A New Chlorinated Phenolic Compound From the Antarctic Lichen,Pertusaria dactylina

Abstract

A new chlorinated phenolic compound, methyl-3-chloro-2-hydroxy-4-methoxy-6-pentylbenzoate (1) and 4 known compounds (2-5) were isolated from the Antarctic lichen, Pertusaria dactylina (Pertusariaceae). The structure of the new compound was determined by means of One-dimensional and two dimensional nuclear magnetic resonance (1D and 2D NMR) and high-resolution fast atom bombardment mass spectrometry (HRFABMS) experiments. The antimicrobial activities of compounds 1 to 5 against Staphylococcus aureus and Candida albicans were evaluated. The results showed that compound 1 exhibited a weak inhibitory effect against C. albicans with an IC₅₀ value of 67 ± 7 μg/mL.

Lichens are symbiotic systems consisting of a mycobiont (the dominant fungal partner), one or more photobionts (algal partner), and a complex microbial consortium comprising a wide array of heterotrophic bacteria and fungi. ^1,2 More than 1000 metabolites have so far been described from lichen sources, displaying various biological activities, such as antibiotic, antimycobacterial, antioxidant, antitumor, antiviral, analgesic, and antipyretic properties. ³ The Pertusaria genus, which contains about 800 species, is globally distributed from the tropics to the Arctic and Antarctic. Phytochemical reports have identified diverse compounds, including xanthones, ⁴ depsides, ⁵ depsidones, ⁶ and fatty acids ⁷ from several Pertusaria species, including Pertusaria amara, Pertusaria albescens, Pertusaria flavicans, Pertusaria pseudocorallina, Pertusaria truncate, and other species from the genus found in China. However, the chemical and biological characteristics of Pertusaria dactylina in its Antarctic distribution have so far been poorly reported. ⁸ In continuation of our research focused on the chemistry and biochemistry of this Antarctic lichen, we isolated a new chlorinated-phenolic compound, together with 4 known mono phenyl derivatives. This paper reports on the isolation and structure elucidation of the new compound (Figure 1).

Figure 1

Chemical structures of compounds 1-5 isolated from Pertusaria dactylina.

The ethyl acetate partition of the Antarctic lichen, P. dactylina, was repeatedly subjected to column chromatography (CC) on silica gel, RP-18 gel, Sephadex LH-20 gel, and semipreparative high performance liquid chromatography (HPLC) to afford a new chlorinated-phenolic compound 1, along with 4 known compounds (2-5).

Compound 1 was obtained as a white amorphous powder. Its molecular formula of C₁₄H₂₀ClO₄ was deduced from an ion at m/z 287.1050 [M+H]⁺ (calcd for C₁₄H₂₀ClO₄, 287.1050) in the positive HRFABMS (Supplemental Figure S1). The ¹H-NMR (Table 1; Supplemental Figures S2, S2-1 and S2-2) spectrum showed a singlet in the aromatic proton signal at δ _H 6.34 (s, H-5); a hydroxyl proton at δ _H 12.10 (s, 2-OH) shifted downfield due to the hydrogen bond with a carbonyl group; 4 methylene protons at δ _H 2.88 to 1.34 (H-1′ to H-4′) and a terminal methyl proton at δ _H 0.91 (t, J = 7.2 Hz, H-5′), indicating the presence of a pentyl group ⁹ ; and 2 methoxy protons at δ _H 3.96 (s, 7-OCH₃) and δ _H 3.94 (s, 4-OCH₃). The ¹³C-NMR spectrum (Supplemental Figure S3) revealed an ester carbonyl carbon at δ _C 171.5 (C-7); 2 oxygen-bearing aromatic carbons at δ _C 159.8 (C-2) and δ _C 158.9 (C-4); 3 fully substituted aromatic carbons at [δ _C 146.3 (C-6), δ _C 107.4 (C-3), and δ _C 106.1 (C-1)], and a protonated aromatic carbon at δ _C 105.9 (C-5) assigned by heteronuclear single quantum coherence spectroscopy (HSQC) analysis (Supplemental Figures S4 and S4-1), including the pentyl group carbons at δ _C 37.2 (C-1′), 32.0 (C-3′), 31.7 (C-2′), 22.5 (C-4′), and δ _C 14.0 (C-5′) confirmed by ¹H-¹³C heteronuclear multiple bond correlation (HMBC) correlations (Supplemental Figure S5-2). The NMR data for 1 were quite similar to that of ethyl 3-chloro-2-hydroxy-4-methoxy-6-pentylbenzoate, a compound that has been reported previously, ⁹ except for a methoxy group at C-7 instead of ethoxy group of ethyl 3-chloro-2-hydroxy-4-methoxy-6-pentylbenzoate. ⁹ The ¹H-¹³C long range HMBC correlations (Supplemental Figures S5, S5-1 and S5-2) from δ _H 3.96 (s, 7-OCH₃) to δ _C 171.5 (C-7), 4-OCH₃/H-5 to C-4, and H-5 to C-3/C-1′ supported the position of the methoxy group at the carbonyl carbon (C-7); the chlorine atom was attached at the C-3 position (Figure 2). ⁸ Thus, compound 1 was elucidated as a new compound, methyl-3-chloro-2-hydroxy-4-methoxy-6-pentylbenzoate.

