'3+1' Mixed-ligand oxotechnetium(V) complexes with affinity for melanoma: Synthesis and evaluation in vitro and in vivo

Article abstract of DOI:10.1021/jm000050e

'3+1' Mixed-ligand [^99mTc]oxotechnetium complexes with affinity for melanoma were synthesized in a one-pot reaction. Complexation of technetium-99m with a mixture of N-R(3- azapentane-1,5-dithiol) [R = Me, Pr, Bn, Et₂N(CH₂)₂] and N-(2- dialkylamino)ethanethiol [alkyl = X = Et, Bu, morpholinyl] using Sn²⁺ as the reducing agent resulted in the formation of '3+1' mixed-ligand technetium-99m complexes [TcO(SN(R)S)(SNX₂)] in high radiochemical yield (60-98%). In vitro uptake studies in B16 murine melanoma cells indicated a moderate tumor-cell accumulation (40%) of compound 1 [R = Me, X = Et] and a higher accumulation (69%) of compound 2 [R = Me, X = Bu] after a 60-min incubation. In vivo evaluation of compounds 1-6 in the C57B16/B16 mouse melanoma model demonstrated tumor localization. Compound 2 displayed the highest accumulation with up to 5% ID/g at 60 min after injection. In vivo, 2 also showed a low blood-pool activity and high melanoma/spleen (4.3) and melanoma/lung (1.9) ratios at 1 h. These results suggest that small technetium-99m complexes could be useful as potential melanoma-imaging agents.

Full text of DOI:10.1021/jm000050e

J . Med. Chem. 2000, 43, 2745-2752
2745
‘3+1’ Mixed -Liga n d Oxotech n etiu m (V) Com p lexes w ith Affin ity for Mela n om a :
Syn th esis a n d Eva lu a tion in Vitr o a n d in Vivo
Matthias Friebe,† Ashfaq Mahmood,† Hartmut Spies,‡ Ralf Berger,‡ Bernd J ohannsen,‡ Ashour Mohammed,§
Michael Eisenhut,§ Cristina Bolzati,†, Alan Davison,| and Alun G. J ones*,†
Department of Radiology, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts 02115,
Institute of Bioinorganic and Radiopharmaceutical Chemistry, Research-Center Rossendorf, Dresden, Germany, Department of
Nuclear Medicine, University of Heidelberg, Heidelberg, Germany, Department of Inorganic Chemistry, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139, and Department of Clinical and Experimental Medicine, University of
Ferrara, Ferrara 44100, Italy
Received February 9, 2000
‘3+1’ Mixed-ligand [99mTc]oxotechnetium complexes with affinity for melanoma were synthesized
in a one-pot reaction. Complexation of technetium-99m with a mixture of N-R(3-azapentane-
1,5-dithiol) [R ) Me, Pr, Bn, Et2N(CH2)2] and N-(2-dialkylamino)ethanethiol [alkyl ) X ) Et,
Bu, morpholinyl] using Sn2+ as the reducing agent resulted in the formation of ‘3+1’ mixed-
ligand technetium-99m complexes [TcO(SN(R)S)(SNX2)] in high radiochemical yield (60-98%).
In vitro uptake studies in B16 murine melanoma cells indicated a moderate tumor-cell
accumulation (40%) of compound 1 [R ) Me, X ) Et] and a higher accumulation (69%) of
compound 2 [R ) Me, X ) Bu] after a 60-min incubation. In vivo evaluation of compounds 1-6
in the C57Bl6/B16 mouse melanoma model demonstrated tumor localization. Compound 2
displayed the highest accumulation with up to 5% ID/g at 60 min after injection. In vivo, 2
also showed a low blood-pool activity and high melanoma/spleen (4.3) and melanoma/lung (1.9)
ratios at 1 h. These results suggest that small technetium-99m complexes could be useful as
potential melanoma-imaging agents.
In tr od u ction
Increase in the incidence of skin cancer is of great
concern.1,2 Nearly all deaths caused by skin malignan-
cies result from malignant melanoma. The significant
mortality of this disease is caused by the high prolifera-
tion rate of melanoma cells and the early occurrence of
F igu r e 1. Iodine-123-labeled N-(2-diethylaminoethyl)-4-io-
dobenzamide ([123I]BZA) and N-(2-diethylaminoethyl)-3-iodo-
4-methoxybenzamide ([123I]IMBA).
metastases. The choice of treatment depends on the
timely detection of the melanoma and any associated
metastases. While positron emission tomography (PET)
using 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG), an 18F-
radiolabeled glucose analogue, has been successfully
anotropin peptides.12,13 Tumor-uptake and biodistribution
studies with these peptide-based radioconjugates gener-
ated favorable results, indicating that these labeled
peptides may be useful for in vivo melanoma scintigra-
phy. However, the search for nonpeptidic small mol-
ecules that possess high affinity for melanoma contin-
ues.
used for melanoma imaging,3-5
a
99mTc-labeled single-
photon-emission computed tomography (SPECT) radio-
pharmaceutical with affinity for melanoma may provide
a cost-effective means of early detection and diagnosis
with widespread availability.
