Evaluation of lipophilicity and antitumour activity of parallel carboxamide libraries

Searching for molecules possessing antitumour activity, a parallel molecule library of aromatic carboxamides has been designed and synthesised. This work resulted in a "thiophene" sub-library containing a thiophene core and of a "furoyl" sub-library with a furoyl core, respectively. In both sub-libraries substitutions were carried out with six different groups resulting in six pairs of compounds differing in only the heteroatom of aromatic ring of the cores. To study the importance of the type of cores and the specific substitutions in relation to their lipophilicity and antitumour activity, lipophilicity of carboxamides was determined by chromatographical data (log k') and by software calculated parameters (CLOGP). Pairs of compounds were tested for their ability to inhibit the proliferation of the A431 cells by MTT assay. The isosteric molecule pairs were successfully separated. Our results showed that the experimentally determined (log k') and the calculated (CLOGP) lipophilicity parameters correlated well with each other. Furthermore, lipophilicity values of the thiophene sub-library were always higher than those in the furoyl sub-library. Moreover, compounds of the thiophene sub-library were more active than their respective furoyl pairs in our MTT antiproliferative assay. From these observations we can conclude that the higher the lipophilicity values the higher the antitumour activity of the carboxamides synthesised. Therefore, determination of lipophilicity by measuring the log k' or by calculating the CLOGP values of the carboxamide sub-libraries may help to predict their biological activities.     

Carbonic anhydrase inhibitors. Novel sulfanilamide/acetazolamide derivatives obtained by the tail approach and their interaction with the cytosolic isozymes I and II, and the tumor-associated isozyme IX

A series of sulfonamides has been obtained by reacting sulfanilamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide with omega-chloroalkanoyl chlorides, followed by replacement of the omega-chlorine atom with secondary amines. Tails incorporating heterocyclic amines belonging to the morpholine, piperidine and piperazine ring systems have been attached to these sulfonamides, by means of an alkanoyl-carboxamido linker containing from two to five carbon atoms. The new derivatives prepared in this way were tested as inhibitors of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic isozymes CA I and II, and the catalytic domain of the transmembrane, tumor-associated isozyme CA IX. Several low nanomolar CA I and CA II inhibitors were detected both in the aromatic and heterocyclic sulfonamide series, whereas the best hCA IX inhibitors (inhibition constants in the range of 22-35 nM) all belonged to the acetazolamide-like derivatives.     

Carbonic anhydrase inhibitors: inhibition of isozymes I, II and IV by sulfamide and sulfamic acid derivatives

Sulfamide and sulfamic acid are the simplest compounds containing the SO2NH2 moiety, responsible for binding to the Zn(II) ion within carbonic anhydrase (CA, EC 4.2.1.1) active site, and thus acting as inhibitors of the many CA isozymes presently known. Here we describe two novel classes of CA inhibitors obtained by derivatizations of the lead molecules mentioned above. The new compounds, possessing the general formula RSO2NH-SO2X (X = OH, NH2), were obtained by reaction of sulfamide or sulfamic acid with alkyl/arylsulfonyl halides or arylsulfonyl isocyanates. A smaller series of derivatives has been obtained by reaction of aromatic aldehydes with sulfamide, leading to Schiff bases of the type ArCH = NSO2NH2. All the new compounds act as strong inhibitors of isozymes I, II and IV of carbonic anhydrase. Their mechanism of CA inhibition is also discussed based on electronic spectroscopic measurements on adducts with the Co(II)-substituted enzyme. These experiments led to the conclusion that the new inhibitors are directly coordinated (in a monodentate manner) to the metal ion within the enzyme active site, similarly to the classical inhibitors, the aromatic/heterocyclic sulfonamides.     

