Tricyclic sulfonamides incorporating benzothiopyrano[4,3-c]pyrazole and pyridothiopyrano[4,3-c]pyrazole effectively inhibit α- And β-carbonic anhydrase: X-ray crystallography and solution investigations on 15 isoforms

Article abstract of DOI:10.1021/jm300878g

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous isozymes involved in crucial physiological and pathological events, representing the targets of inhibitors with several therapeutic applications. In this connection, we report a new class of carbonic anhydrase inhibitors, based on the thiopyrano-fused pyrazole scaffold to which a pendant 4-sulfamoylphenyl moiety was attached. The new sulfonamides 3a-e were designed as constrained analogues of celecoxib and valdecoxib. The most interesting feature of sulfonamides 3 was their predominantly strong inhibition of human (h) CA I and II, as well as those of the mycobacterial β-class enzymes (Rv1284, Rv3273, and Rv3588c), whereas their inhibitory action against hCA III, IV, VA, VB, VI, VII, IX, XII, XIII, and XIV was found to be at least 2 orders of magnitude lower. X-ray crystallography and structural superposition studies made it possible to explain the very distinct inhibition profile of the tricyclic sulfonamides, different from those of celecoxib and valdecoxib.

Full text of DOI:10.1021/jm300878g

Article
pubs.acs.org/jmc
Tricyclic Sulfonamides Incorporating
Benzothiopyrano[4,3‑c]pyrazole and
Pyridothiopyrano[4,3‑c]pyrazole Effectively Inhibit α- and β‑Carbonic
Anhydrase: X‑ray Crystallography and Solution Investigations on 15
Isoforms
Anna M. Marini,*,† Alfonso Maresca,‡ Mayank Aggarwal,§ Elisabetta Orlandini,† Susanna Nencetti,†
Federico Da Settimo,† Silvia Salerno,† Francesca Simorini,† Concettina La Motta,† Sabrina Taliani,†
Elisa Nuti,† Andrea Scozzafava,‡ Robert McKenna,§ Armando Rossello,† and Claudiu T. Supuran*,‡
†Dipartimento di Scienze Farmaceutiche, Universita
‡Universita
degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019
Sesto Fiorentino (Florence), Italy
̀
di Pisa, Via Bonanno 6, 56126 Pisa, Italy
̀
§Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Box 100245, Gainesville, Florida
32610, United States
S
* Supporting Information
ABSTRACT: Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous isozymes involved in
crucial physiological and pathological events, representing the targets of inhibitors with
several therapeutic applications. In this connection, we report a new class of carbonic
anhydrase inhibitors, based on the thiopyrano-fused pyrazole scaffold to which a pendant
4-sulfamoylphenyl moiety was attached. The new sulfonamides 3a−e were designed as
constrained analogues of celecoxib and valdecoxib. The most interesting feature of
sulfonamides 3 was their predominantly strong inhibition of human (h) CA I and II, as
well as those of the mycobacterial β-class enzymes (Rv1284, Rv3273, and Rv3588c),
whereas their inhibitory action against hCA III, IV, VA, VB, VI, VII, IX, XII, XIII, and XIV
was found to be at least 2 orders of magnitude lower. X-ray crystallography and structural
superposition studies made it possible to explain the very distinct inhibition profile of the
tricyclic sulfonamides, different from those of celecoxib and valdecoxib.
enhanced formation of the metal hydroxide, catalytically active
species of the enzyme, shown in Scheme 1 (steps A−D).
Inhibitors generally bind to the metal ion from the enzyme
active site in the deprotonated state (as anions), as shown
schematically in steps E and F of Scheme 1, for a tetrahedrally
bound inhibitor (E) and for one in which the Zn(II) ion is in a
trigonal bipyramidal geometry (F), a case in which a water
molecule, in addition to the inhibitor, is also coordinated to
Zn(II) ion (some inorganic anions bind in this way).19
Although the mechanisms of Scheme 1 are depicted for an α-
CA, they are valid even if another metal ion is present within
the active site cavity, i.e., Cd(II) or Fe(II), as the corresponding
hydroxides have similar nucleophilicity as the zinc hydrox-
ide.3−6 The same is true for a different coordination pattern of
the metal ion (i.e., two Cys and one His residues), as for the β-
and ζ-class CAs.1−19 Sulfonamides and their bioisosteres
(sulfamates, sulfamides),1,2 representing the main class of
pharmacologically relevant CA inhibitors (CAIs),1−4 share with
INTRODUCTION
■
The carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of
metalloenzymes which catalyze the interconversion between
CO2 and bicarbonate by using a metal hydroxide nucleophilic
mechanism.1−19 Five genetically distinct CA families are known
to date, the α-, β-, γ-, δ-, and ζ-class enzymes.1−6 They differ in
their preference for the metal ion used within the active site for
performing the catalysis as well as in the three-dimensional fold
of the protein backbone, constituting a paradigmatic example of
convergent Darwinian evolution at the molecular level. Zn(II)
ions may be used by all five classes mentioned above, but the γ-
CAs are probably Fe(II) enzymes (being active also with bound
Zn(II) or Co(II) ions),3,4 whereas the ζ-class uses Cd(II) or
Zn(II) to perform the physiologic reaction catalysis.5,6 The
inhibition and activation of CAs are well understood processes,
with most classes of inhibitors binding to the metal center1−19
and activators binding at the entrance of the active site cavity.
