Versiquinazolines AâK, Fumiquinazoline-Type Alkaloids from the

Article pubs.acs.org/jnp

Versiquinazolines A−K, Fumiquinazoline-Type Alkaloids from the Gorgonian-Derived Fungus Aspergillus versicolor LZD-14‑1 Zhongbin Cheng,† Lanlan Lou,‡ Dong Liu,† Xiaodan Li,† Peter Proksch,§ Sheng Yin,‡ and Wenhan Lin*,† †

State Key Laboratory of Natural and Biomimetic Drugs, Institute of Ocean Research, Peking University, Beijing, 100191, People’s Republic of China ‡ School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People’s Republic of China § Institute für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany S Supporting Information *

ABSTRACT: Eleven fumiquinazoline-type alkaloids, namely, versiquinazolines A−K (1−11), along with cottoquinazolines B−D, were isolated from the gorgonian-derived fungus Aspergillus versicolor LZD14-1. Their structures were determined by extensive analyses of the spectroscopic data (1D and 2D NMR, HRESIMS), in addition to the experimental and calculated ECD data and X-ray single-crystal diffraction analysis for the assignments of the absolute configurations. Versiquinazolines A, B, and F (1, 2, and 6), bearing a methanediamine or an aminomethanol unit and representing a unique subtype of fumiquinazolines, were found from nature for the first time. Possible biogenetic relationships of the versiquinazolines are postulated. In addition, the structures of cottoquinazolines B (12), D (13), and C (14) should be revised to the enantiomers. Compounds 1, 2, 7, and 11 exhibited inhibitory activities against thioredoxin reductase (IC50 values ranging from 12 to 20 μM).

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was performed with our marine microorganism library. The gorgonian (Pseudopterogorgia sp.)-derived fungus Aspergillus versicolor LZD-14-1 was found to possess inhibitory effects against thioredoxin reductase (TrxR), a potential target for new treatments for human diseases such as cancer, AIDS, and autoimmune diseases.9 In addition, the 1H NMR and ESIMS spectra of the EtOAc extract of the solid fermentation of the fungal strain presented data that suggested the presence of aromatic alkaloids. Extensive chromatographic separation of the large-scale fermentation resulted in the isolation of 14 compounds including 11 new alkaloids (1−11). Herewith we report the structure determination of the new compounds and their inhibitory effects toward TrxR.

aturally occurring fumiquinazoline-type polycyclic alkaloids are a group of natural products with unique scaffolds, which are characterized by two distinct moieties, a pyrazinoquinazolinedione and an imidazoindolone linked by a methylene “bridge”.1−6 The structural variations of the fumiquinazoline-based alkaloids are attributed to the incorporation of distinct amino acids to generate various pyrazinoquinazolinedione and imidazoindolone moieties, in addition to various skeletal rearrangements. Biogenetically, the pyrazinoquinazolinedione unit is considered to be assembled through the condensation of anthranilic acid (AA), tryptophan, and an additional amino acid (alanine, valine, or glycine), whereas the tricyclic imidazoindolone is generated by the condensation of an amino acid (alanine, valine, isoleucine, 2-aminoisobutyric acid, etc.) with the indole ring of tryptophan.7,8 These peptidyl alkaloids are mainly produced by species of the fungal genera Aspergillus, Acremonium, and Neosartorya collected from marine or terrestrial environments. Some of the analogues possess significant bioactivities, such as antifungal, cytotoxic, antifeedant, and antiviral effects. Since fumiquinazolines A−C were first reported in 1992,1 a total of 39 analogues with various subtypes have been isolated and characterized from fungi. The structural novelty and potent pharmaceutical effects of fumiquinazoline-type alkaloids have attracted much attention from chemists and pharmacologists to uncover more structural diversity with potential bioactivities from nature. In our continuing effort to characterize new and/or bioactive natural products from marine microorganisms, a chemical examination © 2016 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Versiquinazoline A (1) has a molecular formula of C24H19N5O4, as determined by the HRESIMS and NMR data, containing 18 degrees of unsaturation. The IR absorptions at 1734, 1686, 1630, and 1608 cm−1 suggested the presence of carbonyl and phenyl groups. The 13C NMR spectrum exhibited a total of 24 carbon resonances, involving 12 aromatic carbons for two phenyl groups and three carbonyl carbons. The 1H NMR and COSY spectra provided two ABCD aromatic spin systems for two ortho-substituted benzene rings. Comprehensive analyses Received: September 2, 2016 Published: November 10, 2016 2941

DOI: 10.1021/acs.jnatprod.6b00801 J. Nat. Prod. 2016, 79, 2941−2952

Journal of Natural Products

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3S*, 14S*, 16R*, 17R*, and 26S*. These assignments agreed with the results of the X-ray single-crystal diffraction experiment (Flack parameter: 0.4(5), Figure 3).10 The absolute configuration of 1 was determined by the experimental and calculated ECD data. On the basis of the TDDFT-ECD method,11,12 the ECD spectra of (3S,14S,16R,17R,26S)-1 and its enantiomer were calculated at the B31YP/6-311++G(2d,2p) level in the gas phase using the B3LYP/6-31G(d) optimized geometries after conformational searches via the MMFF94S force field. Comparison of the experimental ECD data of 1 with the calculated data for the model molecules (Figure 4) indicated 1 to be in agreement with the 3S, 14S, 16R, 17R, and 26S configuration. The molecular formula of versiquinazoline B (2) was established as C25H19N5O4 according to the HRESIMS and NMR data, possessing one carbon atom more than that of 1. Comparison of the NMR data (Table 1) indicated that the structures of both 1 and 2 were closely similar. The difference was attributed to the substitution at C-26 (δC 48.1), in which 2 presented a nonprotonated carbon to replace the methine of 1. In addition, the presence of two vicinal-coupling methylenes, H2-28 (δH 1.04, 1.20) and H2-29 (δH 0.79, 1.53), was observed in the COSY spectrum. The HMBC correlations from both H228 and H2-29 to C-26 and C-27 (δC 172.3) revealed C-26 to be located by a cyclopropane unit. The similar NOE interactions and the comparable Cotton effects of 1 and 2 (Figure 4) allowed the assignment of the absolute configuration of 2 to be the same as that of 1. Versiquinazoline C (3) has a molecular formula of C24H19N5O4, as established by the HRESIMS and NMR data, requiring 18 degrees of unsaturation. Its 1H and 13C NMR data were comparable to those of 2, except for the absence of a methylene group (H2-30). The COSY relationships between NH-2 (δH 9.57)/H-3 (δH 5.65) and NH-25 (δH 4.12)/H-17 (δH 5.19), in association with the HMBC correlations from NH-2 to C-1 (δC 168.6) and C-3 (δC 78.8) and NH-25 to C-26 (δC 46.0) and C-17 (δC 84.9), confirmed that 3 lacks a methylene group (H2-30) between N-2 and N-25. The relative configurations of 3 were determined by the NOESY data. The NOESY relationship between H-17 and H2-15 assigned the same orientation of H-17 and the methylene group H2-15. Comparison of the experimental ECD data of 3 with those calculated for the model molecules of (3S,14S,16R,17R)-3 and (3R,14R,16S,17S)-3 at B3LYP/6-311++G(2d,2p)//B3LYP/631G(d) level (Figure 5), in association with the biogenetic consideration that 2 was derived from 3 by incorporation of a methylene between N-2 and N-25, suggested the absolute configuration of 3 to be the same as that of 2. It was noted that 3 spontaneously converted to the analogue 13 in acetone (Figure 6). The structure of 13 was determined

