Synthesis and SAR of novel Re/99mTc-Labeled benzenesulfonamide carbonic anhydrase IX inhibitors for molecular imaging of tumor hypoxia

Article abstract of DOI:10.1021/jm3015348

Carbonic anhydrase IX (CA-IX) is upregulated in cancer in response to the hypoxic tumor microenvironment, making it an attractive molecular target for the detection of hypoxic solid tumors. A series of small molecule benzenesulfonamide based CA-IX inhibitors containing novel tridentate chelates complexed with the M(CO)₃ core (M = Re or ^99mTc) were designed and synthesized. The in vitro binding affinity of the benzenesulfonamide rhenium complexes yielded IC₅₀ values ranging from 3 to 116 nM in hypoxic CA-IX expressing HeLa cells. One of the most potent compounds, 3d (IC₅₀ = 9 nM), was radiolabeled with technetium tricarbonyl ({^99mTc(CO)₃}⁺) to afford the {^99mTc(CO)₃}⁺ complex in excellent yield and high purity. ^99mTc(CO)₃-3d bound specifically to CA-IX expressing hypoxic HeLa cells. This effort led to the identification of a diverse series of promising high affinity {^99mTc(CO) ₃}⁺ radiolabeled CA-IX inhibitors with the potential to significantly impact diagnosis, staging, and treatment selection of hypoxic solid tumors.

Full text of DOI:10.1021/jm3015348

Article
pubs.acs.org/jmc
Synthesis and SAR of Novel Re/99mTc-Labeled Benzenesulfonamide
Carbonic Anhydrase IX Inhibitors for Molecular Imaging of Tumor
Hypoxia
Genliang Lu, Shawn M. Hillier, Kevin P. Maresca, Craig N. Zimmerman, William C. Eckelman,
John L. Joyal, and John W. Babich*
Molecular Insight Pharmaceuticals, Inc., 160 Second Street, Cambridge, Massachusetts 02142, United States
ABSTRACT: Carbonic anhydrase IX (CA-IX) is upregulated in cancer
in response to the hypoxic tumor microenvironment, making it an
attractive molecular target for the detection of hypoxic solid tumors. A
series of small molecule benzenesulfonamide based CA-IX inhibitors
containing novel tridentate chelates complexed with the M(CO)3 core
(M = Re or 99mTc) were designed and synthesized. The in vitro binding
affinity of the benzenesulfonamide rhenium complexes yielded IC50
values ranging from 3 to 116 nM in hypoxic CA-IX expressing HeLa
cells. One of the most potent compounds, 3d (IC50 = 9 nM), was
radiolabeled with technetium tricarbonyl ({99mTc(CO)3}+) to afford the
{
99mTc(CO)3}+ complex in excellent yield and high purity. 99mTc-
(CO)3−3d bound specifically to CA-IX expressing hypoxic HeLa cells.
This effort led to the identification of a diverse series of promising high
affinity {99mTc(CO)3}+ radiolabeled CA-IX inhibitors with the potential to significantly impact diagnosis, staging, and treatment
selection of hypoxic solid tumors.
non-small cell lung cancers.7−9 An 125I-labeled anti-CA-IX
INTRODUCTION
■
antibody, M75, has been used to monitor tumor hypoxia in
both HeLa10 and HT-296 xenografts in mice. Moreover, a
chimeric 124I-labeled anti-CA-IX antibody, G250, is currently
undergoing clinical trials for the detection of clear cell renal
carcinoma, validating CA-IX as a promising cancer target.11
While intact antibodies such as G250 offer the potential for
tumor targeting with a radioisotope, the long circulating half-life
and poor tissue penetrability limit the effectiveness of
antibodies as radiodiagnostic and radiotherapeutic agents for
solid tumors.12 Radiolabeled small molecule radiotracers that
bind CA-IX offer the preferred approach over antibodies.13,14
While many series of CA-IX inhibitors have been reported,15−22
there are only a few examples of small molecule CA-IX
inhibitors with the potential to be used as cancer diagnostic
agents.23−26 For example, a fluorescent compound, 4-
(sulfamoylphenylethylthioureido)fluorescein, has been shown
to bind hypoxic tumor cells containing CA-IX.23−25 In addition,
the synthesis of membrane-impermeable CA-IX inhibitors that
utilize click chemistry as an efficient method for incorporation
of the radioisotope, fluorine-18 (18F) have been described.26
These analogues are being investigated as positron emission
tomography (PET) imaging agents for characterizing various
cancers.
Carbonic anhydrases (CAs) are a family of enzymes comprised
of 16 isoenzymes that catalyze the hydration of carbon dioxide
to a proton and water represented by the chemical reaction
−
CO2 + H2O ↔ HCO3 + H+ and therefore play an important
role in the regulation of intracellular and extracellular pH.
Specific isozymes are either cytosolic (CA-I, CA-II, CA-III, CA-
VII, and CA-XIII), anchored to the membrane (CA-IV, CA-IX,
CA-XII, and CA-XIV), found within the mitochondria (CA-VA
and CA-VB), or secreted from the cell (CA-VI).1 Inhibitors of
CAs, including acetazolamide, methazolamide, and ethoxazola-
mide, have been utilized for the treatment of a range of human
diseases.2−4 CA-IX is a transmembrane isoform with an
extracellular catalytic domain. It has a limited tissue distribution
and is found at low levels primarily in the gastrointestinal tract.1
Importantly, the expression of CA-IX is under the control of
hypoxia inducible factor-1α and is highly expressed in tumor
cells exposed to hypoxia both in vitro and in vivo.5 Increased
CA-IX expression (greater than 20-fold) has been detected in
selected tumor cell lines in vitro6 and by immunohistochem-
istry in carcinomas of the cervix, ovaries, kidneys, esophagus,
lung, breast, and brain.1 Additionally, CA-IX is constitutively
expressed in the majority of clear cell renal carcinomas as a
result of a mutation in the Von Hippel−Lindau tumor
suppressor.4,5
99mTc has become the mainstay of diagnostic nuclear
medicine employing SPECT and in various chemical forms is
CA-IX has been shown by immunohistochemistry and by a
variety of molecular techniques to be correlated with tumor
progression and poor survival and has been proposed as a
clinical diagnostic and prognostic marker for breast, renal, and
Received: October 19, 2012
© XXXX American Chemical Society
A
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used in more than 85% of the diagnostic scans performed each
year in hospitals.27−29 The preferential use of 99mTc
radiopharmaceuticals reflects the ideal nuclear properties of
the isotope (t1/2 = 6 h, E = 140 keV) as well as its convenient
availability from commercial generator columns.30,31 The
development of 99mTc complexes as radiopharmaceuticals is
facilitated by the use of rhenium, the group VIIB congener of
technetium. Rhenium generally produces complexes with
similar physical properties to those of technetium and is
often used as a nonradioactive alternative to technetium for
structure−activity relationship (SAR) analysis and structural
characterization.
We have recently described a series of tridentate pyridine and
carboxyl functionalized imidazole chelators for the M(CO)3
core (M = Re or 99mTc) which can alter the binding properties
and pharmacokinetics of small molecules and peptides to which
they are attached.32−34 Herein we describe the design,
synthesis, and in vitro evaluation of several novel benzene-
sulfonamide rhenium tricarbonyl complexes (Re(CO)3)
incorporating these chelators derived from 4-(2-Y-alkyl)-
benzenesulfonamide where Y = bis(pyridin-2-ylmethyl)amino
(DPA), (pyridin-2-ylmethyl)(carboxymethyl)amino (PAMA),
bis((1-methyl-1H-imidazol-2-yl)methyl)amino (NMI), bis((1-
(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM), ((1-
(carboxymethyl)-1H-imidazol-2-yl)methyl)((1-(2-(bis-
(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)-
methyl)amino (CIM/TIM), bis((1-(2-(bis(carboxymethyl)-
amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM),
((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-
yl)methyl)((1-(2-((1,5-dicarboxy-3-(2-carboxyethyl)pentan-3-
yl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM/
HIM), and bis((1-(2-((1,5-dicarboxy-3-(2-carboxyethyl)-
pentan-3-yl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)-
amino (HIM) (Figure 1). One of the most active molecules, 4-
(2-TIM-ethyl)benzenesulfonamide rhenium complex 3d, was
successfully radiolabeled with 99mTc in high yield and specific
activity and evaluated for binding to CA-IX expressing cells.
