5-Aryl-1H-pyrazole-3-carboxylic acids as selective inhibitors of human carbonic anhydrases IX and XII

Article abstract of DOI:10.1016/j.bmc.2015.05.052

Inhibitory activity of a congeneric set of 23 phenyl-substituted 5-phenyl-pyrazole-3-carboxylic acids toward human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II, IX and XII was evaluated by a stopped-flow CO₂ hydrase assay. These compounds exerted a clear, selective inhibition of hCA IX and XII over hCAI and II, with K_i in two to one digit micromolar concentrations (4-50 μM). Derivatives bearing bulkier substituents in para-position of the phenyl ring inhibited hCA XII at one-digit micromolar concentrations, while derivatives having alkyl substituents in both ortho- and meta-positions inhibited hCA IX with K_is ranging between 5 and 25 μM. Results of docking experiments offered a rational explanation on the selectivity of these compounds toward CA IX and XII, as well as on the substitution patterns leading to best CA IX or CA XII inhibitors. By examining the active sites of these four isoforms with GRID generated molecular-interaction fields, striking differences between hCA XII and the other three isoforms were observed. The field of hydrophobic probe (DRY) appeared significantly different in CA XII active site, comparing to other three isoforms studied. To the best of our knowledge such an observation was not reported in literature so far. Considering the selectivity of these carboxylates towards membrane-associated over cytosolic CA isoforms, the title compounds could be useful for the development of isoform-specific non-sulfonamide CA inhibitors.

Full text of DOI:10.1016/j.bmc.2015.05.052

Accepted Manuscript
5-Aryl-1H-pyrazole-3-carboxylic acids as selective inhibitors of human car-
bonic anhydrases IX and XII
Ilija N. Cvijetić, Muhammet Tanç, Ivan O. Juranić, Tatjana Ž. Verbić, Claudiu
T. Supuran, Branko J. Drakulić
PII:
S0968-0896(15)00474-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.05.052
BMC 12348
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 April 2015
26 May 2015
29 May 2015
Please cite this article as: Cvijetić, I.N., Tanç, M., Juranić, I.O., Verbić, T., Supuran, C.T., Drakulić, B.J., 5-
Aryl-1H-pyrazole-3-carboxylic acids as selective inhibitors of human carbonic anhydrases IX and XII, Bioorganic
& Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.05.052
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5-Aryl-1H-pyrazole-3-carboxylic acids as selective inhibitors of human
carbonic anhydrases IX and XII
Ilija N. Cvijetić,a Muhammet Tanç,b Ivan O. Juranić, c Tatjana Ž. Verbićd, Claudiu T.
Supuran,b,* Branko J. Drakulićc,**
a Innovation Center of the Faculty of Chemistry, University of Belgrade, Studentski Trg
12-16, 11000 Belgrade, Serbia
b NEUROFARBA Department, Sezione di Scienze Farmaceutiche e Nutraceutiche,
Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence,
Italy
c Department of Chemistry - Institute of Chemistry, Technology and Metallurgy,
University of Belgrade, Njegoševa 12, 11000 Belgrade, Serbia
d Faculty of Chemistry, University of Belgrade, Studentski Trg 12-16, 11000 Belgrade,
Serbia
* Corresponding author. Tel.: 039 055 4573005. E-mail: claudiu.supuran@unifi.it (C.T.
Supuran).
** Corresponding author. Tel.: +381 11 3336741. E-mail: bdrakuli@chem.bg.ac.rs (B.J.
Drakulić)
Abreviations: CA, carbonic anhydrase; ZBG, zinc bnding group; CAI, carbonic
anhydrase inhibitors; pKa, acid dissociation constant; Ki, inhibition constant; PDB,
Protein Data Bank; MIF, Molecular interaction fields; HBD, hydrogen bond donor;
HBA, hydrogen bond acceptor.
Abstract
Inhibitory activity of a congeneric set of 23 phenyl-substituted 5-phenyl-pyrazole-3-
carboxylic acids toward human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II, IX
and XII was evaluated by a stopped-flow CO2 hydrase assay. These compounds exerted a
clear, selective inhibition of hCA IX and XII over hCAI and II, with Ki in two to one
digit micromolar concentrations (4 – 50 µM). Derivatives bearing bulkier substituents in
para-position of the phenyl ring inhibited hCA XII at one-digit micromolar
concentrations, while derivatives having alkyl substituents in both ortho- and meta-
positions inhibited hCA IX with Kis ranging between 5 and 25 µM. Results of docking
experiments offered a rational explanation on the selectivity of these compounds toward
CA IX and XII, as well as on the substitution patterns leading to best CA IX or CA XII
inhibitors. By examining the active sites of these four isoforms with GRID generated
molecular-interaction fields, striking differences between hCA XII and the other three
isoforms were observed. The field of hydrophobic probe (DRY) appeared significantly
different in CA XII active site, comparing to other three isoforms studied. To the best of
our knowledge such an observation was not reported in literature so far. Considering the
selectivity of these carboxylates towards membrane-associated over cytosolic CA

isoforms, the title compounds could be useful for the development of isoform-specific
non-sulfonamide CA inhibitors.
Keywords: Carbonic anhydrase, Phenyl-pyrazole-carboxylic acids, Docking, Molecular
interaction fields, Active sites comparison
1. Introduction:
There are a few reports on biological activity of 5-aryl-pyrazole-3-carboxylic acid in the
literature. Compounds were described as partial agonists of the nicotinic acid receptors,1
as inhibitors of replication protein A protein-protein interactions,2 as scaffolds that lead to
inhibitors of protein tyrosine phosphatase 1B,3 and as an inhibitors of a tissue-nonspecific
alkaline phosphatase.4 Along with this, 5-heteroaryl-pyrazole-3-carboxylic acids, among
other compounds, were recently described as inhibitors of human carbonic anhydrases
(CAs, EC 4.2.1.1) I, II, IX and XII.5
The majority of reported small molecule CA inhibitors (CAIs) comprises a
primary sulfonamide moiety as a zinc binding group (ZBG). The sulfonamide anion (-
SO2NH−) coordinates the active site Zn2+ ion and blocks catalysis. In Protein data bank
more than 300 unique sulfonamide-bearing small molecules co-crystallized with carbonic
anhydrases (mainly human isoform II) can be found. On the other hand, significantly less
non-sulfonamide CAIs, belonging to other classes of small organic molecules, such as for
example carboxylic acids, were investigated so far. It was shown that some molecules
comprising -COOH moiety co-crystallized with different CA isoforms, are bound within
the CA active site, interacting with the active site Zn2+ by water-mediated H-bonding,
while other were found bound to the enzyme but did not interact with the active site Zn2+
ion, or they were bound out of the active site. Best examples of the latter are, probably,
ortho-hydroxy cinnamic acids – mechanism-based inhibitors originated in situ by
hydrolysis of coumarins by Zn2+ - hydroxyl ion couple in the CA active site.6,7,8,9 Isoform
selectivity of this class of compounds depends on substitution pattern on coumarin phenyl
ring. Recently reported 2-benzylsulfinylbenzoic acid10 was found bound to hCA II out of
the active site. This compound constrained the proton-shuttle residue His64 in its ‘out’
conformation by a network of water molecules-mediated H-bonds. Inhibition study of
hCA I and II, as well as mycobaterial and fungal CA with a library of natural products,
found adduct of cinnamic and maleic acid amide, 3-(3-chloro-4-hydroxyphenyl)-2-[(4-
methoxy-4-oxobut-2-enoyl)amino]propanoic acid (CID 72785959, CAS 852690-88-1), as
a low micromolar CA inhibitor.11 Structure of compound co-crystallized with hCA II
revealed that carboxyl group of cinnamic acid moiety is not involved in interaction with
catalytic Zn2+ ion. Inhibition of mycobacterial CA Rv3588c by (+)-mispyric acid, natural
product bearing two carboxyl groups, was also described,12 without additional structural
data. Smaller carboxylate-bearing molecules, such as hydroxy and thio-substituted
benzoic acids, were found bound to the CA active site, forming hydrogen bond to the Zn-
bound water molecule/hydroxide ion.13 In the same crystal structures two more molecules
of substituted benzoic acids were found, occupying protein cavities out of the binding
site. Inhibition of CAs with biologically-relevant aliphatic carboxylates, along with
benzoic and 1,2,5,6-tetrafluoro-benzoic acids were also described, without additional

