Isolation and Structure Elucidation of Cytotoxic Saccharothriolides D to

Jun 22, 2016 - ... of the phenolic hydroxy group at C-2″ and the stereochemistry of C-2 for the inhibition of human fibrosarcoma HT1080 cell growth...
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Isolation and Structure Elucidation of Cytotoxic Saccharothriolides D to F from a Rare Actinomycete Saccharothrix sp. and Their Structure−Activity Relationship Shan Lu,† Shinichi Nishimura,† Masashi Ito,‡ Toshio Tsuchida,‡ and Hideaki Kakeya*,† †

Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan ‡ Bioresource Laboratories, MicroBioPharm Japan Co., Ltd. (MBJ), Iwata, Shizuoka 438-0078, Japan S Supporting Information *

ABSTRACT: Three new 10-membered macrolides, saccharothriolides D−F (1−3), were isolated from a rare actinomycete, Saccharothrix sp. A1506. The planar structures were determined from analysis of extensive NMR and HRESIMS data, and the absolute configurations were established by ECD spectroscopy analysis. Saccharothriolides D (1) and E (2) were determined to be C-2 epimers of saccharothriolides A (4) and B (5), respectively. Saccharothriolide F (3) was identified to be a demethylated congener of saccharothriolides D (1) and A (4) at the C-2 position. The availability of compounds 1−6 enabled a structure−activity relationship study that revealed the importance of the phenolic hydroxy group at C-2″ and the stereochemistry of C-2 for the inhibition of human fibrosarcoma HT1080 cell growth.

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lides, saccharothriolides D−F (1−3) (Figure 1). Unexpectedly, two pairs of C-2 epimers were isolated from the same

xploring natural chemical diversity is an important way for drug leads discovery, since natural products can possess highly potent and/or selective biological activities.1 Over 10 000 bioactive secondary metabolites have been identified from a variety of actinomycetes.2 In any discovery program, compounds identified as active substances from bioassay-guided isolation are often already known.3 To avoid this problem, rare actinomycetes can be explored as potential sources for the discovery of novel classes of bioactive compounds.4 The genus Saccharothrix is considered to be a rare actinomycetes. A growing number of novel bioactive metabolites including antibiotics and cytotoxic compounds have been reported from this genus.5 Chemical screening is an alternative to identify novel metabolites although with no information regarding bioactivity.6 Recently, we identified saccharothriolides A−C (4−6) from Saccharothrix sp. A1506 by LC-MS analysis of the metabolic components of 30 000 culture broths.7 Saccharothriolides A−C (4−6) possessed unique phenyl-substituted 10membered macrolide structures. They could be biosynthesized from an aryl acid, and all sp3 carbons on the lactone ring possess chirality. The results from cytotoxicity evaluation indicated that the activity of saccharothriolides depended on the nature of the functional group at C-7, although the molecular mode of action is not known. To obtain saccharothriolide congeners and thereby investigate the structure−activity relationship (SAR), we further analyzed the LC-MS data of the culture extract of Saccharothrix sp. A1506 and found three new phenyl-substituted 10-membered macro© XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Chemical structures of saccharothriolides D−F (1−3).

actinomycete extract, and the epimers differed considerably in their cytotoxic activities against the human fibrosarcoma HT1080 cells. Herein, we report the isolation, structure elucidation, and biological activities of the three new metabolites 1−3. A substantial SAR of saccharothriolides A− F is also presented. The culture broth of Saccharothrix sp. (6 L) was extracted with n-BuOH and then concentrated to afford a crude residue (5.44 g). This residue was separated by repeated column chromatography on silica gel to give 35 fractions. To identify congeners of saccharothriolides A−C (4−6), we analyzed the LC-MS data of each fraction. Saccharothriolide A (4) had a Received: April 26, 2016

A

DOI: 10.1021/acs.jnatprod.6b00372 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR and 13C NMR Data for Saccharothriolides D−F (1−3) in Methanol-d4 1 position

a

δC, type

1 2

173.3, C 44.4, CH

3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 2″-COOH

73.9, 52.2, 216.3, 42.2, 61.5, 43.8, 74.6, 16.7, 9.0, 19.2, 11.1, 144.4, 104.6, 159.6, 102.3, 159.6, 104.6, 151.5, 117.5, 133.9, 115.5, 134.9, 111.7, 173.3,

