Isocoumarindole A, a Chlorinated Isocoumarin and Indole Alkaloid

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

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Isocoumarindole A, a Chlorinated Isocoumarin and Indole Alkaloid Hybrid Metabolite from an Endolichenic Fungus Aspergillus sp. Minghua Chen,†,‡,⊥ Renzhong Wang,†,§,⊥ Wuli Zhao,† Liyan Yu,† Conghui Zhang,† Shanshan Chang,† Yan Li,† Tao Zhang,† Jianguo Xing,‡ Maoluo Gan,*,† Feng Feng,*,§ and Shuyi Si*,†

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†

NHC Key Laboratory for Microbial Drug Bioengeering, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Key Laboratory for Uighur Medicine, Institute of Materia Medica of Xinjiang Uygur Autonomous Region, Urumqi 830004, People’s Republic of China § Department of Natural Medicinal Chemistry, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: Isocoumarindole A (1), a novel polyketide synthetase−nonribosomal peptide synthetase (PKS−NRPS) hybrid metabolite, was isolated from the endolichenic fungus Aspergillus sp. CPCC 400810. The structure of isocoumarindole A (1) was featured by an unprecedented skeleton containing chlorinated isocoumarin and indole diketopiperazine alkaloid moieties linked by a carbon−carbon bond, which was determined by a combination of spectroscopic analyses, Marfey’s method, and calculations of NMR chemical shifts, ECD spectra, and optical rotation values. Isocoumarindole A showed significant cytotoxicity and mild antifungal activities.

T

400810, which was isolated from the lichen Cetrelia sp. collected from Laojun Mount in Yunnan Province of China, exhibited antifungal activity against Candida albicans. In our preliminary research, 8-methyl-11-chlorodiaporthin (2),13 8methyl-11,11-dichlorodiaporthin (3),13 and cyclo (D-N-methyl Leu-L-Trp) (4)14 were isolated from the ethyl acetate extracts of the solid cultures of strain CPCC 400810 on rice media (Figure 1). To expand the chemical diversity of metabolites, this strain was further cultured in different media using the OSMAC (one strain, many compounds) strategy.15 LC-MS

he endolichenic fungi residing in the thalli of lichens are similar to the endophytes that inhabit the healthy tissues in plants.1 Since the first report by Paranagama et al. in 2007,2 more than 300 secondary metabolites, of which 160 were novel compounds, including polyketides,3 alkaloids,3a,b,4 steroids,5 terpenoids,6 and cyclic peptides,7 have been identified from the endolichenic fungi. These compounds exhibit a range of biological activities, such as cytotoxic, antifungal, antiviral, antibacterial, and anti-Aβ42 aggregation effects.3a,b,i Endolichenic fungi are considered as a treasure trove of unique structured compounds and bioactive metabolites. Isocoumarins are a class of lactonic natural products with significant pharmacological activities. They are abundant in fungi, bacteria, lichens, liverworts, and higher plants.8 Nearly 400 isocoumarins and dihydroisocoumarins, including a few examples of dimeric isocoumarins, have been identified up to now. Indole diketopiperazine alkaloids are a family of important microbial metabolites formed by the condensation of tryptophan with other different amino acids. Most of these alkaloids are characterized by diverse structural scaffolds and ring systems derived from dimerization by two indole units9 and conjugation with another building block, such as a prenyl group10 and other isoprenoids.11 As a part of the ongoing search for novel or/and bioactive natural products from microorganisms,12 chemical investigation of endolichenic fungi was initiated in our laboratory. The extracts of the endolichenic fungus Aspergillus sp. CPCC © XXXX American Chemical Society

