Melanoma uptake of 99mTc complexes containing the N-(2-diethylaminoethyl)benzamide structural element

Article abstract of DOI:10.1021/jm021026z

On the basis of the avid uptake of radioiodinated benzamides by melanoma cells, ^99mTc complexes containing the structural elements of N-(dialkylaminoalkyl)benzamide pharmacophores have been synthesized and evaluated in vitro and in vivo for mel

Full text of DOI:10.1021/jm021026z

5802
J . Med. Chem. 2002, 45, 5802-5805
Brief Articles
Mela n om a Up ta k e of 99m Tc Com p lexes Con ta in in g th e
N-(2-Dieth yla m in oeth yl)ben za m id e Str u ctu r a l Elem en t†
Michael Eisenhut,*,| Ashour Mohammed,§ Walter Mier,§ Frank Scho¨nsiegel,| Matthias Friebe,‡
Ashfaq Mahmood,‡ Alun G. J ones,‡ and Uwe Haberkorn§
German Cancer Research Center and Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany, and
Department of Radiology, Harvard Medical School and Brigham and Women’s Hospital, Harvard University,
Boston, Massachusetts 02115
Received August 16, 2002
On the basis of the avid uptake of radioiodinated benzamides by melanoma cells, 99mTc
complexes containing the structural elements of N-(dialkylaminoalkyl)benzamide pharma-
cophores have been synthesized and evaluated in vitro and in vivo for melanoma uptake. One
of the complexes Tc-12 containing the ligand 4-(S-benzoyl-2-thioacetyl-glycyl-glycylamido)-N-
(2-diethylaminoethyl)benzamide (11) displayed the highest melanoma uptake. The 1-h
melanoma uptake values and the corresponding blood counts indicate an interdependence of
tumor uptake and bioavailability of the 99mTc complexes.
In tr od u ction
complexes formed from ligands containing the N-(2-
diethylaminoethyl)benzamide structural element using
(1) a chelate design that integrates the phenyl ring of
the pharmacophore within the complex, (2) a bis-
(aminothiol) ligand connected via a butylene spacer, and
(3) a hydrazinonicotinamide ligand with tricine as
ancillary coligand.
Based on the melanoma-affinity characteristics of
radioiodinated benzamides,1 the development of 99mTc
complexes containing structural elements of N-(dialkyl-
aminoalkyl)benzamide as pharmacophores has been
pursued. Thus, a series of ‘3 + 1’ mixed-ligand [99mTc]-
oxotechnetium complexes [TcO(SN(R)S)(SNX2)] were
synthesized and studied in B16 murine melanoma cells
as well as in the C57Bl/6-B16 mouse melanoma model.
In biodistribution studies with the compound in which
R ) Me and X ) Bu, up to 5% ID/g and an average
melanoma/nontarget tissue ratio (M/NTT) of 12.6 were
obtained at 1 h postinjection.2 When the [99mTc]oxotech-
netium(V) complexes of tetradentate amine-amide-
dithiol (AADT) chelates containing only tertiary amine
substituents, AADT-CH2-[CH2]n-NR2 (n ) 1, 2; R ) Et,
n-Bu), were tested, melanoma uptake of 7.6% ID/g (M/
NTT 9.5) was obtained at 1 h after administration.3 In
other approaches the complete benzamide pharmaco-
phore N-(2-diethylaminoethyl)benzamide was connected
to bis(aminothiol) ligands (BAT), resulting in [99mTc]-
oxotechnetium [TcO(BAT)] and [99mTc]nitridotechne-
tium [TcN(BAT)] complexes that displayed melanoma
uptake between 0.43 and 1.51%ID/g (M/NTT 4.5 and
1.6) 1 h postinjection in the C57Bl/6-B16 mouse model4
(see also Supporting Information).
Resu lts a n d Discu ssion
Ch em istr y. The syntheses of the four complexes Tc-
7, Tc-12, Tc-15, and Tc-19 were performed as outlined
in Scheme 1. Ligand 6 was synthesized by N-BOC
protection of commercially available 3,4-diaminobenzoic
acid (1), using di-tert-butyl dicarbonate followed by
conjugation with 2-(diethylamino)ethylamine using
HATU as a coupling agent to give the N-BOC-protected
3,4-diamino-N-(2-diethylaminoethyl)benzamide (3). BOC
was removed from 3 with 25% HCl. Then 4 was coupled
with the S-benzoyl-protected thioglycolic acid (5)5 using
DCC as the coupling agent to afford ligand 6. Ligand
11 was synthesized by activating 5 with N-hydroxysuc-
cinimide (NHS) to give 9, which was then conjugated
to Gly-Gly in an aqueous CH3CN solution at 90 °C to
provide 10 in 93% yield. Equimolar amounts of 10 and
4-amino-N-(2-diethylaminoethyl)benzamide (8) were re-
acted with DCC to form 11. Technetium-99m-labeled
complexes Tc-7 and Tc-12 were obtained in 70% radio-
chemical yield by reduction of [99mTc]pertechnetate in
the presence of ligand 6 and ligand 11, respectively, with
SnCl2 at 80 °C for 10 min followed by HPLC purification.
Ligand 14 was synthesized by reacting the heterobi-
functional chelator N-hydroxysuccinimide ester of 6-(4′-
(4′′-carboxyphenoxy)butyl)-2,10-dimercapto-2,10-dimethyl-
4,8-diazaundecane (13), described previously,6 with a
5-fold excess of 2-(diethylamino)ethylamine. Complex-
ation of 14 with technetium-99m was performed with
Herein, we report the synthesis and in vivo evaluation
in the C57Bl/6-B16 mouse melanoma model of 99mTc
* Author for correspondence: Michael Eisenhut, Ph.D., Deutsches
Krebsforschungszentrum Heidelberg, Abt. Radiochemie und Radiop-
harmakologie (E0300), Im Neuenheimer Feld 280, 69120 Heidelberg,
Germany. Fax: 06221-422421. E-mail: m.eisenhut@dkfz.de.
† This article is dedicated to Harald zur Hausen on the occasion of
his retirement as head of the Deutsches Krebsforschungszentrum
Heidelberg.
| German Cancer Research Center, University of Heidelberg.
§ Department of Nuclear Medicine, University of Heidelberg.
‡ Harvard Medical School and Brigham and Women’s Hospital.
[
99mTc]pertechnetate and SnCl2 at ambient temperature
10.1021/jm021026z CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/23/2002