In this regard, high tumor uptake was recently
obtained with 123I-labeled N-(2-diethylaminoethyl)-4-
iodobenzamide ([123I]BZA) and N-(2-diethylaminoethyl)-
3-iodo-4-methoxybenzamide ([123I]IMBA).11,12 The chemi-
cal structures of BZA and IMBA are shown in Figure
1. In vivo investigations with subcutaneously trans-
planted B16 melanoma cells in C57Bl6 mice showed
uptake values ranging from 5% to 20% injected dose/g
(ID/g) tumor.14,15 Subsequent human clinical trials also
indicated adequate uptake by melanoma and good
scintigraphic images.14,16 While recent reports suggest
that the uptake is nonsaturable and may be related to
the formation of melanin within the melanosome,17
structurally similar benzamides containing a piperidinyl
substituent have also been shown to possess σ-receptor
affinity.18,19 Although such radioiodinated benzamides
have entered phase I clinical trials14 for the diagnosis
Previous attempts to image melanoma with radiola-
beled monoclonal antibodies met with little success.6,7
Subsequent use of simpler radiolabeled molecules,
including radioiodinated amino acids8,9 and nucleic
acids,10,11 as false precursors in the melanin formation
cycle either displayed insufficient localization in tumors,
resulting in low tumor-to-nontumor ratios,8-11 or pos-
sessed poor pharmacokinetics.8,11 More promising re-
sults were obtained recently with 99mTc-labeled R-mel-
* Author for correspondence: Alun G. J ones, Harvard Medical
School, Dept. of Radiology, Goldenson Building B-1/242, 220 Longwood
Ave., Boston, MA 02115. Phone: (617)-432-3777. Fax: (617)-432-2419.
E-mail: agjones@hms.harvard.edu.
† Harvard Medical School and Brigham and Women’s Hospital.
‡ Research-Center Rossendorf.
§ University of Heidelberg.
| Massachusetts Institute of Technology.
University of Ferrara.
10.1021/jm000050e CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/21/2000

2746 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Friebe et al.
Sch em e 1. Preparation and Numbering Scheme of
‘3+1’ Mixed-Ligand Complexes
Sch em e 2. Preparation of Tridentate Ligands SN(Me)S
(a ) and SN(R)S [(R ) n-C3H7 (b), CH2-C6H5 (c),
CH2-CH2-N(CH2)2 (d )] and Monodentate SNX2 Ligands
[NX2 ) N(C4H9)2 (e), morpholinyl (f)]a
7
a
(i) CHCl3, SOCl2, 60 °C; (ii) SdC(NH2)2, 100 °C, 30 min; (iii)
NaOH (diluted); (iv, v) toluene, autoclave, 25 °C/2 h, 80 °C/8 h.
Ta ble 1. RP-HPLC Comparison of Technetium-99m Complexes
with Analogous ‘3+1’ Mixed-Ligand Rhenium Complexes
tR (min)
tR (min)
of malignant melanoma, their routine clinical use may
be hampered by the associated disadvantages of iodine-
123, i.e., lack of routine availability and low cost-
effectiveness.
complex
Re
Tc
complex
Re
Tc
1
2
3
5.9
7.1
4.1
6.1
7.0
4.4
4
5
6
17.5
8.9
3.7
16.6
10.0
3.8
The most widely used isotope in clinical nuclear
medicine, technetium-99m, possesses ideal character-
istics (t1/2 ) 6.02 h, 140 keV monoenergetic γ-emission)
for nuclear medicine imaging and is available on
demand via the 99Mo-99mTc generator system. These
properties have led to the search for small technetium-
99m complexes possessing high affinity for melanoma.
Initial attempts to replace the radioiodinated benza-
mides by a series of technetium-99m complexes contain-
ing the N-(2-diethylaminoethyl)benzamide structural
element gave melanoma-uptake values with a maxi-
mum of 1.6% ID/g tumor (1 h postinjection).20-22 While
tumor uptake was low, the values suggest that further
refinement in the molecular structure might lead to a
higher tumor affinity and in vivo uptake. Following this
hypothesis, we envisioned that removal of the aromatic
ring and concomitant integration of the oxotechnetium
core with the N-(2-diethylaminoethyl) part of the phar-
macophore might enhance the melanoma affinity of
these compounds compared with the previously de-
scribed complexes possessing the entire aromatic struc-
ture element.20-22 Various N-alkyl-substituted (SN(R)S)
[R ) Me, Pr, Bn, Et2N(CH2)2]-3-azapentane-1,5-dithiols
as tridentate chelating agents and N-(2-dialkylamino)-
ethanethiols [X ) Et, Bu; NX2 ) morpholinyl] as mono-
dentate pharmacophores were evaluated with regard to
developing a small nonpeptidic technetium-99m com-
plex with affinity for melanoma.
procedure with high radiochemical yield of the final
complex.23 Utilizing the flexibility inherent in this
approach, we synthesized compounds 1-6 as described
in Scheme 1 to obtain a series of ‘3+1’ technetium-99m
complexes.
The required tridentate ligands containing the SNS
metal coordinating donor atoms were prepared as
described in Scheme 2. The monodentate ligands, where
X ) Bu or NX2 ) morpholinyl (complexes 2-4), were
synthesized by autoclaving in a high-pressure reaction
vessel the appropriate secondary amine with ethylene
sulfide in argon-saturated toluene (Scheme 2), while
N-(2-diethylamino)ethanethiol (monodentate ligand for
complexes 1 and 5) and methanethiol (monodentate
ligand for complex 6) were available from commercial
sources.