RESULTS AND DISCUSSION
■
Synthesis of Benzenesulfonamide Rhenium Com-
plexes with Homogeneous Ligands. The preparation of
homogeneous 4-aminoalkylbenzenesulfonamide rhenium tri-
carbonyl complexes 3a−j commenced with double reductive
amination of benzenesulfonamide 1a−d with two molecules of
the appropriate picolinaldehyde or N-substituted imidazole-2-
carbaldehyde to afford the desired 4-aminoalkylbenzenesulfo-
namide ligand 2a−j (Scheme 1).34,35 4-(2-DPA-ethyl) and 4-
(2-NMI-ethyl)benzenesulfonamide rhenium complexes 3a and
3b were prepared by heating a solution of 2a and 2b in
methanol to 90 °C in a pressure tube for 4 h in the presence of
the rhenium tricarbonyl precursor [NEt4]2[ReBr3(CO)3] and
purified by flash chromatography over silica gel with yields
ranging from 96 to 98%. Synthesis of the tert-butyl protected
benzenesulfonamide Re(CO)3 analogues was accomplished by
heating a solution of 2c−j in methanol or acetonitrile to 90 °C
in a pressure tube for 4 h in the presence of the rhenium
tricarbonyl precursor [NEt4]2[ReBr3(CO)3]. The crude tert-
butyl protected benzenesulfonamide rhenium complexes were
not characterized but directly deprotected with TFA/DCM at
room temperature. Upon completion of the deprotection the
reaction was concentrated under a stream of nitrogen to afford
the crude residue, which was purified by HPLC using a C18
column to afford the desired complexes 3c−j.
Figure 1. General structure of benzenesulfonamide rhenium
tricarbonyl complexes.
Synthesis of Benzenesulfonamide Rhenium Com-
plexes with Heterogeneous Ligands. The heterogeneous
ligands 4a−c were prepared using sequential double reductive
alkylation on 4-aminoethylbenzenesulfonamide 1a (Scheme
2).36,37 Addition of 1 equiv of the first aldehyde (picolinalde-
hyde or N-substituted imidazole-2-carbaldehyde) followed by
sodium borohydride afforded the intermediate. Subsequent
treatment of intermediate with t-butylglyoxalate or different N-
substituted imidazole-2-carbaldehyde afforded benzenesulfona-
mide 4a−c. 4-(2-PAMA-ethyl)benzenesulfonamide rhenium
complexes 5a was readily prepared by deprotection of tert-butyl
group with TFA, followed by heating a solution of 4a in
methanol to 100 °C in a pressure tube for 5 h in the presence
of the rhenium tricarbonyl precursor [NEt4]2[ReBr3(CO)3]. 4-
(2-CIM/TIM-ethyl) or 4-(2-TIM/HIM-ethyl)-
benzenesulfonamide rhenium complexes 5b or 5c was readily
prepared by heating a solution of 4b or 4c in acetonitrile to 90
°C in a pressure tube for 4 h in the presence of the rhenium
tricarbonyl precursor [NEt4]2[ReBr3(CO)3] followed by
deprotection of tert-butyl group with TFA.
B
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Scheme 1. Synthesis of Benzenesulfonamide Rhenium Complexes 3a−j with Homogeneous Ligands
To compare free ligands with benzenesulfonamide rhenium
complexes for the binding affinity against CA-IX, the
nonmetalated free ligands 6, 7, 8, and 9 (Figure 2) were
conveniently prepared via removal of tert-butyl group of 2d, 2e,
4c, and 2i by TFA/DCM solution.
CA-IX was observed. The N-methylimidazole complex (Re-
NMI) 3b with hydrophobicity that was intermediate between
Re-DPA 3a and Re-PAMA 5a displayed affinity (IC50 = 53 nM)
for CA-IX that was 2-fold better than the more hydrophobic 3a
but 2-fold poorer affinity than the less hydrophobic 5a. These
results established a working hypothesis that the hydro-
phobicity as measured by HPLC retention times and
experimental partition coefficients (clogP) of similarly related
complexes34 influenced the binding affinity.
Therefore, benzenesulfonamides incorporating polar imida-
zole ligands CIM, CIM/TIM, and TIM were designed and
synthesized. The rhenium complexes demonstrated between a
3- and 29-fold improvement in affinity over the hydrophobic
complexes 3a, 3b, and 5a. Complexes with the highest affinity
for CA-IX in the carboxylic acid substituted imidazole series,
Re-CIM 3c (IC50 = 4 nM), Re-CIM/TIM 5b (IC50 = 3 nM),
and Re-TIM 3d (IC50 = 9 nM) tended to be molecules that
were significantly less hindered than the complexes Re-TIM/
HIM 5c (IC50 = 44 nM) and Re-HIM 3e (IC50 = 109 nM).
An effect of the linker length and composition of the linker
between benzenesulfonamide and chelate portion of the
Competitive Binding to Hypoxic CA-IX Expressing
HeLa Cell. All compounds were evaluated in a competitive
binding assay using the 4-(2-TIM-ethyl)benzenesulfonamide
99mTc(CO)3 complex (99mTc−3d) as the radioligand for
binding to hypoxic CA-IX expressing HeLa cells. Acetazolamide
(AZO) was included in all assays as the positive control. As
illustrated in Table 1, all rhenium analogues demonstrated
moderate to high affinity for CA-IX (IC50 = 3−116 nM).
The SAR of the rhenium complexes displayed several
interesting trends. The evaluation of several different chelates
was first investigated within the 4-(2-Y-ethyl)-
benzenesulfonamide rhenium complexes 3a−e and 5a−c. The
very hydrophobic dipyridylmethylamine complex, Re-DPA 3a
(IC50 = 116 nM), displayed only modest affinity for CA-IX. For
the less hydrophobic pyridylamine monoacetic acid complex
(Re-PAMA) 5a (IC50 = 28 nM), a 4-fold increase in affinity for
C
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Scheme 2. Synthesis of Benzenesulfonamide Rhenium Complexes with Heterogeneous Ligands 5a−c
molecules was also observed. For the polar analogues Re-CIM
3f (IC50 = 51 nM) and Re-TIM 3g (IC50 = 116 nM), a
methylene (−CH2−) linker displayed 13-fold lower affinity
than the corresponding homologues 3c and 3d which
contained the ethylene (−CH2CH2−) linker. Additionally
complex 3h (IC50 = 43 nM) with the longer oxygen containing
(−O(CH2)4−) linker displayed lower affinity than 3d (IC50 = 9
nM) which contained the ethylene (−CH2CH2−) linker.
However, a different trend was observed with the polar chelate
that contained additional carboxylic acids, HIM, where the
longer length linkers (−O(CH2)4−) 3i (IC50 = 35 nM) and
(−O(CH2)6−) 3j (IC50 = 33 nM) provided complexes with
better affinity for CA-IX than complexes like 4-(2-HIM-ethyl)
benzenesulfonamide rhenium complex 3e (IC50 = 109 nM)
with the shorter linker (−CH2CH2−).
structure of the metal complexes compared to the correspond-
ing free ligands.
Radiochemistry. The ability of these novel benzenesulfo-
namide CA-IX inhibitors to be converted to the radioactive
99mTc complexes was demonstrated by employing one of the
highest affinity compounds, 3d (IC50 = 9 nM). The
radiolabeling was accomplished using the fully protected tert-
butyl ester derivative 2d. The radiolabeling of tert-butyl ester
derivative 2d with {99mTc(CO)3}+ to form the desired metal
complex was achieved in two steps utilizing standard method-
ology.36 IsoLink kits (Covidien) were used to generate the
desired intermediate [99mTc(CO)3(H2O)3]+, which following
neutralization with hydrochloric acid, was reacted with 2d in a
sealed vial at 75 °C for 60 min with a ligand concentration of
10−4 M in an equal volume mixture of acetonitrile and water.
After cooling and evaporation of solvent, the tert-butyl ester
protecting groups were removed by treatment with 50% TFA in
DCM at room temperature. Upon completion of the
deprotection, the reaction was analyzed for purity via HPLC
and subsequently purified by HPLC to afford the desired
conjugate 99mTc−3d in 90% RCY and >95% RCP (Figure 3).
All of the nonmetalated free ligands analyzed displayed poor
affinity for CA-IX as demonstrated by 6 (IC50 = 2500 nM), 7
(IC50 = 1600 nM), 8 (IC50 = 2500 nM), and 9 (IC50 = 782
nM). This suggested that the metal complex is required for
high affinity binding to CA-IX. This may be attributed to the
positive charge of the metal complexes or a more constrained
D
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This procedure utilized HPLC to remove excess free ligand.
Because the free ligand has poor affinity for CA-IX compared to
the radiolabeled final product, current research is directed at
production of a “kit” formulation where the product can be
utilized directly without purification from the excess free ligand.
