Novel N-(benzo[d]oxazol-2-yl)alkanamides; synthesis and carbonic anhydrase II inhibition studies

Carbonic anhydrase (CA II) inhibitors are very important therapeutic targets in drug design for treatment of neuropathic pain and in eradication of glaucoma, cancer, epilepsy, ulcer and obesity. In this study, some two2-substituted benzoxazoles ( 3a-j ) were developed as a new family of carbonic anhydrase II inhibitors by employing acyl thiourea chemistry via a simple and expedient protocol and evaluated for CA II inhibitor activity and radical scavenging ability. Compounds 3f and 3j were found to be the most potent inhibitors, with IC₅₀ values of 0.00564 and 0.00596 μM, respectively which are several times better than that of the standard, acetazolamide (IC₅₀ value 0.997 ± 0.0586 μM). Docking experiments were carried out against the carbonic anhydrase II crystal structure to better rationalize the inhibitory activities of these new structures. Moreover, the results of a DPPH radical scavenging assay showed that the antioxidant profile of compound 3i is superior to those of other derivatives. The results have revealed that derivatives 3f and 3j behave as CA-II inhibitors significantly better than standard and 3i has good anti-oxidation potential.     

Fluorescent Silica Nanoparticles with Multivalent Inhibitory Effects towards Carbonic Anhydrases

Multifunctional silica nanoparticles decorated with fluorescent and sulfonamide carbonic anhydrase (CA) inhibitors were prepared and investigated as multivalent enzyme inhibitors against the cytosolic isoforms hCA I and II and the transmembrane tumor‐associated ones hCA IX and XII. Excellent inhibitory effects were observed with these nanoparticles, with K_I values in the low nanomolar range (6.2–0.67 nM ) against all tested isozymes. A significant multivalency effect was seen for the inhibition of the monomeric enzymes hCA I and II compared to the dimeric hCA IX and hCA XII isoforms, where no multivalent effect was observed, suggesting that the multivalent binding is occurring through enzyme clustering.     

A selectivity study of benzenesulfonamide derivatives on human carbonic anhydrase II/IX by 3D-QSAR,Molecular Docking and Molecular Dynamics Simulation

Nowadays, different approaches have been pursued with the intent to develop sulfonamide-like carbonic anhydrase inhibitors that possess better selectivity profiles toward the different human isoforms of the enzyme. Here, we used conventional 3D-QSAR methods, including comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and Topomer CoMFA, to construct three-dimensional quantitative structure-activity relationship (3D-QSAR) models for benzenesulfonamide derivatives as human carbonic anhydrase (hCA) II/IX inhibitors. The theoretical models had good reliability (R²>0.75) and predictability (Q²>0.55), and the contour maps could graphically present the contributions of the force fields for activity and identify the structural divergence between human carbonic anhydrase II inhibitors and human carbonic anhydrase IX inhibitors. Consequently, we explored the selectivity of inhibitor for human carbonic anhydrase II and IX through molecular docking, and the difference of activity coincides with the potential binding mode well. According to the results of the predicted values and the molecule docking, we found that the inhibitors published in the literature had stronger inhibition on the hCA IX; based on the theoretical models, we designed seven new compounds with good potential activity and reasonably good ADMET profile, which could selectively inhibit hCA IX. Molecular Dynamics Simulation showed that newly-designed compound D7 had good selectivity on hCA IX. The findings from 3D-QSAR and docking studies maybe helpful in the rational drug design of isoform-selective inhibitors.     

Novel silver(I)N-heterocyclic carbene complexes bearing 2-(4-hydroxyphenyl)ethyl group: Synthesis,characterization, and enzyme inhibition properties

Herein, novel silver-based N-heterocyclic carbene (NHC) complexes bearing 2-(4-hydroxyphenyl)ethyl group were synthesized. Novel Ag(I)NHC complexes were synthesized from the 2-(4-hydroxyphenyl)ethyl-substituted benzimidazolium salts and silver oxide via in situ deprotonation method. The successful formation of all Ag(I)NHC complexes was proved by using ¹H NMR, ¹³C NMR, FTIR spectroscopy, and elemental analysis techniques. In addition, their inhibitory effects have been investigated of these substances on acetylcholinesterase (AChE), α-glycosidase (α-Gly), human carbonic anhydrase I (hCA I), and human carbonic anhydrase II (hCA II) enzymes. It has been seen that all compounds have a better ability to inhibit compared with existing tried inhibitors. Among these, the best inhibitor against AChE enzyme is 1g (K_i : 9.54 ± 0.98 μM and IC₅₀ : 17.40), and against α-Gly, 1c showed the highest effect (K_i 3.09 ± 0.36 μM and IC₅₀ 7.91). The best inhibitor against hCA I and hCA II enzymes are 1c and 1g compounds. For hCA I and hCA II, IC₅₀ values were calculated as 17.85 and 9.06 μM and K_i values were measured as 5.45 ± 2.02 and 8.99 ± 2.02 μM, respectively.     

