Superacid synthesis of halogen containing N-substituted-4-aminobenzene sulfonamides: New selective tumor-associated carbonic anhydrase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.05.037

A series of new, halogen containing N-substituted 4- aminobenzenesulfonamides were synthesized by using superacid HF/SbF₅ chemistry and investigated as inhibitors of several human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, that is, the cytosolic hCA I and II and, the tumor-associated transmembrane isoforms hCA IX and XII. Despite the substitution of the sulfonamide function, the presence of fluorine atom(s) in β position of the sulfonamide function strongly favors hCA inhibition. A similar effect of the β-fluorinated alkyl substitution on the amino function has been also observed. Among the tested compounds, several chlorinated derivatives have been identified as selective nanomolar, tumor-associated isoforms inhibitors. These non-primary sulfonamides probably bind in the coumarin-binding site, at the entrance of the cavity, and not to the metal ion as the primary sulfonamide inhibitors.

Full text of DOI:10.1016/j.bmc.2012.05.037

Bioorganic & Medicinal Chemistry 21 (2013) 1555–1563
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Superacid synthesis of halogen containing N-substituted-4-aminobenzene
sulfonamides: New selective tumor-associated carbonic anhydrase inhibitors
Guillaume Compain a, Agnès Martin-Mingot a, Alfonso Maresca b, Sebastien Thibaudeau a,
,
⇑
Claudiu T. Supuran b,
⇑
a Superacid group in « Glycochimie, Superacide et Chimie des systèmes » team—Université de Poitiers, CNRS UMR 7285 IC2MP, 4, avenue Michel Brunet,
F-86022 Poitiers 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:
Available online 26 May 2012
A series of new, halogen containing N-substituted 4-aminobenzenesulfonamides were synthesized by
using superacid HF/SbF5 chemistry and investigated as inhibitors of several human carbonic anhydrase
(hCA, EC 4.2.1.1) isoforms, that is, the cytosolic hCA I and II and, the tumor-associated transmembrane
isoforms hCA IX and XII. Despite the substitution of the sulfonamide function, the presence of fluorine
atom(s) in b position of the sulfonamide function strongly favors hCA inhibition. A similar effect of the
b-fluorinated alkyl substitution on the amino function has been also observed. Among the tested com-
pounds, several chlorinated derivatives have been identified as selective nanomolar, tumor-associated
isoforms inhibitors. These non-primary sulfonamides probably bind in the coumarin-binding site, at
the entrance of the cavity, and not to the metal ion as the primary sulfonamide inhibitors.
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Keywords:
Hypoxic tumors
Superacid
Fluorine chemistry
Benzenesulfonamide
Halogen effect
1. Introduction
Structural studies, mainly performed on adducts of benzenesulf-
onamides with hCA II, showed that these inhibitors bind in depro-
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metallo-
enzymes that catalyse the reversible hydratation of carbon dioxide
to bicarbonate and proton.1 At least 16 isoforms were discovered
up to now in humans,2 and many of them constitute interesting
targets for the design of pharmaceutical agents. Their inhibition
or activation can be used to treat disorders along with glaucoma,
obesity, osteoporosis, cancer, etc.3 Recently, transmembrane iso-
forms IX (hCA IX) and XII (hCA XII) have been shown to be over ex-
pressed in many tumors, and associated with cancer progression
and response to therapy,4 making them very attractive anticancer
drug targets.5 As a consequence, there is a clear need to design spe-
cific CA inhibitors for the tumor associated isoforms hCA IX and XII,
which should present lower affinity for the main off target iso-
forms (hCA I and II which have a physiological relevance).6 The pri-
mary benzenesulfonamides constitute the most common and best
tonated form to the active site, coordinating the catalytic Zn2+ ion.
Several other polar and hydrophobic interactions were also ob-
served.7,9 Recently, 4-aminobenzenesulfonamides were evaluated
for their selective inhibition of human carbonic anhydrase iso-
forms.10 The performed structural studies suggested that differ-
ences in the active sites ‘hydrophobicity’ (near the sulfonamide
group) modulate the affinity of the inhibitors. Analogously, a mod-
ification of the amino group in this series can induce a very differ-
ent inhibitory profile of the inhibitors against human carbonic
anhydrases (Table 1, Fig 2).7,11
N-substitution of sulfanilamide (SA) dramatically modifies its
inhibition properties. Whereas sulfanilamide is a medium potency
inhibitor for hCA II and IX, and a poor hCA I inhibitor, its corre-
sponding sulfanylated analogue 1 showed excellent hCA I, II and
IX inhibitory properties. In addition, the SA analogue 2, incorporat-
ing a 1,3,5-triazine moiety is an excellent hCA IX inhibitor, and only
inhibits hCA I at a low micromolar range. However, the non-selec-
tive profile of the tosyl ureido sulfanilamide 3 confirms that the
effect of the nitrogen substitution on the inhibitory properties of
a lead compound is difficult to be predicted. Nevertheless, based
on the comparison of X-ray crystal structures of the adducts of
hCA II with compounds 2 and 3 (Fig 2), it has been recently postu-
lated that the important difference in selectivity for the inhibition
of the cytosolic over transmembrane isoform could be attributed
to different binding modes of the tails nitrogen-containing
characterized class of a
-carbonic anhydrases inhibitors (Fig. 1).7
However, due to their lack of selectivity, the benzenesulfon-
amide drugs targeting CAs are far from being perfect. As a conse-
quence, the quest of selective inhibitors in this family remains an
attractive challenge as shown by the ongoing research in this field.8
⇑
Corresponding authors. Tel.: +33 549454588; fax: +33 549453501 (S.T.); tel.:
+39 055 4573005; fax: +39 055 4573385 (C.T.S.).
E-mail addresses: sebastien.thibaudeau@univ-poitiers.fr (S. Thibaudeau),
claudiu.supuran@unifi.it (C.T. Supuran).
0968-0896/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.037

1556
G. Compain et al. / Bioorg. Med. Chem. 21 (2013) 1555–1563
N
N
S
N
S
N
O
N
SO2NH2
S
O
SO2NH2
SO2NH2
EtO
N
N
H
acetazolamide
methazolamide
ethoxzolamide
SO2NH2
SO2NH2 Cl
Cl
SO2NH2
H2N
n
S
N
NH
Me
A
SO2NH2
O
S
C
HN
Me
n=0,1,2
N
B
Figure 1. Structures of selected classical carbonic anhydrase inhibitors acetazol-
amide, methazolamide, ethoxzolamide and of compounds A–C investigated against
various isoforms.
fragments.11 It has been observed that the nitrogen substitution
of 4-aminobenzene sulfonamides with various tails strongly
influences the inhibition profile and selectivity toward the tumor
associated isoform hCA IX.11b
Fig. 2. View of compounds 2 and 3 (PDB file 1zfk) superposed within the active site
of hCA II as obtained by high resolution X-ray crystallography.11b
Recently, one of our groups observed rather interesting inhibi-
tion of several CA isoforms with primary/secondary sulfonamides,
compounds less investigated until now as CAIs, among which com-
pound 4 is an interesting example.11c This compound and some of
his congeners were submicromolar hCA I and II inhibitors and it is
probable that their inhibition mechanism involves binding of the
bulky substituted sulfonamide not to the metal ion, but within
the coumarin-binding site, situated at the entrance of the active
site cavity.12
Among the SAR studies based on the modification of the sulfa-
nilamide lead compound, the use of fluorine atoms was found to be
a good strategy toward hCA IX selective inhibitors discovery.13 An
example of the success of such a strategy has been recently re-
ported.14 A fluorine substitution effect on cytosolic human car-
bonic anhydrase isoforms inhibition has been shown. This effect
was not only attributed to the modification of both hydrophilic/
lipophilic balance between the inhibitor and the enzyme pocket,
but also to a modification of the molecule direction in the active
site, leading to specific interactions with the enzyme (Fig 3).
