Piperazine-linked bisbenzamidines: A novel class of antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2004.01.009

A series of 13 1,4-diarylpiperazines has been prepared, evaluated for antileishmanial activity and their binding affinity to DNA was measured. Among these compounds, 1,4-bis[4-(1H-benzimidazol-2-yl)phenyl]piperazine (14) emerged as the most active compoun

Full text of DOI:10.1016/j.ejmech.2004.01.009

European Journal of Medicinal Chemistry 39 (2004) 547–553
www.elsevier.com/locate/ejmech
Preliminary communication
Piperazine-linked bisbenzamidines:
a novel class of antileishmanial agents
a
a
a
b,c
Annie Mayence , Jean Jacques Vanden Eynde , Louis LeCour Jr , Larry A. Walker ,
b
a,
Babu L. Tekwani , Tien L. Huang *
a
Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125, USA
b
National Center for Natural Products Research, University of Mississippi, University, MS 38677, USA
c
Department of Pharmacology, University of Mississippi, University, MS 38677, USA
Received 9 September 2003; received in revised form 5 January 2004; accepted 14 January 2004
Available online 06 May 2004
Abstract
A series of 13 1,4-diarylpiperazines has been prepared, evaluated for antileishmanial activity and their binding affinity to DNA was
measured. Among these compounds, 1,4-bis[4-(1H-benzimidazol-2-yl)phenyl]piperazine (14) emerged as the most active compound with an
IC50 value of 0.41 µM which is about sevenfold more potent than pentamidine.
©
2004 Elsevier SAS. All rights reserved.
Keywords: Amphotericin B; Leishmania donovani; 1,4-Diarylpiperazines; DNA binding; Diamidines; Pentamidine
1. Introduction
candidates, we decided to synthesize an expanded series of
,4-diarylpiperazines, and investigated their biological ef-
1
Leishmaniases are parasitic diseases transmitted by the
fects against the Leishmania parasites. In particular, we fo-
cused our attention on the preparation of piperazine-linked
bisbenzamidines and related derivatives, and evaluated the in
vitro antileishmanial activity and DNA binding properties of
these compounds.
bite of the infected female phlebotomine sandfly and mani-
fest with visceral, cutaneous, and mucocutaneous forms.
They are a significant cause of morbidity and mortality in the
developing countries of the world, and they affect about
2
million people per annum mostly in tropical and subtropi-
cal regions, the geographical distribution being limited by the
biotope of the insect vector [1].
2. Chemistry
Chemotherapy for these parasitic diseases is generally
ineffective mainly due to the emergence of drug-resistant
strains and toxicity of the therapeutic agents [2]. The pen-
tavalent antimonials are widely used as primary therapy
whereas alternative drugs include amphotericin B, pentami-
dine, paromomycin, and azoles [3,4]. However, resistance to
antimonials is common [5] and treatments with amphotericin
B and pentamidine are plagued by severe toxic side effects
The key step for the preparation of 1,4-diarylpiperazines
is a double nucleophilic displacement of fluorine in 4-fluoro
derivatives by the nitrogen atoms of piperazine. That reac-
tion, performed in DMF at 120 °C, affords the expected
tricyclic molecules in good yields provided the aromatic
precursor bears a strong electron-withdrawing group in the
para position.
[
3].
Recently, we reported on the synthesis of a series of novel
,4-diarylpiperazines as potential antipneumocystis [6] and
Conversion of the dinitrile 1 into the amidines 3–7
(Scheme 1) was effected by the Pinner reaction [8,9]. Treat-
ment of a solution of 1 in dichloromethane with methanol
and gaseous hydrochloric acid afforded the intermediate bis-
benzenecarboximidic ester (2) which was subsequently re-
acted with ammonia or the appropriate primary amine (me-
thylamine, propylamine, cyclohexylamine, benzylamine).
Action of a diamine (ethylenediamine or propylenediamine)
1
trypanocidal agents [7]. As part of our research program on
the development of novel antifungal and antiparasitic drug
*
Corresponding author.
E-mail address: thuang@xula.edu (T.L. Huang).