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.     

Inhibitory Effect of Polyphenols from the Whole Green Jackfruit Flour against α-Glucosidase, α-Amylase,Aldose Reductase and Glycation at Multiple Stages and Their Interaction: Inhibition Kinetics and Molecular Simulations

For the first time, α-glucosidase, α-amylase, aldose reductase, and glycation at multiple stages inhibitory assays were used to explore the antidiabetic potential of whole unripe jackfruit (peel with pulp, flake, and seed). Two polyphenols (phenolic acids) with strong antihyperglycaemic activity were isolated from the methanol extract of whole jackfruit flour (MJ) using activity-guided repeated fractionation on a silica gel column chromatography. The bioactive compounds isolated were identified as 3-(3,4-Dihydroxyphenyl)-2-propenoic acid (caffeic acid: CA) and 4-Hydroxy-3,5-dimethoxybenzoic acid (syringic acid: SA) after various physicochemical and spectroscopic investigations. CA (IC₅₀: 8.0 and 26.90 µg/mL) and SA (IC₅₀: 7.5 and 25.25 µg/mL) were identified to inhibit α-glucosidase and α-amylase in a competitive manner with low Ki values. In vitro glycation experiments further revealed that MJ and its components inhibited each stage of protein glycation as well as the generation of intermediate chemicals. Furthermore, CA (IC₅₀: 3.10) and SA (IC₅₀: 3.0 µg/mL) inhibited aldose reductase effectively in a non-competitive manner, respectively. The binding affinity of these substances towards the enzymes examined has been proposed by molecular docking and molecular dynamics simulation studies, which may explain their inhibitory activities. The found potential of MJ in antihyperglycaemic activity via inhibition of α-glucosidase and in antidiabetic action via inhibition of the polyol pathway and protein glycation is more likely to be related to the presence of the phenolic compounds, according to our findings.     

Discovery of Novel Sulfonamide‐Based 5‐Arylidenerhodanines as Effective Carbonic Anhydrase (II) Inhibitors: Microwave‐Assisted and Ultrasound‐Assisted One‐Pot Four‐Component Synthesis,Molecular Docking,and Anti‐CA II Screening Studies

The synthesis of N‐substituted‐5‐arylidenerhodanines was carried out by the optimized one‐pot sequential four‐component procedure with the condensation between 4‐aminobenzenesulfonamide, suitable aldehyde, ethyl bromoacetate, and carbon disulfide. In addition to traditional method, microwave‐irradiated and ultrasound‐irradiated techniques were implemented in water at ambitious conditions, and the target compounds were obtained in high yields and purity without purification methods. The enzyme inhibition activity of newly synthesized compounds on carbonic anhydrase (II) was also evaluated. The reference inhibitor molecule was sulfanilamide, the IC₅₀ value of which was 3.5 μM. It was also found that the IC₅₀ values of all examined molecules were in nanomolar level and much smaller than those of sulfanilamide. The inhibition between 93.5 and 99.6% was observed in the presence of new compounds synthesized in the present study at the accessible maximum concentration in the reaction mixtures. 5j , among the tested compounds possessing the lowest IC₅₀ value, was found to be the most potent carbonic anhydrase (II) inhibitor.     

Selective hydrophobic pocket binding observed within the carbonic anhydrase II active site accommodate different 4-substituted-ureido-benzenesulfonamides and correlate to inhibitor potency

4-Substituted-ureido benzenesulfonamides showing inhibitory activity against carbonic anhydrase (CA, EC 4.2.1.1) II between 3.3-226 nM were crystallized in complex with the enzyme. Hydrophobic interactions between the scaffold of the inhibitors in different hydrophobic pockets of the enzyme were observed, explaining the diverse inhibitory range of these derivatives.     

