Artopithecin K,a new phenolic derivative with tyrosinase inhibitory activity from Artocarpus pithecogallus

Abstract

A new phenolic derivative, artopithecin K (1), was isolated from the twigs of Artocarpus pithecogallus. Its structure was elucidated based on extensive spectroscopic analyses. Compound 1 was evaluated for the inhibitory activity against tyrosinase.

Introduction

The genus Artocarpus (Moraceae) is widely distributed in the tropic and subtropic regions of Asia. Among them, about 15 species are found to grow in China and some are usually used as folk medicines. ¹ Previous investigations reported that Artocarpus plants are rich in isoprenylated phenolics possessing diverse bioactivities such as cytotoxic,^2–5 anti-inflammatory,^6–8 antifungal, ⁹ antioxidant, ¹⁰ and tyrosinase inhibitory^11–13 activities. Artocarpus pithecogallus C. Y. Wu, an evergreen tree, is mainly distributed in the southern part of Yunnan province, P. R. China. Recently, our group has reported some prenylated 2-arylbenzofurans and flavones with tyrosinase inhibitory bioactivities isolated from the twigs of A. pithecogallus. ¹⁴ As part of our ongoing efforts to discover novel potential leads from Moraceae plants,^15,16 this plant has been chemically reinvestigated, which led to the isolation of a phenolic derivative possessing an acetal moiety. Herein, we described the isolation and structural elucidation of the new compound and its tyrosinase inhibitory activity.

A 95% aqueous ethanol extract prepared from the twigs of A. pithecogallus was subjected repeatedly to column chromatography and semi-preparative high-performance liquid chromatography (HPLC) to yield artopithecin K (1) (Figure 1). Its ¹H and ¹³C NMR data are listed in Table 1.

Figure 1.

Chemical structure of compound 1.

Table 1.

¹H NMR and ¹³C NMR Data for Compound 1 in Acetone-d₆ (δ in ppm, J in Hz).

No.	1
	δ H	δ C	heteronuclear multiple-bond coherence (HMBC)
2	5.53, 1H, s	99.5	C-4
3		192.6
4		173.8
4a		106.3
5		164.0
6	6.38, 1H, d, 2.4	96.8	C-4, C-7, C-4a
7		168.9
8	6.49, 1H, d, 2.4	105.2	C-7, C-4a, C-8a
8a		150.6
1′		121.1
2′	8.04, 1H, d, 8.4	133.4	C-4′
3′	7.00, 1H, d, 8.4	116.3	C-1′, C-4′
4′		163.5
5′	7.00, 1H, d, 8.4	116.3	C-1′, C-4′
6′	8.04, 1H, d, 8.4	133.4	C-4′

Results and discussion

Artopithecin K (1) was obtained as a pale yellowish amorphous powder. The molecular formula of 1 was determined to be C₁₅H₁₀O₇ by high resolution-electrospray ionization (HR-ESI)-MS, indicating 11 degrees of unsaturation requirement. The ¹H NMR spectrum of 1 (Figure S1, Supplemental material) showed signals attributable to two sets of ortho-coupled aromatic protons at δ_H 8.04 (2H, d, J = 8.4 Hz, H-2′ and H-6′) and 7.00 (2H, d, J = 8.4 Hz, H-3′ and H-5′), two meta-coupled aromatic protons at δ_H 6.38 (¹H, d, J = 2.4 Hz, H-6) and 6.49 (1H, d, J = 2.4 Hz, H-8), and one methine singlet at δ_H 5.53 (¹H, s, H-2). The ¹³C NMR (DEPT) spectrum (Figure S2 and S3, Supplemental material) displayed one conjugated ketone carbonyl (δ_C 192.6), one ester carbonyl (δ_C 173.8), six sp² quaternary carbons, six sp² methines, and one sp³ methine.

As shown in Figure 2, the two aromatic ring systems (A ring and B ring) were easily deduced by ¹H-¹H COSY cross peaks of H-2′(6′)/H-3′(5′) and HMBC correlations from H-8 to C-7, C-8a, and C-4a, H-6 to C-7 and C-4a, H-2′(6′) to C-4′, and H-3′(5′) to C-1′ and C-4′ (Figure S5 and S6, Supplemental material). The ester carbonyl was attached at C-4a as determined by a J⁴ HMBC correlation from H-6 to C-4 (δ_C 173.8), while the ketone carbonyl was fused at C-1′ with ring B according to the downfield shifts of H-2′/H-6′. All aforementioned functionalities occupied 10 out of the total 11 degrees of unsaturation. Thus, the last one unsaturation degree suggested the remaining sp³ methine [δ_H 5.53 (¹H, s, H-2)] connected with C-3, C-4, and C-8a through C–C, C–O, and C–O bonds, respectively, and formed an additional ring (C ring), which was collaborated by the chemical shift of C-2 (δ_C 99.5) together with the HMBC correlation of H-2 with C-4, although no HMBC correlations were observed between H-2 with C-3 and C-1′.

Figure 2.

¹H-¹H COSY (—) and HMBC (H→C) correlations of 1.

