Letter pubs.acs.org/OrgLett
Citrifurans A−D, Four Dimeric Aromatic Polyketides with New Carbon Skeletons from the Fungus Aspergillus sp. Guo-Ping Yin, Ya-Rong Wu, Ming-Hua Yang,* Tian-Xiao Li, Xiao-Bing Wang, Miao-Miao Zhou, Jian-Li Lei, and Ling-Yi Kong* Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China S Supporting Information *
ABSTRACT: Citrifurans A−D (1−4), metabolized by an Aspergillus sp., are unusual dimers of azaphilone and furanone derivatives. Michael addition was thought to be the pivotal procedure in their biosynthesis, and different addition sites generated two new different carbon skeletons. Their structures were elucidated on the basis of spectroscopic methods, single-crystal X-ray diffraction, chemical conversion, and electronic circular dichroism analyses. Compounds 1−3 showed moderate inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages with IC50 values of 18.3, 22.6, and 25.3 μM, respectively.
A
romatic polyketides are a family of structurally diverse natural products produced by fungi, bacteria, and other natural sources.1−3 Assembled by type II polyketide synthases (PKSs), different aromatic carbon skeletons could be constructed through successive rounds of Clasien condensation reactions of short-chain fatty acid units. The varieties in polyketide biosynthetic reactions after that, like reduction, alkylation, and cyclization, further contribute to the tremendous structural diversities.4,5 As an important supplement, polyketide dimerization generates many complex molecular skeletons, like recently reported fistulains A and B,6 curindolizine,7 and naquihexcins A and B.8 The multiple dimerizations not only enrich the structural diversities but also provide compounds with various significant biological activities, such as antimicrobial, cytotoxic, anti-inflammatory, and lipoxygenase inhibitory activities.9−12 In our ongoing endeavor to search for structurally unique and biologically interesting metabolites from fungal resources,13 a chemical investigation was carried out on an Aspergillus sp. It was isolated from centipede intestine by the plate-coating method and cultivated through solid fermentation (Supporting Information). Citrifurans A−D (1−4) (Figure 1), four novel dimers of
aromatic polyketides, were isolated which were the first heterodimers of azaphilone and furanone derivatives. Because of the key Michael addition, new C−C bonds were generated between two polyketide moieties, and the different Michael donors of the furanone derivatives led to two dimeric forms. Moreover, citrifurans A−C (1−3) exhibited moderate inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages. Herein, the details of their isolation, structural elucidation, and bioactivities as well as plausible biogenetic pathways are reported. Citrifuran A (1) was obtained as colorless needle crystals (MeOH−H2O). Its molecular formula, C27H32O7, was established by HRESIMS (m/z 491.2039 [M + Na]+, calcd for 491.2046) and indicated 12 degrees of unsaturation. The 1H NMR data (Table 1) gave one triplet methyl at δH 0.96 (t, J = 7.4 Hz), three secondary methyls at δH 1.12 (d, J = 6.8 Hz), 1.19 (d, J = 6.8 Hz), and 1.28 (d, J = 6.2 Hz), two tertiary methyls at δH 1.46 (s) and 1.98 (s), and a conjugated diene moiety with four olefinic protons at δH 5.62 (d, J = 15.4 Hz), 6.30 (dd, J = 15.4, 10.4 Hz), 6.08 (dd, J = 15.4, 10.4 Hz), and 5.91 (dt, J = 15.4, 6.5 Hz). On the basis of the HSQC spectrum, the 27 carbons were assigned as six methyls, one methylene, 10 methines, and 10 quaternary carbons. More detailed structural information was derived by 2D NMR spectral analyses. The key HMBC correlations from H3-11 to C4a/C-5/C-6, from H-7 to C-5/C-6/C-8/C-8a, from H-4 to C4a/C-8a/C-5, and from OH-6 to C-5/C-6/C-7 suggested the presence of an o-cresol moiety. The 1H−1H COSY correlations of H3-9/H-3/H-4/H3-10 and the key HMBC correlations from H-1 to C-3/C-4a/C-8a/C-8 and from H-4 to C-4a/C-8a
Figure 1. Structures of compounds 1−4.
