Modification and Functionalization of the Guanidine Group by Tailor-made Precursors.

The guanidine group is one of the most important pharmacophoric groups in medicinal chemistry. The only amino acid carrying a guanidine group is arginine. In this article, an easy method for the modification of the guanidine group in peptidic ligands is provided, with an example of RGD-binding integrin ligands. It was recently demonstrated that the distinct modification of the guanidine group in these ligands allows for the selective modulation of the subtype (e.g., between the subtypes αv and α5). Moreover, a formerly unknown strategy for the functionalization via the guanidine group was demonstrated, and the synthetic approach is reviewed in this document. The modifications described here involve terminally (Nω) alkylated and acetylated guanidine groups. For the synthesis, tailor-made precursor molecules are synthesized, which are then subjected to a reaction with an orthogonally deprotected amine to transfer the pre-modified guanidine group. For the synthesis of alkylated guanidines, precursors based on N,N'-Di-Boc-1H-pyrazole-1-carboxamidine are used to synthesize acylated compounds, the precursor of choice being a correspondingly acylated derivative of N-Boc-S-methylisothiourea, which can be obtained in one- and two-step reactions.

The design of full agonists for the cortical muscarinic receptor.

In the present study we describe a novel series of oxadiazole based tertiary amines which include the most efficacious and potent muscarinic ligands known. These compounds possess physicochemical characteristics which enable rapid equilibration into the CNS and are able to fully activate cortical muscarinic receptors. Data obtained from this series have allowed us to propose a pharmacophoric model which distinguishes high and low affinity state binding. This in turn has led us to suggest that agonists and antagonists may bind at two independent sites on the receptor protein and to speculate on the steps putatively involved in agonist-induced receptor activation.

Synthesis and structure-activity studies of a series of spirooxazolidine-2,4-diones: 4-oxa analogues of the muscarinic agonist 2-ethyl-8-methyl-2,8-diazaspiro[4.5]decane-1,3-dione.

A series of spirooxazolidine-2,4-dione derivatives related to the putative M1 agonist 2-ethyl-8-methyl-2,8-diazaspiro[4.5]decane-1,3-dione (RS86; 1) were synthesized. The compounds were evaluated as cholinergic agents in in vitro binding assays and in in vivo pharmacological tests including antiamnesic effects using scopolamine-treated mice, hypothermia, and salivation in mice. Four compounds (5a,c,f and 17a) exhibited affinity for cortical M1 receptors and reversed scopolamine-induced impairment of mouse passive avoidance tasks, as did 1. Among these compounds, only 5a exhibited M1-receptor stimulating activity in pithed rats. Structural requirements for muscarinic activity in this series of spirooxazolidine-2,4-dione derivatives were as strict as those reported for spirosuccinimide derivatives including 1. The antiamnesic dose of 3-ethyl-8-methyl-1-oxa-3,8-diazaspiro[4.5]decane-2,4-dione (5a) was 2 orders of magnitude lower than the doses inducing hypothermia and salivation, in contrast to 1 for which the former dose was only 5-10-fold lower than the latter. These results suggest that the 8-azaspiro[4.5]decane skeleton represents a useful template for designing new muscarinic agonists as antidementia drugs.

A novel class of conformationally restricted heterocyclic muscarinic agonists.

A series of conformationally restricted compounds containing the 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) skeleton, including O-methyl-THPO (10a) and O,5-dimethyl-THPO (11a), were synthesized. The compounds were designed by bioisosteric replacement of the methyl ester groups of the muscarinic cholinergic agonists norarecoline and arecoline by the 3-methoxyisoxazole group, and their interactions with central and peripheral muscarinic receptors were tested in vitro. The compounds 10a, 11a, O-ethyl-THPO (10b), O-propargyl-THPO (10j), and O-ethyl-5-methyl-THPO (11b) were inhibitors of the binding of the muscarinic mustard [3H]PrBCM to rat brain membranes with an increasing order of potency. There was, however, a very low degree of correlation between these binding data and the effects of the compounds on peripheral (ileal) muscarinic receptors, where 11a, 10j, 11b, and 10a were agonists with a decreasing order of potency, whereas O-isopropyl-THPO (10e) showed antagonistic effects. The relatively low pKa values of the compounds (7.5-7.7 for compounds with secondary and 6.1-7.0 for compounds with tertiary amino groups) are likely to allow the compounds to penetrate the blood-brain barrier.

