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Dioxane

Q: What are the concerns regarding Dioxane?

Dioxane has been classified as a possible human carcinogen, and its environmental release is a concern due to its persistence and potential for groundwater contamination. Researchers need to be mindful of these risks when working with Dioxane.

Q: How can PubCompare.ai help with Dioxane research?

PubCompare.ai allows you to screen protocol literature more efficiently and leverage AI to pinpoint critical insights. The platform can help researchers identify the most effective protocols related to Dioxane for their specific research goals. The AI-driven analysis can highlight key differences in protocol effectiveness, enabling you to choose the best option for reproducibility and accracy.

Q: Are there different types or variations of Dioxane?

Dioxane is a specific chemical compound, and there are no major variations or types of Dioxane. However, Dioxane can be found in different purity levels or formulations depending on the industrial or laboratory application.

Q: What are some common applications of Dioxane?

Dioxane is widely used as a solvent in a variety of industries. Some of the key applications include the production of pharmaceuticals, plastics, and textiles. It is also used in laboratory settings for a range of experiments and analysis procedures.

Dioxane is a synthetic organic compound with the chemical formula C4H8O2.
It is a colorless, flammable liquid with a faint ethereal odor.
Dioxane is used as a solvent in a variety of industrial and laboratory applications, including the production of pharmaceuticals, plastics, and textiles.
However, dioxane has been classified as a possible human carcinogen and its environmental release is a concern due to its persistence and potential for groundwater contamination.
Researchers studying dioxane exposure and remediation can optimize their work using the PubCompare.ai tool, which helps locate the best protocols and enhance the reproducibility and accuracy of dioxane-related studies.

Most cited protocols related to «Dioxane»

Automated Derivation of Atomic Charges

Cited 153 times

Geometry optimization and MEP computation were performed using the Gaussian (versions 98 and 03),76 , 77 GAMESS-US [versions 24 Mar 2007 (R3) and January 2009 (R1)],54 and the PC-GAMESS/Firefly (versions 7.1) programs,55 on a 1.67 GHz SGI Altix running the SUSE Linux Enterprise Server 10 operating system, an IBM RS6000 based cluster (AIX 5.2), R5000 and R12000 SGI workstations (IRIX 6.5.22), and/or PC Linux based workstations (Fedora 6.0, 8.0 and CentOS 5.2). RESP and ESP charge fitting was carried out using the RESP program.28 The latter program was modified and recompiled to slightly increase the charge accuracy as well as the maximum number of charges, Lagrange constraints and molecules allowed during the fitting step (the convergence criteria “qtol”, the maximum number of charge values “maxq”, the maximum number of lagrange constraints “maxlgr” and the maximum number of molecules “maxmol” were adjusted to 1.0d-5, 5000, 500 and 200, respectively). The HF method and the 6-31G* basis set were used to optimize molecular geometries.33 -35 MEP were computed based on two different approaches: using either (i) the HF/6-31G* theory level in the gas phase,28 , 29 or (ii) the density functional theory (DFT) method, the B3LYP exchange and correlation functionals, the IEFPCM continuum solvent model (ε = 4) to mimic organic solvent environment, and the cc-pVTZ basis set.48 -50 (link) The HF/STO-3G theory level.34 , 36 , 37 was also tested to calculate MEP since it was used in ESP charge derivation for the Weiner et al. force field.38 , 39 Both the CHELPG and Connolly surface algorithms used in MEP calculation were considered in this work.10 , 15 , 16 Charge derivation and building force field library reported here were carried out by the R.E.D. Tools. Initial structures were constructed using the LEaP or InsightII program.74 , 78 The corresponding optimized geometries and charge values were displayed using the LEaP or VMD program.74 , 79
More than fifty molecular systems have been considered in this work in order to demonstrate the different capabilities of the R.E.D. Tools. Considering the large amount of data generated only few characteristic results will be presented below. The entire set of data is summarized in the Table S4 of the supplementary material, and is available in R.E.DD.B. It includes well-studied structures for which atomic charge values are known allowing for comparisons with published data and creating a benchmark. Several new molecular systems are also reported. The first group of studied structures includes organic molecules such as ethanol (anti and gauche+ conformations),29 , 43 , 47 (link), 80 , 81 dimethylsulfoxide,81 -83 dimethylphosphate (gauche+, gauche+ conformation),40 , 59 trifluoroethanol (anti and gauche+ conformations),84 -86 (link) methoxyethane (anti and gauche+ conformations),40 , 43 , 47 (link), 80 , 87 N-methylacetamide (cis and trans conformations),28 , 29 , 40 , 43 , 80 , 87 , 88 1-4-dioxane (chair and twist-boat conformations),43 , 89 , 90 ethane-1,2-diol (anti anti anti, anti gauche+ anti, gauche+ anti gauche-, gauche+ gauche- gauche+ and gauche+ gauche+ gauche+ conformations),29 , 47 (link), 80 methanol,25 , 28 , 29 , 40 , 47 (link), 80 propanone, ethanoic acid,43 , 80 acetonitrile,25 formamide,25 , 87 methanal,87 furane,87 pyrrole, benzene,40 , 80 toluene,80 chloroforme,81 cyclohexane (chair and twist-boat conformations).43 , 80 , 90 These molecules were involved in explicit solvent MD simulations and/or force field development in the past. The second group of structures studied consists of bio-molecules such as alanine dipeptide (C5, C7ax and C7eq conformations),21 , 25 , 40 , 80 , 91 as well as standard deoxyribonucleosides (i. e. deoxyadenosine, deoxycytidine, deoxyguanosine and thymidine in the C2’-endo and C3’-endo conformations) and ribonucleosides (i. e. adenosine, cytidine, guanosine and uridine in the C3’-endo conformation).40 , 42 (link) Finally, following the strategy proposed by Cieplak et al.,59 charge derivation and force field library building were carried out for various molecular fragments of unusual amino acids as well as for standard nucleic acid nucleotides. The central, H3N(+)-terminal, (-)O2C-terminal molecular fragments (as well as terminal neutral fragments) of alpha-aminoisobutyric acid92 (link), 93 (link) and O-methyl-L-tyrosine residues94 (link) were generated using the corresponding N-acetyl N’-methylamide amino acid (with φ, ψ dihedral angles characteristic for the α-helix and/or β-sheet secondary structures), methylammonium and acetate. The central, 5’-terminal and 3’-terminal fragments of standard nucleic acid nucleotides for DNA and RNA were obtained using dimethylphosphate (gauche+, gauche+ conformation), the four deoxyribonucleosides (C2’-endo and C3’-endo conformations) and/or four ribonucleosides (C3’-endo conformation).
In the following section of the article we will discuss reproducibility of the RESP or ESP charge models. We will first compare the charge values of single conformation molecular systems determined by the same QM program, i. e. using either Gaussian or GAMESS-US. For every molecule, geometry optimization was performed using four different sets of initial Cartesian coordinates selected randomly. Computation of MEP and derivation of charge values were carried out using each optimized Cartesian coordinate set. We will compare results obtained using the Gaussian and GAMESS-US programs. In this context, the role of ab initio threshold criteria during geometry optimization, and the impact of different optimized geometry re-orientation procedures, available in both programs, on the charge values will be addressed. Finally, we will discuss a rigid-body re-orientation algorithm based on the selection of any three atoms, which has been implemented in the R.E.D. source code to provide a general method for reorienting optimized geometries before MEP computation. This approach is independent of the QM program used for calculations. According to this strategy, the first selected atom is translated to the origin of axes, the first two atoms define the (O, X) axis while the third one is used to define the (O, X, Y) plane. The (O, Z) axis is automatically set as the cross-product between the (O, X) and (O, Y) axes.71 , 72 This approach can be used for every optimized molecular geometry, and is the basis for multiple orientation charge fitting.
In the last section, we will demonstrate how multiple orientation and multiple conformation can be combined together during charge derivation. The R.E.D. program provides easy setup for handling MEP computation (using either the Connolly surface or the CHELPG algorithm), single ESP stage, single RESP stage as well as two RESP stage fitting, which makes it an efficient tool for comparing various charge models. In addition, the introduction of intra-molecular charge constraint(s) during charge fit extends the number of charge models and allows building force field libraries of molecular fragments in a similar way as it is done for the central fragment of an amino acid employed for building polypeptide chains.59 Examples of charge derivation involving multiple orientations, multiple conformations and multiple molecules will be then described. Including more than one molecule in charge derivation and introducing inter-molecular charge constraints and inter-molecular charge equivalencing during the charge fitting allows determining atomic charges for a large variety of molecular fragments.47 (link), 59 Thus, inter-molecular charge constraints can be used for defining molecular fusion between two molecules by eliminating groups of atoms with zero sum of charge. This approach is applied in the process of an automatic generation of the force field libraries of molecular fragments, from which larger systems can be built, and is a standard strategy for creating libraries of the central and terminal amino acid and nucleotide fragments. By analogy, this method can be directly extended to other biomolecular systems such as oligosaccharides, glycoconjugates as well as bio-inorganic complexes. In addition to the above features, R.E.D. is capable of generating all-atom or united-carbon atom charge models, and create appropriate force field libraries, which can be readily used for validation in MD simulations.38 , 40 , 59 , 73 (link) The simultaneous formation of an ensemble of force field libraries for a family of structures or FFTopDB is then presented and discussed using standard nucleic acids as an example.

