Bioorganic & Medicinal Chemistry Letters 23 (2013) 6580–6584
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Synthesis, antioxidant capacity, and structure–activity relationships of tri-O-methylnorbergenin analogues on tyrosinase inhibition Yusei Kashima, Mitsuo Miyazawa ⇑ Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, 3-4-1 Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan
a r t i c l e
i n f o
Article history: Received 26 August 2013 Revised 9 October 2013 Accepted 29 October 2013 Available online 7 November 2013 Keywords: Bergenin Tri-O-methylnorbergenin analogues Tyrosinase inhibitor Antioxidant Structure–activity relationship
a b s t r a c t A series of tri-O-methylnorbergenin analogues 1–9 were synthesized and their antioxidant activities and inhibitory effects on tyrosinase were evaluated. Among tested analogues, compound 4 bearing cathechol moiety exhibited greater antioxidant activity and excellent inhibition on tyrosinase with IC50 value of 9.1 lM, comparable to that of corresponding positive controls. The inhibition mechanism analysis of compound 4 demonstrated that it was a mixed-type inhibitor on tyrosinase. These results suggest that these compounds may serve as a useful clue for further designing and development of novel potential tyrosinase inhibitors. Ó 2013 Elsevier Ltd. All rights reserved.
Tyrosinase (EC 1.14.18.1, monophenol or o-diphenol, oxygen oxidoreductase) is a multifunctional copper-containing enzyme that is widely distributed in nature such as animals, plant, and microorganisms.1 It could catalyze two distinct reaction involving the hydroxylation of monophenols and oxidation of o-diphenols to o-quinones using molecular oxygen; the quinones could polymerize spontaneously to form macromolecular dark pigments (e.g., melanin) or react with amino acids and proteins to enhance brown color of the pigment produced.2 The melanin synthesis is altered in many disease states. The accumulation of an excessive level of epidermal pigmentation can cause some dermatological disorders associated with melasma, freckles, and senile lentigines.3 Recently, melanin pigments are found in the mammalian brain, and tyrosinase has been linked to neurodegeneration associated with Parkinson’s and other degenerative diseases.4 In addition, tyrosinase is involved in the insect molting process and adhesion in marine organisms.5 In fruits and vegetables, the enzymatic browning results in quality such as nutritional and commercial value loss.6 Thus, the development of effective tyrosinase inhibitors has become increasingly important in the agricultural, medicinal, and cosmetic industries in relation to hyperpigmentation. So far, numerous natural and synthetic compounds as tyrosinase inhibitors were reported.7 Unfortunately, only a few such as kojic acid and arbutin are used practically as therapeutic agents and cosmetic products. Therefore, it is still necessary to search and discover novel tyrosinase inhibitors with higher activities and lower ⇑ Corresponding author. Tel.: +81 6 6721 2332; fax: +81 6 6727 2024. E-mail address: [email protected] (M. Miyazawa). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.066
side effects. Recently, several phenolic substrates as well-known antioxidants from natural or synthetic source have been targeted for the inhibition of the enzyme.8 Several hydroxybenzoic acid derivatives have been found to possessed tyrosinase inhibitory activities in our laboratory.9 On the other hand, many researchers have shown that the antioxidants of natural or synthetic origin could have a great importance as therapeutic agents in several diseases, caused by oxidative stress.10 Moreover, applications of antioxidants such as preservatives in the food industry and skin-protective source in cosmetics have also attracted much interest.11 Bergenin and the corresponding tri-O-methylnorbergenin (8,10-di-O-methyl derivative) were isolated from various plants,12 and exhibit biological properties, such as antioxidant,13 anti-inflammatory,14 anti-arthritis,15 hypolipidemic,16 antiarrhythmic,17 hepatoprotective,18 antinociceptive,19 and anti-HIV effects.20 Bergenin and its analogues are characterized by a b-D-glucosyl residue C-linked to a hydroxylated or methoxylated phenylcarboxylic acid ortho to the carboxyl group, then, the carboxyl group is esterified with the C2 hydroxyl group of the glucosyl moiety to form a d-lactone bridge. Furthermore, its esterified analogues occur widely in some plants, especially those used in traditional medicines.21 Our investigation demonstrated that bergenin esterified compounds exhibited potent inhibitory activities against tyrosinase and b-secretase.22 However, although the interesting biological activities of this class of natural products, the investigation for bergenin analogues, in particular tri-O-methylnorbergenin analogues, and their biological effect are unsatisfying. Stimulated by these results, in the present investigation, we synthesized a
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series of tri-O-methylnorbergenin analogues and identify their structural importance for the inhibition of tyrosinase23 and antioxidant activity.24 The synthesis pathways are described Scheme 1.25,26 The antioxidative properties of tri-O-methylnorbergenin analogues 1–9,27 tri-O-methylnorbergenin,28 and bergenin against peroxyl radicals were investigated and compared with that of dibutylhydroxytoluene (BHT). As shown on Table 1, some analogues including 2–4, 6, 7, and 9 showed a more active than BHT. Especially, compound 4, which contains catechol moiety, showed significantly enhanced antioxidant activity. Moreover, among benzoic acid derivatives 1a–9a, the ORAC value (lmol of TE/lmol) of compound 4a was high. Since compounds 4 and 4a had relatively potent the activities compared to other compounds, catechol moiety affects the antioxidant activity. Furthermore, although triO-methylnorbergenin did not exhibit the activity, compound 4 had higher activity than 4a. Consequently, tri-O-methylnorbergenin moiety was a potent factor of antioxidant activity. Among triO-methylnorbergenin analogues, compounds 4, 6, and 7 had high ORAC values relatively; therefore, the presence of the substituted group (OH or OMe) at 30 and 40 -positions on the analogues was a great influence on the activities. Next, we evaluated tyrosinase inhibition activity of tri-Omethylnorbergenin analogues, tri-O-methylnorbergenin, and bergenin. From the regression curves of the analogues
OH
11 2
OH O H 10b 10
MeO HO
8
7
3
4a
6
OH
OH
O
H
MeO
OH
OH
OMe O H
a
4
10a
9
concentration against percentage of control activity, IC50 values of each analogue were evaluated and presented in Table 2. They revealed from low to high tyrosinase inhibitory activity. Among the analogues tested, compound 4 showed the most potent inhibitory activity (IC50 = 9.1 lM). Similarly, compounds 2 and 7 exhibited IC50 values of 31.5 and 38.8 lM, respectively and were more potent than arbutin and kojic acid as positive controls (Fig. 1). Several other compounds inhibited tyrosinase to lesser extent, these included compounds 1, 3, and 8 with IC50 values of more than 100 lM. All other compounds including tri-O-methylnorbergenin and bergenin, whereas, exhibited no inhibitions against tyrosinase (Table 2). Further investigations were undertaken to examine the inhibition mechanism of compounds 2, 4, and 7 exhibiting high inhibitory potency against tyrosinase activity. For the type of enzyme inhibition and the inhibition constant for an enzyme-inhibitor complex, the mechanisms were analysed by Dixon plot, which is a graphical method [plot of 1/enzyme velocity (1/V) versus inhibitor concentration (I) with varying concentrations of the substrate] as shown in Figure 2. The Dixon plots were constructed using three different substrate concentrations 1.5, 0.75, 0.375 mM. Each three straight lines representing the uninhibited enzyme and different concentrations of compound 2 and 4 intersected in a single point at the second quadrant (Panels A in Fig. 2). From the results, the inhibition constants were
O
MeO
O
H
OH
O
+
R1
OH
R2 R3
O
R1, R 2, R 3 = H, OH, OMe
R1 3' 2'
7'
2
OMe O H 10b
4
MeO
8
1: 2: 3: 5: 6: 7: 8: 9:
4a
10a 6 7
OH
3
10 9
R3
6'
O
11
MeO
1a-3a, 5a-9a
R2
5'
O
b
4'
O
H
OH
O
R1 =H, R 2=H, R 3=H R1 =H, R 2=OH, R 3=H R1 =H, R 2=OMe, R 3=H R1 =OMe, R 2=OMe, R 3=H R1 =OMe, R 2=OH, R 3=H R1 =OH, R 2=OMe, R 3=OMe R1 =OMe, R 2=H, R 3=OMe R1 =OMe, R 2=OH, R 3=OMe
OH OH O
OH
MeO
OMe O H O
MeO O
O
OH
H
OH
O
+
b OH
c
MeO
O
MeO
BnO OBn
OH
OMe O H H
OH
O 4
Scheme 1. Reagents and conditions: (a) MeI, K2CO3, DMF, rt; (b) DIAD, P(Ph)3, THF, rt; (c) H2, Pd/C, EtOAc, rt.