Synthesis and muscarinic activities of 1,2,4-thiadiazoles.

A series of novel 1,2,4-thiadiazoles bearing a mono- or bicyclic amine at C5 were prepared. Quinuclidine and 1-azabicyclo[2.2.1]heptane derivatives were synthesized by reaction of the lithium enolate of the 3-methoxycarbonyl compounds followed by ester hydrolysis and decarboxylation. The receptor-binding affinity and efficacy of these compounds as muscarinic ligands was assessed by radioligand binding assays using [3H]-N-methylscopolamine and [3H]oxotremorine-M. Optimal agonist affinity was observed for 3'-methyl compounds. Smaller substituents (H) retained efficacy with reduced affinity while larger groups led to substantially lower efficacy. The observed binding affinity was influenced both by the conformational energy of rotation around the C3-C5' bond and the steric requirement of the mono- or bicyclic amine.

Stereochemical analogs of a muscarinic, ganglionic stimulant. 3. 2,3-Substituted bicyclo(2.2.1)hept-5-enes and -heptanes related to 4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343).

Preparation of analogs of 4-[N-(3-chlorophenyl)carbamoyloxy]-2-butynyltrimethylammonium chloride (1, McN-A-343), the isomeric 2-trimethylammoniomethyl-3-[N-(4-chlorophenyl)carbamoyloxymethyl]bicyclo [2.2.1]hept-5-ene iodides (10-13), and the corresponding -bicyclo[2.2.1]heptane iodides (14-17) are reported. None of the compounds demonstrated ganglion-stimulating activity similar to 1 or antagonized the effects of 1.

Beta-lactam analogues of oxotremorine. 3- and 4-methyl-substituted 2-azetidinones.

Four beta-lactam analogues (8-11) of oxotremorine were synthesized and assayed for muscarinic and antimuscarinic activity on the isolated guinea pig ileum. The pharmacological results were compared with those obtained previously with the beta-lactam analogue 7 and the 3-, 4-, and 5-methyl-substituted 2-pyrrolidones 2-6. The new compounds were less potent than the corresponding 2-pyrrolidones, regardless of whether they showed agonist (10 and 11), partial agonist (8), or antagonist properties (9) in the ileum assay. The agonists 10 and 11 were about 200-fold less potent than 7. Compounds 8-11 also were less potent than the similarly substituted 2-pyrrolidones in inhibiting the binding of the muscarinic antagonist (-)-[3H]-N-methylscopolamine in homogenates of the rat cerebral cortex.

Muscarinic activity of the thiolactone, lactam, lactol, and thiolactol analogues of pilocarpine and a hypothetical model for the binding of agonists to the m1 receptor.

Pilocarpine isosteres have been synthesized and characterized with regard to their in vitro muscarinic properties. The results indicate that the carbonyl oxygen of the lactone function of pilocarpine is of primary importance for agonist activity with the ether oxygen being of lesser or secondary importance. An X-ray structure determination for the hydrogen O,O'-ditoluoyltartrate salt of thiolactone pilocarpine isostere 2a has been performed. This compound has an unusual pharmacological profile exhibiting M1-agonist selectivity as well ass presynaptic antagonism. As a result this compound is also viewed as having therapeutic potential for Alzheimer's disease. A model for the binding of pilocarpine and other muscarinic agonists to the third transmembrane helix of the human m1 muscarinic receptor has been developed.

Synthesis and muscarinic activity of quinuclidinyl- and (1-azanorbornyl)pyrazine derivatives.

The synthesis and cortical muscarinic activity of a novel series of pyrazine-based agonists is described. Quinuclidine and azanorbornane derivatives were prepared either by reaction of lithiated pyrazines with azabicyclic ketones, followed by chlorination and reduction, or by reaction of the lithium enolate of the azabicyclic ester with 2-chloropyrazines followed by ester hydrolysis and decarboxylation. Substitution at all three positions of the heteroaromatic ring has been explored. Optimal muscarinic agonist activity was observed for unsubstituted pyrazines in the azanorbornane series. The exo-1-azanorbornane 18a is one of the most efficacious and potent centrally active muscarinic agonists known. Studies on the 3-substituted derivatives have provided evidence of the preferred conformation of these ligands for optimal muscarinic activity. Substitution at C6 gave ligands with increased affinity and reduced efficacy. Moving the position of the diazine ring nitrogens to give pyrimidine and pyridazine derivatives resulted in a significant loss of muscarinic activity.

