Reactive Michael Acceptor Research Articles

Natural sesquiterpene lactones as inhibitors of Myb-dependent gene expression: Structure–activity relationships

c-myb is a proto-oncogene encoding a transcription factor which is highly expressed in hematopoietic progenitor cells. It regulates the expression of genes important for lineage determination, cell proliferation, and differentiation. Deregulation of c-myb expression is known to be involved in the development of human tumors, especially certain types of leukemia and breast and colon cancer. The c-Myb protein has thus been identified as an interesting therapeutic target. We recently discovered that some sesquiterpene lactones suppress Myb-dependent gene expression which is a new mechanism for these natural products' potential anti-cancer activity. We developed a test system to screen compounds for inhibitory activity on Myb-inducible reporter gene activation. Using this system we have now investigated 60 sesquiterpene lactones for their capacity to inhibit c-Myb-dependent gene activation. The IC50 values were in a range between 0.7 and >30 μM. The furanoheliangolide goyazensolide and the pseudoguaianolide helenalin acetate (IC50 = 0.6 and 0.7 μM, respectively) represent the most active inhibitors of c-Myb dependent gene expression found up to present. Control measurements for cell viability (MTS assay) proved that the observed activity on c-Myb dependent gene expression is not a function of cytotoxicity/unspecific cell damage.Structure–activity relationships were investigated by a QSAR approach based on flexible alignment of the most active compounds and a common pharmacophore model. These investigations resulted in a QSAR model which indicates that the potency of inhibitory activity on c-Myb-dependent transcription does not only depend on the presence of reactive Michael-acceptor features but also on their optimal spatial arrangement in the molecule.

Synthesis, Chemical Reactivity as Michael Acceptors, and Biological Potency of Monocyclic Cyanoenones, Novel and Highly Potent Anti-inflammatory and Cytoprotective Agents

Novel monocyclic cyanoenones examined to date display unique features regarding chemical reactivity as Michael acceptors and biological potency. Remarkably, in some biological assays, the simple structure is more potent than pentacyclic triterpenoids (e.g., CDDO and bardoxolone methyl) and tricycles (e.g., TBE-31). Among monocyclic cyanoenones, 1 is a highly reactive Michael acceptor with thiol nucleophiles. Furthermore, an important feature of 1 is that its Michael addition is reversible. For the inhibition of NO production, 1 shows the highest potency. Notably, its potency is about three times higher than CDDO, whose methyl ester (bardoxolone methyl) is presently in phase III clinical trials. For the induction of NQO1, 1 also demonstrated the highest potency. These results suggest that the reactivity of these Michael acceptors is closely related to their biological potency. Interestingly, in LPS-stimulated macrophages, 1 causes apoptosis and inhibits secretion of TNF-α and IL-1β with potencies that are higher than those of bardoxolone methyl and TBE-31.

Crystallization and preliminary X-ray diffraction analysis of the protease from Southampton norovirus complexed with a Michael acceptor inhibitor.

Noroviruses are the predominant cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional parts. Here, the crystallization of the recombinant protease from the Southampton norovirus is described. Whilst the native crystals were found to diffract only to medium resolution (2.9 Å), cocrystals of an inhibitor complex diffracted X-rays to 1.7 Å resolution. The polypeptide inhibitor (Ac-EFQLQ-propenyl ethyl ester) possesses an amino-acid sequence designed to match the substrate specificity of the enzyme, but was synthesized with a reactive Michael acceptor group at the C-terminal end.

Mass Spectral Analyses of Hydroxymethylvinyl Ketone-Hemoglobin Adducts Formed after in Vivo Exposure of Sprague−Dawley Rats to 3-Butene-1,2-diol

