Synthesis and antimicrobial properties of Monensin A esters

Article abstract of DOI:10.1016/j.bmcl.2008.03.038

The esters (2-10) of the ionophore antibiotic Monensin (1) were synthesized by four different methods, which are discussed in detail. These new esters were characterized by various spectroscopic techniques and subsequently tested in the face of their anti

Full text of DOI:10.1016/j.bmcl.2008.03.038

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 2585–2589
Synthesis and antimicrobial properties of Monensin A esters
a
b
a
´
´
Adam Huczynski, Joanna Stefanska, Piotr Przybylski,
Bogumil Brzezinskia,* and Franz Bartlc
aFaculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland
bMedical University, Department of Pharmaceutical Microbiology, Oczki 3, 02-007 Warsaw, Poland
cInstitute of Medical Physics and Biophysics Charite´, Universita¨tsmedizin Berlin Campus Charite´ Mitte,
Ziegelstr. 5/9, 10117 Berlin, Germany
Received 6 February 2008; revised 12 March 2008; accepted 14 March 2008
Available online 16 March 2008
Abstract—The esters (2–10) of the ionophore antibiotic Monensin (1) were synthesized by four different methods, which are
discussed in detail. These new esters were characterized by various spectroscopic techniques and subsequently tested in the face
of their antimicrobial properties. Three derivatives (3, 8 and 10) showed activity against Gram-positive bacteria. Additionally
derivative (10) exhibited a relatively low antifungal activity against Candida in contrast to Monensin A.
Ó 2008 Elsevier Ltd. All rights reserved.
Monensin A (1, Fig. 1) isolated from Streptomyces
cinnamonensis is a well-known polyether antibiotic,
capable to transport monovalent and divalent metal cat-
ions across lipid membranes. Therefore, it belongs to a
group of highly bioactive molecules.1,2 Monensin exhib-
its antibiotic,3 coccidiostatic,4 cardiovascular5 and other
important biological and medical properties.6 Only
recently it was shown that Monensin A is also a highly
effective ionophore for Li+, Rb+ as well as for Pb2+
cations.7 These properties are the basis of many biolog-
ical and pharmaceutical fields of application of this
compound and new ones can be expected for the future.
related with a strong antifungal and/or antibacterial
activity.10,11 Furthermore, our previous results demon-
strated that the esterification of Monensin A influences
its physicochemical properties and consequently these
Monensin derivates have the potential to change the
mode of action.12 These interesting results have moti-
vated our group to optimize the synthesis methods and
to study the biological activities of these new com-
pounds. In the present contribution, we compare four
methods of Monensin A ester synthesis. Furthermore,
we compare the antimicrobial activity of the new ester
with the activity of unmodified Monensin A.
Up to now various derivatives of Monensin were synthe-
sized in order to reduce its toxicity and to extend its
fields of application.8 In previous publications we
reported on the synthesis and the physicochemical prop-
erties of several new Monensin A esters.9 The complex-
ation of mono- and divalent metal cations by Monensin
A esters and the properties of these complexes have been
described in detail. These esters show especially high
affinity towards Na+ and Ca2+ cations.9
The synthesis of carboxylic esters is one of the most fun-
damental methods in organic chemistry to obtain useful
natural and synthetic compounds. However, most ester-
ification procedures require rather harsh conditions such
as the presence of strong acids, bases or other catalysts.
Furthermore, the reactions of this type often proceed
only at high temperatures. However, Monensin A is very
sensitive to acidic conditions and heating. For this rea-
son we attempted to choose the reaction conditions for
the esterification as mild as possible. The new esters of
Monensin A (2–10) were prepared according to four dif-
ferent methods. All esters used for our experiments
including the acyloxy esters are stable under the experi-
mental conditions. This was checked by several spectro-
scopic and spectrometric methods such as FT-IR, NMR
and ESI-MS.
From the literature data it is known that the complexa-
tion ability towards Na+ and Ca2+ cations is often
Keywords: Ionophores; Monensin; Esters; Synthesis; Antimicrobial
activity.
*
Corresponding author. E-mail: bbrzez@amu.edu.pl
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.038

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A. Huczynski et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2585–2589
2586
30-31
monensin A (1): R = H
(2): R = CH2CH3
29
32
(4): R =
13
H2C
H2C
C
CH2
H2C
(3): R =
(5): R =
H
17
O
O
9
O
28
(6): R = H2C
O
21
34
O
5
OH
25
HO
IV
36
33
X
27
Me
26
O
O
OMe
(8): R =
(7): R =
H2C
O
N
O
HO
H2C
O
35
Me
XI
1
O
OR
O
NO2
(9): R = H2C
(10): R = H2C
Figure 1. Chemical structure of Monensin A and its esters.
