Carbonic anhydrase inhibitors; Fluorinated phenyl sulfamates show strong inhibitory activity and selectivity for the inhibition of the tumor-associated isozymes IX and XII over the cytosolic ones I and II

Article abstract of DOI:10.1016/j.bmcl.2009.07.056

A series of fluorinated-phenylsulfamates have been prepared by sulfamoylation of the corresponding phenols and the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic CA I and II (off-targets), and the t

Full text of DOI:10.1016/j.bmcl.2009.07.056

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5082–5085
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Bioorganic & Medicinal Chemistry Letters
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Carbonic anhydrase inhibitors; Fluorinated phenyl sulfamates show strong
inhibitory activity and selectivity for the inhibition of the tumor-associated
isozymes IX and XII over the cytosolic ones I and II
Jean-Yves Winum a, Alessio Innocenti b, Daniela Vullo b, Jean-Louis Montero a, Claudiu T. Supuran b,
*
a Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-UM1-UM2, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier,
8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
b Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 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:
A series of fluorinated-phenylsulfamates have been prepared by sulfamoylation of the corresponding
phenols and the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes,
the cytosolic CA I and II (off-targets), and the transmembrane, tumor-associated CA IX and XII is investi-
gated. Unlike the lead molecule (phenylsulfamate), a very potent CA I and II inhibitor and a modest CA IX/
XII inhibitor, the fluorinated sulfamates were stronger inhibitors of CA IX (KIs of 2.8–47 nM) and CA XII
(KIs of 1.9–35 nM) than of CA I (KIs of 53–415 nM) and CA II (KIs of 20–113 nM). Some of these com-
pounds were selective CA IX over CA II inhibitors, with selectivity ratios in the range of 11.4–12.1, making
them interesting candidates for targeting hypoxic tumors overexpressing CA IX and/or XII.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 5 June 2009
Revised 6 July 2009
Accepted 7 July 2009
Available online 23 July 2009
Keywords:
Carbonic anhydrase
Sulfamate
Fluorophenyl
Tumor-associated isoforms
Selective inhibitor
Sulfamates/bis-sulfamates1–3 were shown recently to act as
inhibitors of several carbonic anhydrase (CA, EC 4.2.1.1) isozymes
involved in fundamental physiologic/pathologic states.4,5 In
mammals, this family of metalloenzymes comprises 16 different
isoforms, of which several are cytosolic (CA I-III, CA VII, and CA
XIII), five are membrane-bound (CA IV, CA IX, CA XII, CA XIV, and
CA XV), two mitochondrial (CA VA and VB), and one (CA VI) is
secreted into saliva/milk.6–13 Three acatalytic forms are also
known, that is, CA VIII, CA X, and CA XI.9 These proteins are
involved in crucial physiological processes connected with respira-
tion and transportation of CO2/bicarbonate between metabolizing
tissues and lungs, pH and CO2 homeostasis, electrolyte secretion
in a variety of tissues/organs, biosynthetic reactions (such as gluco-
neogenesis, lipogenesis, and ureagenesis), bone resorption, calcifi-
cation, tumorigenicity, and several other physiologic/pathologic
processes.5–13 Many CAs are well-established drug targets.4 In fact,
sulfonamide CA inhibitors (CAIs), such as acetazolamide AAZ,
methazolamide MZA, and dorzolamide DZA or brinzolamide BRZ
among others, are clinically used agents for the management of a
variety of disorders connected to CA disbalances, such as glaucoma,
in the treatment of edema due to congestive heart failure,7,8 or for
drug-induced edema,7 in the management of mountain sickness,7,8
or as diuretics.5–7 Other agents of this pharmacological class show
applications as anticonvulsants,12 antiobesity13
drugs/tumor diagnostic agents.5,6,9,11,14
,
or antitumor
H3C
N
N
N
N
CH3CONH
HN
SO2NH2
S
CH3CON
SO2NH2
S
AAZ
MZA
HN
S
SO2NH2
SO2NH2
N
S
S
H3C
S
CH3O(CH2)3
O
