Carbonic anhydrase inhibitors. Interaction of 2-N,N-dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide with 12 mammalian isoforms: Kinetic and X-ray crystallographic studies

Article abstract of DOI:10.1016/j.bmcl.2007.12.022

2-N,N-Dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide was tested for its interaction with the 12 catalytically active mammalian carbonic anhydrase (CA, EC 4.2.1.1) isozymes, CA I-XIV. The compound is a potent inhibitor of CA IV, VII, IX, XII, and XIII (K_Is of 0.61-39 nM), a medium potency inhibitor of CA II and VA (K_Is of 121-438 nM), and a weak inhibitor against the other isoforms (CA III, VB, VI, and XIV), making it a very interesting candidate for situations in which a strong/selective inhibition of certain isozymes is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed interesting interactions between the inhibitor and the enzyme which are quite different from those observed in the adducts of CA II with the structurally related aliphatic derivatives zonisamide, 2-amino-1,3,4-thiadiazolyl-5-difluoromethanesulfonamide, and 2-dimethylamino-5-[sulfonamido-(aminomethyl)]-1,3,4-thiadiazole reported earlier.

Full text of DOI:10.1016/j.bmcl.2007.12.022

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 999–1005
Carbonic anhydrase inhibitors. Interaction of
2-N,N-dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide
with 12 mammalian isoforms: Kinetic and X-ray
crystallographic studiesq
Claudia Temperini,a Alessandro Cecchi,a Nicholas A. Boyle,b Andrea Scozzafava,a
Jaime Escribano Cabeza,c Paul Wentworth, Jr.,c
G. Michael Blackburnb and Claudiu T. Supurana,*
a
`
Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
50019 Sesto Fiorentino (Florence), Italy
bDepartment of Chemistry, University of Sheffield, Sheffield S3 7HF, England, UK
cThe Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
Received 8 November 2007; revised 10 December 2007; accepted 11 December 2007
Available online 15 December 2007
Abstract—2-N,N-Dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide was tested for its interaction with the 12 catalytically
active mammalian carbonic anhydrase (CA, EC 4.2.1.1) isozymes, CA I–XIV. The compound is a potent inhibitor of CA IV,
VII, IX, XII, and XIII (KIs of 0.61–39 nM), a medium potency inhibitor of CA II and VA (KIs of 121–438 nM), and a weak inhib-
itor against the other isoforms (CA III, VB, VI, and XIV), making it a very interesting candidate for situations in which a strong/
selective inhibition of certain isozymes is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed interesting
interactions between the inhibitor and the enzyme which are quite different from those observed in the adducts of CA II with the
structurally related aliphatic derivatives zonisamide, 2-amino-1,3,4-thiadiazolyl-5-difluoromethanesulfonamide, and 2-dimethyl-
amino-5-[sulfonamido-(aminomethyl)]-1,3,4-thiadiazole reported earlier.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread
metalloenzymes in bacteria, archaea, and eukaryotes,
catalyzing a critically important physiologic reaction,
hydration of carbon dioxide to bicarbonate and pro-
tons.1–4 These enzymes are inhibited by several classes
of compounds, such as sulfonamides,1,5–9 sulfamates,1,2
and sulfamides,1,2 some of which have pharmacologic
applications for the treatment of glaucoma,5 obesity,6
cancer,8–12 epilepsy,7 and other neurological disor-
ders,1,2 or as diuretics.5 Bacterial, fungal, and protozoan
CAs belonging to the a-, b-, c-, and/or d-CA gene fam-
ilies, which are present in many pathogens, started also
to be considered recently as potential targets for the
development of inhibitors with therapeutic applica-
tions.13–18 Inhibitors belonging to the chemical classes
mentioned above bind to the catalytic zinc ion within
the enzyme cavity, as shown by means of X-ray crystal-
lographic studies for many representatives, mainly in
complex with the ubiquitous human isoform II (hCA
II).5a,19–24 A number of such derivatives are clinically
used drugs, such as acetazolamide, methazolamide,
ethoxzolamide, dichlorophenamide, dorzolamide, brin-
zolamide, topiramate, zonisamide, sulpiride, sulthiame,
celecoxib, and valdecoxib among others.1,25 Other com-
pounds are in clinical development, such as indisulam
and COUMATE-667.1
Keywords: Carbonic anhydrase; Isozyme selective inhibitor; X-ray
crystallography; Aliphatic sulfonamide.
