Design and synthesis of chalcone derivatives as potential non-purine xanthine oxidase inhibitors

Background Based on some previous research, the chalcone derivatives exhibited potent xanthine oxidase inhibitory activity, e.g. sappanchalcone (7), with IC50 value of 3.9 μM, was isolated from Caesalpinia sappan. Therefore, objectives of this research are design and synthesis of 7 and other chalcone derivatives by Claisen–Schmidt condensation and then evaluate their XO inhibitory activity. Results Fifteen chalcone derivatives were synthesized by Claisen–Schmidt condensation, and were evaluated for XO inhibitory activity. Nine out of 15 synthetic chalcones showed inhibitory activity (3; 5–8; 10–13). Sappanchalcone derivatives (11) (IC50, 2.5 μM) and a novel chalcone (13) (IC50, 2.4 μM) displayed strong xanthine oxidase inhibitory activity that is comparable to allopurinol (IC50, 2.5 μM). The structure–activity relationship of these chalcone derivatives was also presented. Conclusions It is the first research on synthesis sappanchalcone (7) by Claisen–Schmidt condensation. The overall yield of this procedure was 6.6 %, higher than that of reported procedure (4 %). Design, synthesis, and evaluation of chalcone derivatives were carried out. This result suggests that the chalcone derivative can be used as potential non-purine XO inhibitors.Graphical abstract The chalcone derivatives as potential non-purine xanthine oxidase inhibitors Electronic supplementary material The online version of this article (doi:10.1186/s40064-016-3485-6) contains supplementary material, which is available to authorized users.


Background
Xanthine oxidase (XO) is a key enzyme in purine metabolic pathway. This complex metalloflavoprotein catalyzes the oxidation of hypoxanthine into xanthine and then finally into uric acid (Massey et al. 1969). Overproduction or under excretion of uric acid leads to hyperuricemia, a key cause of gout (Scott and Agudelo 2003). Also, hyperuricemia has been identified as an independent risk factor for chronic kidney and cardiovascular diseases (Edwards 2008;Nakagawa et al. 2006); thus, maintaining uric acid at a normal level is an important therapy to prevent gout. In many kinds of research, XO has been targeted as a promising agent for treatment of hyperuricemia. Allopurinol is a potent XO inhibitor with a purine backbone and has been used clinically for more than 40 years (Murata et al. 2009). Unfortunately, this drug has infrequent and severe side effects as in the cause of hypersensitivity syndrome (Hammer et al. 2001), Stevens-Johnson syndrome (Fritsch and Sidoroff 2000), and renal toxicity (Horiuchi et al. 2000). Therefore, there is a need to develop other novel chemical structural types of XO inhibitors.
Chalcones are within a class of chemical compounds that widely exist in a variety of medicinal plants. Claisen-Schmidt condensation, a base catalyzed condensation, was found to be most convenient to synthesize chalcones. Their flexible structure allows them to possess a large number of biological activities including antitumor, antifungal, antiprotozoal, antimitotic, and antiviral (Zhang et al. 2013). Some chalcone derivatives exhibited potent XO inhibitory activity (Beiler and Martin 1951;Niu et al. 2011).
Our preliminary screening to search for XO inhibitory activity of Vietnamese medicinal plants revealed that the methanolic extract of Caesalpinia sappan's heartwood exhibited significant XO inhibitory activity with an IC 50 value of 14.2 μg/mL (Nguyen et al. 2005). The bioactivity-guided fractionation of MeOH extract of C. sappan's heartwood was carried out. Sappanchalcone (7) was isolated from EtOAc-soluble fraction (IC 50 , 12.8 μg/mL); this compound displayed the most potent activity with an IC 50 value of 3.9 μM, comparable to that of allopurinol (IC 50 , 2.5 μM) (Nguyen et al. 2005). To study the possibility of using 7 as gout treatment required a large amount of this compound but the amount of 7 in C. sappan is very low.
The synthesis of 7 was carried out by Heck coupling reaction followed by demethylation (Bianco et al. 2004). Therefore, objectives of this research are design and synthesis of 7 and other chalcone derivatives by Claisen-Schmidt condensation and then evaluate their XO inhibitory activity.