O
O
O
DZA
BRZ
Two of the CA isozymes mentioned above, CA IX and XII, are
overexpressed in many tumor types, contributing to the extracellu-
lar acidification and intracellular alkalinization of these cells.15–17
It has been shown by several groups17,18 that these isoforms play
a role in acidosis, pH regulation, and survival of hypoxic tumor
cells, and that their inhibition with sulfonamide CAIs leads to a
perturbation of these processes, reducing the cell capability to
adapt to hypoxic stress and causing its death. This leads to a strong
antitumor effect of such sulfonamide CA IX/XII inhibitors.15–18
Various classes of sulfonamides and sulfamates have been inves-
tigated to date in the search of potent and isoform-selective CAIs,
* Corresponding author. Tel.: +39 055 457 3005; fax: +39 055 457 3385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.056

J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5082–5085
5083
Table 1
H2NSO2Cl
R-OH
R-OSO2NH2
Inhibition of hCA isoforms I, II, (cytosolic) IX and XII (transmembrane, tumor-
associated) with sulfamates 2–12, the lead (phenylsulfamate) and standard sulfon-
amide inhibitors (AAZ and DZA), by a stopped-flow CO2 hydrase assay.21
1
2-12
DMA
F
F
R:
Compound
KI (nM)#
Selectivity ratio
F
F
F
F
F
hCA Ia
hCA IIa
hCA IXb
hCA XIIb
KI(hCA IX)/KI(hCA II)
5
8
9
2
F
F
Ph-OSO2NH2
2
3
4
5
6
7
8
2.1*
75
85
55
84
56
53
54
54
1.3*
74
24
44
25
33
35
35
20
41
34
113*
12
9
63*
6.5
9.5
17
9.6
9.1
10
21
11
16
56
0.02
11.38
2.52
2.58
2.60
3.62
3.50
1.66
1.81
2.56
12.14
2.40
0.48
0.17
F
F
F
F
F
3.9
5.9
13
6.3
7.4
5.8
14
8.1
3.9
1.9
35
5.7
3.5
11
12
F
F
F
F
F
3
6
F
F
F
F
F
F
F
F
F
9
10
11
12
4
7
10
F
57
61
2.8
47*
25
415*
250
50000
Scheme 1. Preparation of fluorophenyl sulfamates 2–12 by sulfamoylation of the
corresponding phenols 1.
AAZ**
DZA**
52
a
h = human cloned full length enzyme.
b
with potential applications as diagnostic tools/antitumor lead
candidates targeting CA IX and/or XII.4,5,15–18 However, considering
the potential of this class of derivatives to design new therapies for
the management of malignant diseases, much effort is being dedi-
cated by many laboratories to obtain novel such compounds.4 Here
we report a new class of such derivatives based on a very simple lead
molecule, phenyl-sulfamate, investigated earlier by our group.1
A series of fluorophenylsulfamates (2–12) was prepared as pre-
viously described1,2 by reacting the corresponding fluoro-phenol 1
with sulfamoyl chloride in dimethylacetamide (Scheme 1).19 This
procedure proved to be very efficient and the different derivatives
were obtained in high yields after purification either by crystalliza-
tion or by silica-gel chromatography. Sulfamoyl chloride was pre-
pared from chlorosulfonyl isocyanate and formic acid as
described earlier.1,2 The rationale for introducing fluorine atoms
in the molecule of lead 1 is based on our previous observations20
that sulfonamides incorporating functionalities possessing one or
more fluorine atoms bound to an aromatic ring/aliphatic chain
possess enhanced CA inhibitory activity but also lipo- and
hydrosolubilities, compared to structurally similar derivatives
containing hydrogen instead of fluorine. On the other hand, fluo-
rine-containing sulfamates were scarcely investigated, with only
the perfluorophenyl derivative 12 being prepared earlier and stud-
ied for its interaction with isoforms CA I, II, and IX by our group.1a
We thus decided to obtain compounds containing between 1 and 5
fluorine atoms in the aromatic ring of the lead 1, in order to inves-
tigate how the presence of the halogen atoms, their number, and
positions in the various isomers of the difluoro- (5 isomers) and tri-
fluoro-substituted (3 isomers) compounds influences the inhibi-
tion of various CA isozymes. The different compounds 2–12
reported here were obtained in very good yields (>90%) by the
above-mentioned reaction (Scheme 1) and their structures con-
firmed by spectroscopic and spectrometric analyses.19
Catalytic domain of human recombinant enzyme.
