Bioactive Penicipyrrodiether A, an Adduct of GKK1032 Analogue and

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Bioactive Penicipyrrodiether A, an Adduct of GKK1032 Analogue and Phenol A Derivative, from a Marine-sourced Fungus Penicillium sp. ZZ380 Tengfei Song, Mengxuan Chen, Zhi-Wei Ge, Weiyun Chai, Xing-Cong Li, Zhizhen Zhang, and Xiao-Yuan Lian J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02172 • Publication Date (Web): 05 Oct 2018 Downloaded from http://pubs.acs.org on October 5, 2018

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The Journal of Organic Chemistry

Bioactive Penicipyrrodiether A, an Adduct of GKK1032 Analogue and Phenol A Derivative, from a Marine-sourced Fungus Penicillium sp. ZZ380 Tengfei Song,† Mengxuan Chen,† Zhi-Wei Ge,§ Weiyun Chai,† Xing-Cong Li,# Zhizhen Zhang,*,† Xiao-Yuan Lian*,‡ †

Ocean College, Zhoushan Campus, Zhejiang University, Zhoushan 316021, P. R. China

‡

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, P. R. China

§

Analysis Center for Agrobiology and Environmental Sciences, Zhejiang University,

Hangzhou 310058, P. R. China #

National Center for Natural Products Research, Research Institute of Pharmaceutical

Sciences, School of Pharmacy, The University of Mississippi, University 38677, United States S Supporting Information ●

ABSTRACT: Penicipyrrodiether A, an adduct of GKK1032 analogue and phenol A derivative, was isolated from a culture of marine-associated fungus Penicillium sp. ZZ380, and represents the first example of this type of fungal metabolite. Its structure was elucidated by extensive spectroscopic analyses, including 1D- and 2D-NMR, HRESIMS, MS/MS, and electronic circular dichroism calculation as well as single-crystal X-ray diffraction. Penicipyrrodiether A showed antibacterial activity in inhibiting the growth of methicillinresistant Staphylococcus aureus with a MIC value of 5.0 g/mL. Its plausible pathway for biosynthesis has been proposed.

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

INTRODUCTION

Hirsutellones and related compounds are a group of fungal metabolites with a 13-membered macro-ether ring, which is made of three characteristic substructures of decahydrofluorene, para-cyclophane, and pyrrolidinone.1,2 To date, a total of 26 such metabolites have been discovered from fungal origins, including GKK1032 analogues A1A3 and B from Penicillium sp. GKK1032,2a hirsutellones AE from Hirsutella nivea BCC 2594,2b hirsutellone F from Trichoderma sp. BCC 7579,2c pyrrocidines A and B from Cylindrocarpon sp. LL-Cyan426,2d,2e pyrrocidine C from Lewia infectoria SNBGTC2402,2f pyrrospirones A and B, bispyrrocidine, epoxy derivative of pyrrocidine B, 19O-methyl-pyrrrocidine B, 19-O-ethyl-pyrrrocidine B from Neonectria ramulariae Wollenw KS-246,2g,2h and pyrrospirones C-I from Penicillium sp. ZZ3802i. Some of these compounds showed cytotoxicity,2a,2g,2i antifungal activity2a,3 and antibacterial activity against Mycobacterium tuberculosis2b,2c and other Gram-positive bacteria including drug-resistant strains.1,2d,2f,2i The biosynthesis of GKK1032A2 was proposed based on feeding experiments on the fungus Penicillium sp. GKK1032 with isotopically labeled (13C and 2H) precursors.4 The backbone of this compound originates from one L-tyrosine and nine acetate units forming a nonaketide chain flanked with five methyl groups from L-methionine, probably by a polyketide synthase and a nonribosomal peptide synthetase hybrid. The challenging molecular architecture of GKK1032 analogues, hirsutellones, and pyrrocidines with ten or more stereogenic centers and the promising antimicrobial activities (especially the antimycobacterial activity for hirsutellone B) have made them very attractive targets for the synthetic community.1 There has been much work done on the synthesis of these compounds so far.5 The first total synthesis of hirsutellone B was achieved in 2009 by Nicolaou.5a Marine-associated fungi of the genus Penicillium are able to synthesize both known and novel compounds with diverse structures and bioactivities.6 During the course of our ongoing project for the discovery of novel antimicrobial and anti-glioma agents from marine-sourced microorganisms,7 a marine fungus Penicillium sp. ZZ380 was recently isolated from a wild crab Pachygrapsus crassipes. Larger culture of this fungus in BMPM 2


