Synthesis and biological evaluation of 14-alkoxymorphinans. 2. (-)-N-(cyclopropylmethyl)-4,14-dimethoxymorphinan-6-one, a selective mu opioid receptor antagonist.

(-)-N-(Cyclopropylmethyl)-4,14-dimethoxymorphinan-6-one (2) was synthesized with 4,14-dimethoxy-N-methylmorphinan-6-one (1) as starting material. In vivo and in vitro experiments show 2 (cyprodime) to be a pure opioid receptor antagonist. Some of these tests (opioid receptor binding assays, guinea pig ileal longitudinal muscle preparation, rat and mouse vas deferens preparation, acetic acid writhing antagonism test) indicate that 2 is a selective mu opioid receptor antagonist.

Synthesis and biological evaluation of 14-alkoxymorphinans. 3. Extensive study on cyprodime-related compounds.

A series of cyprodime-related compounds (2, 4-12, and 26) has been synthesized and evaluated for opioid agonist and antagonist activity with the mouse vas deferens and guinea pig ileum preparations. None of the changes to cyprodime, including the introduction of a 3-OMe group, increasing and decreasing the size of or completely removing the substituent in position 4, replacing the N-cyclopropylmethyl group with an N-allyl group, or replacing the 14-OMe with an 14-OEt substituent, resulted in an improved mu antagonist profile and most were detrimental either in terms of mu selectivity and potency or increased agonist activity. Increasing the length of the substituent in position 4 resulted in a compound (6a) with a very similar profile to that of cyprodime.

Synthesis and biological evaluation of 14-alkoxymorphinans. 1. Highly potent opioid agonists in the series of (-)-14-methoxy-N-methylmorphinan-6-ones.

A series of eight (-)-14-methoxymorphinan-6-ones was synthesized and biologically evaluated. The morphinanones 3-7 were prepared from 3-desoxy-7,8-dihydro-14-hydroxymorphinone (1). The key step in this synthetic sequence, O-methylation in position 14, was accomplished with dimethyl sulfate. Hydrolysis followed by reductive opening of the 4,5-oxygen bridge afforded the phenol 4, which was O-methylated to give 5. Removal of the 4-OH group yielded the aromatic unsubstituted morphinan 7. The synthesis of 9 and 10 was accomplished by starting from 14-methoxy-7,8-dihydrocodeinone and involved a similar reaction sequence. The compounds 12-15 were synthesized from oxymorphone (11), which was 3-O-benzylated, 6,14-bis-O-methylated with dimethyl sulfate, hydrolyzed, and hydrogenated to yield the oxymorphone 14-O-methyl ether 15. The derivatives 3, 4, 5, 7, 9, 10, 14, and 15 exhibited high antinociceptive potency in the hot-plate assay in mice, after both subcutaneous and oral administration. The most potent derivative in this series (15) showed a potency (sc) about 400 times higher than that of morphine and about 40 times higher than its 14-OH analogue oxymorphone (11). The 14-OCH3 series also exhibited a considerably higher affinity to opioid receptors in binding studies using [3H]naloxone as ligand when compared to their 14-OH analogues.

Synthesis and biological evaluation of 14-alkoxymorphinans. 11. 3-Hydroxycyprodime and analogues: opioid antagonist profile in comparison to cyprodime.

A series of 3-hydroxy-substituted analogues (3-7) of the mu selective opioid antagonist cyprodime has been synthesized in order to evaluate the role of a hydroxy group at C-3 concerning mu opioid antagonist selectivity. Compounds 3-7 were tested in bioassays (electrical stimulated mouse vas deferens preparation and myenteric-plexus longitudinal muscle preparation of the guinea pig ileum) and opioid receptor binding assays. Antagonism of mu receptor-mediated responses induced by the mu selective agonist DAMGO afforded equilibrium dissociation constants in the mouse vas deferens preparation (Ke values) for compounds 3-7 which agreed closely with their affinities as determined by opioid receptor binding assays (Ki values). At kappa and delta receptors differences were apparent. Although the compounds had high affinity for both kappa and delta receptors in opioid receptor binding, they were very poor at antagonizing agonist responses mediated by kappa and particularly delta agonists in the mouse vas deferens preparation. None of the compounds tested showed agonist potency in the mouse vas deferens preparation or the myenteric-plexus longitudinal muscle preparation of the guinea pig ileum.

