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Iakyricidins A#D, antiproliferative piericidin analogs bearing carbonyl group or cyclic skeleton from Streptomyces iakyrus SCSIO NS104 Kunlong Li, Zhi Liang, Weihao Chen, Xiaowei Luo, Wei Fang, Shengrong Liao, Xiuping Lin, Bin Yang, Junfeng Wang, Lan Tang, Yonghong Liu, and Xuefeng Zhou J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Jul 2019 Downloaded from pubs.acs.org on July 26, 2019
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Iakyricidins A‒D, antiproliferative piericidin analogs bearing carbonyl group or cyclic skeleton from Streptomyces iakyrus SCSIO NS104 Kunlong Li,†,‡ Zhi Liang,§ Weihao Chen,†,‡ Xiaowei Luo,†,‡ Wei Fang,⊥Shengrong Liao,† Xiuping Lin,† Bin Yang,† Junfeng Wang,† Lan Tang, *,§ Yonghong Liu,*,†,‡ and Xuefeng Zhou*,†,‡ CAS Key Laboratory of Tropical Marine Bio-resources and Ecology/Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § Biopharmaceutics, Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China †
⊥
Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Wuhan 430064, China.
Corresponding Author * E-mail:
[email protected] (X. Zhou). * E-mail:
[email protected] (Y. Liu). * E-mail:
[email protected] (L. Tang). OH
OH O O
O
OH
O
N
OH OH
N R
R
5/6 R = CH3/H oxidative cleavage
cyclization
OH
OH
O O
2/3 R = CH3/H
O
OH N
O
OH OH
N
O
1
4
ABSTRACT: Iakyricidins A‒D (1‒4), a carbonyl containing piericidin derivate and three novel piericidin analogs bearing cyclic skeleton, were isolated from the mangrove sediment-derived strain Streptomyces iakyrus SCSIO NS104. These structures were established by spectroscopic techniques, Mosher’s method, and ECD calculations. Compounds 2‒4 represent a novel skeleton of piericidins with branched chain C-C cyclization, and their biosynthetic pathways are proposed. Compound 1, the first natural carbonyl containing piericidin derivate, exhibited potent antiproliferative activity against ACHN with an IC50 value of 20 nM.
Actinomycetes are continue to be one of the most prolific sources of novel chemical structures and new antibiotics.1 Piericidins, which feature a 4-pyridinol core linked with a variable methylated polyene side chain, are exclusively produced by actinomycetes, especially members of the genus Streptomyces isolated from soil, insects and marine samples. Before our study, there are more than 40 naturally occurring piericidin analogs discovered, while most of them were reported before the year of 2000.2 Marine-derived actinomycetes have become principal sources of new piericidins in this century.3-6 Besides glycosylation, the structural variations of this class occur mainly on the branched chains, such as common methylation, demethylation, butenylation, and epoxidation.2 Piericidin A, the first discovered and most common piericidin, is known as a potent inhibitor of
NADH−ubiquinone oxidoreductase (complex I) in the mitochondrial electron transport chain.7 In addition to antimicrobial and insecticidal activities as mitochondrial complex I inhibitors,8 some piericidins were also reported to have potential as antitumour agents.2, 9-11 Recently, an efficient total synthesis route of piericidin A was reported, and we expect that it will contribute to the development of this group of antibiotics.12 In order to discover more natural piericidin analogs with diverse structures and biological activities, several marinederived Streptomyces strains were screened for piericidins by dereplication using HPLC/HRMS. As a result, the strain Streptomyces iakyrus SCSIO NS104, isolated from a mangrove sediment sample collected from the Pearl River estuary to South China Sea, was chosen for chemical investigation. A carbonyl containing piericidin derivate,
