Indolediketopiperazine Alkaloids from Eurotium cristatum EN-220, an Endophytic Fungus Isolated from the Marine Alga Sargassum thunbergii

Four new indolediketopiperazine derivatives (1–4), along with nine known congeners (5–13), were isolated and identified from the culture extract of Eurotium cristatum EN-220, an endophytic fungus obtained from the marine alga Sargassum thunbergii. The structures of thesecompounds were elucidated on the basis of extensive spectroscopic analysis and the absolute configurations of compounds 1–4 were established by NOESY experiments and by chiral HPLC analyses of their acid hydrolysates. The absolute configuration of C-8 (a quaternary carbon substituted with a hydroxyl group) in 5 of preechinulin class was firstly determined by electronic circular dichroism (ECD) calculations. All these compounds were evaluatedfor brine shrimp (Artemia salina) lethality and nematicidal activity as well as antioxidativeand antimicrobial potency.


Structural Elucidation of Indolediketopiperazines
The rice culture of E. cristatum EN-220 was exhaustively extracted with EtOAc and the extract was further then purified by a combination of column chromatography on Si gel, Sephadex LH-20, and Lobar LiChroprep RP-18 to obtain subfractions, which were further purified by semi-preparative HPLC (with Elite ODS-BP column) to yield compounds 1-13 ( Figure 1). Compound 1 was obtained as a colorless amorphous powder. The molecular formula C19H23N3O3 was assigned on the basis of HRESIMS ( Figure S2 in the Electronic Supplementary Materials, ESM) and NMR data (Tables S1 and S2 in the ESM). The UV absorptions at λmax 222 and 286 nm suggested the presence of an indole moiety [12]. The 13 C NMR along with the DEPT spectroscopic data (Table S2 in the ESM) revealed the presence of 19 carbon atoms including six quaternary carbons, eight methines (with one olefinic and five aromatic), three methylenes (with one oxygenated), and two methyl groups. Detailed analysis of the NMR spectroscopic data revealed that 1 might be an indolediketopiperazine derivative similar to cyclo(alanyltryptophyl) [23]. However, additional signals atδH4.80/δC44.0 (CH2, C-15), δH 5.62/δC120.2 (CH, C-16), δC140.4 (C, C-17), δH 3.95/δC67.5 (CH2, C-18), and δH 1.84/δC13.9 (Me, C- 19), which corresponding to an isoprenic unit with a terminal hydroxy substitution, were observed in the NMR spectra of 1 (Tables S1 and S2 in the Compound 1 was obtained as a colorless amorphous powder. The molecular formula C 19 H 23 N 3 O 3 was assigned on the basis of HRESIMS ( Figure S2 in the Electronic Supplementary Materials, ESM) and NMR data (Tables S1 and S2 in the ESM). The UV absorptions at λ max 222 and 286 nm suggested the presence of an indole moiety [12]. The 13 C NMR along with the DEPT spectroscopic data (Table S2 in the ESM) revealed the presence of 19 carbon atoms including six quaternary carbons, eight methines (with one olefinic and five aromatic), three methylenes (with one oxygenated), and two methyl groups. Detailed analysis of the NMR spectroscopic data revealed that 1 might be an indolediketopiperazine derivative similar to cyclo(alanyltryptophyl) [23]. However, additional signals atδ H 4.80/δ C 44.0 (CH 2 , C-15), δ H 5.62/δ C 120.2 (CH, C-16), δ C 140.4 (C, C-17), δ H 3.95/δ C 67.5 (CH 2 , C-18), and δ H 1.84/δ C 13.9 (Me, C- 19), which corresponding to an isoprenic unit with a terminal hydroxy substitution, were observed in the NMR spectra of 1 (Tables S1 and S2 in the ESM). This isoprene unit was placed on the nitrogen atom of the indole ring system as evidenced by the observed HMBC correlations from H-15 to C-2 and C-7a ( Figure 2) . The NOE correlations from H-16 to H-18 and from H-15 to H-19 indicated the E-geometry for the C-16 double bond, while the correlation from H-9 to H-12 suggested the cofacial orientation of these protons ( Figure 3). Based on the above evidence, the structure of compound 1 was determined and it was named as N-(4 -hydroxyprenyl)-cyclo(alanyltryptophyl).
Mar. Drugs 2017, 15, 24 3 of 10 ESM). This isoprene unit was placed on the nitrogen atom of the indole ring system as evidenced by the observed HMBC correlations from H-15 to C-2 and C-7a ( Figure 2) . The NOE correlations from  H-16 to H-18 and from H-15 to H-19 indicated the E-geometry for the C-16 double bond, while the  correlation from H-9 to H-12 suggested the cofacial orientation of these protons ( Figure 3). Based on the above evidence, the structure of compound 1 was determined and it was named as N-(4′hydroxyprenyl)-cyclo(alanyltryptophyl).  The HRESIMS data of compound 2 ( Figure S8 in the ESM) demonstrated its molecular formula to be C25H31N3O3, same as that of variecolorin I ( Figure 1) [12], indicating that these two compounds are isomers. Except for signals corresponding to the phenyl nucleus, the NMR data and UV absorptions of these two compounds were very similar. The aromatic proton signals at δH 7.08 (1H, d, J = 8.1 Hz, H-4), 6.83 (1H, d, J = 8.1 Hz, H-5), and 7.20 (1H, s, H-7) indicated that the isoprenic unit was connected at C-6 of the indole moiety of 2, not at C-5 as that of variecolorin I [12]. This deduction was further supported by the HMBC correlations from H-21 to C-5, C-6, and C-7 ( Figure 2). The lower-field-shifted methine proton signal for H-8 at δH 6.99 (1H, s, H-8) of 2 implied that this proton was influenced by the deshielding effect of the C=O group, which suggested the double bond at C-8 has Z-geometry [24]. The zero specific rotation, similar to variecolorin I [12], suggested that 2 was a racemic compound. To further confirm whether compound 2 is a natural product or an artifact due to the use of MeOH during the purification procedures, an experiment simulating the conditions that were used during chromatographic purification procedures was performed. As the diketopiperazine precursor of 2 was not available in our laboratory, the similar compound, variecolorin O (Figure 1), was used in the experiment. A mixture of variecolorin O and Si gel in the solvent CHCl3/MeOH (1:1, v/v) was stirred at room temperature for 72 h and the mixture was checked by HPLC every 24 h. Compound 13, the methylated product of variecolorin O, was analyzed by HPLC at the same time.
