Antiproliferative effects of sulphonamide carbonic anhydrase inhibitors C18, SLC-0111 and acetazolamide on bladder,glioblastoma and pancreatic cancer cell lines

Carbonic anhydrase IX/XII (CA IX/XII), are cell-surface enzymes typically expressed by cancer cells as a form of adaptation to hypoxia and acidosis. It has been widely reported that these proteins play pivotal roles in cancer progression fostering cell migration, aggressiveness and resistance to first line chemo- and radiotherapies. CA IX has emerged as a promising target in cancer therapy and several approaches and families of compounds were characterised in the attempt to find optimal targeting by inhibiting of the high catalytic activity of the enzyme. In the present work, different cell lines representing glioblastoma, bladder and pancreatic cancer have been exploited to compare the inhibitory and antiproliferative effect of primary sulphonamide acetazolamide (AAZ), the Phase Ib/II clinical grade sulphonamide SLC-0111, and a membrane-impermeant positively charged, pyridinium-derivative (C18). New hints regarding the possibility to exploit CA inhibitors in these cancer types are proposed.     

SLC-0111 enaminone analogs, 3/4-(3-aryl-3-oxopropenyl) aminobenzenesulfonamides,as novel selective subnanomolar inhibitors of the tumor-associated carbonic anhydrase isoform IX

SLC-0111, an ureido substituted benzenesulfonamide, is a selective carbonic anhydrase (CA, EC 4.2.1.1) IX inhibitor that is currently in Phase I/II clinical trials for the treatment of advanced hypoxic tumors complicated with metastases. Herein we report the synthesis of two series of 3/4-(3-aryl-3-oxopropenyl) aminobenzenesulfonamides 5a–i and 6a–j as SLC-0111 enaminone congeners. The prepared enaminones were in vitro investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, IV and IX, using a stopped-flow CO₂ hydrase assay. All these isoforms were inhibited by the enaminones reported here in variable degrees. The target tumor-associated isoform hCA IX was undeniably the most affected one (K_Is: 0.21–7.1 nM), with 6- to 21-fold enhanced activity than SLC-0111 (K_I = 45 nM). All the prepared enaminones displayed interesting selectivity towards hCA IX over hCA I (SI: 32 – >35714), hCA II (SI: 2 – 1689) and hCA IV (SI: 11 – >45454). Of particular interest, bioisosteric replacement of phenyl tail with the bulkier 2-naphthyl tail, sulfonamide 6h, achieved the higher II/IX selectivity herein reported with SI of 1689.     

Sulfonamides containing curcumin scaffold: Synthesis,characterization, carbonic anhydrase inhibition and molecular docking studies

Curcumin is a multi-functional pharmacologically safe natural agent with proven cytoprotective effects to healthy human cells. In this study, a new series of sulfonamides with curcumin scaffold were synthesized, characterized and investigated for their carbonic anhydrase isoenzyme I (human) and II (bovine) isoforms. The structures of newly synthesized compounds were described by IR, ¹H NMR and ¹³C NMR spectral data. Compound 14 showed the K_i value of 0.99 µM with highest inhibitory activity among all other synthesized compounds against hCA-I enzyme. Similarly enzyme kinetic studies of compound 14, 16 and 30 against bCAII enzyme showed Ki values of 0.71, 0.67 and 0.71 µM respectively. Our biological assays results showed that most of active compounds have similar inhibitory activities compared to standard acetazolamide drug. The molecular docking predicted binding modes showed that these compounds bind with hCA-1 enzyme in similar fashion.     

