Xiao
Lin‡
abc,
Ling
Chai‡
d,
Hong Rui
Zhu
e,
Yongjun
Zhou
e,
Yaoyao
Shen
e,
Kai Hao
Chen
e,
Fan
Sun
e,
Bu Ming
Liu
d,
Shi Hai
Xu
*b and
Hou Wen
Lin
*e
aInstitute of Marine Drugs, Guangxi University of Chinese Medicine, Nanning 530200, P. R. China
bCollege of Chemistry and Materials, Jinan University, Guangzhou 510632, P. R. China. E-mail: txush@jnu.edu.cn
cCollege of Pharmacy, Jinan University, Guangzhou 510632, P. R. China
dGuangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Guangxi Institute of Traditional Medical and Pharmaceutical Sciences, Nanning, 530022, P. R. China
eResearch Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P. R. China. E-mail: franklin67@126.com
First published on 13th January 2021
LC-HRMS/MS molecular networking enabled the targeted isolation of three new neoantimycin analogs (1, 3, 5) and two known ones (2, 4) from the culture broth of Streptomyces conglobatus RJ8. After derivatization into C1-hydroxyl form compounds (6–10) respectively, the absolute structures of 1–5 were clearly determined by analyzing the hydrolyzed components from 6–10. Compounds 2 and 3 were confirmed to be a pair of epimers with different stereochemistry at C-2, and so were 4 and 5. This is the first report of the isolation and characterization of epimers of NATs. The most abundant eight compounds we obtained were subjected to a cytotoxicity assay, 1 and 6 exhibited excellent cytotoxicity with the lowest IC50 value in the picomolar range against six human carcinoma cell lines while 7 and 8 showed potent cytotoxicity against PC-9 and PC-9/GR cell lines.
Herein, we report the application of MN led to the isolation and subsequent structure elucidation of five antimycin-like depsipeptides, neoantimycin L (1), unantimycin B1 (2), B2 (3), D1 (4), and D2 (5). 1–5 were reduced by NaBH4 to afford compounds 6–10 (Chart 1), which facilitate the absolute structural depiction of 1–5. Biological evaluation of 1–5, as well as the reduced derivatives (6–8), revealed moderate to potent cytotoxic properties against six cancer cells (human colon cancer cell line, human gastric cancer cell line, and human non-small cell lung cancer cell line as well as their respectively drug-resistant cancer cell lines).
Neoantimycin L (1) was isolated as a pale yellow amorphous solid. Comparable HR-ESI-MS and NMR analyses (Tables 1, S1, S2 and Fig. S2†) with the reported NAT-H permitted the structural assignment of 1 (C37H46N2O12). The mass difference of 14.01 Da between 1 and NAT-H and the additional NMR signals for a methylene group CH2-21 and methyl group CH3-35, resulting in resonances at δH 1.03/δC 23.7 and δH 0.77/δC 11.1 respectively, suggested an additional methylene group present in 1. Further analysis of the 1H–1H COSY and HMBC (Fig. 2) indicated that compound 1 had two units of 2-hydroxy-3-methyl valerate, and it was a methylated congener of NAT-H.
