Synthesis, DNA binding and antileishmanial activity of low molecular weight bis-arylimidamides

Article abstract of DOI:10.1016/j.ejmech.2012.06.058

The effects of reducing the molecular weight of the antileishmanial compound DB766 on DNA binding affinity, antileishmanial activity and cytotoxicity are reported. The bis-arylimidamides were prepared by the coupling of aryl S-(2-naphthylmethyl)thioimidates with the corresponding amines. Specifically, we have prepared new series of bis-arylimidamides which include 3a, 3b, 6, 9a, 9b, 9c, 13, and 18. Three compounds 9a, 9c, and 18 bind to DNA with similar or moderately lower affinity to that of DB766, the rest of these compounds either show quite weak binding or no binding at all to DNA. Compounds 9a, 9c, and 13 were the most active against Leishmania amazonensis showing IC₅₀ values of less than 1 μM, so they were screened against intracellular Leishmania donovani, showing outstanding activity with IC ₅₀ values of 25-79 nM. Despite exhibiting little in vitro cytotoxicity these three compounds were quite toxic to mice.

Full text of DOI:10.1016/j.ejmech.2012.06.058

European Journal of Medicinal Chemistry 55 (2012) 449e454
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, DNA binding and antileishmanial activity of low molecular weight
bis-arylimidamides
,
,
,d
Moloy Banerjee a, Abdelbasset A. Farahat a b, Arvind Kumar a, Tanja Wenzler c d, Reto Brun c
**
,
Manoj M. Munde a, W. David Wilson a, Xiaohua Zhu e, Karl A. Werbovetz e , David W. Boykin
,
a,
*
a Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
c Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel CH-4002, Switzerland
d University of Basel, Basel CH-4002, Switzerland
e Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The effects of reducing the molecular weight of the antileishmanial compound DB766 on DNA binding
affinity, antileishmanial activity and cytotoxicity are reported. The bis-arylimidamides were prepared by
the coupling of aryl S-(2-naphthylmethyl)thioimidates with the corresponding amines. Specifically, we
have prepared new series of bis-arylimidamides which include 3a, 3b, 6, 9a, 9b, 9c, 13, and 18. Three
compounds 9a, 9c, and 18 bind to DNA with similar or moderately lower affinity to that of DB766, the rest
of these compounds either show quite weak binding or no binding at all to DNA. Compounds 9a, 9c, and
Received 28 February 2012
Received in revised form
22 June 2012
Accepted 28 June 2012
Available online 14 July 2012
13 were the most active against Leishmania amazonensis showing IC50 values of less than 1 mM, so they
Keywords:
were screened against intracellular Leishmania donovani, showing outstanding activity with IC50 values of
25e79 nM. Despite exhibiting little in vitro cytotoxicity these three compounds were quite toxic to mice.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Leishmaniasis
Arylimidamides
DB766
Antileishmanial agents
1. Introduction
limitations, including the development of resistance, cost, paren-
teral administration, long treatment regimens, and various toxicities
In rural developing countries protozoanparasitic diseases, which
have plagued mankind for centuries, continue to cause significant
public health problems. Leishmaniasis, a potentially fatal protozoal
tropical disease, is caused by parasites of the genus Leishmania
which include as many as 20 species that are pathogenic for humans
[1]. The three main clinical manifestations of leishmaniasis are
cutaneous, mucocutaneous, and visceral leishmaniasis, with
symptoms ranging from skin and mucosal ulceration to a systemic
infection that is fatal if left untreated [2]. An estimated 12 million
people are currently infected with Leishmania, and up to 350 million
people in 88 countries are at risk of infection [3]. Approximately 2
million new cases of leishmaniasis are believed to occur annually,
0.5 million involving visceral leishmaniasis and 1.5 million with
cutaneous leishmaniasis. A number of drugs have been used to treat
leishmaniasis including pentostam, glucantime, amphotericin B,
miltefosine, and pentamidine, but all of these have one or more
[4e8]. From this brief survey of the current status of chemotherapy
for this disease, it is clear that an urgent need exists for development
of new effective and safe therapies for treating leishmaniasis.
In our efforts to find promising antileishmanial agents from
aromatic diamidines and their analogues, we identified a new class
of molecules, arylimidamides I, Fig.1, AIAs; previously referred to as
“reversed” amidines [9,10], that exhibited submicromolar 50%
inhibitory concentrations (IC50s) in the axenic Leishmania donovani
amastigote assay and the L. donovani infected macrophage assay [9].
