Small molecule inhibitors of PSMA incorporating technetium-99m for imaging prostate cancer: Effects of chelate design on pharmacokinetics

Article abstract of DOI:10.1016/j.ica.2012.03.002

Single amino acid chelate (SAAC) systems for the complexation of the M(CO)₃ moiety (M = Tc/Re) have been successfully incorporated into novel synthetic strategies for radiopharmaceuticals and evaluated in a variety of biological applications. However, the lipophilicity of the first generation of ^99mTc(CO)₃ complexes has resulted in substantial hepatobiliary uptake when examined either as lysine derivatives or integrated into biologically active small molecules and peptides. Here, we designed, synthesized, and evaluated novel polar functionalized imidazole derived SAAC systems (SAAC II) which have been chemically modified to promote overall ^99mTc(CO)₃L₃ complex hydrophilicity with the intent of reducing non-target effects and enhancing renal clearance of prostate specific membrane antigen (PSMA) targeting small molecules. The ^99mTc-labeled compounds were prepared, purified, and evaluated for stability, lipophilicity, and tissue distribution in LNCaP xenograft mice. The Glu-urea-Lys-C11 analogs were prepared with a variety of chelators to form (19R,23S)-1-(X)-2-((Y)methyl)-13,21-dioxo-2,14,20,22-tetraazapentacosane-19,23, 25-tricarboxylic acid where X = Y = (methyl)pyridin-2-yl (6), X = Y = (methyl)-1H-imidazol-2-yl (7), X = (methyl)pyridin-2-yl, Y = carboxymethyl (8), X = Y = 1-(carboxymethyl)-1H-imidazol-2-yl (9), X = 1-(carboxymethyl)-1H- imidazol-2-yl, Y = carboxymethyl (10), and X = Y = 1-(1-(2-(bis(carboxymethyl) amino)-2-oxoethyl)-1H-imidazol-2-yl)-2-((1-(2-(bis(carboxymethyl)amino) -2-oxoethyl)-1H-imidazol-2-yl) (11). ^99mTc labeling was achieved at ligand concentrations as low as 10^-6 M and the complexes were stable (>90%) for 24 h. These new SAAC II chelators were evaluated for their influence on binding of the Glu-urea-Lys-C11 analogs to PSMA-positive LNCaP cells and compared to pyridine- and N-methylimidazole-containing SAAC ligands. Tissue distribution of the ^99mTc-complexes containing the more polar chelators, 9 and 11, demonstrated decreased liver (<2% ID/g) and increased LNCaP tumor (>11% ID/g) accumulation at 1 h post-injection.

Full text of DOI:10.1016/j.ica.2012.03.002

Inorganica Chimica Acta 389 (2012) 168–175
Contents lists available at SciVerse ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Small molecule inhibitors of PSMA incorporating technetium-99m
for imaging prostate cancer: Effects of chelate design on pharmacokinetics
Kevin P. Maresca, Shawn M. Hillier, Genliang Lu, John C. Marquis, Craig N. Zimmerman,
⇑
William C. Eckelman, John L. Joyal, John W. Babich
Molecular Insight Pharmaceuticals, Inc., 160 Second Street, Cambridge, MA 02142, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 10 March 2012
Single amino acid chelate (SAAC) systems for the complexation of the M(CO)3 moiety (M = Tc/Re) have
been successfully incorporated into novel synthetic strategies for radiopharmaceuticals and evaluated
in a variety of biological applications. However, the lipophilicity of the first generation of 99mTc(CO)3
complexes has resulted in substantial hepatobiliary uptake when examined either as lysine derivatives
or integrated into biologically active small molecules and peptides. Here, we designed, synthesized,
and evaluated novel polar functionalized imidazole derived SAAC systems (SAAC II) which have been
chemically modified to promote overall 99mTc(CO)3L3 complex hydrophilicity with the intent of reducing
non-target effects and enhancing renal clearance of prostate specific membrane antigen (PSMA) targeting
small molecules. The 99mTc-labeled compounds were prepared, purified, and evaluated for stability, lipo-
philicity, and tissue distribution in LNCaP xenograft mice. The Glu-urea-Lys-C11 analogs were prepared
with a variety of chelators to form (19R,23S)-1-(X)-2-((Y)methyl)-13,21-dioxo-2,14,20,22-tetraazapenta-
cosane-19,23,25-tricarboxylic acid where X = Y = (methyl)pyridin-2-yl (6), X = Y = (methyl)-1H-imidazol-
2-yl (7), X = (methyl)pyridin-2-yl, Y = carboxymethyl (8), X = Y = 1-(carboxymethyl)-1H-imidazol-2-yl
(9), X = 1-(carboxymethyl)-1H-imidazol-2-yl, Y = carboxymethyl (10), and X = Y = 1-(1-(2-(bis(carboxy-
methyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)-2-((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-
imidazol-2-yl) (11). 99mTc labeling was achieved at ligand concentrations as low as 10ꢀ6 M and the
complexes were stable (>90%) for 24 h. These new SAAC II chelators were evaluated for their influence
on binding of the Glu-urea-Lys-C11 analogs to PSMA-positive LNCaP cells and compared to pyridine-
and N-methylimidazole-containing SAAC ligands. Tissue distribution of the 99mTc-complexes containing
the more polar chelators, 9 and 11, demonstrated decreased liver (<2% ID/g) and increased LNCaP tumor
(>11% ID/g) accumulation at 1 h post-injection.
Dedicated to Prof. Jon Zubieta
Keywords:
Technetium-99m
Tc-tricarbonyl
SAAC II
PSMA inhibitors
Chelates
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
the overall pharmacological character and biological fate of the
complex, by altering the overall charge, polarity, hydrophobicity,
Metals have been used in medicine for radiodiagnostic and radi-
otherapeutic applications for decades [1–3]. Of all the radiometals
used in medicine today none has more prominence than the tran-
sition metal, technetium, specifically the radionuclide Tc-99m [4].
Tens of millions of imaging procedures are performed annually
worldwide using this radiometal attached to a variety of mostly
organic ligands that influence the biological fate and protein inter-
actions of the metal–ligand complex in vivo, thereby creating
scintigraphic images that allow physicians to infer diagnostic
information based on morphology and function [1,5].
and steric bulk. As the size of the structural moiety responsible
for biological targeting decreases the influence of the metal-com-
plex generally increases, such that large proteins like immunoglob-
ulins would be considerably less affected by the presence and
nature of a metal–chelate structure than smaller molecules (such
as an enzyme inhibitor or peptide) used for imaging biological
targets such as enzymes or receptors. It is therefore reasonable
to conclude that the chelate, the metal–chelate complex and the
nature of the outer solvation sphere of the metal chelate complex
all directly or indirectly influence the performance of such a radi-
olabeled ligand in regards to its intended pharmacological and
clinical purpose [6–11].
