Sulfocoumarins (1,2-benzoxathiine-2,2-dioxides): A class of potent and isoform-selective inhibitors of tumor-associated carbonic anhydrases

Article abstract of DOI:10.1021/jm301625s

Coumarins were recently shown to constitute a novel class of mechanism-based carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. We demonstrate that sulfocoumarins (1,2-benzoxathiine 2,2-dioxides) possess a similar mechanism of action, acting as effective CA inhibitors. The sulfocoumarins were hydrolyzed by the esterase CA activity to 2-hydroxyphenyl-vinylsulfonic acids, which thereafter bind to the enzyme in a region rarely occupied by other classes of inhibitors. The X-ray structure of one of these compounds in adduct with a modified CA II enzyme possessing two amino acid residues from the CA IX active site, allowed us to decipher the inhibition mechanism. The sulfonic acid was observed anchored to the zinc-coordinated water molecule, making favorable interactions with Thr200 and Pro201. Some other sulfocoumarins incorporating substituted-1,2,3-triazole moieties were prepared by using click chemistry and showed low nanomolar inhibitory action against the tumor-associated isoforms CA IX and XII, being less effective against the cytosolic CA I and II.

Full text of DOI:10.1021/jm301625s

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Article
Sulfocoumarins (1,2-benzoxathiine 2,2-dioxides): a class of potent and
isoform-selective inhibitors of tumor-associated carbonic anhydrases
Kaspars Tars, Daniela Vullo, Andris Kazaks, Janis Leitans, Alons Lends, Aiga
Grandane, Raivis Zalubosvkis, Andrea Scozzafava, and Claudiu T Supuran
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301625s • Publication Date (Web): 17 Dec 2012
Downloaded from http://pubs.acs.org on December 18, 2012
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Sulfocoumarins (1,2-benzoxathiine 2,2-dioxides): a class of potent and isoform-selective
inhibitors of tumor-associated carbonic anhydrases#
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Kaspars Tars,a Daniela Vullo,b Andris Kazaks,a Janis Leitans,a Alons Lends,c Aiga Grandane, c
Raivis Zalubovskis,*c Andrea Scozzafava,b and Claudiu T. Supuran b,d*
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a Biomedical Research and Study Center, Ratsupites 1, LV 1067, Riga, Latvia
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Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della
Lastruccia 3, Iꢀ50019 Sesto Fiorentino (Firenze), Italy.
c Latvian Institute of Organic Synthesis, Aizkraukles 21, LVꢀ1006, Riga, Latvia.
d Università degli Studi di Firenze, Dipartimento di Scienze Farmaceutiche, Via Ugo Schiff 6, Iꢀ
50019 Sesto Fiorentino (Firenze), Italy.
Abstract: Coumarins were recently shown to constitute a novel class of mechanismꢀbased carbonic
anhydrase (CA, EC 4..2.1.1) inhibitors. We demonstrate that sulfocoumarins (1,2ꢀbenzoxathiine
2,2ꢀdioxides) possess a similar mechanism of action, acting as effective CA inhibitors. The
sulfocoumarins were hydrolyzed by the esterase CA activity to 2ꢀhydroxyphenylꢀvinylsulfonic
acids which thereafter bind to the enzyme, in a region rarely occupied by other classes of inhibitors.
The Xꢀray structure of one of these compounds in adduct with a modified CA II enzyme
possessesing two amino acid residues from the CA IX active site, allowed us to decipher the
inhibition mechanism. The sulfonic acid was observed anchored to the zincꢀcoordinated water
molecule, making favorable interactions with Thr200 and Pro201. Some other sulfocoumarins
incorporating substitutedꢀ1,2,3ꢀtriazole moieties, were prepared by using click chemistry, and
showed low nanomolar inhibitory action against the tumorꢀassociated isoforms CA IX and XII,
being less effective against the cytosolic CA I and II.
_______
*Correspondence authors. Tel +371ꢀ67014826; Fax: +371ꢀ67550338; Eꢀmail: raivis@osi.lv (RZ);
Tel: +39ꢀ055ꢀ457 3005; Fax: +39ꢀ055ꢀ4573385; Eꢀmail: claudiu.supuran@unifi.it (CTS).
# The Xꢀray coordinates of the hCA II – 9 adduct are available in PDB with the ID code 4bcw.
Abbreviations: CA, carbonic anhydrase; CAI, CA inhibitor; DIPEA, diisopropylethylamine; hCA,
human CA; MS, mass spectrometry; THF, tetrahydrofuran.
