Novel inhibition of porcine pepsin by a substituted piperidine. Preference for one of the enzyme conformers

Pepsin inhibition by 3-alkoxy-4-arylpiperidine (substituted piperidine; (3R,4R)-3-(4-bromobenzyloxy)-4-[4-(2-naphthalen-1-yl-2-oxo-ethoxy)phenyl]piperidine) has been studied using steady-state kinetic and pre-equilibrium binding methods. Data were compared with pepstatin A, a well known competitive inhibitor of pepsin. Steady-state analysis reveals that the substituted piperidine likewise behaves as a competitive inhibitor. Pre-equilibrium binding studies indicate that the substituted piperidine can displace a fluorescently labeled statine inhibitor from the enzyme active site. Simulation of the stopped-flow fluorescence transients provided estimates of the K(d) values of 1.4 +/- 0.2 microm and 39 +/- 2 nm for the piperidine and the fluorescently labeled statine, respectively. The effects of combinations of these two inhibitors resulted in a series of parallel lines when plotted by the method of Yonetani and Theorell (Yonetani, T., and Theorell, H. (1964) Arch. Biochem. Biophys. 106, 234-251), suggesting that the two inhibitors bind in a mutually exclusive fashion to pepsin. Fitting of the entire data set to the appropriate equation yielded an alpha factor of 8 +/- 1. The magnitude of this factor ( infinity > alpha > 1) can be explained by a conformational distinction between the enzyme species that bind each inhibitor. The effects of pH on the inhibition constants for pepstatin A and the substituted piperidine also suggest that the inhibitors bind to distinct conformational forms of the enzyme. No inhibition by the piperidine was observed at acidic pH, while pepstatin A inhibition is maximal at low pH values. Inhibition by the piperidine was maximal when a group with pK 4.8 +/- 0.2 was deprotonated and another group with pK 5.9 +/- 0.2 was protonated. Most likely these two groups are the catalytic aspartates with perturbed ionization properties as a result of a significant and unique conformational change. Taken together, these data suggest that the enzyme can readily interconvert between two conformers, one capable of binding substrate and pepstatin A and the other capable of binding the substituted piperidine.     

Asymmetric carbon-carbon bond-forming reaction at the 2-position of a piperidine skeleton

An asymmetric carbon-carbon bond-forming reaction at the 2-position of a piperidine skeleton was exploited. This method consisted of a reaction between 1-(4-methoxybenzoyl)-3,4-didehydro-2-methoxypiperidines and dimethyl malonate catalyzed by Cu(II)-chiral 2,2'-isopropylidenebis(4-phenyl-2-oxazoline) to afford a 2-substituted piperidine skeleton with moderate enantioselectivity.     

Absolute configurations and conformations of the opioid agonist and antagonist enantiomers of picenadol

The absolute configurations of the enantiomers of the opiod picenadol [cis-1,3-dimethyl-4-propyl-4-propyl-4-(3-hydroxyphenyl)piperidine; cis-3-methyl, 4-propyl] have been determined by an X-ray crystallographic study of the chloride salt of the (+)-enantiomer. The agonist (+)-enantiomer and the antagonist (?)-enantiomer were found to have the 3R, 4R and 3S, 4S absolute configurations, respectively. The conformational properties of the enantiomers were also examined with MM2–87 calculations. There was good agreement between the computed global minimum and the crystallographic structure with the phenyl ring approximately bisecting the piperidine ring by both methods. This orientation of the phenyl ring differs from that of related opioids such as the phenylmorphans, prodines, meperidine, and ketobemidone in which the phenyl ring tends to eclipse one edge of the piperidine ring. Because the phenyl ring bisects the piperidine ring in picenadol, there is little difference in the three-dimensional orientations of the phenyl rings of the two enantiomers when one superimposes the piperidine rings. The agonist (+)-enantiomer is ambiguous with respect to an opioid ligand model, which suggests that agonist activity requires a specific range of dihedral angles for the phenyl ring. While the global minimum of the agonist is not consistent with the model, a second conformer that is only 1.2 kcal/mol above the global minimum is consistent. An alternative explanation is that agonist or antagonist activity is solely due to the presence of the 3-methyl group on the different edges of the piperidine ring. MM2–87 calculations were also performed on the opioid agonist des-3-methyl analog of picenadol and the closely related trans-1,3,4-trimethyl-4-(3-hydroxyphenyl)piperidines (trans-3-methyl, 4-methyl) in which both enantiomers are opioid antagonists. The conformational properties of these compounds are consistent with the ligand model. © 1995 Wiley-Liss, Inc.     

