Asperorydines AâM: Prenylated Tryptophan-derived Alkaloids with

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Asperorydines A-M: Prenylated Tryptophan-derived Alkaloids with Neurotrophic Effects from Aspergillus oryzae Li Liu, Li Bao, Long Wang, Ke Ma, Junjie Han, Yanlong Yang, Ruixing Liu, Jinwei Ren, Wenbing Yin, Wenzhao Wang, and Hongwei Liu J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02802 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 22, 2017

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The Journal of Organic Chemistry

Asperorydines A−M: Prenylated Tryptophan-derived Alkaloids with Neurotrophic Effects from Aspergillus oryzae Li Liu,†,‡,# Li Bao,†,‡,# Long Wang,†,# Ke Ma,†,‡ Junjie Han,†,‡ Yanlong Yang,† Ruixing Liu,†,‡ Jinwei Ren,† Wenbing Yin,†,‡ Wenzhao Wang,† Hongwei Liu†,‡,*

†

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of

Sciences, Beijing, 100101, People’s Republic of China ‡

Savaid Medical School, University of Chinese Academy of Sciences, Beijing,

100049, People’s Republic of China #

Authors contributed equally to this work.

*

Corresponding Author Tel: +86 10 64806074; E-mail: [email protected] (H−W, Liu)

1

ACS Paragon Plus Environment

The Journal of Organic Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Graphic Abstract

2


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ABSTRACT: As part of our program to discover new bioactive agents from fungi, 13 new alkaloids accompanying 13 known related alkaloids were isolated from a wild strain of Aspergillus oryzae L1020. Compounds 1 and 2 have unprecedented 6/6/5/7/5 and 6/6/6/5/5 chemical skeletons, representing new members of quinoline alkaloids. Compound 3 is a new macrolactam with an unusual 6/5/6/8 ring system. Compounds 4−13 are new α-cyclopiazonic acid related alkaloids. The absolute configurations of 1−4, 8, and 9 were assigned by electronic circular dichroism (ECD) calculation. Compounds 2, 5, 6, 11, 14, 22, and 26 exhibited pronounced neurite outgrowth promoting effects on PC12 cell in the range of 25−100 µM. Keywords: Aspergillus oryzae, quinoline alkaloids, prenylated indole alkaloids, neurotropic effect

3



INTRODUCTION With the lifetime extension, the aging-related neurodegenerative diseases have become the major threat to the public health of aged people. Bioactive compounds with neurotrophic properties are in an urgent need for the discovery and development of new drugs against neurodegenerative diseases. Natural products, such as jiadifenin from the plant Illicium jiadifengpi, huperzine A from Huperzia serrata, erinacines from medicinal mushroom Hericium erinacium, paecilomycine A from the fruiting bodies of Paecilomyces tenuipes, have been demonstrated as a rich source for neurotrophic agents.1-5 Prenylated indole alkaloids have attracted much attention of chemists and biologists due to their complex structures and interesting bioactivities. Great efforts have been endeavored to synthesize and identify new prenylated indole alkaloids. The fungi belonging to the genus of Claviceps, Penicillium and Aspergillus produced a wide array of indole alkaloids, including versicolamides, paraherquamides, fumitremorgins, α-cyclopiazonic acid, and the ergot alkaloids.6−10 With the aim to exploring new prenylated indole alkaloids, the solid culture extracts of different Aspergillus strains were screened by HPLC analysis of the secondary metabolites with the UV characteristics of indole alkaloids. The strains producing rich indole alkaloids were put into further chemical investigation. In our recent report, three highly modified dioxopiperazine alkaloids with novel skeletons were reported from A. tennesseensis.6 Herein, we report the isolation, structure elucidation, neuroprotective and neurotrophic activities of 13 new prenylated alkaloids, asperorydines A−M (1−13), 4


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from the solid culture of A. oryzae L1020 fermented on rice. 22

