Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection.

A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.

Oxabicyclo[3.2.1]octenes in organic synthesis--direct ring opening of oxabicyclo[3.2.1] systems employing silyl ketene acetals in concentrated solutions of lithium perchlorate-diethyl ether: application to the synthesis of the C(19)-C(27) fragment of rifamycin S.

[figure: see text] The direct opening at the bridgehead of oxabicyclo[3.2.1]octenes employing silyl ketene acetals in 4.0-5.0 M lithium perchlorate in diethyl ether has been realized, which gives rise to highly functionalized cycloheptadienes that can be further manipulated for use in natural product synthesis. The bridgehead opening reaction has been employed in the construction of the C(19)-C(27) fragment of Rifamycin S.

Novel esters of glaucarubolone as inducers of terminal differentiation of promyelocytic HL-60 cells and inhibitors of 7,12-dimethylbenz[a]anthracene-induced preneoplastic lesion formation in mouse mammary organ culture.

In an effort to discover new chemotherapeutic/chemopreventive agents from natural sources, brusatol (1) was found to induce HL-60 cellular differentiation, accompanied by strong antiproliferative and cytotoxic effects. A series of natural and semisynthetic quassinoids (1-48) was designed to effect both antiproliferative and differentiation-inducing properties. Compounds were assessed in vitro using the HL-60 promyelocytic cell model. Changes in activity due to structural modification of the core structure glaucarubolone (24) were consistent with activities reported in other cell systems. However, the following were novel SAR findings: (1) semisynthetic analogues with a hydroxylated ring at the beta-position of the ester side chain at C-15 were able to induce cellular differentiation at concentrations lower than those inducing cell growth arrest, and (2) quassinoids inhibiting DNA synthesis with greater efficacy than reducing cellular viability possessed alkyl substitutions at the alpha-position of the C-15 ester side chain. Analogues from this latter group and brusatol (1) and bruceantin (2) inhibited dimethylbenz(a)anthracene-induced preneoplastic lesion formation in a mouse mammary organ culture. The novel finding of 1 and glaucarubolone analogues as potent inducers of differentiation leads to potential novel applications in the field of cancer.

Baeyer-Villiger oxidation promoted by reaction of peracids with cyclic oxocarbenium ions generated in situ from internal hemiketals.

[reaction: see text] Reactive cyclic oxocarbenium ions, generated in situ from internal hemiketals, undergo Baeyer-Villiger oxidation upon exposure to m-chloroperbenzoic acid leading, after hydrolysis of the resultant lactones, to acyclic fragments for use in natural product synthesis.

Non-quassinoid constituents from the twigs and thorns of Castela polyandra.

The structures of two new constituents, a beta-glycoside (1) and a steroid (2), isolated from the twigs and thorns of Castela polyandra, were established by a combination of spectroscopic and single-crystal X-ray analysis.

Quassinoids from the twigs and thorns of Castela polyandra.

The structures of six new C20 quassinoids and one new C19 quassinoid, all isolated from the twigs and thorns of Castela polyandra, were established by a combination of spectroscopic and single-crystal X-ray analysis. Five known quassinoids and one known sterol were also identified.

Anticancer activity of glaucarubinone analogues.

A series of glaucarubinone analogues, obtained from natural sources as well as synthesized by us, were studied both in vitro and in vivo. The focus of the in vitro assessment was to define solid tumor-selective compounds by quantitating differential cytotoxic activity between murine and human solid tumor cells and either murine leukemia or normal cells. Subsequent in vivo studies were aimed at determining the therapeutic efficacy of these analogues against the murine models. Structure-activity analysis consequent to both the in vitro and in vivo studies demonstrated that few changes could be made in the parent glaucarubinone structure (outside of the C-15 position) without abrogating either cytotoxicity or potency. However, significant changes could be made at the C-15 position which modified, either enhanced or diminished, in vitro differential cytotoxicity, potency, human solid tumor selectively, and differential cytotoxicity to a MDR-expressing murine mammary tumor.

Mode of action of the anticancer quassinoids--inhibition of the plasma membrane NADH oxidase.

A plasma membrane-associated NADH oxidase of transformed cells was shown to be inhibited by nanomolar and subnanomolar concentrations of the antitumor quassinoid, glaucarubolone. The inhibition was seen with plasma membrane vesicles of HeLa cells at two log orders less glaucarubolone than with plasma membrane vesicles of rat liver. Assignment of a drug-binding site to the external surface of the HeLa cell plasma membrane was supported by findings where full activity of the glaucarubolone in the inhibition of NADH oxidase activity of isolated plasma membrane vesicles and of growth of HeLa cells was given on a molar glaucarubolone basis by an impermeant conjugate of glaucarubolone in which the glaucarubolone moiety was linked via the C-15 hydroxyl to amino polyethyleneglycol (ave Mr 5,000). The activity of the conjugate, and to a lesser extent, of free glaucarubolone was modulated by the redox environment of the cells and of the plasma membrane vesicles. Activity, both in the inhibition of NADH oxidase activity and in the inhibition of growth, was enhanced by oxidizing conditions in the presence of oxidized glutathione compared to reducing conditions in the presence of reduced glutathione.