Binding to CA-IX. To ensure that the binding of 99mTc−3d
to CA-IX on HeLa cells was specific and saturable, cells were
incubated with 3 nM 99mTc−3d alone or in the presence of 10
μM acetazolamide, a structurally unrelated carbonic anhydrase
inhibitor, after exposure of the cells to hypoxic or normoxic
conditions for 24 h. As illustrated in Figure 4A, 99mTc-3d bound
only to hypoxic HeLa cells and not to HeLa cells that remained
under normoxic conditions. Further, the binding of 99mTc−3d
to the hypoxic HeLa cells was abrogated by coincubation with
10 μM acetazolamide. A dose curve of 1−10000 nM
acetazolamide or nonradiolabeled 3d demonstrated IC50 values
of 7 and 9 nM, respectively (Figure 4B).
CONCLUSIONS
■
In summary, a series of benzenesulfonamide CA-IX inhibitors
incorporating tridentate chelates DPA, NMI, CIM, CIM/TIM,
TIM, TIM/HIM, and HIM with the M(CO)3 core (M = Re or
99mTc) were designed, prepared, and evaluated for binding to
hypoxic CA-IX expressing HeLa cells to generate a SAR. All
rhenium analogues demonstrated moderate to high affinity for
CA-IX (IC50 = 3−116 nM), with compounds containing the
less hindered polar imidazole derivatized chelates, CIM, CIM/
TIM, and TIM, and ethylene linker exhibiting the highest
affinity. This effort has led to the identification of a diverse
series of promising high affinity {99mTc(CO)3}+ radiolabeled
CA-IX inhibitors which, because of the variation in chemical
composition, will likely exhibit divergence in tissue distribution
and pharmacokinetic properties. These compounds will be
further evaluated in animal models of tumor hypoxia to select a
lead compound with adequate tumor uptake and acceptable
tumor-to-background for advancement to clinical trials.
Figure 2. Structure of nonmetal free ligands 6, 7, 8, and 9.
Table 1. Competitive Binding of Benzenesulfonamide
Analogues
EXPERIMENTAL SECTION
■
General Procedures. All reactions were carried out in dry
glassware under an atmosphere of argon unless otherwise noted.
Reactions were purified by flash chromatography under medium
pressure using a Biotage SP4 or by preparative high pressure liquid
compd
L
Y
IC50 (nM)
1
chromatography using a Varian HPLC Prostar 210 Instrument. H
AZO
3a
5a
3b
3c
5b
3d
5c
3e
3f
7
116
28
NMR was recorded on a Bruker 400 MHz instrument. Spectra are
reported as ppm and are referenced to the solvent resonances in
CDCl3, DMSO-d6, or methanol-d4. Low resolution ESI mass spectra
were obtained on Agilent G1956B LC/MS with an Agilent 1100
HPLC system. Higher resolution ESI mass spectra were obtained on
Agilent G6510A Q-TQF LC/MS. All compounds evaluated for
binding affinity toward CA-IX (3c−j, 5a−c, 6, 7, 8, and 9) were
purified on a Varian Prostar 210 preparative HPLC system using a
Vydac C18 reverse-phase column (250 mm × 10 mm × 5 μm)
connected to a Varian Prostar model 340 UV−vis detector monitoring
at 220 nm or Biotage SP4 system over C18 catridge. Final product
purification by HPLC was achieved using a binary solvent gradient of
5−85% mobile phase B where mobile phase A is water containing
0.1% TFA and mobile phase B is acetonitrile containing 0.1% TFA.
The analytical purity of test compounds was obtained on an Agilent
1100 HPLC system with ZORBAX SB-Aq column (4.6 × 50 mm × 5
μm) using a binary solvent gradient of 15−100% mobile phase B
where mobile phase A is water containing 0.1% formic acid and mobile
phase B is acetonitrile containing 0.1% formic acid. The purity of all
compounds evaluated in the biological assay was >95% purity as
judged by HPLC. Analytical HPLC of 99mTc−3d was performed using
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
CH2
Re-DPA
Re-PAMA
Re-NMI
Re-CIM
Re-CIM/TIM
Re-TIM
Re-TIM/HIM
Re-HIM
Re-CIM
Re-TIM
Re-TIM
Re-HIM
Re-HIM
TIM
53
4
3
9
44
109
51
3g
3h
3i
CH2
116
43
O(CH2)4
O(CH2)4
O(CH2)6
(CH2)2
(CH2)2
(CH2)2
O(CH2)4
35
3j
33
6
2500
1600
2500
782
7
HIM
8
TIM/HIM
HIM
9
E
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Figure 3. Radiochromatogram of 99mTc(CO)3−3d (50 μCi injection) demonstrating 98% RCP, 90% RCY (top), and the corresponding UV−visible
chromatogram of the rhenium complex 3d used for confirmation of identity (bottom).
the same method described above with an analytical Vydac C18
reverse-phase column (250 mm × 4.6 mm × 5 μm).
3.26 (s, 6 H), 2.72 (t, J = 6.8 Hz, 2 H), 2.66 (t, J = 6.8 Hz, 2 H). MS
(ESI), 389.1 (M + H)+.
tert-Butyl 2,2′-(2,2′-(4-Sulfamoylphenethylazanediyl)bis-
(methylene)bis(1H-imidazole-2,1-diyl))diacetate (2c). Treatment of
4-(2-aminoethyl)benzenesulfonamide 1a (110 mg, 0.55 mmol), tert-
butyl 2-(2-formyl-1H-imidazol-1-yl)acetate (250 mg, 1.19 mmol), and
AcOH (0.10 mL, 1.75 mmol) in DCE (20 mL) according to the
General Synthesis of Benzenesulfonamide Analogues 2a−j.
A suspension of benzenesulfonamide 1a−d (1 equiv), appropriate
pyridine-2-carboxaldehyde or N-substituted imidazole-2-carbaldehyde
(2 equiv), and AcOH in DCE was stirred at 75 °C for 30 min under
nitrogen. The reaction mixture was cooled to 0 °C and treated with
NaBH(OAc)3 (3 equiv). The reaction mixture was stirred at room
temperature overnight and quenched by methanol. Solvents were
concentrated under reduced pressure to give a residue, which was
purified by flash chromatography over silica gel with a gradient of 0−
50% methanol in DCM to afford desired product 2a−j.
1
general procedure described above afforded 2c (132 mg, 41%). H
NMR (400 MHz, CD3OD) 7.75 (d, J = 8.4 Hz, 2 H), 7.18 (d, J = 8.4
Hz, 2 H), 7.07 (s, 2 H), 6.93 (s, 2 H), 4.58 (s, 4 H), 3.68 (s, 4 H),
2.84−2.74 (m, 4 H), 1.44 (s, 18 H). MS (ESI), 589.4 (M + H)+.
tert-Butyl 2,2′-(2,2′-(4-Sulfamoylphenethylazanediyl)bis-
(methylene)bis(1H-imidazole-2,1-diyl-acetylazanediyl))diacetate
(2d). Treatment of 4-(2-aminoethyl)benzenesulfonamide 1a (100 mg,
0.50 mmol), tert-butyl 2,2′-(2-(2-formyl-1H-imidazol-1-yl)-
acetylazanediyl)diacetate (457 mg, 1.2 mmol), and AcOH (0.10 mL,
1.75 mmol) in DCE (30 mL) according to the general procedure
4-(2-(Bis(pyridin-2-ylmethyl)amino)ethyl)benzenesulfonamide
(2a). Treatment of 4-(2-aminoethyl)benzenesulfonamide 1a (200 mg,
1.0 mmol), pyridine-2-carboxaldehyde (214 mg, 2.0 mmol), and
AcOH (0.05 mL, 0.87 mmol) in DCE (20 mL) according to the
general procedure described above afforded 2a (370 mg, 97%) as a
1
described above afforded 2d (465 mg, 100%). H NMR (400 MHz,
1
yellow solid. H NMR (400 MHz, CDCl3) 8.46 (d, J = 4.0 Hz, 2 H),
DMSO-d6) 7.63 (d, J = 8.0 Hz, 2 H), 7.23−7.21 (m, 4 H), 6.96 (s, 2
H), 6.79 (s, 2 H), 5.00 (s, 4 H), 4.30 (s, 4 H), 3.95 (s, 4 H), 3.59 (s, 4
H), 2.70−2.66 (m, 2H), 2.59−2.55 (m, 2 H), 1.42 (s, 18 H), 1.33 (s,
18 H). MS (ESI), 466.4 (M/2 + H)+.
Tetra-tert-butyl 4,4′-((2,2′-(2,2′-(((4-Sulfamoylphenethyl)-
azanediyl)bis(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis-
(azanediyl))bis(4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate) (2e).
