Synthesis and evaluation of 18F-labeled tertiary benzenesulfonamides for imaging carbonic anhydrase IX expression in tumours with positron emission tomography

Article abstract of DOI:10.1016/j.bmcl.2014.05.021

Three tertiary benzenesulfonamide inhibitors 4a-c were radiolabeled with ¹⁸F and evaluated for imaging carbonic anhydrase IX (CA IX) expression with positron emission tomography. All three inhibitors exhibit <10 nM affinity for CA IX with no me

Full text of DOI:10.1016/j.bmcl.2014.05.021

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3064–3068
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 18F-labeled tertiary
benzenesulfonamides for imaging carbonic anhydrase
IX expression in tumours with positron emission tomography
Joseph Lau a, Jinhe Pan a, Zhengxing Zhang a, Navjit Hundal-Jabal a, Zhibo Liu b, François Bénard a,
Kuo-Shyan Lin a,
⇑
a Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Rm 4-123, Vancouver, BC V5Z1L3, Canada
b Department of Chemistry, University of British Columbia, Vancouver, BC V6T1Z1, Canada
a r t i c l e i n f o
a b s t r a c t
Three tertiary benzenesulfonamide inhibitors 4a–c were radiolabeled with 18F and evaluated for imaging
carbonic anhydrase IX (CA IX) expression with positron emission tomography. All three inhibitors exhibit
<10 nM affinity for CA IX with no measurable affinity for CA II. Despite good affinity/selectivity to CA IX
and excellent stability in plasma, uptake of [18F]4a–c in CA IX-expressing HT-29 tumours was low with-
out significant contrast. [18F]4a,b were excreted rapidly, while [18F]4c exhibited significant in vivo deflu-
orination leading to high bone uptake. Due to minimal uptake in HT-29 tumours compared to normal
organs/tissues, 18F-labeled benzenesulfonamides [18F]4a–c are not suitable as CA IX imaging agents.
Ó 2014 Elsevier Ltd. All rights reserved.
Article history:
Received 12 April 2014
Revised 8 May 2014
Accepted 9 May 2014
Available online 17 May 2014
Keywords:
Carbonic anhydrase IX
Tertiary benzenesulfonamides
Fluorine-18
In vivo defluorination
Positron emission tomography
Carbonic anhydrases (CAs) are a class of zinc metalloenzymes
found in most living organisms.1 Most CAs are efficient catalysts
for the reversible hydration of carbon dioxide to bicarbonate ion
and proton (H2O + CO2 M HCOÀ3 + H+).2,3 To date, 15 human CA iso-
enzymes have been identified,1 with expression of CA IX being
strongly associated with cancer progression. CA IX is not expressed
in normal tissues except in the gastrointestinal mucosa,4 but is
highly expressed in malignancies such as gliomas, cervical, blad-
der, ovary, head and neck, lung, breast, and colon cancers.5–14
tumours.26–29 In imaging clinical trials, cG250 demonstrated great
sensitivity and accuracy for the diagnosis of clear cell renal cell
carcinoma—a cancer subtype that commonly exhibits high
constitutive expression of CA IX due to a mutation in the von
Hippel–Lindau tumour suppressor gene.30 However, imaging
hypoxia-associated CA IX expression with mAbs is less likely to
succeed. Hypoxic niches within tumours are less accessible for
mAbs due to low perfusion rates caused by aberrant vasculature
and increased interstitial pressure. Furthermore, Dubois et al.
reported that anti-CA IX mAbs could not distinguish active CA IX
in hypoxic cells from inactive CA IX in aerobic cells.31 mAb binding
was observed after hypoxic cells were re-oxygenated.31 In contrast,
the binding of sulfonamide inhibitors to CA IX occurred only under
hypoxia exposure, and co-localized with the binding of anti-CA IX
mAbs.31 These findings suggest that CA IX-targeting probes derived
from sulfonamide inhibitors will be more suitable for discriminat-
ing between aerobic and hypoxic regions.