The polyvalent nature of Tc demands that due consideration is
given to the chemical approach used for its incorporation into a
targeting ligand. How the metal is bound can greatly influence
We have previously described a chelate platform incorporating
the epsilon amine of lysine as one of three donor atoms in a triden-
tate chelate system, referred to as single amino acid chelate (SAAC)
[12,13]. This amino acid based radiolabeling platform is
⇑
Corresponding author.
E-mail address: jbabich@molecularinsight.com (J.W. Babich).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.03.002

K.P. Maresca et al. / Inorganica Chimica Acta 389 (2012) 168–175
169
constructed from derivatized amino acids modified to provide
three donor groups (L3) for chelation to the {M(CO)3}+1 core
(M = 99mTc or Re), as described by Alberto and co-workers
[14,15]. SAAC, as exemplified by 2-amino-6-(bis-pyridin-2-yl-
methyl-amino)-hexanoic acid, (dipyridyl-lysine or DPK), demon-
noted. The following common abbreviations are used: AcOH =
acetic acid, DMF = N,N-dimethylformamide, DMSO = dimethylsulf-
oxide, DCM = dichloromethane, DCE = 1,2-dichloroethane, Glu =
glutamic acid, NaOH = sodium hydroxide, HATU = 2-(7-aza-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate, NaBH(OAc)3 = sodium triacetoxyborohydride, EtOAc = ethyl
acetate, TEA = triethylamine, DIPEA = diisopropylethyl amine, ID/
g = injected dose per gram, PBS = phosphate buffered saline,
RCP = radiochemical purity.
strated facile radiolabeling with
{
99mTc(CO)3}+1 and robust
stability of the formed complex [16,17]. In addition, SAAC ligands,
like DPK, may be incorporated into bioactive peptides via standard
solid phase peptide synthesis as the non-radioactive Re complex
({M(CO)3L3}+1) or as the free ligand which is subsequently labeled
with 99mTc or 186/188Re [18,19]. However, our experience to date
has shown that the hydrophobic nature of the dipyridyl metal
complexes has a powerful and unwanted affect on in vivo pharma-
cokinetics. We have recently shown that altering the nitrogen
containing ring structures of SAAC from pyridyl groups to function-
alized imidazolyl groups incorporating polar, hydrophilic groups
such as carboxylates can significantly reduce this affect on peptide
receptor targeting ligands such as octreotide [20].
2.2. Synthesis
2.2.1. General procedure for the alkylations
To a solution of the imidazole-2-carboxaldehyde dissolved in
DMF (1 mL) was added 1 mol equivalent of the alkylbromide,
excess potassium carbonate and a catalytic amount of potassium
iodide. The reactions were heated at 110 °C for 18 h followed by
evaporation to dryness and purified utilizing a Biotage SP4 with
a gradient method of 5–50% methanol in DCM.
Here, we describe the synthesis of a series of SAAC containing glu-
tamate-urea-lysine (Glu-urea-Lys) derivatives, for targeting PSMA,
with a variety of metal donor ligand groups to form (19R,23S)-1-(X)-
2-((Y)methyl)-13,21-dioxo-2,14,20,22-tetraazapentacosane-19,23,25
-tricarboxylic acid where X = Y = (methyl)pyridin-2-yl (6), X = Y =
(methyl)-1H-imidazol-2-yl (7), X = (methyl)pyridin-2-yl, Y = carboxy-
methyl (8), X = Y = 1-(carboxymethyl)-1H-imidazol-2-yl (9), X = 1-
(carboxymethyl)-1H-imidazol-2-yl, Y = carboxymethyl (10), and
X = Y = 1-(1- (2 -(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imida
.3zol-2-yl)-2-(1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-
imidazol-2-yl) (11). The 99mTc/Re complexes of these Glu-urea-Lys-
C11-SAAC ligands were then prepared and evaluated to determine
the influence the chelators had on the in vitro target affinity, and
in vivo tumor localization and normal organ tissue distribution in nude
mice bearing human prostate cancer xenografts.
2.2.2. General procedure for the reductive aminations
To a solution of 1-amino undecanoic acid dissolved in DCE
(2 mL) was added 2.1 equivalence of the aldehyde. The reaction
was heated at 50 °C for one hour whereupon 2 equivalents of so-
dium triacetoxyborohydride was added. The reaction stirred at
room temperature for 12 h and was subsequently evaporated to
dryness and purified using a Biotage SP4 with a gradient method
of 5–50% methanol in DCM afforded the desired compounds.
2.2.3. General synthesis of Glu-urea-Lys(X) analogs
The compounds (6, 7 and 9–11) of the general structure 5 were
prepared in overall yields ranging from 10% to 50% using the gen-
eral route depicted in Scheme 1. The key synthetic intermediate for
all the compounds was formed by reacting the appropriate alde-
hyde at room temperature for 1 h to form the schiff base interme-
diate. The schiff base was not isolated but was reduced in situ with
sodium triacetoxyborohydride to form the bis-derivatized amine
(2). The derivatized amine was coupled to (S)-di-tert-butyl-2-(3-
((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedio-
ate (1) using the terminal carboxylic acid, HATU and base to form
the protected intermediate 3. The synthesis of the Re(I) complexes
(4) was accomplished by the reaction of [NEt4]2[ReBr3(CO)3] with
the appropriate ligand in the ratio of 1:1.2 in 10 mL of methanol.
The reaction was heated at 80 °C for 4 h. Upon cooling the reaction
products were purified using C18 Sep Pak columns with yields
ranging from 20-50%. The tert-butyl ester protecting groups were
removed with 50% TFA in DCM after 12 h at room temperature.
Upon completion of the deprotection, the reactions were concen-
trated on a rotary evaporator and purified by HPLC or flash chro-
matography to afford the desired products (5) in 10–50% yield.
2. Experimental
2.1. General methods
All reactions were carried out in dry glassware under an atmo-
sphere of argon unless otherwise noted. Reactions were purified by
column chromatography under medium pressure using a Biotage
SP4 or by semi-preparative high pressure liquid chromatography
using a Varian Prostar 210 high pressure liquid chromatography
(HPLC) system equipped with a semi-preparative Vydac C18 re-
verse-phase column (250 mm ꢁ 10 mm ꢁ 5
lm) connected to a
Varian Prostar model 320 UV–Vis detector and monitored at a
wavelength of 254 nm. The final technetium complexes were puri-
fied and analyzed using a binary solvent gradient of 5–95% buffer B
over 21 min (buffer A = triethylammonium phosphate (TEAP), pH
3), (buffer B = methanol) or were purified and analyzed with a Vy-
2.2.4. (19R,23S)-tri-tert-butyl-13,21-dioxo-1-(pyridin-2-yl)
-2-(pyridin-2-ylmethyl)-2,14,20,22-tetraazapentacosane-19,23,
25-tricarboxylate (6)
dac C18 reverse phase column (250 mm ꢁ 4.6 mm ꢁ 5
lm)
employing a gradient method of 5–50% buffer B over 30 minutes
(buffer A = water + 0.1% TFA, buffer B = acetonitrile + 0.1% TFA).
