Thioxocoumarins Show an Alternative Carbonic Anhydrase Inhibition Mechanism Compared to Coumarins

Article abstract of DOI:10.1021/acs.jmedchem.5b01720

A series of coumarins and the corresponding 2-thioxocoumarines were prepared and tested for their inhibition profiles against four physiologically relevant human carbonic anhydrases (hCAs, EC 4.2.1.1), isoforms hCA I, II, IX, and XII. The X-ray crystal structure of 6-hydroxy-2-thioxocoumarin bound to hCA II revealed an unprecedented and unexpected inhibition mechanism for this new class of inhibitors when compared to isostructural coumarins. Unlike coumarins which are hydrolyzed by the esterase CA activity to the corresponding 2-hydroxy-cinnamic acid derivatives, the 2-thioxocoumarin was observed intact when bound to hCA II, with its exo-sulfur atom anchored to the zinc-coordinated water molecule, whereas the scaffold establishing favorable contacts with amino acid residues from the active site. This inhibition mechanism is very different from the one observed for hydrolyzed coumarins, which occlude the entrance of the active site cavity. This versatility in the binding mode of coumarins/thioxocoumarins has important consequences for the design of isoform-selective CA inhibitors, some of which are in clinical use or clinical development for various pathologies, among which glaucoma, edema, epilepsy, neuropathic pain, and hypoxic tumors.

Full text of DOI:10.1021/acs.jmedchem.5b01720

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Article
Thioxocoumarins show an alternative carbonic
anhydrase inhibition mechanism compared to coumarins
Marta Ferraroni, Fabrizio Carta, Andrea Scozzafava, and Claudiu T Supuran
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01720 • Publication Date (Web): 19 Dec 2015
Downloaded from http://pubs.acs.org on December 28, 2015
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Thioxocoumarins show an alternative carbonic anhydrase inhibition mechanism compared to
coumarins
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Marta Ferraroni,1 Fabrizio Carta,1 Andrea Scozzafava,1 and Claudiu T. Supuran1,2
*
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1
Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm.
188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
2 Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via
Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
Abstract: A series of coumarins and the corresponding 2ꢀthioxocoumarines were prepared and
tested for their inhibition profiles against four physiologically relevant human carbonic anhydrases
(hCAs, EC 4.2.1.1), isoforms hCA I, II, IX and XII. The Xꢀray crystal structure of 6ꢀhydroxyꢀ2ꢀ
thioxocoumarin bound to hCA II revealed an unprecedented and unexpected inhibition mechanism
for this new class of inhibitors, when compared to isostructural coumarins. Unlike coumarins which
are hydrolyzed by the esterase CA activity to the corresponding 2ꢀhydroxyꢀcinnamic acid
derivatives, the 2ꢀthioxocoumarin was observed intact when bound to hCA II, with its exoꢀsulphur
atom anchored to the zincꢀcoordinated water molecule, whereas the scaffold establishing favorable
contacts with amino acid residues from the active site. This inhibition mechanism is very different
from the one observed for hydrolyzed coumarins, which occlude the entrance of the active site
cavity. This versatility in the binding mode of coumarins/thioxocoumarins has important
consequences for the design of isoformꢀselective CA inhibitors, some of which are in clinical use or
clinical development for various pathologies, among which glaucoma, edema, epilepsy, neuropathic
pain and hypoxic tumors.
Key words: thioxocoumarin, coumarin, metalloenzyme, carbonic anhydrase; isoforms I, II, IX, XII,
Xꢀray crystallography
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Introduction.
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Among the metalloꢀenzymes possessing a crucial physiologic function, the carbonic
anhydrases (CAs, EC 4.2.1.1) represent an interesting case, as they act on very simple substrates,
such as CO2, COS, CS2 or cyanamide1ꢀ3 generating products which are either involved in pH
regulation (bicarbonate and protons), biosynthetic processes (bicarbonate, urea) or in other
important phenomena such as for example chemosensing (in vertebrates and invertebrates),4 sexual
development (in pathogenic fungi),5 pH and CO2ꢀsensing, pathogenicity, and survival in ambient air
of many bacteria, fungi and/or protozoa.6ꢀ8 There are six genetic families encoding such enzymes in
virtually all organisms known to date, the αꢀ, βꢀ, γꢀ, δꢀ, ζꢀ and ηꢀCAs, with the last class reported
very recently.9 All CAs known so far are metal ionꢀdependent enzymes, with a metalꢀhydroxide
species within the enzyme cavity acting as a nucleophile in the catalytic cycle, and a second step
(usually rateꢀdetermining) involving a proton transfer reaction from a water molecule coordinated to
the active site metal ion to the environment, for regenerating the nucleophile.10 Metal ions
employed at the active site of the different CAs include Zn(II) (in all classes), Cd(II) (in ζꢀCAs),
Co(II) (in the δ class) or Fe(II) (for γꢀCAs, in anaerobic conditions).11,12 This pingꢀpong mechanism
makes some of the members of the CA superfamily among the most effective enzymes known in
nature, with kcat/KM values close to the limit of the diffusionꢀcontrolled processes.13
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Only αꢀCAs have been reported in vertebrates, but in most investigated species a large
number of different isoforms were described.1ꢀ3 For example in humans, 15 CA isoforms are
known, CA I ꢀ CA VA, CA VB, CA VI ꢀ CA XIV, with 12 of them being catalytically active and
three (CA VIII, X and XI) devoid of activity but still playing significant functions in tumorigenesis
and other physiologic as well as pathologic processes.14
Due to the fact that the substrates/reaction products of αꢀCAs (CO2, bicarbonate and
protons) are simple molecules/ions involved in a host of physiologic processes, their upꢀ or downꢀ
regulation is associated with a range of diseases.1ꢀ3,15ꢀ18 Indeed, CA inhibitors (CAIs) are clinically
used for decades as diuretics,15b antiglaucoma agents,1b,d,3d antiepileptics,16 or more recently antiꢀ
obesity agents,17 whereas compounds targeting the tumorꢀassociated isoforms CA IX and XII are in
clinical development as anticancer agents/diagnostic tools for hypoxic, metastatic tumors.3,18 CA
activators (CAAs) may have potential for developing agents for Alzheimer’s disease or aging, as in
these pathologies a diminishing of the activity of some physiologically relevant isoforms (such as
CA I and II) has been reported.19
One of the main hurdles connected with the use of CAIs in the treatment of diverse
conditions as those mentioned above, is related to the offꢀtarget inhibition of isoforms other than the
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desired one.1ꢀ3 In fact the various pharmacological applications of the CAIs are due to the high
number of isoforms and their involvement in different pathologies.15ꢀ18
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Recently a number of important advances in the field of designing isoformꢀselective CAIs
targeting various isoforms has been achieved, mainly by using structureꢀbased drug design
approaches.1ꢀ3 Among them the soꢀcalled tail approach is one of the most employed one for such
purposes.20,21 This approach was initially reported for the sulfonamide CAIs,20 and consists in
attaching tails (moieties) able to interact with the middle and the rim part of the active site cavity,
which is the most variable region among the 15 CA isoforms known in humans.1ꢀ3 Thereafter this
approach was extended to all other classes of CAIs, such as the coumarins,22 sulfocoumarins,23 and
dithiocarbamates.24
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It has been demonstrated that coumarins, a class of CAIs reported in 2009, do possess highly
selective CA inhibition profiles, which rely on their particular inhibition mechanism.25 In fact the
coumarin itself acts as a prodrug, whereas its hydrolysis products (formed due to the esterase CA
activity which opens the lactone ring of the coumarin) represents the real inhibitor (Fig. 1).25
Indeed, coumarins A or B in complex with hCA II were crystallized allowing the evidence of their
hydrolysis products A1 and B1 (2ꢀhydroxyꢀcinnamic acid derivatives) bound at the entrance of the
active site cavity, occluding it.25b
OH
O
O
O
O
O
A
B
OH
OH
OH
O
(E)
(Z)
CO
CO
2
2
OH
OH
A1
B1
Figure 1. Superposition of the coumarin A hydrolysis product (transꢀ2ꢀhydroxyꢀcinnamic acid A1
in yellow) with the coumarin B hydrolysis product (cisꢀ 2ꢀhydroxycinnamic acid B1, magenta)ꢀhCA
II adducts (PDB code 3F8E and 5BNL, respectively). The protein backbone is shown as green
(PDB code 3F8E) and grey (PDB code 5BNL) ribbon, the catalytic Zn (II) ion as violet sphere, with
its three protein ligands (His94, 96, and 119) also evidenced.25b
With the aim to pursue the identification of new potent and selective coumarineꢀbased CAIs
we report here a series of coumarins and their corresponding thioxocoumarines 1-19 which were
tested in vitro for their inhibition profiles against four most physiologically important hCAs, such as
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the hCA I, II, IX and XII. The Xꢀray crystal structure adduct of 6ꢀhydroxyꢀ2ꢀthioxocoumarin 8a
bound hCA II at 1.1 Å resolution, is also reported. This data reveals an unprecedented and
unexpected inhibition mechanism of the thioxocoumarins when compared to the structurally related
coumarin scaffold.
