Alkanediamide-linked bisbenzamidines are promising antiparasitic agents

Article abstract of DOI:10.3390/ph9020020

A series of 15 alkanediamide-linked bisbenzamidines and related analogs was synthesized and tested in vitro against two Trypanosoma brucei (T.b.) subspecies: T.b. brucei and T.b. rhodesiense, Trypanosoma cruzi, Leishmania donovani and two Plasmodium falci

Full text of DOI:10.3390/ph9020020

pharmaceuticals
Article
Alkanediamide-Linked Bisbenzamidines Are
Promising Antiparasitic Agents
Jean J. Vanden Eynde 1, Annie Mayence 1, Madhusoodanan Mottamal 2,3, Cyrus J. Bacchi 4,
Nigel Yarlett 5, Marcel Kaiser 6, Reto Brun 6 and Tien L. Huang 1,2,
*
1
College of Pharmacy, Xavier University of Louisiana, New Orleans, LA 70125, USA;
jean-jacques.vandeneynde@ex.umons.ac.be (J.J.V.E.); annie.mayence@condorcet.be (A.M.)
RCMI Cancer Research Center, Xavier University of Louisiana, New Orleans, LA 70125, USA;
mmottama@xula.edu
Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA
Haskins Laboratories and Department of Biological and Health Sciences, Pace University, 1 Pace Plaza,
New York, NY 10038, USA; cbacchi@pace.edu
Haskins Laboratories and Department of Chemistry and Physical Sciences, Pace University, 1 Pace Plaza,
New York, NY 10038, USA; nyarlett@pace.edu
Swiss Tropical and Public Health Institute, Socinstrasse 57, Basel CH-4002, Switzerland;
marcel.kaiser@unibas.ch (M.K.); reto.brun@unibas.ch (R.B.)
2
3
4
5
6
*
Correspondence: thuang@xula.edu; Tel.: +1-504-520-7603; Fax: +1-504-520-7954
Academic Editor: Suzanne Peyrottes
Received: 11 February 2016; Accepted: 13 April 2016; Published: 19 April 2016
Abstract: A series of 15 alkanediamide-linked bisbenzamidines and related analogs was
synthesized and tested in vitro against two Trypanosoma brucei (T.b.) subspecies: T.b. brucei
and T.b. rhodesiense, Trypanosoma cruzi, Leishmania donovani and two Plasmodium falciparum subspecies:
a chloroquine-sensitive strain (NF54) and a chloroquine-resistant strain (K1). The in vitro cytotoxicity
was determined against rat myoblast cells (L6). Seven compounds (5, 6, 10, 11, 12, 14, 15) showed
high potency against both strains of T. brucei and P. falciparum with the inhibitory concentrations for
50% (IC50) in the nanomolar range (IC50 = 1–96 nM). None of the tested derivatives was significantly
active against T. cruzi or L. donovani. Three of the more potent compounds (
in vivo in mice infected with the drug-sensitive (Lab 110 EATRO and KETRI 2002) or drug-resistant
(KETRI 2538 and KETRI 1992) clinical isolates of T. brucei. Compounds and were highly effective
5, 6, 11) were evaluated
5
6
in curing mice infected with the drug-sensitive strains, including a drug-resistant strain KETRI 2538,
but were ineffective against KETRI 1992. Thermal melting of DNA and molecular modeling studies
indicate AT-rich DNA sequences as possible binding sites for these compounds. Several of the tested
compounds are suitable leads for the development of improved antiparasitic agents.
Keywords: antiparasitics; bisbenzamidines; DNA binding; Plasmodium falciparum; Trypanosoma brucei
1. Introduction
We previously reported on the promising anti-Pneumocystis activity of two key
alkanediamide-linked bisbenzamidines when evaluated in an animal model of pneumocystosis [1,2].
Based on the results with the two lead compounds, the synthesis of an expanded series of
alkanediamide-linked bisbenzamidines and the in vitro anti-Pneumocystis and anti-trypanosomal
activities were reported [3]. The in vitro results showed excellent activity against drug-sensitive
and -resistant isolates of Trypanosoma brucei with low cytotoxicity in the A549 human lung
carcinoma cell line. The design strategy of replacing the strong electron-donating ether functions
of the pentyldioxylinker in pentamidine with poor electron-donating amide functions in the lead
compounds (5 and 6, Figure 1) resulted in highly potent antiprotozoal agents with greatly reduced
Pharmaceuticals 2016, 9, 20; doi:10.3390/ph9020020
www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2016, 9, 20
2 of 11
cytotoxicity. In this study, we wish to report on the antiparasitic activity of the expanded series
of alkanediamide-linked bisbenzamidines against several other parasitic protozoa such as T. cruzi,
L. donovani, P. falciparum as well as their in vivo efficacy in several animal models of trypanosomiasis
infected with drug-sensitive (Lab 110 EATRO and KETRI 2002) or -resistant (KETRI 2538 and 1992)
clinical isolates [4] of T.b. rhodesiense.
Figure 1. Chemical structures of pentamidine and lead compounds.
There is a great need for new antiparasitic agents with improved efficacy and low toxicity. Current
chemotherapy is inadequate for many parasitic diseases because of limited efficacy, unacceptable
side effects, and increasing occurrence of resistance. Development of an effective vaccine for these
parasitic diseases have proven ineffective, therefore, chemotherapy with existing drugs is the only
available therapeutic option. Parasitic diseases infect almost one sixth of the world population
(>one billion people), with malaria being the most common. Besides its antimicrobial properties,
the bisbenzamidines class of agents, represented by pentamidine, has demonstrated a wide range
of biological properties [5] including anticancer, antidiabetic, antiviral and antiparasitic properties.