Tetrahydropyridyloxadiazoles: semirigid muscarinic ligands.

Recent studies have described novel azabicycle-based muscarinic agonists which readily penetrate into the central nervous system and are capable of displaying high efficacy at cortical sites. The current paper describes the synthesis and biochemical assessment of semirigid muscarinic ligands which were used to map the requirements of the cortical muscarinic receptor and to study the degree of conformational flexibility required to cause receptor activation. Analogues 6 and 9 provide high-efficacy muscarinic agonists at cortical sites; however, C-alkylation on the tetrahydropyridine ring resulted in more rigid analogues and showed lower predicted efficacy. Molecular mechanics calculations indicated a preference for the E rotameric form. This conformation was also observed in the X-ray crystal structure of ethenyloxadiazole 12. The new compounds were tested in a biochemical assay designed to measure receptor affinity and to predict cortical efficacy.

Design, synthesis, and neurochemical evaluation of 5-(3-alkyl-1,2,4- oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidines as M1 muscarinic receptor agonists.

A series of 5-(3-alkyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidines+ ++ (7a-h) was synthesized for biological evaluation as selective agonists for M1 receptors coupled to phosphoinositide (PI) metabolism in the central nervous system. Each ligand bound with high affinity to muscarinic receptors from rat brain as measured by inhibition of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) binding. 5-(3-Methyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine+ ++ trifluoroacetate (CDD-0098-J;7a) displayed high affinity (IC50 = 2.7 +/- 0.69 microM) and efficacy at muscarinic receptors coupled to PI metabolism in the rat cortex and hippocampus. Increasing the length of the alkyl substituent increased affinity for muscarinic receptors yet decreased activity in PI turnover assays. The hippocampal PI response of 7a was blocked by lower concentrations of pirenzepine (8) or by higher concentrations of either AF-DX 116 (9) or p-fluorohexahydrosiladifenidol (10), suggesting that at low concentrations 7a selectively stimulates PI turnover through M1 receptors.

Central cholinergic agents. 6. Synthesis and evaluation of 3-[1-(phenylmethyl)-4-piperidinyl]-1-(2,3,4,5-tetrahydro-1H-1-benzazepi n-8-yl)-1-propanones and their analogs as central selective acetylcholinesterase inhibitors.

In an attempt to find central selective acetylcholinesterase (AChE) inhibitors, 3-[1-(phenylmethyl)-4-piperidinyl]-1-(2,3,4,5-tetrahydro-1H-1- benzazepin-8-yl)-1-propanones 9 and their analogs were designed on the basis of our working hypothesis of the enzyme's active site. These compounds were prepared by regioselective Friedel-Crafts acylation of 2,3,4,5-tetrahydro-1H-1-benzazepines and related nitrogen heterocycles as a key step. Most compounds showed potent inhibitory activities with IC50s in the 10-300 nM range. In order to estimate their central selectivities, we examined their effects on the apomorphine-induced circling behavior in rats with unilateral striatal lesions. Among compounds with potent AChE inhibition, 3-[1-(phenylmethyl)-4-piperidinyl]-1-(2,3,4,5-tetrahydro-1H-1- benzazepin-8-yl)-1-propanone fumarate (9a, TAK-147) (IC50 of AChE inhibition = 97.7 nM) inhibited the circling behavior at 3 mg/kg po, in which it had no significant effect on peripheral cholinergic effects. This demonstrates that 9a has favorable central selectivity. Furthermore, 9a significantly ameliorated diazepam-induced passive avoidance deficit at 1 mg/kg po. The benzazepine derivative 9a was selected as a candidate for clinical evaluation.

Synthesis and characterization of all four isomers of the muscarinic agonist 2'-methylspiro[1-azabicyclo[2.2.2]octane-3,4'-[1,3]dioxolane].

