[1 1C]Spiroperidol:
Synthesis,
Specific
Activity
Determination,
and
Biodistribution in Mice J. S. Fowler, C. D. AmeU, A. P. Wolf, R. R. MacGregor, E. F. Norton, and A. M. Findley
Brookhaven NatlonalLaboratory, Upton, New York
Carbon-I 1-labeledspiroperldoi—a positron-emittingdopamine-receptorantag onistwith potentialfor use In serial PET studiesof humanneurotransmltterrecep
tors—wassynthesizedfrom cyclotron-producedH11CNIn 40 mm, with a radio chemicalyieldof 20-30%. The specificactivityof the H11CNusedinthe synthesis was determinedusingtwo independentmethods,and the specific activfty of the [11C]spiroperidoi synthesized was determined by radioreceptor assay. The tissue distribution in mice showed a brain concentration of 0.5 % of the injected dose, and this remainedrelativelyconstantfor the first 30 mm. J Nucl Med 23: 437—445,1982 The study of neurotmansmitter receptors is a rapidly expanding area of investigation. One approach to the study of receptors has been the use of radioligands as probes to identify and examine the receptor sites. Ra dioligand binding studies on postmortem tissues from
receptors in the living human brain involves consider ations of biodistnibution, transport kinetics, and me tabolism, in addition to those ofspecificity and affinity inherent to in vitro binding studies.
Spinoperidol (Scheme 1) is a dopamine-receptor an
human brains indicate that specific changes in dopamine receptors @
(either concentration
brain regions
are associated
or affinity)
with a number
in various of diseases.
H@[I@/\@
CH2 CH2
F
Dopamine-meceptombinding was significantly decreased in certain brain regions in patients with Parkinson's disease (1 ,2) or Huntington's disease (3), whereas it was significantly higher in some regions of the brain in pa tients diagnosed as schizophrenic (4,5). Whether on not
these changes in the dopamine receptor are related to the physiological or behavioral manifestations of the disease is open to debate, since it has not been possible to cx amine receptor populations in normal and diseased
human brain in vivo. The successful measurement of regional brain metabolism of glucose in man, using 2deoxy-2-[' 8F]fluomo-D-glucose and positron-emission
C6H,
1
tagonist which, because of its high affinity, is widely used in studiesof dopamine-receptor binding. Radiolabeled spimoperidol has been used extensively as a ligand to label the dopamine receptor in vitro (7,8). In vivo experiments have demonstrated that it is not rapidly metabolized in either rat (9) or mouse (10) brain. A potential use of
labeled spiroperidol for in vivoautoradiography has been demonstrated by Kuham and colleagues using [3H]-
spimopemidolin studies with mats (9). Considerable effort has been devoted to extension of these studies to humans cation of PET to the study of other aspects of human using PET. neurology, such as probing receptor populations in The interpretation of PET studies of neurotransmitter vivo. receptors requires not only the labeling of appropriate Thedesignof a madioligand to labelneurotransmitten ligands with positron-emitting nuclides but also the demonstration of specific receptor binding and appro Received May 14, 1981; revision accepted Jan. 4, 1982. priate mathematical modeling of this complex process. For reprints contact: J. S. Fowler, PhD, Dept. of Chemistry, It is also of considerable importance that the uptake of Brookhaven National Lab., Associated Univs. Inc., Upton, NY 11973. labeled ligand into the brain be sufficiently high to per
tomography (PET) (6), hasgiven impetusto the appli
Volume23,Number 5
437
FOWLER, ARNETT, WOLF, MACGREGOR, NORTON, AND FINDLEY
mit statistically significant imaging studies after the administration of acceptable amounts of radioactivity. Therefore, questions concerning the magnitude of the uptake of receptor ligands into the brain bear signifi
dium carbonate, and 0.7 ml (8.4 mmol) of 3-chlomo-lpropanol in 40 ml of dimethylfonmamide (DMF) was mefluxed for 3 hr. The reaction mixture was diluted with
water and extracted with chloroform. The chloroform
cantly on the feasibility of PET studies, as do the time courses of the specifically bound, nonspecifically bound,
extract was dried (K2CO3, anhydrous) and the solvent removed to give 1.48 g (59%) of crude product (m.p. and free (unbound) tracers in the brain. 180— 195°).A small sample (28 mg) of the crude product was chromatographed over a 10-cm column of silica gel The interest in spiropenidol and spiropenidol analogs to give 11 mg of pure alcohol; this was recrystallized labeled with positron emitters has resulted in the devel opment of synthetic routes to [‘8Flspiropenidol (11, T112 from ethyl acetate to give the analytical sample (m.p. = 1 10 mm) and p-[77Bn]bnomospinopenidol (12, T112 = 204-206°). The NMR spectrum (CDC13) showed 6 57 hr). Because of the shorten C-i I half-life (20 mm), 7.5-6.9 (m, 5H, aromatic H's), 4.8 (s, 2H )NCthNZZ), [I ‘C]spmropenidol
offers
the
potential
for
serial
PET
studies of receptors at short time intervals. Thus an cx penimental subject can serve as its own control before the administration of drugs or other stimuli in studies de
signed to elucidate the contribution of specific receptor binding to the resultant
3.8—4.0(t, 2H, —OCthCH2—), 3.3—2.5(m, 8H), 1.9—1.7(m, 4H). Anal. calcd. for C16H23N3O2: C 66.41, H 8.01, N 14.52.
