Synthesis and characterization of fluorescent ligands for the norepinephrine transporter: Potential neuroblastoma imaging agents

Article abstract of DOI:10.1021/jm9811155

Radiolabeled m-iodobenzylguanidine (MIBG) is a tumor-seeking radioactive drug used in the diagnosis and treatment of pheochromocytomas and neuroblastomas. It is transported into the tumor cells by the neuronal norepinephrine (NE) transporter (NET) which is expressed in almost all neuroblastoma cells. Here, we describe the synthesis and some pharmacological properties of a series of fluorescent compounds structurally related to the NET substrate, MIBG, or to the NET inhibitors, (-)-(2R,3S)-cocaine and nisoxetine. Three of 10 synthesized fluorescent compounds, 1-(1- naphthylmethyl)guanidinium sulfate (1), 1-[2-(dibenz[b,f]azepin-5- yl)ethyl]guanidinium sulfate (2), and (2R,3S)-2β-ethoxycarbonyl-3β-tropanyl 5-(dimethylamino)naphthalene-1-sulfonate (6), exhibited high affinity (IC₅₀ about 50 nM) for the NET. The nisoxetine derivatives 8 (rac-N-[(3- methylamino-1-phenyl)propyl]-5-(dimethylamino)-1-naphthalene-sulfonamide) and 9 (rac-4-[(3-methylamino-1-phenyl)propyl]amino-7-nitro-2,1,3-benzoxadiazole) and especially the guanidine derivative 4 (1-[4-(4-phenyl-l,3- butadienyl)benzyl]guanidinium sulfate) which are characterized by intermediate affinity for the NET (IC₅₀ 370-850 nM) caused significant and nisoxetine-sensitive cell fluorescence. At least the guanidine derivative 4 might represent a potentially useful agent for imaging of neuroblastoma cells.

Full text of DOI:10.1021/jm9811155

J . Med. Chem. 1999, 42, 3101-3108
3101
Syn th esis a n d Ch a r a cter iza tion of F lu or escen t Liga n d s for th e Nor ep in ep h r in e
Tr a n sp or ter : P oten tia l Neu r obla stom a Im a gin g Agen ts
Dirk Hadrich,‡,§ Frank Berthold,† Eberhard Steckhan,‡ and Heinz Bo¨nisch*,§
Kekule´-Institut fu¨r Organische Chemie und Biochemie, Universita¨t Bonn, Gerhard-Domagk-Strasse 1,
D-53121 Bonn, Germany, Kinderklinik, Universita¨t Ko¨ln, J oseph-Stelzmann-Strasse 9, D-50924 Ko¨ln, Germany,
and Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bonn, Reuterstrasse 2b, D-53113 Bonn, Germany
Received November 17, 1998
Radiolabeled m-iodobenzylguanidine (MIBG) is a tumor-seeking radioactive drug used in the
diagnosis and treatment of pheochromocytomas and neuroblastomas. It is transported into
the tumor cells by the neuronal norepinephrine (NE) transporter (NET) which is expressed in
almost all neuroblastoma cells. Here, we describe the synthesis and some pharmacological
properties of a series of fluorescent compounds structurally related to the NET substrate, MIBG,
or to the NET inhibitors, (-)-(2R,3S)-cocaine and nisoxetine. Three of 10 synthesized fluorescent
compounds, 1-(1-naphthylmethyl)guanidinium sulfate (1), 1-[2-(dibenz[b,f]azepin-5-yl)ethyl]-
guanidinium sulfate (2), and (2R,3S)-2â-ethoxycarbonyl-3â-tropanyl 5-(dimethylamino)naph-
thalene-1-sulfonate (6), exhibited high affinity (IC50 about 50 nM) for the NET. The nisoxetine
derivatives 8 (rac-N-[(3-methylamino-1-phenyl)propyl]-5-(dimethylamino)-1-naphthalene-
sulfonamide) and 9 (rac-4-[(3-methylamino-1-phenyl)propyl]amino-7-nitro-2,1,3-benzoxadiazole)
and especially the guanidine derivative 4 (1-[4-(4-phenyl-1,3-butadienyl)benzyl]guanidinium
sulfate) which are characterized by intermediate affinity for the NET (IC50 370-850 nM) caused
significant and nisoxetine-sensitive cell fluorescence. At least the guanidine derivative 4 might
represent a potentially useful agent for imaging of neuroblastoma cells.
In tr od u ction
amounts in patients with neuroblastomas, and elevated
urinary levels are used in the diagnosis of this tumor.4
Neuroblastomas and pheochromocytomas are neural
crest-derived tumors of the sympathetic nervous system.
Neuroblastomas belong to the most common extracra-
nial solid tumors in young children. About 10-20% of
neuroblastomas show spontaneous regression, and few
mature to benign ganglioneuroma, whereas most pa-
tients with metastatic disease have an unfavorable
prognosis for long-term survival.1,2 Although the tumor
responds initially to chemotherapy, it regrows from
residual resistant cells resulting in a fatal outcome for
the patients. Since bone marrow is the predominant
(80% of cases) metastatic site in neuroblastoma, serial
bone marrow aspirations are performed during the
course of the disease to evaluate response to therapy.
In this respect the detection of minimal residual disease
is important. A specific technique to image those cells
would be therefore a powerful tool. m-Iodobenzylguani-
dine (MIBG) is considered a selective agent which is
accumulated by the neuroblastoma cells;3 however, its
accumulation is microscopically not visible. Thus, a
fluorescent substance with a neuroblastoma specificity
similar to that of MIBG would be helpful.
