Neuronal Modulators from the Coral-Associated Fungi Aspergillus candidus.

Three new p-terphenyl derivatives, named 4″-O-methyl-prenylterphenyllin B (1) and phenylcandilide A and B (17 and 18), and three new indole-diterpene alkaloids, asperindoles E-G (22-24), were isolated together with eighteen known analogues from the fungi Aspergillus candidus associated with the South China Sea gorgonian Junceela fragillis. The structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic analysis, and DFT/NMR and TDDFT/ECD calculations. In a primary cultured cortical neuronal network, the compounds 6, 9, 14, 17, 18 and 24 modulated spontaneous Ca2+ oscillations and 4-aminopyridine hyperexcited neuronal activity. A preliminary structure-activity relationship was discussed.

1. Introduction

Fungi of Aspergillus candidus is found to be wide-spread in soil [1] and marine environments [2,3,4], and also co-existing with various animals and plants [5,6,7,8]. The fungi are reported to produce prolific secondary metabolites, including p-terphenyls [9,10,11], terpenes [3], flavonoids [7,12,13], cyclopeptides [6], alkaloids [5], polyketones, and fatty acids [3]. These metabolites display a wide spectrum of biological activities, including cytotoxic [5,8], antibacterial [5,6], antifungal [3,8], antioxidant [6], and immunosuppressive activities [14].

As part of our continuing search for bioactive molecules from marine invertebrates and the associated fungi [15,16,17], a strain of A. candidus was isolated from the internal tissues of the gorgonian coral Junceela fragillis, collected from the Xisha area of the South China Sea. Chemical investigation of the fermentation extract of this fungus resulted in the isolation of three new p-terphenyl derivatives and three new indole-diterpene alkaloids, together with eighteen known analogues. Further, p-Terphenyls are regarded as the main metabolites of A. candidus. More than 230 analogues have been reported up to date, with the chemical diversity being attributed to the substituents on rings A and C [8,18,19]. The indole-diterpene alkaloids are a cluster of characteristic metabolites from this genus and were firstly reported from the titled fungi [20]. These metabolites are structurally constructed with an indole molecule and a saculatane diterpenoid. The biosynthetic process of both types of metabolites were conducted by investigating 6′-hydroxy-4,2′,3′,4″-tetra-methoxy-p-terphenyl [18,19,21] for p-terphenyls, and penitrem [22] and lolitrems [23,24] for the indole-diterpene alkaloids. The bioactivities and the intriguing ring-systems of these molecules also attracted attention for total synthesis [19,25,26]. Herein, we report the isolation, structure elucidation, and neuronal modulatory activity of these compounds.

2. Results

The A. candidus was cultivated on biomalt agar medium and then extracted with EtOAc. The EtOAc extract was subjected to the usual workup for isolation [15,16,27] to yield compounds 1–24 (Figure 1), including eighteen p-terphenyl derivatives and six indole-diterpene alkaloids. Based on the spectroscopic analysis and comparison with the reported data, the known compounds were determined as 3″-hydroxyl-prenylterphenyllin (2), 3-methoxy-4″-deoxyterprenin (3), 3-hydroxyterphenyllin (4) [28], 4-O-methylprenyl-terphenyllin (5) [9], terphenyllin (6) [12,28], 3-methoxyterprenin (7) [14], prenylterphenyllin J (8) [8], 3,3″-dihydroxy-6′-desmethyl terphenyllin (9) [29], deoxyterhenyllin (10) [30], prenylterphenyllin (11) [4], prenylterphenyllin B (12) [11], candidusins A (13) and B (14) [31], prenylcandidusin A (15) [11], 4-methyl-candidusin A (16) [32], and asperindoles A (19), C (20) and D (21) [20]. A complete NMR assignment is presented for the compounds 2 and 3 since there is no report for the NMR data of both the compounds. The assignments are fully supported by those of co-occurring prenylterphenyllin (11) [4] and 3-methoxyterprenin (7) [14], two analogues once reported from the soil-derived strains of the same species.

Figure 1

Structures of compounds 1–24.

Further, 4″-O-methyl-prenylterphenyllin B (1) was obtained as a colorless amorphous solid. Its molecular formula was determined as C26H28O5 by HRESIMS, requiring 13 degrees of unsaturation. The IR spectrum displayed absorptions for hydroxy (3357 cm−1) and substituted benzol (1609, 1462, 834, and 815 cm−1) functionalities. The presence of benzol rings was supported by the strong UV absorptions at 276, 248, and 209 nm. The NMR spectra of 1 displayed resonances for twenty sp2 carbons and six sp3 carbons, taking into account the ten degrees of unsaturation. The remaining three degrees of unsaturation assigned to the ring system of this molecule were in agreement with that of the terphenyl framework. Its NMR data were almost identical to those of the co-isolated prenylterphenyllin B (12) [11], except for the appearance of an additional methoxy group (δH 3.89, 3H, s; δC 55.6, CH3). The methoxy group was assigned as 4″-OMe via its HMBC correlations to C-4″, and further confirmed by its NOE correlation with H-5″ (Figure S1). The structure of compound 1 was therefore determined as 4″-O-methyl-prenylterphenyllin B.

