Synthesis and Evaluation of Aminothiazole-Paeonol Derivatives as Potential Anticancer Agents

In this study, novel aminothiazole-paeonol derivatives were synthesized and characterized using 1H-NMR, 13C-NMR, IR, mass spectroscopy, and high performance liquid chromatography. All the new synthesized compounds were evaluated according to their anticancer effect on seven cancer cell lines. The experimental results indicated that these compounds possess high anticancer potential regarding human gastric adenocarcinoma (AGS cells) and human colorectal adenocarcinoma (HT-29 cells). Among these compounds, N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]-4-methoxybenzenesulfonamide (13c) had the most potent inhibitory activity, with IC50 values of 4.0 µM to AGS, 4.4 µM to HT-29 cells and 5.8 µM to HeLa cells. The 4-fluoro-N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (13d) was the second potent compound, showing IC50 values of 7.2, 11.2 and 13.8 µM to AGS , HT-29 and HeLa cells, respectively. These compounds are superior to 5-fluorouracil (5-FU) for relatively higher potency against AGS and HT-29 human cancer cell lines along with lower cytotoxicity to fibroblasts. Novel aminothiazole-paeonol derivatives in this work might be a series of promising lead compounds to develop anticancer agents for treating gastrointestinal adenocarcinoma.


Introduction
Paeonol, 2-hydroxy-4-methoxy acetophenone (1, Figure 1), is a major component of traditional Chinese medicine. Moutan Cortex, the outer layer of the root of Moutan, is classified in the genus Paeonia and has been used for more than 1000 years. Paeonol is categorized as a flavonoid derivative and exhibits many remarkable biological effects, and it has been applied for anti-inflammatory [1,2], analgesic [2], antioxidant [3], antidiabetic [4], anticancer [5], and antiatherogenic purposes [6]. Paeonol was also found to protect against memory loss after ischemic stroke by reducing amyloid precursor protein (APP), beta-site APP cleaving enzyme (BACE), and apoptosis [7]. Moreover, paeonol derivatives have been reported to show many attractive biological activities. For example, Pan and Hui demonstrated that the donepezil-like paeonol derivative (2, Figure 1) exhibited strong metal-chelating ability for Alzheimer's disease (AD) treatment [8]. Yang presented a copper ion chelating paeonol Schiff-base derivative (3, Figure 1) complexes that possessed high antioxidant activity and moderate DNA-binding activity as well as high tumor cell cytotoxicity [9]. Moreover, Yu reported a paeonol thiosemicarbazone derivative (4, Figure 1), which exhibited potential mushroom tyrosinase inhibitors [10]. Recently, our group found that phenylsulfonyl moieties-conjugated paeonol derivatives were potential anti-Hepatitis B virus leads [11] and could prevent lipid accumulation at lower doses, and they might be prominent antiatherogenic agents [12]. was also found to protect against memory loss after ischemic stroke by reducing amyloid precursor protein (APP), beta-site APP cleaving enzyme (BACE), and apoptosis [7]. Moreover, paeonol derivatives have been reported to show many attractive biological activities. For example, Pan and Hui demonstrated that the donepezil-like paeonol derivative (2, Figure 1) exhibited strong metal-chelating ability for Alzheimer's disease (AD) treatment [8]. Yang presented a copper ion chelating paeonol Schiff-base derivative (3, Figure 1) complexes that possessed high antioxidant activity and moderate DNA-binding activity as well as high tumor cell cytotoxicity [9]. Moreover, Yu reported a paeonol thiosemicarbazone derivative (4, Figure 1), which exhibited potential mushroom tyrosinase inhibitors [10]. Recently, our group found that phenylsulfonyl moieties-conjugated paeonol derivatives were potential anti-Hepatitis B virus leads [11] and could prevent lipid accumulation at lower doses, and they might be prominent antiatherogenic agents [12]. The thiazole ring (5, Figure 2), a five-membered heterocyclic core structure, displays a variety of biological effects, such as antibacterial, antifungal, anti-Human immunodeficiency virus, antiinflammatory, antidiabetic, antioxidant, and anticancer effects [13]. These heterocyclic rings, notably 2-aminothiazole (6, Figure 2), are considered stable and lipophilic bioisosteres of phenol (7, Figure 2) or catechol (8, Figure 2) moieties, which might retain pharmacological action while having improved oral bioavailability [14]. Talipexole (9, Figure 2), a dopamine agonist for Parkinson's disease treatment, was designed on the basis of the bioisosteric effect of phenol and 2-aminothiazole [15]. In addition, the 2-aminothiazole core was found to act as the pharmacophore for antitubercular agents, the activity and the cytotoxicity of which could be improved and reduced with appropriate modification [16]. Introducing a phenylsulfonyl moiety in some molecules may increase the solubility of the molecules and trigger antitumor activity [17][18][19].  