Schisandrol B promotes liver enlargement via activation of PXR and YAP pathways in mice.

BACKGROUND: Schisandrol B (SolB) is one of the bioactive components from a traditional Chinese medicine Schisandra chinensis or Schisandra sphenanthera. It has been demonstrated that SolB exerts hepatoprotective effects against drug-induced liver injury and promotes liver regeneration. It was found that SolB can induce hepatomegaly but the involved mechanisms remain unknown. PURPOSE: This study aimed to explore the mechanisms involved in SolB-induced hepatomegaly. METHODS: Male C57BL/6 mice were injected intraperitoneally with SolB (100 mg/kg) for 5 days. Serum and liver samples were collected for biochemical and histological analyses. The mechanisms of SolB were investigated by qRT-PCR and western blot analyses, luciferase reporter gene assays and immunofluorescence. RESULTS: SolB significantly increased hepatocyte size and proliferation, and then promoted liver enlargement without liver injury and inflammation. SolB transactivated human PXR, activated PXR in mice and upregulated hepatic expression of its downstream proteins, such as CYP3A11, CYP2B10 and UGT1A1. SolB also significantly enhanced nuclear translocation of PXR and YAP in human cell lines. YAP signal pathway was activated by SolB in mice. CONCLUSION: These findings demonstrated that SolB can significantly induce liver enlargement, which is associated with the activation of PXR and YAP pathways.

Structure-immunomodulatory activity relationships of Hedysarum polysaccharides extracted by a method involving a complex enzyme combined with ultrasonication.

A new, more effective and environmentally friendly method involving a complex enzyme combined with ultrasonication was employed to extract and isolate three novel polysaccharides (HPS-MCs: HPS-MC, HPS-MC (50%) and HPS-MC (80%)) of Radix Hedysari. Compared with polysaccharides obtained using a traditional extraction method (hot water extraction, HPS-R), the yields and total carbohydrate contents of HPS-MCs were significantly higher. HPS-MC (80%) exhibited relatively strong immunomodulatory activity and a concentration-dependent dose-response relationship under cyclophosphamide (CP)-induced immunosuppressive conditions in mice models. To more comprehensively investigate the relationships between structural characteristics and immunomodulatory activity, HPS-MC (80%) was fractionated into three major homogeneous polysaccharide fractions (HPS-MC (80%)s: HPS-MC (80%)-1, HPS-MC (80%)-2, and HPS-MC (80%)-3). These three homogeneous polysaccharides had different mass percentages of monosaccharides species (rhamnose, arabinose, mannose, glucose, and galactose) by gas chromatography (GC) and different molecular weights and chain conformations by high-performance gel permeation chromatography coupled with multi-angle laser light scattering (HPGPC-MALLS), and promoted macrophage and splenocyte proliferation to different degrees. These findings indicated that HPS-MC (80%) had a prominent potential immune response, especially HPS-MC (80%)-2 and HPS-MC (80%)-3, and might be suitable candidates for functional foods or potential novel immunomodulators.

Schisandrin B elicits the Keap1-Nrf2 defense system via carbene reactive metabolite which is less harmful to mice liver.