Potent heteroarylpiperidine and carboxyphenylpiperidine 1-alkyl-cyclopentane carboxamide CCR2 antagonists

This report describes replacement of the 4-(4-fluorophenyl)piperidine moiety in our CCR2 antagonists with 4-heteroaryl piperidine and 4-(carboxyphenyl)-piperidine subunits. Some of the resulting analogs retained potency in our CCR2 binding assay and had improved selectivity versus the I(Kr) channel; poor selectivity against I(Kr) had been a liability of earlier analogs in this series.     

Synthesis and structure–activity relationships of N-aryl-piperidine derivatives as potent (partial) agonists for human histamine H3 receptor

4-((1H-Imidazol-4-yl)methyl)-1-aryl-piperazine and piperidine derivatives were designed and synthesized as candidate human histamine type 3 agonists. The piperazine derivatives were found to have low (or no) affinity for human histamine H3 receptor, whereas the piperidine derivatives showed moderate to high affinity, and their agonistic activity was greatly influenced by substituents on the aromatic ring. Among the piperidine-containing compounds, 17d and 17h were potent human histamine H3 receptor agonists with high selectivity over the closely related human H4 receptor. Our results indicate that appropriate conformational restriction, that is, by the piperidine spacer moiety, favors specific binding to the human histamine H3 receptor.     

Heterocyclic rimantadine analogues with antiviral activity

2-(1-Adamantyl)pyrrolidines 6, 7, 2-(1-adamantyl)piperidines 10, 12a–c, 15a,b and 2-(1-adamantyl)hexahydroazepines 19, 21, 22 were synthesized and tested for their antiviral activity against influenza A, B viruses and the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). The synthetic procedure followed for the preparation of the parent piperidine 10 represents a general method for the synthesis of 2-alkyl- or cycloalkyl-substituted piperidine alkaloids. Parent aminoadamantanes 6, 10 and 19 contain the 1-aminoethyl pharmacophore group of rimantadine drug 2, extended into a saturated nitrogen heterocycle: pyrrolidine, piperidine and hexahydroazepine, respectively. The ring size effect in anti-influenza A activity was investigated. Rimantadine analogues 6 and 10 were, respectively, 6- and 4-fold more active than the drug Rimantadine 2, whereas the hexahydroazepine derivative 19 was inactive. Thus, enlargement from a 5-(pyrrolidine)- or 6-(piperidine)- to a 7-(hexahydroazepine)- membered heterocyclic ring dramatically reduced the anti-influenza virus A activity. Substitution of piperidine 10 with a dialkyaminoethyl group led to the active compounds 15a and 15b: compound 15a was active against influenza A virus whereas both 15a and 15b were active against HIV-1.     

Synthesis and structure-activity relationships of a new series of 2alpha-substituted trans-4,5-dimethyl-4-(3-hydroxyphenyl)piperidine as mu-selective opioid antagonists

Structure-activity relationships at the 2alpha-position of the piperidine ring of the trans-4,5-dimethyl-4-(3-hydroxyphenyl)piperidine mu-opioid antagonist series were investigated. This study showed that only small linear alkyl groups (methyl, propyl) are tolerated at the 2alpha-position of the piperidine ring of this series.     

Novel histamine H3 receptor antagonists based on the 4-[(1H-imidazol-4-yl)methyl]piperidine scaffold

We report the discovery of novel histamine H(3) receptor antagonists based on 4-[(1H-imidazol-4-yl)methyl]piperidine. The most potent compounds in the series (e.g., 7) result from the attachment of a substituted aniline amide to the main pharmacophore piperidine via a two-methylene linker.     

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.     

Synthesis of the major metabolites of paroxetine

Paroxetine is a well-known antidepressant, used worldwide in therapeutics. In comparison with other selective serotonin reuptake inhibitors, it exhibits the highest activity in serotonin reuptake inhibition. Paroxetine metabolism initially involves its demethylenation to the catechol intermediate, which is then O-methylated at positions C3 or C4. Herein, the chemistry resulting in the syntheses of these metabolites (3S,4R)-4-(4-fluorophenyl)-3-(hydroxymethyl)piperidine and (3S,4R)-4-(4-fluorophenyl)-3-(4-hydroxy-3-methoxyphenoxymethyl)piperidine is described starting from the common intermediate (3S,4R)-4-(4-fluorophenyl)-3-hydroxymethyl-1-methylpiperidine. Additionally, the common intermediate was used to synthesize paroxetine, which had the same structure and stereochemistry as commercial paroxetine, thereby confirming our synthetic route.     