In this way, they participate in proton shuttling processes
between the metal ion-bound water molecule and the
environment, which is the rate-determining step in the catalytic
cycle of most CA isoforms.20,21 This process leads to the
Received: June 22, 2012
© XXXX American Chemical Society
A
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Scheme 1. Catalytic and Inhibition Mechanisms (with Zinc Ion Binders) of α-CAs (hCA I Amino Acid Numbering of the Zinc
a
Ligands)
a
A similar catalytic/inhibition mechanism is valid also for CAs from other classes (β-, γ-, and ζ-CAs), but either the metal ion is coordinated by other
amino acid residues or a Cd(II) ion is present instead of zinc at the active site. Sulfonamides and dithiocarbamates bind as shown in step E.
dithiocarbamates22−24 and some carboxylates25−27 the inhib-
ition mechanism depicted in step E.
as well as high resolution X-ray crystallography of enzyme−
inhibitor adducts may lead to novel classes of CAIs with the
desired physicochemical and pharmacologic properties.
It should be mentioned here that, recently, other CA
inhibition mechanisms than the binding to the metal center
were reported for α-CAs, which do not directly involve the
metal ion from the enzyme active site.28−33 For example,
polyamines bind to the enzyme by anchoring to the zinc-
coordinated water/hydroxide ion,28 whereas coumarins act as
prodrugs and bind at the entrance of the active site cavity,
rather far away from the metal ion, after being hydrolyzed to 2-
hydroxy-cinnamic acids.29−33
CA inhibition not only has pharmacological applications in
the field of diuretics, antiglaucoma, anticonvulsant, and
anticancer agents1,2,7−11 but is also an emerging target for
designing anti-infectives (antifungal and antibacterial
agents)34−36 with a novel mechanism of action. Sulfonamides,
sulfamates, sulfamides, and dithiocarbamates belonging to
many structural types were reported to possess significant
inhibitory activities against many CA classes but were mostly
investigated as inhibitors of the mammalian isoforms (16 of
which are known in nonprimates and 15 in primates).37,38
Several drug design strategies have been reported ultimately
based on the tail approach39−42 for obtaining sulfonamides/
dithiocarbamates, which exploit more external binding regions
within the enzyme active site (in addition to coordination to
the metal ion), thus leading to isoform-selective com-
pounds.43−47 The most promising data have been obtained
by a combination of X-ray crystallography of enzyme−inhibitor
adducts, with novel synthetic approaches for generating
chemical diversity.1,2,48−56 The exploration of novel scaffolds
Continuing our research in the field, in this paper we explore
for the first time tricyclic sulfonamides 3a−e incorporating the
poorly investigated benzothiopyrano[4,3-c]pyrazole (comp
3a−c) and pyridothiopyrano[4,3-c]pyrazole (comp 3d−e)
systems, as well as the classical benzenesulfonamide moiety
responsible for binding to the catalytically crucial metal ion.
Actually, the lead compounds used in the present drug design
study were the clinically used derivatives celecoxib (CLX) and
valdecoxib (VLX), initially launched as cyclooxygenase 2
(COX-2) specific inhibitors,57−60 and later shown also to act
as potent CAIs.46,47,61 (Chart 1) Both compounds possess a
benzenesulfonamide group linked to a five-membered,
substituted heterocyclic ring. The presence of the SO2NH2
moiety seems not to be necessary for COX-2 inhibition, but it
is essential for the CA inhibition.46,47,57−60 The two
compounds were shown to possess interesting and isoform-
selective CA inhibitory action, and their X-ray crystal structures
in complex with one human (h) CA isoform (hCA II) were
reported by our group.46,47 The rationale for designing the new
compounds 3a−e reported here was to use the benzenesulfo-
namide as a zinc binding moiety connected to a pyrazole
moiety annealed with a bulky heterocyclic ring in order to
explore both alternative chemotypes and the possibility to
further enhance the isoform selectivity observed with celecoxib
and valdecoxib as CAIs.46,47,57−61 Furthermore, compounds
3a−e may be regarded as geometrically constrained analogues
of the two reference leads CLX and VLX.
