Auranthine, a Benzodiazepinone from Penicillium aurantiogriseum

Article Cite This: J. Nat. Prod. 2018, 81, 2177−2186

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Auranthine, a Benzodiazepinone from Penicillium aurantiogriseum: Refined Structure, Absolute Configuration, and Cytotoxicity Svetlana A. Kalinina,†,‡,# Dmitrii V. Kalinin,§,⊥,# Yannick Hövelmann,† Constantin G. Daniliuc,∥ Christian Mück-Lichtenfeld,∥,¶ Benedikt Cramer,† and Hans-Ulrich Humpf*,†,‡ †

Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Straße 10, 48149 Münster, Germany § Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany ⊥ Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany ∥ Organisch-Chemisches Institut, Westfälische Wilhelms-Universität Münster, Corrensstraße 40, 48149 Münster, Germany ¶ Center for Multiscale Theory and Computation, Westfälische Wilhelms-Universität Münster, Corrensstraße 40, 48149 Münster, Germany

J. Nat. Prod. 2018.81:2177-2186. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/26/18. For personal use only.

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S Supporting Information *

ABSTRACT: The structure of the known Penicillium aurantiogriseum-derived secondary metabolite auranthine was refined using a combination of synthetic, spectroscopic, and X-ray diffractometric approaches. Thus, auranthine was shown to be a fused quinazolino benzodiazepinedione (2) bearing an acyclic aliphatic nitrile moiety, thereby significantly differing from the originally proposed structure 1 published in 1986. Its absolute configuration was confirmed by CD spectroscopy and DFT calculations. The cultivation of P. aurantiogriseum was optimized, allowing high production of auranthine. The cytotoxicity profile of auranthine and its semisynthetic analogues is reported. The refined structure of auranthine provides a valid target for the total synthesis of this underexplored natural product and its derivatives.

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Until now the configuration of the asymmetric center of auranthine remains unknown. In this respect, to elucidate the full structure of the molecule and also to gain facile access to this poorly investigated toxin, several synthetic approaches were undertaken toward its total synthesis. In 2002, Witt et al. completed the synthesis of a C-acetyl derivative of auranthine (3), although the original structure of auranthine (1) could not be synthesized in the course of this study.3 Subsequently, Argade and co-workers yielded a racemic mixture of auranthine (1) starting from protected glutamic anhydride and 2aminobenzylamine over seven synthetic steps in 2010.4 Its structure was unambiguously confirmed by 1H NMR as well as 13 C NMR spectroscopy and crucially reinforced by X-ray crystallography. However, the 1H NMR spectrum of the synthetically obtained auranthine (1) was not in agreement with the original structure 1, which was previously assigned and reported in 1986.1,4 Moreover, differences in the solubility

uranthine (1) is a known fungal secondary metabolite that was isolated in 1986 by Mantle and co-workers from Penicillium aurantiogriseum.1 Its name originates from the producing fungus and two proposed precursors from the biosynthetic pathway: anthranilic acid and glutamine. Based on the spectral characteristics and biosynthetic evidence, the structure of auranthine was assigned in favor of a rather unusual quinazoline-based alkaloid possessing a diazepine moiety (1). When its structure was first disclosed, all the reported spectroscopic characteristics appeared to be in agreement with the proposed structure 1 apart from an uncertainty in its fragmentation spectrum.1 The particular mechanism of the molecule breakdown, especially with resect to losses of C3H3N and CH2N, remain unclear. Subsequently, to gain insights into the biosynthetic pathway of auranthine, 14 C-labeled anthranilic acid, glutamine, glutamate, and ornithine were administered to the liquid cultures of P. aurantiogriseum. Based on the results of the aforementioned study it was concluded that auranthine is biosynthesized by condensation of two molecules of anthranilic acid with one molecule of glutamine.2 © 2018 American Chemical Society and American Society of Pharmacognosy

