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Identification of spirobisnaphthalene derivatives with anti-tumor activities from the endophytic fungus Rhytidhysteron rufulum AS21B
Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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Identification of spirobisnaphthalene derivatives with anti-tumor activities from the endophytic fungus Rhytidhysteron rufulum AS21B Ittipon Siridechakorn a, Zongwei Yue a, Yanisa Mittraphab b, Xiaoguang Lei a, Khanitha Pudhom b,⇑ a Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China b Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
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Article history: Received 28 January 2017 Revised 23 February 2017 Accepted 24 February 2017 Available online xxxx Keywords: Endophytic fungi Rhytidhysteron rufulum Spirobisnaphthalene Anti-cancer activity Fungal cultivation
a b s t r a c t The cultivation of the mangrove-derived fungus Rhytidhysteron rufulum AS21B in acidic condition changed its secondary metabolite profile. Investigation of the culture broth extract led to the isolation and identification of two new spirobisnaphthalenes (1 and 2) together with eleven known compounds (3– 13) from the crude extract of the fungus grown under an acidic condition as well as six known compounds (4, 10, 14–17) were isolated from the crude extract of the fungus grown under a neutral condition. Their structures were elucidated on the basis of extensive spectroscopic data. The isolated compounds were evaluated for their cytotoxicity against two human cancer cell lines, Ramos lymphoma and drug resistant NSCLC H1975. Compounds 2 and 10 displayed the most promising anti-tumor activity against both cancer cell lines. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Endophytic fungi are a rich source of secondary metabolites, exhibiting a wide range of pharmacological activities.1–3 Plantderived fungi from mangrove forests have received much attention from pharmaceutical and natural product chemists are interested in this unique ecosystem.4–6 Spirobisnaphthalenes are a group of compounds consisting of two naphthalene-derived C10 units linked together through a spiroketal linkage. Examples of spirobisnaphthalenes include the palmarumycins,7,8 decaspirones,7,8 preussomerins9,10 and spiro-nonadiene,11 which have been mainly isolated from fungi. They have been reported to possess a variety of biological activities such as antibacterial,12 antifungal,12 anticancer,13 and antileishmanial10 activities. Endophytic fungi have been able to adapt themselves to reside in the living tissues of virtually all plants. The metabolomes of endophytic fungi thus display great diversity and adaptability.14,15 Moreover, varying the conditions of the fungal cultivation, such as pH or addition of epigenetic modifiers, had led to the production of higher amounts of some components or even to the production of some new metabolites.16–18 ⇑ Corresponding author. E-mail address: [email protected] (K. Pudhom).
As a result, alteration of culture media conditions may be of great value in the discovery of new compounds. In the course of our ongoing search for new bioactive compounds from mangrove-derived fungi, chemical investigations of the Rhytidhysteron rufulum AS21B fungus under acidic (pH5) and normal cultivation conditions were performed. This led to the isolation and characterization of two new spirobisnaphthalenes, rhytidenones G and H (1 and 2), together with fifteen known compounds. Isolated compounds were further evaluated for their anti-tumor activities against two human cancer cell lines, Ramos and H1975.
2. Results and discussion The EtOAc extracts of the R. rufulum AS21B grown in normal and acidic SDB broth were successively purified by column chromatography. Cultivation in acidic condition led to the isolation of two novel spirobisnaphthalenes, rhytidenones G and H (1 and 2), along with eleven known compounds: deoxypreussomerin B (3),19 palmarumycin CP17 (4),10 1-oxo-1,4-dihydronapthalene-4-spiro-20 -naptho[400 hydroxy-100 ,800 -de][10 ,30 ]-dioxine (5),20 preussomerin EG4 (6),21 CJ-12,371 (7),22 4-O-methyl-CJ-12,371 (8),22 rhytidenone E (9),23 rhytidenone F (10),23 palmarumycin C5 (11),24 rhytidone A (12)25 and 4,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one (13),26 while culture in normal condition (pH 7) gave six known compounds
http://dx.doi.org/10.1016/j.bmc.2017.02.054 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Siridechakorn I., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.054
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Rhytidenone G (1), ½a24 D 92.8 (c 0.10, MeOH), was obtained as a yellow viscous oil. Its molecular formula, C20H16O5, was deduced by HR-ESI-MS which showed the molecular ion peak at m/z 359.0941 [M+Na]+ (calcd. for C20H16O5Na, 359.0895). The IR spectrum showed the hydroxyl functionality at 3389 cm1. The combination of 1H, 13C, and HSQC NMR data for 1 (Table 1) displayed the typical signals expected for a 1,8-disubstituted naphthalene, due to the HMBC correlations between H-30 (7.48, dd, J = 8.0, 1.2 Hz) and C-10 (147.8); C-4a0 (135.2), H-40 (7.57, d, J = 8.0 Hz) and C-20 (110.7); C-50 (121.4); C-8a0 (114.6), H-60 (7.48, dd, J = 8.0, 1.2 Hz) and C-4a0 (135.2); C-8 (148.2), H-70 (6.96, d, J = 8.0 Hz) and C-50 (121.4); C-8a0 (114.6), as well as COSY correlations of H-20 /H-30 , H-30 /H-40 , H-50 /H-60 , and H-60 /H-70 (Fig. 2). Furthermore, the presence of a naphthalene subunit with an epoxide moiety [dH 3.41 (d, J = 4.0 Hz), H-6 and 3.77 (t, J = 4.0 Hz), H-7] that conjugated with a, b-unsaturated ketone at C-5 was established by HMBC correlation between H-6 and C-4a (131.3); C-5 (195.2), H-7 and C-5 (192.5); C8a (45.1) together with COSY correlations of 1 was interpreted the existence of the spin systems corresponding to the C-2 C-4 and C-8 C-8a. According to our previous isolation of compounds from this fungal strain,23 compound 1 was recognized as a spirobisnaphthalene derivative with a spiroketal-linked 1,8-dioxynaphthalene unit via a spiroketal bridge, confirming by HMBC correlation of H-2 (2.19, 1.90, m) with C-1 (103.3). Moreover, the relative configuration of 1 was determined by NOESY correlations of H-8/H-8a and H-6/H-7.
