Discovery of a Dimeric Zinc Complex and Five Cyclopentenone Derivatives from the Sponge-Associated Fungus Aspergillus ochraceopetaliformis.

In devotion to investigating structurally novel and biologically active marine natural products, a dimer of a zinc complex, dizinchydroxyneoaspergillin (1), aspernones A-E (2-6), five cyclopentenone derivatives together with known polyketides (7-10), and neoaspergillic acid analogues (11-14) were isolated from the sponge-associated fungus Aspergillus ochraceopetaliformis SCSIO 41018. Their structures were elucidated on the basis of spectroscopic analysis, electronic circular dichroism (ECD) analysis, and X-ray diffraction. Dizinchydroxyneoaspergillin (1) displayed significant bactericide effects toward methicillin-resistant Staphyloccocus aureus, Staphyloccocus aureus, Enterococcus faecalis, Acinetobacter baumannii, and Klebsiella pneumonia with MIC values of 0.45-7.8 μg/mL and moderate in vitro cytotoxic activities against the K562, BEL-7402, and SGC-7901 cell lines with IC50 values of 12.88 ± 0.14, 15.83 ± 0.23, and 15.08 ± 0.62 μM, respectively. This is the first time to report the dimer of the zinc complex of hydroxyneoaspergillic acid conjunction at Zn-N-4 by a coordination bond. Additionally, compound 1 displayed significant antibacterial and cytotoxic activities, which would be a promising drug lead and could attract much attention from both chemists and pharmacists.

Introduction

Natural products have proven to be a prolific source of novel promising drug leads with various unique chemical structures and multifarious pharmacological properties.[1] Furthermore, metal coordination complexes, a kind of a very limited number of natural products,[2,3] have attracted significant attention from chemists and biologists depending on superior bioactivities,[4,5] especially in oncology and infection treatments. Additionally, metal could influence the physiological activities of pharmaceuticals, for example, enhancing absorption of drug, improving the drug effect, and reducing side effects. As we know, carboplatin[6] as an extensive antineoplastic was approved by FDA in 1989. Furthermore, moxifloxacin is a new fourth generation 8-methoxy fluoroquinolone. The research revealed that moxifloxacin–Au(III) and Ag(I) complexes had showed significant antibacterial activity against the Gram-positive and Gram-negative bacteria.[7] Other metal chelates, such as aluminium neoaspergillin, have also been reported to have antibacterial activities against Enterobacter aerogenes and Staphylococcus aureus.[8] What is more, the discovery of penicillin from Penicillium sp. was a great motive to discover antibiotics from microorganisms. For example, aspergillin, isolated from Aspergillus fumigatus, displayed inhibition against the growth of Mycobacterium tuberculosis. Also, the antibiotic helvolic acid was discovered from Aspergillus filtrates, which exhibited antibacterial activity against Gram-positive bacteria.[9]

As a continuation of our investigation on structurally novel and biologically active metabolites from marine-derived fungi,[10,11] the sponge-associated fungus Aspergillus ochraceopetaliformis SCSIO 41018 was selected for investigation due to an interesting HPLC–UV profile for its extract when grown on a solid rice medium. Herein, we report the isolation, structure elucidation, and antibacterial as well as cytotoxic evaluations of a dimeric zinc complex (1), five cyclopentenone derivatives (2–6), and eight known compounds (7–14) (Figure ).

Figure 1

Structures of compounds 1–14 from Aspergillus ochraceopetaliformis SCSIO 41018.