Synthesis,molecular docking analysis and carbonic anhydrase I-II inhibitory evaluation of new sulfonamide derivatives

New sulfonamide-hydrazone derivatives (3a-3n) were synthesized to evaluate their inhibitory effects on purified human carbonic anhydrase (hCA) I and II. The inhibition profiles of the synthesized compounds on hCA I-II isoenzyme were investigated by comparing their IC₅₀ and K_i values. Acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide, AZA) has also been used as a standard inhibitor. The compound 3e demonstrated the best hCA I inhibitory effect with a K_i value of 0.1676 ± 0.017 µM. Besides, the compound 3m showed the best hCA II inhibitory effect with a K_i value of 0.2880 ± 0.080 µM. Cytotoxicity of the compounds 3e and 3m toward NIH/3T3 mouse embryonic fibroblast cell line was observed and the compounds were found to be non-cytotoxic. Molecular docking studies were performed to investigate the interaction types between active compounds and hCA enzymes. Pharmacokinetic profiles of compounds were assessed by theoretical ADME predictions. As a result of this study a novel and potent class of CA inhibitors were identified with a good activity potential.     

A potentiated cooperation of carbonic anhydrase IX and histone deacetylase inhibitors against cancer

Abstract

The emergence of tumour recurrence and resistance limits the survival rate for most tumour-bearing patients. Only, combination therapies targeting pathways involved in the induction and in the maintenance of cancer growth and progression might potentially result in an enhanced therapeutic efficacy. Herein, we provided a prospective combination treatment that includes suberoylanilide hydroxamic acid (SAHA), a well-known inhibitor of histone deacetylases (HDACs), and SLC-0111, a novel inhibitor of carbonic anhydrase (CA) IX. We proved that HDAC inhibition with SAHA in combination with SLC-0111 affects cell viability and colony forming capability to greater extent than either treatment alone of breast, colorectal and melanoma cancer cells. At the molecular level, this therapeutic regimen resulted in a synergistically increase of histone H4 and p53 acetylation in all tested cell lines. Overall, our findings showed that SAHA and SLC-0111 can be regarded as very attractive combination providing a potential therapeutic strategy against different cancer models.     

Synthesis,biological activity and multiscale molecular modeling studies of bis-coumarins as selective carbonic anhydrase IX and XII inhibitors with effective cytotoxicity against hepatocellular carcinoma

A series of novel bis-coumarin derivatives containing triazole moiety as a linker between the alkyl chains was synthesized and their inhibitory activity against the human carbonic anhydrase (hCA) isoforms I, II, IX and XII were evaluated. In addition, cytotoxic effects of the synthesized compounds on renal adenocarcinoma (769P), hepatocellular carcinoma (HepG2) and breast adeno carcinoma (MDA-MB-231) cell lines were examined. While the hCA I and II isoforms were inhibited in the micromolar range, the tumor-associated isoform hCA IX and XII were inhibited in the high nanomolar range. 4-methyl-7-((1-(12-((2-oxo-2H-chromen-7-yl)oxy)dodecyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (5p) showed the strongest inhibitory activity against hCA IX with the K_i of 144.6 nM and 4-methyl-7-((1-(10-((2-oxo-2H-chromen-7-yl)oxy)decyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (5n) exhibited the highest hCA XII inhibition with the K_i of 71.5 nM. In order to better understand the inhibitory profiles of studied molecules, multiscale molecular modelling approaches were applied. Low energy docking poses of studied molecules at the binding sites of targets have been predicted. In addition, electrostatic potential surfaces (ESP) for binding sites were also generated to understand interactions between proteins and active ligands.     

New thiopyrimidine-benzenesulfonamide conjugates as selective carbonic anhydrase II inhibitors: synthesis,in vitro biological evaluation,and molecular docking studies

