(±) Benzomalvins E isolated from Penicillium sp. SYPF 8411 in the rhizosphere soil of Codonopsis clematidea

Abstract (+) Benzomalvins E (1) and (-) Benzomalvins E (2), a pair of epimeric derivatives, together with three known benzomalvins (3–5), were isolated from solid cultures of a interrhizospheric fungus Penicillium sp. SYPF 8411. The planar structure of (+) Benzomalvins E (1) has been previously reported. While, the absolute configuration of compound 1 was established by X-ray crystallographic analysis for the first time. The planar structure of the new compound 2 were elucidated by detailed interpretation of their HR ESI-TOF MS and NMR spectroscopic data. The absolute configuration of compound 2 was established by Rh2(OCOCF3)4-induced CD spectral data and the electronic circular dichroic (ECD) method. Furthermore, the epimerization induced by pH, temperature and H2O was revealed. Benzomalvins (1–3, 5), a type of indoximod, enhanced the cytotoxic capability of 5-fluorouracil against A549. Graphical Abstract


Results and discussion
(þ) Benzomalvins E (1) was isolated as a white crystal. On the basis of detailed analyses of 1D NMR, HSQC and HMBC (Figures S9-S14), structure 1 (planar) was determined, which was same as Benzomalvins E (Jang et al. 2012). In the previous report, the absolute configuration Benzomalvins E was not confirmed. However, in our report, suitable crystal for X-ray diffraction experiment with Cu Ka radiation was obtained from MeOH after careful recrystallization, which enabled us to determine the absolute configuration of 1 as 19R, 20R [Flack parameter = 0.07(10)] ( Figure S8, Table S2).
(-) Benzomalvins E (2) was obtained as a white amorphous powder, [a]20D -63.8 (c 0.0023, CH 3 OH). A HR ESI-TOF MS [M þ H] þ peak at m/z 398.1496 indicated that the molecular formula of Compound 2 was C 24 H 19 N 3 O 3 with 17 degrees of unsaturation, which was identical to that of 1. Analysis of the 1D NMR data (Table S1) and HMBC spectrum (Figures S19-S22) of 2 led to the conclusion that 2 was an epimeric isomer of 1, and assigned the same gross structure as 1. Detailed inspection of 1 H NMR, 13 C NMR and HMBC spectra indicated that 1 possessed a higher chemical shift for C-19 (d C 75.6 for 1 and d C 60.3 for 2) than 2. And the relative configurations of 1 and 2 were distinguished using the related coupling constants contained in a 1 H NMR spectrum. Generally, adjacent protons of the erythro type have been reported to have smaller coupling constants than those of the threo type in differentd-solvents (Bouaziz et al. 2002;Nakajima et al. 2003;Gan et al. 2008). Likewise, the new epimerides, 1 and 2, were identified as threo-and erythro-configurations, respectively, due to the 9.96 Hz coupling constants between H-19 and 20 in 1 and 6.17 Hz coupling constant for 2 in CDDl 3 , respectively (Table S1, Figure S16). Therefore, the relative configuration of 2 was assigned as 19S Ã , 20R Ã . The absolute configuration of C-20 tertiary alcohol was confirmed by the induced CD of the in situ formed [Rh 2 (OCOCF 3 ) 4 ] complex. The sign of the E band (at ca. 350 nm) was used to correlate the absolute configuration of a tertiary alcohol by applying the bulkiness rule (Chen et al. 2015;Frelek and Szczepek 1999). According to the bulkiness rule, the formed Rh-complex with a "bR" confguration causes a negative sign of the E band and the one with a "bS" confguration causes a positive sign. A negative Cotton effect at 350 nm in the [Rh 2 (OCOCF 3 ) 4 ]-induced CD spectrum ( Figure S24, Table S6) indicated the 20R absolute confguration of 2 (Gerards and Snatzke 1990). In addition, comparison of the calculated ECD spectra data ( Figure S23) with that of experimental data of compound 2 indicated the absolute configuration of compound 2 was assigned as 19S, 20R.
Interestingly, (þ) Benzomalvin E (1) and (-) Benzomalvin E (2) were obtained as an inseparable solid mixture in a ratio of 3:7 ( Figure S1). And the same is true for N-Methylnovobenzomalvin A (4) and (-) benzomalvin A (5) as well. Each of them obtained by column chromatography and HPLC employing a C 18 column could spontaneously gave rise to a mixture of both compounds, indicating that there is a transformation reaction between these four compounds. Moreover, similar epimerization phenomenon has also been observed in synthetic benzomalvin derivatives, which was inevitably synthesized as an inseparable mixture (Sugimori et al. 2010). In our study, the effect of pH, temperature-and H 2 O-dependent epimerization between (þ) Benzomalvin E (1) and (À) Benzomalvin E (2) were further revealed. The chromatographic condition was in accordance with that of the separation of 1 and 2 by reverse phase HPLC at a flow rate of 0.4 mL/min. The epimerization effect of the compounds 1 and 2 were triggered by temperature and H 2 O.
In this study, five benzomalvins (1-5) were evaluated for their cytotoxic activities against human lung cancer cell lines A549 by the MTT method as described previously. While the result indicated no inhibiting activities of benzomalvins (1-5) against A549, using 5-fluorouracil as the positive drug. Then, compounds (1-5), combined with 5-fluorouracil respectively, were tested for the cytotoxic capability against A549. As shown in Figure 2, benzomalvins (1-3, 5) enhanced the cytotoxic capability of 5-fluorouracil against A549 on a different level.