However, pentamidine is clinically used only in the treatment of Pneumocystis jirovecii pneumonia,
early stage human African trypanosomiasis and antimony-resistant leishmaniasis. The development
of a novel prodrug from the bisbenzamidine class, parafuramidine maleate (DB289) against HAT
and Pneumocystis pneumonia, was halted in a phase III sleeping sickness clinical trial because of
nephrotoxicity in healthy volunteers [6]. Despite this setback, there is considerable interest in
developing novel and safer bisbenzamidines [7] and several of these compounds are in various
stages of drug development [8].
2. Results and Discussion
2.1. Chemistry
The syntheses of the targeted compounds are illustrated in Scheme 1. It is to be noted that
the complete synthetic and structural characterization have been described for compounds and
in the literature [ ] but not for the other compounds reported in this study. The appropriate
diacid chlorides were reacted with aminobenzamidine or aminobenzonitrile or aminobenzamide
to obtain the bisbenzamidines ( 10 15), bisbenzonitrile ( ) and bisbenzamides ( ) in fair
yields. The bisbenzamidoxime is readily obtained by reacting with hydroxylamine. Compound
3–6
8
1,3,9
1,
2,
5
–
7,
–
3
8, 9
4
3
4
is specifically designed to function as a prodrug with improved oral bioavailability by masking the
highly basic amidine functions with the less basic amidoxime moieties. We recently demonstrated that
the amidoxime moiety in
4 is readily metabolized to the amidine function following incubation in vitro
with mouse liver microsomes [9].

Pharmaceuticals 2016, 9, 20
3 of 11
Scheme 1. General procedures for the synthesis of compounds 1–15. Reagents and conditions: (a) DMF,
pyridine, reflux, 30 min–2 h; (b) and (c): Dioxane, room temp., stirred overnight; (d) Hydroxylamine,
DMSO, 70 ˝C, 17 h.
2.2. Pharmacology
The structure-activity relationship of 15 alkanediamide-linked bisbenzamidines (including
analogs) and their antiparasitic activity were studied. Several of the bisbenzamidines were
characterized with high potency against the two strains of T. brucei and P. falciparum (Table 1).
Bisbenzamidines linked with the diamide moiety of the type Bza–NHCO–(CH2)n–CONH–Bza where
Bza = benzamidine, n = 3, 4, 5, 6, 8, 10 showed high potency against both strains of T. brucei and
P. falciparum in the low nanomolar range (IC50 = 1–96 nM) irrespective of the length of the methylene
chain. Compound 1 was also highly active (IC50 = 3–8 nM) against both strains of P. falciparum, but
was inactive against the other parasitic protozoa. None of the tested derivatives was active against
T. cruzi or L. donovani. The tested compounds exhibited low cytotoxicity against the non-carcinoma L6
rat skeletal myoblasts cell line.
The selectivity index of these compounds for T. brucei (SIT.b) or P. falciparum (SIP.f) are defined as
the ratio of the L6 cells IC50 to the T. brucei or P. falciparum IC50 values. Based on this definition, the
most selective compounds in this series against T. brucei was compound
6 with SI > 50,000, whereas
compounds and were the most selective against P. falciparum with SI > 33,000. The selectivity
1
5
indices for the reference compounds, pentamidine or chloroquine, could not be calculated since they
were not tested in the L6 cells. The remarkable low cytotoxicity and high selectivity of some of the
compounds, especially
the lung A549 [ ] lung NCI-H460, CNS SF-268 and breast MCF7 carcinoma cell lines [10]. In vivo
evaluation in the immunosuppressed mouse model of Pneumocystosis demonstrated that compound
5 and 6 when compared with pentamidine, were demonstrated earlier with
3
5
was highly efficacious against the infection at 20 mg/kg and 40 mg/kg, with >1000-fold reductions
in burden, and resulted in improved survival curves versus those of pentamidine treated mice at the
same doses [2]. The replacement of the dioxypentyl linker in pentamidine with the diamidealkyl linker
resulted in compounds with reduced toxicity. This might be attributed to the lower lipophilicity (see
Table 2) and different pathways of metabolism of these compounds since it has been suggested that
the phenolic linkage in pentamidine might be involved in the toxicity of the compound [11]. Further
characterization of the metabolites of our compounds would be needed to confirm this hypothesis.

Pharmaceuticals 2016, 9, 20
4 of 11
Table 1. In vitro antiparasitic and cytotoxic properties of alkanediamide-linked bisbenzamidines and analogs.
IC50 (µM) a
Compd.
n
T.b. rhod c
T. cruzi
L. donovani
P. falcipK1 e
L6 Cells
T.b. brucei b
P. falcipNF54 d
1
2
3
4
5
6
7
8
9
1
2
3
3
3
4
4
4
4
4
5
6
7
8
10
p-Am
p-Am
p-CN
p-Am
p-Am
p-CN
ND
9.0
6.50
ND
2.19
7.90
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
99.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
67.4
0.008
ND
ND
0.003
0.29
44.8
>100
28.0
>100
>100
69.0
>100
>100
>100
46.4
>100
43.0
p-C(=NOH)NH2
p-Am
p-C(=NOH)NH2
p-Am
7.30
10.0
ND
3.16
0.009
0.003
0.041
2.80
0.096
0.002
0.021
1.10
0.002
0.004
ND
ND
ND
0.015
0.002
0.002
0.002
0.014
0.032
0.006 i
0.002
0.018
0.38
p-Am
m-Am
p-CONH2
m-CONH2
p-Am
p-Am
p-Am
p-Am
p-Am
p-Am
m-Am
p-CONH2
m-CONH2
m-Am
p-Am
p-Am
p-Am
p-Am
>100
>100
0.004
0.002
0.006
0.008
0.012
0.015
0.18 i
NT
1.97
10
11
12
13
14
15
Ref
a
0.012
0.002
0.003
0.400
0.002
0.008
0.002 f
0.007
0.002
0.001
0.240
0.004
0.007
0.002 f
38.4
49.2
78.6
76.4
68.5
p-Am
p-Am
80.9
10.7
80.2
1.13 g
0.44 h
0.010 j
b,c
Each value is the average of at least two determinations.