Found: C 65.86, H 8.05, N 14.82.
effect of these experimental
treatments. Serial studies may also be of particular im portance in kinetic studies of neurotransmittem
receptor
Preparation of 8-(3-chloropnopyl)- 1-phenyl- 1,3,8tmiazaspimo[4.5]decan-4-one (4)
binding in vivo. We report here the synthesis
of [‘ ‘C]spiropenidol
([@C]-1, Fig. i) from H―CNin relatively high yield, the determination
of its specific activity by radioreceptor
assay using [3Hlspiroperidol
(13), the determination
of
the specific activity of H' ‘CNby two independent methods, and the biodistnibution of [‘ ‘C]spiroperidol in mice over a 30-mm time course.
To 0.55 g (1.9 mmol)of alcohol3 in 15 ml of chlono form was added 0.27 ml of thionyl chloride, and the mixture was mefluxed 1 hr. The solvent was evaporated
and the mixture made basic with 1 M NaOH and cx tracted with chloroform
to give 0.38 g (65%) of crude
product which TLC showed to be one compound. A portion (0.1 g) of the product was purified by silica-gel
chromatography to yield 73 mg of product (m.p. 166MATERIALS AND METHODS
Chemistry. Procedures for the production of the nec essary intermediates for the [I ‘C]spiroperidol synthesis, and the synthesis itself, are described below. Elemental analyses were penfonmed,* and NMR,@ IR,@and UVII spectra were made. Melting points are uncorrected.
Preparation of 8-(3-hydmoxypmopyl)-1-phenyl- 1,3,8tmiazaspino[4.Sjdecan-4-one
(3)
A mixture of 2 g (8.6 mmol) of l-phenyl-l,3,8-tmia zaspimo[4.5]decan-4-onel (2), 0.89 g (8.4 mmol) of so
@
CIcH.CH.CH.UH,
_._@5@4.
[‘)(
[email protected]
169°) which was recrystallized from ethyl acetate to yield the analytical sample (m.p. 173- 175°).The NMR spectrum (CDC13) showed 6 7.6—6.8(m, 5H, aromatic H's), 4.8 (s, 2H, )NCthN(), 3.8—3.6 (t, 2H, ClC@j@—),2.98—2.5(m, 8H), 2.2—1.6(m, 4H). Anal. calcd. for C16H22C1N3O: C 62.42, H 7.20, N 13.65. Found: C 62.46, H 7.26, N 13.50. Preparation of 8-(3-cyanopropyl)- 1-phenyl- I ,3,8tniazaspino[4.5]decan-4-one (5)
To 2 g (8.6 mmol)of 2 in 40 ml of DMF wasadded l.69g(l6 mmol)ofNa2CO3 and 1.4 ml(l5 mmol)of 4-chlomobutynonitmile, and the mixture was refluxed for 3 hr. After cooling, water was added, the mixture was extracted with chloroform, and the chloroform extract dried with K2CO3. Removal of the solvent gave 1.49 g of a colorless solid, which was purified by solution in HC1 and washing with chloroform. The acidic solution was
made basic and the product was extracted with chloro form. After drying (K2CO3), evaporation, and recrys @ >
PIC,HM@K I― CI
FIG.1. Synthesis of[11C]spiroperidol. 438
tallization from ethyl acetate, 0.77 g (30%) of a colorless solid, m.p. 173-174°, was obtained. The NMR spectrum (CDC13) showed 6 7.6-6.9 (m, 5H, aromatic H's), 4.8 (s, 2H, )NCthN(), 3.0-2.4 (m, 1OH), 2.1-1.7 (m, 4H). IR (KBr), 2250 cm@ (—C@N). THE JOURNAL OF NUCLEAR MEDICINE
BASIC SCIENCES RADIOCHEMISTRY AND RADIOPHARMACEUTICALS
Anal. calcd. for C17H22N4O: C 68.43, H 7.43, N 18.78. Found: C 68.24, H 7.36, N 18.32. Reaction of 4 and NaCN
To 39 mg (0. 127 mmol) of 4 in 0.4 ml of DMF was added 12 mg (0.245 mmol) of NaCN, and the mixture was heated at 130°for 10mm. The DMF was evapo
mated,water was added, the mixture extracted with ether, and the ethereal solution dried with K2CO3. Removal
.of theetherleft 30 mgof a colorless solid.TheNMR spectrum
and the TLC were identical to nitmile 5 pre
pared by the reaction of 2 and 4-chlorobutyronitmile. Preparation
ofp-fluorophenylmagnesium
Bromide
A mixture of 2.4 g Mg turnings and 10.98 ml of p fluonobromobenzene in 100 ml of ether was stirred and nefluxed until most of the Mg had dissolved. The Gri gnard reagent was decanted from the residue and stoned in the cold. For use in the spiroperidol synthesis, the ether solvent was replaced by xylene. This was conveniently done by evaporating 5 ml of the Grignard solution under ,vacuum and dissolving the residue in 5 ml of xylene. This
solution is stable at room temperature 1 mo.