Treatment of 4-(2-aminoethyl)benzenesulfonamide 1a (80 mg, 0.40
mmol), di-tert-butyl 4-(2-bromoacetamido)-4-(3-(tert-butoxy)-3-
oxopropyl)heptanedioate (447 mg, 0.81 mmol), and AcOH (0.05
mL, 0.87 mmol) in DCE (20 mL) according to the general procedure
7.69−7.65 (m, 4 H), 7.30−7.20 (m, 6 H), 3.79 (s, 4 H), 2.87 (t, J = 7.0
Hz, 2 H), 2.69 (t, J = 7.0 Hz, 2 H). MS (ESI), 383.1 (M + H)+.
4-(2-(Bis((1-methyl-1H-imidazol-2-yl)methyl)amino)ethyl)-
benzenesulfonamide (2b). Treatment of 4-(2-aminoethyl)-
benzenesulfonamide 1a (200 mg, 1.0 mmol), 1-methyl-1H-imida-
zole-2-carbaldehyde (220 mg, 2.0 mmol), and AcOH (0.05 mL, 0.87
mmol) in DCE (20 mL) according to the general procedure described
1
above afforded 2b (389 mg, 100%) as a yellow solid. H NMR (400
MHz, DMSO-d6) 7.62 (d, J = 8.0 Hz, 2 H), 7.25 (s, 2 H), 7.04 (s, 2
H), 7.02 (d, J = 8.0 Hz, 2 H), 6.79 (d, J = 0.8 Hz, 2 H), 3.64 (s, 4 H),
F
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Tetra-tert-butyl 4,4′-((2,2′-(2,2′-(((4-(4-Sulfamoylphenoxy)butyl)-
azanediyl)bis(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis-
(azanediyl))bis(4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (2i).
Treatment of 4-(4-aminobutoxy)benzenesulfonamide TFA salt (1c)
(216 mg, 0.59 mmol) and di-tert-butyl 4-(3-(tert-butoxy)-3-oxoprop-
yl)-4-(2-(2-formyl-1H-imidazol-1-yl)acetamido)heptanedioate (570
mg, 1.033 mmol) in DCE (20 mL) according to the general
procedure described above afforded 2i (186 mg, 28%). 1H NMR (400
MHz, DMSO-d6) 7.73−7.70 (m, 4 H), 7.17 (s, 2 H), 7.00−6.98 (m, 4
H), 6.78 (s, 2 H), 4.61 (s, 4 H), 3.87 (t, J = 7.2 Hz, 2 H), 3.51 (s, 4 H),
2.50−2.48 (m, 2 H), 2.11 (t, J = 8.0 Hz, 12 H), 1.78 (t, J = 8.0 Hz, 12
H), 1.60−1.35 (m, 58 H). MS (ESI), 658.4 (M/2 + H)+.
Tetra-tert-butyl 4,4′-((2,2′-(2,2′-(((6-(4-Sulfamoylphenoxy)hexyl)-
azanediyl)bis(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis-
(azanediyl))bis(4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate) (2j).
Treatment of 4-((6-aminohexyl)oxy)benzenesulfonamide containing
TFA 1d (346 mg, 0.659 mmol), di-tert-butyl 4-(3-(tert-butoxy)-3-
oxopropyl)-4-(2-(2-formyl-1H-imidazol-1-yl)acetamido)heptanedioate
(727 mg, 1.32 mmol), and AcOH (0.05 mL, 0.87 mmol) in DCE (50
mL) according to the general procedure described above afforded 2j
1
(153 mg, 17%). H NMR (400 MHz, DMSO-d6) 7.76 (s, 2 H), 7.71
(d, J = 8.8 Hz, 2 H), 7.17 (s, 2 H), 7.04 (s, 2 H), 7.02 (d, J = 8.8 Hz, 2
H), 6.82 (s, 2 H), 4.62 (s, 4 H), 3.97 (t, J = 6.6 Hz, 2 H), 3.53 (s, 4 H),
2.44−2.40 (m, 2 H), 2.11 (t, J = 7.8 Hz, 12 H), 1.78 (t, J = 7.8 Hz, 12
H), 1.67−1.19 (m, 62 H). MS (ESI), 672.4 (M/2 + H)+.
Figure 4. Binding of 99mTc−3d to HeLa cells. (A) 99mTc−3d (3 nM)
was incubated with hypoxic or normoxic HeLa cells in the presence or
absence of 10 μM acetazolamide. (B) 99mTc−3d (3 nM) was
incubated with hypoxic HeLa cells in the presence of 1−10000 nM
acetazolamide or nonradiolabeled 3d.
tert-Butyl 2-((Pyridin-2-ylmethyl)(4-sulfamoylphenethyl)amino)-
acetate (4a). A solution of 4-(2-aminoethyl)benzenesulfonamide
(1a) (1.60 g, 8.0 mmol), AcOH (0.30 mL, 5.24 mmol) and 2-
pyridinecarboxaldehyde (0.76 mL, 8.0 mmol) in DCE (50 mL) was
heated at 75 °C for 30 min under nitrogen. The reaction mixture was
cooled to 0 °C and treated sequentially with NaBH(OAc)3 (6.36 g, 30
mmol) and crude tert-butyl glyoxalate37 (2.08 g). The reaction mixture
was stirred at room temperature overnight and quenched by water.
The reaction mixture was extracted with DCM. The organic layer was
dried and concentrated under reduced pressure to give a residue,
which was purified by flash chromatography over silica gel with a
1
described above afforded 2e (322 mg, 63%). H NMR (400 MHz,
DMSO-d6) 7.77 (s, 2 H), 7.64 (d, J = 8.0 Hz, 2 H), 7.23 (s, 2 H), 7.21
(d, J = 8.4 Hz, 2 H), 7.01 (s, 2 H), 6.80 (s, 2 H), 4.57 (s, 4 H), 3.61 (s,
4 H), 2.79−2.62 (m, 4 H), 2.09 (t, J = 8.0 Hz, 12 H), 1.76 (t, J = 8.0
Hz, 12 H), 1.32 (s, 54 H). MS (ESI), 636.5 (M/2 + H)+.
1
gradient of 0−10% methanol in DCM afforded 4a (1.04 g, 32%). H
NMR (400 MHz, DMSO-d6) 8.45−8.43 (m, 1 H), 7.70−7.63 (m, 3
H), 7.33 (d, J = 8.4 Hz, 2 H), 7.27 (s, 2 H), 7.24−7.20 (m, 2 H), 3.86
(s, 2 H), 3.31 (s, 2 H), 2.85−2.77 (m, 4 H), 1.40 (s, 9 H). MS (ESI),
406 (M + H)+.
tert-Butyl 2,2′-(2,2′-(4-Sulfamoylbenzylazanediyl)bis(methylene)-
bis(1H-imidazole-2,1-diyl))diacetate (2f). Treatment of 4-(2-
aminomethyl)benzenesulfonamide hydrochloride (1b) (223 mg, 1.0
mmol) and tert-butyl 2-(2-formyl-1H-imidazol-1-yl)acetate (441 mg,
2.1 mmol) in DCE (20 mL) according to the general procedure
tert-Butyl 2,2′-(2-(2-((((1-(2-tert-Butoxy-2-oxoethyl)-1H-imidazol-
2-yl)methyl)(4-sulfamoylphenethyl)amino)methyl)-1H-imidazol-1-
yl)acetylazanediyl)diacetate (4b). A solution of 4-(2-aminoethyl)-
benzenesulfonamide (1a) (600 mg, 3.0 mmol), AcOH (0.10 mL, 1.75
mmol), and tert-butyl 2,2′-(2-(2-formyl-1H-imidazol-1-yl)-
acetylazanediyl)diacetate (381 mg, 1.0 mmol) in DCE (25 mL) was
stirred at 75 °C for 30 min under nitrogen. The reaction mixture was
cooled to 0 °C and treated with NaBH(OAc)3 (0.422 g, 2.0 mmol).
