Chemical synthesis of an indomethacin ester prodrug and its metabolic activation by human carboxylesterase 1

It is necessary to consider the affinity of prodrugs for metabolic enzymes for efficient activation of the prodrugs in the body. Although many prodrugs have been synthesized with consideration of these chemical properties, there has been little study on the design of a structure with consideration of biological properties such as substrate recognition ability of metabolic enzymes. In this report, chemical synthesis and evaluation of indomethacin prodrugs metabolically activated by human carboxylesterase 1 (hCES1) are described. The synthesized prodrugs were subjected to hydrolysis reactions in solutions of human liver microsomes (HLM), human intestine microsomes (HIM) and hCES1, and the hydrolytic parameters were investigated to evaluate the hydrolytic rates of these prodrugs and to elucidate the substrate recognition ability of hCES1. It was found that the hydrolytic rates greatly change depending on the steric hindrance and stereochemistry of the ester in HLM, HIM and hCES1 solutions. Furthermore, in a hydrolysis reaction catalyzed by hCES1, the V_max value of n-butyl thioester with chemically high reactivity was significantly lower than that of n-butyl ester.     

Synthesis,molecular docking,and biological evaluation of 3-oxo-2-tolylhydrazinylidene-4,4,4-trifluorobutanoates bearing higher and natural alcohol moieties as new selective carboxylesterase inhibitors

To search for effective and selective inhibitors of carboxylesterase (CES), a series of 3-oxo-2-tolylhydrazinylidene-4,4,4-trifluorobutanoates bearing higher or natural alcohol moieties was synthesized via pre-transesterification of ethyl trifluoroacetylacetate with alcohols to isolate transesterificated oxoesters as lithium salts, which were then subjected to azo coupling with tolyldiazonium chloride. Inhibitory activity against porcine liver CES, along with two structurally related serine hydrolases, acetylcholinesterase and butyrylcholinesterase, were investigated using enzyme kinetics and molecular docking. Kinetics studies demonstrated that the tested keto-esters are reversible and selective mixed-type CES inhibitors. Analysis of X-ray crystallographic data together with our IR and NMR spectra and QM calculations indicated that the Z-isomers were the most stable. The kinetic data were well explained by the molecular docking results of the Z-isomers, which showed specific binding of the compounds in the CES catalytic active site with carbonyl oxygen atoms in the oxyanion hole and non-specific binding outside it. Some compounds were studied as inhibitors of the main human isozymes involved in biotransformation of ester-containing drugs, hCES1 and hCES2. Esters of geraniol (3d) and adamantol (3e) proved to be highly active and selective inhibitors of hCES2, inhibiting the enzyme in the nanomolar range, whereas esters of borneol (3f) and isoborneol (3g) were more active and selective against hCES1. Computational ADMET studies revealed that all test compounds had excellent intestinal absorption, medium blood-brain barrier permeability, and low hERG liability risks. Moreover, all test compounds possessed radical-scavenging properties and low acute toxicity. Overall, the results indicate that members of this novel series of esters have the potential to be good candidates as hCES1 or hCES2 inhibitors for biomedicinal applications.     

Utility of ionic liquid for Geotrichum candidum-catalyzed synthesis of optically active alcohols

Ionic liquids have recognized as a solvent for Geotrichum candidum-catalyzed optical resolution and/or deracemization of racemic secondary alcohols, giving optically active alcohols. The immobilized Geotrichum candidum proceeded the enantioselective oxidation of alcohols, producing chiral alcohols in an ionic liquid. Further, deracemization of racemic alcohols was proceeded to give the corresponding chiral alcohols in high yield with excellent stereoselectivity by the Geotrichum candidum–NaBH₄ system in the mixture of MES buffer solution and ionic liquid.     

Purification and Some Properties of Carboxylesterase from Arthrobacter globiformis; Stereoselective Hydrolysis of Ethyl Chrysanthemate

An esterase with excellent stereoselectivity for (+)-trans-ethyl chrysanthemate was purified to homogeneity from Arthrobacter globiformis SC-6-98-28. The purified enzyme hydrolyzed a mixture of ethyl chrysanthemate isomers stereoselectively to produce (+)-trans-acid with 100% stereoisomeric purity. The apparent molecular weight of the purified enzyme was 43,000 on SDS–polyacrylamide gel electrophoresis, and 94,000 on gel filtration chromatography. The optimum conditions for the ester hydrolysis were pH 10.0 at 45°C. The purified esterase hydrolyzed short-chain fatty acid esters, but did not have detectable activity on long-chain water-insoluble fatty acid esters. The enzyme activity was inbibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride.     

