Synthesis and opioid activity of new fentanyl analogs

Three new fentanyl analogs (compounds 3-4-5) have been synthesized and evaluated for antinociceptive properties using the writhing test. The analgesic property of the active compound, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (compound 4), was tested using the hot plate test in mice. Its opioid agonistic activity was characterized using three isolated tissues: guinea pig ileum, mouse vas deferens, and rabbit vas deferens. Compound 4 was as effective as fentanyl or morphine and it showed less antinociceptive potency than fentanyl but it was more potent than morphine. The duration of the antinociception was similar to that of fentanyl. This compound inhibited the electrically evoked contractions of myenteric plexus-longitudinal muscle strips of guinea pig ileum and of mouse vas deferens but not those of rabbit vas deferens. These effects could be reversed by micro selective antagonists (naloxone and/or CTOP) but not by the delta selective antagonist naltrindole, thus indicating that the compound acted as a micro opioid agonist. Finally, the binding data confirmed that compound 4 had high affinity and selectivity for the micro-receptor.     

Structure-activity relationships of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the treatment of cough

A series of 3-axial-aminomethyl-N-benzhydryl-nortropane analogs have been synthesized and identified to bind to the nociceptin receptor with high affinity. Many of these analogs showed high binding selectivity over classic opioid receptors such as mu receptor. The synthesis and structure-activity relationships around the C-3 nortropane substitution are described. Selected compounds with potent oral antitussive activity in the guinea pig model are disclosed.     

Synthesis and evaluation of potential affinity labels derived from endomorphin-2.

In an attempt to identify potential peptide-based affinity labels for opioid receptors, endomorphin-2 (Tyr-Pro-Phe-PheNH2), a potent and selective endogenous ligand for mu-opioid receptors, was chosen as the parent peptide for modification. The tetrapeptide analogs were prepared using standard Fmoc-solid phase peptide synthesis in conjunction with incorporation of Fmoc-Phe(p-NHAlloc) and modification of the p-amino group. The electrophilic groups isothiocyanate and bromoacetamide were introduced into the para position on either Phe3 or Phe4; the corresponding free amine-containing peptides were also prepared for comparison. The peptides bearing an affinity label group and their free amine analogs were evaluated in a radioligand-binding assay using Chinese hamster ovary (CHO) cells expressing mu- and delta-opioid receptors. Modification on Phe4 was better tolerated than on Phe3 for mu-receptor binding. Among the analogs tested, [Phe(p-NH2)4]endomorphin-2 showed the highest affinity (IC50 = 37 nm) for mu-receptors. The Phe(p-NHCOCH2Br)4 analog displayed the highest mu-receptor affinity (IC50 = 158 nm) among the peptides containing an affinity label group. Most of the compounds exhibited negligible binding affinity for delta-receptors, similar to the parent peptide.     

Cyclic, disulfide- and dithioether-containing opioid tetrapeptides: development of a ligand with high delta opioid receptor selectivity and affinity

Tetrapeptides of primary sequence Tyr-X-Phe-YNH2, where X is D-Cys or D-Pen (penicillamine) and where Y is D-Pen or L-Pen, were prepared and were cyclized via the side chain sulfurs of residues 2 and 4 to disulfide or dithioether-containing analogs. These peptides are related to previously reported penicillamine-containing pentapeptide enkephalin analogs but lack the central glycine residue of the latter and were designed to assess the effect of decreased ring size on opioid activity. Binding affinities of the tetrapeptides were determined to both mu and delta opioid receptors. Binding affinity and selectivity in the tetrapeptide series were observed to be highly dependent on primary sequence. For example, L-Pen4 analogs displayed low affinity and were nonselective, while the corresponding D-Pen4 diastereomers were of variable affinity and higher selectivity. Among the latter compounds were examples of potent analogs in which selectivity shifted from delta selective to mu selective as the ring size was increased. The relatively high binding affinity and delta receptor selectivity observed with one of the carboxamide terminal disulfide analogs led to the synthesis of the corresponding carboxylic acid terminal, Tyr-D-Cys-Phe-D-PenOH. This analog displayed delta receptor binding selectivity similar to that of the standard delta ligand, [D-Pen2,D-Pen5]enkephalin (DPDPE), and was found to have a 3.5-fold higher binding affinity than DPDPE. All the tetrapeptides were further evaluated in the isolated mouse vas deferens (mvd) assay and all displayed opioid agonist activity. In general, tetrapeptide potencies in the mouse vas deferens correlated well with binding affinities but were somewhat lower. Receptor selectivity in the mvd, assessed by examining the effect of opioid antagonists on the tetrapeptide concentration-effect curves, was similar to that determined in the binding studies.     

