Ent-homocyclopiamine B, a Prenylated Indole Alkaloid of Biogenetic Interest from the Endophytic Fungus Penicillium concentricum

Ent-homocyclopiamine B (1), a new prenylated indole alkaloid bearing an alicyclic nitro group along with 2-methylbutane-1,2,4-triol (2) were isolated from an endophytic fungus Penicillium concentricum of the liverwort Trichocolea tomentella (Trichocoleaceae). The structure of 1 was elucidated through extensive spectroscopic analyses and comparison with data reported for a structurally related nitro-bearing Penicillium metabolite, clopiamine C (3), which contain an indolizidine ring instead of the quinolizine ring in 1. The new compound, ent-homocyclopiamine B, exhibited slight growth inhibition against Gram-positive bacteria. Based on the reported biosynthesis of related compounds and the isolation of the mevalonic acid derived compound 2-methyl-1,2,4-butanetriol (2), we proposed that ent-homocylopiamine B (1) was biosynthesized from lysine and prenyl group-producing mevalonic pathway.


Introduction
Endophytes are microorganisms (bacteria and fungi) that live within plants without causing apparent pathogenic disease symptoms. Although researchers are seeking to understand the roles of most endophytes in planta, endophytes are believed to protect plant hosts by producing biologically active molecules, reported to secure growth by biosynthesizing plant hormones (e.g., gibberellins), and to balance plant microbial community by counteracting activities of pathogens. It is obvious that plants acquire their endophytes from the environment and thus there should not be any difference between endophytic and non-endophytic microbes of the same species. The only dissimilarity is undoubtedly the surrounding environment where they adapt and acquire starting materials for the biosynthesis of their bioactive metabolites that they use for symbiosis. This adaptation makes endophytes capable to produce wide range of compounds when isolated from the host and fermented using various media. Compounds exhibiting antimicrobial, antiviral and antiproliferative activity have been reported from endophytes [1]. We recently described chemical and pharmacological investigations of extracts prepared from a Penicillium (Trichocomaceae) fungus endophytic to the liverwort Trichocolea tomentella (Trichocoleaceae) that led to the isolation of the antifungal griseofulvin, dehydrogriseofulvin, dechlorogriseofulvin, dehydrodechlorogriseofulvin, griseophenones B and C, griseoxanthones, driniopsin H, norlichexanthonealternariol, ethylene glycol benzoate, 6-chloro-3,8-dihydroxy-1-methylxanthone, epi-epoxydon, gentisyl alcohol, gentisyl quinone, hydroxychlorogentisyl quinone, chlorogentisyl quinone, bromogentisyl alcohol and the identification of compounds exhibiting antiproliferative activity against hormone-dependent breast cancer cell line (MCF-7) and the human colorectal adenocarcinoma cell line HT-29. In the continuation of our systematic studies aiming to understand the metabolic profile of the same endophytic strain and to isolate new and bioactive compounds, we prepared an extract of its fermentation in brown rice supplemented with sodium fluoroacetate and isolated 2-methylbutane-1,2,3-triol (2) [2] and ent-homocyclopiamine B (1), a nitro containing alicyclic alkaloid. The isolation and the structure elucidation of the new compound, the effect of the supplementation of sodium monofluoroacetate to the media, the biosynthetic pathway of (1) as well as the results of its antimicrobial evaluation will be discussed herein.

