New Insights into the Opioid Analgesic Profile of cis-(-)-N-Normetazocine-derived Ligands.

In this work, we report on the in vitro and in vivo pharmacological properties of LP1 analogs to complete the series of structural modifications aimed to generate compounds with improved analgesia. To do that, the phenyl ring in the N-substituent of our lead compound LP1 was replaced by an electron-rich or electron-deficient ring and linked through a propanamide or butyramide spacer at the basic nitrogen of the (-)-cis-N-normetazocine skeleton. In radioligand binding assays, compounds 3 and 7 were found to display nanomolar binding affinity for the µ opioid receptor (MOR) (Ki = 5.96 ± 0.08 nM and 1.49 ± 0.24 nM, respectively). In the mouse vas deferens (MVD) assay, compound 3 showed an antagonist effect against DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin), a highly selective MOR prototype agonist, whereas compound 7 produced naloxone reversible effect at MOR. Moreover, compound 7, as potent as LP1 and DAMGO at MOR, was able to reduce thermal and inflammatory pain assessed by the mouse tail-flick test and rat paw pressure thresholds (PPTs) measured by a Randall-Selitto test.

Novel N-normetazocine Derivatives with Opioid Agonist/Sigma-1 Receptor Antagonist Profile as Potential Analgesics in Inflammatory Pain.

Although opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) are the most common drugs used in persistent pain treatment; they have shown many side effects. The development of new analgesics endowed with mu opioid receptor/delta opioid receptor (MOR/DOR) activity represents a promising alternative to MOR-selective compounds. Moreover, new mechanisms, such as sigma-1 receptor (σ1R) antagonism, could be an opioid adjuvant strategy. The in vitro σ1R and σ2R profiles of previous synthesized MOR/DOR agonists (-)-2R/S-LP2 (1), (-)-2R-LP2 (2), and (-)-2S-LP2 (3) were assayed. To investigate the pivotal role of N-normetazocine stereochemistry, we also synthesized the (+)-2R/S-LP2 (7), (+)-2R-LP2 (8), and (+)-2S-LP2 (9) compounds. (-)-2R/S-LP2 (1), (-)-2R-LP2 (2), and (-)-2S-LP2 (3) compounds have Ki values for σ1R ranging between 112.72 and 182.81 nM, showing a multitarget opioid/σ1R profile. Instead, (+)-2R/S-LP2 (7), (+)-2R-LP2 (8), and (+)-2S-LP2 (9) isomers displayed a nanomolar affinity for σ1R, with significative selectivity vs. σ2R and opioid receptors. All isomers were evaluated using an in vivo formalin test. (-)-2S-LP2, at 0.7 mg/kg i.p., showed a significative and naloxone-reversed analgesic effect. The σ1R selective compound (+)-2R/S-LP2 (7), at 5.0 mg/kg i.p., decreased the second phase of the formalin test, showing an antagonist σ1R profile. The multitarget or single target profile of assayed N-normetazocine derivatives could represent a promising pharmacological strategy to enhance opioid potency and/or increase the safety margin.

Acid-mediated ring-expansion reaction of N-aryl-2-vinylazetidines: synthesis and unanticipated reactivity of tetrahydrobenzazocines.

The aza-Clasen rearrangement of N-aryl-2-vinylazetidines has been explored. N-Aryl-2-vinylazetidines were transformed to corresponding tetrahydrobenzazocines in good yields. Unexpectedly, the tetrahydrobenzazocine was unstable and readily isomerized to vinyltetrahydroquinoline in the presence of acid. The mechanism of this ring contraction was studied in detail.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 8. High affinity ligands for opioid receptors in the picomolar Ki range: oxygenated N-(2-[1,1'-biphenyl]-4-ylethyl) analogues of 8-CAC.

N-[2-(4'-methoxy[1,1'-biphenyl]-4-yl)ethyl]-8-CAC (1) is a high affinity (K(i)=0.084 nM) ligand for the µ opioid receptor and served as the lead compound for this study. Analogues of 1 were made in hopes of identifying an SAR within a series of oxygenated (distal) phenyl derivatives. A number of new analogues were made having single-digit pM affinity for the µ receptor. The most potent was the 3',4'-methylenedioxy analogue 18 (K(i)=1.6 pM).

Asymmetric syntheses of (-)-pentazocine and (-)-eptazocine through an aza-Prins cyclization.