The thiazole ring (5, Figure 2), a five-membered heterocyclic core structure, displays a variety of biological effects, such as antibacterial, antifungal, anti-Human immunodeficiency virus, anti-inflammatory, antidiabetic, antioxidant, and anticancer effects [13]. These heterocyclic rings, notably 2-aminothiazole (6, Figure 2), are considered stable and lipophilic bioisosteres of phenol (7, Figure 2) or catechol (8, Figure 2) moieties, which might retain pharmacological action while having improved oral bioavailability [14]. Talipexole (9, Figure 2), a dopamine agonist for Parkinson's disease treatment, was designed on the basis of the bioisosteric effect of phenol and 2-aminothiazole [15]. In addition, the 2-aminothiazole core was found to act as the pharmacophore for antitubercular agents, the activity and the cytotoxicity of which could be improved and reduced with appropriate modification [16]. Introducing a phenylsulfonyl moiety in some molecules may increase the solubility of the molecules and trigger antitumor activity [17][18][19]. was also found to protect against memory loss after ischemic stroke by reducing amyloid precursor protein (APP), beta-site APP cleaving enzyme (BACE), and apoptosis [7]. Moreover, paeonol derivatives have been reported to show many attractive biological activities. For example, Pan and Hui demonstrated that the donepezil-like paeonol derivative (2, Figure 1) exhibited strong metal-chelating ability for Alzheimer's disease (AD) treatment [8]. Yang presented a copper ion chelating paeonol Schiff-base derivative (3, Figure 1) complexes that possessed high antioxidant activity and moderate DNA-binding activity as well as high tumor cell cytotoxicity [9]. Moreover, Yu reported a paeonol thiosemicarbazone derivative (4, Figure 1), which exhibited potential mushroom tyrosinase inhibitors [10]. Recently, our group found that phenylsulfonyl moieties-conjugated paeonol derivatives were potential anti-Hepatitis B virus leads [11] and could prevent lipid accumulation at lower doses, and they might be prominent antiatherogenic agents [12]. The thiazole ring (5, Figure 2), a five-membered heterocyclic core structure, displays a variety of biological effects, such as antibacterial, antifungal, anti-Human immunodeficiency virus, antiinflammatory, antidiabetic, antioxidant, and anticancer effects [13]. These heterocyclic rings, notably 2-aminothiazole (6, Figure 2), are considered stable and lipophilic bioisosteres of phenol (7, Figure 2) or catechol (8, Figure 2) moieties, which might retain pharmacological action while having improved oral bioavailability [14]. Talipexole (9, Figure 2), a dopamine agonist for Parkinson's disease treatment, was designed on the basis of the bioisosteric effect of phenol and 2-aminothiazole [15]. In addition, the 2-aminothiazole core was found to act as the pharmacophore for antitubercular agents, the activity and the cytotoxicity of which could be improved and reduced with appropriate modification [16]. Introducing a phenylsulfonyl moiety in some molecules may increase the solubility of the molecules and trigger antitumor activity [17][18][19].  Herein, we present a new series of paeonol derivatives combined with the aminothiazole ring as the core structure and further conjugated with the phenylsulfonyl side-chains. With arylsulfonamidothiazole scaffold decoration, the anticancer activity of paeonol may be enhanced through additional hydrogen bonding interactions while retaining or even improving the solubility of paeonol itself [20][21][22]. This new series of aminothiazole-paeonol derivatives was determined to have potential anticancer effects in human gastric adenocarcinoma (AGS), human cervix adenocarcinoma (HeLa), human pancreas adenocarcinoma (PaTu8988t), human colorectal adenocarcinoma (HT-29), human glioblastoma (U87-MG), human lung adenocarcinoma (A549) and mouse colon carcinoma (CT26.WT) cells. Simultaneously, the toxicity of aminothiazole-paeonol derivatives against normal cells was evaluated by embryonic fibroblast (BALB/3T3) cells. The newly synthesized compounds could be structural templates for designing and developing novel anticancer agents.