of the 2D NMR (COSY, HSQC, HSBC) data revealed the planar structure of 1 containing two moieties (units A and B), closely related to fumiquinazoline C.1 In unit A, the COSY relationships connected the aromatic protons from H-7 (δH 7.74) to H-10 (δH 8.22), while the HMBC correlations between H-7 and C-11 (δC 121.0) and from H-10 to C-6 (δC 146.8) and C-12 (δC 158.7) assigned a quinazoline ring (rings A and B). Additional HMBC correlations from H-3 (δH 6.18, s) to C-4 (δC 148.8) and C-1 (δC 170.9) and from H-14 (δH 5.50) to C1, C-4, and C-12 (Figure 1) established a tricyclic pyrazinoquinazolinedione (unit A). Moreover, the HMBC correlations from the proton in the second aromatic ring at H23 (δH 7.62) to C-16 (δC 80.7) and C-19 (δC 138.8) and from H-17 (δH 5.09) to C-19 and C-24 (δC 133.7) demonstrated the presence of an indoline unit. Additional HMBC correlations from H-17 to C-26 (δC 59.5) and C-27 (δC 173.3) in association with the COSY relationship between H3-28 (δH 1.36) and H-26 (δH 3.93) clarified the fusion of an alanine unit to the indoline unit. Thus, unit B was assigned to an imidazoindolone segment. The linkage of units A and B through a methylene group was deduced by the COSY relationship between H2-15 (δH 2.76, 2.92) and H-14 in association with the HMBC correlations from H2-15 to C-16, C-17, and C-24. Subsequently, the connection of C-3 (δC 84.8) with C-16 across an ether bond was evident from the HMBC correlation between H-3 and C-16 (Figure 1). These assignments confirmed the structure of 1 to be closely related to fumiquinazoline C. Besides, the NMR spectra presented an additional methylene group, CH2-30 (δH 4.37, 5.30, d; δC 58.0), which was located between N-2 and N-25 to generate a sevenmembered ring (ring G), based on the HMBC correlations from H2-30 to C-1, C-3, C-17, and C-26, respectively. The NOE relationships from H-17 (δH 5.09) to H3-28 (δH 1.36) and H-15a (δH 2.76) assigned the same orientation of CH3-28 and H-17, while CH2-15 was also on the same face as H-17. Additional NOE interactions from H-30a (δH 4.37) to H3 and H-26 (Figure 2) clarified the same orientation of H-3 and H-26. Therefore, the relative configuration of 1 was assigned as

Figure 1. Key COSY and HMBC correlations of 1, 6, and 7. 2942



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Figure 2. Key NOE correlations of 1, 3, 4, 6−8, and 10.

Figure 3. X-ray crystallographic structure of 1.

Figure 4. Experimental and calculated ECD spectra of 1 and 2.

by an X-ray single-crystal diffraction experiment [Cu Kα radiation (λ = 1.5418 Å); Flack parameter (−0.01(7)] (Figure 7). This finding further confirmed the configurational assignments of 3. In addition, the specific rotations of 1−3 were calculated at the B3lyp/6-31+G(d) level with the polarizable continuum model (PCM) in CH2Cl2. The calculated results afforded the rotations of 1 (+190), 2 (+211), and 3 (+197), which were consistent with those of experimental data for 1−3 ([α]D =

+180, +178, and +150, respectively). These findings provided additional data to support the absolute configurations of 1−3. Versiquinazoline D (4) has a molecular formula of C23H21N5O4 as determined by the HRESIMS and NMR data. The 1H and 13C NMR data (Tables 2 and 3) featured a fumiquinazoline-type analogue, structurally related to fumiquinazoline A.1 Diagnostic 2D NMR data established the planar structure of 4 to be a 3-demethyl analogue of fumiquinazoline A. This assignment was evident from the COSY relationship between H2-3 (δH 4.25, 5.06) and NH-2 (δH 8.81) in addition to the HMBC correlations of H2-3 and NH-2 to C-1 (δC 168.4) 2943



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Table 1. 1H and 13C NMR Data of 1 and 2 and 6 in DMSO-d6a,b 1 no. 1 3

170.9, C 84.8, CH

4 6 7 8 9 10 11 12 14 15

148.8, 146.8, 127.8, 134.8, 127.6, 126.5, 121.0, 158.7, 52.0, 38.8,

C C CH CH CH CH C C CH CH2

16 17 19 20 21 22 23 24 26 27 28 29 30

80.7, 82.3, 138.8, 114.5, 130.8, 124.8, 127.0, 133.7, 59.5, 173.3, 10.7,

C CH C CH CH CH CH C CH C CH3

58.0, CH2

2 δH (J in Hz)

δC, type 6.18, s

7.74, 7.89, 7.63, 8.22,

d (8.0) dd (7.5, 8.0) dd (7.5, 8.0) d (8.0)

5.50, d (5.4) 2.92, d (15.2) 2.76, dd (5.4, 15.2).

5.09, s 7.42, 7.42, 7.12, 7.62,

dd (2.1, 8.0) t (8.0) ddd (2.1, 6.1, 8.0) d (6.1)

3.93, q (6.8) 1.36, d (6.8) 5.30, d (9.4) 4.37, d (9.4)

δC, type 171.0, C 84.7, CH 148.8, 146.8, 127.8, 134.8, 127.6, 126.5, 121.1, 158.7, 52.1, 38.7,


80.8, 83.0, 139.3, 113.9, 131.0, 124.6, 127.1, 133.3, 48.1, 172.3, 6.2, 4.5, 57.5,

C CH C CH CH CH CH C C C CH2 CH2 CH2

6 δH (J in Hz)

6.14, s

7.74, 7.90, 7.64, 8.22,

dd (1.5, 8.0) dt (1.5, 8.0) dt (1.5, 8.0) dd (1.5, 8.0)

5.51, dd (1.2, 5.5) 2.93, dd (1.2, 15.2) 2.83, dd (5.5, 15.2)

5.00, s 7.42, 7.42, 7.13, 7.64,

dd (2.4, 8.0) dt (1.0, 8.0) ddd (2.4, 6.1, 8.0) dd (1.0, 6.1)

1.20,m; 1.04, m 1.53, m; 0.79, m 5.02, d (9.3) 4.19, d (9.3)

NH-2 a

δC, type 167.3, C 44.0, CH2 149.4, 146.7, 126.3, 134.6, 126.5, 126.4, 119.5, 159.8, 52.2, 32.4,


89.0, C 84.8, CH 142.3, C 114.4, CH 130.3, CH 124.3, CH 124.3, CH 131.7, C 49.7, C 171.2, C 14.4, CH2 8.74, CH2 85.1, CH2

δH (J in Hz) 5.13, d (17.4) 4.22, d (5.0, 17.4)

7.54, 7.77, 7.43, 7.85,

d (8.1) dd (7.8, 8.1) t (7.8) d (7.8)

5.27, dd (6.5, 9.1) 2.92, dd (9.1, 14.7) 2.68, dd (6.5, 14.7) 5.84, s 7.15, 7.00, 6.75, 7.41,

d (7.8) dd (7.7, 7.8) t (7.7) d (7.7)

1.80, m; 1.23, m 1.08, m 4.71, d (9.7) 3.96, d (9.7) 8.76, d (5.0)

Recorded at 400 MHz. Chemical shifts are in ppm, coupling constants J in Hz. bRecorded at 100 MHz.