In the present work, a new series of thiopyrimidine-benzenesulfonamide conjugates was designed, synthesized and tested as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Our design strategy was based on the molecular hybridization of the benzenesulfonamide moiety as a zinc binding group (ZBG), an alkylated thiopyrimidine moiety as a spacer and (un)substituted phenyl moieties with various electronic and hydrophobic environments as a tail. The designed and synthesized compounds were evaluated against four human (h) CA isoforms hCA I, hCA II, hCA IX and hCA XII. Series 6 showed promising activity and selectivity toward the cytosolic isoforms hCA I and hCA II versus the membrane bound isoforms hCA IX and hCA XII. Compounds 6e and 6f showed K_i of 0.04 µM against hCA II with a selectivity of 15.8- to 980-fold towards hCA II over hCA I, hCA IX, hCA XII isoforms. Molecular docking in the hCA II active site attributed the promising inhibitory activity of series 6 to the interaction of their sulfonamide moiety with the active site Zn²⁺ ion as well as its hydrogen bonding with the key amino acids Thr199 and Thr200. Through hydrophobic interaction, the benzenesulfonamide and the thiopyrimidine moieties interact with the hydrophobic side chains of the amino acids Val121/Leu198 and Ile91/Phe131, respectively. These results indicated that the designed and synthesized series is an interesting scaffold that can be further optimized for the development of selective antiglaucoma drugs.     

Discovery of new potent inhibitors for carbonic anhydrase IX by structure-based virtual screening

Through structure-based virtual screening, some dozen of benzene sulfonamides with novel scaffolds are identified as potent inhibitors against carbonic anhydrase (CA) IX with IC₅₀ values ranging from 2.86 to 588.34 nM. Among them, compounds 1 and 9 show high selectivity against tumor-target CA IX over CA II (the selectivity ratios are 21.3 and 136.6, respectively). The possible binding poses of hit compounds are also explored and the selectivity is elucidated by molecular docking simulations. The hit compounds discovered in this work would provide novel scaffolds for further hit-to-lead optimization.     

4-Anilinoquinazoline-based benzenesulfonamides as nanomolar inhibitors of carbonic anhydrase isoforms I,II, IX,and XII: design,synthesis, in-vitro,and in-silico biological studies

Human carbonic anhydrase inhibitors (hCAIs) are a key therapeutic class with a multitude of novel applications such as anticonvulsants, topically acting antiglaucoma, and anticancer drugs. Herein, a new series of 4-anilinoquinazoline-based benzenesulfonamides were designed, synthesised, and biologically assessed as potential hCAIs. The target compounds are based on the well-tolerated kinase scaffold (4-anilinoquinazoline). Compounds 3a (89.4 nM), 4e (91.2 nM), and 4f (60.9 nM) exhibited 2.8, 2.7, and 4 folds higher potency against hCA I when compared to the standard (AAZ, V), respectively. A single digit nanomolar activity was elicited by compounds 3a (8.7 nM), 4a (2.4 nM), and 4e (4.6 nM) with 1.4, 5, and 2.6 folds of potency compared to AAZ (12.1 nM) against isoform hCA II, respectively. Structure-activity relationship (SAR) and molecular docking studies validated our design approach that revealed highly potent hCAIs.     

Carbonic anhydrase inhibitors: Synthesis,molecular docking,cytotoxic and inhibition of the human carbonic anhydrase isoforms I,II, IX,XII with novel benzenesulfonamides incorporating pyrrole,pyrrolopyrimidine and fused pyrrolopyrimidine moieties

A series of novel pyrroles, pyrrolopyrimidines, pyrazolopyrrolopyrimidine, triazolopyrrolopyrimidines, tetrazolopyrrolopyrimidine, triazinopyrrolopyrimidines and pyrrolopyrimidotriazepines bearing the biologically active benzenesulfonamide moiety were synthesized by using pyrrole-o-amino-carbonitrile as key intermediate. All the synthesized compounds were evaluated for their in vitro carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects against the human (h) isoforms hCA I, II, IX and XII. Among the tested derivatives, compounds 16, 18 and 20–24 showed potent activity as inhibitors for the tumor associated transmembrane isoforms (hCA IX and XII) in the nanomolar and subnanomolar range, with high selectivity. All compounds underwent cytotoxic activity assays on human breast cancer cell line (MCF-7) showing effective activity, comparable to that of the clinically used drug doxorubicin.     

