Neosartoryadins A and B, Fumiquinazoline Alkaloids from a Mangrove

Dec 29, 2015 - Neosartoryadins A (1) and B (2), both with a unique 6/6/6/5 quinazoline ring system connected directly to a 6/5/5 imidazoindolone ring,...
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Neosartoryadins A and B, Fumiquinazoline Alkaloids from a Mangrove-Derived Fungus Neosartorya udagawae HDN13-313 Guihong Yu, Guoliang Zhou, Meilin Zhu, Wei Wang, Tianjiao Zhu, Qianqun Gu, and Dehai Li* Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China S Supporting Information *

ABSTRACT: Neosartoryadins A (1) and B (2), both with a unique 6/6/6/5 quinazoline ring system connected directly to a 6/5/5 imidazoindolone ring, together with three biogenetically related compounds 3−5, were isolated from the endophytic fungus Neosartorya udagawae HDN13-313. The absolute configurations of new compounds 1−4 were established. Compounds 1 and 2 displayed anti-influenza virus A (H1N1) activities with IC50 values of 66 and 58 μM, respectively (ribavirin as positive control, IC50 = 94 μM).

T

he quinazoline-containing indole alkaloids, which possess pyrimido[2,1-b]quinazoline and imidazo[1,2-a]indole moieties linked by a methylene (and in some cases further linked via additional spiro-bridges), were first isolated from Aspergillus f umigates in 1992 and named fumiquinazolines A− C.1 In the following decades, about 40 fumiquinazoline analogues with these structural characteristics were reported from various fungal genera, including Aspergillus sp.,1 Acremonium sp.,2 and Neosartorya sp.,3 and named fiscalins,3 aniquinazolines,4 quinadolines,5 etc. (Figure S1, Supporting Information, SI). Biogenetically, they are all derived from anthranilic acid (ATA) and tryptophan, together with two other amino acids, including alanine, valine, glycine, leucine, 2aminoisobutyric acid (Aib), etc.6 As an important class of tremorgenic mycotoxins, they show potent biological properties such as antifungal,2 cytotoxic,7 antifeedant,8 and antiviral9 activities. Because of their structural novelty and attractive bioactivities, these compounds have attracted broad interest from chemical and biosynthetic scientists. During our search for novel bioactive secondary metabolites from microorganisms derived from diverse ecological environments,10−12 an endophytic fungus Neosartorya udagawae HDN13-313, isolated from the root of the mangrove plant Aricennia marina, was selected for investigation due to its extract’s interesting HPLC−UV profile and weak cytotoxicity (41% inhibition of P388 cells at 100 μg/mL). Further examination of a bulk culture led to the discovery of four new quinazoline-containing indole alkaloids, named neosartoryadins A and B (1 and 2) and fiscalins E and F (3 and 4), along with a known biogenetic related compound fiscalin C (5) 3 (Figure 1). The absolute configurations of new compounds 1−4 were established on the basis of NOESY spectra, electronic circular dichroism (ECD), or single-crystal X-ray diffraction analysis (for compound 3). Distinguishing 1 and 2 from classic fumiquinazoline alkaloids such as compounds 3−5 is the unprecedented pyrido[2,1-b]© XXXX American Chemical Society

Figure 1. Structures of compounds 1−5.

quinazoline moiety with the quinazoline conjugated to a pyridine (C ring) rather than to a pyrimidine ring and the existence of a unique tetrahydrofuran ring (D ring). Compounds 1 and 2 showed potential antiviral activity against influenza virus A (H1N1). Herein, we report the details for the isolation, structure elucidation, bioactivity, and a plausible biogenetic pathway to 1 and 2. Compounds 1 and 2 were obtained as white amorphous powders. Their molecular formulas were established as C27H26N4O5 and C27H26N4O6 according to the HRESIMSprotonated molecular ions detected at m/z 487.1966 and 503.1924, respectively. The 1D NMR data of 1 and 2 included four methyls, one methylene, 10 methines (including eight aromatic methines), and 12 nonprotonated carbons (including three carbonyls) (Table 1). Analysis of the 1D and 2D NMR data of 1 and 2 revealed two sets of four contiguous sp2 Received: November 26, 2015

