Novel polar single amino acid chelates for technetium-99m tricarbonyl-based radiopharmaceuticals with enhanced renal clearance: Application to octreotide

Article abstract of DOI:10.1021/bc900517x

Single amino acid chelate (SAAC) systems for the incorporation 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 Tc(CO)₃-dipyridyl complexes has resulted in substantial hepatobiliary uptake when either examined as lysine derivatives or integrated into biologically active small molecules and peptides. Here we designed, synthesized, and evaluated novel SAAC systems that have been chemically modified to promote overall Tc(CO)₃L₃ complex hydrophilicity with the intent of enhancing renal clearance. A series of lysine derived SAAC systems containing functionalized polar imidazole rings and/or carboxylic acids were synthesized via reductive alkylation of the ε amino group of lysine. The SAAC systems were radiolabeled with ^99mTc, purified, and evaluated for radiochemical stability, lipophilicity, and tissue distribution in rats. The log P values of the ^99mTc complexes were determined experimentally and ranged from -0.91 to -2.33. The resulting complexes were stable (>90%) for at least 24 h. Tissue distribution in normal rats of the lead ^99mTc complexes demonstrated decreased liver (<1 %ID/g) and gastrointestinal clearance (<1.5%ID/g) and increased kidney clearance (>15 %ID/g) at 2 h after injection compared to the dipyridyl lysine complex (DpK). One of the new SAAC ligands, [^99mTc]bis-carboxymethylimidazole lysine, was conjugated to the N-terminus of Tyr-3 octreotide and evaluated for localization in nude mice bearing AR42J xenografts to examine tissue distribution, tumor uptake and retention, clearance, and route of excretion for comparison to ¹¹¹In-DOTA-Tyr-3-octreotide and ^99mTc-DpK-Tyr-3- octreotide. ^99mTc-bis-(carboxymethylimidazole)-lysine-Tyr-3- octreotide exhibited significantly less liver uptake and gastrointestinal clearance compared to ^99mTc-DpK-Tyr-3-octreotide while maintaining tumor uptake in the same mouse model. These novel chelators demonstrate that lipophilicity can be controlled and organ distribution significantly altered, opening up broad application of these novel SAAC systems for radiopharmaceutical design.

Full text of DOI:10.1021/bc900517x

1032
Bioconjugate Chem. 2010, 21, 1032–1042
Novel Polar Single Amino Acid Chelates for Technetium-99m Tricarbonyl-Based
Radiopharmaceuticals with Enhanced Renal Clearance: Application to Octreotide
Kevin P. Maresca, John C. Marquis, Shawn M. Hillier, Genliang Lu, Frank J. Femia, Craig N. Zimmerman,
William C. Eckelman, John L. Joyal, and John W. Babich*
Molecular Insight Pharmaceuticals, Inc., 160 Second Street, Cambridge, Massachusetts 02142. Received November 23, 2009;
Revised Manuscript Received March 10, 2010
Single amino acid chelate (SAAC) systems for the incorporation 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 Tc(CO)3-dipyridyl complexes has resulted
in substantial hepatobiliary uptake when either examined as lysine derivatives or integrated into biologically
active small molecules and peptides. Here we designed, synthesized, and evaluated novel SAAC systems that
have been chemically modified to promote overall Tc(CO)3L3 complex hydrophilicity with the intent of enhancing
renal clearance. A series of lysine derived SAAC systems containing functionalized polar imidazole rings and/or
carboxylic acids were synthesized via reductive alkylation of the ε amino group of lysine. The SAAC systems
were radiolabeled with 99mTc, purified, and evaluated for radiochemical stability, lipophilicity, and tissue distribution
in rats. The log P values of the 99mTc complexes were determined experimentally and ranged from -0.91 to
-2.33. The resulting complexes were stable (>90%) for at least 24 h. Tissue distribution in normal rats of the
lead 99mTc complexes demonstrated decreased liver (<1 %ID/g) and gastrointestinal clearance (<1.5%ID/g) and
increased kidney clearance (>15 %ID/g) at 2 h after injection compared to the dipyridyl lysine complex (DpK).
One of the new SAAC ligands, [99mTc]bis-carboxymethylimidazole lysine, was conjugated to the N-terminus of
Tyr-3 octreotide and evaluated for localization in nude mice bearing AR42J xenografts to examine tissue distribution,
tumor uptake and retention, clearance, and route of excretion for comparison to 111In-DOTA-Tyr-3-octreotide
and 99mTc-DpK-Tyr-3-octreotide. 99mTc-bis-(carboxymethylimidazole)-lysine-Tyr-3-octreotide exhibited significantly
less liver uptake and gastrointestinal clearance compared to 99mTc-DpK-Tyr-3-octreotide while maintaining tumor
uptake in the same mouse model. These novel chelators demonstrate that lipophilicity can be controlled and
organ distribution significantly altered, opening up broad application of these novel SAAC systems for
radiopharmaceutical design.
the many problems associated with using sulfhydryl containing
radioligands and chelating agents that do not fully occupy the
coordination sites of Tc and Re or form unstable complexes. In
addition, SAAC may be incorporated into bioactive peptides
via standard solid phase peptide synthesis and subsequently
labeled with 99mTc and 186/188Re (8, 9).
During the past decade, the field of technetium coordination
chemistry has seen both a rising interest in the Tc(I) core (10-15)
and diminishing expectations for its utility, as the core and the
subsequent chelators have demonstrated significant deficiencies
(16, 17). These shortcomings, most notably the hydrophobicity
of the metal complexes, are inherent characteristics of the core
itself, which are exaggerated by the very nature of the chelators
that form the robust complexes. Unfortunately, the lipophilicity
of the chelators typically contributes to a poor pharmacokinetic
profile of the radiotracer characterized by hepatic accumulation
or hepatobiliary clearance. The ability to capitalize on the
synthetic flexibility of incorporation, and the stability and
inertness of the Tc(I) core, while decreasing the lipophilicity is
key to the future success of the technology.
INTRODUCTION
The potential to employ the radionuclide technetium-99m with
its optimal decay characteristics into targeting molecules has
been the foremost consideration in developing diagnostic
radiopharmaceuticals (1-3). 99mTc-labeled radiopharmaceuticals
are preferred over other isotopes because of the ideal nuclear
properties of the isotope, as well as its widespread availability
from commercial generator columns. 99mTc emits a 140 keV
γ-ray with 89% abundance, which is ideal for imaging with
commercial γ cameras. Furthermore, the combination of the
medically useful radionuclides, 99mTc and rhenium-186/188
(
186/188Re), which have remarkably similar coordination chem-
istries in terms of the {M(CO)3L3}+ core thereby yielding
isostructural coordination complexes with favorable physical
decay characteristics, is attractive for molecular imaging and
radiotherapy, respectively (4, 5).
We previously described a technology known as the single
amino acid chelate (SAAC) (6), a radiolabeling platform that
has broad utility in radiochemical, fluorescent and dual modality
(radio/fluorescent) molecular imaging. SAAC ligands are novel
bifunctional chelates constructed from derivatized amino acids
modified to provide three donor groups (L3) for chelation to
the {M(CO)3}1+ core (M ) Tc or Re). SAAC derivatives
demonstrate facile labeling with 99mTc and robust complex
stability toward cysteine and histidine challenges, which present
major in vivo competition for these ligands (7). SAAC avoids
One potential solution to overcome the high hepatobiliary
clearance of the complexes is to decrease the lipophilicity
surrounding the metal center. Two possible ways to address this
issue include (i) derivatizing the metal coordinating ligands with
polar, nonlipophilic substituents to counterbalance the lipophilic
metal-chelate core or (ii) decreasing the overall size of the
ligands used to coordinate the metal core (18, 19). The former
may be accomplished by modifying imidazole SAAC systems
* Corresponding author.
10.1021/bc900517x  2010 American Chemical Society
Published on Web 04/19/2010

Chelates for 99mTc Radiopharmaceuticals
Bioconjugate Chem., Vol. 21, No. 6, 2010 1033
with polar, water-solubilizing functional groups such as ethers,
alcohols, and carboxylic acids, which should alter the pharma-
cokinetic properties of the SAAC systems, and the biologically
relevant molecules to which they are conjugated. In addition,
these substitutions will result in metal complexes with neutral,
cationic, or anionic charges of the metal complex, which may
impact the pharmacokinetic profile. Alternatively, replacing one
or more of the ligand donor heteroaromatic rings with a smaller,
less hydrophobic donor, i.e., acetic acid, will reduce the size
and lipophilicity of the complexes. For example, the nitrilotri-
acetic acid chelate (NTA) reported by Lipowska et al. (20)
favored tubular transport rather than hepotobiliary excretion.
