Gunnilactams A–C, Macrocyclic Tetralactams from the Mycelial

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Gunnilactams A−C, Macrocyclic Tetralactams from the Mycelial Culture of the Entomogenous Fungus Paecilomyces gunnii Yongbiao Zheng,*,† Jiyan Zhang,† Liufeng Wei,† Mianmian Shi,† Jifeng Wang,‡ and Jianzhong Huang† †

Engineering Research Center of Industrial Microbiology, College of Life Sciences, Fujian Normal University, Fuzhou 350117, People’s Republic of China ‡ Fudan Institute of Urology, the Fifth People’s Hospital of Shanghai, Fudan University, Shanghai 200240, People’s Republic of China S Supporting Information *

ABSTRACT: Three novel macrocyclic tetralactams, gunnilactam A (1), gunnilactam B (2), and gunnilactam C (3), were isolated from the submerged fermentation broth of Paecilomyces gunnii, an entomogenous fungus identified as the anamorph of Cordyceps gunnii. Their structures were determined using NMR data, HREIMS, and single-crystal X-ray crystallography. Gunnilactam A exhibited selective cytotoxic activity against human prostate cancer C42B cells with an IC50 value of 5.4 μM.

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tyrosinase inhibition activity, were isolated from the liquidcultivated mycelia of P. gunnii.9 In this paper, we report compounds isolated from its submerged liquid fermentation broth. Gunnilactam A (1) was obtained in the form of a white, amorphous gel with [α]25 D +19.7 (c 0.07, CH3OH) and the UV absorption (λmax) at 207 nm. A square crystal of 1 was formed in methanol at room temperature. The molecular formula of 1 was found to be C20H30N4O5 based on the HREIMS and NMR data (Table 1 and Figures S1 and S2). NMR data (Table 1) revealed the presence of three tertiary methyl groups, four methylenes, seven methines (two are sp2 carbons), and six sp3 carbons (including three amide carbonyl carbons and two sp2 carbons) in 1. HMBC correlations from H3-20 to C-3/C-2, from H2-19 to C-13/C-14/C-3/C-2, and from H-14 to C-19/ C-14a/C-18a/C-13 and a 1H−1H COSY cross-peak between H2-19 and H-14, together with the two sp2 quaternary carbons of C-14a (δC 97.0) and C-18a (δC 164.1), allowed establishing the fragment 1a (Figure 1, C-5 to C-11). Similarly, HMBC correlations from H-6/7 to C-5/C-8, from H-10 to C-11, and from H3-22 to C-11/C-10 and 1H−1H COSY cross-peaks between H-10 and H-11 and between H-8 and H-9 allowed the connection of the fragment 1b (Figure 1, C-14a/14/19/2/3/ 20). In addition, the other fragment 1c (Figure 1, C-21/17/18/ 18a) could be obtained from HMBC correlations of H3-21 to C-18 and 1H−1H COSY cross-peaks between H-17 and H-18. Moreover, the chemical shifts of C-5 (δC 164.4), C-13 (δC 170.6), C-15 (δC 165.7), and C-2 (δC 97.8), the bond lengths

ntomogenous fungi are associated with insects. They have been used historically as biological control agents. In highaltitude areas, some entomogenous fungi can complete their life cycle by infecting larvae underground and then under suitable climatic conditions forming stromata. In China, these stromata together with muscardine cadaver have been used as an edible tonic and a medicinal additive since ancient times. This is usually called aweto and includes Cordyceps sinesis, Cordyceps gunnii, Cordyceps takaomontana, and other Cordyceps fungi. However, these so-called caterpillar fungi cannot be artificially cultivated due to a limited knowledge of the mechanism underlying the formation of their stromata, which usually grow in small quantities and only during a short period annually. Overexploitation of these wild aweto, which have considerable market value, can cause ecological problems. To foster the scientific, rational, and sustainable use of these aweto, the biologically active constituents of the fermentation products of their anamorph fungi were here evaluated. Recently, some bioactive compounds have been extracted from these fungi fermentation products; these include one new diketopiperazine and one new 10-membered macrolide from Paecilomyces tenuipes,1 the anamorph of C. takaomontana, and 2,3dihydroxypropyl nonanoate, 4-hydroxy-6-pentyltetrahydropyran-2-one, and 4-methyl-1H-imidazole-5-ethanol from Hirsutella sinensi,2 the anamorph of C. sinesis. Cordyceps gunnii is a wellknown edible tonic distributed in China’s Guizhou, Tibet, and Sichuan Provinces. It has previously been reported that C. gunnii has immunomodulating,3 antitumor,4 antimicrobial,5 analgesic,6 and cytotoxic activity.7 The entomogenous fungi Paecilomyces gunnii Liang is the anamorph of C. gunnii.8 In a previous report, three phenanelones, paecilomycone A−C, all of which have © 2017 American Chemical Society and American Society of Pharmacognosy