The HPLC profiles showed that variecolorin O is very stable and could not be transformed to compound 13 under the experiment conditions ( Figure S26 in the ESM). This result supports that compound 2 is most likely a natural product. Based on the above evidence, the structure of 2 was determined and it was named as isovariecolorin I. ESM). This isoprene unit was placed on the nitrogen atom of the indole ring system as evidenced by the observed HMBC correlations from H-15 to C-2 and C-7a ( Figure 2). The NOE correlations from H-16 to H-18 and from H-15 to H-19 indicated the E-geometry for the C-16 double bond, while the correlation from H-9 to H-12 suggested the cofacial orientation of these protons ( Figure 3). Based on the above evidence, the structure of compound 1 was determined and it was named as N-(4′hydroxyprenyl)-cyclo(alanyltryptophyl).  The HRESIMS data of compound 2 ( Figure S8 in the ESM) demonstrated its molecular formula to be C25H31N3O3, same as that of variecolorin I ( Figure 1) [12], indicating that these two compounds are isomers. Except for signals corresponding to the phenyl nucleus, the NMR data and UV absorptions of these two compounds were very similar. The aromatic proton signals at δH 7.08 (1H, d, J = 8.1 Hz, H-4), 6.83 (1H, d, J = 8.1 Hz, H-5), and 7.20 (1H, s, H-7) indicated that the isoprenic unit was connected at C-6 of the indole moiety of 2, not at C-5 as that of variecolorin I [12]. This deduction was further supported by the HMBC correlations from H-21 to C-5, C-6, and C-7 ( Figure 2). The lower-field-shifted methine proton signal for H-8 at δH 6.99 (1H, s, H-8) of 2 implied that this proton was influenced by the deshielding effect of the C=O group, which suggested the double bond at C-8 has Z-geometry [24]. The zero specific rotation, similar to variecolorin I [12], suggested that 2 was a racemic compound. To further confirm whether compound 2 is a natural product or an artifact due to the use of MeOH during the purification procedures, an experiment simulating the conditions that were used during chromatographic purification procedures was performed. As the diketopiperazine precursor of 2 was not available in our laboratory, the similar compound, variecolorin O ( Figure 1), was used in the experiment. A mixture of variecolorin O and Si gel in the solvent CHCl3/MeOH (1:1, v/v) was stirred at room temperature for 72 h and the mixture was checked by HPLC every 24 h. Compound 13, the methylated product of variecolorin O, was analyzed by HPLC at the same time.
The HPLC profiles showed that variecolorin O is very stable and could not be transformed to compound 13 under the experiment conditions ( Figure S26 in the ESM). This result supports that compound 2 is most likely a natural product. Based on the above evidence, the structure of 2 was determined and it was named as isovariecolorin I. The HRESIMS data of compound 2 ( Figure S8 in the ESM) demonstrated its molecular formula to be C 25 H 31 N 3 O 3 , same as that of variecolorin I ( Figure 1) [12], indicating that these two compounds are isomers. Except for signals corresponding to the phenyl nucleus, the NMR data and UV absorptions of these two compounds were very similar. The aromatic proton signals at δ H 7.08 (1H, d, J = 8.1 Hz, H-4), 6.83 (1H, d, J = 8.1 Hz, H-5), and 7.20 (1H, s, H-7) indicated that the isoprenic unit was connected at C-6 of the indole moiety of 2, not at C-5 as that of variecolorin I [12]. This deduction was further supported by the HMBC correlations from H-21 to C-5, C-6, and C-7 ( Figure 2). The lower-field-shifted methine proton signal for H-8 at δ H 6.99 (1H, s, H-8) of 2 implied that this proton was influenced by the deshielding effect of the C=O group, which suggested the double bond at C-8 has Z-geometry [24]. The zero specific rotation, similar to variecolorin I [12], suggested that 2 was a racemic compound. To further confirm whether compound 2 is a natural product or an artifact due to the use of MeOH during the purification procedures, an experiment simulating the conditions that were used during chromatographic purification procedures was performed. As the diketopiperazine precursor of 2 was not available in our laboratory, the similar compound, variecolorin O (Figure 1), was used in the experiment. A mixture of variecolorin O and Si gel in the solvent CHCl 3 /MeOH (1:1, v/v) was stirred at room temperature for 72 h and the mixture was checked by HPLC every 24 h. Compound 13, the methylated product of variecolorin O, was analyzed by HPLC at the same time. The HPLC profiles showed that variecolorin O is very stable and could not be transformed to compound 13 under the experiment conditions ( Figure S26 in the ESM). This result supports that compound 2 is most likely a natural product. Based on the above evidence, the structure of 2 was determined and it was named as isovariecolorin I. (3) has the molecular formula C 29 H 39 N 3 O 3 as determined by HRESIMS ( Figure S14 in the ESM). The 1D NMR data of 3 (Tables S1 and S2 in the ESM) showed marked similarities to echinulin (11) [23], except that one methyl group at δ C 17.9 (C-30)/δ H 1.80 (H-30) of echinulin disappeared in the NMR spectra of 3. Instead, signals corresponding to an oxymethylene group at δ C 60.0 (C-30)/δ H 4.13 (H-30) were observed in the NMR spectra of 3. The HMBC correlations from H-30 to C-27, C-28, and C-29 verified the structure of 3 ( Figure 2). The double bond at C-27 was determined to have Z-geometry by the observed NOE correlations from H-27 to H-29 and from H-26 to H-30, while NOE from H-9 to H-12 indicated the cis relationship of this proton pair (Figure 3).

