Tyrosinase inhibitory effect of benzoic acid derivatives and their structure-activity relationships

A series of benzoic acid derivatives 1–10 have been synthesised by two different methods. Compounds 1–6 were synthesised by a facile procedure for esterification using N,N’-dicyclohexylcarbodiimide (DCC) as a coupling agent, methylene chloride as a solvent system and dimethylaminopyridine (DMAP). While 7–10 were synthesised by converting benzoic acid into benzoyl chloride by treating with thionyl chloride in the presence of benzene and performing a further reaction with amine in dried benzene. The structures of all the synthesised derivatives of benzoic acid (1–10) were assigned on the basis of extensive NMR studies. All of them showed inhibitory potential against tyrosinase. Among them, compound 7 was found to be the most potent (1.09 μM) when compared with the standard tyrosinase inhibitors of kojic acid (16.67 μM) and L-mimosine (3.68 μM). Finally in this paper, we have discussed the structure–activity relationships of the synthesised molecules.     

Inhibition of Ache: Structure-Activity Relationship Among Conformational Transition of Trp84 and Bimolecular Rate Constant

Abstract

In this study the authors attempt to correlate kinetic constants for carbamylation of AChE, by a series of carbamate inhibitors, with the conformational positioning of Trp84 in transition state complexes of the same carbamates with Torpedo AChE, as obtained by computerized molecular modelling. They present evidence for changes in the distance of the carbamates from the center of the indole ring which can be correlated with the bimolecular rate constants for inhibition. As a result the greater the distance from Trp84, the smaller the bimolecular inhibition constant value, k₁ (= k₂/K_a), becomes. In conclusion, the value of the biinolecular rate constant for selected AChE inhibitors (structural changes that have been hypothesised or natural alkaloids of unknown activity) which possess similar size and rigidity, can be obtained. Under these conditions energy minimization alone seems to be sufficient even to accurately predict protein-substrate interactions that actually occur. Modelling studies also suggest that conformational re-orientation of Trp84 in the transition state could produce an overall movement of the Cys67-Cys94 loop.     

In vitro inhibitory effects of some heavy metals on human erythrocyte carbonic anhydrases

The inhibition of two human carbonic anhydrase (HCA, EC 4.2.1.1) isozymes, the cytosolic HCA I and II, with heavy metal salts of Pb(II), Co(II) and Hg(II)has been investigated. Human erythrocyte CA-I isozyme was purified with a specific activity of 920 EUmg^{? 1} and a yield of 30% and CA-II isozyme was purified with a specific activity of 8000 EUmg^{? 1} and a yield of 40% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 104-fold for HCA-I and 900-fold for HCA-II. The inhibitory effects of different heavy metals (lead, cobalt and mercury) on CA activity were determined at low concentrations using the esterase method under in vitro conditions. K_i values for these metals were calculated from Lineweaver-Burk graphs as 1.0, 3.22 and 1.45 mM for HCA-I and 0.059, 1.382 and 0.32 mM for HCA-II respectively. Lead was a noncompetitive inhibitor for HCA-I and competitive for HCA-II, cobalt was competitive for HCA-I and noncompetitive for HCA-II and mercury was uncompetitive for both HCA-I and HCA-II. Lead was the best inhibitor for both HCA-I and HCA-II.     

Inhibition of jack bean urease by p-benzoquinone: elucidation of the role of thiols and reversibility of the process

p-Benzoquinone (pBQ) was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 °C. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. The influence of the inhibitor concentration and the preincubation time on the enzyme activity was elucidated. It was found that increase in pBQ concentration resulted in a linear decrease of urease activity. The dependence of the enzyme activity on the preincubation time showed that the rate of inhibition rapidly decreased at the beginning of the process in order to achieve the constant value. The inhibition became time independent in the studied time range. This observation is characteristic of a slow binding mechanism of inhibition. The protective experiment proved that the urease active site is involved in the binding of pBQ. High effectiveness of thiol protectors against pBQ inhibition indicates the strategic role of the active site sulfhydryl group in the blocking process. There were two methods used for reactivation of pBQ-inhibited urease. The dilution of the urease-pBQ complex in urea solution did not result in a regain of enzyme activity. Alternatively, the addition of dithiothreitol into the urease-pBQ mixture caused the instant and efficient reactivation of the enzyme. The experiments showed that the nature of the urease-pBQ complex is irreversible but the application of a specific thiol reagent can release the active enzyme from the complex.     

Synthesis of N-substituted indole-2-carboxamides and investigation of their biochemical responses against free radicals

The antioxidant role of novel N-substituted indole-2-carboxamides (I2CDs) was investigated for their inhibitory effects on superoxide anion (O₂^? ) and lipid peroxidation (LP). Among the synthesized I2CDs, 3, 4, 6, 8 and 9 significantly inhibited O₂^{· ?} with an inhibition range at 70–98%. Examination of substituent effects on activity showed that both the ortho- and para-positions of the benzamide residue needs to be dichlorinated in order to get a maximum inhibitory effect on superoxide anion. In general, halogenated derivatives were found more active then the non-halogenated ones. However, none of the I2CDs had a significant inhibitory effects on the level of lipid peroxidation; only compounds 7 and 10 moderately decreased LP levels by over 50% at 10^{? 3} M concentrations.     