B
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sulfamidophenylhydrazine hydrochloride, afforded the new
compounds 3a−e, which were purified by crystallization. The
purity of the target compounds was assessed by TLC analysis
Chart 1
1
and by physicochemical properties, analytical, and H NMR
spectral data, which were in agreement with the proposed
structures and with other previously reported results (see
experimental protocols and Supporting Information Table 1 for
details).65−68
RESULTS AND DISCUSSION
■
CA Inhibition. Compounds 3a−e as well as the lead
molecules CLX and VLX were assayed69 as inhibitors of all
catalytically active human CA isoforms, hCA I-XIV, and of the
three mycobacterial β-class enzymes Rv1284, Rv3273, and
Rv3588c, shown earlier to be interesting drug targets by our
group70−73 (Table 1). The following structure−activity
relationship (SAR) can be evidenced from the data of Table 1:
i. The cytosolic, widespread isoforms hCA I and II were
moderately (hCA I) or effectively (hCA II) inhibited by
compounds 3. The inhibition profile of these com-
pounds, at least for hCA II, is quite similar to that of
celecoxib and valdecoxib (Table 1), whereas the
derivatives 3 were much better hCA I inhibitors
compared to the lead compounds CLX and VLX.
Indeed, the inhibition constants (KIs) of 3a−e against
hCA I were in the range of 65−318 nM (versus 50−54
μM for the lead molecules) and against hCA II in the
range of 16−210 nM (versus 21−43 nM for CLX and
VLX). The presence in the heterocyclic scaffold both of a
benzene or a pyridine leads to effective CAIs. Moreover,
the most effective inhibitors were those bearing the
methoxy (3a) or chlorine (3b) as R moieties, while the
CF3 moiety as R substituent (3c) was associated with a
lower inhibitory activity against both isoforms (Table 1).
ii. The cytosolic slow isoform hCA III was poorly inhibited
by these sulfonamides, with KIs in the range of 6.4−32.0
μM, which are in the same range as for the coxibs CLX
and VLX. This is probably due to the specific active site
architecture of hCA III, which has a bulky Phe residue
(Phe198) in the middle of the cavity, which interferes
with the binding of sterically demanding compounds74
such as 3a−e.
Compounds 3a−e were investigated for the inhibition of 15
CAs of mammalian or bacterial origin, and one of them (3e)
was crystallized in complex with hCA II, affording interesting
hints for the drug design of sulfonamide CAIs belonging to this
class of derivatives. Moreover, a possible dual activity on both
COX-2 and CA isoforms was also assessed by an in vitro assay.
CHEMISTRY
■
The preparation of the 1-(p-sulfonamidophenyl) substituted
pyrazole derivatives 3a−e was performed following the
synthetic route described in Scheme 2. The starting key
intermediates 7-substituted-3-hydroxymethylenebenzothiopyra-
nones 2a−c or 3-hydroxymethylenepyrido thiopyranones 2d−e
were obtained by Claisen condensation of the appropriate
thiopyranone 1a−e with ethyl formate in toluene solution.
Intermediates 1a−e were prepared following previously
described procedures.62−64 Compounds 2a−b and 2d−e have
been described earlier,65−67 whereas 2c was newly synthesized,
following a similar procedure.65−67 Analytical and IR, 1H NMR
spectral data of 2c were in accordance with those of the
analogous compounds 2a−b and 2d−e. Compounds 2a−e
have been obtained in good yields and sufficiently pure to be
utilized in the second step of the synthetic procedure without
crystallization. The condensation of 2a−e, containing the
reactive CH group adjacent to the CO function, with p-
iii. The membrane-anchored isoform hCA IV was moder-
ately inhibited by compounds 3a−e, with inhibition
a
Scheme 2. Synthesis of Sulfonamides 3a−e
a
Reagents and conditions: (i) MeONa/MeOH, HCOOEt/anhydrous toluene, rt, 24 h N2 atm; (ii) p-sulfonamidophenylhydrazine hydrochloride/
refluxing methanol.
C
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Table 1. Inhibition of hCA Isoforms I−XIV and Mycobacterial β-CAs Rv1284, Rv3273, and Rv3588c with Sulfonamides 3a−e,
Celecoxib (CLX), and Valdecoxib (VLX) by a Stopped Flow CO2 Hydrase Assay69
a
KI (nM)
enzyme
3a
65
3b
212
3c
3d
3e
CLX
VLX
hCA I
318
210
193
72
155
49
50000
21
54000
43
hCA II
16
22700
8850
923
29
32000
7200
440
hCA III
hCA IV
hCA VA
hCA VB
hCA VI
hCA VII
hCA IX
hCA XII
hCA XIII
hCA XIV
Rv1284
Rv3273
Rv3588c
28600
7140
327
6400
328
7900
7500
992
7400
880
794
93
78000
1340
912
476
1072
7116
609
3140
9280
602
3250
9340
628
3180
8055
873
3270
8140
912
88
94
572
2170
16
3900
27
2182
4550
938
1845
5620
2810
797
2570
6755
714
2340
5540
4300
715
3250
5870
4630
844
18
13
98
425
931
548
689
10350
7760
713
107
870
412
613
134
115
12970
7810
682
750
316
238
286
241
610
235
357
273
144
a
Mean from three different assays. Errors were in the range of 10 of the reported values (data not shown).