30-Hydroxyechinulin
The HRESIMS of 29-hydroxyechinulin (4) ( Figure S20 in the ESM) gave the same molecular formula C 29 H 39 N 3 O 3 as that of 3, and comparison the NMR data (Tables S1 and S2 in the ESM) with those of 3 revealed that compound 4 possessed the same planar structure as that of 3. However, the E-geometry for the double bond at C-27, differed from that in compound 3, was evidenced by the observed NOE correlations from H-27 to H-29 of the oxygenated CH 2 group and from H-26 to H-30 of the methyl group ( Figure 3). Same as that of 3, H-9 and H-12 also has cis relationship as corroborated by the observed NOE correlation among the two protons.
The alanine residue in the 2,5-dikeopiperazine unit of compounds 1, 3 and 4 was determined to have L-configuration by the results from the chiral HPLC analyses (Figure 4) of the acid hydrolysis products [25], as compared with that of the authentic standards, which indicated that the absolute configuration at C-12 of these compounds was S. The S-configuration at C-9 of compounds 1, 3 and 4 was therefore deduced (Figure 3).  (3) has the molecular formula C29H39N3O3 as determined by HRESIMS ( Figure S14 in the ESM). The 1D NMR data of 3 (Tables S1 and S2 in the ESM) showed marked similarities to echinulin (11) [23], except that one methyl group at δC 17.9 (C-30)/δH 1.80 (H-30) of echinulin disappeared in the NMR spectra of 3. Instead, signals corresponding to an oxymethylene group at δC 60.0 (C-30)/δH 4.13 (H-30) were observed in the NMR spectra of 3. The HMBC correlations from H-30 to C-27, C-28, and C-29 verified the structure of 3 (Figure 2). The double bond at C-27 was determined to have Z-geometry by the observed NOE correlations from H-27 to H-29 and from H-26 to H-30, while NOE from H-9 to H-12 indicated the cis relationship of this proton pair (Figure 3).
The HRESIMS of 29-hydroxyechinulin (4) ( Figure S20 in the ESM) gave the same molecular formula C29H39N3O3 as that of 3, and comparison the NMR data (Tables S1 and S2 in the ESM) with those of 3 revealed that compound 4 possessed the same planar structure as that of 3. However, the E-geometry for the double bond at C-27, differed from that in compound 3, was evidenced by the observed NOE correlations from H-27 to H-29 of the oxygenated CH2 group and from H-26 to H-30 of the methyl group ( Figure 3). Same as that of 3, H-9 and H-12 also has cis relationship as corroborated by the observed NOE correlation among the two protons.
The alanine residue in the 2,5-dikeopiperazine unit of compounds 1, 3 and 4 was determined to have L-configuration by the results from the chiral HPLC analyses (Figure 4) of the acid hydrolysis products [25], as compared with that of the authentic standards, which indicated that the absolute configuration at C-12 of these compounds was S. The S-configuration at C-9 of compounds 1, 3 and 4 was therefore deduced (Figure 3). Compound 5, an indolediketopiperazine derivative of preechinulin class, was also isolated in this study. This compound was very recently characterized from a marine-derived fungus Eurotium rubrum by Chen and co-workers and was named as rubrumline M (5) [26]. A literature searching result showed that only two indolediketopiperazine derivatives of preechinulin class, rubrumline M (5) and arestrictin A [27], had been isolated and characterized with a hydroxyl group substituted at C-8. However, the absolute configuration of C-8 (a quaternary carbon substituted with a hydroxyl group) in 5 as well as in arestrictin A had not been determined. To clarify the absolute configuration, the electronic circular dichroism (ECD) quantum chemical calculations in Gaussian 09 [28], was performed. To obtain minimum energy conformers, geometry optimization of each possible isomer of 5 was conducted, and the time-dependent density functional method was then used at the Compound 5, an indolediketopiperazine derivative of preechinulin class, was also isolated in this study. This compound was very recently characterized from a marine-derived fungus Eurotium rubrum by Chen and co-workers and was named as rubrumline M (5) [26]. A literature searching result showed that only two indolediketopiperazine derivatives of preechinulin class, rubrumline M (5) and arestrictin A [27], had been isolated and characterized with a hydroxyl group substituted at C-8. However, the absolute configuration of C-8 (a quaternary carbon substituted with a hydroxyl group) in 5 as well as in arestrictin A had not been determined. To clarify the absolute configuration, the electronic circular dichroism (ECD) quantum chemical calculations in Gaussian 09 [28], was performed. To obtain minimum energy conformers, geometry optimization of each possible isomer of 5 was conducted, and the time-dependent density functional method was then used at the B3LYP/6-31G(d) level to generate calculated ECD spectra of 5. The experimental and calculated ECD spectra for 5 showed excellent agreement for the 8R, 9S, and 12S-absolute configuration in 5 ( Figure 5). Both the calculated and experimental data spectra showed a strong negative Cotton effect (CE) near 220 nm and positive CE around 275 nm. These close similarities enabled assignment of the absolute configuration of 5 as shown in Figure 1.

