In vitro screening of pentamidine analogs against bacterial and fungal strains

Article abstract of DOI:10.1016/j.bmcl.2014.04.075

A series of linear pentamidine analogs exhibiting low cytotoxicity, active against Pneumocystis carinii, were evaluated for in vitro activities against bacterial and fungal strains. The majority of the tested bis-amidines exhibited marked activities against Gram-positive strains. In view of the fact that the highest potency was found for 1,5-bis(4-amidinophenoxy)-3-thiapentane dihydrochloride 1j with the S atom in the middle of the aliphatic linker, four new pentamidines bearing S atoms were synthesized and also evaluated against MRSA strains. N,N′-Dialkylated pentamidines with S atoms in the linker are the promising lead structures for antimicrobials development.

Full text of DOI:10.1016/j.bmcl.2014.04.075

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro screening of pentamidine analogs against bacterial
and fungal strains
a
a
b
b,c
Dorota Maciejewska a, , Jerzy Zabinski , Paweł Kazmierczak , Karolina Wójciuk , Marcin Kruszewski
,
⇑
_
´
´
Hanna Kruszewska d,
a Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland
b Centre for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland
c Department of Molecular Biology and Translational Research, Institute of Rural Health, Jaczewskiego 2, 20-090 Lublin, Poland
d National Medicines Institute, Department of Antibiotics and Microbiology, 30/34 Chełmska, 00-725 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of linear pentamidine analogs exhibiting low cytotoxicity, active against Pneumocystis carinii,
were evaluated for in vitro activities against bacterial and fungal strains. The majority of the tested
bis-amidines exhibited marked activities against Gram-positive strains. In view of the fact that the high-
est potency was found for 1,5-bis(4-amidinophenoxy)-3-thiapentane dihydrochloride 1j with the S atom
in the middle of the aliphatic linker, four new pentamidines bearing S atoms were synthesized and also
evaluated against MRSA strains. N,N0-Dialkylated pentamidines with S atoms in the linker are the prom-
ising lead structures for antimicrobials development.
Received 26 February 2014
Revised 16 April 2014
Accepted 21 April 2014
Available online xxxx
Keywords:
Linear pentamidine analogs
Anti-bacterial activity
Anti-fungal activity
Anti-MRSA activity
Structure–activity
Ó 2014 Elsevier Ltd. All rights reserved.
The antimicrobial activity of aromatic bis-amidines is well
known, but only pentamidine is clinically used for the treatment
of pneumonia caused by the opportunistic fungus Pneumocystis
jiroveci, against antimony-resistant leishmaniasis, and in the initial
stage of human African trypanosomiasis.1–9 Pneumocystis pneu-
monia (PCP) can be life threatening for patients receiving
chemotherapy and immunosuppressive medication, including
high-dose steroids.10 Since 1984 pentamidine isethionate has been
more commonly used for the treatment of PCP in immunocompr-
omized and immunodeficiency patients. The mechanism of action
is unclear and may vary for different microorganisms. Trypano-
somes actively transport pentamidine intracellularly, where it
may then interfere with DNA biosynthesis, but during interaction
with Pneumocystis sp. pentamidine appears to kill nonreplicating
organisms.11
gondii.13–16 Aromatic bis-amidines which target the DNA minor
groove are intensively tested as anti-infective agents and their sig-
nificant therapeutic potential was highlighted.17 Bis-amidines with
benzimidazole and imidazole moieties which received much inter-
est as concerns the discovery of antibacterial drugs are also men-
tioned as possible inhibitors of
a bacterial two-component
system (a histidine protein kinase and a response regulator) or as
targeting bacterial cell-wall synthesis (the inhibitors of undecapre-
nyl diphosphate synthase).18,19 To be an effective drug, the cationic
bis-amidine compounds which are not lipid soluble, need to reach
the molecular target in the cell, and one can suppose that they
must be carried into the cell via a dedicated carrier or in non-cat-
ionic prodrug forms.20
However, it should be taken into account that the activity of bis-
amidines is associated with high toxicity and low bioavailability in
mammalian host.21 Recently, we have found that newly synthe-
sized pentamidine analogs had little to no cytoxicity in two
in vitro evaluations.22 These analogs which are shown in Figure 1
(see Supplementary data for their chemical names) exhibited no
There are several indications that pentamidine may have a wide
range of antimicrobial, anti-inflammatory and anti-cancer activi-
ties.12 It was found that in vitro pentamidine inhibits Tropheryma
whipplei, Coxiella burnetti, Acanthamoeba keratitis and Toxoplasma
toxicity in the human epithelial cell line A549 (IC50 >100
Moreover, all analogs except 1d, 1e and 1i also exhibited no symp-
toms of toxicity in the rat cell line L2 (IC50 >100
g/ml).22 It seemed
lg/ml).
l
interesting to assess their activities towards other fungi than Pneu-
mocystis sp. and different microorganisms for a comprehensive
study on their structure–activity relationship.
