Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors

Article abstract of DOI:10.1016/j.bmcl.2011.10.058

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.

Full text of DOI:10.1016/j.bmcl.2011.10.058

Bioorganic & Medicinal Chemistry Letters 22 (2012) 653–657
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of near-infrared fluorescent sulfonamide
derivatives for imaging of hypoxia-induced carbonic anhydrase IX
expression in tumors
Kevin Groves a, , Bagna Bao a, Jun Zhang a, Emma Handy a, Paul Kennedy a, Garry Cuneo a,
⇑
Claudiu T. Supuran b, Wael Yared a, Jeffrey D. Peterson a, Milind Rajopadhye a
a PerkinElmer, 549 Albany St., Boston, MA 02118, United States
b Department of Chemistry, Laboratory of Bioinorganic Chemistry, Università degli Studi di Firenze, Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent
dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro,
ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX
showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form
hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA
XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic
conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo
study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide
derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose
was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating
the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 25 August 2011
Revised 14 October 2011
Accepted 18 October 2011
Available online 24 October 2011
Keywords:
Cancer
Hypoxia
Carbonic anhydrase
Fluorescence
Tumor imaging
Human carbonic anhydrase (hCA) IX is a transmembrane cell
surface enzyme which catalyzes the reversible interconversion of
CO2 to bicarbonate and a proton. hCA IX is overexpressed in re-
sponse to tumor hypoxia in many common tumor types and plays
a critical role in hypoxia associated tumor acidosis.1–5 Tumor hy-
poxia and subsequent expression of hCA IX are also correlated to
metastasis, poor prognosis and resistance to therapeutic interven-
tion making hCA IX an important biomarker in the study of hypox-
ia and tumor cell proliferation and a valuable target for both
therapeutics and novel diagnostics.6–8 While hCA inhibitors have
been in clinical use for many decades for conditions such as glau-
coma, seizures, altitude sickness and as a diuretic, recently a num-
ber of inhibitors of hCA IX and the related transmembrane hCA XII
have shown considerable antitumor and antimetastatic activity.9–
A veritable pharmacopeia of small molecule inhibitors for vari-
ous hCAs has been described in the literature with sulfonamide
based inhibitors standing out in particular as a robust class of
hCA active molecules due to their high affinity, availability and
ease of chemical manipulation.11,13–19 As such, diverse arrays of
sulfonamide inhibitors have been synthesized incorporating a vari-
ety of chemical functionalities for modifying properties such as
selectivity among hCAs and cell permeability15,20 and include sev-
eral radiolabeled21,22 and fluorescent derivatives.9,16,23–27 Fluores-
cent sulfonamides have been reported to bind to hCAs on
osteoclasts23 as well as a number of tumor cell lines including CA
IX transfected MDCK28 HeLa, HT-29,29 LS174T, SK-RC-529 and
SiHa26 cells. In vivo studies with fluorescent sulfonamides have
also shown promise for detecting hCA expression in HT-29 and
SK-RC-52 tumor xenografts.9,30 However, to date, in vivo studies
with fluorescent sulfonamides have been performed with fluores-
cein derivatives, whose green excitation and emission wavelengths
are suitable for only ex vivo or superficial in vivo imaging applica-
tions and preclude accurate deep tissue quantification.
11
Furthermore, a CA IX specific antibody, girentuximab, is also
being evaluated in Phase III clinical trials for the treatment of
clear-cell renal cell cancer.12 The ability to image this important
enzyme with a targeted fluorescent agent would be of considerable
value for further investigation of tumor hypoxia and hCA targeted
therapeutics.
In contrast, optical imaging in the near-infrared (NIR) range
allows efficient penetration of photons through living tissue and
minimizes interference from tissue autofluorescence. Combined
with quantitative fluorescence molecular tomography (FMT), NIR
fluorescent agents have emerged as invaluable tools for
Abbreviations: hCA, human carbonic anhydrase; FMT, fluorescence molecular
tomography; NIR, near-infrared.
⇑
Corresponding author. Tel.: +1 617 350 9424; fax: +1 617 350 9295.
