Bafilomycins N and O, novel cytotoxic bafilomycin analogues produced by Streptomyces sp. GIC10-1 isolated from marine sponge Theonella sp.

Tetrahedron 73 (2017) 5170e5175

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Bafilomycins N and O, novel cytotoxic bafilomycin analogues produced by Streptomyces sp. GIC10-1 isolated from marine sponge Theonella sp. Yu-Hsin Chen a, b, Juan-Cheng Yang c, d, Mei-Chin Lu b, e, Ching-Feng Weng a, Yin-Di Su b, Jimmy Kuo b, e, Yang-Chang Wu f, g, h, **, Ping-Jyun Sung b, d, e, f, i, * a

Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, Hualien, 974, Taiwan National Museum of Marine Biology and Aquarium, Pingtung, 944, Taiwan School of Pharmacy, College of Pharmacy, China Medical University, Taichung, 404, Taiwan d Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung, 404, Taiwan e Graduate Institute of Marine Biology, National Dong Hwa University, Pingtung, 944, Taiwan f Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, 807, Taiwan g Research Center for Natural Products and Drug Development, Kaohsiung Medical University, Kaohsiung, 807, Taiwan h Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, 807, Taiwan i Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, 804, Taiwan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 May 2017 Received in revised form 1 July 2017 Accepted 5 July 2017 Available online 8 July 2017

Two novel 16-membered tetraene marcolides, bafilomycins N (1) and O (2), along with two known analogues, JBIR-100 (3) and bafilomycin K (4) were produced from isolate GIC10-1, a Streptomyces sp. strain that was originally cloned from bacterial communities associated with marine sponge Theonella sp. The structures of new bafilomycins 1 and 2 were established by using spectroscopic methods and comparing the spectroscopic data with those of known related metabolites. Compounds 1 and 2 were proven to be the first 14-methylbafilomycins to be isolated. Cytotoxicity of these compounds toward various cancer cell lines also is described. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Bafilomycin Streptomyces Theonella Cytotoxicity

1. Introduction In continuing studies involving screening of fermentation broths to identify marine natural products with cytotoxic activities against cancer cells, an organic extract of bacterium was identified as strain GIC10-1, a Streptomyces sp. isolate that was originally cloned from bacterial communities associated with marine sponge Theonella sp. (family Theonellidae). The results of our study indicated that this extract exhibited significant cytotoxicity towards K562 (human chronic myelogenous leukemia) and MOLT-4 (human acute lymphoblastic leukemia) cells (IC50 ¼ 0.21, 0.02) mg/mL),

* Corresponding author. National Museum of Marine Biology and Aquarium, Pingtung, 944, Taiwan. ** Corresponding author. Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, 807, Taiwan. E-mail addresses: [email protected] (Y.-C. Wu), [email protected] (P.-J. Sung). http://dx.doi.org/10.1016/j.tet.2017.07.009 0040-4020/© 2017 Elsevier Ltd. All rights reserved.

respectively. In addition, we discovered four 16-membered tetraene macrolides, including two novel 14-methylbafilomycins, bafilomycins N (1) and O (2), and their known analogues, JBIR-100 (3)1 and bafilomycin K (4),2 respectively, through bioassay-guided fractionation. Previous research has shown that bafilomycins are a family of 16-membered tetraene macrolides that are produced by actinomycetes via the polyketide pathway,3 and compounds of this type exhibit extensive pharmacological activities. In the current study, we further investigated the fermentation, isolation, structure determination, and cytotoxicity of these bafilomycins derived from a microorganism associated with a marine sponge.

