Diketopiperazine-Type Alkaloids from a Deep-Sea-Derived

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Diketopiperazine-Type Alkaloids from a Deep-Sea-Derived Aspergillus puniceus Fungus and Their Effects on Liver X Receptor α Xiao Liang,†,‡ Xuelian Zhang,§ Xinhua Lu,§ Zhihui Zheng,§ Xuan Ma,† and Shuhua Qi*,†

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†

CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, Institution of South China Sea Ecology and Environmental Engineering, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, Guangdong 510301, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § New Drug Research & Development Co., Ltd, North China Pharmaceutical Group Corporation, Shijiazhuang, Hebei 050015, People’s Republic of China S Supporting Information *

ABSTRACT: Eight new diketopiperazine-type alkaloids including four oxepin-containing diketopiperazine-type alkaloids, oxepinamides H−K (1−4), and four 4-quinazolinone alkaloids, puniceloids A−D (5−8), together with two known analogues (9 and 10), were isolated from the culture broth extracts of the deep-sea-derived fungus Aspergillus puniceus SCSIO z021. Their structures were elucidated by spectroscopic analyses, and their absolute configurations were determined by Marfey’s method along with comparison of their specific rotations and ECD spectra. The absolute configurations of 4 and 5 were further confirmed by a single-crystal X-ray diffraction analysis. Compounds 1−8 showed significant transcriptional activation of liver X receptor α with EC50 values of 1.7−50 μM, and 7 and 8 were the most potent agonists.

L

K (1−4), and four 4-quinazolinone alkaloids, puniceloids A−D (5−8), together with two known alkaloids, oxepinamides F and D (9 and 10),4 were isolated from the culture broth extracts of the fungal strain SCSIO z021. These compounds were evaluated for their transactivation effects on LXRα as well as their enzyme inhibitory activity against IDO1 (indoleamine 2,3-dioxygenase 1), LDHA (lactate dehydrogenase A), and five different phosphatases, including SHP1, SHP2, PTP1B, TCPTP, and MEG2, and antifungal activity. Herein, we describe the isolation and structure elucidation of the new compounds as well as their bioactivities.

iver X receptors (LXRα and LXRβ) are critical modulators of cholesterol and lipid metabolism, inflammatory responses, and innate immunity.1,2 LXRs are ligandactivated transcription factors that belong to a family of hormone nuclear receptors.1,2 Their important regulating roles have led to expanded interest in developing new smallmolecule modulators for these receptors.3 The vast majority of research conducted to find LXR modulators of therapeutic utility has been directed toward developing LXR agonists.3 LXR agonists have been shown to have potential use in the treatment of atherosclerosis, diabetes, anti-inflammation, and Alzheimer’s disease. Currently, the focus of the field is to develop selective liver X receptor modulators to avoid the undesirable side effects caused by the first generation of LXR modulators.3 In order to explore new bioactive alkaloids from marinederived fungi, we studied the secondary metabolites of the fungus Aspergillus puniceus SCSIO z021 isolated from deep-sea sediments. Eight new alkaloids including four oxepincontaining diketopiperazine-type alkaloids, oxepinamides H− © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The molecular formula of oxepinamide H (1) was assigned as C20H19N3O4 according to HRESIMS and NMR data. The 1H NMR data (Table 1) displayed characteristic signals for a monosubstituted benzene ring at δH 7.02 (d, J = 7.0 Hz, 2H), Received: January 20, 2019

A

DOI: 10.1021/acs.jnatprod.9b00055 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Key HMBC and COSY correlations of compounds 1, 2, 4, 6, and 7.