Comparison of physical dependence of ohmefentanyl stereoisomers in mice

Stereo-structural difference of ohmefentanyl stereoisomers on analgesic action and receptor affinity has been studied. To assess the difference of ohmefentanyl stereoisomers in physical dependence, the potency of physical dependence was quantified by estimating the ED50 value of ohmefentanyl stereoisomers in the naloxone-precipitated jumping test in mice. Morphine was used to assess the method and as a drug of comparison. The results indicate that the degree of physical dependence of morphine can been quantified by estimating the ED50 value of morphine withdrawal jumping induced by naloxone. A significant difference was observed in withdrawal jumping ED50 values among ohmefentanyl stereoisomers. Of these isomers, F9202 and F9204 had similarly potent analgesic action, but very significant difference in naloxone precipitated withdrawal response. Dependent potency index of F9204 was 618-fold weaker than that of F9202. It is concluded that a stereo-structural difference in physical dependence is found to exist among ohmefentanyl stereoisomers. Compound F9204 displayed a strong analgesic action and weak physical dependent potency.     

Biological activities of cyclic enkephalin pseudopeptides containing thioamides as amide bond replacements

Analogs of H-Tyr-cyclo(N epsilon-D-Lys-Gly-Phe-Leu) have been prepared which contain thioamides at the 3-4 position (monothio), 3-4 and 5-2 positions (dithio), and 2-3, 3-4, and 5-2 positions (trithio). These compounds have been tested for opioid activity in mu- and delta-receptor selective bio- and binding assays. As the number of sulfurs increased, the biological activities dropped on the guinea pig ileum and fluctuated modestly on the mouse vas deferens assay. Surprisingly, the compounds displayed increasing delta selectivity as the number of sulfurs increased. In the binding assay, the thioamide analogs tended to retain affinity toward the mu receptor. The mono- and dithio-analogs were more mu selective than the parent, while the trithio-analog was more delta selective. These results suggest that the subtle exchange of sulfur for oxygen can have a significant impact on receptor selectivity and affinity, and probably reflect the different conformation/structural requirements for binding vs. the biological transduction event.     

RTI-4614-4: an analog of (+)-cis-3-methylfentanyl with a 27,000-fold binding selectivity for mu versus delta opioid binding sites.

The objective of this study was to determine the binding affinities of (+/-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]- N-phenylpropanamide-HCI (RTI-4614-4), which is an analog of (+)-cis-3-methylfentanyl for opioid receptor subtypes. The Ki values (nM) of this agent for opioid receptor subtypes were as follows: mu (0.0055), delta (148), kappa 1 (84.8), kappa 2a (2275), and kappa 2b (22.3). The selectivity of this agent for the mu binding site was 27,000 vs. the delta binding site, 15,400 vs. the kappa 1 binding site, 413,700 vs the kappa 2a and 4,054 vs the kappa 2b binding site. In contrast, two other fentanyl analogs, N-(2-(4-methylpyridinyl))-N-(1-phenethyl-4-piperidinyl) 2-furamide and N-(2-pyrazinyl)-N-(1-phenethyl-4-piperdinyl)2-furamide had considerably higher Ki values at, and were less selective for, the mu binding site. Since RTI-4614-4 is composed of a mixture of four stereoisomers, the resolution of these isomers should permit identification of an extremely potent and selective agent for the opioid mu receptor.     

beta-endorphin: synthesis and biological activity of shortened peptide chains

Three analogs of beta-endorphin have been synthesized by the solid-phase method: betac-endorphin-(1--5)-(28--31), betac-endorphin-(6--31) and betah-endorphin-(1--5)-(16--31). The analgesic activities of these synthetic peptides relative to that of the parent molecule are reported. All three peptides at high doses exhibit either no or much weaker analgesic activity than beta-endorphin. These data suggest that the entire beta-endorphin molecule is necessary for full in vivo analgesic activity.     

