Evaluation of radiolabeled (Hetero)aromatic analogues of N-(2-diethylaminoethyl)-4-iodobenzamide for imaging and targeted radionuclide therapy of melanoma

Article abstract of DOI:10.1021/jm701424g

Targeted radionuclide therapy using radioiodinated compounds with a specific affinity for melanoma tissue is a promising treatment for disseminated melanoma, but the candidate with the ideal kinetic profile remains to be discovered. Targeted radionuclide therapy concentrates the effects on tumor cells, thereby increasing the efficacy and decreasing the morbidity of radiotherapy. In this context, analogues of N-(2-diethylaminoethyl)-4- iodobenzamide (BZA) are of interest. Various (hetero)aromatic analogues 5 of BZA were synthesized and radioiodinated with ¹²⁵I, and their biodistribution in melanoma-bearing mice was studied after i.v. administration. Most [¹²⁵I]5-labeled compounds appeared to bind specifically and with moderate-to-high affinity to melanoma tumor. Two compounds, 5h and 5k, stood out with high specific and long-lasting uptake in the tumor, with a 7- and 16-fold higher value than BZA at 72 h, respectively, and kinetic profiles that makes them promising agents for internal targeted radionuclide therapy of melanoma.

Full text of DOI:10.1021/jm701424g

J. Med. Chem. 2008, 51, 3133–3144
3133
Evaluation of Radiolabeled (Hetero)Aromatic Analogues of N-(2-diethylaminoethyl)-4-
iodobenzamide for Imaging and Targeted Radionuclide Therapy of Melanoma
Jean-Michel Chezal, Janine Papon, Pierre Labarre, Claire Lartigue, Marie-Josephe Galmier, Caroline Decombat,
Olivier Chavignon, Jean Maublant, Jean-Claude Teulade, Jean-Claude Madelmont, and Nicole Moins*
UniVersity Clermont 1, Clermont-Ferrand F-63001, France, INSERM U484, Clermont-Ferrand F-63005, France, Centre Jean Perrin,
Clermont-Ferrand F-63011, France
ReceiVed NoVember 12, 2007
Targeted radionuclide therapy using radioiodinated compounds with a specific affinity for melanoma tissue
is a promising treatment for disseminated melanoma, but the candidate with the ideal kinetic profile remains
to be discovered. Targeted radionuclide therapy concentrates the effects on tumor cells, thereby increasing
the efficacy and decreasing the morbidity of radiotherapy. In this context, analogues of N-(2-diethylami-
noethyl)-4-iodobenzamide (BZA) are of interest. Various (hetero)aromatic analogues 5 of BZA were
synthesized and radioiodinated with 125I, and their biodistribution in melanoma-bearing mice was studied
after i.v. administration. Most [125I]5-labeled compounds appeared to bind specifically and with moderate-
to-high affinity to melanoma tumor. Two compounds, 5h and 5k, stood out with high specific and long-
lasting uptake in the tumor, with a 7- and 16-fold higher value than BZA at 72 h, respectively, and kinetic
profiles that makes them promising agents for internal targeted radionuclide therapy of melanoma.
Introduction
In recent years, there has been a dramatic worldwide increase
in the incidence of malignant melanoma, which is fast becoming
a major public health problem in many countries.1 It displays a
strong tendency to metastasize, and patients with metastases at
stage IV have an average survival of only a few months.2 There
is therefore an urgent need to find ways to treat disseminated
melanoma. Standard therapies such as external beam therapy
or monochemotherapy with a response rate of around 10% are
of very limited value.3 A higher response rate of up to 40%
can be obtained with combined treatment schedules but without
significant prolonged survival in randomized studies.4 Globally,
the response rate is still rather low, and there has been no
progress in systemic palliative melanoma treatment for close
on three decades.5,6 The lack of specific therapy has steered
research on melanoma treatment toward vaccine-based immu-
notherapy and targeted chemotherapy approaches.7 Targeted
internal radionuclide therapy would be an effective alternative.
The search for a radiopharmaceutical with a selective affinity
for melanoma tissue is therefore of utmost importance both for
the early detection of local recurrences to allow efficient surgical
excision1 and for treatment of in-transit or developed metastases.
Different melanoma targets and carriers for radionuclides are
currently being investigated. Peptide analogues of R-melanocyte
stimulating hormone (R-MSHa) targeting the melanocortin-1
receptor have recently been developed. Using SPECT/CT
equipment, 99mTc and 111In-labeled peptides provide good
quality imaging in melanoma-bearing mice.8 For application in
systemic radionuclide therapy, promising experimental results
in melanoma-bearing mice have been reported with one of them
conjugated with 188Re,9 which is a ꢀ emitter, and with 212Pb,10
Figure 1. Some structures with a high affinity for melanin.
Scheme 1a
a Ar or Het ) naphthalene, pyridine, indole, benzo[b]furan, benzo[b-
]thiophene, imidazo[1,2-a]pyridine, benzimidazole, quinoline, quinolone,
isoquinoline, quinoxaline, 1,6-naphthyridine.
which is an R particle emitting radionuclide11 with even greater
therapeutic efficacy. However, these are only preliminary results,
and high kidney concentrations may result in a problematic renal
toxicity.
Melanin pigment can itself be a specific target for melanoma
tissue. Melanin is an amorphous, irregular polymer composed
of intimate mixtures12 of two separate but biogenetically related
pigments, namely eumelanins and pheomelanins, with different
degrees of oxidation.13 The eumelanin chromophore is formed
by polymerization of dihydroxyindole (DHI) monomer units and
its 2-carboxyl derivative dihydroxyindole-2-carboxylic acid
(DHICA), while the pheomelanin is produced by the tyrosinase-
mediated copolymerization of L-3,4-dihydroxyphenylalanine (L-
dopa) and L-cysteine.14
* To whom correspondence should be addressed. Phone: +(33)4 73 15
08 00. Fax: +(33)4 73 15 08 01. E-mail: moins@inserm484.u-clermont1.fr.
a Abbreviations: BZA, N-(2-diethylaminoethyl)-4-iodobenzamide; R-MSH,
R-melanocyte stimulating hormone; SPECT/CT, single photon emission
computed tomography/computed tomography; PET, positron emission
tomography; DHI, dihydroxyindole; DHICA, dihidroxyindole-2-carboxylic
acid; MTB, methylene blue; SAR, structure–activity relationship; DMAP,
4-dimethylaminopyridine; PBS, phosphate buffer solution.
10.1021/jm701424g CCC: $40.75
2008 American Chemical Society
Published on Web 05/16/2008

3134 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Scheme 2. Synthetic Pathways for Esters 6b,d,g,h,j-la
Chezal et al.
a Reagents and conditions: (i) ethyl chloroformate, NEt3, DMAP, CH2Cl2, 0 °C then reflux; (ii) H2, 10% Pd-C, 1 atm; (iii) (a) NaNO2, HBF4, 0 °C, (b)
KI, H2O, ∆; (iv) (a) AcONa, AcOH, 75 °C, (b) Br2, AcOH, reflux; (v) AgNO3, EtOH, H2O, reflux; (vi) (a) NaNO2, HCl, HBF4, 0 °C, (b) KI, H2O, 80 °C;
(vii) H2SO4, EtOH, reflux; (viii) (a) NaNO2, HBF4, 0 °C, (b) KI, H2O, 50 °C; (ix) PTSA, NIS, -10 °C then rt.
Scheme 3a
a Reagents and conditions: (i) (a) N,N-diethylethylenediamine, AlMe3, CH2Cl2, 0 °C, (b) reflux; (ii) 2N HCl ether, rt.
Many drugs bind to melanin both in vivo and in vitro:
polycyclic aromatic compounds such as methylene blue MTB
(1), chloroquine (2), or acridine orange (3), and especially those
with coplanar fused rings, all bind strongly with melanin15
(Figure 1). This affinity is probably due to the interaction
between the aromatic rings of the drugs and the aromatic rings
of the melanin subunits. Studies performed with labeled MTB
(1) revealed stable, exceptionally high concentrations in pig-

Analogues of BZA for Imaging and Therapy of Melanoma
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3135
Scheme 4. Radiolabeling of Compounds 5a-la
Esterification of 6-iodonicotinic acid32 (7) was performed
using mild conditions33 in the presence of ethyl chloroformate,
4-dimethylaminopyridine (DMAP), and triethylamine to give
nicotinic ester 6b in 76% yield. Ethyl esters 6d and 6g were
obtained in two steps from commercially available compound
8 and nitro derivative 10,34 respectively, following a previously
reported synthetic route developed in the benzimidazole series.35
Catalytic hydrogenation of nitro compounds 8 and 10 yielded
ethyl aminoesters 936 and 11,37 which were diazotized in
fluoroboric acid to give diazonium borofluorides. Decomposition
of these diazonium salts with potassium iodide gave iodoesters
6d and 6g with low yields (21% and 12%, respectively). The
quinoline analogue 6h can be prepared by the method reported
for the preparation of ethyl 6-chloroquinaldinate.38 Typically,
ethyl 6-iodoquinoline-2-carboxylate (6h) was obtained from
6-iodoquinaldine39 (12) in two steps, including bromination to
13 and final conversion to ester compound 6h with silver nitrate
in aqueous ethanol, with an overall yield of 46%. The iodoiso-
quinoline ester 6j was prepared from methyl 5-aminoisoquino-
line-3-carboxylate40 (14) by a diazotization reaction in aqueous
hydrochloric acid followed by treatment with fluoroboric acid
to give the corresponding diazonium borofluoride. This salt was
converted into 6j in 25% yield according to the procedure
described for compounds 6d,g. The usual route was followed
to prepare quinoxaline 6k from 6-nitroquinoxaline-2-carboxylic
acid41 (15). Esterification using a common method gave ethyl
ester 16, which was reduced by catalytic hydrogenation to yield
the amine 17. Diazotization in fluoroboric acid followed by
treatment with potassium iodide gave ethyl 6-iodoquinoxaline-
2-carboxylate (6k). In this case, the diazonium salt was not
isolated. Finally, the iodonaphthyridinic ester 6l was obtained
by treating the ethyl 1,6-naphthyridine-2-carboxylate (19) with
N-iodosuccinimide in the presence of catalytic p-toluenesulfonic
acid. Ester 19 was itself prepared from 1,6-naphthyridine-2-
carboxylic acid (18)42 in 45% yield by the method used for
ester 6b.
