Synthesis, enzyme inhibition, and molecular docking studies of a novel chalcone series bearing benzothiazole scaffold.

This study reports the facile synthesis of a novel series of benzothiazole-chalcones, in addition to their inhibitory profile on important metabolic enzymes including human carbonic anhydrases (hCA-I, hCA-II) and paraoxonase (PON-1). The inhibition parameters, IC50 (concentration for 50% inhibition) and Ki (dissociation constant) values, toward the title enzymes were determined for the studied compounds. As a result, IC50 values of hydratase activity were in the range 4.15-5.47 and 2.56-4.58 µM for hCA-I and hCA-II, respectively. At the same time, IC50 values of esterase activity were in the range 24.91-104.00 and 35.25-97.00 µM, while Ki values were in the range 14.43-59.66 and 26.65-73.34 µM for hCA-I and hCA-II, respectively. In addition, PON-1 enzyme inhibition results showed interesting inhibitory effects, with IC50 values between 13.28 and 16.68 µM. Finally, a comprehensive approach was established for the synthesized compounds based on theoretical calculations, which have been done using B3LYP, PBE0 theories and SVP, TVZP, TVZPP basis sets, followed by docking studies by which the outputs proved the harmonically flows with the experimental results.

Metal ion effects on Polyphenol Oxidase Covalently immobilized on a Bio-Composite.

Biosensors can be developed using different immobilization methods. Interest in immobilization methods have increased because biosensors have been important for science. Polyphenol oxidase (PPO) was used generally in biosensor applications. For this purpose, Polyphenol oxidase from banana was purified and covalently immobilized on chitosan-gelatin bio-composite. The properties of immobilized enzyme were investigated and compared to free enzyme. Various parameters were studied such as pH, temperature and storage stability on immobilized and free enzyme. Kinetic parameters were also evaluated by different substrates on immobilized and free enzyme. Catechol was determined the best substrate for immobilized enzyme with optimum condition. In vitro effects of metal ions were studied on immobilized enzyme. Concentration range of metal ions is 1.0-10.0 x10-6 mol/L. The activity of immobilized PPO was increased by Fe+2 and Ag+1 ion. Co+3 and Cu+1 had very strong inhibitory effects with IC50 values of 19.69x10-3 mol/L and 23.49 x10-3 mol/L, respectively. Inhibition constants (Ki) and inhibition types of metal ions were determined with immobilized enzyme. Zn+2 and Cr+3 ions were showed competitive inhibition and Pb+2 ions were determined non-competitive inhibition with immobilized enzyme. Mixed type inhibition was obtained with Co+3 ion using catechol as substrate with 3.33x10-5 mol/L Ki value on immobilized PPO. Immobilized PPO can be evaluated for biosensor for the purpose of measurements of metal ions.

Synthesis of new series of thiazol-(2(3H)-ylideneamino)benzenesulfonamide derivatives as carbonic anhydrase inhibitors.

Human carbonic anhydrase I and II isoenzymes (hCA I and II) are important metabolic enzymes. In this study, a new series of thiazol-(2(3H)-ylideneamino)benzenesulfonamide derivatives were synthesized and also some inhibition parameters including IC50 (hydratese) and inhibition constant values (Ki , esterase) were determined. All studied compounds exhibited potent inhibition against these enzymes. They inhibited carbonic anhydrases (CAs) with the IC50 values of 113 to 395.8 nM (Ki = 77.38-319.59 nM) for hCA I and 91.9 to 516 nM (Ki = 62.79-425.89 nM) for hCA II. Among the compounds, 5c was found to be the most active one (Ki : 77.38 nM) for hCA I and 5g was found for hCA II with the value of 62.79 nM.

Evaluation of carbonic anhydrase and paraoxonase inhibition activities and molecular docking studies of highly water-soluble sulfonated phthalocyanines.

