Synthesis and preliminary biological characterization of a new potential 125I-radioligand for dopamine and serotonin receptors

Article abstract of DOI:10.1016/S0968-0896(01)00229-2

The synthesis and a preliminary biological characterization of a new class of N-benzyl-aminoalcohols which have serotonin (5-HT₂) and dopamine (D₂) receptor affinity is described. In vitro competition binding studies were conducted with the new molecules and ³H-spiperone on crude membrane preparation from rat striatum and frontal cortex. One of these compounds, 3-benzylamino-1-(4-fluoro-2-iodophenyl)-propan-1-ol (6f), whose IC₅₀ values are in the micromolar range for both the D₂ and 5-HT₂ receptors, was prepared in iodine-125 labelled form (6i) by nucleophilic substitution of the bromine atom of 3-benzylamino-1-(2-bromo-4-fluorophenyl)-propan-1-ol (6d). In the in vivo studies, conducted on rats, the radiolabelled molecule 6i shows a good capacity to cross the blood-brain barrier (BBB) with a mean value of first pass cerebral extraction (E) of ca. 50% when the regional cerebral blood flow, measured with microsphere technique, is in the experimental animal's physiologic range (0.8-1 mL/min/g). A preliminary in vitro autoradiographic distribution on coronal rat brain slices of the radioiodinated molecule showed that it was preferentially localized in the striatum and in the cerebral regions rich in dopamine- and serotonin receptors, even if a high non-specific binding was observed.

Full text of DOI:10.1016/S0968-0896(01)00229-2

Bioorganic & Medicinal Chemistry 9 (2001) 3197–3206
Synthesis and Preliminary Biological Characterization of a New
125
Potential I-Radioligand for Dopamine and Serotonin Receptors
Antonio Guarna,a,* Gloria Menchi,a Giovanna Berti,a Nicoletta Cini,a
Anna Bottoncetti,b Silvia Raspanti,b Alessandro Politib and Alberto Pupib
aDipartimento di Chimica Organica ‘‘U. Schiff’’, e Centro di Studio sulla Chimica e la Struttura dei
`
Composti Eterociclici e loro Applicazioni, CNR, Universita di Firenze, Via G. Capponi 9, Firenze I-50121, Italy
b
`
Dipartimento di Fisiopatologia Clinica, Nuclear Medicine Unit, Universita di Firenze, Viale G. Pieraccini 6,
Firenze I-50134, Italy
Received 16 February 2001; accepted 29 May 2001
Abstract—The synthesis and a preliminary biological characterization of a new class of N-benzyl-aminoalcohols which have ser-
otonin (5-HT2) and dopamine (D2) receptor affinity is described. In vitro competition binding studies were conducted with the new
3
molecules and H-spiperone on crude membrane preparation from rat striatum and frontal cortex. One of these compounds, 3-
benzylamino-1-(4-fluoro-2-iodophenyl)-propan-1-ol (6f), whose IC50 values are in the micromolar range for both the D2 and 5-HT2
receptors, was prepared in iodine-125 labelled form (6i) by nucleophilic substitution of the bromine atom of 3-benzylamino-1-(2-
bromo-4-fluorophenyl)-propan-1-ol (6d). In the in vivo studies, conducted on rats, the radiolabelled molecule 6i shows a good
capacity to cross the blood–brain barrier (BBB) with a mean value of first pass cerebral extraction (E) of ca. 50% when the regional
cerebral blood flow, measured with microsphere technique, is in the experimental animal’s physiologic range (0.8–1 mL/min/g). A
preliminary in vitro autoradiographic distribution on coronal rat brain slices of the radioiodinated molecule showed that it was
preferentially localized in the striatum and in the cerebral regions rich in dopamine- and serotonin receptors, even if a high non-
specific binding was observed. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
by in vitro competition binding studies on crude rat
brain membrane preparations. Moreover we have taken
into account the possibility to introduce, by nucleophilic
substitution, a radioisotopic atom in a functionally sui-
table position of a molecule of this class and to ensure
the possibility for the analytical discrimination and
separation between the radiolabelled molecule and the
non-labelled precursor so as to obtain a radioligand
with high specific activity. The radiolabelled molecule
was then tested in in vivo experiments to value its ability
to cross the blood–brain barrier in the experimental
animals and in vitro autoradiographic assays to
determine its cerebral distribution.
The expanding sector of in vivo imaging devices,1,2 with
the efficient implementation of single photon and posi-
tron emission tomography (SPECT and PET respec-
tively), stimulates radiopharmaceutical research to
synthesize new radiolabelled molecules allowing the
evaluation of brain functions. The use of radioligands to
assess in vivo receptor concentrations has enabled
researchers to investigate the extent to which specific
neurotransmitter systems are involved in the pathogen-
esis and progression of mental illness and has provided
the basis for the design of better therapies and for
quantitative monitoring of therapeutic response.3,4
In this paper we report the synthesis of new molecules,
N-benzyl-aminoalcohol derivatives, and their pre-
liminary characterization as ligands for brain receptors
Results
The synthesis of a series of new secondary N-benzyl-b-
aminoalcohol derivatives of general formula 6a–6h is
shown in Scheme 1. b-Chloroketones 1a–1e, commer-
cially available or prepared by Friedel–Craft acyla-
tion,5,6 were transformed, by reduction with NaBH4,
*Corresponding author. Tel.: +39-055-2757611; fax: +39-055-
2476964; e-mail: guarna@chimorg.unifi.it
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00229-2

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A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 3197–3206
into the corresponding chloroalcohols (2a–2e) which
were coupled with phthalimide in DMF at 120 ꢀC for 8
h in the presence of KF7,8 to give imides 4a–4e. Imide 4f
was obtained from the product 3 prepared by the Mit-
sunobu reaction9 from b-chloroalcohol 2c with 3-iodo-
phenol. Imide derivatives 4a–4f were hydrolyzed to the
corresponding amines (5a–5f) in high yield by hydrazine
in MeOH at reflux in the presence of HCl.6,10 These
amines, sufficiently pure to be used without further
purification, were transformed into the target
compounds 6a–6h by treatment with an equivalent of
benzaldehyde or p-fluorobenzaldehyde in the presence
of NaBH3CN at room temperature for 24 h.11
The capacity of the synthesized molecules to interact
with the D2- and 5-HT2 receptors was evaluated using
inhibition experiments of the specific 3H-spiperone
binding in homogenates of striate and frontal cortex rat
brain tissue, respectively. All the substances were tested
at a final concentration of 10 mM and the inhibition
Scheme 1. Preparation of aminoalcohols 6a–6h: (a) NaBH4 in CH3OH at 0 ꢀC; (b) 3-iodophenol, DEAD/PPh3 in THF, 21 h at rt; (c) phthalimide
and anhydrous KF in DMF at 120 ꢀC for 8 h; (d) hydrazine in H2O/CH3OH/HCl, reflux, 3 h; (e) benzaldehyde or p-fluorobenzaldehyde, NaBH3CN
in CH3OH, 24 h at rt.

A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 3197–3206
3199
data are reported in Table 1. Molecule 6a, which is the
reference molecule for our study, shows poor affinity for
the D2 receptors and a very low affinity towards the 5-
HT2 receptors. Molecules 6c–6f, containing the 3-aryl
group substituted with two halogen atoms, which can be
either fluorine and bromine, or, alternatively fluorine
and iodine atoms, show an affinity increment especially
for 5-HT2 receptors and, when the substituent in posi-
tion 2of the benzene ring is an iodine atom as in 6f,
there is a relevant affinity increment also towards the D2
receptors. The introduction of a fluorine atom on the N-
benzyl ring, as in compounds 6b and 6g, causes a slight
increase of the affinity for the D2 receptor binding site
with respect to the analogous molecules (6a and 6d),
thus reducing their selectivity towards the studied
receptors. Compound 6h, which has a iodophenoxy
group, shows a slightly lower affinity with respect to the
hydroxyl derivative analogue 6d.
