Development of highly active anti-: Pneumocystis bisbenzamidines: Insight into the influence of selected substituents on the in vitro activity

Article abstract of DOI:10.1039/c7md00445a

Here we describe the potency of 21 pentamidine analogues against the fungal pathogen, Pneumocystis carinii, in an ATP bioluminescent assay with toxicity profiles in 2 mammalian cell lines. Reduction of two 5-methyl-1,2,4-oxadiazole rings was applied to the synthesis of acid-labile bisamidines. Anti-Pneumocystis activity is discussed in the context of 3 groups of compounds depending on the main structural changes of the pentamidine lead structure. The groups include: 1) 1,4-bis(methylene)piperazine derivatives 1-5; 2) alkanediamide derivatives 6-10; 3) alkane-derived bisbenzamidines 11-21. IC₅₀ values of 18 compounds were lower than the IC₅₀ of pentamidine. Four bisamidines were active at nanogram concentrations. Introduction of sulfur atoms in the alkane bridge, replacement of the amidino groups with imidazoline rings, or attachment of nitro or amino groups to the benzene rings is responsible for remarkable activity of the new leading structures. The vast majority of compounds, including four highly active ones, can be classified as mild or nontoxic to host cells. These compounds show promise as candidates for new anti-Pneumocystis agents.

Full text of DOI:10.1039/c7md00445a

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abiski, M. Rezler, P. Kamierczak, M. Collins, L. Ficker and M. Cushion, Med. Chem. Commun., 2017, DOI:
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DOI: 10.1039/C7MD00445A
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ARTICLE
Development of highly active anti-Pneumocystis bisbenzamidines:
Insight intothe influence of selected substituentsonthe in vitro activity†
D. Maciejewska,a* J. Żabiński,a M. Rezler,a P. Kaźmierczak,a M. S. Collins,b,c L. Ficker,b,c and M. T. Cushionb,c*
Received 00th January 20xx,
Accepted 00th January 20xx
Here we describe the potency of 21 pentamidine analogues against the fungal pathogen, Pneumocystis carinii, in an ATP
DOI: 10.1039/x0xx00000x
bioluminescent assay with toxicity profiles in
2
mammalian cell lines. Reduction of two
www.rsc.org/
5-methyl-1,2,4-oxadiazole rings was applied to the synthesis of acid-labile bisamidines. Anti-Pneumocystis activity
is discussed in the context of 3 groups of compounds depending on the main structural changes of the pentamidine lead
structure. The groups include: 1) 1,4-bis(methylene)piperazine derivatives 1–5; 2) alkanediamide derivatives 6–10;
3) alkane-derived bisbenzamidines 11–21. IC50 values of 18 compounds were lower than the IC50 of pentamidine.
Four bisamidines were active at nanogram concentrations. Introduction of sulfur atoms in the alkane bridge, replacement
of the amidino groups with imidazoline rings, or attachment of nitro or amino groups to the benzene rings is responsible
for remarkable activity of the new leading structures. The vast majority of compounds, including four highly active ones,
can be classified as mild or nontoxic to host cells. These compounds show promise as candidates for new
anti-Pneumocystis agents.
drugs including azoles and amphotericin B, due to the lack of
an active ergosterol synthesis pathway.10 Only
a few
Introduction
therapeutic choices are effective for treatment of PCP;
primarily trimethoprim-sulfamethoxazole, followed by
secondary regimens such as pentamidine, atovaquone, or
clindamycin-primaquine. Their application is limited
respectively by potential resistance and toxicity.5,11,12
Sustainable, long term in vitro cultivation of any Pneumocystis
species remains elusive, hampering serious development of
alternative therapies and prophylactic agents.13 Moreover, the
mechanism of action of pentamidine on molecular level seems
to be complex. Several molecular targets may be involved in
the overall anti-Pneumocystis activity.14,15 Despite current
searches for the next generation of anti-PCP therapy,
no compound has passed all of the phases of clinical trials.16
Therefore, there is an urgent need for development of new
potent molecules for both prophylaxis and treatment of PCP.
Pentamidine provides an effective treatment of PCP but is
Immunocompromised humans and other mammals are
potential targets for opportunistic infections (OI).1–5 In humans
with debilitated immune systems, infection with Pneumocystis
jirovecii and development of Pneumocystis pneumonia (PCP)
remains a major cause of morbidity and mortality in these
susceptible populations.6,7 Although the incidence of PCP has
decreased in HIV-positive individuals after introduction of
Highly-Active Antiretroviral Therapy (HAART), it remains
a principal AIDS-defining illness.8 Infection and colonization
with P. jirovecii continue to be serious problems in patients
with other types of immunodeficiencies as well. For example,
widespread use of immunosuppressive medications plays
a crucial role in the development of PCP in patients with
autoimmune diseases (rheumatoid arthritis, Crohn’s disease)
and following transplantations.9
While PCP is a significant life-threatening OI, there are still
many problems hindering the development of modern and
dedicated therapies for its treatment and prevention.
P. jirovecii is naturally resistant to the majority of antifungal
considered
a second-line treatment, primarily due to
associated toxicities in treatment populations.6,9
It is our goal to identify analogues of the parent compound
that are equally or more efficacious with reduced toxicity in
the pre-clinical setting. Previous reports of new classes of
bisamidines showed notable in vitro anti-Pneumocystis efficacy
and low toxicity to the mammalian host cell lines.
Leading structures contained alkane17, alkanediamide18,19
or a piperazine linker.20,21 Based on the outstanding properties
of these pentamidine analogues, we decided to investigate
further selected structural modifications of them.
Among the new bisamidines described herein, compounds 1–5
have piperazine linker with loosened rigidity by adding two
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C7MD00445A
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Journal Name
H
3C
O
CH3
methylene groups between the piperazine and phenyl groups.
We wondered if elongation of the distance between the
benzamidine moieties would increase anti-PCP activity. This
hypothesis was based on the examples of highly active
pentamidine analogues with linkers containing more than
4 atoms.19 Analogues 6–10 were modified alkanediamides.
