New analogues of brefeldin A from sediment-derived fungus Penicillium sp. DT-F29

Abstract Four new analogues of brefeldin A named 7, 7-dimethoxybrefeldin C (3), 6β-hydroxybrefeldin C (4), 4-epi-15-epi-brefeldin A (5), 4-epi-8α-hydroxy-15-epi-brefeldin C (6), together with four known analogues (1, 7−9) were isolated from a fermentation of the sediment-derived fungus Penicillium sp. DT-F29. The structures of these compounds were elucidated on the basis of extensive spectroscopic and chemical methods. In the bioactivity assays, only compounds 1 and 8 showed significant inhibitory activities against human lung adenocarcinoma cell. In addition, compound 1 was first reported for the potent ability to reactivate latent HIV with EC50 value of 0.03 μM.


Introduction
Though being potent and life prolonging, the current therapy can not cure cancer and HIV infection completely. Advanced research suggested that the primary obstacle to fixing it is how to kill the cancer stem cells (Colak & Medema 2014) and remaining latent HIV (Kulkosky et al. 2001;Richman et al. 2009), respectively. Therefore, searching for new bioactive compounds for drug-resisted tumour cells and reactivating latent HIV becomes urgent.
Brefeldin A analogues is a family of bioactive metabolites consisting of a 16-membered macrolide and its derivatives. Brefeldin A (1) was first isolated from a Penicillium decumbens in 1958 (Singleton et al. 1958), whose biogenetic precursor is found to be brefeldin C (2) (Sunagawa et al. 1983). Brefeldin A has displayed various biological activities such as antiviral (Tamura et al. 1968;Takatsuki et al. 1969), antifungal (Hayashi et al. 1974), antitumour (Betina et al. 1962) and ABSTRACT Four new analogues of brefeldin A named 7, 7-dimethoxybrefeldin C (3), 6β-hydroxybrefeldin C (4), 4-epi-15-epi-brefeldin A (5), 4-epi-8αhydroxy-15-epi-brefeldin C (6), together with four known analogues (1, 7−9) were isolated from a fermentation of the sediment-derived fungus Penicillium sp. DT-F29. The structures of these compounds were elucidated on the basis of extensive spectroscopic and chemical methods. In the bioactivity assays, only compounds 1 and 8 showed significant inhibitory activities against human lung adenocarcinoma cell. In addition, compound 1 was first reported for the potent ability to reactivate latent HIV with EC 50 value of 0.03 μM.
antimitotic activities (Betina & Montagier 1966). Although brefeldin A exhibits excellent activity, its low bioavailability and poor pharmacokinetics becomes the obstacle to be a drug (Phillips et al. 1993). To solve this problem, a variety of brefeldin A analogues including synthetic analogues has been investigated in continuing exploration, and most of them exhibited significant biological activity (Argade et al. 1998;Anadu et al. 2006;Seehafer et al. 2013).
undoubtedly, marine fungus has become an important source of new natural products and provides many active compounds (Saraiva et al. 2015;Tian et al. 2015). During our approach to identify new bioactive metabolites from sediment-derived fungi, the EtOAc extract of Penicillium sp. DT-F29 was chosen for further investigation due to its good cytotoxic activity against PC3 cell line and activation of latent HIV on J-Lat clones C11 cells (at 20 μg/mL). The chemical research of the crude extract led to the discovery of a series of brefeldin A analogues including four new compounds, named 7, 7-dimethoxybrefeldin C (3), 6α-hydroxybrefeldin C (4), 4-epi-15-epi-brefeldin A (5), 4-epi-8α-hydroxy-15-epi-brefeldin C (6), as well as four known metabolites, brefeldin A (1), 12α-hydroxybrefeldin A (7), 7-epi-brefeldin A (8) and 7-dehydrobrefeldin A (9) (Figure 1). The structures of the compounds were determined by comprehensive spectroscopic analysis. Herein, we describe the details of the separation, structure elucidation, and bioactivity of the isolated substances.

