Agonists of the γ-aminobutyric acid type B (GABAB) receptor derived from ß-hydroxy and ß-amino difluoromethyl ketones.

ß-Hydroxy difluoromethyl ketones represent the newest class of agonists of the GABA-B receptor, and they are structurally distinct from all other known agonists at this receptor because they do not display the carboxylic acid or amino group of γ-aminobutyric acid (GABA). In this report, the design, synthesis, and biological evaluation of additional analogues of ß-hydroxy difluoromethyl ketones characterized the critical nature of the substituted aromatic group on the lead compound. The importance of these new data is interpreted by docking studies using the X-ray structure of the GABA-B receptor. Moreover, we also report that the synthesis and biological evaluation of ß-amino difluoromethyl ketones provided the most potent compound across these two series.

Magnesium-Promoted Additions of Difluoroenolates to Unactivated Imines.

Although there are many synthetic methods to produce fluorinated and trifluoromethylated organic structures, the construction of difluoromethylated compounds remains a synthetic challenge. We have discovered that unactivated imines will react with difluoroenolates under exceedingly mild conditions when using magnesium salts and organic bases. We have applied this approach to the iminoaldol reaction to produce difluoromethylene groups as α,α-difluoro-ß-amino-carbonyl groups. This method provides synthetically useful quantities of difficult to access α,α-difluoro-ß-aminoketones without the need of protecting groups or the use of activated imines. Moreover, we have applied this strategy to create analogues of the dual orexin receptor antagonist, almorexant, in only two synthetic steps.

15-Methylene-Eburnamonine Kills Leukemic Stem Cells and Reduces Engraftment in a Humanized Bone Marrow Xenograft Mouse Model of Leukemia.

Recent studies suggest that leukemia stem cells (LSCs) play a critical role in the initiation, propagation, and relapse of leukemia. Herein we show that (-)-15-methylene-eburnamonine, a derivative of the alkaloid (-)-eburnamonine, is cytotoxic against acute and chronic lymphocytic leukemias (ALL and CLL) and acute myelogenous leukemia (AML). The agent also decreases primary LSC frequency in vitro. The cytotoxic effects appear to be mediated via the oxidative stress pathways. Furthermore, we show that the compound kills AML, ALL, and CLL stem cells. By the use of a novel humanized bone marrow murine model of leukemia (huBM/NSG), it was found to decrease progenitor cell engraftment.

Total Synthesis of Shishijimicin A.

The total synthesis of the rare but extremely potent antitumor agent shishijimicin A has been achieved via a convergent strategy involving carboline disaccharide 3 and hydroxy enediyne thioacetate 4.

Synthesis of 15-methylene-eburnamonine from (+)-vincamine, evaluation of anticancer activity, and investigation of mechanism of action by quantitative NMR.

The biological role of installing a critical exocyclic enone into the structure of the alkaloid, (-)-eburnamonine, and characterization of the new chemical reactivity by quantitative NMR without using deuterated solvents are described. This selective modification to a natural product imparts potent anticancer activity as well as bestows chemical reactivity toward nucleophilic thiols, which was measured by quantitative NMR. The synthetic strategy provides an overall conversion of 40%. In the key synthetic step, a modified Peterson olefination was accomplished through the facile release of trifluoroacetate to create the requisite enone in the presence of substantial steric hindrance.

Fluorinated amino-derivatives of the sesquiterpene lactone, parthenolide, as (19)f NMR probes in deuterium-free environments.

The design, synthesis, and biological activity of fluorinated amino-derivatives of the sesquiterpene lactone, parthenolide, are described. A fluorinated aminoparthenolide analogue with biological activity similar to the parent natural product was discovered, and its X-ray structure was obtained. This lead compound was then studied using (19)F NMR in the presence and absence of glutathione to obtain additional mechanism of action data, and it was found that the aminoparthenolide eliminates amine faster in the presence of glutathione than in the absence of glutathione. The exact changes in concentrations of fluorinated compound and amine were quantified by a concentration-reference method using (19)F NMR; a major benefit of applying this strategy is that no deuterated solvents or internal standards are required to obtain accurate concentrations. These mechanistic data with glutathione may contribute to the conversion of the amino-derivative to parthenolide, the active pharmacological agent, in glutathione-rich cancer cells.