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iakyricidin A (1), and three novel piericidin analogs bearing cyclic skeleton, iakyricidin B‒D (2‒4), were obtained from the liquid cultures, along the known IT-143-A (5), IT-143-B (6) and piericidin A (7) (Figure 1). Iakyricidins B‒D (2‒4) represent new subgroup of piericidin with C-C cyclization and double bonds rearrangements in the polyene side chain. Herein, we present the isolation, structure elucidation, hypothetical biogenesis pathways, and biological evaluation of the four new piericidin analogs. 15
OH
OH
4'
8' O
7' O 2' N
9' 4
6'
1 OH O O 2' N
10
1 21
20
19
O
OH
8
18
17
13
O
17
OH 5 20
O 2' N
1 21
19
4 9' 4
O 2' N
OH
7' O 2' N 6'
B H9
OH
R
10
14
18
17
8
16
OH
1
10
12
14
18
17
O
NOESY 3
11
17
Me Me 19
19
HMBC
H10 12
15 19
20
18
8
12
5 21
HO
18
19
5 R = CH3 6R=H
OH O
R
21
8
OH
OH
OH
8
COSY
10
8
9' 2
9'
O
1 21
16
2 R = CH3 3R=H OH
8
1
OH
4'
O
17
5
O 2' N
14
absolute configuration of 1 (Figure 3A). Thus, the structure was established and assigned the trivial name iakyricidin A (1). This is the first report of carbonylic-containing piericidin analog in nature. A
14 12
15 9'
9'
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erythro
J H9-H10 = 7.4 Hz J H9-C11 = 2.4 Hz 3 J H10-C8 = 2.6 Hz 3 J H10-C18 = 2.9 Hz 2 J H9-C10 = 2.7 Hz 3
Figure 2. A) Key COSY, HMBC and NOESY correlations of 1. B) Newman projection and key NOESY correlations in the side chain in 1, with key interproton and carbon-proton spincoupling constants.
13
7
Figure 1. Structures of compounds 1–7. Iakyricidin A (1) was isolated as a pale yellow gum. Its molecular formula of C27H39NO5 was established by HRESIMS (m/z 458.2909 [M + H]+), which required 9 degrees of unsaturation. The NMR spectra indicated the presence of 9 methyls (including two oxygenated ones), 2 methylenes, 6 methines (including four olefinic and one oxygenated), 9 aromatic/olefinic quaternary carbons and a ketone carbonyl carbon (Table 1). Comparison of its 1H, 13C NMR data with those of piericidin A2 indicated that they shared the same piericidin skeleton.13 The only difference was the replacement of 13-CH3 in piericidin A2 by an acetyl group in 1, which was confirmed by the HMBC correlations between H3-14 (δH 2.21) and C-13 (δC 199.1)/C-12 (δC 124.8), and H-12 (δH 6.25) and C-13. The planar structure of 1 was mainly elucidated by HMBC and COSY correlations (Figure 2A). The configurations of the Δ2, 5, 7, 11 double bonds were all deduced as E, based on the NOESY correlations of H2-1/H3-21, H2-4/H1-6, H1-9/H3-19, and H1-10/H1-12 (Figure 2A). The relative configuration of C-9/C-10 was analyzed by J-based configurational analysis and NOESY correlations. The protonproton spin-coupling constant, 3JH9-H10 (7.4 Hz), and carbonproton spin-coupling constants, 3JH9-C11 (2.4 Hz), 3JH10-C8 (2.6 Hz) and 3JH10-C18 (2.9 Hz) (Figure 2B, Figure S10), indicated anti rotamer of H-9/H-10, rather than gauche one.14, 15 Besides, the NOESY correlations of H-10/H3-18, H3-16/H3-15 supported the H-9/H-10 as erythro rotamer (Figure 2B).14, 15 The absolute configurations of C-9 and C-10 in the known and co-isolated piericidin A (7) were determined to be 9R,10R by the modified Mosher's method (Table S11). As the derivative of piericidin A (7), compound 1 was suggested to have the same absolute configurations, which were also confirmed by ECD calculations based on the time-dependent density functional theory (TD-DFT) (Supporting Information).16 The Boltzmann-weighted ECD curves of 9R,10R-1 and 9S,10S-1 were calculated and compared with the experimental ECD curve, which led to the determination of the 9R,10R
