Total Synthesis and Antibacterial Study of Cyclohexapeptides Desotamide B, Wollamide B and Their Analogs

Article abstract of DOI:10.1002/cbdv.201700414

As natural-product-derived antibiotics, desotamides A?–?D and wollamides exhibit growth inhibitory activity against Gram-posivite bacteria (IC₅₀ 0.6?–?7?μm) and are noncytotoxic to mammalian cells (IC₅₀?>?30?μm). Herein we firstly re

Full text of DOI:10.1002/cbdv.201700414

Article Type: Full Paper
Total synthesis and antibacterial study of cyclohexapeptides Desotamide B, Wollamide B and
their analogs
Yixing Chen1 Chao Liu1 Nan Liu2 Ye Wu3 Qingjie Zhao1 Honggang Hu1 Xiang Li1 Yan
Zou1
ꢀ Xiang Li
xiangli@smmu.edu.cn
ꢀ Yan Zou
zouyan@smmu.edu.cn
1 Department of Organic Chemistry, School of Pharmacy, Second Military Medical University,
Shanghai 200433, China.
2 China State Institute of Pharmaceutical Industry, Shanghai 200433, China.
3 School of Pharmacy, Chengdu Medical Collage, Chengdu 610083, China.
These authors contributed equally to this work.
Abstract As natural-product-derived antibiotics, Desotamides A-D and wollamides exhibit
growth inhibitory activity against Gram-posivite bacteria (IC50 0.6-7 μM) and are
noncytotoxic to mammalian cells (IC50 30 μM). Herein we firstly report the total synthesis
of above two cyclohexapeptides as well as a series of structural variants through solid phase
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201700414
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peptide synthesis (SPPS), of which DE-3 displayed a 2-fold increase of anti-bacterial activity
when compared with the original peptide DE-1. This strategy may offer good improvements
for the synthesis of other cyclic peptides.
Keywords Desotamides Wollamides Total synthesis SPPS Antibacterial
Introduction
In light of emerging drug-resistance among human pathogenic bacteria, bacterial
infection has been proved a critical problem calling for the development of new antibacterial
molecules.[1-3] It is well known that natural products serve as a valuable treasure in drug
discovery and are broadly used in much clinical therapeutics such as treatment of cancer,
inflammation, and hyper cholesterolemia.[4-7] Among them, natural-product-derived
antibiotics, especially the naturally occurring cyclopeptides, play a pivotal role and capture
high attention owing to their intriguing chemical structures and potent biological activities,
such as daptomycin[8], vancomycin[9], tyrocidine-A[10]
.
Figure1. Structure of natural cyclopeptides Desotamides A-F and Wollamides A-B.
The cyclohexapeptide desotamides A-D have been isolated from the deep-sea-derived
Streptomyces scopuliridis SCSIO ZJ46, among which desotamides A and B exhibit notable
antibacterial activities against pathogenic Gram-positive strains of Streptococcus pneumonia
NCTA 7466, Staphylococcus aureus ATCC 29213 and methicillin-resistant Staphylococcus
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epidermidis shhs-E1 (MRSE, a clinical isolate).[11] Another two new homologues
desotamides E-F and wollamides A-B have been isolated from soil-derived Streptomycesnov.
sp. (MST-115088) near Wollogorang Station, Queensland, Australia, which exhibit growth
inhibitory activity against Gram-posivite bacteria (IC50 0.6-7 μM) and are noncytotoxic to
mammalian cells (IC50 30 μM).[12] All these natural desotamides and wollamides contain
L-allo-Ile (Figure 1) other than desotamide B and wollamide B, which show significant
growth inhibitory activity against Staphylococcus aureus (ATCC9144 and ATCC25923 with
IC50 0.9 μM and 0.6 μM).[13]
Figure. 2 Structure of target cyclopeptides.