Recently, in the quest of new hCA activators, SAR studies on
histamine derivatives also showed that halogen substitution can
dramatically alter the activation profile of compounds.15 As a con-
sequence, it appears of interest to explore novel chemical scaffolds,
incorporating halogen containing N-alkylated 4-aminobenezene
sulfonamide core in the quest of selective trans-membrane tu-
mor-associated isoforms hCA IX and XII inhibitors.
The role of fluorine in medicinal chemistry is well recognized.16
For nitrogen containing compounds, the modification of the basicity
Table 1
Inhibition data of selected 4-aminobenzenesulfonamides reported inhibitors against isoforms hCA I, II, IX and XII
*
Compound
Ki* (nm)
Selectivity ratios
hCA Ia
hCA IIa
240c
hCA IXb
hCA XIIb
37c
I/IX
I/XII
II/IX
II/XII
6.5
H2N
H2N
SO2NH2
SA
1
25000c
294c
85.0
675.0
0.8
1.3
HN
SO2
SO2NH2
SO2NH2
164d
46d
34d
ND
4.8
ND
ND
HO
HN
HN
N
N
N
2
1098e
37e
0.75e
1.6e
1464.0
686.2
23.1
9.2
23.1
Cl
Ts
HN
HN
O
SO2NH2
3
4
7e
12e
1.3e
1.5e
5.4
4.7
8.0
HN
SO2
g
g
g
g
g
g
419f
395f
/
/
/
/
/
/
*
Errors in the range of 5% of the reported data from 3 different assays.
a
Recombinant isoforms.29,30
b
Catalytic domain.
c
Values from Ref.7
Values from Ref.11a
d
e
Values from Ref.11b
Values from Ref.11c
f
g
Not determined.

G. Compain et al. / Bioorg. Med. Chem. 21 (2013) 1555–1563
1557
X
S
HN
O
SO2NH2
Ki (hCA II) = 50 nM
Ki (hCA II) = 390 nM
5
X = H
6 X = F
Fig. 3. Superposition of hCA II complexed with 5 (green) and 6 (cyan) and inhibition constants against hCA II isoform. The zinc is depicted as a grey sphere. Fig made by using
PyMOL (DeLano Scientific).14
of the nitrogen containing proximal functions, by using fluorine
strong electron withdrawing effect17 has been largelyused in medic-
inal chemistry SAR studies.18 It is also accepted that through a fluo-
rine gauche effect and through electrostatic interactions, fluorine
atoms can strongly modify the preferred conformation of nitrogen
containing biomolecules.19 To the best of our knowledge, only one
report on perfluoroalkyl(aryl)-substituted derivatives of aromatic/
heterocyclic sulfonamides has been published to date.20 Studying
the effect of the substitution of the nitrogen containing functions
of the 4-aminobenzene sulfonamides with halogenated aliphatic
chains on the inhibitor activities against hCA could be of crucial
importancefor this topic, also considering the recent data onthe sec-
ondary/tertiary sulfonamides mentioned above.11c
In due course of a part of our work focused on the development
of new reactions in superacid21 to access to novel fluorinated nitro-
gen containing compounds, going from high valued building
blocks22 to bioactive elaborated molecules,23 we recently applied
superacid chemistry toward the synthesis of new hCA inhibitors
in the benzenesulfonamide family.24 Herein, we report the synthe-
sis of new halogen containing N-substituted 4-aminobenzene sul-
fonamides in superacid, and their screening for the inhibition of
the tumor associated isoforms hCA IX and XII as well as the offtar-
gets cytosolic hCA I and II.
First, primary N-alkylated benzenesulfonamides 7–9 were syn-
thesized as described in Scheme 1. Starting from the commercially
available 4-fluorobenzenesulfonamide, after nucleophilic aromatic
substitution using an excess of allylamine, the allylic derivative 7
could be selectively formed. Then with allylic substrate 7, by using
dicationic ammonium-carbenium superelectrophilic activation in
superacid,25 the tetrahydroquinoline product 8 was formed. After
hydrofluorination, starting from the same substrate, the b-fluori-
nated analogue 9 could be selectively synthesized.
Then, a series of 4-aminobenzenesulfonamides, substituted on
the sulfonamide function, was synthesized (Scheme 2). After alkyl-
ation with the appropriate derivatives, alkylated products 11, 12,
15, 18 were synthesized in good yields. An excess of 1,3-dichloro-
propene allowed the synthesis of the dialkylated product 21. After
hydrofluorination of substrate 12, the b-fluorinated aminoben-
zenesulfonamide 13 was obtained. Similarly, using compounds
15 and 18 as substrates, the corresponding gem-chlorofluorinated
derivatives 16 and 19 were synthesized after hydrofluorination.
By modifying the superacid media composition,26 the selective for-
mation of the difluorinated analogues 17 and 20 could be achieved
in excellent yields.
2.2. CA inhibition
2. Results and discussion
2.1. Chemistry
As mentioned above, the rationale for the choice to evaluate the
novel 4-aminobenzenesulfonamides scaffolds reported here, was
that the incorporation of alkyl cores (halogenated or not) might
strongly influence the inhibitory profile of this inhibitor family,
through a modification of both the hydrophilic/hydrophobic bal-
ance and a modification of the conformational behavior of the
compounds by using electrostatic intramolecular interactions.27
First, primary N-alkylated benzensulfonamides 7–9 were
screened for the inhibition of the tumor associated isoforms hCA
IX and XII as well as for the offtargets hCA I and II (Table 2).
The following SAR has been observed for the inhibition of these
four isozymes. Activities from nanomolar to low micromolar have
been observed for the four isoforms with derivatives 7–9. It is note-
worthy that by modifying the structure shape, closely to the amino
function, by using a cyclic constrained core (compound 8), the inhi-
bition potency against the four isozymes strongly increased. Even
more interesting is the effect of the fluorine substitution of the ali-
phatic carbon chain. Compound 9 showed an inhibition profile very
similar to the compound 8. This similar inhibition profile seems to
reinforce the following hypothesis: Through particular electrostatic
interactions, the fluorinated nitrogen containing core might adopt a
preferential constrained conformation, essential for hCA inhibition.