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Introduction
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Coumarins such as 1, a natural product isolated from the Australian plant Leionema
ellipticum, P.G. Wilson (Rutaceae), or the simple unsubstituted coumarin 2, were recently
discovered to act as effective inhibitors of the metalloenzyme carbonic anhydrase (CA, EC
4.2.1.1).1ꢀ3 A large number of diversely substitutedꢀcoumarins was subsequently screened for their
inhibitory activity against all the 13 catalytically active mammalian CA isoforms,4ꢀ6 CA IꢀVII, IX,
XIIꢀXV. Many of these isoforms are established drug targets for designing agents with various
applications, such as diuretics, antiglaucoma drugs, anticonvulsants, antiobesity agents or antitumor
drugs/cancer diagnostic tools.7ꢀ9
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CAs are involved in numerous physiological and pathological processes, including
respiration and transport of CO2/bicarbonate between metabolizing tissues and lungs, pH and CO2
homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions (such as
gluconeogenesis, lipogenesis and ureagenesis), bone resorption, calcification, tumorigenicity, and
many other physiological and pathological processes in humans,3,4,9 as well as the growth and
virulence of various fungal/bacterial/protozoan pathogens.10ꢀ12 Many of the isoforms/enzymes
involved in these processes are therapeutic targets with the potential to be inhibited or activated to
treat a wide range of disorders.2,4,9ꢀ11 However a critical barrier to the design of CA inhibitors
(CAIs) as therapeutic agents is related to the high number of isoforms, their diffuse localization in
many tissues/organs, and the lack of isozyme selectivity of the presently available classical
inhibitors (sulfonamides and their bioisosteres).2ꢀ4 The recently discovered coumarins represented a
completely new structural motif for the inhibition of this enzyme class, and also led to the first
isoformꢀselective inhibitors with a very high selectivity ratio for inhibiting transmembrane isoforms
(such as CA IX and XII) over the cytosolic, widespread, offtarget ones (CA I and II).1ꢀ6
Scheme 1 here
Coumarins 1 and 2 were shown to act as “prodrug” inhibitors, being hydrolyzed by the
esterase CA activity to the corresponding 2ꢀhydroxyꢀcinammic acids 3Z and 4E, which are the de
facto CAIs, as shown by kinetic, Xꢀray crystallographic and MS methods (Scheme 1).1,2
Interestingly, these compounds were found bound at the entrance of the CA active site cavity 1,2 in a
region where only CA activators were observed earlier in the many CA – modulators of activity
adducts reported in the literature.7ꢀ9,13 In this region of the enzyme active site, the classical CAIs
such as the sulfonamides,8 sulfamates,8,9 sulfamides,8,9,14 dithiocarbamates15 or other (in)organic
anions16 were never observed. All these classes of CAIs in fact directly coordinate to the
catalytically crucial Zn(II) ion from the active site, by substituting the water molecule/hydroxide ion
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coordinated to it,7ꢀ9 or by adding to the metal ion coordination sphere, leading to trigonal
bipyramidal geometries of the Zn(II).8,9 Thus, coumarins are a relevant class of CAIs because: (i)
they are mechanismꢀbased, “prodrug” inhibitors; (ii) they bind in a totally different active site
region compared to the classical inhibitors (sulfonamides and congeners); and (iii) they led to
highly isoformꢀselective compounds for many mammalian CA isoforms, such as CA IX, XII, XIII
and XIV among others.1ꢀ6
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Chart 1 here
In addition, coumarins or they derivatives are easy to synthesize, being possible to
incorporate in their molecule a large variety of substitution patterns, which lead to the possibility of
exploring a vast chemical space, difficultly accessible for other classes of CAIs. For example,
thiocoumarins,2,16 2ꢀthioxocoumarins,16 dithiocoumarins,16 coumarinꢀNꢀoximes,16 5ꢀ/6ꢀmembered
lactones17 and thiolactones,17, or lactone oximes,17 were recently investigated for their CA
inhibitory properties (Chart 1). “Sulfocoumarins”, i.e., 1,2ꢀbenzoxathiin 2,2ꢀdioxides were reported
recently by one of our groups18 as coumarin bioisosteres, but their CA inhibitory properties were
not yet explored. In this paper we report that sulfocoumarins are indeed a new class of mechanismꢀ
based CAIs, we decipher their mechanism of action at molecular level (which is similar but not
identical to that of the coumarins), and report a series of new such derivatives prepared by using
click chemistry. Some of these compounds showed low nanomolar inhibitory activity against two of
the most interesting CA isoforms, the tumorꢀassociated CA IX and XII (these enzymes are
overexpressed in many hypoxic tumors),9,19 being much weaker inhibitors against the offtarget,
widely spread cytosolic isoforms CA I and II.