7.22 (t, J = 7.0 Hz, 1H), and 7.27 (t, J = 7.0 Hz, 2H), four olefinic protons at δH 5.83 (t, J = 5.5 Hz), 6.24 (dd, J = 11.0, 5.5 Hz), 6.31 (d, J = 5.5 Hz), and 6.59 (d, J = 11.0 Hz), one methoxy group at δH 3.02 (s), and one methyl group at δH 0.32 (d, J = 7.0 Hz). The 13C NMR spectral data (Table 1) showed 20 carbon signals including one methyl, one methoxy, one methylene, one methine, nine olefinic or aromatic methines, one oxygenated nonprotonated carbon, four nonprotonated carbons, and two carbonyl carbons. These data showed great similarity to those of 10.4 The only obvious difference between 1 and 10 was the additional presence of one methoxy group in 1. This was supported by the 2D NMR analysis (Figure 1). The HMBC spectrum showed a correlation of δH 3.02 (s, OCH3) with C-3 (δC 89.2, C), suggesting that the additional methoxy group was attached at C-3. The configuration of the Ala subunit was assigned as D-form by Marfey’s method,5 indicating the C-15 R-configuration. The NOESY spectrum (Figure 2) showing NOE correlations of H-15 with 3-OCH3 and H3-16 with H-2′/H-3′ suggested the methyl group of the Ala unit and the benzyl group at C-3 were on the same side of the ring, which indicated the S-configuration for C-3. Oxepinamide I (2) had the same molecular formula of C20H19N3O4 as 1 according to HRESIMS and NMR data. The 1 H and 13C NMR data for 2 were greatly similar to those for 1,

Figure 2. Key NOESY correlations of compounds 1 and 2.

and the only obvious differences between them were the changes of chemical shifts of H3-16 (2: δH 1.47; 1: δH 0.32), C1 (2: δC 168.5; 1: δC 166.3), and C-17 (2: δC 40.3; 1: δC 46.6). The 2D NMR analysis (Figure 1) proved that 2 had the same planar structure as 1. Comparison of the electronic circular dichroism (ECD) spectra (Figure 3) and specific rotation values of 2 and 1 ([α]25D −235 for 2 and [α]25D +10.7 for 1) suggested they were a pair of stereoisomers. The D-Ala residue was established by Marfey’s method, which determined the C15 R-configuration in 2. Correspondingly, the absolute configuration of C-3 was determined as R based on the NOESY spectrum (Figure 2) showing an NOE correlation between H3-16 and 3-OCH3. Analysis of the 3D structures of 1 and 2 (Figure 2) showed that the methyl group (CH3-16) in 1 was located in the shielded region of the benzene ring, explaining the 0.32 ppm chemical shift of H3-16 in 1. However, the methyl group in 2 was not in the shielded region.

Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data for 1−4 1 position

δC, type

1 2-NH 3 3-OCH3/−OH 4 6 8 9 10 11 12 13 15 16 17

89.2, C 50.4, CH3 154.7, C 162.0, C 143.7, CH 117.3, CH 128.7, CH 125.0, CH 110.4, C 159.5, C 51.6, CH 17.3, CH3 46.6, CH2

1′ 2′, 6′ 3′, 5′ 4′

133.8, 130.6, 128.6, 127.5,

2

δH (J in Hz)

166.3, C

δC, type

δH (J in Hz)

168.5, C 9.29, s

C CH CH CH

3 δC, type

3.02, s

6.31, 5.83, 6.24, 6.59,

d (5.5) t (5.5) dd (11.0, 5.5) d (11.0)

4.55, 0.32, 3.11, 3.48,

q d d d

(7.0) (7.0) (13.0) (13.0)

7.02, d (7.0) 7.27, t (7.0) 7.22, t (7.0)

134.2, 130.7, 127.9, 126.8,

C CH CH CH

δH (J in Hz)

168.8, C 9.57, s

87.3, C 50.3, CH3 153.0, C 161.0, C 143.4, CH 117.4, CH 128.9, CH 124.9, CH 110.7, C 159.7, C 51.8, CH 18.4, CH3 40.3, CH2

4 δC, type

9.31, s

10.62, s

82.5, C 3.31, s

δH (J in Hz)

