Synthesis and inhibition potency of novel ureido benzenesulfonamides incorporating GABA as tumor-associated carbonic anhydrase IX and XII inhibitors

New ureido benzenesulfonamides incorporating a GABA moiety as a linker between the ureido and the sulfonamide functionalities were synthesized and their inhibition potency determined against both the predominant cytosolic (hCA I and II) and the transmembrane tumor-associated (hCA IX and XII) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The majority of these compounds were medium potency inhibitors of the cytosolic isoform hCA I and effective hCA II inhibitors, whereas they showed strong inhibition of the two transmembrane tumor-associated isoforms hCA IX and XII, with K_Is in nanomolar range. Only one derivative had a good selectivity for inhibition of the tumor-associated hCA IX target isoform over the cytosolic and physiologically dominant off-target hCA I and II, being thus a potential tool to develop new anticancer agents.     

Fluoroenesulphonamides: N-sulphonylurea isosteres showing nanomolar selective cancer-related transmembrane human carbonic anhydrase inhibition

After hydrofluorination of ynesulphonamides in superacid or in the presence of hydrofluoric acid/base reagents, a series of α-fluoroenamides has been synthesised and tested for the inhibition of carbonic anhydrase (CA, EC 4.2.1.1) isoforms. This study reveals a new, highly selective family of cancer-related transmembrane human (h) CA IX/XII inhibitors. These original fluorinated ureido isosters do not inhibit the widespread cytosolic isoforms hCA I and II and selectively inhibit the transmembrane cancer-related hCA IX and XII, offering interesting new leads for future studies.     

Inhibition of carbonic anhydrase isoforms I,II, IX and XII with secondary sulfonamides incorporating benzothiazole scaffolds

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO₂ hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of benzothiazole-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I and hCA II and the transmembrane hCA IX and hCA XII. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting hCA IX and hCA XII over the off-target ones hCA I and hCA II. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.     

Aryl derivatives of 3H-1,2-benzoxathiepine 2,2-dioxide as carbonic anhydrase inhibitors

Abstract

A new series of homosulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) possessing various substitution patterns and moieties in the 7, 8 or 9 position of the heterocylic ring were prepared by original procedures and investigated for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the human (h) hCA I, II, IX and XII. The 8-substituted homosulfocoumarins were the most effective hCA IX/XII inhibitors followed by the 7-substituted derivatives, whereas the substitution pattern in position 9 led to less effective binders for the transmembrane, tumour-associated isoforms IX/XII. The cytosolic isoforms hCA I and II were not inhibited by these compounds, similar to the sulfocoumarins/coumarins investigated earlier. As hCA IX and XII are validated anti-tumour targets, with one sulphonamide (SLC-0111) in Phase Ib/II clinical trials, finding derivatives with better selectivity for inhibiting the tumour-associated isoforms over the cytosolic ones, as the homosulfocoumarins reported here, is of crucial importance.     

Vanillin enones as selective inhibitors of the cancer associated carbonic anhydrase isoforms IX and XII. The out of the active site pocket for the design of selective inhibitors?

New C-glycosides and α,β-unsaturated ketones incorporating the 4-hydroxy-3-methoxyphenyl (vanillin) moiety as inhibitors of carbonic anhydrase (CA, EC 4.2.1.1) isoforms have been investigated. The inhibition profile of these compounds is presented against four human CA (hCA) isozymes, comprising hCAs I and II (cytosolic, ubiquitous enzymes) and hCAs IX and XII (tumour associated isozymes). Docking analysis of the inhibitors within the active sites of these enzymes has been performed and is discussed, showing that the observed selectivity could be explained in terms of an alternative pocket out of the CA active site where some of these compounds may bind. Several derivatives were identified as selective inhibitors of the tumour-associated hCA IX and XII. Their discovery might be a step in the strategy for finding an effective non-sulfonamide CA inhibitor useful in therapy/diagnosis of hypoxic tumours or other pathologies in which CA isoforms are involved.     