To fully explore the potential of desotamide B (DE-1) and wollamide B (WO-2) as
candidates of antibacterial drug development and to further investigate their structure-activity
relationship (SAR), the present work directly targeted at the total synthesis of DE-1 and
WO-2 as well as their corresponding analogues (Figure 2), and biological studies of the
antibacterial and cytotoxic properties.
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Results and Discussion
Chemistry
As shown in Scheme 1, we firstly anchored Fmoc-Asp-OAllyl to the Rink Amide AM
resin using HCTU/DIPEA as coupling reagent to obtain resin-bond amino acid 1. Then, the
linear peptide 2 was readily assembled via SPPS with different Fmoc protected amino acids.
Uncapping of Allyl group in the presence of Pd(PPh3)4 and phenylsilane, followed by the
treatment of 20% piperidine/DMF to release the N-terminal amino group, we can afford
linear peptide precursor 3. The subsequent on-resin cyclization step was achieved under the
condition of PyAOP/HOAt/NMM for 12 hours to obtain protected cyclopeptide (4) on resin.
The final cleavage and global deprotection using TFA/Phenol/Water/TIPS (88:5:5:2, v/v/v/v)
cocktail successfully produced target cyclopeptides. The structure of the synthesized
cyclopeptide was confirmed by HRMS and NMR.
Scheme 1. Synthesis route of the desotamide B (DE-1). Conditions and reagents: a)
HCTU, DIPEA, DMF, 1 h, rt; b) i) 20% piperidine/DMF, 5+10 min, rt; ii) Fmoc-AA-OH,
HCTU, DIPEA, DMF, 1 h, rt; c) i) Pd(PPh3)4, phenylsilane, DCM; ii) 20%
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piperidine/DMF, 5+10 min, rt; d) PyAOP, HOAt, NMM, 12 h, rt; e)
TFA/H2O/phenol/TIPs (88:5:5:2, v/v/v/v), 2 h, rt.
The cyclopeptide could be split into single amino acids units and readily assembled to
linear form through standard Fmoc/tBu SPPS strategy. However, the cyclization step is
critical and may depend on the peptide sequence, structure constraints and the resulting ring
size.[14] In previous studies, classical methods used to be cyclized in solution phase after
cleavage of the on-resin linear precursors. Tedious operations and repeated purification were
inevitable for this synthetic method, therefore, leading in some cases to a considerable loss of
product.[15] Thus, on-resin head-to-tail cyclization strategy was regarded as an attractive
alternative in which the final cyclization step was achieved while the peptide still remained
anchoring to the resin. We chose the Allyl as the capping group for the starting amino acid
Asp which was fully orthogonal to Fmoc/tBu SPPS and could be readily released by
employing Pd(PPh3)4 treatment in high chemical yield after the linear peptide was
assembled.[16] [17] The cyclization reagent, PyAOP/HOAt/NMM, was proved to be an
effective condition for the on-resin cyclization of the linear peptide unit with the remarkable
chemical yield of 76.9-88.5% (based on the loading amount of the first coupling amino acid).
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Pharmacological Activity Studies
The synthesized cyclopeptides were subjected to the in vitro cytotoxicity study against the
human tumor cell lines MCF-7 and HepG-2. The cytotoxicity assay was described by
measuring IC50 of the cell lines. The results of the cytotoxic assay were presented in Table 1.
Table 1. Cytotoxic Activity (IC50 μM) for the cyclopeptide
Compound No.
De-1
MCF-7
268.5
766.9
158.0
289.2
372.5
115.4
8.9
HepG-2
329.0
405.0
606.8
318.0
137.6
79.2
De-2
De-3
De-4
Wo-1
Wo-2
Nutlin-3*
5.2
*Nutlin-3 was measured as a positive control.
Next, antibacterial activity was evaluated against the Gram-positive bacteria,
methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus ATCC 25923,
and Gram-negative bacteria, Salmonella typhimurium CMCC 50097, Klebsiella peneumoniae
ATCC 13883, Escherichia coli ATCC 25922, using tetracycline and vancomycin as
antibacterial agent control. The results of the antibacterial studies are exhibited in Table 2.
