Tetrahydrofuran

On the basis of molecular scaffold of sunitinib, we synthesized three novel analogs with chalcone-based hydroxamic acids possessing a central 2, 4-dimethypyrrole linker as recently reported (Yousefian et al., 2024 (link)). Briefly, the synthesis of 2,4-dimethyl-1H-pyrrole-3- carboxylate chalcone derivatives (analogs 1 and 3) was carried out via a reaction between 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (Sigma) and acetophenone derivatives in methanol (Merck). Following overnight stirring of the mixture at 30°C, it was cooled and acidified, and the precipitate was recrystallized in ethanol to yield pure analog 1, (E)-2,4-dimethyl-5-(3-oxo-3-(4-phenoxyphenyl) prop-1-en-1-yl)- 1H-pyrrole-3-carboxylic acid, and analog 3, (E)-5-(3-(4-(benzyloxy)phenyl)- 3-oxoprop-1-en-1-yl)- 2,4- dimethyl-1H-pyrrole-3-carboxylic acid (Figure 1).
For the synthesis of 2,4-dimethyl-1H-pyrrole-3-carboxamide derivative (analog 2), N, N′-carbonyldiimidazole (Sigma) was added to carboxylic acid in tetrahydrofuran (Merck) and stirred. Following the addition of hydroxylamine hydrochloride (Merck), the mixture was diluted with potassium bisulfate and the organic phase was extracted with ethyl acetate. The final analog 2, (E)-N-hydroxy-2,4-dimethyl-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)-1H-pyrrole-3- carboxamide, was washed with petroleum ether.
To monitor the reaction progress and preliminarily assess the homogeneity of the analogs, thin layer chromatography was performed using 250 mm Silica Gel GF Uniplates (Whatman) and dimethyl sulfoxide (DMSO, Merck) and chloroform were used as solvents. Pure analogs were visualized under ultraviolet at 254 and 365 nm. Infrared spectra (Perkin Elmer 1420 spectrometer) and ¹H and ¹³C nuclear magnetic resonance (Bruker FT-300 MHz) were acquired to confirm all structures.

Ethanol (99.5%) and tetrahydrofuran (THF) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Malachite green isothiocyanate (MGITC) was purchased from Invitrogen Corporation (Carlsbad, CA, USA). Ultrapure water (0.055 μs/cmc) was obtained from a Laboratory Water System (Göttingen, Germany). A 125 μm thick polyethylene naphthalate (PEN) polymer substrate (Dupont, Wilmington, DE, USA) was used after removing the protective film. A polymer nano dimple pattern was fabricated on the PEN film by O₂ ion beam bombardment in a linear moving substrate. A linear O₂ ion beam with a width of 300 mm was generated using a linear ion source [30 (link)]. An O₂ flow rate was 70 sccm with the vacuum process chamber pressure of 0.9 mTorr. The PEN substrates were reciprocated at a linear moving speed of 10 mm s⁻¹, followed by 60 scans. The ion dose per scan was 2.3 ± 0.2 × 10¹⁵ cm⁻² using a Faraday cup that reduced secondary electron emission by magnetic fields. The mean ion energy was 700 ± 70 eV measured with an ion energy analyzer [31 (link)]. A 100 nm thick Au layer was deposited directly on PEN nano dimples at a deposition rate of 2.0 Å s⁻¹ using a thermal evaporation system (LAT, Osan, Republic of Korea). The base pressure of the chamber was 9.6 × 10⁻⁶ Torr. Then, the prepared Au/PEN nano-dimple substrates were processed with 97% perfluorodecanethiol (Sigma-Aldrich, St. Louis, MO, USA). A total of 10 μL of 97% perfluorodecanethiol solution was poured into a glass Petri dish, the Petri dish lid was put to the Au/PEN nano dimple substrate, and the lid was closed for 2 h [23 (link)]. Next, an 80 nm thick Au layer was deposited onto the PFDT-treated Au/PEN nano dimple substrate at a deposition rate of 0.3 Ås⁻¹ through a thermal evaporation process (LAT, Osan, Republic of Korea). The base pressure of the chamber was 9.6 × 10⁻⁶ Torr. The deposition rate was watched using a quartz crystal microbalance.
Before the experiment, the substrate was cut into 3 × 3 μm² size and washed with tetrahydrofuran and distilled water. Then, MGITC solution was diluted to the concentration using pure Ethanol as buffer (10⁻⁷ M~5 × 10⁻¹³ M, blank). The cut substrate was added to 200 μL different concentrations of MGITC solution and reacted by shaking (500 rpm) for 1 h. After the reaction, the substrate was added to 200 μL of DW, washed by shaking for 5 min, and fixed on a slide glass to dry. To evaluate reproducibility between substrates, five Au nano popcorn substrates of 3 × 3 μm² size were prepared and reacted in 10⁻⁶ M of MGITC solution for 1 h.
The SERS spectra and Raman mapping images were obtained by an inVia Renishaw Raman microscope system (Renishaw, New Mills, UK). A He-Ne laser operating at 632.8 nm was used as the excitation source. Raman mapping images were obtained using a 20× objective lens and measured with an exposure time of 1 s and a laser power of 10%. To evaluate reproducibility, Raman mapping images were obtained using a 20× objective lens and measured with an exposure time of 0.1 s and laser power of 10%. The characteristic Raman peak of MGITC at 1614 cm⁻¹ was scanned over an area of 48 × 48 μm² range with 2 × 2 μm² mapping steps, for a total of 625 pixels. The baseline correction of Raman spectra was carried out using WiRE V 5.3 software (Renishaw, Newmills, UK). Spectral analysis and Raman mapping image, digital decoding image generation were performed using Origin 2017 64Bit software (OriginLab Corporation, Northampton, MA, USA).
The data were processed using the mean and standard deviation of pixels. An area of 48 × 48 μm² range was measured three times at 2 μm step size, yielding a total of 1875 points of data. For data processing, we acquired the mean and standard deviation of the 1875 points to obtain a calibration curve. Based on the mean and standard deviation of the intensity of the blank (without analyte), the mean + 3 × standard deviation was set as the threshold value, which was set to “0” if it was less than the threshold value and “1” if it was greater than the threshold value. For each concentration, the number of pixels higher than the threshold value was counted and calculated for each concentration.