Carbonic anhydrase inhibitors; phosphoryl-sulfonamides--a new class of high affinity inhibitors of isozymes I and II

A series of phosphorylated aromatic/heterocyclic sulfonamides with the general formula ArSO2NHPO3H2 have been prepared by condensing ArSO2NH2 with phosphorus pentachloride, followed by controlled hydrolysis in the presence of formic acid. The new derivatives generally act as stronger inhibitors of two carbonic anhydrase (CA) isozymes, CA I and CA II, as compared to the parent unsubstituted sulfonamides from which they were obtained. The inhibition mechanism by this new class of CA inhibitors, as well as structure activity correlations for the series of investigated derivatives, are also discussed.     

Carbonic anhydrase inhibitors: metal complexes of a sulfanilamide derived Schiff base and their interaction with isozymes I,II and IV

Metal complexes of aromatic/heterocyclic sulfonamides act as stronger inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) as compared to the uncomplexed sulfonamides from which they are derived. Here we report the synthesis and inhibition studies against the physiologically relevant isozymes CA I, CA II and CA IV, of a series of metal complexes (Co(II), Ni(II) and Cu(II) derivatives) of a Schiff-base ligand, obtained from sulfanilamide and salicylaldehyde. The best activity was observed for the Cu(II) and Co(II) complexes, against CA II and CA IV, for which inhibition constants in the range of 15-39 and 72-108nM, respectively, were seen. The enhanced efficacy in inhibiting the enzyme may be due to a dual mechanism of action of the metal complexes, which interact with CA both by means of the sulfonamide moieties as well as the metal ions present in their molecule.     

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with aromatic and heterocyclic sulfonamides

The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme has been investigated with a series of aromatic and heterocyclic sulfonamides, including the six clinically used derivatives acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide and brinzolamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) were also provided for comparison. A very interesting and unusual inhibition profile against CA IX with these sulfonamides has been observed. Several nanomolar (K(I)-s in the range of 14-50 nM) CA IX inhibitors have been detected, both among the aromatic (such as orthanilamide, homosulfonilamide, 4-carboxy-benzenesulfonamide, 1-naphthalenesulfonamide and 1,3-benzenedisulfonamide derivatives) as well as the heterocylic (such as 1,3,4-thiadizole-2-sulfonamide, etc.) sulfonamides examined. Because CA IX is a highly active isozyme predominantly expressed in tumor tissues with poor prognosis of disease progression, this finding is very promising for the potential design of CA IX-specific inhibitors with applications as anti-tumor agents.     

Carbonic anhydrase inhibitors: synthesis of water-soluble, topically effective intraocular pressure lowering aromatic/heterocyclic sulfonamides containing 8-quinoline-sulfonyl moieties: is the tail more important than the ring?

Reaction of 20 aromatic/heterocyclic sulfonamides containing a free amino, imino, hydrazino or hydroxyl group, with 8-quinoline-sulfonyl chloride afforded a series of water-soluble (as hydrochloride or triflate salts) compounds. The new derivatives were assayed as inhibitors of the zinc enzyme carbonic anhydrase (CA), and more precisely of three of its isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form), involved in important physiological processes. Efficient inhibition was observed against all three isozymes, but especially against CA II (in nanomolar range), which is the isozyme known to play a critical role in aqueous humor secretion within the ciliary processes of the eye. Some of the best inhibitors synthesized were topically applied as 2% water solutions onto the eye of normotensive and glaucomatous albino rabbits, when strong and long-lasting intraocular pressure (IOP) lowering was observed with many of them. This result prompted us to reanalyze the synthetic work done by other groups for the design of water soluble, topically effective antiglaucoma sulfonamides. According to these researchers, the IOP lowering effect is due to the intrinsic nature of the specific heterocyclic sulfonamide considered, among which the thienothiopyran-2-sulfonamide derivatives represent the best studied case. Indeed, the first agents developed for such applications, such as dorzolamide, are derivatives of this ring system. In order to prove that the tail (in this case the 8-quinoline-sulfonyl moiety) conferring water solubility to a sulfonamide CA inhibitor is more important than the ring to which the sulfonamido group is grafted, we also prepared a dorzolamide derivative to which the 8-quinoline-sulfonyl moiety was attached. This new compound is quite water soluble as hydrochloride salt, behaves as a strong CA II inhibitor, and fared better than the parent molecule in lowering IOP in experimental animals. Thus, the tail conferring water solubility to such an enzyme inhibitor is more important for its topical activity as antiglaucoma drug than the heterocyclic/aromatic ring to which the sulfonamido moiety is grafted.     