q
As mentioned above, CA inhibitors (CAIs) are mainly
used in therapy as diuretics and antiglaucoma agents
but some of them also show marked anticonvulsant,
antiobesity, and antitumor effects.1,2,5–11 This is due to
the fact that such inhibitors target different isozymes
The coordinates of the hCA II–sulfonamide adduct have been
deposited in PDB, ID code 3BL0.
*
Corresponding author. Tel.: +39 055 4573005; fax: +39 055
4573385; e-mail: claudiu.supuran@unifi.it
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.022

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C. Temperini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 999–1005
among the 16 presently known in vertebrates.1,2 How-
ever, most of the presently available CAIs show unde-
sired side effects due to indiscriminate inhibition of CA
isoforms other than the target one.1,2,5–14 Thus, many
new CAI classes are being developed in the search of iso-
zyme selective compounds as potential drugs with less
side effects.1,5,25
cal 2(5)-substituted-1,3,4-thiadiazole ring present in
two of the classical CAIs, acetazolamide and methazola-
mide. Compounds 5–7 are representatives of this new
class of CAIs recently described in the literature, but
they were not assayed in detail for the inhibition of dif-
ferent CA isoforms.26 The X-ray crystal structure of the
adducts of hCA II with 6 and 7 was reported recently by
Fisher et al., but no CA inhibitory data were available
for these two derivatives.24 Compounds structurally
similar to 6 and 7 were however reported by Boyle
et al.26 to act as good to moderate CA II inhibitors
(KIs in the range of 15–208 nM). On the other hand,
Cecchi et al.27c investigated similar aliphatic sulfona-
mides possessing CF2SO2NH2 and CH2SO2NH2 ZBGs
attached to benzene, coumarin or steroid scaffolds,
which showed selective inhibition of the mitochondrial
isoform CA VA over the ubiquitous, cytosolic isozymes
CA I and II or the transmembrane, tumor-associated
one CA IX. Thus, this type of relatively little
The most investigated CAIs belong to the sulfonamide
class.1,2,24–27 Aromatic, heterocyclic, and aliphatic such
derivatives were investigated, such as for example aceta-
zolamide 1, its deacetylated precursor 2, methazolamide
3 and zonisamide 4.1,2,24–27 Compounds1, 3, and 4 have
clinical applications as diuretics, antiglaucoma, and
antiepileptic drugs.1,2,26,27 An interesting class of novel
CAIs was recently reported by Blackburn’s group,24,26
consisting
of
aliphatic
derivatives
possessing
CF2SO2NH2 and CH(R)SO2NH2 (where R = H or ami-
no) zinc binding groups (ZBGs), attached to the classi-
Table 1. Inhibition data with the sulfonamide derivatives 1–5 against 12 mammalian a-CA isoforms
O
O
N
N
N N
N
N
N
S
SO2NH2
N
S
SO2NH2
H2N
S
SO2NH2
SO2NH2
SO2NH2
H
1
2
3
SO2NH2
N
N
N
N
SO2NH2
F
H2N
S
N
N
S
N
F
O
6
5
4
N
N
S
NH2
7
Isozymea
KIb (nM)
1
2
3
4
5
6
hCA Id
250
12
2 · 105
74
63
8600
60
50
14
7 · 105
6200
65
56
35
2.2 · 106
8590
20
1710
nt
hCA IId
121
15.1c
nt
hCA IIId
hCA IVd
hCA VAd
hCA VBd
hCA VId
hCA VIId
hCA IXe
hCA XIIe
mCA XIIId
hCA XIVd
1.5 · 105
940
2300
2150
798
5.2
3.6 · 105
23.5
438
nt
nt
54
11
62
10
6033
89
1422
2150
0.61
39
nt
nt
2.5
25
2.1
27
117
5.1
nt
41
33
nt
5.7
17
3.4
19
11,000
430
5250
7.7
20
nt
nt
215
nt
41
43
2900
nt
Data for the inhibition of these CAs with compounds 1–4 are from Refs. 1,28. No inhibition data of compounds 6 and 7 are available in the
literature.26
a h, human; m, murine isozyme.
