Development and biological evaluation of 99mTc-sulfonamide derivatives for in vivo visualization of CA IX as surrogate tumor hypoxia markers

Article abstract of DOI:10.1016/j.ejmech.2013.10.027

In vivo visualization of tumor hypoxia related markers, such as the endogenous transmembrane protein CA IX may lead to novel therapeutic and diagnostic applications in the management of solid tumors. In this study 4-(2-aminoethyl)benzene sulfonamide (AEBS, K_i = 33 nM for CA IX) has been conjugated with bis(aminoethanethiol) (BAT) and mercaptoacetyldiglycine (MAG₂) tetradendate ligands and the conjugates radiolabelled with ^99mTc, to obtain anionic and neutral ^99mTc-labeled sulfonamide derivatives, respectively. The corresponding rhenium analogues were also prepared and showed good inhibitory activities against hCA IX (K _i = 59-66 nM). In addition, a second generation bis AEBS was conjugated with MAG₂ and labeled with ^99mTc, and the obtained diastereomers were also evaluated in targeting CA IX. Biodistribution studies in mice bearing HT-29 colorectal xenografts revealed a maximum tumor uptake of <0.5% ID/g at 0.5 h p.i for all the tracers. In vivo radiometabolite analysis indicated that at 1 h p.i. MAG₂ tetradendate ligands were more stable in plasma (>50% intact) compared to the neutral complex (28% intact). This preliminary data suggest that negatively charged ^99mTc-labeled sulfonamide derivatives with modest lipophilicity and longer circulation time could be promising markers to target CA IX.

Full text of DOI:10.1016/j.ejmech.2013.10.027

European Journal of Medicinal Chemistry 71 (2014) 374e384
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development and biological evaluation of 99mTc-sulfonamide
derivatives for in vivo visualization of CA IX as surrogate tumor
hypoxia markers
Vamsidhar Akurathi a, Ludwig Dubois b, Sofie Celen a, Natasja G. Lieuwes b,
Satish K. Chitneni a, Bernard J. Cleynhens a, Alessio Innocenti c, Claudiu T. Supuran c,
,
Alfons M. Verbruggen a, Philippe Lambin b, Guy M. Bormans a
*
a Laboratory of Radiopharmacy, KU Leuven, Leuven, Belgium
b Department of Radiation Oncology (Maastro Lab), GROW e School for Oncology and Development Oncology, Maastricht University Medical Centre,
Maastricht, The Netherlands
c Universita degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Room 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In vivo visualization of tumor hypoxia related markers, such as the endogenous transmembrane protein
CA IX may lead to novel therapeutic and diagnostic applications in the management of solid tumors. In
this study 4-(2-aminoethyl)benzene sulfonamide (AEBS, Ki ¼ 33 nM for CA IX) has been conjugated with
bis(aminoethanethiol) (BAT) and mercaptoacetyldiglycine (MAG2) tetradendate ligands and the conju-
gates radiolabelled with 99mTc, to obtain anionic and neutral 99mTc-labeled sulfonamide derivatives,
respectively. The corresponding rhenium analogues were also prepared and showed good inhibitory
activities against hCA IX (Ki ¼ 59e66 nM). In addition, a second generation bis AEBS was conjugated with
MAG2 and labeled with 99mTc, and the obtained diastereomers were also evaluated in targeting CA IX.
Biodistribution studies in mice bearing HT-29 colorectal xenografts revealed a maximum tumor uptake
of <0.5% ID/g at 0.5 h p.i for all the tracers. In vivo radiometabolite analysis indicated that at 1 h p.i. MAG2
tetradendate ligands were more stable in plasma (>50% intact) compared to the neutral complex (28%
intact). This preliminary data suggest that negatively charged 99mTc-labeled sulfonamide derivatives with
modest lipophilicity and longer circulation time could be promising markers to target CA IX.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 9 July 2013
Received in revised form
8 October 2013
Accepted 10 October 2013
Available online 30 October 2013
Keywords:
Carbonic anhydrases
Sulfonamides
Technetium-99m
Rhenium
Biodistribution
1. Introduction
and neck [5e10]. The expression of CA IX in these tumors typically
varies from 16 to 100% in ‘perinecrotic’ regions and is consistent
Carbonic anhydrase (CA) type IX, a transmembrane protein
belonging to the class of zinc metalloenzymes, has been identified
to play a key role in the extracellular acidification of hypoxic tu-
mors, due to the hypoxia inducing factor (HIF-1) dependent over
expression [1]. A low extracellular pH (pHe in hypoxic solid tumors
(pH 6e6.5), in contrast to normal tissue (pH 7.4), is associated
with tumorigenic transformation, chromosomal rearrangements,
migration, invasion and overall tumor survival [2e4]. The unique
feature of CA IX lies in its expression, where a limited expression in
the gastro-intestinal tract is observed in contrast to over expression
in carcinomas of kidney, lung, breast, bladder, uterus, cervix, head
with the distribution of hypoxia [11]. In addition, the clinical co-
relevance of metastasis, poor prognosis and resistance to chemo-
and radiotherapy makes CA IX a promising surrogate marker for
tumor hypoxia [12e16]. Recent studies from Divgi et al. reported
that iodine-124 labeled mAb-cG250, a chimeric monoclonal anti-
body, effectively recognizes CA IX and provides excellent in vivo
visualization of clear cell renal cell carcinoma (ccRCC) in a pre-
liminary clinical study. Based on this study, advanced clinical trials
are currently underway for application of 124I-mAb-cG250 in the
diagnosis of ccRCC [17].
However, one of the limitations of 124I-mAb-cG250 is the fact
that no discrimination can be made between hypoxic and aerobic
cells expressing CA IX, since the antibody binding persists in re-
oxygenated tumor cells due to the slow turnover of CA IX (half-
life of CA IX upon re-oxygenation is 38 h) and this precludes im-
aging of periodic or cyclic areas of tumor oxygenation. On the other
* Corresponding author. O&N2, Bus 821, Herestraat 49, BE-3000 Leuven, Belgium.
Tel.: þ32 16 330447; fax: þ32 16 330449.
E-mail address: guy.bormans@pharm.kuleuven.be (G.M. Bormans).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.027

V. Akurathi et al. / European Journal of Medicinal Chemistry 71 (2014) 374e384
375
hand, slower pharmacokinetics of 124I-mAb-cG50 further confine
the imaging protocols for more than six days to obtain a good
contrast image, which is a set-back in diagnostic nuclear medicine.
Various sulfonamide derivatives with high affinity and selectivity
for CA IX and with favourable pharmacokinetics have been re-
ported in literature [12,18]. Studies from Dubois et al. demonstrated
that the CA IX active site is accessible for sulfonamides only under
hypoxic conditions. Thus, radiolabelled sulfonamides may provide
a powerful tool to visualize hypoxic tumors and a promising
approach to patient selection for CA IX-directed in vivo imaging or
therapies based on a functional inhibition of CA IX [19].
concentration at the target site. On the other hand the presence of a
negative charge on a 99mTc-labeled sulfonamide conjugate (anionic
complex) could prevent intracellular binding of the tracer agent to
other CA subtypes, thereby enhancing the specificity of interaction
with membrane bound CA IX. In addition, a second generation
anionic 99mTc-labeled bis-sulfonamide derivative was developed,
formed as two diastereomers after labeling with 99mTc. A recent
report from Morsy et al. further demonstrated that the presence of
two sulfonamide moieties can result in increased affinity for the
active site of CA IX [21]. Biodistribution characteristics of all the
tracer agents synthesized were evaluated in nude mice bearing HT-
29 colorectal xenografts for their potential in SPECT imaging.
