-Disubstituted 4-Sulfamoylbenzoic Acid Derivatives as Inhibitors of Cytosolic Phospholipase A: Synthesis, Aqueous Solubility, and Activity in a Vesicle and a Whole Blood Assay.

Background: Cytosolic phospholipase A2α (cPLA) is the key enzyme that initiates the arachidonic acid cascade through which pro-inflammatory lipid mediators can be formed. Therefore, cPLA is considered an interesting target for the development of anti-inflammatory drugs. Although several effective inhibitors of the enzyme have been developed, none of them has yet reached clinical application.

1-Benzylindoles as inhibitors of cytosolic phospholipase Aα: synthesis, biological activity, aqueous solubility, and cell permeability.

Cytosolic phospholipase Aα (cPLAα) is considered an interesting target for the development of new anti-inflammatory drugs, as it is significantly involved in the formation of pro-inflammatory lipid mediators. Recently, in a ligand-based virtual screening approach, 2,4-dichlorobenzyl-substituted 4-[2-(indol-3-ylmethylene)hydrazineyl]benzoic acid 7 was found to be an inhibitor of cPLAα with micromolar activity. This compound has now been systematically varied to increase inhibitory potency.

ω-(5-Phenyl-2H-tetrazol-2-yl)alkyl-substituted glycine amides and related compounds as inhibitors of the amine oxidase vascular adhesion protein-1 (VAP-1).

Vascular adhesion protein-1 (VAP-1), also known as plasma amine oxidase or semicarbazide-sensitive amine oxidase, is an enzyme that degrades primary amines to aldehydes with the formation of hydrogen peroxide and ammonia. Among others, it plays a role in inflammatory processes as it can mediate the migration of leukocytes from the blood to the inflamed tissue. We prepared a series of ω-(5-phenyl-2H-tetrazol-2-yl)alkyl-substituted glycine amides and related compounds and tested them for inhibition of purified bovine plasma VAP-1.

Synthesis, activity and metabolic stability of propan-2-one substituted tetrazolylalkanoic acids as dual inhibitors of cytosolic phospholipase Aα and fatty acid amide hydrolase.

The serine hydrolases cytosolic phospholipase Aα (cPLAα) and fatty acid amide hydrolase (FAAH) are interesting targets for the development of new anti-inflammatory and analgesic drugs. Structural modifications of a potent dual inhibitor with a propan-2-one substituted tetrazolylpropionic acid moiety led to compounds with also nanomolar activity against both enzymes but better physicochemical properties. The structure-activity relationships showed that the variations had partially divergent effects on the inhibitory activity of the compounds towards cPLAα and FAAH reflecting differences in the binding mode to the enzymes.

Synthesis, activity, metabolic stability and cell permeability of new cytosolic phospholipase Aα inhibitors with 1-indolyl-3-phenoxypropan-2-one structure.

Cytosolic phospholipase Aα (cPLAα), the key enzyme of the arachidonic acid cascade, is considered to be an interesting target for the development of new anti-inflammatory drugs. Potent inhibitors of the enzyme include indole-5-carboxylic acids with propan-2-one residues in position 1 of the indole. Previously, it was found that central pharmacophoric elements of these compounds are their ketone and carboxylic acid groups, which unfortunately are subject to pronounced metabolism by carbonyl reductases and glucuronosyltransferases, respectively.

HPLC fluorescence assay for measuring the activity of NAPE-PLD and the action of inhibitors affecting this enzyme.

N-Acyl phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) is the major enzyme for the biosynthesis of the endocannabinoid anandamide. The role of NAPE-PLD in various physiological and pathophysiological conditions is currently under investigation. For example, the enzyme might be involved in the control of neuronal activity, embryonic development and pregnancy, and prostate cancer.

Metabolism of sumatriptan revisited.

Scientific literature describes that sumatriptan is metabolized by oxidative deamination of its dimethylaminoethyl residue by monoamine oxidase A (MAO A) and not by cytochrome P450 (CYP)-mediated demethylation, as is usual for such structural elements. Using recombinant human enzymes and HPLC-MS analysis, we found that CYP enzymes may also be involved in the metabolism of sumatriptan. The CYP1A2, CYP2C19, and CYP2D6 isoforms converted this drug into N-desmethyl sumatriptan, which was further demethylated to N,N-didesmethyl sumatriptan by CYP1A2 and CYP2D6.

