Penicibrocazines A–E, Five New Sulfide Diketopiperazines from the Marine-Derived Endophytic Fungus Penicillium brocae

Five new sulfide diketopiperazine derivatives, namely, penicibrocazines A–E (1–5), along with a known congener (6), were isolated and identified from the culture extract of Penicillium brocae MA-231, an endophytic fungus obtained from the fresh tissue of the marine mangrove plant Avicennia marina. The structures of these compounds were elucidated by detailed interpretation of NMR and mass spectroscopic data and the structures of compounds 1 and 3 were confirmed by single-crystal X-ray diffraction analysis. All these compounds were examined for cytotoxic and antimicrobial activities. Compounds 2–6 exhibited antimicrobial activity against some of the tested strains with MIC values ranging from 0.25 to 64 μg/mL.


Introduction
Thiodiketopiperazine derivatives, which generally possess a 6-5-6-5-6 diketopiperazine skeleton with a disulfide bridge or S-methyl group(s), have attracted considerable attention due to their diversified OPEN ACCESS structures and potent biological activities [1][2][3].As part of our recently initiated program aimed at the discovery of bioactive secondary metabolites from marine-derived fungi, a series of structurally interesting and biologically active natural products has been described [4][5][6][7][8][9].Very recently, six new cytotoxic bisthiodiketopiperazine derivatives, brocazines A-F, have been isolated from the culture extract of Penicillium brocae MA-231, an endophytic fungus obtained from the fresh tissue of the marine mangrove plant Avicennia marina [10].Further work on the remaining fractions of the fungus resulted in the isolation of five new sulfide diketopiperazine derivatives, namely, penicibrocazines A-E (1-5) and one known analog (6) (Figure 1).The structures of these compounds were determined by detailed analysis of the NMR and mass spectrometric data and compounds 1 and 3 were confirmed by single-crystal X-ray diffraction analysis.All these compounds were examined for cytotoxic and antimicrobial activities.Details of the isolation, structure elucidation, and biological activity of compounds 1-6 are reported herein.

