Synthesis and investigation of inhibition effect of fluorinated sulfonamide derivatives on carbonic anhydrase

Article abstract of DOI:10.1016/j.ejmech.2009.11.052

Series of perfluoroalkanesulfonamides 1, sodium salt of perfluoroalkanesulfonamides 2 and polyfluoroalkanesulfonamides 3 derivatives were synthesized and characterized by ¹H NMR, ¹³C NMR, ¹⁹F NMR, IR and HRMS. Inhibition effects of these compounds on bovine carbonic anhydrase (bCA) and human carbonic anhydrase isoenzyme II (hCA) have been investigated. Comparing IC₅₀ values of the synthesized molecules 1, 2 and 3, it has been found that compound 2b is a more potent inhibitor than acetazolamide on hCA. Moreover 2b does not present cellular toxicity on sheep red globules.

Full text of DOI:10.1016/j.ejmech.2009.11.052

European Journal of Medicinal Chemistry 45 (2010) 1225–1229
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and investigation of inhibition effect of fluorinated sulfonamide
derivatives on carbonic anhydrase
a
b
c
a
a,
*
Zohra Benfodda , Franck Guillen , Bernard Romestand , Abdelkader Dahmani , Hubert Blancou
a
Institut des Biomol e´ cules Max Mousseron (IBMM) UMR CNRS 5247 Universit e´ de Montpellier I et de Montpellier II, CC1706, Place Eug e` ne Bataillon 34095 Montpellier
cedex 05, France
S3F Chimie Universit e´ de Montpellier II, CC1706, Place Eug e` ne Bataillon 34095 Montpellier cedex 05, France
b
c
Laboratoire Ecosyst e` mes lagunaires, UMR 5554 Universit e´ de Montpellier II, CC, Place Eug e` ne Bataillon 34095 Montpellier cedex 05, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 August 2009
Received in revised form
Series of perfluoroalkanesulfonamides 1, sodium salt of perfluoroalkanesulfonamides 2 and poly-
fluoroalkanesulfonamides 3 derivatives were synthesized and characterized by 1H NMR, C NMR,
13
19
F
NMR, IR and HRMS. Inhibition effects of these compounds on bovine carbonic anhydrase (bCA) and
human carbonic anhydrase isoenzyme II (hCA) have been investigated. Comparing IC50 values of the
synthesized molecules 1, 2 and 3, it has been found that compound 2b is a more potent inhibitor than
acetazolamide on hCA. Moreover 2b does not present cellular toxicity on sheep red globules.
Ó 2009 Elsevier Masson SAS. All rights reserved.
20 October 2009
Accepted 27 November 2009
Available online 5 December 2009
Keywords:
Perfluoroalkanesulfonamides
Sodium salt of perfluoroalkanesulfonamides
Carbonic anhydrase
Cellular toxicity
1. Introduction
perfluoroalkanesulfonamides could complex the Zn metal of the
metalloenzyme [10].
Sulfonamides of the type RSO
2
NH
2
occur in numerous biologi-
3 2 2
Moreover, It was shown previously that CF SO NH could
cally active compounds which include antimicrobial drugs, salu-
retics, insulin releasing sulfonamides, anthyroid agents, antitumour
drugs, carbonic anhydrase inhibitors and number of other biolog-
ical activities [1–9]. However investigations on poly-
fluoroalkanesulfonamides or perfluoroalkanesulfonamides with
a long chain have not been carried out in the biological area.
In our laboratory we studied perfluoroalkanesulfonamides with
a long chain and their derivatives in order to inhibit carbonic
anhydrase. Indeed, our working idea is that on one hand the per-
fluoroalkyl chain is very hydrophobic and we thought that per-
fluoroalkanesulfonamides could push back the water which is
formed at the time of the enzymatic catalyse. On the other hand we
showed that perfluoroalkanesulfonamides could bind different
metal ions in particular divalents cations and the hypothesis is that
inhibit carbonic anhydrase, these findings comfort us to prepare
and to evaluate the different perfluoroalkanesulfonamides on
carbonic anhydrase [11,12].
We herein report the synthesis of different perfluoro-
alkanesulfonamides, sodium salts of perfluoroalkanesulfonamides
and polyfluoroalkanesulfonamides in order to study their inhibi-
tion on bovine carbonic anhydrase (bCa) and human carbonic
anhydrase isoenzyme II (hCa). Finally, the cellular toxicity of these
compounds was evaluated on sheep red globules.
