Pallidopenillines: Polyketides from the Alga-Derived Fungus

Article pubs.acs.org/jnp

Pallidopenillines: Polyketides from the Alga-Derived Fungus Penicillium thomii Maire KMM 4675 Maria P. Sobolevskaya,† Elena V. Leshchenko,†,‡ Trinh P. T. Hoai,§ Vladimir A. Denisenko,† Sergey A. Dyshlovoy,†,‡,∥ Natalya N. Kirichuk,† Yuliya V. Khudyakova,† Natalya Yu. Kim,† Dmitrii V. Berdyshev,† Evgeny A. Pislyagin,† Aleksandra S. Kuzmich,† Andrey V. Gerasimenko,⊥ Roman S. Popov,† Gunhild von Amsberg,∥ Alexandr S. Antonov,† and Shamil Sh. Afiyatullov*,† †

G.B. Elyakov Pacific Institute of Bioorganic Chemistry and ⊥Institute of Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospect 100-let Vladivostoku 159, Vladivostok 690022, Russian Federation ‡ Far Eastern Federal University, Suhanova 8, Vladivostok 690950, Russian Federation § Nha Trang Institute of Technology Research and Application, Vietnam Academy of Science and Technology, Hanoi, Vietnam ∥ Department of Oncology, Hematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald-Tumorzentrum, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany S Supporting Information *

ABSTRACT: Eleven new polyketides, pallidopenillines 1−11, were isolated from the alga-derived fungus Penicillium thomii. The structures of these compounds were established based on spectroscopic methods. The absolute configuration of pallidopenilline A (1) as 4R, 5S, 8S, 9R, 10R, 13R was established using a combination of the modified Mosher’s method, X-ray analysis, and NOESY data. The absolute configurations of 2−5 were determined by time-dependent density functional theory calculations of the ECD spectra and ECD and NOESY data. It was shown that 1-acetylpallidopenilline A (2) and pallidopenilline G (10) inhibit the growth of colonies of 22Rv1 cells by 40% at 2 and 1 μM, respectively.

F

Si gel column chromatography and reversed-phase HPLC to yield individual compounds 1−11. The molecular formula of 1 was determined to be C15H24O4 based on the HRESIMS peak at m/z 291.1582 [M + Na]+ and was consistent with 13C NMR data. A close inspection of the 1 H NMR, 13C NMR, DEPT, and HSQC data of 1 (Tables 1 and 2) revealed the presence of a carbonyl group (δC 212.0), two methyl (δH 0.91, 0.93, δC 18.9, 25.7) groups, four methylenes (δC 28.4, 32.8, 49.9, and 56.1), including one that was oxygen-bearing, five methines (δH 1.24, 1.39, 1.63, 2.88, 2.53, δC 40.0, 38.9, 48.4, 61.9, 76.5), including one methine linked to an oxygen atom, one disubstituted double bond (δH 5.85, 5.41, δC 128.9, 135.6), and one oxygenated sp3 (δC 70.4) nonprotonated carbon. The COSY-45 data and the HMBC correlations H-4/C-5 (δC 38.9), C-10 (δC 48.4), C-12 (δC 135.6), C-13 (δC 70.4); H5/C-6 (δC 28.4), C-10, C-13; H-6b/C-7 (δC 32.8), C-8 (δC 40.0), C-10; H-7b/C-5, C-8, C-9; H-10/C-9, C-11 (δC 128.9), C-12; H-12/C-13; H3-15/C-7, C-8, C-9; OH-9/C-8, C-9, C10; and OH-13/C-4, C-13, C-14 revealed the presence of a decalin system in 1 and established a Δ11 double bond and the locations of the methyl group at C-8 and the hydroxy groups at

ungi isolated from marine environments have received great attention as prolific sources of chemically diverse bioactive metabolites.1,2 As part of our ongoing search for structurally novel and bioactive metabolites from marine grassand alga-derived fungi, we have previously isolated 10 new austalide meroterpenoids from Penicillium thomii KMM 4645 and Penicillium lividum KMM 4663 associated with the marine brown alga Sargassum miyabei.3 Some of the austalides exhibited significant inhibitory activity against endo-1,3-β-Dglucanase from a crystalline stalk of the marine mollusk Pseudocardium sachalinensis.3 Four new eudesmane-type sesquiterpenes, thomimarines A−D, which show an inhibitory effect on NO production in LPS-induced RAW 264.7 murine macrophages, were obtained from P. thomii KMM 4667 that was isolated from superficial mycobiota of the rhizome sea grass Zostera marina.4 On the basis of other successful studies like this, further alga-derived fungi were examined. We report herein the isolation, structure determination, and biological assay results of new pallidopenillines 1−11 from the marinederived fungus P. thomii KMM 4675 associated with the brown alga Sargassum pallidum.

■

RESULTS AND DISCUSSION The fungus was cultured for 21 days on rice medium. The EtOAc extract of the culture was purified by a combination of © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 7, 2016