Fig. 1. Compounds isolated from R. rufulum AS21B strain.
including compounds 4 and 10, and other four compounds: rhytidone B (14)25 and C (15),25 MK3018 (16)27 and palmarumycin CR1 (17)20 (Fig. 1). Interestingly, it was found that the culture in acidic medium enhanced the production of compounds 4 and 10, with 4-fold and 8fold increasing, respectively, which are present in minor quantities under normal culture condition. The structures of the known compounds were determined by comparison of their NMR spectroscopic data with those in the literature.
Rhytidenone H (2), ½a24 D 57.4 (c 0.15, MeOH) was obtained as a pale yellow gum. Its molecular formula, C22H18O5, was established by HR-ESI-MS which showed the molecular ion peak at m/z 385.1186 [M+Na]+ (calcd. for C22H18O5Na, 385.1152). The NMR data of 2 (Table 1) were similar to those of 1, except for the upfield shift of carbonyl ketone at C-5 to dC 187.7. This finding was confirmed by HMBC correlations between H-6 (6.17, d, J = 10.0 Hz) and C-4a (131.5); C-8 (63.9), H-7 (6.91, d, J = 10.0 Hz) and C-5 (187.7); C-8a (45.1) (Fig. 2). Furthermore, the presence of an acetyl group [dH 2.11 (s), dC 21.0 and 169.9] instead of hydroxyl at C-8 established a downfield shift of H-8 (dH 5.69, dd, J = 5.6, 3.2 Hz) on the basis of the mesomeric effect of the acetyl group. Moreover, the relative configuration of 2 was determined by NOESY correlation of H-8 with H-8a. The anti-tumor activities of compounds 1–13 were evaluated against Ramos and H1975 cell lines (Table 2). Ramos and H1975 cell lines were derived from a human Burkitt’s lymphoma and non-small cell lung cancer tissue respectively, and is hence representative of this type of neoplasm. Though Burkitt’s lymphoma (BL) is one of the most curable hematological malignancies, this disease is also one of the most aggressive types of cancer, which always occur in adolescents and young adults even as well as in childhood patients.28 Ibrutinib (PCI-32765), a Bruton’s tyrosine kinase inhibitor, was developed by Pharmacyclics partnered with Johnson & Johnson and was granted an accelerated approval by US FDA in November 2013, for the treatment B cell cancers like mantle cell lymphoma. Lung cancer is one of the most frequently diagnosed types of cancer among males and females in China and in Western countries, and it remains the leading cause of cancer mortality in the USA, and non-small-cell lung carcinoma (NSCLC) which is characterized by aberrant activity of EGFR accounts for about 85% of all lung cancers. Though the kinase inhibitors, gefitinib and erlotinib are effective clinical therapies for this disease, approximately 50% of patients who initially respond to therapy will relapse due to emergence of drug resistance H1975 cell line with EGFR mutation L858R/ T790M.29,30 To overcome this drug resistance, new inhibitors effective for H1975 cell line with less toxic and side effect are urgent to be developed. Afatinib (BIBW 2992), an irreversible, tyrosine kinase inhibitor (TKI) of ErbB receptor family members, is effective in the
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I. Siridechakorn et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx Table 1 H (400 MHz) and
1
13
C (100 MHz) NMR data of compounds 1 and 2.