Results and Discussion
Chemistry and CA inhibition. A series of monoꢀ or disubstituted 1,2ꢀbenzoxathiin 2,2ꢀdioxides
possessing various functionalities on the benzene ring, of types 5-12, were reported recently18 by
one of our groups. They were synthesized by an intramolecular aldol cyclization reaction of
mesylsalicyl aldehydes in the presence of strong bases, such as 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene
(DBU), and the Xꢀray structure of one of them, the bromo derivative 9, was reported too.18 These
compounds were stable at room temperature and in the presence of air humidity. 18 Compounds 5-
12 were however not investigated for their possible CA inhibitory properties. This may be expected,
as they are coumarin bioisosteres (Chart 1), and structurally related such compounds were recently
studied as CA inhibitors.1ꢀ6,16,17
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As compounds 5-7 and 9-11 reported earlier18 incorporated simple functionalities, such as
the hydroxyl, methanesulfonyl, benzyloxy, bromo, nitro and amino (in position 6 of the 1,2ꢀ
benzoxathiin 2,2ꢀdioxide ring), or the 5,6ꢀbenzoꢀ 8 and 6,8ꢀdisubstituted derivative 12, it appeared
of interest to incorporate a larger chemical diversity in this ring system, by using click chemistry.20
Thus, the amine 11 reported earlier was diazotized and subsequently converted to the azide 13,
which was then reacted with a series of alkynes, being transformed to the 1,2,3ꢀtriazoles 14-23 by
the classical click chemistry cycloaddition reaction20 (Scheme 2). Compounds 14-23 incorporated
several groups at the triazole ring (such as substituted aryl, carboxyalkyl, substituted silyl,
alkylaminomethyl, etc.), being structurally diverse when compared to compounds 5-12. These
structural variations are essential in order to investigate the structureꢀactivity relationship of this
potentially new class of CAIs. The new compounds 14-23 were fully characterized and their
structure confirmed (see Experimental protocols for details).
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Scheme 2 here
Sulfocoumarins 5-23 have been investigated for their inhibitory activity against four human
(h) CA isoforms, the cytosolic, widespread hCA I and II (offtargets in this case) as well as the
transmembrane, tumorꢀassociated hCA IX and XII (anticancer drug targets).9ꢀ19 Several of these
derivatives were also investigated for the inhibition of an engineerized protein, a hCA II in which
two amino acid residues present in the hCA IX active site have been introduced, denominated here
CA II/IX mimic and reported recently by McKenna’s group.21 This is a double mutant of hCA II
with Ala65 replaced by Ser, and Asn67 replaced by Gln (as these are the corresponding amino acids
found in hCA IX).22 It has been demonstrated earlier that this CA IX mimic may provide a useful
model to design isozymeꢀspecific CA IX inhibitors.21 We have used this CA II/IX mimic in this
work, due to the fact that CA IX is difficult to crystallize, whereas this mimic easily crystallized in
complex with one of the compounds investigated here (see later in the text).
Table 1 here
Table 1 shows inhibition data of sulfocoumarins 5-23 (as well as coumarins 1 and 2 as
standards) against hCA I, II, IX and XII, after a period of 6 hours of incubation of the enzyme and
inhibitor solutions. It should be mentioned that assaying the inhibition with the usual 15 min
incubation period (as for the sulfonamides)23 leads to the measurement of a very weak inhibition
(data not shown). This was also the case with the coumarins.1,2 For this reason, a 6 h incubation
time has been used for assaying all sulfocoumarins as CAIs. Compounds 5-7 and 9 were also
investigated for the inhibition of the CA II/IX mimic mentioned above. The following should be
noted regarding the inhibition data of Table 1:
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(i) The cytosolic isoform hCA I was poorly inhibited by the simple sulfocoumarins 5-12 (KIs in the
range of 91 ꢀ > 100 ꢀM), except the amino derivative 11, which was a medium potency inhibitor
(KI of 6.78 ꢀM). A similar behavior of medium potency inhibitors was also observed for the
remaining sulfocoumarins, incorporating the substituted triazole scaffold, of types 14-23, which
showed KIs in the range of 6.00 ꢀ 8.93 ꢀM (Table 1). It should be noted that the natural product
coumarin 1 was an effective hCA I inhibitor (KI of 80 nM), whereas the simple coumarin 2 was a
less effective inhibitor (KI of 3.10 ꢀM), as reported earlier by us.1b Acetazolamide (5ꢀacetamidoꢀ
1,3,4ꢀthiadiazoleꢀ2ꢀsulfonamide), a clinically used sulfonamide,9 showed an inhibition constant of
0.25 ꢀM against this isoform (Table 1).
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(ii) The physiologically dominant isoform hCA II was also poorly inhibited by sulfocoumarins 5-10
and 12 (KIs > 100 ꢀM) and moderately inhibited by coumarin 2, as well as sulfocoumarins 11, and
14-23, with KIs in the range of 6.33 ꢀ 9.64 ꢀM. Coumarin 1 was a more effective hCA II inhibitor
(KI of 62 nM) whereas acetazolamide was a very effective one (KI of 12 nM).
(iii) Some of the simple sulfocoumarins investigated here, e.g., compounds 5-8, showed
submicromolar hCA IX inhibition, with KIs in the range of 0.275 ꢀ 0.375 ꢀM, being thus much
more effective CAIs compared to the coumarins 1 and 2 (KIs in the range of 54.5 ꢀ > 100 ꢀM).