Biochemical consequences of resistance to a recently discovered IMP dehydrogenase inhibitor, benzamide riboside, in human myelogenous leukemia K562 cells.

Benzamide riboside (BR) exhibits potent antitumor activity in a variety of cultured human tumor cells. The drug is metabolized to benzamide adenine dinucleotide (BAD), which in turn functions as a selective inhibitor of IMP dehydrogenase (IMPDH) activity with a Ki of 0.118 microM. In vitro, BR is a more potent antitumor inhibitor of IMPDH than tiazofurin, another IMPDH inhibitor which has shown significant oncolytic activity in adult patients with end-stage leukemia. To elucidate the mechanism of resistance, a variant of human myelogenous leukemia K562 cells was developed by subculturing sensitive cells in sublethal concentrations of BR over 60 generations. The BR resistant line that emerged exhibited an IC50 (a concentration producing 50% reduction in cell proliferation) of 148 microM, compared to the sensitive line which had an IC50 of 1.6 microM. The activity of the target enzyme, IMPDH, was increased 3-fold in the resistant variant. Studies on BR metabolism revealed that resistant cells formed only 18% of the active metabolite, BAD, compared to sensitive cells. This finding, in turn, correlated with the specific activity of NAD pyrophosphorylase (the enzyme responsible for the synthesis of BAD) which was reduced to undetectable levels in the resistant variant. The basal levels of NAD and guanylates were also significantly decreased to 41% and 48%, respectively, in the resistant line compared to the parent line. Additionally, after treatment with BR a decrease in guanylate level was observed only in the sensitive cells. Sensitive and resistant cells exhibit comparable cytotoxicity to agents outside the tiazofurin family, suggesting that a multidrug resistance was unlikely to explain the resistance to BR. Moreover, BR resistant cells exhibit collatoral sensitivity to 6-aminopurine, cytarabine and 5-fluorouracil, which have different mechanisms of action. In conclusion, these studies establish that the primary mechanism of resistance to BR involves an increase in IMPDH (target enzyme) activity with a concurrent decrease in NAD pyrophosphorylase (BAD synthetic enzyme) activity.

Synthesis and biological evaluation of the methyl esters of (+)-12-fluoro-13,14-dihydroprostaglandin F2alpha and (+)-15-epi-12-fluoro-13,14-dihydroprostaglandin F2alpha.

(+)-12-Fluoro-13,14-dihydroprostaglandin F2alpha methyl ester (2a) and (+)-15-epi-12-fluoro-13,14-dihydroprostaglandin F2alpha methyl ester (2b) were prepared from the readily available (-)-7-fluorospiro[bicyclo[2.2.1]hept-5-ene-2,2'-[1,3]dioxolane]-7-methanol (3). Fluoroprostaglandins 2a and 2b possess truly significant separations of antifertility activity from smooth-muscle stimulating properties. In addition, our studies showed that 2a and 2b were totally inert toward the placental 15-hydroxyprostaglandin dehydrogenase.

Fluoroprostaglandins: synthesis and biological evaluation of the methyl esters of (+)-12-fluoro-, (-)-ent-12-fluoro-, (+)-15-epi-fluoro-, and (-)-ent-15 epi-12-fluoroprostaglandin F2 alpha.

The synthesis and biological activity of the methyl esters of (+)-12-fluoroPGF2 alpha, (+)-15-epi-12-fluoroPGF2 alpha, (-)-ent-12-flurorPGF2 alpha, and (-)-ent-15-epi-12-fluoroPGF2 alpha are described. Each fluoroprostaglandin has been evaluated from pregnancy interruption in the hamster and smooth-muscle stimulating effects on gerbil colon and hamster uterine strips. All fluoroprostaglandins synthesized were shown to be neither substrates for the 15-hydroxyprostaglandin dehydrogenase nor inhibitors of the enzyme.

C(14)-fluorinated prostaglandins: synthesis and biological evaluation of the methyl esters of (+)-14-fluoro-, (+)-15-epi-14-fluoro-, (+)-13(E)-14-fluror-, and (+)-13(E)-15-epi-14-fluoroprostaglandin F2 alpha.

The synthesis and biological evaluation of the methyl esters of (+)-14-fluoroPGF2 alpha, (+)-15-epi-14-fluoroPGF2 alpha, (+)-13(E)-14-fluoroPGF2 alpha, and (+)-13(E)-15-epi-14-fluoroPGF2 alpha are described. Each fluoroprostaglandin has been evaluated for pregnancy interruption in the hamster and smooth-muscle stimulating effects on gerbil colon and hamster uterine strips.

Synthesis of mono- and bifunctional alpha-methylene lactone systems as potential tumor inhibitors.

Synthetic nono- and bifunctional alpha-methylene lactone derivatives including deoxyvernolepin and kihydrodeoxyvernolepin were tested as inhibitors of the growth of CCRF-CEM human lymphoblastic leukemia cells in culture. The range of ID-50 values for compounds 1-7 (ca. 10(-5)-10(-6)M) was roughly comparable to the doses observed earlier in the CCRF-CEM cell system with synthetic alpha-methylene-gamma-butyrolactones. Of significance is that dihydrodeoxyvernolepin and deoxyvernolepin were at least an order of magnitude more active than natural vernolepin.