Moreover, the LC-MS/MS spectrum of 1 (Figure S8, Supplemental material) in negative mode unequivocally confirmed above our deduction as in which a cluster of prominent fragment peaks at m/z 181, 153, 151 was demonstrated due to decomposition of the precursor ion ([M−H]⁻ m/z 301) via two competitive pathways (Figure 3). The flat CD curve and a minor specific optical rotation value ([α]²⁵_D +2.00) indicated 1 was a racemic mixture. However, subsequent effort for chiral resolution performing on two chiral columns (Daicel Chiralpak AD-H and OD-H) was unsuccessful. Consequently, the structure of compound 1 was determined to be 5, 7-dihydroxy-2-(4-hydroxybenzoyl)-4H-benzo[d][1, 3]dioxin-4-one, and named artopithecin K.

Figure 3.

Proposed MS/MS fragmentation pathway of 1.

Compound 1 was evaluated to have tyrosinase inhibitory activity using kojic acid as a positive control. As a result, it exhibited a potent activity with an IC₅₀ value of 14.25 µM, which is comparable with that of kojic acid (IC₅₀ = 17.32 µM).

Experimental

General

NMR spectra were recorded on a Bruker AVANCE 600 NMR spectrometer with tetramethylsilane (TMS) as an internal reference. All HRESIMS spectra were analyzed on a Waters UPLC-QTOF mass spectrometer equipped with ESI source (Waters, Milford, MA, USA). Optical rotations were measured on an Anton Paar MCP-200 polarimeter. CD spectra were measured on a JASCO J-815 spectropolarimeter in MeOH. 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.), and Sephadex LH-20 gel (75−150 µM, GE Healthcare) were used for column chromatography. Precoated silica gel GF254 plates (Qingdao Marine Chemical Plant) were used for thin-layer chromatography (TLC) and spots were visualized by heating silica gel plates sprayed with 8% H₂SO₄ in EtOH. Semipreparative HPLC was performed on an Agilent 1200 system equipped with a VWD G1314B detector and a Zorbax SB-C₁₈ column (250 mm × 10 mm, 5 μm). All solvents used were of analytical grade (Xilong Chemical Reagent Co., Ltd., Guangdong, P. R. China). Both the mushroom tyrosinase and l-DOPA were purchased from the Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Kojic acid was purchased from the Solarbio Science & Technology Co., Ltd (Beijing, P. R. China).

Plant material

The twigs of A. pithecogallus were collected from Mengla County, Yunnan Province, P. R. China, in October 2015, and identified by Professor You-Kai Xu of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, P. R. China. A voucher specimen (HZYD-201510) has been deposited in School of Pharmacy, Nanchang University, P. R. China.

Extraction and isolation

The air-dried powder of twigs (4.0 kg) of A. pithecogallus was extracted with 95% EtOH three times at ambient temperature to yield a crude extract (220.0 g), which was suspended in water and then extracted with EtOAc. The EtOAc extract (146.0 g) was subjected to a MCI gel column chromatography (CC) eluted with CH₃OH/H₂O gradient (3: 7 → 10: 0, v/v) to give 10 fractions, Fr.1−Fr.10. Fr.7 (4.0 g) was chromatographed over a silica gel column (petroleum ether/ ethyl acetate, 2: 1 → 1: 2) to afford eight subfractions (Fr.7A−Fr.7H). Fr.7C (105 mg) was then purified by a Sephadex LH-20 (MeOH) chromatography to give three subfractions Fr.7C₁−Fr.7C₃. Fr.7C₂ (25 mg) was then purified by semi-preparative HPLC (CH₃CN/H₂O, 35: 65, 3.0 mL/min) to afford 1 (t_R = 25.1 min, 3.0 mg).

Artopithecin K (1), pale yellow amorphous powder; UV λ_max (log ε) 269 (3.13), 317 (2.65) nm; ¹H NMR and ¹³C NMR data: see Table 1; (+) HRESIMS m/z 325.0314 [M + Na]⁺ (calcd for C₁₅H₁₀O₇Na⁺, 325.0319); (−) HRESIMS m/z 301.0349 [M − H]⁻ (calcd for C₁₅H₉O₇⁻, 301.0354).

Tyrosinase inhibitory assay

The tyrosinase assay was conducted as reported by us previously. ¹⁴ All of the testing samples were dissolved in dimethyl sulfoxide (DMSO) and used at a concentration series of 100, 50, 25, 10, and 5 μM. The tyrosinase assay was performed in 96-well microplates with 200 μL total testing solution using kojic acid, a known tyrosinase inhibitor, as positive control. The assay mixture consists of 40 μL of sample solution, 40 μL of l-DOPA (2 mmol/L in 0.1 M phosphate buffer pH 6.8), 40 μL of mushroom tyrosinase solution (2 U/mL in 0.1 M phosphate buffer pH 6.8), and 80 μL of 0.1 M phosphate buffer pH 6.8. After incubation at room temperature for 30 min, the assay mixture was measured at an absorbance of 475 nm in triplicate with a microplate reader (Thermo Electron Corporation, CA, USA).

Supplemental Material

Supporting_Information-revised – Supplemental material for Artopithecin K, a new phenolic derivative with tyrosinase inhibitory activity from Artocarpus pithecogallus

Supplemental material, Supporting_Information-revised for Artopithecin K, a new phenolic derivative with tyrosinase inhibitory activity from Artocarpus pithecogallus by Yan Xiong, Xi Liu, Liu-Yun Xu, Qiao-Mei Lin, Xiao-Ru He and Zhi-Wang Zhou in Journal of Chemical Research