(R)-Veliparib (ABT-888) is a poly(ADP-ribose)polymerase (PARP) inhibitor that is being investigated for the treatment of a broad spectrum of oncology indications, including BRCA1/2-mutated breast cancer and other solid tumors. The (R)-veliparib process consists of three stages utilizing two proposed regulatory starting materials, (R)-Boc-2-methylproline and 2,3-diaminobenzamide dihydrochloride, with two isolated intermediates. The drug substance control strategy, which was established based on a combination of analytical tools and uniquely designed manufacturing process and unit operations, provides robust controls for mutagenic and other impurities and ensures that (R)-veliparib drug substance consistently meets all critical quality attributes (CQAs) and acceptance criteria. The purpose of this article is to provide details of how the (R)-veliparib control strategy for the selected CQAs was cross-functionally developed using analytical measurement tools and specially designed unit operations.

( R)-Boc-2-methylproline (3a) was synthesized in good yield with excellent stereochemical control from alanine benzyl ester hydrochloride 11. The process, which is based on a modification of one described by Kawabata, proceeds in four steps and requires no chromatography. The product ( R)-Boc-2-methylproline (3a) was then carried forward in three steps to produce veliparib 1, a poly(ADP-ribose) polymerase inhibitor.

Dasabuvir (1) is an HCV polymerase inhibitor which has been developed as a part of a three-component direct-acting antiviral combination therapy. During the course of the development of the synthetic route, two novel coupling reactions were developed. First, the copper-catalyzed coupling of uracil with aryl iodides, employing picolinamide 16 as the ligand, was discovered. Later, the palladium-catalyzed sulfonamidation of aryl nonaflate 33 was developed, promoted by electron-rich palladium complexes, including the novel phosphine ligand, VincePhos (50). This made possible a convergent, highly efficient synthesis of dasabuvir that significantly reduced the mutagenic impurity burden of the process.

We report the use of biaryl phosphorinanes as ligands for Pd-catalyzed cross-coupling reactions. A modular synthesis was developed that employs a double conjugate addition of primary biaryl phosphines into 1,1,5,5-tetraalkyl penta-1,4-diene-3-ones. Notably, this synthesis does not require the use of copper, a known contaminant in structurally related biaryl phosphane ligands. Using the synthetic strategy described above, we synthesized a library of biaryl phosphorinanes, varying their substitution about phosphorus and the steric and electronic nature of the biaryl motif. We then benchmarked their performance as ligands in Pd-catalyzed cross coupling reactions such as aryl sulfonamidation, aryl alkoxylation, and aryl amination in the presence of soluble organic bases. In each reaction studied, many ligands outperformed biaryl phosphanes known to promote the given transformation. Detailed substrate scopes were determined using high-throughput screening technology. Several biaryl phosphorinanes and their corresponding Pd(II) oxidative-addition complexes were extensively characterized using NMR spectroscopy and X-ray crystallography. General observations support that biaryl phosphorinanes promote reductive elimination and form robust catalysts with palladium. In many cases the use of these biaryl phosphorinanes may be advantageous over the use of biaryl phosphanes with respect to lower catalyst loadings, shorter reaction times, and robustness.

Dietary microRNAs (miRNAs), notably those found in milk, are currently being investigated for their potential to elicit biological effects via canonical binding to human messenger RNA targets once ingested. Besides milk, beef and other bovine tissue-derived ingredients could also be a relevant source of potentially bioactive dietary miRNAs. In this study, we characterized the human homologous miRNA profiles in food-grade, bovine-sourced sirloin, heart and adrenal tissue (raw, cooked, and pasteurized, freeze-dried extracts) via deep-sequencing and quantitative reverse transcription PCR (RT-qPCR). A total of 198 human homologous miRNAs were detected at 10 or more normalized reads in all replicates (n = 3) of at least one preparation method. Tissue origin rather than preparation method was the major differentiating factor of miRNA profiles, and adrenal-based miRNA profiles were the most distinct. The ten most prevalent miRNAs in each tissue represented 71-93% of the total normalized counts for all annotated miRNAs. In cooked sirloin, the most abundant miRNAs were miR-10b-5p, (48.8% of total annotated miRNA reads) along with the muscle-specific miR-1 (24.1%) and miR-206 (4.8%). In dried heart extracts, miR-1 (17.0%), miR-100-5p (16.1%) and miR-99a-5p (11.0%) gave the highest normalized read counts. In dried adrenal extracts, miR-10b-5p (71.2%) was the most prominent followed by miR-143-3p (7.1%) and 146b-5p (3.7%). Sequencing results for five detected and two undetected miRNAs were successfully validated by RT-qPCR. We conclude that edible, bovine tissues contain unique profiles of human homologous dietary miRNAs that survive heat-based preparation methods.