Although several attempts have been made to develop radiola-
beled small CA IX sulfonamide inhibitors for imaging, so far, none
of them has been reported to successfully visualize CA IX-express-
ing tumours in preclinical/clinical settings. Apte et al.32 reported
the preparation of an 18F-labeled sulfonamide derivative
(Fig. 1A), but no biological evaluation data were presented. Akura-
thi et al.33,34 reported the synthesis of 99mTc-labeled sulfonamides
Under hypoxic conditions CA IX is up-regulated by HIF1a, and
transports HCOÀ3 into the cell to maintain pH homeostasis.15 The
remaining extracellular H+ acidifies the tumour microenvironment,
activates metalloproteinases, induces angiogenesis, and facilitates
invasion and metastasis.16–19 Clinically, the expression of CA IX is
associated with resistance to chemo- and radiation therapy,20–22
increased recurrence and reduced survival.23,24 As CA IX is
expressed on the extracellular surface, it is an attractive and acces-
sible imaging biomarker for hypoxic tumours.
Since its development in 1986,25 anti-CA IX monoclonal anti-
body (mAb) cG250 have been labeled with different radioisotopes
including 131I, 111In, 124I, and 89Zr to annotate CA IX expression in
⇑
Corresponding author. Tel.: +1 604 675 8208; fax: +1 604 675 8218.
E-mail address: klin@bccrc.ca (K.-S. Lin).
http://dx.doi.org/10.1016/j.bmcl.2014.05.021
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

J. Lau et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3064–3068
3065
O
N
O
S NH2
O
(B)
(A)
O
H2N S
O
M
O
N
S
HN
O
O
NH
O
H
N
O
H2N
S
O
M
N
S
N
S
N
O
18F
O
N
M= Re or 99mTc
Ki(CA IX) = 58 nM
N
CO
CO
O
M
M= Re or 99mTc
Ki(CA IX) = 66 nM
O
O
M= Re or 99mTc
Ki(CA IX) = 59 nM
CO
O
(C)
(D)
H2N
(E)
O
O S O
NH2
S
NH2
O
S
N
O
H
N
R
11C
H
N
O
HO
O
N
N
N
N
H
S
N
O
O
O
O
H2N
O
HO
O
S
Ki(CA IX) = 0.9 nM (R = 3-NO2)
5.4 nM (R = 4-CH3CO)
O
OH
18F-VM4-037A
O
18F
OC
N
0.3 nM (R = 2-CN)
Ki(CA IX) = 1.3 nM
N
(F)
O O
N
N
O
O
O
H
N
HO
(G)
S
NH2
O
S
N
M
CO
CO
O
NH2
O
O
N
N
S
H
OC
HN
O
O
Re
CO
OH
Ki(CA IX) = 43 nM
CO
Ki(CA IX) = 3.7 nM
M= Re or 99mTc
Ki(CA IX) = 9.0 nM
Re
CO
OC
OC
CO
O
NH2
O
S
H2N
S
F
O
O
O
O
O
N
N
H
H
N
N
H
M= Re or 99mTc
O
H
M= Re or 99mTc
Ki(CA IX) = 5.2 nM
M
CO
M
CO
S
CO
OC
CO
18F-U-104
Ki(CA IX) = 45 nM
NH2
Ki(CA IX) = 7.0 nM
O
Figure 1. Structures of reported CA IX radiotracers derived from sulfonamide-based small molecule inhibitors.
(Fig. 1B), but minimal uptake (up to 0.5%ID/g at 0.5 h post-injection
(pi)) of these tracers was obtained in HT-29 tumour xenografts.
