Synthesis of fluoro-functionalized diaryl-λ3-iodonium salts and their cytotoxicity against human lymphoma U937 cells

Conscious of the potential bioactivity of fluorine, an investigation was conducted using various fluorine-containing diaryliodonium salts in order to study and compare their biological activity against human lymphoma U937 cells. Most of the compounds tested are well-known reagents for fluoro-functionalized arylation reactions in synthetic organic chemistry, but their biological properties are not fully understood. Herein, after initially investigating 18 fluoro-functionalized reagents, we discovered that the ortho-fluoro-functionalized diaryliodonium salt reagents showed remarkable cytotoxicity in vitro. These results led us to synthesize more compounds, previously unknown sterically demanding diaryliodonium salts having a pentafluorosulfanyl (SF5) functional group at the ortho-position, that is, unsymmetrical ortho-SF5 phenylaryl-λ3-iodonium salts. Newly synthesized mesityl(2-(pentafluoro-λ6-sulfanyl)phenyl)iodonium exhibited the greatest potency in vitro against U937 cells. Evaluation of the cytotoxicity of selected phenylaryl-λ3-iodonium salts against AGLCL (a normal human B cell line) was also examined.


Results and Discussion
To begin our investigation related to bioactivity, we randomly selected some fluorinating reagents that we had already developed, including Shibata reagents I [20] and II [21] (trifluoromethylation reagent 1 and trifluoromethylthiolation reagent 2a, respectively), pentafluorophenylating reagent 2b and several hypervalent iodine reagents, i.e., diaryliodonium salts with a mesitylene ligand (3a-o) and a triisopropylphenyl ligand 4a [20][21][22][59][60][61][62][63][64] (Figure 1). We used the Muse TM Annexin V and Dead Cell Assay Kit (FITC), which is a common tool to detect the ability of compounds to induce cell death. U937, a human histiocytic lymphoma cell line (DS Pharma Biomedical EC85011440; Osaka, Japan) was used to examine the ability of our synthesized compounds to induce cell death.
Following this investigation, we found that the ortho-substituted diaryliodonium salts with an ortho-SO 2 CF 3 group 3k and an ortho-CF 3 group 3m displayed impressive results. A common feature of ortho-substitution on the aromatic group is steric demand, which allowed us to analyse the cell death-inducing potency of phenyl aryliodonium salts with a more sterically demanding fluoro-functional group of SF 5 at the ortho-position. As we did not succeed in synthesizing ortho-SF 5 -substituted aryliodonium salts previously [59], we decided to proceed with a further investigation of the synthesis of ortho-SF 5 phenyl aryliodonium salts.
The newly synthesized SF 5 -diaryliodonium salts 3p, 4b, 5a and 6a were characterized spectroscopically. The single crystal X-ray structure of 3p was also analysed. The SF 5 -diaryliodonium salt 3p has a T-shaped geometry at the iodine centre, consistent with the general structure of diaryliodonium salts [33] (Figure 3).
From the above analysis, we selected a series of ortho-fluorinated diaryliodonium salts SO 2 CF 3 type 3k, CF 3 type 3m and SF 5 type 3p and examined their IC 50 values based on an MTT assay (Table 1). While 3k was more potent than 3m at both  concentrations, i.e., 1 μM and 5 μM (Figure 2), 3m has a lower IC 50 value of 0.68 μM than that of 3k (2.45 μM), as evaluated by the MTT assay. The best potency and IC 50 value (0.49 μM) was obtained for SF 5 type 3p, which is quite impressive when compared to the well-known antitumor drug cytosine arabinoside (ara-C), (0.16 μM) [27].
Since, 3k, 3m and 3p exhibited strong cytotoxicity against U937 cells, we finally evaluated their cytotoxicity against normal cells in vitro. AGLCL, a human normal B cell line (DS Pharma Biomedical EC89120566; Osaka, Japan) was chosen for the experiments and investigations were performed at 20 μM ( Figure S2 in Supporting Information File 1) and 5 μM and 1 μM concentrations ( Figure 5) of the compounds. Although 3k, 3m and 3p exhibited cytotoxicity even against AGLCL cells, a remarkable difference was observed. That is, moderate cytotoxicity at 5 μM (51.1%, 51.2% and 62.0%, respectively) and low cytotoxicity at 1 μM concentration (20.4%, 15.8% and 24.9%, respectively) against AGLCL cells were observed. It is noteworthy that the cytotoxicity displayed by 3k, 3m and 3p against U937 cells is much higher than those against AGLCL cells at both concentrations. These results strongly suggested that antitumor drug candidates could be designed by further structural modification of these compounds 3. Moreover, with 3p exhibiting the greatest potency against U937 cells with comparably lower toxicity against AGLCL cells, further biological studies using 3p including in vivo evaluation should be conducted. A mechanistic study that examines the structure-cytotoxicity relationships of a series of diaryliodonium salts 3 will also be conducted.