166.1, C 125.6, C 7.41, s

6.30, 5.83, 6.25, 6.59,

d (5.5) t (5.5) dd (11.0, 5.5) d (11.0)

4.55, 1.47, 3.23, 3.61,

q d d d

(7.0) (7.0) (13.5) (13.5)

7.26, d (7.0) 7.20, t (7.0) 7.15, t (7.0) B

155.4, C 161.5, C 143.4, CH 117.4, CH 128.5, CH 125.0, CH 110.1, C 159.8, C 52.0, CH 18.4, CH3 43.0, CH2 135.0, 130.8, 127.8, 126.6,

C CH CH CH

6.29, 5.81, 6.23, 6.60,

d (5.5) t (5.5) dd (11.0, 5.5) d (11.0)

4.66, 1.53, 3.16, 3.73,

q d d d

(7.0) (7.0) (13.5) (13.5)

7.36, d (7.0) 7.20, t (7.0) 7.14, tt (7.0, 2.5)

150.0, C 162.1, C 143.3, CH 117.4, CH 127.7, CH 125.4, CH 109.5, C 159.8, C 51.7, CH 18.1, CH3 117.7, CH 133.1, 129.7, 128.7, 128.7,

C CH CH CH

6.22, 5.79, 6.19, 6.65,

d (5.5) t (5.5) dd (11.0, 5.5) d (11.0)

5.02, q (7.0) 1.48, d (7.0) 7.14, s

7.62, d (7.5) 7.44, t (7.5) 7.36, t (7.5)

DOI: 10.1021/acs.jnatprod.9b00055 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. Comparison of the experimental ECD spectra of compounds 1−8 and 10 in CH3OH.

Oxepinamide J (3) had a molecular formula of C19H17N3O4 based on HRESIMS and NMR spectra. The 1H and 13C NMR data for 3 were greatly similar to those for 2 with the absence of a methoxy group (δC 50.3, δH 3.31) and the additional presence of an active hydrogen (δH 7.41, s) in 3, suggesting a hydroxy replaced a methoxy group attached at C-3 in 3. The 2D NMR spectra confirmed the planar structure of 3 was the same as that of 10. Comparison of the ECD spectra (Figure 3) and specific rotation values of 3 and 10 ([α]25D −212 for 3 and [α]25D +30.2 for 10) indicated that 3 and 10 were a pair of stereoisomers, which was further supported by the NOESY spectrum showing an NOE correlation of H3-16 and 3-OH in 3. The existence of a D-Ala residue was determined by Marfey’s method. So, the absolute configuration of 3 was determined to be the same as that of 2. The molecular formula of oxepinamide K (4) was determined as C19H15N3O3 by HRESIMS and NMR spectra. The 1H and 13C NMR data for 4 were similar to those for 1−3, suggesting that 4 had the same skeleton as 1−3. The most obvious difference between 4 and 1 was the presence of a trisubstituted double bond (δC 125.6 (C), 117.7 (CH)) and the absence of signals for a methoxy, an oxygenated nonprotonated carbon, and a methylene in 4. The HMBC correlations from H-17 (δH 7.14, s) to C-4/C-2′/C-6′ and from NH (δH 10.62, s) to C-3 (δC 125.6, C) located the double bond between C-3 and C-17. The R-configuration was assigned for C-15 by Marfey’s method, and the double bond between C-3 and C-17 was determined to have the Zconfiguration by a single-crystal X-ray diffraction experiment (Figure 4). Puniceloid A (5) exhibited the same molecular formula of C20H19N3O4 as 1 by analyzing the HRESIMS and NMR spectra. The 1H and 13C NMR data for 5 were highly similar to those for 1, but the UV absorptions of 5 and 1 were significantly different, implying they possessed a different conjugated ring systems. Comparison of the 1H and 13C NMR data for 5 and 1 displayed that the main differences between them were the additional presence of a phenolic hydroxy proton (δH 9.99, br s) and a deshielded nonprotonated carbon (C-7: δC 153.1) in 5 instead of an olefinic methine (CH-8 in 1). Besides that, the chemical shifts and the coupling constants of H-8/H-9/H-10 revealed the presence of a 1,2,3trisubstituted benzene ring instead of an oxepine ring in 5. The HMBC correlations (Figure 1) from H-10 to C-6/C-12, from H-9 to C-7/C-11, and from H-8 to C-6 suggested the phenolic hydroxy group was attached at C-7. The relative