Synthesis,biological evaluation and in silico studies of novel N-substituted phthalazine sulfonamide compounds as potent carbonic anhydrase and acetylcholinesterase inhibitors

The synthesis, characterization and biological evaluation of a series of novel N-substituted phthalazine sulfonamide (5a-l) are disclosed. Phthalazines which are nitrogen-containing heterocyclic compounds are biologically preferential scaffolds, endowed with versatile pharmacological activity, such as anti-inflammatory, cardiotonic vasorelaxant, anticonvulsant, antihypertensive, antibacterial, anti-cancer action. The compounds were investigated for the inhibition against the cytosolic hCA I, II and AChE. Most screened sulfonamides showed high potency in inhibiting hCA II, widely involved in glaucoma, epilepsy, edema, and other pathologies (K_is in the ranging from 6.32 ± 0.06 to 128.93 ± 23.11 nM). hCA I was inhibited with K_is in the range of 6.80 ± 0.10–85.91 ± 7.57 nM, whereas AChE in the range of 60.79 ± 3.51–249.55 ± 7.89 nM. ADME prediction study of the designed N-substituted phthalazine sulfonamides showed that they are not only with carbonic anhydrase and acetylcholinesterase inhibitory activities but also with appropriate pharmacokinetic, physicochemical parameters and drug-likeness properties. Also, in silico docking studies were investigated the binding modes of selected compounds, to hCA I, II, and AChE.     

Synthesis,molecular modelling and QSAR study of new N-phenylacetamide-2-oxoindole benzensulfonamide conjugates as carbonic anhydrase inhibitors with antiproliferative activity

In continuation of our previous studies to optimise potent carbonic anhydrase inhibitors, two new series of isatin N-phenylacetamide based sulphonamides were synthesised and screened for their human (h) carbonic anhydrase (EC 4.2.1.1) inhibitory activities against four isoforms hCA I, hCA II, hCA IX and hCA XII. The indole-2,3-dione derivative 2h showed the most effective inhibition profile against hCAI and hCA II (K_I = 45.10, 5.87 nM) compared to acetazolamide (AAZ) as standard inhibitor. Moreover, 2h showed appreciable inhibition activity against the tumour-associated hCA XII, similar to AAZ showing K_I of 7.91 and 5.70 nM, respectively. The analogs 3c and 3d showed good cytotoxicity effects, and 3c revealed promising selectivity towards lung cell line A549. Molecular docking was carried out for 2h and 3c to predict their binding conformations and affinities towards the hCA I, II, IX and XII isoforms.     

Carbonic anhydrase inhibitory properties of novel benzylsulfamides using molecular modeling and experimental studies

In this study, a series of sulfamoyl carbamates and sulfamide derivatives were synthesized. Six commercially available benzyl amines and BnOH were reacted with chlorosulfonyl isocyanate (CSI) to give sulfamoyl carbamates. Pd–C catalyzed hydrogenolysis reactions of carbamates afforded sulfamides. The inhibition effects of novel benzylsulfamides on the carbonic anhydrase I, and II isoenzymes (CA I, and CA II) purified from fresh human blood red cells were determined by Sepharose-4B-L-Tyrosine-sulfanilamide affinity chromatography. In vitro studies were shown that all of novel synthesized benzylsulfamide analogs inhibited, concentration dependently, both hCA isoenzyme activities. The novel benzylsulfamide compounds investigated here exhibited nanomolar inhibition constants against the two isoenzymes. K_i values were in the range of 28.48 ± 0.01–837.09 ± 0.19 nM and 112.01 ± 0.01–268.01 ± 0.22 nM for hCAI and hCA II isoenzymes, respectively. Molecular modeling approaches were also applied for studied compounds.     

Novel sulphonamides incorporating triazene moieties show powerful carbonic anhydrase I and II inhibitory properties

Abstract

A series of compounds incorporating 3-(3-(2/3/4-substituted phenyl)triaz-1-en-1-yl) benzenesulfonamide moieties were synthesised and their chemical structure was confirmed by physico-chemical methods. Carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects of the compounds were evaluated against human isoforms hCA I and II. K_I values of these sulphonamides were in the range of 21?±?4–72?±?2?nM towards hCA I and in the range of 16?±?6–40?±?2?nM against hCA II. The 4-fluoro substituted derivative might be considered as an interesting lead due to its effective inhibitory action against both hCA I and hCA II (K_Is of 21?nM), a profile rarely seen among other sulphonamide CA inhibitors, making it of interest in systems where the activity of the two cytosolic isoforms is dysregulated.     