Two down more to go: The asymmetric syntheses of (-)-pentazocine and (-)-eptazocine are presented. The highlights of the syntheses are the construction of the core skeleton through an aza-Prins cyclization and intramolecular Friedel-Crafts reaction.

Structures in solutions from joint experimental-computational analysis: applications to cyclic molecules and studies of noncovalent interactions.

The potential of an approach combining nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics (MD) simulations, and quantum mechanical (QM) calculations for full structural characterizations in solution is assessed using cyclic organic compounds, namely, benzazocinone derivatives 1-3 with fused five- and eight-membered aliphatic rings, camphoric anhydride 4, and bullvalene 5. Various MD simulations were considered, using force field and semiempirical QM treatments, implicit and explicit solvation, and high-temperature MD calculations for selecting plausible molecular geometries for subsequent QM geometry optimizations using mainly B3LYP, M062X, and MP2 methods. The QM-predicted values of NMR parameters were compared to their experimental values for verification of the final structures derived from the MD/QM analysis. From these comparisons, initial estimates of quality thresholds (calculated as rms deviations) were 0.7-0.9 Hz for (3)J(HH) couplings, 0.07-0.11 Å for interproton distances, 0.05-0.08 ppm for (1)H chemical shifts, and 1.0-2.1 ppm for (13)C chemical shifts. The obtained results suggest that the accuracy of the MD analysis in predicting geometries and relative conformational energies is not critical and that the final geometry refinements of the structures selected from the MD simulations using QM methods are sufficient for correcting for the expected inaccuracy of the MD analysis. A unique example of C(sp(3))-H···N(sp(3)) intramolecular noncovalent interaction is also identified using the NMR/MD/QM and the natural bond orbital analyses. As the NMR/MD/QM approach relies on the final QM geometry optimization, comparisons of geometric characteristics predicted by different QM methods and those from X-ray and neutron diffraction measurements were undertaken using rigid and flexible cyclic systems. The joint analysis shows that intermolecular noncovalent interactions present in the solid state alter molecular geometries significantly compared to the geometries of isolated molecules from QM calculations.

A novel synthesis of (di)-benzazocinones via an endocyclic N-acyliminium ion cyclisation.

The triflic acid-mediated endocyclic N-acyliminium ion cyclisation provides a facile synthesis of (di)-benzazocinones. On reduction of the 10-phenyl derivative, an unusually non-polar tertiary alkylamine was obtained.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 7: syntheses and opioid receptor properties of cyclic variants of cyclazocine.

A series of 7,8- and 8,9-fused triazole and imidazole analogues of cyclazocine have been made and characterized in opioid receptor binding and [(35)S]GTPgammaS assays. Target compounds were designed to explore the SAR surrounding our lead molecule for this study, namely the 8,9-fused pyrrolo analogue 2 of cyclazocine. Compared to 2, many of the new compounds in this study displayed very high affinity for opioid receptors.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 6: Opioid receptor binding properties of cyclic variants of 8-carboxamidocyclazocine.

A series of 7,8- and 8,9-fused pyrimidinone, aminopyrimidine and pyridone derivatives of 8-carboxamidocyclazocine (8-CAC) have been prepared and evaluated in opioid receptor binding assays. Targets were designed to corroborate a pharmacophore hypothesis regarding the bioactive conformation of the carboxamide of 8-CAC. In addition to the results from this study strongly supporting this pharmacophore hypothesis, a number of novel compounds with high affinity to opioid receptors have been identified.

New benzomorphan derivatives of MPCB as MOP and KOP receptor ligands.

There is considerable interest in developing KOP Opioid receptor ligands as clinically useful analgesics. Moreover, compounds with mixed KOP receptor and mu-opioid peptide (MOP) receptor agonist/antagonist properties could have a better therapeutic potential. The benzomorphan-based synthetic ligands MPCB and CCB have been shown to bind KOP receptors with high affinity and selectivity. We report here a series of compounds synthesized to perform structure-affinity relationship (SAR) studies on MPCB. The aim of this study was to optimise KOP receptor-ligand interaction and to modulate MOP receptor selectivity. In the benzylamide analogue of MPCB (compound 9) the presence of a third aromatic nucleus, at an appropriate distance and conformation with respect to aromatic pharmacophoric residues, increased KOP receptor affinity by about 6-fold compared to MPCB (Ki = 35 nM and Ki = 240 nM, respectively). Instead, compound 28 with a tertiary amino group in the nitrogen substituent displayed a comparable KOP receptor affinity (Ki = 179 nM) but also high MOP receptor affinity (Ki = 45 nM). Thus, the present study shows that in benzomorphan-based ligands the presence of different functional groups in the nitrogen substituent, ranging from a positive charged amine to an additional aromatic ring, is able to promote the correct aligment of aromatic pharmacophoric residues with MOP and KOP receptor types. Evaluation of docking simulations of compounds 9 and 28 into the KOP and MOP receptor displayed selective ligand interactions with the important amino acid residues Tyr320 (TMVII) and Trp318 (TMVII), respectively.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 5. Opioid receptor binding properties of N-((4'-phenyl)-phenethyl) analogues of 8-CAC.