BACKGROUND: Schisandrin B (Sch B) a main active component of Schisandra chinensis, has been shown to act as a liver protectant via activation of the Nrf2 pathway. Nevertheless, it remains unclear whether its reactive metabolite is responsible for Nrf2 activation; also, the effects of its reactive metabolite on liver function are still unknown. METHODS: The present study determined and identifed the carbene reactive metabolite of Sch B in human and mice liver microsomes. Its roles in activating Nrf2 pathway and modifying macromolecules were further explored in human liver microsomes. Moreover the potential cytotoxicity and hepatoxicity of carbene on HepG-2 and mice were also investigated. RESULTS: In the present study, cytochromes P450 (CYP450s) metabolized Sch B to carbene reactive metabolite, which, with the potential to modify peptides, were identifed and observed in human and mice liver microsomes. Moreover, the relevance of carbene in Nrf2 activation was verifed by co-incubation in the presence of CYP450 inhibitors in HepG-2 cells, as well as by molecular docking study of carbene and Keap1. Additionally, the cytotoxicity of Sch B on HepG-2 cells was signifcantly aggravated by CYP450 inducer (with LD50 decreasing from 63 to 21 µM) and signifcantly alleviated by CYP450 inhibitor and glutathione (with LD50 increasing from 63 µM to 200 µM). Besides, after oral administration of mice with Sch B (25-100 mg/kg) for 21 days, only the highest dose induced mild hepatotoxicity, which was accompanied by increasing the aminotransferase activity and centrilobular hepatocellular infltration of lymphocytes. In addition, upregulation of CYP450 activity; Nrf2, NQO-1, and GST expression; and glutathione level was observed in Sch B treatment groups. CONCLUSION: The present study revealed that CYP450s mediate the conversion of Sch B to carbene, which subsequently binds to Keap1 and elicits Nrf2 pathway, which could further increase the elimination of carbene and thus exhibit a less harmful effect on mice liver.

Annonalide and derivatives: Semisynthesis, cytotoxic activities and studies on interaction of annonalide with DNA.

The cytotoxic activity of the pimarane diterpene annonalide (1) and nine of its semisynthetic derivatives (2-10) was investigated against the human tumor cell lines HL-60 (leukemia), PC-3 (prostate adenocarcinoma), HepG2 (hepatocellular carcinoma), SF-295 (glioblastoma) and HCT-116 (colon cancer), and normal mouse fibroblast (L929) cells. The preparation of 2-10 involved derivatization of the side chain of 1 at C-13. Except for 2, all derivatives are being reported for the first time. Most of the tested compounds presented IC50s below 4.0 µM, being considered potential antitumor agents. The structures of all new compounds were elucidated by spectroscopic analyses including 2D NMR and HRMS. Additionally, the interaction of annonalide (1) with ctDNA was evaluated using spectroscopic techniques, and the formation of a supramolecular complex with the macromolecule was confirmed. Competition assays with fluorescent probes (Hoechst and ethidium bromide) and theoretical studies confirmed that 1 interacts preferentially via DNA intercalation with stoichiometric ratio of 1:1 (1:ctDNA). The ΔG value was calculated as -28.24 kJ mol-1, and indicated that the interaction process occurs spontaneously. Docking studies revealed that van der Walls is the most important interaction in 1-DNA and EB-DNA complexes, and that both ligands (1 and EB) interact with the same DNA residues (DA6, DA17 and DT19).

Mechanisms of chromosomal aberrations induced by sesamin metabolites in Chinese hamster lung cells.

Sesamin is a major lignan in sesame seeds and oil. We previously demonstrated that sesamin induces chromosomal aberrations (CA) in Chinese hamster lung (CHL/IU) cells in the presence of a metabolic activation system (S9 mix), although no genotoxicity was detected in vivo. To clarify the mechanism of CA induction by sesamin, we identified its principal active metabolite. A mono-catechol derivative, [2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabi-cyclo[3.3.0]octane (SC-1)], was previously identified in culture medium when sesamin was incubated with S9 mix. In the present study, we show that SC-1 induces CA in CHL/IU cells but not in human hepatoblastoma (HepG2) cells. SC-1 was unstable in culture medium. Addition of glutathione (GSH) to the incubation mixture decreased the rate of decomposition and also suppressed induction of CA in CHL/IU cells. These results indicate that SC-1 itself may not contribute to the induction of CA. Two GSH adducts of SC-1 were identified when SC-1 was incubated with GSH, suggesting that SC-1 was converted to the semiquinone/quinone form and then conjugated with GSH in the culture medium. Sodium sulfite (a quinone-responsive compound) also suppressed CA induction by SC-1. These findings strongly suggest that SC-1 is oxidized to semiquinone/quinone derivatives extracellularly in culture medium, that these derivatives are responsible for the induction of CA in CHL/IU cells, and therefore that the positive results obtained with sesamin in in vitro CA tests using CHL/IU cells may not be relevant to the assessment of in vivo activity.