The Trypanosoma brucei brucei strain was Lab 110 and the Trypanosoma brucei rhodesiense strain was KETRI 243. Data for
both strains are obtained from [
3
]; d,e The Plasmodium falciparum strains NF 54 and K1 are chloroquine-sensitive and -resistant strains respectively; f pentamidine; g benznidazole;
miltefosine; i chloroquine; j podophyllotoxin. ND denotes not done.
h

Pharmaceuticals 2016, 9, 20
5 of 11
Table 2. Calculated physicochemical properties, Lipinski rule of 5 and lead likeness.
a
b
d
H-don
Count
Lipinki
Rule of 5
Lead
Likeness
H-acc
Rot
c
e
Compd. MolWt
LogP
Ring #
LogD
PSA
Count
Bonds
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16 f
338.37
352.40
332.36
398.42
366.42
380.45
380.45
382.42
382.42
380.45
394.48
408.50
422.53
436.56
466.61
340.42
0.38
0.31
2.63
0.75
0.76
1.20
1.20
1.06
1.06
1.20
1.64
2.09
2.53
2.98
3.87
2.32
6
6
2
6
6
6
6
4
4
6
6
6
6
6
6
4
6
6
4
8
6
6
6
6
4
6
6
6
6
6
6
6
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
´4.44
´4.5
6
7
6
8
8
9
9
9
9
157.94
157.94
105.78
175.42
157.94
157.94
157.94
144.38
144.38
157.94
157.94
157.94
157.94
157.94
157.94
118.20
3 of 4
3 of 4
4 of 4
3 of 4
3 of 4
3 of 4
3 of 4
4 of 4
4 of 4
3 of 4
3 of 4
3 of 4
3 of 4
3 of 4
3 of 4
4 of 4
4 of 6
4 of 6
6 of 6
5 of 6
4 of 6
5 of 6
5 of 6
6 of 6
6 of 6
5 of 6
5 of 6
4 of 6
4 of 6
4 of 6
4 of 6
6 of 6
2.63
0.74
´4.06
´3.61
´3.6
1.06
1.06
´3.61
´3.17
´2.72
´2.28
´1.84
´0.95
´2.50
9
10
11
12
13
15
10
All the values reported in this Table were obtained using the Marvin Suite of ChemAxon [12] and the pKa
values were obtained using the Schrodinger Suite [13]. Lipinski Rule of 5 (MolWt
H-donor atoms
ď
500, LogP
ď
5, a Number of
ď
5, b Number of H-acceptor atoms
ď
10). Lead Likeness (MolWt
ď
450, LogD (on pH 7.4) ě ´
4
and ď 4, Number of H-donor atoms ď 5, Number of H-acceptor atoms ď 8, c Number of rings ď 4, d Number
of rotatable bonds ď 10,). Polar Surface Area, f Pentamidine.
e
Because of the promising in vitro activities, several compounds (5, 6, 11) were selected for in vivo
efficacy studies using mice infected with drug-susceptible (Lab 110 EATRO and KETRI 2002) and
drug-resistant (KETRI 2538 and 1992) strains of T. brucei (Table 3). The compounds were administered
via the intraperitoneal route at doses ranging from 1.0 mg/kg/day to 25.0 mg/kg/day for 3 days
unless otherwise noted. Pentamidine was used as the reference compound. All three of the tested
compounds were effective in curing mice infected with the Lab 110 EATRO or KETRI 2002 strains at
several doses. Compound 11 was the most effective against these two strains since it also cured the
mice at lower doses (1.0 or 2.5 mg/kg). Two of the tested compounds, namely 5 and 6, were highly
efficacious in curing mice infected with the drug-resistant KETRI 2538 clinical isolate at several doses.
However, the bisbenzamidines including pentamidine were ineffective in curing mice infected with
the drug-resistant KETRI 1992 isolate, although the tested compound
days of the treated animals to 25 days at the 25 mg/kg dose.
5
prolonged the mean survival
The in vivo results reported here as well as the earlier results with pneumocystosis [
2] indicate
that several of the bisbenzamidines reported here are suitable leads for further optimization to
develop orally active prodrugs. These compounds generally differ from pentamidine in having
lower lipophilicity, greater H-bond donor capability and polar surface area (Table 2). The bisamidine
moieties have similar calculated basicity (pKa~10.68) when compared to pentamidine (pKa~10.94).
Several of them have “lead-like” characteristics based on physicochemical properties. Therefore,
modifying the leads into prodrugs with greater oral bioavailability would be a rational approach to
develop more effective antiparasitic agents. In fact, we have already reported our initial attempts in
this approach [9] by selecting compound 5 as a lead in which the amidine functions were masked
with less basic and more lipophilic moieties that can readily be biotransformed into the active amidine
functions. Further in vitro metabolism study of these potential prodrugs is in progress.