Results and Discussion

Structural Elucidation

Compound 1 was obtained as a white needle and had a molecular formula of C48H76N8O12Zn2 based on its (+)-HRESIMS ion cluster [M + Na]+ at m/z 1107.4058, 1109.4036, and 1111.4025 and X-ray photoelectron spectroscopy (Figure S2), which is clearly indicative of the presence of a zinc atom in the molecule. The 1H NMR spectrum of 1 (Table ) exhibited an olefinic proton at δH 7.55 (s, H-5), oxygenated methine at δH 4.18 (d, J = 2.9 Hz, H-1′), two methines at δH 2.09–2.15 (m, H-2′ and H-2″), methylene at δH 2.55–2.62 (m, H-1″), and four methyls. A detailed comparison of the NMR data of 1 (Table ) to those of hydroxyneoaspergillic acid (13)[12] (Table S1), which was also isolated from this fungus, emphasized their similar structure. However, the methylene (H-1″) from the 1H NMR spectra (Table and Table S1) of 1 and 13 were significantly different, which provided an essential clue as to where the hydroxyneoaspergillic acid might be coordinated to a metal.[13] The methylene protons in 13 (H-1″) (Table S1) appear as double-double-singlets at δH 2.62 (dd, J = 13.7, 7.2 Hz) and 2.56 (dd, J = 13.7, 7.4 Hz). In contrast, due to the chelation of Zn2+, the methylene protons in 1 (Table ) appear as multiplets at δH 2.58. Our structural assignment of 1 was further supported by the corresponding heteronuclear multiple bond correlation (HMBC) and COSY spectra (Figure ). Therefore, the structure of 1 was determined as a zinc complex of hydroxyneoaspergillic acid. In addition, this result was also proved by X-ray diffraction (Figure , Cu Kα radiation, CCDC 1875006, flack parameter 0.050(12)). Interestingly, the compound 1 is a dimer of the zinc complex of hydroxyneoaspergillic acid, in which Zn binds to the N-4 position by a coordination bond. Notably, a number of coordination complexes of Fe(III),[14] Al(III),[15] and Zr(IV)[15] in which hydroxyneoaspergillic acid was as the ligand have been isolated from the marine-derived fungi. However, compound 1 possessed two coordination complexes of Zn(II), which were connected by the coordination bond of Zn–N-4 that has not been reported in any natural products.

Table 1

1H and 13C NMR Data for 1 and 6 (TMS, δ ppm)a,b

no.	1a		no.	6b
	δC	δH (J in Hz)		δC	δH (J in Hz)
2	154.9 C		1	199.9 C
3	146.7 C		2	73.3 C
5	125.2 CH	7.55, s	3	202.1 C
6	141.9 C		4	134.8 C
1′	70.2 CH	4.81, d (2.9)	5	165.8 C
2′	30.7 CH	2.09–2.15, m	6	7.0 CH3	1.93, s
3′	19.6 CH3	0.92, d (6.4)	7	20.3 CH3	1.32, s
4′	16.3 CH3	0.75, d (6.6)	8	60.3 CH3	4.33, s
1″	41.4 CH2	2.55–2.62, m
2″	26.7 CH	2.09–2.15, m
3″	22.5 CH3	0.85, d (6.4)
4″	22.5 CH3	0.85, d (6.4)

1H and 13C NMR data of 1 recorded at 500 MHz and 125 MHz in DMSO-d6, respectively.

1H and 13C NMR data of 2 recorded at 700 MHz and 175 MHz in CDCl3, respectively.

Figure 2

Key 1H–1H COSY (bold) and HMBC (arrows) correlations of 1–6.