structural data.14 A congeneric set of di-methoxy-bromophenyl-substituted 2-benzyl-
cyclopropyl carboxylic acids and derivatives, having the same topological distance
between aryl and carboxyl moieties as in a title compounds, were recently reported as
inhibitors of human CA isoforms I, II, IX and XII.15 Those compounds exerted strong
preferences toward hCA I and II inhibition. Carboxylic acid derivatives briefly described
in the previous lines inhibited CAs with Kis spanning range between the milimolar to
micromolar concentrations, with the exception of 2-benzyl-cyclopropyl carboxylic acids,
found as nanomolar inhibitors.
In this article we report the synthesis and inhibitory activity of a congeneric set of
23 phenyl-substituted 5-phenyl-pyrazole-3-carboxylic acids (1-23) toward human CA
isoforms I, II, IX and XII.
2. Results and discussion:
2.1 Chemistry:
The rationale of this work was to investigate different chemotypes incorporating the
COOH moiety as a ZBG for obtaining CAIs. As heterocyclic carboxylic acids were rarely
investigated as CAIs, we decided to synthesize a congeneric series of such derivatives
incorporating the synthetically easily available pyrazole-3-carboxylic acid moiety.
Acetophenones II were prepared by Fridel-Crafts acylation of the corresponding
aromatic substrates (I), or commercially available substances were used. The aryl-diketo
acids III were prepared following previously reported procedures,16 by base-promoted
condensations of substituted acetophenones (II) and diethyl-oxalate. Compounds 1-23
(IV) were synthesized from aryldiketo acids (III) and hydrazine-hydrate in glacial acetic
acid (Scheme 1).
Scheme 1.
1
13
Identity and purity of compounds was confirmed by H and C NMR spectroscopy and
LC-HR/ESI-MS.
2.2 Tautomeric preferences and protonation states:
Aryl-pyrazole-carboxylic acid could appear in three tautomeric forms (Scheme 2).
Scheme 2.
Tautomers A and B, with protonated pyrazole nitrogens, are more probable than the
tautomer C. In solid state, tautomeric form A was observed.17 To gather more data on
tautomeric preferences of examined compounds in solution, we recorded NOESY spectra
of 4-Me-derivative (1), in DMSO-d6. 4-Me-derivative (1) was chosen because of simple
1H NMR pattern of protons on phenyl ring. Spectra were recorded in DMSO due to good
solubility of examined compounds and its polarity, which preclude possible aggregation
of solute by intramolecular H-bonding, observed in solid state (see below). Clear NOESY

1
signal between broad H peak at 13.38 ppm and doublet at 7.71 ppm (Figure 1) was
observed.
Figure 1.
Doublet at 7.71 ppm belongs to hydrogens in ortho-position of the phenyl ring, while
broad signal at 13.38 ppm belongs to pyrazole NH. Those data suggested that tautomer A
is principal one in DMSO solution. Upon addition of one molar equivalent of piperidine,
to deprotonate carboxyl group, broad signal at 13.38 ppm was shifted to 9.75 ppm and
NOESY signal disappeared (Figure S1 in Supplementary Material). Our preliminary
results suggested that tautomeric form A should not be predominant one when carboxyl
group of title compounds is in its anionic form, although it is well known that tautomeric
preferences are different in different solvents. Tautomeric preferences of title compounds
will be a subject of separate studies. IR spectra of compounds were obtained by ATR
(attenuated total reflectance) from solid samples. In spectra of some compounds we
clearly observed broad IR bands in the region 3100-3250 cm-1, where NH stretching
bands were overlapped with aromatic C-H bands. Such pattern in IR spectra suggested
aggregation via hydrogen bonding of some derivatives. Examples are given in
Supplementary Material, Figure S2. This is in accordance with crystal structure of
unsubstituted derivative,17 where in crystal packing intermolecular H-bonds between
carboxyl group and pyrazole -NH-N= part is observed. We also estimated protonation
states18 of compound 1 (Figure S3 in Supplementary Material). pKa Value of carboxyl
group is predicted to be within the range 3.6 - 4.0; while pKa value of pyrazole nitrogen is
predicted to be within the range 10.2 -10.7, depending on tautomeric state (Figure S4 in
Supplementary Material). pKa1 Value (carboxyl group) of derivative 22 was
experimentally determined using spectrophotometric titration. Three wavelengths used
for spectrophotometric pKa1 determination (270, 280, 290 nm) were selected as
wavelengths where the difference in absorptivity of species existing in the solution is
maximal. The UV/Vis spectra and the graph used for the pKa1 value determination are
shown in Figure 2. For details see Experimental part. The determined pKa1 of derivative
22 is 3.31 0.07.
Figure 2.
2.3 CA inhibition
Inhibitory activity of compounds toward hCA I, II, IX and XII was evaluated by a
stopped-flow CO2 hydrase assay. Results are reported as Ki values (Table 1).
Table 1.
All compounds tested were proved to be inactive towards CA I and II, which were not
inhibited up to concentrations of 50 µM inhibitor in the assay system. Depending on the
substitution pattern on the phenyl ring, different subsets of compounds were active
towards CA IX or towards CA XII. Along with Ki data reported in Table 1, strong

selective inhibition of 1-23 toward CA IX and CA XII over CA I and II can be noted (see
also Supplementary Table 1). Dose-response curves for inhibitory activity of these
compounds toward all CA isoforms appeared rather flat, still percentage of enzyme
activity of CA I and II were between 60 and 70 % even at 1×10-4 M concentrations of
inhibitors. Providing selectivity toward membrane-associated CA isoforms, considered as
a good anti-tumor targets,19 over cytosolic isoforms, the title compounds might be used as
a good starting point for the design of isoform-selective inhibitors incorporating -COOH
ZBGs. Compounds active toward CA IX and CA XII inhibited those two isoforms in
one- to two-digit micromolar concentrations, comparable with the potency of the majority
of so-far reported CA inhibitors bearing this ZBG.
2.4 Structure-activity study
In an attempt to explain selectivity of some of these compounds toward CA IX and CA
XII and to explain preferences of different subsets of compounds toward CA IX or CA
XII, we performed docking studies. For this purpose we chose Protein data bank (PDB)
crystal structures derived from the wild-type CA isoforms and having a high resolution.
CA I was modeled from PDB entry 1HCB, at 1.60 Å resolution20; CA II from 3B4F, at
1.89 Å resolution21; CA IX from 3IAI, at 2.20 Å resolution22 and CA XII from 1JD0, at
1.50 Å resolution.23 Some of those enzymes were co-crystallized with small molecule
inhibitors. For the docking experiments we removed all small molecules and waters,
neutralized proteins with counter-ions, than embedded them in water clusters and relaxed
all structures by molecular dynamics minimization (see Experimental part for details). In
this point reasonable question arise: are minimization of the protein structure upon bound
ligand removal can cause collapse of the active site? We believe that regarding CAs this
possibility is not an issue. CAs poses a wide and conical active site in significant extent
exposed to solvent. During catalytic cycle, i.e. CO2 hydration, small changes of protein
conformation is observed, and such changes mainly can be attributed to the His64 (proton
shuttle) conformation change. Consequently, different from some other enzyme classes,
CAs in their crystal structures are not ‘trapped’ in some local free-energy minimum for
which another, close, local minimum can exist. Additionally, majority of CA inhibitors
are medium-sized molecules, which typically did not significantly change conformation
of active site, comparing to uninhibited protein. By visual inspection of the 3D structures
of very efficient inhibitors (typically nanomolar, from the sulfonamide class)
cocrystallized with the CAs it can be observed that such molecules typically did not
interact with its whole molecular surface with the active site residues. Most probably this
can be attributed to the wide CAs active site, as well as to very high affinity of ZBG to
catalytic Zn2+ ion. On the other hand, from the modelling set-up view, it should be noted
that MD minimization (as far as recent CHARMM force field variants and recent NAMD
releases are used) commonly very limited mobility of backbone appears, while position
of side-chains are usually efficiently relaxed. In all so far reported crystal structures of
CA co-crystallized with small molecules having a -COOH group within enzyme active
site, the water molecule bound to catalytic Zn2+ ion was present. Thus, for the docking
study we added this water molecule. Initial position of the water was estimated from PDB
entry 4E3G,13 para-hydroxybenzoyc acid co-crystallized with CA II, and further refined
by the WaterFLAP protocol. All compounds were treated in their anionic forms, with