Recently we tested compounds with reversed manner of
amide group substitution17, but this resulted in compounds
with decreased activity. In this studies we tested other
modifications in the alkyl linker. The last group of pentamidine
analogues included compounds 11–21 that have various
heteroatoms in the aliphatic linker (O, S, N). The influence of
phenyl ring substituents was also studied taking into account
electrodonating and electrowithdrawing groups. These
modifications expand diversity of the alkane-linked
pentamidine analogues library, and add new data to the
previously reported results.17 In summary, we built a library of
21 unique compounds for a comprehensive structure-activity
relationship study and comparison with the previously
published results.
O
N
N
7
N
CH3
O
O
5
5'
2'
O
(i)
N
4
4'
7'
6
1
6'
1'
Y
N
N
+
O
O
Cl
Cl
3
3'
HN
X
N
X
Y
N
X
2
(iii)
8
bis(5-methyl-1,2,4-oxadiazoles) 6a-10a
8'
6: X = H, Y = CH2OCH2
8'
7: X = H, Y = CH2SCH2
8
(ii)
NH2
NH2
7'
8
8'
8: X = CH3, Y = CH2CH2
Cl
H2
5
5'
2'
Cl
4
4'
3'
6
1
6'
1'
7
N
O
O
NH2
8
9
8'
3
9: X = CH3, Y = CH2CH2CH2
N
X
Y
N
X
2
8
9
9'
10: X = CH3, Y = CH2CH2CH2CH2
8'
Scheme 2 Synthetic route to diamide-linked bisamidines 6–10 with atom numbering
of the newly synthesized compounds. Reagents and conditions:
(i): N-methylmorpholine, CH2Cl2; (ii): 1) H2, 10% Pd/C, AcOH, MeOH, 2) HCl/MeOH;
(iii) 4-(5-methyl-1,2,4-oxadiazol-3-yl)aniline was treated with: 1) paraformaldehyde,
MeOH, 2) NaBH4, MeOH; to give appropriate N-methyl derivative.
Synthesis of compounds
1 and 1a have been published by our
research group together with their structural analysis.22
Intermediate bis(5-methyl-1,2,4-oxadiazoles) 6a–10a were
obtained by substitution of appropriate dicarboxylic acid
Results and discussion
chlorides
with
4-(5-methyl-1,2,4-oxadiazol-3-yl)anilines.
Chemistry
N-Methyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)aniline was obtained
by reductive amination of paraformaldehyde with
4-(5-methyl-1,2,4-oxadiazol-3-yl)aniline24 (see Scheme 2).
Bisnitriles 11a–21a were prepared following well established
The new bisamidines were obtained following known methods
by the modified Pinner reaction17,22 of appropriate bisnitriles
(
1a, 11a–21a) in case of compounds 1–5 (Scheme 1) and
compounds 11–21 (Scheme 3) or by catalytic reduction23 of
bis(5-methyl-1,2,4-oxadiazoles) 6a–10a in case of acid-labile
diamide-linked derivatives 6–10 (Scheme 2). We report a
successful preparation and deprotection strategy for
compounds with two 5-methyl-1,2,4-oxadiazole moieties. This
strategy seems to be a serious alternative to the Pinner
reaction, used widely in the preparation of pentamidine
analogues, especially in case of compounds with sensitive
functional groups.
methods,
including
SNAr
reactions
of
various
4-chlorobenzonitriles (when X = S or NH), or SN2 reactions of
several 4-hydroxybenzonitriles (when X = O) (Scheme 3).25–33
7
H
C2H5
CN
4
CHO
NH2OHxHCl
CN
N
N
Cl
Cl
HO
OH
5
6
3
KNO3
Ph SO2Cl
SOCl2
2
NO2
1
Y
Cl
X1
X1
Cl
Cl
11aS1
19aS1: X1 = Cl, Y= NSO2Ph 21aS1: X1 = Cl, Y= NC2H5
15aS1
( iii )
CN
7
NC
7'
CN
5
5'
7
NC
8'
4
R2
X
R2
X
6
6'
1'
4'
3'
10
9'
6'
5
2
K2CO3 / T
NMP
H
CN
Y
4
+
9
6
1
5'
4'
(i)
N
X1
X1
N
8
8'
1
1'
2'
3
Y
+
R1
R2
7'
CN
3
N
N
2
2'
R1
10'
9
9'
H
BrCH2
X2
X2 = OH, Cl
8
3'
R1
:
X1 = Cl, SH, NH2
bisnitriles 11a-21a
bisnitrile 1a
11 11'
NH CH3
NH2
(ii)
7
7
11: R1= H, R2= H, X= S, Y= O
12: R1= H, R2= H, X= S, Y= O
( i ) ( ii )
2: Am =
1: Am =
3: Am =
Cl
Cl
7'
7'
NH CH3
12 12'
NH2
NH2
H
N
8'
10
6'
13: R1= NO2, R2= H, X= O, Y= O
14: R1= NH3Cl, R2= H, X= O, Y= O
15: R1= NO2, R2= H, X= NH, Y=O
16: R1= NH3Cl, R2= H, X= NH, Y=O
17: R1= CH3, R2= CH3, X= O, Y= CH2
18: R1= CH3, R2= CH3, X= O, Y= O
19: R1= CH3, R2= CH3, X=O, Y= NSO2
20: R1= Br, R2= OCH3, X= O, Y= CH2
5
Am
5'
4
5
9
6
5'
4'
Am
4
R1
R1
X
Am
6
4'
3'
6'
1'
Cl
7
1'
2'
1
11
3
7'
12 13
N
8'
8
1
10'
3
Y
Am
NH CH2CH2CH3
2
9'
8
3'
X
2'
R2
H
2
9
9'
11'
13'
12'
Cl
11 12
2'' 3''
NH2
NH2
R2
1''
7
Cl
x 2Cl
7
4''
H
4: Am =
5: Am =
13
11
NH CH2CH2CH2CH2CH3
11' 12' 13'
7'
14
12 13 14 15
7'
10,10'
NH2
NH2
N
6'' 5''
NH CH2CH2CH2CH
3
7
7'
21: R1= H, R2= H, X= O, Y= NC2H5 xHCl
12 Am =
11, 13-21 Am =
Cl
Cl
11' 12'
14'
15'
13' 14'
NH 11,11'
Scheme 3 Synthetic route to bisamidines 11–21 with atom numbering of the newly
synthesized compounds. Reagents and conditions: (i) HCl/EtOH; (ii) NH3/EtOH,
or NH2CH2CH2NH2/EtOH in case of 12; (iii) 1,5-bis(4-cyanophenoxy)-3-oxapentane33 was
treated with HNO3/AC2O/TFA to give 13a; NO2 groups in 13a and 15a were reduced
with H2/Pd to obtain 14a and 16a.
