Targeting carbonic anhydrases with fluorescent BODIPY-labelled sulfonamides

Article abstract of DOI:10.1002/ejic.201101371

In the pursuit of dual-mode imaging agents of the tumour hypoxia markers carbonic anhydrases (CA) IX and XII, sulfonamides 4-(2-aminoethyl) benzenesulfonamide and 1,3,4-thiadiazole were functionalised with a 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacene (BODIPY) dye to afford fluorescent aromatic sulfonamide conjugates as potential agents for simultaneous positron emission tomography and fluorescence imaging. Both compounds exhibited nanomolar potency as inhibitors of four CA isoforms: the cytosolic CA I and II, and the transmembrane CA IX and XII. Cell uptake experiments with a CA IX positive and null cell line showed no uptake with the 1,3,4-thiadiazole species in either cell line, whilst the benzenesulfonamide species showed uniform internalisation in both cell lines that limited these compounds as selective imaging agents. Copyright

Full text of DOI:10.1002/ejic.201101371

Job/Unit: A11371
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DOI: 10.1002/ejic.201101371
Targeting Carbonic Anhydrases with Fluorescent BODIPY-Labelled
Sulfonamides
Philip A. Waghorn,*[a] Michael W. Jones,[a] Alan McIntyre,[b] Alessio Innocenti,[c]
Daniela Vullo,[c] Adrian L. Harris,[b] Claudiu T. Supuran,[c] and Jonathan R. Dilworth[a]
Keywords: Carbonic anhydrases / Sulfonamides / Fluorescence / BODIPY / Imaging agents
In the pursuit of dual-mode imaging agents of the tumour
hypoxia markers carbonic anhydrases (CA) IX and XII, sulf-
onamides 4-(2-aminoethyl)benzenesulfonamide and 1,3,4-
thiadiazole were functionalised with a 4,4-difluoro-4-borata-
3a-azonia-4a-aza-s-indacene (BODIPY) dye to afford fluores-
cent aromatic sulfonamide conjugates as potential agents for
simultaneous positron emission tomography and fluores-
cence imaging. Both compounds exhibited nanomolar po-
tency as inhibitors of four CA isoforms: the cytosolic CA I and
II, and the transmembrane CA IX and XII. Cell uptake ex-
periments with a CA IX positive and null cell line showed no
uptake with the 1,3,4-thiadiazole species in either cell line,
whilst the benzenesulfonamide species showed uniform in-
ternalisation in both cell lines that limited these compounds
as selective imaging agents.
cence, which offers much higher spatial and temporal reso-
lution, thereby allowing a comprehensive picture of the bio-
distribution of the imaging agent to be produced.[16,17] Such
dual-mode imaging capabilities are commonly achieved
through the combination of the two imaging agents sepa-
rately,[18–20] whilst in principle a single agent that provides
both fluorescent and radioimaging potential would allow
for simpler syntheses and reduced modification of the tar-
geting vector.
Introduction
Whilst fluorine-18 remains the radionuclide of choice in
positron emission tomography (PET),[1–3] new expedient
methods for the 18F labelling of small molecules, peptides
and antibodies that avoid challenging multistep carbon–flu-
orine bond formation remain to be found. To this end, al-
ternative strategies that employ the facile formation of
strong fluorine–metal bonds have been considered, with
particular focus on the 18F labelling of boron, silicon and
aluminium.[1,4–9] Fluorine bonds to boron are some of the
Recently, interest has focussed on the B–F-contain-
ing
4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacene
thermodynamically
most
stable
covalent
bonds
(BODIPY) dyes, which not only provide high photochemi-
cal stability, high absorption coefficients and large fluores-
cence quantum yields,[21,22] but also a potential radio-fluor-
ine labelling site. Initially work with radiolabelling was
achieved through an 18F-labelled boronic ester coupled to
a BODIPY derivative, thus offering the potential for com-
bined PET and fluorescence imaging with the same mole-
cule.[23] Recently, however, Gabbai et al.[24] have made pro-
gress towards the direct labelling of neutral and cationic
BODIPY species with fluoride, thereby achieving 18F label-
ling in water in a 61% radiochemical yield with a specific
activity of Ͼ1.4 Ciμmol–1, which has enabled the first in
vivo and ex vivo dual-mode images of an 18F-labelled
BODIPY species.[25] Work by this group is currently un-
derway to improve on the specific activity of labelling
achieved with these agents.