Humans are exposed to toxins which accumulate in the body, and are detoxified primarily in the liver. Studies have shown that cruciferous vegetables (such as radishes) may be beneficial to health by aiding detoxification of toxins in the liver.

A practical, scalable synthetic process for a sulfonamide was developed featuring a Semmler–Wolff aromatization as the key step. The optimized reaction conditions using HCl in HOAc give directly the desired naphthylamine in high yield as opposed to a naphthylacetamide commonly formed in the Semmler–Wolff reactions. One little known byproduct of anomalous rearrangement, ketoamine, was observed and a mechanism proposed to explain its formation. Employing the optimized process, 360 kg was prepared to support drug development.

Celeriac is an agricultural root crop and source of secondary plant defense compounds called polyacetylenes (PAC's). PAC's inhibit lipid transport enzymes, up regulate phase II liver detoxification enzymes, are cytotoxic to cancer cells, and potent antiinflammatory modulators. SFE of PAC's has been reported, but extraction parameters have not been optimized. A full factorial experiment was designed to test extraction parameters of temperature, pressure, and ethanol co‐solvent addition at a solvent‐to‐ feed ratio of 100. The dried feed stock contained 4.5x's the amount of PAC's compared to purple carrots. Optimal extraction conditions were low temperature (40°C), high pressure (500 bar), and high ethanol co‐solvent (20% w/w). Pressure had a larger effect (F <0.0001) on extraction of falcarinol and falcarindiol than temperature (F = 0.13 and 0.02 respectively), which was also dependent on co‐solvent amount (F = 0.0009). Under optimal extraction conditions, 1.7% of the starting dry weight, or 47% of the total lipids were extracted and up to 43% of falcarindiol was obtained compared to exhaustive ethyl acetate extraction. Ethyl acetate extracts of celeriac inhibited (IC50) pro‐inflammatory cyclooxygenases (Cox‐1 and Cox‐2) at concentrations of 0.26 and 0.06 mg/mL respectively. SFE extraction of dried celeriac root offers potential as an industrial scale enrichment process of antiinflammatory PAC's.

Eight commercial molecular distilled (MD), one commercial fermented, and five crude manufacturer supplied fish (Gadidae) liver oils were analyzed for fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), vitamin A and ester profiles, sterols, and oxidative state (malondialdehyde, MDA). Fatty acids and sterols were determined by GC‐MS, MDA by colorimetric assay, vitamin A by HPLC. Vitamin A ester profiles were determined by UPLC‐MS (QTOF). The ratio of DHA/EPA for MD products was 1.3–1.7 with one exception, while the fermented product (0.5), and crude samples (2.2–3.3) were different. The total concentration of EPA and DHA per 5 mL serving was 944–1200 mg in MD, 957 mg in fermented, and 182–274 mg in unrefined oil. MDA, an indicator of polyunsaturated fatty acid oxidation, was 10‐fold higher in the fermented product, and 2.4‐fold higher in the unrefined oils compared to MD products. Vitamin A concentrations in the MD products were 500–583 IU/mL with the exception of one product (1,086 IU/mL). The fermented product contained 1117 IU/mL, while the unrefined contained 611–2126 IU/mL vitamin A. Vitamin A2, the dehydroretinol form was only detected in fermented and unrefined samples in measurable amounts. Total sterols were lower in the fermented sample. Commercial MD products contain higher amounts of EPA/DHA than crude, while the fermented and crude products contain higher levels of vitamin A.

