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Quinazolinobenzodiazepine Derivatives, Novobenzomalvins A–C: Fibronectin Expression Regulators from Aspergillus novofumigatus

Three new quinazolinobenzodiazepine derivatives, novobenzomalvins A (1), B (2), and C (3), have been isolated as fibronectin expression regulators from Aspergillus novofumigatus CBS117520. The structures of 1 to 3 were established by spectroscopic and physicochemical analysis, and chemical investigation including the total synthesis of 1. Treatment with novo-benzomalvins A (1), B (2), C (3), and N-methylnovobenzomalvin A (5) increased the expression of fibronectin in normal human neonatal dermal fibroblast cells.


Introduction
The fungus Aspergillus fumigatus is known as an important human pathogen, which produces many secondary metabolites. Recently, Hong et al. re-identified the fungus Aspergillus novofumigatus CBS11520 as the new Aspergillus sp., which is closely related to A. fumigatus [1]. We have previously reported the isolation of two diketopiperazines, novoamauromine and ent-cycloechinulin, and a cyclic tripeptide, novofumigatamide, from the MeOH extract of this fungus cultivated on rice [2,3]. Further investigation of this fungal Sci Pharm. 2011; 79: 937-950 metabolite led to the isolation of three new quinazolinobenzoziazepine derivatives: novobenzomalvins A (1), B (2), and C (3) (Figure 1). This report describes the isolation, structure, and biological activity of 1-3.

Results and Discussion
Solid-substrate fermentation cultures of A. novofumigatus CBS117520 grown on rice were extracted with MeOH. The evaporated extract was suspended in water and extracted with EtOAc. The evaporated extract was partitioned with MeCN and n-hexane to yield a MeCNsoluble fraction. The fraction was extracted sequentially with n-hexane, benzene, CHCl 3 , EtOAc, and MeOH. The benzene extract was chromatographed on a Sephadex LH-20 column, followed by medium-pressure liquid column chromatography (MPLC) on silica gel. Thin-layer chromatography (TLC) analysis led to the separation of the compoundcontaining fractions. Further purification of these fractions by high-performance liquid chromatography (HPLC) yielded 1, 2, and 3 along with the known compounds epiaszonalenin A and C, and helvolic acid [4][5][6][7].
The molecular formula of 1 was determined to be C 23 H 17 N 3 O 2 (17 degrees of unsaturation) by high-resolution chemical ionization mass spectrometry (HRCIMS). The presence of NH groups was deduced from the broad absorption at 3437 cm −1 in the compound's IR spectrum. Two carbonyl groups were inferred from its 13  To confirm the stereochemistry of 1, the compound was synthesized from D-phenylalanine by a modified method [9] (Scheme 1), and the optical rotation of the synthetic product (1) was +111° (c 0.73, MeOH). From the above results, the chemical structure of 1 was confirmed to be identical to those as shown in Figure 1.
The molecular formula of 2 was found to be C 23 H 17 N 3 O 3 by HREIMS. The 1 H and 13 C NMR spectra of 2 was similar to those of 1, expect for the downfield shift of the carbon at C-20 from δ 35.2 in 1 to δ 72.9 in 2 and the new appearance of a methine signal (δ 5.32) in 2 instead of a methylene signal (δ 3.29 and δ 3.59) in 1 ( Table 1). Analysis of the 1 H 1 H COSY and HMBC spectra supported the planar structure of 2 being the 20-hydroxy derivative of 1 ( Figure 2). X-ray crystallographic analysis of novobenzomalvin B (2) was performed to confirm the structure, as the crystal of 2 was suitable for X-ray analysis. The result established the absolute configuration of 2 as shown in Figure 3 [10], in light of the Flack parameter [11] of −0.04 (12), using anomalous dispersion of 1263 Friedel pairs [12,13].