for approximately
divided into two portions and the specific activity of one portion was determined as described below, whereas the other portion was used to synthesize [1‘C@spimopemidol, which was used in the radiomeceptom assay of specific activity described below. Polarographic measurements were carried out.1 Samples and standards were prepared in I 0 ml of a so dium hydroxide (0.05 M) and boric acid (0.05-0. 1 M) supporting electrolyte (16), and the anodic waves, owing
to the electrode reaction Hg + 2 CN —@ Hg(CN)2 + 2e, were measured at peak potentials of 0.2 to 0.3 V against the silver/silver chloride electrode. The standards were chosen to bracket the sample in cyanide concen
tration and were aliquots of a dilution of a 0. 1 M sodium hydroxide solution of sodium cyanide,** which was standardized by titration with silver nitrate (18). The limit ofdetection, defined as the concentration of cyanide giving a measurable peak when compared with a blank, is 10 ppm on 0. 1 zg of cyanide per 10 ml. The samples measured for the specific-activity determination con tamed between 1 and 5 .tg of cyanide. The pynidine-pynazolone spectrophotometnic method of Epstein (17) was also used for determination of in active cyanide. The pH of the sample solution was ad justed to 6 to 7 with I M acetic acid, and the color was developed in 10 ml volume using Epstein's procedure. The absombance measurements were made in 1-cm silica
cells at 620 nm using a spectrophotometer. A calibration
Preparation of Spinopenidol from S and p-fluorophenylmagnesium Bromide
was made using the standard
To 56 mg (0. 19 mmol) of nitnile 5 dissolved in 2 ml of
sodium cyanide solution
described above for the polarographic method. The calibration coveredthe range 0.25-2.0 j.@g of cyanide.
xylene (heating required) was added 1.5 ml of 1 M p
Thelimitofdetection equalto3o@ oftheblankis0.02@ig
fluorophenylmagnesium bromide in xylene, and the mixture was heated at I 40°for 10 mm. The reaction mixture was worked up by adding 0.2 ml of 7.1 M NH4CI solution. The xylene solution was decanted from the residue and the residue washed with hot ethyl ace tate. Evaporation of the solvent gave 32 mg (43%) of
of cyanide.
colorless solid, m.p. 189—193°, which was crystallized
At the end of bombardment (EOB) the liquid nitrogen
Synthesis of [‘ ‘C]Spiroperidol Carbon-i i-labeled HCN was prepared and trapped in a trap cooled with liquid nitrogen during irradiation.
was removed, thc trap allowed to warm, and the H' ‘CN was purged (using the target gas mixture) through a onTLC (seebelow)matchedthatof anauthenticsample solution of 0.80 mg of chloride 4, 0.20 ml of 0.05 M NaOH, and 0.1 ml of DMF. The mixture was heated to of spiropenidol. The NMR spectrum (in CDCI3) showed 6 8.06-6.7 (m, 9H, aromatic H's), 4.7 (s, 2H, 140°for 4 mm, cooled, and the solvent evaporated to dryness under vacuum with slight warming. A solution )NCjj@N@3, 3.1-2.4 (m, 1OH), 2.2-1.6 (m, 4H). The of 0.7 ml of I M p-fluorophenylmagnesium bromide in ultraviolet spectrum showed A@H 248 nm ( = xylene was added to the reaction vessel, which was then 23,000). from ethyl acetate to give a sample with m.p. 198-200° [lit. (14) m.p. 190-193.6°]. The NMR spectrum and Rf
heated at 140°for 5 mm. The reaction mixture was cooled and 0.2 ml of 7.1 M NH4C1 solution was added.
El‘C]Cyanide Specific Activity Determination
@
The
The mixture was stirred at 25°for 3 mm, 3 ml of ethyl acetonitnile (4:1) was added, and after two more mm of stirring, the mixture was transferred through a K2CO3 tube onto a dry column (0.75 X 10 cm) of silica gel 60.tt The column was washed with 35 ml of CH3CN:CH3OH (4:1) and 15-20 ml of CH3CN: CH3OH (2:1), which was discarded. The product was
ICN waspreparedaspreviouslydescribed acetate:
(15), trapped
in 0.05 M sodium
hydroxide,
counted,
and
theinactivecyanidewasdetermined bydifferentialpulse polanognaphy (16) or spectrophotometny (1 7). The specific activities of a number of preparations were measured. In two experiments the H' ‘CNproduced was
Volume23,Number 5
439
FOWLER, ARNE11', WOLF, MACGREGOR, NORTON. AND FINDLEY
eluted with 15 ml of CH3CN:CH3OH (2:1) directly into a rotary evaporator by means ofvacuum, with the solvent being removed during the elution [see Chromatography of [1‘Cjspiroperidol, belowl. To the eluate was added 1 ml of 2% HCI in ethanol, and this was evaporated to dryness. The product was dissolved in saline for animal experiments.