A

DOI: 10.1021/acs.jnatprod.6b00624 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Mosher’s method and NOESY correlations (Figure 3). Esterification of 1 with (R)- and (S)-MTPA chloride occurred at the C-9 hydroxy group to give the bis(S)- and (R)-MTPA esters, respectively. The observed chemical shift differences Δδ(δS − δR) (Figure 4, Supporting Information S7-b) revealed the 9R configuration and hence allowed assignment of the absolute stereostructure of 1 with 4R, 5S, 8S, 9R, 10R, 13R configurations. Compound 1 was named pallidopenilline A. The molecular formula of 2 was determined to be C17H26O5 based on the HRESIMS peak at m/z 333.1678 [M + Na]+ and was consistent with the 13C NMR data. The general features of the 1H and 13C NMR spectra (Tables 1 and 2) of 2 showed a close similarity of the carbon chemical shifts to the ones for pallidopenilline A, with the exception of the C-1 and C-2 carbon signals. The HMBC correlations H3-2′ (δH 1.94)/C-1′ (δC 170.2), C-1 (δC 58.8), together with the molecular mass difference of 42 mass units between 1 and 2, indicated the presence of an acetoxy group at C-1 in 2 instead of a hydroxy group. The relative configuration of 2 was defined based on the observed NOESY correlations (Figure 3). The absolute configuration of 2 was established based on the electronic circular dichroism (ECD) data. Because the relative configuration of 2 was established based on the NMR data, the only goal of the ECD investigations was to distinguish between the two enantiomeric forms of 2: 4R,5S,8S,9R,10R,13R or 4S,5R,8R,9S,10S,13S. The conformational analysis for (4R,5S,8S,9R,10R,13R)-2 was performed using the B3LYP method, 6-31G(d) basis set, and the PCM model (Supporting Information S13-a). The trans connection of the A and B rings leads to high rigidity of the structure; hence, only the internal rotations of the two OH groups around the corresponding C− O bonds and different possible internal rotations of the 3acetoxy-1-oxopropyl substituent at C-4 were considered. The most stable conformations of (4R,5S,8S,9R,10R,13R)-2 were found to occur when the substituent at C-4 has a linear orientation. The next most stable conformations are the ones for which the main part of the substituent at C-4 is linear, and

C-9 and C-13. The COSY correlations between H2-1 and OH1, H2-2 along with HMBC correlations H2-1/C-2 (δC 49.9), C3 (δC 212.0); H-2b/C-4; and H3-14/C-4, C-12, C-13 indicated the presence of 3-hydroxy-1-oxopropyl (C-1−C-3 residue numbering) and methyl groups at C-4 and C-13 in 1 (Figure 1). Thus, the planar structure of 1 was elucidated. The molecular structure and relative configuration of 1 were unequivocally confirmed by X-ray crystallographic analysis, which was performed on a single crystal obtained by recrystallization from EtOH (Figure 2, Supporting Information S7-a). The absolute configuration of 1 was established by the modified

Table 1. 13C NMR Spectroscopic Data (176.04 MHz) for Pallidopenillines 1−5a 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1′ 2′ a

2

δC, type 56.1, 49.9, 212.0, 61.9, 38.9, 28.4, 32.8, 40.0, 76.5, 48.4, 128.9, 135.6, 70.4, 25.7, 18.9,

CH2 CH2 C CH CH CH2 CH2 CH CH CH CH CH C CH3 CH3

3

δC, type 58.8, 45.1, 210.2, 62.0, 38.7, 28.3, 32.8, 40.0, 76.5, 48.3, 125.9, 135.5, 70.4, 25.6, 18.8,

4

δC, type


58.8, 45.1, 210.1, 61.9, 40.5, 27.9, 28.8, 36.7, 34.9, 40.6, 129.2, 135.6, 70.8, 25.7, 68.4, 170.4, 20.6, 170.3, 20.5,

170.2, C 20.6, CH3

CH2 CH2 C CH CH CH2 CH2 CH CH2 CH CH CH C CH3 CH2 C CH3 C CH3

5

δC, type 58.8, 45.0, 210.7, 62.1, 41.3, 29.0, 28.3, 40.4, 35.3, 41.0, 129.6, 135.4, 70.8, 25.7, 66.3,

CH2 CH2 C CH CH CH2 CH2 CH CH2 CH CH CH C CH3 CH2

δC, type 56.1, 49.9, 211.9, 61.8, 40.6, 28.2, 28.8, 36.9, 35.1, 40.7, 129.2, 135.8, 71.1, 25.9, 68.5, 170.7, 20.7,

CH2 CH2 C CH CH CH2 CH2 CH CH2 CH CH CH C CH3 CH2 C CH3

170.5, C 20.6, CH3

Chemical shifts referenced to DMSO-d6. B


C

a

δH (J in Hz)

4.40, t (5.2) 4.62, d (6.8) 4.92, s 4.65, d (6.7) 4.98, s

1.94, s

0.93, s 0.91, d (6.4)

d (11.6) dddd (11.6, 11.5, 10.5, 2.8) dddd (12.3, 12.1, 11,5, 3.0) dq (12.3, 3.0) dddd (13.2, 12.1, 3.7, 3.1) m m dt (10.2, 6.0)

0.93 s 0.91, d (6.4)

2.86, 1.41, 0.86, 1.47, 0.96, 1.60, 1.25, 2.54, 1.63, tt (10.4, 2.2) 5.85, dd (10.1, 1.6) 5.42, dd (10.1, 2.6)

d (11.6) dddd (11.6, 11.6, 10.0, 3.0) m dq (11.8, 3.1) m m m dt (7.0, 10.0)

1.63, tt (10.0, 2.3) 5.85, dd (10.0, 1.5) 5.41, dd (10.0, 5.8)

2.88, 1.39, 0.85, 1.47, 0.94, 1.59, 1.24, 2.53,

4.17, m 2.74, ddd (18.0, 6.0, 6.0) 3.01, ddd (18.0, 7.1, 6.0)

δH (J in Hz)

3.60, m 2.61, ddd (16.7, 6.5, 6.5) 2.76, ddd (16.7, 6.5, 6.5)

Chemical shifts referenced to DMSO-d6.