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Results and Discussion
Compound design and synthesis. Coumarins were discovered to act as prodrug inhibitors of the
metalloenzyme carbonic anhydrase by this group.25 The first compound for which such an activity
has been reported was the natural product coumarin B (reported in figure 1), which was isolated
from the Australian plant Leionema elipticum.25a Its Xꢀray crystal structure in adduct with the
ubiquitous cytosolic isoform hCA II surprisingly showed that the lactone ring of the inhibitor was
hydrolysed due to the esterase activity of the CA, with formation of the Zꢀhydroxycinnamic acid
derivative B1, which was observed bound at the entrance of the CA active site thus occluding it.25b
No other inhibitors were ever observed in that region of the CA active site,26 which has been always
associated with the binding of the CA activators.26ꢀ28 A similar behaviour was thereafter observed
for the simple coumarin derivative A, which again by means Xꢀray crystallography, was found
bound in the same active site region as B1, but in the case of A1 the Eꢀhydroxycinnamic acid was
observed (figure 1).25b The very new mechanism of CA inhibition revealed for coumarins inspired
much research in this field, mainly because a large number of such derivatives possessing various
substitution patterns at the coumarin ring proved to act as highly isoformꢀspecific CAIs,25,29
a
phenomenon never observed for the main class of clinically used such agents, the sulfonamides and
their isosteres (sulfamates, sulfamides, etc.).30
In a previous work31a we reported the 2Hꢀchromeneꢀ2ꢀthione 2 (2ꢀthioxocoumarin) as well as the
2Hꢀthiochromenꢀ2ꢀone 3 (thiocoumarin) and the thiochromeneꢀ2ꢀthione 4 (dithiocoumarin) as CAIs
isosters of the simple coumarin 1, which was also used for their preparation as depicted in Scheme
1.
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Lawesson's reagent
4
5
6
7
8
9
O
O
O
O
S
Toluene
2
1
Lawesson's reagent
Toluene
i) SOCl2, DCM, 40°C
ii) PhSH, Pyr
iii) AlCl3, Tol.
OH
S
O
S
S
3
4
10
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O
HO
tBuMe2SiCl, Et3N
HO
O
Lawesson's Reagent
O
Tol. reflux
TBAF 1.0M
THF
S
Si
Si
THF, r.t.
O
O
O
O
S
O
6a: 6ꢀOH
6b: 7ꢀOH
5a: 6ꢀOH
5b: 7ꢀOH
7a: 6ꢀOH
7b: 7ꢀOH
8a: 6ꢀOH
8b: 7ꢀOH
Br
O
HO
O
Lawesson's reagent
Toluene, reflux
O
O
O
O
O
S
Cs2CO3
DMF
5a: 6ꢀOH
5b: 7ꢀOH
5c: 4ꢀOH
9a: 6ꢀOH
9b: 7ꢀOH
9c: 4ꢀOH
10a: 6ꢀOH
10b: 7ꢀOH
10c: 4ꢀOH
(OC)6Co2
(OC)6Co2
O
O
O
O
O
S
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14
Co2(CO)8
THF dry
Co2(CO)8
THF dry
OH
Ph3P, DIAD
THF dry
Sonication
0°Cꢀꢀr.t.
Lawesson's reagent
Toluene, reflux
HO
O
O
O
O
O
O
O
S
11
12
5b
PhN3, Cu0 nanosized
TMACl
PhN3, Cu0 nanosized
TMACl
tertꢀBuOH/H2O
tertꢀBuOH/H2O
N
O
N
O
O
O
O
S
N
N
N
N
15
16
Lawesson's reagent
Toluene, reflux
3 O
O
S
18
3OH
Ph3P, DIAD
THF dry
Sonication
0°Cꢀꢀr.t.
HO
O
O
O
O
O
3
5b
17
(OC)6Co2
Co2(CO)8
THF dry
O
O
O
3
19
Scheme 1. Synthesis of coumarin and thioxocoumarin derivatives 2-19.
Moreover 4ꢀ, 6ꢀ and 7ꢀ substituted derivatives 6-10 were also prepared31b and assayed as CAIs
(Table 1 and Scheme 1). As an extension of our previous studies we report here new derivatives,
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such as compounds 11-19, which were obtained by means of known synthetic procedures. As
shown in Scheme 1, the introduction of a terminal alkyne chain, such as in compounds 11 and 17,
was accomplished by means of sonicatedꢀmediated Mitsunobu coupling reactions, which allow fast
and cleaner reaction procedures when compared to the standard thermal conditions (data not
shown). The presence of a terminal alkyne moiety also allowed us to explore the effect of various
moieties, also considering the liker lengths between the main scaffold and the alkyne functionality.
Thus we investigated lipophylic bulky moieties such as in 13 and 19 or a phenyltriazole moiety
(compound 15), which is expected to interact through hydrogen bonds with amino acid residues
located at the rim of the enzymatic cavity. All obtained compounds were treated with Lawesson’s
reagent to afford the corresponding thioxo derivatives 12, 14, 16 and 18.
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Carbonic anhydrase inhibition. As shown in Table 1, compounds 1-19 were tested in vitro for
their inhibition profiles against four physiologycally relevant hCA enzymes, the cytosolic isoforms
I and II and the transꢀmembrane, tumor associated IX and XII.
Table 1. CA inhibition data against isoforms hCA I, II, IX and XII with compounds 2-19 and
acetazolamide (AAZ) as standard, by a stoppedꢀflow CO2 hydrase assay.32
______________________________________________________________________
Compound
KI (ꢁM)*
hCA IXa
______________________________________________________________________
hCA I
hCA II
hCA XIIa
1**
3.1
9.2
>100
6.7
>100
95.2
26.5
96.0
0.68
0.75
6.30
0.28
0.83
0.31
0.35
0.34
0.32
2
>100
>100
>100
>100
58.4
95.0
8.78
8.32
7.57
8.18
7.17
8.02
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100#
>100
3
0.97
19.6
0.19
0.48
0.41
0.80
0.85
0.86
0.96
0.80
0.78
4
5a***
5b***
5c***
6a***
6b***
7a***
7b***
8a***
8b***
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9a***
9b***
9c***
10a***
10b***
10c***
11
30.3
72.9
43.2
8.51
7.60
9.24
72.0
950
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.012
0.93
0.73
0.21
3.26
3.23
3.04
1.35
41.6
61.2
47.3
0.008
0.004
0.83
0.22
52.3
0.025
0.80
0.64
0.88
1.25
2.83
1.27
0.73
38.9
31.0
30.2
0.005
0.027
0.37
0.41
61.2
0.006
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85.8
>200
>200
>200
124
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18
>200
>200
0.20
19
AAZ
______________________________________________________________________
*Errors in the range of ±5 % of the reported values, from three different assays. ** From Ref.16b
***From Ref.25a ;#A KI of 285 µM has been measured, working with higher concentrations of
inhibitor 8a.a Catalytic domain.
In general all compounds reported showed to be lowꢀ medium potency inhibitors of the slow
cytosolic isoform hCA I (KIs of 7.17 ꢁM or > 100 ꢁM), were inactive against the hCA II isoform
with the only exception represented by the simple coumarin 1, and were highly potent inhibitors of
the tumor associated isoforms hCA IX and XII, with KIs spanning between 0.004ꢀ47 ꢁM (hCA IX)
and 0.005ꢀ95.2 ꢁM (hCA XII), respectively. In particular the following structureꢀactivityꢀ
relationship (SAR) considerations for each hCA tested can be drawn:
i) For the hCA I the replacement of one or both of the oxygen atoms into the simple coumarin 1 (KI
3.1 ꢁM) to afford compounds 2-4 resulted in a complete loss of the inhibitory activity (KIs > 100
ꢁM). The introduction of the phenolic moiety into the coumarin 1 at positions 4, 6 and 7 also
spoiled the inhibition potency with KIs of 95.0, >100 and 58.4 for 5c, 5a and 5b respectively.