3-Butene-1,2-diol (BDD), a known in vivo metabolite of 1,3-butadiene, is oxidized to a reactive Michael acceptor, hydroxymethylvinyl ketone (HMVK). Previously, we characterized the formation of three HMVK-amino acid monoadducts when HMVK was incubated in vitro with N-acetyl-l-cysteine, l-valinamide, and N-acetyl-l-lysine (NAL) at physiological conditions. One HMVK-NAL cyclic diadduct (cyclic diadduct 1) also formed by sequential Michael addition reactions of two HMVK molecules with the epsilon-amino group of NAL followed by enolization and cyclization. Loss of a water molecule and autoxidation convert cyclic diadduct 1 to a more stable cyclic diadduct 2. In the present study, we used multiple mass spectrometry techniques to investigate the formation of HMVK adducts with nucleophilic residues of Hb in vivo after dosing Sprague-Dawley rats with 25 and 200 mg/kg BDD. Trypsin-digested globin peptides with mass shifts consistent with the presence of HMVK monoadducts and cyclic diadducts were detected by LC/electrospray-quadrupole time-of-flight/MS with all rats given BDD. Use of matrix-assisted laser desorption ionization/Fourier transform ion cyclotron resonance provided further evidence for the formation of HMVK monoadducts and cyclic diadducts, and use of LC/MS/MS provided unequivocal evidence for adduction of HMVK with Cys125 of globin beta chains. Because BDD can also be oxidized to 1,2-dihydroxy-3,4-epoxybutane (EBD), the formation of N(2)-(2,3,4-trihydroxybutyl) (THB)-Hb adducts was also investigated in rats given BDD, and several peptides modified by THB were detected. However, because HMVK incubations with red blood cells in vitro also led to the detection of THB-Hb adducts, the THB adducts formed in vivo could be attributed to formation of HMVK, EBD, or both. Collectively, the results provide new insights into the reaction of HMVK with proteins.

Abrogation of ionizing radiation-induced G2 checkpoint and inhibition of nuclear export by Cryptocarya pyrones

G(2) checkpoint inhibitors can force cells arrested in G(2) phase by DNA damage to enter mitosis. In this manner, several G(2) checkpoint inhibitors can enhance killing of cancer cells by ionizing radiation and DNA-damaging chemotherapeutic agents, particularly in cells lacking p53 function. All G(2) checkpoint inhibitors identified to date target protein phosphorylation by inhibiting checkpoint kinases or phosphatases. Using a phenotypic cell-based assay for G(2) checkpoint inhibitors, we have screened a large collection of plant extracts and identified Z-Cryptofolione and Cryptomoscatone D2 as highly efficacious inhibitors of the G(2) checkpoint. These compounds and related pyrones also inhibit nuclear export. Leptomycin B, a potent inhibitor of Crm1-mediated nuclear export, is also a very potent G(2) checkpoint inhibitor. These compounds possess a reactive Michael acceptor site and do not appear promising as a radiosensitizing agents because they are toxic to unirradiated cells at checkpoint inhibitory concentrations. Nevertheless, the results show that inhibition of nuclear export is an alternative to checkpoint kinase inhibition for abrogating the G(2) checkpoint and they should stimulate the search for less toxic nuclear export inhibitors.

Addition of Indole to Methyl 2‐Chloro‐2‐cyclopropylideneacetate en Route to Spirocyclopropanated Analogues of Demethoxyfumitremorgine C and Tadalafil

AbstractIndole readily added across the double bond of the highly reactive Michael acceptor methyl 2‐chloro‐2‐cyclopropylideneacetate (4) to yield 2‐chloro‐2‐(3′‐indolylcyclopropyl)acetate 5 (85%) which was converted in two steps into the racemic tryptophan analogue 7 in 90% yield. This in turn was transformed by a condensation and Pictet−Spengler sequence into the spirocyclopropane analogues, 11, 13 and 15, of both the natural product (demethoxy)fumitremorgine C (3b, 3 steps, 11% yield) and the potent PDE‐5 inhibitor Tadalafil (2, 3 steps, 71% yield) as well as the original lead structure with a hydantoin backbone (1, 2 steps, 79% yield). (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Identification and characterization of novel stable deoxyguanosine and deoxyadenosine adducts of benzo[a]pyrene-7,8-quinone from reactions at physiological pH.