Y.-H. Chen et al. / Tetrahedron 73 (2017) 5170e5175

2. Results and discussion Bafilomycin N (1) was isolated as an amorphous powder that gave an [MþNa]þ ion peak at m/z 711.40813 in the HRESIMS, indicating the molecular formula C39H60O10 (calcd for C39H60O10þNa, 711.40787) (10 of unsaturation). IR absorptions at 3416 and 1667 cm1 suggested the presence of hydroxy and a,bunsaturated lactone groups in 1. The 13C NMR and DEPT spectra of 1 (Table 1) showed that this compound had 39 carbons, including eleven methyls, two sp3 methylenes, twelve sp3 methines (including five oxymethines), seven sp2 methines, one sp3 quaternary carbon, and six sp2 quaternary carbons (including three ester carbonyls). From the 13C NMR data, eight degrees of unsaturation were accounted for, and 1 was deemed to be a bicyclic compound. The gross structure of 1 was determined using 2D NMR studies. From the 1He1H COSY spectrum of 1, four different structural units were identified, which were assembled with the assistance of an HMBC experiment (Table 1 and Fig. 1). The presence of a lactone ring between C-1 (dC 173.8) and C-15 (dC 79.8) via an oxygen atom was deduced from an HMBC correlation between H-15 (dH 4.85)/C1. The C-26, C-27, and C-30 vinyl methyls at C-2 (dC 123.0), C-4 (dC 134.8), and C-10 (dC 140.9) were confirmed by HMBC correlations between H3-26/C-1, C-2, C-3; H3-27/C-3, C-4, C-5; and H3-30/C-9, C-10, C-11; and further supported by allylic couplings between H-3/ H3-26, H-5/H3-27, and H-11/H3-20, respectively. The C-19 hydroxy group was concluded to be a part of a hemiketal constellation on the basis of a characteristic quaternary carbon signal at dC 100.3 (C19). Thus, the remaining hydroxy groups had to be positioned at C-7 and C-17, as indicated by analysis of 1He1H COSY correlations and characteristic NMR signals. Furthermore, the remaining fumaric acid unit was deduced on the basis of a proton coupling between olefin protons H-20 (dH 6.48) and H-30 (dH 6.91); HMBC correlations between H-20 and both carbonyl carbons at C-10 (dC 167.7) and C-40 (dC 172.9); and an HMBC correlation between H-30 and the carbonyl carbon C-10. An HMBC correlation between H-21 (dH 4.99) and the C-10 carbonyl carbon proved that the substitution position of the fumaric acid moiety was at C-21. Based on the data obtained from the aforementioned experiments, the planar structure of 1 was able to be established. The relative configuration of 1 was elucidated on the basis of the results of a NOESY experiment and by vicinal 1He1H proton coupling constant analysis. In the NOESY experiment of 1, the correlations between H-3/H-5, H-11/H-13, and H-12/H3-30, as well as the lack of correlation between H-3/H3-26, H-5/H3-27, and H-11/ H3-30, reflected the E-configurations of the C-2/C-3, C-4/C-5, and C10/C-11 double bonds. The double bonds between C-12/C-13 and C-