Conversely the manipulation of the phenol moiety in compounds 5a-c through the introduction of a
TBDMS or an allyl group restored the inhibition potencies against the hCA I to lowꢀmedium
micromolar values. In particular silylation of 5a and 5b (KIs >100 and 58.4 ꢁM) to afford
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compounds 6a and 6b resulted in a significant reduction of the KI values to 8.78 and 8.32 ꢁM
respectively. The introduction of the allyl group in 5a-c resulted in a marked enhancement of the
inhibition potencies only for the 5a and 5c derivative to afford 9a and 9c (KIs 30.3 and 42.9 ꢁM), as
for the 7ꢀOꢀ allyl susbstituted derivative 9b a 1.25 fold decrease of the inhibitory activity was
observed (KI 72.9 ꢁM). The same trend was also observed when coumarin 5b was propargylated
(compounds 11 and 17, KIs 72 and 124 ꢁM) and the terminal acetylenic moiety was further
elaborated with a metallorganic species (compound 13 KI 85.8 ꢁM) or subjected to a copper
catalyzed click chemistry reaction to afford compound 15 (KI >200 ꢁM). Interestingly, the
introduction of an exoꢀsulfur atom into the species previously discussed strongly influenced the
inhibitory potencies. As reported in Table 1 both silyl derivatives 7a,b, their corresponding phenolic
derivatives 8a,b as well as the allyl substituted compounds 10a-c showed KIs in the low micromolar
range and comprised between 7.17ꢀ9.24 ꢁM, which make them among the most potent compounds
within the series against hCA I. Conversely the substitution of the exoꢀ lactonic oxygen with a
sulphur in 11 to afford compound 12 resulted in a significant increase of the KI (> 100 ꢁM) and all
the other derivatives such as 14, 16 and 18 were inactive.
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ii) Contrary to hCA I, the replacement of one or both oxygen atoms within the coumarin 1 scaffold
to afford the compounds 2-4 resulted in a significant increase of the inhibition activity against hCA
IX with KI values of 6.7, 0.97 and 19.6 ꢁM respectively. Also the introduction of hydroxyl moieties
into the coumarin ring at positions 4, 6 and 7 led to a marked increase of the inhibition potency (KIs
of 0.41, 0.19 and 0.48 ꢁM for 5a-c, respectively). The conversion of the hydroxyl moieties in 5a
and 5b, to the corresponding silylated derivatives, such as 6a and 6b, resulted in a slight increase of
the KIs of up to 0.80 and 0.85 ꢁM, respectively. In analogy, the introduction of an Oꢀallyl group at
position 6 and 7 to afford 9a and 9b resulted in a 4.9 and 1.5ꢀfold reduction of the inhibition
activities, respectively. The only exception in this case was represented by the Oꢀallyl derivative at
4 position (compound 9c), which KI was halved (0.41 ꢁM for 5c and 0.21 ꢁM for 9c). The
introduction of a terminal alkyne moiety in 5b to afford compounds 11 and 17 resulted in a 2.8 and
1.73 KI fold increase of the inhibitory power respectively (compared to the lead 1), which was
further enhanced when the cobalt(II)ꢀbased protection of the terminal alkyne was installed (61.2 and
52.3 ꢁM for compounds 13 and 19 respectively). Interestingly, the introduction of the phenyl
triazole moiety in 11 to afford compound 15 led to a great reduction of the KI to 8.0 nM, thus
making it one of the most active inhibitors against hCA IX within the series herein reported. In
general the replacement of the exoꢀoxygen atom of the coumarins with a sulphur, resulted in
reduction of the inhibitory activity, which make the 2ꢀthioxocoumarins a highly interesting class of
CAIs. As shown in Table 1, the thioxo derivatives 8a and 8b had KIs 4.2 and 1.6ꢀ fold higher when
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compared to the coumarin progenitors 5a and 5b. Analogous inhibition profiles were also observed
for the silyl derivatives 7a and 7b (KI 0.86 and 0.96 ꢁM), for the Oꢀallyl thioxo derivatives 10a-c
(KIs of 3.26, 3.23 and 3.04 ꢁM respectively) and the propargyl derivative 12 (KI 41.6 ꢁM).
Interestingly, a slight improvement in the inhibition potency was reported for the pentyne derivative
18 (KI 0.22 ꢁM), for the bulky protected alkyne 14 (KI 47.3 ꢁM) and for the phenyltriazolyl
derivative 16, which was the most potent inhibitor reported within this series against the hCA IX
(KI 4 nM). The phenyltriazolylꢀcontaining compounds 15 and 16 were even more potent CAIs
compared to the standard sulfonamide acetazolamide (KI 25 nM)
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iii) The inhibition profiles of the compounds reported here against hCA XII isoform were more
intricate compared to what discussed above for the other three investigated isoforms, and a clearꢀcut
structureꢀactivity relationship is rather difficult to draw. As for hCA IX, the introduction of one or
more sulphur atoms within the simple coumarine scaffold 1 (KI >100 ꢁM) accounted for a
restoration of the inhibition activity (KIs 95.2, 26.5 and 96.0 ꢁM for 2-4, respectively). Also the
introduction of the hydroxyl moiety at the 4, 6 and 7 positions of the coumarin ring resulted in a
marked enhancement of the inhibitory activity (KIs 6.30, 0.68 and 0.75 ꢁM for compounds 5c, 5a
and 5b, respectively). The functionalisation of the hydroxyl moieties, as for the silyl derivatives 6a
and 6b, determined different behaviours. Thus the 6ꢀOꢀTBDMS derivative 6a was more active
when compared to its progenitor 5a (KI 0.28 ꢁM for 6a and 0.68 ꢁM for 5a); conversely the 7ꢀOꢀ
TBDMS derivative 6b showed just a modest 1.1 fold decrease of its activity (KI 0.83 ꢁM). The
introduction of the Oꢀallyl moiety into the coumarins 5a-c resulted in a reduction of the inhibition
potency for the 4 and 7ꢀsubstituted derivatives 9b and 9c (KIs 0.64 and 0.88 ꢁM respectively) whilst
the 6ꢀOꢀallyl derivative 6a showed a 1.2 times increase of its inhibitory potency. Conversely to the
hCA I and IX enzymes in which the introduction of the Oꢀpropargyl and Oꢀpentenyl chains at the 7ꢀ
position of the coumarin scaffold determined a decrease of the inhibitory potencies, in the case of
hCA XII a slight KI decrease was observed for compound 11 (KI 0.73 ꢁM) and a 2 fold decrease for
the longerꢀchain derivative 17 (KI 0.37 ꢁM). The protection of the terminal alkyne moieties in 11
and 17, to afford compounds 13 and 19, spoiled their inhibition potencies by a 42.5 and 165.4 fold
KI increase, respectively. Interestingly the introduction of the phenyltriazolyl moiety in 11 to afford
compound 15 resulted in a drastic reduction of the KI up to 5.0 nM, thus making this compound as
the most active in the series (against hCA XII) and comparable to the inhibition value of the
sulfonamide AAZ (KI 6.0 nM), which however is a promiscuous CAI unlike the
coumarins/thioxocoumarins. Introduction of the exoꢀsulphur moiety into the simple hydroxyl
coumarins 5a and 5b, as in compounds 8a and 8b, led to an enhancement of the inhibition potency
(KI 0.34 and 0.32 ꢁM respectively) compared to the corresponding coumarins. As for the silyl
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derivatives 7a and 7b only a slight KI increase was observed for the former (KI 0.31 ꢁM) whereas
the latter showed a 0.42 fold increase of its potency (KI 0.35 ꢁM). The insertion of the sulphur atom
in compounds 9a-c to afford 10a-c, determined a reduction of the inhibition against the hCA XII
(KIs 1.25, 2.83 and 1.27 ꢁM, respectively). In analogy the conversion of 11 and 17 to their
corresponding thioxo derivatives 12 and 18 resulted in reduction of the inhibition potencies (KIs
38.9 and 0.41 ꢁM respectively). Only a small KI reduction was observed for compound 14 when
compared to its oxoꢀanalogue 13 (KI 31.0 ꢁM for 13 and 30.2 ꢁM for 14). Finally the thioxo
derivative of 15, i.e., 16, showed a 5.4 fold increase of its KI, thus making it the second most potent
inhibitor against the hCA XII within this series.
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In summary among all the compounds reported here, the phenyltriazolyl bearing derivatives
15 and 16 were the most potent and selective inhibitors of the tumor associated hCA IX and XII
with KIs comparable or lower of the standard sulfonamide AAZ. In particular the introduction of
the exoꢀsulphur atom within the coumarine scaffold of 15, to afford 16, halved the KI against the
tumor associated isoform hCA IX. Such an inhibition profile was not observed against hCA XII.
However both compounds 15 and 16 were highly active and selective for the tumorꢀassociated
isoforms among the derivatives belonging to this series.
X-ray crystallography. In order to understand the structural elements which led to such interesting
inhibitory profiles, as well as to dissect the inhibition mechanism with thioxocoumarins, we report
here the high resolution (1.1 Å) crystal structure of the adduct of hCA II with thioxocoumarin 8a. A
very interesting binding mode for this compound within the enzyme active site (Figure 2A and B,
and experimental section Table 2) has been thus revealed.
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Figure 2. A: FoꢀFc omit map of 8a and water molecules within the hCA II active site in the hCA II
– 8a adduct; B: Tilted view of the electron density of 8a and water molecules within the hCA II
active site.
