New Anthraquinone Derivatives from Geosmithia lavendula

Abstract

A phytochemical study of Geosmithia lavendula Pitt led to the isolation of three new anthraquinones: 1-acetyl-2,4,6,8-tetrahydroxy-9,10-anthraquinone (1), 2-acetyl-1,4,5,7-tetrahydroxy-9,10-anthraquinone (2), and 1-acetyl-2,4,5,6,7-pentahydroxy-9,10-anthraquinone (3), as well as another new compound named didodecyl thiodipropionate (propionic acid, 3,3-sulfinyl di-1,1′-didodecyl ester) (4), along with ten known compounds: 1-acetyl-2,4,5,7-tetrahydroxy-9,10-anthraquinone (rhodolamprometrin) (5), 1-acetyl-2,4,5,7,8-pentahydroxy-9,10-anthraquinone (6), (22E)-ergosta-6,22-diene-3β,5α,8α-triol, p-hydroxybenzyl alcohol, oleic acid, D-mannitol, palmitic acid, stearic acid, cis-vaccenic acid and 2-decenal. The structures of the isolated metabolites were elucidated based on NMR spectroscopic and mass spectrometric data. Compound 1 exhibited moderate activity against methicillin resistant Staphylococcus aureus with an IC₅₀ value of 16.1 μg/mL.

Geosmithia lavendula Pitt (Ascomycota: Hypocreales) is a dry-spored, lilac colored filamentous fungus and considered as associates built by bark beetles [1-4]. Three anthraquinones: 1,3,6,8-tetrahydroxy-9,10-anthraquinone; 1-acetyl-2,4,5,7-tetra-hydroxy-9,10-anthraquinone; and 1-acetyl-2,4,5,7,8-pentahydroxy-9,10-anthraquinone were previously isolated from this fungus [3,4]. Also, an analytical HPLC method was reported for the possible separation of another ten new anthraquinones and their proposed structures were determined using only FTMS and UV-VIS spectrometry [3]. In this study, when G. lavendula was cultured in Potato Dextrose Broth (PDB), it produced five anthraquinones (1, 2, 3, 5 and 6; Figure 1), of which 1-3 were new. However, when the fungus was cultured in MID media supplemented with 1 g soytone, no anthraquinones were detected, but a new compound (4) (Figure 1), and ten known compounds were isolated.

Figure 1.

Chemical structures of compounds 1- 6.

Compound 1, obtained as a red powder (MeOH), displayed a deprotonated molecular ion [M-H]⁺ at m/z 313.0299 (calcd.: 313.03477) in the negative mode HR-ESI-MS, corresponding to a molecular formula of C₁₆H₁₀O₇. The ¹³C- and ¹H-NMR spectra of 1 (Table 1) displayed resonances for one acetylic methyl [δ_C 30.8/δ_H 2.317 (3H, s)] and three aromatic methines [δ_C 108.4 /δ_H 6.939 (1H, br.s), C-5, δ_C108.3/δ_H 6.469 (1H, br.s), C-7, and δ_C 108.0/δ_H 6.007 (1H, s), C-3]. The ¹³C NMR spectrum, in association with the DEPT spectra, revealed the presence of an anthraquinone moiety: two carbonyl moieties [δ_C 184.8, C-9; δ_C 183.6, C-10], a third carbonyl moiety (δ_C 204.3) corresponding to the carbonyl of an acetyl group, and seven aromatic quaternary carbons [δ_C 132.0, C-1; δ_C 165.0, C-2 and C-4; δ_C 163.5, C-6 and C-8; δ_C 129.4, C-9a; δ_C 102.1, C-4a; δ_C 134.3, C-8a; δ_C 108.5, C- 10a]. By comparing the ¹³C NMR spectral data of compounds 1 and 5 [4], it was clear that the carbonyl carbon at C-10 shifted upfield suggesting that 1 is protonated at position 5. This was supported by the HMBC spectrum, which showed the following correlations (Table 1): from H-3 (δ_H 6.007) to C- 1 (δ_C 132.0), C-4a (δ_C102.1), C-2 and C-4 (δ_C165.0); from H-5 (δ_H6.939) to C-7 (δ_C108.3) and C-10 (δ_C183.6); from H-7 (δ_H6.469) to C- 5 (δ_C108.4), C-6 and C-8 (δ_C 163.5); and from acetyl protons (δ_H 2.31) to acetyl carbonyl (δ_C204.1), and C-1 (δ_C132.0). The correlation from H-5 to the upfield carbonyl moiety C-10 (δ_C 183.6) revealed that the structure is protonated at position 5. Based on the above spectral data, as well as by comparison with closely related published anthraquinones, compound 1 was identified as a new naturally occurring anthraquinone, the structure of which is 1-acetyl-2,4,6,8-tetrahydroxy-9,10-anthraquinone.