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20 /C-30 were designated as being of the E-configurations owing to the detection of large coupling constants between olefinic protons H-12/H-13 (J ¼ 14.8 Hz) and H-20 /C-30 (J ¼ 15.6 Hz), respectively. By comparison of the 13C chemical shifts with those of a related known bafilomycin analogue, bafilomycin C1 (5) (Fig. 2), which possesses a structure similar to that of 1,4e6 it was found that the 13C NMR chemical shifts for the stereogenic centers C-6 (dC 38.3), C-7 (dC 81.4), C-8 (dC 41.4), C-16 (dC 39.5), C-17 (dC 71.8), C-18 (dC 43.4), C-19 (dC 100.3), C-21 (dC 75.6), C-22 (dC 39.4), C-23 (dC 77.2) in 1 were identical to those of bafilomycin C1 [C-6 (dC 38.5), C-7 (dC 81.2), C-8 (dC 41.7), C-16 (dC 39.4), C-17 (dC 72.0), C-18 (dC 43.7), C-19 (dC 100.3), C-21 (dC 75.5), C-22 (dC 39.4), C-23 (dC 77.3)],6 suggesting that these stereogenic centers are of the same configurations as those of 5. It is interesting to note that all bafilomycin analogues possess the same configurations at stereogenic centers C-6, C-7, C8, C-14, C-15, C-16, C-17, C-18, C-19, C-21, C-22, and C-23,116 and in known bafilomycins JBIR-100 (3)1 and bafilomycin K (4),2 which were also obtained in this study from Streptomyces sp. GIC10-1, the Me-31 at C-14 in 1 is of an a-orientation according to a NOESY correlation between H-16 and H3-31. Based on the above findings, the structure of 1 was established and the configurations of the stereogenic centers of 1 were assigned as 6R*, 7S*, 8S*, 14S*, 15S*, 16S*, 17R*, 18S*, 19R*, 21R*, 22S*, and 23R*. Since 1982, when the first bafilomycin marcolide analogue, hygrolidin, was obtained from the Streptomyces hygroscopicus D1166 strain,7,8 all naturally-occurring bafilomycin-based natural marcolides produced by Streptomyces have been found to possess a methyl group at C-16 trans to H-15, and these two groups have been proven to be b- and a-oriented, respectively, by X-ray diffraction analysis.9,10 Based on biosynthetic derivation,3 we suggest that the absolute configurations of the stereogenic centers of 1 are assigned as 6R, 7S, 8S, 14S, 15S, 16S, 17R, 18S, 19R, 21R, 22S, and 23R. Bafilomycin O (2) was found to have the molecular formula C35H58O7, as deduced from HRESIMS at m/z 613.40763 (calcd for C35H58O7þNa, 613.40748). The IR spectrum of 2 showed bands at 3405 and 1667 cm1, which were in agreement with the presence of hydroxy and a,b-unsaturated lactone groups. Carbonyl resonances in the 13C NMR spectrum of 2 at dC 173.8 revealed the presence of a lactone moiety (Table 2). It was found that the NMR data of 2 were similar to those of 1, except that the signals corresponding to the 21-fumarate in 1 were replaced by a hydroxy group in 2. From the NOESY signals of 2, H3-34 was correlated with H-21, indicating that the hydroxy group at C-21 was b-oriented. The results of 1He1H COSY and HMBC correlations fully elucidated the positions of functional groups (Table 2), and hence bafilomycin O (2) was found to be the 21-defumaroxy derivative of 1. Bafilomycins N (1) and O (2) were proven to be rare 14methylbafilomycin analogues. It was obvious that bafilomycins N (1) and O (2) were the precursors of JBIR-100 (3) and bafilomycin K (4), respectively. JBIR-100 (3) and bafilomycin K (4) might be derived from bafilomycins N (1) and O (2) by oxidation, decarboxylation, and methyoxylation to produce 3 and 4, respectively. The cytotoxicities of bafilomycins 1e4 against human cancer cell lines K-562 (chronic myelogenous leukemia), MOLT-4 (acute lymphoblastic leukemia), U-937 (histiocytic lymphoma), SUP-T1 (Tcell lymphoblastic lymphoma), and LNCaP (prostate adenocarcinoma) cells were investigated. The results were as shown in Table 3. Comparison of the cytotoxic activities of the isolated compounds 1 and 2 suggested that the 21-fumarate moiety in compound 1 was critical for the cytotoxic activity. According to the outcome of cytotoxic assays, small structural variations could dramatically influence the biological activities of compounds of this type (see Table 3), and the structure-activity relationship (SAR) warrants further study to inform potential drug development in the future.

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Table 1 1 H and 13C NMR data, 1He1H COSY, and HMBC correlations for 1. C/H 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 26 27 28 29 30 31 32 33 34 35 21-fumarate 10 20 30 40 a b c d

dHa

He1H COSY

dCb 173.8 (C)d 123.0 (C) 147.8 (CH) 134.8 (C) 147.0 (CH) 38.3 (CH) 81.4 (CH) 41.4 (CH) 42.4 (CH2) 140.9 (C) 126.0 (CH) 129.0 (CH) 133.7 (CH) 41.8 (CH) 79.8 (CH) 39.5 (CH) 71.8 (CH) 43.4 (CH) 100.3 (C) 40.5 (CH2) 75.6 (CH) 39.4 (CH) 77.2 (CH) 29.2 (CH) 14.6 (CH3) 14.1 (CH3) 15.4 (CH3) 17.9 (CH3) 22.1 (CH3) 20.1 (CH3) 20.3 (CH3) 10.0 (CH3) 7.1 (CH3) 12.6 (CH3) 21.6 (CH3)