Replacement of the benzamido portion of the BZA
(Figure 1) by the metal chelate unit [TcO(SNS)(S)] was
made possible by using the ‘3+1’ approach. Complexes
1-6 were obtained by mixing [99mTc]pertechnetate, the
tridentate ligand, and the monodentate ligand with Sn2+
as reducing agent and heating the reaction mixture at
45 °C for 20 min. This synthesis was based on an
optimized protocol,23 which was essential for obtaining
high radiochemical yields of ‘3+1’ technetium com-
plexes. In a typical no-carrier-added technetium-99m
synthesis, a 1:10 ratio of tridentate ligand:monodentate
ligand was always maintained. The resulting techne-
tium complexes were purified by RP-HPLC, and the
radiochemical yields ranged between 60% and 98%
(Scheme 2). Since the final concentration of the tech-
netium-99m complexes was generally in the nanomole
to picomole range, the identification of these complexes
was based on chromatographic comparisons with previ-
ously characterized analogous nonradioactive rhenium
complexes (Table 1).24
Resu lts a n d Discu ssion
Ch em istr y. The ‘3+1’ mixed-ligand technetium-99m
complex is generally described as an entity containing
a tridentate ligand and a monodentate ligand surround-
ing an oxotechnetium core (Scheme 1). These complexes
are quite versatile in that the substitution of the
monodentate ligand allows for easy manipulation of the
physicochemical properties and a simple radiolabeling

‘3+1’ Mixed-Ligand Oxotechnetium(V) Complexes
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2747
Ta ble 2. pKa and Lipophilicity Data for Prepared Compounds
‘3+1’ complex
R
R′
pKa
D(pH 7.4)
log D(pH 7.4)
P
log P
1
2
3
4
5
6
-CH3
-CH3
-CH3
-C3H7
-(CH2)2-N(C2H5)2
-(CH2)2-N(C4H9)2
-(CH2)2-N-morpholino
-(CH2)2-N(C4H9)2
-(CH2)2-N(C2H5)2
-CH3
9.7
9.9
7.2
9.9
9.6
8.3
1.0
10.0
9.0
14.0
9.0
0
47.0
1.7
3.5
1.2
3.7
3.6
2.7
1.0
1.0
1.1
0.95
1.6
3210
14.0
4987
4468
530
-CH2-C6H5
-(CH2)2-N(C2H5)2
43.0
Lip op h ilicity a n d p Ka . Since lipid partitioning and
transport of the complexes across cell membranes are
governed not only by lipophilicity of the complex but also
by basicity of the pendant tertiary amine, which can be
protonated at physiologic pH, measurements were made
to determine partition coefficients of the unprotonated
free amine (log P) as well as the partition coefficient at
physiologic pH (log D(pH 7.4)).25,26 This provides a com-
posite parameter that is a function of both lipophilicity
and pKa. The pKa of complexes 1-6 were also deter-
mined using HPLC techniques25,26 and are presented
in Table 2.
As complex 1 contains the N,N′-diethylamino group,
the observed pKa of 9.7 is consistent with >98% of the
complex being protonated at the physiologic pH of 7.4
and the observed log D(pH
value of 0. The presence
7.4)
of a more lipophilic N,N′-dibutylamino group in complex
2 leads to a slightly higher pKa (9.9) and a significantly
higher log D(pH 7.4) (1.0). The replacement of the dialky-
lamine moiety by a morpholino group in complex 3
decreases the pKa of the complex to 7.2 but maintains
a log D(pH 7.4) comparable to that of 2. Thus, roughly 50%
of complex 3 is unprotonated and neutral at pH 7.4.
To maintain the pKa of 2 while altering the lipophi-
licity (log D(pH 7.4)) of the complex, compound 4 was
synthesized, which carries the same monodentate ligand
as 2 but a more lipophilic substituent in the tridentate
part of the molecule, thus yielding a pKa of 9.9 and a
F igu r e 2. In vivo melanoma uptake (% ID/g) of complexes
1-6 at 1 and 6 h postinjection in C57/B16 mice with palpable
hind limb B16 melanoma nodules.
summarized for organs of special interest, including the
lung, liver, and spleen, since these are the likely sites
for the occurrence of metastases.
Compound 1 shows a 1-h tumor uptake of 3.2% ID/g
and a very slow washout from the tumor (0.1%) between
1 and 6 h postinjection (pi). Although uptake in the
spleen and lung approximates that in the tumor at 1 h,
washout from these organs relative to the tumor is
faster and results in a tumor/spleen ratio of 4.5 and a
tumor/lung ratio of 1.6 at 6 h pi.
The highest melanoma uptake of 5.0% ID/g is attained
with compound 2 at 1 h pi. Even at this time point, com-
pared with complex 1, a high tumor/blood ratio (3.9), a
significantly higher tumor/spleen ratio (4.3), and a
slightly higher tumor/lung ratio (1.9) are observed. Over
6 h the tumor/blood ratio increases to 4.2 and the tumor/
lung ratio to 3.1; however, the melanoma/spleen ratio
drops to 3.2. Although there is a significantly faster
washout from the tumor over 6 h (0.8%) compared with
1, the tumor uptake remains high relative to nontarget
organs.