known,[10,11] which has driven the development of radio-
pharmaceuticals that contain B–F bonds, such as the 18F-
labelled tetrafluoroborate for thyroid imaging.[12–14] Several
examples of functionalised 18F-labelled aryl boronic esters
have been established as radiolabelling strategies for label-
ling biomolecules or bifunctional linkers.[15] These include
the labelling of folic acid for folate receptor imaging,[13]
maleimide derivatives for conjugation to cysteine-contain-
ing peptides for imaging integrins[1] and azide precursors
for “click” chemistry assemblies.[1]
A limitation of PET imaging, however, can be its low
spatial resolution (1–2 mm). This can be resolved by a com-
bination with a second imaging technique such as fluores-
[a] Chemistry Research Laboratory, University of Oxford,
12 Mansfield Road, Oxford, OX1 3TA, UK
Fax: +44-1865-285002
One target of interest in oncology is the tumour-associ-
ated transmembrane carbonic anhydrases (CA).[26] CAs are
zinc metalloenzymes that catalyse the interconversion be-
E-mail: philip.waghorn@oncology.ox.ac.uk
[b] Cancer Research UK Growth Factor Group, Weatherall Insti-
tute of Molecular Medicine,
–
John Radcliffe Hospital, Oxford, OX3 9DS, UK
[c] Universita degli Studi di Firenze,
tween CO2 and HCO3 , thereby maintaining a pH balance
in blood plasma and transporting carbon dioxide out of
tissue. Sixteen isoforms of CA have been identified to
Laboratorio di Chimica Bioinorganica, Room 188,
Via della Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italy
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FULL PAPER
date;[27] they are located in the cytosol, mitochondria and
Primary aromatic and heterocyclic sulfonamides are po-
cellular membranes, or present as a secreted form (CA VI). tent inhibitors of CAs. Despite a nonspecific action against
Particular interest surrounds the transmembrane CA IX different CA isozymes, structural modifications can be in-
isozyme, which has been found to be overexpressed in cer- troduced to induce selectivity toward specific iso-
tain cancer lines including renal,[28,29] cervical squamous,[30] forms.[38,42–44] For instance, fluorescent imaging of CA IX
breast,[31,32] colorectal,[33,34] bladder[35] and non-small-cell by the incorporation of a fluorescein group {[(4-sulfamoyl-
lung carcinoma.[36] A strong association between CA IX ex- phenylethyl)thioureido]fluorescein (FTES); Figure 1a} has
pression and intratumoural hypoxia has also been demon- been achieved, with enzyme-modelling studies suggesting
strated[37] as a result of strong transcriptional activation of that the combination of increased bulk and hydrogen-bond-
the CA IX gene by HIF-1α.[28] Studies on non-small-cell ing interactions between a guanidine residue specific to
lung carcinomas have shown CA IX association with pro- CA IX and the carbonyl oxygen atom of the fluorescein tail
teins involved in angiogenesis, apoptosis inhibition and of the inhibitor accounts for the much increased affinity of
cell–cell adhesion disruption, which suggests a strong rela- FTES for CA IX over the ubiquitous CA II enzyme.[45]
tionship of this enzyme with a poor clinical outcome.[38]
Recently, Sando et al. have investigated 8-(4-aminosulf-
Similar interest surrounds CA XII, a second transmem- onylphenyl)-BODIPY (Figure 1b) as a selective inhibitor
brane tumour-associated CA isozyme with an extracellular dye conjugate for “turn-on” fluorescence sensing of CAs.[46]
active site that has been identified in renal cell carci- In the presence of CA, a twofold enhancement in fluores-
noma.[39] As a result, interest lies in the development of CA cence intensity was observed as a result of the increased
inhibitors as an approach to the treatment of cancers in steric constraints imposed by the active site of the enzyme,
which CA IX/XII is overexpressed. Recent work has shown which acts to reduce the nonradiative mechanisms of fluo-
that the inhibition of CA IX has a strong anticancer effect rescent decay. Similarly, Gay et al.[47] have previously inves-
with growth delay of both primary tumours and metasta- tigated an acetazolamide-functionalised BODIPY 556/558
ses.[40,41] As such, the inhibition of these tumour-associated dye (Figure 1c) for CA II binding of osteoclasts. Inhibition
CA isozymes could represent a novel therapeutic approach of bone resorption through reduced H+ release upon sulf-
for the management of hypoxic tumours that overexpress onamide binding was confirmed by reduced fluorescence
these proteins.
emission under competition studies with unlabelled sulfon-
Figure 1. Structures of (a) [(4-sulfamoylphenylethyl)thioureido]fluorescein (FTES); (b) 8-(4-aminosulfonylphenyl)-BODIPY; (c) BODIPY
558/568 acetazolamide; (d) [18F]5-(p-fluorobenzenesulfonamido)-1,3,4-thiadiazole-2-sulfonamide; (e) acetazolamide (AZA); and (f) 4-(2-
aminoethyl)benzenesulfonamide (ABS).
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Carbonic Anhydrases with Fluorescent BODIPY-Labelled Sulfonamides
amide inhibitors. However, no data on the specific CA IX/
Results and Discussion
XII inhibition was reported in either of these studies.
There are relatively few reports that involve the labelling
of sulfonamides with radioisotopes for nuclear imaging.
Synthesis and Chemical Properties
BODIPY conjugates of two well-known CA IX and XII
[18F]5-(p-fluorobenzenesulfonamido)-1,3,4-thiadiazole-2- inhibitors that incorporate the sulfonamide motif were in-
sulfonamide (Figure 1d) has been prepared by an aromatic vestigated.
nucleophilic substitution reaction of the nitro group precur-
ABS (Figure 1f) is an efficient and specific CA IX and
sor with radioactive fluoride and was shown to act as a XII inhibitor but is less selective toward the widely distrib-
nanomolar inhibitor of CA II and CA IV,[48] and Le Bars uted cytoplasmic CA II and the plasma-membrane-an-
et al.[49] have prepared 11C-labelled acetazolamide (AZA) chored CA IV.[38,44] AZA is itself a nonspecific inhibitor of
(Figure 1e) to study lung CA inhibition.
several CAs but has shown selectivity toward CA IX and
Chelators such as ethylenediaminetetraacetic acid CA XII with respect to CA II when structurally modified
(EDTA) or diethylenetriaminepentaacetic acid (DTPA) with fluorescein,[53] DTPA[50] and boroxazolidones.[54]
have been conjugated to sulfonamides with retention of
The target compounds were prepared by the (benzotri-
CA IX inhibition, thus enabling the binding of metal radio- azol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro-
isotopes such as copper-64,[50–52] although radiolabelling phosphate (BOP) mediated peptide coupling of ABS or
studies have not yet been carried out.
AZA-NH2 (1) to BODIPY–CO2H (2) to afford the ABS–
Given the present interest in 18F-labelling of B–F bonds BODIPY (3) and AZA–BODIPY (4) conjugates in 81 and
and the recent progress towards the direct 18F labelling of 73% yield, respectively (Scheme 1b). BODIPY–CO2H (2)
BODIPY adducts in high specific activity, we describe here was prepared according to a literature procedure,[55]
the synthesis and preliminary CA inhibition and fluores- whereas 1 was synthesised from AZA according to an
cence studies of arylsulfonamides functionalised with a adapted literature procedure, as shown in Scheme 1a.[56]
BODIPY dye as analogues of the FTES dye. The identifica-
Fluorescence quantum yields were measured for 3 and
tion of agents as specific CA IX/XII inhibitors will allow 4 in both methanol and a water/methanol (95:5) solution
further modification of the BODIPY boron core, thus al- (Table 1). Addition of the sulfonamide groups caused some
lowing individual 18F radiolabelling strategies to be devel- quenching of fluorescence with respect to the parent com-
oped and optimised in parallel with current established pound 2, though the quantum yields remained high. Com-
methods.[25] This paper describes our preliminary steps parable quantum yields were obtained for measurements
towards dual-mode imaging agents for targeted fluores- made in both H2O and MeOH, with no quenching in H2O,
cence and radioimaging of selective transmembrane CA ex- thereby suggesting their suitability for in vitro fluorescence
pression.
imaging.