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The sulphur (S1) atom of inhibitor 8a is hydrogen bonded to the water coordinating the zinc ion
(Wat348 – S1 2.90 Å). The Wat348 to Zn(II) distance is of 1.91 Å, as in most Xꢀray structures of
CA alone or in complex with inhibitors in which the nonꢀprotein zinc ligand is a water
molecule/hydroxide ion.20 Furthermore, S1 forms a hydrogen bond with the Thr199 peptide
nitrogen (S1···H– N Thr199 of 2.64 Å). This is very different from inhibitors which directly
coordinate to the metal ion, in which a hydrogen bond with the OH of Thr199 is usually
observed.21,22 A disordered second water molecule (Wat394) was observed in the electron density
nearby the Zn(II) coordinated water molecule (Wat348). This is probably due to the fact that the
occupancy of the inhibitor in the adduct is of 50% (also due to its low affinity for isoform hCA II;
see Table 1) and to the high degree of disorder observed for the water molecules in the adduct (see
below). This water is too far from the Zn(II) (Wat394 – Zn 2.58 Å) to be considered as coordinated,
and too close to the S1 atom of the inhibitor (Wat394 – S1 1.71 Å). The bicyclic ring system of
inhibitor 8a resides in a hydrophobic pocket formed by residues Phe131, Val121, Leu198 and
Pro202 (Figures 3, 4). The inhibitor forms two CꢀH···O hydrogen bonds with Leu198 (CB
Leu198···H–OAK, of 2.44 Å, and CD2 Leu198···H–OAK Leu198, and one with Thr200,
OG1Thr200···H–CAL of 2.24 Å). The inhibitor was introduced at 0.5 of occupancy, as mentioned
above (see also Supplementary Table 3 for the B factors of the inhibitor/water molecules). Some
water molecules occupy the same position of the inhibitor and they were also introduced at partial
occupancy. Interestingly they form in the active site the same hydrogen bonding network usually
observed in native hCA II structures.
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Thus, unlike coumarins investigated in detail by Xꢀray crystallography (and kinetic
measurements) the structurallyꢀrelated 2ꢀthioxocoumarins possess a CA binding mode which
resembles the phenols,33a polyamines,33b or sulfocoumarins,34 which all anchor to the zincꢀ
coordinated water molecule/hydroxide ion, with the scaffold participating in supplementary
interactions with the active site, thus stabilizing the enzymeꢀinhibitor adduct. In fact as seen from
Figure 3, where the present structure was superimposed on that of the hCA II – coumarin B adduct,
the active site regions occupied by the two structurally similar inhibitors are quite distinct, with the
main difference being that the coumarin B is hydrolyzed whereas the 2ꢀthioxocoumarin 8a was
observed intact within the enzyme active site.
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Figure 3. Superposition of the hCA II ꢀ 8a adduct (sky blue, 4WL4) with the hCA II ꢀ hydrolyzed
coumarin B1 adduct (5BNL) (silver). The zinc ion, its three His ligands and amino acid residues
involved in the binding of inhibitors are shown.
Conclusions. Coumarins and their isosteres represent very interesting classes of CAIs which led to
highly isoformꢀselective compounds. Such derivatives investigated by Xꢀray crystallography and
kinetic measurements allowed the discovery of new CA inhibition mechanisms, i.e., occlusion of
the active site entrance.1ꢀ3 Here we report that the structurallyꢀrelated sulfur containing coumarin
derivatives, such as the 2Hꢀchromeneꢀ2ꢀ thiones, possess a CA inhibition mechanism different form
the parent oxygenꢀbearing compounds. The hCA IIꢀ8a adduct revealed the exoꢀsulfur atom of the
inhibitor anchored to the zincꢀcoordinated water molecule/hydroxide ion, with the scaffold
participating in supplementary interactions within the active site, thus contributing in stabilizing the
enzymeꢀinhibitor adduct. Thus, the main difference of the binding modes between coumarins and
2Hꢀchromeneꢀ2ꢀthione derivatives is that the first were observed hydrolyzed when bound to the
enzyme, whereas the latter ones are not. This different behavior is amenable to drug design
campaigns, also considering the simplicity of the scaffold of 8a and the relative facility with which
some of its derivatives could be obtained. In fact the click chemistry applied to this class of
compounds afforded low nanomolar inhibition of the tumorꢀassociated isoforms hCA IX/XII with
thioxocoumarins, these compounds being not inhibitory against the offtarget cytosolic isoforms
hCA I and II. As one hCA IXꢀselective sulfonamide inhibitor is in Phase I clinical trials for the
treatment of hypoxic, metastatic solid tumors, we estimate that the present findings may lead to
even more interesting drug candidates for the treatment of this condition.
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Experimental protocols
Chemistry
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General. Anhydrous solvents and all reagents were purchased from SigmaꢀAldrich, Alfa Aesar and
TCI. All reactions involving airꢀ or moistureꢀsensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic
resonance (1HꢀNMR, 13CꢀNMR) spectra were recorded using a Bruker Advance III 400 MHz
spectrometer in DMSOꢀd6. Chemical shifts are reported in parts per million (ppm) and the coupling
constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet; brs, broad singlet; dd, double of doublets. The assignment of
exchangeable protons (OH and NH) was confirmed by the addition of D2O. Analytical thinꢀlayer
chromatography (TLC) was carried out on Merck silica gel Fꢀ254 plates. Flash chromatography
purifications were performed on Merck Silica gel 60 (230ꢀ400 mesh ASTM) as the stationary phase
and ethyl acetate/nꢀhexane were used as eluents. Melting points (mp) were carried out in open
capillary tubes using a Gallenkamp MPD350.BM3.5 apparatus and are uncorrected. 2HꢀChromenꢀ
2ꢀone 1 and transꢀcinnamic acid were commercially available from SigmaꢀAldrich, Milan, Italy. All
compounds reported here were > 98% pure.
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Synthesis of 2Hꢀchromeneꢀ2ꢀthione 2.31a
Lawesson's reagent
O
O
O
S
Toluene, Reflux
1
2
2HꢀChromenꢀ2ꢀone 1 (0.5 g, 1.0 eq) was dissolved in dry toluene (20 ml) and treated with
Lawesson’s reagent (2.0 eq). The reaction mixture was refluxed until consumption of the starting
material (TLC monitoring). Then the solvent was removed under vacuo and the obtained residue
was purified by silica gel column chromatography eluting with 20% v/v ethyl acetate/nꢀhexane to
afford the titled compound 2 as a yellow solid.
2HꢀChromeneꢀ2ꢀthione 2: 60% yield; silica gel TLC Rf 0.27 (ethyl acetate/nꢀhexane 20% v/v); νmax
(KBr) cmꢀ1 1765, 1518, 1220; δH (400 MHz, DMSOꢀd6) 7.31 (1H, d, J 10.0, 3ꢀH), 7.61 (1H, dt, J
7.6, 1.2, 6ꢀH), 7.64 (1H, d, J 8.4, 5ꢀH), 7.74 (1H, dt, J 7.6, 1.2, 7ꢀH), 7.85 (1H, d, J 8.4, 8ꢀH), 7.96
(1H, d, J 10.0, 4ꢀH); δc (100 MHz, DMSOꢀd6), 198.5 (C=S), 157.0, 137.0, 133.6, 130.0, 129.6,
126.8, 121.2, 117.1; Anal. Calc. C, 66.64; H, 3.73; S, 19.77; Anal. Found. C, 66.15; H, 3.43; S,
12.38.
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Synthesis of 2Hꢀthiochromenꢀ2ꢀone 3.31a
6
7
8
9
O
O
AlCl3
Toluene
i) SOCl2, DCM, 40°C
ii) PhSH, Pyr.
OH
S
S
O
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3
Intermediate 1
(E)-S-Phenyl 3-phenylprop-2-enethioate
transꢀCinnamic acid (1.0 g, 1.0 eq) was dissolved in dry DCM (20 ml) and thionyl chloride (10.0
eq) was added dropꢀwise at 0 °C. The solution was refluxed until starting material was consumed
(TLC monitoring), then the solvents were removed under vacuo to give a sticky oily residue that
was dissolved in dry pyridine (10 ml) at 0 °C and thiophenol (0.74 g, 1.0 eq) was added dropꢀwise.
The yellow solution was stirred at r.t. for 2 h, quenched with H2O (30 ml), extracted with ethyl
acetate (3 x 15 ml) and the combined organic layers were dried over Na2SO4, filtered and
concentrated under vacuo to give a residue that was purified by silica gel column chromatography
eluting with 5% v/v ethyl acetate/nꢀhexane to afford intermediate 1 as a pale yellow solid.
(E)ꢀSꢀPhenyl 3ꢀphenylpropꢀ2ꢀenethioate (intermediate 1): 62% yield; 94ꢀ96 °C; silica gel TLC Rf
0.17 (ethyl acetate/nꢀhexane 5% v/v); νmax (KBr) cmꢀ1, 1670 (C=O), 1515 (aromatic); δH (400 MHz,
DMSOꢀd6) 7.16 (1H, d, J 16.0, 2ꢀH), 7.49 (3H, m, 2 x 6ꢀH, 7ꢀH), 7.54 (5H, s, SꢀArꢀH), 7.70 (1H, d,
J 16.0, 3ꢀH), 7.84 (2H, m, 2 x 5ꢀH); δc (100 MHz, DMSOꢀd6), 188.0 (C=O), 142.5, 135.4, 134.6,
132.0, 130.5, 130.3, 130.0, 129.9, 128.2, 125.2.