deprotonated carboxyl group, in accordance with predicted and experimentally
determined pKa values. Both tautomers A and B, Scheme 2, were used for docking. Initial
attempts of docking compounds into the active site of each isoform appeared very
inconclusive, so we proceeded considering the whole enzyme as a target.
The docking study found all compounds bound out of the active site of CA I
(Figure S4 in Supplementary Material), in the cleft lined by the residues Pro13, Asn24,
Thr100 and His103. The carboxylate anion of these compounds makes polar interactions
with Ser99 and Thr100 hydroxyl groups, while the pyrazole nitrogens make polar
interactions with Asn245 side-chain and His103 backbone carbonyl. Larger alkyl
substituents were typically anchored between side chains of Pro13 and Pro247. For CA
II, the docking study also found majority of the compounds out of the active site (Figure
S5 in Supplementary Material). The compounds are clustered in a region lined with
residues: Trp5, Lys170, Asn232 and Pro237. The carboxylate anion makes polar
+
interactions with His4 NH backbone and Lys170 side chain NH3 , whereas the pyrazole
NH was hydrogen bounded with Phe231 backbone carbonyl. For CA I and CA II similar
docking solutions were found irrespective of the position of the His64 (‘in’ or ‘out’).
Such results revealed higher affinity of these compounds toward regions of CA I
and CA II out of enzyme active site and can offer possible explanation of inactivity of 1-
23 toward these cytosolic CA isoforms studied here.
Our docking study found all compounds bound close to the active site Zn2+ ion of
CA IX and CA XII (including those for which Ki > 50 µM were measured). All
compounds were clustered with very similar orientation of the pyrazole-COO– moiety in
the active site of each isoform, but orientation of this part of the molecule appears
different in CA IX and CA XII, respectively. In CA IX, the carboxylate anion makes
polar interactions with the hydroxyl group of Thr199 and Thr200 and with the water
coordinated to zinc. The pyrazole NH makes polar interactions with Gln67 side-chain
carbonyl. As a trend, compounds found as active toward CA IX (Table 1) have alkyl
substituents in ortho- and meta-positions of the phenyl ring. Compounds having bulkier
substituents in para-position appeared to be inactive. Visual inspection of docking results
revealed that ortho- and meta-substituted compounds, as well as those having medium-
sized alkyl substituents in para-position assessed better shape complementarily with the
cleft of active site than the rest of the investigated compounds, which were inactive
toward CA IX. This cleft is lined by backbone of residues Trp5, Asn62 and His64, and
situated distal from the sequence 131-134. (Figure 3 and Figure S6 in Supplementary
Material). These residues were shown earlier to be involved in the binding of various
classes of CAIs or CA activators.24
Figure 3.
This observation could explain the structural features that differentiate active from
inactive compounds towards CA IX. Along with this, we observed that compounds 9
(2,3,5,6-tetra-methyl derivative) and 5 (4-t-butyl derivative) with Ki < 10 µM, have
mutually very similar 3D-dependent whole-molecular properties, particularly apolar
surface area and volume (Table S2 in Supplementary Material).
Docking solutions for CA XII found all compounds close to the active site Zn2+
ion, but in orientations quite different compared to CA IX. The carboxylate anion makes

polar interactions with Tyr7 and Thr199 hydroxyl groups, or Asn62 side-chain NH2. The
pyrazole NH and N make polar interactions with Asn62 side-chain or water-mediated
interactions with the Thr199. Aromatic moiety of the compounds were directed toward
the sequence 131-134 (130’s segment). This part of the active site is specific for CA XII
and it was suggested that can be used for the design of isoform specific inhibitors [23].
Visual inspection of the docking results showed that aryl moiety of all compounds found
active towards CA XII makes van-der Walls contacts with the 130’s segment, while the
same moiety in inactive compounds cannot reach this segment (Figure 4 and Figure S7 in
Supplementary Material). This rationalizes the structure-activity relationship data of 1-23
observed towards CA XII and shown in Table 1.
Figure 4.
It should be noted that we did not observe any regularity between calculated
affinities of compounds (docking scores) and the order of activity toward CA IX or CA
XII. ChemGauss4 scoring function accounts for shape and electrostatic
complementarities, as well as the binding site and the ligand desolvation penalty. Not
overall scoring, nor shape complementarities show any trend with the experimentally
found Ki values. Considering the wide and conical shape of the CA active site, the
congeneric set of examined compounds - different in apolar moieties only, and the
narrow range of Ki values, such an observation is not surprising.
In literature, we found one systematic comparison of CAs I, II, IX and XII active
sites.25 CA active site residues involved in ligand binding were listed and similarity and
differences commented. In order to elucidate more structural information that can explain
selectivity of 1-23 toward membrane-associated isoforms, we studied active sites of CA I,
II, IX and XII by GRID force field.26 Molecular interaction fields (MIF) were calculated
by hydrophobic (DRY), HBD (N1) and HBA (O) probes in the box encompassing active
site residues that interact with small molecules in so far reported crystal structures of CAs
(for details see Experimental part and the Supplementary Material, file 2). Inspection of
the results revealed expected differences of MIFs obtained with HBA and HBD probe (O
and N1) in vicinity of residue 67 (Figure S8 in Supplementary Material). In this position
mutually similar Asn of CA II or Gln of CA IX are replaced with His in CA I or Lys in
CA XII. Less obvious difference appears by comparison of MIFs obtained by
hydrophobic probe (DRY). In all four isoforms studied here, the global minimum of
DRY probe, on about –3 kcal/mol, appears in vicinity of Pro202 - conserved in all four
enzymes. On arbitrary chosen isocountour level of –0.7 to –0.9 kcal/mol, we observed
‘continuous’ isosurface of DRY probe from the region of Pro202 to the region between
Leu198 and Gln92 in CAs I, II and IX, Figure 5.
Figure 5.
In CA XII, DRY isosurface appears only in region of Pro202 and region between Leu198
and Gln92, but those two isosurface are not mutually connected (Figure 5). Such a
difference appears not only because significantly less voluminous, apolar Ala131 in CA
XII, compared with Leu in CA I, Phe in CA II and Val in CA IX in this position; but also
because of differences of residue in position 204, situated between Pro200 and Leu198.

In CA I this is Ala, in CA II Leu, and in CA IX Tyr. All three residues can form
hydrophobic interactions. In CA XII, the residue in position 204 is Asn that can make
preferably polar interactions. To the best of our knowledge such differences among the
four CA isoforms studied in this work were not explicitly described in literature so far,
and can be used for design of isoform selective inhibitors by introduction of suitable
hydrophobic/hydrophilic moiety relatively far from the ZBG. Probably because of
feasibility of CA II crystallization, there are significantly less wild-type CA XII crystal
structures comparing with CA II, or mimics of other isoforms derived by targeted
mutations of CA II. Thus, in the literature so far, mainly differences in the 130’s segment
have been exploited for the design of CA XII selective inhibitors.
From our docking solutions of the four most potent derivatives toward CA XII
(compounds 2, 4, 14, 15), we observed that their phenyl moieties fairly fit to DRY
minima between Leu198 and Gln92 (Figure 6), while the same moieties of inactive
derivatives are displaced from this region.
Figure 6.
Considering the activity of these compounds against CA IX and XII, the results
obtained by our docking study and comparison of the four isoforms active sites suggested
that more flexibility imposed to novel congeners will be desirable for increasing
inhibitory potency, whereas bulkiness of phenyl substituents should be retained in order
to have isoform selectivity toward CA XII. Having two rotatable bonds in the core
structure (Ph-pyrazole-COOH), and substituents on the phenyl ring with the very limited
conformational freedom (except in the 4-n-Bu derivative, 4), our compounds appeared
relatively rigid. Thus, anchored near the Zn-bound water by the -COO– moiety, their
hydrophobic moieties cannot simultaneously reach parts of the active site relatively far
from the Zn2+ ion to make stronger non-covalent interactions. These conclusions may be
useful for the design of novel congeners of CAIs incorporating carboxylate ZBGs.
3 Conclusions
We described the synthesis and inhibitory activity of a congeneric set of 23 phenyl-
substituted 5-phenyl-1H-pyrazole-3-carboxylic acids toward human CA isoforms I, II, IX
and XII. The compounds were active in 4-50 µM range and showed strong preferences
toward CA IX and XII over CA I and II. Due to such a selectivity, 5-aryl-pyrazole-3-
carboxylic acids reported in this work could be used for further development of isoform-
specific inhibitors. The results of our docking study offered possible explanations on the
selectivity of these compounds toward CA IX and XII, as well as on their diverse affinity
depending on the substitution pattern of the aryl moiety. Comparison of active sites of all
four isoforms studied by GRID generated molecular-interaction fields revealed
interesting differences between CA XII and other three isoforms in regions that have high
affinity towards hydrophobic moieties. To the best of our knowledge such an observation
was not reported so far and can foster development of the selective CA XII inhibitors.