Dupradeau F.Y., Pigache A., Zaffran T., Savineau C., Lelong R., Grivel N., Lelong D., Rosanski W, & Cieplak P. (2010). The R.E.D. Tools: Advances in RESP and ESP charge derivation and force field library building. Physical chemistry chemical physics : PCCP, 12(28), 7821-7839.

Publication 2010

Synthesis of Lignin Model Compounds

Cited 15 times

Model compounds were prepared and their NMR spectra were acquired in DMSO to enable the assignments made in a previous paper.² Coniferyl alcohol and sinapyl alcohol were prepared from commercially available coniferaldehyde and sinapaldehyde using borohydride exchange resin.^{105 (link)}p-Coumaryl alcohol was synthesized from p-coumaric acid.¹⁰⁶ Coniferyl alcohol dimers were synthesized from in vitro radical coupling reactions using MnO₂ in dioxane :H₂O (1 : 1, v/v).¹⁰⁷ Sinapyl alcohol dimers were prepared using FeCl₃·6H₂O in dioxane :H₂O (5 : 2, v/v).¹⁰⁸p-Coumaryl alcohol dimers were synthesized with horseradish peroxidase with hydrogen peroxide in acetone :water (1 : 10, v/v) or with FeCl₃·6H₂O in acetone: H₂O (5 : 1, v/v). Each metallic oxidative radical reaction was stirred for 1 to 4 h, and the metal salts were filtered off through a silica gel bed in fine sintered glass filters. The peroxidase reactions were conducted for about 15 h. Reaction solutions were poured into EtOAc, and washed with satd. aqueous NH₄Cl. EtOAc extracts were dried over anhydrous MgSO₄, and concentrated under reduced pressure. Model dimers were separated on preparative TLC plates with CHCl₃–MeOH (10 : 1, v/v). The fully authenticated NMR data for model compounds will be deposited in the “NMR Database of lignin and cell wall model compounds” available via the internet.⁴⁸

Kim H, & Ralph J. (2009). Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5. Organic & biomolecular chemistry, 8(3), 576-591.

Publication 2009

Acetone Borohydrides Cell Wall Chloroform coniferaldehyde coniferyl alcohol Coumaric Acids dioxane Ethanol Horseradish Peroxidase Lignin Metals p-coumaryl alcohol Peroxidase Peroxide, Hydrogen Pressure Resins, Plant Salts Silica Gel sinapaldehyde sinapyl alcohol Sulfate, Magnesium Sulfoxide, Dimethyl

Fabrication of 3D NF-Gelatin Scaffolds

Cited 8 times

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Publication 2009

Bath CD3EAP protein, human Cold Temperature Cyclohexane dioxane Dry Ice Ethanol Freezing Fungus, Filamentous Gelatins n-hexane Paraffin Sodium Chloride Solvents Teflon Vacuum

