Inhibition of tyrosine protein kinases by halomethyl ketones

A chloromethyl ketone derivative of lactic acid was shown to inhibit protein phosphorylation in plasma membranes of Ehrlich ascites tumor cells [Johnson, H. J., Zimniak, A., & Racker, E. (1982) Biochemistry 21, 2984-2989]. We now show that this inhibitor as well as three halomethyl ketone derivatives of amino acids and peptides specifically inhibits tyrosine protein kinase activity in intact plasma membranes and Triton extracts of plasma membrane of A-431 tumor cells. The most effective inhibitor is a bromomethyl ketone derivative of leucine that inhibits the phosphorylation of a protein that migrates to the same position as the EGF receptor in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of phosphorylation took place in the presence or absence of added EGF, and the inhibitor did not interfere with the binding of EGF to the receptor nor with the dephosphorylation of the EGF-stimulated phosphoprotein. EGF-dependent phosphorylation in a Triton extract of plasma membranes from normal placenta was considerably less sensitive to the bromomethyl ketone derivative of leucine. The tyrosine protein kinase activity of the transformation gene product of Fujinami virus was particularly sensitive to the bromomethyl ketone derivative of leucine, while the src gene product of Rous sarcoma virus was comparatively less sensitive. The bromomethyl ketone inhibitor interfered with the phosphorylation of the EGF receptor by [gamma-32P]-8-azido-ATP but much less with the light-sensitive binding. This observation and the lack of interference with EGF binding suggest that the inhibitor interacts with the protein kinase portion of the receptor complex.     

Comparative studies of thyrotropin releasing hormone diazomethyl ketone and chloromethyl ketone analogs

Diazomethyl ketone and chloromethyl ketone analogs of thyrotropin releasing hormones have been synthesized and studied for their inhibitory effects on thyrotropin releasing hormone-induced release of radioactive ¹²⁵I-labelled hormones from the thyroid gland of eight-week old male Long-Evans rats. When Long-Evans rats were pretreated with thyrotropin releasing hormone diazomethyl ketone (TRH-DMK) or the chloromethyl ketone derivative (TRH-CMK), a dose-related inhibition of thyrotropin releasing hormone-induced ¹²⁵I release was observed which could be partially reversed by thyrotropin stimulating hormone (TSH). The diazomethyl ketone was a more effective inhibitor than the chloromethyl ketone. These compounds may act as an active-site directed antagonists whose effects are unique to the hypothalamo-pituitary-thyroid system.     

Structure-activity relationship studies of chloromethyl ketone derivatives for selective human chymase inhibitors

Based on the SAR study of a classical chloromethyl ketone derivative, Z-PheCH2Cl 1, a series of compounds were synthesized. Among all the derivatives, compound 21 was found to be a potent human chymase inhibitor with no inhibitory activity against human leukocyte cathepsin G.     

Correlation between the predicted and the observed biological activity of the symmetric and nonsymmetric cyclic urea derivatives used as HIV-1 protease inhibitors. A 3D-QSAR-CoMFA method for new antiviral drug design

The predicted inhibition constant (Ki) and the predicted inhibitor concentration (IC90) of the HIV-1 protease (HIV-1 PR) inhibitors: symmetric and nonsymmetric - benzyl, ketone, oxime, pyrazole, imidazole, and triazole cyclic urea derivatives, were obtained by the 3D-CoMFA (Comparative Molecular Field Analysis) method. The CoMFA statistical parameters: cross-validate correlation coefficient (q2), higher than 0.5, and the fitted correlation coefficient (r2), higher than 0.90 validated the predicted biological activities. The best predictions were found for the trifluoromethyl ketoxime derivative (log 1/Ki predict = 8.42), the m-pyridineCH2 pyrazole derivative (log 1/Ki predict = 9.77) and the 1,2,3 triazole derivative (log 1/Ki predict = 7.03). We attempted to design a new potent HIV-1 protease inhibitor by addition of o-benzyl to the (p-HOPhCH2) pyrazole 12f derivative inhibitor. A favorable steric area surrounded the o-benzyl, suggesting a possible new potent HIV-1 protease inhibitor.     

Synthesis and absolute configuration assignment of 5-amino-1,3,5-triphenyl-pentane-1,3-diol stereoisomers

Starting from 2,4,6-triphenylpyrylium perchlorate, 5-amino-1,3,5-triphenyl-pentane-1,3-diol stereoisomers 4 were obtained in a simple two-step synthesis: reaction with hydroxylamine, and reduction with LAH of the resulting 2-isoxazoline ketone derivative 2. The eight stereoisomers of 4 were separated in a single shot on a chiral stationary phase cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD-H). The absolute configuration of the title compounds, intermediate 2-isoxazoline ketone 2 and isoxazoline alcohol derivative 3 were determined using a combination of diastereoselective synthesis, affiliation of the sign in chemical interconversion method, and X-ray determination. 2-Isoxazoline ketone 2 enantiomers and isoxazoline alcohol 3 enantiomers were obtained by chiral HPLC on Chiralpak AD column. 2-Isoxazoline ketone 2 enantiomers can be racemized via a retro Michael addition.     