b Errors in the range of 5% of the reported data from three different assays (n = 3), by a stopped flow CO2 hydration method.29
c Boyle N. A. Ph.D., Thesis, Sheffield University, 2001, nt = not tested.
d Recombinant full length enzyme.
e Recombinant enzyme, catalytic domain.

C. Temperini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 999–1005
1001
Table 2. Crystallographic parameters and refinement statistics for the
hCA II–5 adduct
investigated CAIs shows great interest in the search of
compounds with different and possibly improved selec-
tivity/inhibition profiles, as well as enhanced solubility,
as compared to those of the classical, clinically used sul-
fonamide CAIs.
Parameter
Value
Crystal parameter
Space group
P2I
˚
Cell parameters (A)
a = 41.5
b = 42.1
c = 72.4
In this work, we report the first detailed inhibition study
of all 12 catalytically active mammalian CA isoforms
with an aliphatic sulfonamide of this type, that is,
5,26,28 as well as a high resolution X-ray crystal structure
for its adduct with the ubiquitous and physiologically
relevant hCA II.
˚
Data collection statistics (20.0–1.9 A)
No. of total reflections
No. of unique reflections
Completeness (%)a
99,060
21,015
98.0 (96.7)
6.5 (1.3)
17.0 (33.0)
F2/sig (F2)
R-sym (%)
Sulfonamide 5 has been investigated for the inhibition of
the 12 catalytically active mammalian CA isoforms CA
I–XIV (h, human; m, mouse isozyme) (Table 1). Data
for the structurally related, clinically used derivatives
1–4 are also included for comparison, as they are avail-
able in the literature.1,27,29 It may be observed that com-
pound 5 acts as a potent inhibitor of isoforms CA IV,
VII, IX, XII, and XIII, with KIs in the range of 0.61–
39 nM. On the other hand, 5 is a medium potency inhib-
itor of CA II and VA (KIs of 121–438 nM), and a weak
inhibitor against the other isoforms (CA III, VB, VI,
and XIV), with KIs in the range of 1422–3.6 · 105 nM
(Table 1). It is thus clear that the inhibition profile of
5 is completely different from those of the clinically used
sulfonamides 1, 3, and 4 or the acetazolamide precursor
2 with which 5 is structurally related. Indeed, 5 is one of
the most potent CA IV and CA VII inhibitors ever re-
ported, while acting as a much weaker inhibitor of the
ubiquitous isoform CA II, which is often considered as
a house-keeping enzyme, whose inhibition is not de-
sired.1,2,6 This is clearly a very important result, and
one which makes 5 a quite unique CAI in the armamen-
tarium of compounds showing isoform selective inhibi-
tory properties.1,5–7 Furthermore, in addition to the
isozymes mentioned above, 5 shows potent inhibitory
activity only against CA XII and IX, which are tumor-
associated and thus not present in normal tissues,7–10
and against CA XIII, an isoform present in restricted
compartments of the genito-urinary tract in normal tis-
sues.6c All these facts make 5 a selective inhibitor of iso-
forms IV and VII, a profile which is completely new for
any known CAI.1–6 This distinct inhibition profile is in
fact unsimilar both to those of the thiadiazole/thiadiaz-
oline clinically used inhibitors (1 and 3), as well as to
that of the related aliphatic antiepileptic sulfonamide
zonisamide, 4. Unlike 5, compounds 1 and 3 possess a
quite promiscuous inhibitory activity, acting as potent
inhibitors against many isozymes (e.g., acetazolamide
1 and methazolamide 2 are potent inhibitors of all iso-
forms except CA I and III), whereas zonisamide 4 al-
ready shows a more modulated activity, being a potent
inhibitor of only isoforms I, II, VA, VI, and IX.