Phenylcandilide A (17), obtained as a yellow amorphous solid, had a molecular formula of C18H18O5 as deduced from the HRESIMS, indicating 10 degrees of unsaturation. The NMR data of 17 showed similarity to those of 4-methyl-candidusin A (16) [32], regarding the signals for the phenol ring C and benzofuran ring B. The C-2 to C-5 diene fragment of the phenol ring A in 16, however, was degraded to a methyl and a hydroxymethyl groups in 17. The distinct HMBC correlations from H3-2 to C-1′ and C-6, and H2-5 to C-6 and C-1 confirmed the location of 1-Me and 6-CH2OH (Figure 2). The structure of 17 was determined and nominated as phenylcandilide A.

Figure 2

Selected COSY and HMBC correlations of compounds 17 and 18.

Phenylcandilide B (18) was obtained as a yellow amorphous solid. Its molecular formula was established as C20H20O6 on the basis of the HRESIMS. Comparison of its NMR data (Table 1) with those of 17 revealed a similarity in the structures. A difference was recognized for the signals of the hydroxymethyl group in 17 being replaced by a methyl acetate subunit in 18, confirmed by the IR at 1740 cm–1. The location of the methyl acetate subunit was indicated by the diagnostic HMBC correlations from H2-5 to C-4, C-1 and C-6, and 4-OMe to C-4 (Figure 2). The structure of compound 18 was thus determined and nominated as phenylcandilide B.

Table 1

1H and 13C NMR data for 1, 17 and 18.

Position	1 (in CDCl3)		17 (in DMSO)		18 (in DMSO)
	δHa (J in Hz)	δCb, Type	δHc (J in Hz)	δCb, Type	δHa (J in Hz)	δCb, Type
1		125.7, C		111.7, C		112.6, C
2	7.36, d (8.5)	132.2, CH	2.30, s	9.5, CH3	2.25, s	9.4, CH3
3	6.92, d (8.5)	115.2, CH
4		154.8, C				169.4, C
5	6.92, d (8.5)	115.2, CH	4.51, d (5.7)	53.5, CH2	3.93, s	31.6, CH2
6	7.36, d (8.5)	132.2, CH		152.2, C		145.8, C
1′		116.1, C		119.1, C		119.0, C
2′		147.3, C		146.9, C		146.9, C
3′		138.9, C		135.9, C		135.7, C
4′		132.8, C		129.0, C		128.9, C
5′	6.46, s	104.0, CH	6.57, s	105.0, CH	6.58, s	105.2, CH
6′		153.5, C		149.6, C		149.3, C
1″		130.3, C		128.6, C		128.6, C
2″	7.44, d (2.5)	130.0, CH	7.38, d (8.7)	130.3, CH	7.37, d (8.5)	130.3, CH
3″		130.2, C	6.83, d (8.7)	115.0, CH	6.83, d (8.5)	115.0, CH
4″		157.0, C		156.5, C		156.5, C
5″	6.93, d (8.7)	110.3, CH	6.83, d (8.7)	115.0, CH	6.83, d (8.5)	115.0, CH
6″	7.45, dd (8.7, 2.5)	127.2, CH	7.38, d (8.7)	130.3, CH	7.37, d (8.5)	130.3, CH
1‴	3.38, d (7.5)	28.7, CH2
2‴	5.36, t (7.5)	122.5, CH
3‴		132.8, C
4‴	1.73, s	18.0, CH3
5‴	1.75, s	26.0, CH3
4-OMe					3.66, s	52.1, CH3
3′-OMe	3.45, s	60.8, CH3	3.71, s	60.4, CH3	3.68, s	60.3, CH3
6′-OMe	3.74, s	56.1, CH3	3.85, s	55.7, CH3	3.85, s	55.7, CH3
4″-OMe	3.89, s	55.6, CH3
5-OH			5.28, t (5.7)
2′-OH	5.94, s
4″-OH			9.51, s		9.48, s

a 500 MHz. b 125 MHz. c 600 MHz.

Asperindole E (22) was obtained as an optically active, white powder. Its molecular formula was established as C27H31NO5 by HRESIMS, implying 13 degrees of unsaturation. The IR spectrum of 22 displayed absorptions for hydroxy (3360 cm–1) and substituted benzol (1632, 1468, 800, and 742 cm−1) functionalities. The presence of an α,β-unsaturated ketone moiety was suggested by the characteristic IR absorption at 1658 cm−1. The strong UV absorptions at 279, 268, 230 and 210 nm were in agreement with the presence of a benzol ring or an α,β-unsaturated ketone moiety in the structure. The NMR spectra (Table 2) demonstrated a great similarity to the known metabolites of asperindoles A–D, previously obtained from an ascidian-derived fungus Aspergillus sp. [20], suggesting the same indole-diterpene framework for these molecules. Signals for the acetyl group in asperindole B were not observed for 22. The structure of 22 was suggested to be the deacetyl analogues of asperindole B, which was fully confirmed by 2D NMR experiments, particularly HMBC and NOESY (Figure 3). The absolute configurations of 22 were the same as that of asperindoles A–D on the basis of the similar ECD spectra (Figure 4). The structure of compound 22 was thus determined as asperindole E.