Synthesis and Evaluation of Glycyrrhetinic Acid Derivatives

Cited 8 times

Reagents: CDDO-Me 1 was synthesized from oleanolic acid according to a previously described method[5 (link)] with 10 % yield (NMR ¹H and ¹³C data).
18βH-Glycyrrhetinic acid acetate 2, obtained from a licorice extract, was used as starting material (purity∼94 %).[39 ]
General experimental procedures: Melting points were determined on a Hoover melting point apparatus and were uncorrected. The elemental composition of the products was determined from high-resolution mass spectra recorded on a DFS (Double Focusing Sector) Thermo Electron Corporation instrument. ¹H and ¹³C NMR spectra were measured from CDCl₃ solutions on Bruker spectrometers: AM-400 (400.13mhz for ¹H, 100.61mhz for ¹³C) and DRX-500 (500.13mhz for ¹H, 125.76mhz for ¹³C). Chloroform was used as the internal standard (δ_H 7.24 ppm, δ_C 76.90 ppm). The structure of the compounds was determined by NMR from proton spin–spin coupling constants in ¹H,¹H double-resonance spectra, and by analyzing ¹³C NMR proton-selective and off-resonance saturation spectra, 2D ¹³C,¹H correlated spectroscopy on CH constants (COSY, ¹J_C,H=135 Hz; and COLOC, ^{2, 3}J_C,H=10 Hz, correspondingly), and 1D ¹³C,¹H long-range J modulation difference (LRJMD, J_C,H=10 Hz). Flash column chromatography was performed with silica gel (Merck, 60–200 mesh) and neutral alumina (Chemapol, 40–250 mesh).
Methyl 18βH-Glycyrrhetinate acetate (3):[42 ] A solution of diazomethane in ether was added dropwise at 0°C to a stirred suspension of 2 (10 g, 19.0 mmol) in methanol (200 mL) until the originally colorless mixture turned yellow. The resulting mixture was allowed to stand at room temperature overnight. The solvent was removed and the product was purified by crystallization (chloroform/methanol; yield=9.1 g, 89 %). M.p. 303–304°C; ¹H NMR (CDCl₃): δ=0.76 (dd, ³J(H^5a,H^6a)=12.5, ³J(H^5a,H^6e)=1.5 Hz; H^5a), 0.76 (s, 3 H; C28-H₃), 0.84 (s, 6 H; C23-H₃, C24-H₃), 0.97 (dm, ²J(H^16e,H^16a)=13.8 Hz; H^16e), 1.01 (ddd, ²J(H^1a,H^1e)=13.5, ³J(H^1a,H^2a)=13.5, ³J(H^1a,H^2e)=3.7 Hz; H^1a), 1.08 (s, 3 H; C26-H₃), 1.10 (s, 3 H; C29-H₃), 1.12 (s, 3 H; C25-H₃), 1.14 (dm, ²J(H^15e_,H^15a)=13.8 Hz; H^15e), 1.23–1.39 (m, 4 H; H⁷, H^21a, 2 H²²), 1.32 (s, 3 H; C27-H₃), 1.41 (dddd, ²J(H^6a_,H^6e)=13.5, ³J(H^6a_,H^7a)=13.5, ³J(H^6a_,H^5a)=12.0, ³J(H^6a,H^7e)=3.2 Hz; H^6a), 1.51–1.66 (m, 3 H; H^6e, H^2e, H^7′), 1.57 (dd, ²J(H^19a,H^19e)=13.5, ³J(H^19a,H^18a)=13.5 Hz; H^19a), 1.66 (dddd, ²J(H^2a,H^2e)=13.5, ³J(H^2a,H^1a)=13.5, ³J(H^2a,H^3a)=11.7, ³J(H^2a,H^1e)=3.7 Hz; H^2a), 1.78 (ddd, ²J(H^15a,H^15e)=13.8, ³J(H^15a,H^16a)=13.8, ³J(H^15a_,H^16e)=4.5 Hz; H^15a), 1.88 (ddd, ²J(H^19e,H^19a)=13.5, ³J(H^19e,H^18a)=4.2, ⁴J(H^19e,H^21e)=2.7 Hz; H^19e), 1.95 (dm ²J(H^21e,H^21a)=10 Hz; H^21e), 1.98 (ddd, ²J(H^16a,H^16e)=13.8, ³J(H^16a,H^15a)=13.8, ³J(H^16a,H^15e)=4.8 Hz; H^16a), 2.00 (s, 3 H; C33-H₃), 2.04 (dd, ³J(H^18a,H^19a)=13.5, ³J(H^18a,H^19e)=4.2 Hz; H^18a), 2.32 (s, 1 H; H^9a), 2.76 (ddd ²J(H^1e,H^1a)=13.5, ³J(H^1e,H^2a)=3.7, ³J(H^1e,H^2e)=3.0 Hz; H^1e), 3.64 (s, 3 H; OC31-H₃), 4.47 (dd, ³J(H^3a,H^2a)=11.7, ³J(H^3a,H^2e)=4.7 Hz; H^3a), 5.62 (s, 1 H; H¹²); ¹³C NMR (CDCl₃): δ=38.63 (t, C1), 23.41 (t, C2), 80.45 (d, C3), 37.88 (s, C4), 54.88 (d, C5), 17.22 (t, C6), 32.55 (t, C7), 43.03 (s, C8), 61.56 (d, C9), 36.78 (s, C10), 199.85 (s, C11), 128.34 (d, C12), 169.01 (s, C13), 45.23 (s, C14), 26.31 (t, C15), 26.26 (t, C16), 31.67 (s, C17), 48.25 (d, C18), 40.93 (t, C19), 43.87 (s, C20), 30.98 (t, C21), 37.59 (t, C22), 27.89 (q, C23), 16.52 (q, C24), 16.24 (q, C25), 18.52 (q, C26), 23.17 (q, C27), 28.36 (q, C28), 28.15 (q, C29), 176.73 (s, C30), 51.58 (q, C31), 170.77 (s, C32), 21.13 (q, C33); HRMS: m/z calcd for C₃₃H₅₀O₅: 526.7471; found: 526.3658.
Methyl 3 β-Acetoxy-18 βH-olean-12-en-30-oate (4):[43 ] A solution of conc. hydrochloric acid (50 mL) was added dropwise at 10°C to a stirred suspension of 3 (9.1 g, 17.3 mmol) and zinc powder (18.2 g, 280 mmol) in dioxane (300 mL) over 2 h. The reaction mixture was stirred for a further 3 h at 5–10°C, concentrated in a vacuum, diluted with water (1 L), and filtered. The solid was dried and subjected to flash column chromatography (silica gel; benzene followed by chloroform) to give crude 4 (yield=6.8 g, 77 %). This material was used for the next reaction without further purification. An analytically pure sample was obtained by recrystallization from a mixture chloroform/methanol. M.p. 265–267°C; ¹H NMR (CDCl₃): δ=0.74 (s, 3 H; C28-H₃), 0.81 (dd, ³J(H^5a,H^6a)=12.0, ³J(H^5a,H^6e)=1.6 Hz; H^5a), 0.82–0.86 (m, H^16e), 0.83 (s, 3 H; C24-H₃), 0.84 (s, 3 H; C23-H₃), 0.93 (s, 6 H; C25-H₃, C26-H₃), 0.94 (dm ²J(H^15e,H^15a)=13.5 Hz; H^15e), 1.02 (m; H¹), 1.09 (s, 3 H; C29-H₃), 1.10 (s, 3 H; C27-H₃), 1.17–1.35 (m, 4 H; H⁷, H²¹, 2 H²²), 1.39 (m, H^6a), 1.44–1.64 (m, 7 H; H^1′, 2 H², H^6e, H^7′, H^9a, H¹⁹), 1.73 (ddd, ²J(H^15a,H^15e)=13.5, ³J(H^15a,H^16a)=13.5, ³J(H^15a,H^16e) 4.6 Hz; H^15a), 1.79–1.93 (m, 5 H; 2 H¹¹, H¹⁸, H^19′, H^21′), 1.92 (m; H^16a), 2.01 (s, 3 H; C33-H₃), 3.64 (s, 3 H; OC31-H₃), 4.47 (dd, ³J(H^3a,H^2a)=10.0, ³J(H^3a,H^2e)=6.0 Hz; H^3a), 5.23 (t, ²J(H¹²,H¹¹)=3.6 Hz; H¹²); ¹³C NMR (CDCl₃): δ=38.13 (t, C1), 23.42 (t, C2), 80.74 (d, C3), 37.56 (s, C4), 55.13 (d, C5), 18.11 (t, C6), 32.46 (t, C7), 39.65 (s, C8), 47.42 (d, C9), 36.70 (s, C10), 23.34 (t, C11), 122.34 (d, C12), 144.23 (s, C13), 41.38 (s, C14), 25.99 (t; C15), 26.82 (t, C16), 31.79 (s, C17), 48.05 (d, C18), 42.68 (t, C19), 44.12 (s, C20), 31.15 (t, C21), 38.25 (t, C22), 27.89 (q, C23), 16.54 (q, C24), 15.41 (q, C25), 16.64 (q, C26), 25.77 (q, C27), 28.03 (q, C28), 28.38 (q, C29), 177.46 (s, C30), 51.35 (q, C31), 170.77 (s, C32), 21.13 (q, C33); HRMS: m/z calcd for C₃₃H₅₂O₄: 512.7636; found: 512.3866.
Methyl 3β-acetoxy-12-oxo-18βH-olean-30-oate (5): A mixture of hydrogen peroxide (∼30 %, 25 mL) and acetic acid (25 mL) was added dropwise at 80°C to a stirred suspension of 4 (3.0 g, 5.7 mmol) in acetic acid (100 mL) over 1 h. The reaction mixture was stirred for a further 30 min at 80°C, cooled to room temperature, and diluted with water (500 mL). The solid was filtered, washed with water, and dried to give crude 5 (yield=6.8 g, 96 %). This material was used for the next reaction without further purification. An analytically pure sample was obtained by recrystallization from a mixture chloroform/methanol. M.p. 296–299°C; ¹H NMR (CDCl₃): δ=0.79–0.88 (m, 2 H; H^5a, H^16e), 0.82 (s, 3 H; C28-H₃), 0.83 (s, 3 H; C24-H₃), 0.84 (s, 3 H; C23-H₃), 0.86 (s, 3 H; C25-H₃), 0.90 (s, 3 H; C27-H₃), 0.92–1.01 (m, 2 H; H¹, H^15e), 1.09 (s, 3 H; C29-H₃), 1.10 (s, 3 H; C26-H₃), 1.19 (dd, ²J(H^19a,H^19e)=13.4, ³J(H^19a,H¹⁸)=13.4 Hz; H^19a), 1.18–1.27 (m, 2 H; H^21a, H^22e), 1.31–1.48 (m, 4 H; 2 H⁷, H^22a, H⁶), 1.49–1.64 (m, 4 H; H^1′, 2 H², H^6′), 1.64 (dd ³J(H^9a,H^11a)=13.0, ³J(H^9a,H^11e)=5.1 Hz; H^9a), 1.74 (m, H¹⁸), 1.76 (m, H^15a), 1.85 (ddd, ²J(H^16a,H^16e)=13.2, ³J(H^16a,H^15a)=13.2, ³J(H^16a,H^15e)=4.2 Hz; H^16a), 1.91 (dm, ²J(H^21e,H^21a)=13.2 Hz; H^21e), 2.00 (s, 3 H; C33-H₃), 2.12 (dd, ²J(H^11a,H^11e)=17.0, ³J(H^11a,H^9a)=13.0 Hz; H^11a), 2.23 (dd, ²J(H^11e,H^11a)=17.0, ³J(H^11e,H^9a)=5.1 Hz; H^11e), 2.54 (ddd, ²J(H^19e,H^19a)=13.4, ³J(H^19e,H¹⁸)=3.4, ⁴J(H^19e,H^21e)=2.8 Hz; H^19e), 2.72 (d, ³J(H¹³,H¹⁸)=4.4 Hz; H¹³), 3.68 (s, 3 H; OC31-H₃), 4.44 (dd, ³J(H^3a,H^2a)=11.4, ³J(H^3a,H^2e)=4.8 Hz; H^3a); ¹³C NMR (CDCl₃): δ=37.50 (t, C1), 23.26 (t, C2), 80.28 (d, C3), 37.60 (s, C4), 55.04 (d, C5), 18.05 (t, C6), 31.63 (t, C7), 41.41 (s, C8), 49.32 (d, C9), 36.67 (s, C10), 38.28 (t, C11), 211.91 (s, C12), 50.08 (d, C13), 41.90 (s, C14), 25.84 (t, C15), 26.29 (t, C16), 32.01 (s, C17), 38.41 (d, C18), 34.05 (t, C19), 43.97 (s, C20), 31.19 (t, C21), 38.33 (t, C22), 27.75 (q, C23), 16.30 (q, C24), 15.16 (q, C25), 15.95 (q, C26), 20.78 (q, C27), 26.83 (q, C28), 28.59 (q, C29), 177.44 (s, C30), 51.36 (q, C31), 170.70 (s, C32), 21.09 (q, C33); HRMS: m/z calcd for C₃₃H₅₂O₅: 528.7630; found: 528.3815.
