Sterically hindered 5,11-dicarbo analogues of clozapine as potential chiral antipsychotic agents.

Sterically hindered 5,11-dicarbo analogues of clozapine were prepared as potential chiral antipsychotic agents, with the possibility that for a particular analogue the antipsychotic activity of clozapine may reside in one enantiomer of the analogue whereas other unwanted biological effects of clozapine may be caused by the other enantiomer. Variable-temperature proton nuclear magnetic resonance studies showed that although 5-methylene-10-(4-methylpiperazino)-5H-dibenzo[a,d]cycloheptene exists at room temperature as configurational enantiomers, the activation energy for thermal racemization is 19 kcal mol-1 at 105 degrees C, and it is doubtful that the enantiomers of this analogue can be isolated under usual laboratory conditions. The (Z)-5-ethylidene and 5-isopropylidene analogues have activation energies greater than 23 kcal mol-1 at 160 degrees C, and thus there is a possibility that the analogues can be obtained as their respective enantiomers. 5-Methyl-10-(4-methylpiperazino)-5H-dibenzo[a,d]cycloheptene incorporates a chiral center which is not thermally racemized, but it exists at room temperature as two diasteromers with an activation energy for inversion of the 5H-dibenzo[a,d]cycloheptene ring of 21 kcal mol-1. When the 5,11-dicarbo analogues were tested in vitro for biological activity and their activities were compared to that of clozapine, the affinities for muscarinic and dopamine D-1 and D-2 sites were reduced but were still substantial. Thus the respective biological activities of the racemates indicate that the biological activities of the thermally stable enantiomers may be of importance in finding a clozapine derivative with fewer side effects than those shown by clozapine itself. Because of the susceptibility of the enamines to acid-catalyzed hydrolysis, resolution into respective enantiomers is not anticipated.

Chloro-substituted, sterically hindered 5,11-dicarbo analogues of clozapine as potential chiral antipsychotic agents.

Variable-temperature proton nuclear magnetic resonance studies have shown that 5-(2-propylidene)-10-(4-methylpiperazino)-5H-dibenzo[a,d] cycloheptene, a 5,11-dicarbo analogue of the atypical neuroleptic agent clozapine [8-chloro-11-(4-methylpiperazino)-5H-dibenzo[b,e][1,4]diazepine], exists as thermally stable configurational isomers. The presence of the 2-propylidene group at C-5 on the 5H-dibenzo[a,d]cycloheptene moiety did not interfere greatly, as compared to clozapine, with the in vitro affinity of this 5,11-dicarbo analogue of clozapine for muscarinic and dopamine D-1 and D-2 binding sites in rat brain. Since the presence and position of a chloro substituent on the 5H-dibenzo[b,e][1,4]diazepine moiety have a marked influence on the respective binding affinities of 1,4-diazepines related to clozapine, chloro-substituted 5,11-dicarbo analogues of clozapine were prepared in order to further examine structure-activity relationships. Evaluation of these analogues for binding to muscarinic and dopamine binding sites in comparison with clozapine and other 5H-dibenzo[b,e][1,4]diazepine analogues of clozapine shows that the dopamine D-1 and D-2 receptor affinities of both the 5-(2-propylidene)-5,11-dicarbo analogue and its corresponding distal-chloro derivative, 2-chloro-5-(2-propylidene)-10-(4-methylpiperazino)-5H- dibenzo[a,d]cycloheptene, are retained. Because of the susceptibility to acid-catalyzed hydrolysis of these tertiary enamines, however, these compounds serve only as model compounds for their structure-activity evaluation. Since the proximal nitrogen atom of the piperazine ring is redundant for biological activity, 5-(2-propylidene)-10-(1-methyl-4-pyridyl)-5H-dibenzo[a,d]cycloh eptene and its 2-chloro derivative are excellent candidates for resolution into enantiomers as a means to separate antimuscarinic and antidopaminergic activity, respectively, associated with only a single stereoisomer.