Asakawa et al.35 reported C-11 labeling of three potent benzene-
sulfonamides (Fig. 1C) for CA IX imaging, but no biological evalua-
tion data were presented either. Doss et al. reported37 that in a
clinical study VM4-037 (Fig. 1D), a potent 18F-labeled CA IX inhib-
itor developed by Siemens,36 showed intense uptake in kidneys
and liver, which reduces lesion visibility in these two organs. Lu
et al.38 recently reported the preparation of several Re/99mTc-
labeled benzenesulfonamides. Among them, the 99mTc-labeled
compound shown in Figure 1E exhibited good affinity (IC50 = 9 nM)
to CA IX-expressing HeLa cells, but no biological evaluation was
presented. Recently, Can et al.39 reported four sulfonamides
labeled with Re/99mTc via a cyclopentadienyl group (Fig. 1F). These
compounds showed good affinity to CA IX, but no biological evalu-
ation was presented. Previously we reported the preparation of an
18F-labeled CA IX inhibitor U-104 (Fig. 1G).40 Imaging study in mice
showed low uptake in HT-29 tumour xenografts (0.83 0.06%ID/g)
but very high in blood (13.92 3.07%ID/g) at 1 h pi. The high blood
retention is likely due to the binding of U-104 to CA II (Ki = 95 nM,
Table 1) which is abundantly expressed in erythrocytes.41,42 There-
fore, for this study we 18F-labeled three selective tertiary sulfon-
amide CA IX inhibitors 4a–c (Table 1), and evaluated their
potential for imaging CA IX expression in tumours with positron
emission tomography. Compounds 4a–c were among the first
identified benzenesulfonamides that are selective nanomolar CA
IX inhibitors with no measurable affinity for the off target CA II.43
The synthesis of standards 4a–c and their respective radiolabel-
ing tosylate precursors 3a–c are illustrated in Scheme 1. The start-
ing sulfonamides 1a–c were prepared according to published
procedures.43 Coupling of sulfonamides 1a–c with excess propyl-
ene oxide in DMF in the presence of K2CO3 as base afforded the
desired secondary alcohols 2a–c in excellent yield (92–99%). Ini-
tially, we attempted to prepare mesylate precursors for radiolabel-
ing. However, we found out that it was difficult to separate the
desired mesylates from their respective alcohol reactants 2a–c by
Table 1
Reported inhibition constants (Ki, nM) of acetazolamide,44 HS680,47 U-104,44 and
tertiary sulfonamides 4a–c43 to human CA isoenzymes I, II, IX and XII
R
N
F
O
S
O
Compounds
hCA I
hCA II
hCA IX
hCA XII
Acetazolamide
HS680
U-104
4a (R = Me)
4b (R = Ac)
4c (R = Cl)
250
—
5080
73.1
89.1
77.3
12
248
95
Not active
Not active
Not active
25
7.5
45
9.3
9.6
9.1
5.7
35
4.5
33.6
83.8
100
flash column chromatography (data not shown). Therefore, tosyl-
ate precursors were prepared instead as they could be easily sepa-
rated from respective alcohol reactants 2a–c due to the additional
benzene on their structures. By using diisopropylethylamine as the
base and trimethylamine hydrochloride as the catalyst,45 the reac-
tion of alcohols 2a–c with p-toluenesulfonyl chloride proceeded
smoothly in CH2Cl2, and provided the desired tosylate precursors
3a–c in 77–91% yield. Previously, standards 4a–c were prepared
by the reaction of HF/SBF5 with their respective N-allylic interme-
diates.43 However, due to safety concern with HF, different strate-
gies for the preparation of standards 4a–c were investigated. For
the preparation of 4a, tosylate 3a was refluxed in THF in the pres-
ence of excess tetrabutylammonium fluoride (TBAF). However, the
yield (6.5%) of desired product 4a was low due to the formation of
elimination by-product. Therefore, standards 4b,c were prepared
by directly converting alcohols 2b,c to fluorides using diethylami-
nosulfur trifluoride (DAST), and better yields (23–44%) of 4b,c were
obtained.

3066
J. Lau et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3064–3068
(A)
R
N
R
N
OH
OTs
R
NH
S
O
O
O
O
O
S
S
TsCl, TMA.HCl, DIEA
CH2Cl2
O
O
O
DMF
1a-c
2a-c
4a
3a-c
(B)
a: R = Me
b: R= Ac
c: R = Cl
N
OTs
N
F
O
S
S
TBAF
THF
O
O
3a
(C)
R
N
F
R
N
OH
O
O
S
S
DAST
O
O
CH2Cl2
2b-c
4b-c
(D)
18F
O
R
N
R
N
OTs
O
S
TBA[18F]F
DMF
S
O
O
[
18F]4a-c
3a-c
Scheme 1. Syntheses of (A) precursors 3a–c and (B and C) standards 4a–c of tertiary sulfonamide CA IX inhibitors, and (D) their 18F-labeled analogs [18F]4a–c.
