Novel coumarins and benzocoumarins acting as isoform-selective inhibitors against the tumor-associated carbonic anhydrase IX

Abstract

A series of coumarins and benzocoumarins incorporating methyl and hydroxyl moieties in the heterocyclic ring were investigated for the inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). These coumarins were very weak or ineffective as inhibitors of the house-keeping, offtarget isoforms CA I and II, but showed effective, submicromolar inhibition of the transmembrane, tumor-associated isoforms CA IX and to a slightly less extent, CA XII. The nature and position of the groups substituting the coumarin ring influenced CA inhibitory properties. 4-Methyl-5,7-dihydroydroxycoumarin showed K_Is >200?µM against CA I and II, of 0.19?µM against CA IX and of 6.4?µM against CA XII, being thus a selective, efficient inhibitor for the tumor-associated over cytosolic CA isoforms. These compounds are interesting leads for designing isoform-selective enzyme inhibitors.     

Carbonic anhydrase inhibitors. Diazenylbenzenesulfonamides are potent and selective inhibitors of the tumor-associated isozymes IX and XII over the cytosolic isoforms I and II

A series of diazenylbenzenesulfonamides, azo-dye derivatives of sulfanilamide or metanilamide incorporating phenol and amine moieties, were tested for inhibition of the tumor-associated isozymes of carbonic anhydrase (CA, EC 4.2.1.1), CA IX and XII. These compounds showed moderate-low inhibitory activities against the cytosolic isoforms CA I and II (offtargets) and excellent, low nanomolar inhibitory activity against the transmembrane CA IX and XII (K_Is in the range of 3.5–63 nM against CA IX and 5.0–69.4 nM against CA XII, respectively). The selectivity ratio for inhibiting the tumor-associated CA IX over the offtarget CA II was in the range of 15–104 for these diazenylbenzenesulfonamides, making them among the most isoform-selective inhibitors targeting tumor-associated CAs (over the ubiquitous CA II). Since CA IX/XII were recently shown to be both therapeutic and diagnostic targets for hypoxic solid tumors overexpressing these proteins, such compounds held promise for the management of hypoxic tumors, which are largely non-responsible to classical chemo- and radio-therapy.     

Design,synthesis and evaluation of N-substituted saccharin derivatives as selective inhibitors of tumor-associated carbonic anhydrase XII

A series of N-alkylated saccharin derivatives were synthesized and tested for the inhibition of four different isoforms of human carbonic anhydrase (CA, EC 4. 2.1.1): the transmembrane tumor-associated CA IX and XII, and the cytosolic CA I and II. Most of the reported derivatives inhibited CA XII in the nanomolar/low micromolar range, hCA IX with K_Is ranging between 11 and 390 nM, whereas they were inactive against both CA I (K_Is >50 μM) and II (K_Is ranging between 39.1 nM and 50 μM). Since CA I and II are off-targets of antitumor carbonic anhydrase inhibitors (CAIs), the obtained results represent an encouraging achievement for the development of new anticancer candidates without the common side effects of non-selective CAIs. Moreover, the lack of an explicit zinc binding function on these inhibitors opens the way towards the exploration of novel mechanisms of inhibition that could explain the high selectivity of these compounds for the inhibition of the transmembrane, tumor-associated isoforms over the cytosolic ones.     

Carbonic anhydrase inhibitors. Inhibition of the human cytosolic isoforms I and II and transmembrane,tumor-associated isoforms IX and XII with boronic acids

A series of aromatic, arylalkenyl- and arylalkyl boronic acids were assayed as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, and the transmembrane, tumor-associated hCA IX and XII. The best hCA I and II inhibitor was biphenyl boronic acid with, a K_I of 3.7–4.5 μM, whereas the remaining derivatives showed inhibition constants in the range of 6.0–1560 μM for hCA I and of 6.0–1050 μM for hCA II, respectively. hCA IX and XII were effectively inhibited by most of the aromatic boronic acids (K_Is of 7.6–12.3 μM) whereas the arylalkenyl and aryl–alkyl derivatives generally showed weaker inhibitory properties (K_Is of 34–531 μM). The nature of the moiety substituting the boronic acid group strongly influenced the CA inhibitory activity, with inhibitors possessing low micromolar to millimolar activity being detected in this small series of investigated compounds. This study proves that the B(OH)₂ moiety represents a new zinc-binding group for the generation of effective CA inhibitors targeting isoforms with medicinal chemistry applications. The boronic acids probably bind to the Zn(II) ion within the CA active site leading to a tetrahedral geometry of the metal ion and of the B(III) derivative.     

6-Triazolyl-substituted sulfocoumarins are potent,selective inhibitors of the tumor-associated carbonic anhydrases IX and XII

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3-triazol-4-yl-/5-yl moieties were synthesized by employing click chemistry. The new sulfocoumarins incorporated cycloalkyl, tert-butyl and substituted aryl moieties at the triazole ring, and were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The triazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms hCA I and II (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 7.2 to 10.5 nM against hCA IX, and between 5.5 and 17.7 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.     