The
radiochemical
yield
of [1 ‘C]spino
penidol was 20-30%, the radiochemical purity was> 98.5% as determined by TLC in three different solvent systems (see description below), and the specific activity was determined to be 2.6 ±1.9 Ci/.imol (EOB) as de
scnibed below. In addition, 12.5 mg of unlabeled spiro peridol was added to some of the [1‘Cjspiroperidol,and the specific activity (corrected for decay) of five suc cessive necrystallizations of this material remained constant. Expression of yields and specific activity with short@
lived nucides. Unless otherwise specified, the quantities of radioactivity as well as the specific activities described in this manuscript are decay corrected to the end of cy clotnon bombardment (EOB). The use of decay-con rected yields and specific activities eliminates variables in synthesis time, and the values can be used directly in the calculation of radiochemical yields. In the present work the specific activity of the H' ‘CNand the [1‘C]spiroperidol were determined to be 3.8 ±0.9 and 2.6 ± 1.9 Ci/@zmol, respectively. This corresponds to a ratio of carbon-12 to carbon-il of 2500—3600:1,and therefore the change in mass associated with the decay of car bon-1 1 is insignificant. Note that when the C-12/C-1 1 ratio is high, the mass remains essentially constant during the decay process and the specific activity is re
TLC, since applying in saline caused multiple spots to appear, confusing the results. Note that single-spot TLCs could be obtained from samples that had been dissolved in saline by evaporation
of the sample to dryness and
nedissolving in CH3CN:CH3OH. The radioactivity was congruent with UV active spot(s) corresponding to spiroperidol in both the saline and CH3CN:CH3OH solvents. The product was not contaminated by excess chloride 4, as determined by examination of the ultraviolet spectrum as well as TLC. During the development of this synthesis, TLC was used to monitor the reaction at each step. Chloride 4, nitnile 5, and spinoperidol are well
separated in the CH3CN:CH3OH (4:1) solvent system (Rf 0.21, 0.31, and 0.17, respectively), so this system could be used to determine yields and punities at various steps as well as the recovery from the chromatography column. Conditions for the flash chromatography (19) were optimized so that excess unlabeled chloride 4, C-i 1labeled nitnile 5, and unidentified C-i 1 activity that had a higher Rf than [‘ 1C]spiropendol would be eluted in the first fractions with CH3CN:CH3OH (4: 1) and 15-20 ml CH3CN:CH3OH (2:1), and the pure [‘ ‘C]spiro peridol would be eluted in the final fraction [CH3CN:
CH3OH (2:1)]. Column conditions were determined using unlabeled chloride 4, nitnile 5, and spinopenidol, and assaying fractions by UV and TLC. These elution volumes may vary with such factors as silica-gel activity and elution speed, so that monitoring of the UV ab sorption of the column effluent during a synthesis is advisable.
[I 1ClSpiroperidol for specific-activity determination. duced by one-halfwith the passage ofeach half-life. For example, the synthesis of [1‘C]spiropenidol reported here [I ‘C]Spinopenidol was prepared and chromatographed yields a product having a specific activity of 2.6 Ci/@imol as described above except that the CH3CN:CH3OH (EOB). Since the mass is not changing significantly (2:1) column effluent was collected in 5-ml fractions. during decay, the actual specific activity at the end of Each fraction was counted, its radiochemical purity determined by TLC, evaporated to dryness, medissolved synthesis (EOS) is 0.65 Ci/@mol, since the synthesis requires two half-lives. We suggest that in reporting the in 5 ml of 95%EtOH, and the UV absombance at 250nm was recorded. The product was collected in the fractions synthesis of radiotracens labeled with short-lived nuc following elution of RC1 (4), which was eluted in the lides, all quantities of radioactivity or specific activities CH3CN:CH3OH fonecut. In two experiments (Runs I that are reported be followed by EOB (end of born and 2 of Table 1), fractions following the elution of 4 bardment) or EOS (end of synthesis) to reduce am were combined for specific-activity determination by biguities. Chromatographyof[―Cjspiroperidol. A portion of the madioneceptorassay. In another experiment (Run 3), the labeled spiroperidol, with no carrier added (NCA), was C-l 1-labeled nitrile (5) was first separated from the chloride (4) using flash chromatography with added to unlabeled spiropenidol with CH3CN:CH3OH CH3CN:CH3OH (9: 1) as the eluent. The purified C- 11 (4: 1) and applied to silica-gel 60 TLC plates.@*The plates were developed in CH3CN:CH3OH (4:1), CHC13: nitrile was allowed to react with the Grignand reagent. CH3OH:H20 (30:9: 1), and EtOAc:acetone:H20 (70: After the usual workup, the product was chromato ‘C]spinopen 40:5), with the spiroperidol visualized with UV light. graphed and the fraction containing the [‘ Radioactivity was congruent with the mass in each case. idol was retained for nadioreceptor assay. In all cases the The spiropenidol in the three solvent systems gave R@= ethanol was evaporated from the selected fractions and the residue was dissolved in 1 ml of 10-mM HCI for 0.17, 0.80, and 0.18, respectively. specific-activity determination. It was essential to apply the C-l 1 and carrier com Radioreceptor assay (20) ofl' ‘C]spiroperidol.Mem pounds to the plate in CH3CN:CH3OH solution for the
440
THE JOURNAL OF NUCLEAR MEDICINE
BASIC SCIENCES RADIOCHEMISTRY AND RADIOPHARMACEUTICALS
TABLE 1. COMPARISONOF SPECIFIC ACTIVITIES OF H11CN AS DETERMINED BY A
CHEMICAL METhOD AND [11C]SPIROPERIDOL AS DETERMINEDBY RADIORECEPTORASSAY Actlvfty
(CI/@imaI,EOB)UV[“C]
2.00.418(0.050)22.7 1.40.176(0.010)3—
filters,1111 and the filters were washed four times with 5 ml each of ice-cold incubation buffer. The filters were transferred to liquid-scintillation vials, 10 ml of Aquasol
1.2*0.046(0.004)4— 2.711
was added, and the vials were stored overnight to allow
0.058 5.8110.085(0.005)
the filter-trapped material to become distributed throughout the liquid-scintillation cocktail. The samples were then counted for tnitium on a liquid scintillation counter at an efficiency of44%. Nonspecific binding of
—64.2
—73.0 —
Average 3.8 ±0.9 . Total
absorbance
2.6 ±1.9 at
250
tion, 750 zl incubation buffer, 100 @l of the appropriate
concentration of cold spiropenidol, 50 ,ul of [3H]spino peridol (final concentration = 1 nM, specific activity = 39 Ci/mmol)tt, and 100 zl of brain-membrane prepa nation (final protein concentration = 0.4 mg/ml). The tubes were incubated at 37°Cfor 15 mm. The suspen sions were then filtered through prewetted glass-fiber
RunNo.SpecIfic Spiropenidol14.5t H―CN
(0.011)54.5
obtain the desired concentrations for the standard curve. Triplicate incubation tubes contained, in order of addi
nm
of
the
[3Hjspiroperidol (to membrane sites other than receptors fraction
that
was
and to experimental materials such as test tubes and
assayedfor specific activity by radioreceptorassay.Values
filters) was determined by incubating some of the tubes
in parenthesesaretheexpectedabsorbancesdueto spiro
with 1 @M unlabeled spinopenidolto saturate the specific
peridol, based on a calculatIon using the value of H―CN
receptor
specific activityas the absolutevalueof the spiroperidol
peridol cannot bind to the receptors, so the filter-trapped
specific activity. For Runs 3 and 4 the average H―CNape
radioactivity represents nonspecific binding. Total binding was that obtained in the absence of added excess
clflc activitywasused. t In this run the mass of HCN was determined using both
methods(seeMethods). * Ptwlficatlon was carried out at the R'1CN step and the
[“C]spiroperidol step(seeMethods). IITwo separate fractions in the chromatographic ptrifl cation of the [“C]spiroperidol.