Stereoselective disposition of flurbiprofen from a mutual prodrug with a histamine H2-antagonist to reduce gastrointestinal lesions in the rat

The in vitro and in vivo stereoselective hydrolysis characteristics of the mutual prodrug FP-PPA, which is a conjugate of flurbiprofen (FP) with the histamine H₂-antagonist PPA, to reduce gastrointestinal lesions induced by FP were investigated and compared with those of FP methyl ester (rac-FP-Me) and FP ethyleneglycol ester (rac-FP-EG). The rac-FP derivatives were hydrolyzed preferentially to the (+)-S-isomer in plasma and to the (−)-R-isomer in liver and small intestinal mucosa. Interestingly, in the gastric mucosa, the stereoselectivity of hydrolysis of (−)-R-FP-PPA was opposite from that of rac-FP-Me and rac-FP-EG, which suggested that the stereoselective hydrolysis of FP-PPA was helpful in reducing gastric damage induced by (+)-S-FP. However, hydrolysis of all rac-FP derivatives was found to be catalyzed by carboxylesterases in the gastric mucosa. The stereoselective disposition of FP enantiomers early after intravenous administration of rac-FP-PPA could be explained by the stereoselective formation of (−)-R-FP from rac-FP-PPA in the liver. (−)-R-FP-PPA was completely hydrolyzed to form (−)-R-FP in vivo, while 78% of (+)-S-FP-PPA was hydrolyzed to (+)-S-FP, with a corresponding decrease in the area under the curve. Twenty-five percent of (+)-S-FP-PPA might be eliminated as the intact prodrug or its metabolites other than FP. The most important bioconversion of FP-PPA occurred in plasma, and additional hydrolysis of the R-enantiomer in liver resulted in the stereoselectivity observed following both i.v. and p.o. administration. © 1996 Wiley-Liss, Inc.     

Enhanced Resolution of a Secondary Alcohol by Hydrolysis of a Bichiral Ester Catalyzed by Lipase from Candida cylindracea

The effect of a chiral centre in the acyl group on the resolution of esters prepared from a racemic alcohol was investigated. R-2-chloropropionic acid afforded a higher enantiomeric ratio than S-2-chioropropionic acid in the hydrolysis of the corresponding esters of racemic 1-phenylethanol catalyzed by Candida cylindracea lipase. Even when a mixture of esters prepared from racemic acid and racemic alcohol was used for resolution of the alcohol, a noteworthy high enantioselectivity was observed. The hydrolysis of a bichiral ester offers an amplification in the resolution of enantiomers of alcohols by the combination of a chemical diastereoselectivity and an enzymatic enantio- and diastereoselectivity.     

Stereoselective hydrolysis of xenobiotic esters by different cell lines from rat liver and hepatoma and its application to chiral prodrugs for designated growth suppression of cancer cells

Stereoselectivity in the hydrolysis of racemic ethyl 2-phenylacetate derivatives by cultured cells of noncancerous cell lines from rat liver (BRL, BRL 3A, Clone 9, and ARLJ301–3), spontaneously or oncogene transformed rat liver cell lines (ARLJ301–3TR1, Anr4, Anr9–1, and Anr13–1), and cancer cell lines from rat hepatoma (H4-II-E, McA-RH7777, and MH₁C₁) and sarcoma (XC) was studied. A strong (R)-enantiomer preference was found in the hydrolysis of ethyl 2-hydroxy- ( 2c ) and 2-methoxy-2-phenylacetate ( 3c ) by the noncancerous and oncogene-transformed cells and an (S)-enantiomer preference for ethyl N-acylphenylalaninates with all the present cell lines. These inclinations were, however, not recognized with ethyl 2-methoxy-2-phenylpropanoate and ethyl N-difluoroacetyl-or N-trifluoroacetylphenylalaninate. Moreover, the R preference for 3c was reversed in the reaction by hepatoma cells. Thus, the stereoselectivity is influenced by both structure of acyl group and species of cell lines. The hepatoma cells were considerably different from the noncancerous or oncogene-transformed cells in stereoselectivity. This fact was consistent with the order of colony formation in soft agar cultures (index of malignancy) and the resemblance in actively stained esterase patterns in gel electrophoresis. The stereoselective hydrolysis leads to cell-specific activation of anticancer prodrugs. This has been confirmed for the first time by the stereoselectivity of Anr4 and H4-II-E cells in the hydrolysis of a chiral mustard ester, bis(2-chloroethyl)aminophenyl 2-methoxy-2-phenylacetate ( 14 ) and by the difference of IC₅₀ values of (R)- and (S)- 14 against the two cell lines. © 1995 Wiley-Liss, Inc.     