In previous work we reported the preparation of 99mTc(CO)3
labeled 4-(2-aminoethyl)benzene-sulfonamide (Ki ¼ 33 nM for CA
IX) conjugated with an N-(2-picolyl-amine)-N-acetic acid moiety
with reliable (radio)chemical yield and purity. Its rhenium analog
was also prepared and showed a Ki ¼ 58 nM for CA IX. Although
in vitro studies had shown some interesting properties in targeting
CA IX, in vivo studies revealed that the tracer agent retention in
tumor is minimal (<0.2% I.D/g at 0.5 h p.i.) in HT 29 colorectal
xenograft bearing mice. This low retention in the tumor is attrib-
uted to a quick wash out from the blood pool [20]. Studies from
Dubois et al. and Supuran et al. demonstrated that charged sul-
fonamide derivatives have interesting properties with selective
inhibition of membrane bound CA IX over the cytosolic isoform
[1,19,21]. In a recent study of Lu et al. [22] a series of novel Re/99mTc-
labelled benzene sulfonate carbonic anhydrase IX inhibitors were
synthesized and evaluated for molecular imaging of tumor hypoxia.
In their tests they found that 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 (IC50 ¼ 9 nM) was radiolabelled
with 99mTc(CO)þ3 to afford a technetium-99m-tricarbonyl com-
pound in excellent yield and high purity (Fig. 1). The new com-
pound showed specific binding to CA-IX expressing hypoxic HeLa
cells, but no biodistribution data in normal mice or mice bearing a
CA-IX expressing tumor were given.
2. Results and discussion
2.1. Chemistry and radiolabelling of AEBS-BAT
The precursors used for radiolabeling are all derivatives of AEBS
conjugated either to a BAT or a MAG2 ligand. The rationale behind
choosing BAT or MAG2 tetradentate ligands as the 99mTc chelating
moiety lies in their ability to form chemically robust core struc-
tures. The BAT ligand (1) was prepared as reported previously by
our group [22]. The two thiol groups of the BAT ligand were each
protected by a trityl group to prevent oxidation. One of the sec-
ondary amines was protected with a BOC group and the other
amine was derivatized with an acetic acid group allowing conju-
gation with AEBS. The labeling of AEBS-BAT with 99mTc was ach-
ieved in a two-step one-pot reaction (Scheme 1). In a first step, the
trityl and BOC groups of the BAT ligand were removed by heating 2
at 100 ꢀC for 20 min under acidic conditions. In a second step a
cocktail solution of buffering and chelating agents together with
stannous ions and 99mTcO4ꢁ was added. Heating of this reaction
mixture at 100 ꢀC for 10 min resulted in the formation of a neutral
99mTc-3 complex. Complex formation involves coordination of
99mTc with the 2 thiol and 2 amine functional groups, to form a
stable technetium(V)oxo core [24]. The crude reaction mixture was
purified by RP-HPLC with a radiochemical yield of 40% and a
radiochemical purity of ꢂ98% (n ¼ 3). The corresponding rhenium
analog Re-3 was synthesized in a low yield (7%) by deprotection of
precursor 2 followed by a complexation reaction with the use of
TBA[ReOCl4] [23]. Radio-LCeMS was performed to confirm the
identity of 99mTc-3. Due to the minute amounts of technetium-99m
in the preparation (typically in nano to picomolar range), it is
difficult to obtain useful mass spectra of technetium-99m labeled
compounds in no-carrier-added form. Therefore, the labeling re-
Therefore, it is interesting to develop and evaluate 99mTc labeled
sulfonamide derivatives with varying physicochemical properties
(Log D, complexes with neutral or anionic charge and with variation
of molecular mass). The main objective of this study was to explore
whether favorable results are obtained with a lipophilic neutral
99mTc-labeled sulfonamide derivative, which is presumed to result
in a reservoir-binding of the tracer in blood pool through intra-
cellular erythrocyte CAII binding to maintain an effective
action was done after spiking with technetium-99 (b
ꢁ emitter with
COOH
a half life of 2.14 ꢃ 105 years) in the form of ammonium pertech-
netate and the reaction mixture was analyzed by radio-LCeMS.
Fig. 2: depicts a single ion mass chromatogram of 99mTc-3 and
99mTc-3 in radiometric channel, supporting the identity of 99mTc-3.
O
N
HOOC
N
N
2.2. Chemistry and radiolabelling of AEBS-MAG2
M(CO)3
N
The synthesis of AEBS-MAG2 (5) and subsequent radiolabeling
with 99mTcOꢁ4 was carried out as outlined in Scheme 2a, starting
from the S-benzyl MAG2 ligand (4). The conjugation with AEBS was
accomplished in moderate yields via an amide coupling between
the carboxylic acid of MAG2 and the aminoethyl function of AEBS
using a standard peptide coupling agent. The radiolabelling of this
conjugate with 99mTc was performed by a classical one-pot ex-
change labeling reaction. In brief, the deprotection (removal of the
S-benzyl group) of precursor (5) and the exchange labeling with
99mTcOꢁ4 were carried out in the presence of SnCl2.2H2O and
chelating agents at 100 ꢀC for 15 min, leading to the anionic com-
plex 99mTc-8. The negative charge is due to the coordination of 1
N
N
H2NO2S
O
N
HOOC
COOH
M= Re or 99mTc
IC50 against CA-IX: 9nm
Fig. 1. Structure of the radiolabelled technetium/rhenium tricarbonyl compound
evaluated by the group of Lu.

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V. Akurathi et al. / European Journal of Medicinal Chemistry 71 (2014) 374e384
Scheme 1. Synthesis of 99mTc or Re-labelled 3. Reagents and conditions: (a) AEBS, dimethylformamide (DMF), dimethylaminopyridine (DMAP), benzotriazol-1-yl-oxy-
tripyrrolidinophosphonium hexafluorophosphate (PyBOP) at room temperature (RT) for 4 days (b) (i) TBA[ReOCl4], ethanol, HCl gas, 2 h, RT (ii) Et3N 0 ꢀC for 12 h (c) HCl 0.5 M,
100 ꢀC for 20 min, Na2EDTA 0.1 M, SnCl2, 99mTcOꢁ4 , 100 ꢀC for 10 min.
thiol and 3 amide functional groups with 99mTc, to form a stable
technetium(V)oxo core [28]. The crude reaction mixture was pu-
rified using RP-HPLC and 99mTc-8 was obtained with an overall
radiochemical yield of 65% and a radiochemical purity of ꢂ98%
(n ¼ 3).
followed by conjugation with AEBS, S-benzoyl-MAG2-AEBS (7) was
formed in a good yield. The thiol protecting benzoyl group was
removed in alkaline conditions, followed by the reaction with a
rhenium(V)citrate solution. Re-8 was isolated from the reaction
mixture as the tetraphenyl arsonium chloride salt with a chemical
yield of 60%. The identity of 99mTc-3 was supported by co-elution
with Re-3 (Fig. 3). To synthesise a derivative with two AEBS
The corresponding rhenium analog Re-8 was synthesized in 3
steps (Scheme 2b). Starting from S-benzoyl protected MAG2 (6)
Fig. 2. (a) Single ion mass chromatogram of carrier added 99mTc-3 and (b) 99mTc-3 in radiometric channel, eluting with a similar retention time of 10.2 min (ESþ 533.0167 and
533.0173 Da respectively) supporting the identity of 99mTc-3.

V. Akurathi et al. / European Journal of Medicinal Chemistry 71 (2014) 374e384
377
Scheme 2. a. Synthesis of 99mTc-8. Reagents and conditions: (a) AEBS, DMF, DMAP and PyBOP at RT for 16 h, 2 M HCl (b) NaK tartrate, SnCl2, 99mTcOꢁ4 at pH 8, 100 ꢀC for 15 min b.
