Artopithecins A–D, Prenylated 2-Arylbenzofurans from the Twigs of Artocarpus pithecogallus and Their Tyrosinase Inhibitory Activities

Abstract

Four new prenylated 2-arylbenzofurans, namely artopithecins A–D (1–4), together with five known compounds (5–9) were isolated from the twigs of Artocarpus pithecogallus for the first time. Their structures were elucidated based on extensive spectroscopic analysis and in comparison with literature data. All isolates were evaluated for their inhibitory activities against mushroom tyrosinase. Compounds 3 and 4 displayed significant tyrosinase inhibitory activities with IC₅₀ values of 37.09±0.33 and 38.14±0.21 µM, respectively.

Tyrosinase is a key metal enzyme implicated in mammalian melanin biosynthesis, in which melanin protects the skin from photodamage by absorbing UV rays and removing reactive oxygen species.¹⁾ Tyrosinase inhibitors, therefore, can be clinically useful for the treatment of some dermatological disorders associated with melanin hyperpigmentation.²⁾ Usually they were also applicable in cosmetics for whitening and depigmentation after sunburn as well as in food preservation by preventing the undesirable browning reactions.³⁾ Moreover, tyrosinase inhibitors may offer a potential treatment for Parkinson’s disease.⁴⁾ Thus, the development of new tyrosinase inhibitors with high-performance is urgent needed in medicinal, cosmetic, and food products.⁵⁾ Recent studies on plants of the Artocarpus genus (Moraceae) revealed the presence of a number of prenylated phenolics possessing potent tyrosinase inhibitory activity.^6–8) As part of our ongoing efforts to discover novel potential leads from Moraceae plants,^9,10) Artocarpus pithecogallus C. Y. WU, an evergreen tree distributed in the southern part of Yunnan province, P. R. China, has been chemically investigated for the first time. Herein, we describe the structural elucidation of four new prenylated 2-arylbenzofurans as well as the tyrosinase inhibitory activity of all isolated compounds.

Results and Discussion

The air-dried powder of twigs of A. pithecogallus was extracted with 95% EtOH three times at room temperature. The EtOAc-soluble fraction of the EtOH extract was purified using various column chromatographies to afford nine prenylated phenolics (1–9) (Fig. 1). The known compounds were identified as moracin I (5),¹¹⁾ 6-prenylapigenin (6),¹²⁾ artocarpesin (7),¹²⁾ artocarpusin C (8),¹³⁾ morachalcone A (9)¹⁴⁾ by analysis of their spectroscopic data and comparison with literature data.

Fig. 1. Chemical Constituents Isolated from the Twigs of A. pithecogallus

Artopithecin A (1) was obtained as a yellowish amorphous solid. Its molecular formula, C₂₀H₂₀O₄, was deduced from positive high resolution-electrospray ionization (HR-ESI)-MS at m/z 325.1430 [M+H]⁺ (Calcd for C₂₀H₂₁O₄, 325.1434). The IR spectrum showed the typical absorption bands for hydroxy and benzene ring functionalities. The ¹H-NMR spectrum of 1 (Table 1) clearly displayed one set of ABX aromatic protons at 7.43 (1H, d, J=8.4 Hz, H-4), 6.86 (1H, dd, J=8.4, 2.4 Hz, H-5), and 7.07 (1H, d, J=2.4 Hz, H-7), one downfield singlet at δ_H 6.71 (1H, s, H-3), and two meta-coupled protons [δ_H 6.63 and 6.36 (each 1H, d, J=2.4 Hz, H-6′, H-4′)], together with one methoxy signal at δ_H 3.85 (3H, s), and a prenyl group [δ_H 3.44 (2H, d, J=6.4 Hz, H-1″), 5.14 (1H, t, J=6.4 Hz, H-2″), and 1.65 (6H, s, H-4″, H-5″)]. The ¹³C-NMR (Table 1) and heteronuclear single-quantum coherence (HSQC) spectra showed 20 carbon signals consisting of nine sp² quaternary carbons (including five oxygenated ones), seven sp² methines, one methylene, two methyls, and one methoxy. Analysis of the one dimensional (1D)-NMR spectra indicated 1 was a methyl ether of demethylmoracin I.¹⁵⁾ Heteronuclear multiple-bond coherence (HMBC) correlations from the methoxy protons [δ_H 3.85 (3H, s)] to C-6 (δ_C 159.5) easily assigned the methoxy group at C-6 (Fig. 2), which was confirmed by nuclear Overhauser effect spectroscopy (ROESY) correlations of H₃ (δ_H 3.85, 3H, s) with H-5 (δ_H 6.86, dd, J=8.4, 2.4 Hz) and H-7 (δ_H 7.07, d, J=2.4 Hz). The prenyl group was located at C-2′ by HMBC correlations from H₂-1″ to C-1′ (δ_C 132.9), C-3′ (δ_C 157.9), and C-2′ (δ_C 119.3) (Fig. 2). Thus, the structure of 1 was fully elucidated as shown in Fig. 1.