Position
1a
2b
dC, type 1 2 3 4 4a 5 6 7 8 8a 10 20 30 40 4a0 50 60 70 80 8a0 8-OAc 8-OAc a b
103.3 28.8 23.5 139.3 131.3 192.5 55.6 57.2 64.3 43.6 147.8 110.7 128.6 121.7 135.2 121.4 128.5 110.3 148.2 114.6 – –
C CH2 CH2 CH C C CH CH CH CH C CH CH CH C CH CH CH C C – –
dH, mult. (J in Hz)
dC, type
– 2.19, 2.37, 7.10, – – 3.41, 3.77, 5.10, 3.27, – 6.99, 7.48, 7.57, – 7.56, 7.48, 6.96, – – – –
101.2 29.2 22.6 136.7 131.5 187.7 132.9 142.0 63.9 45.1 146.4 109.7 127.8 120.8 134.3 120.8 127.6 109.4 147.0 113.5 169.9 21.0
m1.90, m m dd (6.4, 3.6)
d (4.0) t (4.0) br s br s d (8.0) dd (8.0, 1.2) d (8.0) d (8.0) dd (8.0, 1.2) d (8.0)
dH, mult. (J in Hz) C CH2 CH2 CH C C CH CH CH CH C CH CH CH C CH CH CH C C C@O CH3
– 2.18, 2.49, 7.08, – – 6.17, 6.91, 5.69, 3.31, – 6.86, 7.39, 7.50, – 7.49, 7.43, 6.88, – – – 2.11,
m m2.59, m br m
d (10.0) d (10.0) dd (5.6, 3.2) m d (7.6) t (8.0) d (8.0) d (8.0) t (8.0) d (7.6)
s
Recorded in CDCl3. Recorded in acetone-d6.
Fig. 2. Selected HMBC and COSY correlations of compounds 1 and 2.
Table 2 Anti-tumor activities of compounds isolated from the crude extract of the fungus grown under an acidic condition. Compound
1 2 3 4 5 6 7 9 10 11 12 13 Ibrutinib Afatinib
IC50 (mM) Ramos Cell
H1975 Cell
17.98 0.018 18 33.1 15 82.9 >100 0.461 0.048 31.7 >100 23.1 28.7 ND
7.3 0.252 >100 >100 >100 >100 >100 10.24 1.17 >100 >100 50 ND 1.97
FDA. Among the compounds tested, compound 2 displayed the most promising anti-tumor activity against both Ramos and H1975 cell lines with IC50 values of 0.018 and 0.252 lM, respectively. The second potent one is compound 10, the deacetylated derivative of 2, and it gave IC50 values of 0.048 and 1.17 lM, respectively. Compound 2 and 10 may be more effective in anti-tumor activity against Ramos and H1975 than stand drug Ibrutinib and afatinib, with IC50 values of 28.7 and 1.97 lM. When their structure-activity relationship was considered, it could be concluded that the C-4 a,b-unsaturated ketone moiety was crucially required for their potent antitumor activity against Ramos and H1975 cell lines. This could be seen in the case of compounds 5 and 6, which had the C-2 a,b-unsaturated ketone group. Both compounds exhibited only moderate and weak activity on Ramos, and completely lost activity on H1975 cell line (IC50 > 100 lM). It is worth mentioning that the a,b-unsaturated carbonyl compounds always have cytotoxic activity, most likely through the Michael addition reaction with biomolecules.31 3. Conclusion In this study, we studied the effect of pH of the culture medium on Rhytidhysteron rufulum metabolite profile. The culture in acidic condition (pH 5) significantly enhanced the production of its secondary metabolites, including two new spirobisnaphthalenes (1 and 2). Interestingly, the acidic culture condition also remarkably increased the production of the most two promising metabolites (2 and 10) against Ramos and drug resistant H1975 human cancer cell lines with very low IC50 values. Based on their structure-activity relationship, we found the C-4 a,b-unsaturated ketone moiety was required for their potent anti-tumor activity. These findings suggest that compounds 2 and 10 might be promising leads for leukemia and lung cancer treatments. Their mechanism of action will be further studied and reported in due course.
ND: not determined.
4. Material and method
treatment of lung cancer models, including those with EGF receptor (EGFR) mutations (L185R/T790M) resistant to reversible first-generation EGFR inhibitor like Gefitinib. It was developed and marketed by Boehringer Ingelheim, and was first approved in 2013 by US
4.1. General experiment procedures The UV spectra were recorded on a Biotek Powerwave XS2 (USA). The IR spectra were recorded with a PerkinElmer FTS FT-
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IR spectrophotometer. Optical rotations were measured on a PerkinElmer 341 polarimeter. NMR spectra were acquired on a Varian Mercury-400 Plus NMR spectrometer and 400 MHz Bruker FTNMR Ultra Shield with TMS as internal standard. HRESI-MS was carried out on a micrOTOF-Q II ESI mass spectrometer. HPLC was performed on Thermo Finnigan (TSP) with preparative column Inertsil ODS-3 (GL Science, 5 lm, 20 250 mm). Column chromatography (CC) was carried out on silica gel (Silicycle, SiliaFlash P60 40-63 mm and SiliaFlash F60 40-63 mm). Sephadex LH-20 was also used for CC. Precoated plates of silica gel 60 F254 were used for analytical purposes. 