Iakyricidin B (2) was isolated as a pale yellow oil. The HRESIMS (m/z 486.3223 [M + H]+) data suggested the molecular formula of C29H43NO5, revealing 9 degrees of unsaturation. Comparison of its 1H, 13C NMR data with those of co-isolated IT-143-A (5) revealed that 2 is a piericidin type compound containing a pyridine ring and four double bonds in its side chain. The HMBC correlations from the olefinic methine proton H-6 (δH 6.13) to C-4 (δC 51.7)/C-5 (δC 138.0)/C-7 (δC 148.4)/C-19 (δC 115.0), and from the olefinic methylene protons H2-19 (δH 4.99 and 5.25) to C-7 permitted the establishment of Δ5,6 and Δ7,19 conjugated terminal double bonds moiety (C=CH–C=CH2), which is uncommon in the piericidin family. The HMBC correlations from H-8 (δH 2.94) to C-12 (δC 65.3)/C-13 (δC 136.3), and from H-12 (δH 3.07) to C-7, suggested the carbon bond linkage of C-8/C-12. The novel five-membered ring of C-8/C-9/C-10/C-11/C-12 with C10/C-11-diol, contributing the last unsaturation, was further confirmed by the COSY correlations for the vicinal coupled protons (H-12/H-8/H-9/H-10 and H-9/H3-18) and other HMBC correlations shown in Figure 4A. Therefore, the planar structure of 2 was established. Iakyricidin C (3) was determined to have the molecular formula of C28H41NO5 from the HR-ESIMS data. The similar 1H and 13C NMR data with those of 2 indicated that 3 had the same piericidin skeleton with cyclized side chain. The only difference was the replacement of the olefinic methyl (CH3-20) in 2 by an olefinic protons (H-5, δH 5.81, m) in 3, which was confirmed by the HMBC correlations from H-5 to C-4 (δC 43.3)/C-7 (δC 147.8), as well as the COSY correlations of H4/H-5/H-6 (Figure 4A). So, the planar structure of 3 was defined as a 5-demethylated analog of 2. Iakyricidin D (4), with the same molecular formula as 2, was determined as a piericidin analog with the same fivemembered ring. The HMBC correlations from H2-4 (δH 2.93, s) to C-5 (δC 145.1)/C-6 (δC 128.2) and terminal olefinic C-20 (δC 115.0), from terminal olefinic H2-20 (δH 5.04, s; 5.16, s) to C5/C-6, from H3-19 to C-6/C-7 (δC 140.0) double bond and C-8 (δC 58.3), allowed the assignments of the conjugated Δ5,20 and Δ6,7 , different with the conjugated double bonds in 2 and 3 (Figure 4A). So, the planar structure of 4 was confirmed.
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Figure 3. A) Experimental and calculated ECD spectra of 1. B) Experimental ECD spectra of 2, 3 and calculated ECD spectra of truncated model 2a and 2b. C) Experimental ECD spectrum of 4 and calculated ECD spectra of truncated model 4a and 4b. Table 1. 1H and 13C NMR (δ in ppm) data of iakyricidins A−D (1−4). Pos. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1a δC , type δH (J in Hz) 34.7, CH2 3.47 (d, 6.8) 123.8, CH 5.34 (t, 6.8) 133.9, C 51.2, CH2 2.70 (s) 135.0, C 130.0, CH 5.67 (s) 136.5, C 129.4, CH 5.07 (d, 9.9) 37.3, CH 2.71 (m) 81.5, CH 3.75f (d, 7.4) 156.4, C 124.8, CH 6.25 (s) 199.1, C 32.1, CH3 2.21 (s)
15.1, CH3 14.3, CH3
2.1F2 (s) 0.95 (d, 6.7)
19
17.8, CH3
1.75 (s)
20
17.5, CH3
1.65 (s)