Sub 100-nanomolar IC50 values were found in a few cases against
Leishmania amazonensis intracellular parasites [11] and nanomolar
IC50 values were observed against promastigotes of Leishmania
infantum, Leishmania major and Leishmania tropica [12,13]. DB766
(Fig. 1), a relatively high molecular weight compound, is among the
most potent antileishmanial AIAs, with an IC50 value <0.1 mM. This
in vitro activity is similar to that of amphotericin B and substantially
greater than miltefosine and paromomycin [14]. The AIAs, including
DB766, also have significant activity against Trypanosoma cruzi [15].
Recently, anti-inflammatory and anticancer activity of this class of
compounds has been described [16].
* Corresponding author. Tel.: þ1 404 651 3798; fax: þ1 404 651 1416.
** Corresponding author.
E-mail address: dboykin@gsu.edu (D.W. Boykin).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.058

450
M. Banerjee et al. / European Journal of Medicinal Chemistry 55 (2012) 449e454
diamine 1 with 2.2 equivalents of S-(2-naphthylmethyl)thioimidate
X1
X1
X2
HN
X2
NH
hydrobromide 2a,b [9,10] in anhydrous ethanol/acetonitrile
mixture at room temperature to give the hydrobromide salt. Puri-
fication was achieved by conversion to the free base followed by
making the dihydrochloride salts of 3a,b.
O
NH
I
HN
Scheme
2 outlines the synthesis of target compound 6.
N
N
2-Aminopyridine 5 was allowed to react with dicyanobenzene 4 in
the presence of sodium bis(trimethylsilyl)amide, a strong non-
nucleophilic base, in tetrahydrofuran at ꢀ80 ꢁC [18] followed by
addition of ethanolic hydrochloride to form the hydrochloride salt
of compound 6.
O
O
Scheme 3 outlines the synthesis of target compounds 9aec. The
commercial dinitro compounds 7aec were reduced through reac-
tion with a mixture of hydrazine and Raney nickel in methanol [19]
to give the corresponding diamines 8aec in very good yield
(87e95%). These diamines were allowed to react with S-(2-naph-
thylmethyl)-2-pyridylthioimidate hydrobromide 2a to give the
corresponding arylimidamides 9aec as discussed before.
O
HN
NH
NH
HN
N
N
DB766
Fig. 1. Lead bis-arylimidamides.
Scheme 4 outlines the synthesis of the target compound 13. The
dinitro biaryl compound 11 was achieved by a one-pot Suzuki-
Miyaura homocoupling process [20] using the bromo compound
10, bis(pinacolato)diboron, PdCl2(dppf)2, and potassium acetate in
dimethylformamide as solvent. The dinitro compound 11 was
reduced to the diamine 12 using hydrazine and Raney nickel as
discussed before. The diamine was conveniently converted to the
arylimidamide 13 through reaction with S-(2-naphthylmethyl)-2-
pyridylthioimidate hydrobromide 2a.
Scheme 5 outlines the synthesis of the target compound 18. The
dinitro compound 16 was prepared through alkylation of
4-nitrobenzyl alcohol 14 with p-nitrobenzyl bromide 15 using
sodium hydride as the base and a catalytic amount of sodium iodide
in dimethylformamide as solvent. The diamine 17 was obtained in
good yield through reduction of the dinitro compound 16 using
hydrazine and Raney nickel as discussed before. The arylimidamide
18 was conveniently prepared through reaction of the diamine 17
with S-(2-naphthylmethyl)-2-pyridylthioimidate hydrobromide 2a.
AIAs significantly differ from classical diamidines (pentamidine/
furamidine analogues) as they are much less basic (pKs in the 7e8
range as opposed to 10e12 for amidines) and this property is
thought to give rise to their enhanced oral bioavailability [14]. Little
is known about the mode of action of AIAs, despite the fact that
they were originally conceived as DNA minor groove binders [10].
Their DNA affinity has been found to be highly variable with
structure, ranging from quite weak to moderately strong [10,14,17].
It has been shown that the T. cruzi activity of AIAs is not correlated
with their binding to T. cruzi kinetoplast DNA [17]. Given the potent
antiprotozoal activity and promising pharmacokinetic properties of
AIAs, despite their relatively high molecular weights (e.g. DB766),
we decided to embark upon SAR studies of this class of compounds
by exploring low molecular weight AIAs. In this study, we have
prepared four different types of low molecular weight AIAs
including (i) replacement of the diphenylfuran moiety of DB766
with only a phenyl group (3a, 3b, 6), (ii) removing the furan ring of
DB766 (9a, 13), (iii) replacement of the furan ring of DB766 with
a single atom (9b, 9c), (iv) replacement of the furan ring of DB766
with a 3-atom linker which can approximate the geometry of the
furan ring of DB766 (18).
