Potential Drug Abuse Therapeutics Derived from the Hallucinogenic Natural Product Salvinorin A

Previous structure-activity relationship studies of salvinorin A have shown that modification of the acetate functionality off the C-2 position to a methoxy methyl or methoxy ethyl ether moiety leads to increased potency at KOP receptors. However, the reason for this increase remains unclear. Here we report our efforts towards the synthesis and evaluation of C-2 constrained analogs of salvinorin A. These analogs were evaluated at opioid receptors in radioligand binding experiments as well as in the GTP-γ-S functional assay. One compound, 5, was found to have affinity and potency at κ opioid (KOP) receptors comparable to salvinorin A. In further studies, 5 was found to attenuate cocaine-induced drug seeking behavior in rats comparably to salvinorin A. This finding represents the first example of a salvinorin A analog that has demonstrated anti-addictive capabilities.     

Modification of the furan ring of salvinorin A: Identification of a selective partial agonist at the kappa opioid receptor

In an effort to find novel agents which selectively target the kappa opioid receptor (KOPR), we modified the furan ring of the highly potent and selective KOPR agonist salvinorin A. Introduction of small substituents at C-16 was well tolerated. 12-epi-Salvinorin A, synthesized in four steps from salvinorin A, was a selective partial agonist at the KOPR. No clear SAR patterns were observed for C-13 aryl ketones. Introducing a hydroxymethylene group between C-12 and the furan ring was tolerated. Small C-13 esters and ethers gave weak KOPR agonists, while all C-13 amides were inactive. Finally, substitution of oxadiazoles for the furan ring abolished affinity for the KOPR. None of the compounds displayed any KOPR antagonism or any affinity for mu or delta opioid receptors.     

Synthetic studies of neoclerodane diterpenes from Salvia divinorum: selective modification of the furan ring

A synthetic sequence has been developed to selectively functionalize the furan ring of the natural product salvinorin A (2a). The synthetic routes described convert the furan ring in 2a into an N-sulfonylpyrrole, oxazole or an oxadiazole. In addition, a procedure has been found to remove the furan skeleton completely. Biological results indicate that replacement of the furan ring with an N-sulfonylpyrrole leads to reduced affinity and efficacy at kappa opioid receptors.     

Synthesis and biological evaluation of C-12 triazole and oxadiazole analogs of salvinorin A

Salvinorin A (1), the main active ingredient of Salvia divinorum, is a potent and selective κ-opioid receptor (KOPR) agonist. A series of C-12 triazole analogs and the oxadiazole (4) analog of 1 are synthesized and screened for binding affinity at κ, μ (MOPR), or δ (DOPR). Surprisingly, all triazole analogs have shown negligible binding affinity at opioid receptors and the oxadiazole 4, a reported MOPR and KOPR antagonist, exhibits very low affinities to opioid receptors and no antagonism in our binding assays. These results suggest that electronic factors that may affect either the electron density of hydrogen bond acceptor at C-12 or hydrophobic interactions between C-12 moiety and KOPR are critical to C-12 analog’s affinity for KOPR.     

Buprenorphine‐elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ‐, μ‐opioid and nociceptin receptors

Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ‐ (KOP), μ‐opioid (MOP) and nociceptin/opioid receptor‐like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium‐labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N‐linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U‐69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration‐dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP‐expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration‐dependent AC superactivation elicited by chronic buprenorphine exposure.     

The biological consequences of replacing hydrophobic amino acids in deltorphin I with amphiphilic alpha-hydroxymethylamino acids.

New analogues of deltorphin I (DT I), in which the Phe residue in position 3, and the Val residue in position 5 or 6 are replaced with respective amphiphilic alpha-hydroxymethylamino acid residues (HmAA), were synthesized and tested for receptor affinity and selectivity to mu and delta opioid receptors. The analogue with (R)-HmPhe at position 3 lost receptor selectivity, as a result of a partial decrease of affinity to delta and a significant increase of affinity to mu receptors. In contrast, an analogue with (S)-HmPhe in the same position, was very potent and more specific to delta receptors than parent DT I. The analogue with (R)-HmVal at position 5 expressed higher delta affinity and selectivity than parent DT I. The analogue with other possible isomer (S)-HmVal was less selective for delta opioid receptors, as a result of decreasing affinity to delta and increasing affinity to mu receptors. The analogues with (R)- or (S)-HmVal in position 6 expressed equally low receptor affinity and selectivity. The data obtained support a previously proposed model of active conformation of deltorphins.     