Results
Compound 1 was obtained as white powder and its positive HRESIMS displayed a protonated molecular ion peak at m/z 482.  respectively were concluded to be isolated. The allocation of all functionalities present in 1 was carried out by comparison of its spectroscopic data with those of clopiamine C (3) as well as interpretation of its HMBC spectroscopic data. The presence of a 2,2,-dimethyl-2,3-dihydroquinolin-4(1H)-one was confirmed by the HMBC long-range correlations from H-25 and H-26 to C-4 and C-5, H-5 to C-6 and C-7, OCH 3 to C-8, the orthocoupled methine proton at δ.65 (H-9) to C-7, C-8, and C-11. The presence of 8, (3) was substantiated by the above-mentioned spin network deduced from the COSY spectrum and the HMBC correlations from H-28/29 to C-1, C-13 and C-14, from H-20 to C-22, from H-22 to C-24, C-14, and C-23. The above data together with the HMBC correlation observed from H-10 to the quaternary carbon at δ 62.3 (C-1) and the presence of 12 degree of unsaturation in 1 allowed us to conclude that as in 3, the structure of 1 must also contain a spiro-oxindole moiety.
The relative configurations at C-1, C-14, C-16 and C-23 of 1 were determined by NOE experiment while the absolute configurations of 1 were established by comparison of its optical rotation and CD spectroscopic data with those of 3, the absolute stereostructure of which was deduced by NMR, CD and X-ray diffraction crystallography analyses. The CD spectrum of 1 which displayed negative and positive Cotton effects [(∆ε 356 − 0.92) and (∆ε 242 − 6.08), and (∆ε 265 + 2.82) and (∆ε 229 + 2.8), respectively) is superimposable with those of 3 [3].
Clopiamine C (3) belongs to the rare alkyl nitro group-bearing prenylated spirooxindole alkaloids of Penicillium species. Of this group of compounds, only cyclopiamine and citranilin-type have been reported (Figure 1) [3][4][5][6]. It is worthy to note that clopiamine C (3) and cyclopiamine B (Figure 1) are enantiomers. Their NMR spectroscopic data (although measured in different solvent) are superimposable while their reported optical rotations ([α] D ) have opposite sign (−97.9 • and +117, respectively) [3,4]. From these data, the absolute configurations at C-1, C-14, C-16, and C-22 were thus determined to be S, R, S, and R, respectively and the structure of 1 was deduced to be ent-homocyclopiamine B as depicted in Figure 2.     The isolation of 1 from Penicillium concentricum adds to the list of Penicillium strains that produce cyclopiamines. To determine if supplementation of sodium monofluoroacetate to the rice medium affects production of 1, an ethyl acetate extract of P. concentricum grown on brown rice medium and an ethyl acetate extract of the fungus fermented on brown rice supplemented with sodium monofluoroacetate were analyzed using LC-MS. Ent-homocyclopiamine B and its derivatives have been detected in the LCMS spectra of both extracts showing that the supplementation did not influence the production of compound 1. This was confirmed by positive ion electrospray LC-MS screening of the two extracts, which both displayed the peak at m/z 482.2650 (compound 1). Additionally, LC-MS analyses ( Figure S11) of the extracts also showed a peak corresponding to the characteristic loss of a nitro-radical (m/z 435.2642) from the molecular ion of 1 [3].
The possible of biosynthesis of citranilins and cyclopiamines have been studied using stable isotope ( 13 C) labelling experiments to conclude glucose, anthranilic acid and ornithine as proposed precursors [6]. The present investigation led to the isolation of mevalonic acid derived compound 2-methylbutane-1,2,3-triol (2), suggesting that the prenyl group in 1 may be introduced from the catabolism of glucose through mevalonic pathway. Lysine instead ornithine was may be involved in the biosynthesis of the indolizidine component of 1, analogous to ornithine in clopiamine C. The clear observation of three units of isoprene and a lysine residue in the structure of 1 suggests that further investigation is needed to understand if an aminophenol related compound (apart from the reported anthranilic acid) may be involved in its biosynthesis (Figure 3).   Next, the ethyl acetate fraction was subjected to fluorine NMR experiments ( 1 H coupled and decoupled experiments). As results, the presence of fluorinated compounds was clearly observed in the two experiments performed (Data not shown). However, fluorinated compounds were not isolated during this study due to their apparent instability of during the isolation process.

