Cytotoxic Indole-Diterpenoids from the Marine-Derived Fungus Penicillium sp. KFD28

Four new indole-diterpenoids, named penerpenes K-N (1–4), along with twelve known ones (5–16), were isolated from the fermentation broth produced by adding L-tryptophan to the culture medium of the marine-derived fungus Penicillium sp. KFD28. The structures of the new compounds were elucidated extensively by 1D and 2D NMR, HRESIMS data spectroscopic analyses and ECD calculations. Compound 4 represents the second example of paxilline-type indole diterpene bearing a 1,3-dioxepane ring. Three compounds (4, 9, and 15) were cytotoxic to cancer cell lines, of which compound 9 was the most active and showed cytotoxic activity against the human liver cancer cell line BeL-7402 with an IC50 value of 5.3 μM. Moreover, six compounds (5, 7, 10, 12, 14, and 15) showed antibacterial activities against Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633.


Introduction
Marine fungi have formed different metabolic pathways and adaptation mechanisms within the peculiar marine environment. Hence, marine fungi can produce natural secondary metabolites that are characterized by unique chemical structures and high biological activities [1,2]. Alkaloids derived from marine-derived compounds have received extensive attention in recent years. Indole alkaloids, as an important class of secondary metabolites produced by marine-derived fungi [3], showed excellent biological activities, including cytotoxic [4], antibacterial [5], quorum sensing inhibitory [6], anti-Zika virus [7], and protein tyrosine phosphatase inhibitory activities [8,9].

Structure Elucidation
Compound 1 was obtained as a yellow oil. Its formula was determined as C28H39NO3 on the basis of HRESIMS data (m/z 476.2565 for [M+K] + ), indicating ten degrees of