No. | 1 | No. | 2 | 3 | ||||
---|---|---|---|---|---|---|---|---|
δ C | δ H (J in Hz) | δ C | δ H (J in Hz) | δ C | δ H (J in Hz) | |||
a Carbon resonances are not observed due to signal broadening. | ||||||||
1 | 202.6 | 1 | 202.4 | 202.6 | ||||
2 | 76.7 | 5.67, dd (7.6, 5.6) | 2 | 76.4 | 5.40, dd (10.0, 2.6) | 76.4 | 5.68, dd (7.7, 5.6) | |
3 | 167.0 | 3 | 168.1 | 167.2 | ||||
4 | 75.4 | 5.10, d (5.2) | 4 | 75.5 | 5.02, d (3.0) | 75.9 | 5.05, d (5.4) | |
5 | 168.0 | 5 | 168.5 | 168.2 | ||||
6 | 55.4 | 5.04, br s | 6 | 55.4 | 5.16, dd (9.2, 2.9) | 55.9 | 4.95, dd (8.4, 3.1) | |
7 | 70.8 | 5.59, m | 7 | 70.3 | 5.67, qd (6.4, 3.0) | 70.6 | 5.55, m | |
8 | 167.7 | 8 | 167.7 | 167.7 | ||||
9 | 75.6 | 4.83, d (7.6) | 9 | 75.5 | 5.21, d (8.2) | 75.6 | 4.86, d (7.7) | |
10 | 170.9 | 10 | 170.8 | 171.0 | ||||
11 | 54.1 | 11 | 55.0 | 54.2 | ||||
12 | 37.1 | 3.16, dd (14.2, 5.6) | 12 | 36.6 | 3.16, m | 37.1 | 3.16, dd (14.1, 7.8) | |
3.07, dd (14.2, 7.7) | 2.92, m | 3.06, dd (14.1, 7.8) | ||||||
13 | 135.4 | 13 | 136.2 | 135.4 | ||||
14/18 | 129.6 | 7.20, m | 14/18 | 129.2 | 7.28, overlapped | 129.6 | 7.20, m | |
15/17 | 128.3 | 7.30, t (7.5) | 15/17 | 128.5 | 7.33, overlapped | 128.4 | 7.28, overlapped | |
16 | 126.9 | 7.25, overlapped | 16 | 126.9 | 7.25, m | 127.0 | 7.25, m | |
19 | 36.4 | 1.79, m | 19 | 29.7 | 2.24, m | 30.0 | 2.01, m | |
20 | 14.2 | 0.77, overlapped | 20 | 16.1 | 0.79, d (6.9) | 16.8 | 0.69, d (6.8) | |
21 | 23.7 | 1.03, m | 21 | 18.3 | 0.91, d (6.9) | 17.9 | 0.83, overlapped | |
22 | 169.6 | 22 | 167.9 | 167.8 | ||||
23 | 114.8 | 23 | 135.2 | 135.0 | ||||
24 | N.o.a | 24 | 114.8 | 7.28, overlapped | 114.7 | 7.28, overlapped | ||
25 | 128.3 | 25 | 157.2 | 157.2 | ||||
26 | N.o.a | 8.14, m | 26 | 118.5 | 6.94, dd (8.1, 2.4) | 118.5 | 6.95, dd (7.6, 1.8) | |
27 | N.o.a | 6.57, br s | 27 | 129.2 | 7.28, overlapped | 129.2 | 7.28, overlapped | |
28 | 123.6 | 7.69, br s | 28 | 118.5 | 7.35, overlapped | 118.5 | 7.34, br d (7.8) | |
29 | 15.7 | 1.23, d (6.6) | 29 | 16.1 | 1.21, d (6.5) | 15.6 | 1.23, d (6.3) | |
30 | 35.7 | 1.88, m | 30 | 36.7 | 1.81, m | 35.7 | 1.88, qd (7.6, 3.6) | |
31 | 14.1 | 0.86, d (7.8) | 31 | 13.8 | 0.87, d (6.8) | 14.0 | 0.86, d (6.9) | |
32 | 24.0 | 1.43, m | 32 | 24.0 | 1.45, m | 24.0 | 1.43, m | |
1.14, m | 1.06, m | 1.13, m | ||||||
33 | 21.3 | 1.34, s | 33 | 21.3 | 1.41, s | 21.1 | 1.34, s | |
34 | 21.2 | 1.21, s | 34 | 20.7 | 1.32, s | 21.3 | 1.20, s | |
35 | 11.1 | 0.77 overlapped | 35 | 10.6 | 0.87, t (6.8) | 10.4 | 0.83, overlapped | |
36 | 10.5 | 0.84, t (7.6) | 36 | |||||
6-NH | 6-NH | 8.72, d (9.2) | 8.67, d (8.3) | |||||
24-OH | 25-OH | 9.66, s | 9.68, s | |||||
25-NH | 9.70 br s | |||||||
CHO | 159.8 | 8.31, d (2.0) |
Compounds 2 and 3 were isolated as pale yellow amorphous solid. The same adduct ion and the indistinguishable peak signals in HPLC-MS chromatogram hardly made us considered that they were the same substance. The HR-ESI-MS (Table S1†) of 2 gave an adduct ion [M + H]+ at m/z 654.2924, indicating a molecular formula of C35H43NO11 (calcd for 654.2914 [M + H]+) with 15 degrees of unsaturation. After analyzing the 1H NMR, 13C NMR and HSQC spectra (Tables 1, S3 and Fig. S3†), as well as comparison with the reported spectra of unantimycin B,6 the planar structure of 2 was unambiguously assigned. The molecular formula of 3 was also elucidated as C35H43NO11 based on HR-ESI-MS (Table S1†). The 13C NMR (Tables 1, S4 and Fig. S4†) data of 3 were almost identical with those of 2, and the 1H–1H COSY and HMBC (Fig. 2) also suggested that 3 shared the same planar structure with 2, nevertheless, the 1H NMR chemical shift of the backbone between 2 and 3 showed delicate difference (e.g. δH-2 at 5.68 in 3, 5.40 in 2; δH-9 at 4.86 in 3, 5.21 in 2), hinting that 3 would be a stereoisomer of 2. Considering the structural similarities and isomeric diversities, compounds 2 and 3 were named unantimycin B1 and B2, respectively.