Figure 1. Stereo stick representation of hCA II active site complexed with 3e (pink). The active-site zinc ion is depicted as a gray sphere. The
electron density is represented by a 2σ-weighted 2Fo − Fc Fourier map (gray mesh). Amino acids are as labeled. Figure made using PyMOL (DeLano
Scientific).
constants in the range of 328−8850 nM. The best CA
inhibitor was 3d, which incorporates the 7-unsubstituted
pyridine ring (R = H).
activity, with compound 3c (R = CF3) showing the best
inhibition values. It should be also noted that the coxibs
CLX and VLX are weak hCA VII inhibitors but show a
significantly better inhibition profile against hCA XIII.
vi. The transmembrane isoforms hCA IX, XII and XIV were
modestly inhibited by the new compounds 3a−e, which
usually showed an activity in the low micromolar−
submicromolar range. hCA XIV was the isoform more
prone to be inhibited by the compounds (KIs in the
range of 548−931 nM), followed by hCA IX (KIs in the
range of 1845−3250), with hCA XII that was the least
inhibited one (KIs in the range of 4550−6755 nM).
vii. The newly investigated compounds 3a−e showed
relevant inhibition profiles also toward the mycobacterial
enzymes Rv1284, Rv3273, and Rv3588c (Table 1). For
example, Rv1284 was inhibited with KIs in the range of
115−870 nM. It is interesting to note that the two
pyridine incorporating compounds 3d and 3e were much
more effective than the benzene analogues 3a−c. The
same behavior has been also observed in the inhibition
assays of the remaining mycobacterial enzymes, Rv3273
and Rv3588c (Table 1). Indeed, the last two compounds,
3d and 3e, had KIs in the range of 241−286 nM against
Rv3273 and of 144−273 nM against Rv3588c,
iv. The mitochondrial isoforms hCA VA and VB, as well as
the secreted one hCA VI, were inhibited by these
compounds and by the coxibs, with inhibition constants
in the range of 327−9340 nM (Table 1). Among these
isoforms, hCA VA was the mostly inhibited, followed by
hCA VB and hCA VI. Actually, this may be a useful
inhibition pattern, because many sulfonamide drugs have
side effects due to the inhibition of the secreted, salivary
(hCA VI), or mitochondrial enzymes (hCA VA and
VB).75
v. The remaining cytosolic isoforms, hCA VII and XIII,
were also moderately inhibited by the new compounds 3,
with KIs in the range of 602−912 nM against hCA VII,
and of 714−4630 nM against hCA XIII, respectively
(Table 1). Against hCA VII, the benzo-fused derivatives
3a−c had similar inhibitory activity, whereas the two
pyrido-fused derivatives 3d−e were CAIs slightly less
effective on this isoform. It seems that for this isoform
the nature of the R moiety has less influence on the
inhibitory action. On the contrary, against hCA XIII this
seems to be the most important parameter influencing
D
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Table 2. Crystallographic Data Refinement and Model
Statistics of the hCA II−3e Adduct
respectively. However, it has to be pointed out that also
the benzothiopyrano derivatives 3b and 3c showed
significative inhibitory activities against each of these
enzymes: Rv3588c (KI of 235 nM) and Rv3272 (KI of
238 nM), respectively.
3e
PDB accession
3QYK
Data Collection Statistics
temperature (K)
wavelength (Å)
X-ray Crystallography. To better understand inhibition of
CAs with these compounds, X-ray crystallographic studies were
performed on 3e in complex with hCA II. The crystal structure
was determined to 1.5 Å resolution, using protocols previously
described,76,77 3e refined with an occupancy of 0.80, and B-
factors that were comparable to the solvent within the active
site. Residual density adjacent to 3e was observed in the final Fo
− Fc electron density map, revealing a possible second
conformer, but refinement efforts to fit this weaker binding
site were unsuccessful as the electron density was too diffuse to
fit a reliable ordered second molecule. 3e was buried deep into
the active site and displaced the catalytic zinc-bound solvent,
such that the sulfonamide nitrogen was bound directly to the
zinc ion (distance ∼2.0 Å). The N and O2 atoms of the
sulfonamide were also within hydrogen bond distances (2.9−
3.0 Å) of OG1 and N of Thr199. This observation is consistent
with the results of the other sulfonamide inhibitors complexed
to CAs investigated so far.76−80 The sulfur atom caused a
pucker in the ring geometry but was not directly involved in
any interaction with hCA II. Protruding out of the active site,
the inhibitor’s hydrophobic rings were stabilized predominantly
by hydrophobic residues that line the active site cavity,
engaging good van der Waals interactions with the side chains
of Val121, Phe131, Leu198, Pro202, and His64 (the proton
shuttle residue of hCA II). 3e was buried within the active site
with 407 Å2 (78.1%) of its surface area in contact with hCA II
(Figure 1). The crystallographic parameters and data collection
statistics are shown in Table 2.