2. Results and discussion
2.1. Chemistry
In this study, the perfluoroalkanesulfonamides 1 were prepared
Abbreviations: bCA, Bovine carbonic anhydrase; hCA, Human carbonic anhy-
drase isoenzyme II.
Corresponding author. Institut des Biomol e´ cules Max Mousseron (IBMM) UMR
CNRS 5247 Universit e´ de Montpellier I et de Montpellier II, CC1706, Place Eug e` ne
in good yields by reaction of perfluoroalkanesulfonyl fluorides with
an excess of ammonia without any solvent at room temperature
*
(Scheme 1).
The perfluoroalkanesulfonamides were easily purified by crys-
Bataillon 34095 Montpellier cedex 05, France. Tel.: þ33 4 67 14 39 19; fax: þ 33 4 67
1
tallisation in ethanol/water media. They were characterized by H,
6
3 10 46.
13
19
E-mail address: hubert.blancou@univ-montp2.fr (H. Blancou).
C, F NMR spectroscopy, HRMS and IR.
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.052

1226
Z. Benfodda et al. / European Journal of Medicinal Chemistry 45 (2010) 1225–1229
-
CH O Na
3
+
NH3
-
+
R SO F
R SO NH
R SO NH Na
F 2
F
2
F
2
2
and
Et O
2
FSO (CF ) SO F
2a R = C F
4 9
98%
2
2 4
2
F
F
1
1
1
1
1
a R = C F
4 9
77%
79%
F
2
b R = C F 90%
6
13
17
b R = C F
F
6
13
2
c R = C F
94%
F
8
c R = C F
76%
80%
2
F
7
15
d R = C F
F
8 17
e R = NH SO (CF ) 76%
F
2
2 4
Scheme 1. Synthesis of perfluoroalkanesulfonamides and sodium salts derivatives.
Then, the perfluoroalkanesulfonamides underwent a reaction
with sodium methylate in a Et O/CH OH media to give in excellent
yields with the corresponding sodium sulfonamides (2 as described
in Scheme 1). These compounds were characterized by F NMR
spectroscopy and HRMS.
inhibition constants in the range of 1.901–2.089
than that of acetazolamide (1.351 M).
For the polyfluoroalkanesulfonamides (3) bCA inhibition varied
with the perfluorinated alkyl chain length. The poly-
mM which is less
2
3
m
19
fluoroalkanesulfonamides (3) with the C
inhibitor than those with the C 13 chain (3b), which were also
more inhibitor than those with C chain (3a).
8
F17 chain (3c) were more
The polyfluoroalkanesulfonamides 3 were prepared, in good
6
F
F
4 9
yields by reaction of polyfluoroalkanesulfonyl chlorides with
ꢀ
gaseous NH
3
in AcOEt at 0 C (Scheme 2).
The polyfluoroalkanesulfonamides were easily purified by
crystallisation in ethanol/water media or by sublimation. These
compounds were characterized by H, C, F NMR spectroscopy,
HRMS and IR.
Indeed, we have synthesized two alkanesulfonamides 4 from
corresponding alkanesulfonyl chorides using the same method
2.2.2. Human carbonic anhydrase isoenzyme II (hCA)
hCA II is generally considered as the main therapeutic target of
sulfonamides carbonic anhydrase inhibitors.
To confirm the study model realised on bCA, we have evaluated
the inhibition of hCA II by the compound which present the best
inhibition properties (IC50) on bCA in particular the sodium salt of
1
13 19
(
Scheme 3) in order to compare them with the per-
perfluorohexanesulfonamides (2b, IC50:1.380
m
M). We found that
M) was a more potent inhibitor than
mM) on hCa. (Table 1).
fluoroalkanesulfonamides conterparts in their biological activities.