Syntheses of novel high affinity ligands for opioid receptors

A series of novel high affinity opioid receptor ligands have been made whereby the phenolic-OH group of nalbuphine, naltrexone methiodide, 6-desoxonaltrexone, hydromorphone and naltrindole was replaced by a carboxamido group and the furan ring was opened to the corresponding 4-OH derivatives. These furan ring ‘open’ derivatives display very high affinity for μ and κ receptors and much less affinity for δ. The observation that these target compounds have much higher receptor affinity than the corresponding ring ‘closed’ carboxamides significantly strengthens our underlying pharmacophore hypothesis concerning the bioactive conformation of the carboxamide group.     

Novel Dmt-Tic dipeptide analogues as selective delta-opioid receptor antagonists

A series of Dmt-Tic analogues with substitution on the Tic aromatic ring has been synthesized and evaluated for opioid receptor affinity and activation. Incorporation of large hydrophobic groups at position 7 of Tic did not greatly alter the delta opioid receptor binding affinities of the dipeptides whereas substitution at position 6 substantially diminished their affinity. These modified Dmt-Tic peptides showed binding affinities as low as 2.5 nM with up to 500-fold selectivity for the delta versus mu opioid receptor and proved to be delta receptor antagonists.     

Desulfohaplosamate, a new phosphate-containing steroid from Dasychalina sp., is a selective cannabinoid CB2 receptor ligand

From the polar organic extract of the Indonesian sponge Dasychalina sp. we have isolated haplosamate A (1), a unique C(28) sterol containing a sulfate group at C-3 and a methyl phosphate at C-15, along with its new desulfo analogue 2, whose structure has been secured by detailed NMR investigation. Compounds 1 and 2, as well as their semi-synthetic analogues 3-5, have been evaluated for interaction with CB(1) and CB(2) receptors through a binding test. Desulfohaplosamate (2) showed a selective affinity for CB(2) receptors in the low μM range, while a semi-synthetic derivative with cleaved ring B showed a complete loss of affinity for both receptors, highlighting the importance of an intact steroid nucleus. To our knowledge, haplosamate derivatives represent the first CB receptor ligands belonging to the class of steroids.     

C-nucleoside analogues of furanfurin as ligands to A1 adenosine receptors

Furanfurin (2-beta-D-ribofuranosylfuran-4-carboxamide) derivatives and analogues were synthesized and their affinity for adenosine receptors was determined. The agonistic behavior of furanfurin against A1 receptors is preserved only when the furan ring is substituted with isosteric pentatomic ring systems such as oxazole, thiazole or thiophene, and the carboxamide group is unsubstituted. Replacement of the hydrogen atoms of the carboxamide group with alkyl, cycloalkyl or arylalkyl groups generates compounds endowed with moderate antagonistic activity.     

Influence of solvent and configuration of residues at positions 2 and 3 on distance and mobility of pharmacophore groups at positions 1 and 4 in cyclic enkephalin analogues

The analgesic activity of opioid peptides is mainly connected with their affinity and selectivity for the mu-receptors. The biological activity of cyclic opioid analogues depends on mutual orientation and conformational freedom of aromatic pharmacophore groups at positions 1 and 4. The distance and distance distributions between chromophores at positions 1 [Phe(p-NO(2)), p-nitrophenylalanine] and 4 [Nal, beta-(2-naphthyl)alanine], which constitute an energy donor-acceptor pair, were calculated based on measured fluorescence intensity decays of a donor (Nal). The influence of the solvent and configuration of the residues at position 2 and 3 on donor-acceptor distance distribution and mobility of pharmacophore groups at position 1 and 4 in cyclic enkephalin analogues are discussed.     