A series of halogen-containing 4-aminobenzene sulfonamides
were designed as CA inhibitors.
H2NO2S
NH
b
8
R
NH
a
H2NO2S
F
H2NO2S
7
R =
c
H2NO2S
NH
F
9
Scheme 1. Synthesis of amino-substituted sulfanilamide derivatives 7–9. Reagents
and conditions: (a) Allylamine (exces), sealed tube, 100 °C, 51%; (b) HF/SbF5 (SbF5
mol % = 21.6), 0 °C, 1 h, 92 %; (c) HF/SbF5 (SbF5 mol % = 3.8), ꢀ50 °C, 10 min, 66%.

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G. Compain et al. / Bioorg. Med. Chem. 21 (2013) 1555–1563
Cl
F
HN
H2N
H2N
SO2
e
Cl
16
17
F
F
f
HN
HN
SO2
H2N
H2N
SO2
15
Cl
Cl
d
HN
SO2
N
SO2
a
j
SO2NH2
H2N
H2N
SA
b
11
12
21
F
HN
SO2
HN
SO2
g
c
Cl
H2N
H2N
HN
SO2
13
H2N
18
i
h
F
F
Cl
F
HN
SO2
HN
SO2
H2N
H2N
20
19
Scheme 2. Synthesis of sulfonamido-substituted sulfanilamide derivatives 11–21. Reagents and conditions: (a) Pr-Br, K2CO3, CH3CN, rt, 42%; (b) ClCH2CH(CH3)@CH2, K2CO3,
DMF, rt, 27%; (c) HF/SbF5 (SbF5 mol % = 3.8), ꢀ50 °C, 1 h, 60 %; (d) ClCH2CHCl@CH2, DMF, K2CO3, 50 °C, 16%; (e) HF/SbF5 (SbF5 mol % = 12.1), ꢀ50 °C, 10 min, 37%; (f) HF/SbF5
(SbF5 mol % = 21.6), 0 °C, 10 min, 91%; (g) ClCH2CH@CHCl, K2CO3, CH3CN, rt, 23%; (h) HF/SbF5 (SbF5 mol % = 3.8), ꢀ50 °C, 20 min, 18%; (i) HF/SbF5 (SbF5 mol % = 12.1), ꢀ20 °C,
40 min, 56%; (j) ClCH2CCl@CH2 (2 equiv), DMF, K2CO3, 50 °C, 62%.
As mentioned previously, the selectivity ratio for the inhibition
of the tumor-associated isoforms hCA IX and XII over the cytosolic
isoforms hCA I and II is of crucial importance in the search of new
lead compounds that selectively inhibit the transmembrane tumor
associated isoforms. In the quest of new leads in the 4-aminoben-
zene sulfonamide family by using superacid chemistry, compound
10 was recently identified as a tumor associated inhibitor with
inhibition constants of 136 and 298 nm against hCA IX and XII,
respectively.24 The inhibition profile of this compound strongly in-
trigued us. Benzenesulfonamides are known to bind in deproto-
nated form to the Zn(II) ion from the active site.1,2 However,
despite the polysubstitution of the sulfonamide function, com-
pound 10 still showed nanomolar inhibition activities, even better
than the primary sulfonamide SA. This profile led us to envisage a
new binding site for this class of compounds, as recently reported
for the coumarins.12,28
Thus the hCA inhibition activities of new 4-aminobenzenesulf-
onamide substituted on the sulfonamide function were studied.
Really intrigued by the effect of the b-fluorinated core on the inhi-
bition potency in the previously described series, we focused our
attention on the effect of the substitution of the aliphatic core with
halogen(s). Compounds 11–21 were tested as inhibitors against
hCA I, II, IX and XII. Again, the effect of the b-fluoropropyl substitu-
tion was dramatic. When the N-alkylated 11 or N-allylated deriva-
tive 12 showed inhibition at low micromolar level against the four
tested isoforms, the fluorinated analogue 13 showed a better inhi-
bition with interesting inhibition constants at the nanomolar level
against the tumor-associated isoforms. However, this fluorinated
compound was also effective inhibitor against the cytosolic iso-
forms hCA I and II. It is noteworthy that in the case of sulfanilamide
(with sulfonamide function substitution), the free amino group is
crucial for the inhibition as the corresponding amide 14 was only
a micromolar hCA I, II, IX and XII inhibitor. When the effect of a
geminal chlorine atom is negative for the inhibition (compound
16) with a loss of activity of two orders of magnitude for all the
tested isoforms, a second geminal fluorine atom did not alter the
good inhibition profile (compound 17). At this stage it is difficult
to clearly conclude. Beside a classical effect of the halogen atom(s)
on the sulfonamide pKa through electron withdrawing effect, con-
sidering the strong difference of inhibitory profile between com-
pounds 16 and 17, other parameters must influence the inhibition
in these cases. It might be considered that a conformational prefer-
ence of the b-fluorinated sulfonamide might be surprisingly modi-
fied by the geminal chlorine atom and not by adding the second
fluorine atom, with strong consequences on the enzyme–adduct
interactions. As shown by the inhibition profile of compounds 19
and 20, increasing the distance between the halogen atoms and
the sulfonamide function had a negative effect on the inhibition.
This result which could be attributed to a loss of conformational
preference, due to the longer distance between the halogen atoms
and the sulfonamide function, seems to reinforce our initial hypoth-
esis. Among the tested compounds, the chlorinated allylic deriva-
tives 15 and 21 showed the best inhibitory profile. Compared to
the methyl substituted allylic derivative 12, the substitution of
the methyl group by a chlorine atom completely modifies the inhi-
bition properties of the N-allylic benzenesulfonamide substrate.
Compound 15 showed only micromolar hCA I and II inhibition,
and inhibit the tumor associated isoforms IX and XII at a nanomolar
level. Interestingly, the calculated selectivity ratios showed that
compound 15 can be considered as a good selective inhibitor of
the tumor associated isoforms over cytosolic ones. By substituting
the sulfonamide function by a second chlorinated allylic chain,
the selectivity toward the tumor-associated isoforms hCA IX and
XII can even be increased as shown by the inhibitory profile of com-
pound 21. The low micromolar inhibition of the cytosolic isoforms
and the good nanomolar selective inhibition of hCA IX and XII, could
be correlated to a new binding mode of this inhibitor with the
transmembrane carbonic anhydrases isoforms.