Results and discussion
2.1. Structure elucidation 7, 7-dimethoxybrefeldin C (3) was obtained as a white powder, and its molecular formula was established as C 18 H 28 O 5 by the HRESIMS ion at m/z 671.3766 [2M + Na] + , implying the presence of 5° of unsaturation. The 1 H and 13 C NMR spectra of 3 were very similar to those of brefeldin A (1), revealing that it was an analogue of 1 (Table S1 and S2). A detailed comparison of the data suggested that C-7 hydroxylated methine (δ C 72.7, δ H 4.37) in 1 was replaced by a quaternary carbon (δ C 109.4) in addition to two methoxy group (δ C 49.8 and 48.8, δ H 3.20 and 3.22) in 3, which was further verified by the key HMBC correlations from H 3 -17 and H 3 -18 to C-7 ( Figure S1). The relative configuration of 3 was established on the basis of 1 H− 1 H coupling constants and NOESY correlations. The 9.6 Hz magnitude of 3 J(H-5 to H-9) illustrated the trans-arrangement between H-5 and H-9, coupled with the anti-periplanar signals 10.1 and 9.6 Hz values for the respective 3 J(H-4, H-5) and 3 J(H-9, H-10) coupling constants (Glaser et al. 2000). The presumptions described earlier were confirmed by NOESY correlations from H-4 to H-9 and H-5 to H-10 ( Figure S2). Furthermore, the key coupling constants of H-3/H-4/H-9/H-10/H-15 matched well with those of 1 (Glaser et al. 2000). Thus, the relative configuration of 3 was determined as 2E*, 4R*, 5R*, 9S*, 10E*, 15S*.
4-epi-15-epi-brefeldin A (5) was obtained as a colourless needles, and its molecular formula was determined as C 16 H 24 O 4 on the basis of the HRESIMS peak at m/z 303.1572 [M + Na] + . The detailed interpretation of the COSY and HMBC data of 5 revealed that it was a stereoisomers of 1 ( Figure S1). The relative configuration of 5 was also established on the basis of 1 H− 1 H coupling constants and NOESY correlations ( Figure S2). The coupling constant of a 2.2 Hz 3 J(H-4, H-5) indicated a typically syn-diaxial relationship and 3.4 Hz 3 J (H-15, H 2 -14) implied a contrary configuration of C-15 compared with 1 (Table S1). Furthermore, the key coupling constants of H-9/H-10 matched well with those of 1 (Glaser et al. 2000), and H-9 were approaching to downfield, which can be found in 4-epi-brefeldin A (Zhu et al. 2000). Thus, the relative configuration of 5 was determined as 2E*, 4S*, 5R*, 7S*, 9S*, 10E*, 15R*. 4-epi-8α-hydroxy-15-epi-brefeldin C (6) was obtained as a colourless needles, and its molecular formula was also determined as C 16 H 24 O 4 on the basis of the HRESIMS peak at m/z 303.1567 [M + Na] + . The detailed analysis of the comprehensive spectrum data of 6 revealed that it was a constitutional isomer of 5 (Table S1 and S2). The key COSY correlations between H-8 (δ H 4.13) and H-7 (δ H 1.75)/H-9 (δ H 2.37) indicated hydroxy substituted at C-8, which is consistent with key HMBC correlations between H-10 (δ H 5.41) and C-8 (δ C 72.1) ( Figure S1). The relative configuration of 6 was also established on the basis of 1 H− 1 H coupling constants and NOESY correlations ( Figure S2). The coupling constant of a 4.9 Hz for 3 J (H-4, H-5) and 4.6 Hz for 3 J (H-8, H-9) indicated typically syn-diaxial relationship and 3.5 Hz 3 J (H-15, H 2 -14) implied a similar configuration of C-15 compared with the 5 (Table S1). Furthermore, the key coupling constants of H-7/H-9/H-10 matched well with those of brefeldin A (Glaser et al. 2000), and the signals of H-4 and H-15 were proximate with the corresponding of 5. Thus, the relative configuration of 6 was determined as 2E*, 4S*, 5R*, 8R*, 9S*, 10E*, 15R*.

Bioactivity assays
The cytotoxicity of brefeldin A analogues was evaluated in vitro against H1975 cells and anti-HIV latency was investigated on J-Lat clones C11 cells. The results (Table S3) revealed that 1 and 8 exhibited significant inhibitory against H1975 Cell. As for the ability to reactivate latent HIV, 1 was found to be a powerful activator.

Experimental
Experimental section was supplied as online Supplementary material.

Conclusion and discussion
In summary, four new and four known brefeldin A analogues were identified from the crude extract of Penicillium sp. DT- F29. 1, 3, 4, 8 illustrated the configuration of C-7 is important for its bioactivity, whereas 1, 5 and 6 pointed out the significance of stereoisomerism at C-4 and C-15. The observed is in agreement with the weakening cytotoxicity of (15S)-vinyl-norbrefeldin A (Seehafer et al. 2013) and 4-epi-brefeldin A (Zhu et al. 2000). In addition, we first reported the potent ability of brefeldin A to reactivate latent HIV, which provide a new application of brefeldin A.