Non-deleterious inhibition of endogenous peroxidase activity (EPA) by cyclopropanone hydrate: a definitive approach.

Endogenous peroxidase activity (EPA) poses a serious problem in immunoperoxidase localization of antigens unable to withstand deleterious effects of aldehyde fixatives, alcohols, and various oxidative reagents. This has forced the development of more selective inhibition methods. Of these, phenylhydrazine or azide combined with small amounts of H2O2 have proved quite effective. However, the precise mechanism of the action of these compounds on EPA generating proteins is not understood. Cyclopropanone hydrate is a compound whose inhibitory action on the heme moiety of horseradish peroxidase is well understood. The aim of this study was to investigate the effect of this compound on EPA and to compare its efficiency with that of optimal phenylhydrazine and sodium azide regimens. In addition, any gross deleteriousness of cyclopropanone hydrate towards immunoperoxidase immunolocalization of three of the most delicate lymphocyte surface antigens was investigated. Cyclopropanone hydrate was found to inhibit EPA with progressing strength between 0.15-15 mM. Over this range, H2O2 was found necessary for inhibition only for cyclopropanone hydrate concentrations up to 0.15 mM. Beyond this amount, the compound inhibited EPA equally strongly in the presence or absence of H2O2, reaching near-maximum inhibition at 15 mM. This and the H2O2-requiring regimens were found to cause no gross diminution in immunoperoxidase staining of CD4, CD6, and CD8 antigens in snap-frozen, acetone-fixed human tonsil sections. Cyclopropanone hydrate therefore provides a definitive non-deleterious mode of inhibiting EPA for immunoperoxidase staining of delicate antigens.

HS-599: a novel long acting opioid analgesic does not induce place-preference in rats.

1. When administered subcutaneously HS-599, a new didehydroderivative of buprenorphine (18,19-dehydrobuprenorphine), produced a long-lasting antinociceptive response in rats. Its potency exceeded twice that of buprenorphine. In the tail-flick test it acted as a full agonist but in the plantar test only as a partial agonist. Whereas the mu-opioid antagonists naloxone and naltrexone antagonized HS-599 antinociception the delta-opioid antagonist naltrindole and the kappa-opioid antagonist nor-binaltorphimine did not. 2. Unlike buprenorphine and morphine, HS-599 never induced conditioned place-preference in rats. 3. In radioligand binding assays, compared with buprenorphine HS-599 had 3 fold higher mu-opioid receptor affinity but lower delta- and kappa-opioid receptor affinity. 4. In isolated guinea-pig ileum preparations, HS-599 only partially inhibited the electrically-stimulated contraction, acting as a partial opioid agonist. When tested against the mu-opioid receptor agonist dermorphin, it behaved as a non-equilibrium antagonist. Conversely, in mouse vas deferens (rich in delta-opioid receptors) and rabbit vas deferens preparations (rich in kappa-opioid receptors) HS-599 acted as a pure equilibrium antagonist, shifting the log-concentration-response curves of the delta-opioid agonist deltorphin I and the kappa-opioid agonist U-69593 to the right. 5. In conclusion, HS-599 is a novel buprenorphine derivative with higher affinity, selectivity and potency than the parent compound, for mu-opioid receptors. It produces intense and long-lasting antinociception and does not induce place-preference in rats.

Opioid peptide receptor studies. 9. Identification of a novel non-mu- non-delta-like opioid peptide binding site in rat brain.