2,4-Disubstituted piperidines as selective CC chemokine receptor 3 (CCR3) antagonists: synthesis and selectivity

Linear unselective CCR3 antagonist leads with IC(50) values in the 200 nM range were converted into low nM binding compounds selective at CCR3 by moving the piperidine nitrogen substituent to the carbon at the 2-position of the ring. Substitution of the piperidine nitrogen with simple alkyl and acyl groups was found to improve the selectivity of this new compound class. In particular, N-{3-[(2S, 4R)-1-(propyl)-4-(4-fluorobenzyl)piperidinyl]propyl}-N'-(3-acetylphenyl)urea exhibited single digit nanomolar IC(50) values for CCR3 with >100-fold selectivity against an extensive counter screen panel.     

The Influences of Jasmonic Acid Methyl Ester on Microtubules in Potato Cells and Formation of Potato Tubers

Amides in a CH₂Cl₂ extract from the fruits of Piper retrofractum were detected by HPLC/APCI-MS. Seven new unsaturated amides, together with six known ones, were isolated, and their structures were determined to be N-isobutyl-2E,4E,12Z-octadecatrienamide (1), N-isobutyl-2E,4E,14Z-eicosatrienamide (2), 1-(octadeca-2E,4E,12Z-trienoyl)piperidine (3), 1-(eicosa-2E,4E,14Z-trienoyl)piperidine (4), 1-(octadeca-2E,4E-dienoyl)piperidine (5), 1-(eicosa-2E,4E-dienoyl)piperidine (6), and 1-(eicosa-2E,14Z-dienoyl)piperidine (7) on the basis of chemical and spectroscopic evidence.     

Synthesis and in vitro and in vivo anticancer activity of novel phenylmethylene bis-isoxazolo[4,5-b]azepines

A series of novel phenylmethylene bis-isoxazolo[4,5-b]azepine derivatives (10) have been synthesized from 3-methyl-4-nitro-5-styrylisoxazoles 6. The reaction of 6 with 3,5-dimethyl-4-nitroisoxazole (7) in piperidine afforded the Michael type adducts 8, which on treatment with different substituted chalcones in the presence of piperidine gave the Michael adducts 9. Compounds 9 underwent reductive cyclization on treatment with SnCl(2)-MeOH to afford the title compounds 10. Structure of these compounds was established on the basis of IR, (1)H NMR, (13)C NMR and Mass spectral data. The title compounds 10a-j were evaluated for in vitro and in vivo anticancer activity. Compound 10j exhibited good anticancer activity as that of standard drug Cisplatin.     

Highly potent PDE4 inhibitors with therapeutic potential

The hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system prompted us to design and synthesize a hydrophilic piperidine analog to improve the side effect profile of Ariflo 1, which is an orally active second-generation PDE4 inhibitor. During evaluation of various water-soluble piperidine analogs, 2a-b, 11b-14b, and 17a showed therapeutic potential in cross-species comparison studies. The following three findings were obtained: (1) The hydroxamic acid group, a well known metal chelator, caused a marked increase of inhibitory activity. (2) Water-soluble piperidine analogs lacked the configurational isomerism of Ariflo 1 without loss of inhibitory activity. (3) Replacement of the 4-methoxy residue with a difluoromethoxy residue led to an increase of in vivo potency. Structure-activity relationships are presented. Single-dose rat pharmacokinetic data for 11b, 12b, and 17a are also presented.     

Inhibition of gonadotropin-stimulated adenylate cyclase by dansyl-arginyl-(4'-ethyl)piperidine amide (DAPA)

Protease inhibitors are known to suppress basal, fluoride-, and hormone-stimulated adenylate cyclase activities. The thrombin inhibitor, dansyl-arginyl-(4'-ethyl)piperidine amide (DAPA), also specifically inhibits the binding of gonadotropins to their receptors. Our studies were undertaken to find a concentration of DAPA that would specifically inhibit gonadotropin-stimulated adenylate cyclase without significantly altering basal, fluoride-, isoproterenol-, or prostaglandin E1-stimulated cyclase. Basal adenylate cyclase activity was not inhibited by DAPA in either human chorionic gonadotropin (hCG)- or follicle-stimulating hormone (FSH)-responsive rat ovarian plasma membranes. Human chorionic gonadotropin-stimulated cyclase was completely inhibited by DAPA at a concentration of 2.96 mM; the ID50 was 1.32 mM. Follicle-stimulating hormone-stimulated cyclase was completely inhibited by a DAPA concentration of 4.44 mM, and the ID50 was 1.75 mM. Dansyl-arginyl-(4'-ethyl)piperidine amide (2.96 mM) inhibited isoproterenol-, prostaglandin E1-, and fluoride-stimulated cyclase in hCG-responsive membranes by 11%, 28%, and 35%, respectively. Dansyl-arginyl-(4'-ethyl)piperidine amide (4.44 mM) inhibited fluoride- and prostaglandin-stimulated cyclase in FSH-responsive membranes by 10% and 11%, respectively. The data show that appropriate concentrations of DAPA can antagonize gonadotropin-stimulated adenylate cyclase while only minimally affecting fluoride- and other receptor-activated cyclase activities.     