#
Mean value was from at least three different measurements.21 Errors were in
the range of 5% of the obtained value (data not shown).
*
From Ref. 1a.
From Ref. 4.
**
to a drastic diminution of the inhibitory capacity of this sul-
famate, with a KI of only 415 nM. Thus, in the case of hCA I,
the fluorinated sulfamates behave contrary to many analo-
gous sulfonamide classes, for which perfluoro-substituted
derivatives act as much more potent inhibitors than the
corresponding perhydro derivatives.20 It is impossible to
rationalize this behavior without a thorough X-ray crystallo-
graphic analysis of hCA I-fluorophenyl sulfamate adducts. In
fact Christianson’s group22 reported that some fluorophenyl-
substituted sulfonamides possess a rather variable binding
pattern within the hCA II active site, by means of an elegant
X-ray crystallographic analysis.
(ii) As for hCA I, the second cytosolic isoform hCA II was highly
inhibited by the lead Ph-OSO2NH2 (KI of 1.3 nM) whereas
the fluorinated analogues 2–12 showed weaker potency, with
inhibition constants in the range of 20–113 nM. The best hCA
II inhibitor (inhibition similar to that of the potent, clinically
used compounds AAZ and DZA) was the 2,3,4-trifluorophe-
nyl-sulfamate 9 (KI of 20 nM) together with the difluoroderiv-
atives 3 and 5 (KIs in the range of 24–25 nM). The weakest hCA
II inhibitor was the pentafluorophenylsulfamate 12 (KI of
113 nM) whereas the remaining derivatives showed an inter-
mediate behavior, of medium-potency hCA II inhibitors (KIs of
33–74 nM). Thus, small variations in the number of fluorine
atoms substituting the phenyl ring as well as their positions
strongly influence the hCA II inhibitory activity for this conge-
neric series of sulfamates, with the fluorinated derivatives 2–
12 being 15.4–86.9 times less inhibitory compared to the lead
molecule Ph-OSO2NH2. This feature, difficult to explain with-
out X-ray crystal structures of these adducts, is however a
beneficial one for compounds that should inhibit less the
house-keeping, ubiquitous isozyme hCA II compared to the
target ones involved in tumorigenesis, that is, hCA IX and XII
(see discussion later in the text).
Compounds 2–12, the lead molecule Ph-OSO2NH2 as well as
standard CAIs (acetazolamide AAZ and dorzolamide DZA) were
tested for their inhibitory activity against two cytosolic, wide-
spread isozymes (CA I and II) as well as the two transmembrane,
tumor-associated isoforms CA IX and XII (Table 1).
The following SAR is obvious from the data shown in Table 1:
(i) Whereas the lead molecule Ph-OSO2NH2 is a very potent
inhibitor of the cytosolic isoform hCA I (KI of 2.1 nM), all
the fluorine-containing congeners 2–12 act as much weaker
inhibitors, with inhibition constants in the range of 53–
415 nM. The mono-, di-, tri-, and tetrafluorophenyl-substi-
tuted sulfamates 2–11 show a rather compact behavior of
medium potency hCA I inhibitors (KIs in the range of 53–
84 nM) whereas the five fluorine atoms present in 12 lead
(iii) The transmembrane, tumor-associated isoform hCA IX, was
moderately inhibited by the lead Ph-OSO2NH2 (KI of
63 nM) and was much better inhibited by the fluorinated
sulfamates 2–12, which showed inhibition constants in the
range of 2.8–47 nM. Thus, the situation is completely
reverted for the transmembrane isozyme (hCA IX) compared
to the cytosolic ones hCA I and II discussed above. The best

5084
J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5082–5085
hCA IX inhibitor was the tetrafluorophenylsulfamate 11 (KI
References and notes
of 2.8 nM), whereas several other compounds (e.g., 2, 3, 5,
6, and 7) showed KIs <10 nM, thus being very effective hCA
IX inhibitors (better than the clinically used AAZ and DZA,
which possess inhibition constants of 25–52 nM against this
isozyme). Again the least effective inhibitor was the penta-
fluorophenyl derivative 12 (KI of 47 nM), as for hCA I and
II. The difference of hCA IX inhibitory activity between the
tetra- and pentafluorophenyl derivatives 11 and 12 is 16.8,
which is probably the highest one evidenced so far for two
compounds from a congeneric series differing by only one
atom. It is indeed really puzzling to explain this phenome-
non but in the absence of X-ray crystal structures this is
virtually impossible. The remaining derivatives (4 and 8–
10) showed a compact behavior of strong hCA IX inhibitors,
with inhibition constants of 11–21 nM (Table 1).