˚
Refinement statistics (20.0–1.9 A)
R-factor (%)
R-freeb (%)
Rmsd of bonds from ideality (A)
19.8
24.8
0.009
1.36
˚
Rmsd of angles from ideality (ꢁ)
a Values in parentheses relate to the highest resolution shell (2.0–
˚
1.9 A).
b Calculated using 5% of data.
˚
tion for Ca atoms of only 0.30 A. Examination of the
initially calculated electron density maps in the active-
site region showed clear evidence for the binding of a
single inhibitor molecule within the active site cavity.
The electron density of all moieties of the inhibitor is
in fact very well defined (Fig. 1).
The tetrahedral geometry of the Zn2+ binding site and
the key hydrogen bonds between the SO2NH2 moiety
of 5 and enzyme active site are all retained with re-
spect to other hCA II-sulfonamide/sulfamate/sulfamide
complexes structurally characterized so far (Figs. 1
In order to better understand the interesting inhibitory
activity of 5 and also to learn some lessons for the drug
design of new CAIs based on this ring system/spacer, we
report the X-ray crystal structure of the hCA II–5 ad-
duct (Table 2).30–34 The three-dimensional structure of
the enzyme is very similar to that of hCA II without
any ligand bound,19,21,23–25 as judged by an rms devia-
Figure 1. Electron density map of 5 (in yellow) bound within the hCA
II active site. The Zn(II) ion of the enzyme, its three histidine ligands
(His94, 96, and 119), and residues involved in the binding of the
inhibitor (Thr199, Thr200, and Gln92) are also shown.

1002
C. Temperini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 999–1005
and 2).5,19,21,23–25,35 In particular, the ionized nitrogen
atom of the sulfonamide group of 5 is coordinated to
sulfonamide 2 or zonisamide 4, but the corresponding
distances are of course different.19,21–25,35 Interestingly,
one of the endocyclic nitrogen atoms of the thiadia-
zole ring makes a strong hydrogen bond with the
˚
the zinc ion at a distance of 1.90 A. This nitrogen
atom is also hydrogen bonded to the hydroxyl group
˚
of Thr199 (N–Thr199OG = 2.63 A), which in turn
˚
interacts with the Glu106OE1 atom (2.50 A, data
˚
OH moiety of Thr200, of 3.14 A, an interaction not
observed earlier in any other adduct of hCA II with
1,3,4-thiadiazole-sulfonamide derivatives.35 The scaf-
fold of 5 is accommodated perfectly within the active
site channel, being oriented toward the hydrophobic
half of it, and participating in several favorable inter-
actions with various amino acid residues (Figs. 1 and
2). Thus, the 1,3,4-thiadiazole-methyl scaffold of the
inhibitor makes favorable van der Waals contacts
not shown). One oxygen atom of the coordinated sul-
famoyl moiety is hydrogen bonded to the backbone
˚
amide of Thr199 (ThrN–O2 = 2.70 A), whereas the
˚
second oxygen atom of this moiety is 3.20 A away
from the catalytic Zn2+ ion, being considered as
weakly coordinated to the metal ion.1,19,21,23–25,35 All
these interactions have also been observed in the ad-
ducts of hCA II with other sulfonamide inhibitors,
such as acetazolamide 1, 5-amino-1,3,4-thiadiazole-2-
˚
(around 4 A) with amino acids lining the hydrophobic
half of the hCA II cavity (such as Gln92, Phe131,
Val121, Val143, and Leu198). For example, the dis-
tance between the endocyclic sulfur atom of 5 and
the nitrogen atom of the carboxamido side chain of
A
˚
Gln92 is of 4 A (Fig. 2). It is also interesting to note
that the dimethylamino moiety of 5 does not partici-
pate in any interaction with the enzyme nor with
water molecules from the active site, which may in
fact explain the medium potency inhibitory activity
of this compound. The same is true for the CH2
spacer between the ZBG and the substituted-1,3,4-
thiadiazole ring (Figs. 1 and 2). From this point of
view (i.e., scarcity of interactions between the enzyme
and the moiety in position 2 of the 1,3,4-thiadiazole
ring), 5 is rather similar to 2 (also a medium potency
CA II inhibitor, KI of 60 nM) for which the crystal
structure in adduct with hCA II was recently reported
by our group.35
In order to try to understand the different inhibition
profiles of compound 5 and the structurally related
derivatives 4, 6, and 7 investigated earlier,19,24 we have
also superposed the 3D structures of these four sulfona-
mides complexed within the hCA II active site (Fig. 3).
