Citriperazines A-D produced by a marine algae-derived fungus Penicillium sp. KMM 4672

Abstract Four new diketopiperazine alkaloids, citriperazines A-D were isolated from algae-derived Penicillium sp. KMM 4672. The structures of compounds 1–4 were determined using spectroscopic methods. The absolute configurations of compounds 1 and 4 were established by comparison of calculated and experimental ECD spectra. The cytotoxicity of compounds 1–4 against several human prostate cell lines was evaluated. Graphical Abstract


Introduction
Marine-derived fungi are a prolific source of chemically diverse bioactive metabolites (Blunt et al. 2018). 2,5-Diketopiperazines (DKPs) are one of the most common groups of fungal metabolites (Borthwick 2012;Huang et al. 2014). Many of them exhibited various bioactivities, such as antibiotic (Song et al. 2012), antiviral (Cai et al. 2012), antitumor ) and some others. Remarkably, plinabulin, a synthetic derivative of marine fungal 2,5-DKP phenylahistin, now is under phase III of clinical trials.
During our ongoing search for structurally novel and bioactive metabolites from marine-derived fungi we have investigated the fungus Penicillium sp. KMM 4672 isolated from Vietnamese marine brown algae Padina sp. Recently, we have reported the isolation of several new epidithiodiketopiperazines together with the known alkaloids and polyketides from this strain Yurchenko et al. 2016;Smetanina et al. 2017). Thorough investigation of low-and medium-polar fractions from EtOAc extract allow to isolate some new compounds. Herein, we report the isolation, structure elucidation and biological activity of the new 2,5-DKP alkaloids 1-4 produced by the marine-derived fungus Penicillium sp.