Inhibitors designed for the active site of dihydroorotase

Four new compounds have been synthesized as potential inhibitors of dihydroorotase from Escherichia coli. NMR spectroscopy was used to show that 4,6-dioxo-piperidine-2(S)-carboxylic acid (3), exists in solution as a mixture of the hydrate (7), enol (8), and enolate (9) tautomeric forms. This compound was found to be a competitive inhibitor versus dihydroorotate and thio-dihydroorotate at pH values of 7-9. The K(i) of 76 microM was lowest at pH7.0 where the ketone and hydrate forms of the inhibitor 3 predominate in solution. Compound 3 was reduced to the two diastereomeric 4-hydroxy derivatives (4 and 5) and then dehydrated to yield the alkene derivative, 1,2,3,6-tetrahydro-6-oxopyridine-2(S)-carboxylic acid (6). Compounds 4-6 were competitive inhibitors versus thio-dihydroorotate at pH 8.0 with K(i) values of 3.0, 1.6, and 2.3 mM. Dihydroorotase was unable to dehydrate the 4-hydroxy derivative 4 or 5 to the alkene 6 or catalyze the reverse reaction.     

Design and synthesis of 3-pyrrol-3-yl-3H-isobenzofuran-1-ones as inhibitors of human cytosolic phospholipase A2alpha

A series of 3-pyrrol-3-yl-3H-isobenzofuran-1-ones was synthesized and assessed for the ability to inhibit cytosolic phospholipase A(2)alpha (cPLA(2)alpha). Several of these compounds were found to be active in both a cell based assay and an isolated enzyme assay. The most potent inhibitor was the thiazolidine-2,4-dione substituted derivative 35. With IC(50)-values of 0.7 muM and 7.3 muM in the cellular and isolated enzyme assay, respectively, it possesses similar inhibitory potency as the known cPLA(2)alpha inhibitor arachidonyltrifluoromethyl ketone (AACOCF(3)). Structure-activity relationship studies revealed that the evaluated isobenzofuran-1-ones seem to exert their cellular activities not only by a direct interaction with the enzyme but also by other as yet unknown mechanisms.     

Caspases-3 and -7 are activated in goniothalamin-induced apoptosis in human Jurkat T-cells.

Goniothalamin, a plant styrylpyrone derivative isolated from Goniothalamus andersonii, induced apoptosis in Jurkat T-cells as assessed by the externalisation of phosphatidylserine. Immunoblotting showed processing of caspases-3 and -7 with the appearance of their catalytically active large subunits of 17 and 19 kDa, respectively. Activation of these caspases was further evidenced by detection of poly(ADP-ribose) polymerase cleavage (PARP). Pre-treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) blocked apoptosis and the resultant cleavage of these caspases and PARP. Our results demonstrate that activation of at least two effector caspases is a key feature of goniothalamin-induced apoptosis in Jurkat T-cells.     

Nα-formyl-norleucyl-leucyl-phenylalanyl chloromethylketone

Summary N-formyl-norleucyl-leucyl-phenylalanine-chloromethyl ketone is chemotactic for, and induces lysosomal enzyme release from rabbit peritoneal neutrophils over essentially the same range of concentrations as does the free acid form of the same peptide (Na-formyl-norleucyl-leucyl-phenylalanine-OH). The chloromethyl ketone derivative does however differ from the free acid in respect to its ability to interact with the neutrophil and cause deactivation or desensitization to cytochalasin B. Neutrophils preincubated in the cold with the chloromethyl ketone followed by washing have cytochalasin B sensitivity conferred upon them, as measured by the release of lysosomal enzymes. The degree of release induced by this pre-treatment appears to be related to the initial responsiveness of the cells. This is in contrast to the free acid where no cytochalasin B sensitivity in conferred under any circumstances. Thus, the chloromethyl ketone, unlike the free acid, appears to irreversibly activate the cell. Desensitization to the late addition of cytochalasin B is also significantly retarded when the chloromethyl ketone derivative is compared to the free acid form of the peptide. These studies suggest that the chloromethyl ketone derivative of the peptide may covalently interact with the neutrophil receptor.     

Synthesis of chloromethyl ketone derivatives of fatty acids. Their use as specific inhibitors of acetoacetyl-coenzyme A thiolase, cholesterol biosynthesis and fatty acid synthesis.