Figure 3A and B shows that although the last three com-
pounds have a common 1,3,4-thiadiazolyl-methanesul-
fonamide scaffold, their binding to the enzyme is very
different. Indeed, only the SO2NH2 moieties of the three
inhibitors are superposable, whereas the orientation of
the 1,3,4-thiadiazole rings and of the spacers between
the ring and the ZBG is completely different. For exam-
ple, as shown by Fisher et al.24 in the adduct of 6 with
hCA II the 1,3,4-thiadiazole ring participates in a p-
stacking interaction with the imidazole ring of His94,
an interaction not seen in the adducts of hCA II with
5 or 7 (Fig. 3A). On the other hand, in the adduct of
hCA II with 7, the 1,3,4-thiadiazole ring adopted a
puckered conformation, which is again typical only for
that adduct. On the contrary, the derivative 5 investi-
gated by us here shows an extended conformation of
the thiadiazole-methyl scaffold, which enables it to par-
ticipate in an increased number of hydrophobic interac-
tions (in addition to the polar ones mentioned above)
(Fig. 3B). These data also indicate that as the 2-dimeth-
ylamino-moiety of 5 and 7 or the 2-amino- one in 6 does
not participate in any particular interactions with amino
acid residues in the active site, substituting this moiety
with functionalities which can interact with amino acid
residues situated at the entrance of the cavity, such as,
Gln92
B
HN
O
Phe131
Val121
O
NH2
3.99
N
Val143
Leu198
S
N
N
3.14
O
O
S
H
N
-
3.20
NH
2.70
HN
OH
His94
H
N
N
1.90
Zn2+
N
2.63
His119
O
OH
O
Thr200
N
Thr199
N
H
NH
His96
Figure 2. (A) 3D overview of the hCA II–5 complex. The surface of
the enzyme is colored according to the hydropathy (blue, hydrophilic;
red, hydrophobic residues). Zinc is violet and the inhibitor 5 is green.
(B) Detailed schematic representation for interactions in which
sulfonamide 5 participates when bound to the hCA II active site.
˚
Figures represent distances (in A).

C. Temperini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 999–1005
1003
CH2 spacer is rather different in the two adducts, but
the two ring systems (1,2-benzoxazole in 4, and 1,3,4-
thiadiazole in 5, respectively) lie approximately in the
same region of the CA II active site. In fact, among
derivatives 1–5, the inhibition profile of 5 is the most
similar just to that of zonisamide 4, which can be under-
stood also from these X-ray crystallographic data.
In conclusion, we investigated the interaction of 2-N,N-
dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide
with the 12 catalytically active mammalian CA iso-
zymes, CA I–XIV. The compound is a potent inhibitor
of CA IV, VII, IX, XII, and XIII (KIs of 0.61–39 nM),
a medium potency inhibitor of CA II and VA (KIs of
121–438 nM), and a weak inhibitor against the other
isoforms (CA III, VB, VI, and XIV), making it a very
interesting candidate for situations in which a strong/
selective inhibition of certain isozymes is needed. The
crystal structure of the hCA II adduct of this sulfon-
amide revealed interesting interactions between the
inhibitor and the enzyme which are quite different from
those observed in the adducts of CA II with the structur-
ally related 2-amino-1,3,4-thiadiazolyl-5-difluorome-
thanesulfonamide or 2-dimethylamino-5-[sulfonamido-
(aminomethyl)]-1,3,4-thiadiazole reported earlier.