Table 1. ¹H (400 MHz) and ¹³C (100 MHz) NMR Data for Compounds 1–4 in CD₃OD (δ in ppm, J in Hz)

No.	1		2		3		4
	δH	δC	δH	δC	δH	δC	δH	δC
2		156.9		154.7		154.1		153.3
3	6.71, s, 1H	105.5	6.78, s, 1H	103.0	6.82, s, 1H	102.6	6.50, s, 1H	104.3
3a		123.7		113.6		113.9		113.3
4	7.43, d (8.4), 1H	121.9		152.2		152.1		151.9
5	6.86, dd (8.4, 2.4), 1H	112.8	6.25, d (2.0), 1H	98.0	6.21, d (1.6), 1H	100.1	6.26, d (2.0), 1H	97.8
6		159.5		160.5		160.4		160.0
7	7.07, d (2.4), 1H	96.4	6.59, d (2.0), 1H	88.4	6.51, d (1.6), 1H	88.1	6.56, d (2.0), 1H	88.6
7a		156.7		158.1		158.0		158.3
1′		132.9		133.0		132.9		133.3
2′		119.3		119.1		120.5		121.7
3′		157.9		157.9		160.3		154.6
4′	6.36, d (2.4), 1H	103.8	6.34, d (2.4), 1H	103.6	6.43, d (2.0), 1H	103.4	6.45, s, 1H	104.8
5′		156.9		156.8		157.4		154.6
6′	6.63, d (2.4), 1H	107.7	6.62, d (2.4), 1H	107.6	6.70, d (2.0), 1H	108.1		121.7
1″	3.44, d (6.4), 2H	26.5	3.45, d (6.4), 2H	26.9	3.45, d (6.4), 2H	26.5	3.08, d (6.8), 2H	27.3
2″	5.14, t (6.4), 1H	125.6	5.14, t (6.4), 1H	125.5	5.11, t (6.4), 1H	125.3	5.06, t (6.8), 1H	125.5
3″		131.4		131.5		131.5		130.3
4″	1.65, s, 3H	25.9	1.67, s, 3H	25.9	1.68, s, 3H	25.9	1.55, s, 3H	25.9
5″	1.65, s, 3H	18.1	1.67, s, 3H	18.1	1.66, s, 3H	18.1	1.33, s, 3H	17.8
1‴							3.08, d (6.8), 2H	27.3
2‴							5.06, t (6.8), 1H	125.5
3‴								130.3
4‴							1.55, s, 3H	25.9
5‴							1.33, s, 3H	17.8
6-OMe	3.85, s, 3H	56.1	3.80, s, 3H	56.0	3.81, s, 3H	56.1	3.80, s, 3H	56.1
3′-OMe					3.79, s, 3H	56.0

Fig. 2. Key HMBC (H→C) and NOE (HH) Correlations of 1–4

Artopithecin B (2) was isolated as a tawny amorphous solid. Its ¹H-NMR spectrum (Table 1) was very similar to those of 1 except that the aromatic ABX spin-coupled system in 1 was replaced by a meta-coupled spin system [δ_H 6.25 and 6.59 (each 1H, d, J=2.0 Hz, H-5, H-7)] in 2. The molecular formula, C₂₀H₂₀O₅, deduced from the positive HR-ESI-MS ion peak at m/z 341.1391 [M+H]⁺ (Calcd for C₂₀H₂₁O₅, 341.1389), further indicated a hydroxy group linked to C-4, which was confirmed by the downfield shift of C-4 (δ_C 152.2) and the upfield shifts of C-3a (δ_C 113.6), C-5 (δ_C 98.0), and C-7 (δ_C 88.4), respectively (Table 1). HMBC correlation from H₃ (δ_H 3.80, s) to C-6 (δ_C 160.5) together with ROESY correlations of H₃ (δ_H 3.80, s) with H-5 (δ_H 6.25, d, J=2.0 Hz) and H-7 (δ_H 6.59, d, J=2.0 Hz) indicated the location of the methoxy at C-6 (Fig. 2). The prenyl group was located at C-2′ by HMBC correlations from H₂-1″ to C-1′ (δ_C 133.0), C-2′ (δ_C 119.1), and C-3′ (δ_C 157.9) (Fig. 2). Finally, the structure of 2 was unambiguouslly assigned as depicted by HSQC and HMBC spectra (Fig. 2).