Received: March 1, 2018 Published: October 8, 2018 2177

DOI: 10.1021/acs.jnatprod.8b00187 J. Nat. Prod. 2018, 81, 2177−2186

Journal of Natural Products

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(Figure 1): colony character, diameter, and fungal growth as well as in auranthine production (Supporting Information). Notably, we observed red-colored excretions on P. aurantiogriseum colonies when a high amount of auranthine was produced (Figure 1), which also correlated with the supplementation of the media with certain amino acids. Particularly, being cultivated on media with elevated concentrations of glutamine and glutamate, P. aurantiogriseum showed more intense growth (Supporting Information) as well as a rise in auranthine biosynthesis. Compared to Czapek-Dox agar (CDA) alone, glutamine supplementation (100 mM) led to a nearly 5-fold increase in auranthine production (Figure 2). The observed exceptional role of glutamine and glutamate in auranthine production is in agreement with the previously reported study on its biosynthesis, showing a direct incorporation of these amino acids into the molecule.2 Consequently, for further experiments, auranthine was isolated from P. aurantiogriseum cultivated on modified Czapek−Dox agar supplemented with 100 mM glutamine, and the pH was adjusted to 6 (optimal pH, Supporting Information). This approach allowed us to isolate auranthine on a larger scale (63 mg). Moreover, along with auranthine, another known P. aurantiogriseum-born secondary metabolite, namely, aurantiamine5 (7), was also isolated. Synthesis of Auranthine Derivatives. In order to elucidate the full configuration of auranthine, multiple efforts on its crystallization were undertaken. However, natural auranthine failed to produce diffractable crystals. Therefore, the decision was made to follow a synthetic approach toward the production of semisynthetic analogues of auranthine exhibiting altered physicochemical properties allowing their crystallization. As initially the structure of auranthine was considered to be structure 1,1 we proposed to reduce its cyclic amidine groups to afford derivatives possessing two secondary amino groups. Considering structure 1, theoretically, this type of reduction could lead to the formation of two additional centers of chirality in the molecule, resulting in a mixture of diastereomers. However, our first attempt to treat natural auranthine with a mild reducing agent (NaBH3CN) afforded only one product (8) in 66% yield (Scheme 1). The exact mass of 8 was 333.1377 Da (for [M + H]+), indicating the reduction of only one π-bond, as the exact mass of auranthine is 331.1195 Da (for [M + H]+). Moreover, NaBH3CN-promoted reduction was found to be diastereoselective, yielding only one (S,S)-configured stereoisomer, as evidenced by spectroscopic data, HPLC purity, and X-ray diffractometry. Subsequently, to afford the N-methylated semisynthetic analogue of auranthine, the product of its reduction (8) was treated with a methylating agent (CH3I) under basic conditions, thus regioselectively producing (S,S)-configured N-methyl auranthine (9) in a yield of 61% (Scheme 1). Thus, exclusively methylation of the amide nitrogen was observed, as subsequently evidenced by HMBC spectral data of 9, where its CH3 protons CH3-23 showed correlation with the C-11 and C-16 carbon atoms (Table 1). Structure of Auranthine and its Derivatives. X-ray Crystallography. In contrast to auranthine, its reduced semisynthetic analogue 8 was found to be suitable for the production of X-ray-diffractable crystals. Single crystals of compound 8 were obtained by the slow evaporation of its CH2Cl2−MeOH solution (95:5 volume ratio). To our surprise, according to the crystallography data, compound 8

in benzene-d6 between naturally isolated and synthesized auranthine were observed.4 The lack of knowledge of the full configuration of natural auranthine as well as the inconsistency in its analytical characteristics compared to the synthesized analogue impedes further studies toward its synthesis and bioactivity evaluation. Herein we report semisynthetic analogues of auranthine (8 and 9), the synthesis of which resulted in the unequivocal identification of a refined structure of auranthine (2) and its absolute configuration employing circular dichroism (CD), NMR, and X-ray diffractometry. Moreover, in this study an additional emphasis is placed on the cytotoxicity profile of the isolated auranthine and its semisynthetic analogues.

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RESULTS AND DISCUSSION Production and Isolation of Auranthine. A commercially available strain of P. aurantiogriseum (CBS 112021) was employed for the auranthine production. For this purpose, the fungal culture (70 Petri dishes) was grown for 3 weeks at 22 °C in the dark, followed by mycelium extraction with EtOAc and automated flash-column chromatography (reversed-phase column) to obtain auranthine. The final purification was performed using a normal-phase silica gel column. The applied method provided auranthine of high purity (≥95%, determined by HPLC-UV-ELSD). However, the isolated amount of auranthine (22 mg) was insufficient for further investigations. As our study required substantial amounts of auranthine, we optimized the cultivation conditions of P. aurantiogriseum for the quantitative production of this secondary metabolite. To that end, different abiotic factors, such as media, pH, carbon and nitrogen source, and oxidative stress were studied (Supporting Information). The cultivation of P. aurantiogriseum in various abiotic environments revealed differences in the fungal morphology 2178



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Figure 1. Morphology of P. aurantiogriseum colonies after 7 days of incubation at 22 °C in the dark on modified Czapek−Dox agar (CDA).

Figure 2. Production of auranthine (mg/g of dry mass) after 21 days by P. aurantiogriseum cultured on CDA and modified CDA supplemented with different concentrations (10 and 100 mM) of amino acids. Results are displayed as a mean of five replicates ± SD and given in mg of auranthine related to dry mass (mg/g).