Acknowledgments
This research was financed in part by two grants of the
6th Framework Programme of the European Union
(EUROXY and DeZnIT projects).
References and notes
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Figure 3. Superposition of the: (A) hCA II–5 adduct (in blue) with hCA
II–6 adduct (PDB file 2EU3),24 in yellow; (B) hCA II–5 adduct (in blue)
with hCA II–7 adduct (PDB file 2EU2),24 in gold, and (C) hCA II 5
adduct (in blue) with hCA II–zonisamide 4 adduct (magenta).19
for example, dimethylaminoethyl-carboxamido,36 ben-
zenesulfonamido,37 etc., may lead to analogues with in-
creased affinity for hCA II and probably also with a
diverse inhibition profile versus other isoforms of inter-
est. On the other hand, as shown in Figure 3C, the inhib-
itors 4 and 5 possessing both a methanesulfonamide
ZBG are again not very much superposable, except for
the sulfamoyl moiety. In fact, the orientation of the
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28. 2-N,N-Dimethylamino-5-methyl-1,3,4-thiadiazole. POCl3
(0.82 ml, 8.8 mmol) was added dropwise to an ice-cooled
mixture of glacial acetic acid (0.5 ml, 8.8 mmol) and 4,4-
dimethylthiosemicarbazide (0.50 g, 4.2 mmol), under
argon. The resulting suspension was heated at 70 ꢁC for
3 h, ice-cold water (100 ml) was added, and the residue
dissolved with gentle heating and stirring. The resulting
suspension was filtered and the filtrate was adjusted to pH
8 with saturated NaHCO3. The mixture was extracted
with EtOAc (3· 50 ml) and the combined organic extracts
were washed with brine and dried over MgSO4. Evapo-
ration in vacuo gave the product as a white solid (0.49 mg,
82%); TLC (100% EtOAc): Rf = 0.2; dH (250 MHz;
CDCl3) 3.08 (6H, s, 2· NCH3), 2.53 (3H, s, CCH3); dC
(250 MHz; CDCl3) 172.1 (C3), 154.2 (C2), 41.4 (C4 and 5),
15.8 (C1); MS (EI+) m/z 143 (M+, 100%); HRMS calcd for
Wouters, B. G.; Lambin, P. Radiother. Oncol. 2007, 83,
367.
12. (a) De Simone, G.; Vitale, R. M.; Di Fiore, A.; Pedone,
C.; Scozzafava, A.; Montero, J. L.; Winum, J. Y.;
Supuran, C. T. J. Med. Chem. 2006, 49, 5544; (b) Alterio,
V.; Vitale, R. M.; Monti, S. M.; Pedone, C.; Scozzafava,
A.; Cecchi, A.; De Simone, G.; Supuran, C. T. J. Am.
Chem. Soc. 2006, 128, 8329.
13. Krungkrai, J.; Krungkrai, S. R.; Supuran, C. T. Curr. Top.
Med. Chem. 2007, 7, 909.
14. (a) Nishimori, I.; Minakuchi, T.; Morimoto, K.; Sano, S.;
Onishi, S.; Takeuchi, H.; Vullo, D.; Scozzafava, A.;
Supuran, C. T. J. Med. Chem. 2006, 49, 2117; (b)
Nishimori, I.; Minakuchi, T.; Kohsaki, T.; Onishi, S.;
Takeuchi, H.; Vullo, D.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2007, 17, 3585.
15. Klengel, T.; Liang, W. J.; Chaloupka, J.; Ruoff, C.;
Schroppel, K.; Naglik, J. R.; Eckert, S. E.; Morgensen, E.
G.; Haynes, K.; Tuite, M. F.; Levin, L. R.; Buck, J.;
Muhlschlegel, F. A. Curr. Biol. 2005, 15, 2021.