In an attempt to address the undesirable liver and gastrointes-
tinal uptake and clearance characteristic of many of the SAAC
systems previously designed and evaluated in our laboratory,
we have investigated functionalized polar lysine imidazole
derivatives with the potential to enhance renal clearance. These
novel imidazole SAAC systems can be synthesized with a
variety of hydrophilic R-groups via alkylation of imidazole-2-
carboxaldehyde with available alkyl halides that contain the
desired polar functionality (21). In previous work, we func-
tionalized imidazole ligands with ether-containing moieties to
investigate cardiac imaging (22) resulting in complexes with
very low liver uptake and retention. These same principles,
expanded to include various alcohols, ethers, and acids, were
applied to the SAAC systems to shift the current pharmacoki-
netic profile from hepatobiliary uptake to higher renal clearance.
Here we describe the synthesis and evaluation of a series of
lysine derived SAAC systems with a variety of metal donor
ligand groups to form (2-amino-6-(bis((1-X-1H-imidazol-2-
yl)methyl)amino)hexanoic acid) where X ) methyl (1), 2,2-
dimethoxyethyl (2), 2-ethoxyethyl (3), carboxymethyl (4),
2-hydroxyethyl (5), 3-(diethoxyphosphoryl)propyl (6), as well
as (S)-2-amino-6-((carboxymethyl)(thiazol-2-ylmethyl)amino-
hexanoic acid (7), (S)-2-amino-6-((carboxymethyl)((1-methyl-
1H-imidazol-2-yl)methyl)aminohexanoic acid (8), 2,2′-(5-amino-
5-carboxypentylazanediyl)diacetic acid (9) and compared them
to 2-amino-6-(bis(pyridin-2-ylmethyl)amino)hexanoic acid (DpK)
and 2-amino-6-((carboxymethyl)(pyridin-2-ylmethyl)amino)hex-
anoic acid (PAMAK). Tissue distribution data obtained with
these complexes in normal rats demonstrated greater kidney
clearance and significantly less liver and gastrointestinal uptake
than first generation SAAC ligands. Two SAAC ligands were
subsequently incorporated using solid phase peptide synthesis
into Tyr-3-octreotide yielding, 99mTc-4-Tyr-3-octreotide and
99mTc-DpK-Tyr-3-octreotide. These peptides were evaluated for
localization in nude mice bearing AR42J xenografts to examine
tissue distribution, tumor uptake and retention, clearance, and
route of excretion for comparison to 111In-DOTA-Tyr-3-oct-
reotide, a well established radioimaging agent with excellent
pharmacokinetic properties that targets the type 2 somatostatin
receptor in neuroendocrine tumors (23-25).
SAAC-peptide conjugates were purified and analyzed on Vydac
C18 reverse-phase column (250 mm × 4.6 mm × 5 µm)
employing a gradient method of 5-50% buffer B over 30 min
(buffer A ) water + 0.1% TFA, buffer B ) acetonitrile + 0.1%
1
TFA). H NMR spectra were obtained on a Bruker 400 MHz
instrument. Spectra are reported as ppm and are referenced to
the solvent resonances in chloroform-d (CDCl3), dimethyl
sulfoxide-d6 (DMSO-d6), or methanol-d4 (MeOD). Combustion
analyses were performed by Prevalere Life Sciences, Inc. 99mTc
was used as a solution of Na99mTcO4 in saline, as a commercial
99Mo/99mTc generator eluant (Cardinal Health). The 99mTc-
containing solutions were always kept behind sufficient lead
shielding. The [99mTc(CO)3(H2O)3]+ was prepared from com-
mercially available Isolink kits (Covidien). The 111In was used
as an 111InCl3 solution commercially available from MDS
Nordion. All solvents and reagents were purchased from Sigma
Aldrich (St. Louis, MO), Bachem (Switzerland), Akaal (Long
Beach, CA), or Anaspec (San Jose, CA). The following
abbreviations are used: Fmoc ) fluorenylmethyloxycarbonyl,
DMAP ) N,N-dimethylaminopyridine, DMF ) N,N-dimethyl-
formamide, DMSO ) dimethyl sulfoxide, DCM ) dichlo-
romethane, DCE ) 1,2-dichloroethane, NaOH ) sodium
hydroxide, (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
6-aminohexanoic acid hydrochloride salt ) L-Fmoc-lysine-OH
hydrochloride salt, ID/g ) injected dose per gram, NTA )
nitrilotriacetic acid, PBS ) phosphate buffered saline, RCP )
radiochemical purity, RCY ) radiochemical yield.
Syntheses. General Procedure for the Alkylations. To a
solution of the imidazole-2-carboxaldehyde dissolved in DMF
(1 mL) was added 1 mol equiv of the alkyl bromide, excess
potassium carbonate, and a catalytic amount of potassium iodide.
The mixture was 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.
General Procedure for the ReductiVe Aminations. To a
solution of L-Fmoc-lysine-OH hydrochloride salt (90 mg, 0.19
mmol) dissolved in DCE (2 mL) was added 2.1 equiv of the
aldehyde. The mixture was heated at 50 °C for 1 h whereupon
sodium triacetoxyborohydride (36 mg, 0.19 mmol) was added.
The mixture was stirred at room temperature for 12 h and was
subsequently evaporated to dryness and purified utilizing a
Biotage SP4 with a gradient method of 5-50% methanol in
DCM. The purified compound (24 mg, 0.034 mmol) was
deprotected by treatment with piperidine/DMF 1:1 (1 mL), and
the mixture was stirred at room temperature for 2 h. Following
evaporation to residue, aqueous extraction and washing with
excess DCM afforded the desired compounds as an off-white
solid.
(S)-2-Amino-6-(bis((1-methyl-1H-imidazol-2-yl)methyl)amino)-
hexanoic Acid (1). A solution of Fmoc-Lys-OH ·HCl (1.8 g, 4.5
mmol) and 1-methyl-1H-imidazole-2-carbaldehyde (1.1 g, 10
mmol) in DCE (50 mL) was stirred at 75 °C for 30 min under
nitrogen. The reaction mixture was cooled to 0 °C and treated
with sodium triacetoxyborohydride (3.2 g, 15 mmol). The
reaction mixture was stirred at room temperature overnight and
was 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 flash chroma-
tography over silica gel to afford (S)-2-(((9H-fluoren-9-yl)-
methoxy)carbonylamino)-6-(bis((1-methyl-1H-imidazol-2-yl)-
methyl)amino)hexanoic acid (2.3 g, 92%). MS (ESI), 557 (M
+ H)+. To a solution of (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl-
amino)-6-(bis((1-methyl-1H-imidazol-2-yl)methyl)amino)hexanoic acid
(556 mg, 1.00 mmol) in DMF (4.0 mL) was added piperidine (0.80
mL). The mixture was stirred at room temperature for 2 h. Solvent
was evaporated under reduce pressure to afford a residue, which was
purified by Amberchrom column chromatography to afford (S)-2-
EXPERIMENTAL SECTION
General Methods. All reactions were carried out in dry
glassware under an atmosphere of argon unless otherwise noted.
Reactions were purified by column chromatography under
medium pressure using a Biotage SP4 or by preparative high
pressure liquid chromatography using a Varian Prostar 210
preparative high pressure liquid chromatography (HPLC) system
equipped with a semipreparative Vydac C18 reverse-phase
column (250 mm ×10 mm × 5 µm) connected to a Varian
Prostar model 320 UV-visible detector and monitored at a
wavelength of 254 nm. The final technetium SAAC complexes
were purified 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). The final 99mTc-

1034 Bioconjugate Chem., Vol. 21, No. 6, 2010
Maresca et al.
amino-6-(bis((1-methyl-1H-imidazol-2-yl)methyl)amino)hexanoic acid
(320 mg, 0.958 mmol, 96% yield). 1H NMR (400 MHz, DMSO-d6)
δ 7.94 (s, 1 H), 7.04 (d, J ) 3, 1.2 Hz, 2H), 6.74 (d, J ) 1.2 Hz, 2 H),
3.54 (s, 4 H), 3.98 (brs, 1 H), 2.88 (s, 3 H), 2.72 (s, 3 H), 2.35 (t, J )
6.8 Hz, 2 H), 1.60-1.54 (m, 1 H), 1.43-1.29 (m, 3 H), 1.16-1.11
(m, 2 H). MS (ESI): 335 (M + H)+.
