoxylamides (PIGAs) are effective TSPO modulators and potentially useful therapeutics for the treatment of anxiety, central nervous system pathologies featuring astrocyte loss, and inflammatory-based neuropathologies. For this class of compounds, no correlation exists between the TSPO binding affinity and the corresponding functional efficacy. Rather, their biological effectiveness correlates with the kinetics of the unbinding events and more specifically with the residence time (RT). So far, the structural reasons for the different recorded RT of congeneric PIGAs remain elusive. Here, to understand the different kinetics of PIGAs, their unbinding paths were studied by employing enhanced-sampling molecular dynamics simulations. Results of these studies revealed how subtle structural differences between PIGAs have a substantial effect on the unbinding energetics. In particular, during the egress from the TSPO binding site, slow-dissociating PIGAs find tight interactions with the protein LP1 region thereby determining a long RT. Further support to these findings was achieved by in vivo studies, which demonstrated how the anxiolytic effect observed for the inspected PIGAs correlated with their RT to TSPO.

In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5β1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4. Herein, we present the discovery of compound 7, which inhibits both MDM2/4 and α5β1/αvβ3 integrins. A lead optimization campaign was carried out on 7 with the aim to preserve the activities on integrins while improving those on MDM proteins. Compound 9 turned out to be a potent MDM2/4 and α5β1/αvβ3 blocker. In p53-wild type glioma cells, 9 arrested cell cycle and proliferation and strongly reduced cell invasiveness, emerging as the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in subpopulations of patients with glioblastoma expressing a functional p53 concomitantly with a high level of α5β1 integrin.

Glioblastoma (GBM) is characterized by a poor response to conventional chemotherapeutic agents, attributed to the insurgence of drug resistance mechanisms and to the presence of a subpopulation of glioma stem cells (GSCs). GBM cells and GSCs present, among others, an overexpression of antiapoptotic proteins and an inhibition of pro-apoptotic ones, which help to escape apoptosis. Among pro-apoptotic inducers, the Bcl-2 family protein Bax has recently emerged as a promising new target in cancer therapy along with first BAX activators (BAM7, Compound 106, and SMBA1). Herein, a derivative of BAM-7, named BTC-8, was employed to explore the effects of Bax activation in different human GBM cells and in their stem cell subpopulation. BTC-8 inhibited GBM cell proliferation, arrested the cell cycle, and induced apoptosis through the induction of mitochondrial membrane permeabilization. Most importantly, BTC-8 blocked proliferation and self-renewal of GSCs and induced their apoptosis. Notably, BTC-8 was demonstrated to sensitize both GBM cells and GSCs to the alkylating agent Temozolomide. Overall, our findings shed light on the effects and the relative molecular mechanisms related to Bax activation in GBM, and they suggest Bax-targeting compounds as promising therapeutic tools against the GSC reservoir.

The quinazoline class was exploited to search for a new translocator protein (TSPO) fluorescent probe endowed with improved affinity and residence time (RT). Computational studies on an "in-house" collection of quinazoline derivatives, featuring highly steric demanding groups at the amide nitrogen, suggested that, despite their molecular extension, these ligands are still easily lodged in the TSPO binding site. Binding assays supported this hypothesis, highlighting a low nanomolar/subnanomolar affinity of these ligands, together with a higher RT of the representative compound 11 with respect to our previously reported indole-based fluorescent probe. Thanks to the amenability of the amide nitrogen atom to be substituted with bulky groups, we developed quinazoline-based imaging tools by fluorescently labeling the scaffold at this position. Probes with relevant TSPO affinity, favorable spectroscopic properties, and improved RT were identified. The results from fluorescence microscopy showed that these probes specifically labeled the TSPO at the mitochondrial level in the U343 cell line.

The low binding affinity of the approved anxiolytic drug etifoxine (Stresam) at the steroidogenic 18 kDa translocator protein (TSPO) has questioned the specific contribution of this protein in mediating the etifoxine neurosteroidogenic efficacy. Residence time (RT) at the binding site of the classical TSPO ligand PK11195 is emerging as a relevant neurosteroidogenic efficacy measure rather than the binding affinity. Here etifoxine was evaluated for (i) the in vitro neurosteroidogenic activity in comparison to poorly neurosteroidogenic reference TSPO ligands (PK11195 and Ro5-4864) and (ii) the affinity and RT at [ 3 H]PK11195 and [ 3 H]Ro5-4864 binding sites in rat kidney membranes. Etifoxine shows (i) high neurosteroidogenic efficacy and (ii) low affinity/short RT at the [ 3 H]PK11195 site and low affinity/long RT at the [ 3 H]Ro5-4864 site, at which etifoxine competitively bound. These findings suggest that the long RT of etifoxine at the Ro5-4864 binding site could account for its high neurosteroidogenic efficacy.

Protein homodimers play important roles in physiological and pathological processes, including cancer invasion and metastasis. Recently, MMP-9 natural homodimerization via the PEX domain has been correlated with high migration rates of aggressive cancer cells. Here we propose that bifunctional MMP-9 inhibitors designed to impair natural MMP-9 homodimerization promoted by PEX-PEX interactions might be an effective tool to fight cancer cell invasion. Elaborating a previously described dimeric hydroxamate inhibitor 1 , new ligands were synthesized with different linker lengths and branch points. Evaluation of the modified bifunctional ligands by X-ray crystallography and biological assays showed that 7 and 8 could reduce invasion in three glioma cell lines expressing MMP-9 at different levels. To rationalize these results, we present a theoretical model of full-length MMP-9 in complex with 7 . This pioneering study suggests that a new approach using MMP-9 selective bifunctional inhibitors might lead to an effective therapy to reduce cancer cell invasion.

Recent data have demonstrated a positive correlation between the residence time (RT) and neurosteroidogenic efficacy of a ligand at the translocator protein (TSPO), an attractive anxyolitic target. To explore the potential impact of RT on TSPO ligand anxiolytic activity, the RT and the steroidogenic activity of XBD173, a ligand exerting anxiolytic activity in humans, were retrospectively evaluated. To this aim, XBD173 association and dissociation rate constants were measured (1.23 × 10(7) M(-1) min(-1) and 0.0079 min(-1), respectively). XBD173 resulted to have a long RT (127 min) and to stimulate efficaciously neurosteroidogenesis, in terms of pregnenolone production. The present findings corroborate the importance of TSPO ligand RT to predict their effective neurosteroidogenic activity and promising anxiolytic action. These positive results prompted us to set up a fast and high-throughput kinetic method to improve the efficiency of RT-based TSPO drug-discovery process.

Structure-activity relationships (SARs) within the 4-phenylquinazoline-2-carboxamide series of translocator protein (TSPO) ligands have been explored further by the synthesis and TSPO binding affinity evaluation of N-benzyl-N-ethyl/methyl derivatives variously decorated at the 6-, 2'-, 4'-, and 4″-positions. Most of the compounds showed high affinity with K(i) values in the nanomolar/subnanomolar range. A pharmacophore model was developed and employed to better address SAR data presented by the new TSPO ligands. A subset of the new compounds (5, 8, 12, and 19) were tested for their ability to inhibit the viability of human glioblastoma cell line U343. The observed antiproliferative effect was demonstrated to be specific for compound 19, endowed with the best combination of binding affinity and efficacy. Furthermore, the ability of 19 to induce mitochondrial membrane dissipation (Δψ(m)) substantiated the intracellular pro-apoptotic mechanism activated by the binding of this class of ligands to TSPO.