Cruciferae vegetable contain high levels of chemopreventative glucosinolates. Spanish black radishes (SBR) are unique crucifers that contain 4X more glucosinolates than other crucifers, such as broccoli. This study examined whether feeding mice a diet containing 20 % Spanish black radish for two weeks prior to treatment with the carcinogen 7, 12‐dimethylbenz(a)anthracene (DMBA) could enhance metabolism of this carcinogen and inhibit the DMBA‐mediated bone marrow toxicity. Expression of Phase I and Phase II detoxification enzymes was significantly greater for mice on SBR diet. The blood levels of DMBA in SBR‐fed mice were significantly lower than those on control diets. DMBA significantly reduced BM cells of mice fed control diet, whereas mice on the SBR diet showed diminished effect. Colony forming assays demonstrated that mice on the SBR diet had significantly: 1) less reduction in lymphoid CFU‐preB progenitor cells, 2) greater recovery of CFU‐preB progenitor cells, and 3) less reduction of CFU‐GM progenitor cells. Therefore, mice fed a 20% SBR diet for two weeks had higher expression of detoxification enzymes, faster metabolism of DMBA and a reduction in DMBA‐induced bone marrow toxicity.

Sample preparation for the determination of vitamin D typically involves saponification and/or liquid/liquid solvent extraction. Cod liver oil has substantial amounts of vitamin D and has a traditional use as a vitamin D supplement, however, its oily sample matrix creates difficulty in extraction and remaining lipid residues limit UV detection. Conventional quantification requires two HPLC columns and runs (normal phase preparative and reverse phase analytical) or recently expensive mass spectrometers to measure vitamin D. Here we describe a method using normal and reverse phase SPE to achieve good cleanup of residual lipids. The complete serving size of eight commercially available cod liver oil supplements were prepared using a 2g NH2 SPE column followed by 500mg C‐18 column before analytical quantification. Using this method, a limit of detection of 20IU/serving was achieved.This method provides a simpler alternative for vitamin D detection in cod liver oil samples using SPE and a single HPLC run with UV detection and may have additional use in the quantification vitamin A and tocopherols.

Vitamin D 2 , or ergocalciferol, is a fungal source of vitamin D suitable as a dietary supplement for vegans. Spent brewer's yeast is an inexpensive by‐product of the brewing industry which may be used as a starting raw material for the enrichment of D 2 from the precursor ergosterol. Dried brewer's yeast was reconstituted in water and stirred during exposure to ultraviolet light (UV) for an optimized period of time to convert ergosterol to ergocalciferol. After freeze drying, the D 2 enriched yeast was mixed with rice hulls and wheat germ oil for SFE extraction of D 2 . The SFE parameters consisted of an extraction temperature of 40°C, a solvent to feed of 75 (g CO 2 /g material), and a flow rate of 75 g/min in a 4L extraction vessel. 100 g of material was extracted and separated in a two stage separator (separator 1, 1500 psi, 40°C; separator 2, 750 psi, 40°C). The extraction pressure was experimentally varied (3000, 6000, and 9000 psi) to understand extraction efficiency from the yeast matrix. UV treatment of brewer's yeast led to a D 2 enhancement from non‐detectable levels to 3.8 mg/g (152,000 IU/g). The percentage of D 2 extracted from the yeast at the extraction pressure of 3000, 6000, and 9000 psi was 55, 35, and 31% of the starting concentration respectively. The D 2 concentration was 1.5 fold higher in separator #2 compared to separator #1. SFE extraction of vitamin D 2 from UV enhanced yeast is more efficient at lower extraction pressures.