Anti-fungal effect of berberine on <Emphasis Type="Italic">Candida albicans</Emphasis> by microcalorimetry with correspondence analysis

Using a LKB-2277 bioactivity monitor, stop-flow mode, the power–time curves of Candida albicans growth at 37 °C affected by berberine were measured. The check experiments were studied based on agar cup method to observe the inhibitory diameter and serial dilution method to determine the minimal inhibitory concentration (MIC) of berberine on C. albicans growth. By analyzing the quantitative thermogenic parameters taken from the power–time curves using correspondence analysis (CA), we could find that berberine at a low concentration (5.0 μg mL⁻¹) began to inhibit the growth of C. albicans and at a high concentration (75.0 μg mL⁻¹) completely inhibited C. albicans growth. The anti-fungal activity of berberine could also be expressed as half-inhibitory concentration IC₅₀, i.e., 50% effective in this inhibition. The value of IC₅₀ of berberine on C. albicans was 34.52 μg mL⁻¹. The inhibitory diameters all exceeded 10 mm in test range and the MIC was 500 μg mL⁻¹. Berberine had strong anti-fungal effect on C. albicans growth. This work provided an important idea of the combination of microcalorimetry and CA for the study on anti-fungal effect of berberine and other compounds. Compared with the agar cup method and serial dilution method, microcalorimetry not only offered a useful way for evaluating the bioactivity of drugs, but also provides more information about the microbial growth and all this information was significant for the synthesis and searching of antibiotics.     

Urease Inhibitory Kinetic Studies of Various Extracts and Pure Compounds from Cinnamomum Genus

Urease is an enzyme that plays a significant role in the hydrolysis of urea into carbonic acid and ammonia via the carbamic acid formation. The resultant increase in pH leads to the onset of various pathologies such as gastric cancer, urolithiasis, hepatic coma, hepatic encephalopathy, duodenal ulcers and peptic ulcers. Urease inhibitors can reduce the urea hydrolysis rate and development of various diseases. The Cinnamomum genus is used in a large number of traditional medicines. It is well established that stem bark of Cinnamomum cassia exhibits antiulcerogenic potential. The present study evaluated the inhibitory effect of seven extracts of Cinnamomum camphora, Cinnamomum verum and two pure compounds Camphene and Cuminaldehyde on urease enzyme. Kinetic studies of potential inhibitors were carried out. Methanol extract (IC₅₀ 980 µg/mL) of C. camphora and a monoterpene Camphene (IC₅₀ 0.147 µg/mL) possess significant inhibitory activity. The Lineweaver Burk plot analysis suggested the competitive inhibition by methanol extract, hexane fraction and Camphene. The Gas Chromatography-Mass Spectroscopy (GC–MS) analysis of hexane fraction revealed the contribution of various terpenes. The present study targets terpenes as a new class of inhibitors that have potential therapeutic value for further development as novel drugs.     

Enhancing the Anticancer Potential of Targeting Tumor-Associated Metalloenzymes via VEGFR Inhibition by New Triazolo[4,3-a]pyrimidinone Acyclo C-Nucleosides Multitarget Agents

The role of metalloenzymes in tumor progression had broadened their application in cancer therapy. Of these, MMPs and CAs are validated druggable targets that share some pivotal signaling pathways. The majority of MMPs or CAs inhibitors are designed as single-target agents. Despite their transient efficacy, these agents are often susceptible to resistance. This set the stage to introduce dual inhibitors of correlated MMPs and CAs. The next step is expected to target the common vital signaling nodes as well. In this regard, VEGFR-2 is central to various tumorigenesis events involving both families, especially MMP-2 and CA II. Herein, we report simultaneous inhibition of MMP-2, CA II, and VEGFR-2 via rationally designed hybrid 1,2,4-triazolo[4,3-a]pyrimidinone acyclo C-nucleosides. The promising derivatives were nanomolar inhibitors of VEGFR-2 (8; IC₅₀ = 5.89 nM, 9; IC₅₀ = 10.52 nM) and MMP-2 (8; IC₅₀ = 17.44 nM, 9; IC₅₀ = 30.93 nM) and submicromolar inhibitors of CA II (8; IC₅₀ = 0.21 µM, 9; IC₅₀ = 0.36 µM). Docking studies predicted their binding modes into the enzyme active sites and the structural determinants of activity regarding substitution and regioselectivity. MTT assay demonstrated that both compounds were 12 folds safer than doxorubicin with superior anticancer activities against three human cancers recording single-digit nanomolar IC₅₀, thus echoing their enzymatic activities. Up to our knowledge, this study introduces the first in class triazolopyrimidinone acyclo C-nucleosides VEGFR-2/MMP-2/CA II inhibitors that deserve further investigation.     