(E)ꢀSꢀPhenyl 3ꢀphenylpropꢀ2ꢀenethioate (0.2 g, 1.0 eq) was dissolved in dry toluene (5.0 ml) and
AlCl3 (0.56 g, 5.0 eq) was added. The orange solution was stirred at 70 °C for 5 h (TLC
monitoring), cooled down to r.t., quenched with slush and extracted with ethyl acetate (3 x 20 ml).
The combined organic layers were washed with H2O (2 x 20 ml), dried over Na2SO4, filteredꢀoff
and concentrated under vacuo to give an orange residue that was purified by silica gel column
chromatography eluting with 5% v/v ethyl acetate/nꢀhexane to afford the titled compound 2 as a
pale yellow solid.
2HꢀThiochromenꢀ2ꢀone 3: 55% yield; 77ꢀ78 °C; silica gel TLC Rf 0.11 (ethyl acetate/nꢀhexane 5%
v/v); νmax (KBr) cmꢀ1, 1660 (C=O), 1515 (aromatic); δH (400 MHz, DMSOꢀd6) 6.65 (1H, d, J 10.8,
3ꢀH), 7.64 (3H, m, 5ꢀH, 6ꢀH, 7ꢀH), 7.92 (1H, d, J 8.0, 8ꢀH), 8.12 (1H, d, J 10.8, 4ꢀH); δc (100 MHz,
DMSOꢀd6), 185.1 (C=O), 145.8, 137.2, 133.0, 131.4, 127.8, 126.8, 126.7, 124.4; Anal. Calc. C,
66.64; H, 3.73; S, 19.77; Anal. Found. C, 62.96; H, 3.63; S, 12.08.
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Synthesis of thiochromeneꢀ2ꢀthione 4
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8
Lawesson's reagent
Toluene, reflux
S
O
S
S
3
9
4
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2HꢀThiochromenꢀ2ꢀone 3 (0.03 g, 1.0 eq) was dissolved in dry toluene (10 ml) and treated with
Lawesson’s reagent (2.0 eq). The reaction mixture was refluxed until consumption of the starting
material (TLC monitoring). Then the solvent was removed under vacuo and the obtained residue
was purified by silica gel column chromatography eluting with 10% v/v ethyl acetate/nꢀhexane to
afford the titled compound 4 as a red solid.
2HꢀThiochromeneꢀ2ꢀthione 4: 33% yield; 103ꢀ105 °C; silica gel TLC Rf 0.20 (ethyl acetate/nꢀ
hexane 10% v/v); νmax (KBr) cmꢀ1, 1770, 1520, 1230; δH (400 MHz, DMSOꢀd6) 7.43 (1H, d, J 10.0,
3ꢀH), 7.61 (1H, dt, J 8.0, 1.6, 5ꢀH), 7.28 (2H, m, 6ꢀH, 7ꢀH), 7.90 (1H, d, J 10.0, 4ꢀH), 8.00 (1H, d, J
8.0, 8ꢀH); δc (100 MHz, DMSOꢀd6), 209.7 (C=S), 140.2, 136.9, 136.3, 133.0, 131.9, 129.2, 128.5,
124.6; Anal. Calc. C, 60.63; H, 3.39; S, 35.97; Anal. Found. C, 59.48; H, 3.05; S, 21.27.
Synthesis of 7ꢀ(propꢀ2ꢀynyloxy)ꢀ2Hꢀchromenꢀ2ꢀone 11.
OH
Ph3P, DIAD
THF dry
Sonication
HO
O
O
O
O
O
5b
0°Cꢀꢀr.t.
11
7ꢀHydroxy coumarin 5b (1.0 g, 1.0 eq), propargyl alcohol (1.0 eq) and triphenylphoshine (1.0 eq)
were dissolved in dry THF (90 ml). Then the temperature was lowered to 0 °C and
diisopropylazadicarboxylate (1.1 eq) was added dropꢀwise under sonication. The orange solution
was sonicated at r.t. under a nitrogen atmosphere using a water bath sonication system working at
40 kHz, until starting material was consumed (TLC monitoring). Solvents were removed under
vacuo to give a white solid that was recrystallized from MeOH to give 2 as a white solid.
7ꢀ(Propꢀ2ꢀynyloxy)ꢀ2Hꢀchromenꢀ2ꢀone 11: 67% yield; m.p. 118 °C (Lit.35 120 °C); silica gel TLC
Rf 0.53 (ethyl acetate/nꢀhexane 50% v/v); νmax (KBr) cmꢀ1, 3310 (C≡CꢀH), 2160 (C≡CH), 1765
(C=O), 1604 (Aromatic); δH (400 MHz, DMSOꢀd6) 3.69 (1H, t, J 2.4, 3’ꢀH), 4.97 (2H, d, J 2.4, 1’ꢀ
H2), 6.36 (1H, d, J 9.6, 3ꢀH), 7.03 (1H, dd, J 8.5, 2.3, 6ꢀH), 7.09 (1H, d, J 2.3, 8ꢀH), 7.69 (1H, d, J
8.5, 5ꢀH), 8.03 (1H, d, J 9.6, 4ꢀH); δC (100 MHz, DMSOꢀd6) 161.1 (Cꢀ2), 161.0 (Cꢀ7), 156.0 (Cꢀ8a),
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145.1 (Cꢀ4), 130.4 (Cꢀ5), 113.9 (Cꢀ3), 113.8 (Cꢀ4a), 113.7 (Cꢀ6), 102.7 (Cꢀ8), 79.8 (Cꢀ2’), 79.4 (Cꢀ
3’) and 57.0 (Cꢀ1’). Data in agreement with reported data.35
6
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Synthesis of 7ꢀ(propꢀ2ꢀynyloxy)ꢀ2Hꢀchromeneꢀ2ꢀthione 12
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Lawesson's reagent
Toluene, reflux
O
O
O
O
O
S
11
12
7ꢀ(Propꢀ2ꢀynyloxy)ꢀ2Hꢀchromenꢀ2ꢀone 11 (0.2 g, 1.0 eq) and Lawesson’s Reagent (1.5 eq) were
dissolved in dry toluene (10 ml) and the yellow solution was refluxed until starting material was
consumed (TLC monitoring). Then the solvent was removed under vacuo and the orange residue
was partitioned between H2O and ethyl acetate. The organic phase was washed with H2O (2 x 20
ml), brine (3 x 20 ml), dried over Na2SO4, filteredꢀoff and concentrated under vacuo to give a red
sticky oil that was purified by silica gel column chromatography eluting with 10 % ethyl acetate in
nꢀhexane to afford the title compound 12 as a yellow solid.
7ꢀ(Propꢀ2ꢀynyloxy)ꢀ2Hꢀchromeneꢀ2ꢀthione 12: 72% yield; m.p. 97ꢀ101 °C; silica gel TLC Rf 0.27
(ethyl acetate/nꢀhexane 10% v/v); νmax (KBr) cmꢀ1, 3300 (C≡CꢀH), 2165 (C≡CꢀH), 1601 (Aromatic);
δH (400 MHz, DMSOꢀd6) 3.72 (1H, t, J 2.4, 3’ꢀH), 5.02 (2H, d, J 2.4, 1’ꢀH2), 7.13 (1H, dd, J 9.2,
2.4, 6ꢀH), 7.18 (1H, d, J 9.2, 3ꢀH), 7.31 (1H, d, J 2.4, 8ꢀH), 7.80 (1H, d, J 9.2, 5ꢀH), 7.90 (1H, d, J
9.2, 4ꢀH); δC (100 MHz, DMSOꢀd6) 198.1 (Cꢀ2), 161.8 (Cꢀ7), 158.6 (Cꢀ8a), 137.4 (Cꢀ4), 130.6 (Cꢀ
5), 127.4 (Cꢀ3), 115.7 (Cꢀ4a), 115.6 (Cꢀ6), 102.3 (Cꢀ8), 80.0 (Cꢀ2’), 79.2 (Cꢀ3’) and 57.3 (Cꢀ1’).
Anal. Calc%. C, 66.65; H, 3.73; S, 14.83; Anal. Found. C, 65.36; H, 3.71; S, 9.37.
Synthesis of 7ꢀ(propꢀ2ꢀynyloxy)ꢀ2Hꢀchromenꢀ2ꢀone hexacarbonyldicobalt 13
(OC) Co
6
2
Co (CO)
2
8
THF dry
O
O
O
O
O
O
11
13
7ꢀ(Propꢀ2ꢀynyloxy)ꢀ2Hꢀchromenꢀ2ꢀone 11 (0.1 g, 1.0 eq) was dissolved in THF (10ml) and then
cobalt carbonyl (1.05 eq) was added. The black solution was stirred at r.t. for 40 min. Then SiO2
(0.3 g) was added and solvent removed under vacuo to give a black solid that was purified by silica
gel column chromatography eluting with 20% v/v ethyl acetate/nꢀhexane to afford 6 as a red solid.