Table 2

1H and 13C NMR data for 22–24.

Position	22 (in CDCl3)		23 (in CDCl3)		24 (in DMSO)
	δHa (J in Hz)	δCb, Type	δHc (J in Hz)	δCb, Type	δHa (J in Hz)	δCb, Type
1-NH	7.70, s		7.72, s		10.96, s
2		152.0, C		152.8, C		153.9, C
3		52.0, C		52.1, C		50.4, C
4		39.4, C		39.4, C		42.7, C
5α	1.87, dd(11.5, 9.2)	27.3, CH2	1.84, ov d	27.2, CH2	2.37, dd(17.0, 6.0)	30.6, CH2
5β	2.65, dd (11.5, 9.5)		2.60, ov d		3.02, d (17.0)
6α	2.23, dd (13.0, 9.2)	31.1, CH2	2.12, dd (13.2, 9.0)	30.7, CH2	5.69, m	111.5, CH
6β	2.66, dd (13.0, 9.5)		2.61, ov d
7		94.5, C		94.0, C		145.0, C
9	4.05, d (2.0)	84.0, CH	4.81, d (2.4)	79.2, CH	4.25, d (1.5)	82.1, CH
10		196.5, C		196.0, C		194.7, C
11	6.24, s	120.7, CH	6.22, s	120.4, CH	5.84, s	116.1, CH
12		158.9, C		159.8, C		154.5, C
13		78.9, C		78.8, C		73.8, C
14α	2.00, ovd	33.9, CH2	2.00, ov d	33.8, CH2	1.93, ovd	31.7, CH2
14β	1.97, ovd		1.96, dd (13.2, 4.8)		1.83, ovd
15α	2.08, dd (13.0, 4.0)	21.3, CH2	2.09, ov d	21.3, CH2	1.92, ovd	21.1, CH2
15β	1.82, m		1.82, ov d		1.66, m
16	2.83, m	48.6, CH	2.82, m	48.7, CH	2.72, m	49.3, CH
17α	2.45, dd(13.2, 11.0)	27.7, CH2	2.43, dd(13.2, 10.2)	27.7, CH2	2.33, dd(13.0, 10.5)	26.8, CH2
17β	2.74, dd(13.2, 6.5)		2.71, dd(13.2, 6.0)		2.61, dd(13.0, 7.0)
18		117.4, C		117.5, C		115.3, C
19		125.3, C		123.9, C		123.3, C
20	7.43, dd (6.7, 2.0)	118.7, CH	7.31, d (8.4)	119.3, CH	7.28, d (8.5)	118.9, CH
21	7.08, td (6.7, 2.0)	119.9, CH	7.04, dd (8.4, 1.8)	120.4, CH	6.92, dd (8.5, 2.0)	118.8, CH
22	7.10, td (6.7, 2.0)	120.7, CH		126.4, C		123.9, C
23	7.31, dd (6.7, 2.0)	111.7, CH	7.28, d (1.8)	111.6, CH	7.26, d (2.0)	111.3, CH
24		139.9, C		140.2, C		140.3, C
25	1.38, s	16.4, CH3	1.37, s	16.4, CH3	1.29, s	16.4, CH3
26	1.16, s	24.5, CH3	1.14, s	24.5, CH3	0.99, s	19.7, CH3
27		66.8, C		76.9, C		74.3, C
28α	3.64, dd (12.0, 2.0)	68.2, CH2	4.21, dd (13.2, 2.4)	65.0, CH2	3.88, s	67.9, CH2
28β	3.75, d (12.0)		3.69, d (13.2)
29	1.07, s	18.9, CH3	1.37, s	17.3, CH3	1.30, s	21.9, CH3
1′				176.9, C		175.5, C
2′				72.5, C		71.3, C
3′			1.52, s	27.4, CH3	1.29, s	27.3, CH3
4′			1.53, s	27.3, CH3	1.29, s	27.3, CH3
13-OH					4.98, s
27-OH					5.06, d (1.5)
2′-OH					5.26, s

a 500 MHz. b 125 MHz. c 600 MHz. d overlapped signals.

Figure 3

Selected COSY, HMBC, and NOESY correlations of compounds 22–24.

Figure 4

Experimental ECD data of compounds 20 and 22–24 in MeCN.

