Hydroxide-Mediated SNAr Rearrangement for Synthesis of Novel Depside Derivatives Containing Diaryl Ether Skeleton as Antitumor Agents

A simple and efficient hydroxide-mediated SNAr rearrangement was reported to synthesize new depside derivatives containing the diaryl ether skeleton from the natural product barbatic acid. The prepared compounds were determined using 1H NMR, 13C NMR, HRMS, and X-ray crystallographic analysis and were also screened in vitro for cytotoxicity against three cancer cell lines and one normal cell line. The evaluation results showed that compound 3b possessed the best antiproliferative activity against liver cancer HepG2 cell line and low toxicity, which made it worth further study.


Introduction
Depsides are dimers formed by ester bonds between two aromatic rings, which are the main secondary metabolites of lichens [1]. Previous research reported that depsides had good biological properties, such as antitumor, antioxidant, and antibacterial activities [2][3][4]. Barbatic acid (1, Figure 1) is one kind of depside that was widely discovered from the lichen [5] and was determined to have a variety of biological activities, including anticancer, schistosomicidal, diuretic, and the potential to inhibit the growth of plants and algal [6][7][8][9]. However, to the best of our knowledge, little attention has been paid to the further structural modification of barbatic acid for developing potential antitumor agents. Diaryl ethers are an important class of organic compounds with two aromatic rings and a flexible oxygen bridge, which are widely used in various fields such as medicine and pesticides [10]. For instance, Sorafenib (I, Figure 1) is a highly effective small molecule inhibitor with anticancer properties, which is used to treat advanced renal cancer [11]. Nimesulide (II, Figure 1), a nonsteroidal anti-inflammatory drug, has been popularized for several decades [12]. Difenoconazole (III, Figure 1) and famoxadone (IV, Figure 1) have been utilized as fungicides to protect a wide range of plants, such as rice, cotton, and Diaryl ethers are an important class of organic compounds with two aromatic rings and a flexible oxygen bridge, which are widely used in various fields such as medicine

Results and Discussion
As illustrated in Figure 2, firstly, 3-hydroxy-4-(isopropoxycarbonyl)-2,5-dimethylphenyl 2-hydroxy-4-methoxy-3,6-dimethylbenzoate (2a) was prepared by our previously reported method [20]. Subsequently, 2a reacted with potassium hydroxide in the mixed solvent (DMSO/water = 10/1, v/v) at room temperature. To our delight, the reaction did not undergo hydrolysis to produce corresponding benzoic acid and phenol as we expected but instead underwent a rearrangement reaction to produce diaryl ethers with a yield of 81% (3a). This was proven by comparison with the partial 1 H NMR spectra of compounds 2a and 3a ( Figure 3). Compared to compound 2a, compound 3a had only one phenolic hydroxyl group, and the chemical shift of aromatic signals was shifted from 6.37 ppm to 5.77 ppm due to the steric effects. There was no significant change in the other chemical shifts. Moreover, the X-ray crystal structure ( Figure 4) of compound 3a further demonstrated this conclusion. Diaryl ethers are an important class of organic compounds with two aromatic rings and a flexible oxygen bridge, which are widely used in various fields such as medicine and pesticides [10]. For instance, Sorafenib (I, Figure 1) is a highly effective small molecule inhibitor with anticancer properties, which is used to treat advanced renal cancer [11]. Nimesulide (II, Figure 1), a nonsteroidal anti-inflammatory drug, has been popularized for several decades [12]. Difenoconazole (III, Figure 1) and famoxadone (IV, Figure 1) have been utilized as fungicides to protect a wide range of plants, such as rice, cotton, and cereals, from diseases [13,14]. Usually, diaryl ethers are mainly achieved through the coupling of phenols and aryl halides under the action of catalysts to form C-O bonds. These methods have certain deficiencies, such as high temperatures, expensive catalysts, or toxic solvents [15][16][17][18][19].
Herein, we reported an interesting means of converting some barbatic acid esters to novel diaryl ethers through a hydroxide-mediated SNAr rearrangement reaction. The synthesized compounds were also tested for antitumor activity, and some compounds demonstrated good effects.