Compounds 4 and 5 were also a pair of epimers. The analyses of 1H NMR, 13C NMR, 1H–1H COSY, HSQC, HMBC, and ROESY spectra (Tables 2, S5, S6,†Fig. 2, S5 and S6†) in DMSO-d6 suggested that they are structurally related to 2 and 3 with the same characteristic scaffold except for a 2-hydroxyisovalerate unit tethered at C-9 (2-hydroxy-3-methyl valerate unit in both 2 and 3). 4 and 5 were named unantimycin D1 and D2, respectively.
No | 4 | 5 | ||
---|---|---|---|---|
δ C | δ H (J in Hz) | δ C | δ H (J in Hz) | |
a Values are interchangeable. | ||||
1 | 202.5 | 202.6 | ||
2 | 76.4 | 5.37, dd (10.0, 2.6) | 76.4 | 5.69, dd (7.8, 5.6) |
3 | 168.1 | 167.2 | ||
4 | 75.5 | 5.01, d (2.9) | 76.0 | 5.05, d (5.3) |
5 | 168.6 | 168.3 | ||
6 | 55.4 | 5.15, overlapped | 55.9 | 4.94, dd (8.3, 3.1) |
7 | 70.2 | 5.66, qd (6.4, 4.0) | 70.6 | 5.54, qd (6.4, 4.0) |
8 | 167.7 | 167.8 | ||
9 | 76.8 | 5.15, overlapped | 76.7 | 4.80, d (7.2) |
10 | 170.9 | 171.0 | ||
11 | 55.2 | 54.2 | ||
12 | 36.6 | 3.15, dd (14.9, 2.7) | 37.2 | 3.15, dd (14.1, 5.6) |
2.91, dd (14.9, 2.7) | 3.06, dd (14.0, 7.8) | |||
13 | 136.3 | 135.4 | ||
14/18 | 129.3 | 7.28, overlapped | 129.6 | 7.20, m |
15/17 | 128.6 | 7.34, overlapped | 128.4 | 7.28, overlapped |
16 | 127.0 | 7.25, m | 127.0 | 7.25, m |
19 | 29.7 | 2.25, m | 30.0 | 2.03, overlapped |
20 | 16.1 | 0.80, d (6.9) | 16.8 | 0.69, d (6.8) |
21 | 18.3a | 0.92, d (6.9) | 17.6a | 0.82, d (6.9) |
22 | 168.0 | 167.6 | ||
23 | 135.2 | 135.0 | ||
24 | 114.8 | 7.28, overlapped | 114.8 | 7.28, overlapped |
25 | 157.2 | 157.3 | ||
26 | 118.5 | 6.94, ddd (8.0, 2.5, 1.1) | 118.6 | 6.95, ddd (8.0, 2.6, 1.1) |
27 | 129.3 | 7.28, overlapped | 129.3 | 7.28, overlapped |
28 | 118.5 | 7.35, overlapped | 118.5 | 7.34, m |
29 | 16.2 | 1.21, d (6.4) | 15.7 | 1.23, d (6.5) |
30 | 30.7 | 1.99, m | 29.8 | 2.03, overlapped |
31 | 17.6a | 0.88, d (8.4) | 17.7a | 0.88, dd (6.8, 2.6) |
32 | 18.0a | 0.90, d (8.4) | 17.9a | 0.88, dd (6.8, 2.6) |
33 | 21.3 | 1.43, s | 21.3 | 1.34, s |
34 | 20.6 | 1.33, s | 21.2 | 1.21, s |
6-NH | 8.70, d (9.2) | 8.65, d (8.3) | ||
25-OH | 9.70, br s | 9.76, br s |
Strong curiosity leads us to further explore the stereochemical difference between compounds 2 and 3, 4 and 5. The configurational establishment of 1–5 required confirming the stereochemistry of each degraded components from 1–5. To prevent isomerization at C-2 the keto group at C-1 was reduced.17 In our previous published work, we found the ketoreductase, NatE, could specifically convert the ketone moiety of NATs to a hydroxyl group in vitro and in vivo,5 and we also successfully applied NatE to facilitate the stereochemical elucidation of two NATs with C-1 keto (NAT-J and K).10 However, this conversion didn't work for 2 and 4 when we intended to adopt the same method. More interestingly, ketone (2) and C1 hydroxyl type (8) of UAT-B were