Despite the relevance of carbohydrates as cues in eliciting specific biological responses, the covalent surface modification of collagen-based matrices with small carbohydrate epitopes has been scarcely investigated. We report thereby the development of an efficient procedure for the chemoselective neoglycosylation of collagen matrices (patches) via a thiol-ene approach, between alkene-derived monosaccharides and the thiol-functionalized material surface. Synchrotron radiation-induced X-ray photoelectron spectroscopy (SR-XPS), Fourier transform-infrared (FT-IR), and enzyme-linked lectin assay (ELLA) confirmed the effectiveness of the collagen neoglycosylation. Preliminary biological evaluation in osteoarthritic models is reported. The proposed methodology can be extended to any thiolated surface for the development of smart biomaterials for innovative approaches in regenerative medicine.

New arylthioindole derivatives having different cyclic substituents at position 2 of the indole were synthesized as anticancer agents. Several compounds inhibited tubulin polymerization at submicromolar concentration and inhibited cell growth at low nanomolar concentrations. Compounds 18 and 57 were superior to the previously synthesized 5. Compound 18 was exceptionally potent as an inhibitor of cell growth: it showed IC₅₀ = 1.0 nM in MCF-7 cells, and it was uniformly active in the whole panel of cancer cells and superior to colchicine and combretastatin A-4. Compounds 18, 20, 55, and 57 were notably more potent than vinorelbine, vinblastine, and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines, which overexpress P-glycoprotein. Compounds 18 and 57 showed initial vascular disrupting effects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage. Derivative 18 showed water solubility and higher metabolic stability than 5 in human liver microsomes.

A series of novel 4-phenylquinazoline-2-carboxamides (1-58) were designed as aza-isosters of PK11195, the well-known 18 kDa translocator protein (TSPO) reference ligand, and synthesized by means of a very simple and efficient procedure. A number of these derivatives bind to the TSPO with K(i) values in the nanomolar/subnanomolar range, show selectivity toward the central benzodiazepine receptor (BzR) and exhibit structure-affinity relationships consistent with a previously published pharmacophore/topological model of ligand-TSPO interaction.

Synthesis of a library of pyrrolo[2,1-c][1,4]-benzodiazepines derived from spiro bicyclic D- or L-proline analogues containing a D- or L-fructose moiety was developed. The L-fructose moiety was obtained by using a new synthetic pathway starting from L-arabinose through a six steps synthesis in 18% overall yield. Molecular modeling calculations and DNMR studies showed that D- and L-fructose-based pyrrolobenzodiazepines exhibit a rigid (P)- and (M)-helical conformation, respectively, in which the C-11a substituent was always pseudoequatorial. Additionally, pyrrolobenzodiazepines functionalized with a chloride, bromide, nitro, or amino group in the benzene ring, with or without N-methylation and with or without protection of sugar alcohol groups, allowed a relationship between the molecular structure and biological activity to be established. The conformation of the diazepam ring was not the sole key player influencing binding affinities, and the sugar moiety can in some cases increase the binding activity, possibly by participating in the binding event. Finally, these compounds have increased the understanding of the differential recognition of (M)-/(P)-helical benzodiazepines on GABA(A) receptor.

Anxioselective agents may be identified among compounds binding selectively to the alpha(2)beta(x)gamma(2) subtype of the gamma-aminobutyric acid-A (GABA(A))/central benzodiazepine receptor (BzR) complex and behaving as agonists or among compounds binding with comparable potency to various BzR subtypes but eliciting agonism only at the alpha(2)beta(x)gamma(2) receptor. Because of subtle steric differences among BzR subtypes, the latter approach has proved much more successful. A biological screening within the class of indol-3-ylglyoxylamides 1-3 allowed us to identify compounds 1c and 2b as potential anxiolytic/nonsedative agents showing alpha(2) selective efficacy in vitro and anxioselective effects in vivo. According to molecular modeling studies, and consistently with SARs accumulated in the past decade, 5-NO(2)- and 5-H-indole derivatives would preferentially bind to BzR by placing the indole ring in the L(Di) and the L(2) receptor binding sites, respectively.

New glycolipids and a benzylammonium lipid were rationally designed by varying the chemical structure of a D-glucose-derived hit compound active as lipid A antagonist. We report the synthesis of these compounds, their in vitro activity as lipid A antagonists on HEK cells, and the capacity to inhibit LPS-induced septic shock in vivo. The lack of toxicity and the good in vivo activity suggest the use of some compounds of the panel as hits for antisepsis drug development.

Novel N,N-disubstituted indol-3-ylglyoxylamides (1-56), bearing different combinations of substituents R 1-R 5, were synthesized and evaluated as ligands of the translocator protein (TSPO), the 18 kDa protein representing the minimal functional unit of the "peripheral-type benzodiazepine receptor" (PBR). Most of the new compounds showed a nanomolar/subnanomolar affinity for TSPO and stimulated steroid biosynthesis in rat C6 glioma cells with a potency similar to or higher than that of classic TSPO ligands such as PK 11195. Moreover, when evaluated in vivo by means of the elevated-plus-maze (EPM) paradigm in the rat, compound 32, the best-performing derivative in terms of TSPO affinity and pregnenolone production, showed clear anxiolytic effects. The results of this study suggested that the novel N,N-disubstituted indol-3-ylglyoxylamides may represent a promising class of compounds potentially suited for the treatment of anxiety disorders.

In our pursuit to identify selective ligands for Bz/GABA(A) receptor subtypes, a novel pyrazolo[1,5-a]pyrimidine derivative (4), the azaisostere of zolpidem, was synthesized and evaluated in vitro on bovine brain homogenate and on recombinant benzodiazepine receptors (alphaxbeta2/3gamma2, x = 1-3, 5) expressed in HEK293 cells. Compound 4 displayed affinity only for alpha1beta2gamma2 subtype (K(i) = 31 nM), and in an in-depth, in vivo study it revealed sedative and anxiolytic-like properties without any amnesic and myorelaxant effects in rodents.

A series of 2-phenyl[1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-diones (PTBTs), VII, were prepared and tested at the central benzodiazepine receptor (BzR). The skeleton of these compounds was designed by formally combining the N-C=O moieties of the known BzR ligands, triazoloquinoxalines (IV) and triazinobenzimidazoles (ATBIs) (VI). Most of the PTBTs displayed submicromolar/nanomolar potency at the BzR. The 9-chloro derivatives (45-49) were generally found to be more potent than their 9-unsubstituted counterparts (37-44). Compound 45 turned out to be the most potent of the PTBTs (K(i) 2.8 nM). A subset of compounds (37, 42, 45, 49), when tested for their affinity on recombinant rat alpha1beta2gamma2, alpha2beta2gamma2, and alpha5beta3gamma2 GABA(A)/Bz receptor subtypes, showed enhanced affinities for the alpha1beta2gamma2 isoform, with compounds 45 and 49 exhibiting the highest selectivity. Moreover, compounds 45 and 49 were found to display a full agonist efficacy profile at alpha1 and alpha2 receptor subtypes, and an antagonist efficacy at alpha5-containing receptors.