O 24

O 21 22 19

H 11

8 9 17

16

13

15 14

4

NH

2

1

1

11

H

11

14

5 4

19

15

18 17

N

2

N

N

O

15

16

N 7

17

O

O

8

4

13 14

O

O

N H1

14 13

O

17 R=

3

15

O

O

H

15

O

22 R=

12

19 10

17

1

O

O N

H

H

HO

N H

N

H

N H

H O H

NH 12

O H N H

N

N

H

H

14 R= 24 R=H

N

N OH N

O OH

H

O

H O H R O

O

N 16 R=OH 18 R=H O

O

OH

H OH

H O

19

N H 15 R= H 20 R= H

O

OH

N R O

O O

N

O

O

H H

N

13 O

OH

N

H

O H OH

H O H H OH O

N

N

N

H

H O H OH O

O

R

O

N

H

N

O

NH

11

H

9 R=CH3 10 R=OCH3

O H

O

NH

O

NH

2

N

H

O

O

O NH

O

N

H H

4

H

H

N

H

14

O

H

R

16

O 5

1 13

18

17

6

3

12

O

8

6

9

11

15

N

8

H

7

NH

2

25 R=H

7

H

3

O

4 R= H 5 R= H O O

20

H O

5

4

N1

18

18 19

6

4

16

O

N

11

6

17

OH 8

N

H

OH

7 R=

9

10 21

N

5

16

22

O

R 2

1419

3

O

R

15

16

20 19

4 3

O

N1

17

7 5

12

O

3

13

9

13

4

2

11

19

5

2

18

8

N

20

6

18

14

O

7

8

10 11

21 12

O H HH OH

O

O

21 10

HN

20

1

16

2 12

N

H

O HH OH

H

3

H

O

O 9

H

N 21 H

7

6 12

20

8

N

5

6

9

13

6

3

20

10

O

18

O

H N H

21

H

23

7

10 12

20

O

9

10

H

N

N H H

H

N

N

H O H

H

HN

H O H

H

O

O N

N H

21

23

N H

N H 26

Figure 1. Structures of 1−26 from Aspergillus oryzae.

RESULTS AND DISCUSSION The fungus A. oryzae was isolated from the surface of an unidentified leaf collected in Tiantanshan Mountain (Shanxi, China), and identified on the basis of the morphological characteristics and the ITS gene sequences. The fungus was fermented 5



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on rice medium and extracted with EtOAc. The EtOAc extract was subjected to silica gel, ODS, Sephadex LH-20, and HPLC chromatography to afford new alkaloids 1−13 and known α-cyclopiazonic acid derivatives (Figure 1) including speradine D (14),11 α-cyclopiazonic acid (CPA) (15),9 3-OH-speradine A (16),12 speradine H (17),13 speradine A (18),14 cyclopiamide E (19),15 iso-α-cyclopiazonic acid (20),16 speradine B (21),11 speradine E (22),17 α-cyclopiazonic acid imine (23),18 cyclopiamide H (24),15 cyclopiamide (25),19 and bissecodehydrocyclopiazonic acid (26).18

O

O

O O

N

O

NH2

HN

O OH

NH

O O

N H

N

1

N

O 3

2 1

HMBC

H-1H COSY

Figure 2. Key HMBC and 1H−1H COSY correlations of 1−3.

Asperorydine A (1) has a molecular formula of C20H20N2O3 with 12 unsaturation degrees, as determined from HRESIMS at m/z [M+H]+ 337.1551. The 1H,

13

C, and

HSQC NMR spectra (Table 1 and Table 3) of 1 showed the presence of a 1, 2, 3-trisubstituted benzene, three singlet methyls, two exchangeable hydrogens, a trisubstituted double bond, three carbonyl carbons, one methylene, three methines, and two quaternary carbons. The exchangeable hydrogens were deduced to be secondary amines in accordance with its molecular formula.

1

H−1H COSY

correlations of H-1−H-2, H-12−H-13−H-14, and H2-10−H-9−H-17, as well as key 6


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HMBC correlations of H-2 with C-3, C-15, and C-17, H-9 with C-3, C-10, C-11, and C-17, H2-10 with C-9, C-11, C-12, C-16, and C-17, H-17 with C-2, C-3, C-9, C-10, and C-16, and NH-1 with C-2, C-3, C-15, and C-16 confirmed the presence of cyclopenta[de]quinoline

moiety

(Figure

2).