2b δC , type δH (J in Hz) 35.8, CH2 3.64 (d, 6.8) 125.2, CH 5.73 (t, 6.8) 134.4, C 51.7, CH2 2.89 (s) 138.0, C 126.5, CH 6.13 (s) 148.4, C 56.2, CH 2.97 (t, 10.8) 40.3, CH 2.74 (m) 83.5, CH 4.36 (d, 5.5) 83.3, C 65.3, CH 3.07 (d, 10.8) 136.3, C 122.4, CH 5.54 (q, 6.7) 14.3, CH3 1.65 (d, 6.7) 16.2, CH3 1.97 (s) 22.3, CH3 1.66 (s) 13.9, CH3 1.36 d (6.9) 5.25 (s) 115.0, CH2 4.99 (s)
3a δC , type δH (J in Hz) 34.7, CH2 3.36 (d, 7.0) 122.4, CH 5.36 (t, 7.0) 134.8, C 43.3, CH2 2.76 (d, 7.0) 128.8, CH 5.81 (m) 132.2, CH 6.00 (d, 15.5) 147.8, C 49.9, CH 2.59 (t, 10.7) 40.9, CH 2.22 (m) 82.2, CH 3.85 (d, 6.7) 82.7, C 63.8, CH 2.51 (d, 10.7) 133.5, C 123.1, CH 5.23 (q, 6.6) 13.7, CH3 1.55 (d, 6.6) 15.2, CH3 1.57 (s) 20.4, CH3 1.14 (s) 12.9, CH3 0.93 (s) 5.00 (s) 112.6, CH2 4.83 (br.s)
17.9, CH3 1.84 (s)
21 16.0, CH3 1.68 (s) 16.6, CH3 1.87 (s) 16.7, CH3 2’ 153.7, C 156.0, C 153.7, C 3’ 127.9, C 130.1, C 127.9, C 4’ 154.1, C 157.4, C 154.1, C 5’ 112.1, C 114.6, C 112.1, C 6’ 151.1, C 151.3, C 151.1, C 7’ 53.2, CH3 4.06 (s) 53.5, CH3 4.07 (s) 53.2, CH3 8’ 60.8, CH3 3.88 (s) 60.6, CH3 3.75 (s) 60.8, CH3 9’ 10.6, CH3 2.11(s) 11.7, CH3 2.38 (s) 10.6, CH3 a Data were measured in CDCl b Data were measured in pyridine-d . 3. 5
δC , type 35.8, CH2 125.0, CH 134.3, C 49.5, CH2 145.1, C 128.2, CH 140.0, C 58.3, CH 39.6, CH 83.5, CH 83.3, C 64.7, CH 136.6, C 122.4, CH 14.3, CH3 15.9, CH3 22.3, CH3 13.8, CH3 14.8, CH3 115.0, CH2
1.73 (s)
3.94 (s) 3.86 (s) 2.08 (s)
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16.7, CH3 155.9, C 130.0, C 157.3, C 114.5, C 151.3, C 53.5, CH3 60.5, CH3 11.7, CH3
4b δH (J in Hz) 3.65 (d, 6.9) 5.73 (t, 6.9) 2.93 (s) 5.95 (s) 2.89 (t, 10.8) 2.73 (m) 4.35 (d, 4.8) 3.07 (d, 10.8) 5.52 (q, 6.6) 1.64 (d, 6.6) 1.96 (s) 1.67 (s) 1.31 d (6.9) 1.99 (s) 5.16 (s) 5.04 (s) 1.85 (s)
4.08 (s) 3.75 (s) 2.40 (s)
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methyltransferases. Compound 1 might be derived from 5 by oxidative cleavage between C-13 and C-14. In the plausible biosynthetic pathways of 2‒4, the most important step was the C8-C12 cyclization from IT-143-A or B (5 or 6), under the certain kind of enzyme-catalysis.20 The opposite configuration of C-9 may be caused by the rotation of the branched chain. The different rearrangements of the double bonds Δ5 and Δ7,19 led to the formations of positional isomers, like 2 and 4 with different conjugated double bonds. Above all, iakyricidins B‒D (2‒4), represent a novel skeleton of piericidin family with a five-membered ring in side chain, were considered to be biosynthesized by uncommon C-C cyclizations and rearrangements of the double bonds in the polyene side chain. Scheme 1. Plausible biogenetic pathways of 1–4. OH
OH
O 6
Figure 4. A) Key COSY and HMBC correlations of part structure of 2−4. B) Key NOESY correlations of part structure of 2/3 and 4. The E configurations of the Δ2, 5, 13 in 2, 3 and Δ2, 6, 13 in 4 were established by analysis of NOESY data. Additionally, the NOESY correlations of H-10 with H-12/H3-17/H3-18, and H-8 with H3-17/H3-18, demonstrated that H-8/H-10/H-12/H317/H3-18 were co-facial, whereas H-9/10-OH/11-OH occupied the opposite face in the five-membered ring of 2-4 (Figure 4B). Thus, the relative configurations of the five-membered ring moiety in 2−4 were established as (8S*,9S*,10R*,11S*,12R*). The modified Mosher's method was used to determine the absolute configuration of C-10 in 2.17 Treatment of 2 with (R)and (S)-MTPA-Cl yielded the (S)- and (R)-MTPA ester