The present work reports the synthesis of trans-2-amino-5(6)-fluoro-6(5)-hydroxy-1-phenyl-2,3-dihydro-1H-indenes (4a-f, 5a-f) as a continuation of our studies to better understand the significance of the halo substituent in the trans-1-phenyl-2-aminoindane series and to extend knowledge of the monophenolic ligands of DA receptors. The affinity of the new compounds and related methoxylated precursors (10-15 and 18-23) was estimated in vitro by displacement of [(3)H]SCH23390 (for D(1)-like receptors) or [(3)H]YM-09-151-2 (for D(2)-like receptors) from homogenates of porcine striatal membranes. The results indicate that unsubstituted amines 4a, 5a, 10, and 11 are poorly effective at DA receptors. The introduction of two n-propyl groups on the nitrogen atom (compounds 14, 15, 4c, and 5c) and N-allyl-N-methyl- or N-methyl-N-propyl- substitution (compounds 20-23, 4e, 4f, 5e, 5f) increased the D(2)-like affinities and selectivity. The D(2)-like agonistic activity of selected compounds 15, 20, 21, 4e, 5c, and 5e was proved by evaluating their effects on the cyclic guanosine monophosphate (cGMP) content in rat neostriatal membranes. All tested compounds displayed a potential dopamine D(2)-like agonist profile decreasing basal levels of cGMP. The selective D(2)-like agonism of compounds 20 and 5e was proved by their effects on basal striatal adenylyl cyclase activity.

A number of 3'-C-methyl analogues of selective adenosine receptor agonists such as CPA, CHA, CCPA, 2'-Me-CCPA, NECA, and IB-MECA was synthesized to further investigate the subdomain of the receptor that binds the ribose moiety of the ligands. Affinity data at A(1), A(2A), and A(3) receptors in bovine brain membranes showed that the 3'-C-modification in adenosine resulted in a decrease of the affinity at all three receptor subtypes. When this modification was combined with N(6)-substitution with groups that induce high potency and selectivity at A(1) receptor, the affinity and selectivity were increased. However, all 3'-C-methyl derivatives proved to be very less active than the corresponding 2'-C-methyl analogues. The most active compound was found to be 3'-Me-CPA which displayed a K(i) value of 0.35 microM at A(1) receptor and a selectivity for A(1) vs A(2A) and A(3) receptors higher than 28-fold. 2'-Me-CCPA was confirmed to be the most selective, high affinity agonist so far known also at human A(1) receptor with a K(i) value of 3.3 nM and 2903- and 341-fold selective vs human A(2A) and A(3) receptors, respectively. In functional assay, 3'-Me-CPA, 3'-Me-CCPA, and 2-Cl-3'-Me-IB-MECA inhibited forskolin-stimulated adenylyl cyclase activity with IC(50) values ranging from 0.3 to 4.9 microM, acting as full agonists. A rhodopsin-based model of the bovine A(1)AR was built to rationalize the higher affinity and selectivity of 2'-C-methyl derivatives of N(6)-substituted-adenosine compared to that of 3'-C-methyl analogues. In the docking exploration, it was found that 2'-Me-CCPA was able to form a number of interactions with several polar residues in the transmembrane helices TM-3, TM-6, and TM-7 of bA(1)AR which were not preserved in the molecular dynamics simulation of 3'-Me-CCPA/bA(1)AR complex.

We report the synthesis and the affinity data at both the peripheral (PBR) and the central benzodiazepine receptors of a series of N,N-dialkyl-2-phenylindol-3-ylglyoxylamide derivatives III, designed as conformationally constrained analogues of 2-phenylindole-3-acetamides II such as FGIN-1-27. Most of the new compounds showed a high specificity and affinity for PBR, with K(i) in the nanomolar to subnanomolar range. The most potent ligands (4-7, 9, 13-27) stimulated steroid biosynthesis in rat C6 glioma cells with a potency similar to or higher than that of classical ligands. The SARs of this new class of compounds are discussed.

New 3-aryl-6-(3-thienyl)pyrazolo[1,5-a]pyrimidin-7-ones (2a-j) are synthesized and evaluated in vitro on Bz/GABA(A) receptors and on recombinant benzodiazepine receptors (alpha x beta 2/3 gamma 2; x = 1-3, 5) expressed in HEK293 cells. SAR studies on the new compounds are conducted and molecular modeling is accomplished to better investigate requirements leading to subtype selectivity. Some of the synthesized compounds are tested in vivo to explore their pharmacological effect as a consequence of their high alpha 1 beta 2 gamma 2 subtype selectivity observed in vitro.

The synthesis and binding study of new 8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 3-ester compounds are reported. A pharmacological evaluation of the high-affinity ligands 1-4 belonging to the 3-heteroarylester series is made. The 3-(2-thienylmethoxycarbonyl) derivative 4 stands out from the other heteroarylesters and is found, using nine different behavioral methods, to be a functionally selective ligand in vivo: it shows anxiolytic-like activity in the conflict models (light-dark box and plus maze test) similarly to diazepam, without any sedative and amnesic properties or interference from alcohol.

1'-C-Methyl analogues of adenosine and selective adenosine A(1) receptor agonists, such as N-[(1R)-1-methyl-2-phenylethyl]adenosine ((R)-PIA) and N(6)-cyclopentyladenosine, were synthesized to further investigate the subdomain that binds the ribose moiety. Binding affinities of these new compounds at A(1) and A(2A) receptors in rat brain membranes and at A(3) in rat testis membranes were determined and compared. It was found that the 1'-C-methyl modification in adenosine resulted in a decrease of affinity, particularly at A(1) and A(2A) receptors. When this modification was combined with N(6) substitutions with groups that induce high potency and selectivity at A(1) receptors, the high affinity was in part restored and the selectivity was increased. The most potent compound proved to be the 1'-C-methyl analogue of (R)-PIA with a K(i) of 23 nM for the displacement of [(3)H]CHA binding from rat brain A(1) receptors and a > 435-fold selectivity over A(2A) receptors. In functional assays, these compounds inhibited forskolin-stimulated adenylate cyclase with IC(50) values ranging from 0.065 to 3.4 microM, acting as full agonists. Conformational analysis based on vicinal protonminus signproton J-coupling constants and molecular mechanics calculations using the MM2 force field proved that the methyl group on C1' in adenosine has a pronounced impact on the furanose conformation by driving its conformational equilibrium toward the north, gamma+, syn form.

