Aporphines are attractive candidates for imaging D2 receptor function because, as agonists rather than antagonists, they are selective for the receptor in the high affinity state. In contrast, D2 antagonists do not distinguish between the high and low affinity states, and in vitro data suggests that this distinction may be important in studying diseases characterized by D2 dysregulation, such as schizophrenia and Parkinson’s disease. Accordingly, MCL-536 (R-(−)-N-n-propyl-2-(3-[18F]fluoropropanoxy-11-hydroxynoraporphine) was selected for labeling with 18F based on in vitro data obtained for the non-radioactive (19F) compound. Fluorine-18-labeled MCL-536 was synthesized in 70% radiochemical yield, >99% radiochemical purity and specific activity of 167 GBq/μmol (4.5 Ci/μmol) using p-toluenesulfonyl (tosyl) both as a novel protecting group for the phenol and a leaving group for the radiofluorination.
aporphines; D2 agonist; neurological disorders; positron emission tomography; dopamine receptors; D2high; D2low; tosyl group; phenol protection; fluorine-18
PET has been used to examine changes in neurotransmitter concentrations in the living brain. Pioneering PET studies on the dopamine system have used D2 and D3 receptor (D2/D3) antagonists such as 11C-raclopride. However, more recently developed agonist radioligands have shown enhanced sensitivity to endogenous dopamine. A limitation of available agonist radioligands is that they incorporate the short-lived radioisotope 11C. In the current study, we developed the 18F-labeled D2/D3 receptor agonist (R)-(−)-2-18F-fluoroethoxy-N-n-propylnorapomorphine (18F-MCL-524).
In total, 10 PET measurements were conducted on 5 cynomolgus monkeys. Initially, the binding of 18F-MCL-524 was compared with that of 11C-MNPA in 3 monkeys. Second, the specificity of 18F-MCL-524 binding was examined in pretreatment studies using raclopride (1.0 mg/kg) and D-amphetamine (1.0 mg/kg). Third, a preliminary kinetic analysis was performed using the radiometabolite-corrected arterial input function of the baseline studies. Finally, 2 whole-body PET measurements were conducted to evaluate bio-distribution and radiation dosimetry after intravenous injection of 18F-MCL-524.
18F-MCL-524 entered the brain and provided striatum-to-cerebellum ratios suitable for reliable quantification of receptor binding using the multilinear reference tissue model. Mean striatal nondisplaceable binding potential (BPND) values were 2.0 after injection of 18F-MCL-524 and 1.4 after 11C-MNPA. The ratio of the BPND values of 18F-MCL-524 and 11C-MNPA was 1.5 across striatal subregions. After administration of raclopride and D-amphetamine, the 18F-MCL-524 BPND values were reduced by 89% and 56%, respectively. Preliminary kinetic analysis demonstrated that BPND values obtained with the 1-tissue- and 2-tissue-compart-ment models were similar to values obtained with the multilinear reference tissue model. Estimated radiation doses were highest for gallbladder (0.27 mSv/MBq), upper large intestine (0.19 mSv/MBq), and small intestine (0.17 mSv/MBq). The estimated effective dose was 0.035 mSv/MBq.
The 18F-labeled agonist 18F-MCL-524 appears suitable for quantification of D2/D3 receptor binding in vivo, and the results encourage extension to human studies. The longer half-life of 18F makes 18F-MCL-524 attractive for studies on modulation of the dopamine concentration—for example, in combination with simultaneous measurement of changes in blood-oxygen-level–dependent signal using bimodal PET/functional MRI.
18F-MCL-524; agonist; dopamine; monkey; PET
A series of levo-
and dextromorphinan pairs have been synthesized and evaluated for
their affinities to the mu, kappa, and delta opioid receptors, the N-methyl-d-aspartate (NMDA) channel, and sigma
1 and 2 receptors. It was found that levo isomers tended to have higher
affinities at the opioid receptors and moderate to high affinities
to the NMDA and sigma receptors, while dextro isomers tended to have
lower affinities to the opioid receptors but comparatively higher
affinities to the NMDA and sigma receptors. This series of compounds
have interesting and complex pharmacological profiles, and merit further
investigation as potential therapies for drug abuse treatment.