2.2. Biology
Table 1 contains the DNA binding affinities for the new lower
molecular weight AIAs as well as the in vitro activity for these
compounds against Leishmania spp. For comparative purposes,
similar data for DB766 are included. The thermal melting increase
2. Results and discussion
2.1. Chemistry
D
Tm (Tm of complex ꢀ Tm of free DNA) is a rapid and reliable
method for ranking binding affinities for large numbers of aryl-
diamidines and arylimidamides [21]. The Tm values for the
Scheme 1 outlines our approach to the synthesis of the target
compounds 3a,b. Compounds 3a,b were prepared by stirring the
D
complexes between poly (dA ꢀ dT) and the new analogues range
from 0 to 6.0 ꢁC. The three smallest AIAs (3a, 3b, 6) essentially do
NH
X2
S
H2N
NH2
+
H2N
X1
+
NC
CN
HBr
N
1
2a,b
4
5
X1
N
X2
CH
N
(a)
(a)
a
b
N
HCl
X2
HCl
X2
HCl
N
HCl
HN
X1
NH
X1
NH
HN
NH
HN
NH
HN
N
3a,b
6
Scheme 1. Reagents and conditions (a) i-EtOH, CH3CN, NaOH; ii-EtOH/HCl.
Scheme 2. Reagents and conditions (a) (i) NaN(TMS)2, THF, ꢀ80 ꢁC, (ii) EtOH/HCl.

M. Banerjee et al. / European Journal of Medicinal Chemistry 55 (2012) 449e454
451
X
X
(a)
(b)
H2N
NH2
O2N
NO2
8a-c
7a-c
a
b
c
X = Nil CH2
O
X
HCl
HCl
HN
NH
N
H
N
H
N
N
9a-c
Scheme 3. Reagent and conditions (a) Raney Ni, NH2NH2, MeOH; (b) (i) S-(2-naphthylmethyl)-2-pyridylthioimidate HBr, EtOH, CH3CN, NaOH; (ii) EtOH/HCl.
not bind to DNA. The parent biphenyl analogue 9a binds with the
same affinity as DB766, however, the bis-i-propoxybiphenyl
analogue 13 essentially does not bind to DNA, likely due to the quite
twisted biphenyl unit. The two analogues with one atom linkers
the three most active compounds 9a, 9c, and 13 are 594, 178 and
916, respectively. Given this activity and selectivity, these
compounds (9a, 13, 9c) were advanced to animal studies. Unfor-
tunately, in preliminary toxicity evaluations all three of these
compounds caused severe tremors in uninfected animals 10 min
between the two phenyl groups 9b and 9c give
DTm values of 2.1
and 5.2 ꢁC, respectively. The
DTm differences are also likely to be
after administered intraperitoneally at a dose of 30 mg/kg.
due to differences in the twist of the two molecules. The molecule
Considering the potency of these compounds against L. donovani
in vitro we tested one of these compounds (9a) for its toxicity at
a dose of 10 mg/kg, again by the intraperitoneal route. However,
similar adverse reactions were also observed 40 min after
administration of this lower dose of 9a. Due to their high in vivo
toxicity, these compounds could not be evaluated in the murine
visceral leishmaniasis model.
with a three atom linker 18 exhibits modest DNA binding with
a D
Tm value of 4.0 ꢁC.
Also in Table 1, the activity of the new AIAs against L. ama-
zonensis in vitro and their cytotoxicity to L6 rat myoblast cells is
presented. The lowest molecular weight analogues in this study 3a,
6, and 3b are, in general, the least active against L. a., giving IC50
values ranging from 5 to >10
mM. The biphenyl analogues 9a and
13 show promising activity with IC50 values of less than 1
mM. The
two analogues 9c and 9b, which have the two AIA separated by
3. Conclusion
a single atom linker, show unexpectedly divergent IC50 values of
0.67 and >10
can approximate the geometry of the furan ring of DB766, gives an
IC50 value of 1.1 M. To further evaluate the antileishmanial
potential of low molecular weight AIAs, the three most active
compounds (9a, 13, 9c) with submicromolar IC50 values against L.
amazonensis were screened against intracellular L. donovani (see
Table 1). These compounds apparently enter macrophages rather
well as the IC50 values for (9a, 13, 9c) are 79, 25 and 28 nM,
respectively. The selectivity indices (SI ¼ IC50cytotox/IC50 L. d.) of
m
M. The compound 18, with a 3-atom linker which
Four small series of low molecular weight AIAs derived from
DB766 have been synthesized and three compounds from two of
the series were found to have excellent activity versus intracellular
L. donovani and acceptable selectivity indices. Our studies show
that relatively low molecular weight AIAs can retain high activity
against Leishmania sp. However, these molecules were highly toxic
to mice and additional efforts are required to determine if struc-
tural modifications can be found which retain antileishmanial
activity and eliminate toxicity.
m
O
O
Br
(a)
(b)
O
O2N
NO2
HCl
H2N
NH2
O
O
NO2
10
12
11
HCl
NH
O
HN
NH
c
N
HN
N
O
13
Scheme 4. Reagents and conditions (a) bis(pinacolato)diboron, PdCl2(dppf), KOAc, DMF; (b) Raney Ni, NH2NH2, MeOH; (c) (i) S-(2-naphthylmethyl)-2-pyridylthioimidate HBr, EtOH,
CH3CN, NaOH; (ii) EtOH/HCl.