Synthesis of Re-8. Reagents and conditions: (a) AEBS, DMF, DMAP and PyBOP at RT for 16 h, 2 M HCl (b) 0.1 M NaOH, 90 ꢀC for 10 min, rhenium(V)citrate solution, 90 ꢀC for 60 min.
moieties, a glutamine spacer (glu) was introduced (Scheme 3). First
N-BOC-L-Glu was reacted with two molecules of AEBS followed by
deprotection of the amine with TFA. Coupling tothe S-benzoyl MAG2
precursor (6) was done in the same conditions as for the synthesis of
7 with a yield of 55%. The radiolabelling of the conjugate was carried
out by a classical one pot exchange reaction followed by purification
with RP-HPLC. Because of the chiral carbon atom in the glutamic
acid spacer and due to the syn and anti orientation of the 99mTc ¼ O
group, two diastereomers 99mTc-11A and 99mTc-11B were formed
with a radiochemical yield of 52% and 36%, respectively (Fig. 4).
Attempts to make the oxo rhenium (V) reference compounds of
99mTc-11A and 99mTc-11B were not successful.
Fig. 3. Analytical RP-HPLC chromatogram showing the co-elution of 99mTc-8 with the Re-8 at about 4 min, supports the identity of 99mTc-8.

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V. Akurathi et al. / European Journal of Medicinal Chemistry 71 (2014) 374e384
Scheme 3. Synthesis of the diastereomers of 99mTc-11. Reagents and conditions: (a) AEBS, acetonitrile, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl (b) trifluoroacetic acid
(c) MAG2, DMF, 4-dimethylaminopyrimidine, PyBOP at RT for 16 h (d) NaK tartrate, SnCl2 and 99mTcO4- at pH 8, 100 ꢀC for 15 min.
2.3. Log D and inhibition studies (determination of Ki)
Table 1
The log D is a very useful parameter that can be used to un-
derstand the behavior of drug molecules and predict the distribu-
Inhibition constants of reference compounds Re-3, Re-8 and AEBS against carbonic
anhydrase isozymes hCA I, II, IX and XII for the CO2 hydration reaction at 25 ꢀC.
tion of a drug compound in a biological system [23]. By a shake flask
Compound
hCA Ib
hCA IIb
Ki (nM)a
hCA XIIb
Selectivity ratio
hCA II/IXc
method, log D
of the tracer agents
(1ꢁoctanol/phosphate buffer pH 7.4)
hCA IXb
99mTc-3, 99mTc-8 and 99mTc-11A/B was 1.0 ꢄ 0.02, 0.5 ꢄ 0.02
and ꢁ1ꢄ 0.07 (mean ꢄ S.D, n ¼ 6), respectively, indicating that the
tracer agents 99mTc-3 and 99mTc-8 are modest in lipophilicity, and
the diasteromers 99mTc-11A and 99mTc-11B are hydrophilic in na-
ture. The inhibition of the catalytically active and physiologically
relevant hCA I, II, IX and XII by the corresponding rhenium analogs
Re-3 and Re-8 was studied by assaying the CA-catalyzed CO2 hy-
dration activity [24]. The inhibition activity (Ki) against hCA IX was
found to be 59 nM for Re-3 and 66 nM for Re-8 (Table 1). Despite
the presence of a bulky chelating group (BAT or MAG2), the rhenium
Re-3
Re-8
AEBS
268
4875
2100
165
47
160
59
66
33
76
57
3.2
2.8
0.7
4.8
a
Standard errors of the mean in the range of 5e10% of the reported value (n ¼ 3,
different assays).
b
h e Human (cloned) isozymes41
Ki ratios are indicative of selectivity.
.
c
Fig. 4. Semipreparative RP-HPLC chromatogram showing diastereomers of 99mTc-11A and 11B eluting at about 18 min and 21 min, respectively.

V. Akurathi et al. / European Journal of Medicinal Chemistry 71 (2014) 374e384
379
Table 2b
analogs show a good inhibitory activity, comparable to that of the
parent moiety AEBS (Ki ¼ 33 nM). The selectivity ratios of the
rhenium analogs for CA IX over cytosolic isoforms of hCA II were
found to be modest when compared to the parent moiety.
Biodistribution of 99mTc-8 in CA IX expressing tumor bearing mice at 0.5, 1, 2 and
4 h p.i. Data are expressed as mean þ/ꢁ standard error (n ¼ 3).
99mTc-8
Organs [% ID ]
0.5 h
1 h
2 h
7.2 ꢄ 3.6
4 h
4.4 ꢄ 0.9
2.4. In vivo studies
Kidneys
Urine
Liver
21.6 ꢄ 3.0
6.9 ꢄ 1.8
38.6 ꢄ 3.6
0.1 ꢄ 0.0
0.4 ꢄ 0.0
0.1 ꢄ 0.0
23.3 ꢄ 5.6
0.8 ꢄ 0.1
0.0 ꢄ 0.0
1.5 ꢄ 0.4
0.1 ꢄ 0.0
17.5 ꢄ 2.0
6.1 ꢄ 2.6
35.9 ꢄ 3.6
0.1 ꢄ 0.0
0.2 ꢄ 0.0
0.0 ꢄ 0.0
35.2 ꢄ 3.0
0.1 ꢄ 0.1
0.0 ꢄ 0.0
1.1 ꢄ 0.0
0.2 ꢄ 0.0
18.5 ꢄ 5.0
18.9 ꢄ 6.9
0.1 ꢄ 0.1
0.1 ꢄ 0.0
0.0 ꢄ 0.0
52.0 ꢄ 6.7
1.1 ꢄ 0.5
0.0 ꢄ 0.0
0.8 ꢄ 0.1
0.2 ꢄ 0.0
19.4 ꢄ 7.0
12.0 ꢄ 2.1
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.0 ꢄ 0.0
62.4 ꢄ 2.9
0.6 ꢄ 0.2
0.0 ꢄ 0.0
0.5 ꢄ 0.0
0.1 ꢄ 0.1
The biodistribution data of 99mTc-3, 99mTc-8 and diasteromers
99mTc-11A and 99mTc-11B in CA IX expressing HT 29 colorectal
xenograft mice are summarized in Tables 2 and 3, respectively. Data
are expressed as percentage of the injected dose (% ID) and per-
centage of the injected dose per gram of tissue (% ID/g) for selected
body parts. All tracer agents were cleared mainly via the hep-
atobiliary pathway with ꢂ70% of the ID in intestines at 4 h p.i. A
smaller fraction was cleared via the renal pathway (ꢅ19% ID in
urine at 4 h p.i.). Surprisingly, the clearance of diastereomers 99mTc-
11A and 11B occurred also via the hepatobiliary pathway despite of
their negative charge. Polar compounds bearing a negative charge
are considered to be hydrophilic in nature and expected to clear
more via the renal pathway [30].
