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This study focused on formulating conjugate vaccines targeting oxycodone and heroin for technology transfer, good manufacturing practice (GMP), and clinical evaluation. Lead vaccines used the highly immunogenic carrier protein keyhole limpet hemocyanin (KLH), which poses formulation problems because of its size. To address this barrier to translation, an oxycodone-based hapten conjugated to GMP-grade subunit KLH (OXY-sKLH) and adsorbed on alum adjuvant was studied with regard to carbodiimide coupling reaction time, buffer composition, purification methods for conjugates, conjugate size, state of aggregation, and protein/alum ratio. Vaccine formulations were screened for post-immunization antibody levels and efficacy in reducing oxycodone distribution to the brain in rats. While larger conjugates were more immunogenic, their size prevented characterization of the haptenation ratio by standard analytical methods and sterilization by filtration. To address this issue, conjugation chemistry and vaccine formulation were optimized for maximal efficacy, and conjugate size was measured by dynamic light scattering prior to adsorption to alum. An analogous heroin vaccine (M-sKLH) was also optimized for conjugation chemistry, formulated in alum, and characterized for potency against heroin in rats. Finally, this study found that the efficacy of OXY-sKLH was preserved when co-administered with M-sKLH, supporting the proof of concept for a bivalent vaccine formulation targeting both heroin and oxycodone. This study suggests methods for addressing the unique formulation and characterization challenges posed by conjugating small molecules to sKLH while preserving vaccine efficacy.

Procedures were developed for the synthesis of exo-(2‘-chloro-5-pyridinyl)-7-(endo and exo)-amino[2.2.1]heptanes (3a and 3b). The compounds were evaluated for binding to the α4β2 and α7 nicotinic acetylcholine receptors (nAChRs), for pharmacological activity in the mouse tail-flick and hot-plate assays, and for hypothermia and locomotor activity. Compounds 3a and 3b possessed α4β2 nAChR binding properties similar to those of (−)-nicotine and were nAChR agonists in all four mouse assays.

Synthetic procedures were developed for the synthesis of 2β,3β- and 2α,3α-diaryltropanes. These compounds are analogues of the 3-aryltropane-2β-carboxylic acid methyl ester class of monoamine uptake inhibitors, where the 2β-carbomethoxy group has been replaced by an aryl group. The compounds were evaluated for inhibition of radioligand binding at the dopamine, norepinephrine, and serotonin transporters (DAT, NET, and 5-HTT, respectively). The results showed that the replacement of the 2β-carbomethoxy group in the 3-aryltropane class with a 2β-aryl group led to compounds possessing very similar monoamine transporter binding properties. However, the 2β,3β-diaryltropanes tended to be more potent at the DAT and more selective for the DAT relative to the NET and 5-HTT. One of the most interesting compounds was 3β-(4-methylphenyl)-2β-(4-methylphenyl)tropane (3d), which showed an IC50 of 1.23 nM at the DAT with 289- and 185-fold selectivity for the DAT relative to the NET and 5-HTT. The 2α,3α-diaryltropanes were much less potent at all three transporters than 2β,3β-diaryltropanes.

3α-(Substituted phenyl)nortropane-2β-carboxylic acid methyl esters (8a−h) showed high affinity for the norepinephrine transporter (NET). The most potent and selective compound was 3α-(3-fluoro-4-methylphenyl)nortropane-2β-carboxylic acid methyl ester (8d), with a Ki of 0.43 nM at the NET and 21- and 55-fold selectivity relative to binding at the dopamine and serotonin transporters. The development of 8d makes available compounds selective for all three transporters from the same structural class.

3β-(3‘-Methyl-4‘-chlorophenyl)tropane-2-carboxylic acid methyl ester (3b, RTI-112) is a 3-phenyltropane analogue that has high affinity for both the dopamine and serotonin transporters (DAT and 5-HTT, respectively). Compound 3b shows significant reduction of cocaine self-administration in rhesus monkeys, yet fails to maintain robust drug self-administration. PET studies revealed that unlike more DAT selective analogues such as GBR 12 909 and 3-(4-chlorophenyl)tropane-2-carboxylic acid phenyl ester (RTI-113), 3b shows no detectible DAT occupancy when dosed at its ED50 for reduction of cocaine self-administration. In contrast, it highly occupies the 5-HTT at this dose. In this study, we report the synthesis and monoamine transporter binding potency of several new 3-(3‘,4‘-disubstituted phenyl)tropane-2-carboxylic acid methyl esters (3c−k), which have binding properties very similar to 3b. With the exception of the 3‘,4‘-dimethyl analogue 3k, all of the compounds possess subnanomolar IC50 and Ki values at the DAT and 5-HTT, respectively. The 3‘-chloro-4‘-bromo analogue 3e (IC50 = 0.12 nM) and the 3‘-bromo-4‘-iodo analogue 3i (Ki = 0.14 nM) are the most potent analogues at the DAT and 5-HTT, respectively. These compounds will be useful to further characterize the highly interesting behavioral profile of 3b.