10 11 12 13 14 15 16 17 1′ 2′ 1-OH 9-OH 13-OH 14-OH

8 9

7

3 4 5 6

1 2

position

2

1

4.20, m 5.04, brs

1.96, s

5.03, s

1.95, s

4.35, brs

d (11.6) m m m m m m q (12.5) m m d (9.9) dd (9.9, 2.7)

2.01, s

2.86, 1.33, 0.86, 1.68, 1.59, 0.86, 1.44, 0.66, 1.77, 1.77, 5.32, 5.38, 0.95, s 3.19, brs

m q (12.3) dq (12.3, 2.9) m dd (9.9, 1.4) dd (9.9, 2.7)

d (11.6) dddd (11.6, 11.5, 11.2, 3.1) dddd (12.3, 11.5, 11.0, 3.0) dq (12.3, 3.0) m

δH (J in Hz)

4 4.18, m 2.71, ddd (18.0, 6.0, 6.0) 3.01, ddd (18.0, 6.9, 6.0)

0.97, s 3.83, ddd (10.8, 6.4, 6.2)

1.69, 0.77, 1.77, 1.81, 5.33, 5.41,

2.87, 1.35, 0.90, 1.62, 1.67,

4.20, m 2.75, ddd (18.0, 6.9, 5.9) 3.03, ddd (18.0, 7.0, 6.0)

δH (J in Hz)

3

Table 2. 1H NMR Spectroscopic Data (700.13 MHz) for Pallidopenillines 1−6a

d (11.4) dddd (11.4, 11.0, 10.3, 2.5) m m m m m q (11.9) m m dd (9.7, 1.5) dd (9.7, 2.6)

4.9,7 s

4.41, t (6.3)

2.00, s

0.95, s 3.82, dd (6.0, 1.6)

2.87, 1.33, 0.89, 1.61, 0.91, 1.66, 1.68, 0.77, 1.75, 1.80, 5.31, 5.39,

3.61, m 2.61, ddd (16.9, 6.3, 6.3) 2.76, ddd (16.9, 6.3, 6.3)

δH (J in Hz)

5

d (11.7) dddd (11.7, 11.6, 11.3, 2.6) dddd (12.5, 12.5, 11.6, 2.6) dq (12.5, 3.2) dddd (12.8, 12.6,12.5, 3.2) m tt (12.6, 3.6) q (12.6) brd (12.6) m dd (9.9, 1.2) dd (9.9, 2.6) 0.95, s

2.87, 1.33, 0.90, 1.62, 1.23, 1.85, 2.28, 1.05, 1.94, 1.81, 5.32, 5.40,

3.61, m 2.61, ddd (16.7, 6.5, 6.5) 2.76, ddd (16.7, 6.5, 6.5)

δH (J in Hz)

6

Journal of Natural Products Article



Article

absolute configuration 4R,5S,8S,9R,10R,13R for 2. Compound 2 was named 1-acetylpallidopenilline A. The molecular formula of 3 was determined to be C19H28O6 based on HRESIMS and 13C NMR data. The general features of the 1H and 13C NMR spectra (Tables 1 and 2) of 3 resembled the spectra of 2 with the exception of the C-5−C-10 and C-14 carbon signals. The COSY and HSQC spectra of 3 revealed the partial connectivity sequence of the protons in the A ring as CH2(15)−CH(8)−CH2(9)−CH(10). These data and the HMBC correlations H-8 (δH 1.69)/C-9 (δC 34.9), C-10 (δC 40.6), C-15 (δC 68.4); H2-15 (δH 3.83)/C-16 (δC 170.4); and H3-17 (δH 2.01)/C-15, C-16 established an A ring structure lacking the hydroxy group at C-9, but with an acetoxymethyl group at C-8. The NOESY correlations H-4/H-10, OH-13; H5/H3-14; and H-8/H-10 indicated the trans-ring fusion of the A and B rings, a β-orientation of CH3-14, an acetoxymethyl group at C-8, and the α-orientation for OH-13. The absolute configuration of 3 was established based on the ECD data. The experimental ECD spectra for 3 and 2 were qualitatively similar (Experimental Section). We calculated the theoretical statistically averaged ECD spectrum for 3 using the same procedure as for 2. The only exception was the larger basis set used to calculate the ECD spectra for individual rotameric forms: B3LYP/6-311+G(d,p)//B3LYP/6-31G(d) (Supporting Information S19-a). Thus, the absolute configuration of 3 was established as 4R,5S,8S,10R,13R. Compound 3 was named pallidopenilline B. The molecular formula of 4 was determined to be C17H26O5 based on the HRESIMS peak at m/z 333.1677 [M + Na]+ and 13 C NMR data. The 1H and 13C NMR data observed for 4 matched the data reported for pallidopenilline B (3) with the exception of the C-8 and C-15 proton and carbon signals (Tables 1 and 2). These data, together with the molecular mass difference of 42 mass units between 3 and 4, indicated the presence of a hydroxymethyl group at C-8 in 4. Compound 4 showed characteristic Cotton effects (CEs) at λ192 +0.26, λ204 −0.72, and λ294 −0.85 in the ECD spectrum in hexane solution, which were in good agreement with the results for 2 and 3. The only exception concerns the existence of a low-intensity broad band, corresponding to a positive Cotton effect, in the region 215 ≤ λ ≤ 250 nm. These data made it possible to establish the absolute configuration of 4 as 4R,5S,8S,10R,13R. Compound 4 was named 15-deacetylpallidopenilline B. The molecular formula of 5 was determined to be C17H26O5 based on the HRESIMS peak at m/z 333.1677 [M + Na]+ and the 13C NMR data. The structure and location of the 3hydroxy-1-oxopropyl group at C-4 in 5 were established based on the HMBC and COSY correlation as for 1. Compound 5 showed characteristic CEs at λ192 +0.46, λ204 −0.15, and λ294 −0.25 in the ECD spectrum in hexane solution, which were in good agreement with the results for 2, 3, and 4. These data made it possible to establish the absolute configuration of 5 as 4R,5S,8S,10R,13R. Compound 5 was named 1-deacetylpallidopenilline B. The molecular formula of 6 was determined to be C15H22O5 based on HRESIMS and 13C NMR data. The NMR data (Tables 2 and 3) observed for 6 closely resembled the data for 5 with the exception of the proton and carbon signals at the C8 position. The HMBC correlations H-9 (δH 1.05)/C-8 (δC 42.5) and H-8/C-14(δC 176.4) revealed the structure had a carboxylic acid at the C-8 position. The relative configuration of 6 was defined based on the observed NOESY correlations. Compound 6 was named pallidopenilline C.