Compound 2 was isolated as yellow crystals with the molecular formula of C14H22O5 from its protonated molecular ion at m/z 271.1551 [M + H]+ by high-resolution electrospray ionization mass spectrometry (HRESIMS), indicating four degrees of unsaturation. The 1H NMR spectroscopic data (Table ) of 2 exhibited signals for four methyls at δH 0.82 (3H, t, J = 7.4 Hz, H3-4′), 0.99 (3H, d, J = 7.0 Hz, H3-5′), 1.26 (3H, d, J = 6.3 Hz, H3-7), 1.58 (3H, s, 2-CH3); one methoxyl at δH 3.14 (3H, s, 5-OCH3); two methylenes at δH 2.48 (1H, d, J = 18.0 Hz, H2-4), 2.36 (1H, d, J = 18.0 Hz, H2-4), 1.44–1.51 (1H, m, H2-3′), 1.33–1.39 (1H, m, H2-3′); and two methines at δH 5.20 (1H, q, J = 6.3 Hz, H-6), δH 2.17–2.22 (1H, m, H-2′). The 13C NMR together with distortionless enhancement by polarization transfer (DEPT) spectra (Table ) revealed 12 carbon resonances comprising four methyls, one methoxyl, two sp3 methylenes, two sp3 methines including an oxygenated carbon at δC 70.8 (C-6), one sp3 oxygenated quaternary carbon (δC 84.3, C-5), one olefinic carbon (δC 117.5, C-2), and one ester group (δC 176.4, C-1′), which indicated that two carbons were absent in the 13C NMR spectrum compared with its molecular formula C14H22O5, and one of them was determined as olefinic carbon (C-3) according to only C-2. Further analysis of 1H–1H COSY and HMBC spectra (Figure ) led to the elucidation of the planar structure of 2. According to the 1H–1H COSY correlations of H3-4′/H2-3′/H-2′/H3-5′ and the HMBC correlations from H2-3′, H-2′, and H3-4′ to C-1′, 2-methyl butyrate side-chain was deduced. In the HMBC spectrum (Figure ), the key correlations of H2-4 with C-1 (δC 198.4, the ketone group was absent in the 13C NMR spectrum), C-2, C-5, and C-6; 2-CH3 with C-2 and C-1 (δC 198.4); H-6 with C-4 and C-5 together with the 1H–1H COSY correlations (Figure ) of H3-7/H-6 indicated the presence of cyclopentenone, which was connected with the 2-methyl butyrate side chain by C-6. Considering the molecular formula C14H22O5 of 2, we speculated there was a hydroxyl linked with C-3. Consequently, the planar structure of 2 was elucidated as a cyclopentenone derivative as shown and is named as aspernone A. After trying to recrystallize by various solvent systems, compound 2 is furnished as a high-quality crystal in methanol at room temperature. Hence, the X-ray diffraction study (Cu Kα radiation) of 2 was successfully performed (Figure , CCDC 1914669, flack parameter −0.03(11)), which not only reconfirmed the planar structure of 2 but also ascertained its absolute configuration (5S,6S,2′R), unambiguously.

Table 2

1H and 13C NMR Data for 2, 3, 4, and 5 (700, 175 MHz, TMS, δ ppm)a,b

	2a		3b		4a		5a
no.	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
1	198.4 C		201.7 C		204.0 C		201.6 C
2	117.5 C		116.5 C		120.8 C		115.6 C
3	188.3 C		183.0 C		190.0 C		179.8 C
4	36.3 CH2	2.63, d (18.0)	31.7 CH2	2.83, dq (17.5, 1.7)	31.8 CH2	3.06, d (18.4)	73.6 CH	5.01, q (1.4)
		2.48, d (18.0)		2.60, dq (17.5, 1.7)		3.06, d (18.4)
5	84.3 C		83.0 C		84.4 C		86.3 C
6	70.8 C	5.20, q (6.3)	70.3 CH	5.20, q (6.3)	71.7 CH	5.12, q (6.4)
7	15.3 CH3	1.26, d (6.3)	14.8 CH3	1.31, d (6.3)	14.9 CH3	1.29, d (6.4)
1′	176.4 C		175.0 C		176.5 C		177.4 C
2′	42.7 CH	2.17–2.22, m	41.3 CH	2.21–2.26, m	42.4 CH	2.21–2.26, m	42.1 CH	2.38–2.41, m
3′	27.6 CH2	1.44–1.51, m	26.7 CH2	1.51–1.58, m	27.6 CH2	1.49–1.53, m	27.8 CH2	1.65–1.68, m
		1.33–1.39, m		1.34–1.40, m		1.36–1.40, m		1.47–1.51, m
4′	11.9 CH3	0.82, t (7.4)	11.7 CH3	0.85, t (7.4)	11.9 CH3	0.84, t (7.4)	11.9 CH3	0.97, t (7.4)
5′	17.2 CH3	0.99, d (7.0)	16.8 CH3	1.03, d (7.0)	17.0 CH3	1.01, d (7.0)	16.8 CH3	1.14, d (7.0)
5-OMe/Me	51.3 CH3	3.14, s	52.0 CH3	3.23, s	52.0 CH3	3.20, s	19.5 CH3	1.32, s
2-Me/CH2OH	5.5 CH3	1.58, s	6.0 CH3	1.63, s	51.6 CH2	4.14, d (1.96)	6.1 CH3	1.69, d (1.5)
3-OH/OMe			57.1 CH3	4.01, s	58.8 CH3	4.17, s	59.2 CH3	4.18, s

Recorded in CD3OD.