Scheme 1. Synthesis of Auranthine Analogues 8 and 9

was shown to be a fused quinazolino benzodiazepinedione possessing an acyclic aliphatic nitrile moiety, thereby exhibiting a significantly different structure from the formula 1 proposed.1 The absolute configuration of compound 8 was assigned as 2S, 11S and is shown in Figure 3. Detailed crystallographic data are presented in the Supporting Information. The X-ray crystallography data of auranthine derivative 8 raised the question about the original structure of auranthine. It was concluded that it is unlikely to obtain derivative 8 via treatment of the original structure of auranthine 1 with a reducing agent such as NaBH3CN. Therefore, the structure of auranthine needs to be revised. NMR Spectroscopy. The structure of natural auranthine (2) as well as its semisynthetic analogues 8 and 9 were analyzed

using NMR spectroscopy; the obtained data are summarized in Table 1. In general, the 1H, 13C, and HSQC spectra of natural auranthine (2) in DMSO-d6 revealed signals of 19 carbon atoms attributed to two methylene groups, one sp3-hybridized methine group, eight aromatic methine C atoms, and C atoms bearing no hydrogens, among which two C atoms at 160.9 ppm (C-4) and 166.9 ppm (C-16) were assigned as carbonyl C atoms. Further analysis of COSY and HMBC (Table 1) data showed that the structure of auranthine could be easily misinterpreted in favor of structure 1. Thus, theoretically the signals of two methylene groups and one methine group of the piperidine moiety of structure 1 could be very similar to those of the propanenitrile moiety of structure 2. However, the X-ray data of compound 8 was fully consistent with its NMR spectra, 2179



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Table 1. NMR Data for Refined Auranthine (2) in DMSO-d6 and Its Semisynthetic Analogues 8 in DMSO-d6 and 9 in CD3OD

known fungal-born quinazolino benzodiazepinediones such as asperlicin (4) and sclerotigenin (5) isolated from A. alliaceus and P. sclerotigenum, respectively7−11 as well as with auranthine’s closest analogue auranomide C (6), recently isolated from P. aurantiogriseum.12 Also, similarly to what was reported by Argade and co-workers,4 our recorded NMR spectra for natural auranthine (2) were not in agreement with published NMR spectra of synthetically obtained 1. IR Spectroscopy. Apart from the absorption bands typical for sp2 C−H, CO, and N−H bond stretching, IR spectra of auranthine (2) as well as its semisynthetic analogues 8 and 9 showed a sharp absorption band at 2249 cm−1 of medium intensity, which is characteristic for CN stretching vibrations (Supporting Information). This observation serves as additional evidence in favor of the refined structure 2 of auranthine possessing a nitrile moiety. HPLC-MS/MS Analysis of Auranthine. To the best of our knowledge, there is a lack of data concerning MS/MS analysis of auranthine. Therefore, its structure was further investigated based on HPLC-MS/MS using an Orbitrap mass spectrometer by application of higher-energy collisional dissociation (HCD). Data from theoretical calculations of 2-pyrrolidinone fragmented by collision-induced dissociation (CID) in ESIMS/MS instruments revealed that the N-protonated system shows a H−N−CO bond length longer than usual, confirming a heterolytic cleavage at this position opening the five-membered lactam and consequently transferring the charge to the new acylium group.13 This example supports the possible protonation of the secondary amide function at the sevenmembered lactam ring of auranthine (2), followed by heterolytic cleavage of the amide bond and resulting in the opening of the lactam ring. In this case, two characteristic neutral eliminations driven by an E2-like mechanism14 are theoretically possible starting from the ring-opened ion,

Figure 3. X-ray crystal structure of (S,S)-configured semisynthetic analogue of auranthine 8. Thermal ellipsoids are drawn at the 50% probability level.

which are similar to the NMR data of auranthine (2). Thus, auranthine appeared to be a fused quinazolino benzodiazepinedione (2) bearing an acyclic aliphatic nitrile moiety. In the 13 C NMR spectrum of 2, the carbon at 120.1 ppm (C-14) (Table 1) was assigned as a carbon of the cyano group (C N). This fact is supported by the typical chemical shifts in various aliphatic nitriles (∼120.0 ppm)6 as well as by the chemical shift of the cyano group carbon of 8 at 119.9 ppm (C14). According to HMBC data of 2, methylene protons H2-12 and H2-13 showed correlation with C-14 of the cyano group, further suggesting the presence of a propanenitrile moiety. The carbon chemical shift assignments and the character of HMBC correlations for 2 are consistent with spectral data for the other 2180



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Scheme 2. Proposed Fragmentation Pathway toward Fragment Ions [11]+, [12]+, and [13]+ Derived from MS/MS Data of Auranthine (2) Based on HCD

Scheme 3. Proposed Products of Auranthine Hydrolysis

fragments as well as the corresponding (proposed) fragmentation pathways. Hydrolysis of Auranthine. In particular cases basic and/or acidic hydrolysis of natural compounds can be a useful tool, as products of hydrolysis can be identified by their physicochemical properties, e.g., by exact mass measurement, providing thereby valuable information helping to identify the parent natural product. Apart from the hydrolysis of esters and amides, the hydrolysis of nitriles can be utilized. Thus, nitriles are known to undergo a stepwise hydrolysis under base treatment, affording the amides, which are usually contaminated with subsequently formed carboxylates.15 To further verify the presence of a nitrile moiety in the structure of auranthine, its basic hydrolysis was performed. For this, auranthine was treated with potassium hydroxide solution in ethanol under reflux conditions for 10 min, resulting in a mixture of products that were identified by HPLC-MS