Opioid activity of C8813, a novel and potent opioid analgesic

Compound trans-4-(p-bromophenyl)-4-(dimethylamino)-1-(2-thiophen-2-yl-ethyl)-cyclohexanol (C8813), structurally unrelated to morphine, is a novel analgesic. The present study examined the antinociception, opioid receptor selectivity and in vitro activity of C8813. The antinociceptive activity was evaluated using mouse hot plate and acetic acid writhing tests. In mouse hot plate test, the antinociceptive ED(50) of C8813 was 11.5 microg/kg, being 591 times and 3.4 times more potent than morphine and fentanyl respectively. In mouse writhing test, the antinociceptive ED(50) of C8813 was 16.9 microg/kg, being 55 times and 2.3 times more active than morphine and fentanyl respectively. In the opioid receptor binding assay, C8813 showed high affinity for mu-opioid receptor (K(i) = 1.37 nM) and delta-opioid receptor (K(i) = 3.24 nM) but almost no affinity for kappa-opioid receptor (at 1 microM). In the bioassay, the inhibitory effect of C8813 in the guinea-pig ileum (GPI) was 16.5 times more potent than in the mouse vas deferens (MVD). The inhibitory effects of C8813 in the GPI and MVD could be antagonized by mu-opioid receptor antagonist naloxone and delta-opioid receptor antagonist ICI174,864 respectively. However, the inhibitory effect of C8813 in the rabbit vas deferens was very weak. These results indicated that C8813 was a potent analgesic and a high affinity agonist for the mu- and delta-opioid receptors.     

Novel Aza-resveratrol analogs: Synthesis,characterization and anticancer activity against breast cancer cell lines

Novel Aza-resveratrol analogs were synthesized, structurally characterized and evaluated for cytotoxic activity against MDA-MB-231 and T47D breast cancer cell lines, which exhibited superior inhibitory activity than parent resveratrol compound. The binding mechanism of these compounds with estrogen receptor-α was rationalized by molecular docking studies which indicated additional hydrogen binding interactions and tight binding in the protein cavity. Induction of Beclin-1 protein expression in breast cancer cell lines after treatment with newly synthesized resveratrol analogs indicated inhibition of growth of these cell lines through autophagy. The study highlighted the advantage of introducing the imino-linkage in resveratrol motif in enhancing the anticancer potential of resveratrol suggesting that these analogs can serve as better therapeutic agents against breast cancer and can provide starting point for building more potent analogs in future.     

Structural-functional relations of short analogs of enkephalin

The similarity of action of narcotic analgesics and opioid peptides is due to activation of a common opiate receptor as the primary step in initiating biochemical chains responsible for diverse morphine-like effects. The most widely used assays for opioid and analgesic activities are presented and evaluated. Approximately 180 short enkephalin analogues (di-, tri- and tetrapeptides), described in the literature, are systematized and their opioid and systemic analgesic activities compared with methionine-enkephalin and morphine as the reference compounds, respectively. The analysis of structure-opioid activity relationships among these enkephalin analogues substantiates the hypothesis that only a limited N-terminal region of the peptide molecule is essential for the binding of opioid peptides to the subclass of opiate receptors interacting with narcotic alkaloids (mu-receptors). An attempt has been made to identify minimal structural elements responsible for the mu-receptor activation. Shortening of the molecule and modification of its elements are examined with regard to the mu- and delta-receptor selectivity. It is emphasized that the aromatic structure of the C-terminal region of the peptide is not obligatory for the mu-receptor binding. Modifications of short enkephalin analogues which might confer them antagonistic properties are reviewed. The correlation between the ability of short enkephalin analogues to interact with mu-receptors and their antinociceptive properties is discussed along with some structural features pertinent to the analgesic effect after systemic administration of peptides. On the basis of this analysis, peptides containing no more than four amino acids are considered as the most probable morphine-like analgesics.     

Discovery of novel N-acylsulfonamide analogs as potent and selective EP3 receptor antagonists

A series of novel N-acylsulfonamide analogs were synthesized and evaluated for their binding affinity and antagonist activity for the EP3 receptor subtype. Representative compounds were also evaluated for their inhibitory effect on PGE₂-induced uterine contraction in pregnant rats. Among those tested, a series of N-acylbenzenesulfonamide analogs were found to be more potent than the corresponding carboxylic acid analogs in both the in vitro and in vivo evaluations. The structure activity relationships (SAR) are also discussed.     

Inhibition of analgesia by C-terminal deletion analogs of human beta-endorphin

Human beta-endorphin (beta h-EP) analogs of variable chain lengths have been investigated for their potency in inhibiting analgesia induced by beta h-EP or by the potent opiate etorphine. It was found that beta h-EP-(1-28) inhibits the analgesic effect of beta h-EP and etorphine when co-injected intracerebroventricularly into mice. Antagonism by competition at same opioid receptor subtypes is suggested from parallel shifts of the dose-response curve of etorphine or beta h-EP in the presence of increasing doses of beta h-EP-(1-28). On a molar basis, beta h-EP-(1-28) is nearly 10 times more potent than naloxone. The reduction of the chain length from residues 1-28 to 1-27 lowered the antagonist potency while further reduction of the peptide chain led to a complete loss of inhibitory activity. From comparison of the opioid-receptor binding affinity, analgesic activity and antagonist potency, it is concluded that the C-terminus of beta-EP is critical to the biological efficacy of the molecule and that the antagonist activity of C-terminal deletion analogs is probably mediated through residues 27 and 28.     