Drug abuse; opioid receptor; sigma receptor; NMDA receptor; enantiomer; ICSS
Previous studies with aminothiazolomorphinans suggested that this class of opioid ligands may be useful as a potential pharmacotherapeutic to decrease drug abuse. Novel aminothiazole derivatives of cyclorphan were prepared in order to evaluate a series of aminothiazolomorphinans with varying pharmacological properties at the MOR and KOR. This study was focused on exploring the regioisomeric analogs with the aminothiazole on the C-ring of the morphinan skeleton. Receptor binding and [35S]GTPγS binding assays were used to characterize the affinity and pharmacological properties of the aminothiazolomorphinans. Intracranial self-stimulation (ICSS) was used to compare effects of a representative aminothiazolomorphinan with the morphinan mixed KOR/MOR agonist butorphan (MCL-101) on brain stimulation reward.
A series of 3-benzylamino-3-desoxymorphinan (I) and 3-benzylamino-3-desoxymorphine (II) derivatives were synthesized and evaluated for their binding affinities and functional activity data are presented at MOR, KOR, and DOR. Some of these ligands were found to have high binding affinity at MOR and KOR, and displayed increased selectivity at MOR over KOR and DOR compared to butorphan or cyclorphan. The most selective compound, 3-(3′-hydroxybenzyl)amino-17-methylmorphinan (4g) (24-fold MOR to KOR and 1700-fold MOR to DOR) also showed high binding affinity (0.42 nM to MOR), and was a full agonist in the [35S]GTPγS binding assay. 2-(3′-Hydroxybenzyl)amino-17-cyclopropylmethylmorphinan (17) was found to be a KOR-selective ligand (150-fold over MOR and >10,000-fold over the DORs). Most 3-benzylaminomorphinan derivatives were partial agonists at MOR and full agonists at KOR in the [35S]GTPγS binding assay.
Sulfur-containing analogues of 8-substituted xanthines were prepared in an effort to increase selectivity or potency as antagonists at adenosine receptors. Either cyclopentyl or various aryl substituents were utilized at the 8-position, because of the association of these groups with high potency at A1-adenosine receptors. Sulfur was incorporated on the purine ring at positions 2 and/or 6, in the 8-position substituent in the form of 2- or 3-thienyl groups, or via thienyl groups separated from an 8-aryl substituent through an amide-containing chain. The feasibility of using the thienyl group as a prosthetic group for selective iodination via its Hg2+ derivative was explored. Receptor selectivity was determined in binding assays using membrane homogenates from rat cortex [[3H]-N6-(phenylisopropyl) adenosine as radioligand] or striatum [[3H]-5′-(N-ethylcarbamoyl)adenosine as radioligand] for A1- and A2-adenosine receptors, respectively. Generally, 2-thio-8-cycloalkylxanthines were at least as A1 selective as the corresponding oxygen analogue. 2-Thio-8-aryl derivatives tended to be more potent at A2 receptors than the oxygen analogue. 8-[4-[(Carboxymethyl)oxy]phenyl]-1,3-dipropyl-2-thioxanthine ethyl ester was >740-fold A1 selective.
Investigation of bivalent ligands at μ, δ and κ opioid receptors is now focused on the preparation of ligands containing κ agonist and μ agonist/antagonist pharmacophores at one end joined by a linking chain containing the μ antagonists pharmacophores (naltrexone, naloxone or nalbuphine) at the other end. These ligands were evaluated in-vitro by their binding affinity at μ, δ and κ opioid receptors and their relative efficacy in the [35S]GTPγS assay.
Ligands 6-8 displayed better or the same affinity at κ and μ receptors compared to the monovalent ligands 1-5. Ligands 6-8 generally showed reduced affinity at δ receptor compared to the monovalent ligands 1-5. Functional assays showed that the ligand 6 was a κ agonist/antagonist and μ antagonist whereas ligands 7 and 8 were κ agonists and μ agonists/antagonists.
A novel series of homo- and heterodimeric ligands containing κ/μ agonist and μ agonist/antagonist pharmacophores joined by a 10-carbon ester linker chain were synthesized and evaluated for their in vitro binding affinity at κ, μ, and δ opioid receptors and their functional activities were determined at κ and μ receptors in [35S]GTPγS functional assays. Most of these compounds had high binding affinity at μ and κ receptors (Ki values less than 1 nM). Compound 15b, which contains butorphan (1) at one end of linking chain and butorphanol (5) at the other end, was the most potent ligand in this series with binding affinity Ki values of 0.089 nM at the μ receptor and 0.073 nM at the κ receptor. All of the morphinan-derived ligands were found to be partial κ and μ agonists; ATPM-derived ligands 12 and 11 were found to be full κ agonists and partial μ agonists.