Synthesis of 6-tetrazolyl-substituted sulfocoumarins acting as highly potent and selective inhibitors of the tumor-associated carbonic anhydrase isoforms IX and XII

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3,4-tetrazol-5-yl moieties were synthesized by reaction of 6-iodo-sulfocoumarin and the corresponding tetrazole via the CH activation reaction. The new sulfocoumarins incorporating alkyl and substituted aryl moieties at the 1-position of the tetrazole, were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The tetrazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 6.5 to 68.6 nM against hCA IX, and between 4.3 and 59.8 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.     

Synthesis and carbonic anhydrase inhibitory properties of sulfamides structurally related to dopamine

A series of novel sulfamides incorporating the dopamine scaffold were synthesized. Reaction of amines and tert-butyl-alcohol/benzyl alcohol in the presence of chlorosulfonyl isocyanate (CSI) afforded sulfamoyl carbamates, which were converted to the title compounds by treatment with trifluoroacetic acid or by palladium-catalyzed hydrogenolysis. Inhibition of six α-carbonic anhydrases (CAs, EC 4.2.1.1), that is, CA I, CA II, CA VA, CA IX, CA XII and CA XIV, and two β-CAs from Candida glabrata (CgCA) and Mycobacterium tuberculosis (Rv3588) with these sulfamides was investigated. All CA isozymes were inhibited in the low micromolar to nanomolar range by the dopamine sulfamide analogues. K_is were in the range of 0.061–1.822 μM for CA I, 1.47–2.94 nM for CA II, 2.25–3.34 μM for CA VA, 0.041–0.37 μM for CA IX, 0.021–1.52 μM for CA XII, 0.007–0.219 μM for CA XIV, 0.35–5.31 μM for CgCA and 0.465–4.29 μM for Rv3588. The synthesized sulfamides may lead to inhibitors targeting medicinally relevant CA isoforms with potential applications as antiepileptic, antiobesity antitumor agents or anti-infective.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, VII,IX and XII with benzene sulfonamides incorporating 4,5,6,7-tetrabromophthalimide moiety

A series of 4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzene sulfonamide derivatives with 4,5,6,7-tetrabromophthalic anhydride moiety. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I, II and VII and the transmembrane tumor-associated isoform hCA IX and XII. The new compounds were good hCA I inhibitors (Kis in the range of 143 to >10,000 nM), but were moderately effective, as hCA II inhibitors (Kis of 47–190 nM) and poor hCA VII inhibitors (Kis in the range of 54–175 nM) compared to acetazolamide. The tumor-associated hCA IX was effectively inhibited with Kis ranging between 8.5 and 234 nM and hCA XII with inhibition constants in the range of 6.1–197 nM with high selectivity ratio. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking study of compounds was performed to rationalize the SAR reported over here.     

Substituted benzene sulfonamides incorporating 1,3,5-triazinyl moieties potently inhibit human carbonic anhydrases II,IX and XII

A series of benzene sulfonamides incorporating 1,3,5-triazinyl moieties were synthesized using cyanuric chloride as starting material. Inhibition studies against human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms were performed with the new compounds. hCA I was modestly inhibited (K_Is in the range of 87 nM–4.35 μM), hCA II was moderately inhibited by most of the new compounds (K_Is in the range of 12.5–130 nM), whereas the tumor associated isoforms were potently inhibited, with K_Is in the range of 1.2–34.1 nM against hCA IX and of 2.1–33.9 against hCA XII, respectively. Docking studies of some of the new compounds showed an effective binding mode within the enzyme active site, as demonstrated earlier by X-ray crystallography for structurally-related sulfonamides incorporating 1,3,5-triazinyl functionalities.     

Carbonic anhydrase inhibitors; Fluorinated phenyl sulfamates show strong inhibitory activity and selectivity for the inhibition of the tumor-associated isozymes IX and XII over the cytosolic ones I and II

A series of fluorinated-phenylsulfamates have been prepared by sulfamoylation of the corresponding phenols and the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic CA I and II (off-targets), and the transmembrane, tumor-associated CA IX and XII is investigated. Unlike the lead molecule (phenylsulfamate), a very potent CA I and II inhibitor and a modest CA IX/XII inhibitor, the fluorinated sulfamates were stronger inhibitors of CA IX (K_Is of 2.8–47 nM) and CA XII (K_Is of 1.9–35 nM) than of CA I (K_Is of 53–415 nM) and CA II (K_Is of 20–113 nM). Some of these compounds were selective CA IX over CA II inhibitors, with selectivity ratios in the range of 11.4–12.1, making them interesting candidates for targeting hypoxic tumors overexpressing CA IX and/or XII.     