452
M. Banerjee et al. / European Journal of Medicinal Chemistry 55 (2012) 449e454
(a)
(b)
O
OH
Br
O2N
NO2
O2N
O2N
16
14
15
HCl
NH
HCl
HN
O
(c)
O
N
H
N
H
H2N
NH2
N
N
17
18
Scheme 5. Reagents and conditions (a) NaH, NaI, DMF; (b) Raney Ni, NH2NH2, MeOH; (c) (i) S-(2-naphthylmethyl)-2-pyridylthioimidate HBr, EtOH, CH3CN, NaOH; (ii) EtOH/HCl.
4. Experimental
4.2. Chemistry
4.1. Biology
All commercial solvents and reagents were used without puri-
fication. Melting points were determined on a Mel-Temp 3.0
melting point apparatus, and are uncorrected. TLC analysis was
carried out on silica gel 60 F254 precoated aluminium sheets using
UV light for detection. 1H and 13C NMR spectra were recorded on
a Bruker 400 MHz spectrometer using the indicated solvents. Mass
spectra were obtained from the Georgia State University Mass
Spectrometry Laboratory, Atlanta, GA. The compounds reported as
salts frequently analyzed correctly for fractional moles of water
and/or other solvents; in each case 1H NMR spectra were consistent
with the presence of water or organic solvent(s). Elemental anal-
yses were performed by Atlantic Microlab Inc., Norcross, GA.
4.1.1. In vitro efficacy studies
The antileishmanial efficacy of the compounds against intra-
cellular L. amazonensis parasites was measured as described by
Delfin et al. [22]. Efficacy of the compounds against intracellular L.
donovani was assessed by the method of Zhu et al. [23].
4.1.2. Cytotoxicity measurements
The in vitro cytotoxicity of the compounds was determined as
described by Bakunov et al. [24].
4.1.3. Tm measurements
Thermal melting experiments were conducted with a Cary 300
spectrophotometer. Cuvettes for the experiment were mounted in
a thermal block and the solution temperatures monitored by
a thermistor in the reference cuvette. Temperatures were main-
tained under computer control and increased at 0.5 ꢁC/min. The
experiments were conducted in 1 cm path length quartz cuvettes in
CAC 10 buffer (cacodylic acid 10 mM, EDTA 1 mM, NaCl 100 mM
with NaOH added to give pH ¼ 7.0). The concentrations of DNA
were determined by measuring its absorbance at 260 nm. A ratio of
0.3 mol compound per mole of DNA was used for the complex and
4.3. General procedure for the synthesis of the arylimidamides
dihydrochloride: 3a,b, 6, 9aec, 13,18
S-(2-Naphthylmethyl)-2-pyridyl or pyrimidylthioimidate hydro-
bromide 2a,b (2.2 mmol) was added to a cooled solution of the
diamine (1 mmol) in a mixture of dry ethanol (30 mL) and dry
acetonitrile (10 mL) in an ice bath. The reaction mixture was allowed
to come to room temperature and was stirred overnight. After the
disappearance of the starting material, the organic solvent was
evaporated under reduced pressure to yield a crude oil product. Dry
ether (50 mL) was added to the crude material and the mixture was
stirred at room temperature for 4 h. The red precipitate was filtered
and washed with dry ether. The solid was dissolved in ethanol
(5 ml); the solution was cooled to 0 ꢁC in an ice bath and 10% NaOH
was added until the pH reached approximately 10. The free base was
extracted with ethyl acetate (3 ꢂ 100 mL). The organic layer was
washed with distilled water, dried over dry K2CO3, filtered and
concentrated under reduced pressure. The resulting suspensionwas
crystallized by adding dry ether and then filtered. The free base was
suspended in dry ethanol (10 mL) and cooled to 0 ꢁC in an ice bath.
Freshly prepared anhydrous HCleethanol (2 mL) was added to the
suspension and the mixture was stirred at room temperature
overnight. The resulting red solution was concentrated under
reduced pressure. The red crude solid was crystallized from a dry
ethanol/ether mixture and filtered.
DNA alone was used as a control [21]. DTm values were determined
by the peak in first derivative curves (dA/dT).