©
2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.01.009

548
A. Mayence et al. / European Journal of Medicinal Chemistry 39 (2004) 547–553
R
1
CN
H N
N H
+ 2
F
R
R
N
N
R
1
0
5
CHO
CO C H
2 5
1
2
1, 10, 15
ClH.HN
NH.HCl
OCH3
CH OH / HCl g / CH Cl
3
2
2
N
N
1
H CO
3
2
R
H
RNH2
ClH.HN
RHN
NH.HCl
NHR
3
4
5
6
7
2
N
N
CH3
C H
3
7
cyclo-C H
3
- 7
6
11
CH -C H
2
6
5
-
Cl H
H
Cl-
H N-CH -(CH ) -NH
+
N+
2
2
2 n
2
N
2
n
n
N
N
n
8
9
1
N
H
N
H
2
8
, 9
Scheme 1. Synthesis of compounds 1–10 and 15.
on 2 enabled to prepare 8 and 9 bearing an imidazoline or a
tetrahydropyrimidine system as the end groups (Scheme 1).
From the dialdehyde 10 (Scheme 2) and phenylhydrazine,
we obtained the hydrazone 11, by azeotropic distillation of
water in boiling toluene. Compound 12, bearing dimeth-
ylimidazolidinyl rings was prepared similarly. However, 13,
containing diphenylimidazolidinyl rings, could not be ob-
tained in a pure state by that procedure. Compound 13 was
synthesized by refluxing 10 and dianilinoethane in a mixture
of ethanol and acetic acid [10]. The bisbenzimidazole com-
pound 14, on the other hand, could not be synthesized from
the bisulfite adduct [11] of 10 and phenylenediamine. It was,
however, conveniently isolated after activation of the alde-
hyde group with pyridine and thionyl chloride and subse-
quent reaction with the diamine [12,13]. The dicarboxhy-
drazide compound 16 (Scheme 2) was obtained by
hydrazinolysis of the diester 15.
trophotometric
3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo-
xymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS)-
based assay [16], a radiorespirometric microtest [17], and the
fluorometric/colorimetricAlamar blue test [18] which is used
in this study. The Alamar blue test measures the effect of the
compounds on the growth of the promastigotes in culture.
Assessment of effects of potential drugs on the intracellular
survival of amastigotes requires amastigote-macrophage cul-
tures [19] or axenic amastigote cultures [14] and typically
enables investigation of drug sensitivity at various stages of
the life of the parasite.
4
. Results and discussion
The data (Table 1) suggests that several 1,4-diaryl-
piperazines represent interesting leads as antileishmanial
agents, and their activity are dramatically dependent on the
nature of the aryl substituents. Among this series of com-
pounds, diester 15 and dicarboxhydrazide 16 have no inhibi-
tory effect on the L. donovani promastigotes, whereas weak
inhibition was observed with the dihydrazone 11. Clearly,
piperazines 1,4-disubstituted by 4-benzamidine groups (e.g.
3, 4, 6 and 7) or related moieties (e.g. 8 and 14) emerged as
the most promising compounds. The parent diamidine 3,
however, exhibits moderate inhibition and it appears that the
inhibitory activity can be modulated by the introduction of an
alkyl group on a nitrogen atom of the amidine function (4–7).
An alkyl group such as propyl (5) is detrimental to activity,
whereas a smaller alkyl group such as methyl (4) or large
cyclic moieties such as cyclohexyl (6) or benzyl (7) increased
3. Biological evaluation
In the sandfly vector, Leishmania parasites exist as extra-
cellular promastigotes, while in the mammalian hosts they
exist primarily as intracellular amastigotes within phagoly-
sosomes of macrophages. There are different methods to
evaluate the in vitro leishmaniacidal activity of potential
drugs. Most of them are based on testing the action of the
compounds on viability, metabolic activity, or proliferation
of the promastigotes. Those methods involve microscopic
counting of live promastigotes in control and treated culture
[14], quantification of intracellular ATP levels [15], the spec-

A. Mayence et al. / European Journal of Medicinal Chemistry 39 (2004) 547–553
549
H C -HN-N
N-NH-C H
5
6
6
5
C H -NH-NH
2
6
5
N
N
1
0
H
H
1
1
R
N
R
N
RHN-CH -CH -NHR
R
2
2
1
0
N
N
12 CH3
N
N
R
13 C H
6
5
R
1
2, 13
NH2
NH2
/
SOCl2
N
N+
Cl
N+
Cl
Cl-
Cl-
10
N
N
N
Cl-
Cl-
+H
H
N
+
N
N
N
N
H
H
14
O
O
NH NH
2
2
1
5
N
N
H NHN
NHNH2
2
1
6
Scheme 2. Synthesis of compounds 11–14 and 16.
potency by 1.8–5.0-fold based on the IC50 values. The benzyl
derivative (7) has an inhibitory profile comparable to that of
pentamidine, a drug clinically used in the treatment of Leish-
maniasis. More interestingly, the most potent compound (14)
is a derivative in which both nitrogen atoms are included in a
five-membered cyclic ring. Compound 14 was about seven-
fold more potent than pentamidine and almost as potent as
amphotericin B in the in vitro study. The size or the rigidity of
the cyclic ring seems to play an important role in the mode of
action of the agent, since going from a five-membered ring
However, compounds that showed moderate inhibition of the
parasite, namely 3, 4, 6, 7 were good binders of DNA. This
suggests that binding to DNA may not be the major mode of
antileishmanial action of this class of compounds and that
other mechanisms of action such as the disruption of
polyamine metabolism [20,21], inhibition of topoisomerase
II [22], and inhibition of serine proteases [23] may be in-
volved. In fact, diamidines have been found to be pleotropic
(multitarget) compounds, which effect different cellular
functions. Uptake of diamidines by Leishmania and trypano-
some cells occurs through arginine/nucleoside/polyamine
transporters [24,25]. Therefore, relative uptake of different
diamidine molecules and their effect on other targets func-
tions may also be important for their leishmanicidal action.
Based on this observation, it is likely that the compounds
reported here may inhibit the parasite by binding to several
macromolecular targets in the parasite.
(8, 14) to a six-membered ring (9) leads to a complete
disappearance of the antileishmanial effect. It should be
noted that compounds substituted by a saturated five-
membered ring, namely the imidazolidinyl-containing com-
pounds 12 and 13 were devoid of activity. It is likely that
steric hindrance of the methyl or phenyl groups around the
nitrogen atoms may prevent binding of the compounds to the
macromolecular target in the parasite.
The binding of these compounds to calf thymus DNA and
poly(dA-dT) was determined (see Table 1) in order to inves-
tigate the relationship between DNA binding and antileish-
manial activity. A direct correlation was not apparent for this
series of compounds. It is interesting to note that the most
active compound, 14, was one of the weakest DNA binder.
5. Conclusions
In summary, a series of 1,4-diarylpiperazines were syn-
thesized and evaluated for their in vitro inhibitory activity
against the Leishmania parasite. The DNA binding affinity of

550
A. Mayence et al. / European Journal of Medicinal Chemistry 39 (2004) 547–553
Table 1
Antileishmanial activity and DNA binding affinity of 1,4-diarylpiperazines
R
N
N
R
a
Compound
Number
3
Antileishmanial activity
IC50 (µM)
21.5 ± 4.3
DNA binding (DT , °C)
Calf thymus DNA
16.8
m
R
IC90 (µM)
>100.0
Poly (dA-dT)
25.3
NH
NH2
4
5
NH
NH CH
8.5 ± 2.1
>100.0
–
15.3
17.0
22.2
26.0
3
b
NH
NH
NA
C
3
H
7
6
7
NH
11.6 ± 2.6
4.3 ± 1.0
21.4 ± 5.7
15.7 ± 3.6
15.5
18.0
23.6
25.1
NH
NH
NH CH2
N
8
9
6.7 ± 2.5
10.0 ± 1.3
15.8
18.3
N
H
b
b
N
NA
NA
12.2
–0.2
17.9
0.0
HN
11
33.7 ± 5.9
>100.0
>100.0
N
NH
H
1
1
2
3
H C
–
2.4
1.8
0.0
3
N
N
H C
3
b
NA
–
–1.2
N
N
1
4
5
N
0.41 ± 0.07
0.63 ± 0.20
0.1
0.6
2.0
0.0
N
H
b
1
O
NA
–
O
C
2
H
5
(
continued on next page)

A. Mayence et al. / European Journal of Medicinal Chemistry 39 (2004) 547–553
551
Table 1
continued)
(
a
Compound
Number
Antileishmanial activity
IC50 (µM)
DNA binding (DT , °C)
m
R
IC90 (µM)
–
Calf thymus DNA
Poly (dA-dT)
0.0
b
16
O
NA
0.1
NH NH
2
Pentamidine
2.9 ± 0.4
10.1 ± 2.0
14.0
–
26.0
–
Amphotericin B
0.14 ± 0.03
0.58 ± 0.12
a
Values are given as mean ± S.D. of at least three observations.