Results and discussion
As outlined in Scheme 1, some known and novel chalcone analogs (group I: the hydroxyl groups attached to one of two aromatic rings of chalcones; and group II: both two aromatic rings carried the hydroxy groups) were prepared via Claisen Schmidt condensation reactions between appropriate benzaldehydes and aryl methyl ketones. The reaction was monitored by thinlayer chromatography (TLC). The reaction mixture after aldol condensation was acidified and cooled to obtain the crude product. Pure chalcone was purified by recrystallization and structure elucidation was determined by NMR spectroscopy. The overall yield of the reaction was then measured by HPLC-UV/260 nm.
For the purpose of simplifying the synthesis, the protecting group was not carried out, so the concentration of aqueous alkaline base was critical in Claisen-Schmidt condensation. Therefore, typical reactions affording 3,4-dihydroxychalcone (3) and 3,4,2ʹ,4ʹtetrahydroxychalcone (5) were investigated in the presence of different concentrations of the aqueous solution of KOH at room temperature 30 °C (Table 1).
The synthesis of 3, in the presence of 1.00 mL of MeOH as the solvent, and aqueous base with different concentrations from 6 to 14 M, together with ultrasound-assisted (UA), afforded the highest yield of 3 (39.7 %) when the reaction was carried out at KOH 10 M (Table 1, entry 3). When comparing the synthesis of 5 and that of 3, both were synthesized from 3,4-dihydroxybenzaldehyde (1a), differed only in acetophenone derivatives. In this case, we used 2ʹ,4ʹ-dihydroxyacetophenone (2b), a more polar substrate than acetophenone (2a). The use of MeOH solvent was not necessary because both substrates were dissolved in alkaline solution well; and highest yield of 5 (33.4 %) was afforded when KOH 14 M (Table 1, entry 10) was used.
From results in Tables 2 and 3, the yield of these typical reactions increased up to a period and then stopped changing. The reaction time may vary depending on different activation methods i.e. conventional heating (entry 1-4 in Tables 2 or 3) or ultrasound-assisted (entry 5-11 in Tables 2 or 3). The reaction temperature was significantly impacted yield of the synthesis of 3 and 5; it can be seen that the optimal reaction temperatures were 70 °C ( Scheme 1 Synthesis of chalcones in group I and group II. Reagents and conditions: a KOH aq , MeOH, ultrasound-assisted; b KOH aq , ultrasoundassisted respectively. Due to limited solubility in the aqueous base of acetophenone (2a), using a suitable organic solvent and appropriate volume is crucial to synthesize 3. Therefore, under these optimal conditions, an investigation on the effect of volume of MeOH (Table 2, entry 13, 16-19) was carried out.
Compound 7, both two aromatic rings carried the hydroxy groups, so it was classified as group II. However, with above optimal conditions, the desired product was not observed. In compound 2c, the methoxyl group at position C(2ʹ) was less polar than hydroxyl group, then changed the reactivity of compound 2c comparing to compound 2b. Therefore, the KOH concentration was again investigated while other optimal parameters have remained the same as in the synthesis of chalcone in group II (Table 1, entry 12-15).
Consequently, according to the above results, the structure-activity relationship of some synthetic chalcone derivatives (compound 3-18) was evaluated. In all cases, the carbonyl group plays a major role in the XO inhibition activity of these compounds; it acts as a reactive oxygen species acceptor (Ponce et al. 2000). Likewise, the presence of hydroxyl groups composes another important bioactive region. That are mainly involved in dispersion interactions with an aromatic aminoacidic residue of the enzyme (Costantino et al. 1996). So, the activity of chalcones increases with increasing numbers of hydroxyls. The tetrahydroxychalcones (5, 6) are more active than either of the dihydroxychalcones (3, 4); and the non-substituted chalcone (18) was not displayed xanthine oxidase inhibitory activity. Moreover, the presence of hydroxyl groups at C(2′), C(4′), and C(4) plays an important role in the inhibition of XO (5 > 6 ≫ 4), these hydroxyl groups increase the activity through an increment in the stabilization of the aromatic ring due to inductive effect (Ponce et al. 2000). So, the methylation or acetylation of the hydroxyl groups generally decreases the inhibition activity (3 > 14 ≈ 15; 5 > 10 > 8 > 9 ≈ 16;  , 15-17) has an extreme reducing effect on inhibitory activity. The presence of hydroxyl group at C(2′) may allow ring closure in solution, thus reducing the effective concentration of the compound in its chalcone form (Beiler and Martin 1951). Thus, the methylation of C(2′) hydroxyl group causes an increase in activity (7 > 5, 11 > 10). However, the presence of methoxyl groups at both C(2′) and C(4′) increases the activity (11 > 7 > 5 > 10) due to the activation of the keto group by oxygens on ring A (Beiler and Martin 1951).
Compound 13, a dimer-like compound of 10, showed the most potent active due to additional a carbonyl and a catechol group.