Chemistry
The synthetic methods of preparing the paeonol-2-aminothiazole-phenylsulfonyl derivatives are outlined in Scheme 1. The 2-aminothiazole scaffold was obtained by treating paeonol with thiourea and iodine; the condensation-cyclization of thiourea initiated by iodine afforded compound 11. To produce various paeonol-phenylsulfonyl derivatives, we treated 2-aminothiazole-paeonol 11 with substituted phenylsulfonyl chloride 12 to yield the final desired compounds 13. All these products were obtained in sufficient yield and purified by using recrystallization for anticancer assays. Herein, we present a new series of paeonol derivatives combined with the aminothiazole ring as the core structure and further conjugated with the phenylsulfonyl side-chains. With arylsulfonamidothiazole scaffold decoration, the anticancer activity of paeonol may be enhanced through additional hydrogen bonding interactions while retaining or even improving the solubility of paeonol itself [20][21][22]. This new series of aminothiazole-paeonol derivatives was determined to have potential anticancer effects in human gastric adenocarcinoma (AGS), human cervix adenocarcinoma (HeLa), human pancreas adenocarcinoma (PaTu8988t), human colorectal adenocarcinoma (HT-29), human glioblastoma (U87-MG), human lung adenocarcinoma (A549) and mouse colon carcinoma (CT26.WT) cells. Simultaneously, the toxicity of aminothiazole-paeonol derivatives against normal cells was evaluated by embryonic fibroblast (BALB/3T3) cells. The newly synthesized compounds could be structural templates for designing and developing novel anticancer agents.

Chemistry
The synthetic methods of preparing the paeonol-2-aminothiazole-phenylsulfonyl derivatives are outlined in Scheme 1. The 2-aminothiazole scaffold was obtained by treating paeonol with thiourea and iodine; the condensation-cyclization of thiourea initiated by iodine afforded compound 11. To produce various paeonol-phenylsulfonyl derivatives, we treated 2-aminothiazole-paeonol 11 with substituted phenylsulfonyl chloride 12 to yield the final desired compounds 13. All these products were obtained in sufficient yield and purified by using recrystallization for anticancer assays.

Anticancer Activity and Structure Activity Relationship Analysis
The antitumor effects of the new synthesized compounds against AGS, HeLa, PaTu8988t, HT-29, U87-MG, A549, CT26.WT and BALB/3T3 are described in Table 1 Table 2), which arose from the F and OCH3 groups at the para-positions of the phenylsulfonyl terminals. On the other hand, compound 13g was the most inactive compound. In the HT-29 treatment, the activity of the parent compound 13a was between those of the most potent compound 13c and the most inactive compound 13g. Comparing the structures of 13c and 13g, compound 13c was substituted with the OCH3 group and compound 13g was replaced with the NO2 group at the para-positions of the phenylsulfonyl terminals. In general, the NO2 group manifest Scheme 1. Synthesis of the aminothiazole-paeonol derivatives.

Anticancer Activity and Structure Activity Relationship Analysis
The antitumor effects of the new synthesized compounds against AGS, HeLa, PaTu8988t, HT-29, U87-MG, A549, CT26.WT and BALB/3T3 are described in Table 1 Table 2), which arose from the F and OCH 3 groups at the para-positions of the phenylsulfonyl terminals. On the other hand, compound 13g was the most inactive compound. In the HT-29 treatment, the activity of the parent compound 13a was between those of the most potent compound 13c and the most inactive compound 13g. Comparing the structures of 13c and 13g, compound 13c was substituted with the OCH 3 group and compound 13g was replaced with the NO 2 group at the para-positions of the phenylsulfonyl terminals. In general, the NO 2 group manifest electron-withdrawing and lipophilic properties in the structure, which could conceivably alter the pharmacokinetic behaviors of compounds. Moreover, the electron-donating OCH 3 contributed an additional oxygen atom to the structure, making it more hydrophilic and thus increasing the water solubility. Some pharmacokinetic properties are listed in Table 2 [23,24]. All the compounds meet Lipinski's Rule of Five, with the molecular weights under 500 and the log p-values lower than five. In summary, compound 13c presented the most potent activity against AGS and HT-29 cells with IC 50 values of 4.0 and 4.4 µM, respectively, which was superior to 5-FU (IC 50 values: 43.8 and 7.2 µM against AGS and HT-29). The compound 13c was less toxic against fibroblast cells than 5-FU; therefore, compound 13c might be a promising lead compound for developing an anticancer agent against gastrointestinal tract-related adenocarcinoma.
Melting points were obtained with a Fargo MP-2D melting point apparatus (melting point range up to~400˝C). Analytical thin layer chromatography (TLC) was performed on precoated plates (silica gel 60 F-254), purchased from Merck Inc. Infrared (IR) spectra were measured on a Perkin-Elmer Model Spectrum 100 spectrophotometer. Absorption intensities are recorded by the following abbreviations: s = strong; m = medium; and w = weak. Proton NMR spectra were obtained on a Varian Mercury-400 (400 MHz) spectrometer or Bruker AC-400 (400 MHz) spectrometer by use of chloroform-d (CDCl 3 ) and dimethylsulfoxide-d 6 (DMSO-d 6 ) as the solvents. Proton NMR chemical shifts were referenced to residual protonated solvents (δ 7.24 for chloroform and δ 2.49 for dimethylsulfoxide). Carbon-13 NMR spectra were obtained on a Varian Mercury-400 (100 MHz) spectrometer by use of chloroform-d (CDCl 3 ) and dimethylsulfoxide-d 6 (DMSO-d 6 ) as the solvents. Carbon-13 chemical shifts are referenced to the center of the CDCl 3 triplet (δ 77.0 ppm) and DMSO septet (δ 39.5 ppm). Multiplicities are recorded by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; J, coupling constant (hertz). High-resolution mass spectra were obtained by means of a JEOL JMS-700 mass spectrometer.

