6-Substituted 1,2-benzoxathiine-2,2-dioxides are isoform-selective inhibitors of human carbonic anhydrases IX, XII and VA

Article abstract of DOI:10.1039/c4ob02155j

A series of 6-substituted 2-benzoxathiine-2,2-dioxides were synthesized starting from 2,5-dihydroxybenzaldehyde, and then screened in vitro for their inhibition properties against five human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. All the compounds

Full text of DOI:10.1039/c4ob02155j

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DOI: 10.1039/C4OB02155J
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6-Substituted
1,2-benzoxathiine-2,2-dioxides
are
Isoform-Selective Inhibitors Towards Human Carbonic
Anhydrases IX, XII and VA
Cite this: DOI: 10.1039/x0xx00000x
Muhammet Tanc,a,b Fabrizio Carta,b* Andrea Scozzafavab and Claudiu T. Supurana,b*
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of 6-substituted 2-benzoxathiine-2,2-dioxides were
synthesized starting from 2,5-dihydroxybenzaldehyde, and
then screened in vitro for their inhibition properties against
five human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms.
All the compounds showed excellent selectivity against the
mitochondrial (hCA VA) and the tumor associated (hCA IX
and XII) enzymes.
H2O
SO3H
R
R
O
S
R
SO3H
O
OH
O
OH
Reaction intermediated
(not isolated)
Sulfocoumarin
Hydrolyzed sulfocoumarin
A
The 1,2ꢀbenzoxathiine 2,2ꢀdioxides, also called
sulfocoumarines, are bioisosters of the coumarines and were recently
reported as potent and isoform selective carbonic anhydrase
inhibitors (CAIs).1 In analogy to the coumarine class, their inhibition
mechanism starts with the CAꢀmediated hydrolysis of the
intramolecular sulfonic acid ester to give, upon geometrical
isomerisation, the trans vinyl sulfonic acid, which in turn binds to
the zincꢀcoordinated water molecule.2
CAIs of the sulfonamide type are in clinical use for the
treatment of glaucoma, obesity, epilepsy and as diuretics for almost
60 years.3ꢀ5 The use of CAIs for pharmaceutical applications relies
on the diverse distribution of the CAs in physio/pathological
conditions within the tissues. For instance the antiglaucoma drugs
mainly target CA II, IV and XII; diuretics CA II, IV, XII and XIV;
antiepileptics CA VII and XIV, whereas the isoforms IX and XII are
strictly correlated to the tumors.6ꢀ10 The main drawback associated to
their use is the lack of selectivity in inhibiting the various enzymatic
isoforms, which results in a plethora of side effects.3ꢀ10
B
Fig. 1: CA inhibition mechanism of sulfocoumarines. A) The
sulfocoumarin undergoes an enzymeꢀmediated hydrolysis with
formation of the transꢀ2ꢀhydroxyꢀphenylꢀωꢀethenylsulfonic acid. B.
The sulfonic acid binds to the CA II active site, by anchoring of the
sulfonic acid group to the zincꢀcoordinated water molecule. The
Zn(II) ion (central larger sphere), its three His ligands (His94, 96 and
119), water molecule coordinated to the zinc (small sphere) as well
as active site residues Thr200 and Pro201 involved in the binding of
the hydrolyzed sulfocoumarin are shown, as determined by Xꢀray
crystallography (PDB file 4BCW).1
a
Università degli Studi di Firenze, NEUROFARBA Department, Section of
Pharmaceutical Chemistry, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence);
Italy. Eꢀmail: claudiu.supuran@unifi.it
b
Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica
Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence);
Italy. Eꢀmail: fabrizio.carta@unifi.it
† Electronic supplementary information (ESI) available: Analytical data and spectra
(1H, 13C NMR) for all products.