Structure Elucidation of the New Compounds
Compound 1 was obtained as colorless crystals.Its molecular formula was determined as C19H24N2O6S on the basis of HRESIMS, implying nine degrees of unsaturation.Detailed analyses of the 1 H and 13 C NMR data (Tables 1 and 2, Supplementary Figures S1 and S2) indicated the presence of one methyl, six methylenes, seven sp 3 methines, and five quaternary carbons (including two ketones and two ester/amide carbonyl carbons).The NMR data of 1 revealed some structural similarities to epicoccin G, a disulfide diketopiperazine isolated from the fungus Epicoccum nigrum [2,11].However, the signal for a quaternary carbon resonating at δC 71.6 (C-2') in epicoccin G was absent in the 13 C NMR spectrum of 1. Instead, resonances for a methine group at δH 4.57 (H-2') and δC 59.6 (C-2') were observed in the NMR spectra of 1 (Tables 1 and 2).Moreover, signals for one of the two S-methyl groups (δH 1.90/δC 14.2, CH3) in epicoccin G [2] was absent in the NMR spectra of 1, indicating the lack of a S-methyl group in 1.This deduction was confirmed by the 1 H-1 H COSY correlations from H2-3' to H-2' and H-4' as well as by the observed HMBC correlations from H-2' and H-3' to C-1' and from H-4' to C-2', as shown in Figure 2.
Table 1. 1 H NMR (500 MHz) data of compounds 1-5 (δ in ppm, J in Hz).The relative configuration of compound 1 was determined by analysis of its 1 H-1 H coupling constants and NOESY experiment.The coupling constants for H-4 and H-9 (JH-4/H-9 = 8.0 Hz) as well as for H-4' and H-9' (JH-4'/H-9' = 12.8 Hz), revealed the cis-relationship between H-4 and H-9 and trans-relationship of H-4' and H-9'.The NOE correlations from SCH3-2 to H-8, H-2', and H-8' as well as from H-8' to H-2' and H-4' indicated the same orientation of these groups (Figure 3).An X-ray crystallographic experiment (Figure 4) confirmed the structure and relative configuration of 1.The Cu/Kα radiation allowed the assignment of the absolute configuration of all the stereogenic centers in 1 as 2R, 4R, 8S, 9S, 2'S, 4'S, 8'S, and 9'S.The Electronic Circular Dichroism (ECD) spectrum of 1 showed negative Cotton Effect (CE) at 254 nm and positive CE at 218 nm (Supplementary Information).The negative CE around 254 nm is indicative for the 2R configuration of TDKPs [12].Based on the above data, the structure of 1 was determined and it was named as penicibrocazine A.
Compound 2 was obtained as yellowish solid.The HRESIMS experiment established its molecular formula C19H22N2O5S (10 degrees of unsaturation).The 1 H and 13 C NMR chemical shifts for the left portion of 2 were identical to those of 6 (phomazine B) [13], whereas the right portion closely matched to that of 1. Detailed analysis of the 1 H-1 H COSY and HMBC correlations (Figure 2) confirmed the planar structure of 2. The relative configuration of compound 2 was determined by analysis of the NOESY spectrum.The NOE correlation from 8-OH to H-9 indicated the opposite orientation of H-8 and H-9, whereas correlations from 8'-OH to H-4' and H-9' and from 2-SMe to H-8, H-2' and H-9' indicated the same face of these groups (Figure 3).The absolute configuration of 2 was determined by comparison of its CD spectrum with that of phomazine B [13].Three chromophoric groups including the S-methyl group, skewed diene, and an allylic hydroxy group contributed to the long-wavelength band around 270 nm [12].Compound 2 showed similar CEs to those of phomazine B, indicating that the absolute configuration of the left portion at C-2, C-8, and C-9 of 2 are the same as those of phomazine B. These data in conjunction with chemical shifts and NOESY correlations, as compared with those of 1 from C-1' to C-9', allowed the assignment of the absolute configuration of all the stereogenic centers in 2 as 2R, 8S, 9S, 2'S, 4'S, 8'R, and 9'R.The structure of 2 was thus elucidated and it was named as penicibrocazine B.   Penicibrocazine C (3) was obtained as colorless crystals and its molecular formula was determined as C20H26N2O6S2 (nine degrees of unsaturation) on the basis of HRESIMS data.Its 1 H and 13 C NMR spectroscopic data (Tables 1 and 2) revealed the presence of one methyl, one methylene, six methines (with two olefinic and three oxygenated/nitrogenated) and two quaternary carbons (with one amide carbonyl).These data only accounted for half of the elemental composition, implying a symmetrical feature for 3.The structure of 3 was deduced from exhaustive analyses of the 1 H-1 H COSY and HMBC spectra.In the 1 H-1 H COSY spectrum, correlations from H-3 to H-4, from H-4 to H-5 and H-9, from H-8 to H-9, and from H-6 to H-7 were observed, while in the HMBC spectrum, a full set of all possible 2 J and 3 J correlations, such as from H-3 to C-1, C-2, C-5, and C-9, from H-6 to C-4 and C-8, from H-7 to C-5, and from 5-OH to C-5, allowed for construction of the C-1 through C-9 fragment.The chemical shift of the methyl group (δH/δC 2.15/14.3)and the HMBC cross-peak from the methyl protons to C-2 indicated that the methyl group was connected to the sulfur atom attached to C-2.These data in conjunction with symmetrical feature indicated the planar structure of 3 as shown in Figure 1.
The relative configuration of compound 3 was determined by analysis of its 1 H-1 H coupling constants and NOESY data.The large coupling constants between H-4 and H-9 (J = 11.9Hz) revealed their trans relationship.NOE correlations from H-9 to OH-8 and H-5 in the NOESY spectrum indicated the cofacial orientation of these groups, while the observed correlations from H-8 to H-4 and 2-SMe placed these groups on the opposite face.An X-ray crystallographic experiment (Figure 4) confirmed the structure and relative configuration of 3 as depicted.The presence of sulfur in the molecule and the Flack parameter 0.02(9) allowed the assignment of the absolute configuration of all the stereogenic centers as 2R, 4S, 5S, 8S, and 9S.
Penicibrocazine D (4) was isolated as colorless solid.Its molecular formula was determined as C20H26N2O6S2 by HRESIMS data, having one SCH2 unit more than that of 1. Detailed comparison of the NMR data of 4 with those of 1 suggested that compound 4 had the same basic 6-5-6-5-6 diketopiperazine skeleton as 1.The NMR spectroscopic data (Tables 1 and 2) differed from those of 1 mainly in the absence of the methine signal at δH 4.57 and δC 59.6 (C-2').Instead, one S-methyl signal at δH 2.07 and δC 14.3 (2'-SMe) and one quaternary carbon signal at δC 72.7 (C-2') were observed in the NMR spectra of 4. The substitution of a S-methyl group at C-2' of 4 was evidenced by the HMBC correlation from 2'-SMe to C-2'.
The relative configuration of 4 was assigned by J-coupling constants and NOESY spectrum.The NOE correlations from 2-SMe to H-4 and H-9 and from H-8 to H-4 as well as the coupling patterns for H-4 (J = 8.0 Hz) and H-9 (J = 8.0 Hz) indicated the cofacial relationship of these groups.The large coupling constant between H-8' and H-9' (J = 13.2Hz) revealed their trans relationship.The J coupling constant between H-9' and H-4' (J = 8.8 Hz) and NOE correlations from H-9' to H-4' and OH-8' indicated the cofacial orientation of these protons, while the correlation from H-8' to 2'-SMe placed these protons on the opposite face.As the CD spectrum of 4 exhibited negative CEs at approximately 259 nm, the absolute configuration of the α/α' chiral centers were determined to be 2R/2'R [12].Therefore, the absolute configuration of compound 4 was assigned as 2R, 4S, 8S, 9R, 2'R, 4'R, 8'S, and 9'S.
Penicibrocazine E (5) had the molecular formula C20H24N2O6S2 as determined by HRESIMS data, with two hydrogen atoms less than 4. Detailed interpretation of the NMR data indicated that the 1 H and 13 C NMR chemical shifts for the left portion of 5 were nearly identical to those of 4. For the right portion, the primary difference in the NMR spectroscopic data was that the signals for two alphatic methylenes at δC 36.7/δH2.59 and 2.27 (CH2-6') and at δC 33.7/δH 1.58 and 2.27 (CH2-7') in 4 disappear in the NMR spectra of 5. Instead, two olefinic methine signals at δC 129.1/δH 6.01 (CH-6') and δC 152.6/δH 6.86 (CH-7') were observed in the NMR spectra of 5.These observations were supported by relevant 1 H-1 H COSY and HMBC correlations (Figure 2).Furthermore, the resonance for C-5' was more upfield (−10.4 ppm) in the 13 C NMR spectrum of 5, due to the incorporation of the conjugated double bond at C-6'.
The relative configuration of 5 was also determined by analysis of J-coupling constants and the NOESY spectrum.The coupling patterns for the relevant protons on the left portion of 5 were similar to their counterparts of 4, indicating that the left motif of 5 possessed the same relative configuration as that of 4. The large coupling constant between H-4' and H-9' (J = 13.0Hz) revealed their trans relationship.NOE correlations from H-9' to 8'-OH and 2'-SMe placed these protons on the same face of the molecule, while the NOE correlation from H-8' to H-4' placed these protons on the opposite face.As might be expected, the CD spectrum of 5 exhibited negative CEs at approximately 260 nm, similar as that of 4, indicating the absolute configuration of 5 to be 2R, 4S, 8S, 9R, 2'R, 4'R, 8'R, and 9'R.