Pharmaceuticals 2016, 9, 20
6 of 11
Table 3. In vivo trypanocidal activity of selected compounds in mice a.
Dosage
(mg/kg/Day)
Mean Survival
(Days)
No. of Mice
Cured/Total (%)
Clinical Isolate
Compd.
None
-
5.0
0/3
Pentamidine
1.0, 2.5, 5, 10
>30
5/5 (100) c
1.0
2.5
5
10
15
6.0
11.3
10
>30
>30
0/3
0/3
2/3 (66)
3/3 (100)
3/3 (100)
5
6
Lab 110 EATRO b
1.0
2.5
5
10
15
6.7
10.0
>30
>30
>30
0/3
0/3
3/3 (100)
3/3 (100)
3/3 (100)
11
1.0, 2.5, 5, 10
-
>30
9.0
3/3 (100) c
None
0/3
Pentamidine
1.0, 5, 10
10, 15, 25
10, 15, 25
1.0, 2.5, 5, 10
-
>30
>30
>30
>30
4.3
5/5 (100) c
3/3 (100) c
3/3 (100) c
3/3 (100) c
0/3
KETRI 2002 b
KETRI 2538 b
5
6
11
None
Pentamidine
1.0, 5, 10
10, 15, 25
5, 10, 15
-
>30
>30
>30
7.4
5/5 (100) c
3/3 (100) c
3/3 (100) c
0/5
5
6
None
1
5
10
12.6
17.0
22.2
0/3
0/3
0/3
Pentamidine
KETRI 1992 b
10
15
25
17.5
14.5
25
0/3
0/3
0/3
5
a
In vivo efficacy of compounds given via i.p route vs. several clinical isolates of T. brucei. Mice were infected
with 250,000 parasites and dosing commenced 24 h post infection. Mice were separated into groups of three
and injected i.p once a day for 3 days unless otherwise noted. Infected untreated controls were used for each
experiment. Mice were considered cured if surviving more than 30 days beyond death of the last control without
parasites in tail vein blood smears. Mean survival (in days) of animals dying of trypanosomiasis is exclusive of
b
cured animals. Trypanosome strains. T.b. brucei Lab 110 EATRO strain is susceptible to standard trypanocides
including the diamidines. The following are clinical isolates of T.b. rhodesiense: KETRI 243, 2002, 2538 and 1992.
Strain 2002 is susceptible to standard trypanocides including the diamidines. Strains refractory to DFMO are
KETRI 243 and 2538. Strains refractory to arsenical drugs are KETRI 243, 1992 and 2538. Strains refractory to
c
diamidines are KETRI 243 and 1992 (see [
4
] for details). All doses cured. Groups of 3 or 5 animals used for
all doses.
Bisbenzamidines such as pentamidine, propamidine and furamidine have been recognized
as promising antimicrobial agents against bacteria, protozoa, fungi and amoeba [ ]. This class of
compounds has demonstrated excellent activity against Pneumocystis, Trypanosoma spp., Leishmania
spp., and Plasmodium spp. [ 14 15]. These compounds have been proposed to act by initially
binding to DNA at AT-rich sites followed by inhibition of one or more of several DNA dependent
enzymes (e.g., topoisomerases and nucleases) or by direct inhibition of the transcription process [16 17].
7
2,3, ,
,
In kinetoplastid parasites such as African trypanosomes, the bisbenzamidines are postulated to be
taken up by transporters and concentrate in the mitochondrion leading to destruction of the kinetoplast

Pharmaceuticals 2016, 9, 20
7 of 11
DNA [
However, other possible modes of action cannot be ruled out. These include the binding of these
compounds to ferriprotoporphyrin IX and inhibiting the formation of hemozoin [15 19 20], disruption
of polyamine metabolism [21], and interactions with a host of non-conventional biomolecular targets [ ].
7]. In the case of P. falciparum, the nuclear DNA has been implicated as the bioreceptor target [18].
,
,
5
These observations suggest that dicationic diamidines are pleotropic compounds that are capable of
interacting with multiple targets.
In addition to the determination of the potency of the synthesized compounds against different
strains of cell lines (Table 1), thermal melting experiments of these compounds were also conducted
with poly(dA-dT) and calf thymus DNA (Table S1, supplementary file). In general, the
∆Tm values
are higher for poly(dA-dT) and a correlation was obtained between the Tm of poly(dA-dT) and the
∆
pIC50 values of these compounds in T.b. brucei (r = 0.73), T.b. rhodesiense (r = 0.79) (see Figure S1A,B,
supplementary file) and P. falciparum K1 (r = 0.67) (Figure S1C, supplementary file). The weaker binding
to DNA (lower ∆Tm values) compared to pentamidine might be attributed to the lower lipophilicity
and higher PSA properties of these compounds since they would be expected to bind less favorably
in the less polar environment of the minor grove of DNA as was reported for the aza-analogues of
furamidine [11]. Our results suggest that the antiparasitic activities of the benzamidine analogs against
T.b. brucei, T.b. rhodesiense and P. falciparum K1 are associated with their ability to bind to AT rich
oligomers, as reported in the literature [7,20,22]. However, it should be noted that compound 5 and
several bisbenzamidines with various linkers were shown to interfere with hemozoin formation at
micromolar concentrations in ex vivo experiments [15]. Therefore, ability of these compounds to act at
other potential targets cannot be ruled out.
