Selective cholinesterase inhibition by lanostane triterpenes from fruiting bodies of Ganoderma lucidum

Two new lanostane triterpenes, named methyl ganoderate A acetonide (1) and n-butyl ganoderate H (2), were isolated from the fruiting bodies of Ganoderma lucidum together with 16 known compounds (3-18). Extensive spectroscopic and chemical studies established the structures of these compounds as methyl 7β,15α-isopropylidenedioxy-3,11,23-trioxo-5α-lanost-8-en-26-oate (1) and n-butyl 12β-acetoxy-3β-hydroxy-7,11,15,23-tetraoxo-5α-lanost-8-en-26-oate (2). Because new compounds exhibiting specific anti-acetylcholinesterase activity are being sought as possible drug candidates for the treatment of Alzheimer's and related neurodegenerative diseases, compounds 1-18 were examined for their inhibitory activities against acetylcholinesterase and butyrylcholinesterase. All of the compounds exhibited moderate acetylcholinesterase-inhibitory activity, with IC(50) values ranging from 9.40 to 31.03μM. In contrast, none of the compounds except lucidadiol (13) and lucidenic acid N (14) exhibited butyrylcholinesterase-inhibitory activity at concentrations up to 200μM. These results indicate that these lanostane triterpenes are preferential inhibitors of acetylcholinesterase and may be suitable drug candidates.     

Quantitative structure-activity relationships for the pre-steady state acetylcholinesterase inhibition by carbamates

4-Nitrophenyl-N-substituted carbamates (1) are characterized as pseudosubstrate inhibitors of acetylcholinesterase. The first step is formation of the enzyme-inhibitor tetrahedral intermediate with the inhibition constant (Ki), the second step is formation of the carbamyl enzyme with the carbamylation constant (kc), and the third step is hydrolysis of the carbamyl enzyme with decarbamylation constant (kd). According to pre-steady state kinetics the Ki step is divided further into two steps: (1) formation of the enzyme-inhibitor complex with the dissociation constant (KS) and (2) formation of the enzyme-inhibitor tetrahedral intermediate from the complex with the equilibrium constant (k2/k-2). Since the inhibitors are protonated in pH 7.0 buffer solution, the virtual dissociation constant (KS') of the enzyme-protonated inhibitor complex can be calculated from the equation, -log KS'=-log KS-pKa + 14. The -logKS, -log KS', log k2, and log k-2 values are multiply linearly correlated with the Jave equation (log(k/k0)=rho*sigma* + deltaEs + psi pi). For -log KS'-sigma*-Es)pi-correlation, the rho* value of -0.4 indicates that the enzyme-protonated inhibitor complexes have more positive charges than the protonated inhibitors, the delta value of 0.44 suggests that the bulkily substituted inhibitors lessen the reaction due to the difficulty of the inhibitors to enter the narrow enzyme active site gorge, and the psi value of 0.27 implies that the inhibitors with hydrophobic substituents accelerate the inhibitors entering the active site gorge of the enzyme. For log k2/k-2,-sigma*-Es-pi-correlation, the rho* value of 1.1 indicates that the enzyme-protonated inhibitor tetrahedral intermediates have more negative charges than the enzyme-protonated inhibitor complexes, the delta value of 0.15 suggests that the bulkily substituted inhibitors are difficult to bind into a small acyl binding site of the enzyme, and the psi value of -0.3 implies that the inhibitors with hydrophobic substituents resist binding to the hydrophilic acyl binding site of the enzyme.     

Inhibition of ricin A-chain with pyrrolidine mimics of the oxacarbenium ion transition state