Synthesis and exploration of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives for their effects against carbonic anhydrase I,II, IX and XII isoforms as a non-sulfonamide class of inhibitors

Novel series of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives (8a–s) have been synthesized and explored as a non-sulfonamide class of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The newly synthesized molecules were evaluated for their CA inhibitory potency against four isoforms: the cytosolic isozyme hCA I, II as well as trans-membrane tumor associated isoform hCA IX and hCA XII taking acetazolamide (AAZ) as standard drug. The results revealed that most of the compounds showed good activity against hCA II, IX, and XII whereas none of them were active against hCA I (K_i >100 μM). It is observed that the physiologically most important cytosolic isoform hCA II was inhibited by these molecules in the range of K_i 9.3–77.7 μM. It is also found the both the transmembrane isoforms hCA IX and XII were also inhibited with K_is ranging between 54.7–96.7 μM and 4.6–8.8 μM, respectively. The binding modes of the active compounds within the catalytic pockets of hCA II, IX and XII were evaluated by docking studies. This new non-sulfonamide class of selective inhibitors of hCA II, IX and XII over the hCA I isoform may be used for further understanding the physiological roles of some of these isoforms in various pathologies.     

Synthesis of N′-phenyl-N-hydroxyureas and investigation of their inhibitory activities on human carbonic anhydrases

A series of N′-phenyl-N-hydroxyureas has been prepared by reacting hydroxylamine with aromatic isocyanates. These compounds were investigated as inhibitors of human carbonic anhydrases (hCAs, EC 4.2.1.1), considering four physiologically relevant isoforms, the cytosolic isoforms hCA I and II, and tumor associated, transmembrane isoforms hCA IX and XII. The new compounds reported here did not inhibit the widespread cytosolic isoforms hCA I and II, but they inhibited the tumor associated isoforms with interesting potencies. The most effective inhibitors showed K_Is ranging between 72.8 and 78.9 nM against hCA IX and between 6.9 and 7.2 against hCA XII, making them of interest as candidates for antitumor studies.     

A class of carbonic anhydrase IX/XII – selective carboxylate inhibitors

Abstract

A small series of 2,4-dioxothiazolidinyl acetic acids was prepared from thiourea, chloroacetic acid, aromatic aldehydes, and ethyl-2-bromoacetate. They were assayed for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms of human (h) origin, the cytosolic hCA I and II, and the transmembrane hCA IX and XII, involved among others in tumorigenesis (hCA IX and XII) and glaucoma (hCA II and XII). The two cytosolic isoforms were not inhibited by these carboxylates, which were also rather ineffective as hCA IX inhibitors. On the other hand, they showed submicromolar hCA XII inhibition, with K_Is in the range of 0.30–0.93?µM, making them highly CA XII-selective inhibitors.     

Open saccharin-based secondary sulfonamides as potent and selective inhibitors of cancer-related carbonic anhydrase IX and XII isoforms

A large number of novel secondary sulfonamides based on the open saccharin scaffold were synthesized and evaluated as selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). They were obtained by reductive ring opening of the newly synthesized N-alkylated saccharin derivatives and were shown to be inactive against the two cytosolic off-target hCA I and II (K_is?>?10?µM). Interestingly, these compounds inhibited hCA IX in the low nanomolar range with K_is ranging between 20 and 298?nM and were extremely potent inhibitors of hCA XII isoenzyme (K_is ranging between 4.3 and 432?nM). Since hCA IX and XII are the cancer-related isoforms recently validated as drug targets, these results represent an important goal in the development of new anticancer candidates. Finally, a computational approach has been performed to better correlate the biological data to the binding mode of these inhibitors.     

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.     

Synthesis of novel benzenesulfonamide bearing 1,2,3-triazole linked hydroxy-trifluoromethylpyrazolines and hydrazones as selective carbonic anhydrase isoforms IX and XII inhibitors

A series of twenty four hydroxy-trifluoromethylpyrazoline-carbonyl-1,2,3-triazoles and four hydrazones bearing benzenesulfonamide moieties was obtained by condensation of carboxyhydrazides with substituted 1,3-diketones. All the newly synthesized compounds were investigated as inhibitors of physiologically and pharmacologically relevant human (h) carbonic anhydrsae (CA, EC 4.2.1.1) cytosolic isoforms hCA I and II, as well as transmembrane tumor-assosciated isoforms hCA IX and XII. These compounds exhibited excellent CA inhibitory potency against the four CA isoenzymes as compared to clinically used reference drug acetazolamide (AAZ). Some compounds bearing bulkier group at C-5′ position of 1,2,3-triazoles ring were weaker inhibitors of hCA I. Inhibition assay against hCA II indicates, that several derivatives exhibited upto 27-fold more effective inhibitory activity compared to AAZ. Five of the assayed compounds displayed low nanomolar potency (K_i ≤ 10 nM) against hCA IX, whereas five compounds were found to be endowed with excellent inhibitory potencies (K_i ≤ 5 nM) against hCA XII. The biological activity profile presented herein will be useful for designing new leads and provide candidates for preclinical investigations.     