Table 1.

¹H- and ¹³C-NMR and HMBC spectral data (400 MHz, δ in ppm, J values in Hz) for compounds 1-3, 5, and 6 in DMSO-d₆

No.	1			2			3			5		6
	δ C	δ H	HMBC	δ C	δ H	HMBC	δ C	δ H	HMBC	δ C	δ H	δ C	δ H
1	132.0			164.2			125.7			126.9		125.9
2	165.0			128.8			163.6			161.2		160.8
3	108.0	6.007 s	C- 1, 4a, 2, 4	108.04	6.369 s	C- 4a, 2, 1	108.5	6.616 s	C- 4a, 1, 2, 4	108.2	6.655 s	108.4	6.694 s
4	165.0			164.9			163.6			163.6		163.6
4a	102.1			105.5			108.9			107.9		108.5
5	108.4	6.939 br.s	C-7, 10	164.9			160.9			164.1		159.4
6	163.5			108.2	6.530 br.s	C- 5, 7	151.8			108.3	6.602 br.s	110.1	6.637 s
7	108.3	6.469 br.s	C- 5, 6, 8	163.8			160.9			165.1		156.8
8	163.5			108.5	6.993 d, J=2.4 Hz	C- 5, 6	109.5	6.468 s	C-10a, 6, 7	108.8	7.051 br.s	149.6
8a	134.3			134.4			112.0			134.5		112.2
9	184.8			182.6			184.4			181.9		186.4
9a	129.4			130.2			130.7			130.8		130.5
10	183.6			187.0			186.4			188.6		186.2
10a	108.5			108.08			102.6			108.5		103.9
Ac-C=O	204.3			203.0			201.7			201.2		201.2
Ac-CH3	30.8	2.317 s	C-1, Ac-C=O	30.7	2.354 s	C- 2, Ac-C=O	31.2	2.418 s	C-1, Ac-C=O	30.7	2.382 s	30.9	2.424 s

Compound 2, obtained as a red powder (MeOH), displayed a deprotonated molecular ion [M-H]⁺ at m/z 313.0357 (calcd.: 313.03477) in the negative mode HR-ESI-MS, corresponding to a molecular formula of C₁₆H₁₀O₇. The ¹³C- and ¹H-NMR spectra of compound 2 (Table 1) displayed resonances for one acetylic methyl [δ_C 30.7/δ_H 2.354 (3H, s)] and three aromatic methines [δ_C 108.04/δ_H 6.369 (1H, br.s), C-3, δ_C108.2/δ_H 6.530 (1H, br.s), C-6, and δ_C 108.5/δ 6.993 (1H, d, J=2.4 Hz), C-8]. The ¹³C NMR spectrum, in association with the DEPT spectra, revealed the presence of an anthraquinone moiety: two carbonyl moieties [δ_C 182.6, C-9; δ_C 187.0, C-10], a third carbonyl moiety (δ_C 203.0) corresponding to the carbonyl of an acetyl group, and eight aromatic quaternary carbons [δ_C 164.2, C-1; δ_C 128.8, C-2; δ_C 164.9, C-4 and 5; δ_C 163.8, C-7; δ_C 105.5, C-4a; δ_C 134.4, C-8a; δ_C 130.2, C-9a; δ_C 108.08, C-10a]. The HMBC spectrum showed the following correlations (Table 1): from H-3 (δ_H 6.369) to C- 4a (δ_C 105.5), C-2 (δ_C128.8), and C-1 (δ_C 164.2); from H-6 (δ_H 6.530) to C- 8 (δ_C 108.5), and C-7 (δ_C 163.8); from H-8 (δ_H 6.993) to C-6 (δ_C 108.2), C-5 (δ_C 164.9) and C-9 (δ_C 182.6); and from the acetyl protons (δ_H 2.354) to an acetyl carbonyl (δ_C 203.0) and C-2 (δ_C 128.8). The strong correlation from H-3 to C-1 (δ_C 164.2) and the moderate one from H-3 to C-2 (δ_C 128.8) revealed that the structure is protonated at position 3 and hydroxylated at position 1. Based on the above spectral data, as well as by comparison with published anthraquinones that are closely related, compound 2 was identified as a new naturally occurring anthraquinone, the tentative structure of which is 2-acetyl-1,4,5,7- tetrahydroxy-9,10-anthraquinone.