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DOI: 10.1021/acs.orglett.5b02964 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters Table 1. 1H (500 MHz), 13C (125 MHz) and 15N (50 MHz) NMR Data of Compounds 1 and 2 in DMSO-d6 (δ ppm) 1 no.

a

δC/N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

106.2 50.5 192.8 144.2 281.7b 146.8 129.3 135.2 129.0 126.7 122.8 161.1 264.0b 61.7 40.1

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1-OH 18-OH

85.6 82.4 277.9b 65.3 176.0 289.2b 138.8 115.3 130.4 125.5 126.9 137.0 16.4 21.0 25.6 26.3

(δH 2.36) to C-16/C-17/C-30 (δC 25.6)/C-31 (δC 26.3), and the 1H−15N HMBC correlations from H-17 to N-18 (δN 277.9), and from H3-30/H3-31 to N-18. The chemical shifts of the 6/5/5 imidazo[1,2-a]indole moiety also agree with those reported for the fumiquinazolines.1 The imidazo[1,2-a]indole and the pyrido[2,1-b]quinazoline moieties were linked by C-15 (δC 40.1) based on HMBC correlations from H2-15 (δH 3.52, 2.63) to C-1 and C-14 in ring C and C-16, C-17 and C-27 in F and from H-14 to C-16 and the 1H−15N HMBC correlation from H2-15 to N-13 (δN 264.0). Finally, the planar structure of 1 was completed by a furan ring (D ring) evidenced by the chemical shifts of C-16 (δC 85.6) and the distinctive hemiketal carbon (δC 106.2, C-1), as well as the required degrees of unsaturation. The NMR data of 2 were very similar to those of 1. The major differences between them included the multiplicity of H17, which was changed from a doublet in 1 to a singlet in 2, and the replacement of nitrogen-linked H-18 (δH 2.36) in 1 by a hydroxyl group (δH 5.94, 18-OH), which was further supported by the HMBC correlation from 18-OH to C-17 (Figure S2, SI), and the chemical shifts of C-17 and C-19 similar to those in the reported tryptoquivaline L.13 The relative configurations of 1 and 2 were established on the basis of NOESY and NOE spectral data (Figure 2 and

2

δH (J, Hz)

δC

δH (J, Hz)

106.2 50.6 193.0 144.6

7.90−7.93a 7.90−7.93a 7.66, m 8.23, d (7.9)

4.93, d (6.6) 3.52, dd (15.3, 6.8) 2.63, d (15.3) 4.98, d (9.8) 2.36, d (9.8)

146.7 129.4 135.1 128.9 126.6 122.6 161.1 61.9 42.5 85.3 87.0

7.85−7.91a 7.85−7.91a 7.65, t (7.6) 8.22, d (8.0)

4.98, d (6.8) 3.48, dd (15.3, 6.9) 2.76, d (15.5) 4.74, s

71.2 171.8

7.29, 7.37, 7.24, 7.63,

d (7.6) t (7.6) t (7.6) d (7.6)

1.18, 1.11, 1.06, 1.03, 6.92,

s s s s s

137.8 114.8 130.3 125.4 127.3 137.3 16.4 20.9 17.3 23.4

7.31−7.37a 7.31−7.37a 7.23, t (7.9) 7.71, d (7.6) 1.20, 1.14, 1.01, 1.09, 6.90, 5.94,

s s s s s s

Figure 2. Key COSY, HMBC, NOESY, and NOE correlations of 1.

