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: Synthesis of Sulfonamides Incorporating 2, 4, 6–Trisubstituted-Pyridinium-Ethylcarboxamido Moieties Possessing Membrane-Impermeability and in Vivo Selectivity for the Membrane-Bound (CA IV) Versus the Cytosolic (CA I and CA II) Isozymes

A new approach is proposed for the selective in vivo inhibition of membrane-bound versus cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes with a class of positively-charged, membrane-impermeant sulfonamides. Aromatic/heterocyclic sulfonamides acting as strong (but unselective) inhibitors of this zinc enzyme were derivatized by the attachment of trisub-stituted-pyridinium-ethylcarboxy moieties (obtained from 2, 4, 6–trisubstituted-pyrylium salts and β-alanine) to the amino, imino, hydrazino or hydroxyl groups present in their molecules. Efficient in vitro inhibition (in the nanomolar range) was observed with some of the new derivatives against three investigated CA isozymes, i.e., hCA I, hCA II (cytosolic forms) and bCA IV (membrane-bound isozyme; h = human; b = bovine isozyme). Due to their salt-like character, the new type of inhibitors reported here, unlike the classical, clinically used compounds (such as acetazolamide, methazolamide, ethoxzolamide), are unable to penetrate biological membranes, as shown by CJ vivo and in vivo perfusion experiments in rats. The level of bicarbonate excreted into the urine of the experimental animals perfused with solutions of the new and classical inhibitors suggest that: (i) when using the new type of positively-charged sulfonamides. only the membrane-bound enzyme (CA IV) was inhibited. whereas the cytosolic isozymes (CA I and II) were not affected, (ii) in the experiments in which the classical compounds (acetazolamide, bcn-zolamíde. etc.) were used. unselective inhibition of all CA isozymes (I. II and IV) occurred.     

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: 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.     

Carbonic Anhydrase Inhibitors. Schiff Bases of some Aromatic Sulfonamides and Their Metal Complexes: Towards More Selective Inhibitors of Carbonic Anhydrase Isozyme IV

Abstract

Reaction of three aromatic sulfonamides possessing a primary amino group, i.e., sulfanilamide, homosulfanilamide and p-aminoethyl-benzenesulfonamide with heterocyclic and aromatic aldehydes afforded a series of Schiff bases. Metal complexes of some of these Schiff bases with divalent transition ions such as Zn(II), Cu(II), Co(II) and Ni(II) have also been obtained. The new compounds were assayed as inhibitors of three isozymes of carbonic anhydrase (CA). Several of the new compounds showed a modest selectivity for the membrane-bound (bovine) isozyme CA IV (bCA IV) as compared to the cytosolic human isozymes hCA I and II, in contrast to classical inhibitors which generally possess a 17-33 times lower affinity for bCA IV. This greater selectivity toward bCA IV is due mainly to a slightly decreased potency against hCA II relative to classical inhibitors. However, metal complexes of these Schiff bases possessed an increased affinity for hCA II, being less inhibitory against bCA IV. The first type of compounds reported here (i.e., the Schiff bases of aromatic sulfonamides with heterocyclic aldehydes) might thus lead to the development of low molecular weight isozyme specific CA IV inhibitors. The difference in affinity for the three isozymes of the inhibitors reported by us here is tentatively explained on the basis of recent X-ray crystallographic studies of these isozymes and their adducts with substratesiinhibitors     

Inhibition of carbonic anhydrase isoforms I,II, IV,VII and XII with carboxylates and sulfonamides incorporating phthalimide/phthalic anhydride scaffolds

We report a panel of carboxylates and sulfonamides incorporating phthalic anhydride and phthalimide moieties in their structure and their interaction with the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). They were synthesized from substituted anthranilic acids and trimellitic anhydride chloride, followed by reaction with primary amines and were tested for the inhibition of five physiologically relevant CA isoforms, the human (h) hCA I, II, IV, VII and XII, some of which are involved in serious pathologies (CA II, IV and XII in glaucoma; CA VII in epilepsy; CA XII in some solid tumors). The carboxylic acids were generally poor inhibitors of isoforms hCA I, II and IV but were highly effective, low nanomolar inhibitors of hCA VII and XII. The sulfonamides inhibited all isoforms significantly, and some of them were sub-nanomolar hCA VII inhibitors, although their isoform selectivity was lower compared to the carboxylates. This study proves that carboxylic acids incorporating a phthalic anhydride/phthalimide based scaffold may lead to isoform-selective inhibitors by applying the tail approach, mostly used up until now for obtaining sulfonamide, sulfamide and sulfamate CA inhibitors.     