Figure 5. Experimental and calculated ECD spectra of 3.

and C-4 (δC 149.4). The NOE interactions between H3-28 (δH 1.32)/H-17 (δH 5.63), OH-16 (δH 5.46)/NH-25 (δH 3.56), and H-17/H2-15 (Figure 2) determined the same orientation of CH3-28 and H-17, which were on the opposite face relative to OH-16. On the basis of the exciton chirality ECD method,13 the negative Cotton effect at 250 nm and the positive Cotton effect at 225 nm reflected the exciton coupling between the transition moments of the two chromophores, the pyrazino-

Figure 6. Conversion of 3 to 13. (a) 2 h in acetone-d6 (3 and 13 in a ratio of 2:1); (b) 4 h in acetone-d6 (3 and 13 in a ratio of 1:1); (c) 6 h in acetone-d6 (3 and 13 in a ratio of 1:2); (d) 12 h in acetone-d6 (13).

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Figure 7. Structures of 12−14 and the X-ray crystallographic structures of 12 and 13.

Table 2. 1H NMR Data of 3−5 and 7−9 in DMSO-d6a no. 3 4 7 8 9 10 14 15 17 20 21 22 23 26 28 29 30 31 OMe-1 OMe-3 OH-7 OH-16 NH-2 NH-25 a

3 5.65, d (4.9)

7.67, d (8.0) 7.89, t (8.0) 7.66, t (8.0) 8.26, d (8.0) 5.44, t (4.0) 2.80, dd (4.0, 15.8) 2.72, dd (4.0, 15.8) 5.19, d (9.2) 7.31, d (7.9) 7.28, dt (2.0, 7.9) 6.80, t (7.9) 6.82, dd (2.0, 7.9) 1.14, m 1.02, m

4

5

7

5.06, d (17.3) 4.25, dd (5.2, 17.3)

5.07, d (17.4) 4.24, dd (17.4, 5.1)

7.56, d (8.0) 7.77, ddd (1.2, 7.6, 8.0) 7.42, dd (7.6, 8.0) 7.85, dd (1.2, 8.0) 5.38, dd (6.6, 8.6) 2.64, dd (6.6, 14.7) 2.50, dd (8.6, 14.7) 5.63, brs 7.18, d (7.6) 6.97, dd (7.5, 7.6) 6.66, dd (7.5, 7.6) 7.22, d (7.6) 3.77, q (7.1) 1.32, d (7.1)

7.56, d (8.0) 7.78, dd (7.6, 8.0) 7.42, dd (7.6, 7.9) 7.86, d (7.9) 5.38, dd (6.2, 8.7) 2.66, dd (6.2, 14.8) 2.55, dd (7.8, 14.8) 5.53, d (9.6) 7.21, d (7.7) 7.00, dd (7.5, 7.7) 6.71, t (7.5) 7.28, d (7.5) 0.89, m 0.99, m; 0.88, m

8

9

5.06, s

7.73, d (8.1) 7.87, dd (7.6, 8.1) 7.58, dd (7.6, 8.0) 8.10 d (8.0) 6.49, d (9.1) 3.22, d (14.7) 2.24, dd (9.1, 14.7) 5.17, s 7.33, d (8.0) 7.32, t (8.0) 7.16, dd (7.4, 8.0) 7.46, d (7.4) 1.21, m; 1.15, m 1.65, m; 0.85, m

3.81, dd (4.6, 16.9) 3.10, d (16.9) 8.29, s 7.71, d (8.1) 7.87, dd (7.6, 8.1) 7.59, dd (7.6, 8.1) 8.18, d (8.1) 4.83, dd (5.3, 8.6) 3.05, dd (8.6, 13.8) 2.78, dd (5.3, 13.8) 5.81, s 7.48, d (7.8) 7.44, t (7.8) 7.27, dd (7.6, 7.8) 7.55, d (7.6) 4.27, d (9.5) 2.24, m 1.73, m 1.28, m 0.97, t (7.4) 1.12, d (6.8)

7.20, dd (1.2, 7.8) 7.36, dd (7.8, 8.0) 7.63, dd (1.2, 8.0) 5.26, t (4.7) 3.44, dd (4.7, 14.0) 3.45, dd (4.7, 14.0) 6.87, d (2.3) 7.32, d (7.8) 7.02, dd (7.6, 7.8) 6.82, dd (7.5, 7.6) 7.31, d (7.5)

3.62, s 3.33, s 9.67, s 9.57, d (4.9) 4.12, d (9.2)

5.46, s 8.81, d (5.2) 3.56, brs

5.61, s 8.78, d (5.1) 3.72, d (9.6)

5.92, s

6.31, s 8.29, d (4.6) 10.9, d (2.3)

Recorded at 400 MHz, chemical shifts are in ppm, coupling constants J are in Hz.

experimental Cotton effect (CE) of 4 (positive CE at 260 nm) with the calculated data for the model molecules (Figure 8) assigned 4 to have the 14S, 16R, 17R, and 26S configuration. The NMR data of versiquinazoline E (5) closely resembled those of 4 (Tables 2 and 3), except for the absence of a methyl substitution at C-26 (δC 46.5), which was replaced by a

quinazolinedione and imidazoindolone groups. These data agreed with a left-handed screw rule (Figure 8), indicating the 14S configuration. In regard to the absolute configuration of the imidazoindolone group, the ECD spectra of the molecules for (14S,16R,17R,26S)-4 and its enantiomer were calculated using the quantum chemical TDDFT method. Comparison of the 2945



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Table 3. 13C NMR Data of 3−5 and 7−9 in DMSO-d6a

a

no.

3

1 3 4 6 7 8 9 10 11 12 14 15 16 17 19 20 21 22 23 24 26 27 28 29 30

168.6, 78.8, 149.3, 147.0, 128.0, 135.1, 127.7, 126.7, 120.8, 158.8, 51.5, 34.8, 80.9, 84.9, 139.1, 115.5, 130.2, 124.7, 124.6, 136.9, 46.0, 176.3, 13.9, 13.2,

4 C CH C C CH CH CH CH C C CH CH2 C CH C CH CH CH CH C CH C CH2 CH2

168.4, 44.2, 149.4, 146.7, 126.4, 134.5, 126.5, 126.3, 119.7, 159.9, 52.1, 36.2, 75.3, 80.8, 138.4, 114.9, 129.0, 124.1, 124.0, 137.3, 60.1, 175.5, 17.9,

5 C CH2 C C CH CH CH CH C C CH CH2 C CH C CH CH CH CH C CH C CH3

168.3, 44.2, 149.5, 146.7, 126.3, 134.5, 126.5, 126.3, 119.9, 160.0, 52.0, 36.4, 75.0, 79.8, 138.7, 114.4, 129.2, 124.1, 123.9, 136.8, 46.5, 174.1, 14.0, 11.6,

7 C CH2 C C CH CH CH CH C C CH CH2 C CH C CH CH CH CH C C C CH2 CH2

169.5, 90.6, 150.1, 146.2, 127.3, 134.9, 127.8, 126.3, 121.1, 161.1, 53.6, 34.4, 74.7, 81.3, 137.2, 115.0, 129.4, 125.0, 124.3, 138.5, 51.4, 168.9, 8.9, 8.5, 52.5, 53.6,

C CH C C CH CH CH CH C C CH CH2 C CH CH CH CH CH CH C C C CH2 CH2 OCH3 OCH3

8

9

164.6, C 146.9, 147.5, 127.1, 134.7, 127.3, 126.1, 121.5, 159.8, 55.6, 34.0, 73.8, 84.8, 140.5, 114.2, 130.1, 125.2, 124.6, 134.6, 68.4, 174.1, 33.8, 26.1, 10.7, 14.9,

CH C CH CH CH CH C C CH CH2 C CH CH CH CH CH CH CH CH C CH CH2 CH3 CH3

167.6, C 43.8, CH2 147.4, C 136.2, C 152.4, C 118.6, CH 127.2, CH 116.0, CH 120.8, C 159.9, C 56.5, C 26.5, CH2 107.8, C 124.4., CH 136.0, C 111.4, CH 121.2, CH 118.7, CH 117.8, CH 127.2, C

Recorded at 100 MHz, chemical shifts are in ppm.