Scheme 1 Synthetic route to 1,4-bis(methylene)piperazine-linked bisamidines 1–5
with atom numbering of the newly synthesized compounds. Reagents and conditions:
(i) K2CO3, DMF; (ii) 1) HCl/EtOH, 2) RNH2/EtOH, 3) HCl/EtOH.
2 | J. Name., 2012, 00, 1-3
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ARTICLE
Anti-Pneumocystis activity
(15 and 16). Similar observations have been made
previously.17,38 Compounds 13 and 14, with their activity at
nanogram concentration, should be considered as next two
leading structures. With the exception of bisamidine 19
mentioned above, introduction of four methyl substituents at
ortho positions (never studied before) produced compounds:
17 and 18. Methyl substituents at the aromatic rings seems to
be superior to methoxy groups, because all respective
bisamidines with methoxy substituents are less active than
pentamidine.17 When bromine atoms are introduced together
with methoxy groups, as in compound 20, activity increased
significantly in comparison to activity of bisamidines with only
methoxy17 or only bromine38 substituents. Perhaps,
heteroatoms in amino, nitro, methoxy and bromo substituents
in compounds 13, 14, 15 and 20 favourably interact with the
molecular target increasing activities of these compounds, and
only combination of different types of the substituents
improves anti-Pneumocystis activity. Compound 21 with
N-ethyl group in the central part of the linker shows better
activity (IC50 < 0.1 μg/ml, < 0.209 µM) than the analogue with
N-methyl group (IC50 = 0.82 μg/ml, 1.73 µM)17, what means
that increasing length of alkyl group in the middle of linker can
be favourable. Introducing N instead of O atoms have
pronounced consequences. A nitrogen atom in the centre of
Compounds 1–21 were first tested at a single high dose
to screen out ineffective products. With the exception of
compound
2, all studied pentamidine derivatives were
effective at reducing the viability of P. carinii in vitro
at 100 µg/ml screening concentration. In our discovery
pipeline, these compounds would then be selected for in vitro
evaluation using P. carinii organisms and the ATP
bioluminescent assay to identify the 50% inhibitory
concentration (IC50) as a means to determine efficacy (Table 1).
IC50 values of 18 bisamidines were lower than 0.30 µg/ml (0.50
µM) given for pentamidine. Compound
1 which showed “very
marked” activity is related to the piperazine linked derivatives
described before20,21. Inserting methylene groups between
piperazine and benzene rings increased anti-Pneumocystis
activity only in case of compounds without bulky groups
at amidine part: 1 (IC50 = 0.062 μg/ml, 0.109 µM), 3 (IC50 = 1.10
μg/ml, 1.685 µM). However we also observed an increase of
activity when the longest alkyl chains were introduced
(compounds
disubstitued bisamidine was evaluated in this assay
(compound ), but this resulted in total loss of activity. The
4 and 5). For the first time, an acyclic N,N'-
2
loss of activity was likely due to a steric hindrance, where the
two methyl groups which are apart from each other form an
unfavourable trans conformation. This is in opposition to the
cyclic analogue (compound 12) with fixed cis conformation and
high anti-Pneumocystis activity.
the pentamidine bridge introduces
a positive charge
(compounds 1–5 and 21), while at the benzene ring it does not
(compounds 15 and 16). Further studies are necessary to
validate the impact of additional positive charges on anti-
Pneumocystis activity.
The alkanediamide linked bisamidines previously have been
tested18,19, with an IC50 of 0.001 μg/ml (0.003 μM) for
a compound with a propylene linker. Replacement of the
middle methylene group in the connecting propylene chain
Toxicity
Mammalian cell toxicity testing was assessed in 2 cell lines.
The A549 cell line is a human-derived adenocarcinomic
alveolar basal epithelial cell line and the L2 was derived from
rat lung and is an adherent epithelial cell line. For compounds
1–21 the 50% inhibitory concentration (IC50) was determined
and pentamidine isethionate was run as a positive control (see
Table 2). Only compounds 11 (with bridge S atoms), 17 and 18
(with methyl groups), and 20 (with bromine and methoxy
substituents) were found to have mild to moderate toxicity to
both A549 and L2 cells. Compounds 12 (with bridge S atoms)
and 13 (with nitro groups) were found to be mildly toxic to
only one cell line, A549 or L2 respectively. All piperazine and
alkanediamide derivatives were found to be non-toxic. Similar
observations were made in previous studies.17–21 Closing of
both amidine groups into imidazoline ring decreased the
toxicity of compound 11 with heteroatoms O and S in the
linker, and compound 12 became only mildly toxic to A549
cells. Toxicity of 13 with two nitro groups at benzene ring
disappeared without decreasing anti-Pneumocystis activity
after reducing the nitro to amino groups. Introducing
with heteroatoms O and S (compounds
6 and 7) decreased
activities against P. carinii, though significant activities were
retained. This is consistent with data obtained earlier for
alkane-linked bisamidines17. Substitution of amide nitrogen
atoms with methyl groups (giving new compounds 8–10
)
modulated the potency of these compounds. The high activity
at nanogram concentration of 10 was retained only for the
butylene chain (0.006 μg/ml, 0.012 μM). This probably
indicates that additional steric hindrance is not desirable
in case of bisamidines with short alkanediamide linker.
The best results were obtained for compounds 11–21 with
variations of bridge heteroatoms and additional substituents
at phenyl rings. They demonstrated higher potency than that
of the parent compound, pentamidine, apart from compound
19, which had an aromatic substituent at central N atom. All
other structural changes caused improvement of activity.