Antibacterial Activity of Ent-homocyclopiamine B
Prenylated indole alkaloids have been reported to wide-ranging bioactivities including antibacterial, cytotoxic, and insecticidal properties. The antibacterial activity of 1 was tested against a panel of Gram-positive and -negative strains. Although 1 inhibited growth of Bacillus subtilis ATCC 6633 and Mycobacterium smegmatis NRRL B-14646 on agar plates, zones were significantly smaller than from an equivalent amount of kanamycin ( Figure S11). Compound 1 showed no activity against Escherichia coli K12, Salmonella LT2, Micrococcus luteus ATCC 4698, Pseudomonas putida PRS2000, and Serratia marcescens NRRL B-2544 in this assay.
B. subtilis ATCC 6633, Rhodococcus jhostii RHA1, and Corynebacterium glutamicum NRRL B-2784 were partially inhibited in microbroth dilution assays with 100 µM of 1 (30% inhibition compared with background controls), but not with any concentrations below. No detectable activity was observed against any of the other tested strains.
In summary, inhibition was observed against some (but not all) of the tested Gram-positive strains. None of the tested Gram-negative bacteria were susceptible to 1.

Materials and Methods
1 H and 13 C-NMR spectra were recorded at 25 • C with a Bruker Avance III 400 HD NMR spectrometer and Bruker Avance III HD Ascend 700 MHz. High-resolution mass spectra were acquired with a Thermo LTQ Orbitrap, specifications; analyzer: ITMS and FTMS, mass range: 50-4000 m/z, resolution: 7500-100,000. Optical rotations were determined on an Anton Paar MCP 150 polarimeter. Ultraviolet spectra (UV) were recorded using a Hitachi U-2910 UV/Vis double-beam spectrophotometer (Hitachi High-Technologies America, Schaumburg, IL, USA). Circular dichroism was measured on a Jasco J-810 spectropolarimeter. Thermo LTQ Orbitrap and Agilent 1100 HPLC were used to measure LC-MS spectra.

Fungal Source
Penicillium concentricum, the endophytic fungus used in this study was isolated from healthy liverwort Trichocolea tomentella and identified as previously described [1].

LC-MS Method
LC-MS experiment was performed using Agilent 1100 HPLC binary and Thermo LTQ Orbitrap Mass spectrometer. For the HPLC, Beckman ODS column (5 µm, 4.6 mm, 25 cm, Part#235329) heated at 25 • C (oven temperature). Sample was injected to a water (containing 0.1% formate)-Acetonitrile (containing 0.1% formate) gradient from 5% to 95% (v/v) over 20 min with a 5 min hold at 95% (v/v) for 5 min. The column was then reequilibrated to 5% (v/v) for 3 min. The flow rate was kept at 1 mL/min. Mass spectrometry aquisitions were performed using instrument settings as described in SI.
Growth inhibition was evaluated on plates inoculated with lawns of the test strains. 50 µL of an overnight culture of bacteria was added to 5 mL of broth and grown until the culture reached a turbidity equal to 1 × 10 7 -2 × 10 8 CFU/mL (based on a previously determined calibration curve). 100 µL was then evenly spread onto plates using sterile glass beads to dryness. 50 nmoles ent-homocyclopiamine B (dissolved in 5 µL 25% DMSO), 50 nmoles kanamycin (dissolved in 5 µL water), or diluent controls (5 µL of sterile water or 5 µL 25% DMSO) were applied, allowed to dry, and then incubated for 16-20 h at the growth temperature appropriate for each strain.
The broth microdilution assay was performed as outlined by the Clinical and Laboratory Standards Institute (CLSI) [7]. Briefly, 2 µL of a 50X stock of ent-homocyclopiamine B (dissolved in 25% DMSO) was added to wells of a 96-well plate. Live and background controls received 2 µL of diluent. 88 µL of broth was added to all wells except background control wells which received 98 µL. 50 µL of an overnight culture of bacteria was added to 5 mL of broth and grown until the culture reached a turbidity equal to 1 × 10 7 -2 × 10 8 CFU/mL (based on a previously determined calibration curve). The culture was diluted to 5 × 10 6 CFU/mL and 10 µL added to each well except background controls to a final concentration of 5 × 10 5 CFU/mL. Plates were incubated at 30 • C or 37 • C (as appropriate for each strain) for 16-20 h. Absorbance was then recorded on a Bio-Rad xMark microplate spectrophotometer at λ = 600nm. Minimal inhibitory concentration (MIC) values were defined as the lowest concentration resulting in ≥ 90% growth inhibition.