compound 2b (IC50: 0.122
m
acetazolamide (IC50: 0.152
It was known that F-alkylated chain has two main properties,
they are very hydrophobic (from C chain) and they have an
2.2. Carbonic anhydrase inhibition
4 9
F
electron-attracting effect (–I) very important and relatively
constant with the length of the F-alkylated chain studied. These
properties can play two roles on one hand the electron-acceptor
effect (–I) stabilize the sodium salt of perfluoroalkanesulfonamide
Among the zinc enzymes extensively studied recently, the
carbonic anhydrases (CAs) have a special place by the reasons that
these enzymes are ubiquitous in all kingdoms. They catalyse the
þ
ꢁ
3
)
reversible hydration of carbon dioxide (H
2
O þ CO
2
¼ H þ HCO
(2) and this ion seems to be very good complexing metal ion by its
and as such are vital to many biological and physical functions. The
inhibition of these enzymes may be clinically exploited in the
treatment or prevention of a variety of disorders [13,14].
delocalization of charge. On the other hand the hydrophobic
F-alkylated chain could have a favorable positioning in the enzy-
matic site (interaction between the hydrophobic F-alkylated chain
with the hydrophobic pocket of the enzyme) with the stabilization
possibility by hydrogen bond.
2
.2.1. Bovine carbonic anhydrase (bCA) inhibition
As can be seen from data in Table 1, there is a very sharp
difference in inhibition activity on bCA, between the per-
fluoroalkanesulfonamides 1a–e, the sodium salts of per-
fluoroalkanesulfonamides 2a–c, the polyfluoroalkanesulfonamides
2
.2.3. Cellular toxicity on sheep red globules
We knew that some perfluoroalkanesulfonamides in particular
d present toxicity [10,12]. We decided to evaluate the cellular
2
3
a–c on the one hand and the carbohydrogenated compounds 4a–b
toxicity on sheep red globules of some sulfonamides. The results
were summarized in the Graph 1.
The sodium salt of perfluorohexanesulfonamide 2b did not
present a cellular cytotoxicity whatever the concentration studied.
Trifluoromethanesulfonamide and perfluorohexanesulfonamide
on the other hand. We can deduce that the substitution of hydrogen
by fluorine increases the inhibitory activity of these compounds.
Thus, perfluoroalkanesulfonamides CF SO NH , 1a,b,d and the
3 2 2
sodium salt of perfluorohexanesulfonamide 2b showed that inhi-
bition constants (IC50) are comparable to that of acetazolamide
1
b present a cellular cytotoxicity at concentration of 1000 mM. The
(
a clinically used sulfonamide) e.g in the range of 1.380–1.585
mM
sodium salt of perfluorobutanesulfonamide (2a) and the per-
fluorobutanesulfonamide (1b) (data not shown) did not present
a cellular cytototoxicity whatever the concentration studied.
The sodium salt of perfluorooctanesulfonamide 2c afford to the
and 1.351 M.
m
In our experiment, the sodium salt of perfluoro-
alkanesulfonamides (2a–c) were solubilized in water; they did not
need the addition of any cosolvents (DMSO in particular) for the
determination of the inhibition activity on bCA comparatively to
the others synthesized compounds. The perfluoroalkane-
sulfonamides 1c,e and the polyfluoroalkanesulfonamide 3a,b have
shown rather weak inhibitory properties against this enzyme with
hemolysis at concentration of 31.25
nesulfonamide 1d present a cellular cytotoxicity at concentration of
2.5 M.
These results seems to be promising to continue to explore the
sodium salt of perfluoroalkanesulfonamide and their derivatives in
mM. The perfluoroocta-
6
m
NH3
R (CH ) SO NH
NH3
AcOEt/ 0°C
R (CH ) SO Cl
F
2 2
2
2
F
2 2
2
R SO NH
H 2 2
R SO Cl
H
2
AcOEt/ 0°C
3
3
3
a R = C F
4 9
90%
F
b R = C F 89%
4a R = C H
4 9
92%
88%
F
6
13
H
c R = C F
91%
4b R = C H
H 8
F
8
17
17
Scheme 2. Synthesis of polyfluoroalkanesulfonamides.
Scheme 3. Synthesis of alkanesulfonamides.

Z. Benfodda et al. / European Journal of Medicinal Chemistry 45 (2010) 1225–1229
1227
Table 1
Inhibitor
chlorides were synthesized according to a new method devel-
oped in our laboratory (publication under review). Per-
fluoroalkanesulfonyl fluorides were a gift from Elf Atochem.
Solvents were distilled from the appropriate drying agents imme-
diately prior to use.