⇑
Corresponding author. Tel.: +48 225720643.
E-mail addresses: dmaciejewska@wum.edu.pl (D. Maciejewska).
Co-correspondence. h.kruszewska@nil.gov.pl (H. Kruszewska).
http://dx.doi.org/10.1016/j.bmcl.2014.04.075
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Maciejewska, D.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.075

2
D. Maciejewska et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
The emergence of microbial resistance to the most commonly
atoms in the linker (Fig. 2). In order to determine the influence
of alkyl substituents, the N,N0-dialkyl derivatives 3a–3c of 1j were
prepared, whereas to determine the role of the S atoms localiza-
tion, the derivative 3d was synthesized with two S atoms which
replaced the O atoms of pentamidine. Compounds 1j, and 3a–3d
were tested additionally against five MRSA strains.
The present study is focused on biological evaluation of low
toxic bis-amidines in two in vitro assays. We explored the possibil-
ity that linear pentamidine analogs, presented in Figures 1 and 2,
have potency against fungal and bacterial strains, and wanted to
find out if the selected compounds 1j, and 3a–3d can also be
effective against MRSA strains. Several yeast strains, a mold and
used drugs is an increasing problem in medicine,23 and the earlier
mentioned pentamidine analogs with low toxicity in two in vitro
assays could be good candidates as alternative substances. Some
earlier studies24–26 supported these predictions because the
screening of aromatic bis-amidines identified them as the potent
anti methicilin-resistant Staphylococcus aureus (MRSA) and vanco-
mycin-resistant Enterococccus faecium agents.
Out of fifteen derivatives (Fig. 1) tested in the first course, the
1,5-bis(4-amidinophenoxy)-3-thiapentane dihydrochloride 1j with
the S atom in the aliphatic linker was the most potent one. There-
fore, we synthesized four new pentamidine analogs bearing S
Am
Am
O
O
Pent
pentamidine dihydrochloride
Am
Am
CH3
N
1a
O
O
Am
Am
2a Am
SO2
N
1bAm
OCH3
O
H3CO
O
Am
O
O
CH3
N
Am
OCH3
O
H3CO
O
OCH3
O
H3CO
O
Am
Am
1cAm
SO2
N
CH3
N
2b
OCH3
OCH3
Am
1d
N
N
Am
OCH3
O
H3CO
O
Am
SO2
N
CH3
CH3
2c
Am
Am
Am
OCH3
OCH3
1e
O
O
N
H
N
H
NHCOCH3
Am
1f
O
O
Am
OCH3
O
H3CO
SO2
Am
2d
2e
N
O
1g Am
OCH3
O
H3CO
O
Am
Am
O
O
NH2
Am
OCH3
O
H3CO
O
1h
Am
OCH3
O
H3CO
SO2
Am
N
OCH3
OCH3
OCH3
OCH3
O
OCH3
OCH3
Am
OCH3
O
H3CO
O
Am
Am
1i
NH2
Am
Am
=
C
Cl
1j
NH2
S
O
O
Figure 1. Chemical formulas of tested pentamidine analogs 1a–1j and 2a–2e.
Please cite this article in press as: Maciejewska, D.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.075

D. Maciejewska et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
HCl.NH
HN
NH.HCl
3a
S
NH
H3C
CH3
O
O
HCl.NH
HN
NH.HCl
3b
NH
S
O
O
N.HCl
HCl.N
3c
S
NH
NH
O
O
HCl.NH
NH.HCl
3d
H2N
NH2
O
S
S
Figure 2. Chemical formulas of tested pentamidine analogs 3a–3d.