(S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-[125I]iodo- 2-methoxybenzamide hydrochloride, a new selective radioligand for dopamine D-2 receptors.

From salicyclic acid, the two enantiomers of N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2-methoxybenzamide (6b) were prepared in a five-step synthesis. With use of Heindel's triazene method for introduction of the radionuclide, the iodine-125-labeled substituted benzamide was obtained with a calculated specific activity of 136 Ci/mmol and 14% radiochemical yield. For the preparation of the iodine-125-labeled benzamide with higher specific activity, this method was unsuccessful and utilization of the corresponding tri-n-butyltin derivative was required. Treatment of the latter in dilute hydrochloric acid with sodium iodide-125 and chloramine-T gave [125I](S)-6b in 56% radiochemical yield and at least 97% radiochemical purity. The displacement of [125I](S)-6b and [3H](S)-sulpiride from their respective binding sites in striatal rat brain homogenates using various neuroleptic agents showed that (S)-6b has the same binding profile but more potent binding for dopamine D-2 receptors than has sulpiride. These experiments also indicate that the S enantiomer of 6b is a specific ligand (KD = 1.2 nM) for the D-2 receptor. Further, the octanol-water partition coefficient of (S)-6b as determined by reverse-phase high-performance liquid chromatography was found to be 40 times greater than that for sulpiride. Thus (S)-6b has a lipophilicity that will allow a relatively higher uptake into the brain compared to sulpiride. In vivo experiments with rats show that [125I](S)-6b penetrates readily into the brain and is preferentially localized in the striatum as compared to the cerebellum, the ratio of uptake being 7.2 to 1, 60 min after injection. These observations of good brain penetration and high affinity and selectivity for D-2 receptors indicate that the corresponding iodine-123-labeled benzamide may be a useful ligand for the noninvasive visualization study of dopamine D-2 receptor sites in vivo by single photon emission computed tomography.

Potential antipsychotic agents 5. Synthesis and antidopaminergic properties of substituted 5,6-dimethoxysalicylamides and related compounds.

A series of 3-substituted 5,6-dimethoxysalicylamides III (9-13 and 15) has been synthesized from the corresponding 2,5,6-trimethoxybenzoic acids. Relaxation times T1 and carbon chemical shifts of the methoxy groups in III showed that the 6-methoxy group adopts a nearly perpendicular orientation and the 5-methoxy group takes on a more coplanar orientation with respect to the ring plane in solution. The salicylamides III display a very high and stereoselective affinity for the [3H]spiperone and [3H]raclopride binding sites in vitro. Regioisomeric salicylamides IV also exhibit pronounced, but lower than III, affinity for the [3H]spiperone binding site. The structural requirements were further assessed by studies of the related amino analogues 23 and 24 and hydroxy analogue 27. The 3-bromo compound 11 (FLB 463) was studied in various in vivo models and compared with the dopamine-D2 antagonists sulpiride, raclopride, eticlopride, and haloperidol. The high potency of 11 to selectively block dopamine-D2 receptors in vitro and in vivo combined with indications on a low potential for motor side effects makes it a very interesting new member of the class of substituted salicylamides.

Potential antipsychotic agents. 7. Synthesis and antidopaminergic properties of the atypical highly potent (S)-5-bromo-2,3-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]benzamide and related compounds. A comparative study.