Structure Elucidation
Compound 1 was obtained as a yellow oil. Its formula was determined as C 28  or aromatic carbons (four protonated) for a 2,3-disubstituted indole moiety, four methyls, eight sp 3 methylenes with one oxygenated, four sp 3 methines with two oxygenated, and four non-protonated sp 3 carbons with one oxygenated. These NMR data allowed the construction of the carbon skeleton of indole-diterpenoid. Careful contrast of the similar NMR spectral data between 1 and paspaline (11) [20] revealed that they had the same planar structure except that the methyl group at C-12 (δ C 36.6) in paspaline was oxidized to oxymethylene in 1. Consistent with the introduction of the hydroxyl at C-30 (δ C 58.5), it showed a distinctive deshielding of the C-12 signal (δ C 48.8) in 1 compared to those of paspaline (δ C 36.6). The linkage of a hydroxyl at C-30 was further supported by the COSY correlation of 30-OH/H 2 -30 ( Figure 2; Supplementary Material, Figure S8) and ROESY correlation of H 3 -26/H 2 -30 (Supplementary Material, Figure S9). Detailed analysis of 2D NMR data further confirmed that 1 and paspaline share the same indole-diterpenoid skeleton. The relative configuration of 6/6/6 tricyclic rings in indole-diterpenoid were determined by the ROESY spectrum (Figure 3), in which the sequential correlations of H-16/H 3 -26/H 2 -30/H-10β/H 3 -28 suggested the same face of H-16, CH 3 -26, CH 2 -30, and C-27 in the 6/6/6 tricyclic ring system, while the correlations of H 3 -25/H-13/H-7/H-9 suggested CH 3 -25, H-13, H-7, and H-9 were on the opposite face of this system. It has been reported that the strong Cotton effect (CE) around 220 nm was related to the absolute configurations of the chiral carbons around the indole chromophore in the paxilline-type indole-diterpene [17]. Thus, the strong negative CE at 223 nm in the experimental ECD spectrum of 1 ( Figure 4) suggested its (3S,4S,7S,9S,12S,13R,16S)-1 absolute configuration [17]. As for the absolute configuration of 1, the ECD spectrum of (3S,4S,7S,9S,12S,13R,16S)-1 was calculated; this deduction was further supported, establishing the absolute configuration of 1 as presented in Figure 1.
Compound 2 was isolated as a yellow oil. Its formula was determined as C 27 H 35 NO 4 on the basis of HRESIMS data, indicating 11 degrees of unsaturation. Its 13 C NMR data showed a total of 27 carbon signals comprising eight aromatic or olefinic carbons for an indole moiety, four methyls, six sp 3 methylenes, four sp 3 methines with two oxygenated, and five non-protonated sp 3 carbons with three oxygenated. Analysis of the NMR spectra (Tables 1 and 2, Supplementary Materials, Figures S12-S18) of 2 suggested that its structure was related to that of 4a-demethylpaspaline-3,4,4a-triol [17], and the main difference being C-9 (δ C 109.8), C-11 (δ C 43.7), and C-12 (δ C 84.6) in 2 instead of C-9 (δ C 82.6), C-11 (δ C 73.5), and C-12 (δ C 76.1) in 4a-demethylpaspaline-3,4,4a-triol, indicating the replacement of two oxygenated carbons by a methylene (C-11) and an acetal carbon (C-9) in 2. The HMBC correlations (Supplementary Material, Figure S16) from H-11, H-7, H-29, and H-28 to C-9 confirmed this deduction. The presence of an indole moiety, together with the HRESIMS data (Supplementary Material, Figure S19), indicated that 2 has a heptacyclic ring system. At this point, one more ring was required to fulfill the 11 double-bond equivalents, and an oxygen bridge was proposed according to the molecular formula. The oxygen bridge was assigned to connect C-9 and C-12 as deduced from distinctive deshielding of the C-12 (δ C 84.6) and C-9 (δ C 109.8) in 2 compared to corresponding C-12 (δ C 76.1) and C-9(δ C 96.4-96.5) [25,26] with a free hydroxyl group. In the ROESY spectrum (Supplementary Material, Figure S18), correlations of H-16/H 3 -26/H-5β, and H-5α/H 3 -25/H-13/H-7/H-10/H 3 -28 determined the relative configuration of 2, as shown in Figure 3. In addition, the β orientation of the oxygen bridge was also proved by the ROESY correlation of H-7/H 2 -11 with the aid of the 3D ball-and-stick molecular model. Thus, the planar structure of compound 2 was established and named penerpene L. The ECD curve ( Figure 4) of compound 2 is similar to 1, indicating that the absolute configurations for the chiral carbons C-3, C-4, C-16, and C-13 in 1 were the same as those of 2. The ECD calculation experiment also confirmed the above deduction (  Figures S22 and S23) displayed 28 carbon signals, including ten aromatic or olefinic carbons (six protonated), five methyls, seven sp 3 methylenes, three sp 3 methines with one oxygenated, three non-protonated sp 3 carbons with one oxygenated. These NMR spectra data indicated that 3 was very similar to emeniveol (16) [24] except that a methyl in C-26 (δ C/H 16.3/1.11) and an oxygenated non-protonated sp 3 carbon C-14 (δ C 76.4) in 3 replaced two olefinic carbons in emeniveol, suggesting that the exocyclic double bond in emeniveol changed to a methyl and an oxygenated non-protonated sp 3 carbon. This obvious difference was supported by the HMBC correlations (Supplementary Material, Figure S25) from the H 3 -26 to C-14, C-13 (δ C 42.0) and C-9 (δ C 42.9). The relative configuration of 3 was determined by the ROESY spectrum ( The molecular formula of compound 4 was established as C 27 H 33 NO 4 on the basis of HRESIMS data, indicating 12 degrees of unsaturation. Analysis of the 1 H and 13 C NMR spectra (Tables 1 and 2, Supplementary Materials, Figures S30 and S31) of 4 suggested that its structure was closely related to that of penerpene G [9], a previously reported indole diterpene with an unusual 6/5/5/6/6/7 hexacyclic ring system bearing a 1,3-dioxepane ring. The main difference between them was the replacement of an oxygenated nonprotonated sp 3 Figure S22 and S23) displayed 28 carbon signals, including ten aromatic or olefinic carbons (six protonated), five methyls, seven sp 3 methylenes, three sp 3 methines with one oxygenated, three non-protonated sp 3 carbons with one oxygenated. These NMR spectra data indicated that 3 was very similar to emeniveol (16) [24] except that a methyl in C-26 (δC/H 16.3/1.11) and an oxygenated non-protonated sp 3 carbon C-14 (δC 76.4) in 3 replaced two olefinic carbons in emeniveol, suggesting that the exocyclic double bond in emeniveol changed to a methyl and an oxygenated non-protonated sp 3 carbon. This obvious difference was supported by the HMBC correlations (Supplementary Material, Figure S25) from the H3-26 to C-14, C-13 (δC 42.0) and C-9 (δC 42.9). The relative configuration of 3 was determined by the ROESY spectrum (Figure 3), and the correlations of 14-OH/H3-25/H3-27/17-OH and H-9/H3-25 suggested the same face of these protons, while the correlation of H-12/H-17 indicated that these protons were on the opposite face to 17-OH. Thus, the planar structure of 3 was assigned, as shown in Figure 1. The absolute configuration of 3 was established as (9S,12S,13S,14S,17S,18S)-3 by comparison of its experimental ECD spectrum with the calculated ECD curves (Figure 4).
The molecular formula of compound 4 was established as C27H33NO4 on the basis of HRESIMS data, indicating 12 degrees of unsaturation. Analysis of the 1 H and 13 C NMR spectra (Tables 1 and 2, Supplementary Materials, Figure S30 and S31) of 4 suggested that its structure was closely related to that of penerpene G [9], a previously reported indole diterpene with an unusual 6/5/5/6/6/7 hexacyclic ring system bearing a 1,3-dioxepane ring. The main difference between them was the replacement of an oxygenated non-protonated sp 3