Fungal Material
The fungus Penicillium brocae MA-231 was isolated from the fresh tissue of the marine mangrove plant Avicennia marina.The fungus was identified by sequence analysis of the ITS region of its rDNA as described previously [10,14].The resulting sequence data obtained from the fungal strain have been deposited in GenBank (with accession no KM191342).A BLAST search result indicated that the sequence was most similar (99%) to the sequence of Penicillium brocae (compared to AF484393).The strain is preserved at the China General Microbiological Culture Collection Center.

Cytotoxicity Assay
The cytotoxic activity of the isolated compounds against nine tumor cell lines including Du145 (human carcinoma of prostate cell line), HeLa (human cervix carcinoma cell line), HepG2 (human liver hepatocellular cells), MCF-7 (human breast carcinoma cell line), NCI-H460 (human large cell lung carcinoma cell line), SGC-7901 (human gastric carcinoma cell line), SW1990 (human pancreatic cancer cell line), SW480 (human colon carcinoma cancer), and U251 (human glioma cells) were determined according to previously reported methods [19].

Antimicrobial Assay
Antimicrobial assay against human-and aqua-pathogenic microbes Aeromonas hydrophilia, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, Vibrio arveyi, V. parahaemolyticus and plant pathogenic fungi Alternaria brassicae, Colletotrichum gloeosporioides, Fusarium graminearum, and Gaeumannomyces graminis was carried out using the well diffusion method [20].Chloromycetin was used as a positive control for the bacteria, while amphotericin B was used as a positive control for the fungi.

Conclusions
Five new thiodiketopiperazine derivatives (1-5), along with one known analog (6), were isolated from the marine mangrove-derived endophytic fungus P. brocae MA-231.The structures and relative configurations were determined on the interpretation of the NMR data and the absolute configurations of all compounds were determined by CD comparison, while the structures of compounds 1 and 3 were confirmed by single-crystal X-ray diffraction analysis.Compounds 2-6 exhibited activity against some of the tested microbial strains.

Figure 1 .
Figure 1.Structures of the isolated compounds 1-6 and reference compound epicoccin G.