1-(2,2-Dimethoxyethyl)-1H-imidazole-2-carbaldehyde (2A).
To a solution of the imidazole-2-carboxaldehyde (410 mg, 4.3
mmol) dissolved in DMF (1 mL) was added 1.1 equiv of
2-bromo-1,1-dimethoxyethane (800 mg, 4.7 mmol), potassium
carbonate, and a catalytic amount of potassium iodide. The
mixture was 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 to yield the desired
compound (250 mg, 1.4 mmol, 32% yield). 1H NMR (400 MHz,
DMSO-d6) δ 9.9 (s, H), 7.85 (s, H), 7.55 (s, H), 5.82 (m, H),
4.75 (d, 2H), 3.45 (s, 6H).
NMR (400 MHz, DMSO-d6) δ 7.67 (d, 2H), 7.35 (m, 4H), 7.30
(m, 2H), 7.05 (s, H), 6.7 (s, H), 4.70 (s, 4H), 4.2 (m, 4H), 3.4
(d, 2H), 2.4 (m, 2H), 1.8 (s, 2H), 1.39 (s, 18H). 1.2 (m, 2H).
ESMS m/z: 758 (M + H)+. The purified compound was
deprotected by treatment with piperidine/DMF 1:1 (1 mL), and
the mixture was stirred at room temperature for 18 h. Following
evaporation to a residue, aqueous extraction from DCM afforded
the desired product (25 mg, 0.047 mmol, 25% yield) as an off-
white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.0 (s, 2H), 6.65
(s, H), 4.70 (s, 4H), 4.2 (m, 4H), 3.2 (d, 2H), 2.4 (m, 2H), 1.8
(s, 2H), 1.39 (s, 18H). 1.15 (m, 2H). ESMS m/z: 535 (M +
H)+.
(S)-2-Amino-6-(bis((1-(2-hydroxyethyl)-1H-imidazol-2-yl)methyl)-
amino)hexanoic Acid (5). To a solution of 3 (25 mg, 0.055 mmol)
dissolved in DCM (10 mL) was added 1.0 M boron tribromide
in DCM (83 mg, 0.333 mmol) at -20 °C. The mixture was
stirred at -20 °C for 2 h and was subsequently quenched with
water and washed with excess DCM. The water portion was
evaporated to dryness and purified as the product utilizing a
Biotage SP4 with a gradient method of 5-50% methanol in
(S)-2-Amino-6-(bis((1-(2,2-dimethoxyethyl)-1H-imidazol-2-yl)-
methyl)amino)hexanoic Acid (2). Following the same procedure
as utilized in the preparation of 1 yielded the desired compound
(93 mg, 0.132 mmol, 54% yield). 1H NMR (400 MHz, DMSO-
d6) δ 7.90 (d, 2H), 7.72 (d, 2H), 7.41 (m, 2H), 7.30 (m, 2H),
7.05 (s, H), 6.75 (s, H), 4.45 (m, 3H), 4.2 (m, 4H), 3.95 (d,
2H), 3.80 (m, H), 3.55 (s, 2H), 3.2 (s, 6H), 2.3 (m, 2H), 1.60
(m, H), 1.35 (m, H) 1.15 (m, 2H). ESMS m/z: 705 (M + H)+.
The purified compound was deprotected by treatment with
piperidine/DMF 1:1 (1 mL), and the mixture was stirred at room
temperature for 18 h. Following evaporation to residue, aqueous
extraction from DCM afforded the desired product (44 mg, 0.093
1
DCM (15 mg, 0.038 mmol, 34% yield). H NMR (400 MHz,
DMSO-d6) δ 8.40 (m, 2H), 8.16 (d, 2H), 7.70 (d, 2H), 5.50 (s,
2H), 4.20 (s, 2H), 4.05 (m, 4H), 3.68 (d, 2H), 3.15 (m, 5H),
2.46 (s, 2H), 1.70 (m, 2H), 1.65 (m, 2H). 1.21 (m, 2H). ESMS
m/z: 395 (M + H)+.
Diethyl 3-(2-Formyl-1H-imidazol-1-yl)propylphosphonate (6A).
Following the same procedure as utilized in the preparation of
2A, the target compound was prepared from diethyl (3-
bromopropyl)phosphonate (2.96 g, 11.4 mmol) and imidazole-
2-carboxaldehyde to yield the desired compound (466 mg, 1.69
mmol, 16% yield). ESMS m/z: 275 (M + H)+.
1
mmol, 76% yield) as an off-white solid. H NMR (400 MHz,
DMSO-d6) δ 7.98 (s, H), 7.05 (s, H), 6.85 (s, H), 4.45 (s, 2H),
3.95 (m, 4H), 3.55 (s, 2H), 3.2 (s, 6H), 2.85 (m, 2H), 2.15 (m,
2H), 1.40 (m, 2H). 1.15 (m, 2H). ESMS m/z: 483 (M + H)+.
1-(2-Ethoxyethyl)-1H-imidazole-2-carbaldehyde (3A). Following
the same procedure as utilized in the preparation of 2A, the
target compound was prepared from 1-bromo-2-ethoxyethane
(3.51 g, 22 mmol) and imidazole-2-carboxaldehyde to yield the
(S)-2-Amino-6-(bis((1-(3-(diethoxyphosphoryl)propyl)-1H-imi-
dazol-2-yl)methyl)amino)hexanoic Acid (6). Following the same
procedure as utilized in the preparation of 1 yielded the desired
Fmoc-protected product (63 mg, 0.071 mmol, 21% yield). ESMS
m/z: 443 (M/2). The purified compound (53 mg, 0.060 mmol)
was deprotected by treatment with piperidine/DMF 1:1 (1 mL),
and the mixture was stirred at room temperature for 18 h.
Following evaporation to a residue, aqueous extraction from
DCM afforded the desired product (32 mg, 0.048 mmol, 80%
yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ
7.1 (s, 2H), 6.78 (s, 2H), 3.98 (m, 4H), 3.8 (m, 4H), 3.6 (s,
4H), 2.85 (m, 4H), 2.75 (s, H), 2.38 (s, 2H), 1.77 (m, 4H), 1.55
(m, 4H), 1.39 (s, 2H), 1.18 (t, 12H). ESMS m/z: 664 (M +
H)+.
(S)-2-Amino-6-((carboxymethyl)(thiazol-2-ylmethyl)amino)hex-
anoic Acid (7). A suspension of Fmoc-Lys-OH ·HCl (6.1 g, 15
mmol) and thiazole-2-carbaldehyde (1.697 g, 15 mmol) in DCE
(100 mL) was refluxed for 30 min under nitrogen. The reaction
mixture was cooled to 0 °C and treated sequentially with sodium
triacetoxyborohydride (7.9 g, 38 mmol) and crude tert-butyl
glyoxalate (3.53 g, 27 mmol) as previously reported (26). The
reaction mixture was stirred at room temperature 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 flash chroma-
tography over silica gel to afford (S)-1-(9H-fluoren-9-yl)-14,14-
dimethyl-3,12-dioxo-10-(thiazol-2-ylmethyl)-2,13-dioxa-4,10-
diazapentadecane-5-carboxylic acid (1.85 g, 21%). MS (ESI),
580 (M + H)+. To a solution of (S)-1-(9H-fluoren-9-yl)-14,14-
dimethyl-3,12-dioxo-10-(thiazol-2-ylmethyl)-2,13-dioxa-4,10-
diazapentadecane-5-carboxylic acid (72.5 mg, 0.125 mmol) in
DMF (1.0 mL) was added piperidine (0.20 mL). The mixture
was stirred at room temperature for 2 h. Solvent was evaporated
under reduced pressure to afford a residue, which was purified
by flash chromatography over silica eluting with 10% MeOH
in DCM to afford (S)-2-amino-6-((2-tert-butoxy-2-oxoethyl)(thi-
1
desired compound (580 mg, 3.56 mmol, 17% yield). H NMR
(400 MHz, DMSO-d6) δ 9.63 (s, H), 7.6 (s, H), 7.21 (s, H),
4.45 (dd, 2H), 3.62 (dd, 2H), 3.38 (m, 2H), 1.05 (t, 3H).
