Herein described are radiolabeled compounds and their precursors useful to image D4 receptors in vivo, of the formula: ##STR1## wherein R is selected from iodo, tri(loweralkyl)tin and a radioisotopically labeled iodide and R.sup.1 is selected from H and alkoxycarbonyl, with the proviso that R is not iodo when R.sup.1 is H. The radiopharmaceutical compounds are useful particularly to image localization of D4 receptor in the human brain, and can therefore aid in the diagnosis of schizophrenia and other medical conditions in which the D4 receptor is implicated.

This invention is in the field of medical diagnostics, and relates to 
radiolabeled compounds useful to image D4 receptor loci in brain tissue. 
BACKGROUND TO THE INVENTION 
Neuronal cell receptors that bind the neurotransmitter dopamine constitute 
a group of at least five structurally distinct proteins that can now be 
produced using recombinant DNA techniques. These techniques have been 
applied to construct cell lines that incorporate the dopamine receptor in 
their membranes, to provide regenerable and homogeneous substrates with 
which chemical libraries can be screened to identify potential CNS-active 
drugs. 
Recent evidence strongly implicates the dopamine receptor classified as D4 
in the etiology of schizophrenia. It has been suggested that compounds 
capable of interfering with the function of this receptor, which is 
present in schizophrenics at levels that are six times normal, would be 
useful in the treatment of this disease (Seeman et at, Nature, 1993, 
365:441). Therefore, it would be desirable to provide compounds that 
exhibit a high degree of affinity for the D4 receptor. 
Some dopamine receptor ligands currently sold as pharmaceuticals exhibit 
the desired affinity and antagonism for the D4 receptor, yet interact 
non-selectively with related dopamine receptors, particularly the D2 
receptor type, which results in significant side effects that include 
altered motor function and tachycardia. In the context of medical 
diagnostics, this non-selective binding at the D4 receptor prevents the 
generation of an accurate image of the localization and prevalence 
specifically of the D4 type of dopamine receptor. It would therefore be 
desirable to provide compounds that, in their radiolabeled state, bind at 
the D4 receptor with affinity and selectivity appropriate for diagnostic 
imaging purposes. When used in combination with such diagnostic imaging 
techniques as single photon emission tomography (SPECT), such radiolabeled 
compounds would be useful particularly to diagnose schizophrenia and other 
medical conditions associated with D4 receptor anomalies. 
SUMMARY OF THE INVENTION 
According to one aspect of the present invention, there are provided 
compounds of Formula (I): 
##STR2## 
wherein R is selected from iodo, tri(loweralkyl)tin and a 
radioisotopically labeled iodide and R.sup.1 is selected from H and 
alkoxycarbonyl, with the proviso that R is not iodo when R.sup.1 is H. 
According to another aspect of the invention, there is provided a process 
for preparing compounds of Formula I, wherein R is a radioisotopic iodide 
and R.sup.1 is H, comprising the step of treating a compound of Formula I, 
wherein R is tri(loweralkyl)tin with a radioisotopically labelled iodide 
source in the presence of an oxidizing agent and an acid. 
According to another aspect of the invention, there is provided another 
process for preparing compounds of Formula I, wherein R is a radioisotopic 
iodide and R.sup.1 is H, comprising the steps of treating a compound of 
Formula I, wherein R is tri(loweralkyl)tin and R.sup.1 is alkoxycarbonyl 
with a radioisotopically labeled iodide source in the presence of an 
oxidizing agent and an acid, followed by removal of the alkoxycarbonyl 
protecting group under acidic deprotection conditions in the same reaction 
vessel. 
According to another aspect of the invention, there is provided a 
radiopharmaceutical composition comprising a compound of Formula I wherein 
R is a radioisotopic iodide and R.sup.1 is H and a pharmaceutically 
acceptable carrier, such as physiological buffered saline. 
