One Pot and Metal-Free Approach to 3-(2-Hydroxybenzoyl)-1-aza-anthraquinones

Herein, a direct strategy to synthesize 3-(2-hydroxybenzoyl)-1-aza-anthraquinones with excellent efficiency, mild conditions, and benign functional group compatibility was reported. A variety of 3-formylchromone compounds were employed as compatible substrates and this protocol gave the 3-(2-hydroxybenzoyl)-1-aza-anthraquinone derivatives in good to excellent yields without inert gas and expensive transition metal catalysts. Some compounds displayed good anti-proliferative activities.


Results and Discussion
3-Formylchromones are representative building blocks, which are usually employed to prepare varieties of heterocyclic systems with enamine-type compounds [33][34][35][36][37][38]. Accordingly, we chose 3-formylchromone (1a) and 2-amino-naphthalene-1,4-dione (2a) as the template substrates to optimize the reaction conditions (Table 1). Surprisingly, the initial attempt gave the targeting product 3a with 35% yield after stirring in acetic acid for 24 h at 80 • C ( Table 1, entry 1). Based on this result, different reaction temperatures were examined ( Table 1, entries 1 to 3). By the increasing reaction temperature, the yield was remarkably improved and the reaction time was shortened to 4 h under reflux. Then, a variety of solvents were screened for the reaction and the results showed that acetic acid was superior to other solvents (Table 1, entries 4 to 8). In addition, further investigations were conducted on the ratio of reactants, finding that 1.2 eq of 3-formylchromone gave a better yield (Table 1, entry 3, entries 9 to 12). Therefore, after the screening of the temperature, solvent, and reagents' ratio, the optimized reaction conditions were established (Table 1, entry 10). 3-Formylchromones are representative building blocks, which are usually employed to prepare varieties of heterocyclic systems with enamine-type compounds [33][34][35][36][37][38]. Accordingly, we chose 3formylchromone (1a) and 2-amino-naphthalene-1,4-dione (2a) as the template substrates to optimize the reaction conditions (Table 1). Surprisingly, the initial attempt gave the targeting product 3a with 35% yield after stirring in acetic acid for 24 h at 80 °C (Table 1, entry 1). Based on this result, different reaction temperatures were examined (Table 1, entries 1 to 3). By the increasing reaction temperature, the yield was remarkably improved and the reaction time was shortened to 4 h under reflux. Then, a variety of solvents were screened for the reaction and the results showed that acetic acid was superior to other solvents (Table 1, entries 4 to 8). In addition, further investigations were conducted on the ratio of reactants, finding that 1.2 eq of 3-formylchromone gave a better yield (Table 1, entry 3, entries 9 to 12). Therefore, after the screening of the temperature, solvent, and reagents' ratio, the optimized reaction conditions were established (Table 1, entry 10). With the optimized reaction conditions in hand, we next explored the substrates' scope and the generality of the reaction with different 3-formylchromones. To our delight, this protocol was applicable to a series of 3-formylchromones under the optimized reaction conditions, providing the corresponding products 3a-3t with moderate to excellent yields ( Table 2, 40-95%). All substituted 3-formylchromones with electron-donating groups (methyl, methoxyl groups) or electron-withdrawing groups (chloro, bromo, fluoro, cyano, and nitro groups) could be transformed into the desired compounds (3a-3q, 40-90%). When there was an electron-donating group on C6-position of 3-formylchromones, the yield was decreased (3m, 3q vs. 3a). Meanwhile, the electron-donating mono-methoxyl group on 3-formylchromones were suitable substrates, affording the corresponding products in excellent yields (3j-3l, 82-85%). Then, several di-substituted 3-formylchromones were also investigated and gave good results (3g, 3n-3p, 40-75%). Interestingly, we found that two electron-donating groups on the benzene ring gave lower yields (3n, 3p vs. 3g, 3o). Notably, naphthyl-substituted 3-formylchromones were well-tolerated under the optimized reaction conditions, which afforded the desired products excellent yields up to 95% (3r, 3s). To further explore the substrate scope of this protocol, 2-amino-6,7-dimethoxynaphthalene-1,4-dione was also employed in this reaction, and successfully provided the corresponding product with a good yield (3t, 68%). The single crystal structure of 3t as trifluoroacetic acid salt hydrate was confirmed by X-ray crystallographic analysis ( Figure 3).
Based on the isomerization of 2-amino-naphthalene-1,4-dione, we proposed two possible mechanisms (Scheme 1). In path A, the reaction initiates nucleophilic attack of 3-formylchromone by the nitrogen atom of 2a to give intermediate A. Then, intramolecular nucleophilic substitution of A generates intermediate B, which terminates by ring cleavage and aromatization to give the final product 3a. In path B, the C3-position of 2a becomes more nucleophilic than the nitrogen atom because of the conjugative effect. Subsequently, like path A, after intramolecular nucleophilic substitution, ring cleavage, and aromatization, the final product 3a was obtained.   a Reaction conditions: 1 (0.6 mmol), 2 (0.5 mmol), and AcOH (6.0 mL) in air refluxed for 4 h. b Isolated yields. c 1a (0.6 mmol), 2-amino-6,7-dimethoxynaphthalene-1,4-dione (0.5 mmol) was used.
a Reaction conditions: 1 (0.6 mmol), 2 (0.5 mmol), and AcOH (6.0 mL) in air refluxed for 4 h. b Isolated yields. c 1a (0.6 mmol), 2-amino-6,7-dimethoxynaphthalene-1,4-dione (0.5 mmol) was used. Subsequently, some products were evaluated for anti-proliferative activity against human cervical cancer cell line Hela and colon cancer cell line HT-29 (Table 3). Some compounds displayed moderate to good anti-proliferation activity (such as 3j, 3l, 3n, 3p, 3q). Although these compounds were less potent than YCH337 and CA-4 to the Hela cells, compound 3q exhibited potent antiproliferative activity towards HT-29 cells.  Subsequently, some products were evaluated for anti-proliferative activity against human cervical cancer cell line Hela and colon cancer cell line HT-29 (Table 3). Some compounds displayed moderate to good anti-proliferation activity (such as 3j, 3l, 3n, 3p, 3q). Although these compounds were less potent than YCH337 and CA-4 to the Hela cells, compound 3q exhibited potent antiproliferative activity towards HT-29 cells. Subsequently, some products were evaluated for anti-proliferative activity against human cervical cancer cell line Hela and colon cancer cell line HT-29 (Table 3). Some compounds displayed moderate to good anti-proliferation activity (such as 3j, 3l, 3n, 3p, 3q). Although these compounds were less potent than YCH337 and CA-4 to the Hela cells, compound 3q exhibited potent antiproliferative activity towards HT-29 cells.