Gram-positive and Gram-negative bacteria from American Type
Culture Collection (ATCC), and MRSA hospital strains from WUM
Museum Collection were chosen for the investigation. Moreover,
cytotoxicity assay of new analogs 3a–3d was established on the
human lung cell carcinoma cell line A549 (ATCC CCL-185) and
the human cervix adenocarcinoma cell line HeLa (ATCC CCL-2).
The syntheses and structural studies of 15 compounds were
reported previously. For compounds 1a–1c, 1g–1j, 2a–2d data
are in,22 for 1d in,27 for 1e and 1f in,28 and for 2e in.29 The method
of synthesis for new bis-amidines 3a–3d generally followed the
established procedures30–32 which involved the preparation of
bis-nitriles and their conversion into bis-amidines. Compounds
3a–3c were synthesized from 1,5-bis(4-cyanophenoxy)-3-thiapen-
tane22 which was treated with appropriate amines (butylamine,
cyclohexylamine, ethane-1,2-diamine) (Scheme 1). The synthesis
of compound 3d required a three-step procedure (Scheme 1). All
synthetic details and the spectroscopic data are given in Supple-
mentary data.
middle of the aliphatic linker, and varying numbers of the OCH3
groups. The methoxy groups were inserted because of their pres-
ence in trimethoprim molecule and the sulfonamide groupings
were inserted because of their known anti-bacterial efficacy.33
All compounds, apart from 1a–1c which were analyzed as trihy-
drochlorides, were tested as dihydrochlorides. Pentamidine dihy-
drochloride (Pent) was used as
biological assays methods34 are presented in Supplementary data.
The MIC values ( mol/ml) of pentamidine analogs for the tested
a reference compound. The
l
bacterial and fungal strains are presented in Tables 1 and 2 and
their chemical structures are shown in Figures 1 and 2 to facilitate
the discussion about structure–activity relationship.
In the first course (Tables 1 and 2), all pentamidine analogs
showed the activity against the tested Gram-positive strains. The
analogs 1d, 1f, 1i, 1j, 2a, 2b, 2c, 2e showed a broad activity spec-
trum. They inhibited the growth of all tested strains at a concentra-
tion ranging from 0.0028 to 7.4 lmol/ml. In this series, the higher
activity was associated with the presence of the sulfonamide group
(2c) and four methoxy substituents on the benzene rings (2c and
1i). Three active compounds 1d, 1f, 1j have various heteroatoms
in the aliphatic linker. The derivatives 1d, 1j, 2a and 2b also
showed considerable activity against the tested yeast (MIC 0.07–
From the structural point of view, the tested compounds belong
to two series. Series I includes 14 compounds 1a–1j, 3a–3d with
different heteroatoms in the aliphatic linker (N, O and S), and 0,
2-, and 4-OCH3 groups on benzamidine moieties. Series II includes
5 compounds of 2a–2e bearing the sulfonamide substituents in the
0.04
l
mol/ml). The introduction of the S atom in the middle of
NC
CN
CN
O
S
+
HS
SH
Cl
O
O
1) HCl/C2H5OH, 24h rt
2) appropriate amine/C2H5OH
24h rt, reflux 3h
K2CO3,DMSO
115-120 oC, 2h
3) C2H5OH/HCl, short reflux
5
NC
7
4
CN
6
5
4
8
R
R
6
1
1
3
O
S
S
8
3
9
2
S
3d-nit
O
O
2
9
1) HCl/C2H5OH, 24h rt
2) NH3/C2H5OH, 24h rt
3) C2H5OH/HCl, short reflux
3a
3b
10
10
NH.HCl
NH.HCl
13 14
10
NH.HCl
14
12
7
7
12
NH.HCl
NH
11
CH3
15
15
NH
11
5
13
7
4
6
17 16
NH2
H2N
11
8
1
3
13
10
HCl.N
7
O
S
S
R =
2
9
12 3c
NH
11
3d
Scheme 1. Synthesis of new pentamidine analogs 3a–3d. Reagents and conditions together with the atoms numbering.