NA: no activity (no effect on growth of Leishmania cells up to the highest concentration (50 µg ml ) tested).
b
–1
these compounds was also measured. A correlation between
the in vitro antileishmanial activity and DNA binding affinity
of the diamidines reported here was not observed. The anti-
leishmanial effect was dramatically dependent on the nature
of the substituents attached on the aryl groups. The bisami-
dine moiety is essential for activity, although the activity can
be readily modulated by introduction of substituents on the
nitrogen atoms of the amidine functions. Such modifications
led to the identification of 1,4-bis[4-(1H-benzimidazol-2-
yl)phenyl]piperazine (14) as a promising candidate since it
was about sevenfold more potent than pentamidine and al-
most equipotent to amphotericin B. Further studies on com-
pound 14 and optimization of its structure leading to novel
analogues with superior biological properties are on going in
our laboratories.
boximidamide), dihydrochloride salt (4). The compound
was obtained by treatment of the crude imidate with methy-
lamine (50 mmol; 3.9 ml of an aqueous solution at 40%) in
ethanol (50 ml) at reflux for 30 min. The precipitate was
filtered from the hot mixture and washed with ethanol.
Yield: 40%.
M.p.: >300 °C.
NMR (DMSO-d ): 9.2 (br, 6H); 7.7 (d, 4H, J = 9 Hz); 7.1
6
(d, 4H, J = 9 Hz); 3.6 (s, 8H); 3.0 (s, 6H) ppm.
–
1
IR: 3124; 2847; 1668; 1505; 1452; 1232 cm .
C H N ·2HCl (423.38). Calc.: C, 56.74; H, 6.67; N,
2
0 26 6
19.85. Found: C, 56.82; H, 6.53; N, 20.02.
6.1.1.2. 4,4′-(1,4-Piperazinediyl)bis(N-propyl benzenecar-
boximidamide), dihydrochloride salt (5). The compound
was obtained by treatment of the crude imidate with propy-
lamine (20 mmol; 1.7 ml) in ethanol (50 ml) at room tem-
perature for 3 days. The reaction medium was concentrated
under reduced pressure and the residue was washed with
ether.
6. Experimental protocols
6.1. Chemistry
Yield: 30%.
M.p.: >300 °C.
1
H NMR spectra were obtained using a Varian Inova
instrument (500 MHz), chemical shifts (d) are given in ppm
using TMS as internal reference. IR spectra were recorded on
a Perkin–Elmer Spectrum One instrument operating in the
diffuse reflectance mode. Solvents and reagents are commer-
cially available (Aldrich, Acros Organics, Fisher Scientific,
Sigma Chemical Co.) and were used without further purifi-
cation.
NMR (DMSO-d ): 9.6 – 8.6 (br, 6H); 7.7 (d, 4H,
6
J = 9 Hz); 7.1 (d, 4H, J = 9 Hz); 3.5 (s, 8H); 3.4 (t, 4H); 1.6
(sext, 4H, J = 7 Hz); 0.9 (t, 6H, J = 7 Hz) ppm.
–
1
IR: 3048; 1672; 1606; 1515; 1385 cm .
C H N ·2HCl (479.49). Calc.: C, 60.12; H, 7.57; N,
2
4 34 6
17.53. Found: C, 59.94; H, 7.32; N, 17.61.
Compounds 1 [26], 3 [6,27], 8 [6,27], 10 [28], and 11 [29]
have been described in the literature.
Elemental analyses were performed by M-H-W Laborato-
ries, Phoenix, AZ.
6.1.1.3. 4,4′-(1,4-Piperazinediyl)bis(N-cyclohexyl benzen-
ecarboximidamide), dihydrochloride salt (6). The com-
pound was obtained by treatment of the crude imidate with
cyclohexylamine (20 mmol; 2.3 ml) in boiling ethanol
(50 ml) for 30 min. The solid was filtered and successively
6
.1.1. General procedures for compounds 4–7 and 9
A mixture of 4,4′-(1,4-piperazinediyl)bisbenzonitrile (1)
washed with water, ethanol, and ether.
