Stereoselective synthesis of a new muscarinic M3 receptor antagonist, J-104129.

A diastereoselective synthesis of J-104129 (1) was developed. A key step of this synthesis was Michael addition of enolate generated from cis-chiral dioxolane 2 to cyclopentenone, followed by hydrogenolysis of the resultant enol triflate 4. A mixture of cyclopentyldioxolane (5, 6) was hydrolyzed with sodium hydroxide to yield carboxylic acid 7 in 86% ee.     

Design and synthesis of fluorescent SGLT2 inhibitors

The design and synthesis of the first fluorophore-conjugated SGLT2 inhibitors is described. The mode of linking the fluorophore to the SGLT2 pharmacophore was found to be crucial in achieving optimum potency. Superior potency to phlorizin was provided by examples containing TAMRA, BODIPY, Cy3B and NBD fluorophores.     

Design and synthesis of an irreversible dopamine-sparing cocaine antagonist

Cocaine is a powerful reinforcer and stimulant that binds to specific recognition sites associated with monoamine transporters in the mammalian brain. The search for a functional antagonist to the addictive properties of cocaine has focused on the discovery of a molecule that can inhibit cocaine binding to the dopamine transporter (DAT) but continue to allow dopamine transport by the DAT. No such dopamine-sparing cocaine antagonist has been reported and it is becoming evident that dopamine-sparing antagonism of the pharmacological effects of cocaine by a classical antagonist may not be possible. Herein we present a new concept for the design of dopamine-sparing cocaine antagonists. A unique approach is utilized to deliver an inhibitor that binds irreversibly to the DAT, then cleaves and leaves behind a small fragment attached to the DAT that blocks access by cocaine but permits dopamine transport. The design of these compounds takes advantage of a cysteinyl sulfhydryl group in the DAT. This group is hypothesized to attack the incoming inhibitor and lead to selective inhibition of the cocaine binding site while sparing dopamine transport. This concept of a mechanism based irreversible dopamine-sparing cocaine antagonist has now been demonstrated to be viable and, as example, the unsaturated 6 showed inhibition of cocaine (63%) at the DAT after 24h incubation, while at that point considerably less inhibition of dopamine is manifested (23%). In contrast, the epoxide 7 showed a greater inhibition of dopamine reuptake than cocaine binding at 24h (68% versus 18%).     

Design and synthesis of cell-permeable fluorescent nitrilotriacetic acid derivatives

Fluorescence labeling of the target molecules using a small molecule-based probe is superior than a method using genetically expressed green fluorescence protein (GFP) in terms of convenience in its preparation and functionalization. Fluorophore-nitrilotriacetic acid (NTA) conjugates with several ester protecting groups were synthesized and evaluated for their cell membrane permeability by fluorescence microscopy analysis. One of the derivatives, acetoxymethyl (AM)-protected NTA conjugate is hydrolyzed, resulting in intracellular accumulation, thus providing localized fluorescence intensity in cells. This modification is expected as an effective method for converting a non-cell membrane permeable NTA-BODIPY conjugates to a cell membrane permeable derivatives.     

Design and synthesis of fluorescent probes for GPR54

Kisspeptins are neuropeptides that induce the secretion of gonadotropin-releasing hormone via the activation of the cognate receptor, G-protein coupled receptor 54 (GPR54). The kisspeptin–GPR54 axis is associated with the onset of puberty and the maintenance of the reproductive system. In this study, several fluorescent probes have been designed and synthesized for rat GPR54 through the modification of the N-terminus of rat kisspeptins to allow for the visualization of the expression and localization of kisspeptin receptor(s) in living cells and native tissues. The tetramethylrhodamine (TMR) and rhodamine green (RG)-labeled kisspeptins exhibited good binding and agonistic activities towards GPR54, and the results of the application studies demonstrated that these fluorescent probes could be used effectively for the detection of GPR54 receptors in flow cytometry and confocal microscopy experiments.     