Kinetic and X-ray crystallographic investigations on carbonic anhydrase isoforms I,II, IX and XII of a thioureido analog of SLC-0111

SLC-0111 (4-(4-fluorophenylureido)-benzenesulfonamide) is the first carbonic anhydrase (CA, EC 4.2.1.1) IX inhibitor to reach phase I clinical trials as an antitumor/antimetastatic agent. Here we report a kinetic and X-ray crystallographic study of a congener of SLC-0111 which incorporates a thioureido instead of ureido linker between the two aromatic rings as inhibitor of four physiologically relevant CA isoforms. Similar to SLC-0111, the thioureido derivative was a weak hCA I and II inhibitor and a potent one against hCA IX and XII. X-ray crystallography of its adduct with hCA II and comparison of the structure with that of other five hCA II—sulfonamide adducts belonging to the SLC-0111 series, afforded us to understand the particular inhibition profile of the new sulfonamide. Similar to SLC-0111, the thioureido sulfonamide primarily interacted with the hydrophobic side of the hCA II active site, with the tail participating in van der Waals interactions with Phe131 and Pro202, in addition to the coordination of the deprotonated sulfonamide to the active site metal ion. On the contrary, the tail of other sulfonamides belonging to the SLC-0111 series (2-isopropyl-phenyl; 3-nitrophenyl) were orientated towards the hydrophilic half of the active site, which was correlated with orders of magnitude better inhibitory activity against hCA II, and a loss of selectivity for the inhibition of the tumor-associated CAs.     

Carbohydrazones as new class of carbonic anhydrase inhibitors: Synthesis,kinetics, and ligand docking studies

Discovery and development of carbonic anhydrase inhibitors is crucial for their clinical use as antiepileptic, diurectic and antiglaucoma agents. Keeping this in mind, we have synthesized carbohydrazones 1–27 and evaluated them for their in vitro carbonic anhydrase inhibitory potential. Out of twenty-seven compounds, compounds 1 (IC₅₀ = 1.33 ± 0.01 µM), 2 (IC₅₀ = 1.85 ± 0.24 µM), 3 (IC₅₀ = 1.37 ± 0.06 µM), and 9 (IC₅₀ = 1.46 ± 0.12 µM) have showed carbonic anhydrase inhibition better than the standard drug zonisamide (IC₅₀ = 1.86 ± 0.03 µM). Moreover, compounds 4 (IC₅₀ = 2.32 ± 0.04 µM), 5 (IC₅₀ = 3.96 ± 0.35 µM), 7 (IC₅₀ = 2.33 ± 0.02 µM), and 8 (IC₅₀ = 2.67 ± 0.01 µM) showed good inhibitory activity. Cheminformatic analysis has shown that compounds 1 and 2 possess lead-like properties. In addition, kinetic and molecular docking studies were also performed to investigate the binding interaction between carbohydrazones and carbonic anhydrase enzyme. This study has identified a novel and potent class of carbonic anhydrase inhibitors with the potential to be investigated further.     

1,2,4-Trisubstituted imidazolinones with dual carbonic anhydrase and p38 mitogen-activated protein kinase inhibitory activity

Various 1,2,4 trisubstituted imidazolin-5-one derivatives were synthesized and evaluated for their inhibitory activity against p38 mitogen-activated protein kinase (p38MAPK) and carbonic anhydrase (CA) enzymes aiming to explore potential dual inhibitors. Results revealed that compounds 3c, 3g, 3h, 4a, 6c and 6d were the most effective derivatives against p38αMAPK (IC₅₀ = 0.14, 0.14, 0.056, 0.14, 0.13 and 0.14 μM, respectively) compared to sorafenib (IC₅₀ = 1.58 μM) as standard drug. On the other hand, compound 4a revealed the best inhibitory activity against all the tested carbonic anhydrase isoforms CA I, II, IV and IX with K_i values of 95.0, 0.83, 6.90 and 12.4 nM, respectively compared to acetazolamide with K_i values 250, 12.1, 74 and 12.8 nM, respectively. Therefore, compound 4a can be considered as a potent dual p38αMAPK/CA inhibitor.     