Nanosilver resonance scattering spectral method for determination of hydroxyl radical and its application

Silver nanoparticles were prepared under a microwave high-pressure condition using citric acid sodium as a reducer while the excess citrate was removed under high speed centrifugation. There is a resonance scattering (RS) peak at 470 nm for silver nanoparticles. In a pH 4.0 HAc-NaAc buffer solution, hydroxyl radicals from the Fenton reaction can oxidize silver nanoparticles to Ag , resulting in the RS intensity decreasing. The decreased RS intensity at 470 nm (△I 470 nm) is linear with respect to the concentration of H2O2 (C) in the range of 0.27-7.56 μmol/L with a detection limit of 0.23 μmol/L. Its regression equation is △I 470 nm = 24.3 C 13.8 with a correlation coefficient of 0.9959. This method was applied to screening the antioxidants with satisfactory results.     

Microcalorimetric investigation of the effect of berberine alkaloids from <Emphasis Type="Italic">Coptis chinensis</Emphasis> Franch on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> growth

The inhibitory effects of three berberine alkaloids (BAs) from rhizome of Coptis chinensis Franch, a traditional Chinese medicinal (TCM) herb, on Staphylococcus aureus growth were investigated by microcalorimetry. The power-time curves of S. aureus with and without BAs were acquired; meanwhile the extent and duration of inhibitory effects on the metabolism were evaluated by studying the growth rate constant (k), half inhibitory ratio (IC₅₀), maximum heat-output power (P _max), peak time of maximum heat-output power (t _p) and total heat production (Q _t). The value of k of S. aureus in the presence of the three BAs decreased with the increasing concentrations of BAs. Moreover, P _max was reduced and the value of t _p increased with increasing concentrations of the three drugs. The inhibitory activity varied with different drugs. The values of IC₅₀ of the three BAs are respectively, 101.4 μg/mL for berberine, 241.0 μg/mL for palmatine and 792.3 μg/mL for jateorrhizine. The sequence of antimicrobial activity of the three BAs is: berberine > palmatine > jateorrhizine. It is suggested that the functional group methylenedioxy or methoxyl at C2 on the phenyl ring could possibly improve antimicrobial activity more strongly than hydroxyl at C2 on the phenyl ring. Supported by the National Natural Science Foundation of China (Grant No. 30625042)     

A Novel α-Glucosidase Inhibitor Protein from the Rhizomes of <Emphasis Type="Italic">Zingiber ottensii</Emphasis> Valeton

The objective of this study was to investigate a new protein with α-glucosidase inhibitory activity from the rhizomes of Zingiber ottensii. With a simple salting-out technique followed by single-step anion-exchange purification, the protein was successfully purified from the rhizomes. This protein was found to have three likely sub-unit types, 32.5, 15.2, and 13.8 kDa, as revealed by native and reducing SDS-PAGE analysis. Determination of the kinetics of the inhibition of α-glucosidase from Saccharomyces cerevisiae by standard enzymatic methods indicated the maximum percent inhibition; IC₅₀ and K _i of this protein were 77.5%, 30.15 μg/ml, and 140 μmol, while the K _m and V _max were 2.35 μmol and 0.11 mM/min, respectively. The inhibitory action was pH-independent within the pH range 2–10, but was potentially affected by buffer salts, and was relatively temperature-stable between 4–35 °C, with a maximum activity at 65 °C. The amino acid sequence of an internal fragment of this purified Z. ottensii rhizomal protein had a similarity to the sequence from the plant cysteine proteinase family. Although this α-glucosidase inhibitory protein was purified from Z. ottensii rhizomes and preliminarily characterized, further studies are needed prior to firm applications being envisaged.     

Synthesis, Characterization and the Antioxidative Activity of Copper(II), Zinc(II) and Nickel(II) Complexes with Naringenin

New complexes of Cu^(II), Zn^(II) and Ni^(II) with naringenin have been synthesized and characterized on the basis of elemental analyses, molar conductivities, ¹H-n.m.r., i.r. spectra, u.v. spectra, thermal analyses, and fluorescence spectra. In addition, the suppression ratio for O₂⁻· (a) and OH· (b) of the complexes were studied by spectrophotometric methods. The results show that the effect of the Cu^(II)-complex IC₅₀ (a) = 0.003 μm, IC₅₀ (b) = 0.06 μm is the most remarkable, and the average scavenger ability of the complexes (IC₅₀=0.06–2.67μm) against OH· is higher than that of the ligand (IC₅₀ = 28.5 μm). Taken together, these results indicate that the scavenger effect can be enhanced by the formation of metal-ligand coordination complexes, and the transition-metal ions may have differential and selective roles.     