Compound 3, obtained as a violet-reddish powder (MeOH), displayed a protonated molecular ion [M+H]⁺ at m/z 331.0413 (calcd.: 331.04533) in the positive mode HR-ESI-MS, corresponding to a molecular formula of C₁₆H₁₀O₇. The ¹³C- and ¹H-NMR spectra of compound 3 (Table 1) displayed resonances for one acetylic methyl [δ_C 31.2 /δ_H 2.418 (3H, s)] and two aromatic methines [δ_C108.5 /δ_H 6.616 (1H, s), C-3, and δ_C 109.5 /δ_H 6.468 (1H, s), C-8]. The ¹³C NMR spectrum, in association with the DEPT spectra, revealed the presence of an anthraquinone moiety: two carbonyl moieties [δ_C 184.4, C-9; δ_C 186.4, C-10], a third carbonyl moiety (δ_C 201.7) corresponding to the carbonyl of an acetyl group, and seven aromatic quaternary carbons [δ_C 125.7, C-1; δ_C 163.6, C-2 and C-4; δ_C 160.9, C-5 and C-7; δ_C 151.8, C-6; δ_C 130.7, C-9a; δ_C 108.9, C-4a; δ_C 112.0, C-8a; δ_C 102.6, C-10a]. The HMBC spectrum showed the following correlations (Table 1): from H-3 (δ_H 6.616) to C-1 (δ_C 125.7), C-4a (δ_C112.0), C-2 and C-4 (δ_C163.6); from H-8 (δ_H6.468) to C-6 (δ_C151.8), C-10a (δ_C102.6), and C-7 (δ_C 160.9); and from acetyl protons (δ_H 2.418) to an acetyl carbonyl (δ_C201.7), C-1 (δ_C125.7). By comparing the ¹³C NMR spectral data of 3 and 6 [4], it was clear that the carbonyl carbon at C-9 shifted upfield suggesting that 3 is protonated at position 8. Also, this was supported by the correlation from H-8 to C-10a (δ_C 102.6) and C-6 (δ_C 151.8). Based on the above spectral data, as well as by comparison with closely related published anthraquinones, compound 3 was identified as a new naturally occurring anthraquinone with the structure 1-acetyl-2,4,5,6,7-pentahydroxy-9,10-anthraquinone.

Compound 4, obtained as colorless needles, displayed a protonated molecular ion [M+H]⁺ at m/z 531.47260 (calcd.: 531.50247) in the positive mode HR-ESI-MS, corresponding to a molecular formula of C₃₀H₅₈O₅S. The ¹³C NMR spectrum (Table 2), in association with the DEPT spectrum, displayed one quaternary carbon [δ_C 171.3, C-1,1′] corresponding to an ester carbonyl, one methyl [δ_C 14.2,C-12″, 12‴], 13 methylenes, 11 of which corresponded to the dodecyl moiety [δ_C 22.8, 26.0, 27.2, 28.6, 29.5, 29.6, 29.7, 29.78, 29.79, 29.89, 32.0], one oxygenated methylene [δ_C 65.6, C-1″,1‴] and another downfield methylene [δ_C 47.3, C-2,2′]. The HMBC spectrum showed the following correlations (Table 2): from H-1″,1‴ (δ_H 4.127) to C- 1,1′ (δ_C 171.3); from H-2,2′ (δ_H2.945, 3.069) to C-3,3′ (δ_C27.2) and C-1,1′ (δ_C171.3); from H-3,3′ (δ_H 2.854) to C-2,2′ (δ_C47.3) and C-1,1′ (δ_C171.3); and from H-12″,12‴ (δ_H 0.896) to 10″,10‴ (δ_C 32.0). Based on the above spectral data, as well as by comparison with published data [5], compound 4 was identified as a new naturally occurring compound, didodecyl thiodipropionate. This was previously synthesized [5], but this is the first report of its isolation from a natural source.