Tumors of neuroectodermal origins such as neuro-
blastomas have preserved the ability of neuronal cells
to take up NE through the neuronal NE transporter
(NET), a specific transport system (also termed “uptake-
1”) which is located in the neuronal plasma membrane
(see below). MIBG is a metabolically stable NE analogue
derived from the adrenergic neuron-blocking agents
bretylium and guanethidine. MIBG accumulates in
neuroendocrine tissues and thereby competes with NE
for uptake and storage.4 Due to these features, MIBG
labeled with either 131I or 123I has been used for about
15 years in the scintigraphic visualization (diagnosis)
and therapy of pheochromocytomas and neuroblast-
omas.5-7
Uptake of MIBG has been studied in human neuro-
blastoma cell lines such as SK-N-SH-SY5Y cells.8-10 The
sodium- and chloride-dependent NET is inhibited by
(-)-(2R,3S)-cocaine, nisoxetine, or desipramine.11-12
Fluorescent ligands are useful tools to study recep-
tors.13 Fluorescent ligands with affinity for the NET,
which is often overexpressed in neural crest tumors such
as neuroblastomas, could represent potentially useful
agents for the diagnosis of these tumors. In the present
study, it was our goal to synthesize a series of fluores-
cent compounds structurally related to MIGB (1-5), (-)-
(2R,3S)-cocaine (6, 7), and nisoxetine (8-10) as potential
ligands for the NET. Using the human neuroblastoma
cell line SK-N-SH-SY5Y, we also investigated their
affinities for the NET. As fluorescent markers, we
selected condensed aromatic groups (1), aryl olefins such
as stilbene derivatives (2, 3), 1,4-diphenyl-1,3-butadiene
As a tumor of the sympathetic nervous system,
neuroblastomas have many properties in common with
this system, such as synthesis, storage, release, and
reuptake of norepinephrine (NE). Thus, NE and its
metabolites are renally excreted in larger than normal
* Corresponding author. Phone: +49-228-735431. Fax: +49-228-
735404. E-mail: boenisch@uni-bonn.de.
‡ Universita¨t Bonn, Gerhard-Domagk-Str. 1.
† Universita¨t Ko¨ln.
§ Universita¨t Bonn, Reuterstr. 2b.
10.1021/jm9811155 CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/16/1999

3102 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Hadrich et al.
were due to their low solubility in all solvents except
DMSO; this also complicated their purification and
analysis.
The fluorescent (-)-(2R,3S)-cocaine derivatives (2R,-
3S)-2â-ethoxycarbonyl-3â-tropanyl 5-(dimethylamino)-
naphthalene-1-sulfonate (6) and (2R,3S)-O-(2â-ethoxy-
carbonyl-3â-tropanyl)-4-(dimethylamino)azobenzole-4′-
thiocarbamide (7) were synthesized from ethylecgonine
by condensation with dansyl chloride (for 6) or 4-(dim-
ethylamino)-4’-isocyanoazobenzene (for 7). Ethylecgo-
nine was obtained through hydrolysis of (-)-(2R,3S)-
cocaine in ethanolic HCl (Scheme 2).
The fluorescent nisoxetine derivatives rac-N-[(3-
methylamino-1-phenyl)propyl]-5-(dimethylamino)-1-naph-
thalenesulfonamide (8), rac-4-[(3-methylamino-1-phe-
nyl)propyl]amino-7-nitro-2,1,3-benzoxadiazole (9), and
rac-N-[4-(4-(dimethylamino)phenylazo)phenyl]-N′-[(3-
methylamino-1-phenyl)propyl]thiourea (10) were syn-
thesized in seven steps from 3-chloropropiophenone. The
hydroxy function, which was obtained by reduction of
the carbonyl group, was replaced by an amino function
using the Mitsunobu reaction with potassium phthal-
imide followed by hydrazinolysis. In the following, the
fluorescent groups were introduced by reaction of the
amine function with dansyl chloride (for 8), NBD
chloride (for 9), or 4-(dimethylamino)-4’-isocyanoazoben-
zene (for 10) (Scheme 3).
Biologica l Stu d ies
To obtain a measure of affinity (potency) of the
various ligands for the NET, IC50 values of the com-
pounds for inhibition of uptake of [3H]NE in human SK-
N-SH-SY neuroblastoma cells were determined. All
compounds inhibited [3H]NE uptake with Hill coef-
ficients of the inhibition curves not significantly differ-
ent from unity. As shown in Table 2, the rank order of
potency for inhibition of NE uptake was 2 > 1 ) 6 > 3
> 8 ) 4 > 5 ) 9 > 10 > 7. The affinities of the guanidine
derivatives were all in the submicromolar range and
thus similar to that of MIBG.8,9 This indicates that the
replacement of the benzyl group of MIBG by a naphth-
ylmethyl residue (1) or a dibenzoazepine (cis-stilbene
structure) residue (2) or the addition of a styryl group
in the para-position of benzylguanidine (3) caused no
dramatic affinity change in these MIBG derivatives
(Table 2). It remains to be shown whether these
fluorescent MIBG derivatives are transported by the
NET. Due to their relatively high affinity for the NET,
they might represent potential candidates for fluores-
cent labeling of cells expressing the NET such as
neuroblastoma cells.
F igu r e 1. Chemical structures and code numbers of fluores-
cent norepinephrine transporter ligands (for chemical names,
see Table 1).
(4), and 1,6-diphenyl-1,3,5-hexatriene (5), the dansyl
group (6, 8), azobenzene functions (7, 10), and the
7-nitro-2,1,3-benzoxadiazole (NBD) group (9).
Ch em istr y
Figure 1 shows the chemical structures and the code
numbers of the synthesized fluorescent derivatives of
guanidine (1-5), (-)-(2R,3S)-cocaine (6, 7), and nisox-
etine (8-10). In Table 1 their chemical names together
with wavelengths for optimal excitation and maximal
fluorescence emission in aqueous solution are given. All
10 substances are new, i.e., they have not yet been
described before. However, from 1-(1-naphthylmethyl)-
guanidinium sulfate (1), a corresponding iodide has
already been synthesized earlier.14
The (-)-(2R,3S)-cocaine IC50 values for inhibition of
NE uptake by means of the NET are at about 1 µM.11,17
The introduction of a fluorescent 4-(dimethylamino)-
azobenzene group into the (-)-(2R,3S)-cocaine molecule
(7) caused only a slight decrease in the affinity for the
NET. However, the (-)-(2R,3S)-cocaine derivative with
a fluorescent dansyl group (6) exhibited even increased
affinity for the NET (Table 2) compared to (-)-(2R,3S)-
cocaine. The high affinity to the NET of this fluorescent
compound indicates that it might be used for fluorescent
imaging of NET expressing tumors.