Yield: 25%.
(
(
2 mmol; 0.6 g) in dichloromethane (250 ml) and methanol
10 ml) was saturated with HCl gas and the reaction medium
M.p.: >300 °C.
NMR (DMSO-d ): 9.2 (br, 4H); 9.1 (br, 2H); 7.7 (d, 4H,
6
was left at room temperature for 4 days. The precipitate was
filtered and washed with acetone. The crude imidate was
used without further purification and treated with the appro-
priate (di)amine.
J = 9 Hz); 7.1 (d, 4H, J = 9 Hz); 3.5 (m, 2H); 3.5 (s, 8H); 1.9
(d, 4H, J = 8 Hz); 1.8 (d, 4H, J = 8 Hz); 1.6 (d, 2H, J = 12 Hz);
1.4 (m, 8H); 1.1 (m, 2H) ppm.
–
1
IR: 3053; 1673; 1606; 1516; 1234 cm .
C H N ·2HCl (559.62). Calc.: C, 64.39; H, 7.92; N,
3
0 42 6
6.1.1.1. 4,4′-(1,4-Piperazinediyl)bis(N-methyl benzenecar-
15.02. Found: C, 64.46; H, 7.72; N, 15.15.

552
A. Mayence et al. / European Journal of Medicinal Chemistry 39 (2004) 547–553
6.1.1.4. 4,4′-(1,4-Piperazinediyl)bis[N-(phenylmethyl) ben-
The final product was recrystallized from a mixture (1:1)
zenecarboximidamide], dihydrochloride salt (7). The com-
pound was obtained by treatment of the crude imidate with
benzylamine (20 mmol; 2.2 ml) in ethanol (50 ml) at reflux
for 30 min. The precipitate was filtered from the hot mixture
and washed with water, ethanol, and ether.
Yield: 50%.
of ethanol and dioxane.
Yield: 75%.
M.p.: 250–255 °C (decomposition).
NMR (DMSO-d ): 7.4 (d, 4H, J = 8 Hz); 7.1 (t, 8H,
6
J = 8 Hz); 6.8 (d, 4H, J = 8 Hz); 6.7 (d, 8H, J = 8 Hz); 6.6 (t,
4H, J = 8 Hz); 6.1 (s, 2H); 3.9 (m, 4H); 3.7 (m, 4H); 3.6 (s,
protons from the dioxane of recrystallization); 3.1 (s, 8H)
ppm.
M.p.: >300 °C.
NMR (DMSO-d ): 10.0 (s, 2H); 9.3 (s, 2H); 8.9 (s, 2H);
6
–
1
7
4
.8 (d, 4H, J = 8 Hz); 7.4 (m, 8H); 7.3 (t, 2H, J = 7 Hz); 7.1 (d,
H, J = 8 Hz); 4.7 (s, 4H); 3.6 (s, 8H) ppm.
IR: 3099; 1668; 1607; 1518; 1384; 1235 cm .
IR: 3071; 2963; 2826; 1599; 1482; 1236; 750 cm .
C H N ·1/2C H O (726.40). Calc.: C, 79.31; H, 6.93;
4
6
46
6
4 8 2
–1
N, 11.56. Found: C, 79.67; H, 7.08; N, 11.54.
C H N ·2HCl (575.57). Calc.: C, 66.78; H, 6.30; N,
3
2 34 6
14.60. Found: C, 66.98; H, 6.50; N, 14.41.
6.1.4. 1,4-Bis[4-(1H-benzymidazol-2-yl)phenyl]piperazine,
dihydrochloride salt (14)
6
.1.1.5. 1,4-Bis [4-(1,4,5,6-tetrahydropyrimidin-2-yl) phe-
A solution of 4,4′-(1,4-piperazinediyl)bisbenzaldehyde
(10) (10 mmol; 2.94 g) in dichloromethane (100 ml) was
added dropwise to a mixture of thionyl chloride (24 mmol;
1.8 ml) and pyridine (24 mmol; 2 ml) in dichloromethane
(100 ml). The reaction medium was stirred for 2 h at room
temperature. 1,2-Phenylenediamine (60 mmol; 6.5 g) was
slowly added and the mixture was stirred overnight. The
precipitate was filtered and washed successively with water,
ethanol, and dichloromethane.
nyl] piperazine, dihydrochloride salt (9). The compound
was obtained by treatment of the crude imidate with 1,3-
diaminopropane (30 mmol; 2.5 ml) in ethanol (50 ml) at
reflux for 90 min. After cooling, the precipitate was filtered
and washed with dimethylformamide (5 ml) and ether.