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Table 1 Antioxidant activity of bergenin derivatives by ORAC assay Compound tested
Trolox-equiv
Compound tested
Trolox-equiv
1 2 3 4 5 6 7 8 9 Tri-O-methylnorbergenin Bergenin
0.05 ± 0.01 0.44 ± 0.03 0.17 ± 0.06 3.11 ± 0.12 0.09 ± 0.01 2.53 ± 0.21 1.92 ± 0.17 0.09 ± 0.01 1.46 ± 0.12 0.02 ± 0.01 1.69 ± 0.11
1a 2a 3a 4a 5a 6a 7a 8a 9a
0.05 ± 0.02 1.84 ± 0.88 0.02 ± 0.01 2.24 ± 0.32 0.02 ± 0.01 1.17 ± 0.09 1.17 ± 0.08 0.10 ± 0.01 1.22 ± 0.11
BHT
0.12 ± 0.01
Results are presented as the mean (n = 3) ± standard deviation (SD).
Table 2 Inhibitory effects of tri-O-methylnorbergenin analogues and benzoic acid analogues against tyrosinase IC50a (lM)
Compound tested 1 2 3 4 5 6 7 8 9 Tri-O-methylnorbergenin Bergenin a b c
b
>100 (7.9%) 31.5 ± 0.1 >100 (7.9%) 9.1 ± 0.04 >100 (0.4%) >100 (0.7%) 38.8 ± 0.9 >100 (16.0%) >100 (0.6%) >100 (1.9%) >100 (0.6%)
Compound tested
IC50a (lM)
1a 2a 3a 4a 5a 6a 7a 8a 9a Arbutinc Kojic acidc
>100 (12.7%) >100 (13.2%) >100 (12.4%) >100 (7.9%) >100 (3.2%) >100 (2.4%) >100 (1.1%) >100 (3.6%) >100 (2.4%) 194.5 ± 11.6 46.6 ± 3.9
The results are the means ± SE of three experiments. Inhibition (%) at 100 lM. Arbutin and Kojic acid were used as the positive control.
100
2 4 7
Tyrosinase activity (%)
80
arbutin kojic acid
60
40
20
0 0
50
100
150
200
250
Concentration (µM) Figure 1. Dose-dependent inhibitory effects of compounds 2, 4, and 7 on tyrosinase activity. Tyrosinase activity was measured using L-tyrosine as the substrate. Values are mean ± SE of n = 3 determinations.
determined to be Ki values of compounds 2 and 4 with 74.8 and 4.5 lM, respectively. The Dixon plot is useful method to determine the inhibition mechanism of tri-O-methylnorbergenin analogues; nevertheless this method is limited by the fact that it does not discriminate unambiguously between competitive and mixed types. Moreover, for mixed or uncompetitive inhibitors, the Dixon plots exhibit no measure of Ki0 , the dissociation constant of the enzyme-substrate-inhibitor complex. Therefore, for characterizing the types of inhibition, the Cornish–Bowden plots, which is complementary to Dixon plots, were undertaken with Dixon plots. Figure 2 suggested that compounds 2 and 4 were mixed inhibition because the intersection is above the xaxis in Dixon plots and below the x-axis in Cornish–Bowden plot. In other words, the dissociation constants of enzyme-inhibitor complex (Ki) for these compounds were not equal to Ki0 . On the other hand, compound 7 was typical uncompetitive inhibition by Figure 2 with Ki0 values of 107.4 lM (Table 3). From the structural–activity point of view, compound 1, which is fundamental skeleton lacking substitutions on the benzene ring of benzoic moiety, showed low inhibitory effect (7.9%). The activities of compounds 2 and 3, with an OH or OMe functional group at the 40 -position, showed more inhibition effect relative to compound 1, however, the introduction of another OMe group at the 30 -position (compounds 5 and 6) seems to have no influence. On the other hand, compounds 4 and 7, with an additional OH group at the 30 -position of compounds 2 and 3, exhibited significant increased inhibition. These results showed that functional substitutions on the benzoic acid moiety selectively enhance or decrease inhibition of tyrosinase activity, and the introduction an OH group at the 30 -position as well as the 40 -position seems to be critical factor for the inhibitory activity. Especially, the catechol moiety of compound 4 might play an important role for the activity. The result was consistent with our previous investigation that the catechol moiety of 11-O-protocatechuoylbergenin (8,10-di-hydroxy derivative) exhibited increase inhibition on tyrosinase.29 Comparison of the inhibitory activities of compound 4 and 11-O-protocatechuoylbergenin showed that the potency increased in the order of 4 >11-O-protocatechuoylbergenin (IC50 values: 9.1 vs 17.5 lM). This observation revealed that the presence of two OMe groups at 8 and 10positions led to significantly higher activity than the presence of the OH groups. Furthermore, we evaluated the inhibitory activities of benzoic acid analogues (1a–9a) and tri-O-methylnorbergenin. These compounds exhibited relatively poor inhibitory activities; therefore, we considered that the ester linkage of ‘triO-methylnorbergenin moiety’ and ‘benzoic acid moiety’ is essential for the tyrosinase inhibitory activity. In conclusion, we synthesized a series of tri-O-methylnorbergenin analogues and evaluated their antioxidant properties and tyrosinase inhibitory activities. Compound 4, 6, and 7 containing the functional group (OH or OMe) at 30 and 40 -positions showed potent antioxidant activities in ORAC assay and surpassed positive controls. In the structure-tyrosinase inhibition activity study, some of the analogues indicated tyrosinase inhibitory activities in the micromolar range. The presence of catechol moiety of compound 4 is important for higher inhibitory activity in the tyrosinase assay. The results of the structure–activity relationship revealed that the ester linkage as well as tri-O-methylnorbergenin moiety and benzoic acid moiety affect the two activities. These results suggest that these compounds may serve as structural templates for the design and development of novel tyrosinase inhibitors.
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500
(2-A)
350
(2-B)
400
S/V
1/V
250 300
150
200 100
-100
-50
50 0
50
100
150
-150
-100
-50
50
100
150
-50
[I, µM]
[I, µM]
600
(4-A)
(4-B)
450
S/V
1/V
500 400
350
300
250
200
150
100
50 -10
-5
0
[I, µM]
5
10
15
-10
400
(7-A)
-5
-50
[I, µM]
5
10
15
300
(7-B)
S/V
1/V
300
200
200
100 100
-120
-60
0
60
120
-120
-60
[I, µM]
0
60
120
[I, µM]
Figure 2. Dixon plots (panels A) and Cornish–Bowden plots (panels B) for inhibition of 2, 4, and 7 on tyrosinase activity. The substrate used were 1.5 (), 0.75 (j), and 0.375 mM (N). Table 3 Kinetic parameters for inhibition tyrosinase activity by compounds 2, 4, and 7 Compounds
Ki (lM)
Ki0 (lM)
Type of inhibition
2 4 7
74.8 4.5 —
120.9 7.3 107.4
Mixed Mixed Uncompetitive
Acknowledgment This work was supported by Grant-in Aid from the Japan Society for the Promotion of Science (No. 24658055).