Table 1
DNA binding, in vitro antileishmanial and cytotoxicity data for low molecular weight
arylimidamides.
a
b
Code
IC50 L. a. (
mM)
IC50 L. d. (
m
M)
IC50c cytotox (
mM)
D
Tmd (ꢁC)
DB766
3a
3b
6
9a
9b
9c
0.087e
>10
ND
0.036e
3.0
6.0
0.0
0.0
0.5
6.0
2.1
5.2
0.1
4.0
NDf
>212
>207
>202
46.9
93.5
5.0
ND
ND
5.1
0.76
>10
0.67
0.14
1.1
0.079
ND
0.028
0.025
ND
13
18
22.9
21.6
a
4.3.1. N,N0-(1,4-Phenylene)dipicolinimidamide hydrochloride (3a)
White solid, yield: 68%; mp 200e202 ꢁC; 1H NMR (DMSO-d6),
IC50 values obtained against intracellular L. a. [14].
IC50 values obtained against intracellular L. d. [14].
Cytotoxicity (IC50) was evaluated using cultured L6 rat myoblast cells [24,25].
Increase in thermal melting of poly(dAꢀdT)n in ꢁC [21].
Values taken from [14].
b
c
d
e
f
d
(ppm): 7.65 (brs, 4H), 7.89 (dd, J ¼ 4.0, 8.0 Hz, 2H), 8.23e8.26 (m,
2H), 8.53 (d, J ¼ 8.0 Hz, 2H), 8.93 (d, J ¼ 4.0 Hz, 2H), 9.41 (s, 2H),
10.24 (s, 2H), 12.01 (s, 2H); 13C NMR (DMSO-d6),
(ppm): 124.7,
ND, not determined.
d

M. Banerjee et al. / European Journal of Medicinal Chemistry 55 (2012) 449e454
453
129.1, 129.2, 135.4, 139.2, 144.6, 150.3, 160.8; ESI-MS: m/z calc. for
4.4.3. 4,40-Oxydianiline (8c)
C18H16N6: 316.36, found: 316.20 (base Mþ); Anal. Calc. for C18H16N6.
2HCl. 1.55H2O. 0.1Et2O: C, 52.04; H, 5.24; N, 19.79. Found: C, 51.68;
H, 4.98; N, 19.44.
Yield: 87%, mp 190e191 ꢁC; 1H NMR (CDCl3),
d
(ppm): 6.86 (d,
J ¼ 8.8 Hz, 4H), 6.97 (br s, 4H), 7.41 (d, J ¼ 8.8 Hz, 4H) 13C NMR
(CDCl3),
d (ppm): 119.9, 120.7, 152.4, 154.1; ESI-MS: Calc. for
C12H12N2O: 200.1, found 201.2 (M þ Hþ).
4.3.2. N,N0-(1,4-Phenylene)dipyrimidine-2-carboximidamide
hydrochloride (3b)
4.4.4. N,N0-([1,10-Biphenyl]-4,40-diyl)dipicolinimidamide
White solid, yield: 59%; mp 220e222 ꢁC; 1H NMR (DMSO-d6),
hydrochloride (9a)
d
(ppm): 7.64e7.65 (m, 4H), 8.00e8.06 (m, 2H), 9.19e9.24 (m, 4H),
Yield: 97%; mp 210e212 ꢁC; 1H NMR (DMSO-d6),
d (ppm): 6.89
9.53 (s, 2H), 10.25 (s, 2H), 12.20 (s, 2H); 13C NMR (DMSO-d6),
(d, J ¼ 8.2 Hz, 4H), 7.84e7.87 (m, 2H), 7.89 (d, J ¼ 8.2 Hz, 4H),
8.22e8.25 (m, 2H), 8.68 (br s, 2H), 8.89 (d, J ¼ 4.0 Hz, 2H), 10.21 (br
s, 2H), 11.41 (br s, 2H), 12.05 (br s, 2H); 13C NMR (DMSO-d6),
d
(ppm): 125.5,129.0,135.4,153.5,158.5,158.9; ESI-MS: m/z calc. for
C16H14N8: 318.34, found: 319.20 (base Mþ þ Hþ); Anal. Calc. for
C16H14N8. 2HCl. 1.75H2O. 0.25EtOH: C, 45.63; H, 4.87; N, 25.80.