brane preparation. The membrane preparation and in
binding
sites. In these samples
the [3H]spiro
unlabeled spinopenidol. Specific receptor binding was determined by subtracting the nonspecific binding from the total binding of [3H]spiropenidol. Under the condi tions of this assay, the specific receptor binding observed
was 80% of total binding of [3Hjspiropenidol. Specific Activity of [‘ ‘C]Spimopemidol by Radioreceptor Assay
cubation conditions were slightly modified from those
used by Burt et al. (21 ). Six female Swiss albino mice (BNL strain) were killed by cervical dislocation. The brains were rapidly removed and homogenized (Brink mann polytnon setting 6 for 10 sec) in 10 volumes of ice-cold 50 mM TRIS HC1 buffer, pH 7.4 at 20°C.The homogenate was centrifuged at 4°Cand i000g for iO
Aliquots of the i-ml solution of [‘ ‘C]spinopemidol in iO mM HCI, described above, were assayed for radio activity with a NaI(Tl) well counter. After allowing the
mm. The supernate was centrifuged at 10,000g for 20 mm. The pellet from this centnifugation was resuspended (Teflon-glass homogenizer) in 10 volumes ofTRIS HC1
C-i 1 to decay, successive dilutions of this solution were made in incubation buffer just before aliquoting for determination of the concentration of spinoperidol in each dilution. The competitive binding of each dilution of the [I ‘C]spiropenidol(and its carrier) with the [3Hjspinopenidol was determined in triplicate as de
buffer and centrifuged at iO,000g for 20 mm. This
scribed for the standard
washed P2 pellet was resuspended in 4.5 ml (1.7 volumes)
producing 20—80%inhibition of specific binding were used, since they represent the most accurate values
curve. Only those dilutions
of the incubation buffer (which consisted of 50 mM TRIS HC1, pH 7.4 at 20°C,i 20 mM NaCl, 5 mM KC1, (20). 2 mM CaCl2, 1 mM MgCl2, and 10 pM pargyline) and Tissue distribution of[―Cjspiroperidolin mice. Female either kept on ice or frozen and stored on dry ice until Swiss albino mice (BNL strain), 25-32 g, were injected used.
in a lateral
tail vein with
5—20 @Ciof [‘ ‘C]spiropemidol
dissolved in 100 @l of saline. The mice were killed by Standard Curve for Spimopenidol Unlabeled spinopenidol at a concentration of 1 mM in 10 mM HC1 was prepared as a stock solution. This solution was then diluted further in incubation buffer to
Volume 23, Number 5
cervical dislocation
1, 10, or 30 mm later, tissues were
rapidly excised, blotted, and placed in tamed counting vials. A small sample of blood was taken from the tho racic cavity following removal of the heart. Samples of intestine were stripped of their contents before counting. 441
FOWLER. ARNETT, WOLF, MACGREGOR, NORTON, AND FINDLEY
The samples were counted and weighed, and the activity expressed as percent of injected dose per organ or percent injected dose pen gram of tissue. E 0
RESULTS
z
Spiroperidol synthesis. The known routes to spiro peridol (22,23) were not applicable to labeling with
carbon-i 1, especially when one considers the limited number of C-i I precursors available and the short half-life of the nuclide. The synthesis reported here was developed specifically to allow the rapid introduction of
.J
2 Id 0. In I
W ‘CN intothemolecule, andrequiredthesynthesis and characterization
of compounds 3, 4, and 5 before
applying this route to [‘ ‘C]spiroperidol (Fig. I ). These compounds had not been reported previously except for
4, which was mentioned in the patent literaturewithout details (24). The major synthetic strategy used here is the conversion of a nitnile to a ketone using a Grignard
reagent, a general reaction in organic synthesis (25) that
[sPIR0PERID0L] (M)
FiG.2. Radioreceptor assaycompetition curveforspecific [3H]spiroperidolbindk@g. 0 Standardconcentra@ons of nOnradiOaCtive spiroperidol. a = Dilutionsof the [“C]spiroperidol batth solution (A X5,000; B X2,000; C X1,000).
