Two new rakicidin derivatives from marine Micromonospora chalcea FIM-R160609

Abstract The establishment of structure activity relationship (SAR) for rakicidin derivatives is pretty vital to develop rakicidins as a new type of anti-cancer agents. Herein, two novel rakicidin derivatives, compounds B1-1 (1) and B1-2 (2), a cyclic depsipeptide and a chain lipopeptide, respectively, were isolated from culture broth of Micromonospora chalcea FIM-R160609, and their structures were elucidated clearly by extensive NMR and HR-ESI-MS analyses. Following, their cytotoxic activities were evaluated against HCT-8 and PANC-1 human cancer cell lines under hypoxic and normoxic conditions. Their activities were significantly decreased when compared with that of rakicidin B1. These results demonstrated that the double bond located on the position 9 and 10 of conjugated diene unit and cycle-type structure plays an important role in keeping the biological activity of rakicidins. Furthermore, the positive effect of double bond and cycle form on the anti-bacterial activities were also confirmed by testing their inhibitory activities against gram positive bacteria. This work will definitely diversify the SAR of rakicidins and provide the guidance for the design of new potent rakicidin analogues. Graphical Abstract


Introduction
Rakicidins, as a type of 15-membered depsipeptide composed of four unusual amino acids, were initially isolated from the extract of Micromonospora sp. in 1995 (McBrien et al. 1995).In recent years, rakicidin and its derivatives were reported to possess good biological activities in various areas, such as cytotoxicity, anti-bacterial activity and etc. (Yamazaki et al. 2007;Takeuchi et al. 2011;Lin et al. 2016;Lin et al. 2017).Thereafter, the development of new promising rakicidins analogues has attracted increasing attention of researchers, and great efforts have been devoted to establishing the structural activity relationships of rakicidins.
Among these works, Poulsen and co-workers generated a structure (WY1) encompassing only the APD unit and lipopeptide chain, and the cell-based evaluation revealed that this minimal APD-cyclolipodepsipeptide (APD-CLD) was sufficient to afford hypoxia selective growth inhibition of PANC-1 cells (Tsakos et al. 2016).In addition, Igarashi and co-workers hydrogenated the APD group of isolated rakicidin A to obtain tetrahydrorakicidin A, which was significantly (>100-fold) less active in PANC-1 cells as compared with that of rakicidin A (Tsakos et al. 2016), these results demonstrated that the fragment 4-amino-2,4-pentadienolate (APD) was highly relevant to its potent anti-CML activities (Oku et al. 2014;Sang et al. 2014).More specifically, Chen and co-workers synthesised a library of rakicidin precursors, which exhibited decreased activities against K562 and K562/G þ cells when compared with those compounds containing a conjugated diene moiety (Sang et al. 2016), and the SAR study revealed that the carbon-carbon double bonds on the position 11 and 12 has positive effect on the activities.Our group has been focusing on developing new rakicidin analogues as a new type of anti-cancer agents, and different series of new rakicidin analogues were reported, including rakicidins A, B (Jiang et al. 2006), B1 (Lin et al. 2016), E, and G-I (Chen et al. 2018).Collectively, these studies underscored the importance of the APD units and long lengthy alkyl chain on the biological activities.
Based on the above researches, a preliminary SAR of rakicidins has been established.However, to the best of our knowledge, the modification of some chemical active site on the position 9 and 10 has not yet been carried out and the effect on the activity has not been investigated.It is with high demand to exploit the virgin area for further diversifying the SAR of rakicidins.Herein, two new rakicidin derivatives 1 and 2 (Figure 1) were discovered, and their isolation, structural elucidation, and activities of two compounds are described.Compound 1 (Figure 1) was a cyclic depsipeptide in which the conjugated double bonds were replaced by epoxy groups at positions 9 and 10.Compound 2 (Figure 1) was a chain lipopeptide, a ring-opened compound, which may be formed by the cleavage of the amide bond at position 11 of rakicidin B1.Cytotoxicity results showed both compounds exhibited relatively (>10fold) weak activity against HCT-8 and PANC-1 human tumor cell lines as compared with that of rakicidins, including under hypoxic state (>20-fold).Meanwhile, we found that the partial antibacterial activities of the two compounds were also less active than those of rakicidin B1.These results indicated that the double bonds of diene unit and cyclic lipopeptide structure have great effect on the unique activities of rakicidins.The investigation of two compounds will be useful to enrich the SAR of rakicidins, which is beneficial to guide the reasonable molecular design of new rakicidin analogues.

Hypoxia-selective cytotoxicity
In this investigation, the cytotoxic activities in vitro against HCT-8 and PANC-1 human tumor cell lines were determined in order to evaluate the cytotoxicities of the isolated compounds 1 and 2. Rakicidin B1, well-known antitumor agent and hypoxia-selective cytotoxin (Lin et al. 2016), was used as the positive control in the meantime.Initial results indicated that compounds 1 and 2 have weak cytotoxicities against HCT-8 and PANC-1 human tumor cell lines with IC 50 values of more than 1 lg/mL, lower than that of rakicidin B1.And the IC 50 values further exhibited slightly more cytotoxicity under the hypoxic conditions compared to normoxic conditions (Figures S23-S26, Supplementary material).We could deduce from these preliminary researches that the loss of conjugated diene unit of compound 1 and ring-opening structure of compound 2 have a significant impact on their activities, and those two factors would dampen their activities.