Conclusion
In conclusion, we have analysed a series of fluorinating reagents and diaryliodonium salts for their applicability in in- ducing cell death based on U937 (a human histiocytic lymphoma cell line). We have also successfully synthesized novel ortho-SF 5 phenylaryl-λ 3 -iodonium salts. As expected, several fluorinated diaryliodonium salts exhibited cytotoxicity. Among the series, the newly synthesized ortho-SF 5 salt 3p displayed the greatest potency. The ortho-fluorinated diaryliodonium salts 3k, 3m and 3p were also examined for comparison studies with AGLCL (a normal human B cell line).
Although the values were rather low, selectivity was indeed observed against U937 cells. Also, even though the results are in a preliminary stage of biological evaluation, this is the first report to highlight the cytotoxicity of diaryliodonium salts against U937 cells. Since diaryliodonium salts are fundamentally oxidizing agents, there might be a stronger correlation between cytotoxicity and the oxidation potential of these salts. We will continue the biological investigation of 3 in this direction. From the view point of organic synthesis, the newly synthesized ortho-SF 5 -substituted unsymmetrical iodonium salts 3p, 4b, 5a and 6a have potential use as electrophilic SF 5 -phenylation reagents for a range of nucleophiles such as alcohols, amines, thiols, and active methylene nucleophiles [59][60][61]. The application of these ortho-SF 5 -substituted diaryliodonium salts in organic synthesis, as well as their detailed bioactive behaviour, will be reported in due course.

Experimental
Biological assay Statistical analysis: Data were analyzed using Excel software.
Results are expressed as the mean ± SD of three independent replicates.
MTT assay: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate cell viability by diaryliodonium salt compounds using the MTT cell proliferation assay kit (Cayman Chemical Company, Ann Arbor, USA). U937 cells were incubated in solutions containing the diaryliodonium salts (3k, 3m or 3p). After this treatment, the U937 cells were seeded in culture medium (100 μL) in a 96-well plate (Becton and Dickinson, at a density of 2 × 10 5 cells/well) and incubated at 37 °C for 24 h. MTT reagent (10 μL) was added to each well. After mixing gently, the cells were incubated for 3 h at 37 °C in a CO 2 incubator. The culture medium was aspirated and the crystal-dissolving solution (100 μL) was added to each well and mixed. Finally, the optical density was measured (550 nm) using a microplate reader (BIO-RAD, Benchmark, Hercules, USA).

General information
All reactions were performed in oven-dried glassware under positive pressure of nitrogen or argon unless mentioned otherwise. Solvents were transferred via a syringe and were introduced into reaction vessels though a rubber septum. All reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm Merck silica gel (60-F254). The TLC plates were visualized with UV light (254 nm). Column chromatography was carried out on columns packed with silica gel (60N spherical neutral size 63-210 μm). The 1 H NMR (300 MHz), 19 F NMR (282 MHz), and 13 C NMR (125 MHz) spectra for solution in CDCl 3 or (CD 3 ) 2 CO were recorded on Varian Mercury 300 and Bruker Avance 500 spectrometers. Chemical shifts (δ) are expressed in ppm downfield from TMS (δ = 0.00) or C 6 F 6 [δ = −162.2 (CDCl 3 ) or −163.5 ((CD 3 ) 2 CO)] as an internal standard. Mass spectra were recorded on a Shimadzu GCMS-QP5050A (EIMS) and Shimazu LCMS-2020 (ESIMS) spectrometer. The solvent CH 2 Cl 2 was dried and distilled before use.

Preparation of pentafluoro(2-iodophenyl)-λ 6 -sulfane (7):
The preparation of 7 was based on a modified procedure in the literature [67]. To 4 mL of HBF 4 in a round bottomed flask, 1.1 g of 2-(pentafluoro-λ 6 -sulfanyl)aniline was added and heated until a clear solution formed. The solution was cooled to 0 °C and a cold solution of NaNO 2 (380 mg in 2.5 mL distilled water) was added dropwise. The reaction was allowed to stir at 0 °C for 15 min after which it was added dropwise to a cold stirred solution of KI (1.33 g in 10 mL distilled water) at 0 °C. The reaction was allowed to warm to room temperature then stirred for 20 min. The reaction mixture was extracted with diethyl ether (3 × 20 mL). The combined organic extract was washed with NaHCO 3 solution and Na 2 S 2 O 3 solution and dried over Na 2 SO 4 . The solvent was concentrated under reduced pressure to give a crude product which was purified using silica gel column chromatography General procedure A: preparation of diaryliodonium salts I These salts were prepared according to a modified procedure in the literature [64,65]. m-CPBA (assume 70 wt %, 1.1 equiv) was dried in vacuo at room temperature for 1 h before the addition of 7 (1.0 equiv) and CH 2 Cl 2 (6.0 mL/mmol ArI) in a round-bottomed flask. The solution was cooled to 0 °C followed by the dropwise addition of TfOH (1.7 equiv). The resulting mixture was stirred at room temperature for 2 h. It was then cooled to 0 °C and the arene (1.1 equiv) was added dropwise. The mixture was warmed to room temperature and stirred for 18 h. The solvent was then removed under reduced pressure. The resulting crude product was precipitated by the addition of Et 2 O. The precipitate was filtered and dried in vacuo to give 3-6 as an off-white to white solid.

Supporting Information
Supporting Information File 1 Cytotoxicity data, copies of 1 H, 19 F and 13 C NMR spectra of 7, 3p, 4b, 5a and 6a and the ORTEP diagram of 3p.