The cytotoxic effects of diketopiperaizes against Leishmania donovani promastigotes and amastigotes

Article abstract of DOI:10.1007/s00044-012-0355-9

A series of diketopiperazine derivatives (1-12) were evaluated for their in vitro cytotoxic activity against Leishmania donovani promastigotes and amastigotes. Cytotoxicity study revealed that the number and types of the substituents in the phenyl rings h

Full text of DOI:10.1007/s00044-012-0355-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3452–3458
DOI 10.1007/s00044-012-0355-9
ORIGINAL RESEARCH
The cytotoxic effects of diketopiperaizes against Leishmania
donovani promastigotes and amastigotes
•
Arindam Maity Abhijit Hazra Partha Palit
•
•
•
Shymal Mondal Sanchaita Lala Nirup B. Mondal
•
Received: 3 July 2012 / Accepted: 8 November 2012 / Published online: 23 November 2012
Ó Springer Science+Business Media New York 2012
Abstract A series of diketopiperazine derivatives (1–12)
were evaluated for their in vitro cytotoxic activity against
Leishmania donovani promastigotes and amastigotes.
Cytotoxicity study revealed that the number and types of
the substituents in the phenyl rings have valuable influence
on cytotoxic activity. Compounds 1, 3, 4, 11, and 12
demonstrated appreciable cytotoxic activities with the
mean IC50 values 1.4, 1.1, 1.02, 1, 0.7 lg/ml on extra
cellular promastigotes and 1.6, 1.8, 0.61, 0.53, 1.1 lg/ml
on intracellular amastigotes, respectively. The results sug-
gested that diketopiperazine derivatives 4, 11, and 12 could
be envisaged as new entrants in the domain of antileish-
manial agents.
Introduction
The protozoan parasites of the genus Leishmania (Chan-
Bacab and Penia-Rodriguez, 2001) are the causative agents
of the disease leishmaniasis (WHO, 2010). The disease
manifests mainly in three clinical forms: visceral leishman-
iasis (VL), cutaneous leishmaniasis (CL), and mucocutane-
ous leishmaniasis (MCL). It currently threatens about 350
million people in 88 countries, particularly in subtropical and
tropical regions and is estimated to afflict 12 million people
worldwide with 2 million new cases affected annually
(WHO, 2002, 1999). The co-existence of human immuno-
deficiency virus (HIV) coupled with leishmaniasis has
worsened the incidence more and more and contributed a
new profile to the disease (Wolday et al., 1999; WHO, 1998).
The treatment of the disease was based on the pentavalent
antimonials, pentamidine, and amphotericin-B as first and
second line of drugs since their discovery in 1940s (Berman,
1997) and 1950s (Olliaro and Bryceson, 1993). The use of
these drugs and their various formulations are associated with
various toxic effects like cardiac abnormalities, hypotension,
dysglycemia, and renal disfunction (Vande Wa and Tracy,
1993). Miltefosine, an oral alkyl phospholipid has shown
promising results in the treatment of VL. But, it cannot be used
in children and pregnant women as gastrointestinal toxicity
and teratogenicity were evident from the clinical trials carried
out in India (Jha et al., 1999; Sundar et al., 2000).
Keywords Leishmania donovani ꢀ Diketopiperazines ꢀ
In vitro ꢀ In vivo studies
A. Maity ꢀ A. Hazra ꢀ S. Mondal ꢀ N. B. Mondal (&)
Division of Chemistry, Indian Institute of Chemical Biology,
Council of Scientific & Industrial Research, 4 Raja S C Mullick
Road, Jadavpur, Kolkata 700 032, India
e-mail: nirup@iicb.res.in
P. Palit
Division of Infectious Diseases, Indian Institute of Chemical
Biology, Council of Scientific & Industrial Research, 4 Raja
S C Mullick Road, Jadavpur, Kolkata 700 032, India
The other drugs employed for the treatment of leish-
maniasis also suffer from various shortcomings, viz.
unacceptable adverse effects, poor efficacy, limited
accessibility due to high cost, and poor compliance, as they
require parenteral administration and long treatment regi-
mens (Nwaka and Hudson, 2006). Moreover, the emer-
gence of drug resistance, particularly towards pentavalent
antimonials in the Indian subcontinent (Peters, 1981), and
S. Lala
Department of Zoology, Sarsuna, Kolkata, West Bengal, India
Present Address:
P. Palit
Dr. B. C. Roy College of Pharmacy and AHS, Durgapur 713206,
West Bengal, India
123

Med Chem Res (2013) 22:3452–3458
3453
R1
2''
resurgence of VL with HIV as a co-infection (Savioli et al.,
2006) has broaden the complexity of the treatment.