E-mail address: kevin.groves@perkinelmer.com (K. Groves).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.058

654
K. Groves et al. / Bioorg. Med. Chem. Lett. 22 (2012) 653–657
Table 1
quantitative, deep tissue imaging of a complex array of biological
processes across a range of important areas of disease research
including oncology,31,32 inflammation,33–35 bone metabolism,36,37
angiogenesis38 and atherosclerosis.39–41
Optical and charge properties of NIR fluorescent sulfonamidesa
Compounds
R =
Amax/Emax (nm)
Net charge
1a, 2a
VivoTag-S680
VivoTag-680
VivoTag-680XL
VivoTag-671
674/692
670/686
670/687
667/683
À3
À5
À5
À3
The aim of the present work was to prepare new NIR fluorescent
hCA IX inhibitors for non-invasive in vivo detection and quantifica-
tion of hCA IX expression. A series of NIR fluorescent sulfonamides
was synthesized based on the well characterized inhibitors aceta-
zolamide (AAZ) and benzenesulfonamide 1. The compounds were
screened for inhibition of hCA IX and isoforms II, XII and XIV. Lead
compounds were then visualized on hypoxic cells in vitro by fluo-
rescence microscopy and binding to hypoxic cells quantified by
flow cytometry. Preliminary in vivo bio-distribution and tumor
imaging was demonstrated in HT-29 tumor bearing mice.
The sulfonamide derivatives 2 and 3 were synthesized from
acetazolamide by hydrolysis in refluxing ethanolic HCl followed
by coupling to Boc-aminomethylbenzoic acid or FMOC-b-alanine
using EDC and HOBT in DMF for 24 h as shown in Scheme 1. The
protected intermediates were purified by HPLC42 followed by
deprotection with TFA/water or diethylamine, respectively. The
resulting amines, as well as 1 and benzylamine 4 as a non-hCA
binding control, were coupled to the succinimidyl esters of one
or more of four commercially available hydrophilic indocyanine
NIR fluorochromes (NIRF-OSu) to give the corresponding amide
linked compounds 1a–d, 2a–d, 3c and 4b. The fluorochromes used
in this study all have absorption maxima of about 665–675 nm and
emission maxima of about 680–695 nm, ideal for in vivo imaging,
and a significant net negative charge of À3 to À5 to reduce mem-
brane permeability and nonspecific uptake in cells (Table 1). The
compounds were purified by preparative HPLC and characterized
by LCMS.43
1b, 2b, 3b
1c, 2c, 4c
1d, 2d
a
All dyes were obtained from PerkinElmer as reactive succinimidyl esters.
Absorbance and emission maxima were measured in 1XPBS.
acetazolamide (AAZ) and 4-aminomethylbenzenesulfonamide 1
are shown in Table 2. Also shown are the selectivity ratios relative
to hCA IX.
Six of the new compounds tested showed less than 10 nM inhi-
bition potencies toward hCA IX, including all of the acetazolamide
derivatives and one of the benzenesulfonamides. Inhibition of hCA
IX was generally slightly impaired for the fluorophore conjugated
benzenesulfonamide derivatives 1a–d relative to the parent inhib-
itor 1, with the exception of 1d, which had a Ki of less than 10 nM,
three times lower than 1 and about 10 times lower than benzene-
sulfonamides 1a–c. In contrast, hCA IX inhibition by the AAZ deriv-
atives appeared insensitive to both the fluorophore structure and
the linking moiety with all of the acetazolamide derivatives show-
ing a Ki of 7-10 nM, an approximately threefold improvement over
the parent AAZ.