Carbonic anhydrase inhibitors: synthesis of sulfonamides incorporating dtpa tails and of their zinc complexes with powerful topical antiglaucoma properties

Reaction of diethylenetriamino pentaacetic acid (dtpa) dianhydride with aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded bis-sulfonamides containing metal-complexing, polyamino-polycarboxylic acid moieties in their molecule. The corresponding mono-sulfonamide derivatives of dtpa were also obtained by an alternative method, from the free acid. Zn(II) complexes of these new sulfonamides were then prepared. Many of these derivatives showed nanomolar affinity towards isozymes I, II and IV of carbonic anhydrase (CA). Some of the best inhibitors were applied as 2% water solutions/suspensions into the eye of normotensive or glaucomatous albino rabbits, when strong and long-lasting intraocular pressure (IOP) lowering was observed.     

Carbonic anhydrase inhibitors: design of thioureido sulfonamides with potent isozyme II and XII inhibitory properties and intraocular pressure lowering activity in a rabbit model of glaucoma

A new series of thioureido-substituted sulfonamides were prepared by reacting 4-isothiocyanato- or 4-isothiocyanatoethyl-benzenesulfonamide with amines, hydrazines, or amino acids bearing moieties that can lead to an enhanced hydrosolubility, such as 2-dimethylamino-ethylamine, fluorine-containing aromatic amines/hydrazines, an aminodiol, heterocyclic polyamines (derivatives of morpholine and piperazine), 4-aminobenzoic acid, or natural amino acids (Gly, Cys, Asn, Arg, and Phe). The new compounds showed good inhibitory properties against three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, with K(I)s in the range of 24-324 nM against the cytosolic isoform CA I, of 6-185 nM against the other cytosolic isozyme CA II, and of 1.5-144 nM against the transmembrane isozyme CA XII. Some of the new derivatives were also very effective in reducing elevated intraocular pressure in hypertensive rabbits as a glaucoma animal model. Considering that this is the first study in which potent CA II/CA XII inhibitors are designed and investigated in vivo, it may be assumed that the target isozymes of the antiglaucoma sulfonamides are indeed the cytosolic CA II and the transmembrane CA XII.     

Carbonic anhydrase inhibitors. Preparation of potent sulfonamides inhibitors incorporating bile acid tails

Reaction of TBDMS-protected bile acids (cholic, chenodeoxycholic, deoxycholic, lithocholic, ursodeoxycholic acids) or dehydrocholic acid with aromatic/heterocyclic sulfonamides possessing free amino/hydroxy moieties, in the presence of carbodiimides, afforded after deprotection of the OTBDMS ethers, a series of sulfonamides incorporating bile acid moieties in their molecules. Many such derivatives showed strong inhibitory properties against three isozymes of carbonic anhydrase (CA, EC 4.2.1.1), that is CA I, II and IV, zinc enzymes playing critical roles in many pathologies, and which represent interesting targets for developing diverse pharmacological agents. Some of the most active derivatives, incorporating 1,3,4-thiadiazole-2-sulfonamide or benzothiazole-2-sulfonamide functionalities in their molecules, showed low nanomolar affinity for CA II and CAIV. Furthermore, the bioavailability of these derivatives in rabbits is comparable to that of acetazolamide, being in the range of 85-90%, showing them as promising candidates for systemically acting CA inhibitors.     