Introducing two S atoms at the benzene ring and O atom in the
middle of chain (compounds 11 and 12) yielded very active
compounds. Remarkably, closing both amidine groups of 11
into imidazoline rings resulted in the new leading structure 12
,
benzenesulfonyl group in the middle of linker (compound 19
)
with IC50 = 0.005 μg/ml. This is in consent with similar
results.35–38 Addition of two nitro or two amino groups ortho to
the linker in benzene rings is also favourable (compounds
13–16). Among them, compounds with oxygen atoms in the
linker (13 and 14), showed better activity than aza analogues
decreased toxicity of compounds 17 and 18 with four methyl
groups at benzene ring, but had an adverse impact on
anti-P. carinii activity. In general, substituents at the benzene
rings make bisamidines more toxic, with exception of amino
substituents. Tidwell et al. reported the same relationship.38
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Table 1 Anti-Pneumocystis activity (IC50) and activity ranking of compounds 1–21
No.
Chemical formula
IC50 in μg/ml (μM)
Activity ranking
Pentamidine
0.30 (0.50)
Marked
H2N
H2N
NH2
NH2
CH2
CH2
CH2
CH2
CH2
N
N CH2
H
1
2
0.062 (0.109)
Very Marked
H
x 4 Cl
H3C HN
H3C HN
NH CH3
NH CH3
N
N CH2
H
—
—
H
x 4 Cl
H2N
NH2
N
N CH2
H
3
1.10 (1.685)
0.052 (0.083)
0.027 (0.041)
0.066 (0.146)
0.27 (0.568)
0.035 (0.076)
0.15 (0.303)
0.006 (0.012)
Moderate
Very Marked
Very Marked
Very Marked
Marked
CH3CH2CH2 HN
NH CH2CH2CH3
H
x 4 Cl
H2N
NH2
N
N CH2
H
4
CH3CH2CH2CH2 HN
NH CH2CH2CH2CH3
H
x 4 Cl
H2N
NH2
N
N CH2
H
5
CH3CH2CH2CH2CH2 HN
NH CH2CH2CH2CH2CH3
H
x 4 Cl
O
N
O
N
H2N
H2N
CH2
O
CH2
NH2
NH2
6
H
H
x 2 Cl
O
O
H2N
H2N
CH2
S
CH2
NH2
NH2
7
N
N
H
H
x 2 Cl
O
O
H2N
H2N
CH2 CH2
CH3 H3C
NH2
NH2
8
Very Marked
N
N
x 2 Cl
O
N
O
N
H2N
H2N
CH2 CH2 CH2
CH3
NH2
NH2
9
Marked
H3C
x 2 Cl
O
O
H2N
H2N
CH2 (CH2)2 CH2
CH3 H3C
NH2
NH2
10
Highly Active
N
N
x 2 Cl
4 | J. Name., 2012, 00, 1-3
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Table 1 (continued)
No.
Chemical formula
IC50 in μg/ml (μM)
Activity ranking
NH2
NH2
NH2
x 2 Cl
O
H2N
11
12
13
14
15
16a
17
18
19
20
21
< 0.1 (< 0.223)
0.005 (0.010)
0.007 (0.014)
0.006 (0.011)
0.031 (0.062)
< 0.1 (< 0.175)
< 0.1 (< 0.213)
0.084 (0.175)
1.2 (1.519)
Very Marked
S
S
N
N
x 2 Cl
O
HN
NH
Highly Active
Highly Active
Highly Active
Very Marked
Very Marked
Very Marked
Very Marked
Moderate
S
S
NH2
NH2
NH2
NH2
x 2 Cl
O
NO2
O
O2N
O
H2N
H2N
H2N
NH2
NH2
NH2
x 4 Cl
O
NH3
O
H3N
O
NH2
NH2
x 2 Cl
O
NO2
NH
O2N
HN
NH2
NH2
NH2
x 4 Cl
O
NH3
NH
H3N
HN
H2N
NH2
NH2
NH2
x 2 Cl
CH3
O
H3C
O
H2N
CH3
CH3
NH2
NH2
NH2
x 2 Cl
O
CH3
O
H3C
O
H2N
CH3
CH3
NH2
NH2
NH2
Ph
SO2
N
CH3
O
H3C
O
H2N
CH3
CH3
x 2 Cl
NH2
NH2
NH2
OCH3
O
H3CO
O
H2N
0.025 (0.036)
Very Marked
Very Marked
Br
Br
x 2 Cl
NH2
NH2
NH2
x 3 Cl
H2N
C2H5
NH
< 0.1 (< 0.209)
O
O
a Quench effect of 15% at 100 µg/ml.
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Journal Name
Table 2 Toxicity IC50 results in A549 and L2 cell linesa
General procedure for the preparation of bisamidines 1–5.
Procedure is based on the previously published synthesis of 1 15
.
No.
Average IC50 [µg/ml ± SD, (µM)]
Toxicity
19.6 g (0.1 mol) 4-cyanobenzyl bromide, 13.8 g K2CO3 (0.1 mol),
4.3 g piperazine (0.05 mol) and 235 ml DMF were stirred for 1 h
at room temperature (RT) and then for 5 h at 80–90°C. The
reaction mixture was cooled to RT, 700 ml ice-water was added,
and stirring was continued for 0.5 h at 0–5 °C. The white
precipitate was filtered off, washed with cold water and dried
in vacuo. Crystallization from acetone yielded 27.2 g (86%) of
fine colourless crystals of 1,4-bis(4-cyanobenzyl)piperazine (1a).