O
R2
2
3
1'
1
N
1''
4''
4'
N
The matter of great concern is that the progress in an-
tileishmanial drug discovery has never got momentum as
the sufferers are the poorest of the poor, mostly of third
world countries, and can not afford the cost for treatment.
These afflicted people are being considered as neglected
and consequently the disease has been branded as neglec-
ted disease by the West. The lack of profitability debars the
pharmaceutical houses to give impetus (Pecoul, 2004) to
antileishmanial drug discovery program.
2'
R1
5
R2
O
8. R1 = H, R2 = 4-OMe
9. R1 = h, R2 =3-COCH3
1. R1 = H, R2 = H
2. R1 = OMe, R2 = 5-Cl
3. R1 = H, R2 = 4-Me
4. R1 = Cl, R2 = H
5. R1 = Cl, R2 = 3-Cl
6. R1 = Cl, R2 = 4-Cl
7. R1 = Cl, R2 = 5-Cl
10. R1 = Cl, R2 =4,6-Cl
11. R1 = H, R2 =3-Cl
O
N
N
These issues emphasize an imperative need to carry out
drug discovery programs that would significantly acceler-
ate and facilitate the identification of novel and safer
chemotherapeutic agents against leishmaniasis.
O
12
Fig. 1 Diketopiperazine derivatives
In recent years, piperazine (O’Reilly et al., 2009, 2010)
motifs are prevalent in scientific literature due to high
number of positive hits encountered, in biological evalua-
tion with this heterocycle and its congeners (Dinsmore and
Beshore, 2002; Doemling, 2005). Among these, diketopi-
perazine (DKP) derivatives constitute a family of second-
ary metabolites that are produced by microorganisms
(Lautru et al., 2002). Innumerable molecules bearing
piperazine-2,5-dione-cycle have been isolated from culture
and received much attention due to diverse and interesting
biological activities (Wang et al., 1995; Ikeda et al., 1983;
Chai et al., 1997). In recent years piperazine derivatives
(Mayence et al., 2004) show good antileishmanial activity
but interestingly, no detail studies have yet been appeared
in the literature with DKPs. As a part of our continuation
searches for antileishmanial agents (Chakrabarti et al.,
1999; Pal et al., 2002; Sahu et al., 2002, 2004, Palit et al.,
2012) the diversified biological activity of piperazine-2,5-
dione derivatives intrigued us to explore the possibility of
the privileged scaffolds for their antileishmanial activity. It
is to be noted that we have reported the chemical synthesis
of symmetrically 1,4-disubstituted piperazine-2,5-diones
(Fig. 1) in a one-pot sequence (Hazra et al., 2007).
sequence in presence of sodium hydride under nitrogen
atmosphere. The substrate was taken in a three-necked
reaction flask fitted with a condenser, a dropping funnel and
a stopcock connected with nitrogen cylinder. Sodium
hydride was taken in the reaction flask (half of the mole
proportion of the substrate) and washed free from mineral
oils by dry petroleum ether. Dry dimethylsulphoxide
(DMSO) was added to the flask. Heating and stirring con-
tinued till the temperature reaches 40–42 °C. The substrate
was then added and stirring continued for 1 h at 60 5 °C.
The reaction mixture was poured into ice-cold water,
extracted with CHCl3, washed free from alkali, dried over
anhydrous sodium sulphate, concentrated under reduced
pressure and crystallized from CH3OH (Hazra et al., 2007).
Spectral data of the effective compounds
1,4-Bis-(2-chlorophenyl)piperazine-2,5-dione (4)
M.p. 134–136 °C. IR: 1694, 1594, 1536, 1445 cm-1
.