In silico prediction of human carboxylesterase-1 (hCES1) metabolism combining docking analyses and MD simulations

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted in developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas little has been done to predict the hydrolytic activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES1. The study involves both docking analyses of known substrates to develop predictive models, and molecular dynamics (MD) simulations to reveal the in situ behavior of substrates and products, with particular attention being paid to the influence of their ionization state. The results emphasize some crucial properties of the hCES1 catalytic cavity, confirming that as a trend with several exceptions, hCES1 prefers substrates with relatively smaller and somewhat polar alkyl/aryl groups and larger hydrophobic acyl moieties. The docking results underline the usefulness of the hydrophobic interaction score proposed here, which allows a robust prediction of hCES1 catalysis, while the MD simulations show the different behavior of substrates and products in the enzyme cavity, suggesting in particular that basic substrates interact with the enzyme in their unprotonated form.     

Digestion in vitro of erythritol esters by rat pancreatic juice enzymes

The mechanism of the digestion of erythritol esters was determined using rat pancreatic juice and purified pancreatic lipase (EC 3.1.1.3). Conditions of hydrolysis were used that would selectively activate or inactivate nonspecific lipase or lipase. It was shown that erythritol tetraoleate was hydrolyzed by nonspecific lipase but not by lipase. The initial digestion product was a triester, predominantly erythritol-1,2,3-trioleate. Thus, nonspecific lipase preferentially hydrolyzed the ester of a primary alcohol. In contrast to the results obtained with the tetraester, lipase could remove a fatty acid from the triester but the resulting erythritol-2,3-dioleate was not hydrolyzed by lipase. The selectivity of this hydrolysis and the inability to hydrolyze the diester are attributed to the known specificity of this enzyme to act only on esters of primary alcohols. Nonspecific lipase completely hydrolyzed erythritol tetraoleate to free erythritol in a stepwise manner. The relative rates of these reactions were tetraester --> triester --> diester --> monoester --> erythritol Because of the specificity of pancreatic lipase and the lack of specificity of nonspecific lipase it is likely that this latter enzyme is the primary agent for the hydrolysis of erythritol esters in the intact animal.     

Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.     

Kinetic resolution of amino acid esters catalyzed by lipases

Racemic amino acids were resolved by lipase via hydrolysis of their esters. Lipases (Pseudomonas lipase from Amano PS, Rhizopus lipase from Serva, and porcine pancrease lipase from Sigma) could selectively hydrolyze the L-amino acid esters in aqueous solution with high reactivities and selectivities. The effect of the structural changes in the ester moiety on the stereoselectivity of the lipases was also investigated using D ,L -homophenylalanine as a model. Procedures were developed for the resolution of natural and unnatural amino acids. © 1996 Wiley-Liss, Inc.     

Formate ester formation in amide solutions

Simple aliphatic alcohols, deoxynucleosides and nucleosides undergo reaction with formamide yielding formate esters. Formate ester formation was observed to occur slowly at 100°C and more rapidly at 130°C. As expected, formate esters were hydrolyzed to the alcohol and formic acid upon heating in aqueous solution. It was proposed to study the possibility that formate esters are formed initially in amide solvents, followed by displacement of formate by dihydrogen phosphate ion to form monophosphate esters. Experiments are described which demonstrate the formation and hydrolysis of formate esters, as well as their lack of reaction with hydrogen phosphate ion. Formate esters are not intermediates in the phosphorylation of nucleosides in formamide. Their formation has been observed and such an esterification is a side reaction during the phosphorylation of nucleosides in formamide.     

Hydrolysis of fully esterified alcohols containing from one to eight hydroxyl groups by the lipolytic enzymes of rat pancreatic juice

The enzymatic hydrolysis in vitro of the esters of methanol, ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, and sucrose in which all alcohol groups were esterified with oleic acid was studied. Various preparations of rat pancreatic juice, including pure lipase, were used as the sources of enzymes. Lipase (EC 3.1.1.3) did not hydrolyze compounds that contained more than three ester groups. Compounds containing four and five ester groups were hydrolyzed by certain preparations of pancreatic juice; this activity is attributed to the enzyme, nonspecific lipase. This enzyme also hydrolyzed esters of primary alcohols. The compounds containing six (sorbitol) and eight (sucrose) ester groups were not hydrolyzed.     

Stereoselectivity of the carbonyl reduction of dolasetron in rats,dogs, and humans