2.3. Molecular Modeling Studies
In order to examine how these compounds bind to the minor groove of AT-rich DNA duplex and
their preferred affinities for specific AT-rich binding sites, we have done docking studies as described
in the supplementary section. Docking scores and correlation coefficients (r and p) between the pIC50
and dockings scores are tabulated in Tables S1 and S2 (supplementary file). Correlation data indicates
that our compounds have high preference for binding to the minor groove of a single G or GC inserted
AT-rich DNA duplexes. In the active compound 15, the terminal amidines displayed three H-bonds and
the linker diamide formed one H-bond with the nucleotides of DNA. The detailed binding interactions
of compounds 11 and 15 with the DNA can be found in the supplementary section (Figure S2). In the
crystal structure of pentamidine in complex with AT-rich DNA, one of the terminal amidines formed
one H-bond with N3 of A5 and a close contact to O41 of A6, and the other terminal amidine formed
one H-bond with N3 of A17, a close contact to O41 of A18 and one water mediated contact to O41 of
G10 [23]. While it may not be a direct comparison, as in the crystal structure our model also exhibited
at least two H-bond interactions between the terminal amidines and the nucleotide. Thus, the observed
hydrogen bond interactions and the appropriate curvature of the compounds to match the convex
shape of the DNA minor groove may be responsible for the higher affinity of active compounds. This
study shows that our compounds better bind to G or GC inserted AT-rich sites. While these models
provide a tool to study the trend in binding of our compounds to different oligomers, for an exact
representation of the drug-DNA interactions X-ray or NMR structures are more desirable.
3. Experimental Section
3.1. Chemistry
1H-NMR spectra were obtained using a Varian Inova instrument (500 MHz) (Agilent Technologies,
Santa Clara, CA, USA), and chemical shifts (δ) are given in ppm using TMS as internal reference and
coupling constants (J) in Hz. IR spectra were recorded on a Perkin-Elmer Spectrum One spectrometer
(PerkinElmer, Waltham, MA, USA) operating in the diffuse reflectance mode. High resolution mass
spectra (HRMS) were recorded on an Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham,

Pharmaceuticals 2016, 9, 20
8 of 11
MA, USA) with electrospray ionization (ESI). Solvents and reagents were commercially available
(Aldrich, St. Louis, MO, USA; Fisher Scientific, Pittsburg, PA, USA) and were used without further
purification. Elemental analysis was carried out by M-H-W laboratories, Phoenix, AZ. Analysis of C,
H, N were within
described in the literature.
˘
0.4% of theoretical values. Compounds 3 [3], 4 [9], 5 [3], 6 [1] and 8 [3] have been
General Synthesis of Bisbenzamidines 1, 2, 7, 10–15
A mixture of 4-aminobenzamidine and/or 3-aminobenzamidine (10 mmol), pyridine (8 mL,
100 mmol), appropriate diacid chloride (5 mmol), in N,N-dimethyl formamide (40 mL) was refluxed
for 30–120 min. The resultant precipitate was filtered and washed with the appropriate solvents.
1
N,N -bis[4-(aminoiminomethyl)phenyl] ethane-1,2-dicarboxamide (
1
). The product was successively washed
with water and acetone to yield 10% solid, m.p. > 300 C; IR:3130, 1674, 1611, 1514, 1412 cm´1; 1H-NMR:
(DMSO-d6) 11.1 (s, br, 2H); 9.2 (s, br, 8H); 8.0 (d, 4H, J = 9 Hz), 7.9 (d, 4H, J = 9 Hz) ppm. Anal. Calcd.
for C16H16N6O2 2HCl 1.5 H2O (424.28): C, 45.29; H, 4.98; N, 19.80. Found: C, 45.55; H, 5.12; N, 19.84.
˝
δ
¨
¨
1
N,N -bis[4-(aminoiminomethyl)phenyl] butane-1,4-dicarboxamide ( ). The product was successively washed
with ethanol and ether to yield 40% solid, m.p. > 300 C; IR:3030, 1669, 1602, 1541, 1415 cm´1
1H-NMR:(DMSO-d6)
10.5 (s, 2H); 9.2 (s, 4H); 8.8 (s, 4H); 7.8 (m, 8H); 2.7 (s, br, 4H) ppm. Anal. Calcd.
for C18H20N6O2¨ 2HCl (424.28): C, 50.83; H, 5.21; N, 19.76. Found: C, 50.77; H, 5.44; N, 19.58.
N,N1-bis[3-(aminoiminomethyl)phenyl] hexane-1,6-dicarboxamide (
). The product was washed twice
with acetone to yield 57% solid, m.p. > 300 ˝C; IR:3054; 1668; 1564; 1482; 1411; 1314 cm´1
1H-NMR:(DMSO-d6)
10.5 (s, 2H); 9.3 (s, 4H); 9.1 (s, 4H); 8.1 (s, 2H); 7.8 (d, 2H); 7.5 (t, 2H); 7.3
2
˝
;
δ
7
;
δ
(d, 2H); 2.4 (t br, 4H); 1.7 (t br, 4H) ppm. Anal. Calcd. for C20H24N6O2
N, 18.54. Found: C, 52.74; H, 5.99; N, 18.45.
¨
2HCl (453.37): C, 52.98; H, 5.78;
N-[3-(aminoiminomethyl)phenyl] N1-[4-(aminoimino methyl)phenyl] hexane-1,6-dicarboxamide (10). Oily
liquid, % yield unknown. IR:3086; 1667; 1601; 1481; 1315; 1264 cm´1; 1H-NMR:(DMSO-d6)
10.7 (d,
δ
1H); 10.5 (d, 1H); 9.4 (s, 2H); 9.2 (s, 2H); 9.1 (s, 4H); 9.0 (s, 4H); 8.2 (s, 1H); 7.8 (m, 5H); 7.5 (t, 1H); 7.4 (d,
1H); 2.4 (m br, 4H); 1.6 (m br, 4H) ppm. HRMS: found 381.2057, calcd. 381.2039 for C20H24N6O2.