A series of N-substituted and N′-substituted aminothiazole-derived morphinans (5) were synthesized for expanding the structure-activity relationships of aminothiazolo-morphinans. Although their affinities were somewhat lower than their prototype aminothiazolo-N-cyclopropylmorphinan (3), 3-aminothiazole derivatives of cyclorphan (1) containing a primary amino group displayed high affinity and selectivity at the κ and μ opioid receptors. [35S]GTPγS binding assays showed that the aminothiazolomorphinans were κ agonists with mixed agonist and antagonist activity at the μ opioid receptor. These novel N′-monosubstituted aminothiazole-derived morphinans may be valuable for the development of drug abuse medications.
The 2-fluoroalkoxy substituted catechol-aporphines 6, 8a-f and 11-monohydroxyaporphines 11a-e were synthesized and found to have high in vitro affinity and selectivity for the dopamine D2 receptors. The catechol aporphines, 8b and 8d, and the monohydroxy aporphines, 11a-d, were identified as candidates for development as potential PET ligands.
aporphine; D2 agonist; neurological disorders; positron emission tomography; dopamine receptors
The 2-fluoroalkoxy-substituted catechol-aporphines 6, 8a−f and 11-mono-hydroxyaporphines 11a−e were synthesized and found to have high in vitro affinity and selectivity for the dopamine D2 receptors. The catechol aporphines, 8b and 8d, and the monohydroxy aporphines, 11a−d, were identified as candidates for development as potential PET ligands.
Aporphine; D2 agonist; neurological disorders; positron emission tomography; dopamine receptors
Bifunctional ligands containing an ester linkage between morphine and the δ-selective pharmacophore Dmt-Tic were synthesized, and their binding affinity and functional bioactivity at the μ, δ and κ opioid receptors determined. Bifunctional ligands containing or not a spacer of β-alanine between the two pharmacophores lose the μ agonism deriving from morphine becoming partial μ agonists 4 or μ antagonists 5. Partial κ agonism is evidenced only for compound 4. Finally, both compounds showed potent δ antagonism.
A new synthetic method for the removal of the 4, 5-bridged ether moiety of several opioids has been developed. This process offers a faster, simpler synthetic route to obtain the morphinone scaffold in high yields without the need for protection of the ketone moiety.
morphinone; opioid; 4, 5-ether bridge; triflate reduction
A series of bivalent hydroxy ether butorphan ligands were prepared and their binding affinities at the opioid receptors determined. Addition of a hydroxy group to a hydrocarbon chain can potentiate binding affinity up to 27 and 86 fold at the mu and kappa opioid receptors, respectively. Two bivalent ligands with sub-nanomolar binding affinity at the mu and kappa opioid receptors were discovered.
Based on a renewed importance recently attributed to bi- or multifunctional opioids, we report the synthesis and pharmacological evaluation of some analogues derived from our lead μ agonist / δ antagonist, H-Dmt-Tic-Gly-NH-Bzl. Our previous studies focused on the importance of the C-teminal benzyl function in the induction of such bifunctional activity. The introduction of some substituents in the para position of the phenyl ring (-Cl, -CH3, partially −NO2, inactive -NH2) was found to give a more potent μ agonist / antagonist effect associated with a relatively unmodified δ antagonist activity (pA2 = 8.28-9.02). Increasing the steric hindrance of the benzyl group (using diphenylmethyl and tetrahydroisoquinoline functionalities) substantially maintained the μ agonist and δ antagonist activities of the lead compound. Finally and quite unexpectedly D-Tic2, considered as a wrong opioid message now; inserted into the reference compound in lieu of L-Tic, provided a μ agonist / δ agonist better than our reference ligand (H-Dmt-Tic-Gly-NH-Ph) and was endowed with the same pharmacological profile.