Scheme 1. (a) Synthesis of 1. (b) Synthesis of 3 and 4.
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Table 1. Fluorescent quantum yield data for 2, 3 and 4.
BODIPY
λabs [nm]
λem [nm]
ε [105 mol–1 dm3 cm–1]
Φ (MeOH)[a,b]
Φ (H2O)[a,b]
2
3
4
500
499
499
509
511
511
0.98
0.83
0.87
0.46
0.39
0.39
0.44
0.36
0.35
[a] λexc = 496 nm. [b] Referenced against fluorescein in 0.1 m NaOH, Φ = 0.95,[57] λexc = 496 nm. All Φ values are corrected for changes
in refractive index.
X-ray Crystallography
Table 2. Selected bond lengths [Å] and angles [°] for the X-ray
structures of 3 and 4.
Red crystals of 3 and 4 were grown by slow infusion of
diethyl ether or hexane into solutions of the respective com-
pounds in THF (Figure 2), with the structures broadly sim-
ilar to previously structurally characterised BODIPY deriv-
atives.[58,59]
3
4
3
4
B1–F1
B1–F2
1.399(4) 1.387(2)
1.398(5) 1.406(3)
B1–N1 1.549(6) 1.534(3)
B1–N2 1.527(6) 1.543(3)
N1–C89 1.403(5) 1.402(2)
N2–C79 1.400(5) 1.401(2)
S1–O3
S1–O4
S1–N7
S1–O2
S1–O3
S1–N4
–
–
–
1.442(3)
1.423(3)
1.600(3)
1.442(3)
1.423(3)
1.600(3)
–
–
–
C8–C89 1.391(5) 1.397(3) N1–B1–N2 107.3(3) 107.3(3)
C8–C79 1.407(5) 1.404(2) F1–B1–F2 108.0(3) 108.0(3)
teractions that exist between adjacent sulfonamide groups
with bonding distances of 2.137 Å for N4–H42···O3* (Fig-
ure 3a).
Adjacent pairs are aligned in a head-to-tail formation by
hydrogen-bonding interactions between the adjacent sulfon-
amide amine and amide carbonyl groups O1···H41–N4*
(1.978 Å) (Figure 3a). These infinite chains are further held
together through three short-range B–F···H–C bonding in-
teractions (2.303–2.573 Å) (Figure 3b). Further hydrogen
bonding is present between the amide nitrogen atom and
solvent molecule (diethyl ether) oxygen atom (2.505 Å)
(Figure 3a).
Compound 4 was co-crystallised with two THF mole-
cules, and hydrogen bonding exists between the solvent
THF oxygen atoms with the sulfonamide nitrogen atoms
(1.935 Å) and the carboxyphenyl amide nitrogen atoms
(2.044 Å). A third severely disordered THF molecule that
was not hydrogen-bonded was unable to give a sensible
model and hence removed by SQUEEZE.[61–63] Hydrogen-
bonding interactions are also present between the thiadi-
azole nitrogen atom and the amide nitrogen atom on adja-
cent molecules (2.008 Å) (Figure 3c). In 4 short-range inter-
actions were observed between the B–F unit and the sulfon-
amide amine groups (2.022 Å) and between the B–F unit
and the aromatic ring (2.573 Å) (Figure 3d).
Figure 2. ORTEP representation of the structures of 3 (top) and 4
(bottom) with thermal ellipsoids at 30% probability. Minor disor-
dered components and solvent molecules have been omitted for
clarity.
The central six-membered ring in both structures lies co-
planar with the adjacent five-membered rings to ensure a
planar BODIPY framework with π-electron delocalisation
over the indacene core, with the BF2 unit perpendicular to
this core. The dihedral angle between the phenyl group at
the meso position and the BODIPY indacene skeleton is
85.88° for 3 and 82.88° for 4, and it is indicative of the
restriction that the methyl groups of the dipyrromethene
frame impart on the free rotation of the phenyl substituents
at the meso position. These interactions are integral to the
reduced loss of energy from electronically excited states
through non-irradiative molecular relaxation and hence the
increased fluorescence quantum yield of this class of com-
pounds.[60] Selected bond lengths and angles for 3 and 4 are
given in Table 2.
In Vitro Studies of the BODIPY–Sulfonamide Conjugates
Preliminary in vitro uptake experiments were performed
on a HeLa cell line to ascertain whether or not fluorescence
A number of C–H···F short-range interactions and S/ emission could be observed. Complex 3 showed strong uni-
C=O···H intermolecular hydrogen-bonding interactions are form distribution in the cytoplasm of the HeLa cell line
present in the packing of each structure. In the solid state after only 30 min of incubation, with no nuclear staining
of 3, the molecules are aligned in pairs in a tail-to-tail ar- observed. No difference in uptake pattern was observed be-
rangement as a result of short-range hydrogen-bonding in- tween experiments conducted at either 4 or 37 °C, thereby
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Carbonic Anhydrases with Fluorescent BODIPY-Labelled Sulfonamides
To establish the effect and selectivity on cell uptake of
the two conjugates in the presence of the CA IX enzyme,
cell-uptake experiments were conducted on an HCT116 co-
lon carcinoma cell line transfected to overexpresses the
CA IX enzyme [HCT116 (CA9+)]. Control uptake experi-
ments were performed on the HCT116 null CA IX empty
vector cell line [HCT116 (EV)], with fluorescence-activated
cell sorting (FACS) analysis performed on a fluorescently
labelled CA IX antibody in parallel to confirm the presence
or lack of CA IX expression in the two cell lines.