Carbonic anhydrase inhibitors: synthesis of sulfonamides incorporating 2,4,6-trisubstituted-pyridinium-ethylcarboxamido moieties possessing membrane-impermeability and in vivo selectivity for the membrane-bound (CA IV) versus the cytosolic (CA I and CA II) isozymes

A new approach is proposed for the selective in vivo inhibition of membrane-bound versus cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes with a class of positively-charged, membrane-impermeant sulfonamides. Aromatic/heterocyclic sulfonamides acting as strong (but unselective) inhibitors of this zinc enzyme were derivatized by the attachment of trisubstituted-pyridinium-ethylcarboxy moieties (obtained from 2,4,6-trisubstituted-pyrylium salts and beta-alanine) to the amino, imino, hydrazino or hydroxyl groups present in their molecules. Efficient in vitro inhibition (in the nanomolar range) was observed with some of the new derivatives against three investigated CA isozymes, i.e., hCA I, hCA II (cytosolic forms) and bCA IV (membrane-bound isozyme; h = human; b = bovine isozyme). Due to their salt-like character, the new type of inhibitors reported here, unlike the classical, clinically used compounds (such as acetazolamide, methazolamide, ethoxzolamide), are unable to penetrate biological membranes, as shown by ex vivo and in vivo perfusion experiments in rats. The level of bicarbonate excreted into the urine of the experimental animals perfused with solutions of the new and classical inhibitors suggest that: (i) when using the new type of positively-charged sulfonamides, only the membrane-bound enzyme (CA IV) was inhibited, whereas the cytosolic isozymes (CA I and II) were not affected, (ii) in the experiments in which the classical compounds (acetazolamide, benzolamide, etc.) were used, unselective inhibition of all CA isozymes (I, II and IV) occurred.     

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. Inhibition of Cytosolic Isozymes I and II and Transmembrane,Cancer-associated Isozyme IX with Lipophilic Sulfonamides

A series of new compounds was obtained by reaction of aromatic/heterocyclic sulfonamides incorporating amino groups with N,N-diphenylcarbamoyl chloride and diphenylacetyl chloride. These sulfonamides were assayed for the inhibition of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes: the cytosolic CA I and CA II, and the transmembrane, cancer-associated isozyme CA IX. Good inhibitors against all these isoforms were detected, and the inhibition profile of the newly investigated isozyme IX was observed to be different from that of the cytosolic isozymes, I and II. This may lead to the development of novel anticancer therapies based on the selective inhibition of CA IX.     

Carbonic anhydrase inhibitors: the first selective, membrane-impermeant inhibitors targeting the tumor-associated isozyme IX

The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme possessing an extracellular active site has been investigated with a series of positively-charged, pyridinium derivatives of sulfanilamide, homosulfanilamide and 4-aminoethylbenzenesulfonamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) were also provided for comparison. A very interesting inhibition profile against CA IX with these sulfonamides has been observed. Several nanomolar (K(i)'s in the range of 6-54 nM) CA IX inhibitors have also been detected. Because CA IX is a highly active isozyme predominantly expressed in tumor tissues with bad prognosis of disease progression, this finding is very promising for the potential design of CA IX-specific inhibitors with applications as anti-tumor agents. This is the first report of inhibitors that may selectively target CA IX, due to their membrane-impermeability and high affinity for this clinically relevant isozyme.     