The initial step in the metabolism of dolasetron or MDL 73, 147EF [(2α,6α,8α,9aβ)-octahydro-3-oxo-2,6-methano-2H-quinolizin-8-yl 1H-indol-3-carboxylate, monomethanesulfonate] is the reduction of the prochiral carbonyl group to give a chiral secondary alcohol “reduced dolasetron.” An HPLC method, using a chiral column to separate reduced dolasetron enantiomers, has been developed and used to measure enantiomers in urine of rats, dogs, and humans after dolasetron administration. In all cases, the reduction was enantioselective for the (+)-(R)-enantiomer, although the dog showed lower stereoselectivity, especially after iv administration. An approximate enantiomeric ratio (+/?) of 90:10 was found in rat and human urine. The contribution of further metabolism to this enantiomeric ratio was considered small as preliminary studies showed that oxidation of the enantiomeric alcohols by human liver microsomes demonstrated only minor stereoselectivity. Further evidence for the role of stereoselective reduction in man was obtained from in vitro studies, where dolasetron was incubated with human whole blood. The enantiomeric composition of reduced dolasetron formed in human whole blood was the same as that found in human urine after administration of dolasetron. Enantioselectivity was not due to differences in the absorption, distribution, metabolism, or excretion of enantiomers, as iv or oral administration of rac-reduced dolasetron to rats and dogs lead to the recovery, in urine, of essentially the same enantiomeric composition as the dose administered. It is fortuitous that the (+)-(R)-enantiomer is predominantly formed by carbonyl reductase, as it is the more active compound. © 1995 Wiley-Liss, Inc.     

Green synthesis of enantiopure quinoxaline alcohols using Daucus carota

Green chemistry comprises a new approach in the synthesis of biologically active compounds using biocatalysts, thus diminishing the hazards for human health and environmental pollution. Asymmetric bioreduction is one of the most widely employed strategies in chemoenzymatic synthesis to produce enantiomerically pure chiral alcohols. The present study highlights the use biocatalyst Daucus carota for selective bioreduction of quinoxaline ketones 1a‐6a to their corresponding optically pure alcohols 1b‐6b in high yields (up to 84%) and good enantioselectivity (up to 98%). The absolute configuration of the chiral product (R)‐1‐(3‐methyl 7‐nitroquinoxalin‐2‐yl) ethan‐1‐ol 2b was confirmed by X‐ray crystallography studies. The chiral R‐configuration of the products obtained was confirmed by absolute configuration studies and was assigned following anti‐Prelogs rule. Quinoxaline pharmacophores form a part of well‐known potent drug molecules; hence, the chiral products were studied for determination of their molecular properties using SwissADME property analyser. All the chiral products show no Lipinski rule violations and are expected to have good oral bioavailability. As per the molecular properties prediction studies, the compound 6b (R)‐1‐(6,7‐dichloro‐3‐ methylquinoxalin‐2‐yl) ethanol is observed to show the best physicochemical properties to be a good lead molecule. Thus, the sustainable methodology was developed, and it confirms the synthesis of novel quinoxaline chiral alcohols in a simple, inexpensive, and eco‐friendly condition using D carota.     

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).     

Isolation of a Bacillus strain producing ketone reductase with high substrate tolerance

Target reaction-oriented screening from soil samples yielded a ketone reductase-producing Bacillus sp., strain ECU0013, which exhibits excellent stereoselectivity, high substrate tolerance and is capable of regenerating the required cofactor with glucose as a co-substrate. Whole-cells catalyzed the asymmetric reduction of 2-chloro-1-phenylethanone (50 mM) to (R)-2-chloro-1-phenylethanol with a 93.3% conversion rate and 99% e.e. (enantiomeric excess). A variety of ketones were enantioselectively reduced by resting cells, giving corresponding chiral alcohols with good to excellent e.e. values. These results suggest the potential of this strain for the industrial production of chiral halogenated aromatic alcohols.     

Enantioresolution by the chiral phthalic acid method: absolute configurations of substituted benzylic alcohols

Substituted benzylic alcohols were enantioresolved by the chiral phthalic acid method as follows; 1) esterification of racemic alcohols with chiral phthalic acid, 2) separation of a diastereomeric mixture of the esters formed by HPLC on silica gel, and 3) recovery of enantiopure alcohols from the separated esters. The absolute configurations of chiral phthalic acid esters of benzylic alcohols were unambiguously determined by the X-ray crystallography using the campharsultam moiety as the internal standard of absolute configuration.     

Enzymatic Preparation of (R)-3-Hydroxy-2-Methylpropyl Butyrate by Asymmetric Hydrolysis

(R)-3-Hydroxy-2-methylpropyl butyrate was formed by asymmetric hydrolysis of the corresponding prochiral diester with lipase P (Amano) in high enantiomeric excess. Various physical and chemical reaction parameters were altered in order to optimize the stereoselectivity of the enzymatic reaction; low temperature (0d`C) combined with the application of salting-in salts or (polyhydric) alcohols turned out to be the most suitable systems providing the monobutyrate in 96% ee. Attempts towards chiral monobutyrate by enzymatic esterification of the corresponding prochiral diol were unsuccessful.     

<Emphasis Type="Italic">Pseudomonas putida</Emphasis> esterase contains a GGG(A)X-motif confering activity for the kinetic resolution of tertiary alcohols

An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni–NTA-assisted affinity chromatography, the kinetic parameters K _M and k _cat were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer–Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1.