100
1.5418
P21
space group
unit cell parameters (Å)
a = 42.3
b = 41.4
c = 72.3
β = 104.2
39832
no. of unique reflections
e
resolution (Å)
50.0−1.4 (1.52−1.47)
5.6 (19.0)
a
Rsym (%)
I/σ(I)
23.75 (7.2)
completeness
redundancy
96.0 (92.0)
4.9 (4.8)
Final Model Statistics
b
Rcryst (%)
0.157
0.177
4−261
2288
c
Rfree (%)
residue numbers
d
no. of protein atoms
no. of drug atoms
24
no. of H2O molecules
rmsd bond lengths (Å)
rmsd bond angles (deg)
265
0.01
1.40
Ramachandran statistics (%): Most favored,
allowed, and outliers
88.4, 11.6, 0.0
average B factors (Å2): main chain, side chain,
12.1, 14.9, 13.3, 29.2
compound, solvent
a
b
Rsym = ∑ |I − ⟨I⟩|/∑ ⟨I⟩. Rcryst = (∑ |Fo| − |Fc|/∑ |Fobs|) × 100.
c
Rfree is calculated in same manner as Rcryst, except that it uses 5% of
d
the reflection data omitted from refinement. Includes alternate
conformations. Values in parentheses represent highest resolution
e
The superposition of the hCA II−3e structure was carried
out with celecoxib (CLX, PDB: 1OQ5)46 and valdecoxib (VLX,
PDB: 2AW1),47 also in complex with hCA II using Coot.81 The
most striking feature of this comparison, given that all the
inhibitors are tethered to the active site zinc, is the capacity of
their tail groups to occupy very different surface locations of the
active site (Figure 2). Notably, the hydrophobic phenyl ring of
the ligand VLX pushes the Phe131 out of the hydrophobic
pocket compared to inhibitors 3e and CLX (Figure 2B
compared to parts A and C). In a similar manner, the pendant
hydrophobic phenyl ring of CLX forces Asn67 to change
conformation differently from inhibitors 3e and VLX (Figure
2C compared to parts A and B). In addition, CLX has both a
fluorine rich hydrophilic region and a hydrophobic phenyl ring
at its terminus. Interestingly, the fluorine rich group is located
in the hydrophilic pocket (Leu204, Pro202, Phe131, and
Val135) and the hydrophobic phenyl ring is positioned in the
hydrophilic pocket (Asn62, Asn67, Glu69, and Gln92). This
unusual orientation could be attributed to the bulky nature of
the ligand, bearing a large phenyl group in the 5-position of the
heterocyclic ring, which may cause a steric hindrance in the
relatively small hydrophobic pocket in the active site of hCA II
(Figure 2B). On the other hand, the hydrophilic nitrogen atom
in the fused pyridine ring of 3e may be involved in hydrogen
bonding with the bulk solvent and hence does not need to stay
in the Phe131 hydrophobic pocket. Despite the different
conformational changes in hCA II side chains induced by the
ligand binding (Figure 2) and their distinctly different
orientations within the active site (Figure 2), all three
bin.
inhibitors, 3e, CLX, and VLX bury approximately the same
amount of surface area of the protein: 407, 451, and 409 Å2
respectively.
Structural Superposition. The crystal structure determi-
nation of 3e complexed to hCA II raised several other questions
related to the CAI’s specificity. Although the overall structures
of hCA I, II, IX, and XII are similar, compound 3e behaves as a
highly effective hCA I and II inhibitor, with reduced affinity
against hCA IX and XII (Table 1). To investigate the reasons of
such binding profile, the crystal structures of hCA I (PDB ID:
2NMX),82 hCA IX (PDB ID: 3IAI),83 and hCA XII (PDB ID:
1JCZ)84 were superimposed with the structure of hCA II in
complex with 3e (PDB ID: 3QYK), highlighting that the
differences in affinities could be attributed to a reduction in
hydrophobicity in the hydrophobic pockets within the active
sites of hCA IX and XII. Namely, Val131 and Ala204 in hCA IX
and Ala131 and Asn204 in hCA XII cause a decrease in
hydrophobicity compared to their equivalent amino acids in
hCA I and II (Figure 3A, Table 3). Moreover, Arg62 in hCA
IX, and residues Lys67, Thr91, Ser135, and Asn204 in hCA XII
impart hydrophilic properties to the otherwise hydrophobic
region in hCA I and hCA II (Figure 3B, Table 3). hCA II (PDB
ID: 3QYK) had an rmsd (main chain) of 0.86 Å with hCA I
(PDB ID: 2NMX),82 1.52 Å with hCA IX (PDB ID: 3IAI),83
and 1.14 Å with hCA XII (PDB ID: 1JCZ).84
E
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Figure 2. Stick figure of inhibitors (A) 3e (pink), (B) valdecoxib VLX (cyan), and (C) celecoxib CLX (green), superimposed on the active site of
hCA II. View looking into the active site of hCA II. Amino acids are depicted in yellow sticks as labeled. The active-site zinc is represented as a gray
sphere. Red solid arrows indicate hCA II conformational change on inhibitor binding. Figure made using PyMOL (DeLano Scientific).