The reaction mixture was stirred at room temperature overnight and
quenched with water. Solvent were concentrated under reduced
pressure to give a residue, which was purified by flash chromatography
over silica gel eluting with 1% MeOH in DCM to 20% MeOH in
DCM to afford tert-butyl 2,2′-(2-(2-((4-sulfamoylphenethylamino)-
methyl)-1H-imidazol-1-yl)acetylazanediyl)diacetate (191 mg). To a
solution of the above product, tert-butyl 2,2′-(2-(2-((4-
sulfamoylphenethylamino)methyl)-1H-imidazol-1-yl)acetylazanediyl)-
diacetate (191 mg, 0.33 mmol), AcOH (0.10 mL, 1.75 mmol), and
tert-butyl 2-(2-formyl-1H-imidazol-1-yl)acetate (105 mg, 0.50 mmol)
in DCE (10 mL) at 0 °C was added NaBH(OAc)3 (0.212 g, 1.0
mmol). The reaction mixture was stirred at room temperatureover-
night and quenched by water. Solvent was concentrated under reduced
pressure to give a residue, which was purified by flash chromatography
over silica gel eluting with DCM to 10% MeOH in DCM afforded 4b
(143 mg, 19% over 2 steps). 1H NMR (400 MHz, DMSO-d6) 7.63 (d,
J = 8.4 Hz, 2 H), 7.23 (s, 2 H), 7.21 (d, J = 8.0 Hz, 2 H), 7.03 (s, 1 H),
6.97 (s, 1 H), 6.78 (s, 2 H), 4.93 (s, 2 H), 4.72 (s, 2 H), 4.26 (s, 2 H),
3.94 (s, 2 H), 3.62 (s, 2 H), 3.57 (s, 2 H), 2.71−2.56 (m, 4 H), 1.41 (s,
18 H), 1.34 (s, 9 H). MS (ESI), 760.3 (M + H)+.
1
described above afforded 2f (569 mg, 99%) as a yellow oil. H NMR
(400 MHz, CDCl3) 7.83 (d, J = 8.4 Hz, 2 H), 7.41 (d, J = 8.4 Hz, 2
H), 6.97 (s, 2 H), 6.82 (s, 2 H), 4.66 (s, 2 H), 4.43 (s, 2 H), 3.83 (s, 1
H), 3.73 (s, 1 H), 3.61 (s, 2 H), 3.48 (s, 4 H), 1.39 (s, 18 H). MS
(ESI), 575.3 (M + H)+.
tert-Butyl 2,2′-(2,2′-(4-Sulfamoylbenzylazanediyl)bis(methylene)-
bis(1H-imidazole-2,1-diyl-acetylazanediyl))diacetate (2g). Treat-
ment of 4-(2-aminomethyl)benzenesulfonamide hydrochloride (1b)
(111 mg, 0.50 mmol) and tert-butyl 2,2′-(2-(2-formyl-1H-imidazol-1-
yl)acetylazanediyl)diacetate (419 mg, 1.1 mmol) in DCE (40 mL)
according to the general procedure described above afforded 2g (322
mg, 70%). 1H NMR (400 MHz, DMSO-d6) 7.71 (d, J = 8.4 Hz, 2 H),
7.46 (d, J = 8.4 Hz, 2 H), 7.24 (s, 2 H), 6.94 (s, 2 H), 6.80 (s, 2 H),
5.04 (s, 4 H), 4.30 (s, 4 H), 3.92 (s, 4 H), 3.64 (d, J = 16.8 Hz, 1 H),
3.60 (d, J = 16.8 Hz, 1 H), 3.42 (s, 4 H), 1.42 (s, 18 H), 1.34 (s, 18 H).
MS (ESI), 459.3 (M/2 + H)+.
Tetra-tert-butyl 2,2′,2″,2‴-((2,2′-(2,2′-(((4-(4-Sulfamoylphenoxy)-
butyl)azanediyl)bis(methylene))bis(1H-imidazole-2,1-diyl))bis-
(acetyl))bis(azanetriyl))tetraacetate (2h). Treatment of 4-(4-
aminobutoxy)benzenesulfonamide TFA salt (1c) (437 mg, 1.0
mmol), tert-butyl 2,2′-(2-(2-formyl-1H-imidazol-1-yl)acetylazanediyl)-
diacetate (838 mg, 2.2 mmol), and AcOH (0.10 mL, 1.75 mmol) in
DCE (40 mL) according to the general procedure described above
afforded 2h (560 mg, 57%). 1H NMR (400 MHz, DMSO-d6) 7.70 (d,
J = 8.8 Hz, 2 H), 7.17 (s, 2 H), 6.99−6.97 (m, 4 H), 6.82 (s, 2 H), 5.06
(s, 4 H), 4.31 (s, 4 H), 3.96 (s, 4 H), 3.87 (t, J = 6.2 Hz, 2 H), 3.38 (s,
4 H), 2.42 (t, J = 6.6 Hz, 2 H), 1.56−1.47 (m, 4 H), 1.44 (s, 18 H),
1.36 (s, 18 H). MS (ESI), 975.3 (M + H)+.
Di-tert-butyl 4-(2-(2-((((1-(2-(Bis(2-(tert-butoxy)-2-oxoethyl)-
a m i n o ) - 2 - o x o e t h y l ) - 1 H - i m i d a z o l - 2 - y l ) m e t h y l ) ( 4 -
G
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Journal of Medicinal Chemistry
Article
sulfamoylphenethyl)amino)methyl)-1H-imidazol-1-yl)acetamido)-
4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (4c). A solution of 4-
(4-aminobutoxy)benzenesulfonamide (2.40, 12.0 mmol), AcOH (0.40
mL, 7.0 mmol), and tert-butyl 2,2′-(2-(2-formyl-1H-imidazol-1-yl)-
acetylazanediyl)diacetate (1.524 g, 4.0 mmol) in DCE (100 mL) was
stirred at 75 °C for 30 min under nitrogen. The reaction mixture was
cooled to 0 °C, treated with NaBH(OAc)3 (1.64 g, 8.0 mmol), and
stirred at room temperature overnight and quenched by water. Solvent
was concentrated under reduced pressure to give a residue, which was
purified by flash chromatography over silica gel with a gradient of 0−
20% methanol in DCM afforded di-tert-butyl 2,2′-((2-(2-(((4-
sulfamoylphenethyl)amino)methyl)-1H-imidazol-1-yl)acetyl)-
2,2′-(2,2′-(4-Sulfamoylphenethylazanediyl)bis(methylene)bis(1H-
imidazole-2,1-diyl))diacetic Acid Rhenium Tricarbonyl Complex
(3c). Treatment of 2c (65 mg, 0.11 mmol) and [NEt4]2[ReBr3(CO)3]
(92.4 mg, 0.12 mmol) in methanol (3.0 mL) according to the general
procedure described above afforded 3c (60 mg, 53%) as a white solid.
1H NMR (400 MHz, DMSO-d6) 7.81 (d, J = 8.0 Hz, 2 H), 7.60 (d, J =
8.4 Hz, 2 H), 7.30 (s, 2 H), 7.23 (d, J = 1.2 Hz, 2 H), 7.08 (d, J = 1.2
Hz, 2 H), 4.91 (s, 4 H), 4.72 (s, 4 H), 3.89−3.85 (m, 2 H), 3.18−3.14
(m, 2 H). HRMS (ESI) calcd for C23H24N6O9ReS (M)+, 747.0883;
found, 747.0873.
2,2′-(2,2′-(4-Sulfamoylphenethylazanediyl)bis(methylene)bis(1H-
imidazole-2,1-diyl-acetylazanediyl))diacetic Acid Rhenium Tricar-
bonyl Complex (3d). Treatment of 2d (32 mg, 0.034 mmol) and
[NEt4]2[ReBr3(CO)3] (30 mg, 0.039 mmol) in methanol (3.0 mL)
according to the general procedure described above afforded 3d as a
white solid (33 mg, 27%). 1H NMR (400 MHz, DMSO-d6) 7.79 (d, J
= 8.4 Hz, 2 H), 7.58 (d, J = 8.0 Hz, 2 H), 7.29 (s, 2 H), 7.13 (s, 2 H),
7.06 (s, 2 H), 5.06 (s, 4 H), 4.65 (d, J = 16.4 Hz, 2 H), 4.40 (d, J =
16.4 Hz, 2 H), 4.29 (s, 4 H), 4.08 (d, J = 17.6 Hz, 2 H), 4.02 (d, J =
17.6 Hz, 2 H), 3.87−3.83 (m, 2 H), 3.11−3.08 (m, 2 H). HRMS (ESI)
calcd for C31H34N8O15ReS (M)+, 977.1416; found, 977.1411.