G. Compain et al. / Bioorg. Med. Chem. 21 (2013) 1555–1563
1559
Table 2
Inhibition data of selected amino substituted 4-aminobenzenesulfonamides against isoforms hCA I, II, IX and XII
Ki* (nm)
Selectivity ratios
*
Compound
hCA Ia
hCA IIa
hCA IXb
hCA XIIb
37c
I/IX
I/XII
II/IX
II/XII
c
c
H2NO2S
NH2
SA
7
25000c
240
294
85.0
675.0
2.1
0.8
6.5
2.4
2078
192
2340
78.9
57.6
1398
50.5
43.5
965.7
80.7
1.5
3.8
4.0
1.7
1. 6
1.3
H2NO2S
H2NO2S
H2NO2S
NH
NH
NH
8
9
2.4
2.4
1.0
0.8
175
73.9
F
d
d
d
637d
5142
7136
431
136
298
4.7
2.5
2.1
2.1
2.8
5.6
3.2
1.7
1.4
1.4
1.9
3.7
N
O2S
10
11
12
NH2
NH
O2S
3455
4708
2078
3462
1841
1277
NH2
NH
O2S
NH2
NH2
F
13
14
16
17
19
20
15
174
75
83
39.7
1100d
1521
79.4
1947
1145
96
2.1
1.1
2.1
3.2
2.7
2.0
62
4.4
4.4
3.4
1.9
3.5
3.7
58,6
0.9
1.0
0.6
1.8
0.3
2.0
57.5
1. 9
4.2
0.9
0.6
0.4
3.7
54
NH
O2S
F
4800d
5179
151
4600d
1400
85.3
4400d
2471
47.0
2471
2123
90
NH
O2S
NHAc
NH2
F
F
Cl
F
NH
O2S
NH
O2S
NH2
F
6764
4224
5626
799.1
4235
5176
Cl
NH
O2S
NH2
F
F
NH
O2S
NH2
Cl
NH
O2S
NH2
Cl
Cl
21
6796
4995
73.2
79.8
92.8
85.2
68.2
62.6
N
H2N
SO2
*
Errors in the range of 5% of the reported data from 3 different assay.
a
Recombinant isoforms.29,30
b
Catalytic domain.
Values from Ref. 11a
c
d
Values from Ref. 24
substitution on the amino function has been shown allowing the
identification of new nanomolar fluorinated lead inhibitors in this
chemical family. In addition, despite the substitution of the sulfon-
amide function, the presence of fluorine atom(s) in b position of
the sulfonamide function has been shown to strongly favor hCA
inhibition. Further studies on halogen containing N-substituted-
4-aminobenzenesulfonamides, including modeling studies, are
3. Conclusion
In conclusion, new halogen containing N-substituted 4-amino-
benzenesulfonamides were synthesized by using superacid HF/
SbF5 chemistry and investigated as inhibitors of cytosolic carbonic
anhydrases isoforms I and II and of tumor-associated transmem-
brane isoforms hCA IX and XII. The effect of the b-fluorinated alkyl

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G. Compain et al. / Bioorg. Med. Chem. 21 (2013) 1555–1563
currently in due course to evaluate conformational considerations.
Among the tested compounds, new chlorinated derivatives have
been identified as selective nanomolar tumor-associated isoforms
inhibitors, probably correlated with a new binding mode of the
inhibition with these isoforms, making these halogen containing
4-aminobenzenesulfonamides new leads for further investigations.
(CD3COCD3, 400 MHz, ppm) d: 3.85 (td, CH2, J = 5.5 Hz, J = 1.7 Hz,
0
0
H1 ), 5.12 (ddt, 1H, J = 10.3 Hz, J = 1.6 Hz, J = 1.6 Hz, H3 ), 5.26 (ddt,
0
1H, J = 17.2 Hz, J = 1.8 Hz, J = 1.8 Hz, H3 ), 5.89 (broad s, 1H, NH),
0
5.93 (m, CH, H2 ), 6.16 (s, SO2NH2), 6.69 (d, 2CH, J = 8.9 Hz, H3
and H5), 7.60 (d, 2CH, J = 8.9 Hz, H2 and H6). 13C NMR (CD3COCD3,
0
100 MHz, ppm) d: 46.1 (CH2, C1 ), 112.2 (2CH, C3 and C5), 116.1
0
0
(CH2, C3 ), 128.7 (2CH, C2 and C6), 131.7 (C1), 136.1 (CH, C2 ),
152.5 (C4). MS (IES+, ACN): m/z 213 [M+H]+. HRMS (ESI, CH3OH):
m/z 235.0518 (m/z theoretical: 235.05172) [M+Na]+.
4. Experimental part
4.1. Chemistry
4.1.5. Formation of compound 8:31 1,2,3,4-tetrahydroquinoline-
6-sulfonamide
4.1.1. General procedure
This compound was obtained from substrate
8 (107 mg,
The authors draw the reader’s attention to the dangerous fea-
tures of superacidic chemistry. Handling of hydrogen fluoride
and antimony pentafluoride must be done by experienced chem-
ists with all the necessary safety arrangements in place.
Reactions performed in superacid were carried out in a sealed
TeflonÒ flask with a magnetic stirrer. No further precautions have
to be taken to prevent mixture from moisture (test reaction
worked out in anhydrous conditions leads to the same results as
expected).
0.5 mmol) following the general procedure at 0 °C for 1 h with a
mixture of HF/SbF5 (21.6 mol% SbF5, 2 mL). The reaction crude
was purified with the eluent petroleum ether/ethylacetate: 25/75
to 35/65, thereby obtaining compound 8 (98 mg, 92 %) as a white
solid (mp: 142 °C). 1H NMR (CD3OD, 400 MHz, ppm) d: 1.90 (tt,
CH2, J = 6.1 Hz, J = 5.4 Hz, H3), 2.76 (t, CH2, J = 6.3 Hz, H4), 3.32 (m,
CH2, H2), 6.49 (d, CH, J = 9.1 Hz, H8), 7.40 (m, 2CH, H5 and H7).
13C NMR (CD3OD, 100 MHz, ppm) d: 22.3 (CH2, C3), 28.2 (CH2,
C4), 42.2 (CH2, C2), 113.4 (CH, C8), 121.0 (C10), 126.5 (CH, C7),
128.5 (CH, C5), 129.5 (C6), 150.2 (C9). MS (IES+, ACN): m/z 235
[M+Na]+. HRMS (ESI, CH3OH): m/z 235.0516 (m/z theoretical:
235.05172) [M+Na]+.
Yields refer to isolated pure products.
1H, 13C and 19F NMR were recorded at respectively, 400, 100 and
376 MHz on Bruker Advance DPX spectrometer using CD3COCD3 or
CD3OD as solvent. COSY 1H–1H and 1H–13C experiments were used
to confirm the NMR peaks assignments.
4.1.6. Formation of compound 9: 4-(2-fluoropropylamino)
benzenesulfonamide
Melting points were determined in a capillary tube with a de-
vice Büchi melting point B-545 and were uncorrected.
Mass Spectra (MS) were performed with coupled liquid chro-
matography (electronic impact).