derivatives, respectively. Calculation of the 1H NMR Δ δS-R values for the mono-MTPA esters of 2 indicated the 10Rconfiguration (Figure 5, Table S10). Because of the limited amounts of obtained 3 and 4 to take the Mosher's method, the ECD calculations with the truncated models 2a/2b and 4a/4b were used. The Boltzmann-weighted ECD spectra of 8S,9S,10R,11S,12R-2a (Figure 3B) and 8S,9S,10R,11S,12R-4a (Figure 3C) gave the best agreement, which led to the determination of the 8S,9S,10R,11S,12R absolute configurations of 2−4, with agreement with the configuration established for 2 by Mosher's method. The inconsistent ECD curve trend between 2 and 4 may be caused by the different preferential conformations of them, because of the different conjugated double bonds (Figure 4B). Finally, the absolute configurations of 2−4 were assigned as 8S,9S,10R,11S,12R. -0.03
-0.01 -0.01 +0.02 +0.07
-0.04 -0.18
OH -0.03
+0.06 +0.03
OR
2A R = (S)-MTPA 2B R = (R)-MTPA
+0.11
Figure 5. Δ δH values (δS − δR, in ppm) for 2A and 2B. The known compounds IT-143-A (5), IT-143-B (6) and piericidin A (7) were identified by comparison of their spectral data with literature data.18, 19 The plausible biosynthetic pathways of 1‒4 were proposed (Scheme 1). The known IT-143-B (6) was originated from piericicidin A (7) by common polyketide synthase (PKS) pathway.5 The C-20 methyl residue in 1, 2, 4 and 5 was considered to be formed by methylation under some
O
8
N
12
8
13 15
6 methylation OH
OH O
6
O
O
OH
10
20
13 17
5
O
N
oxidative O cleavage O [O]
O
12 6
O
OH cyclization
N
OH
O [O]
O
OH
N 4
O
12 6
N R
OH
O
13
1
O
rearra ngem OH ent O O
13
OH
8
21 R R = CH3 / H
12
10
N
OH OH
OH
10
7
OH
10
N
PKS
8
OH
R = CH3 / H OH OH
N R 2 R = CH3 3R=H
Iakyricidins A−D (1−4) were evaluated for inhibition of mitochondrial complex I activity. As a result, 1 showed inhibitory activity towards complex I with an IC50 value of 17 μM, and 2−4 showed the weak inhibition rate of 12%-17% at 100 μM, comparing to the well-known potent complex I inhibitor piericidin A with IC50 value of 0.5 μM (Table 2). In the cell antiproliferative test of 1−6 against three human renal carcinoma cell lines, ACHN, 786-O, and OS-RC-2, 1 showed the strongest and selective antiproliferative activities towards ACHN cells with IC50 value of 20 nM, determined by the dose-response curve (Figure 6A). Iakyricidins B−D (2−4) showed weak or moderate activities against the above three cell lines, whereas 2 exhibited more effective (IC50 values of 13-31 μM) than the other two (IC50 values of 21−84 μM) (Table 2). It is suggested that the terminal α, β-unsaturated ketone group in 1 as a Michael acceptor might improve the inhibitory activity against ACHN cell, while the fivemembered ring moiety in 2‒4 might contribute little on the antiproliferative and complex I inhibitory activities. Table 2. Cell antiproliferative and mitochondrial complex I inhibitory activities of 1–6 (IC50, μM). Comp. ACHN 786-O OS-RC-2 Complex I 1 0.02 89 30 17 2 13 31 13 >100 3 69 84 21 >100 4 42 62 31 >100 5 22 61 21 – 6 98 >100 22 – Control 3.4a 13a 14a 0.5b a Sorafenib; b piericidin A
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The Journal of Organic Chemistry