Spleen þ pancreas
Lungs
Heart
Intestines
Stomach
Brain
Blood
Tumor
99mTc-8
Organs [% ID/g]
0.5 h
1 h
2 h
4 h
Kidneys
Liver
37.6 ꢄ 4.1
17.5 ꢄ 1.6
0.2 ꢄ 0.1
0.9 ꢄ 0.1
0.4 ꢄ 0.1
0.0 ꢄ 0.0
1.3 ꢄ 0.3
0.5 ꢄ 0.1
0.4 ꢄ 0.0
37.7 ꢄ 2.4
17.3 ꢄ 1.8
0.2 ꢄ 0.0
0.6 ꢄ 0.1
0,3 ꢄ 0.0
0.0 ꢄ 0.0
0.9 ꢄ 0.1
0.4 ꢄ 0.0
0.4 ꢄ 0.1
12.1 ꢄ 5.2
7.3 ꢄ 2.5
0.2 ꢄ 0.1
0.3 ꢄ 0.1
0.1 ꢄ 0.0
0.0 ꢄ 0.0
0.7 ꢄ 0.2
0.2 ꢄ 0.0
0.3 ꢄ 0.1
6.8 ꢄ 1.4
5.6 ꢄ 2.3
0.1 ꢄ 0.0
0.2 ꢄ 0.0
0.1 ꢄ 0.0
0.0 ꢄ 0.0
0.4 ꢄ 1.0
0.1 ꢄ 0.0
0.5 ꢄ 0.0
Spleen þ pancreas
Lungs
The retention of the tracer agents 99mTc-3, 99mTc-8 and 99mTc-
11A and 99mTc-11B in the blood pool was about 13%, 1.5% and 0.1%
ID at 0.5 h p.i, respectively, indicating the strategy to attain high
concentration of 99mTc-3 in blood pool was accomplished with 10e
130 fold higher retention in the blood pool, when compared to
99mTc-8 and diastereomers of 99mTc-11A and 99mTc-11B complexes.
In vitro incubation studies performed on human whole blood with
99mTc-3 showed that about 13% of activity was retained in RBCs
after 10 min incubation at RT (data not shown). Thus, the higher
activity in the blood pool associated with tracer 99mTc-3 is due to
diffusion through RBC membrane and subsequent interaction with
the CA II isozyme located in erythrocytes [9e12]. The tracer agents
99mTc-8 and diastereomers 99mTc-11A and 99mTc-11B are presum-
ably confined to the extracellular space due to their anionic charge.
All other organs (spleen, pancreas, lungs, heart and brain)
showed negligible tracer retention, similar to the previously re-
ported 99mTc-(CO)3 labeled AEBS [24]. The concentration of tracer
99mTc-3 in the tumors was found to be ꢅ0.2% ID/g, for 99mTc-
Heart
Brain
Blood
Tumor
Tumor/blood
8 ꢅ 0.5% ID/g, and for diastereomers 99mTc-11A and 99mTc-11B a
negligible retention of ꢅ0.2% ID/g in the tumors was observed at
0.5e4 h p.i. indicating that overall retention of these tracer agents
in the tumors was minimal.
However, with 99mTc-8 a 2.5 fold higher retention is observed as
compared to the three other radioligands. This slightly higher tu-
mor uptake of 99mTc-8 can probably be attributed to its interaction
with the active site of CA IX in a distinct manner. The anionic charge
and the modest lipophilicity associated with this complex pre-
sumably restrict the tracer to the extracellular space, thereby pro-
moting its interaction with the active site of CA IX. On the other
hand, the diastereomers 99mTc-11A and 99mTc-11B have a low
retention in tumors due to their quick wash out from the blood
pool. Further, the tumor to blood ratios with the 99mTc-8 and the
diastereomers 99mTc-11A and 99mTc-11B has been found to be
modest with values ranging from 0.3 to 1 and with 99mTc-3 it is
zero. The polar surface area (PSA), another important parameter
that can provide information about the hydrogen bonding capacity
of the molecules, was found to be 109 Å2, 138 Å2 for 99mTc-3 and
99mTc-8 respectively and for the diastereomers 99mTc-11A and
99mTc-11B it was 256 Å2. Generally a PSA of <60 Å2 is considered to
be a requirement for an efficient cellular diffusion of a molecule.
This is in line with the higher blood pool retention observed for
99mTc-3. Studies from Gidal et al. have shown that erythrocytes play
an important role in the circulatory transport of sulfonamide de-
rivatives and extend the in vivo half-life of the antiepileptic drugs
such as zonisamide and topiramate [25]. Using a similar strategy of
erythrocyte mediated carrier to extend the in vivo half-life of tracer
agent 99mTc-3 is intriguing. However, the in vivo results of 99mTc-3
indicated a reduced uptake in the tumors.
Table 2a
Biodistribution of 99mTc-3 in CA IX expressing tumor bearing mice at 0.5, 2 and
4 h p.i. Data are expressed as mean þ/ꢁ standard error (n ¼ 3).
99mTc-3
Organs [% ID ]
0.5 h
8.9 ꢄ 1.3
2 h
1.3 ꢄ 0.0
4 h
0.8 ꢄ 0.0
Kidneys
Urine
Liver
11.4 ꢄ 0.5
25.2 ꢄ 4.8
0.6 ꢄ 0.0
1.3 ꢄ 0.1
0.4 ꢄ 0.1
31.9 ꢄ 3.7
1.6 ꢄ 0.2
0.1 ꢄ 0.0
12.7 ꢄ 0.5
0.3 ꢄ 0.0
14.9 ꢄ 1.5
18.4 ꢄ 0.3
0.2 ꢄ 0.0
0.4 ꢄ 0.1
0.2 ꢄ 0.0
54.2 ꢄ 2.5
0.8 ꢄ 0.1
0.0 ꢄ 0.0
8.3 ꢄ 0.1
0.2 ꢄ 0.0
19.4 ꢄ 4.2
11.4 ꢄ 2.1
0.1 ꢄ 0.0
0.2 ꢄ 0.1
0.1 ꢄ 0.0
61.3 ꢄ 5.5
1.0 ꢄ 0.2
0.0 ꢄ 0.0
3.5 ꢄ 1.1
0.1 ꢄ 0.0
Spleen þ pancreas
Lungs
Heart
Intestines
Stomach
Brain
Blood
Tumor
99mTc-3
Organs [% ID/g]
0.5 h
2 h
4 h
The reason for the limited tumor retention of 99mTc-8 is possibly
due to its fast elimination during the passage of blood through the
metabolising organs such as the liver or due to in vivo instability.
Further the low perfusion of the hypoxic tumor limits the tracer
delivery to the tumor. Ex vivo western blot analysis confirmed that
the CA IX expression levels of the tumors that were evaluated in
biodistribution studies, and the signal intensity was comparable
with that in HT-29 cells exposed to hypoxia in vitro (Fig. 5).
Kidneys
Liver
15.3 ꢄ 1.5
11.6 ꢄ 2.8
1.3 ꢄ 0.1
3.7 ꢄ 0.1
2.2 ꢄ 0.6
0.2 ꢄ 0.0
10.7 ꢄ 2.0
0.2 ꢄ 0.0
0.0 ꢄ 0.0
2.2 ꢄ 0.1
9.8 ꢄ 0.1
0.7 ꢄ 0.1
1.6 ꢄ 0.4
1.1 ꢄ 0.2
0.1 ꢄ 0.0
6.8 ꢄ 0.1
0.1 ꢄ 0.0
0.0 ꢄ 0.0
1.5 ꢄ 0.1
6.8 ꢄ 1.8
0.3 ꢄ 0.0
0.8 ꢄ 0.3
0.5 ꢄ 0.1
0.1 ꢄ 0.0
3.4 ꢄ 1.4
0.1 ꢄ 0.0
0.0 ꢄ 0.0
Spleen þ pancreas
Lungs
Heart
Brain
Blood
Tumor
Tumor/blood

380
V. Akurathi et al. / European Journal of Medicinal Chemistry 71 (2014) 374e384
Table 3a
On the other hand there was no marked accumulation of tracer
agents in the stomach, indicating that there was no free 99mTcO4ꢁ
present. In addition, the reduced plasma stability of 99mTc-3 could
also explain the reduced retention in the tumors.
Biodistribution of 99mTc-11A in CA IX expressing tumor bearing mice at 0.5, 1, 2 and
4 h p.i. Data are expressed as mean þ/ꢁ standard error (n ¼ 3).