1.26 g (4 mmol) of 1,4-bis(4-cyanobenzyl)piperazine 1a was
suspended in 40 ml of anhydrous ethanol. To this mixture dry
hydrogen chloride was passed at 0–5 °C until saturation. The
reaction vessel was stoppered tightly and stirred for 2 weeks at
RT. After 1 week, additional re-saturation of HCl at 0–5 °C has
been done. The solvent was evaporated near to dryness under
reduced pressure below 40 °C. To the residue 100 ml of dry
diethyl ether was added with stirring. A white hygroscopic
precipitate of crude bisimidate was formed, quickly filtered off
and stored for 24 h in a vacuum desiccator over sodium
hydroxide granules. It was then added to a solution of an
appropriate aliphatic amine in absolute ethanol at RT. Reagents
were stirred in tightly stoppered flask at RT for 24 h. Volatiles
were evaporated in vacuo almost to dryness. The residue was
suspended in the solution of 1.0 g NaOH in 40 ml of water, and
stirred for 15 min. The formed precipitate was filtered, washed
thoroughly with water and dried under reduced pressure over
NaOH granules. Obtained dry powder was mixed with 10 ml of
anhydrous EtOH, acidified with 5 ml of saturated ethanolic HCl
and refluxed for 0.5 h. After cooling, 30 ml of dry Et2O was
added slowly with stirring. The formed precipitate was filtered
off, washed with 10 ml of dry Et2O and dried in vacuo over
NaOH granules. The crude product was purified by
crystallization or column chromatography. In the case of the
A549
13.9 ± 2.4
(23.5±4.3)
> 100
L2
20.0 ± 7.9
(33.8±13.3)
> 100
Pentamidine
Mild
1
2
None
—
NDb
NDb
3
> 100
> 100
None
None
None
None
None
None
None
None
4
> 100
> 100
5
> 100
> 100
6
> 100
> 100
7
> 100
> 100
8
> 100
> 100
9
> 100
> 100
10
> 100
> 100
22.8 ± 4.6
(50.9±10.3)
15.3 ± 5.1
(30.6±10.2)
> 100
11.8 ± 3.3
(26.4±7.4)
11
12
Mild
Mild
> 100
13
14
15
16
89 ± 19 (176±38)
> 100
Mild
None
None
None
> 100
> 100
> 100
> 100
> 100
15.1 ± 2.5
(32.2±5.3)
36.4 ± 2.3
(75.8±4.8)
> 100
8.9 ± 2.8
(19.0±6.0)
8.0 ± 2.7
(16.7±5.6)
> 100
17
Mild to moderate
18
19
20
21
Mild to moderate
None
Mild
18.0 ± 3.6
(26.3±5.2)
> 100
20.2 ± 5.6
(29.5±8.2)
> 100
None
a IC50 – average of 3 separate assays.
b ND – not done due to lack of efficacy against P. carinii.
synthesis of compound 2, 40 ml of 33% methylamine solution
in absolute ethanol was used in the second step of Pinner
reaction. In other cases (compounds 3–5), 4 eq. of aliphatic
amine dissolved in anhydrous ethanol was used.
Experimental
Representative data for compound
Characterizations of compounds 2–5 are given in ESI.
Bis[4-(N-pentylamidino)benzyl]piperazine tetrahydrochloride ( ).
5 are given below.
Chemistry
5
Materials and methods. All chemicals were purchased from
chemical suppliers as high or highest purity grade and used
without any further purification. Melting points were
determined with an Electrothermal 9001 Digital Melting Point
apparatus. The chemical structure of the synthesized
compounds were confirmed by their spectral data. 1H NMR
and 13C NMR 1D and 2D spectra in solution were recorded with
Varian 300 VNMRS spectrometer and chemical shifts δ (ppm)
in solutions were referenced to TMS. Their purity was verified
by elemental analyses using C, H, N, S Elementar GmbH Vario
EL III apparatus. For thin layer chromatography (TLC)
precoated plates Merck Kieselgel 60 F254 were used.
For column chromatography Merck Silica gel 60,
230–400 mesh ASTM (0.040–0.063 mm) was used.
Following the general procedure, after column chromatography
(CH2Cl2/MeOH/AcOH/H2O – 70/20/5/5), a light beige powder
was obtained with yield 23%; MP = 279.5–280.5 °C (decomp.).
1H NMR (299.87 MHz, D2O) δ in ppm: 0.91–0.96 (t; J = 6.9 Hz; 6H;
H-15, H-15'), 1.37–1.47 (m; 8H; H-13, H-14, H-13', H-14'),
1.74–1.84 (p; J = 6.9 Hz; 4H; H-12, H-12'), 3.48–3.52 (m; 12H; H-9,
H-10, H-11, H-9', H-10', H-11'), 4.46 (br s; 4H; H-8, H-8'), 7.71–7.73
(pd; J = 7.8 Hz; 4H; H-2, H-6, H-2', H-6'), 7.82–7.84 (pd; J = 7.8 Hz;
4H; H-3, H-5, H-3', H-5'). 13C NMR (75.40 MHz, D2O) δ in ppm: 13.1
(C-15, C-15'), 21.6 (C-14, C-14'), 26.4 (C-13, C-13'), 28.2 (C-12,
C-12'), 43.0 (C-11, C-11'), 48.7 (C-9, C-10, C-9', C-10'), 59.6 (C-8,
C-8'), 128.5 (C-3, C-5, C-3', C-5'), 130.7 (C-4, C-4'), 131.8 (C-2, C-6,
C-2', C-6'), 133.5 (C-1, C-1'), 163.5 (C-7, C-7'). 1H NMR
(299.87 MHz, DMSO-d6) δ in ppm: 0.87–0.92 (t; J = 6.9 Hz; 6H;
H-15, H-15'), 1.28–1.39 (m; 8H; H-13, H-14, H-13', H-14'),
1.60–1.69 (p; J = 6.9 Hz; 4H; H-12, H-12'), 3.29 (br s; 8H; H-9,H-10,
6 | J. Name., 2012, 00, 1-3
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H-9', H-10'), 3.38–3.44 (q; J = 6.6 Hz; 4H; H-11, H-11'), 4.29 (br s; H-5, H-3', H-5'), 9.18 (br s; 4H; 4 × NH), 9.41 (br s; 4H; 4 × NH).