All separations were done under flash-chromatography condi-
tions on silica gel (15–40 lm) or by using a Combiflash™ auto-
This compound was obtained from substrate
7 (296 mg,
1.39 mmol) following the general procedure at ꢀ50 °C for 10 min
with a mixture of HF/SbF5 (3.8 mol% SbF5, 4 mL). The reaction
crude was purified with a combi-flash by using a linear gradient
of the eluent petroleum ether/ethylacetate: 100/0 to 50/50, there-
by obtaining compound 9 (216 mg, 66 %) as a white solid (mp:
110 °C). 1H NMR (CD3OD, 400 MHz, ppm) d: 1.40 (dd, CH3,
mated chromatographic system.
High Resolution Mass Spectrometry (HRMS) spectra were per-
formed at the Centre régional de mesures physiques de l’Ouest of
the University of Rennes 1, France.
0
0
J = 23.6 Hz, J = 6.3 Hz, H3 ), 3.37 (m, CH2, H1 ), 4.85 (dd, CHF,
0
J = 3.6 Hz, J = 49.1 Hz, H2 ), 6.73 (d, 2CH, J = 8.9 Hz, H3 and H5),
7.67 (d, 2CH, J = 8.9 Hz, H1 and H6). 13C NMR (CD3OD, 100 MHz,
4.1.2. Preparation of HF/SbF5 mixture
0
0
ppm) d: 18.8 (d, CH3, J = 22 Hz, C3 ), 49.2 (d, CH2, J = 22 Hz, C1 ),
After liquid condensation of the desired quantity of hydrogen
fluoride in a TeflonÒ reactor at ꢀ30 °C, antimony pentafluoride
was slowly added to the reactor at the same temperature and
the reactor is then sealed and maintained at reaction temperature.
In these conditions, the SbF5 molar percentage is determined
accordingly: For example, 1 mL of SbF5 (13.8ꢁ10ꢀ3 mol) was added
to 7 mL of anhydrous liquid HF (0.35 mol) to give a HF/SbF5 solu-
tion with a SbF5 molar percentage of 3.8 mol %.
0
90.4 (d, CHF, J = 168 Hz, C2 ), 112.4 (2CH, C3 and C5), 128.9 (2CH,
C2 and C6), 130.8 (C1), 153.3 (C4). 19F{1H} NMR (CD3OD,
376 MHz, ppm) d: ꢀ179.18. MS (IES+, ACN): m/z 255 [M+Na]+.
HRMS (ESI, CH3OH): m/z 255.0578 (m/z theoretical: 255.05795)
[M+Na]+.
4.1.7. Formation of compound 11: 4-amino-N-(propyl)benzene
sulfonamide
To a solution of 172 mg of sulfanilamide (1 mmol) and 304 mg
of K2CO3 (2.2 mmol) in 2.5 mL of DMF was added dropwise 91 lL
4.1.3. General procedure for superacid reaction
To a mixture of HF/SbF5 maintained at the desired temperature,
was added nitrogen derivative. The mixture was magnetically stir-
red at the same temperature for the required time. The reaction
mixture was then neutralized with water-ice–Na2CO3, extracted
with ethylacetate (ꢁ3). The combined organic phases were dried
(MgSO4) and concentrated in vacuo. Products were isolated by col-
umn chromatography over silica gel.
of 1-bromopropane (1 mmol). The mixture was stirred at room
temperature for 5 days. Then, DMF was evaporated and the crude
was dissolved with water and the solution was extracted with eth-
ylacetate (ꢁ3). The combined organic phases were dried (MgSO4)
and concentrated in vacuo. The crude mixture was purified by
combi-flash with the eluent petroleum ether/ethylacetate: 70/30,
thereby obtaining compound 11 (90 mg, 42%) as a white solid
(mp: 80–82 °C). 1H NMR (CDCl3, 400 MHz, ppm) d: 0.83 (t, CH3,
4.1.4. Synthesis
0
J = 7.4 Hz, H3 ), 1.45 (qt, CH2, J = 7.3 Hz, J = 7.2 Hz, H2’), 2.78 (dd,
Formation of compound 7: 4-(allylamino)benzenesulfonamide/
100 mg of 4-fluorobenzensulfonamide (0.71 mmol) dissolved with
allylamine (434 lL, 5.71 mmol) in a sealed tube was heated at
110 °C for 14 days. Then, the mixture was cooled to room temper-
ature and was diluted with MeOH. The solvent was evaporated and
the crude mixture was purified by combi-flash with the eluent
petroleum ether/ethylacetate: 70/30, thereby obtaining compound
7 (62 mg, 51%) as an orange solid (mp: 74-76 °C). 1H NMR
0
CH2, J = 7.1 Hz, J = 6.3 Hz, H1 ), 5.39 (broad s, 2H, SO2NH2), 6.02 (t,
1H, J = 6.1 Hz, NH), 6.74 (d, 2CH, J = 8.8 Hz, H3 and H5), 7.53 (d,
2CH, J = 8.7 Hz, H2, H6). 13C NMR (CDCl3, 100 MHz, ppm) d: 11.5
0
0
0
(CH3, C3 ), 23.4 (CH2, C2 ), 45.5 (CH2, C1 ), 113.9 (2CH, C3 and C5),
127.9 (C1), 129.5 (2CH, C2 and C6), 153.0 (C4). MS (IES+, ACN):
m/z 215 [M+H]+, 137 [M+Na]+. HRMS (ESI, CH3OH): m/z 237.0672
(m/z theoretical: 237.06737) [M+Na]+.

G. Compain et al. / Bioorg. Med. Chem. 21 (2013) 1555–1563
1561
4.1.8. Formation of compound 12: 4-amino-N-(2-methylallyl)
benzenesulfonamide
To a solution 1.722 g of sulfanilamide (10 mmol) and 3.04 g of
K2CO3 in 25 mL of DMF was added dropwise 984 lL of 2-methyl-
petroleum ether/ethylacetate/dichloromethane: 65/30/5, allowed
the separation of pure compound 16 (62 mg, 37 %) as a white solid
(mp: 140–142 °C). 1H NMR (CD3COCD3, 400 MHz, ppm) d: 1.92 (d,
3H, J = 19.6 Hz, 30), 3.40 (m, 2H, 10), 5.49 (s, 2H, NH2), 6.75 (d, 2H,
J = 8.8 Hz, 3, 5), 6.76 (m, 1H, SO2NH), 7.56 (d, 2H, J = 8.8 Hz, 2, 6).
13C NMR (CD3COCD3, 100 MHz, ppm) d: 28.1 (d, 1C, J = 24 Hz, 30),
53.7 (d, 1C, J = 27 Hz, 10), 113.4 (d, 1C, J = 244 Hz, 20), 114.0 (s, 2C,
3, 5), 128.0 (s, 1C, 1), 129.7 (s, 2C, 2, 6), 153.5 (s, 1C, 4). 19F{1H}
NMR (CD3COCD3, 376 MHz, ppm) d: ꢀ106.88. MS (IES+, ACN):
m/z 267 [M+H]+. HRMS (ESI, CH3OH): m/z 267.0376 (m/z theoreti-
cal: 267.03703) [M+H]+.
