Synthesis and evaluation of a-Glucosidase and tyrosinase inhibitory activities of ester derivatives of usnic acid

Science & Technology Development Journal, 23(3):585-592 Open Access Full Text Article Research Article 1Department of Chemistry, Ho Chi Minh City University of Education, District 5, Ho Chi Minh City, Viet Nam 2Institute of Fundamental and Applied Sciences, Duy Tan University, Ho Chi Minh City 700000, Vietnam 3Faculty of Natural Sciences, Duy Tan University, Da Nang, 550000, Vietnam Correspondence Nguyen Van Kieu, Institute of Fundamental and Applied Sciences, Duy Tan University, Ho Chi Minh City 700000, Vietnam Faculty of Natural Sciences, Duy Tan University, Da Nang, 550000, Vietnam Email: nguyenvankieu2@duytan.edu.vn History  Received: 2020-03-25  Accepted: 2020-07-12  Published: 2020-07-27 DOI : 10.32508/stdj.v23i3.1850 Copyright © VNU-HCM Press. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Synthesis and evaluation of a-glucosidase and tyrosinase inhibitory activities of ester derivatives of usnic acid PhamDuc Dung1, Duong Thuc Huy1, Nguyen Van Kieu2,3,* 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, anticancerHowever, 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 a-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 a-glucosidase activity with IC50 26.70.57 and 68.80.15 mM. 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 againsta-glucosidase and tyrosinase. Key words: Acetyl chloride, benzoyl chloride, ester derivatives, a-glucosidase, tyrosinase, usnic acid INTRODUCTION Isolated compounds from lichens exhibited a wide range of biological properties, such as antimicro- bial, antiviral, anti-inflammatory, anticaner1. Us- nic 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, Strep- tococcus, and antifungal2. Futhermore, it also has an- tiviral, anti-inflammatory, antipyretic activities 2. In vitro experiments showed that usnic acid could in- hibit many human cancer cell lines growth 3. How- ever, toxicity with liver and low solubility in water of usnic acid has limited application of it in cancer treat- ment. This attracts interests of many researchers to overcome the limit. The first research of usnic acid derivatives synthe- sis was carried out by Takai in 1979, the solubility of products were improved by preparing glycoside and imine derivatives of usnic acid4. Recently, many researchs showed that usnic acid bearing triazole, enamine, pyrazole and benzylidene groups had strong antiviral and anticancer activities5–8. 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 a-glucosidase and tyrosinase inhibitory activities. MATERIALS ANDMETHODS Materials (+)-Usnic acid isolated from lichen. Acetyl chloride, benzoyl chloride (Sigma-Aldrich). Silica gel 60 (HiMedia, India). Bruker Advance III (400 MHz for 1H NMR and 100 MHz for 13C NMR) spectrometer with TMS as inter- nal standard recorded NMR spectra. The HR–ESI–MS were recorded on a HR–ESI–MS Bruker microTOF Q-II. Column chromatography was performed with silica gel 60. General experimental procedure A mixture of (+)-usnic acid (0.250 g, 0.727 mmol) in CHCl3 (5.0 mL) was stirred at room temperature for 5 minutes. Acetyl chloride (0.341 g, 4.350 mmol) was added, followed by pyridine (3.5 mL, 43.502 mmol) and stirred at room temperature for 6 h. Then, the organic layer was extracted with water and saturated Cite this article : DungPD,HuyDT, KieuNV.Synthesisandevaluationofa-glucosidaseand tyrosinase inhibitory activities of ester derivatives of usnic acid. Sci. Tech. Dev. J.; 23(3):585-592. 