The

structural

fragment

of

3-acetylpyrrolidine-2,4-dione was deduced on the basis of 1H−1H COSY correlations of H-4 with NH-5, as well as HMBC correlations of H-4 with C-3, C-6, C-7, C-17, and C-18, NH-5 with C-4, C-6, C-7, and C-18, H3-20 with C-7 and C-19. HMBC correlations of H-21 (22) with C-7, C-8, and C-9 finally connected the moieties of cyclopenta[de]quinoline and 3-acetylpyrrolidine-2,4-dione to form an unusual pentacyclic ring system of 6/6/5/7/5. The relative configuration of 1 was determined by ROESY experiments. The NOE correlations of H-4 with H-17 and H3-20, H3-21 with H-10α and H-17, H-9 with NH-5 and H-10β, and H3-22 with H-10β together with the larger coupling constant of 10.5 Hz between H-9 and H-17 supported the relative configurations of 1 as described in Figure 3. To determine the absolute configuration in 1, ECD calculation method by time-dependent density functional theory (TDDFT) at B3LYP/6-311G(d,p) level was first applied. Unfortunately, the simulated ECD curves obtained by B3LYP method did not match with the experimental ECD for 1. Next, we attempted the BP86 method that had been successfully used to determine the absolute configuration of bistramide C, bisnicalaterines, chisomicines, eucophylline.20−23 A comparison of the experimental ECD spectrum for 1 with the calculated ECD spectra for the 1a and 1b enantiomers obtained by BP86 method is shown in Figure 4. The overall ECD spectra for 1a 7



obtained by BP86 method matched well with the experimental ECD for 1 (Figure 4). Thus, the absolute configuration of 1 was determined to be 4S, 7S, 9S, 17S.

Figure 3. Key NOE correlations of 1−3.

Asperorydine B (2) was obtained as a mixture of two interchangeable isomers. Its molecular formula was determined to be C22H23N3O4 on the basis of the HRESIMS. The 1H and 13C NMR data (Table 1 and Table 3) of 2 showed the presence of two sets of signals, each containing three exchangeable hydrogens, four methyls (one attached to nitrogen), one methylene, three methines, and fourteen quaternary carbons including three carbonyl groups, ten olefinic carbons, and one sp3 hubridized carbon. The 1D NMR data of 2 showed similarity with those of cyclopiazonic acid imine (23)18 except that additional signals of N-CH3 and carbonyl group were present in 2. Compound 23 was reported as a pair of isomers with the ration of 3:2 due to the existence of tautomeric forms of ketamine and enimine.18 Comparison of 1H NMR spectrum between 2 and 23 indicated that the exchangeable protons at δH 8.95 ppm (two protons), 9.59 ppm, and 9.72 ppm in 2 were ascribed to hydrogens in the ketamine and enimine systems. Further interpretation of 1H−1H COSY and HMBC spectra confirmed a new type of quinoline alkaloid with unusual ring system of 6/6/6/5/5 (Figure 2). A hydroxyl group (2A: δH 9.33; 2B: δH 9.14) was attached at C-3 8


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on the basis of the HMBC correlation between OH-3 and C-4, and the consideration of the chemical shift of C-3 (2A: δC 142.6; 2B: δC 142.5). In the ROESY spectrum of 2, the NOE correlations of H-12 with H-5 and H-13α, and H-6 with H-13β assigned the relative configuration of 2 (Figure 3). The similar Cotton effects at 224 and 259 nm between 2 and 23 assigned the absolute configuration of 6S in 2 (Figure S11).9 The absolute configuration of 2 was further determined by ECD calculation. The calculation was conducted in methanol by using TDDFT at B3LYP/6-311G(d,p) basis set level. Considering the relative configuration of 2, two possible stereoisomers 2a and 2b were deduced. The experimental ECD spectrum of 2 was similar to the calculated ECD spectrum of 2b (Figure 4). Accordingly, the absolute configuration of 2 was determined to be 5R, 6S, 12R.

9



Figure 4. Experimental CD spectra of 1 and 2 and the calculated ECD spectra of 1a, 1b, 2a, and 2b.

