Synthesis and evaluation of novel radioiodinated benzamides for malignant melanoma

Article abstract of DOI:10.1021/jm0701627

The imaging potential of a series of [¹²³I]benzamides was studied in mice bearing B16F0 melanoma tumors. Compound [¹²³I]25 exhibited tumor uptake >8 %ID/g at 1 h, while that of [¹²³I]14d and [¹²³I]25 reached a maximum of 9-12 %ID/g at 6 h. Standardized uptake values of [¹²³I]14d were higher than 100 between 24 and 72 h after injection. In haloperidol treated animals, the tumor uptake of [ ¹²³I]14d was not significantly different to controls, while significant reduction of [¹²³I]25 uptake was observed, supporting that [¹²³I]14d uptake relates to melanin interaction, whereas part of the mechanism of [¹²³I]25 uptake is related to its σ₁-receptor affinity. Benzamides 14d and 25, which display rapid and high tumor uptake, appear to be promising imaging agents for melanoma detection, while 14d, which displays a long lasting and high melanoma/nontarget ratio, is more suitable for evaluation as a potential radiotherapeutic.

Full text of DOI:10.1021/jm0701627

J. Med. Chem. 2007, 50, 3561-3572
3561
Synthesis and Evaluation of Novel Radioiodinated Benzamides for Malignant Melanoma
Tien Q. Pham, Ivan Greguric, Xiang Liu, Paula Berghofer, Patrice Ballantyne, Janette Chapman, Filomena Mattner,
Branko Dikic, Timothy Jackson, Christian Loc’h, and Andrew Katsifis*
Radiopharmaceuticals Research Institute, Australian Nuclear Science and Technology Organisation, PMB 1 Menai N.S.W. 2234,
Sydney, Australia
ReceiVed February 13, 2007
The imaging potential of a series of [123I]benzamides was studied in mice bearing B16F0 melanoma tumors.
Compound [123I]25 exhibited tumor uptake >8 %ID/g at 1 h, while that of [123I]14d and [123I]25 reached a
maximum of 9-12 %ID/g at 6 h. Standardized uptake values of [123I]14d were higher than 100 between 24
and 72 h after injection. In haloperidol treated animals, the tumor uptake of [123I]14d was not significantly
different to controls, while significant reduction of [123I]25 uptake was observed, supporting that [123I]14d
uptake relates to melanin interaction, whereas part of the mechanism of [123I]25 uptake is related to its
σ1-receptor affinity. Benzamides 14d and 25, which display rapid and high tumor uptake, appear to be
promising imaging agents for melanoma detection, while 14d, which displays a long lasting and high
melanoma/nontarget ratio, is more suitable for evaluation as a potential radiotherapeutic.
Introduction
Malignant melanoma is a very aggressive cancer, with a high
rate of metastasis brought about by excessive UV exposure.
Despite the increasing incidence of this disease and compared
to advances in other areas of cancer, there are still no effective
treatments available. Radiopharmaceuticals that can target the
random metastatic dissemination of melanoma tumors may also
offer opportunities for imaging and staging the disease as well
as potential radiotherapeutic applications.1
Several biochemical targeting systems incorporating a variety
of diagnostic and therapeutic radionuclides have been investi-
gated as potential imaging and radiotherapeutic agents, including
monoclonal antibodies,2 iodothiouracils,3 melanocortin-1 recep-
tor targeting peptides,1,4 iodoquinolines,5 methylene blue dye,6
and iodobenzamides.7
Figure 1. Structures of the known benzamides BZA, BZA2, 1, and
IBP.
confirmed the efficacy of radioiodinated benzamides as selective
imaging agents in patients with cutaneous and ocular melanoma
based on the selective high affinity binding to melanin contain-
ing melanocytes. These findings have provided the basis for
further developments in melanin-based radiopharmaceuticals.
A key feature of melanoma tumors is the extensive pigmenta-
tion present in most melanoma tumors cells, thus making it a
very attractive target for both diagnosis and treatment. To date
there has been a considerable number of iodinated benzamide
derivatives exhibiting good uptake in melanoma tissue.7 Pre-
clinical investigations with a number of melanin targeting radio-
pharmaceuticals demonstrated selective uptake in melanoma
tumor bearing mice.8,9 It was also shown that the uptake of the
radioiodinated benzamides N-(2-diethylaminoethyl)-4-[123I]iodo-
benzamide) ([123I]BZAa) and the ortho derivative ([123I]BZA2)
in cells was dependent on the melanin content10 and not on a
receptor-based mechanism, as demonstrated by similar compounds
such as IBP, which were sigma-receptor related11 (Figure 1).
In the current work, our aim was to design and optimize a
series of iodinated benzamides that could display high melanoma
tumor uptake and rapid clearance from the body suitable for
scintigraphy and radiotherapeutic applications. This optimization
program involved the incorporation of a variety of alkyl- or
piperidinyl side chains to a series of iodinated benzamides. The
strategy in this work was to simultaneously take advantage of
the optimized lipophilic side chains developed previously14,15
and the optimized substitution pattern on the aromatic ring.16
To further enhance the tumor uptake and retention of the
radiotracers through sigma binding, substitution with piperidine
as in IBP,11 was also examined. Here we report the synthesis
of a series of [123I]iodobenzamides and their biological evalu-
ation in melanoma tumor bearing mice.
The radioiodinated benzamides [123I]BZA12 and [123I]BZA2
13,14
have been prepared and evaluated in melanoma patients,
resulting in excellent detection of melanoma and its metastases
with high sensitivity and selectivity. These studies have
Chemistry. Access to the alkyl amino benzamides was via
condensation of a benzoic acid derivative with an appropriate
amine. The butyldiamines 5a, 5b, 5d, and 5e were produced
by alkylation of a variety of secondary amines (2a, 2b, 2d, and
2e) with 4-bromobutyronitrile (3) using K2CO3 in 1-butanol
followed by reduction of the resulting nitriles (4a, 4b, 4d, and
4e) with lithium aluminum hydride (Scheme 1). Butyldiamine
5c was prepared by literature methods.17
* To whom correspondence should be addressed. Tel.: +61 2 9717 9094.
Fax: +61 2 9717 9262. E-mail: akx@ansto.gov.au.
a Abbreviations: SUV, standardized uptake value; BZA, N-(2-diethyl-
aminoethyl)-4-iodobenzamide; BZA2, N-(2-diethylaminoethyl)-2-iodoben-
zamide; IBP, N-(1-benzylpiperidin-4-yl)-4-iodobenzamide; EDC, 1-ethyl-
3-(3-dimethylaminopropyl)-carbodiimide; HOBT, N-hydroxybenzotriazole;
NMM, N-methylmorpholine; CAT, chloramine-T, that is, [sodium chloro-
(tosyl)amide)]; RP-HPLC, reverse phase high pressure liquid chromatog-
raphy; DTG, 1,3-di-o-tolylguanidine; SIMS, secondary ion mass spectros-
copy; SPECT, single photon emission computed tomography.
10.1021/jm0701627 CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/28/2007

3562 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Scheme 1. Chemical Synthesis of Butyldiaminesa
Pham et al.
chemical purity of the [123I]iodobenzamides, as assessed by
analytical RP-HPLC, was >95% and the specific activity was
>2 GBq/nmol.
Lipophilicity Values. The lipophilicity values of the iodo-
benzamides were determined by RP-HPLC in buffered solutions
at pH 7.5 as a representation of physiological conditions. The
results are summarized in Table 2. The logP7.5 values of the
studied benzamides are in the range of 0.75-2.6. The 2-meth-
oxy-5-iodobenzamides 1, 14b, 14c, 14d, 14e, 14f, 19m, and
19n exhibited logP values between 0.8 and 1.95 depending on
the length of the N-alkyl chains and the nature of the amino
function on the benzene ring. Generally, with identical N-alkyl
chains, the 4-iodobenzamides, 25, 27, 30, and 33, displayed
higher logP values than the corresponding 2-methoxy-5-iodo-
benzamides, illustrated by 30 and 14c.
Receptor Binding Studies. The “benzamide”-type structure
of the compounds described here have biological activity that
may result from a number of competing mechanisms, that is,
melanin uptake (cf. BZA2) or affinity to the σ-receptor (cf. IBP),
which is highly expressed in melanoma tumors. The potency of
unlabeled benzamides to inhibit the specific binding of [3H]-
(+)-pentazocine to σ1-receptors and of [3H]DTG to σ2-receptors
from guinea pig brain membranes were determined in competi-
tive binding assays at 10-5 M concentration of unlabeled iodo-
benzamides. For compounds able to inhibit more than 90% of
the tritiated ligands, the IC50 values were determined in full
competition experiments. The Ki values are reported in Table
2. High to moderate affinities for σ1-receptors were obtained with
the 4-piperidines 25 and 27, which are structural analogs of IBP,
with Ki values of 6 and 140 nM, respectively, and with 33, with
Ki ) 32 nM. Compounds 1, 14b, 14c, 14d, 14e, 14f, 19m, 19n,
and 30 exhibited Ki values higher than 600 nM. For σ2-receptors,
only 33 presented a moderate affinity with Ki ) 75 nM.
Biodistribution. The [123I]iodobenzamides were injected in
melanoma tumor bearing mice to evaluate their potential as
tumor markers. Postinjection points at 1, 6, and 24 h were
chosen for determination of the distribution of each compound
in various organs and tissues. For those benzamides with high
uptake in the tumor, 3, 48, and 72 h time points were added to
determine long-term retention.
To visualize melanoma tumors, a potential radiotracer needs
to meet two criteria: high uptake in the tumor and simultaneous
high contrast to other tissues. A comparison between organ
distribution and body clearance was made for all the [123I]-
iodobenzamides studied.
a Reagents and conditions: (a) K2CO3, KI, 1-butanol, reflux 20 h; (b)
LiAlH4, ether, 0 °C, 1 h room temperature.
The 4-acetamido-2-methoxybenzamide 7, prepared by acetyl-
ation of the amine 618 with acetic anhydride (Scheme 2), not
only served as a lead fragment with a precedence in tumor
targeting, but was also amenable to iodination in the 5 position.16
The corresponding iodinated derivatives were synthesized by
treatment with iodine monochloride at 50 °C in acetic acid. Ester
hydrolysis of 7 and its iodinated analogue 8 under basic
conditions also resulted in deacetylation to give the free amines
9 and 10. Subsequent reacetylation of the amine with acetic
anyhydride produced the required benzoic acids 11 and 12.
The benzoic acids 11 and 12 were condensed with amines
5a-5e via an acid chloride intermediate, prepared from thionyl
chloride, to form the benzamides 13a-13e and 14a-14e
(Scheme 2). The known compound 1 was synthesized by the
same method using the commercially available diethylamino-
ethylamine. Benzamides 13a and 14a were treated with saturated
HCl in ethyl acetate at room temperature to give the benzamides
13f and 14f. The 4-mesylamino and 4-tosylamino analogues,
18m,n and 19m,n, were provided by first, deacetylating 1 with
KOH in methanol and second, treating the deprotected amine
with methanesulphonyl chloride or p-toluenesulphonyl chloride
with pyridine in dichloromethane.
The synthesis of the conformationally restricted IBP analo-
gous benzamides began with an alkylation of 4-BOC-aminopi-
peridine with either 1-bromo-2-fluoroethane or bromoethanol
(Scheme 3). Subsequent BOC-deprotection of the resulting
alkylpiperidines 20 and 21 with neat TFA gave amines 22 and
23 that were suitable for condensation with the appropriate
4-halobenzoic acids, using EDC (1-ethyl-3-(3-dimethylamino-
propyl)-carbodiimide) and HOBT (N-hydroxybenzotriazole)
with NMM (N-methylmorpholine) in DMF, to give benzamides
24, 25, 26, and 27.
The condensation of 4-halobenzoic acids with amines 5c and
5e, gave the known benzamide 3015 and its analogues 31, 33,
and 34. The bromobenzamides, 24, 26, 31, and 34, were treated
with hexamethylditin and a catalytic amount of Pd(PPh3)4 in
refluxing toluene or dioxane to yield the stannyl derivatives 28,
29, 32, and 35 to be used for electrophilic radioiodination.
Radiochemistry. The benzamides were radioiodinated with
no carrier added [123I]iodine using two electrophilic substitution
methods, as shown in Scheme 4. Compounds 13b-13f, 15, and
18m,n were regioselectively radioiodinated with 123I via the
corresponding thallium bis trifluoroacetate intermediate,16 by
first treating the benzamide with Tl(TFA)3 in acetic acid
followed by reaction with [123I]INa to give the corresponding
radioiodinated products in 40-55% radiochemical yields (Table
1). The trialkyl stannane derivatives 28, 29, 32, and 35 were
labeled with 123I via standard electrophilic iododestannylation
reactions in the presence of chloramine-T (CAT) as the oxidant
to afford the corresponding [123I]iodobenzamides in 50-95%
radiochemical yields. After purification of the radiolabeled
benzamides, by semipreparative C18 RP-HPLC, the radio-
In the liver, compounds 30 and 33 displayed the highest
uptake (40-45 %ID) at 1 h, which cleared with a 6 h biological
half-life. Liver uptake of 14d, 27, and 25 was lower (15-20
%ID), with a faster clearance (biological half-life: 1-1.5 h).
At that time, for all other [123I]iodobenzamides, the liver uptake
was between 2 and 5 %ID, comparable to the value obtained
with [123I]BZA2. In the kidney, the highest concentrations were
found at 1 h for 14c, 14d, and 33 (3-5 %ID), followed by
14b, 30, and 25 (1.2-2 %ID), which were similar to [123I]BZA2.
From this organ, 14d cleared quickly, with a biological half-
life of less than 1 h, while other compounds had half-life
clearances ranging from 1.5 to 2 h for methoxy-5-iodobenza-
mides 1, 14b, 14c, 14e, 14f, 19m, and 19n to 3-5 h for the
4-iodobenzamides, 25, 27, 30, and 33.
In the intestine, higher amounts of radioactivity were observed
at 1 h for the 2-methoxy-5-iodobenzamides 1, 14b, 14c, 14d,
14e, 14f, 19m, and 19n (30-40 %ID) than that for the
4-iodobenzamides, 25, 27, 30, 33, and BZA2 (7-15 %ID).
However, at 24 h postinjection, the radioactivity of both the