M)a
IC50
(
m
bCAb
hCA IIc
a
a
Ac e´ tazolamide
CF SO NH
1.351
1.495
1.486
1.473
1.722
1.585
1.735
1.670
1.380
1.789
2.089
1.901
1.658
0.152
a
a
a
a
a
a
a
a
a
a
a
a
e
3
2
2
ND
1
H, F, and 13C NMR spectra were recorded at 300.13 MHz,
19
e
1
1
1
1
1
2
2
2
3
3
3
4
4
a
b
c
ND
e
ND
282.37 MHz and 75.46 MHz respectively with a Brucker Avance 300
spectrometer; the chemical shifts are given in ppm relative to
e
ND
e
d
e
a
b
c
a
b
c
ND
1
13
19
4 3
Me Si for the H and C, and CCl F for F, as internal standards.
e
ND
e
Coupling constants are given in Hz. Mass spectra and HRMS were
ND
a
recorded on a Jeol SX 102 spectrometer. IR Spectra were obtained
0.122
e
ꢁ1
ND
using a Nicolet 205 FT-IR instrument (
n
cm ). Compounds 4a and
e
ND
4b were subjected to a quantitative elemental (C, H, N) analysis by
e
ND
a Flash EA 1112 (Thermo Finnigan) instrument. Melting points were
recorded at atmospheric pressure unless otherwise stated on
a Stuart scientific SMP3 apparatus and were uncorrected.
e
ND
d
e
a
b
NI
NI
ND
d
e
ND
a
Mean ꢂ Errors in the range 5–10% of the reported value (from 3 different
assays).
4.1.1. General procedure for the synthesis of perfluoro-
alkanesulfonamides 1a–e
b
Bovine carbonic anhydrase.
c
d
e
Human carbonic anhydrase isoenzyme II.
NI: non inhibitor at these inhibition concentrations.
ND: not determined.
A
typical procedure for perfluoroalkanesulfonamides is
described. In 100 mL dried stainless steel bomb, per-
a
fluoroalkanesulfonyl fluoride (1 mol) was placed with magnetic
ꢀ
particular 2b because of their inhibition properties on carbonic
stirrer, at 25 C. Then ammonia (3 mol) was transferred into the
anhydrase and the absence of cellular toxicity.
bomb through the vacuum line within 1 h. The excess of ammonia
was then removed with a flow of dry N
2
. The solid product
F) was acidified with an aqueous HCl fol-
lowed by the addition of Et O. The organic layer was extracted and
dried over anhydrous Na SO and concentrated under reduced
pressure. The crude product was purified by recrystallization from
EtOH/H O (90/10, V/V) to give white crystals of the corresponding
3
. Conclusion
ꢁ
þ
(R
F
SO
2
NH NH and NH
4
4
2
In summary, series of perfluoroalkanesulfonamides 1, sodium salt
2
4
of perfluoroalkanesulfonamide 2, polyfluoroalkanesulfonamides 3
were synthesized and their inhibitory activities on carbonic anhy-
drase have been evaluated. All compounds demonstrated potent
inhibition against bovine carbonic anhydrase. Moreover, compound
2
sulfonamides.
The synthesis of perfluorobutanesulfonamide 1a and per-
fluorooctanesulfonamide 1c were described previously [15].
2
b was also evaluated on human carbonic anhydrase isoenzyme II
and it presented better inhibitory properties than the commercial
drug acetazolamide. Finally the cellular toxicity of these compounds
was evaluated on sheep red globules. We found that 2b did not
present cellular cytotoxicity.
4.1.1.1. Synthesis of 1,1,2,2,3,3,4,4,5,5,6,6,6-tridecafluoro-n-hexane-1-
sulfonamide 1b. Yield: 79%; IR (KBr): 3458 (
n
NH2), 1375 (
-acetone):
-acetone):
ꢁ125.85 (m,
CF (CF
)), ꢁ121.45 (m,
(CF CF CF )),
)), ꢁ80.77 (m, 3F, (CF (CF )); MS
13HNO S:
n
SO2), 1147
ꢀ
1
(
n
CF); Mp: 119–121 C; H NMR (300.13 MHz, d
6
d
4.90
19
(m, 2H, SO
2
NH
(CF
(CF
113.79 (m, 2F, (CF
2
); F NMR (282.37 MHz, d
)), ꢁ122.37 (m, 2F, (CF CF
CF (CF
6
d
4
. Experimental
2
2
F, (CF
3
CF
2
2
)
4
3
2
2
2 3
)
F, (CF
3
2
)
2
2
2
)
2
)), ꢁ119.79 (m, 2F, (CF
3
2
)
3
2
2
4.1. Chemistry
ꢁ
3
(CF
2
)
4
CF
2
3
2 5
)
ꢁ
þ
(
FAB , NBA): [M ꢁ H ] ¼ 398; HRMS calcd for
C
6
F
2
Moisture sensitive reactions were carried out under dry
397.9520, found: 397.9521.