Figure 6. A) Dose-response curve of antiproliferative activity of 1 against ACHN cells. B) Cell cycle in ACHN cells treated with 1 (20 and 40 nM) for 24 and 72 h. C) Apoptosis in ACHN cells induced by 1 (20 and 40 nM) for 24 and 72 h. Notably, although 1 exhibited inhibitory activity at a very low concentration, it is hard to exert more than 50% inhibition, even at the concentrations of up to 1 μM, as revealed by the dose-response curve (Figure 6A). Moreover, the ability to arrest the cell cycle and cell apoptosis effects induced by 1 were further investigated in ACHN cells. After 72 h treatment with 1 at the concentrations of 20 and 40 nM, no obvious alteration on cell cycle was found (Figure 6B), and only very few apoptotic cells were detected by flow cytometry using AV-FITC/PI double staining (Figure 6C). So, it suggested that iakyricidin A (1) could significantly inhibit the proliferation of ACHN cell, rather than killing or inducing apoptosis of ACHN cell. In our recent study, 27 piericidins were obtained in the strain Streptomyces psammoticus SCSIO NS126.21 Here, iakyricidins B−D (2−4), three novel piericidin analogs with a five-membered ring moiety were obtained and considered to be biosynthesized by uncommon C-C cyclization and double bonds rearrangement in the polyene side chain. This is the first reported three cases of piericidin derivatives probably from a relatively complex post-modification process on the branch chain, which is worthy of further study. In addition, oxidized side chain piericidin analog iakyricidin A (1) displayed potent antiproliferative activity against ACHN cell at nanomolar concentrations, which possessed the potential for further development as an antitumor agent. Experimental Section General experimental procedures. Optical rotations were acquired using a PerkinElmer MPC 500 (Waltham) polarimeter. UV spectra were recorded on a Shimadzu UV2600 PC spectrometer (Shimadzu). ECD spectra were measured with a Chirascan circular dichroism (Applied Photophysics). IR spectra were measured on an IR Affinity1spectrometer (Shimadzu, Beijing, China). The NMR spectra were obtained on a Bruker Avance spectrometer (Bruker) operating at 700 MHz for 1H NMR and 175 MHz for 13C NMR, using tetramethylsilane as an internal standard. HRESIMS spectra were collected on a Bruker miXis TOF-Q Ⅱ mass spectrometer (Bruker). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10−40 μm) and over silica gel (200−300 mesh) (Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Biosciences), respectively. All solvents employed were of analytical grade (Tianjin Fuyu Chemical and Industry Factory). The semipreparative HPLC was performed on an HPLC (Hitachi-L2130, diode array detector, Hitachi L-2455, Tokyo, Japan) using a Phenomenex ODS column (250 mm × 10.0 mm i.d., 5 μm; Phenomenex,
USA). The artificial sea salt was a commercial product (Guangzhou Haili Aquarium Technology Company). Collection and phylogenetic analysis of the strain NS104. The strain NS104 was isolated from a mangrove sediment sample collected from the Pearl River estuary (E 113°33′11.15′′, N22°53′40.16′′) to South China Sea in May 2015, by incubation at 28 °C for two weeks on ISP-2 medium (yeast extract 4 g, malt extract 4 g, glucose 4 g, crude sea salt 30 g, agar powder 20 g, distilled water 1000 mL, pH, 7.2−7.4). The strain was identified as Streptomyces iakyrus by the 16S rRNA gene sequence analysis (Supporting Information). It was deposited at the China Center for Type Culture Collection (Wuhan) as CCTCC M2017186 (Streptomyces iakyrus SCSIO NS104). Cultivation and extraction. The strain NS104 was fermented in a total volume of 30 L. A few loop of cells of the strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed medium (mannitol 1 g, soya