Ricin A-chain (RTA) catalyzes the hydrolytic depurination of a specific adenosine at position 4324 of 28S rRNA. Kinetic isotope effects on the hydrolysis of a small 10mer stem-tetraloop oligonucleotide substrate established the mechanism of the reaction as D(N)*A(N), involving an oxacarbenium ion intermediate in a highly dissociative transition state. An inhibitor with a protonated 1,4-dideoxy-1,4-imino-D-ribitol moiety, a 4-azasugar mimic, at the depurination site in the tetraloop of a 14mer oligonucleotide with a 5 bp duplex stem structure had previously been shown to bind to RTA with a K(d) of 480 nM, which improved to 12 nM upon addition of adenine. Second-generation stem-tetraloop inhibitors have been synthesized that incorporate a methylene bridge between the nitrogen of a 1-azasugar mimic, namely, (3S,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidine, and substituents, including phenyl, 8-aza-9-deazaadenyl, and 9-deazaadenyl groups, that mimic the activated leaving group at the transition state. The values for the dissociation constants (K(i)) for these were 99 nM for the phenyl 10mer, 163 and 94 nM for the 8-aza-9-deazaadenyl 10- and 14mers, respectively, and 280 nM for the 9-deazaadenyl 14mer. All of these compounds are among the tightest binding molecules known for RTA. A related phenyl-substituted inhibitor with a deoxyguanosine on the 5'-side of the depurination site was also synthesized on the basis of stem-loop substrate specificity studies. This molecule binds with a K(i) of 26 nM and is the tightest binding "one-piece" inhibitor. 8-Aza-9-deaza- and 9-deazaadenyl substituents provide an increased pK(a) at N7, a protonation site en route to the transition state. The binding of these inhibitors is not improved relative to the binding of their phenyl counterpart, however, suggesting that RTA might also employ protonation at N1 and N3 of the adenine moiety to activate the substrate during catalysis. Studies with methylated adenines support this argument. That the various stem-loop inhibitors have similar potencies suggests that an optimal one-piece inhibitor remains to be identified. The second-generation inhibitors described here incorporate ribose mimics missing the 2-hydroxy group. On the basis of inhibition data and substrate specificity studies, the 2'-hydroxyl group at the depurination site seems to be critical for recruitment as well as catalysis by RTA.     

Cage amines as the stopper inhibitors of cholinesterases

Cage amines 1-4 are potent peripheral anionic site-bound reversible inhibitors of both acetylcholinesterase and butyrylcholinesterase. Cage amines 1-3 are selective butyrylcholinesterase inhibitor versus acetylcholinesterase. For both enzymes, the -log K(i) values linearly correlate with the difference of substituted phenyl radius of cage amines (-log K(i)=5.4+3.4Deltagamma for acetylcholinesterase, -log K(i)=5.9+3.2Deltagamma for butyrylcholinesterase). Moreover, the relationship between the enzymes and cage amines mimics that between bottles and stoppers.     

Fluorinated aldehydes and ketones acting as quasi-substrate inhibitors of acetylcholinesterase.

1. The inhibition of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) by compounds containing trifluoromethyl-carbonyl groups was investigated and related to the effects observed with structurally similar, non-fluorinated chemicals. 2. Compounds that in aqueous solution readily form hydrates inhibit acetylcholinesterase in a time-dependent process. On the other hand non-hydrated, carbonyl-containing compounds showed rapid and reversible, time-independent enzyme inactivation when assayed under steady state conditions. 3. m-N,N,N-Trimethylammonium-acetophenone acts as a rapid and reversible, time-independent, linear competitive inhibitor of acetylcholinesterase (Ki = 5.0 . 10(-7) M). 4. The most potent enzyme inhibitor tested in this series was N,N,N,-trimethylammonium-m-trifluoroacetophenone. It gives time-dependent inhibition and the concentration which inactivates eel acetylcholinesterase to 50% of the original activity after 30 min exposure is 1.3 . 10(-8) M. The bimolecular rate constant for this reaction is 1.8 . 10(6) 1 . mol-1 . min-1. The enzyme-inhibitor complex is very stable as the inhibited enzyme after 8 days of dialysis is reactivated to 20% only. This compound represents a quasi-substrate inhibitor of acetylcholinesterase.     

Binding of the factor IX gamma-carboxyglutamic acid domain to the vitamin K-dependent gamma-glutamyl carboxylase active site induces an allosteric effect that may ensure processive carboxylation and regulate the release of carboxylated product

Propeptides of the vitamin K-dependent proteins bind to an exosite on gamma-glutamyl carboxylase; while they are bound, multiple glutamic acids in the gamma-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41). In addition, we characterized the effect of the Gla domain and carboxylation on propeptide and substrate binding. For the propeptide of factor IX (proFIX18), FIXproGla41, and carboxylated FIXproGla41, the Kd values were 50, 2.5, and 19.7 nM and the koff values were 273 x 10(-5), 9 x 10(-5), and 37 x 10(-5) s(-1), respectively. The koff of proFIX18 is reduced 3-fold by FLEEL and 9-fold by the Gla domain (residues 1-46) of FIX. The pre-steady state rate constants for carboxylation of FIXproGla41 was 0.02 s(-1) in enzyme excess and 0.016 s(-1) in substrate excess. The steady state rate in substrate excess is 4.5 x 10(-4) s(-1). These results demonstrate the following. 1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.     