As illustrated in Scheme 1D, the preparation of [18F]4a–c was
performed via aliphatic nucleophilic substitution reactions
between tosylate precursors 3a–c and TBA[18F]F in DMF at
125 °C. After HPLC purification, [18F]4a–c were obtained in 7.1–
43% decay-corrected yields with >99% radiochemical purity, and
Table 2
Biodistribution data (%ID/g at 1 h pi; N = 4) of [18F]4a–c in NSG mice bearing HT-29
human colorectal tumour xenografts
Tissues/organs
Radiotracers
18F]4b
[
18F]4a
[
[
18F]4c
>740 GBq/lmole specific activity at the end of synthesis
Blood
Fat
0.58 0.49
0.76 0.55
7.47 7.46
1.35 2.21
0.68 0.53
9.77 8.43
0.81 0.83
1.68 1.66
1.22 0.90
0.59 0.46
0.52 0.47
0.91 0.70
0.32 0.28
0.51 0.45
0.37 0.11
1.48 0.53
16.7 5.85
1.25 0.98
0.82 0.23
3.35 1.72
0.75 0.26
2.03 0.65
1.46 1.07
0.48 0.11
0.51 0.18
2.72 0.57
0.20 0.06
0.59 0.29
0.98 0.36
0.94 0.51
2.64 0.26
0.40 0.16
0.81 0.35
3.57 1.80
0.61 0.20
2.34 1.04
1.51 0.42
0.93 0.38
0.92 0.50
(ꢀ100 min). The stability of [18F]4a–c was determined in mouse
plasma, and no detectable metabolites of [18F]4a–c were observed
by HPLC analysis after 2 h incubation at 37 °C. The logD7.4 (D: dis-
Intestine
Stomach
Spleen
Liver
Pancreas
Kidney
Lungs
Heart
Muscle
Bone
tribution coefficient) values of
[
18F]4a–c were 3.45 0.04,
3.05 0.01, and 3.51 0.27, respectively, which were measured
using the traditional shake flask method. These values were used
to assess if [18F]4a–c could cross cell membrane freely, and poten-
tially bind to intracellular off targets CA I and II which are
expressed in erythrocytes.41,42 Since the molecular weights (MW)
and logD7.4 values of 4a–c are in the range of 321–349 Daltons,
and 3.05–3.51, respectively, they are likely to enter cells freely
according to the Lipinski’s rule of five (MW < 500 and
logD7.4 < 5).46
12.61 5.18
0.33 0.05
0.98 0.48
Brain
Tumour
To evaluate the potential of [18F]4a–c for imaging CA IX expres-
sion, biodistribution and PET imaging studies were performed in
mice bearing HT-29 human colorectal tumour xenografts. HT-29
tumour model is commonly used in literature for evaluating CA
IX-targeting tracers as it constitutively expresses CA IX.33,34,40,47
As shown in Table 2, fast blood clearance of [18F]4a–c was observed
with only 0.58 0.49, 0.37 0.11, and 0.98 0.36%ID/g, respec-
tively, retained in blood at 1 h pi. These numbers are substantially
lower than the previously reported 13.92 3.07%ID/g obtained
with 18F-labeled U-10440 indicating no significant binding of
stage why it was [18F]4c but not [18F]4b that resulted in massive
in vivo defluorination. Based on the structures of 4a–c, the 4-acetyl
group in 4b is the most electron-withdrawing group followed by
the 4-chloro group in 4c, and then the 4-methyl group in 4a.
Presumably, the 4-acetyl group would make the nitrogen of sulfon-
amide 4b more electron deficient, and the adjacent fluoro a better
leaving group, resulting in a higher degree of in vivo defluorination.
Although bone uptake of
[ [
18F]4b was higher than 18F]4a
(2.72 0.57 vs. 0.91 0.70%ID/g at 1 h pi), these numbers were
much lower than 12.61 5.18%ID/g obtained by using [18F]4c sug-
gesting other factors might contribute to in vivo defluorination of
these compounds. Since the uptake of [18F]4a–c in HT-29 tumours
was not significantly higher than the uptake values in the sur-
rounding tissues (blood, fat, muscle and bone), no clear tumour
visualization was obtained from the PET images (Fig. 2).
Due to the large number of human CA isoenzymes and their
highly conserved catalytic domain, it is very challenging to design
potent inhibitors specifically targeting an individual CA isoform.1
[
18F]4a–c to the intracellular off target CA II. However, the uptake
of [18F]4a–c in HT-29 tumours did not improve as only 0.51 0.45,
0.59 0.29, and 0.98 0.48%ID/g, respectively, were observed at
1 h pi. The majority of [18F]4a,b radioactivity was excreted via
the hepatobiliary pathway reflecting the lipophilic nature of these
two radiotracers. For
[
18F]4c very high bone uptake
(12.61 5.18%ID/g at 1 h pi) was observed which indicated mas-
sive defluorination of [18F]4c in vivo. However, it is unclear at this

J. Lau et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3064–3068
3067
and would prevent the radiolabeled sulfonamide conjugates from
entering cells. Another promising alternative is the use of a multi-
valent design. This approach combines several CA-targeting sulfon-
amides into one single molecule, which could potentially enhance
binding affinity to CA IX and afford cell impermeability by accumu-
lation of MW.
