Synthesis and biological evaluation of crown ether acyl derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.09.066

A set of crown ethyl acyl derivatives based on 18-crown-6 moiety was synthesized and evaluated for biological activity. In vitro antiproliferative profiling demonstrated significant activities against HBL-100, HeLa, SW1573 and WiDr human cell lines. The m

Full text of DOI:10.1016/j.bmcl.2016.09.066

Accepted Manuscript
Synthesis and biological evaluation of crown ether acyl derivatives
Martín Febles, Sofía Montalvão, Guillermo Díaz Crespín, Manuel Norte, José
M. Padrón, Päivi Tammela, José J. Fernández, Antonio Hernández Daranas
PII:
S0960-894X(16)31015-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.09.066
BMCL 24290
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Bioorganic & Medicinal Chemistry Letters
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15 April 2016
26 September 2016
27 September 2016
Please cite this article as: Febles, M., Montalvão, S., Crespín, G.D., Norte, M., Padrón, J.M., Tammela, P.,
Fernández, J.J., Daranas, A.H., Synthesis and biological evaluation of crown ether acyl derivatives, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.09.066
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Graphical Abstract
Synthesis and biological evaluation of crown
ether acyl derivatives
Leave this area blank for abstract info.
Martín Febles, Sofía Montalvão, Guillermo Díaz Crespín, Manuel Norte, José M. Padrón, Päivi Tammela, José
J. Fernández, Antonio Hernández Daranas

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis and biological evaluation of crown ether acyl derivatives
Martín Febles a, †, Sofía Montalvão b, †, Guillermo Díaz Crespín a, Manuel Norte a, José M. Padrón a, Päivi
Tammela b,*, José J. Fernández a,*, Antonio Hernández Daranas a,*∗
a Instituto Universitario de Bio-Organica Antonio Gonzalez (IUBO AG), Universidad de La Laguna, Avenida Francisco Sánchez 2, 38205 La Laguna, Spain
b Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, P.O. Box 56, FI-00014 University of Helsinki, Finland
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A set of crown ethyl acyl derivatives based on 18-crown-6 moiety was synthesized and
evaluated for biological activity. In vitro antiproliferative profiling demonstrated significant
activities against HBL-100, HeLa, SW1573 and WiDr human cell lines. The most active
compound exhibited GI50 values in the range of 3.7-5.6 µM. Antimicrobial evaluation showed
that three polyaromatic compounds were active against Staphylococcus aureus (MIC90 values
from 8.3 µM to 50 µM), whereas a (decyloxy)benzene substitution exhibited moderate activity
against Candida albicans (MIC90 values 36 µM). According to SAR evaluation, the size of the
crown ether and the acyl side chain had a significant effect on the bioactivity. Aromatic moieties
close to the acyl group led to improved bioactivity as exemplified by some of the tested
compounds. These results provide further evidence on the potential of crown ethyl structure as a
scaffold for developing new biological probes and lead candidates for drug development.
Keywords:
Crown ether
Antiproliferative
Antimicrobial
Structure-activity
18-Crown-6
2009 Elsevier Ltd. All rights reserved.
———
∗Corresponding authors. Tel.: +34 922 318587; e-mail: adaranas@ull.es, jjfercas@ull.es, paivi.tammela@helsinki.fi
† These authors contributed equally to the work