4,4′-((2,2′-(2,2′-(((4-Sulfamoylphenethyl)azanediyl)bis-
(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis(azanediyl))-
bis(4-(2-carboxyethyl)heptanedioic Acid Rhenium Tricarbonyl
Complex (3e). Treatment of 2e (167 mg, 0.131 mmol) and
[NEt4]2[ReBr3(CO)3] (116 mg, 0.15 mmol) in methanol (3.0 mL)
according to the general procedure described above afforded 3e (75
mg, 48%) as a white solid. 1H NMR (400 MHz, DMSO-d6) 12.10 (brs,
6 H), 7.78 (d, J = 8.4 Hz, 2 H), 7.71 (s, 2 H), 7.55 (d, J = 8.0 Hz, 2 H),
7.27 (s, 2 H), 7.17 (s, 2 H), 7.03 (s, 2 H), 4.72 (s, 4 H), 4.64 (d, J =
16.4 Hz, 2 H), 4.52 (d, J = 16.4 Hz, 2 H), 3.86−3.82 (m, 2 H), 3.13−
3.09 (m, 2 H), 2.14 (t, J = 8.0 Hz, 12 H), 1.85 (t, J = 8.0 Hz, 12 H).
HRMS (ESI) calcd for C43H54N8O19ReS (M)+, 1205.2778; found,
1205.2776.
1
azanediyl)diacetate as a white solid (547 mg, 24%). H NMR (400
MHz, DMSO-d6) 7.68 (d, J = 8.0 Hz, 2 H), 7.32 (d, J = 8.0 Hz, 2 H),
7.24 (s, 2 H), 6.94 (s, 1 H), 6.72 (s, 1 H), 4.95 (s, 2 H), 4.25 (s, 2 H),
3.95 (s, 2 H), 3.61 (s, 2 H), 2.70−2.67 (m, 4 H), 1.43 (s, 9 H), 1.35 (s,
9 H). MS (ESI), 566.2 (M + H)+. To a solution of above product, di-
tert-butyl 2,2′-((2-(2-(((4-sulfamoylphenethyl)amino)methyl)-1H-
imidazol-1-yl)acetyl)azanediyl)diacetate (200 mg, 0.353 mmol), di-
tert-butyl 4-(3-(tert-butoxy)-3-oxopropyl)-4-(2-(2-formyl-1H-imidazol-
1-yl)acetamido)heptanedioate (195 mg, 0.353 mmol), and AcOH
(0.10 mL, 1.75 mmol) in DCE (10 mL) at 0 °C was treated with
NaBH(OAc)3 (148 mg, 0.70 mmol). The reaction mixture was stirred
at 0 °C for 30 min and at room temperature overnight and quenched
by water. The reaction mixture was concentrated under reduced
pressure to give a residue, which was purified by flash chromatography
over silica gel with a gradient of 0−10% methanol in DCM afforded 4c
1
(237 mg, 61%) as yellow solid. H NMR (400 MHz, DMSO-d6) 7.76
(s, 1 H), 7.66 (d, J = 8.4 Hz, 2 H), 7.23 (d, J = 8.4 Hz, 2 H), 7.22 (s, 2
H), 7.01 (d, J = 1.2 Hz, 1 H), 6.99 (d, J = 1.6 Hz, 1 H), 6.83 (d, J = 0.8
Hz, 1 H), 6.80 (d, J = 0.8 Hz, 1 H), 5.02 (s, 2 H), 4.55 (s, 2 H), 4.31
(s, 2 H), 3.97 (s, 2 H), 3.63 (s, 2 H), 3.60 (s, 2 H), 2.77−2.73 (m, 2
H), 2.66−2.60 (m, 2 H), 2.10 (t, J = 8.2 Hz, 6 H), 1.78 (t, J = 8.2 Hz, 6
H), 1.36 (s, 45 H). MS (ESI), 551.4 (M/2 + H)+.
General Procedure for Synthesis of Rhenium Tricarbonyl
Complexes 3c−j, 5b, and 5c. A solution of 2c−i, 4b, or 4c and
[NEt4]2[ReBr3(CO)3] in methanol or acetonitrile in sealed pressure
tube was stirred at 90 °C for 4 h. The solvent was evaporated to give a
residue, which was dissolved in DCM (1.0−3.0 mL) and TFA (1.0−
3.0 mL) and the reaction mixture was stirred at room temperature
overnight. The solvent was evaporated and afforded the crude product,
which was purified by HPLC using a binary solvent gradient of 0−50%
mobile phase B where mobile phase A is water containing 0.1% TFA
and mobile phase B is acetonitrile containing 0.1% TFA and
lyophilized to afford the desired product 3c−j, 5b, or 5c.
2,2′-(2,2′-(4-Sulfamoylbenzylazanediyl)bis(methylene)bis(1H-
imidazole-2,1-diyl))diacetic Acid Rhenium Tricarbonyl Complex
(3f). Treatment of 2f (40 mg, 0.070 mmol) and [NEt4]2[ReBr3(CO)3]
(60 mg, 0.077 mmol) in methanol (3.0 mL) according to the general
procedure described above afforded 3f (23 mg, 45%) as a white solid.
1H NMR (400 MHz, DMSO-d6) 7.93 (d, J = 8.0 Hz, 2 H), 7.86 (d, J =
8.4 Hz, 2 H), 7.47 (s, 2 H), 7.14 (d, J = 1.2 Hz, 2 H), 7.06 (d, J = 1.2
Hz, 2 H), 4.92 (s, 2 H), 4.79 (d, J = 16.0 Hz, 2 H), 4.76 (s, 4 H), 4.20
(d, J = 16.0 Hz, 2 H). HRMS (ESI) calcd for C22H22N6O9ReS (M)+,
733.0726; found, 733.0715.
2,2′,2″,2‴-((2,2′-(2,2′-(((4-Sulfamoylbenzyl)azanediyl)bis-
(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis(azanetriyl))-
tetraacetic Acid Rhenium Tricarbonyl Complex (3g). Treatment of
2g (75 mg, 0.082 mmol) and [NEt4]2[ReBr3(CO)3] (75 mg, 0.097
mmol) in methanol (4.0 mL) according to the general procedure
4-(2-(Bis(pyridin-2-ylmethyl)amino)ethyl)benzenesulfonamide
Rhenium Tricarbonyl Complex (3a). A solution of 2a (107 mg, 0.279
mmol) and [NEt4]2[ReBr3(CO)3] (216 mg, 0.279 mmol) in methanol
(5.0 mL) in sealed pressure tube was stirred at 90 °C for 4 h. The
solvent was evaporated to give a residue, which was purified by flash
chromatography over silica gel with a gradient of 0−10% methanol in
1
described above afforded 3g (56 mg, 70%) as a white solid. H NMR
(400 MHz, DMSO-d6) 7.91 (d, J = 8.4 Hz, 2 H), 7.87 (d, J = 8.4 Hz, 2
H), 7.43 (s, 2 H), 7.09 (d, J = 1.6 Hz, 2 H), 7.06 (d, J = 1.6 Hz, 2 H),
5.03−4.92 (m, 6 H), 4.75 (d, J = 15.6 Hz, 2 H), 4.21 (s, 4 H), 4.08−
3.92 (m, 6 H). HRMS (ESI) calcd for C30H32N8O15ReS (M)+,
963.1260; found, 963.1255.
2,2′,2″,2‴-((2,2′-(2,2′-(((4-(4-Sulfamoylphenoxy)butyl)azanediyl)-
bis(methylene))-bis(1H-imidazole-2,1-diyl))bis(acetyl))bis-
(azanetriyl))tetraacetic Acid Rhenium Tricarbonyl Complex (3h).
Treatment of 2h (97.4 mg, 0.10 mmol) and [NEt4]2[ReBr3(CO)3] (80
mg, 0.10 mmol) in acetonitrile (5.0 mL) according to the general
procedure described above afforded 3h as a white solid (70 mg, 69%).
1H NMR (400 MHz, DMSO-d6) 7.73 (d, J = 9.2 Hz, 2 H), 7.18 (s, 2
H), 7.11 (d, J = 2.4 Hz, 2 H), 7.10 (d, J = 9.2 Hz, 2 H), 7.03 (d, J = 1.6
Hz, 2 H), 5.00 (s, 4 H), 4.39 (d, J = 16.4 Hz, 2 H), 4.29 (d, J = 16.4
Hz, 2 H), 4.23 (s, 4 H), 4.12 (t, J = 6.0 Hz, 2 H), 4.02 (s, 4 H), 3.79−
3.75 (m, 2 H), 1.92−1.80 (m, 4 H). HRMS (ESI) calcd for
C33H38N8O16ReS (M)+, 1021.1684; found, 1021.1676.