Sulfocoumarins 9 and 10, were slightly less effective as hCA IX inhibitors, with inhibition
constants in the range of 3.77 – 6.83 ꢀM. Among the simple sulfocoumarins, only the amino
derivative 11 was an effective hCA IX inhibitor with a KI of 46 nM, whereas all triazoleꢀcontaining
derivatives were quite effective inhibitors of this isoform, with nanomolar inhibition constants
ranging between 18 – 95 nM. Acetazolamide has a KI of 25 nM against hCA IX. The SAR is thus
quite interesting both for the simple derivatives 5-12 as well as for the triazoleꢀsubstituted ones 14-
23. For the later derivatives, the 6ꢀOH moiety and its derivatization (as sulfonic ester or ether, such
as in compounds 5-7) induced an effective, submicromolar inhibition of isoform hCA IX. 6ꢀBromoꢀ
9 and 6ꢀnitroꢀderivatives 10 were on the other less beneficial substitution patterns, leading to an
order of magnitude loss of efficacy, whereas the 6ꢀamino moiety restored and enhanced hCA IX
inhibitory activity 82 times compared to the corresponding 6ꢀnitro derivative 10. For the
compounds incorporating the substituted 1,2,3ꢀtriazole scaffold in position 6 of the sulfocoumarin
ring, again SAR was quite interesting. The best substitution patterns at the triazole incorporated the
phenyl (compound 14), diethylaminomethyl (19) and 3ꢀ and 4ꢀsubstituted phenyl (20-23) moieties.
These compounds showed inhibition constants in the range of 18ꢀ49 nM (Table 1). The least
effective triazole derivative as hCA IX inhibitor was 15 which incorporated a methoxycarbonyl
moiety (KI of 0.95 ꢀM) but its congener with an extra CH2 group, compound 16, was 11 times more
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effective as an inhibitor (KI of 86 nM). It is obvious that very small structural changes in the
scaffold of sulfocoumarins lead to a dramatic change in inhibitory activity against hCA IX.
(iv) Rather similar SAR with what discussed above for hCA IX has been observed for the inhibition
of the second transmembrane isoform, hCA XII. Thus, several sulfocoumarins, such as 9, 10 and 12
were low micromolar hCA XII inhibitors (inhibition constants in the range of 2.93 – 4.51 ꢀM). The
remaining sulfocoumarins were much more effective inhibitors of this isoform, with KIs in the
range of 7 nM ꢀ 0.717 ꢀM. The simple sulfocoumarins 5-8 were the least effective inhibitors in this
subseries (KIs in the range of 0.234 – 0.717 ꢀM) whereas all the derivatives incorporating 1,2,3ꢀ
triazole moieties, of types 14-23, were low nanomolar hCA XII inhibitors (KIs in the range of 7ꢀ39
nM). In this case all substitution patterns at the triazole ring were highly effective in inducing
excellent inhibition (Table 1). It should be also noted that coumarins 1 and 2 are ineffective hCA
XII inhibitors whereas acetazolamide is a low nanomolar one.
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(v) The CA II/IX active site mimic was inhibited in the submicromolar – low micromolar range by
the sulfocoumarins 5-7 and 9, with inhibition constants of 0.80 – 2.37 ꢀM. Considering the limited
number of compounds tested against this hybrid enzyme, it can be assessed that the inhibition
profile of the CA II/IX active site mimic more closely resembles that of CA IX.
X-Ray Crystallography.
To understand the inhibition mechanism with this new class of CAI, we resolved the Xꢀray crystal
structure (at a resolution of 1.5 Å) of the CA II/IX mimic enzyme complexed with sulfocoumarin 9.
This is due to the fact that hCA IX is difficultly crystallisable, whereas CA II, which easily forms
crystals appropriate for Xꢀray investigations, has a low affinity for most of the simple
sulfocoumarins investigated here. The statistics for the refinement and data collection of the CA
II/IX – 9 adduct are shown in Table 2.