Carbonic Anhydrase Inhibitors: Metal Complexes of a Sulfanilamide Derived Schiff base and their Interaction with Isozymes I,II and IV

Metal complexes of aromatic/heterocyclic sulfonamides act as stronger inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) as compared to the uncomplexed sulfonamides from which they are derived. Here we report the synthesis and inhibition studies against the physiologically relevant isozymes CA I, CA II and CA IV, of a series of metal complexes (Co(II), Ni(II) and Cu(II) derivatives) of a Schiff-base ligand, obtained from sulfanilamide and salicylaldehyde. The best activity was observed for the Cu(II) and Co(II) complexes, against CA II and CA IV, for which inhibition constants in the range of 15-39 and 72-108 nM, respectively, were seen. The enhanced efficacy in inhibiting the enzyme may be due to a dual mechanism of action of the metal complexes, which interact with CA both by means of the sulfonamide moieties as well as the metal ions present in their molecule.     

Relationship between lipophilicity and antitumor activity of molecule library of Mannich ketones determined by high-performance liquid chromatography, clogP calculation and cytotoxicity test

A series of Mannich ketones were synthesized in order to study the relative importance of structure and specific substitutions in relation to their lipophilicity and antitumor activity. Substitutions were carried out with morpholinyl, pirrolidinyl, piperidyl and tetrahydro-isoquinolyl groups in various positions on three different skeletons. Lipophilicity of Mannich ketones was characterised by chromatography data (log k') and by software calculated parameters (clogP). Compounds were tested on their ability to inhibit the proliferation of the A431 human adenocarcinoma cell line evaluated by MTT and apoptosis assays. The results suggest that the higher the lipophilicity values (log k' and clogP), the higher the antitumor and apoptotic activity of Mannich ketones. Determination of lipophilicity by measuring the log k' or by calculating the clogP values of the compounds may help to predict their biological activities.     

Carbonic Anhydrase Inhibitors: Inhibition of Isozymes I,II and IV by Sulfamide and Sulfamic Acid Derivatives

Abstract

Sulfamide and sulfamic acid are the simplest compounds containing the SO₂NH₂ moiety, responsible for binding to the Zn(II) ion within carbonic anhydrase (CA, EC 4.2.1.1) active site, and thus acting as inhibitors of the many CA isozymes presently known. Here we describe two novel classes of CA inhibitors obtained by derivatizations of the lead molecules mentioned above. The new compounds, possessing the general formula RSO₂NH-SO₂X (X = OH, NH₂), were obtained by reaction of sulfamide or sulfamic acid with alkyl/arylsulfonyl halides or aryl-sulfonyl isocyanates. A smaller series of derivatives has been obtained by reaction of aromatic aldehydes with sulfamide, leading to Schiff bases of the type ArCH=NSO₂NH₂. All the new compounds act as strong inhibitors of isozymes I, II and IV of carbonic anhydrase. Their mechanism of CA inhibition is also discussed based on electronic spectroscopic measurements on adducts with the Co(II)-substituted enzyme. These experiments led to the conclusion that the new inhibitors are directly coordinated (in a monodentate manner) to the metal ion within the enzyme active site, similarly to the classical inhibitors, the aromatic/heterocyclic sulfonamides.     

Inhibition of carbonic anhydrase isoforms I,II, IX and XII with novel Schiff bases: Identification of selective inhibitors for the tumor-associated isoforms over the cytosolic ones