Radioiodinated Benzamides for Malignant Melanoma
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3563
Scheme 2. Synthesis of Iodobenzamides and Precursors for Radiolabelinga
a Reagents and conditions: (a) acetic anhydride, 50 °C, 2 h; (b) ICl, acetic acid, 50 °C, 6 h, room temperature, 24 h; (c) KOH, methanol, reflux, 2-18
h; (d) SO2Cl, THF, 4 h, addition of amine, room temperature, 4 h; (e) 3 M HCl in ethyl acetate, 45 min; (f) mesyl chloride or tosyl chloride, pyridine, DCM,
0 °C, 30 min, room temperature, 24 h.
[
123I]-2-methoxy-5-iodobenzamides and the [123I]-4-iodobenza-
reference compounds [123I]BZA2, [123I]1, and [123I]30, which
were prepared and studied under our experimental conditions.
The benzamides 1, 14c, and 25 exhibited the highest tumor
uptake at 1 h (>8 %ID/g), followed by 14b, 14d, 30, 19m, and
27, with uptake similar to BZA2 (∼5-7 %ID/g). For com-
pounds 14d, 25, and 27, the uptake increased with time and
reached a maximum at 6 h. For benzamides 1, 14c, 14d, 30,
25, and 27, the tumor uptake was still higher than 5 %ID/g at
24 h.
A comparison between the uptake values of the 2-methoxy-
5-iodobenzamide derivatives against the corresponding amine
side chain structure indicated higher and longer retention of
activity in the tumor for the N-dipropyl (14c) and N-methyl-
butyl (14d) substituents compared to other linear, branched, or
cyclic derivatives. The higher uptake displayed by these two
benzamides strongly correlates with the highest lipophilicity
values of the 2-methoxy-5-iodobenzamides (Figure 2). The
effects of further structural modifications in the para postion
of these 2-methoxy-5-iodobenzamide derivatives are reflected
in the tumor uptake of [123I] benzamides 1, 19m, and 19n. In
comparison to 1, a significant decrease in the earlier tumor
uptake and an increased wash out from tissue was observed
with the 4-mesylamino- and 4-tosylamino-analogues (19m,n).
The N-piperidin-4-yl analogues of IBP, 25, and 27 presented
high and long lasting tumor uptake that peaked at more than
10 %ID/g at 6 h.
mides in the intestine was almost eliminated (ID < 0.6%). These
results seemed to indicate that the [123I]iodobenzamide excretion
route is greatly influenced by the number and nature of
substituents on the benzene ring of the molecules.
The thyroid uptake of the [123I]benzamides was low and
increased slightly during the study from 0.1 %ID at 1 h to reach
a plateau of ∼1 %ID from 6 to 24 h. During the time course of
the study, compounds 25, 27, and 33 indicated a particularly
low thyroid uptake, < 0.2 %ID. This is probably due to the
fact that they are bound to sigma enriched tissues, reducing their
rate of deiodination. The low thyroid uptake of the studied [123I]-
iodobenzamides is an indication that these tracers are relatively
stable to in vivo deiodination.
Significant uptake of the [123I]benzamides was also observed
in the eyes (0.5 %ID), supporting a melanin-related uptake
mechanism that has been described by Moins et al.14 for [123I]-
BZA. Recent studies, using secondary ion mass spectroscopy
(SIMS), confirmed that BZA was found to locate in pigmented
tissues of skin and grafted melanomas and in the pigmented
structures of the eye: choroidal melanocytes and retinal pigment
cells of C57BL/6J black mice.19
As a consequence of simultaneous low organ uptakes and
rapid clearance, less than 1.4 %ID of the radioactivity remained
in the mouse body 24 h after administration of radioiodinated
14d. For compounds 1, 14b, 14c, 14e, and 25, this value was
less than 6 %ID. In contrast, 19m, 19n, 30, 33, and 27 had
higher organ uptake and slower clearance, with more than 10%
of the radioactivity remaining in the mouse body.
In the brain, at 1 h, the uptake of 25 and 27 was 3.1 %ID/g
and 1 %ID/g, respectively. This uptake is explained by the logP
values (2.57 and 1.93, respectively), allowing the tracers to cross
the blood-brain barrier with relatively good affinity of these
compounds for σ-receptors.
The values of tumor and organ concentration, expressed in
%ID/g, are summarized in Table 3 and were compared to

3564 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Scheme 3. Synthesis of Trimethylstannyl Precursorsa
Pham et al.
a Reagents and conditions: (a) K2CO3, ACN, 5 h; (b) TFA, 1 h; (c) EDC, HOBT, NMM, DMF, 24 h; (d) SO2Cl, THF 4 h; (e) (Me3Sn)2, Pd(PPh3)4,
toluene or dioxane, reflux 24 h.
Scheme 4. Radiolabeling of [123I]iodobenzamides
tive σ1-σ2 inhibitor, was examined over 24 h in selected organs
and tumor tissue. As seen in Table 4, at 1 h p.i., the tumor and
main organ uptakes were not significantly different in treated
animals compared to controls with [123I]14d. At 24 h p.i., a
significant decrease was observed in the kidney, but the uptake
was found to be unchanged in the tumor, eyes, and in organs
such as liver, lung, and brain. With [123I]25, at 1 h postinjection,
decreases in the uptake of radioactivity were observed in the
kidney, liver, and brain (-25 to -45%, p < 0.01), while an
increase of radioactivity concentration was observed in the blood
(+50%, p < 0.01). At 24 h p.i., a significant reduction of the
uptake of the radioactivity occurred in the tumor (-33%, p <
0.01) in the brain, lung, and kidney (-62 to -68%, p < 0.01).
Sigma receptors are present in the central nervous system as
well as tissues such as the liver, kidneys, and lungs.20 Halo-
peridol, which is known to inhibit the binding at both σ1 and
σ2 receptor subtypes with high affinity,21 was able to decrease
the uptake of [123I]25 in these organs and in the tumor. These
findings confirmed the in vitro results and supports that part of
the mechanism of tumor uptake of [123I]25 may be due to its
σ-receptor affinity. Compared to the control animals, the uptake
of [123I]14d and of [123I]25 in the eyes of haloperidol-treated
animals was not statistically different. This indicated that the
uptake in the eyes of these [123I]iodobenzamides is related to
pigmented cells and is not σ-receptor mediated.
Imaging Studies. The whole body distribution of [123I]14d
and [123I]25 in C57BL/6J mice was followed over 48 h using
SPECT imaging. Figure 3 shows typical SPECT images of mice
acquired 24 h after the radiotracer injection. In these images,
the highest radioactivity was observed in the tumor, followed
by the eyes. The radioactivity observed in the thyroid of the
mouse injected with [123I]14d was higher than those injected
with [123I]25. In contrast, the activity remaining in the body of
[
123I]Iodobenzamide body versus tumor contrast was ascer-
tained by calculating the standardized uptake values (SUV) at
the time of animal sacrifice. These values are reported in Table
3. Due to the higher radioactive concentration remaining in the
body, SUV24 was lower than 6 for compounds 19m, 19n, and
33. For compounds 14f, 14b, 14c, 14e, 30, 25, 27, and BZA2,
SUV24 ranged from 10 to 20, despite a tumor concentration
higher than 5 %ID/g for 25 and 27. The benzamide 1 presented
a high contrast with SUV greater than 40 between 24 and 72 h.
For 14d, due to simultaneous high uptake and long retention in
the tumor and low body concentration, SUV24-72 was higher
than 100.
Competition Studies. To assess the tumor uptake mechanism,
in vivo competition studies were performed on the two lead
compounds [123I]14d and [123I]25, having the highest tumor
uptake but different in vitro pharmacological profile. The
blocking effect on the tracer uptake of haloperidol, a nonselec-

Radioiodinated Benzamides for Malignant Melanoma
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3565
Table 1. Radiolabeling Data for Benzamides
radiolabeling
method
flow rate
(mL/min)
retention
time (min)
RCYg
(%)
[
123I]-cmpd
precursor
solventc
1
15
Tl(TFA)3
40/60d
40/60d
40/60d
50/50d
60/40d
40/60d
40/60d
40/60d
40/60e
70/30e
55/45d
70/30d
42.5/57.5f
1.5
1.5
1.5
2.0
2.0
1.5
1.5
1.5
2.0
1.5
3.0
2.0
4.0
17
16
26
17
24
15
21
16
14
13
17
21
11
55
44
50
50
40
48
46
42
91
96
50
72
85
14b
14c
14d
14e
14f
19m
19n
25
27
30
33
BZA2
13b
13c
13d
13e
13f
18m
18n
28
29
32
35
Br-BZA2
Tl(TFA)3
Tl(TFA)3
Tl(TFA)3
Tl(TFA)3
Tl(TFA)3
Tl(TFA)3
Tl(TFA)3
CAT
CAT
CAT
CAT
a
halogen-exchangeb
a Bromo analogue of BZA2. b Halogen-exchange method, as described by Moins et al.15 c Acetonitrile/(0.1 M) ammonium acetate, %/%, v/v. d Alltech
Alphabond column (C18, 10 µm, 300 × 7.5 mm). e Phenomenex Bondclone column (C18, 10 µm, 300 × 7.8 mm). f Alltech Econosil (C18, 10 µm, 250 ×
10 mm). g Radiochemical purity for each radiotracer was greater than 95%.
Table 2. Receptor Binding Data and logP7.5 Values for Benzamides
a
b
Ki σ1
Ki σ2
X
Y
Z
R
(nM)
(nM)
logPc
1
I
I
I
I
I
I
I
I
H
H
H
H
NHAc
NHAc
NHAc
NHAc
NHAc
NHAc
NHmesyl
NHtosyl
I
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
2-(N,N-diethylamino)ethyl
4-(N-i-propyl-N-methylamino)butyl
4-(N,N-dipropylamino)butyl
4-(N-butyl-N-methylamino)butyl
4-(2-azanorborn-2-yl)butyl
4-(N-butylamino)butyl
2-(N,N-diethylamino)ethyl
2-(N,N-diethylamino)ethyl
N-(2-fluoroethyl)piperidin-4-yl
N-(2-hydroxyethyl)piperidin-4-yl
4-(N,N-dipropylamino)butyl
4-(2-azanorborn-2-yl)butyl
1650
5200
7800
1600
680
2500
800
650
6
>100 000
5400
>100 000
2300
3900
6600
45000
>100 000
2600
1.54 ( 0.03
0.75 ( 0.02
1.65 ( 0.04
1.57 ( 0.03
0.80 ( 0.02
1.05 ( 0.02
0.82 ( 0.02
1.95 ( 0.03
2.57 ( 0.04
1.93 ( 0.03
2.46 ( 0.04
1.63 ( 0.03
14b
14c
14d
14e
14f
19m
19n
25
27
30
33
I
I
I
H
H
H
140
1300
32
1700
1000
75
a Assays were carried out by Novascreen Biosciences using [3H]-(+)-pentazocine for σ1-receptors. b [3H]-DTG for σ2-receptors and the conditions provided
in literature.31 c Assessed by HPLC using a literature method.30
the mouse injected with [123I]25 is significantly higher than of
the mouse injected with [123I]14d. These observations confirmed
the quantitative results obtained in biodistribution studies.
(Ki ) 6 nM). In vivo, haloperidol, a nonspecific σ1-σ2 receptor
inhibitor, was able to prevent uptake of [123I]25 in organs
possessing a high density of σ-receptors and also in the B16
melanoma tumor. As the B16 melanoma cell line has also been
reported to express σ-receptors,25 it is likely that the tumor
uptake of [123I]25 is partially due to its sigma receptor profile.
No competitive effect of haloperidol was observed in the tumor
uptake of the aminoalkylbenzamide [123I]14d, suggesting no
interaction with σ-receptors. In addition, [123I]14d had significant
uptake in the eyes, confirming a mainly melanin-related uptake
mechanism.
From these results, compound 14d displayed a 7-fold higher
contrast value than BZA2 with similar uptake in tumors, while
compound 25 exhibited a 50% higher uptake in tumors than
BZA2 over a 24 h period. These qualities suggest that 14d and
25 appear to be promising imaging agents for melanoma
detection and 14d, with a high melanoma/nontarget ratio, is
more suitable for evaluation as a potential radiotherapeutic.
In conclusion, two series of iodobenzamides have been
developed for their ability for tumor scintigraphy and for studies
in potential radiotherapeutic applications. The biodistribution
and pharmacokinetic profile of the [123I]iodobenzamides studied
was greatly influenced by the substituents on the benzene ring,
whereas the uptake values in the tumor correlated with the
molecule’s lipophilicity through the nature of the aminoalkyl
side chain.
Summary
In the development of novel iodobenzamides for melanoma
scintigraphy and radiotherapy, a better understanding of the
pharmacology and pharmacokinetics of the radiotracer is
required. The uptake mechanism of radioiodinated benzamides
into melanoma cells has been extensively studied. In vitro
studies showed that the uptake of aminoalkylbenzamide deriva-
tives is related to the melanin content of cells and is not receptor
mediated.9,10,22 Moreover, the in vivo localization of BZA in
the cytoplasm of tumor cells and its association with intracy-
toplasmic pigments was confirmed by secondary ion mass
spectrometry (SIMS) supporting BZA’s specific interaction with
melanin.23 On the other hand, 4-piperidinyl-iodobenzamide
derivatives are known to bind to sigma receptors in a variety
of tumor cells, including melanoma.11,24
From our results, the uptake mechanisms of iodobenzamides
in melanoma tumors is dependent on the nature of the chemical
moiety associated with the benzamide structure.
According to the in vitro assays for the determination of
inhibition constants for sigma receptors, only piperidinyl-
iodobenzamides show a σ-receptor characteristic, and of these,
compound 25 showed the highest affinity for the σ1-receptor