Schisandrin B inhibits cell growth and induces cellular apoptosis and autophagy in mouse hepatocytes and macrophages: implications for its hepatotoxicity.

A number of drugs and herbal compounds have been documented to cause hepatoxicity. Schisandrin B (Sch B) is an active dibenzocyclooctadiene isolated from Schisandrae fructus, with a wide array of pharmacological activities. However, the potential hepatotoxicity of Sch B is a major safety concern, and the underlying mechanism for Sch B-induced liver toxic effects is not fully elucidated. In the present study, we aimed to investigate the liver toxic effects and the molecular mechanisms of Sch B in mouse liver and macrophage cells. The results have shown that Sch B exhibits potent grow inhibitory, proapoptotic, and proautophagic effects in AML-12 and RAW 264.7 cells. Sch B markedly arrested cells in G1 phase in both cell lines, accompanied by the down-regulation of cyclin dependent kinase 2 (CDK2) and cyclin D1 and up-regulation of p27 Kip1 and checkpoint kinase 1. Furthermore, Sch B markedly increased the apoptosis of AML-12 and RAW 264.7 cells with a decrease in the expression of B-cell lymphoma-extra-large and (Bcl-xl) B-cell lymphoma 2 (Bcl-2), but an increase in the expression of B-cell lymphoma 2-associated X protein (Bax). Sch B promoted the cleavage of caspase 3 and poly-adenosine diphosphate-ribose polymerase (PARP) in both cell lines. Additionally, Sch B significantly induced autophagy of AML-12 and RAW 264.7 cells. Sch B inhibited the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, as indicated by their altered phosphorylation, contributing to the proautophagic effect of Sch B. Taken together, our findings show that the inducing effects of Sch B on cell cycle arrest, apoptosis, and autophagy may contribute to its liver toxic effects, which might provide a clue for the investigation of the molecular toxic targets and underlying mechanisms for Sch B-induced hepatotoxicity in herbal consumers. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Sch B for clinical use.

Effects of the brominated flame retardant TBCO on fecundity and profiles of transcripts of the HPGL-axis in Japanese medaka.

The novel brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO) is an additive flame retardant which is marketed under the trade name Saytex BCL-48. TBCO has recently been investigated as a potential alternative to the major use brominated flame retardant, hexabromocyclododecane (HBCD), which could have major implications for significant increases in amounts of TBCO used. Yet there is a lack of information regarding potential toxicities of TBCO. Recently, results of in vitro experiments have demonstrated the potential of TBCO to modulate endocrine function through interaction with estrogen and androgen receptors and via alterations to the synthesis of 17-ß-estradiol and testosterone. Further research is required to determine potential endocrine disrupting effects of TBCO in vivo. In this experiment a 21-day fecundity assay with Japanese medaka (Oryzias latipes) was conducted to examine endocrine disrupting effects of TBCO in vivo. Medaka were fed a diet containing either 607 or 58µg TBCO/g food, wet mass (wm). Fecundity, measured as cumulative deposition of eggs and fertilization of eggs, as well as abundances of transcripts of 34 genes along the hypothalamus-pituitary-gonadal-liver (HPGL) axis were measured as indicators of holistic endocrine disruption and to determine mechanisms of effects, respectively. Cumulative fecundity was 18% lesser by medaka exposed to 58µg TBCO/g, wm food. However, fecundity of medaka exposed to 607µg TBCO/g, wm food was not significantly different from that of controls. Organ-specific and dose-dependent alterations to abundances of transcripts were observed in male and female medaka. A pattern of down-regulation of expression of genes involved in steroidogenesis, metabolism of cholesterol, and regulatory feedback mechanisms was observed in gonads from male and female medaka which had been exposed to the greater concentration of TBCO. However, these effects on expression of genes were not manifested in effects on fertilization of eggs or fecundity. In livers from male and female medaka exposed to the lesser concentration of TBCO greater expression of genes that respond to exposure to estrogens, including vitellogenin II, choriogenin H, and ERα, were observed. The results reported here confirm the endocrine disrupting potential of TBCO and elucidate potential mechanisms of effects which include specific patterns of alterations to abundances of transcripts of genes in the gonad and liver of medaka.