4 Experimental
4.1 Chemistry:
All chemicals were purchased from Fluka, Aldrich, or Merck, having >98% purity, and
were used as received. For the thin-layer chromatography, silica gel pre-coated plates
with fluorescent indicator (254 nm) were used. Melting points were determined in open
capillary tubes on Stuart SMP-10 apparatus and are uncorrected. ESI-MS (Electrospray
ionization mass spectrometry) analysis was performed in methanol on an Agilent
Technologies 6210-1210 TOF-LC-ESI-MS instrument in positive mode. IR (Infra-red)
spectra were recorded on a Thermo Nicolet 6700 FT-IC spectrophotometer, by ATR
1
13
(Attenuated total reflectance). H, C NMR (Nuclear magnetic resonance) and NOESY
(Nuclear Overhauser effect spectroscopy) spectra were recorded in DMSO-d6 on Bruker
AVANCE 500/125 MHz or on a Varian Gemini2000 200/50 MHz instruments. Chemical
shifts are reported in parts per million (ppm) relative to tetramethylsilane (TMS) and spin
multiplicities are given as follows: s (singlet), d (doublet), t (triplet), q (quartet), qn
(quintet), sx (sextet), h (heptet) m (multiplet), br (broad). UV/Vis Spectra were recorded
on Thermo Scientific Evolution 60S spectrophotometer (Thermo Fisher Scientific Inc,
Waltham, Massachusetts, USA). pH Values were measured using CRISON pH-Burette
24 2S equipped with CRISON 50 29 micro-combined pH electrode (Crison Instruments,
S.A. Spain). The electrode was calibrated by standard CRISON buffer solutions (pH
4.01, 7.00, and 9.21).
4.1.1 Typical experimental procedure for synthesis of 1-23
5-(4-Methylphenyl)-1H-pyrazole-3-carboxylic acid (1): 0.4 g of 4-(4-methylphenyl)-2,4-
dioxobutanoic acid [1] was dissolved in 20 ml of glacial acetic acid in 50 ml round-
bottom flask. In a stirred solution, 280 µL (3 eq.) of hydrazine monohydrate (N2H4·H2O)
was added dropwise. The color of solution was changed to pale yellow immediately after
addition of first amount of hydrazine. The progress of reaction was monitored by TLC.
After completion, reaction mixture was poured in 50 ml H2O and left to stir overnight.
White precipitate was collected, dried on air and recrystallized from the mixture of
hexane/EtOH (~ 9:1), giving 0.34 g of product (87 % of theoretical yield).
4.1.2 Characterization of compounds 1-23
5-(4-Methylphenyl)-1H-pyrazole-3-carboxylic acid. (1) White solid; m.p. = 253-254 °C
(hexane/EtOH); C11H10N2O2, Mw = 202.21; ESI-MS: Calculated for C11H11N2O2 [M+H]+:
203.08150, measured: 203.08063; IR (ν, cm-1): 3264 (N–H), 1696 (C=O), 1500, 1198; 1H
NMR (500 MHz, DMSO-d6) δ (ppm): 2.30 (s, 3 H), 7.14 (s, 1 H), 7.22 (d, J = 7.83 Hz, 2
H), 7.71 (d, J = 8.31 Hz, 2 H), 13.38 (br). 13C NMR (125 MHz, DMSO-d6) δ (ppm):
20.85, 104.91, 125.25, 128.13, 129.48, 137.58, 140.09, 147.44, 162.04.
5-(4-Ethylphenyl)-1H-pyrazole-3-carboxylic acid. (2) Starting from 0.625g of 4-(4-
ethylphenyl)-2,4-dioxobutanoic acid, 0.55 g of product was obtained (89% of theoretical
yield). White solid; m.p. = 229-230 °C (hexane/EtOH); C12H12N2O2, Mw = 216.24; ESI-

MS: Calculated for C12H13N2O2 [M+H]+: 217.09715, measured: 217.09634; IR (ν, cm-1):
3265 (N–H), 1692 (C=O), 1498, 1237; 1H NMR (200 MHz, DMSO-d6) δ (ppm): 1.17 (t,
J = 7.30 Hz, 3 H), 2.60 (q, J = 7.30 Hz, 2 H), 7.14 (s, 1 H) 7.26 (d, J=7.86 Hz, 2 H), 7.74
(d, J = 8.42 Hz, 2 H), 12.93 (br); 13C NMR (50 MHz, DMSO-d6) δ (ppm): 15.74, 28.19,
105.19, 125.60, 128.57, 140.48, 144.19, 147.67, 162.32.
5-(4-Isopropylphenyl)-1H-pyrazole-3-carboxylic acid. (3) Starting from 0.095 g of 4-(4-
isopropylphenyl)-2,4-dioxobutanoic acid, 0.078 g of product was obtained (83% of
theoretical yield). White solid; m.p. = 241-243 °C (hexane/EtOH); C13H14N2O2, Mw =
230.26; ESI-MS: Calculated for C13H15N2O2 [M+H]+: 231.11280, measured: 231.11197.
1
IR (ν, cm-1): 3229 (N–H), 1691 (C=O), 1499, 1194; H NMR (200 MHz, DMSO-d6) δ
(ppm): 1.22 (d, J = 6.74 Hz, 6 H) 2.91 (h, J = 6.74 Hz, 1 H) 7.14 (s, 1 H) 7.31 (d, J =
13
7.86 Hz, 2 H) 7.76 (d, J = 8.42 Hz, 2 H) 13.05 (br). C NMR (50 MHz, DMSO-d6) δ
(ppm): 23.99, 33.43, 105.11, 125.58, 127.05, 128.78, 148.70, 162.27.
5-(4-n-Butylphenyl)-1H-pyrazole-3-carboxylic acid. (4) Starting from 0.52 g of 4-(4-n-
butylphenyl)-2,4-dioxobutanoic acid, 0.435 g of product was obtained (85% of
theoretical yield). White solid; m.p. = 203-205 °C, dec. (hexane/EtOH); C14H16N2O2, Mw
= 244.29; ESI-MS: Calculated for C14H17N2O2 [M+H]+: 245.12845, measured:
1
245.12744. IR (ν, cm-1): 3320 (N–H), 1665 (C=O), 1455, 1265; H NMR (200 MHz,
DMSO-d6) δ (ppm): 0.85 (t, J = 7.30 Hz, 3 H), 1.26 (sx, J1,2= 7.30 Hz, 2 H), 1.52 (qn, J
= 7.30 Hz, 2 H), 2.55 (t, J = 7.30 Hz, 2 H), 7.14 (s, 1 H), 7.22 (d, J = 8.42 Hz, 2 H), 7.73
(d, J = 7.86 Hz, 2 H), 13.09 (br); 13C NMR (50 MHz, DMSO-d6) δ (ppm): 14.03, 22.02,
33.31, 34.87, 105.19, 125.54, 128.62, 129.09, 138.12, 142.77, 147.79, 162.38.
5-(4-tert-Butylphenyl)-1H-pyrazole-3-carboxylic acid. (5) Starting from 0.62 g of 4-(4-
tert-butylphenyl)-2,4-dioxobutanoic acid, 0.48 g of product was obtained (79% of
theoretical yield). White solid; m.p. = 257-259 °C, dec. (hexane/EtOH); C14H16N2O2, Mw
= 244.29; IR (ν, cm-1): 3315 (N–H), 3145, 1723 (C=O), 1584, 1210; ESI-MS: Calculated
for C14H17N2O2 [M+H]+: 245.12845, measured: 245.12888; 1H NMR (200 MHz, DMSO-
d6) δ (ppm): 2.50 (s, 9 H, overlapped with the solvent signal), 8.31 (s, 1 H), 8.65 (d, J =
13
7.86 Hz, 2 H), 8.98 (d, J = 7.86 Hz, 2 H), 9.94 (br); C NMR (50 MHz, DMSO-d6) δ
(ppm): 31.29, 34.56, 104.70, 125.29, 125.82, 129.20, 141.27, 148.25, 150.65, 162.78.
5-(2,4-di-Methylphenyl)-1H-pyrazole-3-carboxylic acid. (6) Starting from 0.58 g of 4-
(2,4-di-methylphenyl)-2,4-dioxobutanoic acid, 0.49 g of product was obtained (86% of
theoretical yield). White solid, m.p. = 248-249 °C (hexane/EtOH); C12H12N2O2, Mw =
216.24; ESI-MS: Calculated for C12H13N2O2 [M+H]+: 217.09715, measured: 217.09704;
IR (ν, cm-1): 3099 (N–H, overlapped with C–H), 2975, 2917, 1695 (C=O), 1494, 1423,
1279; 1H NMR (200 MHz, DMSO-d6) δ (ppm): 3.51 (s, 3 H), 3.58 (s, 3 H), 8.11 (s, 1 H),
8.24 - 8.36 (m, 3 H; overlapped ortho-H doublet with meta-H signals (doublet and
splitted singlet)), 8.62 (d, J = 7.86 Hz, 4 H), 14.29 (br). 13C NMR (50 MHz, DMSO-d6) δ
(ppm): 20.93, 108.01, 126.94, 127.78, 129.08, 131.79, 135.56, 137.89, 140.56, 146.72,
162.69.