Figure 4. X-ray crystallographic structure of compound 4.

configuration of 5 was established by the NOESY correlations between H3-15 and H-2′/H-3′, which indicated CH3-15 and the benzyl group were located on the same side of the ring. The absolute configuration of C-14 was determined as R by Marfey’s method, and correspondingly, C-3 was assigned as the S-configuration. These assignments were further confirmed by a single-crystal X-ray diffraction analysis (Figure 5). Puniceloid B (6) had the molecular formula C19H17N3O4 on the basis of HRESIMS and NMR spectra. The 1H and 13C

Figure 5. X-ray crystallographic structure of compound 5. C

DOI: 10.1021/acs.jnatprod.9b00055 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Data for 5−8 5 position 1 2-NH 3 3-OCH3/-OH 4 6 7 7-OH 8 9 10 11 12 14 15 16 1′ 2′, 6′ 3′, 5′ 4′

δC, type

6 δH (J in Hz)

166.8, C

δC, type 165.6, C

9.24, s 89.5, C 50.0, CH3 147.2, C 135.6, C 153.1, C 119.0, CH 128.3, CH 115.8, CH 120.8, C 159.2, C 50.9, CH 17.9, CH3 46.1, CH2 134.7, 130.9, 128.3, 127.1,

C CH CH CH

7 δH (J in Hz)

δC, type 169.8, C

9.22, s 6.82, br s

br s dd (8.0, 1.5) t (8.0) dd (8.0, 1.5)

4.73, 0.35, 3.12, 4.06,

q d d d

(7.0) (7.0) (12.5) (12.5)

7.06, d (7.0) 7.18, t (7.0) 7.15, m

118.4, CH 128.0, CH 115.8, CH 120.5, C 159.3, C 51.2, CH 17.8, CH3 47.4, CH2 135.2, 130.8, 128.2, 127.0,

C CH CH CH

δH (J in Hz)

166.6, C 10.52, s

83.0, C

126.0, C 7.22, br s

150.8, C 135.6, C 152.7, C 9.99, 7.32, 7.42, 7.54,

δC, type

9.19, s

84.3, C 2.98, s

8 δH (J in Hz)

148.2, C 136.0, C 153.1, C 9.76, 7.30, 7.40, 7.52,

br s dd (8.0, 1.5) t (8.0) dd (8.0, 1.5)

4.61, 0.44, 3.12, 3.91,

q d d d

(7.0) (7.0) (12.5) (12.5)

7.06, d (7.0) 7.18, t (7.0) 7.15, m

118.8, CH 128.1, CH 115.8, CH 120.9, C 159.6, C 51.8, CH 18.3, CH3 43.1, CH2 135.3, 131.4, 127.5, 126.3,

C CH CH CH

144.1, C 136.1, C 152.8, C 10.00, br s 7.30, dd (8.0, 1.5) 7.39, t (8.0) 7.55, dd (8.0, 1.5)

4.83, 1.59, 3.17, 4.26,

q d d d

(7.0) (7.0) (13.5) (13.5)

7.53, d (7.5) 7.12, t (7.5) 7.07, tt (7.5, 2.5)

118.4, CH 128.1, CH 116.0, CH 120.4, C 159.5, C 51.2, CH2 18.2, CH3 117.2, CH 134.0, 129.4, 128.7, 127.6,

C CH CH CH

9.75, 7.26, 7.35, 7.58,

s dd (8.0, 1.0) t (8.0) dd (8.0, 1.0)

