Carbonic anhydrase inhibitors. Novel sulfanilamide/acetazolamide derivatives obtained by the tail approach and their interaction with the cytosolic isozymes I and II, and the tumor-associated isozyme IX

Article abstract of DOI:10.1016/j.bmcl.2004.10.070

A series of sulfonamides has been obtained by reacting sulfanilamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide with ω-chloroalkanoyl chlorides, followed by replacement of the ω-chlorine atom with secondary amines. Tails incorporating heterocyclic amines belonging to the morpholine, piperidine and piperazine ring systems have been attached to these sulfonamides, by means of an alkanoyl-carboxamido linker containing from two to five carbon atoms. The new derivatives prepared in this way were tested as inhibitors of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic isozymes CA I and II, and the catalytic domain of the transmembrane, tumor-associated isozyme CA IX. Several low nanomolar CA I and CA II inhibitors were detected both in the aromatic and heterocyclic sulfonamide series, whereas the best hCA IX inhibitors (inhibition constants in the range of 22-35 nM) all belonged to the acetazolamide-like derivatives.

Full text of DOI:10.1016/j.bmcl.2004.10.070

Bioorganic & Medicinal Chemistry Letters 15 (2005) 367–372
Carbonic anhydrase inhibitors. Novel
sulfanilamide/acetazolamide derivatives obtained by the
tail approach and their interaction with the cytosolic
isozymes I and II, and the tumor-associated isozyme IX
Hasan Turkmen,a,* Mustafa Durgun,a Serpil Yilmaztekin,a Mahmut Emul,a
Alessio Innocenti,b Daniela Vullo,b Andrea Scozzafavab andClaudiu T. Supuran
b,
*
aDepartment of Chemistry, Faculty of Science and Arts, Harran University, 63300 Sßanlıurfa, Turkey
b
`
Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188,
Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
Received24 September 2004; revised18 October 2004; accepted21 October 2004
Available online 13 November 2004
Abstract—A series of sulfonamides has been obtained by reacting sulfanilamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide with x-
chloroalkanoyl chlorides, followed by replacement of the x-chlorine atom with secondary amines. Tails incorporating heterocyclic
amines belonging to the morpholine, piperidine and piperazine ring systems have been attached to these sulfonamides, by means of
an alkanoyl-carboxamido linker containing from two to five carbon atoms. The new derivatives prepared in this way were tested as
inhibitors of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic isozymes CA I andII, andthe catalytic domain of
the transmembrane, tumor-associatedisozyme CA IX. Several low nanomolar CA I andCA II inhibitors were detectedboth in the
aromatic andheterocyclic sulfonamide series, whereas the best hCA IX inhibitors (inhibition constants in the range of 22–35nM) all
belonged to the acetazolamide-like derivatives.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Several sulfonamide CAIs are used clinically, such as
acetazolamide 2, methazolamide 3, dichlorophenamide
4, ethoxolamide 5, dorzolamide 6 andbrinzolamide 7.1,2
Inhibition of the zinc enzyme carbonic anhydrase (CA,
EC 4.2.1.1) with sulfonamides may be exploited clini-
cally for the treatment andprevention of a multitude
of diseases.1,2 With the early report that sulfanilamide
13 acts as an inhibitor of CA, a great scientific adventure
initiated, leading to the development of several classes of
drugs based on the sulfonamide motif, such as the anti-
hypertensives of the benzothiadiazine and high-ceiling
diuretics types, the CA inhibitors (CAIs) initially used
as antiglaucoma agents, some anti-thyroiddrugs, hypo-
glycemic sulfonamides and ultimately to some novel
Recently, the interest in this class of pharmacological
agents has been revivedby the finding that some of
the 15 presently known CA isozymes in humans, are
1,2,4–8
mainly foundin tumors,
andthat their inhibition
may leadto tumor cell growth inhibition by complex
mechanisms, that involve change of the tumor pH
among others.4–9 Very recently, Robertson et al.9c dem-
onstratedthe critical role playedby CA IX in tumor
growth andsurvival by using RNA interference proto-
cols. One of our groups on the other handreported
inhibitors of one of the isozymes involvedin tumorigen-
1,2,4–8
types of anticancer andantiviral agents.