585 Science & Technology Development Journal, 23(3):585-592 with aqueous NaHCO3, respectively, and dried over anhydrous Na2SO4. 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 CHCl3 (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. Theproducts, UE 5 were purified by subjecting to silica gel column. A mixture of UE3 (0.280 g, 0.727 mmol) in CHCl3 (5.0 mL) was stirred at room temperature for 5 min- utes. 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. Theproducts, UE 6 were purified by subjecting to silica gel column. Biological activities investigation These inhibitory activities were evaluated according to9. Enzymatic activity was calculated by measur- ing absorbance at 405 nm (ALLSHENG micro plate reader AMR-100). All samples were analyzed in trip- licate at various concentrations to obtain the IC50 value of each compound. The mean values and stan- dard deviation were also identified. Structure determination of products The products were verified structures by 1H and 13C NMR method using CDCl3 as solvent and HR-ESI- MS method. UE1: Light yellow powder, m = 0.0342 g, yield: 10 %; 1HNMR (CDCl3, 400 MHz) dH 6.38 (1H, s), 2.65 (3H, s), 2.40 (3H, s), 2.35 (3H, s), 2.23 (3H, s), 2.22 (3H, s), 2.19 (3H, s), 2.02 (3H, s). 13C NMR (CDCl3, 100 MHz) dC 203.0, 202.9, 195.0, 169.1x2, 168.5, 151.2, 147.8, 145.7, 145.5, 144.5, 121.5, 120.3, 115.5, 113.7, 108.5, 47.0, 31.8, 29.5, 21.1, 20.7, 20.5, 9.7, 9.2. HR-ESI-MS m/z [M+H]+ calcd. for C24H23O10 : 471.1291; found: 471.1297. UE2: Light yellow powder, m = 0.1055 g, yield: 34 %; 1HNMR (CDCl3, 400 MHz) dH 5.90 (1H, s), 2.60 (3H, s), 2.54 (3H, s), 2.46 (3H, s), 2.33 (3H, s), 1.98 (3H, s), 1.81 (3H, s). 13CNMR (CDCl3, 100MHz) dC 198.6, 195.0, 192.8, 190.9, 177.8, 168.9, 168.8, 153.7, 149.0, 148.5, 123.6, 118.9, 116.1, 106.2, 98.8, 59.5, 32.1, 31.1, 26.2, 21.4, 20.8, 10.4. UE3: Light yellow powder, m = 0.0420 g, yield: 15 %; 1H NMR (CDCl3, 400 MHz) dH 13.22 (1H, s), 5.91 (1H, s), 2.74 (3H, s), 2.54 (3H, s), 2.45 (3H, s), 2.03 (3H, s), 1.78 (3H, s). 13CNMR (CDCl3, 100MHz) dC 201.9, 198.4, 193.3, 190.9, 178.1, 168.6, 163.3, 155.7, 151.5, 117.7, 111.1, 106.3, 105.4, 98.8, 59.4, 32.0, 31.2, 26.0, 21.4, 9.3. UE4: Light yellow powder, m = 0.0505 g, yield: 18 %; 1H NMR (CDCl3, 400 MHz) dH 11.07 (1H, s), 5.97 (1H, s), 2.66 (3H, s), 2.57 (3H, s), 2.35 (3H, s), 2.06 (3H, s), 1.80 (3H, s). 13CNMR (CDCl3, 100MHz) dC 201.9, 197.8, 194.0, 191.8, 179.3, 169.2, 155.5, 154.2, 149.7, 117.4, 110.0, 109.9, 105.4, 98.5, 59.1, 32.4, 32.0, 28.0, 20.9, 8.9. UE5: Light yellow powder, m = 0.3250 g, yield: 81 %; 1H NMR (CDCl3, 400 MHz) dH 13.32 (1H, s), 10.52 (1H, s), 8.01 (2H, d, J = 8.0 Hz), 7.88 (2H, d, J = 8.0 Hz), 7.66 (2H, t, J = 8.0 Hz), 7.53 (2H, t, J = 8.0 Hz), 7.46 (1H, t, J = 8.0 Hz), 7.32 (1H, t, J = 8.0 Hz), 6.03 (1H, s), 5.43 (1H, d, 1.2), 5.24 (1H, d, 1.2), 2.65 (3H, s), 2.12 (3H, s), 1.88 (3H, s). 13C NMR (CDCl3, 100 MHz) dC 200.9, 200.5, 174.0, 165.1, 164.5, 164.0, 163.0, 157.5, 156.4, 143.5, 134.6, 133.6, 130.7, 130.1, 128.9, 128.5, 128.4, 127.9, 114.6, 109.8, 109.2, 104.0, 101.9, 96.6, 60.7, 31.3, 31.1, 7.7. UE6: Light yellow powder, m = 0.2529 g, yield: 71 %; 1HNMR (CDCl3, 600 MHz) dH 8.18 (2H, d, 8.0), 7.66 (1H, t, 8.0), 7.53 (2H, t, 8.0), 5.92 (1H, s), 2.60 (3H, s), 2.56 (3H, s), 2.48 (3H, s) 2.04 (3H, s), 1.85 (3H, s). 