The ¹H- and ¹³C-NMR data (Table 1) of artopithecin C (3) highly resembled those of 2 except for the presence of one extra methoxy signal. This was consistent with the molecular formula C₂₁H₂₂O₅ as determined by HR-ESI-MS at m/z 355.1542 [M+H]⁺ (Calcd 355.1545). The HMBC correlations from H₃ (δ_H 3.81, s) to C-6 and H₃ (δ_H 3.79, s) to C-3′ assigned these two methoxy groups at C-6 and C-3′ successively (Fig. 2), which was also corroborated by ROESY correlations (Fig. 2). The linkage between the prenyl with C-2′ was verified by HMBC correlations from H₂-1″ to C-1′ (δ_C 132.9), C-2′ (δ_C 120.5), and C-3′ (δ_C 160.3) (Fig. 2). Thus, the structure of 3 was proposed as drawn by using two dimensional (2D)-NMR techniques.

Artopithecin D (4) exhibited a molecular formula of C₂₅H₂₈O₅ by HR-ESI-MS at m/z 409.2017 [M+H]⁺ (Calcd for C₂₅H₂₉O₅, 409.2015). The ¹H-NMR spectrum (Table 1) also displayed distinctive prenylated 2-arylbenzofuran signals just like 2 but the right meta-coupled spin system in 4 degenerated into one aromatic singlet. Moreover, two sets of identical proton resonances corresponding to two prenyls were also observed in the ¹H-NMR spectrum and indicated two prenyl residues symmetrically distributed at C-2′ and C-6′, which was unambiguously supported by analysis of the ¹³C-NMR, HSQC, and HMBC correlations (Fig. 2). Finally, the methoxy group was assigned to C-6 based on HMBC correlation from H₃ (δ_H 3.80, s) to C-6 (δ_C 160.0) and ROESY correlations of H₃ (δ_H 3.80, s) with H-5 (δ_H 6.26, d, J=2.0 Hz) and H-7 (δ_H 6.56, d, J=2.0 Hz), respectively (Fig. 2). Therefore, the structure of 4 was completely established as shown.

All isolates (1–9) was evaluated for the inhibitory activity against mushroom tyrosinase (Table 2). Kojic acid, a well-known tyrosinase inhibitor currently used as a cosmetic skinwhitening agent, was used as a positive control. As a result, morachalcone A (9) exhibited the most potent inhibitory effect against tyrosinase (IC₅₀=0.77±0.01 µM), which had been reported previously as a strong tyrosinase inhibitory activity.^7,16) Compound 6 showed a similar inhibitory activity (IC₅₀=24.29±0.12 µM) with the positive control. Moreover, compounds 3 and 4 exhibited a significant but slightly weaker inhibitory effect than kojic acid with IC₅₀ values of 37.09±0.33 and 38.14±0.21 µM, respectively, followed by compound 8 (IC₅₀=54.52±0.47 µM). However, compounds 1, 2, 5 and 7 showed null inhibitory activity with IC₅₀ values over 100 µM.

Table 2. Tyrosinase Inhibitory Activity of the Isolated Compounds

Compound	IC50/(µM)	Compound	IC50/(µM)a)
1	>100	6	24.29±0.12
2	>100	7	>100
3	37.09±0.33	8	54.52±0.47
4	38.14±0.21	9	0.77±0.01
5	>100	Kojic acid	17.32±0.24

a) Each value represents the mean±S.D. of three determinations.

Experimental

General Experimental Procedures

The 1D- and 2D-NMR were recorded on a Bruker AVANCE 400 NMR spectrometer with tetramethylsilane (TMS) as an internal reference. All HR-ESI-MS spectra were measured on an AB Sciex 5600 LC/MS/MS system. UV spectra were obtained on a Shimadzu UV-2550 Spectrophotometer. IR spectra were made in thin polymer films on a Shimadzu FTIR-8400 spectrometer. Silica gel (300–400 mesh, Qingdao Marine Chemical Plant, Qingdao, P. R. China), C₁₈ reversed-phase silica gel (150–200 mesh, Merck), MCI gel (CHP20P, 75–150 µM, Mitsubishi Chemical Industries Ltd., Japan), and Sephadex LH-20 gel (75–150 µM, GE Healthcare, U.S.A.) were used for column chromatography. Precoated silica gel GF254 plates (Qingdao Marine Chemical Plant) were used for TLC. Semipreparative HPLC was performed on an Agilent 1200 system equipped with a VWD G1314B detector and a Zorbax SB-C₁₈ column (250×10 mm, 5 µm). All solvents used were of analytical grade (Xilong Chemical Reagent Co., Ltd., Guangdong, P. R. China).