General Information
All reagents were purchased from commercial suppliers and used without further purification. The progress of all of the reactions was monitored by thin layer chromatography with standard TLC silica gel plates, and the developed plates were visualized under UV light. All of the compounds were purified by column chromatography. Chromatography was performed on silica gel (100-200 mesh). Nuclear magnetic resonance spectra ( 1 H, 13 C NMR) were recorded on Varian Mercury-300/400 and Varian Mercury-400/500 spectrometers and TFA-d was used as the solvent. NMR peaks were calibrated by reference to standard peaks of TFA at 11.50 ppm for 1 H and 116.60 and 164.20 ppm for 13 C. For peak descriptions, the following abbreviations were used: s (singlet), d (doublet), t (triplet), dd (doublet of doublets), td (triplet of doublets), dt (doublet of triplets), pt (pseudo-triplet), ddd (double of doublets of doublets). EI-HRMS were recorded using a Thermo DFS mass spectrometer. The NMR spectra of the obtained compounds and some additional experimental details can be found in the Supplementary Materials.

General Procedure for the Synthesis of Compounds 1a-1t
To a cooled (0 • C) solution of 2'-hydroxyacetophenone (1 g, 1 eq) in DMF (30 mL), phosphorus oxychloride (5 eq) was added. The mixture was stirred at 64 • C for 4 h until the 2'-hydroxyacetophenone was consumed completely (TLC). The reaction was quenched with glacial water (100 mL), and the mixture was stirred for an additional 30 min. Then, the mixture was extracted three times with dichloromethane (100 mL). The solvent was evaporated in vacuo and the crude product was purified by column chromatography on silica gel.

General Procedure for the Synthesis of Compounds 3a-3t
To a solution of 2-amino-naphthalene-1,4-dione (0.5 mmol, 1 eq) in AcOH (6 mL), 3-formylchromone (0.6 mmol, 1.2 eq) was added. The resulting reaction mixture was heated to reflux for 4 h. Upon completion (determined by TLC), the reaction mixture was cooled to room temperature, diluted with water (30 mL), and extracted three times with dichloromethane (10 mL). The organic phase was dried over Na 2 SO 4 , concentrated in vacuo, and purified by column chromatography on silica gel. Several compounds were collected by filtration for poor solubility.  13 13 13 13 13 13

Conclusions
We have developed a facile, efficient, and mild synthetic strategy to assemble 3-(2-hydroxybenzoyl)-1-aza-anthraquinone derivatives from 3-formylchromones and 2-amino-naphthalene-1,4-dione. Most of the substrates bearing diverse substituents worked well with this procedure, and a great variety of 3-(2-hydroxybenzoyl)-1-aza-anthraquinone derivatives were successfully obtained in moderate to excellent yields, which are potentially useful skeletons in pharmaceutical discovery research. Moreover, this reaction could proceed without metal catalysis and be insensitive to air and moisture.