Design and synthesis of novel delta opioid receptor agonists and their pharmacologies

We re-examined the accessory site of the 4,5-epoxymorphinan skeleton by camdas conformational analysis in an effort to deign novel δ opioid receptor antagonists. We synthesized three novel compounds (SN-11, 23 and 28) with a 10-methylene bridge and without a 4,5-epoxy ring. Among them, compounds SN-23 (17-isobutyl derivative) and SN-28 (17-methyl derivative) showed very strong agonist activity (over 10 times more than TAN-67). SN-28 also showed high δ selectivity. The δ agonist activity of SN-23 was weaker than that of SN-28, but in terms of the δ selectivity, SN-23 was higher than that of SN-28. These unexpected results indicated that the 4,5-epoxy ring, but not the 10-methylene bridge, was an accessory site in δ opioid receptor agonists.     

In-vitro investigation of oxazol and urea analogues of morphinan at opioid receptors

A series of 2-amino-oxazole (7 and 8) analogs and 2-one-oxazole analogs (9 and 10) were synthesized from cyclorphan (1) or butorphan (2) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors and compared with their 2-aminothiozole analogs 5 and 6. Ligands 7-10 showed decreased affinities at kappa and mu receptors. Urea analogs (11-14) were also prepared from 2-aminocyclorphan (3) or 2-aminobutorphan (4) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors. The urea derived opioids retained their affinities at mu receptors while showing increased affinities at delta receptors and decreased affinities at kappa receptors. Functional activities of these compounds were measured in the [35S]GTPgammaS binding assay, illustrating that all of these ligands were kappa agonists. At the mu receptor, compounds 11 and 12 were mu agonist/antagonists.     

Peripheral orphanin FQ/nociceptin analgesia in the mouse.

Orphanin FQ/Nociceptin (OFQ/N) administered peripherally was an effective analgesic in the tailflick test in mice (ED50 16.3 microg). It had a peak effect at 5 min and lasted up to 30 min. The kappa3 analgesic naloxone benzoylhydrazone was also active peripherally (ED50 3.8 microg). The analgesic actions of both agents were blocked by naloxone. Neither OFQ/N(1-11) nor OFQ/N(1-7) had appreciable peripheral activity. Antisense mapping both compounds against the murine orphan opioid receptor (KOR-3) confirmed the importance of this clone in their actions. Antisense probes targeting the second and third coding exons significantly lowered the analgesic effects of both compounds. However, the antisense targeting the first coding exon blocked only the actions of OFQ/N and not kappa3 analgesia.     

Truncated atrial natriuretic peptide analogs. Comparison between receptor binding and stimulation of cyclic GMP accumulation in cultured vascular smooth muscle cells

A series of truncated atrial natriuretic peptide analogs were examined as a means of defining the structural requirements for receptor occupancy and stimulation of cyclic GMP accumulation in bovine aortic smooth muscle cells. It was determined that deletion of amino acids from the carboxyl and/or amino termini of the peptides diminished their ability to increase cyclic GMP levels. Deletion of amino acids from the carboxyl terminus had the greatest effect, and atrial natriuretic peptide analogs lacking the carboxyl-terminal phenylalanyl-arginyl-tyrosine tripeptide were 100-1000-fold less active than parent compounds in stimulating intracellular cyclic GMP accumulation. In marked contrast to the cyclic GMP effects, deletion of amino- and/or carboxyl-terminal amino acids had only minor effects on the affinity of the peptides for specific smooth muscle cell-associated receptors. Peptide analogs lacking the phenylalanyl-arginyl-tyrosine tripeptide bound to receptors with an affinity only 1.1-5-fold weaker than the parent compounds. Thus, there was no correlation between apparent receptor binding affinity of atrial natriuretic peptide analogs and potency of these same peptides for stimulating intracellular cyclic GMP accumulation. Furthermore, analogs that bound to receptors and failed to elicit significant cyclic GMP responses did not antagonize or modulate increases in cyclic GMP induced by parent compounds. These data are most consistent with the existence of multiple subpopulations of atrial natriuretic peptide receptors on aortic smooth muscle cells.     