CH CH C CH CH CH CH CH3 CH3 CH3 CH3 C CH C CH C CH C C CH CH CH CH C

2 δH, m, J (Hz)

δC, type

2.47, dq, 9.7, 6.9

173.3, C 44.3, CH

4.40, dd, 9.7, 2.9 3.26, qd, 6.3, 3.4 3.51, 3.67, 2.10, 5.37, 1.45, 1.09, 1.32, 1.05,

q, 6.9 brs qd, 7.5, 2.9 brs d, 6.9 d, 6.3 d, 6.9 d, 7.5

5.87, s 6.05, s 5.87, s

7.93, 6.55, 7.27, 6.62,

d, 6.3 t, 6.9 t, 7.5 d, 8.6

73.8, 52.3, 217.4, 42.1, 62.7, 43.2, 74.5, 16.7, 8.9, 19.3, 11.3, 144.5, 104.7, 159.5, 102.3, 159.5, 104.7, 138.3, 146.1, 115.1, 117.3, 121.6, 110.9,

CH CH C CH CH CH CH CH3 CH3 CH3 CH3 C CH C CH C CH C C CH CH CH CH

3 δH, m, J (Hz) 2.47, dq, 9.7, 6.9 4.40, dd, 9.7, 3.4 3.25, qd, 6.9, 3.4 3.48, 3.53, 2.16, 5.38, 1.45, 1.08, 1.35, 1.02,

qd, 7.5, 1.7 brs qd, 6.9, 2.9 brs d, 7.5 d, 6.9 d, 7.5 d, 6.9

5.89, d, 1.7 6.06, t, 2.3 5.89, d, 1.7

6.73, 6.46, 6.69, 6.44,

dd, 8.0, 1.2 td, 7.5, 1.2 td, 7.5, 1.2 d, 8.0

position

δC, type

1 2a 2b 3 4 5 6 7 8 9 10 11 12

169.8, C 39.0, CH2 68.8, 51.9, 216.9, 42.8, 61.6, 43.6, 75.4, 8.7, 19.0, 11.1,

1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 2″-COOH

144.4, C 104.6, CH 159.5, C 102.2, CH 159.5, C 104.6, CH 151.2, C n.i.a 133.9, CH 115.5, CH 134.6, CH 111.7, CH 172.7, C

CH CH C CH CH CH CH CH3 CH3 CH3

δH, m, J (Hz) 2.78, 2.47, 4.81, 3.28,

dd, 15.8, 4.3 dd, 15.8, 11.2 d, 10.3 overlap

3.50, 3.68, 2.12, 5.41, 1.10, 1.33, 1.05,

m brs m brs d, 6.9 d, 6.9 d, 7.5

5.89, s 6.04, s 5.89, s

7.93, 6.55, 7.26, 6.63,

d, 4.6 t, 5.7 t, 7.5 d, 8.6

Noninformation.

4″/H-5″/H-6″ (Figure 2). HMBC correlations from H-6/CH311/CH3-12 to C-5 connected C-4 and C-6 through a ketone

characteristic UV absorption around 345 nm, attributed to the presence of an anthranilic acid. In the fractions that contained metabolite 4, we detected two metabolites (1 and 3) that showed UV absorption similar to that of metabolite 4, but were less hydrophobic (Figure S1). Metabolite 1 exhibited an MS signal (m/z 486.2107 [M + H]+) that was identical with compound 4 (m/z 486.2134 [M + H]+), and 3 exhibited an MS signal (m/z 472.1981 [M + H]+) 14 mass units less than 4. In the same manner, compound 2 (m/z 458.2167 [M + H]+) was identified as a saccharothriolide congener because its UV absorption and MS signal were similar to that of saccharothriolide B (5) (m/z 458.2162 [M + H]+). LC-MS-guided fractionation was carried out by silica gel column chromatography and HPLC to yield three metabolites, 1 (4.98 mg), 2 (0.90 mg), and 3 (1.38 mg). Saccharothriolide D (1) was obtained as a light yellow oil. The molecular formula was determined to be C26H31NO8 by HRESIMS, the same as that of saccharothriolide A (4). The characteristic UV absorption at 341 nm revealed the presence of an anthranilic acid moiety, also present in 4.7 The 1H and 13C NMR data were also similar to those of 4, except for slight differences in chemical shifts for atoms in the right part of the lactone ring (Table 1). The 1H NMR chemical shifts corresponding to H-2 and CH3-11 in compound 1 were shifted upfield, while those for H-3 and CH310 were shifted downfield compared with compound 4 (Figure S2). Comparison of the 13C NMR spectra of compounds 1 and 4 revealed the differences in the chemical shifts of C-3, C-5, CH3-10, and CH3-11 (Figure S2). The 1H−1H COSY data revealed the presence of three spin systems: CH3-10/H-2/H-3/ H-4/CH3-11, CH3-12/H-6/H-7/H-8/CH3-13, and H-3″/H-