The investigation of carbonic anhydrase and paraoxonase enzyme inhibition properties of water-soluble zinc and gallium phthalocyanine complexes ( 1 and 2 ) are reported for the first time. The binding of p-sulfonylphenoxy moieties to the phthalocyanine structure favors excellent solubilities in water, as well as providing an inhibition effect on carbonic anhydrase (CA) I and II isoenzymes and paraoxonase (PON1) enzyme. According to biological activity results, both complexes inhibited hCA I, hCA II, and PON1. Whereas 1 and 2 showed moderate hCA I and hCA II (off-target cytosolic isoforms) inhibitory activity (Ki values of 26.09 µM and 43.11 µM for hCA I and 30.95 µM and 33.19 µM for hCA II, respectively), they exhibited strong PON1 (associated with high-density lipoprotein [HDL]) inhibitory activity (Ki values of 0.37 µM and 0.27 µM, respectively). The inhibition kinetics were analyzed by Lineweaver-Burk double reciprocal plots. It revealed that 1 and 2 were noncompetitive inhibitors against PON1, hCA I, and hCA II. These complexes can be more advantageous than other synthetic CA and PON inhibitors due to their water solubility. Docking studies were carried out to examine the interactions between hCA I, hCA II, and PON1 inhibitors and metal complexes at a molecular level and to predict binding energies.

Association of human serum paraoxonase-1 with some respiratory drugs.

In this study, we investigated the effects of certain respiratory drugs, which are mainly used on human serum paraoxonase-1 (hPON1; EC 3.1.8.1). hPON1 was purified from human serum, with 354.91 fold and 45% yield by using two simple step procedures including, first, ammonium sulfate precipitation, then, Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis showed a single protein band belonging to hPON1 with 43 kDa. All the pharmaceutical compounds inhibited the PON1 enzyme highly at the micromolar level. The obtained IC50 values for nine different pharmaceutics ranged from 0.219 µM (salbutamol sulfate) to 67.205 µM (montelukast sodium). So, all drugs could be considered as potent hPON1 inhibitors. Ki values and inhibition types were determined by Lineweaver-Burk graphs. While varenicline tartrate and moxifloxacin hydrochloride inhibited the enzyme in a noncompetitive manner, others inhibited it in a mixed manner.

Synthesis of carbazole bearing pyridopyrimidine-substituted sulfonamide derivatives and studies their carbonic anhydrase enzyme activity.

The synthesis of carbazole containing pyridopyrimidine-substituted sulfonamide derivatives (3a-i) and their inhibitory effects on human carbonic anhydrase (hCA) I and II were studied. Spectral data and elemental analysis confirmed the structures of the compounds synthesized. The results show that all the synthesized compounds inhibited the CA I and II activities. Among them, 3a was found to be the most active ( K i : 14 µM) for hCA I and 3f ( K i : 126 µM) for hCA II.

Microwave-assisted synthesis of 1-substituted-1H-benzimidazolium salts: Non-competitive inhibition of human carbonic anhydrase I and II.

A series of 1-substituted-1H-benzimidazolium p-toluenesulfonate salts were synthesized in good yields by the reaction of 1-substituted benzimidazole derivatives and p-toluenesulfonic acid under microwave irradiation. Two iodide salts were synthesized by the anion exchange reaction of the corresponding p-toluenesulfonate salt and NaI. All compounds were characterized by 1 H NMR, 13 C NMR, IR, LC-MS spectroscopic methods, and elemental analyses. The crystal structure of 1-methoxyethyl-1H-benzimidazolium p-toluenesulfonate 2d showed that cation and anion are interconnected by N-H···O and C-H···O hydrogen bonds. All compounds were examined as inhibitor of human carbonic anhydrase (hCA) I and II, and all of them inhibited hCA I and hCA II. Kinetic investigation results revealed that these compounds inhibit hCA I and hCA II in a non-competitive manner. The iodide salts had higher inhibitory activity than their corresponding p-toluenesulfonate salts.

Development of carbazole-bearing pyridopyrimidine-substituted urea/thiourea as polyphenol oxidase inhibitors: synthesis, biochemistry, and theoretical studies.