Due to the cytosolic location of hCA II and the expected low
membrane permeability of these highly charged fluorescent inhib-
itors, in vitro and in vivo selectivity is expected to be driven to-
wards the membrane form hCA IX. However, hCA II is of
considerable importance due to the ubiquitous expression of this
enzyme in cells and the generally low selectivity of sulfonamide
inhibitors and was thus also examined in this study. Four of the
compounds tested, 1d, 2a–b and 3b showed selectivity ratios for
hCA IX over hCA II of greater than 30, a marked improvement over
the parent inhibitors, especially AAZ, which actually shows a slight
preference for hCA II. All of the benzenesulfonamides 1a–d had
The inhibition potency and selectivity of the new fluorescent
inhibitors towards various hCAs was characterized by measuring
inhibition of CO2 hydration using the stopped flow method.44 Inhi-
bition data for four recombinant, purified hCA isozymes, hCA IX,
the cytosolic form hCA II and two other transmembrane forms,
hCA XII and XIV, along with that for the parent inhibitors,
O
NH2
HCl/MeOH
R
HN
H
N
reflux
O
O
NH2
O
S
O
S
1a: R = VivoTag-S680
1b: R = VivoTag-680
1c: R = VivoTag-680XL
1d: R = VivoTag-671
NIRF-OSuc
NIRF-OSuc
S
S
O
S
O
S
N
H2N
H2N
N
O
N
H2N
N
H2N
O
O
AAZ
AZ
1a-d
1
O
N-BOC-4-
aminomethyl
benzoic acid
EDC/HOBT
DMF
BOC
HN
NH2
R
HN
H
N
H
N
TFA/water
H
N
O
O
O
O
S
S
O
O
S
2a: R = VivoTag-S680
2b: R = VivoTag-680
2c: R = VivoTag-680XL
2d: R = VivoTag-671
AZ
S
S
S
N
O
N
O
H2N
H2N
N
N
N
O
H2N
N
2a-d
2
N-FMOC-β-Ala
EDC/HOBT
DMF
H
N
H
H
FMOC
NH
NIRF-OSuc
NIRF-OSuc
O
O
Diethylamine
O
O
R
H
N
N
NH2
N
S
S
O
O
S
S
S
AZ
S
O
N
O
N
O
H2N
H2N
N
N
N
O
H2N
N
3b: R = VivoTag-680
4c: R = VivoTag-680
3b
3
O
NH2
R
HN
4
4c
Scheme 1. Synthesis of NIR fluorescent sulfonamide derivatives.

K. Groves et al. / Bioorg. Med. Chem. Lett. 22 (2012) 653–657
655
Table 2
CA II, IX, XII and XIV inhibition data with sulfonamides 1a–d, 2a–d, 3b, 4c as well as parent inhibitors AAZ and 1 by stopped-flow CO2 hydration method. Ki ratios relative to CA IX
and Kd values to hypoxic HeLa cells for selected compounds are also shown
b
Compound
Kia (nM)
Ki Ratios
Kd (nM) HeLa
hCA II
hCA IX
hCA XII
hCA XIV
hCA II/IX
hCA XII/IX
hCA XIV/IX
AAZc
1d
1a
1b
1c
1d
2a
2b
2c
2d
3b
4c
12
25
33
88.1
116
103
9.9
8.4
7.9
7.6
7.7
5.7
3.2
297
75
8.1
40
57
43
36
40
41
—
407
9.9
60
69
77
45
57
0.5
4.8
3.8
6.6
5.5
50.7
34.3
34.3
1.3
3.5
33.1
1.0
0.2
0.1
3.4
0.6
0.1
4.0
6.8
5.4
4.7
5.2
4.7
1.0
1.6
—
—
—
—
—
—
—
—
52
49
—
8.3
—
160
336
765
564
502
288
271
9.8
4.6
0.1
0.6
7.0
9.2
5.7
7.5
10.9
8.8
1.0
27.1
248
>100,000
84
66
>100,000
7.5
>100,000
35
>100,000
a
b
c
Mean from three different determinations. Errors in the range of 10% of the reported value.
Dissociation constants for hypoxic HeLa cells minus normoxic background.
From Ref. 6.
From Ref. 20.
d
A
3b:
CAIX Ab
4c: (control)
1d:
2a:
2b:
i
ii
iii
iv
v
vi
vii
viii
ix
x
xi
xii
B
2b
2c
3b
12000
10000
8000
6000
4000
2000
0
7500
6000
4500
3000
1500
0
7500
6000
4500
3000
1500
0
Kd = 52 nM
Kd = 49 nM
Kd = 8.3 nM
0
0.5
1
0
0.5
1
0
0.5
1
Concentration (µM)
Concentration (µM)
Concentration (µM)
Figure 1. (A) Fluorescence microscopy of the uptake in normoxic (i–vi) or hypoxic (vii–xii) HeLa cells cultured with a CAIX antibody (i, vii), nonbinding control compound 4c
(ii, viii), 1d (iii, ix), 2a (iv, x), 2b (v, xi), and 3b (vi, xii). Magnification is 40Â and exposure times are all 4000 ms except F and L, 6000 ms. Agent signal is shown in red, cell
nuclear stain DAPI is shown in blue. (B) Fluorescence signal by flow cytometry for hypoxic (d) and normoxic (N) HeLa cells for compounds 2b, 2c and 3b over a range of
concentrations from 10 nM to 1 lM along with curve fits for specific (hypoxic) and nonspecific (normoxic) binding.