Figure 1. Structures of compounds 1−4. Received: January 30, 2019

A

DOI: 10.1021/acs.orglett.9b00385 Org. Lett. XXXX, XXX, XXX−XXX

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Information). By comparison with those of 2, the proton resonances at δH 6.65 (d), 6.68 (d), and 6.72 (s) could be easily assigned to H-8′, H-6′, and H-4′, respectively, of the 6′,8′-disubstituted isocoumarin chromophore. This moiety was corroborated by HMBC correlations of H-4′ with C-3′, C-5′, C-6′, and C-10′, of H-8′ with C-6′, C-7′, C-9′, and C-10′, of OCH3-9′ (δH 3.91) with C-9′, and of OCH3-7′ (δH 3.93) with C-7′. HMBC correlations of the methine proton H-11′ with C3′, C-4′, C-12′, and C-13′ demonstrated the presence of the 13′-chloro-12′-oxopropyl side chain located at C-3′ of the isocoumarin moiety, leading to the establishment of partial structure unit A. The 1H−1H COSY correlations of H-9/H10/H-11/H-12, H2-15/H-16, H-3/H2-17/H-18/H3-19, and H3-20 revealed three independent spin systems in 1. The HMBC correlations of the α-amino proton H-3 (δH 3.79) with C-4 and C-17 and of the methyl protons at δ 2.82 with C-3 led to the identification of the N-methyl leucine residue. In addition, a dihydrotryptophan moiety was established by HMBC correlations of H-6 with C-8, C-14, and C-15; of H-15 with C-6, C-13, and C-14; and of H-16 with C-1 and C-15. HMBC correlations of H-3 with C-1 and H-16 with C-4 revealed that the leucine and dihydrotryptophan moieties formed a diketopiperazine ring. Furthermore, the HMBC correlation of H-6 with C-16, along with the consideration of the required degrees of unsaturation and the characteristic chemical shift of C-6 (δC 80.6),17 indicated the formation of a pyrrole ring between C-6 and C-16, resulting in the construction of the fragment B. Finally, HMBC correlations of H-11′ with C-6, C-14, and C-15 allowed the connectivity of fragments A and B via the C-14−C-11′ bond (Figure 2). As a result, the planar structure of 1 was established as illustrated in Figure 2. The relative configuration of 1 was determined by analysis of NOESY data (Figure 3). The obvious NOESY correlations of

analysis revealed that one of the metabolites from the Potato Dextrose Broth (PDB) liquid cultures displayed a similar UV spectrum as those of chlorodiaporthin analogues (2, 3). However, its molecular weight suggested by the ESIMS ion at m/z 608 [M + H]+ was much higher than those of 2 and 3 (Figure S1 in the Supporting Information). This inspired us for further chemical investigation. LC-MS-guided isolation ultimately resulted in the identification of a novel polyketide− nonribosomal peptide hybrid metabolite, named isocoumarindole A (1). Isocoumarindole A represents the first example of an indole diketopiperazine alkaloid linking an isocoumarin unit via a carbon−carbon bond. Herein, the isolation, structure elucidation, bioactivities, and possible biogenetic pathway of isocoumarindole A (1) are discussed. Isocoumarindole A (1) was obtained as an optically active light-yellow gum ([α]20D + 105.5, MeOH). The postitive ESIMS spectrum displayed a pseudomolecular ion peak cluster at m/z 608/610 [M + H]+ with a ratio of 3:1, suggesting one chlorine atom in the molecule. The molecular formula of 1 was determined to be C32H34O7N3Cl by HRESIMS at m/z 608.2185 [M + H]+ (calcd for C32H35O7N3Cl, 608.2158), corresponding to 17 degrees of unsaturation. The IR spectrum of 1 indicated the presence of amino (3358 cm−1), carbonyl (1717 and 1668 cm−1), and aromatic ring (1600 and 1560 cm−1) functionalities. The UV spectrum (λmax 245, 285, 295, and 325 nm) was very similar to those of 2 and 3, indicating the presence of the diaporthin chromophore in 1.16 The 1H NMR spectrum (Table S1 in the Supporting Information) of 1 in the lower field displayed signals attributable to an orthodisubstituted benzene ring at δH 7.18 (1H, d), 6.60 (1H, dd), 7.00 (1H, dd), and 6.62 (1H, d), a 1,3,4,5-tetrasubstituted benzene ring at δH 6.65 (1H, d) and 6.68 (1H, d), and an isolated olefinic proton at δH 6.72 (1H, s). Three methyl (δH 0.88, 0.85, and 2.82) and two aromatic methoxy (δH 3.91 and 3.93) singlet signals were observed in the higher field. In addition, 11 one-proton multiplet signals were assigned to five methine groups (δH 6.33, 3.84, 3.79, 4.78, and 1.71) and three methylene groups (δH 2.92, 2.28, 1.45, 1.38, 4.58, and 4.47). The 13C NMR and DEPT spectra indicated the presence of 32 carbon signals, including the signals corresponding to the above units and additional four carbonyls at δC 157.0, 167.3, 167.6, and 197.6 and a sp3 carbon at δC 57.2 (Table S1). Further analysis of the 1H and 13C NMR data revealed that partial data in 1 closely resembled those of 2 and 3,13,16a indicating that 1 was a conjugate of the chlorinated diaporthin with another structural moiety. The full structure of 1 was elucidated by 2D NMR data analyses (Figure 2 and Figures S13−S15 in the Supporting

Figure 3. Key NOESY correlations of 1.