Asperindole F (23), obtained as an optically active, white powder, has a molecular formula of C31H36ClNO7 as determined by HRESIMS. The presence of a chlorine atom in the molecule was indicated by the isotopic peaks at m/z 568/570 [M − H]− with a ratio of 3:1. The NMR spectra of 23 (Table 2) resembled those of asperindole C (20) [20] (Table S1), except for the acetyl group which is absent, showing the same difference pattern as that between asperindole E (22) and B. Compound 23 is the deacetylated derivative of asperindole C, and was named as asperindole F. This assignment was further confirmed by NMR and ECD experiments (Figure 3 and Figure 4).

Asperindole G (24) was obtained as an optically active, white powder. Its HRESIMS gave the same molecular formula as that of asperindole F (23). As expected, the NMR data of 24 (Table 2) showed similarity to those of 23. However, two sp3 carbon atoms (δ 94.0, C; δ 30.7, CH2) in 23 were replaced by two sp2 carbon atoms (δ 145.0, C; δ 111.5, CH) in 24, suggesting a cleavage of the ether bridge of ring G in 23 to form a C-6 to C-7 double bond, and a 28-hydroxymethyl group in 24. The assignment for the C-6 to C-7 double bond was confirmed by the HMBC correlations of H-6 with C-4 and C-12, and H-9 with C-7. In addition, the α-hydroxyisobutyrate moiety was attached to the C-28 methylene group of the side-chain instead of the C-27 of the bridged 1,3-dioxane ring (as those in 19–23), as confirmed by the diagnostic HMBC correlations between H2-28 and the ester carbonyl carbon (δ 175.5, C). Compound 24 was expected to have the same stereochemistry as that of 19–23 due to their obvious correlations in biogenetic origin, even though the ECD spectrum of 24 was significantly different from those of 20–23. However, a very weak NOE correlation was observed between H-9 and H3-26 in the NOESY spectrum of 24. This suggested that an epimerization might occur at C-9, which is adjacent to the C-10 ketone group. In order to determine the absolute configuration of C-9, TDDFT-ECD [33] and DFT-NMR calculations [34] were performed on the (3S,4R,9R,13S,16S,27S) and (3S,4R,9S,13S,16S,27S) epimers. Ring F, containing an α,β-unsaturated carbonyl chromophore, was expected to have a major impact on the high-wavelength ECD transitions, and thus a large difference was expected between the ECD spectra of the two epimers. The initial Merck molecular force field (MMFF) conformational search resulted in 353 and 156 conformers in a 21 kJ/mol energy window for (3S,4R,9R,13S,16S,27S)-24 and (3S,4R,9S,13S,16S,27S)-24, respectively. The re-optimization of these conformers yielded 15 and 17 low-energy conformers over the 1% Boltzmann distribution at the ωB97X/TZVP [35] PCM/MeCN level (Figures S83 and S84). Unexpectedly, the Boltzmann-weighted ECD spectra of both epimers computed at various levels reproduced well the major transitions of the experimental ECD spectrum, indicating that the contribution of ring E is dominant. However, the (9R) epimer could reproduce the 225 nm positive Cotton effect (CE) with a blue shift (Figure 5), while the (9S) epimer had only a negative computed CE below 250 nm (Figure 6). This small difference suggested that 24 had (9R) absolute configuration, and the difference in the experimental ECD spectra of 20–23 and 24 derives from the different chromophore systems and different planar structures. It is well-documented that even small structural changes can result in markedly different or mirror-image ECD spectra for homochiral derivatives by changing the preferred conformation or electronic properties of the molecule [36]. The (9R) and (9S) epimers were further distinguished by 13C NMR DFT calculations, which has been proven an efficient method to distinguish diastereomeric natural products [17,34]. For the NMR calculations, the above MMFF conformers were re-optimized at the B3LYP/6-31 + G(d,p) level yielding 16 and 14 low-energy structures above the 1% Boltzmann distribution, respectively. Despite the DMSO solvent and the presence of the halogen, both causing larger deviations in the computed data, the calculated 13C NMR shifts of the (9R) epimer had a slightly smaller MAE average value than those of the (9S) epimer, and the DP4+ statistical analysis [35,37] resulted in an 83.75% confidence for the (9R) epimer (Table S2). Since both the computed ECD and NMR data suggested (9R) configuration, the H-9 and H3-26 NOE cross-peak must be an artefact, and the absolute configuration of 24 was determined to be (3S,4R,9R,13S,16S,27S). The interatomic distance of H-9 and H3-26 is 3.7 Å in the (9S) epimer and it is above 5.0 Å in the (9R) epimer.

Figure 5

Experimental ECD spectrum of 24 in MeCN compared with the Boltzmann-weighted CAM-B3LYP/TZVP PCM/MeCN ECD spectrum of (3S,4R,9R,13S,16S,27S)-24. Level of optimization: ωB97X/TZVP PCM/MeCN. Bars represent the rotatory strength values of the lowest-energy conformer.