Figure 2. 1H−1H COSY (left, bold) correlations and selected HMBC (left, arrow) and NOESY (right) correlations in saccharothriolide D (1).

group. HMBC correlations from H-2/CH3-10 to carbonyl C-1, and from CH3-13 to C-9, combined with the downfield shifted chemical shift of H-9 (δH 5.37), connected C-2 and C-9 through an ester bond, leading to the formation of the 10membered lactone ring (Figure 2). The meta-disubstituted benzene ring was connected to C-9 based on the HMBC correlation from aromatic protons H-2′/6′ to C-9. Taken together, the planar structure of 1 was revealed to be identical to that of 4, and thus compound 1 is a diastereomer of saccharothriolide A (4). B

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The relative stereochemistry of 1 was determined from the NOESY and 3JH−H data (Figure 2). NOESY cross-peaks between H-3 and H-4/H-6/CH3-10/CH3-13, and between H6 and CH3-13, suggested that they were placed in the same αface. The NOESY correlations between H-7 and H-6/H-8/ CH3-12/CH3-13 indicated that H-7 also has an α-configuration, while the β-orientation of H-9 was revealed by the correlation between the aromatic proton H-6′ and H-8/CH3-13. The NOESY correlations described above were also observed for metabolite 4. However, contrary to the case of 4, no NOESY correlation was detected between H-2 and H-4/aromatic proton H-2′. Instead, H-2 exhibited a NOESY correlation with CH3-11, and the aromatic proton H-2′ and CH3-10 showed a NOESY correlation, which led to the identification of the β-orientation of H-2 and the α-orientation of CH3-10 (Figure 2). Thus, 1 was revealed to be a C-2 epimer of saccharothriolide A (4). This result was supported by the 3JH−H values: values in the left half of the lactone ring were similar between metabolites 1 and 4, whereas there was an apparent difference in the 3JH2−H3 value (9.7 Hz in 1 and 3.4 Hz in 4). The absolute stereochemistry of 1 was determined by comparing the ECD spectra of 1 and 4. The ECD spectrum of 1 showed characteristic Cotton effects at 201 (Δε, +19.2), 227 (Δε, −27.4), and 347 (Δε, +5.1) nm, which were also observed in the spectrum of 4 (Figure 3). Thus, the absolute configuration of 1 was established to be 2S, 3R, 4S, 6R, 7R, 8R, 9S.