1H NMR spectra were obtained on a Bruker 400 MHz instrument.
Spectra are reported as d (ppm) and are referenced to the solvent
resonances in chloroform-d (CDCl3), dimethylsulfoxide-d6
(DMSO-d6) or methanol-d4 (MeOD). 99mTc was obtained from a
commercial 99Mo/99mTc generator eluant (Cardinal Health) and
was used as a solution of Na99mTcO4 in saline. The 99mTc containing
solutions are kept behind a lead shield that offers sufficient protec-
tion. The [99mTc(CO)3(H2O)3]+ utilized in the formation of the 99mTc
complexes was prepared from commercially available Isolink™
kits (Mallinckrodt/Covidien). Solvents and reagents were pur-
chased from Sigma Aldrich (St. Louis, MO), Bachem (Switzerland),
Akaal (Long Beach, CA), or Anaspec (San Jose, CA) unless otherwise
The
(19R,23S)-tri-tert-butyl-13,21-dioxo-1-(pyridin-2-yl)-2-
(pyridin-2-ylmethyl)-2,14,20,22-tetraazapentacosane-19,23,25-
tricarboxylate (6) was prepared following the same general proce-
dure as shown in Scheme 1, using previously prepared and
protected
(S)-di-tert-butyl-2-(3-((S)-6-amino-1-(tert-butoxy)-1-
oxohexan-2-yl)ureido)pentanedioate (1). The reaction mixture
was purified by reverse phase HPLC 10–100% buffer B in buffer A
as eluent to afford 6 (151 mg, 0.17 mmol, 11%) as a white solid.
1H NMR (400 MHz, DMSO-d6) d 8.47 (dd, 2H), 7.81 (m, 2H), 7.70
(s, 1H), 7.45 (m, 2H), 7.37 (m, 2H), 6.25 (m, 2 H), 4.25 (m, 2H),
3.95 (s, 4H), 2.98 (m, 2H), 2.30 (m, 2 H), 2.20 (m, 4H), 2.05 (m,
2H), 1.90 (m, 2H), 1.60 (m, 4H), 1.38 (s, 27H), 1.20 (m, 16 H); MS
(ESI): 854(M+H)+.

170
K.P. Maresca et al. / Inorganica Chimica Acta 389 (2012) 168–175
L
N
L
(CO)3
M
n
L
O
L
OH
N
L
n
N
L
n
NH2
O
2
O
O
HN
O
O
O
HN
[NEt4]2[ReBr3(CO)3]
O
O
O
O
O
O
HATU
triethylamine
O
N
H
N
H
O
O
O
O
N
H
N
H
O
O
N
H
N
H
4
L = heterocyclic or
O
O
O
O
aliphatic donor groups
1
3
(CO)3
M
L
N
L
n
O
TFA
HN
M = Re or 99m
Tc
O
OH
O
HO
OH
N
H
N
H
O
O
5
Scheme 1. General pathway for the synthesis of 99mTc or Re-Glu-urea-Lys-X analogs (5).
2.2.5. [Re(CO)3{((19R,23S)-13,21-dioxo-1-(pyridin-2-yl)-2-(pyridin-
2-ylmethyl)-2,14,20,22-tetraazapentacosane-19,23,25-tricarboxylic
acid}] (6R)
H), 7.03 (d, 2H), 6.33 (m, 2H), 5.72 (s, 4H), 4.60 (dd, 4H), 4.00 (m,
2H), 3.6 (s, 6H), 2.98 (d, 2H), 2.26 (m, 2H), 2.39 (t, 2H), 2.24 (m,
4H), 2.01 (t, 4H), 1.83 (m, 2H), 1.60–1.35 (m, 4H), 1.30 (m, 16H);
MS (ESI): 962 (M+H)+.
The rhenium complex was prepared following the same proce-
dure as described in the general rhenium experimental methods
using 6 to yield the desired product 6R (6 mg, 0.0063 mmol,
7.4%) as an off-white solid. 1H NMR (400 MHz, d6-DMSO) d 8.77
(dd, 2H), 7.98 (t, 2H), 7.70 (s, H), 7.47 (d, 2H), 7.38 (m, 2 H), 6.25
(m, 2H), 4.84 (m, 2H), 3.75 (m, 4H), 3.0 (m, 2H), 2.25 (m, 2H),
2.20 (m, 4H), 2.05 (m, 2H), 1.85 (m, 2H), 1.55 (m, 4H), 1.22 (m,
16 H); MS (ESI): 955 (M+H)+.
2.2.8. (19R,23S)-13,21-dioxo-2-(pyridin-2-ylmethyl)-2,14,20,22-tetra
azapentacosane-1,19,23,25-tetracarboxylic acid (8)
The (19R,23S)-13,21-dioxo-2-(pyridin-2-ylmethyl)-2,14,20,22-
tetraazapentacosane-1,19,23,25-tetracarboxylic acid (8) was pre-
pared employing the same general procedure as shown in
Scheme 1, using previously prepared and protected (S)-di-tert-
butyl-2-(3-((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)-
pentanedioate (1). The reaction mixture was purified by Biotage
SP4 with a gradient method of 5–50% methanol in DCM as eluent
to afford 8 (3 mg, 0.005 mmol, 9%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) d 8.45 (d, H), 7.95 (t, H), 7.75 (m, 2H), 7.65
(m, 2H), 6.40 (m, 2H), 4.60 (m, 2H), 4.05 (s, 2H), 3.62 (s, 2H), 3.0
(m, 2H), 2.65 (m, 2H), 2.30 (m, 2H), 2.15 (m, 4H), 1.98 (m, 2H),
1.50 (m, 4H), 1.3 (m, 16H); MS (ESI): 652 (M+H)+.
2.2.6. (19R,23S)-tri-tert-butyl-1-(1-methyl-1H-imidazol-2-yl)-2-((1-
methyl-1H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,20,22-tetraazape
ntacosane-19,23,25-tricarboxylate (7)
(19R,23S)-tri-tert-butyl-1-(1-methyl-1H-imidazol-2-yl)-2-((1-
methyl-1H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,20,22-tetra-
azapentacosane-19,23,25-tricarboxylate (7) was prepared follow-
ing the same general procedure as shown in Scheme 1, using
previously prepared and protected (S)-di-tert-butyl-2-(3-((S)-6-
amino-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedioate (1).