Figure S3, SI). The NOESY and NOE spectra displayed correlations from 1-OH to H-14 and H-26 and between H-15a (δH 3.52 in 1 and δH 3.48 in 2) and H-14, indicating that 1-OH, H-14, H-15a, and H-26 were on the same side of the furan ring. The correlations between H-15b (δH 2.63 in 1 and δH 2.76 in 2) and H-17 suggested that H-17 was on the other side. The absolute configuration of 1 was determined by comparing experimental and calculated ECD spectra using time-dependent density-functional theory (TDDFT). The DFT reoptimization of the initial MMFF minima of the arbitrarily selected (1R,14R,16S,17R)-1, which was performed at the B3LYP/TZVP level with a PCM solvent model for MeOH, resulted in only one optimized conformer (Figure S4, SI). Subsequently, the absolute configuration of compound 1 was established from close agreement of the TDDFT-calculated ECD spectrum of (1R,14R,16S,17R)-1 with that determined experimentally (Figure 3). Since compounds 1 and 2 exhibited almost identical ECD spectra, the absolute configuration of 2 was also established as 1R,14R,16S,17S (Figure 3). Compounds 3 and 4 had the same molecular formula of C28H31N5O5 based on the HRESIMS ions detected at m/z 518.2393 [M + H]+ and 518.2398 [M + H]+, respectively. Their 1D NMR data (Table S2, SI) indicated the presence of five methyls (including a methoxy), one methylene, 11 methines (including eight aromatic methines), and 11 non-

Signals were overlapped. bObtained from 1H−15N HMBC spectrum.

aromatic proton signals (δH 8.24−7.22), indicating the presence of two ortho-disubstituted aromatic rings. Similar to the fumiquinazolines, the presence of a quinazoline ring system (A and B rings) in 1 was deduced from characteristic 13C NMR resonances (Table 1; C-4 to C-12),1 along with the HMBC correlations from H-10 (δH 8.23) to C-6 (δC 146.8)/C-12 (δC 161.1), from H-9 (δH 7.66) to C-11 (δC 122.8) and the 1H−15N HMBC correlation from H-7 to N-5 (δN 281.7). The quinazoline ring was further fused with a pyridine (C ring) to form a pyrido[2,1-b]quinazoline moiety which was established from HMBC correlations between H-14 (δH 4.93) to C-1 (δC 106.2)/C-4 (δC 144.2), H3-28 (δH 1.18)/ H3-29 (δH 1.11) to C-1/C-2 (δC 50.5)/C-3 (δC 192.8), and from 1-OH (δH 6.92) to C-1/C-2/C-14 (δC 61.7). The HMBC correlations from H-26 (δH 7.63) to C-16 (δC 85.6) and C-22 (δC 138.8), from H-23 (δH 7.29) to C-27 (δC 137.0), from H-17 (δH 4.98) to C-16, and the 1H−15N HMBC correlation from H-17 to N-21 (δN 289.2) suggested the presence of a substituted indole moiety. The imidazo[1,2a]indole moiety (E, F, and G rings) was further constructed on the basis of the HMBC correlations from H3-30 (δH 1.06)/H331 (δH 1.03) to C-19 (δC 65.3)/C-20 (δC 176.0), from 18-NH B

DOI: 10.1021/acs.orglett.5b02964 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

Figure 3. Experimental ECD spectra of compounds 1 and 2 and the calculated spectrum for (1R,14R,16S,17R)-1.

Figure 5. Experimental ECD spectra of compounds 3 and 4.

protonated carbons (including three carbonyls) and suggested that they possessed quinazoline-containing indole alkaloid skeleton, similar to epi-fiscalin C and fiscalin C (5).3,13 Further analysis of the 2D NMR data revealed that compounds 3 and 4 had the same planar structure and the main differences to 5 were the replacement of H-3 in 5 by methoxy group in 3 and 4 (Figure S2, SI). The absolute configuration of 3 was determined unambiguously by the X-ray diffraction (CCDC 1413444) as 3R,14R,16S,17R (Figure 4), with Flack parameter −0.05 (17).

form the pyrido[2,1-b]quinazoline unit. Similar to the reported biogenetic pathway for fumiquinazoline A,1,14 compounds 1 and 2 are also speculated to be biosynthesized from Ltryptophan, ATA, L-valine, and 2-aminoisobutyric acid (Aib) (Scheme 1). Different from the formation of 3 and 4,15 Scheme 1. Hypothetical Biogenetic Pathway of Compounds 1−4

Figure 4. X-ray ORTEP diagram of compound 3.

