Synthesis and Biological Evaluation of New 99mTc-N2S2 or 99mTc-N2SO Complexes as Radiopharmaceuticals

Full text of DOI:10.1002/jlcr.481

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 521–528.
DOI: 10.1002/jlcr.481
Synthesis of the [3H] labelled potent and
selective peripheral benzodiazepine receptor
ligand CB 34
A. Latrofa1, G. Trapani1,*, M. Franco1, M.J. Harris2, M. Serra3,
G. Biggio3 and G. Liso1
1 Dipartimento Farmaco-Chimico, Facolta" di Farmacia, Universita" degli Studi di
Bari, Via Orabona 4, 70125 Bari, Italy
2 Ligand Development Services, Amersham Pharmacia Biotech, Laboratories
Forest Farm Estate, Whitchurch, Cardiff CF4 7YT, UK
3 Dipartimento di Biologia Sperimentale, Sezione di Neuroscienze,
"
Universita di Cagliari, Cittadella Universitaria Monserrato,
SS 554 Km 4.5 Monserrato (Cagliari), Italy
Summary
The synthesis of the [3H] labelled peripheral benzodiazepine receptor (PBR)
ligand CB 34, useful forbinding assay, tissue distirbution studies, and
elucidation of the physiological role of PBR, is described. Catalytic reduction
3
with H2 gas and 10% Pd/C catalyst of the key intermediate 2 afforded the
product at a specific activity of 111 Ci/mmol. Copyright # 2001 John Wiley &
Sons, Ltd.
Key Words: PBR ligand; 2-phenyl-imidazo[1,2-a]pyridines; CB 34; tritiation;
binding assay
Introduction
The peripheral benzodiazepine receptor (PBR) is present in most tissues
such as adrenals, kidney and heart, as well as in the brain.1–3 It is
pharmacologically distinct from the central benzodiazepine receptor
"
*Correspondence to: G. Trapani, Dipartimento Farmaco-Chimico, Facolta di Farmacia,
Universita degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy. E-mail: trapani@farmchim.
"
uniba.it (G. Trapani)
Copyright # 2001 John Wiley & Sons, Ltd.
Received 19 February 2001
Revised 16 March 2001
Accepted 27 March 2001
Published online \ \ \ \

522
A. LATROFA ET AL.
which is associated with GABAA receptors and mediates classical
sedative, anxiolytic and anticonvulsant properties of benzodiazepines.4
Conversely, the physiological role of PBR is still unclear, although
several studies have shown that PBR may be involved in calcium
homeostatis, lipid metabolism, mitochondrial oxidation, cell prolifera-
tion, and steroidogenesis.5–7 Particularly interesting is the finding that
PBR expression is selectively increased in human brain tumors.8
Selective ligands forPBR may be useful as tools forelucidating the
physiological role of PBR and therapeutic agents, but only a few
examples of such compounds are known. These include benzodiazepine
compounds (Ro 5-4864),9 isoquinoline carboxamides (PK 11195),10
indoleacetamide derivatives (FGIN-1-27, FGIN-1-44),11 phenoxyphe-
nylacetamides (DAA1097 and DAA1106).12 To examine the role of PBR,
we studied the receptor binding and behavioral profiles of 2-phenyl-
imidazo[1,2-a]pyridine derivatives as potent and selective ligands for
peripheral benzodiazepine receptors.13–15 Ourbinding data demon-
strated that substitution at the C-8 position in the imidazopyridine
compounds is a key factor for improving affinity and selectivity towards
peripheral binding sites. In fact, members of the 8 and 6,8-disubstituted
imidazopyridines show PBR selectivity >103–105. Among these ligands,
the 6,8-dichloro derivative (1, CB34) has been shown to be a particularly
interesting compound because of its affinity, selectivity, and stimulation
of the steroidogenesis.15 In this paperwe describe the synthesis of labelled
CB34, a useful tool for radioligand binding assay, tissue distribution
studies, and elucidation of the physiological role of PBR.