Cell uptake experiments were repeated with 3 and 4 over
a 24 h incubation period and imaged by epifluorescence mi-
croscopy with the fluorescein–sulfonamide conjugate FTES
run in parallel as a positive control. After 24 h, 4 showed
no uptake either in the membrane or internalised in either
cell line, whereas 3 showed uptake in both the CA IX posi-
tive and empty-vector cell lines (Figure 4). By comparison,
the FTES standard stained only the positive control with
no fluorescein fluorescence emission associated with the
empty-vector cell line.
Figure 4. Epifluorescence uptake studies of FTES, 3 and 4 in
HCT116 (CA9+) and HCT116 (EV) cell lines co-stained with 4Ј,6-
diamidino-2-phenylindole (DAPI; blue stain). FACS analysis using
a fluorescein-isothiocyanite labelled CA IX antibody were con-
ducted concomitantly as a second positive control of CA IX ex-
pression. Each field of view is 100 by 80 μm.
Whereas fluorescein is impermeable to the cell membrane
and hence promotes the selective transmembrane CA IX
binding, the BODIPY dye is internalised rapidly into
cells[64] by a process of diffusion, which, in the case of com-
Figure 3. Packing diagrams depicting symmetry elements of
3·Et2O, showing (a) hydrogen-bonding interactions and (b) short- pound 3, prevents significant interactions with the mem-
range interactions with the B–F unit (Et2O omitted for clarity).
brane-bound CA IX and hence an observed lack of specific-
Packing diagrams depicting symmetry elements of 4·2THF, show-
ity with uptake in both the CA IX positive and control
ing (c) hydrogen-bonding interactions and (d) short-range interac-
empty-vector HCT116 cell lines. Conversely to this and to
tions with The B–F unit.
the work of Gay et al.,[47] despite the strong cell uptake of
both the BODIPY dye alone and AZA itself (as shown by
3
suggesting that the mode of internalisation is by passive dif- Gay et al. through H-labelling studies[65,66]) their conju-
fusion. Conversely, compound 4 showed no uptake in the gate, 4, showed no cellular uptake, which suggests that the
HeLa cell line after 30 min of incubation.
thiadiazole group once conjugated makes this BODIPY de-
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Table 3. Inhibition of CA isoforms human CA (hCA) I, II, IX and XII with sulfonamides 3 and 4 compared to the starting materials
ABS and AZA and FTES. Errors in the range of Ϯ5–10% of the reported value, from three different determinations.
Inhibitor Ki* [nm]
Selectivity ratios
hCA I[a] hCA II[a] hCA IX[b] hCA XII[c] hCA IX/hCA I hCA IX/hCA II hCA XII/hCA I hCA XII/hCA II
3
4
301
90
31
67
160
12
45
63
70
33
25
24
78
89
3.2
5.7
5.0
4.8
1.3
636.4
10.0
54.2
0.5
1.0
4.9
0.5
1.9
3.9
1.0
6563
43.9
260
0.4
0.8
50
2.1
9.0
ABS[38,44] 21000
AZA[42]
250
1300
FTES[42]
[a] Human (cloned) isozymes. [b] Catalytic domain of the human, cloned isozyme. [c] Catalytic domain of the human recombinant
isozyme, CO2 hydrase assay method.
rivative cell impermeable. Whereas cell impermeability in
the empty-vector cell line is preferable to ensure enhanced
contrast, unlike the FTES species, no CA IX binding was
observed. Estimates of the partition coefficient (logP) val-
ues of 3 and 4 were made to attempt to ascertain an expla-
nation for the lack of cell permeability of compound 4.
Comparable results were obtained for both 3 and 4 with
Conclusion
Despite the favourable fluorescence properties and recent
routes towards 18F radiolabelling of BODIPY dyes, both
BODIPY–sulfonamide conjugates lack selectivity for the
transmembrane CA IX and CA XII isozymes as measured
by the stopped-flow binding-affinity assay. The comparable
size of the BODIPY dyes with the fluorescein species
logP values of 1.57Ϯ0.15 and 1.43Ϯ0.11, respectively.
should parallel CA IX selectivity over CA II binding, but –
These results are in keeping with the HPLC retention times
whereas the fluorescein adduct provides secondary binding
interaction with the CA IX enzyme active site – the BOD-
and suggest that both species are lipophillic with complex
4 showing an increased hydrophilicity, though this is not
IPY indacene core does not provide similar hydrogen-bond-
sufficient to explain the observed differences in cell perme-
ing interactions and therefore lacks enzyme selectivity. In
ability of these two complexes, which remains uncertain.
the case of 3, uptake is dominated by the BODIPY dye
To attempt to understand the lack of CA IX binding by 4
entity, and – whereas with 4 cellular uptake in the control
samples was minimised – the weak CA IX binding affinity
resulted in similarly negligible CA IX interaction in the pos-
itive cell line.
under in vitro studies, the binding affinities of the BODIPY
sulfonamides 3 and 4 compared with FTES were carried
out with the CA isoforms in isolation. Inhibition data
against four CA isozymes (CA I, CA II, CA IX and
Future modifications to make the BODIPY dye charged
CA XII) was measured, and the results are given in Table 3
and cell-impermeable and the use of a greater library of
together with the data for the parent sulfonamides AZA
sulfonamides may yet yield selective contrast agents for
and ABS. The data from the enzyme inhibition suggests
dual-mode PET and fluorescence imaging based on the
BODIPY motif.
that the binding affinities of the BODIPY derivatives to the
transmembrane CA IX and XII isozymes are significantly
weaker than those of FTES and the AZA and ABS groups
alone, and the selectivity for CA IX and CA XII with re-
spect to the ubiquitous CA enzymes CA I and CA II is
Experimental Section
General: All reactions were carried out in oven-dried glassware un-
der nitrogen. Nitrogen gas was dried by passage through silica gel.
All solvents were dried according to standard procedures. All rea-
gents were purchased from Aldrich and used without further purifi-
cation. Column chromatography was performed on silica gel 60m
poor by comparison.
Compound 4 binds to all four CA enzymes investigated
with uniform affinity with a threefold and eighteenfold
weaker binding affinity toward the CA IX enzyme and
CA XII enzyme, respectively, relative to that of FTES.
Compound 3 shows a weak binding affinity for CA I with
a selectivity ratio of 4.8 for CA IX/CA I and 3.9 for
CA XII/CA I, although its binding affinity for the trans-
membrane CAs is less than half that of the affinity for the
1
(mesh 230–400) from Macherey–Nagel. H NMR spectra were re-
corded with a Varian Mercury VX300 (300 MHz) spectrometer at
298 K and referenced to residual nondeuterated solvent peaks.
Chemical shifts are quoted in ppm. Coupling constants (J) are
measured to the nearest 0.1 Hz and are presented as observed. 13
C
ubiquitous CA II isoform. The comparable sizes of the NMR spectra were recorded with a Varian Mercury VX300
(75.5 MHz) spectrometer at 298 K and were referenced to the sol-
vent peak.[67] 19F NMR spectra were recorded with a Varian Mer-
cury VX300 (282 MHz) at 298 K and were referenced to a fluoro-
benzene spike (δ = –113.15 ppm). NMR spectra were processed by
using MestReC software. Mass spectrometry was performed with
a Bruker Micromass LCT time-of-flight mass spectrometer under
electrospray ionisation (ESI-MS) conditions. Accurate masses are
reported to four decimal places by using tetraoctylammonium
bromide (466.5352 Da) as an internal reference. Values are reported
as a ratio of mass to charge in Daltons. HPLC characterisation
(analytical HPLC) of compounds was performed with a Waters C-
BODIPY dyes with the fluorescein compound would sug-
gest favourable CA IX selectivity for the BODIPY conju-
gates as a result of the increased size of the CA IX cavity
relative to the ubiquitous CA II isozyme.[43] However, the
lack of secondary hydrogen-binding interactions between
these BODIPY-functionalised sulfonamides and the CA IX
enzyme as observed for the FTES derivative hinders selec-
tive inibition, thus leading to weaker binding affinity and
poor CA selectivity and the apparent lack of fluorescence
emission for 4 in the HCT116 (CA9+) cell line.
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Carbonic Anhydrases with Fluorescent BODIPY-Labelled Sulfonamides
18 column (4.6ϫ250 mm) and UV/Vis detection at λobs = 254 and
410 nm with a 1.0 mLmin–1 gradient elution method (solvent A:
acetonitrile with 0.1% TFA, v/v; solvent B: water with 0.1% TFA,
4,4-Difluoro-8-(4Ј-carboxyphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-
diaza-s-indacene (2): Complex 2 was synthesised according to a lit-
erature procedure.[55] Pyrrole (1.35 mL, 19.4 mmol) and 4-form-
v/v): start with 5% of A, gradient over 23 min to reach 95% of A, ylbenzoic acid (1.30 g, 8.63 mmol) were dissolved in CH2Cl2
hold at 25 min at 95% of A, reverse gradient for 27 min to reach
5% A, then hold for 30 min at 5% A. Elemental analyses were
performed either by the microanalysis service in the Inorganic
Chemistry Department at the University of Oxford or by Mr. S.
Boyer at the London Metropolitan University for electronic ab-
sorption spectroscopy. UV/Vis spectroscopy was performed with a
Perkin–Elmer Lambda 19 spectrometer that was running UV Win-
lab software. Spectra were measured by using 1.00 cm quartz cu-
vettes. Fluorescence spectra were recorded in 1.00 cm quartz cu-
vettes with a Hitachi F-4500 fluorescence spectrometer that was
running FL Solutions software. Relative quantum yields were de-
termined by comparison to fluorescein in 0.1 m NaOH (ΦR = 0.95
at 496 nm) by using the formula: ΦS = ΦR·(DS/DR)·(AR/AS)·(IR/IS)·
(ηS/ηR)2 in which the variable Φ is the relative quantum yield, D
is the integrated area of the fluorescence emission peak, A is the
absorption of the solutions at the excitation wavelength, I is the
flux at the excitation wavelength used, and η is the solution refrac-
tive index. R and S subscripts refer to the reference and sample,
respectively.
(1.00 L) under nitrogen. Two drops of TFA were added, and the
solution was stirred at room temp. for 1 h. 2,3-Dichloro-5,6-dicy-
ano-1,4-benzoquinone (DDQ; 1.96 g, 8.63 mmol) was added to
CH2Cl2 (20.0 mL), and the solution was stirred for 1 h, after which
NEt3 (20.0 mL) and BF3·OEt2 (20.0 mL) were added dropwise. The
reaction mixture was stirred at room temp. for 2 h before being
quenched by the addition of water (500 mL) and extracted with
CH2Cl2 (3ϫ200 mL). The combined organic extracts were dried
with anhydrous MgSO4, filtered and concentrated under reduced
pressure. The crude oil was purified by silica gel chromatography
(0–8% MeOH in CH2Cl2) to yield a red solid after recrystallisation
from THF/hexanes (701 mg, 1.90 mmol, 22%). 1H NMR
(300 MHz, [D6]DMSO, 25 °C): δ = 13.25 (br. s, 1 H, COOH), 8.10
(d, J = 7.9 Hz, 2 H, ArH), 7.53 (d, J = 8.2 Hz, 2 H, ArH), 6.20 [s,
2 H, NC(CH3)CHC(CH3)], 2.46 [s, 2ϫ 3 H, NC(CH3)CHC(CH3)],
1.33 [s, 2ϫ 3 H, NC(CH3)CHC(CH3)] ppm. 19F NMR (282 MHz,
[D6]DMSO, 25 °C): δ = –143.7 [q, J(B,F1) = 34 Hz, 2 F, BF] ppm.