has not been applied previously to the synthesis of C11-labeled ketones. The success of this reaction depends
ontheselectivereactionof theGnignardreagentwitha
was used to determine the spimopemidolconcentration
nitnile in the presence of an amide. In this particular case
each of these three dilutions. Together with the original
no productsresulting from the addition of the Grignard reagent to the amide carbonyl are observed. This may be attributed to proton abstraction from the amide by the Grignand reagent, resulting in a compound that is protected from further reaction with the Grignard me agent. This synthetic strategy may be applicable to the synthesis of other C- 1i-neuroleptics
of the butyrophe
noneclassif there are no functional groupsin the mole cule that undergo competing reactions at the displace
radioactivity
determinations
of
of the batch preparation,
the average of these three values was used to determine the specific activity (calculated to EOB) of the batch, as listed in Table 1. The average of five separate deter minations was 2.6 Ci/@mol (EOB). This means that only
one molecule in
3,000 contained C-I 1 (maximum
theoretical specific activity of C-l 1 is 9.2 X 106 Ci/ mmol). Thus the C-i I-labeled spiropemidol molecules
did not contribute significantly to the mass of spimopem
ment step on at the Gnignard step. Each of the two major steps in the synthesis—the displacement of the alkyl chloride 4 with [‘ ‘C]cyanide and the reaction of the resulting [‘ ‘C]nitnile with the Gnignard reagent to
idol in the batch. Therefore, the mass of spimoperidol in the batch was not changing significantly owing to car bon- 11 decay before or during the assay.
produce[‘ ‘C]spiropenidol—pnoceeds in about50%yield,
H―CNAnalysis
resulting
in an overall
yield of 20—30%. Theme is about
15% variation in yield from run to run. Therefore,
from
300 mCi (EOB) of H' ‘CNone can obtain 15-22 mCi (EOS) of no-cannier-added [‘ ‘C]spimopenidol of 98.5% madiochemical purity at the end of a 40-mm synthesis. In addition, all steps before the chromatography are
carried out in a single reaction vessel.
The specific activity of the H' ‘CNwas determined by determining the mass of HCN associated with a measured amount of radioactivity. The results of the specific activity determinations on several runs using the polarogmaphic method (16) are shown in Table i . In one
experiment the HCN mass was also determined using a spectrophotometnic
The competitive binding of each dilution of the [I ‘C]spiroperidol
solution
was
compared
with
method (1 7), and the value agreed
with that obtained by the polarographic method within
Specific Activity of [1‘C]Spinoperidol
that
of a
standard competitive binding curve made up of a series of concentrations of nonmadioactive spiroperidol. A
3%. In two of these runs the H ‘ ‘CNwas divided into two portions. One portion was used for HCN mass deter
mination, and the other was used for [‘ ‘C]spimopemidol synthesis for madioreceptor assay of specific activity. In these runs the specific activity of the H' ‘CNand
typical curve is shown in Fig. 2. On this plot, three of the
[I ‘C]spiroperidol
[I ‘C]spinopenidol
mating inter-run variables in cyclotron beam and flow
dilutions
were
within
the
range
of
20-80% inhibition of specific binding. A log-probability
plot of the normalized competitive binding curve (26) 442
should
be directly
comparable,
elim
rate. A summary of specific-activity determinations by the two methods is given in Table 1. THE JOURNAL OF NUCLEAR MEDICINE
BASIC SCIENCES RADIOCHEMISTRY AND RADIOPHARMACEUTICALS
TABLE 2. TISSUE DiSTRIBUTION OF [11C]SPIROPERIDOL IN MiCE Time after in (mm)1030% 1 Tissue
% dose/g
Brain
% dose/organ
0.92 (0.82—1.00)
0.42 (0.37—0.46)
1.2 (1.1—1.3)
0.51 (0.47—0.55)
1.7
1.3 (1.1—1.4)
Blood
dose/organ
% dose/gJection
% dose/g 1.1 (1.0—1.3)
% dose/organ 0.50 (0.44—0.58)
1.6 (1.3—1.7)
(1.3—2.2)
2.4 0.54 0.26 (0.48—0.65) (1.9—3.2)(0.23—0.29)
Heart
16 (13—18)
1.8 (1.5—2.0)
4.4 (3.8—5.0)
Lungs
52 (45—57)
7.0 (6.2—7.5)
28 (21—36)
4.3 (2.6—5.7)
Liver
4.8 (4.3—5.7)
6.8 (5.7—7.6)
8.0 (6.7—9.0)
11 (10—13)
6.4 9.0 (5.8—7.2) (8.0—10.1)
Spleen
5.0 (4.5—5.7)
0.84 (0.74—0.90)
8.3 (6.2—10.1)
1.3 (1.0—1.5)
6.6 1.3 (4.6—8.6) (0.7—2.2)
Sm. Intestine
5.2 (4.6—6.0)
6.3 (5.5—7.3)
(5.4—9.2)
Ovaries
Mean
23 (21—25) of
four
determinations,
with
8.9 (8.2—10.0) range
in
6.9
5.6 (4.8—6.8)
0.25 (0.15—0.38)
O.l9@ (0.09—0.28)
Kidneys
4
6.9 (5.5—8.4)
14 (9—18)
23 (18—27)
9.1 (8.8—9.6)
2.0 (1.4—2.4)
7.3 (5.5—8.5) 0.12 (0.05—0.20)
18 (17—21)
6.8 (6.3—7.2)
parentheses.
t n = 3.
of
duced depends on the flow rate and duration of flow. The
[I ‘C]spinoperidolin selected tissues of mice at I, 10, and
activity associated with this mass is variable, depending on beam current and length of irradiation (15). The
Tissue distribution.
