Synthesis and biological evaluation of benzimidazolyl substituted aloe-emodin derivatives

Benzimidazolic derivatives of the natural product aloe-emodin were prepared in good yields via aerobic condensation of the corresponding aldehydes with diamines in the presence of potassium iodide as auxiliary reagent. The required aldehydes were easily obtained by oxidation of aloe-emodin or its dimethyl-protected analogue. In vitro antimicrobial activity and cytotoxicity of the synthesized compounds were assessed. Most of the compounds showed either low to moderate antiprotozoal activity, although it was quite aspecific. 3-(1H-Benzo[d]imidazol-2-yl)-1,8-dihydroxyanthracene-9,10-dione showed the highest specificity against Leishmania infantum with IC 50 = 4.06 µM and a selectivity index of 10.29.

One of the general methods for the synthesis of benzimidazoles is the coupling of o-aryldiamines with carboxylic acids or their derivatives under dehydrating conditions. 23Also the reaction of o-phenylenediamines with aldehydes in the presence of oxidants and catalysts has been described.Herein, we report the synthesis of benzimidazoles derived from aloe-emodin carbaldehyde and o-phenylenediamines via aerobic oxidation in the presence of potassium iodide as auxiliary reagent, following the conditions described by Chen et al. 24

Results and Discussion
Aloe-emodin 1 was first methylated at the phenolic hydroxyl groups using dimethyl sulfate as reported in literature to afford compound 5 (Scheme 1).It was observed that an excess of dimethyl sulfate is required to allow an efficient dimethylation because of the low solubility of aloe-emodin in most organic solvents.In our hands optimal conditions used 9.8 equiv of Me2SO4 (added in two portions) at 0.04 M concentration of aloe-emodin in acetone.Stirring the mixture longer than 24h in attempts to increase the yield resulted in significant amounts of the over-methylated compound 6 as side-product.Fortunately, anthraquinone 6 is much more soluble in acetone than the desired product, which can be purified by recrystallization from cold acetone.
The synthesis of the required aldehydes 7a and 7b has been reported in literature using different oxidants, including chromium reagents like pyridinium chlorochromate (PCC), TEMPO/trichloroisocyanuric acid, VO(acac)2/DABCO/O2 and others. 25,15,26,12Yang-Ming et al. reported the oxidation of aloe-emodin 1 to aldehyde 7a in the presence of manganese dioxide in refluxing acetone. 27We synthesized aldehydes 7a and 7b by oxidation of aloe-emodin 1 or its dimethyl-protected analogue 5 with an excess of manganese dioxide in ethyl acetate (See Scheme 1) at room temperature, which resulted in the desired aldehyde 7a (gram scale reaction) within 8 h in high yield (91 %).The same procedure was followed using dimethylated compound 5 to give 7b but in lower yield.Having in hand the aldehydes 7a,b, the corresponding benzimidazoles were prepared by reaction with aromatic diamines using potassium iodide as auxiliary reagent.A mechanism of this reaction was proposed by Chen et al. 24 According to these authors, KI promotes the cyclization of N-(o-aminoaryl)imines by attacking the C=N bond giving a tetrahedral intermediate which can undergo 5-exo-tet cyclization instead of a direct but less plausible a 5-endo-trig cyclization on the parent imine (Scheme 2).Scheme 2. Mechanism proposed by Chen et al. 24 A mixture of the corresponding aldehyde and diamine was heated at 80 °C in DMF in the presence of 1 equiv of KI for 24 hours.Although KI is regenerated and could act as catalyst, it was found that raising the amount to 1 equiv gave superior results.The use of catalytic amounts of KI (10mol%) resulted in a longer reaction time and poor yields.The reaction using aldehyde 7a resulted in the corresponding benzimidazolyl derivatives 8a-c in 46-66% yield, while using aldehyde 7b gave compounds 8d-g in 50-72% yield (See Table 1).All compounds were purified by recrystallization or column chromatography, depending on their solubility.Compounds 8a and 8b have a poor solubility in common organic solvents such as chloroform and acetone, while compounds 8d and 8f were more soluble.Table 1.Oxidative cyclization of aldehydes 7a-b with aromatic diamines affording benzimidazoles 8a-g The synthesis of benzimidazole 8d was previously reported using sodium metabisulfite. 17The reaction was conducted at 120°C for 2 hours, no yield was reported.Compound 8a was obtained by demethylation of compound 8d with concentrated hydrobromic acid in acetic acid.Only the melting point of both compounds was reported.Cytotoxicity against MRC-5 cells (human fetal lung fibroblasts) was evaluated as well as to assess selectivity.Antibacterial activity was tested against both Gram-positive and Gram-negative bacteria, S. aureus and E. coli respectively.Compound 7a (IC50 = 2.06 µM) was the most active against S. aureus, followed by compounds 8f (IC50 = 16.11µM) and 8b (IC50 = 33.37 µM) but still much less active than the standard doxycycline.
Antiprotozoal activity evaluation of the synthesized compounds against Leishmania infantum, Trypanosoma cruzi, Trypanosoma brucei and Trypanosoma brucei rhodesiense was also conducted.In general, compounds with the highest antiprotozoal activity (7a, 8f and 8d) also showed the highest cytotoxicity on MRC-5 cells, revealing this activity to be quite aspecific.Compound 8a showed good activity (IC50 = 4.06 µM) against L. infantum, with a Selectivity Index (ratio of the cytotoxicity on MRC-5 (IC50) to the antimicrobial activity (IC50)) of 10.29.