In a further aspect of the invention, there is provided a method for 
imaging D4 receptors in vivo, comprising the step of administering 
systemically to a patient an effective amount of the radiopharmaceutical 
composition, and then imaging the composition following its accumulation 
at D4 receptor sites in the brain.

DETAILED DESCRIPTION OF THE INVENTION 
The term lower alkyl as used herein means straight chain alkyl radicals 
containing from one to six carbon atoms and branched chain alkyl radicals 
containing three to six carbon atoms and includes methyl, n-butyl, 
1-methylethyl and the like. 
The term alkoxycarbonyl as used herein means straight and branched chain 
alkyl carbonates containing from two to six carbon atoms and includes 
methoxycarbonyl, ethoxycarbonyl and t-butoxycarbonyl and the like. 
Compounds of the present invention are those of Formula I in which R and 
R.sup.1 are as defined above. In embodiments of the invention R is 
tri(loweralkyl)tin. In a preferred embodiment, R is tributyltin or 
trimethyltin. 
In another embodiment of the invention R is a radioisotopic iodide 
including .sup.123 I, .sup.125 I and .sup.131 I. In a preferred 
embodiment, R is .sup.123 I. 
In an embodiment of the invention, R.sup.1 is selected from H and 
alkoxycarbonyl. In a preferred embodiment, R.sup.1 is selected from H and 
t-butoxycarbonyl. In a more preferred embodiment, R.sup.1 is 
t-butoxycarbonyl. 
Compounds of Formula I, wherein R.sup.1 is H and R is a radioisotopically 
labeled iodide, can be prepared by reacting compounds of Formula I, 
wherein R is tri(loweralkyl)tin and R.sup.1 is H, with radioisotopic 
iodide source, for example a solution of radioisotopically labeled sodium 
iodide (e.g. as a solution in 1N NaOH), in the presence of an acid and an 
oxidizing agent in an alcoholic solvent. Preferred conditions are hydrogen 
peroxide and hydrochloric acid in ethanol. 
In a preferred method, compounds of Formula I, wherein R.sup.1 is H and R 
is a radioisotopically labeled iodide, are prepared by reacting a compound 
of Formula I, wherein wherein R is tri(loweralkyl)tin and R.sup.1 is 
alkoxycarbonyl, with a radioisotopic iodide source as described above, 
followed by removal of the alkoxycarbonyl protecting group under acidic 
conditions in the same reaction vessel. The preferred acid is hydrochloric 
acid. 
To generate compounds of Formula I wherein R.sup.1 is H and R is 
tri(loweralkyl)tin, an appropriately substituted piperazine derivative is 
coupled with the 1H-pyrrolo2,3-b!pyridine in the presence of formaldehyde 
in an aqueous buffer solution, for example, aqueous sodium acetate and 
acetic acetate. The 1H-pyrrolo2,3-b!pyridine is commercially available 
and the piperazines are either commercially available or can be prepared 
by methods known to one skilled in the art. Thus 
1-4-(triloweralkyltin)phenyl!-piperazines can be prepared from 
1-(4-iodophenyl)-piperazine by reaction with hexa(loweralkyl)ditin 
reagents under standard palladium-catalyzed cross coupling conditions, for 
example, in the presence of a catalytic amount of 
tetrakis(triphenylphosphine)palladium (0) in an inert solvent such as 
1,2-dimethoxyethane at temperatures ranging from 50.degree.-100.degree. 
C., preferably at about 85.degree. C. The 1-(4-iodophenyl)-piperazine can 
be prepared from 1-phenylpiperazine by reaction with an electrophilic 
iodine reagent under acidic aqueous conditions, for example, by treating 
1-phenylpiperazine with iodine monochloride in acetic acid/water (3:1) 
according to Hanson et al. J. Heterocyclic Chem., 1985, 22:47. 