Saccharothriolide F (3) was obtained as a light yellow oil. HRESIMS data for 3 showed an ion peak at m/z 472.1981 [M + H]+, indicating the molecular formula C25H29NO8, which was smaller than saccharothriolide D (1) by a CH2 unit. The UV absorption at 343 nm suggested that metabolite 3 also had an anthranilic acid. The 1H and 13C NMR spectra of 3 were similar to those of 1 (Table 1, Figure S2), except for the absence of signals corresponding to CH-2 and CH3-10 in 1 and the presence of a methylene signal (δH 2.78 ppm; δC 39.0 ppm). These data suggested that 3 should be a demethylated analogue of 1. The relative configuration of six stereocenters in the 10membered lactone ring was established on the basis of the 1 H−1H coupling constant values and NOESY data (Figure S4). The absolute stereochemistry of 3 was determined as 3S, 4S, 6R, 7R, 8R, 9S, since the ECD spectrum (Figure S5) of 3 showed high similarity to that of 1. We previously reported that only saccharothriolide B (5) showed moderate cytotoxicity (13.9 μM) against human fibrosarcoma HT1080 cells among the saccharothriolides A− C (4−6). Neither saccharothriolide A (4) nor C (6) was active even at 100 μM, indicating the importance of the functional group substituted on C-7. To further analyze the structure− activity relationship of these 10-membered macrolides, we examined the cytotoxicity of saccharothriolides D−F (1−3) against HT1080 cells (Figure 4). First, saccharothriolide E (2) showed weak toxicity (IC50 value, 29.2 μM), but it was still less potent than the C-2 epimer (5). Saccharothriolides D (1) and F (3), both of which have an anthranilic acid substituted at C-7, showed no (no effect at 100 μM) and weak (IC50 value, 66.4 μM) toxicity, respectively. These results confirmed the importance of the phenolic hydroxy group at C-2″ and revealed the involvement of the stereochemistry of C-2 in the cytotoxicity. In conclusion, three new 10-membered macrolides, saccharothriolides D−F (1−3), were isolated from Saccharothrix sp. A1506, expanding the members of the saccharothriolide family. Two of them were C-2 epimers of the previously reported saccharothriolides A (4) and B (5). The SAR study indicated that the phenolic hydroxy group at C-2″ is important for cytotoxicity, while the stereochemistry of C-2 also plays an important role. As these metabolites have novel chemical scaffolds, further investigations on their biological activity and SAR are ongoing in our laboratory.

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Figure 3. Experimental ECD spectra of 1 (solid line) and 4 (dotted line).

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using the sodium D line (589 nm) at 20 °C in methanol. UV spectra were recorded in methanol on a spectrophotometer. IR spectra were measured using an FTIR spectrometer equipped with a ZnSe ATR plate. High-resolution ESIMS spectra were recorded on a LC-IT-TOF MS. NMR spectra were recorded on a 500 MHz instrument. 1H and 13C chemical shifts are shown relative to the residual solvent: δH 3.31 and δC 49.15 for methanol-d4. Chemical shifts (δ) are shown in parts per million (ppm), and coupling constants (J) are in hertz (Hz). CD spectra were recorded using a CD spectrometer with a 1 mm path length cell. Fermentation, Extraction, and Isolation. Saccharothrix sp. A1506 was isolated from a soil sample as described previously.7 Whole culture broth (6 L) was extracted with n-BuOH to afford a crude residue (5.44 g) after concentration in vacuo. The residue was subjected to column chromatography on silica gel and eluted with CHCl3/MeOH (50:1, 20:1, 10:1, 5:1, 2:1, and 1:10 v/v) to give 35 fractions. Fraction 18 was separated by silica gel column chromatography with CHCl3/MeOH (50:1, 20:1, 10:1, and 5:1 v/v) to give six subfractions. Subfraction 5 was subjected to RP-HPLC (PEGASIL

Saccharothriolide E (2) was obtained as a light yellow oil. The molecular formula was determined by HRESIMS to be C25H31NO7, the same as that of saccharothriolide B (5). The 1 H and 13C NMR data of 2 resembled those of 5, while differences were observed for the chemical shifts of H-2, H-3, CH3-10, and CH3-11 (Table 1, Figure S2). Additionally, the chemical shifts of the lactone ring were very similar to those of 1. From these results, 2 was deduced to be an epimer of 5 at C2. The proposed structure was confirmed by detailed analysis of the NOESY spectrum of 2 (Figure S3). The relative configuration of the C-2/C-3 was proven to be the same as that of 1 on the basis of the similar NOESY correlations and coupling constants. Therefore, the relative configuration of 2 was deduced to be 2S*, 3R*, 4S*, 6R*, 7R*, 8R*, 9S*. The ECD spectrum of 2 was almost identical to that of 5 (Figure S5), indicating the absolute stereochemistry was 2S, 3R, 4S, 6R, 7R, 8R, 9S.8 C

DOI: 10.1021/acs.jnatprod.6b00372 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 4. Cytotoxicity of saccharothriolides A−F against human fibrosarcoma HT1080 cells.