namely, the loss of NH3 or the side chain bearing the proposed nitrile moiety (Scheme 2). In accordance with the latter, the MS/MS analysis of auranthine revealed the presence of both ions resulting from the described neutral eliminations (Scheme 2, Supporting Information) and thus strongly supports the proposed structure. Furthermore, an additional ion was observed at m/z 83.0598 with a very high intensity (Supporting Information). Its occurrence can be explained by the formation of a new five-membered lactam ring after the initial protonation and ring opening as described above, induced by the lone pair of the respective nitrogen atom and reaction of the acylium group (Scheme 2). In this case, the neutral elimination of the major core of the molecule is observed, yielding a diagnostic ion at m/z 83.0604 (found: m/ z 83.0598). In conclusion, the MS/MS analysis of auranthine further supports the proposed structure (2), based on the observed 2181



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Conformers with ΔG(298)solv > +1.0 kcal/mol occur with a ratio less than 5% in the equilibrium (Boltzmann distribution, pi = exp(−ΔGi/(RT))/[∑j(exp(−ΔGi/(RT))]) and have therefore been neglected. The obtained calculated CD spectrum fits very well with the experimental spectrum (Figure 4) and supports the proposed S-configuration of 2. Cytotoxicity of the Compounds. Although auranthine (2) was first isolated in 1986,1 there are no cytotoxicity data available. However, P. aurantiogriseum was previously reported to be highly toxicogenic and was suspected to be involved in Balkan Endemic Nephropathy.19,20 Therefore, evaluation of toxicity profiles of P. aurantiogriseum-born secondary metabolites is of particular interest. In order to gain insights into the cytotoxic properties of auranthine (2) and its derivatives 8 and 9, compounds were tested in human liver cancer cells (HepG2) and human kidney epithelial cells (IHKE). Additionally, cytotoxicity of coisolated aurantiamine (7) was tested. In general, auranthine (2) as well as its semisynthetic analogues 8 and 9 showed low cytotoxicity against both cell lines tested (Figure 6). However, IHKE cells were found to be slightly more sensitive toward methylated derivative 9, showing a dose-dependent decrease of cell viability in concentrations above 10 μM. Interestingly aurantiamine (7), the other isolated metabolite from P. aurantiogriseum, demonstrated a more profound cytotoxicity profile. Thus, 7 decreased the viability of HepG2 cells (up to 56% at 100 μM) in a dose-dependent manner. A higher toxicity effect, with an IC50 of 25 μM against IHKE cells, was found for aurantiamine (7) (Figure 6). In conclusion, employing synthetic, spectroscopic, and X-ray diffractometry approaches, the structure of P. aurantiogriseumborn secondary metabolite auranthine (2) was refined. The use of CD spectroscopy and DFT calculations confirmed the absolute configuration of auranthine (2). The low toxicity profile of isolated auranthine (2) and its semisynthetic analogues 8 and 9 underlines their potential use as biologically active substances. A refined structure of auranthine (2) opens a venue for the total synthesis of this natural product and its derivatives.

(Scheme 3). Performed analysis of the reaction mixture revealed three main products with distinct retention times and exact masses [M + H]+ of m/z 349.2, 350.2, and 378.2, which were proposed to be the known P. aurantiogriseum secondary metabolites auranomide C (6), auranthic acid (14), and auranthic acid ethyl ester (15), respectively (Supporting Information). This finding is in line with known nitrile hydrolysis pathways, first affording an amide followed by its conversion into a carboxylic acid, which then can be esterified to some extent, producing the ethyl ester. CD of Auranthine. It has been previously reported that natural auranthine exhibits a negative value of the optical 1 rotation [α]25 D = −164.0 (1% w/v, C2H5OH). Our experiment also demonstrated a similar value of its specific rotation, [α]20 D = −113.2 (c 1.35, CH2Cl2). However, the configuration of the asymmetric center of proposed auranthine 2 remained unverified. Chiroptical techniques such as CD, especially in combination with quantum-mechanical calculations, are commonly employed for the elucidation of chirality.16,17 Therefore, to verify the absolute configuration of auranthine (2), additional experiments employing CD spectroscopy and density functional theory (DFT) calculations were performed. For this purpose, the CD spectrum of auranthine (2) was recorded in acetonitrile. The CD spectrum of 2 showed a negative CD couplet with a negative first Cotton effect (CE) at 230 nm and a positive second CE at 210 nm resulting from the exciton chirality interaction18a between the two intramolecular chromophores of 2 (Figure 4). The obtained negative CD couplet indicates a counterclockwise orientation of the two chromophores and therefore an S-configuration.18b

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined with an MP50 melting point apparatus (Mettler Toledo, Giesen, Germany) and are uncorrected. Optical rotation, α [deg], was determined with a UniPol L1000 polarimeter (Schmidt+Haensch, Berlin, Germany), path length 1 dm, wavelength 589 nm (sodium D −1 −1 line); the unit of specific rotation [α]20 D [deg·mL·dm ·g ] is omitted; the concentrations of the sample c [mg·mL−1] and solvent used are given in brackets. UV spectra were recorded on a Jasco V750 spectrophotometer (Jasco Labor- and Datentechnik, GroßUmstadt, Germany). CD spectra were recorded on a Jasco J-600

Figure 4. Experimental and calculated CD spectra of auranthine (2).