The best ligand, amongst those containing an iodine
atom, turned out to be the compound 3-benzylamino-1-
(4-fluoro-2-iodophenyl)-propan-1-ol (6f) (Table 1), that
in the in vitro assays displayed a dose-related inhibition
3
of H-spiperone specific binding with IC50 values in the
micromolar range (about 20 mM in the 5-HT2 assay,
frontal cortex homogenates, and about 30 mM in the D2
assay, striate homogenates).
To study the in vivo cerebral extraction of this new
ligand, we prepared the compound 6f in the 125I-iodine
labelled form [3-benzylamino-1-(4-fluoro-2-[125I]-iodo-
phenyl)-propan-1-ol (6i)], by Cu(I)-assisted nucleophilic
substitution of the bromine atom with 125I-iodine on
3-benzylamino-1-(2-bromo-4-fluorophenyl)-propan-1-ol
6d (Scheme 2). This procedure allowed the separation of
the radiolabelled ligand 6i from the precursor 6d. The
exchange of bromine with the 125I radioisotope was
performed by heating compound 6d at 100 ꢀC for 90
min in an ethanol/water mixture containing Na125I and
catalyzed by an organo-copper (I) complex formed in
situ.12À14 The resulting mixture was separated from the
unreacted 125I-iodide by elution on an ion-exchange
column and then, compound 6i was separated by
reversed-phase HPLC from the starting compound 6d,
obtaining the radiochemically and chemically pure com-
pound 6i (Fig. 1) (specific activity 2.2 Ci/mmol) which
was used for in vivo rat brain uptake measurement.
Table 1. Compounds 6a–6h were tested at concentrations of 10 mM
against 3H-spiperone (2.5 nM). D2 receptor assay was performed on
rat striate tissue homogenates added with ketanserine (4Â10À8 M fin),
to block 5-HT2 receptors, and with sulpiride (10À5 M fin), to deter-
mine the non-specific binding. 5-HT2 receptor assay was performed on
rat frontal cortex tissue homogenates added with sulpiride (10À6
M
fin) to block the D2 receptors, and with ketanserine (10À5 M), to
determine the non-specific binding
Ligand
D2
5-HT2
The octanol/water partition coefficient experimentally
determined according to Leo et al.15 was shown to be
(6a)
(6b)
(6c)
(6d)
(6e)
(6f)
(6g)
6Æ5
2 Æ8
4
9
11Æ1
2 Æ3
60 Æ7
48Æ7
Æ10
2 Æ9
6
19Æ5
18Æ248
51Æ14
39Æ1246
50Æ10
Æ5
Figure 1. Radiogram and ultraviolet chromatograms to document the
radiochemical and chemical purity of compound 6i. (A): UV chroma-
togram of a mixture of 6f and 6d; ( B) and (C): Radiogram and UV
chromatogram of 6i after HPLC purification.
(6h)
14Æ4
42Æ6
Scheme 2. Preparation of aminoalcohol 6i: Na125I, Cu(I)-organo complexes in EtOH–H2O, 100 ꢀC, 90 min.

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A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 3197–3206
4.10 which is a value in the range useful for a good
extraction in the brain.16
for both the 5-HT2 and D2 receptor populations. The
corresponding 125I-iodine labelled compound 6i, pre-
pared radiochemically and chemically pure, showed a
high first-pass cerebral extraction (E) value, according
to its octanol/water partition coefficient value of 4.10.
Indeed the molecule easily reaches the cerebral tissue
crossing the BBB after its iv administration. The ability
to cross the BBB is of fundamental importance for a
cerebral tracer, but the useful range of this transfer is
defined also by the other kinetic and pharmacological
characteristics of the tracer. Indeed, if a molecule with a
very high affinity toward a given cerebral binding site
has also a very high capacity to cross the BBB, then the
tracer uptake will reflect more cerebral blood flow
dependent delivery than the density of its cerebral
binding sites.22 On the other hand, also a molecule with
high affinity and low cerebral extraction may have
kinetics which are too slow to reach a sufficient con-
centration in the brain to be actually used in an emis-
sion tomographic study. As a matter of fact, the
extraction values shown by 6i (mean about 50%) are in
a range similar to that of IBZM, a benzamide derivative
clinically used for imaging the D2 dopamine receptors
(Fig. 2).
The cerebral extraction fraction (E), calculated as the
ratio between the unidirectional influx constant Ki (mL/
min/g) and the regional cerebral blood flow rCBF (mL/
min/g), was 0.56Æ0.14 (meanÆS.D.) (a range of Ki
values between 0.6 and 2.1 mL/min/g, were obtained
varying the rCBF values between 0.8 and 4.8 mL/min/g
with hypercapnia). The experimental data of the mole-
cule 6i tested in different tissues were compared with
those previously obtained17 using three well-known
receptor-binding tracers, IBZM,18 Epidepride19 and
NCQ298,20 by fitting each tracer data-set in a 4-para-
meter converging function (based on a saturable
recruitment model of the cerebral capillaries21) (Fig. 2).
The cerebral extraction fraction of 6i is close to that of
IBZM, the most diffusible molecule in the group. In
vitro autoradiography performed on rat coronal brain
slices showed accumulation of the radioligand in those
regions of the rat brain known to be rich in dopamine-
and serotonin receptors, thus confirming the affinity
towards this type of receptors already observed in the in
vitro binding experiments. However it is present a high
non-specific binding, even after extensive washing of the
slices, which does not allow to have a well definite
auotradiographic image.
A preliminary in vitro autoradiographic study on rat
cerebral coronal section revealed an accumulation of the
radioligand 6i in those cerebral regions rich in dopa-
mine and serotonin receptors (about 15% of activity
was displaced from the specific binding sites by adding
(+)-butaclamol and ketanserine); however there was
also a very high fraction of radioactivity not specifically
bound. This non-specific binding was higher than that
obtained in the rat brain homogenate assays. We hypo-
thesize that this fact could be attributed to a binding of
the radioligand 6i with tissue components that are dis-
carded during the homogenate preparation procedure.
Indeed the high lipophilicity of the molecule, which
allows a better penetration through the BBB, also could
increase the non-specific binding interaction with tissue
components.23
Discussion and Conclusions
We report the synthesis of the compounds 6a–6h, which
belong to a class of N-benzyl aminoalcohols; the mole-
cules (6c, 6d, 6g and 6h), containing a bromine atom on
the benzene ring, can be substituted with a radioactive
iodine atom obtaining pure labelled compounds.
In the preliminary in vitro characterization studies as
brain receptor ligand, compound 6f, tested in racemic
form, showed a detectable affinity at micromolar level
The main characteristic usually required for an in vivo
receptor-binding tracer is to have a high specificity and
a high affinity for the receptor population type under
study, but that is not the case of 6i. Nevertheless it is
worthwhile to mention that the increasing sensitivity of
PET and SPECT instrumentation is encouraging
researchers to study not only the receptor population
concentrations and their state of affinity, but also the
different receptor populations interaction, and, actually,
also the influence of the endogenous ligand; such stud-
ies, allowing the elucidation of the action mechanism of
drugs which are active on different type of
receptors,24À27 or the monitoring of pharmacological
therapies, can motivate tracers with multiple specificity
or moderate affinity, respectively.
For this reason a ligand having moderate affinity and
specificity as those showed by 6i, could have a role in
the in vivo studies where the presence of the endogenous
transmitter and the complexity of the neuronal trans-
mission should be addressed; however the very high
Figure 2. First pass cerebral extraction (experimental data fitting
based on the capillaries recruitment model) of compound 6i, IBZM,
Epidepride and NCQ298.