These synthesis pathways summarized in Scheme 1 are
the only efficient ones, albeit with different yields.
the presence of DCC, PPy and p-TSA (p-toluenesulfonic
acid monohydrate). This method was quite efficient and
esters (3), (7) and (8) could be obtained in high yields
(up to 70%). However, under the same reaction condi-
tions, the Monensin benzyl ester (4) was only obtained
with a yield of 35%.14
In the first method DCC (N,N0-dicyclohexylcarbodiim-
ide) was used as a coupling agent (Scheme 1, method
a).13,14 This procedure results in low to moderate chem-
ical yields, for example, the yield of Monensin ethyl ester
(2) was only 20%.
The fourth reliable strategy of Monensin (1) esterifica-
tion is based on the direct alkylation of carboxylate ions.
This method uses the corresponding alkyl bromides with
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as an effective
nucleophilic catalysts. Under these reaction conditions
the yield was above 75% (4, 6). Note that the use of alkyl
bromides instead of alkyl chlorides significantly
increases the yields of the respective esters. Interestingly,
this esterification method shows also a remarkable
solvent dependence. Toluene was the most appropriate
of all solvents tested, probably because of an optimal
solubility of reactants and products in this solvent
(Scheme 1, method d).14
When PPy (4-pyrrolidinopyridine), a very effective acyl-
ation catalyst, was additionally used in catalytic
amounts, the yield of compound (2) could be drastically
increased to 71%. However, for the synthesis of the
other Monensin esters such as (3), (7) and (8), this ester-
ification method was unsatisfactory because only low
yields of these compounds could be achieved (Scheme
1, method b).14
The third method for the synthesis of new Monensin
esters (Scheme 1, method c) is based on the reaction
between Monensin A and the appropriate alcohol in
All esters (2–10) can easily be purified by column chro-
matography on silica gel. The structures of the esters
were determined on the basis on elemental analysis,
FT-IR, 1H, 13C NMR, ESI-MS and semiempirical
(PM5) methods.14,15
(4) (89%*); (5) (30%**);
(6) (80%*); (9) (45%**);
(10) (76%*)
Monensin A (1) as well as the new esters of Monensin A
(2–10) were tested in vitro in the face of their antibacte-
rial and antifungal activity. The Gram-positive cocci,
Gram-negative rods and yeasts-like micro-organisms
used in the tests are collected in Table 1.
d)
c)
a)
(3) (67%); (4) (35%);
(7) (72%); (8) (69%)
monensin A
(2) (20%)
(1)
Hospital strains of S. aureus were isolated from different
biological materials of patients of the Warsaw Medical
University Hospital. 10 of these strains were methicil-
lin-susceptible (MSSA) and 10 other strains were methi-
cillin-resistant (MRSA). Due to this resistance these
strains were tested concerning their sensitivity towards
the new Monensin A esters. The other micro-organisms
used here were provided by the Department of Pharma-
ceutical Microbiology, Medical University of Warsaw,
Poland.16,17
b)
(2) (71%); (3) (32%);
(7) (37%); (8) (15%)
Scheme 1. Synthetic access to Monensin A ester derivatives. Reagents
and conditions: (a) EtOH, DCC (N,N0-dicyclohexylcarbodiimide),
CH2Cl2, 0 °C to rt; (b) R-OH (corresponding alcohols), DCC, PPy (4-
pyrrolidinopyridine), CH2Cl2, 0 °C to rt; (c) R-OH, DCC, PPy, p-TSA
(p-toluenesulfonic acid monohydrate), CH2Cl2, 0 °C to rt; (d) R-X
(bromides* or chlorides**), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene),
toluene, 90–100 °C, 5 h.
The data concerning the antimicrobial activity of the
compounds are summarized in Table 1.