Figure 2

Key HMBC correlations of compound 1.

Table 1

¹H (600 MHz) and ¹³C (150 MHz) NMR Data for 1 (CDCl₃, δ, ppm, J/Hz).

C atom	1
	δ C	δ H
1	106.1
2	159.8
3	107.4
4	158.9
5	105.9	6.34 (s)
6	146.3
7	171.5
1′	37.2	2.88 (m)
2′	31.7	1.54 (m)
3′	32.0	1.34 (m)
4′	22.5	1.34 (m)
5′	14.0	0.91 (t, J = 7.2)
4-OCH3	56.2	3.83 (s)
7-COOCH 3	52.3	3.82 (s)

The physical and spectral properties of the known compounds we isolated were compared with the published values, and identified as methyl 2,4-dihydroxy-6-methylbenzoate (2), ¹⁰ methyl 2-hydroxy-6-methoxy-4-methylbenzoate (3), ¹¹ methyl 2,4-dihydroxy-6-methoxybenzoate (4), ¹¹ and methyl 3,6-dihydroxy-2,4-dimethylbenzoate (5). ¹² To the best of our knowledge, compounds 2 to 5 have been isolated for the first time from this species.

Antimicrobial activities of compounds 1 to 5 against Staphylococcus aureus and Candida albicans, respectively, were evaluated. Among the isolated compounds, compound 1 exhibited a weak inhibitory effect against C. albicans with an IC₅₀ value of 67 ± 7 µg/mL, when compared to the positive control constant (Nystatin, IC₅₀ value of 1.4 ± 0.1 µg/mL). However, compounds 1 to 5 showed no inhibitory activity against S. aureus (Supplemental Table S1; Supplemental Figure S1).

Experimental

General

Optical rotations were measured on a Rudolph Research Autopol IV multiwavelength polarimeter. UV spectra were recorded on a Shimadzu PharmaSpec-1700 Ultraviolet (UV)-visible spectrophotometer. Infrared (IR) spectra were measured on a Bruker Tensor-27 spectrophotometer. 1D and 2D NMR spectra were recorded on a Bruker AVANCE (600 MHz) spectrometer. High-resolution electrospray ionization mass spectra (HREIMS) were obtained with an Agilent 6530 liquid chromatography quadrupole time-of-flight (LC-qTOF) High Mass Accuracy mass spectrometer operated in the positive- and negative-ion modes. Thin-layer chromatography (TLC) was performed on silica gel 60 F₂₅₄ (0.25 mm, Merck, Germany). Silica gel (230-400 mesh, Merck, Germany) and C-18 (YMC·GEL ODS-A, 12 nm, S-150 μm) were used for CC. Semipreparative HPLC was conducted on YL9100 HPLC system (Young Lin, South Korea) equipped with a UV/Vis detector using an Alltech reversed-phase YMC-Pak C-18 column (10 µm, 20 × 250 mm) with a flow rate of 2 mL/min.