nitrogen. Polyfluoroalkanesulfonyl chlorides and alkanesulfonyl
4
.1.1.2. Synthesis of 1,1,2,2,3,3,4,4,5,5,6,6,7,7,7-pentadecafluoro-n-
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
1
1
2
d
b
b
heptane-1-sulfonamide 1c. Yield: 76%; IR (KBr): 3460 (
n
NH2), 1380
-acetone):
ꢁ126.73
ꢀ
1
(
d
n
SO2),1147 (
8.2 (m, 2H, SO
(m, 2F, CF CF (CF
(CF
); MS (FAB , NBA): [M ꢁ H ] ¼ 448; HRMS calcd for
S: 447.9488, found: 447.9500.
n
CF);Mp: 128–130 C; H NMR (300.13 MHz, d
6
1
9
NH
2
); F NMR (282.37 MHz, d
), ꢁ123.31 (m, 2F, CF CF CF
(CF (CF
), ꢁ114.34 (m, 2F, CF
6
-acetone): d
CF3SO2NH2
2
2
c
3
2
)
2 5
3
2
2
(CF
2
)
CF
4
), ꢁ122.29 (m,
4
3
C
F, CF
F, CF
15HNO
3
(CF
2
)
2
)
2 2
2
)
2
3
)
2 5
2
), ꢁ81.72 (m,
ꢁ
þ
3
(CF
2 6
)
7
F
2
4.1.1.3. Synthesis of 1,1,2,2,3,3,4,4 octafluoro-n-butane-1-4-bis-
sulfonamide 1e. Yield: 76%; IR (KBr): 3462 ( NH2), 1375 ( SO2), 1145
8.10 (m,
n
n
ꢀ
1
(n
CF); Mp: 114–116 C; H NMR (300.13 MHz, d
6
-acetone):
d
1
9
4
H, (SO
F,
2
NH
NSO
CF (CF
HRMS calcd for C
2
)
2
); F NMR (282.37 MHz, d
CF (CF CF SO NH );
CF SO NH
6
-acetone):
d
ꢁ114.24 (m,
4
H
H
2
2
2
2
)
2
2
2
2
ꢁ120.76
(m,
4F,
ꢁ
þ
2
NSO
2
2
2
)
2
2
2
2
); MS (FAB , NBA): [M ꢁ H ] ¼ 359;
4 8 3 2 4 2
F H N O S : 358.9407, found: 358.9390.
4.1.2. General procedure for the synthesis of sodium
Concentration (µM)
perfluoroalkanesulfonamides (2a–c)
A solution of perfluoroalkanesulfonamides (1 mol), dissolved in
anhydrous diethyl ether was added dropwise to a suspension of
3 2 2
Graph 1. Determination of cellular toxicity of sulfonamides (CF SO NH , 1b, 1d, 2b, 2c)
on sheep red globules.

1228
Z. Benfodda et al. / European Journal of Medicinal Chemistry 45 (2010) 1225–1229
sodium methylate (95%) in anhydrous methanol (0.9 mol). The
mixture was refluxed for 3–5 h and then filtered. The filtrate was
concentrated in vacuo and the residue was washed three times
with diethyl ether to remove the excess of perfluoroalk-
anesulfonamide and then dried under vacuum.