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7. Yan, Q.; Cao, R.; Yi, W.; Yu, L.; Chen, Z.; Ma, L.; Song, H. Bioorg. Med. Chem. Lett. 2009, 19, 4055. 8. (a) Kim, Y. J.; Uyama, H. Cell. Mol. Life. Sci. 2005, 62, 1707; (b) Takahashi, T.; Miyazawa, M. Bioorg. Med. Chem. Lett. 1983, 2011, 21. 9. Miyazawa, M.; Oshima, T.; Koshio, K.; Itsuzaki, Y.; Anzai, J. J. Agric. Food Chem. 2003, 51, 6953. 10. Laguerre, M.; Lecomte, J.; Villeneuve, P. Prog. Lipid Res. 2007, 46, 244. 11. Winkler, C.; Frick, B.; Schroecksnadelm, K.; Schennach, H.; Fuchs, D. Food Chem. Toxicol. 2003, 2006, 44. 12. (a) Piacente, S. J. Nat. Prod. 1996, 59, 565; (b) Singh, D. P.; Srivastava, S. K.; Govindarajan, R.; Rawat, A. K. S. Acta Chromatogr. 2007, 19, 246; (c) Arfan, M.; Amin, H.; Karamac´, M.; Kosin´ska, A.; Wiczkowsk, W.; Amarowicz, R. Czech J. Food Sci 2009, 27, 109. 13. Kumar, R.; Patel, D. K.; Prasad, S. K.; Laloo, D.; Krishnamurthy, S.; Hemalatha, S. Fitoterapia 2012, 83, 395. 14. Swarnalakshmi, T.; Sethuraman, M. G.; Sulochana, N.; Arivudainambi, R. Curr. Sci. 1984, 53, 917. 15. Nazir, N.; Koul, S.; Qurishi, M. A.; Taneja, S. C.; Ahmad, S. F.; Bani, S.; Qazi, G. N. J. Ethnopharmacol. 2007, 112, 401. 16. Jahromi, M. A. F.; Chansouria, J. P. N.; Ray, A. B. Phytother. Res. 1992, 6, 180. 17. Pu, H. L.; Huang, X.; Zhao, J. H.; Hing, A. Planta Med. 2002, 68, 372. 18. Lim, H. K.; Kim, H. S.; Choi, H. S.; Oh, S.; Choi, J. J. Ethnopharmacol. 2000, 72, 469. 19. DeOliveira, C. M.; Nonato, F. R.; DeLima, F. O.; Couto, R. D.; David, J. P.; David, J. M.; Soares, M. B. P.; Villareal, C. F. J. Nat. Prod. 2011, 74, 2062. 20. Piacente, D.; Pizza, C.; DeTommasi, N.; Mahmood, N. J. Nat. Prod. 1996, 59, 565. 21. Fuji, M.; Miyaichi, Y.; Tomimori, T. Nat. Med. (Tokyo, Jpn.) 1996, 50, 404. 22. Kashima, Y.; Miyazawa, M. J. Oleo Sci. 2013, 62, 391. 23. Method of tyrosinase assay: The tyrosinase assay was performed using Ltyrrosine as the substrate. 100 lL of 0.1 M phosphate buffer (pH 7.0), 36 lL of 1.5 mM L-tyrosine, and 10 lL of sample solution containing DMSO+0.05% TweenÒ 20 to dissolve the sample were added to each well of a 96-well plate and then incubated at 37 °C for 10 min. Then, 16 lL of mushroom tyrosinase (500 units/mL, 0.1 M phosphate buffer at pH 7.0) was added, and the assay mixture was incubated at 37 °C for 20 min. Before and after incubation, the amount of dopachrome produced in the reaction mixture was measured at 492 nm in a microplate reader (Corona Electric Co. Ltd). Arbutin and kojic acid were used as a positive control. The extent of tyrosinase inhibition by the different compounds added was calculated and expressed as the percentage necessary for 50% inhibition concentration (IC50). The percentage of tyrosinase activity was calculated as follows: Tyrosinase activity (%) = [(CD)/ (AB)] 100, where A is the absorbance at 492 nm without test sample, B is the absorbance at 492 nm without test sample and substrate, C is the absorbance at 492 nm with test sample, D is the absorbance at 492 nm with test sample, but without substrate. All data are the mean of three experiments. 24. The ORAC assay was carried out on a microplate reader (Corona Electric Co. Ltd) with fluorescence filters for an excitation wavelength of 480 nm and emission wavelength of 540 nm. The measurements were made in aplate with 96 black flat-bottom wells. The samples were dissolved in 50% acetone and incubated for 1 h at room temperature. The reaction was performed in 75 mM phosphate buffer (pH 7.4) and the final assay mixture (200 lL) contains fluorescein (160 lL, 63 nM final concentration) as an oxidizable substrate, AAPH (20 lL, 12.8 mM final concentration), and trolox (20 lL, 5–20 lM final concentration) or samples (20 lL, 5–20 lM final concentration). The reaction was performed at 37 °C, and fluorescence was recorded every minute for 120 min. A blank using phosphate buffer instead of the sample was carried out in each experiment. BHT was used as a positive control. The ORAC value was calculated as follows: ORAC value = [CTrolox (AUCSampleAUCBlank)]/ [CSample (AUCTroloxAUCBlank)], where CTrolox is the concentration of trolox, CSample is the concentration of the sample, and AUC is the area below the fluorescence decay curve of the sample, blank and trolox, respectively, calculated as follows: AUC = 1 + f1/f0 + f2/f0 + f3/f0. . . + fn/f0, where f0 is the initial fluorescence and fn is the fluorescence at time n. All data are the mean of three experiments. 25. General procedure for the synthesis of tri-O-methylnorbergenin analogues (1– 3, 5–9): Methylation of bergenin with iodomethane (5 equiv) in the presence of potassium carbonate (5 equiv) in DMF produced tri-O-methylnorbergenin (97% yield). To a solution of tri-O-methylnorbergenin in dry THF (2 mL/mmol), corresponding benzoic acids (1 equiv) and Ph3P (2 equiv) were dissolved. DIAD (1.7 equiv) was added the solution under 0 °C. The mixture was stirred for 3 h on rt under nitrogen and monitored by TLC. It was concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/EtOAc, 2:8) to afford the analogue as a white power (72–93% total yield). 26. General procedure for the synthesis of 11-O-protocatechuoyl-tri-Omethylnorbergenin (4): The procedure mentioned ref 25 was carried out using 3,4-bis(benzyloxy)benzoic acid (1 equiv) to afford monoester (77% yield). Further, the monoester was debenzylated-using Pd-C/H2 in EtOAc. The reaction mixture was filtered, concentrated, and the residue was purified by column chromatography (CH2Cl2/MeOH, 4:1) to afford pure compound 4 (74% yield). 27. 11-O-Benzoyl-tri-O-methylnorbergenin (1): This compound was obtained with a yield of 76%; amorphous powder; mp: 202.7 °C; IR (KBr) 3478, 1714, 1593 cm1; 1H NMR (400 MHz, DMSO-d6) d 8.02 (2H, m, H-20 , 60 ), 7.69 (1H, m, H-40 ), 7.56 (2H, m, 30 , 50 ), 7.35 (1H, s, H-7), 4.94 (1H, d, J = 10.4 Hz, H-10b), 4.68 (1H, d, J = 12.0 Hz, H-11b), 4.40 (1H, dd, J = 12.0, 6.4 Hz, H-11a), 4.02 (1H, dd, J = 10.4, 9.6 Hz, H-4a), 3.83 (1H, m, H-2), 3.73 (1H, m, H-4), 3.43 (1H, m, H3), 3.85, 3.80, 3.63 (9H, 3s, H-8, 9, 10-OMe). 13C NMR (100 MHz, DMSO-d6) d
