Synthesis and Antiproliferative Screening Of Novel Analogs of Regioselectively Demethylated Colchicine and Thiocolchicine

Colchicine, a pseudoalkaloid isolated from Colchicum autumnale, has been identified as a potent anticancer agent because of its strong antimitotic activity. It was shown that colchicine modifications by regioselective demethylation affected its biological properties. For demethylated colchicine analogs, 10-demethylcolchicine (colchiceine, 1) and 1-demethylthiocolchicine (3), a series of 12 colchicine derivatives including 5 novel esters (2b–c and 4b–d) and 4 carbonates (2e–f and 4e–f) were synthesized. The antiproliferative activity assay, together with in silico evaluation of physicochemical properties, confirmed attractive biological profiles for all obtained compounds. The substitutions of H-donor and H-acceptor sites at C1 in thiocolchicine position provide an efficient control of the hydration affinity and solubility, as demonstrated for anhydrate 3, hemihydrate 4e and monohydrate 4a.


S2
The error of the chemical shift value was 0.01 ppm. The 13 C NMR spectra were recorded at the operating frequency 101.25 MHz. The error of chemical shift value was 0.1 ppm. All spectra were locked to deuterium resonance of CDCl3. The ESI (Electrospray Ionisation) mass spectra were recorded on a Waters/Micromass (Waters Corporation, Manchester, UK) ZQ mass spectrometer equipped with a Harvard Apparatus syringe pump. The samples were prepared in dry acetonitrile (5 x10-5 mol dm -3 ). The sample was infused into the ESI source using a Harvard pump at a flow rate of 20 ml min-1. The ESI source potentials were: capillary 3 kV, lens 0.5 kV, extractor 4 V. The standard ESI mass spectra were recorded at the cone voltages: 10 and 30 V. The source temperature was 120 °C and the desolvation temperature was 300 °C. Nitrogen was used as the nebulizing and desolvation gas at flow-rates of 100 dm 3 h -1 . Mass spectra were acquired in the positive ion detection mode with unit mass resolution at a step of 1 m/z unit. The mass range for ESI experiments was from m/z = 100 to m/z = 1000, as well as from m/z = 200 to m/z = 1500 The antiproliferative activity

The Antiproliferative Assays In Vitro
Twenty-four hours before adding the tested compounds, all cell lines were seeded in 384-well plates (Sarstedt, Nümbrecht, Germany) in appropriate media with 1x10 3 or 1,5x10 3 or 3x10 3 cells per well. All cell lines were exposed to each tested agent at four different concentrations in the range 100-0.001 μg/mL for 72 h. Cells were also exposed to the Compounds at each concentration were tested in triplicates in single experiment and each experiment was repeated at least three times independently.

X-ray measurements
The X-ray diffraction measurements at RT of 3, 4a and 4e, were carried out on two different diffractometers. First one equipped with MoKα (λ =0.7108 Å) source, Eos Xcalibur, and the second one, with microfocus CuKα (λ =1.5418 Å) radiation source, SuperNova Agilent diffractometer. For data collections and their preliminary reduction, CrysAlisPro 171.37.31 software was used 3 . Crystal structure was solved by direct methods with Shelxs and further refined with the Shelxl 4,5 ; with the Olex2 interface 6 .The position of hydrogen atoms were located at their best geometrical positions. In the structure 4e disorder on C9 carbonyl group and C10 thiomethoxy group was refined with half occupancy in two positions accordingly.
The details of crystal data, data collection, structure solution and refinement are given in

Synthesis and characterization of 10-demethylcolchicine (colchiceine, 1)
A mixture of colchicine (500 mg, 1.25 mmol), glacial acetic acid (0.5 mL), and hydrochloric acid (0.1N, 30 mL) was stirred for 6h at 100 °C. After that time crystals start to precipitate, and X-ray analysis, which was in consistence with results obtained by Mackey et al. [33], confirmed them to be the desired products. 1 was obtained as yellow-brown crystals (435 mg, 90%) and directly used for next steps. 1

Synthesis and characterization of thiocolchicine
To a mixture of colchicine (500 mg, 1.25 mmol) in MeOH/water (1/1, v/v, 5 mL), the sodium methanethiolate (solution 21% in H2O, 0.83 mL, 2.5 mmol) was added. The mixture was stirred in at RT for 24 h. Reaction was controlled by TLC. After that time, the reaction mixture was quenched by the addition of water (150 mL). The whole mixture was extracted four times with CH2Cl2, and the combined organic layers were dried over MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by CombiFlash® (hexane/EtOAc (1/1), then EtOAc/MeOH, increasing concentration gradient) to give thiocolchicine as amorphous yellow solid with yield 78% . 1

Synthesis and characterization of 1-demethylthiocolchicine (3)
To the solution of thiocolchicine (1.0 g, 2.41 mmol) in dichloromethane (DCM, 50 ml) cooled to the 0 °C temperature, an acetyl chloride (3 mL) and tin tetrachloride (1mL) were added. The mixture was first stirred at 0 °C temperature for 1h, and then for the next 48h at RT. After that time TLC analysis showed no presence of thiocolchicine, and the DCM was evaporated to dryness under reduced pressure. The residue was dissolved in MeOH/water (5/1, v/v, 60 mL), then lithium hydroxide (excess) was added and the mixture was stirred at RT for 1h. Reaction time was determined by TLC. The solvent was evaporated to dryness under reduced pressure and the residue was purified by CombiFlash® (chloroform/acetone, increasing concentration gradient) to give product as amorphous yellow solid with 40% yield.               S22 Figure S26. The 13 C NMR spectrum of 4c in DMSO-d 6 . Figure S27. The ESI-MS spectrum of 4c.