Bioactivities of Indolediketopiperazines
Brine shrimp (Artemia salina), an aquatic species featuring with the highly sensitive to toxic and easily culturable to researchers, could be used as a model organism for a preliminary and quick screening of the insecticidal activity [30,31]. Previous research of E. cristatum EN-220 showed that the fermentation extract exhibited a significant lethality to brine shrimp [3]. Therefore, in order to search the insecticidal leading compounds, 1-13 were evaluated for the lethal activity against brine shrimp [32,33], and furthermore, nematicidal activity against Panagrellus redivivus [11,34] was evaluated as well (Table 1). In the brine shrimp assay, compounds 2, 8, 9, and 10 showed lethal activity with LD50 values of 19.4, 70.1, 19.8, and 27.1 μg/mL, respectively, while compounds 3, 4, and 7 exhibited weaklethal activities. The nematicidal assay showed that compounds 2, 9, and 10 exhibited weak activity with LD50 values of 110.3, 106.7, and 126.4 μg/mL, respectively. Compounds 9 and 10 were notablely more active against brine shrimp and Panagrellus redivivusthan 7 and 8, which was probably due to the number and position of the isoprenic unit. This deduction was also proved by the structure differences between compounds 2 and 13 compared with their activities. The insecticidal activities of compounds 9 and 10 might also be related to the exocyclic double bond in the 2,5-diketopiperazine moiety compared to the structures of 11 and 12, which did not show any activity. n.a.: no activity. In addition to the isolation of indolediketopiperazine derivatives 1-5, eight other congeners including rubrumazine B (6) [9], neoechinulin B (7) [24], neoechinulin C (8) [24], alkaloid E-7 (9) [29], didehydroechinulin (10) [13], echinulin (11) [23], dehydroechinulin (12) [7], and variecolorin H (13) [12], were also isolated and identified. The structures of these compounds were determined by detailed analysis of their spectroscopic data and by comparison with that reported in the literature.