Lichen Material

The lichen, P. dactylina, was collected in January 2017 from King George Island, Antarctica, (62°12′53.69″ S; 58°55′23.87″W), and identified by Dr Ji Hee Kim and Miss Jae Eun So. A voucher specimen (no. Ant-053) was deposited at the Natural Product Chemistry Laboratory of the Korea Polar Research Institute.

Extraction and Isolation

An air-dried and powdered lichen, P. dactylina (80 g), was used for extraction by maceration in methanol (MeOH) (3 × 0.5 L) at room temperature. The solvent was concentrated in vacuo, yielding 3.0 g of a crude extract which was then suspended in distilled water (0.2 L) and extracted successively with n-hexane (2 × 0.2 L), ethyl acetate (EtOAc) (2 × 0.2 L), and n-butanol (2 × 0.2 L). The ethyl acetate partitions (1.2 g) were separated by CC over a silica gel column and eluted with Hex:EtOAc (90:10-50:50) to obtain 15 subfractions (Es1-Es15). The subfractions from Es4 to Es7 (250 mg) were combined based on their TLC patterns, subjected to a Sephadex LH-20 (30 g) gel CC using a solvent mixture (MeOH:H₂O, 50:50). They were then purified with HPLC on a semipreparative C-18 gel column, using an MeOH:H₂O mixture (60:40), and yielded 5 (2.0 mg, t _R 70 minutes), 2 (1.5 mg, t _R 78 minutes), and 4 (4.0 mg, t _R 83 minutes). The combined subfractions Es11 and Es12 (100 mg) were purified over a C-18 gel column, using MeOH:H₂O mixtures (10:90-80:20) as the solvent system, and yielded 5 subfractions (Es11r1-Es11r5). Subfraction Es11r2 (30 mg) was separated on a semipreparative C₁₈ gel column by HPLC, using MeOH:H₂O mixtures (10:90-80:20) as the solvent system, yielded compounds 1 (2.0 mg, t _R 75 minutes) and 3 (3.0 mg, t _R 80 minutes).

Methyl-3-Chloro-2-Hydroxy-4-Methoxy-6-Pentylbenzoate (1)

White amorphous powder.

UV (MeOH) λ _max (log ε): 262 (4.0) nm.

¹H- (600 MHz, CDCl₃) and ¹³C-NMR (150 MHz, CDCl₃) data, see Table 1.

HRFABMS: m/z 287.1050 [M+H]⁺ (calcd for C₁₄H₂₀ClO₄, 287.1050)

Antibacterial Assays

The antimicrobial activities were tested using S. aureus KCTC 3881 (bacteria) and C. albicans KCTC 27242 (fungi) (Korean Collection for Type Cultures, Daejeon, Korea) in a 96-well plate. The cell culture was diluted up to 0.5 McFarland Standard with sterilized media. For C. albicans, the culture broth was 100 times more diluted before use. Each well was filled with 95 μL of culture broth. The compounds dissolved in DMSO were added until the final concentrations (0.5, 1, 2, 5, 10, 20, and 50 ug/mL), and the final volume of each well was 100 μL. ¹³ The plate was incubated at 25°C for 16 hours. Cell inhibition was measured at 600 nm (for S. aureus) and 530 nm (for C. albicans) using Multiskan GO Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, United States). The IC₅₀ value was calculated using an exponential trend line in Excel software (Microsoft, Redmond, WA, United States), and the values are mean ± standard errors of 3 determinations. Kanamycin and nystatin were used as positive controls against the bacterium and yeast, respectively.

Supplemental Material

Supplementary material - Supplemental material for A New Chlorinated Phenolic Compound From the Antarctic Lichen, Pertusaria dactylina

Supplemental material, Supplementary material, for A New Chlorinated Phenolic Compound From the Antarctic Lichen, Pertusaria dactylina by Man H. Koo, Min J. Kim, Jae E. Seo, Ji H. Kim, Se J. Han, Il C. Kim, Jun H. Lee and Ui J. Youn in Natural Product Communications