The four separated isomers of the muscarinic agonist 1, previously known as AF30, have been synthesized by a route that has allowed the absolute stereochemistry of each isomer to be assigned. With the chirality of (-)-camphanic acid known, X-ray analysis of the more crystalline intermediate diastereomeric camphanate 5A allowed the absolute stereochemistry at the quinuclidine chiral center to be determined. Each diastereomer was separately transformed into the spirodioxolane with concomitant introduction of the second chiral center. Chromatographic separation followed by a second crystal structure determination revealed the absolute stereochemistry of all four isomers of 1. Detailed biological evaluation of each isomer indicated that while the 3(R),2'(S) isomer was the most active in binding studies, it was the 3(R),2'(R) isomer that displayed the largest functional selectivity between ganglion (M-1 site) and heart (M-2 site). With the same internal chiral standard, the absolute configuration of the more active enantiomer of 3 was shown to be S, confirming earlier literature reports.

Muscarinic ganglionic stimulants: conformationally restrained analogues related to [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride.

The synthesis of a series of tertiary and quaternary cyclic analogues (isoarecolinol, dihydroisoarecolinol, arecolinol, and 3-pyrroline-3-carbinol derivatives) of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343) (1), a selective stimulant of muscarinic receptors in sympathetic ganglia (so-called M1 receptors), is reported. The compounds 3-10 were tested for muscarinic ganglion-stimulating activity by recording blood pressure responses in pithed rats. All tertiary compounds tested had no ganglion-stimulating activity. Among the series of quaternary derivatives, only the isoarecolinol analogues 4a and 4b showed considerable ganglion-stimulating effects, whereas the dihydroisoarecolinol (8), the arecolinol (6a, 6b), and the 3-pyrroline-3-carbinol derivatives (10) were much less potent. Our experiments therefore demonstrate that in this series a quaternary nitrogen atom, unsaturation at C2 of the ammonium side chain, and a certain spatial arrangement of the ammonium and the phenylcarbamate groups are essential for potent M1-receptor stimulating activity.

Functionalized congener approach for the design of novel muscarinic agents. Synthesis and pharmacological evaluation of N-methyl-N-[4-(1-pyrrolidinyl)-2-butynyl] amides.

A functionalized congener approach was used to design ligands for muscarinic cholinergic receptors (mAChRs). A series of omega-functionalized alkyl amides of N-methyl-4-(1-pyrrolidinyl)-2-butynamine (22) were prepared as functionalized analogues of UH 5 [N-methyl-N-[4-(1-pyrrolidinyl)-2-butynyl]acetamide], a muscarinic agonist related to oxotremorine. Intermediate 22 was coupled to a series of Boc-protected omega-amino acids, and the resulting amides were deprotected and acylated. Intermediate 22 was also acylated with succinic anhydride and derivatized. The synthetic intermediates and final compounds were evaluated in vitro for their effects on the turnover of phosphatidylinositides in SK-N-SH human neuroblastoma cells that express m3AChRs, and on the production of cyclic AMP in NG108-15 neuroblastoma x glioma cells that express only m4AChRs. The displacement of [3H]-N-methylscopolamine was also measured in membrane preparations from each of these cell lines. Conjugates of glycine and beta-alanine were agonists at m4AChRs, having little or no activity at m3AChRs. The potency in displacement of [3H]-N-methylscopolamine from both m3- and m4AChRs generally increased with increasing chain lengths of the omega-aminoalkyl congeners. The amides of 7-aminoheptanoic acid and 8-aminooctanoic acid, and their Boc-protected derivatives, had comparable affinities to UH 5 (Ki = 5.0 and 4.5 microM at m3AChRs and at m4AChRs, respectively) at both receptors but lacked any agonist effects.

Synthesis and biological activity of 1,2,4-oxadiazole derivatives: highly potent and efficacious agonists for cortical muscarinic receptors.

The synthesis and biochemical evaluation of novel 1,2,4-oxadiazole-based muscarinic agonists which can readily penetrate into the CNS is reported. Efficacy and binding of these compounds are markedly influenced by the structure and physicochemical properties of the cationic head group. In a series of azabicyclic ligands efficacy and affinity are influenced by the size of the surface area presented to the receptor, at the active site, and the degree of conformational flexibility. The exo-1-azanorbornane 16a represents the optimum arrangement, and this compound is one of the most efficacious and potent muscarinic agonists known. In a series of isoquinuclidine based muscarinic agonists efficacy and affinity are influenced by the geometry between the cationic head.group and hydrogen bond acceptor pharmacophore and steric bulk in the vicinity of the base. The anti configuration represented by 22a is optimal for muscarinic activity. Ligands with pKa below 6.5 show poor binding to the muscarinic receptor as exemplified by the diazabicyclic derivative 42.