Compounds of Formula I wherein R.sup.1 is alkoxycarbonyl and R is iodo or 
tri(loweralkyl)tin, can be prepared by reacting compounds of Formula I 
wherein R.sup.1 is H and R is iodo or tri(loweralkyl)tin with 
dialkoxydicarbonate reagents in the presence of a base in an inert solvent 
at temperatures in the range of 0.degree. C. to 50.degree. C., preferably 
at around room temperature. Suitable bases include sodium or potassium 
hydroxide or triethylamine. Suitable inert solvents include chloroform, 
dichloromethane or acetonitrile. Preferred conditions are potassium 
hydroxide in dichloromethane. The dialkoxydicarbonate compounds are 
readily available protecting group reagents. 
Compounds of Formula I wherein R.sup.1 is alkoxycarbonyl and R is 
tri(loweralkyl)tin, can also be prepared by reacting compounds of Formula 
I wherein R is iodo and R.sup.1 is alkoxycarbonyl with 
hexa(loweralkyl)ditin reagents under standard palladium-catalyzed cross 
coupling conditions as described above. 
In preferred embodiments of the invention, the compounds are selected from 
3-4-(4-.sup.123 I-phenyl)piperazin-1-yl!methyl-1H-pyrrolo2,3-b!pyridine; 
3-4-(4-.sup.125 I-phenyl)piperazin-1-yl!methyl-1H-pyrrolo2,3-b!pyridine; 
3-4-(4-.sup.131 I-phenyl)piperazin-1-yl!methyl-1H-pyrrolo2,3-b!pyridine; 
3-{4-4-(tributyltin)phenyl!piperazin-1-yl 
}methyl-1H-pyrrolo2,3-b!pyridine; 
3-4-(4-iodophenyl)piperazin-1-yl!methyl-1-(t-butoxycarbonyl)-pyrrolo2,3-b 
!pyridine; 
3-{4-4-(trimethyltin)phenyl!piperazin-1-yl}methyl-1-(t-butoxycarbonyl)-pyr 
rolo2,3-b!pyridine; and 
3-{4-4-(trimethyltin)phenyl!piperazin-1-yl}methyl-1H-pyrrolo2,3-b!pyridin 
e. 
In more preferred embodiments of the invention, the compounds are selected 
from 
3-4-(4-.sup.123 I-phenyl)piperazin-1-yl!methyl-1H-pyrrolo2,3-b!pyridine; 
3-{4-4-(tributyltin)phenyl!piperazin-1-yl}methyl-1H-pyrrolo2,3-b!pyridine 
; 
3-4-(4-iodophenyl)piperazin-1-yl!methyl-1-(t-butoxycarbonyl)-pyrrolo2,3-b 
!pyridine; 
3-{4-4-(trimethyltin)phenyl!piperazin-1-yl}methyl-1-(t-butoxycarbonyl)-pyr 
rolo 2,3-b!pyridine; and 
3-{4-4-(trimethyltin)phenyl!piperazin-1-yl}methyl-1H-pyrrolo2,3-b!pyridin 
e. 
The compounds of the invention wherein R is a radioisotopic iodide are 
formulated as radiopharmaceutical compositions together with any 
physiologically and radiologically tolerable vehicle appropriate for 
administering the compound systemically. Included among such vehicles are 
phosphate buffered saline solutions, buffered for example to pH 7.4. 
It is contemplated that the present compounds will be administered to 
patients by intravenous injection or infusion at doses suitable (e.g. 
between 1 and 10 mCi) to generate an image of the compound as localized 
within the brain, using for example a gamma camera. It is further 
contemplated that the method of the present invention can usefully be 
applied diagnose to patients suspected of suffering from schizophrenia. 
For these patients, diagnosis can be aided or confirmed by determining the 
intensity of radiolabeled compound relative to the brain of a healthy 
patient; greater image intensity is indicative of an overabundance of D4 
receptor, and is hence indicative of a schizophrenic condition. 