The bioactive metabolites of glucosinolates, such as isothiocyanates, contained in cruciferous vegetables have been shown to reduce the risk of cancers through the induction of detoxification enzymes. However, cruciferous vegetables are commonly processed before consumption, significantly altering the phytochemical composition of these vegetables. Compared to freeze-dried Brussels sprouts, oven-dried Brussels sprouts contain low concentrations of glucosinolates (22.14 and 0.85 mu mol/g, respectively) and isothiocyanates (3.68 and 0.15 mu mol/g, respectively). The effect of oven-dried Brussels sprouts on the expression of detoxification enzymes was evaluated in vitro and in vivo. Treatment of immortalized human hepatoma cells with the aqueous extract from oven-dried Brussels sprouts significantly increased quinone activity (0.5 and 1.5 mg/mL) and the activity of the antioxidant response element (EC50 = 2.39 mg/mL) and xenobiotic response element (EC50 2.92 mg/mL). C3H/HeJ mice fed a diet containing 20% oven-dried Brussels sprout diets for 2 wk demonstrated significantly higher expression than animals fed a nutrient-matched control diet of CYP1A1, CYP1A2, and epoxide hydrolase in the liver and CYP1A1, CYP1A2, CYP1B1, epoxide hydrolase, UGT1A1, thioredoxin reductase, and heme oxygenase in the lungs. The low concentrations of glucosinolates and isothiocyanates in oven-dried Brussels sprouts suggest that other compounds, such as the Maillard reaction products that are produced during heating, are responsible for the induction of detoxification enzymes in vitro and in vivo.Practical ApplicationThe manner in which cruciferous vegetables are processed prior to consumption has significant effects on what compounds people are exposed to. The presence of glucosinolates or isothiocyanates can be a good indicator of the ability of cruciferous vegetables to induce detoxification enzymes. However, the data presented here demonstrate that while heat processing of Brussels sprouts greatly reduced the concentrations of glucosinolates and isothiocyanates, their ability to induce detoxification enzymes in vitro and in vivo was retained.

UNLABELLED The biological activity of cruciferous vegetables is hypothesized to be due to the metabolites of a class of phytochemicals called glucosinolates. The chemical properties of these metabolites, including isothiocyanates, determine the biological activity of these compounds and thus their effects on human health. The 2 primary radish (Raphanus sativus L.) glucosinolates, glucoraphasatin, and glucoraphenin, were isolated using solid phase extraction followed by preparative HPLC purification. In an aqueous environment, 77.6% of the maximum amount of sulforaphene produced by the metabolism of glucoraphenin was present after 24 h. Under the same conditions raphasatin, the isothiocyanate metabolite of glucoraphasatin and the oxidized counterpart of sulforaphene, was highly unstable with a half-life of less than 30 min and no raphasatin was detectable after 24 h. In HepG2 cells, raphasatin-induced quinone reductase activity and the RNA expression of several phase 1 and 2 detoxification enzymes by a significantly greater amount than the degradation products of raphasatin. Raphasatin, but not its degradation products, activated the antioxidant response element (ARE) in a stably-transfected reporter cell line. Mice fed a diet consisting of 20% freeze dried radishes for 2 wk had significantly higher liver expression of cytochrome P450 (CYP) 1A1, 1A2, quinone reductase, microsomal epoxide hydrolase, and glutathione S-transferase α2 than mice fed a nutritionally-matched control diet. PRACTICAL APPLICATION   Glucoraphasatin, the primary glucosinolate in radishes, is metabolized into an isothiocyanate (raphasatin) that has biological activity but is also unstable in an aqueous environment. Despite the instability of raphasatin, dietary exposure to radishes produced significant induction of detoxification enzymes. Understanding the chemical properties of raphasatin, both in terms of biological activity and instability, could help develop processing methods to retain the most activity from radishes, glucoraphasatin, and raphasatin.

Radishes (Raphanus sativus L.) are members of the cruciferous vegetable family that contain many classes of biologically active phytochemicals. This study determined the phytochemical composition of the sprouts and mature taproots of 8 radish varieties. Radish sprouts contained significantly greater concentrations of glucosinolates (3.8-fold) and isothiocyanates (8.2-fold) than the mature radish taproot and also contained significantly greater concentrations of phenolics (on average 6.9-fold). The anthocyanin concentrations of the mature radish taproot were significantly greater than in the sprouts of red, pink, and purple varieties. The primary anthocyanidins present in the red and pink radish varieties were pelargonidin and delphinidin, while the primary anthocyanidin in the purple radish variety was cyanidin. Radish sprouts were between 9- and 59-fold more potent than the corresponding mature taproot at activating the antioxidant response element (ARE) in a stably transfected hepatoma cell line. The ARE activity of the radish sprouts and mature taproots was significantly correlated with the total isothiocyanate concentration of the radishes. Practical Application: Understanding the influence variety and developmental stage has on the biological activity of cruciferous vegetables provides important information for further studies examining the in vivo effects of radish treatment and foundation for providing recommendations to reduce the risk of chronic disease through dietary intervention.