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable ‘enzyme-product’ complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (K_b) values between 10.23?×?10³ and 6.52?×?10³ M^?1. The binding reactions were enthalpy driven with ΔH_b values between ?50.99 and ?46.07 kJ mol^?1. From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.     

Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza

In this study we investigated the superoxide radicals scavenging effect and xanthine oxidase inhibitory activity by magnesium lithospermate B, which was originally isolated from the roots of Salvia miltiorrhiza (also named Danshen or Dansham), an important herb in Oriental medicine. Superoxide radicals were generated both in β-NADH/PMS system and xanthine/ xanthine oxidase system. Magnesium lithospermate B significantly inhibited the reduction of NBT induced by superoxide radicals with an IC₅₀ of 29.8 μg/mL and 4.06 μg/mL respectively in the two systems. Further study suggested that magnesium lithospermate B can directly inhibit xanthine oxidase and exhibits competitive inhibition. Magnesium lithospermate B was also found to have the hypouricemic activity in vivo against potassium oxonate-induced hyperuricaemia in mice. After oral administration of magnesium lithospermate B at doses of 10, 20 and 30 mg/kg, there was a significant decrease in the serum urate level when compared to the hyperuricemia control. In addition, magnesium lithospermate B significantly protected HL-60 cells from superoxide radicals-induced apoptosis in the xanthine/ xanthine oxidase reactions. This study provided evidence that magnesium lithospermate B exhibits direct superoxide radicals scavenging and xanthine oxidase inhibitory activity.     

Calpain inhibitory flavonoids isolated from Orostachys japonicus

The n-butanol (n-BuOH) fraction of Orostachys japonicus A. Berger (Crassulaceae) significantly inhibited calpain activity. Through the activity-guided isolation from the n-BuOH fraction, herbacetin 8-O-α-D-ribopyranoside (1), kaempferol (2), quercetin (3), afzelin (4), astragalin (5), isoquercetin (6) and quercitrin (7) were obtained. Their structures were determined by spectroscopic techniques. Among them, compound 3 and 5 had significant calpain inhibitory activities.     

Synthesis of monooxime-monocarbamoyl bispyridinium compounds bearing (E)-but-2-ene linker and evaluation of their reactivation activity against tabun- and paraoxon-inhibited acetylcholinesterase

Six AChE monooxime-monocarbamoyl reactivators with an (E)-but-2-ene linker were synthesized using modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by the nerve agent tabun and insecticide paraoxon was tested in vitro. The reactivation efficacies of pralidoxime, HI-6, obidoxime, K048, K075 and the newly prepared reactivators were compared. According to the results obtained, one reactivator seems to be promising against tabun-inhibited AChE and two reactivators against paraoxon-inhibited AChE. The best results were obtained for bisquaternary substances with at least one oxime group in position four.     

Purification of carbonic anhydrase from dog erythrocytes and investigation of in vitro inhibition by various compounds

The enzyme carbonic anhydrase (E.C. 4.2.1.1) has a stimulatory effect on glaucoma, an eye disease that has a risk to dogs, which are models for the human eye disease, that is similar to that in humans.

In this study, some sulfonamide derivatives, 2-(3-cyclohexene-1-carbamido)-1,3,4-thiadiazole-5-sulfonamide (CCTS), 4-(3-cyclohexene-1-carbamido) methyl-benzenesulfonamide (CCBS), 2-(9-octadecenoylamido)-1,3,4-thiadiazole-5-sulfonamide (ODTS), 2-(4,7,10-trioxa-tetradecanoylamido)-1,3,4-thiadiazole-5-sulfonamide (TDTS), and 2-(8-methoxycoumarine-3-carbamido)-1,3,4-thiadiazole-5-sulfonamide (MCTS), as well as some anionic compounds (perchlorate and chloride) and existing medicines (dorzolamide-HCl, gentamicine sulphate, tropicamide, and procaine-HCl) were assayed for their inhibition of dog carbonic anhydrase (dCA), which was purified from erythrocytes on an affinity gel of L-tyrosine-sulfonamide-Sepharose 4B. ODTS showed the highest potency amongst the synthetic compounds with IC₅₀ value 1.18 × 10^{? 5} M. Amongst the medicines tested, only dorzolamide showed inhibition with IC₅₀ value 5.05 × 10^{? 4} M. Procaine and tropicamide actually showed an activatory effect, whereas gentamicine sulfate had no significant effect. The inhibitory effects of anionic compounds such as perchlorate and chloride were also investigated; whereas perchlorate showed inhibition, chloride did not.