1-chloropropane (10 mmol). The was stirred at room temperature
for 1 day. Then, 25 mL of water was added and the solution was ex-
tracted with the mixture ethylacetate/toluene: 2/1 (ꢁ3). The com-
bined organic phases were dried (MgSO4) and concentrated in
vacuo. The crude mixture was purified by column chromatography
with the eluent petroleum ether/ethylacetate: 70/30, thereby
obtaining compound 12 (619 mg, 27 %) as a white solid (mp: 78–
81 °C). 1H NMR (CD3COCD3, 400 MHz, ppm) d: 1.67 (CH3, H1 ),
00
0
0
0
3.39 (d, CH2, J = 6.5 Hz, H1 ), 4.76 (s, 1H, H3 ), 4.87 (s, 1H, H3 ),
5.43 (broad s, 2H, NH2), 6.17 (t, 1H, J = 6.0 Hz, SO2NH), 6.74 (d,
2CH, J = 8.7 Hz, H3 and H5), 7.53 (d, 2CH, J = 8.6 Hz, H2 and H6).
4.1.12. Formation of compound 17: 4-amino-N-(2,2-difluoro
propyl)benzenesulfonamide
This compound was obtained from substrate 15 (61 mg,
0.25 mmol) following the general procedure at 0 °C for 10 min with
a mixture of HF/SbF5 (21.6 mol % SbF5, 1 mL). The reaction crude
was purified with the eluent petroleum ether/ethylacetate: 50/
50, thereby obtaining compound 15 (54 mg, 86%) as a white solid
(mp: 151–152 °C). 1H NMR (CD3OD, 400 MHz, ppm) d: 1.58 (t,
13C NMR (CD3COCD3, 100 MHz, ppm) d: 20.3 (CH3, C1 ), 49.6
00
0
0
(CH2, C1 ), 112.1 (CH2, C3 ), 113.9 (2CH, C3 and C5), 128.2 (C1),
0
129.7 (2CH, C2 and C6), 142.7 (C2 ), 153.1 (C4). MS (IES+, ACN): m/
z 227 [M+H]+. HRMS (ESI, CH3OH): m/z 249.0674 (m/z theoretical:
249.06737) [M+Na]+.
0
0
CH3, J = 18.7 Hz, H3 ), 3.12 (t, CH2, J = 12.8 Hz, H1 ), 6.69 (d, 2CH,
4.1.9. Formation of compound 13: 4-amino-N-(2-fluoro-2-meth
ylpropyl)benzenesulfonamide
J = 8.8 Hz, H3 and H5), 7.53 (d, 2CH, J = 8.8 Hz, H2 and H6). 13C
0
NMR (CD3OD, 100 MHz, ppm) d: 21.3 (t, CH3, J = 26 Hz, C3 ), 48.9
0
This compound was obtained from substrate 12 (226 mg,
1 mmol) following the general procedure at ꢀ50 °C for 1 h with a
mixture of HF/SbF5 (3.8 mol% SbF5, 4 mL). The reaction crude was
purified with the eluent petroleum ether/ethylacetate: 60/40 to
55/45, thereby obtaining compound 13 (147 mg, 60%) as a white
solid (mp: 124 °C). 1H NMR (CD3OD, 400 MHz, ppm) d: 1.31 (d,
2CH3, J = 21.2 Hz, H3 and H1 ), 2.89 (d, CH2, J = 18.6 Hz, H1 ), 6.70
(d, 2CH, J = 8.8 Hz, H3 and H5), 7.53 (d, 2CH, J = 8.8 Hz, H2 and
H6). 13C NMR (CD3OD, 100 MHz, ppm) d: 24.8 (d, 2CH3, J = 24 Hz,
(t, CH2, J = 33 Hz, C1 ), 114.5 (2CH, C3 and C5), 123.5 (t, CF2,
0
J = 239 Hz, C2 ), 127.5 (C1), 129.9 (2CH, C2 and C6), 154.2 (C4).
19F{1H} NMR (CD3OD, 376 MHz, ppm) d: ꢀ97.29. MS (IES+, ACN):
m/z 273 [M+Na]+. HRMS (ESI, CH3OH): m/z 273.0486 (m/z theoret-
ical: 273.04853) [M+Na]+.
0
00
0
4.1.13. Formation of compound 18: 4-amino-N-(3-chloroallyl)
benzenesulfonamide
To a solution 3.44 g of sulfanilamide (20 mmol) and 6.08 g of
K2CO3 in 50 mL of acetonitrile was added dropwise 1.82 mL of
1,3-dichloropropane (20 mmol). The mixture was stirred at room
temperature for 8 days. Then, DMF was evaporated and the crude
was dissolved with 40 mL of water and the solution was extracted
with ethylacetate (ꢁ3). The combined organic phases were dried
(MgSO4) and concentrated in vacuo. The crude mixture was puri-
fied by column chromatography with the eluent petroleum
ether/ethylacetate: 65/35, thereby obtaining compound 18 as a
mixture of isomers (1.11 g, 23%, with a NMR ratio E/Z: 67/33), as
a brown solid. 1H NMR (CD3COCD3, 400 MHz, ppm) d: 3.52 (td,
0
00
0
C3 and C1 ), 52.5 (d, CH2, J = 26 Hz, C1 ), 95.2 (d, CF, J = 168 Hz,
0
C2 ), 114.5 (2CH, C3 and C5), 127.8 (C1), 129.9 (2CH, C2 and C6),
154.0 (C4). 19F{1H} NMR (CD3OD, 376 MHz, ppm) d: ꢀ145.03. MS
(IES+, ACN): m/z 269 [M+Na]+. HRMS (ESI, CH3OH): m/z 269.0735
(m/z theoretical: 269.0736) [M+Na]+.
4.1.10. Formation of compound 15: 4-amino-N-(2-chloroallyl)
benzenesulfonamide
To a solution 1.722 g of sulfanilamide (10 mmol) and 3.04 g of
K2CO3 in 25 mL of DMF was added dropwise 925 lL of 1,2-dichlo-
0
ropropane (10 mmol). The mixture was heating at 50 °C for 1 day.