Carbonic anhydrase inhibitors. Selective inhibition of human tumor-associated isozymes IX and XII and cytosolic isozymes I and II with some substituted-2-mercapto-benzenesulfonamides

A series of 2-mercapto-substituted-benzenesulfonamides has been prepared by a unique two-step procedure starting from the corresponding 2-chloro-substituted benzenesulfonamides. Compounds bearing an unsubstituted mercapto group and the corresponding S-benzoyl derivatives were investigated as inhibitors of four isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), i.e., the cytosolic, ubiquitous isozymes CA I and II, as well as the transmembrane, tumor associated isozymes CA IX and XII. These derivatives were medium potency hCA I inhibitors (K_Is in the range of 1.5–5.7 μM), two derivatives were strong hCA II inhibitors (K_Is in the range of 15–16 nM), whereas the others showed weak activity. These compounds inhibited hCA IX with inhibition constants in the range 160–1950 nM and hCA XII with inhibition constants in the range 1.2–413 nM. Some of these derivatives showed a certain degree of selectivity for inhibition of the tumor-associated over the cytosolic isoforms, being thus interesting leads for the development of potentially novel applications in the management of hypoxic tumors which overexpress CA IX and XII.     

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.     

Metronidazole-coumarin conjugates and 3-cyano-7-hydroxy-coumarin act as isoform-selective carbonic anhydrase inhibitors

Reaction of 6-/7-hydroxycoumarin with metronidazole afforded conjugates which incorporate two interesting chemotypes which may inhibit carbonic anhydrases (CAs, EC 4.2.1.1) due to the presence of the coumarin moiety and possess radiosensitizing effects due to the presence of the nitroazole. Another dual action compound, which may act both as CA inhibitor as well as monocarboxylate transporter inhibitor, is 3-cyano-7-hydroxy-coumarin. These compounds have been investigated as inhibitors of 11 human CA isoforms. Submicromolar inhibition was observed against hCA VA, hCA VB, hCA VI, hCA VII, hCA IX, hCA XII and hCA XIV, whereas isoforms hCA I, II and XIII were not inhibited by these compounds. These coumarins thus act as isoform-selective CA inhibitors with the possibility to target isoforms involved in pathologies such as obesity (CA VA/VB) or cancer (CA IX and XII) without inhibiting the physiologically dominant, highly abundant hCA I and II.     

Synthesis of rhodamine B-benzenesulfonamide conjugates and their inhibitory activity against human α- and bacterial/fungal β-carbonic anhydrases

A series of fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors were obtained by attaching rhodamine B moieties to the scaffold of benzenesulfonamides. The new compounds have been investigated for the inhibition of 12 human α-CA isoforms (hCA I-hCA XIV), three bacterial and one fungal β-class enzymes from the pathogens Mycobacterium tuberculosis and Candida albicans. All types of inhibitory activities have been detected, with several compounds showing low nanomolar inhibition against the transmembrane isoforms hCA IX, XII (cancer-associated) and XIV. The β-CAs were inhibited in the micromolar range by these compounds which may have applications for the imaging of hypoxic tumors or bacteria due to their fluorescent moieties.     

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: 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.     

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.     

Benzoxepinones: A new isoform-selective class of tumor associated carbonic anhydrase inhibitors

Benzoxepinones (“homocoumarins”) are identified as a new class of selective inhibitors for tumor associated human carbonic anhydrases (hCA, EC 4.2.1.1) isoforms IX and XII. Similar to coumarins, they do not inhibit or poorly inhibit cytosolic human (h) isoforms hCA I and II, but act as nanomolar inhibitors of the trans-membrane, tumor associated isoforms hCA IX and XII.     

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.