N alpha-dansyl-L-arginine 4-phenylpiperidine amide. A potent and selective inhibitor of horse serum cholinesterase

The relationship between chemical modifications of arginine derivatives and inhibitory activity to horse serum cholinesterase (BuChE) was investigated. It provided a new insight into the topography of the active site of BuChE. 1) BuChE has the hydrophobic binding pocket, the depth of which corresponds to the length of ethylpiperidine. 2) In the opposite side to the hydrophobic binding pocket, BuChE has a certain entity which repulses carboxyl group at the 2-position of piperidine of L-arginine piperidine amide. 3) The P site of BuChE can allow 4-propyl and 4-phenyl group attached to piperidine. Comparison of the results with those of thrombin and trypsin clearly revealed similarities and dissimilarities among BuChE, trypsin, and thrombin in the active site topography, and hence, we introduce a new selective inhibitor for BuChE, N alpha-dansyl-L-arginine 4-phenylpiperidine amide. It inhibits BuChE strongly (Ki = 0.016 microM), whereas it inhibits trypsin, thrombin, plasmin, and glandular kallikrein only weakly and shows actually no inhibition on acetylcholinesterase from the human erythrocyte. In addition, the new inhibitor becomes highly fluorescent when bound with BuChE, indicating that the compound is an ideal probe of the interactions of BuChE as well as a titrant of it.     

The measurement of nanogram amounts of piperidine in tissues by gas chromatography.

A gas chromatographic method is presented which allows the determination of piperidine in tissue in nanogram quantities. Reaction of the perchloric acid tissue extracts with 3,5-dinitro-4-chloro-benzotrifluoride followed by extraction with hexane, results in a derivative of piperidine suitable for electron capture detection. The specificity of the procedure is confirmed by combined gas chromatography - mass spectrometry. Using this procedure the mean concentration of piperidine in whole mouse brain was found to be 219 pmoles per gram of tissue. No significant difference between the concentration of piperidine in the brains of active and behavioral sleeping mice could be found.     

Piperidine levels in the brain and peripheral organs in rats during cold exposure

Piperidine is one of biogenic amines possessing nicotine-like synaptotropic actions on the nervous systems. Since piperidine produces multiplex pharmacological actions, a role for the amine as a modulator in neuroendocrine as well as neuronal functions has been supposed. In the present study, piperidine levels in the brain and peripheral organs in rats during cold exposure were examined by use of a mass fragmentographic technique and it was found that piperidine concentrations in the brain and peripheral endocrine glands significantly increased at 180 min following exposure to 4°C. The significance of the findings is discussed with respect to the neurohormone-releasing effect of piperidine.     

Core modification of substituted piperidines as Novel inhibitors of HDM2–p53 protein–protein interaction

The discovery of 3,3-disubstituted piperidine 1 as novel p53–HDM2 inhibitors prompted us to implement subsequent SAR follow up directed towards piperidine core modifications. Conformational restrictions and further functionalization of the piperidine core were investigated as a strategy to gain additional interactions with HDM2. Substitutions at positions 4, 5 and 6 of the piperidine ring were explored. Although some substitutions were tolerated, no significant improvement in potency was observed compared to 1. Incorporation of an allyl side chain at position 2 provided a drastic improvement in binding potency.     

Syntheses of 3-carbomethoxy-4-(aryl)piperidines and in vitro and in vivo pharmacological evaluation: identification of inhibitors of the human dopamine transporter

A series of 3-carbomethoxy-4-(aryl-substituted)piperidines with various aryl groups were synthesized and examined for binding and reuptake inhibition at the human dopamine transporter, the human serotonin transporter, and the human norepinephrine transporter. The binding potency and reuptake inhibition efficacy was compared with that of (-)-cocaine to determine the significance of removing the two-carbon bridge of the cocaine nucleus on the inhibition of transporter binding and reuptake. Of the transporters examined, the substituted piperidines were relatively selective for the human dopamine transporter. In all cases examined, the cis-diastereomer of the 3-carbomethoxy-4-(aryl-substituted)piperidine was observed to be a more potent inhibitor of the human dopamine transporter than the trans diastereomer. Based on the K(i) (binding) and IC(50) (reuptake inhibition) values obtained, the most potent inhibitor of the series was cis-3-carbomethoxy-4-(4'-chlorophenyl)piperidine, and this compound suppressed spontaneous- and cocaine-induced stimulation in non-habituated male Swiss-Webster mice. The conclusion is that substantial portions of the cocaine structure can be dissected away to provide compounds with significant binding and reuptake inhibition of the human dopamine transporter.