3566 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Table 3. Biodistribution of [123I]Benzamides in Mice
Pham et al.
cmpd
time (h)
melanomaa
livera
kidneya
lunga
hearta
braina
blooda
SUVb
1
1
6
24
48
72
9.6 ( 2.9
8.5 ( 1.2
5.2 ( 0.5
3.4 ( 1.0
2.2 ( 0.7
9.0 ( 0.7
2.3 ( 0.3
1.3 ( 0.2
0.4 ( 0.2
0.09 ( 0.03
0.07 ( 0.02
nd
0.79 ( 0.05
0.31 ( 0.05
0.12 ( 0.01
0.07 ( 0.01
0.18 ( 0.08
0.13 ( 0.06
0.02 ( 0.01
0.01 ( 0.01
0.01 ( 0.01
nd
0.98 ( 0.05
0.35 ( 0.15
0.08 ( 0.02
0.03 ( 0.01
0.06 ( 0.02
3.7 ( 1.1
9.5 ( 2.6
38 ( 3
48 ( 18
51 ( 11
3.0 ( 0.3
0.44 ( 0.07
0.08 ( 0.02
0.04 ( 0.01
0.06 ( 0.05
1.1 ( 0.1
0.51 ( 0.07
0.28 ( 0.06
14b
14c
14d
1
6
24
5.5 ( 1.6
6.0 ( 1.5
3.7 ( 0.3
9.5 ( 3.6
1.5 ( 0.3
0.42 ( 0.09
5.7 ( 1.2
1.5 ( 0.3
0.84 ( 0.22
0.30 ( 0.02
0.07 ( 0.02
0.07 ( 0.02
0.03 ( 0.02
0.07 ( 0.01
1.3 ( 0.3
1.6 ( 0.3
4.0 ( 1.8
20 ( 4
0.69 ( 0.12
0.06 ( 0.01
0.50 ( 0.08
0.06 ( 0.01
0.68 ( 0.12
0.02 ( 0.01
1
6
24
8.0 ( 2.8
6.8 ( 0.6
5.1 ( 0.7
7.3 ( 0.8
1.8 ( 0.3
0.64 ( 0.10
14.1 ( 3.5
1.0 ( 0.2
0.18 ( 0.06
1.7 ( 0.1
0.80 ( 0.16
0.14 ( 0.06
0.93 ( 0.11
0.49 ( 0.08
0.14 ( 0.04
0.07 ( 0.01
0.05 ( 0.01
0.01 ( 0.01
1.7 ( 0.3
1.1 ( 0.3
0.15 ( 0.06
2.4 ( 0.8
3.6 ( 0.5
19 ( 3
1
3
6
24
48
72
5.6 ( 1.5
9.7 ( 3.1
8.4 ( 1.5
6.1 ( 0.9
4.6 ( 0.9
2.2 ( 0.3
16.8 ( 1.7
5.0 ( 0.8
1.8 ( 0.1
0.47 ( 0.04
0.38 ( 0.04
0.25 ( 0.03
21.6 ( 5.8
3.9 ( 0.5
1.2 ( 0.1
0.12 ( 0.07
0.07 ( 0.02
0.10 ( 0.02
1.9 ( 0.4
0.98 ( 0.13
0.54 ( 0.12
0.75 ( 0.82
0.12 ( 0.05
0.13 ( 0.03
0.06 ( 0.09
0.07 ( 0.01
0.04 ( 0.01
0.03 ( 0.00
0.01 ( 0.01
0.01 ( 0.00
0.01 ( 0.01
1.2 ( 0.2
1.7 ( 0.5
4.3 ( 1.4
6.6 ( 2.6
108 ( 32
163 ( 28
99 ( 22
0.95 ( 0.16
0.56 ( 0.07
0.10 ( 0.05
0.08 ( 0.02
0.05 ( 0.02
0.95 ( 0.21
0.71 ( 0.06
0.11 ( 0.08
0.04 ( 0.01
0.05 ( 0.00
14e
14f
19m
19n
25
1
6
24
3.4 ( 0.5
2.8 ( 0.4
1.4 ( 0.1
7.1 ( 1.4
0.8 ( 0.2
0.19 ( 0.03
3.7 ( 0.5
2.6 ( 0.4
0.94 ( 0.15
0.3 ( 0.1
0.08 ( 0.01
0.03 ( 0.01
nd
1.92 ( 0.19
0.78 ( 0.35
0.02 ( 0.01
1.0 ( 0.1
2.0 ( 0.8
15 ( 4
0.67 ( 0.03
0.03 ( 0.01
0.68 ( 0.3
0.04 ( 0.01
0.03 ( 0.01
1
6
24
3.4 ( 0.7
3.4 ( 0.8
2.2 ( 0.3
6.0 ( 0.7
1.3 ( 0.1
0.45 ( 0.01
3.26 ( 0.85
0.46 ( 0.09
0.05 ( 0.01
2.0 ( 0.1
0.45 ( 0.13
0.04 ( 0.01
0.64 ( 0.09
0.28 ( 0.15
0.06 ( 0.01
0.04 ( 0.01
0.02 ( 0.01
nd
0.84 ( 0.05
0.47 ( 0.08
0.05 ( 0.01
1.1 ( 0.2
2.1 ( 0.8
20 ( 6
1
6
24
5.4 ( 0.4
3.6 ( 0.7
1.7 ( 0.2
2.3 ( 0.3
1.2 ( 0.2
0.28 ( 0.03
1.30 ( 0.15
0.49 ( 0.09
0.06 ( 0.01
0.67 ( 0.10
0.38 ( 0.05
0.03 ( 0.01
0.42 ( 0.06
0.19 ( 0.06
0.04 ( 0.01
0.05 ( 0.01
0.02 ( 0.01
nd
0.77 ( 0.10
0.49 ( 0.16
0.01 ( 0.01
2.2 ( 0.2
2.6 ( 0.5
3.1 ( 0.5
1
6
24
4.2 ( 0.8
2.6 ( 0.2
1.4 ( 0.3
3.8 ( 0.9
1.5 ( 0.4
0.21 ( 0.02
1.2 ( 0.1
0.36 ( 0.03
0.06 ( 0.01
0.62 ( 0.05
0.22 ( 0.02
0.05 ( 0.01
0.28 ( 0.04
0.11 ( 0.01
0.03 ( 0.01
0.03 ( 0.01
nd
nd
0.74 ( 0.08
0.33 ( 0.03
0.05 ( 0.01
1.8 ( 0.4
2.2 ( 0.3
2.7 ( 0.6
1
6
24
10.9 ( 0.8
12.5 ( 1.9
5.5 ( 2.3
19.2 ( 2.4
3.8 ( 0.3
0.29 ( 0.05
8.7 ( 0.9
3.4 ( 0.3
0.22 ( 0.02
10.8 ( 0.8
6.4 ( 0.4
0.33 ( 0.08
3.0 ( 0.1
0.93 ( 0.04
0.09 ( 0.01
3.1 ( 0.3
0.48 ( 0.04
0.09 ( 0.01
1.00 ( 0.06
0.34 ( 0.02
0.04 ( 0.01
3.3 ( 0.4
9.3 ( 1.8
17 ( 2
27
1
6
24
5.9 ( 0.7
11.7 ( 2.0
9.2 ( 0.9
17.1 ( 1.2
9.2 ( 4.3
5.4 ( 0.5
10.6 ( 1.2
3.2 ( 0.9
1.2 ( 0.1
17.8 ( 2.8
4.7 ( 2.2
1.44 ( 0.25
3.1 ( 0.2
0.93 ( 0.45
0.49 ( 0.06
1.0 ( 0.1
0.46 ( 0.16
0.15 ( 0.01
1.0 ( 0.1
0.21 ( 0.06
0.04 ( 0.01
1.7 ( 0.2
6.6 ( 1.0
10 ( 2
30
1
6
24
6.2 ( 1.5
7.5 ( 0.9
6.9 ( 0.5
49.1 ( 2.0
33.0 ( 1.8
2.9 ( 0.4
8.6 ( 0.7
5.4 ( 0.8
0.52 ( 0.13
8.2 ( 0.9
3.9 ( 0.4
0.39 ( 0.86
1.9 ( 0.3
0.81 ( 0.12
0.12 ( 0.01
0.50 ( 0.05
0.17 ( 0.02
0.04 ( 0.01
0.52 ( 0.04
0.35 ( 0.02
0.08 ( 0.01
1.4 ( 0.9
3.1 ( 0.2
15 ( 2
33
1
6
24
3.8 ( 0.6
3.5 ( 0.8
3.4 ( 0.3
43.2 ( 5.0
27.0 ( 6.5
3.5 ( 0.65
15.2 ( 2.5
4.9 ( 0.9
0.59 ( 0.04
6.9 ( 0.5
2.1 ( 0.3
0.40 ( 0.06
1.05 ( 0.12
0.44 ( 0.04
0.13 ( 0.02
0.10 ( 0.01
0.06 ( 0.01
0.01 ( 0.00
0.48 ( 0.06
0.24 ( 0.01
0.03 ( 0.00
0.9 ( 0.1
1.7 ( 0.5
6.5 ( 1.4
BZA2
1
6
24
7.1 ( 1.3
8.8 ( 1.3
3.0 ( 1.2
6.8 ( 0.8
0.73 ( 0.19
0.10 ( 0.01
6.8 ( 1.2
0.76 ( 0.25
0.06 ( 0.01
4.5 ( 0.7
0.82 ( 0.26
0.09 ( 0.06
2.0 ( 0.4
0.40 ( 0.11
0.07 ( 0.06
1.3 ( 0.2
0.11 ( 0.03
0.01 ( 0.01
1.9 ( 0.3
1.2 ( 0.5
0.06 ( 0.02
2.7 ( 0.5
8.7 ( 2.8
16 ( 2
a Data are the means of %ID/g of tissue ( SD, n ) 5. b Standardized uptake values (SUVt) are calculated by dividing the tumor radioactivity concentration
by the mean radioactive concentration remaining in the mouse at time t.
methoxybenzoate (6)16,18,26 and N′,N′-dipropylbutyl-1,4-diamine
(5c)17 were synthesized by literature methods. 123I was produced
by the National Medical Cyclotron, Sydney, Australia, using the
124Xe(p, 2n) reaction and was delivered as [123I]INa in 0.02 M
NaOH at a concentration of 37 GBq/mL. Melting points were
determined in open capillary tubes using a Gallenkamp melting
point apparatus and are uncorrected. NMR spectra were performed
on a Bruker Avance DPX 400 operating at 400 MHz for 1H NMR
spectra and 100 MHz for 13C NMR spectra. Elemental Analysis
was determined by The Campbell Microanalytical Laboratory,
Department of Chemistry, University of Otago, New Zealand.
LRMS was performed on a Micromass ZMD quadrupole mass
spectrometer, while HRMS was performed by the University
Analytical Laboratory (University of New South Wales) using a
Bruker Daltonics BioApex-II 7T FTICR mass spectrometer equipped
with an off-axis analytical ESI source. Preparative HPLC purifica-
tion was performed on a system equipped with a Waters 600
gradient pump and 486 UV detector using a C18 column (Alltech
Econosphere, 10 µm, 250 × 22 mm). Semipreparative radio-HPLC
was performed on a Berthold LB501 system equipped with variable
UV (linear) and a γ-detector using either an Alltech Alphabond
Figure 2. Relationship between tumor uptake (%ID/g) at 24 h and
the lipophilicty (logP7.5) of the 2-methoxy-5-iodobenzamides.
Experimental Section
All commercially available chemicals were purchased from
Sigma-Aldrich, Lancaster, or Microchemistry Building Block
(Russia) and used without further purification. Methyl 4-amino-2-

