Synthesis and muscarinic cholinergic receptor affinities of 3-quinuclidinyl alpha-(alkoxyalkyl)-alpha-aryl-alpha-hydroxyacetates.

Seven analogues of 3-quinuclidinyl benzilate (QNB) in which one phenyl ring was replaced by an alkoxyalkyl moiety were synthesized and their affinities for the muscarinic cholinergic receptor determined. An oxygen in the beta-position of the moiety was not well-tolerated. By contrast, an oxygen in the gamma-position did not change the affinity for the muscarinic receptor. However, when a bromine was placed on the remaining phenyl ring, the affinity was significantly reduced in striking contrast to results obtained on halogenation of QNB.

Synthesis and structure-activity relationship of some 5-[[[(dialkylamino)alkyl]-1-piperidinyl]acetyl]-10,11-dihydro-5H- benzo[b,e][1,4]diazepin-11-ones as M2-selective antimuscarinics.

A series of 5-[[[(dialkylamino)alkyl]-1-piperidinyl]acetyl]- 10,11-dihydro-5H-dibenzo[b,e][1,4]-diazepin-11-ones were prepared as potential M2-selective ligands. The compounds were evaluated for their affinity and selectivity for the muscarinic cholinergic receptor. The best M2-selective antimuscarinic agent studied is 5-[[4-[4-diethylamino)butyl]-1- piperidinyl]acetyl]-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-11- one, which is approximately 10 times more potent at M2 receptors than previously known compounds such as 11-[[4-[4-(diethylamino)butyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepin-6-one (AQ-RA 741).

In vitro and in vivo characteristics of [iodine-125] 3-(R)-quinuclidinyl (S)-4-iodobenzilate.

The radioiodinated muscarinic acetylcholine receptor antagonist, [125I] 3-quinuclidinyl 4-iodobenzilate, has two high affinity diastereomeric forms, the (R,R) and (R,S)-isomers. The (R,S)-diastereomer is only threefold lower in affinity than the (R,R)-isomer, but the kinetic properties are considerably different--the dissociation rate constant is 13-fold faster for the (R,S)-isomer and the association rate constant is two to threefold faster. The calculated affinity is therefore only fourfold lower. In vivo, the clearance of (R,S)-4IQNB from receptor-rich tissue is also more rapid than that of the (R,R)-isomer, that is a reflection of the more rapid in vitro kinetic properties since the physicochemical properties and the metabolic clearance of the diastereomers is the same.

In vivo competition studies of Z-(-,-)-[125I]IQNP against 3-quinuclidinyl 2-(5-bromothienyl)-2-thienylglycolate (BrQNT) demonstrating in vivo m2 muscarinic subtype selectivity for BrQNT.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Until recently, emission tomographic study of the loss of m2 receptors in AD has been limited by the absence of available m2-selective radioligands that can penetrate the blood-brain barrier. We now demonstrate the in vivo m2 selectivity of an analog of (R)-QNB, 3-quinuclidinyl 2-(5-bromothienyl)-2-thienylglycolate (BrQNT), by dissection and autoradiographic studies of the in vivo inhibition of radioiodinated Z-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenyl-acetate (Z-(-,-)-[125I]IQNP) binding by unlabeled BrQNT in rat brain. In the absence of BrQNT, Z-(-,-)-[125I]IQNP labels brain regions containing muscarinic receptors, with an enhanced selectivity for the m2 subtype. In the presence of 60-180 nmol of co-injected racemic BrQNT, Z-(-,-)-[125I]IQNP labeling in those brain regions containing predominantly m2 subtype is reduced to background levels, while levels of radioactivity in areas not enriched in the m2 subtype do not significantly decrease. We conclude that BrQNT is m2-selective in vivo, and that [76Br]BrQNT, or a radiofluorinated analog, may be of potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD. In addition, a radioiodinated analog may be of potential use in single photon emission tomographic (SPECT) studies.

A novel m2-selective muscarinic antagonist: binding characteristics and autoradiographic distribution in rat brain.

Although several m2-selective muscarinic antagonists have been described, they are not particularly potent. Thus, the development of potent m2-selective compounds remains an important goal. We now report that a bio-isoster of AQ-RA 741 is both one order of magnitude more potent and slightly more selective than previously described compounds. DIBA, a di-benzo derivative of AQ-RA 741, in which the pyridine of the tricycle is replaced with a benzene ring, had Ki values of 4, 0.3, 11 and 2 nM at m1 through m4 receptors, respectively. These values were determined in competition studies with [3H]N-methylscopolamine ([3H]NMS) in membranes from transfected A9 L cells (m1 and m3), rat heart (m2) and NG108-15 cells (m4). AQ-RA 741 had Ki values of 34, 4, 86 and 15 nM at each of these receptors. The autoradiographic distribution of DIBA binding sites was determined by competition studies of [3H]NMS in rat brain. At low concentration, DIBA reduced [3H]NMS binding most significantly from superior colliculi, thalamus, hypothalamus, pontine nucleus, and interpeduncular nucleus, and not appreciably from caudate nucleus, cerebral cortical regions, or hippocampus, consistent with its binding to m2 receptors. These data indicate that DIBA is the most potent, m2-selective muscarinic antagonist yet described. DIBA should therefore become a useful probe in future studies of muscarinic function.

