Synthesis and evaluation of α-glucosidase and tyrosinase inhibitory activities of ester derivatives of usnic acid

Use your smartphone to scan this QR code and download this article ABSTRACT Introduction: Usnic acid isolated from lichenwas a potential bioactivity compound. It has a broad spectrum bioactivity, including antiviral, anti-inflammatory, anticancer...However, low solubility in water limited its application. Many researchs have done to overcome the restriction. Recent results showed that usnic acid derivatives bearing triazole, enamine, pyrazole and benzylidene groups had strong antiviral and anticancer activities. Thus, investigation of usnic acid derivatives synthesis was an attractive aspect due to the diversity of bioactivities of usnic acid derivatives. Methods: Usnic acid was isolated from lichen, six ester derivatives of usnic acid were synthesized from usnic acid with acetyl chloride and benzoyl chloride under stirring at room temperature. The products were evaluated α-glucosidase and tyrosinase inhibitory activities. Results: All the ester derivatives were createdwith good yields. All derivatives exhibited the same or higher activity comparingwith usnic acid. Ester of usnic acid bearing benzoyl group showed excellent α-glucosidase activity with IC50 26.7±0.57 and 68.8±0.15 μM. Conclusion: Among the ester derivatives, UE1 and UE6 were reported as as new compounds. Interestingly, all products displayed the same or higher biological activity than the startingmaterial, usnic acid when evaluated againstα-glucosidase and tyrosinase.


INTRODUCTION
Isolated compounds from lichens exhibited a wide range of biological properties, such as antimicrobial, antiviral, anti-inflammatory, anticaner… 1 . Usnic acid, a dibenzofuran derivative found only in lichens was a remarkable substance. Usnic acid has a broad spectrum of bioactivity, especially against gram-positive bacteria such as Staphylococcus, Streptococcus, and antifungal 2 . Futhermore, it also has antiviral, anti-inflammatory, antipyretic… activities 2 .
In vitro experiments showed that usnic acid could inhibit many human cancer cell lines growth 3 . However, toxicity with liver and low solubility in water of usnic acid has limited application of it in cancer treatment. This attracts interests of many researchers to overcome the limit. The first research of usnic acid derivatives synthesis was carried out by Takai in 1979, the solubility of products were improved by preparing glycoside and imine derivatives of usnic acid 4 . Recently, many researchs showed that usnic acid bearing triazole, enamine, pyrazole and benzylidene groups had strong antiviral and anticancer activities [5][6][7][8] . The diversity of bioactivities of usnic acid derivatives showed that they could be a potential drugs in medicinal treatments.
Herein, we described a procedure of ester derivatives synthesis from usnic acid, these compounds were evaluated of α-glucosidase and tyrosinase inhibitory activities.
with aqueous NaHCO 3 , respectively, and dried over anhydrous Na 2 SO 4 . The mixture was filtered and evaporated using rotatory vacuum evaporator. The products, UE1-4 were purified by subjecting to silica gel column. A mixture of (+)-usnic acid (0.250 g, 0.727 mmol) in CHCl 3 (5.0 mL) was stirred at room temperature for 5 minutes. enzoyl chloride (0.611 g, 4.350 mmol) was added, followed by pyridine (3.5 mL, 43.502 mmol) and stirred at room temperature for 6 h. The products, UE 5 were purified by subjecting to silica gel column. A mixture of UE3 (0.280 g, 0.727 mmol) in CHCl 3 (5.0 mL) was stirred at room temperature for 5 minutes. enzoyl chloride (0.611 g, 4.350 mmol) was added, followed by pyridine (3.5 mL, 43.502 mmol) and stirred at room temperature for 6 h. The products, UE 6 were purified by subjecting to silica gel column.

Biological activities investigation
These inhibitory activities were evaluated according to 9 . Enzymatic activity was calculated by measuring absorbance at 405 nm (ALLSHENG micro plate reader AMR-100). All samples were analyzed in triplicate at various concentrations to obtain the IC 50 value of each compound. The mean values and standard deviation were also identified.

Structure determination of products
The products were verified structures by 1 Figure 1 showed esterification of usnic acid with acetyl chloride and benzoyl chloride. Six ester derivatives (UE1-6) were synthesized from usnic acid. Table 1 showed the results in the synthesis of six ester derivatives of usnic acid. Yields of the reactions using acetyl chloride or benzoyl chloride were good (> 70%). Proposed mechanism of UE3 synthesis from usnic acid was shown in Scheme 1. Table 2 and Table 3 summarized data of nuclear magnetic resonance spectra of these ester products. These signals demonstrated that six ester derivatives had been synthesized successfully. α-glucosidase and tyrosinase inhibitory activities of UE1-6 were listed in Table 4. All derivatives exhibited the same or higher activity comparing with starting material (usnic acid).

Ester derivatives synthesis from usnic acid
There are three hydroxy groups in usnic acid structure at C-3, C-8 and C-10 could be esterified. In the         reaction, we use large amounts of acetyl chloride in order to react at three hydroxy groups completely. However, the reaction produced four ester derivatives (UE1-4) depending on the number and position of hydroxy groups that participated in the reaction when acetyl chloride was used as a reactant. Besides, only one product (UE5) was created when benzoyl chloride was used. Moreover, the ester product (UE6) was also generated when UE3 product reacted with benzoyl chloride in the same conditions ( Figure 1). The synthesis results were listed in Table 1 below showed that yields of the reactions using acetyl chloride or benzoyl chloride were good (> 70%    11 , is elucidated as shown in Figure 1.
The examination of the 1 H and 13 C NMR spectra of UE4 revealed the similar spectra to those of UE3, excepted for the lack of 8-OH and the occurrence of 10-OH that indicated the reaction occurred at 8-OH. Thus, UE4, 8-O-acetylusnic acid 11 , is established as shown in Figure 1.  Figure 1.

Biological activities of usnic acid derivatives
Six usnic acid derivatives including via esterification (UE1-6) were further tested with α-glucosidase and tyrosinase inhibitory activities. From the results, all derivatives exhibited the same or higher activity comparing with starting material (usnic acid: >200 µM and no activity (NA) for α-glucosidase and tyrosinase, respectively). Especially, UE5 and UE6 showed excellent α-glucosidase activity with IC 50 26.7±0.57, and 68.8±0.15 µM, respectively. These compounds not only displayed higher activity than that of usnic acid, but also with that of a positive control, acarbose (IC 50 : 93.6±0.49 µM) as shown in Table 4. In this case, UE5 displayed the strongest activity (IC 50 : 26.7±0.57 µM).

CONCLUSION
From usnic acid, six derivatives were synthesized via esterification reactions (UE1-6). Their chemical structures were elucidated by NMR and HRES-IMS as well as comparison with those from literature. Among them, UE1 and UE6 were reported as as new compounds. Interestingly, all products displayed the same or higher biological activity than the starting material, usnic acid when evaluated against α-glucosidase and tyrosinase. In the α-glucosidase assay, UE5 and UE6 showed excellent activity (IC 50 26.7±0.57, and 68.8±0.15 µM, respectively). On the other hand, all tested compounds revealed weak or no inhibitory activity in the tyrosinase assay.