Development of 3-(4-aminosulphonyl)-phenyl-2-mercapto-3H-quinazolin-4-ones as inhibitors of carbonic anhydrase isoforms involved in tumorigenesis and glaucoma

A series of heterocyclic benzenesulfonamides incorporating 2-mercapto-3H-quinazolin-4-one tails were prepared by condensation of substituted anthranilic acids with 4-isothiocyanato-benzenesulfonamide. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA IX and XII (trans-membrane, tumor-associated enzymes). They acted as medium potency inhibitors of hCA I (K_Is of 81.0–3084 nM), being highly effective as hCA II (K_Is in the range of 0.25–10.8 nM), IX (K_Is of 3.7–50.4 nM) and XII (K_Is of 0.60–52.9 nM) inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of three isoforms (CA II, IX and XII) is dysregulated.     

A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms

Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were synthesized and tested as atypical and selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of them inhibited hCA XII in the low nanomolar range, hCA IX with K_Is ranging between 19 and 2482 nM, whereas they were poorly active against hCA II (K_Is >10 μM) and hCA I (K_Is ranging between 318 nM and 50 μM). Since hCA I and II are ubiquitous off-target isoforms, whereas the cancer-related isoforms hCA IX and XII were recently validated as drug targets, these results represent an encouraging achievement in the development of new anticancer candidates. Moreover, the lack of a classical zinc binding group in the structure of these inhibitors opens innovative, yet unexplored scenarios for different mechanisms of inhibition that could explain the high inhibitory selectivity. A computational approach has been carried out to further rationalize the biological data and to characterize the binding mode of some of these inhibitors.     

7,8-disubstituted- but not 6,7-disubstituted coumarins selectively inhibit the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic ones I and II in the low nanomolar/subnanomolar range

Two series of disubstituted coumarins incorporating ether and acetyl/propionyl moieties in positions 6,7- and 7,8- of the heterocyclic ring were synthesized investigated for the inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). All these coumarins were very weak or ineffective as inhibitors of the housekeeping, offtarget isoforms CA I and II. The 6,7-disubstituted series showed ineffective inhibition also for the transmembrane tumor-associated isoforms CA IX and XII, whereas the corresponding isomeric 7,8-disubstituted coumarins showed nanomolar/subnanomolar inhibition of CA IX/XII. The nature and position of the groups substituting the coumarin ring in the 7,8-positions greatly influenced CA inhibitory properties, with C1-C4 alkyl ethers being the most effective inhibitors.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties

A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (K_is in the range of 295–10,000 nM), but were more effective hCA II inhibitors (K_is of 1.7–887 nM). The tumor-associated hCA IX was also inhibited, with K_is in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90–3746 nM. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors.     

Coumarinyl-substituted sulfonamides strongly inhibit several human carbonic anhydrase isoforms: solution and crystallographic investigations

We investigated a series of coumarinyl-substituted aromatic sulfonamides as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms with medical applications, the cytosolic hCA I, and II, and the transmembrane, tumor-associated hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4-yl-acetamide-tails and benzenesulfonamide and benzene-1,3-disulfonamide scaffolds showed medium potency inhibition of hCA I (K_Is of 73–131 nM), effective hCA II inhibition (K_Is of 9.1–36 nM) and less effective hCA IX and XII inhibition (K_Is of 55–128 nM). Only one compound, the derivatized 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide with the coumarinyl tail, showed effective inhibition of the transmembrane isoforms, with K_Is of 5.9–14.2 nM, although it was less effective as hCA I and II inhibitor (K_Is of 36–120 nM). An X-ray crystal structure of hCA II in complex with 4-(7-methoxy-coumarin-4-yl-acetamido)-benzenesulfonamide (K_I of 9.1 nM against hCA II) showed the intact inhibitor coordinated to the zinc ion from the enzyme active site by the sulfonamide moiety, and participating in a edge-to-face stacking with Phe131, in addition to other hydrophobic and hydrophilic interactions with water molecules and amino acid residues from the active site. Thus, sulfonamides incorporating coumarin rings have a distinct inhibition mechanism compared to the coumarins, and may lead to compounds with interesting inhibition profiles against various α-CAs found in mammals or parasites, such as Plasmodium falciparum.     

Flow synthesis and biological activity of aryl sulfonamides as selective carbonic anhydrase IX and XII inhibitors

A series of secondary and tertiary aryl sulfonamides were synthesized under flow conditions and evaluated for their ability to selectively inhibit tumor-associated carbonic anhydrase isoforms IX and XII. The tested compounds revealed to be highly potent CA IX inhibitors in nanomolar range, and to inhibit CA XII activity with different ranks of potencies. Remarkably, 4-methyl-N-phenyl-benzenesulfonamide was a selective nanomolar CA IX inhibitor with an IC₅₀ of 90 nM.     

Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties

Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA XII (a transmembrane, tumor-associated enzyme also involved in glaucoma-genesis). The new sulfonamides acted as medium potency inhibitors of hCA I (K_Is of 28.5–2954 nM), being highly effective as hCA II (K_Is in the range of 0.62–12.4 nM) and XII (K_Is of 0.54–7.11 nM) inhibitors. All substitution patterns present in these compounds (e.g., halogens, methyl and methoxy moieties, in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring) led to highly effective hCA II/XII inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of isoforms CA II and XII is dysregulated.     

Sulfonamides incorporating heteropolycyclic scaffolds show potent inhibitory action against carbonic anhydrase isoforms I,II, IX and XII

Three series of polycyclic compounds possessing either primary sulfonamide or carboxylic acid moieties as zinc-binding groups were investigated as inhibitors of four physiologically relevant CA isoforms, the cytosolic hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides reported here showed excellent inhibitory effects against isoforms hCA II, IX and XII, but no highly isoform-selective inhibition profiles. On the other hand, the carboxylates selectively inhibited hCA IX (K_Is ranging between 40.8 and 92.7 nM) without inhibiting significantly the other isoforms. Sulfonamides/carboxylates incorporating polycyclic ring systems such as benzothiopyranopyrimidine, pyridothiopyranopyrimidine or dihydrobenzothiopyrano[4,3-c]pyrazole may be considered as interesting candidates for exploring the design of isoform-selective CAIs with various pharmacologic applications.     

5-Substituted-(1,2,3-triazol-4-yl)thiophene-2-sulfonamides strongly inhibit human carbonic anhydrases I,II, IX and XII: Solution and X-ray crystallographic studies

We report here a series of 2-thiophene-sulfonamides incorporating 1-substituted aryl-1,2,3-triazolyl moieties, prepared by click chemistry from 5-ethynylthiophene-2-sulfonamide and substituted aryl azides. The new sulfonamides were investigated as inhibitors of the zinc metalloenzyme CA (EC 4.2.1.1), and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated ones hCA IX and XII: The new compounds were medium–weak hCA I inhibitors (K_Is in the range of 224–7544 nM), but were compactly, highly effective, low nanomolar hCA II inhibitors (K_Is of 2.2–7.7 nM). The tumor-associated hCA IX was inhibited with K_Is ranging between 5.4 and 811 nM, whereas hCA XII with inhibition constants in the range of 3.4–239 nM. The X-ray crystal structure of the adducts of two such compounds bound to hCA II (one incorporating 1-naphthyl, the other one 3-cyanophenyl moieties) evidenced the reasons of the high affinity for hCA II. Highly favorable, predominantly hydrophobic interactions between the sulfonamide scaffold and the hCA II active site were responsible for the binding, in addition to the coordination of the sulfamoyl moiety to the zinc ion. The tails of the two inhibitors adopted very diverse orientations when bound to the active site, with the naphthyltriazolyl moiety orientated towards the hydrophobic half of the active site, and the 3-cyanophenyl one pointing towards the hydrophilic half. These data may be used for the structure-based drug design of even more effective hCA II inhibitors, with potential use as antiglaucoma agents or as diuretics.     

Carbonic anhydrase inhibitors. Comparison of chlorthalidone,indapamide, trichloromethiazide,and furosemide X-ray crystal structures in adducts with isozyme II,when several water molecules make the difference

Thiazide and high ceiling diuretics were recently shown to inhibit all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1) with a very different profile as compared to classical inhibitors, such as acetazolamide, methazolamide, and ethoxzolamide. Some of these structurally related compounds have a very different behavior against the widespread isozyme CA II, with chlorthalidone, trichloromethiazide, and furosemide being efficient inhibitors against CA II (K_Is of 65–138 nM), whereas indapamide is a much weaker one (K_I of 2520 nM). Furthermore, some of these diuretics are quite efficient (low nanomolar) inhibitors of other isoforms, for example, chlorthalidone against hCA VB, VII, IX, and XIII; indapamide against CA VII, IX, XII, and XIII, trichloromethiazide against CA VII and IX, and furosemide against CA I and XIV. Examining the four X-ray crystal structures of their CA II adducts, we observed several (2–3) active site water molecules interacting with the chlorthalidone, trichloromethiazide, and furosemide scaffolds which may be responsible for this important difference of activity. Indeed, indapamide bound to CA II has no interactions with active site water molecules. Chlorthalidone bound within the CA II active site is in an enolic (lactimic) tautomeric form, with the enolic OH also participating in two strong hydrogen bonds with Asn67 and a water molecule. The newly evidenced binding modes of these diuretics may be exploited for designing better CA II inhibitors as well as compounds with selectivity/affinity for various isoforms with medicinal chemistry applications.