A series of aryl-containing N-monosubstituted analogues of the lead compound 8-[N-((4'-phenyl)-phenethyl)]-carboxamidocyclazocine were synthesized and evaluated to probe a putative hydrophobic binding pocket of opioid receptors. Very high binding affinity to the mu opioid receptor was achieved though the N-(2-(4'-methoxybiphenyl-4-yl)ethyl) analogue of 8-CAC. High binding affinity to mu and very high binding affinity to kappa opioid receptors was observed for the N-(3-bromophenethyl) analogue of 8-CAC. High binding affinity to all three opioid receptors were observed for the N-(2-naphthylethyl) analogue of 8-CAC.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 4. Opioid receptor binding properties of 8-[N-(4'-phenyl)-phenethyl)carboxamido] analogues of cyclazocine and ethylketocycalzocine.

The synthesis and evaluation of a series of aryl-containing N-monosubstituted analogues of the lead compound 8-carboxamidocyclazocine were performed to probe a putative hydrophobic binding pocket of opioid receptors. High binding affinity to mu, kappa, and delta opioid receptors was observed for the 8-[N-(4'-phenyl)-phenethyl)carboxamido] analogue.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 3: 8-Thiocarboxamido and 8-thioformamido derivatives of cyclazocine.

8-Position variants of cyclazocine have been made where the phenolic 8-OH was replaced by thioamide, amidine, guanidine, urea and thiourea groups. High affinity for opioid receptors was observed for the 8-CSNH2 and 8-NHCHS analogues indicating that these groups are isosteric with not only the 8-OH but with the previously synthesized 8-CONH2 and 8-NHCHO cyclazocine derivatives.

Effects of the mixed-action kappa/mu opioid agonist 8-carboxamidocyclazocine on cocaine- and food-maintained responding in rhesus monkeys.

The present study evaluated the effects of 8-carboxamidocyclazocine (8-CAC), a novel mixed-action kappa/mu agonist with a long duration of action, on food- and cocaine-maintained responding in rhesus monkeys to assess the potential utility of 8-CAC as a medication for the treatment of cocaine dependence. The effects of acute and chronic (10 days) 8-CAC were examined in rhesus monkeys responding under a multiple schedule for both cocaine and food reinforcement. Acute 8-CAC (0.032-0.56 mg/kg, i.m.) dose-dependently eliminated cocaine-maintained responding in all three monkeys. However, doses of 8-CAC that decreased cocaine self-administration typically also decreased food-maintained responding, and 8-CAC-induced decreases in cocaine self-administration diminished during chronic 8-CAC treatment. These results confirm that 8-CAC acutely decreases cocaine self-administration. However, non-selective effects of 8-CAC on food-maintained responding and tolerance to 8-CAC effects on cocaine self-administration may limit its potential for the treatment of cocaine dependence.

Cyclazocine: comparison to hydromorphone and interaction with cocaine.