A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 3197–3206
3201
non-specific binding of the compound 6i does not allow
yet to propose its use as in vivo radiotracer.
phase was washed with aqueous 5% NaOH and brine,
dried over Na2SO4. Removal of the solvent gave a 2:3
mixture of 1b and 1c as a light yellow oil (21.14 g).
Purification by flash chromatography (petroleum ether–
EtOAc, 20:1) afforded 1b (7.48 g, 31%, Rf 0.34) and 1c
(9.98 g, 42%, Rf 0.48). 1b: GC–MS m/z 229 (M–Cl)+;
1H NMR: d 7.80 (t, J=7.3 Hz, 1H), 7.30–7.41 (m, 2H),
3.87 (t, J=6.6 Hz, 2H), 3.40 (td, J=6.6, 3.3 Hz, 2H);
13C NMR: d 193.7 (s, JF=3.7 Hz), 161.7 (s, JF=259.1
Hz), 131.8 (d, JF=3.7 Hz), 128.8 (s, JF=10.0 Hz), 128.3
(d, JF=3.7 Hz), 123.8 (s, JF=12.8 Hz), 120.4 (d,
JF=27.5 Hz), 46.0 (t, JF=8.2Hz), 38.1 (t, JF=2.8 Hz).
In conclusion, although radiolabelled ligand 6i doesn’t
possess all the desired characteristics for its use as in vivo
cerebral receptor tracer, the chemical structure of the
class to which it belongs together with the other com-
pounds of this study, has several promising features,
mainly due to the fact that it could be easily modified
and labelled with radioisotopes. Future efforts will be
devoted to extend this synthetic approach to new com-
pounds of this class, in order to optimize their affinity
and specificity and to reduce their non-specific binding,
using appropriate structural changes.
1
1c: GC–MS m/z 229 (M–Cl)+; H NMR: d 7.47 (dd,
J=8.4, 5.9 Hz, 1H), 7.30 (dd, J=8.1, 2.5 Hz, 1H), 7.05
(td, J=8.1, 2.2 Hz, 1H), 3.84 (t, J=6.4 Hz, 2H), 3.38 (t,
J=6.4 Hz, 2H); 13C NMR: d 200.7 (s), 163.5 (s,
JF=256.4 Hz), 130.9 (d, JF=9.1 Hz), 128.3 (s), 121.2 (d,
JF=24.7 Hz), 120.5 (s, JF=18.3 Hz), 114.7 (d, JF=11.0
Hz), 44.9 (t), 38.5 (t).
Experimental
Chemistry
Melting points were determined with a Buchi 510 appa-
ratus without corrections. Chromatographic separations
were performed under pressure on silica gel using flash-
column techniques; Rf values refer to TLC carried out
on 25 mm silica gel plates (Merck F254), with the same
eluent indicated for column flash chromatography.
3-Chloro-1-(2-fluoro-4-iodiophenyl)-propan-1-one (1d)
and 3-Chloro-1-(4-fluoro-2-iodophenyl)-propan-1-one
(1e). Prepared as reported for 1b and 1c. Starting from
1-fluoro-3-iodiobenzene (13.23 g, 59.6 mmol), a 2:3
mixture of 1d and 1e as a light yellow oil (18.0 g) was
recovered. Purification by flash chromatography (pet-
roleum ether–EtOAc, 20:1) afforded 1d (4.63 g, 26%, Rf
0.33) and 1e (7.8 g, 44%, Rf 0.51). 1d: GC–MS m/z 314
(M+, 30%) and 312(M +, 70%); 1H NMR: d 7.62–7.46
(m, 3H), 3.88 (t, J=6.2Hz, 2H), 3.42(td, J=6.6, 3.4 Hz,
2H); 13C NMR: d 194.2(s, JF=0 Hz), 161.4 (s,
JF=255.9 Hz), 135.4 (s), 134.5 (d, JF=3.0 Hz), 131.8 (d,
JF=3.0 Hz), 124.4 (d, JF=26.2 Hz), 100.9 (s, JF=8.6
Hz), 46.3 (t, JF=8.2Hz), 38.3 (t, JF=2.5 Hz). 1e: GC–
MS m/z 314 (M+, 30%) and 312(M +, 70%); 1H NMR:
d 7.68 (dd, J=5.8, 2.6 Hz, 1H), 7.50 (dd, J=5.8, 2.9 Hz,
1H), 7.11 (td, J=8.0, 2.5 Hz, 1H), 3.88 (t, J=6.4 Hz,
2H), 3.37 (t, J=6.4 Hz, 2H); 13C NMR: d 200.4 (s),
164.4 (s, JF=257.8 Hz), 140.3 (s), 131.4 (d, JF=9.0 Hz),
129.7 (d, JF=23.7 Hz), 116.7 (d, JF=21.2 Hz), 93.0 (s,
JF=8.0 Hz), 45.3 (t), 39.9 (t).
1H and 13C NMR spectra were recorded in CDCl3 at
200 and 50.33 MHz, respectively, unless otherwise sta-
ted, on a Varian Gemini instrument. EI mass spectra
were carried out at 70 eV ionizing voltage with a Carlo
Erba QMD 1000 spectrometer. IR spectrum was recor-
ded with a Perkin–Elmer 881 spectrophotometer. HPLC
analyses were performed on a Shimadzu instrument,
model SCL-6B, equipped with a Waters Spherisorb
S5C8 column (4.6Â250 mm) and with a UV detector,
model UVICORD VW 2251-Pharmacia, operating at
254 nm and radioactivity detector Gaby-Raytest. Ana-
lytical data is reported as retention time (tR) in minutes.
Dowex 1X8–200 ion-exchange resin (Sigma–Aldrich),
previously activated, was used to eliminate the inorganic
salts and the unreacted Na125I.
3-Chloro-1-phenyl-propan-1-ol (2a). NaBH4 (2.00 g,
53.37 mmol) was slowly added to a stirred solution of 3-
chloropropiophenone (1a) (5.0 g, 29.65 mmol) in MeOH
(100 mL) cooled at 0 ꢀC. The reaction mixture was left 10
min at room temperature and was then adjusted to pH 7
with a saturated solution of NH4Cl. The organic phase
was extracted with Et2O, washed with brine and dried
over Na2SO4. Pure 2a (3.65 g, 72%) was obtained by
flash chromatography (CH2Cl2–petroleum ether 2:1, Rf
0.30). GC–MS m/z 170 (M+); 1H NMR: d 7.42–7.23 (m,
5H), 4.74 (d, J=7.2 Hz, 1H), 3.87–3.65 (m, 2H), 2.31–
2.17 (m, 2H); 13C NMR: d 148.5 (s), 130.6 (d), 128.3 (d),
126.1 (d), 56.4 (d), 41.7 (t), 39.4 (t).
THF was distilled from Na/benzophenone. DMF was
distilled under a nitrogen atmosphere (bp. 102 ꢀC, 100
mmHg).
3-Chloropropiophenone (1a) was an Aldrich product.
Na125I in 0.2N NaOH solution was purchased from
Nycomed Amersham Sorin. (10 mL, 3.7–18.5 GBq (100–
500 mCi)/mL, 125I 97%, 126I < 3%, radiochemical purity
> 99%, carrier free).
1-(4-Bromo-2-fluorophenyl)-3-chloro-propan-1-one (1b) and
1-(2-Bromo-4-fluorophenyl)-3-chloropropan-1-one
(1c).
Anhydrous AlCl3 (21.0 g, 157.5 mmol) was added to a
solution of 1-bromo-3-fluorobenzene (15.67 g, 89.5
mmol) and 3-chloropropionyl chloride (15.96 g, 126
mmol) in CS2 (40 mL). The mixture was refluxed for 8
h, stirred at room temperature overnight and then
added to ice (100 g) and adjusted to pH 2with HCl. The
resulting solution was extracted with Et2O; the organic
1-(4-Bromo-2-fluorophenyl)-3-chloro-propan-1-ol
(2b).