a Reagents and conditions: (i) (a) citrate buffer solution, 0.5% aq CuSO4,
[
125I]NaI, ∆, 30-60 min, (b) 2N HCl ether, rt.
mented melanoma in vivo.16 Radiohalogenated MTB (iodine-
123 or iodine-131, γ emitters) was used with some success in
a clinical trial for early detection of melanoma metastases,17
while R emitting astatine-211 MTB has proven particularly
effective in inhibiting growth of human cutaneous pigmented
melanomas, such as xenografts in athymic mice, and their
spontaneous lymph node metastases.18 However, with R-parti-
cles delivering densely ionizing radiation, their decay should
result in highly localized energy deposition which needs a highly
specific accumulation in tumor tissue.19 Another class of
aromatic compounds with a cationic site such as N-(2-diethy-
laminoethyl)iodobenzamide (4) were developed as potent mela-
noma-seeking agents. Two of these compounds 4a,b, named
respectively BZA and BZA2, have been clinically evaluated as
suitable imaging agents for use in nuclear medicine with very
encouraging results for metastases imaging.20 The intracellular
localization of BZA within melanin-containing structures has
been clearly visualized with analytical imaging by secondary
ion mass spectrometry.21 Furthermore, interaction studies on
BZA and synthetic melanin revealed the presence of two classes
of binding sites, one ionic and the other hydrophobic.22
We have previously reported on spermidine benzamide
derivatives, which possess high in vitro affinity for melanin but
poor in vivo tumor concentration.23 More recently, various
iodinated benzamides were evaluated24 by taking into account
the optimized lipophilic side chain (already reported by us)25
and the substitution pattern on the benzene ring developed
previously.26 These studies showed original in vivo distribution
profiles with certain molecules showing a higher, more specific
tumor uptake that was promising for imaging but still needed
improvement for radionuclide therapy.
Here, within the framework of our continuing efforts to
discover new molecular derivatives for targeted radionuclide
therapy with strong, specific, and long-lasting uptake in
melanoma tissues, we examined the structure-activity relation-
ships (SAR) and biological properties in B16 melanoma-bearing
mice of aromatic or heteroaromatic BZA analogues. Our strategy
involved the incorporation of aromatic or heteroaromatic
structures in place of the benzene moiety to take advantage of
polycyclic aromatic compounds15 that display a strong affinity
for melanin, while the lipophilic side chain was kept identical
to the parent compound BZA. For these studies, various
iodo(hetero)aromatic fused ring systems with a carboxamide
part 5 were easily synthesized from corresponding iodoesters 6
(Scheme 1). To facilitate the radiolabeling, iodine was preferably
introduced on a benzene ring when possible.
The synthesis of the final compounds 5a-l is depicted in
Scheme 3. Esters 6a-l were coupled with N,N-diethylethyl-
enediamine in the presence of trimethylaluminium to give the
corresponding amides 20a-l. To facilitate pharmacological
studies, all the amides 20a-l were converted into their
hydrochloride salts 5a-l for better solubility in water, using a
2N HCl/ether solution.
Radiolabeling. The radiolabeling of compounds 5a-l with
no-carrier-added [125I]NaI was performed using an isotopic
exchange reaction in acidic medium as previously described for
BZA43 (Scheme 4) to give, after purification through an Extrelut
column, the radioiodinated compounds [125I]5a-l in 22-92%
radiochemical yields. Specific activity was in the range 4.7-36.3
MBq/µmol. Reaction conditions and radiochemical data are
reported in Table 1.
In Vitro Binding to Melanin and Partition Coefficients.
As indicated, our main challenge was to enhance affinity for
melanin. Except for compound 5b, the (hetero)aromatic ana-
logues 5 synthesized displayed a strong affinity (45-88%) for
synthetic melanin in water, as reported in Table 2. When
melanin was suspended in PBS, these values decreased strongly
for compound 5f and were comparable to the values obtained
with BZA,25 probably due to the ionic strength of PBS.22 For
the other compounds, molecular binding to melanin was less
affected in PBS, ranging from 35% to 59%. A plausible
explanation for these variations in molecule binding in PBS
observed with identical N-alkyl side chains is a π stacking
interaction independent of both pH and ionic strength. The
Results and Discussion
Synthesis. The following iodoarylesters were prepared ac-
cording to the literature: methyl 6-iodo-2-naphthoate (6a),27
ethyl 5-iodoindole-2-carboxylate (6c),28 methyl 4-iodobenzo[b]-
thiophene-2-carboxylate (6e),29 ethyl 6-iodoimidazo[1,2-a]py-
ridine-2-carboxylate (6f),30 and ethyl 1,4-dihydro-6-iodo-4-
oxoquinoline-3-carboxylate (6i).31 The synthetic methods for
preparation of ester intermediates 6b,d,g,h,j-l are illustrated
in Scheme 2.

3136 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Table 1. Chemical and Radiochemical Data for Compounds [125I]5a-l
Chezal et al.
a Radiochemical yields were calculated by dividing the radioactivity in the final filtered product by the initial amount of radioactive sodium iodide. b Radiochemical
purities were determined by TLC (Al2O3, CH2Cl2/EtOH (97/3, v/v)). c TLC conditions (Al2O3, CH2Cl2/EtOH (9/1, v/v)). d TLC conditions (Al2O3, EtOAc).

Analogues of BZA for Imaging and Therapy of Melanoma
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3137
Table 2. In Vitro Binding to Melanin and log P of [125I]5a-l
Derivatives Compared with BZA
compounds
% bound to synthetic melanin H2O/PBS
log Pa
BZAb
5a
5b
5c
5d
5e
78.6
80.8
28.1
79.8
87.0
87.6
75.3
84.5
87.4
86.7
87.0
67.4
44.8
/
/
/
/
/
/
/
/
/
/
/
/
/
13.8
50.9
3.5
1.34
1.33
-0.29
1.69
1.57
1.54
0.71
1.47
1.87
1.30
1.65
1.12
0.51
43.1
45.2
56.1
11.6
47.1
43.2
59.0
45.2
47.3
35.3
5f
5g
5h
5i
5j
5k
5l
a P: partition coefficient between n-octanol and phosphate buffer solution
(pH 7.4). b BZA values were obtained from previous work.25
Table 3. Urinary and Fecal Excretionsa of Radioactivity Following
Injection of [125I]5a-l Derivatives in B16 Melanoma-Bearing Mice
Compared with BZA
Figure 2. Concentrations of four new [125I] aromatic compounds
compared with BZA in selected tissues after i.v. administration in B16
melanoma-bearing mice. (A) at 3 h. (B) at 72 h. Mean ( SD (two
mice, n determinations for each compound at each time). Absence of
value: organ concentration was equal to background value.
compounds
urinary %ID 0-72 h
fecal %ID 0-72 h
BZAb
5a
5b
5c
5d
5e
83.1
43.7
67.3
21.5
21.4
52.6
36.8
17.1
80.7
23.8
49.9
70.2
68.9
4.8
26.7
5.3
61.7
73.0
46.7
43.5
40.7
11.4
69.2
21.2
18.0
29.4
5l exhibited remarkable in vivo behavior, with significant tumor
uptake and very high specificity illustrated by a rapid release
from nontarget tissues, leading to high values of tumor to
nontarget organ activity ratios and rapidly unquantifiable ratios
when organ concentration was within the range of the back-
ground value (Figure 2, Table 5). These promising profiles made
these compounds good candidates for scintigraphic imaging.
Compounds 5h and 5k, which possessed a quinoline moiety
and a quinoxaline moiety, respectively, demonstrated very long-
lasting tumor concentrations, respectively 7-fold and 16-fold
higher values than BZA at 72 h, lending them a kinetic profile
favorable for application as a vector for radionuclide therapy.
5f
5g
5h
5i
5j
5k
5l
a Cumulative excretions (two mice for each compound). b BZA values
were obtained from previous work.25
lipophilicity values (log P) of the studied compounds, which
are summarized in Table 2, seem to some extent related to
melanin binding affinity in water. However, the influence of
PBS on the melanin binding differed between compounds, with
no correlation to lipophilicity values.
Pharmacokinetic Profiles. Compound excretion showed a
broad panel of variations (Table 3). First, in terms of rapidity,
although elimination was almost total after 72 h for most of
the compounds (range 90-100%), around 40% of the radioac-
tivity of compound 5g was still present in the mouse body at
this time point. Second, concerning the elimination route, some
compounds exhibited mainly urinary elimination, like BZA.25
This was the case for the remarkable compounds 5h and 5k.
For derivatives 5c, 5d, 5g, and 5i, the major route appeared to
be faecal excretion, while 5e and 5f showed balanced distribu-
tion between the two routes.
Current radionuclide therapy in humans is based almost
exclusively on high-energy ꢀ particle-emitting isotopes44 such
as 32P,89Sr, 90Y,131I, 177Lu, 153Sm,166Ho,186Re, and 188Re. The
most widely used radionuclide is iodine-131 (T1/2 8.02 days),
which emits both a γ ray of 365 keV for diagnostic purposes
and a ꢀ particle of 606 keV for treatment. With a maximum
tissue range of 2-3 mm due to its relatively low ꢀ energy, 131
I
could be used for neoadjuvant or adjuvant treatment of
melanoma metastases. For some of our new compounds, the
biological half-life calculations showed significantly higher
values compared with BZA, leading to high values of tumor
irradiation when extrapolated for 131I use (Table 4). For
compound 5k, an administered dose of 37 MBq will allow the
delivery of 49 Gy to the tumor, i.e., a usefully high dose. As
illustrated in Figure 2, these high affinities for tumor tissue were
particularly selective, given the low amounts of activity in other
major organs after 3 h. For compound 5k, no measurable activity
in liver, lung, or brain tissues was detected at 72 h. Such profiles
offer high tumor/nontarget organ ratios (Table 5).
For all the [125I] labeled compounds evaluated in melanoma-
bearing mice, tissue distribution demonstrated a significant tumor
uptake (Table 4). For all the studied compounds, there was
accumulation in B16 melanoma as from 1 h postinjection (p.i.)
