Long-acting contraceptive agents: in vitro hydrolysis of esters of norethisterone and levonorgestrel

The hydrolysis of 108 esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) was studied in vitro using a rabbit liver preparation. Introduction of a double or triple bond into a straight-chain ester did not inhibit hydrolysis but a marked reduction in hydrolysis was produced on replacement of a methylene group by an oxygen atom. Hydrolysis was inhibited by substituents at C2 of the ester chain except in short chain esters. Cyclopropylcarboxylate and cyclobutylcarboxylate were readily hydrolysed and introduction of a furan ring into the side-chain did not affect hydrolysis. No hydrolysis occurred with a cholesteryl carbonate ester or with a pentamethyldisilyloxy ether. Forty-nine esters of levonorgestrel (13 beta-ethyl-17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one) were also studied. In general, the pattern of hydrolysis for these esters was similar to that of the norethisterone esters. However, with few exceptions the levonorgestrel esters were hydrolyzed more slowly. For those esters for which information regarding the biological activity was available, there was no correlation between the potency of the esters and their rate of hydrolysis in vitro.     

Plant sterol and stanol substrate specificity of pancreatic cholesterol esterase

Consumption of plant sterols or stanols (collectively referred to as phytosterols) and their esters results in decreased low-density lipoprotein cholesterol, which is associated with decreased atherosclerotic risk. The mechanisms by which phytosterols impart their effects, however, are incompletely characterized. The objective of the present study is to determine if pancreatic cholesterol esterase (PCE; EC 3.1.1.13), the enzyme primarily responsible for cholesterol ester hydrolysis in the digestive tract, is capable of hydrolyzing various phytosterol esters and to compare the rates of sterol ester hydrolysis in vitro. We found that PCE hydrolyzes palmitate, oleate and stearate esters of cholesterol, stigmasterol, stigmastanol and sitosterol. Furthermore, we found that the rate of hydrolysis was dependent on both the sterol and the fatty acid moieties in the following order of rates of hydrolysis: cholesterol>(sitosterol=stigmastanol)>stigmasterol; oleate>(palmitate=stearate). The addition of free phytosterols to the system did not change hydrolytic activity of PCE, while addition of palmitate, oleate or stearate increased activity. Thus, PCE may play an important but discriminatory role in vivo in the liberation of free phytosterols to compete with cholesterol for micellar solubilization and absorption.     

Inhibition of the proteolytic activity of thrombin by methyl esters of arginine-containing oligopeptides

The kinetics of thrombin-catalyzed hydrolysis of the esters of arginine-containing di- and tripeptides at pH 8.5 and the inhibition of fibrinogen-thrombin reaction by these compounds are studied. The experimentally obtained values of Km, kcat, I50 and the suggestion about the competitive character of the antithrombin action of the peptides allowed the individual kinetic parameters to be calculated. The data obtained indicate that the efficiency of the thrombin-catalyzed hydrolysis of the peptides or a degree of the retardation of the proteolytic activity of thrombin by the esters under investigation depend on the hydrophobicity of the amino acid residues located at subsides P2 and P3 of the peptides. An increase of the hydrophobicity has induced a decrease in the rate constants k2 and k3 and an increase in the inhibition power of the oligopeptides.     

Leishmanicidal activity of phenylene bridged C2 symmetric glycosyl ureides

A number of phenylene bridged C2 symmetric glycosyl uerides with ester (3a-f), alcohol (4a-c) and acid (5a-d) functionalities were prepared by addition of glycosyl amino esters with phenyl diisocyanates and their further reaction with LiAlH(4) or hydrolysis with LiOH. All the compounds were screened for their in vitro and in vivo antileishmanial activity. Most of the compounds exhibited good activity while two of the compounds 3e and 3f reduced the clinical dose of standard drug SSG.     

Hydrolysis of tocopheryl and retinyl esters by porcine carboxyl ester hydrolase is affected by their carboxylate moiety and bile acids

The objective of this study was to examine the in vitro hydrolysis of vitamin E esters (alpha-tocopheryl acetate, alpha-tocopheryl succinate and alpha-tocopheryl nicotinate) by pancreatic carboxyl ester hydrolase (CEH) at the concurrent presence of different bile acids at different concentrations. The assay was performed by measuring the amount of alpha-tocopherol released by porcine pancreatic juice upon addition to different solutions of alpha-tocopheryl esters, which were dispersed in bile acid mixed micelles at 37 degrees C, pH 7.4. The CEH activity was 10 U in the final assay, and the optimal concentration of cholate in this in vitro-system was determined to 30 mM for the hydrolysis of alpha-tocopheryl acetate. The hydrolysis of alpha-tocopheryl esters required presence of pancreatic juice and bile acids, and the results showed furthermore that the ability of pancreatic CEH towards hydrolysis of different alpha-tocopheryl esters increased with increasing lipophility, irrespective of the type or concentration of bile acid present in the assay. Likewise, retinyl palmitate was hydrolyzed at a faster rate than retinyl acetate. The structure of the bile acid influenced the rate of hydrolysis. Thus, cholate followed by glycodeoxy- and glycochenodeoxycholate were the most effective activators of CEH among the bile acids tested in this assay. The presence of gamma-tocopherol or all-trans-retinyl acetate in the assay showed a non-competitive inhibition of the hydrolysis rate of alpha-tocopheryl acetate.     