Figure 6

Experimental ECD spectrum of 24 in MeCN compared with the Boltzmann-weighted CAM-B3LYP/TZVP PCM/MeCN ECD spectrum of (3S,4R,9S,13S,16S,27S)-24. Level of optimization: ωB97X/TZVP PCM/MeCN. Bars represent the rotatory strength values of the lowest-energy conformer.

All the isolated compounds were evaluated for their neuronal modulatory activities by testing their effect on spontaneous Ca2+ oscillations (SCOs), and the seizurogenic agent 4-aminopyridine (4-AP) induced hyperexcitation in primary cultured neocortical neurons (Table 3). SCOs play a crucial role in mediating neuron development, and are closely associated with neuronal excitable and inhibitory neuronal transmission [38,39,40]. The compounds with modulatory activity on SCOs may have potential in drug candidates for treating neurological diseases such as epilepsy, pain and depression [41].

Table 3

Compounds influence SCOs and 4-AP-induced SCOs.

Compounds	SCOsEC50 (μM, Mean ± SEM)		4-AP-induced SCOsEC50 (μM, Mean ± SEM)
	Amplitude	Frequency	Amplitude	Frequency
6	10.28 ± 1.22	6.96 ± 0.73	28.45 ± 1.65	27.08 ± 2.94
17	3.86 ± 0.06	2.32 ± 0.67	3.70 ± 2.11	1.90 ± 1.04
18	1.85 ± 0.21	2.68 ± 0.04	3.88 ± 0.09	3.67 ± 0.01
24	5.62 ± 1.39	4.77 ± 0.14	6.05 ± 0.83	3.49 ± 0.51
9	7.48 ± 0.09	5.32 ± 3.92 I	N/T	N/T
14	2.40 ± 0.57	0.26 ± 0.08 I	N/T	N/T

Data represent mean values of five independent experiments; “N/T” means not tested. ”I” indicates increase in the SCO frequency.

In the present study, we found that four compounds 6, 17, 18 and 24 inhibited SCO activity, and 4-AP induced hyperexcitability by decreasing the SCO amplitude and frequency in the primary cultured cortical neuronal network. However, compounds 9 and 14 produced a more complicated Ca2+ response. Their concentration dependently increased the SCO frequency with the concurrent suppression of the SCO amplitude at concentrations below 10 μM and 3 μM, respectively, and transiently increased the intracellular Ca2+ concentration which recovered to basal level within 5 min at concentrations of 30 μM and 10 μM, respectively (Table 3, and Figures S85–S90). For the cluster of p-terphenyl derivatives, all the active compounds have hydroxyls for both R2 and R4 and those with hydrogens for both R1 and R3 displayed the strongest activity. Substitution for one of the R2/R4 pair of hydroxyls or one of the R1/R3 pair of hydrogens will decrease the activity. Interestingly, the degradation of ring A to a hydroxymethyl group may lead to an increase in activity. For the cluster of indole-diterpene alkaloids, ring cleavage on the ether bridge of ring G seems critical for the activity since all those compounds that have ring G are not active.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were determined with a Rudolph Autopol VI polarimeter. UV spectra were recorded on a Shimadzu UV-2700 spectrophotometer. IR spectra were recorded on a Bruker TENSOR II spectrophotometer. The ECD spectra were measured with a Jasco-715 spectropolarimeter. NMR spectra were recorded on Bruker DRX-600 and DRX-500 spectrometers, and the signals of residual CHCl3 (δH 7.26 ppm; δC 77.0 ppm), DMSO (δH 2.50 ppm; δC 39.6 ppm) and CH3OH (δH 3.31 ppm; δC 49.0 ppm) were used as references for chemical shifts. The HR-ESI-MS data were recorded on an Agilent 1290-6545 UHPLC-Q-TOF-MS spectrometer. Semipreparative HPLC was performed using an Agilent Technology 1100 system with an ODS column (YMC Pack ODS-A, 10 × 250 mm, 5 μM). Column chromatography (CC) was performed with Sephadex LH-20 gel (SE-751 84 Uppsala, GE Healthcare Bio-Sciences AB) and silica gel (200–300 mesh, 400–600 mesh; Yantai, China), respectively. The thin-layer chromatography (TLC) experiments were conducted with silica gel plates (HSGF-254, Yantai, China), and detected by heating after spraying with anisaldehyde sulfuric acid reagent.

3.2. Fungal Material

The fungal strain SG-8-⑤ was isolated from the internal tissues of the gorgonian coral J. fragillis, which was collected from the Xisha area of the South China Sea, and identified as A. candidus by 18sRNA sequence (GenBank accession number AB008396.1). The fungus was deposited in Tongji University, Shanghai, China.