Received: June 15, 2017 Published: July 20, 2017
© 2017 American Chemical Society
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DOI: 10.1021/acs.orglett.7b01823 Org. Lett. 2017, 19, 4058−4061
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Organic Letters Table 1. NMR Spectroscopic Data of Compounds 1−4 1a δH
no.
a1
4.86, d (10.7) 3.53, m 2.70, m
2a δC
1 3 4 4a 5 6 7 8 8a 9 10 11 12 2′ 3′ 4′ 5′ 6′ 7′
1.19, d (6.8) 1.12, d (6.8) 1.98, s
71.6 78.5 37.2 138.3 114.6 156.1 98.9 149.8 111.0 21.2 19.2 11.1
3.95, brs
90.6 197.6 109.8 194.0 163.2 46.1
8′ 9′
4.46, dq (12.6, 6.2) 1.28, d (6.2)
71.9 18.5
10′ 11′ 12′ 13′ 14′ 15′ 16′ 5-OH 6-OH
1.46, s 5.62, d (15.4) 6.30, dd (15.4, 10.4) 6.08, dd (15.4, 10.4) 5.91, dt (15.4, 6.5) 2.08, m 0.96, t (7.4)
21.6 125.5 132.0 127.8 139.2 25.1 13.2
6.18, s
3a
δH
δC
4.69, brs 3.55, m 2.48, m
δH
4b δC
δH
δC
4.56, dd (12.7, 5.9) 3.67, m 2.83, m
1.16, d (6.1) 1.23, d (6.8) 1.41, s
70.8 77.3 38.2 157.0 76.9 203.7 99.0 165.0 121.5 20.0 16.0 29.8
68.6 78.9 38.1 146.0 118.8 161.9 97.8 148.5 111.8 21.7 19.6 11.2 170.9 94.4 202.8 107.3 193.8 163.6 36.4
1.15, d (6.4) 1.20, d (6.8) 1.28, s
68.9 76.5 37.0 155.2 77.2 201.7 99.4 165.0 120.9 20.6 18.2 29.1
4.40, brs
89.5 200.1 110.0 189.8 163.5 46.3
4.50, brs
89.3 200.8 109.1 189.2 163.0 45.4
74.5 18.5
4.59, brs 1.28, brs
74.3 18.4
5.36, s
4.69, brs 1.29, d (6.2) 1.42, s 5.57, d (15.5) 6.26, dd (15.5, 10.4) 6.06, dd (15.5, 10.4) 5.87, dt (15.5, 6.5) 2.07, m 0.96, t (7.4) 5.42, brs
21.7 126.1 131.4 127.8 138.8 25.1 13.2
4.71, brs 3.54, m 2.53, m
5.30, s
1.41, s 5.56, d (15.5) 6.25, m 6.06, m 5.87, brs 2.07, m 0.95, t (7.5) 5.38, brs
21.7 126.3 131.6 127.9 138.8 25.1 13.2
1.34, d (6.1) 1.19, d (7.0) 2.12, s
3.50, dd (14.3, 8.5) 3.78, brd (14.3) 4.87, m 1.87, q (12.7) 2.50, dd (12.7, 5.9) 1.65, s 5.51, d (15.5) 6.25, dd (15.5,10.3) 5.93,dd (15.5, 10.3) 5.71, dt (15.5, 6.6) 2.07, m 0.96, t (7.5)
74.9 33.7 22.2 123.0 134.0 127.2 141.4 25.8 13.2
9.35, s
H (600 MHz) and 13C (150 MHz) NMR of 1−3 in DMSO-d6;
b1
H (500 MHz) and 13C (125 MHz) NMR of 4 in CDCl3.