detected simultaneously in the fermentation extract of S. conglobatus RJ2 (a wide type NAT yielding strain but losing conglobatin production), which we previously supposed was attributed to the lower activity of NatE toward the substrates of NAT skeleton containing a 3-HBA moiety at our last work,6 it now seems the reason was not the lower conversion capacity but the stereoselectivity of NatE. Hence, NaBH4 was applied to reduce C1-keto of 1–5 respectively in the presence of MnC12 (Scheme 1), which was reported to give the highest selectivity for the stereoselective reduction of α-allyl β-keto esters into syn α-allyl β-hydroxy esters.18 It turned out as expected that only one main reduced product was afforded for 1–5 respectively during chemical reduction. After purification using the semi-HPLC method, five reduced products, 6–10 (Chart 1), from 1–5 correspondingly were obtained.
Compound 6 converted from 1 were confirmed to be the known NAT-G by comparison of OR and NMR (Tables 3, S7 and Fig. S7†) with those previously reported data.3 Compound 7 and 8, converted from 2 and 3 respectively, have different retention characteristics (for 7tR = 12.3 min; for 8tR = 9.0 min in 75% aqueous acetonitrile with 0.1% formic acid) on the chromatogram but share the same molecular formula as C35H45NO11 based on HR-ESI-MS (Table S1†). The spectroscopic values of 8 closely matched those reported in our previous work,6 which confirmed the stereoselectivity of NatE and explained the co-appearance of C1 hydroxyl and keto form of NAT in the broth of RJ2. After analyzing and comparing their 1H NMR, 13C NMR, HSQC and HMQC spectra (Tables 3, S8, S9, Fig. S8 and S9†) with those of 2 and 3, the structures of 7–8 were deduced to be C-1 hydroxyl form of 2–3 and allocated the names unantimycin C1 and C2 respectively. The structure of 9 and 10 converted from 4 and 5 respectively were deduced by HR-ESI-MS (Table S1†) and 1H-NMR data (ESI Fig. S10 and S11†), and named unantimycin E1 and E2 respectively.
No. | 6 | No. | 7 | 8 | |||
---|---|---|---|---|---|---|---|
δ C | δ H (J in Hz) | δ C | δ H (J in Hz) | δ C | δ H (J in Hz) | ||
a Values are interchangeable. | |||||||
1 | 77.8 | 3.29, d (10.5) | 1 | 75.8 | 3.48, overlapped | 77.9 | 3.29, d (10.6) |
2 | 71.6 | 5.45, dd (10.2, 4.6) | 2 | 76.7 | 5.42, t (7.0) | 71.8 | 5.38, dd (10.4, 4.4) |
3 | 167.6 | 3 | 168.4a | 168.1 | |||
4 | 75.7 | 5.34, d (3.7) | 4 | 76.9 | 5.17, d (4.6) | 75.4 | 5.29, d (3.4) |
5 | 167.6 | 5 | 168.5a | 167.8 | |||
6 | 55.3 | 5.14, dd (8.7, 3.3) | 6 | 55.9 | 5.15, dd (9.0, 3.1) | 55.5 | 5.05, dd (9.1, 3.4) |
7 | 70.7 | 5.56, m | 7 | 70.7 | 5.80, qd (6.4, 2.9) | 71.0 | 5.50, qd (6.4, 3.4) |
8 | 168.1 | 8 | 170.4 | 168.1 | |||
9 | 74.6 | 4.57, d (8.3) | 9 | 74.9 | 5.01, d (7.9) | 74.4 | 4.57, d (8.6) |
10 | 174.8 | 10 | 174.2 | 174.9 | |||
11 | 45.4 | 11 | 45.4 | 45.3 | |||