13CNMR(CDCl3, 150MHz) dC 202.5, 198.7, 195.0, 190.9, 177.9, 168.9, 164.6, 153.5, 148.9, 148.5, 134.2, 130.6, 128.9, 128.7, 119.1, 116.7, 114.5, 114.0, 98.9, 59.6, 32.0, 29.8, 26.2, 21.5, 10.6. HR-ESI-MSm/z [M+Na]+ calcd. for C27H22O9Na: 513.1162; found 513.1122. RESULTS Figure 1 showed esterification of usnic acid with acetyl chloride and benzoyl chloride. Six ester deriva- tives (UE1-6) were synthesized from usnic acid. Ta- ble 1 showed the results in the synthesis of six ester derivatives of usnic acid. Yields of the reactions us- ing 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 mag- netic resonance spectra of these ester products. These signals demonstrated that six ester derivatives had been synthesized successfully. a-glucosidase and tyrosinase inhibitory activities of UE1-6 were listed in Table 4. All derivatives exhib- ited the same or higher activity comparing with start- ing material (usnic acid). DISCUSSION Ester derivatives synthesis from usnic acid There are three hydroxy groups in usnic acid struc- ture at C-3, C-8 and C-10 could be esterified. In the 586 Science & Technology Development Journal, 23(3):585-592 Table 2: 1HNMR data of ester derivatives Position Usnic acid (dH J, Hz) UE1 (dH J, Hz) UE2 (dH J, Hz) UE3 (dH J, Hz) UE4 (dH J, Hz) UE5 (dH J, Hz) UE6 (dH J, Hz) 1 - - - - - - - 2 - - - - - - - 3 - - - - - - - 4 5.97 s 6.38 s 5.90 s 5.91 s 5.97 s 6.03 s 5.92 s 5 - - - - - - - 6 - - - - - - - 7 - - - - - - - 8 - - - - - - - 9 - - - - - - - 10 - - - - - - - 11 - - - - - - - 12 - - - - - - - 13 1.76 s 2.02 s 1.81 s 1.78 s 1.80 s 1.88 s 1.85 s 14 - - - - - - - 15 2.66 s 2.40 s 2.54 s 2.54 s 2.57 s 5.43 d (1.2) 5.24 d (1.2) 2.56 s 16 2.11 s 2.35 s 2.46 s 2.45 s 2.35 s 2.12 s 2.48 s 17 - 18 2.68 s 2.65 s 2.60 s 2.74 s 2.66 s 2.65 s 2.60 s 3-OH - - - - - - - 8-OH 13.29 s - - 13.22 s - 13.32 s - 10-OH 11.01 s - - - 11.07 s 10.52 s - 2’ 2.23 s 2.33 s 2.03 s 2.06 s - - 2” 2.22 s 1.98 s - - 2.04 s 2”’ 2.19 s - - - - - 3’,7’ 8.01 d (8.0) 8.18 d (8.0) 3”,7” 7.88 d (8.0) - 4’-6’ 7.66 t (8.0) 7.66 t (8.0) 4”-6” 7.53 t (8.0) - 5’ 7.46 t (8.0) 7.53 t (8.0) 5” 7.32 t (8.0) - 587 Science & Technology Development Journal, 23(3):585-592 Table 3: 13C NMR data of ester derivatives Position Usnic acid (dC) 9 UE1 (dC) UE2 (dC) UE3 (dC) UE4 (dC) UE5 (dC) UE6 (dC) 1 198.1 195.0 192.8 193.3 194.0 200.5 195.0 2 105.3 120.3 118.9 111.1 110.0 109.2 116.7 3 191.7 151.2 190.9 190.9 191.8 165.1 190.9 4 98.3 108.5 98.8 98.8 98.5 96.6 98.9 5 179.4 147.8 177.8 178.1 179.3 174.0 177.9 6 155.2 145.5 149.0 155.7 154.2 156.4 148.5 7 101.6 113.7 106.2 105.4 105.4 101.9 114.0 8 163.9 145.7 153.7 163.3 155.5 157.5 153.5 9 109.4 121.5 123.6 117.7 117.4 114.6 119.1 10 157.5 144.5 148.5 151.5 149.7 143.5 148.9 11 103.9 115.5 116.1 106.3 109.9 104.0 114.5 12 59.1 47.0 59.5 59.4 59.1 60.7 59.6 13 7.5 9.2 10.4 9.3 8.9 7.7 10.6 14 200.3 203.0 198.6 201.9 201.9 163.0 202.5 15 27.8 29.5 31.1 31.2 32.0 109.8 29.8 16 32.2 9.7 26.2 21.4 28.0 31.1 26.2 17 201.7 202.9 195.0 198.4 197.8 200.9 198.7 18 31.2 31.8 32.1 32.0 32.4 31.3 32.0 1’ 169.1 168.9 168.6 169.2 164.5 168.9 1” 169.1 168.8 164.0 164.6 1”’ 168.5 - - - 2’ 21.1 21.4 26.0 20.9 127.9 128.7 2” 20.7 20.8 128.4 21.5 2”’ 20.5 - - - 3’ 130.7 130.6 3” 130.1 - 4’ 128.9 128.9 4” 128.5 - 5’ 134.6 134.2 5” 133.6 - 6’ 128.9 128.9 6” 128.5 - 7’ 130.7 130.6 7” 