165.7 163.5, 153.0, 150.8, 147.8, 133.4, 129.7, 129.2, 128.8, 126.2, 119.0, 109.2, 80.0, 78.2, 73.6, 71.1, 70.4, 64.5, 61.0, 60.6, 56.0; MS (ESI): m/z 459 [MH]. 11-O-p-Hydroxybenzoyl-tri-O-methylnorbergenin (2): This compound was obtained with a yield of 84%; amorphous powder; mp: 235.7 °C; IR (KBr) 3362, 1726, 1592 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.86 (2H, d, J = 8.8 Hz, H-20 , 60 ), 7.35 (1H, s, H-7), 6.87 (2H, d, J = 8.8 Hz, H-30 , 50 ), 4.93 (1H, d, J = 10.0 Hz, H-10b), 4.62 (1H, d, J = 12.0 Hz, H-11b), 4.32 (1H, dd, J = 12.0, 6.4 Hz, H-11a), 4.01 (1H, dd, J = 10.4, 9.6 Hz, H-4a), 3.83 (1H, m, H-2), 3.75 (1H, t, J = 8.8 Hz, H-4), 3.46 (1H, m, H-3), 3.86, 3.80, 3.64 (9H, 3s, H-8, 9, 10-OMe). 13 C NMR (100 MHz, DMSO-d6) d 165.5, 163.5, 162.1, 153.0, 150.9, 147.8, 131.5, 126.2, 120.2, 119.0, 115.4, 109.2, 80.0, 78.4, 73.6, 71.1, 70.5, 64.1, 61.0, 60.6, 56.0; MS (ESI): m/z 475 [MH]. 11-O-p-Methoxybenzoyl-tri-O-methylnorbergeninamorphous (3): This compound was obtained with a yield of 84%; mp: 210.8 °C; IR (KBr) 3421, 1730, 1609 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.96 (2H, d, J = 8.0 Hz, H-20 , 60 ), 7.35 (1H, s, H-7), 7.08 (2H, d, J = 8.0 Hz, H-30 , 50 ), 4.94 (1H, d, J = 10.4 Hz, H10b), 4.64 (1H, d, J = 11.6 Hz, H-11b), 4.34 (1H, dd, J = 11.6, 6.3 Hz, H-11a), 4.01 (1H, dd, J = 10.4, 9.6 Hz, H-4a), 3.85 (1H, m, H-2), 3.75 (1H, m, H-4), 3.46 (1H, m, H-3), 3.86, 3.80, 3.64 (9H, 3s, H-8, 9, 10-OMe). 13C NMR (100 MHz, DMSOd6) d 165.4, 163.5, 163.2, 153.0, 150.9, 147.8, 131.3, 126.2, 121.8, 119.1, 114.1, 109.2, 80.0, 78.3, 73.6, 71.1, 70.5, 64.3, 61.0, 60.7, 56.0, 55.5; MS (ESI): m/z 489 [MH]. 11-O-Protocatechuoyl-tri-O-methylnorbergenin (4): This compound was obtained with a yield of 74%; mp: 230.7 °C; IR (KBr) 3407, 1716, 1608 cm1; 1 H NMR (400 MHz, DMSO-d6) d 7.40 (1H, s, H-20 ), 7.39 (1H, d, J = 8.4 Hz H-60 ), 7.35 (1H, s, H-7), 6.83 (1H, d, J = 8.4 Hz, H-50 ), 4.93 (1H, d, J = 10.0 Hz, H-10b), 4.58 (1H, d, J = 11.6 Hz, H-11b), 4.30 (1H, dd, J = 11.6, 6.4 Hz, H-11a), 4.00 (1H, dd, J = 10.0, 9.6 Hz, H-4a), 3.85 (1H, m, H-2), 3.75 (1H, dd, J = 9.6, 8.8 Hz, H-4), 3.37 (1H, d, 9.2, 8.8 Hz, H-3), 3.86, 3.80, 3.65 (9H, 3s, H-8, 9, 10-OMe). 13C NMR (100 MHz, DMSO-d6) d 165.7, 163.5, 153.0, 150.9, 150.8, 147.8, 145.2, 126.3, 121.9, 120.3, 119.1, 116.3, 115.3, 109.2, 80.0, 78.4, 73.6, 71.1, 70.5, 64.0, 61.0, 60.7, 56.1; MS (ESI): m/z 491 [MH]. 11-O-(30 ,40 -Dimethoxybenzoyl)-tri-O-methylnorbergeninmorphous (5): This compound was obtained with a yield of 72%; mp: 158.2 °C; IR (KBr) 3411, 1715, 1596 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.65 (1H, dd, J = 8.4, 2.0 Hz, H-60 ), 7.50 (1H, d, J = 2.0 Hz, H-20 ), 7.35 (1H, s, H-7), 7.11 (1H, d, J = 8.4 Hz, H50 ), 4.94 (1H, d, J = 10.4 Hz, H-10b), 4.69 (1H, d, J = 12.0 Hz, H-11b), 4.31 (1H, dd, J = 12.0, 7.2 Hz, H-11a), 4.01 (1H, dd, J = 10.4, 10.0 Hz, H-4a), 3.85 (1H, m, H2), 3.75 (1H, m, H-4), 3.38 (1H, m, H-3), 3.86, 3.80, 3.65 (9H, 3s, H-8, 9, 10OMe), 3.85 (3H, s, 40 -OMe), 3.82 (3H, s, 30 -OMe). 13C NMR (100 MHz, DMSO-d6) d 164.4, 163.5, 153.1, 153.0, 150.9, 148.4, 147.8, 126.2, 123.2, 121.8, 119.1, 111.8, 111.2, 109.2, 80.0, 78.4, 73.6, 71.1, 70.7, 64.5, 61.0, 60.6, 56.0, 55.7, 55.5; MS (ESI): m/z 519 [MH]. 11-O-Vanilloyl-tri-O-methylnorbergenin (6): This compound was obtained with a yield of 89%; mp: 162.9 °C; IR (KBr) 3224, 1706, 1592 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.53 (1H, d, J = 8.0 Hz, H-60 ), 7.50 (1H, s, H-20 ), 7.35 (1H, s, H-7), 6.90 (1H, d, J = 8.0 Hz, H-50 ), 4.94 (1H, d, J = 10.0 Hz, H-10b), 4.66 (1H, d, J = 12.0 Hz, H-11b), 4.29 (1H, dd, J = 12.0, 7.2 Hz, H-11a), 4.01 (1H, dd, J = 10.0, 9.6 Hz, H-4a), 3.85 (1H, m, H-2), 3.75 (1H, m, H-4), 3.38 (1H, m, H-3), 3.86, 3.80, 3.65 (9H, 3s, H-8, 9, 10-OMe), 3.83 (3H, s, 30 -OMe). 13C NMR (100 MHz, DMSOd6) d 165.5, 163.5, 153.0, 151.7, 150.9, 147.8, 147.4, 126.2, 123.5, 120.5, 119.1, 115.2, 112.6, 109.2, 80.0, 78.4, 73.7, 71.1, 70.6, 64.3, 61.0, 60.6, 56.0, 55.6; MS (ESI): m/z 505 [MH]. 11-O-Isovanilloyl-tri-O-methylnorbergenin (7): This compound was obtained with a yield of 77%; mp: 164.4 °C; IR (KBr) 3420, 1715, 1609 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.49 (1H, d, J = 8.8 Hz, H-60 ), 7.42 (1H, s, H-20 ), 7.35 (1H, s, H-7), 7.05 (1H, d, J = 8.8 Hz, H-50 ), 4.94 (1H, d, J = 10.0 Hz, H-10b), 4.62 (1H, d, J = 12.0 Hz, H-11b), 4.32 (1H, dd, J = 12.0, 6.4 Hz, H-11a), 4.00 (1H, dd, J = 10.0, 9.2 Hz, H-4a), 3.85 (1H, m, H-2), 3.75 (1H, m, H-4), 3.37 (1H, m, H-3), 3.86, 3.80, 3.65 (9H, 3s, H-8, 9, 10-OMe), 3.84 (3H, s, 40 -OMe). 13C NMR (100 MHz, DMSOd6) d 165.6, 163.5, 153.0, 152.0, 150.9, 147.8, 146.3, 126.2, 121.9, 121.6, 119.1, 115.8, 111.5, 109.2, 80.0, 78.3, 73.6, 71.1, 70.5, 64.2, 61.0, 60.7, 56.0, 55.7; MS (ESI): m/z 505 [MH]. 11-O-(30 ,50 -Dimethoxybenzoyl)-tri-O-methylnorbergenin (8): This compound was obtained with a yield of 89%; mp: 179.1 °C; IR (KBr) 3446, 1719, 1596 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.35 (1H, s, H-7), 7.12 (2H, d, J = 2.4 Hz, H-20 , 60 ), 6.81 (1H, d, J = 2.4 Hz, H-40 ), 4.94 (1H, d, J = 10.4 Hz, H-10b), 4.71 (1H, d, J = 12.0 Hz, H-11b), 4.33 (1H, dd, J = 12.0, 6.8 Hz, H-11a), 4.01 (1H, dd, J = 10.4, 9.6 Hz, H-4a), 3.83 (1H, m, H-2), 3.75 (1H, m, H-4), 3.38 (1H, m, H3), 3.86, 3.80, 3.65 (9H, 3s, H-8, 9, 10-OMe), 3.81 (6H, s, 30 , 50 -OMe). 13C NMR (100 MHz, DMSO-d6) d 165.3, 163.5, 160.5, 153.0, 151.0, 147.8, 131.6, 126.1, 119.1, 109.2, 107.0, 105.1, 80.0, 78.2, 73.6, 71.1, 70.6, 64.8, 61.0, 60.6, 56.0, 55.5; MS (ESI): m/z 519 [MH]. 11-O-Syringyl-tri-O-methylnorbergenin (9): This compound was obtained with a yield of 77%; mp: 226.5 °C; IR (KBr) 3379, 1718, 1618 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.33 (1H, s, H-7), 7.26 (2H, s, H-20 , 60 ), 4.93 (1H, d, J = 9.6 Hz, H10b), 4.71 (1H, d, J = 11.6 Hz, H-11b), 4.23 (1H, dd, J = 12.0, 6.4 Hz, H-11a), 4.00 (1H, t, J = 9.6 Hz, H-4a), 3.83 (1H, m, H-2), 3.73 (1H, m, H-4), 3.37 (1H, m, H-3), 3.84, 3.80, 3.77, 3.64 (15H, 4s, H-8, 9, 10, 30 , 50 -OMe). 13C NMR (100 MHz, DMSO-d6) d 165.6, 163.5, 153.0, 151.0, 147.8, 147.6, 141.0, 126.1, 119.1, 109.2, 107.0, 80.0, 78.4, 73.7, 71.1, 70.8, 64.0, 61.1, 60.6, 56.0; MS (ESI): m/z 535 [MH]. 28. Sakamaki, S.; Kawanishi, E.; Nomura, S.; Ishikawa, T. Tetrahedron 2012, 68, 5744. 29. Kashima, Y.; Miyazawa, M. Arch. Pharmacal Res. 2012, 35, 1533.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis, antioxidant capacity, and structure–activity relationships of tri-O-methylnorbergenin analogues on tyrosinase inhibition Yusei Kashima, Mitsuo Miyazawa ⇑ Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, 3-4-1 Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan
a r t i c l e
i n f o
Article history: Received 26 August 2013 Revised 9 October 2013 Accepted 29 October 2013 Available online 7 November 2013 Keywords: Bergenin Tri-O-methylnorbergenin analogues Tyrosinase inhibitor Antioxidant Structure–activity relationship
a b s t r a c t A series of tri-O-methylnorbergenin analogues 1–9 were synthesized and their antioxidant activities and inhibitory effects on tyrosinase were evaluated. Among tested analogues, compound 4 bearing cathechol moiety exhibited greater antioxidant activity and excellent inhibition on tyrosinase with IC50 value of 9.1 lM, comparable to that of corresponding positive controls. The inhibition mechanism analysis of compound 4 demonstrated that it was a mixed-type inhibitor on tyrosinase. These results suggest that these compounds may serve as a useful clue for further designing and development of novel potential tyrosinase inhibitors. Ó 2013 Elsevier Ltd. All rights reserved.