3.3. Culture, Extraction and Isolation

The fungal strain A. candidus was cultivated on 20.0 L of 5% w/v biomalt (Villa Natura, Germany) solid agar medium (20.0 g/L biomalt, 15.0 g/L agar, 800 mL/L artificial seawater) at room temperature for 28 days. The fungal mycelia with the medium were extracted with EtOAc (4.0 L × 5) under ultrasonic conditions. The EtOAc extract was concentrated under vacuum (13.6 g) and was separated into thirteen fractions (Fr.1–13) by silica gel CC, eluting with a gradient CH2Cl2/MeOH (v/v 100:0 to 4:1). Fraction 2 (52.7 mg) was subjected to a Sephadex LH-20 CC (CH2Cl2/MeOH 2:1) and then purified by HPLC (MeOH/H2O 89:11, 1.5 mL/min) to give 3 (1.3 mg, tR 18 min). Fraction 5 (29.9 mg) was subjected to a Sephadex LH-20 CC (CH2Cl2/MeOH 2:1) and HPLC (MeOH/H2O 80:20, 2.0 mL/min) to afford 5 (3.1 mg, tR 24 min). Fraction 6 (644.9 mg) was subjected to a Sephadex LH-20 CC (CH2Cl2/MeOH 2:1) to give eight subfractions (Fr.6a to Fr.6h). The Fr.6e (215.8 mg) was separated by silica gel CC using a gradient petroleum (PE) in acetone (39:1 to 6:4), then split by HPLC to yield 7 (1.2 mg, MeOH/H2O 79:21, 1.5 mL/min, tR 18 min), 8 (0.9 mg, MeOH/H2O 79:21, 1.5 mL/min, tR 21 min), 1 (1.2 mg, MeOH/H2O 80:20, 2.0 mL/min, tR 33 min), 19 (1.8 mg, MeOH/H2O 80:20, 2.0 mL/min, tR 50 min), 20 (2.3 mg, MeOH/H2O 80:20, 2.0 mL/min, tR 63 min), 21 (1.1 mg, MeOH/H2O 80:20, 2.0 mL/min, tR 36 min). Fr.6f (54.7 mg) was separated by silica gel CC (PE-EtOAc 9:1 to 1:1) and purified by HPLC (MeOH/H2O 80:20, 2.0 mL/min) to afford 23 (1.2 mg, tR 44 min) and 24 (0.7 mg, tR 25 min). Fr.6g (18.7 mg) was purified by HPLC (MeOH/H2O 70:30, 2.0 mL/min) to afford 10 (0.8 mg, tR 23 min) and 22 (0.9 mg, tR 64 min). Fr.6h (52.4 mg) was purified by HPLC (MeOH/H2O 65:35, 2.0 mL/min) to yield 16 (1.6 mg, tR 30 min), 11 (1.5 mg, tR 48 min), and 12 (1.0 mg, tR 51 min). Fraction 8 (164.7 mg) was subjected to a Sephadex LH-20 CC (CH2Cl2/MeOH 2:1) to yield thirteen subfractions (Fr.8a to Fr.8m). Fr.8h (19.8 mg) was further purified by HPLC (MeOH/H2O 70:30, 1.5 mL/min) to afford 6 (3.6 mg, tR 13 min), 2 (0.5 mg, tR 20 min), and 15 (3.1 mg, tR 41 min). Fr.8j (33.4 mg) was purified by HPLC (MeOH/H2O 65:35, 2.0 mL/min) to afford 13 (13.9 mg, tR 18 min). Fraction 9 (364.2 mg) was subjected to a Sephadex LH-20 CC (CH2Cl2/MeOH 2:1) to yield 4 (50.0 mg). Fraction 10 (649.6 mg) was subjected to a Sephadex LH-20 CC (CH2Cl2/MeOH 2:1) to furnish fourteen subfractions (Fr.10a to Fr.10n) and pure compound 14 (11.6 mg). Fr.10g (29.3 mg) was further purified by HPLC (MeOH/H2O 70:30, 2.0 mL/min) to afford 18 (2.0 mg, tR 27.5 min). Fr.10i (84.5 mg) was separated by silica gel CC with the eluent of gradient petroleum ether-ethyl acetate from 9:1 to 1:1, followed by the purification of HPLC (MeOH/H2O 58:42, 2.0 mL/min) to yield 17 (2.5 mg, tR 42 min). Fraction 11 (201.0 mg) was applied on a Sephadex LH-20 CC (CH2Cl2/MeOH 2:1) and purified by HPLC (MeOH/H2O 55:45, 2.0 mL/min) to afford 9 (13.0 mg, tR 11 min).

The 4″-O-methyl-prenylterphenyllin B (1): colorless, amorphous solid; UV (MeCN) λmax (log ε) 276 (3.39), 248 (3.12), 209 (3.64) nm; IR (film) νmax 3357, 2922, 2852, 1659, 1609, 1462, 1246, 1073, 1029, 834, 815 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 419.1870 [M − H]− (calcd for C26H27O5, 419.1864).