Complexes 3-6 all display lower tumor uptake and
lower tumor/nontumor ratios with no significant im-
provement over a 6-h period relative to complex 2 (Table
3). Due to the lipophilic nature of these complexes, the
liver uptake of all compounds investigated is in the
range of 7% (6) to 43% (2).
log D(pH
of 1.1. In an analogous manner, the pKa of
7.4)
complex 5 (9.6) is very similar to that of complex 1, but
due to the introduction of a benzyl group in the triden-
tate portion, 5 is more lipophilic than 1 and comparable
with 2 and 3. Additional modification of the pKa and
lipophilicity has been achieved by altering the position
of the disubstituted amino group from the monodentate
portion of the complex to the tridentate side as in
complex 6. Here the pKa of the disubstituted amine is
tempered by being attached at the â-position to the
metal-coordinating nitrogen in the tridentate ligand.
This results in both a lower lipophilicity (log P) and a
lower pKa (8.3) relative to 1 and 5. The lowering of the
pKa consequently results in a significantly higher log
D(pH
as less of the amine is protonated.
7.4)
Mela n om a -Up ta k e Stu d ies. To evaluate whether
the complexes possess melanoma affinity, initial in vivo
tumor-uptake studies of the technetium-99m complexes
were performed in the murine C57Bl6/B16 mouse tumor
model.15 The tumor uptake of complexes 1-6 (Figure
2) and the biodistribution of these complexes (Table 3)
are calculated as percentage injected dose per gram (%
ID/g). In addition, the melanoma/organ ratios are also
To elucidate some of the parameters necessary for
melanoma targeting of these ‘3+1’ complexes, correla-
tion of tumor uptake with molecular parameters, in-
cluding lipophilicity, pKa, and position of the pharma-

2748 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Friebe et al.
Ta ble 3. Biodistribution and Tumor/Nontumor Ratios of Compounds 1-6 at 1 and 6 h Postinjection
1
2
3
4
5
6
organ
1 ha
6 h
1 h
6 h
1 h
6 h
1 h
6 h
1 h
6 h
1 h
6 h
blood
heart
lung
2.08b
(0.41)c
1.84
(1.11)
2.73
1.35
(0.23)
0.84
(0.11)
1.96
1.26
(0.33)
0.66
(0.06)
2.60
0.98
(0.54)
0.46
(0.12)
1.35
2.36
(0.52)
1.26
(0.21)
5.75
1.44
(0.32)
0.83
(0.09)
2.91
1.34
(0.41)
1.17
(0.05)
3.27
1.44
(0.24)
0.73
(0.08)
2.07
3.28
(0.57)
2.09
(0.41)
13.2
1.30
(0.33)
0.86
(0.07)
5.50
3.49
(0.93)
1.74
(0.25)
5.73
2.05
(0.30)
1.15
(0.23)
3.40
(1.32)
3.44
(1.04)
27.3
(0.31)
0.70
(0.27)
18.7
(0.32)
1.15
(0.08)
43.3
(0.52)
1.30
(1.00)
23.0
(0.75)
(0.36)
1.53
(0.21)
11.3
(0.25)
(0.38)
1.25
(0.51)
17.7
(2.46)
(0.48)
(0.59)
(0.50)
1.66
(0.21)
6.10
spleen
liver
4.02
3.35
6.04
1.44
2.39
(1.22)
19.4
(1.34)
35.3
(1.47)
16.6
(0.73)
7.23
(0.22)
7.11
(3.12)
17.9
(1.40)
0.57
(0.02)
0.18
(2.72)
17.9
(1.60)
0.39
(0.10)
0.14
(4.00)
30.4
(2.24)
0.24
(0.04)
0.08
(5.80)
19.3
(4.56)
0.20
(0.07)
0.07
(2.37)
(1.74)
13.3
(1.43)
0.26
(0.01)
0.12
(3.65)
(3.46)
11.7
(2.20)
0.26
(0.04)
0.08
(3.49)
(1.18)
(0.49)
(1.10)
10.1
(1.59)
0.39
(0.11)
0.15
kidney
muscle
brain
16.0
15.3
24.0
12.9
13.0
(0.79)
0.64
(0.10)
0.20
(2.33)
0.53
(0.07)
0.13
(1.80)
0.57
(0.08)
0.32
(0.15)
0.26
(0.02)
0.13
(1.08)
0.78
(0.21)
0.24
(0.08)
3.23
(0.10)
3.13
(0.01)
4.95
(0.01)
4.12
(0.11)
2.16
(0.02)
1.53
(0.01)
3.26
(0.01)
3.23
(0.06)
2.88
(0.03)
1.39
(0.05)
2.55
(0.01)
2.83
melanoma
(0.46)
1.6
<1
1.2
<1
(0.37)
2.3
(1.00)
3.9
(0.80)
4.2
(0.09)
(0.32)
1.1
(0.10)
(0.74)
2.2
(1.10)
(0.02)
(0.17)
(0.85)
1.4
melanoma/blood
melanoma/spleen
melanoma/lung
melanoma/liver
<1
2.4
<1
1.1
<1
1.1
<1
<1
4.5
4.3
3.2
<1
<1
<1
1.0
<1
1.0
<1
2.6
<1
<1
<1
<1
<1
<1
1.7
1.6
<1
1.9
<1
3.1
<1
<1
<1
1.6
<1
<1
<1
a
b
n ) 3 animals per time point. Values represent % ID/g wet tissue. c Standard deviation in parentheses.