4.1.3.5. Synthesis of n-octanesulfonamide 4b. Yield: 82%; IR (KBr):
ꢀ
1
3465 (
(300.13 MHz, d
n
NH2), 1365 (
-acetone):
), 1.42 (m, 2H, CH
CH ), 2.0 (m, 2H, SO
n
SO2), 2975 (
0.9 (t, 3H, CH
(CH CH
NH
-acetone):
n
CH); Mp: 76–78 C; H NMR
(CH ), 1.30 (m, 8H,
(CH ), 1.91 (m, 2H,
), 3.05 (m, 2H,
14.34 (CH ),
CH S), 28.60
6
d
3
2 5
)
)
2 2
CH
CH
CH
3
3
3
(CH
(CH
(CH
2
2
2
)
4
)
5
)
6
(CH
2
)
3
3
2
)
4
2
CH
CH
2
2
2
2
2
1
3
The synthesis of sodium perfluorobutanesulfonamide 2a, sodium
perfluorohexanesulfonamide 2b, and perfluorooctanesulfonamide
); C NMR (75.46 MHz, d
6
d
3
23.29, 29.78, 29.89 et 32.47 (CH
(CH CH (CH , 55.69 (CH
HRMS calcd for C
2
)
4
, 24.84 (CH
2
2
ꢁ
þ
2
c were described previously [15,16].
2
)
2
2
2
)
2
2
S); MS (FAB , NBA): [M ꢁ H ] ¼ 192;
8
H18NO
2
S: 192.1058; found: 192.1054. Anal. Calcd.
4
.1.3. General procedure for the synthesis of polyfluoroalkanesulfon-
for C
7.26
8 2
H19NO S: C, 49.71; H, 9.91; N, 7.25. Found: C, 49.60; H, 9.95; N,
amides and alkanesulfonamides (3 and 4)
Polyfluoroalkanesulfonyl chlorides or alkanesulfonyl chlorides
(
1 mol) was dissolved in AcOEt and gaseous NH
3
was bubbled
4.2. Biological
ꢀ
through the solution for 30 min at 0 C. The mixture was stirred at
room temperature for 15 min, then filtered and evaporated. The
residue was dissolved with AcOEt and washed with water and
Bovine carbonic anhydrase (bCA) and human carbonic isoen-
zyme 2 (hCA) were purchased as a lyophilised powder from Sigma
brine. The organic layer was dried over anhydrous Na
concentrated under reduced pressure. The crude product was
purified by recrystallization from EtOH/H O (90/10) to give white
crystals of the corresponding sulfonamides.
2
SO
4
and
Chemical Co. All reagents used were of analytical grade. CF
and acetazolamide were purchased from aldrich.
3 2 2
SO NH
2
4.2.1. Evaluation of inhibition activity on bCA and hCA
Native enzyme concentrations were determined from the
absorbance at 280 nm, using molar absorbance of
4
3
9
3
.1.3.1. Synthesis of 3,3,4,4,5,5,6,6,6-nonafluoro-n-hexanesulfonamide
a. Yield: 90%; IR (KBr): 3478 ( NH2), 1380 ( SO2), 1147 ( CF); Mp:
-acetone): 2.72 (m, 2H, C CH ),
); F NMR (282.37 MHz,
(CF
), ꢁ124.88 (m, 2F,
(CF CF
), ꢁ82.01 (m, 3F,
-acetone): 22.28 (m, 2H, C CH ),
); MS (FAB , NBA): [M ꢁ H ] ¼ 326;
S: 325.9897, found: 325.9888.
a
4
ꢁ1
ꢁ1
4
ꢁ1
ꢁ1
n
n
n
5.7 ꢃ 10 M cm for bCA and 5.5 ꢃ10 M cm for the hCA. All
ꢀ
1
2ꢁ
6–98 C; H NMR (300.13 MHz, d
.45 (m, 2H, C CH CH ), 8.1 (m, 2H, SO
-acetone): CF
ꢁ126.72 (m, 2F, CF
CF CF CF CF
), ꢁ114.44 (m, 2F, CF
CF (CF
6.81 (m, 2H, CH
HRMS calcd for C
6
d
4
F
9
2
enzyme preparations were stored in a mixture of 0.05 M TrisSO
4
/
19
ꢀ
4
F
d
9
2
2
2
NH
2
1 mM mercaptoethanol pH 8.7 at 4 C. Enzymes concentrations
ꢁ
6
ꢁ6
d
6
3
2
2
)
2
were 2.9 ꢃ 10 M for the bCA and 0.82 ꢃ 10 M for the hCA.
Initial rate of 4-nitrophenyl acetate (4-NPA) hydrolysis was
estimated by a modification of the method of Verpoorte et al.