Table 2.

¹H- and ¹³C-NMR and HMBC spectral data (400 MHz, δ in ppm, J values in Hz) for compound 4 in CDCl₃

No.	4
	δ C	δ H	HMBC
1,1′	171.3
2,2′	47.3	2.945, 3.069 (2H, m)	C- 3,3′ and C-1,1′
3,3′	27.2	2.854 (2H, m)	C-2,2′ and C-1,1′
1″,1‴	65.6	4.127 (2H, t, J= 6.8)	C-1,1′
2″-11″ and 2‴-11‴	22.8, 26.0, 28.6, 29.5, 29.6, 29.7, 29.78, 29.79, 29.89, 32.0	1.269 – 1.645
12″,12‴	14.2	0.896 (3H, t, J= 7.2, 6)	C- 10″,10‴

Compound 1 exhibited moderate activity against methicillin resistant Staphylococcus aureus with an IC₅₀ value of 16.1 μg/mL. Six known compounds were also isolated from G. lavendula and identified by comparing their spectral data with those published as; 1-acetyl-2,4,5,7- tetrahydroxy-9,10-anthraquinone [4], 1-acetyl-2,4,5,7,8-pentahydroxy-9,10-anthraquinone [4], (22E)-ergosta-6,22-diene-3β,5α,8α-triol [6], p-hydroxybenzyl alcohol, oleic acid [7,8] and D-mannitol [9].

Three known fatty acids and an aldehyde were detected by using GC/MS and identified as: palmitic acid, stearic acid, cis-vaccenic acid, and 2-decenal.

Experimental

General experimental procedures

High resolution mass spectra were measured using a Bruker BioApex spectrometer. 1D and 2D NMR spectra were recorded on a Varian AS 400 MHz spectrometer. GC/MS analysis was carried out on a HP 6890 series GC, equipped with a split/ splitless capillary injector, a HP 6890 Series injector auto sampler, and a DB-5 ms column (30 m × 0.25 mm × 0.25 μm, Agilent). The GC was interfaced to a HP 5973 quadrupole mass selective detector through a transfer line set at 240°C. The injector temperature was 250°C, and 1 μL injections were performed in the split (1:10) mode. Column flow was set at a constant pressure of 5.66 psi, giving an initial flow of 0.7 mL/min, using helium as carrier gas. The oven temperature was raised from 70°C to 200°C at a rate of 2°C/min, and then continued at 200°C for 15 min. The total run was 80 min. Incubator shakers (New Brunswick Scientific, innova 4430) were used for incubating fungi. Sephadex LH-20 (Mitsubishi Kagaku, Tokyo, Japan) and silica gel (60-120 mesh, Merck) were used for CC. Solid phase extraction (SPE) cartridges (supelco, silica, 2 gm, 5 gm and 10 gm) were used under vacuum. Fractions from CC were monitored using precoated aluminum sheets [silica 60 F254, 0.25 mm (Merck, Darmstadt, Germany)] with detection provided by UV light (254 and 366 nm) and by spraying with 1% vanillin-sulfuric acid reagent followed by heating for 5-10 min (105°C). Diaion ^® HP-20 (250 μm, Sigma Aldrich) was used for the separation of metabolites from the liquid media. Analytical HPLC was carried out using a Waters Delta Prep 4000 with a Waters 2487 dual λ absorbance detector and Polymer Laboratories Evaporative Light Scattering (PL-ELS 1000) detector attached, using a Luna 5 μ C18 (2) 100A, 150 × 4.6 mm column with isocratic systems: (a) acetonitrile: H₂O: formic acid (24:76:0.1) at a flow rate of 0.8 mL/min over a 90 min time span, and (b) acetonitrile: H₂O: formic acid (22:78:0.1) at a flow rate of 1 mL/min over a 100 min time span. This was scaled up for preparative HPLC using a Luna 5 μ C18 (2), 100A, 250 × 21.2 mm column at a flow rate of 35.4 mL/min and 28.3 mL/min, respectively with the same solvent systems that were used on the analytical column. HPLC-grade reagents were used as standards (Sigma Chemical Co., St. Louis, MO). Solvents were filtered through a 0.45-μm membrane filter and degassed under vacuum.