Since the identification of the sulfonamides and their bioisosters, the
sulfamates and the sulfonates, as CAIs many efforts have been made
for the development of specific inhibitors. In this contest the “tail
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C4OB02155J
approach” was the preferred one. It consisted of the chemical Table 1. hCA I, II, VA, IX and XII inhibition data with
sulfocoumarins 4–11, by a stoppedꢀflow CO2 hydrase assay. 19
manipulation of a compound at its tailꢀend, which resulted in a
modification of its physical/chemical properties.3,4 In addition
several and novel CAI scaffolds have been identified such as the
Entry
Ki *
polyamines,11 phenols,12 dithiocarbamates,13 xanthates,14 the
coumarines2 and their structural derivatives such as the
thiocoumarines, 2ꢀthioxoꢀcoumarines and coumarine oximes.6a,15
The sulfocoumarines represent the latest class of CAIs identified and
show high selectivity profiles.1,7 The inhibition mechanisms of many
of these compounds were determined by Xꢀray crystallography of
the enzymeꢀinhibitor adducts.2, 11ꢀ14
hCAI(µM)
hCA II(µM) hCAVA(µM) hCAIX(nM) hCAXII(nM)
4
>100
>100
91**
>100
>100
>100
>100
>100
>100
0.012
89
83.7
36.2
300**
26.8
50.3
11.5
25
36.2
16.5
204**
10.4
684
9.8
5
42
7
96
In our previous reports we explored the CA kinetic profiles
8
>100
>100
>100
0.25
0.06
42
of
a small series of 6ꢀtetrazolyl and 6ꢀtriazolylꢀsubstituted
sulfocoumarines as well as the effects derived by the introduction of
different substituents at the 7 position.1,7 As extension herein we
report the synthesis, characterization and in vitro CA inhibition of a
small series of 6ꢀsubstituted sulfocoumarines.
9
11
AAZ
87
0.06
5.7
O
O
O
BnBr, K2CO3
BnO
MgBr2
HO
BnO
Acetone, Reflux
50% yield
Benzene/Ether 7/1
90 % yield
OH
OBn
OH
2
1
* Errors in the range of ±5 % of the reported values, from three different assays.
** Data retrieved from reference 1.
MsCl, Et3
DCM, quant.
N
O
OH
OH
BnO
BnO
HO
O
S
DBU
10% PdꢀC
The obtained compounds 4, 5, 7-9 and 11 were screened in
vitro as inhibitors against the most abundant and cytosolic CAs
(hCAI and II), the mitochondrial (hCA VA) and the transmembrane
tumorꢀassociated hCA IX and XII in comparison with acetazolamide
(AAZ) as a standard. In consideration of the previous reports1,7 we
can summarize that the main influencing factor for isozyme
selectivity is the substitution at the phenyl ring. In particular all the
compounds showed to be ineffective inhibitors of the cytosolic hCAs
I and II. (Table 1) Herein and for the first time we report the kinetic
profiles of the 6ꢀsubstituted sulfocoumarines 7-9, 11 and their
precursors 4 and 5 against the mitochondrial hCA VA, with KIs in
the range of 0.06ꢀ96 µM (Table 1). Such an isoform is raising much
attention as it was proved to be a suitable target for the development
of new and effective antiobesity agents.5b,18 As preliminary
StructureꢀActivityꢀRelationship (SAR) here we report that the
introduction at 7ꢀposition in the sulfocoumarine scaffold of the
ethanolic moiety (as for the compound 8) resulted in a significant
reduction of the Ki for the hCA VA (0.06 µM). Further
manipulations at the alcoholic terminal such as in compounds 9 and
11, or elimination of the alkyl chain as for 7, suppressed any isoform
selectivity (Table 1). Currently an exhaustive SAR investigation on
this kind of compounds is ongoing. All the compounds of the series
are potent inhibitors of the transmembrane and tumorꢀassociated
isoform hCAs IX and XII with KIs spanning between 9.8ꢀ684 nM
(Table 1). As for the hCA VA isoform a defined SAR is not feasible
at the moment. However some key elements are evident: i) the
introduction of an alkyl substituent, as for the compound 8,
drastically reduced the Ki values (26.8 and 10.4 nM for the hCA IX
and XII respectively). Such a behaviour is analogous to the
previously reported for the mitochondrial isoform. ii) The
introduction of the lipophilic and bulky substituent Bocꢀmoiety at
the end of the chain, as for the compound 11, accounted for
selectivity against the tumor associated CAs. Such an effect was not
observed when the tosyl group was introduced instead (compound
9). Probably hydrophobic interactions occurring between the Bocꢀ
group of 11 with specific aminoacids present at the rim of the
enzymatic cavities particularly contribute to the stabilization of the
inhibitorꢀenzyme complex.
O
O
S
EtOAc, quant.
S
O
O
O
DCM, 90 % yield
O
O
O
5
4
3
TfCl, Et3N
THF
HO
Cl
O
O
HO
O
Et4NOH
K2CO3, KI
S
O
F3
C
O
S
O
S
O
O
S
1,4ꢀDioxane 79% yield
O
DMF 60 °C; 73% yield HO
O
O
O
O
7
6
8
O
OH 10
O
N
H
TsCl
Pyr. ; 55 % yield
Ph3P, DIAD
THF, r.t. 46 % yield
O
O
O
O
O
S
O
S
TsO
O
N
O
O
O
H
11
9
Scheme 1:Synthesis of the 6ꢀsubstituted sulfocoumarines 7-9 and 11.