Bioactivities of Indolediketopiperazines
Brine shrimp (Artemia salina), an aquatic species featuring with the highly sensitive to toxic and easily culturable to researchers, could be used as a model organism for a preliminary and quick screening of the insecticidal activity [30,31]. Previous research of E. cristatum EN-220 showed that the fermentation extract exhibited a significant lethality to brine shrimp [3]. Therefore, in order to search the insecticidal leading compounds, 1-13 were evaluated for the lethal activity against brine shrimp [32,33], and furthermore, nematicidal activity against Panagrellus redivivus [11,34] was evaluated as well (Table 1). In the brine shrimp assay, compounds 2, 8, 9, and 10 showed lethal activity with LD 50 values of 19.4, 70.1, 19.8, and 27.1 µg/mL, respectively, while compounds 3, 4, and 7 exhibited weaklethal activities. The nematicidal assay showed that compounds 2, 9, and 10 exhibited weak activity with LD 50 values of 110.3, 106.7, and 126.4 µg/mL, respectively. Compounds 9 and 10 were notablely more active against brine shrimp and Panagrellus redivivus than 7 and 8, which was probably due to the number and position of the isoprenic unit. This deduction was also proved by the structure differences between compounds 2 and 13 compared with their activities. The insecticidal activities of compounds 9 and 10 might also be related to the exocyclic double bond in the 2,5-diketopiperazine moiety compared to the structures of 11 and 12, which did not show any activity. Based on the reported typical bioactivity of indolediketopiperazines, the isolated compounds were further assessed for antioxidative and antimicrobial activities, with the purpose of enriching the bioactive diversity of these compounds. Compounds 2 and 6-13 were evaluated for antioxidative activities against DPPH and superoxide anion radicals [8,9] (Table 2). Compound 12 showed potent radical scavenging activity against DPPH with IC 50 value of 6.4 µg/mL, which was comparable to that of the positive control ascorbic acid (IC 50 2.0 µg/mL). The other compounds (2, 6-12) exhibited moderate antioxidative activities with IC 50 values ranging from 10.1 µg/mL to 28.5 µg/mL. However, all the tested compounds did not show the superoxide anion radical scavenging activity. Compounds 1-13 were also evaluated for the antimicrobial activities [3,9] against six pathogenic bacteria (Escherichia coli, Staphyloccocus aureus, Bacillus subtilis, Micrococcus luteus, Salmonella enteric, and Bacillus pumilus) and nine plant-pathogenic fungi (Alternaria brassicae, Valsa mali, Physalospora obtuse, Alternaria solania, Sclerotinia miyabeana, Magnaporthe grisea, Fusarium oxysporium, Botryosphaeria dothidea, and Colletotrichum gloeosporioides). Compounds 11, 12, and 13 showed weak activity against S. aureus with the same MIC value of 256 µg/mL, while compound 6 exhibited moderate activity against Magnaporthe grisea with the MIC value of 64 µg/mL.
Indole diketopiperazine alkaloids are characterized by condensation of the tryptophan with a second amino acid such as L-alanine, L-proline, or L-tryptophan, forming the ring of diketopiperazine unit [10]. The isolated compounds of E. cristatum EN-220, featuring with the second amino acid of L-alanine, showed potent brine shrimp lethality and antioxidative activity against DPPH. However, the isolated compounds did not exhibit any antimicrobial activities, which indicated that indolediketopiperazines having alanine residue might have the low relevance to the antimicrobial activity. Bioactive compounds with potent brine shrimp lethality also showed weak nematicidal activity, further proving the application of brine shrimp as a preliminary screening of insecticidal activity.