(g4.6% ID/g), with the three compounds 5h, 5k, and 5l,
demonstrating a very high tumor uptake (g17% ID/g). We noted
a similar radioactivity uptake profile in the pigmented structures
of the eyes (uvea, see Supporting Information). These results
were consistent with a melanin binding uptake mechanism
previously described for BZA.21 The compound 5i exhibited
the lowest tumor concentration for short times, but the value
was noticeably constant during the study and remained signifi-
cant after 72 h. Tumor labeling continued to be measurable for
all compounds at this 72 h time point. Compounds 5e, 5f, and
The scintigraphic imaging performed on one mouse after i.v.
injection of 5k is reported in Figure 3. The images illustrate
the specific affinity for the tumor and a rapid clearance from
nontarget organs, allowing a well-defined imaging of the tumor
until 72 h postadministration.
On the basis of these results, compound 5k presented the
most favorable in vivo behavior as a vector for application in
radionuclide therapy, displaying high and long-lasting tumor

3138 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Chezal et al.
Figure 3. In vivo kinetic of compound [125I]5k in a B16 melanoma-bearing mouse illustrated by repeated planar scintigraphic images using a
dedicated γ imager for small animals at 1 h (A), 3 h (B), 6 h (C), 24 h (D), 48 h (E), and 72 h (F) after i.v. injection of a 3 MBq dose (acquisition
time 10 min).
Table 4. Tumor Uptake in B16 Melanoma-bearing Mice of [125I]5a-l Compared with BZA at Various Times after i.v. Injection (%ID/g),a Tumor
Biological Half-Life, and Delivered Dose Calculated for a 131I Labeling
%ID/g of B 16 at different times
tumor parameters
cmpds
1 h
3 h
6 h
24 h
72 h
biological half-life (h)
delivered doseb Gy/MBq
BZAc
5a
5b
5c
5d
5e
9.5 ( 1.9
13.0 ( 2.7
15.5 ( 1.7
8.3 ( 3.1
8.8 ( 3.4
12.5 ( 4.8
5.7 ( 3.0
14.8 ( 4.8
19.6 ( 9.0
4.7 ( 1.2
7.3 ( 2.4
17.0 ( 11.1
23.2 ( 4.3
9.1 ( 1.6
11.4 ( 2.2
9.3 ( 1.5
10.2 ( 1.4
7.5 ( 4.3
10.0 ( 4.3
7.1 ( 1.9
16.1 ( 2.9
25.4 ( 9.6
4.1 ( 0.8
7.1 ( 5.5
27.7 ( 7.0
13.9 ( 2.7
7.7 ( 3.25
9.0 ( 0.72
7.0 ( 0.96
9.8 ( 0.9
7.8 ( 1.8
8.7 ( 4.1
8.1 ( 3.4
9.9 ( 3.8
17.2 ( 4.6
5.8 ( 1.9
7.9 ( 2.2
40.7 ( 7.8
11.4 ( 3.0
3.7 ( 1.2
7.0 ( 3.9
2.7 ( 0.7
8.2 ( 4.4
5.0 ( 0.5
7.3 ( 2.3
8.0 ( 2.4
7.1 ( 2.0
12.1 ( 5.2
2.7 ( 1.1
7.1 ( 2.4
21.7 ( 10.8
8.4 ( 2.1
0.8 ( 0.3
1.5 ( 0.5
0.9 ( 0.4
0.8 ( 0.3
2.0 ( 0.4
1.7 ( 0.5
2.7 ( 1.1
4.5 ( 2.1
5.9 ( 1.7
2.4 ( 0.8
3.1 ( 1.9
12.5 ( 1.6
3.3 ( 1.0
19.6
24.0
19.0
20.1
34.0
26.7
52.6
42.9
38.0
66.3
56.3
66.1
30.2
0.27
0.44
0.42
0.24
0.40
0.46
0.37
0.82
1.00
0.37
0.50
1.33
0.96
5f
5g
5h
5i
5j
5k
5l
a (SD (two mice, n determinations for each compound at each time). b Dosimetry parameter determined for 131I using the MIRD program. c BZA values
were obtained from previous work.25
Table 5. Melanoma/Organ Ratios of [125I]Compound Uptake in B16 Melanoma-Bearing Mice at Different Times After the Injection
compounds
time (h)
BZAa
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
melanoma /liver
melanoma /lung
melanoma /brain
1
3
6
24
72
1.60
2.76
2.94
8.79
1.37
2.40
3.28
3.01
3.08
3.33
0.70
1.96
2.71
5.07
1.38
1.36
2.74
4.41
5.64
7.98
1.43
7.21
1.13
2.26
2.93
1.80
2.85
2.42
5.39
7.26
0.91
2.20
18.16
39.14
1.88
3.52
5.14
1.29
3.12
5.40
5.92
6.10
7.69
11.20
16.40
14.54
1
3
6
24
72
1.72
4.49
7.75
1.10
2.57
4.22
2.04
2.34
2.17
0.58
2.95
5.79
1.72
5.90
13.30
3.38
2.34
2.86
4.31
2.09
4.54
3.23
2.28
4.60
38.73
68.50
1.96
2.58
3.86
2.17
4.87
9.13
9.86
7.41
9.03
22.05
22.89
1
3
6
24
72
6.20
10.97
2.97
14.60
35.80
24.20
42.14
58.08
1.69
3.91
5.38
6.00
31.29
15.40
10.02
30.54
46.51
67.0
nd
72.6
68.5
23.5
24.23
79.11
7.26
26.87
27.14
32.18
a BZA values were obtained from previous work.25 Absence of value: very high ratio, the organ concentration being equal to background value.
concentrations. Compound 5k emerges as the leading candidate
for further development.
images to be obtained promptly after administration. The
quinoline compound 5h and the quinoxaline derivative 5k stood
out due to their specific affinity for the tumor and the highest
and the most long-lasting tumor uptake. They present very
favorable biological half-life values and dosimetry parameters
for clinical application in radionuclide therapy of disseminated
melanoma, for which they appear to be very promising agents.
Conclusion
To discover innovative melanoma-targeting vectors, a dozen
BZA derivatives of varying aromatic ring moieties were
designed, successfully synthesized, and evaluated in terms of
their biodistribution in melanoma-bearing mice. All the com-
pounds in the series exhibited an affinity for melanoma but with
different pharmacokinetic profiles. Compounds 5e and 5l, with
their rapid clearance from nontarget organs, would be good
candidates for scintigraphic imaging, allowing high-contrast
Experimental Section
Materials. Column chromatography was performed with Merck
neutral aluminum oxide 90 standardized (63-200 µm) or silica gel
A normal phase (35-70 µm). Thin layer chromatography was

Analogues of BZA for Imaging and Therapy of Melanoma
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3139
performed on SDS neutral aluminum oxide 60 A F254 plates or
SDS silica gel 60F254 60 A-15 µm plates. The plates were
visualized with UV light (254 nm) and by exposure to iodine vapor.
Melting points were determined on an electrothermal IA9300
(capillary) or a Reichert-Jung-Koffler apparatus and are not
by filtration and washed successively with water (10 mL), methanol
(10 mL), and ether (10 mL) to yield 2-ethoxycarbonylbenzo[b]fu-
ran-5-diazonium borofluoride as a brown precipitate (2.20 g, 7.21
mmol). To a solution of this diazonium salt (2.10 g, 6.88 mmol) in
water (96 mL) was added a solution of potassium iodide (1.15 g,
6.88 mmol) in water (8 mL). The reaction mixture was heated at
50 °C until no more gas was evolved. After cooling to room
temperature, the solution was extracted with CH2Cl2 (3 × 35 mL).
The combined organic extracts were washed with a 5% aqueous
NaHSO3 solution (2 × 75 mL), dried (MgSO4), filtered, and
evaporated under reduced pressure. The residual material was
purified by chromatography (Al2O3, cyclohexane/EtOAc (8/2, v/v))
to give the white solid 6d (1.00 g, 3.16 mmol). Yield: 21%; Rf )
0.69 (Al2O3, cyclohexane/EtOAc (8/2, v/v)); mp 77-79 °C. 1H
NMR (400 MHz, CDCl3) δ: 1.45 (t, 3H, J ) 7.5 Hz), 4.42 (q, 2H,
J ) 7.5 Hz), 7.35 (d, 1H, J ) 9 Hz), 7.46 (s, 1H), 7.70 (dd, 1H, J
) 9, 1.5 Hz), 8.05 (d, 1H, J ) 1.5 Hz). 13C NMR (100 MHz,
CDCl3) δ: 14.3, 61.7, 87.3, 112.5, 114.3, 129.5, 131.5, 136.1, 146.4,
154.9, 159.1. IR (KBr) ν cm-1: 1178, 1735. MS m/z: 316 (M+,
100), 288 (53), 271 (37), 161 (18), 88 (20), 62 (17).
1
corrected. NMR spectra (400 or 200 MHz for H and 100 or 50
MHz for 13C) were recorded on a Bruker Avance 400 or Bruker
AM 200 instrument using CDCl3 or DMSO-d6 as solvent. Infrared
spectra were recorded in KBr pellets or in CCl4 on an FTIR Nicolet
Impact 410 or an FT Vector 22 instrument (ν expressed in cm-1).
Mass spectral analyses were performed on a Hewlett-Packard
5989A instrument. Microanalyses were performed by the Analytical
Laboratory of the CNRS (Vernaison, France) for the elements
indicated and were within 0.4% of the theoretical values unless
indicated. All air-sensitive reactions were run under argon atmo-
sphere. All solvents were dried using common techniques.45
Materials for Radiolabeling and in Vivo Distribution.
[
125I]NaI (3.7 GBq/mL, 644 MBq/µg) for labeling was obtained
from Amersham Int. plc (Little Chalfont, Buckinghamshire, UK).
Extrelut and citrate buffer solution (pH ) 4) were purchased from
Merck (Darmstadt, Germany). The radio TLC strips (Merck neutral
aluminum oxide 60F254 plates) were developed with CH2Cl2/EtOH
(97/3, v/v) and measured on an AMBIS 400 (Scanalytics, CSPI,
San Diego, CA). All radiolabeled compounds were shown by TLC
or HPLC to be identical to the authentic nonradioactive material
and to be free of significant chemical and radiochemical impurities.