Synthesis and pharmacokinetic properties of novel cPLAα inhibitors with 1-(carboxyalkylpyrrolyl)-3-aryloxypropan-2-one structure.

Indole-5-carboxylic acids with 3-aryloxy-2‑oxopropyl residues in position 1 have been shown to be potent inhibitors of cytosolic phospholipase Aα (cPLAα), an enzyme involved in the formation of pro-inflammatory lipid mediators. Unfortunately, in animal experiments, only very low plasma concentrations could be achieved after peroral administration of this type of compound. Since insufficient metabolic stability was suspected as the cause, structural modifications were made to optimize this property.

HPLC fluorescence assay for measuring the activity of diacylglycerol lipases and the action of inhibitors thereof.

1,2-Diacylglycerol lipases (DAGLs) are the most important enzymes for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG), and their role in various pathophysiological conditions is currently under investigation. We synthesized a new 1,2-diacylglycerol substrate for these enzymes with a fluorogenic 4-(pyren-1-yl)butanoyl residue in sn-2 position. Using the fluorescent substrate, we measured DAGL activity in rat liver S9 fraction and brain microsomes.

ω-(5-Phenyl-2H-tetrazol-2-yl)alkyl-substituted hydrazides and related compounds as inhibitors of amine oxidase copper containing 3 (AOC3).

Amine oxidase copper containing 3 (AOC3), also known as plasma amine oxidase, semicarbazide-sensitive amine oxidase, or vascular adhesion protein-1, catalyzes the oxidative deamination of primary amines to aldehydes using copper and a quinone as cofactors. Because it is involved in the transmigration of inflammatory cells through blood vessels into tissues, AOC3 is thought to play an important role in inflammatory diseases. Therefore, inhibitors of this enzyme could lead to new therapeutics for the treatment of inflammation-related diseases.

Contribution of cyclooxygenase-1-dependent prostacyclin synthesis to bradykinin-induced dermal extravasation.

Background: Non-allergic angioedema is a potentially life-threatening condition caused by accumulation of bradykinin and subsequent activation of bradykinin type 2 receptors (B2). Since COX activity plays a pivotal role in B2 signaling, the aim of this study was to determine which prostaglandins are the key mediators and which COX, COX-1 or COX-2, is predominantly involved.

Methods: We used Miles assays to assess the effects of inhibitors of COX, 5-lipoxygenase, epoxyeicosatrienoic acid generation, cytosolic phospholipase Aα and a variety of prostaglandin receptor antagonists on bradykinin-induced dermal extravasation in C57BL/6 and COX-1-deficient mice (COX-1).

Hexafluoroisopropyl Carbamates as Selective MAGL and Dual MAGL/FAAH Inhibitors: Biochemical and Physicochemical Properties.

A series of hexafluoroisopropyl carbamates with indolylalkyl- and azaindolylalkyl-substituents at the carbamate nitrogen was synthesized and evaluated for inhibition of the endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). The synthesized derivatives with butyl to heptyl spacers between the heteroaryl and the carbamate moiety were inhibitors of both enzymes. For investigated compounds in which the alkyl chain was partially incorporated into a piperidine ring, different results were obtained.

Aryl -[ω-(6-Fluoroindol-1-yl)alkyl]carbamates as Inhibitors of Fatty Acid Amide Hydrolase, Monoacylglycerol Lipase, and Butyrylcholinesterase: Structure-Activity Relationships and Hydrolytic Stability.

A series of aryl -[ω-(6-fluoroindol-1-yl)alkyl]carbamates with alkyl spacers of varying lengths between the indole and the carbamate group and with differently substituted aryl moieties at the carbamate oxygen were synthesized and tested for inhibition of the pharmacologically interesting serine hydrolases fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), butyrylcholinesterase (BuChE), and acetylcholinesterase (AChE). Furthermore, the chemical stability in an aqueous solution and the metabolic stability toward esterases in porcine liver homogenate and porcine blood plasma were determined. While most of the synthesized derivatives were potent inhibitors of FAAH, a considerable inhibition of MAGL and BuChE was elicited only by compounds with a high carbamate reactivity, as evidenced by a significant hydrolysis of these compounds in an aqueous solution.