7.29 br s c

5.95 br d (8.4) 2.55 m 3.22 dd (6.4, 2.0) 1.83 m 2.00e1.89 m (2H) 5.64 6.33 5.18 2.81 4.85 1.86 4.10 1.81

d (10.8) dd (14.8, 10.8) dd (14.8, 8.8) m m m dd (10.4, 1.6) qd (7.2, 1.6)

2.29 4.99 1.62 3.57 1.89 0.81 2.02 1.96 1.06 0.90 1.83 1.02 0.83 0.97 0.83 0.94

dd (12.0, 4.8); 1.26 m ddd (11.2, 11.2, 4.8) m dd (10.0, 2.0) m d (7.2) d (0.8) d (1.2) d (7.2) d (6.4) s d (6.8) d (6.8) d (7.2) d (6.4) d (6.8)

167.7 129.0 143.2 172.9

6.48 d (15.6) 6.91 d (15.6)

(C) (CH) (CH) (C)

1

HMBC

H-5, H3-26

C-1, C-2, C-5, C-27

H-3, H-5, H-6, H-7, H-8,

C-3, C-5, C-5, C-6, C-7,

H-6, H3-27 H-7, H3-28 H-8 H2-9, H3-29 H-11

C-27 C-28 C-28, C-29 C-9, C-10 C-8, C-30

H2-9, H-12, -30 H-11, H-13 H-12, H-14 H-13, H-15, H3-31 H-14, H-16 H-15, H-17, H3-32 H-16, H-18 H-17, H3-33

C-30 C-14 C-11 C-12, C-15 C-1, C-14, C-25, C-31, C-32 C-17, C-32 C-18, C-33 C-19, C-33

H-21 H2-20, H-22 H-21, H-23, H3-34 H-22, H-24 H-23, H3-25, H3-35 H-24 H-3 H-5 H-6 H-8 H-11 H-14 H-16 H-18 H-22 H-24

C-19, C-21, C-22 C-10 C-21, C-23 C-25, C-34 C-25 C-23, C-24, C-35 C-1, C-2, C-3 C-3, C-4, C-5 C-5, C-6, C-7 C-7, C-8, C-9 C-9, C-10, C-11 C-13, C-14, C-15 C-15, C-16, C-17 C-17, C-18, C-19 C-21, C-22, C-23 C-23, C-24, C-25

H-30 H-20

C-10 , C-40 C-10

Spectrum recorded at 400 MHz in CD3OD. Spectrum recorded at 100 MHz in CD3OD. J values (in Hz) in parentheses. Multiplicity deduced from DEPT and HSQC spectra and incidated by the usual symbols.

25

35

1 23

O HO

21 1'

4'

2

4

28

OH

34

: HMBC

27

O

24

: 1H-1H COSY

26

O

OH

O

29 15

19

10

O OH

O

33

32

31

30

Fig. 1. 1He1H COSY and selective HMBC correlations that show the main carbon skeleton of 1.

3. Experimental 3.1. General experimental procedures Melting points of all the compounds were determined using a Fargo apparatus, and the measured values were uncorrected. Optical rotations were measured using a digital polarimeter (Jasco P1010). IR spectra were obtained using a spectrophotometer (Thermo, Nicolet FT-IR iS5); peaks are reported in cm1. NMR

spectra were recorded on a FT-NMR spectrometer (Bruker, AVIII HD700X) operating at 700 MHz for 1H and 175 MHz for 13C, or by FT-NMR (Varian, Mercury Plus 400) operating at 400 MHz for 1H and 100 MHz for 13C, respectively, in CD3OD or CDCl3 using the residual signal as the internal standard; coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded using a Bruker 7 FTMS system. Column chromatography was performed on normalphase silica gel (230e400 mesh, Merck) and C18 (17%) reversephase silica gel (230e400 mesh, SiliCycle). TLC was carried out on

Y.-H. Chen et al. / Tetrahedron 73 (2017) 5170e5175

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Fig. 2. The structure of bafilomycin C1 (5).

