2. Results and Discussion
An EtOAc extract prepared from a mass culture of
Penicillium sp. ZZ380 in PDB medium was separated by ODS column chromatography, followed by HPLC purification, to give compounds
9 and
11–
18. Compound
10 was isolated from a previous culture of ZZ380 in BMPM medium [
2]. On the basis of the nuclear magnetic resonance (NMR) data and specific rotation, as well as the comparison with the reported data, eight known compounds were identified as 2,4,5-trimethylresorcinol (
11) [
8], stoloniferol B (
12) [
9], coniochaetone E (
13) [
10], pinselin (
14) [
11], quinolactacin A
1 (
15) [
12], ergosterol (
16) [
13], penicitrinol A (
17) [
14] and B (
18) [
15]. Their
13C NMR data are presented in
Tables S1 and S2 (Supplementary Materials).
Compound
9 was obtained as colorless block crystals from MeOH and has a molecular formula C
32H
41NO
5 deduced from its high resolution electrospray ionization mass spectroscopy (HRESIMS) ions at
m/
z [M + H]
+ 520.3063 and [M + Na]
+ 542.2877 as well as its
13C NMR data. Analyses of its
1H,
13C,
1H-
1H COSY, HSQC and HMBC spectra (
Figures S1–S17) indicated that the structural parts of rings A–C, F and G for
9 and pyrrospirones C–I (
1–
7) are the same. However, the structures of
9 and pyrrospirones have significant differences. The first difference is the ring D with a five-membered ether ring for
9 and a cyclohexanone for pyrrospirones. The second difference is that
9 has no spiro junction for rings D and E. The five-membered ether ring D and its connections with rings C and E were confirmed by
1H-
1H COSY correlation (
Table 1 and
Figure 2) of H-8 (
δH 3.12, m) with H-13 (
δH 3.78, d,
J = 5.7 Hz) and HMBC correlations (
Table 1 and
Figure 2) of H-13 with C-8 (
δC 48.9), C-9 (
δC 87.5), C-11 (
δC 126.8), C-12 (
δC 48.6), C-14 (
δC 102.1), C-16 (
δC 139.2) and C-31 (
δC 22.0), H-15 (
δH 4.35, q,
J = 6.4 Hz) with C-11 and C-31 and H-19 (
δH 6.95, d,
J = 1.8 Hz) with C-14, C-17 (
δC 172.7) and C-18 (
δC 88.1). The locations of CH
3-31, CH
3-32 and OH-14 were also established by HMBC correlations of H
3-31 (
δH 1.35, s) with C-11, C-12, C-13 (
δC 56.3) and C-15 (
δC 79.4) and H
3-32 (
δH 1.26, d,
J = 6.4 Hz) with C-12 and C-15, as well as OH-14 (
δH 6.25, s) with C-14 and C-16. The planner structure of
9 was further confirmed by X-ray diffraction analysis (Cu Kα, CCDC deposition number 1868814, crystallized in MeOH,
Figure 3).
The relative stereochemistry of
9 was established based on the analyses of NOESY spectrum (
Figures S18 and S19) and coupling constants. As depicted in
Figure 2, NOE correlations of H-9 with H-5 and H-13, H
3-31 with H-7, H-13 and H-15, βH-20 with NH-17 and H-26 and H-25 with H-9 and H-26 indicated a β-orientation for these protons. Similarly, NOE correlations of H
3-29 with H-2, H-4 and H-8, H-8 with OH-14 and H-22 with αH-20 and H-23 were suggestive of an α-orientation for these protons. The large coupling constants of
3J4,
5 (11.3 Hz) and
3J7,
8 (13.2 Hz) also confirmed the
trans-juncture for A/B and B/C rings [
16]. NOE correlations of H-11 with H
3-30 and H-19 with OH-14 indicated a
Z-geometry for both of the double bonds at C
10–11 and C
16–19. The absolute configuration of
9 was determined as 2
R,4
S,5
R,6
S,7
S,8
S,9
S,12
S,13
R,14
R,18
R based on the result of the X-ray diffraction analysis (Cu Kα, Flack parameter 0.02). A computational method was also applied to assign the absolute stereochemistry of
9 by comparing its experimental electronic circular dichroism (ECD) spectrum with the calculated ECD spectra. The result (
Figure 4) showed that the calculated ECD spectrum of model molecule
9a was close to the experimental curve of
9. Based on the foregoing evidence, the structure of
9 was elucidated as a new alkaloid, named penicipyrroether A. Its
13C and
1H NMR data (
Table 1) were assigned based on a combination of HSQC,
1H-
1H COSY, HMBC and NOE spectroscopic analyses.