comparatively low potencies toward hCA II in the range of 336–
765 nM, 2–5 times less effective than 1. Among the AAZ series
2a–b and 3b, the increase in selectivity for hCA IX is completely de-
rived from a decrease in inhibition potency toward hCA II, possibly
due to steric effects from the comparatively bulky (>1000 Da) fluo-
rophores. However, acetazolamide derivatives 2c and 2d, had inhi-
bition potencies not significantly different than the parent
inhibitor AAZ.
hCA XII is another transmembrane CA isoform that is also asso-
ciated with hypoxia and tumor growth that has been identified as a
potential therapeutic target.45 The parent inhibitors used both
have single digit nM inhibition constants toward hCA XII, whereas
the fluorescent derivatives synthesized in the current study were
somewhat less potent. The AAZ series was 5–10 times less potent
and the benzenesulfonamide series 10–100 times than the corre-
sponding parent inhibitors. The exception was benzenesulfon-
amide 1c which, with a Ki of 8.1 nM, had the highest potency
toward hCA XII and also the highest selectivity for hCA XII among
all of all the compounds tested.
For hCA XIV, the AAZ derivatives were all within a factor of two
of the potency of the parent (45–84 nM), while the potencies for
the benzenesulfonamide series varied more widely from 9.9 to
407 nM. The most selective hCA XIV inhibitor of the series was
1b. As expected, the non-sulfonamide 4c, which shares
a

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K. Groves et al. / Bioorg. Med. Chem. Lett. 22 (2012) 653–657
A
Bio-distribution in Mice Bearing HT-29 Tumors @24h
4c
3b
0.60
0.40
0.20
0.00
FMT Quantification @ 24h
Tumors
D
B
C
500
*
4c
3b
400
300
200
100
0
10%
I.D.
Kidney
Tumors
Kidney
4c
3b
Figure 2. (A) Ex vivo bio-distribution of control compound 4c and CA IX inhibitor 3b in HT-29 tumor bearing mice 24 h post i.v. injection (4 nmol). (B) Ex vivo fluorescence
reflectance images of HT-29 tumors and selected organs. (C) Noninvasive in vivo FMT tomographic images of 4c and 3b in HT-29 tumor bearing mice 24 h post injection and
(D) FMT in vivo quantitation in pmol indicating 10% injected dose (400 pmol of 4 nmol injected) of 3b accumulating in tumors relative <1% (35 pmol) for the control 4c.
fluorochrome with 1c, 2c, and 3c, had no inhibition potency to-
ward any of the hCAs tested and thus provides an excellent control
for background cell and tissue uptake properties of this class of
fluorescent compounds.
Compounds 1d, 2a–b, 3b and control compound 4c were se-
lected for in vitro uptake in hypoxic and normoxic HeLa cells. HeLa
cells were chosen for in vitro screening of the NIR fluorescent
inhibitors due to their very robust upregulation of hCA IX under
hypoxic culture conditions and low background expression under
normoxic conditions.46
was nearly undetectable in both normoxic and hypoxic cells (pan-
els ii and viii), reflecting the expected membrane impermeability
of this highly charged molecule. In contrast, compound 2a (net
charge À3) showed a relatively high background signal in both
hypoxic and normoxic cells, saturating the microscope under the
exposure settings even in the normoxic cells (panels iv and x).
Compounds 2b (panels v and xi), 3b (panels vi and xii), each with
a net charge of À5, showed comparable or better fluorescence sig-
nal in hypoxic cells to that of the fluorescent CA IX antibody, with
very low background in the normoxic cells similar to the control
compound. 1d (panels iii and ix), which bears a net charge of À3,
also had low background and exhibited a modest preference for
hypoxic cells, but the overall brightness was less than 2b and 3b.
The dissociation constants (Kd) of compounds 2b, 2c, and 3b to
live, hypoxic HeLa cells were also quantified by flow cytometry
HeLa cells were seeded in 8-well slide chambers and cultured
under normoxia (air with 5% CO2) for 24 h before hypoxic cul-
ture.47 The five hCA IX targeted compounds 1d, 2a–b and 3b and
the control compound 4c (1 lM final concentration in a well) were
added to pre-designated well chambers during the last hour of cul-
ture. Anti-hCA IX antibody48 was used as positive control for the
assays. Slide chambers were then taken out from cultures and cell
wells were rinsed with cold PBS after discarding the culture media.