Biological Assay
The cytotoxic activities of compounds 1-16 were conducted by the MTT assay method [27] using cisplatin as the positive control. All compounds were tested against the human cervical cancer cell line HeLa, human gastric cancer cell line SGC-7901, human lung carcinoma cell line A549, and human liver cancer cell line BeL-7402. The results (Table 3) indicated that compound 9 exhibited the most pronounced activity against BeL-7402 with an IC 50 value of 5.3 µM and was comparable to that of positive control cisplatin (IC 50 4.1 µM). Compound 9 also displayed moderate cytotoxic activity against A549. While compounds 4 showed low cytotoxicity against HeLa. Compound 15 displayed mild inhibitory activity against HeLa, A549, and BeL-7402 (IC 50 = 24.4-40.6 µM). The remaining compounds were found to be inactive against the three cell lines. All the tested compounds 1-16 were inactive against the cell line SGC-7901. The loss of a hydroxyl at C-14 suggested being a determinant of cytotoxicity shown by compound 4 against HeLa (4 vs. penerpene G [9]). It is worth mentioning that it was the first-time report of the cytotoxicity of epipaxilline (9) [18]. Arintari et al. [19] and Sallam et al. [28] demonstrated the cytotoxicities of emindole SB (15) against human breast cell line MCF-7, murine lymphoma cell line L5178Y, and the human embryonic kidney cell line HEK-293, which provides emindole SB (15) a meaningful pharmacophore for further biological studies. Compounds 1-16 were also tested for their antibacterial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, Listeria monocytogenes ATCC 1911, and Bacillus subtilis ATCC 6633 using the 96-well microtiter plates method [29] reported previously and using ampicillin as a positive control. The results (Table 4) revealed that six compounds 5, 7, 10, 12, 14, and 15 showed moderate inhibitory activity against S. aureus ATCC 6538. Emindole SB (15) displayed reported selectivity toward S. aureus ATCC 33591 (MIC = 6.25 µg/mL) [21]. Compound 7 showed reasonable antibacterial activity against B. subtilis ATCC 6633 (MIC = 16 µg/mL), but compounds 5, 10, and 12 exhibited lower inhibitory. The results of the rest ten compounds (1-4, 6, 8-9, 11, 13, and 16) did not show remarkable antibacterial activities (MIC > 128 µg/mL) against S. aureus and B. subtilis. In this assay, none of these compounds showed inhibitory activity against E. coli ATCC 25922 and L. monocytogenes ATCC 1911 (MIC > 128 µg/mL).

Fungus Material
The fungus Penicillium sp. KFD28 (GenBank accession No. MK934323) was isolated from a bivalve mollusk, Meretrix lusoria, collected from Haikou Bay, Hainan province, in China. A reference culture of Penicillium sp. KFD28 is deposited in our laboratory and maintained at −80 • C.

Culture Conditions
The fungus Penicillium sp. KFD28 was cultured in 200 × 1000 mL Erlenmeyer flasks containing 100 g rice and 100 mL of water (33 g sea salt, 5 g L-tryptophan per liter pure water). The fungus was cultured in the medium and incubated at room temperature for thirty days.
Fr. 3 (4.2 g) was applied to ODS silica gel with gradient elution of MeOH-H 2 O

Conclusions
To summarize, based on the OSMAC culture strategy, four new indole-diterpenoids were isolated from the marine-derived fungus Penicillium sp. KFD28 secondary metabolites. The absolute configurations of new compounds 1-3 were determined by spectroscopic methods coupled with experimental and calculated ECD. New compound 4 showed mild cytotoxicity against the human cervical cancer cell line HeLa. Notably, compound 9 exhibited strong cytotoxic activity against the human liver cancer cell line BeL-7402 with IC 50 values of 5.3 µM, indicating that compound 9 deserves further study for its therapeutic potential to develop new anti-hepatoma drugs. Compound 7 showed pronounced antibacterial activity against Bacillus subtilis with MIC values of 16 µg/mL, which had the potential to become an antibiotic. However, the mechanisms causing cytotoxicity and bacteria restraint were unknown and require further study. In general, this study expanded the application of the OSMAC method to increase the chemical and biological diversity of natural products isolated from the Penicillium sp. KFD28.