The green synthesis and molecular docking of novel N-substituted rhodanines as effective inhibitors for carbonic anhydrase and acetylcholinesterase enzymes

Recently, inhibition effects of enzymes such as acetylcholinesterase (AChE) and carbonic anhydrase (CA) has appeared as a promising approach for pharmacological intervention in a variety of disorders such as epilepsy, Alzheimer’s disease and obesity. For this purpose, novel N-substituted rhodanine derivatives (RhAs) were synthesized by a green synthetic approach over one-pot reaction. Following synthesis the novel compounds, RhAs derivatives were tested against AChE and cytosolic carbonic anhydrase I, and II (hCAs I, and II) isoforms. As a result of this study, inhibition constant (Ki) were found in the range of 66.35 ± 8.35 to 141.92 ± 12.63 nM for AChE, 43.55 ± 14.20 to 89.44 ± 24.77 nM for hCA I, and 16.97 ± 1.42 to 64.57 ± 13.27 nM for hCA II, respectively. Binding energies were calculated with docking studies as −5.969, −5.981, and −9.121 kcal/mol for hCA I, hCA II, and AChE, respectively.     

Synthesis,biological activity and multiscale molecular modeling studies for coumaryl-carboxamide derivatives as selective carbonic anhydrase IX inhibitors

New coumaryl-carboxamide derivatives with the thiourea moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and their inhibitory activity against the human carbonic anhydrase (hCA) isoforms hCA I, II, VII and IX were evaluated. While the hCA I, II and VII isoforms were not inhibited by the investigated compounds, the tumour-associated isoform hCA IX was inhibited in the high nanomolar range. 2-Oxo-N-((2-(pyrrolidin-1-yl)ethyl)carbamothioyl)-2H-chromene-3-carboxamide (e11) exhibited a selective inhibitory action against hCA IX with the K_i of 107.9?nM. In order to better understand the inhibitory profiles of studied molecules, multiscale molecular modeling approaches were used. Different molecular docking algorithms were used to investigate binding poses and predicted binding energies of studied compounds at the active sites of the CA I, II, VII and IX isoforms.     

Acetylcholinesterase and carbonic anhydrase inhibitory properties of novel urea and sulfamide derivatives incorporating dopaminergic 2-aminotetralin scaffolds

In the present study a series of urea and sulfamide compounds incorporating the tetralin scaffolds were synthesized and evaluated for their acetylcholinesterase (AChE), human carbonic anhydrase (CA, EC 4.2.1.1) isoenzyme I, and II (hCA I and hCA II) inhibitory properties. The urea and their sulfamide analogs were synthesized from the reactions of 2-aminotetralins with N,N-dimethylcarbamoyl chloride and N,N-dimethylsulfamoyl chloride, followed by conversion to the corresponding phenols via O-demethylation with BBr₃. The novel urea and sulfamide derivatives were tested for inhibition of hCA I, II and AChE enzymes. These derivatives exhibited excellent inhibitory effects, in the low nanomolar range, with K_i values of 2.61–3.69 nM against hCA I, 1.64–2.80 nM against hCA II, and in the range of 0.45–1.74 nM against AChE. In silico techniques such as, atomistic molecular dynamics (MD) and molecular docking simulations, were used to understand the scenario of the inhibition mechanism upon approaching of the ligands into the active site of the target enzymes. In light of the experimental and computational results, crucial amino acids playing a role in the stabilization of the enzyme–inhibitor adducts were identified.