Similarly, the binding affinities of compound 3e toward hCA
I and II, different from those of the reference compounds CLX
and VLX, were investigated at molecular level. The inhibition
data show that compound 3e is a better inhibitor of hCA I than
CLX and VLX, although they bind with similar affinities with
hCA II. This can be attributed to the reason that residue His67
in hCA I (PDB ID: 2NMX),82 in place of Asn67 in hCA II, has
fewer degrees of conformational freedom and therefore causes a
steric clash with CLX. The possible rotamers of His67 were
examined, but none allowed the binding of CLX without a
steric clash. Conversely, compound 3e binds in a different
orientation within the active site and hence His67 does not
affect its binding (Figure 4). Regarding VLX, when modeled
into hCA I, it is surrounded by residues that are different from
those of hCA II, and this difference in the environment could
be a potential reason for the difference in its inhibition
constants. Residues Ala121, Phe91, and Leu131 in hCA I, in
place of Val, Ile, and Phe in hCA II, have a slightly reduced
hydrophobicity and Leu131 seems to cause a putative clash
with VLX. These differences in amino acid residues do not
affect compound’s 3e binding due to its different orientation
within the active site (Figure 5).
A complete listing of amino acid differences that may
contribute to the effect on the binding affinities of the
compounds with hCA I, II, IX and XII, is reported in Table 3.
COX-2 Inhibition. As mentioned above, both reference
sulfonamido-type coxibs, CLX and VLX, demonstrate action as
potent inhibitors of several CA isoforms, with activities in the
same order of magnitute as those of clinically used CAIs.46,47,61
This inhibition profile suggested that also our sulfonamide
containing derivatives 3a−e might possess COX-2 inhibition
properties, thus, the inhibitory activity of compounds 3a−e
against ovine COX-2 was determined. The inhibitory effect of
the new derivatives was routinely estimated at a concentration
of 100 μM by the Colorimetric COX inhibitor screening assay,
which exploits the peroxidase component of cyclooxygenases.85
None of the assayed compound showed any inhibitory effect
(data not shown).
Consistent with these results, the newly investigated
compounds 3a−e demonstrated to behave as CAs selective
inhibitors.
F
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Figure 5. (A) Surface view of inhibitors 3e (pink), celecoxib CLX
(green), and valdecoxib VLX (cyan) superimposed in the active site of
hCA II. hCA II is depicted as a surface representation (gray), and the
zinc is depicted as a blue surface. (B) close-up of the active site. Amino
acids are as labeled. Figure made using PyMOL (DeLano Scientific).
CONCLUSIONS
■
The ubiquitous metallo-enzymes CAs play important functions
in crucial processes connected with respiration and CO2/
bicarbonate transport between metabolizing tissues and lungs,
thus affecting many physiological or pathological events. This
can explain why several inhibitors, CAIs, of these enzymes are
clinically efficient agents, marketed as antiglaucoma, diuretic, or
antiepilectic drugs. In addition, besides the α-class CAs, the β-
CAs are the most abundant catalysts in metabolically diverse
species such as bacteria or pathogenic fungi, thus becoming the
potential target of CAI-based antibiotics/antifungals.
Figure 3. Stick representation of compound 3e (pink) shown in the
active sites of hCA I (yellow, PDB ID: 2NMX82) and hCA II (yellow,
PDB ID: 3QYK) compared with (A) hCA IX (PDB ID: 3IAI83) amino
acids labeled (gray) and (B) hCA XII (PDB ID: 1JCZ84) amino acids
labeled (gray). Compare with Table 3. hCA II numbering. Zn is shown
as a gray sphere. Figure made using PyMOL (DeLano Scientific).
