Design and Synthesis of Thiourea-Conjugating Organic Arsenic D-Glucose with Anticancer Activities

Organic arsenic compounds such as p-aminophenylarsine oxide (p-APAO) are easier for structural optimization to improve drug-like properties such as pharmacokinetic properties, therapeutic efficacy, and target selectivity. In order to strengthen the selectivity of 4-(1,3,2-dithiarsinan-2-yl) aniline 7 to tumor cell, a thiourea moiety was used to strengthen the anticancer activity. To avoid forming a mixture of α/β anomers, the strategy of 2-acetyl’s neighboring group participation was used to lock the configuration of 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate from 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide. 1-(4-(1,3,2-dithiarsinan-2-yl) aniline)-2-N-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranos-1-yl)-thiourea 2 can increase the selectivity of human colon cancer cells HCT-116 (0.82 ± 0.06 μM vs. 1.82 ± 0.07 μM) to human embryonic kidney 293T cells (1.38 ± 0.01 μM vs. 1.22 ± 0.06 μM) from 0.67 to 1.68, suggesting a feasible approach to improve the therapeutic index of arsenic-containing compounds as chemotherapeutic agents.


Introduction
Arsenic trioxide (As, +III) and its derivatives have been used for various purposes for more than 2000 years [1][2][3].In the 1970s, arsenic trioxide was used to heal acute promyelocytic leukemia (APL) in clinical application by Zhang Tingdong [1].Subsequently, arsenic trioxide (Trade Name: Trisenox) has been approved for the treatment of relapsed or refractory APL by the U.S. Food and Drug Administration since 2000 and European Medicines Agency since 2017, respectively.The mechanisms of action of arsenic trioxide have revealed that the drug is able to promote the catabolic degradation of an oncogenic fusion protein [4][5][6], disrupt the mitochondrial function [7], downregulate Bcl-2 expression [8], and reactivate mutant p53 for tumor suppression [9].Despite the remarkable success of arsenic trioxide in the treatment of APL, limitations of inorganic arsenic compound as a chemotherapeutic was the systemic toxicity, associated with its poor pharmacokinetic properties.This may be attributed to the rapid renal clearance of arsenic trioxide metabolites [2,10].
Urea cycle dysregulation (UCD) in cancer is a prevalent phenomenon in multiple cancers.It is also associated with a worse prognosis but a better response to immune therapy [19].Small organic molecules with a thiourea moiety have been widely used in the treatment of anticancer [20][21][22][23].
Combined with the previous basis of the design and synthesis of small molecules containing urea structure and based on the principle of bioisosterism, we proposed the research topic of the design, synthesis and bioactivity of small-molecule compounds containing a thiourea structure.Here, the moiety of thiourea-conjugating organic arsenic was induced to anomeric carbon of D-glucose derivatives.
Based on the derivatives of D-glucosamine, it could reduce the toxicity of trivalent arsenic compound and other pharmacophore [24,25].Actually these D-glucosamine linker trivalent arsenic compounds and other pharmacophore a the mixture of α/β anomeric isomers in dynamic equilibrium.To avoid the effect of enantiomer on biological activity, here, a novel glucose conjugated arsenic compound was designed as the follows: thiourea was used to strengthen the anticancer biological activities; the anomeric group's steric configuration of D-glucosamine derivative was locked by the reaction of 2-acetyl's neighboring group participation via five-membered glycosyl oxocarbenium ion (Figure 2) to avoid the effect of enantiomer on biological activity [25,26].2,3,4, 6-tetra-O-acetyl-β-Dglucopyranosyl isothiocyanate was designed as precursor of 1-amine-β-D-glucose to avoid the effect of enantiomer on biological activity.Isothiocyanate's strong electrophilicity was used to react with amine in compound 6 or 7 to form derivatives of 1-amine β-D-glucose 1 and 2, respectively.