A series of N-(arylalkyl)indol-3-ylglyoxylylamides (4-8) was synthesized as ligands of the benzodiazepine receptor (BzR) and tested for their ability to displace [(3)H]flumazenil from bovine brain membranes. The new compounds, bearing a branched (4) or a geometrically constrained benzyl/phenylethyl amide side chain (5-8), represent the continuation of our research on N-benzylindol-3-ylglyoxylylamides 1 (Da Settimo et al., 1996), N'-phenylindol-3-ylglyoxylohydrazides 2 (Da Settimo et al., 1998), and N-(indol-3-ylglyoxylyl)alanine derivatives 3 (Primofiore et al., 1989). A few indoles belonging to the previously investigated benzylamides 1 and phenylhydrazides 2 were synthesized and tested to enrich the SARs in these two series. The affinities and the GABA ratios of selected compounds for clonal mammalian alpha(1)beta(2)gamma(2), alpha(3)beta(2)gamma(2), and alpha(5)beta(3)gamma(2) BzR subtypes were also determined. It was hypothesized that the reduced flexibility of indoles 4-8 would both facilitate the mapping of the BzR binding cleft and increase the chances of conferring selectivity for the considered receptor subtypes. In the series of indoles 4, the introduction of a methyl group on the benzylic carbon with the R configuration improved affinity of the 5-substituted (5-Cl and 5-NO(2)) derivatives, whereas it was detrimental for their 5-unsubtituted (5-H) counterparts. All S enantiomers were less potent than the R ones. Replacement of the methyl with hydrophilic substituents on the benzylic carbon lowered affinity. The isoindolinylamide side chain was tolerated if the 5-position was unsubstituted (K(i) of 5a = 123 nM), otherwise affinity was abolished (5b, c). All the 2-indanylamides 6 and (S)-1-indanylamides 8 were devoid of any appreciable affinity. The 5-Cl and 5-NO(2) (R)-1-indanylamides 7b (K(i) 80 nM) and 7c (K(i) 28 nM) were the most potent among the indoles 5-8 geometrically constrained about the side chain. The 5-H (R)-1-indanylamide 7a displayed a lower affinity (K(i) 675 nM). The SARs developed from the new compounds, together with those collected from our previous studies, confirmed the hypothesis of different binding modes for 5-substituted and 5-unsubstituted indoles, suggesting that the shape of the lipophilic pocket L(1) (notation in accordance with Cook's BzR topological model) is asymmetric and highlighted the stereoelectronic and conformational properties of the amide side chain required for high potency. Several of the new indoles showed selectivity for the alpha(1)beta(2)gamma(2) subtype compared with the alpha(3)beta(2)gamma(2) and alpha(5)beta(3)gamma(2) subtypes (e.g.: 4t and 7c bind to these three BzR isoforms with K(i) values of 14 nM, 283 nM, 239 nM, and 9 nM, 1960 nM, 95 nM, respectively). The GABA ratios close to unity exhibited by all the tested compounds on each BzR subtype were predictive of an efficacy profile typical of antagonists.

A series of analogues related to 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (2) and 1-¿2-[bis(4-fluorophenyl)methoxy]ethyl¿-4-(3-phenylpropyl)piperazine (3) (GBR 12935 and GBR 12909, respectively), in which the piperazine moiety was replaced by bridged piperazines for structural rigidity, has been designed, synthesized, and evaluated for their ability to bind to the dopamine transporter (DAT) and to inhibit the uptake of (3)H-labeled dopamine (DA). The binding data indicated that compounds 7 and 11, the N-methyl- and N-propylphenyl-3,8-diaza[3.2. 1]bicyclooctane analogues of 3, showed high affinity for the DAT (IC(50) = 8.0 and 8.2 nM, respectively), and 7 had high selectivity at the DAT relative to the serotonin transporter (SERT) (88- and 93-fold for binding and reuptake, respectively). They also displayed linear activity in DA uptake inhibition, possessing a similar binding and reuptake inhibition profile to 3. The N-indolylmethyl analogue 16 showed the highest affinity (IC(50) = 1.4 nM) of the series, with a 6-fold increase over its corresponding N-phenypropyl derivative 11. Interestingly, this compound exhibited a high ratio (29-fold) of IC(50) for the inhibition of DA reuptake versus binding to the DAT. Replacing the piperazine moiety of 2 and 3 with (1S, 4S)-2,5-diazabicyclo[2.2.1]heptane resulted in compounds 23-26, which showed moderate to poor affinity (IC(50) = 127-1170 nM) for the DAT. Substitution of the homopiperazine moiety of 4 with a more rigid 3,9-diazabicyclo[4.2.1]nonane gave compounds 28-33. However, the binding data showed that compound 32 displayed a 10-fold decrease in affinity at the DAT and a 100-fold decrease in selectivity at the DAT relative to the SERT compared to its corresponding homopiperazine compound 4.

The synthesis of new 3-heteroaryl-8-chloropyrazolo[5,1-c][1,2, 4]benzotriazine 5-oxides and their binding activities at the central benzodiazepine receptor (BZR) are reported. The derivatives substituted at the 3-position with electron-rich five-membered rings, such as pyrrole 11, 2-thiophene 13c, or 3-thiophene 13d, showed good affinity values for BZR. In in vivo tests the 3-(thien-3-yl)-8-chloropyrazolo[5,1-c][1,2,4] benzotriazine 5-oxide (13d) showed selective anticonvulsant activity.

The design, synthesis, and biological evaluation of compounds related to the dopamine (DA) uptake inhibitors: 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (1) and 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (2) (GBR 12395 and GBR 12909, respectively), directed toward the development and identification of new ligands interacting with high potency and selectivity at the dopamine transporter (DAT) is reported. The substitution of the piperazine ring in the GBR structure with other diamine moieties resulted in the retention of the high affinity of new ligands for the DAT. Some of the modified GBR analogs (e.g. 8, 10, (-)-49, or (-)-50) displayed substantially higher selectivity (4736- to 693-fold) for the dopamine (DA) versus the serotonin (5HT) reuptake site than the parent compounds. The bis(p-fluoro) substitution in the (diphenylmethoxy)ethyl fragment slightly increased the affinity of the ligands at the DA reuptake site but reduced their selectivity at this site (e.g. 9 and 8, 11 and 10, or 17 and 16, respectively). Congeners, such as the series of monosubstituted and symmetrically disubstituted piperazines and trans-2,5-dimethylpiperazines, which lack the (diphenylmethoxy)ethyl substituent lost the affinity for the DAT yet exhibited very high potency for binding to the sigma receptors (e.g.28). The chiral pyrrolidine derivatives of 1, (-)-49, and (+)-49, exhibited an enantioselectivity ratio of 181 and 146 for the inhibition of DA reuptake and binding to the DAT, respectively.