10


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position 1(N) 1N-CH3 2 3-OH 4 5 6 7

1a 10.76 s

2Aa, e

2Ba, e

Table 1. 1H NMR data of 1−7 δH mult. (J in Hz) 3a 4b

3.70 s

3.71 s

3.37 s

3.45 s

3.44 s

9.33 brs

9.14 brs

3.66 o 4.01 d (11.0)

3.66 o 4.01 d (11.0)

5.44 s

6.62 s

6.54 s

2.20 d (15.9) 2.82 d (15.9)

4.43 m

4.61 m

2.61 dd (15.7, 10.4) 2.70 dd (15.7, 5.6)

2.40 dd (16.6, 5.6) 2.70 dd (16.6, 7.2)

7.14 o 4.64 s 9.38 s

9

1.82 td (10.5, 3.1)

10

2.82 dd (15.4, 10.5) 2.95 dd (15.4, 3.1)

14 15 16 17 18 18-OH 19 20 21 22 23 24 a

6.74 d (7.0) 6.98 dd (8.1, 7.0)

2.37 o 3.01 d (5.6) 3.17 o

2.35 o 2.98 d (5.6) 3.17 o

7.14 o 7.36 o 7.36 o

7.14 o 7.36 o 7.36 o

7.13 o 3.27 d (10.5)

2.03 s 1.13 s 1.33 s

6b 8.09 s 7.15 od

7a 3.38 s

4.12 d (7.9)

8

11 12 13

5b

8.95 s, 9.59s 2.33 s 1.51 s 1.49 s

8.95 s, 9.72 s 2.35 s 1.51 s 1.49 s

8.11 s 7.60 d (8.4) 7.53 dd (8.4, 7.0) 7.13 d (7.0)

1.91 s 1.49 s 1.92 s

7.84 s

7.84 s

7.55 d (8.5) 7.50 dd (8.5, 6.9) 6.90 d (6.9)

7.64 d (8.5) 7.49 dd (8.5, 6.9) 6.89 d (6.9)

1.83 s 1.93 s 1.57 d (6.2)

1.80 s 1.96 s 1.38 d (6.3)

2.70 m 2.95 dd (16.9, 6.0) 3.12 dd (16.9, 6.0)

8.52 s

6.90 d (6.8) 7.17 o 7.19 d (6.8)

7.68 od 7.68 o 7.19 d (7.5)

3.93 d (16.4) 4.02 d (16.4)

3.11 dd (15.7, 5.7) 3.23 dd (15.7, 7.1) 4.17 m 4.64 d (5.3) 1.17 d (6.2) 1.82 s 1.82 s

2.24 s 1.25 s 1.69 s

Recorded in DMSO-d6; b Recorded in CDCl3; c “m” means multiplet with other signals; d “o” means overlapped with other signals; e A: ketamine-form; B: enimine-form.

11



The molecular formula of asperorydine C (3) was determined to be C20H20N2O3 with 12 degrees of unsaturation, based on the HRESIMS at m/z 337.1547 for [M+H]+. The 1H, 13C and HSQC NMR spectra (Table 1 and Table 3) of 3 revealed the presence of four methyl groups, one methylene, one oxygenated methine, two quaternary carbons, one of which is oxygenated carbon, one naphthalene system, and two carbonyl groups. A detailed comparison of the proton and carbon signals of 3 and 25 indicated the presence of a 4, 5-disubstituted-1-methylbenzo[cd]indol-2(1H)-one moiety, which was further supported by HMBC experiments. The HMBC correlations of H-5 with C-2, C-3, C-4, C-11, and C-19, H2-7 with C-5, C-6, C-8, and C-19, H3-19 with C-5, C-6, C-7 and C-8 indicated the connection of 3-methylbutanoid acid related moiety to C-4 (Figure 2). The attachment of an isopropyl moiety to C-11 was confirmed by the HMBC correlations of H3-20 (21) with C-10 and C-11. Considering the molecular formula of 3, the unsaturation degree requirement, the chemical shifts of C-5, C-6, C-8, and C-10, an amide linkage between C-8 and C-10 and one oxirane ring incorporating with C-5 and C-6 were deduced. Based on the evidence, the planar structure of 3 was established. The NOE correlations of H-5 with H3-19 and H3-20 indicated that the H-5, CH3-19, and CH3-20 were on the same side of azocan-2-one ring (Figure 3). Computer simulation of the ECD spectra for 3a and 3b was performed by using TDDFT at B3LYP/6-31G(d) basis set level in methanol. The spectrum for 3a is in good accordance with the experimental spectrum of 3 (Figure 5). Therefore, the absolute configuration of 3 was assigned as 5R, 6S.