Fungal Material
The endophytic fungus Eurotium cristatum EN-220 was isolated from the marine alga Sargassum thunbergii collected from the coast of Qingdao, China, in November 2009. The fungus was identified by analysis of the ITS region of the rDNA, as described in our previous report [3]. The sequence data derived from the fungal strain was deposited at GenBank, with accession No. JQ743649. The strain is preserved at the Institute of Oceanology, Chinese Academy of Sciences.

Fermentation, Extraction, and Isolation
For chemical investigations, the fungal strain was statically fermented at r.t. for 30 days on sterilized solid medium containing rice (100 g/flask), peptone (0.6 g/flask), and sea water (100 mL/flask) in 1-L Fernbach flasks (×100). The rice culture of the fungal strain was exhaustively extracted with EtOAc to give a crude extract, which was dried and fractionated by Si gel vacuum liquid chromatography (VLC) using different solvents of increasing polarity from petroleum ether (PE) to MeOH to yield 12 fractions (Fractions (Frs.) 1-12) based on TLC analysis.
Detailed 1 H and 13 C NMR data of compounds 1-4 were shown in Tables S1 and S2 of the ESM, and the main physical and chemical properties could also be viewed in Table S3 of the ESM.

ECD Calculation of Compound 5
Conformational searches for 5 were performed via the molecular mechanics using MM + method in HyperChem 8.0 software (Beijing HuanZhongRuiChi Technology Co., Ltd., Daxing, Beijing, China), and the geometries were further optimized at B3LYP/6-31G(d) level via Gaussian 09 software (Gaussian Inc., Wallingford, CT, USA) to give the energy-minimized conformers. Then, the optimized conformers were subjected to the calculations of ECD spectra using TD-DFT at B3LYP/6-31G(d) level; solvent effects of the MeOH solution were evaluated at the same DFT level using the SCRF/PCM method [28].

Acidic Hydrolysis of Compounds 1 and 3-6
Compound 1 (1 mg) was dissolved in 3 mL of 6 N HCl and heated in a sealed tube at 110 • C for 24 h. The hydrolysate was dried, reconstituted in H 2 O (1 mL), and then subjected to chiral HPLC analysis. The retention times of the authentic amino acids were as follows: D/L-Ala, t R 11.27/8.23 min. The retention time of amino acid in hydrolysate of 1 was 8.23 min, indicating that the amino acid in 1 was L-Ala. By the same procedure, compounds 3-6 gave the same results that the amino acids in these compounds were all L-Ala.

Verification of Compound 2 Being Not an Artifact
As the probable oxidized diketopiperazine precursor of 2 was not isolated in our experiment, the similar compound, variecolorin H (13), as well as its oxidized precursor, variecolorin O [12], were used in the experiment. A sample of variecolorin O (2 mg) was mixed with Si gel (1 g) in 5 mL solvent (CHCl 3 :MeOH = 1:1, v/v) and the mixture was stirred at r.t. for 72 h. The mixture was analyzed using HPLC every 24 h and the retention time was consistent to variecolorin O with 28.5 min, while variecolorin H with 30.9 min.

Conclusions
Thirteen indolediketopiperazine derivatives (1-13), including four new compounds (1-4), were isolated and identified from the culture extract of the marine algal-derived endophytic fungus Eurotium cristatum EN-220. The relative and absolute configurations of compounds 1-4 were established by NOESY experiments and chiral HPLC analyses of their acid hydrolysates. The absolute configuration of 8-quarternary carbon substituted with a hydroxyl group in 5 of preechinulin class was firstly determined by ECD calculations. Compounds 2, 9, and 10 exhibited potent lethal activity against brine shrimp and weak nematicidal effect against Panagrellus redivivus. This is the first report for the brine shrimp inhibition activity of compounds 2, 9, and 10. In addition, compound 12 showed potent radical scavenging activity against DPPH radicals.
Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/15/2/24/s1, 1D and 2D NMR spectra and synthetic tables of the NMR data of compounds l-4 including their physical properties and HPLC profiles for checking the artificial nature of variecolorin O.