Benzo[a]pyrene (B[a]P) is an archetypal member of the family of polycyclic aromatic hydrocarbons (PAHs) and is a widely distributed environmental pollutant. B[a]P is known to induce cancer in animals, and B[a]P-containing complex mixtures are human carcinogens. B[a]P exerts its genotoxic and carcinogenic effects through metabolic activation forming reactive intermediates that damage DNA. DNA adduction by B[a]P is a complex phenomenon that involves the formation of both stable and unstable (depurinating) adducts. One pathway by which B[a]P can mediate genotoxicity is through the enzymatic formation of B[a]P-7,8-quinone (BPQ) from B[a]P-7,8-diol by members of the aldo-keto-reductase (AKR) family. Once formed, BPQ can act as a reactive Michael acceptor that can alkylate cellular nucleophiles including DNA and peptides. Earlier studies have reported on the formation of stable and depurinating adducts from the reaction of BPQ with DNA and nucleosides, respectively. However, the syntheses and characterization of the stable adducts from these interactions have not been addressed. In this study, the reactivity of BPQ toward 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) nucleosides under physiological pH conditions is examined. The identification and characterization of six novel BPQ-nucleoside adducts obtained from the reaction of BPQ and dG or dA in a mixture of phosphate buffer and dimethylformamide are reported. The structures of these adducts were determined by ultraviolet spectroscopy, electrospray mass spectrometry, and NMR experiments including (1)H, (13)C, two-dimensional COSY, one-dimensional NOE, ROESY, HMQC, HSQC, and HMBC. The reaction of BPQ with dG afforded four unique Michael addition products: two diastereomers of 8-N(1),9-N(2)-deoxyguanosyl-8,10-dihydroxy-9,10-dihydrobenzo[a]pyren-7(8H)-one (BPQ-dG(1,2)) and two diastereomers of 10-(N(2)-deoxyguanosyl)-9,10-dihydro-9-hydroxybenzo[a]pyrene-7,8-dione (BPQ-dG(3,4)). The BPQ-dG(1,2)( )()adducts suggest a 1,6-Michael addition reaction of dG, an oxidation of the hydroquinone to the quinone, a 1,4-Michael addition of water, and an internal cyclization. The BPQ-dG(3,4)( )()adducts suggest a 1,4-Michael addition reaction of dG, an oxidation of the hydroquinone to the quinone, and a 1,6-Michael addition of water. Under similar but extended reaction conditions, the reaction of BPQ with dA produced only one diastereomeric pair of adducts identified as 8-N(6),10-N(1)-deoxyadenosyl-8,9-dihydroxy-9,10-dihydrobenzo[a]pyren-7(8H)-one (BPQ-dA(1,2)). The BPQ-dA(1,2)( )()adducts suggest a 1,4-Michael addition reaction of dA, an oxidation of the hydroquinone to the quinone, a 1,6-Michael addition of water, and an internal cyclization. As considerable efforts have been placed in documenting the genotoxic effects of BPQ, this first report of the identification and characterization of these stable adducts of BPQ formed under physiological pH conditions is expected to contribute significantly to the area of BPQ-mediated genotoxicity and carcinogenesis.

A metabolic activation mechanism of 7H-dibenzo[c,g]carbazole via o-quinone. Part 2: covalent adducts of 7H-dibenzo[c,g]carbazole-3,4-dione with nucleic acid bases and nucleosides.

7H-dibenzo[c,g]carbazole (DBC) is a potent multispecies, multisite carcinogen present in the environment. The metabolic activation pathways of DBC are not completely known. It is hypothesized that DBC may be metabolically activated by oxidation to the reactive Michael acceptor o-quinones, which can form stable and depurinating DNA adducts. The synthesis of DBC-3,4-dione has been previously reported by this research group. In the present article, we describe the synthesis and chemical structural elucidation of nine DBC-nucleic acid adducts produced from reactions of DBC-3,4-dione with Ade, Cyt, 2'-deoxyguanosine (dGuo), 2'-deoxycytidine (dCyd), and Guo. Adducts were isolated from reaction mixtures by HPLC and analyzed using MS including elemental compositions and collision-activated dissociation (CAD), (1)H NMR, and two-dimensional chemical shift correlation spectroscopy (COSY) NMR. The adducts, 7-[3,4-dione-DBC-1-yl]-Ade, N4-[3,4-dione-DBC-1-yl]-Cyt, 5-[3,4-dione-DBC-1-yl]-Cyt, two conformational isomers of N2-[3,4-dihydroxy-DBC-1-yl]-dGuo, and two conformational isomers of N2-[3,4-dihydroxy-DBC-1-yl]-Guo, were characterized. Two adducts from reactions of DBC-3,4-dione with dCyd were identified by MS but not fully characterized by NMR due to instability of the adducts. Under similar conditions, the reactions of DBC-3,4-dione with Gua and 2'-deoxyadenosine (dAdo) did not result in an identifiable adduct. Liver DNA adducts from mice treated topically with DBC-3,4-dione (100 microg) in dimethyl sulfoxide/acetone (15/85, 100 microL) were identified with 32P-postlabeling. The major adduct chromatographically matched one of the adducts formed from livers of DBC-treated mouse (adduct 3) using identical conditions.