(S)-5-Bromo-2,3-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]benzamide (6) and some related compounds, i.e. the R isomer 7, the 3-hydroxy analogue 8, the desbromo derivative 9, the monomethoxy compound 10, and the 2,4-dimethoxy analogue 11, have been synthesized from the corresponding benzoic acids. The benzamides, lacking o-hydroxy groups, were evaluated for their affinity for the [3H]spiperone binding site and for their inhibition of apomorphine-induced behavioral responses in relation to the effect of the corresponding salicylamides. Besides the 2-hydroxy-3-methoxybenzamide 12 and the related 1,4-benzodioxane (13) and 2,3-dihydrobenzofuran (14), carboxamides were investigated in order to evaluate the stereoelectronic requirements on the 2-methoxy group for the receptor interaction. The study supports the view that the o-methoxy group may adopt coplanar, as well as perpendicular orientations, and maintain the intramolecular hydrogen bonding required in the bioactive conformation. The benzamide 6 was found to be equipotent with the analogous highly active salicylamide 3 (FLB 463) both in vitro and in vivo. In addition, 6 displayed a preferential inhibition of the hyperactivity component of the behavioral syndrome, which is regarded to indicate a low tendency to induce extrapyramidal side effects in man at antipsychotically effective doses. The benzamide class of compounds (6-10) were found to be somewhat more sensitive to the structural modifications than the salicylamide class, i.e. the o-hydroxy-substituted benzamides (2-5). The potent and selective benzamide 6 (FLB 457) is highly suitable for investigations of dopamine D-2 mediated responses and, in radiolabeled form, for receptor binding studies in vitro and in vivo.

Potential antipsychotic agents. 9. Synthesis and stereoselective dopamine D-2 receptor blockade of a potent class of substituted (R)-N-[(1-benzyl-2-pyrrolidinyl)methyl]benzamides. Relations to other side chain congeners.

A number of substituted N-[(1-benzyl-2-pyrrolidinyl)methyl]benzamides and -salicylamides have been prepared and investigated as dopamine D-2 receptor antagonists in vitro and in vivo. The affinity was found to be confined to the R enantiomer, in contrast to the corresponding N-ethyl or N-allyl derivatives. The X-ray structure of one of the compounds (15) confirmed the R stereochemistry. This compound (15) was found to adopt a solid-state conformation in which the 4-fluorobenzyl group is folded over the salicylamide moiety. Benzamides having a 2,3-dimethoxy substitution pattern (24 and 26) or salicylamides with a 5,6-dimethoxy grouping (21 and 22) were especially potent, in that they inhibited [3H]spiperone binding to rat striatal dopamine D-2 receptors in vitro with IC50 values of about 1 nM. The new compounds' ability to block apomorphine-induced stereotypies correlated with the affinity for the [3H]spiperone binding site. Higher dose levels were necessary to induce catalepsy than to block the apomorphine-induced responses. The influence of the aromatic substituents on the potency of substituted benzamides with three types of side chains, i.e. (R)-(1-benzyl-2-pyrrolidinyl)methyl, (S)-(1-ethyl-2-pyrrolidinyl)methyl and 1-benzyl-4-piperidinyl, was compared. The 3-bromo-5,6-dimethoxysalicylamide substitution pattern was found to be the most general since it gave very potent compounds in all series. The substituted (R)-N-[(1-(4-fluoro-benzyl)-2-pyrrolidinyl)methyl]benzamides (26) and -salicylamides (22) are suitable for development into 18F radioligands without altering the parent structure.

Potential neuroleptic agents. 3. Chemistry and antidopaminergic properties of substituted 6-methoxysalicylamides.

A series of substituted 6-methoxysalicylamides were synthesized from their corresponding 2,6-dimethoxybenzamides by demethylation of one methoxy group with boron tribromide. Substituted 6-methoxysalicylamides having a lipophilic aromatic substituent in the 3-position para with respect to the methoxy group, e.g. a bromo or an iodo atom or an ethyl or a propyl group, and having an (S)-N-(1-alkyl-2-pyrrolidinyl)methyl moiety as the side chain were found to be potent blockers of [3H]spiperone binding in vitro and potent inhibitors of the apomorphine syndrome in the rat. Similar to remoxipride but in contrast to haloperidol, some of the substituted salicylamides show a 10-20-fold separation between the dose that inhibits hyperactivity and that which inhibits stereotypy. It was concluded that, besides the requirement of a lipophilic substituent in the position para to the methoxy group for antidopamine activity in vivo, the formation of a coplanar six-membered pseudoring involving the amide moiety and the methoxy group is a structural requirement for activity in vitro.