EXAMPLE 1 
Prepartion of 1-(4-iodophenyl)piperazine 
The title compound was prepared by adding a suspension of iodine 
monochloride (1.2 g, 7.4 mmol) in acetic acid/water (3:1, 7 ml) to a 
solution of 1-phenylpiperazine (1.09 g, 6.7 mmol) in acetic acid/water 
(3:1, 5 ml) at 50.degree. C. The reaction was stirred and heated at 
55.degree. C. for 1 hour, then at room temperature for 1 hour. The 
solution was poured into 100 mL of crushed ice and the pH adjusted to 13 
with 4N NaOH. The product was then extracted into dichloromethane 
(2.times.100 mL) and the combined organic layers were dried over sodium 
sulfate, filtered and concentrated to provide the title compound as a pale 
yellow solid (1.4 g, 72.5%). 
EXAMPLE 2 
Preparation of 1-4-(triloweralkyltin)phenyl!piperazines 
To a solution of 1-(4-iodophenyl)piperazine (50 mg, 0.174 mmol, 1 eq) and 
hexa(loweralkyl)ditin (0.208 mmol, 1.2 eq) in 1,2-dimethoxyethane (7.5 mL) 
under argon was added tetrakis(triphenylphosphine)palladium(0) (0.1 eq). 
The reaction was refluxed for 4-8 hours followed by filtration through 
celite and washing with methanol (10 mL). The organic layer was 
concentrated and the corresponding products were purified by preparative 
thin layer chromatography on silica gel using 
dichloromethane/methanol/aqueous ammonium hydroxide (50:7:1) as eluent to 
provide the title compounds as white solids. 
Using the above procedure the following compound was prepared: 
(a) 1-4-(Tributyltin)phenyl!piperazine, from hexa-(n-butyl)ditin. 
EXAMPLE 3 
Preparation of 3-4-(4- 
substituted-phenyl)piperazin-1-yl!methyl-1H-pyrrolo2,3-b!pyridines 
A solution of 1-4-(substituted)phenyl!piperazine (0.25 mmol, 1 eq), 
formaldehyde (0.3 mmol, 1.2 eq) and sodium acetate (0.25 mmol, 1 eq) in 
acetic acid/water (300 .mu.L, 2:1) was allowed to stir at room temperature 
for 10 minutes then 1H-pyrrolo2,3-b!pyridine (0.25 mmol, 1 eq) was added 
and the solution stirred overnight. Ammonium hydroxide (aq, 30%, 1 mL) was 
then added and the product extracted into ethyl acetate. The organic layer 
was dried over sodium sulfate and concentrated. The corresponding product 
was purified by preparative thin layer chromatography on silica gel using 
dichloromethane/methanol/aqueous ammonium hydroxide (50:7:1) as eluent. 
Using the above procedure the following compound was prepared: 
a) 3-4-(4-iodophenyl)-piperazin-1-yl!methyl-1H-pyrrolo2,3-b!pyridine, 
from 1-(4-iodophenyl)piperazine (Example 1), yellow solid (51 mg, 49%). 
EXAMPLE 4 
Synthesis of 
3-4-(4-iodophenyl)piperazin-1-yl!methyl-1-(t-butoxycarbonyl)pyrrolo2,3-b 
!pyridine 
To 3-4-(4-iodophenyl)piperazin-1-yl!methyl-1H-pyrrolo2,3-b!pyridine 
(Example 3a, 0.5 mmol, 209 mg) in dichloromethane (5 mL) under argon was 
added powdered KOH (1.0 mmol, 56 mg), followed by di-t-butyl dicarbonate 
(0.55 mmol, 120 mg). The reaction was stirred for 1 hour at room 
temperature. The solution was filtered and concentrated to a yellow oily 
solid. The product was purified by flash chromatography on silica gel 
using 30 to 50% ethyl acetate in hexanes (with 1% triethylamine) as 
eluent. The pooled fractions were concentrated to yield the title compound 
as a white solid (190 mg, 73%). MH.sup.+ 519.40. 