Fungal material

Geosmithia lavendula Pitt (Ascomycota: Hypocreales) was provided by Assiut University Mycological Center, Assiut, Egypt (Accession No. 1004). This fungus (strain No. CBS 344.48, ATCC^® number: 10463) was purchased from the fungal Biodiversity Center, Utrecht, The Netherlands.

Culture media

Fungi were grown on Potato Dextrose Agar (PDA) plates at room temperature ~ 28°C for 14 days. Plates were kept in a refrigerator and used when needed. The fungus was grown on 2 different media: a) Potato dextrose broth (PDB) and b) MID medium (supplemented with 1 g soytone) [10].

Extraction and isolation of bioactive metabolites

Metabolites obtained from PDB medium

Geosmithia lavendula was cultured in 2800 mL Erlenmeyer flasks containing 1 L of PDB medium, which had been prepared by dissolving 24 g of PDB in 1L distilled water and then autoclaved. The fungi were grown at 30°C using shakers (160 rpm) for 14 days. After incubating the fungal culture, the fungal cells were filtered through sterile cotton using vacuum filtration and the filtrate extracted with activated ion exchange resin (Diaion^® HP-20) by adding 100 g of resin to each 1L of filtrate before being returned to the shakers and left overnight. The contents of the flasks were then filtered and the HP-20 was washed with distilled water to remove salts and sugars. Then, the resin was eluted with MeOH and acetone. The MeOH and acetone eluates were combined and dried under vacuum to yield a viscous residue, which was dissolved in water and successively extracted with n-hexane, dichloromethane, and EtOAc. Each solvent was separately concentrated under vacuum to afford 120 mg (n-hexane), 350 mg (DCM) and 537 mg (EtOAc).

The DCM and EtOAc fractions were combined (887 mg) and subjected to silica gel (60-120 mesh, 20 gm) CC (50 × 3.8 cm). Stepwise gradient elution was carried using CHCl₃ and MeOH. Fractions of 200 mL each were collected and concentrated. Similar fractions were combined according to their TLC profiles and concentrated to dryness to afford 4 main groups [1-4]: group 1 (35.3 mg), group 2 (131.7 mg), group 3 (303.2 mg) and group 4 (104.4 mg). Group 1 was subjected to solid phase separation using a SPE cartridge (silica, 5 g) under vacuum and eluted with n-hexane and EtOAc mixtures of increasing polarities. The fraction eluted with (40% EtOAc in n-hexane) yielded 5 (7 mg). Group 2 was subjected to Sephadex LH-20 CC (50 × 2.5 cm) and eluted with MeOH (100%) yielding 1 (7.8 mg). Group 3 (100 mg) was subjected to HPLC (Luna 5 μ C18 (2) 100A, 250, 21.2 mm) and eluted with isocratic system (a) yielding 6 (t_R 75.4 min, 4.3 mg) and 2 (t_R 84.5 min, 8.6 mg). Group 4 was subjected to HPLC (Luna 5 μ C18 (2) 100A, 250, 21.2 mm) and eluted with isocratic system (b) yielding 3 (t_R 67.5 min, 8 mg).