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Table 2. Antibacterial Activity (MIC ug/mL) for the cyclopeptides
DE-1
MRSA2 >128
MRSA4 >128
DE-2
>128
>128
>128
>128
>128
>128
>128
DE-3
128
32
DE-4
>128
>128
>128
>128
>128
>128
>128
WO-1 WO-2
VAN
64
TET
---
>128
>128
>128
>128
>128
>128
>128
32
32
<8
---
MRSA5
S. aura)
S. typ
128
64
32
<8
---
>128
>128
>128
>128
64
64
<8
---
>128
>128
>128
128
128
64
---
<8
<8
<8
K. pen
E. coli
---
---
Abbreviations: S. aur: Staphylococcus aureus ATCC 25923; S. typ: Salmonella typhimurium
CMCC 50097; K. pen: Klebsiella peneumoniae ATCC 13883; E. coli: Escherichia coli
ATCC 25922. VAN: vancomycin; TET: tetracycline.
To our delight, DE-3 displayed an inhibitory potency for MRSA2, MRSA4, MRSA5, and
Staphylococcus aureus with an increase (at least 2 fold) compared to DE-1, suggesting that
the replacement of Val with Ile resulted in an improved bioactivity of DE-1. Furthermore,
the loss activity of DE-2 and DE-4 indicated that the DLeu could be indispensable for
antibacterial activity in DE-1. On the other hand, it is worth of noting that the total
deprivation of activity of WO-1 may suggest that the DOrn could be a determining factor
for the antibacterial activity of WO-2. In addition, almost all the target cyclopeptides lack
cytotoxicities (IC50>100 μM against two human tumor cell line MCF-7 and HepG-2. WO-1
showed lower cytotoxicity than WO-2, indicating that D-Orn is critical for antibacterial
activity but may increase cytotoxicity.
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In summary, a series of desotamide and wollamide analogues were designed and
successfully synthesized, with reasonable yield via the on-resin head-to-tail cyclization
strategy. This strategy may be potentially applicable to the synthesis of other cyclic peptides.
The biological evaluation suggested that DE-3 displayed a 2-fold increase of antibacterial
activity when compared with the original peptide DE-1. Of note, we have demonstrated that
the DOrn could be a determining factor for the antibacterial activity of WO-2. Further
synthetic and SAR studies are currently in progress in our laboratory.
Materials and methods
All reagents were purchased from J&K Chemical Ltd., Adamas and Innochem Chemical
Reagent. Amino acids were commercial available from GL Biochem Shanghai Co. Ltd. All
solvents used were bought from Sinopharm Chemical Reagent Co. Ltd. Dichloromethane
(DCM) and N, N-Dimethylformamide (DMF) were distilled over calcium hydride (CaH2)
under argon atmosphere and stored in flask containing 4 Å molecular sieves. All reaction
vessels were oven-dried before use. High resolution mass spectra were measured on a Waters
Xevo G2 QTOF mass spectrometer. 1H- and 13C-NMR spectra were recorded on a Bruker
Avance 600 MHz instrument. Chemical shifts (δ) were reported relative to TMS (0 ppm) for
1H-NMR and 13C-NMR spectra. Reactions were monitored by visualized by ninhydrin and/or
phosphomolybdic acid. The crude peptides were dissolved with CH3CN/H2O and analyzed or
purified by analytical or semi-preparative RP-HPLC, respectively. A Vydac C4 or C18
column (5 µm, 4.6 mm×250 mm) with a 1 mL/min flow rate was used for analytical
RP-HPLC, and a Vydac C4 column (10 µm, 10mm×250 mm or 22 mm×150 mm) with a 3-6
mL/min flow rate was used for semi-preparative RP-HPLC. The solvents systems were buffer
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A (0.1% TFA in CH3CN), buffer B (0.1% TFA in water). Data were recorded and analyzed
using the software system LC Solution.