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liquid medium resulted in the isolation and identification of novel pyrrospirones C-I with antiglioma and antibacterial activities2i and an unidentified compound due to the limited amount. This compound showed GKK1032 analogues-like NMR signals with a GKK1032 analogues-unlike HRESIMS ion at m/z [M+H]+ 712.3843. Therefore, a targeted accumulation of this compound (9.0 mg) from the culture of strain ZZ380 was conducted, resulting in the identification of penicipyrrodiether A (1), in which a derivative of phenol A (2) fused to the pyrrolidinone core via the addition of a five-membered ether ring (Figure 1). To the best of our knowledge, penicipyrrodiether A is the first example of this type of hirsutellones and related compounds. Herein, we report the isolation, structural elucidation, bioactive evaluation, and plausible biogenetic pathway of penicipyrrodiether A.

Figure 1. Structures of penicipyrrodiether A (1), phenol A (2), and GKK1032A2 

RESULTS AND DISCUSSION

Penicipyrrodiether A (1) was obtained as colorless plate crystals and has a molecular formula C43H53NO8 deduced from its HRESIMS ions at m/z [M+H]+ 712.3843 and [M+Na]+ 734.3661 as well as

13

C NMR data. The 1H NMR spectrum showed four heteroatom

attached protons at H 8.10 (s), 7.89 (s), 6.31 (s), and 6.20 (s); four aromatic protons at H 7.01 (dd, J = 8.5, 2.0 Hz), 6.86 (dd, J = 8.5, 2.4 Hz), 6.80 (dd, J = 8.1, 2.0 Hz), and 6.59 (dd, J = 8.1, 2.4 Hz); four olefinic protons at H 5.44 (dd, J = 17.6, 10.8 Hz), 5.35 (br s), 4.68 (dd, J = 17.6, 1.1 Hz), and 4.40 (dd, J = 10.8, 1.1 Hz); four tertiary methyls at H 1.85 (s), 1.72 (s), 1.10 (s), and 1.02 (s); four secondary methyls at H 1.29 (d, J = 6.1 Hz), 1.22 (d, J = 6.8 Hz), 1.06 (d, J = 6.2 Hz), and 0.89 (d, J = 6.3 Hz). Analysis of its 13C and HSQC NMR spectra indicated that the 43 carbons of 1 (Table 1) were attributed to two carbonyls (C 204.3, 168.4), seven pairs of double bonds (C 162.0, 159.6, 158.2, 145.4, 135.5, 131.7, 3