The reaction mixture was purified by use of a Biotage SP4 purifica-
tion system with a gradient of 5–50% methanol in DCM as eluent to
afford 7 (57 mg, 0.066 mmol, 9%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) d 7.70 (s, H), 7.10 (d, 2 H), 6.84 (d, 2H), 6.23
(m, 2H), 4.00 (m, 2H), 3.6 (s, 2H), 3.44 (s, 2 H), 3.07 (s, 6H), 3.02
(m, 2H), 2.39 (t, 2H), 2.24 (m, 4H), 2.05 (t, 2H), 1.60 (m, 4H), 1.35
(s, 27H), 1.15 (m, 16H); MS (ESI): 860(M+H)+.
2.2.9. [Re(CO)3{(19R,23S)-13,21-dioxo-2-(pyridin-2-ylmethyl)-
2,14,20,22-tetraazapentacosane-1,19,23,25-tetracarboxylic acid}]
(8R)
The rhenium complex was prepared employing the same proce-
dure as described in the general rhenium experimental. The com-
pound was deprotected using the previously described methods
to yield the desired product 8R (3.0 mg, 0.003 mmol, 75%) as an
off-white solid. MS (ESI): 922 (M+H)+.
2.2.7. [Re(CO)3{(19R,23S)-1-(1-methyl-1H-imidazol-2-yl)-2-((1-methyl-
1H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,20,22-tetraazapentacosane-
19,23,25-tricarboxylic acid}] (7R)
The rhenium complex was prepared following the same proce-
dure as described in the general rhenium experimental methods to
yield the desired product 7R (3.2 mg, 0.003 mmol, 5.2%) as an off-
white solid. 1H NMR (400 MHz, DMSO-d6) d 7.70 (s, H), 7.10 (d, 2
2.2.10. (19R,23S)-tri-tert-butyl-1-(1-(2-tert-butoxy-2-oxoethyl)-1H-
imidazol-2-yl)-2-((1-(2-tert-butoxy-2-oxoethyl)-1H-imidazol-2-
yl)methyl)-13,21-dioxo-2,14,20,22-tetraazapentacosane-19,23,25-
tricarboxylate (9)
The (19R,23S)-tri-tert-butyl-1-(1-(2-tert-butoxy-2-oxoethyl)-
1H-imidazol-2-yl)-2-((1-(2-tert-butoxy-2-oxoethyl)-1H-imidazol-
2-yl)methyl)-13,21-dioxo-2,14,20,22-tetraazapentacosane-19,23,

K.P. Maresca et al. / Inorganica Chimica Acta 389 (2012) 168–175
171
25-tricarboxylate was prepared following the same general
procedure as shown in Scheme 1, using previously prepared and
protected (S)-di-tert-butyl-2-(3-((S)-6-amino-1-(tert-butoxy)-1-
above deprotected product in water (1.0 mL) that was adjusted to pH
9 with 2 N NaOH was added Re(CO)3(H2O)OTf (0.50 mL, 0.10 mL/
mmol). The reaction mixture were stirred at room temperature over-
night and purified by HPLC 10–100% buffer B buffer in buffer A as eluent
to afford 10R (4.0 mg, 0.004 mmol, 19%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) d 7.70 (t, 1H), 7.33 (s, 1H), 7.13 (s, 2H), 6.29 (d,
1H), 6.26 (d, 1H), 4.96 (d, 2H), 4.56 (d, 1H), 4.12 (d, 1H), 4.07–3.90 (m,
2H), 3.70 (d, 1H), 3.40 (d, 1H), 2.98–2.94 (m, 4H), 2.21 (q, 2H), 1.99 (t,
2H), 1.70–1.22 (m, 24H); MS (ESI): 485.2 (M/2+H)+.
oxohexan-2-yl)ureido)pentanedioate (1) to afford
9 (234 mg,
0.22 mmol, 61%) as a white solid. 1H NMR (400 MHz, DMSO-d6) d
8.25 (s, 1H), 7.20 (s, 2H), 6.95 (s, 2H), 6.22 (m, 2H), 4.80 (d, 4H),
4.11 (m, 2H), 3.80 (s, 4H), 2.99 (m, 2H), 2.46 (m, 2H), 2.30 (m,
4H), 2.1 (m, 2H), 1.90 (m, 2H), 1.55 (m, 4H), 1.30 (s, 45H), 1.2 (m,
16H). MS (ESI): 530(M/2).
2.2.11. [Re(CO)3(19R,23S)-1-(1-(carboxymethyl)-1H-imidazol-2-yl)-
2-((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)-13,21-dioxo-
2,14,20,22-tetraazapentacosane-19,23,25-tricarboxylic acid}] (9R)
The rhenium complex was prepared employing the same proce-
dure as described in the general rhenium experimental to yield the
desired product 9R (7.0 mg, 0.006 mmol, 24%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) d 7.8 (s, H), 7.2 (s, 2H), 7.0 (2, 2H), 6.3
(s, 2H), 4.8 (s, 4H), 4.55 (d, 2H), 4.1 (m, 4H), 2.9 (m, 2H), 2.2 (m, 4H),
2.05 (m, 4H), 1.7 (m, 2H), 1.45 (m, 4H), 1.3 (m, 16H); MS (ESI): 525
(M/2).
2.2.15. (19S,23S)-tri-tert-butyl-1-(1-(2-(bis(2-tert-butoxy-2-oxoethyl)
amino)-2-oxoethyl)-1H-imidazol-2-yl)-2-((1-(2-(bis(2-tert-butoxy-2-
oxo ethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)-13,21-dioxo-
2,14, 20,22-tetraazapentacosane-19,23,25-tricarboxylate (11)
(19S,23S)-1-(1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-
imidazol-2-yl)-2-((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-
1H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,20,22-tetraazapenta-
cosane-19, 23, 25-tr icarboxylic acid was prepared following the
same general procedure as shown in Scheme 1, using previously
prepared and protected (S)-di-tert-butyl-2-(3-((S)-6-amino-1-
(tert-butoxy)-1-oxohexan-2-yl)ureido) pentanedioate (1).