It has been reported that compound
20, a dehydro-derivative of GKK1032A
2 (
19) (
Figure 5), might be a precursor of the fungal metabolites of GKK1032s, pyrrospirones and penicipyrrodiether A [
1,
2,
3,
17]. Similarly, penicipyrroether A (
9) might be also derived from
20. A proposed biosynthetic pathway for penicipyrroether A (
9) was depicted in
Figure 5. After oxidation of the vinyl group of
20, the epoxide derivative (
21) was transformed into
22 and then an intramolecular hemiketal formation [
3,
15] of
22 forms penicipyrroether A (
9).
Compound
10 was obtained as a colorless amorphous powder and has a molecular formula C
30H
37NO
6 deduced from its HRESIMS ions at
m/
z [M + H]
+ 508.2711 and [M + Na]
+ 530.2531 as well as its
13C NMR data, which showed two fewer carbons, when compared to the structure of penicipyrroether A (
9). Detailed interpretation of
1H,
13C,
1H-
1H COSY, HSQC and HMBC spectra (
Figures S23–S38) demonstrated that compound
10 is an analogue of penicipyrroether A (
9) with the same structural part of rings A, B, F and G but possesses an epoxy moiety at C-10 and C-11 and a different five-membered ether ring D fused with ring E through a spiro carbon of C-15. In addition, the dehydro-pyrrolidinone moiety (ring E) in
9 was replaced by a pyrrolidinone moiety in
10. The presence of the epoxy moiety was confirmed by HMBC correlations (
Table 2 and
Figure 6) of H-7 (
δH 1.55, d,
J = 14.3 Hz) with C-10 (
δC 58.8) and C-11 (
δC 63.5), H-11 (
δH 2.46, s) with C-10, C-12 (
δC 81.3), C-13 (
δC 44.5), C-29 (
δC 21.0) and C-30 (
δC 26.0) and H
3-29 (
δH 1.20, s) with C-7 (
δC 49.9), C-10 and C-11. Similarly, the ether ring D and its connections with rings C and E was indicated by HMBC correlations of H-8 (
δH 2.79, m) with C-13 and C-14 (
δC 180.9), H-11 with C-12 and C-13, H-13 (
δH 3.21, d,
J = 8.0 Hz) with C-8 (
δC 39.2), C-11, C-12, C-14 and C-30, H-18 (
δH 1.99, 2.45, d,
J = 12.1Hz, each) with C-14, C-15 (
δC 79.7), C-16 (
δC 172.1), C-17 (
δC 86.2) and C-19 (
δC 45.0), H
3-30 (
δH 1.59, s) with C-11, C-12 and C-13 and NH-16 (
δH 8.76, s) with C-15, C-16, C-17 and C-18 (
δC 40.9).