Ta ble 4. Molecular Mass and MS and 1H NMR Data for Prepared Ligands
MW
(g/mol) m/z [M + H]+
MS dataa
ligand
1H NMR δ
SN(Me)S (a )
151
152
2.83-2.84 (s, 1H, HS-CH2-), 2.84-2.87 (m, 2H, -S-CH2-), 2.86-2.87 (s, 3H, N-CH3),
3.30-3.35 (m, 2H, N-CH2-CH2-)
SN(Pr)S (b)
SN(Bn)S (c)
179
180
0.84-0.87 (t, 3H, -CH2-CH3), 1.39-1.46 (sext, 2H, -CH2-CH2-CH3), 1.68-1.73 (s, 2H,
HS-CH2-), 2.34-2.37 (t, 2H, CH3-CH2-CH2-N), 2.53-2.56 (t, 4H, N-CH2-CH2-S),
2.58-2.61 (t, 4H, N-CH2-CH2-S)
227
228
2.57-2.62 (m, 4H, S-CH2-CH2-), 2.68-2.74 (m, 4H, N-CH2-CH2-), 3.61-3.64 (s, 2H,
aryl-CH2-N), 7.30-7.35 (m, 5H, aryl)
SN(NR2)S (d )
SNBu2 (e)
212
189
213
190
1.02 (t, -CH2-CH3(ethyl)), 1.87 (s, SH), 2.6 (m, (-CH2)2 + -CH2-CH3(ethyl))29
0.84-0.92 (t, 6H, -(CH2-CH3)2), 1.28-1.38 (sext, 2H, -CH2-CH2-CH3), 1.55-1.64 (m, 2H,
-CH2-CH2-CH2-CH3), 2.74-2.77 (m, 2H, -S-CH2-CH2), 3.30-3.80 (m, 4H, -N(CH2)2-CH2),
3.22-3.26 (m, 2H, -CH2-CH2-NR2)
S-morpholinyl (f)
147
148
1.73-1.84 (s, 1H, HS-CH2-), 2.41-2.48 (m, 4H, -N<(CH2-CH2)2>O), 2.53-2.58 (m, 2H,
HS-CH2-CH2-), 2.59-2.66 (m, 2H, -S-CH2-CH2-N<), 3.67-3.73 (m, 4H,
-N<(CH2-CH2)2>O)
a
Ionization technique: fast atom bombardment (FAB) in glycerol matrix and positive polarity.
cophore, was undertaken. Complexes 2 and 4 both
contain the same amine-bearing pharmacophore in the
monodentate ligand portion of the complex. This results
in both complexes having identical pKa values with
differing lipophilicity. If there is a simple correlation
between tumor uptake and lipophilicity, the slightly
more lipophilic complex 4 should have a slightly higher
or comparable accumulation. In fact, a lower melanoma
uptake is observed for 4. On the other hand, compound
5 also displays a lower melanoma uptake compared with
complex possesses a more basic amine in the side chain
and a higher lipophilicity caused by the N-dibutyl group.
A further increase in lipophilicity, however, does not
improve in vivo tumor uptake (see 4, Tables 2 and 3).
Although it is not clear what other parameters or
structural features may affect the in vivo tumor uptake,
complex 2 possesses a basic amine group with a pKa of
9.9 and a stronger H-bond donor character at physiologic
pH which seem to favor high in vivo tumor uptake.
In Vitr o Tu m or -Up ta k e Stu d ies. To gain a better
understanding of the tumor-uptake mechanisms, com-
pounds 1 and 2 were further examined in vitro in the
B16 melanoma and in an amelanotic C3H 10T1/2
fibroblast cell line. The B16 melanoma tumor-cell-
uptake kinetics of compound 2 at 37 °C reveal that the
complex is readily taken up and 68% of the complex is
cell-associated in 60 min (Figure 3a). Consistent with
the in vivo results, compound 1 displays a significantly
lower uptake at 37 °C with 38% total uptake in 60 min
(Figure 3c). To differentiate the active and passive
components of the uptake, experiments were performed
at 4 °C. Under these conditions a 65% reduction in the
total cell uptake of 1 (Figure 3c) and a 30% reduction
in that of 2 (Figure 3a) were observed. To confirm that
the reduced cellular uptake at 4 °C was due to reduced
complex 2, even though the log D(pH
for both is
7.4)
equivalent. Thus, a simple correlation between mela-
noma uptake and lipophilicity (log D(pH 7.4)) does not
seem to exist. This is consistent with earlier reports on
a series of radioiodinated benzamide derivatives.14 The
presence of a â-oxygen atom in the cyclic amine (3) or a
variation of the pharmacophore position in the molecule
(6) leads to a less basic tertiary amine nitrogen and
results in decreased melanoma uptake. Thus a more
constrained molecule caused by either the cyclic amine
(3) or by positioning on the tridentate ligand (6) and a
pKa lower than 9.7 cause decreased melanoma uptake
in this class of compounds.