4–9
esis, that is, CA IX,4–8 basedboth on the sulfonamide
as well as sulfamate types of compounds,10,11 showing
that such agents may be useful for the imaging of tum-
ors or for inhibiting their growth both alone, or in com-
bination with other anticancer agents.9 Thus, CA IX
represents a new andattractive target for the develop-
ment of novel anti-tumor agents possessing different
*
Corresponding authors. Tel.: +90 414 3440020/1203; fax: +90 414
3440051 (H.T.); tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S);
e-mail addresses: hturkmen@harran.edu.tr; claudiu.supuran@unifi.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.070

368
H. Turkmen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 367–372
SO2NH2
H
S
N
SO2NH2
H3C
N
N
2
O
Ethanol/HCl
Reflux
73%
NH2
(1)
H
S
S
S
H2N
SO2NH2
18
N
SO2NH2
N
SO2NH2
H3C
H3C
N
N
N
N
N
N
O
O
H3C
NEM/THF
-13 oC
Acyl Chloride
(2)
62%
()n
(3)
H2N
SO2NH2
1
SO2NH2
H
Cl(CH2)nCOCl
THF/NEM
S
N
SO2NH2
19
CH3CH2O
S
Cl
N
N
SO2NH2
O
H
Cl
SO2NH2
N
N
()n
Cl
Cl
SO2NH2
(5)
NEM/THF
R2NH
O
(4)
8
R2NH
O2
H3C
S
H
O
O
S
CH3O(CH2)3
S
S
S
H
SO2NH2
N
SO2NH2
N
N
()n
()n
SO2NH2
R2N
R2N
N
N
SO2NH2
O
O
NHC2H5
NHC2H5
20-24
9-17
(6)
(7)
Scheme 1. Synthesis of the new derivatives 9–17 and 20–24 reportedin
the paper.
mechanisms of action.1,2,9 Here we extendthis research
in the design of such enzyme inhibitors, and present
the synthesis andCA IX inhibition data for two series
of sulfonamides based on the sulfanilamide and acet-
azolamide motifs. These compounds have also been
testedfor the inhibition of the major cytosolic isozymes
I and II, widely distributed in the human body, and
participating in important physiological/pathological
processes.1,2
(TEA) or N-ethyl-morpholine (NEM). The chloro-sub-
stituted amide key intermediates 8 were then treated
with secondary amines such as methylpiperazine,
morpholine, benzyl-piperidine, benzyl-piperazine and
methyl-piperidine in dry THF, in order to obtain the de-
siredtertiary amines 9–17 (Table 1).
Our secondaim was to synthesize novel acetazolamide
derivatives of type 20–24, by the same approach de-
scribed above for the corresponding sulfanilamide com-
pounds 9–17 (Scheme 1). Acetazolamide 2 was usedas
starting material, being deacetylated with concentrated
hydrochloric acid, when 5-amino-1,3,4-thiadiazole-2-
sulfonamide 18 was obtained.14 The secondstep of the
synthesis consistedin the reaction of amine 18 with chlo-
roacyl chlorides (i.e., 2-chloroethanoyl chloride or 3-
chloropropanoyl chloride), leading to the second key
intermediates, of types 19, which have been further deri-
vatizedas shown above for the corresponding sulfanil-
amide compounds. The new sulfonamides 20–24 were
thus obtainedin goodyield( Table 2 and Scheme 1).15
2. Chemistry
In previous work12 from one of our groups, some ana-
logues of acetazolamide and a-fluorobenzyl sulfon-
amides were synthesized. In vitro inhibitory activity of
such compounds showed them to be potent inhibitors
of isozyme CA II. Considering the fact that a general
approach for the synthesis of strong CAIs with different
applications both as antiglaucoma or anti-tumor agents
among others has recently been described, that is, the
tail approach,13 in this paper we report the application
of the tail approach for the synthesis of CAIs incorpo-
rating protonatable heterocyclic moieties in the sulfanil-
amide and acetazolamide scaffolds, in order to design
putative CA IX inhibitors with enhancedaffinity for
the enzyme andeventually increasedwater solubility.
