Substrate specificity of human matriptase-2.

Human matriptase-2 is an enzyme that belongs to the family of type II transmembrane serine proteases. So far there is a limited knowledge regarding its specificity and protein substrate(s). One of the identified natural substrates is hemojuvelin, a protein involved in the control of iron homeostasis. In this work, we describe the synthesis and evaluation of internal quenched substrates using a combinatorial approach. The iterative deconvolution of two libraries to define the specificity of matriptase-2 yielded to the identification of the substrate ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Gly-Tyr(3-NO2)-NH2 with a k(cat)/K(m) value of 4.5 × 10(5) M(-1) × s(-1), i.e. the highest specificity constant reported so far for matriptase-2.

In vitro synthesis and characterisation of three fenoterol sulfoconjugates detected in fenoterol post-administration urine samples.

Fenoterol, a fast-acting ß2-adrenergic agonist, is used in the therapy of obstructive pulmonary diseases and for the inhibition of premature labour obstetrics. Doping control for ß2-agonists, which are prohibited in sports by the World Anti-Doping Agency, is commonly performed by liquid chromatography/mass spectrometry after hydrolysis of phase II metabolites. The continuing development of analytical procedures has led to direct injection of urine samples without sample preparation becoming a viable tool. For the detection of substances without sample preparation, including hydrolysis, detailed information of the phase II metabolism of the substances is essential. In this study, human S9 fractions of different tissues and two recombinant sulfotransferases were investigated for their potential to form fenoterol sulfoconjugates, which were characterised in detail. Two mono-sulfoconjugates and one bis-sulfoconjugate were synthesised and their structures confirmed by liquid chromatography­high-resolution/high-accuracy mass spectrometry. All of the metabolites were identified as esterified phenolic compounds. Excretion studies with orally and inhalatively administered fenoterol proved the occurrence of the sulfoconjugates in vivo. Inhalatively administered fenoterol resulted in the detection of the two monosulfoconjugates in low amounts in urine due to the lower inhalation dose of fenoterol compared to the oral dose. After oral uptake of fenoterol, the two mono-sulfoconjugates and a fenoterol bis-sulfoconjugate were detected in urine. This is the first report of the bis-sulfoconjugate.

Defining the role of MRP-mediated efflux and glutathione in detoxification of oxaliplatin.

Albeit platinum complexes are widely used in cancer chemotherapy, their cellular processing has not been completely elucidated so far. In this study the effects of modulating multidrug resistance-associated protein (MRP)-mediated efflux and glutathione (GSH) depletion on the cytotoxicity of oxaliplatin were assessed in a human ileocecal colorectal adenocarcinoma cell line and its oxaliplatin-resistant variant. Upon oxaliplatin exposure, DNA platination was elevated by co-incubation with Gü83, a MRP1 and MRP2 inhibitor, but cytotoxicity was not increased. Addition of oxaliplatin did not alter the cellular GSH content. Following GSH depletion, platinum accumulation was unchanged but cytotoxicity was increased in oxaliplatin-sensitive cells. In conclusion, modulation of MRP-mediated efflux did not affect oxaliplatin cytotoxicity in the investigated cell lines. Intracellular GSH depletion seems to sensitize the cells but does not overcome resistance.

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 (ß = 0.24).

Terbutaline sulfoconjugate: characterization and urinary excretion monitored by LC/ESI-MS/MS.

Terbutaline is a fast-acting beta(2)-adrenergic agonist used in the treatment of obstructive pulmonary diseases. Doping control for beta(2)-agonists, which are forbidden in sports by the World Anti-doping Agency (WADA), is performed in screening by liquid chromatography/mass spectrometry after hydrolysis of phase-II metabolites. In this study, the mono-sulfoconjugated phase-II metabolite of terbutaline was synthesized and the chemical structure was characterized by (1)H-nuclear magnetic resonance spectrometry and high resolution/high accuracy Orbitrap mass spectrometry. The metabolite was designated as the phenolic esterified compound, which has been mentioned in most literature reports but has not been verified so far. The benzylic esterified compound was also synthesized and characterized by high-resolution/high accuracy Orbitrap mass spectrometry but was not detectable in urine samples from an excretion study performed after a single application of one terbutaline capsule (7.5 mg terbutaline sulfate salt). The phenolic sulfate of terbutaline was detected for two to four days after administration, whereas the unchanged terbutaline was detected for four to five days. A glucuronidated, disulfated or trisulfated phase-II metabolite of terbutaline was not found. The measurement of phase-II metabolites is planned to be incorporated into existing screening procedures to allow a faster sample preparation.