Nucleophile recognition as an alternative inhibition mode for benzoic acid based carbonic anhydrase inhibitors

A series of hydroxybenzoic acid derivatives have shown inhibitory activity against carbonic anhydrase (CA). X-ray crystallography shows that these molecules inhibit not by binding the active site metal ion but by strong hydrogen bonding to the metal-bound water nucleophile. The binding mode observed for these molecules is distinct when compared to other non-metal-binding CA inhibitors.     

Phenolics Profiling of Carpobrotus edulis (L.) N.E.Br. and Insights into Molecular Dynamics of Their Significance in Type 2 Diabetes Therapy and Its Retinopathy Complication

Adverse effects associated with synthetic drugs in diabetes therapy has prompted the search for novel natural lead compounds with little or no side effects. Effects of phenolic compounds from Carpobrotus edulis on carbohydrate-metabolizing enzymes through in vitro and in silico methods were assessed. Based on the half-maximal inhibitory concentrations (IC₅₀), the phenolic extract of the plant had significant (p < 0.05) in vitro inhibitory effect on the specific activity of alpha-amylase (0.51 mg/mL), alpha-glucosidase (0.062 mg/mL) and aldose reductase (0.75 mg/mL), compared with the reference standards (0.55, 0.72 and 7.05 mg/mL, respectively). Molecular interactions established between the 11 phenolic compounds identifiable from the HPLC chromatogram of the extract and active site residues of the enzymes revealed higher binding affinity and more structural compactness with procyanidin (−69.834 ± 6.574 kcal/mol) and 1,3-dicaffeoxyl quinic acid (−42.630 ± 4.076 kcal/mol) as potential inhibitors of alpha-amylase and alpha-glucosidase, respectively, while isorhamnetin-3-O-rutinoside (−45.398 ± 4.568 kcal/mol) and luteolin-7-O-beta-d-glucoside (−45.102 ± 4.024 kcal/mol) for aldose reductase relative to respective reference standards. Put together, the findings are suggestive of the compounds as potential constituents of C. edulis phenolic extract responsible for the significant hypoglycemic effect in vitro; hence, they could be exploited in the development of novel therapeutic agents for type-2 diabetes and its retinopathy complication.     

Antioxidant peptides from <Emphasis Type="Italic">Cicer arietinum</Emphasis> of Xinjiang,China

Four peptides of molecular weights 1.148, 4.68, 5.41, and 9.086 kDa with antioxidant activity were isolated from chickpea sprout using two-step ion-exchange chromatographic and HPLC techniques for the first time. The partial N-terminal amino acid sequences of the peptide named YZDBCM-1 (9.086 kDa) was determined as H₂N^1-15-H₂N-Ala-Ile-Thr-Cys-Gly-Arg-Val-Ser-Ala-Ala-Leu-Ala-Pro-Pro-Leu using the Edman automated sequencing apparatus, which was a new peptide rich in alanine. It was shown that the antioxidant activity of the peptide YZDBCM-1 was IC₅₀ 156.2 μg/mL (17.2 μmol/L) to the free radical of ABTS.     

Synthesis,characterization, and urease inhibitory activity of two copper(II) complexes of cyclohexanecarboxylate

The crystal structures of two copper(II) complexes of the cyclohexanecarboxylate ligand, namely [Cu(C₆H₁₁CO₂)₂(H₂O)₂]·H₂O (1) and [Cu(dpyam)₂(C₆H₁₁CO₂)](NO₃)·H₂O (2) (C₆H₁₁CO₂H = cyclohexanecarboxylic acid; dpyam = di-2-pyridylamine), have been determined by single-crystal X-ray analysis. Complex 1 contains the square-planar trans-CuO₄ chromophore, while 2 shows the square pyramidal cis-distorted octahedral CuN₄OO′ chromophore. Both complexes were found to show strong inhibitory activity against jack bean urease (IC₅₀ = 1.75 and 8.57 μM for 1 and 2, respectively), when compared with acetohydroxamic acid (IC₅₀ = 63.12 μM).