In cruciferous vegetables, myrosinase metabolizes the relatively inactive glucosinolates into isothiocyanates and other products that have the ability to increase detoxification enzyme expression. Thus, maintaining myrosinase activity during food preparation may be critical to receiving the maximum benefit of consumption of Brussels sprouts or other cruciferous vegetables. To test the importance of maintaining myrosinase activity for maximizing bioactivity, experimental diets containing 20% unblanched (active myrosinase) or 20% blanched (inactivated myrosinase) freeze-dried Brussels sprouts and a nutrient-matched control diet were evaluated in vitro and in vivo for their ability to induce detoxification enzymes. Treatment of immortalized HepG2 human hepatoma cells with the unblanched Brussels sprout diet caused a greater increase quinone activity compared to the blanched Brussels sprout diet. C3H/HeJ mice fed the unblanched Brussels sprout diets for 2 wk had significantly higher plasma sulforaphane concentrations. Liver expression of CYP1A1 and epoxide hydrolase, measured using real-time PCR, was correlated with the plasma concentration of sulforaphane. In the lung, expression of epoxide hydrolase, thioredoxin reductase, UDP glucuronosyltransferase, quinone reductase, heme oxygenase, CYP1A1, CYP1A2, and CYP1B1 were also correlated with the plasma concentration of sulforaphane. Together these data demonstrate that, as predicted by the in vitro experiment, in vivo exposure to Brussels sprouts with active myrosinase resulted in greater induction of both phase I and phase II detoxification enzymes in the liver and the lungs that correlated with plasma sulforaphane concentrations.

Fenugreek and cayenne contain bioactive phytochemicals that have both been shown to affect glucose handling. The purpose of this study was to determine the effect of these two foods on glucose handling in a model of diabetes, as well as whether there would be a synergistic effect of these two foods combined. C57BL/KsJ‐db/db (leptin receptor deficient) diabetic mice were fed a control diet or experimental diets containing 1% ground fenugreek seed, 1% dried cayenne pepper powder or the combination of 1% fenugreek and 1% cayenne for two weeks. Inclusion of fenugreek and/or cayenne did not significantly reduce food consumption, nor did it affect the body weight of the animals. At the end of two weeks, glucose handling was measured by i.p. glucose tolerance test (IPGTT). Mice fed either the fenugreek or cayenne alone diets did not have significant improvement in IPGTT in comparison with control. However, the mice fed the combination of fenugreek and cayenne demonstrated significantly improved glucose handling (115% of baseline after 120 minutes) over control (blood glucose 167% of baseline after 120 minutes). Values for circulating adipokines and hemoglobin A1c are also reported.

Mushrooms have traditionally been used for the modulation of immune function. Hot water, ethyl acetate and hexane extracts from Coriolus versicolor (turkey tail), Hericium erinaceus (bearded tooth), Ganoderma lucidum (reishi), Grifola frondosa (maitake), Lentinula edodes (shiitake), and Cordyceps sinensis (caterpillar fungus) were compared for their ability to reduce T lymphocyte activation. Jurkat immortalized T lymphocytes were pretreated with 1, 5 and 7.5 mg of mushroom extract per mL of media for 1 hour prior to activation by phytohemagglutinin (PHA) and phorbol 12‐myristate 13‐acetate (PMA). IL‐2 secretion by Jurkat cells was measured 24 hours after stimulation. Hot water extracts from all mushroom varieties reduced IL‐2 concentrations in a dose‐dependent manner, and extracts from Cordyceps sinensis and Grifola frondosa were the most potent. The ethyl acetate extracts from the mushrooms had similar effects as the hot water extracts, although in general they were less potent. Hexane extracts from all mushroom varieties had the least effect on IL‐2 production. Together these data demonstrate that water soluble compounds in the mushrooms tested have the greatest anti‐inflammatory bioactivity.