Design and synthesis of 3alpha-helix peptides forming a cavity for a fluorescent ligand

As a model of receptor protein, a series of 3alpha-helix bundle peptides constructed on a template peptide were designed so as to possess a hydrophobic cavity. The size of cavity was modulated by simple replacements of Leu residues to Ala residues in the hydrophobic core. Binding abilities to 8-anilino-1-naphthalenesulfonic acid (ANS) were estimated by the increase of fluorescence intensity. The peptide having three or four Ala residues in the hydrophobic core remarkably increased the binding ability for ANS, though the peptide having two Ala residues gave an inefficient cavity for ANS. The peptide having six Ala residues decreased the binding ability due to crucial destabilization of the helix bundle structure. This scaffold can be utilized to a receptor model, while further tuning of the sequence is necessary.     

Design and total synthesis of a fluorescent phorboxazole A analog for cellular studies

To enable studies to elucidate the intracellular processing and targeting of the potent cytostatic/apoptotic anticancer natural products phorboxazoles A and B, a fluorescent derivative has been developed. This involved the total syntheses of the terminal alkyne 33-O-Me-45,46-dehydrobromophorboxazole A (MDHBPA) and a terminal vinyl iodide derivative of the blue fluorescent dye N,N,-dimethyl-7-aminocoumarin (DMC). Sonogashira coupling of these partners provided enyne DMC-MDHBPA in high yield.     

Hemodynamic consequences of chronic parasympathetic blockade with a peripheral muscarinic antagonist

Whereas the sympathetic nervous system has a well-established role in blood pressure (BP) regulation, it is not clear whether long-term levels of BP are affected by parasympathetic function or dysfunction. We tested the hypothesis that chronic blockade of the parasympathetic nervous system has sustained effects on BP, heart rate (HR), and BP variability (BPV). Sprague-Dawley rats were instrumented for monitoring of BP 22-h per day by telemetry and housed in metabolic cages. After the rats healed from surgery and a baseline control period, scopolamine methyl bromide (SMB), a peripheral muscarinic antagonist, was infused intravenously for 12 days. This was followed by a 10-day recovery period. SMB induced a rapid increase in mean BP from 98 +/- 2 mmHg to a peak value of 108 +/- 2 mmHg on day 2 of the SMB infusion and then stabilized at a plateau value of +3 +/- 1 mmHg above control (P < 0.05). After cessation of the infusion, the mean BP fell by 6 +/- 1 mmHg. There was an immediate elevation in HR that remained significantly above control on the last day of SMB infusion. SMB also induced a decrease in short-term (within 30-min periods) HR variability and an increase in both short-term and long-term (between 30-min periods) BPV. The data suggest that chronic peripheral muscarinic blockade leads to modest, but sustained, increases in BP, HR, and BPV, which are known risk factors for cardiovascular morbidity.     

PD 142676 (CI 1002), a novel anticholinesterase and muscarinic antagonist

Inhibition of brain acetylcholinesterase (AChE) can provide relief from the cognitive loss associated with Alzheimer's disease (AD). However, unwanted peripheral side effects often limit the usefulness of the available anticholinesterases. Recently, we identified a dihydroquinazoline compound, PD 142676 (CI 1002) that is a potent anticholinesterase and a functional muscarinic antagonist at higher concentrations. Peripherally, PD 14276, unlike other anticholinesterases, inhibits gastrointestinal motility in rats, an effect consistent with its muscarinic antagonist properties. Centrally, the compound acts as a cholinomimetic. In rats, PD 142676, decreases core body temperature. It also increases neocortical arousal, as measured by quantitative electroencephalography, and cortical acetylcholine levels, measured by in vivo microdialysis. The compound improves the performance of C57/B10j mice in a water maze task and of aged rhesus monkeys in a delayed match-to-sample task involving short-term memory. The combined effect of AChE inhibition and muscarinic antagonism distinguishes PD 142676 from other anticholinesterases and may be useful in treating the cognitive dysfunction of AD and produce fewer peripheral side effects.     

Design and synthesis of a near-infrared fluorescent nanofiber precursor for detecting cell-secreted urokinase activity

Abnormal proteolysis is often observed during disease progression. Up-regulation of certain tumor-associated proteases such as urokinase plasminogen activator (uPA) can be a hallmark of malignant transformation. Here we report the design and synthesis of a near-infrared nanofiber precursor (NIR–NFP) for detecting uPA activity. NIR–NFP, which is optically silent in its native state, is composed of multiple self-assembled peptide units (PEG₅₄-BK(NIR664)SGRSANA-kldlkldlkldl-CONH₂). On uPA activation, NIR–NFP releases peptide fragments (PEG₅₄-BK(NIR664)SGR-CONH₂) that contribute to a significant fluorescence amplification at 684 nm. NIR–NFP was able to detect cell-secreted uPA from human cancer cells (SKBR-3, PANC-1, MCF-7, SKOV-3, MDA-MB-231, PC-3, and HT-1080) expressing various levels of uPA. Fluorescence changes were uPA dependent, as confirmed with both Western blot analysis and enzyme activity assay. Our data suggest that an optimized preparation may be useful for imaging uPA activity in vivo.     