99mTc-11A
Organs [% ID ]
0.5 h
7.8 ꢄ 0.1
1 h
2.6 ꢄ 1.6
2 h
1.5 ꢄ 0.5
4 h
0.2 ꢄ 0.1
3. Conclusions
Kidneys
Urine
Liver
1.3 ꢄ 1.0
28.3 ꢄ 2.9
0.1 ꢄ 0.1
0.1 ꢄ 0.0
0.1 ꢄ 0.0
57.4 ꢄ 2.9
0.9 ꢄ 0.1
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.2 ꢄ 0.0
1.0 ꢄ 0.6
8.3 ꢄ 5.0
0.1 ꢄ 0.0
0.1 ꢄ 0.0
0.1 ꢄ 0.3
80.2 ꢄ 1.7
0.7 ꢄ 0.6
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.0 ꢄ 0.0
0.4 ꢄ 0.3
8.9 ꢄ 3.0
0.1 ꢄ 0.0
0.0 ꢄ 0.0
0.0 ꢄ 0.0
85.5 ꢄ 2.4
1.2 ꢄ 0.7
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.1 ꢄ 0.0
6.2 ꢄ 1.2
14.3 ꢄ 8.9
0.0 ꢄ 0.0
0.0 ꢄ 0.0
0.0 ꢄ 0.0
78.9 ꢄ 7.8
1.9 ꢄ 0.4
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.1 ꢄ 0.0
The sulfonamide derivative AEBS, an inhibitor for CA IX was
conjugated with the BAT and MAG2 ligand, and radiolabelled with
99mTc. The resulting radiolabelled neutral and anionic complexes
were obtained in high radiochemical yields and purity. The corre-
sponding rhenium analogs showed a good inhibitory constant
against CA IX. Biodistribution studies in mice bearing HT-29 tumor
xenografts revealed a limited retention in tumors (ꢅ0.5% I.D at 0.5e
4 h p.i.) for all tracer agents. The anionic tracer 99mTc-8 showed a
modest tracer retention (0.5% I.D/g at 0.5 h), whereas with the
diastereomeric bisesulfonamides complexes 99mTc-11A and 11B
where a dual interaction with CA IX is anticipated showed low
tumor uptake (ꢅ0.2% I.D/g) primarily due to a fast clearance from
the blood pool. The strategy using the blood pool as a reservoir for
the sulfonamide derivative 99mTc-3 did not result in an increased
retention in the tumor. It is concluded that screening of other sul-
fonamide derived radiolabelled complexes possessing a charge, a
slow clearance, and high affinity and selectivity for CA IX is
necessary. This will contribute to the exploration of the potential of
CA IX inhibitors in diagnosis and treatment of hypoxic tumors,
which are highly non-responsive to classical chemo- and
radiotherapy.
Spleen þ pancreas
Lungs
Heart
Intestines
Stomach
Brain
Blood
Tumor
99mTc-11A
Organs [% ID/g]
0.5 h
1 h
2 h
4 h
Kidneys
Liver
25.5 ꢄ 6.4
22.6 ꢄ 3.6
0.6 ꢄ 0.6
0.5 ꢄ 0.1
1.0 ꢄ 0.2
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.1 ꢄ 0.0
1.0 ꢄ 0.0
5.8 ꢄ 4.4
5.3 ꢄ 3.3
0.4 ꢄ 0.1
0.3 ꢄ 0.2
0.7 ꢄ 0.4
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.1 ꢄ 0.0
1.0 ꢄ 0.0
4.6 ꢄ 1.2
6.9 ꢄ 2.3
0.3 ꢄ 0.1
0.1 ꢄ 0.0
0.2 ꢄ 0.0
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.0 ꢄ 0.0
0.0 ꢄ 0.0
0.4 ꢄ 0.1
11.1 ꢄ 6.8
0.5 ꢄ 0.3
0.1 ꢄ 0.1
0.2 ꢄ 0.1
0.0 ꢄ 0.0
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.1 ꢄ 0.0
Spleen þ pancreas
Lungs
Heart
Brain
Blood
Tumor
Tumor/blood
Another possible reason for a lower retention of the tracer
agents could be a low Bmax (concentration of CA IX in tumor tissue)
and Kd (dissociation constant for the ligand-CA IX complex). Ideally
a Bmax/Kd ratio should be ꢂ2 for an efficient binding and imaging
[26]. Finally, the in vivo stability of the new complexes was studied
by determination of the percentage of parent tracer in plasma of
NMRI mice (n ¼ 2) at 1 h p.i. using RP-HPLC. This was 28% for the
neutral complex 99mTc-3 and 77% for 99mTc-8 respectively. For the
diastereomers 99mTc-11A and 99mTc-11B about 65% and 52% was
found to be intact, indicating that these anionic complexes were
relatively stable when compared to the neutral 99mTc-3 complex.
4. Experimental procedures
4.1. Materials and methods
Reagents and solvents were obtained commercially from Acros
(Geel, Belgium), Aldrich, Fluka, Sigma (SigmaeAldrich, Bornem,
Belgium), Merck (Darmstadt, Germany), TCI-Europe (Zwijndrecht,
Belgium) or Fischer Bioblock Scientific (Tournai, Belgium) and used
without further purification unless otherwise specified. 99mTcO4ꢁ
was eluted from a 99Mo/99mTc generator (UltraTechnekowÒ, Covi-
dien, Petten, The Netherlands). 1H NMR spectra were recorded on a
Bruker 400 MHz spectrometer (Brussels, Belgium). Chemical shifts
are reported in parts per million (ppm) relative to tetramethylsilane
Table 3b
Biodistribution of 99mTc-11B in CA IX expressing tumor bearing mice at 0.5, 1, 2 and
4 h p.i. Data are expressed as mean þ/ꢁ standard error (n ¼ 3).
(d
¼ 0). Coupling constants are reported in hertz (Hz). Splitting
99mTc-11B
patterns are defined by s (singlet), d (doublet), t (triplet) and m
(multiplet). HPLC analysis was performed on a LaChrom Elite HPLC
system (Hitachi, Darmstadt, Germany) connected to a UV spec-
trometer set at 254 nm. For analysis of radiolabelled compounds,
the HPLC eluate after passage through the UV detector was directed
over a 3-inch NaI(Tl) scintillation detector connected to a single
channel analyzer (Gabi box, Raytest, Straubenhardt, Germany).