Plant Material

The twigs of A. pithecogallus was collected at Mengla County, Yunnan Province, P. R. China, in August 2014, and identified by Professor You-kai Xu of the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, P. R. China. A voucher specimen (AP201408) has been deposited in School of Pharmacy, Nanchang University.

Extraction and Isolation

The dry powder of twigs (5.0 kg) of A. pithecogallus was extracted with 95% EtOH three times at ambient temperature to yield a crude extract (553.0 g), which was suspended in water and then extracted with EtOAc. The EtOAc extract (246.0 g) was subjected to MCI gel column chromatography eluted with CH₃OH–H₂O (3 : 7 to 1 : 0, v/v) to give six fractions, Fr. 1–Fr. 6. Fraction 2 (4.6 g) was purified by Sephadex LH-20 (MeOH) and semi-preparative HPLC (CH₃CN–H₂O, 20 : 80) to afford 1 (t_R=58.4 min, 1.8 mg), 2 (t_R=10.4 min, 2.1 mg), 3 (t_R=48.9 min, 2.3 mg), and 9 (t_R=18.2 min, 1.4 mg). Fraction 3 (19.8 g) was purified by C₁₈ reversed-phase (RP-18) silica gel chromatography (MeOH–H₂O, 35 : 65–40 : 60) and semi-preparative HPLC (CH₃CN–H₂O, 20 : 80) to afford 4 (t_R=22.5 min, 2.8 mg), 5 (t_R=26.9 min, 2.0 mg), 6 (t_R=42.1 min, 2.2 mg), 7 (t_R=44.6 min, 1.7 mg), and 8 (t_R=54.6 min, 1.6 mg).

Artopithecin A (1)

Yellow amorphous solid; UV λ_max (MeOH) nm (log ε): 212 (4.47), 276 (4.05, sh), 308 (4.30); IR ν_max (Film) cm⁻¹: 3364, 2924, 1626, 1598, 1488, 1145; ¹H- and ¹³C-NMR data: see Table 1; (+) HR-ESI-MS m/z 325.1430 [M+H]⁺ (Calcd for C₂₀H₂₁O₄, 325.1434).

Artopithecin B (2)

Tawny amorphous solid; UV λ_max (MeOH) nm (log ε): 215 (4.48), 301 (4.32); IR ν_max (Film) cm⁻¹: 3329, 2929, 1625, 1586, 1489, 1438, 1115; ¹H- and ¹³C-NMR data: see Table 1; (+) HR-ESI-MS m/z 341.1391 [M+H]⁺ (Calcd for C₂₀H₂₁O₅, 341.1389).

Artopithecin C (3)

Tawny amorphous solid; UV λ_max (MeOH) nm (log ε): 234 (4.07), 299 (4.31); IR ν_max (Film) cm⁻¹: 3427, 2917, 1625, 1602, 1489, 1440, 1145; ¹H- and ¹³C-NMR data: see Table 1; (+) HR-ESI-MS m/z 355.1542 [M+H]⁺ (Calcd for C₂₁H₂₃O₅, 355.1545).

Artopithecin D (4)

Tawny amorphous solid; UV λ_max (MeOH) nm (log ε): 262 (4.01), 292 (4.00); IR ν_max (Film) cm⁻¹: 3377, 2969, 2917, 1618, 1602, 1491, 1154; ¹H- and ¹³C-NMR data: see Table 1; (+) HR-ESI-MS m/z 409.2017 [M+H]⁺ (Calcd for C₂₅H₂₉O₅, 409.2015).

Tyrosinase Inhibitory Assay

The tyrosinase assay was conducted as described by Zhang et al.¹⁶⁾ with minor modifications. Both the mushroom tyrosinase and L-3,4-dihydroxyphenylalanine (L-DOPA) were purchased from the Sigma-Aldrich Co. LLC, St. Louis, MO, U.S.A. All the samples were first dissolved in dimethyl sulfoxide (DMSO) and used at concentrations of 100, 50, 25, 10, 5 µg/mL. The tyrosinase inhibitory activity assay was performed in 96-well microplates with 200 µL total testing solution. The assay mixtures consists 40 µL of sample solution, 40 µL L-DOPA (2 mmol/L in 0.1 M phosphate buffer pH 6.8), and 40 µL of mushroom tyrosinase solution (2 U/mL in 0.1 M phosphate buffer pH 6.8). The assay mixture was incubated at room temperature for 30 min, and the absorbance at 475 nm was measured in triplicate with a microplate reader (Thermo Electron Corporation, CA, U.S.A.). Kojic acid, a known tyrosinase inhibitor, was used as positive control.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Nos. 21362023 and 30901855), the Natural Science Foundation of Jiangxi Province, China (Nos. 20142BAB215021 and 20151BAB205083), and the Project of Jiangxi Provincial Education Department (No. 14097).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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