Yield: 25%.
M.p.: >300 °C.
NMR (DMSO-d ): 9.5 (br, 4H); 7.6 (d, 4H, J = 9 Hz); 7.1
6
(d, 4H, J = 9 Hz); 3.5 (s, 8H); 3.4 (t, 8H, J = 5 Hz); 1.9 (mult,
Yield: 85%.
4
H, J = 5 Hz) ppm.
IR: 3271, 1638, 1602, 1516, 1231 cm .
M.p.: >300 °C.
NMR (DMSO-d ): 8.1 (d, 4H, J = 9 Hz); 7.6 (dd, 4H,
–1
6
C H N ·2HCl·1.8H O (507.90). Calc.: C, 56.76; H,
.07; N, 16.55. Found: C, 56.91; H, 7.35; N, 16.67.
J = 6 and 3 Hz); 7.3 (dd, 4H, J = 6 and 3 Hz); 7.2 (d, 4H,
J = 9 Hz); 3.7 (s, 8H) ppm.
2
4
30
6
2
7
–
1
IR: 3600–2200 (br); 1603; 1508; 1229; 1034; 745 cm .
C H N ·2HCl (543.49). Calc.: C, 66.30; H, 5.19; N,
6
.1.2. 1,4-Bis(1,3-dimethylimidazolidine-2-yl)piperazine
3
0 26 6
(
12)
A mixture of 4,4′-(1,4-piperazinediyl)bisbenzaldehyde
10) (2.9 g, 10 mmol), N,N′-dimethylethylenediamine (85%,
.1 g, 20 mmol), and p-toluenesulfonic acid (100 mg) in
15.46. Found: C, 66.05; H, 5.32; N, 15.30.
(
2
6.1.5. 4,4′-(1,4-Piperazinediyl)bisbenzoic acid ethyl ester
(15)
benzene (100 ml) was heated under reflux in a Dean Stark
apparatus for 5 h. After cooling the solvent was evaporated
under reduced pressure to afford a solid.
The final product was recrystallized from acetonitrile.
Yield: 70%.
A mixture of piperazine (0.85 g, 10 mmol), ethyl
4-fluorobenzoate (3.36 g, 3.0 ml, 20 mmol), and potassium
carbonate (2.8 g, 20 mmol) in dimethylformamide (10 ml)
was heated under reflux for 8 h. After cooling, water (20 ml)
was added to the reaction mixture to precipitate the final
product that was filtered and washed with water and ethanol.
The final product was recrystallized from acetone.
Yield: 40%.
M.p.: 204–206 °C.
NMR (CDCl ): 7.4 (d, 4H, J = 9 Hz); 7.0 (d, 4H, J = 9 Hz);
3
3
.4 (m, 4H); 3.3 (s, 8H); 3.2 (s, 2H); 2.5 (m, 4H); 2.2 (s, 6H)
ppm.
IR: 2940; 2826; 1611; 1515; 1449; 1227; 1043;
M.p.: 199–200 °C.
NMR (CDCl ): 7.9 (d, 4H, J = 8 Hz); 6.9 (d, 4H, J = 8 Hz);
3
–
1
1032 cm .
4.4 (quad, 4H, J = 9 Hz); 3.5 (s, 8H); 1.4 (t, 6H, J = 9 Hz)
ppm.
C H N (434.62). Calc.: C, 71.85; H, 8.81; N, 19.34.
2
6 38 6
Found: C, 72.05; H, 8.71; N, 19.50.
IR: 2981; 2907; 2846; 1710; 1688; 1601; 1519; 1446;
402 cm .
–
1
1
6
(
.1.3. 1,4-Bis(1,3-diphenylimidazolidine-2-yl)piperazine
13)
A mixture of 4,4′-(1,4-piperazinediyl)bisbenzaldehyde
10) (2.9 g, 10 mmol), dianilinoethane (8.5 g, 40 mmol), and
C H N O (382.45). Calc.: C, 69.09; H, 6.85; N, 7.32.