As mentioned above, the highest melanoma uptake
is obtained with compound 2. Unlike compound 1, this

‘3+1’ Mixed-Ligand Oxotechnetium(V) Complexes
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2749
a
b
c
d
F igu r e 3. In vitro cell uptake in B16 murine melanoma (5 × 106 cells/mL) at 37, 4, and 37 °C with DTG (90 µM) of compounds
1 and 2: (a) uptake kinetics of 2, (b) dose-dependent uptake inhibition of 2 at 37 °C with DTG, (c) uptake kinetics of 1, (d)
dose-dependent uptake inhibition of 1 at 37 °C with DTG.
metabolism and not cell death, following a 4 °C (60 min)
cell-uptake experiment, the cells were reincubated at
37 °C (60 min). This restored the uptake to the original
values obtained at 37 °C (data not shown), thus indicat-
ing a significant active component of the total tumor-
cell uptake. This active component is consistent with
the lipophilicity measurements and indicates a greater
nonspecific uptake component in 2 and, conversely, a
greater active uptake component for the less lipophilic
1. Similar cellular uptake kinetic studies for complexes
1 and 2 in rapidly dividing, non-melanoma murine C3H
10T1/2 fibroblasts at 37 and 4 °C displayed significantly
less accumulation, with a maximum uptake of only 15-
18% at 60 min (data not shown) compared with 68%
(for 2) and 37% (for 1) in the B16 melanoma cells.
Although the exact nature and mechanism of uptake
are not known for these complexes, the reduced uptake
at 4 °C may be related to a decreased incorporation of
these complexes in melanin synthesis as has been
postulated for the parent iodinated benzamides.17 Since
structural analogues of the parent benzamides have
been shown previously to possess σ-receptor affinity,18,19
in vitro tumor-uptake studies of these technetium-99m
complexes were performed in the presence of either
N-(2-diethylaminoethyl)-4-iodobenzamide (BZA) or the
structurally different 1,3-di-o-tolylguanidine (DTG),
both known σ-ligands with differing σ-affinity (Ki ) 2.1
and 28 nM, respectively).18 A 30-min pretreatment of
the tumor cells with either DTG or BZA (90 µM),
followed by a 60-min incubation at 37 °C with the
technetium-99m complexes, demonstrates a 43% and
44% reduction in the tumor-cell uptake of complexes 1
and 2, respectively (Figure 3c,a). Consistent with the
σ-receptor affinity of the inhibitors BZA and DTG, in
vitro dose-response experiments at 37 °C also show a
50% inhibition of the specific cellular uptake of complex
1 at 18.50 µM DTG (Figure 3d) and complex 2 at 2.50
µM DTG (Figure 3b) in B16 cells. Likewise, BZA, a
higher-affinity σ-receptor ligand of a different structural
class, causes a 50% reduction of the cellular uptake of
1 at 1.57 µM and 2 at 1.32 µM (data not shown).
Inhibition experiments with either DTG or BZA
performed at 4 °C indicate no dose-dependent decrease
in the cell uptake of compounds 1 and 2. Since a lower
temperature should not decrease the binding of the
technetium-99m complexes to the cell surface σ-receptor,
given the lipophilic nature of these complexes, it is
possible that specific σ-receptor binding may be a very
small fraction of the total binding at 4 °C and, therefore,
not observable in this whole-cell assay.
While binding to the σ-receptor by these technetium
complexes may contribute to cellular accumulation and
cannot be excluded, an alternative mechanism may
involve the inhibitory effect of σ-ligands on cell growth

2750 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Friebe et al.
and proliferation27,28 and thus indirectly affect the active
uptake observed for these complexes.
temperature for 3-4 days whereupon the precipitated oil
began to crystallize. Addition of methyl tert-butyl ether
completed the crystallization and the isothiouronium salt was
isolated by filtration of the precipitate followed by vacuum-
drying over P2O5: yield 15.479 g, 88%.
The isothiouronium salt (6.810 g, 30.0 mmol) was dissolved
in 20 mL of water, sodium hydroxide (3.600 g, 90.0 mmol) was
added, and the reaction mixture was heated briefly at 100 °C
for 3-5 min. The solution was quickly cooled to 20 °C in an
ice bath and extracted with 3 × 10 mL of diethyl ether. The
dried ether extract (MgSO4) was filtered and reduced in volume
to a pale yellow oil. The pure product was obtained as a
colorless oil by subsequent distillation (Kugelrohr): yield 2.050
g, 13.6 mmol, 45%.
Con clu sion s
‘3+1’ Mixed-ligand technetium-99m complexes with
significant affinity for malignant melanoma were syn-
thesized by incorporating into the monodentate portion
of the complex N-(2-dialkylaminoalkyl) fragments that
are known to contribute to the melanoma affinity of
radioiodinated benzamides.15 The effect of pKa, log D(pH
7.4), and log P on the melanoma uptake of this series of
complexes indicates that, although there is no correla-
tion between uptake and lipophilicity (log P) of the ‘3+1’
technetium-99m complexes, a pKa of 9.9 and a log
2-[(2-Mercaptoethyl)propylamino]ethanethiol(b),2-[Ben-
zyl(2-m er ca p toeth yl)a m in o]eth a n eth iol (c), a n d 2-[(2-
Dieth yla m in oeth yl)(2-m er ca p toeth yl)a m in o]eth a n eth i-
ol (d ) (Com p lexes 4-6). The appropriate primary amine (n-
propylamine, benzylamine, N-(2-diethylamino)ethyleneamine)
(41.0 mmol) was dissolved in 80 mL of toluene and saturated
with dry argon in an autoclave bottle. To this solution was
added ethylene sulfide (5.850 g, 80.0 mmol) dropwise while
stirring under argon. The reaction mixture was tightly sealed
and stirred for 2 h at room temperature followed by heating
at 80 °C for 8 h. The slightly yellowish solution was filtered
to remove a small amount of a solid white impurity. The
filtrate was then concentrated to a pale yellow oil by evapora-
tion and subsequently purified by vacuum distillation: yield
(b) 6.444 g (88%), (c) 5,770 g (62%), (d) 5.737 g (66%).