All the compounds were prepared according to the general
strategy of Zalubovskis’s group16 by reacting the commercially
available 2,5ꢀdihydroxybenzaldehyde with benzyl bromide to afford
1 which was treated with magnesium bromide for selective deꢀ
benzylation.17 Then mesylation of the phenol 2 was followed by
intramolecular
cyclisation
in
the
presence
of
1,8ꢀ
diazabicyclo[5.4.0]undecꢀ7ꢀene (DBU) to afford the racemic 4ꢀ
hydroxyꢀ6ꢀbenzyloxyꢀ3,4ꢀdihydrosulfocoumarin 4, which was
deprotected under PdꢀC catalyzed hydrogenation and then
dehydrated by means of its triflate derivative 6. As expected such a
compound proved to be unstable and was directly subjected to
treatment with tetraethylammonium hydroxide to afford the 6ꢀ
hydroxyꢀsubstituted sulfocoumarine 7. At this point the hydroxyl
group in 7 was alkylated with 2ꢀchloroethanol through an in situ
Filkenstein reaction in the presence of potassium carbonate as a base
to afford 8, which in turn was treated with tosylchloride in pyridine
to give 9. Finally compound 11 was obtained via the Mitsunobu
coupling reaction of 7 with the freshly prepared Bocꢀprotected 2ꢀ
aminoethanol 10 (Scheme 1).
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C4OB02155J
c)S. Pastorekova, S. Parkkila, J. Pastorek and C.T. Supuran, J.
Enzyme Inhib. Med. Chem. 2004, 19, 199.
Conclusions
Herein we reported a series of 6ꢀsubstituted sulfocoumarines
obtained by intramolecular cyclization of the corresponding
methanesulfonic acid esters. We investigated compounds 4, 5,
10. a)C.T. Supuran, Bioorg. Med. Chem
.
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Supuran, Future Med. Chem. 2011,
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Boone, B. Kondeti and R. McKenna, J. Enzyme Inhib. Med. Chem
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isoforms.
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Christianson, J. Med. Chem. 1994, 37, 2100; e) L. Baranauskiene, M.
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As expected for the 6ꢀsubstituted sulfocoumarines, the
compounds showed to be ineffective in inhibiting the cytosolic
hCAs I and II, whereas showed interesting profiles against the
tumor associated hCA IX and XII with Kis in a range of 9.8ꢀ
684 nM.
For the first time we report the 6ꢀsubstituted sulfocoumarines as
effective inhibitors of the mitochondrial hCA VA isoform, with
11. F. Carta, C. Temperini, A. Innocenti, A. Scozzafava, K. Kaila and
C.T. Supuran, J. Med.Chem. 2010, 53, 5511.
compound
8 as the strongest in the series (Ki 0.06 ꢁM).
These findings are of particular importance for the future
development of new and effective inhibitors against the
mitochondrial CA isoforms having antiobesity pharmacological
applications. In particular the lack of safe and effective drugs
for the treatment of obesity and/or obesityꢀrelated pathologies
makes these compounds particularly interesting for developing
new therapeutics.
12. S.K. Nair, P.A. Ludwig and D.W. Christianson, J. Am. Chem. Soc
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14. F. Carta, A. Akdemir, A. Scozzafava, E. Masini and C.T. Supuran, J.
Med. Chem. 2013, 56, 4691.
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This work was financed by two FP7 EU grants (METOXIA and
DYNANO).
.
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19. An Applied Photophysics stoppedflow 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 10–100 s. The CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the kinetic
parameters and inhibition constants. For each inhibitor at least six
traces of the initial 5–10% of the reaction have been used for
determining the initial velocity. The uncatalyzed rates were
determined in the same manner and subtracted from the total
observed rates. Stock solutions of inhibitor (0.1 mM) were prepared
in distilled–deionized water and dilutions up to 0.01 nM were done
thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 15 min—6 h at room temperature (15 min)
or 4 °C (6 h) prior to assay, in order to allow for the formation of the
E–I complex. Data from Table 1 were obtained after 6 h incubation of
enzyme and inhibitor, as for the sulfocoumarins and coumarins
reported earlier.1,7 The inhibition constants were obtained by nonꢀ
linear leastsquares methods using PRISM 3, as reported earlier,8a and
represent the mean from at least three different determinations. All
CA isofoms were recombinant ones obtained inꢀhouse as reported
earlier.1,7
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