Polyphenol oxidase (Tyrosinase, PPO) has received considerable attention, since it is the key enzyme in melanin biosynthesis. In this study, we investigated prepared novel carbazole-containing pyridopyrimidine-substituted with urea and thiourea derivatives and their PPO activities on the diphenolase activity of banana tyrosinase. The structures of the compounds synthesized were confirmed by 1 H NMR, 13 C NMR, FTIR and elemental analysis. PPO enzyme was purified from banana on an affinity gel comprised of Sepharose 4B-L-tyrosine-p-amino benzoic acid. For evaluating the enzyme activity, the synthesised compounds were subjected to tyrosinase inhibition assay using catechol as substrate. While some of the compounds (6, 7, 8f, 8h, 8i, 8j) showed enzyme inhibitor effect, some of them (8a, 8b, 8c, 8d, 8e, 8g, 8k) activated the PPO enzyme activity. Gaussian software was used for the molecular calculations to explain the results for the prepared compounds.

Synthesis and carbonic anhydrase inhibitory properties of new spiroindoline-substituted sulphonamide compounds.

New spiroindoline-substituted sulphonamide compounds were synthesised and their inhibitory effects on the activity of purified human carbonic anhydrase I and II were evaluated. Human carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of the 14 synthesised sulphonamides (6a-n) on esterase activities of these isoenzymes were studied in vitro. In relation to these activities, the inhibition equilibrium constants (Ki) were determined. The results showed that all the synthesised compounds inhibited the carbonic anhydrase (CA) isoenzyme activity. Among them, 6b was found to be the most active (Ki: 0.042 µM) for hCA I and 6a (Ki: 0.151 µM) for hCA II.

Functionalized imidazolium and benzimidazolium salts as paraoxonase 1 inhibitors: Synthesis, characterization and molecular docking studies.

Paraoxonase (PON) is a key enzyme in metabolism of living organisms and decreased activity of PON1 was acknowledged as a risk for atherosclerosis and organophosphate toxicity. The present study describes the synthesis, characterization, PON1 inhibitory properties and molecular docking studies of functionalized imidazolium and benzimidazolium salts (1a-5g). The structures of all compounds were elucidated by IR, NMR, elemental analysis and structures of compounds 2b and 2c were characterized by single-crystal X-ray diffraction. Compound 1c, a coumarin substituted imidazolium salt showed the best inhibitory effect on the activity of PON1 with good IC50 value (6.37 µM). Kinetic investigation was evaluated for this compound and results showed that this compound is competitive inhibitor of PON1 with Ki value of 2.39 µM. Molecular docking studies were also performed for most active compound 1c and one of least active compound 2c in order to determine the probable binding model into active site of PON1 and validation of the experimental results.

Some coumarins and benzoxazinones as potent paraoxonase 1 inhibitors.

In this study, we aimed to investigate the effect of some coumarin and benzoxazinone derivatives on the activity of human PON1. Human serum paraoxonase 1 was purified from fresh human serum blood by two-step procedures that are ammonium sulfate precipitation (60-80%) and then hydrophobic interaction chromatography (Sepharose 4B, L-tyrosine and 1-napthylamine). The enzyme was purified 232-fold with a final specific activity of 27.1 U/mg. In vitro effects of some previously synthesized ionic coumarin or benzoxazinone derivatives (1-21) on purified PON1 activity were investigated. Compound 14 (1-(2,3,4,5,6)-pentamethylbenzyl-3-(6,8-dimethyl-2H-chromen-2-one-4-yl))benzimidazolium chloride was found out as the strongest inhibitor (IC50 = 7.84 µM) for PON1 among the compounds. Kinetic investigation and molecular docking study were evaluated for one of the most active compounds (compound 12) and obtained data showed that this compound is competitive inhibitor of PON1 and interact with Leu262 and Ser263 in the active site of PON1. Moreover, coumarin derivatives were found out as the more potent inhibitors for PON1 than benzoxazinone derivatives.

Synthesis and evaluation of sulfonamide-bearing thiazole as carbonic anhydrase isoforms hCA I and hCA II.