Radioiodinated Benzamides for Malignant Melanoma
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3567
Table 4. Effect of Haloperidol on the Organ Uptake of [123I]14d and [123I]25a
time (h)
1
tumor
eye
liver
kidney
lung
blood
brain
14d
25
control
treated
control
treated
control
treated
control
treated
6.8 ( 1.2
6.1 ( 0.8
4.5 ( 0.4
4.5 ( 0.5
9.5 ( 1.9
10.5 ( 2.1
4.9 ( 0.2
3.3 ( 0.9b
18.9 ( 1.4
16.9 ( 3.3
18.8 ( 3.5
18.7 ( 2.8
27.8 ( 7.7
34.8 ( 3.1
24.7 ( 3.6
24.6 ( 0.9
15.9 ( 1.4
15.6 ( 2.1
0.34 ( 0.05
0.30 ( 0.03
26.0 ( 2.7
10.1 ( 0.7b
0.36 ( 0.06
0.12 ( 0.03b
17.3 ( 1.6
17.3 ( 1.9
0.07 ( 0.02
0.03 ( 0.01#
9.7 ( 1.5
1.67 ( 0.07
1.54 ( 0.09
0.03 ( 0.01
0.02 ( 0.01
10.3 ( 1.5
9.7 ( 1.3
1.13 ( 0.15
1.15 ( 0.06
0.03 ( 0.01
0.02 ( 0.01
0.97 ( 0.13
1.46 ( 0.08b
0.04 ( 0.01
0.03 ( 0.01
0.07 ( 0.01
0.06 ( 0.03
0.004 ( 0.001
0.002 ( 0.001
2.83 ( 0.22
1.61 ( 0.25b
0.08 ( 0.01
0.06 ( 0.01b
24
1
7.3 ( 0.7b
0.26 ( 0.02
0.10 ( 0.02b
24
0.33 ( 0.02
0.14 ( 0.03b
a The values are expressed as the average of %ID/g of tissue ( SD; n ) 5. b p < 0.01.
1 h followed by the work up according to Micovic and Mihailovic28
gave the N,N-dialkylaminobutylamines (5).
N′-tert-Butoxycarbonyl-N′-butylbutan-1,4-diamine (5a). 4-(Bu-
tylamino)butyronitrile (13 g, 0.1 mol), synthesized via general
procedure 1, in anhydrous acetonitrile (20 mL) and di-tert-butyl
dicarbonate (21.6 g, 0.1 mol) in acetonitrile (60 mL) was stirred at
room temperature for 15 h. Removal of all volatiles in vacuo gave
4-(N-tert-butoxycarbonyl-N-butylamino)butyronitrile (4a) as a col-
orless oil (16.5 g, 70%) that was converted to the diamine (5a) as
a colorless oil (77%) using general procedure 2. 1H NMR (CDCl3)
δ 0.81 (t, J ) 7.4 Hz, 3H), 1.19 (m, 2H), 1.22-1.51 (m, 15H),
2.60 (q, J ) 6.7 Hz, 2H), 3.06 (m, 4H). 13C NMR (CDCl3) δ 13.6,
19.8, 28.0, 28.1, 28.2, 30.7, 42.0, 46.6, 78.9, 155.3. MS ES (+ve)
m/z: 245 (M + 1)+ (60%), 189 (M + 1 - Ot-Bu)+, 145 (M + 1
- Boc)+.
N′-Isopropyl-N′-methylbutan-1,4-diamine (5b). See general
procedure 2. Colorless oil (82%). 1H NMR (CDCl3) δ 0.96 (s, 3H),
0.99 (s, 3H), 1.49 (m, 4H), 2.20 (s, 3H), 2.41 (m, 2H), 2.86 (m,
2H), 3.21 (m, 1H). 13C NMR (CDCl3) δ 19.0, 26.5, 32.9, 38.1,
43.3, 54.4. MS ES (+ve) m/z: 145 (M + 1)+. HRMS calcd for
C8H21N2, 145.1699; found, 145.1697
N′-Butyl-N′-methylbutane-1,4-diamine (5d). See general pro-
cedure 2. Colorless oil (72%). 1H NMR (CDCl3) δ 0.91 (t, J ) 7.2
Hz, 3H), 1.31 (m, 2H), 1.45 (m, 5H), 1.78 (m, 3H), 2.18 (s, 1.5H,
NCH3 of rotamer 1), 2.20 (s, 1.5H, NCH3 of rotamer 2), 2.32 (m,
3H), 2.44 (m, 2H), 2.70 (m, 1H). 13C NMR (CDCl3) δ 15.2, 15.3,
15.9, 21.7, 21.9, 24.7, 25.9, 30.6, 32.9, 43.3, 43.4, 56.8, 58.7, 58.9.
MS ES (+ve) m/z: 159 (M + 1)+. HRMS calcd for C9H23N2 (M
+ 1)+, 159.1855; found, 159.1854.
Figure 3. Scaled SPECT images of the distribution of [123I]14d (A)
and [123I]25 (B) in C57BL/6J female mice bearing a B16F0 murine
melanoma tumor, acquired 24 h after injection of 10 MBq of radiotracer.
The scale indicated the highest value in count/pixel in the image. High
concentration of radioactivity was observed in the tumors and the eyes
of the animals.
column (C18, 10 µm, 300 × 7.5 mm), a Phenomenex Bondclone
column (C18, 10 µm, 300 × 7.8 mm), or Alltech Econosil column
(C18, 10 µm, 250 × 10 mm). For quality control and stability
measurements, the radioiodinated tracer solution was ascertained
on an analytical HPLC system using a Phenomenex Luna column
(C18, 5 µm, 150 × 4.6 mm) eluting with 45% acetonitrile-55%
ammonium acetate (0.1 M) at 1 mL/min.
4-(2-Azanorborn-2-yl)butan-1-amine (5e). See general proce-
1
dure 2. Colorless oil (76%). H NMR (CDCl3) δ 1.23 (m, 2H),
General Procedure 1 for the Preparation of Nitriles (4).27
A
1.38-1.52 (m, 4H), 1.62-1.73 (m, 4H), 2.14 (m, 1H), 2.30 (m,
1H), 2.39 (m, 1H), 2.46 (m, 1H), 2.69 (m, 2H), 2.85 (m, 1H), 3.47
(m, 1H). 13C NMR (CDCl3) δ 27.0 (CH2), 27.7 (CH2), 29.9 (CH2),
32.9 (CH2), 37.4 (CH2), 38.8 (CH), 43.3 (NHCH2), 55.7 (NCH2),
60.9 (NCH2), 61.8 (NCH). MS ES (+ve) m/z: 169 (M + 1). HRMS
calcd for C10H21N2, 169.1699; found, 169.1700.
solution of the secondary amine (1 equiv) in 1-butanol, anhydrous
K2CO3 (1 equiv), KI (0.1 equiv), and 4-bromobutyronitrile (1 equiv)
was heated to reflux for 20 h. Filtration, followed by an acid (2 N
HCl) and base (pH 10, conc NaOH) workup gave the corresponding
3-cyanopropylamine.
4-(N-Isopropyl-N-methylamino)butyronitrile (4b). See general
procedure 1 (82%). 1H NMR (CDCl3) δ 0.97 (s, 3H), 0.99 (s, 3H),
1.76 (m, 2H), 2.15 (s, 3H), 2.40 (t, J ) 7.2 Hz, 2H), 2.46 (t, J )
6.8 Hz, 2H, CH2), 2.79 (m, 2H). 13C NMR (CDCl3) δ 15.8, 18.9,
25.0, 37.8, 52.2, 54.9, 121.2. MS ES (+ve) m/z: 141 (M + 1)+.
HRMS calcd for C8H17N2, 141.1386; found, 141.1384.
4-(N-Butyl-N-methylamino)butyronitrile (4d). See general
procedure 1 (84%). 1H NMR (CDCl3) δ 0.91 (t, J ) 7.2 Hz, 3H),
1.31 (m, 2H), 1.42 (m, 2H), 1.78 (m, 2H), 2.18 (s, 3H), 2.33 (t, J
) 7.2 Hz, 2H), 2.41 (m, 4H). 13C NMR (CDCl3) δ 15.2, 16.0, 21.7,
24.6, 30.6, 43.1, 56.9, 58.8, 121.1. MS ES (+ve) m/z: 155 (M +
1). HRMS calcd for C9H19N2 (M + 1)+, 155.1542; found, 155.1544.
4-(2-Azanorborn-2-yl)butyronitrile (4e). See general procedure
1 (79%). 1H NMR (CDCl3) δ 1.25 (m, 2H), 1.41 (m, 1H), 1.53 (m,
1H), 1.60 (m, 1H), 1.70-1.78 (m, 3H), 2.14 (m, 1H), 2.32 (m,
1H), 2.44 (m, 3H), 2.56 (m, 1H), 2.61 (m, 1H), 2.86 (m, 1H), 3.28
(m, 1H). 13C NMR (CDCl3) δ 16.1, 26.1, 27.5, 29.8, 37.4, 38.9,
54.1, 60.9, 62.3, 121.2. MS ES (+ve) m/z: 165 (M + 1)+. HRMS
calcd for C10H17N2, 165.1386; found, 165.1384.
Methyl 4-Acetamido-2-methoxybenzoate (7). Methyl 4-amino-
2-methoxybenzoate (6; 20 g, 0.11 mol) was treated with acetic
anhydride (12 g, 0.12 mol) in absolute ethanol (150 mL) at 50 °C
for 2 h. Normal workup followed by crystallization from hot ethanol
1
yielded (7) as a tan colored solid (22 g, 89%). H NMR (DMSO-
d6) δ 2.07 (s, 3H), 3.73 (s, 3H), 3.83 (s, 3H), 7.18 (d, J ) 8.8 Hz,
1H), 7.46 (s, 1H), 7.65 (d, J ) 8.8 Hz, 1H). 13C NMR (CDCl3) δ
24.4, 51.8, 55.6, 102.9, 110.5, 114.1, 132.6, 143.7, 160.3, 166.2,
169.3. MS ES (+ve) m/z: 246 (M + Na)+. HRMS calcd for C11H14-
NO4 (M + 1)+, 224.0923; observed (M + 1)+, 224.0921.
Methyl 4-Acetamido-5-iodo-2-methoxybenzoate (8). Methyl
4-acetamido-2-methoxybenzoate (7; 10 g, 0.045 mol) in acetic acid
(50 mL) at 50 °C was treated with iodine monochloride (8.14 g,
0.05 mol) in acetic acid (50 mL). The reaction was heated for 6 h
at 50 °C with a further addition of iodine monochloride (0.5 g)
after 4 h. After stirring overnight at room temperature, the resulting
white solid was filtered and washed with acetic acid (30 mL) and
1
H2O (100 mL) to yield a white solid (12.1 g, 77%). H NMR
(CDCl3) δ 2.11 (s, 3H), 3.77 (s, 3H), 3.78 (s, 3H), 7.44 (s, 1H),
8.07 (s, 1H), 9.37 (s, 1H). 13C NMR (CDCl3) δ 25.1, 52.0, 56.1,
76.1, 104.4, 116.6, 141.3, 142.6, 160.7, 164.6, 168.5. MS ES (+ve)
General Procedure 2 for the Preparation of the Butylamines
(5). Reaction of LiAlH4 and the nitrile (4) in anhydrous ether for

3568 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Pham et al.
m/z: 372 (M + Na)+. HRMS calcd for C11H13INO4 (M + 1)+,
349.9889; observed (M + 1)+, 349.9914.
(CDCl3) δ 0.85 (t, J ) 7.35 Hz, 6H), 1.40-1.63 (m, 8H), 2.20 (s,
3H), 2.33-2.45 (m, 6H), 3.46 (m, 2H), 6.82 (dd, J ) 1.9, 8.52 Hz,
1H), 7.88 (m, 1H), 7.90 (m, 1H, superimposed), 8.03 (d, J ) 8.52
Hz, 1H), 8.78 (s, 1H). 13C NMR (CDCl3) δ 13.15, 21.38, 25.86,
25.98, 28.83, 40.93, 54.99, 57.16, 57.42, 103.91, 112.65, 117.76,
133.54, 144.04, 159.42, 166.48, 170.58. MS ES (+ve) m/z: 364
(M + 1)+. Anal. Calcd for C20H33N3O3‚1H2O‚1TFA: C, 53.32; H,
7.34; N, 8.48. Found: C, 53.70; H, 7.68; N, 9.07.
4-Acetamido-2-methoxybenzoic Acid (11). Methyl 4-aceta-
mido-2-methoxybenzoate (7; 15.0 g, 0.067 mol) in methanol (100
mL), H2O (40 mL), and KOH (10 g) was heated to reflux for 2 h.
The methanol was evaporated, H2O (50 mL) was added, and the
mixture was cooled to 5 °C. Acidification to pH 5.0 with 6 M HCl
precipitated 9 as a white solid. The solid (9) and acetic anhydride
(11.6 g, 0.11 mol) in acetic acid (150 mL) was stirred for 2 h at 50
°C. The reaction was cooled to room temperature and stirred
overnight. The resulting precipitate was filtered, washed with acetic
acid (20 mL), and dried to give 11 as a white solid of the title
4-Acetamido-N-(4-(N-butyl-N-methylamino)butyl)-2-meth-
oxybenzamide (13d). Benzamide 13d, prepared according to
general procedure 3, was purified by column chromatography on
silica with 1:9 methanol/ethyl acetate to give a white solid (1.2 g,
1
1
compound (10.2 g, 73%). H NMR (CDCl3) δ 2.01 (s, 1H), 3.77
72%). H NMR (CDCl3) δ 0.90 (t, J ) 7.29 Hz, 3H) 1.28-1.69
(s, 3H), 7.16 (dd, J ) 1.6, 8.4 Hz, 1H), 7.45 (d, J ) 1.6 Hz, 1H),
7.65 (d, J ) 8.4 Hz), 10.17 (s, 1H). 13C NMR (CDCl3) δ 25.8,
57.1, 104.0, 111.7, 116.2, 134.0, 145.7, 161.1, 168.1, 170.6. MS
ES (-ve) m/z: 208 (M - 1)-.
(m, 8H), 2.20 (s, 6H), 2.35 (m, 4H), 3.46 (m, 2H), 3.92 (s, 3H),
6.80 (dd, J ) 1.89, 8.51 Hz, 1H), 7.86 (m, 1H), 7.90 (t, J ) 5.61
Hz, 1H), 8.04 (d, J ) 8.51 Hz, 1H), 8.56 (s, 1H). 13C NMR (CDCl3)
δ 15.24, 21.90, 25.88, 26.01, 28.80, 30.54, 40.87, 43.39, 57.19,
58.53, 58.80, 103.92, 112.62, 117.84, 133.56, 143.94, 159.42,
166.47, 170.49. MS ES (+ve) m/z: 350 (M + 1)+. HRMS calcd
for C19H32N3O3 (M + Na)+, 372.2258; observed (M + Na)+,
372.2256.
4-Acetamido-N-(4-(2-azanorborn-2-yl)butyl)-2-methoxyben-
zamide (13e). Benzamide 13e, prepared according to general
procedure 3, was purified by column chromatography on silica with
1:4 methanol/chloroform to give a white solid (0.76 g, 71%) as a
1:1 mixture of endo and exo isomers. 1H NMR (CDCl3) δ 1.2-1.9
(m, 10H), 2.60 (m, 5H), 3.40 (m, 3H, superimposed), 7.46 (d, J )
8.4, 2H), 7.67 (d, J ) 8.4, 2H), 7.78 (s, br, 1H). 13C NMR (CDCl3)
δ 26.11, 26.36, 27.97, 29.12, 37.27, 38.26, 40.43, 54.45, 60.67,
62.69, 126.96, 130.14, 132.76, 134.94, 168.02. MS ES (+ve) m/z:
360 (M + 1)+. HRMS calcd for C20H30N3O3 (M + 1)+, 360.2282;
observed (M + 1)+, 360.2279.
4-Acetamido-N-(4-(butylamino)butyl)-2-methoxybenzamide
(13f). 4-Acetamido-N-(4-(N-tert-butoxycarbonyl-N-butylamino)-
butyl)-2-methoxybenzamide (13a; 0.7 g, 1.6 mmol) in anhy-
drous ethyl acetate (5 mL) was treated with saturated HCl in
anhydrous ethyl acetate (1 mL) for 45 min at room temperature to
give a white solid of 13f as its hydrochloride salt (0.55 g, 92%).
1H NMR (DMSO-d6) δ 0.88 (t, J ) 7.2 Hz, 3H), 1.31 (m, 2H),
1.55 (m, 4H), 1.59 (m, 2H), 2.01 (s, 3H), 2.83 (br m, 2H), 2.87
(br m, 2H), 3.28 (m, 2H), 3.87 (s, 3H), 7.20 (d, J ) 8.8 Hz, 1H),
7.53 (s, 1H), 7.74 (d, J ) 8.8 Hz, 1H), 8.10 (t, J ) 5.6 Hz, 1H),
8.88 (br s, 2H), 10.31 (br s). 13C NMR (CDCl3) δ 15.2, 21.0, 24.7,
25.8, 28.1, 29.1, 39.9, 48.1, 57.4, 103.6, 112.3, 118.5, 133.0, 144.8,
159.2, 166.1, 170.5. MS ES (+ve) m/z: 336 (M + 1)+. HRMS
calcd for C18H30N3O3 (M + 1)+, 336.2287; observed (M + 1)+,
336.2281.
4-Acetamido-5-iodo-2-methoxybenzoic Acid (12). Methyl 4-
acetamido-5-iodo-2-methoxybenzoate (8; 2.0 g, 5.72 mmol) in
methanol (15 mL), H2O (5 mL) and KOH (1.5 g) was reacted
according to the above procedure to give 10 as a white solid.
Treatment of 10 (1.5 g) in acetic acid (20 mL) with acetic anhydride
(1.04 g, 10 mmol) at 50 °C for 2 h followed by stirring overnight
precipitated a tan solid that was filtered, washed with acetic acid
1
(10 mL), and dried to yield 12 as a white solid (1.5 g, 78%). H
NMR (DMSO-d6) δ 2.10 (s, 3H), 3.77 (s, 3H), 7.40 (s, 1H), 8.01
(s, 1H), 9.35 (s, 1H). 13C NMR (DMSO-d6) δ 25.2, 57.6, 83.8 ,
111.8, 121.2, 142.2, 145.5, 160.4, 167.0, 170.3. MS ES (+ve) m/z:
358 (M + Na)+. HRMS calcd for C10H11INO4 (M + Na)+,
355.9733; observed (M + Na)+, 355.9827.
General Procedure 3 for the Preparation of Benzamides 1,
13a-13e, 14a-14e, 15, 30, 31, 33, and 34. Each of 4-acetamido-
2-methoxybenzoic acid (11), 4-acetamido-5-iodo-2-methoxybenzoic
acid (12), 4-bromobenzoic acid or 4-iodobenzoic acid (∼1.0 g),
and thionyl chloride (4 equiv) in anhydrous THF (50 mL) was
refluxed for 2 h and then dried under vacuum. The resulting acid
chloride was redissolved in THF (50 mL) and treated with the
amines [5a-5e or N,N-diethylethylenediamine (1 equiv)]. After 5
min, triethylamine (4 equiv) was added and it was stirred for 2 h
before normal workup. The resulting benzamides were purified by
column chromatography on silica.
4-Acetamido-N-(4-(N-tert-butoxycarbonyl-N-butylamino)but-
yl)-2-methoxybenzamide (13a). Benzamide 13a, prepared accord-
ing to general procedure 3, was purified by column chromatography
on silica with 3:1 ethyl acetate/petroleum spirit to give a white solid
1
(1.4 g, 68%). H NMR (CDCl3) δ 0.90 (t, J ) 7.2 Hz, 3H), 1.26
(m, 2H), 1.44 (s, 9H) 1.51 (m, 2H), 1.59 (m, 4H), 2.20 (s, 3H),
3.14 (br m, 2H), 3.20 (br m, 2H), 3.46 (m, 2H), 3.94 (s, 3H), 6.80
(dd, J ) 1.6, 8.5 Hz, 1H), 7.86 (br m, 2H), 8.06 (d, J ) 8.5 Hz,
1H), 8.41 (br s, 1H). 13C NMR (CDCl3) δ 15.1, 21.2, 25.9, 26.1,
27.1, 28.3, 29.7, 33.3, 40.6, 47.8, 48.1, 57.2, 80.3, 103.9, 112.6,
117.8, 133.7, 143.8, 156.9, 159.5, 166.3, 170.3. MS ES (+ve) m/z:
436 (M + 1)+. HRMS calcd for C23H38N3O5 (M + 1)+, 436.2811;
observed (M + 1)+, 436.2791.
4-Acetamido-N-(4-(N-tert-butoxycarbonyl-N-butylamino)bu-
tyl)-5-iodo-2-methoxybenzamide (14a). Benzamide 14a, prepared
according to general procedure 3, was purified by column chro-
matography on silica with ethyl acetate to give a white solid (1.2
1
g, 72%). H NMR (CDCl3) δ 0.91 (t, J ) 7.2 Hz, 3H), 1.27 (m,
2H), 1.44 (s, 9H), 1.48 (m, 2H), 1.59 (m, 4H), 2.27 (s, 3H), 3.15
(br m, 2H), 3.21 (br m, 2H), 3.46 (m, 2H), 3.98 (s, 3H), 7.64 (br
s, 1H), 7.77 (br s, 1H), 8.24 (s, 1H), 8.56 (s, 1H). 13C NMR (CDCl3)
δ 15.1, 21.2, 26.3, 27.2, 28.2, 29.7, 32.3, 40.7, 47.8, 48.1, 57.5,
79.1, 80.2, 105.1, 122.2, 142.8, 156.8, 159.6, 164.7, 169.8. MS ES
(+ve) m/z: 562 (M + 1)+. HRMS calcd for C23H37IN3O5 (M +
1)+, 562.1778; observed (M + 1)+, 562.1876.
4-Acetamido-N-(4-(N-isopropyl-N-methylamino)butyl)-2-meth-
oxybenzamide (13b). Benzamide 13b, prepared according to
general procedure 3, was purified by column chromatography on
silica with 1:9 methanol/ethyl acetate to give a white solid (1.28 g,
76%). 1H NMR (CDCl3) δ 0.98 (s, 3H), 0.99 (s, 3H) 1.60 (m, 4H),
2.18 (s, 3H), 2.20 (s, 3H), 2.39 (t, J ) 7.27 Hz, 2H), 2.82 (dq, J )
6.55, 6.56 Hz, 1H), 3.46 (dt, J ) 6.74, 5.79 Hz, 2H), 3.97 (s, 3H),
6.80 (dd, J ) 8.51, 1.91 Hz, 1H), 7.86 (m, 1H), 7.90 (t, J ) 5.79
Hz, 1H), 8.05 (d, J ) 8.51 Hz, 1H), 8.37 (s, 1H). 13C NMR (CDCl3)
δ 19.02, 25.9, 26.74, 28.83, 38.16, 40.92, 54.07, 54.54, 57.2, 103.89,
112.54, 118.00, 133.64, 143.76, 159.44, 166.38, 170.32. MS ES
(+ve) m/z: 336 (M + 1)+. HRMS calcd for C18H30N3O3 (M +
1)+, 336.2287; observed (M + 1)+, 336.2301.
4-Acetamido-N-(4-(N-isopropyl-N-methylamino)butyl)-5-iodo-
2-methoxybenzamide (14b). Benzamide 14b, prepared according
to general procedure 3, was purified by column chromatography
on silica with 1:8 methanol/ethyl acetate to give a white solid (0.99
1
g, 72%). H NMR (CDCl3) δ 0.99 (s, 3H), 1.01 (s, 3H), 1.52-
1.66 (m, 4H), 2.20 (s, 3H), 2.27 (s, 3H), 2.41 (t, J ) 7.2 Hz, 2H),
2.82 (dq, J ) 6.55, 6.56 Hz, 1H), 3.45 (dt, J ) 6.4, 5.6 Hz, 2H),
3.97 (s, 3H), 7.64 (br s, 1H), 7.79 (t, J ) 5.6 Hz, 1H), 8.22 (s,
1H), 8.55 (s, 1H). 13C NMR (CDCl3) δ 18.9, 26.4, 26.6, 28.8, 38.1,
40.9, 54.0, 54.5, 57.5, 79.2, 105.1, 120.1, 142.7, 159.5, 164.7, 170.3.
MS ES (+ve) m/z: 462 (M + 1)+. Anal. Calcd for C18H28IN3O3:
C, 46.86; H, 6.12; N, 9.11. Found: C, 46.74; H, 6.00; N, 8.98.
4-Acetamido-N-(4-(dipropylamino)butyl)-2-methoxybenza-
mide (13c). Benzamide 13c, prepared according to general proce-
dure 3, was purified by column chromatography on silica with 1:9
methanol/ethyl acetate to give a white solid (1.28 g, 75%). 1H NMR

Radioiodinated Benzamides for Malignant Melanoma
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3569
4-Acetamido-N-(4-(dipropylamino)butyl)-5-iodo-2-methoxy-
benzamide (14c). Benzamide 14c, prepared according to general
procedure 3, was purified by column chromatography on silica with
1:1 methanol/ethyl acetate to give a white solid (0.65 g, 44%). 1H
NMR (CD3OD) δ 1.02 (t, J ) 7.36, 6H), 1.75 (m, 8H), 2.22 (s,
3H), 3.12 (m, 4H), 3.22 (m, 2H), 3.48 (m, 2H), 3.96 (s, 3H), 7.60
(s, 1H) 8.33 (s, 1H). 13C NMR (CDCl3) δ 12.04, 18.50, 22.05, 26.34,
27.99, 39.14, 54.96, 56.50, 57.61, 81.56, 105.43, 118.84, 142.41,
143.44, 160.16, 166.00, 169.95. MS ES (+ve) m/z: 490 (M + 1)+.
Anal. Calcd for C20H32IN3O3‚3H2O‚1.5TFA: C, 38.66; H, 5.57;
N, 5.88. Found: C, 38.15; H, 5.16; N, 5.55.
4-Amino-N-(2-(diethylamino)ethyl)-5-iodo-2-methoxybenza-
mide (17). Treatment of 4-acetamido-N-(2-(diethylamino)ethyl)-
5-iodo-2-methoxybenzamide (1; 0.45 g, 1.04 mmol) with KOH
(0.23 g, 4.17 mmol) in methanol (5 mL) and water (1 mL) yielded
1
benzamide 17 as a white solid (0.37 g, 91%). H NMR (DMSO-
d6) δ 0.97 (t, J ) 7.2 Hz, 6H), 2.49 (m, 6H), 3.29 (m, 2H), 3.81 (s,
3H), 5.74 (br s, 2H), 6.46 (s, 1H), 8.08 (m, 2H). MS ES (+ve)
m/z: 392 (M + 1)+.
N-(2-(Diethylamino)ethyl)-2-methoxy-4-(mesylamino)benza-
mide (18m). 4-Amino-N-(2-(diethylamino)ethyl)-2-methoxyben-
zamide (16; 59 mg, 0.22 mmol) in anhydrous DCM (10 mL) and
dry pyridine (190 µL) was treated with methanesulfonyl chloride
(28 mg, 0.24 mmol) at 0 °C for 30 min followed by 48 h at room
temperature. Aqueous workup followed by preparative HPLC
purification on a C18 column eluting with acetonitrile: (0.1 M)
ammonium acetate (65:35) at 15 mL/min gave the benzamide 18m
(Rt ) 16 min) as a colorless oil (40 mg, 53%). 1H NMR (CD3OD)
δ 1.20 (t, J ) 7.06, 6H), 2.92 (q, J ) 7.06 Hz, 4H), 2.95 (t, J )
6.14 Hz, 2H), 3.06 (s, 3H), 3.62 (t, J ) 6.14 Hz, 2H), 3.99 (s, 3H),
6.91 (dd, J ) 1.85 Hz, 8.57, 1H); 7.02 (d, J ) 1.85 Hz, 1H); 7.95
(d, 1H, J ) 8.54 Hz). 13C NMR (CD3OD) δ 10.92, 37.71, 39.73,
49.32, 52.81, 56.54, 102.92, 111.77, 117.30, 133.77, 144.96, 160.33,
168.00. MS ES (+ve) m/z: 344 (M + 1)+. HRMS calcd for
C15H26N3O4S (M + 1)+, 344.1641; observed (M + 1)+, 344.1640.
N-(2-(Diethylamino)ethyl)-2-methoxy-4-(tosylamino)benza-
mide (18n). 4-Amino-N-(2-(diethylamino)ethyl)-2-methoxybenza-
mide (16; 0.1 g, 0.38 mmol) in anhydrous DCM (10 mL) and dry
pyridine (0.2 mL) was treated with p-toluenesulphonyl chloride
(0.08 g, 0.42 mmol) at 0 °C for 30 min then stirred at room
temperature for 18 h. Aqueous workup followed by preparative
RP-HPLC purification with acetonitrile ammonium acetate (0.1 M;
80:20) as the eluent at 16 mL/min gave benzamide 18n (Rt ) 19
min) as a colorless oil (49 mg, 31%). 1H NMR (CD3OD) δ 1.34 (t,
J ) 7.30 Hz, 6H), 2.38 (s, 3H), 3.31 (m, 4H), 3.37 (t, J ) 6.02 Hz,
2H), 3.76 (t, J ) 6.02 Hz, 2H), 3.92 (s, 3H); 6.80 (dd, J ) 2.00
Hz, 8.54, 1H) 6.92 (d, J ) 2.00 Hz, 1H), 7.33 (d, 2H, J ) 8.01
Hz), 7.75 (d, 2H, J ) 8.01 Hz), 7.83 (d, 1H, J ) 8.54 Hz). 13C
NMR (CD3OD) δ 9.25, 21.48, 36.53, 49.32, 53.31, 56.58, 103.25,
112.23, 116.98, 128.38, 130.81, 138.00, 144.60, 145.59, 160.20,
169.00. MS ES (+ve) m/z: 420 (M + 1)+. HRMS calcd for C21H30-
N3O4S (M + 1)+, 420.1954; observed (M + 1)+, 420.1956.
N-(2-(Diethylamino)ethyl)-5-iodo-2-methoxy-4-(mesylamino)-
benzamide (19m). Treatment of 4-amino-N-(2-(diethylamino)-
ethyl)-5-iodo-2-methoxybenzamide (17; 50 mg, 0.13 mmol) with
methanesulphonyl chloride, as in the synthesis of 18n, gave, after
column chromatography on silica and eluting with (1:1) MeOH/
ethyl acetate, benzamide 19m as a colorless oil (28 mg, 47%). 1H
NMR (CD3OD) δ 1.30 (t, J ) 7.2 Hz, 6H), 3.05 (s, 3H), 3.21 (q,
J ) 7.2 Hz, 4H), 3.29 (t, J ) 6.0 Hz, 2H), 3.75 (t, J ) 6.0 Hz,
2H), 3.97 (s, 3H), 7.28 (s, 1H), 8.35 (s, 1H). MS ES (+ve) m/z:
470 (M + 1)+. HRMS calcd for C15H25N3O4SI (M + 1)+, 470.0611;
observed (M + 1)+, 470.0626.
4-Acetamido-N-(4-(N-butyl-N-methylamino)butyl)-5-iodo-2-
methoxybenzamide (14d). Benzamide 14d, prepared according to
general procedure 3, was purified by column chromatography on
silica with 1:9 methanol/ethyl acetate to give a white solid (0.97 g,
1
69%). H NMR (CDCl3) δ 0.90 (t, J ) 7.28, 3H), 1.28-1.63 (m,
8H), 2.20 (s, 3H), 2.27 (s, 3H), 2.35 (m, 4H), 3.46 (m, 2H), 3.97
(s, 3H), 7.64 (s, 1H), 7.68 (t, J ) 5.61, 1H), 8.24 (s, 1H), 8.56 (s,
1H). 13C NMR (CDCl3) δ 15.26, 21.92, 26.06, 26.35, 28.77, 30.64,
40.93, 43.47, 57.46, 58.56, 58.82, 79.19, 105.11, 120.12, 142.75,
142.75, 159.50, 164.66, 169.84. MS ES (+ve) m/z: 476 (M + 1)+.
HRMS calcd for C19H31N3O3I (M + 1)+, 476.1405; observed (M
+ 1)+, 476.1404. Anal. Calcd for C19H31N3O3I: C, 48.00; H, 6.37;
N, 8.84. Found: C, 48.10; H, 6.26; N, 8.81.
4-Acetamido-N-(4-(2-azanorborn-2-yl)butyl)-5-iodo-2-meth-
oxybenzamide (14e). Benzamide 14e, prepared according to general
procedure 3, was purified by column chromatography on silica with
ethyl acetate to give a white solid (0.94 g, 65%) as a 1:1 mixture
1
of endo and exo isomers. H NMR (CDCl3) δ 0.90 (t, J ) 7.29
Hz, 3H) 1.28-1.69 (m, 8H), 2.20 (s, 6H), 2.35 (m, 4H), 3.46 (m,
2H), 3.92 (s, 3H), 6.80 (dd, J ) 1.89, 8.51 Hz, 1H), 7.86 (m, 1H),
7.90 (t, J ) 5.61 Hz, 1H), 8.04 (d, J ) 8.51 Hz, 1H), 8.56 (s, 1H).
13C NMR (CDCl3) δ 21.49, 21.66, 22.68, 25.11, 25.56, 25.78, 26.33,
26.77, 26.87, 27.59, 34.89, 35.99, 36.61, 37.05, 38.38, 38.46, 50.71,
55.59, 56.30, 58.49, 59.47, 62.44, 64.98, 67.92, 77.19, 77.98,
103.95, 118.28, 118.30, 141.39, 141.42, 141.78, 141.82, 158.48,
163.98, 164.03, 168.62. MS ES (+ve) m/z: 486 (M + 1)+. HRMS
calcd for C20H29IN3O3 (M + 1)+, 486.1248; observed (M + 1)+,
486.1248.
4-Acetamido-N-(4-(butylamino)butyl)-5-iodo-2-methoxyben-
zamide (14f). 4-Acetamido-N-(4-(N-tert-butoxycarbonyl-N-butyl-
amino)butyl)-5-iodo-2-methoxybenzamide (14a; 0.7 g, 1.25 mmol)
was treated with saturated HCl in anhydrous ethyl acetate to give
14f as its hydrochloric salt (0.54 g, 93%). 1H NMR (DMSO-d6) δ
0.89 (t, J ) 7.2 Hz, 3H), 1.31 (m, 2H), 1.56 (m, 4H), 1.62 (m,
2H), 2.09 (s, 3H), 2.83 (br m, 2H), 2.87 (br m, 2H), 3.28 (m, 2H),
3.85 (s, 3H), 7.36 (s, 1H), 8.10 (s, 1H), 8.21 (t, J ) 6.0 Hz, 1H),
8.79 (br s, 2H), 9.41 (br s). 13C NMR (CDCl3) δ 15.17, 20.99,
24.61, 25.14, 27.94, 29.09, 40.09, 48.04, 57.84, 85.0, 111.74,
123.66, 141.53, 144.47, 158.82, 164.94, 170.30. MS ES (+ve)
m/z: 462 (M + 1)+. Anal. Calcd for C18H28IN3O3‚3H2O: C, 41.94;
H, 6.65; N, 8.15. Found: C, 41.67; H, 6.11; N, 8.09.
N-(2-(Diethylamino)ethyl)-5-iodo-2-methoxy-4-(tosylamino)-
benzamide (19n). Treatment of 4-amino-N-(2-(diethylamino)ethyl)-
5-iodo-2-methoxybenzamide (17; 0.1 g, 0.26 mmol) with p-tolu-
enesulphonyl chloride as above gave after column chromatography
on silica and eluting with (1:1) MeOH/ethyl acetate gave benzamide
19n as a colorless oil (0.06 g, 43%). 1H NMR (CD3OD) δ 1.31 (t,
J ) 7.2 Hz, 6H), 2.38 (s, 3H), 2.24 (q, J ) 7.2 Hz, 4H), 3.29 (t,
J ) 6.0 Hz, 2H), 3.73 (t, J ) 6.0 Hz, 2H), 3.89 (s, 3H), 7.16 (s,
1H), 7.29 (d, J ) 8.0 Hz, 2H), 7.68 (d, J ) 8.0 Hz, 2H), 8.24 (s,
1H). 13C NMR (CD3OD) δ 9.1, 21.3, 36.3, 49.4, 52.9, 56.7, 82.3,
108.8, 120.5, 128.4, 130.6, 138.1, 142.8, 143.1, 145.6, 159.6, 167.8.
MS ES (+ve) m/z: 546 (M + 1)+. HRMS calcd for C21H29N3O4SI
(M + 1)+, 546.0924; observed (M + 1)+, 546.0891.
1-(2-Fluoroethyl)piperidin-4-amine (22). N-tert-Butoxycarbon-
ylpiperidine (2 g, 10 mmol), bromofluoroethane (1.5 g, 12 mmol),
and K2CO3 (11 g, 80 mmol) in acetonitrile (40 mL) were heated to
reflux for 5 h. Aqueous workup gave the BOC-protected amine 20
as a light yellow solid (2 g, 91%), which was subsequently stirred
with TFA (20 mL) for 1 h to give amine 22 (1.8 g, 95%). 1H NMR
4-Acetamido-N-(2-(diethylamino)ethyl)-2-methoxybenza-
mide (15). Benzamide 15, prepared according to general procedure
3, was purified by column chromatography on silica eluting with
1
ethyl acetate to give 15 as a white solid (1.2 g, 66%). H NMR
(CDCl3) δ 1.05 (t, J ) 7.2 Hz, 6H), 2.20 (s, 3H), 2.58 (q, J ) 7.2
Hz, 4H), 2.64 (t, J ) 6.0 Hz, 2H), 3.55 (dt, J ) 4.8, 6.0 Hz, 1H),
3.92 (s, 3H), 6.83 (dd, J ) 1.8, 8.5 Hz, 1H), 7.85 (br s, 1H), 8.05
(d, J ) 8.5 Hz, 1H). 8.44 (t, J ) 4.8 Hz, 1H), 8.73 (br s, 1H). MS
ES (+ve) m/z: 308 (M + 1)+.
4-Amino-N-(2-(diethylamino)ethyl)-2-methoxybenzamide (16).
Treatment of 4-acetamido-N-(2-(diethylamino)ethyl)-2-methoxy-
benzamide (15; 0.5 g, 1.63 mmol) with KOH (0.37 g, 6.5 mmol)
in methanol (5 mL) and water (1 mL) yielded benzamide 16 as a
1
pale cream solid (0.32 g, 74%). H NMR (DMSO-d6) δ 1.21 (t, J
) 7.2 Hz, 6H), 3.05 (q, J ) 7.2 Hz, 4H-), 3.12 (t, J ) 6.8 Hz,
2H), 3.61 (dt, J ) 6.0, 6.8 Hz, 1H), 5.79 (br s, 2H), 6.18 (dd, J )
1.6, 8.4 Hz, 1H), 6.23 (d, J ) 1.6 Hz, 1H), 7.61 (d, J ) 8.4 Hz,
1H), 8.26 (t, J ) 6.0 Hz). MS ES (+ve) m/z: 266 (M + 1)+.