A series of 3‘-substituted deschloroepibatidine analogues (3a−g and 4) showed high affinity for α4β2 binding and relatively weak affinity for α7 nAChRs. The 3‘-ethynyl (3g) and 3‘-fluoro (3a) analogues with Ki values of 0.02 and 0.037 nM, respectively, were the most potent. Even though the α4β2 binding affinity of several of the analogues were equal to that of epibatidine, all of the compounds were weak agonists in the antinociceptive, hypothermia, and spontaneous activity test in mice. In contrast, all of the compounds were functional antagonists of nicotine-induced antinociception. In general, compounds 3a−g and 4 were more potent in the tail-flick assay than the hot-plate test. For example, the 3‘-fluoro analogue 3a and the N-methyl-3‘-iodo analogue 4 showed AD50 values of 0.07 and 0.04 μg/kg, respectively, in the tail flick test and only 35 and 0% inhibition at 20 and 10 μg/kg in the hot-plate assay, respectively. These results suggest that these compounds will be highly useful for identifying which specific receptor subtypes are involved in each of nicotine's pharmacological effects. The high affinity of the N-methyl-3‘-iodo analogue 4 combined with its weak agonist and potent antagonist activity suggests that carbon-11 and iodine-123 analogues may be useful as PET and SPECT ligands, respectively, for studying nAChRs in vivo.

The monoamine transporter binding properties, gross behavior, and locomotor activity effects in mice and drug discrimination results in cocaine-trained rats of the 2β,3β-, 2β,3α-, 2α,3β-, and 2α,3α-isomers of several 3-(4-substituted-phenyl)tropane carboxylic acid methyl esters were compared (2a−f, 3a−f, 4a−f, and 5b,c). The 2β,3β-isomer showed the highest affinity for the dopamine transporter (DAT), and the 2β,3α-isomer showed the next highest affinity. The order of potency for the 2β,3β-isomer is 4‘-chloro (2c) = 4‘-iodo (2e) > 4‘-bromo (2d) = 4‘-methyl (2f) > 4‘-fluoro (2b) > 4‘-hydrogen (2a). In the case of the 2β,3α-isomer, the order of affinity was 4‘-bromo (3d) > 4‘-iodo (3e) = 4‘- chloro (3c) > 4‘-methyl (3f) > 4‘-fluoro (3b) > 4‘-hydrogen (3a). The 4‘-hydrogen, 4‘-fluoro, and 4‘-methyl 2α,3β-isomers, 4a, 4b, and 4f, had the lowest affinity for the DAT. While most of the compounds showed their highest affinity at the DAT, none were selective relative to the other two monoamine transporters. In general, the 2α,3α- and 2α,3β-isomers were more toxic (death and convulsions) than the 2β,3β- and 2β,3α-isomers. With the exception of the 2α,3α-isomers, all compounds produced the locomotor activity stimulation typical of dopaminergic drugs. The ED50 ranges for the 2β,3β- (2a−f), 2β,3α- (3a−f), and 2α,3β-isomers (4a−f) in the locomotor assay were 0.1−1.2, 6.6−21.8, and 2.4−11.7 mg/kg, respectively. With the exception of the 2α,3α-isomer, all compounds generalized to cocaine. The 2β,3β-isomers were at least 10-fold more potent than cocaine and the other three sets of isomers in this test.

A series of 2‘-fluoro-3‘-(substituted phenyl)deschloroepibatidine analogues (5a−k) showed high affinity for α4β2 binding with no affinity at α7 nAChRs. The most potent compound was 2‘-fluoro-3‘-(4-nitrophenyl)deschloroepibatidine (5g) which possessed a Ki value of 0.009 nM. Surprisingly, none of the compounds showed agonist effects in pain tests and body temperature changes in mice even when tested at 10−15 mg/kg with the exception of 5b, which showed only very weak agonist effects. In contrast, all the compounds were potent functional antagonists of nicotine-induced antinociception. Interestingly, the 3‘-substituted phenyl analogues 5b−k were 10−870-fold more effective as antagonists in the tail-flick test versus the hot-plate procedure. They failed to antagonize nicotine-induced hypothermia. The 4-chlorophenyl analogue (5e) (AD50 = 0.0003 in the tail-flick test) was the most potent and selective analogue. These results suggest that these compounds will be highly useful for identifying which specific receptor subtypes are involved in each of nicotine's pharmacological effects. These compounds also deserve consideration as potential pharmacotherapies for treatment of smoking cessation.

In vitro characterization and comparison of JDTic, its dehydroxy analogue and nor-BNI, and its dehydroxy analogue demonstrates that the N-substituted 3,4-dimethyl-(3-hydroxyphenyl)piperidine-derived antagonist, JDTic, relies more heavily on its phenol address group for affinity and antagonist activity relative to the corresponding naltrexone derived antagonists, nor-BNI. The structural flexibility of the former class of compound relative to the latter is postulated to underlie the difference.