Evaluation of inhibitors of the arachidonic acid cascade with intact platelets using an on-line dilution and on-line solid phase extraction HPLC-MS method.

An automatic on-line dilution/on-line solid phase extraction (SPE) system has been developed for the detection of metabolites of the arachidonic acid cascade in platelets. The method allows the direct injection of larger quantities of centrifugates from cell suspensions previously treated with an equal volume of an acetonitrile/methanol mixture for protein precipitation. The method was used to study the effect of inhibitors of platelet arachidonic acid cascade enzymes (cytosolic phospholipase Aα, cyclooxygenase-1, thromboxane synthase, 12-lipoxygenase) and related targets (cyclooxygenase-2, microsomal prostaglandin E synthase-1, 5-lipoxygenase) in intact platelets after stimulation with calcium ionophore A23187.

Acylated 1-1,2,4-Triazol-5-amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action.

We herein report the conventional and microscale parallel synthesis of selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a 1,2,4-triazol-5-amine scaffold. Structural variations of this scaffold allowed identifying derivative , a potent 29 nM inhibitor of FXIIa, with improved selectivity over other tested serine proteases and also finding compound with 27 nM inhibitory activity toward thrombin. For the first time, acylated 1,2,4-triazol-5-amines were proved to have anticoagulant properties and the ability to affect thrombin- and cancer-cell-induced platelet aggregation.

HPLC-UV assay for the evaluation of inhibitors of plasma amine oxidase using crude bovine plasma.

Recently, we have described a method for evaluation of plasma amine oxidase (PAO) inhibitors, which monitors the formation of 6-(5-phenyl-2H-tetrazol-2-yl)hexanal from the corresponding amine substrate by HPLC with UV-detection using purified bovine PAO. We now investigated, whether crude bovine plasma can be used as enzyme source in this assay instead of the purified enzyme. With the aid of specific inhibitors, it was ensured that there was no detectable activity of other important amine oxidases in the plasma, namely monoamine oxidase (MAO) A and B and diamine oxidase (DAO).

Tetrazolylpropan-2-ones as inhibitors of fatty acid amide hydrolase: Studies on structure-activity relationships and metabolic stability.

A series of derivatives of 1-(4-octylphenoxy)-3-(2H-tetrazol-2-yl)propan-2-one (3) and 1-(4-octylphenoxy)-3-(1H-tetrazol-1-yl)propan-2-one (4) was synthesized and tested for fatty acid amide hydrolase (FAAH) inhibitory potency and phase I metabolic stability. Introduction of certain substituents like 4-chlorophenyl, 4-methoxycarbonylphenyl and carboxyl in position 5 of the tetrazole ring of 3 led to a significant increase of the metabolic stability of the scissile ketone pharmacophore, while the high activity towards FAAH was not affected markedly. In contrast, substituents in position 5 of the heterocyclic system of 4 did not have a considerable impact on the undesired ketone reduction.

HPLC-UV assays for evaluation of inhibitors of mono and diamine oxidases using novel phenyltetrazolylalkanamine substrates.

Recently, we have described an HPLC-UV assay for the evaluation of inhibitors of plasma amine oxidase (PAO) using 6-(5-phenyl-2H-tetrazol-2-yl)hexan-1-amine (4) as a new type of substrate. Now we studied, whether this compound or homologues of it can also function as substrate for related amine oxidases, namely diamine oxidase (DAO), monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B). Among these substances, 4 was converted by DAO with the highest rate.

Non-canonical Escherichia coli transcripts lacking a Shine-Dalgarno motif have very different translational efficiencies and do not form a coherent group.

Translation initiation in 50-70 % of transcripts in Escherichia coli requires base pairing between the Shine-Dalgarno (SD) motif in the mRNA and the anti-SD motif at the 3' end of the 16S rRNA. However, 30-50 % of E. coli transcripts are non-canonical and are not preceded by an SD motif.