Figure 8. Experimental and calculated ECD spectra of 4−6 and the exciton coupling of 4.

distinction was attributed to the presence of the additional methylene group CH2-30 (δH 4.71, 3.96; δC 85.1) and the absence of OH-16 and NH-25 protons. The HMBC correlations of H2-30 to C-16 (δC 89.0), C-17 (δC 84.8), and C-26 (δC 49.7) (Figure 1) connected the methylene group CH2-30 to C-16 by an ether bond and to N-25 to generate a tetrahydrooxazole ring. The NOE interaction between H-17 (δH 5.84, s) and H2-15 (δH 2.68, 2.92) clarified the same orientation of H-17 toward H2-15. The similar Cotton effects of 6 and 4 (Figure 8) as induced by the exciton coupling indicated that 6 possesses the same absolute configuration as that of 4. Versiquinazoline G (7) has a molecular formula of C26H24N4O6, as established by the HRESIMS and NMR data, requiring 17 degrees of unsaturation. The 1H and 13C NMR data (Tables 2 and 3) indicated that 7 presented the substructure of a quinazoline and a 26-cyclopropane bearing imidazo[1,2-a]indoline, which were comparable to those of 5.

nonprotonated carbon. The observation of the COSY relationship between two methylene H2-28 (δH 0.89) and H2-29 (δH 0.88, 0.99) in association with the HMBC correlations from both H2-28 and H2-29 to C-26 and C-27 (δC 174.1) deduced a cyclopropane unit to be positioned at C-26, as for 2. The NOE correlations between OH-16/NH-25 and H-17/H2-15 indicated 5 possessed the same relative configuration as 4. The similar Cotton effects of both 5 and 4 (Figure 8) suggested C16 of 5 to be in the S configuration. In addition, reductive conversion 3 to 5 by NaBH4/MeOH (Supporting Information, Figure S90)14,15 confirmed that both 5 and 3 have the same absolute configurations. The molecular formula of versiquinazoline F (6) was determined as C25H21N5O4 on the basis of the HRESIMS and NMR data, containing a carbon atom more than that of 5 and requiring 18 degrees of unsaturation. The NMR data of 6 closely resembled those of 5 (Tables 2 and 3), whereas the 2946



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HPLC chromatography (tR 31 min for L-Ile and tR 39 min for D-Ile) (Figure S65) indicated an L-isoleucine for 8. Versiquinazoline I (9) has the molecular formula C20H16N4O3, as determined by the HRESIMS data. Analyses of 2D NMR data indicated that 9 was composed of an anthranilic acid, a tryptophan, and a glycine residue, structurally close to glyantrypine.18 The difference was found by the substitution of C-7, in which a hydroxy group at C-7 (δC 152.4) in 9 was deduced by the HMBC correlations of the phenol proton at δH 9.67 (s) to C-6 (δC 136.2), C-7, and C-8 (δC 118.6). Because the specific rotation of 9 was contributed by the sole stereogenic center at C-14, the similar specific rotations of 9 ([α]20D +150) and (+)-glyantrypine ([α]25D +150.1) assigned 9 to have a 14S configuration. In addition, the experimental ECD curve of 9 exhibiting a positive Cotton effect at around 230 nm was consistent with that calculated for the model molecule with the 14S configuration, whereas the calculated ECD curve for (14R)-9 showed a negative Cotton effect at 230 nm (Figure S93). These data further supported the configurational assignment of 9. Versiquinazoline J (10) has a molecular formula of C21H25N3O4, as established by the HRESIMS and NMR data, indicating 11 degrees of unsaturation. The 1H NMR spectrum exhibited an ABCD spin system for a 1,2-disubstituted aromatic ring, the protons for a terminal vinyl group, and five methyl groups including two methoxy groups. The 13C NMR spectrum showed 21 resonances including six aromatic carbons, four olefinic carbons for two double bonds, and two carbonyl carbons. Analyses of the 2D NMR data revealed the structure of 10 to be closely related to oxaline, a fungal alkaloid with a tetracyclic nucleus.19 The conjunction of an α-methoxy-α,βunsaturated δ-lactam to a indoline ring at C-2 and C-3 was established by the HMBC correlations from H-10 (δH 5.17) to C-2 (δC 101.1), C-3 (δC 51.6), and C-12 (δC 157.7), in association with the correlation between a MeO (δH 3.47) and C-11 (δC 145.8). A 1,1-dimethylallyl (“reverse-isopentenyl”) group, as deduced by the HMBC correlations from the methyl singlets H3-19 (δH 1.25) and H3-20 (δH 1.22) to C-17 (δC 144.2) and C-16 (δC 42.1), was substituted at C-3 based on an additional HMBC correlation between H3-19/H3-20 and C-3. Moreover, an alanine unit fused to the parent nucleus was deduced by the HMBC correlations from H3-15 (δH 1.44) to C-13 (δC 52.5) and C-14 (δC 173.3) and from H-13 (δH 4.22, q) to C-2 and C-12. The second methoxy group was positioned at N-1, as evident from the absence of an NH-1 proton and the observation of an NOE interaction between H-8 and the MeO protons. The NOE interaction between the amide proton and H3-19/H3-20 indicated the cis fusion between rings B and C, while the NOE relationship between H3-20 and H3-15 assigned the same orientation for the 1,1-dimethylallyl group and H3-15. The absolute configuration of 10 was determined by analyses of the X-ray single-crystal diffraction data [Flack parameter (−0.04 (6)], revealing the 2S, 3R, and 13S configuration (Figure 9). The structure of versiquinazoline K (11) was determined to be a 1-O-demethyl analogue of 10 based on the closely similar NMR data of 10 with 11, lacking a methoxy group at N-1 (Table 4). The HMBC correlations of N-OH with C-9 and C-2 indicated N-1 to be substituted by a hydroxy group. The relative configuration of 11 was in agreement with that of 10 based on the similar NOE interactions of both compounds. In addition, the similar Cotton effects of 10 and 11 in the ECD spectra assigned 11 with the same absolute configuration as 10 (Figure S94).