Sulfonamide-bearing thiazole compounds were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase I and II were evaluated. Human carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of the 12 synthesized sulfonamide (5a-l) on the hydratase and esterase activities of these isoenzymes (hCA-I and hCA-II) were studied in vitro. In relation to these activities, the inhibition equilibrium constants (Ki) were determined. The results showed that all the synthesized compounds inhibited the CA isoenzyme activity. Among them 5b was found to be the most active (IC50 = 0.35 µM; Ki: 0.33 µM) for hCA I and hCA II.

Is there a relation between genetic susceptibility with cancer? A study about paraoxanase (PON1) enzyme activity in breast cancer cases.

Human serum paraoxonase 1 (PON1, EC 3.1.1.2) is a high density lipoprotein (HDL)-associated antioxidant enzyme that not only decreases oxidative stress, but it is also implicated in development of many cancers. Genetic information provides a means of identifying people who have an increased risk of cancer, thus this knowledge of cancer genetics helps to identify the ability to characterize malignancies leading to the development of new therapeutic approaches. Because of this reason, in this preliminary study we aimed to investigate the role of human serum PON1 enzyme activity and phenotypic distribution in 32 breast cancer (BC) patients (age range 28-82) and 35 cancer free (CF) control group (age range 21-67). PON1 enzyme was prepared from the serum pool of BC patients using hydrophobic interaction chromatography on L-tyrosine-9-aminophenanthrene-coupled Sepharose 4Bgel. The PON1 enzyme activity towards paraoxon substrate was quantified spectrophotometrically. The basal activity of PON1 was statistically decreased in cancer cases compared to the control group. In addition, individuals were classified according to phenotyping of human PON1 Q and R types. In the cohort of BC patients, an increase in the frequency of the PON homozygote Q (AA) genotype was observed (31% in the BC group versus 14% in the CF controls). The frequency of the PON heterozygote QR (AB) genotype was 34.5% in the patients with BC and 37% in the CF group. The same trend was observed in PON homozygote R (BB) genotype frequency (BC cases 34.5% versus controls 49%). We determined that the kinetic parameters of the purified enzyme by Lineweaver-Burk method. We obtained Km and Vmax values of 0.227 mM and 62 U/mL min for the BC enzyme, compared with 0.775 mM and 206 U/mL min for the CF control enzyme. As a conclusion, it is clear from our results that while the PON1 AA allele frequency in BC cases is much higher, that of BB allele is much lower, in comparison with the control group. The most significant finding of this study is AA allele activity which is low in BC cases was found high. We concluded that decreased AA allele PON1 activity might have a relation with BC.

Synthesis, antioxidant and carbonic anhydrase I and II inhibitory activities of novel sulphonamide-substituted coumarylthiazole derivatives.

New secondary benzenesulphonamide-substituted coumarylthiazole derivatives were synthesized and their inhibitory effects on purified carbonic anhydrase I and II were evaluated using CO2 as a substrate. The result showed that all the synthesized compounds exhibited inhibitory activity on both hCA I and hCA II with N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)naphthalene-2-sulphonamide (5f, IC50 value of 5.63 and 8.48 µM, against hCA I and hCA II, respectively) as the strongest inhibitor revealed from this study. Structure-activity relationship revealed that the inhibitory activity of the synthesized compounds is related to the type of the halogen and bulky substituent on the phenyl ring. In addition, the cupric reducing antioxidant capacities (CUPRAC) and ABTS cation radical scavenging abilities of the synthesized compounds were assayed. 4-methoxy-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)benzenesulphonamide (5e) exhibited the strongest ABTS and CUPRAC activity with IC50 value of 48.83 µM and A0.50 value of 23.29 µM, respectively.

Coumarin or benzoxazinone based novel carbonic anhydrase inhibitors: synthesis, molecular docking and anticonvulsant studies.