13C NMR (75.5 MHz, [D6]DMSO, 25 °C): δ = 167.3, 155.7, 150.2,
143.1, 138.8, 135.5, 131.9, 130.6, 128.8, 122.0, 14.7, 14.5 ppm. ESI-
MS: calcd. for C20H18BF2N2O2 [M
367.1442. HPLC: tR = 14.72 min.
–
H]– 367.1435; found
2-Oxo-2-(5-sulfamoyl-1,3,4-thiadiazol-2-ylamino)ethanaminium Tri-
fluoroacetate (1)
General Amide Coupling Procedure: N,N-Diisopropylethylamine
(DIPEA; 1.50 equiv.) was added to a stirred solution of BODIPY–
CO2H (2) (1.00 equiv.) in DMF (5.00 mL) and the solution was
cooled to 0 °C in an ice bath. BOP (1.50 equiv.) was added, and
the solution was stirred at 0 °C for 30 min. Amine (1.20 equiv.) was
added, and the solution was warmed to room temperature and
stirred for 12 h. The DMF was removed under reduced pressure.
The crude product was dissolved in CHCl3 (25.0 mL) and washed
sequentially with 1 m HCl (20.0 mL), saturated NaHCO3 (aq.)
(100 mL), water (100 mL), brine (100 mL) and was dried with an-
hydrous MgSO4. The CHCl3 was removed under reduced pressure,
and the crude residue was purified by silica gel chromatography.
(a) 5-Amino-1,3,4-thiadiazole-2-sulfonamide:[56] A mixture of acet-
azolamide (1.00 g, 4.50 mmol), concentrated HCl (2.00 mL) and
ethanol (15.0 mL) was heated under reflux conditions for 3 h. The
solvent was evaporated in vacuo to near dryness, and the remaining
suspension was allowed to cool slowly to room temp. The solid was
collected by filtration and dried in vacuo (528 mg, 2.93 mmol,
1
65%). H NMR (300 MHz, [D6]DMSO, 25 °C): δ = 8.05 (s, 2 H,
SO2NH2), 7.79 (s, 2 H, thiadiazole-NH2) ppm. ESI-MS: calcd. for
C2H4N4O2S2 [M – H]– 178.9776; found 178.9758.
(b) tert-Butyl [2-Oxo-(5-sulfamoyl-1,3,4-thiadiazol-2-ylamino)ethyl]-
carbamate:
tert-Butoxycarbonyl
(Boc)
glycine
(87.5 mg,
0.500 mmol) and NEt3 (70 μL, 0.500 mmol) in MeCN (1.50 mL)
was cooled to –10 °C, and isobutyl chloroformate (65.3 μL,
0.500 mmol) was added with stirring. After 20 min at –10 °C, a
shaken suspension of 5-amino-1,3,4-thiadiazole-2-sulfonamide
(90.0 mg, 0.500 mmol) in MeCN (1.50 mL) that contained NEt3
(70.0 μL, 0.500 mmol) was added. The mixture was stirred at room
temp. overnight and the solvent evaporated. The solid was recrys-
tallised from a water/EtOH mixture to give a crystalline white solid
(102 mg, 0.300 mmol, 61%). 1H NMR (300 MHz, [D6]DMSO,
25 °C): δ = 13.09 (s, 1 H, CONH-thiadiazole), 8.28 (s, 2 H,
SO2NH2), 7.31 (t, J = 6.3 Hz, 1 H, BocNHCH2CONH), 3.87 (d, J
= 6.1 Hz, 2 H, BocNHCH2CONH), 1.32 (s, 9 H, BocNH) ppm.
ESI-MS: calcd. for C9H15N5O5S2 [M – H]– 336.0515; found
336.0524.
4,4-Difluoro-8-[(4-sulfamoylphenethyl)benzamido]-1,3,5,7-tetra-
methyl-4-bora-3a,4a-diaza-s-indacene (3): Compound 3 was synthe-
sised by employing the general amide coupling procedure with BOP
(180 mg, 0.407 mmol), 2 (100 mg, 0.272 mmol), DIPEA (52.6 mg,
70.9 μL, 0.407 mmol) and ABS (65.4 mg, 0.326 mmol). Flash
chromatography of the residue by using an elution gradient of 0–
5 % MeOH in CHCl3 afforded the title product as a red solid
(121 mg, 2.20 mmol, 81 %). 1H NMR (300 MHz, [D6]DMSO,
25 °C): δ = 8.78 (t, J = 5.5 Hz, 1 H, CONH), 7.99 (d, J = 8.2 Hz,
2 H, ArH), 7.75 (d, J = 8.3 Hz, 2 H, ArH), 7.48 (d, J = 8.3 Hz, 2
H, ArH), 7.44 (d, J = 8.4 Hz, 2 H, ArH), 7.30 (s, 2 H, SO2NH2),
6.17 [s, 2 H, NC(CH3)CHC(CH3)], 3.53 (m, 2 H, NHCH2CH2),
2.94 (t, J = 7.3 Hz, 2 H, NHCH2CH2), 2.44 [s, 2ϫ 3 H, NC(CH3)-
CHC(CH3)], 1.32 [s, 2 ϫ 3 H, NC(CH3)CHC(CH3)] ppm. 13C
NMR (75.5 MHz, [D6]DMSO, 25 °C): δ = 166.0, 155.6, 144.2,
143.1, 142.5, 141.5, 137.2, 135.4, 130.9, 129.6, 128.5, 128.5, 126.2,
122.0, 41.0, 35.2, 14.7, 14.6 ppm. 19F NMR (282 MHz, [D6]DMSO,
25 °C): δ = –143.7 [q, J(B,F1) = 34 Hz, 2 F, BF] ppm. ESI-MS:
calcd. for C28H29BF2NaN4O3S [M + Na]+ 573.1919; found
573.1915. HPLC: tR = 13.06 min. C28H29BF2N4O3S (550.20):
calcd. C 61.1, H 5.3, N 10.2; found C 60.8, H 5.2, N 10.0.