Probing the peripheral anionic site of acetylcholinesterase with quantitative structure activity relationships for inhibition by biphenyl-4-acyoxylate-4'-N-Butylcarbamates

Biphenyl-4-acyoxylate-4'-N-butylcarbamates 1-8 are synthesized from 4,4'-biphenol and are characterized as the pseudosubstrate inhibitors of acetylcholinesterase. In other words, the inhibitors bind to the enzyme and react with the enzyme to form the tetrahedral intermediates for the K(i) steps, and then the tetrahedral intermediates exclude the leaving groups to form a common N-butycarbamyl enzyme intermediate for the k(c) steps. Due to a linear character of the 4,4'-biphenyl moiety, the 4'-N-butylcarbamate moieties of the inhibitors react with the Ser200 residue of the enzyme while the 4-acyoxylate moieties of the inhibitors, on the other hand, should fit in the peripheral anionic site of the enzyme, which is located at the mouth of the deep active site gorge. Thus, carbamates with varied acyl substituents at the 4-position of the biphenyl ring are good candidates for probing the quantitative structure activity relationships for the peripheral anionic site of the enzyme. The fact that the pK(i), log k(c), and log K(i) values are correlated with neither the Taft substituent constant (sigma*) nor the Taft steric constant (E(s)) indicates that the 4-acyoxylate moieties of the inhibitors are too far away from the reaction center. However, the pK(i), log k(c), and log K(i) values are linearly correlated with the Hansch hydrophobicity constant, pi. The intensity constants (psi) for these correlations are 0.16, -0.035, and 0.13, respectively. These results indicate that interactions between the 4-acyoxylate groups of the inhibitors and the peripheral anionic site of the enzyme are mainly hydrophobic ones. The correlation results are slightly improved by using the two-parameter correlations with the Taft substituent steric constant, E(s), and pi. For pK(i), log k(c), and log K(i)-E(s)-pi correlations, the psi values are 0.21, -0.021, and 0.19, respectively; the intensity constants for steric effect (delta) are 0.08, 0.022, and 0.10, respectively. Besides hydrophobic interactions, the two-parameter correlations also suggest that little steric hindrance occurs for the bulkier inhibitors to pass by the peripheral anionic site of the enzyme.     

QSAR for phospholipase A2 inhibitions by 1-acyloxy-3-N-n-octylcarbamyl-benzenes

1-Acyloxy-3-N-n-octylcarbamyl-benzenes are potent reversible competitive inhibitors of Naja mocambique mocambique phospholipase A2 with the Ki values from 9.6 to 119 microM. The pKi values are correlated to both Taft substituent constant sigma* and Hansch hydrophobicity constant pi. The pre-steady state inhibition studies indicate that the pK(S) values for the first inhibition step are linearly correlated to sigma* alone with the rho* of -0.09 for this correlation. Thus, the first inhibition step may involve the insertion of the inhibitor to hepta-coordinated Ca2+ ion of the enzyme to form the octa-coordinated Ca2+ ion of the enzyme. The log(k2/k(-2)) values for the second inhibition step are linearly correlated to pi alone, and the psi value for this correlation is 0.13. Therefore, the second step inhibition step may involve the van der Waals' interaction between the acyl group of the inhibitor and Tyr 69 of the enzyme.     

Compounds with the dioxopyrimidine cycle inhibit cholinesterases from different groups of animals