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7ꢀ(Propꢀ2ꢀynyloxy)ꢀ2Hꢀchromenꢀ2ꢀonehexacarbonyldicobalt 13: 94% yield; silica gel TLC Rf 0.22
(ethyl acetate/nꢀhexane 20% v/v); νmax (KBr) cmꢀ1 1752 (C=O), 1600 (Aromatic); δH (400 MHz,
DMSOꢀd6) 5.50 (2H, s, 1’ꢀH2), 6.35 (1H, d, J 9.4, 3ꢀH), 6.89 (1H, s, 3’ꢀH), 7.00 (1H, dd, J 8.8, 2.4,
6ꢀH), 7.11 (1H, d, J 2.4, 8ꢀH), 7.70 (1H, d, J 8.8, 5ꢀH), 8.04 (1H, d, J 9.4, 4ꢀH); δC (100 MHz,
DMSOꢀd6) 200.9 (C=O), 161.7 (Cꢀ2), 161.0 (Cꢀ7), 156.2 (Cꢀ8a), 145.1 (Cꢀ4), 130.5 (Cꢀ5), 113.7,
113.6, 113.4, 102.4 (Cꢀ8), 90.8 (Cꢀ3’), 73.9 and 69.4.
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Synthesis of 7ꢀ(propꢀ2ꢀynyloxy)ꢀ2Hꢀchromeneꢀ2ꢀthione hexacarbonyldicobalt 14
(OC)6Co2
Co2(CO)8
THF dry
O
O
S
O
O
S
12
14
7ꢀ(Propꢀ2ꢀynyloxy)ꢀ2Hꢀchromeneꢀ2ꢀthione 12 (0.1 g, 1.0 eq) was dissolved in THF (10ml) and then
cobalt carbonyl (1.05 eq) was added. The black solution was stirred at r.t. for 40 min. Then SiO2
(0.3 g) was added and solvent removed under vacuo to give a black solid that was purified by silica
gel column chromatography eluting 10% v/v ethyl acetate/nꢀhexane to afford 14 as a red solid.
7ꢀ(propꢀ2ꢀynyloxy)ꢀ2Hꢀchromeneꢀ2ꢀthione hexacarbonyldicobalt 14: 79% yield; silica gel TLC Rf
0.18 (ethyl acetate/nꢀhexane 10% v/v); νmax (KBr) cmꢀ1 1775 (C=O), 1530 (aromatic); δH (400 MHz,
DMSOꢀd6) 5.55 (2H, s, 1’ꢀH2), 6.90 (1H, s, 3’ꢀH), 7.09 (1H, dd, J 8.8, 2.4, 6ꢀH), 7.18 (1H, d, J 9.2,
3ꢀH), 7.36 (1H, d, J 2.4, 8ꢀH), 7.80 (1H, d, J 8.8, 5ꢀH), 7.90 (1H, d, J 9.2, 4ꢀH); δc (100 MHz,
DMSOꢀd6), 200.7 (C≡O), 198.3 (C=S), 166.5, 162.4, 158.9, 137.2, 130.0, 127.1, 115.4, 101.9, 73.9,
69.7, 57.4 ; Anal. Calc. C, 44.12; H, 2.14; S, 6.20; Anal. Found. C, 44.75; H, 2.08; S, 3.94.
Synthesis of 7ꢀ[(1ꢀphenylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone 15
PhN3, Cu0 nanosized
TMACl
N
tertꢀBuOH/H2O
O
O
O
O
O
O
N
N
15
11
7ꢀ(Propꢀ2ꢀynyloxy)ꢀ2Hꢀchromenꢀ2ꢀone 11 (0.08 g, 1.0 eq) and phenylazide (1.1 eq) were dissolved
in tertꢀButOH/H2O (1/1, 2.0 ml) and then tetramethylamonium chloride (1.0 eq) and copper
nanosize (5% mol) were added. The mixture was vigorously stirred at r.t. until starting material was
consumed (TLC monitoring). Solvents were removed under vacuo (temperature has not to exceed
40 °C) and the brown residue was purified by silica gel column chromatography eluting with 25%
v/v ethyl acetate/nꢀhexane to afford 15 as a white solid.
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7ꢀ[(1ꢀPhenylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone 15: 54% yield; m.p. 170ꢀ174 °C
silica gel TLC Rf 0.11 (ethyl acetate/nꢀhexane 25% v/v); νmax (KBr) cmꢀ1 1750 (C=O), 1602
(Aromatic); δH (400 MHz, DMSOꢀd6) 5.40 (2H, s, 1’ꢀH2), 6.35 (1H, d, J 9.6, 3ꢀH), 7.10 (1H, dd, J
9.6, 2.4, 6ꢀH), 7.24 (1H, d, J 2.4, 8ꢀH), 7.55 (1H, tt, J 7.6, 1.2, ArꢀH), 7.65 (2H, d, J 7.6, 2 x ArꢀH),
7.7 (1H, d, J 9.6, 5ꢀH), 7.95 (2H, d, J 7.6, 2 x ArꢀH), 8.04 (1H, d, J 9.6, 4ꢀH), 9.04 (1H, s, 3’ꢀH); δC
(100 MHz, DMSOꢀd6) 162.0 (Cꢀ2), 161.2 (Cꢀ7), 156.2 (Cꢀ8a), 145.2 (Cꢀ2’), 144.1 (Cꢀ4), 138.0,
130.9, 130.5, 129.8, 124.1, 121.2, 113.8, 113.7, 113.6, 102.6 (Cꢀ8) and 63.0 (Cꢀ1’).
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Synthesis of 7ꢀ[(1ꢀphenylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methoxy]ꢀ2Hꢀchromeneꢀ2ꢀthione 16
PhN3, Cu0 nanosized
TMACl
N
tertꢀBuOH/H2O
O
O
S
O
O
S
N
N
16
12
7ꢀ(Propꢀ2ꢀynyloxy)ꢀ2Hꢀchromeneꢀ2ꢀthione 12 (0.1 g, 1.0 eq) and phenylazide (1.1 eq) were
dissolved in tertꢀButOH/H2O (1/1, 2.0 ml). Then tetramethylamonium chloride (1.0 eq) and copper
nanosize (10% mol) were added. The mixture was vigorously stirred at r.t. until starting material
was consumed (TLC monitoring). Solvents were removed under vacuo (temperature has not to
exceed 40 °C) and the brown residue was purified by silica gel column chromatography eluting
with 50% v/v ethyl acetate/nꢀhexane to afford 16 as a yellow solid.
7ꢀ[(1ꢀPhenylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methoxy]ꢀ2Hꢀchromeneꢀ2ꢀthione 16: silica gel TLC Rf 0.50
(ethyl acetate/nꢀhexane 10% v/v); νmax (KBr) cmꢀ1, 1604 (Aromatic); δH (400 MHz, DMSOꢀd6) 5.50
(2H, s, 1’ꢀH2), 7.12 (1H, dd, J 9.6, 2.4, 6ꢀH), 7.26 (1H, d, J 9.6, 3ꢀH), 7.35 (1H, d, J 2.4, 8ꢀH), 7.58
(1H, tt, J 7.6, 1.2, ArꢀH), 7.70 (2H, d, J 7.6, 2 x ArꢀH), 7.72 (1H, d, J 9.6, 5ꢀH), 7.95 (2H, d, J 7.6, 2
x ArꢀH), 8.02 (1H, d, J 9.6, 4ꢀH), 9.01 (1H, s, 3’ꢀH); δC (100 MHz, DMSOꢀd6) 198.0 (Cꢀ2), 162.0
(Cꢀ7), 157.0 (Cꢀ8a), 146.3 (Cꢀ2’), 144.0 (Cꢀ4), 136.0, 132.0, 131.0, 1230, 124.6, 121.0, 115.0,
114.0, 113.7, 103.0 (Cꢀ8) and 63.0 (Cꢀ1’).
Synthesis of 7ꢀPentꢀ4ꢀynyloxyꢀchromenꢀ2ꢀone 17
3OH
Ph3P, DIAD
THF dry
Sonication
0°Cꢀꢀr.t.
HO
O
O
O
O
O
5b
17
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7ꢀHydroxy coumarin 5b (1.0 g, 1.0 eq), pentꢀ4ꢀynꢀ1ꢀol (1.0 eq) and triphenylphoshine (1.0 eq) were
dissolved in dry THF (90 ml). Then the temperature was lowered to 0 °C and
diisopropylazadicarboxylate (1.1 eq) was added dropꢀwise under sonication. The orange solution
was sonicated at r.t. under a nitrogen atmosphere until starting material was consumed (TLC
monitoring). Solvents were removed under vacuo to give a white solid that was purified by silica
gel column chromatography eluting with 50% v/v ethyl acetate/nꢀhexane to afford the title
compound 17 as white solid.