Table 2 gives the distribution
30 mm after intravenous injection. The results are means and ranges for four animals pen time interval, except as noted. The bulk of radioactivity appears in liver, kidney, and lung tissues at these early time points. Although only about 0.5% of the injected dose appeared in the brain, its uptake there is rapid and the brain concentration appears
to be constant
for this time period,
suggesting
significant retention by brain tissue. These results are similar to those found for [3H]spinopemidol uptake and
retention by rat brain (9,27). DISCUSSION
An accurate determination of the specific activity of a receptor madioligandis of considerable consequence if dynamic measurements of association and dissociation are to be made in vivo. Furthermore, the paucity of me ceptor sites requires high specific activity in order to avoid saturation
effects. The synthesis of [I ‘C]spiro
peridol was performed without added carrier HCN, so that the absolute specific activity of the product (com nected for decay) is the same as that of the precursor, H' ‘CN.Cyclotron-produced H' ‘CNis not carrier free because of small amounts of hydrocarbon contaminants in the target gases. The mass of unlabeled HCN pro
Volume 23, Number 5
determination
ofthe specific activity of [‘ ‘C]spinoperidol
at no-carrier-added (NCA) levels required the accurate measurement
ofvery small amounts ofcompound.
One
assay technique that has picomole sensitivity for spiro peridol is the radioreceptor assay using tritiated spiro peridol (13), and this method was used to measure the concentration of spiropenidol in several batch prepama tions of [‘ ‘C]spiropenidol.
In the present work we have determined the specific activity of H' ‘CN—andtherefore the absolute specific activity of [I ‘C]spiropenidol—tobe 3.8 ±0.9 Cifizmole
(EOB) (Table 1). The effective specific activity of [I IC]spinopenidol
was shown by radionecepton
assay to
be 2.6 ±1.9 Ci @imole(EOB) (Table I ). In the two cx peniments (Runs 1 and 2, Table 1) where the specific
activity was determined using both methods on the same run, the values of the madiomeceptom assay were lower than those obtained by assay of the HCN. The lower values obtained by the former assay could possibly reflect some undetected contaminants that compete for binding sites during the radiomeceptom assays. We feel that the
presence of contaminants that lower the apparent spe cific activity is unlikely, since the IC50 (the concentration
of the unlabeled compound that reduces the binding of 443
FOWLER, ARNE11', WOLF, MACGREGOR, NORTON, AND FINDLEY
the labeled ligand by 50%) of the alkyl chloride 4 shows it to have about one thousandth
of the potency of spiro
** B & A reagent grade. tt Merck, No. 9385.
It Merck, No. 5775.
penidol (unpublished results). We have removed com pound 4 during the purification, as determined by monitoring the ultraviolet absorbance of the eluent during chromatography. The ultraviolet absorbance associated with the [‘ ‘C]spiroperidolfraction is gener ally less than 0.2 units at 250 nm. The alkyl chloride (4) elutes with the forecut under our chromatography con
We wishto thank Dr. Arnold Friedmanfor a preprint of his work (/2), JanssenPharmaceutics for supplyingunlabeledspiroperidol, and Abbott Laboratoriesfor supplyingpargylineHCI. We also ac
ditions. Although
knowledge discussions with Dr. Tibor Farkas in the initial stages of
we have not identified
the source of the
small ultraviolet absorbance in the product, its presence does not lower the effective specific activity of the [I ‘C]spiropenidol, as can be seen by comparing
Runs 1
II WhatmanGF/C. II New EnglandNuclear. ACKNOWLEDGMENTS
thiswork. This research was carried out at Brookhaven National Laboratory
under Contract No. DE-ACO2-76CH00016with the U.S. Dept. of Energy, and supported by its Office of Basic Energy Sciences and
and 2 in Table I . Furthermore, when a two-step purifi
Offices of Health and Environmental Research, and also under NIH
cation scheme was used, in which 4 was removed before
Grant No. NS15638.
the Grignard reaction, there was no appreciable differ ence in specific activity (Run 3, Table 1). Note that 5-20% of each UV absorbance value in Table 1 is due to
1. REISINEID, FIELDSJZ, YAMAMURAHI, et al: Neuro
spiropenidol, as determined by calculation using the specific activity of HCN as the absolute specific activity
Sc! 21:335—343, 1977 2. LEE I, SEEMANP, RAJPUTA, et al: Receptorbasisfor
of spinoperidol. The value for the H' ‘CN specific activity has been relatively constant for five runs (see Table 1), so that intencomparisons of specific activities determined by radiorecepton assay in Table i should be valid. In one experiment
(Run 4, Table 1), two fractions
with different
UV absorbances gave different specific-activity values by nadioreceptor assay. Although there appears to be a reasonably lange variation in specific-activity values determined by radioneceptor assay, when the values are averaged the specific activities obtained by the two methods
differ by less than an order of magnitude,
with
the value obtained by the radioreceptor assay being consistently lower. The utility of C- i 1-labeled spiropenidol will depend on the time courseof its distribution and whether a high specific-to-nonspecific
binding
ratio is attained
at early
times after injection. Although it is possible that the optimal time for imaging may exceed the half-life ne
striction imposed by carbon-i i (T,,2 = 20 mm), the potential for doing serial studies makes [‘ ‘C]spiroperidol
an attractive candidate as a radioligand for PET studies of human brain neumotransmitten receptors. The uptake in the mouse brain is relatively low (0.5%/organ), al
though it is constant throughout the time course of the present study. If the activity taken up by the brain is associated with dopamine receptors at early times, then
PET studies may be feasible. Studies to determine the time course of specific binding in vivo are in progress.