5.22, q (7.0) 1.50, d (7.0) 7.74, s

7.65, d (7.5) 7.47, t (7.5) 7.37, m

acid, TFA) as the eluting solvent. However, after concentration under reduced pressure, the four compounds could interconvert, and finally they could transform into 4, which was a stable compound under the acidic conditions. In addition, 5−7 also exhibited a similar phenomenon, and they could slowly transform into 8 in the acidic solution. As we know, chloroform can decompose under light to generate hydrogen chloride and create an acidic environment. When the 1H and 13 C NMR spectroscopic data for 5 were recorded in CDCl3, about 11 h later, it was observed that 5 almost completely converted into 8 in the NMR tube (Figure S72). It is speculated that 4 and 8 might be artificial products derived from 1−3, 10, and 5−7, respectively. As CH3OH was used in the isolation process and compounds 10 and 3 are hemiaminals, there is a strong possibility that 1 and 2 are isolation artifacts, with 10 and 3 being the original natural products. A similar situation exists for compound 5. So, 1, 2, and 5 are also likely artifacts derived from 10, 3, and 6, respectively. The oxepin-containing alkaloids 1−3, 9, and 10, quinazolinone alkaloids 5−7, and their elimination products 4 and 8 exhibited three distinctive UV spectra, respectively (Figures S73 and S74). It is easy to differentiate the skeletons of these kinds of compounds according to their UV characteristics. The known compounds 9 and 10 had been reported to show moderate transcriptional activation of LXRα.6 Therefore, the analogues 1−10 were tested for their transactivation effects on LXRα by the same bioassay methods as reported in the literature.6 The results (Table 3) showed that 1−3 and 5−8 had significant transcriptional activation on LXRα with EC50 values of 1.7−16 μM, and 7 and 8 were the most potent agonists, with EC50 values of 1.7 μM. It was obvious that the transactivation effects of quinazolinone alkaloids 5−8 were more potent than those of oxepine-containing alkaloids 1−4, 9, and 10. The values of corresponding maximum fold induction of 1−8 were from 3.0 to 4.2. Comparison of the structures and transactivation effects on LXRα of 1−10 indicated that when the benzene ring moiety was converted to an oxepin unit in

NMR data for 6 were similar to those for 5 except for the additional presence of a hydroxy proton and the loss of a methoxy group in 6. Further analysis of the HMBC and COSY spectra suggested that 6 had the same planar structure as 5 except for a hydroxy group instead of a methoxy group attached at C-3. The NOE correlations between H3-15 and H2′/H-3′ in the NOESY spectrum of 6 indicated that CH3-15 and the benzyl group were on the same side of the ring. The configuration of C-14 in 6 was determined to be R by Marfey’s method. Correspondingly, the configuration of C-3 in 6 was inferred to be S. Puniceloid C (7) shared the same molecular formula of C19H17N3O4 as 6 on the basis of HRESIMS data. The 1H and 13 C NMR data for 6 and 7 showed great similarity. Analysis of the HMBC and COSY spectra suggested 6 and 7 had the same planar structure. However, the NOESY spectrum of 7 showed an NOE correlation of H3-15 with 3-OH, which indicated that CH3-15 and 3-OH were cofacial. The absolute configuration of C-14 in 7 was also determined to be R by Marfey’s method. Correspondingly, the configuration of C-3 in 7 was inferred to be R. Thus, 6 and 7 were a pair of epimers at C-3. Puniceloid D (8) had the molecular formula C19H15N3O3 based on HRESIMS data. The 1H and 13C NMR data for 8 were similar to those for 5, with the additional presence of a trisubstituted double bond between C-3 (δC 126.0, C) and C16 (δC 117.2, CH) and the absence of signals for a methoxy, an oxygenated nonprotonated carbon, and a methylene in 8. The planar structure of 8 was confirmed as shown by analysis of the HMBC and COSY spectra. The NOESY correlation of H-16 with 7-OH indicated that the double bond between C-3 and C-16 was also of the Z-configuration as that in 4. The configuration of C-14 was determined to be R by Marfey’s method. Compounds 1−3 and 10 were relatively stable in neutral solvent, while they were unstable in acidic solvent. We had attempted to isolate them by preparative HPLC with an ODS column using CH3OH/H2O (containing 0.03% trifluoroacetic D