The synthesis and antibacterial activity of totarol derivatives. Part 2: Modifications at C-12 and O-13

Alterations of the C-12 and C-13 aromatic ring substituents of totarol (1) afforded the series of derivatives 2-14, and introduction of substituents at C-12 gave exclusively 2a-14a. The majority of these analogues were tested in vitro against the following organisms: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and multiresistant Klebsiella pneumoniae. The results were evaluated in terms of structure-activity relationship which reveals that: (a) the phenolic moiety at C-13, in general, is essential for antibacterial activity at < 32 microg/mL against gram-positive species, and (b) derivatization at C-12 has an undesirable effect on the antibacterial activity of this class of compounds, while (c) all compounds tested are ineffective against the gram-negative Klebsiella pneumoniae.     

Dermorphin tetrapeptide analogues with 2',6'-dimethylphenylalanine (Dmp) substituted for aromatic amino acids have high mu opioid receptor binding and biological activities

To investigate the value of the 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid substitution, we prepared analogues of the mu opioid receptor-selective dermorphin tetrapeptide Tyr-D-Arg-Phe-betaAla-NH(2) (YRFB) in which Dmp or its D-isomer replaced Tyr(1) or Phe(3). Replacing Phe(3) with Dmp essentially tripled mu receptor affinity and the receptor's in vitro biological activities as determined with the guinea pig ileum (GPI) assay but did not change delta receptor affinity. Despite an inversion of the D configuration at this position, mu receptor affinity and selectivity remained comparable with those of the L-isomer. Replacing the N-terminal Tyr residue with Dmp produced a slightly improved mu receptor affinity and a potent GPI activity, even though the substituted compound lacks the side chain phenolic hydroxyl group at the N-terminal residue. Dual substitution of Dmp for Tyr(1) and Phe(3) produced significantly improved mu receptor affinity and selectivity compared with the singly substituted analogues. Subcutaneous injection of the two analogues, [Dmp(3)]YRFB and [Dmp(1)]YRFB, in mice produced potent analgesic activities that were greater than morphine in the formalin test. These lines of evidence suggest that the Dmp residue would be an effective aromatic amino acid surrogate for both Tyr and Phe in the design and development of novel opioid mimetics.     

Synthesis and binding properties of deltorphin I analogues containing (R) and (S)-alpha-hydroxymethylnaphtylalanine

New analogues of deltorphin I (DT I), in which the phenylalanine residue in position 3 is substituted with amphiphilic alpha,alpha-disubstituted amino acid enantiomers, (R) and (S)-alpha-hydroxymethylnaphtylalanine, were synthesized and tested for mu and delta opioid receptor affinity and selectivity. Although both analogues have lower affinity to delta receptors than DT I, they both expressed specificity to delta receptors.     

Synthesis of 7 alpha- and 7 beta-carboxymethyl-9(11)-ene derivatives of estrone and estradiol

As part of a search for derivatives designed to conjugate to amino groups, either of a protein carrier for antibody production or of an immobilized side-chain on a polymer for affinity chromatography, functionalized estrone and estradiol analogues were prepared. These modified steroids were obtained via the introduction of a carboxymethyl side-chain at the C-7 alpha and C-7 beta position on an adrenosterone derivative and were then aromatized on the A ring. These new compounds are unsaturated at the C-9 (11) position, which could be useful for a second modification.     

Neuroprotective Potential of Biphalin,Multireceptor Opioid Peptide,Against Excitotoxic Injury in Hippocampal Organotypic Culture

Biphalin is a dimeric opioid peptide that exhibits affinity for three types of opioid receptors (MOP, DOP and KOP). Biphalin is undergoing intensive preclinical study. It was recognized that activation of δ-opioid receptor elicits neuroprotection against brain hypoxia and ischemia. We compare the effect of biphalin and morphine and the inhibition of opioid receptors by naltrexone on survival of neurons in rat organotypic hippocampal cultures challenged with NMDA. Findings: (1) 0.025–0.1 μM biphalin reduces NMDA-induced neuronal damage; (2) biphalin neuroprotection is abolished by naltrexone; (3) reduced number of dead cells is shown even if biphalin is applied with delay after NMDA challenge.     

14-membered cyclic opioids related to dermorphin and their partially retro-inverso modified analogues. I. Synthesis and biological activity.