EXAMPLE 5 
Synthesis of 
3-{4-4-(trimethyltin)phenyl!piperazin-1-yl}methyl-1-(t-butoxycarbonyl)pyr 
rolo2,3-b!pyridine 
To 
3-4-(4-iodophenyl)piperazin-1-yl!methyl-1-(t-butoxycarbonyl)pyrrolo2,3-b 
!pyridine (Example 4, 0.10 mmol, 52 mg) and hexamethylditin (0.105 mmol, 
34.5 mg) in dry dimethoxyethane (3 mL) under argon was added 
tetrakis(triphenylphosphine)palladium (0) (0.010 mmol, 12 mg). The 
reaction was fitted with a condensor and heated at 60.degree. C. for 1 
hour. The solution was cooled to room temperature, filtered through a 
short silica gel bed and washed with ethyl acetate. The product was 
concentrated and purified on a preparative silica chromatography plate 
developed with 50% ethyl acetate in hexanes with 1% TEA to yield the title 
compound as a pale yellow oil (30 mg, 54%). MH.sup.+calc 555.38, found 
555.44. 
EXAMPLE 6 
Preparation of 
3-{4-4-(radioisotopic-iodo)phenyl!piperazin-1-yl}methyl-1H-pyrrolo2,3-b! 
pyridine 
Method A: 
A solution of the 
3-{4-4-(triloweralkyltin)phenyl!piperazin-1-yl}methyl-1H-pyrrolo2,3-b!py 
ridine (50-200 .mu.g) in dichloromethane (300 .mu.L) is concentrated to an 
oil by passing argon over the solution through a septa in a 2 mL vial. 
Ethanol (300 .mu.L) is then added and the resulting solution swirled to 
ensure complete dissolution. A solution of radioisotopically labeled 
sodium iodide (6 .mu.L in 0.1N NaOH) in ethanol (300 .mu.L) is prepared in 
a reactivial and to this is added sequentially, the solution of 
3-{4-4-(triloweralkyltin)phenyl!-piperazin-1-yl}methyl-1H-pyrrolo2,3-b!p 
yridine, hydrochloric acid (0.3N, 17 .mu.L) and hydrogen peroxide (3%, 100 
.mu.L). The resulting solution is swirled, adequately shielded and left 
stoppered for 15 minutes at room temperature. After venting in the 
fumehood, aqueous solutions of sodium metabisulfite (150 mg/mL, 100 
.mu.L), sodium carbonate (saturated, 60 .mu.L) and saline (100 .mu.L) are 
added and the product extracted into dichloromethane (300 .mu.L). The 
organic later is separated and analysed and purified by HPLC. 
Method B: 
A solution of 
3-{4-4-(triloweralkyltin)phenyl!piperazin-1-yl}methyl-1-(alkoxycarbonyl)- 
pyrrolo2,3-b!pyridine (200 ug) was dissolved in ethanol (100 uL). To the 
Na.sup.123 I solution (2 mCi, in 0.1N NaOH) in a 3 mL reactivial was added 
ethanol (200 uL). Sequentially added were hydrochloric acid (1N, 100 uL), 
the solution of 
3-{4-4-(triloweralkylltin)phenyl!-piperzin-1-yl}methyl-1-(alkoxycarbonyl) 
-pyrrolo2,3-b!pyradine and hydrogen peroxide (3%, fresh, 100 uL). The vial 
was swirled, adequately shielded, and left to react stoppered for 15 
minutes at room temperature. The iodination reaction was complete after 15 
minutes, however, the protecting group hydrolysis required an hour to 
finish. The septum was then vented in the fumehood and sodium 
metabisulfite (150 mg/mL, 100 uL), sodium carbonate (sat, 200 uL) and 
saline (100 uL) were added. The product was extracted into dichloromethane 
(300 uL) which was removed carefully by syringe. The organic layer 
contained the desired product. HPLC analysis was used to confirm the 
results by comparing the product retention time to that of the authentic 
cold product as well as the starting material and intermediate. 