Found: C, 45.54; H, 4.66; N, 25.47.
d
(ppm): 114.8, 123.1, 132.8, 138.3, 142.8, 144.5, 148.1, 149.9, 153.4,
159.2; ESI-MS: Calc. for C24H20N6: 392.2, found 393.3 (base
M þ Hþ); Anal. Calc. for C24H20N6. 3.5HCl.1H2O: C, 53.57; H, 4.77; N,
15.61. Found: C, 53.20; H, 4.66; N, 15.61.
4.3.3. N1,N4-Di(pyridin-2-yl)terephthalimidamide hydrochloride (6)
Sodium bis(trimethylsilyl)amide 1 M solution in tetrahydro-
furan (1.58 g, 9.48 ml, 8.59 mmol) was added dropwise to a solu-
tion of 2-aminopyridine (0.8 g, 8.59 mmol) in tetrahydrofuran
(10 mL) at ꢀ80 ꢁC. Stirring was continued for 1 h, then 1,4-
dicyanobenzene (0.5 g, 3.9 mmol) in tetrahydrofuran was added
dropwise at ꢀ80 ꢁC. The reaction mixture was allowed to come to
room temperature and was stirred overnight. The reaction mixture
was then cooled to 0 ꢁC and HCl saturated ethanol (3 ml) was
added. The mixture was stirred for 5 h, diluted with ether and the
resultant solid was collected by filtration. Purification was via free
base formation by neutralization with 1 N sodium hydroxide
solution followed by filtration of the resultant solid that was
washed with water. Finally, the dry free base was stirred with
saturated ethanolic HCl for 8 h, diluted with ether, and the solid
which formed was filtered and dried to give the target compound
as hydrochloride salt.
4.4.5. N,N0-(Methylenebis(4,1-phenylene))dipicolinimidamide
hydrochloride (9b)
Yield: 98%; mp 220e223 ꢁC; 1H NMR (DMSO-d6),
d (ppm): 3.74
(s, 2H), 6.78 (d, J ¼ 8.0 Hz, 4H), 7.70 (d, J ¼ 8.0 Hz, 4H), 7.84 (br s, 2H),
8.05e8.06 (m, 2H), 8.42 (d, J ¼ 7.6 Hz, 2H), 8.91 (d, J ¼ 4.0 Hz, 2H),
9.37 (br s, 2H), 10.07 (br s, 2H), 11.73 (br s, 2H); 13C NMR (DMSO-d6),
d
(ppm): 39.6, 114.3, 128.5, 132.9, 137.8, 138.3, 144.2, 145.3, 149.3,
155.0, 158.8;
ESI-MS: Calc. for C25H22N6: 406.2, found 407.3 (base M þ Hþ);
Anal. Calc. for C25H22N6. 4HCl. 0.5H2O: C, 53.49; H, 4.84; N, 14.97.
Found: C, 53.15; H, 4.90; N, 14.58.
4.4.6. N,N0-(Oxybis(4,1-phenylene))dipicolinimidamide
hydrochloride (9c)
Yield: 87%; mp 280 ꢁC (dec.); 1H NMR (DMSO-d6),
d (ppm): 7.01
White solid, yield: 42%; mp 190e192 ꢁC; 1H NMR (DMSO-d6),
(d, J ¼ 8.1 Hz, 4H), 7.56 (d, J ¼ 8.1 Hz, 4H), 7.86 (br s, 2H), 8.21e8.23
d
(ppm): 7.43e7.46 (m, 2H), 7.83 (d, J ¼ 8.0 Hz, 2H), 8.08 (d,
J ¼ 8.0 Hz, 2H), 8.25 (br s, 4H), 8.55e8.56 (br s, 2H), 11.30 (s, 2H),
11.95 (s, 2H), 12.85 (s, 2H); 13C NMR (DMSO-d6),
(ppm): 124.7,
(m, 2H), 8.43 (d, J ¼ 7.6 Hz, 2H), 8.90 (d, J ¼ 4.0 Hz, 2H), 9.39 (br s,
2H), 10.27 (br s, 2H), 11.71 (br s, 2H); 13C NMR (DMSO-d6),
d (ppm):
d
119.4, 123.1, 149.7, 150.3, 153.4, 153.9, 155.0, 156.8, 159.4; ESI-MS
(þ): Calc. for C24H20N6O: 408.2, found 409.3 (base M þ Hþ); Anal.
Calc. for C24H20N6O. 3.5HCl. 1.4H2O: C, 51.35; H, 4.72; N, 14.97.
Found: C, 51.60; H, 4.67; N, 14.47.
127.6, 129.3, 135.4, 139.2, 144.6, 149.7, 163.1; ESI-MS: m/z calc. for
C18H16N6: 316.36, found: 316.20 (base Mþ); Anal. Calc. for C18H16N6.
3HCl. 1H2O: C, 48.71; H, 4.76; N, 18.93. Found: C, 48.71; H, 4.63; N,
18.58.
4.4.7. 2,20-Di-isopropoxy-4,40-dinitro-1,10-biphenyl (11)
4.4. General procedure for the reduction of the dinitro compounds:
8aec, 12, 17
1-Bromo-2-isopropoxy-4-nitrobenzene (2 g, 7.69 mmol) was
dissolved in 20 mL of dry DMF and potassium acetate (1.5 g,
15.38 mmol), bis(pinacolato)diboron (1.95 g, 7.69 mmol) and
PdCl2(dppf)2 (5 mol %) was added and heated for 8 h at 90 ꢁC. The
black solution was cooled, passed through celite and washed with
ethyl acetate. The ethyl acetate filtrate was washed with ice cold
water, concentrated and the desired biphenyl compound was
purified from it as a light yellow solid by column chromatography
with hexaneeethyl acetate as the eluent.
