Morphine Alkaloids Research Articles

Gindarudine, a novel morphine alkaloid from Stephania glabra

A novel morphine alkaloid, named gindarudine 1 has been isolated from ethanol extract of Stephania glabra tubers, together with four known alkaloids, palmatine, dehydrocorydalmine, stepharanine, and 8-(4′-methoxybenzyl)-xylopinine. Compound 1 was elucidated as 3,6-O,N-detrimethyl-10-hydroxy-1-methoxy-thebaine by means of spectroscopic data including 2D NMR studies.

Direct Subunit-Dependent Multimodal 5-Hydroxytryptamine3Receptor Antagonism by Methadone

Homomeric 5-hydroxytryptamine (5-HT)(3A) and heteromeric 5-HT(3AB) receptors mediate rapid excitatory responses to serotonin in the central and peripheral nervous systems. The alkaloid morphine, in addition to being a mu-opioid receptor agonist, is a potent competitive inhibitor of 5-HT(3) receptors. We examined whether methadone, an opioid often used to treat morphine dependence, also exhibited 5-HT(3) receptor antagonist properties. Racemic (R/S)-methadone inhibited currents mediated by human homomeric 5-HT(3A) receptors (IC(50) = 14.1 +/- 2.5 microM). Incorporation of the 5-HT(3B) subunit into heteromeric 5-HT(3AB) receptors reduced the potency of inhibition by (R/S)-methadone (IC(50) = 41.1 +/- 0.9 microM). (R/S)-Methadone also increased apparent desensitization of both 5-HT(3) receptor subtypes. The inhibition of the 5-HT(3A) receptor was competitive; however, incorporation of the 5-HT(3B) subunit caused the appearance of inhibition that was insurmountable by 5-HT. In the absence of rapid desensitization, when dopamine was used as an agonist of 5-HT(3AB) receptors, the inhibition by (R/S)-methadone was voltage-dependent. The antagonist and desensitization-enhancing effects of (R/S)-methadone were shared by pure (R)- and (S)-methadone enantiomers, which had similar actions on 5-HT-evoked currents mediated by 5-HT(3) receptors. However, (R)-methadone exhibited a larger voltage-dependent inhibition of dopamine-evoked currents mediated by 5-HT(3AB) receptors than did (S)-methadone. Inhibition of 5-HT(3A) receptors by (R/S)-methadone was not influenced by voltage. Thus, methadone displays multimodal subunit-dependent antagonism of 5-HT(3) receptors.

Biotransformations of morphine alkaloids by fungi: N-demethylations, oxidations, and reductions

Morphine alkaloids and some of its derivatives (morphine, codeine, thebaine, oripavine, hydrocodone, and oxycodone) were subjected to fermentations with six fungal strains. The alkaloids were transformed to a variety of products via biological oxidations, reductions, and oxidative demethylations. The strain Cunninghamella echinulata proved to be the most effective for demethylations of all of the above compounds, except for morphine. The time profile of the conversion of 3-[14CH3]thebaine to 3-[14CH3]northebaine by C. echinulata cultures was also determined.

Comparison of physicochemical properties and biological activities of opioid morphinans interacting with mu opioid receptors

Morphinans play an important role among therapeutically valuable opioids. They include powerful pain relieving agents such as naturally occurring alkaloids (e.g. morphine, codeine), semisynthetic derivatives (e.g. oxycodone, oxymorphone, buprenorphine), and synthetic analogues (e.g. levorphanol, butorphanol). These opioid drugs produce their biological actions through three receptor types, μ, δ and κ, belonging to the family of seven transmembrane G protein-coupled receptors. All three opioid receptors are located in the central and peripheral nervous systems of many species including human. Morphine and related opioids currently prescribed as potent analgesics for the treatment of severe pain produce their analgesic activity primarily through their agonist action at μ opioid receptors, which is the main receptor type targeted for pharmacotherapy of pain. Despite their widespread use, opioid drugs have a number of severe side effects including respiratory depression, emesis, sedation, constipation, tolerance, and physical dependence. Consequently, a great deal of effort was spent trying to develop safer, more efficacious and non-addicting compounds, with the goal of an improved therapeutic index. Optimal physicochemical properties are often an important consideration for the development of bioactive molecules as therapeutic agents. Therefore, a significant aim in medicinal chemistry research and drug development is to predict the behavior of targeted molecules based on their physicochemical features. This task, however, cannot be accomplished without correlating and understanding the differences in the physicochemical and pharmacological properties of existing opioid morphinans. Toward this goal, we performed a comparative study on physicochemical properties and biological activities of well-known opioid morphinans such as the naturally occurring alkaloid morphine, and semisynthetic analogues e.g. oxymorphone. Besides the classical and clinically relevant opioid analgesics, synthetic morphinans developed by our group were included in the study, the 14-O-methyl derivative of oxymorphone and its 5-methyl substituted analogue, 14-methoxymetopon. The major physicochemical parameters (pKa, logP and logD) have been experimentally determined using a Sirius PCA200/Cheqsol Instrument. In vitro binding affinities and selectivities to opioid receptors were determined in rodent brain membranes using binding assays. Analgesic potencies were evaluated after subcutaneous administration to mice using tail-flick and hot-plate tests. The structure-activity relationship observations, combining physicochemical and biological data, will be discussed. Altogether, the present study indicate that specific structural elements are required to confer high affinity/selectivity for μ opioid receptors and potent agonist and analgesic activity in the morphinan class of opioids, and are important in drug development.