Recorded in CDCl3.

Figure 3

ORTEP drawings of compounds 1 and 2.

Compound 3, yellow oil, was determined to have the molecular formula C15H24O5 with four degrees of unsaturation. The 1H and 13C NMR data (Table ) of 3 were nearly identical to those of compound 2, except for the presence of an additional methoxyl group (δC/H 57.1/4.01, C/H-16) in 3, which was in conjunction with C-3 according to the HMBC correlation (Figure ) from 3-CH3 to C-3 (δC 183.0). Hence, the planar structure of 3 were established as shown.

Compound 4 had the molecular formula C15H24O6 in accordance with HRESIMS data. Analysis of the NMR data (Table ) of 4 revealed that it had structural resemblance with compound 3. A methylene (δC/H 51.6/4.14) at C-14 bearing a hydroxyl in 4 instead of the methyl in 3 was observed, which was further substantiated by the HMBC correlations (Figure ) from 3-CH2OH to C-1 (δC 204.0), C-2 (120.8), and C-3 (190.0) together with its molecular formula C15H24O6.

Compound 5, isolated as yellow oil, showed the molecular formula C13H20O5, with four degrees of unsaturation on the basis of HRESIMS. The 1H NMR spectroscopic data of 5 (Table ) revealed the presence of four methyls at δH 0.97 (3H, t, J = 7.4 Hz, H3-4′), 1.14 (3H, d, J = 7.0 Hz, H3-5′), 1.32 (3H, s, 5-CH3), 1.69 (3H, d, 2-CH3); one methoxyl at δH 4.18 (3H, s, 3-OCH3); one methylene at δH 1.65–1.68, 1.47–1.51 (2H, m, H2-3′); two methines at δH 2.38–2.41 (1H, m, H-2′), 5.01 (1H, q, J = 1.4, H-4); and one hydroxyl hydrogen at δH 6.08 (1H, d, J = 1.4 Hz, 4-OH) in DMSO-d6. The 13C NMR and DEPT spectra (Table ) of 5 indicated 13 carbon resonances including four methyls, one methoxyl, one sp3 methylene, two sp3 methines (one oxygenated methine), one sp3 oxygenated quaternary carbon, two olefinic carbons, an ester group, and a ketone group. Upon analysis on the NMR data (Table ) of 5, we inferred that it was also a cyclopentenone derivative. According to the 1H–1H COSY correlations (Figure ) of H3-4′/H2-3′/H-2′/H3-5′ and the HMBC correlations from H2-3′, H-2′, and H3-5′ to C-1′, a 2-methyl butyrate side chain was deduced. In the HMBC spectrum, the correlations of H-4 with C-2, C-3, and C-5; 5-CH3 with C-1, C-4, and C-5; 2-CH3 with C-1, C-2, and C-3; and 3-OCH3 with C-3 proved the presence of cyclopentenone. Additionally, the 2-methyl butyrate side chain was connected with C-5 according to C-5 as an oxygenated quaternary carbon (δC 86.3). What else, hydroxyl hydrogen was linked with C-4. Therefore, the planer structure of 5 was confirmed.

Compound 6 had the molecular formula of C8H10O4, with four degrees of unsaturation. The 1H NMR spectrum (Table ) of 6 displayed signals attributable to two methyls at δH 1.93 (3H, s, H3-6) and 1.32 (3H, s, H3-7), and one methyoxyl at δH 4.33 (3H, s, H3-8). The 13C NMR data (Table ) of 6 revealed eight carbon signals, classified by DEPT and heteronuclear single quantum coherence (HSQC) spectra as two methyls, one methyoxyl, one sp3 oxygenated quaternary carbon, two olefinic carbons, and two ketone groups. The above NMR data of 6 closely resembled to those of cyclopentenedione,[16] except for that the C-2 was hydroxylated in 6, and the hydroxyl of C-4 in cyclopentenedione was displaced by a methyl in 6, which was proved by the HMBC correlations (Figure ) from H3-6 to C-3, C-5, and C-2 (δC 73.3) as an oxygenated quaternary carbon.