(c) 2-Oxo-2-(5-sulfamoyl-1,3,4-thiadiazol-2-ylamino)ethanaminium
Trifluoroacetate: tert-Butyl [2-oxo-(5-sulfamoyl-1,3,4-thiadiazol-2-
ylamino)ethyl]carbamate (122 mg, 0.362 mmol) was added to TFA
(5.00 mL) at room temp. and stirred for 3 h. The TFA was evapo-
rated under reduced pressure and the residue dried in vacuo
(110 mg, 0.313 mmol, 87%). 1H NMR (300 MHz, [D6]DMSO,
25 °C): δ = 13.46 (br. s, 1 H, CONH-thiadiazole), 8.38 (s, 2 H,
SO2NH2), 5.24 (m,
3
H, NH3CH2CONH), 3.96 (m,
2
H, 4,4-Difluoro-8-[2-oxo-2-(5-sulfamoyl-1,3,4-thiadiazol-2-ylamino)eth-
NH3CH2CONH) ppm. 13C NMR (75.5 MHz, [D6]DMSO, 25 °C): yl]benzamido-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (4):
δ = 167.5, 165.7, 164.7, 161.5, 116.5, 41.8 ppm. ESI-MS: calcd. for
Compound 4 was synthesised by employing the general amide cou-
pling procedure with BOP (180 mg, 0.407 mmol), 2 (100 mg,
C4H8N5O3S2 [M – CF3CO2]+ 238.0063; found 238.0081.
Eur. J. Inorg. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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P. A. Waghorn et al.
repeat measurements of identical and Laue equivalent reflections.
FULL PAPER
0.272 mmol), DIPEA (52.6 mg, 70.9 μL, 0.407 mmol) and
1
(115 mg, 0.326 mmol). Flash chromatography of the residue by Structure solution was carried out by using direct methods with
using an elution gradient of 0–7% MeOH in CHCl3 afforded the
title product as a red solid (116 mg, 0.198 mmol, 73%). H NMR
the programs SIR92[70] within the CRYSTALS software suite.[71] In
general, coordinates and anisotropic displacement parameters of
1
(300 MHz, [D6]DMSO, 25 °C): δ = 13.28 (s, 1 H, CONH-thiadi- all non-hydrogen atoms were refined freely except where disorder
azole), 9.23 (t, J = 5.6 Hz, 1 H, CONHCH2), 8.35 (s, 2 H, necessitated the use of “same-distance restraints” together with
SO2NH2), 8.07 (d, J = 8.3 Hz, 2 H, ArH), 7.53 (d, J = 8.3 Hz, 2
H, ArH), 6.19 [s, 2 H, NC(CH3)CHC(CH3)], 4.27 (d, J = 5.5 Hz,
thermal similarity and vibrational restraints to maintain sensible
geometry/displacement parameters. Hydrogen atoms were generally
2 H, CONHCH2), 2.44 [s, 2ϫ 3 H, NC(CH3)CHC(CH3)], 1.34 [s, visible in the difference map and refined with soft restraints prior
2ϫ 3 H, NC(CH3)CHC(CH3)] ppm. 13C NMR (75.5 MHz, [D6]-
DMSO, 25 °C): δ = 170.0, 166.9, 165.3, 161.8, 155.9, 143.3, 141.7,
137.9, 134.8, 131.1, 128.9, 128.4, 122.1, 43.5, 14.9, 14.7 ppm. 19F
NMR (282 MHz, [D6]DMSO, 25 °C): δ = –143. 7 [q, J(B,F) =
34 Hz, 2 F, BF] ppm. ESI-MS: calcd. for C24H24BF2NaN7O4S2 [M
to inclusion in the final refinement by using a riding model.[62] In
the case of compound 4, the difference Fourier map indicated the
presence of diffuse electron density believed to be a molecule of
disordered THF. PLATON/SQUEEZE[61–63] was used and left a
void from which the electron density was removed. CCDC-871337
(3) and -871338 (4) contain the supplementary crystallographic
data for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/data_request/cif. Table 4 offers a summary of the crystal and
refinement data for the X-ray structures.
+
Na]+ 610.1290; found 610.1298. HPLC: tR
= 11.93 min.
C24H24BF2N7O4S2 (587.14): calcd. C 49.1, H 4.1, N 16.7; found C
48.9, H 4.0, N 16.7.
X-ray Structures: Single-crystal X-ray diffraction data were ob-
tained for compounds 3 and 4. In each case, a typical crystal was
mounted by using the oil drop technique in perfluoropolyether oil
at 150(2) K with a Cryostream N2 open-flow cooling device.[68] Dif-
fraction data were collected by using graphite-monochromated
Mo-Kα radiation (λ = 0.71073 Å) with a Nonius Kappa CCD dif-
fractometer. For all data collections, series of ω scans were per-
formed in such a way as to collect a complete data set to a maxi-
mum resolution of 0.77 Å. Data reduction including unit-cell re-
finement and interframe scaling was carried out with DENZO-
SMN/SCALEPACK.[69] Intensity data were processed and cor-
rected for absorption effects by the multiscan method, based on
Fluorescence Microscopy: HeLa cells and HCT116 cells were pro-
vided by Professor Adrian Harris, Weatherall Institute for Molecu-
lar Medicine, University of Oxford. Cells were seeded as mono-
layers in T75 tissue culture flasks, and cultured in Dulbecco’s modi-
fied Eagle medium (DMEM) supplemented with 10% foetal bovine
serum, l-glutamine, penicillin and streptomycin. Cells were main-
tained at 37 °C in a 5% CO2 humidified atmosphere and grown to
approximately 85% confluence before being split by using 2.5%
trypsin. For microscopy, cells were seeded onto chambered coverg-
lass slides and incubated for 12 h to ensure adhesion. BODIPY
Table 4. Crystallographic data for compounds 3 and 4.