Figure 1. COSY and selected HMBC correlations in 1.

Figure 2. Molecular structure of compound 1 (α-C15H24O4). Displacement ellipsoids are shown at the 50% probability level.

Figure 3. Selected NOESY correlations in 1 and 2.

Figure 4. Δδ (δS − δR) values (in ppm) for the bis-MPTA ester of 1.

only the acyl group is rotated by approximately θ ≈ ±90° around the C-1−C-2 bond. On the basis of this conformational analysis, the rotameric forms of 4R,5S,8S,9R,10R,13R, for which the calculated Gibbs free energies are in the region ΔGim ≤ 3 kcal·mol−1, were selected for ECD calculations. The experimental and calculated statistically averaged ECD spectra for 2 are compared in Figure 5. Both spectra are qualitatively similar in the representative region above 180 nm, which proves the D



Article

Figure 5. Experimental and calculated ECD spectra of 2.

Table 3. 13C NMR Spectroscopic Data (176.04 MHz) for Pallidopenillines 6−11a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1′ 2′ a

6

7

8

9

10

11

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

56.1, 49.9, 212.3, 61.8, 40.2, 28.0, 28.5, 42.5, 34.5, 40.5, 129.0, 136.1, 70.9, 25.8, 176.4,

CH2 CH2 C CH CH CH2 CH2 CH CH2 CH CH CH C CH3 C

58.9, 45.1, 210.6, 62.1, 40.3, 24.5, 38.4, 67.7, 44.6, 36.4, 130.1, 135.6, 70.9, 25.8, 31.4,


170.7, C 20.6, CH3

58.9, 45.3, 210.0, 60.1, 33.8, 29.6, 32.0, 39.3, 73.9, 45.2, 61.4, 76.9, 71.2, 23.9, 18.9,


170.3, C 20.5, CH3

59.1, 45.5, 211.5, 59.9, 34.4, 30.5, 34.3, 32.2, 37.0, 39.3, 73.1, 77.2, 72.4, 22.2, 22.8,


170.2, C 20.9, CH3

59.0, 37.8, 210.2, 62.8, 32.1, 30.5, 32.0, 38.9, 72.9, 48.5, 60.6, 128.4, 132.2, 20.8, 19.2,


55.9, 42.4, 211.5, 62.9, 32.7, 30.4, 31.1, 36.2, 28.4, 40.9, 64.8, 128.2, 132.9, 21.1, 68.9, 170.4, 20.0,

CH2 CH2 C CH CH CH2 CH2 CH CH2 CH CH, CH C CH3 CH2 C CH3

170.2, C 20.6, CH3


isotopic peaks for M+2 indicates the presence of the chlorine atom in 8 (for example, the ratio of the peak intensities I(397.1197)/I(399.1171): 1.55, calcd for C17H27Cl2O6: 1.49). The HMBC correlations established the structure of the A ring as well as the linkage of a 3-acetoxy-1-oxopropyl moiety at C-4 as in 1-acetylpallidopenilline A (2). The coupling constants of H-11 (δH 4.57, t, J = 3.0 Hz) and H-12 (δH 3.53, dd, J = 4.0, 3.0 Hz), deshielded chemical shifts of C-11 (δC 61.4) and C-12 (δC 76.9), and HMBC correlations H-12 (δH 3.53)/C-10 (δC 45.2), C-11 (δC 61.4), C-13 (δC 71.2), C-14 (δC 23.9); OH-12 (δH 5.78)/C-11, C-12 (δC 76.9), C-13; and OH-13 (δH 4.48)/ C-4 (δC 60.1), C-13, C-14 established the structure of the B ring, including the C-11 location for the chlorine atom and the positions of the methyl and hydroxy groups at C-13 and the hydroxy group at C-12. The NOESY correlations H-4/H-10,

The molecular formula of 7 was determined to be C17H26O5 based on HRESIMS and 13C NMR data. The 1H and 13C NMR spectra (Tables 3 and 4) for this compound were very similar to those obtained for pallidopenilline B (3) with the exception of the C-6−C-10 carbon and proton signals. The HMBC correlations H-9 (δH 1.56)/C-8 (δC 67.7), C-10 (δC 36.4) and H3-14 (δH 1.07)/C-7 (δC 38.4), C-8, C-9 (δC 44.6) established the structure of 7 including the hydroxy and methyl groups at the C-8 position. The relative configuration of 7 was defined based on the observed NOESY correlations. Compound 7 was named pallidopenilline D. The molecular formula of 8 was deduced to be C17H27ClO6 based on the [M − H]− ion peak at m/z 361.1427 and the [M + Cl]− ion peak at m/z 397.1179 in the (−)HRESIMS spectrum and on the 13C NMR data. The intensity of the E



Article

Table 4. 1H NMR Spectroscopic Data (700.13 MHz) for Pallidopenillines 7−11a 7

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1′ 2′ 1-OH 9-OH 11-OH 12-OH 13-OH 14-OH a