130.6, 130.0, 129.8, 126.4, 121.0, 118.3, 109.1, 108.3), three non-protonated carbons linked to heteroatoms (C 106.0, 100.9, 99.3), two oxymethine carbons (C 88.5, 80.9), two nonprotonated carbons (C 40.8, 42.2), nine methines (C 63.3, 59.9, 52.3, 48.3, 47.8, 46.4, 42.9, 27.5, 26.7), three methylenes (C 48.0, 45.0, 43.6), and eight methyls (C 26.5, 22.7, 20.3, 19.4, 18.8, 15.8, 15.1, 11.4). The two carbonyls and seven pairs of double bonds accounted for nine out of the 18 degrees of unsaturation required by the molecular formula and the remaining nine suggested the presence of nine rings in 1. Detailed analysis of the 1H-1H COSY and HMBC correlations (Figure 2) revealed that 1 and GKK1032A2 (Figure 1) shared a similar structural part of rings A-D, H, and I. The only difference was that the ketone at C-14 in GKK1032A2 was replaced by an enol in 1. The remaining structural part of 1 was further established by 2D NMR correlations. The neighbor fused rings D-F were suggested by the 1H-1H COSY correlation of H-18 (H 2.59, d, J = 8.8 Hz) with H-19 (H 2.51, d, J = 8.8 Hz) and the HMBC correlations of H-18 with C-14, C-15, C-16, C-17, and C-24, and H-19 with C-15, C-18, C-20, C-21, and C-24. The HMBC correlations of OH-20 (H 6.20, s) with C-19, C-20, and C-21, and OH-21 (H 6.31, s) with C-20, C-21, and C-22 determined the positions of the two hydroxyl groups. In the same way, the neighbor fused rings F and G and the positions of the three methyls were established by the HMBC correlations of H-25 with C-22, C-23, C-42, and C-43, H3-41 (H 1.72, s) with C-22, C-23, and C-24, H3-42 (H 1.22, d, J = 6.8 Hz) with C-22, C-25, and C-26, H3-43 (H 1.29, d, J = 6.1 Hz) with C-25 and C-26, and the 1H-1H COSY correlations of H-25 with H-26 and H342, and H-26 with H3-43, as well as the NOE correlations (Figure 3) of OH-20 with H-25 (H 2.54, m), and OH-21 with H-26 (H 3.97, m).

Figure 2. Key HMBC and 1H-1H COSY correlations of penicipyrrodiether A (1) 4


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Figure 3. Key NOE correlations of penicipyrrodiether A (1) Table 1. 13C (125 MHz) and 1H (500 MHz) NMR Data of Penicipyrrodiether A (1, in DMSO-d6) No.

C, type

H (J in Hz)

No.

C, type

H (J in Hz)

1

26.7, CH

1.78, m

25

42.9, CH

2.54, m

2

45.0, CH2

H: 0.61, m; H: 1.75, m

26

80.9, CH

3.97, m

3

27.5, CH

1.80, m

27

19.4, CH3

1.06, d (6.2)

4

48.0, CH2

H: 0.85, m; H: 1.86, m

28

22.7, CH3

0.89, d (6.3)

5

40.8, C



29

15.8, CH3

1.10, s

6

59.9, CH

1.18, dd (11.3, 7.8)

30

158.2, C



7

88.5, CH

4.44, dd (7.8, 5.0)

31

121.0, CH

6.59, dd (8.1, 2.4)

8

48.3, CH

2.36, m

32

130.6, CH

6.80, dd (8.1, 2.0)

9

52.3, CH

2.0, d (12.5)

33

129.8, C



10

135.5, C



34

131.7, CH

7.01, dd (8.5, 2.0)

11

130.0, CH

5.35, br s

35

118.3, CH

6.86, dd (8.5, 2.4)

12

42.2, C



36

43.6, CH2

H: 2.90, d (12.4); H: 2.97, d (12.4)

13

46.4, CH

3.59, d (8.5)

37

20.3, CH3

1.85, s

14

159.6, C



38

26.5, CH3

1.02, s

15

108.3, C



39

145.4, CH

5.44, dd (17.6, 10.8)

16

168.4, C



40

109.1, CH2

4.40, dd (10.8, 1.1); 4.68, dd (17.6, 1.1)

17

99.3, C



41

11.4, CH3

1.72, s

18

47.8, CH

2.59, d (8.8)

42

15.1, CH3

1.22, d (6.8)

19

63.3, CH

2.51, d (8.8)

43

18.8, CH3

1.29, d (6.1)