(S)-2-Amino-6-(bis((1-(2-ethoxyethyl)-1H-imidazol-2-yl)methyl)-
amino)hexanoic Acid (3). Following the same procedure as
utilized in the preparation of 1 yielded the desired Fmoc-
protected product (141 mg, 0.210 mmol, 44% yield). 1H NMR
(400 MHz, DMSO-d6) δ 7.67 (d, 2H), 7.35 (m, 4H), 7.30 (m,
2H), 7.05 (s, H), 6.75 (s, H), 3.95 (m, 4H), 3.58 (d, 4H), 3.55
(s, 4H), 3.3 (s, 4H), 2.30 (m, 2H), 2.15 (m, 2H), 1.50 (m, 2H).
1.15 (s, 2H), 1.05 (t, 6H). ESMS m/z: 674 (M + H)+. The
purified compound was deprotected by treatment with piperi-
dine/DMF 1:1 (1 mL), and the mixture was stirred at room
temperature for 18 h. Following evaporation to residue, aqueous
extraction from DCM afforded the desired product (31 mg, 0.069
1
mmol, 91% yield) as an off-white solid. H NMR (400 MHz,
DMSO-d6) δ 8.35 (s, H), 7.98 (s, H), 7.05 (s, H), 6.75 (s, H),
3.95 (m, 4H), 3.58 (d, 4H), 3.55 (s, 4H), 3.3 (s, 4H), 2.30 (m,
2H), 2.15 (m, 2H), 1.50 (m, 2H). 1.15 (s, 2H), 1.05 (t, 6H).
ESMS m/z: 451 (M + H)+.
tert-Butyl 2-(2-Formyl-1H-imidazol-1-yl)acetate (4A). Following
the same procedure as utilized in the preparation of 2A, the
target compound was prepared from tert-butyl 2-bromoacetate
and imidazole-2-carboxaldehyde to yield the desired compound
(850 mg, 4.03 mmol, 39% yield). 1H NMR (400 MHz, DMSO-
d6) δ 7.6 (s, H), 7.23 (s, H), 5.15 (s, 2H), 1.40 (s, 9H).
(S)-2,2′-(2,2′-(5-Amino-5-carboxypentylazanediyl)bis(methylene)-
bis(1H-imidazole-2,1-diyl))diacetic Acid (4). Following the same
procedure as utilized in the preparation of 1 yielded the desired
Fmoc-protected product (155 mg, 0.205 mmol, 42% yield). 1H

Chelates for 99mTc Radiopharmaceuticals
Bioconjugate Chem., Vol. 21, No. 6, 2010 1035
azol-2-ylmethyl)amino)hexanoic acid (25 mg, 0.083 mmol,
66%). H NMR (400 MHz, CD3OD) δ 7.70 (d, J ) 3.6 Hz, 1
H), 7.52 (d, J ) 3.6 Hz, 1 H), 4.15 (s, 2 H), 3.52 (dd, J ) 7.2,
5.2 Hz, 1 H), 3.39 (s, 2 H), 2.73 (t, J ) 7.2 Hz, 2 H), 1.91-1.76
(m, 2 H), 1.60-1.57 (m, 2 H), 1.55-1.47 (m, 11 H). MS (ESI):
358 (M + H)+.
acetonitrile and water in a sealed vial. The sealed vial was heated
at 100 °C for 30 min, and upon cooling, the mixture was
analyzed for purity via reverse-phase HPLC. Purification by
HPLC resulted in “carrier-free” products, and the RCP was
determined via HPLC and shown to be consistently g95% pure.
The radiolabeling can be achieved at concentrations as low as
10-6 M at 75 °C with RCY e 80% and with RCY > 95% when
conducted at 10-4 M at 75 °C.
1
(S)-2-Amino-6-((2-tert-butoxy-2-oxoethyl)((1-methyl-1H-imida-
zol-2-yl)methyl)amino)hexanoic Acid (8). The title compound was
prepared by following the same procedure as described in the
preparation of 7 except 1-methyl-1H-imidazole-2-carbaldehyde
The 99mTc(I)(CO)3 radiolabeling of the SAAC-conjugated
+
peptides was achieved and purified in a similar manner with
the exception of the heating temperature, which was reduced
to 60 °C. The 111In-DOTA-Tyr-3-octreotide was prepared from
111InCl3 in an aqueous solution (10-4 M of the peptide) by
heating at 100 °C for 30 min.
1
was used in place of thiazole-2-carbaldehyde. H NMR (400
MHz, CDCl3) δ 7.88 (d, J ) 7.2 Hz, 2 H), 7.71 (dd, J ) 7.2,
2.4 Hz, 2 H), 7.55 (d, J ) 8.0 Hz, 1 H), 7.40 (t, J ) 7.6 Hz, 2
H), 7.31 (t, J ) 7.6 Hz, 4 H), 7.01 (s, 1 H), 6.71 (s, 1 H),
4.27-4.18 (m, 3 H), 3.88-3.83 (m, 1 H), 3.72 (s, 2 H), 3.14
(s, 2 H), 1.62-1.50 (m, 2 H), 1.38 (s, 9 H), 1.33-1.21 (m, 4
H). MS (ESI): 577 (M + H)+. To a solution of (S)-1-
(9H-fluoren-9-yl)-14,14-dimethyl-10-((1-methyl-1H-imidazol-
2-yl)methyl)-3,12-dioxo-2,13-dioxa-4,10-diazapentadecane-5-
carboxylic acid (190 mg, 0.33 mmol) in DMF (2 mL) was added
piperidine (0.4 mL). The mixture was stirred at room temper-
ature for 2 h. Solvent was evaporated under reduce pressure to
afford a residue, which was purified by Amberchrom column
chromatography to afford (S)-2-amino-6-((2-tert-butoxy-2-oxo-
ethyl)((1-methyl-1H-imidazol-2-yl)methyl)amino)hexanoic acid
Determination of log P Values. The log P values of the
99mTc(I) complexes were determined using the following
procedure: The 99mTc radiotracer was prepared and purified by
reverse-phase HPLC. The collected mobile phase was evapo-
rated and the residue dissolved in an equal volume mixture of
n-octanol and 25 mM phosphate buffer (pH 7.4). The samples
were extracted as follows: the samples were vortexed for 20
min and centrifuged at 8000 rpm for 5 min, and then aliquots
were removed from both the aqueous phosphate buffer and the
organic n-octanol layers and counted separately in a γ counter
(LKB model 1282, Wallac Oy, Finland). The extraction was
repeated twice for a total number of three extractions to ensure
full extraction of all the organic components. The partition
coefficients were then calculated using the equation P ) (activity
concentration in n-octanol)/(activity concentration in aqueous
layer). The samples were analyzed in triplicate and the log P
values determined from the average of the three different
measurements.
Rat Tissue Distribution of SAAC Complexes. Tissue distri-
bution studies of the 99mTc SAAC systems were performed in
separate groups of male Sprague-Dawley rats (n ) 5/time
point) administered as a bolus intravenous injection (ap-
proximately 10 µCi/rat) in a constant volume of 0.1 mL.
Animals were euthanized by carbon dioxide at 5, 30, 60, and
120 min after injection. Blood, heart, lungs, liver, spleen,
kidneys, adrenals, stomach (with contents), large and small
intestines (with contents), testes, skeletal muscle, bone, brain,
and adipose were excised, weighed wet, transferred to plastic
tubes, and counted in an automated γ counter (LKB model 1282,
Wallac Oy, Finland). Aliquots of the injected dose were also
measured to convert the counts per minute in each tissue sample
to percent-injected dose per organ. Tissue radioactivity levels,
expressed as %ID/g, were determined by converting the decay
corrected counts per minute to the percent dose and dividing
by the weight of the tissue or organ sample.
1
(115 mg, 0.32 mmol, 97%). H NMR (400 MHz, DMSO) δ
7.27 (brs, 1 H), 7.04 (s, 1 H), 6.72 (s, 1 H), 3.73 (s, 2 H), 3.64
(s, 3 H), 3.15 (s, 2 H), 3.04 (dd, J ) 6.8, 5.2 Hz, 1 H), 2.47 (t,
J ) 7.2 Hz, 2 H), 1.65-1.46 (m, 2 H), 1.39 (s, 9 H), 1.30-1.21
(m, 4 H). MS (ESI): 355 (M + H)+.