Cycloaddition Across the Benzofuran Ring as an Approach to the Morphine Alkaloids

The intramolecular Diels-Alder reaction of several amidofurans tethered onto a benzofuran ring was examined as a strategy for the synthesis of morphine. Bromo substitution on the furan ring did not provide sufficient activation to allow the cycloaddition to take place across the aromatic benzofuran. However, the presence of a large o-methylbenzyl group on the amido nitrogen atom causes the reactive s-trans conformation of the amidofuran to be highly populated, thereby facilitating its Diels-Alder cycloaddition across a tethered benzofuran.

The Presence of Endogenous Morphine Signaling in Animals

Recent empirical findings have contributed valuable mechanistic information in support of a regulated de novo biosynthetic pathway for chemically authentic morphine and related morphinan alkaloids within animal cells. Importantly, we and others have established that endogenously expressed morphine represents a key regulatory molecule effecting local circuit autocrine/paracrine cellular signaling via a novel mu(3) opiate receptor coupled to constitutive nitric oxide production and release. The present report provides an integrated review of the biochemical, pharmacological, and molecular demonstration of mu(3) opiate receptors in historical linkage to the elucidation of mechanisms of endogenous morphine production by animal cells and organ systems. Ongoing research in this exciting area provides a rare window of opportunity to firmly establish essential biochemical linkages between dopamine, a morphine precursor, and animal biosynthetic pathways involved in morphine biosynthesis that have been conserved throughout evolution.

Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae

The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches.

Carbonate derivatives of 14β-hydroxycodeine

Carbonate derivatives of 14β-hydroxycodeine, viz., 14β-hydroxy-6-O-(methoxycarbonyl)codeine, 6-O-methoxycarbonyl-14β-(methoxycarbonyloxy)codeine, and 14β-acetoxy-6-O-methoxy-carbonylcodeine, potential substrates for ring C modification in morphinane alkaloids, were synthesized for the first time

Enantioselective addition of diethylzinc to aromatic aldehydes catalyzed by 14-hydroxylsubstituted morphinans

Enantioselective addition reactions of diethylzinc to aromatic aldehydes were performed with catalytic amounts of 14-hydroxylsubstituted morphine alkaloids. The reaction conditions such as catalyst loading, time and temperature were optimized to obtain the highest enantiomeric excess. Optically active secondary alcohols were synthesized with ee up to 95%.

A Practical Asymmetric Synthesis of the ACNO Fragment of Morphine Alkaloids

Asymmetric total synthesis of the ACNO skeleton of morphine alkaloids has been achieved in excellent overall yields and optical purities using the Ru-catalyzed asymmetric transfer hydrogenation, Pd-catalyzed cyclization, and Pt-catalyzed hydrogenation as key steps.

Quantitative 1H nuclear magnetic resonance metabolite profiling as a functional genomics platform to investigate alkaloid biosynthesis in opium poppy.