demonstrated the presence of an azaphilone moiety with two methyls at C-3 and C-4. The 1H−1H COSY correlations of H39′/H-8′/H-7′/H-1, along with the chemical shift of C-8′ (δC 71.9), indicated that a 2-hydroxypropyl group was fused to the azaphilone at C-1, giving unit A as shown in Figure 2. The E,Ehexa-1,3-diene given by 1H−1H COSY correlations showed HMBC correlations from H-11′ to C-2′/C-3′/C-10′, forming the unit B. In addition, there were three residual quaternary carbons (δC 109.8, 163.2, 194.0) that were deduced as an α,βunsaturated acid fragment by their characteristic chemical shifts; however, no HMBC correlations were found to verify the assumption, even using different deuterated solvents. Units A and B and the residual could not be assembled on the basis of data obtained so far. To solve this problem, compound 1 was methoxylated at C-3′, and 1A was afforded (Figure 3A; Supporting Information, Table S3). The key HMBC correlations from H-3′ to C-2′/C-4′/C-5′/ C-10′/C-6′ determined the furancarboxylic acid group. Meanwhile, the C-5′ of furan ring had HMBC correlations with both H-7′ and H-1, which ambiguously confirmed the derivative 7,8dihydonivefuranone15 moiety in 1. However, there was still one degree of unsaturation left, and the consequent deduction was the oxo bridging between C-8, C-8′, and C-6′. Since no useful NMR data was found, methylation product (1B; Supporting Information, Table S3) was hoped to define the uncyclized
Figure 2. Key 1H−1H COSY and HMBC correlations of 1-3, 1A, and 4A.
hydroxyl. The key HMBC correlations from methoxyl at δH 3.80 to C-6′ in 1B, combined with the HRESIMS data, confirmed the 4059
DOI: 10.1021/acs.orglett.7b01823 Org. Lett. 2017, 19, 4058−4061
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Organic Letters
revealed their closely related structures. However, alcoholic hydroxyls instead of phenolic hydroxyl (OH-6) in 1 were present in 2 and 3 at δH 5.42 and 5.38, respectively. HMBC analysis gave correlations from OH-5 to C-4a/C-5/C-6/C-11 and from H-7/ OH-5/H3-11 to the carbonyl at C-5; therefore, C-6 and C-5 were oxidized to carbonyl and tertiary alcohol, respectively. Further analysis of 2D NMR (Figure 2) confirmed the same planar structure of 2 and 3. However, H3-11 showed an NOE effect with H-1/H3-10 in 2 but with H-4 in 3, which assigned its different configurations in 2 and 3. Other NOE correlations of 2 and 3 (H1 with H-3/H-8′/H3-10 and H-7′ with H3-9′) were very similar to those of 1, indicating the same configurations. For further verification, we performed a one-step synthetic reaction using Dess−Martin reagent to afford the 2 and 3 epimers from 1 (Figure 3B). Accordingly, the absolute configurations of 2 and 3 were determined to be 1S,3R,4S,5S,2′R,7′R,8′R and 1S,3R,4S,5R,2′R,7′R,8′R, respectively. Citrifuran D (4) was obtained as a red oil. Its molecular formula was established as C28H32O9 by HRESIMS (m/z 535.1939 [M + Na]+, calcd for 535.1944), indicating 13 degrees of unsaturation. Comparison of its NMR data with those of 1 revealed their very similar structures. The main differences were the existence of two methylenes (C-7′/C-9′) in 4 instead of the methine and secondary methyl in 1. Meanwhile, the disappearing H-7 and the additional carboxyl carbon at δC 170.9 in 4 gave rise to the speculation of carboxyl substitution at C-7. However, some carbon singlets were broad in the 13C NMR spectrum, which caused certain key signal loss in 2D NMR. A methyl derivative 4A (Supporting Information, Table S4) was therefore obtained to solve this uncertainty. Analysis of the HRESIMS data showed that two carboxyl groups were esterified in 4A, which was supported by the HMBC correlations from H3-6″ to C-6′ and from H3-12″ to C-12. Fully analyzing 2D NMR data of 4A revealed two similar aromatic polyketide moieties like 1 but with different dimeric positions. HMBC correlations from H2-7′ to C4′/C-5′/C-9′ and from H2-9′ to C-8a/C-1 confirmed the C-1/C9′ bond, which was also supported by the 1H−1H COSY correlations of H-1/H2-9′/H-8′/H2-7′. The remained one degree of unsaturation demanded the oxo-bridge between C-8 and C-8′. Thus, the planar structure of 4 was elucidated as displayed (Figure 