A general route for the synthesis of chloromethyl ketone derivatives of fatty acids is described. 5-Chloro-4-oxopentanoic acid, 7-chloro-6-oxoheptanoic acid, 9-chloro-8-oxononanoic acid and 11-chloro-10-oxoundecanoic acid were synthesized by this method and tested as covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The K1 decreased by approx. 20-fold for each pair of methylenes added to the chain length, showing that the initial stage in inhibitor binding occurs at a non-polar region of the protein. This region is probably located at the enzyme active site, since inhibition was prevented by acetoacetyl-CoA or acetyl-CoA but not by CoA. The site of modification by chloromethyl ketone derivatives of fatty acids is restricted to a thiol group, since inactivation of the enzyme was prevented by reversible thiomethylation of the active-site thiol. In contrast, an amino-directed reagent, citraconic anhydride, still inactivated the enzyme, even when the active-site thiol was protected. Evidence that the enzyme thiol was particularly reactive came from studies on the pH-dependence of the alkylation reaction and thiol-competition experiments. Inhibition of the enzyme proceeded suprisingly well at acidic pH values and a 10(5) molar excess of external thiol over active-site thiol was required to prevent inhibition by 0.3 mM-9-chloro-8-oxononanoic acid. In addition to inhibiting isolated acetoacetyl-CoA thiolase, in hepatocytes the chloromethyl ketone derivatives of fatty acids also inhibited chloresterol synthesis, which uses this enzyme as an early step in the biosynthetic pathway. In isolated cells, the chloromethyl ketone derivatives of fatty acids were considerably less specific in their inhibitory action compared with 3-acetylenic derivatives of fatty acids, which act as suicide inhibitors of acetoacetyl-CoA thiolase. However, 9-chloro-8-oxononanoic acid was also an effective inhibitor of both hepatic cholesterol and fatty acid synthesis in mice in vivo, whereas the acetylenic fatty acid derivative, dec-3-ynoic acid, was completely ineffective. The effective inhibitory dose of 9-chloro-8-oxononanoic acid (2.5-5 mg/kg) was substantially lower than the estimated LD50 for the inhibitor (100 mg/kg).     

Phosphorylation and dephosphorylation of the Ca2+ pump of human red cells in the presence of monovalent cations

A chloromethyl ketone derivative of pyroglutamic acid was newly synthesized and its reactivity with bacterial pyroglutamyl aminopeptidase (L-pyroglutamyl-peptide hydrolas, EC 3.4.11.8) as an affinity labelling reagent was examined. The compound was found to inactivate the enzyme markedly and rapidly at very low concentrations, though the enzyme was resistant to N-tosyl-phenylalanyl chloromethyl ketone. The rate of the enzyme inactivation by pyroglutamyl chloromethyl ketone was retarded in the presence of a poor substrate, pyroglutamyl valine. The enzyme inactivated by treating with p-chloromercuribenzoate failed to react with pyroglutamyl chloromethyl ketone. These results strongly suggest an active site-directed mechanism for the enzyme inactivation by pyroglutamyl chloromethyl ketone. This compound was shown to be useful as a titrant for the catalytically active protein of pyroglutamyl aminopeptidase.     

Inactivation of cathepsin B1 by diazomethyl ketones

Benzyloxycarbonyl-phenylalanyl diazomethyl ketone and benzyloxy-carbonyl-phenylalanyl-phenylalanyl diazomethyl ketone, which have been shown to inactivate the thiol protease papain by a mechanism different from that of substrate chloromethyl ketone derivatives, have now been examined as inhibitors of cathepsin B₁ of beef spleen. The dipeptide derivative irreversibly inactivates this protease rapidly, apparently by affinity labeling.     

17,20-lyase inhibitors. Part 4: design, synthesis and structure-activity relationships of naphthylmethylimidazole derivatives as novel 17,20-lyase inhibitors

A novel series of naphthylmethylimidazole derivatives and related compounds have been investigated as selective 17,20-lyase inhibitors. Optimization of the substituent at the 6-position on the naphthalene ring was performed to yield a methylcarbamoyl derivative, which exhibited potent inhibitory activity against human 17,20-lyase and promising selectivity (>200-fold) for 17,20-lyase over CYP3A4. Further modifications of the methylcarbamoyl derivative led to the discovery of the corresponding tricyclic compound, which showed highly potent activity against human 17,20-lyase (IC(50) 19 nM) and good selectivity (>1000-fold) for inhibition of 17,20-lyase over CYP3A4. Additional biological evaluation revealed that the tricyclic compound had potent in vivo efficacy in monkeys and favorable pharmacokinetic profiles when administered in rats. Asymmetric synthesis of the selective tricyclic inhibitor was also achieved using a chiral α-hydroxy ketone.     