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(iv) hCA XII was also moderately inhibited by the lead Ph-
OSO2NH2 (KI of 56 nM) and was much better inhibited by
the fluorinated compounds 2–12 (KIs in the range of 1.9–
35 nM). Thus there is a close parallelism between the inhibi-
tion of hCA XII and IX with this class of inhibitors. Indeed,
the best hCA XII inhibitor was again the tetra-
fluorophenylsulfamate 11 (KI of 1.9 nM) whereas the least
effective one was the pentafluorophenylsulfamate 12 (KI of
35 nM). Compounds 2, 3, 5–7, and 10 also showed highly
effective hCA XII inhibitory properties (KIs in the range of
3.9–7.4 nM), whereas 4, 8, and 9 were slightly less effective
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(v) Many of the effective CA IX inhibitors reported so far1-5 have
as the main drawback the lack of selectivity for inhibiting
the tumor-associated isozyme (CA IX) over the cytosolic,
ubiquitous one CA II. Data in Table 1 show, for example, that
the selectivity ratios CA IX/CA II for the two clinically used,
standard drugs AAZ and DZA are in the range of 0.17–0.48,
thus the two sulfonamides being much better CA II inhibitors
than CA IX inhibitors. The same is true for the lead compound,
for which this ratio is even less favorable (i.e., of 0.02). How-
ever, many of the fluorinated sulfamates reported here show
selectivity ratios >2.5 for inhibiting CA IX over CA II. Thus,
the most CA IX-selective inhibitors were the monofluorophe-
nyl derivative 2 and the tetrafluoro-substituted one 11 (selec-
tivity ratios of 11.38–12.52), whereas the remaining
derivatives had this parameter in the range of 1.66–3.62.
16. (a) Brahimi-Horn, M. C.; Pouysségur, J. Essays Biochem. 2007, 43, 165; (b) Stock,
C.; Schwab, A. Pflugers Arch., in press.
17. (a) Ebbesen, P.; Pettersen, E. O.; Gorr, T. A.; Jobst, G.; Williams, K.; Kienninger,
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In conclusion, we report here a series of simple fluorinated
phenylsulfamates which have been investigated for the inhibition
of four physiologically relevant CA isozymes, the cytosolic CA I
and II (off-targets) and the transmembrane, tumor-associated CA
IX and XII. Unlike the lead molecule (phenylsulfamate), a very
potent CA I and II inhibitor and a modest CA IX/XII inhibitor, the
fluorinated sulfamates act as stronger inhibitors of CA IX (KIs of
2.8–47 nM) and CA XII (KIs of 1.9–35 nM) than of CA I (KIs of 53–
415 nM) and CA II (KIs of 20–113 nM). Some of these compounds
are selective CA IX inhibitors over CA II inhibitors, with selectivity
ratios in the range of 11.4–12.1, making them interesting candi-
dates for targeting hypoxic tumors overexpressing CA IX and/or
XII.