Alkylating partial muscarinic agonists related to oxotremorine. N-[4-[(2-haloethyl)methylamino]-2-butynyl]-5-methyl-2-pyrrolidones.

N-[4-[(2-Chloroethyl)methylamino]-2-butynyl]-5-methyl-2-pyrrolidone (3) and N-[4-[(2-bromoethyl)methylamino]-2-butynyl]-5-methyl-2- pyrrolidone (4) were synthesized. Compounds 3 and 4 cyclized in neutral aqueous solution to an aziridinium ion (4A). The rate constants for the cyclization of 3 and 4 at 37 degrees C were 0.025 and 0.89 min-1, respectively. The aziridinium ion was equipotent with carbachol as a muscarinic agonist on the isolated guinea pig ileum. It was more potent than the corresponding 2-pyrrolidone derivative (2A) in alkylating muscarinic receptors in homogenates of the rat cerebral cortex. This higher potency was due to greater receptor affinity of 4A as compared to 2A rather than to greater rate constant for alkylation of muscarinic receptors. These properties of 3 and 4 and their low toxicity should make them valuable tools for receptor inactivation studies in vivo and in vitro.

Derivatives of the muscarinic agent N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide.

A series of tertiary and quaternary analogues (acyclic imides, sulfonimides, N-acetyl sulfonamides, and trifluoroacetamides) of the selective partial muscarinic agonist N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide (BM 5,35) was synthesized. The compounds were found to be muscarinic agonists, partial agonists, or antagonists in the isolated guinea pig ileum. Replacement of the acetyl group or the N-methyl group of 35 and its analogues by a methanesulfonyl group abolished efficacy and decreased affinity at ileal muscarinic receptors. Trifluoroacetamide analogues of 35 also had lower affinity and efficacy than 35. Substitution of an acetyl group for the N-methyl group in compounds related to 35 decreased efficacy, but had no appreciable effect on affinity. Most of the tertiary amines showed central antimuscarinic activity as they antagonized oxotremorine-induced tremors in mice.

2-Methyl-1,3-dioxaazaspiro[4.5]decanes as novel muscarinic cholinergic agonists.

Many nonquaternary ammonium muscarinic agonists have been developed over the last few years, but most of the existing compounds (e.g., arecoline, RS-86, AF-30) behave as weak partial agonists at cholinergic receptors in tissues of limited receptor reserve. The current paper describes the synthesis and biochemical assessment of analogues of AF-30 designed to have sufficient conformational freedom to allow greater receptor flexibility and hence activation. The new compounds and important standards were tested in a new biochemical assay designed to measure both receptor affinity and intrinsic activity of each compound and for their ability to stimulate phosphatidylinositol turnover in rat cerebral cortex. Two azaspirodecanes (5a and 5b) were shown to have far greater predicted efficacy than AF-30.

Synthesis and pharmacological investigation of the enantiomers of muscarone and allomuscarone.

A strategy based on the use of (R)- and (S)-lactic ester as starting materials allowed the synthesis of the two enantiomers of muscarone [(-)-1 and (+)-1] and allomuscarone [(-)-5 and (+)-5] in greater than 98% enantiomeric excess. The compounds were examined for their ability to bind to membranes from cerebral cortex (M1), heart (M2), and salivary glands (M3) and to recognize affinity agonist states of the muscarinic receptors. The two pairs of enantiomers were also tested in five functional assays, and their muscarinic potency was determined. In both binding and functional tests, (-)-1 (2S,5S) and (-)-5 (2R,5S) were the eutomers of muscarone and allomuscarone, respectively. The eudismic ratio of muscarone, evaluated in the functional tests, spanned a range of 280-440. These values are substantially different from ones (2.4-10.1) reported in the literature. From a stereochemical point of view, muscarone behaves as muscarine and all other major muscarinic agonists; as a consequence, the hypotheses advanced to account for the anomalies of muscarone no longer have reason to exist.