Results and Discussion
As illustrated in Figure 2, firstly, 3-hydroxy-4-(isopropoxycarbonyl)-2,5-dimethylphenyl 2-hydroxy-4-methoxy-3,6-dimethylbenzoate (2a) was prepared by our previously reported method [20]. Subsequently, 2a reacted with potassium hydroxide in the mixed solvent (DMSO/water = 10/1, v/v) at room temperature. To our delight, the reaction did not undergo hydrolysis to produce corresponding benzoic acid and phenol as we expected but instead underwent a rearrangement reaction to produce diaryl ethers with a yield of 81% (3a). This was proven by comparison with the partial 1 H NMR spectra of compounds 2a and 3a ( Figure 3). Compared to compound 2a, compound 3a had only one phenolic hydroxyl group, and the chemical shift of aromatic signals was shifted from 6.37 ppm to 5.77 ppm due to the steric effects. There was no significant change in the other chemical shifts. Moreover, the X-ray crystal structure ( Figure 4) of compound 3a further demonstrated this conclusion.  In order to test the reliability of the method, compounds 3b-j continued to be synthesized with a 70-95% yield under the same reaction conditions, as shown in Figure 5. The structures of all target compounds were characterized using 1 H NMR, 13 C NMR, and HRMS. The stereochemistry of 3d was further confirmed by X-ray crystallographic analysis ( Figure 6).
In addition, a probable reaction mechanism for this rearrangement was proposed and is illustrated in Figure 7. The sequence began with a simple acid-base reaction wherein hydroxide deprotonated the hydroxyl group, as shown in pink in structure 2. The resulting phenoxide then participated in an intramolecular S N Ar cyclization onto the second ring, as illustrated by the conversion of structure 4 to the spirocyclic intermediate 5. The reestablishment of aromaticity was accompanied by the formation of the carboxylate anion shown in structure 6, and then acidification delivered the target structures 3. Molecules 2023, 28, x FOR PEER REVIEW 3 of 11  In order to test the reliability of the method, compounds 3b-j continued to be synthesized with a 70-95% yield under the same reaction conditions, as shown in Figure 5. The structures of all target compounds were characterized using 1 H NMR, 13 C NMR, and   In order to test the reliability of the method, compounds 3b-j continued to be synthesized with a 70-95% yield under the same reaction conditions, as shown in Figure 5. The structures of all target compounds were characterized using 1 H NMR, 13 C NMR, and In addition, a probable reaction mechanism for this rearrangement was proposed and is illustrated in Figure 7. The sequence began with a simple acid-base reaction wherein hydroxide deprotonated the hydroxyl group, as shown in pink in structure 2. The resulting phenoxide then participated in an intramolecular SNAr cyclization onto the second ring, as illustrated by the conversion of structure 4 to the spirocyclic intermediate 5. The re-establishment of aromaticity was accompanied by the formation of the carboxylate anion shown in structure 6, and then acidification delivered the target structures 3.   In addition, a probable reaction mechanism for this rearrangement was proposed and is illustrated in Figure 7. The sequence began with a simple acid-base reaction wherein hydroxide deprotonated the hydroxyl group, as shown in pink in structure 2. The resulting phenoxide then participated in an intramolecular SNAr cyclization onto the second ring, as illustrated by the conversion of structure 4 to the spirocyclic intermediate 5. The re-establishment of aromaticity was accompanied by the formation of the carboxylate anion shown in structure 6, and then acidification delivered the target structures 3.    Compounds 3a-j were screened for their antitumor activities against three human cancer cell lines (lung cancer A549 cells, liver cancer HepG2 cells, and prostatic cancer 22RV1 cells) in vitro by the standard MTT method, and the IC50 values are presented in Table 1. The experimental results showed that compounds 3a, 3c, 3e, and 3f exhibited moderate cytotoxic activities against A549 cells with IC50 values of 2.61, 1.43, and 2.21 mmol/L, respectively. Compounds 3a-d, i-j induced high cytotoxic activity against HepG2 cells, which exhibited excellent activities with IC50 values of 0.41-1.56 mmol/L. Compound 3d afforded the best antiproliferation activities toward 22RV1 cells, with an IC50 value of 0.78 mmol/L. The results demonstrated that diaryl ethers synthesized in this Compounds 3a-j were screened for their antitumor activities against three human cancer cell lines (lung cancer A549 cells, liver cancer HepG2 cells, and prostatic cancer 22RV1 cells) in vitro by the standard MTT method, and the IC 50 values are presented in Table 1 Selective killing of cancer cells without affecting normal cell growth is an important feature that must be considered in cancer chemotherapy. Therefore, the target compounds 3a-j were estimated for cytotoxicity toward normal madin-daby canine kidney cells (MDCK) and as shown in Figure 8. Compared to the blank control, most compounds could influence the growth of normal MDCK cells with cell survival rates below 90%, except for compound 3b (97.47%), suggesting that 3b had almost no toxicity to normal cells and could selectively inhibit the growth of liver cancer HepG2 cells.

Chemistry
All reagents and solvents were of reagent grade or were purified according to standard methods before use. Analytical thin-layer chromatography (TLC) was performed with silica gel plates using silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Melting points were determined on an XT-4 digital melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) and were uncorrected. Proton nuclear magnetic resonance spectra ( 1 H NMR) were recorded on a Bruker Avance DMX 400 MHz instrument (Bruker, Bremerhaven, Germany) in CDCl3 or DMSO-d6 using TMS (tetramethylsilane) as the internal standard. High-resolution mass spectrometry (HRMS) was carried out with a Xevo G2-SQTOF instrument (Waters, Milford, MA, USA).

Chemistry
All reagents and solvents were of reagent grade or were purified according to standard methods before use. Analytical thin-layer chromatography (TLC) was performed with silica gel plates using silica gel 60 GF 254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Melting points were determined on an XT-4 digital melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) and were uncorrected. Proton nuclear magnetic resonance spectra ( 1 H NMR) were recorded on a Bruker Avance DMX 400 MHz instrument Molecules 2023, 28, 4303 6 of 10 (Bruker, Bremerhaven, Germany) in CDCl 3 or DMSO-d 6 using TMS (tetramethylsilane) as the internal standard. High-resolution mass spectrometry (HRMS) was carried out with a Xevo G2-SQTOF instrument (Waters, Milford, MA, USA).

Synthesis of Intermediates 2a-j
Compounds 2a-j were prepared, as described in our previous publication [20], and their spectra data are shown below.

Synthesis of Target Compounds 3a-j
To a stirred solution of potassium hydroxide (67.3 mg, 1.2 mmol) in a mixed solvent (20 mL, DMSO/water = 10/1, v/v) at room temperature, 2a-j (0.6 mmol) was added. The reaction mixture was stirred for 1-2 h. Subsequently, the pH of the reaction mixture was adjusted to 1-2 with 1 mol/L hydrochloric acid, and the crude solid was collected by filtration before it was recrystallized with ethanol to afford 3a-j in 70-95%.