Prepared as reported for 2a. Starting from 1b (1.54 g,
4.91 mmol), after purification by flash chromatography
(CH2Cl2–petroleum ether 2:1, Rf 0.35), pure 2b (0.69 g,
53%) was obtained. GC–MS m/z 268 (M+, 30%) and 2 66
1
(M+, 70%); H NMR: d 7.71 (dd, J=8.8, 6.3 Hz, 1H),
7.35–7.30 (m, 1H), 7.22–7.17 (dd, 1 H), 5.19 (t, J=6.4 Hz,

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A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 3197–3206
1H), 3.77–3.45 (m, 2H), 2.10–2.00 (m, 2H); 13C NMR: d
159.6 (s, JF=250.8 Hz), 130.1 (s, JF=13.6 Hz), 128.7 (d,
JF=50.0 Hz), 128.0 (d, JF=3.5 Hz), 121.8 (s, JF=10.1
Hz), 119.4 (d, JF=5.2Hz), 65.6 (d), 41.5 (t), 40.2(t).
sphere, to a solution of 2a (2.5 g, 14.64 mmol) in anhy-
drous DMF (120 mL). The reaction mixture was heated
at 120 ꢀC for 6 h and stirred overnight at 25 ꢀC, then 240
mL of H2O was added and the resulting solution
extracted with Et2O. The organic phase was washed
with H2O, with aqueous 5% NaOH solution and brine
and dried over Na2SO4. After evaporation of the sol-
vent, the crude product was first washed with Et2O and
then crystallized from acetone recovering pure com-
pound 4a (2.1 g, 54%). GC–MS m/z 281 (M+); 1H
NMR: d 7.80 (m, 4H), 7.45–7.15 (m, 5H), 4.85 (d,
J=8.2 Hz, 1H), 3.98–3.71 (m, 2H), 2.18–1.98 (m, 2H);
13C NMR: d 166.3 (s), 156.1 (s), 148.9 (s), 132.6 (d),
129.5 (d), 127.1 (d), 59.8 (d), 34.5 (t), 32.5 (t).
1-(2-Bromo-4-fluorophenyl)-3-chloro-propan-1-ol
(2c).
Prepared as reported for 2a. Starting from 1c (2.80 g,
10.56 mmol), after flash chromatography (CH2Cl2–pet-
roleum ether 2:1, Rf 0.35), pure 2c (1.8 g, 64%) was
recovered. GC–MS m/z 268 (M+, 30%) and 266 (M+,
70%); 1H NMR: d 7.55 (dd, J=8.8, 6.0 Hz, 1H), 7.30 (d,
J=2.5 Hz, 1H), 7.07 (td, J=8.5, 2.6 Hz, 1H), 5.27 (d,
J=8.8 Hz, 1H), 3.82–3.69 (m, 2H), 2.15 (d, J=3.7 Hz,
1H), 2.10–2.02 (m, 2H); 13C NMR: d 161.6 (s, JF=249.4
Hz), 138.8 (s), 128.3 (d, JF=11.0 Hz), 121.4 (s, JF=9.1
Hz), 119.8 (d, JF=24.6 Hz), 114.9 (d, JF=21.0 Hz), 69.5
(d), 41.5 (t), 38.8 (t).
2-[3-(4-Bromo-2-fluorophenyl)-3-hydroxypropyl]-isoin-
dole-1,3-dione (4b). Prepared as reported for 4a. Start-
ing from 2b (0.600 g, 2.24 mmol), after crystallization
from acetone pure 4b (0.246 g, 29%) was collected. GC–
MS m/z 378 (M+); 1H NMR: d 7.80 (m, 4H), 7.42(t, J=8.0
Hz, 1H), 7.29 (d, 1H), 7.15 (dd, J=9.5, 1.8 Hz, 1H), 4.90 (d,
J=8.8 Hz, 1H), 3.96–3.69 (m, 2H), 2.12–1.94 (m, 2H); 13C
NMR: d 168.9 (s), 159.1 (s, JF=248.5 Hz), 134.2 (d), 132.0
(s), 130.0 (s, JF=13.7 Hz), 128.4 (d, JF=4.5 Hz), 127.7 (d,
JF=2.7 Hz), 123.4 (d), 121.1 (s, JF=9.1 Hz), 118.8 (d,
JF=24.6 Hz), 64.9 (d, JF=1.8 Hz), 36.5 (t), 34.6 (t).
3-Chloro-1-(2-fluoro-4-iodiophenyl)-propan-1-ol
(2d).
Prepared as reported for 2a. Starting from 1d (0.63 g,
2.02 mmol), purification by flash chromatography
(CH2Cl2–petroleum ether 2:1, Rf 0.37) gave pure 2d
(0.44 g, 70%). GC–MS m/z 316 (M+, 30%) and 314
1
(M+, 70%); H NMR d 7.50 (dd, J=8.0 Hz, 1H), 7.40
(d, J=9.8 Hz, 1H), 7.20 (t, J=7.6 Hz, 1 H), 5.18 (t,
J=6.2 Hz, 1H), 3.81–3.55 (m, 2H), 2.24–2.20 (m, 2H).
3-Chloro-1-(4-fluoro-2-iodophenyl)-propan-1-ol (2e). Pre-
pared as reported for 2a. Starting from 1e (4.05 g, 12.97
mmol), purification by flash chromatography (CH2Cl2–
petroleum ether 2:1, Rf 0.33) gave pure 2e (2.73 g, 67%).
2-[3-(2-Bromo-4-fluorophenyl)-3-hydroxypropyl]-isoin-
dole-1,3-dione (4c). Prepared as reported for 4a. Start-
ing from 2c (1.08 g, 4.03 mmol), after crystallization
from acetone pure compound 4c (0.71 g, 47%) was
recovered. GC–MS m/z 378 (M+); 1H NMR: d 7.81 (m,
4H), 7.59 (dd, J=8.4, 6.2Hz, 1H), 7.20 (dd, J=8.4, 2.6
Hz, 1H), 7.05 (td, J=8.4, 2.6 Hz, 1H), 4.94 (d, J=11.4
Hz, 1H), 4.08–3.84 (m, 2H), 3.05 (d, J=4.4 Hz, 1H),
2.23–2.08 (m, 1H), 1.89–1.72 (m, 1H); 13C NMR: d
169.1(s), 163.0 (s, JF=255.4 Hz), 137.8 (s), 134.4 (d),
132.2 (s), 128.4 (d, JF=8.6 Hz), 123.6 (d), 119.8 (d,
JF=25.3 Hz), 115.1 (d, JF=21.2 Hz), 99.2 (s), 69.7 (d),
36.8 (t), 35.0 (t).
1
GC–MS m/z 316 (M+, 30%) and 314 (M+, 70%); H
NMR: d 7.56–7.50 (m, 2H), 7.11 (td, J=8.4, 2.6 Hz,
1H), 5.11 (dt, J=9.5, 3.5 Hz, 1H), 3.89–3.66 (m, 2H),
2.10–2.00 (m, 2H); 13C NMR: d 161.7 (s, JF=251.8 Hz),
141.8 (s, JF=3.0 Hz), 128.0 (d, JF=8.0 Hz), 126.4 (d,
JF=23.7 Hz), 116.0 (d, JF=20.6 Hz), 96.2 (s, JF=8.0
Hz), 74.2(d), 41.7 (t), 40.3 (t).