The relative stereochemistry of
10 was assigned by analyses of the NOESY spectrum (
Figures S39 and S40) and coupling constants. NOE correlations (
Figure 6) of H-5 (
δH 1.22) with βH-3 (
δH 0.51) and H-9 (
δH 4.82), H-7 (
δH 1.55) with βH-1 (
δH 0.81) and H
3-30 (
δH 1.59), H-9 with H-13 (
δH 3.21) and H-24 (
δH 6.94), H-13 with H
3-30, NH-16 (
δH 8.76) with βH-19 (
δH 2.99) and H-25 (
δH 6.78) and H-25 with βH-19 and H-24 suggested a β-orientation for these protons; while NOE correlations of H-8 (
δH 2.79) with H
3-28 (
δH 1.05), H
3-28 with αH-1 (
δH 1.80), H-2 (
δH 1.82), H-4 (
δH 1.78) and H
3-29 (
δH 1.20), H
3-29 with H-11 (
δH 2.46), H-21 (
δH 6.92) with αH-19 (
δH 2.69) and H-22 (
δH 6.82) and H-18 (
δH 1.99) with OH-17 (
δH 6.24) proved an α-orientation for these protons. The
trans-juncture for A/B and B/C rings was also confirmed by the large coupling constants of
3J4,
5 (11.6 Hz) and
3J7,
8 (14.3 Hz) [
16]. The absolute configuration of
10 was determined as 2
R,4
S,5
R,6
R,7
R,8
S,9
S,10
R,11
R,12
R,13
R,15
R,17
R based on the result from ECD calculation (
Figure 7). Unfortunately, several efforts to obtain single crystal for X-ray diffraction were not successful because the structure of
10 was changed during the process of crystallization.
It was noted that the ketonic carbonyl at C-14 of
10 resonated at 180.9 ppm, which is unusual. In order to confirm this possibility, we conducted a computational calculation of its
13C chemical shifts [
18,
19,
20]. The calculated
13C NMR data (
Table S7) of
10 were in good agreement with its experimental values, with the corrected mean absolute error (CMAE) of 0.28 ppm and the correlation coefficient (
R2) of 0.9982 (
Figure 8) and the calculated
13C chemical shift for C-14 was 184.1 ppm, which was close to the experimental value of 180.9 ppm. Based on the foregoing evidence, the structure of
10 was determined as a new alkaloid, named pyrrospirone J. The full assignment of
13C and
1H NMR data (
Table 2) was made based on HSQC,
1H-
1H COSY, HMBC and NOE spectroscopic analyses.
New alkaloids—penicipyrroether A (
9) and pyrrospirone J (
10)—were evaluated for their activities in inhibiting the proliferation of human glioma U87MG and U251 cells through sulforhodamine B (SRB) assay [
21]. Doxorubicin (DOX, a chemotherapeutic drug) was used as a positive control. It has been found that both compounds had activity against different glioma cells with IC
50 values of 1.64–5.50 μM for
9 and 10.52–17.92 μM for
10 (
Table 3). The cytotoxicities of penicipyrroether A (
9) and DOX against normal human astrocytes were also tested and showed CC
50 values of 23.28 ± 1.05 μM for
9 and 8.57 ± 0.16 μM for DOX. The data indicated that the antiglioma activity of
9 was equivalent to (or slightly stronger than) the activity of the positive control DOX and the selective index (CC
50/IC
50) of 4.2–14.2 for
9 was higher than that of 1.1–7.1 for DOX.
The antibacterial activities of penicipyrroether A (
9) and pyrrospirone J (
10) against MRSA and
E.
coli were also determined by the micro broth dilution method [
22]. Gentamicin (an antibiotic against both Gram-positive and Gram-negative bacteria) and vancomycin (an antibiotic against MRSA) were used as positive controls. The results (
Table 3) showed that penicipyrroether A (
9) had good antibacterial activities with MIC values of 1.7 μg/mL against MRSA and 3.0 μg/mL against
E. coli. However, pyrrospirone J (
10) was inactive at a concentration of 50 μg/mL.