The radioactivity contained in the slices was analyzed using an
AMBIS 4000 detector (Scanalytics, CSPI, San Diego, CA), which
is a computer-controlled multiwire proportional counter previously
described and validated for the evaluation of iodinated agents in
mice.46 Scintigraphic imaging of B16 melanoma bearing C57BL6
mice was performed using a dedicated γ IMAGER for small animal
imaging (Biospace, Paris, France). The B16-F0 murine melanoma
was obtained from ATCC (no. CRL-6322) and C57BL6 male mice
from Charles River, L’Arbresle, France.
Ethyl 5-Amino-1(3)H-benzimidazole-2-carboxylate (11).37 The
title compound was synthesized in the same manner as described
for the preparation of amino compound 9. From ethyl 5-nitro-1(3)H-
benzimidazole-2-carboxylate (10),34 reaction time 18 h. The residual
material was purified by chromatography (Al2O3, CH2Cl2/EtOH (97/
3, v/v)) to afford compound 11. Yield: 36%; Rf ) 0.25 (Al2O3,
CH2Cl2/EtOH (97/3, v/v)); mp 147-149 °C. 1H NMR (200 MHz,
CDCl3) δ: 1.41 (t, 3H, J ) 7 Hz), 4.46 (q, 2H, J ) 7 Hz), 6.76 (m,
2H), 7.58 (d, 1H, J ) 9 Hz). IR (KBr) ν cm-1: 1269, 1701, 3370.
MS m/z: 205 (M+, 53), 159 (100), 133 (52), 131 (44), 105 (35),
83 (22), 78 (20), 52 (21).
Ethyl 5-Iodo-1(3)H-benzimidazole-2-carboxylate (6g). To a
solution of ethyl 5-amino-1(3)H-benzimidazole-2-carboxylate (11)37
in 50% aqueous fluoroboric acid solution (40 mL), at 0 °C, was
added dropwise, a solution of sodium nitrite (750 mg, 11.0 mmol)
in water (3 mL). The reaction mixture was stirred for further hour
at 0 °C and filtered. The corresponding diazonium borofluoride (3.26
g, 10.7 mmol) was dissolved in water (67 mL) and a solution of
potassium iodide (2.79 g, 16.8 mmol) in water (14 mL) was added.
The reaction mixture was heated at 70 °C until no more gas was
evolved (2 h). After cooling to room temperature, the mixture was
made alkaline with a saturated aqueous Na2CO3 solution (pH )
8-9) and extracted with CH2Cl2 (2 × 80 mL). The combined
organic layers were washed with a 5% aqueous NaHSO3 solution
(2 × 40 mL), dried (MgSO4), filtered, and concentrated under
Vacuum. The brown residue was purified by chromatography
(Al2O3, CH2Cl2/EtOH (99/1, v/v)) to give iodo compound 6g (407
mg, 1.29 mmol). Yield: 12%; Rf ) 0.57 (Al2O3, CH2Cl2/EtOH (99/1
Ethyl 6-Iodonicotinate (6b).47 To a solution of 6-iodonicotinic
acid32 (7) (250 mg, 1.00 mmol) in dry CH2Cl2 (25 mL), was added
successively, at 0 °C, triethylamine (167 µL, 1.19 mmol), ethyl
chloroformate (0.70 mL, 7.32 mmol), and 4-dimethylaminopyridine
(DMAP) (0.45 g, 3.68 mmol). After cooling to room temperature,
the reaction mixture was refluxed for 2 h. Evaporation of the solvent
followed by chromatography (Al2O3, CH2Cl2) gave ester 6b (211
mg, 0.76 mmol). Yield: 76%; Rf ) 0.92 (Al2O3, CH2Cl2); mp 46-48
1
°C. H NMR (200 MHz, CDCl3) δ: 1.32 (t, 3H, J ) 3 Hz), 4.32
(q, 2H, J ) 7 Hz), 7.78 (m, 2H), 8.82 (d, 1H, J ) 2 Hz). 13C
NMR (50 MHz, CDCl3) δ: 14.2, 61.6, 123.3, 125.7, 134.7, 138.0,
151.4, 164.7. IR (KBr) ν cm-1: 1268, 1292, 1577, 1711, 3082.
MS m/z: 277 (M+, 61), 232 (21), 204 (20), 150 (100), 127 (16),
77 (43), 51 (22).
1
Ethyl 5-Aminobenzo[b]furan-2-carboxylate (9).36 A solution
of ethyl 5-nitrobenzo[b]furan-2-carboxylate (8) (3.92 g, 16.7 mmol)
in ethyl acetate (320 mL) was hydrogenated (1 atm) at room
temperature over 10% Pd/C (395 mg) for 16 h. The black
suspension was filtered through celite 521 and the filtrate was
concentrated under reduced pressure to give amino compound 9
(3.25 g, 15.9 mmol), which was used without further purification.
Yield: 95%; Rf ) 0.78 (Al2O3, CH2Cl2/EtOH (97/3, v/v)); mp
v/v)); mp 166-168 °C. H NMR (200 MHz, DMSO-d6) δ: 1.40
(t, 3H, J ) 7 Hz), 4.50 (q, 2H, J ) 7 Hz), 7.51 (d, 1H, J ) 9 Hz),
7.66 (dd, 1H, J ) 9, 2 Hz), 8.09 (d, 1H, J ) 2 Hz). IR (KBr) ν
cm-1: 1246, 1310, 1515, 1697, 3287. MS m/z: 316 (M+, 56), 270
(30), 244 (100), 117 (38), 90 (25), 63 (19).
2-Tribromomethyl-6-iodoquinoline (13). 6-Iodo-2-methylquin-
oline39 (12) (7.12 g, 26.5 mmol) and sodium acetate (12.1 g) were
dissolved in acetic acid (15 mL). The reaction mixture was heated
at 75 °C for 10 min and a solution of bromine (4.45 mL, 86.6 mmol)
in acetic acid (13 mL) was added dropwise. After the addition, the
solution was refluxed for 2 h. The mixture was poured onto ice
(200 g) and stirred for 18 h. The solid product was filtered, washed
with water (60 mL), and dried to yield compound 13, which was
used without purification (9.10 g, 18.0 mmol). Yield: 68%; Rf )
1
68-70 °C (literature:36 59-60 °C). H NMR (400 MHz, CDCl3)
δ: 1.42 (t, 3H, J ) 7 Hz), 3.70 (m, 2H), 4.43 (q, 2H, J ) 7 Hz),
6.84 (dd, 1H, J ) 9, 2.5 Hz), 6.89 (d, 1H, J ) 2.5 Hz), 7.37 (m,
2H). 13C NMR (100 MHz, CDCl3) δ: 14.3, 61.4, 106.2, 112.7,
113.3, 117.5, 127.8, 142.9, 145.9, 150.3, 159.2. IR (KBr) ν cm-1
:
1225, 1559, 1724. MS m/z: 205 (M+, 87), 177 (100), 160 (19),
133 (24), 104 (44), 77 (23), 52 (22), 51 (23).
1
0.86 (Al2O3, cyclohexane/EtOAc (8/2, v/v); mp 124-126 °C. H
NMR (400 MHz, CDCl3) δ: 7.92 (d, 1H, J ) 8.5 Hz), 8.03 (d, 1H,
J ) 8.5 Hz), 8.14 (d, 1H, J ) 9 Hz), 8.26 (m, 2H). 13C NMR (100
MHz, CDCl3) δ: 40.8, 94.5, 118.6, 129.1, 131.7, 135.9, 136.4,
139.3, 144.0, 159.0. IR (KBr) ν cm-1: 1293, 1480, 1584. MS m/z:
509 (M++6, 1), 507 (M++4, 2), 505 (M++2, 3), 503 (M+, 1),
428 (40), 426 (77), 424 (45), 348 (81), 346 (81), 255 (100), 140
(97), 127 (90), 82 (90), 57 (89).
Ethyl 5-Iodobenzo[b]furan-2-carboxylate (6d). Ethyl 5-ami-
nobenzo[b]furan-2-carboxylate36 (9) (3.25 g, 15.9 mmol) was
suspended in water (24 mL) and concentrated hydrochloric acid
was added (3.7 mL). The mixture was cooled to 0 °C, and diazotized
by slow addition of sodium nitrite (1.07 g, 15.9 mmol) in water
(17 mL). The solution was stirred for 0.5 h at 0 °C and a solution
of 50% fluoroboric acid (2 mL) was added. The solid was removed

3140 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Chezal et al.
Ethyl 6-Iodoquinoline-2-carboxylate (6h). A solution of silver
nitrate (9.05 g, 53.8 mmol) in water (83 mL) was added carefully
with stirring to a solution of tribromo compound 13 (9.10 g, 18.0
mmol) in EtOH (132 mL). The mixture was refluxed for 30 min
and then allowed to stand at room temperature, filtered, and acidified
with aqueous hydrochloric acid (1.5 M, 5 mL). The resulting
solution was concentrated under vacuum to 100 mL. The solution
was made alkaline with a saturated aqueous Na2CO3 solution (100
mL), extracted with ether (3 × 30 mL), dried (MgSO4), and
evaporated. The crude product was purified by chromatography
(Al2O3, EtOAc/cyclohexane (8/2, v/v)) to give ester 6h (3.96 g,
12.1 mmol). Yield: 67%; Rf ) 0.97 (Al2O3, EtOAc/cyclohexane
(8/2, v/v)); mp 119-121 °C. 1H NMR (200 MHz, CDCl3) δ: 1.42
(t, 3H, J ) 7 Hz), 4.46 (q, 2H, J ) 7 Hz), 7.97 (d, 1H, J ) 9 Hz),
8.16 (m, 2H), 8.55 (d, 1H, J ) 9 Hz), 8.64 (s, 1H). 13C NMR (100
MHz, CDCl3) δ: 14.4, 62.4, 95.0, 121.8, 130.7, 132.2, 135.6, 136.4,
139.1, 146.4, 148.7, 165.0. IR (KBr) ν cm-1: 1307, 1714. MS m/z:
327 (M+, 17), 283 (19), 255 (100), 127 (28), 100 (11).