130.1 - 588 Science & Technology Development Journal, 23(3):585-592 Figure 1: Esterification of usnic acid Table 4: a-Glucosidase and tyrosinase inhibitory activities of usnic acid derivatives Entry Compound a-Glucosidase IC50 (mM) Tyrosinase IC50 (mM) 1 UE1 >200 NA 2 UE2 >200 >200 3 UE3 >200 NA 4 UE4 >200 >200 5 UE5 26.7 0.57 >200 6 UE6 68.8 0.15 NA 7 Usnic acid >200 NA 8 Acarbose 93.60.49 9 Kojic acid 36.1 1.07 589 Science & Technology Development Journal, 23(3):585-592 Figure 2: Proposed mechanism of UE3 synthesis from usnic acid Table 1: Ester derivatives synthesis of usnic acid Entry Ester com- pound Yield (%)a 1 UE1 10 2 UE2 34 3 UE3 15 4 UE4 18 5 UE5 81 6 UE6 71 a Isolated yields 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 reactionwhen acetyl chloride was used as a reactant. Besides, only one product (UE5) was created when benzoyl chlo- ride was used. Moreover, the ester product (UE6) was also generated when UE3 product reacted with ben- zoyl 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%). The 1H NMR spectrum of UE1 showed an olefin proton at dH 6.38, and seven methyl groups at dH 2.65, 2.40, 2.35, 2.23, 2.22, 2.19 and 2.02. The 13C NMR spectrum of UE1 displayed twenty-three car- bon signals, including three ketone carbons at dC 203.0, 202.9 and 195.0, three carboxyl carbons at dC 169.1x2 and 168.5, ten olefin carbons in the range of dC 155.0-100.0, one tertiary carbon at dC 47.0 and sevenmethyl carbons at dC 31.8, 29.5, 21.1, 20.7, 20.5, 9.7 and 9.2. The lack of 8- and 10-OH signal in us- nic acid along with the appearance of seven methyl groups (usnic acid has only four methyl groups10) in- dicated the esterification reaction occurred on 3-, 8-, and 10-OH of usnic acid. Thus, UE1 is established as 3,8,10-triacetoxyusnic acid. The 1HNMR spectrum of UE2 showed an olefin pro- ton at dH 5.90, and six methyl groups at dH 2.60, 2.54, 2.46, 2.33, 1.98 and 1.81. The lack of both of 10- OH and 8-OH in usnic acid along with the appear- ance of only two acetoxycarbonyl groups (dH 2.33 and 1.98; dC 168.9 and 168.8) indicated the esteri- fication reaction occurred on both of 10-OH and 8- OH of usnic acid. Thus, the structure of UE2, 8,10- O-diacetylusnic acid 10, is elucidated as shown in Fig- ure 1. The 1H NMR spectrum of UE3 showed a singlet of hydroxy chelated signal at dH 13.22, an olefin proton at dH 5.91, and five methyl groups at dH 2.74, 2.54, 2.45, 2.03, and 1.78. Similar toUE2, the lack of 10-OH in usnic acid 10 along with the appearance of only one acetoxycarbonyl group (dH 2.03, dC 168.6 and 26.0) indicated the esterification reaction occurred on 10- OH of usnic acid. Thus, the structure of UE3, 10-O- acetylusnic acid11, is elucidated as shown in Figure 1. The examination of the 1H and 13C NMR spectra of UE4 revealed the similar spectra to those of UE3, ex- cepted for the lack of 8-OH and the occurrence of 590 Science & Technology Development Journal, 23(3):585-592 10-OH that indicated the reaction occurred at 8-OH. Thus, UE4, 8-O-acetylusnic acid 11, is established as shown in Figure 1. The 1H NMR of UE5 displayed the presence of two chelated hydroxyl groups at dH 13.32 and 10.52, ten aromatic protons at dH 7.00-8.50, three olefin protons at dH 6.03, 5.43, and 5.24, and three methyl groups at dH 2.65, 2.12 and 1.88. Comparison with those of usnic acid indicated the hydroxyl groups at dH 13.32 and 10.52 belonging to 8-OH