All five guanidines were synthesized from their cor-
responding amines by treatment with S-methylthiou-
ronium sulfate. These compounds are not iodinated in
the meta-position such as in MIBG, but they contain a
successively enlarged π-system, which is responsible for
their fluorescent properties as described for diphenyl-
butadiene compounds.15,16 The conjugated CdC double
bonds in 1-[4-(4-phenyl-1,3-butadienyl)benzyl]guanidin-
ium sulfate (4) and 1-[4-(6-phenyl-1,3,5-hexatrienyl)-
benzyl]guanidinium sulfate (5) were obtained through
a Wittig reaction using cinnamaldehyde as the main
building block (Scheme 1). The main difficulties in the
synthesis of the fluorescent guanidine derivatives 3-5
Nisoxetine is known to bind with high affinity (KD
about 5 nM) to the NET.18,19 However, and in contrast

Potential Neuroblastoma Imaging Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3103
Ta ble 1. Fluorescent Derivatives of Guanidine, (-)-(2R,3S)-Cocaine, and Nisoxetine and Their Optimal Excitation and Maximal
Fluorescence Emission Wavelengths
code
no.
excit/emiss
derivatives of
guanidine
chemical name
1-(1-naphthylmethyl)guanidinium sulfate
1-[2-(dibenz[b,f]azepin-5-yl)ethyl]guanidinium sulfate
1-(trans-stilben-4-ylmethyl)guanidinium sulfate
1-[4-(4-phenyl-1,3-butadienyl)benzyl]guanidinium sulfate
1-[4-(6-phenyl-1,3,5-hexatrienyl)benzyl]guanidinium sulfate
(2R,3S)-2â-ethoxycarbonyl-3â-tropanyl 5-(dimethylamino)naphthalene-1-sulfonate
(2R,3S)-O-(2â-ethoxycarbonyl-3â-tropanyl)-4-(dimethylamino)azobenzole-4′-thiocarbamide
rac-N-[(3-methylamino-1-phenyl)propyl]-5-(dimethylamino)-1-naphthalenesulfonamide
rac-4-[(3-methylamino-1-phenyl)propyl]amino-7-nitro-2,1,3-benzoxadiazole
rac-N-[4-(4-(dimethylamino)phenylazo)phenyl]-N′-[(3-methylamino-1-phenyl)propyl]thiourea
(nm)
1
2
3
4
5
6
7
8
9
313/334, 672
384/440
342/458
340/384
352/448
356/518
340/681
402/504
481/521
339/684
(-)-(2R,3S)-cocaine
nisoxetine
10
Sch em e 1
Sch em e 2
to the corresponding (-)-(2R,3S)-cocaine derivatives, the
fluorescent dansyl (8) or 4-(dimethylamino)azobenzene
(10) derivatives of nisoxetine exhibited a relatively low
affinity for the NET (Table 2) compared to nisoxetine.
This also holds true for the niosoxetine derivative with
a 7-nitro-2,1,3-benzoxadiazole group (9). A prerequisite
for a useful fluorescent ligand for labeling NET-express-
ing cells is either to be transported and intracellularly
accumulated or to bind to the NET with high affinity
and thus dissociate slowly from the target protein.
Since the fluorescent nisoxetine derivatives are presum-
ably not transported and exhibit relatively low affinity
for the NET, they are probably less useful imaging
ligands.
For microscopic observation of fluorescence, the emit-
ted fluorescence of the substance should be strong and
in the visible range (380-800 nm). Determination of the
emission wavelengths of the various compounds (Table
1) showed that compounds 1-3 caused in the visible
range only relatively weak fluorescence intensities. The

3104 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Hadrich et al.
Sch em e 3
Ta ble 2. IC50 Values of the Various Ligands for Inhibition of
[3H]NE Uptake into Human SK-N-SH-SY5Y Neuroblastoma
Cellsa
fluorescence in cells incubated in the absence of the NET
inhibitor nisoxetine than in its presence. In addition,
only in cells incubated with one of these five compounds
the extracts of cells incubated in the absence of nisox-
etine (compared to those in its presence) showed a
higher content of the fluorescent compound; this is
illustrated in Figure 2 for the guanidine derivative 4.
log IC50
IC50
compd
(-log M)
(µM)
2
1
6
3
8
4
5
9
10
7
7.52 ( 0.35
7.20 ( 0.34
7.18 ( 0.45
6.92 ( 0.33
6.43 ( 0.07
6.40 ( 0.19
6.07 ( 0.54
6.07 ( 0.43
5.99 ( 0.28
5.88 ( 0.12
0.03
0.06
0.06
0.12
0.37
0.40
0.85
0.85
1.02
1.32
For cells incubated with compounds 5, 6, 8, 9, and 4,
the substance-specific fluorescence peak was by a factor
of 1.12, 1.29, 2.22, 2.56, and 2.70, respectively, higher
in the extracts from cells incubated in the absence of
nisoxetine than in its presence. Thus, the guanidine
derivative 4 showed the highest nisoxetine-sensitive
cellular retention which presumably was due to NET-
mediated uptake of this compound. However, also the
nisoxetine derivatives 8 and 9 which are not expected
to be transported but to bind to the NET exhibited a
more than 2-fold difference in the substance-specific
fluorescence peaks. Since net rates of uptake of trans-
ported substrates decrease with time and initial rates
of specific uptake relative to “uptake” by diffusion are
highest at nonsaturating substrate concentrations, a
reduction in the incubation time (to measure initial
uptake rates) and a reduction of the substrate concen-
tration (below IC50 or Km) should result in an increase
in the specific uptake of a substrate compared to
nonspecific diffusion. Whether this holds true for the
guanidine derivative 4 remains to be shown.
a
SK-N-SH-SY5Y cells were incubated with 10 nM [3H]NE in
the presence of the indicated ligands in 5 different concentrations
and in the absence or presence of 10 µM nisoxetine (to determine
nonspecific uptake). Shown are means ( SEM from 3-4 determi-
nations of IC50 values for inhibition of specific [3H]NE uptake.
other synthesized compounds showed clearly stronger
fluorescence peaks.