System A was equipped with an XTerra analytical C18 column
(4.6 mm ꢃ 250 mm, Waters, Milford, MA, USA) eluted in an iso-
cratic way with 0.5 M ammonium acetate pH 6.7: ethanol (60:40 V/
V) at a flow rate of 1 mL/min. System B employed an analytical
XBridge C18 column (4.6 mm ꢃ 150 mm, Waters) eluted isocrati-
cally with a mobile phase consisting of 0.01 M disodium hydrogen
phosphate buffer pH 8.0: ethanol (75:25 V/V) at a flow rate of 1 mL/
min. System C was equipped with the same column of system B but
used an isocratic mobile phase consisting of 0.05 M ammonium
acetate buffer pH 6.6:ethanol (82:18 V/V). Combined radioliquid
chromatography and mass spectrometry (radio-LCeMS) analysis
was performed on a system consisting of a Waters Alliance 2690
Organs [% ID ]
0.5 h
8.1 ꢄ 1.9
1 h
1.3 ꢄ 0.3
2 h
0.5 ꢄ 0.2
4 h
0.0 ꢄ 0.0
Kidneys
Urine
Liver
2.3 ꢄ 1.4
28.9 ꢄ 1.6
0.1 ꢄ 0.0
0.1 ꢄ 0.0
0.0 ꢄ 0.0
55.5 ꢄ 11.6
1.9 ꢄ 0.4
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.3 ꢄ 0.1
2.9 ꢄ 2.0
12.0 ꢄ 1.3
0.1 ꢄ 0.0
0.1 ꢄ 0.0
0.0 ꢄ 0.0
62.4 ꢄ 2.9
0.6 ꢄ 0.2
0.0 ꢄ 0.0
0.5 ꢄ 0.0
0.1 ꢄ 0.1
1.3 ꢄ 1.0
15.7 ꢄ 3.6
0.1 ꢄ 0.1
0.1 ꢄ 0.1
0.0 ꢄ 0.0
78.2 ꢄ 2.2
1.4 ꢄ 0.8
0.0 ꢄ 0.0
0.1 ꢄ 0.0
0.2 ꢄ 0.1
1.7 ꢄ 1.4
2.4 ꢄ 0.1
0.0 ꢄ 0.0
0.0 ꢄ 0.0
0.0 ꢄ 0.0
94.7 ꢄ 2.5
0.1 ꢄ 0.0
0.0 ꢄ 0.0
0.0 ꢄ 0.0
0.2 ꢄ 0.1
Spleen þ pancreas
Lungs
Heart
Intestines
Stomach
Brain
Blood
Tumor
99mTc-11B
Organs [% ID/g]
0.5 h
1 h
2 h
4 h
Kidneys
Liver
24.0 ꢄ 5.5
24.6 ꢄ 8.6
0.3 ꢄ 0.0
0.2 ꢄ 0.1
0.2 ꢄ 0.0
0.0 ꢄ 0.0
0.5 ꢄ 0.1
0.1 ꢄ 0.0
0.2 ꢄ 0.0
4.4 ꢄ 0.1
11.1 ꢄ 1.7
0.3 ꢄ 0.1
0.3 ꢄ 0.1
0.1 ꢄ 0.0
0.0 ꢄ 0.0
0.2 ꢄ 0.0
0.2 ꢄ 0.0
1.0 ꢄ 0.2
1.8 ꢄ 0.6
15.1 ꢄ 3.7
0.7 ꢄ 0.6
0.6 ꢄ 0.5
0.1 ꢄ 0.0
0.0 ꢄ 0.0
0.2 ꢄ 0.0
0.2 ꢄ 0.1
1.0 ꢄ 0.1
0.1 ꢄ 0.0
2.0 ꢄ 0.8
0.0 ꢄ 0.0
0.1 ꢄ 0.1
0.0 ꢄ 0.0
0.0 ꢄ 0.0
0.1 ꢄ 0.1
0.1 ꢄ 0.0
1.0 ꢄ 0.0
Spleen þ pancreas
Lungs
Heart
Brain
Blood
Tumor
separation module connected to an XTerra MS C18 column (3.5 mm,
2.1 mm ꢃ 50 mm; Waters) eluted with gradient mixtures of 0.01%
Tumor/blood
formic acid and acetonitrile (0 min 100:0 V/V, linear gradient for

V. Akurathi et al. / European Journal of Medicinal Chemistry 71 (2014) 374e384
381
Fig. 5. In vitro and in vivo assessment of CA IX expression in HT-29 cell lines under normoxic and hypoxic (0.2% O2) conditions by densitometric analysis of the western blots.
Upregulation of CA IX expression was observed in the tumors of mice that were injected with 99mTc-3 or 99mTc-8.
6 min reaching 50:50 V/V, followed by isocratic elution with
50:50 V/V to 10 min) at a flow rate of 0.2 mL/min. The eluent was
monitored for UV absorbance (Waters 2487 Dual wavelength
absorbance detector) and radioactivity (3-inch NaI(Tl) detector).
The time-of-flight mass spectrometer (Micromass LCT, Manchester,
UK) was equipped with an electrospray ionization (ESI) source.
Data acquisition and processing was performed with Masslynx
software (version 3.5, Waters). Quantification of radioactivity in
samples of biodistribution studies and in vivo stability analyses was
done using an automated gamma counter equipped with a 3-inch
NaI(Tl) well crystal coupled to a multichannel analyzer (Wallac
1480 Wizard, Wallac, Turku, Finland). The results were corrected
for background radiation and physical decay during counting. The
polar surface area (PSA) of the reference compounds is calculated
by using Marvin space software from Chemaxon (Budapest,
Hungary). All animal experiments were conducted according to the
Belgian code of practice for the care and use of animals, after
approval from the university ethics committee for animals. The
mice were housed in individually ventilated cages in a thermo-
regulated (w22 ꢀC), humidity-controlled facility under a 12 h/12 h
light/dark cycle with access to food and water ad libitum. The CA IX
expressing HT-29 tumor cells were cultured as previously
described [19]. The HT-29 cells were resuspended in Basement
Membrane Matrix (MatrigelÔ BD, Biosciences, Breda, The
Netherlands) and injected subcutaneously into a lateral flank of the
animal. When tumors reached an average volume of 400 mm3 the
animals were used for the biodistribution studies.
column chromatography with gradient mixtures of dichloro-
methane and methanol (100:0 to 95:5). The purified product was
obtained as white foam with a yield of 66% (0.2 mmol, 200 mg).
C
59H64N4O5S3 ESI-MS: [M þ H]þ calcd 1004.4, found 1004.4. 1H
NMR, 400 MHz (DMSO ed6) d 7.19e7.29 (m, 34H), 2.87 (t, 4H), 2.80
(s, 2H), 2.77 (s, 2H), 2.67e2.71 (m, 4H, J ¼ 8.10), 2.31 (t, 2H), 2.2 (t,
4H), 1.2 (s, 9H).
4.2.3. Synthesis of complex of 2 with rhenium (Re-3)
The formation of the complex of rhenium with the BAT analogue
2 proceeded in two steps. In a first step the precursor TBA[ReOCl4]
was synthesized as reported earlier [28]. Briefly, under stirring, HCl
gas was bubbled slowly through a solution of TBA[ReO4] (tetrabu-
tylammonium perrhenate, 2.2 mmol, 1.1 g) in ethanol (20 mL). The
solution initially turned to an intense yellow color. Addition of HCl
was continued until complete saturation, observed by a change of
the color to dark yellow. The volume of the solution was reduced to
50% with a stream of nitrogen. The precursor crystallized out after
12 h at 0 ꢀC and was filtered off. Yield 60 mg (0.1 mmol, 5%). In a
second step, the protecting groups of the BAT ligand (0.1 mmol,
100 mg) were removed by addition of 0.5 M HCl (2 mL) and heating
at 100 ꢀC for 20 min, followed by neutralization by addition of 0.1 M
NaOH. To this solution, TBA[ReOCl4] (0.1 mmol, 59 mg) and NEt3
(2 mmol, 0.25 mL) dissolved in methanol (10 mL) were added at
0
ꢀC under a stream of nitrogen and the reaction mixture was
stirred for 12 h at room temperature. The solvent was removed
under reduced pressure. The residue was purified by preparative
reversed phase high pressure liquid chromatography (RP-HPLC) on
4.2. Synthesis
an XTerra column (10
gradient mixtures of acetonitrile and water (0 min 20:80 V/V;
m
m, 10 mm ꢃ 250 mm; Waters) eluted with
4.2.1. S,S0-bis-triphenylmethyl-N-(BOC)eN0-(acetic acid)-1,2-
ethylenedi-cysteamine (S,S0-bis-trityl-N-BOC-N0-(acetic acid)BAT, 1)
The title compound was synthesized as previously reported by
our group [27] with a yield of 97%. C51H54N2O4S2 ESI-MS: [M þ H]þ
calcd 822.3, found 822.5.
20 min 90:10 V/V) at a flow rate of 3 mL/min. UV detection was
performed at 254 nm. Yield 7% (0.003 mmol,
C
2
mg).