Tyrosinase (EC 1.14.18.1, monophenol or o-diphenol, oxygen oxidoreductase) is a multifunctional copper-containing enzyme that is widely distributed in nature such as animals, plant, and microorganisms.1 It could catalyze two distinct reaction involving the hydroxylation of monophenols and oxidation of o-diphenols to o-quinones using molecular oxygen; the quinones could polymerize spontaneously to form macromolecular dark pigments (e.g., melanin) or react with amino acids and proteins to enhance brown color of the pigment produced.2 The melanin synthesis is altered in many disease states. The accumulation of an excessive level of epidermal pigmentation can cause some dermatological disorders associated with melasma, freckles, and senile lentigines.3 Recently, melanin pigments are found in the mammalian brain, and tyrosinase has been linked to neurodegeneration associated with Parkinson’s and other degenerative diseases.4 In addition, tyrosinase is involved in the insect molting process and adhesion in marine organisms.5 In fruits and vegetables, the enzymatic browning results in quality such as nutritional and commercial value loss.6 Thus, the development of effective tyrosinase inhibitors has become increasingly important in the agricultural, medicinal, and cosmetic industries in relation to hyperpigmentation. So far, numerous natural and synthetic compounds as tyrosinase inhibitors were reported.7 Unfortunately, only a few such as kojic acid and arbutin are used practically as therapeutic agents and cosmetic products. Therefore, it is still necessary to search and discover novel tyrosinase inhibitors with higher activities and lower ⇑ Corresponding author. Tel.: +81 6 6721 2332; fax: +81 6 6727 2024. E-mail address: [email protected] (M. Miyazawa). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.066
side effects. Recently, several phenolic substrates as well-known antioxidants from natural or synthetic source have been targeted for the inhibition of the enzyme.8 Several hydroxybenzoic acid derivatives have been found to possessed tyrosinase inhibitory activities in our laboratory.9 On the other hand, many researchers have shown that the antioxidants of natural or synthetic origin could have a great importance as therapeutic agents in several diseases, caused by oxidative stress.10 Moreover, applications of antioxidants such as preservatives in the food industry and skin-protective source in cosmetics have also attracted much interest.11 Bergenin and the corresponding tri-O-methylnorbergenin (8,10-di-O-methyl derivative) were isolated from various plants,12 and exhibit biological properties, such as antioxidant,13 anti-inflammatory,14 anti-arthritis,15 hypolipidemic,16 antiarrhythmic,17 hepatoprotective,18 antinociceptive,19 and anti-HIV effects.20 Bergenin and its analogues are characterized by a b-D-glucosyl residue C-linked to a hydroxylated or methoxylated phenylcarboxylic acid ortho to the carboxyl group, then, the carboxyl group is esterified with the C2 hydroxyl group of the glucosyl moiety to form a d-lactone bridge. Furthermore, its esterified analogues occur widely in some plants, especially those used in traditional medicines.21 Our investigation demonstrated that bergenin esterified compounds exhibited potent inhibitory activities against tyrosinase and b-secretase.22 However, although the interesting biological activities of this class of natural products, the investigation for bergenin analogues, in particular tri-O-methylnorbergenin analogues, and their biological effect are unsatisfying. Stimulated by these results, in the present investigation, we synthesized a
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series of tri-O-methylnorbergenin analogues and identify their structural importance for the inhibition of tyrosinase23 and antioxidant activity.24 The synthesis pathways are described Scheme 1.25,26 The antioxidative properties of tri-O-methylnorbergenin analogues 1–9,27 tri-O-methylnorbergenin,28 and bergenin against peroxyl radicals were investigated and compared with that of dibutylhydroxytoluene (BHT). As shown on Table 1, some analogues including 2–4, 6, 7, and 9 showed a more active than BHT. Especially, compound 4, which contains catechol moiety, showed significantly enhanced antioxidant activity. Moreover, among benzoic acid derivatives 1a–9a, the ORAC value (lmol of TE/lmol) of compound 4a was high. Since compounds 4 and 4a had relatively potent the activities compared to other compounds, catechol moiety affects the antioxidant activity. Furthermore, although triO-methylnorbergenin did not exhibit the activity, compound 4 had higher activity than 4a. Consequently, tri-O-methylnorbergenin moiety was a potent factor of antioxidant activity. Among triO-methylnorbergenin analogues, compounds 4, 6, and 7 had high ORAC values relatively; therefore, the presence of the substituted group (OH or OMe) at 30 and 40 -positions on the analogues was a great influence on the activities. Next, we evaluated tyrosinase inhibition activity of tri-Omethylnorbergenin analogues, tri-O-methylnorbergenin, and bergenin. From the regression curves of the analogues
OH
11 2
OH O H 10b 10
MeO HO
8
7
3
4a
6
OH
OH
O
H
MeO
OH
OH
OMe O H
a
4
10a
9
concentration against percentage of control activity, IC50 values of each analogue were evaluated and presented in Table 2. They revealed from low to high tyrosinase inhibitory activity. Among the analogues tested, compound 4 showed the most potent inhibitory activity (IC50 = 9.1 lM). Similarly, compounds 2 and 7 exhibited IC50 values of 31.5 and 38.8 lM, respectively and were more potent than arbutin and kojic acid as positive controls (Fig. 1). Several other compounds inhibited tyrosinase to lesser extent, these included compounds 1, 3, and 8 with IC50 values of more than 100 lM. All other compounds including tri-O-methylnorbergenin and bergenin, whereas, exhibited no inhibitions against tyrosinase (Table 2). Further investigations were undertaken to examine the inhibition mechanism of compounds 2, 4, and 7 exhibiting high inhibitory potency against tyrosinase activity. For the type of enzyme inhibition and the inhibition constant for an enzyme-inhibitor complex, the mechanisms were analysed by Dixon plot, which is a graphical method [plot of 1/enzyme velocity (1/V) versus inhibitor concentration (I) with varying concentrations of the substrate] as shown in Figure 2. The Dixon plots were constructed using three different substrate concentrations 1.5, 0.75, 0.375 mM. Each three straight lines representing the uninhibited enzyme and different concentrations of compound 2 and 4 intersected in a single point at the second quadrant (Panels A in Fig. 2). From the results, the inhibition constants were
O
MeO
O
H
OH
O
+
R1
OH
R2 R3
O
R1, R 2, R 3 = H, OH, OMe
R1 3' 2'
7'
2
OMe O H 10b
4
MeO
8
1: 2: 3: 5: 6: 7: 8: 9:
4a
10a 6 7
OH
3
10 9
R3
6'
O
11
MeO
1a-3a, 5a-9a
R2
5'
O
b
4'
O
H
OH
O
R1 =H, R 2=H, R 3=H R1 =H, R 2=OH, R 3=H R1 =H, R 2=OMe, R 3=H R1 =OMe, R 2=OMe, R 3=H R1 =OMe, R 2=OH, R 3=H R1 =OH, R 2=OMe, R 3=OMe R1 =OMe, R 2=H, R 3=OMe R1 =OMe, R 2=OH, R 3=OMe
OH OH O
OH
MeO
OMe O H O
MeO O
O
OH
H
OH
O
+
b OH
c
MeO
O
MeO
BnO OBn
OH
OMe O H H
OH
O 4
Scheme 1. Reagents and conditions: (a) MeI, K2CO3, DMF, rt; (b) DIAD, P(Ph)3, THF, rt; (c) H2, Pd/C, EtOAc, rt.