19. 4-Fluorophenylsulfamate 2: mp 78–80 °C; 1H NMR (DMSO-d6, 400 MHz) d 8.01
(s, 2H), 7.32 (s, 2H), 7.3 (d, 2H, J = 2.8 Hz); 2,6-difluorophenylsulfamate 4: mp
99–101 °C; 1H NMR (DMSO-d6, 400 MHz) d 8.42 (s, 2H), 7.39 (m, 1H), 7.27 (m,
2H); 2,4-difluorophenylsulfamate 5: mp 83–85 °C; 1H NMR (DMSO-d6,
400 MHz)
d
8.26 (s, 2H), 7.49 (m, 2H), 7.16 (m, 1H); 3,5-
difluorophenylsulfamate 6: mp 68–70 °C; 1H NMR (DMSO-d6, 400 MHz) d
8.25 (s, 2H), 7.30 (m, 1H), 7.08 (m, 2H); 3,4-difluorophenylsulfamate 7: mp 69–
72 °C; 1H NMR (DMSO-d6, 400 MHz) d 8.12 (s, 2H), 7.32 (s, 2H), 7.56 (m, 1H),
7.42 (m, 1H), 7.16 (m, 1H); 2,3,4-trifluorophenylsulfamate 9: mp 70–72 °C; 1H
NMR (DMSO-d6, 400 MHz) d 8.50 (s, 2H), 7.57 (m, 1H), 7.43 (m, 1H); 3,4,6-
trifluorophenylsulfamate 10: mp 86–88 °C; 1H NMR (DMSO-d6, 400 MHz) d
8.46 (s, 2H), 7.41 (t, 2H, J = 9.2 Hz), 7.3 (d, 2H, J = 2.8 Hz); 2,3,5,6-
tetrafluorophenylsulfamate 11: mp 68–70 °C; 1H NMR (DMSO-d6, 400 MHz)
d 8.97 (s, 2H), 7.21 (m, 1H); pentafluorophenylsulfamate 12: mp 104À106 °C;
1H NMR (DMSO-d6, 250 MHz) 9.2 (s, 2H); 13C NMR (DMSO-d6, 400 MHz) d
138.3, 136, 133.6, 131.6; IR (KBr) 3381, 3305, 3094, 1527, 1370, 1223,
1141 cmÀ1; MS m/z 262 (MÀH)À.
Acknowledgments
This research was financed in part by a grant of the 6th Frame-
work Programme (FP) of the European Union (DeZnIT project), and
by a grant of the 7th FP of EU (Metoxia project).

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21. Khalifah, R. G.. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics (Oxford,
UK) stopped-flow instrument has been used for assaying the CA catalyzed CO2
hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes
(pH 7.5) as buffer, 0.1 M Na2SO4 (for maintaining the ionic strength constant),
following the CA-catalyzed CO2 hydration reaction. The CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the kinetic parameters and
inhibition constants. For each inhibitor at least six traces of the initial 5–10% of
the reaction have been used for determining the initial velocity. The
uncatalyzed rates were determined in the same manner and subtracted from
the total observed rates. Stock solutions of inhibitor (10 mM) were prepared in
distilled–deionized water with 5–10% (v/v) DMSO (which is not inhibitory at
these concentrations) and dilutions up to 0.1 nM were done thereafter with
distilled–deionized water. The inhibitors were tested in concentrations from
0.01 nM to 100 lM, in subsequent 10-fold dilutions. Inhibitor and enzyme
solutions were preincubated together for 15 min at room temperature prior to
assay, in order to allow for the formation of the E–I complex. The inhibition
constants were obtained by nonlinear least-squares methods using PRISM 3,
and represent the mean from at least three different determinations. CA
isozymes were recombinant ones obtained as reported earlier.14,15,18 Enzyme
concentrations used in these assays were hCA I: 10.4 nM; hCA II: 12 nM; hCA
IX: 8.9 nM: hCA XII: 15.3 nM.
22. Kim, C.-Y.; Chang, J. S.; Doyon, J. B.; Baird, T. T.; Fierke, C. A.; Jain, A.;
Christianson, D. W. J. Am. Chem. Soc. 2000, 122, 12125.