Several 3β-(substituted phenyl)-2β-(3-substituted isoxazol-5-yl)tropanes (3a−t) were evaluated for their ability to inhibit radioligand binding at the DAT, 5-HTT, and NET as well as in gross observation and locomotor activity in mice and in rats trained to discriminate cocaine. All compounds showed high affinity for the DAT. The IC50 values ranged from 0.5 to 26 nM. With the exception of 3e and 3f, which have no substituent on the 2β-(1,2-isoxazole) ring, all compounds were selective for the DAT relative to the 5-HTT and NET. No compound showed death when dosed at 100 mg/kg; however, most compounds did show signs typical of dopamine activity. The ED50 values for 2β-(1,2-isoxazoles) that caused locomotor stimulation ranged from 0.2 to 12.8 mg/kg. Most compounds showed slower on-set and longer duration of action relative to cocaine. Surprisingly, 3β-(4-methylphenyl)-2β-[3-(4‘-chlorophenyl)isoxazol-5-yl]tropane (3p) and 3β-(4-methylphenyl)-2β-[3-(4‘-methylphenyl)isoxazol-5-yl]tropane (3r) did not produce significant increases in locomotor activity. In the cocaine discrimination test, all analogues showed full or at least 50% generalization with the exception of 3p, which did not show generalization. Importantly, both the locomotor activity and the cocaine discrimination ED50values were correlated with the DAT binding but not 5-HTT and NET binding. This provides further support for the dopamine hypothesis of cocaine abuse. High DAT affinity and selectivity, increased locomotor activity with slow onset and long duration of action, and generalization to cocaine shown by the 3β-(substituted phenyl)-2β-(3-substituted isoxazol-5-yl)tropanes are properties thought necessary for a pharmacotherapy for treating cocaine abuse.

A structurally novel opioid δ receptor selective antagonist has been identified. This compound, (+)-5-(3-hydroxyphenyl)-4-methyl-2-(3-phenylpropyl)-2-azabicyclo[3.3.1]non-7-yl-(1-phenyl-1-cyclopentane)carboxamide [(+)-KF4, (+)-4], showed a Ke value of 0.15 nM in the [³⁵S]GTPγS functional assay. (+)-KF4 is also a δ inverse agonist with an IC50 value of 1.8 nM. To our knowledge, this is the first potent and selective δ opioid receptor antagonist from the 5-phenylmorphan class of opioids.

(3R)-7-Hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic) was identified as a potent and selective κ opioid receptor antagonist. Structure−activity relationship (SAR) studies on JDTic analogues revealed that the 3R,4R stereochemistry of the 3,4-dimethyl-4-(3-hydroxyphenyl)piperidine core structure, the 3R attachment of the 7-hydroxy-1,2,3,4-tetrahydroisoquinoline group, and the 1S configuration of the 2-methylpropyl (isopropyl) group were all important to its κ potency and selectivity. The results suggest that, like other κ opioid antagonists such as nor-BNI and GNTI, JDTic requires a second basic amino group to express potent and selective κ antagonist activity in the [³⁵S]GTPγS functional assay. However, unlike previously reported κ antagonists, JDTic also requires a second phenol group in rigid proximity to this second basic amino group. The potent and selective κ antagonist properties of JDTic can be rationalized using the “message−address” concept wherein the (3R,4R)-3,4-dimethyl-4-(hydroxyphenyl)piperidinyl group represents the message, and the basic amino and phenol group in the N substituent constitutes the address. It is interesting to note the structural commonality (an amino and phenol groups) in both the message and address components of JDTic. The unique structural features of JDTic will make this compound highly useful in further characterization of the κ receptor.

A number of 2‘,3‘-disubstituted epibatidine analogues were synthesized and evaluated in vitro for potency at nicotinic acetylcholine receptors (nAChRs) and in vivo for antinociception activity in the tail-flick and hot-plate models of acute pain and for their ability to affect core body temperature. Compounds that possessed electron-withdrawing groups (F, Cl, Br, and I) in both the 2‘- and the 3‘-positions showed affinities at the nAChR similar to epibatidine. However, in vivo efficacy did not correlate with affinity. 2-exo-(3‘-Amino-2‘-chloro-5‘-pyridinyl)-7-azabicyclo[2.2.1]heptane (2i), an epibatidine analogue possessing an electron-releasing amino group in the 3‘-position, produced the highest affinity. Compound 2i was also the most selective epibatidine analogue with a Ki of 0.001 nM at αβ nAChRs, which is 26 times greater than that of epibatidine, and a αβ/α7 Ki ratio of 14 000, twice that of epibatidine. In vivo testing revealed that this compound potently inhibited nicotine-induced antinociception with AD50 values below 1 μg/kg. Surprisingly, this same compound was also an agonist at higher doses (ED50 ~20 μg/kg). Thus, the addition of the 3‘-amino group to epibatidine confers potent antagonist activity to the compound with little effect on agonist activity. 2,3-Disubstituted epibatidine analogues possessing a 2‘-amino group combined with a 3‘-bromo or 3‘-iodo group showed in vitro and in vivo nAChR properties similar to nicotine.