Table 2 here
Scheme 3 and Figs 1, 2 here
Inspection of the electron density maps (Figure 1) at various stages of the refinement,
showed features compatible with the presence of one molecule of inhibitor bound within the active
site, but compound 9 could not be fitted in the observed electron density, as in the case of the
coumarins 1 and 2 investigated earlier.1b,2 Instead, its hydrolysis product, 2ꢀdihydroxyꢀ5ꢀ
bromophenylꢀvinyl sulfonic acid 24 (Scheme 3) perfectly fitted within this electron density (Figure
1). It should be mentioned that compound 9 (as well as other derivatives investigated here of type 5-
23) have been incubated for 6ꢀ24 h with the buffer of the enzyme assay (see above and
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Experimental protocols) in order to see whether the hydrolysis process depicted in Scheme 3 takes
place. No hydrolysis products (of type 24 in the case of 9 could be detected (by means of HPLC)
after this incubation time, which proves that the hydrolysis of the sulfocoumarin to the vinyl
sulfonic acid is mediated by the zinc hydroxide nucleophile from the CA active site, as for the
coumarins which are transformed to 2ꢀhydroxycinamic acids.1,2
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As seen from Fig. 1, the electron density of 24E is perfectly defined for all its atoms. It is
probable that the sulfocoumarin 9 is initially hydrolyzed (by the zinc hydroxide, nucleophilic
species of the enzyme) to the cis vinyl sulfonic acid 24E which subsequently isomerizes
spontaneously to the trans derivative 24E (Scheme 3) which is observed in the electron density of
the CA II/IX – 9 adduct (Fig. 1). Interestingly, the sulfonic acid moiety (presumably as a sulfonate
group, at the pH of 7.8 at which the crystallization was done) is not coordinated to the Zn(II) ion but
it is anchored to the zincꢀbound water molecule/hydroxide ion (Fig. 1). This binding mode was
initially observed for phenol,12a then for spermine,24 a carboxylic acid methyl ester,25 and recently
for several other phenols and aromatic carboxylic acids.26 With the present structure, it is obvious
that anchoring to the zincꢀcoordinated water molecule may be considered as a quite general CA
inhibition mechanism (we prefer to use the term “anchoring to the zincꢀcoordinated water” for the
inhibition mechanism of these compounds, as originally proposed by Christianson,12a over the more
recent and unhappy term “nucleophile recognition” proposed by Martin and Cohen26). The distance
between the zincꢀcoordinated water/hydroxide ion and an oxygen atom of the sulfonate moiety of
24E was of 2.5 Å. Another oxygen of this moiety was at 2.8 Å from the metal ion, being “halfꢀ
coordinated”. The organic scaffold of 24E did not participate in other interactions with residues
from the enzyme active site, except for the OH moiety in ortho to the ethenylsufonate group. This
moiety participates in a bifurcated hydrogen bond with the hydroxyl of Thr200 (of 3.3 Å) and
through a bridging water molecule, with the carbonyl oxygen of Pro201 (of 2.2 Å). The relatively
few interactions between the inhibitor scaffold and the active site may in fact explain the not so
good inhibitory power of this compound against the CA II/IX mimic (KI of 0.93 ꢀM, Table 1). We
stress this again, that in contrast to structurally similar sulfamides (such as acetazolamide which
bind to the metal ion, Fig. 2), the hydrolyzed sulfocoumarins did not replace the Znꢀcoordinating
water molecule, but merely interact with it (Fig. 2). The hydrolyzed compound 9 also did not make
any interactions with the mutated residues within the active site. From the superposition showed in
Fig 2 between the hCA II – acetazolamide adduct and the adduct of the hydrolyzed sulfocoumarin 9
with the CA II/IX mimic, it can be observed the very different inhibition mechanism of the new
class of CAIs reported here, the sulfocoumarins, compared to the widely investigated sulfonamide
inhibitors.
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We demonstrate in this paper that sulfocoumarins, i.e., 1,2ꢀbenzoxathiine 2,2ꢀdioxides, possess a
similar mechanism of CA inhibition as the coumarins, acting as effective inhibitors of this enzyme.
The sulfocoumarins were hydrolyzed by the esterase CA activity to 2ꢀhydroxyphenylꢀvinylsulfonic
acids which thereafter bound within the enzyme active site, in a region rarely occupied by other
classes of inhibitors, such as the sulfonamides or the dithiocarbamates which are Zn(II) ion binders.
The Xꢀray crystal structure of one of the investigated 6ꢀhydroxyꢀ1,2ꢀbenzoxathiine 2,2ꢀdioxides in
adduct with a modified CA II enzyme (possessing two amino acid residues from the CA IX active
site), allowed us to decipher the inhibition mechanism. The vinylsulfonic acid formed by hydrolysis
of the initial sulfocoumarin was observed anchored to the zincꢀcoordinated water molecule, making
favorable interactions with Thr200 and Pro201. Some other sulfocoumarins incorporating
substitutedꢀ1,2,3ꢀtriazole moieties, were prepared by means of click chemistry. The simple
sulfocoumarins were usually micromolar inhibitors of hCA IX and XII, being ineffective against
hCA I and II. The compounds incorporating 1,2,3ꢀtriazole moieties showed low nanomolar
inhibitory action against the tumorꢀassociated isoforms hCA IX and XII, being less effective against
the cytosolic hCA I and II. Sulfocoumarins, similar to coumarins, constitute a versatile, effective
and isoformꢀselective novel class of inhibitors targeting the tumorꢀassociated isoforms, hCA IX and
XII.