4H; H-8, H-8'), 7.83 (br s; 8H; H-2, H-3, H-5, H-6, H-2', H-3', H-5', 13C NMR (75.40 MHz, DMSO-d6) δ in ppm: 24.3 (C-9, C-9'), 33.3 (C-8,
H-6'), 9.16 (br s; 2H; 2 × NH), 9.54 (br s; 2H; 2 × NH–C5H11), 9.89 C-8'), 36.7 (2 × H3C–N), 125.8 (C-4, C-4'), 127.1 (C-2, C-6, C-2', C-6'),
(br s; 2H; 2 × NH). Element. Anal. C30H46N6 × 4HCl × 1.5H2O 128.6 (C-3, C-5, C-3', C-5'), 147.5 (C-1, C-1'), 164.7 (C-7, C-7'), 171.3
(663.59 g/mol) calcd. (%): C = 54.30, H = 7.99, N = 12.67, (2 × C=O). Element. Anal. C22H28N6O2 × 2HCl (481.42 g/mol) calcd. (%):
Cl = 21.42; found (%): C = 54.24, H = 7.79, N = 12.73, Cl = 21.15.
C = 54.89, H = 6.24, N = 17.46, Cl = 14.76; found (%): C = 54.74,
H = 5.96, N = 17.40, Cl = 14.67.
General procedure for the preparation of bisamidines 6–10.
Bis(5-methyl-1,2,4-oxadiazoles) 6a–10a as intermediates were General procedure for the preparation of bisamidines 11–21.
obtained as follows (see Scheme 2). 5 mmol of an appropriate Bisbenzonitriles 11a–21a were obtained with two types of
aliphatic dicarboxylic acid chloride in 20 ml of dichloromethane was reactions: O-alkylation of appropriate 4-hydroxybenzonitriles with
cooled to 0–5 °C. Then it was added dropwise to a stirred, bis(2-chloroethyl) derivates or by nucleophilic aromatic substitution
ice-cooled solution of 4-(5-methyl-1,2,4-oxadiazol-3-yl)aniline of appropriate 4-chlorobenzonitriles and bis(2-mercaptoethyl)ether
or N-methyl derivative (10 mmol) and of N-methylmorpholine or bis(2-aminoethyl)ether. Synthetic procedures were based on
(10 mmol) in 20 ml of CH2Cl2. After addition, the reaction mixture previously published papers17,28–33, but several modifications were
was stirred at RT for 12 h. The solvent was evaporated under implemented. Characterizations of compounds 11a–21a are given
reduced pressure to dryness and the residue was washed in ESI. The appropriate bisbenzonitrile 11a–21a (1–5 mmol) was
consecutively with 1 M NaHCO3, water, 1 M HCl and again with stirred in a sealed flask with a saturated solution of HCl in
water. The rinsed precipitate was dried under reduced pressure anhydrous ethanol (15–25 ml) for 24 h at RT. Dry diethyl ether was
over anhydrous CaCl2 yielding pure bis(5-methyl-1,2,4-oxadiazoles). added until a solid was precipitated or solvent was evaporated in
N-Methyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)aniline was synthesized vacuo. The solid was filtered, washed with diethyl ether and dried
by reductive amination of paraformaldehyde with 4-(5-methyl- under vacuum over anhydrous CaCl2 for 2–6 h. The crude imidate
1,2,4-oxadiazol-3-yl)aniline24 before preparation of intermediates obtained in high yield was added to a saturated solution of NH3 in
8a–10a. Representative data for compound 10a are given below. anhydrous ethanol (15–30 ml). The mixture was stirred at RT for
Characterizations of compounds 6a–10a are given in ESI. 24–48 h in a sealed vessel. The solvent was evaporated in vacuo,
N1,N6-Dimethyl-N1,N6-bis[4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl]-
and the solid residue was mixed with 2–4% aq NaOH
hexanediamide (10a). Following the general procedure, a white (10–20 ml). The precipitate of free bisamidines was filtered, washed
powder of pure 10a was obtained. Yield 99%; MP = 143.5–145.5 °C. with water, and dried under reduced pressure over anhydrous
1H NMR (299.87 MHz, DMSO-d6) δ in ppm: 1.39 (br s; 4H; H-9, H-9'), CaCl2. The solid of free bisamidine was suspended in anhydrous ethanol
2.04 (br s; 4H; H-8, H-8'), 2.68 (s; 6H; 2 × H3C–C), 3.18 (s; 6H; (5–10 ml) and saturated solution of HCl in anhydrous ethanol (0.5–1 ml)
2 × H3C–N), 7.44–7.46 (pd; J = 8.4 Hz; 4H; H-2, H-6, H-2', H-6'), was added and boiled for a few min to obtain the appropriate
7.99–8.02 (pd; J = 8.4 Hz; 4H; H-3, H-5, H-3', H-5'). 13C NMR hydrochloride. Representative data for compound 13 are given below.
(75.40 MHz, DMSO-d6) δ in ppm: 12.0 (2 × H3C–C), 24.2 (C-9, C-9'), Characterizations of compounds 11–21 are given in ESI.
33.0 (C-8, C-8'), 36.7 (2 × H3C–N), 124.8 (C-4, C-4'), 127.8 (C-2, C-6, 1,5-Bis(4-amidino-3-nitrophenoxy)-3-oxapentane dihydrochloride
C-2', C-6'), 128.0 (C-3, C-5, C-3', C-5'), 146.5 (C-1, C-1'), 167.0 (C-7, (13). After the general procedure, the solid residue of was filtered
C-7'), 171.3 (2 × C=O), 177.5 (2 × C–CH3). 2.5 mmol of the appropriate off, washed with ethanol and dried. The crude product was
bis(5-methyl-1,2,4-oxadiazole) was dissolved in 25 ml of glacial acetic suspended in acetone (25 ml) and intensively stirred under reflux
acid. In some cases quick heating was needed for complete for 10 minutes, then cooled to RT, filtered off and dried to give a
dissolution. The resultant clear solution was diluted with 100 ml of pale yellow solid of 13; yield 41%; MP. 237.0–238.5 °C (decomp.).
