Victor I. Cohen

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.

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

Alzheimers 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.

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 α-hydroxy-α-(1-iodo-1-propen-3-yl)-α-phenylacetate (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.

Autoradiographic Evidence That QNB Displays in Vivo Selectivity for the m2 Subtype

Alzheimers 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.

Novel potent and m2-selective antimuscarinic compounds which penetrate the blood-brain barrier

Summary A series of 5-[[[(dialkylamino)alkyl]-1-phenyl]acetyl]-10,11-dihydro-5 H -dibenzo[ b,e ][1,4]diazepin-11-ones 1 were prepared as potential m 2 -selective ligands. The binding affinities and selectivities of these compounds for the muscarinic cholinergic receptor subtypes were determined. The best m 2 -selective antimuscarinic agent studied was 5-[[4-[4-(diisobutylamino)butyl]-1-phenyl] acetyl]-10,11-dihydro-5 H -dibenzo[ b,e ][1,4]diazepin-11-one 1h (DIBD), which caused a significant reduction in (R,R)-3-quinuclidinyl-[ 125 I]-4-iodobenzilate ((R,R)-[ 125 I]-4IQNB) binding in brain regions known to contain a high percentage of m 2 -receptors. Thus DIED penetrates the blood-brain barrier and exhibits in vivo selectivity for the m 2 subtype. In contrast, neither DIBA, AF-DX 116, nor AQ-RA 741 caused a significant m 2 -selective reduction in (R,R)-[ 125 I]-4IQNB binding in the brain regions studied.

Autoradiographic Evidence That 3-Quinuclidinyl-4-fluorobenzilate (FQNB) Displaysin VivoSelectivity for the m2 Subtype

Alzheimers 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.

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 new synthesis of 3,6‐dialkyl, diaryl and diheterocyclic‐1,2‐dihydro‐l, 2,4,5‐tetrazines

Some 3,6-dialkyl, diaryl and diheterocyclic-1,2-dihydro-1,2,4,5-tetrazines have been synthesized from the action of hydrazine on aliphatic selenonesters, and aromatic and heterocyclic selenoamides.

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

Alzheimers 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 (</=5 nM) and high m2/m1 selectivity (>/=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.

Autoradiographic Evidence That Quinuclidinyl 4-(Bromophenyl)-2-thienylglycolate (QBPTG) Displays in Vivo Selectivity for the Muscarinic m2 Subtype

Alzheimers 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.