Found: C, 69.18; H, 6.77; N, 7.45.
2
2 26 2 4
(
6.1.6. 4,4′-(1,4-Piperazinediyl)bisbenzenecarboxhydrazide
(16)
A mixture of 4,4′-(1,4-piperazinediyl)bisbenzoic acid
ethyl ester (15) (0.4 g: 1 mmol), water (0.5 ml), ethanol
acetic acid (10 ml) in ethanol (100 ml) was heated under
reflux for 16 h. After cooling, the precipitate was filtered and
washed with acetone.

A. Mayence et al. / European Journal of Medicinal Chemistry 39 (2004) 547–553
553
(2.5 ml), and hydrazine hydrate (5 ml) was heated under
Acknowledgements
reflux for 8 h. After cooling, the precipitate was filtered and
retreated under the same experimental conditions for a fur-
ther 8 h period. After cooling, the precipitate was filtered and
washed with water.
The study was funded by NIH grants DA07970,
DA13546, and 2S06GM08008 to T.L.H., L.A.W. and B.L.T.
were supported by CDC Cooperative agreement grant U50-
CCU418839 on EIDs and USDA cooperative agreement
Yield: 80%.
M.p.: >300 °C.
5
8-6408-2-0009 to NCNPR.
NMR (DMSO-d ): 9.6 (br, 2H); 7.9 (d, 4H, J = 8 Hz); 7.0
6
(
d, 4H, J = 8 Hz); 4.5 (br, 4H); 3.5 (s, 8H) ppm.
References
–
1
IR: 3307; 2982; 2848; 1690; 1640; 1602; 1278; 940 cm .
C H N O (354.41). Calc.: C, 61.00; H, 6.26; N, 23.71.
1
8
22
6
2
[1] P.J. Guerin, P. Olliaro, S. Sundar, M. Boelaert, S.L. Croft, P. Desjeux,
M.K. Wasunna, A.D. Bryceson, Lancet Infect. Dis. 2 (2002) 494–501.
Found: C, 61.26; H, 6.38; N, 23.71.
[
2] J.M. Perez-Victoria, A. Di Pietro, D. Barron, A.G. Ravelo, S. Cast-
anys, F. Gamarro, Curr. Drug Targets 3 (2002) 311–333.
6
.2. Antileishmanial activity
[
[
3] J.D. Berman, Clin. Infect. Dis. 24 (1997) 684–703.
4] S.L. Croft, V. Yardley, Curr. Pharm. Des. 8 (2002) 319–342.
The compounds were dissolved in DMSO at the concen-
[5] R. Lira, S. Sundar, A. Makharia, R. Kenney, A. Gam, E. Saraiva,
D. Sacks, J. Infect. Dis. 180 (1999) 564–567.
–
1
tration of 2 mg ml and diluted with the culture medium to
obtain the desired final concentrations. The solutions were
added, in a 96 well microplate assay, to the L. donovani
[
6] B. Tao, T.L. Huang, Q. Zhang, L. Jackson, S.F. Queener, I.O. Donkor,
Eur. J. Med. Chem. 34 (1999) 531–538.
[
7] I.O. Donkor, T.L. Huang, B. Tao, D. Rattendi, S. Lane, M. Vargas,
B. Goldberg, C. Bacchi, J. Med. Chem. 46 (2003) 1041–1048.
6
promastigotes culture (2 × 10 cell per ml). Controls with
corresponding dilutions of DMSO indicated that growth of
the parasite was not altered under those experimental condi-
tions. The plates were incubated at 26 °C for 72 h and growth
of Leishmania promastigotes was determined by Alamar
blue assay [18]. Pentamidine and amphotericin B were used
as the standard antileishmanial agents. IC50 and IC90 values
for each compound was computed from the growth inhibition
curve.
[8] R.L. Shriner, F.W. Neumann, Chem. Rev. 35 (1944) 351–425.
[
[
[
9] R. Roger, D.G. Neilson, Chem. Rev. 61 (1961) 179–211.
10] H.-W. Wanzlich, W. Lochel, Chem. Ber. 86 (1953) 1463–1466.
11] H.F. Ridley, R.G. Spickett, G.M. Timmis, J. Heterocycl. Chem. 2
(
1965) 453–456.
[12] J.J. Vanden Eynde, A. Mayence, A. Maquestiau, E. Anders, Bull. Soc.