methanol and cooled. Pd/C (10% Pd, 25% w/w) was then added. The 1H NMR (299.87 MHz, DMSO-d6) δ in ppm: 3.88 (t; J = 4.2 Hz; 4H; H-
suspension was vigorously stirred at RT under hydrogen atmosphere 9, H-9'), 4.44 (t; J = 4.2 Hz; 4H; H-8, H-8'), 7.62 (d; J = 4.5 Hz; 2H; H-6,
(4 bar) for 12 h. After completion of the reaction the catalyst was H-6'), 8.16 (dd; J1 = 2.1 Hz, J2 = 8.4 Hz; 2H; H-5, H-5'), 8.45 (d; J = 2.1
filtered through thick filter paper. Filtrate was evaporated nearly to Hz; 2H; H-3, H-3'), 9.31 (br s; 4H; 4 × NH), 9.54 (br s; 4H; 4 × NH). 13
C
dryness and dissolved in 10 ml of methanol. 10 ml of saturated NMR (75.40 MHz, DMSO-d6) δ in ppm: 68.6 (C-9,C-9'), 69.9 (C-8, C-
methanolic HCl was added to this solution. After 1 h of stirring at RT, 8'), 115.6 (C-6,C-6'), 119.6 (C-4, C-4'), 125.5 (C-3, C-3'), 134.2 (C-5, C-
the solvent was evaporated. The residue was triturated with dry 5'), 138.9 (C-2, C-2'), 154.9 (C-1, C-1'), 163.3 (C-7, C-7'). Element.
acetone. The formed suspension was quickly filtered, washed with Anal. C18H20N6O7 × 2HCl (505.31 g/mol) calcd. (%): C = 42.78, H =
dry diethyl ether and dried immediately under reduced pressure over 4.39, N = 16.63; found (%): C = 42.63, H = 4.51, N = 16.72.
NaOH granules yielding pure (TLC) bisamidines as dihydrochlorides.
Anti-Pneumocystis activity
Representative data for compound 10 are given below.
Organism sources. Pneumocystis carinii organisms were
obtained from male CD rats (125–145 g) (Charles River,
Portage, MI). Animals were housed under barrier conditions
at the Cincinnati Veteran’s Affairs Veterinary Medical Unit.
After a week of acclimation, the immune systems of the rats
were suppressed by weekly subcutaneous injections
of methylprednisolone (Pharmacia & Upjohn, Kalamazoo, MI)
Characterizations of compounds 6–10 are given in ESI.
N1,N6-Dimethyl-N1,N6-bis(4-amidinophenyl)hexanediamide
dihydrochloride (10). After the general procedure, a white powder
was obtained with yield 88%; MP = 233.5–235.5 °C (decomp.).
1H NMR (299.87 MHz, DMSO-d6) δ in ppm: 1.40 (br s; 4H; H-9, H-9'),
2.15 (br s; 4H; H-8, H-8'), 3.21 (s; 6H; 2 × H3C–N), 7.55–7.58 (pd;
J = 8.6 Hz; 4H; H-2, H-6, H-2', H-6'), 7.87–7.90 (pd; J = 8.6 Hz; 4H; H-3,
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20 mg/kg body weight. After the second injection, rats were a slight decrease in the overall lost in ATP. All other compounds
inoculated with 2 × 107 cryopreserved P. carinii by intranasal did not exhibit a quenching effect.
inoculation. Seven to nine weeks after inoculation, moribund Data analysis. Based on the IC50 values, each agent was
animals were sacrificed by CO2 inhalation. Lungs were classified by using an activity scale of 5 rankings ranging from
aseptically removed, homogenized, strained, and centrifuged “Highly active” (compounds with an IC50 of < 0.010 µg/ml),
at 2,400 × g, 10 min., 4 °C. Red blood cells were lysed using “Very marked” (IC50 values of 0.011 to 0.099 µg/ml), “Marked”
0.85% NH4Cl pH 6.8, 37 °C, 10 min. Cells were washed with PBS (IC50 values from 0.10 to 0.99 µg/ml), “Moderate” (IC50 values
pH 7.0 and prepared for cryopreservation in RPMI-1640 from 1.0 to 9.99 µg/ml), “Slight” (IC50 values from 10.0 to 49.9
(Invitrogen-Gibco, Grand Island, NY) with 10% Fetal Bovine µg/ml), and “None” (i.e., inactive; IC50 values of ≥ 50 µg/ml).8
Serum (Atlanta Biologicals, Lawrenceville, GA) and 7.5% A549 and L2 toxicity assay. A549 and L2 cell lines were purchased
dimethylsulfoxide (Sigma-Aldrich, St. Louis, MO) and stored from American Type Culture Collection, Manassas, Virginia, USA.
in liquid nitrogen. Nuclei were enumerated by microscopic The A549 cells were provided with DMEM media and L2
analysis of HEMA-3 (Fisher Scientific, Kalamazoo, MI) stained cells with F12 media for mammalian cell toxicity testing.
slides. Isolates were screened for bacterial contamination Both were supplemented with 10% fetal calf sera, 1% MEM
using Luria-Bertani agar, Columbia Blood Agar (Fisher vitamins, and 1% NEAA. Cultured cells were plated at 2 ×
Scientific, Cincinnati, OH), and MacConkey II plates 105/ml and grown to confluent monolayers. Media was
(Becton-Dickinson, Sparks, MD).
removed and replaced with fresh media containing controls
ATP assay. Cryopreserved P. carinii was thawed and suspended and test compound dilutions. First, all compounds were
in RPMI-1640 media containing 20% donor horse serum tested at a single high dose, 100 µg/ml. Assays of 3 time
(Atlanta Biologicals, Lawrenceville, GA), 1% Non-essential points (24, 48, 72 hours) with duplicate wells were tested
amino acids solution (NEAA), 1% MEM vitamin solution, for viability. Media was aspirated from the wells, adherent
2% penicillin/streptomycin (Invitrogen-Gibco, Grand Island, cells were lysed with 0.1 M NaOH, and a portion of the
NY) to 1 × 108 nuclei/ml. 0.25 ml of the cell suspension was lysate was assayed for ATP using the Perkin Elmer ATP-liteM
distributed into 48-well tissue culture plates (Corning, Inc. luciferin-luciferase assay as described above. Background
Corning, NY). Experimental and control compounds ampicillin luminescence was subtracted and replicate well readings of were
and pentamidine isethionate (Sigma-Aldrich, St. Louis, MO) averaged. For each day’s readings, % reduction in ATP for all
were suspended in the RPMI 1640 media at 2 × concentration groups
was
calculated:
media control 100.