As a continuation of our program to study structure-activity relationships of opiate peptides, we report the syntheses and biological activities of a series of 14-membered cyclic dermorphin analogues closely related to enkephalin analogue Tyr-c[D-A2bu-Gly-Phe-Leu] incorporating a phenylalanine at the third position in place of glycine. In addition to two parent dermorphin analogues Tyr-c[D-A2bu-Phe-Phe-(L and D)-Leu], four stereoisomeric retro-inverso modified analogues Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu] with a reversed amide bond between residues four and five, and Tyr-c[D-Glu-Phe-gPhe-(L and D)-rLeu] with two reversed amide bonds between residues four and five, and between residue five and the side chain of residue two have been synthesized. The results from the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays show that all analogues are superactive at either one or both opiate receptors and in general display higher activities as compared to the corresponding enkephalin analogues with a glycine at the third position. Results from the in vitro biological assays and conformational analysis using 1H-NMR spectroscopy (adjoining paper) will provide useful information to understand the role of the Phe3 aromatic side chain in dermorphin, and that of the Phe4 aromatic side chain in enkephalin, on opiate activity since these cyclic dermorphin analogues contain two Phe residues at both the third and fourth positions.     

Synthesis and evaluation of a series of homologues of lobelane at the vesicular monoamine transporter-2

A series of lobelane homologues has been synthesized and evaluated for their [(3)H]DTBZ binding affinity at the vesicular monoamine transporter-2 (VMAT2). The structure-activity relationships (SAR) indicate that for retention of binding affinity at VMAT2, the lengths of the methylene linkers should be no shorter than one methylene unit at C-6 of the piperidine ring, and no shorter than two methylene units at C-2 of the piperidine ring. These results indicate that the intramolecular distances between the piperidine ring and two phenyl rings in lobelane analogues are an important criterion for retention of high affinity at VMAT2.     

Synthesis and SAR studies of chiral non-racemic dexoxadrol analogues as uncompetitive NMDA receptor antagonists

A series of chiral non-racemic dexoxadrol analogues with various substituents in position 4 of the piperidine ring was synthesized and pharmacologically evaluated. Only the enantiomers having (S)-configuration at the 2-position of the piperidine ring and 4-position of the dioxolane ring were considered. Key steps in the synthesis were an imino-Diels-Alder reaction of enantiomerically pure imine (S)-13, which had been obtained from d-mannitol, with Danishefsky's Diene 14 and the replacement of the p-methoxybenzyl protective group with a Cbz-group. It was shown that (S,S)-configuration of the ring junction (position 2 of the piperidine ring and position 4 of the dioxolane ring) and axial orientation of the C-4-substituent ((4S)-configuration) are crucial for high NMDA receptor affinity. 2-(2,2-Diphenyl-1,3-dioxolan-4-yl)piperidines with a hydroxy moiety ((S,S,S)-5, K(i)=28nM), a fluorine atom ((S,S,S)-6, WMS-2539, K(i)=7nM) and two fluorine atoms ((S,S)-7, K(i)=48nM) in position 4 represent the most potent NMDA antagonists with high selectivity against σ(1) and σ(2) receptors and the polyamine binding site of the NMDA receptor. The NMDA receptor affinities of the new ligands were correlated with their electrostatic potentials, calculated gas phase proton affinities (negative enthalpies of deprotonation) and dipole moments. According to these calculations decreasing proton affinity and increasing dipole moment are correlated with decreasing NMDA receptor affinity.     

Exploring the biological consequences of conformational changes in aspartame models containing constrained analogues of phenylalanine

The dipeptide aspartame (Asp-Phe-OMe) is a sweetener widely used in replacement of sucrose by food industry. 2′,6′-Dimethyltyrosine (DMT) and 2′,6′-dimethylphenylalanine (DMP) are two synthetic phenylalanine-constrained analogues, with a limited freedom in χ-space due to the presence of methyl groups in position 2′,6′ of the aromatic ring. These residues have shown to increase the activity of opioid peptides, such as endomorphins improving the binding to the opioid receptors. In this work, DMT and DMP have been synthesized following a diketopiperazine-mediated route and the corresponding aspartame derivatives (Asp-DMT-OMe and Asp-DMP-OMe) have been evaluated in vivo and in silico for their activity as synthetic sweeteners.