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Table 1 Antioxidant activity of bergenin derivatives by ORAC assay Compound tested
Trolox-equiv
Compound tested
Trolox-equiv
1 2 3 4 5 6 7 8 9 Tri-O-methylnorbergenin Bergenin
0.05 ± 0.01 0.44 ± 0.03 0.17 ± 0.06 3.11 ± 0.12 0.09 ± 0.01 2.53 ± 0.21 1.92 ± 0.17 0.09 ± 0.01 1.46 ± 0.12 0.02 ± 0.01 1.69 ± 0.11
1a 2a 3a 4a 5a 6a 7a 8a 9a
0.05 ± 0.02 1.84 ± 0.88 0.02 ± 0.01 2.24 ± 0.32 0.02 ± 0.01 1.17 ± 0.09 1.17 ± 0.08 0.10 ± 0.01 1.22 ± 0.11
BHT
0.12 ± 0.01
Results are presented as the mean (n = 3) ± standard deviation (SD).
Table 2 Inhibitory effects of tri-O-methylnorbergenin analogues and benzoic acid analogues against tyrosinase IC50a (lM)
Compound tested 1 2 3 4 5 6 7 8 9 Tri-O-methylnorbergenin Bergenin a b c
b
>100 (7.9%) 31.5 ± 0.1 >100 (7.9%) 9.1 ± 0.04 >100 (0.4%) >100 (0.7%) 38.8 ± 0.9 >100 (16.0%) >100 (0.6%) >100 (1.9%) >100 (0.6%)
Compound tested
IC50a (lM)
1a 2a 3a 4a 5a 6a 7a 8a 9a Arbutinc Kojic acidc
>100 (12.7%) >100 (13.2%) >100 (12.4%) >100 (7.9%) >100 (3.2%) >100 (2.4%) >100 (1.1%) >100 (3.6%) >100 (2.4%) 194.5 ± 11.6 46.6 ± 3.9
The results are the means ± SE of three experiments. Inhibition (%) at 100 lM. Arbutin and Kojic acid were used as the positive control.
100
2 4 7
Tyrosinase activity (%)
80
arbutin kojic acid
60
40
20
0 0
50
100
150
200
250
Concentration (µM) Figure 1. Dose-dependent inhibitory effects of compounds 2, 4, and 7 on tyrosinase activity. Tyrosinase activity was measured using L-tyrosine as the substrate. Values are mean ± SE of n = 3 determinations.
determined to be Ki values of compounds 2 and 4 with 74.8 and 4.5 lM, respectively. The Dixon plot is useful method to determine the inhibition mechanism of tri-O-methylnorbergenin analogues; nevertheless this method is limited by the fact that it does not discriminate unambiguously between competitive and mixed types. Moreover, for mixed or uncompetitive inhibitors, the Dixon plots exhibit no measure of Ki0 , the dissociation constant of the enzyme-substrate-inhibitor complex. Therefore, for characterizing the types of inhibition, the Cornish–Bowden plots, which is complementary to Dixon plots, were undertaken with Dixon plots. Figure 2 suggested that compounds 2 and 4 were mixed inhibition because the intersection is above the xaxis in Dixon plots and below the x-axis in Cornish–Bowden plot. In other words, the dissociation constants of enzyme-inhibitor complex (Ki) for these compounds were not equal to Ki0 . On the other hand, compound 7 was typical uncompetitive inhibition by Figure 2 with Ki0 values of 107.4 lM (Table 3). From the structural–activity point of view, compound 1, which is fundamental skeleton lacking substitutions on the benzene ring of benzoic moiety, showed low inhibitory effect (7.9%). The activities of compounds 2 and 3, with an OH or OMe functional group at the 40 -position, showed more inhibition effect relative to compound 1, however, the introduction of another OMe group at the 30 -position (compounds 5 and 6) seems to have no influence. On the other hand, compounds 4 and 7, with an additional OH group at the 30 -position of compounds 2 and 3, exhibited significant increased inhibition. These results showed that functional substitutions on the benzoic acid moiety selectively enhance or decrease inhibition of tyrosinase activity, and the introduction an OH group at the 30 -position as well as the 40 -position seems to be critical factor for the inhibitory activity. Especially, the catechol moiety of compound 4 might play an important role for the activity. The result was consistent with our previous investigation that the catechol moiety of 11-O-protocatechuoylbergenin (8,10-di-hydroxy derivative) exhibited increase inhibition on tyrosinase.29 Comparison of the inhibitory activities of compound 4 and 11-O-protocatechuoylbergenin showed that the potency increased in the order of 4 >11-O-protocatechuoylbergenin (IC50 values: 9.1 vs 17.5 lM). This observation revealed that the presence of two OMe groups at 8 and 10positions led to significantly higher activity than the presence of the OH groups. Furthermore, we evaluated the inhibitory activities of benzoic acid analogues (1a–9a) and tri-O-methylnorbergenin. These compounds exhibited relatively poor inhibitory activities; therefore, we considered that the ester linkage of ‘triO-methylnorbergenin moiety’ and ‘benzoic acid moiety’ is essential for the tyrosinase inhibitory activity. In conclusion, we synthesized a series of tri-O-methylnorbergenin analogues and evaluated their antioxidant properties and tyrosinase inhibitory activities. Compound 4, 6, and 7 containing the functional group (OH or OMe) at 30 and 40 -positions showed potent antioxidant activities in ORAC assay and surpassed positive controls. In the structure-tyrosinase inhibition activity study, some of the analogues indicated tyrosinase inhibitory activities in the micromolar range. The presence of catechol moiety of compound 4 is important for higher inhibitory activity in the tyrosinase assay. The results of the structure–activity relationship revealed that the ester linkage as well as tri-O-methylnorbergenin moiety and benzoic acid moiety affect the two activities. These results suggest that these compounds may serve as structural templates for the design and development of novel tyrosinase inhibitors.
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500
(2-A)
350
(2-B)
400
S/V
1/V
250 300
150
200 100
-100
-50
50 0
50
100
150
-150
-100
-50
50
100
150
-50
[I, µM]
[I, µM]
600
(4-A)
(4-B)
450
S/V
1/V
500 400
350
300
250
200
150
100
50 -10
-5
0
[I, µM]
5
10
15
-10
400
(7-A)
-5
-50
[I, µM]
5
10
15
300
(7-B)
S/V
1/V
300
200
200
100 100
-120
-60
0
60
120
-120
-60
[I, µM]
0
60
120
[I, µM]
Figure 2. Dixon plots (panels A) and Cornish–Bowden plots (panels B) for inhibition of 2, 4, and 7 on tyrosinase activity. The substrate used were 1.5 (), 0.75 (j), and 0.375 mM (N). Table 3 Kinetic parameters for inhibition tyrosinase activity by compounds 2, 4, and 7 Compounds
Ki (lM)
Ki0 (lM)
Type of inhibition
2 4 7
74.8 4.5 —
120.9 7.3 107.4
Mixed Mixed Uncompetitive
Acknowledgment This work was supported by Grant-in Aid from the Japan Society for the Promotion of Science (No. 24658055).