2-Bromo-1-[3-chloro-1-(3-iodophenoxy)-propyl]-4-fluoro-
benzene (3). To a stirred solution of 2c (1.8 g, 6.73
mmol), 3-iodophenol (1.6 g, 7.27 mmol) and triphenyl-
phosphine (1.94 g, 7.4 mmol) in anhydrous THF (50
mL), at 0 ꢀC and under nitrogen atmosphere, diethyl
azodicarboxylate (DEAD) (1.4 g, 8.04 mmol) was
added. The mixture was stirred at 0 ꢀC for 1 h and for
24 h at room temperature; the solvent was distilled off
under reduced pressure and the residue was washed with
petroleum ether and then purified by flash chromato-
graphy (petroleum ether, Rf 0.30) to give pure com-
pound 3 (1.4 g, 44%). GC–MS m/z 470 (M+, 30%) and
2-[3-(2-Fluoro-4-iodophenyl)-3-hydroxypropyl]-isoindole-
1,3-dione (4d). Prepared as reported for 4a. Starting
from 2d (0.59 g, 1.88 mmol), crystallization from ace-
tone gave pure 4d (0.40 g, 50%). GC–MS m/z 425
1
(M+); H NMR: d 7.79 (m, 4H), 7.46 (dd, J=8.5, 1.9
Hz, 1H), 7.34 (d, J=1.8 Hz, 1H), 7.32(d, J=1.4 Hz,
1H), 4.90 (m, 1H), 3.95–3.88 (m, 2H), 3.17 (d, J=4.7
Hz, 1H), 2.10–1.93 (m, 2H); 13C NMR: d 168.9 (s),
158.8 (s, JF=249.5 Hz), 134.2 (d), 133.6 (d, JF=2.7 Hz),
131.9 (s), 130.8 (s, JF=13.6 Hz), 128.7 (d, JF=4.5 Hz),
124.4 (d, JF=23.6 Hz), 123.4 (d), 91.8 (s, JF=8.5 Hz),
64.9 (d), 36.5 (t), 34.6 (t).
1
468 (M+, 70%); H NMR: d 7.41–7.30 (m, 2H), 7.22
(m, 2H), 7.00 (td, J=8.1, 2.6 Hz, 1H), 6.90 (t, J=7.7 Hz,
1H), 6.71–6.65 (m, 1H), 5.65 (t, J=5.9 Hz, 1H), 3.87–
3.65 (m, 2H), 2.31–2.17 (m, 2H); 13C NMR: d 161.9 (s,
JF=250.0 Hz), 157.8 (s), 135.3 (s, J=3.5 Hz), 130.9 (d),
130.6 (d), 128,5 (d, JF=8.2 Hz), 125.4 (d), 121.7 (s,
2-[3-(4-Fluoro-2-iodophenyl)-3-hydroxypropyl]-isoindole-
1,3-dione (4e). Prepared as reported for 4a. Starting
from 2e (0.77 g, 2.56 mmol), after crystallization from
acetone pure 4e (0.31 g, 28%) was collected. GC–MS m/
JF=9.1 Hz), 120.2 (d, JF=24.6 Hz), 115.6 (d, JF=21.9
Hz), 114.4 (d), 94.4 (s), 75.0 (d), 40.8 (t), 39.6 (t).
1
z 425 (M+); H NMR: d 7.79 (m, 4H), 7.55–7.41 (m,
2H), 7.07 (td, J=8.4, 2.6 Hz, 1H), 4.74 (d, J=8.4 Hz,
1H), 4.01–3.85 (m, 2H), 3.04 (d, J=4.0 Hz, 1H), 2.15–
2.03 (m, 1H), 1.79–1.67 (m, 1H); 13C NMR: d 169.0 (s),
161.2(s, JF=249.4 Hz), 141.4 (d, JF=2.75 Hz), 134.2
2-[3-Hydroxy-3-phenyl-propyl]-isoindole-1,3-dione- (4a).
Phthalimide (4.31 g, 29.3 mmol) and anhydrous KF
(10.7 g, 73.2mmol) were added, under nitrogen atmo-

A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 3197–3206
3203
(d), 132.0 (s), 127.5 (d, JF=8.2Hz), 125.8 (d, JF=22.8
Hz), 123.4 (d), 115.7 (d, JF=20.0 Hz), 96.0 (s, JF=9.2
Hz), 73.7 (d), 36.8 (t), 34.7 (t).
MS m/z 295 (M+); 1H NMR: d 7.56 (dd, J=9.5, 3.0 Hz,
1H), 7.48 (dd, J=8.0, 5.5 Hz, 1H), 7.10 (td, J=8.4, 2.2
Hz, 1H), 5.05 (dd, J=8.8, 2.2 Hz, 1H), 3.14 (t, J=4.4
Hz, 1H), 3.07–2.95 (td, J=9.9, 2.9 Hz, 1H), 1.98–1.87
(m, 1H), 1.60–1.46 (m, 1H); 13C NMR: d 161.3 (s,
JF=250.0 Hz), 142.7 (s), 128.3 (d, JF=8.1 Hz), 126.0 (d,
JF=23.2 Hz), 115.6 (d, JF=20.7 Hz), 95.9 (s, JF=8.1
Hz), 78.8 (d), 40.9 (t), 38.0 (t).
2-[3-(2-Bromo-4-fluorophenyl)-3-(3-iodophenoxy)-propyl]-
isoindole-1,3-dione (4f). Prepared as reported for 4a.
Starting from 3 (1.4 g, 2.98 mmol), after purification by
flash chromatography (petroleum ether–ethyl acetate
10:1, 0.1% NEt3, Rf 0.50), pure compound 4f (1.1 g,
1
63%) was obtained. GC–MS m/z 580 (M+); H NMR:
3-(2-Bromo-4-fluorophenyl)-3-(3-iodophenoxy)-propyla-
mine (5f). Prepared as reported for 5a Starting from 4f
(0.90 g, 1.55 mmol), after purification by flash chroma-
tography (CH2Cl2–CH3OH 10:1, NEt3 0.1%, Rf 0.20),
5f as oil (0.345 g, 49%) was recovered. GC–MS m/z 451
d 7.77 (m, 4H), 7.38–7.30 (m, 2H), 7.20–7.17 (m, 1H),
7.00 (s, 1H), 6.96–6.91 (m, 1H), 6.84 (t, J=8.1 Hz, 1H),
6.57–6.53 (m, 1H), 5.49 (dd, J=8.8, 3.0 Hz, 1H), 4.06–
3.85 (m, 2H), 2.29–2.12 (m, 2H).
1
(M+); H NMR: d 7.41–7.28 (m, 2H), 7.24–7.20 (m,
3-Amino-1-phenyl-propan-1-ol (5a). A suspension of 4a
(1.3 g, 4.87 mmol) and hydrazine (0.47 mL, 0.49 g, 9.74
mmol) in MeOH (30 mL) was stirred at reflux for 3 h.
After cooling at 0 ꢀC, H2O (30 mL) and HCl concd to
adjust the pH to 2–3, was added. The resulting mixture
was refluxed for 1 h, the white solid formed was filtered
off and the pH of the solution was adjusted to 9 with
aqueous 50% NaOH. The obtained solution was
extracted with CH2Cl2 and dried over Na2SO4.
Removal of the solvent gave 5a (0.59 g, 80%) as an oil
sufficiently pure to be used without further purification.
2H), 6.99 (td, J=8.0, 2.2 Hz, 1H), 6.89 (t, J=8.5 Hz,
1H), 6.67 (m, 1H), 5.53 (t, J=6.0 Hz, 1H), 2.99–2.93 (m,
2H), 2.02–1.99 (m, 2H); 13C NMR: d 161.7 (s, JF=250.9
Hz), 157.9 (s), 136.0 (s, JF=2.8 Hz), 130.8 (d), 130.3 (d),
128.5 (d, JF=8.2 Hz), 125.3 (d), 121.6 (s, JF=10.1 Hz),
120.0 (d, JF=27.7 Hz), 115.6 (d, JF=21.1 Hz), 114.5
(d), 94.4 (s), 76.2(d), 40.0 (t), 38.5 (t).