5-(3,4-di-Methylphenyl)-1H-pyrazole-3-carboxylic acid. (7) Starting from 0.38 g of 4-
(3,4-di-methyl phenyl)-2,4-dioxobutanoic acid, 0.33 g of product was obtained (88% of
theoretical yield). White solid, m.p. = 276-277 °C (hexane/EtOH); C12H12N2O2, Mw =
216.24; ESI-MS: Calculated for C12H13N2O2 [M+H]+: 217.09715, measured: 217.09685;
1
IR (ν, cm-1): 3107 (N–H, C–H), 2917, 1697 (C=O), 1508, 1469, 1270; H NMR (200
MHz, DMSO-d6) δ (ppm): 2.24 (s, 3 H), 2.27 (s, 3 H), 7.15 (s, 1 H), 7.20 (d, J = 7.86 Hz,
1 H), 7.57 (d, J = 7.86 Hz, 1 H), 7.65 (s, 1 H), 13.38 (br); 13C NMR (50 MHz, DMSO-d6)
δ (ppm): 19.43, 19.71, 105.08, 122.99, 126.67, 128.58, 130.22, 136.63, 137.01, 140.60,
147.62, 162.45.
5-(2,4,5-tri-Methylphenyl)-1H-pyrazole-3-carboxylic acid. (8) Starting from 0.35 g of 4-
(2,4,5-tri-methylphenyl)-2,4-dioxobutanoic acid, 0.28 g of product was obtained (81% of
theoretical yield). White solid, m.p. = 227-228 °C (hexane/EtOH); C13H14N2O2, Mw =
230.26; ESI-MS: Calculated for C13H15N2O2 [M+H]+: 231.11280, measured: 231.11234;
1
IR (ν, cm-1): 3163, 3109 (N–H, overlapped with C–H), 1696 (C=O), 1514, 1277. H
NMR (200 MHz, DMSO-d6) δ (ppm): 2.22 (s, 6 H), 2.33 (s, 3 H), 3.91 (pyrazole CH2 of
tautomer C (Scheme 2), ~ 34% in respect to main tautomer), 6.88 (s, 1 H), 7.07 (s, 1 H),
13
7.31 (s, 1 H), 13.33 (br); C NMR (50 MHz, DMSO-d6) δ (ppm): 19.20, 20.40, 51.64
(pyrazole CH2 of tautomer C (Scheme 2)), 107.86, 127.80, 130.11, 132.32, 132.73,
133.90, 136.56, 140.69, 146.57, 162.72.
5-(2,3,5,6-tetra-Methylphenyl)-1H-pyrazole-3-carboxylic acid. (9) Starting from 0.37 g
of 4-(2,3,5,6-tetra-methylphenyl)-2,4-dioxobutanoic acid, 0.33 g of product was obtained
(91 % of theoretical yield). White solid, m.p. = 246-248 °C, dec. (hexane/AcOEt);
C14H16N2O2, Mw = 244.29; ESI-MS: Calculated for C14H17N2O2 [M+H]+: 245.12845,
measured: 245.12844; IR (ν, cm-1): 3244 (N–H), 3123, 2927, 1718 (C=O), 1471, 1410,
1242; 1H NMR (500 MHz, DMSO-d6) δ (ppm): 1.88 (s, 6 H), 2.20 (s, 6 H), 6.57 (s, 1 H),
7.05 (s, 1 H), 13.06 (br). 13C NMR (125 MHz, DMSO-d6) δ (ppm): 16.66, 19.69, 108.14,
130.47, 131.65, 133.19, 142.40, 144.52, 163.03.
5-(2,4,6-tri-Ethylphenyl)-1H-pyrazole-3-carboxylic acid. (10) Starting from 0.41 g of 4-
(2,4,6-tri-ethylphenyl)-2,4-dioxobutanoic acid, 0.33 g of product was obtained (82% of
theoretical yield). White solid, m.p. = 221-224 °C, dec. (hexane/AcOEt); C16H20N2O2,
Mw = 272.34; ESI-MS: Calculated for C16H21N2O2 [M+H]+: 273.15975, measured:
1
273.15938; IR (ν, cm-1): 3145 (N–H, broad), 2967, 1712 (C=O), 1460, 1263; H NMR
(200 MHz, DMSO-d6) δ (ppm): 0.98 (t, J = 7.30 Hz, 6 H), 1.20 (t, J= 7.30 Hz, 4 H), 2.30
(q, J1,2= 7.30 Hz, 4 H), 2.60 (q, J = 7.30 Hz, 2 H) 6.65 (s, 1 H) 7.00 (s, 2 H) 13.07 (br);
13C NMR (50 MHz, DMSO-d6) δ (ppm): 15.88, 16.01, 26.63, 28.37, 108.77, 125.56,
126.84, 143.48, 143.90, 144.97, 163.23.
5-(2,4-di-Isopropylphenyl)-1H-pyrazole-3-carboxylic acid. (11) Starting from 0.35 g of
4-(2,4-di-isopropylphenyl)-2,4-dioxobutanoic acid, 0.28 g of product was obtained (79%
of theoretical yield). White solid, m.p. = 213-216 °C, dec. (hexane/AcOEt); C16H20N2O2,
Mw = 272.34; ESI-MS: Calculated for C16H21N2O2 [M+H]+: 273.15975, measured:
1
273.15940; IR (ν, cm-1): 3232 (N–H), 2962, 1704 (C=O), 1465, 1273; H NMR (200
MHz, DMSO-d6) δ (ppm): 1.16 (d, J=6.74 Hz, 6 H), 1.22 (d, J=6.74 Hz, 6 H), 2.91 (h,