16H25N4O4ReS3 ESI-MS: [M þ H]þ calcd 619.7, found 619.9.
4.2.4. S-Benzylmercaptoacetylglycylglycine, S-Bn-MAG2 (4)
The title compound was synthesized as previously reported by
our group [29] with a yield of 71% as a white solid. C13H15N2O4SNa
ESI-MS [M þ Na]þ calcd. 318.3, found 318.7.
4.2.2. Conjugation of 4-(2-aminoethyl)benzene sulfonamide (AEBS)
with S,S0-bis-trityl-N-BOC-N0-(acetic acid)BAT (2)
To a solution of AEBS (0.3 mmol, 60 mg) in DMF (5 mL), DMAP
(0.6 mmol, 73 mg), PyBOP (0.6 mmol, 303 mg) and 1 (0.3 mmol,
250 mg) were added and the mixture was stirred at room tem-
perature (RT) for 2 h. DMF was evaporated under reduced pressure,
water (50 mL) was added and this solution was extracted twice
with CH2Cl2 (2 ꢃ 50 mL). The organic phases were combined,
washed with water and brine, dried using MgSO4 and evaporated
under reduced pressure. The residue was purified using silica gel
4.2.5. Conjugation of AEBS with S-benzyl-MAG2 (5)
The conjugation reaction was performed using similar reaction
conditions as reported by Zhao et al. [30]. To a solution of 4-(2-
aminoethyl)benzenesulfonamide (1.54 mmol, 308 mg) in 5 mL
DMF, Bn-MAG2 (1.54 mmol, 453 mg), DMAP (3.08 mmol, 375 mg)
and PyBOP (3.08 mmol,1.56 g) were added. The mixture was stirred
overnight at room temperature and poured into 2 M HCl (50 mL)

382
V. Akurathi et al. / European Journal of Medicinal Chemistry 71 (2014) 374e384
saturated with NaCl. The white precipitate formed was filtered off,
washed with water and recrystallized from ethanol/water, with a
yield of 66% (1 mmol, 0.5 g) of a white solid. C21H25N4O5S2Na ESI-
MS: [M þ Na]þ calcd 500.5, found 500.6. 1H NMR, 400 MHz (DMSO
(0.25 mmol, 70 mg), DMAP (0.5 mmol, 60 mg) and PyBOP
(0.25 mmol, 260 mg). Stirring was continued at RT for 4 days. The
obtained white precipitate was filtered off, washed with water and
recrystallized from methanol-water to yield 70 mg (0.1 mmol, 36%)
of a white solid. C34H43N7O9S3 ESI-MS: [M þ H]þ calcd 789.2, found
ed6)
d
8.29 (s, 1H), 8.18 (s, 1H), 7.91 (s, 1H), 7.73 (d, 2H, J ¼ 7.96 Hz),
7.38 (d, 2H, J ¼ 7.88 Hz), 7.24e7.31 (m, 6H, J ¼ 3.89 Hz), 3.81 (s, 2H),
3.74 (d, 2H, J ¼ 5.16 Hz), 3.66 (d, 2H, J ¼ 5.28 Hz), 3.36 (m, 3H), 3.11
(s, 2H), 2.79 (t, 2H, J ¼ 6.66 Hz).
789.2. 1H NMR, 400 MHz (DMSO ed6)
d 8.29 (m, 1H), 8.18 (m, 1H),
7.91 (m, 3H, J ¼ 10.6 Hz), 7.72 (m, 4H, J ¼ 8.2 Hz), 7.37 (m, 4H), 7.29
(m, 8H, J ¼ 3.6 Hz), 4.13 (m, 1H), 3.78 (m, 4H), 3.28 (m, 6H), 3.08 (m,
2H), 2.75 (m, 4H), 2.04 (m, 2H), 1.85 (s, 1H), 1.68 (m, 2H).
4.2.6. S-Benzoylmercaptoacetylglycylglycine (S-Bz-MAG2, 6)
The title compound was synthesized as reported by our group
[24] with a yield of 72%. C13H13N2O5SNa ESI-MS: [M þ Na]þ calcd
332.3, found 332.6.
4.2.11. 99mTc-BAT-AEBS; 99mTc-3
99mTc-3 was synthesized by a two-step one-pot reaction. In a
first step, compound 2 (0.2 mg in 0.2 mL acetonitrile) and 0.5 M HCl
(60 m
L) were mixed in a reaction vial which was heated at 100 ꢀC for
4.2.7. Conjugation of AEBS with S-benzoyl-MAG2 (7)
20 min. After cooling down to RT, a cocktail solution (0.2 mL)
consisting of buffering and chelating agents [0.5 M phosphate
buffer pH 7.0 (5 mL), 0.1 M Na2EDTA (2.5 mL) and NaK tartrate
(100 mg)] was added followed by addition of SnCl2.2H2O (0.015 mL
of a 4 mg/mL solution in 0.05 M HCl) and 99mTcO4- solution (400e
600 MBq/0.5 mL). The reaction mixture was heated at 100 ꢀC for
10 min. After cooling to RT, an aliquot (0.5 mL) of the reaction
mixture was purified by RP HPLC using system A.
The conjugation was performed using a similar procedure as for
the preparation of (5). To a solution of AEBS (308 mg, 1.54 mmol) in
acetonitrile (20 mL) were added S-Bz-MAG2 6 (477 mg, 1.54 mmol),
DMAP (375 mg, 3.08 mmol) and PyBOP (1.60 g, 3.08 mmol). DMF
(2 mL) was added until a clear solution was obtained, and the
mixture was stirred at room temperature for 4 days. The obtained
white precipitate was filtered off, washed with water and recrys-
tallized from methanol/water with a yield of 66% (1.1 mmol, 0.5 g).
C
21H23N4O6SNa ESI-MS: [M þ Na]þ calcd 514.5, found 514.6. 1H
4.2.12. 99mTc-MAG2-AEBS; 99mTc-8
NMR, 300 MHz (DMSO ed6)
d
8.56 (t, 1H, J ¼ 5.68 Hz), 8.19 (t, 1H,
To a solution of compound 5 (0.5 mg/0.5 mL 0.5 M phosphate
buffer, pH 8.0), NaK tartrate (10 mg, 0.25 mL in H2O), stannous
J ¼ 5.3 Hz), 7.91 (t, 3H, J ¼ 7.4 Hz), 7.71 (t, 3H, J ¼ 9.2 Hz), 7.56 (t, 2H,
J ¼ 7.5 Hz), 7.37 (d, 2H, J ¼ 8.0 Hz), 7.30 (s, 2H), 3.90 (s, 2H), 3.77 (d,
2H, J ¼ 5.3 Hz), 3.66 (d, 2H, J ¼ 5.5 Hz), 3.17 (m, 2H, J ¼ 6.0 Hz), 2.77
(t, 2H, J ¼ 7.0 Hz).
chloride dihydrate (12 mg/mL in 0.05 M HCL, 25 m
L) and 99mTcOꢁ4
solution (400e600 MBq/0.5 mL) were added consecutively. The
reaction mixture was heated at 100 ꢀC for 15 min. After cooling to
RT, an aliquot (0.5 mL) of the reaction mixture was purified by RP
HPLC using system B. Radiochemical purity was assessed by RP
4.2.8. Complexation of 7 with rhenium (Re-8)
The complexation was carried out in 2 steps. In a first step, 7
(0.57 mmol, 280 mg) was deprotected by dissolving the product in
0.1 M NaOH (20 mL) and heating the solution for 10 min at 90 ꢀC
under N2. In a second step, rhenium (V) citrate was prepared by
mixing sodium perrhenate (NaReO4, 0.4 mmol, 109 mg) and
SnCl2.2H2O (0.4 mmol, 7.78 mg in 0.5 M citric acid (10 mL). This
mixture was added to the solution of deprotected 7 obtained in the
first step. The pH was adjusted to 10 by the addition of 0.1 M NaOH
and the mixture was stirred for 1 h at 90 ꢀC. After cooling to RT,
HPLC (X BridgeÔ C18 column, 3.5
a flow rate of 0.6 mL/min.
m
m, 3 mm ꢃ 100 mm, Waters) at
4.2.13. 99mTc-MAG2-Glu-bis-AEBS; 99mTc-11A and 99mTc-11B
The tracer agents were prepared according to the same proce-
dure used for the preparation of 99mTc-8. After cooling to RT, an
aliquot (0.5 mL) of the reaction mixture was purified by RP-HPLC
using system C.