In a preliminary series of experiments, the fluorescent
compounds listed in Table 1 were studied for their
ability to label human SK-N-SH-SY neuroblastoma cells
due to transport by or binding to the NET and for their
specific fluorescence qualities. After 30 min of incuba-
tion of the cells with one of the fluorescent compounds
(at a concentration of 10 µM and in the absence or
presence of nisoxetine), nisoxetine-sensitive fluorescence
of the cells was examined qualitatively by fluorescence
microcopy and quantitatively by measuring spectro-
fluorimetrically the cell extract. Inspection of the cells
under the fluorescence microscope showed that only five
of the compounds (4-6, 8, and 9) caused a clearly higher
intensity of substance-induced blue or yellow cellular
Con clu sion s
Methods have been developed for the preparation of
10 fluorescent derivatives of the NET ligands MIBG,
(-)-(2R,3S)-cocaine, and nisoxetine. All 10 compounds
inhibited uptake of [3H]NE in human neuroblastoma

Potential Neuroblastoma Imaging Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3105
an Ploemopak illuminator and with Leitz filters A (450-490
nm) or G (350-460 nm). Flash chromatography was performed
with silica gel (0.04-0.063 mm) from Merck Darmstadt. All
chemicals for syntheses were obtained from Aldrich Chemical
Co., except NBD-chloride which was obtained from Sigma and
(-)-(2R,3S)-cocaine HCl which was obtained from Merck
Darmstadt. Dansyl chloride had to be separated from the acid
by flash chromatography, eluting with dichloromethane. If not
stated otherwise, all substances used for biological tests were
obtained from Sigma.
B. Syn th eses. 1-(1-Na p h th ylm eth yl)gu a n id in iu m Su l-
fa te (1). A solution of 1-(aminomethyl)naphthalene (0.79 g)
and S-methylthiouronium sulfate (0.7 g) was heated under
reflux for 24 h in 15 mL of boiling 50% ethanol. The solvent
was removed in vacuo followed by addition of another portion
of the 50% ethanol which was subsequently again removed in
vacuo. This procedure was repeated three times. The precipi-
tated product was recrystallized from water to give 0.71 g
(57%) of 1 as a white solid. Mp: 245-247 °C. 1H NMR (200
MHz, DMSO-d6): 4.68 (2 H, s, CH2), 7.3-8.1 (7 H, m, arom),
7.9 (4 H, s, (H2N)2C), 8.96 (1 H, s, HNC2). 13C NMR (50.3 MHz,
DMSO-d6): 42.28 (1 C, CH2), 123.27 (1 C, C8), 123.98 (1 C,
C3), 125.50 (1 C, C6), 125.83 (1 C, C7), 126.21 (1 C, C4), 127.54
(1 C, C2), 128,. 5 (1 C, C5), 130.45 (1 C, C8a), 132.95 (1 C, C4a),
133.21 (1 C, C1), 157.54 (1 C, CN3).
1-[2-(Dib en z[b,f]a zep in -5-yl)et h yl]gu a n id in iu m Su l-
fa te (2). 1.44 mL of bromoacetonitrile was added to a mixture
of 500 mg of 5H-dibenz[b,f]azepine and 2 g of sodium bicarbon-
ate in 20 mL of dichloromethane. After heating under reflux
for 48 h, the 5-(cyanomethyl)dibenz[b,f]azepine was separated
by flash chromatography (silica, petroleum ether (PE):ethyl
acetate, 10:1). A solution of 494.5 mg of 5-(cyanomethyl)dibenz-
[b,f]azepine in 10 mL of THF was added to 161.5 mg of lithium
aluminum hydride in 20 mL of THF. After heating under
reflux for 2 h, the cooled solution was treated slowly with 0.3
mL of water. This mixture was heated under reflux for 2 h,
after cooling the precipitated solid was filtered off, and the
5-(aminoethyl)dibenz[b,f]azepine was purified by chromatog-
raphy, eluting with ethanol. The guanidinium sulfate was
prepared from 181.0 mg of this amine by the procedure
described above to give 163.5 mg (65%) of 2 as a light-yellow
F igu r e 2. Excitation spectra (left) and emission spectra (right)
of extracts of human SK-N-SH-SY neuroblastoma cells incu-
bated with the fluorescent guanidine derivative 4 in the
absence (uppermost curves) or presence (middle curves) of
nisoxetine; spectra of cells incubated in the absence of the
fluorescent compound (lowest curves).
cells with IC50 values ranging from 30 to 1320 nM. Two
of the five synthesized fluorescent MIBG derivatives,
1-(1-naphthylmethyl)guanidinium sulfate (1) and 1-[2-
(dibenz[b,f]azepin-5-yl)ethyl]guanidinium sulfate (2), as
well as one of the two fluorescent (-)-(2R,3S)-cocaine
analogues, (2R,3S)-2â-ethoxycarbonyl-3â-tropanyl 5-(dim-
ethylamino)naphthalene-1-sulfonate (6), inhibited [3H]-
NE uptake with high potency (IC50 ) 30 and 60 nM,
respectively). However, only the nisoxetine derivatives
rac-N-[(3-methylamino-1-phenyl)propyl]-5-(dimethylami-
no)-1-naphthalenesulfonamide (8) and rac-4-[(3-methy-
lamino-1-phenyl)propyl]amino-7-nitro-2,1,3-benzoxadi-
azole (9) and especially the guanidine derivative 1-[4-
(4-phenyl-1,3-butadienyl)benzyl]guanidinium sulfate (4)
caused more than 2-fold (nisoxetine-sensitive) cellular
retention and, when inspected under the fluorescence
microscope, nisoxetine-sensitive cell fluorescence. After
improvement of some experimental conditions, at least
the guanidine derivative 4 might represent a potentially
useful diagnostic agent for labeling of neuroblastoma
tumor cells.