Inflammatory cytokines and antioxidants in midtrimester amniotic fluid: correlation with pregnancy outcome.

OBJECTIVE: Elevated interleukin-6 (IL-6) level in midtrimester amniotic fluid is associated with preterm delivery. We hypothesized that, in patients with elevated IL-6, vitamin C and alpha-fetoprotein may provide protection from spontaneous preterm delivery through antioxidant functions. STUDY DESIGN: Antioxidant potential of alpha-fetoprotein was assessed in vitro. Amniotic fluid was collected from a prospective cohort of patients who underwent midtrimester amniocentesis. In patients with IL-6 >600 pg/mL, alpha-fetoprotein, vitamin C, tumor necrosis factor-alpha, tumor necrosis factor receptors, and antioxidant capacity were compared between subjects with spontaneous preterm and term deliveries. RESULTS: Alpha-fetoprotein demonstrated 75% the antioxidant capacity of albumin in vitro. Of 388 subjects, 73 women had elevated IL-6 levels. Among these subjects, alpha-fetoprotein, but not vitamin C, was significantly lower in 9 women with preterm birth. Antioxidant capacity correlated with vitamin C and tumor necrosis factor receptors, but not with alpha-fetoprotein or pregnancy outcome. CONCLUSION: Amniotic fluid alpha-fetoprotein, but not vitamin C, may protect against preterm birth in patients with elevated midtrimester IL-6 levels.

Prevention of PPROM: current and future strategies.

Our understanding of the pathophysiologic processes leading to preterm premature rupture of membranes (PPROM) has grown tremendously in recent years. Evidence suggests that there may be a genetic susceptibility to PPROM and that genetic and environmental elements are important cofactors in its development. A number of risk-based protocols have been proposed in an attempt to identify those women at highest risk for PPROM. While we have made advances in the area of predicting PPROM, treatments based on current risk-based systems have failed to distinguish a specific, effective preventive therapy for PPROM. The concept that genetic factors increase susceptibility or decrease resistance to disease has stimulated new work in the field of PPROM. Several maternal and fetal gene polymorphisms have been identified that are associated with an increased risk for PPROM. Patients with 'susceptible' genotypes may also have clinical risk factors for PPROM resulting in a synergistic increase in the risk for PPROM, a so-called gene-environment interaction. The concept that these gene-environment interactions represent new targets for our efforts to prevent PPROM is explored.

Effects of maternal antioxidant supplementation on maternal and fetal antioxidant levels: a randomized, double-blind study.

OBJECTIVE: We sought to determine whether vitamins C and E could be delivered to the fetal-placental unit through maternal oral supplementation. STUDY DESIGN: In a randomized, double-blind study, 20 women received a daily prenatal vitamin with or without 400 IU of vitamin E and 500 mg of vitamin C, starting at 35 weeks' gestation. At randomization, a nutritional questionnaire, plasma vitamin C and E and red blood cell (RBC) vitamin E levels were determined. At delivery, concentrations of maternal and fetal plasma vitamin C and E, maternal and fetal RBC vitamin E, amniotic fluid vitamin C, and chorioamnion vitamin E and tensile strength were determined. RESULTS: Maternal plasma vitamin E levels increased in the supplemented women but not in the control subjects. No changes in maternal vitamin C levels were noted. Maternal plasma vitamin C concentrations at delivery correlated closely with amniotic fluid vitamin C levels. Similarly, maternal plasma vitamin E levels at delivery correlated with the chorioamnion concentration of vitamin E. CONCLUSIONS: Maternal plasma vitamin E levels are increased by oral supplementation. Maternal plasma vitamin C and E concentrations correlate with the concentration of vitamin C in the amniotic fluid and vitamin E in the chorioamnion, respectively.

The effect of labor on maternal and fetal vitamins C and E.