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotation, ultraviolet-visible (UV) and electronic circular dichroism (ECD) were measured on an Autopol I polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA), a METASH UV-8000 spectrometer (Shanghai METASH Instruments Co. Ltd., Shanghai, China) and a JASCO J-815 spectropolarimeter (JASCO Co. Tokyo, Japan), respectively. Infrared radiation (IR) spectra were recorded on a Bruker TENSOR II high performance FT-IR spectrometer (Bruker, Karlsruhe, Germany). HRESIMS data was obtained from an Agilent 6230 time-of-flight liquid chromatography–mass spectrometry (TOF LC-MS, Agilent, CA, USA). NMR spectra were acquired on a Bruker 500 spectrometer and chemical shifts were expressed in δ (ppm). Octadecyl silane (ODS, Cosmosil 75C18-Prep, Nacalai Tesque Inc., Kyoto, Japan) was applied for column chromatography. HPLC separation was conducted on a SHIMADZU LC-20AP prepared HPLC system with column A (Welch-20, 250 × 21 mm, 5 μm, XB-C18) or column B (CT-30, 280 × 30 mm, 10 μm, Fuji-C18). All solvents were ordered from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Human glioma U87MG (JDS-2568) and U251 (XB-0439) cells were purchased from the Cell Bank of the Chinese Academy of Sciences and normal human astrocytes (HA, Cat. No. 1800) from the ScienCell. Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 and Escherichia coli ATCC 25922 were gifts from Drs. Zhongjun Ma and Pinmei Wang, respectively. Gentamicin (99.6%) and vancomycin (>98.0%) were bought from Meilune Biotechnology Co. Ltd. (Dalian, China), doxorubicin (DOX, >98.0%) from Sigma-Aldrich, PDA (potato dextrose agar) from Baisi Biotechnology Co. Ltd. (Hangzhou, China) and MHB (Mueller-Hinton Broth) from Oxoid Ltd. PDB medium (potato dextrose broth, potato 100 g, glucose 10 g, sea salt 35 g, tap water 1000 mL) was made in the authors’ laboratory.
3.2. Marine Strain ZZ380
Strain ZZ380 was previously isolated from marine crab
Pachygrapsus crassipes and assigned as
Penicillium sp. ZZ380 by ITS DNA sequence analysis [
2].
3.3. Mass Culture of Strain ZZ380
Colonies of the strain ZZ380 from the PDA slant were inoculated into 250 mL of PDB liquid medium in a 500 mL Erlenmeyer flask and then incubated at 28 °C for three days on a rotary shaker (180 rpm) to produce seed broth. The seed broth of 5 mL was inoculated into a 500 mL Erlenmeyer flask, containing 250 mL of PDB medium and then incubated at 28 °C for 30 days under stationary state. A total of 55 L of culture were prepared for this study.
3.4. Isolation of Compounds 9–18
The 55 L culture of ZZ380 in PDB medium was centrifuged to give mycelia and broth. The mycelia were extracted with MeOH three times to get a MeOH extract. The broth was partitioned with EtOAc three times to give an EtOAc extract. A mixture (23.0 g) of the MeOH and EtOAc extract was fractionated by an ODS column eluting successively with 40%, 60%, 80% and 100% MeOH to afford four fractions (Frs. 1–4). Fr. 1 was further separated by an ODS column eluting with 35%, 45% and 55% MeOH to give three sub fractions (SFrs. 1a–1c). Through purification using prepared HPLC with column A at a flow rate of 12 mL/min, compounds 11 (4.2 mg, tR: 22.0 min, mobile phase: MeOH/H2O, 38/62, detector: 254 nm) and 12 (6.7 mg, tR: 31.0 min, mobile phase: MeOH/H2O, 38/62, detector: 254 nm) were obtained from SFr. 1a; compounds 15 (4.0 mg, tR: 33.0 min, mobile phase: MeOH/H2O, 45/55, detector: 210 nm), 14 (20.5 mg, tR: 37.0 min, mobile phase: MeOH/H2O, 53/47, detector: 254 nm), 18 (7.0 mg, tR: 37.0 min, mobile phase: MeOH/H2O, 66/34, detector: 254 nm) were obtained from SFr. 1b, SFr. 1c and Fr. 2, respectively. Fr. 3 and Fr. 4 were also separated by ODS column eluting with 85% or 95% MeOH to furnish SFrs. 3a and 3b or SFrs. 4a and 4b. Through purification using prepared HPLC with column B at a flow rate of 15 mL/min, compounds 13 (43.0 mg, tR: 19.0 min, mobile phase: MeOH/H2O, 80/20, detector: 210 nm) and 17 (9.5 mg, tR: 45.0 min, mobile phase: MeOH/H2O, 83/17, detector: 254 nm) were obtained from SFrs. 3a and 3b, respectively; while compounds 9 (3.1 mg, tR: 41.0 min, mobile phase: MeOH/H2O, 91/9, detector: 210 nm) and 16 (50.0 mg, tR: 62.0 min, mobile phase: MeOH/H2O, 98/2; 280 nm; tR 62 min; 50.0 mg) were obtained from SFrs. 4a and 4b, respectively.