The above studies of compounds 2–5 have suggested that they have the same 3,5-dihydroxy-cyclopentenone core skeleton. Fortunately, quality crystals of 2 were obtained from recrystallization in methanol, and the X-ray diffraction study (Cu Kα radiation) of 2 was successfully performed (Figure , CCDC 1914669), which not only confirmed the planar structure of 2 but also determined its absolute configuration (5S,6S,2′R) unambiguously. However, the same attempt on compounds 3–5 did not succeed to obtain the quality crystals. The experimental CD curves were just the opposite between 2 and compounds 3/4, indicating a 5R-cyclopentenone core skeleton in 3 and 4. This assignment was further confirmed by comparing the calculated ECD spectrum with experimental values of compound 4 (Figure ). In order to use the modified Mosher’s method to determine the absolute configuration of C-6 of compounds 3 and 4, the hydrolysis experiment on compound 3 was carried out. Unfortunately, the reaction failed. Since 3, 4, and 5 were isolated as co-metabolites with 2 whose absolute configuration was determined by the X-ray diffraction study, we tentatively proposed the same configuration at C-2′ (2′R) among 3–5 on biosynthetic grounds.[17,18] The relative configuration of 5 was determined by the NOESY correlations. The NOSEY correlation of H-4/5-CH3 suggested that these protons were cofacial. Similarly, by comparison of the experimental ECD spectra of 5 and 6 with the corresponding calculated ones (Figure ), the absolute configurations of 5 and 6 were confirmed as (4R,5R)-5 and (2S)-6.

Figure 4

Comparison between calculated and experimental ECD spectra of 3–6.

In addition, the known polyketides and neoaspergillic acid derivatives were elucidated as dihydropenicillic acid (7),[19] phomapyrone C (8),[20] botryoisocoumarin A (9),[21] wasabidienone-E (10),[22] ferrineoaspergillin (11),[14] deoxy-β-hydroxyneoaspergillic acid (12),[23] hydroxyneoaspergillic acid (13),[12] and 3,6-diisobutyl-2(1H)-pyrazinone (14) by comparing their NMR data and mass spectra with the reported literature values.

The Activities

Compounds 1–14 were evaluated for their cytotoxic activities against K562, BEL-7042, SGC-7901, A-549, and HeLa lines, and 1 and 11–14 were tested for antimicrobial activities (three Gram-positive bacteria, methicillin-resistant Staphyloccocus aureus, Staphyloccocus aureus, Enterococcus faecalis, and three Gram-negative bacteria, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumonia). Among the tested compounds, compounds 1 and 13 exhibited different antibacterial activities as shown in Table . It is worth noting that compounds 1 and 13 showed potent inhibitory activities against A. baumannii with MIC values of 0.45 and 0.45 μg/mL, respectively. More interestingly, the metal coordination of Zn(II) (1) exhibited moderate in vitro cytotoxic activities against the K562, BEL-7402, and SGC-7901 cell lines with IC50 values of 12.88 ± 0.14, 15.83 ± 0.23, and 15.08 ± 0.62 μM, respectively (Table S2).

Table 3

Inhibitory Effects of Tested Compounds against Pathogenic Bacteria

	pathogenic bacteria (MIC, μg/mL)
comp.	MRSA	S. aureus	E. faecalis	A. baumannii	E. coli	K. pneumonia
1	7.8	7.8	0.9	0.45	62.5	7.8
13	3.9	3.9	0.9	0.45	125	3.9
ampicillin	0.65	0.325	0.04	—	—	—
gentamicin	—	—	—	0.33	1.25	0.16