Crown ethers, first described by Pedersen et al.1 are
macrocyclic polyethers well known for their noncovalent ion
binding properties. Their common names include the number and
type of atoms in the polyether ring. The ring size of these
molecules controls their binding selectivity for a range of metal
ions, thus 18-crown-6 has high affinity for potassium while 15-
crown-5 selects sodium cations. To explain this, a high complex
stability has been associated with greater penetration of the metal
cation into the polyether cavity.
do not comply with the druglike, “rule-of-five” properties,6 the
potential of macrocycles for drug discovery has attracted
considerable attention in the recent years.7
Crown ether macrocycles are known in all ring sizes from 9 to
at least 60, leading to great variety in structures (exceeding 10000
examples).2 Therefore, crown ethers have been extensively
studied from different aspects, including sensor applications,
biological model systems (for example, abiotic ion channels)3
and biological functionality, especially for anticancer and
antimicrobial effects.2,4,5 Crown ether derivatives function
similarly to natural ionophores (such as gramicidin) and have
thus been used to study several biological processes, in particular
due to their channel forming and ion transport capabilities
through lipid membranes.5 Despite, large macrocylic compounds
Figure 1. Structure of the crown ether acyl derivatives.
Table 1. Antiproliferative activity (GI50) against human solid
tumour and non-tumour cellsa
BJ-hTert
cLogPb
Compound
HBL-100 (breast)
HeLa (cervix)
SW1573 (lung)
WiDr (colon)
(fibroblast)
1a
1b
1c
>100
>100
>100
>100
-0.96
0.86
3.27
>100
>100
>100
>100
72.0 ( 13.0)
>100
71.0 ( 9.3)
>100
51.0 ( 6.0)
>100
33.0 ( 0.8)
>100
2c
3c
1d
3.46
3.66
2.04
>100
>100
>100
>100
>100
>100
69.0 ( 23.0)
52.0 ( 3.4)
1e
2.56
64.0 ( 24.0)
>100
79.0 ( 25.0)
>100
78.0 ( 5.4)
>100
>100
1f
1g
>100
1.46
4.63
24 ( 4.0)
20.0 ( 6.2)
>100
41.0 ( 28.0)
>100
24.0 ( 2.5)
>100
66.0 ( 12.0)
>100
1h
1i
1j
1.50
-0.59
1.20
>100
>100
>100
>100
57.0 ( 2.1)
48.0 ( 7.6)
48.0 ( 5.9)
>100
1k
1l
>100
1.12
3.51
2.40
2.34
3.94
3.10
2.51
50.0 ( 2.6)
6.4 ( 0.4)
40.0 ( 3.4)
29.0 ( 2.6)
21.0 ( 1.0)
5.0 ( 0.9)
56.0 ( 4.5)
7.8 ( 6.5)
63.0 ( 7.3)
35.0 ( 2.2)
17.0 ( 0.4)
3.7 ( 0.7)
54.0 ( 3.2)
4.4 ( 0.7)
54.0 ( 3.2)
36.0 ( 2.9)
18.0 ( 4.4)
3.8 ( 0.1)
56.0 ( 4.5)
6.2 ( 3.8)
56.0 ( 4.5)
24.0 ( 6.5)
19.0 ( 1.4)
5.6 ( 1.3)
8.2 ( 0.1)
>100
1m
1n
1o
1p
1q
22 ( 9.6)
19 ( 1.5)
5.4 ( 2.4)
18 ( 3.8)
11.0 ( 0.6)
1.9 ( 0.2)
1.4 ( 0.1)
12.0 ( 1.3)
2.0 ( 0.3)
3.3 ( 1.6)
8.8 ( 1.0)
3.0 ( 0.4)
15.0 ( 1.5)
12.0 ( 1.4)
26.0 ( 5.3)
23.0 ( 3.1)
CDDP
VP-16
a Values are given in μM and are means of two to three experiments; standard deviation is given in parentheses.
b cLogP values were calculated using the Schrodinger small-molecule drug discover suite conditions.