The use of visible absorption spectra to evaluate different color reactions for the detection of cyclic ureides.

Modifications of the Zwikker- and Parri color detection tests were investigated and compared according to their ability to distinguish between nine different barbituric acids and hydantoins. Solutions of the resulting complexes in 50% DMSO were analyzed spectrophotometrically. 350 spectra have been analyzed and criteria for their assessment have been defined. The evaluation based upon the occurrence of a peak in the visible absorption spectra, in comparison with the spectrum of the blank solution. The results were in accordance to those obtained in the visual assessment using a color palette formerly introduced. Cobalt(II) nitrate and methanolic solution of piperidine, or cyclohexylamine, respectively, were the suitable components to get unmistakable results.

Degradation of quercetin and luteolin by Eubacterium ramulus.

The degradation of the flavonol quercetin and the flavone luteolin by Eubacterium ramulus, a strict anaerobe of the human intestinal tract, was studied. Resting cells converted these flavonoids to 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid, respectively. The conversion of quercetin was accompanied by the transient formation of two intermediates, one of which was identified as taxifolin based on its specific retention time and UV and mass spectra. The structure of the second intermediate, alphitonin, was additionally elucidated by (1)H and (13)C nuclear magnetic resonance analysis. In resting-cell experiments, taxifolin in turn was converted via alphitonin to 3,4-dihydroxyphenylacetic acid. Alphitonin, which was prepared by enzymatic conversion of taxifolin and subsequent purification, was also transformed to 3,4-dihydroxyphenylacetic acid. The coenzyme-independent isomerization of taxifolin to alphitonin was catalyzed by cell extract or a partially purified enzyme preparation of E. ramulus. The degradation of luteolin by resting cells of E. ramulus resulted in the formation of the intermediate eriodictyol, which was identified by high-performance liquid chromatography and mass spectrometry analysis. The observed intermediates of quercetin and luteolin conversion suggest that the degradation pathways in E. ramulus start with an analogous reduction step followed by different enzymatic reactions depending on the additional 3-hydroxyl group present in the flavonol structure.

Aza analogues of thalidomide: synthesis and evaluation as inhibitors of tumor necrosis factor-alpha production in vitro.

A synthetic entry to derivatives of the new classes of 5-phthalimidouracils and 5-phthalimidobarbituric acids is reported. These 5-phthalimidopyrimidines as well as phthalimido-2,4-difluorobenzenes were designed as analogues of thalidomide, a well known inhibitor of TNF-alpha production. A preliminary in vitro investigation of the compounds as inhibitors of the TNF-alpha production was performed. Among the compounds of the present series, 5-ethyl-1-phenyl-5-(tetrafluorophthalimido)barbituric acid and 2-(2,4-difluorophenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione were proved to be potent inhibitors. Both compounds showed inhibitory activity in the lower micromolar range on the LPS-induced TNF-alpha production in human monocytes.

Inhibition of human chymase by 2-amino-3,1-benzoxazin-4-ones.

A series of 2-sec.amino-4H-3,1-benzoxazin-4-ones was evaluated as acyl-enzyme inhibitors of human recombinant chymase. The compounds were also assayed for inhibition of human cathepsin G, bovine chymotrypsin, and human leukocyte elastase. Introduction of an aromatic moiety into the 2-substituent resulted in strong inhibition of chymase, cathepsin G, and chymotrypsin. Extension of the N(Me)CH2Ph substituent by one methylene unit was unfavourable to inhibit these proteases. Towards chymase, 2-(N-benzyl-N-methylamino)-4H-3,1-benzoxazin-4-one (32) and 2-(N-benzyl-N-methylamino)-6-methyl-4H-3,1-benzoxazin-4-one (33) were found to exhibit Ki values of 11 and 17 nM, respectively, and form stable acyl-enzymes with half-lives of 53 and 25 min, respectively. Benzoxazinone 33 also inhibited the human chymase-catalyzed formation of angiotensin 11 from angiotensin I.

2-(diethylamino)thieno1,3oxazin-4-ones as stable inhibitors of human leukocyte elastase.

A series of 2-(diethylamino)thieno1,3oxazin-4-ones was synthesized and evaluated in vitro for inhibitory activity toward human leukocyte elastase (HLE). The Gewald thiophene synthesis was utilized to obtain several ethyl 2-aminothiophene-3-carboxylates. These precursors were subjected to a five-step route to obtain thieno2,3-d1,3oxazin-4-ones bearing various substituents at positions 5 and 6. Both thieno2,3-d and thieno3,2-d fused oxazin-4-ones possess extraordinary chemical stability, which was expressed as rate constants of the alkaline hydrolysis. The kinetic parameters of the HLE inhibition were determined. The most potent compound, 2-(diethylamino)-4H-1benzothieno2,3-d1,3oxazin-4-one, exhibited a K(i) value of 5.8 nM. 2-(Diethylamino)thieno1, 3oxazin-4-ones act as acyl-enzyme inhibitors of HLE, similar to the inhibition of serine proteases by 4H-3,1-benzoxazin-4-ones. The isosteric benzene-thiophene replacement accounts for an enhanced stability of the acyl-enzyme intermediates.