Based on a renewed importance recently attributed to bi- or multifunctional opioids, we report the synthesis and pharmacological evaluation of some analogues derived from our lead μ agonist/δ antagonist, H-Dmt-Tic-Gly-NH-Bzl (Dmt = 2′,6′-dimethyl-l-tyrosine, Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, Bzl = benzyl). Our previous studies focused on the importance of the C-teminal benzyl function in the induction of such bifunctional activity. The introduction of some substituents in the para position of the phenyl ring (−Cl, −CH3, partially −NO2, inactive −NH2) was found to give a more potent μ agonist/antagonist effect associated with a relatively unmodified δ antagonist activity (pA2 = 8.28−9.02). Increasing the steric hindrance of the benzyl group (using diphenylmethyl and tetrahydroisoquinoline functionalities) substantially maintained the μ agonist and δ antagonist activities of the lead compound. Finally and quite unexpectedly d-Tic2, considered as a wrong opioid message now, inserted into the reference compound in lieu of l-Tic provided a μ agonist/δ agonist better than our reference ligand (H-Dmt-Tic-Gly-NH-Ph; Ph = phenyl) and was endowed with the same pharmacological profile.
Bifunctional opioids; Dmt−Tic pharmacophore; opioid peptides; opioid receptors, angiogenesis, tolerance
The phenolic group of the potent μ and κ opioid morphinan agonist/antagonists cyclorphan and butorphan was replaced by phenylamino and benzylamino groups including compounds with p-substituents in the benzene ring. These compounds are highly potent μ and κ ligands, e. g. p-methoxyphenylaminocyclorphan showing a Ki of 0.026 nM at the mu and a Ki of 0.03 nM at the kappa receptor. Phenyl carbamates and phenylureas were synthesized and investigated. Selective o-formylation of butorphan and levorphanol was achieved. This reaction opened the way to a large set of 2-substituted 3-hydroxymorphinans, including 2-hydroxymethyl-, 2-aminomethyl-, and N-substituted 2-aminomethyl-3-hydroxymorphinans. Bivalent ligands bridged in the 2-position were also synthesized and connected with secondary and tertiary aminomethyl groups, amide bonds or hydroxymethylene groups, respectively. Although most of the 2-substituted morphinans showed considerably lower affinities compared to their parent compounds, the bivalent ligand approach led to significantly higher affinities compared to the univalent aminomethylmorphinans.
Bivalent morphinan compounds containing ester linkers were synthesized and their binding affinities at the μ, δ, and κ opioid receptors determined. Addition of methyl groups adjacent to the hydrolytically labile ester linkage increased stability while only partially affecting binding affinity. The resulting bivalent ligands with optimized spacer-length and structure show potent binding profiles with the most potent compound (4b) having Ki values of 0.47 nM for both the μ and κ opioid receptors, and 4a having Ki values of 0.95 and 0.62 nM for the μ and κ receptors, respectively. Both 4a and 4b were partial agonists at the κ and μ receptors in the [35S]GTPγS binding assay.
To investigate the effects of ATPM-ET [(−)-3-N-Ethylaminothiazolo [5,4-b]-N-cyclopropylmethylmorphinan hydrochloride] on physical dependence and behavioral sensitization to morphine in mice.
The pharmacological profile of ATPM-ET was characterized using competitive binding and GTPγS binding assays. We then examined the antinociceptive effects of ATPM-ET in the hot plate test. Morphine dependence assay and behavioral sensitization assay were used to determine the effect of ATPM-ET on physical dependence and behavior sensitization to morphine in mice.
The binding assay indicated that ATPM-ET ATPM-ET exhibited a high affinity to both κ- and μ-opioid receptors with Ki values of 0.15 nmol/L and 4.7 nmol/L, respectively, indicating it was a full κ-opioid receptor agonist and a partial μ-opioid receptor agonist. In the hot plate test, ATPM-ET produced a dose-dependent antinociceptive effect, with an ED50 value of 2.68 (2.34–3.07) mg/kg. Administration of ATPM-ET (1 and 2 mg/kg, sc) prior to naloxone (3.0 mg/kg, sc) injection significantly inhibited withdrawal jumping of mice. In addition, ATPM-ET (1 and 2 mg/kg, sc) also showed a trend toward decreasing morphine withdrawal-induced weight loss. ATPM-ET (1.5 and 3 mg/kg, sc) 15 min before the morphine challenge significantly inhibited the morphine-induced behavior sensitization (P<0.05).