12 | 39.2 | 3.04, dd (14.2, 10.2) | 12 | 37.3 | 3.04, dd (13.6, 5.7) | 39.2 | 3.04, dd (14.0, 10.4) |
2.97, dd (14.2, 4.6) | 2.90, dd (13.7, 8.3) | 2.95, dd (14.0, 4.4) | |||||
13 | 137.5 | 13 | 137.4 | 137.6 | |||
14/18 | 129.0 | 7.22, overlapped | 14/18 | 129.3 | 7.29, overlapped | 129.1 | 7.23, overlapped |
15/17 | 128.3 | 7.28, m | 15/17 | 128.4 | 7.30, overlapped | 128.3 | 7.28, overlapped |
16 | 126.4 | 7.20, overlapped | 16 | 126.4 | 7.20, overlapped | 126.4 | 7.20, overlapped |
19 | 36.6 | 1.50, m | 19 | 31.5 | 1.99, overlapped | 30.3 | 1.70, m |
20 | 14.4 | 0.60, overlapped | 20 | 17.0 | 0.67, d (7.0) | 15.9 | 0.30, d (6.9) |
21 | 22.9 | 0.8, m | 21 | 18.2 | 0.67, d (7.0) | 18.4 | 0.67, d (6.9) |
22 | 170.0 | 22 | 167.6 | 167.8 | |||
23 | 114.7 | 23 | 135.1 | 135.2 | |||
24 | N.o.a | 24 | 114.7 | 7.29, overlapped | 114.8 | 7.28, overlapped | |
25 | 127.4 | 25 | 157.3 | 157.2 | |||
26 | N.o.a | 8.19, d (7.8) | 26 | 118.4a | 6.96, dd (8.1, 2.4) | 118.5 | 6.94, ddd (8.0, 2.5, 1.0) |
27 | N.o.a | 6.81, br s | 27 | 128.0 | 7.29, overlapped | 129.2 | 7.28, overlapped |
28 | 123.6 | 7.87, d (7.2) | 28 | 118.6a | 7.36, d (7.7) | 118.5 | 7.34, br d (7.7) |
29 | 15.6 | 1.21, d (6.4) | 29 | 15.9 | 1.28, d (6.4) | 15.4 | 1.20, d (6.4) |
30 | 35.4 | 1.85, m | 30 | 35.8 | 1.93, overlapped | 35.4 | 1.84, m |
31 | 14.0 | 0.87, d (7.1) | 31 | 14.7 | 0.92, d (6.9) | 13.9 | 0.86, d (6.9) |
32 | 24.3 | 1.50, m | 32 | 24.1 | 1.49, m | 24.2 | 1.49, m |
1.17, m | 1.19, m | 1.16, m | |||||
33 | 26.2 | 1.31, s | 33 | 23.7 | 1.10, s | 26.3 | 1.30, s |
34 | 22.0 | 1.25, s | 34 | 22.6 | 1.32, s | 21.9 | 1.26, s |
35 | 11.1 | 0.60 overlapped | 35 | 10.9 | 0.88, t (7.3) | 10.2 | 0.83, t (7.5) |
36 | 10.3 | 0.84, d (7.6) | 36 | ||||
1-OH | 4.39, d (10.4) | 1-OH | 4.85, br d (10.3) | 4.38, d (10.5) | |||
6-NH | 6-NH | 8.77, d (8.9) | 8.65, d (9.1) | ||||
24-OH | 12.7, s | 25-OH | 9.67 br, s | ||||
25-NH | 9.78 br s | ||||||
CHO | 159.8 | 8.32, d (1.7) |
The reduced derivatives 6–10 were used to facilitate the configurational assignment of 1–5 according to the reported methods.3,10,17 First, the relative configurations between C-1 and C-2, as well as C-9 and C-30 in each compound were determined from the HETLOC and ROESY assay. The small coupling constant constants for 3J1H–2H (∼0 Hz), 2J1C–2H (∼0 Hz) and 2J2C–1H (∼2 Hz) reasonably depicted the threo configuration between C-1 (R*) and C-2 (R*) (Fig. S12A†). On the other hand, the large 3J9H–30H coupling constant and small 3J9H–31C and 3J9H–32C coupling constants constructed two possible relative configurations of C-9 and C-30. The discrimination between these two possibilities was completed by further analyses of ROESY between the methyl signal of H-29 and that of H-31, which reasonably explained the relative configuration at C-9 and C-30 to be S* and S*, respectively (Fig. S12B†).