A series of 4‘-substituted 3β-phenyltropane-2β-carboxylic acid methyl esters were synthesized and evaluated for binding at the dopamine transporter (DAT) in order to better define the pharmacophore for the cocaine binding site on the DAT. Results from the study of 3β-[(4‘-phenylalkyl)phenyl]tropane-2β-carboxylic acid methyl esters (5a−c and 6a,b) revealed strong evidence of a previously unknown remote binding domain. The 3β-[(4‘-phenylethyl)phenyl]tropane-2β-carboxylic acid methyl ester (5a), which has a two methylene linker between the 3β-phenyl group and the remote phenyl group, has an IC50 value of 5.14 nM at the DAT. The 3β-[4‘-(benzyl)phenyl] and 3β-[4‘-(phenylpropyl)phenyl] analogues 6b and 5b, respectively, are 102- and 68-fold less potent than 5a at the DAT. Compound 5a also has good affinity for the serotonin and norepinephrine transporters (Ki = 21 and 6.5 nM, respectively) and is thus a nonselective monoamine uptake inhibitor. Electrostatic effects make a significant contribution to the DAT binding affinity of the 3β-[(4‘-phenylalkenyl)phenyl]tropane-2β-carboxylic methyl esters (6c, 7a,b, and 8) and 3β-[(4‘-phenylalkynl)phenyl]tropane-2β-carboxylic acid methyl esters (4a−e). However, the results from the DAT binding on these compounds suggest that there may be another binding domain even further remote from the 4‘-position on the 3β-phenyl group. In both cases, steric barriers have to be overcome before potent binding to the DAT is observed. 3β-(4‘(3-Phenyl-1-propynyl)phenyl)tropane-2β-carboxylic acid methyl ester (4b), with an IC50 value of 1.82 nM, was the most potent compound studied. This compound possessed Ki values of 1.19 and 16.5 nM for the serotonin and norepinephrine transporter and is thus a nonselective monoamine uptake inhibitor.

A library of compounds biased toward opioid receptor antagonist activity was prepared by incorporating N-phenylpropyl-4β-methyl-5-(3-hydroxyphenyl)morphans as the core scaffold using simultaneous solution phase synthetic methodology. From this library, N-phenylpropyl-4β-methyl-5-(3-hydroxyphenyl)-7α-[3-(1-piperidinyl)propanamido]morphan [(−)-3b] was identified as the first potent and selective κ opioid receptor antagonist from the 5-phenylmorphan class of opioids.

The binding characteristics of [125I]3β‐(4‐ethyl‐3‐iodophenyl)nortropane‐2β‐carboxylic acid methyl ester ([125I]EINT), a high‐affinity selective ligand for the serotonin transporter (5‐HTT), and its binding characteristics to rat brain membranes were determined. [125I]EINT binding to rat cerebral cortex membranes was saturable and reversible, and its specific binding represented approximately 90% of the total binding. [125I]EINT labeled a single site with Kd= 0.22 ± 0.03 nM and Bmax= 583 ± 38 fmol/mg protein. Kinetic analysis revealed a t1/2for association and dissociation of 20 and 24 min, respectively. Pharmacological characterization of [125I]EINT confirmed its high specificity for the 5‐HTT. The pattern of brain region distribution in vivo of intravenously administered [125I]EINT indicated greater accumulation of the radioligand in 5‐HTT‐rich brain regions. However, the signal‐to‐background ratio was low. Thus, [125I]EINT appears to be a useful radioligand for studying the 5‐HTT in vitro, but it may not be a good in vivo ligand. Synapse 41:241–247, 2001. © 2001 Wiley‐Liss, Inc.

A structurally novel opioid κ receptor selective ligand has been identified. This compound, (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic, 10) demonstrated high affinity for the κ receptor in the binding assay (κ Ki = 0.3 nM) and highly potent and selective κ antagonism in the [³⁵S]GTP-γ-S assay using cloned opioid receptors (κ Ki = 0.006 nM, μ/κ ratio = 570, δ/κ ratio > 16600).

A convenient, high-yield synthesis of 7-tert-butoxycarbonyl-7-azabicyclo[2.2.1]hept-2-ene (5), which involved the addition of tributyltin hydride to 7-tert-butoxycarbonyl-2-p-toluenesulfonyl-7-azabicyclo[2.2.1]hept-2-ene (4) followed by elimination of the tributyltin and p-tolylsulfonyl groups using tetrabutylammonium fluoride was developed. The addition of 2-amino-5-iodopyridine to 5 under reductive Heck conditions provided 7-tert-butoxycarbonyl-2-exo-(2‘-amino-5‘-pyridinyl)-7-azabicyclo[2.2.1]heptane (6). Compound 6 was the key intermediate used to prepare epibatidine analogues where the 2‘-chloro group on the pyridine ring was replaced with a fluorine (1b), bromine (1c), iodine (1d), hydroxy (1e), amino (1f), dimethylamino (1g), trifluoromethanesulfonate (1h), and hydrogen (1i) group. (+)- and (−)-Epibatidine and compounds 1b−d and 1i all possess similar binding affinities at the α4β2 nAChR receptors labeled by [³H]epibatidine. Compound 1f has affinity similar to nicotine, whereas compounds 1e, 1g, and 1h have much lower affinity. The binding affinity appears to be dependent upon the electronic nature of the substituent. However, other factors are also involved. None of the compounds possesses appreciable affinity for the α7 nAChR labeled by [¹²⁵I]iodo-MLA. With the exception of 1f and 1g, all the epibatidine analogues are full agonists (tail flick test) in producing antinociception after intrathecal injection in mice.