H-6 with H-16 and H-11′ indicated that they were all positioned on the same face of the hexahydropyrrolo[2,3b]indole ring. In addition, the correlations of H-16 with H2-17, H-18, and H3-20 were suggestive of a cis orientation for these groups in the diketopiperazine ring, thus a trans configuration between H-16 and H-3. Marfey’s analysis allowed the assignment of D-configuration for the N-methyl leucine residue (Figure S17).18 Therefore, the absolute configurations of C-3, C-6, C-14, and C-16 were assigned as R, R, S, and S. In order to determine the configuration of C-11′, we initially attempted to obtain a suitable crystal for X-ray analysis under different conditions but failed. Thus, we performed ab initio

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

DOI: 10.1021/acs.orglett.9b00385 Org. Lett. XXXX, XXX, XXX−XXX

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elucidated by Hertweck and co-workers.13 It is supposed that O-methylation and chlorination of the precursor 5 by the hybrid enzyme produces the chlorinated isocoumarin 6. Subsequent chlorination and reduction of the ketone group of 6 leads to 2 and 3, respectively. In addition, the cyclization and dimerization mechanisms of the dimeric diketopiperazine alkaloid (−)-ditryptophenaline were proved to be catalyzed by cytochrome P450 through a radical route.9c Since the connection of the two units and the diketopiperazine ring skeleton in 1 is very close to those of (−)-ditryptophenaline, it is very likely that the hybridation of the isocoumarin and the diketopiperazine units in 1 was also mediated by a similar free radical mechanism. The abstraction of the proton NH-10 in 4 by cytochrome P450 produces a radical at N-10. The radical migrates to C-3 by ring closure between N10−C2 to produce the intermediate 8, which then conjugates with the intermediate 7 via C-3 and C-11′, resulting in the formation of 1 (Scheme 1). In the in vitro antimicrobial bioassay, isocoumarindole A (1) exhibited mild inhibitory activity against Candida albicans with a minimum inhibitory concentration (MIC) of 32.0 μg/mL (positive control: caspofungin, MIC 0.03 μg/mL). In addition, compound 1 displayed significant cytotoxicity against human pancreatic adenocarcinoma cell lines MIA-PaCa-2 and AsPC-1 with IC50 values of 1.63 and 5.53 μM, respectively, comparable to the positive control gemcitabine (IC50: 1.02 and 20.10 μM, respectively). In summary, although the polyketide synthetase−nonribosomal peptide synthetase (PKS−NRPS) hybrid metabolites are not uncommon in microbial natural products,21 isocoumarindole A (1) represents the first example of a PKS− NRPS conjugate composed of isocoumarin and diketopiperazine units through a carbon−carbon bridge bond. The discovery of isocoumarindole A expands the structural diversity of the PKS−NPRS biosynthetic pathway and also provides a challenging target for synthetic chemistry.

NMR calculations of the possible diastereomers, 3R,6R,14S,16S,11′S-1 (1a) and 3R,6R,14S,16S,11′R-1 (1b) (Figure 4) at the PCM/mPW1PW91/6-311+G(d,p) level

Figure 4. Comparison of the experimental and calculated ECD spectra of 1.

using the GIAO method.19 The calculated 13C NMR chemical shifts of 1a showed a better agreement with the experimental values with a higher correlation coefficient (R2, 1a: 0.9976; 1b: 0.9969, Figures S4 and S6). In addition, DP4+ probability analysis19b based on both 1H and 13C NMR data predicated 1a as the correct structure with 99.92% probability (Tables S19 and S20). To confirm the configuration of C-11′, the ECD spectra and optical rotation (OR) data12a of 1a and 1b were further computed using the time-dependent density functional theory (TDDFT) methods. The theory-calculated ECD curve for stereoisomer 1a showed a good agreement with the experimental spectrum of 1 (Figure 4). Moreover, the experimental OR value of 1 (+105.5) was similar to the predicted OR value of 1a (+480.5) but was opposite in sign to that of 1b (−138.9) in methanol (Tables S21 and S22). Consequently, the absolute configuration of 1 was unambiguously determined to be 3R,6R,14S,16S,11′S. Isocoumarindole A (1) was identified as a new type of isocoumarin−indole diketopiperazine alkaloid hybrid metabolite. A plausible biosynthetic pathway of isocoumarindole A (1) is proposed as illustrated in Scheme 1. The isocoumarin moiety originated from the polyketide synthetase (PKS) pathway.20 The mechanism of chlorination and O-methylation leading to dichlorodiaporthin, which was catalyzed by a bifunctional hybrid enzyme in Aspergillus oryzae, was recently