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Experimental Protocols
Chemistry
Commercial reagents were purchased from Sigma Aldrich, Acros or AlfaAesar and used without
further purification. Compounds 5-12 were prepared as reported earlier.18 Reactions were monitored
using thinꢀlayer chromatography (TLC) on EMD Merck Silica Gel 60 F254 plates. Visualization of
the developed plates was performed under UV light (254 nm). Flash chromatography was carried
out using Merck silica gel (230ꢀ400 mesh). Organic solutions were concentrated under reduced
pressure on a Büchi rotary evaporator. 1H and 13C NMR spectra were recorded on a Varian Mercury
400 spectrometer. 1H and 13C NMR spectra were internally referenced to the residual solvent signal.
Elemental analyses were performed on a CarloErba CHNSeO EAꢀ1108 apparatus. High resolution
mass spectra (HRMS) were obtained on a QꢀTOF Micro Spectrometer (ESI). Melting points were
determined on an OptiMelt Automated Melting Point System. Purity of the new compounds was
assessed by HPLC and was > 98 %.
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6-Azido-1,2-benzoxathiine 2,2-dioxide (13)
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A solution of amine 11 (2.50 g, 12.7 mmol) in trifluoric acetic acid (17 mL) was cooled to 0 °C and
NaNO2 (0.964 g, 13.8 mmol) was added. The reaction was stirred at 0 °C for 30 minutes. NaN3
(0.826 g, 12.7 mmol) solution in water (4.5 mL) was cooled to 0 °C and added to reaction mixture
and stirred for 1 hour at 0 °C. Precipitate was collected by filtration and dried under vacuum to
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yield 2.52 g (89 %) of 13 as orange crystalline solid: mp 113.5 – 114.5 °C (dec.); H NMR (400
MHz, DMSOꢀd6, δ): 7.31 (dd, J = 2.8, 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 2.8 Hz,
1H), 7.59 (d, J = 10.4 Hz, 1H), 7.69 (d, J = 10.4 Hz, 1H); 13C NMR (100 MHz, DMSOꢀd6, δ):
119.9, 120.0, 120.1, 122.9, 123.6, 135.8, 137.5, 147.6. Anal. Calcd for C8H5N3O3S: C, 43.05; H,
2.26; N, 18.83. Found: C, 42.73; H, 2.32; N, 18.69.
General Procedure for the Synthesis of 1,2,3-Triazolyl Derivatives
To a solution of 13 (1.0 equiv) in dry THF (1mL per mmol of 13) DIPEA (50 equiv), the
appropriate alkyne (1.1 or 2.0 or 5.0 equiv) and CuI (2 equiv) were added. The resulting mixture
was stirred at room temperature under an argon atmosphere for 20 h then poured into the mixture of
ice and water and extracted with EtOAc, dried over Na2SO4, and the solvent was evaporated to
obtain a crude product.
1-(2,2-dioxido-1,2-benzoxathiin-6-yl)-4-phenyl-1H-1,2,3-triazole (14)
Compound 14 was obtained from 13 (0.142 g, 0.64 mmol), phenylacetylene (0.08 mL, 0.70 mmol),
DIPEA (5.57 mL, 32 mmol) and CuI (0.244 g, 1.28 mmol). The crude product was recrystallized
1
from EtOH to yield 0.111 g (53 %) of 14 as yellow crystalline solid: mp 239 – 240 °C; H NMR
(400 MHz, DMSOꢀd6, δ): 7.38 – 7.43 (m, 1H), 7.49 – 7.55 (m, 2H), 7.71 (d, J = 10.4 Hz, 1H), 7.76
(d, J = 8.8 Hz, 1H), 7.85 (d, J = 10.4 Hz, 1H), 7.92 – 7.97 (m, 2H), 8.15 (dd, J = 8.8, 2.8 Hz, 1H),
8.41 (d, J = 2.8 Hz, 1H), 9.35 (s, 1H); 13C NMR (100 MHz, DMSOꢀd6, δ): 119.7, 119.8, 120.0,
121.2, 123.5, 123.8, 125.2, 128.2, 128.9, 129.8, 134.1, 135.7, 147.3, 149.9; HRMSꢀESI (m/z) calcd
for C16H12N3O3S [M + H]+ 326.0599, found 326.0641.
Protein production and purification. The CA II gene with the two CA IX mutations in active
site21 was ordered in Genscript and sequence cloned in modified pET14b vector (Novagen) without
a 6xHisꢀtag. The plasmid was transformed in E. coli strain BL21(DE3) and cells grown in LB
medium containing 50 mg/L ampicillin at 37oC until OD590 reached 0.5. IPTG was added to 1mM
concentration, cells grown for another 4h and harvested by centrifugation. Cells were reꢀsuspended
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in a buffer, containing 40 mM TrisꢀHCl pH 8.0, 200 mM NaCl, 20 mM MgSO4, 0.1% TritonꢀX100,
0.1 mg/mL DNAse, and 1 mg/mL lysozyme and additionally lysed by sonification. The lysate was
clarified by centrifugation and loaded on a His Trap HP affinity column (GE Healthcare). Most of
the CA II mutant protein was adsorbed on the column due to two naturally occuring histidines (His3
nd 4) close to the Nꢀterminus. The protein was eluted by using 300 mM imidazole in 40 mM trisꢀ
HCl buffer and further purified by gelꢀfiltration on Superdex 200 gelꢀfiltration and MonoꢀQ ion
exchange columns (GE Healthcare).