Synthesis and biological activity of abscisic acid esters

16 ABA esters including 11 new compounds were prepared by two different esterification routes. All the structures of ABA esters were confirmed by ¹H NMR, ¹³C NMR and HRMS. Their biological activity and hydrolysis stability were investigated. Fortunately, there were 15 and 9 compounds which displayed much better or nearly the same inhibition activity for rice seedling growth and Arabidopsis thaliana seed germination compared to ABA, respectively. Especially, compounds 2d and 2g showed better biological activities than ABA in the three tests. Moreover, we found that chemical hydrolysis ability of the esters in vitro had little relationship to their biological activity.     

Synthesis of heparin-like antithrombotics having perphosphorylated thrombin binding domains.

The synthesis of three heparin analogues (i.e. compounds VI-VIII) having perphosphorylated thrombin binding domains (TBDs) is reported. These compounds were tested in vitro for their antithrombin III (ATIII)-mediated anti-Xa and antithrombin activities. Conjugates VI and VIII show a remarkable increase in antithrombin activity compared to the structurally related conjugates with persulfated TBDs (i.e. compounds IV and V), whereas compound VII displays a diminished activity.     

A retinyl ester hydrolase activity intrinsic to the brush border membrane of rat small intestine.

Retinol esterified with long-chain fatty acids is a common dietary source of vitamin A. Hydrolysis of these esters in the lumen of the small intestine is required prior to absorption. Bile salt-stimulated retinyl esterase activity was present with purified rat intestinal brush border membrane, with the maximum rate of ester hydrolysis at approximately pH 8, the physiological luminal pH. Taurocholate, a trihydroxy bile salt, stimulated hydrolysis of short-chain fatty acyl retinyl esters more than hydrolysis of long-chain fatty acyl esters. Deoxycholate, a dihydroxy bile salt, primarily stimulated hydrolysis of long-chain esters. Calculated Kms of 0.74 microM for retinyl palmitate (16:0) hydrolysis and 9.6 microM for retinyl caproate (6:0) hydrolysis suggested the presence of two separate activities. Consistent with that, the activity responsible for retinyl caproate hydrolysis could be inactivated to a greater degree than retinyl palmitate hydrolysis by preincubation of the brush border membrane at 37 degrees C for extended times. Brush border membrane from animals who had undergone common duct ligation 48 h prior to tissue collection showed little ability to hydrolyze retinyl caproate but retained 70% of retinyl palmitate hydrolytic activity, compared to sham-operated controls. Thus, two distinguishable retinyl esterase activities were recovered with purified brush border membranes. One apparently originated from the pancreas, was stimulated by trihydroxy bile salts, and preferentially hydrolyzed short-chain retinyl esters, properties similar to cholesterol ester hydrolase, known to bind to the brush border. The other was intrinsic to the brush border, stimulated by both trihydroxy and dihydroxy bile salts, and preferentially hydrolyzed long-chain retinyl esters, providing the majority of activity of the brush border against dietary retinyl esters.     

Acyloxymethyl as a drug protecting group. Part 7: Tertiary sulfonamidomethyl ester prodrugs of benzylpenicillin: chemical hydrolysis and anti-bacterial activity

Tertiary sulfonamidomethyl esters of benzylpenicillin (4) were synthesised and evaluated as a new class of potential prodrugs for beta-lactam antibiotics. Their hydrolysis in aqueous buffers was studied by HPLC and reveal a U-shaped pH rate profile with a pH-independent process extending from ca. pH 2 to ca. pH 10. This pathway is characterised by kinetic data that are consistent with a unimolecular mechanism involving rate-limiting iminium ion formation and penicillinoate expulsion. Benzylpenicillin and the corresponding sulfonamide are the ultimate products detected and isolated, indicating that beta-lactam ring opening is much slower than ester hydrolysis. As expected from the high reactivity, benzylpenicillin esters (4) displayed similar in vitro antibacterial activity to benzylpenicillin itself. Compared to the benzylpenicillin derivatives, sulfonamidomethyl esters of benzoic, clofibric and valproic acids display a much higher stability, giving rise to a Br?nsted beta1g value of -0.96 and suggesting that tertiary sulfonamidomethyl esters may be useful prodrugs for carboxylic acid drugs with pKa > 4.     

