Absolute Stereostructures and Biogenetic Relationships of

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Absolute Stereostructures and Biogenetic Relationships of Phomopsols A and B, Including the First Axially Chiral PolyketideDerived Alkaloid, from the Mangrove Endophytic Fungus Phomopsis sp. xy21 Wan-Shan Li,† Han-Bo Hu,‡ Zhong-Hui Huang,† Ren-Jie Yan,† Li-Wen Tian,*,† and Jun Wu*,† †

School of Pharmaceutical Sciences, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou 510515, P. R. China Marine Drugs Research Center, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, P. R. China

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‡

S Supporting Information *

ABSTRACT: A polyketide-derived alkaloid featuring a rotationally hindered C-9−C-9a axial bond and a unique 3,4dihydro-2H-indeno[1,2-b]pyridine-1-oxide motif, named phomopsol A (1), and a highly oxidized polyketide containing a new 3,5-dihydro-2H-2,5-methanobenzo[e][1,4]dioxepine moiety, named phomopsol B (2), were isolated from the Thai mangrove endophytic fungus Phomopsis sp. xy21, together with the known compound 3. The absolute stereostructures of 1−3 were unambiguously established by single-crystal X-ray diffraction analysis (Cu Kα). Biosynthetic origins of 1−3 are proposed.

P

olyketide-derived alkaloids originate from various polyketides and diverse amination patterns. To date, 11 classes of these alkaloids have been reported.1−3 However, only rogersonins A and B are polyketide-derived alkaloid-N-oxides, which can be biosynthesized from a mixed polyketide−amino acid pathway.4 Alkaloid-N-oxides are well-known for their diverse frameworks and a wide range of bioactivities. To date, 33 classes of alkaloid-N-oxides, such as β-carboline-,5−8 crinane-,9−11 indole-,12,13 isoquinoline-,14−16 and pyrazine-Noxides,17 have been identified, mainly from plants, sponges, fungi, and bacteria. From the perspective of biosynthetic origins, all of these alkaloid-N-oxides, except for rogersonins A and B,4 can be traced back to mixed amino acid−mevalonate pathways. Mangrove endophytic fungi of the genus Phomopsis are a prolific source for the production of structurally unique and biologically diverse metabolites.18,19 In order to search for bioactive natural products with novel skeletons, a new polyketide-derived alkaloid named phomopsol A (1) and a highly oxidized polyketide named phomopsol B (2) were isolated from the mangrove endophytic fungus Phomopsis sp. xy21, associated with leaves of the Thai Xylocarpus granatum, together with compound 3 as a pair of C-9 epimers of the known polyketide 3-(2,6-dihydroxyphenyl)-4-hydroxy-6-methylisobenzofuran-1(3H)-one20 (Figure 1). Phomopsol A (1) contains a rotationally hindered C-9−C-9a axial bond and a unique 3,4-dihydro-2H-indeno[1,2-b]pyridine-1-oxide motif; whereas phomopsol B (2) consists of a novel 6/6/5-fused tricyclic 3,5-dihydro-2H-2,5-methanobenzo[e][1,4]dioxepine © XXXX American Chemical Society

Figure 1. Structures of phomopsols A (1) and B (2) and compound 3. The * denotes an axis with a stable configuration.

moiety. Herein we report the isolation and absolute stereostructure identification of 1−3. Neuroprotective effects of 1−3 against corticosterone-induced injury in PC12 cells were evaluated. Phomopsol A [1, [α]25 D = +41.3 (c 0.15, MeOH)] was obtained as light-red crystals. The molecular formula C19H17NO4 with 12 degrees of unsaturation was established by the HR-ESI-MS negative ion at m/z 322.1088 ([M − H]−, calcd 322.1085). According to the 1H and 13C NMR spectroscopic data for 1 (Table S1), eight degrees of unsaturation are due to seven CC bonds and a CN bond. Therefore, the molecule has to be tetracyclic. Two substructures, viz., resorcinol (ring A) and 3,4-dihydro2H-indeno[1,2-b]pyridine (rings B−D), were established by analysis of 1H−1H COSY, HSQC, and HMBC correlations of Received: May 1, 2019

A

DOI: 10.1021/acs.orglett.9b01536 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters 1 (Figure 2). The presence of the resorcinol moiety (from C-1 to C-4, 4a, and 9a) was corroborated by three successive ortho-