Conclusions
Benzimidazolic derivatives of aloe-emodin were obtained in good to moderate yields using a simple procedure.In vitro antimicrobial activity of the synthesized compounds was tested.The compounds showed no activity against E. coli, C. albicans or A. fumigatus.Compounds 7a and 8f were the most active against S. aureus but showed high cytotoxicity on MRC-5 cells.Most of the compounds showed either low to moderate antiprotozoal activity, although it was quite aspecific.Compound 8a showed the highest specificity against Leishmania infantum with IC50 = 4.06 µM and selectivity index of 10.29.

Experimental Section
General.Column chromatography purifications were conducted on silica gel 60 (40-63 μm; Grace Davisil).TLC was carried out on plastic TLC sheets, precoated with silica gel 60F254 (Merck); the spots were visualized under UV light (λ = 254nm).Melting points were acquired on a Buchi Melting Point B-540.IR absorption spectra were recorded on a Thermo Nicolet Avatar 370 FT-IR spectrophotometer. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance DRX 250 console and a Bruker Avance II 500 console at 250 or 63 MHz and 500 or 125 MHz, respectively.The deuterated solvent is mentioned in the analysis section and tetramethylsilane was used as an internal standard.Chemical shifts (δ) are given in parts per million (ppm).High Resolution Mass Spectrometry was conducted on a Waters Micromass QTof in ES+ mode, using reserpine as a reference.All reagents were purchased and used without further purifications.Unless explicitly mentioned, reactions were performed without specific drying of solvents or use of an inert atmosphere.Ethyl acetate for chromatography purposes was distilled prior to use.

Synthesis of 3-(hydroxymethyl)-1,8-dimethoxyanthracene-9,10-dione (5).
This product was synthesized similarly to a literature procedure. 11A solution of aloe-emodin (1 g, 3.7 mmol) in acetone (200 mL) was added with K2CO3 (2.5 g, 18.1 mmol) and dimethyl sulfate (1.73 mL, 18.2 mmol), and then refluxed overnight.The reaction mixture was added then with more K2CO3 (2.5 g) and dimethyl sulfate (1.73 mL) and refluxed for 6 hours.The reaction was monitored by TLC.After the completion of reaction, the yielded mixture was cooled to room temperature and filtered.The filtrate was dried under vacuum to afford a yellow solid.Recrystallization of the yellow solid from acetone gave compound 2 as yellow needles, yield 75%; m.p. 224-225 °C; 1  General procedure for the synthesis of the aldehydes 7a-b.To a mixture of aloe-emodin (1 g, 3.7 mmol) (or compound 2) and manganese dioxide (16.09 g, 185 mmol) was added 1000 mL of ethyl acetate.The reaction mixture was stirred for 8 hours, filtered, and the solvent was evaporated in vacuo.The product was purified by recrystallization from acetone.General procedure for the synthesis of the benzimidazoles 8a-g.An ortho-phenylenediamine derivative (1.0 mmol; 1.0 equiv) and an aldehyde 7 (1.0 mmol; 1.0 equiv) were dissolved in DMF (20 mL).Potassium iodide (1.0 mmol; 1.0 equiv) was added.The resulting reaction mixture was stirred at 80 °C in an open flask, and the reaction progress was monitored by TLC.On the completion of the reaction, the mixture was cooled to room temperature, poured into water (150 mL) and extracted with ethyl acetate (3 x 75 mL).The crude product obtained was purified by column chromatography on silica gel to afford the corresponding benzimidazole 8; except compounds 8a-c, which were purified by recrystallization due to their low solubility in common organic solvents.Antimicrobial activity.9][30][31] The IC50 values were determined from five 4-fold dilutions.The following positive controls were used: Tamoxifen (MRC-5, human fetal lung fibroblasts, ECACC 84100401) IC50 8. Statistical analysis.IC 50 calculations were performed using regression analysis [% inhibition vs. log (concentration)] using Sigma plot 13.0.

Table 2 .
Activities of synthesized compounds against S. aureus, E. coli, C. albicans, and A. fumigatus and cytotoxicity against Human Lung Fibroblasts (MRC-5) a Doxycycline.bFlucytosine.cTerbinafine.dTamoxifen.The antimicrobial activity and cytotoxicity of the synthesized compounds were tested in vitro.Results of antibacterial and antifungal activities are shown in Table2.