3. CA inhibition
Inhibition data against three CA isozymes, that is, hCA
I, II andIX, with the new sulfonamides reportedhere
Our first aim was to synthesize novel sulfanilamide
derivatives of type 9–17 (Scheme 1). Sulfanilamide 1
was reactedwith 3-chloropropionyl chloride (or the con-
generic derivatives with one, two or four carbon atoms
in their molecule) in the presence of triethylamine
andsome standardCAIs, of types
Table 3.16
1–6, are shown in
The following should be noted regarding data of Table
3: (i) derivatives 9–17 and 20–24 reportedhere generally

H. Turkmen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 367–372
369
Table 1. Sulfanilamide derivatives 9–17 preparedin this study
Table 3. Inhibition data for sulfonamides 9–24 investigatedin the
present paper and standard sulfonamide CAIs 1–6, against isozymes
16
hCA I, II andIX
O
R2N
Inhibitor
KIa (nM)
Selectivity ratio
SO2NH2
N
H
n
hCA Ib
hCA IIb
hCA IXc
KI (hCA II)/
KI (hCA IX)
Sulfanilamide Derivatives
1d
2d
3d
4d
5d
6d
9
28,000
900
780
300
12
294
25
1.02
0.48
0.52
0.23
0.76
0.05
0.70
0.44
0.97
0.87
1.04
0.48
2.94
0.42
0.47
1.46
0.04
0.10
0.02
0.05
0.05
n
Compdno
R
2N–
Yield%
15
14
27
34
2
9
H3C
N
N
25
8
38
1200
50,000
644
50
52
9
2
2
2
2
10
11
12
13
75
65
72
62
O
N
165
82
235
184
240
197
248
181
90
10
11
12
13
14
15
16
17
18d
20
21
22
23
24
9.4
549
381
752
234
173
258
87
N
N
N
9.6
820
371
265
104
77
H3C
N
246
163
41
7.9
8600
14.0
60
0.9
3.8
1.8
1.6
1.9
22
35
8.2
7.6
9.6
7.1
67
31
33
1
3
3
14
15
16
70
64
65
O
N
a Errors in the range of 5–10% of the reportedvalue (from three dif-
ferent assays).
b Human (cloned) isozymes, by the CO2 hydration method.
H3C
O
N
c Catalytic domain of human, cloned isozyme, by the CO2 hydration
method.23
d From Ref. 24.
N
N
act as better inhibitors against all three investigatediso-
zymes, as comparedto the parent sulfonamides from
which they were prepared, that is, sulfanilamide 1 in
the case of the first derivatives, and aminothiadiazole-
sulfonamide 18, in the case of the acetazolamide-like
derivatives 20–24; (ii) against hCA I, three sulfanil-
amides, that is, 10, 14 and 17, andall the acetazol-
amide-like derivatives 20–24, act as very potent
inhibitors, with inhibition constants in the range of
7.1–14.0nM, being much more effective than the two
leadcompounds 1 and 18, which showed KI values in
the range of 8600–28,000nM. These compounds are also
much better hCA I inhibitors as comparedto the clini-
cally usedderivatives 1–6 (Table 3). It is very interesting
to note that for the sulfanilamide derivatives 10, 14 and
17, all these potent inhibitors incorporate the morpho-
line moiety in their tail, whereas for the heterocyclic ser-
ies 20–24, the corresponding morpholine derivative (20)
was the least active. It is quite difficult to rationalize
these results at the present moment. The number of ali-
phatic carbon atoms separating this heterocyclic amine
moiety from the sulfanilamide scaffold in derivatives
10, 14 and 17 seems to be less important for these aro-
matic CAIs, since both the mono-, two- or four-carbon
atoms derivatives showed quite comparable enzyme
inhibitory activity. On the other hand, the other aro-
matic derivatives, that is, 9, 11–13, 15 and 16, showed
moderate hCA I inhibitory properties, with KI values
in the range of 371–820nM; (iii) against hCA II, the
five heterocyclic sulfonamides 20–24 showedexcellent
4
17
65
O
Table 2. Acetazolamide-like derivatives 20–24 synthesizedin this study
H
S
N
SO2NH2
n
R2N
N
N
O
n
Compdno
R
2N–
Yield%
70
O
1
20
N
1
1
21
22
60
60
N
N
N
CH
2
N
N
1
2
23
24
50
65
N
N

370
H. Turkmen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 367–372
inhibitory properties, with KI values in the range of 0.9–
3.8nM, being thus much better inhibitors than the clini-
