Luna c18 column

The purity of the final product 3 was determined by HPLC using the LUNA C18 column on a Hitachi system
equipped with a diode array detector set at 224 nm. The HPLC method
followed that of the USP method for amodiaquine hydrochloride. Compounds
were dissolved in water (15 mg/100 mL) and injected through a loop.
Retention time (t_R) was obtained at a
flow rate of 1.2 mL/min using an isocratic run of 78% eluent A (potassium
phosphate buffer) and 22% eluent B (MeOH) for a period of 0 to 15
min. The purity of the sample was determined based on the pharmacopoeia
standard by preparing two standard solutions (15 mg in 100 mL of water),
one with six injections and the other with two injections, to determine
standard recovery with acceptable criteria of 97–103. After
different drying conditions were evaluated, the oven-dried product
(15 mg) was dissolved in 100 mL of water and injected (10 μL)
into the specified column with a runtime of 15 min.

All of the raw materials
and solvents purchased were used without further purification. Thin
layer chromatography (TLC) was performed on Macherey-Nagel 0.2 mm
silica gel 60 F254 packed aluminum plates observed under UV light
at 254 nm. The synthesized compounds were analyzed by FT-IR spectroscopy,
NMR spectroscopy with the residual solvent peak as an internal reference
(DMSO-d₆ = 2.50 and 39.5 ppm and CDCl₃ = 7.26 and 77.16 ppm for ¹H and ¹³C
NMR spectra, respectively), and gas chromatography mass spectroscopy
(GC–MS). The purity of the final product 3 was
determined by using high-performance liquid chromatography (HPLC)
on a Hitachi system equipped with a LUNA C18 column and a diode array
detector set at 224 nm.
Thermal analyses of the final ADQ products
(3) were conducted using thermogravimetric analysis (TGA),
TGA-TA 5500, and differential scanning calorimetry (DSC), DSC-TA 2500,
under a nitrogen atmosphere. The TGA and DSC thermograms were analyzed
by TRIOS 5.3.0.48151 version and Origin2018. Isothermal experiments
were performed with a TRIOS 5.3.0.48151 version calorimeter with a
nitrogen flow rate of 50 mL/min.

UV spectra were measured on a UV-2550 UV-visible spectrophotometer (Shimadzu, Shimane-ken, Japan). IR spectra were recorded on a 380 FT-IR spectrometer (Thermo Nicolet, Waltham, MA, USA). The optical rotations were measured on an AutoPol IV automatic polarimeter (Rudolph Research, Wilmington, MA, USA) at room temperature. 1D and 2D NMR data were recorded on a 600 MHz instrument (Varian, Palo Alto, CA, USA) with TMS as internal standard. HRESIMS data were acquired using a Triple TOF 6600 mass spectrometer (AB Sciex, Framingham, MA, USA). Semi-preparative HPLC separations were performed on a Chromaster system (Hitachi, Tokyo, Japan) consisting of a 5110 pump, 5210 autosampler, 5310 column oven, 5430 diode array detector and a Phenomenex Luna C₁₈ column (250 × 10 mm, S-5 μm), all operated using EZChrom Elite software. All solvents were of ACS or HPLC grade, and were obtained from Tansoole (Shanghai, China) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Silica gel (300–400 mesh), C₁₈ reverse-phased Silica gel (150-200 mesh, Merck, Darmstadt, German), and MCI gel (CHP20P, 75–150 μM, Mitsubishi Chemical Industries Ltd., Tokyo, Japan) were used for column chromatography (CC), and pre-coated Silica gel GF254 plates (Qingdao Marine Chemical Plant, Qingdao, China) were used for TLC.