The 1H and 13C NMR spectra of 3 and 4 were very similar but not identical. The most significant difference between them in the 13C NMR spectra (Table S2, SI) was the signals of C-28 (δC 30.5 in 3 and δC 37.6 in 4), which indicated that they are epimers with different absolute configuration at C-3.7,9,13 The relative configuration of 4 was further deduced as 3S*,14R*,16S*,17R* according to the NOESY correlation between H3-29 (δH 0.91) and H-15b (δH 2.57), H-17 (δH 5.19) and H-15b, and H-14 (δH 5.33) and OH-16 (δH 5.80) (Figure S3, SI). The absolute configuration of compound 4 was finally determined as 3S,14R,16S,17R based on the similar ECD curve between 3 and 4 (Figure 5).7,9 In addition, the similar ECD curves of compounds 3 and 4 to fiscalin C suggested that they shared the same absolute configuration at C-14, C-16, and C17, which indicated that the configuration of C-3 has almost no effect on the ECD spectra of them.3,7,9,13 Compounds 1 and 2 represent a new class of quinazolinecontaining indole alkaloids with a unique 6/6/6/5 quinazoline ring system connected directly to a 6/5/5 imidazoindolone ring system, requiring a different process from the classic ones to

compounds 1 and 2 are generated by further modification of the key intermediate 5 including oxidation, hydrolysis, nucleophilic attack by water, dehydration, deprotonation, and subsequently aldol reaction to form the unprecedented C ring.6 The cytotoxicity of 1−5 was tested using the methylthiazoletrazolium (MTT) method against the HL-60 cancer cell line,16 but no activity was detected (IC50 > 50 μM). The antiviral activity of them was also evaluated against influenza A virus C

DOI: 10.1021/acs.orglett.5b02964 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters (H1N1) using the cytopathic effect (CPE) inhibition assay.17 Compounds 1 and 2 exhibited inhibitory effects with IC50 values of 66 μM and 58 μM, respectively (ribavirin as positive control, IC50 = 94 μM).

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(13) Buttachon, S.; Chandrapatya, A.; Manoch, L.; Silva, A.; Gales, L.; Bruyère, C.; Kiss, R.; Kijjoa, A. Tetrahedron 2012, 68, 3253−3262. (14) Ames, B. D.; Haynes, S. W.; Gao, X.; Evans, B. S.; Kelleher, N. L.; Tang, Y.; Walsh, C. T. Biochemistry 2011, 50, 8756−8769. (15) Snider, B. B.; Zeng, H. B. J. Org. Chem. 2003, 68, 545−563. (16) Du, L.; Zhu, T. J.; Liu, H. B.; Fang, Y. C.; Zhu, W. M.; Gu, Q. Q. J. Nat. Prod. 2008, 71, 1837−1842. (17) Peng, J. X.; Jiao, J. Y.; Li, J.; Wang, W.; Gu, Q. Q.; Zhu, T. J.; Li, D. H. Bioorg. Med. Chem. Lett. 2012, 22, 3188−3190.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.5b02964. Experimental details, 1D and 2D NMR spectra of 1−4, structures of fumiquinazoline analogues, and computational calculation details (PDF) X-ray data for compound 3 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21372208), The Shandong Provincial Natural Science Fund for Distinguished Young Scholars (JQ201422), the Program for New Century Excellent Talents in University (NCET-12-0499), the National High Technology Research and Development Program of China (2013AA092901), and NSFC−Shandong Joint Fund for Marine Science Research Centers (U1406402). This work also supported by Fundamental Research Funds for the Central Universities. We also thank Dr. R. A. Keyzers (Victoria University of Wellington) for help with preparation of the manuscript.

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DOI: 10.1021/acs.orglett.5b02964 Org. Lett. XXXX, XXX, XXX−XXX