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Method of tyrosinase assay: The tyrosinase assay was performed using Ltyrrosine as the substrate. 100 lL of 0.1 M phosphate buffer (pH 7.0), 36 lL of 1.5 mM L-tyrosine, and 10 lL of sample solution containing DMSO+0.05% TweenÒ 20 to dissolve the sample were added to each well of a 96-well plate and then incubated at 37 °C for 10 min. Then, 16 lL of mushroom tyrosinase (500 units/mL, 0.1 M phosphate buffer at pH 7.0) was added, and the assay mixture was incubated at 37 °C for 20 min. Before and after incubation, the amount of dopachrome produced in the reaction mixture was measured at 492 nm in a microplate reader (Corona Electric Co. Ltd). Arbutin and kojic acid were used as a positive control. The extent of tyrosinase inhibition by the different compounds added was calculated and expressed as the percentage necessary for 50% inhibition concentration (IC50). The percentage of tyrosinase activity was calculated as follows: Tyrosinase activity (%) = [(CD)/ (AB)] 100, where A is the absorbance at 492 nm without test sample, B is the absorbance at 492 nm without test sample and substrate, C is the absorbance at 492 nm with test sample, D is the absorbance at 492 nm with test sample, but without substrate. All data are the mean of three experiments. 24. The ORAC assay was carried out on a microplate reader (Corona Electric Co. Ltd) with fluorescence filters for an excitation wavelength of 480 nm and emission wavelength of 540 nm. The measurements were made in aplate with 96 black flat-bottom wells. The samples were dissolved in 50% acetone and incubated for 1 h at room temperature. The reaction was performed in 75 mM phosphate buffer (pH 7.4) and the final assay mixture (200 lL) contains fluorescein (160 lL, 63 nM final concentration) as an oxidizable substrate, AAPH (20 lL, 12.8 mM final concentration), and trolox (20 lL, 5–20 lM final concentration) or samples (20 lL, 5–20 lM final concentration). The reaction was performed at 37 °C, and fluorescence was recorded every minute for 120 min. A blank using phosphate buffer instead of the sample was carried out in each experiment. BHT was used as a positive control. The ORAC value was calculated as follows: ORAC value = [CTrolox (AUCSampleAUCBlank)]/ [CSample (AUCTroloxAUCBlank)], where CTrolox is the concentration of trolox, CSample is the concentration of the sample, and AUC is the area below the fluorescence decay curve of the sample, blank and trolox, respectively, calculated as follows: AUC = 1 + f1/f0 + f2/f0 + f3/f0. . . + fn/f0, where f0 is the initial fluorescence and fn is the fluorescence at time n. All data are the mean of three experiments. 25. General procedure for the synthesis of tri-O-methylnorbergenin analogues (1– 3, 5–9): Methylation of bergenin with iodomethane (5 equiv) in the presence of potassium carbonate (5 equiv) in DMF produced tri-O-methylnorbergenin (97% yield). To a solution of tri-O-methylnorbergenin in dry THF (2 mL/mmol), corresponding benzoic acids (1 equiv) and Ph3P (2 equiv) were dissolved. DIAD (1.7 equiv) was added the solution under 0 °C. The mixture was stirred for 3 h on rt under nitrogen and monitored by TLC. It was concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/EtOAc, 2:8) to afford the analogue as a white power (72–93% total yield). 26. General procedure for the synthesis of 11-O-protocatechuoyl-tri-Omethylnorbergenin (4): The procedure mentioned ref 25 was carried out using 3,4-bis(benzyloxy)benzoic acid (1 equiv) to afford monoester (77% yield). Further, the monoester was debenzylated-using Pd-C/H2 in EtOAc. The reaction mixture was filtered, concentrated, and the residue was purified by column chromatography (CH2Cl2/MeOH, 4:1) to afford pure compound 4 (74% yield). 27. 11-O-Benzoyl-tri-O-methylnorbergenin (1): This compound was obtained with a yield of 76%; amorphous powder; mp: 202.7 °C; IR (KBr) 3478, 1714, 1593 cm1; 1H NMR (400 MHz, DMSO-d6) d 8.02 (2H, m, H-20 , 60 ), 7.69 (1H, m, H-40 ), 7.56 (2H, m, 30 , 50 ), 7.35 (1H, s, H-7), 4.94 (1H, d, J = 10.4 Hz, H-10b), 4.68 (1H, d, J = 12.0 Hz, H-11b), 4.40 (1H, dd, J = 12.0, 6.4 Hz, H-11a), 4.02 (1H, dd, J = 10.4, 9.6 Hz, H-4a), 3.83 (1H, m, H-2), 3.73 (1H, m, H-4), 3.43 (1H, m, H3), 3.85, 3.80, 3.63 (9H, 3s, H-8, 9, 10-OMe). 13C NMR (100 MHz, DMSO-d6) d
165.7 163.5, 153.0, 150.8, 147.8, 133.4, 129.7, 129.2, 128.8, 126.2, 119.0, 109.2, 80.0, 78.2, 73.6, 71.1, 70.4, 64.5, 61.0, 60.6, 56.0; MS (ESI): m/z 459 [MH]. 11-O-p-Hydroxybenzoyl-tri-O-methylnorbergenin (2): This compound was obtained with a yield of 84%; amorphous powder; mp: 235.7 °C; IR (KBr) 3362, 1726, 1592 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.86 (2H, d, J = 8.8 Hz, H-20 , 60 ), 7.35 (1H, s, H-7), 6.87 (2H, d, J = 8.8 Hz, H-30 , 50 ), 4.93 (1H, d, J = 10.0 Hz, H-10b), 4.62 (1H, d, J = 12.0 Hz, H-11b), 4.32 (1H, dd, J = 12.0, 6.4 Hz, H-11a), 4.01 (1H, dd, J = 10.4, 9.6 Hz, H-4a), 3.83 (1H, m, H-2), 3.75 (1H, t, J = 8.8 Hz, H-4), 3.46 (1H, m, H-3), 3.86, 3.80, 3.64 (9H, 3s, H-8, 9, 10-OMe). 13 C NMR (100 MHz, DMSO-d6) d 165.5, 163.5, 162.1, 153.0, 150.9, 147.8, 131.5, 126.2, 120.2, 119.0, 115.4, 109.2, 80.0, 78.4, 73.6, 71.1, 70.5, 64.1, 61.0, 60.6, 56.0; MS (ESI): m/z 475 [MH]. 