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

position aromatic protons [δH 6.40 (2H, d, J = 7.8 Hz, H-2 and H-4) and 7.00 (1H, t, J = 7.8 Hz, H-3) as well as 1H−1H COSY cross-peaks between H-2/H-3 and H-3/H-4] and HMBC correlations between H-4/C-4a, H-4/C-9a, H-2/C-1, H-2/C-9a, H-3/C-1, and H-3/C-4a (Figure 2). The 3,4-dihydro-2H-indeno[1,2-b]pyridine moiety was elucidated by starting from a 1,2,3,5-tetrasubstituted benzene ring (ring B, from C-5 to C-8, C-8a, and C-10a) and was corroborated by two meta-oriented aromatic protons [δH 7.68 (1H, br s, H-5) and 6.57 (1H, br s, H-7)] and HMBC correlations between H-5/C-8a, H-5/C-7, H-7/C-8, and H-7/ C-8a. HMBC correlations from protons of a methyl group (δH 2.28, s, H3-11) to C-5, C-6, and C-7 placed it at C-6. The existence of the 8-OH group was evidenced by the downshifted C-8 (δC 152.2, qC). The presence of a 2,3,4,5tetrahydropyridine ring (ring D, from C-1′ to C-4′, C-10, and N) was confirmed by the proton spin system H2-1′−H2-2′− H 2 -3′, which was deduced from the 1 H− 1 H COSY correlations, along with HMBC correlations between H2-1′/ C-10, H2-1′/C-3′, H2-3′/C-10, and H2-3′/C-4′. The connection of rings B and D through the C-10−C-10a bond was corroborated by the key HMBC correlation from H5 to C-10; whereas the existence of the C-4′C-9 bond was confirmed by HMBC correlations from H2-3′ to C-4′ and C-9. Two quaternary carbons, viz., C-8a (δC 126.5, qC) and C-9 (δC 133.7, qC), and the remaining one degree of unsaturation of 1 inferred the presence of the five-membered ring C (C-8a, C-9, C-4′, C-10, and C-10a), which is fused with rings B and D via the C-8aC-10a and C-10−C-4′ bonds, respectively, to form the 3,4-dihydro-2H-indeno[1,2-b]pyridine moiety (rings B− D) (Figure 2). In addition, the existence of the N-oxide group was corroborated by the downshifted C-1′ (δC 61.8, CH2) and the upshifted C-10 (δC 149.0, qC).21 Finally, on the basis of the remaining quaternary carbons C-9 and C-9a, it was concluded that the above two substructures had to be connected through the C-9−C-9a axial bond, despite the lack of direct NMR spectroscopic evidence. However, the chirality of the C-9−C-9a axial bond remained to be determined. In order to establish the reliable constitution and absolute configuration of 1, particularly the chirality of the C-9−C-9a bond, single-crystal X-ray diffraction (XRD) analysis was carried out. After considerable efforts, suitable crystals of 1 were obtained from a 3:1 methanol/acetone solvent mixture. Finally, the above-mentioned constitution of 1 was solidified, and the absolute configuration of 1 as an axially chiral polyketide-derived alkaloid-N-oxide containing an M-configured C-9−C-9a axial bond was unambiguously identified by single-crystal XRD analysis conducted with Cu Kα radiation (Figure 3, CCDC 1903463).

Figure 3. ORTEP of the X-ray crystal structure of 1. Ellipsoids are given at the 30% probability level.

Phomopsol B (2) was isolated as colorless crystals. Its molecular formula was determined to be C15H16O6 with eight degrees of unsaturation by the HR-ESI-MS negative ion at m/z 291.0875 ([M − H]−, calcd 291.0874). According to 1H and 13 C NMR spectroscopic data for 2 (Table S2), four degrees of unsaturation are due to a lactone function and three CC bonds. Thus, the molecule has to be tetracyclic. HSQC and DEPT 135 experiments revealed the presence of a methyl group, two methylene groups, seven methine groups (three olefinic and three oxygenated), and five quaternary carbons (one carbonyl, one oxygenated, and three olefinic). Three substructures, viz., 2a (from C-1 to C-4, C-4a, and C9a), 2b (C-9, C-10, C-10a, and C-8a), and 2c (from C-5 to C8 and C-11), were deduced and connected by 1H−1H COSY and HMBC correlations of 2 (Figure 4). Substructure 2a was elucidated as a 1,2,3-trisubstituted benzene ring by three successive ortho-position aromatic protons [δH 6.34 (br d, J = 8.0 Hz, H-2), 6.96 (t, J = 8.0 Hz, H-3), and 6.26 (d, J = 8.0 Hz, H-4) as well as 1H−1H COSY cross-peaks between H-2/H-3 and H-3/H-4] and HMBC correlations between H-2/C-9a, H4/C-9a, H-2/C-1, and H-4/C-4a (Figure 4). The existence of

Figure 4. Partial structures, 1H−1H COSY and HMBC correlations, and the NOE interaction that are indicative of the gross structure of phomopsol B (2). Blue arrows indicate correlations within the fragments and green ones between them. B