Anthroylcholine bromide: a fluorescent ligand for the muscarinic receptor.

The action of anthroylcholine bromide, a new fluorescent probe, has been studied at the cellular (contraction of intestinal muscle) and subcellular levels (binding of 3H-quinuclidinyl benzilate to brain cortex membranes, fluorescence and enzyme activity) with the following results: 1. Anthroylcholine bromide competitively antagonized the contractile effect of acetylcholine in isolated rat duodenum (pA2 = 6.12), but had no effect either on the concentration response curves to histamine or to noradrenaline in isolated guinea pig ileum and rat vas deferens. 2. Anthroylcholine bromide displaced competitively 3H-quinuclidinyl benzilate from brain cortex membranes (Ki = 0.77 mumol/l). 3. Direct binding to the muscarinic site could be measured by exploiting the fluorescence properties of the probe. Binding displaceable by atropine (approximately 20% change in fluorescence) had an apparent affinity constant similar to that found with indirect methods. In contrast, d-tubocurarine did not displace the probe from its site, and atropine- or d-tubocurarine-sensitive binding of anthroylcholine bromide to Torpedo marmorata electric organ membranes, rich in nicotinic receptors, was not observed. These properties suggest the applicability of the probe to study the distribution, structure and/or kinetic properties of the muscarinic receptor.     

Design and synthesis of fluorescent substrates for human tyrosyl-DNA phosphodiesterase I

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that acts upon protein–DNA covalent complexes. Tdp1 hydrolyzes 3′-phosphotyrosyl bonds to generate 3′-phosphate DNA and free tyrosine in vitro. Mutations in Tdp1 have been linked to patients with spinocerebellar ataxia, and over-expression of Tdp1 results in resistance to known anti-cancer compounds. Tdp1 has been shown to be involved in double-strand break repair in yeast, and Tdp1 has also been implicated in single-strand break repair in mammalian cells. Despite the biological importance of this enzyme and the possibility that Tdp1 may be a molecular target for new anti-cancer drugs, there are very few assays available for screening inhibitor libraries or for characterizing Tdp1 function, especially under pre-steady-state conditions. Here, we report the design and synthesis of a fluorescence-based assay using oligonucleotide and nucleotide substrates containing 3′-(4-methylumbelliferone)-phosphate. These substrates are efficiently cleaved by Tdp1, generating the fluorescent 4-methylumbelliferone reporter molecule. The kinetic characteristics determined for Tdp1 using this assay are in agreement with the previously published values, and this fluorescence-based assay is validated using the standard gel-based methods. This sensitive assay is ideal for kinetic analysis of Tdp1 function and for high-throughput screening of Tdp1 inhibitory molecules.     

Design, synthesis and structure-activity relationship of N-substituted tropane muscarinic acetylcholine receptor antagonists

A novel series of N-substituted tropane derivatives was characterized as potent muscarinic acetylcholine receptor antagonists (mAChRs). Kinetic washout studies showed that the N-endosubstituted analog 24 displayed much slower reversibility at mAChRs than the methyl-substituted parent molecule darotropium. In addition, it was shown that this characteristic appeared to translate into enhanced which duration of action in a mouse model of bronchonstriction.     

The discovery of AZD9164, a novel muscarinic M3 antagonist

The optimization of a new series of muscarinic M(3) antagonists is described, leading to the identification of AZD9164 which was progressed into the clinic for evaluation of its potential as a treatment for COPD.     