Methyl 3β-acetoxy-12-oxo-18βH-olean-9(11)-en-30-oate (6): Compound 6 was synthesized according to a known method.[44 ] Briefly, a solution of bromine (0.5 mL, 9.8 mmol) in glacial acetic acid (50 mL) was added dropwise at 80°C to a stirred solution of 5 (4.0 g, 7.6 mmol) in glacial acetic acid (150 mL) over 1 h. The reaction mixture was stirred for a further 1 hour at 80°C, cooled to room temperature, diluted with water (1.5 L) and filtered. The solid was washed with water, dried, and subjected to flash column chromatography (silica gel, chloroform) to give a solid 6 (yield=3.0 g, 75 %). This material was used for the next reaction without further purification. An analytically pure sample was obtained by recrystallization from a mixture chloroform/methanol. M.p. 298°C; ¹H NMR (CDCl₃): δ=0.84 (m; H^16e), 0.85 (s, 3 H; C24-H₃), 0.86 (s, 6 H; C23-H₃, C28-H₃), 0.91 (s, 3 H; C27-H₃), 0.94 (dm, ³J(H^5a,H^6a)=10 Hz; H^5a), 1.01 (dm, ²J(H^15e,H^15a)=13.2 Hz; H^15e), 1.07 (s, 3 H; C29-H₃), 1.16 (s, 3 H; C25-H₃), 1.20 (dd, ²J(H^19a,H^19e)=13.3, ³J(H^19a,H¹⁸)=13.3 Hz; H^19a), 1.16–1.27 (m, 2 H; H^21a, H^22e), 1.32 (s, 3 H; C26-H₃), 1.35–1.44 (m, 2 H; H^1a, H^7e), 1.46 (ddd, ²J(H^22a,H^22e)=14.0, ³J(H^22a,H^21a)=14.0, ³J(H^22a,H^21e)=4.2 Hz; H^22a), 1.55–1.73 (m, 5 H; 2 H⁶, H^7a, 2 H²), 1.77 (m; H^15a), 1.83 (m; H^16a), 1.90 (m, 2 H; H^1e, H^21e), 1.93 (dm, ³J(H¹⁸,H^19a)=13.3; H¹⁸), 2.00 (s, 3 H; C33-H₃), 2.16 (ddd, ²J(H^19e,H^19a)=13.3, ³J(H^19e,H¹⁸)=3.4, ⁴J(H^19e,H^21e)=2.8 Hz; H^19e), 2.92 (d, ³J(H¹³,H¹⁸)=4.7 Hz; H¹³), 3.69 (s, 3 H; OC31-H₃), 4.43 (dd, ³J(H^3a,H^2a)=11.7, ³J(H^3a,H^2e)=4.5 Hz; H^3a), 5.72 (s; H¹¹); ¹³C NMR (CDCl₃): δ=35.86 (t, C1), 23.66 (t, C2), 79.49 (d, C3), 37.95 (s, C4), 50.08 (d, C5), 17.67 (t, C6), 32.66 (t, C7), 45.29 (s, C8), 177.61 (s, C9), 39.59 (s, C10), 122.89 (d, C11), 201.14 (s, C12), 47.70 (d, C13), 41.61 (s, C14), 26.03 (t, C15), 26.03 (t, C16), 31.87 (t, C17), 37.81 (d, C18), 33.69 (t, C19), 43.88 (s, C20), 31.10 (t, C21), 38.13 (t, C22), 27.77 (q, C23), 16.46 (q, C24), 23.79 (q, C25), 23.85 (q, C26), 21.84 (q, C27), 26.90 (q, C28), 28.45 (q, C29), 177.22 (s, C30), 51.32 (q, C31), 170.59 (s, C32), 21.01 (q, C33); HRMS: m/z calcd for C₃₃H₅₀O₅: 526.7471; found: 526.3658.
Methyl 3β-hydroxy-12-oxo-18βH-olean-9(11)-en-30-oate (7): A mixture of 6 (6.2 g, 11.8 mmol) and KOH (41 g, 732 mmol) in methanol (400 mL) was heated under reflux for 1.5 h. The resulting solution was cooled to room temperature, concentrated in vacuo, and 10 % aqueous hydrochloric acid solution was added. The mixture was extracted with chloroform/ethyl acetate (1:4, 3×75 mL). The combined organic layers were washed with saturated sodium hydrogen carbonate solution (3×50 mL) and brine (3×50 mL), and dried over magnesium sulfate. The solvent was removed to give a solid 7 (yield=5.13 g, 90 %). This material was used for the next reaction without further purification. An analytically pure sample was obtained by recrystallization from a mixture chloroform/methanol. M.p. 202–203°C; ¹H NMR: δ=0.80 (s, 3 H; C24-H₃), 0.89 (s, 3 H; C28-H₃), 0.94 (s, 3 H; C27-H₃), 1.01 (s, 3 H; C23-H₃), 1.08 (s, 3 H; C29-H₃), 1.16 (s, 3 H; C25-H₃), 1.35 (s, 3 H; C26-H₃), 0.83–0.91 (m, 2 H; H^5a, H^16e), 1.04 (dm, ²J(H^15e,H^15a)=12.8 Hz; H^15e), 1.23 (dd, ²J(H^19a,H^19e)=13.2, ³J(H^19a,H¹⁸)=13.2 Hz; H^19a), 1.18–1.25 (m; H^21a), 1.27 (dm, ²J(H^22e,H^22a)=14.0 Hz; H^22e), 1.31 (m; H^1a), 1.44 (dm, ²J(H^7e,H^7a)=9.8 Hz; H^7e), 1.49 (ddd, ²J(H^22a,H^22e)=14.0, ³J(H^22a,H^21a)=14.0, ³J(H^22a,H^21e)=4.2 Hz; H^22a), 1.55–1.76 (m, 5 H; 2 H⁶, H^7a, 2 H²), 1.80 (m, H^15a), 1.85 (m; H^16a), 1.89–2.00 (m, 3 H; H^1e, H^21e, H¹⁸), 2.19 (ddd, ²J(H19^e,19^a)=13.2, ³J(H^19e,H¹⁸)=3.2, ⁴J(H^19e,H^21e)=2.7 Hz; H^19e), 2.95 (d, ³J(H¹³,H¹⁸) 4.7 Hz; H¹³), 3.18 (dd ³J(H^3a,H^2a)=11.7, ³J(H^3a,H^2e)=4.4 Hz; H^3a), 3.71 (s, 3 H; OC31-H₃), 5.76 (s, H¹¹); ¹³C NMR (CDCl₃): δ=36.22 (t, C1), 27.38 (t, C2), 77.83 (d, C3), 39.11 (s, C4), 50.06 (d, C5), 17.87 (t, C6), 32.79 (t, C7), 45.39 (s, C8), 178.10 (s, C9), 39.81 (s, C10), 122.85 (d, C11), 201.36 (s, C12), 47.76 (d, C13), 41.70 (s, C14), 26.14 (t, C15), 26.12 (t, C16), 31.95 (s, C17), 37.88 (d, C18), 33.79 (t, C19), 43.95 (s, C20), 31.17 (t, C21), 38.21 (t, C22), 27.96 (q, C23), 15.44 (q, C24), 23.81 (q, C25), 23.87 (q, C26), 21.99 (q, C27), 26.96 (q, C28), 28.52 (q, C29), 177.33 (s, C30), 51.43 (q, C31); HRMS: m/z calcd for C₃₁H₄₈O₄: 484.7104; found: 484.3553.
Methyl 3,12-Dioxo-18βH-olean-9(11)-en-30-oate (8): Jones reagent (5 mL), prepared from Na₂Cr₂O₇⋅2 H₂O in dilute sulfuric acid (33 %)[45 ] was added dropwise to a solution of 7 (5.13 g) in acetone (500 mL) at 0°C over 30 min till the brown color persisted. The mixture was stirred for further 2.5 h at room temperature, and ethanol (10 mL) was added. The resulting mixture was concentrated in a vacuum (∼100 mL), and water (1 L) was added. The solid was filtered and dried. The ketone 8 was purified by column chromatography (neutral alumina, chloroform) (yield=4.7 g, 94 %). This material was used for the next reaction without further purification. An analytically pure sample was obtained by recrystallization from a mixture chloroform/methanol. M.p. 189–192°C; ¹H NMR (CDCl₃): δ=0.88 (s, 3 H; C28-H₃), 0.94 (s, 3 H; C27-H₃), 1.05 (s, 3 H; C24-H₃), 1.07 (s, 3 H; C29-H₃), 1.08 (s, 3 H; C23-H₃), 1.27 (s, 3 H; C25-H₃), 1.38 (s, 3 H; C26-H₃), 0.89 (m, H^16e), 1.06 (m, H^15e), 1.21 (dd, ²J(H^19a,H^19e)=13.3, ³J(H^19a,H¹⁸)=13.3 Hz; H^19a), 1.17–1.29 (m, 2 H; H^21a, H^22e), 1.43–1.50 (m, 2 H; H^5a, H⁷), 1.48 (ddd, ²J(H^22a,H^22e)=14.0, ³J(H^22a,H^21a)=14.0, ³J(H^22a,H^21e)=4.3 Hz; H^22a), 1.60–1.72 (m, 3 H; 2 H⁶, H^7′), 1.75 (m, H^1a), 1.80 (m; H^15a), 1.85 (m, H^16a), 1.92 (m, H^21e), 1.96 (ddd, ³J(H¹⁸,H^19a)=13.3, ³J(H¹⁸,H¹³)=4.7, ³J(H¹⁸,H^19e)=3.2 Hz; H¹⁸), 2.16 (m, H^1e), 2.17 (ddd, ²J(H^19e,H^19a)=13.3, ³J(H^19e,H¹⁸)=3.2, ⁴J(H^19e,H^21e)=2.8 Hz; H^19e), 2.44 (ddd, ²J(H^2e,H^2a)=15.8, ³J(H^2e,H^1a)=7.2, ³J(H^2e,H^1e)=3.8 Hz; H^2e), 2.60 (ddd, ²J(H^2a,H^2e)=15.8, ³J(H^2a,H^1a)=11.5, ³J(H^2a,H^1e)=7.3 Hz; H^2a), 2.98 (d, ³J(H¹³,H¹⁸)=4.7 Hz; H¹³), 3.70 (s, OC31-H₃), 5.78 C(H¹¹); ¹³C NMR (CDCl₃): δ=36.80 (t, C1), 33.97 (t, C2), 215.57 (s, C3), 47.35 (s, C4), 50.73 (d, C5), 18.97 (t, C6), 31.90 (t, C7), 45.46 (s, C8), 176.36 (s, C9), 39.23 (s, C10), 124.10 (d, C11), 200.78 (s, C12), 47.86 (d, C13), 41.78 (s, C14), 26.13 (t, C15), 26.11 (t, C16), 31.90 (s, C17), 37.82 (d, C18), 33.76 (t, C19), 43.90 (s, C20), 31.12 (t, C21), 38.14 (t, C22), 26.10 (q, C23), 21.25 (q, C24), 23.69 (q, C25), 23.75 (q, C26), 21.81 (q, C27), 26.94 (q, C28), 28.45 (q, C29), 177.19 (s, C30), 51.37 (q, C31); HRMS: m/z calcd for C₃₁H₄₆O₄: 484.6945; found: 484.3396.