Metabolites obtained from MID media (supplemented with 1 g soytone)

Geosmithia lavendula was cultured in 2 L Erlenmeyer flasks containing 500 mL of MID medium supplemented with 1 g soytone. The fungi were grown at 24°C under still conditions for 21 days. After incubating the fungal culture, fungal cells were separated by filtration through 4-layered cheesecloth. The filtrate was extracted with an equal volume of EtOAc, 3 times, and the solvent was then evaporated under reduced pressure to dryness. The EtOAc fraction (2.1 g) was subjected to vacuum liquid chromatography (VLC) on flash silica gel (200 g, 13.5 × 13.5) eluted with n-hexane and EtOAc mixtures of increasing polarities and then with EtOAC and MeOH gradients. Fractions of 250 mL were collected and concentrated. Similar fractions were combined according to their TLC profiles and concentrated to dryness to afford 9 groups [A-I]. Group A (eluted with 100% n-hexane, 70 mg) was subjected to solid phase separation using a SPE cartridge (silica, 2 g) under vacuum and eluted with n-hexanes and EtOAc gradients. Fractions of 10 mL were collected and concentrated to afford 4 subfractions. Subfractions 1 and 2 were subjected to GC/MS leading to the detection and identification of palmitic acid, stearic acid, cis-vaccenic acid and decenal. Subfraction 3 yielded oleic acid (7 mg). Group C (eluted with 50% EtOAc in n-hexane, 92 mg) was subjected to solid phase separation using a SPE cartridge (silica, 2 g) and eluted with CHCl₃ and MeOH gradients. Fractions of 10 mL were collected and concentrated in vacuo. The fraction eluted with 2% MeOH in CHCl₃ yielded p-hydroxybenzyl alcohol (10.3 mg). Group D (eluted with 75% EtOAc in n-hexanes, 430 mg) was subjected to solid phase separation using a SPE cartridge (silica, 10 g) and eluted with n-hexane and EtOAc gradients, yielding 4 subfractions (1-4); subfraction 1 (150 mg), subfraction 2 (203 mg), subfraction 3 (27 mg) and subfraction 4 (14 mg). (22E)-Ergosta-6,22-diene-3β,5α,8α-triol (8 mg) and compound 4 (1.5 mg) were isolated from subfractions 3 and 4, respectively by precipitation from MeOH. Groups G-I (eluted with EtOAc – MeOH, 50%, 75% and 100%) were collected together yielding a residue (1.3 g) that was subjected to solid phase using a SPE cartridge (silica, 10 g) and eluted with EtOAc and MeOH gradients. Fractions of 200 mL were collected and concentrated. D-mannitol (2.5 mg) was precipitated from the 35% MeOH / 65% EtOAc fraction.

Alkaline hydrolysis of compound 4

1 mg of compound 4 was dissolved in 5% methanolic KOH (1 mL) and refluxed for 4 h. The reaction mixture was extracted with n-hexane, and the n-hexane layer was subjected to GC/MS analysis, which yielded 1-dodecanol.

Antimicrobial bioassay

Crude extracts and pure compounds were tested for antimicrobial activity against the fungi Candida albicans ATCC 90028 (Ca), C. glabrata ATCC 90030 (Cg), C. krusei ATCC 6258 (Ck), and Aspergillus fumigates ATCC 90906 (Af), and the bacteria Staphylococcus aureus ATCC 33591 (MRSA), Cryptococcus neoformans ATTC 90113 (Cn), Staphylococcus aureus ATTC 29213 (Sa), Escherichia coli ATCC 35218 (Ec), Pseudomonas aeruginosa ATCC 27853 (Pa), and Mycobacterium intracellulare ATCC 23068 (Mi) [11]. Amphotericin B (ICN Biomedicals, Ohio) for fungal and ciprofloxacin (ICN Biomedicals, Ohio) for bacterial bioassays were respectively used as positive controls.

Antimalarial bioassay

Antimalarial activity of the crude extracts and the pure compounds were determined in vitro on chloroquine sensitive (D6, Sierraleon) and resistant (W2, Indo-China) strains of Plasmodium falciparum using a previously reported method [11]. Chloroquine and artemisinin were included in each assay as positive controls.

Antileishmanial bioassay

The antileishmanial activity was evaluated in vitro against a culture of L. donovani promastigotes [12,13]. Pentamidine (IC₅₀ 1.01 and IC₉₀ 2.03 μg/mL) and amphotericin B (IC₅₀ 0.47 and IC₉₀ 0.65) were used as the controls.