Sch. 1.
Total synthesis of novobenzomalvin A (1) The molecular formula (C 23 H 15 N 3 O 3 ) of 3 suggested the loss of two protons from that of 2 (C 23 H 17 N 3 O 3 ) and thus indicated the replacement of a hydroxyl group in 2 with a carbonyl group in 3. The 1 H and 13 C NMR spectra of 3 and 2 showed only the difference of a carbonyl sp 2 carbon signal (δ 188.9) in 3 instead of a secondary hydroxyl group (δ 72.9, δ 5.32) in 2 ( Table 1). The analysis of the 1 H 1 H COSY, and HMBC spectra supported 3 having a planar structure as the 20-didehydro derivative of 2, that is, the 20-oxo derivative of 1 ( Figure 2). Novobenzomalvin C (3) was prepared by treatment of 2 with pyridinium chlorochromate (PCC). The optical rotation of 3 derived from 2 was −329° (c 0.14, MeOH), whereas that of naturally occurring 3 was −256° (c 0.18, MeOH). Therefore, the absolute structure of 3 was determined to be the one shown in Figure 1.
A wide variety of quinazolinobenzodiazepine alkaloids have been recently reported as natural products from several filamentous fungi, for example: sclerotigenin, an anti-insect active compound isolated from the sclerotia of Penicillium sclerotigenum [14]; asperlicins A-E, potent nonpeptidal cholecystokinin antagonists isolated from Aspergillus alliaceus [15][16][17]; benzomalvins, substance-P inhibitors isolated from Penicillium sp. [8]; and circumdatins A-I isolated from Aspergillus ochraceus [18][19][20][21] and Exphiala sp. [22]. These alkaloids which have been recently reported as natural products consist of two anthranilic acids and a L-amino acid (e.g., benzomalvin A contains L-phenylalanine, asperlicin C contains L-tryptophan, and circumdatin C, F, G, and I contain L-alanine, etc.) and the all C-19 configuration of them is the same as that of L-amino acids. Interestingly, the C-19 configuration of 1-3 is opposite that of other quinazolinobenzodiazepine alkaloids, and this fact suggests that the only A. novofumigatus in genus Aspergillus utilized D-amino acid in its biosynthesis. Therefore, it might be possible to utilize novobenzomalvins for good chemotaxonomic markers of Aspergillus section Fumigati. We studied effects of novobenzomalvins A (1)-C (3) and N-methylnovobenzomalvin A (5) on the expression of fibronectin (FN). Normal human neonatal dermal fibroblast (NHDFneo) cells were incubated with novobenzomalvins (each 10 µM), or TGF-β (10 ng/mL) as a positive control for 24 h. Treatments of cells with all of the novobenzomalvins increased the expression of fibronectin, as well as that of TGF-β (10 ng/mL) as a positive control ( Figure 4). Fibronectin is an important ingredient of the extracellular matrix proteins in the skin, and is often used as an index for evaluating the extent of matrix accumulation [23].
Although previous reports have shown that some alkaloids such as berberine and cepharanthine effectively reduced fibronectin protein expression [24,25], little is known about stimulation of fibronectin expression by other alkaloids. In the present study, our data revealed that fibronectin increases in NHDF-neo cultured in the treatment with novobenzomalvins, indicating novobenzomalvins could activate the skin function through stimulating extracellular matrix accumulation. To our knowledge, this is the first reported example that quinazolinobenzodiazepine alkaloids increased the expression of fibronectin. We would like to examine their functional mechanism in future research. In addition, the antifungal and cytotoxic activities of 1-3 were studied using a previously reported method [26]. 1-3 showed non-specific antifungal activity against four human pathogenic fungi (Aspergillus niger, A. fumigatus, Candida albicans, and Cryptococcus neoformans) tested at 100 μg per disk, and these compounds did not inhibit the cell proliferation in A549, Hela, and LNCap cells at 100 μM. Ltd., Kyoto, Japan), equipped with a YRD-883 RI detector (Shimamuratech Ltd., Tokyo, Japan). Microwave irradiation reactions were performed in a microwave synthesizer (Initiator 1, Biotage Japan Ltd., Tokyo, Japan).