Then, 25 mL of water was added and the solution was extracted
with the mixture ethylacetate/toluene: 2/1 (ꢁ3). The combined or-
ganic phases were dried (MgSO4) and concentrated in vacuo. The
crude mixture was purified by column chromatography with the
eluent pentane/ethylacetate: 70/30 up to 60/40, thereby obtaining
compound 21 (945 mg, 38 %) as a white solid (mp: 76–78 °C) fol-
lowed by compound 15 (338 mg, 16%) as a white solid (mp: 86–
88 °C). 1H NMR (CD3OD, 400 MHz, ppm) d: 3.60 (t, CH2, J = 1.1 Hz,
CH2, J = 6.4 Hz, J = 1.6 Hz, H1 E), 3.68 (td, CH2, J = 6.1 Hz, J = 1.9 Hz,
H1 Z), 5.47 (broad s, NH2), 5.83 (dt, CH, J = 7.1 Hz, J = 6.2 Hz, H2 E),
5.86 (dt, CH, J = 6.6 Hz, J = 6.6 Hz, H2 Z), 6.21 (td, 1H, J = 7.1 Hz,
0
0
0
0
J = 1.9 Hz, H3 Z), 6. 28 (broad s, NHE), 6.30 (td, 1H, J = 13.3 Hz,
J = 1.5 Hz, H3 E), 6.35 (broads, NHZ), 6.75, 7.53. 13C NMR (CD3COCD3,
0
100 MHz, ppm) d: 40.8, 43.7, 114.0, 120.2, 121.0, 127.2, 127.6,
129.6, 129.8, 129.9, 131.0, 153.4 (2C). MS (IES+, CH3OH): m/z 269
[M+Na]+. HRMS (ESI, CH3OH): m/z 269.0128 (m/z theoretical:
269.01275) [M+Na]+.
0
0
C1 ), 5.23 (td, 1H, J = 1.7 Hz, J = 0.9 Hz, C3 ), 5.41 (dd, 1H,
0
J = 3.0 Hz, J = 1.4 Hz, C3 ), 6.70 (d, 2CH, J = 8.9 Hz, C3 and C5), 7.52
4.1.14. Formation of compound 19: 4-amino-N-(3-chloro-3-
fluoropropyl)benzenesulfonamide
(d, 2CH, J = 8.9 Hz, C2 and C6). 13C NMR (CD3OD, 100 MHz, ppm)
0
0
d: 49.7 (CH2, C1 ), 114.1 (CH2, C3 ), 114.4 (2CH, C3 and C5), 127.7
This compound was obtained from substrate 18 (256 mg,
1.04 mmol) following the general procedure at ꢀ50 °C for 20 min
with a mixture of HF/SbF5 (3.8 mol % SbF5, 4 mL). The reaction
crude was purified with the eluent dichloromethane/methanol:
100/0 to 99.5/0.5. A further purification on TLC plate with the elu-
ent dicloromethane/NH3aq: 99/1, allowed the separation of pure
compound 19 (49 mg, 18%) as a white solid (mp: 92–94 °C) beside
starting material remaining. 1H NMR (CD3OD, 400 MHz, ppm) d:
0
(C1), 130.0 (2CH, C2 and C6), 139.6 (C2 ), 154.2 (C4). MS (IES+,
ACN): m/z 269 [M+Na]+. HRMS (ESI, CH3OH): m/z 269.0129 (m/z
theoretical: 269.01275) [M+Na]+.
4.1.11.Formation of compound 16: 4-amino-N-(2-chloro-2-fluor
opropyl)benzenesulfonamide
This compound was obtained from substrate 15 (155 mg,
0.63 mmol) following the general procedure at ꢀ50 °C for 10 min
with a mixture of HF/SbF5 (12.1 mol% SbF5, 9 mL). The reaction
crude was purified with the eluent dichloromethane/methanol:
99.5/0.5. A further purification on TLC plate with the eluent
0
0
2.19 (m, CH2, H2 ), 2.97 (t, CH2, J = 6.8 Hz, H1 ), 6.34 (dt, CHFCl,
0
J = 50.6 Hz, J = 5.5 Hz, H3 ), 6.71 (d, 2CH, J = 8.8 Hz, H3 and H5),
7.52 (d, 2CH, J = 8.8 Hz, H2 and H6). 13C NMR (CD3OD, 100 MHz,
0
0
ppm) d: 39.2 (d, CH2, J = 6 Hz, C1 ), 40.5 (d, CH2, J = 20 Hz, C2 ),

1562
G. Compain et al. / Bioorg. Med. Chem. 21 (2013) 1555–1563
0
102.2 (d, CHF, J = 240 Hz, C3 ), 114.5 (2CH, C3 and C5), 126.9 (C1),
130.0 (2CH, C2 and C6), 154.2 (C4). 19F{1H} NMR (CD3OD,
376 MHz, ppm) d: ꢀ134.23. MS (IES+, ACN): m/z 267 [M+H]+. HRMS
(ESI, CH3OH): m/z 273.0485 (m/z theoretical: 273.04853) [M+H]+.
Acknowledgments
This work was financed in part by an FP7 EU grant (METOXIA)
to CTS and from the CNRS, the University of Poitiers and the Region
Poitou-Charentes (grant to GC).
4.1.15. Formation of compound 20: 4-amino-N-(3,3-difluoro
propyl)benzenesulfonamide
References and notes
This compound was obtained from substrate 18 (247 mg,
1 mmol) following the general procedure at ꢀ20 °C for 40 min with
a mixture of HF/SbF5 (12.1 mol % SbF5, 3 mL). The reaction crude
was purified with the eluent dichloromethane/methanol: 99.5/0.5
to 96/4, thereby obtaining compound 20 (141 mg, 56%) as a white
solid (mp: 72–75 °C). 1H NMR (CD3OD, 400 MHz, ppm) d: 1.97 (m,
1. (a) Krishnamurthy, V. M.; Kaufman, G. K.; Urbach, A. R.; Gitlin, I.; Gudiksen, K.
L.; Weibel, D. B.; Whitesides, G. M. Chem. Rev. 2008, 108, 946; (b) Supuran, C. T.
Nat. Rev. Drug. Discovery 2008, 7, 168; (c) Supuran, C. T. Future Med. Chem. 2011,
3, 1165.
2. Supuran, C. T. In Drug Design of Zinc-Enzyme Inhibitors Functional, Structural, and
Disease Applications; Supuran, C. T., Winum, J. Y., Eds.; Wiley: Hoboken (NJ),
2009; pp 15–38.
3. (a) Marquis, R. E.; Whitson, J. T. Drugs Aging 2005, 22, 1; (b) Vullo, D.; Innocenti,
A.; Nishimori, I.; Pastorek, J.; Scozzafava, A.; Pastorekova, S.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2005, 15, 963; (c) Santos, M. A.; Marques, S.; Gil, M.;
Tegoni, M.; Scozzafava, A.; Supuran, C. T.; Enzyme Inhib, J. Med. Chem. 2003, 18,
233; (d) Loiselle, F. B.; Morgan, P. E.; Alvarez, B. V.; Casey, J. R. Am. J. Physiol. Cell.
Physiol. 2004, 286, 1423; (e) Rivera, C.; Voipio, J.; Kaila, K. J. Physiol. 2005, 562,
27; (f) Vullo, D.; Voipio, J.; Innocenti, A.; Rivera, C.; Ranki, H.; Scozzafava, A.;
Kaila, K.; Supuran, C. T.; Bioorg, C. T. Med. Chem. Lett. 2005, 15, 971; (g) Lyons, K.