DOI: 10.1021/acs.jnatprod.9b00055 J. Nat. Prod. XXXX, XXX, XXX−XXX

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instrument. Optical rotations were measured with an MCP 500 polarimeter (Anton Paar). ECD and UV spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd.). IR spectra were recorded with an IR Affinity-1 Fourier transform infrared spectrophotometer (Shimadzu). 1H, 13C, and 2D NMR spectra were acquired with a Bruker AV-500 MHz NMR spectrometer with tetramethylsilane as reference. ESIMS and HRESIMS spectroscopic data were acquired with an amaZon SL ion trap mass spectrometer and MaXis quadrupole-time-of-flight mass spectrometer (Bruker), respectively. The crystallographic data were collected on a Rigaku MicroMax 007 diffractometer equipped with Cu Kα radiation and a graphite monochromator. Semipreparative reversed-phase (SP-RP) HPLC was performed on a Shimadzu LC20A preparative liquid chromatography system with a YMC-Pack ODS column, 250 × 20 mm, S-5 μm, 12 nm. RP-MPLC (reversedphase medium-pressure preparative liquid chromatography) was carried out using the CHEETAH MP200 system (Agela Technologies) and Claricep Flash columns filled with ODS (40−63 μm, YMC). Sephadex LH-20 (GE Healthcare) was used for column chromatography (CC). Silica gel (200−300 mesh) for CC and GF254 for thinlayer chromatography (TLC) were obtained from Yantai Jiangyou Silica Gel Development Co., Ltd. Sea salts were obtained from Guangzhou Hai Li Aquarium Technology Co., Ltd. Fungal Materials. The fungus Aspergillus puniceus SCSIO z021 was isolated from deep-sea sediments collected from Okinawa Trough (27°34.01′ N and 126°55.59′ E, ∼1589 m depth) that was about 4.7 km away from active hydrothermal vents.8 The strain was identified according to the ITS rDNA sequence (GenBank accession number KX258801) and deposited in the RNAM Center, South China Sea Institute of Oceanology, Chinese Academy of Science. Fermentation and Extraction. The strain was cultured on a potato dextrose agar (PDA) plate containing 3% sea salt at 28 °C for 6 days. Then the spores were collected and mixed with liquid medium to make a spore suspension, which was transferred into 210 × 1 L Erlenmeyer flasks, each containing 300 mL of culture medium (2% glucose, 20% potato, 3% sea salt). Static fermentation was carried out at 26 °C for 25 days; after that, the broth and mycelia were separated by cheesecloth. The broth was extracted with XAD-16 resin, successively eluting with H2O and EtOH. The EtOH fraction was collected and concentrated under vacuum to obtain a broth extract (11 g). The mycelia were extracted with acetone. The acetone fraction was evaporated under reduced pressure to afford an aqueous solution, which was further extracted with EtOAc three times to yield a mycelia extract (77 g). HPLC analysis (Figure S77) showed that the chemical profiles of the broth and mycelia extracts were similar, but the main components were obviously different. In order to enrich the trace components, the extracts from the broth and mycelia were combined for further processing. Isolation and Purification. The extracts (88 g) were fractionated on a normal-phase column using a stepped gradient elution with CH2Cl2/CH3OH (v/v, 100:0, 98:2, 95:5, 92:8, 90:10, 80:20, 70:30) to obtain nine subfractions (Fr.1−Fr.9). Fr.5 (2.7 g) was filtered to remove most of the insoluble white crystals, and the remaining residue was subjected to an ODS column eluting with CH3OH/H2O/ TFA (v/v/v, from 27:73:0.03 to 100:0:0.03) to give 14 fractions (Fr.5-1−Fr.5-14). Fr.5-7 was separated by Sephadex LH-20 eluting with CH2Cl2/CH3OH (v/v, 1:1) to give four fractions, and then Fr.57-1 was further purified by preparative HPLC eluting with CH3OH/ H2O/TFA (v/v/v, 64:36:0.03, 5 mL/min) to give 4 (11.2 mg, tR = 35.0 min) and 8 (6.9 mg, tR = 30.1 min). Fr.5-8 was separated by preparative HPLC eluting with CH 3 OH/H 2 O/TFA (v/v/v, 66:34:0.03, 5 mL/min) to give 9 (40.2 mg, tR = 33.1 min). Fr.6 (10 g) was subjected to a silica gel column using CH2Cl2/acetone (v/ v, 100:0, 95:5, 90:10, 80:20, 70:30, 50:50) to give nine fractions (Fr.61−Fr.6-9). Fr.6-6 was separated on an ODS column eluting with CH3OH/H2O/TFA (v/v/v, from 18:82:0.03 to 100:0:0.03) to afford 19 fractions. Fr.6-6-10 was purified by preparative thin-layer chromatography (prep-TLC) using CH2Cl2/CH3OH (v/v, 50:3) as the developing solvent to yield 2 (5.3 mg, Rf = 0.92), 1 (3.3 mg, Rf = 0.67), 3 (4.2 mg, Rf = 0.53), and 10 (4.0 mg, Rf = 0.25). Fr.6-6-11 was