A study of the effect of transposition of the internal nitrogen atom for the adjacent benzylic carbon atom in δ-selective agonists such as BW373U86 (1) and SNC-80 (2) has been undertaken. It was shown that high-affinity, fully efficacious, and δ opioid receptor-selective compounds can be obtained from this transposition. In addition to the N,N-diethylamido group needed as the δ address, the structural features identified to promote δ receptor affinity in the set of compounds studied included a cis relative stereochemistry between the 3- and 4-substituents in the piperidine ring, a trans-crotyl or allyl substituent on the basic nitrogen, the lack of a 2-methyl group in the piperidine ring, and either no substitution or hydroxyl substitution in the aryl ring not substituted with the N,N-diethylamido group. Structural features found to be important for μ affinity include hydroxyl substitution in the aryl ring, the presence of a 2-methyl group in a cis relative relationship to the 4-amino group as well as N-substituents such as cyclopropylmethyl. It was also determined that μ receptor affinity could be increased while maintaining δ receptor affinity, especially when hydroxyl-substituted compounds are considered. Additionally, it was discovered that the somewhat lower μ/δ selectivities observed for the piperidine compounds relative to the piperazine-based ligands appear to arise as a consequence of the carbon−nitrogen transposition which imparts an overall lower δ and higher μ affinity to the piperidine-based ligands. This higher affinity for the μ receptor, apparently intrinsic to the piperidine-based compounds, suggests that ligands of this class will more easily be converted to μ/δ combination agonists compared to the piperazine ligands such as 1. This is particularly important since analogues of 1, which show both μ- and δ-type activity, are now recognized as important for their strong analgesia and cross-canceling of many of the side effects found in agonists operating exclusively from either the δ or μ opioid receptor.

A convenient enantioselective synthesis of (1R,5R,9R)- and (1S,5S,9S)-9-alkyl-2-benzyl-6,7-benzomorphans (2a−c) which starts with naphthaldehyde is described. These compounds were designed to gain additional information on the structure−σ binding relationship of the 6,7-benzomorphan class of σ ligands. In contrast to pentazocine and most 6,7-benzomorphans, the (1R,5R,9R)-isomers of 2a−c showed greater affinity for the σ1 receptor than the (1S,5S,9S)-isomers. Despite reversal of enantioselectivity at the σ1 sites, moderate affinity and enantioselectivity at the σ2 sites [greater affinity for (1R,5R,9R)-isomers than (1S,5S,9S)-isomers] were maintained. A comparison of the binding affinities of 2a−c to the more conformationally flexible trans-2-alkyl-1-benzaminoethyl-1,2-dihydronaphthalenes (10a−c) suggested that the relatively rigid structure of 2a−c played an important part in their σ1 binding properties. These compounds, particularly (1R,5R,9R)-2-benzyl-9-methyl-6,7-benzomorphan [(−)-2a], which has a Ki value of 0.96 nM, will be useful in further characterization of the σ1 receptor.

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the κ opioid receptor selective ligand [³H]U69,593 led to the discovery of a novel κ opioid receptor selective ligand, N-{(2‘S)-[3-(4-hydroxyphenyl)propanamido]-3‘-methylbutyl}-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure−activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the κ receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the μ receptor and an 18-fold increase in affinity for the κ receptor relative to the μ-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [³⁵S]GTPγS at all three opioid receptors, compound 8 behaves as a μ/κ opioid receptor pure antagonist with negligible affinity for the δ receptor.

The present study addressed the hypothesis that there exist multiple sites/states associated with the dopamine (DA) transporter ligands. The authors used [3H](1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) (GBR12935) and [3H]-N-(1-(2-benzo[b]thiophenyl)cyclohexyl)piperidine ([3H]BTCP) to label binding sites present in striatal membranes and conducted experiments under nearly identical assay conditions, i.e., 18- to 24-hr incubations at 4 degrees C in 55.2 mM sodium phosphate buffer, pH 7.4, with a protease inhibitor and antioxidant cocktail. To obtain data suitable for quantitative curve fitting, it was necessary to repurify the [3H]ligands periodically by high-performance liquid chromatography. Under these conditions, greater than 90% specific binding was observed. The method of binding surface analysis was used to characterize the interaction of GBR12935, BTCP, mazindol and 2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane with binding sites labeled by the [3H]ligands. Nonlinear least-squares curve fitting of the data to one- and two-site binding models demonstrated that, for both [3H]ligands, the two-site model fit the data far better than did the one-site model. The results indicated that [3H]GBR12935 labeled two binding sites, with higher (GBR site 1) and lower affinity (GBR site 2) for BTCP (Ki values of 5.84 nM and 1394 nM). [3H]BTCP labeled two sites with high affinity (Kd values of 9.3 nM and 6.3 nM) at which GBR12935 also had high affinity (Ki values of 8.9 nM and 0.98 nM). Intrastriatal 6-hydroxy-DA lesions decreased the density of both [3H]GBR12935 binding sites but affected site 1 much more than site 2, which indicated that a greater portion of GBR site 1 is localized on striatal nerve terminals. By contrast, the 6-hydroxy-DA lesions decreased BTCP site 2 without significantly decreasing BTCP site 1, which indicated that BTCP site 1 is not located on DA nerve terminals. The i.c.v. administration of 5,7,-dihydroxytryptamine did not decrease GBR site 1 or 2, which indicated that neither is located on serotonin striatal nerve terminals. Viewed collectively with other reports, these data support the hypothesis that DA transporter ligands label multiple binding sites in caudate membranes. The identification of selective agents for these sites may be valuable tools for identifying drugs that might modulate the effects of cocaine.