3-Benzylamino-1-phenyl-propan-1-ol (6a). NaBH3CN
(0.067 g, 1.066 mmol), in small amounts, was added to a
solution of 5a (0.126 g, 0.836 mmol) and benzaldehyde
(0.085 mL, 0.087 g, 0.835 mmol) in MeOH (10 mL) at
0 ꢀC; the pH of the solution was adjusted to 6 by adding
glacial acetic acid. After 24 h at 25 ꢀC, H2O (20 mL) was
added and solid Na2CO3 until a basic pH was reached.
After saturation with solid NaCl, the aqueous phase
was extracted with CH2Cl2 and dried over Na2SO4. The
crude product was purified by flash chromatography
(CH2Cl2–CH3OH 10:1, NEt3 0.1%, Rf 0.35) thus giving
1
GC–MS m/z 151 (M+); H NMR: d 7.38–7.27 (m, 5H),
4.85–4.93 (m, 1H), 3.02 (m, 2H), 1.87–1.82 (m, 2H); 13C
NMR: d 145.1 (s), 128.3 (d), 127.2 (d), 125.7 (d), 53.8
(d), 40.3 (t), 37.3 (t).
3-Amino-1-(4-bromo-2-fluorophenyl)-propan-1-ol
(5b).
Prepared as reported for 5a. Starting from 4b (0.195 g,
0.56 mmol), pure 5b (0.10 g, 71%) was recovered. GC–
MS m/z 249 (M+); 1H NMR: d 7.48 (t, J=8.0 Hz, 1H),
7.27 (d, J=10.0 Hz, 1H), 7.15 (d, J=10.0 Hz, 1H), 5.20 (d,
J=8.0 Hz, 1H), 3.08 (m, 2H), 1.93 (m, 1H), 1.65 (m, 1H).
pure 6a (0.052g, 62%). GC–MS
m/z 241 (M+); 1H
NMR: d 7.33 (s, 10H), 4.95 (dd, J=8.0, 3.5 Hz, 1H),
3.82 (s, 2H), 2.99–2.89 (m, 2H), 1.91–1.81 (m, 2H), 13C
NMR: d 144.9 (s), 138.9 (s), 128.6 (d), 128.4 (d), 128.3
(d), 127.4 (d), 127.0 (d), 125.6 (d), 75.6 (d), 53.7 (t), 47.7
(t), 37.1 (t); IR (cmÀ1, CDCl3): 3200 (br), 1610, 1512,
1238. Anal. calcd for: C16H19NO: C, 79.69; H, 7.94; N,
5.80. Found: C, 79.42; H, 7.80; N, 5.95.
3-Amino-1-(2-bromo-4-fluorophenyl)-propan-1-ol
(5c).
Prepared as reported for 5a. Starting from 4c (0.61 g,
1.61 mmol), pure 5c as oil (0.35 g, 87%) was obtained.
GC–MS m/z 249 (M+); 1H NMR: d 7.64 (dd, J=8.8, 6.6
Hz, 1H), 7.20 (d, J=2.2 Hz, 1H), 7.03 (td, J=8.4, 2.5
Hz, 1H), 5.20 (dd, J=8.8, 2.2 Hz, 1H), 3.20–2.90 (m,
2H), 1.99–1.87 (m, 1H), 1.60–1.40 (m, 1H); 13C NMR: d
161.5 (s, JF=247.0 Hz), 140.0 (s, JF=3.0 Hz), 128.8 (d,
JF=8.0 Hz), 121.3 (s, JF=9.5 Hz), 119.7 (d, JF=24.5
Hz), 114.8 (d, JF=20.6 Hz), 74.3 (d), 40.8 (t), 37.7 (t).
3-(4-Fluorobenzylamino)-1-phenyl-propan-1-ol (6b). Pre-
pared as reported for 6a. Starting from 5a (0.100 g,
0.661 mmol) and p-fluorobenzaldehyde (0.071 mL,
0.082g, 0.661 mmol), after purification by flash chro-
matography (CH2Cl2–CH3OH 10:1, NEt3 0.1%, Rf
0.40), 6b (0.078 g, 45%) was recovered. GC–MS m/z
259 (M+); 1H NMR: d 7.33 (m, 7H), 7.02(t, J=8.6 Hz,
2H), 4.93 (dd, J=7.7, 2.6 Hz, 1H), 3.78 (s, 2H), 2.95–
2.90 (m, 2H), 1.90–1.83 (m, 1H), 1.76–1.68 (m, 1H); 13C
NMR: d 162.3 (s, JF=245.3 Hz), 144.7 (s), 133.9 (s),
130.2(d, JF=8.2 Hz), 128.3 (d), 127.1 (d), 126.6 (d),
115.5 (d, JF=21.1 Hz), 75.1 (d), 52.8 (t), 47.3 (t), 36.9
(t); IR (cmÀ1, CDCl3): 3200 (br), 3088, 1602, 1504,
1233. Anal. calcd for: C16H18NOF: C, 74.16; H, 7.00; N,
5.40. Found: C, 74.42; H, 7.12; N, 5.52.
3-Amino-1-(2-fluoro-4-iodophenyl)-propan-1-ol (5d). Pre-
pared as reported for 5a. Starting from 4d (0.31 g, 0.74
mmol), pure 5d as oil (0.19 g, 86%) was recovered. GC–
1
MS m/z 295 (M+); H NMR: d 7.48 (dd, J=8.4 Hz,
1H), 7.35 (d, J=7.7 Hz, 1H), 7.33 (dd, J=9.5, 1.5 Hz,
1H), 5.20 (d, J=6.6 Hz, 1H), 3.21–3.04 (m, 2H), 2.03–
1.81 (m, 1H), 1.70–1.61 (m, 1H); 13C NMR: d 159.2(s,
JF=251.0 Hz), 133.6 (d, JF=3.6 Hz), 132.5 (s, JF=13.7
Hz), 129.3 (d, JF=5.0 Hz), 124.4 (d, JF=24.0 Hz), 91.3
(s, JF=8.0 Hz), 69.8 (d), 40.8 (t), 37.9 (t).
3-Benzylamino-1-(4-bromo-2-fluorophenyl)-propan-1-ol
(6c). Prepared as reported for 6a. Starting from 5b (0.040
g, 0.16 mmol), purification by flash chromatography
(CH2Cl2–CH3OH 10:1, NEt3 0.1%, Rf 0.40) gave pure
3-Amino-1-(4-fluoro-2-iodophenyl)-propan-1-ol (5e). Pre-
pared as reported for 5a. Starting from 4e (0.21 g, 0.47
mmol), pure 5e as oil (0.132g, 96%) was obtained. GC–

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A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 3197–3206
1
6c (0.045 g, 83%). GC–MS m/z 337 (M+); H NMR: d
7.40–7.37 (m, 1H), 7.32(m, 5H), 7.22(m, 1H), 7.15 (dd,
J=10.1, 1.5 Hz, 1H), 5.19 (dd, J=8.0, 2.5 Hz, 1H), 3.81
(s, 2H), 2.96–2.90 (m, 2H), 2.03–1.94 (m, 1H), 1.76–1.68
(m, 1H); 13C NMR: d 159.1 (d, JF=250.0 Hz), 138.6 (s),
131.2(s, JF=12.8 Hz), 128.7 (d), 128.5 (d), 127.6 (d), 127.4
(d, JF=3.7 Hz), 125.3 (s, JF=25.6 Hz), 120.4 (d, JF=9.1
Hz), 118.5 (d, JF=24.7 Hz), 69.3 (d, JF=1.8 Hz), 53.6 (t),
47.4 (t), 35.1 (t); IR (cmÀ1, CDCl3): 3180 (br), 3082, 1598,
1500, 1228. Anal. calcd for: C16H17NOBrF: C, 57.02; H,
7.94; N, 5.08. Found: C, 56.94; H, 7.86; N, 5.18.
purification by flash chromatography (CH2Cl2–CH3OH
15:1, NEt3 0.1%, Rf 0.40), 6g (0.130 g, 76%) was
recovered. GC–MS m/z 357 (M+); 1H NMR: d 7.55 (m,
1H), 7.33–7.25 (m, 3H), 7.04 (t, J=8.6 Hz, 3H), 5.18 (t,
J=8.4 Hz, 1H), 3.79 (s, 2H), 2.95 (m, 2H), 2.00 (m, 1H),
1.63 (m, 1H); 13C NMR: d 162.2 (s, JF=245.0 Hz),
161.3 (s, JF=249.0 Hz), 139.5 (s), 134.7 (s, JF=2.8 Hz),
130.0 (d, JF=7.4 Hz), 128.6 (d, JF=8.3 Hz), 121.1 (s,
JF=9.1 Hz), 119.5 (d, JF=24.87 Hz), 115.5 (d, JF=21.1
Hz), 114.6 (d, JF=21.1 Hz), 74.3 (d), 53.0 (t), 47.6 (t),
35.0 (t); IR (cmÀ1, CDCl3): 3200 (br), 3076, 1609, 1360,
1221. Anal. calcd for: C16H16NOBrF2: C, 54.08; H,
4.54; N, 3.94. Found: C, 53.94; H, 4.66; N, 3.87.