2.2.12. (19S,23S)-tetra-tert-butyl-2-((1-(2-tert-butoxy-2-oxoethyl)-
1H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,20,22-tetraazapenta-
cosane-1,19,23,25-tetracarboxylate (10)
2.2.16. Step 1. tert-butyl-2,20-(2-bromoacetylazanediyl)diacetate
To
a
solution of tert-butyl-2,20-azanediyldiacetate (3.00 g,
A suspension of 11-aminoundecanoic acid (603 mg, 3.0 mmol), 2-
pyridinecarboxaldehyde (630 mg, 3.0 mmol) and AcOH (0.20 mL) in
DCE (20 mL) was heated to reflux for 30 min. The reaction mixture
was cooled to 0 °C, and treated sequentially with NaBH(OAc)3 (1.9 g,
9.0 mmol) and crude tert-butyl glyoxalate (1.50 g, 11.5 mmol). The
reaction mixture was stirred at room temperature for overnight and
decomposed with water. The reaction mixture was extracted with
DCM. The organic layer was dried and concentrated under reduced
pressure. The residue was purified by Biotage SP4 5-50% methanol in
DCM as eluent to afford 11-((2-tert-butoxy-2-oxoethyl)((1-(2-tert-
butoxy-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino)undecanoic acid
(343 mg, 0.67 mmol, 22%) as a yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.00 (d, 1H), 6.87 (d, 1H), 5.30 (s, 1H), 5.07 (s, 1H), 4.67 (s, 2H), 4.66
(s, 2H), 3.83 (s, 1H), 3.17 (s, 1H), 2.41–2.32 (m, 2H), 1.66–1.63 (m,
2H), 1.47 (s, 9H), 1.45 (s, 9H), 1.42–1.10 (m, 14H); MS (ESI): 510
(M+H)+.
12.24 mmol) and 2-bromoacetyl bromide (3.23 g, 1.39 mL,
16.00 mmol) in DCM (100 mL) was added TEA (2.0 mL) at room
temperature. The reaction mixture was stirred at room tempera-
ture for 2 h then diluted with DCM (300 mL), washed with water,
and dried over sodium sulfate. Solvent was evaporated under re-
duce pressure to afford a residue, which was purified by biotage
eluting with 10% hexanes in EtOAc to 50% hexanes in EtOAc to
tert-butyl-2,20-(2-bromoacetylazanediyl)diacetate (4.68 g, 100%).
1H NMR (400 MHz, CDCl3) d 4.09 (s, 2H), 4.07 (s, 2H), 3.86 (s,
2H), 1.49 (s, 9H), 1.46 (s, 9H); MS (ESI): 388, 390 (M+Na)+.
2.2.17. Step 2. tert-butyl-2,20-(2-(2-formyl-1H-imidazol-1-
yl)acetylazanediyl)diacetate
A solution of tert-butyl-2,20-(2-bromoacetylazanediyl)diacetate
(4.55 g, 12.43 mmol), 1H-imidazole-2-carbaldehyde (1.54 g,
16.0 mmol), DIPEA (5.0 mL), and potassium iodide (0.64 g,
4.0 mmol) was stirred at 80 °C overnight. The solvent was evapo-
rated under reduced pressure, the reaction mixture was diluted
with DCM, washed with water and dried. The solvent was evapo-
rated under reduce pressure to afford a residue, which was purified
by Biotage SP4 DCM to 3% MeOH in DCM as eluent to afford the de-
sired product tert-butyl-2,20-(2-(2-formyl-1H-imidazol-1-yl)acety-
lazanediyl)diacetate (3.96 g, 84%). 1H NMR (400 MHz, CDCl3) d 9.76
(s, 1H), 7.31 (s, 1H), 7.25 (s, 1H), 5.30 (s, 2H), 4.14 (s, 2H), 4.07 (s,
2H), 1.51 (s, 9H), 1.43 (s, 9H); MS (ESI): 382 (M+H)+.
2.2.13. Step 2. (19S,23S)-tetra-tert-butyl-2-((1-(2-tert-butoxy-2-
oxoethyl)-1H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,20,22-
tetraazapentacosane-1,19,23,25-tetracarboxylate
A solution of (S)-di-tert-butyl-2-(3-((S)-6-amino-1-(tert-butoxy)-
1-oxohexan-2-yl)ureido)pentanedioate (1) (85 mg, 0.175 mmol),
11-((2-tert-butoxy-2-oxoethyl)((1-(2-tert-butoxy-2-oxoethyl)-1H-
imidazol-2-yl)methyl)amino)undecanoic acid (89 mg, 0.175
mmol), EDCI (38 mg, 0.20 mmol), HOBt (26 mg, 0.20 mmol) and DI-
PEA (0.30 mL) in DCM (5.0 mL) was stirred at rt for 3 days. The
reaction mixture was purified by Biotage SP4 5–50% methanol in
DCM as eluent to afford (19S,23S)-tetra-tert-butyl-2-((1-(2-tert-
butoxy-2-oxoethyl)-1H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,
20,22-tetraazapentacosane-1,19,23,25-tetracarboxylate (111 mg,
0.11 mmol, 65%) as a yellow oil. MS (ESI): 490.5 (M/2+H)+.
2.2.18. Step 3. 11-(bis((1-(2-(bis(2-tert-butoxy-2-oxoethyl)amino)-2-
oxoethyl)-1H-imidazol-2-yl)methyl)amino)undecanoic acid
A solution of 11-aminoundecanoic acid (100 mg, 0.50 mmol),
tert-butyl-2,20-(2-(2-formyl-1H-imidazol-1-yl)acetylazanediyl)
diacetate (381 mg, 1.0 mmol) and AcOH (0.02 mL) in DCE (30 mL)
was stirred at 75 °C for 30 min under nitrogen. The reaction mix-
ture was cooled to 0 °C, and treated with NaBH(OAc)3 (0.3165 g,
1.5 mmol). The reaction mixture was stirred at room temperature
overnight and decomposed with water. The solvent was evapo-
rated under reduce pressure to afford a residue, which was purified
by Biotage SP4 1–10% MeOH in DCM as eluent to afford the desired
product 11-(bis((1-(2-(bis(2-tert-butoxy-2-oxoethyl)amino)-2-oxo
ethyl)-1H-imidazol-2-yl)methyl)amino)undecanoic acid (368 mg,
79%). 1H NMR (400 MHz, DMSO-d6) d 6.93 (s, 2H), 6.76 (s, 2H),
5.02 (s, 4H), 4.29 (s, 4H), 3.93 (s, 4H), 3.44 (s, 4H), 2.30 (t,
2.2.14. [Re(CO)3{(19R,23S)-2-((1-(carboxymethyl)-1H-imidazol-2-
yl)methyl)-13,21-dioxo-2,14,20,22-tetraazapentacosane-1,19,23,25-
tetracarboxylic acid}] (10R)
A solution of (19S,23S)-tetra-tert-butyl-2-((1-(2-tert-butoxy-2-oxo-
ethyl)-1H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,20,22-tetraaza penta
cosane-1,19,23,25-tetracarboxylate (18.8 mg, 0.019 mmol) in TFA
(1.0 mL)/DCM (1.0 mL) was stirred at room temperature overnight.