In addition, we performed a conformational search for auranthine (2) with the semiempirical (tight binding DFT) GFN-xTB method followed by the calculation of the CD spectra. The four most stable conformers of auranthine (2) are shown in Figure 5, and the energies are given in Table 2.

Figure 5. DFT-optimized preferred conformations of auranthine (2) in acetonitrile solution. In parentheses: relative concentration (in percent) according to the Boltzmann distribution. 2182



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Table 2. Relative Energies of Conformers s1−s3 and s6 As Calculated with DFTa E(TPSS-D3)

GRRHO298

Gsolv298 (MeCN) COSMO-RS

E(PW6B95-D3)

ΔG(298)solv

conformer

[Eh]

[kcal/mol]

[kcal/mol]

[Eh]

[kcal/mol]b

s1 s2 s3 s6

−1102.604657 −1102.602973 −1102.602731 −1102.601319

155.40 155.28 155.18 155.11

−20.13 −21.02 −20.93 −21.68

−1103.782853 −1103.781302 −1103.781047 −1103.779774

0.04 0.00 0.15 0.13

All calculations were performed with the def2-TZVP basis set. bΔG(298)solv is the relative free energy with respect to conformer s2.

a

Figure 6. Cellular viability of HepG2 (A) and IHKE (B) cells after the treatment with tested compounds. n ≥ 9; * statistically significant (p ≤ 0.05); ** statistically highly significant (p ≤ 0.01); positive control T-2 toxin (10 μM). temperature of the ELSD was 40 °C, and 2.5 bar of pressurized air was used. The column oven was set to 40 °C. The gradient with MeCN (solvent A) and H2O (solvent B) of the ELSD measurement starts with 0−5 min 5% of A; 6−25 min, 5−100% A, followed by a washing step of 5 min with 100% A. HPLC-MS/MS Analysis of Auranthine. An LTQ Orbitrap XL mass spectrometer with an HESI II source (Thermo Fisher) attached to an LC system consisting of an LC-20AD XR pump with a DGU20A5R degasser, a SIL-20AC XR autosampler, and a CTO-10AS column oven and controlled by a CBM-20A controller (all Shimadzu) was used for the analysis. Electrospray ionization was applied in positive mode. The sheath gas flow, the aux gas flow, and the sweep gas flow were set to 40, 20, and 10, respectively. The vaporizer and the capillary temperature were both 350 °C. In the positive mode the vaporizer temperature was set to 350 °C. The source voltage, capillary voltage, and tube lens voltage were set to 4 kV, 20 V, and 125 V, respectively. The fragmentation was performed in the HCD cell with a normalized collision energy of 60% and an activation time of 30 ms. For the fragmentation experiments, the resolution was set to 7500. Chromatography was performed using a Nucleodur C18 gravity column (100 mm × 2 mm i.d., 3 μm, Macherey-Nagel) by application of a binary gradient consisting of MeCN (solvent A) and H2O

spectropolarimeter. IR spectra were recorded on a Prestige-21 (Shimadzu, Duisburg, Germany). A full set (H, C, gHMBC, gHSQC, and COSY) of NMR spectra were recorded on an Agilent DD2 600 MHz spectrometer and 400 MHz spectrometer (Agilent Technologies, Waldbronn, Germany); δ is in ppm related to the solvent signal. High-resolution mass spectra were obtained on an LTQ Orbitrap XL (Thermo Fisher Scientific, Bremen, Germany) in electrospray ionization mode by direct infusion of the purified compounds. The sheath gas flow is in arbitrary units; the aux gas flow in 5 arbitrary units. In the positive mode the vaporizer temperature was set to 300 °C and capillary temperature to 270 °C. The source voltage was 4.1 kV, capillary voltage 5 V, and tube lens 135 V. Thinlayer chromatography was performed on silica gel 60 F254 plates (Merck, Darmstadt, Germany). Automatic flash column chromatography was performed with Isolera One (Biotage, Uppsala, Sweden) (RP SNAP 30 g cartridge). Flash chromatography was done with silica gel 60, 40−64 μm (Macherey-Nagel, Düren, Germany). Purity measurements of all isolated and synthesized compounds were performed on an HPLC-UV-ELSD with an LC-20AT system (Shimadzu). A Reprosil-Pur, C18-AQ (150 mm × 4 mm i.d., 3 μm) (Dr. Maisch GmbH, Ammerbuch, Germany) column was employed. The wavelength of the UV detector was set to 236 nm, the 2183