The whole CAIs group includes a number of structurally
related agents whose most distinctive feature is a sulfonamido
moiety responsible for the coordination bond to the Zn2+ ion of
the enzymes. On the basis of the above considerations, we
investigated a new class of CAIs based on the benzo- or pyrido-
fused thiopyrano[4,3-c]pyrazole scaffold, characterized by a
pendant 4-sulfamoylphenyl moiety. The new compounds 3a−e,
obtained by an original synthesis, have been designed by using
as lead molecules the CAIs celecoxib and valdecoxib, both
possessing an arylsulfonamide group in their structure. The
most interesting feature of this new class of sulfonamides was
their capacity to predominantly exert strong inhibition of only
hCA I and II, as well as of the mycobacterial β-class enzymes
(Rv1284, Rv3273, and Rv3588c), whereas their inhibitory
activity against hCA III, IV, VA, VB, VI, VII, IX, XII, XIII, and
XIV was at least 2 orders of magnitude lower. The combination
of X-ray crystal structure of the hCAII−compound 3e adduct,
and homology modeling allowed to explain this peculiar
inhibition profile, which is also quite different from those of the
reference coxibs, CLX and VLX. Thus, the benzenesulfona-
mides 3a−e constitute a highly interesting class of compounds
which, inhibiting only a restricted number of physiologically
relevant CA isoforms among the 12 catalytically active human
such enzymes, should lead to fewer side effects. In addition, the
good inhibition profile of some of the new derivatives (3d−e)
against mycobacterial CAs is also to be considered as relevant
because these enzymes are less inhibited by other classes of
sulfonamides.
Table 3. Amino Acid Differences in the Active Site of hCA I,
II, IX, and XII (hCA II Numbering)
residue no.
CA I
CA II
CA IX
CA XII
62
Val
His
Phe
Ala
Leu
Ala
Tyr
Asn
Asn
Ile
Arg
Gln
Leu
Val
Val
Leu
Ala
Asn
Lys
Thr
Val
Ala
Ser
Asn
67
91
121
131
135
204
Val
Phe
Val
Leu
Figure 4. Stick representation of compounds 3e (pink), celecoxib
CLX (green), and valdecoxib VLX (cyan shown in the active sites of
hCA I (yellow) compared with hCA II (gray). Note for hCA I His67
and Leu131 can cause a putative clash with CLX and VLX,
respectively. hCA II amino acid in parentheses. Zn is shown as a
gray sphere. Figure made using PyMOL (DeLano Scientific).
Finally, the sulfonamido type CAIs CLX and VLX are known
to act also as potent COX-2 inhibitors, with serious concerns
about their cardiovascular side effects. Consequently, the high
selectivity toward hCA isoforms of compounds 3a−e, which do
not show any inhibitory activity against COX-2, may be
regarded as an important clinical advantage.
G
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using HKL2000.87 Starting phases were calculated from Protein Data
Bank entry 3KS388 with waters removed. Refinement using Phenix
package,89 with 5% of the unique reflections selected randomly and
excluded from the refinement data set for the purpose of Rfree
calculations, was alternated with manual refitting of the model and
solvent placement in Coot.81 The validity of the final model was
assessed by PROCHECK.90 Complete refinement statistics and model
quality are included in Table 2.
EXPERIMENTAL SECTION
Chemistry. The uncorrected melting points were determined using
■
a Reichert Kofler hot-stage apparatus. IR spectra were obtained on a
̈
1
NICOLET/AVATAR 360 FT spectrophotometer by Nujol mulls. H
NMR spectra were recorded on a Varian Gemini 200 spectrometer in
dimethyl-d6 sulfoxide solution. The coupling constants are given in
Hertz. Elemental analyses were performed by our Analytical
Laboratory and were within 0.4%. Magnesium sulfate was used as
the drying agent. Evaporations were made in vacuo (rotating
evaporator). Analytical TLC were carried out on Merck 0.2 mm
precoated silica gel aluminum sheets (60 F-254). Reagents, starting
materials, and solvents were purchased from commercial suppliers and
used as received. According to the methods described previously, the
following substrates were obtained: 7-trifluoromethyl-2,3-dihydro-4H-
1-benzothiopyran-4-one (1c),63 7-methoxy- (2a) and 7-chloro- (2b)
2,3-dihydro-3-hydroxymethylene-1-benzothiopyran-4(4H)-ones,65 2,3-
dihydro-3-hydroxymethylenethiopyrano[2,3-b]pyridin-4(4H)-one
(2d),67 7-methyl-2,3-dihydro-3-hydroxymethylenethiopyrano[2,3-b]-
pyridin-4(4H)-one (2e).65 The preparation of compound 7-trifluor-
omethyl-2,3-dihydro-3-hydroxymethylene-1-benzothiopyran-4(4H)-
one (2c) is described in Supporting Information.
7-Substituted-1-(p-sulfonamidophenyl)-1,4-dihydro-
benzothiopyrano[4,3-c]pyrazoles 3a−c and 7-Substituted-1-
(p-sulfonamidophenyl)-1,4-dihydro-pyrido[3′,2′:5,6]-
thiopyrano [4,3-c]pyrazoles 3d−e. General Procedure. p-
Sulfonamidophenylhydrazine hydrochloride (5.40 mmol) was added
to a solution of the appropriate hydroxymethylene derivative 2a−e
(4.50 mmol) in 50 mL of methanol, and the reaction mixture was
stirred at room temperature for 24 h and then refluxed for 7 h. After
cooling, the yellow solid, when present, was collected and the solution
was evaporated under reduced pressure. The solid and the residue
were washed with an aqueous potassium carbonate saturated solution
to give crude pyrazoles 3a−e, which were purified by crystallization
from ethanol (see Supporting Information Tables 1−2 for physical,
analytical, and spectral data).