(S)-2,2′-(5-Amino-5-carboxypentylazanediyl)diacetic Acid (9).
(S)-10-(2-tert-Butoxy-2-oxoethyl)-1-(9H-fluoren-9-yl)-14,14-di-
methyl-3,12-dioxo-2,13-dioxa-4,10-diazapentadecane-5-carbox-
ylic acid was prepared from a solution of Fmoc-Lys-OH (1.5
g, 4.0 mmol) and crude tert-butyl glyoxalate (3.6 g, 27.6 mmol)
in DCE (50 mL), and the mixture was stirred at 75 °C for 30
min under nitrogen. The reaction mixture was cooled to 0 °C
and treated with sodium triacetoxyborohydride (2.1 g, 10 mmol).
The reaction mixture was stirred at room temperature for 3 h
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 flash chroma-
tography over silica gel to afford (S)-10-(2-tert-butoxy-2-
oxoethyl)-1-(9H-fluoren-9-yl)-14,14-dimethyl-3,12-dioxo-2,13-
dioxa-4,10-diazapentadecane-5-carboxylic acid (1.70 g, 3.51
1
mmol 71%). H NMR (400 MHz, CDCl3) δ 7.76 (d, J ) 7.2
Hz, 2 H), 7.60 (d, J ) 7.2 Hz, 2 H), 7.40 (t, J ) 7.2 Hz, 2 H),
7.30 (t, J ) 7.2 Hz, 2 H), 6.63 (d, J ) 7.6 Hz, 1 H), 4.40-4.34
(m, 3 H), 4.22 (t, J ) 7.2 Hz, 1 H), 3.49 (s, 4 H), 2.83-2.64
(m, 4 H), 1.96-1.77 (m, 4 H), 1.40 (s, 18 H). MS (ESI): 598
(M + H)+. The purified compound (12 mg, 0.21 mmol) was
deprotected by treatment with piperidine/DMF 1:1 (1 mL), and
the mixture was stirred at room temperature for 18 h. Following
evaporation to a residue, aqueous extraction from DCM afforded
the desired product 9 (3 mg, 0.11 mmol, 55% yield) as an off-
white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.9 (s, 2H), 3.5
(m, H), 3.3 (d, 2H), 2.4 (m, 2H), 1.8 (s, 2H), 1.45 (m, 2H),
1.15 (m, 2H). ESMS m/z: 263 (M + H)+.
Peptide Synthesis. Tyr-3-octreotide analogues {4-Tyr-3-
octreotide} and {DpK-Tyr-3-octreotide} with the lysine SAAC
incorporated at the amino terminus were synthesized by
AnaSpec, Inc. (San Jose, CA) using solid phase peptide synthesis
employing standard Fmoc coupling techniques with cleavage
and deprotection using TFA. Following synthesis, all peptides
were purified using reverse phase HPLC and were lyophilized
with purity profiles of >90%.
Tumor Cell Lines. AR42J cells were obtained from American
Type Culture Collection (Rockville, MD). The cells were grown
in Kaighn’s F-12 media supplemented with 20% fetal bovine
serum, sodium pyruvate, and gentamicin in a humidified
atmosphere of 5% CO2/95% air at 37 °C.
General Preparation of 99mTc Complexes and Peptides. The
99mTc(I)(CO)3+ radiolabeling of the SAAC systems to form the
metal complexes was accomplished on either the free R-amino
acids or the R-N-Fmoc protected lysine derivative utilizing
similar methodology. 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 system
(Figure 2) at 10-6-10-4 M in an equal volume mixture of
Tissue Distribution in AR42J Bearing Mice. Uptake of
radiolabeled peptides in rat tumor xenograft models were
performed according to published methods. Briefly, AR42J cells
were trypsinized, counted, and suspended in a solution contain-
ing 50% Dulbecco’s PBS (D-PBS) (with 1 mg/mL D-glucose

1036 Bioconjugate Chem., Vol. 21, No. 6, 2010
Maresca et al.
heterocyclic aldehydes coupled with the ease of synthesis has
led to the preparation of several classes of polar SAAC systems
that form cationic 99mTc complexes with enhanced renal
clearance. In addition, incorporation of one acetic acid “arm”
onto the ε amine of lysine (R1) in lieu of the second heterocyclic
ligand affords SAAC systems of reduced size with a neutral
metal center.
Ligand Design. As illustrated in Figure 1, both first generation
(SAAC I) and second generation (SAAC II) SAAC systems
were derived from the amino acid lysine. First generation 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 of the chelate ring systems through the
modification of heterocyclic imidazole ring substituents for R2.
The effort focused on the SAAC ligands that form cationic
homogeneous tridentate metal complexes as well as the smaller
heterogeneous monoacetic acid derivatized compounds which
form neutral metal complexes.
Figure 1. General structures of the first and second generation of
heterocyclic SAAC systems.
and 36 µg/mL sodium pyruvate) and 50% Matrigel (BD
Biosciences, Franklin Lakes, NJ). Male NCrnu/nu mice were
anesthetized by intraperitoneal injection of 0.5 mL of Avertin
(20 mg/mL) (Sigma-Aldrich, St. Louis, MO) and 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. Tissue
biodistribution studies were conducted by administering, via the
tail vein, a bolus injection of 2 µCi/mouse in a constant volume
of 0.05 mL of D-PBS containing 1 mg/mL bovine serum
albumin. Groups of five animals were euthanized by asphyxi-
ation with carbon dioxide at 1 and 4 h after injection. All tissues
from Figure 4, including the tumor, were excised, weighed wet,
and transferred to plastic tubes, and radioactivity present was
counted in an automated γ-counter (LKB model 1282, Wallac
Oy, Finland). Tissue time - radioactivity levels were expressed
as % injected dose per gram tissue (%ID/g).
Preparation of Homogeneous Tridentate Ligands as Second
Generation Lysine Imidazole Derivatived SAAC Systems. The
derivatized imidazole SAAC systems were prepared via the
alkylation of the commercially available imidazole-2-carbox-
aldehyde (10) with the desired alkyl halide (11) to afford the
N-substituted imidazole-2-carboxaldehyde analogues (12). Re-
ductive amination of N-Fmoc lysine with sodium triacetoxy-
borohydride produces the N-Fmoc protected SAAC system (13)
which following deprotection with piperidine affords the desired
SAAC systems (14) in good yields as illustrated in Scheme 1.
Preparation of Heterogeneous SAAC Systems: Monoacetic
Acid Analogues. Several compounds from the homogeneous
series described above which exhibited promising pharmaco-
kinetic profiles were prepared as their corresponding monoacetic
acid analogues, shown in Figure 2. These new heterogeneous
99mTc-SAAC complexes were smaller in size, were less hindered
complexes, and were uncharged at the metal center, in contrast
to the positive charged metal centers of the homogeneous
complexes.
RESULTS AND DISCUSSION
We previously described a technology known as SAAC, a
radiolabeling platform that has broad utility in radiochemical,
fluorescent, and dual modality (radio/fluorescent) molecular
imaging (6, 9) and is particularly convenient for developing
radiolabeled peptides. One limitation of the Tc(CO)3-dipyridyl
complex, characteristic of the original SAAC systems, is the
extremely hydrophobic nature of the radiolabeled complexes
which has often lead to the liver accumulation and/or hepato-
biliary clearance of the bioconjugates. This undesirable phar-
macokinetic profile is not unique to the original SAAC systems
but a characteristic shared by many bioconjugates (27, 28). The
goal of this work was to develop a second generation series of
hydrophilic lysine-based SAAC systems that have been modified
to favor renal clearance of the 99mTc-SAAC complexes and thus
facilitate the development of novel 99mTc-labeled radiopharma-
ceuticals that clear the body more readily. This was ac-
complished, as shown in Figure 1, by modifying the lysine core
of SAAC systems in two ways to create novel tridentate
chelators with reduced lipophilicity: the addition of polar
substituents and the reduction of the ligand size. The N-protected
lysine building block of the SAAC systems was derivatized at
the ε amine with substituted heterocyclic rings (R1) that
contained distinct polar functional groups (R2) in an attempt to
reduce the lipophilicity of the resulting 99mTc complexes. The
ε amine of lysine allows for the facile synthesis of large numbers
of structural variants of SAAC systems with the potential of
diverse physiochemical properties. The chemical synthesis of
the bifunctional SAAC systems was accomplished via reductive
alkylation of N-Fmoc protected lysine with the appropriate
heterocyclic aldehyde. The availability of a diverse set of
The compounds in this series were prepared using the double
reductive alkylation sequence on Fmoc-lysine. Addition of the
first aldehyde followed by reduction with sodium borohydride
affords a monoalkylated intermediate as shown in Scheme 2.