Table 2 1 H and 13C NMR data, 1He1H COSY, and HMBC correlations for 2. C/H 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 26 27 28 29 30 31 32 33 34 35 a b c d e

d Ha

He1H COSY

dCb

1

HMBC

H3-26

C-1, C-2, C-5, C-26

H-6, H-5, H-6, H-7, H-8,

C-3, C-27 C-4, C-5, C-28 C-5, C-29 C-10 C-7, C-8, C-10, C-11

d

173.8 (C) 123.0 (C) 147.9 (CH) 134.8 (C) 147.0 (CH) 38.3 (CH) 81.4 (CH) 41.4 (CH) 42.4 (CH2) 140.9 (C) 126.0 (CH) 129.0 (CH) 133.7 (CH) 41.8 (CH) 79.8 (CH) 39.5 (CH) 71.8 (CH) 43.5 (CH) 100.4 (C) 43.9 (CH2) 71.3 (CH) 42.1 (CH) 77.6 (CH) 29.2 (CH) 14.7 (CH3) 14.1 (CH3) 15.4 (CH3) 18.0 (CH3) 22.1 (CH3) 20.1 (CH3) 20.3 (CH3) 10.0 (CH3) 7.1 (CH3) 12.5 (CH3) 21.7 (CH3)

7.29 s 5.95 2.55 3.22 1.86 2.00

d (9.1)c m dd (7.7, 2.1) m m; 1.93 m

5.64 6.33 5.17 2.80 4.84 1.92 4.10 1.77

d (11.2) dd (14.7, 11.2) dd (14.7, 9.1) m m m dd (10.5, 2.1) qd (7.0, 2.1)

2.18 3.52 1.30 3.42 1.89 0.78 2.02 1.97 1.06 0.89 1.83 1.02 0.83 0.99 0.92 0.92

dd (11.9, 4.9); 1.16 dd (11.9, 11.9) ddd (11.9, 10.5, 4.9) m dd (9.8, 2.1) m d (7.0) s s d (7.0) d (7.0) s d (6.3) d (7.0) d (7.0) d (7.0) d (7.0)

H3-27 H-7, H3-28 H-8 H2-9, H3-29 H-11

H2-9, H-12, H3-30 H-11, H-13 H-12, H-14 H-13, H-15, H3-31 H-14, H-16 H-15, H-17, H3-32 H-16, H-18 H-17, H3-33

C-9, C-13, C-30 C-14 C-11 C-13 C-1, C-14, C-31, C-32 C-17 C-33 C-19, C-33

H-21 H2-20, H-22 H-21, H-23, H3-34 H-22, H-24 H-23, H3-25, H3-35 H-24 H-3 H-5 H-6 H-8 H-11 H-14 H-16 H-18 H-22 H-24

C-21 n.o.e n.o. C-25 n.o. C-23, C-24, C-35 C-1, C-2, C-3 C-3, C-4, C-5 C-5, C-6, C-7 C-7, C-8, C-9 C-9, C-10, C-11 C-13, C-14, C-15 C-15, C-16, C-17 C-17, C-18, C-19 C-21, C-22, C-23 C-23, C-24, C-25

Spectrum recorded at 700 MHz in CD3OD. Spectrum recorded at 175 MHz in CD3OD. J values (in Hz) in parentheses. Multiplicity deduced from DEPT and HSQC spectra and incidated by the usual symbols. n.o. ¼ not observed.

Table 3 Cytotoxic effects of bafilomycins 1e4 on different human tumor cell lines. Compounds

1 2 3 4 doxorubicina

Cell lines IC50 (nM) K-562

MOLT-4

U-937

SUP-T1

LNCaP

31.8 54.2 9.9 305.1 85.5

0.01 389.6 N. A. 5.00 17.2

N.A.b N.A. N.A. N.A. 224.1

6.0 64.4 0.9 42.9 70.7

3.9 118.6 89.4 3.3 155.2

a Doxorubicin (¼adriamycin) was used as a reference compound for cytotoxic activity. b N.A. ¼ not active at 2.0 mg/mL.

precoated Kieselgel 60 F254 (0.25 mm; Merck), and spots were visualized by spraying with 10% H2SO4 solution followed by heating. Normal-phase HPLC (NP-HPLC) was performed using a system comprised of a pump (Hitachi, L-7100), an injection port (Rheodyne, 7725), and a semi-preparative normal-phase column (Supelco Ascentis Si, Cat #:581514-U, 25 cm  10 mm, 5 mm; SigmaAldrich). Reverse-phase HPLC (RP-HPLC) was performed using a system comprised of a pump (Hitachi, L-2130), a photodiode array detector (Hitachi, L-2455;), an injection port (Rheodyne, 7725), and a reverse-phase column (Luna 5 mm, C18(2) 100 Å AXIA Packed, 250  21.2 mm).