We firstly synthesized derivatives of 6-methyluracil, alloxazine, and xanthine, containing omega-tetraalkylammonium (TAA) groups at the N(1) and N(3) atoms in a pyrimidine cycle and assayed their anticholinesterase activities. Compounds with triethylpentylammoniumalkyl groups behaved as typical reversible inhibitors of acetylcholinesterase (AChE) (pI(50) 3.20-6.22) and butyrylcholinesterase (BuChE) (pI(50) 3.05-5.71). Compounds, containing two ethyl residues and a substituted benzyl fragment in the tetraalkylammonium group at N(3) atoms or two similar TAA groups at N(1) and N(3) atoms, possessed very high anticholinesterase activity. Although these compounds displayed the activity of typical irreversible AChE inhibitors (a progressive AChE inactivation; k(i) 7.6x10(8) to 3.5x10(9)M(-1)min(-1)), they were reversible inhibitors of BuChE (pI(50) 3.9-6.9). The efficiency of AChE inhibition by some of these compounds was more than 10(4) times higher than the efficiency of BuChE inhibition. Several synthesized TAA derivates of 6-methyluracil reversibly inhibited electric eel and cobra venom AChEs and horse serum BuChE. However, depending on their structure, the tested compounds possessed the time-progressing inhibition of mammalian erythrocyte AChE, typically of irreversible inhibitors. As shown upon dialysis and gel-filtration, the formed mammalian AChE-inhibitor complex was stable. Thus, a new class of highly active, selective, and irreversible inhibitors of mammalian AChE was described. In contrast to classical phosphorylating or carbamoylating AChE inhibitors, these compounds are devoid of acylating functions. Probably, these inhibitors interact with certain amino acid residues at the entrance to the active-site gorge.     

A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis

The F(0)F(1)-ATP synthase couples the functions of H(+) transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located gamma, epsilon, and c subunits. To understand the gamma subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F(1)-ATPase enzyme that uniquely fits the pre-steady state and steady state data ( Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797 ). Here we directly test the model using two single cysteine mutants, betaD380C and betaE381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved gammaCys-87. In the pre-steady state, the gamma-beta cross-linked enzyme at high Mg.ATP conditions retained the burst of hydrolysis but was not able to release P(i). These data show that the rate-limiting rotation step, k(gamma), occurs after hydrolysis and before P(i) release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the betaGlu-381 residue for proper formation of the rate-limiting transition state involving gamma subunit rotation.     

QSAR studies and pharmacophore identification for arylsubstituted cycloalkenecarboxylic acid methyl esters with affinity for the human dopamine transporter

Data from a series of 29 monoamine transport inhibitors were used to generate 2D and 3D QSAR models for their binding affinity to the human dopamine transporter (hDAT). Among the inhibitors were many non-nitrogen containing compounds. The 2D QSAR analysis resulted in the equation -logK(i)=4.00-3.93E(LUMO)-0.67E(HOMO)-3.24sigma(p), which predicted the importance of electron withdrawing groups in the aromatic moiety. However, the model failed to predict the observed poor binding of nitro-substituted compounds. In contrast, a derived 3D QSAR model was capable of predicting these more correctly.     

Hyperbolic mixed-type inhibition of acetylcholinesterase by tetracyclic thienopyrimidines

A series of tetracyclic thienopyrimidines (7–14) was prepared and investigated as inhibitors of acetylcholinesterase from Electrophorus electricus acetylcholinesterase (EeAChE), as well as human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE). A new synthetic procedure was employed for the synthesis of the angularly fused heterocycles 7–10. Among them, the presence of a tetrahydropyrido ring with a benzyl rest at the basic nitrogen was required for EeAChE inhibition. A detailed kinetic analysis of the hyperbolic mixed-type inhibition of EeAChE by 9–14 was performed. These heterocyclic compounds inhibited EeAChE with K_i values of less than 3 µM. Most α values were relatively close to 1, indicating a similar affinity of the inhibitor to the free enzyme and the enzyme-substrate complex. Inhibitor 10 displayed a rather uncompetitive pattern of inhibition (α?=?0.47) and a relatively high residual activity of a postulated ternary enzyme-substrate-inhibitor complex (β?=?0.24).     

Kinetics and structure-activity relationship studies on pregnane-type steroidal alkaloids that inhibit cholinesterases

The mechanism of inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) enzymes by 23 pregnane-type alkaloids isolated from the Sarcococca saligna was investigated. Lineweaver-Burk and Dixon plots and their secondary replots showed that the majority of these compounds, that is 1, 4, 5, 6, 9, 10, 12, 13, 15-19, and 21 were found to be noncompetitive inhibitors of both enzymes. Compounds 8, 20, 22, and 23 were determined to be uncompetitive inhibitors of BChE, while compounds 11 and 14 were found to be uncompetitive and linear mixed inhibitors of AChE, respectively. Ki values were found to be in the range of 2.65-250.0 microM against AChE and 1.63-30.0 microM against BChE. The structure-activity relationship (SAR) studies suggested that the major interaction of the enzyme-inhibitor complexes are due to hydrophobic and cation-pi interactions inside the aromatic gorge of these cholinesterases. The effects of various substituents on the activity of these compounds are also discussed in details.     