Results and Discussion
On the basis of a retrosynthetic analysis, the target compound [3H]-1
should be obtained via tritiation of the unsaturated diallylamide 2
(Scheme 1). This compound, in turn, can be derived by coupling the
diallylamine with the carboxylic acid 4. Therefore, the key intermediate
of the synthesis is the diallylamide 2, which was prepared in two steps
starting from the ethyl ester 3.13 Hydrolysis of 3 with ethanolic NaOH
gave the sodium salt of acid 4 from which this last compound was
obtained, afterthe usual wokrup, in 85% yield. Amidification of the
acid with diallylamine was attempted by using several condensing
agents. We were able to obtain compound 2 in satisfactory yield (60%)
by using carbonyl diimidazole (CODIm) as dehydrating agent.
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SYNTHESIS OF THE [3H] LABELLED
523
Scheme 1. (a) NaOH, ethanol, RT, 4 h; (b) Diallylamine ꢀ HCl, CODIm,
CH2Cl2, THF, RT; (c) H2, ethyl acetate, 10% Pd/C RT
3
Compound 2 was obtained as a solid and its purity was checked by TLC
and 1H-NMR spectroscopy. To obtain the final labelled compound, the
3
unsaturated amide 2 was tritiated by using H2 in the presence of 10%
Pd/C as catalyst.
Specific [3H] CB34 binding to rat brain was characterized (Serra et al.,
submitted); the data obtained indicated that [3H] CB34 was rapid,
reversible, saturable and of high affinity. Figure 1 shows that this
binding exhibits discrete regional distribution in the rat central nervous
system, being highest in the hypothalamus, whereas the cerebral cortex
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A. LATROFA ET AL.
Figure 1. Representative plots of [3H] CB34 binding to selected rat brain areas
membranes. Membranes from hypothalamus (A), tuberculum olfactorium (B),
hippocampus (C) and cerebral cortex (D) were incubated with [3H] CB34 in
concentrations from 0.05 to 16 nM for 120 min
showed the lowest amount of binding among those areas studied.
Scatchard plot analysis indicate that the regional differences found in
[3H] CB34 binding are due primarily to variation in receptor density
while the affinity of [3H] CB34 forthese ecrognition sites is
approximately equal in all areas studied. Mean values of Kd and Bmax
are: hypothalamus 0.17 ꢁ 0.02 nM and 700 ꢁ 43 fmol/mg protein;
tuberculum olfactorium 0.14 ꢁ 0.03 nM and 524 ꢁ 14 fmol/mg protein;
hippocampus 0.19 ꢁ 0.02 nM and 265 ꢁ 12 fmol/mg protein; cerebral
cortex 0.19 ꢁ 0.02 nM and 188 ꢁ 8 fmol/mg protein.
In summary, we have developed a short synthesis of [3H] CB34 ([3H]-
1) starting from the ester 3 with the unsaturated amide 2 as key
intermediate. Tritiation of 2 proceeds smoothly to provide the title
compound. [3H] CB34 ([3H]-1) was used successfully to establish a
radioligand binding assay at PBR.
Experimental
.
Melting points were determined in open capillary tubes with a Buchi
apparatus and are uncorrected. IR spectra were obtained on a
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SYNTHESIS OF THE [3H] LABELLED
525
1
Perkin-Elmer 283 spectrophotometer (KBr pellets). H NMR spectra
were determined on a Varian or Bruker instrument, operating at 90 or
300 MHz, respectively. Chemical shifts are given in d values downfield
from Me4Si as internal standard. Mass spectra were recorded on a
Hewlett Packard 5995c GC-MS low-resolution spectrometer. All
compounds showed appropriate IR, 1H NMR and mass spectra.
Elemental analyses were carried out with a Carlo Erba model 1106
analyser and results were within ꢁ 0.40% of the theoretical values.
Silica gel 60 (Merck 70–230 mesh) was used for column chromato-
graphy. HPLC analyses were performed under the following conditions:
column: Beckman Ultrasphere C18 (250 ꢂ 10 mm); eluent: 0.1%
trifluoroacetic acid in water: acetonitrile 1:1; flow rate: 3 ml/min;
detection: 254 nM and radioactive; sample injection: 200 mCi in HPLC
eluent (2 ml).