The solvent was evaporated to give (19S,23S)-2-((1-(carboxymethyl)-1
H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,20,22-tetraazapentacosane -
1,19,23,25-tetracarboxylic acid as a colorless oil. To a solution of the

172
K.P. Maresca et al. / Inorganica Chimica Acta 389 (2012) 168–175
J = 7.6 Hz, 2H), 2.09 (t, J = 7.6 Hz, 2H), 1.43 (s, 18H), 1.35 (s, 18H),
protected tert-butyl ester derivatives. The radiolabeling was
accomplished in two steps using commercially available IsoLink™
kits (Covidien) to form the [99mTc(CO)3(H2O)3]+ intermediate,
which was neutralized and reacted with the appropriate SAAC or
SAAC II system (Fig 2) at a concentration of 10ꢀ6 M in an equal vol-
ume mixture of acetonitrile and water in a sealed vial. The sealed
vial was heated at 100 °C for 30 min and upon cooling the reaction
was analyzed for purity via reverse-phase HPLC. The tert-butyl es-
ter protecting groups were removed by treatment with 50% TFA in
DCM for 45 min at room temperature. Upon completion of the
deprotection, the reactions were concentrated on a rotary evapora-
tor and purified by HPLC. The excess chelating agent was separated
from the final 99mTc-products to the extent that <5% of the original
10ꢀ6 M chelate remained. The RCP was determined via HPLC and
shown to be consistently P90% pure.
1.29–1.00 (m, 16H); MS (ESI): 466.9 (M/2+H)+.
2.2.19. Step 4. (19S,23S)-tri-tert-butyl-1-(1-(2-(bis(2-tert-butoxy-2-
oxoethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)-2-((1-(2-(bis(2-tert-
butoxy-2-oxoethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)-
13,21-dioxo-2,14,20,22-tetraazapentacosane-19,23,25-tricarboxylate
A solution of (S)-di-tert-butyl-2-(3-((S)-6-amino-1-(tert-butoxy)-
1-oxohexan-2-yl)ureido)pentanedioate (1) (85 mg, 0.174 mmol), 11-
(bis((1-(2-(bis(2-tert-butoxy-2-oxoethyl)amino)-2-oxoethyl)-1H-imi
dazol-2-yl)methyl)amino)undecanoicacid(118 mg, 0.127 mmol), EDC
(38 mg, 0.20 mmol), HOBt (26 mg, 0.20) and DIPEA (0.30 mL) in DCM
(5.0 mL) was stirred at rt overnight. The reaction mixture was purified
by Biotage SP4 1–10% MeOH in DCM as eluent to afford (19S,23S)-tri-
tert-butyl-1-(1-(2-(bis(2-tert-butoxy-2-oxoethyl)amino)-2-oxoethyl)-
1H-imidazol-2-yl)-2-((1-(2-(bis(2-tert-butoxy-2-oxoethyl)amino)-
2-oxoethyl)-1H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,20,22-
tetraaza pentacosane-19,23,25-tricarboxylate (38 mg, 0.03 mmol,
21%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d 6.95 (d,
J = 1.2 Hz, 2H), 6.83 (d, J = 0.80 Hz, 2H), 5.97 (s, 1H), 5.28 (d,
J = 7.6 Hz, 1H), 5.23 (d, J = 8.4 Hz, 1H), 4.94 (s, 4H), 4.33–4.25 (m,
2H), 4.12 (s, 4H), 4.03 (s, 4H), 3.63 (s, 4H), 3.25–3.16 (m, 2H), 2.53
(t, J = 7.4 Hz, 2H), 2.33–2.24 (m, 2H), 2.15 (t, J = 7.6 Hz, 2H), 2.08–
2.03 (m, 2H), 2.02–1.20 (m, 85H); MS (ESI): 701.6 (M/2+H)+.
2.5. Tissue distribution in LNCaP bearing mice
Uptake of radiolabeled complexes in LNCaP xenografts was per-
formed according to published methods [23]. Briefly, LNCaP cells
were trypsinized, counted, and suspended in a solution containing
50% Dulbecco’s PBS (with 1 mg/mL D-glucose and 36 lg/mL sodium
pyruvate) and 50% Matrigel (BD Biosciences, Franklin Lakes, NJ).
Male NCrnu/nu mice were anesthetized by intraperitoneal injection
of 0.5 mL Avertin (20 mg/mL) (Sigma-Aldrich, St. Louis, MO) then
inoculated subcutaneously into the hind flank with 2 ꢁ 106 cells
in a 0.25 mL suspension volume. Studies of tumor uptake were
conducted when the tumors reached a size of 600–800 mm3. Tis-
sue distribution studies were conducted by administering, via the
2.2.20. [Re(CO)3{(19S,23S)-1-(1-(2-(bis(carboxymethyl)amino)-
2-oxoethyl)-1H-imidazol-2-yl)-2-((1-(2-(bis(carboxymethyl)amino)-
2-oxoethyl)-1H-imidazol-2-yl)methyl)-13,21-dioxo-2,14,20,22-
tetraazapentacosane-19,23,25-tricarboxylic acid}] (11R)
The rhenium complex was prepared following the same proce-
dure as described in the general rhenium experimental to yield the
desired product (17.6 mg, 0.014 mmol, 69% over 2 steps) as a white
solid. 1H NMR (400 MHz, DMSO-d6) d 7.70 (t, J = 4.8 Hz, 1H), 7.10 (s,
2H), 7.03 (s, 2H), 6.29 (d, J = 8.4 Hz, 1H), 6.26 (d, J = 8.4 Hz, 1H),
5.02 (s, 4H), 4.37–3.97 (m, 14H), 3.60–3.57 (m, 2H), 3.01–2.94
(m, 2H), 2.24–1.22 (m, 28H); MS (ESI): 640.3 (M/2+H)+.
tail vein, a bolus injection of 2
lCi/mouse in a constant volume
of 0.05 mL -PBS. Groups of five animals were euthanized by
D
asphyxiation with carbon dioxide at 1 h post injection. All tissues
from Table 2, including the tumor were excised, weighed wet,
transferred to plastic tubes and radioactivity assayed in an auto-
mated
c-counter (LKB Model 1282, Wallac Oy, Finland). Tissue
time-radioactivity levels were expressed as % injected dose per
gram tissue (%ID/g).
2.3. In vitro screening
3. Results and discussion
LNCaP human prostate cancer cells were obtained from Ameri-
can Type Culture Collection, Rockville, MD, and maintained in
RPMI-1640 medium supplemented with 10% fetal bovine serum
(FBS). Competitive binding of the cold Re derivatives to LNCaP cells
was performed as previously described [21]. Briefly, cells were pla-
ted in 12-well plates at approximately 4 ꢁ 105 cells/well and incu-
bated for 48 h in a humidified incubator at 37 °C/5% carbon dioxide
prior to addition of compound. Each Glu-urea-X derivative was
prepared and diluted in serum-free cell culture medium containing
0.5% bovine serum albumin (BSA) in combination with 3 nM
123I-(S)-2-(3-((S)-1-carboxy-5-(4-iodobenzylamino)pentyl)ureido)-
pentanedioic acid (MIP-1072) or N-[N-[(S)-1,3-dicarboxypro-
We previously described the SAAC platform as a labeling tech-
nology for use with Tc(CO)3 and Re(CO)3, that has broad utility in
radiochemical, fluorescent and dual modality (radio/fluorescent)
molecular imaging [24,25]. This platform is particularly convenient
for developing 99mTc(CO)3 – based radiolabeled small molecules.