Received: January 19, 2017 Published: May 31, 2017 1935

DOI: 10.1021/acs.jnatprod.7b00060 J. Nat. Prod. 2017, 80, 1935−1938

Journal of Natural Products

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Table 1. NMR Spectroscopic Data (500 MHz, DMSO-d6) for 1 position

97.8, 74.6, 164.4, 117.9, 155.1, 67.2, 27.7,

C CH C CH CH CH CH2

10 11 13 14 14a 15 17 18 18a 19

34.1, 71.5, 170.6, 34.3, 97.0, 165.7, 68.4, 27.9, 164.1, 31.7,

CH2 CH C CH C C CH CH2 C CH2

20 21 22 a

δC, type

2 3 5 6 7 8 9

13.1, CH3 18.6, CH 20.2, CH

δH (J in Hz) 4.72, oa 5.79, 6.77, 4.51, 1.79, 1.48, 2.54, 4.47,

d (16) d (16) o m o o o

3.45, o

4.75, o 1.46, o 2.30, 2.00, 1.30, 0.98, 1.35,

Figure 2. X-ray crystal structure of 1.

dd (14.3, 6.5) dd (14.3, 8.0) d (6.4) d (6.2) d (6.2)

carbons), and seven sp3 carbons (including six carbonyl carbons and one sp2 carbon) (Table 2). The difference between 2 and 3 was that one nitro- and oxygenated sp3 carbon (C-2) in 2 was substituted by a carbonyl carbon (C-5) in 3. Comparison of NMR data of 1 and 2 revealed that compounds 1 and 2 were analogues. Compound 2 was the dehydrogenation derivative of 1 at C-17 and C-18. A single crystal of gunnilactam B (2) with a melting point of 193−194 °C was obtained from the aqueous acetone extract of a mixture of 2 and 3. In this way, the structure of 2 was confirmed by its X-ray diffraction analysis (Figure 3). Gunnilactams A−C are novel macrocyclic tetralactams with an unusual skeleton produced by an as yet undescribed biosynthesic pathway. In recent years, many lactam-containing natural products showing a variety of bioactivities have been reported.10−14 On the basis of previous data concerning P. gunnii, the isolated pure compound gunnilactam A (1) was evaluated for cytotoxic activity. Gunnilactam A displayed selective inhibitory activity against C42B cells with an IC50 value of 5.4 μM, but the inhibition rate against DU-145 cell was 4.5% at a concentration of 2 μM. Compounds 1−3 may explain in part the pharmaceutical application of P. gunnii.

o = overlapped.

Figure 1. Chemical structures of compounds 1−3.

of N-3−C-5 (1.354 Å), N-12−C-13 (1.329 Å), N-16−C-15 (1.365 Å), and N-1−C-2 (1.464 Å) measured in a single crystal, and the prominent absorptions for a lactam group (3428 and 1720 cm−1) and a −CN stretched bond (1384 cm−1) in the IR spectra indicated the existence of three amide groups and one reduced amide group. These lactam groups allow the connections of fragments 1a, 1b, and 1c. On the basis of the above data, the core structure of 1 was formed. The stereochemistry of 1 was decided by X-ray diffraction analysis of a single crystal obtained from methanol (Figure 2). Gunnilactam B (2) and gunnilactam C (3) were obtained together as a mixture of an amorphous, white gel and are here regarded as a pure compound revealed by TLC detections. However, 1H and 13C NMR data indicated it should be a mixture of two compounds with the ratio of ca. 1:2 (2:3) based on the proton signal integrations. Detailed analysis of the NMR spectroscopic data (1H and 13C NMR, DEPT, HSQC, HMBC, 1 H−1H COSY) revealed 2 and 3 to be isomeric compounds (Figure 1 and Figures S3 and S4). The NMR data (Table 2) revealed the presence of three tertiary methyl groups, three methylenes, seven methines (including three oxymethines and three sp2 carbons), and seven sp3 carbons (including five carbonyl carbons and two sp2 carbons) in 2. The same NMR data also revealed the presence of compound 3, which contained three tertiary methyl groups, three methylenes, seven methines (including three oxymethines and three sp2