1
solid. Mp: 157-158 °C. H NMR (200 MHz, DMSO-d6): 3.10
(2 H, t, CH2NAr2), 3.75 (2 H, t, CH2NH), 6.8-7.3 (10 H, m,
arom), 7.10 (4 H, s, (H2N)2C), 8.00 (1 H, s, HNC2). 13C NMR
(50.3 MHz, DMSO-d6): 31.38 (1 C, CH2NAr2), 49.00 (1 C, CH2-
NH), 120.48-13.,30 (12 C, arom), 147.80 (1 C, CN3), 149.75 (2
C, C-N).
1-(tr a n s-Stilben -4-ylm eth yl)gu a n id in iu m Su lfa te (3).
571.2 mg of 4-(chloromethyl)-trans-stilbene was treated with
463.8 mg of potassium phthalimide in 10 mL of dimethylfor-
mamide. After stirring for 1 h, the mixture was heated (10
°C/min) to 120 °C. The solution was then extracted with
trichloromethane, which was washed with aqueous sodium
bicarbonate. The trichloromethane solution was dried, and the
solvent was removed in vacuo. Crystallization from ethyl
acetate gave 4-(phthalimidomethyl)-trans-stilbene (758.5 mg)
as a white solid, which was dissolved in 20 mL of methanol,
treated with 0.434 mL of hydrazine monohydrate, and then
heated under reflux for 4 h. After removal of methanol and
addition of 50 mL of water and 8 mL of 6 M HCl, the solution
was heated under reflux for 3 h. Basification and extraction
with trichloromethane gave 425.1 mg of the amine, which was
transformed into the guanidinium sulfate by the procedure
described above to yield 274.2 mg (45%) of 3 as a white solid.
Mp: 260-262 °C. 1H NMR (200 MHz, CD3OD/TFA-d): 4.07
(2 H, s, CH2), 7.03-7.60 (11 H, m, arom). 13C NMR (50.3 MHz,
CD3OD/TFA-d): 39.83 (1 C, CH2), 122.31-129.79 (11 C),
132.96 (2 C, C1), 135.13 (1 C, C4), 154.74 (1C, CN3).
Exp er im en ta l Section
A. Gen er a l Meth od s. Melting points were determined with
a Reichert Kofler-Heiztisch microscope and are uncorrected.
Thin-layer chromatography was performed on 0.2-mm silica
gel 60 F254 sheets (Merck, Darmstadt). 1H and 13C NMR spectra
of all compounds were consistent with the assigned structure.
NMR spectra were recorded on a Bruker AC 200 instrument,
and chemical shifts are reported in ppm (δ) relative to the
residual signal on the deuterated solvent (CHCl3, δ 7.26; CHD2-
OD, δ 3.30; (CD3)2SO, δ 2.49). Purity and structures of
precursors were checked by gas chromatography combined
with mass spectrometry (GC-5890 and MSD-5970 from Hewlett-
Packard). Excitation and emission spectra were recorded on
a Shimadzu spectrophotometer RF-5000. For observation of
the fluorescence, a Leitz Diavert microscope was applied with
1-[4-(4-P h en yl-1,3-bu tadien yl)ben zyl]gu an idin iu m Su l-
fa te (4). 6.10 g of (4-cyanobenzyl)triphenylphosphonium bro-
mide was prepared by heating 4.09 g of triphenylphosphine
and 2.84 g of 4-(brommethyl)benzonitrile in 110 mL of p-xylene
at 130 °C for 24 h followed by washing with diethyl ether. The
phosphonium bromide was added at -78 °C to a solution of

3106 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Hadrich et al.
3.28 mL of diisopropylamine, 17.47 mL of butyllithium-
hexane solution (1.6 M), and 25 mL of THF. After the resulting
red solution was stirred for 1 h, 1.68 mL of cinnamaldehyde
in 9 mL of THF was added slowly. The reaction was quenched
with 1 mL of water the next morning, and the product was
purified by flash chromatography (silica, cyclohexane:ethyl
acetate, 10:1) to afford 2.66 g of the light-yellow 1-(4-cyanophe-
nyl)-4-phenyl-1,3-butadiene. This was reduced with a 3-fold
excess of diisobutylaluminum hydride in 30 mL of THF under
reflux for 1.5 h. The resulting amine was purified by flash
chromatography (silica, dichloromethane:ethanol, 30:1) to yield
2.06 g. Finally, the amine was transformed into the guani-
dinium sulfate by the procedure described above to give 2.24
g (40%) of 4 as a light-yellow solid. Mp: 241-243 °C. 1H NMR
(200 MHz DMSO-d6/TFA-d): 4.01 (2 H, s, CH2), 6.4-6.7 (4 H,
m, dCH), 7.1-7.5 (9 H, m, arom). 13C NMR (50.3 MHz, DMSO-
d6/TFA-d): 41.9 (CH2), 125.9 (2 CH), 126.1 (2 CH), 126.2 (2
CH), 127.0, 128.0 (2 CH), 128.1 (2 CH), 128.2 (2 CH), 137.3,
138.6, 139.4, 172.2 (CN3). Compound 4 is a mixture of E- and
Z-isomers. From the gas chromatography data one can see that
the all-trans,E-configurated isomer is predominant (longer
retention times). One can also see that the amount of the all-
trans-configurated product increases with time. Therefore,
clear NMR assignments to the different isomers are not
possible.