One limitation of the original SAAC platform which emphasized
the Tc(CO)3-dipyridyl complex as the prototype SAAC system, is
the extremely hydrophobic nature of the radiolabeled complexes
which often lead to high liver accumulation and/or hepatobiliary
clearance of the bioconjugates derived from them [26]. Subse-
quently, we refined the platform to include imidazoles that would
pyl]carbamoyl]-S-3-iodo-L-tyrosine
(
123I-DCIT). Plates were
incubated at room temperature for 1 h. Cells were removed from
the plates by gently pipeting and transferred to eppendorff tubes.
Samples were microcentrifuged for 15 s at 10 K ꢁ g. The medium
was aspirated and the pellet was washed twice by dispersal in
fresh assay medium followed by microcentrifugation. Cell binding
SAAC (First Generation) Ligand
SAAC II (Second Generation) Ligand
N
of
123I-(S)-2-(3-((S)-1-carboxy-5-(4-iodobenzylamino)pentyl)
N
R1
N
R1
N
ureido)pentanedioic acid or 123I-DCIT was determined by counting
the cell pellet in an automated gamma counter.
N
R2
2.4. General preparation of 99mTc-complexes
R1 = imidazole (symmetric)
or COOH
R1 = 2-pyridyl (Dp derivative)
1 = COOH (PAMA derivative)
R
R2 = polar functionality
The 99mTc(CO)3 radiolabeling of the SAAC and SAAC II systems
+
to form the desired 99mTc metal complexes was accomplished
utilizing standard methodology [22] from the free acids or the
Fig. 1. General structure of the first (SAAC) and second generation (SAAC II) derived
ligand systems.

K.P. Maresca et al. / Inorganica Chimica Acta 389 (2012) 168–175
173
O
NH
OH
X
O
OH
O
HO
N
H
N
H
O
O
HO
N
O
O
O
O
OH
OH
O
HO
O
N
N
N
N
N
N
N
N
N
N
N
N
X = 6=
N
N
N
N
N
7 =
10 =
11 =
9 =
8 =
N
N
N
OH
OH
N
N
N
O
O
N
O
O
OH
O
OH
OH
Fig. 2. Structures of the chelates employed to form the 99mTc-PSMA inhibitors derived from SAAC (6–8) and SAAC II (9–11) chelators.
allow pendant groups that would add charge and polarity to the
overall metal-chelate complex independent of the coordinating
donor atom set. This second generation SAAC, enabled more
favorable pharmacokinetics and increased renal clearance of the
99mTc-labeled complexes. These changes in physicochemical
properties yielded strong influence over pharmacokinetics and
excretion. In addition, SAAC II systems could be successfully incor-
porated into the design of small ligands and low molecular weight
peptides such as octreotide without detrimentally influencing
receptor/enzyme interaction or pharmacokinetics and thus facili-
tate the development of novel 99mTc-labeled radiopharmaceuticals
that possess more favorable biological distributions.
This was accomplished by derivatization of the imidazole rings
[27] with polar carboxylic acid functional groups. The SAAC and
SAAC II chelate systems were then examined on the Glu-urea-
Lys-C11-X moiety (where X = SAAC or SAAC II derived chelate)
for determination of their influence on the affinity and pharmaco-
kinetic profiles of the complexes.
as previously described [16]. Deprotection employing trifluoroace-
tic acid (TFA) to yield the desired free ligand, 8. Complexation to
form the rhenium complex 8R followed as described above for
the homogenous SAAC and SAAC II systems.
3.2. In vitro binding and preliminary SAR of Glu-urea-X analogs
A series of Glu-urea-C11-SAAC/SAAC II heterodimers comprised
of a Glu linked through a urea functionality to the
a-amine of a
modified Lys derivative were prepared as shown in Fig 2. The Glu
residue contributes two of the three carboxylic acids important
for binding to the PSMA active site [28–30]. The modified Lys res-
idue contributes the third free carboxylic acid functionality and the
e-amine was utilized as the synthetic handle that led to the
efficient synthesis of this series of C11 compounds which aided
the rapid establishment of the SAR surrounding the chelator.
The compounds were evaluated in a competitive binding
assay using 123I-(S)-2-(3-((S)-1-carboxy-5-(4-iodobenzylamino)
pentyl)ureido)pentanedioic acid or 123I-DCIT as the radioligand
for binding to PSMA on LNCaP cells.
3.1. Chelate design
One of the first Glu-urea-X heterodimers prepared and evalu-
ated was 6. The modest affinity of the rhenium complex 6R (IC50
value 113 nM) shown in Table 1 demonstrated and confirmed that
the Glu-urea-Lys recognition sequence could be derivatized with a
metal chelate to afford active inhibitors with bulky metal centers
extended away from the active site with a tether [31–33]. Similar
binding affinity was observed with the rhenium analog of the
bis-dimethylimidazole complex 7R (IC50 value 183 nM) showing
that the N-methyl imidazole appears to be a tolerated substitution
for the 2-pyridyl ring in this series. The less bulky monoacetic acid
derived neutral complex 8R (IC50 value 696 nM) was significantly
less potent possibly indicating a loss of a beneficial hydrophobic
interaction with a secondary site on PSMA distant from the active
site.
As illustrated below in Fig 1, first generation SAAC ligands
incorporated either two pyridine rings or a pyridine and an acetic
acid for R1. The design of the novel second generation bifunctional
chelates explored the concept of increasing the water solubility
and polarity of the chelate ring systems through the modification
of the imidazole ring substituents, R2.
As previously reported, the homogenous polar derivatized imid-
azole SAAC II systems were prepared via the alkylation of the com-
mercially available imidazole-2-carboxaldehyde with the desired
alkyl halide to afford the N-substituted imidazole-2-carboxalde-
hyde analogs in moderate yields [20]. Reductive amination of 11-
aminoundecanoic acid using sodium triacetoxyborohydride as
the reducing agent produced the C11 derived SAAC/SAAC II inter-
mediates 2 in good yield. Amide bond formation of 1 with 2 affor-
ded the desired Glu-urea-Lys-C11-SAAC/SAAC II systems 3 in good
yields as illustrated in Scheme 1. Subsequent treatment with the
rhenium starting material [NEt4]2[ReBr3(CO)3] followed by purifi-
cation using C18 Sep Pak columns and subsequent deprotection
with trifluoroacetic acid (TFA) afforded the desired series of metal
complexes (5) in 5–60% yield.
Table 1
Summary of in vitro cell binding data of the Re-Glu-urea-Lys-C11 derivatives.