20

106.0, C



NH-16



7.89, s

21

100.9, C



OH-14



8.10, s

22

162.0, C



OH-20



6.20, s

23

126.4, C



OH-21



6.31, s

24

204.3, C



5



MS-MS analysis was also applied to elucidate the structure of 1. As shown in Figure S28 (Supporting Information), a series of fragment ions, including three ions at m/z 271.2414, 193.0853 and 107.0498 for the three main structure units of decahydrofluorene (calcd for C20H31+ 271.2420), phenol A derivative (calcd for C11H31O3+ 193.0859) and paracyclophane (calcd for C7H7O+ 107.0491), were observed in the MS-MS spectrum and the possible structures (Figure S29) for thirteen fragment ions have been proposed. So far, the planar structure of 1 has been determined, which was further confirmed by the result from X-ray diffraction (Cu Kα) analysis (Figure 4, CCDC deposition number 1846569, crystallized in MeOH). The relative stereochemistry of 1 was deduced from NOE correlations, coupling constants, and X-ray crystallography. Three double bonds of C10-11, C14-15, and C22-23 were assigned to be Z-, E-, and Z-geometry, respectively, based on the NOE cross-peaks of H-11 (H 5.35, br s) with H3-37 (H 1.84, s), OH-14 (H 8.10, s) with H-18, and H3-41 with H342. As shown in Figure 3, the key NOE correlations of H-6 (H 1.18, dd, J = 11.3 Hz, 7.8Hz) with H-7 (H 4.44, dd, J = 7.8 Hz, 5.0 Hz) and H-9 (H 2.0, d, J = 12.5 Hz), H-7 with H-13 (H 3.59, d, J = 8.5 Hz), H-9 with H-4b (H 0.85, m) and H3-38 (H 1.02, s), and H-13 with H3-38 indicated a -orientation for all these protons. The NOE correlations of H3-29 (H 1.10, s) with H-1 (H 1.78, m), H-3 (H 1.80, m), and H-4a (H 1.86, m) were suggestive of an -orientation for these protons. The large coupling constants of 3J1,6 (11.3 Hz) and 3J8,9 (12.5 Hz) also confirmed the trans-juncture for A/B and B/C rings.2g,2i Similarly, the same -orientation for the protons of NH-16 (H 7.89, s), H-19, OH-20, H-25, H-34 (H 7.01, dd, J = 8.5 Hz, 2.0 Hz ), H-35 (H 6.86, dd, J = 8.5 Hz, 2.4 Hz), and H-36b (H 2.90, d, J = 12.4 Hz) was supported by the NOE correlations of NH-16 with H-34 and H-36b, OH-20 with H-19 and H-25, H-25 with H3-43, H-34 with H-35 and H-36b, and H-35 with H-7; whereas the NOE cross-peaks of H-18 with OH-14 and H-32(H 6.80, dd, J = 8.1 Hz, 2.0 Hz), OH21 with H-26 and H-36a (H 2.97, d, J = 12.4 Hz), H-26 with H3-42, and H-32 with H-31 (H 6.59, dd, J = 8.1 Hz, 2.4 Hz) and H-36a revealed their -orientations. The relative stereochemistry of 1 was thus assigned as 1S*,3R*,5S*,6R*,7S*,8S*,9S*,12S*,13R*,17R*, 18R*,19R*,20R*,21R*,25S*,26R*. The data from X-ray diffraction analysis further 6


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confirmed these assignments.

Figure 4. X-ray crystal structure of penicipyrrodiether A (1, Cu K radiation, and displacement ellipsoids are drawn at the 30% probability level) The Cu K data of X-ray diffraction analysis may not be useful in assigning the absolute configuration because the Flack parameter is 0.1. Several attempts to develop high quality crystals were unachievable, most likely due to the limited amount of the sample material. As a result, a computational method was applied to clarify the absolute configuration of 1 by comparing the experimental ECD spectrum with the time-dependent density-functional theory (TDDFT) calculated ECD spectra.8 The geometry of 1 assigned from the X-ray analysis was re-optimized using DFT at the b3lyp/6-31+g(d, p) level in MeOH using the conductor-like polarizable calculation model (CPCM) by the GAUSSIAN 09 program.9a The energies, oscillator strengths, and rotational strengths (velocity) of the first 30 electronic excitations were calculated using the TDDFT methodology at the b3lyp/6-311+g(d,p) level in MeOH. The ECD spectrum was simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height,  = 0.2).9b The calculated ECD spectrum of model molecule 1a showed good agreement with the experimental curve of 1 (Figure 5). Therefore, the absolute configuration of 1 was determined to be 1S,3R,5S,6R,7S,8S,9S,12S,13R,17R,18R, 19R,20R,21R,25S,26R. Based on the foregoing evidence, the structure of 1 was elucidated as shown in Figure 1, named as penicipyrrodiether A. The full assignments of 13C and 1H NMR data (Table 1) of 1 were made based on a combination of COSY, HMBC, and NOE spectroscopical analyses. 7