Studies on 2-benzyloxy-4H-3,1-benzoxazin-4-ones as serine protease inhibitors.

The class of 3,1-benzoxazin-4-ones includes potent inhibitors of various serine proteases. Structural investigation on three 2-benzyloxy-4H-3,1-benzoxazin-4-ones (1-3) are described with respect to their reactivity to alkaline hydrolysis. The 13C NMR data of 2-benzyloxy-5-methyl-4H-3,1-benzoxazin-4-one 3 are discussed. This peri substituted compound was subjected to a crystal structure analysis. The heterocyclic skeleton together with the carbonyl oxygen and the methyl carbon is planar, and only small angle distortions occurred. The inhibition of neutrophil serine proteases by 1-3 is reported. The different reactivity of the 5-methyl derivative 3 towards serine proteases is mainly influenced by specific interactions within the active sites. Thus, 3 was found to rapidly acylate human leukocyte proteinase 3 and exhibited a Ki value of 1.8 nM.

Novel thieno[2,3-d][1,3]oxazin-4-ones as inhibitors of human leukocyte elastase.

A series of thieno[2,3-d][1,3]oxazin-4-ones was synthesized and evaluated in vitro for inhibitory activity toward human leukocyte elastase. New synthetic routes to 2-alkoxy-, 2-alkylthio-, and 2-sec-amino-substituted derivatives are reported. This study demonstrates the versatility of 2-aminothiophenes prepared by Gewald reaction as a synthetic entry to serine protease-inhibiting, fused 1,3-oxazin-4-ones. Introduction of ethoxy, n-propoxy, and ethylthio groups at C-2 delivered the most potent inhibitors of this series with Ki values lower than 11 nM. Kinetic studies and product analyses revealed the formation of acyl-enzymes as a result of the attack of the active site serine at the carbon C-4 and subsequent deacylation. This mode of action is similar to the inhibition of serine proteases by 4H-3,1-benzoxazin-4-ones. Replacement of the benzene ring in benzoxazinones by a (substituted) thiophene led to improved hydrolytic stability and retained inhibitory potency.

Inhibition of cathepsin G by 4H-3,1-benzoxazin-4-ones.

A series of 4H-3,1-benzoxazin-4-ones is reported that inhibit the serine proteases human cathepsin G and bovine chymotrypsin. The synthesis and kinetic parameters of the alkaline hydrolysis is described. These compounds act as acyl-enzyme inhibitors of both enzymes. The reaction of cathepsin G with 2-benzylamino-4H-3,1-benzoxazin-4-one (20) was studied in detail. A partition in deacylation of the initially formed acyl-enzyme was observed, leading to the formation of 2-(3-benzylureido)benzoic acid (26) and 3-benzylquinazoline-2,4-(1H,3H)-dione (27). A 6-methyl substitution strongly increased the acylation rate of both proteases. Introduction of an aryl moiety into the 2-substituent led to compounds with Ki values towards cathepsin G in the nanomolar range. Their inhibitory potency is stronger than that of other synthetic inhibitors of cathepsin G.

[Bis((2,4-dioxo-1,2,3,4-tetrahydroquinazolin-3-yl)alkyl)disulfane and 3-(mercaptoalkyl)quinazolin-2,4(1H,3H)-dione: synthesis by ring transformation and antiviral activity. 42. Heterocyclic azines with heteroatoms in the 1- and 3- positions]. / Bis[(2,4-dioxo-1,2,3,4-tetrahydrochinazolin-3-yl)alkyl]disulfane and 3-(Mercaptoalkyl)chinazolin02,4,(1H,3H)-dione: Synthese durch Ringtransformation and antivirale Aktivität. 42. Mitteilung: Mehrcyclische Azine mit Heteroatomen in 1- and 3-Stellung.