A series of new Schiff bases was obtained from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide and aromatic/heterocyclic aldehydes incorporating both hydrophobic and hydrophilic moieties. The obtained sulfonamides were investigated as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, as well as the transmembrane, tumor-associated CA IX and XII. Most derivatives were medium potency or weak hCA I/II inhibitors, but several of them showed nanomolar affinity for CA IX and/or XII, making them an interesting example of isoform-selective compounds. The nature of the aryl/hetaryl moiety present in the initial aldehyde was the main factor influencing potency and isoform selectivity. The best and most CA IX-selective compounds incorporated moieties such as 4-methylthiophenyl, 4-cyanophenyl-, 4-(2-pyridyl)-phenyl and the 4-aminoethylbenzenesulfonamide scaffold. The best hCA XII inhibitors, also showing selectivity for this isoform, incorporated 2-methoxy-4-nitrophenyl-, 2,3,5,6-tetrafluorophenyl and 4-(2-pyridyl)-phenyl functionalities and were also derivatives of 4-aminoethylbenzenesulfonamide. The sulfanilamide and 3-fluorosulfanilamide derived Schiff bases were less active compared to the corresponding 4-aminoethyl-benzenesulfonamide derivatives. As hCA IX/XII selective inhibition is attractive for obtaining antitumor agents/diagnostic tools with a new mechanism of action, compounds of the type described here may be considered interesting preclinical candidates.     

Carbonic anhydrase inhibitors: allylsulfonamide,styrene sulfonamide,N-allyl sulfonamides and some of their Si,Ge, and B derivatives

Unsubstituted aromatic, heterocyclic and perfluoroalkylic sulfonamides possessing the general formula RSO2NH2 act as powerful inhibitors of the zinc enzyme carbonic anhydrase (CA). Unsaturated primary/substituted sulfonamides have never been investigated for their interaction with the enzyme. Here it is shown that such compounds, and more precisely allyl-sulfonamide and trans-styrene sulfonamide possessing the above general formula (with R=CH2=CH-CH2- and C6H5-CH=CH-, respectively) behave as nanomolar inhibitors of the physiologically relevant isozymes CAI and CAII. Some other derivatives of these two leads (incorporating Si(IV), Ge(IV) and B(III) moieties among others) were also synthesized and investigated for their interaction with CA, but showed decreased affinity for both isozymes. The structure-activity relationship for this class of CA inhibitors is discussed. Furthermore, it was observed that allylsulfonyl chloride is a strong CA inactivator, probably by reacting with amino acid residues critical for the catalytic cycle.     

Carbonic anhydrase inhibitors: 4-sulfamoyl-benzenecarboxamides and 4-chloro-3-sulfamoyl-benzenecarboxamides with strong topical antiglaucoma properties.

Reaction of 4-carboxy-benzenesulfonamide or 4-chloro-3-sulfamoyl benzoic acid with carboxy-protected amino acids/dipeptides, or aromatic/heterocyclic sulfonamides/mercaptans afforded the corresponding benzene-carboxamide derivatives. These were tested as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II and IV. Some of the new derivatives showed affinity in the low nanomolar range for isozymes CA II and IV, involved in aqueous humor secretion within the eye, and were tested as topically acting anti-glaucoma agents, in normotensive and glaucomatous rabbits. Good in vivo activity and prolonged duration of action has been observed for some of these derivatives, as compared to the clinically used drugs dorzolamide and brinzolamide. Some of the 4-chloro-3-sulfamoyl benzenecarboxamides reported here showed higher affinity for CA I than for the sulfonamide avid isozyme CA II.     

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with fluorine-containing sulfonamides. The first subnanomolar CA IX inhibitor discovered

Polyfluorinated CAIs show very good inhibitory properties against different carbonic anhydrase (CA) isozymes, such as CA I, II, and IV, but such compounds have not been tested for their interaction with the transmembrane, tumor-associated isozyme CA IX. Thus, a series of such compounds has been obtained by attaching 2,3,5,6-tetrafluorobenzoyl- and 2,3,5,6-tetrafluorophenylsulfonyl- moieties to aromatic/heterocyclic sulfonamides possessing derivatizable amino moieties. Some of these compounds showed excellent CA IX inhibitory properties and also selectivity ratios favorable to CA IX over CA II, the other physiologically relevant isozyme with high affinity for sulfonamide inhibitors. The first subnanomolar and rather selective CA IX inhibitor has been discovered, as the 2,3,5,6-tetrafluorobenzoyl derivative of metanilamide showed an inhibition constant of 0.8 nM against hCA IX, and a selectivity ratio of 26.25 against CA IX over CA II. Several other low nanomolar CA IX inhibitors were detected among the new derivatives reported here. The reported derivatives constitute valuable candidates for the development of novel antitumor therapies based on the selective inhibition of tumor-associated CA isozymes.     