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L), glucose (30.0 g/L)], and potato dextrose [potato dextrose (20 g/ L)] media were employed. To obtain preculture, liquid media was autoclaved in 500 mL Erlenmeyer flasks and inoculated with approximately 0.25 cm2 of agar plates covered with P. aurantiogriseum strain. The flasks were cultivated in an incubator at 25 °C in the dark. After 3 days Penicillium colonies were subcultured onto modified Czapek−Dox agar for 3 weeks. To analyze the auranthine production, the modified CDA was supplemented with different nitrogen and carbon sources33 (see Supporting Information). Several amino acids in concentrations of 10 and 100 mM were examined: glycine (0.75 and 7.5 g/L), glutamine (1.74 and 17.4 g/L), glutamate (1.47 and 14.7 g/L), and proline (1.15 and 11.5 g/L). Analysis of Auranthine in Fungal Cultures. A slightly modified method of microscale extraction described by Smedsgaard was used to analyze the production of auranthine.34 Three plugs (diameter 1.3 cm) from five agar plates were cut from the fungal colonies. The agar plug was transferred to 2 mL Eppendorf tubes and extracted with 1 mL of EtOAc with the help of an Innova 44 Eppendorf (WesselingBerzdorf, Germany) laboratory shaker for 25 min at 250 rpm. The supernatant was removed, and the extraction was repeated. Combined supernatants after two extractions were transferred into a vial and evaporated to dryness under a gentle nitrogen stream at 40 °C. Thereafter, the residue was dissolved in 1 mL of MeCN−H2O (50:50, v/v) and passed through a 15 mm × 0.45 μm membrane filter (Phenomenex, Aschaffenburg, Germany). The Jasco HPLC system, Jasco Labor and Datentechnik (Gross-Umstadt, Germany), equipped with an FLD (FP-1520), DAD (MD-2010 Plus), and autosampler (AS-2057 Jasco) for the quantitative determination of auranthine, was used. The FLD wavelength was set to excitation at 292 nm and emission at 342 nm. The flow rate was 0.8 mL/min, and the injection volume 30 μL. The binary gradient consisting of MeCN (solvent A) and H2O (solvent B), both containing 0.1% formic acid, was used for the separation of analytes. Starting conditions of the gradient were as follows: 0−20 min, 20−100% A; 20−23 min held at 100% A, followed by reequilibrating the column under starting conditions (20% A) for 3 min. A Reprosil-Pur RP-18 column, 150 mm × 4 mm i.d., 3 μm (Dr. Maisch GmbH, Ammerbuch, Germany), was used. Obtained data were analyzed with ChromPass Cromatography (Jasco Labor and Datentechnik, Gross-Umstadt, Germany). The limit of quantitation for auranthine was 0.1 μg/mL. Auranthine content was normalized to biomass and expressed as mg per g dry mass. Extraction and Isolation of Auranthine and Aurantiamine. For the isolation of auranthine and aurantiamine, P. aurantiogriseum was cultivated on agar plates with modified CDA (supplemented with 17.4 g/L of glutamine) during 3 weeks in the dark at 22 °C. The fungal cultures were extracted with EtOAc and concentrated in vacuo. The residue was subjected to flash chromatography on an automated flash chromatograph (Biotage, Isolera One, Uppsala, Sweden) employing an RP SNAP 25 g cartridge (MeCN−H2O gradient). Afterward, the fractions containing metabolites of interest were concentrated in vacuo to provide about 80 mg of a mixture of auranthine and aurantiamine. The final separation of auranthine and aurantiamine was achieved employing an NP silica gel column with a gradient of cyclohexane−EtOAc to yield 63 mg of auranthine and 12 mg of aurantiamine. Auranthine (2): colorless powder; mp 156−158 °C; [α]20 D −113.2 (c 1.35, CH2Cl2); UV (MeCN) λmax 225, 271, 314 nm, IR (neat) νmax 3179, 3071, 2249, 1663, 1616, 1697, 1454, 1381, 1254, 768, 694; 1H NMR (DMSO-d6, 600 MHz, Table 1); 13C NMR (DMSO-d6, 150 MHz, Table 1); HRMS m/z [M + H]+ 331.1182 (calcd for C19H15N4O2, 331.1195). Aurantiamine (7): pale yellow powder; mp 235−237 °C; [α]20 D −120 (c 1.01, MeOH); 1H NMR (DMSO-d6, 400 MHz) δ [ppm] 0.83 (3H, d, J = 6.8 Hz), 0.93 (3H, d, J = 7.1 Hz), 1.42 (6H, s), 2.20− 2.10 (1H, m), 3.90 (1H, t, J = 2.9 Hz), 5.06 (2H, ddd, J = 18.4, 13.9, 1.0 Hz), 6.05 (1H, dd, J = 17.4, 10.5 Hz), 6.69 (1H, s), 7.80 (1H, s), 8.24 (1H, d, J = 2.5 Hz), 11.84 (1H, brs), 12.23 (1H, brs); HRMS m/ z [M + H]+ 303.1828 (calcd for C16H23N4O2, 303.1821). Synthesis. 3-((7S,7aS)-5,13-Dioxo-5,6,7,7a,8,13hexahydrobenzo[6,7][1,4]diazepino[2,1-b]quinazolin-7-yl)-