3570 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Pham et al.
(CD3OD) δ 2.03 (m, 2H), 2.27 (d, J ) 13.3 Hz, 2H), 3.24 (t, J )
14.0 Hz, 2H), 3.45 (m, 1H), 3.50 (dt, J ) 4.4, 27.7 Hz, 2H), 3.75
(d, J ) 13.3 Hz, 2H), 4.85 (dm, J ) 47.2 Hz, 2H.). 13C NMR
(CDCl3) δ 32.18, 47.00, 52.71, 58.04, 58.24, 81.10, 82.76, 126.03,
128.48, 131.74, 133.50, 165.88. MS ES (+ve) m/z: 147 (M + 1)+.
1-(2-Hydroxyethyl)piperidin-4-amine (23). N-tert-Butoxycar-
bonylpiperidine (2 g, 10 mmol), 2-bromoethanol (0.85 mL, 12
mmol), and K2CO3 (11.0 g, 80 mmol) were treated as in the
synthesis of 22. Purification by column chromatography on silica
and eluting with 1:9 MeOH/DCM gave the BOC-protected amine
mide (24; 0.4 g, 1.2 mmol), hexamethylditin (0.6 g, 1.82 mmol),
and a catalytic amount of Pd(PPh3)4 (5 mg) in anhydrous toluene
(18 mL) was heated to reflux for 48 h, with further addition of
hexamethylditin (0.8 mg) and Pd(PPh3)4 (20 mg) added after 24 h.
The crude black solid was purified by column chromatography on
silica eluting with 1:20 MeOH/CHCl3 to give 28 as a yellow oil
1
(200 mg, 40%). H NMR (CD3OD) δ 0.32 (s, 9H), 1.72 (m, 2H),
1.96 (m, 2H), 2.25 (m, 2H), 2.75 (dm, J ) 28.6 Hz, 2H), 3.05 (m,
2H), 3.92 (m, 1H), 4.60 (dm, 2H, J ) 47.6 Hz), 7.60 (d, J ) 8.2
Hz, 2H), 7.77 (d, J ) 8.2 Hz, 2H). 13C NMR (CD3OD) δ 9.96,
32.25, 48.49, 54.10, 59.10, 59.15, 82.20, 83.80, 127.51, 135.50,
136.80, 145.70, 170.00. MS ES (+ve) m/z: 415 (M + 1)+. HRMS
calcd for C17H27FN2O116Sn (M + 1)+, 411.1203; observed (M +
1)+, 411.1203.
1
21 as a colorless oil (2.3 g, 94%). H NMR (CDCl3) δ 1.44 (s,
9H), 1.52 (m, 2H), 1.94 (m, 2H), 2.26 (m, 2H), 2.59 (t, J ) 5.26,
2H), 2.92 (m, 2H), 3.48 (m, 1H), 3.65 (t, J ) 5.26, 2H), 4.76 (d,
J ) 7.92, 1H). MS ES (+ve) m/z: 267 (M + Na)+. Stirring 21
(1.1 g, 4.5 mmol) in TFA (2 mL) at room temperature for 1 h gave
23 as a light yellow solid (1.1 g, 95%). 1H NMR (CD3OD) δ 2.05
(m, 2H), 2.28 (d, J ) 13.6 Hz, 2H), 3.17 (br m, 2H), 3.30 (m, 2H,
superimposed), 3.45 (m, 1H), 3.73 (m, 2H), 3.88 (m, 2H). MS ES
(+ve) m/z: 145 (M + 1)+.
N-(1-(2-Hydroxyethyl)piperidin-4-yl)-4-(trimethylstannyl)-
benzamide (29). N-(1-(2-hydroxyethyl)piperidin-4-yl)-4-bromoben-
zamide (26; 0.15 g, 0.46 mmol) hexamethylditin (0.22 g, 0.69
mmol), and Pd(PPh3)4 (3 mg) were treated as above to give 29 as
1
a yellow oil (59 mg, 32%). H NMR (CD3OD) δ 0.32 (s, 9H),
1.88 (m, 2H), 2.12 (m, 2H), 2.82 (m, 2H), 2.96 (t, J ) 5.9 Hz,
2H), 3.40 (m, 2H), 3.82 (t, J ) 5.9 Hz, 2H), 4.08 (m, 1H), 7.60 (d,
J ) 8.2 Hz, 2H), 7.78 (d, J ) 8.2 Hz, 2H). 13C NMR (CD3OD) δ
10.09, 31.92, 48.20, 53.90, 59.62, 60.89, 127.32, 135.26, 136.61,
148.43, 169.87. ES (+ve) m/z: 413 (M + 1)+. HRMS calcd for
C17H28FN2O2SnNa (M + Na)+, 435.1077; observed (M + Na)+,
435.1076.
N-(4-(Dipropylamino)butyl)-4-iodobenzamide (30). Compound
30 was prepared according to literature methods.15 1H NMR (400
MHz, CDCl3) δ 0.86 (t, J ) 7.2 Hz, 6H), 1.40-1.50 (m, 4H), 1.58-
1.70 (m, 4H), 2.43 (m, 4H), 2.52 (t, J ) 6.7 Hz, 2H), 3.45 (dt, J
) 5.8, 6.2 Hz, 2H), 7.21 (br, 1H), 7.52 (d, J ) 8.5 Hz, 2H), 7.77
(d, J ) 8.5 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 11.8, 19.2,
24.3, 27.2, 39.68, 53.2, 55.6, 97.9, 128.6, 134.3, 137.4, 166.8. MS
ES (+ve) m/z: 403 (M + 1)+.
General Procedure 4 for the Preparation of Benzamides 24,
25, 26, and 27. To a solution of 4-iodobenzoic acid or 4-bro-
mobenzoic acid (1 mmol) in DMF (10 mL) was added 1-hydroxy-
benzotriazole hydrate (HOBT; 1.2 mmol), N-methylmorpholine (4
mmol), triethylamine (2 mmol), either 1-(2-fluoroethyl)piperidin-
4-amine (22; 1 mmol) or 1-(2-hydroxyethyl)piperidin-4-amine (23;
1 mmol; as their TFA salts) followed by 1-(3-dimethylaminoprop-
yl)-3-ethylcarbodiimide hydrochloride (EDC; 1.2 mmol). The
reaction was stirred at room temperature for 20 h before it was
filtered and evaporated to dryness. Aqueous workup followed by
recrystallization gave the corresponding benzamides.
4-Bromo-N-(1-(2-fluoroethyl)piperidin-4-yl)benzamide (24).
Benzamide (24), prepared according to procedure 4, was triturated
1
with DCM and filtered to give a white solid (0.55 g, 68%). H
NMR (CDCl3) δ 1.6 (m, 2H), 2.05 (m, 2H), 2.27 (dt, J ) 2.0,
11.7, 2H), 2.72 (dm, J ) 28.1 Hz, 2H), 2.96 (m, 2H), 4.00 (m,
2H), 4.68 (dm, J ) 47.6 Hz, 2H), 4.63 (dd, 1H, J ) 5.0 Hz), 5.90
(m, 1H), 7.57 (d, J ) 8.8 Hz, 2H), 7.62 (d, J ) 8.8 Hz, 2H). MS
ES (+ve) m/z: 351 (M + Na)+. Anal. Calcd for C14H18FBrN2O:
C, 51.08; H, 5.51; N, 8.51. Found: C, 51.34; H, 5.45; N, 8.30.
N-(1-(2-Fluoroethyl)piperidin-4-yl)-4-iodobenzamide (25). Ben-
zamide (25), prepared according to procedure 4, was recrystallized
4-Bromo-N-(4-(dipropylamino)butyl)benzamide (31). See gen-
eral procedure 3. Purification by column chromatography on silica
1
with ethyl acetate gave a colorless oil (1.33 g, 75%). H NMR
(CDCl3) δ 0.84 (t, J ) 7.4 Hz, 6H), 1.43 (m, 4H), 1.55 (m, 2H),
1.62 (m, 2H), 2.38 (m, 4H), 2.46 (t, J ) 6.8 Hz, 2H), 3.40 (dt, J
) 5.8, 6.0 Hz, 2H), 7.51 (d, J ) 8.4 Hz, 2H), 7.65 (d, J ) 8.4 Hz,
2H). 13C NMR (CDCl3) δ 11.8, 19.3, 24.5, 27.3, 39.8, 53.3, 55.8,
125.6, 128.6, 131.4, 133.7, 166.6. MS ES (+ve) m/z: 355 (M +
1)+. HRMS calcd for (M + 1)+, 355.1385; observed (M + 1)+,
355.1384.
N-(4-(Diethylamino)butyl)-4-(trimethylstannyl)benzamide (32).
4-Bromo-N-(4-(dipropylamino)butyl)benzamide (31; 0.5 g, 1.4
mmol) was treated with hexamethylditin (0.46 mg, 1.4 mmol) and
a catalytic amount of Pd(PPh3)4 (10 mg) in refluxing toluene as
described in the synthesis of 28. Purification by column chroma-
tography, eluting with 1:9 methanol/chloroform, gave the title
compound as a colorless oil (0.3 g, 52%). 1H NMR (CDCl3) δ 0.33
(s, 9H), 1.02 (t, J ) 7.2 Hz, 6H), 1.70-1.87 (m, 8H), 3.06 (m,
4H), 3.17 (m, 2H), 3.49 (t, J ) 6.5 Hz, 2H), 7.62 (d, J ) 8.2 Hz,
2H), 7.83 (d, J ) 8.2 Hz, 2H). 13C NMR (CDCl3) δ 9.8, 11.4,
18.6, 22.6, 27.8, 39.9, 54.1, 56.0, 127.5, 135.2, 136.9, 148.8, 170.5.
MS ES (+ve) m/z: 441 (M + 1)+. HRMS calcd for C20H37N2OSn
(M + 1)+, 441.1928; observed (M + 1)+, 441.1898.
1
from DCM to give a white solid (260 mg, 30% yield). H NMR
(CD3OD) δ 1.75 (m, 2H), 1.98 (m, 2H), 2.28 (dt, J ) 2.3, 12.0
Hz, 2H), 2.76 (dm, J ) 28.6 Hz, 2H), 3.05 (m, 2H), 3.90 (m, 2H),
4.65 (dm, J ) 47.6 Hz, 2H), 7.60 (d, J ) 8.6 Hz, 2H), 7.62 (d, J
) 8.6 Hz, 2H). 13C NMR (CD3OD) δ 32.0, 48.4, 53.8, 58.2, 59.1,
81.5, 83.2, 98.8, 129.9, 135.2, 138.6, 168.8. MS ES (+ve) m/z:
399 (M + 1)+. Anal. Calcd for C14H18FIN2O: C, 44.70; H, 4.82;
N, 7.45. Found: C, 44.73; H, 5.03; N, 7.26.
4-Bromo-N-(1-(2-hydroxyethyl)piperidin-4-yl)benzamide (26).
Benzamide (26), prepared according to procedure 4, was recrystal-
1
lized from ethyl acetate to yield a white solid (117 mg, 40%). H
NMR (CDCl3) δ 1.60 (m, 2H), 2.05 (m, 2H), 2.29 (dt, J ) 2.0,
11.98 Hz, 2H), 2.58 (t, J ) 5.3 Hz, 2H), 2.96 (m, 2H), 3.61 (t, J
) 5.3 Hz, 2H), 4.00 (m, 1H), 6.10 (m, 1H), 7.56 (d, J ) 8.8 Hz,
2H), 7.64 (d, J ) 8.8, 2H). 13C NMR (CDCl3) δ 31.98, 48.63, 53.92.
59.79, 61.01, 126.71, 130.01, 132.52, 134.70, 168.49. MS ES (+ve)
m/z: 327 (M + 1)+. Anal. Calcd for C14H19FBrN2O2: C, 51.39;
H, 5.85; N, 8.56. Found: C, 51.61; H, 5.79; N, 8.30.
N-(4-(2-Azanorborn-2-yl)butyl)-4-iodobenzamide (33). See
general procedure 3. Purification by column chromatography on
silica with 1:4 methanol/chloroform gave 33 as a colorless oil (0.88
N-(1-(2-Hydroxyethyl)piperidin-4-yl)-4-iodobenzamide (27).
Benzamide (27), prepared according to procedure 4, was recrystal-
1
g, 55%). H NMR (CDCl3) δ (1.6-1.9, m, 10H), 2.05 (m, 1H),
1
lized from DCM to yield a white solid (180 mg, 50%). H NMR
2.65-3.4 (m, 4H), 3.45 (m, 2H), 4.0 (m, 1H), 7.60 (d, J ) 8.58
Hz, 2H), 7.87 (d, J ) 8.58 Hz). 13C NMR (CDCl3) δ 22.07, 23.13,
24.06, 26.58, 27.43, 27.47, 27.60, 27.65, 30.51, 34.98, 37.41, 37.75,
37.98, 39.69, 51.73, 56.52, 59.97, 61.49, 64.79, 66.94, 99.06,
129.78, 134.91, 138.80, 169.40. MS ES (+ve) m/z: 399 (M + 1)+.
HRMS: 39.9, 54.1, 56.0, 127.5, 135.2, 136.9, 148.8, 170.5. MS
ES (+ve) m/z: 441 (M + 1)+. HRMS calcd for C17H24N2OI (M +
1)+, 399.0933; observed (M + 1)+, 399.0924.
(CDCl3) δ 1.55 (m, 2H), 2.0 (m, 2H), 2.28 (dt, J ) 2.0, 11.7, 2H),
2.57 (t, J ) 5.3 Hz, 2H), 2.92 (m, 2H), 3.62 (t, J ) 5.3 Hz, 2H),
4.00 (m, 1H), 5.95 (m, 1H), 7.47 (d, J ) 8.5 Hz, 2H), 7.78 (d, J )
8.5 Hz, 2H). 13C NMR (CDCl3) δ 33.56, 48.35, 53.37, 59.10, 60.33,
99.57, 129.68, 135.26, 139.00, 167.28. MS ES (+ve) m/z: 375 (M
+ 1)+. Anal. Calcd for C14H19FIN2O2: C, 44.93; H, 5.12; N, 7.49.
Found: C, 45.21; H, 5.22; N, 7.40.
N-(1-(2-Fluoroethyl)piperidin-4-yl)-4-(trimethylstannyl)ben-
zamide (28). 4-Bromo-N-(1-(2-fluoroethyl)piperidin-4-yl)benza-
N-(4-(2-Azanorborn-2-yl)butyl)-4-bromobenzamide (34). See
general procedure 3. Purification by column chromatography on

Radioiodinated Benzamides for Malignant Melanoma
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3571
silica with ethyl acetate gave 34 as colorless oil (1.1 g, 63%) and
as a 1:1 mixture of endo and exo isomers. H NMR (CDCl3) δ
σ1-receptors of [3H]-(+)-pentazocine and to σ2-receptors of [3H]-
1
DTG were determined in competitive binding assays at 10-5
M
1.2-1.9 (m, 10H), 2.35-2.84 (m, 5H), 3.40 (m, 3H), 7.46 (d, J )
8.4, 2H), 7.67 (d, J ) 8.4, 2H), 7.78 (s br, 1H). 13C NMR (CDCl3)
δ 26.11, 26.36, 27.97, 29.12, 37.27, 38.26, 40.43, 54.45, 60.67,
62.69, 126.96, 130.14, 132.76, 134.94, 168.02. MS ES (+ve) m/z:
351 (M + 1)+. HRMS calcd for C17H24N2OBr (M + 1)+, 351.1072;
observed (M + 1)+, 351.1062.
concentration of unlabeled benzamides. Then the inhibition constant
(IC50) for σ receptors of selected benzamides was determined by
incubating, in duplicate, aliquots of diluted guinea pig brain
membrane preparation at 25 °C for 2 h with concentrations of
benzamide ranging from 10-10 to 10-6 M in 50 nM Tris-HCl (pH
8.0) with [3H]-(+)-pentazocine (2 nM) for σ1-receptors or with [3H]-
DTG (2 nM) and (+)-pentazocine (100 nM) for σ2-receptors. In
both cases, nonspecific binding was determined in the presence of
haloperidol (1 µM). Then incubations were terminated by rapid
filtration through Whatman GF/B glass fiber. Filters were im-
mediately washed with ice-cold buffer and measured in a â-scintil-
lation counter to determine the amount of bound radioactivity. The
IC50 values were then converted to apparent Ki values using the
Cheng-Prussof equation and radioligand Kd values.
N-(4-(2-Azanorborn-2-yl)butyl)-4-trimethylstannylbenza-
mide (35). N-(4-(2-Aza-bicyclo[2.2.1] heptan-2-yl)butyl)-4-bro-
mobenzamide (34; 0.46 g, 1.31 mmol) was treated with hexa-
methylditin (0.64 g, 1.97 mmol) and a catalytic amount of Pd(PPh3)4
(10 mg) in refluxing dioxane as described above. Purification by
column chromatography on silica, using 2% MeOH in CHCl3, gave
35 as a pale yellow oil (0.42 g, 64%) and as a 1:1 mixture of endo
1
and exo isomers. H NMR (CDCl3) δ 0.29 (s, 9H), 1.61 (m, 2H),
Biodistribution Studies. The animal experiments were per-
formed in compliance with the NHMRC Australian Code of Practice
for the care and use of animals for scientific purposes. Female
C57BL/6J mice were obtained from Animal Resources Centre,
Western Australia. Biodistribution time-course studies were per-
formed in these mice bearing the B16F0 murine melanoma tumor
model. For inoculation, melanoma cells, obtained from European
Collection of Cell Cultures (UK), were resuspended in Ca2+ and
Mg2+ free PBS at 3 × 106 viable cells per mL and 0.1 mL was
subcutaneously injected in the left flank of 6-7 week old mice.
Ten days later, 98-100% of the animals had developed tumors.
The [123I]-labeled benzamide derivatives (0.37-0.74 MBq, 100
µL) were injected intravenously via the tail vein into mice (15-18
g). Time points of 1, 3, 6, 24, 48, and 72 h after injection were
chosen for determining the distribution of each compound in various
organs and tissues. At defined times postinjection, groups of mice
(n ) 5) were weighed, sacrificed by CO2 administration followed
by cervical dislocation and dissected. Selected organs were weighed
and their radioactivity measured with a γ-counter. The remaining
activity in the carcass was also determined to obtain the total activity
in the mouse at defined time points. The fraction of injected activity
(%ID) in the organ was calculated by comparison with suitable
dilutions of the injected dose. Then the radioactivity concentration
in the organ (%ID/g) was found by dividing the %ID for each organ
by the weight of the organ. The SUV in the tumor at the time of
sacrifice (SUVt) was calculated for each animal by dividing the
tumor concentration by the radioactive concentration in the mouse
according to the following formula:
1.72-1.77 (m, 4H), 1.90-1.97 (m, 2H), 2.08 (m, 1H), 2.29 (br m,
1H), 2.65 (br s, 1H), 2.99 (m, 2H), 3.15 (m, 2H), 3.50 (m, 2H),
3.95 (br s, 1H), 7.55 (d, J ) 8.0 Hz, 2H), 7.79 (t, J ) 5.6 Hz, 1H),
9.22 (d, J ) 8.0 Hz, 2H). 13C NMR (CDCl3) δ -8.3, 23.1, 27.5,
37.4, 39.2, 61.2, 65.8, 127.8, 134.6, 137.1, 148.7, 169.3. MS ES
(+ve) m/z: 437 (M + 1)+. HRMS calcd for C20H33N2OSn (M +
1)+, 437.1623; observed (M + 1)+, 437.1622.
Radiolabeling using Tl(TFA)3. To a solution of the benzamide
precursor (13b-13f, 15, 18m, and 18n; 0.25 mg, ∼0.8 µmol) in
TFA (100 µL) was added Tl(TFA)3 (26 M in TFA, 150 µL). After
20 min, the resulting thallium complex solution was added to a
dried sample of [123I]INa (400-500 MBq) in a Wheaton conical
vial. The reaction mixture was allowed to stand for 15 min before
it was neutralized with concentrated NH3 (1 mL) and then purified
by solid-phase extraction (RP-SPE). The labeled compound was
washed off the cartridge with EtOH (1 mL), was dried in vacuo,
and then mobile phase (500 µL) was added in preparation for further
HPLC purification with conditions, as described in Table 1. The
radiolabeled compound was collected, dried in vacuo, and then
formulated in saline for biological evaluation.
Radiolabeling using Chloramine-T (CAT). To a solution of
the trimethylstannyl precursor (28, 29, 32, or 35; 0.25 mg, 0.6 µmol)
in ethanol (200 µL) was added [123I]INa (400-500 MBq), CAT
(4.5 mM, 100 µL), and HCl (1 M, 100 µL). After 5 min at room
temperature, Na2S2O5 (260 mM, 100 µL), NaHCO3 (650 mM, 100
µL), followed by HPLC mobile phase (350 µL) were added. The
resulting solution was then purified by HPLC with conditions, as
described in Table 1. The radiolabeled compound was collected,
dried in vacuo, and then formulated in saline for biological
evaluation.
SUVt ) [activity at time (t)/tumor (g)]/
Radiolabeling of BZA2. No carrier added [123I]BZA2 was
synthesized using a similar procedure as described in the radio-
synthesis of carrier added [125I]BZA2.14 A solution of 2-bromo-N-
(2-(diethylamino)ethyl)benzamide29 (1.3 mg), synthesized from
2-bromobenzoyl chloride and N,N-diethylethylenediamine using
step 2 of general procedure 3, in sodium acetate buffer (200 µL of
0.16 M NaOAc in 0.7% acetic acid) was added to a dried sample
of [123I]INa (800 MBq) in a Wheaton conical vial and heated to
130 °C for 30 min. The reaction was cooled and mobile phase
added. The resulting solution was then purified by HPLC with
conditions as described in Table 1. The radiolabeled compound
was collected, dried in vacuo, and then formulated in saline for
biological evaluation.
Lipophilicity. The lipophilicity of the compounds was assessed
using RP-HPLC by determining the logP7.5 value using literature
procedures.30 Samples, dissolved in methanol, were analyzed using
a C18 column (RP C18, Xterra, 5 µm; 4.6 × 150 mm) and a mobile
phase consisting of MeOH and phosphate buffer (0.1 M, pH 7.5),
50:50 v/v with a flow rate of 1 mL/min. The logP7.5 of a studied
compound was estimated by a comparison of its retention time to
that of standards of known log P values.
[remaining activity at time (t)/weight of animal (g)]
Competition Studies. Blocking studies were performed to
examine the in vivo uptake mechanism of the [123I]benzamides in
the tumor. The blocking effect of haloperidol, a nonselective σ1-σ2
inhibitor, on the tracer uptake over 24 h was examined in various
organs and tumor tissues. Groups (n ) 5) of mice bearing B16F0
murine melanomas were injected intravenously with 1 mg/kg of
haloperidol 5 min prior to injection of each radiotracer (0.37-0.74
MBq) dose. The mice were sacrificed at 1 and 24 h postinjection,
and tissues were handled as described for the biodistribution studies.
Radioactive concentrations in the organs and the tumor of treated
animals were compared to the controls. Statistical significance was
evaluated using one-way ANOVA. The criterion for significance
was p < 0.01.
SPECT Imaging. SPECT imaging was performed using a high
resolution γ-camera (X-SPECT, Gamma Medica Inc., U.S.A.)
designed for laboratory animals equipped on an array of discrete 2
× 2 × 6 mm NaI(Tl) crystals optically isolated from each other
and a high-resolution parallel hole collimator that has a 12.5 ×
12.5 cm field of view. The mouse was anaesthetized via inhalant
isoflurane (Forthane brand) in 200 mL/min oxygen via a nose cone
fitted to the animal bed and imaged for 10-20 min at 1, 24, and
48 h after injection of 7-10 MBq of the studied [123I]14d and [123I]-
25.
Ligand Binding Assays. The σ1 and σ2 binding affinities of
unlabeled benzamides were determined in in vitro binding assays
(Novascreen Biosciences, Baltimore, U.S.A.) according to literature
methods.31 The percentage of inhibition of the specific binding to