N,N1-bis[4-(aminoiminomethyl)phenyl] heptane-1,7-dicarboxami˝de (11). The product was successively
´1
washed with ethanol and ether to yield 45% solid, m.p. > 300 C; IR:3117, 1675, 1616, 1541, 1496 cm
;
1H-NMR:(DMSO-d6)
δ10.5 (s, 2H); 9.2 (s, 4H); 8.9 (s, 4H); 7.8 (m, 8H); 2.3 (t, 4H, J = 7 Hz); 1.6 (q, 4H,
J = 7 Hz); 1.3 (q, 2H, J = 7 Hz) ppm. Anal. Calcd. for C21H30N6O2 2HCl.0.2H2O) (503.42): C, 50.10; H,
¨
6.40; N, 16.69. Found: C, 50.38; H, 6.03; N, 16.51.
N,N1-bis[4-(aminoiminomethyl)phenyl] octane-1,8-dicarboxamide (12).˝ The product was successively
washed with water, ethanol and ether to yield 10% solid, m.p. > 300 C; IR:3063, 1674, 1609, 1541, 1338,
1308 cm´1; 1H-NMR:(DMSO-d6) δ10.5 (s, 2H); 9.2 (s, 4H); 8.9 (s, 4H); 7.8 (m, 8H); 2.3 (t, 4H, J = 7 Hz);
1.6 (m, br, 4H); 1.3 (m, br, 4H) ppm. Anal. Calcd. for C22H28N6O2
17.46. Found: C, 55.12; H, 6.25; N, 17.28.
¨
2HCl) (481.42): C, 54.89; H, 6.28; N,
N,N1-bis[4-(aminoiminomethyl)phenyl] nonane-1,9-dicarboxamide (13). After refluxing for 2 h, the cooled
solution was poured into acetone (100 mL). The product was successively washed with acetone and
water to yield 50% solid, m.p. > 300 ˝C; IR:2935, 1696, 1674, 1541, 1330 cm´1; 1H-NMR:(DMSO-d6)
δ
10.4 (s, 2H); 9.3 (s, br, 8H); 7.8 (m, 8H); 2.3 (t, 4H, J = 7 Hz); 1.6 (q, br, 4H); 1.3 (q, br, 6H, J = 7 Hz) ppm.
Anal. Calcd. for C23H32N6O2
H, 6.61; N, 15.96.
¨
2HCl 0.5C2H5OH) (518.48): C, 55.60; H, 6.80; N, 16.21. Found: C, 55.98;
¨
N,N1-bis[4-(aminoiminomethyl)phenyl] decane-1,10-dicarboxamide (14). After refluxing for 2 h, thecooled
solution was poured into acetone (100 mL˝). The product was successively washed with acetone
and water to yield 50% solid, m.p. > 265 C decomposed; IR:2931, 1667, 1599, 1559, 1487 cm´1
;

Pharmaceuticals 2016, 9, 20
9 of 11
1H-NMR:(DMSO-d6)
δ
10.4 (s, 2H); 9.1 (s, br, 8H); 7.8 (m, 8H); 2.4 (t, 4H, J = 7 Hz); 1.6 (br m, 4H); 1.3
(br m, 8H, J = 7 Hz) ppm. HRMS: found 437.2672, calcd. 437.2665 for C24H32N6O2.
N,N1-bis[4-(aminoiminomethyl)phenyl] dodecane-1,12-dicarboxamide (15). The product was successively
washed with water, ethanol and ether to yield 35% solid, m.p. > 300 ˝C; IR:3093, 1669, 1602, 1541,
1411 cm´1; 1H-NMR:(DMSO-d6)
δ 10.4 (s, 2H); 9.2 (s, 4H); 8.9 (s, 4H); 7.8 (m, 8H); 2.3 (t, 4H, J = 8 Hz);
1.6 (q, br, 4H); 1.3 (m, br, 12H) ppm. HRMS: found 465.2985, calcd. 465.2978 for C26H36N6O2.
3.2. Biology
3.2.1. In Vitro Antiparasitic Activity
The two clinical isolates of T. brucei used for the in vitro study were T. b. brucei Lab 110 EATRO
(pentamidine-sensitive) and T. b. rhodesiense KETRi 243 (melarsoprol, pentamidine- and diminazene
diaceturate-resistant) [14]. The in vitro assays with the two isolates of T. brucei were conducted
according to previously described procedures [14]. The anti-T. cruzi activity was determined using
intracellular amastigote forms of T. cruzi Tulahuen strain C2C4 in rat skeletal myoblasts (L6 cells)
as described previously [24]. The anti-L. donovani activity was determined using the amastigotes of
L. donovani strain MHOM-ET-67/L82 as described previously [24]. The anti-plasmodial activity against
the two strains of P. falciparum NF54 (chloroquine-sensitive) and K1 (chloroquine-resistant) were
determined using the erythrocytic stage of P. falciparum as described previously [24]. The cytotoxicity
assays against rat skeletal myoblast L6-cells were performed as described previously [24].
3.2.2. In Vivo Anti-Trypanosomal Activity in Mice
The in vivo studies were conducted according to the procedures described previously [4,14].
3.2.3. DNA Binding Affinity Measurements
The DNA binding affinity of the tested compounds was determined by measuring the change in
mid-point of the thermal denaturation curves for a 1:5 compound to DNA base pair ratio as described
previously [15]. Each Tm value reported in Table S1 (supplementary file) represents the mean of at
least two experimental determinations.