Time and dose relationships between schisandrin B- and schisandrae fructus oil-induced hepatotoxicity and the associated elevations in hepatic and serum triglyceride levels in mice.

BACKGROUND: Schisandrin B (Sch B), a dibenzocyclooctadiene compound, is isolated from schisandrae fructus (SF). This study was conducted to compare the time- and dose-response between Sch B- and SF oil (SFO)-induced changes in hepatic and serum parameters in mice. METHODS: Institute of Cancer Research (ICR) mice were given a single oral dose of Sch B (0.125-2 g/kg) or SFO (0.3-5 g/kg). Serum alanine aminotransferase (ALT) activity, hepatic malondialdehyde, and triglyceride (TG) levels were measured at increasing time intervals within 6-120 hours postdosing. RESULTS: Serum ALT activity was elevated by 60%, with maximum effect (E(max)) = 45.77 U/L and affinity (K(D)) = 1.25 g/kg at 48-96 hours following Sch B, but not SFO, treatment. Sch B and SFO treatments increased hepatic malondialdehyde level by 70% (E(max) =2.30 nmol/mg protein and K(D) =0.41 g/kg) and 22% (E(max) = 1.42 nmol/mg protein and K D = 2.56 g/kg) at 72 hours postdosing, respectively. Hepatic index was increased by 16%-60% (E max = 11.01, K(D) = 0.68 g/kg) and 8%-32% (E(max) = 9.88, K D = 4.47 g/kg) at 12-120 hours and 24-120 hours after the administration of Sch B and SFO, respectively. Hepatic TG level was increased by 40%-158% and 35%-85%, respectively, at 12-96 hours and 6-48 hours after Sch B and SFO treatment, respectively. The values of E max and K D for Sch B/SFO-induced increase in hepatic TG were estimated to be 22.94/15.02 µmol/g and 0.78/3.03 g/kg, respectively. Both Sch B and SFO increased serum TG (up to 427% and 123%, respectively), with the values of E(max) = 5.50/4.60 mmol/L and K D = 0.43/2.84 g/kg, respectively. CONCLUSION: The findings indicated that Sch B/SFO-induced increases in serum/hepatic parameters occurred in a time-dependent manner, with the time of onset being serum TG level < hepatic TG level < hepatic index < serum ALT activity. However, the time of recovery of these parameters to normal values varied as follow: serum TG level < hepatic TG level and liver injury < hepatic index. The E max and affinity of Sch B on tissue/enzyme/receptor were larger than those of SFO.

Effects of novel brominated flame retardants on steroidogenesis in primary porcine testicular cells.

Brominated flame retardants are chemicals with fire quenching properties which are extensively used in manufacturing. Historically, less regulated use of legacy brominated flame retardants (BFRs) for a number of years has resulted in ubiquitous contamination of the environment. As a result, some of the more persistent BFRs have been phased out and are being replaced by a next generation of brominated compounds for which there is little toxicological data. The study investigated effects of 2-ethylhexyl tetrabromobenzoate (TBB), 1,2,5,6-tetrabromocyclooctane (TBCO), and bis-(2-ethylhexyl) tetrabromophthalate (TBPH)on steroidogenesis in a porcine primary testicular cell model. TBB did not affect sex-steroid production in this cell model; rather the data suggest a flux towards synthesis of aldosterone and cortisol via up-regulation of CYP21A2. At the greatest concentrations of TBCO and TBPH tested greater production of sex hormones testosterone (T) and estradiol (E2) was observed. Effects were mediated by regulation of multiple molecular targets in the steroidogenesis pathway; CYP11A in the case of TBPH and CYP17A1 in the case of TBCO. This investigation is the first of its kind to use a testicular mixed population cell model to investigate mechanism(s) of action of three chemically diverse compounds currently used in commercial fire retardants.

Fragrance material review on 1-[5(or 6)-methyl-7(or 8)-1-(methylethyl)bicyclo[2.2.2]oct-5-en-2-yl]ethan-1-one.