9

10

11

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

4.19, m 2.75, ddd (18.0, 6.0, 6.0) 3.01, ddd (18.0, 6.0, 6.0)

4.16, m 2.74, ddd (18.0, 6.2, 6.2) 3.02, ddd (18.0, 6.2, 6.2)

4.15, m 2.67, ddd (17.8, 6.4, 6.4) 2.97, ddd (17.8, 6.4. 6.4)

4.17, m 2.66, ddd (18.0, 6.0, 6.0) 2.87, ddd (18.0, 6.0, 6.0)

3.60, m 2.49, ddd (17.1, 6.2, 6.2) 2.64, ddd (17.1, 6.2, 6.2)

2.86, 1.30, 1.20, 1.30, 1.20, 1.50,

d (11.4) m m m m dt (12.6, 2.5)

1.01, 1.56, 2.19, 5.25, 5.38,

t (13.0) dt (13.0, 2.7) m dd (9.8, 1.7) dd (9.8, 2.8)

2.80, 1.65, 0.85, 1.39, 0.85, 1.53, 1.26, 2.95,

2.64, 1.83, 0.97, 1.46, 0.88, 1.56, 1.26, 3.07,

1.70, m 4.57, t (3.0) 3.53, dd (4.0, 3.0)

2.69, 1.49, 1.42, 0.75, 0.72, 1.49, 1.33, 0.75, 1.42, 1.41, 3.48, 3.25,

0.96, m 4.15, m 5.78, m

2.55, 1.75, 0.97, 1.60, 1.20, 1.53, 1.62, 0.86, 1.62, 1.17, 3.72, 5.78,

1.11, s 0.92, d (6.3)

1.02, s 0.85, d (6.3)

1.53, s 0.93, d (6.3)

1.51, s 3.83, dd (6.3, 1.3)

position 1 2

8

0.95, s 1.07, s

d (10.7) m m m m m m dt (9.4, 6.8)

d (11.5) m m m m m m m m m m d (3.0)

δH (J in Hz)

d (11.0) dddd (11.8, 11.3, 11.0, 3.6) m m m m m m

1.96, s 1.94, s

δH (J in Hz)

d (10.5) dddd (11.3, 11.3, 10.5, 3.5) m m q (12.0) m m m m m brs m

2.00, s 1.93, s

1.93, s 4.48, brs

4.61, d (6.8) 5.78, d (4.0) 4.48, s

4.58, brd (3.0) 4.88, brs 4.25, brs

4.22, d (6.5) 4.34, d (5.2)

4.50, brs


the structure of the B ring, including the position of the double bond Δ12 and the location of the hydroxy and methyl groups at C-11 and C-13. The relative configuration of 10 was elucidated based on the NOESY correlations H-5/H-9, H3-14; H-4/H-10; and H-8/H-10. Compound 10 was named pallidopenilline G. The molecular formula of 11 was determined to be C17H26O5 based on the HRESIMS peak at m/z 333.1672 [M + Na]+ and 13 C NMR data. The structures of the B ring and the side chain were established based on HMBC correlations as for pallidopenilline G (10). The HMBC correlations assigned the structure of the A ring and indicated an acetoxymethyl group at C-8. The relative configuration of 11 was elucidated based on NOESY correlations. Compound 11 was named pallidopenilline H. Compounds 1, 5, and 7−11 were examined for their cytotoxic activity against the human prostate cancer 22Rv1, PC3, and LNCaP cell lines. Pallidopenilline G (10) exhibited cytotoxicity against the 22Rv1 cell line with an IC50 value of 9.8 μM. The activity of these pallidopenillines with respect to the formation and growth of 22Rv1 cell colonies was also studied using the soft agar method. The compounds 1-acetylpallidopenilline A (2) and pallidopenilline G (10) were shown to inhibit the colony growth of these cells by 40% at 2 and 1 μM, respectively. Pallidopenillines 1−5 were assayed for their cytotoxic activity against the MEL-28 and HCT-116 cell lines. None of the compounds exhibited cytotoxity at concentrations up to 100 μM). The compounds 15-deacetylpallidopenilline B (4), pallidopenilline E (8), and pallidopenilline G (10) at a concentration

OH-12, OH-13; H-5/H-9, H3-14; H-10/OH-9; and OH-9/H8, H-11 indicated the trans-ring fusion of the A and B rings, a βorientation of the chlorine atom, CH3-14, and CH3-15, and an α-orientation of OH-9, OH-12, and OH-13 in 8. Compound 8 was named pallidopenilline E. The molecular formula of 9 was determined to be C17H28O6 based on HRESIMS and 13C NMR data. The correlations observed in the COSY and HSQC spectra and HMBC H-7/C15 and H3-15/C-8, C-9 established the structure of the A ring and indicated the presence of a methyl group at C-8. The HMBC correlations H-4/C-5 (δC 34.4), C-13 (δC 72.4), C-14 (δC 22.2); H-12/C-4 (δC 59.9), C-10 (δC 40.2), C-11 (δC 73.1), C-13, C-14; OH-11/C-10; OH-13/C-14; and H3-14/C-13 proved the structure of the B ring and confirmed the locations of the hydroxy groups at C-11, C-12, and C-13 and a methyl group at C-13. The structure and position of the 3-acetoxy-1oxopropyl group at C-4 in 9 were established based on the HMBC and COSY correlation, as for 8. The relative configurations of the C-4, C-5, C-8, C-10, C-11, C-12, and C-13 stereogenic centers in 9 were the same as in 8 based on NOESY correlations. Compound 9 was named pallidopenilline F. The molecular formula of 10 was determined to be C17H26O5 based on HRESIMS and 13C NMR data. The structures of the A ring and a 3-acetoxy-1-oxopropyl moiety in 10 were established to be the same as in pallidopenilline E (8). The COSY-45 data and the HMBC correlations H-4/C-13 (δC 132.2); H-12/C-4 (δC 62.8), C-10 (δC 48.5), C-11 (δC 60.6), C-14 (δC 20.8); and H3-14/C-4, C-13 (δC 132.2) established F