Neutral and acid retinyl ester hydrolases associated with rat liver microsomes: relationships to microsomal cholesteryl ester hydrolases

We recently reported the presence of a neutral, bile salt-independent retinyl ester hydrolase (REH) activity in rat liver microsomes and showed that it was distinct from the previously studied bile salt-dependent REH and from nonspecific carboxylesterases (Harrison, E. H., and M. Z. Gad. 1989. J. Biol. Chem. 264: 17142-17147). We have now further characterized the hydrolysis of retinyl esters by liver microsomes and have compared the observed activities with those catalyzing the hydrolysis of cholesteryl esters. Microsomes and microsomal subfractions enriched in plasma membranes and endosomes catalyze the hydrolysis of retinyl esters at both neutral and acid pH. The acid and neutral REH enzyme activities can be distinguished from one another on the basis of selective inhibition by metal ions and by irreversible, active site-directed serine esterase inhibitors. The same preparations also catalyze the hydrolysis of cholesteryl esters at both acid and neutral pH. However, the enzyme(s) responsible for the neutral REH activity can be clearly responsible for the neutral REH activity can be clearly differentiated from the neutral cholesteryl ester hydrolase(s) on the basis of differential stability, sensitivity to proteolysis, and sensitivity to active site-directed reagents. These results suggest that the neutral, bile salt-independent REH is relatively specific for the hydrolysis of retinyl esters and thus may play an important physiological role in hepatic vitamin A metabolism. In contrast to the neutral hydrolases, the activities responsible for hydrolysis of retinyl esters and cholesterol esters at acid pH are similar in their responses to the treatments mentioned above. Thus, a single microsomal acid hydrolase may catalyze the hydrolysis of both types of ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in the activity of Rhizopus delemar lipase on water-soluble esters by its binding with phosphatidylcholine

Rhizopus (Rh.) delemar (ATCC 34612) C-lipase was found to exhibit a slight activity towards water-soluble esters. The hydrolytic reaction of this lipase on alpha-naphthyl acetate was competitively inhibited by the presence of olive oil or Tween 80. This finding showed that both substrates, insoluble triglyceride and water-soluble ester, were hydrolyzed at the same site on the enzyme. The activities on water-soluble esters (alpha-naphthyl acetate, beta-naphthyl acetate, methyl acetylsalicylate and Tween 80) increased on binding of lipase with phosphatidylcholine (PC), although the activity on olive oil did not change. The increase in activity on water-soluble esters was due to the increase in the Vmax for its hydrolysis. It appears that local structural change of the catalytic site on lipase occurred on binding of PC to the lipase molecule and resulted in an increase in the activity on water-soluble esters. The temperature dependence of the hydrolysis of water-soluble esters demonstrated that the activation energy was lowered on binding of PC to the lipase molecule, and this resulted in an increase in the activity.     

Nonspecific cleavage of proteins using graphene oxide

In this article, we report the intrinsic catalytic activity of graphene oxide (GO) for the nonspecific cleavage of proteins. We used bovine serum albumin (BSA) and a recombinant esterase (rEstKp) from the cold-adapted bacterium Pseudomonas mandelii as test proteins. Cleavage of BSA and rEstKp was nonspecific regarding amino acid sequence, but it exhibited dependence on temperature, time, and the amount of GO. However, cleavage of the proteins did not result in complete hydrolysis into their constituent amino acids. GO also invoked hydrolysis of p-nitrophenyl esters at moderate temperatures lower than those required for peptide hydrolysis regardless of chain length of the fatty acyl esters. Based on the results, the functional groups of GO, including alcohols, phenols, and carboxylates, can be considered as crucial roles in the GO-mediated hydrolysis of peptides and esters via general acid–base catalysis. Our findings provide novel insights into the role of GO as a carbocatalyst with nonspecific endopeptidase activity in biochemical reactions.     

The influence of esters and carboxylic acids as the N-substituent of opioids. Part 1: Benzomorphans

To investigate the effects of carboxylic ester and acid moieties as the N-substituent of opioids, a short series of racemic N-substituted normetazocines was prepared. The introduction of both groups as the normetazocine N-substituent produced compounds which displayed low potency in vitro and in vivo, with the esters displaying the greater activity. The pharmacology of the compounds is discussed with implications resulting from potential in vivo metabolic hydrolysis.     