Synthesis of tricyclic analogs of stephaoxocanidine and their evaluation as acetylcholinesterase inhibitors

The synthesis of simplified analogs of the novel isoquinoline alkaloid stephaoxocanidine, carrying the oxazaphenalene ABC-ring system of the natural product, and their activity as inhibitors of the enzyme acetylcholinesterase, are reported. 5,6-Dimethoxy-7H -8-oxa-1-aza-phenalen-9-one (5) was as active as a Narcissus extract enriched in galantamine.     

Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures

Synthetic bivalent thrombin inhibitors comprise an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. Possible nonpolar interactions of the P1' and P3' residues of the linker with thrombin S1' and S3' subsites, respectively, were identified using the "Methyl Scan" method [Slon-Usakiewicz et al. (1997) Biochemistry 36, 13494-13502]. A series of inhibitors (4-tert-butylbenzenesulfonyl)-Arg-(D-pipecolic acid)-Xaa-Gly-Yaa-Gly-betaAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala- (be ta-cyclohexylalanine)-(D-Glu)-OH, in which nonpolar P1' residue Xaa or P3' residue Yaa was incorporated, were designed and improved the affinity to thrombin. Substitution of the P3' residue with D-phenylglycine or D-Phe improved the K(i) value to (9.5 +/- 0.6) x 10(-14) or 1.3 +/- 0.5 x 10(-13) M, respectively, compared to that of a reference inhibitor with Gly residues at Xaa and Yaa residues (K(i) = (2.4 +/- 0.5) x 10(-11) M). Similarly, substitution of the P1' residue with L-norleucine or L-beta-(2-thienyl)alanine lowered the K(i) values to (8.2 +/- 0.6) x 10(-14) or (5.1 +/- 0.4) x 10(-14) M, respectively. The linker Gly-Gly-Gly-betaAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvement of the K(i) values to (3.8 +/- 0.6) x 10(-14) or (1.7 +/- 0.4) x 10(-14) M, respectively. These K(i) values are equivalent to that of natural hirudin (2.2 x 10(-14) M), yet the size of the synthetic inhibitors (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with L-norleucine or L-beta-(2-thienyl)alanine at the P1' residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human alpha-thrombin. The crystal structures of these complexes were solved and refined to 2.1 A resolution. The Lys(60F) side chain of thrombin moved significantly and formed a large nonpolar S1' subsite to accommodate the bulky P1' residue.     

alpha,beta-Dehydrophenylalanine choline esters, a new class of reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase

Our goal was to design, synthesize, and evaluate new cholinesterase inhibitors. Fourteen dehydroamino acids esterified to choline and to its ternary analog were synthesized by a new method that gave a yield of 84-93%. The potency of the amino acid ester derivatives was tested by measuring K(i) values for inhibition of human red cell acetylcholinesterase and human plasma butyrylcholinesterase. The most potent compound was a choline ester of dehydrophenylalanine where the amine group of the amino acid was derivatized with a benzoyl group containing a methoxy in the 2-position, CH(3)O(C(6)H(4))CONHC(CHC(6)H(5))COOCH(2)CH(2)N(+)(CH(3))(3). This compound was a strong inhibitor of both human acetylcholinesterase and human butyrylcholinesterase, with K(i) values of 10 microM and 0.08 microM, respectively. These K(i) values are comparable to that of Rivastigmine. Docking of the most potent compound into the active site of human butyrylcholinesterase showed that the lowest energy model had two benzene rings oriented towards Trp 82 and Tyr 332 whereas the positively charged nitrogen group was stabilized by Trp 231. This orientation placed the ester group 3.89 A from the active site Ser 198, a distance too far for covalent bonding, explaining why the esters are inhibitors rather than substrates. This class of anticholinesterase agents has the potential for therapeutic utility in the treatment of disorders of the cholinergic system.     