Synthesis of 4-sulfamoylphenyl-benzylamine derivatives with inhibitory activity against human carbonic anhydrase isoforms I,II, IX and XII

Imine derivatives were obtained by condensation of sulfanilamide with substituted aromatic aldehydes. The Schiff bases were thereafter reduced with sodium borohydride, leading to the corresponding amines, derivatives of 4-sulfamoylphenyl-benzylamine. 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 (transmembrane, tumor-associated enzymes). We noted that the compounds incorporating secondary amine moieties showed a better inhibitory activity against all CA isozymes compared to the corresponding Schiff bases. Low nanomolar CA II, IX and XII inhibitors were detected, whereas the activity against hCA I was less potent. The secondary amines incorporating sulfonamide or similar zinc-binding groups, poorly investigated chemotypes for designing metalloenzyme inhibitors, may offer interesting opportunities in the field due to the facile preparation and possibility to explore a vast chemical space.     

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.     

Carbonic Anhydrase Inhibitors. Inhibition of Cytosolic Isozymes I and II and Transmembrane,Cancer-associated Isozyme IX with Anions

Except for sulfonamides, metal complexing anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the transmembrane, tumor-associated isozyme CA IX with anions is reported here. Inhibition data of the cytosolic isozymes CA I and CA II with a large number of anionic species such as halides, pseudohalides, bicarbonate, nitrate, hydrosulfide, arsenate, etc., are also provided for comparison. Isozyme IX has an inhibition profile by anions different in some aspects from those of CA I and CA II, that may have interesting physiological consequences.     

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.     

(R)-/(S)-10-camphorsulfonyl-substituted aromatic/heterocyclic sulfonamides selectively inhibit mitochondrial over cytosolic carbonic anhydrases

A series of aromatic and heterocyclic sulfonamides incorporating R- and S-camphorsulfonyl moieties were synthesized and investigated for the inhibition of several mammalian isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The new sulfonamides selectively inhibited the mitochondrial isozymes hCA VA and VB (h = human isoform) over the cytosolic, off-target ones hCA I and II, with inhibition constants in the low nanomolar range. The chirality and position of the groups substituting the sulfonamide scaffold greatly influenced CA inhibitory properties. These compounds are excellent leads for designing isoform-selective enzyme inhibitors targeting mitochondrial CAs involved in lipogenesis and obesity.     

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: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, IX, and XII with N-hydroxysulfamides--a new zinc-binding function in the design of inhibitors

A small library of N-hydroxysulfamides was synthesized by an original approach in order to investigate whether this zinc-binding function is efficient for the design of inhibitors targeting the cytosolic (hCA I and II) and transmembrane, tumor-associated (hCA IX and XII) isozymes of carbonic anhydrase (CA, EC 4.2.1.1). The parent derivative, N-hydroxysulfamide was a more potent inhibitor as compared to sulfamide or sulfamic acid against all isozymes, with inhibition constants in the range of 473 nM-4.05 microM. Its substituted n-decyl-, n-dodecyl-, benzyl-, and biphenylmethyl-derivatives were less inhibitory against hCA I (K(I)s in the range of 5.8-8.2 microM) but more inhibitory against hCA II (K(I)s in the range of 50.5-473 nM). The same situation was true for the tumor-associated isozymes, with K(I)s in the range of 353-790 nM against hCA IX and 372-874 nM against hCA XII. Some sulfamides/sulfamates possessing similar substitution patterns have also been investigated for the inhibition of these isozymes, being shown that in some particular cases sulfamides are more efficient inhibitors as compared to the corresponding sulfamates. Potent CA inhibitors targeting the cytosolic or tumor-associated CA isozymes can thus be designed from various classes of sulfonamides, sulfamides, or sulfamates and their derivatives, considering the extensive interactions in which the inhibitor and the enzyme active site are engaged, based on recent X-ray crystallographic data.     