Discussion

Metal coordination complexes of Zn as natural products are not very common in the nature. It had been reported that imidazole alkaloids and their zinc complexes with anti-inflammatory activity and cytotoxicity were isolated from the sponge.[13,24] What is more, hydroxyneoaspergillic acid is a series of fungal metabolites,[23,25] which as a chelating agent tend to form metal coordination complexes such as Fe(III), Al(III) and Zr(IV).[17] Interestingly, it should be pointed out that compound 1 possessed two coordination complexes of Zn(II), which were connected by a coordination bond of Zn–N-4 that has not been reported in any natural products. What is more, the novelty of new compounds 2–6, cyclopentenones, is mainly due to the side chain and configuration. Also, as the report shows, the cyclopentenone group is an ideal chemical device to direct an attack to a pathogenetic protein.[26] Therefore, the biological activities of 2–6 are worth to study deeply. Additionally, compound 1 displayed significant antibacterial and cytotoxic activities, which could be a promising drug lead and would attract much attention from both chemists and pharmacists.

Materials and Methods

General Experimental Procedures

NMR spectra were obtained on a Bruker Avance spectrometer (Bruker) operating at 500 and 700 MHz for 1H NMR 125 and 175 MHz and for 13C NMR with TMS as the internal reference. HRESIMS spectra data were measured with a Bruker maXis quadrupole-time-of-flight mass spectrometer (Bruker). Optical rotations were recorded on a PerkinElmer MPC 500 (Waltham) polarimeter. UV spectra were acquired using a Shimadzu UV-2600 PC spectrometer (Shimadzu). ECD spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics). X-ray diffraction intensity data were collected on a CrysAlis PRO CCD area detector diffractometer with graphite-monochromated Cu Kα radiation (λ = 1.54184). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10–40 μm) and over silica gel (200–300 mesh) (Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Biosciences), respectively. Spots were detected on TLC (Qingdao Marine Chemical Factory) under a 254 nm UV light. All solvents employed were of analytical grade (Tianjin Fuyu Chemical and Industry Factory). Semipreparative HPLC was carried out using an octadecyl-silica (ODS) column (YMC-pack ODS-A, YMC Co. Ltd., 10 × 250 mm, 5 μm, 2 mL/min). The artificial sea salt was a commercial product (Guangzhou Haili Aquarium Technology Company).

Fungal Material

This strain was stored on methylene blue (MB) agar slants (malt extract 15 g, agar 18 g, sea salt 10 g, pH 7.4–7.8, distilled water 1.0 L) at 4 °C and then deposited at the Marine Microbial collection center of CAS Key Laboratory of Tropical Marine Bio-resources and Ecology. It was identified as a member of the genus Aspergillus ochraceopetaliformis SCSIO 41018 on the basis of its internal transcribed spacer (ITS) phylogenetic analysis of the rDNA and the scanning electron microscopic image (Figure S1) as described in the Supporting Information. (NCBI Gen Bank accession number MH109740.1).

Fermentation

The strain Aspergillus ochraceopetaliformis SCSIO 41018 stored on MB ager slants at 4 °C was cultured on MB agar plants and incubated at 25 °C for 7 days. Inoculum (malt extract 15 g, sea salt 10 g, distilled water 1.0 L) was inoculated with Aspergillus ochraceopetaliformis SCSIO 41018 and incubated at 25 °C for 2 days on a rotating shaker (180 rpm, 25 °C). Then, the solid rice medium was cut into small pieces and extracted with EtOAc three times to afford 220 g of crude extract. Flasks were incubated at 25 °C under static condition and fermented for 60 days.