General procedure for the synthesis of chalcones in group I (compounds
General procedure for the synthesis of chalcones in group II (compounds 5 and 6) 2.0 mmol of benzaldehyde derivatives [275.9 mg of 3,4-dihydroxybenzaldehyde (1a); 276.3 mg of 2,4-dihydroxy benzaldehyde (1c)] and ~1.0 mmol of 2ʹ,4ʹdihydroxyacetophenone (2b) (152.1 mg) were dissolved in 1.00 mL H 2 O, then 1.00 mL KOH 14 M was added. The flask containing the resulting mixture was suspended in the ultrasonic water bath at 80 °C for 8 h.

Assessment of xanthine oxidase inhibitory activity
Briefly, the XO inhibitory activity was assayed spectrophotometrically under aerobic conditions (Nguyen et al. 2005). The assay mixture consisting of 50 μL of test solution, 35 μL of 70 mM phosphate buffer (pH 7.5), and 30 μL of enzyme solution (0.01 units/mL in 70 mM phosphate buffer, pH 7.5) was prepared immediately before use. After preincubation at 25 °C for 15 min, the reaction was initiated by the addition of 60 μL of substrate solution (150 μM xanthine in the same buffer). The assay mixture was incubated at 25 °C for 30 min. The reaction was stopped by adding 25 μL of HCl 1 N, and the absorbance at 290 nm was measured with a Shimadzu UV-1800. A blank was prepared in the same way, but the enzyme solution was added to the assay mixture after adding HCl 1 N. One unit of xanthine oxidase is defined as the amount of enzyme required to produce 1 μmol of uric acid/min at 25 °C. XO inhibitory activity was expressed as the percentage inhibition of XO in the above assay system, calculated as (1 − B/A) × 100, where A and B are the activities of the enzyme without and with the test material. IC 50 values were calculated from the mean values of data from four determinations. Allopurinol, a known inhibitor of XO, was used as a positive control.

Conclusions
It is the first research on synthesis sappanchalcone (7) by Claisen-Schmidt condensation. This procedure was simple and generated fewer by-products than Heck coupling reaction followed by demethylation (Bianco et al. 2004). The overall yield of this procedure was 6.6 %, higher than that of reported procedure (4 %) (Bianco et al. 2004). Nine out of fifteen synthetic chalcones showed inhibitory activity (3; 5-8; 10-13). Compound 5, 7, 11 and 13 with IC 50 values ranging from 2.4 to 4.3 μM displayed potent activity, comparing to allopurinol (IC 50 , 2.5 μM). This result suggests that these chalcone derivatives can be used as potential non-purine xanthine oxidase inhibitors. Structure-activity relationship was also proposed.