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5803
Sch em e 1a
a
Reaction conditions: (i) (BOC)2O, DIPEA; (ii) 2-(diethylamino)ethylamine, HATU; (iii) 25% HClaq; (iv) S-benzoylthioglycolic acid 5,
DCC; (v) Na99mTcO4, 0.9% NaCl, tartaric acid, SnCl2, 80 °C; (vi) NHS, DCC, 0 °C; (vii) GlyGly, H2O, 90 °C; (viii) 4-amino-N-(2-
diethylaminoethyl)benzamide 8, DCC; (ix) 2-(diethylamino)ethylamine; (x) Na99mTcO4, 0.9% NaCl, tartaric acid, SnCl2; (xi) TFA/thioanisol/
H2O (92/6/2); (xii) tricine and (x). If not stated otherwise the reactions were performed in DMF and at ambient temperature.
to yield Tc-15 in 59% isolated radiochemical yield after
HPLC purification.
Control measurements with distinctly dianionic and
monocationic complexes, [99mTc-MAG3]2- and [99mTc-
MIBI]+, indicated migration of the radioactive spots
toward the anode and cathode, respectively. The behav-
ior of Tc-7 and Tc-12 complexes can be viewed as
zwitterionic as the negative charge resulting from the
complexation of 99mTc by the chelate is also accompanied
by the protonation of the tertiary amino group at pH
7.4 resulting in a net neutral charge. The neutral
complexes Tc-15 and Tc-19 become positively charged
under these conditions and hence display migration
toward the cathode.
Ligand 18 was prepared using a previously described
6-BOC-hydrazinopyridine-3-carboxylic acid (16) precur-
sor, which was synthesized via the reaction of 6-hy-
drazinonicotinic acid with BOC anhydride.7 The result-
ing compound 16 without further purification was
coupled with 2-(diethylamino)ethylamine using HATU,
affording the 6-hydrazinonicotinamide 17. Unexpectedly
BOC deprotection with TFA yielded almost exclusively
the trifluoroacetylated product TFA-17. TFA cleavage
was performed by overnight treatment of TFA-17 with
25% HCl resulting in ligand 18 as an HCl salt (23%
yield). Complexation of 18 was achieved by reduction
of [99mTc]pertechnetate in the presence of the coligand
tricine with SnCl2 followed by complexation with 18 to
give complex Tc-19 in 75% radiochemical yield following
HPLC purification.
The octanol-buffer partition coefficients for 99mTc
complexes were measured at pH 7.4. With respect to
log D(pH 7.4), the four complexes can be divided into two
groups: (1) the negatively charged complexes Tc-7 (log
D(pH
1.81 ( 0.01) and Tc-12 (1.89 ( 0.01) with
7.4)
medium lipophilicity, and (2) the neutral Tc-15 (2.53
( 0.02) and Tc-19 (2.68 ( 0.03) complexes exhibiting
high lipophilicity. Although the log D(pH 7.4) of Tc-7 and
Tc-12 was similar to values found for the radioiodinated
benzamides,11,12 there is no meaningful correlation of
While the tracer concentrations of the 99mTc com-
plexes Tc-7, Tc-12, Tc-15, and Tc-19 (Scheme 1)
preclude macroscopic characterization, the proposed
99m
structures are in accordance with DADS and MAG3
-
Tc complexes of similar ligands.8,9 Similarly, the struc-
ture of the 99mTc complexes Tc-15 and Tc-19 are
analogous to the neutral technetium complexes formed
with the bis(aminoethanethiol)10 and HYNIC7 ligands.
Electrophoresis was employed to determine the net
charge of the complexes at pH 7.4. At this physiological
pH, the electrophoretic mobility of complex Tc-7 and
Tc-12 was zero while that of complex Tc-15 and Tc-19
displayed migration of the spots toward the cathode.
log D(pH
compounds.
with melanoma accumulation in these
7.4)
In Vit r o Mela n om a -Up t a k e St u d ies. Tumor-cell
uptake of the 99mTc complexes was perfomed with
cultured murine B16 melanoma cells which were also
used for the in vivo studies. For comparison, cell uptake
of [131I]IMBA was determined as well. As shown in
Figure 1a, two of the benzamide complexes, Tc-12 and
Tc-15, display high uptake which exceeded the [131I]-