Binding of 5H-dibenzo[b,e][1,4]diazepine and chiral 5H-dibenzo[a,d]cycloheptene analogues of clozapine to dopamine and serotonin receptors.

5H-Dibenzo[b,e][1,4]diazepine, dibenz[b,f]oxepin, and 5H-dibenzo[a,d]cycloheptene analogues of clozapine [8-chloro-11-(4-methylpiperazino)-5H- dibenzo[b,e][1,4]diazepine] were evaluated for their binding affinity to dopamine D-1, D-2, and D-4 and serotonin S-2A (5-HT2A), S-2C (5-HT2C) and S-3 (5-HT3) receptors. The diazepine analogues display selective binding to the dopamine D-4 and serotonin S-2A receptors similar to that of clozapine, but none has a dopamine D-4 selectivity (Ki for the dopamine D-2A receptor/Ki for the dopamine D-4 receptor) greater than that of clozapine. All of the oxepin analogues also show substantial binding to the dopamine D-4 and serotonin S-2A receptors with 10-(4-methylpiperazino)dibenz[b,f]oxepin having a dopamine D-4 selectivity greater than that of clozapine. Some of the 5H-dibenzo-[a,d]cycloheptene analogues also show strong binding to both the dopamine D-4 and serotonin S-2A receptors, 5-methyl-10-(4-methylpiperazino)-5H-dibenzo[a,d]cycloheptene having a dopamine D-4 selectivity of 7.8 as compared to 10 for clozapine but a serotonin S-2A selectivity (Ki for the dopamine D-2 receptor/Ki for the serotonin S-2A receptor) of 2.0 as compared to 28 for clozapine. The serotonin S-2A selectivity of 2-chloro-10-(4-methylpiperazino)-5H-dibenzo[a,d]-cycloheptene++ + is 200. As an extension of these studies, chiral 5-substitute 10-(1,2,3,6-tetrahydro-1-methyl-4-pyridinyl)-5H-dibenzo[a,d]cyclohept ene analogues show a substantial enantiospecificity toward dopamine and serotonin receptor subtypes, (R)-(-)-5-methyl compound having a 2-fold higher dopamine D-4 selectivity than its (S)-(+) enantiomer as the result of enhanced binding to the dopamine D-4 receptor rather than diminished binding to the dopamine D-2 receptor. (pRa,pSb)-(+)-5-(2-Propylidene)-10-(1,2,3,6-tetrahydro-1-met hyl- 4-pyridinyl)-5H-dibenzo[a,d]cycloheptene is 17 times more active in binding to the dopamine D-4 receptor than is its pSa,pRb enantiomer while being only 1.5 times more active in binding to the dopamine D-2 receptor.

Potential neuroleptic agents. 4. Chemistry, behavioral pharmacology, and inhibition of [3H]spiperone binding of 3,5-disubstituted N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-methoxysalicylamides.

A series of 3,5-disubstituted N-[(1-ethyl-2-pyrrolidinyl) methyl]-6-methoxysalicylamides was synthesized, starting from the 2,6-dimethoxybenzoic acids, by boron tribromide demethylation of the corresponding 3,5-disubstituted 2,6-dimethoxybenzamides and separation of the two positional isomers. The correct structure assignments were based on selective decoupling studies on their 13C NMR spectra. The salicylamide derivatives were tested for antidopamine activity in vivo by their ability to inhibit the apomorphine syndrome in the rat and in vitro by their ability to displace [3H]spiperone from striatal preparations of the rat brain. The activity seems to reside exclusively in the S enantiomer. Several compounds were considerably more potent than haloperidol, particularly those having an ethyl group in the 3-position and a halogen atom in the 5-position of the aromatic ring. The corresponding 5-alkyl-3-halogen-substituted compounds were much less active. A low acute toxicity was found for the most potent compounds. Some of the salicylamides displayed a 10-20-fold separation between the dose which blocks apomorphine-induced hyperactivity and that which blocks apomorphine-induced stereotypy. One compound, S-(-)-3,5-dichloro-N-[(1-ethyl-2-pyrrolidinyl) methyl]-6-methoxysalicylamide (raclopride, FLA 870) (13) had a stereotypy--hyperactivity separation more than twice that of sulpiride while being 100 times more potent in blocking the apomorphine effects. On this basis, 13 was selected for clinical trials against schizophrenia.