Herein we report the synthesis of a series of crown ether acyl
derivatives and the evaluation of their antimicrobial and
antiproliferative properties. The antimicrobial activity was tested
against Gram (+) and Gram (-) bacteria as well as against the
model yeast Candida albicans. Previous reports describe
dialkyldiaza-18-crown-6 ethers as inhibitors of bacterial
growth.8,9 In addition, the antiproliferative profile was evaluated
in vitro against a panel of human solid tumour cell lines. Thus we
first synthetized compounds 1c, 2c and 3c where the same
cyclohexyl side-chain was attached to 18-6, 15-5 and 12-4 crown
ether moieties. Based on their antiproliferative activity (Table 1)
we selected 18-crown-6 derivatives for further development,
changing the length of the side chain and the size of the attached
cyclic moiety.
Table 2. Antimicrobial primary screening of compounds. Ciprofloxacin and amphotericin B were used as positive controls. Data is
presented as average inhibition% ( standard deviation, n = 3) at 50 µM concentration. Inhibition results >90% are in bold
Compound
C. albicans ATCC 90028
E. faecalis ATCC 29212
S. aureus ATCC 25923
E. coli ATCC 25922
1a
1b
1c
2c
3c
1d
1e
1f
nt
nt
nt
nt
9.4 ( 5.5)
4.7 ( 5.9)
6.9 ( 0.6)
4.3 ( 3.6)
0
0
0
0
0
0
0
61.0 ( 8.6)
0
0
0
0
0
3.1 ( 2.7)
4.9 ( 2.2)
5.0 ( 2.8)
5.8 ( 4.2)
8.1 ( 3.6)
0
5.5 ( 17.0)
11.0 ( 23.0)
1.6 ( 16.0)
101.0 ( 0.1)
nt
0.3 ( 1.6)
0
3.5 ( 4.6)
1g
1h
1i
2.1 ( 4.2)
29.0 ( 20.0)
nt
nt
nt
24.0 ( 9.6)
36.7 ( 13.0)
0
6.8 ( 4.8)
0
6.2 ( 1.7)
5.9 ( 1.0)
7.3 ( 7.4)
6.5 ( 4.3)
0
1j
3.3 ( 2.7)
3.4 ( 18.0)
0
0
0
1k
1l
1.0 ( 26.0)
98.0 ( 0.1)
1m
1n
1o
1p
1q
0
0
0
0
0
0
3.6 ( 2.5)
0
0
0
101.0 ( 0.1)
4.3 ( 18.0)
99.0 ( 0.3)
98.0 ( 0.7)
7.8 ( 5.4)
0.1 ( 1.1)
0
0
0
The general synthetic approach used included two
alternatives: first the “Steglich esterification” using the acid of
the selected side chain and a crown ether alcohol, utilizing
dicyclohexylcarbodiimide as coupling agent and 4-
dimethylaminopyridine as catalytic base.10 The second approach
consisted in the usage of the corresponding acyl chloride as
described in the supporting information. Using this simple
strategy we obtained the corresponding derivatives 1a-q with
yields ranging 60-95% (Fig. 1).
antimicrobial testing by using the broth microdilution method
according EUCAST and CLSI guidelines.12,13 All samples were
initially tested at 50 µM. As can be seen from the primary
screening results in Table 2, compounds 1g and 1o were highly
active against C. albicans ATCC 90028 and compounds 1l, 1o
and 1p displayed significant activity against Staphylococcus
aureus ATCC 25923, fully inhibiting the growth of this Gram-
positive bacterium. Confirmatory dose-response experiments
were carried out for these compounds.
The in vitro antiproliferative activity of the crown ether acyl
derivatives was determined in HBL-100, HeLa, SW1573 and
WiDr human solid tumor cells. Table 1 shows the results
(expressed as GI50) using the SRB assay.11 The standard
anticancer drugs cisplatin (CDDP) and etoposide (VP-16) were
used as positive controls. The most active compound of the series
was 1p and exhibited GI50 values against all cells in the range
3.7-5.6 µM. When compared to CDDP and VP-16, compound 1p
showed an improved biological activity in the resistant cell line
WiDr. In addition, the non-tumor cell line BJ-hTert (telomerase-
immortalized human foreskin fibroblasts) was used to study the
effect on a subset of the most potent crown ether acyl derivatives
of the series. The results show that compounds 1k and 1m are
inactive (GI50 > 100 µM), thus indicating some selectivity,
whilst the other derivatives showed no discrimination between
tumor and non-tumor cell lines. Further experiments will be
necessary to explain this outcome.
Compounds 1l, 1o and 1p showed MIC90 values of 25 µM, 8.3
µM and 50 µM, respectively, against S. aureus (Table 3). Against
C. albicans 1g and 1o exhibited MIC90 values of 36 and 42 µM,
respectively. All the compounds were inactive against the Gram-
negative E. coli, possibly due to its outer membrane structure.
Antimicrobial properties of crown ether derivatives have
previously been studied by Leevy et al.4 who demonstrated that
activity against Escherichia coli is altered by the spacer length.
Furthermore, Gram-positive Bacillus subtilis has been
demonstrated to be in general more susceptible to different crown
ether derivatives than E. coli.4,14 Interestingly, the activity of
compound 1p, the most active compound in the antiproliferative
experiments, was about 4-fold lower against S. aureus based on
comparison between the GI50 and MIC50 values (Table 1, Table
3). On the other hand, compound 1o with MIC50 of 7.2 µM
against S. aureus was only moderately active against the cancer
cells (GI50 values in the range of 17-21 µM, Table 1).
Furthermore, the antimicrobial activity of the compounds was
evaluated against a set of strains typically used in clinical