11-O-p-Methoxybenzoyl-tri-O-methylnorbergeninamorphous (3): This compound was obtained with a yield of 84%; mp: 210.8 °C; IR (KBr) 3421, 1730, 1609 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.96 (2H, d, J = 8.0 Hz, H-20 , 60 ), 7.35 (1H, s, H-7), 7.08 (2H, d, J = 8.0 Hz, H-30 , 50 ), 4.94 (1H, d, J = 10.4 Hz, H10b), 4.64 (1H, d, J = 11.6 Hz, H-11b), 4.34 (1H, dd, J = 11.6, 6.3 Hz, H-11a), 4.01 (1H, dd, J = 10.4, 9.6 Hz, H-4a), 3.85 (1H, m, H-2), 3.75 (1H, m, H-4), 3.46 (1H, m, H-3), 3.86, 3.80, 3.64 (9H, 3s, H-8, 9, 10-OMe). 13C NMR (100 MHz, DMSOd6) d 165.4, 163.5, 163.2, 153.0, 150.9, 147.8, 131.3, 126.2, 121.8, 119.1, 114.1, 109.2, 80.0, 78.3, 73.6, 71.1, 70.5, 64.3, 61.0, 60.7, 56.0, 55.5; MS (ESI): m/z 489 [MH]. 11-O-Protocatechuoyl-tri-O-methylnorbergenin (4): This compound was obtained with a yield of 74%; mp: 230.7 °C; IR (KBr) 3407, 1716, 1608 cm1; 1 H NMR (400 MHz, DMSO-d6) d 7.40 (1H, s, H-20 ), 7.39 (1H, d, J = 8.4 Hz H-60 ), 7.35 (1H, s, H-7), 6.83 (1H, d, J = 8.4 Hz, H-50 ), 4.93 (1H, d, J = 10.0 Hz, H-10b), 4.58 (1H, d, J = 11.6 Hz, H-11b), 4.30 (1H, dd, J = 11.6, 6.4 Hz, H-11a), 4.00 (1H, dd, J = 10.0, 9.6 Hz, H-4a), 3.85 (1H, m, H-2), 3.75 (1H, dd, J = 9.6, 8.8 Hz, H-4), 3.37 (1H, d, 9.2, 8.8 Hz, H-3), 3.86, 3.80, 3.65 (9H, 3s, H-8, 9, 10-OMe). 13C NMR (100 MHz, DMSO-d6) d 165.7, 163.5, 153.0, 150.9, 150.8, 147.8, 145.2, 126.3, 121.9, 120.3, 119.1, 116.3, 115.3, 109.2, 80.0, 78.4, 73.6, 71.1, 70.5, 64.0, 61.0, 60.7, 56.1; MS (ESI): m/z 491 [MH]. 11-O-(30 ,40 -Dimethoxybenzoyl)-tri-O-methylnorbergeninmorphous (5): This compound was obtained with a yield of 72%; mp: 158.2 °C; IR (KBr) 3411, 1715, 1596 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.65 (1H, dd, J = 8.4, 2.0 Hz, H-60 ), 7.50 (1H, d, J = 2.0 Hz, H-20 ), 7.35 (1H, s, H-7), 7.11 (1H, d, J = 8.4 Hz, H50 ), 4.94 (1H, d, J = 10.4 Hz, H-10b), 4.69 (1H, d, J = 12.0 Hz, H-11b), 4.31 (1H, dd, J = 12.0, 7.2 Hz, H-11a), 4.01 (1H, dd, J = 10.4, 10.0 Hz, H-4a), 3.85 (1H, m, H2), 3.75 (1H, m, H-4), 3.38 (1H, m, H-3), 3.86, 3.80, 3.65 (9H, 3s, H-8, 9, 10OMe), 3.85 (3H, s, 40 -OMe), 3.82 (3H, s, 30 -OMe). 13C NMR (100 MHz, DMSO-d6) d 164.4, 163.5, 153.1, 153.0, 150.9, 148.4, 147.8, 126.2, 123.2, 121.8, 119.1, 111.8, 111.2, 109.2, 80.0, 78.4, 73.6, 71.1, 70.7, 64.5, 61.0, 60.6, 56.0, 55.7, 55.5; MS (ESI): m/z 519 [MH]. 11-O-Vanilloyl-tri-O-methylnorbergenin (6): This compound was obtained with a yield of 89%; mp: 162.9 °C; IR (KBr) 3224, 1706, 1592 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.53 (1H, d, J = 8.0 Hz, H-60 ), 7.50 (1H, s, H-20 ), 7.35 (1H, s, H-7), 6.90 (1H, d, J = 8.0 Hz, H-50 ), 4.94 (1H, d, J = 10.0 Hz, H-10b), 4.66 (1H, d, J = 12.0 Hz, H-11b), 4.29 (1H, dd, J = 12.0, 7.2 Hz, H-11a), 4.01 (1H, dd, J = 10.0, 9.6 Hz, H-4a), 3.85 (1H, m, H-2), 3.75 (1H, m, H-4), 3.38 (1H, m, H-3), 3.86, 3.80, 3.65 (9H, 3s, H-8, 9, 10-OMe), 3.83 (3H, s, 30 -OMe). 13C NMR (100 MHz, DMSOd6) d 165.5, 163.5, 153.0, 151.7, 150.9, 147.8, 147.4, 126.2, 123.5, 120.5, 119.1, 115.2, 112.6, 109.2, 80.0, 78.4, 73.7, 71.1, 70.6, 64.3, 61.0, 60.6, 56.0, 55.6; MS (ESI): m/z 505 [MH]. 11-O-Isovanilloyl-tri-O-methylnorbergenin (7): This compound was obtained with a yield of 77%; mp: 164.4 °C; IR (KBr) 3420, 1715, 1609 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.49 (1H, d, J = 8.8 Hz, H-60 ), 7.42 (1H, s, H-20 ), 7.35 (1H, s, H-7), 7.05 (1H, d, J = 8.8 Hz, H-50 ), 4.94 (1H, d, J = 10.0 Hz, H-10b), 4.62 (1H, d, J = 12.0 Hz, H-11b), 4.32 (1H, dd, J = 12.0, 6.4 Hz, H-11a), 4.00 (1H, dd, J = 10.0, 9.2 Hz, H-4a), 3.85 (1H, m, H-2), 3.75 (1H, m, H-4), 3.37 (1H, m, H-3), 3.86, 3.80, 3.65 (9H, 3s, H-8, 9, 10-OMe), 3.84 (3H, s, 40 -OMe). 13C NMR (100 MHz, DMSOd6) d 165.6, 163.5, 153.0, 152.0, 150.9, 147.8, 146.3, 126.2, 121.9, 121.6, 119.1, 115.8, 111.5, 109.2, 80.0, 78.3, 73.6, 71.1, 70.5, 64.2, 61.0, 60.7, 56.0, 55.7; MS (ESI): m/z 505 [MH]. 11-O-(30 ,50 -Dimethoxybenzoyl)-tri-O-methylnorbergenin (8): This compound was obtained with a yield of 89%; mp: 179.1 °C; IR (KBr) 3446, 1719, 1596 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.35 (1H, s, H-7), 7.12 (2H, d, J = 2.4 Hz, H-20 , 60 ), 6.81 (1H, d, J = 2.4 Hz, H-40 ), 4.94 (1H, d, J = 10.4 Hz, H-10b), 4.71 (1H, d, J = 12.0 Hz, H-11b), 4.33 (1H, dd, J = 12.0, 6.8 Hz, H-11a), 4.01 (1H, dd, J = 10.4, 9.6 Hz, H-4a), 3.83 (1H, m, H-2), 3.75 (1H, m, H-4), 3.38 (1H, m, H3), 3.86, 3.80, 3.65 (9H, 3s, H-8, 9, 10-OMe), 3.81 (6H, s, 30 , 50 -OMe). 13C NMR (100 MHz, DMSO-d6) d 165.3, 163.5, 160.5, 153.0, 151.0, 147.8, 131.6, 126.1, 119.1, 109.2, 107.0, 105.1, 80.0, 78.2, 73.6, 71.1, 70.6, 64.8, 61.0, 60.6, 56.0, 55.5; MS (ESI): m/z 519 [MH]. 11-O-Syringyl-tri-O-methylnorbergenin (9): This compound was obtained with a yield of 77%; mp: 226.5 °C; IR (KBr) 3379, 1718, 1618 cm1; 1H NMR (400 MHz, DMSO-d6) d 7.33 (1H, s, H-7), 7.26 (2H, s, H-20 , 60 ), 4.93 (1H, d, J = 9.6 Hz, H10b), 4.71 (1H, d, J = 11.6 Hz, H-11b), 4.23 (1H, dd, J = 12.0, 6.4 Hz, H-11a), 4.00 (1H, t, J = 9.6 Hz, H-4a), 3.83 (1H, m, H-2), 3.73 (1H, m, H-4), 3.37 (1H, m, H-3), 3.84, 3.80, 3.77, 3.64 (15H, 4s, H-8, 9, 10, 30 , 50 -OMe). 13C NMR (100 MHz, DMSO-d6) d 165.6, 163.5, 153.0, 151.0, 147.8, 147.6, 141.0, 126.1, 119.1, 109.2, 107.0, 80.0, 78.4, 73.7, 71.1, 70.8, 64.0, 61.1, 60.6, 56.0; MS (ESI): m/z 535 [MH]. 28. Sakamaki, S.; Kawanishi, E.; Nomura, S.; Ishikawa, T. Tetrahedron 2012, 68, 5744. 29. Kashima, Y.; Miyazawa, M. Arch. Pharmacal Res. 2012, 35, 1533.