In conclusion, we successfully synthesized and radiolabeled ter-
tiary benzenesulfonamides 4a–c, and evaluated their potential as
CA IX imaging agents. Despite their good affinity and selectivity
for CA IX, imaging and biodistribution data showed only minimal
tumour uptake in xenografted mice relative to normal tissues.
Therefore, [18F]4a–c are not suitable for CA IX targeted molecular
imaging.
Acknowledgments
This work was supported by the Canadian Institutes of Health
Research. We are thankful to Milan Vuckovic, Wade English, and
Julius Klug for operating the cyclotron and providing 18F-fluoride
for radiolabeling experiments.
Figure 2. Representative PET images of [18F]4a–c at 1 h pi in NSG mice bearing CA
IX-expressing HT-29 human colorectal tumour xenografts. White arrows point to
HT-29 tumours.
Supplementary data
Compounds 4a–c were explored for imaging CA IX expression in
HT-29 tumours because they were reported to have nanomolar
binding affinity for CA IX, and most importantly to be the first sul-
fonamides to show no interaction with CA II in CO2 hydration
assays (Table 2). The binding of 4a–c to CA XII could be beneficial
as CA XII, a transmembrane protein, is also up-regulated in hypoxic
tumours.48 Cytosolic CA I and II are expressed in large quantities in
erythrocytes41,42 and the binding of CA IX-targeting tracers to CA I
and II would reduce imaging contrast. Previously, we observed
slow blood clearance of 18F-labeled U-104 presumably due to bind-
ing with CA II (Ki = 95 nM, Table 2).40 In this study, we did not
observe much retention of [18F]4a–c in blood at 1 h pi despite their
suitable physical characteristics (neutral, lipophilic, and with
MW < 500 Daltons) to cross cell membrane, and good binding
affinity to CA I (Ki = 73.1–89.1 nM, Table 2). Possible explanations
for the low tumour uptake and blood retention of [18F]4a–c could
be due to relatively fast metabolism and/or blood clearance of
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.05.021.
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these tracers, and in the case of
defluorination.
[
18F]4c, rapid in vivo
Although no small sulfonamide-based radiotracers have been
reported to visualize CA IX-expressing tumours in preclinical imag-
ing studies, a successful optical imaging probe HS680 is already
commercially available. HS680, a conjugate of acetazolamide and
the near-infrared fluorochrome VivoTag 680 developed by Perkin-
Elmer, showed impressive 10% injected dose accumulation in HT-
29 tumour xenografts in mice at 24 h pi.47 Acetazolamide is a pro-
miscuous CA inhibitor, and therefore the derivatives of acetazola-
mide including HS680 are expected to have good binding affinity
to most CAs including the intracellular isoenzymes I and II
(Table 2). However, the conjugation of acetazolamide with the
highly charged and bulky (MW > 1000 Daltons) VivoTag 680 pre-
vents it from crossing cell membrane and binding to intracellular
off targets. The success of HS680 could be a valuable lesson for
the future design of radiotracers for imaging CA IX expression with
positron emission tomography or single photon emission com-
puted tomography. Instead of radiolabeling CA IX-specific inhibi-
tors which may be difficult to synthesize, the design of cell-
impermeable sulfonamide-based radiotracers represents an easier
and quicker solution. This could be achieved, for example, by con-
jugating the CA-targeting sulfonamide moiety with a polyamino-
carboxylate chelator for labeling with radiometals such as 68Ga,
64Cu, or 111In for imaging. The radiometal–polyaminocarboxylate-
chelator complexes are generally highly hydrophilic and charged,
25. Oosterwijk, E.; Ruiter, D. J.; Hoedemaeker, P. J.; Pauwels, E. K. J.; Jonas, U.;
Zwartendijk, J.; Warnaar, S. O. Int. J. Cancer 1986, 38, 489.

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