Compound
10 was isolated from a previous culture of strain ZZ380 in BMPM medium [
2]. A crude extract was fractionated on an ODS column eluting successively with 80% MeOH, 90% MeOH and 100% MeOH to give six fractions (Frs. 1–6) based on the results of TLC analysis. Fr. 2 was re-separated by an ODS column eluting with 80% MeOH to give Frs. 2A and 2B. Fr. 2A was further separated by prepared HPLC with column B using mobile phase of acetonitrile/water (68:32) at a flow rate of 10 mL/min to give
10 (2.0 mg, t
R: 60.0 min) and Frs. 2A
1–2A
3.
Penicipyrroether A (
9): Colorless block crystals; molecular formula C
32H
41NO
5; [
α]
+ 71.4° (
c 0.10, MeOH); ECD (10 mg/L, MeOH) λ
max (Δε) 208 (−57.22), 233 (+64.65), 286 (+34.08) nm; UV (MeOH) λ
max (log ε) 202 (4.20), 229 (3.66), 278 (2.67) nm; IR (MeOH)
νmax 3375, 2924, 1672, 1437, 1408, 1239, 1098, 1013, 951 cm
−1;
13C (125 MHz) and
1H (500 MHz) NMR data, see
Table 1; HRESIMS
m/
z [M + H]
+ 520.3063 (calcd. for C
32H
42NO
5, 520.3063), [M + Na]
+ 542.2877 (calcd. for C
32H
41NNaO
5, 542.2882).
Pyrrospirone J (
10): Colorless amorphous powder; molecular formula C
30H
37NO
6; ECD (10 mg/L, MeOH) λ
max (Δε) 204 (−55.93), 223 (+59.59), 256 (−11.80), 293 (−17.92) nm; UV (MeOH) λ
max (log ε) 211 (4.40), 230 (4.21), 284 (3.36) nm; IR (MeOH)
νmax 3347, 2923, 2851, 1748, 1686, 1505, 1460, 1371, 1239, 1169, 1074, 937 cm
−1;
13C (125 MHz) and
1H (500 MHz) NMR data, see
Table 2; HRESIMS
m/
z [M + H]
+ 508.2711 (calcd. for C
30H
38NO
6 508.2699) and [M + Na]
+ 530.2531 (calcd. for C
30H
37NNaO
6 530.2519).