Results and Discussion
Our target compounds, the derivatives of 4-aminophenylarsenous acid (As, +III) linking to the anomer of D-glucose to form β-configuration D-glucose-arsenate compounds were designed as follows: the synthesis routes were started from commercial chemical 4-aminophenylarsonic acid (As, +V) 3. It was reduced to 4-aminophenylarsenic acid (As, +III) 4 by sulfur dioxide under a catalyst of minor iodine.The solution of 4 in ethanol was heated to reflux with propane-1,3-dithiol to obtain compound 7 [27][28][29].Compound 4 was also dissolved in concentrated HCl to produce intermediate 5.It further reacted with ethane-1,2-dithiol at room temperature to obtain compound 6 under aqueous sodium carbonate [18].2,3,4,6-O-tetraacetyl-α-D-glucopyranosyl bromide 8 reacted with potassium thiocyanate through nucleophilic substitution to form β-anomer of D-glucose derivative 9 under the catalyst of a 4 Å molecular sieve and (n-Bu)4NBr as a phase transfer catalyst.With the aid of 2-acetyl's neighboring group participation, the configuration of SCN formed the β-anomer via a five-membered glycosyl oxocarbenium ion to replace the original α-anomer [30].The chemical shift of anomeric H in compound 8 is 6.62-6.61ppm.J1,2 (coupling constant) of anomeric H of compound 8 is 4.0 Hz (Figure 3A), which is in the J1,2 range of α-D-glucose (3-5 Hz), while the peak at 5.14-5.12ppm is anomeric H of compound 9. Anomeric H's J1,2 (coupling constant) of compound 9 is 8.0 Hz, which is in the J1,2 range of β-D-glucose (6-9 Hz) in 1 H NMR (Figure 3B).The anomeric H's coupling constants (J1,2) of compound 1 and 2 are 8.0 Hz and 8.0 Hz, respectively.They are also in the anomeric H's J1,2 of β-D-glucose (6-9 Hz) range in 1 H NMR [31].These results showed that the anomeric H has been changed from α configuration (compound 8) to β one (compound 9, 1 and 2).In the following steps, compounds 6 and 7 reacted with 9 involving a nucleophilic attack to reach our target compounds 1 and 2 at room temperature with yields of 81.9% and 71.5%, respectively (Scheme 1).