Mon od en ta te Liga n d s. N-(2-Dibu tyla m in o)eth a n eth iol
(e) a n d 2-Mor p h olin -4-yleth a n eth iol (f) (Com p lexes 2-4).
The procedure described above was used with the following
stoichiometric equivalents of the reactants: 16.9 mmol of
secondary dibutylamine or morpholine and 16.50 mmol of
ethylene sulfide: yield (e) 1.933 g (62%), (f) 2.183 g (90%).
‘3+1’ Rh en iu m Com p lexes. These were synthesized and
characterized by procedures described in detail elsewhere.24
Gen er a l P r oced u r e for th e Syn th esis of ‘3+1’ Tech n e-
tiu m -99m Com p lexes (1-6). The complexes were prepared
by mixing [99mTc]pertechnetate (13.5-27 mCi in 0.5-1.0 mL
of saline), propylene glycol (400 µL), the appropriate mono-
dentate ligand (e-h ) (∼0.5 mg in 100 µL of methanol) and
the appropriate tridentate ligand (a -d ) (∼0.05 mg in 100 µL
of methanol) with 20 µL of stannous chloride solution (1.0-
2.0 mg SnCl2 dissolved in 5 mL of 0.1 N HCl) and heating the
reaction mixture at 45 °C for 20 min. After cooling to room
temperature the radio-metalated complexes were purified by
HPLC (vide supra). Yields generally ranged from 60% to 98%
(Scheme 1).
Deter m in a tion of Lip op h ilicity a n d p Ka Va lu es. The
lipophilicity and pKa values of all complexes were determined
using HPLC methods described earlier.25,26 The log P, log D(pH
7.4) and pKa values were determined on a Perkin-Elmer HPLC
system 1020 using a PRP-1 (250 × 4.1 mm, 10 µm; Hamilton)
reversed-phase column run under isocratic conditions with a
flow rate of 1.5 mL/min at room temperature. The mobile
phase was acetonitrile:phosphate buffer (0.01 M), 3:1, and
pKa’s were determined between 3 and 11.25,26
The pKHPLC values were obtained as the fitted points of
inflection from the sigmoidal log DHPLC/pH profiles. The
aqueous ionization constants pKa were calculated from the
pKHPLC values after correction with a predetermined correction
factor obtained using standard amine compounds.25,26 Log P
values of the neutral complexes were estimated from the
respective upper plateau of the sigmoidal log D/pH curve in
the alkaline range.
D(pH
of 1 may be optimum for in vivo melanoma
7.4)
uptake of these complexes. The high uptake of com-
pound 2 compared with other small benzamide-contain-
ing, nonpeptidic technetium-99m complexes20-22 indi-
cates that such integrated complexes can lead to higher
melanoma uptake and lends credence to the develop-
ment of such SPECT imaging agents for the early
diagnosis of malignant melanoma.
Exp er im en ta l Section
All chemicals used were of analytical grade and were
obtained from Sigma, Aldrich, or Fluka. Yields displayed are
values obtained without attempts at optimization. Mass
spectra were recorded on a MS device FINNIGAN MAT-95
using the “fast atom bombardment” (FAB) ionization tech-
1
nique. 400-MHz H NMR measurements were carried out on
a VARIO-400 device (Varian). Elemental analyses were per-
formed on
a LECO-CHNS-932 elemental analyzer. N-(2-
Diethylamino)ethanethiol (g) and sodium methanethiolate (h ),
the monodentate ligands for compounds 1, 5, and 6, were
obtained from Fluka, and N-(2-diethylaminoethyl)-4-iodoben-
zamide was synthesized according to the literature.18 Phos-
phate buffer (PB), 0.01 M, pH 7.4, contained Na2HPO4 and
KH2PO4, while phosphate-buffered saline (PBS) contained 0.01
M PB (pH 7.4) with 0.12 M NaCl and 0.0027 M KCl.