5804 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
Brief Articles
Ta ble 1. Biodistribution of 99mTc-Complexes Tc-7, Tc-12, Tc-15, and Tc-19 in C57Bl/6 Mice with B16 Melanoma
mean tissue concentrations (%ID/g, n ) 3)a
compd time (h, pi) M/NTTb
melanoma
blood
heart lungs spleen
liver
kidneys muscle brain adrenals
Tc-7
1
6
1
6
1
6
1
6
4.7
11.0
2.6
7.3
3.2
5.2
5.4
4.0
0.16 ( 0.01 0.26 ( 0.20
0.09 ( 0.02 0.03 ( 0.01
3.42 ( 0.30 3.44 ( 0.38
4.35 ( 0.92 3.20 ( 0.73
0.84 ( 0.17 0.55 ( 0.13
0.45 ( 0.15 0.28 ( 0.12
1.24 ( 0.31 0.75 ( 0.20
0.89 ( 0.26 0.68 ( 0.21
0.11
0.02
1.55
1.32
0.86
0.33
0.48
0.47
0.30
0.05
1.96
1.73
1.96
0.67
0.97
1.01
0.08
0.04
1.30
0.88
1.73
0.46
0.25
0.48
1.12
0.39
1.55
1.31
25.59
6.65
3.11
2.16
1.84
0.39
11.90
1.93
8.74
4.92
2.84
2.49
0.02
0.00
0.90
0.60
0.26
0.07
0.28
0.20
0.01
0.00
0.52
0.15
0.05
0.02
0.05
0.05
0.17
0.04
1.22
1.19
1.97
1.36
0.89
0.87
Tc-12
Tc-15
Tc-19
a
b
To simplify the table, standard deviations are shown only for melanoma and blood data. M/NTT: average melanoma/nontarget
tissue ratio (excluding adrenals).
Tc-19 were lower, with Tc-7 showing the lowest value.
In this context it is noteworthy that only the melanoma
uptake of complex Tc-12 displayed an increase in
accumulation between 1 and 6 h while all other com-
plexes washed out from the tumor. Prominent uptake
in nontarget tissues was observed for Tc-12 (kidneys)
and Tc-15 (kidneys, liver). Low values throughout all
tissues were observed after injection of Tc-7, and this
may be the result of its rapid excretion.
The differences in the biodistribution data reflect
differences in the route of metabolic degradation and
the rate of tissue and renal clearance of metabolites,
which competes with melanoma uptake by reducing
intravascular concentrations.11 A correlation of the 1-h
melanoma uptake values with the corresponding blood
counts shows that the degree of melanoma uptake is
proportional to the concentration of the 99mTc complex
circulating in the blood. Figure 2 (Supporting Informa-
tion) demonstrates a linear correlation (r ) 0.977) of
both parameters indicating that the melanoma uptake
of these complexes is distinctly related to their bioavail-
ability. A similar bioavailability relationship has re-
cently been described in a series of radioiodinated
benzamides.11 The results obtained earlier with [99mTc]-
oxotechnetium(V) complexes of tetradentate amine-
amide-dithiol (AADT) chelates3 also fit this bioavail-
ability/melanoma uptake model, displaying significant
correlation between the 1-h blood and melanoma values
F igu r e 1. (a) In vitro cell uptake of complexes Tc-7, Tc-12,
(r ) 0.951). The ‘3 + 1’ mixed-ligand [99mTc]oxotechne-
tium complexes,2 however, do not follow this pattern,
with the correlation points scattered.
Tc-15, Tc-19, and [131I]IMBA in B16 melanoma cells. (b) In
vivo melanoma uptake values of Tc-7, Tc-12, Tc-15, and
Tc-19.
In addition to the net accumulation of the 99mTc
complex in melanoma tissue, another important param-
eter for an imaging agent is the melanoma/nontarget-
tissue ratio (M/NTT), which is comprehensively ex-
pressed here as the average M/NTT of eight organs
(adrenals were excluded because of their small size).
Aside from Tc-19 (M/NTT ) 5.4 and 4.0 at 1 and 6 h
postinjection, respectively), all complexes displayed
ratios that increased from 1 to 6 h postinjection indicat-
ing specific melanoma binding. Although the highest
M/NTT ratio at 6 h was obtained with Tc-7 (4.0 and
11.0), this ligand exhibits the lowest melanoma uptake.
The M/NTT ratio for Tc-12 (2.6 and 7.3) was the second
highest, and this compound displayed the highest
melanoma uptake value among these complexes, indi-
cating more favorable characteristics as an imaging
agent.
IMBA values, while Tc-7 and Tc-19 accumulated much
less in melanoma cells.
With the exception of complex Tc-15, these results
are in accordance with the melanoma uptake values
obtained from biodistribution studies (Figure 1b). An
explanation for the low in vivo melanoma uptake of
Tc-15 cannot be given.
Biod istr ibu tion Stu d ies. The animal experiments
were performed to assess organ uptake, target/nontarget
ratios and pharmacokinetics of the 99mTc complexes, Tc-
7, Tc-12, Tc-15, and Tc-19. Following HPLC separa-
tion, approximately 5 MBq of complex was intra-
venously injected into C57Bl/6 mice bearing subcutane-
ously transplanted B16 melanoma tumors. The animals
were sacrificed 1 and 6 h after injection, and 99mTc
activity in selected organs and tissues was analyzed.
The biodistribution data are summarized in Table 1.
The complex Tc-12 exhibited the highest melanoma
uptake at both time points. As illustrated in Figure 1b,
the 1-h uptake values of compounds Tc-7, Tc-15, and
Con clu sion s
Four new 99mTc complexes derived from N-(2-diethyl-
aminoethyl)benzamide have been evaluated in the