1). The relative configuration of 4 was determined from the ROESY spectrum of 4A. The correlations of H-1/H-3, H-1/H8′, and H-1/H3-10 suggested that H-1, H-3, H3-10, and H-8′ were β-oriented, whereas H-4 and H3-9 were α-oriented. Because of the unsettled relative stereochemistry of C-2′, there could be four possible stereoisomers of 4. Yet only the calculated ECD curves of 1S,3R,4S,2′R,8′S well matched with the experimental one (Figure 6). Thus, the absolute configuration of 4 was established. Along with the above four novel dimers of aromatic polyketides, two biosynthetically related monomers (citrinin16 and 7,8-dihydonivefuranone15) were also isolated in this work. Therefore, compounds 1−4 are postulated to be the heterodimers of azaphilone and furanone derivatives. Herein, the plausible biosynthetic pathways for 1−4 are postulated in Scheme 1. To begin, the important presumed precursors 5 (citrinin) and 8 are biosynthesized through a polyketide synthase pathway, originating from acetate and S-adenosyl methionine.17 Then decarbonylation of 5 and oxidation18 of 8 afford intermediates 12 and 9, respectively. Afterward, deprotonation of 9 affords two kinds of enolate anions 10 and 11, which subsequently heterodimerize with 12 and 5 through a Michael
Figure 3. (A) Derivatives of 1A and 1B from 1. (B) Chemical conversion of 2 and 3 from 1.
unsubstituted carboxyl in 1, evidencing the 8,8′-ether bond. Thus, the planar structure of 1 was established (Figure 2). ROESY cross-peaks (Figure 4) were observed between H-1/H-3,
Figure 4. Key ROESY correlations of 1 and 4.
H-1/H-8′, H-7′/H3-9′, and H-1/H3-10, but no correlations of H7′/H-1 and H-7′/H-8′ were found. H-1, H-3, H-8′, and H3-10 were therefore assigned as the same orientation, whereas H-4, H7′, H3-9′, and H3-9 hold the other orientation. Finally, a singlecrystal X-ray diffraction experiment with Cu Kα radiation of 1 was performed,14 which was consistent with the above deduction and unambiguously confirmed the absolute configuration as 1S,3R,4S,2′R,7′R,8′R (Figure 5), with Flack parameter 0.04(4). Citrifurans B (2) and C (3) were two isomers with the same molecular formula, C27H32O8, by HRESIMS (m/z 507.1987 and 507.1988 [M + Na]+, respectively, calcd for 507.1995). Comparison of their NMR spectra (Table 1) with those of 1
Figure 5. X-ray crystallographic analysis of 1. 4060
DOI: 10.1021/acs.orglett.7b01823 Org. Lett. 2017, 19, 4058−4061
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Letter
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Ling-Yi Kong: 0000-0001-9712-2618 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research work was funded by the National Natural Science Foundation of China (81503218), the Program for Changjiang Scholars and Innovative Research Team in University (IRT15R63), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Fundamental Research Funds for the Central Universities (2016ZZD010).
Figure 6. Calculated and experimental ECD spectra of 4.
Scheme 1. Plausible Biosynthetic Pathways of 1−4
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addition reaction19 to form adduct 13 and 6, respectively. A subsequent intramolecular nucleophilic addition and reduction involving 13 construct a dihydropyran rings in 1, which further performed oxidize to form the epimers 2 and 3. Similarly, the intramolecular dehydration involving 6 could produce 4. In addition, compounds 1−4 were evaluated for nitric oxide production inhibitory effects on lipopolysaccharide-activated RAW 264.7 macrophage cells. Compounds 1−3 showed moderate inhibition, with IC50 values of 18.3, 22.6, and 25.3 μM, respectively, but the others were inactive at 50 μM. The cytotoxicity against RAW 264.7 cells was also evaluated for 1−4, yet no significant cytotoxicity was found (50 μM).
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REFERENCES
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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.orglett.7b01823. Crystallographic data for 1 (CIF) Details of isolation, synthesis, and biological experimental procedures; HRESIMS, UV, ECD, and NMR data for new compounds (PDF) 4061
DOI: 10.1021/acs.orglett.7b01823 Org. Lett. 2017, 19, 4058−4061