Crystal data of penicipyrroether A (
9): Colorless crystals of penicipyrroether A (
9) was obtained from MeOH. X-ray diffraction analysis was performed on an Xcalibur Atlas Gemini Ultra diffractometer (Agilent Technologies, CA, USA) with Cu Kα radiation (λ = 1.54184 Å) at 100 K. Structure was solved by direct method (SHELXL-2018) and refined with full-matrix least-squares on
F2 (ShelXL, Sheldrick, 2015). All non-hydrogen atoms were refined anisotropically and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters [
2]. Crystal data of penicipyrroether A (
9): C
32H
41NO
5 (M = 519.68), orthorhombic crystal (0.13 × 0.12 × 0.09 mm), space group P212121 (no. 19), unit cell dimensions
a = 6.32760(10) Å,
b = 19.8687(2) Å,
c = 25.0617(3) Å,
V = 3150.79(7) Å
3,
α = 90°,
β = 90°,
γ = 90°;
Z = 4;
Dcalced = 1.231 g/cm
3;
μ = 0.684 mm
−1; 18406 reflection measured (5.676° ≤ 2θ ≤ 147.088°); 6237 unique (
Rint = 0.0244,
Rsigma = 0.0209) which were used in all calculation; the final refinement (
I ≥ 2
σ (
I)) gave
R1 = 0.0418 and
wR2 = 0.1116 (all data); Flack parameter = 0.02 (5). Crystallographic data of penicipyrroether A (
9) have been deposited at the Cambridge Crystallographic Data Centre (deposition number: CCDC 1868814). Copies of the crystallographic data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, U.K. [fax (+44)1223-336-033; or e-mail:
[email protected]).
3.5. ECD Calculation
Monte Carlo conformational searches of compound
10 were conducted with the Spartan’ 10 software (v1.1.0, x 86, Wavefunction Inc., Irvine, CA, USA) using Merck Molecular Force Field (MMFF) and three conformers were obtained for ECD calculations. The X-ray CIF profile of
9 and the three conformer of
10 (
Tables S3 and S4 and Figures S44 and S45) were initially optimized at B3LYP/6-31g (d,p) level in MeOH using the conductor-like polarizable continuum model (CPCM). The theoretical ECD calculation was carried out in MeOH using Time-dependent Density functional theory (TD-DFT) at the B3LYP/6-31+g (d,p) level for all conformers of
9 and
10. Rotatory strengths for a total of 60 excited states for
9 (or 30 for
10) were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.2 eV for
9 (or 0.3 eV for
10).
3.6. 13C NMR Calculation
The previously described methods [
19,
20,
21] were used for
13C NMR calculation. Briefly, Monte Carlo conformational searches were carried out by means of Spartan’s 10 software using MMFF. Four conformers (
Table S8 and Figure S46) of compound
10 were obtained for NMR calculations. The conformers were initially optimized at B3LYP/6-31g (d,p) level in DMSO using the CPCM calculation model. Gauge-independent atomic orbital (GIAO) calculations of
13C NMR chemical shifts were performed by DFT at the mPW1PW91-SCRF (DMSO)/6-311+g(d,p) level with the CPCM calculation model in Gaussian 09 software (G09w, D01, Gaussian Inc., Wallingford, CT, USA). The calculated
13C NMR data of the lowest energy conformers for
10 were averaged based on the Boltzmann distribution theory and their relative Gibbs free energy.
3.7. Sulforhodamine B (SRB) Assay
The activity of the tested compounds in inhibiting the proliferation of human glioma U87-MG and U251 cells was determined by SRB assay [
22]. Doxorubicin (DOX) was used as a positive control. U87MG cells were cultured in MEM (Minimum Essential Medium, Gibco, Grand Island, NY, USA) with 10% FBS (Fetal Bovine Serum, PAA Laboratories Inc., Toronto, ON, Canada), U251 in DMEM (Dulbecco’s Modified Eagle Medium, Gibco) and normal human astrocytes (HA) in AM (Astrocyte Medium, ScienCell, Cat. No. 1801). All cells were incubated at 37 °C in a 5% CO
2 humidified incubator. The cultured cells after the third generation were used for experiment.
3.8. Antibacterial Activive Assay
The previously described micro broth dilution method [
3,
22] was used to evaluate the antibacterial activity of the tested compounds against the growth of MRSA and
E. coli. Vancomycin (an antibiotic against MRSA) and gentamicin (an antibiotic against Gram-positive and Gram-negative bacteria) were used as positive controls. The microorganisms were cultured in MHB medium in 96-well plates at a concentration of 1 × 10
6 CFU/mL. The MIC was determined after 12 h incubation at 37 °C with tested compounds.