A toxicologic and dermatologic review of 1-[5(Or 6)-Methyl-7(or 8)-1-(methylethyl)bicyclo[2.2.2]oct-5-en-2-yl]ethan-1-one when used as a fragrance ingredient is presented. 1-[5(Or 6)-Methyl-7(or 8)-1-(methylethyl)bicyclo[2.2.2]oct-5-en-2-yl]ethan-1-one is a member of the fragrance structural group Alkyl Cyclic Ketones. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for 1-[5(Or 6)-Methyl-7(or 8)-1-(methylethyl)bicyclo[2.2.2]oct-5-en-2-yl]ethan-1-one were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, mucous membrane (eye) irritation, skin sensitization, phototoxicity, photoallergy, and genotoxicity, data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al., Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013. A Toxicologic and Dermatologic Assessment of Alkyl Cyclic Ketones When Used as Fragrance Ingredients. (submitted for publication)). for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances.

In vitro endocrine disruption and TCDD-like effects of three novel brominated flame retardants: TBPH, TBB, & TBCO.

The novel brominated flame retardants (NBFRs), 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB), Bis(2-ethylhexyl)-2,3,4,5-tetrabromophtalate (TBPH), and 1,2,5,6-tetrabromocyclooctane (TBCO) are components of flame retardant mixtures including Firemaster 550 and Saytex BC-48. Despite the detection of these NBFRs in environmental and biotic matrices, studies regarding their toxicological effects are poorly represented in the literature. The present study examined endocrine disruption by these three NBFRs using the yeast YES/YAS reporter assay and the mammalian H295R steroidogenesis assay. Activation of the aryl hydrocarbon receptor (AhR) was also assessed using the H4IIE reporter assay. The NBFRs produced no TCDD-like effects in the H4IIE assay or agonistic effects in the YES/YAS assays. TBB produced a maximal antiestrogenic effect of 62% at 0.5mgL(-1) in the YES assay while TBPH and TBCO produced maximal antiandrogenic effects of 74% and 59% at 300mgL(-1) and 1500mgL(-1), respectively, in the YAS assay. Significant effects were also observed in the H295R assay. At 0.05mgL(-1), 15mgL(-1), and 15mgL(-1) TBB, TBPH, and TBCO exposures, respectively resulted in a 2.8-fold, 5.4-fold, and 3.3-fold increase in concentrations of E2. This is one of the first studies to demonstrate the in vitro endocrine disrupting potentials of TBB, TBPH, and TBCO.

Neglschisandrins E-F: two new lignans and related cytotoxic lignans from Schisandra neglecta.

Phytochemical investigation of an ethanolic extract of stems of Schisandra neglecta led to the isolation and identification of two new dibenzocyclooctadiene lignans, designated neglschisandrins E (1) and F (2), and thirteen known lignans. All structures and stereochemistries were determined by spectroscopic methods, including 2D-NMR techniques. The isolates were evaluated for in vitro cytotoxic activity. Among them, compounds 2-6 exhibited moderate to weak cytotoxicity against the human colorectal carcinoma HCT-8 cell line with EC50 values of 7.33~19.8 µg/mL. In addition, compounds 2-4 also exhibited marginal cytotoxicity against the human lung carcinoma A549 cell line with EC50 values of 11.8~15.0 µg/mL.

Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response.

ATM and ATR protein kinases play a crucial role in cellular DNA damage responses. The inhibition of ATM and ATR can lead to the abolition of the function of cell cycle checkpoints. In this regard, it is expected that checkpoint inhibitors can serve as sensitizing agents for anti-cancer chemo/radiotherapy. Although several ATM inhibitors have been reported, there are no ATR-specific inhibitors currently available. Here, we report the inhibitory effect of schisandrin B (SchB), an active ingredient of Fructus schisandrae, on ATR activity in DNA damage response. SchB treatment significantly decreased the viability of A549 adenocarcinoma cells after UV exposure. Importantly, SchB treatment inhibited both the phosphorylation levels of ATM and ATR substrates, as well as the activity of the G2/M checkpoint in UV-exposed cells. The protein kinase activity of immunoaffinity-purified ATR was dose-dependently decreased by SchB in vitro (IC(50): 7.25 muM), but the inhibitory effect was not observed in ATM, Chk1, PI3K, DNA-PK, and mTOR. The extent of UV-induced phosphorylation of p53 and Chk1 was markedly reduced by SchB in ATM-deficient but not siATR-treated cells. Taken together, our demonstration of the ability of SchB to inhibit ATR protein kinase activity following DNA damage in cells has clinical implications in anti-cancer therapy.