13

C, 1H, HSQC, 1H-1H


Figure 5. Experimental ECD spectrum of penicipyrrodiether A (1, 200400 nm) in MeOH and the calculated ECD spectra of 1a and 1b at the b3lyp/6-311+g(d,p) level in MeOH (1a: 1S,3R,5S,6R,7S,8S,9S,12S,13R,17R,18R,19R,20R,21R,25S,26R of 1; 1b: 1R,3S,5R,6S,7R, 8R,9R,12R,13S,17S,18S,19S,20S,21S,25R,26S of 1). Biogenetically, penicipyrrodiether A (1) might be derived from two monomers of phenol A (2) and a dehydro-derivative (3) of GKK1032A2 (Scheme 1). Phenol A (2), a fungal metabolite10, was also isolated from the culture of strain ZZ380 in this study and 3 was a precursor of the fungal metabolites of GKK1032 analogues1,4 and pyrrospirones.2i The biosynthetic pathway of 3 and GKK1032A2 has been well elucidated.4 Intramolecular hemiketal formation10b of 2a, an oxidative product of 2, leads to the formation of 2b, and then intermolecular Michael addition11 of 3 with 2c (a tautomer of 2b) produces 3a. Finally, intramolecular hemiketal formation of 3a forms penicipyrrodiether A (1). Penicipyrrodiether A was tested for its antimicrobial activity in inhibiting the growth of methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans by micro broth dilution method.7c The results showed that penicipyrrodiether A exhibited antimicrobial activity with MIC values of 5.0 g/mL (7.02 M) for MRSA and 34.0 g/mL for E. coli. The positive control gentamicin showed activity with MIC values of 0.7 g/mL (1.47 M) for MRSA and 0.9 g/mL (1.88 M) for E. coli. It was reported that pyrrocidines AC had antimicrobial activity against S. aureus with MIC values in a 8


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range from 0.25 to 8.0 g/mL2d,2f and pyrrospirones CF, H, and I showed inhibitory activity against MRSA with MIC values of 2.019.0 g/mL.2i Scheme 1. Hypothetical Biosynthetic Pathways for 1

Penicipyrrodiether A was also evaluated for its activity against the proliferation of human glioma U87-MG, U251, SHG-44 cells, and rat glioma C6 cells by sulforhodamine B (SRB) assay.7a,7e Penicipyrrodiether A showed moderate antiglioma activity with IC50 values of 11.32 to 29.10 M, while the positive control doxorubicin (DOX) had activity with IC50 values of 0.70 to 9.61 M. 

CONCLUSION Penicipyrrodiether A, the first example of this type of fungal metabolite, was isolated

from a culture of marine-derived fungus Penicillium sp. ZZ380 in BMPM liquid medium. Its structure was unambiguously determined based on extensive NMR spectroscopic analyses, HRESIMS data, ECD calculation, and single-crystal X-ray diffraction. Penicipyrrodiether A might derive from two monomers of phenol A and a GKK1032 analogue through the key biosynthetic steps of oxidation, hemiketal formation, and Michael 9



addition. Penicipyrrodiether A showed good antibacterial activity against the drug-resistant pathogen MRSA. 

EXPERIMENTAL SECTION General experimental procedures. Optical rotation was measured on an Autopol I