Molecular recognition by acetylcholinesterase at the peripheral anionic site: structure-activity relationships for inhibitions by aryl carbamates

Substituted phenyl-N-butyl carbamates (1-9) are potent irreversible inhibitors of Electrophorus electricus acetylcholinesterase. Carbamates 1-9 act as the peripheral anionic site-directed irreversible inhibitors of acetylcholinesterase by the stop-time assay in the presence of a competitive inhibitor, edrophonium. Linear relationships between the logarithms of the dissociation constant of the enzyme inhibitor adduct (Ki), the inactivation constant of the enzyme-inhibitor adduct (k2), and the bimolecular inhibition constant (k(i)) for the inhibition of Electrophorus electricus acetylcholinesterase by carbamates 1-9 and the Hammett substituent constant (sigma), are observed, and the reaction constants (ps) are -1.36, 0.35 and -1.01, respectively. Therefore, the above reaction may form a positive charged enzyme-inhibitor intermediate at the peripheral anionic site of the enzyme and may follow the irreversible inactivation by a conformational change of the enzyme.     

Synthesis, characterization and inhibitory potency of two oxono and thiono analogues of phosphoramidate compounds on acetylcholinesterase

Two novel structurally related phosphoramidate compounds, 1 and 2, with likely beta-diketone system were synthesized and characterized by 1H, 13C, 31P NMR, IR spectroscopy and elemental analysis. Compound 2 exhibited a 31P NMR signal which was significantly shielded (8 ppm) relative to compound 1. Determination of human erythrocyte acetylcholinesterase (hAChE) inhibitory activity was carried out according to Ellman's modified kinetic method and the IC50 values of compounds 1 and 2 were 1.567 and 2.986 mM, respectively. The k(i) values of 1 and 2 were 1.39 to 2.65 min(-1) respectively. A comparison of the bimolecular rate constant (k(i)) and IC50 values for the irreversible inhibitors 1 and 2 revealed that the oxono analogue has greater affinity for hAChE than the thiono compound. Furthermore effects of two conventional oximes paralidoxime (A) and obidoxime (B) on reactivation of the inhibited hAChE were studied but low reactivity was shown by both the oximes.     

Synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine and its bromo-derivative as dual cholinesterase inhibitors

Alkaloids have always been a great source of cholinesterase inhibitors. Numerous studies have shown that inhibiting acetylcholinesterase as well as butyrylcholinetserase is advantageous, and have better chances of success in preclinical/ clinical settings. With the objective to discover dual cholinesterase inhibitors, herein we report synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine (1) and its bromo-derivative 2. Our study has shown that cryptolepine (1) and its 2-bromo-derivative 2 are dual inhibitors of acetylcholinesterase and butyrylcholinesterase, the enzymes which are involved in blocking the process of neurotransmission. Cryptolepine inhibits Electrophorus electricus acetylcholinesterase, recombinant human acetylcholinesterase and equine serum butyrylcholinesterase with IC₅₀ values of 267, 485 and 699 nM, respectively. The 2-bromo-derivative of cryptolepine also showed inhibition of these enzymes, with IC₅₀ values of 415, 868 and 770 nM, respectively. The kinetic studies revealed that cryptolepine inhibits human acetylcholinesterase in a non-competitive manner, with ki value of 0.88 µM. Additionally, these alkaloids were also tested against two other important pathological events of Alzheimer’s disease viz. stopping the formation of toxic amyloid-β oligomers (via inhibition of BACE-1), and increasing the amyloid-β clearance (via P-gp induction). Cryptolepine displayed potent P-gp induction activity at 100 nM, in P-gp overexpressing adenocarcinoma LS-180 cells and excellent toxicity window in LS-180 as well as in human neuroblastoma SH-SY5Y cell line. The molecular modeling studies with AChE and BChE have shown that both alkaloids were tightly packed inside the active site gorge (site 1) via multiple π-π and cation-π interactions. Both inhibitors have shown interaction with the allosteric “peripheral anionic site” via hydrophobic interactions. The ADME properties including the BBB permeability were computed for these alkaloids, and were found within the acceptable range.     

Isolation and cholinesterase-inhibition studies of sterols from Haloxylon recurvum

Haloxysterols A-D (1-4), new C-24 alkylated sterols, have been isolated from the chloroform soluble fraction of Haloxylon recurvum, along with five known sterols 5-9, which are reported for the first time from this species. Their structures were determined by means of 1D- and 2D-NMR techniques. Compounds 1-9 inhibited cholinesterase enzymes in a concentration-dependent manner with K(i) values ranging between 0.85-25.5 and 1.0-19.0 microM against acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) enzymes, respectively. Lineweaver-Burk, Dixon plots and their secondary replots indicated that compounds 1-9 are non-competitive inhibitors of both AChE and BChE enzymes.