Article

Pallidopenilline B (3): colorless solid, [α]20D −29 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 195 (2.78), nm; ECD (c 0.05 mg/mL, hexane) λmax (Δε) 192 (+1.38), 204 (−0.79), 294 (−0.68) nm; IR (CHCl3) νmax 3597, 1735, 1714, 1602, 1382, 1369, 1258 cm−1; 13C and 1H NMR data, Tables 1 and 2; HRESIMS m/z 375.1787 [M + Na]+ (calcd for C19H28O6Na, 375.1778). 15-Deacetylpallidopenilline B (4): colorless solid, [α]20D −96 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 195 (3.49) nm; ECD (c 0.05 mg/mL, hexane) λmax (Δε) 192 (+0.26), 204 (−0.72), 222 (+0.15), 294 (−0.85) nm; IR (CHCl3) νmax 3598, 1730, 1725, 1383, 1369, 1603, 1259, 1073 cm−1; 13C and 1H NMR data, Tables 1 and 2; HRESIMS m/z 333.1677 [M + Na]− (calcd for C17H26O5Na, 333.1672). 1-Deacetylpallidopenilline B (5): colorless solid, [α]20D −49 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 194 (2.89) nm; ECD (c 0.05 mg/mL, hexane) λmax (Δε) 192 (+0.46), 204 (−0.15), 294 (−0.25) nm; IR (CHCl3) νmax 3593, 1726, 1703, 1658, 1604, 1389, 1367, 1264, 1058 cm−1; 13C and 1H NMR data, Tables 1 and 2; HRESIMS m/z 333.1689 [M + Na]+ (calcd for C17H26O5Na, 333.1672). Pallidopenilline C (6): colorless solid, [α]20D −28 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 195 (3.28) nm; ECD (c 0.15 mg/mL, MeOH) λmax (Δε) 220 (−1.31), 243 (+0.39), 295 (−1.96) nm; IR (CHCl3) νmax 3604, 1739, 1703, 1602, 1456, 1251, 1054 cm−1; 13C and 1H NMR data, Tables 2 and 3; HRESIMS m/z 317.1135 [M + Cl]− (calcd for C15H2235ClO5, 317.1154), 305.1363 [M + Na]+ (calcd for C15H22O5Na, 305.1359). Pallidopenilline D (7): colorless solid, [α]20D −10 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 195 (2.92) nm; ECD (c 0.38 mg/mL, MeOH) λmax (Δε) 212 (+0.14), 224 (−0.34), 295 (−1.36) nm; IR (CHCl3) νmax 3605, 1734, 1707, 1602, 1253, 1054 cm−1; 13C and 1H NMR data, Tables 3 and 4; HRESIMS m/z 333.1681 [M + Na]+ (calcd for C17H26O5Na, 333.1672). Pallidopenilline E (8): colorless solid, [α]20D −52 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 195 (2.78) nm; ECD (c 0.55 mg/mL, MeOH) λmax (Δε) 202 (+0.42), 224 (−0.43), 293 (−3.04) nm; IR (CHCl3) νmax 3606, 1734, 1716, 1602, 1415, 1254, 1051 cm−1; 13C and 1H NMR data, Tables 3 and 4; HRESIMS m/z 397.1197 [M + Cl]− (calcd for C17H2735Cl2O6, 397.1190); m/z 361.1427 [M − H]− (calcd for C17H2635ClO6, 361.1423). Pallidopenilline F (9): colorless solid, [α]20D −6 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 197 (3.48) nm; ECD (c 0.41 mg/mL, MeOH) λmax (Δε) 218 (+0.51), 258 (−0.33), 293 (−0.46) nm; IR (CHCl3) νmax 3604, 1732, 1703, 1602, 1456, 1251, 1054 cm−1; 13C and 1H NMR data, Tables 3 and 4; HRESIMS m/z 351.1778 [M + Na]+ (calcd for C17H28O6Na, 351.1764). Pallidopenilline G (10): colorless solid, [α]20D −45 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 195 (3.02) nm; ECD (c 0.26 mg/mL, MeOH) λmax (Δε) 195 (−2.83), 213 (+1.41), 243 (−0.05), 293 (+1.31) nm; IR (CHCl3) νmax 3606, 1735, 1713, 1670, 1602, 1255, 1052 cm−1; 13C and 1H NMR data, Tables 3 and 4; HRESIMS m/z 333.1680 [M + Na]+ (calcd for C17H26O5Na, 333.1672). Pallidopenilline H (11): colorless solid, [α]20D −4 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 195 (3.02), 280 (2.47) nm; ECD (c 0.51 mg/mL, MeOH) λmax (Δε) 197 (−5.50), 213 (+1.47), 295 (+2.36) nm; (CHCl3) νmax 3603, 1735, 1714, 1602, 1382, 1369, 1257 cm−1; 13 C and 1H NMR data, Tables 3 and 4; HRESIMS m/z 333.1672 [M + Na]+ (calcd for C17H26O5Na, 333.1658). Preparation of Bis(S)-MTPA and (R)-MTPA Esters of Pallidopenilline A (1). To a solution of 1 (2.0 mg) in pyridine were added 4-dimethylaminopyridine (a few crystals) and (R)-MTPACl (20 μL) at room temperature, and the mixture was stirred for 24 h. After evaporation of the solvent, the residue was purified by HPLC on an Ultrasphere Si (5 μm, 4.6 × 250 mm) column eluting with n-hexane− EtOAc (3:1) to afford the bis(S)-MTPA ester of 1 (1a). The bis(R)MTPA ester of 1 (1b) was prepared similarly using (S)-MTPACl. 1 H NMR data of bis(R, S)-MTPA esters of 1, Supporting Information S7-b; HRESIMS (1a) m/z 723.2328 [M + Na]+ (calcd for C35H38F6O8Na, 723.2363); HRESIMS (1b) m/z 699.2367 [M − H]− (calcd for C35H37F6O8, 699.2398).