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3.2. Marine bacteria isolation, culture conditions, and extract preparation Marine bacterium strain GIC10-1 was isolated from marine sponge Theonella sp., which was collected off the coast of Kenting, south Taiwan on February 9, 2012, at a depth of 5 m. The bacterium strain GIC10-1 was 98.3% identical to Streptomyces sp. OPMA00072 (Genebank accession no. AB896819) on the basis of the 16S rRNA gene sequence. The marine bacterium was cultured in 2.5 L flasks containing 1 L M1 broth (1% starch, 0.4% yeast extract, 0.2% peptone; not containing agar) with 80% seawater. The bacterial culture was incubated at 25  C on an orbital shaker at a rotation speed of 120 rpm. After five days of incubation, extraction of the culture broth (50.4 L) with ethyl acetate (EtOAc, 2  50.4 L) yielded 3.91 g of crude extract in total. The extracts obtained was then stored at 20  C until further use. 3.3. Separation The crude extract was separated on Sephadex LH-20 and eluted using MeOH to yield 9 fractions 1e9. Fraction 1 was separated on C18 reverse-phase silica gel and eluted using MeOH/H2O (stepwise, 3:2epure MeOH) to yield 10 subfractions 1Ae1J. Fraction 1D was purified by RP-HPLC, using a mixture of solvent (MeOH:H2O ¼ 17:3) as a mobile phase to afford 4 subfractions 1D1e1D4. Fraction 1D2 was repurified by RP-HPLC, using a mixture of solvent (acetonitrile:H2O ¼ 3:1) as a mobile phase to afford bafilomycin N (1). Fraction 2 was chromatographed on silica gel and eluted using n-hexane/acetone (stepwise, 100:1eacetone) to yield 17 subfractions 2Ae2Q. Fraction 2Q was separated on C18 reverse-phase silica gel and eluted using MeOH/H2O (stepwise, 1:1epure MeOH) to yield 17 subfractions 2Q1e2Q17. Fraction 2Q5 was purified by RP-HPLC, using a mixture of MeOH and H2O (17:3) as a mobile phase to afford 5 subfractions 2Q5Ae2Q5E. Fraction 2Q5B was repurified by RP-HPLC, using a mixture of MeOH and H2O (3:1) as a mobile phase to obtain JBIR-100 (3). Fraction 3 was separated on silica gel and eluted using n-hexane/acetone (stepwise, 100:1eacetone) to yield 25 subfractions 3Ae3Y. Fraction 3Q was purified by NP-HPLC, using a mixture of solvent (nhexane:acetone ¼ 12:5) as a mobile phase to afford bafilomycin O (2). Fraction 3R was purified by NP-HPLC, using a mixture of solvent (n-hexane:acetone ¼ 12:5) as a mobile phase to afford 4 subfractions 3R1e3R4. Fraction 3R3 was purified by RP-HPLC, using a mixture of MeOH and H2O (4:1) as a mobile phase to afford 8 subfractions 3R3Ae3R3H. Fraction 3R3E was purified by RP-HPLC, using a mixture of acetonitrile and H2O (4:1) as a mobile phase to afford bafilomycin K (4). 3.3.1. Bafilomycin N (1) White powder (0.9 mg); mp 129e130  C; e12 (c 0.06, CHCl3); IR 13 (neat) nmax 3416, 1667 cm1; 1H (CD3OD, ½a25 C D 400 MHz) and (CD3OD, 100 MHz) NMR data (see Table 1); ESIMS: m/z 711 (M þ Na)þ; HRESIMS: m/z 711.40813 (calcd for C39H60O10þNa, 711.40787). 3.3.2. Bafilomycin O (2) White powder (1.1 mg); mp 78e79  C; ½a25 D þ7 (c 0.05, CHCl3); IR (neat) nmax 3405, 1667 cm1; 1H (CD3OD, 700 MHz) and 13C (CD3OD, 175 MHz) NMR data (see Table 2); ESIMS: m/z 613 (M þ Na)þ; HRESIMS m/z 613.40763 (calcd for C35H58O7þNa, 613.40748). 3.3.3. JBIR-100 (3) White powder (8.8 mg); mp 144  C; ½a26 D e24 (c 0.41, CHCl3) (ref.1, ½a25 e15.7 (c 0.1, MeOH)); IR (neat) n 3409, 1671 cm1; 1H max D