3-Benzylamino-1-(2-bromo-4-fluorophenyl)-propan-1-ol
(6d). Prepared as reported for 6a. Starting from 5c
(0.245 g, 0.98 mmol), after purification by flash chro-
matography (CH2Cl2–CH3OH 10:1, NEt3 0.1%, Rf
0.38), 6d (0.26 g, 79%) was recovered. GC–MS m/z 337
Benzyl-[3-(2-bromo-4-fluorophenyl)-3-(3-iodophenoxy)-
propyl]-amine (6h). Prepared as reported for 6a. Starting
from 5f (0.324, 0.72 mmol), purification by flash chro-
matography (CH2Cl2–CH3OH 10:1, CH3OH–NEt3
100:1, Rf 0.32) gave pure 6h (0.310 g, 79%). 1H NMR: d
7.38–7.24 (m, 8H), 7.20–7.16 (m, 2H), 7.02–6.94 (m,
1H), 6.92(t, J=8.1 Hz, 1H), 5.54 (t, J=6.3 Hz, 1H),
3.80 (m, 2H), 2.87 (t, J=7.0 Hz, 2H), 2.06 (m, 2H); 13C
NMR: d 161.7 (s, JF=250.9 Hz), 158.1 (s), 140.1 (s),
136.3 (s, JF=3.6 Hz), 130.8 (d), 130.3 (d), 128.7 (d,
JF=7.0 Hz), 128.4 (d), 128.2 (d), 127.0 (d), 125.4 (d),
121.6 (s, JF=10.0 Hz), 120.0 (d, JF=24.0 Hz), 115.5 (d,
JF=20.5 Hz), 114.5 (d), 94.4 (s), 76.6 (d), 53.8 (t), 45.4
(t), 37.2(t); IR (cm À1, CDCl3): 3029, 2856, 1709, 1357.
Anal. calcd for: C22H20NOBrFI: C, 49.03; H, 3.74; N,
2.60. Found: C, 49.24; H, 3.56; N, 2.77.
(M+); H NMR: d 7.56 (dd, J=8.4, 6.2Hz, 1H), 7.34
1
(m, 5H), 7.21 (dd, J=8.4, 2.6 Hz, 1H), 7.01 (td, J=8.4,
2.2 Hz, 1H), 5.19 (d, J=8.0 Hz, 1H), 3.83 (s, 2H), 2.98–
2.92 (m, 2H), 2.07–1.98 (m, 1H), 1.68–1.54 (m, 1H); 13C
NMR: d 160.9 (s, JF=250.3 Hz), 139.6 (s, JF=2.8 Hz),
139.1 (s), 128.8 (d), 128.7 (d), 128.5 (d, JF=7.6 Hz),
127.8(d), 121.0 (d, JF=7.8 Hz); 119.5 (d, JF=22.3 Hz),
114.6 (s, JF=21.0 Hz), 74.3 (d), 53.9 (t), 47.7 (t), 35.0
(t); IR (cmÀ1, CDCl3): 3090 (br), 2990, 1598. Anal.
calcd for: C16H17NOBrF: C, 57.02; H, 7.94; N, 5.08.
Found: C, 57.24; H, 7.98; N, 5.23.
3-Benzylamino-1-(2-fluoro-4-iodophenyl)-propan-1-ol (6e).
Prepared as reported for 6a. Starting from 5d (0.110 g,
0.373 mmol), after chromatography (CH2Cl2–CH3OH
10:1, CH3OH–NEt3 100:1, Rf 0.45), 6e (0.108 g, 75%)
was obtained. GC–MS m/z 385 (M+); 1H NMR: d
7.51–7.44 (m, 2H), 7.34 (m, 5H), 7.03 (td, J=8.4, 2.6 Hz,
1H), 5.00 (dd, J=8.4, 2.3 Hz, 1H), 3.81 (s, 2H), 3.03–
2.91 (m, 2H), 2.03–1.94 (m, 1H), 1.70–1.47 (m, 1H); 13C
NMR: d 161.3 (s, JF=250.3 Hz), 142.5 (s, JF=3.0 Hz),
138.8 (s), 128.9 (d), 128.6 (d), 128.3 (d, JF=7.5 Hz),
127.8 (d), 126.0 (d, JF=23.7 Hz), 115.6 (d, JF=20.6
Hz), 95.9 (s, JF=8.0 Hz), 78.6 (d), 53.9 (t), 47.8 (t), 35.4
(t); IR (cmÀ1, CDCl3) 3240 (br), 3078, 1590, 1496, 1233.
Anal. calcd for: C16H17NOFI: C, 49.91; H, 4.45; N,
3.64. Found: C, 50.24; H, 4.56; N, 3.77.
3-Benzylamino-1-(4-fluoro-2–125iodophenyl)-propan-1-ol
125
(
I-6i). To a solution of 6d (1.070 mg, 3.164 mmol) in
EtOH (350 mL), under nitrogen atmosphere, 320 mL of a
solution A were added. The solution A was prepared by
adding into a vial, under a nitrogen atmosphere, 1.2mg
of copper (II) sulphate, 1.2mg of tin (II) sulphate, 30
mg of gentisic acid, 42mg of citric acid, 30 mL of glacial
acetic acid and 2.7 mL of deionized water. After 10 min
10 mL of Na125I in 0.2N NaOH (10 mL, 1 mCi) and 10
mL of deionized water were added. The pH was adjusted
at 1–2with glacial acetic acid and the reaction mixture
heated at 100 ꢀC for 90 min under a stream of N2; the
residue was cooled down and taken up in 500 mL of
deionized water. The solution was eluted through a
Dowex 1X8–200 ion-exchange column to eliminate the
inorganic salts and the unreacted Na125I.
3-Benzylamino-1-(4-fluoro-2-iodophenyl)-propan-1-ol(6f).
Prepared as reported for 6a. Starting from 5e (0.040 g,
0.133 mmol), purification by flash chromatography
(CH2Cl2-CH3OH 10:1, NEt3 0.1%, Rf 0.50) gave pure
HPLC analyses (eluting with a linear gradient of 25–
90% acetonitrile in 0.1% aqueous TFA over 50 min at a
flow rate of 1 mL/min) showed two peaks: the first
having tR 33 min (UV detector) as 6d compound, the
latter having tR 40 min (radioactivity detector) as 3-
Benzylamino-1-(4-fluoro-2–125iodophenyl)-propan-1-ol.
Fractions containing the chemically pure 125I-6i were
pooled together (130 mCi, yield 13%) and, after eva-
poration of the solvent, used for in vivo experiments.