Synthesis of clozapine analogues and their affinity for clozapine and spiroperidol binding sites in rat brain.

Analogues of clozapine, some prepared by a novel, shorter synthesis than those described previously, were evaluated as potential antipsychotic agents using clozapine binding sites in rat forebrain that are nonmuscarinic and nondopaminergic in nature and from which [3H]clozapine is displaced by known antipsychotic agents. The binding of clozapine to muscarinic sites is inhibited in the presence of atropine. Displacement of [3H]clozapine by an analogue of clozapine in the presence of atropine represents nonmuscarinic binding, while displacement in the absence of atropine represents muscarinic (cholinergic) plus nonmuscarinic binding. The relative affinity of the analogues for dopamine binding sites was determined by their ability to displace [3H]spiroperidol from binding sites in rat caudate nuclei. To the extent which binding affinity for nonmuscarinic clozapine sites in rat forebrain reflects the antipsychotic potential of a particular drug, dibenzo-5H-cycloheptene analogues of clozapine are as effective as clozapine itself. Strong binding to nonmuscarinic clozapine sites is not dependent on the presence of a chlorine atom on th tricyclic system. One or both of the nitrogen atoms in the dibenzo-5H-[1,4]diazepine ring of clozapine appear to be necessary for the strong inhibition of clozapine binding to spiroperidol sites in rat caudate nuclei. Anticholinergic activity is substantially higher for clozapine and its dibenz[1,4]oxazepine analogue than for its benzo-5H-cycloheptene analogue.

Binding of 5H-dibenzo[a,d]cycloheptene and dibenz[b,f]oxepin analogues of clozapine to dopamine and serotonin receptors.

Series of 5,11-dicarbo- and 11-carbo-5-oxy-10-(1-alkyl-1,2,3,6-tetrahydro-4 pyridinyl) analogues and a 11-carbo-5-oxy-10-(1-methyl-4-piperidinyl) analogue of the atypical antipsychotic agent clozapine were prepared and tested for binding to the dopamine D-2L and D-4 and serotonin S-2A and S-2C receptors. Some of these analogues were found to have dopamine D-2L and D-4 and serotonin S-2A and S-2C receptor binding activities as high as or higher than those of clozapine, indicating that neither the diazepine structure nor the piperazine ring present in clozapine is essential for high antidopamine activity and or for high dopamine D-4 selectivity (Ki for the dopamine D-2L receptor/Ki for the dopamine D-4 receptor). Increasing in the effective size of the alkyl substituent at the tertiary amine nitrogen atom in the 1,2,3,6-tetrahydro-4-pyridinyl moiety in the 5H-dibenzo[a,d]cycloheptene series reduces the affinity for the dopamine D-4 receptor, but in the dibenz[b,f]oxepin series, no significant change in binding affinity to the dopamine D-4 receptor was observed. Equal or slightly higher affinity for the serotonin S-2A and S-2C receptors was observed for the 10-(1-ethyl-1,2,3,6-tetrahydro-4- pyridinyl) analogues in both series, but for the 10-[1,2,3,6-tetrahydro-1-(2-propenyl)-4- pyridinyl] analogues, any favourable steric factor is overshadowed by an unfavorable electronic effect as a result of change in the basicity of the tertiary amino group in the pyridinyl moiety. Replacement of three of the four nitrogen atoms in clozapine with three carbon or two carbon atoms and an oxygen atom and removal of the chlorine atoms gives 10-(1,2,3,6-tetrahydro-1- methyl-4-pyridinyl)dibenzo[a,d]cycloheptene and 10-(1-methyl-4-piperidinyl)dibenz[b,f]oxepin, each having twice the binding activity to the dopamine D-4 receptor as does clozapine and a dopamine D-4 selectivity equal to that of clozapine.