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7ꢀPentꢀ4ꢀynyloxyꢀchromenꢀ2ꢀone 17: 52% yield; m.p. 112 °C; silica gel TLC Rf 0.50 (ethyl
acetate/nꢀhexane 50% v/v); δH (400 MHz, DMSOꢀd6) 1.96 (2H, pent, J 6.4), 2.38 (2H, m), 2.89 (1H,
t, J 2.8, ≡CH), 4.18 (2H, t, J 6.4), 6.32 (1H, d, J 9.6, ArꢀH), 6.98 (1H, d, J 9.6, ArꢀH), 7.03 (1H, d, J
2.4, 8ꢀH), 7.66 (1H, d, J 9.6, ArꢀH), 8.01 (1H, d, J 9.6, ArꢀH); δC (100 MHz, DMSOꢀd6) 15.3, 28.4,
67.7, 72.6, 84.4, 102.1, 113.3, 113.4, 113.6, 130.4, 145.2, 156.3, 161.2, 162.6.
Synthesis of 7ꢀpentꢀ4ꢀynyloxyꢀchromeneꢀ2ꢀthione 18
Lawesson's reagent
Toluene, reflux
O
O
S
O
O
O
17
18
7ꢀPentꢀ4ꢀynyloxyꢀchromenꢀ2ꢀone 17 (0.2 g, 1.0 eq) and Lawesson’s Reagent (1.5 eq) were
dissolved in dry toluene (10 ml) and the yellow solution was refluxed until starting material was
consumed (TLC monitoring). Then the solvent was removed under vacuo and the orange residue
was partitioned between H2O and ethyl acetate. The organic phase was washed with H2O (2 x 20
ml), brine (3 x 20 ml), dried over Na2SO4, filteredꢀoff and concentrated under vacuo to give a red
sticky oil that was purified by silica gel column chromatography eluting with 10% v/v ethyl
acetate/nꢀhexane to afford the title compound 18 as a yellow solid.
7ꢀPentꢀ4ꢀynyloxyꢀchromeneꢀ2ꢀthione 18: 35% yield; silica gel TLC Rf 0.54 (ethyl acetate/nꢀhexane
50% v/v); δH (400 MHz, DMSOꢀd6) 1.94 (2H, pent, J 6.4), 2.40 (2H, m), 2.76 (1H, t, J 2.8, ≡CH),
4.19 (2H, t, J 6.4), 6.99 (1H, s, ArꢀH), 7.04 (1H, dd, J 8.8, 2.4, ArꢀH), 7.19 (1H, d, J 9.2, ArꢀH),
7.35 (1H, d, J 2.4, ArꢀH), 7.80 (1H, d, J 8.8, ArꢀH), 7.92 (1H, d, J 9.2, ArꢀH); δC (100 MHz,
DMSOꢀd6) 15.3, 28.4, 67.7, 72.6, 84.4, 102.1, 113.3, 113.4, 113.6, 130.4, 145.2, 156.3, 161.2, 198
(C=S).
Synthesis of 7ꢀ(pentꢀ4ꢀynyloxy)ꢀ2Hꢀchromenꢀ2ꢀone hexacarbonyldicobalt 19
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Co2(CO)8
THF dry
(OC)6Co2
O
O
O
O
O
O
17
19
6
7
8
9
7ꢀ(Pentꢀ4ꢀynyloxy)ꢀ2Hꢀchromenꢀ2ꢀone 17 (0.05 g, 1.0 eq) was dissolved in THF (10 ml) and then
cobalt carbonyl (1.05 eq) was added. The black solution was stirred at r.t. for 40 min. Then SiO2
(0.3 g) was added and solvent removed under vacuo to give a black solid that was purified by silica
gel column chromatography eluting with 20% v/v ethyl acetate/nꢀhexane to give 19 as a red solid.
7ꢀ(Pentꢀ4ꢀynyloxy)ꢀ2Hꢀchromenꢀ2ꢀone hexacarbonyldicobalt 19: 92 % yield; silica gel TLC Rf 0.20
(ethyl acetate/nꢀhexane 20% v/v); νmax (KBr) cmꢀ1 1762 (C=O), 1530 (aromatic); δH (400 MHz,
DMSOꢀd6) 2.10 (2H, quint, J 6.8, 2’ꢀH2), 3.09 (2H, t, J 6.8, 3’ꢀH2), 4.28 (2H, t, J 6.8, 1’ꢀH2), 6.33
(1H, d, J 9.6, 3ꢀH), 6.84 (1H, s, 5’ꢀH), 7.01 (1H, dd, J 8.8, 2.0, 6ꢀH), 7.06 (1H, d, J 2.0, 8ꢀH), 7.66
(1H, d, J 8.8, 5ꢀH), 8.03 (1H, d, J 9.6, 4ꢀH); δc (100 MHz, DMSOꢀd6), 200.9 (C≡O), 162.7 (C=O),
161.3, 156.5, 145.4, 130.6, 113.8, 113.5, 102.3, 98.5, 75.5, 72.5, 68.4, 31.8, 31.0; ; Anal. Calc. C,
47.66; H, 2.86; Anal. Found. C, 46.74; H, 2.27.
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Co-crystallization and X-ray data collection. Crystals of native hCA II were obtained using the
hanging drop vapor diffusion method. 2 µl of the protein solution were mixed with 2 µl of a
solution of 1.6 M sodium citrate, 50 mM Tris pH 8.0 and were equilibrated against the same
solution at 296 K. Protein concentration was 0.4 mM in 50 mM Tris pH=8.0. Crystals of the
complex with 8a were obtained by soaking the hCAII crystals in a saturated solution of the
compound dissolved in 1.2 M sodium citrate, 50 mM Tris pH 8.0 and 15% glycerol.
A crystal of the complex was harvested from this solution and flashꢀfrozen at 100K. A data set on a
crystal of the complex hCAIIꢀinhibitor 8a was collected to a maximum resolution of 1.10 Å, using
synchrotron radiation at the ID23ꢀ1 beamline at ESRF (Grenoble, France) with a wavelength of
1.000 Å and a DECTRIS Pilatus 6M detector. Data were integrated and scaled using the program
XDS.36 Data processing statistics are showed in Table 1.
Structure determination. The crystal structure of hCA II (PDB accession code: 3P58) without
solvent molecules and other heteroatoms was used to obtain initial phases of the structures using
Refmac537 5% of the unique reflections were selected randomly and excluded from the refinement
data set for the purpose of Rfree calculations. Inspection of the difference electronꢀdensity maps
indicated the presence of an inhibitor molecule bound to the water that coordinate the catalytic zinc
ion. Atomic models for the inhibitor were calculated and energy minimized using the program
JLigand 1.0.39. A fractional occupancy factor of 0.5 was attributed to all the inhibitor atoms. After
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the introduction of the inhibitor positive residual densities were present in the difference electronꢀ
density maps close to the inhibitor and were attributed to disordered water molecules (occupancy
factors 0.5).
During the refinement anisotropic temperature factors were introduced and hydrogen atoms were
added to the model. Manual building of the atomic model were carried out using COOT38 Solvent
molecules were introduced automatically using the program ARP39 working in the default solvent
building mode. The quality of the final models were assessed with PROCHECK.40 Crystal and
refinement data are summarized in Table 2. Graphical representations were generated with
Chimera.41
8
9
10
11
12
13
14
15
16
17
18
19
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21
22
23
24
25
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28
29
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31
32
33
34
35
36
37
38
39
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Table 2. Summary of Data Collection and Atomic Model Refinement Statistics.*
hCA II+ 8a
PDB ID
4WL4
Wavelength (Å)
Space Group
1.000
P21
Unit cell (a,b,c, ) (Å, °)
Limiting resolution (Å)
Unique reflections
Rsym (%)
42.26, 41.37, 72.28, 104.21
29.11ꢀ1.10 (1.17ꢀ1.10)
78925 (3784)
4.8 (42.1)
Redundancy
3.5 (2.1)
Completeness overall (%)
<I/(I)>
80.7 (27.2)
12.90 (1.73)
Refinement statistics
Resolution range (Å)
Unique reflections, working/free
Rfactor (%)
29.11ꢀ1.10
75094 (3784)
10.90
Rfree(%)
12.99
No. of protein atoms
No. of water molecules
4682
395
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8
No. of compound atoms
r.m.s.d. bonds(Å)
18
0.0066
1.311
r.m.s.d. angles (°)
9
Ramachandran statistics (%)
Most favored
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48
49
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96.5
3.5
0
additionally allowed
generously allowed regions
Average B factor (Å2)
mainꢀchain protein atoms
side chain protein atoms
compound
12.72
14.88
15.80
33.69
solvent
*Values in parentheses are for the highest resolution shell.