Extraction and Isolation of Compounds

Strain cultures were harvested after 60 days. Then, the solid rice medium was cut into small pieces and immersed into acetone for 2 days, sonicated for 10 min, and filtered using gauze, yielding the rice solid medium and water phases, which were extracted with EtOAC (6 × 1 L and 6 × 10 L, respectively). Both organic extracts were combined to gain 220 g of crude extract. All of crude EtOAC extract was subjected to silica gel column chromatography and eluted with petroleum ether/CH2Cl2 in gradient eluent (100:0–0:100) and followed by CH2Cl2/MeOH in gradient eluent (99:1–0:100) to yield eight fractions (fractions 1–8). Fraction 3 (38.0 g) was applied to a C-18 reverse-phase column (H2O/MeOH, 95:5–0:100), gaining seventeen subfractions (Fr.3–1 to Fr.3–17). Fr.3–3 was further purified by semipreparative reverse-phase HPLC (10% MeCN/H2O, 2 mL/min) to yield 6 (3.8 mg, tR 21 min). Fr.3–16 was separated by semipreparative reverse-phase HPLC (92% MeOH/H2O, 2 mL/min) to yield 11 (62.8 mg, tR 24 min). Fraction 4 (15.4 g) was applied to a C-18 reverse-phase column (H2O/MeCN, 95:5–0:100), gaining eighteen subfractions (Fr.4–1 to Fr.4–18). Fr.4–4 was further purified by semipreparative reverse-phase HPLC (45% MeCN/H2O, 2 mL/min) to yield 1 (20 mg, tR 24 min) and 5 (2.0 mg, tR 17 min). Fr.4–6 was separated by semipreparative reverse-phase HPLC (35% MeOH/H2O, 2 mL/min) to yield 4 (2.3 mg, tR 55 min) and 12 (10.8 mg, tR 31 min). Fr.4–9 was separated by semipreparative reverse-phase HPLC (50% MeCN/H2O, 2 mL/min) to yield 14 (4.6 mg, tR 15 min). Fr.4–10 was separated by semipreparative reverse-phase HPLC (45% MeCN/H2O, 2 mL/min) to yield 2 (12.0 mg, tR 14 min) and 3 (2.2 mg, tR 20 min). Fraction 5 was applied to C-18 Sephadex LH-20 (MeOH), gaining four subfractions (Fr.5–1 to Fr.5–4). Then, Fr.5–3 was applied to a C-18 reverse-phase column (H2O/MeOH, 95:5–0:100), gaining ten subfractions (Fr.5–3-1 to Fr.5–3-10). Fr.5–3-1 was further purified by semipreparative reverse-phase HPLC (20% MeCN/H2O, 2 mL/min) to yield 10 (12.1 mg, tR 45 min). Fr.5–3-2 was separated by semipreparative reverse-phase HPLC (20% MeCN/H2O, 2 mL/min) to yield 8 (4.5 mg, tR 11 min). Fr.5–4 was separated by semipreparative reverse-phase HPLC (40% MeOH/H2O, 2 mL/min) to yield 7 (14.7 mg, tR 12 min), 9 (6.1 mg, tR 20 min), and 13 (42.9 mg, tR 29 min).

Dizinchydroxyneoaspergillin (1): white crystal; [α]25 D – 42.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 330 (2.62), 231 (3.97), 205 (3.80) nm; ECD (0.19 mg/mL, MeOH) λmax (Δε): 335 (−5.22), 281 (−1.14), 266 (0.35), 242 (4.11), 228 (−6.95), 220 (−3.43), 210 (−9.03), 201 (−0.17) nm; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, Table ; HRESIMS at m/z 1107.4058, 1109.4036, 1111.4025 [M + Na]+ calcd for C48H39N8O12Zn2; found 1107.4058.

X-ray crystallographic data of 1: Dizinchydroxyneoaspergillin (1) was crystallized from methanol to give white crystal; crystal data: monoclinic, space group P21 with a = 11.8599 (10) Å, b = 24.2844 (3) Å, c = 29.9249 (3) Å, V = 8618.52 (16) Å3, Z = 2, Pcalc = 1.217 g/cm3, R = 0.0923, wR2 = 0.2505; flack parameter, 0.050(12). Crystallographic data (excluding structure factors) for structure 1 in this paper have been deposited to Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1875006. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, U.K. [fax: +44 (0)-1223-336033 or email: deposit@ccdc.cam.ac.uk].

Aspernone A (2): yellow crystal; [α]25 D – 23.1 (c 0.1 MeOH); UV (MeOH) λmax (log ε): 252 (3.60), 201 (3.55) nm; ECD (0.3 mg/mL, MeOH) λmax (Δε) 285 (−2.18), 247 (+1.66), 210 (−0.59) nm; 1H (CD3OD, 700 MHz) and 13C NMR (CD3OD, 175 MHz) data, Table ; HRESIMS at m/z 271.1551 [M + H]+ calcd for C14H23O5; found, 217.1540.