High affinity dopamine D2 receptor radioligands. 1. Regional rat brain distribution of iodinated benzamides.

Five 125I-labeled substituted benzamides, which are close structural analogues of (S)-sulpiride, eticlopride, and isoremoxipride, were evaluated for their selective in vivo uptake into dopamine D2 receptor rich tissue of the rat brain. "Iodopride" (KD 0.88 nM), an iodine substituted benzamide structurally related to sulpiride, displayed a maximal striatum: cerebellar uptake ratio of 7.6. Demonstration of saturation of the receptor with [125I]iodopride in striatum required uptake in frontal cortex to be used, rather than cerebellar uptake, to define nonspecific binding. Two other ligands structurally related to eticlopride, "iclopride" (KD 0.23 nM) and "itopride" (KD 0.16 nM), displayed maximal striatal: cerebellar uptake ratios of 9.8 and 3.3, respectively. The most potent ligands, "epidepride" (KD 0.057 nM) and "ioxipride" (KD 0.070 nM) showed striatal:cerebellar uptake ratios of 234 and 65, respectively. The observed uptake ratios correlated poorly with the affinity constants for the dopamine D2 receptor alone, but were highly correlated (r = 0.92) with the product of the receptor dissociation constant (KD) and the apparent lipophilicity (kw), as determined by reverse-phase HPLC at pH 7.5. Total striatal uptake also appeared dependent on lipophilicity, with maximal uptake occurring for ligands having log kw 2.4-2.8.

Dosimetry of iodine-123-epidepride: a dopamine D2 receptor ligand.

UNLABELLED: Substituted benzamides have been shown to have very high affinity and specificity for the dopamine D2 receptor. One of these is radiolabeled epidepride, an iodine-substituted benzamide currently under evaluation as a SPECT imaging agent. Detailed estimates of the radiation absorbed dose to 26 organs and the whole body from [123I]epidepride have been calculated. METHODS: The dosimetry calculations use a combination of in vivo uptake and biodistribution data from one rhesus monkey and seven humans to estimate residence times in eight organs. The computer program MIRDOSE2 was used to calculate the dosimetry. RESULTS: Results indicate that 75% of the radioactivity is cleared through the urinary tract while the remaining radioactivity clears through the gallbladder and intestinal tract. The radiation absorbed dose can be minimized by administering a high lipid content meal 1.5 hr postinjection to empty the gallbladder and by giving large volumes of fluids throughout the study to induce increased urinary output. CONCLUSION: By emptying the gallbladder and urinary bladder, the lower large intestine becomes the critical organ, 0.102 mGy/MBq (0.38 rad/mCi) followed by the upper large intestine, 0.092 mGy/MBq (0.34 rad/mCi). The effective dose equivalent is 0.025 mSv/MBq (0.092 rem/mCi).

M-100907 (Aventis).

M-100907 is a highly selective 5-HT2A antagonist that is being developed by Aventis Pharmaceuticals, formerly Hoechst Marion Roussel (HMR), for the potential treatment of schizophrenia. M-100907 is in phase III trials for chronic schizophrenia [307936], [307942], [307940]. In August 1999, development was discontinued for acute schizophrenia (schizoaffective disorder) on the basis of poor results [335083]. M-100907 is a potent antagonist in every putative animal behavioral model of schizophrenia that involves activation of 5-HT2A receptors [181713]. Interestingly, M-100907 is also active in animal models involving blockade of NMDA glutamatergic channel receptors, an effect known to resemble some behavioral symptoms of schizophrenia in man [390328]. M-100907 belongs to a series of piperidine derivatives, which were originally disclosed in the associated patent, EP-00208235. M-100907 is specifically claimed in a later patent, EP-00531410. This patent describes superior in vivo potency for M-100907 and its claims include the use of M-100907 for the treatment of thromboembolic disorders. The use of M-100907 for the treatment of various developmental neurological disorders such as autism and attention deficit hyperactivity disorder is disclosed in WO-09956750. In 1996, this product was designated one of HMR's nine top priority products, serving an unmet medical need and addressing a potential market in excess of US $500 million per year [221118]. In January 1999, BT Alex Brown predicted sales of US $30 million in 2000 rising to US $220 million in 2002 [318220]. In April 1999, ABN Amro predicted annual sales of DM 50 million in 2000, rising to DM 150 million in 2002 [328676].