(Indolylalkyl)piperidine carbamates as inhibitors of fatty acid amide hydrolase (FAAH).

A series of phenyl 4-[(indol-1-yl)alkyl]piperidine carbamates was synthesized and tested for inhibition of the endocannabinoid degrading enzyme fatty acid amide hydrolase (FAAH) and for metabolic stability in rat liver S9 fractions and porcine blood plasma. Structure-activity relationship studies revealed that variation of the length of the alkyl spacer connecting the indole and the piperidine heterocycle, introduction of substituents into the indole ring, replacement of the piperidine by a piperazine scaffold as well as opening of the piperidine ring system affect activity significantly. The metabolic stability of this compound class proved to be significantly higher than that of corresponding phenyl -(indol-1-ylalkyl)carbamates.

Effects of Kasugamycin on the Translatome of Escherichia coli.

It is long known that Kasugamycin inhibits translation of canonical transcripts containing a 5'-UTR with a Shine Dalgarno (SD) motif, but not that of leaderless transcripts. To gain a global overview of the influence of Kasugamycin on translation efficiencies, the changes of the translatome of Escherichia coli induced by a 10 minutes Kasugamycin treatment were quantified. The effect of Kasugamycin differed widely, 102 transcripts were at least twofold more sensitive to Kasugamycin than average, and 137 transcripts were at least twofold more resistant, and there was a more than 100-fold difference between the most resistant and the most sensitive transcript.

1-Heteroarylpropan-2-ones as inhibitors of fatty acid amide hydrolase: Studies on structure-activity relationships and metabolic stability.

The serine hydrolase fatty acid amide hydrolase (FAAH) catalyzes the degradation of the endocannabinoid anandamide, which possesses analgesic and anti-inflammatory effects. A new series of 1-heteroarylpropan-2-ones was synthesized and evaluated for FAAH inhibition. Structure-activity relationship studies revealed that 1H-benzotriazol-1-yl, 1H-7-azabenzotriazol-1-yl, 1H-tetrazol-1-yl and 2H-tetrazol-2-yl substituents have the highest impact on inhibitory potency.

Structure-activity relationship studies on 1-(2-oxopropyl)indole-5-carboxylic acids acting as inhibitors of cytosolic phospholipase Aα: Effect of substituents at the indole 3-position on activity, solubility, and metabolic stability.

Cytosolic phospholipase Aα (cPLAα) is a key enzyme in the biosynthesis of pro-inflammatory lipid mediators and therefore represents an attractive target for the development of new anti-inflammatory drugs. Recently, we have found that 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (4) is a potent inhibitor of the enzyme. In this work, we evaluate the effect of butanoyl- and hexanoyl-substituents in position 3 of the indole scaffold of this compound bearing terminal groups of varying polarity.

1-(5-Carboxyindazol-1-yl)propan-2-ones as dual inhibitors of cytosolic phospholipase Aα and fatty acid amide hydrolase: bioisosteric replacement of the carboxylic acid moiety.

Indazole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 were previously reported to be potent dual inhibitors of cytosolic phospholipase Aα (cPLAα) and fatty acid amide hydrolase (FAAH). In continuation of our structure-activity studies on cPLAα and FAAH inhibitors, a number of derivatives of these substances characterized by bioisosteric replacement of the carboxylic acid functionality by inverse amides, sulfonylamides, carbamates and ureas were prepared. The biological evaluation of the obtained compounds showed that the carboxylic acid functionality of the lead compounds is of special importance for a pronounced inhibition of cPLAα and FAAH.

HPLC-UV method for evaluation of inhibitors of plasma amine oxidase using derivatization of an aliphatic aldehyde product with TRIS.

Plasma amine oxidase (PAO), which is also designated as semicarbazide-sensitive amine oxidase (SSAO), copper-containing amine oxidase 3 (AOC3), or vascular adhesion protein-1 (VAP-1), catalyzes the oxidative deamination of primary amines to aldehydes using copper and a quinone as cofactors. Because it participates in the transmigration of inflammatory cells through the blood vessels into the tissue, PAO is attributed an important role in inflammatory diseases. Therefore, inhibitors of this enzyme could lead to new therapeutics for the treatment of inflammation-related conditions.