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EXPERIMENTAL SECTION

General Experimental Procedures. The melting point was measured on an SGWX-4 microscopic melting point determinator (Shanghai Optical Instrument Factory). Optical rotations were performed on a Jasco P-1020 automatic polarimeter (Tokyo, Japan). UV spectra were measured on a Shimadzu UV-2600 spectrophotometer (Tokyo, Japan). IR spectra were recorded on a Bruker Tensor-27 FT-IR spectrophotometer (Ettlingen, Germany). NMR spectra were recorded at 500 MHz for 1H NMR and 150 MHz for 13C NMR on a Bruker AMX-500 spectrometer (Bruker BioSpin Group, Zurich, Switzerland). HREI-MS spectra were recorded on a Waters AutoSpec Premier P776 mass spectrometer (Milford, MA, USA). Column chromatography was performed with silica gel (Qingdao Marine Chemical Company, Qingdao, China), reversed-phase octadecyl-silica (Merck, Darmstadt, Germany), and Sephadex LH20 (Amersham Biosciences, Piscataway, NJ, USA). Thin-layer chromatography was performed on precoated silica gel GF254 plates (Qingdao Marine Chemical Company, Qingdao, China). Fungal Material. The strain P. gunnii was supplied by Professor Liang Zhong-Qi, and its identity was confirmed using 18S rDNA (GenBank accession number KY986928, Figure S5). The strain 1936

DOI: 10.1021/acs.jnatprod.7b00060 J. Nat. Prod. 2017, 80, 1935−1938

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Table 2. NMR Spectroscopic Data (500 MHz, DMSO-d6) for 2 and 3 2 position 2 3 5 6 7 8 9

97.8, C 73.87, CH 164.8, C 118.8, CH 154.8, CH 67.4, CH 27.3, CH2

10

26.9, CH2

11 13 14 14a 15 17 18 18a 19 20 21 22 a

δC, type

68.6, 170.7, 34.5, 95.3, 162.7, 161.5, 100.0, 162.5, 31.3,

CH C CH C C C CH C CH2

12.6, CH3 19.2, CH3 17.7, CH3

3 δH (J in Hz) 4.81, oa 5.84, 6.63, 4.49, 1.78, 1.36, 1.46, 1.28, 4.94,

d (15.7) d (15.7) s o o o o s

δC, type

5 6 8 9 10 11 12

208.5, C 73.89, CH 164.1, C 117.0, CH 154.1, CH 68.8, CH 32.0, CH2

13

28.9, CH2

14 2 3 3′ 2′ 6′ 5′ 4′ 4

3.44, o

6.15, s 2.13, 2.02, 1.40, 2.21, 0.98,

position

o o d (6.4) o d (6.4)

15 7′ 16

72.2, 169.7, 36.5, 97.7, 166.1, 161.7, 99.5, 163.4, 38.2,

CH C CH C C C CH C CH2

17.0, CH3 19.3, CH3 20.4, CH3

δH (J in Hz) 5.19, o 5.94, 7.51, 4.23, 2.04, 1.21, 1.87, 1.54, 4.86,

o s o o o o o o

3.95, o

5.98, o 3.90, 2.47, 1.32, 2.16, 1.12,

o o d (6.9) o d (6.1)