[17,18]. The increase in absorbance was followed at 348 nm for
3
2
2
2
3
3
2
)
2
2
1
3
2
)
3
); C NMR (75.46 MHz, d
), 110–120 (C
NO
6
d
F
4 9
2
-
þ
4
2
4 9
F
6
F
9
H
5
2
approximatively 20 min. Steady state measurements were made at
ꢀ
2
5 C in a Kontron Uvikon 860 spectrophotometer. Solutions of
4
.1.3.2. Synthesis of 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-n-octane-
sulfonamide 3b. Yield: 89%; IR (KBr): 3478 ( NH2), 1377 ( SO2), 1147
CF); Mp: 131–133 C; H NMR (300.13 MHz, d -acetone): 2.72
m, 2H, C 13CH ), 3.42 (m, 2H, C 13CH CH ), 8.2 (m, 2H,
); F NMR (282.37 MHz, d -acetone):
ꢁ126.81 (m, 2F,
(CF CF CF (CF ), ꢁ123.50
), ꢁ123.93 (m, 2F, CF
(CF (CF (CF CF CF ), ꢁ114.19
), ꢁ122.49 (m, 2F, CF
m, 2F, CF (CF CF (CF
), ꢁ81.79 (m, 3F, CF
75.46 MHz, d -acetone): 26.65 (m, 2H, C
), 110–120 (C
13); MS (FAB , NBA): [M ꢁ H ] ¼ 426; HRMS
calcd for C NO S: 425.9912, found: 425.9905.
substrate were prepared in acetone/H
2
0 media; the substrate
ꢁ3
ꢁ4
n
n
concentrations varied from 1.04 ꢃ10 M to 1.875 ꢃ10 M for the
ꢀ
1
ꢁ4
(
n
6
d
bCA and 9.4 ꢃ10 M for hCA. A molar absorption coefficient
3 of
3
ꢁ1
ꢁ1
(
SO
CF
6
F
2
6
F
2
2
16.3 ꢃ 10 M cm was used for the 4-nitrophenolate formed by
hydrolysis, in the conditions of the experiments (pH 8). Non
enzymatic hydrolysis rates were always subtracted from the
observed rates. Triplicate experiments were done for each substrate
concentration and for each inhibitor concentration. Solutions of
inhibitor were prepared in distilled–deionized water for
1
9
2
NH
CF
2
6
d
3
2
2
)
4
3
2
2
2 3
)
(
(
(
CF
3
2
)
2
CF
2
2
)
2
3
2
)
3
2
2
1
3
3
2
)
4
2
3
2
)
5
);
C NMR
6
d
F13CH ), 46.39 (m, 2H,
6 2
-
þ
ꢁ þ
CH
2
6
F
R SO NH Na (2) and in distilled–deionized water with 10% (v/v)
F 2
8
F
13
H
5
2
of DMSO (which is not inhibitory at these concentrations) for the
others inhibitors (1,3,4). Dilutions were done thereafter with
distilled–deionized water; the inhibitor concentrations varied
4
.1.3.3. Synthesis of 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadeca-
ꢁ
6
ꢁ6
fluoro-n-decanesulfonamide 3c. Yield: 91%; IR (KBr): 3475 (
n
NH2),
between 2.07 ꢃ 10
M
to 0.27 ꢃ 10
M
for the bCA and
ꢀ
1
ꢁ7
ꢁ7
1
377 (
acetone):
m, 2H, SO
nSO2), 1150 (
n
CF); Mp: 152–154 C; H NMR (300.13 MHz, d
17CH ); 3.41 (m, 2H, C 17CH CH ), 8.2
); F NMR (282.37 MHz, d -acetone):
ꢁ126.80 (m,
); ꢁ123.84 (m, 2F, CF CF CF (CF ); ꢁ123.33 (m,
CF (CF (CF (CF CF );
114.16 (m, 2F, CF (CF (CF ); C NMR
75.46 MHz, d -acetone): ); 46.10 (m, 2H,
CH ); 110–120 (C
calcd for C10 NO
6
-
6.75 ꢃ10 M to 0.675 ꢃ10 M for the hCA. Inhibitor and enzyme
solutions were preincubated together for 5 min at room tempera-
ture prior to assay. Acetazolamide was used as a specific inhibitor in
our studies, its inhibitory effectiveness was determined using
d
2.73 (m, 2H, C
8
F
2
8
F
2
2
19
(
2
2
2
NH
(CF
(CF
2
6
d
F, CF
3
CF
2
2
)
6
3
2
2
2 5
)
ꢁ
6
ꢁ6
F, CF
3
2
)
2
2
2
)
4
); ꢁ122.32 (m, 6F, CF
3
2
)
3
2
)
3
2
2.9 ꢃ 10 M of bCA, 0.82 ꢃ 10 M of hCA and varying acetazol-
1
3
ꢁ6
ꢁ7
ꢁ6
ꢁ
3
2
)
6
CF
2
); ꢁ81.70 (m, 3F, CF
3
2
)
7
amide concentration from 64 ꢃ 10 M to 0.27 ꢃ 10 M for the
7
(
6
d
26.68 (m, 2H, C F17CH
8 2
bCA and 6.75 ꢃ10ꢁ to 0.675 ꢃ10 M for the hCA.