1
6f (0.033 g, 64%). GC–MS m/z 385(M+); H NMR: d
7.44 (d, J=8.4 Hz, 1H), 7.32(m, 7H), 5.17 (d, J=5.6
Hz, 1H); 3.78 (s, 2H), 2.91 (m, 2H), 1.93 (m, 1H), 1.72–
1.66 (m, 1H); 13C NMR: d 158.9 (s, JF=250.9 Hz),
138.2 (s), 133.4(d), 129.5 (s), 129.0 (s), 128.7 (d), 128.5
(d, JF=23.7 Hz), 127.6 (d), 124.4 (d), 123.9 (d), 69.3 (d),
53.5 (t), 47.3 (t), 34.9 (t); IR (cmÀ1, CDCl3): 3180 (br),
3076, 1590, 1510, 1240. Anal. calcd for: C16H17NOFI: C,
49.91; H, 4.45; N, 3.64. Found: C, 49.82; H, 4.32; N, 3.82.
1-(2-Bromo-4-fluorophenyl)-3-(4-fluorobenzylamino)-pro-
pan-1-ol (6g). Prepared as reported for 6a. Starting
from 5c (0.120 g, 0.486 mmol) and p-fluoro-
benzaldehyde (0.052mL, 0.060 g, 0.486 mmol), after
Biological Studies
Animal care, surgical preparation and experimental
procedures were carried out in compliance with the

A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 3197–3206
3205
Italian Law (D.L. 116/92) regulating detention and
practice with experimental animals.
competition experiments28 following previously descri-
bed procedures.29,30 Briefly, the D2 receptor assays were
performed on rat striate tissue homogenates, to which
were added ketanserine (4.10À8 M fin), to block 5-HT2
receptors, and (+)-butaclamol (10À6 M fin) and /or
sulpiride (10À5 M fin), to determine the non-specific
binding. The 5-HT2 receptor assays were performed on
rat frontal cortex tissue homogenates, to which was
added sulpiride (10À6 M fin) to block the D2 receptors,
and ketanserine (10À5 M fin), to determine the non-
specific binding. In the 5-HT2 assays the non-specific
binding was determined also in presence of (+)-buta-
clamol (10À6 M fin) obtaining non-specific binding
values similar to those obtained using ketanserine alone.
After 60 min of incubation at 20–22 ꢀC, 0.5 mL of each
sample were vacuum-filtered through Whatman GF/B
filters and washed with 2Â5 mL of Tris–HCl buffer. The
filters were dissolved in a scintillation cocktail and
counted the day after. All the experiments were carried
out in triplicate.
The experimental animals studies were all conducted on
male Sprague-Dawlley rats (350–400
g b/w). To
measure the cerebral extraction albumin microspheres,
Sferotec-S (Sorin Biomedica, Italy), were used to pre-
pare 99mTc labelled microspheres (MI). 131I-Hippuran
(Sorin Biomedica, Italy) specific activity=4 MBq/mL
was used to evaluate the tested molecule intravascular
content. Briefly, the rats were rapidly tracheotomized
under light ether anaesthesia and local anaesthesia
(lidocaine 2%), paralyzed with tubocurarine (0.5 mg/Kg
iv) and passively ventilated with a mixture of halothane
and O2 (3 mL halothane in 32L of O ) by a small-ani-
2
mal respirator (Basile, Milano Italy). Atropine (0.5 mg/
Kg) was injected iv to minimize tracheobronchial secre-
tion and to control arterial hypotension due to halo-
tane. Body temperature was monitored by a rectal
probe and maintained between 37–39 ꢀC with a heating
lamp. With the animal positioned under the guillotine,
both femoral arteries were catheterized; one catheter
was connected to a pression transducer for monitoring
arterial pressure and heart rate, the other one to a peri-
staltic pump (Pharmacia) set to withdraw blood at a
rate of 0.5 mL/min, and utilised like the ‘reference
organ’. Eparinization was performed with 100 I.U. iv of
eparine, and the left ventricle was catheterised via the
right common carotid artery with a catheter (0.96–0.58
mm external and inner diameter respectively) connected
with a pression transducer to ascertain its positioning into
the left heart ventricle. A range of rCBF values were
obtained varying the ventilatory rate and the tidal volume
(between 58–89 minÀ1 and 2.5–6 mL respectively).
In vivo kinetic studies
Data from three male Sprague–Dawley rats (300–350 g)
are reported.
The cerebral extraction of the molecule was obtained
measuring, simultaneously, the regional cerebral blood
flow, rCBF (mL/min/g), and the blood to brain uni-
directional influx constant, Ki (mL/min/g). Indeed, the
Ki of a freely diffusible blood-brain barrier molecule is
.
related to the rCBF by the following: Ki=rCBF E,
where E is the single pass cerebral extraction of the
molecule.
For the in vitro binding studies [3H]spiperone (103 Ci/
mmol) was obtained from Amersham (UK) and used at
0.25 nM final concentration; (+)-butaclamol, ketanser-
ine and sulpiride were purchased from Alexis Biochem-
ical (Switzerland). For the membrane preparations and
the reagents dilutions Tean buffer (Tris–HCl buffer 15
mM, NaEDTA 5mM, ascorbic acid 1.1 mM, nialamide
12.5 mM) was used. Tissue samples were measured in a
1282-Compugamma CS-Wallac.
The used method17,31 calls for the injection of a mixture
of tracers: 99mTc-labelled albumin microspheres (to
measure rCBF), 131I-Hippuran (to estimate the intra-
vascular content of the tracer) and the 125I-labelled test
molecule, into the left heart ventricle.
Briefly, under strict control of physiological parameters,
arterial blood was withdrawn at a rate of 0.5 mL/min
using a peristaltic pump and, with the withdrawal pump
on, the injection mixture was loaded into the cardiac
catheter and rapidly flushed (<0.5 s) into the left ven-
tricle with a bolus of saline. Six seconds following the
injection, the rat was decapitated and the withdrawing
pump simultaneously stopped by a switch triggered by
the guillotine. The brain was rapidly removed and dis-
sected on ice. Samples of cortex, cerebellum and striate
(about 100 mg each) were weighed and solubilized
(Protosol, Dupont, USA). Blood and tissue samples
were counted in a g-counter and the measured activity
corrected for the spill-over.
For the in vitro autoradiography the rat brain slices
were incubated in buffer Tris–HCl 50 mM pH 7.4 added
of 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and mole-
cule 6i at the concentration of 2.25 nM final concentra-
tion. Adjacent sections were incubated in the same
medium containing (+)-butaclamol and ketanserine
(both at 1mM final concentration) additionally. Once
dried, the slides were put in contact with ScopixRD
(Agfa) films which was developed in a Curix 242S
(Agfa-Geavert). Slice autoradiographic images were
qualitatively analyzed using the scanner ArcusII-Agfa
and an image analyzing system: ImageJ 1.18x (National
Institutes of Health, USA).
In vitro autoradiography
In vitro autoradiography was performed on serial cor-
onal rat brain section of 20 mm thickness prepared by
criocutting as previously described.32 Sections were
thaw-mounted onto glass slides and incubated with a
solution of 6i (Specific Activity 2.2 Ci/mmol) dissolved
In vitro binding assays
The new molecules were tested in vitro on rat brain
membrane preparations in 3H-spiperone specific binding

3206
A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 3197–3206
in ethyl alcohol and diluted in buffer Tris–HCl 50 mM
pH 7.4 added of 120 mM NaCl, 5 mM KCl, 2 mM
CaCl2. Adjacent sections were incubated in the same
medium containing (+)-butaclamol and ketanserine (1
mM final concentration) additionally. After 1 h of incu-
bation, at room temperature, slides were given 3 rinses
of 20 s each in ice cold buffer followed by a short dip in
distilled water to remove salts and finally dried in a
stream of dry air. Ultimately the slides were put in con-
tact with films which were developed at several times of
exposure. Slice autoradiographic images were qualitatively
analyzed using an image analyzing system.
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Acknowledgements
The authors thank the Ministry of University and Sci-
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(National Research Council)—Italy for financial support.
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