(C6H5)4AsCl.H2O (20
m
mol, 8.7 mg) was added and the precipitate
4.3. Log D1ꢁoctanol/phosphate buffer pH 7.4 determination
formed was filtered off and dried in vacuum. Yield 233 mg
(0.4 mmol, 60%). C14H16N4O6S2Re ESI-MS: [M þ H]þ calcd 586.6,
found 586.0.
Determination of the distribution coefficient was carried out by
a shake flask method [31]. An aliquot (25 m
L) of 99mTc-5, 99mTc-8 or
99mTc-11A and 99mTc-11B (185 kBq/mL) was added to a poly-
propylene tube (5 mL) (Sarstedt, Nümbrecht, Germany) containing
2 mL of 0.025 M sodium phosphate buffer pH 7.4 and 2 mL 1-
octanol. The tubes were shaken for 2 min and then centrifuged at
3000 rpm for 10 min (Eppendorf centrifuge 5810, Eppendorf,
4.2.9. Conjugation of AEBS with N-BOC-L-Glu (BOC-Glu-bis-AEBS,
9)
To a solution of N-BOC-L-Glu (1 mmol, 247 mg) in acetonitrile
(150 mL) at 0 ꢀC HOBT (2 mmol, 270 mg), EDC HCl (2 mmol,
383 mg) and AEBS (2 mmol, 400 mg) were successively added. The
reaction mixture was stirred at 0 ꢀC for 1 h and at RT for 72 h. The
solvent was removed under reduced pressure and the residue was
crystallized from a methanol-water mixture. Yield 540 mg (1 mmol,
100%). C26H37N5O8S2 calcd 611.7, found 611.8. 1H NMR, 400 MHz
Westbury, USA). Aliquots of 50 mL 1-octanol and 500 mL of phos-
phate buffer phases were pipetted out into separate tared eppen-
dorf tubes with adequate care to avoid cross-contamination
between the two phases. The samples were weighed and radioac-
tivity was quantified using an automated gamma counter. The ex-
periments were carried out six times.
(DMSO ed6)
d
7.92 (Br s, 2H), 7.73 (m, 4H, J ¼ 4.1 Hz), 7.34 (m, 4H,
J ¼ 19.4 Hz), 3.49 (signal overlap with H2O), 3.28 (m, 6H), 2.76 (m,
3H), 2.03 (Br s, 1H), 1.65 (m, 2H), 1.36 (s, 9H).
4.4. Inhibition studies (determination of Ki)
4.2.10. Conjugation of BOC-Glu-bis-AEBS (9) with S-Bn-MAG2 (10)
Under a stream of N2, TFA (1.5 mL) was added dropwise to a
solution of 9 (0.25 mmol, 120 mg) in CH2Cl2 (20 mL) at 0 ꢀC and the
solution was stirred for 4 h at RT. After removal of the solvent under
reduced pressure, the residue was dissolved in toluene (5 mL) and
the mixture concentrated to remove residual traces of TFA. A
mixture of acetonitrile-DMF (10:1, 20 mL) was added, followed by 4
The inhibition constants (Ki) of the rhenium reference analogs
Re-3 and Re-8 against hCA I, hCA II, hCA IX and hCA XII isozymes
were determined by assaying the CA-catalyzed CO2 hydration ac-
tivity using an applied photophysics stopped flow instrument [32].
Phenol red (at a concentration of 0.2 mM) was used as indicator.
The conditions included working at the absorbance maximum of
557 nm with 10 mM Hepes buffer (pH 7.5) and 0.1 M Na2SO4 (for

V. Akurathi et al. / European Journal of Medicinal Chemistry 71 (2014) 374e384
383
maintaining constant ionic strength) and following the CA-
catalyzed CO2 hydration reaction for a period of 10e100 s. The
CO2 concentrations ranged from 1.7 to 17 mM for the determination
of the kinetic parameters and inhibition constants at 25 ꢀC. The
uncatalyzed rates were determined in the same manner and sub-
tracted from the total observed rates. Stock solutions of inhibitor
(0.1 mM) were prepared in distilled water. Inhibitor (I) and enzyme
(E) solutions were pre-incubated together for 15 min at room
temperature prior to assay, this in order to allow for the formation
of the EeI complex. The inhibition constants were obtained by non-
linear least square methods using PRISM 3 (GraphPad Software, La
Jolla, CA, USA) and represent the mean from at least three different
determinations [33].
rate), the mice were decapitated and blood was collected at the
above mentioned time points in lithium heparin containing tubes
(4.5 mL LH PST tubes; BD vacutainer, Franklin Lakes, USA) and
stored on ice. Next, the blood was centrifuged for 10 min at
3000 rpm to separate the plasma. The plasma (0.5 mL) was injected
onto an HPLC system consisting of a ChromolithÔ Perfomance
column (3.0 mm ꢃ 100 mm, Merck), eluted with gradient mixtures
of 0.01 M Na2HPO4, pH 8.0 (A) and acetonitrile (B) (0e4 min: 0% B,
flow rate 0.5 mL/min; 4e20 min: linear gradient to 10% A and 90% B,
flow rate 1 mL/min). The HPLC-eluate was collected as 1-mL frac-
tions of which the radioactivity was measured using a gamma
counter. The peak corresponding to the intact tracer was identified
by comparing the retention time with the retention time of the
intact tracers 9 and 10 analyzed on the same HPLC system.
4.5. Biodistribution studies
Acknowledgments
Colorectal (HT-29, ATCC HTB-38) adenocarcinoma cells were
cultured as mentioned in our earlier report [19]. The cells were
resuspended in Basement Membrane Matrix (MatrigelÔ BD Bio-
sciences, Breda, The Netherlands) and injected subcutaneously into
the lateral flanks of NMRI e nu (nu/nu) mice. When the tumors
reached an average volume of 400 mm3 the mice were used for
biodistribution studies. Each mouse received a dose of 0.1 MBq/
0.15 mL of the tracers 99mTc-5, 99mTc-8 or 99mTc-11A and 99mTc-11B
via a tail vein under isoflurane anesthesia (2% isoflurane in O2 at 1 L/
min flow rate). The mice were sacrificed by decapitation at 0.5, 1, 2
or 4 h post injection (p.i., n ¼ 3/time point). Blood was collected and
all major organs (heart, lungs, kidneys, liver, spleen, pancreas,
brain, intestines, stomach and tumors) were excised and weighed.
The radioactivity in each organ was quantified using a gamma
counter, corrected for background radioactivity and expressed as
percentage of the injected dose (% ID) or as percentage of the
injected dose per gram tissue (% ID/g). For calculation of total
radioactivity in blood, blood mass was assumed to be 7% of the total
body mass.
We thank Peter Vermaelen and Ann Vansantvoort for excellent
technical assistance and this work is supported by in vivo molecular
imaging research (IMIR), K.U. Leuven, Belgium.
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