Materials and Methods
All the chemical reagents and solvents were purchased from Sinopharm Group Company limited (Shanghai, China).They were used without further purification, unless specified otherwise.4-Aminophenylarsonic acid was bought from Tokyo Chemical Industry (Shanghai) Development Co., Ltd.(Shanghai, China).All anhydrous reactions were performed under nitrogen atmosphere.Organic phases were dried over anhydrous Na 2 SO 4 and removed under pressure during work-up.
Purities of the intermediates were established by silica gel (200-300 mesh) column chromatography.Thin-layer chromatography (TLC) was carried out by silica gel GF254, both of which were obtained from Qingdao Ocean Chemicals (Qingdao, China).In all experiments, water used was distilled and purified by the Milli-Q system (Millipore, Mississauga, Canada). 1 H NMR and 13 C NMR spectra of final compounds were recorded on a Bruker Ultra-shield 400 MHz Plus spectrometer (Bruker, Rheinstetten, Germany) using TMS as the internal standard (see Supplementary Materials).All chemical shifts are reported in the standard δ notation of parts per million.High-Resolution Mass Spectra were obtained using Waters UPLC Class I/XevoG2Q-Tof (Waters, Milford, MA, USA).Compound 9 (200 mg, 0.5136 mmol) and compound 6 (134 mg, 0.5136 mmol) were dissolved in 10 mL anhydrous dichloromethane.It was stirred overnight.The reaction was monitored by TLC (thin-layer chromatography (PE:EA = 2:1, R f = 0.28)).When the reaction was over, the solvent was removed under reduced pressure.The residue was purified by preparation of thin-layer chromatography with eluant (PE:EA = 1:1).The product was obtained as a white solid (273.6 mg, 81.9%).The product was characterized by IR, 1 H NMR, 13  Compound 9 (200 mg, 0.5136 mmol) and compound 7 (140.4mg, 0.5136 mmol) were dissolved in 10 mL anhydrous dichloromethane.This was stirred overnight.The reaction was monitored by TLC (thin-layer chromatography (PE:EA = 2:1, R f = 0.48)).When the reaction was over, the solvent was removed under reduced pressure.The residue was purified by preparation of thin-layer chromatography with eluant (PE:EA = 1:1).The product was obtained as a white solid (243 mg, 71.5%).The product was characterized by IR, 1 H NMR, 13    6) [24,26] Compound 6 was synthesized with a yield of 87% as described in previous literature.It was characterized by IR, 1 H NMR, 13  Compound 4 (1 g, 5.4654 mmol) and propane-1,3-dithiol (615 µL mg, 6.1376 mmol) were dissolved in ethanol (15 mL).The solution was refluxed under N 2 for 5 h.It was monitored by TLC (PE: EA = 4:1, R f = 0.45).The reaction was cooled to room temperature.The solution was evaporated under reduced pressure to concentrated residue.Then, it was transferred to a refrigerator overnight.It was filtered.The filtered cake was washed with 5 mL cold ethanol twice.Compound 7 was obtained as an off-white solid (1.1761 g, 78.8%).It was characterized by IR, 1 H NMR, 13  3.1.5.2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (9) [30] Potassium thiocyanate (473 mg, 4.863 mmol), a 4Å molecular sieve (3.48 g) and tetrabutylammonium bromide (784 mg, 2.432 mmol) were added to 30 mL anhydrous acetonitrile.It was stirred under N 2 at room temperature for 3 h.2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide 8 (1000 mg, 2.432 mmol) was added to the solution.The reaction was heated to reflux under N 2 for 4 h.It was monitored by thin-layer chromatography with eluant (PE:EA = 3:1).When the reaction was over, the solution was cooled to the room temperature.The solvent was removed under reduced pressure.The residue was purified by silica gel chromatography with eluant (PE:EA = 3:1).The product was obtained as a off-white solid with a yield of 76.3% (690.8 mg).It was characterized by IR, 1 H NMR, 13   [25] HeLa and HepG2 cells were obtained from Stem Cell Bank, Chinese Academy of Sciences.The human colon cancer HCT-116 cells and human embryonic kidney cells 293T were purchased from the American Type Culture Collection (ATCC).Cells were cultured in high-glucose DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% (v/v) FBS (Gibco, Grand Island, NE, USA) and 1% (v/v) penicillin-streptomycin (PS) (Gibco, Grand Island, NE, USA), and maintained at 37 • C under 5% CO 2 humidified atmosphere.Only cells in logarithmic phase were used in all following experiments.

MTT Assay [25]
The cytotoxicity of compounds 1, 2, 6 and 7 in Hela, HepG2, HCT 116 and 293T cells was evaluated by MTT assay.Cells were seeded onto a 96-well plate (Corning, Corning, NY, USA) with a density of 3000 cells/well for 24 h in high-glucose DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% (v/v) FBS (Gibco, Grand Island, NE, USA) and 1% (v/v) penicillin-streptomycin (PS) (Gibco, Grand Island, NE, USA), and then treated with tested compounds at various concentrations for 72 h.MTT solution was added to each well for 4 h, and the absorbance was measured at 495 nm by using Varioskan Flash Multimode Reader (Thermo, Waltham, MA, USA).The values of IC 50 were calculated by GraphPad Prism Software 9.0.

3 .
It was reduced to 4-aminophenylarsenic acid (As, +III) 4 by sulfur dioxide under a catalyst of minor iodine.The solution of 4 in ethanol was heated to reflux with propane-1,3-dithiol to obtain compound 7 [27-29].Compound 4 was also dissolved in concentrated HCl to produce intermediate 5.It further reacted with ethane-1,2-dithiol at room temperature to obtain compound 6 under aqueous sodium carbonate [18].2,3,4,6-O-tetraacetyl-α-D-glucopyranosyl bromide 8 reacted with potassium thiocyanate through nucleophilic substitution to form β-anomer of D-glucose derivative 9 under the catalyst of a 4 Å molecular sieve and (n-Bu) 4 NBr as a phase transfer catalyst.