In vitro and in vivo m2 muscarinic subtype selectivity of some dibenzodiazepinones and pyridobenzodiazepinones.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype receptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD has been limited by the absence of available m2-selective radioligands, which can penetrate the blood-brain barrier. We now report on the in vitro and in vivo m2 muscarinic subtype selectivity of a series of dibenzodiazepinones and pyridobenzodiazepinones determined by competition studies against (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) or [3H]QNB. Of the compounds examined, three of the 5-[[4-[(4-dialkylamino)butyl]-1-piperidinyl]acetyl]-10, 11-dihydro-5-H-dibenzo[b,e][1,4]diazepin-11-ones (including DIBA) and three of the 11-[[4-[4-(dialkylamino)butyl]-1-phenyl]acetyl]-5, 11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepin-6-ones (including PBID) exhibited both high binding affinity for the m2 subtype (/=10). In vivo rat brain dissection studies of the competition of PBID or DIBD against (R,S)[125I]IQNB or [3H]QNB exhibited a dose-dependent preferential decrease in the binding of the radiotracer in brain regions that are enriched in the m2 muscarinic subtype. In vivo rat brain autoradiographic studies of the competition of PBID, BIBN 99, or DIBD against (R,S)[125I]IQNB exhibited an insignificant effect of BIBN 99 and confirmed the effect of PBID and DIBD in decreasing the binding of (R,S)[125I]IQNB in brain regions that are enriched in the m2 muscarinic subtype. We conclude that PBID and DIBD are potentially useful parent compounds from which in vivo m2 selective derivatives may be prepared for potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD.

Specific binding component of the "inactive" stereoisomer (S,S)-[125I] IQNB to rat brain muscarinic receptors in vivo.

In vivo nonspecific binding can be estimated using the inactive stereoisomer of a receptor radioligand. However, the binding of the inactive stereoisomer may be partially specific. Specific binding of the inactive (S,S)-[125I]IQNB was estimated from the inhibition induced by a competing nonradioactive ligand. This technique differed from the usual approach, since it was used to study the inactive rather than the active stereoisomer. The results indicate that there is substantial specific binding for (S,S)-[125I]IQNB.

Muscarinic receptor subtype selectivity of novel heterocyclic QNB analogues.

In an effort at synthesizing centrally-active subtype-selective antimuscarinic agents, we derivatized QNB (quinuclidinyl benzilate), a potent muscarinic antagonist, by replacing one of the phenyl groups with less lipophilic heterocyclic moieties. The displacement of [3H]-N-methyl scopolamine binding by these novel compounds to membranes from cells expressing m1-m4 receptor subtypes was determined. Most of the novel 4-bromo-QNB analogues were potent and slightly selective for m1 receptors. The 2-thienyl derivative was the most potent, exhibiting a 2-fold greater potency than BrQNB at m1 receptors, and a 4-fold greater potency at m2 receptors. This compound was also considerably less lipophilic than BrQNB as determined from its retention time on C18 reverse phase HPLC. This compound may therefore be useful both for pharmacological studies and as a candidate for a radioiodinated SPECT imaging agent for ml muscarinic receptors in human brain.

A novel muscarinic receptor ligand which penetrates the blood brain barrier and displays in vivo selectivity for the m2 subtype.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in the posterior parietal cortex of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. In our efforts to prepare such a radioligand, we have used competition studies against currently existing muscarinic receptor radioligands to infer the in vitro and in vivo properties of a novel muscarinic receptor ligand, 5-[[4-[4-(diisobutylamino)butyl]-1-phenyl]acetyl]-10,11-dihydro-5H - -dibenzo [b,e][1,4]diazepin-11-one (DIBD). In vitro competition studies against [3H](R)-3-quinuclidinylbenzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]NMS), using membranes derived from transfected cells expressing only m1, m2, m3, or m4 receptor subtypes, indicate that DIBD is selective for m2/m4 over m1/m3. In vivo competition studies against (R,R)-[125I]IQNB indicate that DIBD crosses the blood brain barrier (BBB). The relationship of the regional percentage decrease in (R,R)-[125I]IQNB versus the percentage of each of the receptor subtypes indicates that DIBD competes more effectively in those brain regions which are known to be enriched in the m2, relative to the m1, m3, and m4, receptor subtype; however, analysis of the data using a mathematical model shows that caution is required when interpreting the in vivo results. We conclude that a suitably radiolabeled derivative of DIBD may be of potential use in emission tomographic study of changes in m2 receptors in the central nervous system.