Quantitative binding studies resolved two high-affinity [3H][D-Ala2,D-Leu5]enkephalin binding sites in rat brain membranes depleted of mu binding sites by pretreatment with the irreversible agent BIT. The two binding sites had lower (delta ncx-2, Ki = 96.6 nM) and higher (delta ncx-1, Ki = 1.55 nM) affinity for DPDPE. The ligand-selectivity profile of the delta ncx-1 site was that of a classic delta binding site. The ligand-selectivity profile of the delta ncx-2 site was neither mu- or delta-like. The Ki values of selected agents for the delta ncx-2 site were: [pCl]DPDPE (3.9 nM), DPLPE (140 nM), and DAMGO (2.6 nM). Under these assay conditions, [3H][D-Ala2,D-Leu5]enkephalin binding to the cells expressing the cloned mu receptor is very low and pretreatment of cell membranes with BIT almost completely inhibits [3H]DAMGO and [3H][D-Ala2,D-Leu5]enkephalin binding. Intracerebroventricular administration of antisense DNA to the cloned delta receptor selectively decreased [3H][D-Ala2,D-Leu5]enkephalin binding to the delta ncx-1 site. Administration of buprenorphine to rats 24 h prior to preparation of membranes differentially affected mu, delta ncx-1, and delta ncx-2 binding sites. Viewed collectively, these studies have identified a novel non-mu- non-delta-like binding site in rat brain.

Highly potent novel opioid receptor agonist in the 14-alkoxymetopon series.

The newly synthesized 14-alkoxymetopon derivatives, 14-methoxymetopon, 14-ethoxymetopon, 14-methoxy-5-methyl-morphinone, exhibit high affinity for the naloxone binding sites in rat brain. A substantial decrease in affinity was observed, in the presence of NaCl indicating a high degree of agonist activity. All three 14-alkoxymetopon derivatives displayed high affinity for [3H][D-Ala2,(Me)Phe4,Gly-ol5]enkephalin ([3H]DAMGO) binding sites, much less potency toward delta sites and were the least effective at kappa sites. Isolated tissue studies using the guinea pig ileum preparation confirmed their high agonist potency. Following administration the new compounds produced naloxone reversible antinociceptive effects and were 130-300 times more potent than morphine in the acetic acid induced abdominal constriction model in the mouse, and the hot plate and tail flick tests in the rat. The compounds also produced dose-dependent muscle rigidity, and potentiated barbiturate-induced narcosis. The in vivo apparent pA2 values for naloxone against 14-ethoxymetopon and morphine were similar in analgesia, suggesting an interaction with the same (mu) receptor site. The dependence liability of 14-alkoxymetopon derivatives in the withdrawal jumping test was less pronounced than that of morphine in either rats or mice, similar to tolerance to the their analgesic action. It is concluded that the 14-alkoxymetopon derivatives studied are selective and potent agonists at mu opioid receptors, with reduced dependence liability.

Mu-opioid receptor specific antagonist cyprodime: characterization by in vitro radioligand and [35S]GTPgammaS binding assays.

The use of compounds with high selectivity for each opioid receptor (mu, delta and kappa) is crucial for understanding the mechanisms of opioid actions. Until recently non-peptide mu-opioid receptor selective antagonists were not available. However, N-cyclopropylmethyl-4,14-dimethoxy-morphinan-6-one (cyprodime) has shown a very high selectivity for mu-opioid receptor in in vivo bioassays. This compound also exhibited a higher affinity for mu-opioid receptor than for delta- and kappa-opioid receptors in binding assays in brain membranes, although the degree of selectivity was lower than in in vitro bioassays. Cyprodime has recently been radiolabelled with tritium resulting in high specific radioactivity (36.1 Ci/mmol). We found in in vitro binding experiments that this radioligand bound with high affinity (K(d) 3. 8+/-0.18 nM) to membranes of rat brain affording a B(max) of 87. 1+/-4.83 fmol/mg. Competition studies using mu, delta and kappa tritiated specific ligands confirmed the selective labelling of cyprodime to a mu-opioid receptor population. The mu-opioid receptor selective agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO) was readily displaced by cyprodime (K(i) values in the low nanomolar range) while the competition for delta- ([D-Pen(2), D-Pen(5)]enkephalin (DPDPE)) and kappa- (5alpha,7alpha, 8beta-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl]-benzene-acetamide (U69,593)) opioid receptor selective compounds was several orders of magnitude less. We also found that cyprodime inhibits morphine-stimulated [35S]GTPgammaS binding. The EC(50) value of morphine increased about 500-fold in the presence of 10 microM cyprodime. These findings clearly indicate that cyprodime is a useful selective antagonist for mu-opioid receptor characterization.