All complexes were purified by HPLC using a Perkin-Elmer
device consisting of a Turbo LC System with a quaternary
pump (series 200 LC Pump), a programmable UV/VIS detector
model 785A, and a homemade γ-detector (Bohrloch NaI(Tl)
crystal) connected in series with the UV detector. All HPLC’s
were performed using a Hypersil-ODS column (250 × 8 mm,
10 µm; Knauer) and run under isocratic conditions with a 80:
20 solvent mixture of methanol/phosphate buffer at a flow rate
of 2 mL/min as the mobile phase. The column effluent was
monitored simultaneously by both UV (254 nm) and γ-detec-
tion. The identity of the technetium-99m complexes was
established by HPLC comparison with previously character-
ized analogous rhenium compounds (Table 1).24
Tr is-Ch ela tin g Liga n d s. N-Meth yl-3-a za p en ta n e-1,5-
d ith iol (a ) (Com p lexes 1-3). N-Methyl-2,2′-aminodiethanol
(12.000 g, 0.10 mol) was first converted to the hydrochloride
salt by dissolving it in 30 mL of trichloromethane and treating
it with HCl-saturated diethyl ether. The hydrochloride salt was
then dissolved in 30 mL of trichloromethane, and thionyl
chloride (17.800 g, 0.15 mol) was slowly added while stirring
under nitrogen at room temperature. After the reaction
mixture turned yellow and became homogeneous, an additional
volume of thionyl chloride (17.800 g, 0.15 mol) was added and
the reaction mixture was refluxed for 2 h. After cooling the
mixture to room temperature and removing excess thionyl
chloride by evaporation, the precipitated bis(2-chloroethyl)-
methylamine hydrochloride was isolated via filtration and
dried under vacuum: yield 19.260 g, 99%.
An im a l Stu d ies. All animal experiments were performed
in compliance with the Principles of Laboratory Animal Care
(NIH Publication #85-23, revised 1985). Biodistribution studies
and tumor-uptake measurements were performed in C57B16
mice (15-20 g) bearing the B16 murine melanoma on the hind
limb.14-17 The tumor cells (B16/F0), obtained from the German
Cancer Research Centre (Heidelberg), were washed with PBS
and transplanted subcutaneously on the left hind flank by an
A mixture of bis(2-chloroethyl)methylamine hydrochloride
(9.677 g, 50.40 mmol) and thiourea (8.252 g, 0.106 mol) was
refluxed in 35 mL of ethanol for 5 h. After cooling the mixture
to room temperature, the solution was allowed to stand at room

‘3+1’ Mixed-Ligand Oxotechnetium(V) Complexes
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2751
inoculation of 0.5 × 106 cells (0.1 mL). Ten to 14 days later
the animals developed palpable tumor nodules 3-5 mm in
diameter. The biodistribution studies were carried out by tail-
vein injection of 25-30 µCi (0.05-0.1 mL) of the 99mTc-labeled
‘3+1’ derivative. At designated times postinjection, the animals
were weighed and sacrificed. The harvested organs and tumors
were blotted dry when appropriate, weighed, and counted in
a γ-counter along with technetium-99m standards of the
injected dose. The results are expressed as % ID/g tissue
(Figure 2, Table 3).
In Vitr o Cell Stu d ies. Murine B16/F0 melanoma cells
(ATCC) were grown in T-75 flasks in 14 mL of Dulbecco’s
modified Eagle medium (D-MEM; Gibco, Life Technology,
Gaithersburg, MD) containing 4500 mg/L D-glucose, L-gluta-
mine, and pyridoxine hydrochloride, 110 mg/L sodium pyru-
vate, 10% fetal bovine serum (FBS), 0.2% gentamicin and 0.5%
penicillin-streptomycin solution as the cell culture medium.
C3H 10T1/2 fibroblast cells (ATCC) were cultivated in T-75
flasks in Basal medium Eagle (BME; Gibco) supplemented
with 10% defined fetal bovine serum (Hyclone, cat. no.
SH30070, matched lots AGM7413 and AFC5040) and 25 µg/
mL gentamicin. All cells were harvested from cell culture
flasks by trypsinization with 1 mL trypsin-EDTA solution
(0.25% trypsin, 1 mM EDTA‚4Na) (Gibco). After being washed
with 12 mL of Dulbecco’s phosphate-buffered saline (PBS)
(Gibco), pH 7.2 (Ca2+- and Mg2+-free; g/L KCl, 0.20; KH2PO4,
0.20; NaCl, 8.00; Na2HPO4, 1.15), the cells were counted and
resuspended in 8 mL of S-MEM (Gibco) (Ca2+-free, with
reduced Mg2+content) and stored at 4 °C until use.
For in vitro tumor-cell-accumulation studies, 5 × 106 cells
were incubated at 37 or 4 °C in polypropylene test tubes with
intermittent agitation with 1-2 µCi (5 µL) technetium-99m
complex (1-6) in a total volume of 350 µL of S-MEM. At
appropriate time intervals the tubes were vortexed and 8-µL
samples were layered on 350 µL of cold FBS in a 400-µL
Eppendorf microcentrifuge tube. After centrifugation at 15000
rpm for 2 min, the tubes were frozen in a dry ice-acetone bath.
While still frozen, the bottom tip of the microcentrifuge tube
containing the cell pellet was cut and placed in a counting tube.
The remaining portion of the tube with the supernatant was
placed in a separate counting tube. Both fractions were
counted for radioactivity in a γ-counter (Wallac, 1480 WIZARD
3′′). The amount of supernatant in the cell pellet was deter-
mined to be <1% in separate experiments. The percentage cell
uptake of the technetium-99m complex was calculated as:
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% uptake ) [cpm (pellet)]/[cpm (pellet) +
cpm (supernatant)] × 100
The effect of the inhibitors (DTG, BZA) on cell uptake of
these complexes was studied by addition of the inhibitors at
various concentrations to the cell suspension 30 min prior to
addition of the 99mTc complexes. Fresh DTG stock solutions
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of PBS and 0.12 mL of hydrochloric acid (0.1 N) and subjecting
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added to the cell suspension such that the final concentration
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