General method for the cyclohexapeptides synthesis
Synthesis was performed in a peptide synthesizer under anhydrous conditions.[18] Rink
Amide AM resin 1 g was swelled with DCM (5 mL) for 20 min. The resin was treated with
20% piperidine/DMF twice (5 min and 10 min) to deprotect the Fmoc group, followed by
washing with DCM and DMF 5 times respectively. The first amino acid (Fmoc-Asp-OAllyl,
3 equiv, 1 mmol) was loaded on the Rink Amide resin by treatment with HCTU (0.9 mmol),
DIPEA (2 mmol) and DMF (6 mL). After 1 h, the resin was washed with DCM and DMF 5
times respectively. The deprotection, washing, coupling and washing steps were repeated
until all the amino acid residues were assembled. Then the Allyl group was deprotected in the
presence of Pd(PPh3)4, phenylsilane in DCM, followed by the deprotection of Fmoc. The
cyclization was carried out on resin by treatment with 5 equiv PyAOP, 5 equiv HOAt, 10
equiv NMM in pure NMP for 12 h. The cyclopeptide was cleaved from the resin by treating
with regent B (88% TFA, 5% H2O, 5% phenol, 2% TIPS) for 4 h at room temperature. After
completion of the cleavage reaction, TFA was evaporated by blowing with Ar. The crude
cyclopeptides were obtained by precipitation with 40 mL of cold diethyl ether and
centrifugation at 3500 r/min for 3 min (3 times). The supernatant diethyl ether was decanted
from the centrifuge tube and the crude peptides were allowed to air dry. The crude peptides
were purified by semi-preparative RP-HPLC.
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Cyclo-(Asn-Val-DLeu-Leu-Trp-Gly) (DE-1): 1H-NMR (600 MHz, d-DMSO) δ: 10.85 (1H,
s), 8.32-8.31 (1H, d, J=6 Hz), 8.28-8.27 (1H,d, J=6 Hz), 8.24-8.23 (1H, d, J=6 Hz), 7.92-7.90
(1H, t), 7.67-7.66 (2H, t), 7.61 (1H,s), 7.54-7.53 (1H,d, J=6 Hz), 7.35-7.34 (1H, d, J=6 Hz),
7.16(1H, s), 7.10-7.07(2H, s), 7.01-6.99 (1H, t), 4.55-4.51 (1H, dd, J=6 Hz), 4.39-4.37 (3H,
t), 4.04-4.02 (1H, t), 3.94-3.90 (1H, dd, J=6 Hz), 3.37-3.34 (1H, dd, J=6 Hz), 3.18-3.15 (1H,
dd, J=6 Hz), 3.02-2.98 (1H, dd, J=6 Hz), 2.83-2.80 (1H, dd, J=6 Hz), 2.69-2.65 (1H, dd, J=6
Hz), 2.25-2.19 (1H, m), 1.59-1.55 (2H, t), 1.50-1.45 (4H,m), 0.93-0.85 (18H, m). 13C-NMR
(600 MHz, d-DMSO) δ: 173.48, 172.49, 172.10, 171.42, 171.38, 171.25, 169.60, 136.59,
127.51, 124.01, 121.39, 118.84, 118.57, 111.83, 110.47, 59.27, 55.81, 52.10, 51.19, 49.88,
43.67, 41.86, 37.13, 24.89, 24.60, 23.08, 22.99, 22.85, 22.62, 19.58, 17.87. White lyophilized
powder; Yield 79.8%; Retention time17.749 min (Gradient: 20-80% of buffer B in 30 min);
HRMS: C34H50N8O7, Calc. for 683.3875, found (M+H)+: 683.3885.