Synthesis and binding potencies of cyclohexapeptide somatostatin analogs containing naphthylalanine and arylalkyl peptoid residues

We report the synthesis, binding affinities to the recombinant human somatostatin receptors, and structure‐activity relationship studies of compounds related to the cyclic hexapeptide, c‐[Pro⁶‐Phe⁷‐D‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹], L‐363,301 (the numbering in the sequence refers to the position of the residues in native somatostatin). The Pro residue in this compound is replaced with the arylalkyl peptoid residues Nphe (N‐benzylglycine), (S)βMeNphe [(S)‐N‐[(α‐methyl)benzyl]glycine] or (R)βMeNphe [(R)‐N‐[(α‐methyl)benzyl]glycine] and l ‐1‐naphthylalanine is incorporated into either position 7 or 11 of the parent compound. The synthesis and binding data of the Nnal⁶ ([N‐naphthylmethyl]glycine) analog of L‐363,301 is also reported. The incorporation of the Nnal residue into position 6 of L‐363,301 resulted in an analog with weaker binding affinities to all hsst receptors but enhanced selectivity towards the hsst2 receptor compared with the parent compound. The other compounds bind effectively to the hsst2 receptor but show some variations in the binding to the hsst3 and hsst5 receptors resulting in different ratios of binding affinities to the hsst5 and hsst2 or hsst3 and hsst2, respectively. The incorporation of the Nphe residue into position 6 and the Nal residue into position 7 of L‐363,301 led to a compound which binds potently to the hsst2 and has increased selectivity towards this receptor (weaker binding to hsst3 and hsst5 receptors) compared with the parent compound. The analogs with β‐methyl chiral substitutions in the aromatic peptoid side chain and Nal in position 7 or 11 bind effectively to the hsst2 and hsst5 receptors. They exhibit similar ratios of binding affinities to the hsst5 and hsst2 receptors as observed for L‐363,301. There are however minor differences in binding to the hsst3 receptor among these analogs. These studies allow us to investigate the influence of additional hydrophobic groups on the binding activity to the isolated human somatostatin receptors and the results are important for the design of other somatostatin analogs. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Protein synthesis by mammalian cells treated with C-3-modified analogs of the 12,13-epoxytrichothecenes T-2 and T-2 tetraol.

Modification at the C-3 position of the trichothecenes T-2 and T-2 tetraol affected their ability to inhibit protein synthesis in African green monkey kidney (Vero) and mouse erythroleukemia cells. Replacement of the 3-hydroxyl of T-2 with hydrogen caused a 24-fold decrease in activity, whereas acetylation resulted in a 500-to 1,000-fold decrease. Protection of the 3-hydroxyl with a tetrahydropyranyl moiety gave an analog that was 37-fold more inhibitory to Vero than to mouse erythroleukemia cells; with the other analogs a similar effect on protein synthesis was found for both types of cells. The analogs obtained after alkaline hydrolysis were much less potent than the parent trichothecenes. The 3-tetrahydropyranyl-modified analog was equivalent in potency to T-2 tetraol, while the deoxygenated species was at least threefold less potent. All T-2 analogs caused some degree of polysome "runoff," thereby demonstrating that these species inhibit protein synthesis at the chain initiation stage when added at their 50% infective dose concentrations or lower. From these results, we suggest that the 3-hydroxyl moiety is essential for T-2 to exhibit such high activity on eucaryotic cell protein synthesis and that modification at the C-3 position decreases but does not eliminate this activity.     

Protein synthesis by mammalian cells treated with C-3-modified analogs of the 12,13-epoxytrichothecenes T-2 and T-2 tetraol

Modification at the C-3 position of the trichothecenes T-2 and T-2 tetraol affected their ability to inhibit protein synthesis in African green monkey kidney (Vero) and mouse erythroleukemia cells. Replacement of the 3-hydroxyl of T-2 with hydrogen caused a 24-fold decrease in activity, whereas acetylation resulted in a 500-to 1,000-fold decrease. Protection of the 3-hydroxyl with a tetrahydropyranyl moiety gave an analog that was 37-fold more inhibitory to Vero than to mouse erythroleukemia cells; with the other analogs a similar effect on protein synthesis was found for both types of cells. The analogs obtained after alkaline hydrolysis were much less potent than the parent trichothecenes. The 3-tetrahydropyranyl-modified analog was equivalent in potency to T-2 tetraol, while the deoxygenated species was at least threefold less potent. All T-2 analogs caused some degree of polysome "runoff," thereby demonstrating that these species inhibit protein synthesis at the chain initiation stage when added at their 50% infective dose concentrations or lower. From these results, we suggest that the 3-hydroxyl moiety is essential for T-2 to exhibit such high activity on eucaryotic cell protein synthesis and that modification at the C-3 position decreases but does not eliminate this activity.