Kappa-opioid agonists produce neurobiological and behavioral effects opposite to those of cocaine and may be useful for the treatment of cocaine dependence. To evaluate the kappa- and mu-agonist effects of cyclazocine and to test whether cyclazocine pretreatment would attenuate the effects of cocaine, healthy, male and female, experienced opiate and cocaine users (n = 13) were enrolled in a two-phase study. In Phase 1, placebo, cyclazocine (0.2, 0.4 and 0.8 mg) and the mu-agonist hydromorphone (5 and 15 mg) were administered orally in six 4.5-hour sessions separated by at least 72 h. In Phase 2, cocaine (100 mg intranasal) was given 2 h after oral pretreatment with cyclazocine (0, 0.1, 0.2, 0.4, 0.8 and 0 mg, in that order) in each of six sessions conducted daily Monday to Friday and the following Monday. Physiological, subjective and behavioral measures were collected in each session. Nine participants completed Phase 1; eight completed Phase 2. Hydromorphone (15 mg) produced prototypic mu-agonist effects. Cyclazocine exhibited only modest kappa-like effects. Cyclazocine also had only modest, non-dose-related effects on response to cocaine. However, cocaine effects were consistently lower on the last administration (cyclazocine 0 mg pretreatment) following 4 days of cyclazocine pretreatment, compared to the first administration (0 mg pretreatment). This finding is unlikely to be fully attributable to cocaine tolerance and is not accounted for by pharmacokinetic changes; plasma concentrations of cocaine were not altered by cyclazocine. This study is suggestive but not strongly supportive for the use of kappa-opiate drugs to diminish acute effects of cocaine administration or for the use of these kappa agonists in drug abuse treatment applications.

A laboratory study of hydromorphone and cyclazocine on smoking behavior in residential polydrug users.

The effects of cyclazocine and hydromorphone on spontaneous and laboratory cigarette smoking were compared in a double-blind, placebo-controlled, crossover study. Participants (seven men, one woman) received oral doses of placebo, cyclazocine (0.2, 0.4, and 0.8 mg) and hydromorphone (5 and 15 mg) in a randomized order on experimental days. Spontaneous smoking was recorded during two intervals on the experimental days: a 3-h period 5-8 h after drug administration (Interval 1), and the rest of the day (Interval 2). Measures of smoking topography and subjective and physiologic effects of a single cigarette were obtained on the experimental days. Neither hydromorphone nor cyclazocine significantly changed spontaneous smoking when compared to the placebo condition; however, compared to hydromorphone (5 mg), cyclazocine (0.4 and 0.8 mg) decreased spontaneous smoking during Interval 1. Hydromorphone (5 and 15 mg) and cyclazocine (0.4 and 0.8 mg) diminished smoking-induced increases in heart rate. Compared to the placebo condition, cyclazocine (0.2 and 0.4 mg) reduced exhaled carbon monoxide (CO) boost, a measure of smoke exposure. Further studies of the effects of kappa opioid agonists on smoking behavior may lead to a better understanding of the role of opiates in smoking behavior.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 2: 8-formamidocyclazocine analogues.

High affinity binding for mu and kappa opioid receptors has been observed in analogues of cyclazocine, ethylketocyclazocine and naltrexone where the prototypic (of opiates) phenolic OH group was replaced with a formamide (-NHCHO) group. For the 8-formamide analogue of cyclazocine, binding is highly enantiospecific (eudismic ratios approximately 2000 for mu and kappa) with K(i) values

Cardiovascular effects of intravenous pentazocine and cyclazocine in conscious, curarized-conscious, and anesthetized dogs.

The cardiovascular effects of intravenous pentazocine and cyclazocine in dogs were studied under conscious, curarized-conscious (paralyzed by gallamine), and anesthetized states. In the conscious state, blood pressure and heart rate were dose-dependently increased by pentazocine (1, 2, 3 mg/kg) and to a lesser extent by cyclazocine (0.3 mg/kg). In all subsequent experiments on dogs, the results were obtained using 3 mg/kg pentazocine and 0.3 mg/kg cyclazocine. Pentazocine accelerated breathing, peaking at about 10 min, whereas cyclazocine reduced breathing to a minimum in 1 min, followed by a gradual recovery thereafter. In the curarized-conscious state, the blood pressure response to pentazocine was biphasic, namely an initial decrease followed by an increase; chronotrophic activity was stimulated. Pretreatment with either ganglionic or alpha andrenergic blocking agents not only significantly antagonized the pressory responses to the drug but also potentiated the initial decreases in blood pressure and unmasked a bradycardic component, but these parameters were not altered by 0.3 mg/kg naxalone. In open-chest anesthetized dogs, blood pressure, heart rate, contractility, and mean peripheral vascular resistance were simultaneously decreased by both pentazocine and cyclazocine, initially accompanied by increases in aortic blood flow. During the later stages of drug action, only the blood pressure and contractility were increased above control levels (biphasic effect). A comparison of blood pressure and heart rate responses to pentazocine in dogs kept under differing experimental conditions revealed that conscious dogs were more sensitive than curarized conscious and anesthetized animals to pentazocine action. In isolated guinea pig atria, the effect of adrenaline (0.1, 0.3, or 1 mg/mL) on the spontaneous breathing rate was significantly augmented by 10 mg/mL pentazocine (p < 0.02 for 0.3 g/mL; p < 0.01 for 0.1 g/mL adrenaline). In dogs, however, adrenaline (1 mg/kg)-induced increases in heart contractility, aortic blood flow, and blood pressure remained almost unaltered in the presence of pentazocine. We concluded that the abovementioned cardiovascular responses to pentazocine and cyclazocine are a consequence of the sum of the two following opposing effects: (i) an indirect reflex activation of sympathetic neuromediation in the periphery, and (ii) a direct membrane effect on the heart leading to bradycardia and a depression in myocardial contractility.