Methyl 2-hydroxymethylene-3,12-dioxo-18βH-olean-9(11)-en-30-oate (9): Ethyl formate (3.75 mL, 39.5 mmol) and sodium methylate (2.1 g, 38.9 mmol) were added to a solution of ketone 8 (4.5 g, 9.3 mmol) in dry benzene (50 mL). The mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with a mixture of chloroform/diethyl ether (1:3, 100 mL), and 5 % HCl was added to achieve pH<7. The organic layer was separated, and the aqueous layer was extracted with chloroform/diethyl ether (1:3, 3×50 mL). The combined organic layers were washed with saturated sodium hydrogen carbonate solution (3×50 mL), and brine (3×50 mL), and dried over magnesium sulfate. The solvent was removed to give an amorphous solid 9 (yield=4.5 g, 95 %). This material was used for the next reaction without further purification. An analytically pure sample was obtained by flash column chromatography (silica gel; hexane/ethyl acetate (9:1) followed by hexane/ethyl acetate (3:1)). ¹H NMR (CDCl₃): δ=0.90 (s, 3 H; C28-H₃), 0.96 (s, 3 H; C27-H₃), 1.09 (s, 3 H; C29-H₃), 1.13 (s, 3 H; C24-H₃), 1.15 (s, 3 H; C25-H₃), 1.20 (s, 3 H; C23-H₃), 1.38 (s, 3 H; C26-H₃), 0.91 (m, H^16e), 1.07 (m, H^15e), 1.23 (dd, ²J(H^19a,H^19e)=13.3, ³J(H^19a,H¹⁸)=13.3 Hz; H^19a), 1.18–1.31 (m, 3 H; H^21a, H^22e, H^5a), 1.49 (ddd, ²J(H^22a,H^22e)=14.0, ³J(H^22a,H^21a)=14.0, ³J(H^22a,H^21e)=4.1 Hz; H^22a), 1.47–1.53 (m, H⁷), 1.59–1.67 (m, 3 H; 2 H⁶, H^7′), 1.82 (m, H^15a), 1.86 (m, H^16a), 1.94 (dddd ²J(H^21e,H^21a)=13.3, ³J(H^21e,H^22a)=4.1, ³J(H^21e,H^22e)=3.4, ⁴J(H^21e,H^19e)=2.7 Hz; H^21e), 1.99 (ddd, ³J(H¹⁸,H^19a)=13.3, ³J(H¹⁸,H¹³)=4.6, ³J(H¹⁸,H^19e)=3.3 Hz; H¹⁸), 2.21 (ddd, ²J(H^19e,H^19a)=13.3, ³J(H^19e,H¹⁸)=3.3, ⁴J(H^19e,H^21e)=2.7 Hz; H^19e), 2.26 (d, ²J(H¹,H^1′)=14.5 Hz; H¹) and 2.58 (d, ²J(H^1′,H¹)=14.5 Hz; H^1′)—AB-system, 3.02 (d, ³J(H¹³,H¹⁸)=4.6 Hz; H¹³), 3.71 (s, 3 H; OC31-H₃), 5.90 (s, H¹¹), 8.70 (d, ³J(H³²_,OH)=2.4 Hz; H³²), 14.81 (d, ³J (OH,H³²)=2.4 Hz; OH); ¹³C NMR (CDCl₃): δ=36.85 (t, C1), 104.80 (s, C2), 188.08 (s, C3), 40.32 (s, C4), 48.07 (d, C5), 18.80 (t, C6), 31.25 (t, C7), 45.55 (s, C8), 175.36 (s, C9), 38.89 (s, C10), 124.37 (d, C11), 200.73 (s, C12), 47.86 (d, C13), 41.80 (s, C14), 26.22 (t, C15), 26.14 (t, C16), 31.93 (s, C17), 37.82 (d, C18), 33.74 (t, C19), 43.93 (s, C20), 31.14 (t, C21), 38.18 (t, C22), 28.14 (q, C23), 20.65 (q, C24), 23.36 (q, C25), 23.23 (q, C26), 21.82 (q, C27), 26.96 (q, C28), 28.48 (q, C29), 177.22 (s, C30), 51.42 (q, C31), 189.65 (d, C32); HRMS: m/z calcd for C₃₂H₄₆O₅: 510.7046; found: 510.3345.
Methyl 12-oxoisoxazolo[4,5-b]-18βH-olean-9(11)-en-30-oate (10): Hydroxylamine hydrochloride (6.0 g, 86.0 mmol) was added to a solution of 9 (4.4 g, 8.6 mmol) in ethanol (120 mL) and water (12 mL). The mixture was heated under reflux for 2 h, cooled to room temperature, concentrated in vacuo, and water (100 mL) was added. The mixture was extracted with ethyl acetate (3×70 mL). The combined organic layers were washed with water (3×50 mL), brine (3×50 mL) and dried over magnesium sulfate. The solvent was evaporated and the solid was purified by column chromatography (silica gel; hexane/ethyl acetate (3:1)) to give 10 (yield=3.2 g, 73 %). ¹H NMR (CDCl₃): δ=0.91 (s, 3 H; C28-H₃), 0.98 (s, 3 H; C27-H₃), 1.10 (s, 3 H; C29-H₃), 1.15 (s, 3 H; C25-H₃), 1.24 (s, 3 H; C24-H₃), 1.32 (s, 3 H; C23-H₃), 1.40 (s, 3 H; C26-H₃), 0.91 (m, H^16e), 1.06–1.11 (m, H^15e), 1.24 (dd, ²J(H^19a,H^19e)=13.2, ³J(H^19a,H¹⁸)=13.2 Hz; H^19a), 1.19–1.34 (m, 2 H; H^21a, H^22e), 1.44–1.57 (m, 3 H; H^5a, H^22a, H⁷), 1.64–1.79 (m, 3 H; 2 H⁶, H^7′), 1.84 (m, H^15a), 1.88 (m, H^16a), 1.95 (dddd, ²J(H^21e,H^21a)=13.3, ³J(H^21e,H^22a)=4.2, ³J(H^21e,H^22e)=3.2, ⁴J(H^21e,H^19e)=2.7 Hz; H^21e), 2.00 (ddd ³J(H¹⁸,H^19a)=13.2, ³J(H¹⁸,H¹³)=4.6, ³J(H¹⁸,H^19e)=3.2 Hz; H¹⁸), 2.21 (ddd, ²J(H^19e,H^19a)=13.2, ³J(H^19e,H¹⁸)=3.2, ⁴J(H^19e,H^21e)=2.7 Hz; H^19e), 2.38 (d, ²J(H¹,H^1′)=15.0 Hz; H¹) and 2.75 (d, ²J(H^1′,H¹)=15.0 Hz; H^1′)—AB system, 3.04 (d, ³J(H¹³,H¹⁸)=4.6 Hz; H¹³), 3.73 (s, 3 H; OC31-H₃), 5.89 (s, H¹¹), 8.04 (s, H³²); ¹³C NMR (CDCl₃): δ=33.42 (t, C1), 108.38 (s, C2), 171.94 (s, C3), 35.01 (s, C4), 49.53 (d, C5), 18.16 (t, C6), 31.24 (t, C7), 45.75 (s, C8), 175.87 (c, C9), 41.09 (s, C10), 124.65 (d, C11), 200.77 (s, C12), 47.90 (d, C13), 41.79 (s, C14), 26.27 (t, C15), 26.13 (t, C16), 31.95 (s, C17), 37.85 (d, C18), 33.79 (t, C19), 43.95 (s, C20), 31.16 (t, C21), 38.18 (t, C22), 28.65 (q, C23), 21.26 (q, C24), 24.52 (q, C25), 23.28 (q, C26), 21.87 (q, C27), 26.99 (q, C28), 28.51 (q, C29), 177.26 (s, C30), 51.47 (q, C31), 150.06 (d, C32); HRMS: m/z calcd for C₃₂H₄₅NO₄: 507.7040; found: 507.3349.
The mixture of tautomers (11): Sodium methylate (11 g, 204 mmol) was added at 0°C to a solution of isoxazole 10 (3.0 g, 5.9 mmol) in methanol (85 mL) and diethyl ether (170 mL). The mixture was stirred at room temperature for 1 h. The resulting mixture was diluted with a mixture of chloroform/diethyl ether (1:3; 100 mL), and 5 % HCl was added to achieve pH<7. The organic layer was separated, and the aqueous layer was extracted with chloroform/diethyl ether (1:3; 3×50 mL). The combined organic layers were washed with saturated sodium hydrogen carbonate solution (3×50 mL) and brine (3×50 mL), and dried over magnesium sulfate. The solvent was removed to give a mixture of tautomers 11 (yield=3.0 g, 100 %). This material was used for the next reaction without further purification. HRMS: m/z calcd for C₃₂H₄₅NO₄: 507.7040; found: 507.3349.
Methyl 2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-oate (12): Mixture 11 (2.8 g, 5.5 mmol) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (1.5 g, 6.5 mmol) in dry benzene (160 mL) were heated under reflux for 4 h. Insoluble matter was removed by filtration, and the filtrate was evaporated in a vacuum to give a solid. The solid was subjected to flash column chromatography (silica gel; benzene followed by benzene/acetone (10:1)) to give crude 12. The crude product was purified by recrystallization from methanol/chloroform to give crystals 12 (yield 1.7 g, 61 %). M. p. 247–249°C; ¹H NMR (CDCl₃): δ=0.90 (s, 3 H; C28-H₃), 0.96 (s, 3 H; C27-H₃), 1.09 (s, 3 H; C29-H₃), 1.14 (s, 3 H; C24-H₃), 1.22 (s, 3 H; C23-H₃), 1.44 C (s, 3 H; C26-H₃), 1.47 (s, 3 H; C25-H₃), 0.93 (dm, ²J(H^16e,H^16a)=13.3 Hz; H^16e), 1.08 (m, H^15e), 1.20 (dd, ²J(H^19a,H^19e)=13.2, ³J(H^19a,H¹⁸)=13.2 Hz; H^19a), 1.18–1.32 (m, 2 H; H^21a, H^22e), 1.48 (ddd, ²J(H^22a,H^22e)=14.0, ³J(H^22a,H^21a)=14.0, ³J(H^22a,H^21e)=4.2 Hz; H^22a), 1.55 (dm, ²J(H^7e,H^7a)=13.5 Hz; H^7e), 1.67–1.79 (m, 4 H; H^5a, 2 H⁶, H^7a), 1.82 (m, H^15a), 1.87 (m, H^16a), 1.94 (dddd, ²J(H^21e,H^21a)=13.3, ³J(H^21a,H^22a)=4.2, ³J(H^21e,H^22e)=3.2, ⁴J(H^21e,H^19e)=2.8 Hz; H^21e), 2.02 (ddd, ³J(H¹⁸,H^19a)=13.2, ³J(H¹⁸,H¹³)=4.7, ³J(H¹⁸,^19e)=3.2 Hz; H¹⁸), 2.17 (ddd, ²J(H^19e,H^19a)=13.2, ³J(H^19e,H¹⁸)=3.2, ⁴J(H^19e,H^21e)=2.8 Hz; H^19e), 3.02 (d, ³J(H¹³,H¹⁸)=4.7 Hz; H¹³), 3.72 (s, 3 H; OC31-H₃), 5.97 (s, H¹¹), 8.01 (s, H¹); ¹³C NMR (CDCl₃): δ=165.65 (d, C1), 114.46 (s, C2), 196.42 (s, C3), 44.86 (s, C4), 47.57 (d, C5), 18.12 (t, C6), 31.58 (t, C7), 45.82 (s, C8), 168.18 (s, C9), 42.36 (s, C10), 124.17 (d, C11), 199.52 (s, C12), 48.04 (d, C13), 42.10 (s, C14), 26.03 (t, C15), 26.00 (t, C16), 31.88 (s, C17), 37.75 (d, C18), 33.61 (t, C19), 43.91 (s, C20), 31.11 (t, C21), 38.14 (t, C22), 26.88 (q, C23), 21.40 (q, C24), 26.61 (q, C25), 24.77 (q, C26), 21.81 (q, C27), 26.96 (q, C28), 28.46 (q, C29), 177.13 (s, C30), 51.48 (q, C31), 114.22 (s, C32); HRMS: m/z calcd for C₃₂H₄₃NO₄: 505.6881; found: 505.3192.