In addition, two methoxy resonances were observed in the NMR spectra. On the basis of the COSY relationship between H2-15 (δH 2.24, 3.22) and H-14 (δH 6.49), in combination with the HMBC correlations from H-14 to C-4 (δC 150.1), C-16 (δC 74.7), H2-15 to C-16 and C-17 (δC 81.3), H-3 (δH 5.06, s) to C-4 and C-17, and H-17 (δH 5.17) to C-3 (δC 90.6) and C-16, a diazocane ring connecting the quinazoline unit and the imidazo[1,2-a]indoline unit was established. Additional HMBC correlations between the methoxy protons at δH 3.33 (3H, s) and C-3 allowed the linkage of a MeO group at C-3, while the HMBC correlations from H2-15, H-14, and the methoxy protons at δH 3.62 (3H, s) to the carbonyl carbon C-1 (δC 169.5) were ascribed to a methyl ester at C-14 (δC 53.6) (Figure 1). In addition, the position of a hydroxy group at C-16 was deduced by the HMBC correlation of a hydroxy proton at δH 5.92 (s) to C-15, C-16, C-17, and C-24. The NOE correlations between H-14/OH-16 (δH 5.92), H-14 (δH 6.49)/ OMe-3 (δH 3.33), and H-17/H-3 (Figure 2) confirmed the same orientation of H-14 as OH-16 and OMe-3, whereas H-3 and H-17 were on the opposite face. Thus, the relative configuration of 7 was assigned as 3S*, 14S*, 16R*, and 17R*. Comparison of the experimental ECD data with those of calculated ECD data for the model molecule with the 3S, 14S, 16R, and 17R configuration and its enantiomer allowed the assignment of the 3S, 14S, 16R, and 17R configuration for 7 (Figure S91). The molecular formula of versiquinazoline H (8) was determined as C25H24N4O4 by the HRESIMS and NMR data. Analyses of the 2D NMR data revealed that the structure of 8 bears two moieties, one of which was attributed to a quinazoline unit. The second moiety was determined as an indoline-based tetracyclic ring. The COSY relationship between H2-15 (δH 2.78, 3.05) and H-14 (δH 4.83), in addition to the HMBC correlations from H2-15 to C-1 (δC 164.6), C-16 (δC 73.8), C-17 (δC 84.8), and C-24 (δC 134.6), revealed a δ-lactam ring fused to the indoline. The presence of an isoleucine unit was recognized by the HMBC correlations from a methyl triplet (δH 0.97) to C-28 (δC 33.8) and C-29 (δC 26.1) and a methyl doublet (δH 1.12) to C-26 (δC 68.4), C-28, and C-29. This unit was fused to N-18 and N-25 according to the additional HMBC correlations from H-17 (δH 5.81) to C-26 (δC 68.4) and C-27 (δC 174.1). In addition, a hydroxy group linked to C-16 was evident from the HMBC correlation of OH (δH 6.31) to C-15, C-16, C-17, and C-24. Thus, the planar structure of 8 was established. It was closely related to isochaetominine C3, with the difference due to an isoleucine in place of a valine unit in the known analogue. The NOE correlations between H-17 and OH-16, from H-17 to H-28 and H-15b (δH 3.05), and from H-4 (δH 8.29) to H-17 (Figure 2) determined the same face for OH-16 and H-17, whereas H-14 and H-26 were on the opposite face relative to H-17. The absolute configuration of 8 was determined to be 14S, 16S, 17S, and 26R, based on the closely similar ECD and specific rotation data of both 8 and isochaetominine C,16 while these were further confirmed by the comparison of the experimental ECD data with those of calculated model molecules of 8 and its enantiomer (Figure S92). In addition, the configuration of the isoleucine unit was determined following acid hydrolysis of 8 and subsequent derivatization according to the advanced Marfey’s method.17 Comparison of the resulting L-FDAA (1fluoro-2,4-dinitrophenyl-5-L-alanine amide) amino acid derivatives with those of appropriate standard amino acids using 2947



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while the absolute configuration of cottoquinazoline D (13) was unambiguously assigned as 3S, 14S, 16S, and 17R. In addition, the comparable specific rotations of 12 ([α]20D + 333) and 13 ([α]20D +613) with the data calculated for the molecules (+412 for 12 and +460 for 13) at the B3lyp/6-31+G(d) level with the PCM in MeOH, along with the comparison of the experimental ECD data with the calculated data for 12 and 13 (Figure S95), provided additional evidence to support the configurational assignments. Cottoquinazoline C (14) possessing the same absolute configuration as that of 12 was evident from the similar ECD data, which were further supported by the ECD calculation (Figure S95). However, these assignments are opposite the absolute configurations originally assigned to cottoquinazolines B (12), D (13), and C (14).5,20 The relative configurations for cottoquinazolines B and D were originally assigned by X-ray crystallography (Mo Kα X-ray source), and the relative configuration for cottoquinazoline C was established by similarities to cottoquinazoline B. The absolute configurations for cottoquinazolines B and C were then established by acidic hydrolysis and examination of the released amino acids by HPLC on a chiral-phase column. It is not clear why the original analyses did not reveal the correct absolute configurations, but our determinations of the absolute configurations using X-ray crystallography, confirmed by ECD experiments, have firmly assigned the absolute configurations for 12 and 13, and the configuration for 14 was assigned from similar ECD data to those for 12 and 13 (Figure S95). Therefore, the revised structures of cottoquinazolines B (12), D (13), and C (14) are the enantiomers of the originally reported structures.5 Biogenetically, (+)-glyantrypine, an alkaloid generated by the condensation of anthranilic acid, glycine, and tryptophan, was considered as a precursor to yield 9 by 7-hydroxylation and to form 4 and 5 through additional intermolecular condensation

Figure 9. X-ray crystallographic structure of 10.

The 1H and 13C NMR data of 12−14 were virtually identical to those reported for cottoquinazolines B, D, and C.5 In addition, compounds 12−14 showed melting points and positive values of the specific rotations similar to those of cottoquinazolines B, D, and C, respectively. These data indicated that 12−14 are identical to cottoquinazolines B (12), D (13), and C (14). The same HPLC retention time by co-injection of 12 with authentic cottoquinazoline B on an ODRH chiral-phase column further supported the assignments. Analyses of the X-ray single-crystal diffraction data by Cu Kα irradiation (Figure 7) using the Flack parameters (0.00(4) for 12 and −0.01(7) for 13) resulted in the absolute configuration of cottoquinazoline B (12) to be 3S, 14S, 16S, 17R, and 26S,

Table 4. 1H and 13C NMR Data of Compounds 10 and 11 in DMSO-d6 10 no. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe-1 OMe-11 NH a

11

δC, type 101.1, 51.6, 126.0, 124.9, 122.6, 127.8, 110.8, 146.7, 107.8, 145.8, 157.7, 52.5, 173.3, 14.0, 42.1, 144.2, 113.0, 24.3, 23.5, 64.5, 55.1,

C C C CH CH CH CH C CH C C CH C CH3 C CH CH2 CH3 CH3 CH3 CH3

δH (J in Hz)

7.55, 7.00, 7.22, 6.90,

δC, typeb

a

d (7.7) t (7.7) t (7.7) d (7.7)

5.17, s

4.22, q (6.8) 1.44, d (6.8) 6.14, 5.07, 1.25, 1.22, 3.68, 3.47, 9.27,

dd (17.6, 10.6) d (17.6), 5.01, d (10.6) s s s s s

102.1, 51.7, 126.6, 124.4, 121.7, 127.8, 110.8, 145.9, 107.8, 144.4, 158.1, 52.9, 173.8, 14.1, 42.1, 144.4, 112.9, 24.4, 23.5,