Methyl 5-Iodoisoquinoline-3-carboxylate (6j). Methyl 5-ami-
noisoquinoline-3-carboxylate40 (14) (500 mg, 2.47 mmol) was
suspended in water (4 mL) and concentrated hydrochloric acid was
added (576 µL). The mixture was cooled to 0 °C and diazotized
by slow addition of sodium nitrite (166 mg, 2.47 mmol) in water
(3 mL). The solution was stirred for 0.5 h at 0 °C and a solution of
50% fluoroboric acid (311 µL) was added. The solid was removed
by filtration, dissolved in water (12 mL), and a solution of potassium
iodide (453 mg, 3.10 mmol) in water (5 mL) was added. The
reaction was heated at 80 °C until no more gas was evolved (2 h).
After cooling to room temperature, the mixture was extracted with
CH2Cl2 (2 × 80 mL). The combined organic layers were washed
with a 5% aqueous NaHSO3 solution (40 mL), dried (MgSO4),
filtered, and concentrated under vacuum. The residue was purified
by chromatography (Al2O3, CH2Cl2/EtOH (99/1, v/v)) to give
compound 6j (190 mg, 0.61mmol). Yield: 25%; Rf ) 0.64 (Al2O3,
CH2Cl2/EtOH (99/1, v/v)); mp 165-167 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 4.00 (s, 3H), 7.65 (t, 1H, J ) 8 Hz), 8.32 (d, 1H, J
) 8 Hz), 8.50 (d, 1H, J ) 8 Hz), 8.55 (s, 1H), 9.37 (s, 1H). 13C
NMR (100 MHz, DMSO-d6) δ: 52.6, 99.6, 126.1, 128.5, 130.5,
131.3, 136.4, 142.2, 142.8, 153.8, 165.2. IR (KBr) ν cm-1: 1249,
1304, 1710. MS m/z: 313 (M+, 22), 283 (20), 255 (100), 127 (41),
100 (18), 74 (11).
Ethyl 6-Nitroquinoxaline-2-carboxylate (16). To a solution of
6-nitroquinoxaline-2-carboxylic acid41 (15) (5.00 g, 22.8 mmol)
in dry EtOH (50 mL) was added, under argon, concentrated sulfuric
acid (750 µL). After stirring at reflux for 7 h, the reaction mixture
was cooled to room temperature and a saturated aqueous Na2CO3
solution (50 mL) was added. The solution was extracted with
CH2Cl2 (3 × 30 mL) and the combined organic layers were dried
(MgSO4), filtered, and evaporated to dryness to give ester 16 (3.79
g, 15.3 mmol). Yield: 67%; mp 221-223 °C. 1H NMR (200 MHz,
CDCl3) δ: 1.58 (t, 3H, J ) 7 Hz), 4.68 (q, 2H, J ) 7 Hz), 8.53 (d,
1H, J ) 9 Hz), 8.68 (dd, 1H, J ) 2.5, 9 Hz), 9.14 (d, 1H, J ) 2.5
Hz), 9.72 (s, 1H). IR (KBr) ν cm-1: 1282, 1347, 1531, 1741. MS
m/z: 248 (M++1, 4), 203 (36), 175 (100), 128 (23), 101 (32), 75
(24).
Ethyl 6-Aminoquinoxaline-2-carboxylate (17). To a flask
containing the nitro compound 16 (2.93 g, 11.9 mmol) in EtOH
(500 mL) was added 10% Pd/C (300 mg). The mixture was
degassed and stirred under a H2 atmosphere for 3 h 45. The catalyst
was removed by filtration through celite 545, washed with EtOH
(50 mL), and the filtrate was evaporated. The resulting crude product
was purified by chromatography (Al2O3, CH2Cl2/EtOH (99/1, v/v))
to give the title product 17 (1.69 g, 7.79 mmol). Yield: 66%; Rf )
0.55 (Al2O3, CH2Cl2/EtOH (99/1, v/v)); mp 181-183 °C. 1H NMR
(200 MHz, CDCl3) δ: 1.49 (t, 3H, J ) 7 Hz), 4.38 (m, 2H), 4.56
(q, 2H, J ) 7 Hz), 7.16 (d, 1H, J ) 2.5 Hz), 7.26 (dd, 1H, J ) 2.5,
9 Hz), 8.05 (d, 1H, J ) 9 Hz), 9.35 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ: 14.4, 62.1, 106.9, 123.1, 131.9, 136.6, 138.4, 145.6,
145.9, 150.4, 164.7. IR (KBr) ν cm-1: 1299, 1487, 1613, 1701,
3202, 3431. MS m/z: 217 (M+, 23), 145 (100), 117 (19), 90 (16),
63 (14).
Ethyl 6-Iodoquinoxaline-2-carboxylate (6k). To a mixture of
ethyl 6-aminoquinoxaline-2-carboxylate (17) (1.37 g, 6.31 mmol)
in 50% aqueous fluoroboric acid solution (10 mL), at 0 °C, was
added dropwise a solution of sodium nitrite (480 mg, 6.96 mmol)
in water (3 mL). The reaction mixture was stirred for further hour
at 0 °C and a solution of potassium iodide (1.57 g, 9.46 mmol) in
water (5 mL) was added. The reaction was stirred at 0 °C for 1 h
and then warmed at 50 °C until no more gas was evolved (1 h).
After cooling to room temperature, the mixture was made alkaline
with a saturated aqueous Na2CO3 solution (60 mL) and extracted
with CH2Cl2 (3 × 40 mL). The combined organic layers were
washed with a 5% aqueous NaHSO3 solution (2 × 30 mL), dried
(MgSO4), filtered, and concentrated under vacuum. The crude
product was purified by chromatography (Al2O3, CH2Cl2) to give
iodo compound 6k (0.68 g, 2.07 mmol). Yield: 33%; Rf ) 0.80
1
(Al2O3, CH2Cl2); mp 160-162 °C. H NMR (400 MHz, CDCl3)
δ: 1.51 (t, 3H, J ) 7 Hz), 4.59 (q, 2H, J ) 7 Hz), 7.99 (d, 1H, J
) 9 Hz), 8.10 (dd, 1H, J ) 2, 9 Hz), 8.62 (d, 1H, J ) 2 Hz), 9.51
(s, 1H). 13C NMR (100 MHz, CDCl3) δ: 14.3, 62.7, 99.2, 131.6,
138.5, 140.0, 140.7, 143.0, 144.1, 145.7, 163.9. IR (KBr) ν cm-1
:
1103, 1151, 1230, 1237, 1717. MS m/z: 328 (M+, 19), 284 (43),
256 (100), 128 (29), 101 (50), 75 (30).
Ethyl 1,6-Naphthyridine-2-carboxylate (19). Compound 19
was prepared according to the procedure developed for ester 6b
from 1,6-naphthyridine-2-carboxylic acid42 (18) using 3.8 equiv
of triethylamine, 6.8 equiv of ethyl chloroformiate, and 3.4 equiv
of DMAP. The reaction mixture was refluxed for 5 h. After
evaporation of the solvent, the crude product was purified by
chromatography (Al2O3, EtOAc) to give ester 19. Yield: 45%; Rf
1
) 0.86 (Al2O3, EtOAc); mp 106-108 °C. H NMR (400 MHz,
CDCl3) δ: 1.41 (t, 3H, J ) 7 Hz), 4.49 (q, 2H, J ) 7 Hz), 8.02 (d,
1H, J ) 6 Hz), 8.21 (d, 1H, J ) 8.5 Hz), 8.39 (d, 1H, J ) 8.5 Hz),
8.75 (d, 1H, J ) 6 Hz), 9.29 (s, 1H). 13C NMR (100 MHz, CDCl3)
δ: 14.3, 62.0, 122.4, 122.7, 124.0, 137.2, 147.5, 149.7, 152.3, 152.9,
164.6. IR (KBr) ν cm-1: 1256, 1734. MS m/z: 202 (M+, 3), 158
(18), 130 (100), 102 (15), 75 (22), 51 (16).
Ethyl 8-Iodo-1,6-naphthyridine-2-carboxylate (6l). To a solu-
tion of ester 19 (517 mg, 2.56 mmoles) in THF (50 mL) were added,
at -10 °C, N-iodosuccinimide (691 mg, 3.07 mmoles), and
p-toluenesulfonic acid (176 mg, 1.02 mmol). The mixture was
stirred at -10 °C for 10 min and then at room temperature for
24 h. More N-iodosuccinimide (576 mg, 2.56 mmoles) was added
and the solution was stirred for a further 21 h at room temperature.
The solvent was evaporated under reduced pressure and the crude
product was dissolved in saturated aqueous Na2CO3 solution (50
mL) and extracted with EtOAc (3 × 20 mL). The organic layers
were dried (MgSO4), filtered, and evaporated to dryness. The residue
was purified by chromatography (Al2O3, EtOAc/cyclohexane (7/3,
v/v)) to afford iodoester 6l (527 mg, 1.61 mmol). Yield: 63%; Rf
) 0.83 (Al2O3, EtOAc/cyclohexane (7/3, v/v)); mp 135-137 °C.
1H NMR (200 MHz, CDCl3) δ: 1.51 (t, 3H, J ) 7 Hz), 4.55 (q,
2H, J ) 7 Hz), 8.32 (d, 1H, J ) 8 Hz), 8.39 (d, 1H, J ) 8 Hz),
9.23 (s, 1H), 9.28 (s, 1H). 13C NMR (100 MHz, CDCl3) δ: 14.2,
62.7, 100.9, 123.4, 125.2, 137.9, 149.3, 152.8, 153.2, 155.2, 164.3.
IR (CCl4) ν cm-1: 1126, 1274, 1742. MS m/z: 328 (M+, 18), 284
(18), 256 (100), 228 (8), 129 (29), 101 (52), 75 (25).
General Procedure for Preparation of Amides 20a-l. Trim-
ethylaluminium (2 M in toluene) (7.2 mL, 14.4 mmol) was added
dropwise at 0 °C to a solution of N,N-diethylethylenediamine (d )
0.827, 2 mL, 14.1 mmol) in dry methylene chloride (110 mL). After
addition of the appropriate ester 6 (10 mmol) in dry methylene
chloride (88 mL), the mixture was refluxed for the reported times
and monitored by TLC. After cooling to room temperature, water
(130 mL) was added and the mixture was extracted with CH2Cl2
(3 × 100 mL). The organic layers were dried (MgSO4) and
concentrated under vacuum. The crude product was chromato-
graphed (Al2O3, CH2Cl2/EtOH, 97/3) to give amides 20.