ATPM-ET may have potential as a therapeutic agent for the treatment of drug abuse.
behavioral sensitization; mu opioid receptors; morphine; kappa opioid receptors; physical dependence; addiction
Once opioid receptor dimers were postulated, a goal has been to synthesize and screen novel opioids, with the hope of furthering our knowledge of the structure-activity relationship of opioid ligands with the opioid receptors. The aim of the current study was to address whether two isomeric bivalent ligands would have pharmacological differences after central administration, in vivo. The two compounds, (−) bis(N-cyclobutylmethyl-morphinan-3-yl) sebacoylate dihydrochloride (MCL-144) and 1-((+)N-cyclobutylmethylmorphinan-3-yl)-10-((−) N-cyclobutylmethylmorphinan-3-yl)sebacolyate (MCL-193) are each linked by a 10-carbon chain ester. The active (−) enantiomer for both ligands is 3-hydroxy-N-cyclobutylmethyl morphinan ((−)MCL-101), a N-cyclobutylmethyl analogue of cyclorphan (1). MCL-144 contains two active levo rotatory (−)(−) pharmacophores, while MCL-193 contains one active (−) and one inactive (+) pharmacophore of MCL-101. In vitro analysis demonstrated that all three compounds, (−)(−) MCL-144, (+)(−) MCL-193 and (−)MCL-101 were κ agonists and μ partial agonists. (−)(−)MCL-144 and (−)MCL-101 had much higher affinity for both the μ and κ opioid receptors compared to (+)(−)MCL-193. In vivo, (−)(−)MCL-144 and (+)(−)MCL-193 produced full dose-response curves, in the 55°C tail-flick test, with each compound having an ED50 value of 3.0 nmol after intracerebroventricular (i.c.v.) administration. The analgesic properties of both compounds were antagonized by the μ-selective antagonist, β-funaltrexamine and the κ-selective antagonist nor-binaltorphimine. Concomitant, i.c.v., administration of either (−)(−)MCL-144 or (+)(−)MCL-193 with morphine, did not significantly antagonize morphine-induced antinociception at any dose tested. In antinociceptive tests, (−)(−)MCL-144 and (+)(−)MCL-193 had the same pharmacological properties, demonstrating that having two active pharmacophores separated by a 10-carbon spacer group did not increase the antinociceptive efficacy of the compound. Additionally, it was also of interest to compare (−)(−)MCL-145 and (−)(−)MCL-144, as the only difference between these bivalent ligands is the spacer region connecting the two pharmacophores, yet (−)(−)MCL-145 produced an ED50 value 10-fold lower than (−)(−)MCL-144 (ED50 values = 0.3 nmol and 3.0 nmol, respectively).
opioid dimers; mu opioid; partial agonist; enantiomers
We synthesized several hydrophobic esters and ethers of butorphanol and assessed their affinities at opioid receptors in CHO cell membranes. Tested compounds displayed moderate to high affinities to the mu and kappa receptors. The findings accord with previous evidence of a lipophilic binding pocket in the opioid receptors that can be accessed to afford good binding affinity without the need for a phenolic hydrogen-bond donor group. The most potent (Ki = 61 pM at μ and 48 pM at κ) novel agent was (-)-N-cyclobutylmethylmorphinan-3-yl-14-ol phenoxyacetate (4d).
A series of 2-amino-oxazole (7 & 8) analogs and 2-one-oxazole analogs (9 &10) were synthesized from cyclorphan (1) or butorphan (2) and evaluated in-vitro by their binding affinity at μ, δ and κ opioid receptors and compared with their 2-aminothiozole analogs 5 & 6. Ligands 7–10 showed decreased affinities at κ and μ receptors.
Urea analogues (11–14) were also prepared from 2-aminocyclorphan (3) or 2-aminobutorphan (4) and evaluated in-vitro by their binding affinity at μ, δ and κ opioid receptors. The urea derived opioids retained their affinities at μ receptors while showing increased affinities at δ receptors and decreased affinities at κ receptors. Functional activities of these compounds were measured in the [35S]GTPγS binding assay, illustrating that all of these ligands were κ agonists. At the μ receptor, compounds 11 and 12 were μ agonist/antagonists.