C C C CH CH CH CH C CH C C CH C CH3 C CH CH2 CH3 CH3

55.5, CH3

δH (J in Hz)

7.50, 6.93, 7.19, 6.80,

dd (7.6, 1.0) ddd (7.6, 7.6, 1.0) ddd (7.6, 7.6, 1.0) dd (7.6, 1.0)

5.14, s

4.20, q (6.8) 1.42, d (6.8) 6.17, 5.07, 1.26, 1.23,

dd (17.5, 10.5) d (17.5), 5.01, d (10.5) s s

3.45, s 9.15, s

Recorded at 400 MHz. bRecorded at 100 MHz. 2948



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Scheme 1. Hypothetical Biogenetic Relationships among 1−7 and 9

with an alanine or a 1-aminocyclopropanecarboxylic acid (ACC) residue. Oxidation-induced21 formation of an ether bond from 4 or 5, respectively, could generate 3 or related compound a. Reaction of compounds 3, a, and 5 with formaldehyde could generate 1, 2, and 6. Compound 7 could be produced by a ring rearrangement and methyl esterification of a followed by additional trapping with MeOH at C-3 (Scheme 1). Compound 8 is structurally related to chaetominine, whose biosynthetic pathway has been depicted.8,22 The pathway to 8 would be similar, with Ile in place of Ala (Scheme S2). Compounds 10 and 11 can be derived by condensation of tryptophan and alanine to form a diketopiperazine followed by reaction with dimethylallyl diphosphate and cyclization.23 Hydroxylation at C-11 with subsequent cleavage of the bond between C-11 and the N atom could yield a nine-membered ring with a carbonyl group at C11. Oxidation to an imine could precede attack by the amide N to generate a tetracyclic nucleus. The N-OH group at the indoline unit could be generated by a P450 monooxygenase, while methylation at OH-11 could yield 11, and additional methylation at the N-OH group of 11 could produce 10 (Scheme S3). All compounds were tested for their inhibitory activities against the tumor cell lines A549 (lung adenocarcinoma) and A2780 (human ovarian cancer), showing weak inhibition with IC50 values greater than 50 μM. However, compounds 1, 2, 7, and 11 exhibited significant inhibitory effects against thioredoxin reductase9 with IC50 values ranging from 12 to 20 μM (Table 5). In summary, this work reports a profile of new fumiquinazoline-related alkaloids with diverse subtypes. It is noted that the fumiquinazoline-type alkaloids, such as the fumiquinazolines,

Table 5. TrxR Inhibitory Activity of Compounds 1−14

a

no.

% inhibition (50 μM)

IC50 (μM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 curcumina

64 83 47 27 28 51 83 62 55 13 76 38 17 43 25 ± 2

20 ± 1 12 ± 2

13 ± 0

13 ± 0

Positive control.

neosartoryadins,2 and fiscalins,24 possess the 14R configuration. The fumiquinazoline analogues in this work exclusively showed the 14S configuration, which has been rarely reported. Compounds 1, 2, and 6 represent new subtype scaffolds that incorporate an additional ring via a methylene group. The biogenetic relationships of the new alkaloids were postulated. Although diverse secondary metabolites from the fungus Aspergillus versicolor have been reported in literature, the present work indicates that the same fungal species from different environments provides natural products with distinct chemical diversity due to each strain activating distinctive biosynthetic pathways. In addition, four compounds exhibited a 2949



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1 (tR = 28.4 min, 4.3 mg). SF3b4 (43 mg) was separated by RP-HPLC (MeCN/H2O = 6:4, 2 mL/min) to get 7 (tR = 33.0 min, 6 mg) and 8 (tR = 27.5 min, 12 mg). Versiquinazoline A (1): colorless crystals (CH2Cl2/MeOH); mp 286−287 °C; [α]20D +180 (c 0.1, CH2Cl2); UV (MeOH) λmax (log ε) 204 (4.61), 227 (4.32), 278 (3.19), 314 (2.43) nm; ECD (1.8 × 10−4 M, CH2Cl2) λmax (Δε) 212 (+0.84), 224 (−36.33), 242 (+6.72), 250 (−0.76), 264 (10.93); IR (KBr) νmax 2923, 2853, 1734, 1686, 1630, 1608, 1485, 1390 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 442.1515 [M + H]+ (calcd for C24H20N5O4, 442.1510). Versiquinazoline B (2): white powder; [α]20D +178 (c 0.17, CH2Cl2); UV (MeOH) λmax (log ε) 204 (4.63), 227 (4.33), 278 (3.21), 314 (2.44) nm; ECD (2.1 × 10−4 M, CH2Cl2) λmax (Δε) 224 (−31.56), 268 (+16.54); IR (KBr) νmax 2920, 2850, 1727, 1688, 1392, 1019 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 454.1503 [M + H]+ (calcd for C25H20N5O4, 454.1510). Versiquinazoline C (3): white powder; [α]20D +150 (c 0.4, CH2Cl2); UV (MeOH) λmax (log ε) 203 (4.42), 227 (4.25), 303 (3.37) nm; ECD (1.4 × 10−4 M, CH2Cl2) λmax (Δε) 218 (+11.43), 238 (+9.89), 250 (−2.89), 270 (+13.17); IR (KBr) νmax 3268, 2925, 2854, 1717, 1686, 1609, 1483, 1468, 1051, 1026 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 442.1512 [M + H]+ (calcd for C24H20N5O4, 442.1510). Versiquinazoline D (4): white powder; [α]20D +110 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.36), 233 (4.14), 306 (3.08) nm; ECD (1.3 × 10−4 M, CH2Cl2) λmax (Δε) 226 (24.4), 248 (−8.30), 272 (+3.74); IR (KBr) νmax 3240, 1680, 1608, 1485, 1217 cm−1; 1H and 13 C NMR data, Tables 2 and 3; HRESIMS m/z 432.1664 [M + H]+ (calcd for C23H22N5O4, 432.1666). Versiquinazoline E (5): white powder ; [α]20D +130 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.38), 233 (4.15), 305 (3.09) nm; ECD (1.3 × 10−4 M, CH2Cl2) λmax (Δε) 226 (+31.46), 248 (−10.17), 278 (+4.50); IR (KBr) νmax 3253, 2923, 1678, 1606, 1471 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 444.1672 [M + H]+ (calcd for C24H22N5O4, 444.1666). Versiquinazoline F (6): white powder; [α]20D +130 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.35), 229 (4.12), 305 (3.08) nm; ECD (1.6 × 10−4 M, CH2Cl2) λmax (Δε) 226 (+19.28), 244 (−3.19), 262 (+9.23); IR (KBr) νmax 2915, 2827, 1682, 1608, 1473, 1296 cm−1; 1 H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 456.1671 [M + H]+ (calcd for C25H22N5O4, 456.1666), 478.1492 [M + Na]+ (calcd for C25H21N5O4Na, 478.1486). Versiquinazoline G (7): colorless oil; [α]20D −230 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 206 (4.41), 228 (4.31), 305 (3.14) nm; ECD (2.2 × 10−4 M, CH2Cl2) λmax (Δε) 208 (+31.5), 228 (−32.5), 306 (−10.29); IR (KBr) νmax 2953, 2926, 2852, 1727, 1684, 1594, 1485, 1082 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 489.1770 [M + H]+ (calcd for C26H25N4O6, 489.1774). Versiquinazoline H (8): colorless oil; [α]20D −100 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.61), 226 (4.43), 302 (3.31) nm; ECD (3.5 × 10−4 M, MeOH) λmax (Δε) 202 (−47.87), 218 (+18.42), 244 (−12.94), 274 (2.00), 300 (−2.72); IR (KBr) νmax 3324, 2925, 1730, 1679, 1608, 1608, 1476 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 445.1871 [M + H]+ (calcd for C25H25N4O4, 445.1870). Versiquinazoline I (9): colorless oil; [α]20D +150 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 215 (4.62), 239 (4.13), 281 (3.71) nm; ECD (1.5 × 10−4 M, MeOH) λmax (Δε) 214 (−5.22), 234 (+10.53), 332 (+2.0); IR (KBr) νmax 3247, 2917, 2858, 1681, 1607, 1485, 1404, 1199, 988 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 361.1298 [M + H]+ (calcd for C20H17N4O3, 361.1301). Versiquinazoline J (10): colorless blocks (MeOH); mp 138−139 °C; [α]20D +120 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 201 (4.38), 230 (4.22), 290 (3.33) nm; ECD (7.7 × 10−4 M, MeOH) λmax (Δε) 230 (−13.8), 252 (4.95), 278 (−2.06); IR (KBr) νmax 2937, 1737, 1688, 1628, 1460, 1318, 1239, 1030 cm−1; 1H and 13C NMR data, Table 4; HRESIMS m/z 384.1924 [M + H]+ (calcd for C21H26N3O4, 384.1918); 406.1741 [M + Na]+ (calcd for C21H25N3O4Na, 406.1737). Versiquinazoline K (11): colorless oil; [α]20D +100 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 200 (4.39), 230, (4.23) 290 (3.35) nm;