N-(2-Diethylaminoethyl)-6-iodo-2-naphthamide (20a). From
methyl 6-iodo-2-naphthoate (6a),27 reaction time: 16 h. Yield: 40%;
Rf ) 0.58 (Al2O3, CH2Cl2/EtOH (97/3, v/v)); mp 105-107 °C. 1H
NMR (400 MHz, CDCl3) δ: 1.05 (t, 6H, J ) 7 Hz), 2.58 (q, 4H,

Analogues of BZA for Imaging and Therapy of Melanoma
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3141
J ) 7 Hz), 2.68 (t, 2H, J ) 6 Hz), 3.53 (q, 2H, J ) 6 Hz), 7.40 (m,
1H), 7.54 (d, 1H, J ) 8.5 Hz), 7.68 (d, 2H, 8.5 Hz), 7.83 (d, 1H,
J ) 8.5 Hz), 8.18 (s, 1H), 8.23 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ: 11.8 (2C), 37.3, 46.6 (2C), 51.1, 93.6, 124.4, 127.0,
127.3, 130.2, 131.1, 132.3, 135.0, 135.7, 136.2, 166.9. IR (KBr) ν
cm-1: 1537, 1614, 1630, 3303. MS m/z: 281 (M+ -115, 6), 126
(9), 86 (100), 58 (14).
1H, J ) 8.5 Hz), 8.26 (d, 1H, J ) 2 Hz), 8.30 (d, 1H, J ) 8.5 Hz),
8.57 (brs, 1H). 13C NMR (100 MHz, CDCl3) δ: 12.1 (2C), 37.5,
47.2 (2C), 51.7, 93.7, 119.6, 130.7, 131.8, 136.1, 136.5, 139.4,
145.4, 150.5, 164.1. IR (KBr) ν cm-1: 1526, 1663, 2963, 3400.
MS m/z: 386 (M+, 1), 86 (100), 58 (11).
N-(2-Diethylaminoethyl)-1,4-dihydro-6-iodo-4-oxoquinoline-3-
carboxamide (20i). From ethyl 1,4-dihydro-6-iodo-4-oxoquinoline-
3-carboxylate (6i),31 reaction time: 5 h. Yield: 81%; Rf ) 0.04
(Al2O3, CH2Cl2/EtOH (97/3, v/v)); mp 234-236 °C. 1H NMR (400
MHz, DMSO-d6) δ: 1.13 (t, 6H, J ) 7 Hz), 2.85 (m, 6H), 3.69 (q,
2H, J ) 6 Hz), 7.33 (d, 1H, J ) 8.5 Hz), 7.87 (d, 1H, J ) 8.5 Hz),
8.49 (s, 1H), 8.67 (s, 1H), 10.32 (brs, 1H). 13C NMR (100 MHz,
DMSO-d6) δ: 11.4 (2C), 36.4, 46.6 (2C), 51.4, 89.6, 111.3, 121.5,
N-(2-Diethylaminoethyl)-6-iodonicotinamide (20b). From ethyl
6-iodonicotinate (6b), reaction time: 20 h. Yield: 98%; Rf ) 0.60
1
(Al2O3, CH2Cl2/EtOH (97/3, v/v)) as an oil. H NMR (200 MHz,
CDCl3) δ 0.83 (t, 6H, J ) 7 Hz), 2.37 (q, 4H, J ) 7 Hz), 2.47 (t,
2H, J ) 6 Hz), 3.28 (q, 2H, J ) 6 Hz), 7.38 (m, 1H), 7.78 (m,
2H), 8.73 (m, 1H). 13C NMR (50 MHz, CDCl3) δ: 11.7 (2C), 37.2,
46.4 (2C), 50.8, 120.9, 129.5, 134.5, 136.1, 148.6, 164.4. IR (CCl4)
ν cm-1: 1538, 1630, 2927, 2965, 3301. MS m/z: 348 (M+ +1, 1),
86 (100), 77 (8), 58 (16).
127.9, 133.8, 138.7, 140.5, 144.0, 164.2, 174.4. IR (KBr) ν cm-1
:
1507, 1551, 1633, 2965, 3061. MS m/z: 413 (M+, 1), 86 (100), 58
(10).
N-(2-Diethylaminoethyl)-5-iodoindole-2-carboxamide (20c). From
ethyl 5-iodoindole-2-carboxylate (6c),28 reaction time: 3 h. Yield:
50%; mp 208-210 °C; Rf ) 0.53 (Al2O3, CH2Cl2/EtOH (97/3, v/v)).
1H NMR (200 MHz, DMSO-d6) δ: 1.03 (t, 6H, J ) 7 Hz), 2.57
(m, 6H), 3.37 (m, 2H), 7.11 (s, 1H), 7.42 (AB system, 2H, J ) 8.5
Hz), 8.09 (s, 1H), 8.52 (t, 1H, J ) 6 Hz), 11.82 (s, 1H). 13C NMR
(50 MHz, DMSO-d6) δ: 11.9 (2C), 37.3, 46.8 (2C), 51.5, 83.3,
101.2, 114.7, 129.8, 129.9, 131.0, 132.8, 135.3, 160.6. IR (KBr) ν
cm-1: 1411, 1547, 1635, 2801, 2965, 3250, 3418. MS m/z: 385
(M+, 2), 86 (100), 58 (2).
N-(2-Diethylaminoethyl)-5-iodoisoquinoline-3-carboxamide (20j).
From methyl 5-iodoisoquinoline-3-carboxylate (6j), reaction
time: 4 h. Yield: 72%; Rf ) 0.47 (Al2O3, CH2Cl2/EtOH (97/3,
1
v/v)) as an oil. H NMR (400 MHz, CDCl3) δ: 1.08 (t, 6H, J )
7 Hz), 2.64 (q, 4H, J ) 7 Hz), 2.74 (t, 2H, J ) 7 Hz), 3.62 (q,
2H, J ) 7 Hz), 7.40 (t, 1H, J ) 8 Hz), 8.00 (d, 1H, J ) 8 Hz),
8.31 (d, 1H, J ) 8 Hz), 8.57 (m, 1H), 8.76 (s, 1H), 9.05 (s,
1H). 13C NMR (100 MHz, CDCl3) δ: 11.9 (2C), 37.5, 47.3 (2C),
51.8, 99.4, 123.8, 128.3, 129.7, 130.6, 138.1, 142.0, 145.8, 152.0,
164.5. IR (KBr) ν cm-1: 1516, 1677, 2971. MS m/z: 397 (M+,
1), 86 (100), 58 (7).
N-(2-Diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide (20k).
From ethyl 6-iodoquinoxaline-2-carboxylate (6k), reaction time: 7 h.
Yield: 77%; Rf ) 0.64 (Al2O3, CH2Cl2/EtOH (97/3, v/v)); mp
60-62 °C. 1H NMR (400 MHz, CDCl3) δ: 1.00 (t, 6H, J ) 7 Hz),
2.54 (q, 4H, J ) 7 Hz), 2.64 (t, 2H J ) 6 Hz), 3.49 (q, 2H, J ) 6
Hz), 7.71 (d, 1H, J ) 9 Hz), 7.96 (dd, 1H, J ) 2, 9 Hz), 8.31 (m,
1H), 8.49 (d, 1H, J ) 2 Hz), 9.55 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ: 12.1 (2C), 37.4, 47.1 (2C), 51.5, 97.9, 130.8, 138.5,
139.5, 139.6, 144.1, 144.3, 144.6, 162.9. IR (KBr) ν cm-1: 1517,
1661, 3397. MS m/z: 398 (M+, 1), 86 (100), 58 (15).
N-(2-Diethylaminoethyl)-8-iodo-1,6-naphthyridine-2-carboxam-
ide (20l). From ethyl 8-iodo-1,6-naphthyridine-2-carboxylate (6l),
reaction time: 3 h. The crude product was purified by chromatog-
raphy (Al2O3, EtOAc). Yield: 73%; Rf ) 0.67 (Al2O3, EtOAc); mp
44-46 °C. 1H NMR (200 MHz, CDCl3) δ: 1.07 (t, 6H, J ) 7 Hz),
2.60 (q, 4H, J ) 7 Hz), 2.70 (t, 2H, J ) 6 Hz), 3.57 (q, 2H, J )
6 Hz), 8.35 (d, 1H, J ) 8.5 Hz), 8.44 (d, 1H, J ) 8.5 Hz), 8.83 (m,
1H), 9.17 (s, 1H), 9.19 (s, 1H). 13C NMR (50 MHz, CDCl3) δ:
12.3 (2C), 37.4, 46.9 (2C), 51.4, 100.2, 121.5, 125.3, 138.1, 148.1,
152.8, 154.7, 154.8, 162.8. IR (KBr) ν cm-1: 1460, 1516, 1689,
2801, 2926, 2965, 3300-3400. MS m/z: 398 (M+, 1), 86 (100),
58 (15).
General Procedure for Preparation of Hydrochloride salts
5a-k. To a stirred solution of the appropriate amide (2.52 mmol)
in dry methylene chloride (22 mL), under argon, was added a
solution of 2N anhydrous hydrochloric acid in ether (22 mL). The
solution was stirred at room temperature for 10 min and evaporated
under vacuum. The residue was taken up in anhydrous ether (50
mL) and stirred under argon for 24 h. The resulting precipitate was
filtered to give hydrochloride salt 5.
N-(2-Diethylaminoethyl)-5-iodobenzo[b]furan-2-carboxamide
(20d). From ethyl 5-iodobenzo[b]furan-2-carboxylate (6d), reaction
time: 14 h. Yield: 81%; Rf ) 0.65 (Al2O3, CH2Cl2/EtOH (97/3,
1
v/v)) as an oil. H NMR (200 MHz, CDCl3) δ 1.06 (t, 6H, J ) 7
Hz), 2.59 (q, 4H, J ) 7 Hz), 2.66 (m, 2H), 3.49 (q, 2H, J ) 7 Hz),
7.29 (d, 1H, J ) 8 Hz), 7.36 (s, 1H), 7.67 (dd, 1H, J ) 8, 2 Hz),
8.01 (d, 1H, J ) 2 Hz). 13C NMR (100 MHz, CDCl3) δ: 10.8 (2C),
35.9, 45.8 (2C), 50.2, 86.0, 107.8, 112.7, 129.1, 130.2, 134.1, 148.7,
152.8, 157.2. IR (CCl4) ν cm-1: 1501, 1675, 2972. MS m/z: 386
(M+, 1), 271 (5), 86 (100), 58 (10).