4.2. Extraction and isolation The fungus R. rufulum AS21B was isolated from leaves of Azima sarmentosa, collected from the mangrove forest in Samutsakhon province, Thailand in July 2008. The strain AS21B was grown on potato dextrose agar (PDA) plate at room temperature for 7 days. Five pieces (5 5 mm2) of mycelia agar plugs were inoculated into 1 L Erlenmeyer flasks (100) containing 200 mL of sabouraud dextrose broth (SDB) in acidic condition (pH 5) as well as the normal SDB. The cultivation was kept at room temperature under static conditions for 21 days. The filtration was used to separate the mycelia and broth from each other. The filtrate was extracted with EtOAc 3 times and evaporated under reduced pressure to give an acidic condition crude extract (5.53 g). The crude extract was subjected to column chromatography (CC) over silica gel, eluting with a gradient of ethyl acetate (EtOAc)-hexane (100% hexane to 100% EtOAc), providing eight fractions (A–H). Fraction A (235.8 mg) was recrystallized by hexane to give solid and mother liquor (ML) fraction. The ML (221.1 mg) was further purified by CC with 30% CH2Cl2-hexane giving compound 3 (11.4 mg). Fraction B (98.4 mg) was subjected to CC with 50% CH2Cl2-hexane to yield compound 4 (15.8 mg) together with three subfractions (BA-BC). Compound 6 (2.7 mg) was purified from subfraction BC (10.0 mg) by CC with 25% EtOAc-hexane. The recrystallization of fraction C (140.7 mg) from methanol (MeOH) provided compound 5 (2.7 mg) and ML fraction. The ML (138.0 mg) was further separated by CC with 50% CH2Cl2hexane yielding six subfractions (CA-CF). Compounds 2 (1.9 mg), 8 (1.0 mg) and 9 (3.5 mg) were derived from subfraction CD (24.4 mg) by CC with 50% CH2Cl2-hexane. Fraction E (372.5 mg) was subjected to CC eluted with 20% EtOAc-hexane, providing twelve subfractions (EA-EL). Compound 11 (1.5 mg) was derived from subfraction EE (7.6 mg) by CC with 40% CH2Cl2-hexane while compound 7 (3.2 mg) was obtained from subfraction EG (27.5 mg) by CC with 10% EtOAc-benzene. The purification of subfraction EI (13.1 mg) by preparative HPLC with isocratic condition of 65% acetonitrile-H2O yielded compound 13 (2.5 mg). Compounds 1 (10.4 mg) and 10 (50.1 mg) were derived from subfraction EK (95.6 mg) by CC with 10% EtOAc-benzene. Fraction H (120.6 mg) was subjected to passage over Sephadex LH-20 using MeOH as solvent to afford four subfractions (HA-HD). Compound 12 (6.1 mg) was derived from subfraction HB (25.3 mg) by preparative HPLC with isocratic condition of 75% acetonitrile-H2O. The neutral condition crude extract (5.89 g) was also subjected to passage over Sephadex-LH20 using MeOH as a solvent to afford eight fractions (I-P). Fraction K (1.58 g) was purified by CC over silica gel with 30% acetone-hexane to give nine subfractions (KA-KI). Compounds 10 (6.7 mg) and 15 (77.4 mg) were derived from subfraction KD (97.4 mg) by CC with 25% EtOAc-benzene. The recrystallization of subfraction KF (142.1 mg) from MeOH provided compound 16 (89.6 mg). Subfraction KG (206.1 mg) was further purified by CC with 20% acetone-hexane to afford compound 14 (188.0 mg). Compound 17 (19.8 mg) was derived from subfraction KI (60.3 mg) by recrystallization from MeOH. Fractionation of frac-
tion L (193.0 mg) by CC Sephadex-LH20 with MeOH to give five subfractions (LA-LE). Compound 4 (4.1 mg) was derived from subfraction LD (101.3 mg) by CC with 25% acetone-hexane. Rhytidenone G (1): yellow viscous oil, ½a24 92.8 (c 0.10, D MeOH); UV (MeOH) kmax (log e) 250 (2.42), 260 (2.43) nm; IR (neat) mmax 3389 cm1; 1H and 13C NMR (acetone-d6, 400 MHz), see Table 1; HRESIMS m/z 359.0941 [M+Na]+ (calcd for C20H16O5Na, 359.0895). Rhytidenone H (2): pale yellow gum; ½a24 D 57.4 (c 0.15, MeOH), UV (MeOH) kmax (log e) 230 (2.44), 250 (2.45), 260 (2.46) nm; 1H and 13C NMR (CDCl3, 400 MHz), see Table 1; HRESIMS m/z 385.1186 [M+Na]+ (calcd for C20H16O5Na, 385.1152). 4.3. Anti-tumor assay Human lymphoma Ramos cells and NSCLC H1975 cells were cultured to the exponential growth phase in RPMI 1640 supplemented with 10% (v/v) fetal calf serum in a humidified atmosphere containing 5% CO2. Cytotoxicity against Ramos and H1975 cells was assessed as follows: 4 104 cells seeded onto 96-well plates were incubated with compounds at the indicated concentrations at 37 °C for 24 h (Ramos) or 72 h (H1975). Cell viability was determined using measurement ATP level in living cells method by CellTiter-Glo kit according to the manufacturer’s instructions. Ibrutinib and afatinib were used as reference drug for Ramos and H1975 cell, respectively. Acknowledgments This work was supported by the Thailand Research Fund (TRF) (Grant No. BRG5980002) and the National Natural Science Foundation of China (Grant No. 21561142002). A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2017.02.054. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
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Contents lists available at ScienceDirect
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Identification of spirobisnaphthalene derivatives with anti-tumor activities from the endophytic fungus Rhytidhysteron rufulum AS21B Ittipon Siridechakorn a, Zongwei Yue a, Yanisa Mittraphab b, Xiaoguang Lei a, Khanitha Pudhom b,⇑ a Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China b Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
a r t i c l e
i n f o
Article history: Received 28 January 2017 Revised 23 February 2017 Accepted 24 February 2017 Available online xxxx Keywords: Endophytic fungi Rhytidhysteron rufulum Spirobisnaphthalene Anti-cancer activity Fungal cultivation