3
4
Empirical formula
Mr
Temperature [K]
λ [Å]
Crystal system
Space group
a [Å]
b [Å]
c [Å]
α [°]
β [°]
C28H29BF2N4O3S·0.85C4H10O
C24H24O4BF2N7S2·2THF
731.65
150
613.76
150
0.71073
monoclinic
C2/c
14.9330(3)
25.7615(7)
17.3381(5)
90
105.1383(11)
90
6438.4(3)
8
0.71073
triclinic
¯
P1
9.23680(10)
12.6383(10)
12.3105(10)
71.4912(4)
86.8518(4)
86.8556(3)
1904.21(3)
2
γ [°]
V [Å3]
Z
Dcalcd. [gcm–3]
μ [mm–1]
F(000)
1.27
0.153
2591.0
1.28
0.199
768
Size [mm]
Crystal description
θ range collected [°]
Index ranges
0.14ϫ0.19ϫ0.36
orange block
5.0ՅθՅ27.5
–19ՅhՅ19
–32ՅkՅ33
–22ՅlՅ22
37242
0.20ϫ0.38ϫ0.40
orange block
5.0ՅθՅ27.5
–11ՅhՅ12
–17ՅkՅ18
0ՅlՅ25
49579
Reflections measured
Unique reflections
13161
8645
Rint
0.047
7036
0.89, 0.98
474
0.015
8644
0.88, 0.96
451
Reflections observed [IϾ3σ(I)]
Transmission coefficients (min., max.)
Refined parameters
R or R1 (reflections observed)
wR or R2 (all data)
R = 0.117
wR = 0.253
0.95
R = 0.056
wR = 0.096
1.03
GoF
Residual electron (min., max.) [eÅ3]
–0.67, 0.86
–0.50, 0.65
8
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Carbonic Anhydrases with Fluorescent BODIPY-Labelled Sulfonamides
complexes were prepared as 10 mm solution in DMSO and diluted
to 10 μm with DMEM, and incubated with the cells at 37 °C. After
30 min or 24 h, the BODIPY solution was discarded, and the cells
were washed with fresh DMEM medium before imaging. BODIPY
complexes were imaged by laser-scanning confocal microscopy with
a Zeiss LSM 510 META microscope irradiating at 488 nm, and
emissions were filtered between 505 and 535 nm and with an
Axiovert 135 inverted microscope (Zeiss, USA) visualised by using
Axiovision software (Zeiss, USA) irradiating at 488 nm with emis-
sions filtered above 500 nm.
(MRC) for funding. A. M. and A. L. H. thank the Cancer Research
UK and the EU Framework 7 Metoxia for funding. The authors
also wish to thank Dr. Amber L. Thompson for helpful discussions.
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each compound in 1-octanol was kept at 60 °C for 1 h. A UV spec-
trum was then recorded, and the value of absorbance at the maxi-
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phosphate buffer solution (PBS; 100 μL), at a concentration of
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enzymes. An Applied Photophysics stopped-flow instrument was
used for the assay of the CA-catalysed CO2 hydration activity.
Phenol red (0.2 mm) was used as an indicator, working at the ab-
sorbance maximum of 557 nm, with N-(2-hydroxyethyl)piperazine-
NЈ-ethanesulfonic acid (10 mm, pH = 7.5) used as buffer, and
Na2SO4 (0.1 m) used to maintain constant ionic strength, all at
25 °C. The CA-catalysed CO2 hydration reaction was monitored
over 10–100 s (the uncatalysed reaction needed around 60–100 s
under assay conditions, whereas the catalysed reactions required
between 6 and 10 s). Each inhibitor was tested over the concentra-
tion range of 0.01 nm to 100 μm. Stock solutions of inhibitor
(1 mm) were prepared in distilled, deionised water with 10–20% (v/
v) DMSO (which is not inhibitory at these concentrations). These
stock solutions were diluted up to 0.01 nm with distilled, deionised
water. Inhibitor and enzyme solutions were preincubated together
at room temp. for 15 min prior to assay, which facilitated the for-
mation of the enzyme–inhibitor complex. The inhibition constants
and catalytic activity (in the absence of inhibitor) of these enzymes
were calculated and represent the mean from three separate experi-
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hCA I, 10.5 nm for hCA II, 8.5 and 14.6 nm for hCA IX. The col-
lected data from the stopped-flow assay was plotted as an inhibi-
tion percentage of the enzyme, against inhibitor concentrations, by
using a sigmoidal dose–response curve. From this data the IC50
(the concentration of BODIPY compound required to inhibit the
turnover of CO2 by the specific CA enzyme by 50%) was obtained
by using Prism 4.0 software.[72] The simplified Michaelis–Menton
equation for steady state was then used to calculate the specific
inhibitor constant Ki for the BODIPY compound with that par-
ticular CA enzyme.
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Acknowledgments
P. A. W. and M. W. J. thank the Engineering and Physical Sciences
Research Council (EPSRC) and the Medical Research Council
Eur. J. Inorg. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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P. A. Waghorn et al.
FULL PAPER
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Received: December 8, 2011
Published Online: ᭿
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 0000, 0–0

Job/Unit: A11371
/KAP1
Date: 23-04-12 11:45:21
Pages: 11
Carbonic Anhydrases with Fluorescent BODIPY-Labelled Sulfonamides
Imaging
4,4-Difluoro-4-borata-3a-azonia-4a-aza-s-
indacene (BODIPY)-functionalised sulfon-
amides have been tested for their carbonic
anhydrase (CA) selectivity by binding-af-
finity assays against CA IX and CA XII. In
vitro studies in a CA IX transfected cell
line have highlighted the importance of
both the nature of the fluorophore and the
presence of a secondary binding interaction
for selective CA inhibition.
P. A. Waghorn,* M. W. Jones,
A. McIntyre, A. Innocenti, D. Vullo,
A. L. Harris, C. T. Supuran,
J. R. Dilworth ................................. 1–11
Targeting Carbonic Anhydrases with
Fluorescent BODIPY-Labelled Sulfon-
amides
Keywords: Carbonic anhydrases / Sulfon-
amides / Fluorescence / BODIPY / Imaging
agents
Eur. J. Inorg. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
11