Procedure for the Preparation of Aminothiazole-Paeonol (11)
To obtain aminothiazole-paeonol (11), paeonol (1) (1.0 equiv) was reacted with iodine (1.1 equiv) and thiourea (3.0 equiv) in ethanol under reflux condition for 12.0-16.0 h. Then, the reaction mixture was quenched with NaOH (aq) (2.0 equiv) and the ethanol was removed under reduced pressure. The residue was extracted with ethyl acetate and the combined organic layer were washed the brine and dried over MgSO 4(s) . After being filtered and condensed under reduced pressure, the crude product was purified by column chromatography on silica gel (ethyl acetate and hexane as eluent) to give compound 11.

Standard Procedure for the Preparation of Aminothiazole-Paeonol Derivatives (13)
To a solution containing aminothiazole-paeonol (11, 1.0 equiv) in anhydrous THF (2.0-3.0 mL) was added potassium carbonate (1.3 equiv) and a sulfonyl chloride 12 (1.1 equiv). After the reaction mixture was stirred at 25˝C for 2.0-3.0 h, it was diluted with dichloromethane (5.0 mL). Inorganic solids were filtered off and the filtrate was concentrated under reduced pressure to afford the residue. It was then purified by use of column chromatography on silica gel (various ratio of methanol to dichloromethane) to give the desired conjugates 13.  13

Cell Culture
Cell lines, including human gastric adenocarcinoma (AGS), human cervix adenocarcinoma (HeLa), human pancreas adenocarcinoma (PaTu8988t), human colorectal adenocarcinoma (HT-29), human glioblastoma (U87-MG), human lung adenocarcinoma (A549), mouse colon carcinoma (CT26.WT) cells and embryonic fibroblast (BALB/3T3) were purchased from Bioresource Collection and Research Center (BCRC, Taiwan) to evaluate anticancer activity of aminothiazole-paeonol derivatives. Five cell lines were cultured in different media from Gibco (Life Technologies, Grand Island, NY, USA) and Hyclone (GE Healthcare Life Science). Cells were cultured in 90% medium mixed with 10% serum at 37˝C in humidified atmosphere with 5% CO 2 and grown in T-75 flask with a feeding cycle of two to three days. The composition and procedures of preparing culture media for different cell lines were followed by instruction manual of suppliers [5,33].

Drug Treatment and Cell Viability Assay
The different cell lines were seeded into 96-well tissue culture plates at a concentration of 5ˆ10 3 cells/100 µL/well overnight. Subsequently, the cells were treated with serial concentrations of seven paeonol derivatives. After 48 h of incubation, cell viability of each cell line was determined by MTT colorimetric assay. Briefly, 100 µL of 2 mg/mL MTT reagent (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 4 h at 37˝C. Later, the medium was aspirated and 100 µL of dimethyl sulphoxide (DMSO) added to each well; finally, the OD 595 of each well was measured by ELISA reader (TECAN, Wien, Austria).

Conclusions
Various phenylsulfonyl side-chains were directly conjugated to paeonol, the major component of a traditional Chinese medicine, Moutan cortex, through chemical synthesis to generate a new series of aminothiazole-paeonol derivatives. The substituents on the phenylsulfonyl side-chain included F, Cl, Br, NO 2 , Me, and OMe. All the synthesized compounds were characterized using 1 H-NMR, 13 C-NMR, and mass spectra data. The cytotoxic effects of all compounds were evaluated against fibroblast cells (BALB/3T3) and seven cancer cell lines, including AGS, HeLa, PaTu8988t, HT-29, U87-MG, A549 and CT26.WT. We observed that the thiazole-paeonol-phenylsulfonyl derivatives demonstrated cytotoxic effects against the tested cancer cell lines. We also discussed the structural activity relationship on the basis of screening results and pharmacokinetic properties. Results indicated that 13c exhibited the most potent activity against AGS and HT-29 cells with IC 50 values of 4.0 and 4.4 µM, respectively, which is superior to 5-FU (IC 50 values: 43.8 and 7.2 µM against AGS and HT-29) and may be a promising lead compound to develop an anticancer agent for gastrointestinal tract-related adenocarcinoma.