Using Method B, the following compound was prepared: 
3-4-(4-.sup.123 I-phenyl)piperazin-1-yl!methyl-1H-pyrrolo2,3-b!pyridine, 
from 
3-{4-4-(trimethyltin)phenyl!piperazin-1-yl}methyl-1-(t-butoxycarbonyl)pyr 
rolo2,3-b!pyridine (Example 5). 
EXAMPLE 7 
Receptor Binding Affinities 
D2 and D4 receptor-binding affinities of the compounds of Example 3 were 
evaluated as described in WO95/17400 (incorporated herein by reference) 
for their ability to reduce binding of .sup.3 H-spiperone as compared to 
the reference compound clozapine. The potency of the test compound to 
reduce .sup.3 H-spiperone binding directly correlated to its binding 
affinity for the receptor. 
Briefly, the D4 receptor was utilized in the form of membrane preparations 
obtained from HEK 298 cells stably transfected with human D4 receptor 
(D4.2 sub-type). D2 receptor was utilized in the form of membrane 
preparations obtained from GH.sub.4 C.sub.1 (rat pituitary) cells stably 
transfected with the human D2 receptor (short isoform). The total 
spiperone binding assay was started by the addition of 500 .mu.l (50 .mu.g 
protein) membrane homogenate to a solution of 900 .mu.l incubation buffer 
and 100 .mu.l (0.25 nM final conc.) .sup.3 H-spiperone. The binding 
reaction was stopped and the samples were filtered under vacuum and 
filters were then washed 3 times with 5 ml ice cold 50 mM Tris buffer (pH 
7.4). Individual filter disks were put in scintillation vials (Biovials, 
Bechman). Ready Protein Plus liquid scintillant (5 ml, Beckman) was added 
and the vials counted by liquid scintillation spectrophotometry (Beckman 
LSC 6500) after equilibrating for three hours at room temperature to 
determine total binding (B.sub.r). 
Non-specific binding for D4 was assayed by incubating membrane homogenate, 
.sup.3 H-spiperone and fresh dopamine. Filtrate was counted using the same 
procedure as in the total binding assay described above to give the 
non-specific binding value (NSB). Non-specific binding for D2 was 
similarly assessed, with the exception that (-)-sulpiride was used in 
place of dopamine. 
To assess displacement, membrane homogenate was incubated with .sup.3 
H-spiperone and test compound dissolved in DMSO. Filtrate was counted 
using the same procedure as in the total binding assay described above, to 
give the displacement binding value (B.sub.D). 
The test compounds were initially assayed at 1 and 0.1 .mu.M and then at a 
range of concentrations chosen such that the middle dose would cause about 
50% inhibition of .sup.3 H-spiperone binding. Specific binding in the 
absence of test compound (B.sub.0) was the difference of total binding 
(B.sub.r) minus non-specific binding (NSB) and similarly specific binding 
(in the presence of test compound) (B) was the difference of displacement 
binding (B.sub.D) minus non-specific binding (NSB). IC.sub.50 was 
determined from an inhibition response curve, logit-log plot of % 
B/B.sub.0 vs concentration of test compound. 
Ki was calculated by the Cheng and Prustoff transformation: 
EQU Ki=IC.sub.50 /(1+L!/K.sub.D) 
where L! is the concentration of .sup.3 H-spiperone used in the assay and 
K.sub.D is the dissociation constant of .sup.3 H-spiperone determined 
independently under the same binding conditions. 
Assay results (Ki) are reported in the following Table, and show clearly 
the D4 selectivity of compounds of the invention: 
______________________________________ 
Compound D4 (nM) D2 (nM) 
______________________________________ 
3-4-(4-iodophenyl)-piperazin-1-yl!methyl 
0.16 2366 
1H-pyrrolo2,3-b!pyridine 
______________________________________