-
þ
2
8
F
17); MS (FAB , NBA): [M ꢁ H ] ¼ 526; HRMS
F
17
H
5
2
S: 525.9848, found: 525.9860.
4.2.2. Evaluation of cellular toxicity on sheep red globules
To determine cellular toxicity we used an hemolytic assay
described [19]. The hemolytic activity of the compounds was
determined using sheep blood cells. The cells were washed three
times in buffered saline (PBS solution pH 7.4) just prior to the assay.
The final cell concentration used was 5% in the same buffer. The cell
4
3
.1.3.4. Synthesis of n-hexanesulfonamide 4a. Yield: 90%; IR (KBr):
ꢀ
1
460 (
n
NH2), 1360 (
-acetone):
), 1.45 (m, 2H, CH
CH ), 2.1 (m, 2H, SO
n
SO2), 2970 (
0.85 (t, 3H, CH
(CH CH
NH
-acetone):
n
CH); Mp: 46–48 C; H NMR
(CH ), 1.32 (m, 4H,
(CH ), 1.85 (m, 2H,
), 3.05 (t, 2H,
14.30 (CH ),
CH (CH
(
300.13 MHz, d
6
d
3
2 5
)
)
2 2
CH
CH
CH
2
5
C
3
(CH
3
(CH
3
(CH
2
2
2
)
2
)
3
)
4
(CH
2
)
3
3
2
)
2
2
CH
CH
2
2
2
2
suspension 10
m
L and 100
ml of varying amounts of inhibitors
1
3
2
); C NMR (75.46 MHz, d
6
d
3
solution (concentration 750
m
M–0.125 M in PBS) were added in
m
3.07 et 32.10 (CH
5.67 (CH
2
)
2
, 24.77 (CH
2
CH
2
S), 28.64 (CH
2
)
2
2
2
)
2
,
each tube. The resulting suspension was incubated for 20 min at
ꢁ
þ
ꢀ
2
S); MS (FAB , NBA): [M ꢁ H ] ¼ 164; HRMS calcd for
37 C, then centrifugated (5 min at 1000 rpm at room temperature).
6
H14NO
2
S: 164.0745, found: 164.0745. Anal. Calcd. for C
6
H15NO
2
S:
After centrifugation 50
ml of each supernatant was introduced in
C, 43.61; H, 9.15; N, 8.48. Found: C, 43.50; H, 9.25; N, 8.30
the plates Nunc Elisa and the absorbance monitored at 405 nm.

Z. Benfodda et al. / European Journal of Medicinal Chemistry 45 (2010) 1225–1229
1229
Two controls 0% and 100% of hemolysis were determined using the
supernatant after centrifugation of 10 L of the erythrocyte stock
suspension added respectively with PBS or distillate water and
analyzed after the same conditions.
[4] D. Mandloi, S. Joshi, P.V. Khadikar, N. Khosla, Bioorg. Med. Chem. Lett. 17
2005) 15–40.
(
m
[5] D. Vullo, B. Steffansen, B. Brodin, C.T. Supuran, A. Scozzafava, C.U. Nielsen,
Bioorg. Med. Chem. 14 (2006) 2418–2428.
[6] A. Sherif, F. Rostom, Bioorg. Med. Chem. 14 (2006) 6475–6485.
[7] M. A Santos, S.M. Marques, T. Tuccinardi, P. Carelli, L. Panelli, A. Rossello,
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Acknowledgements
[
[
7
67–787.
We gratefully acknowledge the « Minist e` re de l’Education
Nationale et de la Recherche » for its financial support (Ph.D. grant)
and the CNRS.
[
[
10] H. Blancou, F. Guillen, WO2004046093.
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