(solvent B), both containing 0.1% formic acid. After keeping the starting conditions of 5% A for 1.5 min, the percentage of A was linearly increased to 95% at 11 min. These conditions were kept constant for 2 min, followed by re-equilibrating the column under starting conditions for 5 min. The temperature of the column oven was set to 40 °C, and the flow rate was 350 μL/min with an injection volume of 10 μL. X-ray Diffraction. For compound 8 data sets were collected with a D8 Venture Dual Source 100 CMOS diffractometer. Programs used: data collection: APEX3 V2016.1-0 (Bruker AXS Inc., 2016, Madison, WI, USA); cell refinement: SAINT V8.37A (Bruker AXS Inc., 2015); data reduction: SAINT V8.37A (Bruker AXS Inc., 2015); absorption correction, SADABS V2014/7 (Bruker AXS Inc., 2014); structure solution SHELXT-2015;21 structure refinement SHELXL-201521 and graphics, XP (Bruker AXS, 1998). R-values are given for observed reflections, and wR2 values are given for all reflections. For the X-ray crystal structure analysis of 8 (dan8896), a colorless needle-like specimen of C19H16N4O2, approximate dimensions 0.065 mm × 0.088 mm × 0.331 mm, was used. The X-ray intensity data were measured. A total of 1095 frames were collected. The total exposure time was 20.72 h. The frames were integrated with the Bruker SAINT software package using a wide-frame algorithm. The integration of the data using a monoclinic unit cell yielded a total of 9062 reflections to a maximum θ angle of 66.59° (0.84 Å resolution), of which 2741 were independent (average redundancy 3.306, completeness = 99.5%, Rint = 9.96%, Rsig = 9.81%) and 2185 (79.72%) were greater than 2σ(F2). The final cell constants of a = 5.7735(3) Å, b = 13.1860(7) Å, c = 10.8190(5) Å, β = 104.311(3)°, and volume = 798.08(7) Å3 are based upon the refinement of the XYZ-centroids of 2986 reflections above 20σ(I) with 8.434° < 2θ < 135.3°. Data were corrected for absorption effects using the multiscan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.830. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.7880 and 0.9520. The structure was solved and refined using the Bruker SHELXTL software package, using the space group P21, with Z = 2 for the formula unit, C19H16N4O2. The final anisotropic full-matrix least-squares refinement on F2 with 234 variables converged at R1 = 6.53% for the observed data and wR2 = 12.84% for all data. The goodness-of-fit was 1.066. The largest peak in the final difference electron density synthesis was 0.289 e−/Å3, and the largest hole was −0.292 e−/Å3 with an RMS deviation of 0.066 e−/Å3. On the basis of the final model, the calculated density was 1.383 g/cm3 and F(000) was 348 e−. The hydrogen atoms at N1 and N15 were refined freely; others were calculated and refined as riding atoms. The Flack parameter was refined to −0.2(3). CCDC number: 1825504. DFT Calculations. A conformational search of auranthine 2 was performed with the semiempirical (tight binding DFT) GFN-xTB method.22 Geometry optimizations of the identified 17 conformers were performed without any geometry constraints using the TPSS functional23 and an atom-pairwise dispersion correction (D3).24a,b A flexible triple-ζ basis set (def2-TZVP)25 was used in all calculations. For the calculation of free enthalpy contributions of internal rotations and vibrations (GRRHO298), a rotor approximation was applied for vibrational modes with wave numbers below 100 cm−1.26 Single-point calculations were performed with the hybrid functional PW6B95(D3).27 Free energies of solvation were obtained with the COSMO-RS model28a,b at 298 K. All geometry optimizations and single-point calculations were performed with the TURBOMOLE 7.2 program.29 The calculation of the CD spectra was performed with ORCA(4.0)30 using the CAM-B3LYP31 functional and the def2-TZVP basis set. We calculated the spectra in vacuo and with a solvent model (CPCM32 for the MeCN). The TDDFT calculations involved the determination of 40 roots. Fungi and Media. P. aurantiogriseum CBS 112021 was purchased from the CBS Fungal Biodiversity Center (Utrecht, The Netherlands). Liquid Czapek−Dox [sucrose (30.0 g/L), NaNO3 (2.0 g/L), K2HPO4 (1.0 g/L), MgSO4·7H2O (0.5 g/L), KCl (0.5 g/L), FeSO4· 7H2O (0.01 g/L), yeast extract (5.0 g/L)], malt extract [malt extract (30.0 g/L), mycological peptone (5 g/L)], YES [yeast extract (5.0 g/ 2184