a b s t r a c t The cultivation of the mangrove-derived fungus Rhytidhysteron rufulum AS21B in acidic condition changed its secondary metabolite profile. Investigation of the culture broth extract led to the isolation and identification of two new spirobisnaphthalenes (1 and 2) together with eleven known compounds (3– 13) from the crude extract of the fungus grown under an acidic condition as well as six known compounds (4, 10, 14–17) were isolated from the crude extract of the fungus grown under a neutral condition. Their structures were elucidated on the basis of extensive spectroscopic data. The isolated compounds were evaluated for their cytotoxicity against two human cancer cell lines, Ramos lymphoma and drug resistant NSCLC H1975. Compounds 2 and 10 displayed the most promising anti-tumor activity against both cancer cell lines. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Endophytic fungi are a rich source of secondary metabolites, exhibiting a wide range of pharmacological activities.1–3 Plantderived fungi from mangrove forests have received much attention from pharmaceutical and natural product chemists are interested in this unique ecosystem.4–6 Spirobisnaphthalenes are a group of compounds consisting of two naphthalene-derived C10 units linked together through a spiroketal linkage. Examples of spirobisnaphthalenes include the palmarumycins,7,8 decaspirones,7,8 preussomerins9,10 and spiro-nonadiene,11 which have been mainly isolated from fungi. They have been reported to possess a variety of biological activities such as antibacterial,12 antifungal,12 anticancer,13 and antileishmanial10 activities. Endophytic fungi have been able to adapt themselves to reside in the living tissues of virtually all plants. The metabolomes of endophytic fungi thus display great diversity and adaptability.14,15 Moreover, varying the conditions of the fungal cultivation, such as pH or addition of epigenetic modifiers, had led to the production of higher amounts of some components or even to the production of some new metabolites.16–18 ⇑ Corresponding author. E-mail address: [email protected] (K. Pudhom).
As a result, alteration of culture media conditions may be of great value in the discovery of new compounds. In the course of our ongoing search for new bioactive compounds from mangrove-derived fungi, chemical investigations of the Rhytidhysteron rufulum AS21B fungus under acidic (pH5) and normal cultivation conditions were performed. This led to the isolation and characterization of two new spirobisnaphthalenes, rhytidenones G and H (1 and 2), together with fifteen known compounds. Isolated compounds were further evaluated for their anti-tumor activities against two human cancer cell lines, Ramos and H1975.
2. Results and discussion The EtOAc extracts of the R. rufulum AS21B grown in normal and acidic SDB broth were successively purified by column chromatography. Cultivation in acidic condition led to the isolation of two novel spirobisnaphthalenes, rhytidenones G and H (1 and 2), along with eleven known compounds: deoxypreussomerin B (3),19 palmarumycin CP17 (4),10 1-oxo-1,4-dihydronapthalene-4-spiro-20 -naptho[400 hydroxy-100 ,800 -de][10 ,30 ]-dioxine (5),20 preussomerin EG4 (6),21 CJ-12,371 (7),22 4-O-methyl-CJ-12,371 (8),22 rhytidenone E (9),23 rhytidenone F (10),23 palmarumycin C5 (11),24 rhytidone A (12)25 and 4,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one (13),26 while culture in normal condition (pH 7) gave six known compounds
http://dx.doi.org/10.1016/j.bmc.2017.02.054 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Siridechakorn I., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.054
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I. Siridechakorn et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Rhytidenone G (1), ½a24 D 92.8 (c 0.10, MeOH), was obtained as a yellow viscous oil. Its molecular formula, C20H16O5, was deduced by HR-ESI-MS which showed the molecular ion peak at m/z 359.0941 [M+Na]+ (calcd. for C20H16O5Na, 359.0895). The IR spectrum showed the hydroxyl functionality at 3389 cm1. The combination of 1H, 13C, and HSQC NMR data for 1 (Table 1) displayed the typical signals expected for a 1,8-disubstituted naphthalene, due to the HMBC correlations between H-30 (7.48, dd, J = 8.0, 1.2 Hz) and C-10 (147.8); C-4a0 (135.2), H-40 (7.57, d, J = 8.0 Hz) and C-20 (110.7); C-50 (121.4); C-8a0 (114.6), H-60 (7.48, dd, J = 8.0, 1.2 Hz) and C-4a0 (135.2); C-8 (148.2), H-70 (6.96, d, J = 8.0 Hz) and C-50 (121.4); C-8a0 (114.6), as well as COSY correlations of H-20 /H-30 , H-30 /H-40 , H-50 /H-60 , and H-60 /H-70 (Fig. 2). Furthermore, the presence of a naphthalene subunit with an epoxide moiety [dH 3.41 (d, J = 4.0 Hz), H-6 and 3.77 (t, J = 4.0 Hz), H-7] that conjugated with a, b-unsaturated ketone at C-5 was established by HMBC correlation between H-6 and C-4a (131.3); C-5 (195.2), H-7 and C-5 (192.5); C8a (45.1) together with COSY correlations of 1 was interpreted the existence of the spin systems corresponding to the C-2 C-4 and C-8 C-8a. According to our previous isolation of compounds from this fungal strain,23 compound 1 was recognized as a spirobisnaphthalene derivative with a spiroketal-linked 1,8-dioxynaphthalene unit via a spiroketal bridge, confirming by HMBC correlation of H-2 (2.19, 1.90, m) with C-1 (103.3). Moreover, the relative configuration of 1 was determined by NOESY correlations of H-8/H-8a and H-6/H-7.