Use of selective antagonists and antisense oligonucleotides to evaluate the mechanisms of BUBU antinociception.

Evidence suggests that the antinociceptive effects of selective delta-opioid receptor agonists may involve an activation of the mu-receptor in some experimental conditions. The aim of this study was to clarify the receptors involved in the antinociceptive responses of the selective and systemically active delta-opioid receptor agonist Tyr-D-Ser-(O-tert-butyl)-Gly-Phe-Leu-Thr-(O-tert-butyl) (BUBU). The antinociception induced by systemic (i.v.) or central (i.c.v.) administration of BUBU was measured in the hot plate (jumping and paw lick latencies) and tail immersion tests in mice. In both tests, the responses were more intense when BUBU was administered by central route. The pre-treatment with the mu-opioid receptor antagonist cyprodime blocked the effects induced by central BUBU in the hot plate and tail immersion tests. The delta-opioid receptor antagonist naltrindole had no effect on BUBU-induced antinociception in the hot plate but decreased BUBU responses in the tail immersion test. Further evidence for this dual receptor action of BUBU was demonstrated by using antisense oligodeoxynucleotides. Thus, a reduction in central BUBU-induced antinociception was observed in the tail immersion test after the administration of antisense probes that selectively blocked the expression of mu- or delta-opioid receptors. These findings clearly indicate using a dual pharmacological and molecular approach that BUBU mediates its antinociceptive effects via activation of both mu- and delta-opioid receptors.

Inverse agonism by Dmt-Tic analogues and HS 378, a naltrindole analogue.

The potent delta-opioid receptor antagonist H-2',6-L-tyrosine(Dmt)-1, 2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic-OH) exhibited partial inverse agonism (EC(50)=6.35 nM, E(max)=-18.87%) for [35S]GTPgammaS binding and H-Dmt-Tic-NH(2) was a neutral antagonist (no effect up to 30 microM). In contrast N,N(CH(3))(2)-Dmt-Tic-NH(2) was a full inverse agonist (EC(50)=2.66 nM, E(max)=-35.95%) similar to ICI 174864 ([N,N-diallyl-Tyr(1),Aib(2,3),Leu(5)]enkephaline) but with a 3.5-fold higher EC(50). In comparison, naltrindole was a neutral antagonist while its analogue HS 378 was a partial inverse agonist (E(max)=-12.99%).

Antinociceptive activity of a novel buprenorphine analogue.

HS-599 is a didehydroderivative of buprenorphine that displays high affinity and good selectivity for mu-opioid receptors. We studied its antinociceptive properties after s.c. injection in mice with the tail-flick and hot-plate tests. In the tail-flick test HS-599 (AD50 = 0.2801 micromol/kg s.c.) behaved as a full agonist and was twice as potent as buprenorphine (AD50=0.4569 micromol/kg s.c.) and 50 times more potent than morphine (AD50 = 13.3012 micromol/kg s.c.). Whereas the mu-opioid receptor antagonists naloxone (1-10 mg/kg s.c.) and naltrexone (5-15 mg/kg s.c.) antagonized HS-599 induced analgesia, the delta-opioid receptor antagonist naltrindole (20 mg/kg s.c.) and the kappa-opioid receptor antagonist nor-binaltorphimine (20 mg/kg s.c.) did not. With the hot-plate test at 50 degrees C, HS-599 (AD50 = 0.0359 micromol/kg s.c.) was a full agonist about 130 times more potent than morphine (AD50 = 4.8553 micromol/kg s.c.). With a high intensity nociceptive stimulus (55 degrees C) HS-599 (AD50 = 1.0382 micromol/kg s.c.) remained 7 times more potent than morphine (AD50 = 7.0210 micromol/kg s.c.) but never exceeded the 55% of the maximum possible effect, behaving as a partial agonist able to antagonize morphine antinociception in a dose-dependent manner. HS-599 promises to be a potent and safe new analgesic, preferentially acting at spinal level.