Chemical synthesis and in vitro biological evaluation of a phosphorylated bisubstrate inhibitor of type 3 17beta-hydroxysteroid dehydrogenase

Type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD) catalyzes the last step in the biosynthesis of the potent androgen testosterone (T) by selectively reducing the C17 ketone of 4-androstene-3,17-dione (delta4-dione), with NADPH as cofactor. This enzyme is thus an interesting therapeutic target for androgen-sensitive diseases. Using an efficient convergent chemical approach we synthesized a phosphorylated version of the best delta4-dione/adenosine hybrid inhibitor of type 3 17beta-HSD previously reported. An appropriately protected C2' phosphorylated adenosine was first prepared and linked by esterification to the steroid delta4-dione bearing an alkyl spacer. After three deprotection steps, the phosphorylated bisubstrate inhibitor was obtained. The inhibitory potency of this compound was evaluated on homogenated HEK-293 cells overexpressing type 3 17beta-HSD and compared to the best non-phosphorylated bisubstrate inhibitor. Unexpectedly, the phosphorylated derivative was slightly less potent than the non-phosphorylated bisubstrate inhibitor of type 3 17beta-HSD. Two hypotheses are discussed to explain this result: 1) the phosphorylated adenosine moiety does not interact optimally with the cofactor-binding site and 2) the bisubstrate inhibitors, phosphorylated or not, interact only with the substrate-binding site of type 3 17beta-HSD.     

A novel ketone derivative of artemisinin biotransformed by Streptomyces griseus ATCC 13273

A novel ketone derivative of artemisinin, artemisitone-9, was produced by the biotransformation of cultured Streptomyces griseus ATCC 13273. The structure of the ketone product was fully elucidated by various spectroscopic techniques, and the mechanism of generating such novel metabolite is also discussed.     

Kinetics of inhibition of Aeromonas aminopeptidase by leucine methyl ketone derivatives

A number of methyl ketones have been prepared from l-leucine and found to be competitive inhibitors of Aeromonas aminopeptidase. These inhibitors were leucine methyl ketone (K_i 18 μm), leucine chloromethyl ketone (K_i 0.67 μm), and leucine bromomethyl ketone (K_i 0.20 μm), and the corresponding succinimido derivative (K_i 170 μm), succinamic acid derivative (K_i 6.9 μm) and phthalimido derivative (K_i 140 μm). Reversible inhibition was observed for all of the inhibitors tested, indicating that the active site of this enzyme is not alkylated or acylated by the nucleophile-sensitive components of some of the inhibitors.The chloromethyl ketones derived from l-leucine and l-phenylalanine were found to have the same relative binding constants as the substrates, l-leucinamide and l-phenylalaninamide.     

Bottromycin derivatives: Efficient chemical modifications of the ester moiety and evaluation of anti-MRSA and anti-VRE activities

Novel bottromycin derivatives were synthesized from bottromycin A₂ via a hydrazide derivative as a common intermediate. Seventeen derivatives were subjected to in vitro evaluation against drug-resistant gram-positive bacteria. Some compounds showed potent anti-MRSA and anti-VRE activity, as did bottromycin A₂. Notably, a propyl ketone derivative exhibited good antibacterial activity with excellent metabolite stability.     

Synthesis of histidine-containing dipeptide affinity-labelling agents. Relative inactivation rates of cathepsins B and L.

Peptidyl diazomethyl ketones and fluoromethyl ketones containing histidine in the C-terminal position were synthesized to determine their properties as proteinase inactivators. These were examined chiefly with derivatives of Z-Ala-His. The protection of histidine during conversion of the C-terminal residue to the diazomethyl ketone required unblocking conditions which avoid acid due to the lability of this function. This was achievable with a Cbz-imidazole derivative since aminolysis provided deblocking without disturbance of the diazomethyl ketone function. In the case of the fluoromethyl ketone synthesis using fluoroacetic anhydride (Dakin-West procedure), the desired product could be isolated without ring blocking. The Z-Ala-His products showed enhanced selectivity for inactivation of cathepsin B over L when compared to analogous dipeptide inhibitors.     

Synthesis of A 3-oxo-4(S)-amino acid analog of pepstatin. A new inhibitor of carboxyl (acid) proteases

A ketone analog of pepstatin, in which the 3S hydroxyl group is oxidized to a ketone group, has been synthesized and shown to be a potent inhibitor of pepsin. Kinetics of inhibition of pepsin provide evidence that the ketone pepstatin analog binds to pepsin differently than pepstatin. The relationship of these complexes to crystal complexes of pepstatin-carboxyl proteases is discussed.     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.