and 10-OH, respectively. Moreover, the appearance of ten aromatic protons at dH 7.00-8.50 ppm along with a couple gem olefin proton at dH 5.43 (1H, d, J = 1.2 Hz) and 5.24 (1H, d, J = 1.2 Hz) implied the disubstitution on C-14 and C-3. Finally, UE5 is established as benzoic acid 1-(6- acetyl-3-benzoyloxy-7,9-dihydroxy-8,9b-dimethyl- 1-oxo-1,9b-dihydro-dibenzofuran-2-yl)-6inyl ester as shown in Figure 111. The 1H NMR spectrum of UE6 showed five aro- matic protons at dH 8.5-7.5, that implied monoben- zoyl chloride reacted with UE3. A singlet signal at dH 5.86 (1H, s), belonging to H-4 in starting mate- rial, and five methyl groups at dH 2.60, 2.56, 2.48, 2.04 and 1.85. The examination of the 13CNMR spec- trum revealed some important structural differences from UE3 including the occurrence of five aromatic carbons at dC 134.2, 130.6 x2 and 128.9x2 confirmed the addition of monobenzoyl chloride. Moreover, the lack of chelated hydroxyl proton 8-OH at dH 13.22 (UE3) identificated that the reaction occurred at 8- OH. Finally, the structure of UE6 was established as shown in Figure 1. Biological activities of usnic acid deriva- tives Six usnic acid derivatives including via esterification (UE1-6) were further tested with a-glucosidase and tyrosinase inhibitory activities. From the results, all derivatives exhibited the same or higher activity com- paring with starting material (usnic acid: >200 mM and no activity (NA) for a-glucosidase and tyrosinase, respectively). Especially,UE5 andUE6 showed excel- lent a-glucosidase activity with IC50 26.70.57, and 68.80.15 mM, respectively. These compounds not only displayed higher activity than that of usnic acid, but also with that of a positive control, acarbose (IC50: 93.60.49 mM) as shown in Table 4. In this case, UE5 displayed the strongest activity (IC50: 26.70.57 mM). CONCLUSION From usnic acid, six derivatives were synthesized via esterification reactions (UE1-6). Their chemi- cal structures were elucidated by NMR and HRES- IMS as well as comparison with those from litera- ture. Among them, UE1 and UE6 were reported as as new compounds. Interestingly, all products dis- played the same or higher biological activity than the starting material, usnic acid when evaluated against a-glucosidase and tyrosinase. In the a-glucosidase as- say, UE5 and UE6 showed excellent activity (IC50 26.70.57, and 68.80.15 mM, respectively). On the other hand, all tested compounds revealed weak or no inhibitory activity in the tyrosinase assay. ABBREVIATIONS 1HNMR: Proton nuclear magnetic resonance; 13C NMR: Carbon-13 nuclear magnetic resonance; s: singlet; d: doublet; t: triplet. CONFLICTS OF INTEREST The authors declare that they have no competing fi- nancial interest. AUTHOR CONTRIBUTION All authors contributed in conducting experiments, acquisition of data, interpretation of data, search- ing the bibliography and gave final approval of the manuscript to be submitted. ACKNOWLEDGEMENT Theauthors are indebted toDr. WarinthornChavasiri and Mrs. Asshaima Paramita Devi (Center of Excel- lence in Natural Products Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn Uni- versity, Thailand) for performing the enzyme in- hibitory against a–glucosidase and tyrosinase. REFERENCES 1. Boustie J, Tomashi S, Grube M. Bioactive lichen metabolites: alpine habitats as an untapped source. 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