Muhammet
Tanc
ab,
Fabrizio
Carta
*b,
Andrea
Scozzafava
b and
Claudiu T.
Supuran
*ab
aUniversità degli Studi di Firenze, NEUROFARBA Department, Section of Pharmaceutical Chemistry, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy. E-mail: claudiu.supuran@unifi.it
bUniversità degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy. E-mail: fabrizio.carta@unifi.it
First published on 27th October 2014
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.
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Fig. 1 CA inhibition mechanism of sulfocoumarins. (A) The sulfocoumarin undergoes an enzyme-mediated hydrolysis with formation of the trans-2-hydroxy-phenyl-ω-ethenylsulfonic acid. (B) The sulfonic acid binds to the CA II active site, by anchoring of the sulfonic acid group to the zinc-coordinated water molecule. The Zn(II) ion (central larger sphere), its three His ligands (His94, 96 and 119), the 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 X-ray crystallography (PDB file 4BCW).1 |
CAIs of the sulfonamide type have been in clinical use for the treatment of glaucoma, obesity, epilepsy and as diuretics for almost 60 years.3–5 The use of CAIs for pharmaceutical applications relies on the diverse distribution of the CAs under 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, and antiepileptics CA VII and XIV, whereas the isoforms IX and XII are strictly correlated with the tumors.6–10 The main drawback associated with their use is the lack of selectivity in inhibiting the various enzymatic isoforms, which results in a plethora of side effects.3–10
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 context the “tail approach” was the preferred one. It consisted of the chemical manipulation of a compound at its tail-end, which resulted in a modification of its physical/chemical properties.3,4 In addition several novel CAI scaffolds have been identified such as the polyamines,11 phenols,12 dithiocarbamates,13 xanthates,14 the coumarins2 and their structural derivatives such as the thiocoumarins, 2-thioxo-coumarins and coumarin oximes.6a,15 The sulfocoumarins 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 X-ray crystallography of the enzyme-inhibitor adducts.2,11–14
In our previous reports we explored the CA kinetic profiles of a small series of 6-tetrazolyl and 6-triazolyl-substituted sulfocoumarins 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 6-substituted sulfocoumarins.
All the compounds were prepared according to the general strategy of Zalubovskis's group16 by reacting the commercially available 2,5-dihydroxybenzaldehyde with benzyl bromide to afford 1 which was treated with magnesium bromide for selective de-benzylation.17 Then mesylation of the phenol 2 was followed by intramolecular cyclisation in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to afford the racemic 4-hydroxy-6-benzyloxy-3,4-dihydrosulfocoumarin 4, which was deprotected under Pd-C 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 6-hydroxy-substituted sulfocoumarin 7. At this point the hydroxyl group in 7 was alkylated with 2-chloroethanol 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 Boc-protected 2-aminoethanol 10 (Scheme 1).
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 tumor-associated 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).
Entry | K i | ||||
---|---|---|---|---|---|
hCAI (μM) | hCA II (μM) | hCAVA (μM) | hCAIX (nM) | hCAXII (nM) | |
a Errors in the range of ±5% of the reported values, from three different assays. b Data retrieved from ref. 1. | |||||
4 | >100 | >100 | 89 | 83.7 | 36.2 |
5 | >100 | >100 | 42 | 36.2 | 16.5 |
7 | 91b | >100 | 96 | 300b | 204b |
8 | >100 | >100 | 0.06 | 26.8 | 10.4 |
9 | >100 | >100 | 42 | 50.3 | 684 |
11 | >100 | >100 | 87 | 11.5 | 9.8 |
AAZ | 0.25 | 0.012 | 0.06 | 25 | 5.7 |
Herein for the first time we report the kinetic profiles of the 6-substituted sulfocoumarins 7–9, 11 and their precursors 4 and 5 against the mitochondrial hCA VA, with Kis in the range of 0.06–96 μM (Table 1). Such an isoform is receiving much attention as it was proved to be a suitable target for the development of new and effective antiobesity agents.5b,18 As a preliminary structure–activity-relationship (SAR) study here we report that the introduction at the 7-position in the sulfocoumarin 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 manipulation 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 compound is ongoing. All the compounds of the series are potent inhibitors of the transmembrane and tumor-associated isoform hCAs IX and XII with Kis spanning between 9.8 and 684 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 compound 8, drastically reduced the Ki values (26.8 and 10.4 nM for the hCAs IX and XII respectively). Such a behaviour is analogous to that previously reported for the mitochondrial isoform. (ii) The introduction of the lipophilic and bulky substituent Boc-moiety 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 Boc-group of 11 and specific amino acids present at the rim of the enzymatic cavities particularly contribute to the stabilization of the inhibitor-enzyme complex.
As expected for the 6-substituted sulfocoumarins, the compounds showed to be ineffective in inhibiting the cytosolic hCAs I and II, whereas they showed interesting profiles against the tumor associated hCAs IX and XII with Kis in the range of 9.8–684 nM.
For the first time we report the 6-substituted sulfocoumarins as effective inhibitors of the mitochondrial hCA VA isoform, with compound 8 as the strongest in the series (Ki 0.06 μM).
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 obesity-related pathologies makes these compounds particularly interesting for developing new therapeutics.
Footnote |
† Electronic supplementary information (ESI) available: Analytical data and spectra (1H, 13C NMR) for all products. See DOI: 10.1039/c4ob02155j |
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