Antibacterial activity
Compounds 1 and 2 were also evaluated for their anti-gram positive bacteria activities in vitro against Methicillin resistant S. aureus 15-1, Methicillin resistant S. aureus 15-2, Methicillin resistant S. aureus 15-3, Methicillin sensitive S. aureus 15-1, Methicillin sensitive S. aureus 15-2, Vancomycin intermediate S. aureus ATCC700699, Vancomycin-resistant E. faecalis ATCC51575, Vancomycin-resistant E. faecalis ATCC700802, Clostridium difficile 18-2, Clostridium difficile ATCC9689, and rakicidin B1 and vancomycin were used as the positive control.Compounds 1 and 2 showed weak activity against most tested strains in the different concentration ranges with MIC values of more than 16 or 32 lg/mL, and we also found the activities of compound 2 against Methicillin sensitive S. aureus 15-1 and 15-2 with MIC values of 8 lg/mL (Table S4, Supplementary material).

General experimental procedures
1 H NMR, 13 C NMR, DEPT, 1 H-1 H COSY, HSQC, HMBC and NOESY spectra were acquired with a Bruker AV-500 spectrometer using DMSO-d 6 as the solvent.ESI-MS and HR-FAB-MS were performed on Agilent 1100 SL series MSD Trap and Bruker Hct Esquire 3000 mass spectrometers, respectively.The UV spectrum was measured on a JASCO V-550 UV/vis spectrometer.The analytical (5 lm, 250 mm Â 4.6 mm, YMC) and semi-preparative (5 lm, 250 mm Â10 mm, Welch Material) RP-HPLC were conducted on a Shimadzu 20 A series.

Hypoxia-selective cytotoxicity
The compound 1 and 2 were dissolved in DMSO to prepare 1 lg/mL of initial concentration, and two-fold (normoxic condition) or three-fold (hypoxic condition) diluted progressively to six concentrations.HCT-8 and PANC-1 tumor cells were seeded into a pair of 96-well microplates at a density of 5.0 Â 10 4 /mL and 4.0 Â 10 4 /mL, with 100 lL medium per well (The 96-well microplates were put in 37 C incubator after 30 min of anaerobic ventilation, under hypoxic condition), and cultured for 24 h to assure complete adherence of the cells to the plates.The cultured cells were treated with different concentrations of compound 1 and 2 and then placed in an incubator containing 5% (V/V) CO 2 (30 min of anaerobic ventilation, under hypoxic condition).After 48 h of incubation, the cytotoxic effects were measured by MTT method under normoxic or hypoxic conditions.All experiments were carried out in triplicate, with rakicidin B1 as the positive control.

Antimicrobial test of Staphylococcus and Enterococcus
Compound 1 and 2 were dissolved in DMSO to prepare 16 lg/mL of initial concentration, and two-fold diluted progressively to 13 concentrations.Staphylococcus and Enterococcus were inoculated in culture medium consisting of broth (Staphylococcus: MH broth, Enterococcus: brain heart broth) and plated in 96 well microplates, 1 Â 10 5 CFU/mL/well in a volume of 100 lL.The MICs were measured with rakicidin B1 and vancomycin as the positive control.

Anti-anaerobic bacteria activity assay
The anaerobic bacteria were inoculated in culture medium consisting of brucella broth supplemented with hemin (5 mg/mL), vitamin K1 (1 mg/mL) and defibered sheep blood (5%, V/V) and plated in 96 well microplates, 1 Â 10 5 CFU/mL/well in a volume of 100 lL.Compound 1 and 2 were dissolved in DMSO and diluted step by step to concentrations of 0.008-64 lg/mL, and then added to the above wells at specified concentration in a volume of 100 lL, and incubated with the anaerobic bacteria at 35 C for 48 h.The MICs were measured under anaerobic conditions, with rakicidin B1 and vancomycin as the positive control.

Conclusions
In summary, two new rakicidin derivatives B1-1 (1) and B1-2 (2) were obtained from culture broth of Micromonospora chalcea FIM-R160609.The results showed the preliminary cytotoxic activities of the two compounds were lower than that of rakicidin B1 against HCT-8 and PANC-1 human tumor cell lines under hypoxic and normoxic conditions, and further indicated that the conjugated diene unit and cycle-type structure are essential for the unique activities of rakicidins.Meanwhile, compound 1 and 2 were weakly active against most of gram positive bacteria, except that compound 2 had certain activities against Methicillin sensitive S. aureus 15-1 and 15-2.The above results also confirmed that the structural fragment APDA and cycle-type of rakicidins were closely related to their cytotoxic activities and antibacterial activities.

Disclosure statement
No potential conflict of interest was reported by the authors.
showed HMBC correlations to a methine carbon C-10 (d C 79.9) and a quaternary carbon C-11 (d C 157.2), while a methine proton (d H 4.02) at C-9 coupled to a carbonyl carbon C-8 (d C 172.0), which revealed the partial structure (C-8 to C-12) containing epoxy groups.The 1 H-13 C long-range correlations from H-7 (d H 3.01) to C-8 (d C 172.0) and C-6 (d C 51.1), and from H-6 (d H 4.73, 3.69) to C-8 (d C 172.0) and a carbonyl carbon C-5 (d C 171.3) confirmed a connection between the C-8 to C-12 moiety and C-5 to C-7 moiety, but the double bond at 9, 10 position were replaced by epoxy groups.The HMBC correlations from methine proton (d H 4.36, H-3) to a carbonyl carbon C-4 (d C 173.3) and from methyl protons (d H 0.79, H-31) to a carbonyl carbon C-13 (d C 171.8) revealed that the carbonyl carbons were located at positions adjacent to C-3 and C-14, respectively.The 1 H-13 C long-range correlations from H-30 (d H 0.84) to C-28 (d C 34.2) and C-29 (d C 29.4), and H-33 (d H 0.83) to C-27 (d C 36.5), C-28 (d C 34.2) and C-29 (d C 29.4) (Figure S4, Supplementary material).These analyses