Synthesis and antituberculosis activity of thiocarboxamide derivatives of Schiff bases.

Synthesis of some N-(4-thiocarboxamidobenzylidene)arylamines and N-(substituted benzylidene)-p-thiocarboxamidoanilines from various arylaldehydes and arylamines is described. Fourteen representative compounds were tested in vitro on the ground H37RV.

Synthesis and structure-activity relationships of new muscarinic antagonists.

In an attempt to develop more selective muscarinic acetylcholine receptor (m-AcChR) antagonists, (R)-1-azabicyclo[2.2.2]oct-3-yl-thioxanthene-9-carboxylate, (R,S)-thiochromane-4-carboxylate, and (R,S)-chromane-4-carboxylate were synthesized. Evaluation of the binding affinities of these compounds to muscarinic receptors indicates that replacing the oxygen by sulfur in the xanthenyl and chromanyl moieties does not significantly change selectivity, but does reduce the affinity of 5 and enhance the affinity of 9a.

Preparation and properties of (R)-(-)-1-azabicyclo[2.2.2]oct-3-yl- (R)-(+)-alpha-hydroxy-alpha-(4-[125I]iodophenyl)-alpha-phenyl acetate and (R)-(-)-1-azabicyclo[2.2.2]oct-3-yl-(S)-(-)-alpha-hydroxy-alpha- (4-[125I]iodophenyl)-alpha-phenyl acetate as potential radiopharmaceuticals.

rac-4-Nitrobenzilic acid was synthesized and resolved with quinidine and quinine to give the corresponding (R)- and (S)-salts. The resolved diastereomeric salts were converted to (R)- and (S)-4-nitrobenzilic acids and subsequent esterification gave their corresponding ethyl esters. Transesterification with (R)-(-)-3-quinuclidinol afforded (R)-(-)-1-azabicyclo[2.2.2]oct-3-yl-(R)-(+)-alpha-hydroxy-alpha- (4-nitrophenyl)-alpha-phenyl acetate and (R)-(-)-1-azabicyclo[2.2.2]oct-3-yl-(S)-(-)-alpha-hydroxy- alpha-(4-nitrophenyl)-alpha-phenyl acetate. After hydrogenation, the (R,R)- and (R,S)-amines were converted to the respective triazene derivatives. The triazene derivatives reacted with sodium [125I]iodide to give (R)-(-)-1-azabicyclo[2.2.2]oct-3-yl-(R)-(+)- alpha-hydroxy-alpha-(4-[125I]iodophenyl)-alpha-phenyl acetate and (R)-(-)-1-azabicyclo[2.2.2]oct-3-yl-(S)-(-)-alpha-hydroxy- alpha-(4-[125I]iodophenyl)-alpha-phenyl acetate. The evaluation of their affinities to muscarinic acetylcholine receptors (MAcChR) shows that (R)-(-)-1-azabicyclo[2.2.2]oct-3-yl-(S)-(-)-alpha-hydroxy-alpha-(4- [125I]iodophenyl)-alpha-phenyl acetate exhibits an affinity for the MAcChR from corpus striatum that is approximately threefold lower than that of (R)-(-)-1-azabicyclo[2.2.2]oct-3-yl-(R)-(+)-alpha-hydroxy-alpha-(4- [125I]iodophenyl)-alpha-phenyl acetate.

Synthesis and receptor affinities of new 3-quinuclidinyl alpha-heteroaryl-alpha-aryl-alpha-hydroxyacetates.

Five analogues of 3-quinuclidinyl benzilate were prepared in which one phenyl ring was substituted by a heterocycle; a bromine was included on either the remaining phenyl or the heterocycle to provide information relating to the affinity of potential radiohalogenated derivatives. Their affinities for the muscarinic cholinergic receptor were determined. Replacing a phenyl ring with either the 2- or 3-furyl moiety or the 2- or 3-thienyl moiety did not significantly alter the affinity to the muscarinic receptor compared with 3-quinuclidinyl 4-bromobenzilate.

Molecular modeling of the interaction of diagnostic radiopharmaceuticals with receptor proteins: m2 antagonist binding to the muscarinic m2 subtype receptor.