Cell culture and glycyrrhetinic acids derivatives: Human KB-3-1 epidermoid carcinoma cell line, HeLa cervical epithelioid carcinoma cell line, MCF-7 breast adenocarcinoma cell line, SKNMC neuroblastoma cell line (Russian Cell Culture Collection, St. Petersburg), KB-8-5 multidrug resistant cancer cell line (kindly provided by Professor M. Gottesman (NIH, USA)), were cultured in DMEM supplemented with 10 % (v/v) heat-inactivated fetal bovine serum, penicillin (100 U mL⁻¹; ICN Biomedicals, Inc), streptomycin (100 μg mL⁻¹) and amphotericin (250 μg mL⁻¹). Cells were maintained in a humidified atmosphere (5 % CO₂, 37°C). The KB-8-5 cell line was incubated in the additional presence of vinblastine (300 nmoll⁻¹).
Glycyrrhetinic acids derivatives were dissolved in DMSO (10 mmoll⁻¹), and stock solution were stored at −20°C.
After treatments, both floating and adherent scraped cells were collected by centrifugation, and used for further analysis.
Cell viability analysis by MTT assay: Cancer cells, growing in log phase, were seeded in triplicate 96-well plates at a density of 5×10³ cells per well for HeLa cells, 7×10³ for KB-3-1, KB-8-5 and MCF-7 cells, and 30×10³ for SKNMC cells. The plates were incubated at 37°C in humidified 5 % CO₂ atmosphere. Cells were allowed to adhere to the surface for 24 h, then treated with varying doses of the compounds for 24 h. Aliquots of [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) solution (10 μL, 5 mg mL⁻¹) were added to each well, and the incubation was continued for an additional 3 h. The dark blue formazan crystals (formed within healthy cells) were solubilized with DMSO, and the absorbance was measured at 570 nm in a Multiscan RC plate reader (Thermo LabSystems, Finland). The IC₅₀ was determined as the compound concentration required to decrease the A₅₇₀ to 50 % of the control (no compound, DMSO), and was determined by interpolation from dose-response curves.
Analysis of antioxidant effect on the cytotoxicity of compound 12: KB-3-1 cells growing in the log phase were seeded in triplicate in 96-well plates (7×10³ cells per well). The plates were incubated at 37°C in a humidified 5 % CO₂ atmosphere. Cells were allowed to adhere to the surface for 24 h, then treated with GSH (1, 5, 15 or 45 mm) or with ascorbic acid (1, 3 or 5 mm), both alone and in combination with 12 (1 μm). Cells were incubated with the compounds for 24 h and cell viability was analyzed by the MTT assay as described above.
Morphological observation of nuclear change: KB-3-1 cells were seeded into 24-well plates (10⁵ cells per well) containing glass cover slips. Cells were allowed to adhere to the surface for 24 h. Cells were treated with 12 (1 μm) or with DMSO (0.1 % (v/v)) for 6, 18 or 24 h at 37°C in a humidified 5 % CO₂ atmosphere. After incubation, cells were fixed with 4 % formaldehyde for 15 min, and then stained for 30 min with Hoechst 33258 (200 ng mL⁻¹). Cells were analyzed for the presence of fragmented nuclei and condensed chromatin by fluorescent microscopy.
Apoptosis detection by Annexin V staining: Log-phase KB-3-1 cells in six-well plates (5×10⁵ cells per well) were treated with 12 (0.3 μm or 1 μm) or with DMSO (0.1 % (v/v)) for 4, 18 or 24 h. The cells were stained with Annexin V-FITC and propidium iodide by using the ApopNexin-FITC apoptosis detection kit (Chemicon Millipore) according to the manufacturer's instructions. Briefly, cells were collected by scraping, washed twice with cold PBS, and centrifuged (400 g, 5 min). Cells were resuspended in binding buffer (1 mL) at a concentration of 1×10⁶ cells per mL, then a sample (200 μL) was transferred to a 5 mL culture tube, and Annexin V-FITC (3 μL) and 100×PI (2 μL) were added. Cells were incubated for 15 min at room temperature in the dark. Finally, binding buffer (300 μL) was added to each tube, and the quantity of apoptotic cells in samples was analyzed by flow cytometry (FC500, Beckman Coulter, USA). For each sample, 10 000 ungated events were acquired. Annexin V⁺/PI⁻ cells represented early apoptotic populations. Annexin V⁺/PI⁺ cells represented either late apoptotic or secondary necrotic populations.
Mitochondria depolarization analysis: Mitochondria involvement in apoptosis was measured by the mitochondrial depolarization that occurs early during the onset of apoptosis. KB-3-1 cells were treated with 1 (1 μm), 12 (1 μm) or DMSO (0.1 % (v/v)) for 6 h, and loss of mitochondrial potential was determined by using the mitochondrial potential sensor JC-1 (Molecular Probes, Invitrogen).
Flow cytometry assay: Cells were incubated for the appropriate time with the compounds, then collected, incubated in complete media in the dark with JC-1 (5 μg mL⁻¹) at 37°C for 15 min, and washed with PBS. At the end of the incubation period the cells were washed twice with cold PBS, and resuspended in PBS (400 μL). J-aggregate and J-monomer fluorescence were recorded in the channesl 2 (FL2) and 1 (FL1), respectively, of an FC500 flow cytometer. Necrotic fragments were electronically gated out, on the basis of morphological characteristics on the forward light scatter versus side light scatter dot plot.
Fluorescent microscopy assay: Cells were plated into 24-well plates (10⁵ cells per well) containing glass cover slips, and allowed to adhere to the surface for 24 h. Cells were incubated for the appropriate time with the compounds. After incubation the cell culture media was removed and replaced with JC-1 reagent (5 μm) diluted in PBS. Cells were incubated at 37°C in a 5 % CO₂ incubator for 15 min, and analyzed by fluorescence microscopy.
Cytofluorimetric analysis of DNA content: Exponentially growing KB-3-1 cells in 6-well plates (5×10⁵ cells per well) were treated with 12 (0.3 μm, or 1 μm) or DMSO (0.1 % (v/v)) for 18 h. After incubation, the cells were collected by centrifugation (400 g, 10 min), fixed with ice-cold 70 % ethanol for at least 1 h at 4°C and treated with RNase A from bovine pancreas (1 mg mL⁻¹; Sigma) for 30 min at 37°C. PI (50 μg mL⁻¹) was then added to the solution and the DNA content was quantitated by a flow cytometry. Cells in sub-G1 phase were considered apoptotic.
Analysis of caspase activation: After treatment of KB-3-1 cells with 1 (0.3 μm, 1 μm), or 12 (0.3 μm, 1 μm), or DMSO (0.1 % (v/v)) for 18 h, caspase activation was assayed by using the CaspACE FITC-VAD-FMK in situ marker (Promega).
Flow cytometry assay: Cells were incubated for the appropriate time in the presence of the compounds, collected, suspended in PBS (0.5 mL), and FITC-VAD-FMK (1 μL, 5 mm) was added. The cells were gently mixed and incubated for 20 min at RT in the dark. Cells were washed twice with PBS, and the pellets resuspended in PBS (0.5 mL). Flow cytometry was conducted within 10 min.
Fluorescent microscopy assay: Cells were seeded (10⁵ cells per well) into 24-well plates containing glass cover slips, and allowed to adhere to the surface for 24 h. After incubation for the appropriate time with the compounds, the cell culture medium was removed and replaced with JC-1 reagent (5 μm) diluted in PBS. Cells were incubated at 37°C in a 5 % CO₂ incubator for 15 min. The cells were washed twice with PBS, and caspase activation was analyzed by fluorescence microscopy within 10 min.