E.; Pahwa, R.; Comella, C. L.; Eisa, M. S.; Elble, R. J.; Fahn, S.; Jankovic, J.; Juncos,
J. L.; Koller, W. C.; Ondo, W. G.; Sethi, K. D.; Stern, M. B.; Tanner, C. M.; Tintner,
R.; Watts, R. L. Drug Saf. 2003, 26, 461; (h) Garske, L. A.; Brown, M. G.; Morrison,
S. C. J. Appl. Physiol. 2003, 94, 991; (i) Hori, K.; Ishida, S.; Inoue, M.; Shinoda, K.;
Kawashima, S.; Kitamura, S.; Oguchi, Y. Retina 2004, 24, 481; (j) Winum, J. Y.;
Scozzafava, A.; Montero, J. L.; Supuran, C. T. Med. Res. Rev. 2005, 25, 186; (k)
Supuran, C. T. Expert Opinion on Therapeutic Patents 2003, 13, 1545.
4. (a) Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3467; (b) Neri, D.; Supuran,
C. T. Nat. Rev. Drug. Discovery 2011, 10, 767.
0
CH2, H2’), 2.94 (t, CH2, J = 7.0 Hz, H1 ), 5.91 (tt, CHF2, J = 56.5 Hz,
0
J = 4.5 Hz, H3 ), 6.71 (d, 2CH, J = 8.7 Hz, H3 and H5), 7.53 (d, 2CH,
J = 8.7 Hz, H2 and H6). 13C NMR (CD3OD, 100 MHz, ppm) d: 35.3
0
0
(t, CH2, J = 22 Hz, C2 ), 37.8 (t, CH2, J = 7 Hz, C1 ), 114.5 (2CH, C3
0
and C5), 117.3 (t, CHF2, J = 237 Hz, C3 ), 126.8 (C1), 129.9 (2CH, C2
and C6), 154.0 (C4). 19F{1H} NMR (CD3OD, 376 MHz, ppm) d:
ꢀ119.02. MS (IES+, ACN): m/z 273 [M+Na]+. HRMS (ESI, CH3OH):
m/z 273.0484 (m/z theoretical: 273.04853) [M+Na]+.
4.1.16. Formation of compounds 21: 4-amino-N-bis(2-chloro
allyl)benzenesulfonamide
To a solution 172 mg of sulfanilamide (1.0 mmol) and 304 mg of
K2CO3 in 2.5 mL of DMF was added dropwise 0.2 mL of 1,2-dichlo-
ropropane (2.1 mmol). The mixture was heating at room tempera-
ture for 6 days. Then, 25 mL of water was added and the solution
was extracted with ethylacetate (ꢁ3). The combined organic
phases were dried (MgSO4) and concentrated in vacuo. The crude
mixture was purified by column chromatography with the eluent
petroleum ether/ethylacetate: 70/30, thereby obtaining compound
21 (498 mg, 62%). 1H NMR (CD3OD, 400 MHz, ppm) d: 4.01 (s,
5. Thiry, A.; Supuran, C. T.; Masereel, B.; Dogné, J.-M. J. Med. Chem. 2008, 51,
3051.
6. Supuran, C. T. Carbonic Anhydrase, Its Inhibitors and Activators; CRC Press:
London, 2004. pp. 67–147.
7. Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. In Drug
Design of Zinc-Enzyme Inhibitors Functional, Structural, and Disease Applications;
Supuran, C. T., Winum, J. Y., Eds.; Wiley: Hoboken (NJ), 2009; p 73.
8. (a) Rami, M.; Maresca, A.; Smaine, F.-Z.; Montero, J.-L.; Scozzafava, A.; Winum,
J.-Y.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2011, 21, 2975; (b) Capkauskaite, E.;
Baranauskiene, L.; Golovenko, D.; Manakova, E.; Grazulis, S.; Tumkevicius, S.;
Matulis, D. Bioorg. Med. Chem. 2010, 18, 7357; (c) Mader, P.; Brynda, J.; Gitto, R.;
Agnello, S.; Pachl, P.; Supuran, C. T.; Chimiri, A.; Rezacova, P. J. Med. Chem. 2011,
54, 2522; (d) Mincione, F.; Benedini, F.; Biondi, S.; Cecchi, A.; Temperini, C.;
Formicola, G.; Pacileo, I.; Scozzafava, A.; Masini, E.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2011, 21, 3216; (e) Bertucci, A.; Innocenti, A.; Scozzafava, A.;
Tambutté, S.; Zoccola, D.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2011, 21, 710.
9. Di Fiore, A.; Maresca, V.; Alterio, C. T.; De Supuran, G.; De Simone Chem.
Commun. 2011, 47, 11636.
0
0
0
2CH2, C1 ), 5.34 (d, 2H, J = 1.5 Hz, C3 ), 5.44 (d, 2H, J = 1.2 Hz, C3 ),
6.69 (d, 2CH, J = 8.7 Hz, C3 and C5), 7.52 (d, 2CH, J = 8.8 Hz, C2 and
137
0
C6).
C NMR (CD3OD, 100 MHz, ppm) d: 54.3 (2CH2, C1 ), 114.3
0
(2CH, C3 and C5), 116.4 (2CH2, C3 ), 126.5 (C1), 130.6 (2CH, C2 and
0
C6), 138.2 (C2 ), 154.7 (C4). MS (IES+, ACN): m/z 343 and 345
[M+Na]+. HRMS (ESI, CH3OH): m/z 343.0050 (m/z theoretical:
343.00507) [M+Na]+.
10. Hen, N.; Bialer, M.; Yagen, B.; Maresca, A.; Aggarwal, M.; Robbins, A. H.;
McKenna, R.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2011, 54,
3977.
4.2. CA Inhibition
11. (a) Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J.; Scozzafava, A.; Pastorekova, S.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2003, 13, 1005; (b) Carta, F.; Garaj, V.;
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therein; (c) Alp, C.; Maresca, A.; Alp, N. A.; Gultekin, M. S.; Ekinci, D.;
Scozzafava, A.; Supuran, C. T. J. Enz. Inhib. Med. Chem. 2012. http://dx.doi.org/
10(3109/14756366), 2012, 658788.
12. Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S.-A.; Scozzafava, A.;
Quinn, R. J.; Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057.
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An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity.32 Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM
Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 for maintaining con-
stant the ionic strength, following the initial rates of the CA-cata-
lyzed CO2 hydration reaction for a period of 10–100 s. The CO2
concentrations ranged from 1.7 to 17 mM for the determination
of the kinetic parameters and inhibition constants. For each inhib-
itor 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 to-
tal observed rates. Stock solutions of inhibitor (10 mM) were pre-
pared in distilled-deionized water and dilutions up to 0.01 nM
were done thereafter with the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min at room temper-
ature prior to assay, in order to allow for the formation of the E–I
complex. The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3, whereas the kinetic param-
eters for the uninhibited enzymes from Lineweaver–Burk plots, as
reported earlier,8–10 and represent the mean from at least three dif-
ferent determinations. The four CA isoforms were recombinant
ones, obtained in-house as reported earlier.8–10
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