of 10 μM induced a significant down-regulation of ROS production in macrophages stimulated with lipopolysaccharide (LPS). The ROS level in these cells was decreased by 27 ± 1 (p < 0.01), 37 ± 3 (p < 0.01), and 36 ± 2 (p < 0.01) percent, respectively, compared to control cells pretreated with LPS.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 343 polarimeter, and melting points were determined with a Leica Galen III instrument. UV spectra were recorded on a Shimadzu UV-1601PC spectrometer in MeOH. ECD spectra were measured with a Chirascan-Plus CD spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 and DMSO-d6 on Bruker Avance-500 and Avance III-700 spectrometers operating at 500.13 and 125.77 MHz and 700.13 and 176.04 MHz, respectively. Spectra were referenced to DMSO solvent signals (1H 2.50 ppm, 13C 39.6 ppm). HRESIMS spectra were measured on an Agilent 6510 QTOF LC mass spectrometer. Low-pressure liquid column chromatography was performed using Si gel L (40/100 μm, Sorbpolimer). Glass plates (4.5 × 6.0 cm) precoated with Si gel (5−17 μm, Sorbfil) were used for thin-layer chromatography. Preparative HPLC was performed on a BeckmanAltex chromatograph, using a YMC-Pack ODS-AM (5 μm, 10 × 250 mm) column with an RIDK-122 refractometer. Fungal Strain. The fungal strain Penicillium thomii KMM 4675 was isolated from superficial mycobiota of the brown alga Sargassum pallidum (Novik Bay, Russky Island, The Sea of Japan) and identified based on morphological evaluation by one of the authors (N.N. K.). The strain is stored at the Collection of Marine Microorganisms, G.B. Elyakov Pacific Institute of Bioorganic Chemistry (PIBOC), Vladivostok, Russia. Cultivation of P. thomii. The fungus was grown in stationary culture at 22 °C for 21 days in 20 Erlenmeyer flasks (500 mL), each flask containing 20 g of rice, 20 mg of yeast extract, 10 mg of KH2PO4, and 40 mL of natural seawater from the Marine Experimental Station of PIBOC, Troitsa (Trinity) Bay, Sea of Japan. Extraction and Isolation. At the end of the incubation period, the mycelium and medium were homogenized and extracted with EtOAc (8 L). The fungal extract was concentrated to dryness. The residue was dissolved in 20% MeOH−H2O (1 L) and extracted with n-hexane (0.5 L × 3) and EtOAc (0.5 L × 3). After evaporation of the EtOAc layer, the residual material (2.1 g, P. thomii) was passed over a silica column (4 × 20 cm), which was eluted first with n-hexane (1.5 L) followed by a step gradient from 5% to 100% EtOAc in n-hexane (total volume 18 L). Fractions of 300 mL each were collected and combined on the basis of TLC (Si gel, toluene−2-propanol, 6:1 and 3:1, v/v). The n-hexane−EtOAc (4:1, 0.8 L, 55 mg; 3:1, 0.6 L, 125 mg; and 7:3, 0.8 L,75 mg) eluates were purified by RP HPLC on a YMC-Pack ODS-AM C-18 column eluting with MeOH−H2O (65:35) to yield 2 (58.8 mg), 3 (4.0 mg), 4 (1.6 mg), 5 (12.4 mg), 7 (3.8 mg), 8 (2.8 mg), and 10 (3.5 mg). The n-hexane−EtOAc (13:7, 0.8 L, 68 mg and 3:2, 0.6 L, 120 mg) eluates were purified by RP HPLC on a YMC-Pack ODS-AM C-18 column eluted with MeOH−H2O (55:45) to yield 6 (4.5 mg), 9 (1.5 mg), and 11 (5.5 mg). Crystallization of the dried residue of the n-hexane−EtOAc (3:2) eluate from EtOH gave colorless needles of 1 (60 mg). Pallidopenilline A (1): colorless crystals (EtOH), mp 173−175 °C; [α]20D −21 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 195 (3.21) nm; ECD (c 0.05 mg/mL, hexane) λmax (Δε) 207 (+0.24), 293 (−0.06) nm; IR (CHCl3) νmax 3595, 1726, 1713, 1382, 1370, 1601, 1260, 1069 cm−1; 13C and 1H NMR data, Tables 1 and 2; HRESIMS m/z 291.1582 [M + Na]+ (calcd for C15H24O4Na, 291.1567). 1-Acetylpallidopenilline A (2): colorless crystals (EtOH), mp 157− 159 °C; [α]20D −7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 195 (+2.47) nm; ECD (c 0.05 mg/mL, hexane) λmax (Δε) 192 (+0.27), 204 (−0.31), 294 (−0.25) nm; IR (CHCl3) νmax 3593, 1738, 1728, 1713, 1383, 1369, 1603, 1259, 1073 cm−1; 13C and 1H NMR data, Tables 1 and 2; HRESIMS m/z 333.1678 [M + Na]+ (calcd for C17H26O5Na, 333.1672). G



Article

Quantum-Chemical Modeling. See Supporting Information S13-

ORCID

a.