Fig. 1. Compounds isolated from R. rufulum AS21B strain.
including compounds 4 and 10, and other four compounds: rhytidone B (14)25 and C (15),25 MK3018 (16)27 and palmarumycin CR1 (17)20 (Fig. 1). Interestingly, it was found that the culture in acidic medium enhanced the production of compounds 4 and 10, with 4-fold and 8fold increasing, respectively, which are present in minor quantities under normal culture condition. The structures of the known compounds were determined by comparison of their NMR spectroscopic data with those in the literature.
Rhytidenone H (2), ½a24 D 57.4 (c 0.15, MeOH) was obtained as a pale yellow gum. Its molecular formula, C22H18O5, was established by HR-ESI-MS which showed the molecular ion peak at m/z 385.1186 [M+Na]+ (calcd. for C22H18O5Na, 385.1152). The NMR data of 2 (Table 1) were similar to those of 1, except for the upfield shift of carbonyl ketone at C-5 to dC 187.7. This finding was confirmed by HMBC correlations between H-6 (6.17, d, J = 10.0 Hz) and C-4a (131.5); C-8 (63.9), H-7 (6.91, d, J = 10.0 Hz) and C-5 (187.7); C-8a (45.1) (Fig. 2). Furthermore, the presence of an acetyl group [dH 2.11 (s), dC 21.0 and 169.9] instead of hydroxyl at C-8 established a downfield shift of H-8 (dH 5.69, dd, J = 5.6, 3.2 Hz) on the basis of the mesomeric effect of the acetyl group. Moreover, the relative configuration of 2 was determined by NOESY correlation of H-8 with H-8a. The anti-tumor activities of compounds 1–13 were evaluated against Ramos and H1975 cell lines (Table 2). Ramos and H1975 cell lines were derived from a human Burkitt’s lymphoma and non-small cell lung cancer tissue respectively, and is hence representative of this type of neoplasm. Though Burkitt’s lymphoma (BL) is one of the most curable hematological malignancies, this disease is also one of the most aggressive types of cancer, which always occur in adolescents and young adults even as well as in childhood patients.28 Ibrutinib (PCI-32765), a Bruton’s tyrosine kinase inhibitor, was developed by Pharmacyclics partnered with Johnson & Johnson and was granted an accelerated approval by US FDA in November 2013, for the treatment B cell cancers like mantle cell lymphoma. Lung cancer is one of the most frequently diagnosed types of cancer among males and females in China and in Western countries, and it remains the leading cause of cancer mortality in the USA, and non-small-cell lung carcinoma (NSCLC) which is characterized by aberrant activity of EGFR accounts for about 85% of all lung cancers. Though the kinase inhibitors, gefitinib and erlotinib are effective clinical therapies for this disease, approximately 50% of patients who initially respond to therapy will relapse due to emergence of drug resistance H1975 cell line with EGFR mutation L858R/ T790M.29,30 To overcome this drug resistance, new inhibitors effective for H1975 cell line with less toxic and side effect are urgent to be developed. Afatinib (BIBW 2992), an irreversible, tyrosine kinase inhibitor (TKI) of ErbB receptor family members, is effective in the
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I. Siridechakorn et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx Table 1 H (400 MHz) and
1
13
C (100 MHz) NMR data of compounds 1 and 2.