Carbonic anhydrase inhibitors: aromatic sulfonamides and disulfonamides act as efficient tumor growth inhibitors

Aromatic/heterocyclic sulfonamides generally act as strong inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). Here we report the unexpected finding that potent aromatic sulfonamide inhibitors of CA, possessing inhibition constants in the range of 10(-8)-10(-9) M (against all the isozymes), also act as efficient in vitro tumor cell growth inhibitors, with GI50 (molarity of inhibitor producing a 50% inhibition of tumor cell growth) values of 10 nM-35 microM against several leukemia, non-small cell lung cancer, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines. The investigated compounds were sulfanilyl-sulfanilamide-, 4-thioureido-benzenesulfonamide- and benzene-1,3-disulfonamide-derivatives. The mechanism of antitumor action with these sulfonamides is unknown, but it might involve either inhibition of several CA isozymes (such as CA IX, CA XII, CA XIV) predominantly present in tumor cells, a reduced provision of bicarbonate for the nucleotide synthesis (mediated by carbamoyl phosphate synthetase II), the acidification of the intracellular milieu as a consequence of CA inhibition or uncoupling of mitochondria and potent CA V inhibition among others. A combination of several such mechanisms is also plausible. Optimization of such derivatives from the SAR point of view, might lead to the development of effective novel types of anticancer agents/therapies.     

Review Article

Carbonic anhydrases (CAs, EC 4.2.1.1) are wide-spread enzymes, present in mammals in at least 14 different isoforms. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA IX, CA XII and CA XIV), CA V is mitochondrial and CA VI is secreted in the saliva and milk. Three cytosolic acatalytic forms are also known (CARP VIII, CARP X and CARP XI). The catalytically active isoforms, which play important physiological and patho-physiological functions, are strongly inhibited by aromatic and heterocyclic sulfonamides. The catalytic and inhibition mechanisms of these enzymes are understood in great detail, and this greatly helped the design of potent inhibitors, some of which possess important clinical applications. The use of such CA inhibitors (CAIs) as antiglaucoma drugs are discussed in detail, together with the recent developments that led to isozyme-specific and organ-selective inhibitors. A recent discovery is connected with the involvement of CAs and their sulfonamide inhibitors in cancer: many potent CAIs were shown to inhibit the growth of several tumor cell lines in vitro and in vivo, thus constituting interesting leads for developing novel antitumor therapies. Future prospects for drug design of inhibitors of these ubiquitous enzymes are dealt with. Although activation of CAs has been a controversial issue for some time, recent kinetic, spectroscopic and X-ray crystallographic experiments offered an explanation of this phenomenon, based on the catalytic mechanism. It has been demonstrated recently, that molecules that act as carbonic anhydrase activators (CAAs) bind at the entrance of the enzyme active site participating in facilitated proton transfer processes between the active site and the reaction medium. In addition to CA II-activator adducts, X-ray crystallographic studies have been also reported for ternary complexes of this isozyme with activators and anion (azide) inhibitors. Structure-activity correlations for diverse classes of activators is discussed for the isozymes for which the phenomenon has been studied, i.e, CA I, II, III and IV. The possible physiological relevance of CA activation/inhibition is also addressed, together with recent pharmacological/biomedical applications of such compounds in different fields of life sciences.     

Carbonic anhydrase inhibitors: sulfonamides as antitumor agents?

Novel sulfonamide inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) were prepared by reaction of aromatic or heterocyclic sulfonamides containing amino, imino, or hydrazino moieties with N,N-dialkyldithiocarbamates in the presence of oxidizing agents (sodium hypochlorite or iodine). The N,N-dialkylthiocarbamylsulfenamido-sulfonamides synthesized in this way behaved as strong inhibitors of human CA I and CA II (hCA I and hCA II) and bovine CA IV (bCA IV). For the most active compounds, inhibition constants ranged from 10(-8) to 10(-9) M (for isozymes II and IV). Three of the derivatives belonging to this new class of CA inhibitors were also tested as inhibitors of tumor cell growth in vitro. These sulfonamides showed potent inhibition of growth against several leukemia, non-small cell lung, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines. With several cell lines. GI50 values of 10-75 nM were observed. The mechanism of antitumor action with the new sulfonamides reported here remains obscure, but may involve inhibition of CA isozymes which predominate in tumor cell membranes (CA IX and CA XII), perhaps causing acidification of the intercellular milieu, or inhibition of intracellular isozymes which provide bicarbonate for the synthesis of nucleotides and other essential cell components (CA II and CA V). Optimization of these derivatives from the SAR point of view, might lead to the development of effective novel types of anticancer agents.