3.3. Molecular Modeling Studies
We used Surflex-Dock to study the DNA binding affinities of 16 bisbenzamidine compounds
to the minor groove of AT-rich sites and an AT absent site. Computational details are described in
supplementary section.
4. Conclusions
This study reports on the synthesis of a series of alkanediamide-linked bisbenzamidines (and
analogs) and their in vitro and in vivo evaluation as potential antiparasitic agents. Potential cytotoxicity
of the compounds in a mammalian cell line, as well as their interactions with DNA via thermal
denaturation and molecular modeling studies, have been determined. Seven of the compounds (
10 11 12 14 15) were highly potent (IC50 = 1–96 nM) against drug-sensitive as well as drug-resistant
cell lines of T. brucei and P. falciparum. Three compounds ( 11) were highly effective in curing
mice infected with the drug-sensitive strains (Lab 110 EATRO and KETRI 2002) of T. brucei, whereas
and were also efficacious in curing mice infected with the drug-resistant KETRI 2538 clinical
isolate. Thermal denaturation studies with DNA showed that these compounds have stronger binding
to poly(dA-dT) versus calf thymus DNA and a good correlation was observed between the Tm of
5, 6,
,
,
,
,
5, 6,
5
6
∆
poly(dA-dT) and the antiparasitic activity of these compounds against T. brucei and P. falciparum.
Molecular modeling studies indicated that these compounds exhibited high preference for binding to

Pharmaceuticals 2016, 9, 20
10 of 11
the minor groove of a single G or GC inserted AT-rich (AAAGTTT or AAAGCTTT) DNA duplexes
and they correlated well with the pIC50 values of the compounds against T. brucei and P. falciparum.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/9/2/20/s1,
Figure S1: Correlation between the experimental pIC50 in different cell lines and the ∆Tm with poly(dA-dT),
Figure S2: Detailed views from the minor groove of the hydrogen bond interactions between compounds 11 and 15
and the central –AAAGTTT- sites of 5’-d(CCAAAGTTTGC)-3’ duplex, Table S1: Docking of alkanediamide-linked
bisbenzamidines and analogs to various DNA duplexes with specific central sequences, in vitro antiparasitic
properties (pIC50) and thermal melting data (
∆
Tm) of tested analogs with poly(dA-dT) and CT-DNA, Table S2:
Tm of poly(dA-dT) and the
Pearson’s correlation (r) between the experimental pIC50 values of three cell lines or
∆
docking scores for different central minor groove of sequences. Details of molecular modeling studies.
Acknowledgments: The work described in this manuscript has been funded in part by the (NIMHD-NIH) under
grant number 2G12MD007595.
Author Contributions: J.J.V.E. and T.L.H. conceived and designed the study; J.J.V.E. and A.M. synthesized the
compounds; C.B., N.Y., M.K., and R.B. performed the in vitro studies; C.B. and N.Y. performed the in vivo studies;
M.M. performed the molecular modeling studies; J.J.V.E., M.M. and T.L.H. wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Vanden Eynde, J.J.; Mayence, A.; Huang, T.L.; Collins, M.S.; Rebholz, S.; Walzer, P.D.; Cushion, M.T.
Novel bisbenzamidines as potential drug candidates for the treatment of Pneumocystis carinii pneumonia.
Bioorg. Med. Chem. Lett. 2004, 14, 4545–4548. [CrossRef] [PubMed]
2.
Cushion, M.T.; Walzer, P.D.; Ashbaugh, A.; Rebholz, S.; Brubaker, R.; Vanden Eynde, J.J.; Mayence, A.;
Huang, T.L. In vitro selection and in vivo efficacy of piperazine- and alkanediamide-linked bisbenzamidines
against Pneumocystis pneumonia in mice. Antimicrob. Agents Chemother. 2006, 50, 2337–2343. [CrossRef]
[PubMed]
3.
4.
Huang, T.L.; Vanden Eynde, J.J.; Mayence, A.; Collins, M.S.; Cushion, M.T.; Rattendi, D.; Londono, I.;
Mazumder, L.; Bacchi, C.J.; Yarlett, N. Synthesis and SAR of alkanediamide-linked bisbenzamidines with
antitrypanosomal and anti-Pneumocystis activity. Bioorg. Med. Chem. Lett. 2009, 19, 5884–5886. [CrossRef]
[PubMed]
Bacchi, C.J.; Nathan, H.C.; Livingston, T.; Valladares, G.; Saric, M.; Sayer, P.D.; Njogu, A.R.; Clarkson, A.B., Jr.
Differential susceptibility to DL-α-difluoromethylornithine in clinical isolates of Trypanosoma brucei
rhodesiense. Antimicrob. Agents Chemother. 1990, 34, 1183–1188. [CrossRef] [PubMed]
Huang, T.L.; Mayence, A.; Vanden Eynde, J.J. Some non-conventional biomolecular targets for diamidines.
A short survey. Bioorg. Med. Chem. 2014, 22, 1983–1992. [CrossRef] [PubMed]
Paine, M.F.; Wang, M.Z.; Generaux, C.N.; Boykin, D.W.; Wilson, W.D.; De Koning, H.P.; Olson, C.A.;
Pohlig, G.; Burri, C.; Brun, R.; et al. Diamidines for human African trypanosomiasis. Curr. Opin.
Investig. Drugs 2010, 11, 876–883. [PubMed]
5.
6.
7.
8.