Carbonic Anhydrase Inhibitors. Arylsulfonylureido-and Arylureido-Substituted Aromatic and Heterocyclic Sulfonamides: Towards Selective Inhibitors of Carbonic Anhydrase Isozyme I

Abstract

Reaction of twenty aromatic/heterocyclic sulfonamides containing a free amino, imino, hydra-zino or hydroxyl group, with tosyl isocyanate or 3,4-dichlorophenyl isocyanate afforded two series of derivatives containing arylsulfonylureido or diarylureido moieties in their molecule respectively. The new derivatives were assayed as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form). Potent inhibition was observed against all three isozymes but especially against CA I, which is generally 10-75 times less susceptible to inhibition by the classical sulfonamides in clinical use as compared to the other major red cell isozyme, CA II, or the membrane-bound one, CA IV. The derivatives obtained from tosyl isocyanate were generally more potent than the corresponding ones obtained from 3,4-dichlorophenyl isocyanate. This is the first reported example of selective inhibition of CA I and might lead to more selective drugs/diagnostic agents from this class of pharmacologically relevant compounds.     

QSAR studies for the inhibition of the transmembrane isozymes XII and XIV of human carbonic anhydrase with a series of sulfonamides

A diverse series of aromatic/heterocyclic sulfonamides possessing inhibitory action against the human transmembrane isoforms XII (cancer-associated) and XIV of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) has been used to develop QSAR models. Including all the 55 investigated sulfonamides in the calibration set, the predictive qualities of the QSAR equations were weak (r(2)=0.1771, F=5.70) for CA XII and good for CA XIV inhibition (r(2)=0.8222, F=57.04 before eliminating the outliers, and r(2)=0.8911, F=67.07 after eliminating them). The obtained models suggest a slightly different inhibition mechanism for the two isoforms. 3-Halogeno-4-amino-benzenesulfonamides were outliers for scaffold hopping for the inhibition of CA XIV. CA XIV inhibitory activity was proportional to the degree of molecular surface rugosity. For compounds of the type X-Ar-SO(2)NH(2) and Ar'-Ar-SO(2)NH(2) type, best inhibitors were detected when Ar/Ar' incorporates a heterocyclic moiety. These studies may be helpful for the design of more specific CA XII/XIV inhibitors, since this is the first QSAR model investigating them.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozyme XIII with aromatic and heterocyclic sulfonamides: a novel target for the drug design

The inhibition of the newly discovered cytosolic carbonic anhydrase isozyme XIII (CA XIII) has been investigated with a series of aromatic and heterocyclic sulfonamides, including some of the clinically used derivatives, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide, and valdecoxib. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and the tumor associated isozyme IX (transmembrane) were also provided for comparison. A very interesting and unusual inhibition profile against CA XIII with these sulfonamides has been observed. The clinically used compounds (except valdecoxib, which was a weak CA XIII inhibitor) potently inhibit CA XIII, with Ki's in the range of 17-23 nM, whereas sulfanilamide, halogenated sulfanilamides, homosulfanilamide, 4-aminoethylbenzenesulfonamide, and orthanilamide were slightly less effective, with Ki's in the range of 32-56 nM. Several low nanomolar (Ki's in the range of 1.3-2.4 nM) CA XIII inhibitors have also been detected, all of them belonging to the sulfanilyl-sulfonamide type of inhibitors, of which aminobenzolamide is the best known representative. Because CA XIII is an active isozyme predominantly expressed in salivary glands, kidney, brain, lung, gut, uterus, and testis, where it probably plays an important role in pH regulation, its inhibition by sulfonamides may lead to novel therapeutic applications for this class of pharmacological agents.