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5805
with Dialkylaminoalkyl Substituents as Potential Diagnostic
Probes for Malignant Melanoma. J . Med. Chem. 2001, 44, 3132-
3140.
C57Bl/6-B16 mouse model. Two of theses complexes, Tc-
12 and Tc-15, showed high melanoma uptake in vitro.
For complex Tc-12 this result was confirmed in vivo
with M/NTT ratios (imaging contrast) increasing with
time. A comparison with earlier results indicates that
the melanoma uptake values of Tc-12 are (1) well above
those obtained with 99mTcO and 99mTcN bis(aminothiol)
complexes carrying the N-(2-diethylaminoethyl)benza-
mide pharmacophore,4,13,14 (2) comparable with ‘3 + 1’
mixed-ligand TcO complexes,2 and (3) lower than the
amine-amide-di-thiol (AADT) 99mTcO complexes both of
which contain dialkylamino-alkyl substituents.3 Similar
relationships were found for the melanoma/nontarget-
tissue ratios with the exception of 99mTcO-AADT com-
plexes.3 Especially at 6 h after injection 99mTcO-AADT
complexes have higher M/NTT ratios indicating faster
excretion from background tissue. The in vitro and in
vivo melanoma uptake of Tc-12 also suggests that
appropriate incorporation of the benzamide structure
within the 99mTc-complexes can result in 99mTc-complex-
es that display significant melanoma uptake.
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