(CDCl3, 400 MHz) and 13C (CDCl3, 100 MHz) NMR data were found to be in complete consistent with the finding reported formerly1; ESIMS: m/z 727 (M þ Na)þ. 3.3.4. Bafilomycin K (4) White powder (1.3 mg); mp 104e105  C; ½a26 D þ62 (c 0.07, CHCl3); IR (neat) nmax 3447, 1717 cm1; 1H (CDCl3, 400 MHz) and 13C (CDCl3, 100 MHz) NMR data were found to be in complete consistent with the finding reported formerly2; ESIMS: m/z 629 (M þ Na)þ. 3.4. MTT antiproliferation assay MOLT-4, K-562, U-937, SUPT-1, and LNCaP cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin) at 37  C in a humidified atmosphere of 5% CO2. Cells were seeded at 4  104 per well in 96-well culture plates before treatment with different concentrations of the tested compounds. The compounds used for cytotoxic assay were all dissolved in dimethyl sulfoxide and diluted to 2, 0.4, 0.08, 0.016, and 0.0032 mg/mL prior to the experiments (the final dimethyl sulfoxide concentrations were lower than 0.02%). Cells were incubated with different concentrations of the compounds for 72 h, then the cytotoxicities of the tested compounds were measured using a colorimetric MTT assay (thiazolyl blue tetrazolium bromide, M2128; Sigma Aldrich) to determine the cell numbers after treatment. In the MTT assay, mitochondrial dehydrogenases produced in viable cells reduces MTT to a formazan product of a purple color. After using dimethyl sulfoxide to dissolve the MTT-formazan product, light absorbance values (OD¼OD570eOD620) of the wells were recorded at wavelengths of 570 and 620 nm using an ELISA reader (Anthos labtec Instrument, Salzburg, Austria). The data were used to calculate the concentration that caused 50% inhibition (IC50), i.e., the concentration that resulted in the OD value of cells treated with the compound being half that of cells treated with vehicle control. Acknowledgments This research was supported by grants from the National Museum of Marine Biology and Aquarium; the National Dong Hwa University; the National Sun Yat-sen University; and the National Research Program for Biopharmaceuticals, Ministry of Science and Technology (Grant Nos. MOST 105-2325-B-291-001, 105-2811-B291-003, 104-2320-B-291-001-MY3, and 104- 2325-B-291-001), Taiwan, awarded to P.-J.S. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tet.2017.07.009. References 1. Ueda J-Y, Hashimoto J, Yamamura H, Hayakawa M, Takagi M, Shin-ya K. J Antibiot. 2010;63:627e629. 2. Zhang D-J, Wei G, Wang Y, et al. J Antibiot. 2011;64:391e393. 3. Zhang W, Fortman JL, Carlson JC, et al. ChemBioChem. 2013;14:301e306. € nberg G. J Am Chem Soc. 4. Hensens OD, Monaghan RL, Huang L, Albers-Scho 1983;105:3672e3679. 5. Werner G, Hagenmaier H, Albert K, Kohlshorn H. Tetrahedron Lett. 1983;24: 5193e5196. 6. Moon S-S, Hwang W-H, Chung YR, Shin J. J Antibiot. 2003;56:856e861. 7. Seto H, Akao H, Furihata K, Otake N. Tetrahedron Lett. 1982;23:2667e2670. 8. Corey EJ, Ponder JW. Tetrahedron Lett. 1984;25:4325e4328.

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