J=6.74 Hz, 1 H), 3.20 (h, J= 6.74 Hz, 1 H), 6.75 (s, 1 H), 7.12 (d, splited J1,2 = 1.68 Hz,
J1,3 =7.86 Hz, 1 H), 7.25 (d, b, J = 7.86 Hz, 1 H), overlapped with 7.28 (s, 1H), 13.26
(br); 13C NMR (50 MHz, DMSO-d6) δ (ppm): 29.28, 33.74, 108.21, 123.74, 123.96,
127.55, 130.17, 146.99, 149.43, 162.70
5-(2,4,6-tri-Isopropylphenyl)-1H-pyrazole-3-carboxylic acid. (12) Starting from 0.32 g of
4-(2,4,6-tri-isopropylphenyl)-2,4-dioxobutanoic acid, 0.21 g of product was obtained
(66% of theoretical yield). ESI-MS: Pale yellow solid, m.p. = 217-219 °C, dec.
(hexane/AcOEt); C19H26N2O2, Mw = 314.42; Calculated for C19H27N2O2 [M+H]+:
315.20670, measured: 315.20693; IR (ν, cm-1): 3154 (N–H, C–H), 2961, 1700 (C=O),
1567, 1461; 1H NMR (200 MHz, DMSO-d6) δ (ppm): 1.08 (d, J=6.74 Hz, 12H), 1.24 (d,
J=6.74 Hz, 6 H), 2.49 (h, J=6.74 Hz, 1H, overlapped with DMSO-d6 signal), 2.92 (h,
J=6.74 Hz, 1H), 3.82 (pyrazole CH2 of tautomer C (Scheme 2), ~ 36% in respect to main
13
tautomer), 6.65 (s, 1H), 7.10 (s, 2H), 13.09 (br, 2H); C NMR (50 MHz, DMSO-d6) δ
(ppm): 23.98, 30.20, 33.83, 51.53 (pyrazole CH2 of tautomer C (Scheme 2)), 108.83,
120.40, 125.49, 148.16, 149.63, 163.13.
5-(5,6,7,8-Tetrahydronaphthalen-2-yl)-1H-pyrazole-3-carboxylic acid.(13) Starting from
0.51 g of 4-(5,6,7,8-tetrahydronaphthalen-2-yl)-2,4-dioxobutanoic acid, 0.41 g of product
was obtained (82% of theoretical yield). White solid, m.p. = 234-236, dec.
(hexane/EtOH); C14H14N2O2, Mw = 242.27; ESI-MS: Calculated for C14H15N2O2 [M+H]+:
243.11280, measured: 243.11257; IR (ν, cm-1): 3104 (N–H, broad), 2925, 1697 (C=O),
1
1430, 1270; H NMR (500 MHz, DMSO-d6) δ (ppm): 1.72 (m, b, 4 H) 2.70 (s, b, 2 H),
2.74 (s, b, 2H) 7.07 - 7.09 (m, overlapped pyrazole CH singlet and meta-H doublet, 2 H)
7.50 - 7.52 (m, overlapped ortho-Hs singlet and doublet, 2 H), 13.32 (br); 13C NMR (126
MHz, DMSO-d6) δ (ppm): 22.71, 28.63, 28.87, 104.77, 122.54, 125.75 127.95, 129.42,
136.82, 137.11, 140.38, 147.35, 162.12.
5-(Naphthalen-2-yl)-1H-pyrazole-3-carboxylic acid. (14) Starting from 0.58 g of 4-(
naphthalen-2-yl)-2,4-dioxobutanoic acid, 0.49 g of product was obtained (82% of
theoretical yield). White solid, m.p. = 274-276 °C, dec. (hexane/AcOEt); C14H10N2O2,
Mw = 238.24; ESI-MS: Calculated for C14H11N2O2 [M+H]+: 239.08150, measured:
1
239.08065; IR (ν, cm-1): 3206 (N–H), 1683 (C=O), 1501, 1274; H NMR (200 MHz,
DMSO-d6) δ (ppm): 7.23 (s, 1 H), 7.50 (t, J1,2= 3.93 Hz, 2 H), 7.89 (d, overlapped with
doublet at 7.95, 1 H), 7.95 (d, J= 8.42 Hz, 2 H), 8.04 (d, J = 8.42 Hz, 1 H), 8.39 (s, 1 H);
13C NMR (50 MHz, DMSO-d6) δ (ppm): 104.77, 123.81, 124.00, 126.23, 126.71, 127.93,
128.31, 128.55, 130.40, 132.77, 133.50, 141.89, 149.21, 163.07.
5-((1,1'-Biphenyl)-4-yl)-1H-pyrazole-3-carboxylic acid. (15) Starting from 0.50 g of 4-
((1,1'-biphenyl)-2-yl)-2,4-dioxobutanoic acid, 0.42 g of product was obtained (85% of
theoretical yield). White solid, m.p. = 286-287 °C, dec. (hexane/AcOEt); C16H12N2O2,
Mw = 264.28; ESI-MS: Calculated for C16H13N2O2 [M+H]+: 265.09715, measured:
1
265.09653; IR (ν, cm-1): 3220 (N–H), 1657 (C=O), 1451, 1275; H NMR (200 MHz,
DMSO-d6) δ (ppm): 7.28 (s, 1H), 7.36 (t, J = 7.30 Hz, 1 H), 7.46 (t, J = 7.86 Hz, 2 H),
7.69 (d, 2 H, overlapped with doublet at 7.74), 7.74 (d, J = 7.86 Hz, 2 H), 7.95 (d, J =

7.86 Hz, 2 H), 13.21 (br); 13C NMR (50 MHz, DMSO-d6) δ (ppm): 105.66, 126.13,
126.84, 127.38, 127.89, 129.28, 130.40, 139.82, 140.00, 147.66, 162.14.
5-(4-(Pyrrolidin-1-yl)phenyl)-1H-pyrazole-3-carboxylic acid. (16) Starting from 0.2 g of
4-(4-(pyrrolidin-1-yl)phenyl)-2,4-dioxobutanoic acid, 0.16 g of product was obtained
(81% of theoretical yield). Pale red solid, m.p. = 276-279 °C, dec. (hexane); C14H15N3O2,
Mw = 257.29; ESI-MS: Calculated for C14H16N3O2 [M+H]+: 258.12370, measured:
1
258.12359; IR (ν, cm-1): 3207 (N–H), 2963, 1711(C=O), 1616, 1515, 1189; H NMR
(200 MHz, DMSO-d6) δ (ppm): 1.94 (br, t, 2 H), 3.24 (br, t, 2 H), 6.54 (d, J = 8.42 Hz, 2
13
H), 6.91 (s, 1 H), 7.60 (d, J=8.42 Hz, 2 H), 8.91 (br); C NMR (50 MHz, DMSO-d6) δ
(ppm): 25.17, 47.48, 103.04, 111.91, 118.28, 126.46, 142.10, 147.67, 147.94, 163.24.
5-(4-Fluorophenyl)-1H-pyrazole-3-carboxylic acid. (17) Starting from 0.58 g of 4-(4-
fluorophenyl)-2,4-dioxobutanoic acid, 0.37 g of product was obtained (65% of theoretical
yield). White solid, m.p. = 227-228 °C (hexane/AcOEt); C10H7FN2O2, Mw = 206.17;
ESI-MS: Calculated for C10H8FN2O2 [M+H]+: 207.05643, measured: 207.05643; IR (ν,
1
cm-1): 3334 (N–H), 3091, 1659 (C=O), 1453, 1240; H NMR (200 MHz, DMSO-d6) δ
(ppm): 7.24 (s, 1 H), 7.30 (t, 3JHH=8.42 Hz, 3JHF = 8.99 Hz, 2H), 7.92 (dd, 3JHH = 8.42 Hz,
4JHF = 5.05 Hz, 2 H) 11.81 - 14.42 (s, br); 13C NMR (50 MHz, DMSO-d6) δ (ppm):
105.57, 115.84, 116.28, 127.56, 127.73, 128.20, 139.38, 147.74, 159.84, 161.92, 164.71.
5-(4-Chlorophenyl)-1H-pyrazole-3-carboxylic acid. (18) Starting from 0.27 g of 4-(4-
chlorophenyl)-2,4-dioxobutanoic acid, 0.23 g of product was obtained (87% of
theoretical yield). White solid, m.p. = 256-257 °C (hexane/AcOEt); C10H7ClN2O2, Mw =
222.63; ESI-MS: Calculated for C10H8ClN2O2 [M+H]+: 223.02688, measured:
1
223.02694; IR (ν, cm-1): 3188 (N–H), 1679 (C=O), 1488, 1271; H NMR (200 MHz,
DMSO-d6) δ (ppm): 8.47 (s, 1 H) 8.71 (d, J=8.42 Hz, 2 H) 9.10 (d, J=8.99 Hz, 2 H); 13C
NMR (50 MHz, DMSO-d6) δ (ppm): 105.86, 127.29, 129.18, 130.62, 132.88, 161.76,
two low-intensity signals from pyrazole C=N and C–NH not observed.
5-(3-Bromophenyl)-1H-pyrazole-3-carboxylic acid. (19) Starting from 0.57 g of 4-(3-
bromo phenyl)-2,4-dioxobutanoic acid, 0.45 g of product was obtained (80% of
theoretical yield). White solid, m.p. = 249-251 °C (hexane/AcOEt); C10H7BrN2O2, Mw =
267.08; ESI-MS: Calculated for C10H8BrN2O2 [M+H]+: 266.97637, measured:
1
266.97587; IR (ν, cm-1): 3246 (N–H), 1694 (C=O), 1462, 1304; H NMR (200 MHz,
DMSO-d6) δ (ppm): 7.35 (s, 1 H), 7.42 (t, J = 7.30 Hz, 1 H), 7.55 (d, J = 7.30 Hz, 1 H),
13
7.90 (d, J=7.86 Hz, 1 H), 8.09 (s, 1 H), 13.43 (br); C NMR (50 MHz, DMSO-d6) δ
(ppm): 106.23, 122.61, 124.58, 125.58, 128.00, 129.20, 131.04, 131.33, 161.70.
5-(4-Hydroxyphenyl)-1H-pyrazole-3-carboxylic acid. (20) Starting from 0.46 g of 4-(4-
hydroxyphenyl)-2,4-dioxobutanoic acid, 0.39 g of product was obtained (87% of
theoretical yield). White solid, m.p. = 282-283 °C, dec. (hexane/AcOEt); C10H8N2O3, Mw
= 204.18; ESI-MS: Calculated for C10H9N2O3 [M+H]+: 205.06077, measured: 205.05983;
1
IR (ν, cm-1): 3290 (N–H), 1679 (C=O), 1616, 1514, 1265; H NMR (200 MHz, DMSO-
d6) δ (ppm): 6.85 (d, J = 8.42 Hz, 2 H), 7.04 (s, 1 H), 7.67 (d, J = 8.42 Hz, 2 H), 9.80