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The plant is widely cultivated as the only commercial source of the narcotic analgesics morphine and codeine. Variations in plant secondary metabolism as a result of genetic diversity are often associated with perturbations in other metabolic pathways. As part of a functional genomics platform, we used (1)H nuclear magnetic resonance (NMR) metabolite profiling for the analysis of primary and secondary metabolism in opium poppy. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principle component analysis of the (1)H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety and a high-thebaine, low-morphine cultivar. Distinction was also made between pharmaceutical-grade opium poppy cultivars and a condiment variety. Such phenotypic differences were not observed in root extracts. Loading plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Quantification of 34 root and 21 latex metabolites, performed using Chenomx NMR Suite version 4.6, showed major differences in the accumulation of specific alkaloids in the latex of the low-alkaloid and high-thebaine, low-morphine varieties. Relatively few differences were found in the levels of other metabolites, indicating that the variation was specific for alkaloid metabolism. Exceptions in the low-alkaloid cultivar included an increased accumulation of the alkaloid precursor tyramine and reduced levels of sucrose, some amino acids, and malate. Real-time polymerase chain reaction analysis of 42 genes involved in primary and secondary metabolism showed differential gene expression mainly associated with alkaloid biosynthesis. Reduced alkaloid levels in the condiment variety were associated with the reduced abundance of transcripts encoding several alkaloid biosynthetic enzymes.

Recent and projected increases in atmospheric carbon dioxide and the potential impacts on growth and alkaloid production in wild poppy (Papaver setigerum DC.)

In the current study, we quantified changes in the growth and alkaloid content of wild poppy, (Papaver setigerum) as a function of recent and projected changes in global atmospheric carbon dioxide concentration, [CO2]. The experimental [CO2] values (300, 400, 500 and 600μmol mol−1) correspond roughly to the concentrations that existed during the middle of the twentieth century, the current concentration, and near and long-term projections for the current century (2050 and 2090), respectively. Additional carbon dioxide resulted in significant increases in leaf area and above ground biomass for P. setigerum at all [CO2] relative to the 300μmol mol−1 baseline. Reproductively, increasing [CO2] from 300 to 600μmol mol−1 increased the number of capsules, capsule weight and latex production by 3.6, 3.0 and 3.7×, respectively, on a per plant basis. Quantification of secondary compounds (i.e. those not involved in primary metabolism) included the alkaloids morphine, codeine, papaverine and noscapine. The amount of all alkaloids increased significantly on a per plant basis, with the greatest relative increase occurring with recent increases in atmospheric carbon dioxide (e.g. from 300 to 400μmol mol−1). Overall, these data suggest that as atmospheric [CO2] continues to increase, significant effects on the production of secondary plant compounds of pharmacological interest (i.e. opiates) could be expected.

Isolation and antimalarial activity of new morphinan alkaloids on Plasmodium yoelii liver stage

Decoction of Strychnopsis thouarsii is used in the Malagasy traditional medicine to combat malaria. We have shown that this traditional remedy prevents malaria infection by targeting Plasmodium at its early liver stage. Bioassay-guided fractionation of S. thouarsii stem barks extracts, using a rodent Plasmodium yoelii liver stage parasites inhibition assay, led to isolate the new morphinan alkaloid tazopsine ( 1) together with sinococuline ( 2) and two other new related morphinan analogs, 10- epi-tazopsine ( 3) and 10- epi-tazoside ( 4). Structures were characterized by 2D NMR, MS, and CD spectral analysis. Compounds 1– 3 were found to fully inhibit the rodent P. yoelii liver stage parasites in vitro.

Endogenous morphine synthetic pathway preceded and gave rise to catecholamine synthesis in evolution (Review)