The 3″-hydroxyl-prenylterphenyllin (2): colorless, amorphous solid; UV (MeCN) λmax (log ε) 277 (3.28), 250 (3.13), 210 (3.65) nm; IR (film) νmax 3346, 2922, 2853, 1665, 1607, 1461, 1260, 1093, 799, 722 cm−1; 1H NMR (CD3OD, 500 MHz): δH 7.11 (1H, d, J = 2.2 Hz, H-2″), 7.05 (1H, d, J = 2.2 Hz, H-2), 7.01 (1H, dd, J = 8.2, 2.2 Hz, H-6), 6.96 (1H, dd, J = 8.2, 2.2 Hz, H-6″), 6.83 (1H, d, J = 8.2 Hz, H-5″), 6.77 (1H, d, J = 8.2 Hz, H-5), 6.42 (1H, s, H-5′), 5.37 (1H, m, H-2‴), 3.67 (3H, s, 6′-OMe), 3.41 (3H, s, 3′-OMe), 3.32 (2H, m, H-1‴), 1.73 (3H, s, H-5‴), 1.72 (3H, s, H-4‴); 13C NMR (CD3OD, 125 MHz): δC 154.8 (C, C-4), 149.1 (C, C-2′), 146.1 (C, C-3″), 145.9 (C, C-4″), 140.8 (C, C-3′), 134.2 (C, C-4′), 133.2 (CH, C-2), 132.7 (C, C-3‴), 131.7 (C, C-1″), 130.3 (CH, C-6), 128.2 (C, C-3), 126.3 (C, C-1), 124.2 (CH, C-2‴), 121.5 (CH, C-6″), 119.0 (C, C-1′), 117.2 (CH, C-2″), 116.2 (CH, C-5″), 115.1 (CH, C-5), 105.0 (CH, C-5′), 60.8 (CH3, 3′-OMe), 56.5 (CH3, 6′-OMe), 29.3 (CH2, C-1‴), 26.0 (CH3, C-5‴), 17.9 (CH3, C-4‴); HRESIMS m/z 423.1787 [M + H]+ (calcd for C25H27O6, 423.1802).

The 3-methoxy-4″-deoxyterprenin (3): colorless, amorphous solid; UV (MeCN) λmax (log ε) 275 (3.27), 250 (3.14), 202 (3.68); IR (film) νmax 3356, 2924, 2853, 1664, 1601, 1461, 1266, 1075, 829, 771 cm−1; 1H NMR (CDCl3, 500 MHz): δH 7.64 (2H, dd, J = 7.5, 1.5 Hz, H-6″), 7.46 (2H, td, J = 7.5, 1.5 Hz, H-5″), 7.39 (1H, tt, J = 7.5, 1.5 Hz, H-4″), 7.02 (1H, dd, J = 8.7, 2.0 Hz, H-6), 7.02 (1H, d, J = 2 Hz, H-2), 6.98 (1H, d, J = 8.7 Hz, H-5), 6.50 (1H, s, H-5′), 5.93 (1H, s, 2′-OH), 5.57 (1H, m, H-2‴), 4.64 (2H, d, J = 7 Hz, H-1‴), 3.88 (3H, s, 3-OMe), 3.76 (3H, s, 6′-OMe), 3.46 (3H, s, 3′-OMe), 1.79 (3H, s, H-5‴), 1.75 (3H, s, H-4‴); 13C NMR (CDCl3, 125 MHz): δC 153.6 (C, C-6′), 149.1 (C, C-3), 147.8 (C, C-4), 147.4 (C, C-2′), 139.0 (C, C-3′), 138.2 (C, C-1″), 137.6 (C, C-3‴), 133.0 (C, C-4′), 128.9 (CH, C-2″, 6″), 128.6 (CH, C-3″, 5″), 127.7 (CH, C-4″), 125.4 (C, C-1), 123.0 (CH, C-6), 120.3 (CH, C-2‴), 117.0 (C, C-1′), 114.4 (CH, C-2), 112.6 (CH, C-5), 104.2 (CH, C-5′), 65.8 (CH2, C-1‴), 61.1 (CH3, 3′-OMe), 56.2 (CH3, 6′-OMe), 56.1 (CH3, 3-OMe), 26.0 (CH3, C-5‴), 18.4 (CH3, C-4‴); HRESIMS m/z 421.2027 [M + H]+ (calcd for C26H29O5, 421.2010).

Phenylcandilide A (17): yellow, amorphous solid; UV (MeCN) λmax (log ε) 276 (3.41), 256 (3.16), 242 (3.45), 227 (3.33), 209 (3.50), 203 (3.49) nm; IR (film) νmax 3359, 2922, 2852, 1658, 1469, 1266, 1206, 1091, 831, 738 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 337.1055 [M + Na]+ (calcd for C18H18NaO5, 337.1046).

Phenylcandilide B (18): yellow, amorphous solid; UV (MeCN) λmax (log ε) 275 (3.58), 255 (3.37), 240 (3.61), 227 (3.54), 212 (3.64) nm; IR (film) νmax 3354, 2924, 2853, 1740, 1660, 1497, 1267, 1213, 1172, 1104, 830, 671 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 357.1349 [M + H]+ (calcd for C20H21O6, 357.1333).