Reaction of N-(sulfonyloxy)phtalimide derivatives 1, 2, with cystamine and homocystamine, respectively, affords bis[2,4-dioxol-1,2,3,4-tetra-hydroquinazolin-3-yl)alkyl] disulfanes [sequence: see text] 3, which could be reduced to 3-(mercaptoalkyl)quinazoline-2,4(1H,3H)-diones. 5. (3-(2-Mercaptoethyl)quinazoline-2,4(1H,3H)-dione (5a) was also obtained in a one-pot reaction from 1 or 2 and cysteamine. 2-Ethoxy-4H-3,1-benzoxazin-4-ones 4a-c were converted with cysteamine to 3-(mercaptoethyl)quinazoline-2,4-diones 5a-c by a new ringtransformation reaction. 3-(2-Mercaptoethyl)quinazoline-2,4(1H,3H)dione (5a) and the corresponding disulfane 3a were evaluated for antiviral activity in vitro. Compounds 3a and 5a showed significant antiviral activity against some DNA- and RNA-viruses (vaccinia-, herpes simplex virus type 1; influenza A virus) at concentrations that were nontoxic to the host cell cultures.

[Polycyclic azines with heteroatoms in the 1- and 3-position. 40. Synthesis of heterocyclic immunomodulators. 2. 3-Mercaptoalkylthieno(2,3-d)pyrimidine-2,4(1H,3H)-diones: synthesis and testing of immunostimulant activity]. / Mehrcyclische Azine mit Heteroatomen in 1- und 3-Stellung, 40. Mitt.; Synthese heterocyclischer Immunmodulatoren, 2. Mitt.: 3- Mercaptoalkylthieno[2,3-d]pyrimidin-2,4(1H,3H)-dione: Synthese und Prüfung auf immunstimulatorische Wirksamkeit.

A series of 3-mercaptoalkylthieno[2,3-d]pyrimidine-2,4(1H,3H)-diones 3 was prepared and their immuno-stimulating activity was examined. The title compounds were obtained conveniently by hydrolytic ring cleavage of fused thiazolo- or 1,3-thiazino-thienopyrimidines 1 under alkaline or acidic reaction conditions. The ms fragmentation of the thieno[2,3-d]pyrimidine-2,4-diones 3 is discussed. In the delayed type hypersensitivity (DTH) test some compounds 3 showed immuno-stimulating activities in the range of isoprinosine.

[Polycyclic azines with heteroatoms in the 1- and 3-position. 40. Synthesis of heterocyclic immunomodulators. 3. Synthesis of n-1 substituted 3-(2-mercaptoethyl)quinazoline-2,4-(1H,3H)-diones from bis(2-(2-aminobenzoylamino)ethyl)disulfane and testing of immunostimulant activity]. / Mehrcyclische Azine mit Heteroatomen in 1- und 3-Stellung. 41. Mitt.; Synthese heterocyclischer Immunmodulatoren. 3. Mitt.: Darstellung von N-1-substituierten 3-(2-Mercaptoethyl)chinazolin-2,4(1H,3H)-dionen über Bis[2-(2-amino-benzoylamino)ethyl]disulfane und Prüfung auf immunstimulatorische Wirksamkeit.

A 3-step synthesis, starting from substituted isatoic anhydride was used to prepare substituted 3-(2-mercaptoethyl)quinazoline-2,4(1H,3H)-diones 4. Reaction of 1 with cystamine afforded bis[2-(2-amino-benzoyl-amino)ethyl]disulfanes 2. Reaction of 2 with ethyl chloroformate and subsequent reduction of the heterocyclic disulfanes 3 gave mercaptoethylquinazoline-2,4-diones 4a-f.N-1 methyl and benzyl substituted derivatives 4b and 4c, respectively, show immuno-stimulating activity in various tests.

N-(sulfonyloxy)phthalimides and analogues are potent inactivators of serine proteases.

A series of 2-(sulfonyloxy) and 2-(acyloxy)-1H-isoindole-1,3(2H)-diones and analogous 1H-benz[de]isoquinoline-1,3(2H)-diones was prepared, and their potential to inactivate chymotrypsin was investigated. The N-(sulfonyloxy) and N-(acyloxy)phthalimides were found to be potent inactivators of chymotrypsin and related serine proteinases. For the most active compounds, N-(dansyloxy)phthalimide and N-(tosyloxy)phthalimide, the second-order rate constant of chymotrypsin inactivation was in the range of 250,000 m-1 s-1. N-(Mesyloxy)-phthalimide was the most active compound for inactivation of leukocyte elastase. It was shown that these compounds act as true suicide substrates. Enzyme-catalyzed opening of the heterocyclic ring results in the formation of an acyl-enzyme with attached O-acyl or O-sulfonylhydroxamic acid moiety. Subsequent Lossen rearrangement leads to the formation of a highly reactive isocyanate, which irreversibly modifies the target protease.