Pharmacological characterization of morphine-6-sulfate and codeine-6-sulfate.

Morphine-6-sulfate (M6S) and codeine-6-sulfate (C6S) are mu-selective opiates which have been isolated from brain. M6S is an effective analgesic, with a 30-fold greater potency than morphine in the mouse radiant heat tailflick assay and similar to the active morphine metabolite morphine-6beta-glucuronide (M6G). M6S analgesia is reversed by 3-methoxynaltrexone at low antagonist doses which are inactive against morphine, suggesting that M6S may be acting through the same mechanisms as M6G. Consistent with this possibility, antisense mapping of the MOR-1 clone revealed that M6S analgesia was lowered by probes targeting exon 2 and not by targeting exon 1, a sensitivity profile similar to that of M6G and not morphine. C6S also has analgesic activity at doses approximately 10-fold greater than M6S. However, its characterization was impeded by the appearance of seizures at doses below full analgesic activity. Thus, M6S is a potent analgesic with pharmacological properties similar to M6G. C6S has limited utility due to its high level of toxicity.

Identification of extrastriatal dopamine D2 receptors in post mortem human brain with [125I]epidepride.

The regional distribution of striatal and extrastriatal dopamine D2 receptors in human brain was studied in vitro with (S)-N-[(1-ethyl-2- pyrrolidinyl)methyl]-5-[125I]iodo-2,3-dimethoxybenzamide, [125I]epidepride, using post mortem brain specimens from six subjects. Scatchard analysis of the saturation equilibrium binding in twenty-three regions of post mortem brain revealed highest levels of binding in the caudate (16.5 pmol/g tissue) and putamen (16.6 pmol/g tissue) with lower levels seen in the globus pallidus (7.0 pmol/g tissue), nucleus accumbens (7.2 pmol/g tissue), hypothalamus (1.8 pmol/g tissue), pituitary (1.3 pmol/g tissue), substantia innominata (1.0 pmol/g tissue), and amygdala (0.87 pmol/g tissue). Of note was the presence of dopamine D2 receptors in the four thalamic nuclei studied, i.e. anterior nucleus (1.0 pmol/g tissue), dorsomedial nucleus (0.96 pmol/g tissue), ventral nuclei (0.72 pmol/g tissue), and pulvinar (0.86 pmol/g tissue), at levels comparable to the amygdala (0.87 pmol/g tissue) and considerably higher than levels seen in anterior cingulate (0.26 pmol/g tissue) or anterior hippocampus (0.36 pmol/g tissue). The frontal cortex had very low levels of dopamine D2 receptors (0.17-0.20 pmol/g tissue) while the inferior and medial temporal cortex had relatively higher levels (0.31-0.46 pmol/g tissue). Inhibition of [125I]epidepride binding by a variety of neurotransmitter ligands to striatal, ventral thalamic and inferior temporal cortical homogenates demonstrated that [125I]epidepride binding was potently inhibited only by dopamine D2 ligands. The present study demonstrates that dopamine D2 receptors are present in basal ganglia, many limbic regions, cortex and in the thalamus. The density of thalamic D2 receptors is comparable to many limbic regions and is considerably higher than in cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Visualization of extrastriatal dopamine D2 receptors in the human brain.

[123I]Epidepride, a potent and selective dopamine D2 radioligand, was administered to a 27 year old normal male volunteer. Single photon tomography revealed that peak striatal uptake occurred at 4 h after injection with a striatal:cerebellar ratio of 7.8 rising to over 100 at 18 h post injection. Uptake above the levels seen in cerebellum was also noted in the thalamus, pituitary, hypothalamus and temporal lobe, particularly medially. Single photon tomography with [123I]epidepride allows visualization of extrastriatal dopamine D2 receptors in man.