Earlier work characterized the binding of the high-affinity cocaine analog 3 beta-(4-125iodophenyl)-tropane-2-carboxylic acid methyl ester ([125I]RTI-55) to membranes prepared from rat caudate. That investigation demonstrated that [125I]RTI-55-labeled serotonin (5-HT) transporters in addition to dopamine (DA) transporters and resolved [125I]RTI-55 binding to 5-HT transporters into two distinct components. In the present study, we characterized [125I]RTI-55 binding to membranes prepared from whole rat brain minus caudate. The first series of experiments established that [125I]RTI-55 labels both DA and 5-HT transporters and that 50 nM paroxetine and either 1000 nM 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)homopiperazine (LR1111) or 500 nM (RTI-120) could be used to block [125I]RTI-55 binding to the 5-HT and DA transporters, thereby generating selective assay conditions for the DA and 5-HT transporters, respectively. Selective lesioning of dopaminergic and serotonergic neurons with intracerebroventricular 6-hydroxydopamine and 5,7-dihydroxytryptamine selectively decreased [125I]RTI-55 binding to DA and 5-HT transporters, respectively, thereby confirming the selectivity of the assay conditions. The ligand-selectivity pattern of the whole brain minus caudate 5-HT transporter correlated significantly with that of the caudate 5-HT transporter, although there were some striking differences for selected test agents. Additional experiments resolved [125I]RTI-55 binding to the 5-HT transporter into two components. A ligand-selectivity analysis of the two components failed to identify a highly selective test agent. In summary, the major findings of the present study are that [125I]RTI-55 labels both DA and 5-HT transporters in membranes prepared from whole brain minus caudate, that 50 nM paroxetine and either 1000 nM LR1111 or 500 nM RTI-120 can be used as a blocking agent to generate selective assay conditions for the DA and 5-HT transporters, respectively, and that [125I]RTI-55 binding to the 5-HT transporter can be resolved into two similar components.

Dopamine Transporter (DAT) binding sites in medial temporal lobe structures are much less dense than in striatum and have been difficult to image and quantitate. The recently synthesized compound [125I]RTI-121 ([125I]2 beta-carboxylic acid isopropyl ester-3 beta-(4-iodophenyl)tropane) has demonstrated high specificity and affinity for the DAT in preliminary animal studies. The present experiments were designed to delineate the pharmacological and anatomical characteristics of [125I]RTI-121 binding sites in the striatum and medial temporal lobe of normal humans. A series of saturation experiments performed with striatal membrane preparations generated a one-site model with a KD averaging 1.49 +/- 0.06 nM, and a Bmax that was comparable to those of earlier reports. Competition experiments confirmed the selectivity of [125I]RTI-121 for DAT binding sites. After the assay conditions for autoradiography were optimized, [125I]RTI-121 binding was visualized, pharmacologically characterized and quantitated in human temporal lobe structures. In the hippocampus, specific binding was distributed in the CA4 and CA3 pyramidal cell layers, the outer stratum radiatum and the dentate molecular layer, but not in the dentate granule cells or outer pyramidal cells. In the amygdala specific binding was limited to the basolateral nuclei. Dopamine nerve terminals, as identified with [125I]RTI-121 binding, also displayed a discrete and homologous innervation pattern in the amygdala and hippocampus of several other mammalian species. In summary, the kinetic, saturation, competition and autoradiographic experiments demonstrated that [125I]RTI-121 can be used to identify DAT binding sites and to assess their functional state in post mortem human brain samples.

RTI-121 AND RTI-122 are 3β-substituted phenyltropane analogs of cocaine that have high, selective binding affinity for dopamine transporters. [123I]RTI-121 and [123I]RTI-122 bind to dopamine transporters in vivoafter intravenous administration and permit imaging of the transporters.

A practical synthesis of epibatidine (1) was developed. The key step involved selective reduction of the 5,6-double bond of 7-(tert-butyloxy-carbonyl)-2-(2-chloro-5-pyridyl)-3-(phenylsulfonyl)-7-azabicyclo[2.2.1]-heptane-2,5-diene (4) using nickel boride.

