Synthesis and biological activity of salinomycin-hydroxamic acid conjugates

Article abstract of DOI:10.1016/j.bmcl.2017.01.080

Several salinomycin-hydroxamic acid conjugates were designed and synthesized. Most conjugates showed better antiproliferative activities than salinomycin in HT-29 colon cancer, HGC-27 gastric cancer, and especially in MDA-MB-231 triple-negative human breast cancer cells. These conjugates are stable in cell culture media, and they showed much better biological activities than the 1:1 physical mixture with hydroxamic acids and salinomycin. The better membrane permeability and hydrolysis rate of the conjugates may lead to the activity improvements.

Full text of DOI:10.1016/j.bmcl.2017.01.080

Accepted Manuscript
Synthesis and biological activity of salinomycin-hydroxamic acid conjugates
Bo Li, Wenxuan Zhang, Jun Wu, Qi Song Zhou Wu
PII:
S0960-894X(17)30092-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.080
BMCL 24656
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
18 October 2016
23 December 2016
26 January 2017
Please cite this article as: Li, B., Zhang, W., Wu, J., Zhou Wu, Q.S., Synthesis and biological activity of salinomycin-
hydroxamic acid conjugates, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/
j.bmcl.2017.01.080
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Synthesis and biological activity of salinomycin-hydroxamic acid conjugates
Bo Li,a# Wenxuan Zhang,a#* Jun Wu,b Qi Zhoub, Song Wua*
a. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical
College and Chinese Academy of Medical Sciences, Beijing 100050, China
b. State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
#1These two authors contributed equally to this work.
Corresponding Author*email: wxzhang@imm.ac.cn; ws@imm.ac.cn
Key words: salinomycin, hydroxamic acid, conjugates, anti-cancer
ABSTRACT: Several salinomycin-hydroxamic acid conjugates were designed and synthesized. Most conjugates showed better
antiproliferative activities than salinomycin in HT-29 colon cancer, HGC-27 gastric cancer, and especially in MDA-MB-231
triple-negative human breast cancer cells. These conjugates are stable in cell culture media, and they showed much better biological
activities than the 1:1 physical mixture with hydroxamic acids and salinomycin. The better membrane permeability and hydrolysis
rate of the conjugates may lead to the activity improvements.
Salinomycin (1, Fig 1) is a kind of polyether antibiotics
isolated from Streptomyces albus, which can transfer alkali
metal ions such as Na+ and K+ through cytomembrane.1 It has
been used in broiler batteries and other livestock as an
anticoccidial drug and growth promoters for many decades.2
Recent studies proved salinomycin also effectively kills cancer
stem cells (CSCs) and differentiated cancer cells that display
efficient mechanisms of resistance to cytotoxic drugs and
radiation, including leukemia, breast cancer, gastric cancer,
lung adenocarcinoma, osteosarcoma, colorectal cancer,
squamous cell carcinoma, prostate cancer in vitro and in vivo.3
Several possible mechanisms of salinomycin were illuminated,
such as induction of apoptosis and cell death, interference with
ABC transporters, inhibition of oxidative phosphorylation and
inhibition of the Wnt/β-catenin signaling pathway, but the exact
mechanisms were still not fully elucidated.4
hydroxyl acylated salinomycin analogs, which efficiently
decreased the CSC population at a 50 nM concentration.6,16
Their following study found the anti-cancer activity of
C20-deoxy-saliomycin is reduced, which emphasizes the
importance of substitution at C20 for the activity.8 Our group
synthesized several salinomycin diastereoisomers and their
acylated derivatives, and we found the stereochemistry has
important influences on the biological activities.17-18 These
results together suggest that the alkali metal ion transport
ability of salinomycin is crucial to its biological activities.
Compared to C20-modified derivatives, which showed
obviously higher pharmacological activities, the C1-ester,
amide and conjugates with other active substances such as
amino acid11, floxuridine12 and silybin13 almost showed much
lower antitumor activities. A possible explanation is that extra
steric hindrance on the head (C1) affects the combination and
transport of the sodium ions, because salinomycin need form
“head to tail” type of intramolecular hydrogen bonds as a
pseudo-cyclic structure.19 Nevertheless, some C1-modified
salinomycin derivatives which have a readily hydrolyzed
connecting bond showed better antitumor activities such as
compound 29 and 35 (Fig 2).
Figure 1. The structure of salinomycin
To study the structure–activity and structure–toxicity
relationship of salinomycin, medicinal chemists have
synthesized several C1 and C20-modified salinomycin
derivatives, some of which showed better antitumor
activities.5-15 Daniel Strand’s group synthesized several C20
Figure 2. The structure of salinomycin derivatives.