N-(2-Diethylaminoethyl)-4-iodobenzo[b]thiophene-2-carboxam-
ide (20e). From methyl 4-iodobenzo[b]thiophene-2-carboxylate
(6e),29 reaction time: 24 h. Yield: 72%; Rf ) 0.58 (Al2O3, CH2Cl2/
EtOH (97/3, v/v)); mp 76-78 °C. 1H NMR (400 MHz, CDCl3) δ:
1.09 (t, 6H, J ) 7 Hz), 2.60 (q, 4H, J ) 7 Hz), 2.68 (t, 2H, J ) 7
Hz), 3.50 (q, 2H, J ) 7 Hz), 7.08 (brs, 1H), 7.11 (t, 1H, J ) 8
Hz), 7.80 (m, 3H). 13C NMR (100 MHz, CDCl3) δ: 12.2 (2C),
37.5, 46.9 (2C), 51.1, 91.4, 122.7, 127.2, 128.4, 134.9, 139.5, 140.0,
142.2, 161.7. IR (KBr) ν cm-1: 1560, 1625, 2963, 3287. MS m/z:
402 (M+, 1), 86 (100), 58 (7).
N-(2-Diethylaminoethyl)-6-iodoimidazo[1,2-a]pyridine-2-car-
boxamide (20f). From ethyl 6-iodoimidazo[1,2-A-a]pyridine-2-
carboxylate (6f),30 reaction time: 72 h. Yield: 83%; Rf ) 0.55
1
(Al2O3, CH2Cl2/EtOH (97/3, v/v)) as an oil. H NMR (200 MHz,
CDCl3) δ: 1.03 (t, 6H, J ) 7 Hz), 2.57 (q, 4H, J ) 7 Hz), 2.66 (t,
2H, J ) 6.5 Hz), 3.51 (q, 2H, J ) 6.5 Hz), 7.34 (m, 2H), 7.68 (m,
1H), 8.06 (s, 1H), 8.39 (t, 1H, J ) 1.5 Hz). 13C NMR (50 MHz,
CDCl3) δ: 12.2 (2C), 37.3, 47.2 (2C), 51.9, 76.5, 113.4, 118.9,
130.9, 133.5, 140.2, 142.6, 161.8. IR (CCl4) ν cm-1: 1218, 1251,
1562, 1670, 2971, 3430. MS m/z: 99 (M+ -287, 11), 86 (100),
58 (11).
N-(2-Diethylaminoethyl)-5-iodo-1(3)H-benzimidazole-2-carboxa-
mide (20g). From ethyl 5-iodo-1(3)H-benzimidazole-2-carboxylate
(6g), reaction time: 6 h. Yield: 82%; Rf ) 0.38 (Al2O3, CH2Cl2/
EtOH (97/3, v/v)); mp 136-138 °C. 1H NMR (200 MHz, CDCl3)
δ: 1.04 (t, 6H, J ) 7 Hz), 2.64 (q, 4H, J ) 7 Hz), 2.76 (m, 2H),
3.62 (m, 2H), 7.36 (d, 1H, J ) 8.5 Hz), 7.52 (d, 1H, J ) 8.5 Hz),
7.96 (brs, 1H), 8.28 (m, 1H). IR ν cm-1: 1420, 1551, 1664, 2966,
3175. MS m/z: 386 (M+, 1), 86 (100), 58 (11).
N-(2-Diethylaminoethyl)-6-iodoquinoline-2-carboxamide (20h).
From ethyl 6-iodoquinoline-2-carboxylate (6h), reaction time: 6 h.
Yield: 69%; Rf ) 0.56 (Al2O3, CH2Cl2/EtOH (97/3, v/v)); mp
75-77 °C. 1H NMR (400 MHz, CDCl3) δ: 1.09 (t, 6H, J ) 7 Hz),
2.63 (q, 4H, J ) 7 Hz), 2.73 (t, 2H, J ) 7 Hz), 3.58 (q, 2H, J )
7 Hz), 7.81 (d, 1H, J ) 9 Hz), 7.98 (dd, 1H, J ) 9, 2 Hz), 8.16 (d,
N-(2-Diethylaminoethyl)-6-iodo-2-naphthamide, hydrochloride
salt (5a). From N-(2-diethylaminoethyl)-6-iodo-2-naphthamide
(20a). Yield: 94%; mp 151-152 °C. 1H NMR (200 MHz, DMSO-
d6) δ: 1.22 (t, 6H, J ) 7 Hz), 3.19 (q, 4H, J ) 7 Hz), 3.44 (m,
4H), 7.83 (m, 2H), 7.97 (m, 2H), 8.47 (m, 2H), 9.03 (m, 1H, NH),
9.77 (m, 1H). IR (KBr) ν cm-1: 1297, 1533, 1655, 3252. Anal.
(C17H21IN2O, HCl, H2O) C, H, N.
N-(2-Diethylaminoethyl)-6-iodonicotinamide, dihydrochloride
salt (5b). From N-(2-diethylaminoethyl)-6-iodonicotinamide
(20b). Yield: 70%; mp 127-129 °C. 1H NMR (200 MHz,
DMSO-d6) δ: 1.26 (t, 6H, J ) 7 Hz), 3.25 (m, 6H), 3.68 (m,
4H), 8.00 (m, 2H), 8.87 (m, 1H), 9.25 (m, 1H), 10.24 (m, 1H).
IR (KBr) ν cm-1: 1278, 1529, 1588, 1661, 2661, 3232. Anal.
(C12H18IN3O, 2HCl) C, H, N.

3142 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Chezal et al.
N-(2-Diethylaminoethyl)-5-iodoindole-2-carboxamide, hydro-
chloride salt (5c). From N-(2-diethylaminoethyl)-5-iodoindole-2-
carboxamide (20c). Yield: 81%; mp 217-219 °C. H NMR (400
MHz, CDCl3) δ: 1.29 (m, 6H), 3.28 (m, 6H), 3.72 (m, 2H), 7.22
(s, 1H), 7.31 (d, 1H, J ) 8.5 Hz), 7.48 (d, 1H, J ) 8.5 Hz), 8.06
(s, 1H), 9.16 (brs, 1H), 10.59 (brs, 1H), 11.94 (s, 1H). IR (KBr) ν
cm-1: 1544, 1646, 2468, 2569, 3228. Anal. (C15H20IN3O, HCl) H,
N. C: calcd, 42.72; found, 43.47.
N-(2-Diethylaminoethyl)-8-iodo-1,6-naphthyridine-2-carboxam-
ide, dihydrochloride salt (5l). From N-(2-diethylaminoethyl)-8-
iodonaphthyridine-2-carboxamide (20l). Yield: 72%; mp 215-217
°C. 1H NMR (200 MHz, DMSO-d6) δ: 1.27 (t, 6H, J ) 7 Hz), 3.25
(m, 6H), 3.82 (m, 2H), 8.34 (d, 1H, J ) 8.5 Hz), 8.81 (d, 1H, J ) 8.5
Hz), 8.95 (m, 1H), 9.29 (s, 1H), 9.29 (s, 1H), 10.75 (brs, 1H). IR (KBr)
ν cm-1: 1450, 1528, 1624, 1681, 2043, 2240-2750, 2939, 3045,
3250-3550. Anal. (C15H19IN4O, 2HCl, 1.5H2O): C, H, N.
1
N-(2-Diethylaminoethyl)-5-iodobenzo[b]furan-2-carboxamide, hy-
drochloride salt (5d). From N-(2-diethylaminoethyl)-5-iodoben-
zo[b]furan-2-carboxamide (20d). Yield: 81%; mp 207-209 °C. 1H
NMR (200 MHz, DMSO-d6) δ: 1.27 (t, 6H, J ) 7 Hz), 3.26 (m,
6H), 3.70 (m, 2H), 7.56 (d, 1H, J ) 9 Hz), 7.61 (s, 1H), 7.78
(d, 1H, J ) 9 Hz), 8.24 (s, 1H), 9.16 (brs, 1H), 10.0 (m, 1H).
IR (KBr) ν cm-1: 1560, 1670, 2586, 3209. Anal. (C15H19IN2O2,
HCl) C, H, N.
N-(2-Diethylaminoethyl)-4-iodobenzothiophene-2-carboxam-
ide, hydrochloride salt (5e). From N-(2-diethylaminoethyl)-4-
iodobenzo[b]thiophene-2-carboxamide (20e). Yield: 72%; mp
163-165 °C. 1H NMR (200 MHz, DMSO-d6) δ: 1.28 (t, 6H, J )
7 Hz), 3.25 (m, 6H), 3.70 (m, 2H), 7.26 (t, 1H, J ) 8 Hz), 7.92 (d,
1H, J ) 8 Hz), 8.00 (d, 1H, J ) 8 Hz), 8.22 (s, 1H), 9.42 (m, 1H),
10.29 (m, 1H). IR (KBr) ν cm-1: 1535, 1648, 3249. Anal.
(C15H19IN2OS, HCl) C, H, N.
Preparation of Radioiodinated Compounds [125I]5a-l. To a
solution of the appropriate compound 5 (2-3 mg) in citrate buffer
pH ) 4 (500 µL) were added, in a closed vial [125I]NaI (20-30
µL, 2-3 mCi), and an aqueous copper sulfate solution (0.5 mg,
100 µL) used as catalyst. The reaction mixture was heated at
120-150 °C for 30-60 min. The residue was taken up in water
(500 µL) and a 1N aqueous NaOH solution (100 µL) was added.
The vial cap and septum were removed. The resulting suspension
was run through an Extrelut column and eluted with CH2Cl2 (5 ×
3 mL). The eluting solution was concentrated under reduced
pressure, diluted with anhydrous CH2Cl2 (2 mL), and treated with
2N anhydrous hydrochloric acid in ether (5 mL). The resulting
hydrochloride solution was evaporated to dryness, and the dry
residue was then dissolved in physiological saline for animal
experiments. Radiochemical yields based on TLC of exchange
reaction mixture and radiochemical purities are given in Table 1.