In the next, mosher's and Marfey's chromatographic analysis after hydrolysis of 6–10 were applied. Inferred from their planar structures, the microscale hydrolysis of 6–10 would inevitably yield the residues of isoleucic acid (Ila), 2-hydroxyisovaleric acid (Hia), 5-benzyl-4-hydroxy-3,3-dimethyldihydrofuran-2-one (Bhdo), and threonine (Thr). Hence, L-and D-Ila (prepared from L-and D-isoleucine respectively) and (2R)- and (2S)-Hia (purchased from Shanghai yuanye Bio-Technology Co., Ltd) were derivatized with the R enantiomer of Mosher's reagent respectively to yield authentic standards of the (S)-MTPA esters. (4R, 5R)-Bhdo obtained from hydrolysate of NAT-H in our previous work10 were derivatized with (R)- and (S)-MTPA-Cl to yield (S)- and (R)-MTPA esters, respectively, and (R)-MTPA ester of (4R, 5R)-Bhdo was regarded as the chromatographically equivalent surrogate for the (S)-MTPA esters of (4S, 5S)-Bhdo.3 The alkaline hydrolysates of 6–10 were respectively reacted with (R)-MTPA-Cl and subsequently dissolved in MeOH, as were the sample solution. The eleven sets of standards and samples mentioned above were subjected to C18 Mosher analysis, and the results clarified that the absolute configurations of all of the Ila moieties in 6–10 were the L-form (Fig. S13†), and the Hia residues were confirmed as S configuration (Fig. S13†). Meanwhile, Bhdo residues in 7 and 9 were determined to be 4S and 5S, 6, 8 and 10 incorporate the (4R, 5R)-Bhoa (Fig. S14†). In addition, the Thr residues in 6–10 were confirmed by advanced Marfey's analysis. L-threonine and L-allo-threonine were reacted with L- and D-FDLA (1-fluoro-2,4-dinitrophenyl-5-leucinamide), respectively. The retention times of the acid hydrolysates of 6–10 derivatized with L-FDLA were consisted with that of the L-FDLA L-threonine conjugates (m/z 412 [M–H]−), indicating the presence of L-threonine (Fig. S15†).
Therefore, the absolute configurations of 1–10 were eventually determined as shown in Chart 1: 1 as 2R, 4S, 6S, 7R, 9S, 19S, 30S; 2 and 4 as 2S, 4S, 6S, 7R, 9S, 30S; 3 and 5 as 2R, 4S, 6S, 7R, 9S, 30S; 6 as 1R, 2R, 4S, 6S, 7R, 9S, 19S, 30S; 7 and 9 as 1S, 2S, 4S, 6S, 7R, 9S, 30S; 8 and 10 as 1R, 2R, 4S, 6S, 7R, 9S, 30S.
Considering most of the NATs exhibited outstanding and broadspectrum tumor cell inhibitory activities, 1–8 were evaluated for their in vitro cytotoxicity. CCK8 bioassays were performed with the human gastric cancer cell line SGC7901, the human colon cancer cell line HCT-8, the human non-small cell lung cancer cell line PC-9, as well as their respective drug-resistant cancer cells SGC7901/DDP (resistant to cisplatin), HTC-8/T (resistant to taxol) and PC-9/GR (resistant to gefitinib). The cells were placed in 96-well plates and treated with various concentrations of the compounds for 72 h with cisplatin, taxol, and gefitinib as positive control respectively. The results (Table 4) revealed that the compounds featuring 3-formamidosalicylate unit, like 1 and 6, showed extraordinary anti-proliferative activities against all six cell lines with half-maximal inhibitory concentration (IC50) values in the range from 0.02 to 9690.0 nM. Though compounds 2–5, 7 and 8 exhibited much weaker cytotoxicity than 1 and 6, they still displayed potent and efficacious activity, especially 7 and 8 against the human non-small cell lung cancer cell lines (PC-9 and PC-9/GR) with IC50 in the range of 122.6–4228.0 nM. Besides, it may be inferred that the stereochemistry at C-1 and C-2 also plays an important role, which was reflected by the IC50 values between three pairs of diastereoisomers (2vs.3, 4vs.5, 7vs.8) against the PC-9 and PC-9/GR. Furthermore, the compounds with hydroxyl at C-1 (6–8) exhibited stronger bioactivity than their respective C-1 keto type (1–3), reconfirming the importance of the hydroxyl at C-1 position, which was demonstrated previously.10