Table 3. Transcriptional Activation of LXRα of 1−10 LXRα compound

EC50 (μM)

maximum fold induction

1 2 3 4 5 6 7 8 9 10 TO901317

15 15 16 50 5.1 5.3 1.7 1.7 41 47 0.53

3.2 3.7 3.4 3.0 4.2 4.0 3.9 3.6 2.7 3.5 19.3

this kind of alkaloid, their bioactivities would decrease to a certain extent, implying the quinazolinone skeleton plays an important role in the bioactivity. Previous studies reported that quinazolinones LN6500 and IMB-170 were potent LXRα agonists.2,7 This study further confirms that the quinazolinone skeleton can be a model for synthesizing LXR agonists. Compounds 1−10 were also tested for their enzyme inhibitory activity toward five different phosphatases including TCPTP, SHP1, MEG2, SHP2, and PTP1B and two other enzymes, IDO1 and LDHA. The results (Table S2) showed that 8−10 selectively exhibited inhibition against the six individual enzymes, with IC50 values of 14−87 μM, while 1−7 did not show inhibition activity, and no compounds showed inhibition activity against LDHA. Comparison of the structures and inhibitory activities between 1−4, 9, and 10 suggested that the substituents on the oxepin-containing diketopiperazine skeleton could significantly affect their enzyme inhibitory activity. Furthermore, antifungal activities of compounds 1−10 and fractions A and B that mainly contained 1−8 and 10 were also evaluated. Four phytopathogenic fungi including Curvularia australiensis, Colletotrichum gloeosporioides, Fusarium oxysporum, and Pyricularia oryzae were used as indicator strains. A preliminary antifungal assay of fractions A and B was performed by a standard disc diffusion method. Fraction B exhibited obvious antifungal activity against P. oryzae at 100 μg/disc, while fraction A did not show antifungal activity. HPLC analysis profiles of fractions A and B, tested at the same concentration (Figure S78), displayed that the contents of 5−8 in fraction B were higher than in fraction A. It was speculated that 5−8 might play important roles in the antifungal activity of fraction B. Thus, compounds 1−10 and fractions A and B were further evaluated for their antifungal activities against the above indicator strains by the microbroth dilution method in a 96-well culture plate at a concentration of ∼0.6 mM and 200 μg/mL, respectively. However, these compounds showed low percent inhibition (