o = overlapped. further subjected to Sephadex LH-20 (200 g) eluted with acetone to afford Fr.3 (61 mg). Fr.3 was finally subjected to silica gel chromatography using a CHCl3−MeOH solvent gradient to yield 1 (2.2 mg) and 39.4 mg of a mixture of 2 and 3. Gunnilactam A (1): square crystal, mp 196−197 °C; [α]25 D +19.9 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 207 (4.18); IR νmax 3428, 1720, 1384 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data (Table 1); HREIMS m/z 406.2202 (calcd for C20H30N4O5, 406.2216). Gunnilactam B/C (2/3): [α]D25 −11.8 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 209 (3.81); IR νmax 3446, 1708, 1451, 1098 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data (Table 2); HREIMS m/z 404.2064 (calcd for C20H30N4O5, 406.2060). X-ray Single-Crystal Diffraction for 1 and 2. X-ray single-crystal diffraction was performed on an Oxford Gemini S Ultra single-crystal diffractometer. A suitable crystal was selected and subjected to λ(Mo Kα) = 0.71073 Å at 273.15 K. The structure was determined using the direct method with Olex2 and refined with full-matrix least-squares calculations on F2 using Olex2. Crystallographic data for the reported structures have been deposited with the Cambridge Crystallographic Data Center (CCDC 1486887 for 1 and CCDC 1486888 for 2). Copies of the data can be obtained, free of charge, upon application to the Director, CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: +44-(0) 1223-336033) or e-mail: [email protected]]. Crystallographic data (CCDC 1486887) for 1: C20H30N4O5, square crystal, size 0.1 × 0.1 × 0.05 mm3, orthorhombic, space group P212121, a = 9.8604(16) Å, b = 12.364(2) Å, c = 15.752(3) Å, V = 1920.4(5) Å3, Z = 38, T = 273.15 K, Dc = 1.4058 g cm −3, F(000) = 872.4, T = 273.15 K, Flack parameter = −0.4(11). Crystallographic data CCDC 1486888 for 2: C20H28N4O5, square crystal, size 0.18 × 0.08 × 0.05 mm3, orthorhombic, space group P212121, a = 9.4557(13) Å, b = 12.8247(17) Å, c = 15.429(2) Å, V = 1871.04(4) Å3, Z = 37, Dc = 1.4214 g cm −3, F(000) = 848.4, T = 273.15 K, Flack parameter = −0.2(11). MTT Cell Viability Assay. An MTT cell viability assay was used to evaluate the inhibitory effect of compound 1 on the human prostate cancer DU-145 and C42B cells. The cells were cultured in Dulbecco’s modified Eagle’s media complemented with 10% fetal bovine serum

Figure 3. X-ray crystal structure of 2. P. gunnii Liang has been deposited in the Engineering Research Centre of Industrial Microbiology, College of Life Sciences, Fujian Normal University. Fermentation and Isolation. The strain P. gunnii was cultured by the submerged liquid fermentation in a fermentor with the media of potato dextrose peptone (30 L) at 28 °C for 100 h. The fermented broth was condensed to 10 L on a rotary evaporator in vacuo at 40 °C. Then the condensed broth was extracted with EtOAc. The organic layer was dried over Na2SO4 (anhydrous), and the solvent evaporated under reduced pressure to afford 6.34 g of a crude organic extract (dark brown oil). The organic crude was subjected to MPLC over RP18 silica gel (170 g) using a stepwise gradient of pure water, 30%, 50%, 70%, and 100% (v/v) MeOH in water and to afford Fr.1 (852.3 mg) obtained from 30% MeOH. Fr.1 was further subjected to Sephadex LH-20 (170 g) eluted with MeOH to afford Fr.2 (213.6 mg). Fr.2 was 1937

DOI: 10.1021/acs.jnatprod.7b00060 J. Nat. Prod. 2017, 80, 1935−1938

Journal of Natural Products

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and 2 mM L-glutamine. The cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The cells (1 × 105/ well) were cultured for 72 h in 96-well plates (Falcon, CA, USA) in the presence of various concentrations of compounds. Cisplatin (Shanghai Haoyuan Chemexpress Co., Ltd.) was used as positive control. All wells (100 μL each) were incubated with 20 μL of 5 mg/ mL MTT solution for 3 h at 37 °C. Then 100 μL of Triplex solution (10% SDS, 5% isobutanol, 12 mM HCl) was added into each well and incubated overnight at 37 °C. Absorbance was read at 595 nm on a microplate reader (M-3350, Bio-Rad) with 655 nm as a reference. IC50 values were calculated by SPSS 18.0.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00060. Experimental section, 1H and 13C NMR spectrum of 1− 3, 18S rDNA Sequence of P. gunnii (PDF) Crystal data for gunnilactam A (CIF) Crystal data for gunnilactam B (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-13959187966; +86-591-22868193. E-mail: [email protected]. ORCID

Yongbiao Zheng: 0000-0003-1557-7542 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are deeply indebted to Prof. Liang Zhong-Qi, Guizhou University, who supplied the strain of P. gunnii for this research. This work was supported by the Key Program of Science and Technology Plan of Fujian Province (No. 2016Y0030), the Major Research Plan of Xiamen Southern Ocean Research Center (No. 14GYY74NF38), and Science Fund of National Health and Family Planning Commission of China (No. WKJ-FJ-20).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.7b00060 J. Nat. Prod. 2017, 80, 1935−1938