cally usedcompounds 1–6 or the leadmolecule 18. In
the aromatic subseries, again 10, 14 and 17 were the best
inhibitors, with KI values in the range of 77–87nM,
whereas the other derivatives (9, 11–13, 15 and 16)
showedweaker inhibition, with KI values in the range
of 104–265nM. Thus, SAR is relatively simple, since
heterocyclic, acetazolamide-like derivatives act as excel-
lent inhibitors irrespective of the heterocyclic moiety
incorporatedinto the tail, whereas for the sulfanil-
amide-like compounds 9–17, best CA inhibitory proper-
ties are induced by morpholine-containing tails. Other
heterocyclic tails (piperazine, benzyl/methyl-piperazine,
methylpiperidine, etc.) and diverse length of the spacer
between the heterocyclic moiety andthe sulfanilamide
scaffold(from C1 to C4), influence less the biological
activity of these derivatives; (iv) against the tumor-asso-
ciatedisozyme hCA IX, best inhibitory activity was
shown by four of the heterocyclic compounds, that is,
20, 21, 23 and 24, which possessed KI values in the range
of 22–35nM, being more effective inhibitors than the
leadmolecule 18 (KI of 41nM) andhaving activity in
the same range as the clinically usedderivatives 2–6 (Ta-
ble 3). Weaker inhibitors were then 22 andthe aromatic
sulfonamide 15 (KI values in the range of 67–90nM),
whereas the other sulfanilamide-like compounds (i.e.,
9–14 and 16, 17) showedmoderate hCA IX inhibitory
properties, with inhibition constants in the range of
163–248nM; (v) very few of the new derivatives reported
here showedany selectivity for the tumor-associated
over the cytosolic isozyme II. Thus, only compound 15
has a selectivity ratio close to 3, derivative 13 has this
parameter close to the unity, whereas all other deriva-
tives, similarly to the clinically usedsulfonamides, act
as better CA II than CA IX inhibitors (Table 3).
andby grants from Harran University Research Finan-
cial Centre (HUBAK Project No: 348, 295 and505). We
wouldlike to thank Prof. Dr. Nurettin Yayli (KTU,
1
TURKEY) for H/13C NMR spectral analyses. Some
of us are grateful to Prof. Dr. G. M. Blackburn (Shef-
fieldUniversity, UK) for helpful discussions. Special
thanks are addressed to Professor Raffaello Giannini
andDr. Cristina Vettori (CNR, IGV Department, Flor-
ence, Italy) for their invaluable help.
References and notes
1. (a) Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C.
T. J. Enzym. Inhib. Med. Chem. 2004, 19, 199–229; (b)
Supuran, C. T.; Vullo, D.; Manole, G.; Casini, A.;
Scozzafava, A. Curr. Med. Chem. Cardiovasc. Hematol.
Agents 2004, 2, 49–68.
2. Carbonic Anhydrase—its Inhibitors and Activators; Supu-
ran, C. T., Scozzafava, A., Conway, J., Eds.; CRC (Taylor
andFrancis Group): Boca Raton, FL, 2004; pp 1–363,
andreferences citedtherein.
3. Mann, T.; Keilin, D. Nature 1940, 164, 146–148.
4. (a) Supuran, C. T.; Scozzafava, A. Expert Opin. Ther. Pat.
2000, 10, 575–600; (b) Supuran, C. T.; Scozzafava, A.
Expert Opin. Ther. Pat. 2002, 12, 217–242; (c) Supuran, C.
T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23,
146–189; (d) Scozzafava, A.; Mastrolorenzo, A.; Supuran,
C. T. Expert Opin. Ther. Pat. 2004, 14, 667–702.
5. Supuran, C. T.; Casini, A.; Scozzafava, A. Development
of Sulfonamide Carbonic Anhydrase Inhibitors (CAIs). In
Carbonic Anhydrase––its Inhibitors and Activators; Supu-
ran, C. T., Scozzafava, A., Conway, J., Eds.; CRC: Boca
Raton, FL, USA, 2004; pp 67–148.
6. (a) Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran,
C. T. Curr. Med. Chem. 2003, 10, 925–953; (b) Casini, A.;
Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Curr.
Cancer Drug Targets 2002, 2, 55–75; (c) Abbate, F.;
Casini, A.; Owa, T.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2004, 14, 217–223.