Signal transduction efficacy of the highly potent mu opioid agonist 14-methoxymetopon.

In search of a truly high-efficacy (i.e., tau > 100) mu opioid analgesic, we determined the efficacy (tau) and apparent in vivo affinity (KA) of the high-potency alkoxymorphinan 14-methoxymetopon. However, in the present study, 14-methoxymetopon's efficacy proved to be only 1.5-fold higher than that of morphine (tau, 19 vs. 12). KA values were 2,900 nmol/kg for 14-methoxymetopon and 46,000 nmol/kg for morphine (Ki for [3H]DAMGO binding, 0.33 vs 3.4 nmol/l). Thus, the 24-fold higher potency of methoxymetopon could be fully accounted for by its 16-fold higher apparent in vivo affinity and its only 1.5-fold higher efficacy. Furthermore, the 10-fold higher affinity of 14-methoxymetopon for the mu opioid receptor - as previously determined in radioligand binding assays - was confirmed in the present behavioral tests of thermal antinociception.

Binding, pharmacological and immunological profiles of the delta-selective opioid receptor antagonist HS 378.

HS 378 is a recently developed indolomorphinan with high selectivity and antagonist potency at the delta-opioid receptor. The present study was performed to characterize the opioid binding properties and pharmacological and immunological activity of HS 378 and to compare them with those of two well-known delta-opioid receptor antagonists, naltrindole (NTI) and naltriben (NTB). In vitro opioid receptor binding profiles were determined in rat brain homogenates. HS 378 showed 4.7- and 2.4-fold higher mu/delta selectivity compared to NTI and NTB, respectively. In the [35S]GTPgammaS functional assay carried out in cell lines expressing cloned human opioid receptors, HS 378 was found to be a pure delta-opioid receptor antagonist. In vitro, exposure of HS 378 resulted in an apparent dose-related suppression of concanavalin A induced rat T-lymphocyte proliferation with an IC50 value of 0.54 microM. NTI showed also immunosuppression with an IC50 value of 6.93 microM, whereas NTB had no effect. The IC50 of HS 378 was 13 times lower than that of NTI and 8 times higher than that of cyclosporin A. Taken together, our findings indicate that the small molecule HS 378 has properties that may be of therapeutic value in the setting of human inflammatory diseases.

Opioid binding profiles of new hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxymorphinans.

Several hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxycodeinones and 14-alkoxydihydrocodeinones were synthesised [1] and characterised in in vitro radioligand binding assays in rat brain membrane preparations. The tested compounds show the highest affinity for the mu opioid binding sites and most of them have agonist character. Subtype analysis of the binding shows mu2 specificity. However, some of these ligands are able to block partially (40-60%) the high affinity (putative mu1) opioid binding sites while all of them act as reversible ligands at the low affinity (putative mu2) sites.

Involvement of the kappa-opioid receptor in the anxiogenic-like effect of CP 55,940 in male rats.

We have studied the possible interaction between three selective opioid-receptor antagonists, nor-binaltorphimine (NB: kappa) (5 mg/kg), cyprodime (CY: mu) (10 mg/kg) and naltrindole (NTI: delta) (1 mg/kg), and the cannabinoid receptor agonist CP 55,940, in the modulation of anxiety (plus-maze) and adrenocortical activity (serum corticosterone levels by radioimmunoassay) in male rats. The holeboard was used to evaluate motor activity and directed exploration. CP 55,940 (75 microg/kg, but not 10 microg/kg) induced an anxiogenic-like effect, which was antagonised by NB. The other effects of CP 55,940 (75 microg/kg), a decreased holeboard activity and stimulation of adrenocortical activity, were not antagonised by any of the three opioid receptor antagonists. CY and NTI, when administered alone, induced marked reductions in motor activity, anxiogenic-like effects and stimulation of adrenocortical activity. The selective kappa-opioid receptor antagonist NB, on its own, did not modify the level of anxiety but stimulated adrenocortical activity. We provide the first pharmacological evidence about the involvement of the kappa-opioid receptor in the anxiogenic-like effect of CP 55,940.