polarimeter (Rudolph Research Analytical). CD spectrum was obtained on a JASCO J-815 spectropolarimeter. UV and IR spectra were recorded on a METASH UV-8000 spectrometer (Shanghai METASH Instruments Co. Ltd., China) and a NICOLET iS 10 FT-IR spectrometer (Thermo Scientific), respectively. HRESIMS and MS-MS data were obtained from an Agilent 6230 TOF LC/MS spectrometer and an AB Triple TOF 5600plus System (AB SCIEX, Framingham, USA), respectively. NMR spectra were acquired on a Bruker 500 spectrometer using standard pulse programs and acquisition parameters and chemical shifts were expressed in  (ppm). Octadecyl-functionalized silica gel (ODS, Cosmosil 75C18-Prep, Nacalai Tesque Inc., Japan) was used for column chromatography. HPLC separation was performed on a SHIMADZU LC-20AP prepared HPLC system with column A (CT-30, 280  30 mm, 10 m, Fuji-C18), or Agilent 1260 HPLC system with column B (Agilent Zorbax, 250 × 9.2 mm, 5 μm, SB-C18). All solvents used for this study were purchased from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Human glioma U87-MG (JDS-2568), U251 (XB-0439), SHG-44 (RX-J150) cells and rat glioma C6 (XB-003) cells were obtained from the Cell Bank of the Chinese Academy of Sciences. Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Escherichia coli ATCC 25922, and Candida albicans were gifts from Drs. Zhongjun Ma, Pinmei Wang, and Bin Wu, respectively. Doxorubicin (DOX, 98.0%) was ordered from Sigma-Aldrich, and gentamicin (99.6%) and amphotericin B (95.0%) from Meilune Biotechnology Co. Ltd. (Dalian, China). Fungal material. Strain ZZ380 was previously isolated from a sample of wild crab (Pachygrapsus crassipes) and identified as Penicillium sp. ZZ380 based on its ITS DNA sequence analysis.2i Culture of strain ZZ380. Colonies of the strain ZZ380 from the PDA (Potato Dextrose Agar) solid medium in plates were inoculated into a 500 mL Erlenmeyer flask containing 10


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250 mL of PDB (Potato Dextrose Broth) liquid medium and then incubated at 28°C for three days on a rotary shaker (180 rpm) to produce seed broth. The seed broth (5 mL) was inoculated into a 500 mL Erlenmeyer flask, which contains 250 mL of BMPM medium (glucose 20 g, glycerol 20 g, soy flour 10 g, cotton seed embryo meal 10 g, (NH4)2SO4 1 g, CaCO3 10 g, sea salt 35 g, tap water 1000 mL). All flasks were left in stationary state at 28°C for 30 days for incubation and 40.0 L of culture was prepared for this study. Isolation of penicipyrrodiether A (1) and phenol A (2). The 40.0 L culture was centrifuged to give fermentation broth and mycelia. The mycelia were extracted with MeOH three times to get a mycelia extract. The fermentation broth was partitioned with EtOAc three times to give an EtOAc extract. A mixture (10.0 g) of the mycelia extract and the EtOAc extract was fractionated by an ODS column eluting successively with 80% MeOH, 90% MeOH, and 100% MeOH (MeOH: H2O, v/v) to give six fractions (Frs. 1-6) based on the results of TLC analysis. Phenol A (2, 50.0 mg, tR 38.0 min) was obtained from Fr. 1 (0.51 g) by prepared HPLC separation using column A (MeOH: H2O, 45:55, v/v; flow rate 10 mL/min; UV 210 nm). Fr. 6 (0.35 g) was separated with column A (MeOH: H2O, 90:10, v/v; flow rate 15 mL/min; UV 235 nm) to give subfraction Fr. 6A (tR 40.5 min), which was purified by an Agilent 1260 HPLC system with column B (MeOH: H2O, 97:3, v/v; flow rate 1.5 mL/min; UV 235 nm) to afford penicipyrrodiether A (1, 9.0 mg, tR 21.5 min). Penicipyrrodiether A (1). Colorless plate crystals; molecular formula C43H53NO8; [α]20D +30.2° (c 0.20, MeOH); ECD (10 g/mL, MeOH) max () 229 (+66.28), 251 (42.13), 280 (+9.75), 318 (+2.73) nm; UV (MeOH) max (log ) 203 (4.46), 233 (4.24), 251 (4.15) nm; IR (CHCl3) max 3260, 2947, 2922, 2854, 1660, 1631, 1504, 1454, 1376, 1304, 1260, 1237, 1221, 1091, 1040, 1004, 961, 903, 808, 755, 641 cm-1; HRESIMS m/z [M+H]+ 712.3843 (calcd for C43H54NO8, 712.3849), [M+Na]+ 734.3661 (calcd for C43H53NNaO8, 734.3669), and [M+K]+ 750.3410 (calcd for C43H53KNO8, 750.3408). 13C (125 MHz) and 1

H (500 MHz) NMR data, see Table. 1. Phenol A (2): Colorless powder, [α]23D 32.1° (c 1.00, EtOH) {ref.