12


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Figure 5. Experimental CD spectra of 3−5 and the calculated ECD spectra of 3a, 3b, 4a, and 4b.

Asperorydine D (4) and E (5) possess the same molecular formula of C20H20N2O3, as determined by HRESIMS (4: m/z 337.1546; 5: m/z 337.1547). The 1H and

13

C

NMR spectra (Table 1 and Table 3) of 4 and 5 resembled with those of 3 and 25, suggesting the presence of the same substituted N-Me benzoindolone moiety as in 3 and 25. The 1H−1H COSY correlations of H2-8−H-7−H3-22, as well as the HMBC correlations of H-5 with C-7, H-7 with C-5, C-8, and C-22, H-8 with C-7, C-9, and C-22, and H3-22 with C-7 and C-8 confirmed the linkage of a butanoic acid moiety at C-5 via an ether bond in 4 (Figure S1). Finally, the connection between C-9 and N-10 was proposed to satisfy its molecular formula and the unsaturation degrees. The NOE cross peak between H-5 and H3-22 indicated that H3-22 and H-5 are orientated to the 13



same side of 1, 3-oxazinan-4-one ring in 4 (Figure S1). The absolute configuration of

4 was determined as 5S, 7S by the comparison of experimental and calculated ECD curves (Figure 5). Compound 5 was determined to possess the same planar structure as that of 4 by detailed analysis of its 2D NMR data. In the ROESY spectrum of 5, the NOE cross peak between H-5 and H-7 supported that these protons were on the same face of 1, 3-oxazinan-4-one ring (Figure S2). The CD spectrum of 5 was completely opposite to that of 4 (Figure 5), which helped us assign the 5R configuration in 5. With the relative configuration determined in 5, the absolute configuration of 5 was assigned as 5R and 7S.

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Table 2. 1H NMR data of 8−12 position 1(N) 1N-CH3 5 6 9 10

8b 9.32 m 2.91 d (4.6) 3.16 dd (9.9, 7.0) 4.34 d (9.9)

12 13

2.70 m 2.64 m 2.96 d (5.1)

14 15 16

a

9a 9.00 q (5.1) 2.87 d (5.1) 3.96 d (2.5) 2.65 mc 2.64 m 2.93 dd (10.6, 2.5) 6.47 d (7.1) 7.34 dd (8.6, 7.2) 6.61 d (8.6)

6.40 d (7.3) 7.30 t (8.0)

17 18 19

6.55 d (8.6)

20 21 22

1.42 s 1.60 s

2.45 s

3.83 d (16.8) 3.98 d (16.8) 2.11 s 1.44 s 1.52 s

δH mult. (J in Hz) 11Aa, e 9.15 q (5.0) 2.92 d (5.0) 2.86 d (6.4) 3.75 d (6.8) 2.97 dd (10.0, 6.4) 4.16 d (10.0) 2.64 m 2.79 m 2.95 m 6.39 d (7.2) 2.59 o 7.30 dd (8.6, 7.2) 2.90 o 3.07 m 6.56 d (8.6) 6.46 d (8.5) 3.80 d (16.8) 7.32 t (8.5) 4.02 d (16.8) 6.57 d (8.5) 3.68 s 1.54 s 8.84 d (5.0) 9.50 d (5.0) 1.65 s 2.32 s 1.44 s 1.28 s 10b

11Ba, e 9.15 q (5.0) 2.86 d (6.4) 2.97 dd (10.0, 6.4) 4.24 d (10.0)

12Aa, e 9.32 brs 2.86 s

12Ba, e 9.32 brs 2.86 s

2.59 o 2.90 o 3.07 o

3.44 d (5.4) 2.86 m 2.92 t (14.0)