Fungal material and fermentation
The fungal strain used in this work was isolated from the rhizosphere soil of Codonopsis clematidea, which was collected from Tacheng, Xinjiang Uygur Autonomous Region of China in July 2015. The fungus was identified on the basis of the DNA sequence of the internal transcribed spacer (ITSI-5.8S-ITS2 region). The sequence data derived from the fungal strain has been submitted and deposited in GeneBank with the accession number MF588880. A voucher specimen (No. SYPF 8411) was deposited at the School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University. The strain was cultured on PDA (potato 20%, glucose 2% and agar 2%) medium in Petri dishes at 28 C for 3 days, and then inoculated in a 500 mL Erlenmeyer flask containing 200 mL medium (mannitol 2%, glucose 2%, yeast cream 0.5%, corn steep liquor 0.1%, protein frozen 1%, KH 2 PO 4 0.05%, MgSO 4 .7H 2 O 0.03%). After incubation at 28 C and in a shaker at 170 rpm for 4 days, the large scale fermentation was carried out in two hundred 500 mL Erlenmeyer flasks each containing 80 g of rice, 110 mL of distilled H 2 O. After sterilization, each flask was inoculated with 15 to 20 mL of the culture medium and incubated at room temperature for 40 days.
3.4.2. Temperature-dependent epimerization effect (þ) Benzomalvin E (1) and (À) Benzomalvin E (2) were dissolved in methanol and heated for two hour at À20, 4, 20, 30 C, respectively. No obvious variation was observed under 4 C. After heated for 36 hours at 4 C and À20 C, no transformation was detected for 2. As temperature increased, the speed of epimerization accelerated. Then compounds 1 and 2 dissolved in methanol were continuously heated for 60 h at 20 C, respectively, to reach an equilibrium state ( Figure S4).
3.4.3. H2O-dependent epimerization effect (-) Benzomalvin E (2) dissolved in H 2 O and continuously heated at 28 C, which was the exact temperature for the large scale fermentation of Penicillium sp. SYPF 8411.The influence of different ratios of H 2 O to MeOH was subsequently performed. The constant-temperature heating experiment ( Figure S5) suggested that H 2 O could prevent epimerization. As the amount of H 2 O increased, the speed of epimerization also decreased ( Figure S6 and Figure S7). Based on the above results, the epimerization of 1 and 2 can be induced by temperature and H 2 O.

Cytotoxic assay in vitro
The cytotoxic activities of isolated Compounds 1-5 were tested against human lung cancer cell lines A549, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl -2-H-tetrazolium bromide (MTT) method in vitro. A549 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (10% Fetal bovine serum, 100 IU/mL penicillin, and 100 g/mL streptomycin) in 5% CO 2 at 37 C. Then, the cells were cultured in 96-well plates for 48 h with 100 lL complete medium, 5-fluorouracil was added with concentration of 50 lM, and the compounds were added with varying concentrations of 100, 50, 25, 10 and 1 lM. MTT (20 lL) with 5 mg/mL phosphate buffer saline was added for another 4 h in the 96well plates, then dissolved in dimethyl sulfoxide (DMSO) and measured by Multiskan Spectrum Microplate Reader (Thermo, USA) at 490 nm. The cytotoxicity of the compounds against A549 was calculated and expressed as the value of OD (Figure 2).

Conclusions
Our current study describes the discovery of two pair of epimeric derivatives, (þ) Benzomalvin E (1), (À) Benzomalvin E (2), N-Methylnovobenzomalvin A (4) and (À) benzomalvin A (5), from a interrhizospheric fungus Penicillium sp. SYPF 8411. The unambiguous determination of the absolute configurations of compounds 1 and 2 will provide valuable data for future research on this type of quinazolinobenzodiazepine derivatives. Furthermore, the successful separation of compounds 1 and 2 and the epimerization effect triggered by temperature and H 2 O will be valuable for the study of this type of inseparable epimeric mixture. Meanwhile, benzomalvins (1-3, 5), a type of indoleamine 2,3-dioxygenase (IDO) inhibitor, could enhance the cytotoxic capability of 5-fluorouracil against A549 significantly.

Conflicts of interest
The authors declare no conflict of interest.