Position
1a
2b
dC, type 1 2 3 4 4a 5 6 7 8 8a 10 20 30 40 4a0 50 60 70 80 8a0 8-OAc 8-OAc a b
103.3 28.8 23.5 139.3 131.3 192.5 55.6 57.2 64.3 43.6 147.8 110.7 128.6 121.7 135.2 121.4 128.5 110.3 148.2 114.6 – –
C CH2 CH2 CH C C CH CH CH CH C CH CH CH C CH CH CH C C – –
dH, mult. (J in Hz)
dC, type
– 2.19, 2.37, 7.10, – – 3.41, 3.77, 5.10, 3.27, – 6.99, 7.48, 7.57, – 7.56, 7.48, 6.96, – – – –
101.2 29.2 22.6 136.7 131.5 187.7 132.9 142.0 63.9 45.1 146.4 109.7 127.8 120.8 134.3 120.8 127.6 109.4 147.0 113.5 169.9 21.0
m1.90, m m dd (6.4, 3.6)
d (4.0) t (4.0) br s br s d (8.0) dd (8.0, 1.2) d (8.0) d (8.0) dd (8.0, 1.2) d (8.0)
dH, mult. (J in Hz) C CH2 CH2 CH C C CH CH CH CH C CH CH CH C CH CH CH C C C@O CH3
– 2.18, 2.49, 7.08, – – 6.17, 6.91, 5.69, 3.31, – 6.86, 7.39, 7.50, – 7.49, 7.43, 6.88, – – – 2.11,
m m2.59, m br m
d (10.0) d (10.0) dd (5.6, 3.2) m d (7.6) t (8.0) d (8.0) d (8.0) t (8.0) d (7.6)
s
Recorded in CDCl3. Recorded in acetone-d6.
Fig. 2. Selected HMBC and COSY correlations of compounds 1 and 2.
Table 2 Anti-tumor activities of compounds isolated from the crude extract of the fungus grown under an acidic condition. Compound
1 2 3 4 5 6 7 9 10 11 12 13 Ibrutinib Afatinib
IC50 (mM) Ramos Cell
H1975 Cell
17.98 0.018 18 33.1 15 82.9 >100 0.461 0.048 31.7 >100 23.1 28.7 ND
7.3 0.252 >100 >100 >100 >100 >100 10.24 1.17 >100 >100 50 ND 1.97
FDA. Among the compounds tested, compound 2 displayed the most promising anti-tumor activity against both Ramos and H1975 cell lines with IC50 values of 0.018 and 0.252 lM, respectively. The second potent one is compound 10, the deacetylated derivative of 2, and it gave IC50 values of 0.048 and 1.17 lM, respectively. Compound 2 and 10 may be more effective in anti-tumor activity against Ramos and H1975 than stand drug Ibrutinib and afatinib, with IC50 values of 28.7 and 1.97 lM. When their structure-activity relationship was considered, it could be concluded that the C-4 a,b-unsaturated ketone moiety was crucially required for their potent antitumor activity against Ramos and H1975 cell lines. This could be seen in the case of compounds 5 and 6, which had the C-2 a,b-unsaturated ketone group. Both compounds exhibited only moderate and weak activity on Ramos, and completely lost activity on H1975 cell line (IC50 > 100 lM). It is worth mentioning that the a,b-unsaturated carbonyl compounds always have cytotoxic activity, most likely through the Michael addition reaction with biomolecules.31 3. Conclusion In this study, we studied the effect of pH of the culture medium on Rhytidhysteron rufulum metabolite profile. The culture in acidic condition (pH 5) significantly enhanced the production of its secondary metabolites, including two new spirobisnaphthalenes (1 and 2). Interestingly, the acidic culture condition also remarkably increased the production of the most two promising metabolites (2 and 10) against Ramos and drug resistant H1975 human cancer cell lines with very low IC50 values. Based on their structure-activity relationship, we found the C-4 a,b-unsaturated ketone moiety was required for their potent anti-tumor activity. These findings suggest that compounds 2 and 10 might be promising leads for leukemia and lung cancer treatments. Their mechanism of action will be further studied and reported in due course.
ND: not determined.
4. Material and method
treatment of lung cancer models, including those with EGF receptor (EGFR) mutations (L185R/T790M) resistant to reversible first-generation EGFR inhibitor like Gefitinib. It was developed and marketed by Boehringer Ingelheim, and was first approved in 2013 by US
4.1. General experiment procedures The UV spectra were recorded on a Biotek Powerwave XS2 (USA). The IR spectra were recorded with a PerkinElmer FTS FT-
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IR spectrophotometer. Optical rotations were measured on a PerkinElmer 341 polarimeter. NMR spectra were acquired on a Varian Mercury-400 Plus NMR spectrometer and 400 MHz Bruker FTNMR Ultra Shield with TMS as internal standard. HRESI-MS was carried out on a micrOTOF-Q II ESI mass spectrometer. HPLC was performed on Thermo Finnigan (TSP) with preparative column Inertsil ODS-3 (GL Science, 5 lm, 20 250 mm). Column chromatography (CC) was carried out on silica gel (Silicycle, SiliaFlash P60 40-63 mm and SiliaFlash F60 40-63 mm). Sephadex LH-20 was also used for CC. Precoated plates of silica gel 60 F254 were used for analytical purposes. 