A series of N,N'-substituted piperazine and homopiperazine derivatives have been synthesized with the objective of producing compounds that interact with polyamine modulatory sites on N-methyl-D-aspartate (NMDA) receptors. These novel compounds exhibited polyamine-like actions, enhancing [3H]MK-801 binding to NMDA receptors in rat forebrain membranes. The potencies of N,N'-bis(2-aminoacetyl)homopiperazine (15), N,N'-bis(N-methyl-4-aminobutyl)-piperazine (7), and N,N'-bis(3-aminopropyl)homopiperazine (11) (EC50 18.0, 21.3, and 24.4 microM, respectively) to enhance [3H]MK-801 binding were comparable to that of spermine (EC50 5.2 microM). However, the efficacies of 15, 7, and 11 in this measure were lower (by approximately 40%, 32%, and 24%, respectively) than spermine, which may be indicative of partial agonist actions. Like spermine, the ability of these piperazine and homopiperazine derivatives to enhance [3H]MK-801 binding could be inhibited by both a competitive polyamine antagonist (arcaine) and a specific, noncompetitive polyamine antagonist (conantokin-G). However, unlike endogenous polyamines, high concentrations (up to 1 mM) of these novel polyamine-like compounds did not inhibit [3H]MK-801 binding. N,N'-Aminoalkylated and aminoacylated piperazine and homopiperazine derivatives may prove useful for studying polyamine recognition sites associated with NMDA receptors.

A series of polyamines based on the high affinity sigma receptor ligand N-[2-(3,4-dichlorophenyl)-ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (3) were developed and evaluated for their binding characteristics at sigma-1 and sigma-2 receptor subtypes. The data indicated that a considerable degree of structural variation is possible while still retaining nanomolar affinity at sigma receptors. As the structure of the polyamines was varied, their binding at sigma-1 and sigma-2 subtypes showed quite different and in some cases opposite trends, supporting the belief that these are pharmacologically distinct entities. Polyamines containing two nitrogen atoms showed optimal binding at both sigma-1 and sigma-2 receptor subtypes. Although additional nitrogen atoms resulted in decreased affinity at sigma-1 and sigma-2 subtypes, an increase in selectivity for sigma-2 subtypes was evident; the parent 3 showed greater selectivity for sigma-1 subtypes. Internitrogen spacings had a large effect on binding affinity and subtype selectivity. For example, the difference between N-[3-(1-pyrrolidinyl)propyl]-N'-(3,4-dichlorobenzyl)-N,N'- dimethylethylenediamine (8) [K(i) = 29.9 nM at sigma-1 receptor and 18.3 nM at sigma-2 receptor] to N-[3-(1-pyrrolidinyl)propyl]-N'-(3,4-dichlorobenzyl)- N,N'-dimethylethylenediamine (10) [K(i) = 1.49 nM at sigma-1 receptor and 12.1 nM at sigma-2 receptor] illustrates the importance of internitrogen spacing. Triamines 11 and 13 [Ki(sigma-2)/K(i)(sigma-1) = 0.19 and 0.10, respectively] containing the N-N-N-Ar spacings 3-3-2 and 4-4-2, proved to be the most sigma-2 subtype selective of the 15 polyamines examined in this study. The N-N-N spacings appear to be an important factor in their sigma-2 subtype selectivity. These compounds will serve as templates in the design of still further sigma-2 subtype selective ligands. The pyrrolidine ring (present in most of the polyamines tested in this series) proved to be an important recognition site for sigma receptor binding activity. Furthermore, alkyl substitution also appears to be important since the stripped down polyamines N-[2-(3,4-dichlorophenyl)ethyl]ethylenediamine (15) and N1-[2-(3,4-dichlorophenyl)ethyl]diethylenetriamine (16) exhibited relatively low binding affinity.

We previously reported (J. Med. Chem. 1993, 36, 1188-1193) that changes to the ring size of the piperidine and cyclohexyl rings of the high-affinity and selective dopamine (DA)-uptake inhibitor 1-[1-(2-benzo[b]thienyl)cyclohexyl]piperidine (BTCP, 2) caused different, and in some cases opposite, changes in affinity for sites on the DA transporter labeled by [3H]BTCP and [3H]-cocaine. These results suggested that the radioligands label different sites on the transporter. In the present study, we extend the structure-activity relationships (SAR) of BTCP by studying the binding characteristics of a series of N,N-disubstituted 1-(2-benzo[b]thienyl)cyclohexylamines 7-32 at the DA transporter. Cyclohexyl was selected as opposed to other ring sizes since it corresponds to BTCP. The binding results indicate that a considerable degree of structural variation is permitted for the N-substituents, while still retaining nanomolar affinity for sites on the transporter (studied in rat forebrain homogenates). As observed in our earlier study, the differential effects of structural change on binding to sites on the DA transporter labeled by these radioligands suggests that they are different and distinct binding sites. In general, and up to a point, increasing the size and lipophilicity of the N substituents resulted in improvements in binding but appeared to have less predictable effects on DA-uptake inhibition (as measured in rat brain synaptosomes). The binding of these compounds to sites labeled by [3H]BTCP appeared to correlate best with IC50 for DA-uptake inhibition. To our surprise, the monoalkyl N-substituted BTCP derivatives displayed the highest affinity for the DA transporter of all the compounds in this series. For example, the N-(cyclopropylmethyl) derivative 14 displayed IC50's = 23 nM ([3H]cocaine) and 1 nM ([3H]-BTCP), and the N-butyl derivative 10 showed IC50's = 60 nM ([3H]cocaine) and 0.3 nM ([3H]-BTCP). BTCP exhibited IC50's of 39 nM ([3H]cocaine) and 5 nM ([3H]BTCP) in this assay. The observation that N,N-dibutyl derivative 31 exhibited low ratios of IC50 [3H]cocaine/IC50 DA reuptake and IC50 [3H]BTCP/IC50 DA reuptake suggests that it may be a potential candidate for cocaine antagonism studies. The effect of additional amino, amide, and aromatic groups on the N-substituents was examined, and the results are discussed. The failure of all of the compounds in this series to bind phenycyclidine receptors coupled with their high affinity and range of selectivities at the DA transporter identifies many of them as useful tools for probing the mode of action of BTCP at this site.

As a continuation of our earlier study (J. Med. Chem. 1992, 35, 4334-4343) we conformationally restricted the sigma-receptor ligand 2-(1-pyrrolidinyl)-N-[2-(3,4-dichlorophenyl)ethyl]-N-methylethylamine (1) by incorporating it into a series of homologous piperazines 3-9 and homopiperazines 10 and 11, diazabicyclononanes and decanes, bridgehead bicyclooctanes and nonanes as well as other miscellaneous compounds. sigma-Receptor binding affinities were obtained using [3H](+)-pentazocine in guinea pig brain membrane sigma 1 sites. The studies suggest that the nitrogen lone pair orientation found in the piperazines affords the strongest binding interaction. Other nitrogen lone pair orientations or compounds representing unlikely staggered conformations of 1 [ as in 4-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[3.2.2]nonane (16)] show very weak sigma interaction. Comparison of the binding data of different N-substituted homologues of 1 with those of the 1-[2-(3,4-dichlorophenyl)ethyl]-4-alkylpiperazines suggests that the two nitrogen atoms of 1 are working in opposition to one another in terms of their sensitivity to steric bulk. The high binding affinity of the 1,4-diazabicyclo[4.3.0]nonanes 12 suggests that these may approximate the methyl and pyrrolidine ring conformations found in 1 when it is bound to the sigma receptor. Compound 12 exhibited a 4-fold enantioselectivity favoring (+)-12. The synthesis of 6,7-dichloro-2-[[2-(1-pyrrolidinyl)ethyl]amino]tetralin (19) and its desmethyl derivative 20 permitted constraint of the 3,4-dichlorophenyl and N-methyl moieties of 1 into a gauche orientation. The binding data suggests that this conformation in 1 favors strong binding interaction at sigma-receptors. sigma-Receptor Ki's ranged from 0.55 nM for 1-[2-(3,4-dichlorophenyl)ethyl]-4-n-butylpiperazine (7) to 654 nM for 16. Overall comparison of the results indicate that 1 is subject to considerable conformational freedom and suggests that the sigma receptor is not subject to rigid stereochemical restraints with 1. These results add to our earlier study where we restrained 1 using simple monocyclic heterocycles.