All the reagents were obtained from Aldrich, except for 10% Pd/C
(from Fluka) and ethyl acetate (from Rathburn), and used as received.
[2-(4-chlorophenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)] -
acetic acid (4)
To a solution of the ethyl ester 313 (0.38 g, 1.0 mmol) in ethanol 958
(50 ml), an aqueous sodium hydroxide solution, freshly prepared by
dissolving NaOH pellets (0.040 g, 1.0 mmol) in water(2 ml), was added
dropwise. The mixture was stirred at room temperature and under a
nitrogen atmosphere for 4 h. Then, the solvent was evaporated under
reduced pressure, the resulting residue was dissolved in water (20 ml)
and the aqueous solution washed with ethyl ether(6 ꢂ 30 ml). The water
phase was cooled and acidified with dilute HCl. The resulting white
solid was the essentially pure carboxylic acid 4, which was collected by
filtration. M.p. 260–2628C. IR (KBr) 3400–2500 broad absorption,
1695 cmꢃ1, 1H-NMR (90 MHz) (DMSO d6)d: 3.90 (s, 2H, CH2), 7.1–7.6
(m, 5H, Ar), 8.49 (s, 1H, Ar); MS m/z 310 (M+-CO2, base). Anal.
(C15H9Cl3N2O2) C, H, N.
N,N-Di-allyl-[2-(4-chlorophenyl)-6,8-dichloroimidazo[1,2-a]pyridin-
3-yl)]acetamide (2)
To a suspension of the acid 4 (1.5 g, 4.2 mmol) in an anhydrous mixture
of dichloromethane–tetrahydrofuran (3:1 v/v) (40 ml), maintained under
a nitrogen atmosphere, carbonyl diimidazole (CODIm, 0.80 g,
4.9 mmol) was added in small portions. Then, after 15 min, diallylamine
hydrochloride (0.73 g, 5.5 mmol) was added dropwise. The mixture was
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A. LATROFA ET AL.
stirred for about 2 h at room temperature, then filtered and the filtrate
evaporated under reduced pressure. The resulting residue was purified
by silica gel column chromatography [light petroleum ether/ethyl
acetate 8/2 (v/v) as eluent)] to give the amide 2 (1.10 g, 60% yield).
1
M.p. 179–1818C. IR(KBr) 1650 cmꢃ1; H-NMR (300 MHz) (CDCl3) d:
3.81 (d, 2H, CH2), 4.00 (d, 2H, CH2), 4.04 (s, 2H, CH2), 5.0–5.2 (m, 4H,
¼ CH2), 5.5–5.8 (m, 2H, ¼ CH), 7.3–7.6 (m, 5H, Ar), 8.12 (s, 1H, Ar);
MS m=z 433 (M+, 12), 309 (base). Anal. (C21H18Cl3N2O) C, H, N.
N,N-Di-2; 3-[3H]-n-propyl-[2-(4-chlorophenyl)-6,8-dichloroimida-
zo[1,2-a]pyridin-3-yl)]acetamide. (3H-1), ([3H] CB34)
Compound 2 (4 mg) was transferred into a tritiation flask with ethyl
acetate (2 ml) and 10% Palladium on charcoal catalyst (15 mg). The
mixture was stirred under tritium gas (10 Ci) for approximately 30 min
when the uptake of gas had slowed. The solution was filtered and the
labile activity was removed by rotary evaporation with methanol
(3 ꢂ 5 ml). The residue was dissolved in methanol (45 ml). Yield:
1094 mCi. Approximately 200 mCi of the crude product was rotary
evaporated to dryness, reconstituted in HPLC eluent and purified by
HPLC. The fractions corresponding to the major UV and radioactive
peaks were collected, pooled and rotary evaporated to dryness and
dissolved in ethanol. The solution was rotary evaporated and
redissolved in ethanol twice more and the solution was counted. Yield:
26 mCi.