(-)-N,N'-but-2-ene-1,4-diylbimorphinans.

N,N'-But-2-ene-1,4-diylbimorphinans 1-3 have been synthesized and pharmacologically evaluated. Bimorphinans 1 and 3 show opioid antagonist activity and preference for mu and kappa opioid receptors in mouse vas deferens and guinea pig ileum preparations.

Recent advances in the development of 14-alkoxy substituted morphinans as potent and safer opioid analgesics.

Morphine and other opioid morphinans produce analgesia primarily through µ opioid receptors (MORs), which mediate beneficial but also non-beneficial actions. There is a continued search for efficacious opioid analgesics with reduced complications. The cornerstone in the development of 14-alkoxymorphinans as novel analgesic drugs was the synthesis of the highly potent MOR agonist 14-O-methyloxymorphone. This opioid showed high antinociceptive potency but also the adverse effects associated with morphine type compounds. Further developments represent the introduction of a methyl and benzyl group at position 5 of 14-O-methyloxymorphone leading to the strong opioid analgesics 14-methoxymetopon and its 5-benzyl analogue, which exhibited less pronounced side effects than morphine although interacting selectively with MORs. Introduction of arylalkyl substituents such as phenylpropoxy in position 14 led to a series of extremely potent antinociceptive agents with enhanced affinities at all three opioid receptor types. During the past years, medicinal chemistry and opioid research focused increasingly on exploring the therapeutic potential of peripheral opioid receptors by peripheralization of opioids in order to minimize the occurrence of centrally-mediated side effects. Strategies to reduce penetration to the central nervous system (CNS) include chemical modifications that increase hydrophilicity. Zwitterionic 6-amino acid conjugates of 14-Oalkyloxymorphones were developed in an effort to obtain opioid agonists that have limited access to the CNS. These compounds show high antinociceptive potency by interacting with peripheral MORs. Opioid drugs with peripheral site of action represent an important target for the treatment of severe and chronic pain without the adverse actions of centrally acting opioids.

Inverse agonism and neutral antagonism at wild-type and constitutively active mutant delta opioid receptors.

The delta opioid receptor modulates nociceptive and emotional behaviors. This receptor has been shown to exhibit measurable spontaneous activity. Progress in understanding the biological relevance of this activity has been slow, partly due to limited characterization of compounds with intrinsic negative activity. Here, we have used constitutively active mutant (CAM) delta receptors in two different functional assays, guanosine 5'-O-(3-thio)triphosphate binding and a reporter gene assay, to test potential inverse agonism of 15 delta opioid compounds, originally described as antagonists. These include the classical antagonists naloxone, naltrindole, 7-benzylidene-naltrexone, and naltriben, a new set of naltrindole derivatives, H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-TicPsi[CH2N]Cha-Phe-OH [TICP(Psi)], as well as three 2',6'-dimethyltyrosine-1,2,3,4-tetrahydroquinoline-3-carboxylate (Dmt-Tic) peptides. A reference agonist, SNC 80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide], and inverse agonist, ICI 174864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu), were also included. In a screen using wild-type and CAM M262T delta receptors, naltrindole (NTI) and close derivatives were mostly inactive, and TIPP behaved as an agonist, whereas Dmt-Tic-OH and N,N(CH3)2-Dmt-Tic-NH2 showed inverse agonism. The two latter compounds showed negative activity across 27 CAM receptors, suggesting that this activity was independent from the activation mechanism. These two compounds also exhibited nanomolar potencies in dose-response experiments performed on wild-type, M262T, Y308H, and C328R CAM receptors. TICP(Psi) exhibited strong inverse agonism at the Y308H receptor. We conclude that the stable N,N(CH3)2-Dmt-Tic-NH2 compound represents a useful tool to explore the spontaneous activity of delta receptors, and NTI and novel derivatives behave as neutral antagonists.