peptone 0.5 g, soya-bean oil 0.125 g, K2HPO3 0.02 g, pH 7.0, 50 mL distilled water), and then cultivated on a rotary shaker at 120 rpm, 28 °C for 48 h as seed culture. Then, 2 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 100 mL media (cotton seed meal 2.5 g, soluble starch 1 g, glucose 1 g, yeast extract 0.3 g, CaCO3 0.5 g, sea salt, 0.2 g; in 100 mL distilled water, pH 7.2). After cultivation on a rotary shaker at 180 rpm and 28 °C for 120 h, each bacterial culture broth was broken with ultrasonic treatment apparatus for 10 min. Then each culture broth was extracted with an equal volume of ethyl acetate three times. The organic extract was then concentrated under vacuum to afford the crude extract (15.5 g). Extraction and purification. The EtOAc extract was subjected to silica gel vacuum liquid chromatography using step gradient elution of petroleum ether (PE)–CH2Cl2 (1:0, 2:1, 0:1), CH2Cl2–MeOH (200:1, 100:1, 50:1, 30:1, 0:1) to yield eight fractions according to TLC profiles (Frs.A1–A8). The subsequent purification of Frs.A2 using Sephadex LH-20 with CH2Cl2/MeOH (1:1, v/v) and semipreparative HPLC(72% MeOH/H2O, 2 ml/min, 210 nm) afforded 7 (165 mg, tR 34 min). From Frs.A3, 1 (5.2 mg, tR 33 min) was further purified through a Sephadex LH-20 with CH2Cl2/MeOH (1:1, v/v) and semipreparative HPLC (65% MeOH/H2O, 2 ml/min, 210 nm). Frs.A5 was separated into four subfractions (Frs.A5-1–5-4) by ODS silica gel chromatography eluting with MeCN/H2O (5%100%). Frs.A5-2 was directly separated by semipreparative HPLC (60% MeCN/H2O, 2 ml/min, 210 nm) to yield 3 (1.86 mg, tR 34 min). Frs.A5-3 was directly separated by semipreparative HPLC (65% MeCN/H2O, 2 ml/min, 210 nm) to provide 2 (22.18 mg, tR 32 min), 4 (1.51 mg, tR 29 min).
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Frs.A5-4 was chromatographed on silica gel, and 5 (68.2 mg, tR 28 min) and 6 (29.1 mg, tR 27 min) were isolated. 25
Iakyricidin A (1). Pale yellow oil; [α] D 6.9 (c 0.2, MeOH); IR (film) vmax 3392, 2927, 1681, 1587, 1464, 1217, 1126, 1024, 998, 669 cm-1; For 1H and 13C NMR spectroscopic data, see Table 1; HRMS m/z: [M + H]+ Calcd for C27H40NO5 458.2901; Found 458.2909 25
Iakyricidin B (2). Pale yellow oil; [α] D 7.1 (c 0.582, MeOH); IR (film) vmax 3393, 2927, 1585, 1462, 1412, 1385, 1190, 1125, 1024, 771, 667 cm-1; For 1H and 13C NMR spectroscopic data, see Table 1; HRMS m/z: [M + H]+ Calcd for C29H44NO5 486.3214; Found 486.3223. 25
Iakyricidin C (3). Pale yellow oil; [α] D 9.7 (c 0.1, MeOH). IR (film) vmax 3393, 2927, 1585, 1470, 1412, 1385, 1250, 1190, 1124, 1041, 978, 773 cm-1; For 1H and 13C NMR spectroscopic data, see Table 1; HRMS m/z: [M + H]+ Calcd for C28H41NO5 472.3057; Found 472.3057. 25
Iakyricidin D (4). Pale yellow oil; [α] D 2.6 (c 0.171, MeOH); IR (film) vmax 3375, 2928, 1470, 1412, 1190, 1124, 1022, 893, 667, 600 cm-1; For 1H and 13C NMR spectroscopic data, see Table 1; HRMS m/z: [M + H]+ Calcd for C29H44NO5 486.3214; Found 486.3223 Mono-MTPA esters of 2. Iakyricidin B (2, 2.0 mg) was dissolved in freshly distilled dry pyridine (2 mL) with drycrystals of dimethylaminopyridine (DMAP 0.2 mg). Treatment with (R)-MTPA-Cl at room temperature yielded the S-MTPA ester after 16 hours.17 The reaction mixture was purified by semipreparative HPLC (95% CH3CN in H2O) to afford S-MTPA ester (2A) at 35 min. R-MTPA ester (2B) was prepared with S-MTPA-Cl in the same manner. ∆δS-R values for mono-S- and R-MTPA esters of iakiricidin B were recorded in ppm in