Table 3. Occupancy and B factors of the zinc ion, the inhibitor 8a atoms and water molecules in the
active site of the hCA II complex.
atom
Zn
occupancy
1.0
B isotropic
7.36
S1
0.50
16.09
13.79
15.56
14.05
13.93
13.97
15.08
15.99
16.21
CAF
CAI
HAI
CAH
HAH
CAN
CAM
HAM
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
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OAK
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
15.45
CAO
14.83
15.48
15.39
16.35
35.87
18.95
38.00
33.12
12.46
22.01
20.66
22.75
18.95
12.08
CAL
HAL
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17
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CAG
Wat343
Wat344
Wat349
Wat350
Wat351
Wat352
Wat353
Wat354
Wat344
Wat394
Accession Codes. Coordinates and structure factors for CA II complexes with 8a have been
deposited in the Protein Data Bank (PDB) accession code: 4WL4.
Acknowledgements. This research was financed by two EU grants of the 7th framework program
(Metoxia and Dynano projects to AS and CTS).
Nonstandard abbreviations. CA, carbonic anhydrase; CAI, CA inhibitor; KI, inhibition constant;
TBDMS, tertꢀbutyldimethylsylil.
*Corresponding Author: Prof. Claudiu T. Supuran (CTS) Phone: +39ꢀ055ꢀ4573005; fax: +39ꢀ
055ꢀ4573385; Eꢀmail: claudiu.supuran@unifi.it
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from the pathogenic bacterium Vibrio cholera. Acta Crystall. D 2015, 71, 2449ꢀ2456.
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A patent and literature review. Expert Opin. Ther. Pat. 2013, 23, 693ꢀ704; c) Maresca, A.; Vullo,
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396; d) Maresca, A.; Scozzafava, A.; Vullo, D.; Supuran, C.T. Dihalogenated sulfanilamides and
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inhibition studies of the αꢀcarbonic anhydrase from the protozoan pathogen Trypanosoma cruzi, the
causative agent of Chagas disease. Bioorg. Med. Chem. 2013, 21, 4472ꢀ4476; b) GüzelꢀAkdemir,
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16. a) Thiry, A.; Dognè, J.M.; Supuran, C.T.; Masereel, B. Anticonvulsant sulfonamides/
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17. a) Scozzafava, A.; Supuran, C.T.; Carta, F. Antiobesity Carbonic Anhydrase Inhibitors: A
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patent review (2008ꢀ2013). Expert Opin. Ther. Pat. 2013, 23, 737ꢀ749; b) Ward, C.; Langdon, S.P.;
Mullen, P.; Harris, A.L.; Harrison, D.J.; Supuran, C.T.; Kunkler, I. New strategies for targeting the
hypoxic tumour microenvironment in breast cancer. Cancer Treatm. Rev. 2013, 39, 171ꢀ179; c)
Lock, F.E.; McDonald, P.C.; Lou, Y.; Serrano, I.; Chafe, S.C.; Ostlund, C.; Aparicio, S.; Winum,
J.Y.; Supuran, C.T.; Dedhar, S. Targeting Carbonic Anhydrase IX depletes breast cancer stem cell
within the hypoxic niche. Oncogene 2013, 32, 5210–5219; d) Ebbesen, P.; Pettersen, E.O.; Gorr,
T.A.; Jobst, G.; Williams, K.; Kienninger, J.; Wenger, R.H.; Pastorekova, S.; Dubois, L.; Lambin,
P.; Wouters, B.G.; Supuran, C.T.; Poellinger, L.; Ratcliffe, P.; Kanopka, A.; Görlach, A.; Gasmann,
M.; Harris, A.L.; Maxwell, P.; Scozzafava, A. Taking advantage of tumor cell adaptations to
hypoxia for developing new tumor markers and treatment strategies. J. Enzyme Inhib. Med. Chem.
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Carbonic Anhydrase Inhibitors. Synthesis of WaterꢀSoluble, Topically Effective, Intraocular
PressureꢀLowering Aromatic/Heterocyclic Sulfonamides Containing Cationic or Anionic Moieties:
Is the Tail More Important than the Ring? J. Med. Chem. 1999, 42, 2641ꢀ2650; b) Winum, J.ꢀY.;
Poulsen, S.ꢀA.; Supuran, C.T., Therapeutic applications of glycosidic carbonic anhydrase inhibitors.
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A.; Vullo, D.; Supuran, C. T.; Poulsen, S.ꢀA., Carbonic Anhydrase Inhibitors: Inhibition of
Isozymes I, II, and IX with TriazoleꢀLinked OꢀGlycosides of Benzene Sulfonamides. J. Med. Chem.
2007, 50, 1651ꢀ1657; d) Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Supuran,
C. T.; Poulsen, S.ꢀA., A novel class of carbonic anhydrase inhibitors: Glycoconjugate benzene
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21. a) Scozzafava A., Supuran C.T.; Carbonic anhydrase inhibitors. Arylsulfonylureidoꢀ and
arylureidoꢀsubstituted aromatic and heterocyclic sulfonamides: towards selective inhibitors of
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Nuti, E.; Vullo, D.; Rossello, A.; Carta, F.; Scozzafava, A.; Supuran, C.T. Combining the tail and
the ring approaches for obtaining potent and isoformꢀselective carbonic anhydrase inhibitors:
solution and Xꢀray crystallographic studies. Bioorg Med. Chem. 2014, 22, 334ꢀ340.
22. a) Bonneau, A.; Maresca, A.; Winum, J.Y.; Supuran, C.T. Metronidazoleꢀcoumarin conjugates
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investigations. Chem. Commun. 2012, 48, 1868ꢀ1870; b) Avram, S.; Milac, A.L.; Carta, F.;
Supuran, C.T. More effective dithiocarbamate derivatives inhibiting carbonic anhydrases, generated
by QSAR and computational design. J. Enzyme Inhib. Med. Chem. 2013, 28, 350ꢀ359; c) Carta, F.;
Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Masini, E.; Supuran, C.T.
Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo. J.
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25. a) Maresca, A.; Temperini, C.; Vu, H.; Pham, N.B.; Poulsen, S.A.; Scozzafava, A.; Quinn, R.J.;
Supuran, C.T. Nonꢀzinc mediated inhibition of carbonic anhydrases: coumarins are a new class of
suicide inhibitors. J. Am. Chem. Soc. 2009, 131, 3057ꢀ3062; b) Maresca, A.; Temperini, C.; Pochet,
L.; Masereel, B.; Scozzafava, A.; Supuran, C.T. Deciphering the mechanism of carbonic anhydrase
inhibition with coumarins and thiocoumarins. J. Med Chem. 2010, 53, 335ꢀ344.
26. a) Capasso, C.; Supuran, C.T. An overview of the alphaꢀ, betaꢀ and gammaꢀcarbonic anhydrases
from Bacteria: can bacterial carbonic anhydrases shed new light on evolution of bacteria?. J.
Enzyme Inhib. Med. Chem. 2015, 30, 325ꢀ332; b) Tanc, M.; Carta, F.; Scozzafava, A.; Supuran,
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27. a) Saada, M.C.; Montero, J.L.; Vullo, D.; Scozzafava, A.; Winum, J. Y.; Supuran, C.T.
Carbonic anhydrase activators: gold nanoparticles coated with derivatized histamine, histidine, and
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28. Temperini, C.; Scozzafava, A.; Vullo, D.; Supuran, C.T. Carbonic anhydrase activators.
Activation of isozymes I, II, IV, VA, VII, and XIV with lꢀ and dꢀhistidine and crystallographic
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selectively inhibit the transmembrane, tumorꢀassociated carbonic anhydrase isoforms IX and XII
over the cytosolic ones I and II. Bioorg. Med. Chem. Lett. 2010, 20, 4511ꢀ4514; b) Maresca, A.;
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inhibit the transmembrane, tumorꢀassociated carbonic anhydrase isoforms IX and XII over the
cytosolic ones I and II in the low nanomolar/subnanomolar range. Bioorg. Med. Chem. Lett. 2010,
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Aromatic/heterocyclic sulfonamides incorporating phenacetylꢀ, pyridylacetylꢀ and thienylacetylꢀ
tails act as potent inhibitors of human mitochondrial isoforms VA and VB. Bioorg. Med. Chem.
2009, 17, 4894–4899; c) Avvaru, B.S.; Wagner, J.M.; Maresca, A.; Scozzafava, A.; Robbins, A.H.;
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isoform II in adduct with an adamantyl analogue of acetazolamide resides in a new hydrophobic
binding pocket. Bioorg. Med. Chem. Lett. 2010, 20, 4376ꢀ4381; d) Pacchiano, F.; Carta, F.;
McDonald, P.C.; Lou, Y.; Vullo, D., Scozzafava, A.; Dedhar, S.; Supuran, C.T. Ureidoꢀsubstituted
benzenesulfonamides potently inhibit carbonic anhydrase IX and show antimetastatic activity in a
model of breast cancer metastasis. J. Med. Chem. 2011, 54, 1896ꢀ1902; e) Abbate, F.; Winum, J.Y.;
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inhibitors: X ray crystallographic structure of the adduct of human isozyme II with a EMATE, a
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