Cyclo-(Asn-Val-Leu-Leu-Trp-Gly) (DE-2): 1H-NMR (600MHz, d-DMSO) δ: 10.83 (1H, s),
8.22-8.19 (2H, t), 7.94-7.89 (2H, dd, J=6 Hz), 7.59-7.56 (2H, dd, J=6 Hz), 7.41 (1H, s),
7.31-7.29 (1H, d, J=12 Hz), 7.16 (1H, s), 7.05-7.03 (1H, t), 6.97-6.95 (2H, t), 6.91 (1H, s),
4.47-4.43 (1H, dd, J=6 Hz), 4.24-4.18 (2H, m), 4.11-4.07 (1H, dd, J=6 Hz), 4.01-3.99 (1H, t)
,3.75-3.71 (1H, dd, J=6 Hz), 3.22-3.19 (1H, dd, J=6 Hz), 2.98-2.94 (1H, dd, J=6 Hz),
2.69-2.65 (1H, dd, J=6 Hz), 2.59-2.55 (1H, dd, J=6 Hz), 2.04-2.01 (1H, dd, J=6 Hz),
0.88-0.87 (4H, d, J=6 Hz), 0.86-0.81 (15H, m), 0.78-0.77 (5H, d, J=6 Hz). 13C-NMR
(600MHz, d-DMSO) δ: 172.33, 171.70, 171.44, 170.80, 170.64, 170.61, 168.47, 136.01,
134.29, 127.14, 123.57, 120.81, 119.25, 118.21, 118.14, 111.27, 109.84, 58.07, 54.30, 52.92,
51.64, 50.87, 42.81, 40.91, 35.74, 30.17, 26.77, 24.23, 23.16, 22.69, 21.50, 21.08, 18.98,
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17.92. White lyophilized powder; Yield 76.9%; Retention time18.127 min (Gradient: 20-80%
of buffer B in 30 min); HRMS: C34H50N8O7, Calc. for 683.3875, found (M+H)+: 683.3879.
Cyclo-(Asn-Ile- DLeu-Leu-Trp-Gly) (DE-3): 1H-NMR (600 MHz, d-DMSO) δ: 10.82 (1H, s),
8.29-8.28 (1H, d, J=6 Hz), 8.26-8.24 (1H, d, J=12 Hz), 8.17-8.16 (1H, d, J=6 Hz), 7.86-7.84
(1H, t), 7.65-7.64 (2H, d, J=6 Hz), 7.59 (1H, s), 7.49-7.48 (1H, d, J=6 Hz), 7.31-7.29 (1H, d,
J=6 Hz), 7.12 (1H, s), 7.06-7.03 (2H, t), 6.97-6.95 (1H, t), 4.48-4.45 (1H, dd, J=6
Hz),4.35-4.30 (3H, m), 4.02-4.00 (1H, t), 3.88-3.85 (1H, dd, J=6 Hz), 3.13-3.09 (1H, dd, J=6
Hz), 2.97-2.93 (1H, t), 2.79-2.75 (1H, dd, J=6 Hz), 2.64-2.61 (1H, dd, J=6 Hz), 1.45-1.39
(4H, m), 1.21(6H, s), 0.88-0.86 (6H, s), 0.83-0.79 (12H, m). 13C-NMR (600MHz, d-DMSO)
δ: 173.00, 171.98, 171.53, 170.95, 170.87, 170.68, 169.06, 136.06, 129.60, 126.98, 123.50,
120.8 6, 118.31, 118.04, 111.31, 109.93, 58.12, 55.32, 51.50, 50.67, 49.39, 43.14, 41.24,
36.48,35.28, 35.06, 29.03, 28.97, 28.76, 28.67, 28.63, 28.52, 27.21,26.55, 26.51, 25.05,
24.38, 24.08, 24.05, 22.56, 22.51, 22.30, 22.05, 15.52, 11.46. White lyophilized powder;
Yield 78.5%; Retention time17.026 min (Gradient: 20-80% of buffer B in 30 min); HRMS:
C35H52N8O7, Calc.for 697.4032, found (M+H)+: 697.4039.