7. (a) Weber, A.; Casini, A.; Heine, A.; Kuhn, D.; Supuran,
C. T.; Scozzafava, A.; Klebe, G. J. Med. Chem. 2004, 47,
550–557; (b) Pastorekova, S.; Casini, A.; Scozzafava, A.;
Vullo, D.; Pastorek, J.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2004, 14, 869–873; (c) Casey, J. R.; Morgan,
P. E.; Vullo, D.; Scozzafava, A.; Mastrolorenzo, A.;
Supuran, C. T. J. Med. Chem. 2004, 47, 2337–
2347.
4. Conclusions
A series of sulfonamides has been obtained by applying
the tail approach to the sulfanilamide and 5-amino-
1,3,4-thiadiazole-2-sulfonamide scaffolds. Tails incorpo-
rating heterocyclic amines belonging to the morpholine,
piperidine and piperazine ring systems have been at-
tachedto such sulfonamides, by means of an alkanoyl-
carboxamido linker containing from two to five carbon
atoms. The new derivatives prepared in this way were
testedas inhibitors of three CA isozymes, the cytosolic
major isozymes I andII; andthe transmembrane, tu-
mor-associatedisozyme IX. Several low nanomolar
CA I andCA II inhibitors were detectedboth in the aro-
matic andheterocyclic sulfonamide series, whereas the
best hCA IX inhibitors (inhibition constants in the
range of 22–35nM) all belongedto the acetazolamide-
like derivatives.
8. (a) Supuran, C. T. Expert Opin. Invest. Drugs 2003, 12,
283–287; (b) Brown, J. M.; Wilson, W. R. Nat. Rev.
Cancer 2004, 4, 437–447.
ˇ
´
´
´
´
ˇ
´
9. (a) Svastova, E.; Hulıkova, A.; Rafajova, M.; Zatꢀovicova,
´
M.; Gibadulinova, A.; Casini, A.; Cecchi, A.; Scozzafava,
A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S. FEBS
´
Lett., in press; (b) Pastorekova, S.; Pastorek, J. Cancer-
relatedCarbonic Anhydrase Isozymes andtheir Inhibi-
tion. In Carbonic Anhydrase—its Inhibitors and Activators;
Supuran, C. T., Scozzafava, A., Conway, J., Eds.; CRC:
Boca Raton, FL, USA, 2004; pp 255–281; (c) Robertson,
N.; Potter, C.; Harris, A. L. Cancer Res. 2004, 64, 6160–
6165.
10. (a) Winum, J.-Y.; Vullo, D.; Casini, A.; Montero, J.-L.;
Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2003, 46,
2197–2204; (b) Winum, J.-Y.; Vullo, D.; Casini, A.;
Montero, J.-L.; Scozzafava, A.; Supuran, C. T. J. Med.
Chem. 2003, 46, 5471–5477.
Acknowledgements
This research was financedin part by a 6th frame EU
project (EUROXY), by the Turkish Government Plan-
ning Organisation (DPT Project No: 2003K120590)
11. Winum, J.-Y.; Scozzafava, A.; Montero, J.-L.; Supuran,
C. T. Expert Opin. Ther. Pat. 2004, 14, 1273–1308.

H. Turkmen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 367–372
371
12. (a) Zhang, X.-R.; Blackburn, G. M. Ph.D. Thesis, The
University of Sheffield, UK, 1995; (b) Turkmen, H.;
Blackburn, G. M. Ph.D. Thesis, The University of
Sheffield, UK, 1998.
13. (a) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Brig-
anti, F.; Mincione, G.; Supuran, C. T. J. Med. Chem.
1999, 42, 2641–2650; (b) Scozzafava, A.; Briganti, F.;
Mincione, G.; Menabuoni, L.; Mincione, F.; Supuran, C.
T. J. Med. Chem. 1999, 42, 3690–3700.
14. (a) Supuran, C. T.; Briganti, F.; Tilli, S.; Chegwidden, W.
R.; Scozzafava, A. Bioorg. Med. Chem. 2001, 9, 703–714;
(b) Supuran, C. T.; Scozzafava, A. J. Enzym. Inhib. 2000,
15, 597–610; (c) Supuran, C. T.; Scozzafava, A. Eur. J.
Med. Chem. 2000, 35, 867–874.