Please cite this article in press as: Maciejewska, D.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.075

4
D. Maciejewska et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1
Activities of pentamidine analogs against bacterial strains in MIC [lmol/ml]
MIC
l
mol/ml
Compd no
Micrococcus luteus
ATCC 9341 ATCC 10240
Bacillus subtilis
Bacillus cereus
Staphylococcus aureus
Escherichia Coli
ATCC 8739 ATCC 10536
21.1
8.13
Pseudomonas aeruginosa
ATCC 6633
ATCC 11778
ATCC 6538
21.1
8.13
ATCC 6538P
ATCC 15442
ATCC 9027
1a
1b
1c
1d
1e
1f
1g
1h
1i
1.06
1.06
2.11
0.81
1.52
0.098
0.59
0.55
2.10
0.42
0.089
0.14
0.082
0.042
0.070
0.72
0.32
0.067
1.06
0.10
7.61
0.059
0.59
0.55
1.05
16.96
0.11
0.0695
0.016
0.042
0.088
0.072
0.32
10.6
8.13
7.61
0.12
0.59
0.55
0.52
0.21
0.054
0.14
0.049
0.042
0.070
0.36
0.32
0.067
21.1
16.26
15.22
9.78
11.84
0.55
10.52
16.96
0.22
0.58
0.21
0.74
7.04
3.58
6.41
0.28
>21.1
>16.26
>15.22
>19.55
>23.68
11.08
>21.04
>16.96
4.46
>21.1
>16.26
>15.22
>19.55
>23,68
11.08
>21.04
>16.96
1.78
0.2
0.41
0.76
1.52
7.61
0.059
0.59
7.61
9.78
2.37
0.55
2.10
16.96
0.22
0.14
0.21
0.37
7.04
3.58
6.41
0.28
0.0039
0.024
0.0044
2.10
0.0195
0.071
0.011
0.52
0.55
0.26
0.21
0.017
0.0035
0.0046
0.10
0.015
0.0028
0.072
0.038
0.0011
0.051
0.018
0.023
0.016
0.042
0.007
0.089
0.064
0.0044
0.054
0.0695
0.049
0.042
0.070
0.14
1j
2.32
1.74
2a
2b
2c
2d
2e
Pent
1.64
1.64
7.38
7.38
7.04
7.04
7.16
7.16
0.32
0.022
6.41
6.41
0.0011
1.67
1.11
Table 2
Activities of pentamidine analogs against fungal strains in MIC [
lmol/ml]
Compd no
Candida albicans
Candida parapsilosis
Aspergillus brasiliensis
Zygosaccharo-myces rouxi
Saccharomyces cerevisiae
ATCC 10231
ATCC 2091
ATCC 22019
ATCC 16404
ATCC 28253
ATCC 9763
1a
1b
1c
1d
1e
1f
1g
1h
1i
>21.11
>16.3
>15.2
0.12
>21.11
>16.3
>15.2
0.12
10.6
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
0.12
5.92
>22.16
10.52
N.T.
0.11
0.070
0.01
0.18
7.04
1.43
>12.83
0.067
N.T.
N.T.
N.T.
>16.3
>15.2
0.020
0.071
0.066
5.26
>16.96
0.89
0.023
0.10
0.37
7.04
0.36
>12.83
0.0044
9.78
5.92
>22.16
21.04
N.T.
8.92
0.070
8.22
7.38
7.04
0.24
11.84
11.08
10.52
N.T.
4.46
0.29
0.21
0.18
7.04
1.43
>12.83
0.28
5.92
5.92
>22.16
10.52
>16.96
0.89
0.14
0.21
0.092
7.04
0.18
>12.83
0.11
>22.16
10.52
>16.96
1.78
0.14
0.21
0.37
7.04
1.43
>12.83
0.11
1j
2a
2b
2c
2d
2e
Pent
7.16
>12.83
0.022
the aliphatic linker (compound 1j) resulted in the activity against
all tested bacterial and fungal strains including A. brasiliensis sim-
ilar to that of pentamidine. The introduction of the NCH3 group
in the aliphatic linker modified the biological activity of the tested
compounds. The analog 1d in which the O atoms in the aliphatic
linker of pentamidine were replaced by NCH3 groups revealed
remarkable antifungal activity against Gram-positive strains
whereas the analogs 1a, 1b, 1c with the NCH3 group in the middle
of the aliphatic linker showed no antifungal activity. Moreover, the
change of the two distal oxygens to NCH3 yielded the compound
1d with the loss of potency against Gram-negative strains, and
with the change of cytotoxicity. It exhibited low toxicity on human
cell line A549, but some toxic effect was found on rat cell line L2.22
The most potent compound 1j, with limited cytotoxicity, has
the S atom in the aliphatic linker and served as the lead compound
for the synthesis of four pentamidine analogs 3a–3d reported in
this Letter. The synthesized compounds were tested against
drug-resistant strains MRSA and compared with MIC values of 1j
and pentamidine. The obtained results are collected in Table 3. In
the case of N,N0-disubstituted 3a–3c derivatives, the transforma-
tion of amidine groups in 4,5-dihydro-2-imidazoyl rings (com-
pound 3c) reduced the anti-MRSA activity as compared with the
parent compound 1j against three MRSA lines (572/12, 19 K/11
and 573/12), or only slightly increased the activity against 15K/
11 and 495/11 MRSA strains. Introduction of butyl and cyclohexyl
groups had the pronounced consequences, and compounds 3a and
3b were found to be more potent by factor ten against all
MRSA strains. Compounds 3a and 3b showed MIC values in the
range 0.012–0.23 lmol/ml, and exhibited the highest activities
against the MRSA 573/12 strain. Compound 3d with the second S
atom showed the potency equivalent to that of 1j and
pentamidine.