Compd | IC50 (nM) | ||||||
---|---|---|---|---|---|---|---|
SGC7901 | SGC7901/DDP | HCT-8 | HCT-8/T | PC-9 | PC-9/GR | NCM460 | |
a Cisplatin, taxol, and gefitinib served as positive controls. | |||||||
1 | 1.5 ± 0.2 | 1.7 ± 0.2 | 30.3 ± 1.1 | 9690.0 ± 47.0 | 30.8 ± 1.0 | 0.5 ± 0.0 | >50000 |
2 | 16663.3 ± 483.3 | >20000 | >20000 | >20000 | 4091.0 ± 48.1 | 9126.3 ± 189.0 | >50000 |
3 | 17116.7 ± 432.9 | >20000 | >20000 | >20000 | 7866.3 ± 63.0 | 11560.0 ± 835.9 | >50000 |
4 | >20000 | >20000 | >20000 | >20000 | 5101.0 ± 212.3 | 9614.5 ± 623.8 | >50000 |
5 | >20000 | >20000 | >20000 | >20000 | 13821.3 ± 505.8 | 17960.1 ± 817.9 | >50000 |
6 | 0.02 ± 0.01 | 1.0 ± 0.1 | 11.8 ± 0.1 | 1036.7 ± 17.8 | 5.1 ± 0.1 | 779.3 ± 130.1 | >50000 |
7 | 9947.7 ± 353.2 | 18726.7 ± 790.3 | 18708.6 ± 376.6 | 13633.3 ± 357.2 | 122.6 ± 4.3 | 196.2 ± 2.0 | >50000 |
8 | 14983.3 ± 573.9 | 17103.3 ± 285.0 | 12266.7 ± 305.3 | 19256.7 ± 249.9 | 613.6 ± 7.3 | 4228.0 ± 82.3 | >50000 |
cisplatina | 927.0 ± 77.6 | 5028.7 ± 198.7 | — | — | — | — | 4799.3 ± 230.9 |
taxola | — | — | 4603.4 ± 421.6 | >20000 | — | — | — |
gefitiniba | — | — | — | — | 86.1 ± 3.0 | 8994.0 ± 105.7 | — |
The production culture broth (12 L) was adjusting pH to 6 with formic acid before extracted with an equal volume of EtOAc. The EtOAc layer was concentrated under vacuum to afford an extract (8.1 g), which was then dissolved in methanol and degreased with hexane three times. The entire MeOH-soluble extract (6.6 g) was subjected to vacuum liquid chromatography (VLC) on silica gel (200–300 mesh) using a stepwise gradient elution of dichloromethane–MeOH (from 50:1 to 0:1, v/v) to yield six fractions (B1–B6). Fraction B1 (2.5 g) was subsequently passed through an ODS chromatography column eluted with a gradient of aqueous acetonitrile (from 30% to 100%) to give fourteen fractions (B1A–B1N). Fraction B1J (1.2 g) was further fractionated over a silica flash column eluted with petroleum ether (PE)–EtOAc–MeOH (from 8:1:0 to 0:0:1, v/v/v) to give ten subfractions (B1J1-J7). Subfraction B1J5 was further purified by semi-preparative HPLC (Waters Xbridge C18, 10 × 250 mm, 3 mL min−1) under isocratic conditions using 70% aqueous acetonitrile with 0.1% formic acid as the buffer to yield neoantimycin L (1) (7.9 mg, tR = 23.6 min). Subfraction B1J6 (0.7 g) was subjected to a preparative HPLC (YMC-Park C18, 20 × 250 mm, 5 μm, 8 mL min−1) and a subsequent semi-preparative HPLC (Waters Xbridge C18, 10 × 250 mm, 3 mL min−1) to afford unantimycin B1 (2) (11.3 mg, tR = 25.6 min), B2 (3) (10.1 mg, tR = 26.7 min), unantimycin D1 (4) (3.2 mg, tR = 23.2 min), and unantimycin D2 (5) (3.6 mg, tR = 24.6 min) using 65% aqueous acetonitrile with 0.1% formic acid.
Footnotes |
† Electronic supplementary information (ESI) available: HR-ESI-MS and NMR spectra of compounds 1–10. Advanced Marfey's and C18 Mosher analysis. See DOI: 10.1039/d0ra09388b |
‡ These authors contributed equally to this work. |
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