4 hydroxycoumarin

Here, 4-Hydroxycoumarin (Merck), formaldehyde (Chemical Company), ethanol (Chemical Company), piperidine (Merck), bovine serum albumin (Fluka), and standard flat samples of PVC and tetrahydrofuran were used without further purification.

All chemical substances have been used as bought from Sigma-Aldrich and Merck Companies. Tetraethyl orthosilicate (tetraethoxysilane) (98% w/w), ethanol (99.5% w/w), (3-aminopropyl) triethoxysilane (95% w/w), HNO₃ (65% w/w), and silver nitrate (≥ 99.0%), potassium persulphate (99.99%), hydrazine hydrate (24–26% in H₂O (RT)), isatin (97%), acenaphthene quinone (97%), malononitrile (98%), ethyl cyanoacetate (98%), dimedone (95%), barbituric acid ethyl acetoacetate (99%), 4-hydroxycoumarin (98%), three-methyl-1H-pyrazole-five (4H)-one (98%), α-naphthol (99%), β-naphthol (99%), n-hexane (99%), ethyl acetic acid (99%), and desired derivations have been provided from the Sigma-Aldrich Company. The stem and roots of the Spear Thistle were purchased from a local shop in Tehran (in Iran). The leaves were purchased from a local shop in Tehran. The plant we used in this work is a plant that is found in abundance in local shops and is not wild and endangered. This study complies with relevant institutional, national, and international guidelines and legislation.

Sorafenib (Nexavar, BAY 43-9006) (1 µM), osthole (150 µM), and hydroxycoumarins: 4-hydroxycoumarin (Sigma), umbelliferone, and esculin (Sigma) at the final concentrations of 200 µM were used in the experiments. The drugs were dissolved in DMSO (Sigma) to the final concentration not exceeding 0.01%. The doses were chosen based on previous studies [32 (link),33 (link)]. The cancer cells were treated with the coumarins or with Sorafenib separately or in combination for 24 h. As controls, T98G and MOGGCCM cells were incubated only with 0.01% of DMSO.

Cell viability was assessed using, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described^{38 (link)}. In summary, cancer cell lines or F180 were seeded in complete medium at the density of 3 × 10³/well and 5 × 10³/well respectively in 96 well flat-bottom plates. After 24 h, cells were incubated with the candidate compounds at the indicated concentration for (24, 48 or 72 h). DMSO was used as vehicle control and 4-Hydroxycoumarin was purchased from (Sigma-Aldrich, St. Louis, MO, USA) and Cisplatin from Hospira UK Ltd. At the end of treatment, the media were aspirated from each well and incubated with 200 µL of MTT tetrazolium dye (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 0.5 mg/ml for 2 h at 37^oC. The reduced MTT crystals were solubilized in 200 µl/well of DMSO and the absorbance was measured at 570 nm using a microplate reader (Thermo-Scientific, Vantaa, Finland).

Benzoyl-CoA, malonyl-CoA, naringenin and 4-hydroxy­coumarin were all purchased from Sigma (USA); 4-coumaroyl-CoA was purchased from TransMIT (Germany). 3,5-Dihydroxybi­phenyl was prepared as described previously by Nilsson & Norin (1963 ▸ ) and was purified with a Waters preparative LCMS FractionLynx system using mass and UV-directed fractionation on an XBridge Prep OBD C18 5 µm 30 × 100 mm column, with a gradient from 5:95:0.1(v:v:v) water:acetonitrile:formic acid to acetonitrile (0.1% formic acid) over 8 min with a flow rate of 30 ml min⁻¹. Its identity was confirmed by comparison of its ¹H NMR spectrum in MeOD-d₄ and m/z values with those reported in the literature (Liu et al., 2004 ▸ ). Salicoyl-CoA (2-hydroxyBenzoyl-CoA) was prepared as described elsewhere (Sidenius et al., 2004 ▸ ) and was purified via preparative LCMS as described above. The identity of salicoyl-CoA was confirmed by comparison of its ¹H NMR spectrum in MeOD-d₄ and m/z values with those reported in the literature (Guo et al., 2009 ▸ ).

3-hydroxy-4-methoxy-aminophenol, 4-hydroxy-3-methoxy-aminophenol, 4-hydroxycoumarin, acetic acid, phosphoryl chloride, methanol, ethanol, toluene, acetone, and potassium tetrachloridopaladate(II) were purchased from Sigma Aldrich in Germany. A Vario EL III C, H, and N Elemental Analyzer was used to conduct the elemental analyses for the elements C, H, and N. A Perkin-Elmer Spectrum One FT-IR spectrometer was used to record infrared spectra (KBr) (4000–400 cm^–1).
The NMR spectra were recorded on a Bruker AV600 spectrometer with a 5 mm probe head at the Ruđer Bošković Institute (Zagreb, Croatia). ¹H and ¹³C APT NMR spectra were acquired at 600.130 and 150.903 MHz, respectively. Chemical shifts (δ / ppm) for the ¹H and ¹³C spectra were referred to as the DMSO-d₆ signals (¹H: δ = 2.50 ppm; ¹³C: δ = 39.51 ppm), which were purchased from Euriso-Top, France. The assignments of ¹H and ¹³C signals in the NMR spectra of compounds were confirmed by cross peaks obtained in the 2D spectra of ¹H-¹³C Heteronuclear Multiple Bond Correlation (HMBC).
Silica gel (Silica gel 60, layer 0.20 mm, Alugram Sil G, Mashery-Nagel, Germany) was used for the analytical TLC, and a UV lamp (VL-4.LC, 365/254, Vilber Lourmat, France) was used for the visualization of TLC plates.

3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), Oil Red O, dimethyl sulfoxide (DMSO), m-hydroxyphenylglycine, p-aminophenol, ellagic acid, and 4-hydroxycoumarin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Erycibelline was purchased from ChemFaces (Wuhan, China). 5-Hydroxy-2-pyridinemethanol was purchased from IS Chemical Technology (Shanghai, China). Dulbecco's Modified Eagle's Medium (DMEM), bovine serum (BS), and fetal bovine serum (FBS) were purchased from Invitrogen Inc. (Grand Island, NY, USA). All other chemicals were analytical reagent grade.