A sample of the product was analysed by HPLC and mass
spectrometry. The radiochemical purity was shown to be 99.3%, the
active material co-eluting with the inactive reference. The specific
activity was determined as 111 Ci/mmol by mass spectrometry. The
spectrum of the radioactive was consistent with the mass spectrum of
the inactive reference. [3H] CB34 could be diluted to 1 mCi/ml with
ethanol and dispensed.
Binding assays
Females Sprague–Dawley CD rats (Charles River, Como, Italy) was
used. After killing, the brain areas were rapidly removed, dissected and
stored at ꢃ808C until assayed. The tissues were thawed and
homogenized in 50 volumes of Dulbecco’s phosphate-buffered saline
(PBS, pH 7.4) at 48C with a Polytron PT 10 (setting 5 for 20 s). The
homogenate was centrifuged at 40 000 g for 30 min, and the resulting
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SYNTHESIS OF THE [3H] LABELLED
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pellet was resuspended in 50 volumes of PBS and recentrifuged. The
new pellet was resuspended in 10 volumes of PBS and used for the
assay. [3H] CB34 binding was determined in a final volume of 1000 ml,
comprising 100 ml of membranes (0.35–0.40 mg of protein), 100 ml of
[3H] CB34 (127 Ci mmolꢃ1; Amersham Pharmacia Biotech) at a final
assay concentrations of 0.05–16 nM and 800 ml of PBS. The incubations
were performed at 258C for120 min, initiated by addition of membranes
and terminated by rapid filtration through glass-fibre strips (Whatman
GF/B) presoaked with 0.3% polyethyleneimine in a Cell Harvester
filtration manifold (Brandel). The filters were washed five times with
4 ml of ice-cold PBS, and filter-bound radioactivity was quantified by
liquid scintillation spectrometry. Nonspecific binding was defined as
binding in the presence of 10 mM unlabelled PK 1195. Specific binding
was determined by subtracting nonspecific from total binding and was
approximately 70–80% of total binding.
Acknowledgements
This work was supported by a grant from Fondi di Ateneo ex 60%
"
dell’Universita degli Studi di Bari.
References
1. Anholt RR, De Souza EB, Oster-Granite ML, Snyder SH. J Pharmacol
Exp Ther 1985; 233: 517–526.
2. De Souza EB, Anholt RR, Murphy KMM, Snyder SH, Kuhar MJ.
Endocrinology 1985; 116: 567–573.
3. Anholt RRH, Murphy KMM, Mack GE, Snyder SH. J Neurosci 1984; 4:
593–603.
4. Andrews JS, Stephents DN. In Psychopharmacology of Anxiolytic and
Antidepressants, File SE (ed.). Pergamon Press: New York, 1991; 107–130.
5. Auta J, Romeo E, Kozikowski A, Ma D, Costa E, Guidotti A. J
Pharmacol Exp Ther 1993; 265: 649–656.
6. Barbaccia ML, Roscetti G, Trabucchi M, et al. Neuropharmacol 1996; 35:
1299–1305.
7. Baulieu E-E, Robel PJ. J Steroid Biochem Mol Biol 1990; 37: 395–403.
8. Brown RC, Degenhardt B, Kotoula M, Papadoupoulos V. Cancer Lett
2000; 156: 125–132.
9. Marangos PJ, Patel J, Boulenger JP, Clark-Rosemberg R. Mol Pharmacol
1982; 22: 26–32.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 521–528

528
A. LATROFA ET AL.
10. Le Fur G, Perrier ML, Vaucher N, et al. Life Sci 1983; 32: 1839–1847.
11. Romeo E, Auta J, Kozikowski A, et al. J Pharmacol Exp Ther 1992; 262:
971–978.
12. Okuyama S, Chaki S, Yoshikawa R, et al. Life Sci 1999; 64: 1455–1464.
13. Trapani G, Franco M, Ricciardi L, et al. J Med Chem 1997; 40: 3109–3118.
14. Trapani G, Franco M, Latrofa A, et al. J Med Chem 1999; 42: 3934–3941.
15. Serra M, Madau P, Chessa MF, et al. Br J Pharmacol 1999; 127: 177–187.
Copyright # 2001 John Wiley & Sons, Ltd.
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