Isolation of metabolites from Aspergillus novofumigatus CBS117520
Polished rice (Akitakomachi, 24 kg) was soaked in water for 30 min and then sterilized with an autoclave. A. novofumigatus CBS117520 was cultivated for 14 days in Roux flasks, each containing 140 g of moist rice. The cultivated rice was extracted with MeOH, and the extract was concentrated in vacuo. The residue was suspended in water and extracted with EtOAc. The EtOAc extract (52 g) was partitioned between n-hexane and MeOH to yield a MeCN-soluble mixture. The MeCN extract (29.4 g) was extracted sequentially with n-hexane, benzene, CHCl 3 , EtOAc, and MeOH (100 mL each). The benzene extract (18 g

Methylation of novobenzomalvin A (1)
Methyl iodide (2 mL) was added to a solution of 1 (8 mg) and KOH (10 mg) in DMSO (2 mL) and the mixture was stirred at room temperature (rt) for 1 h. The reaction mixture was added to H 2 O (3 mL) and extracted three times with CHCl 3 . The obtained CHCl 3 solution was washed several times with H 2 O. After addition of anhydrous Na 2 SO 4 , the filtered solution was evaporated in vacuo. The resulting residue (9.1 mg) was purified by HPLC [benzene/acetone (18:1)] to give N-methylnovobenzomalvin A (5: 3.8 mg).

Total synthesis of novobenzomalvin A (1)
The total synthesis of novobenzomalvin A (1) was accomplished via triflate-catalyzed dehydrative cyclization promoted by microwave irradiation, following the report of Chu et al. (Scheme 1) [8].

Methyl 2-[(2-aminobenzoyl)amino]benzoate (7)
Anthranilic acid (6: 5 g, 0.024 mol) and isatoic acid (8 g, 0.049 mol) was added to H 2 O (80 mL) at rt, and the mixture was stirred at reflux for 2 h. Then, the H 2 O layer was evaporated to give a crude residue. The residue was added to a solution of H 2 SO 4 /MeOH (1:20 v/v, 94.5 mL), and the mixture was stirred at reflux for 2 d. The reaction was quenched by the addition of saturated NaHCO 3 solution. The aqueous layer was extracted with CHCl 3 . The combined organic layer was evaporated to give a crude residue, which was purified by MPLC [benzene/acetone (4:1)] to afford 7 (3.98 g, 62%) as a colorless amorphous solid. 1

Structure solution and refinement
The structure was solved by a direct method using SIR92 [12] and expanded using Fourier techniques with DIRDIF99 [13] and refined by the full-matrix least-squares method. The non-hydrogen atoms were refined anisotropically and hydrogen atoms were refined using the riding model. The final cycle of full-matrix least squares refinement on F 2 was based on 8714 observed reflections and 280 variable parameters. The final R and Rw values were 0.0337 and 0.0940, respectively. The absolute structure was deduced based on the Flack parameter [11] −0.04 (12), refined using 1263 Friedel pairs [10]. For more detailed information, see Supporting Information available online.

Oxidation of novobenzomalvin B (2) with PCC
The CH 2 Cl 2 solution (0.5 mL) of novobenzomalvin B (2: 9 mg) was added to a CH 2 Cl 2 suspension (1 mL) of PCC (50 mg) and the mixture was refluxed for 29 h with stirring. The reaction mixture was filtered, and the filtrate was evaporated in vacuo. The obtained residue was purified by LPLC with CHCl 3 /acetone (15:1) to give novobenzomalvin C (3: 2 mg). Synthetic 3 was identical to naturally occurring 3 in comparisons of the 1 H, 13  The samples were resolved on 7.5% Tris-Glycine polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membrane (Bio-Rad Bioscience; Hercules, CA). The membranes were blocked for 1 h in TBS containing 0.05% Tween-20 (TBS-T) and 5% fatfree milk and then incubated overnight at 4 °C with the primary antibody. The membranes were washed with TBS-T four times followed by incubation for 1 h at rt with HRPconjugated anti-mouse IgG and the immunoreactivity was assessed by chemiluminescence.

Statistical methods
Densitometry analysis was performed using ImageJ 1.43 on immunoblots from three independent experiments. A t-test was performed with SYSTAT software (Hulinks Inc., Tokyo, Japan).

Antifungal assay
The antifungal assay was performed by using a previously reported method [23]. The antifungal assay was performed using the paper disk method against A. niger IFM 41398, A. fumigatus IFM 41362, C. albicans IFM 40009 and C. neoformans ATCC 90112 as test organisms. Novobenzomalvin A (1) to C (3) were applied to the paper disk (diameter: 8 mm) at 100 μg/disk, and the disks were placed on the assay plates. The test organisms were cultivated in potato dextrose agar (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) at 25 °C. After 48-72 h of incubation, zones of inhibition (the diameter measured in millimeters) were recorded.