3.47 d (5.4) 2.86 m 2.92 t (14.0)

6.47 d (8.5) 7.32 t (8.5)

6.45 d (7.1) 7.28 t (8.5, 7.1)

6.45 d (7.1) 7.28 t (8.5, 7.1)

6.57 d (8.5)

6.59 d (8.5)

6.59 d (8.5)

8.94 d (5.0) 9.74 d (5.0) 2.34 s 1.41 s 1.31 s

9.03 brs 9.75 brs 2.36 s 1.36 s 1.61 s

9.00 brs 9.13 brs 2.36 s 1.38 s 1.61 s

Recorded in DMSO-d6; b Recorded in CDCl3; c “m” means multiplet with other signals; d “o” means overlapped with other signals; e A: ketamine-form; B: enimine-form.

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Table 3. 13C NMR data of 1−12 position 1N-CH3 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 a

1a 118.1 111.7 62.0 169.6 74.5 45.1 46.3 27.3 130.3 115.2 122.1 108.7 133.8 127.0 41.3 205.6 200.2 30.0 16.6 21.6

2Aa, c 29.9 157.7 142.6 117.4 35.9 70.1 194.3 97.8 174.9

2Ba, c 29.9 157.6 142.5 116.8 36.0 68.9 196.0 99.2 172.2

61.7 49.0 27.5 133.7 122.5 126.6 112.4 134.2 117.0 168.2

61.5 49.0 27.4 133.7 122.6 126.6 112.4 134.1 117.0 168.1

18.7 24.7 24.7

17.8 24.7 24.8

3a 26.2 166.4 121.3 134.1 74.5 78.2 51.2 172.7 62.6 153.3 123.9 128.7 120.0 128.7 104.9 139.3 124.4 25.3 29.7 26.0

4b 26.5 166.5 122.6 131.8 83.5

5b 26.5 166.4 122.7 132.0 87.1

69.0 41.8 169.1

71.1 40.1 167.9

66.7 150.4 123.6 130.3 120.6 129.5 105.0 140.8 125.7 28.4 28.4 21.8

66.6 149.9 123.7 130.3 120.5 129.5 105.0 140.7 125.8 28.7 28.1 22.9

δC 7a 26.5 120.0 164.0 106.6 124.2 40.6 125.7 176.4 164.2 6b

66.2 43.2 25.3 128.4 116.7 123.5 108.7 133.5 126.2 168.6 54.4 201.7 30.3 22.2 26.7

65.3 152.4 124.3 131.7 120.2 131.5 105.6 140.9 125.5 172.4 47.6 62.9 23.7 26.4 26.4

8b 29.5 153.3 113.8 195.8 48.8 68.1 193.3 105.0 175.6 64.2 51.3 28.8 143.3 114.4 136.1 109.6 185.4 19.8 24.1 25.2

Recorded in DMSO-d6; b Recorded in CDCl3. c A: ketamine-form; B: enimine-form.