Partition Coefficient Measurements. For each compound, the
partition coefficient between n-octanol and phosphate buffer was
determined. The measurement was performed by shaking 2 mL of
N-(2-Diethylaminoethyl)-6-iodoimidazo[1,2-a]pyridine-2-car-
boxamide, dihydrochloride salt (5f). From N-(2-diethylaminoethyl)-
3-iodoimidazo[1,2-a]pyridine-6-carboxamide (20f). Yield: 80%; mp
208-210 °C. 1H NMR (200 MHz, DMSO-d6) δ: 1.21 (t, 6H, J )
7 Hz), 3.17 (m, 6H), 3.58 (m, 2H), 7.57 (m, 2H), 8.41 (s, 1H),
9.00 (t, 1H, J ) 5 Hz), 9.09 (s, 1H), 9.95 (m, 1H). IR (KBr) ν
[
125I] compound solution (50 µM in phosphate buffer solution, PBS,
pH 7.4) with 2 mL of n-octanol. The activity of each phase was
counted in the γ counter (Packard, Minaxi γ 5530, Rungis, France).
P was calculated as the ratio of activities (n-octanol/buffer), and
its logarithm was determined to express lipophilicity.
cm-1
: 1289, 1600, 1659, 2650, 3100-2900, 3426. Anal.
(C14H19IN4O, 2HCl, 2H2O) C, H, N.
N-(2-Diethylaminoethyl)-5-iodo-1(3)H-benzimidazole-2-carboxa-
mide, dihydrochloride salt (5g). From N-(2-diethylaminoethyl)-5-
iodobenzimidazole-2-carboxamide (20 g). Yield: 82%; mp 217-219
In Vitro Binding to Melanin. An in vitro experiment was
performed to evaluate the binding affinity of new compounds to
melanin using synthetic tyrosine-melanin (Sigma) suspended in two
different media: water and PBS. The general procedure used was
as follows: [125I]compound was added to a melanin suspension (0.5
mg/10 mL). The suspension was incubated at room temperature
for 24 h with stirring. After incubation, the tubes were centrifuged
at 35000g for 20 min, and aliquots of the supernatants were counted.
In Vivo Distribution. All the experiments were carried out in
compliance with French laws governing animal experimentation.
The biodistribution of radioiodinated compounds was studied in
C57BL6 male mice bearing the B16-F0 murine melanoma. Stock
cell cultures were maintained as monolayers in Glutamax (Eagle’s
minimum essential medium with glutamine) supplemented with
10% fetal calf serum, vitamins, sodium pyruvate, nonessential amino
acids, and gentamycin and passaged by trypsination. The cells were
grown in a humidified 37 °C incubator containing 5% CO2. Early
passages were frozen and stored in liquid nitrogen. For transplanta-
tion, an aliquot was grown in a monolayer culture to confluence;
thereafter cells were trypsinized and washed with phosphate buffer
saline (PBS). They were resuspended in PBS, and each mouse
received 0.1 mL subcutaneously (5 × 105 cells) on the left flank.
Ten days later, the tumors became palpable with a percentage of
tumor take of 98-100%.
1
°C. H NMR (200 MHz, DMSO-d6) δ: 1.27 (t, 6H, J ) 7 Hz),
3.22 (m, 6H), 3.74 (m, 2H), 7.52 (d, 1H, J ) 8.5 Hz), 7.64 (dd,
1H, J ) 8.5, 1 Hz), 8.03 (d, 1H, J ) 1 Hz), 9.36 (m, 1H), 10.45
(m, 1H). IR (KBr) ν cm-1: 1593, 1683, 2979. Anal. (C14H19IN4O,
2HCl, H2O) C, H, N.
N-(2-Diethylaminoethyl)-6-iodoquinoline-2-carboxamide, dihy-
drochloride salt (5h). From N-(2-diethylaminoethyl)-6-iodoquino-
line-2-carboxamide (20h). Yield: 69%; mp 104-106 °C. 1H NMR
(200 MHz, DMSO-d6) δ: 1.28 (t, 6H, J ) 7 Hz), 3.24 (m, 6H),
3.77 (q, 2H, J ) 7 Hz), 7.92 (d, 1H, J ) 9 Hz), 8.21 (m, 2H), 8.57
(d, 1H, J ) 9 Hz), 8.65 (s, 1H), 9.34 (m, 1H), 10.08 (m, 1H). IR
ν cm-1: 1384, 1523, 1684, 3415. Anal. (C16H20IN3O, 2HCl,
2.5H2O) C, H, N.
N-(2-Diethylaminoethyl)-1,4-dihydro-6-iodo-4-oxoquinoline-3-
carboxamide, dihydrochloride salt (5i). From N-(2-diethylamino-
ethyl)-1,4-dihydro-6-iodo-4-oxoquinoline-3-carboxamide (20i). Yield:
92%; mp 164-166 °C. 1H NMR (200 MHz, DMSO-d6) δ: 1.27 (t,
6H, J ) 7 Hz), 3.24 (m, 6H), 3.73 (m, 2H), 7.62 (d, 1H, J ) 9
Hz), 8.11 (dd, 1H, J ) 9, 1.5 Hz), 8.51 (d, 1H, J ) 1.5 Hz), 8.80
(d, 1H, J ) 6.5 Hz), 10.07 (m, 2H). IR (KBr) ν cm-1: 1521, 1618,
1654, 3028, 3514. Anal. (C16H20IN3O2, 2HCl, 1.5H2O) C, H, N.
N-(2-Diethylaminoethyl)-5-iodoisoquinoline-3-carboxamide, di-
hydrochloride salt (5j). From N-(2-diethylaminoethyl)-5-iodoiso-
quinoline-2-carboxamide (20j). Yield: 72%; mp 152-154 °C. NMR
1H (200 MHz, DMSO-d6) δ: 1.22 (t, 6H, J ) 7 Hz), 3.25 (m, 6H),
3.74 (m, 2H), 7.63 (t, 1H, J ) 8 Hz), 8.33 (d, 1H, J ) 8 Hz), 8.49
(d, 1H, J ) 8 Hz), 8.56 (s, 1H), 9.38 (m, 2H), 9.76 (m, 1H). IR
(KBr) ν cm-1: 1526, 1684, 3447, 3927. Anal. (C16H20IN3O, 2HCl)
C, H, N.
Study of the Biodistribution on Animal Slices. The [125I]com-
pound was administered intravenously via a tail vein (0.1 µmol,
0.74-0.92 MBq/animal) in 10 animals for each compound. Two
injected mice were sacrificed by CO2 inhalation and quickly frozen
in liquid nitrogen at different times after administration (1, 3, 6,
24, or 72 h). Frozen animals were cryosectioned using the technique
described.48 Slices 40 µm thick were obtained using a Reichert-Jung
cryopolycut (Leica Instruments, Rueil Malmaison, France) at -22
°C and dehydrated for 48 h in the cryochamber. Eight slices per
mouse and per time point were selected from a total number of
more than 500 slices for the analysis corresponding to distant slices
and the section of the organs of interest. Measurements were
performed on an AMBIS 4000 detector (Scanalytics, CSPI, San
Diego, CA) following an acquisition time of 1000 min. The
radioactivity of different organs was quantified after contouring
N-(2-Diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide, di-
hydrochloride salt (5k). From N-(2-diethylaminoethyl)-6-iodoqui-
1
noxaline-2-carboxamide (20k). Yield: 76%; mp 201-203 °C. H
NMR (200 MHz, DMSO-d6) δ: 1.28 (t, 6H, J ) 7 Hz), 3.28 (m,
6H), 3.78 (m, 2H), 7.97 (d, 1H, J ) 9 Hz), 8.30 (d, 1H, J ) 9 Hz),
8.68 (s, 1H), 9.47 (m, 2H), 10.43 (brs, 1H). IR (KBr) ν cm-1
1516, 1674, 2459, 3326. Anal. (C15H19IN4O, 2HCl) C, H, N.
:

Analogues of BZA for Imaging and Therapy of Melanoma
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3143
melanosomes in malignant melanoma. Cancer Res. 1984, 44, 1128–
1134. (b) Hunt, G.; Kyne, S.; Ito, S.; Wakamatsu, K.; Todd, C.; Thody,
A. Eumelanin and phaeomelanin contents of human epidermis and
cultured melanocytes. Pigment Cell Res. 1995, 8, 202–208. (c) Ancans,
J.; Tobin, D. J.; Hoogduijn, M. J.; Smit, N. P.; Wakamatsu, K.; Thody,
A. J. Melanosomal pH controls of melanogenesis, eumelanin/phae-
omelanin ratio and melanosome maturation in melanocytes and
melanoma cells. Exp. Cell Res. 2001, 268, 26–35.
suitable zones on the two-dimensional image of the slice displayed
on the computer screen for analysis. Radioactivity per unit area
(net cpm/mm2) was converted into radioactive concentration (kBq/
g) and expressed as percentage of injected dose/g of tissue (%ID/
g). For comparison of the tumor (T) uptake with other tissues, ratios
of radioactive concentrations (T/organ) were determined, illustrating
the image contrast. For two animals, urine and feces were collected
up to 72 h and counted to determine the cumulative urinary and
fecal excretions.
Scintigraphic Imaging. For each selected compound, the in vivo
kinetic profile was followed in two mice by repeated planar
scintigraphic imaging (3 MBq, acquisition time 10 min) using a
dedicated γ camera for small animal imaging (Biospace, Paris,
France). This study allowed a follow-up in the same animal, namely
the tumor uptake kinetics.
Dosimetry Parameters. From the biodistribution values, the
biological tumor half-life was calculated for each [125I]compound
using an adaptation of the MIRD program to the mouse. With a
view to using the [131I]labeled analogue and taking into account
its physical properties, the effective half-life of [131I]compounds
and the tumor delivered doses (Gy/injected MBq) were calculated.
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on Pheomelanin. 2. J. Am. Chem. Soc. 1984, 106, 5884–5888.
(15) Ings, R. M. The melanin binding of drugs and its implications. Drug
Metab. ReV. 1984, 15, 1183–1212.
(16) Link, E. M.; Lukiewicz, S. A new radioactive drug selectively
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Supporting Information Available: Table of elemental analysis
and biodistribution data of compounds 5a-l. This material is
available free of charge via the Internet at http://pubs.acs.org.
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