The biological presence and regulatory function of the plant alkaloid morphine in relatively simple and complex integrated animal systems has previously been shown. The pivotal role of dopamine as a chemical intermediate in the morphine biosynthetic pathway in plants establishes a functional basis for its expansion into an essential role as the progenitor catecholamine signaling molecule. In invertebrate neural systems, dopamine serves as the preeminent catecholamine signaling molecule, with the emergence and limited utilization of norepinephrine and its biosynthetic enzyme dopamine beta-hydroxylase in newly defined adaptational chemical circuits required by a rapidly expanding set of physiological demands. In vertebrates, epinephrine emerges as the major end of the catecholamine synthetic pathway consistent with a newly incorporated regulatory modification, i.e. N-methylation of norepinephrine. Given the striking similarities between the enzymatic steps in the morphine biosynthetic pathway and those driving the evolutionary adaptation of catecholamine chemical species to accommodate an expansion of interactive but distinct signaling systems, we surmise that the evolutionary emergence of catecholamine systems required conservation and selective 'retrofit' of specific enzyme activities, i.e. catechol O-methyl transferase and phenylethanol-amine N-methyl transferase, drawn from cellular morphine expression. This hypothesis is further supported by the critical recruitment of enzymatically synthesized tetrahydrobiopterin (BH4) both as an essential cofactor for tyrosine hydroxylase-mediated dopamine production and as a secondary electron donor for nitric oxide synthase-mediated nitric oxide (NO) production. The establishment of a reciprocal regulatory linkage between NO and catecholaminergic processes, as mediated by BH4, subserves a pivotal capacity to promote autocrine and paracrine regulation of signaling molecules. In summary, ongoing development and adaptation of catecholamine signaling pathways in animals appear to be related to their mobile lifestyle associated with complex feeding, sexual and protective processes, which also generate free radicals, thus requiring morphinergic signaling coupling to NO release.

Enhancement of alkaloid production in opium and California poppy by transactivation using heterologous regulatory factors

Genes encoding regulatory factors isolated from Arabidopsis, soybean and corn have been screened to identify those that modulate the expression of genes encoding for enzymes involved in the biosynthesis of morphinan alkaloids in opium poppy (Papaver somniferum) and benzophenanthridine alkaloids in California poppy (Eschscholzia californica). In opium poppy, the over-expression of selected regulatory factors increased the levels of PsCOR (codeinone reductase), Ps4'OMT (S-adenosyl-l-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase) and Ps6OMT [(R,S)-norcoclaurine 6-O-methyltransferase] transcripts by 10- to more than 100-fold. These transcriptional activations translated into an enhancement of alkaloid production in opium poppy of up to at least 10-fold. In California poppy, the transactivation effect of regulatory factor WRKY1 resulted in an increase of up to 60-fold in the level of EcCYP80B1 [(S)-N-methylcoclaurine 3'-hydroxylase] and EcBBE (berberine bridge enzyme) transcripts. As a result, the accumulations of selected alkaloid intermediates were enhanced up to 30-fold. The transactivation effects of other regulatory factors led to the accumulation of the same intermediates. These regulatory factors also led to the production of new alkaloids in California poppy callus culture.

Design for Morphine Alkaloids by Intramolecular Heck Strategy: Chemoenzymatic Synthesis of 10-Hydroxy-14-epi-dihydrocodeinone via C-D-B Ring Construction

Enzymatic dihydroxylation of b-bromoethylbenzene provided a homochiral diene diol that served as starting material for the synthesis of the complete morphinan skeleton via an intramolec- ular Heck cyclization.

Metabolic engineering of morphinan alkaloids by over‐expression and RNAi suppression of salutaridinol 7‐O‐acetyltransferase in opium poppy

We demonstrate that both over-expression and suppression of the gene encoding the morphinan pathway enzyme salutaridinol 7-O-acetyltransferase (SalAT) in opium poppy affects the alkaloid products that accumulate. Over-expression of the gene in most of the transgenic events resulted in an increase in capsule morphine, codeine and thebaine on a dry-weight basis. The transgenic line with the highest alkaloid content had 41%, 37% and 42% greater total alkaloids than the control in three independent trials over 3 years. DNA-encoded hairpin RNA-mediated suppression of SalAT resulted in the novel accumulation of the alkaloid salutaridine at up to 23% of total alkaloid; this alkaloid is not detectable in the parental genotype. Salutaridine is not the substrate of SalAT but the substrate of the previous enzyme in the pathway, salutaridine reductase. RNA transcript analysis of 16 primary T0 transformants and their segregating T1 progeny revealed an average reduction in SalAT transcript to about 12% of the control. Reduction in SalAT transcript was evident in both leaves and latex. Reverse transcriptase PCR and high-performance liquid chromatography analyses confirmed cosegregation of the expressed transgene with the salutaridine accumulating phenotype.