15. An example of the new compounds prepared: The key
intermediate 4-(3-Chloropropionylamino)-benzenesulfon-
amide (8): Sulfanilamide 1 (5.40g, 0.03mol) andNEM
(3.80g, 0.03mol) were stirredin THF (200mL) until most
of the starting material had dissolved. 3-Chloropropanoyl
chloride (7.71g, 0.06mol) in THF was slowly added to the
reaction mixture. The reaction was stirredat À15°C for
4h under anhydrous conditions. After warming to room
temperature the white precipitate of NEMÆHCl salt
filteredoff. The THF was removedin vacuo andthe
resulting white solididssolvedin ethyl acetate. The
organic extract was washedwith 3M hydrochloric acid
(20mL) then with saturatedsodium bicarbonate solution
(20mL) andfinally with brine. Drying over magnesium
cDNA of the catalytic domain of hCA IX (isolated as
described by Pastorek et al.22) was amplifiedby using PCR
andspecific primers for the glutathione S-transferase
(GST)-Gene Fusion Vector pGEX-3X. The obtained
fusion construct was insertedin the pGEX-3X vector
andthen expressedin E. coli BL21 Codon Plus bacterial
strain (from Stratagene). The bacterial cells were soni-
cated, then suspended in the lysis buffer (10mM Tris
pH7.5, 1mM EDTA pH8, 150mM NaCl and0.2% Triton
X-100). After incubation with lysozime (approx. 0.01g/L)
the protease inhibitors CompleteTM were added to a final
concentration of 0.2mM. The obtainedsupernatant was
then appliedto a prepackedGlutathione Sepharose 4B
column, extensively washedwith buffer andthe fusion
(GST-CA IX) protein was elutedwith a buffer consisting
of 5mM reduced glutathione in 50mM Tris–HCl, pH8.0.
Finally the GST part of the fusion protein was cleaved
with thrombin. The advantage of this method over the
previous one,9,10 is that CA IX is not precipitatedin
inclusion bodies from which it has to be isolated by
denaturing–renaturing in the presence of high concentra-
tions of urea, when the yields in active protein were rather
low, andthe procedure much longer. The obtainedCA IX
was further purifiedby sulfonamide affinity chromato-
graphy,19 the amount of enzyme being determined by spec-
trophotometric measurements andits activity by stopped-
flow experiments, with CO2 as substrate.23 The
specific activity of the obtainedenzyme was the same as
the one previously reported,9,10 but the yields in active
protein were 5–6 times higher per litre of culture medium.
An SX.18MV-R AppliedPhotophysics stoppe-dflow
instrument has been usedfor assaying the CA-catalyzed
CO2 hydration activity.23 Phenol red(at a concentration
of 0.2mM) has been usedas indicator, working at the
absorbance maximum of 557nm, with 10mM Hepes
(pH7.5) as buffer, 0.1M Na2SO4 (for maintaining con-
sulfate andevaporation yieldeda white solid(
8) which
was recrystallizedfrom water to give the title compound.
(70%), mp 228–230°C; kmax (KBr, cmÀ1) 1672 (NHCO),
1186 (SO2NH2), 1532 (C@N), 661 (C–Cl); dH (DMSO-d6)
10.3 (1H, s, –CONH), 7.8 (4H, m, –Ar–H), 7.0 (2H, s,
SO2NH2), 3.87 (2H, t, J 6Hz, ClCH2), 2.88 (2H, t, J 6Hz,
–CH2CO); dC (DMSO-d6) 172.84 (C@O), 140.75 (CNH–),
136.92 (C–SO2NH2), 125.7 (C-2 Aryl), 125.7 (C-2 Aryl),
117.99 (C-3 Aryl), 117.99 (C-3 Aryl), 38.7 (CH2Cl), 37.87
(CH2CO); m/z EI+ 264 [M]+. 4-(3-Methylpiperazinopro-
pionylamino)-benzenesulfonamide (9): To a stirredsolution
of an excess of methylpiperazine (3equiv) in tetrahydrofu-
ran (20mL) was added compound 8 (1.00g, 3.80mmol)
over 30min at 0°C. The reaction was allowedto warm to
room temperature andstirredat 40 °C for 48h. The
impurities were removedby flash column chromatogra-
phy (ethyl acetate/methanol, 6:1) to give the title com-
pound(65%), mp 199–200 °C; kmax (KBr, cmÀ1) 1681
(NHCO), 1152 (SO2NH2); dH (DMSO-d6) 10.78 (1H, s,
–CONH), 7.84 (4H, m, –Ar–H) 7.7 (2H, s, SO2NH2), 2.74
(2H, t, J 6Hz, –NCH2), 2.58 (4H, t, J 6Hz, CH2NCH2),
2.55 (4H, t, J 6Hz, CH2NCH2), 2.28 (2H, s, J 6Hz,
CH2CO), 2.15 (3H, s, CH3N–); dC (DMSO-d6) 172.27
(C@O), 141.14 (CNH–), 134.99 (C–SO2NH2), 126.12 (C-2
Aryl), 126.12 (C-2 Aryl), 118.12 (C-3 Aryl), 118.12
(C-3 Aryl), 53.97 (CH2NCH2), 52.55 (CH2NCH2),
51.27 (CH2N–), 44.87 (CH3N–), 32.47 (CH2CO); m/z
EI+ 327 [M]+.