(br) 12.90 (br); 13C NMR (50 MHz, DMSO-d6) δ (ppm): 104.31, 115.95, 122.01, 127.07,
141.07, 147.50, 157.90, 162.63.
5-(2-Methoxyphenyl)-1H-pyrazole-3-carboxylic acid. (21) Starting from 0.40 g of 4-(2-
methoxyphenyl)-2,4-dioxobutanoic acid, 0.29 g of product was obtained (74% of
theoretical yield). White solid, m.p. = 234-235 °C (hexane/AcOEt); C11H10N2O3, Mw =
218.21; ESI-MS: Calculated for C11H11N2O3 [M+H]+: 219.07642, measured: 219.07644;
1
IR (ν, cm-1): 3270, (N–H), 1708 (C=O), 1470, 1179; H NMR (200 MHz, DMSO-d6) δ
(ppm): 3.91 (s, 3 H), 7.05 (t, J = 7.30 Hz, 1 H) 7.13 (d, overlapped with singlet at 7.17, 1
H), 7.17 (s, 1 H), 7.37 (t, J = 8.42, 1 H), 7.83 (dd, J1,2 = 7.86 Hz, J1,3 =1.68 Hz, 1 H),
13.27 (br). 13C NMR (50 MHz, DMSO-d6) δ (ppm): 55.76, 108.19, 112.18, 118.79,
120.97, 127.98, 129.91, 141.22, 142.86, 156.19, 162.98.
5-(4-Methoxyphenyl)-1H-pyrazole-3-carboxylic acid. (22) Starting from 0.50 g of 4-(4-
methoxyphenyl)-2,4-dioxobutanoic acid, 0.45 g of product was obtained (92% of
theoretical yield). White solid, m.p. = 226-228 °C, dec. (hexane/AcOEt); C11H10N2O3,
Mw = 218.21; ESI-MS: Calculated for C11H11N2O3 [M+H]+: 219.07642, measured:
1
219.07540; IR (ν, cm-1): 3309, 3249 (N–H), 1679 (C=O), 1456, 1206; H NMR (200
MHz, DMSO-d6) δ (ppm): 3.77 (s, 3 H) 6.99 (d, J = 8.99 Hz, 2 H), 7.09 (s, 1 H), 7.76 (d,
13
J = 8.42 Hz, 2 H), 13.21 (br); C NMR (50 MHz, DMSO-d6) δ (ppm): 55.45, 104.75,
114.60, 123.70, 127.00, 140.62, 147.45, 159.57, 162.43.
5-(4-Methoxy-2,5-dimethylphenyl)-1H-pyrazole-3-carboxylic acid. (23) Starting from
0.62 g of 4-(4-methoxy-2,5-dimethylphenyl)-2,4-dioxobutanoic acid, 0.54 g of product
was obtained (89% of theoretical yield). Pale yellow solid, m.p. = 232-234 °C, dec.
(hexane/AcOEt); C13H14N2O3, Mw = 246.26; ESI-MS: Calculated for C13H15N2O3
[M+H]+: 247.10772, measured: 247.10769; IR (ν, cm-1): 3304, 3164 ( N–H), 1695
(C=O), 1510, 1430, 1238; 1H NMR (200 MHz, DMSO-d6) δ (ppm): 2.14 (s, 3 H), 2.36 (s,
13
3 H), 3.81 (s, 3 H), 6.81 (s, 1 H), 6.87 (s, 1 H), 7.27 (s, 1 H), 13.08 (br); C NMR (50
MHz, DMSO-d6) δ (ppm): 15.77, 20.96, 55.56, 107.68, 112.89, 122.74, 123.27, 131.17,
134.65, 140.82, 146.27, 157.42, 162.83.
Representative 1H and 13C NMR spectra are provided in Supplementary Material, Figures
S10-S31.
4.1.3 Determination of acidity constant
The acidity constant (pKa1) value of 4-MeO derivative (22) was determined using
spectrophotometric titration at 25 1 °C. The 50.0 ml of 1×10-4 M working solution was
prepared in 10 mM phosphate buffer, pH=1.73, I=0.1 M (NaCl) and titrated with 1M
KOH till pH 6 was reached (total volume change was less than 1% at the end of titration).
UV/Vis spectra were recorded in 220-420 nm range against the phosphate buffer as a
blank. pKa1 Value was calculated according to equation:27

A
λ(A- )
− Aλ
Aλ =
⋅ Κa1 + Αλ(HA)
[Η3O+ ]
Where A
is the absorbance of pure deprotonated compound (-COO–), Αλ(ΗΑ) is
λ(A− )
absorbance of molecular form (-COOH), and Aλ is the absorbance of solutions at different
pH values where both forms of compound 22 exist in the solution.
4.2 CA Inhibition
An SX.18MV-R Applied Photophysics stopped-flow instrument was used for assaying
the CA-catalyzed CO2 hydration activity by using the method of Khalifah.28 Inhibitor and
enzyme were preincubated for 15 min prior to assay. IC50 Values were obtained from
dose response curves working at seven different concentrations of test compound (from
0.1 nM to 100 µM), by fitting the curves using PRISM (www.graphpad.com) and non-
linear least squares methods. The obtained values represent the mean of at least three
different determinations, as described earlier by us.29,30 The inhibition constants (Ki) were
then derived by using the Cheng-Prusoff equation, as follows: Ki = IC50/(1 + [S]/Km),
where [S] represents the CO2 concentration at which the measurement was carried out,
and Km the concentration of substrate at which the enzyme activity is at half maximal. All
enzymes used were recombinant, produced in E.coli as reported earlier.31,32,33,34 The
concentrations of enzymes used in the assay were: hCA I, 12.3 nM; hCA II, 7.7 nM; hCA
IX, 9.9 nM and hCA XII, 12.3 nM.
4.3 Molecular modeling
Structures of compounds 1-23, in its anioic form, were prepared in SMILES notation
(simplified molecular-input line-entry system). Both tautomers A and B (Scheme 2) were
35
considered. Conformers of the each compound was generated in OMEGA 2.4.3, using
MMFF94s force field (build and search) and default settings.36 With the exception of the
4-n-Bu derivative (4), conformation assemblies per molecule did not exceed 50
conformations. The use MMFF94s force filed without Coulomb interaction (NoEstat);
and/or decrease of root-mean-square (rms) value of atomic positions between conformers
that should be included in conformational assembly, comparing to OMEGA default
settings, did not produced more conformers per molecule. Carbonic anhydrases were
modeled from the following PDB entries: CA I - 1HCB, CAII - 3BF4, CAIX - 3IAI and
CAXII - 1JD0. The one subunit (subunit A), of the CAIX and CAXII was used, and
water and other small molecules were removed from all structures used for modelling.
Protonation states of His residues was adopted from PDB entry 4G0C,37 obtained by
neutron diffraction. His64 was treated as a neutral, with protonated delta N. In the input
PDB files, protonation states of histidine residues were specified by appropriate residue
names, following CHARMM convention (HSD or HSE). Hydrogens of other atoms are
ascribed automatically during proteins preparation using CHARMM-GUI.38 Protonation
states of ionizable residues other than His was subsequently visually checked and
compared with protonation states of 4G0C structure. Each protein was neutralized by
addition of counter-ions and embedded in water sphere surrounding protein by 10 Å layer