3572 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Pham et al.
(16) Eisenhut, M.; Hull, W. E.; Mohammed, A.; Mier, W.; Lay, D.; Just,
W.; Gorgas, K.; Lehmann, W. D.; Haberkorn, U. Radioiodinated
N-(2-diethylaminoethyl)benzamide derivatives with high melanoma
uptake: structure-affinity relationships, metabolic fate, and intrac-
ellular localization. J. Med. Chem. 2000, 43, 3913-3922.
(17) Seguin, H.; Gardette, D.; Moreau, M. F.; Madelmont, J. C.; Gramain,
J. C. A general method for the synthesis of N,N-dialkylaminobutyl-
amines. Synth. Commun. 1998, 28, 4257-4272.
Supporting Information Available: Table of analytical analysis
and HPLC purity test of target compounds 14b, 14c, 14d, 14e, 14f,
19m, 19n, 25, 27, and 33. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) Chen, J.; Giblin, M. F.; Wang, N.; Jurisson, S. S.; Quinn, T. P. In
vivo evaluation of 99mTc/188Re-labeled linear alpha-melanocyte
stimulating hormone analogs for specific melanoma targeting. Nucl.
Med. Biol. 1999, 26, 687-693.
(2) Allen, B. J.; Rizvi, S.; Li, Y.; Tian, Z.; Ranson, M. In vitro and
preclinical targeted alpha therapy for melanoma, breast, prostate, and
colorectal cancers. Crit. ReV. Oncol. Hematol. 2001, 39, 139-146.
(3) Coderre, J. A.; Packer, S.; Fairchild, R. G.; Greenberg, D.; Laster,
B.; Micca, P.; Fand, I. Iodothiouracil as a melanoma localizing agent.
J. Nucl. Med. 1986, 27, 1157-1164.
(4) Miao, Y.; Owen, N. K.; Whitener, D.; Gallazzi, F.; Hoffman, T. J.;
Quinn, T. P. In vivo evaluation of 188Re-labeled alpha-melanocyte
stimulating hormone peptide analogs for melanoma therapy. Int. J.
Cancer 2002, 101, 480-487.
(5) Boyd, C. M.; Lieberman, L. M.; Beierwaltes, W. H.; Varma, V. M.
Diagnostic efficacy of a radioiodinated chloroquine analog in patients
with malignant melanoma. J. Nucl. Med. 1970, 11, 479-486.
(6) Link, E. M.; Carpenter, R. N. 211At-methylene blue for targeted
radiotherapy of human melanoma xenografts: Treatment of cutaneous
tumors and lymph node metastases. Cancer Res. 1992, 52, 4385-
4390.
(7) Michelot, J. M.; Moreau, M. F.; Labarre, P. G.; Madelmont, J. C.;
Veyre, A. J.; Papon, J. M.; Parry, D. F.; Bonafous, J. F.; Boire, J.
Y.; Desplanches, G. G.; Bertrand, S. J.; Meyniel, G. Synthesis and
evaluation of new iodine-125 radiopharmaceuticals as potential tracers
for malignant melanoma. J. Nucl. Med. 1991, 32, 1573-1580.
(8) Labarre, P.; Papon, J.; Moreau, M. F.; Moins, N.; Veyre, A.;
Madelmont, J. C. Evaluation in mice of some iodinated melanoma
imaging agents using cryosectioning and multi-wire proportional
counting. Eur. J. Nucl. Med. 1999, 26, 494-498.
(9) Coenen, H. H.; Brandau, W.; Dittmann, H.; Dutschka, K.; Niehoff,
T.; Pulawski, P.; Zo¨lzer, F.; Sciuk J.; Streffer, C. Evaluation of
melanoma seeking N-(dialkylamino)-alkyl [123,131I]iodobenzamides
by animal and cell-culture studies J. Labelled Cmpd. Radiopharm.
1995, 37, 260-262.
(18) Marakarmi, M.; Inukai, N.; Koda, A.; Nakano, K. An improved
synthesis of metoclopramide. Chem. Pharm. Bull. 1971, 19, 1696-
1699.
(19) Chehade, F.; De Labriolle-Vaylet, C.; Michelot, J.; Moins, N.;
Moreau, M. F.; Hindie, E.; Papon, J.; Escaig, F.; Galle, P.; Veyre,
A. Distribution of I-BZA (N-2-diethylaminoethyl-4-iodobenzamide)
in grafted melanoma and normal skin: A study by secondary ion
mass spectroscopy. Cell. Mol. Biol. 2001, 47, 529-534.
(20) Hellewell, S. B.; Bruce, A.; Feinstein, G.; Orringer, J.; Williams,
W.; Bowen, W. D. Rat liver and kidney contain high densities of
sigma-1 and sigma-2 receptors; characterization by ligand binding
and photoaffinity labeling. Eur. J. Pharmacol. 1994, 268, 9-18.
(21) Quirion, R.; Bowen, W. D.; Itzhak, Y.; Junien, J. L.; Musacchio, J.
M.; Rothman, R. B.; Tsung-Ping, S.; Tam, S. W.; Taylor, D. P. A
proposal for the classification of sigma binding sites. Trends
Pharmacol. Sci. 1992, 13, 85-86.
(22) Mansard, S.; Papon, J.; Moreau, M. F.; Miot-Noirault, E.; Labarre,
P.; Bayle, M.; Veyre, A.; Madelmont, J. C.; Moins, N. Uptake in
melanoma cells of N-(2-diethylaminoethyl)-2-iodobenzamide (BZA2),
an imaging agent for melanoma staging: Relation to pigmentation.
Nucl. Med. Biol. 2005, 32, 451-458.
(23) Chehade, F.; de Labriolle-Vaylet, C.; Moins, N.; Moreau, M. F.;
Papon, J.; Labarre, P.; Galle, P.; Veyre, A.; Hindie, E. Secondary
ion mass spectrometry as a tool for investigating radiopharmaceutical
distribution at the cellular level: The example of I-BZA and (14)C-
I-BZA. J. Nucl. Med. 2005, 46, 1701-1706.
(24) John, C. S.; Vilner, B. J.; Geyer, B. C.; Moody, T.; Bowen, W. D.
Targeting sigma receptor-binding benzamides as in vivo diagnostic
and therapeutic agents for human prostate tumors. Cancer Res. 1999,
59, 4578-4583.
(10) Dittmann, H.; Coenen, H. H.; Zolzer, F.; Dutschka, K.; Brandau,
W.; Streffer, C. In vitro studies on the cellular uptake of melanoma
imaging aminoalkyl-iodobenzamide derivatives (ABA). Nucl. Med.
Biol. 1999, 26, 51-56.
(11) John, C. S.; Bowen, W. D.; Saga, T.; Kinuya, S.; Vilner, B. J.;
Baumgold, J.; Paik, C. H.; Reba, R. C.; Neumann, R. D.; Varma, V.
M.; McAfee, J. G. A malignant melanoma imaging agent: Synthesis,
characterization, in vitro binding and biodistribution of iodine-125-
(2-piperidinylaminoethyl)-4-iodobenzamide. J. Nucl. Med. 1993, 34,
2169-2175.
(12) Michelot, J. M.; Moreau, M. F.; Veyre, A. J.; Bonafous, J. F.; Bacin,
F. J.; Madelmont, J. C.; Bussiere, F.; Souteyrand, P. A.; Mauclaire,
L. P.; Chossat, F. M.; Papon, J. M.; Labarre, P. G.; Kauffmann, Ph.;
Plagne, R. J. Phase II scintigraphic clinical trial of malignant
melanoma and metastases with iodine-123-N-(2-diethylaminoethyl
4-iodobenzamide). J. Nucl. Med. 1993, 34, 1260-1266.
(13) Brandau, W.; Niehoff, T.; Pulawski, P.; Jonas, M.; Dutschka, K.;
Sciuk, J.; Coenen, H. H.; Schober, O. Structure distribution relation-
ship of iodine-123-iodobenzamides as tracers for the detection of
melanotic melanoma. J. Nucl. Med. 1996, 37, 1865-1871.
(14) Moins, N.; D’Incan, M.; Bonafous, J.; Bacin, F.; Labarre, P.; Moreau,
M. F.; Mestas, D.; Noirault, E.; Chossat, F.; Berthommier, E.; Papon,
J.; Bayle, M.; Souteyrand, P.; Madelmont, J. C.; Veyre, A. 123I-N-
(2-diethylaminoethyl)-2-iodobenzamide: A potential imaging agent
for cutaneous melanoma staging. Eur. J. Nucl. Med. Mol. Imaging
2002, 29, 1478-1484.
(25) Waterhouse, R. N.; Chapman, J.; Izard, B.; Donald, A.; Belbin, K.;
O’Brien, J. C.; Collier, T. L. Examination of four 123I-labeled
piperidine-based sigma receptor ligands as potential melanoma
imaging agents: Initial studies in mouse tumor models. Nucl. Med.
Biol. 1997, 24, 587-593.
(26) Moreau, M. F.; Michelot, J.; Papon, J.; Bayle, M.; Labarre, P.;
Madelmont, J. C.; Parry, D.; Boire, J. Y.; Moins, N.; Seguin, H.;
Veyre, A.; Mauclaire, L. Synthesis, radiolabeling, and preliminary
evaluation in mice of some (N-diethylaminoethyl)-4-iodobenzamide
derivatives as melanoma imaging agents. Nucl. Med. Biol. 1995, 22,
737-747.
(27) Alonso Garrido, D. O.; Buldain G.; Frydman B. Concerning the
synthesis of N-methylputrescine and its homologs. J. Org. Chem.
1984, 49, 2021-2023.
(28) Micovic, V. M.; Mihailovic, M. L. The reduction of acid amides
with lithium aluminium hydride. J. Org. Chem. 1953, 18, 1190-
1200.
(29) Cassebaum, H.; Uhlig, K. Die Reaktion von N-(â-Halogena¨thyl)-
carbonsa¨ureamiden mit Dia¨thylamin. J. Prakt. Chem. (Weinheim,
Ger.) 1973, 315, 1057-1066.
(30) Waterhouse, R. N. Determination of lipophilicity and its use as a
predictor of blood-brain barrier penetration of molecular imaging
agents. Mol. Imaging Biol. 2003, 5, 376-389.
(31) Chaki, S.; Tanaka, M.; Muramatsu, M.; Otomo, S. NE-100, a novel
potent sigma ligand, preferentially binds to sigma 1 binding sites in
guinea pig brain. Eur. J. Pharmacol. 1994, 251, R1-2.
(15) Moins, N.; Papon, J.; Seguin, H.; Gardette, D.; Moreau, M. F.;
Labarre, P.; Bayle, M.; Michelot, J.; Gramain, J. C.; Madelmont, J.
C.; Veyre, A. Synthesis, characterization and comparative biodistri-
bution study of a new series of p-iodine-125 benzamides as potential
melanoma imaging agents. Nucl. Med. Biol. 2001, 28, 799-808.
JM0701627