Table 3. Minimum inhibitory concentrations (MIC90, MIC50) for
the most active compounds against S. aureus ATCC 25923 and
C. albicans ATCC 90028. Ciprofloxacin (MIC90 against S.
aureus 1.4 mg/mL) and amphotericin B (MIC90 against C.
albicans 0.3 mg/mL) were used as positive controls
anticancer and antimicrobial agents based on crown ether
moieties.
Acknowledgments
Co-financed by the EU Research Potential (FP7-REGPOT-
2012-CT2012-31637-IMBRAIN) European Regional Develop-
ment Fund (FEDER), Spanish MINECO (SAF2011-28883-C03-
01, CTQ2014-55888-C03-01). The research leading to these
results has also received funding from the European Union
Seventh Framework Program (FP7/2007-2013) under grant
agreement FP7-KBBE-2009-3-245137 as well as from the
Academy of Finland (Grant 277001).
C. albicans
S. aureus
MIC90
a
Compound
MIC90
MIC50
MIC50
1g
1l
16.7
n.d.
21.7
n.d.
n.d.b
n.d.
20.1
n.d.
n.d.
11.3
4.3
n.d.
6.8
1o
1p
3.7
27.8
11.1
a Values are given in mg/mL.
b Not determined.
References and notes
The structure-activity relationship (SAR) study clearly
demonstrated that the size of the crown ether and the acyl side
chain influence the bioactivity. Initially, it was established that
the preferred crown ether was [18]-crown-6. The selection was
based on the GI50 values obtained for derivatives 1c, 2c and 3c in
the antiproliferative profiling (Table 1). Then, further efforts
were devoted to study the influence of the acyl side chain on the
antiproliferative activity. A clear trend could not be inferred from
the biological data. For instance, a larger aliphatic linker
improves the antiproliferative activity of 1g when compared to
1f. The side-chain length affects significantly the hydrophobicity
of the compound, and thus calculated logP values for 1f and 1g
are markedly different (1.46 and 4.63, respectively, Table 1).
According to Supek et al.,14 logP of the molecule is the most
important molecular descriptor in determining the biological
activity of 18-crown-6 ethers, and not generally affected by
features such as the side chain length or molecular symmetry. For
our compounds, the calculated logP values vary from -0.96 to
4.63 (Table 1). The most active compounds have logP values >3,
but some of the moderately active or inactive compounds have
similar values, and thus the logP does not seem to be clearly
correlated with biological activity according to our data. Based
on our results, improved activity was obtained when the aromatic
structure was close to the acyl group, as exemplified with
compounds 1l, 1p and 1q. Further experiments are necessary to
discern if the observed differences correlate to diverse
mechanisms of action.
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In conclusion, in this study we described an efficient strategy
for synthesizing a series of crown ether acyl derivatives. Of the
crown ether moieties 18-6, 15-5 and 12-4, 18-crown-6 ether core
structure showed initially the best potential based on
antiproliferation assays on human cancer cell lines and was thus
chosen for synthesising a series of acyl derivatives. Compounds
were evaluated for antiproliferative activity against HBL-100,
HeLa, SW1573 and WiDr human solid tumor cell lines, and the
most active compound 1p displayed GI50 values in the range of
3.7-5.6 µM. Antimicrobial evaluation yielded compounds active
against S. aureus and C. albicans; most active was compound 1o
against S. aureus with MIC90 value of 8.3 µM. These results will
be helpful in understanding the biological effects of crown ether
derivatives, and may thus aid the development of novel
Supplementary Material
Supplementary material associated with this article including
compound preparation and characterization as well as
experimental details on biological assays, can be found in the
online version.