Different from ester and amide, carboxylic acid and
hydroxamic acid conjugates are relatively more easily to be
hydrolyzed. In additions, some hydroxamic acids also have
ion-binding ability, which provides central functionality in a
number of metalloproteinase inhibitors, such as histone
deacetylase inhibitors vorinostat and panobinostat.20 Coupling
the salinomycin with hydroxamic acid should be an interesting
attempt. We hope these conjugates can improve the membrane
permeability and antitumor activities.
triple-negative MDA-MB-231 breast cancer cells using MTT
assay (Table 1). These conjugates were proved to be stable for
72h in cell culture media. Most conjugates showed better
antiproliferative activities than salinomycin against these
cancer cell lines, especially in MDA-MB-231 cell. Acetyl and
2, 4-hexadiene acyl derivatives (4b and 6b) showed
comparative activities with salinomycin, while octanoyl
derivative (5b) is a little more potent (1.5-2.5 fold higher than
salinomycin). Compounds 7b-9b with 0-2 carbons between
carbonyl and phenyl group showed similar activities as
octanoyl derivative (5b) (1.5-7 fold higher than salinomycin),
indicating the length of the carbon chain has little influence on
the biological activities. Salicyloyl derivative (11b) showed
comparative activities with benzoyl derivative (7b), but
brominated products (12b and 13b) were more potent,
especially compound 12b showed 7-fold better activities in
HGC-27 gastric cancer and MDA-MB-231 triple negative
human breast cancer cells. However, the cinnamoyl derivative
with an unsaturated bond (10b) showed the reduced activities.
To study the effect of the substituent groups, ten
hydroxamic acids 4a-13a were purchased or prepared from N,
N’-carbonyldiimidazole (CDI) actived carboxylic acids and
21
unprotected hydroxylamine as Usachova’s report,
which
were directly coupled with salinomycin at carboxy group by the
dicyclohexylcarbodiimide (DCC), affording
salinomycin-hydroxamic acid conjugates 4b-13b (Scheme 1).
1
The H NMR (~11.0 ppm, an intramolecular hydrogen bond
with N-H and C=O(C1)), 13C NMR and HRMS-ESI confirmed
the structures of the conjugates and the X-ray of the
monocrystal 11b in CH3CN clearly demonstrated the
three-dimensional structure (CCDC 1490679, Fig 3).
Table 1. Antiproliferative activities in three cancer cell lines
(IC50, [M]a)
Scheme 1. The synthesis of the salinomycin-hydroxamic
acid conjugates
Compd
HT-29
HGC-27
MDA-MB-231
Sal
4b
5b
6b
7b
1.43±0.47
2.32±0.22
0.96±0.02
2.31±0.26
0.85±0.02
2.57±0.12
2.25±0.08
1.35±0.39
2.63±0.08
0.92±0.04
6.91±0.76
3.79±0.48
2.69±0.16
3.82±1.13
2.26±0.05
8b
9b
0.90±0.06
0.80±0.10
7.23±1.39
0.88±0.05
0.70±0.13
0.80±0.01
1.29±0.34
1.05±0.15
7.27±0.88
0.92±0.01
0.51±0.21
0.76±0.08
1.01±0.02
1.89±0.24
9.64±0.02
2.78±0.25
0.94±0.04
0.93±0.04
10b
11b
12b
13b
aIC50 values are the mean (±SE) for 50% reduction of MTT compared to
control. For all entries, n = 3.
Then some hydroxamic acids as well as the 1:1 mixture
with salinomycin were assayed in MDA-MB-231 cell lines. All
the tested hydroxamic acids did not show antiproliferative
biological activities at 10 M. The physical mixture performed
weaker biological activities than single salinomycin (Table 2).
These results indicated salinomycin and simple hydroxamic
acids together did not show a potent synergistic effect in tumor
cells. By contrast, our salinomycin-hydroxamic acid conjugates
showed better biological activities, especially in MDA-MB-231
cell lines, possibly because they have better membrane
permeability and could be readily cleaved to release
salinomycin. It seems that the efficacy of these compounds are
due to their membrane permeability and hydrolysis rate of
linkage within cells. Octanoyl derivative (5b) has a longer
carbon chain than Acetyl derivative (4b), and the former is
more potent. Among these derivatives, the phenyl substituted
compounds especially which have electron-withdrawing group
(12b and 13b) could be cleaved faster after they get
inside the cells, and they all showed better antiproliferative
activities. The derivatives including conjugated double bond
(6b and 10b) are relatively harder to be cleaved, so they showed
weaker activities.
Figure 3. The X-ray structure of salinomycin-salicylhydroxamic
acid conjugate (11b)
The
antiproliferative
activities
of
salinomycin-hydroxamic acid conjugates were evaluated in
HT-29 colorectal cancer, HGC-27 gastric cancer and
2

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Compd
MDA-MB-231
Compd
MDA-MB-231
8.98
6.53
7.89
9.85
9.72
1+10a
1+12a
1+13a
Taxol
Sal
>10.0
1+6a
1+7a
1+8a
(11)
(12)
(13)
(14)
>10.0
0.08
In summary, we have synthesized and evaluated the
biological activities of several salinomycin-hydroxamic acid
conjugates. Most conjugates showed better antiproliferative
activities than salinomycin in HT-29 colon cancer, HGC-27
gastric cancer, and especially in triple-negative MDA-MB-231
human breast cancer cells. These conjugates are stable in cell
culture media, and they showed much better biological
activities than the 1:1 physical mixture with hydroxamic acids
and salinomycin. The better membrane permeability and
hydrolysis rate of the conjugates lead to the activity
improvements. Further studies on these conjugates are
currently under way.
(15)
(16)
(17)
(18)
(19)
Supporting Information
The Supporting Information is available including the preparation
and the characterization of the compounds 4b-13b, as well as the
methods of the cell assay and the stability study.
ACKNOWLEDGMENT
We thank The National Natural Science Fund (81602958), Beijing
Natural Science Foundation（7164282）, the Fundamental Research
Funds for the Central Universities (2016ZX350006) and 973
program (2015CB964800) for financial support.
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3

Graphical and textual abstract
Salinomycin-hydroxamic acid conjugates showed better antiproliferative activities
than salinomycin in HT-29, HGC-27, and especially in MDA-MB-231 cancer cells.
4