from the edges. CHARMM36 parameters were ascribed by CHARMM-GUI. Each
protein was submitted to three cycles of minimization (minimization - heating-
minimization…) during 20 ps each, without any restrains, in NAMD2.9.39 Conjugate
gradient minimizer was used. In this way obtained protein structures were used for
docking studies and for comparison of active sites. For docking studies Zn-bound water
molecule was added. Docking of compounds 1-23 to all four proteins were performed by
FRED 3.0.140,41 with docking resolution set to high, without any constraint. The whole
protein, considered as the receptor, was prepared in OpenEye ‘MakeReceptor’ graphical
user interface. Both His64 ‘in’ and ‘out’ conformation was considered in each protein, in
separate docking runs. Ten top-ranked docking solutions were used for analysis. For the
comparison of active sites proteins are aligned by their α-traces using DaliLight web
interface.42,43 GRID26 molecular interaction fields (MIFs) with hydrophobic (DRY), HBD
(N1), and HBA (O) probes, were calculated in FLAP2.1.44,45 Grid resolution was set to
0.5 Å. MIFs were calculated in the box encompassing following residues (1JD0 residues
numbering): Trp5, Thr60, Asn62, His64, Lys67, Asn69, Gln92, His94, His96, His119,
Val121, Ala131, Ser132, Thr133, Ala134, Val143, Leu198, Thr199, Thr200, Pro202 and
the Zn2+ ion, for the each protein and having exactly the same size. Box used for the
GRID calculation, along with the important residues labeled, on the example of hCA XII,
is depicted in Supplementary Material, Figure S9. The same box and the field of DRY
probe on isocountour level of –0.7 kcal/mol is enclosed as a PyMol state file
(Supplementary Material, file 2). The 3D-dependent whole molecular properties (Surface
area, Polar surface area, Apolar surface area, Volume and VirtualLogP 46) of compounds
1-23 in its anionic forms were calculated in VegaZZ 3.0.547 from structures optimized by
semiempirical molecular-orbital PM6 method,48 as applied in MOPAC2012.49 For the
calculation of 3D-dependent molecular properties, probe with radius of 0 Å was used.
VegaZZ were also used for handling of protein structures and for set-up and analysis of
MD calculations. MD and docking calculations were performed on Linux-based multi-
node clusters equipped with the Intel Xeon E5345, or X5560 processors.50 Figures from
FLAP or FRED outputs were prepared in PyMol 1.7.051 or VIDA 4.2.1. 2D depictions
provided in Supplementary Material were made by OpenEye OEDepictTK.
Acknowledgements
Ministry of Education, Science, and Technological Development of Serbia (Grant No.
172035) and EU FP7 grants (Metoxia and Dynano projects for CTS) supported this work.
Authors gratefully acknowledge OpenEye Scientific Software, Santa Fe, NM, for the
free, academic, licensing of software tools. Authors gratefully acknowledge
computational time provided on HPCG and PARADOX-III high-performance computing
facilities. HPCG cluster is located at the Institute of Information and Communication
Technologies of the Bulgarian Academy of Science (IICT-BAS). PARADOX-III is
located at the Scientific Computing Laboratory, Institute of Physics, Belgrade, and
supported by national and EU grants.
Appendix A. Supplementary data
Supplementary data related to this article can be found in the online version, at doi:

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Table, Schemes and Figures Captions:
Table 1. Structures of compounds 1-23 and inhibitory activities (given as Ki) toward four
CA isoforms.
Scheme 1. Synthesis of 5-aryl-1H-pyrazole-3-carboxylic acids. (a) CH3COCl,
AlCl3/CH2Cl2, 6h, 0°C to rt; (b) 2 eq. Na/CH3OH, 15h, rt, then conc. HCl, (c)
N2H4·H2O/CH3COOH, 8h, rt.
Scheme 2. Tautomers of aryl-pyrazole-carboxylic acids
Figure 1. NOESY spectrum of 4-Me-derivative (1) in DMSO. Signal between ortho-Ph-
hydrogens and pyrazole NH is illustrated.
Figure 2. a) UV/Vis Spectra of compound 22 in pH range 1.73-5.68 used for pKa1
determination (t=25 1 °C); wavelength range used for calculations is magnified; b)
Determination of Ka1 at 280 nm according to Equation 1; slope = 4.0 0.2×10-4, intercept
= 0.764 0.003, r2 = 0.973.
Figure 3. Best-ranked solutions of compounds 9 (a) and 11 (b) docked into CAIX.
Figure 4. Best-ranked solutions of compounds 15 (a) and 14 (b) docked into CAXII.
Figure 5. Isosurface of DRY molecular-interaction field in the active site of CAI, II, IX
and XII, depicted on –0.7 kcal/mol. a) Comparison between CAXII (blue solid field, grey
sticks) and CAI (red mesh field, yellow sticks). b) Comparison between CAXII (blue
solid field, grey sticks) and CAII (red mesh field, orange sticks). c) Comparison between
CAXII (blue solid field, grey sticks) and CAIX (red mesh field, green sticks). Pro202,
conserved in the each CA isoform studied, and residues in position 204 are labeled.
Among other residues shown, residues of 130s’ segment (residues 131-134) on the left
side and His residues which coordinates active site Zn2+ ion (94, 96 and 119) on the right
side are given in stick presentation, without explicit labels; while active site Zn2+ ion is
given as the orange sphere.
Figure 6. Best-ranked docking solutions for compounds 4 (yellow sticks), 14 (green
sticks) and 15 (red sticks) overlapped with the isosurface of DRY probe (–0.7 kcal/mol)
in the CA XII active site. Pro202 and Asn204, given in stick presentation, are labeled.
Among other residues shown, residues of 130s’ segment (residues 131-134) on the left
side and His residues which coordinates active site Zn2+ ion (94, 96 and 119) on the right
side are given in stick presentation, without explicit labels; while active site Zn2+ ion is
given as a red sphere.

Table 1. Structures of compounds 1-23 and inhibitory activities (given as Ki) toward four
CA isoforms.
Ki (µM)*
Compound
R-
hCA I hCA II hCA IX hCA XII
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
42
>50
4.5
>50
5.5
21
1
2
4-Me-
4-Et-
>50
39
3
4-i-Pr-
4
4-n-Bu-
5
5
4-t-Bu-
45
>50
>50
>50
>50
>50
>50
39
6
2,4-di-Me-
3,4-di-Me-
2,4,5-tri-Me-
2,3,5,6-tetra-Me-
2,4,6-tri-Et-
2,4-di-i-Pr-
2,4,6-tri-i-Pr-
β-Tetralinyl-
β-Naphtyl-
4-Ph-
48
7
23.5
5
8
9
>50
16
10
11
12
13
14
15
16
17
18
19
20
21
22
23
39
>50
>50
>50
>50
>50
>50
23.5
>50
>50
49.5
22.5
43.5
5.5
4
33.5
38.5
50
4-Pyrrolidine-
4-F-
4-Cl-
>50
41
3-Br-
4-OH-
50
2-OMe-
>50
>50
4-OMe-
4-OMe-2,5-di-Me-
/
0.25
0.012
0.025
0.006
AAZ**
*Mean from three different assays, errors in the range of 5-10% of the reported values (data not
shown).
**Acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide, AAZ) was used as a standard
inhibitor.

Scheme 1. Synthesis of 5-aryl-1H-pyrazole-3-carboxylic acids. (a) CH3COCl,
AlCl3/CH2Cl2, 6h, 0°C to rt; (b) 2 eq. Na/CH3OH, 15h, rt, then conc. HCl, (c)
N2H4·H2O/CH3COOH, 8h, rt.
Scheme 2. Tautomers of aryl-pyrazole-carboxylic acids
Figure 1. NOESY spectrum of 4-Me-derivative (1) in DMSO. Signal between ortho-Ph-
hydrogens and pyrazole NH is illustrated.

Figure 2. a) UV/Vis Spectra of compound 22 in pH range 1.73-5.68 used for pKa1
determination (t=25 1 °C); wavelength range used for calculations is magnified; b)
Determination of Ka1 at 280 nm according to Equation 1; slope = 4.0 0.2×10-4, intercept
= 0.764 0.003, r2 = 0.973.
a)
b)
Figure 3. Best-ranked solutions of compounds 9 (a) and 11 (b) docked into CAIX.

a)
b)
Figure 4. Best-ranked solutions of compounds 15 (a) and 14 (b) docked into CAXII.
a)
b)

c)
Figure 5. Isosurface of DRY molecular-interaction field in the active site of CAI, II, IX
and XII, depicted on –0.7 kcal/mol. a) Comparison between CAXII (blue solid field, grey
sticks) and CAI (red mesh field, yellow sticks). b) Comparison between CAXII (blue
solid field, grey sticks) and CAII (red mesh field, orange sticks). c) Comparison between
CAXII (blue solid field, grey sticks) and CAIX (red mesh field, green sticks). Pro202,
conserved in the each CA isoform studied, and residues in position 204 are labeled.
Among other residues shown, residues of 130s’ segment (residues 131-134) on the left
side and His residues which coordinates active site Zn2+ ion (94, 96 and 119) on the right
side are given in stick presentation, without explicit labels; while active site Zn2+ ion is
given as the orange sphere.

Figure 6. Best-ranked docking solutions for compounds 4 (yellow sticks), 14 (green
sticks) and 15 (red sticks) overlapped with the isosurface of DRY probe (–0.7 kcal/mol)
in the CA XII active site. Pro202 and Asn204, given in stick presentation, are labeled.
Among other residues shown, residues of 130s’ segment (residues 131-134) on the left
side and His residues which coordinates active site Zn2+ ion (94, 96 and 119) on the right
side are given in stick presentation, without explicit labels; while active site Zn2+ ion is
given as a red sphere.

Graphical abstract