Sergey A. Dyshlovoy: 0000-0002-7155-9245 Shamil Sh. Afiyatullov: 0000-0002-5223-2132

Cytotoxicity Assay. The cytotoxic activities against the human prostate cancer cell lines 22Rv1, PC-3, and LNCaP and the human cancer cell lines MEL-28 and HCT-116 were determined according to previously reported methods.5,6 Effects of Compounds on ROS Levels in RAW 264.7 Murine Macrophages. The protocol was previously reported.7 X-ray Crystallographic Analysis of 1. Experimental intensity data for C15H24O4 were collected at T = 293 K (α-C15H24O4) and T = 170 K (β-C15H24O4) on a Bruker Kappa APEX2 diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.710 73 Å). Intensity data were corrected for absorption using the multiscan method. The structures were solved using direct methods and refined by leastsquares calculation in anisotropic approximation for non-hydrogen atoms. Hydrogen atoms were placed at idealized positions and refined using a riding model. The asymmetric unit of the structure αC15H24O4 contains one molecule of 1 (Figure 2). In the temperature range 235−233 K compound 1 undergoes a reversible phase transition, accompanied by a lowering of the symmetry from the orthorhombic (α-C15H24O4, space group P212121) to the monoclinic (β-C15H24O4, space group P21), increasing the unit cell volume almost twice and pseudomerohedral twinning of the low-temperature structure (twin law [001/0−10/100] and the BASF parameter refined to 0.446(1)). The periods of the monoclinic unit cell can be obtained from 1 by the following transformations: a ≈ [0 1 1]; b ≈ [1 0 0]; c ≈ [0 1 −1]. General views of the β-phase molecules are illustrated in Figure 2 and Supporting Information 7-a. In both structures the molecules of 1 are linked into a three-dimensional framework via O− H···O and C−H···O hydrogen bonds. Data collection and editing, as well as refinement of unit cell parameters, were performed with the APEX2 program packages.8 Structure solution and refinement were performed with the SHELXTL/PC program packages.9 Complete crystallographic details are included in the Supporting Information 7-a. Selected bond lengths and hydrogen bonds are listed in Supporting Information 7-a. Supplementary crystallographic data (accession numbers CCDC 1490151 and 1490152) can be obtained free of charge from the Cambridge Crystallographic Data Center via http://www.ccdc.cam.ac. uk/data_request/cif (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, U.K.; fax: + 44 1223 336 033 or email: [email protected]). Crystallographic data for α-C15H24O4: Mr = 268.34, orthorhombic, space group P212121 with a = 9.2372(1) Å, b = 10.9057(1) Å, c = 14.6437(2) Å; Z = 4, Dcal = 1.208 g/cm3, and F(000) = 584. Crystallographic data for β-C15H24O4: Mr = 268.34; monoclinic; space group P21 with a = 18.1759(5) Å, b = 9.1998(3) Å, c = 18.1799(5) Å, β = 106.594(1)°; Z = 8; Dcal = 1.224 g/cm3, and F(000) = 1168.

■

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are grateful to the Far Eastern Center of Structural Researches for the X-ray investigation. This study was supported by the program grant from the Russian Foundation for Basic Research (RFBR Nos. 14-04-00910-a and 15-2902572/15) and by the Far Eastern Branch of the Russian Academy of Science No. VANT 16-004).

■

REFERENCES

(1) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2016, 33, 382−431. (2) Rateb, J. W.; Ebel, R. N. Nat. Prod. Rep. 2011, 28, 290−344. (3) Zhuravleva, O. I.; Sobolevskaya, M. P.; Leshchenko, E. V.; Kirichuk, N. N.; Denisenko, V. A.; Dmitrenok, P. S.; Dyshlovoy, S. A.; Zakharenko, A. M.; Kim, N. Y.; Afiyatulov, Sh. Sh. J. Nat. Prod. 2014, 77, 1390−1395. (4) Afiyatulov, Sh. Sh.; Leshchenko, E. V.; Sobolevskaya, M. P.; Denisenko, V. A.; Kirichuk, N. N.; Khudyakova, Y. V.; Hoai, T. P. T.; Dmitrenok, P. S.; Menchinskaya, E. S.; Pislyagin, E. A.; Berdyshev, D. V. Phytochem. Lett. 2015, 14, 209−214. (5) Barltrop, J. A.; Owen, T. C.; Cory, A. H.; Cory, J. G. Bioorg. Med. Chem. Lett. 1991, 1, 611−614. (6) Dyshlovoy, S. A.; Venz, S.; Shubina, L. K.; Fedorov, S. N.; Walther, R.; Jacobsen, C.; Stonik, V. A.; Bokemeyer, C.; Balabanov, S.; Honecker, F. J. Proteomics 2014, 96, 223−239. (7) Kicha, A. A.; Kalinovsky, A. I.; Malyarenko, T. V.; Ivanchina, N. V.; Dmitrenok, P. S.; Menchinskaya, E. S.; Yurchenko, E. A.; Pislyagin, E. A.; Aminin, D. L.; Huong, T. T. T.; Long, P. Q.; Stonik, V. A. J. Nat. Prod. 2015, 78, 1397−1405. (8) APEX2; Bruker AXS Inc.: Madison, WI, USA, 2010. (9) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Adv. 2015, A71, 3−8.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00624. Crystallographic data (CIF) Crystallographic data (CIF) )1H, 13C, COSY 45, HSQC, HMBC, and NOESY spectra for compounds 1−11, quantum chemical modeling details, X-ray data tables, calculated and experimental ECD spectra for 3 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel: 7(423)2311168. Fax: 7(423)2314050. E-mail: afiyat@ piboc.dvo.ru. H


Pallidopenillines: Polyketides from the Alga-Derived Fungus

Recommend Documents