mL of dichloromethane. Dansyl chloride (158.2 mg, 1.02 mmol)
and dry pyridine (0.17 mL, 2.08 mmol) were added to this
solution at 0 °C, and then the entire mixture was slowly
warmed to reflux and heated at reflux for 2 h followed by
stirring 16 h at room temperature. A small amount of silica
was added, and the solvent was removed. The crude product
adsorbed on silica was purified by flash chromatography (silica,
first ethyl acetate followed by ethyl acetate:acetone, 1:5) to
1
afford 6 (207.8 mg, 46%) as a yellow fluorescent oil. H NMR
(200 MHz, CDCl3/CD3OD, 1:1): 1.29 (7.1 Hz, 3 H, t, CH3),
1.92-2.08 (6 H, m, CH2), 2.94 (3 H, s, NCH3), 3.43 (6 H, s,
N(CH3)2), 3.61 (1 H, m, CHCOOR), 4.04 (7.1 Hz, 2 H, q, OCH2),
4.12 (2 H, m, CHNR2), 5.83 (1 H, m, CHOSO2), 6.9-7.3 (3 H,
m, arom. C6, C7, C8), 7.8-8.4 (3 H, m, arom. C2, C3, C4). 13C
NMR (50.3 MHz, CDCl3/CD3OD, 1:1): 18.42 (CH3), 21.36
(CH2), 26.27 (CH2), 33.83 (CH2), 36.98 (NCH3), 43.52 (N(CH3)2),
46.01 (CHCOOR), 58.31 (CHN), 61.43 (CHN), 62.75 (CHSO3),
63.11 (OCH2), 117.06, 120.17, 123.85, 124.62, 126.09, 126.83,
130.21, 131.12, 136.19 (CSO3), 146.24 (CN(CH3)2), 170.06
(COOR).
(2R,3S)-O-(2â-Eth oxyca r bon yl-3â-tr op a n yl)-4-(d im eth -
yla m in o)a zoben zole-4′-th ioca r ba m id e (7). Under a nitro-
gen atmosphere, ethyl 3â-tropanol-2â-carboxylate (205 mg,
0.97 mmol) was treated with sodium hydride (29 mg, 1.2 mmol)
in dry THF. After 15 min, 4-(dimethylamino)azobenzene 4′-
isothiocyanate (273 mg, 0.97 mmol) was added, and the
solution was stirred 16 h at room temperature. Then, 1 mL of
water was added, the THF was removed, and the solution was
extracted with trichloromethane. Purification by flash chro-
matography (silica, first dichlomethane followed by dichlo-
romethane:ethanol, 100:1 then 25:1) afforded 7 (180 mg, 37%)
1-[4-(6-P h en yl-1,3,5-h exa t r ien yl)b en zyl]gu a n id in iu m
Su lfa te (5). 15.88 g of 2-(bromomethyl)-1,3-dioxolane and
25.10 g of triphenylphosphine were heated over calcium
chloride for 48 h at 100 °C. After the crude orange product
was washed with diethyl ether, the light-yellow solid was dried
in vacuo and added under nitrogen to a solution of 0.6 g of
lithium in 50 mL of dried methanol. After addition again of
50 mL of dried methanol, a clear red solution was obtained to
which 9.14 mL of cinnamaldehyde was added dropwise. The
solution was then heated under reflux for 1 h and stirred at
room temperature for a further 12 h. Reaction with 20 mL of
water followed by removing the solvent resulted in a brown
oil, which was turned into the corresponding aldehyde by
reaction with a 10% solution of hydrochloric acid for 16 h. After
basification and extraction with PE/trichloromethane (10:1),
the aldehyde was purified by flash chromatography (silica, PE:
trichloromethane, 1:1). The aldehyde thus obtained was then
added dropwise to a solution of 0.26 g of lithium and 14.55 g
of (4-cyanobenzyl)triphenylphosphonium bromide (starting
material for compound 4) in 80 mL of dried methanol. After
heating under reflux for 3 h and stirring 16 h at room
temperature, a yellow-green solid was filtered off, washed with
methanol, and purified by flash chromatography (silica, PE:
trichloromethane, 1:3) affording 3.84 g of the nitrile. This was
reduced by diisobutylaluminum hydride as in the case of the
nitrile precursor of compound 4 to give 3.19 g of the amine.
This was purified and finally transformed into the guani-
dinium sulfate by the procedure described above to afford 1.60
g (74%) of 5 as a light-yellow solid. Mp: 271-274 °C. 1H NMR
(200 MHz, DMSO-d6 /TFA-d): 3.98 (2 H, s, CH2), 6.5-6.7 (6
H, m, dCH), 7.0-7.5 (9H, m, arom). 13C NMR (50.3 MHz,
DMSO-d6/TFA-d): 42.9 (CH2), 127.0 (2 CH), 127.1 (2 CH),
128.3 (2 CH), 129.0, 129.3 (2 CH), 129.6 (2 CH), 129.8 (2 CH),
130.0 (2 CH), 135.0, 137.8, 138.2, 172.0 (CN3). Compound 5 is
a mixture of E- and Z-isomers. From the gas chromatography
data one can see that the all-trans,E-configurated isomer is
predominant (longer retention times). One can also see that
the amount of the all-trans-configurated product increases
with time. Therefore, clear NMR assignments to the different
isomers are not possible.
1
as a red oil. H NMR (200 MHz, CDCl3/CD3OD, 1:1) 1.27 (7.0
Hz, 3 H, t, CH3), 1.85-2.99 (6 H, m, CH2), 3.08 (3 H, s, NCH3),
3.12 (6 H, s, N(CH3)2), 3.59 (1 H, m, CHCOOR), 4.09 (7.0 Hz,
2 H, q, OCH2), 4.19 (2 H, m, CHNR2), 5.82 (1 H, m, CHOCS),
7.8-6.8 (8 H, m, arom). 13C NMR (50. 3 MHz, CDCl3/CD3OD,
1:1): 14.25 (CH3), 24.11 (CH2), 25.28 (CH2), 35.27 (CH2), 37.77
(NCH3), 40.29 (N(CH3)2), 41.08 (CHCOOR), 49.67 (CHN), 60.12
(CHN), 61.65 (OCH2), 63.11 (CHOCS), 111.47, 121.00, 122.93,
124.68, 143.61, 150.21, 152.33, 152.39, 170.48 and 171.50
(COOR or CS).
r a c-N-[(3-Met h yla m in o-1-p h en yl)p r op yl]-5-(d im et h -
yla m in o)-1-n a p h th a len esu lfon a m id e (8). 3-Chloro-1-phe-
nylpropanone (2 g, 11.9 mmol) was reduced by a borane-THF
solution (1 M, 35.7 mL) to give the respective alcohol (1.98 g,
11.7 mmol), which was dissolved in 15 mL of ethanol and
dropped into 57 mL of aqueous 40% methylamine solution.