Piperidine and cyclohexyl ring homologues of the high-affinity dopamine (DA) uptake inhibitor 1-[1-(2-benzo[b]thienyl)cyclohexyl]piperidine (BTCP, 3) were each prepared in four steps from the appropriate cycloalkanones. These compounds were tested for their ability to displace [3H]BTCP and [3H]cocaine and to inhibit [3H]DA uptake in rat striatal homogenates. The ratios IC50([3H]cocaine)/IC50([3H]BTCP) ranged from 62 for BTCP to 1.5 for 1-[2-(benzo[b]thienyl)-cyclopentylamine (17); cocaine gave a ratio of 0.6. This indicates that BTCP is the most selective of all the compounds tested for sites labeled by [3H]BTCP whereas cocaine is most selective for sites labeled by [3H]cocaine. The wide differences in the relative abilities of these compounds to displace [3H]BTCP and [3H]cocaine suggests that these two radioligands are labeling different sites on the transporter. In general, the compounds structurally related to BTCP exhibited greater selectivity for sites labeled by [3H]BTCP. However, several of the BTCP-related derivatives showed greater (compared with BTCP and cocaine) ability to displace [3H]cocaine. Most notably, 1-[1-(2-benzo[b]thienyl)cyclohexyl]pyrrolidine (7) exhibited a 3.4-fold greater affinity for these sites compared with BTCP and a 9-fold greater affinity at these sites than cocaine. Most of the BTCP homologues displayed greater ability to inhibit [3H]DA uptake in rat forebrain synaptosomes than cocaine. BTCP and 7 were the most potent of all the compounds tested in terms of their ability to inhibit uptake of [3H]DA. IC50 ratios for [3H]cocaine binding/[3H]DA uptake ranged from 0.47 for 1-[1-(2-benzo[b]thienyl)cyclopentyl]homopiperidine (11) to 8.8 for 1-(2-benzo[b]thienyl)cyclohexylamine (4). The importance of this ratio remains unclear in terms of identification of potential cocaine antagonists. As for BTCP, all of the compounds tested showed Ki values > 10,000 nM for displacement of [3H]TCP from rat brain homogenates. These compounds were able to displace the highly selective sigma receptor probe [3H]-(+)-pentazocine from guinea pig brain homogenates with Ki values ranging from 125 to 9170 nM. The significance of their sigma-binding activity in light of their dopaminergic properties is unclear. The diverse binding properties of these compounds at the DA-uptake site and their spectrum of inhibitory activities for [3H]DA uptake identifies them as a useful base for the development of subtype selective probes at this site. These compounds will allow further study of the structure and function of the "cocaine" receptor as well as the development of potential cocaine antagonists.

A series of imidazo[1,5-a][1,4]benzodiazepine esters have been synthesized with varying ester side chains and 8-position substituents. The affinities of these compounds were evaluated at both "diazepam-insensitive" (DI) and diazepam-sensitive (DS) subtypes of the benzodiazepine receptor (BZR). A profound steric effect of the 3-position ester side chain moiety was observed on ligand affinity at DI. In contrast, ester size had a less robust effect on ligand affinity at DS. The tert-butyl ester compound 8 displayed the highest affinity (Ki = 1.7 nM) for DI within a series of 8-chloro esters. Furthermore, halogens at the 8-position resulted in an enhancement of both ligand affinity and selectivity at DI among the series of tert-butyl esters examined. The 8-nitro derivative 23 and 8-isothiocyanato congener 25 had high affinities for both DI and DS but exhibited little subtype selectivity (10.8 and 2.7 nM at DI versus 14 and 3.7 nM at DS, respectively). The 8-azido tert-butyl ester 29 exhibited a significantly higher affinity (Ki = 0.43 nM) and selectivity (DI/DS ratio of 0.2) than the corresponding ethyl ester, the prototypic DI ligand 1 (Ro 15-4513). Among the compounds synthesized, 29 is the highest affinity ligand for DI described to date while its 8-bromo analog 18 is the most selective ligand (DI/DS ratio of 0.17) for this novel BZR subtype.

2-, 3-, and 4-idophenyl derivatives of the high-affinity sigma ligand N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (1) were synthesized in two to four steps starting from N-methyl-2-(1-pyrrolidinyl)ethylamine. These compounds were evaluated for their capacity to label both sigma 1 and sigma 2 subtypes in vitro. sigma-1 binding affinity was determined by measuring competition with [3H]-(+)-pentazocine binding to guinea pig brain membranes while sigma 2 binding was evaluated through competition with [3H]DTG binding to rat liver membranes in the presence of excess dextrallorphan. The binding data revealed that N-[2-(3-iodophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (2) and N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (3) displayed almost identical binding affinity at sigma 1 sites to the parent compound 1. This suggests that the 3- or 4-iodo group can effectively substitute for the 3,4-dichloro substituents of 1. In this series of compounds, Ki's at the sigma 1 site varied from 2.0 nM for N-(4-iodobenzyl)-N-methyl-2-(1-pyrrolidinyl)ethylamine (6) to 26.6 nM for N-(2-iodobenzyl)-N-methyl-2-(1-pyrrolidinyl)ethylamine (4). Ki's for sigma 2 site ranged from 8.1 nM for 1 to 220 nM for N-(3-bromobenzyl)-N-methyl-2-(1-pyrrolidinyl)ethylamine (11) while the sigma 2/sigma 1 ratio varied from 1.8 for 4 to 25 for 11. Comparing halogen substitution, the trend Cl = I > Br > F was observed for binding affinity at sigma 1 sites; no such trend was observed at sigma 2 sites. On the basis of the binding data, compounds 2 and 3 were selected for labeling with 123I. Thus, treatment of the corresponding 3- and 4-(tributylstannyl) intermediates (7 and 8) with Na123I in the presence of excess CH3CO3H furnished [123I]-2 and [123I]-3 in up to 70% radiochemical yield. Preliminary in vitro binding with [123I]-3 indicated up to 97% specific binding with guinea pig brain membranes.