´
A. Huczynski et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2585–2589
2587
Table 1. Antimicrobial activity of Monensin A (1) and its esters: (3), (8), (10); diameter of the growth inhibition zone (GIZ) [mm] and minimal
inhibitory concentration (MIC) [lg/ml]16,17
Growth inhibition zone (GIZ) [mm] and Minimal inhibitory concentration (MIC) [lg/ml]
(1)
(3)
(8)
(10)
GIZ
MIC
GIZ
MIC
GIZ
MIC
GIZ
MIC
S. aureus NCTC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
B. subtilis ATCC 6633
B. cereus ATCC 11778
E. hirae ATCC 10541
M. luteus ATCC 9341
M. luteus ATCC 10240
E. coli ATCC 10538
22
22
20
18
15
22
18
—
12
12
na
na
na
na
na
na
na
na
na
na
na
2
13
11
13
13
—
15
14
—
—
11
na
na
na
na
na
na
na
na
na
na
na
100
100
100
100
100
12.5
12.5
>400
100
50
19
19
17
17
13
20
19
—
11
13
na
na
na
na
na
na
na
na
na
na
na
100
50
23
20
23
25
24
27
25
13
22
18
na
na
na
na
na
na
na
na
18
17
14
12.5
6.25
12.5
6.25
12.5
6.25
6.25
50
1
2
100
50
1
2
100
25
1
2
50
12.5
4
>400
200
50
25
12.5
2
E. coli ATCC 25922
E. coli NCTC 8196
P. vulgaris NCTC 4635
P. aeruginosa ATCC 15442
P. aeruginosa NCTC 6749
P. aeruginosa ATCC 27853
B. bronchiseptica ATCC 4617
C. albicans ATCC 10231
C. albicans ATCC 90028
C. parapsilosis ATCC 22019
200
200
400
na, no activity in disc diffusion test—denotes lack of the growth inhibition zone.
Among the tested compounds (Table 1), only Monensin
A (1) as well as three ester derivatives (3, 8 and 10)
showed detectable but different activities against
Gram-positive bacteria. Only one of the derivatives
(10) exhibited relatively low antifungal activity against
Candida while Monensin A was inactive against these
fungi. Compounds (2, 4–7 and 9) were inactive towards
all micro-organisms tested.16
ring moiety, since it was previously observed that vari-
ous compounds containing this substituent show moder-
ate antibacterial and strong antifungal activities.18 The
low antibacterial activity of compound 3 is probably
connected with the presence of allyl group, which has
a significant influence on the antimicrobial activity.19
Drug-resistant Gram-positive bacterial pathogens
including methicillin-resistant S. aureus (MRSA) cause
serious chemotherapeutic problems in hospitals.20 Our
studies reveal that Monensin A clearly shows antimicro-
bial activity against both MRSA (methicillin-resistant)
and MSSA (methicillin-susceptible) strains of Staphylo-
coccus aureus at doses MIC = 1–2 lg/ml, whereas from
among the Monensin A esters studied only compound
10 shows interesting activity at the doses MIC = 6.25–
12.5 lg/ml (Supplementary material, Table 1S).
The interactions between the oxygen atoms of Monensin
A or of the Monensin A esters with mono- and di-valent
metal cations lead to the formation of pseudo-cyclic struc-
tures which are additionally stabilized by intramolecular
hydrogen bonds.2,7,9 In previous investigations we could
show that the mode of complex formation with Na+ cat-
ions is very similar for the majority of Monensin A deriv-
atives and rather independent of the nature of the
respective ester groups. This suggests that the ester groups
are not engaged in the coordination process.7,9 Thus, the
differences in the biological activities between Monensin
A and the Monensin A derivatives described here are
not based on a different capability of complex formation
but on other parameters such as size and chemical nature
of the substituent. One of these parameters is potentially
the lipophilic character of the substituent, which evokes
lower solubility in aqueous solutions. Furthermore, the
presence of aromatic substituent in the ester group, such
as phenyl (compounds 4 and 9) or naphthalene rings
(compounds 5 and 6), might decrease the mobility of
Monensin A esters in the lipid bilayers.
In the present work, we synthesized nine new esters
(2–10) of Monensin A using four different synthesis
pathways. We provide evidence that three Monensin
esters (3, 8 and 10) show antibacterial activity against
human pathogenic bacteria, including antibiotic-resis-
tant S. aureus. Concerning the Monensin A esters, only
compound (10) shows relatively low antifungal activity.
Acknowledgments
´
Adam Huczynski thanks the Foundation for Polish
Science for fellowship. Financial assistance of the Polish
Ministry of Science and Higher Education—Grant No.
N204 056 32/1432 is gratefully acknowledged by
P. Przybylski.
The highest antibacterial activity of compound 10
among the ester derivates and its slight antifungal activ-
ity is probably related to the presence of the morpholine

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A. Huczynski et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2585–2589
2588
Nagatsu, A.; Mizukami, H.; Ogihara, Y.; Sakakibara, J.
Supplementary data
Chem. Pharm. Bull. 2001, 49, 711; (e) Tsukube, H.;
Sohmiya, H. J. Org. Chem 1991, 56, 875; (f) Maruyama,
K.; Sohmiya, H.; Tsukube, H. J. Chem. Soc., Chem.