Results and discussion
The EtOAc extract of the fungal culture was suspended in H 2 O-EtOH (4:1) and partitioned successively with hexane, EtOAc, and n-BuOH. The EtOAc fraction was subjected to repeated column chromatography over silica gel and then separated by normal and reverse phase HPLC to yield individual compounds 1-4 as white solids ( Figure 1).
The molecular formula of compound 1 was determined as C 13 H 16 N 2 O 2 S 2 from a HRESIMS peak at m/z 319.0551 [M þ Na] þ which was in accordance with 13 C NMR data. A close inspection of the 1 H and 13 C NMR data (Supplementary material,  (d C 130.5, 2 C, 127.8, 2 C, 126.8, d H 7.26, 2 H, 7.23, 3H), and sp 3 -methine (d C 57.9, d H 4.52). The remaining functional groups corresponding to the carbon signals at d C 165.0 (C), 164.4 (C), 135.2 (C) and 65.3 (C) suggested the presence of two amide groups, one substituted sp 2 -carbon and one quaternary sp 3 -carbon.
A direct comparison of 1 H and 13 C NMR spectra of 1 with those of known diketopiperazine alkaloid fusaperazine A (Usami et al. 2002) showed similarities with the exception of aromatic ring signals. The molecular mass difference of 16 mass units between 1 and fusaperazine A and the chemical shifts values in the aromatic ring suggested that 1 is the dehydroxy derivative of fusaperazine A. Compound 1 was named citriperazine A.
The absolute configuration of known fusaperazine A was reported earlier as 3S, 6S (Usami et al. 2002). The similarity of NMR data of 1 and fusaperazine A together with optical rotation values differences (þ47.1 for 1 and À110.8 for fusaperazine A) suggested the 3 R, 6 R configurations for 1. This was proved by ECD spectroscopy using methodology described in Experimental section (Supplementary material, p. 25). First, thermodynamically most stable conformations of 1 in methanol were selected based on conformational analysis, performed at B3LYP/6-311G(d,p)_PCM level of theory ( Figure S1).
Then, the ECD spectra for each of these conformations were calculated using TDDFT approach and B3LYP/6-311G(d,p)_PCM level of theory. The rotatory strengths of 65 lowest electronic transitions were calculated and accounted for. The bandwidth was chosen to be D ¼ 0.34 eV. The value of the UV shift Dk ¼ þ 3 nm was chosen to reproduce well the position of the intensive band in the UV spectrum in the longwave region (k exp ¼ 281 nm).
The comparison of the experimental and statistically averaged theoretical ECD spectra of 1 is presented on Figure S2.
The theory reproduces well all main qualitative features of the experimental ECD spectrum. Since ECD spectrum for the enantiomer of 1 must be mirror-imaged to ECD spectrum, presented on Figure S2, we could undoubtedly conclude, that the stereo configurations of chiral centers of 1 are 3 R, 6R.
The molecular formula of citriperazine B (2) was determined as C 13 H 16 N 2 O 2 S 2 , the same as 1, based on a HRESIMS and 13 C NMR analysis. The NMR data (Table S1, Figures S26-S29) for 2 were very similar to those obtained for citriperazine A (1) with maximal differences at CH-3 (d C 57.3, d H 5.02) and CH 3 -15 (d C 9.2, d H 1.13). These data together with significant 3 J H3-H4 coupling constants differences between 2 (1.7 Hz) and 1 (3.4 Hz) proposed that 2 was a C-3 epimer of 1 according to published data for fusaperazines A and B and related compounds (Usami et al. 2002). Thus, the stereoconfigurations of chiral centers of 2 are 3S, 6R.
Citriperazine C (3) was isolated as colorless amorphous solid. The molecular formula of 3 was determined to be C 19 H 18 N 2 O 5 S from a HRESIMS peak at m/z 409.0832 [M þ Na] þ which was supported by the 13 C NMR spectrum.
The molecular formula of citriperazine D (4) was determined as C 18 H 16 N 2 O 6 by a HRESIMS peak at m/z 355.0936 [M-H]and by 13 C NMR analysis. The general features of the 1 H and 13 C NMR spectra (Table S2, Figures S36-S42) of 4 resembled those of 3 with the lacking of S-methyl signals and chemical shifts differences at C-2 and its close surrounding (2-NH, C-1 and C-3). The HMBC correlations from both H-3 (d H 3.41, 2.91) to C-1 (d C 167.1), C-2 (d C 81.9), C-4 (d C 134.7) and C-5/9 (d C 130.4), from 2-NH (d H 9.13) to C-1 and C-2 0 (d C 90.8), and from 2 0 -NH (d H 8.00) to C-2 and C-1 0 (d C 164.8)  were identical with those of 3 and suggested the planar structure of 4 as 2hydroxy-2-dethiomethyl derivative of citriperazine (3).
All possible combinations of three stereocenters were investigated in silico, and the obtained results were compared with the experimental data. The total conformational analysis was carried out using B3LYP/6-31G(d)_PCM, B3LYP/6-311þþG(d,p)_PCM and B3LYP/cc-pVTZ_PCM methods and following calculations of ECD spectra for individual conformers of all possible stereoisomers of 4 were performed using TDDFT approach at the same levels of theory (Supplementary material, pages 10-22). The data for RRR stereoisomer are presented on Figure S11.
Furthermore, the modelling of real solvation shells for RRR stereoisomer was performed using B3LYP/6-311þþG(d,p)_PCM method. As a result the qualitative agreement in the short wave region (200 nm < k < 240 nm) between calculated and experimental ECD spectra was achieved.
Additionally the optical rotation values ([a] D 25 ) were calculated for all stereoisomers ( Thus, the obtained data undoubtedly confirmed the 2R2 0 R3 0 R absolute configurations for 4. Based on similar optical rotation values and CD data (Figures S17, S18) of citriperazines ([a] D 20 À59.6) and D ([a] D 20 À65.6), we suggested the identical absolute configurations for 3 and 4. The effect of the compounds 1-4 on viability and apoptosis induction of human prostate cancer cells was investigated. All isolated compounds did not exhibit cytotoxic activity against human prostate cancer cells at the concentrations up to 100 mM. No significant effect on cell cycle progression was observed for any compound at the concentrations up to 100 mM.

Conclusion
A spirobenzofurandiketopiperazine scaffold is rare for natural compounds and was described only for three metabolites of Aspergillus spp (Berg et al. 1976;Sakata et al. 1987;Guo et al. 2013). Two more compounds with this moiety were isolated as artificial products formed by cyclization of hydroxylated bis-N-norgliovictin from Aspergillus fumigatus (Forseth et al. 2011). Finally, recent studies described similar moiety in the metabolites of sponge-derived Penicillium adametzioides (Liu et al. 2015) and mangrove-derived P. brocae (Meng et al. 2016). To the best of our knowledge, this report is the third case of isolation spirobenzofurandiketopiperazine alkaloids from Penicillium species.