The novel semirigid derivatives (+)-cis-1-[2-phenyl-2-bicyclo[3.1.0]hexyl]piperidine [(+)-8], its enantiomer (-)-8, and (+-)-trans-1-[2-phenyl-2-bicyclo[3.1.0]hexyl]piperidine [(+/-)-9] were synthesized as probes to investigate the mode of interaction of phencyclidine (PCP) with its binding site on the N-methyl-D-aspartate receptor complex. Each target compound was obtained in five steps starting from cyclopent-2-enone. (+)- and (-)-8 were obtained in greater than 98% optical purity through three recrystallizations from ethanol of the (S)-(+)- and (R)-(-)-mandelate salts of intermediate (+-)-cis-2-phenyl-2-bicyclo[3.1.0]hexylamine ([(+/-)-16]. Crystallization of the (R)-(-)-mandelate salt afforded (1R,2R,5S)-(-)-16, whereas the (S)-(+)-mandelate salt afforded (1S,2S,5R)-(+)-16; the absolute configuration was determined by single-crystal X-ray analysis of (-)-16.(R)-(-)-mandelate. Single-crystal X-ray analysis of (+/-)-9-picrate confirmed its trans configuration and provided conformational data. (+)- and (-)-8 and (+/-)-9 were examined for their ability to interact with PCP and sigma binding sites in vitro using [3H]TCP and [3H]pentazocine as radioligands. The binding was compared with that of PCP and contrasted with the rigid symmetrical phencyclidine derivatives cis- and trans-1-[3-phenyl-3-bicyclo[3.1.0]hexyl]piperidines (6 and 7). The results of the study indicated that the conformations of PCP represented by 6-9 are not optimal for potent interaction at either of these sites. Affinities ranged from 582 nM [(+/-)-9] to 29,000 nM [(+)-8] at PCP binding sites and from 1130 nM [(-)-8] to 16,300 nM (7) at sigma sites. In this assay, PCP exhibited affinities of 64.5 nM at PCP and 1090 nM at sigma sites. Qualitative correlation between the sigma and PCP binding data suggests some similarities between these binding sites. An axial phenyl and equatorial piperidine ring with the nitrogen lone pair of electrons antiperiplanar to the phenyl ring has been postulated as the receptor-active conformation of PCP-like ligands at the PCP binding site. Comparison of the binding data of 7-9 with that of the previously described methylcyclohexyl-PCP derivatives allowed its rationalization in terms of this model. It is likely that the lowered affinity in this bicyclo[3.1.0]hexane series is a consequence of nonoptimal geometry (pseudoequatorial phenyl or pseudoboat) for binding as opposed to the presence of steric bulk which proved deleterious in the methylcyclohexyl-PCP derivatives.

The synthesis and sigma receptor affinity of a series of conformationally restricted derivatives of 2-(1-pyrrolidinyl)-N-[2-(3,4-dichlorophenyl)ethyl]-N-methylethylenedi amine (1) is described. The pyrrolidinyl (or N,N-dialkyl),ethylenediamine,N-alkyl, and phenylethyl portions of this sigma receptor pharmacophore were restricted by its incorporation into 1,2-cyclohexanediamine-, pyrrolidine-, piperidine-, homopiperidine-, and tetrahydroisoquinoline-containing ligands. The sigma receptor binding affinities of these compounds were determined using [3H](+)-pentazocine in guinea pig brain homogenates. The synthesis of all but one class was achieved by acylation and alane reduction of the appropriate diamine precursors whose synthesis is also reported. sigma receptor affinities ranged from 1.34 nM for 6,7-dichloro-2-[2-(1-pyrrolidinyl)ethyl]tetrahydroisoquinoline (12) to 455 nM for (1R,2R)-trans-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2- (1-pyrrolidinyl)cyclohexylamine [(-)-4]. In this displacement assay, (+)-pentazocine exhibited a Ki of 3.1 nM while DTG and haloperidol showed Ki values of 27.7 and 3.7 nM, respectively. The conformationally free parent compound 1 exhibited a Ki value of 2.1 nM. Comparison of both the sigma receptor affinities and nitrogen atom geometry of the compounds revealed that a gauche relation of the nitrogen atoms of cis-1,2-cyclohexanediamines is not imperative for high affinity as we had previously thought. It is highly likely that nitrogen lone pair orientations and steric factors on the aliphatic portions of these ligands play a major role in the sigma receptor binding of this pharmacophore.

The epimeric 6 beta- and 6 alpha-iodo-3,14-dihydroxy-17-(cyclopropylmethyl)-4,5 alpha-epoxymorphinans (1, ioxy) and (2, epioxy), respectively, were each synthesized in five steps starting with naltrexone. The configuration of the 6-iodo group of 1 was unequivocally determined to be beta-based on single crystal X-ray analysis of its precursor 3-acetoxy-6 beta-iodo-14-hydroxy-17-(cyclopropylmethyl)-4,5 alpha-epoxymorphinan (10). Both 1 and 2 as well as their corresponding 3-O-acetates 10 and 11 were found to readily cross the blood-brain barrier and completely reverse the analgesic effects of a 10 mg/kg intraperitoneal dose of morphine sulfate as determined by the paw withdrawal latency test. Compounds 1 and 2 were found to bind with high affinity to mu, delta, and kappa receptors in vitro. In general, 1 and 2 exhibited higher affinity for mu and kappa receptors than naltrexone while the 6 beta-iodo epimer 1 (ioxy) was more potent than its epimer 2. In a comparison of the 6 beta-halogen substituent on binding affinity across opioid receptor subtypes, it was generally found that I greater than Br greater than F. On the basis of the results of in vitro and in vivo testing, 1 was selected as a target for radioiodination and evaluation as a potential single photon emission computed tomography imaging agent for opioid receptors. Carrier-free [125I]-1 was synthesized in near quantitative yield by the sequence of reaction of excess 3-acetoxy-6 alpha-[[(trifluoromethyl)sulfonyl]oxy]-14-hydroxy-17- (cyclopropylmethyl)-4,5 alpha-epoxymorphinan (8) with anhydrous Na125I in dry acetonitrile for 90 min at 76 degrees C followed by deacetylation of the product with 1:1 aqueous ammonia/acetonitrile at 25 degrees C. The potential of [125I]-1 as an in vivo imaging agent for opioid receptors is evaluated and discussed.

Unlabeled fluoro- and iodo-substituted ligands exhibiting very high affinity and selectivity for sigma receptors were synthesized based on three different structural classes of sigma receptor ligands. These compounds were evaluated for sigma receptor affinity and specificity in order to assess their potential as PET/SPECT imaging agents. Thus, (+)- and (-)-N-(5-fluoro-1-pentyl)normetazocines [(+)- and (-)-4] based on the (+)-benzomorphan class of sigma ligands were synthesized via N-alkylation of optically pure (+)- and (-)-normetazocine with 5-[(methylsulfonyl)oxy]-1-pentyl fluoride (11). (+)- and (-)-4 displaced [3H](+)-3-PPP with Ki values of 0.29 and 73.6 nM and [3H](+)-pentazocine with Ki values of 10.5 and 38.9 nM, respectively. The second class of PET/SPECT ligands was based upon the N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamine class of sigma ligands; N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-(3-fluoro-1-propyl)-2-(1- pyrrolidinyl)ethylamine (5) was obtained via N-alkylation of N-[2-(3,4-dichlorophenyl)-1-ethyl]-2-(1-pyrrolidinyl)ethylamine (14) with 3-fluoropropyl p-toluenesulfonate. 5 exhibited Ki values of 4.22 and 5.07 nM for displacement of [3H](+)-3-PPP and [3H](+)-pentazocine, respectively, comparable with the parent N-propyl compound. Attempts to synthesize N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-[3- [(methylsulfonyl)oxy]-1-propyl]-2-(1-pyrrolidinyl)ethylamine (26), a precursor to 5 that could conceivably be converted to [18F]-5 by treatment with 18F-, proved unsuccessful. The sequence of regioselective nitration, catalytic hydrogenation, and diazotization followed by NaI quench of N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine (2) afforded the iodinated ethylenediamine N-[2-(2-iodo-4,5-dichlorophenyl)-1-ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine (8), a potential SPECT ligand for sigma receptors. This compound showed an affinity of 0.54 nM ([3H](+)-3-PPP) comparable with the parent compound 2 (Ki = 0.34 nM, [3H](+)-3-PPP). Ligand 8 exhibited a similar potency against [3H](+)-pentazocine. The third class of high-affinity sigma receptor ligands was rationalized based on rearrangement of the bonds in ethylenediamine 2 to give 1-[2-(3,4-dichlorophenyl)-1-ethyl]-4-(1-propyl)piperazine (3). This compound exhibited very high affinity (Ki = 0.31 nM, [3H](+)-3-PPP) and selectivity for sigma receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