Cyclo-(Asn-Ile- DIle-Leu-Trp-Gly) (DE-4): 1H-NMR (600 MHz, d-DMSO) δ: 10.80 (1H, s),
8.29-8.27 (3H, dd, J=6 Hz), 7.88-7.87 (1H, t), 7.66-7.62 (3H, m), 7.49-7.47 (1H, d, J=12 Hz),
7.30-7.29 (1H, d, J=6 Hz), 7.10 (2H, s), 7.05-7.02 (1H, t), 6.97-6.94 (1H, t), 4.51-4.46 (1H,
m), 4.40-4.39 (1H, m), 4.13-4.10 (1H, dd, J=6 Hz), 4.07-4.06 (1H, dd, J=6 Hz), 3.85-3.82
(1H, dd, J=6 Hz), 3.30-3.27 (1H, dd, J=6 Hz), 3.11-3.08 (1H, dd, J=6 Hz), 2.96-2.92 (1H, dd,
J=6 Hz), 2.84-2.80 (1H, dd, J=6 Hz), 2.64-2.61 (1H, dd, J=6 Hz), 1.96-1.92 (1H, m),
1.72-1.67 (1H, m), 1.56-1.54 (1H, t), 1.45-1.39 (3H, m), 1.34-1.28 (1H, m), 1.25-1.18 (1H,
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m), 1.16-1.11 (1H, m), 0.88-0.87 (3H, d, J=6 Hz), 0.83-0.78 (16H, m). 13C-NMR (600 MHz,
d-DMSO) δ: 172.72, 172.12, 171.36, 170.90, 170.87, 170.79, 168.77, 136.05, 126.99, 123.43,
120.84, 118.30, 118.05, 111.30, 110.03, 58.21, 57.34, 55.24, 50.67, 49.17, 43.21, 41.55,
36.62, 35.34, 34.70, 27.40, 24.66, 24.43, 23.87, 22.68, 22.36, 15.76, 14.98, 11.56, 10.14.
White lyophilized powder; Yield 81.3%; Retention time 18.277 min (Gradient: 20-80% of
buffer B in 30 min); HRMS: C35H52N8O7, Calc. for 697.4032, found (M+H)+: 697.4030.
Cyclo-(Asn-Val-DLeu-Leu-Trp-Orn) (WO-1): 1H-NMR (600 MHz, d-DMSO) δ: 10.81 (1H,
s), 8.38-8.33 (2H, dd, J=6Hz), 8.26-8.24 (1H, d, J=12Hz), 7.66-7.65 (1H, d, J=6Hz),
7.57-7.54 (3H, d), 7.51-7.47 (2H, dd, J=6Hz), 7.36-7.35 (1H, d, J=6Hz), 7.31-7.30 (1H, d,
J=6Hz), 7.11 (1H, s), 7.05-7.03 (1H, t), 6.99-6.95 (2H,m), 4.60-4.56 (1H, dd, J=6Hz),
4.43-4.39 (1H, dd, J=6Hz), 4.28-4.25 (2H, m), 4.17-4.13 (1H, dd, J=6Hz), 4.01-3.99 (1J, dd,
J=6Hz), 3.18-3.15 (1H, dd, J=6Hz), 2.95-2.91 (1H, dd, J=6Hz), 2.64-2.61 (3H, dd, J=6Hz),
2.26-2.23 (1H, m), 1.56-1.36 (10H, m), 0.90-0.87 (6H, t), 0.84-0.82 (13H, t). 13C-NMR (600
MHz, d-DMSO) δ: 173.96, 172.23, 172.07, 171.57, 171.14, 171.10, 136.57, 127.46, 124.07,
121.37, 118.82, 118.62, 111.80, 110.47, 59.01, 55.68, 52.37, 52.25, 50.86, 49.98, 42.42,
38.72, 37.51, 29.19, 27.60, 27.00, 24.88, 24.54, 23.62, 23.01, 22.97, 22.85, 22.56, 19.56,
17.43. White lyophilized powder; Yield 86.3%; Retention time18.216 min (Gradient: 20-80%
of buffer B in 30 min); HRMS: C37H57N9O7, Calc. for 740.4454, found (M+H)+:740.4456.