Chim. Belges 102 (1993) 357–364.
[
13] E. Anders, K. Wermann, J.J. Vanden Eynde, Adv. Heterocycl. Chem.
7 (2000) 183–219.
7
[
[
14] S. Habtemariam, Pharmacology 3 (2003) 1–5.
15] J.M.N. Luque-Ortega, O.M. Rivero-Lezcano, S.L. Croft, L. Rivas,
Antimicrob. Agents Chemother. 45 (2001) 1121–1125.
6.3. DNA binding affınity measurements[6,30,31]
[
[
[
16] K. Berg, L. Zhai, M. Chen, A. Kharazmi, T.C. Owen, Parasitol. Res.
8
0 (1994) 235–239.
17] J.E. Jackson, J.D. Tally, D.B. Tang, Am. J. Trop. Med. Hyg. 41 (1989)
18–330.
18] J. Mikus, D. Steverding, Parasitol. Int. 48 (2000) 265–269.
The thermal denaturation temperatures were determined
in 5.0 mM, pH 7.55, Tris–HCl buffer containing 50 µM of
pure EDTA, 5% DMSO, and the appropriate concentration of
the test compounds dissolved in DMSO. Calf thymus DNA
and poly(dA-dT) were used at an initial absorbance of about
3
[19] S.L. Croft, Parasitol. Today 80 (1994) 235–239.
[
[
20] M. Calonge, R. Johnson, R. Balana-Fouce, D. Ordonez, Biochem.
Pharmacol. 52 (1996) 835–841.
21] M. Basselin, M.A. Badet-Denisot, F. Lawrence, M. Robert-Gero, Exp.
Parasitol. 85 (1997) 274–282.
T. Shapiro, P. Englund, Proc. Natl. Acad. Sci. USA 87 (1990) 950–
954.
23] R. Morty, L. Troeberg, R. Pike, R. Jones, P. Nickel, J. Lonsdale-
Eccles, T. Coetzer, FEBS Lett. 433 (1998) 251–256.
24] M. Kandpal, B.L. Tekwani, P.M. Chauhan, A.P. Bhaduri, Life Sci. 59
(1996) PL75–PL80.
0
.3 at 260 nm. The melting curves were recorded on a
Beckman DU Series 640 spectrometer, with a jacketed six
cuvettes Tm cell holder. The cell holder was heated by the
Peltier Temperature Controller that was programmed at a
[22]
–1
[
[
constant rate of 0.5 °C min . The cell temperature was
simultaneously recorded by a temperature probe embedded
into the cell block. Data were collected by using the Tm
analysis accessory software installed on the spectrophotom-
eter and the program recorded absorbance, temperature, and
cuvette number. Each sample was read for 0.10 s, and
[25] P.G. Bray, M.P. Barrett, S.A. Ward, H.P. de Koning, Trends Parasitol.
19 (2003) 232–239.
[
[
26] S.S. Berg, J. Chem. Soc. (1960) 5172–5176.
27] B. Tao, T.L. Huang, T.A. Sharma, I.J. Reynolds, I.O. Donkor, Bioorg.
Med. Chem. Lett. 9 (1999) 1299–1304.
28] N.S. Thampi, F.W. Schueler, V.B. Haarstad, B. Vernon, J. Med. Chem.
10 (1967) 111–112.
5
.0 mM, pH 7.55, Tris–HCl buffer containing 50 µM of pure
EDTA and 5% DMSO was used as the blank. At the end of
[
each experiment, T results were calculated from either the
m
first derivative or the two-points fit calculation. Under these
[29] T. Suzuki, H. Ono, M. Yokoyama, Chem. Abstr. 105 (1986) 15213g.
[30] M. Cory, D.D. McKee, J. Kagan, D.W. Henry, J.A. Miller, J. Am.
Chem. Soc. 107 (1985) 2528–2536.
conditions, the typical T values for calf thymus DNA and
m
poly(dA-dT) were 60.1 and 36.1 °C, respectively. Each DTm
value reported in the table represents the mean of at least two
experimental determinations.
[
31] T.A. Fairley, R.R. Tidwell, I.O. Donkor, N.A. Naiman, K.A. Ohe-
meng, R.J. Lombardy, J.A. Bentley, M. Cory, J. Med. Chem. 36
(1993) 1746–1753.