By synthesizing and testing a part-structure, N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (3), derived from our previously reported high affinity sigma receptor ligands (1S,2R)-(-)-N-[2-(3,4-dichlorophenyl)-ethyl]-N-methyl-2-(1- pyrrolidinyl)cyclohexylamine [(-)-2] and (+)-2, we have identified a novel class of superpotent (subnanomolar affinity) sigma ligands specific for the sigma receptor labeled by [3H]-(+)-3-PPP. When 3 was tested for its capacity to displace [3H]-(+)-3-PPP from guinea pig brain membranes, it exhibited a Ki of 0.34 nM, which is better than either of its parent compounds (-)-2 (Ki = 1.3 nM) and (+)-2 (Ki = 6.0 nM). Other compounds related to 3 such as N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-homopiperidinyl)ethy lamine (19) exhibited Ki = 0.17 nM [( 3H]-(+)-3-PPP). The determinants for high sigma receptor affinity of 3 were examined by manipulation of this structure in a number of different ways. The high efficacy of these compounds for the sigma receptor, their relative chemical simplicity and ease of synthesis, and their high degree of selective identifies N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (3) and related compounds as a highly promising base for determination of the functional role of sigma receptors as well as the development of novel therapeutic agents.

N-Alkyl-substituted derivatives of (+)- and (-)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)cyclohexylamin e have been synthesized in nine steps in a stereospecific manner starting from cyclohexene oxide. The key step in the reaction sequence involved catalytic hydrogenation of oxime 8 in the presence of PtO2 and AcOH to give the cis diamine (+/-)-7. Most of the compounds in this series exhibited very high affinity at sigma receptors when tested against [3H]-(+)-3-PPP, and in general it was observed that the 1R,2S enantiomers bound more potently to sigma receptors than their corresponding 1S,2R enantiomers. The most potent sigma ligand found in this class was the unsubstituted derivative (1R,2S)-(-)-4, which exhibited an affinity constant of 0.49 nM. This compound was also found to be very selective for sigma receptors. It exhibited little or no affinity for kappa opioid, PCP, and dopamine-D2 receptors. It was also demonstrated that the cis configuration as opposed to the trans configuration of (+)- and (-)-5 was necessary for a higher sigma receptor affinity.

p-, m-, and o-isothiocyanate derivatives (1-3, respectively) of tert-butylbicycloorthobenzoate (TBOB) were synthesized from 3-tert-butyloxetane-3-methanol (4) as the starting material. While 2 was readily obtained in four steps via catalytic hydrogenation of the m-nitro-tert-butylbicycloorthobenzoate (9) intermediate, 1 and 3 could not be obtained this way. 1 and 3 were instead synthesized by an alternative four-step approach while made use of the stability of the isothiocyanate moiety to strong Lewis acids such as boron trifluoride etherate, conditions that would isomerize isothiocyanato oxetane ester intermediates to their corresponding orthoesters. The p-isothiocyanate derivative of TBOB, compound 1, inhibited [35S]-tert-butylbicyclophosphorothionate (TBPS) binding to rat cortical membranes with a potency (IC50 62 nM) comparable to the parent compound while 2 and 3 were approximately 10-fold less potent (IC50 values 570 and 609 nM, respectively). Preincubating tissue with radioligand further reduced the potencies of 2 and 3 by approximately 1 order of magnitude (IC50 values 5400 and 7500 nM, respectively) while the potency of 1 (IC50 90 nM) was only marginally affected by this procedure. Pretreatment of membranes with 1 and 2 followed by extensive washing resulted in a concentration-dependent inhibition of [35S]TBPS binding. In contrast, preincubating tissues with up to 2.4 microM of 3 did not elicit an apparent acylation of [35S]TBPS binding sites. Molecular modeling of the effective diameters of 1-3 in their thermodynamically most stable conformations indicates a relationship between these diameters and their relative efficacies as site-directed acylators; the smaller the diameter, the more potent the acylator. This hypothesis explains both the relative potencies of these compounds and their differential abilities to acylate the TBPS binding site.

Certain benzeneacetamides [(-)- and (+)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]benzeneacetamide] were recently reported to be potent sigma receptor ligands. In order to determine whether efficacy for the sigma receptor could be improved, a series of compounds related to the benzeneacetamides, N-substituted cis-2-(1-pyrrolidinyl)-N-methylcyclohexylamines, were synthesized and their structure-activity requirements were determined. The compounds were synthesized by starting with the previously reported (+/-)-, 1S,2R-(+)-, and 1R,2S-(-)-cis-2-(1-pyrrolidinyl)-N-methylcyclohexylamines. Analysis of sigma ([3H](+)-3-PPP), kappa ([3H]bremazocine and [3H]U69,593), dopamine-d2 ([3H](-)-sulpiride), and phencyclidine (PCP) ([3H]TCP) receptor binding in guinea pig brain revealed a number of highly potent and selective sigma receptor ligands. Notably, 1S,2R-cis-(-)-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-(2-naphthyl) acetamide [(-)-29] (Ki = 8.66 +/- 0.35 nM), (+/-)-cis-2-amino-4,5-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide [(+/-)-17] (Ki = 11 +/- 3 nM), 1S,2R-(-)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl ) cyclohexylamine [(-)-44] (Ki = 1.3 +/- 0.3 nM), and 1R,2S-(+)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl ) cyclohexylamine. [(+)-44] (Ki = 6 +/- 3 nM) exhibited very high affinity at sigma receptors, by displacement of [3H]-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [( 3H]-(+)-3-PPP). These compounds showed insignificant affinity for kappa, dopamine, or PCP receptors, making them valuable tools for the study of sigma receptors. Furthermore, these compounds also exhibited enantioselectivity ranging from 5-fold for (+)- and (-)-44 to 160-fold for (+)- and (-)-29. Several other compounds showed equivalent selectivity but displayed lower sigma receptor affinity.