stant the ionic strength), following the CA-catalyzedCO
2
hydration reaction for a period of 10–100s. Saturated CO2
solutions in water at 20°C were usedas substrate. 23 Stock
solutions of inhibitor (1mM) were prepared in distilled–
deionized water with 10–20% (v/v) DMSO (which is not
inhibitory at these concentrations) anddilutions up to
0.01nM were done thereafter with distilled–deionized
water. Inhibitor andenzyme solutions were preincubated
together for 15min at room temperature prior to assay, in
order to allow for the formation of the E–I complex.
Triplicate experiments were done for each inhibitor
concentration, andthe values reportedthroughout the
paper are the mean of such results. The activity of these
enzyme preparations seem to be rather similar with the in
vivo activity of CA IX in tumors as recently proven by CA
inhibitor binding studies and disturbance of tumor pH
following CA IX inhibition as comparedto normal tissue
pH9.
17. Lindskog, S.; Behravan, G.; Engstrand, C.; Forsman, C.;
Jonsson, B. H.; Liang, Z.; Ren, X.; Xue, Y. Structure–
Function Relations in Human Carbonic Anhydrase II as
Studied by Site-Directed Mutagenesis. In Carbonic Anhyd-
rase—From Biochemistry and Genetics to Physiology and
16. Human CA I andCA II cDNAs were expressedin
Escherichia coli strain BL21 (DE3) from the plasmids
pACA/hCA I and pACA/hCA II described by Lindskog
et al.17 Cell growth conditions were those described in Ref.
18 andenzymes were purifiedby affinity chromatography
according to the method of Khalifah et al.19 Enzyme
concentrations were determined spectrophotometrically at
280nm, utilizing a molar absorptivity of 49mMÀ1 cmÀ1
for CA I and54mM À1 cmÀ1 for CA II, respectively, based
on Mr = 28.85kDa for CA I, and29.3kDa for CA II,
respectively.20,21 A variant of the previously published9,10
CA IX purification protocol has been usedfor obtaining
high amounts of hCA IX needed in these experiments. The
`
Clinical Medicine; Botre, F., Gros, G., Storey, B. T., Eds.;
VCH: Weinheim, 1991; pp 1–13.
18. Behravan, G.; Jonsson, B. H.; Lindskog, S. Eur. J.
Biochem. 1990, 190, 351–357.
19. Khalifah, R. G.; Strader, D. J.; Bryant, S. H.; Gibson, S.
M. Biochemistry 1977, 16, 2241–2247.
20. Lindskog, S.; Coleman, J. E. Proc. Natl. Acad. Sci. U.S.A.
1964, 70, 2505–2508.
21. Steiner, H.; Jonsson, B. H.; Lindskog, S. Eur. J. Biochem.
1975, 59, 253–259.

372
H. Turkmen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 367–372
22. Pastorek, J.; Pastorekova, S.; Callebaut, I.; Mornon, J. P.;
Zelnik, V.; Opavsky, R.; Zatovicova, M.; Liao, S.;
Portetelle, D.; Stanbridge, E. J.; Zavada, J.; Burny, A.;
Kettmann, R. Oncogene 1994, 9, 2877–2888.
23. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561–2573.
24. Vullo, D.; Franchi, M.; Gallori, E.; Pastorek, J.; Scozza-
fava, A.; Pastorekova, S.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2003, 13, 1005–1009.