After 16 h of stirring, methylamine and ethanol were removed
in vacuo, and the resulting 3-(methylamino)-1-phenylpropanol
(1.89 g, 11.46 mmol) was extracted with dichloromethane. The
protecting group was introduced by dissolving the secondary
amine in 20 mL of methanol, treatment with 2.75 mL of di-
tert-butyl dicarbonate and 1.6 g of sodium monocarbonate, and
sonication for 8 h. Methanol was removed in vacuo, dichlo-
romethane was added, and the salt was filtered off. Then, 35
mL of water were added, and the protected aminopropanol
(2.54 g, 9.61 mmol) was separated by extraction with dichlo-
romethane. The hydroxy group of this compound was converted
into an amino group by a Mitsunobu reaction. Thus, under a
nitrogen atmosphere, the aminopropanol was dissolved in 10
mL of dry THF, and diethyl azodicarboxylate (1.51 mL, 9.61
mmol), triphenylphosphine (2.52 g, 9.61 mmol), and phthal-
imide (1.41 g, 9.61 mmol) were added. After 16 h of stirring at
room temperature, the solvent was removed in vacuo, and the
product was separated by flash chromatography (silica, first
PE:dichloromethane, 3:1, followed by dichloromethane) to give
the phthalimide (822 mg, 2.09 mmol). This was dissolved in
ethanol, treated with 8.88 mL of hydrazine monohydrate, and
heated for 1 h under reflux to give a light-yellow suspension.
Ethanol was removed in vacuo, diluted sodium bicarbonate
solution was added, and the mixture was extracted three times
with dichloromethane. The organic solvent was twice extracted
with 5% citric acid. To the aqueous citric acid solution was
added dichloromethane followed by basification with sodium
(2R,3S)-2â-E t h oxyca r b on yl-3â-t r op a n yl
5-(Dim et h -
yla m in o)n a p h th a len e-1-su lfon a te (6). (-)-(2R,3S)-Cocaine
hydrochloride (400 mg, 1.18 mmol) was dissolved in 5 mL of
ethanol and 2 mL of 6 M HCl and refluxed under a continuous
stream of dry gaseous HCl for 8 h. After removing the solvent
the residue was made alkaline with aqueous sodium bicarbon-
ate and extracted with trichloromethane. (2R,3S)-Ethyl-3â-
tropanol-2â-carboxylate (218 mg, 1.02 mmol) was isolated by
flash chromatography (silica, ethanol). It was dissolved in 7

Potential Neuroblastoma Imaging Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3107
bicarbonate under cooling with ice and vigorous stirring. The
dichloromethane phase was separated and dried with sodium
sulfate. By removing the dichloromethane in vacuo, rac-3-
(N-methyl-N-(tert-butyloxycarbonyl)amino)-1-phenylprop-
ylamine, the starting material for the formation of 8-10, was
obtained as a colorless oil (1.03 g, 3.84 mmol). This oil was
dissolved in 40 mL of dichloromethane, and then 5.4 mL of
dry pyridine and 1.04 g (3.84 mmol) of dansyl chloride were
added. After 16 h of stirring, dichloromethane was removed
in vacuo and the residual yellow fluorescent oil was purified
by flash chromatography (silica, first dichloromethane followed
by dichloromethane:ethanol, 100:1 then 25:1) to give 1.07 g
(2.14 mmol) of the product. The BOC group was removed by
reaction with 150 mL of TFA at 0 °C. The mixture was allowed
to reach room temperature within 6 h, followed by basification
with aqueous sodium bicarbonate solution. After extraction
with dichloromethane, the product was separated by flash
chromatography (silica, first ethanol followed by ethanol:
dichloromethane, 1:1) to give 8 (567 mg, 1.4 mmol, 65%) as a
yellow fluorescent oil. 1H NMR (200 MHz, CDCl3/CD3OD
1:1): 1.25 (2 H, m, CH2), 2.09 (3 H, s, NCH3), 2.22 (5.9 Hz, 2
H, t, CH2N), 3.21 (6 H, s, N(CH3)2), 4.64 (5.8 Hz, 1 H, t,
CHNHSO2), 6.9-7.3 (8 H, m, arom), 7.7-8.5 (3 H, m, arom).
13C NMR (50.3 MHz, CDCl3/CD3OD, 1:1): 33.11 (NCH3), 38.03
(CH2), 40.97 (N(CH3)2), 46.32 (CH2N), 47.31 (CHN), 116.53,
122.24, 122.65, 124.72, 125.04, 126.22, 126.98, 129.17 (2 CH),
129.50 (2 CH), 129.87, 132.34, 137.31 (CSO2), 140.01, 147.13
(CN(CH3)2).
on 24-multiwells (Falcon) and grown at 37° C (5% CO2
incubator) in Dulbecco’s modified Eagle’s medium supple-
mented with 10% fetal calf serum. Before use for uptake
experiments, the culture medium was removed, and the cells
were washed three times with Krebs-Ringer-Hepes buffer
(KRH) of the following composition (mM): 125 NaCl, 2.4 K2-
SO4, 1.2 KH2PO4, 1.2 MgSO4, 5.6 D-(+)-glucose, 1 ascorbic acid,
25 Hepes/Tris (pH 7.4).
Affin ity a ssa y: To test the affinity for the NET of the
synthesized compounds, IC50 values for inhibition of uptake
of [3H]norepinephrine ([3H]NE) into the neuroblastoma cells
were determined essentially as described earlier.17 Cells were
preincubated for 10 min in KRH containing 10 µM pargyline
(to inhibit monoamine oxidase), 10 µM U-0521 (3,4-dihydroxy-
2-methylpropiophenone (Upjohn); to inhibit catechol O-meth-
yltransferase), 1 g/L bovine serum albumin, and various
concentrations of the test compound (except for controls).