Synthesis of stavudine amino acid ester prodrugs with broad-spectrum chemotherapeutic properties for the effective treatment of HIV/AIDS

A series of prodrugs of stavudine were synthesized in an effort to enhance spectrum of chemotherapeutic properties for the effective treatment of HIV/AIDS. The 5'-OH function of stavudine was esterified with ciprofloxacin, norfloxacin, isoniazide, pyrazinamide, piperazine and dimethylamine acetic acid. The anti-HIV-1 activity of the esters was determined in CEM cell line and stavudine ester bearing piperazine acetic acid was found to be the most potent compound with a selective index of >15,723. Stavudine prodrug bearing ciprofloxacin and norfloxacin acetic acid showed 100% inhibition against Mycobacterium tuberculosis H(37)Rv at 6.25 microg/mL. The prodrugs also exhibited antibacterial activity against 24 pathogenic bacteria. In vitro hydrolysis of the various esters in human plasma indicated that these agents were relatively stable toward plasma esterases with t(1/2) ranging from 20-240 min.     

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.     

Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease.

The hydrolysis of cholesteryl esters contained in plasma low density lipoprotein was reduced in cultured fibroblasts derived from a patient with cholesteryl ester storage disease, an inborn error of metabolism in which lysosomal acid lipase activity is deficient. While these mutant cells showed a normal ability to bind low density lipoprotein at its high affinity cell surface receptor site, to take up the bound lipoprotein through endocytosis, and to hydrolyze the protein component of the lipoprotein in lysosomes, their defective lysosomal hydrolysis of the cholesteryl ester component of the lipoprotein led to the accumulation within the cell of unhydrolyzed cholesteryl esters, the fatty acid distribution of which resembled that of plasma lipoprotein. When the cholesteryl ester storage disease cells were incubated with low density lipoprotein, the reduced rate of liberation of free cholesterol by these mutant cells was associated with a delay in the occurrence of two lipoprotein-mediated regulatory events, suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and activation of endogenous cholesteryl ester formation. In contrast to their defective hydrolysis of exogenously derived lipoprotein-bound cholesteryl esters, the choleseryl ester storage disease cells showed a normal rate of hydrolysis of cholesteryl esters that had been synthesized within the cell. These data lend support to the concept that in cultured human fibroblasts cholesteryl esters entering the cell bound to low density lipoprotein are hydrolyzed within the lysosome and that one of the functions of this intracellular organelle is to supply the cell with free cholesterol.     

Synthesis of glycosylated beta-amino hydroxamates as new class of antimalarials

Glycosylated β-amino acids (3–18, 38, 39), obtained by hydrolysis of glycosylated β-amino esters on reaction with hydroxylamine hydrochloride in presence of DIC/DCC afforded glycosyl β-amino hydroxamates (19–34, 40, 41) in fair to good yields. Compounds (19–34, 40, 41) were screened against human malarial parasite Plasmodium falciparum in vitro for their schizontocidal activity. Compounds (19, 24, 26, 28, 40 and 41) exhibited good activity at 2 μg/mL concentrations.     

Assay and inhibition of diacylglycerol lipase activity

A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-(14)C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-(14)C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-(14)C]arachidonic acid.     

In Vitro Antioxidant Profile of Phenolic Acid Derivatives

Several caffeic acid esters isolated from propolis exhibit interesting antioxidant properties, but their in vivo use is compromised by hydrolysis of the ester bond in the gastrointestinal tract. Therefore, a series of caffeic acid amides were synthesized and their in vitro antioxidant profile was determined. A series of hydroxybenzoic acids, hydroxycinnamic acids, and the synthesized caffeic acid amides were tested for both their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and microsomal lipid peroxidation-inhibiting activity. Some of the highly active antioxidants were further tested by means of electron paramagnetic resonance for their hydroxyl radical scavenging activity. Since a promising antioxidant compound should show a lipid peroxidation-inhibiting activity at micromolar level and a low cytotoxicity, the cytotoxicity of the phenolic compounds was also studied. In all the assays used, the caffeic acid anilides and the caffeic acid dopamine amide showed an interesting antioxidant activity.     

In vitro antioxidant profile of phenolic acid derivatives

Several caffeic acid esters isolated from propolis exhibit interesting antioxidant properties, but their in vivo use is compromised by hydrolysis of the ester bond in the gastrointestinal tract. Therefore, a series of caffeic acid amides were synthesized and their in vitro antioxidant profile was determined. A series of hydroxybenzoic acids, hydroxycinnamic acids, and the synthesized caffeic acid amides were tested for both their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and microsomal lipid peroxidation-inhibiting activity. Some of the highly active antioxidants were further tested by means of electron paramagnetic resonance for their hydroxyl radical scavenging activity. Since a promising antioxidant compound should show a lipid peroxidation-inhibiting activity at micromolar level and a low cytotoxicity, the cytotoxicity of the phenolic compounds was also studied. In all the assays used, the caffeic acid anilides and the caffeic acid dopamine amide showed an interesting antioxidant activity.