Affinity profile of the novel muscarinic antagonist, guanylpirenzepine

The study reports the functional affinity of an amidino derivative of pirenzepine, guanylpirenzepine, for muscarinic receptors mediating relaxation of rat duodenum, inhibition of rabbit vas deferens twitch contraction (both receptors previously classified as M1), guinea pig negative inotropism (M2) and ileal contraction (M3). Unlike pirenzepine, guanylpirenzepine discriminated between duodenum and vas deferens receptors, with a 30-fold greater affinity for the former subtype. The unique selectivity pattern of guanylpirenzepine (duodenum greater than vas deferens greater than ileum greater than atrium) renders it a promising tool for the classification of muscarinic receptor subtypes.     

Isomerization of the muscarinic receptor . antagonist complex.

The mechanism of binding of two antagonists, 3-quinuclidinyl benzilate and N-methyl-4-piperidinyl benzilate, to the muscarinic receptor was studied. The pseudo-first order rate constant of association showed a hyperbolic dependence on the concentration of the antagonist(s) indicating that the interaction involves two equilibria. The first binding equilibrium is reached rapidly and is characterized by dissociation constants 2.7 +/- 0.4 nM and 6.7 +/- 2.5 nM in phosphate buffer (0.05 M, pH = 7.4) for 3-quinuclidinyl benzilate and N-methyl-4-piperidinyl benzilate, respectively. The first binding equilibrium is followed by a slower isomerization step of the receptor . antagonist complex. The equilibrium constants for the isomerization step of the complex for both ligands were about 0.15. The overall constant of binding obtained as the product of the above constants shows good agreement with the results of equilibrium binding studies.     

Design, synthesis, and structure-activity relationship studies of himbacine derived muscarinic receptor antagonists

A parallel synthesis of racemic himbacine analogs was carried out by N-alkylation of various commercially available cyclic amine derivatives with the alkylating agent 4 which bears the tricyclic unit of himbacine. Several of these analogs have potency comparable to that of himbacine, albeit lacking the desired selectivity. Structure-activity relationship studies support the existence of a hydrophobic pocket in the receptor where the piperidine ring of dihydrohimbacine binds.     

Design and synthesis of labeled analogs of PhTX-56, a potent and selective AMPA receptor antagonist

Polyamines and polyamine toxins are biologically important molecules, having modulatory effects on nucleotides and proteins. The wasp toxin, philanthotoxin-433 (PhTX-433), is a non-selective and uncompetitive antagonist of ionotropic receptors, such as ionotropic glutamate receptors and nicotinic acetylcholine receptors. Polyamine toxins are used for the characterization of subtypes of ionotropic glutamate receptors, the Ca2+-permeable AMPA and kainate receptors. A derivative of the native polyamine toxin, philanthotoxin-56 (PhTX-56), has recently been shown to be an exceptionally potent and selective antagonist of Ca2+-permeable AMPA receptors. PhTX-56 and its labeled derivatives are promising tools for structure-function studies of the ion channel of the AMPA receptor. We now describe the design and synthesis of 3H-, 13C-, and 15N-labeled derivatives of PhTX-56 for molecular level studies of AMPA receptors. [3H]PhTX-56 was prepared from a diiodo-precursor with high specific radioactivity, providing the first radiolabeled ligand binding to the pore-forming part of AMPA receptors. For advanced biological NMR studies, 13C and 15N-labeled PhTX-56 were synthesized using solid-phase synthesis. These analogs can provide detailed information on the ligand-receptor interaction. In conclusion, synthesis of labeled derivatives of PhTX-56 provides important tools for future studies of the pore-forming region of AMPA receptors.     

Different antagonist binding properties of rat pancreatic and cardiac muscarinic receptors

The antagonist binding properties of rat pancreatic and cardiac muscarinic receptors were compared. In both tissues pirenzepine (PZ) had a low affinity for muscarinic receptors labelled by (3H)N-methylscopolamine [3)NMS) (KD values of 140 and 280 nM, respectively, in pancreatic and cardiac homogenates). The binding properties of pancreatic and cardiac receptors were, however, markedly different. This was indicated by different affinities for dicyclomine, (11-([(2-[diethylamino)-methyl)-1-piperidinyl] acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4) benzodiazepin-6-on) (AFDX-116), 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP) and hexahydrosiladifenidol (HHSiD). Pancreatic and cardiac muscarinic receptors also showed different (3H)NMS association and dissociation rates. These results support the concept of M2 receptor heterogeneity and confirm that M2 receptor subtypes have different binding kinetic properties.