Fluorine-18 labeled norchlorofluoroepibatidine (NFEP), a high-affinity nicotinic acetylcholine receptor ligand, was prepared by a one-pot, two-step synthesis: nucleophilic heteroaromatic substitution of a tert-Boc protected precursor (7-tert-butyloxycarbonyl-exo-2-(2'-N,N,N-trimethylammonium-5'-pyridinyl)-7-azabicyclo[2.2.1]heptane iodide) using no-carrier-added [¹⁸F]fluoride followed by deprotection with trifluoroacetic acid. Subsequent reductive N-methylation with formaldehyde and sodium cyanoborohydride afforded fluorine-18 labeled N-methyl-norchlorofluoroepibatidine (N-methyl-NFEP). The unusually high radiochemical yield for the first step (70%) and the quantitative conversions in the deprotection and N-methylation steps afforded overall radiochemical yields of 55–65% (decay corrected based on starting [¹⁸F]fluoride) for [¹⁸F]NFEP (synthesis time 65 min) and 45–55% for [¹⁸F]N-methyl-NFEP (synthesis time 75 min), with a specific activity of 2–9 Ci/μmole (EOB). © 1997 John Wiley & Sons, Ltd.

The iodine-125 analog of the dopaminergically selective cocaine analog 3 beta-(4-iodophenyl)tropane-2 beta-carboxylic acid isopropyl ester (RT1-121) was evaluated as a probe for the dopamine transporter in rat striatum. Saturation and kinetic studies indicated that [125I]RTI-121 binds to both high and low affinity components. The Kd of the high affinity component was 0.14 +/- 0.01 nM (mean +/- standard error), whereas the low affinity component demonstrated an affinity of 1.59 +/- 0.09 nM. The corresponding numbers of striatal binding sites labeled by [125I]RTI-121 were 295 +/- 6 and 472 +/- 59 pmol/g of tissue (original wet weight), respectively. Intrastriatal injections of 6-hydroxydopamine eliminated > 90% of specific [125I]RTI-121 binding in the striatum. The pharmacological profile of specific [125I]RTI-121 binding in the rat striatum was consistent with that of the dopamine transporter. There was a strong (r = 0.98, p < 0.0001) correlation between the potencies of drugs that displaced specific [125I]RTI-121 binding and the potencies of these drugs to inhibit the uptake of [3H]dopamine. In contrast, no correlation was found for the potencies of drugs to inhibit the uptake of either [3H]norepinephrine or [3H]serotonin. Autoradiographs produced using [125I]RTI-121 demonstrated a distribution of label consistent with the distribution of dopaminergic neurons in rat brain. Because of its high affinity and high selectivity for the dopamine transporter, [125I]RTI-121 may be an extremely useful ligand for the dopamine transporter.

A study of the binding site requirements associated with the N-substituent of (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) derivatives was undertaken using a set of rigid vs flexible N-substituents. The study showed that compounds 7−9 bearing the trans-cinnamyl N-substituent most closely reproduced the potency at the opioid receptor of the flexible N-propylphenyl or N-propylcyclohexyl analogues previously reported. Neither the N-substituted cis-cinnamyl nor the cis-phenylcyclopropylmethyl compounds 10 and 11, respectively, showed high affinity for the opioid receptor. However, the N-trans-phenylcyclopropylmethyl compound 12 closely approximated the affinity of compounds 7−9. Additionally, we found that free rotation of the phenyl ring is necessary for high affinity binding and μ receptor subtype selectivity as the planar N-substituted thianaphthylmethyl and benzofuranylmethyl compounds 13 and 14 had significantly lower binding affinities. Altogether, these findings suggest that the high binding affinity, selectivity, and antagonist potency of N-propylphenyl or N-propylcyclohexyl analogues of (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) are achieved via a conformation wherein the connecting chain of the N-substituents is extended away from piperidine nitrogen with the appended ring system rotated out-of-plane relative to the connecting chain atoms. This conformation is quite similar to that observed in the solid state for 5, as determined by single crystal X-ray analysis. Additionally, it was found that, unlike naltrexone, N-substituents bearing secondary carbons attached directly to the piperidine nitrogen of 4 suffer dramatic losses of potency vs analogues not substituted in this manner. Using a functional assay which measured stimulation or inhibition of [³⁵S]GTP-γ-S binding, we show that the trans-cinnamyl analogues of (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) retain opioid pure antagonist activity and possess picomolar antagonist potency at the μ receptor.