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Crystallization and data collection. The protein was concentrated to 8 mg/ml in 20 mM trisꢀHCl
pH 8.0 using 10kDa cutoff Amicon concentrator. Crystallization was done by the sitting drop
technique in 96ꢀwell MRC plates (Molecular Dimensions). 1ꢁL of protein solution was mixed with
1 ꢁL of bottom solution, (3M (NH4)2SO4, 100 mM trisꢀHCl pH 8.0). and 0.2 ꢁL of 100 mM
compound 9 disolved in 100% DMSO. The obtained crystals were soaked in the mother liquor
containing 30% glycerol and flashꢀfrozen in liquid nitrogen. Data were collected at beamline I911ꢀ
2, MAXꢀlab synchrotron, in Lund, Sweden.
Structure determination. Images were processed by MOSFLM28 and scaled by SCALA.29 The
30,31
structure was refined by REFMAC
using unliganded CA II mutant (PDB code 3DC9)21 as an
initial model. The parameter files for compound 9 were generated by LIBCHECK.30,31 Ligand was
fitted in electron density by using COOT,32 followed by further REFMAC runs.33 Data scaling,
refinement, and validation statistics are listed in Table 2.
CA inhibition. An Applied Photophysics stoppedꢀflow instrument has been used for assaying the
CA catalysed 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 (pH 7.5) as buffer,
and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the
CAꢀcatalyzed CO2 hydration reaction for a period of 10ꢀ100 s. The CO2 concentrations ranged from
1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each
inhibitor at least six traces of the initial 5ꢀ10% 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 distilledꢀdeionized
water and dilutions up to 0.01 nM were done thereafter with distilledꢀdeionized water. Inhibitor and
enzyme solutions were preincubated together for 15 min ꢀ 24 h at room temperature (15 min) or 4
°C (all other incubation times) prior to assay, in order to allow for the formation of the EꢀI complex
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or for the eventual active site mediated hydrolysis of the inhibitor. The inhibition constants were
obtained by nonꢀlinear leastꢀsquares methods using PRISM 3, as reported earlier,1b,2 and represent
the mean from at least three different determinations. Data of Table 1 represent the values after 6 h
inhibition of enzymde and inhibitor. All CA isoforms were recombinant ones obtained inꢀhouse as
reported earlier.1b,2,21
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Acknowledgments. This research was financed in part by two grants of the 6th and 7th Framework
Programs of the European Union (DeZnIT and Metoxia projects), and by the European Social Fund
(Latvia). Thanks are addressed to Dr. A. Maresca for technical assistance.
Supporting Information: The detailed physicoꢀchemical characterization of compounds 15-23
reported in the paper is provided. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Candida glabrata. Bioorg. Med. Chem. Lett. 2012, 22, 859ꢀ862; c) Carta, F.; Aggarwal, M.;
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inhibit carbonic anhydrases and show antiglaucoma action in vivo. J. Med. Chem. 2012, 55, 1721ꢀ
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22. Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C.T.; Pan, P.; Parkkila, S.; Scaloni, A.; Pastorek,
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and Fungal Carbonic Anhydrases. J. Med. Chem. 2011, 54, 1682ꢀ1692.
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Table 1: Inhibition of human (h) isozymes hCA I, II, IX, XII and a CA II/IX active site mimic with
coumarins 1, 2, and sulfocoumarins 5-23, by a stoppedꢀflow, CO2 hydration assay method.23
6
7
8
9
N
N
R
N
R
O
S
O
O
S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
O
5-12
14-23
Compound
KI (ꢀM)#, a
R
hCA Ib hCA II b
hCA IX c
hCA XIIc
CA II/IX mimic
1
2
ꢀ
ꢀ
0.080 0.062
3.10 9.20
54.5
48.6
ꢀ
>100
0.300
0.324
0.275
0.375
6.83
>100
0.234
0.254
0.219
0.717
4.51
ꢀ
5
6ꢀOH
91
99
93
>100
>100
>100
0.80
6
6ꢀMeSO3
6ꢀBnO
2.03
7
2.37
8
5,6ꢀBenzo
6ꢀBr
>100 >100
>100 >100
ꢀ
9
0.93
10
11
12
14
15
16
17
18
19
20
21
22
23
6ꢀO2N
92
>100
3.77
3.16
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
6ꢀH2N
6.78 8.89
>100 >100
6.86 7.76
8.05 6.33
8.88 9.21
6.00 7.20
7.20 9.29
8.11 9.37
8.43 9.64
8.93 9.35
6.71 7.72
7.47 8.61
0.25 0.012
0.046
3.26
0.023
2.93
6,8ꢀCl2
Ph
0.029
0.095
0.086
0.060
0.058
0.025
0.074
0.018
0.048
0.049
0.025
0.032
0.012
0.013
0.009
0.016
0.007
0.014
0.039
0.013
0.021
0.005
COOMe
COOEt
Me3Si
HOCH2
Et2NCH2
4ꢀF3COꢀC6H4
4ꢀMeOꢀC6H4
3ꢀF3CꢀC6H4
3ꢀMeOꢀC6H4
Acetazolamide
ꢀ
a Preꢀincubation of 6 h between enzyme and inhibitor; b Cytosolic full length, recombinant enzyme;
c Catalytic domain, transmembrane, recombinant isoform.