Basic pharmacology of buprenorphine

Buprenorphine is a derivative of the morphine alkaloid thebaine. The plasma elimination half-life of buprenorphine is three times longer than that of the active metabolite, norbuprenorphine. Although the elimination of norbuprenorphine from the body is more rapid than that of buprenorphine, the plasma concentration of norbuprenorphine was observed to exceed that of buprenorphine with chronic administration of buprenorphine. This plasma level of norbuprenorphine is based on the proportional increase in norbuprenorphine-glucuronide observed after one enterohepatic circulation of buprenorphine due to the first-pass metabolism. When buprenorphine and norbuprenorphine were administered intraventricularly, the analgesic effect of norbuprenorphine was approximately one-fourth that of buprenorphine. After intravenous administration of buprenorphine or norbuprenorphine in rats, the analgesic effect of norbuprenorphine was approximately 1/50th that of buprenorphine. The remarkably weak analgesic effect of norbuprenorphine after intravenous administration may be due to the low permeability of norbuprenorphine into the brain. Therefore, the plasma concentration of norbuprenorphine with chronic administration of buprenorphine has little analgesic effect. The most dangerous side effect of opioid therapy is respiratory depression. In our study of the respiratory effects after intravenous infusion in rats, norbuprenorphine was ten times more potent than the parent drug. However, it is possible that the plasma levels of norbuprenorphine with chronic administration of buprenorphine are not sufficiently high to depress respiratory function. Overall, buprenorphine is a safe and highly effective analgesic opioid for the treatment of chronic pain.

Pharmacological analysis of paregoric elixir and its constituents: In vitro and in vivo studies

Paregoric elixir is a phytomedicinal product which is used widely as an analgesic, antispasmodic and antidiarrheal agent. Here, we investigated the pharmacological actions and some of the mechanisms of action of paregoric elixir and compared its action with some of its components, the alkaloids morphine and papaverine. The paregoric elixir given orally to mice did not present relevant toxic effects, even when administered in doses up to 2000-fold higher than those used clinically. However, it showed an antinociceptive action that was more potent, but less efficacious, than morphine. In contrast to morphine, its effect was not dose-dependent and not reversed by the non-selective opioid antagonist naloxone. Moreover, paregoric elixir produced tolerance, but did not cause cross-tolerance, with the antinociceptive actions of morphine. When assessed in the gastrointestinal motility in vivo, paregoric elixir elicited graduated reduction of gastrointestinal transit. Finally, like morphine and papaverine, paregoric elixir concentration-dependently inhibited electrically-induced contraction of the guinea pig isolated ileum. In vivo and in vitro gastrointestinal actions of paregoric elixir were not reversed by naloxone. Collectively, the present findings lead us to suggest that the pharmacological actions produced by paregoric elixir are probably due to a synergic action of its constituents.

A Plant-Derived Morphinan as a Novel Lead Compound Active against Malaria Liver Stages

BackgroundThe global spread of multidrug–resistant malaria parasites has led to an urgent need for new chemotherapeutic agents. Drug discovery is primarily directed to the asexual blood stages, and few drugs that are effective against the obligatory liver stages, from which the pathogenic blood infection is initiated, have become available since primaquine was deployed in the 1950s.Methods and FindingsUsing bioassay-guided fractionation based on the parasite's hepatic stage, we have isolated a novel morphinan alkaloid, tazopsine, from a plant traditionally used against malaria in Madagascar. This compound and readily obtained semisynthetic derivatives were tested for inhibitory activity against liver stage development in vitro (P. falciparum and P. yoelii) and in vivo (P. yoelii). Tazopsine fully inhibited the development of P. yoelii (50% inhibitory concentration [IC50] 3.1 μM, therapeutic index [TI] 14) and P. falciparum (IC50 4.2 μM, TI 7) hepatic parasites in cultured primary hepatocytes, with inhibition being most pronounced during the early developmental stages. One derivative, N-cyclopentyl-tazopsine (NCP-tazopsine), with similar inhibitory activity was selected for its lower toxicity (IC50 3.3 μM, TI 46, and IC50 42.4 μM, TI 60, on P. yoelii and P. falciparum hepatic stages in vitro, respectively). Oral administration of NCP-tazopsine completely protected mice from a sporozoite challenge. Unlike the parent molecule, the derivative was uniquely active against Plasmodium hepatic stages.ConclusionsA readily obtained semisynthetic derivative of a plant-derived compound, tazopsine, has been shown to be specifically active against the liver stage, but inactive against the blood forms of the malaria parasite. This unique specificity in an antimalarial drug severely restricts the pressure for the selection of drug resistance to a parasite stage limited both in numbers and duration, thus allowing researchers to envisage the incorporation of a true causal prophylactic in malaria control programs.