Commun. 1989, 864; (g) Nagatsu, A.; Tabunoki, Y.;
Nagai, S.; Ueda, T.; Sakakibara, J.; Hidaka, H. Chem.
Pharm. Bull. 1997, 45, 966; (h) Nagatsu, A.; Sakakibara, J.
Yakugaku Zasshi. 1997, 117, 583.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2008.
03.038.
´
9. (a) Huczynski, A.; Przybylski, P.; Brzezinski, B.; Bartl, F.
Biopolymers 2006, 81, 282; (b) Huczynski, A.; Przybylski,
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(Fluka) was dissolved in dichloromethane and stirred
vigorously with a layer of aqueous sulphuric acid (pH 1.5).
The organic layer containing MONA was washed with
distilled water, and dichloromethane was evaporated
under reduced pressure to dryness.
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14. (a) General procedure for the synthesis of Monensin A
esters (2–10): (method a, Scheme 1): To a mixture of (1)
(500 mg, 0.75 mmol) in dichloromethane (15 ml) the
following compounds were added: DCC (206 mg,
1.0 mmol), EtOH (5 mmol). The mixture was first stirred
at a temperature below 0 °C for 24 h and then for further
24 h at room temperature. Subsequently, the solvent was
evaporated under reduced pressure to dryness. The residue
was then suspended in hexane and filtered off. The filtrate
was evaporated under reduced pressure and the residue
purified chromatographically on silica gel (Fluka type 60)
to give (2) (20% yield) as a colourless oil showing a
tendency to form the glass state; (b) (method b, Scheme 1):
To a mixture of MONA (500 mg, 0.75 mmol) in dichlo-
romethane (15 ml) the following compounds were added:
DCC (206 mg, 1.0 mmol), PPy (50 mg, 0.33 mmol), cor-
responding alcohol (5.0 mmol). The mixture was first
stirred at a temperature below 0 °C for 24 h and then for
further 24 h at room temperature. After this time the
solvent was evaporated under reduced pressure to dryness.
The residue was suspended in hexane and filtered off. The
filtrate was evaporated under reduced pressure and the
residue was purified chromatographically on silica gel
(Fluka type 60) to give corresponding esters (2–3, 7–8)
(yield from 15% to 71%) as a colourless oil showing a
tendency to form the glass state; (c) (method c, Scheme 1):
To a mixture of MONA (500 mg, 0.75 mmol) in dichlo-
romethane (15 ml) the following compounds were added:
DCC (206 mg, 1.0 mmol), PPy (50 mg, 0.33 mmol), cor-
responding alcohol (5 mmol) and p-TSA (28.5 mg,
0.15 mmol). The mixture was first stirred at a temperature
below 0 °C for 24 h and then for further 24 h at room
temperature. The solvent was subsequently evaporated
´
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A. Huczynski et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2585–2589
2589
under reduced pressure to dryness. The residue was
suspended in hexane and filtered off. The filtrate
was evaporated under reduced pressure and the residue
was purified chromatographically on silica gel (Fluka type
60) to give (3–4, 7–8) (yield from 35% to 73%) as a
colourless oil showing a tendency to form the glass state;
(d) (method d, Scheme 1): A mixture of alkyl bromide or
chloride (1.45 mmol), MONA (500 mg, 0.75 mmol), and
DBU (175 mg, 1.15 mmol) and 40 ml toluene was heated
at 90 °C for 5 h. After cooling, the precipitate DBU-
hydrohalide (DBUHX) was filtered and washed hexane.
The filtrate and the washing were combined and evapo-
rated under reduced pressure. The residue was purified by
chromatography on silica gel (Fluka type 60) to give the
corresponding ester (4–6, 9–10) (yield from 30% to 89%) as
a colourless oil showing a tendency to form the glass
state.
For the disc diffusion method, sterile filter paper discs
(9 mm diameter, Whatman No. 3 chromatography paper)
were dripped with the compound solutions tested (in
ethanol) to load 400 lg of a given compound per disc. Dry
discs were placed on the surface of an appropriate agar
medium. The results (diameter of the growth inhibition
zone) were read after 18 h of incubation at 35 °C.