Among many others, coumarin derivatives are known to show human carbonic anhydrase (hCA) inhibitory activity. Since hCA inhibition is one of the underlying mechanisms that account for the activities of some antiepileptic drugs (AEDs), hCA inhibitors are expected to have anti-seizure properties. There are also several studies reporting compounds with an imidazole and/or benzimidazole moiety which exert these pharmacological properties. In this study, we prepared fifteen novel coumarin-bearing imidazolium and benzimidazolium chloride, nine novel benzoxazinone-bearing imidazolium and benzimidazolium chloride derivatives and evaluated their hCA inhibitory activities and along with fourteen previously synthesized derivatives we scanned their anticonvulsant effects. As all compounds inhibited purified hCA isoforms I and II, some of them also proved protective against Maximal electroshock seizure (MES) and ScMet induced seizures in mice. Molecular docking studies with selected coumarin derivatives have revealed that these compounds bind to the active pocket of the enzyme in a similar fashion to that previously described for coumarin derivatives.

In vitro inhibition effect of some coumarin compounds on purified human serum paraoxonase 1 (PON1).

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro effect of some hydroxy and dihydroxy ionic coumarin derivatives (1-20) on purified PON1 activity was investigated. Among these compounds, derivatives 11-20 are water soluble. In investigated compounds, compounds 6 and 13 were found the most active (IC50 = 35 and 34 µM) for PON1, respectively. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.

An alternative purification method for human serum paraoxonase 1 and its interactions with anabolic compounds.

In this study, an alternative purification method for human paraoxonase 1 (hPON1) enzyme was developed using two-step procedures, namely, ammonium sulfate precipitation and Sepharose-4B-L-tyrosine-3-aminophenantrene hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent M(W) of 43 kDa. The enzyme was purified 219-fold with a final specific activity of 4,408,400 U/mg and a yield of 10%. Furthermore, we examined the in vitro effects of some anabolic compounds, such as zeranol, 17 ß-estradiol, diethylstilbestrol, oxytocin, and trenbolone on the enzyme activity to understand the better inhibitory properties of these molecules. The five anabolic compounds dose dependently decreased the activity of hPON1 with inhibition constants in the millimolar-micromolar range. The results show that these compounds exhibit inhibitory effects on hPON1 at low concentrations with IC50 values ranging from 0.064 to 16.900 µM.

SYNTHESIS AND EVALUATION OF NEW PHTHALAZINE SUBSTITUTED ß-LACTAM DERIVATIVES AS CARBONIC ANHYDRASE INHIBITORS.

A new series of phthalazine substituted ß-lactam derivatives were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA I and II) were evaluated. 2H-Indazolo[2,1-b]phthala- zine-trione derivative was prepared with 4-nitrobenzaldehyde, dimedone, and phthalhydrazide in the presence of TFA in DMF, and the nitro group was reduced to 13-(4-aminophenyl)-3,3-dimethyl-3,4-dihydro- 2H-indazolo[1,2-b]phthalazine-1,6,11(13H)-trione with SnCl2 · 2H2O. The reduced compound was re- acted with different aromatic aldehydes, and phthalazine substituted imines were synthesized. The imine compounds undergo (2+2) cycloaddition reactions with ketenes to produce 2H-indazolo[2,1-b]phthala-zine-trione substituted ß-lactam derivatives. The ß-lactam compounds were tested as inhibitors of the CA isoenzyme activity. The results showed that all the synthesized compounds inhibited the CA isoenzyme activity. 1-(4-(3,3-dimethyl- 1,6,1 1-trioxo-2,3,4,6,11,13-hexahydro-1H-indazolo[1,2-b]phthalazin-13- yl)phenyl)-2-oxo-4-p-tolylazetidin-3-yl acetate (IC50 = 6.97 µM for hCA I and 8.48 µM for hCA II) had the most inhibitory effect.

Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel.

A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150 kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60 °C and incubated for 60 min. These results indicated that the enzyme was heat stable.

A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme.

In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. L-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-L-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-L-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants K(M) and V(Max) for BTH were determined by using hyaluronic acid as a substrate. K(M) and V(Max) values obtained from the Lineweaver-Burk graph were found to be 2.23 mM and 19.85 U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, ß-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH.