Penerpenes A–D, Four Indole Terpenoids with Potent Protein Tyrosine

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

Penerpenes A−D, Four Indole Terpenoids with Potent Protein Tyrosine Phosphatase Inhibitory Activity from the Marine-Derived Fungus Penicillium sp. KFD28 Fan-Dong Kong,†,⊥ Peng Fan,‡,⊥ Li-Man Zhou,‡,⊥ Qing-Yun Ma,† Qing-Yi Xie,† Hai-Zhou Zheng,§ Zhi-Hui Zheng,§ Ren-Shuai Zhang,∥ Jing-Zhe Yuan,† Hao-Fu Dai,† Du-Qiang Luo,*,‡ and You-Xing Zhao*,†

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Research and Development of Natural Product from Li Folk Medicine, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agriculture Sciences, Haikou 571101, China ‡ College of Life Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China § New Drug Research and Development Center, North China Pharmaceutical Group Corporation, Shijiazhuang 050015, China ∥ Qingdao Cancer Institute, the Affiliated Hospital of Qingdao University, Qingdao 266061, China S Supporting Information *

ABSTRACT: Four unusual indole-terpenoids, penerpenes A−D (1−4), along with two known ones paxilline (5) and emindole SB (6), were isolated from the marine-derived fungus Penicillium sp. KFD28. The absolute structures of 1−4 were elucidated on the basis of spectroscopic data and ECD spectra analysis along with quantum ECD calculations. Compounds 1 and 2 showed potent inhibitory activity toward protein tyrosine phosphatases (PTP1B and TCPTP). Plausible biosynthetic pathways of compounds 1−4 are proposed.


he paxilline-type indole-terpenoids, such as paxilline,1 thiersinines,2 lolicines,3 shearinines,4 and 2,18-dioxo2,18-seco-paxilline,4 represent a class of fungal metabolites with a common core structure consisting of a cyclic diterpene fusing with an indole moiety skeleton derived from geranylgeranyl diphosphate (GGPP) and indole-3-glycerol phosphate.5 They are one of the largest classes of fungal indole-terpenoids with diverse structures, and nearly 100d have been discovered until now. Many of them showed anti-H1N1,6 antibacterial,7 cytotoxic,8b and ion channel antagonistic activity,8 which have attracted attention of pharmacists as potential drug lead compounds. In the course of our ongoing search for structurally new and biologically active metabolites from marine-derived fungi,9 the fungus Penicillium sp. KFD28 was isolated and identified from a bivalve mollusk, Meretrix lusoria, collected from Haikou Bay, China. Subsequent chemical investigation on the EtOAc extract of the fermentation broth led to the isolation and identification of four novel paxilline-type indole-terpenoids (Figure 1), penerpenes A−D (1−4), along with two known ones paxilline (5)1 and emindole SB (6).6 Compound 1 is a unique spiro indole-diterpene bearing an 1,4-dihydro-2Hbenzo[d][1,3]oxazine motif. Compound 2 represents the first indolediterpene with a unique pyridine-containing heptacyclic ring system. Compounds 3 and 4 are nor-indole-diterpenoids with new carbon skeletons that may be derived from paxilline by loss of five (C-21/22/23/24/25) and four (C-22/23/24/ 25) carbons, respectively. Among them, 1 and 2 showed © XXXX American Chemical Society

Figure 1. Chemical structures of compounds 1−6.

potent inhibitory activity toward protein tyrosine phosphatases (PTP1B and TCPTP). Herein, the isolation, structure elucidation, and bioactivities of these compounds are described. Compound 1 has the molecular formula C28H35NO6 as established from its HRESIMS and 13C NMR data (Table S1). The 1H and 13C NMR data of 1 (Table S1), with the aid of an HSQC spectrum (see the Supporting Information), showed a total of 28 carbon signals comprising two ketone carbonyls, eight olefinic or aromatic carbons with five protonated, six sp3 Received: May 18, 2019


DOI: 10.1021/acs.orglett.9b01751 Org. Lett. XXXX, XXX, XXX−XXX


Organic Letters methylenes with one oxygenated, three sp3 methines with two oxygenated, five sp3 nonprotonated carbons with three oxygenated, and four methyls. A comparison of the NMR data of 1 with those of paxilline (5)1 indicated that they shared a similar substructure (unit A, Figure 2), except that the

was replaced by a conjugated ketone carbonyl in 2. The two methyls CH3-24 and CH3-25 and oxygenated nonprotonated carbon C-23 structural fragments are also retained as that in 5 based on the HMBC correlations (Figure 2). The remaining five nonprotonated aromatic carbons with characteristic chemical shifts (Table S1), combined with that an additional nitrogen atom was present in the molecular formula compared to 5, suggested the presence of a fully substituted pyridine ring in the structure, the position of which was determined by HMBC correlations from H2-12 to C-13, H3-26 to C-14, and H-22 to C-21 and C-20 (Figure 2). On the basis of the above data, another ring is still required to fulfill the 15 double-bond equivalent. The chemical shift of C-20 at δC 152.7 suggested it was an oxygenated aromatic carbon. Thus, a connection between C-23 and C-20 via an oxygen atom to form a fivemembered cyclic ether was proposed, which was supported by absence of OH-23 proton signal and the downfield-shifted C23 signal in the NMR spectra compared to those of 1 (Table S1). The ROESY correlations (Figure 2) of H-11/H3-26 and H-16/H-27 assigned the relative configuration of all the asymmetric carbons except C-22, which was far from the others in the structure. In order to verify the planar structure and determine the relative configuration of C-22, the 13C NMR calculations of 2 and 22-epi-2 were performed. As shown in Figure S3, compared to those of 22-epi-2, the calculated NMR chemical shifts of 2 coincided better with the experimental data with R2 = 0.9981 (Figure S3), thus defining the relative structure of 2. To further verify the above structural assignment and determine the absolute configuration of 2, the ECD spectra for four possible stereoisomers [(10R,11S,15S,22S)-2; (10S,11R,15R,22R)-2; (10R,11S,15S,22R)-2; (10S,11R,15R,22S)-2] of 2 according to the ROESY data were calculated and compared with the experimental one. The results (Figure 4) showed that the

Figure 2. Selected HMBC, COSY, and ROESY correlations of 1−4.

aromatic nonprotonated C-9 in 5 was replaced by a ketone carbonyl, as confirmed by HMBC correlations from H3-27 to C-9, C-10, C-11, and C-15. In addition, the presence of the 1,4-dihydro-2H-benzo[d][1,3]oxazine motif (unit B, Figure 2) was revealed by analysis of the remaining carbon and proton signals and their related 2D NMR data including sequential COSY correlations of H-2/H-3/H-4/H-5 and NH-6/H2-28 and HMBC correlations of H-3 and NH-6 to C-1, H-4 to C-6, and H-28 to C-6 and C-8. The fusion of units A and B via the spiro carbon C-8 was demonstrated by HMBC correlations from H2-7 to C-8. The relative configuration of 1 were deduced from the ROESY correlations (Figure 2) of H-28/H26/H-11, H-2/H-27/H-16/H-18/H-22, and OH-14/H-18. The calculated ECD curve for 1 matched well with the experimental one (Figure 3), assigning the 8R,10S,11S,14S,15R,18S,22R configuration of 1. Compound 2 was determined to have the molecular formula C27H28N2O3 based on the positive HRESIMS data, indicating 15 degrees of unsaturation. Detailed analysis of the NMR data disclosed that 2 possessed a similar substructure (unit A, Figure 2) as that of 5, except that the oxymethine C-18 in 5

Figure 4. Experimental and calculated ECD curves for 2.

calculated ECD curve for (10S,11R,15R,22R)-2 matched well with the experimental spectrum, which further confirmed the above relative structure and also assigning the10S,11R,15R,22R absolute configuration of 2. The molecular formula of compound 3 was established as C22H21NO4 by HRESIMS, with five carbons less than 5. The double-bond equivalent of 3 was calculated to be 13. The presence of 10 olefinic or aromatic carbons and two carbonyl groups in the 13C NMR spectrum (Table S1) indicated that 3 has a hexacyclic ring system. Analysis of HMBC and COSY data (Figure 2) of 3 suggested the presence of the same substructures C-1 to C-12 and C-15 to C-17 as well as two methyls (CH3-27 and CH3-26) at C-10 and C-15 as those of 5. The locations of the hydroxylated quaternary carbon C-13 and the α,β-unsaturated ketone moiety C-14/C-19/C-18 were determined by HMBC correlations (Figure 2) from H3-26 to C-14; OH-13 to C-13, C-14, and C-12; and H2-17 to C-18. At

Figure 3. Experimental and calculated ECD curves for 1. B

DOI: 10.1021/acs.orglett.9b01751 Org. Lett. XXXX, XXX, XXX−XXX


Organic Letters

of the ECD exciton chirality models (Figure 5), the absolute configuration of 3 and 4 were determined to be (10S,11R,13S,15R) and (10S,11S,14S,15R,18S), respectively. Protein tyrosine phosphatases (PTPs) are thought to be promising targets for drug discovery and have recently attracted much attention.11 The isolated six compounds were tested for their inhibitory activities against PTPs including nontransmembrane PTPs (PTP1B and TCPTP), receptor-like PTP (PTPsigma), and dual-specificity phosphatases (VHR). Compounds 1, 2, and 6 showed activities with potencies comparable to that of Na3VO4 against PTP1B and TCPTP (Table 1). In addition, the cytotoxicity of 1−6 against four

this point, all structural features were assigned except for an ester group, one degree of unsaturation, and two connection sites at C-13 and C-19, respectively. The chemical shift of C-13 at δC 101.3 (Table S1) suggested that it was a hemiacetal carbon. Thus, the remaining unassigned C-20 ester carbon must be located at C-19 and connect with the hemiacetal carbon C-13 via an oxygen atom to complete the hexacyclic ring system, which was also supported by the significant downfield-shifted C-14 carbon signal due to the electron absorption effect of the ester group. The relative configuration of 3 was determined by ROESY correlations (Figure 2) of H11/H-26/OH-13 and H-12/H-27. The molecular formula of compound 4 was established as C23H25NO3 by HRESIMS. Analysis of the NMR spectra suggested that the structure of 4 was closely related to that of 5 with the differences being the missing of the C-22/23/24/25 moiety to form the five-membered lactone ring in 4, as corroborated by HMBC correlations (Figure 2) and the downfield-shifted H-18 signal compared to that of 1 (Table S1). Thus, the planar structure of 4 was assigned as shown in Figure 1. The relative configurations of compound 4 were determined to be the same as 5 by ROESY correlations (Figure 2) of H-11/H-26/H-13 and H-27/H-16/H-18/OH-14. The structures of 3−6 were similar, all containing an indole and an α,β-unsaturated ester or ketone carbonyl chromophore. Comparison of the ECD data (Figure 5) between 5 and 6

Table 1. Inhibitory Activity of Compounds 1−6 toward Protein Phosphatases (PTP1B, TCPTP, PTPsigma, and VHR) PTPs (IC50, μM) compd





1 2 3 4 5 6 Na3VO4

1.7 2.4 >30.0 >30.0 10.6 0.7 1.6

5.0 4.5 >30.0 >30.0 25.3 1.4 2.4

29.0 >30.0 >30.0 >30.0 29.3 16.6 9.7

>30.0 >30.0 >30.0 >30.0 >30.0 >30.0 13.2

tumor cell lines (SGC-7901, K562, Hela, and A549) was also evaluated using the MTT method.12 The results showed that only 6 exhibited weak cytotoxicity against the K562 cell line with an IC50 value of 18.8 μM. The above data suggested that compounds 1 and 2 have great potential as new PTPs inhibitors with medicinal use. A positive effort was made to explain the activity of 1 and 2 against PTP1B by performing molecular docking.13 Docking results (Figure 6) implied that 1 Figure 5. Experimental ECD spectra for 3−6 and ECD exciton chirality models (with protons hidden) for 3 and 4.

seems to suggest that the strong positive CEs (Cotton effects) around 230 nm and the negative one 240 nm in the ECD curve of 5 could be originated from the indole chromophore and the α,β-unsaturated carbonyl chromophore, respectively, if taking them as two isolated chromophores that do not interfere with each other in the contribution of ECD spectrum. Based on the above deduction, the absolute configurations of 3 and 4 were intended to be assigned by comparing their ECD data with those of 5 and 6 at first. However, when the ECD curves of 3 and 4 were added into comparison (Figure 5), it was found that the presence of the α,β-unsaturated carbonyl chromophores in 3 and 4 could result significant blueshifts of the short-wave strong CEs (217 nm in 3 and 218 nm in 4) compared to that (near 230 nm) of 6, which initially were thought to be originated solely from the indole chromophore. The above data contradicted the previous inferences and suggested that the α,β-unsaturated carbonyl and the indole moiety in 3 and 4 were not acting alone but interacting through space with each other to generate exciton ECDs.10 According to the ECD spectra, a positive couplet in the ECD spectrum of 3 and negative couplet in that of 4 around 225 nm were observed, which are generated by the interaction between the 1Bb transition moment of indole and π−π *transition moment of the α,β-unsaturated carbonyl.10 Thus, on the basis

Figure 6. (A) Model of compounds 1 (cyans) and 2 (green) bound to PTP1B (PDB: 1QXK). (B) Interactions between 1 (cyans), 2 (green), and PTP1B. The key amino acids were shown in yellow.

binds deep in the active site pocket and form H-bonds with Asp-181 and Gln-262 (Figure 6A), while interestingly, 2 with a larger ring structure did not bind at the active site but instead interacted with Phe-30 in the so-called secondary bonding site of PTP1B (Figure 6B). Actually, the secondary bonding site was found as an additional noncatalytic binding site early in 1997.14 Thus, their similar activity against PTP1B is not surprising. The carbon skeletons of previously reported paxilline-type indole terpenoids are highly conserved. The main skeletal changes are the degradation of the methyl located at C-19 in most cases and the C(8)C(9) carbon bond cleavage as C

DOI: 10.1021/acs.orglett.9b01751 Org. Lett. XXXX, XXX, XXX−XXX


Organic Letters exemplified by 2,18-dioxo-2,18-seco-paxilline.4 In the present study, the discovery of 1−2 with unique ring systems and 3−4 with new carbon skeletons derived from unprecedented carbon degradation pattern prompted us to investigate its plausible biosynthetic pathways (Scheme 1). Paxilline (5) was assumed

Author Contributions ⊥

F. K., P. F., and L.Z. contributed equally to this work.


The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of Hainan Province (2019CXTD411), Natural Science Foundation of China (41606088, 31672070), China Agriculture Research System (CARS-21), Financial Fund of the Ministry of Agriculture and Rural Affairs, P.R. of China (NFZX2018), and Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (17CXTD-15, 1630052016008). We thank Yaqin Fan from the School of Medicine and Pharmacy, Ocean University of China, for docking experiments.

Scheme 1. Hypothetical Biogenetic Pathway of Compounds 1−4

to be the biosynthetic precursor of 1, 3, and 4 for their structural similarity. Paxilline underwent methylation and oxidation to produce the epoxy intermediate (A), which then underwent the C-28−O−C-8 ring closure and C-9−N ring opening to generate 1 with a spiro skeleton. Paxilline underwent Baeyer−Villiger oxidation reaction to generate intermediate B and I. B then underwent ring cleavage, dehydration, and oxidation to afford intermediate C, which formed 3 by C-20−O−C-13 ring closure. Compound 4 was formed from intermediate I through the loss of a C4 unit and C-18−O−C-21 ring closure. The biosynthetic precursor of 2 was proposed to be emindole SB (6), which underwent a sequential reaction to generate the amino intermediate E. Subsequent Schiff base formation and oxidation would afford intermediate F, which underwent enolization and C-20−O−C23 ring closure to afford 2.


S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b01751. Experimental details, calculation details, HRESIMS, IR, and NMR spectra (PDF)


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Corresponding Authors

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

You-Xing Zhao: 0000-0002-8107-2510 D

DOI: 10.1021/acs.orglett.9b01751 Org. Lett. XXXX, XXX, XXX−XXX