For MICs determination, all compounds were dissolved in
DMSO. Concentrations of the agents tested in solid
medium ranged from 3.125 to 400 lg/ml. The final
inoculum of all organisms studied was 104 CFU mLꢀ1
(colony forming units per ml), except the final inoculums
for E. hiraeATCC 10541, which was 105 CFU mLꢀ1. A
control test was also performed for DMSO which was
found inactive in the culture medium. Minimal inhibitory
concentrations were read off after 18 h (for bacteria) and
24 h (for yeasts) of incubation at 35 °C. Ionophore
antibiotic—Monensin A was used as a control for bacteria
and fluconazole (for the disc diffusion method 25 lg per
disc has been used) for yeast (C. albicans ATCC 10231
GIZ = 22 mm, MIC = 1 lg/ml; C. albicans ATCC 90028
GIZ = 32 mm, MIC = 1 lg/ml; C. parapsilosis ATCC
22019 GIZ = 22 mm, MIC = 2 lg/ml).
15. Selected spectra data for (5): ESI-MS (m/z): 834 (M+Na+);
1H NMR (d ppm in CD3CN): 2.69 (1H, m, 2-H), 2.78 (1H,
t, J = 6.6 Hz, O(11)-H), 3.11 (3H, s, 35-H), 3.33(2H, lt,
J = 6.6 Hz, 26-H), 3.50 (1H, t, J = 4.3 Hz, 3-H), 3.58 (1H,
overlapped, 3-H), 3.60 (1H, overlapped, 13-H), 3.62 (1H,
overlapped, 21-H), 3.84 (1H, d, J = 4.4 Hz, 17-H), 3.92
(1H, s, O(10)-H), 4.0 (1H, dd, J = 2.2 Hz, 7.1 Hz, 5-H),
4.18 (1H, d, J = 8.8 Hz, O(4)-H), 4.21 (1H, m, 20H), 5.57,
5.64 (each 1H, both d, J = 12.6 Hz, OCH2-Ar), 7.45–8.11
(9H, Ar), 0.70–2.28 pattern of 45 protons; 13C NMR (d
ppm in CD3CN): 175.9, 134.6, 132.8, 132.4, 129.9, 129.4,
128.3, 127.5, 126.9, 126.3, 127.7, 108.5, 97.9, 88.1, 86.9,
86.4, 84.4, 81.8, 77.9, 77.3, 71.9, 68.7, 67.5, 65.2, 58.2, 41.5,
39.7, 37.7, 36.8, 35.9, 35.1, 34.9, 34.6, 34.0, 32.6, 32.0, 30.1,
28.4, 26.3, 17.8, 16.5, 16.1, 12.5, 12.2, 11.1, 8.3; IR(KBr):
1734 cmꢀ1 (mC@O); Elemental analysis: (%): Calcd for
C47H70O11: C, 69.60; H, 8.70; Found: C, 69.40; H, 8.89.
Compounds (3–4 and 7) as well as their complexes with
monovalent cations were characterized by us in Refs. 9c–e,
respectively.
16. Antimicrobial activity was examined by the disc diffusion
and MIC method under standard conditions using Muel-
ler–Hinton II agar medium (Becton Dickinson) for bac-
teria and RPMI agar with 2% glucose (Sigma) for yeasts,
according to CLSI (previously NCCLS) guidelines.17
The compounds giving some growth inhibition zone in
disc diffusion assay were tested by the twofold serial agar
dilution technique to determine their minimal inhibitory
concentration (MIC) values.
17. (a) Clinical and Laboratory Standards Institute. Perfor-
mance Standards for Antimicrobial Disc Susceptibility
Tests; Approved Standard M2-A9. Clinical and Labora-
tory Standards Institute, Wayne, PA, USA, 2006; Clinical
and Laboratory Standards Institute. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically; Approved Standard M7-A7. Clinical and
Laboratory Standards Institute, Wayne, PA, USA, 2006.
18. (a) Mercer, E. I. Biochem. Soc. Trans. 1991, 19, 788; (b)
Hiratani, T.; Asagi, Y.; Matsusaka, A.; Uchida, K.;
Yamaguchi, H. Jpn. J. Antibiot. 1991, 44, 993; (c) Isenring,
H. P. In Recent Trends in the Discovery, Development and
Evaluation of Antifungal Agents; Fromtling, R. A., Ed.;
Prous Science J.R. Publishers: S.A., 1987; pp 543–554.
19. (a) Sehi, L.; Woo, Y.; Kyung, K. H. J. Microbiol.
Biotechnol. 2006, 16, 1236; (b) Choi, K.; Kyung, K. H.
J. Food Sci. 2005, 70, 305.
20. (a) Athanassa, Z.; Siempos, I. I.; Falagas, M. E. Eur.
Respir. J. 2008, 31, 625; (b) Izumida, M.; Nagai, M.; Ohta,
A.; Hashimoto, S.; Kawado, M.; Murakami, Y.; Tada, Y.;
Shigematsu, M.; Yasui, Y.; Taniguchi, K. J. Epidemiol.
2007, 17, S42; (c) Klein, E.; Smith, D. L.; Laxminarayan,
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