significant inhibitory effect against thioredoxin reductases with low cytotoxic activities toward tumor cell lines, suggesting that their inhibitory effects toward TrxR may be related to the microenvironmental regulation of tumor progression and metastasis.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an X-5 micromelting point apparatus (Kexian Co.). Specific rotations were measured by an Autopol III automatic polarimeter (Rudolph Research Co., Ltd.). UV spectra were measured on a Cary 300 spectrometer. ECD spectra were measured on a JASCO J-810/J-815 spectropolarimeter. IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer. The 1H and 13C NMR spectra were recorded on a Bruker Avance-400FT NMR spectrometer with TBI probe (triple resonance broad band probe). HRESIMS spectra were obtained on a Bruker APEX IV 70 eV FT-MS spectrometer and on a Waters Xevo G2 Q-TOF spectrometer fitted with an ESI source (acquisition range: 100−1000, acquisition: start time 0 and end time 4, voltages: ESI+ 2 kV and ESI− 1.5−2 kV, external standard: HCOONa). HF254 silica gel for TLC was obtained from Qingdao Marine Chemistry Co. Ltd. Sephadex LH-20 (18−110 μm) was purchased from Pharmacia. Semipreparative HPLC was performed on an Alltech 426 pump using a UV detector, and the Prevail C18 column (semipreparative, 5 μm) was used for separation. The Gaussian 9.0 version software and Sybyl Software version X 2.0 used for the calculation of specific rotation and ECD spectra were purchased from Gaussian Inc. and Tripos Associates Inc., respectively. Fungal Strain and Identification. The fungus Aspergillus versicolor LZD-14-1 was isolated from the gorgonian Pseudopterogorgia sp. (LZD-14), which was collected from the South China Sea, in May 2015. The strain was identified by comparing the morphological characteristics and analysis of the ITS region of the rDNA sequence with those of standard records (GenBank KX254916). The morphological examination was performed by scrutinizing the fungal culture, the mechanism of spore production, and the characteristics of the spores. For inducing sporulation, the fungal strains were separately inoculated onto potato dextrose agar. All experiments and observations were repeated at least twice, leading to the identification of the strain LZD-14-1 as A. versicolor. The strain LZD-14-1 was deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China. Fermentation. The large-scale fermentation was carried out in Fernbach flasks (50 × 500 mL), each containing 80 g of rice. Distilled artificial seawater (NaCl 26.726 g, MgCl2 2.26 g, MgSO4 3.248 g, CaCl2 1.153 g, NaHCO3 0.198 g, KCl 0.721 g, NaBr 0.058 g, H3BO3 0.058 g, Na2SiO3 0.0024 g, Na2Si4O9 0.0015 g, H3PO4 0.002 g, Al2Cl6 0.013 g, NH3 0.002 g, LiNO3 0.0013 g, H2O 1 L) (100 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 40 days. Extraction and Isolation. The fermented material was extracted successively with EtOAc (3 × 500 mL). After evaporation under vacuum, the EtOAc extract (4.0 g) was subjected to a vacuum liquid chromatography (silica gel, 200−300 mesh) with petroleum ether/ EtOAc (from 5:1 to 0:1, gradient) as an eluent to obtain four fractions (F1 to F4). Fraction F3 (2.0 g) was chromatographed on a Sephadex LH-20 column eluting with MeOH to collect five subfractions (SF3a− SF3e). SF3b1 (130 mg) was purified by a semipreparative reversedphase (RP) HPLC using MeCN/H2O = 45:55 (2 mL/min) as a mobile phase to obtain 12 (tR = 21.2 min, 32.0 mg), 13 (tR = 23.6 min, 33.5 mg), 4 (tR = 25.8 min, 6.2 mg), 10 (tR = 28.4 min, 10.4 mg), and 5 (tR = 27.0 min, 4.0 mg). SF3b2 (176 mg) was subjected to RPHPLC with a mobile phase of MeCN/H2O = 5:5 (2 mL/min) to yield 3 (tR = 25.2 min, 16 mg), 11 (tR = 28.4 min, 132 mg), 14 (tR = 32.1 min, 4 mg), 6 (tR = 26.1min, 4 mg), and 9 (tR = 24.1 min, 1.5 mg). SF3b3 (52 mg) was purified on an RP-HPLC column with MeCN/ H2O = 6:4 as a mobile phase to obtain 2 (tR = 31.9 min, 10.2 mg) and 2950