Ceramidastin, a novel bacterial ceramidase inhibitor, produced by Penicillium sp. Mer-f17067.

Decrease of ceramide in the skin is one of the aggravating factors of atopic dermatitis. The skin is often infected by ceramidase-producing bacteria, such as Pseudomonas aeruginosa. The bacterial ceramidase then degrades ceramide in the skin. To develop anti-atopic dermatitis drugs, we searched for ceramidase inhibitors, which led to the discovery of ceramidastin, a novel inhibitor of bacterial ceramidase, from the culture broth of Penicillium sp. Mer-f17067. Ceramidastin inhibited the bacterial ceramidase with an IC(50) value of 6.25 microg ml(-1). Here we describe the isolation, physicochemical properties, structure determination and biological activity of ceramidastin.

Nephrotoxicity of 4-cycloocten-1-carbaldehyde.

Natural and synthetic chemicals are often used in the fragrance industry. A toxicological profile of the synthetic fragrance booster, 4-cycloocten-1-carbaldehyde, was generated using a test program including the following methods: acute oral toxicity, acute dermal toxicity, acute skin and eye irritation, skin sensitization, subchronic toxicity, and mutagenicity. The substance was strongly irritating to the skin but only weakly irritating to the eye. It gave a clear indication of having skin-sensitizing properties. Based on the comprehensive data from a mutagenicity test battery, 4-cycloocten-1-carbaldehyde was assessed to be nonmutagenic. Although its acute toxicological profile shows no toxicity after oral or dermal application, 4-cycloocten-1-carbaldehyde displays a complex toxicological response after repeated dosing over 13 weeks. 4-Cycloocten-1-carbaldehyde or its metabolites show clear nephrotoxic properties focusing on tubular cells of the kidney. In view of these data no no-effect level can be derived from this study with 4-cycloocten-1-carbaldehyde. A broad interaction of the test substance with various tissue types and cell parameters together with severe and irreversible organic defects even at low doses leads to the conclusion that 4-cycloocten-1-carbaldehyde is unsuitable for the intended use in industrial fragrance formulations.

GABA receptor antagonists and insecticides: common structural features of 4-alkyl-1-phenylpyrazoles and 4-alkyl-1-phenyltrioxabicyclooctanes.

Fipronil [5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulfinylpyrazole] is one of the most important insecticides. Structure-activity studies described here reveal that fipronil retains its very high binding potency at the human beta3 and house fly gamma-aminobutyric acid (GABA) receptors and toxicity to house flies on replacing the pyrazole trifluoromethylsulfinyl moiety with tert-butyl or isopropyl and the phenyl trifluoromethyl substituent with ethynyl, trifluoromethoxy, bromo or chloro. Among the compounds studied, those with other alkyl groups at the 4-position of the pyrazole, as well as phenyl substitution without one or both of the 2,6-dichloro groups, are less effective. 5-Amino-4-tert-butyl-3-cyano-1-(2,6-dichloro-4-ethynylphenyl)pyrazole is highly effective and almost isosteric with 4-tert-butyl-3-cyano-1-(4-ethynylphenyl)-2,6,7-trioxabicyclo[2.2.2]octane (the most potent 4-alkyl-1-phenyltrioxabicyclooctane) as a noncompetitive GABA antagonist and insecticide. These findings are interpreted as three binding subsites in the GABA receptor: a hydrophobic site undergoing steric interaction with the tert-butyl or equivalent group; a hydrogen bonding site to pyrazole N-2; a pi bonding site to the face of the phenyl moiety; with supplemental enhancement by the 3-cyano and 4-ethynyl substituents.