4,4′-((2,2′-(2,2′-(((4-(4-Sulfamoylphenoxy)butyl)azanediyl)bis-
(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis(azanediyl))-
bis(4-(2-carboxyethyl)heptanedioic acid) Rhenium Tricarbonyl
Complex (3i). Treatment of 2i (114 mg, 0.0867 mmol) and
1
DCM afforded 3a (174 mg, 96%) as a yellow solid. H NMR (400
MHz, DMSO-d6) 8.82 (d, J = 5.2 Hz, 2H), 8.02 (td, J = 7.8, 1.2 Hz, 2
H), 7.83 (d, J = 8.0 Hz, 2 H), 7.63 (d, J = 8.4 Hz, 2 H), 7.60 (d, J = 8.4
Hz, 2 H), 7.41 (t, J = 6.6 Hz, 2 H), 7.32 (s, 2 H), 5.18 (d, J = 16.8 Hz,
2 H), 5.04 (d, J = 16.4 Hz, 2 H), 3.97−3.92 (m, 2 H), 3.29−2.20 (m, 2
H). HRMS (ESI) calcd for C23H22N4O5ReS (M)+, 653.0868; found,
653.0866.
4-(2-(Bis((1-methyl-1H-imidazol-2-yl)methyl)amino)ethyl)-
benzenesulfonamiderhenium Tricarbonyl Complex (3b). A solution
of 2b (79.7 mg, 0.205 mmol) and [NEt4]2[ReBr3(CO)3] (158 mg,
0.205 mmol) in methanol (5.0 mL) in a sealed pressure tube was
stirred at 90 °C for 4 h. The solvent was evaporated to give a residue,
which was purified by flash chromatography over silica gel eluting with
0−10% methanol in DCM afforded 3b (118 mg, 98%) as a yellow
solid. 1H NMR (400 MHz, DMSO-d6) 7.82 (d, J = 8.4 Hz, 2H), 7.62
(d, J = 8.0 Hz, 2 H), 7.31 (s, 2 H), 7.29 (d, J = 1.6 Hz, 2 H), 7.07 (d, J
= 1.6 Hz, 2 H), 5.05 (d, J = 16.4 Hz, 2 H), 4.83 (d, J = 16.4 Hz, 2 H),
3.70 (s, 6 H), 2.50−2.48 (m, 4 H). HRMS (ESI) calcd for
C21H24N6O5ReS (M)+, 659.1086; found, 659.1084.
H
dx.doi.org/10.1021/jm3015348 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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[NEt4]2[ReBr3(CO)3] (66.8 mg, 0.0867 mmol) in acetonitrile (5.0
using a binary solvent gradient of 0−50% mobile phase B where
mobile phase A is water containing 0.1% TFA and mobile phase B is
50% acetonitrile and 50% water containing 0.1% TFA and lyophilized
to give the desired product 6 (7, 8 or 9).
mL) according to the general procedure described above afforded 3i as
1
a white solid (57.7 mg, 53%). H NMR (400 MHz, DMSO-d6) 12.11
(brs, 6 H), 7.73 (d, J = 8.8 Hz, 2 H), 7.71 (s, 2 H), 7.18 (s, 2 H), 7.16
(d, J = 1.6 Hz, 2 H), 7.06 (d, J = 9.2 Hz, 2 H), 7.03 (d, J = 1.6 Hz, 2
H), 4.72 (d, J = 17.6 Hz, 2 H), 4.67 (d, J = 16.8 Hz, 2 H), 4.45 (d, J =
16.4 Hz, 2 H), 4.36 (d, J = 16.4 Hz, 2 H), 4.09 (t, J = 6.0 Hz, 2 H),
3.78−3.70 (m, 2 H), 2.13 (t, J = 7.8 Hz, 12 H), 1.90−1.70 (m, 16 H).
HRMS (ESI) calcd for C45H58N8O20ReS (M)+, 1249.3046; found,
1249.3031.
2,2′,2″,2‴-((2,2′-(2,2′-(((4-Sulfamoylphenethyl)azanediyl)bis-
(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis(azanetriyl))-
tetraacetic Acid (6). Treatment of 2d (753 mg, 0.809 mmol) in TFA
(5.0 mL) and DCM (5.0 mL) according to the general procedure
1
described above afforded 6 (584 mg, 99%) as a white solid. H NMR
(400 MHz, DMSO-d6) 7.68 (d, J = 7.6 Hz, 2 H), 7.61 (s, 2 H), 7.60 (s,
2 H), 7.32 (d, J = 8.0 Hz, 2 H), 7.26 (s, 2 H), 5.34 (s, 4 H), 4.35 (s, 4
H), 4.07 (s, 8 H), 2.71−2.68 (m, 4H). HRMS (ESI) calcd for
C28H35N8O12S (M + H)+, 707.2095; found, 707.2093.
4,4′-((2,2′-(2,2′-(((4-Sulfamoylphenethyl)azanediyl)bis-
(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis(azanediyl))-
bis(4-(2-carboxyethyl)heptanedioic Acid) (7). Treatment of 2e (400
mg, 0.314 mmol) in TFA (4.0 mL) and DCM (4.0 mL) according to
the general procedure described above afforded 7 (180 mg, 61%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) 7.70 (s, 2 H), 7.66 (d, J =
8.4 Hz, 2 H), 7.23 (d, J = 8.8 Hz, 2 H), 7.22 (s, 2 H), 7.02 (s, 2 H),
6.82 (s, 2 H), 4.58 (s, 4 H), 3.64 (s, 4 H), 2.74−2.64 (m, 4 H), 2.13 (t,
J = 8.0 Hz, 12 H), 1.83 (t, J = 7.8 Hz, 12 H). HRMS (ESI) calcd for
C40H55N8O16S (M + H)+, 935.3457; found, 935.3452.
4,4′-((2,2′-(2,2′-(((6-(4-Sulfamoylphenoxy)hexyl)azanediyl)bis-
(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis(azanediyl))-
bis(4-(2-carboxyethyl)heptanedioic acid) Rhenium Tricarbonyl
Complex (3j). Treatment of 2j (81 mg, 0.060 mmol) and
[NEt4]2[ReBr3(CO)3] (47 mg, 0.060 mmol) in acetonitrile (5.0
mL) according to the general procedure described above afforded 3j as
1
a white solid (12.2 mg, 16%). H NMR (400 MHz, DMSO-d6) 12.11
(brs, 6 H), 7.72 (d, J = 8.8 Hz, 2 H), 7.71 (s, 2 H), 7.18 (s, 2 H), 7.16
(d, J = 1.6 Hz, 2 H), 7.05 (d, J = 8.8 Hz, 2 H), 7.03 (d, J = 1.6 Hz, 2
H), 4.73 (d, J = 16.8, 2 H), 4.67 (d, J = 16.8, 2 H), 4.42 (d, J = 16.8
Hz, 2 H), 4.34 (d, J = 16.4 Hz, 2 H), 4.04 (t, J = 6.6 Hz, 2 H), 3.64−
3.60 (m, 2 H), 2.13 (t, J = 7.8 Hz, 12 H), 1.84 (t, J = 7.8 Hz, 12 H),
1.76−1.35 (m, 8 H). HRMS (ESI) calcd for C47H62N8O20ReS (M)+,
1277.3359; found, 1277.3351.
2-((Pyridin-2-ylmethyl)(4-sulfamoylphenethyl)amino)acetic Acid
Rhenium Tricarbonyl Complex (5a). To a solution of 4a (150 mg,
0.37 mmol) in DCM (3.0 mL) and TFA (3.0 mL) was stirred at room
temperature overnight. Solvent was removed under reduced pressure
to give a residue. To a solution of the above residue in MeOH (6.0
mL) was added [NEt4]2[ReBr3(CO)3] (384 mg, 0.50 mmol) and
K2CO3 (60 mg). The reaction mixture was stirred at 100 °C for 5 h at
a pressure tube. Solvent was evaporated under reduced pressure to a
residue, which was purified by HPLC afforded 5a (38 mg, 17%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) 8.77 (d, J = 5.6 Hz, 1 H),
8.17 (t, J = 7.8 Hz, 1 H), 7.79 (d, J = 8.0 Hz, 2 H), 7.74 (d, J = 7.6 Hz,
1 H), 7.59−7.56 (m, 3 H), 7.29 (s, 2 H), 4.92 (d, J = 16.0 Hz, 1 H),
4.77 (d, J = 16.0 Hz, 1 H), 4.10 (d, J = 16.4 Hz, 1 H), 3.74−3.68 (m, 1
H), 3.64−3.58 (m, 1 H), 3.53 (d, J = 16.8 Hz, 1 H), 3.14−3.08 (m, 2
H). HRMS (ESI) calcd for C19H19N3O7ReS (M + H)+, 620.0501;
found, 620.0496.