16


9a 29.1 152.1 114.6 190.6 53.6 170.6

10b 29.5 153.1 113.8 190.4 54.0 170.8

63.1 42.7 28.3 143.4 113.2 136.1 109.6 168.2 53.8 202.1 29.9 23.7 22.8

64.0 43.9 29.2 143.2 114.7 136.4 109.9 167.7 45.8 168.1 52.5 24.4 23.1

11Aa, c 29.1 152.2 113.4 196.8 49.1 66.5 193.5 97.3 175.2

11Ba, c 29.1 152.3 113.3 196.7 49.1 65.4 195.9 98.9 172.4

12Aa, c 29.6 153.0 116.4 184.6 117.3 117.7 178.1 100.8 168.6

12Ba, c 29.6 153.0 116.4 184.5 117.3 117.7 176.0 99.3 168.8

62.4 50.7 28.0 144.1 114.5 135.5 108.9 167.9

62.1 51.0 28.1 144.2 114.5 135.5 108.9 168.2

62.7 56.3 31.4 143.5 115.1 135.2 110.0 166.5

62.4 56.4 31.5 143.5 115.1 135.2 110.0 166.5

18.6 24.2 24.7

17.8 24.2 24.8

18.5 20.2 28.1

19.4 20.3 28.1

Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Asperorydine F (6) and G (7) were determined to have the molecular formula of C19H20N2O3 and C20H20N2O4 by HRESIMS, respectively. The 1H and 13C NMR data (Table 1 and Table 3) comparison of 6 with 14 and 15 indicated that it shares the same perhydro-benzoindole moiety with 15, and the rest of the structure with 14. The NOE correlations of H3-21 with H-4 and H-8 supported the same orientation for H-4 and H-8 (Figure S3). The negative Cotton effect at 274 nm and the positive Cotton effect at 303 nm arising from the indole ring 9 in the CD spectrum of 6 were in accordance with the corresponding Cotton effects of 15 (Figure S99), which speculated the 4R and 8R absolute configuration in 6. The 1D NMR data (Table 1 and Table 3) of 7 show much similarity with those of 17 except for the absence of a ketone carbon in 17 and the presence of an oxygenated methine in 7. The hydroxyl group was assigned at C-18 by the 1H−1H COSY correlations of H2-17−H-18−H3-19 and OH-18−H-18, as well as the HMBC correlations of H2-17 with C-16, C-18, and C-19, H-18 and HO-18 with C-16, C-17, and C-19, and H3-19 with C-17 and C-18 (Figure S4). Further analysis of 2D NMR data assigned the structure of 7. The configuration of C-18 was left unsolved due to the small quantity of 7.

17



Page 18 of 33

Figure 6. Experimental CD spectra of 8−10 and the calculated ECD spectra of 8a, 8b, 9a, and 9b.

Asperorydine H (8) was assigned the molecular formula C20H22N2O4 by the HRESIMS at m/z 355.1656 for [M+H]+. The 1H and

13

C NMR spectra (Table 2 and

Table 3) of 8 were similar with those of 16, except for the loss of an oxygenated quaternary carbon. Detailed analysis of 1H−1H COSY and HMBC spectral data completed the planar structure of 8 (Figure S5). Compound 8 possesses a new skeleton

of

5,5a,6,11,11a,11b-hexahydro-1H-benzo[f]pyrrolo[2,1-a]isoindole-1,3(2H)-dione. The α configurations of H-5 and H-12 and the β configuration of H-6 were determined from the NOE cross peaks of H3-21 with H-5 and H-12, and H3-20 with H-6 (Figure S5). The experimental ECD spectrum of 8 was almost consistent with the calculated 18


Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ECD spectrum for (5S, 6S, 12R)-8b (Figure 6). Thus, the absolute configuration of 8 was assigned as 5S, 6S, 12R. The HRESIMS spectra of asperorydine I (9) and J (10) indicated the molecular formula of C19H22N2O4 and C19H22N2O5, respectively. A detailed comparison of NMR data of 9 with those of 6 and 8 revealed the presence of a naphthalen-1(2H)-one derived moiety and a 1-(2,2-dimethyl-5-oxopyrrolidin-1-yl)butane-1,3-dione moiety. Further HMBC spectrum analysis established the structure of 9 (Figure S6). The 1D NMR data of 10 was similar to those of 9, except for the replacement of the methyl keton of 9 with a carbomethoxy moiety in 10. HMBC correlation of the methoxyl group with C-17 indicated the attachment of a methoxyl group at C-17 in 10 (Figure S7). In the ROESY spectrum of 9, the NOE correlation of H3-19 with H-5 and H-9 confirmed the same orientation of H-5 and H-9 (Figure S6). Computer simulations of the ECD spectra for 5S, 9S- and 5R, 9R-9 were conducted. The absolute configurations at C-5 and C-9 were assigned as 5R and 9R by comparison of the experimental and simulated ECD curves (Figure 6). The absolute configuration of 10 was determined to be the same as that of 9 by similar Cotton effects in the CD spectrum between 9 and 10 (Figure 6). Asperorydines K−M (11−13) were isolated as the interchangeable isomer mixture, similar to 2 and 23. The structural assignments of 11−13 were achieved by careful interpretation of their 2D NMR spectra (Figures S8−S10). Compound 8 can be transformed into 11 with the addition of ammonia, which further confirmed the presence of imine moiety in 11. As described for 23,18 compounds 2, and 11−13 could 19