Subsequent treatment with tert-butyl glyoxalate followed by
deprotections with piperidine and trifluoroacetic acid (TFA)
afforded the desired series of heterogeneous neutral ligands (26).
Radiolabeling of 99mTc-SAAC. Radiolabeling of the SAAC
systems was accomplished on either the free R-amino acids or
as the appropriately N-protected amino acid derivative utilizing
similar methodology to form 99mTc-1-9, demonstrating the
flexibility of the radiolabeling and the ease of preparation of
these novel SAAC metal complexes. The 99mTc(I)(CO)3
+
radiolabeling was accomplished in two steps using the com-
mercially available IsoLink kits (Covidien) to form the
[
99mTc(CO)3(H2O)3]+ intermediate, which was reacted with the
appropriate SAAC ligand (ligand concentration of 10-6-10-4
M) in an equal volume mixture of 1:1 acetonitrile and phosphate
buffer. The sealed vial was heated at 100 °C for 30 min, and
upon cooling, the mixture was analyzed for purity via HPLC.
The RCP after HPLC purification resulted in “carrier-free”
products, with purity determined via HPLC to be consistently
g95%. While possible to radiolabel at concentrations as low
as 10-6 M, the RCY at 75 °C was e80%. To achieve RCY >
95% at 75 °C, the ligand concentration needed to be increased
to 10-4 M. This was true for all of the compounds with the
exception of 2,2′-(5-amino-5-carboxypentylazanediyl)diacetic
acid (9) which required 10-3 M ligand to achieve >95% RCY.

Chelates for 99mTc Radiopharmaceuticals
Bioconjugate Chem., Vol. 21, No. 6, 2010 1037
Scheme 1. Preparation of Homogeneous Bifunctional Imidazole SAAC Systems
SAACsystems2,2′-(2,2′-(5-amino-5-carboxypentylazanediyl)-
bis(methylene)-bis(1H-imidazole-2,1-diyl))diacetic acid (4) and
2-amino-6-(bis((1-(2-hydroxyethyl)-1H-imidazol-2-yl)methyl)-
amino)hexanoic acid (5) could be radiolabeled with the ester
Figure 2. Structures of the novel lysine based SAAC systems.

1038 Bioconjugate Chem., Vol. 21, No. 6, 2010
Maresca et al.
Scheme 2. Preparation of the Heterogeneous SAAC Systems
protecting groups present on the bis-imidazole derivative or on
the fully deprotected ligand. For example, the 99mTc complex
of ligand 4 was prepared in 30 min followed by simple
deprotection of the tert-butyl ester protecting groups using TFA
at room temperature to produce 99mTc-4. The radiolabeling and
subsequent deprotection could be followed by HPLC over time,
as shown in Figure 3.
The 99mTc complex of 2-amino-6-(bis((1-(2-hydroxyethyl)-
1H-imidazol-2-yl)methyl)amino)hexanoic acid (5) could be
prepared by radiolabeling 2-amino-6-(bis((1-(2-ethoxyethyl)-1H-
imidazol-2-yl)methyl)amino)hexanoic acid (3) followed by
subsequent deprotection of the ethyl ethers using BBr3 to afford
the desired metal complex 99mTc-5 (Scheme 3). Confirmation
of the formation of the 99mTc-5 complex was established by
independent radiolabeling studies of the compounds 3 and 5
followed by the radiolabeling of 3 with conversion to the 99mTc-5
complex.
Rat Tissue Distribution Studies of SAAC Complexes. Rat
tissue distribution studies were performed with eight of the novel
SAAC complexes, [99mTc(CO)3{η3-((S)-2-amino-6-(bis((1-meth-
yl-1H-imidazol-2-yl)methyl)amino)hexanoic acid)}] (99mTc-1),
[
99mTc(CO)3{η3-(2-amino-6-(bis((1-(2,2-dimethoxyethyl)-1H-
imidazol-2-yl)methyl)amino)hexanoic acid)}] (99mTc-2), [99mTc-
(CO)3{η3-(2-amino-6-(bis((1-(2-ethoxyethyl)-1H-imidazol-2-
yl)methyl)amino)hexanoic acid)}] (99mTc-3), [99mTc(CO)3{η3-
(2,2′-(2,2′-(5-amino-5-carboxypentylazanediyl)bis(methylene)-
bis(1H-imidazole-2,1-diyl))diacetic acid)}] (99mTc-4), [99mTc-
(CO)3{η3-(2-amino-6-(bis((1-(2-hydroxyethyl)-1H-imidazol-
Figure 3. Radiochromatograms of 99mTc-4-di-tert-butyl ester: crude reaction after heating at 75 °C for 30 min (blue) showing multiple radioactive
species; after TFA incubation (red) showing predominantly fully deprotected product (99mTc-4); after reverse phase HPLC purification (green)
demonstrating 99mTc-4 > 98% RCP.

Chelates for 99mTc Radiopharmaceuticals
Bioconjugate Chem., Vol. 21, No. 6, 2010 1039
Scheme 3. Radiolabeling of (2-Amino-6-(bis((1-(2-ethoxyethyl)-1H-imidazol-2-yl)methyl)amino)hexanoic acid (3) and Subsequent
Deprotection To Form the Metal Complex 99mTc-5
Table 1. Comparison of the Rat Tissue Distribution Results of
2-yl)methyl)amino)hexanoic acid)}]
(
99mTc-5),
[
99mTc(CO)3-
99mTc-SAAC Complexes in Blood and Excretory Tissuesa
{η3-((S)-2-amino-6-((carboxymethyl)(thiazol-2-ylmethyl)amino)-
hexanoic acid (99mTc-7), [99mTc(CO)3{η3-((S)-2-amino-6-((2-
tert-butoxy-2-oxoethyl)((1-methyl-1H-imidazol-2-yl)methy-
lamino)hexanoic acid (99mTc-8), and [99mTc(CO)3{η3-(2,2′-
(5-amino-5-carboxypentylazanediyl)diacetic acid)}] (99mTc-
time (min)
compd tissue
5
30
60
120
DpK
blood
liver
0.58 ( 0.05 0.07 ( 0.01 0.025 ( 0.005 0.013 ( 0.001
3.36 ( 0.44 2.75 ( 0.11 2.59 ( 0.08
2.12 ( 0.06
3.89 ( 0.42
2.73 ( 0.57
9). Only
[
99mTc(CO)3{η3-(2-amino-6-(bis((1-(3-(dietho-
kidney 6.05 ( 1.03 4.95 ( 0.11 4.93 ( 0.43
GI 0.49 ( 0.08 0.89 ( 0.07 1.46 ( 0.09
xyphosphoryl)propyl)-1H-imidazol-2-yl)methyl)amino)hex-
anoic acid)}] (99mTc-6) was omitted because of the high
lipophilicity of the complex. In addition, for comparison, rat
tissue distribution studies were performed with previously
published SAAC systems (6), the cationic bis-pyridine
PAMAK blood
liver
0.63 ( 0.06 0.19 ( 0.02 0.07 ( 0.002 0.03 ( 0.01
1.25 ( 0.14 1.34 ( 0.20 0.56 ( 0.05
0.20 ( 0.03
0.48 ( 0.05
kidney 6.38 ( 0.70 3.55 ( 0.35 1.14 ( 0.14
GI
0.18 ( 0.02 1.22 ( 0.24 1.80 ( 0.175 2.50 ( 0.59
1
2
3
4
5
7
8
9
blood
liver
1.18 ( 0.18 0.28 ( 0.03 0.11 ( 0.02
0.8 ( 0.28 1.03 ( 0.21 0.86 ( 0.16
0.03 ( 0.01
0.74 ( 0.14
25.5 ( 3.4
1.12 ( 0.37
0.04 ( 0.01
0.16 ( 0.02
complex
[
99mTc(CO)3(η3-{2-amino-6-(bis(pyridin-2-ylme-
kidney 6.65 ( 2.06 22.2 ( 0.3.9 25.4 ( 1.7
GI
blood
liver
kidney 13.82 ( 2.81 34.1 ( 5.59 40.25 ( 5.17 33.18 ( 2.75
GI
blood
liver
kidney 6.86 ( 0.86 12.13 ( 2.36 12.54 ( 1.00 12.54 ( 0.81
GI
blood
liver
kidney 6.58 ( 1.85 10.7 ( 2.3
GI
blood
liver
kidney 8.55 ( 1.54 18.7 ( 1.2
GI
blood
liver
kidney 10.84 ( 1.25 9.07 ( 0.66 4.19 ( 0.57
GI
blood
liver
kidney 10.79 ( 1.97 17.05 ( 3.17 9.52 ( 2.54
GI
blood
liver
kidney 12.8 ( 1.31 5.68 ( 1.99 2.38 ( 0.45
GI 0.12 ( 0.02 0.36 ( 0.11 0.72 ( 0.11
thyl)amino)hexanoic acid}] [99mTc-DpK] and the neutral
pyridine amine monoacetic acid complex [99mTc(CO)3(η3-{2-
amino-6-((carboxymethyl)(pyridin-2-ylmethyl)amino)hex-
anoic acid} [99mTc-PAMAK]. A summary of the uptake of
all of the SAAC complexes examined in selected organs is
shown in Table 1.