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273:59—61, 1978 3. REISINE ID, FIELDSJZ, STERN LZ, et al: Alterations in dopaminergic receptors in Huntington's disease. Life Sc! 21:1123—1128, 1977 4. OwEN F, CROW TJ, POULTER M, et al: Increased dopa
mine-receptorsensitivityin schizophrenia.Lancet 11:223—226, I978
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7. FIELDSJZ, REISINEID, YAMAMURAHI: Biochemical
demonstrationof dopaminergicreceptorsin rat and human brain using [3H)spiroperidol. Bra!n Res 136:578—584, 1977
8. LEYSENJE, GOMMERENW, LADURONPM: Spiperone: a ligand of choice for neuroleptic receptors. 1. Kinetics and characteristics of in vitro binding. Biochem Pharmacol 27: 307—316, 1978 9. KUHAR MJ, MURRIN LC, MALOUF AT, et al: Dopamine receptor binding in vivo: The feasibility of autoradiogmaphic
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studies. I Label Comp Rad!opharm 18:102-103, 1981 (abet)
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a potential tool for gamma ray imaging. Life Sc! 28:1911-
FOOTNOTES
1916,1981 * Schwarzkopf
Microanalytical
Laboratory,
Woodside,
NY.
t JEOL MH 100 NMR spectrometer. t Perkin Elmer
@
Model 735B spectrophotometer.
13. CREESE1,SNYDER SH: A simpleand sensitiveradioreceptor assay for antischizophrenic drugs in blood. Nature 270: 180—182, 1977
NVarian Model 635 spectrophotometer. I Aldrich ChemicalCompany.
14. JANSSEN PAJ: 1-Oxo-2,4,8-tr!azasp!ro[4.5ldecanes. US
I PAR Model 374 polarographic analyzer and a Model 303 SMDE
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dropping-mercuryelectrode(PrincetonAppliedResearch).
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nide, carbon dioxide, carbon monoxideand methane via the ‘4N(p,a)'‘Creaction (XV). liii J App! Rad!at Iso: 26: 435-442,1975 16. CANTERFORD DR: Simultaneous determination of cyanide
and sulfide with rapid direct current polarography. Ana! Chem47:88—92, 1975 /7. EPSTEINJ: Estimationof microquantitiesof cyanide.Ana! Chem19:272-274,1947 18. KOLTHOFF IM, SANDELL EB: Textbook of Quantitailve !norgan!c Analys!s, 3rd ed. New York, The Macmillan
Company, 1952, pp 546-547 19. STILL WC, KAHN M, MITRA A: Rapid chromatographic technique for preparative separations with moderate resolu tion. J Org Chem 43:2923-2925, 1978
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and drugs. In Neurotransmitter
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Bind!ng. Yamamura HI, EnnaSJ, Kuhar MJ, Eds.New and [3H]dopamine
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23. JANSSENPAJ: 2,4,8-Tr!azasp!ro[4.5]dec-2-enes. US Pat 3,155,669, Nov 3, 1964;Chem Abstr 62:9142e, 1965 24. MARUYAMAI, NAKAOM, SASAJIMAK, et al: 8-Pheny!thioa!ky!-I ,3,8-triazaspiro[4,5]decane derivatives and their acid addition salts. Jap Pat 74 46,316, Dec 9, 1974; C/tern Abstr82:170954z, 1975
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27. BISCHOFF 5, BITTIGERH, KRAUSSJ:Invivo[3H]spiperone
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Society of Nuclear Medicine 7th Annual Western Regional Meeting October 7-10, 1982
Town and Country Hotel
San Diego, California
Announcement and Call for Abstracts for Scientific Program The Scientific Program Committee welcomes the submission of abstracts of original contributions in Nuclear Mod icine from members and nonmembers of the Society of Nuclear Medicine for the 7th Annual Western Regional Meet
ing.Physicians,scientists,andtechnologists—members andnonmembers—are invitedto participate.Theprogram will be structured to permit the presentation of papers from all areas of interest in the specialty of Nuclear Medicine. Abstracts submitted by technologists are encouraged and will be presented at the Scientific Program. Abstracts for the Scientific Program will be published as a Journal Supplement and will be availableto all registrants atthe meeting.
Guidelines for Submitting Abstracts The abstracts will be printed from camera-ready copy provided by the authors. Therefore, only abstracts prepared on the official abstract form will be considered. These abstract forms will be available from the Western Regional Chapter office (listed below) after March 1, 1982.Abstract forms will be sent to members ofthe Southern California, Northern California, Pacific Northwest, and Hawaii Chapters in a regular mailing in early May, 1982.All other requests will be sent on an individual basis. All participants will be required to register and pay the appropriate fee. Please send the original abstract form, sup porting data, and seven copies to: Justine J. Parker, Administrator 7th Western Regional Meeting P.O. Box 40279 San Francisco, CA 94140 The 7th Annual Western Regional Meeting will have commercial exhibits and all interested companies are invited. Please contact the Western Regional SNM office (address above). Tel: (415)647-0722 or 647-1668. DeadlIne for abstract submIssion: Postmark by mIdnight, July 2, 1982.
Volume 23, Number 5
445