pyridine-d5 (Table S10). Mono-MTPA esters of 7. Piericidin A (7, 3.0 mg) was dissolved in freshly distilled dry pyridine (0.5 mL) with drycrystals of dimethylaminopyridine (DMAP 0.3 mg). Treatment with (R)-MTPA-Cl at room temperature yielded the S-MTPA ester after 20 hours.17 The reaction mixture was purified by semipreparative HPLC (90% CH3CN in H2O) to afford S-MTPA ester (7A) at 34 min. R-MTPA ester (7B) was prepared with S-MTPA-Cl in the same manner. ∆δS-R values for mono-S- and R-MTPA esters of piericidin A were recorded in ppm in CDCl3 (Table S11). Cell culture and antiproliferative bioassay. ACHN, OSRC-2 and 786-O cells were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. ACHN cells were grown and maintained in MEM medium with 10% FBS, while OSRC-2 and 786-O cells were grown in RPMI1640 medium with 10% FBS. Cell viability was determined by the CCK-8 (Dojindo) assay.16 The cells were seeded at a density of 400 to 800 cells/well in 384-well plates and then treated with various concentrations of compounds or solvent control. Sorafenib and Gemcitabine were used as positive controls. After 72 h of incubation, CCK-8 reagent was added, and absorbance of the triplicate tests were measured at 450 nm by an Envision 2104 multi-label reader (Perkin Elmer). Dose response curves were plotted to determine IC50 using Prism 5.0 (GraphPad Software Inc.). Cell cycle and apoptosis assay. Cell cycle arrest was analyzed by propidium iodide (PI) DNA staining using flow cytometry.16 Briefly, after treatment with 1 for 24, 48 and 72 h, respectively, cells were harvested, prepared, and then fixed
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overnight. The fixed cells were harvested, washed, resuspended, and finally stained with PI (Sigma-Aldrich). Cell cycle distribution was studied using an Accuri C6 (BD) flow cytometer. Cell apoptosis was analyzed using a FITC Annexin V Apoptosis Detection Kit (BD), according to the manufacturer’s protocol. Cells were treated with 1 for 24, 48 and 72 h, stained with annexin V-FITC and PI solution, examined and analyzed quantitatively using an Accuri C6 (BD) flow cytometer.22 Inhibition of mitochondria complex I. The mitochondrial complex I inhibition test of the compounds were evaluated by Mitocheck Complex I Activity Assay Kits (Cayman, 70093096), without the need to isolate mitochondria or pre-incubate with antibodies. The rate of NADH oxidation is measured by a decrease in absorbance at 340 nm and is proportional to the activity of complex I. Piericidin A were used as positive controls. According to the specification of assay kit, the samples are assayed in at least duplicate. Dose response curves were plotted to determine IC50.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: The 16S rRNA sequence of the strain; ECD calculations of 1, 2 and 4; the NMR, HRESIMS, UV and IR spectra of 1−4 (PDF).
AUTHOR INFORMATION ORCID Xuefeng Zhou: 0000-0001-9601-4869 Yonghong Liu: 0000-0001-8327-3108
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by grants from the Guangdong MEPP Funds (GDME-2018C010, GDOE[2019]A28, 2019017), National natural science foundation of China (81673677, 21772210), Drug Innovation Major Project of China (2018ZX09735001-002-003), and project from Institution of South China Sea Ecology and Environmental Engineering, CAS (No. ISEE2018PY04). We are grateful to the analytical facilities (Z. Xiao, A. Sun, Y. Zhang) in SCSIO.
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