Logashenko E.B., Salomatina O.V., Markov A.V., Korchagina D.V., Salakhutdinov N.F., Tolstikov G.A., Vlassov V.V, & Zenkova M.A. (2011). Synthesis and Pro-Apoptotic Activity of Novel Glycyrrhetinic Acid Derivatives. Chembiochem, 12(5), 784-794.

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Publication 2011

Synthesis of PEG-b-DB Block Copolymers

Cited 7 times

Butyl methacrylate (BMA), poly(ethylene glycol) 4-cyano-4-(phenylcarbonothioylthio)pentanoate, M_n=2,000 Da and M_n=10,000 Da (PEG-CPADB), 4-cyano-4-(phenylcarbonothioylthio)pentanoate (CPADB), N,N’-Dicyclohexylcarbodiimide (DCC), 4-(Dimethylamino)pyridine (DMAP), DL-Dithiothreitol (DTT), 4,4’-azobis(4-Cyanovaleric acid) (V501), dichloromethane, 1,4-dioxane, and poly(ethylene glycol) methyl ether (M_n = 5,000 Da) were purchased from Sigma-Aldrich. 2-(Diethylamino)ethyl methacrlate (DEAEMA) was procured from TCI Chemicals, and 2,2’-Azobis(4-methoxy-2,4-dimethylvaleronitrile) (V70) was purchased from Wako Chemicals. Pyridyl disulfide ethyl methacrylate (PDSMA) was synthesized according to a previously reported procedure.⁵⁴ BHT inhibitor was removed from methacrylate monomers before further use by gravity chromatography using basic alumina (Sigma).
For synthesis of PEG-b-DB polymers, the appropriate PEG-CPADB macroRAFT chain transfer agent (mCTA) was dissolved in anhydrous dioxane with purified BMA, DEAEMA, and V501 at a 60:40 molar ratio of BMA:DEAEMA, sealed with septa, purged with N₂ for 20 minutes, and polymerized at 70˚C for 18 h. An initiator to mCTA (I:mCTA) ratio of 0.2:1 was used with a combined monomer and mCTA to dioxane weight ratio of 0.4. Polymers were precipitated 2x in cold pentane and vacuum dried. Polymer composition were characterized via ¹H-NMR in CDCl₃ on a Bruker AV400 spectrometer (Supplementary Figure S10). Molecular weight and polydispersity index (PDI) were quantified using gel permeation chromatography (Agilent) with DMF containing 0.1M LiBr as the mobile phase and in line light scattering (Wyatt) and refractive index (Agilent) detectors. PEG_5kDa-CPADB or PEG_10kDa-CPADB mCTAs were synthesized as previously described.⁵⁵ PEG_2kDa-DBP_4.5kDa was synthesized with similar conditions, substituting V70 for V501 and a reaction temperature of 30°C for 24h.