Characterization of desamino-5-[125I]iodo-3-methoxy-zacopride ([125I]MIZAC) binding to 5-HT3 receptors in the rat brain.

1. Antagonists at 5-HT3 receptors have shown activity in animal models of mental illness, however, few radiolabeled 5-HT3 ligands are available for preclinical studies. MIZAC, an analogue of the selective 5-HT3 antagonist, zacopride, binds with high affinity (1.3-1.5 nM) to CNS 5-HT3 sites. The authors report here the selectivity of MIZAC for these sites in rat brain homogenates. 2. Ninety-seven percent of total specific binding of [125I]MIZAC (0.1 nM) of was displaced by bemesetron (3 microM), a selective 5-HT3 antagonist. Competition studies using ligands with known affinities for 5-HT3 sites give a high correlation with reported pKi values (r2 0.98). Bemesetron displaceable binding has a regional distribution consistent with that of the 5-HT3 receptor, i.e. highest in cortex and hippocampus, and lowest in striatum and cerebellum. 3. Potent antagonists present at concentrations sufficient to occupy 95% of other 5-HT receptor populations (1A, 1B, 1D, 2A, 2B, 2C, 5A, 5B, 6, and 7) showed minimal ability to displace [125I]MIZAC binding (3 nM). Specificity studies using radioligand binding assays selective for 5-HT4, 5-HT6, and 5-HT7 receptors, and for binding sites of other neurotransmitters indicate a high degree of selectivity of [125I]MIZAC for the 5-HT3 receptor. 4. [125I]MIZAC binds to an apparent low affinity (benzac) site having a unique pharmacology. Low affinity binding was displaceable by benztropine, but not by other muscarinic agents nor inhibitors of dopamine uptake. The regional distribution of the low affinity site differed markedly from that of the high affinity site. The apparent affinity of [125I]MIZAC for the benzac site is two orders of magnitude lower than for the 5-HT3 receptor. Given its high selectivity for 5-HT3 binding sites, [125I]MIZAC appears to be a promising ligand for labeling 5-HT3 receptors in vitro and in vivo.

Synthesis and 5-HT-3 receptor binding of 2- and 3-(halo)alkoxyl substituted (S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chlorobenzamides as potential radioligands.

In an effort to develop selective, high-affinity radioligands for the 5-HT-3 receptor, a series of homologues of 5-chloro-2,3-dimethoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide (2b) was prepared in which individual methoxy groups were replaced by ethoxyl, (2-fluoroethoxyl), allyloxyl, propargyloxyl, or (3-iodoallyl)oxyl groups. Affinities for the 5-HT-3 receptor were determined by displacement of the binding of [125I]MIZAC (2a), a selective 5-HT-3 receptor antagonist radioligand, in rat brain homogenates. The 3-substituted homologues were more potent than the lead compound, 2b. The homologue having the largest 3-substituent, i.e., E-(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chloro-3-(3-iodo-2-propenyl)oxy- 2-methoxybenzamide (3b, THIZAC), had one of the highest affinities, Ki 0.08 nM. The 2-substituted homologues were equipotent with 2b, having Ki 0.2-0.3 nM, regardless of the size of the substituent. The corresponding iodoallyl derivative, E-(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chloro-2-(3-iodo-2-propenyl)oxy- 3-methoxybenzamide (4, LIZAC), displayed a Ki of 0.29 nM. Saturation binding of [125I]-4 gave a KD of 0.31 +/- 0.04 nM and a Bmax of 2.36 +/- 0.10 fmol/mg of entorhinal cortex. In vivo biodistribution of [125I]-4 in the rat brain showed increased accumulation in hippocampus relative to that in cerebellum. Both the high-affinity ligands [125I]-3b and [125I]-4 are potentially useful radioligands for studying the 5-HT-3 receptor.

High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.