Morphinan; Urea; Oxazol; Opioid
(±) SKF83959, like many other arylbenzazepines, elicits powerful neuroprotection in vitro and in vivo. The neuroprotective action of the compound was found to partially depend on its D1-like dopamine receptor agonistic activity. The precise mechanism for the (±) SKF83959-mediated neuroprotection remains elusive. We report here that (±) SKF83959 is a potent blocker for delayed rectifier K+ channel. (±) SKF83959 inhibited the delayed rectifier K+ current (IK) dose-dependently in rat hippocampal neurons. The IC50 value for inhibition of IK was 41.9±2.3 µM (Hill coefficient = 1.81±0.13, n = 6), whereas that for inhibition of IA was 307.9±38.5 µM (Hill coefficient = 1.37±0.08, n = 6). Thus, (±) SKF83959 is 7.3-fold more potent in suppressing IK than IA. Moreover, the inhibition of IK by (±) SKF83959 was voltage-dependent and not related to dopamine receptors. The rapidly onset of inhibition and recovery suggests that the inhibition resulted from a direct interaction of (±) SKF83959 with the K+ channel. The intracellular application of (±) SKF83959 had no effects of on IK, indicating that the compound most likely acts at the outer mouth of the pore of K+ channel. We also tested the enantiomers of (±) SKF83959, R-(+) SKF83959 (MCL-201), and S-(−) SKF83959 (MCL-202), as well as SKF38393; all these compounds inhibited IK. However, (±) SKF83959, at either 0.1 or 1 mM, exhibited the strongest inhibition on the currents among all tested drug. The present findings not only revealed a new potent blocker of IK , but also provided a novel mechanism for the neuroprotective action of arylbenzazepines such as (±) SKF83959.
Dopamine D1-like partial receptor agonists such as SKF 83959 have been proposed as potential candidates for the treatment of cocaine addiction. The present studies were conducted to further characterize SKF 83959 by pharmacologically evaluating effects of its R-(+)- and S-(−) enantiomers, MCL 202 and MCL 201, respectively, on overt behavior (eye blinking) and schedule-controlled performance in squirrel monkeys. MCL 202, like the D1 full receptor agonist SKF 82958, produced dose related increases in eye blinking and decreases in rates of fixed-ratio responding. However, the magnitude of effects of MCL 202 on eye blinking was less than observed with SKF 82958. In contrast to the effects of its R-(+) enantiomer, MCL 201 was relatively devoid of behavioral activity up to doses that were approximately 10-fold greater than MCL 202. Pretreatment with the selective D1-like receptor antagonist SCH 39166 dose-dependently antagonized increases in eye blinking produced by MCL 202, confirming the involvement of D1 mechanisms in its effects. A dose-ratio analysis of the antagonism of effects of MCL 202 by SCH 39166 revealed an apparent pA2 value of 7.675 with a slope of −0.78 ± 0.04. In further studies, pretreatment with MCL 202 antagonized the effects of SKF 82958 on eye blinking and, like SCH 39166, schedule-controlled behavior in a dose-related manner. A dose-ratio analysis of the antagonist effects of MCL 202 on the SKF 82958-induced increases in eye blinking revealed ratios of 2.7, 4.8 and 31.1 for 0.1, 0.3 and 1.0 mg/kg dose of the antagonist, respectively, indicative of a significant change in the potency of SKF 82958. These results suggest that MCL 202, like its parent compound SKF 83959, has both D1 receptor-mediated agonist and antagonist properties, consistent with its characterization as a partial agonist at the D1-like receptor. In addition, the inactivity of MCL 201, the S-(−)-enantiomer, suggests that the behavioral effects of SKF 83959 can be attributed primarily to the activity of its R-(+)-enantiomer.
Dopamine D1-like receptors; Enantiomers of SKF 83959; Partial agonist; Eye blinking; Schedule-controlled behavior; SKF 82958; Squirrel monkeys
Here we report the facile synthesis of a designed multi-pharmacophore ligand derived from the linkage of a delta selective peptide antagonist (Dmt-Tic) and a mu / kappa morphinan agonist butorphan (MCL 101) through a two methylene spacer. The new compound MCL 450 maintains the same characteristics as the two reference compounds. MCL 450 represents a useful starting point for the synthesis of other multiple opioid ligands endowed with analgesic properties with low tolerance and dependence.