The commercially available or synthesized dinitro compounds
(1 mmol) were dissolved in methanol and 4 equivalents of hydra-
zine were added followed by a pinch of Raney nickel and the
mixture was heated to 50 ꢁC. A vigorous evolution of hydrogen gas
ensued and the solution slowly turned colourless in 2e3 h time. It
was cooled and passed through a pad of celite and washed with
ethyl acetate, dried and concentrated to obtain the diamine which
was subsequently washed with hexane to obtain sufficiently pure
compounds to use directly in the next step as judged from 1H NMR.
Yield: 46%; mp 156e158 ꢁC; 1H NMR (CDCl3),
d (ppm): 1.34 (d,
J ¼ 6.4 Hz, 12H), 4.63 (septet, J ¼ 6.4 Hz, 2H), 7.31 (d, J ¼ 8.1 Hz, 2H),
7.76 (d, J ¼ 2.0 Hz, 2H), 7.80 (dd, J ¼ 8.1 Hz, 2.0 Hz, 2H); 13C NMR
(CDCl3),
d (ppm): 22.0, 71.7, 109.1, 116.1, 129.2, 130.9, 147.6, 155.2.
4.4.1. Benzidine (8a)
ESI-MS: Calc. for C18H20N2O6: 360.1, found 361.2 (M þ Hþ). This
compound was used in the next step without further
characterization.
Yield: 95%; mp 128e130 ꢁC; 1H NMR (CDCl3),
d (ppm): 3.56 (br s,
4H), 6.68 (d, J ¼ 8.2 Hz, 4H), 7.21 (d, J ¼ 8.2 Hz, 4H); 13C NMR
(CDCl3),
d (ppm): 115.2, 122.4, 132.2, 143.1; ESI-MS: Calc. for
C12H12N2: 184.1, found 185.2 (M þ Hþ).
4.4.8. 2,20-Di-isopropoxy-[1,10-biphenyl]-4,40-diamine (12)
Yield: 91%; mp 123-125 ꢁC; 1H NMR (CDCl3),
d (ppm): 1.23 (d,
4.4.2. 4,40-Methylenedianiline (8b)
J ¼ 6 Hz, 12H), 3.63 (br s, 4H), 4.34 (septet, J ¼ 6.0 Hz, 2H), 6.31 (d,
Yield: 93%; mp 97e99 ꢁC; 1H NMR (DMSO-d6),
d (ppm): 3.54 (br
J ¼ 2.2 Hz, 2H), 6.35 (dd, J ¼ 8.0 Hz, 2.2 Hz, 2H), 7.10 (d, J ¼ 8.0 Hz,
s, 4H), 3.79 (s, 2H), 6.61 (d, J ¼ 8.2 Hz, 4H), 6.99 (d, J ¼ 8.2 Hz, 4H);
2H); 13C NMR (CDCl3),
d (ppm): 22.3, 71.0, 102.8, 108.2, 113.4,
13C NMR (DMSO-d6),
d
(ppm): 40.0, 115.1, 129.4, 131.7, 144.1; ESI-
122.6, 146.7, 148.1, 150.8, 151.7, 152.9, 155.9, 156.1, 159.2; ESI-MS:
MS: Calc. for C13H14N2: 198.1, found 199.2 (M þ Hþ).
Calc. for C18H24N2O2: 300.2, found 301.3 (M
þ
Hþ). This

454
M. Banerjee et al. / European Journal of Medicinal Chemistry 55 (2012) 449e454
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characterization.
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4.4.9. N,N0-(2,20-Di-isopropoxy-[1,10-biphenyl]-4,40-diyl)
dipicolinimidamide hydrochloride (13)
Yield: 95%; mp 261e264 ꢁC; 1H NMR (DMSO-d6),
d (ppm): 1.28
(d, J ¼ 6.0 Hz, 12H), 4.41 (septet, J ¼ 6.0 Hz, 2H), 6.68 (d, J ¼ 2.2 Hz,
2H), 6.71 (dd, J ¼ 8.0 Hz, 2.2 Hz, 2H), 7.28 (d, J ¼ 8.0 Hz, 2H), 7.78 (m,
2H), 8.24 (m, 2H), 8.66 (d, J ¼ 7.8 Hz, 2H), 8.91 (d, J ¼ 4.0 Hz, 2H),
10.32 (br s, 2H), 11.36 (br s, 2H), 12.01 (br s, 2H); 13C NMR (DMSO-
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d (ppm): 23.1, 72.1, 105.9, 111.8, 116.5, 124.2, 137.2, 138.8, 145.2,
B
treatment for Indian visceral leishmaniasis: conventional versus lipid
147.2, 150.3, 156.2, 157.1, 159.3; ESI-MS: Calc. for C30H32N6O2 (base):
508.3, found 509.4 (M þ Hþ); Anal. Calc. for C30H32N6O2. 2HCl.