(3S,4R)-4-(4-Fluorophenyl)-3-[[3,4-(methylenedioxy)phenoxy]methyl]piperidine [(3S,9R)-3, paroxetine] is a selective serotonin reuptake inhibitor (SSRI) used as an antidepressant in humans. In previous studies, we reported that certain (1R)-3β-(substituted phenyl)nortropane-2β-carboxylic acid methyl esters (2a) exhibited high affinity and reasonable selectivity for the serotonin transporter (5-HTT). The major structural differences between 2a and (3S,4R)-3 are that 2a possesses a different absolute stereochemistry and has an ethylene bridge not present in 3. In addition, 2a possesses a carbomethoxy substituent adjacent to the aryl ring, whereas (3S,4R)-3 contains a [3,4-(methylenedioxy)phenoxy]methyl group. In this study, we present the synthesis and biological evaluations of six of the possible eight isomers of 3-(4-fluorophenyl)-2-[[3,4-(methylenedioxy)phenoxy]methyl]nortropane (4). The data for inhibition of [³H]paroxetine binding show that (1R)-2β,3α-4c, which has the same stereochemistry as paroxetine, has the highest affinity at the 5-HTT. Strikingly, the most potent compounds for inhibition of [³H]WIN-35,428 binding were not the (1R)-2β,3β-isomers but rather (1R)-2β,3α-4c and (1S)-2β,3α-4f. Conformational analyses show that these isomers exist in a flattened boat conformation with pseudoequatorial substituents. Thus, the binding data show that this conformation is recognized by the DAT-associated binding site and also suggest that this conformation of paroxetine is recognized by the 5-HTT-associated binding site.

New methods for the synthesis of 3β-(4‘-alkyl-, 4‘-alkenyl-, and 4‘-alkynylphenyl)nortropane-2β-carboxylic acid methyl esters 2−4, respectively, were developed. These methods involved coupling of the appropriate organometallic reagents to 3β-(4‘-iodophenyl)tropane-2β-carboxylic acid methyl ester (6a, RTI-55) or to an N-protected derivative of 6a followed by N-demethylation or removal of the protecting group. Some analogs were prepared by catalytic reduction of the alkene and alkyne analogs 3 and 4 or by isomerization of the alkenes 3. The analogs 2−4 were evaluated for inhibition of radioligand binding to the serotonin (5-HT), dopamine (DA), and norepinephrine (NE) transporters. 3β-(4‘-Isopropenyl- and 4‘-cis-propenylphenyl)nortropane-2β-carboxylic acid methyl esters (3b,d), which possessed IC50 values of 0.6 and 1.15 nM, respectively, were the most potent analogs at the 5-HT transporter, and with NE/5-HT IC50 ratios of 240 and 128 nM, respectively, they were selective for the 5-HT relative to the NE transporter. Since interaction with the serotonin transporter may modulate the pharmacological effects resulting from binding to the dopamine transporter, 3β-(4‘-isopropenylphenyl)tropane-2β-carboxylic acid methyl ester (11b) which has good affinity for both the 5-HT and DA transporters but low affinity at the NE transporter may be useful for studying this interaction.

A set of 3β-(4‘-substituted phenyl)-2β-heterocyclic tropanes was designed, synthesized, and characterized. We discovered that these compounds can function as bioisosteric replacements for the corresponding WIN 35,065-2 analogs which possess a 2β-carbomethoxy group. Several of the compounds showed high affinity and selectivity for the dopamine transporter (DAT) relative to the serotonin and norepinephrine transporters. From the structure−activity relationship study, the 3β-(4‘-chlorophenyl)-2β-(3‘-phenylisoxazol-5-yl)tropane (5d) emerged as the most potent and selective compound. The binding data for 2β-heterocyclic tropanes were found to show a high correlation with molecular electrostatic potential (MEP) minima near one of the heteroatoms in the 2β-substituents. In contrast, low correlations were found for other MEP minima near the 2β-substituent as well as for calculated log P or substituent volume. These quantitative structure−activity relationship studies are consistent with an electrostatic contribution to the binding potency of these WIN 35,065-2 analogs at the DAT.

Sphingosine is a naturally occurring long-chain amino diol with potent inhibitory activity against protein kinase C in vitro and in cell systems. The use of sphingosine as a pharmacological tool to probe the activity of protein kinase C has been hampered by its amphiphilicity, possible contamination of its commercial preparations, and the existence of other targets for its action. To address these problems, high-purity D-erythro-sphingosine was prepared and employed to develop an approach for the use of sphingosine as a pharmacological agent. The addition of synthetic D-erythro-sphingosine to intact human platelets resulted in quick uptake and preferential partitioning into the particulate fraction. It was rapidly metabolized by intact platelets, 60% being degraded within 1 min after addition. Sphingosine was found to be a potent inhibitor of gamma-thrombin-induced aggregation and secretion of washed human platelets. Multiple criteria indicated that this effect is probably mediated through the inhibition of protein kinase C: (1) sphingosine inhibited protein kinase C activity in intact platelets with a similar dose/response to its inhibition of platelet aggregation and secretion; (2) sphingosine inhibited phorbol binding to intact platelets under identical conditions and with a similar dose-dependence; (3) exogenous dioctanoylglycerol overcame sphingosine's inhibition of platelet activation. The effectiveness of sphingosine in inhibiting platelet activation was primarily determined by the ratio of sphingosine to total number of platelets. These data are discussed in relation to a general approach for the use of sphingosine and other parameters for determining biological activities of protein kinase C.