Synthesis of new anthraquinone compounds and evaluation of their considerable xanthine oxidase inhibitory activities

A series of anthraquinone derivatives ( 1-11 ) was prepared using alkyl halides, quinizarin

Here, the newly synthesized anthraquinone derivatives were compared with allopurinol (scheme) as a standard. Because it is a well-known gout drug and structurally contains an oxo group similar to anthraquinones.

Results and Discussion
The Synthesis of compounds (1-11) was achieved using quinizarin (1,4-dihydroxyanthraquinone), chrysazin (1,8-dihydroxyanthraquinone), and alkyl halides with substitution reaction (Figure) apart from compound 5. However, compound 5 was obtained by a Suzuki-Miyaura cross-coupling type reaction in the presence of a Pd(OAc)2/PPh3 catalyst. The yield of the compounds was calculated in the range of 51-79% and their melting points were measured between 153-273 °C. The structural evaluation for compounds was performed using 1 H, 13 C NMR, IR, and UV-Vis spectroscopic methods. The aromatic group peaks belonging to compounds were observed in the range of 8.26-6.93 ppm in the 1 H-NMR spectra. In addition, the chemical shift values of the benzylic CH2 groups were observed at 5.19, 5.22, 5.20, 5.12, 5.33, 5.27, 5.44, 5.30, 5.32 ppm (for the compounds 1, 2, 3, 4, 5, 7, 8, 9, and 10) respectively. Also, peaks of quinone C=O groups between 183.5-181.9 ppm and benzylic peaks at the range of 71.9-69.8 ppm were observed in 13 C NMR spectra. Moreover, the peaks of C=O groups were observed at 1667, 1668, 1665, 1666, 1668, 1664, 1666, 1668, 1666, 1668, and 1676 cm-1, respectively. The stretching vibrations values of the CN groups in compounds 3, 6, and 8 were determined as 2227, 2241, and 2228 cm-1. These values are in agreement with the literature. 41 The UV-Visible spectral analysis also was performed for anthraquinone derivatives. The lower band (280-364 nm) in the electronic spectra of anthraquinones corresponds to π σ* transitions. The π π* transitions were observed in the visible area for compounds at λmax There are synthetic or natural many drugs that have an anthraquinone core. For example, rhein has a kidney protective effect; emodin is known to have antitumoral, antimicrobial, and antioxidant effects, as well as a protective effect on the liver and kidneys. In addition, chrysophanol and physcion have a protective effect on the nervous system. 43,44 In a study recorded in 1987, the XO inhibition effect of nine anthraquinone compounds was observed in vitro by Noro et al. Here, it was recorded that quinizarin and chrysazine had weak inhibitory activity when compared with other anthraquinone derivatives. 45 In this work, the XO enzyme inhibition abilities were tested of the novel quinizarin and danthron alkoxy derivatives, and their IC50 values were determined between 0.339-1.015 μM (Table). IC50 is used to mean halfmaximal inhibitory concentration. It is the most common measure of the effectiveness of a drug. IC50 values indicate the essential amount of inhibitor for half-inhibition to occur. If the IC50 value is smaller, it is the more effective inhibitor. The IC50 value of allopurinol, which is used as a standard drug, was observed as 1.967 μM. Therefore, it is understood that all anthraquinone compounds have a higher inhibition effect than allopurinol. The IC50 values of compounds 3 and 4 (1,4-bis(4-cyanobenzyl)oxy)anthracene-9,10-dione and 1,4-bis(4bromobenzyl)oxy)anthracene-9,10-dione) are 0.339 and 0.395 μM. But the inhibition values of 1,8disubstituted anthraquinone derivatives with the same functional groups (compounds 8 and 9) were measured as 0.803 and 0.748 μM. These values show that quinizarin derivatives with the same functional groups have higher inhibitory power than danthron derivatives. Also, the structure of ethylisoindoline-1,3dione used for the synthesis of compound 11 contains two oxo groups, it was used to observe in which direction the inhibitory activity of the compound would change. It was seen from the measurement results that compound 11 has one of the highest activity (IC50 = 0.365 μM).
In previous studies, XO inhibition activities of some groups of compounds were reported as IC50 for XO; such as pyridine salts between 0,394-0,623 μM 46 , N-heterocyclic carbene ligands between 0.58-1.69 μM 47 , and new pyrrole carboxamide derivatives between 4.608-7.084 μM. 48 When the IC50 values of the inhibitory agents in the mentioned literature sources are compared with the anthraquinones, it is understood that the new anthraquinone derivatives are more remarkable inhibitors than other compound groups, because of IC50 values (0.339, 0.365, and 0.395 μM). Due to these results, the most active compounds (3, 4, and 11) can be an alternative to allopurinol.

Experimental Section
General conditions and materials. The reactions were performed in atmospheric conditions. Starting materials and reagents used in reactions were supplied commercially from Aldrich or Merck Chemical Co. 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were recorded using a Bruker Avenced III 400 MHz FT NMR spectrometer. Infrared (IR) spectra were recorded using an ATR unit from 4000 to 200 cm -1 on a Perkin-Elmer FT-IR spectrophotometer. Elemental analyses were performed with a LECO CHNS-932 elemental analyzer. UV-Vis spectra were measured on a Perkin-Elmer Lambda 35 spectrophotometer. Melting points were recorded using an electrothermal-9200 melting point apparatus and are uncorrected.

General synthesis procedure
Anthraquinone derivatives (1-4, 6-11) were synthesized from 1,4-dihydroxyanthraquinone, 1,8dihydroxyanthraquinone and alkyl halides in N,N-dimethylformamide (DMF) in the presence of K2CO3. Unlike other compounds, compound 5 was obtained as a result of the C-C coupling reaction of compound 4 and benzeneboronic acid in presence of a catalyst system consisting of Pd(OAc)2/PPh3. (Figure).

In vitro inhibition of xanthine oxidase (XO) activity
The enzyme activity were assayed by measuring the uric acid formation at 294 nm at 37 °C. The in vitro XO inhibitory assay method was reported by Sweeney A.P. et al. 49 For calculation IC50 value of XO inhibition different concentrations of anthraquinone derivatives were added to the reaction mixture. In brief, the enzyme assay protocol contains phosphate buffer (50 mM, pH = 7.4), XO (0.2 U), and xanthine (1 mM). The enzyme was pre-incubated for (10 min) with tested compounds, then the reaction was started by adding xanthine to the reaction mixture. All the experiments were performed in triplicates, and values were expressed as means of three experiments. Allopurinol was used as a positive control. The IC50 values were determined for based compounds were measured as percent inhibition of XO was studied in terms of decrease in uric acid formation as compared to the product formation in the absence of inhibitor. The percent inhibition (IC50 of XO) activities of compounds were calculated related to this formula. Inhibition (%) = (A-B)/A×100; A = the absorbance at 294 nm without the test compound, B = the absorbance at 294 nm with the test compound.