# Errors in the range of ± 10 % of the reported data, from three different assays.
.
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Table 2. Data processing, refinement and validation statistics of the CA II/IX – 9 adduct.
4
5
6
7
8
Space group
Cell dimensions
a (Å)
P21
42.2
41.5
72.0
104.1
20ꢀ1.5
1.6ꢀ1.5
b (Å)
9
c (Å)
β (ο)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Resolution (Å)
Highest resolution shell
(Å)
Number of reflections
Number of reflections in
test set
Completeness (%)
Rmerge
<I/σI>
Average multiplicity
Rꢀfactor
34350
1805
93 (80*)
0.07 (0.32)
7.8 (2.0)
2.0 (1.2)
0.18 (0.30)
0.22 (0.36)
13
Rfree
Average B factor (Å2)
Average B factor for
inhibitor (Å2)
15
<B> from Wilson plot (Å2) 12
Number of protein atoms
Number of inhibitor atoms 14
Number of solvent
molecules
2070
253
r.m.s. deviations from ideal values
Bond lengths (Å)
Bond angles (
Outliers in Ramachandran 0.0
plot
0.011
1.48
̊)
*Values in parenthesis are for the high resolution bin
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1
2
3
4
5
6
7
8
9
OH
OH
O
-
HO
-
COO
O
O
O
OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3Z
-
COO
NaOH
-Na+
-
COO
O
O
OH
4Z
OH
2
4E
Scheme 1: Active site hydrolysis of coumarins 1 and 2 when bound to CA, affords the
hydroxycinnamic acids 3Z or 4E as evidenced by Xꢀray crystallography or enzymeꢀinhibitor
adducts.1a,2
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Journal of Medicinal Chemistry
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2
3
4
R
5
6
7
O
O
S
O
Lactones
(6-membered)
8
9
Thiocoumarin
O
S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-Thioxocoumarin
O
S
Thioactones
(6-membered)
R
S
S
O
O
R
COUMARIN
Dithiocoumarin
O
NOH
Lactone oximes
O
O
O
N OH
Lactones
(5-membered)
Coumarin-oximes
R
O
S
O
O
"Sulfocoumarin"
Chart 1: Compounds investigated as CAIs considering the coumarin scaffold as lead. The
“sulfocoumarins” are reported in the present work as a novel class of CAIs.
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2
3
4
N
R
N
N
NaNO2
NaN3/H2O
H2N
N3
R
DIPEA, CuI
5
6
7
O
O
S
S
O
TFA
0 oC, 1 h
THF
O
S
O
O
O
O
rt, 20 h
O
8
9
11
13
14-23
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2: Preparation of derivatives 14-23 by reaction of azide 13 with alkynes.
Br
Br
H2O
Br
SO3H
O
S
SO3H
O
OH
O
OH
9
24Z
24E
Scheme 3: Active site, CAꢀmediated hydrolysis of 9 to 24E.
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9
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Fig. 1. Binding of hydrolyzed compound 9 within the active site of the CA II/IX mimic. The 2FoꢀFc
map was calculated in the absence of ligand and contoured at 1σ. For the sake of clarity, the map is
shown only around the ligand (in black), zinc ion (grey sphere) and water molecules (red spheres).
Polar interactions are shown with dashed lines and distances are indicated in Å. The Figure was
generated by using PyMol.27
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9
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Fig 2. Comparison of binding modes of hydrolysed sulfocoumarin (compound 9, thick lines with
black carbons) and the sulfonamide CAI acetazolamide (PDB code 2HNC, thin lines with light blue
carbons and dark blue nitrogens). The Zn(II) ion (grey sphere) and its coordinating histidines
(His94, 96 and 119, stick representation, in blue and green) are superposable in both cases. It should
be noted that the sulfonamide inhibitor displaces the Znꢀcoordinating water molecule (red sphere),
whereas the sulfocoumarin anchores to it. The interactions between the inhibitor, the zinc ion and
the water molecule coordinated to zinc are shown as dotted lines.
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