Asperindole E (22): white powder; +50.00 (c 0.03, CHCl3); UV (MeCN) λmax (log ε) 279 (2.78), 268 (2.75), 230 (3.45), 210 (3.24) nm; ECD (0.045 mM, MeCN) λmax (Δε) 238 (−20.91) nm; IR (film) νmax 3360, 2920, 2851, 1720, 1658, 1632, 1468, 1260, 1016, 800, 742 cm−1; 1H and 13C NMR data see Table 3; HRESIMS m/z 472.2086 [M + Na]+ (calcd for C27H31NO5Na, 472.2094).

Asperindole F (23): white powder; +40.56 (c 0.03, CHCl3); UV (MeCN) λmax (log ε) 285 (2.89), 267 (2.76), 236 (3.63), 209 (3.23) nm; ECD (0.035 mM, MeCN) λmax (Δε) 242 (−39.92) nm; IR (film) νmax 3358, 2920, 2851, 1730, 1659, 1633, 1467, 1260, 1013, 799, 704 cm−1; 1H and 13C NMR data see Table 3; HRESIMS m/z 568.2120 [M − H]− (calcd for C31H35ClNO7, 568.2108).

Asperindole G (24): white powder; +31.11 (c 0.03, CHCl3); UV (MeCN) λmax (log ε) 303 (2.92), 293 (2.90), 286 (2.91), 270 (2.85), 236 (3.56), 211 (3.33) nm; ECD (0.035 mM, MeCN), λmax (Δε) 373 (−8.91), 362 (−7.78), 322 (+15.46), 269 (+2.49), 241 (−20.56), 227 (+11.34) nm; IR (film) νmax 3349, 2920, 2851, 1730, 1659, 1462, 1260, 1018, 798 cm−1; 1H and 13C NMR data see Table 3; HRESIMS m/z 592.2068 [M + Na]+ (calcd for C31H36ClNO7Na, 592.2073).

3.4. Neuronal Modulatory Activity Assay In Vitro

The neuronal modulatory activities of 1–24 were evaluated by testing the effect on spontaneous Ca2+ oscillations (SCOs) and seizurogenic agent 4-aminopyridine (4-AP)-induced hyperactive SCOs frequency and amplitude in primary cultured neocortical neurons as described previously [16,38]. Neocortical neurons at 9 days in vitro (DIV) were used to investigate the influence of tested compounds on intracellular Ca2+ concentration ([Ca2+]i). Briefly, the neurons were loaded with Fluo-4 for 1 h at 37 °C in Locke’s buffer. After recording the baseline spontaneous Ca2+ oscillations for 5 min, different concentrations of compounds were added to the corresponding well, and the [Ca2+]i was monitored for 15 min using FLIPRtetra®. To test anti-epileptic potential of the meroterpenoids, 4-AP (10 μM) was added and the monitoring for [Ca2+]i was continued for an additional 10 min. The presented data were values of F/F0, where F is the fluorescence intensity at any time point whereas F0 is the basal fluorescence. An event with Δ F/F0 over 0.1 unit was considered to be an SCO. The frequency and amplitude of SCOs were quantified using Origin software (V7.0) from a time period of 5 min after first or second addition of compound or vehicle (0.1% DMSO).

3.5. Computational Section

Mixed torsional/low-frequency mode conformational searches were carried out by means of the Macromodel 10.8.011 software by using the Merck molecular force field (MMFF) with an implicit solvent model for CHCl3 [42]. Geometry re-optimizations were carried out at the B3LYP/6-31 + G(d,p) level in vacuo and the ωB97X/TZVP level with the PCM solvent model for MeCN. TDDFT-ECD calculations were run with various functionals (B3LYP, BH&HLYP, CAMB3LYP, and PBE0) and the TZVP basis was set as implemented in the Gaussian 09 package, with the same or no solvent model as in the preceding DFT optimization step [43]. ECD spectra were generated as sums of Gaussians with 3000 and 2700 cm−1 widths at half-height, using dipole-velocity-computed rotational strength values [44]. NMR calculations were performed at the mPW1PW91/6-311 + G(2d,p) level [45]. Computed NMR shift data were corrected with I = 185.2853 and S = −1.0306 [46]. Boltzmann distributions were estimated from the B3LYP and ωB97X energies. The MOLEKEL software package was used for visualization of the results [47].

4. Conclusions

From the fungi A. candidus, associated with the South China Sea gorgonian Junceela fragillis, twenty-four metabolites having p-terphenyl and indole-diterpene frameworks were obtained with their structures and absolute configurations being elucidated on the basis of spectroscopic analysis and computational calculations. It was found that small structural changes can result in a markedly different ECD spectra, and 13C NMR DFT calculations are an efficient method to distinguish diastereomeric natural products. Six compounds could modulate SCOs and 4-aminopyridine hyperexcited neuronal activity in the in vitro biotest. A preliminary structure–activity relationship was discussed, which may give a reference for further investigation or chemical optimization of SCO modulators.