CA Inhibition. 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 maximum of 557 nm, with 20 mM Hepes/
TRIS (pH 7.5 for α-CAs, and 8.4, for β-CAs) as buffer and 20 mM
Na2SO4 (for maintaining constant the ionic strength), following the
initial rates of the CA-catalyzed CO2 hydration reaction for a period of
10−100 s.69 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 0.01
nM were done thereafter with the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min at room temperature
prior to assay in order to allow for the formation of the E−I complex.
The inhibition constants were obtained by nonlinear least-squares
methods using PRISM 3, as reported earlier,22−24 and represent the
mean from at least three different determinations. All CA isofoms were
recombinant ones obtained in house as reported earlier.29,30,70−73
X-ray Crystal Structure Determination. Co-crystals for the hCA
II−3e complex were obtained using the hanging drop vapor diffusion
method.86 Drops of 10 μL (0.4 mM hCA II, 0.8 mM 3e, 0.1%
dimethyl sulfoxide (DMSO), 0.8 M sodium citrate, 50 mM Tris-HCl,
pH 7.8) were equilibrated against the precipitant solution (1.6 M
sodium citrate; 50 mM Tris-HCl; pH 7.8) at room temperature (∼20
°C). Crystals were observed after 5 days. A crystal was cryoprotected
by quick immersion into 20% sucrose precipitant solution and flash-
cooled by exposing to a gaseous stream of nitrogen at 100 K. The X-
ray diffraction data was collected using an R-AXIS IV+2 image plate
system on a Rigaku RU-H3R Cu rotating anode operating at 50 kV
and 22 mA, using Osmic Varimax HR optics. The detector−crystal
distance was set to 76 mm. The oscillation steps were 1° with a 5 min
exposure per image. Indexing, integration, and scaling were performed
Structural Superposition. To compare the differences in amino
acid residues (among four isoforms of hCA) involved in interactions
with 3e, the crystal structure of hCA II in complex with 3e (PDB ID:
3QYK) was superposed with highest resolution (1.5 Å) crystal
structure of hCA I (PDB ID: 2NMX),82 the only available crystal
structure of hCA IX (PDB ID: 3IAI)83 and the only uncomplexed
crystal structure available of hCA XII (PDB ID: 1JCZ),84 using the
Secondary Structure Matching (SSM) tool in Coot.81 Additionally, to
investigate the differential binding of CLX and VLX (as compared to
3e) with hCA I and hCA II, the crystal structures of hCA II in complex
with CLX (PDB ID: 1OQ5),46 VLX (PDB ID: 2AW1),47 and 3e
(PDB ID: 3QYK) were superposed with hCA I (2NMX).82
COX-2 Inhibitory Assay. The inhibitory activity of compounds
3a−e against ovine COX-2 was determined by the Colorimetric COX
inhibitor screening assay. Ovine COX-2 and Colorimetric COX
inhibitor screening assay, catalogue no. 760111, were from Cayman
Chemical (Ann Arbor, MI, USA). Test compounds 3a−e (2.2 mM, 10
μL) were incubated at 25 °C for 5 min and under shaking, with ovine
COX-2 enzyme solution (10 μM), heme (10 μL), and assay buffer (0.1
M Tris-HCl, pH 8, 150 μL). Arachidonic acid (20 μL) and the
colorimetric substrate solution (N,N,N′,N′-tetramethyl-p-phenylenedi-
amine, TMPD, 20 μL) were then added, and the resulting mixture was
incubated at 25 °C for 5 min. At the end, the residual activity of COX-
2 enzyme was determined colorimetrically by monitoring the
appearance of the oxidized TMPD, measured at 590 nm with a
PerkinElmer Lambda 25.
All the test compounds 3a−e were dissolved into dilute assay buffer,
and their solubility was facilitated by using DMSO, whose
concentration never exceeded 5% in the final reaction mixture. The
inhibitory effect of the derivatives was routinely estimated at a
concentration of 100 μM.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedure of compound 2c. Tables including
physical, analytical, and spectral data of compounds 2c and
3a−e. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +39 050 2219555. Fax: +39 050 2219605. E-mail:
marini@farm.unipi.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was financed in part by a grant of the 7th
Framework Programme of the European Union (METOXIA
project).
ABBREVIATIONS USED
■
CAs, carbonic anhydrases; hCAII, human carbonic anhydrase
isoform II; CAIs, carbonic anhydrase inhibitors; CLX,
celecoxib; VLX, valdecoxib; COX-2, cyclooxygenase-2
H
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