[10a]

36.8° (c 1.07,

EtOH)}. 1H NMR (500 MHz, DMSO-d6)  6.14a (1H, d, J = 2.3 Hz, H-4), 6.13a (1H, d, J = 2.3 Hz, H-6), 2.90 (1H, m, H-7), 3.66 (1H, m, H-8), 0.95 (3H, d, J = 6.3 Hz, H-9), 1.97 (3H, 11



s, H-10), 1.04 (d, J = 7.1 Hz, H-11), 8.91b (1H, s, OH-1), 8.76b (1H, s, OH-5), 4.36 (1H, d, J = 4.3 Hz, OH-8);

13

C NMR (125 MHz, DMSO-d6) C 155.6c (C, C-1), 112.7 (C, C-2),

144.6 (C, C-3), 104.7d (C-4), 155.2c (C, C-5), 100.0d (C-6), 41.4 (CH, C-7), 69.3 (CH, C-8), 19.4 (CH3, C-9), 10.7 (CH3, C-10), 15.6 (CH3, C-11). a-d Data with the same labels may be interchanged. Antimicrobial assay. The micro broth dilution method as described in the previous study7c was applied to determine the antimicrobial activity of 1 against the growth of MRSA, E. coli, and C. albicans. Gentamicin (an antibiotic against both Gram-positive and Gramnegative bacteria) and amphotericin B (an antifungal drug) were used as positive controls. The microorganism were cultured in MHB (Mueller-Hinton Broth, OXOID LTD.) medium in 96-well plates at a concentration of 1×106 CFU/mL. The MIC was determined after 12 h incubation at 37°C with tested compounds. Anti-glioma assay. Glioma U87-MG cells were cultured in MEM (Minimum Essential Medium, Gibco) with 10% FBS (Fetal Bovine Serum, PAA Laboratories Inc.), U251 and C6 cells in DMEM (Dulbeccos Modified Eagle Medium, Gibco), and SHG-44 cells in RPMI-1640 Medium (Roswell Park Memorial Institution 1640 Medium, Gibco). All cells were incubated at 37C in a humidified incubator with 5% CO2. Cells after the third generation were used for experiments. Sulforhodamine B (SRB) assay7a,7e was used to evaluate the activity of 1 suppressing the proliferation of glioma U87MG, U251, SHG44, and C6 cells. Doxorubicin (DOX) was used as a positive control. 

ASSOCIATED CONTENT

S Supporting Information ●

The Supporting Information is available free of charge on the ACS Publications website at DOI: Full spectroscopic spectra (NMR, HRESIMS, UV, and IR) of penicipyrrodiether A (1), 1H and 13C NMR spectra of phenol A (2) (PDF), X-ray crystallographic analyses of 1 (CIF), the optimized conformer and optimized Z-matrixes of 1a from ECD calculation. Accession Code

12


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CCDC 1846569 contains the supplementary crystallographic data for this paper. The data can be obtained by contacting the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033 or free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected]. 

AUTHOR INFORMATION

Corresponding Authors * Phone: +86-13675859706. E-mail: [email protected] or [email protected] ORCID Zhizhen Zhang: 0000-0002-8290-3507 Notes The authors declare no competing financial interest. 

ACKNOWLEDGMENTS

This research work was supported by the National Key R&D Program of China (No. 2017YFE0102200) and the National Natural Science Foundation of China (Nos. 81773587, 81773769, and 81273428). We thank Dr. Jianyang Pan (Pharmaceutical Informatics Institute of Zhejiang University) for performing the NMR spectrometry. 

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Bioactive Penicipyrrodiether A, an Adduct of GKK1032 Analogue and

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