Complex
Chelator (X)
IC50 (nM)
6
7
8
9
10
11
Di-pyridine-2-amine
Di-(N-methylimidazole)
Picoline-amine mono-acetic acid
Di-(N-carboxymethyl-imidazole) (CIM)
Mono-CIM-mono-acetic acid
113
183
696
33
10
4
The heterogeneous monoacetic acid SAAC ligand 8, Fig 1 is
smaller in size and can form less hindered complexes with a neu-
tral uncharged metal center. The compounds in this series were
prepared using the double reductive alkylation sequence on the
appropriate protected amine followed by deprotection with TFA
Di-(N-dicarboxymethylamido-imidazole (TIM)

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K.P. Maresca et al. / Inorganica Chimica Acta 389 (2012) 168–175
Fig. 3. Radiochromatograms of the 99mTc-11 demonstrating >95% RCP (top), and the corresponding UV–Vis chromatogram of the rhenium complex used for identity
confirmation (bottom).
The polar substituted SAAC II complexes 9R–11R exhibited a
substantial improvement in binding affinity for PSMA. The metal
complexes, 9R (IC50 value 33 nM), and 10R at (IC50 value 10 nM)
both have a pendant free carboxylic acid group attached to the
imidazole donor rings. Although these complexes (9R and 10R)
were originally designed with the intention to drastically alter
the tissue distribution and pharmacokinetic profiles of the conju-
gates, it appeared that they gained additional binding interactions,
likely outside of the PSMA active site, due to the presence of the
pendant carboxylic acid groups not present in the SAAC complexes
6R–8R. Further increases in the number of pendant free acid func-
tional groups through the use of the tetra-carboxy imidazole (TIM)
chelator to form 11R (IC50 value 4 nM) clearly demonstrated that
the additional carboxylic acids appear to participate in favorable
binding interactions that further improve affinity for PSMA.
Tissue distribution of the series of 99mTc-complexes was evalu-
ated in LNCaP tumor bearing mice at 1 h post injection. The results
of the tissue distribution studies, Table 2, demonstrated a clear
pattern present for all the original SAAC derived complexes
99mTc-6, 99mTc-7, 99mTc-8, with characteristically higher hepatobil-
iary uptake. Four of the complexes, 99mTc-6, 99mTc-7, 99mTc-8 and
99mTc-10 displayed >10%ID/g in the intestines at 1 h. These results
were as predicted for 99mTc-6, 99mTc-7, and 99mTc-8 with the very
long C11 attached to the lipophilic cationic SAAC derived metal
complexes. Surprisingly complex 99mTc-10 also showed high intes-
tinal uptake at 1 hour despite having a pendant carboxylic acid,
which was intended to lower clearance via the hepatobiliary route.
The unanticipated high intestinal uptake for 99mTc-10 may be re-
lated to its neutral charge on the metal center or possessing only
one pendant free carboxylic acid group may not be sufficient to
compensate for the lipophilic character of the C11 backbone. It is
clear that despite some of these complexes being rapidly cleared
from the blood, most notably 99mTc-7 and 99mTc-10 (0.27 0.05
and 0.23 0.04%ID/g at 1 h, respectively) a significant portion of
their excretion was via the hepatobiliary route, hindering the po-
tential application of these chelators in the development of radio-
pharmaceuticals for prostate cancer. These limitations severely
restrict the potential development of cancer imaging agents, where
the ability to visualize metastatic lesions in the abdominal cavity
would be confounded by the high background signal present in
the liver and GI. Despite 99mTc-10 possessing high affinity (IC50
value 10 nM), the tumor uptake for 99mTc-10 was poor
(3.29 0.78%ID/g), possibly due to the lipophilic nature of the
complex.
3.3. Synthesis of 99mTc-SAAC/SAAC II ligands
Radiolabeling of the SAAC systems was accomplished on either
the free acids or as the tert-butyl protected derivatives utilizing
similar methodology, to form 99mTc-6–11, in excellent radiochem-
ical yield. The 99mTc(CO)3 radiolabeling was accomplished in two
+
steps using the commercially available IsoLink™ kits (Covidien) to
generate the intermediate [99mTc(CO)3(H2O)3]+, which was reacted
with the appropriate Glu-urea-Lys-C11-SAAC/SAAC II ligands
(10ꢀ6 M) in an equal volume mixture of 1:1 acetonitrile: phosphate
buffer in a sealed vial heated to 100 °C for 30 min. Upon cooling,
the reaction was analyzed for purity via HPLC. RCP after deprotec-
tion and HPLC purification resulted in ‘‘no carrier added’’ products,
with purity determined via HPLC to be consistently P90%, as
shown for 99mTc-11 in Fig. 3.
The 99mTc-SAAC complex, 99mTc-9, which contains two carbox-
ylic acid functionalized imidazole rings exhibited very low liver
Table 2
Tissue distributions of 99mTc-Glu-urea-C11-SAAC/SAACII derived complexes at 1 h post injection in LNCaP xenograft mice.
Tissue
Complex
6
7
8
9
10
11
0.99 0.16
0.25 0.06
0.52 0.03
1.4 0.23
1.44 0.38
Blood
Heart
Lungs
Liver
Spleen
Kidneys
Stomach
Intestine
Sk. muscle
Tumor
1.02 1.19
0.21 0.04
0.5 0.05
2.13 0.24
0.48 0.23
0.27 0.05
0.13 0.04
1.77 2.55
2.3 1.41
0.34 0.11
23.8 9.7
0.18 0.11
18.9 2.2
0.04 0.02
3.17 0.82
2.28 2.38
0.29 0.24
0.91 0.72
2.54 0.18
0.42 0.34
3.3 1.3
0.87 0.55
36.2 9.8
0.05 0.04
0.72 0.23
1.06 0.15
1.39 0.45
2.64 0.47
1.61 0.17
15.68 4.02
147 37
0.23 0.04
0.24 0.04
0.75 0.09
3.73 1.81
3.05 1.67
17
2
28
6
61
4
0.61 0.1
38.9 2.2
0.08 0.05
1.24 0.24
0.88 0.2
1.74 0.61
24.1 3.1
0.19 0.06
3.29 0.78
0.27 0.12
0.66 0.17
0.72 0.16
11.19 3.13
6.59 1.76
0.57 0.09
14.42 5.93
Data are %ID/g, expressed as mean SD.

K.P. Maresca et al. / Inorganica Chimica Acta 389 (2012) 168–175
175
and gastrointestinal accumulation (1.61 0.17 and 1.76 0.57%ID/
at 1 h, respectively) and high tumor and kidney uptake
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This work was supported in part by a Grant (J.W.B.) from the
National Institutes of Health, Grant # 1R41A1054080-01.
Appendix A. Supplementary material
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2012.03.002.