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propanenitrile (8). Under a N2 atmosphere, sodium cyanoborohydride (33.5 mg, 0.53 mmol) was added to a solution of auranthine (2) (44 mg, 0,13 mmol) in MeOH (5 mL) followed by the addition of 0.1 N HCl (2 mL). The reaction mixture was stirred at ambient temperature for 24 h. Then a saturated aqueous solution of NaHCO3 (30 mL) was added, and the mixture was extracted with ethyl acetate (3×). The combined organic layers were washed with water, dried over Na2SO4, and filtered, and the solvent was removed in vacuo. The residue was purified by flash column chromatography (CH2Cl2− MeOH from 95:5 to 90:10) to give 8 as colorless needles (29 mg, 0.09 mmol, 66%): mp 250 °C (dec); [α]20 D +196.2 (c 1.0, MeOH); IR (neat) νmax 3318, 2978, 2249, 1674, 1632, 1609, 1481, 1393, 1377, 1319, 1157, 764, 718; 1H NMR (DMSO-d6, 600 MHz, Table 1); 13C NMR (DMSO-d6, 150 MHz, Table 1); HRMS m/z: [M + H]+ 333.1377 (calcd for C19H17N4O2, 333.1352). 3-((7S,7aS)-6-Methyl-5,13-dioxo-5,6,7,7a,8,13-hexahydrobenzo[6,7][1,4]diazepino[2,1-b]quinazolin-7-yl)propanenitrile (9). Methyl iodide (0.3 mL) was added to a mixture of 8 (22 mg, 0.066 mmol) and potassium carbonate (23 mg, 0.17 mmol) in dry dimethylformamide (2 mL). The reaction mixture was stirred at 35 °C for 24 h. As thin-layer chromatography analysis revealed an incomplete consumption of the starting material at this time point, 0.3 mL of methyl iodide was added additionally. After stirring for another 24 h, water (30 mL) was added and the mixture was extracted with ethyl acetate (3×). The combined organic layers were dried over Na2SO4 and filtered, and the solvent was removed in vacuo. The residue was purified by flash column chromatography (CH2Cl2−MeOH from 99:1 to 95:5) to give 9 as a colorless solid (14 mg, 0.04 mmol, 61%): mp 149−151 °C; IR (neat) νmax 3314, 3291, 2249, 1636, 1612, 1489, 1396, 756, 694; 1H NMR (CD3OD, 600 MHz, Table 1); 13C NMR (CD3OD, 150 MHz, Table 1); HRMS m/z [M + H]+ 347.1527 (calcd for C20H19N4O2, 347.1508). Cytotoxicity Test. In order to examine the cytotoxicity, 1.0 mg of each test compound was dissolved in MeOH. Human liver cancer cells (HepG2, HB-8065) (ATCC, Manassas, VA, USA) were cultivated in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES buffer), 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, and 10% (v/v) fetal calf serum (FCS) using standardized culture conditions (37 °C, 5% CO2, saturated humidified atmosphere). Human kidney epithelial cells (kindly provided by S. Møllerup, National Institute of Occupational Health, Norway) were cultivated in DMEM/Ham’s-F12 medium supplemented with 3.57 g/L HEPES, 5 mg/L human apo-transferrin, 5 mg/L bovine insulin, 5 μg/L Na-selenite, 10 μg/L EGF, 36 μg/L hydrocortisone, 5 mL/L penicillin/streptomycin/glutamine solution, and 1% fetal calf serum. Both culture media were changed twice a week. Subcultivation of cells was performed after trypsination when reaching a microscopic confluence of approximately 80%. Cytotoxic effects of compounds were evaluated in both cell lines with the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Tokyo, Japan). The assay was performed according to the manufacturer’s instructions and our previous studies.35−37 The results for compound-treated cells were normalized to the values of a solvent-treated control (1% MeOH). Studies were performed in triplicate with each cell line from three independent passages (n ≥ 9). The data are shown as the mean ± standard deviation (SD). The IC50 values were calculated by loglinear regression, and the significance indicated refers to the significance level as compared to the solvent-treated control (1% MeOH) calculated with the OriginPro 2016G (64-bit) Sr2 b9.3.2.303 (SF8T5-3089-7901139) (OriginLab Corporation, Northampton, MA, USA). T-2 toxin at a concentration of 10 μM was used as a positive control. The effect of different concentrations of compounds was analyzed by analysis of variance (ANOVA) and Student’s t test; * statistically significant (p ≤ 0.05), ** statistically highly significant (p ≤ 0.01).

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00187. Additional information (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 251 83 33391. Fax: +49 251 83 33396. E-mail: [email protected]. ORCID

Svetlana A. Kalinina: 0000-0001-7564-8213 Christian Mück-Lichtenfeld: 0000-0002-9742-7400 Hans-Ulrich Humpf: 0000-0003-3296-3058 Author Contributions #

S. A. Kalinina and D. V. Kalinin contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank NRW Graduate School of Chemistry and Cells-inMotion Cluster of Excellence (EXC 1003-CiM), University of Münster, Germany. We thank Dr. M. Behrens for the help with interpretation of the cytotoxicity data and Prof. N. P. Lopes and Dr. F. Hü b ner for the discussions on the MS fragmentation spectra.

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REFERENCES

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Auranthine, a Benzodiazepinone from Penicillium aurantiogriseum

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