X-ray crystallographic data of 2: Aspernone A (2) was crystallized from methanol to give white crystal; crystal data: monoclinic, space group P21 with a = 10.3118 (2) Å, b = 6.4245 (10) Å, c = 11.7079 (2) Å, V = 746.90 (2) Å3, Z = 2, Pcalc = 1.202 g/cm3, R1 = 0.0380, wR2 = 0.0980; flack parameter, −0.03(11). Crystallographic data (excluding structure factors) for structure 2 in this paper have been deposited to Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1914669. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, U.K. [fax: +44 (0)-1223-336033 or email: deposit@ccdc.cam.ac.uk].

Aspernone B (3): yellow oil; [α]25 D – 2.6 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 260 (3.44), 202 (3.54) nm; ECD (0.6 mg/mL, MeOH) λmax (Δε) 305 (+0.86), 262 (−2.07), 211 (+1.55); 1H (CDCl3, 700 MHz) and 13C NMR (CDCl3, 175 MHz) data, Table ; HRESIMS at m/z 301.1524 [M + Na]+ calcd for C15H24NaO5; found, 307.1516.

Aspernone C (4): yellow oil; [α]25 D – 16.3 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 256 (4.00), 201 (3.68) nm; ECD (0.3 mg/mL, MeOH) λmax (Δε) 306 (+1.30), 256 (−5.57), 212 (3.61); 1H (CD3OD, 700 MHz) and 13C NMR (CD3OD, 175 MHz) data, Table ; HRESIMS at m/z 301.1643 [M + H]+ calcd for C15H25O6; found, 301.1646.

Aspernone D (5): yellow oil; [α]25 D + 67.3 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 254 (4.15), 219 (3.33) nm; ECD (0.2 mg/mL, MeOH) λmax (Δε) 287 (−5.75), 255 (+17.47), 202 (−5.90); 1H (CD3OD, 700 MHz) and 13C NMR (CD3OD, 175 MHz) data, Table ; HRESIMS at m/z 257.1390 [M + H]+ calcd for C13H21O5; found, 257.1384.

Aspernone E (6): yellow oil; [α]25 D + 7.1 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 273 (3.64), 211 (3.56) nm; ECD (0.2 mg/mL, MeOH) λmax (Δε) 320 (−0.87), 266 (+3.23), 245 (−4.20), 229 (−1.61), 210 (1.91); 1H (CD3OD, 700 MHz) and 13C NMR (CD3OD, 175 MHz) data, Table ; HRESIMS at m/z 171.0631 [M + H]+ calcd for C8H11O4; found, 171.0652.

Biological Assay

Antibacterial Assays

The antibacterial activities against three Gram-positive bacteria, methicillin-resistant Staphyloccocus aureus, Staphyloccocus aureus, and Enterococcus faecalis and three Gram-negative bacteria, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumonia were evaluated by an agar dilution method.[23] The tested strains were cultivated in Luria broth (LB) agar plates for bacterial growth at 37 °C. The dimeric zinc complex and neoaspergillic acid analogs (1 and 11–14), and positive controls (ampicillin, gentamicin) were dissolved in MeOH at the concentration of 100 μg/mL. A 10 μL quantity of test solution was absorbed by a paper disk (5 mm diameter) and placed on the assay plates. After 24 h incubation, zones of inhibition (mm in diameter) were recorded. If the inhibition zone was observed, the compounds are diluted to different concentrations by the continuous twofold dilution methods. The minimum inhibitory concentrations (MICs) were defined as the lowest concentration at which no microbial growth could be observed.

Cytotoxic Assays

Cytotoxic activities (five human cancer cell lines: K562, BEL-7042, SGC-7901, A549, and HeLa cells) were evaluated using the CCK-8 method as described previously.[11]

ECD Calculations

The theoretical calculation of new compounds 4–6 was performed as described previously.[27,28]