Cyclo-(Asn-Val-DLeu-Leu-Trp-DOrn) (WO-2): 1H-NMR (600 MHz, d-DMSO) δ: 10.81 (1H,
s), 8.38-8.33 (2H, dd, J=6 Hz), 8.26-8.24 (1H, d, J=12 Hz), 7.66-7.64 (1H, d, J=12 Hz), 7.54
(3H, s), 7.51-7.48 (2H, t), 7.36-7.35 (1H, d, J=6 Hz), 7.31-7.30 (1H, d, J=6 Hz), 7.11 (1H, s),
7.05-7.03 (1H, t), 7.00-6.95 (2H, t), 4.60-4.56 (1H, dd, J=6 Hz), 4.43-4.40 (1H, dd, J=6 Hz),
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4.30-4.25 (2H, m), 4.17-4.14 (1H, dd, J=6 Hz), 4.01-3.99 (1H, dd, J=6 Hz), 3.18-3.15 (1H,
dd, J=6 Hz), 2.96-2.92 (1H, dd, J=6 Hz), 2.65-2.58 (4H, m), 2.26-2.23 (1H, m), 1.45-1.37
(5H, m), 1.21 (3H, s), 0.99-0.88 (6H, t), 0.85-0.82 (14H, t). 13C-NMR (600 MHz, d-DMSO)
δ: 173.48, 171.75, 171.59, 171.08, 170.65, 136.09, 129.61, 126.98, 123.58, 120.89, 118.34,
118.14, 111.32, 109.99, 58.54, 55.21, 51.89, 51.77, 50.38, 49.50, 41.92, 38.26, 37.03, 24.41,
24.06, 22.53, 22.49, 22.37, 22.10, 19.09, 16.96, 13.90. White lyophilized powder; Yield
88.5%; Retention time 18.312 min (Gradient: 20-80% of buffer B in 30 min); HRMS:
C37H57N9O7, Calc. for 740.4454, found (M+H)+: 740.4465.
Biological Evaluation
Cytotoxic Screening
The cell proliferation inhibitory activities of target cyclopeptides against human cell lines
MCF-7 and HepG-2 were tested using the previously published methods.[13]
Antibacterial assay
The antibacterial activities of compounds DE-1-DE-4, WO-1 and WO-2 were assessed
using sequential 2-fold serial dilutions of each agent in MH broth according to the previously
reported and a standard method (Broth Microdilution Procedure) provided by clinical and
laboratory Standards Institute (CLSI).[14] Vancomycin and tetracycline were used as control.
Gram-positive bacteria (methicillin-resistant Staphylococcus aureus (MRSA Clinical
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isolated strain), Staphylococcus aureus ATCC 25923) and Gram-negative bacteria
(Salmonella typhimurium CMCC 50097, Klebsiella peneumoniae ATCC 13883, Escherichia
coli ATCC 25922) were provided by Huashan Hospital and China State Institute of
Pharmaceutical Industry. These strains were grown in Mueller Hinton (MH) broth media at
37 ºC overnight. The cultures were diluted to 1:20 in the same broth, cultured at 37 ºC until
absorbance 0.4-0.5 at 600 nm and diluted in MH broth to 1:200. 10 μL of the serially 2-fold
diluted peptides (80 μg to 128 μg) in MH broth were mixed with 90 μL of the above bacterial
suspension, then incubated 24 h at 37 ºC in an incubator. The MIC was defined as the lowest
concentration of the peptide at which there was no growth of bacterial.
Acknowledgments
We are grateful to National Natural Science Foundation of China (No. 21402235) and
foundation of our school (No. 2016JS11), Instumental Analysis center of our university for
NMR spectroscopic and mass spectrometric analyses and China State Institute of
Pharmaceutical Industry for biological evaluation.
Conflict of Interest
We declare that we have no financial and personal relationships with other people or
organizations that can inappropriately influence our work.
This article is protected by copyright. All rights reserved.

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