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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.9b00385. Experimental procedures, as well as physical-chemical properties, calculated NMR chemical shifts, ECD spectra and OR data, 1D and 2D NMR, HRMS, and IR spectra for 1 (PDF)

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Scheme 1. Postulated Biogenetic Pathway of 1

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Minghua Chen: 0000-0001-5634-4599 Liyan Yu: 0000-0002-8861-9806 Maoluo Gan: 0000-0002-3089-8654 Author Contributions ⊥

M.C. and R.W. contributed equally to this work.

Notes

The authors declare no competing financial interest. C

DOI: 10.1021/acs.orglett.9b00385 Org. Lett. XXXX, XXX, XXX−XXX

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Yu, L.; Hong, B.; Jiang, W.; Si, S. Bioorg. Med. Chem. Lett. 2015, 25, 562−565. (c) Chen, M. H.; Wu, Y. X.; Xu, Y. N.; Liu, P.; Yu, L. Y.; Hong, B.; Jiang, W.; Si, S. Y. J. Asian Nat. Prod. Res. 2015, 17, 676− 682. (13) Chankhamjon, P.; Tsunematsu, Y.; Ishida-Ito, M.; Sasa, Y.; Meyer, F.; Boettger-Schmidt, D.; Urbansky, B.; Menzel, K. D.; Scherlach, K.; Watanabe, K.; Hertweck, C. Angew. Chem., Int. Ed. 2016, 55, 11955−11959. (14) Klausmeyer, P.; McCloud, T. G.; Tucker, K. D.; Cardellina, J. H.; Shoemaker, R. H. J. Nat. Prod. 2005, 68, 1300−1302. (15) Bode, H. B.; Bethe, B.; Hofs, R.; Zeeck, A. ChemBioChem 2002, 3, 619−627. (16) (a) Almeida, C.; Perez-Victoria, I.; Gonzalez-Menendez, V.; de Pedro, N.; Martin, J.; Crespo, G.; Mackenzie, T.; Cautain, B.; Reyes, F.; Vicente, F.; Genilloud, O. J. Nat. Prod. 2018, 81, 1488−1492. (b) Tanahashi, T.; Takenaka, Y.; Nagakura, N.; Hamada, N.; Miyawaki, H. Heterocycles 2000, 53, 723−728. (17) Tadano, S.; Sugimachi, Y.; Sumimoto, M.; Tsukamoto, S.; Ishikawa, H. Chem. - Eur. J. 2016, 22, 1277−1291. (18) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596. (19) (a) Lodewyk, M. W.; Siebert, M. R.; Tantillo, D. J. Chem. Rev. 2012, 112, 1839−1862. (b) Grimblat, N.; Zanardi, M. M.; Sarotti, A. M. J. Org. Chem. 2015, 80, 12526−12534. (20) Sorensen, J. L.; Nielsen, K. F.; Sondergaard, T. E. Fungal Genet. Biol. 2012, 49, 613−618. (21) (a) Li, X. W.; Ear, A.; Nay, B. Nat. Prod. Rep. 2013, 30, 765− 782. (b) Miyanaga, A.; Kudo, F.; Eguchi, T. Nat. Prod. Rep. 2018, 35, 1185−1209. (c) Boettger, D.; Hertweck, C. ChemBioChem 2013, 14, 28−42.

ACKNOWLEDGMENTS Financial support from the CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-2-002), National Natural Science Foundation of China (NNSFC, 81302675, 81621064, 81630089, and 81872781), the Drug Innovation Major Project of China (Grant Nos. 2018ZX09735001-002 and 2018ZX09711001-007), Tianshan Cedar Project in Xinjiang Uygur Autonomous Region (2017XS10), and National Infrastructure of Microbial Resources (NIMR 2018-3) is acknowledged.

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DOI: 10.1021/acs.orglett.9b00385 Org. Lett. XXXX, XXX, XXX−XXX