1.4H2O: C, 59.45; H, 6.12; N, 13.87. Found: C, 59.55; H, 6.05; N,13.64.
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4.4.10. 4,40-(Oxybis(methylene))bis(nitrobenzene) (16)
4-Nitrobenzyl alcohol (1.2 g, 7.84 mmol) was dissolved in 5 mL
of dry DMF, cooled in an ice bath and NaH (60% dispersion in
mineral oil) (0.41 g, 10.2 mmol) was added. After 10e15 min,
a solution of 4-nitrobenzyl bromide (1.54 g, 7.13 mmol) in 2 mL of
DMF was added followed by a catalytic amount of NaI. After
allowing the reaction mixture to warm to room temperature over
a period of 4 h, it was quenched by the addition of water to obtain
a yellow solid which was washed with ether and recrystallized
from ethanol.
Yield: 52%; mp 231e233 ꢁC; 1H NMR (DMSO-d6),
d (ppm): 4.51
(s, 4H), 7.84 (d, J ¼ 8.0 Hz, 2H), 8.10 (d, J ¼ 8.0 Hz, 2H); 13C NMR
(DMSO-d6), d (ppm), 74.1, 119.4, 126.1, 143.1, 144.5; ESI-MS: Calc. for
C14H12N2O5: 288.1, found 289.2 (M þ Hþ). This compound was used
in the next step without further characterization.
4.4.11. 4,40-(Oxybis(methylene))dianiline (17)
Yield: 92%; mp 178e180 ꢁC; 1H NMR (DMSO-d6),
d (ppm): 3.45
(br s, 4H), 4.59 (s, 4H), 6.93 (d, J ¼ 8.1 Hz), 7.14 (d, J ¼ 8.1 Hz); 13C
NMR (DMSO-d6),
d (ppm): 75.2, 116.3, 126.9, 128.9, 146.1; ESI-MS:
Calc. for C14H16N2O: 228.1, found 229.2 (M þ Hþ); This compound
was used in the next step without further characterization.
4.4.12. N,N0-((Oxybis(methylene))bis(4,1-phenylene))
dipicolinimidamide hydrochloride (18)
Yield: 73%; mp 280e282 ꢁC (dec); 1H NMR (DMSO-d6),
d (ppm):
4.51 (s, 4H), 7.01 (d, J ¼ 8.2 Hz, 4H), 7.42 (d, J ¼ 8.2 Hz, 4H), 7.81 (m,
2H), 8.24 (dd, J ¼ 7.8, 6.8 Hz, 2H), 8.39 (d, J ¼ 7.8 Hz, 2H), 8.89
(d, J ¼ 4.0 Hz, 2H), 10.26 (br s, 2H), 11.38 (br s, 2H), 12.02 (br s, 2H).
13C NMR (DMSO-d6),
d (ppm): 74.4, 118.2, 126.2, 132.4, 144.1,
147.6, 150.7, 152.3, 152.8, 158.2, and 159.5; ESI-MS: Calc. for
C26H24N6O (base): 436.2, found 437.3 (free base M þ Hþ); Anal.
Calc. for C26H24N6O. 3.5 HCl. 0.5H2O: C, 54.48; H, 5.01; N, 14.66.
Found: C, 54.35; H, 5.04; N, 14.44.
Acknowledgements
This work was supported by an award from the Bill and Melinda
Gates Foundation (RB, WDW, KAW, DWB) and NIH grant AI064200
(WDW, DWB). The sponsors had no role in study design; in the
collection, analysis and interpretation of data; in the writing of this
report; nor in the decision to publish.
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A.S. Bridges, O. Zhirnova, J.E. Hall, T. Wenzler, R. Brun, R.R. Tidwell, Synthesis
and antiprotozoal activity of cationic 1,4-diphenyl-1H-1,2,3-triazoles, J. Med.
Chem. 53 (2010) 254e265.
[25] A.A. Farahat, E. Paliakov, A. Kumar, A.-E.M. Bargash, F.E. Goda, H.M. Eisa,
T. Wenzler, R. Brun, Y. Liu, W.D. Wilson, D.W. Boykin, Exploration of larger
central ring linkers in furamidine analogues: synthesis and evaluation of their
DNA binding, antiparasitic and fluorescence properties, Bioorg. Med. Chem. 19
(2011) 2156e2167.
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