16. Mogensen, E. G.; Janbon, G.; Chaloupka, J.; Steegborn,
C.; Fu, M. S.; Moyrand, F.; Klengel, T.; Pearson, D. S.;
Geeves, M. A.; Buck, J.; Levin, L. R.; Muhlschlegel, F. A.
Eukaryot. Cell 2006, 5, 103.
C5H9N3S: 143.0517. Found: 143.0518; IR (KBr, cmÀ1
3414, 1554, 1420, 1337.
2-N,N-Dimethylamino-1,3,4-thiadiazole-5-methanesulfon-
)
17. Bahn, Y. S.; Muhlschlegel, F. A. Curr. Opin. Microbiol.
2006, 9, 572.
amide 5. BuLi (0.84 ml, 2.1 mmol) was added to 2-N,N-
dimethylamino-5-methyl-1,3,4-thiadiazole (0.3 g,
18. Suarez Covarrubias, A.; Larsson, A. M.; Hogbom, M.;
Lindberg, J.; Bergfors, T.; Bjorkelid, C.; Mowbray, S. L.;
Unge, T.; Jones, T. A. J. Biol. Chem. 2005, 280, 18782.
19. De Simone, G.; Di Fiore, A.; Menchise, V.; Pedone, C.;
Antel, J.; Casini, A.; Scozzafava, A.; Wurl, M.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2005, 15, 2315.
20. (a) Cecchi, A.; Hulikova, A.; Pastorek, J.; Pastorekova, S.;
Scozzafava, A.; Winum, J.-Y.; Montero, J.-L.; Supuran,
C. T. J. Med. Chem. 2005, 48, 4834; (b) Winum, J. Y.;
Temperini, C.; El Cheikh, K.; Innocenti, A.; Vullo, D.;
Ciattini, S.; Montero, J. L.; Scozzafava, A.; Supuran, C.
2.1 mmol) in anhydrous THF (10 ml), under argon, at
À78 ꢁC. Sulfur dioxide was introduced into the solution at
À78 ꢁC for 1 h, causing a precipitate and the solution to
turn purple. The mixture was concentrated in vacuo and
the residue dissolved in aq sodium acetate (20 ml, 1.1 M).
Hydroxylamine-O-sulfonic acid (1.13 g, 10 mmol) was
added and the solution stirred at rt overnight. The
solution was adjusted to pH 7 with saturated NaHCO3,
extracted with EtOAc (3· 35 ml), and the combined
organic extracts washed with brine, dried (MgSO4), and
evaporated. The resulting pale yellow solid was suspended
in warm CHCl3, collected by filtration, and washed with
CHCl3 to give the product as an off-white solid (0.12 g,
25%); TLC (100% EtOAc): Rf = 0.2 (stained yellow with
ninhydrin); mp 191–193 ꢁC (decomposition); dH
(250 MHz; DMSO-d6) 7.16 (2H, s, NH2), 4.62 (2H, s,
CH2), 3.09 (6H, s, 2· NCH3); dC (250 MHz; DMSO)-d6
172.8 (C3), 147.4 (C2), 55.3 (C1), 41.0 (C4 and 5); MS
(EI+) m/z 222 (M+, 20%); HRMS calcd for C5H10N4O2S2:
222.0245. Found: 222.0246; IR (KBr, cmÀ1) 3332, 1568,
1330, 1164, 1124.
T. J. Med. Chem. 2006, 49, 7024; (c) Gruneberg, S.;
¨
Stubbs, M. T.; Klebe, G. J. Med. Chem. 2002, 45, 3588.
21. (a) Boriack, P. A.; Christianson, D. W.; Kingery-Wood,
J.; Whitesides, G. M. J. Med. Chem. 1995, 38, 2286; (b)
Kim, C. Y.; Chang, J. S.; Doyon, J. B.; Baird, T. T.;
Fierke, C. A.; Jain, A.; Christianson, D. W. J. Am. Chem.