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ECD (4.1 × 10−4 M, MeOH) λmax (Δε) 228 (−17.1), 244 (7.64), 278 (−4.09); IR (KBr) νmax 3256, 2955, 2852, 1729, 1674, 1629, 1465, 1239, 1030 cm−1; 1H and 13C NMR data, Table 4; HRESIMS m/z 370.1767 [M + H]+ (calcd for C20H24N3O4, 370.1761); 392.1584 [M + Na]+ (calcd for C20H23N3NaO4, 392.1581). Cottoquinazoline B (12): mp 276−277 °C,; [α]20D +330 (c 0.4, MeOH) (lit. mp 273 °C and [α]20D +83).5 Cottoquinazoline D (13): mp 284−285 °C; [α]20D +610 (c 0.3, MeOH) (lit. mp 270 °C and [α]20D +78).5 Cottoquinazoline C (14): mp 260−261 °C; [α]20D = +290 (c 0.3, MeOH) (lit. mp 263 °C and [α]20D +31).5 X-ray Crystallographic Analysis. Crystal data were obtained on a Rigaku MicroMax 002+ single-crystal X-ray diffractometer. Cell parameter measurements and data collection were performed with a Bruker APEX2 CCD diffractometer using the wavelength for Cu Kα (λ = 1.5418 Å) radiation. Compounds were crystallized in polar space groups. The crystal structures of 1, 10, 12, and 13 were solved by direct methods (SHELXS-97) and subsequent Fourier difference techniques (SHELEX-97, version 6.10, Bruker AXS Inc.). The crystallographic data for the structures of 1, 10, 12, and 13 have been deposited in the Cambridge Crystallographic Data Center (CCDC numbers: 1479702, 1479895, 1479703, and 1482678). These data can be obtained free of charge from CCDC via the Internet at www.ccdc.cam.ac.uk/conts/retrieving.html. Colorless crystals of 1 were obtained from CH2Cl2/MeOH (1:1) using the vapor diffusion method. Crystal data for 1, colorless needles, C24H19N5O4, M = 441.44, triclinic, space group P1 (no. 1), a = 9.2133(3) Å, b = 10.4265(2) Å, c = 10.8149(3) Å, α = 75.823(2)°, β = 88.455(2)°, γ = 89.850(2)°, V = 1006.89(5) Å3, Z = 1, T = 293 K, μ(Cu Kα) = 0.844 mm−1, Dcalc = 1.456 g/cm3, 37 760 reflections measured (8.748° ≤ 2θ ≤ 137.832°), 4688 unique (Rint = 0.1260, Rsigma = 0.0646), which were used in all calculations. The final R1 was 0.1160 (I > 2σ(I)) and wR2 was 0.3634 (all data). Flack parameter = 0.4(5). Colorless crystals of 10 were obtained from MeOH. C22H29N3O5· CH3OH (M = 415.48 g/mol): monoclinic (0.40 × 0.35 × 0.35), space group P21 (no. 4), a = 9.2929(7) Å, b = 27.4668(12) Å, c = 9.3388(7) Å, α = 90°, β = 117.638(10)°, V = 2111.7(3) Å3, Z = 4, T = 102.7 K, μ(Cu Kα) = 0.764 mm−1, Dcalc = 1.307 g/cm3, 16 335 reflections measured (6.436° ≤ 2θ ≤ 142.486°), 8036 unique (Rint = 0.0241, Rsigma = 0.0325), which were used in all calculations. The final R1 was 0.0355 (I > 2σ(I)) and wR2 was 0.0937 (all data), Flack parameter = −0.04(6). Colorless crystals of 12 were obtained from acetone/MeOH (1:1). C23H20N5O4 (M = 430.44 g/mol): orthorhombic (0.45 × 0.19 × 0.15), space group P212121 (no. 19), a = 7.53013(6) Å, b = 14.32913(9) Å, c = 17.60029(10) Å, α = 90°, β = 90°, γ = 90°, V = 1899.07(2) Å3, Z = 4, T = 113 K, μ(Cu Kα) = 0.877 mm−1, Dcalc = 1.505 g/cm3, 15 081 reflections measured (7.956° ≤ 2θ ≤ 136.106°), 3394 unique (Rint = 0.0238, Rsigma = 0.0120), which were used in all calculations. The final R1 was 0.0269 (I > 2σ(I)) and wR2 was 0.0703 (all data), Flack parameter = −0.01(4). Colorless crystals of 13 were obtained from acetone/MeOH (1:1). C24H19N5O4 (M = 441.44 g/mol): orthorhombic (0.37 × 0.15 × 0.11), space group P22121 (no. 18), a = 7.249(3) Å, b = 14.463(7) Å, c = 18.856(9) Å, α = 90°, β = 90°, γ = 90°, V = 1977.0 (16) Å3, Z = 4, T = 293 K, μ(Cu Kα) = 0.860 mm−1, Dcalc = 1.483 g/cm3, 15 657 reflections measured (7.704° ≤ 2θ ≤ 133.944°), 3466 unique (Rint = 0.0441, Rsigma = 0.0263), which were used in all calculations. The final R1 was 0.0296 (I > 2σ(I)) and wR2 was 0.0819 (all data), Flack parameter = −0.01(7). Reduction of 3. To the MeOH (0.5 mL) solution of 3 (2.0 mg) was added NaBH4 (1.0 mg) with stirring at room temperature (rt) for 5 min. The reaction mixture was subjected to analytical HPLC. One of the products possessed the same tR as that of 5 and showed [α]20D +130 (c 0.1, MeOH) and ESIMS m/z 444.17 [M + H]+. The 1H NMR spectrum of the product was identical to that of 5. Conversion of 3 to 13. Compound 3 (5 mg) was dissolved in acetone-d6 (0.5 mL) in an NMR tube. The 1H NMR spectrum was measured at the time intervals of 2, 4, 6, and 12 h. As shown in Figure

6, compound 3 completely converted to a product in 12 h, whose 1H NMR spectrum was superimposable with that of 13. In addition, the specific rotation ([α]20D +606 (c 0.1, MeOH)) and the ESIMS (m/z 442.45 [M + H]+) data were measured, while the HPLC retention time of the product was the same as that of 13. Marfey’s Method. Compound 8 (0.5 mg) was placed in a 5 mL conical vial containing HCl (6 M, 1 mL), and the sealed vials were heated at 110 °C for 20 h. After evaporation of the solvent, H2O (100 μL) was added. Then NaHCO3 (1 M, 50 μL) and L-FDAA (1%, 100 μL) in acetone were added to the hydrolysis solution, and the sealed vial was heated at 40 °C for 2 h. Then HCl (2 M, 20 μL) was added to the reaction mixture to stop the reaction. After evaporation, the reaction products were dissolved in MeOH for HPLC analysis on a Thermo BDS Hypersil C18 column (150 mm × 4.6 mm, 5 μm). MeOH/H2O (0.5% H3PO4) gradient: 0 min: 30% MeOH/H2O; 40 min: 70% MeOH/H2O, with a flow rate of 1 mL/min. UV detection was performed at a wavelength of 340 nm. In this protocol, L-Ile and Lallo-Ile are not distinguished. Calculation of Specific Rotations and ECD Data. Specific rotations were calculated at the B3lyp/6-31+G(d) level with the PCM in CH2Cl2 for 1−3 and in MeOH for 12 and 13. Conformational analyses were carried out via random searching in the Sybyl-X 2.0 version using the MMFF94S force field with an energy cutoff of 3.0 kcal/mol. The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, 0.16−0.3 eV). The simulated spectra of the two lowest energy conformers for each structure were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (ΔG). Theoretical ECD spectra of the corresponding enantiomers were obtained by directly inversing the ECD spectra of the above-mentioned compounds, respectively. Evaluation of the TrxR Inhibitory Activities. The TrxR inhibition assay was performed as described.25 Cytotoxic Assay. A tetrazolium-based colorimetric assay (MTT assay) was used for testing the compounds against the tumor cell lines A549 and A2780.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00801. NMR, HRESI, and IR spectra of 1−11 and the theoretical calculation details of ECD spectra and specific rotations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: ++86-10-82806188. E-mail (W. Lin): [email protected]. cn. ORCID

Wenhan Lin: 0000-0002-4978-4083 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the grants from the 973 Program (2015CB755906), NSFC-Shangdong Join Fund for Marine Science (U1406402), and NSFC (81630089, 41376127). We thank Dr. W. Zhu for providing an authentic sample of cottoquinazoline B. 2951



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REFERENCES

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