4-(2-(2-((((1-(2-(Bis(carboxymethyl)amino)-2-oxoethyl)-1H-imi-
dazol-2-yl)methyl)(4-sulfamoylphenethyl)amino)methyl)-1H-imida-
zol-1-yl)acetamido)-4-(2-carboxyethyl)heptanedioic Acid (8). Treat-
ment of 4c (65.5 mg, 0.0595 mmol) in TFA (1.0 mL) and DCM (1.0
mL) according to the general procedure described above afforded 8
1
(38.1 mg, 78%) as a white solid. H NMR (400 MHz, DMSO-d6)
12.43 (brs, 5 H), 7.89 (s, 1 H), 7.68 (d, J = 8.0 Hz, 2 H), 7.55 (s, 1 H),
7.49 (s, 2 H), 7.33 (s, 1 H), 7.32 (d, J = 8.4 Hz, 2 H), 7.26 (s, 2 H),
5.29 (s, 2 H), 4.90 (s, 2 H), 4.34 (s, 2 H), 4.09 (s, 2 H), 4.07 (s, 2 H),
4.02 (s, 2 H), 2.74 (s, 4 H), 2.17 (t, J = 8.2 Hz, 6 H), 1.85 (t, J = 8.0
Hz, 6 H). HRMS (ESI) calcd for C34H45N8O14S (M + H)+, 821.2776;
found, 821.2768.
4,4′-((2,2′-(2,2′-(((4-(4-Sulfamoylphenoxy)butyl)azanediyl)bis-
(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis(azanediyl))-
bis(4-(2-carboxyethyl)heptanedioic Acid) (9). Treatment of 2i (23
mg, 0.0175 mmol) in TFA (1.0 mL) and DCM (1.0 mL) according to
the general procedure described above afforded 9 (17.6 mg, 100%) as
a white solid. 1H NMR (400 MHz, DMSO-d6) 12.12 (brs, 6 H), 7.87
(s, 2 H), 7.71 (d, J = 8.8 Hz, 2 H), 7.52 (brs, 4 H), 7.17 (s, 2 H), 7.00
(d, J = 8.8 Hz, 2 H), 4.88 (s, 4 H), 3.96 (s, 4 H), 3.94 (t, J = 6.4 Hz, 2
H), 2.59−2.56 (m, 2 H), 2.16 (t, J = 8.0 Hz, 12 H), 1.84 (t, J = 7.6 Hz,
12 H), 1.65−1.49 (m, 4 H). HRMS (ESI) calcd for C42H59N8O17S (M
+ H)+, 979.3719; found, 979.3716.
2,2′-(2-(2-((((1-(Carboxymethyl)-1H-imidazol-2-yl)methyl)(4-
sulfamoylphenethyl)amino)methyl)-1H-imidazol-1-yl)-
acetylazanediyl)diacetic Acid Rhenium Tricarbonyl Complex (5b).
Treatment of 4b (91 mg, 0.12 mmol) and [NEt4]2[ReBr3(CO)3] (93
mg, 0.12 mmol) in methanol (5.0 mL) according to the general
procedure described above afforded 5b as a white solid (72 mg, 70%).
1H NMR (400 MHz, DMSO-d6) 7.80 (d, J = 8.4 Hz, 2 H), 7.58 (d, J =
+
Preparation of 99mTc−3d. The 99mTc(I)(CO)3 radiolabeling of
2d was accomplished in two steps using commercially available
IsoLink kits (Covidien) and [Na][99mTcO4] in 1 mL of 0.9% sodium
chloride solution (50 mCi/mL) to form the [99mTc(CO)3(H2O)3]+
intermediate, which was neutralized and reacted with 2d at a
concentration of 10−4 M in an equal volume mixture of acetonitrile
and water in a sealed vial. The sealed vial was heated in an oil bath at
75 °C for 60 min. After cooling and evaporation of solvent by a stream
of nitrogen, the tert-butyl ester protecting groups were removed by
treatment with 50% TFA in DCM (4 mL) at room temperature for 30
min. After subsequent evaporation and formulation in 0.9% sodium
chloride solution, the reaction was analyzed for purity via reverse-
phase HPLC. Purification by HPLC resulted in the desired conjugate
99mTc−3d in 90% RCY and >95% RCP as a “carrier free” product. The
final formulation was composed of 10% ethanol in 0.9% sodium
chloride solution.
Cell Culture. The human cervical cancer cell line, HeLa, was
obtained from the American Type Culture Collection. HeLa cells were
maintained in Minimal Essential Medium supplemented with 10%
fetal bovine serum (Hyclone), 4 mM glutamine, 1 mM sodium
pyruvate, and 50 μg/mL gentamicin. Media components were from
Invitrogen unless otherwise noted. Cells were grown in a humidified
incubator at 37 °C/5% CO2 and removed from flasks for passage or
for transfer to 12-well assay plates by incubating them with 0.25%
trypsin/EDTA (Invitrogen).
8.0 Hz, 2 H), 7.29 (s, 2 H), 7.22 (d, J = 1.2 Hz, 1 H), 7.15 (d, J = 1.2
Hz, 1 H), 7.07 (d, J = 1.2 Hz, 1 H), 7.04 (d, J = 1.2 Hz, 1 H), 5.09 (s, 2
H), 4.86 (s, 2 H), 4.74 (d, J = 16.4 Hz, 1 H), 4.60 (dd, J = 16.0, 6.4 Hz,
2 H), 4.42 (d, J = 16.0 Hz, 1 H), 4.29 (s, 2 H), 4.10 (d, J = 17.6 Hz, 1
H), 4.02 (d, J = 17.2 Hz, 1 H), 3.85−3.80 (m, 2 H), 3.15−3.07 (m, 2
H). HRMS (ESI) calcd for C27H29N7O12ReS (M)+, 862.1147; found,
862.1111.
4-(2-(2-((((1-(2-(Bis(carboxymethyl)amino)-2-oxoethyl)-1H-imi-
dazol-2-yl)methyl)(4-sulfamoylphenethyl)amino)methyl)-1H-imida-
zol-1-yl)acetamido)-4-(2-carboxyethyl)heptanedioic Acid Rhenium
Tricarbonyl Complex (5c). Treatment of 4c (80 mg, 0.0726 mmol)
and [NEt4]2[ReBr3(CO)3] (58 mg, 0.075 mmol) in acetonitrile (5.0
mL) according to the general procedure described above afforded 5c
as a white solid (62.8 mg, 80%). 1H NMR (400 MHz, DMSO-d6) 7.80
(d, J = 8.4 Hz, 1 H), 7.71 (s, 1 H), 7.58 (d, J = 8.4 Hz, 2 H), 7.29 (s, 2
H), 7.20 (d, J = 1.6 Hz, 1 H), 7.14 (d, J = 1.2 Hz, 2 H), 7.14−7.05 (m,
2 H), 5.09 (s, 2 H), 4.71 (s, 2 H), 4.67−3.86 (m, 10 H), 3.13−3.11
(m, 2 H), 2.19 (t, J = 8.2 Hz, 6 H), 1.86 (t, J = 8.2 Hz, 6 H). HRMS
(ESI) calcd for C37H44N8O17ReS (M)+, 1091.2103; found, 1091.2090.
General Procedure for Synthesis of Compounds 6, 7, 8, and
9. A solution of 2d (2e, 4c, or 2i) in TFA and DCM was stirred at
room temperature overnight. Solvent was removed under a stream of
nitrogen to give a residue, which was purified by medium pressure
column chromatography utilizing Biotage SP4 over C18 cartridge
I
dx.doi.org/10.1021/jm3015348 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Binding to CA-IX. The ability of the CA-IX inhibitors to compete
with 99mTc−3d for binding to hypoxic HeLa cells was examined. HeLa
cells were plated in 12-well plates at approximately 2.5 × 105 cells/well
and allowed to adhere to the plate for 24 h. Cells were then incubated
under hypoxic conditions (0.1% O2/5% CO2 at 37 °C) for an
additional 24 h. The cells were then removed from hypoxia and
incubated for 1 h in Hank’s Balanced Salt Solution (HBSS) with 0.5%
BSA and 3nM 99mTc−3d in the presence of 1−10000 nM test CA-IX
inhibitor, or acetazolamide. The assay media was then removed and
the cells were washed 2× with cold HBSS/0.5% BSA, collected by
adding 0.25 mL of 1% SDS, and transferred to a 1.5 mL tube for
counting the amount of radioactivity bound in a Wallac 1282
automated gamma counter. IC50 values were determined by nonlinear
regression using GraphPad Prism software.
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: (617) 492-5554. Fax: (617) 492-5664. E-mail:
jbabich@molecularinsight.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by the National Institutes of
Health, grant no. 1R41A1054080-01.
ABBREVIATIONS USED
■
RCY, radiochemical yield; RCP, radiochemical purity; SPECT,
single-photon emission computed tomography
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