OBJECTIVE: This study was conducted to compare maternal and fetal plasma, amniotic fluid, and chorioamnion levels of vitamins C and E in term (>38 weeks' gestation) subjects undergoing elective repeat cesarean section (CS) without labor with values of subjects of similar gestational age and dietary intake undergoing labor and vaginal delivery (VD). STUDY DESIGN: Healthy women undergoing elective repeat CS (n = 5) or uncomplicated VD (n = 5) at term (>38 weeks' gestation) were studied. For CS patients, maternal and fetal (cord) blood, amniotic fluid, and chorioamnion samples were collected at time of surgery. For VD patients, maternal blood and amniotic fluid were obtained at 5 cm cervical dilation and fetal cord blood and chorioamnion were collected at delivery. Each patient completed a nutritional questionnaire. Plasma and membrane vitamin E concentrations were determined by reversed-phase high-performance liquid chromatography and standardized to cholesterol or membrane protein, respectively. Vitamin C was determined with the use of the 2,4-DNPH method. RESULTS: Dietary intakes for vitamins C and E as well as maternal and fetal vitamin E plasma concentrations were similar for CS and VD patients. In both groups, maternal levels were higher than fetal levels(P <.05). Chorioamnion membrane vitamin E measurements in both groups were similar. Vitamin C concentrations in CS and VD patients were highest in amniotic fluid, lower in fetal plasma, and lowest in maternal plasma. However, mean vitamin C concentrations in maternal plasma, amniotic fluid, and fetal plasma of VD patients were significantly lower, being only 20% +/- 6%, 29% +/- 11%, and 22% +/- 2% of values obtained from CS patients. CONCLUSION: During labor in healthy women at term, uterine contractile activity may generate reactive oxygen species (ROS) through the process of repetitive ischemia and reperfusion. With the significant depletion of vitamin C during labor, we speculate that water-soluble vitamin C scavenges ROS in the aqueous phase and recycles lipid-soluble vitamin E to combat ROS-induced tissue damage.

Physical properties of the chorioamnion throughout gestation.

OBJECTIVE: Our purpose was to assess the tensile strength of the chorioamnion at various gestational ages. STUDY DESIGN: Segments of chorioamnion were obtained from 35 patients delivered at gestational ages ranging from 17 to 41 weeks. Clinical information including gestational age, chorioamnionitis, premature rupture of membranes, and onset of labor was recorded. Tensile strength (grams to burst and deflection at rupture) was measured on 2 to 16 specimens per patient. Tensile strength of several commercial products was used for comparison. RESULTS: Tensile strength increases up to 20 weeks of gestation and then plateaus until 39 weeks of gestation, when it falls dramatically. Clinical chorioamnionitis alone did not affect tensile strength, but gross membrane inflammation resulted in reduced tensile strength. CONCLUSIONS: Tensile strength of the chorioamnion varies with gestational age. This baseline information will be useful in assessing the effects of various conditions and therapies on membrane strength and may provide insight into spontaneous rupture of membranes.

Preterm premature rupture of the membranes and antioxidants: the free radical connection.

AIM: To discuss the role of oxidant stress in preterm, premature rupture of the membranes (PPROM). RESULTS: There is evidence to suggest that preterm, premature rupture of the membranes occurs secondary to focal collagen damage in the fetal membranes. CONCLUSION: Oxidant stress caused by increased ROS formation and/or antioxidant depletion may disrupt collagen and cause premature membrane rupture. We propose that supplementation with vitamins C and E may synergistically protect the fetal membranes, and decrease the risks of PPROM.

Maternal and Transplacental Effects of Cocaine.

Pregnancy is a dynamic process, and maternal as well as fetal risks from cocaine use in pregnancy may differ as pregnancy progresses. Three areas of biology offer opportunities for reevaluating cocaine's effects in pregnancy: (1) Maternal cardiovascular and neurologic responses to cocaine hydrochloride are enhanced when compared with responses in nonpregnant subjects to the same dose per kilogram or to metabolites of crack cocaine. (2) During first trimester placental implantation, oxygen availability to the fetus may normally be limited. Cocaine-induced uterine artery vasoconstriction may lead to reperfusion and oxygen toxicity to the fetus from released reactive oxygen species. (3) Cocaine transport in the first and early second trimester may, in part, be across the placental chorion-amnion. Lacking a skin barrier, the fetus at mid-pregnancy may come in direct contact with high concentrations of cocaine in amniotic fluid, a reservoir that clears cocaine slowly, thereby prolonging exposure during critical periods of fetal neurotransmitter formation. Exploring these three areas of biology may offer new approaches to understanding the ultimate impact of prenatal cocaine exposure on maternal and fetal biology.