0.15 ( 0.02 0.45 ( 0.11 0.84 ( 0.1
1.46 ( 0.21 0.47 ( 0.05 0.14 ( 0.04
1.06 ( 0.34 0.45 ( 0.04 0.24 ( 0.04
0.34 ( 0.12 1.05 ( 0.2
0.83 ( 0.09 0.09 ( 0.02 0.002 ( 0.001 0.01 ( 0.001
1.61 ( 0.32 0.35 ( 0.08 0.28 ( 0.07 0.14 ( 0.02
1.39 ( 0.24
1.21 ( 0.27
99mTc-SAAC imidazole complexes 1, 2, 3, and 5 exhibited
rapid clearance from the blood (maximum of 0.14 %ID/g at
2 h for 99mTc-5), relatively low liver uptake (maximum of 1.61
( 0.32 %ID/g at 5 min for 99mTc-3), and low liver retention
(maximum of 0.74 ( 0.14 %ID/g at 2 h for 99mTc-1). In addition,
these complexes exhibited a shift from hepatobiliary clearance
to a predominantly renal uptake pattern compared to 99mTc-DpK
and 99mTc-PAMAK. However, the high renal accumulation was
in the absence of substantial renal clearance over the 2 h time
period of the study. The four complexes displayed >12 %ID/g
in the kidneys at the earliest (0.5 h) and latest (2 h) time points
tested, demonstrating very little clearance over time. It is not
clear whether the undesirable kidney retention was due to
nonspecific binding of the complexes in the kidneys or a result
of amino acid recycling. Since the goal is to develop chelators
that exhibit rapid clearance from all nontarget tissues, the
extended retention in the kidneys can be viewed as a significant
liability for the complexes and may mitigate their potential
application to biologically relevant molecules.
0.57 ( 0.13 2.33 ( 0.61 3.62 ( 0.3
1.33 ( 0.2 1.14 ( 0.23 0.86 ( 0.17
0.52 ( 0.07 0.55 ( 0.07 0.42 ( 0.08
16.8 ( 4.8
2.96 ( 0.44
0.72 ( 0.12
0.39 ( 0.07
10.2 ( 2.5
1.22 ( 0.27
0.14 ( 0.02
0.46 ( 0.14
23.2 ( 5.0
0.97 ( 0.37
0.15 ( 0.02 0.44 ( 0.08 0.76 ( 0.31
1.3 ( 0.25 0.52 ( 0.03 0.24 ( 0.03
0.88 ( 0.24 0.95 ( 0.13 0.65 ( 0.13
22.1 ( 3.5
0.19 ( 0.05 0.52 ( 0.09 0.76 ( 0.24
0.94 ( 0.19 0.22 ( 0.06 0.09 ( 0.001 0.03 ( 0.01
1.16 ( 0.09 1.06 ( 0.35 1.04 ( 0.18
0.72 ( 0.16
1.63 ( 0.51
2.27 ( 0.59
0.09 ( 0.01
0.44 ( 0.14
3.94 ( 0.43
2.53 ( 0.70
0.14 ( 0.02
0.18 ( 0.03
1.09 ( 0.17
0.69 ( 0.19
0.16 ( 0.03 1.16 ( 0.4
1.03 ( 0.14 0.45 ( 0.1
1.93 ( 0.37
0.22 ( 0.02
0.95 ( 0.14 3.02 ( 0.59 0.98 ( 0.22
0.16 ( 0.01 1.96 ( 0.49 1.64 ( 0.64
1.00 ( 0.14 0.35 ( 0.05 0.19 ( 0.03
0.68 ( 0.06
0.5 ( 0.14 0.26 ( 0.04
a Data are %ID/g, expressed as mean ( SD.
99mTc-SAAC complex 4, which contains carboxylic acid
functionalized imidazole rings, also exhibited very low liver
and gastrointestinal accumulation (0.39 ( 0.07 and 1.22 ( 0.27
%ID/g at 2 h) and high kidney uptake that did not clear over
the time course of the study (10.7 ( 2.3 and 10.2 ( 2.5 %ID/g
at 0.5 and 2 h, respectively). This complex exhibited the least
initial liver uptake (0.52 ( 0.07 %ID/g at 5 min) of any chelate
tested. Interestingly, 4 cleared the blood more slowly than any
of the chelators examined, potentially because of increased
binding to blood proteins.
The complexes of compounds 7 and 8, the monothiazole and
monomethylimidazole analogues, exhibited high kidney uptake,
9.07 ( 0.66 and 17.05 ( 3.17 %ID/g at 0.5 h, respectively,
and rapid clearance of 1.63 ( 0.51 and 3.94 ( 0.43 %ID/g at
2 h, respectively. However, these complexes also demonstrated
increased accumulation in the gastrointestinal tract (GI)
(>2 %ID/g at 2 h) over the 2 h study period. Interestingly, these
neutral complexes are similar in nature to 99mTc-PAMAK, with
one heterocyclic chelating moiety and one acetic acid chelating
moiety, and displayed a similar clearance pattern to 99mTc-
PAMAK as well.
99mTc-SAAC complex 9, the diacetic acid derivative, exhibited
high initial kidney uptake with rapid renal clearance (5.68 (
1.99 and 1.09 ( 0.17 %ID/g at 0.5 and 2 h, respectively) and
the least liver and GI accumulation of any of the chelators tested
after the initial time point (0.18 ( 0.03 and 0.69 ( 0.19 %ID/g
at 2 h, respectively). This complex exhibited an ideal pharma-
cokinetic clearance profile for development into radiopharma-
ceuticals; however, this chelator was not studied further because
of lower RCY.

1040 Bioconjugate Chem., Vol. 21, No. 6, 2010
Maresca et al.
Table 2. Comparisons of the Partition Coefficients (log P) for the
99mTc-SAAC Complexes
compd
DpK
PAMA-K
1
2
3
4
5
6
log P
-1.89
-1.80
-2.00
-1.72
-1.10
-2.33
-1.61
-0.91
-1.75
-1.84
-2.20
-2.88
7
8
9
NTA controla
a Lipowska et al. (20).
It is clear that despite the rapid clearance from the blood of
the first generation of SAAC complexes, 99mTc-DpK and 99mTc-
PAMAK, a significant portion of their excretion was via the
hepatobiliary route, hindering the potential application of these
chelators in the development of radiopharmaceuticals. These
limitations are realized in the development of cancer imaging
agents where the ability to visualize metastatic lesions in the
abdominal cavity with a DpK containing chelator is confounded
by the high background signal present in the liver and GI.
Through the preparation of a variety of novel polar and/or
hydrophilic SAAC systems that are inherently less lipophilic,
the pharmacokinetic profiles have been successfully altered to
favor renal clearance.
Determination of Partition Coefficients (log P) as a
Measure of Lipophilicity. The log P values (Table 2) of the
complexes were determined using the HPLC purified 99mTc-
SAAC complexes by the shake flask method utilizing n-octanol
and 25 mM phosphate buffer (pH 7.4) as the solvents. NTA
was tested as a control based on the published results that the
99mTc-NTA complex undergoes rapid and efficient renal clear-
ance (20).