Soeiro, M.N.; Werbovetz, K.; Boykin, D.W.; Wilson, W.D.; Wang, M.Z.; Hemphill, A. Novel amidines and
analogues as promising agents against intracellular parasites: A systematic review. Parasitology 2013, 140,
929–951. [CrossRef] [PubMed]
Thuita, J.K.; Wolf, K.K.; Murilla, G.A.; Bridges, A.S.; Boykin, D.W.; Mutuku, J.N.; Liu, Q.; Jones, S.K.;
Gem, C.O.; Ching, S.; et al. Chemotherapy of second stage human African trypanosomiasis: Comparison
between the parenteral diamidine DB829 and its oral prodrug DB868 in vervet monkeys. PLoS Negl. Trop. Dis.
2015, 9, e0003409. [CrossRef] [PubMed]
9.
Kode, N.R.; Vanden Eynde, J.J.; Mayence, A.; Wang, G.; Huang, T.L. Design and synthesis of
N1,N5-bis[4-(5-alkyl-1,2,4-oxadiazol-3-yl)phenyl]glutaramides as potential antifungal prodrugs. Molecules
2013, 18, 11250–11263. [CrossRef] [PubMed]
10. Eynde, J.J.V.; Mayence, A.; Johnson, M.T.; Huang, T.L.; Collins, M.S.; Rebholz, S.; Walzer, P.D.; Cushion, M.T.;
Donkor, I.O. Antitumor and anti-Pneumocystis carinii activities of novel bisbenzamidines. Med. Chem. Res.
2005, 14, 143–157. [CrossRef]
11. Wilson, W.D.; Tanious, F.A.; Mathis, A.; Tevis, D.; Hall, J.E.; Boykin, D.W. Antiparasitic compounds that
target DNA. Biochimie 2008, 90, 999–1014. [CrossRef] [PubMed]
12. Chemaxon. Available online: https://www.chemaxon.com (accessed on 18 April 2016).

Pharmaceuticals 2016, 9, 20
11 of 11
13. Schrodinger suite. Available online: http://www.schrodinger.com (accessed on 18 April 2016).
14. Donkor, I.O.; Huang, T.L.; Tao, B.; Rattendi, D.; Lane, S.; Vargas, M.; Goldberg, B.; Bacchi, C. Trypanocidal
activity of conformationally restricted pentamidine congeners. J. Med. Chem. 2003, 46, 1041–1048. [CrossRef]
[PubMed]
15. Mayence, A.; Vanden Eynde, J.J.; Krogstad, F.M.; Krogstad, D.J.; Cushion, M.T.; Huang, T.L. Parallel
solution-phase synthesis of conformationally restricted congeners of pentamidine and evaluation of their
antiplasmodial activities. J. Med. Chem. 2004, 47, 2700–2705. [CrossRef] [PubMed]
16. Dykstra, C.C.; McClernon, D.R.; Elwell, L.P.; Tidwell, R.R. Selective inhibition of topoisomerases
from Pneumocystis carinii compared with that of topoisomerases from mammalian cells.
Antimicrob. Agents Chemother. 1994, 38, 1890–1898. [CrossRef] [PubMed]
17. Henderson, D.; Hurley, L.H. Molecular struggle for transcriptional control. Nat. Med. 1995, 1, 525–527.
[CrossRef] [PubMed]
18. Purfield, A.E.; Tidwell, R.R.; Meshnick, S.R. The diamidine DB75 targets the nucleus of Plasmodium falciparum.
Malar. J. 2009, 8, 104. [CrossRef] [PubMed]
19. Stead, A.M.; Bray, P.G.; Edwards, I.G.; DeKoning, H.P.; Elford, B.C.; Stocks, P.A.; Ward, S.A. Diamidine
compounds: Selective uptake and targeting in Plasmodium falciparum. Mol Pharmacol 2001, 59, 1298–1306.
[PubMed]
20. Rodriguez, F.; Rozas, I.; Kaiser, M.; Brun, R.; Nguyen, B.; Wilson, W.D.; Garcia, R.N.; Dardonville, C. New
bis(2-aminoimidazoline) and bisguanidine DNA minor groove binders with potent in vivo antitrypanosomal
and antiplasmodial activity. J. Med. Chem. 2008, 51, 909–923. [CrossRef] [PubMed]
21. Basselin, M.; Badet-Denisot, M.A.; Lawrence, F.; Robert-Gero, M. Effects of pentamidine on polyamine level
and biosynthesis in wild-type, pentamidine-treated, and pentamidine-resistant leishmania. Exp. Parasitol.
1997, 85, 274–282. [CrossRef] [PubMed]
22. Dardonville, C.; Barrett, M.P.; Brun, R.; Kaiser, M.; Tanious, F.; Wilson, W.D. DNA binding affinity of
bisguanidine and bis(2-aminoimidazoline) derivatives with in vivo antitrypanosomal activity. J. Med. Chem.
2006, 49, 3748–3752. [CrossRef] [PubMed]
23. Edwards, K.J.; Jenkins, T.C.; Neidle, S. Crystal structure of a pentamidine-oligonucleotide complex:
Implications for DNA-binding properties. Biochemistry 1992, 31, 7104–7109. [CrossRef] [PubMed]
24. Mayence, A.;
Vanden Eynde, J.J.;
Kaiser, M.;
Brun, R.;
Yarlett, N.;
Huang, T.L.
Bis(oxyphenylene)benzimidazoles: A novel class of anti-Plasmodium falciparum agents. Bioorg. Med. Chem.
2011, 19, 7493–7500. [CrossRef] [PubMed]
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).