Well chambers were separated and the slides were dried at room
temperature for 4–5 min. The cell nuclear staining marker (Dapi,
Invitrogen) was dropped on the slides that were then covered with
microscope slide cover glass. The slides were examined under fluo-
rescence microscopy and images were acquired using an equal
exposure time of 4000 ms for comparisons.49 The fluorescence
microscope images are shown in Figure 1A.
over a range of agent concentrations from 10 nM to 1 lM, using
the normoxic background as a measure of nonspecific uptake.50
The Kd values thus obtained51 are shown in Figure 1B and Table 1.
For compounds 2b and 2c, the Kd values of 52 nM and 49 nM,
respectively, to live, hypoxic cells were about sixfold lower than
the Ki values obtained for inhibition of the purified active site of
the enzyme. The 8.3 nM Kd measured for compound 3b, however,
was essentially equal to the Ki of 7.5 nM toward purified hCA IX.
Due to its strong binding to hypoxic HeLa cells, compound 3b
was selected for a pilot in vivo experiment.
As shown in Figure 1A, fluorescence from the hCA IX antibody
staining was significant for hypoxic cells, but low for normoxic
cells (panels i and vii). The control compound 4c (net charge À5)
To demonstrate the in vivo efficacy of the new fluorescent
inhibitors, lead compound 3b and control compound 4c (4 nmol
each) were injected i.v. into HT-29 tumor bearing mice followed

K. Groves et al. / Bioorg. Med. Chem. Lett. 22 (2012) 653–657
657
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41. Nahrendorf, M.; Waterman, P.; Thurber, G.; Groves, K.; Rajopadhye, M.; Panizzi,
P.; Marinelli, B.; Aikawa, E.; Pittet, M. J.; Swirski, F. K.; Weissleder, R.
Arterioscler. Thromb. Vasc. Biol. 2009, 29, 1444.
Disclosure statement: KG, BB, JZ, EH, PK, GC, WY, JDP, and MR are
employed by PerkinElmer.
References and notes
42. HPLC purification used
a linear gradient of 10–35% acetonitrile in
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triethylammonium bicarbonate buffer (25 mM, pH 8.5, 5% acetonitrile) and a
21 Â 250 mm C18 column with a flow rate of 20 mL/min.
43. LCMS analyses determined on a Waters Micromass ZQ: Compound 1a: calcd
1206.2 (M+H+), m/z = 1206.3; compound 1b: calcd 1308.1 (M+H+), m/
z = 1308.2; compound 1c: calcd 1421.2 (M+H+), m/z = 1421.1; compound 1d:
calcd 1120.2 (M+H+), m/z = 1120.2; compound 2a: calcd 1333.2 (M+H+), m/
z = 1333.1; compound 2b: calcd 1435.1 (M+H+), m/z = 1435.1; compound 2c:
calcd 1548.2 (M+H+), m/z = 1548.3; compound 2d: calcd 1247.2 (M+H+), m/
z = 1247.3; compound 3b: calcd 1373.1 (M+H+), m/z = 1373.2; compound 4c:
calcd 1342.2 (M+H+), m/z = 1342.1.
44. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
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46. As determined by flow cytometry using anti-CA IX antibody (rabbit polyclonal
Anti-CA IX antibody, Santa Cruz Biotechnology) that was detected by Alexa
Fluor 488 goat anti Rabbit IgG (Invitrogen).
47. To induce hypoxia, slide chambers were placed into a Modular Incubator
Chamber (MIC-101, Billups-Rothenberg, Inc. CA) that was flushed with mixed-
low oxygen gas (1.0% O2, 5.0% CO2, and 94% N2). The modular chamber was
then placed into an incubator and cultured for 24 h along with normoxic
culture slide that was placed directly into incubator.
48. Phycoerythrin-conjugated Anti-Human Carbonic Anhydrase IX (hCA IX)
monoclonal antibody (FAB2188P, R&D Systems.
49. Except compound 3b, 6000 ms.
50. For flow cytometry, cells were harvested, centrifuged at 1000 rpm for 10 min,
resuspended in 0.5 mL PBS and fluorescence quantified using a BD LSR2 flow
cytometer (BD Biosciences, Rockville, MD) equipped with a solid-state 660 nm
(60 mW) red laser and 712/21 nm bandpass filter.
51. Kd values of test agents were calculated using GraphPad Prism Curve Fitting
Software; binding values to normoxic cells were considered as non-specific
binding and binding values to hypoxic cells were used as total binding.