Altered levels of mRNA expression and pharmacological reactivity of alpha1-adrenergic receptor subtypes in the late-pregnant rat myometrium.

The adrenergic system plays a major role in the regulation of the uterine contractility during pregnancy. Our previous studies have shown the significance of the alpha1-adrenergic receptors (ARs) in the control of pregnant uterine contractility both in vitro and in vivo. Our present aim was to determine the changes in mRNA expression and pharmacological reactivity of the alpha1-ARs on days 18, 20, and 22 of pregnancy. To demonstrate the expressions of alpha1-AR subtype mRNA, we used a reverse transcription-polymerase chain reaction (RT-PCR); the pharmacological reactivity was tested by electric field stimulation (EFS). The expression of alpha1A-AR mRNA increased from day 18 to 22, while no alpha1B-AR mRNA was detectable. We found a small increase in the expression of alpha1D-AR mRNA on day 20, which was not followed by a significant change in pharmacological reactivity. The alpha1D-receptor expression and pharmacological reactivity decreased significantly up to day 22. EFS studies revealed that the alpha1A-AR antagonist 5-methylurapidil had EC50 values (1.9 x 10(-6)-6.3 x 10(-6) M) about one order of magnitude lower than those of the alpha1D-AR antagonist BMY 7378 (4 x 10(-6)-3.6 x 10(-5) M). However, the alpha1B-AR antagonist cyclazosine exerted only a slight effect on the stimulated contractions. Strong correlations were found between the alpha1A-mRNA expression and the EC50 of 5-methylurapidil (r(2) =0.9712), and between the alpha1D-AR mRNA expression and the EC50 of BMY 7378 (r(2) = 0.9937). Our findings suggest that both alpha1A- and alpha1D-ARs are involved in the regulation of the pregnant uterine contractility. The density and pharmacological reactivity indicate that the alpha(1A)-AR seems to play the major role in late-pregnant myometrial contraction.

8-Carboxamidocyclazocine: a long-acting, novel benzomorphan.

To obtain benzomorphans with a longer duration of action that may be potential therapeutics for treating cocaine abuse, 8-carboxamidocyclazocine was synthesized. The pharmacological properties of 8-carboxamidocyclazocine were compared with the parent compound cyclazocine. Changing the 8-hydroxyl group on cyclazocine to an 8-carboxamido group resulted in only a 2-fold decrease in the affinity of the compound for the kappa-receptor, and no change in the affinity for the mu-opioid receptor, with both compounds having K(i) values of less than 1 nM, based on radioligand binding assays. In the guanosine 5'-O -(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assay, the two compounds produced moderate stimulation of GTP binding to the human kappa- and mu-receptors. When given by i.c.v. injection, the compounds produced less than 60% antinociception in the mouse 55 degrees C warm-water tail-flick test. However, in the mouse writhing test, the compounds had high potency in producing antinociception. Antinociception induced by either 8-carboxamidocyclazocine or cyclazocine was mediated by both kappa- and mu-opioid receptors. Cyclazocine acted as a mu-antagonist in addition to its agonist properties at the mu-receptor, as measured by the inhibition of morphine-induced antinociception. In contrast, 8-carboxamidocyclazocine did not inhibit morphine-induced antinociception, demonstrating that it was not a mu-opioid receptor antagonist in this assay. An i.p. injection of an ED(70) dose of 8-carboxamidocyclazocine produced antinociception that lasted for 15 h in contrast to cyclazocine, which produced antinociception, lasting 2 h. 8-Carboxamidocyclazocine is a novel, long-acting benzomorphan, which possesses pharmacological properties that are distinct from the properties of cyclazocine.