The lipophilicity of the complexes as measured by the octanol/
phosphate buffer (pH 7.4) extinction coefficients followed the
general trend of the rat tissue distribution pharmacokinetic
profiles whereby the complexes with more negative log P values
displayed a more desirable pharmacokinetic profile (high kidney/
low hepatobiliary clearance). For example, 4 with log P of
-2.33 cleared primarily by renal excretion, while 7 with a less
negative log P of -1.75 cleared by combined renal and
hepatobiliary routes. However, the correlation between the log P
measurements and renal uptake and clearance of the radioactive
complexes was not perfect because complexes like 3 with a
log P of -1.10 still have high kidney uptake. Therefore, the
log P values should only be considered as one of many
physiochemical properties of the complexes that govern the in
vivo distribution, uptake, metabolism, and excretion.
Incorporation of 99mTc-SAAC Complexes into Tyr-3-oct-
reotide. One of the most important applications for the SAAC
technology is the incorporation of the SAAC ligands directly
into clinically relevant peptides. This is readily accomplished
using standard solid phase peptide synthesis strategies (8, 29).
To that end, we have incorporated two SAAC ligands, DpK
and 4, at the N-terminus of Tyr-3-octreotide, a somatostatin
receptor 2 (SSTR2) selective peptide agonist. Following com-
plexation with 99mTc, we evaluated the tissue distribution for
comparison to 111In-DOTA-Tyr-3-octreotide, a well established
peptide based radiopharmaceutical for the detection of carcinoid
and neuroendocrine tumors (30-33). The SAAC ligand, 4, was
selected for evaluation based on the favorable pharmacokinetic
profile in rats demonstrating kidney uptake and GI/liver clear-
ance of the 99mTc-SAAC complex, high RCY, and prolonged
Figure 4. Tissue distribution of SSTR2 receptor mediated 111In-DOTA-
Tyr-3-octreotide (top) verses 99mTc-DpK-Tyr-3-octreotide (middle) and
99mTc-4-Tyr-3-octreotide (bottom) in AR42J mouse tumor model.
chemical stability. The incorporation of DpK was used for a
direct comparison of a first generation SAAC with similar size.
The results of the tissue distributions are shown in Figure 4.
While both 99mTc-DpK-Tyr-3-octreotide and 111In-DOTA-Tyr-
3-octreotide demonstrate specific uptake and retention in target
tissues, tumor and pancreas, the liver and GI uptake of 99mTc-
DpK-Tyr-3-octreotide is significantly greater than that of 111In-
DOTA-Tyr-3-octreotide, thus highlighting the necessity of
developing advanced SAAC ligands with a pharmacokinetic
profile that favors renal clearance. In contrast, 99mTc-4-Tyr-3-
octreotide exhibited a very similar tissue distribution to the 111In-
DOTA-Tyr-3-octreotide with high renal clearance and signifi-
cantly reduced liver uptake of 4.63 ( 0.65 %ID/g at 1 h
compared to 23.31 ( 3.5 %ID/g at 1 h for 99mTc-DpK-Tyr-3-
octreotide (p < 0.001). Likewise, both the large and small
intestines of the 99mTc-4-Tyr-3-octreotide showed reduced
radioactivity at both time points examined compared to 99mTc-
DpK-Tyr-3-octreotide. 99mTc-4-Tyr-3-octreotide also exhibited
slightly higher tumor uptake at 1 h (15.15 ( 7.92 %ID/g)
compared to the 111In-DOTA-Tyr-3-octreotide (8.08 ( 5.17
%ID/g) and significantly higher tumor uptake at 4 h compared
to 111In-DOTA-Tyr-3-octreotide (14.85 ( 6.41 and 5.99 ( 1.65
%ID/g, respectively) (p < 0.03). The tumor-to-blood ratio of
99mTc-4-Tyr-3-octreotide was an encouraging 6.7:1 at 1 h and
reached a maximum at 4 h with an impressive 35:1 ratio. Thus,
the utility of this class of chelators is accentuated by the ability

Chelates for 99mTc Radiopharmaceuticals
Bioconjugate Chem., Vol. 21, No. 6, 2010 1041
Automated solid-phase synthesis, NMR characterization, and in
vitro screening of fMLF(SAAC)G and fMLF[(SAAC-Re-
(CO)3) +]G. Bioconjugate Chem. 15 (1), 128–136.
to maintain high tumor uptake while driving preferential renal
clearance of molecules, which would otherwise be dominated
by hepatobiliary localization and excretion.
(9) Stephenson, K. A., Banerjee, S. R., Besanger, T., Sogbein,
O. O., Levadala, M. K., McFarlane, N., Lemon, J. A., Boreham,
D. R., Maresca, K. P., Brennan, J. D., Babich, J. W., Zubieta,
J., and Valliant, J. F. (2004) Bridging the gap between in vitro
and in vivo imaging: isostructural Re and 99mTc complexes for
correlating fluorescence and radioimaging studies. J. Am. Chem.
Soc. 126 (28), 8598–8599.
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C., Tarallo, L., Arra, C., Barbieri, A., Romanelli, A., and Aloj,
L. (2008) In vivo and in vitro characterization of CCK8 bearing
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A. P. (1999) Basic aqueous chemistry of [M(OH2)3(CO)3]+
(M)Re, Tc) directed towards radiopharmaceutical application.
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Ortner, K., Abram, U., and Schubiger, P. A. (2000) Influence of
the denticity of ligand systems on the in vitro and in vivo
behavior of 99mTc(I)-tricarbonyl complexes: a hint for the future
functionalization of biomolecules. Bioconjugate Chem. 11, 345–
351.
In summary, our ongoing research into the development of
novel 99mTc chelators has led to significant improvements of a
key characteristic of the already robust proprietary SAAC
radiolabeling platform. The pharmacokinetic profile of the 99mTc-
SAAC metal complex has been significantly altered through the
design and synthesis of SAAC systems with the potential to
enhance renal clearance and diminish hepatobiliary accumulation
and/or clearance. The benefit of the second generation 99mTc-
SAAC technology was realized upon incorporation of 99mTc-
SAAC (4) into Tyr-3-octreotide, resulting in a significant
decrease of the 99mTc-SAAC complex in nontarget and hepa-
tobiliary tissues, thus affording the potential to design and
develop a superior 99mTc-SAAC-Tyr-3-octreotide radioimaging
agent with the potential to image cancer with improved whole
body clearance. Future efforts will focus on applying the novel
polar SAAC chelates to additional biologically relevant peptides
and small molecules for the development of novel radiophar-
maceuticals to image and potentially treat a variety of diseases.
ACKNOWLEDGMENT
(13) Pietzsch, H.-J., Gupta, A., Reisgys, M., Drews, A., Seifert,
S., Syhre, R., Spies, H., Alberto, R., Abram, U., and Schubiger,
P. A. (2000) Chemical and biological characterization of tech-
netium(I) and rhenium(I) tricarbonyl complexes with dithioether
ligands serving as linkers for coupling the Tc(CO)3 and Re(CO)3
moieties to biologically active molecules. Bioconjugate Chem.
11 (3), 414–424.
(14) Bourkoula, A., Paravatou-Petsotas, M., Papadopoulos, A.,
Santos, I., Pietzsch, H.-J., Livaniou, E., Pelecanou, M., Papa-
dopoulos, M., and Pirmettis, I. (2009) Synthesis and character-
ization of rhenium and technetium-99m tricarbonyl complexes
bearing the 4-[3-bromophenyl]quinazoline moiety as a biomarker
for EGFR-TK imaging. Eur. J. Med. Chem. 44 (10), 4021–4027.
(15) Maria, L., Fernandes, C., Garcia, R., Gano, L., Paulo, A.,
Santos, I. C., and Santos, I. (2009) Tris(pyrazolyl)methane 99mTc
tricarbonyl complexes for myocardial imaging. Dalton Trans.
(4), 603–606.
This work was supported in part by grants (J.W.B.) from the
Department of Energy (Grant D2-FG02-99ER62791) and the
National Institutes of Health (Grant 1R41A1054080-01).
Supporting Information Available: Radiolabeling summary
for 99mTc-1-9 SAAC complexes as well as 99mTc-4-Tyr-3-
octreotide, 99mTc-DpK-Tyr-3-octreotide, and 111In-DOTA-Tyr-
3-octreotide including representative radiochromatograms. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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