and added to the P. carinii at 0.25 ml/well. Plates were media control
–
experimental
/
×
incubated at 36 °C, 100% humidity, 5% CO2 for 24, 48, and The compounds that were found to be toxic in the initial
72 h. At each time point, plates were agitated and 10% volume screen (that reduced the ATP content by > 50% after 72 h)
transferred to 96-well opaque white plates (USA Scientific, were tested at 100, 10, 1, and 0.1 µg/ml and the 50%
Ocala, FL). ATP content of samples was determined using inhibitory concentration (IC50) was calculated by linear
ATP-lite-M (Perkin-Elmer Inc., Waltham, MA) and evolved light regression (InSTAT and GraphPad Prism v.6 software). Based
was measured with a PolarStar Optima luminometer (BMG on the scale using 72-hour IC50 values, mammalian cell toxicity
Labtechnologies, Durham, NC). For each group, background of tested compounds was classified as non-toxic (compounds
luminescence was subtracted and triplicate well readings of with IC50s > 100µg/ml), as mildly toxic (compounds with IC50
duplicate assays were averaged. For each day’s readings, values of 10.11 to 99.9 µg/ml), moderately toxic (compounds
%
reduction in ATP for all groups was calculated: with IC50 values from 1.1–10.0 µg/ml), highly toxic (compounds
media control – experimental / media control × 100. with IC50 values < 1µg/ml).
50% inhibitory concentration (IC50) was calculated using
INSTAT linear regression program. Data are shown after 3 days
of exposure to the test compounds. Prior to testing, all
Conclusions
compounds, except compounds 12
solubilized in sterile deionized water for a 20 mg/ml stock
solution. Compounds 12 15, and 16 were solubilized in sterile
, 15, and 16, were
A
detailed SAR study with 21 pentamidine analogues was
successfully undertaken. High potency against Pneumocystis carinii
was identified for most of the analogues using an assay to measure
the effects on viability (ATP levels) after exposure to the
compounds. The highest activities were found for each group of
compounds as follows: among N,N'-dimethylenepiperazine
analogues of pentamidine, compound 5, with pentyl substituents at
amidine groups (IC50=0.027 μg/ml, 0.041 μM); from the series with
,
DMSO. Dilutions of 100 µg/ml were made for each compound
in RPMI-1640 containing 20% calf serum, 1% MEM vitamin
solution, 1% MEM non-essential amino acids, 2,000 units/ml
penicillin-streptomycin, and 50 µg/ml vancomycin. Negative
controls were media alone, DMSO vehicle control, and
10 µg/ml ampicillin. Positive control was 1 µg/ml pentamidine
alkanediamide bridges, compound 10 with
(IC50= 0.006 μg/ml, 0.012 μM); and from the series with variation of
a butyl chain
isethionate. Compounds 1–21 were tested for luciferin/luciferase heteroatoms, compound 12 with S atoms (IC50= 0.005 μg/ml,
0.010 μM). The majority of the compounds can be classified as
non-toxic or much less toxic than the parent compound
reaction interference at 100 µg/ml concentration. Compound 16
resulted in a 15% quench effect, which may have accounted for
8 | J. Name., 2012, 00, 1-3
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21 M. T. Cushion, P. D. Walzer, A. Ashbaugh, S. Rebholz,
R. Brubaker, J. J. Vanden Eynde, A. Mayence, T. L. Huang,
Antimicrob. Agents Chemother., 2006, 50, 2337-2343.
22 M. Rezler, T. Żołek, I. Wolska, D. Maciejewska, Acta
Crystallogr. B, 2014, 70, 820-827.
23 M. Vogler, U. Koert, K. Harms, D. Dorsch, J. Gleitz,
P. Raddatz, Synthesis, 2004, 8, 1211-1228.
24 S. Chandrappa, H. Chandru, A. C. Sharada, K. Vinaya, C. S. Ananda
Kumar, N. R. Thimmegowda, P. Nagegowda, M. Karuna Kumar, K.
Rangappa, Med. Chem. Res., 2010, 19, 236-249.
pentamidine, apart from compounds 17 and 18 with four methyl
substituents at the ortho positions in benzene rings, which had mild
to moderate toxicity as evidenced by the IC50 values between
8.0 and 36.4 µg/ml (16.7 – 75.8 µM).
Presence of amide groups or sulfur atoms in the linker, imidazoline
rings instead the amidine groups, and amino groups at the benzene
rings are factors responsible for high activity of tested bisamidines.
Four new leading structures (10 and 12–14) were identified. Their
structural properties should be taken into account in further lead
optimization.
25 J. N. Ashley, H. J. Barber, A. J.; Ewins, G. Newbery, A. D. Self,
J. Chem. Soc., 1942, 103-116.
26 S. S. Berg, G. Newbery, J. Chem. Soc., 1949, 642-648.
27 I. Francesconi, W. D. Wilson, F. A. Tanious, J. E. Hall, B. C. Bender,
Conflicts of interest
R. R. Tidwell, D. McCurdy, D. W. Boykin, J. Med. Chem., 1999, 42
2260-2265.
,
The authors declare no competing interests.
28 J. Żabiński, I. Wolska, D. Maciejewska, J. Mol. Stuct., 2007,
833, 74-81.
29 T. A. van Es, J. Chem. Soc., 1965, 1564.
30 F. Jung, C. Delvare, D. Boucherot, A. Hamon, J. Med. Chem.,
1991, 34, 1110-1116.
31 J. Żabiński, D. Maciejewska, P. Kaźmierczak, J. Mol. Stuct.,
2009, 923, 132-140.
32 J.Y. Lee, Y.H. Lee, Y.G. Byun,J. Chromat.A, 2012,1227, 163-173.
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