Models of the m2 muscarinic receptor have been built and acetylcholine and an antagonist of the quinuclidinyl benzilate family docked to the putative active site. We have incorporated aspects of homology, site-directed mutagenesis studies and structure-activity studies of specific lead compounds in the construction of our receptor models with a primary focus on the structure of the binding sites. We have observed a deep pocket binding of 5-BrQNT, suggesting a plausible explanation for the observation that agonists and antagonists do not bind competitively. The results of these computational studies are interpreted within the context of the observed in vitro results. Our goal is to assist in the development of subtype receptor selective radiopharmaceuticals for use in PET and SPECT.

Comparison of the in vivo rat brain regional pharmacokinetics of [3H]QNB, (R,S)-[125I]-4IQNB, and (R,R)-[125I]-4IQNB binding to the muscarinic acetylcholine receptor in relationship to the regional subtype composition.

We have used the dissection of selected rat brain regions to compare the in vivo pharmacokinetics of [3H]QNB, (R,S)-[125I]-4IQNB, and (R,R)-[125I]-4IQNB binding to the muscarinic acetylcholine receptor (mAChR). [3H]IQNB is distributed in accordance with the m2 subtype concentration, (R,S)-[125I]-4IQNB is distributed in accordance with the total mAChR concentration, and (R,R)-[125I]-4IQNB is distributed in accordance with the m1/m4 subtype concentration. Although the cerebellum is relatively poor in mAChR (composed almost exclusively of the m2 subtype), the [3H]QNB concentration in the cerebellum is nearly equal to that in the other brain regions and is predominantly composed of specific binding. In contrast, the (R,S)-[125I]-4IQNB and (R,R)-[125I]-4IQNB concentrations in the cerebellum are relatively low and are predominantly or exclusively composed of nonspecific binding. These results dramatically demonstrate the in vivo m2 selectivity of [3H]QNB. All three radioligands exhibit large population standard deviations, with a substantial reduction of the between-animal variability resulting from normalization to each individual animal's corpus striatum value. Thus, the large population standard deviations arise from variability in radioligand delivery (variations in global cerebral blood flow, radioligand binding to serum proteins, loss of parent radioligand through conversion to metabolites, and blood-brain barrier transport.

Autoradiographic evidence that 3-quinuclidinyl-4-fluorobenzilate (FQNB) displays in vivo selectivity for the m2 subtype.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. We now demonstrate the in vivo m2 selectivity of a fluorine derivative of QNB (FQNB), by studying autoradiographically the in vivo inhibition of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) binding by unlabeled FQNB. In the absence of FQNB, (R,S)-[125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 30.0 nmol of racemic FQNB, (R,S)-[125I]IQNB labeling in those brain regions containing predominantly the m2 subtype is reduced to background levels. We conclude that FQNB is m2-selective in vivo and that [18F]FQNB or a closely related analogue may be of potential use in positron emission tomographic study of the loss of m2 receptors in AD.

Autoradiographic evidence that quinuclidinyl 4-(bromophenyl)-2-thienylglycolate (QBPTG) displays in vivo selectivity for the muscarinic m2 subtype.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. We now demonstrate the in vivo m2 selectivity of an analogue of QNB, 4-(bromophenyl)-2-thienylglycolate (QBPTG), by studying autoradiographically the in vivo inhibition of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) binding by unlabeled QBPTG in rat brain. In the absence of QBPTG, (R,S)-[125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 37.5 nmol of racemic QBPTG, (R,S)-[125I]IQNB labeling in those brain regions containing predominantly the m2 subtype is reduced to background levels. We conclude that QBPTG is m2-selective in vivo and that [76Br]QBPTG, or a radiofluorinated analogue, may be of potential use in positron emission tomographic study of the loss of m2 receptors in AD. In addition, a radioiodinated analogue may be of potential use in single photon emission tomographic studies.

Autoradiographic evidence that QNB displays in vivo selectivity for the m2 subtype.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. We have previously reported the results of in vivo dissection studies, using both carrier-free and low specific activity [3H]QNB, which show that [3H]QNB exhibits a substantial in vivo m2 selectivity. Because of the expense of the radioligand and the long exposure time required for the X-ray film, performing a large number of direct in vivo autoradiographic studies using [3H]QNB is precluded. Therefore, we now confirm these results autoradiographically by studying the in vivo inhibition of radio-iodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) binding by unlabeled QNB. In the absence of QNB, (R,S)-[125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 15 nmol QNB, (R,S,)-[125I]IQNB labeling in those brain regions containing predominantly m2 subtype is reduced to background levels. We conclude that QNB is m2-selective in vivo and that a suitably radiolabeled derivative of QNB, possibly labeled with 18F, may be of potential use in positron emission tomographic study of the loss of m2 receptors in AD.