Thereafter, cells were incubated for 5 min in the additional
presence of 10 nM [3H]NE (NEN-DuPont; specific activity: 52
Ci/mmol). Nonspecific uptake of [3H]NE was determined in
parallel control experiments carried out in the presence of 10
µM nisoxetine. At the end of the experiment, cells were rinsed
three times with ice-cold KRH and then solubilized in 1.5 mL
of 0.1% Triton-X-100. Radioactivity of the solubilized cells was
determined by scintillation counting. Specific uptake was
defined as the difference between total [3H]NE uptake and
uptake in the presence of nisoxetine. Data for specific uptake
were related to cell protein which was determined according
to the Lowry method.20 Experiments were carried out in
duplicate and are presented as means ( SEM. IC50 values were
determined using the commercial software program GRAPH-
PAD INPLOT.
F lu or escen t la belin g: The fluorescent compounds (Table
1) were examined for their ability to label human SK-N-SH-
SY neuroblastoma cells. Cells were grown until confluent in
small tissue culture flaks (25 cm2) under the conditions
descibed above. After removal of the culture medium and after
two washes with KRH (10 mL each), cells were preincubated
for 10 min (at 37 °C) in KRH (in the absence or presence of 10
µM nisoxetine) and then incubated for 30 min (at 37 °C) in 5
mL of KRH containing either 10 µM nisoxetine (to obtain blank
values) or 10 µM of the fluorescent compound (to obtain total
retention) or 10 µM of the fluorescent compound together with
10 µM nisoxetine (to measure nonspecific retention). There-
after the incubation medium was completely aspirated, and
the cells were washed with 3 × 10 mL of 0.9% sodium chloride
solution. After removal of the washing solution, one set of cells
was inspected under the fluorescence microscope. To the other
set of cells was added 400 µL of distilled water, and the cells
(in closed flasks) were frozen at -70 °C for 24 h and thereafter
sonicated for 10 s. The resulting suspension was transferred
to small centrifuge tubes and centrifugated for 10 min at
10000g. From the clear supernatant excitation and fluores-
cence spectra were recorded using a Shimadzu RF-5000
spectrofluorophotometer. Specific cell retention of the fluores-
cent compound was determined from the difference between
the maximum peak of substance-specific fluorescence intensity
obtained in the absence and presence of the specific NET
inhibitor nisoxetine.
r a c-4-[(3-Meth yla m in o-1-p h en yl)p r op yl]a m in o-7-n itr o-
2,1,3-ben zoxa d ia zole (9). rac-3-(N-Methyl-N-(tert-butoxy-
carbonyl)amino)-1-phenylpropylamine (110 mg, 0.42 mmol)
was dissolved in 15 mL of dichloromethane followed by
addition of 89 mg of sodium bicarbonate and 83.3 mg (0.42
mmol) of 4-chloro-7-nitro-2,1,3-benzoxadiazole under a nitro-
gen atmosphere. The mixture was stirred 16 h at room
temperature. The dichloromethane was removed in vacuo, and
the product precipitated. After the residue was dissolved again
in dichloromethane, the sodium bicarbonate was filtered off.
Purification by flash chromatography as described for the
precursor of compound 8 gave the product (127.3 mg, 0.30
mmol) as a yellow-green fluorescent oil. The BOC group was
removed, and the product was purified according to the
procedure described above to give 9 (71.6 mg. 0.22 mmol, 73%)
1
as a yellow-green fluorescent oil. H NMR (200 MHz, CDCl3/
CD3OD, 1:1): 1.20 (2 H, m, CH2), 1.98 (3 H, s, NCH3), 2.21
(5.9 Hz, 2 H, t, CH2N), 4.86 (5.7 Hz, 1 H, t, CHNH), 7.1-7.35
(6 H, m, arom), 8.22 (9 Hz, 1 H, d, CHCNO2). 13C NMR (50. 3
MHz, CDCl3/CD3OD, 1:1): 35.32 (NCH3), 35.67 (CH2), 48.45
(CH2N), 58.19 (CHN), 101.28 (CNH, CNO2), 123.06 (CH),
126.93 (2 CH), 128.68 (2 CH), 129.72 (2 CH), 137.53 (C), 145.32
(2 CN).
r a c-N-[4-(4-(Dim eth yla m in o)p h en yla zo)p h en yl]-N′-[(3-
m et h yla m in o-1-p h en yl)p r op yl]t h iou r ea (10). rac-3-(N-
Methyl-N-(tert-butoxycarbonyl)amino)-1-phenylpropylamine (108
mg, 0.41 mmol) was dissolved in 15 mL of dry THF followed
by addition of sodium hydride (10.8 mg, 0.45 mmol). After 15
min, 4-(dimethylamino)azobenzole 4′-isothiocyanate (116 mg,
0.41 mmol) was added under a nitrogen atmosphere. After 16
h of stirring at room temperature, 1 mL of water was added,
THF was removed, and the solution was extracted three times
with dichloromethane. Purification by flash chromatography
as described for the precursor of compound 8 gave the product
(109.2 mg, 0.20 mmol) as a red oil. The BOC group was
removed, and the product was purified according to the
procedure described above to give 10 (58.8 mg, 0.13 mmol,
66%) as a red oil. 1H NMR (200 MHz, CDCl3/CD3OD, 1:1): 1.28
(2 H, m, CH2), 2.00 (3 H, s, NCH3), 2.19 (6 Hz, 2 H, t, CH2N),
4.68 (6 Hz, 1 H, t, CHNH), 6.8-7.8 (13 H, m, ArH). 13C NMR
(50.3 MHz, CDCl3/CD3OD, 1:1): 29.05 (NHCH3), 39.23 (CH2),
40.43 (N(CH3)2), 46.77 (CH2NH), 53.45 (CHNH), 112.09,
125.66, 125.91, 126.20, 128.04, 129.25, 129.58, 140.09, 143.70,
152.20, 153,0.1, 153.48, 170.31 (CS).
Ack n ow led gm en t. We thank Petra Breiden for
skillful technical assistance. This work was supported
by a grant from the German Neuroblastoma Research
Foundation (F.B.).
Su p p or tin g In for m a tion Ava ila ble: FAB-MS respec-
tively MALDI-TOF-MS of compounds 1-15. This material is
available free of charge via the Internet at http://pubs.acs.org.
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