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ODS SP100, ⦶ 10 × 250 mm, 40% MeCN, 2.0 mL/min) to yield metabolite 2 (0.90 mg, 22.2 min). Fractions 22 to 28 were combined and fractionated by silica gel column chromatography with CHCl3/ MeOH (20:1, 10:1, 5:1, and 2:1 v/v), to give 12 subfractions. Subfractions 10 and 11 were combined and subjected to RP-HPLC (YMC Carotenoid, ⦶ 20 × 250 mm, 40% MeCN, 8.0 mL/min) to give metabolites 1 (4.98 mg, 15.3 min) and 3 (1.38 mg, 14.0 min), respectively. Saccharothriolide D (1): light yellow oil; [α]20D +95.8 (c 0.33, MeOH); UV (MeOH) λmax (log ε) 259 (4.32), 341 (3.87) nm; CD (c 6.84 × 10−4 M, MeOH) λmax (Δε) 201 (+19.2), 227 (−27.4), 347 (+5.1) nm; IR (neat) νmax 3336, 2978, 2930, 1736, 1677, 1607, 1573, 1508, 1455, 1384, 1264, 1237, 1162 cm−1; 1H and 13C NMR data, see Table 1; HRMS (ESI) [M + H]+ m/z 486.2107 (calcd for C26H32NO8, 486.2128). Saccharothriolide E (2): light yellow oil; [α]20D −6.3 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 247 (4.46) nm; CD (c 6.56 × 10−4 M, MeOH) λmax (Δε) 210 (−14.6), 252 (+7.8) nm; IR (neat) νmax 3394, 2978, 2940, 1742, 1684, 1607, 1518, 1461, 1268, 1161 cm−1; 1H and 13C NMR data, see Table 1; HRMS (ESI) [M + H]+ m/z 458.2167 (calcd for C25H32NO7, 458.2179). Saccharothriolide F (3): light yellow oil; [α]20D +19.8 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 259 (4.27), 343 (3.81) nm; CD (c 6.52 × 10−4 M, MeOH) λmax (Δε) 200 (+17.8), 227 (−23.9), 345 (+4.3) nm; IR (neat) νmax 3392, 2926, 1736, 1677, 1650, 1611, 1571, 1547, 1512, 1461, 1389, 1268, 1168 cm−1; 1H and 13C NMR data, see Table 1; HRMS (ESI) [M + H]+ m/z 472.1981 (calcd for C25H30NO8, 472.1971). Cytotoxicity Assay. Cytotoxicity of metabolites 1−3 against human fibrosarcoma HT1080 cells was evaluated by a WST-8 colorimetric assay (Cell Counting Kit-8, Dojindo). Briefly, cells were cultured in 96-well plates (1500 cells/well) for 24 h followed by exposure to metabolites 1−3 for 72 h, and then the viability was assessed by WST-8. Adriamycin, a control reagent, had an IC50 value of 0.38 μM.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-75-753-4524. Fax: +81-75-753-4591. E-mail: [email protected] (H. Kakeya). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by research grants from the Japan Society for the Promotion of Science (JSPS), the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and the Japan Agency for Medical Research and Development (AMED).

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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.6b00372. HPLC chromatograms and HRESIMS, UV, ECD, and NMR spectra of compounds 1−3 (PDF) D

DOI: 10.1021/acs.jnatprod.6b00372 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Sarkar, A.; Li, J.; Ziemert, N.; Wang, M.; Bandeira, N.; Moore, B. S.; Dorrestein, P. C.; Jensen, P. R. Chem. Biol. 2015, 22, 460−471. (7) Lu, S.; Nishimura, S.; Hirai, G.; Ito, M.; Kawahara, T.; Izumikawa, M.; Sodeoka, M.; Shin-ya, K.; Tsuchida, T.; Kakeya, H. Chem. Commun. 2015, 51, 8074−8077. (8) The difference of the stereochemistry of C-2 had a negligible effect on the conformation of the chromophores. This was supported by NOESY correlations and 3JH‑H values. Metabolites 2 and 5 exhibited similar NOESY correlations and coupling constants for atoms in the left half of the macrolide.

E

DOI: 10.1021/acs.jnatprod.6b00372 J. Nat. Prod. XXXX, XXX, XXX−XXX