According to the table 2C from CLSI35 the susceptibility of
Staphylococcus spp. to linezoid and vancomycin is defined as
60.012
whereas the resistance of Staphylococcus spp. to linezoid and van-
comycin is defined as P0.024 mol/ml and P0.022 mol/ml,
lmol/ml and 60.0028–0.011 lmol/ml, respectively;
l
l
respectively. N,N0-Dialkylated pentamidine analogs with the S
atoms in the middle of the aliphatic linker constitute a promising
structure for further research towards potential medicines.
In order to get a more complete view on the potency of com-
pounds 3a–3d they were screened against other bacterial and fun-
gal strains (see Table 4 for results), and their cytotoxicity was also
evaluated (see Supplementary data). The potency of N,N0-dialkyl
substituted compounds 3a and 3b approached 2.6–9.8 nmol/ml
values for Gram-positive M. luteus bacterial strains and C. albicans
and C. parapsilosis yeast strains. Compounds 3a and 3b also showed
considerable activity against the mold strain A. brasiliensis.
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D. Maciejewska et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
Table 3
Activities of pentamidine analogs against MRSA strains in MIC [
l
mol/ml]
Compd no
MRSA 15K/11
MRSA 495/11
MRSA 572/12
MRSA 19 K/11
MRSA 573/12
1j
3a
3b
3c
3d
Pent
1.16
0.23
0.10
0.98
1.07
1.11
2.32
0.23
0.20
1.96
1.07
1.11
0.29
0.23
0.10
1.96
0.27
0.56
0.14
0.11
0.012
1.96
0.13
0.14
0.29
0.014
0.012
1.96
0.13
0.14
values (0.012–0.23 lmol/ml) against MRSA strains as compared
Table 4
Activities of analogs 3a–3d against bacterial and fungal strains in MIC [
l
mol/ml]
with pentamidine, linezolid and vancomycin. We hypothesize
that N,N0-dialkylated bis-amidines with the S atom in the middle
of the aliphatic linker are the promising template molecules
for future studies, especially towards anti-fungal and anti-MRSA
medicines.
Compd no
3a
3b
3c
3d
Micrococcus luteus
ATCC 9341
0.0026
0.0032
0.0098
0.0043
Micrococcus luteus
ATCC 10240
Bacillus subtilis
ATCC 6633
0.0036
0.014
0.45
0.00081
0.012
0.0098
0.24
0.0043
0.13
0.54
0.13
0.13
0.54
1.07
2.15
1.07
1.07
0.27
2.15
0.54
Supplementary data
Bacillus cereus
0.10
0.98
ATCC 11178
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
04.075.
Staphylococcus aureus
ATCC 6538
Staphylococcus aureus
ATCC 6538P
Escherichia coli
ATCC 8739
0.014
0.027
0.11
0.0032
0.0032
0.10
0.98
0.98
References and notes
0.98
Pseudomonas aeruginosa
ATCC 9027
Pseudomonas aeruginosa
ATCC 15422
Candida albicans
ATCC 10231
Candida albicans
ATCC 2091
Candida parapsilosis
ATCC 22019
Aspergillus brasiliensis
ATCC 16404
Saccharomyces cerevisiae
ATCC 9763
>1.81
>1.81
0.0036
0.0036
0.0036
0.014
0.027
>1.63
>1.96
>1.96
0.24
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