Hydroxymethylvinyl ketone: a reactive Michael acceptor formed by the oxidation of 3-butene-1,2-diol by cDNA-expressed human cytochrome P450s and mouse, rat, and human liver microsomes.

The metabolic fate of 3-butene-1,2-diol (BDD), a secondary metabolite of the industrial carcinogen, 1,3-butadiene, is unclear. The current study characterizes BDD oxidation to hydroxymethylvinyl ketone (HMVK), a reactive Michael acceptor. Because of its instability in aqueous medium, HMVK was trapped by conjugation with GSH, a reaction that occurred readily at physiological conditions (pH 7.4, 37 degrees C) to yield 1-hydroxy-2-keto-4-(S-glutathionyl)butane. The results show that BDD was oxidized to HMVK by mouse, rat, and human liver microsomes and by cDNA-expressed human cytochrome P450s. Eadie-Hofstee plots demonstrated biphasic kinetics of BDD oxidation with mouse and rat liver microsomes and one of three individual human liver microsomes; BDD oxidation by the other two human liver microsomal samples was best described by monophasic kinetics. Of the human P450 enzymes examined, only P450 2E1 exhibited activity at 1 mM BDD. P450 3A4 was capable of catalyzing the reaction at a high BDD (10 mM) concentration; P450 1A1, 1A2, 1B1, 2D6-Met, and 2D6-Val produced only trace amounts of HMVK-GSH whereas P450 2A6, 2C8, 2C9, and 4A11 had no detectable activity. Detection of HMVK or the HMVK-GSH conjugate was dependent on reaction time, protein, and BDD concentrations, and the presence of NADPH. Collectively, the results provide clear evidence for BDD bioactivation to yield HMVK. Because mouse, rat, and human liver microsomes exhibited K(m) values of 50-80 microM, the results also suggest that HMVK could be formed after rodent or human exposure to BDD or its parent compound, BD.

Substituted glycals as probes of glycosidase mechanisms

d-Glucal and a series of substituted derivatives have been tested as substrates, inhibitors and inactivators of the Agrobacterium faecalis β-glucosidase in order to probe structure/function relationships in this enzyme. d-Glucal is shown to be a substrate ( k cat = 2.3 min −1, K m = 0.85 mM) undergoing hydration with stereospecific protonation from the α-face to yield 2-deoxy-β- d-glucose. 1-Methyl- d-glucal surprisingly serves as only a poor substrate ( k cat = 0.056 min −1, K m = 57 mM), also undergoing protonation from the α-face. 2-Fluoro- d-glucal, however, is completely inert, as a result of inductive destabilisation of the oxocarbenium ion-like transition state for protonation, and functions only as a relatively weak ( K i = 24 mM) inhibitor. Similar behaviour was seen with almond β-glucosidase and yeast α-glucosidase and for the interaction of 2-fluoro- d-galactal with Escherichia coli β-galactosidase. A series of of α,β-unsaturated glucal derivatives was also synthesised and tested as potential substrates, inhibitors or inactivators of A. faecalis β-glucosidase. Of these only 1-nitro- d-glucal functioned as a time dependent, irreversible inactivator ( k i = 0.011 min −1, K i = 5.5 mM), presumably acting as a Michael acceptor. Electrospray mass spectrometric analysis revealed mutliple labeling of the enzyme by this inactivator, lessening its usefulness as an affinity label. Less reactive Michael acceptor glycals which might have been more specific (1-cyano-, 2-cyano-, 1-carboxylic acid, 1-carboxylic acid methyl ester) unfortunately did not function as inactivators or substrates, only as relatively weak reversible inhibitors ( K i = 3–96 mM).

An Useful Synthetic Method for 3-Substituted δ-Lactones. Synthesis of (±)-Secocrispiolide

Abstract 2-Phenylthio-2-penten-5-olide (1) acted as a reactive Michael acceptor toward some typical carbon neucleophiles to give 3-substituted 2-phenylthio-5-pentanolides (2) which were convertible both to 3-substituted 5-pentanolides by reductive desulfurization and to 3-substituted 2-penten-5-olides by sulfenic acid syn elimination of the sulfoxides 4 derived from 2. The Pummerer rearrangement of 4 provided 3-substituted 2-phenylthio-2-penten-5-olides and/or 2-hydroxy-2-penten-5-olides. (±)-Secocrispiolide was synthesized via the adduct obtained by the Michael reaction of 1 with the Grignard reagent prepared from 2,6-dimethylbenzyl bromide and magnesium.