Page 20 of 33

be derived from the reaction between the CPA-related metabolites and ammonia produced in the growth of fungi. Accordingly, the absolute configurations of 11−13 were determined to be 5S, 6S, 12R-11, 12R-12, and 3R, 4S, 5S, 11R-13, respectively. In this study, we reported two new quinoline alkaloids with unusual skeletons, eleven new CPA derivatives, and thirteen known CPA analogues from a strain of A. oryzae. Compounds 1−13 are structurally related to each other, composing of a tryptophan residue, a prenyl moiety, and one polyketide unit. After the first report of CPA in 1968,9 a total of 30 CPA-type alkaloids have been discovered from species of Aspergillus and Penicillium. CPA and its analogues showed interesting bioactivities, such as the inhibitory activity against Ca2+-ATPase,14,

24

histone deacetylase,14

antitobacco mosaic virus,25 and toxicity against brine shrimp.15 The CPA biosynthesis gene cluster composed of 7 genes, cpaR, cpaA, cpaD, cpaO, cpaH, cpaM, and cpaT, has been described in A. oryzae.26 The functions of cpaA, cpaD, cpaO, cpaH, and cpaM were demonstrated in the biosynthesis of CPA, 2-oxo CPA, and speradine A.26 These genes are supposed to be partly involved in the biosynthesis of new compounds

1−13. We evaluated the neuroprotective effect and neurite promoting activity of CPA analogues using PC12 cells. Compound 15 that showed cytotoxicity towards PC12 cells at 100 µM was not tested. The cytoprotective activities of compounds 1−14,

16−26 against H2O2 induced oxidative stress were evaluated using PC12 cells. After incubation with 400 µM H2O2 for 24 h, only 47.29% of cultured cells survived. Compounds 4, 5, 6, 9, 10, 14, 17, 22, and 26 showed protective capabilities with the 20


Page 21 of 33

survival rate from 70.16 to 90.85% at 100 µM (Figure 7). Luteolin is used as positive control with survival rate of 91.61% at 100 µM. The effects of compounds 1−26 on the neurite outgrowth of undifferentiated PC12 cells were further evaluated by morphological observations and a quantitative analysis of neurite-bearing cells and neurite length. Compounds 2, 5, 6, 11, 14, 22, and 26 showed significant neurotrophic effects in the range of 25−100 µM (Figure 8, Figure S13−S18), as compared with control group. The percentage of neurite-bearing cells for cells treated with compounds 2, 5, 6, 11, 14, 22, and 26 at 100 µM reached 18.58 ± 1.77%, 18.62 ± 1.87%, 20.07 ± 1.05%, 26.85 ± 1.31%, 18.64 ± 1.47%, 20.14 ± 1.34%, and 15.2 ± 1.35%, respectively. NGF is used as positive control with neurite-bearing cells of 22.06 ± 1.24% at the concentration of 20 ng/mL. The percentage of neurite-bearing cells in the negative group was 4.83 ± 0.65%. Compounds 6 and 14 showed stronger neuroprotective and neurotrophic activities than 15 and 16, respectively, indicating that the substitution of a butanoic acid side chain is more beneficial to the neuroprotective and neurotrophic activities than the formation of a tetramic acid moiety. 100

cell survival rate (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 N P

Figure 7. Effect of compounds 1−26 on H2O2 induced PC12 cell death. 1−26: compounds 1−26 100 µM; N: 400 µM H2O2 as negative control; P: 100 µM luteolin as positive control 21



A (Control)

B

C

Page 22 of 33

D

Figure 8. Neurite outgrowth of PC12 cells after 24 h treatment with NGF (B: 20 ng/mL) or 2 (C: 50µµM). Cell with one or more neurites whose lengths were at least twice the diameter of the cell body were scored as positive. The percentage of positive neurite-bearing cells was determined from at least three different regions of interest in three independent experiments (D). Values were considered significant at ***P

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