Shae D., Becker K.W., Christov P., Yun D.S., Lytton-Jean A.K., Sevimli S., Ascano M., Kelley M., Johnson D.B., Balko J.M, & Wilson J.T. (2019). Endosomolytic Polymersomes Increase the Activity of Cyclic Dinucleotide STING Agonists to Enhance Cancer Immunotherapy. Nature nanotechnology, 14(3), 269-278.

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Publication 2024

Fluorescence-based Characterization of PD-CNDs

Primary alcohols and mixtures of alcohol and 1,4-dioxane with different ratios (0 to 100%, v/v, with an increment of 10%) were prepared. To this, 0.05 mg mL⁻¹ of powdered PD-CNDs was added and dispersed by sonication. The shift in the emission peak maximum of CNDs in various alcohols and alcohol–1,4-dioxane mixtures was monitored using fluorescence spectroscopy.

V. N., Gopal R., C. A., T. P. A., K. K. A., Praveen V.K, & Kizhakayil R.N. (2024). p-Phenylenediamine-derived carbon nanodots for probing solvent interactions. Nanoscale Advances, 6(5), 1535-1547.

Publication 2024

Quantifying 1,4-Dioxane in Environmental Samples

Not available on PMC !

1,4-dioxane and 1,4-dioxane-d 8 were purchased as solutions in methanol (1,000 to 10,000 µg/mL) from Dr Ehrenstorfer GmbH (Augsburg, Germany) via LGC Standards (Molsheim, France). Methanol ULC-MS (MeOH) and ethyl acetate LC-MS (EtAc) were purchased from Biosolve (Dieuze, France). Dichloromethane Pestinorm® (DCM) was purchased from VWR International (Rosny-sous-Bois, France). Ultrapure pure water was produced by a Millipore Milli-Q® Integral 10 water purification system (Milford, MA, USA).
Intermediate stock solutions of 1,4-dioxane at concentrations of 10 and 100 mg/L were prepared in MeOH using volumetric flasks and then transferred to 2 mL amber glass vials and stored at -18 °C to limit evaporation.
Calibration points from 5 to 500 µg/L were prepared in a solvent mixture (v/v) of 80% DCM and 20% EtAc. An internal standard calibration was carried out using 1,4-dioxane-d 8 at 100 µg/L in water samples.

Bach C., Boiteux V, & Dauchy X. (2024). France-Wide Monitoring of 1,4-Dioxane in Raw and Treated Water: Occurrence and Exposure Via Drinking Water Consumption. Archives of environmental contamination and toxicology.

Publication 2024

Determination of 1,4-Dioxane in Water

Not available on PMC !

Procedural blanks were prepared using ultrapure water in 1 L amber collection bottles and stored at 4 °C. For each sample batch, 500 mL of this water was analysed using the SPE procedure described above. These procedural blanks were used to check for possible contamination from the sample containers and the entire analytical procedure.
Within-run and intra-sample controls were systematically performed for each sample batch. The within-run controls consisted of calibration check standards inserted throughout the sample batch at 50 µg/L DCM/EtAc (80:20) for 1,4-dioxane. To validate the batch, the bias between the experimental and the theoretical concentration must be ≤ 20%. Intra-sample controls consisted of spiking some of the collected water samples at 0.15 µg/L (LOQ) and at 1 µg/L with 1,4-dioxane in order to check the trueness of the method. These intrasample controls were considered valid if their recoveries were between 70 and 120% according to ISO 21253-2:2019 (ISO 2019).
Identification of 1,4-dioxane was confirmed according to the requirements of ISO 21253-1:2019: (i) the relative retention time of the target compounds must match that of the calibration points with a tolerance of ± 2.5%, and (ii) the abundance ratio (based on peak area) between samples and calibration points of two different transitions must not exceed 30%.

Publication 2024

Reliable SPE-GC/MSMS Method for 1,4-Dioxane

Not available on PMC !

The SPE-GC/MSMS method with a LOQ of 0.15 µg/L was validated during our study by applying the requirements of French standard NF T 90-210 (NF 2018) using natural representative matrices (surface water, groundwater and tap water) under intermediate precision to demonstrate the reliability of the analytical data. The method was accredited by the French Accreditation Committee (COFRAC) in 2020.
The range of the calibration curve was studied with five calibration points ranging from 15 to 500 µg/L in a solvent mixture (v/v) of 80% DCM and 20% EtAc for 1,4-dioxane. The second order nonlinear internal standard calibration function was performed six times (on different days) from standard solutions freshly prepared each day. The correlation coefficients (r 2 ) obtained were ≥ 0.98. The back-calculated concentrations between the experimental and the nominal values must be within ± 15% for all calibration points, and within ± 20% for the calibration point corresponding to the LOQ at 0.15 µg/L. The results were acceptable and are presented in Fig. S2.
The limit of detection (LOD) was not considered in this study due to the low limit of quantification (LOQ) of 0.15 µg/L. The LOQ was defined as the lowest concentration of the analyte that can be determined with acceptable precision according to the French standard NF T 90-210 (NF 2018). The LOQ for 1,4-dioxane was validated under intermediate precision conditions in natural matrices. Six water samples (two groundwater, two surface water and two drinking water samples) were spiked at the pre-established LOQ. Inter-day precision was performed by analysing six series of duplicate extractions on six different days. To ensure the accuracy (trueness and precision), a maximum allowed tolerance (MAT) between the theoretical and the experimental values at the LOQ must not exceed ± 60%. The MAT was fixed as requested by the NF T90-210 standard, and its calculation was described in detail by Lardy-Fontan et al. (2018) (link) and Mirmont et al. (2023) (link). The LOQ of 0.15 µg/L for 1,4dioxane was validated.
Following the same procedure, the accuracy of the method was also evaluated for two intermediate concentrations of the calibration range (0.75 and 4 µg/L). In this case, the MAT did not exceed ± 40% for these two concentration levels.
The relative recovery study was carried out by spiking groundwater, surface water and drinking water at 0.15 µg/L, 0.75 µg/L and 4 µg/L in duplicates. Mean recoveries were calculated using the three matrices for each spiking concentration. As shown in Table 3S, the mean recoveries for 1,4-dioxane ranged from 117 to 114% for the three 1,4-dioxane concentrations studied. In addition, during the sampling campaign, several water samples of each batch were spiked with 1,4-dioxane at 1 µg/L in order to evaluate recoveries in the experimental conditions with different real matrix samples. Recovery results with their standard deviation are listed in Table 4S. It is worth noting that average recoveries are calculated with 92 different matrix samples (treated water, surface and groundwater) and over a period of 1 year and 4 months (reproducibility conditions).These experimental recoveries were within the limits (70% and 120%) set by ISO 21253-2:2019 (ISO 2019).
The relative uncertainty (U) was calculated in order to compare the measured results. The uncertainty was extended by a coverage factor (k) of 2 (95% confidence level). The measured uncertainty was 47% for the LOQ and 30% for the two intermediate concentrations in the calibration range.

Publication 2024

Top products related to «Dioxane»

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1,4-dioxane is a colorless organic compound with the chemical formula (CH2)2O2. It is commonly used as a solvent and chemical intermediate in the production of various industrial and laboratory products.

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Dioxane is a laboratory chemical used as a solvent. It has a cyclic structure and a molecular formula of C4H8O2. Dioxane is commonly used in various chemical applications, including as a solvent for resins, fats, oils, waxes, and other organic compounds.

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What is Dioxane and how is it used?

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More about "Dioxane"

Dioxane, also known as 1,4-dioxane, is a synthetic organic compound with the chemical formula C4H8O2.
It is a colorless, flammable liquid with a faint ethereal odor.
Dioxane is widely used as a solvent in various industrial and laboratory applications, including the production of pharmaceuticals, plastics, and textiles.
However, dioxane has been classified as a possible human carcinogen, and its environmental release is a concern due to its persistence and potential for groundwater contamination.
Researchers studying dioxane exposure and remediation can optimize their work using the PubCompare.ai tool, which helps locate the best protocols and enhance the reproducibility and accuracy of dioxane-related studies.
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By using this tool, you can unlock greater insights and improve the overall quality of your dioxane-related studies.
In addition to dioxane, researchers may also be interested in related compounds such as DMSO, methanol, ethanol, acetonitrile, and N,N-dimethylformamide, which are commonly used as solvents in various applications.
The Boston Prime C18 column is another related term, as it is often used for the separation and analysis of dioxane and other organic compounds.
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