Soc. 2000, 122, 12125; (c) Antel, J.; Weber, A.; Sotriffer, C.
A.; Klebe, G. Multiple binding modes observed in X-ray
structures of carbonic anhydrase inhibitor complexes and
other systems: consequences for structure-based drug
design. In Carbonic Anhydrase—Its Inhibitors and Activa-
tors; Supuran, C. T., Scozzafava, A., Conway, J., Eds.;
CRC Press: Boca Raton, 2004; pp 45–65.
22. Kim, C. Y.; Whittington, D. A.; Chang, J. S.; Liao, J.;
May, J. A.; Christianson, D. W. J. Med. Chem. 2002, 45,
888.
23. Boriack-Sjodin, P. A.; Zeitlin, S.; Chen, H. H.; Crenshaw,
L.; Gross, S.; Dantanarayana, A.; Delgado, P.; May, J. A.;
Dean, T.; Christianson, D. W. Protein Sci. 1998, 7,
2483.
O
O
N
N
3
5
2
1
S
N
4
NH2
S
29. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561, An
Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalyzed CO2 hydration
activity. Phenol red (at a concentration of 0.2 mM) has
been used as indicator, working at the absorbance

C. Temperini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 999–1005
1005
maximum of 557 nm, with 10 mM Hepes (pH 7.5) as
buffer, 0.1 M Na2SO4 (for maintaining constant the ionic
strength), following the CA-catalyzed 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
inhibitor at least six traces of the initial 5–10% of the
reaction have been used for determining the initial
velocity. The uncatalyzed rates were determined in the
same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (0.1 mM) were prepared
in distilled-deionized water and dilutions up to 0.1 nM
were done thereafter with distilled-deionized water. Inhib-
itor and enzyme solutions were preincubated together for
15 min at room temperature prior to assay, in order to
allow for the formation of the E-I complex. The inhibition
constants were obtained by non-linear least-squares meth-
ods using PRISM 3, as reported earlier,12–14 and represent
the mean from at least three different determinations.
Enzyme concentrations in the assay system were in the
The structure was analyzed by difference Fourier
technique, using the PDB file 1CA2 as starting model.
The refinement was carried out with the program
REFMAC532; model building and map inspections were
performed using the COOT program.33 The final model of
the complex hCA II–5 had an R-factor of 19.8% and
˚
R-free 24.8% in the resolution range 20.0–1.9 A, with a
˚
rms deviation from standard geometry of 0.009 A in bond
lengths and 1.36ꢁ in angles. The correctness of stereo-
chemistry was finally checked using PROCHECK.34
Coordinates and structure factors have been deposited
with the Protein Data Bank (accession code 3BL0).
Crystallographic parameters and refinement statistics are
summarized in Table 2.
31. Oxford Diffraction. CrysAlis RED, 2006, Version
1.171.32.2. Oxford Diffraction Ltd, UK.
32. Jones, T. A.; Zhou, J. Y.; Cowan, S. W.; Kjeldoard, M.
Acta Crystallogr. 1991, A47, 110.
33. Emsley, P.; Cowtan, K. Acta Crystallogr. 2004, D60, 2126.
34. Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.;
Thornton, J. M. J. Appl. Crystallogr. 1993, 26, 283.
35. Menchise, V.; De Simone, G.; Di Fiore, A.; Scozzafava,
A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2006, 16,
6204.
range of 7.1– 13 nM.12–14
.
30. The hCA II–5 complex was crystallized as previously
described. Diffraction data were collected under cryogenic
conditions (100 K) on a CCD Detector KM4 CCD/
˚
Sapphire using CuKa radiation (1.5418 A). The unit cell
36. Abbate, F.; Casini, A.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2004, 14, 2357.
37. Casini, A.; Abbate, F.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2003, 13, 2759.
˚
˚
˚
dimensions were: a = 41.5 A, b = 42.1 A, c = 72.41 A and
a = c = 90ꢁ, b = 104.25ꢁ in the space group P21. Data were
processed with CrysAlis RED (Oxford Diffraction 2006).31