Letter

Organic Letters the 1-OH group was evidenced by the downshifted C-1 (δC 155.7, qC). Substructure 2b could be assigned as a tetrasubstituted tetrahydrofuran ring by the 1H−1H COSY cross-peak between H-8a/H-9 and HMBC correlations between H-9/C-10, H-8a/C-10a, H-10/C-9, H-10/C-8a, and H-10/C-10a (Figure 4). The presence of the 8a-OH group was indicated by the chemical shift of C-8a (δC 73.7, CH). The connection of substructures 2a and 2b through the C-4a−O− C-10a and C-9a−C-9 bonds was corroborated by the chemical shifts of C-4a (δC 154.8, qC) and C-10a (δC 89.2, qC) and HMBC correlations from H-9 to C-1, C-4a, and C-9a, respectively. Substructure 2c was identified as a β-methyl-γlactone ring on the basis of the proton spin system H-5−H6(H3-11)−H2-7, which was deduced from the 1H−1H COSY correlations, along with HMBC correlations between H3-11/ C-5, H3-11/C-7, H-5/C-8, H-6/C-8, and H2-7/C-8. The connection of substructures 2b and 2c via the C-5−C-10a bond was confirmed by the HMBC correlation between H-5 and C-10a. Taken together, these data elucidated the constitution of 2 (Figure 4). In addition, the NOE interaction between H-5 and H3-11 revealed their cofacial relationship (Figure 4). In order to establish the absolute configuration of 2, singlecrystal XRD analysis was conducted with Cu Kα radiation. Finally, the absolute configuration of 2 was unequivocally determined as 5S,6S,8aR,9R,10aR (Flack parameter = 0.00(4); Figure 5, CCDC 1903464).


CoA (MCoA) (Scheme 1). The keto−enol tautomerization of the carbonyl group at C-3′ of A and then the aldol Scheme 1. Proposed Biosynthetic Origin of 1


Compound 3 was isolated as colorless crystals. Its molecular formula was determined to be C15H12O5 by the HR-ESI-MS negative ion at m/z 271.0610 ([M − H]−, calcd 271.0612). The 1H and 13C NMR spectroscopic data for 3 (Table S3) were the same as those of 3-(2,6-dihydroxyphenyl)-4-hydroxy6-methylisobenzofuran-1(3H)-one, previously obtained from the fungus Aspergillus nidulans.20 However, the specific optical rotation of 3 was found to be zero, implying the presence of a pair of epimers. This deduction was further confirmed by the P1̅ space group of 3 observed in single-crystal XRD analysis conducted with Cu Kα radiation (Figure 6, CCDC 1903465). Therefore, the known polyketide 3 was concluded for the first time to be a mixture of a pair of C-9 epimers. The biosynthetic origin of 1 could be traced back to two building blocks, viz., A and B, among which A could originate from one unit of acetyl-CoA (ACoA) and one unit of malonyl-

condensation of A at C-10 of B would generate intermediate I (Int I). Similarly, the keto−enol tautomerization of the carbonyl group at C-3′ of Int I could produce Int II, of which the intramolecular aldol condensation of the A unit at C-9 of the B moiety would afford the crucial Int III. Subsequent oxidation of Int III at C-10 and release of carrier protein could yield Int IV, decarboxylation of which could afford Int V. Dehydration of Int V would generate Int VI, and subsequent reductive release of carrier protein and then amination could produce Int VII. Finally, cyclization between the amino group and the keto carbonyl group at C-10 followed by dehydration, reduction at C-3′, and oxidation at the nitrogen atom could C


Letter

Organic Letters

Province, Thailand) for providing the mangrove leaves used in this work.

give 1 (Scheme 1). The above building block B was also proposed as the crucial precursor for the biosynthesis of 2 and 3 (Scheme S1). The neuroprotective effects of 1−3 against corticosteroneinduced injury were evaluated by the MTT assay in PC12 cells.22 Compounds 1 and 3 exhibited neuroprotective activity in a concentration-dependent manner in the range of 5.0−40.0 μM. The cell viabilities for 1 and 3 at 40.0 μM are 76% and 96%, respectively, whereas that for corticosterone at 200.0 μM is 60% (see the Supporting Information). In conclusion, phomopsol A (1), representing the first axially chiral polyketide-derived alkaloid with an unusual Noxide group, was obtained from the Thai mangrove endophytic fungus Phomopsis sp. xy21, together with an intriguing polyketide, phomopsol B (2), containing a novel 3,5dihydro-2H-2,5-methanobenzo[e][1,4]dioxepine motif. The absolute configurations of 1 and 2 were unequivocally determined by single-crystal XRD analysis. Biogenetic relationships of 1−3 have been proposed. This work demonstrates that mangrove endophytic fungi of the genus Phomopsis harbor polyketides with new frameworks.

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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.9b01536. (1 H and 13C NMR spectroscopic data of 1−3, experimental section, and proposed biosynthetic origins of 2 and 3 PDF) Copies of UV and ECD spectra of 1 and 2 and copies of HR-ESI-MS and 1D and 2D NMR spectra of 1−3 (PDF) Accession Codes

CCDC 1903463−1903465 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by e-mailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.-W.T.) *E-mail: [email protected] (J.W.) ORCID

Jun Wu: 0000-0003-0807-5229 Author Contributions

The manuscript was written through contributions of all authors. All of the authors approved the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from the National Natural Science Foundation of China (U1501221 and 81661148049). We thank Dr. Patchara Pedpradab (Rajamangala University of Technology Srivijaya, Trang D


Absolute Stereostructures and Biogenetic Relationships of

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