4.2. Extraction and isolation The fungus R. rufulum AS21B was isolated from leaves of Azima sarmentosa, collected from the mangrove forest in Samutsakhon province, Thailand in July 2008. The strain AS21B was grown on potato dextrose agar (PDA) plate at room temperature for 7 days. Five pieces (5 5 mm2) of mycelia agar plugs were inoculated into 1 L Erlenmeyer flasks (100) containing 200 mL of sabouraud dextrose broth (SDB) in acidic condition (pH 5) as well as the normal SDB. The cultivation was kept at room temperature under static conditions for 21 days. The filtration was used to separate the mycelia and broth from each other. The filtrate was extracted with EtOAc 3 times and evaporated under reduced pressure to give an acidic condition crude extract (5.53 g). The crude extract was subjected to column chromatography (CC) over silica gel, eluting with a gradient of ethyl acetate (EtOAc)-hexane (100% hexane to 100% EtOAc), providing eight fractions (A–H). Fraction A (235.8 mg) was recrystallized by hexane to give solid and mother liquor (ML) fraction. The ML (221.1 mg) was further purified by CC with 30% CH2Cl2-hexane giving compound 3 (11.4 mg). Fraction B (98.4 mg) was subjected to CC with 50% CH2Cl2-hexane to yield compound 4 (15.8 mg) together with three subfractions (BA-BC). Compound 6 (2.7 mg) was purified from subfraction BC (10.0 mg) by CC with 25% EtOAc-hexane. The recrystallization of fraction C (140.7 mg) from methanol (MeOH) provided compound 5 (2.7 mg) and ML fraction. The ML (138.0 mg) was further separated by CC with 50% CH2Cl2hexane yielding six subfractions (CA-CF). Compounds 2 (1.9 mg), 8 (1.0 mg) and 9 (3.5 mg) were derived from subfraction CD (24.4 mg) by CC with 50% CH2Cl2-hexane. Fraction E (372.5 mg) was subjected to CC eluted with 20% EtOAc-hexane, providing twelve subfractions (EA-EL). Compound 11 (1.5 mg) was derived from subfraction EE (7.6 mg) by CC with 40% CH2Cl2-hexane while compound 7 (3.2 mg) was obtained from subfraction EG (27.5 mg) by CC with 10% EtOAc-benzene. The purification of subfraction EI (13.1 mg) by preparative HPLC with isocratic condition of 65% acetonitrile-H2O yielded compound 13 (2.5 mg). Compounds 1 (10.4 mg) and 10 (50.1 mg) were derived from subfraction EK (95.6 mg) by CC with 10% EtOAc-benzene. Fraction H (120.6 mg) was subjected to passage over Sephadex LH-20 using MeOH as solvent to afford four subfractions (HA-HD). Compound 12 (6.1 mg) was derived from subfraction HB (25.3 mg) by preparative HPLC with isocratic condition of 75% acetonitrile-H2O. The neutral condition crude extract (5.89 g) was also subjected to passage over Sephadex-LH20 using MeOH as a solvent to afford eight fractions (I-P). Fraction K (1.58 g) was purified by CC over silica gel with 30% acetone-hexane to give nine subfractions (KA-KI). Compounds 10 (6.7 mg) and 15 (77.4 mg) were derived from subfraction KD (97.4 mg) by CC with 25% EtOAc-benzene. The recrystallization of subfraction KF (142.1 mg) from MeOH provided compound 16 (89.6 mg). Subfraction KG (206.1 mg) was further purified by CC with 20% acetone-hexane to afford compound 14 (188.0 mg). Compound 17 (19.8 mg) was derived from subfraction KI (60.3 mg) by recrystallization from MeOH. Fractionation of frac-
tion L (193.0 mg) by CC Sephadex-LH20 with MeOH to give five subfractions (LA-LE). Compound 4 (4.1 mg) was derived from subfraction LD (101.3 mg) by CC with 25% acetone-hexane. Rhytidenone G (1): yellow viscous oil, ½a24 92.8 (c 0.10, D MeOH); UV (MeOH) kmax (log e) 250 (2.42), 260 (2.43) nm; IR (neat) mmax 3389 cm1; 1H and 13C NMR (acetone-d6, 400 MHz), see Table 1; HRESIMS m/z 359.0941 [M+Na]+ (calcd for C20H16O5Na, 359.0895). Rhytidenone H (2): pale yellow gum; ½a24 D 57.4 (c 0.15, MeOH), UV (MeOH) kmax (log e) 230 (2.44), 250 (2.45), 260 (2.46) nm; 1H and 13C NMR (CDCl3, 400 MHz), see Table 1; HRESIMS m/z 385.1186 [M+Na]+ (calcd for C20H16O5Na, 385.1152). 4.3. Anti-tumor assay Human lymphoma Ramos cells and NSCLC H1975 cells were cultured to the exponential growth phase in RPMI 1640 supplemented with 10% (v/v) fetal calf serum in a humidified atmosphere containing 5% CO2. Cytotoxicity against Ramos and H1975 cells was assessed as follows: 4 104 cells seeded onto 96-well plates were incubated with compounds at the indicated concentrations at 37 °C for 24 h (Ramos) or 72 h (H1975). Cell viability was determined using measurement ATP level in living cells method by CellTiter-Glo kit according to the manufacturer’s instructions. Ibrutinib and afatinib were used as reference drug for Ramos and H1975 cell, respectively. Acknowledgments This work was supported by the Thailand Research Fund (TRF) (Grant No. BRG5980002) and the National Natural Science Foundation of China (Grant No. 21561142002). A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2017.02.054. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
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