NMR(CDCl3): 1H NMR d 4.34 (s, 4H, H-3 and 6), 7.09 (t,
J = 8 Hz, 2H, H-40, 400), 7.30 (t, J = 8 Hz, 2H, H-50, 500),
7.38 (d, J = 7.8 Hz, 2H, 60, 600), 8.39 (d, J = 7.8 Hz, 2H,
H-30, 300); 13C spectra: (CDCl3) d 71.9 (t, C-3, 6), 122.0 (d,
C-60, 600), 123.6 (s, C-20, 200), 125.7 (d, C-40, 400), 128.2 (d,
C-30, 300), 129.5 (d, C-50, 500), 133.9 (s, C-10, 100), 166.4 (s,
C-2, 5). MS:(MALDI-TOF, positive ion): m/z 373[M?K]?.
In this investigation, we disclose in vitro and in vivo
evaluation of the synthesized products against Leishmania
donovani promastigotes and amastigotes, the causative agent
of leishmaniasis. To the best ofour knowledge, this is the first
detail report of DKP derivatives as antileishmanial agents.
Materials and methods
Chemistry
1,4-Bis-(-chlorophenyl)piperazine-2,5-dione (11)
M.p. 234–236 °C. IR: 1671, 1576, 1523 cm-1. NMR
1
(CDCl3): H NMR d 4.33 (s, 4H, H-3 and 6), 6.99 (d,
General method of preparation
J = 7.8 Hz, 2H, H-60, 600), 7.13 (d, J = 7.8 Hz, 2H, H-20,
200), 7.26 (m, 4H, H-40, 400 and 50, 500), 13C spectra: (CDCl3)
d 53.2 (t, C-3, 6), 118.7 (d, C-60, 600), 120.8 (d, C-20, 200),
124.6 (d, C-40, 400), 130.2 (d, C-50, 500), 134.1 (s, C-30, 300),
The DKPs were prepared by self-condensation of differ-
ently substituted a-chlorophenyl acetamides in a one-pot
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Med Chem Res (2013) 22:3452–3458
142.0 (s, C-10, 100) 166.3 (s, C-2, 5). MS:(MALDI-TOF,
isolation. Approximately 2 9 106/ml macrophages were
allowed to adhere to glass cover slips (20 9 3.25 mm2) in
RPMI-1640 (Gibco Laboratories) supplemented with 10 %
FBS and cultured for overnight at 37 °C in 5 % CO2 before
in vitro infection with L. donovani. Stationary phase L.
donovani promastigotes (2 9 107/ml) were added to each
cover slip and incubated for 6 h at 37 °C in 5 % CO2.
Cover slips were washed (39) with 10 % FBS-supple-
mented RPMI-1640 to remove uningested parasites and
incubated for 2 days in the presence or the absence of
graded concentrations of 1, 3 and 10 lg/ml DKP ana-
logues. Infected macrophage cultures were washed with
PBS, fixed with methanol in slides, stained with Giemsa,
and examined microscopically under oil immersion. At
least 200 target macrophages were examined for each
cover slip. Antileishmanial activity was determined by
calculating the number of amastigotes per 200 macro-
phages in comparison to untreated control. All 50 %
inhibitory concentrations (IC50s) of these derivatives
against promastigotes and amastigotes were determined by
linear regression from the percentages of killing compared
to untreated vehicle controls (0.5 % DMSO).
positive ion): m/z 373[M?K]?.
1,4-Dicylohexylpiperazine-2,5-dione (12)
M.p. 224–226 °C. IR: 1636, 1472, 1450, 1324 cm-1. NMR
1
(CDCl3): H NMR d 1.10–1.75 (m, 20H), 3.79–3.83 (m,
2H), 4.99 (s, 4H, H-3 and 6), 13C spectra: (CDCl3) d 21.9
(t, C-30, 300 and 50, 500), 27.4 (t, C-40, 400), 30.2 (t, C-20, 200
and 60, 600) 46.2 (d, C-10, 100), 50.1 (t, C-3, 6), 169.3 (s, C-2,
5). MS:(MALDI-TOF, positive ion): m/z 317[M?K]?.
Parasite culture and growth conditions
Leishmania donovani strain AG 83 was originally obtained
from an Indian kala-azar patient (Ghosh et al., 1983) and
maintained in golden hamsters. Amastigotes were isolated
from spleens of L. donovani infected golden hamsters as
described (Jaffe et al., 1984). The spleen was rinsed in ice-
cold phosphate-buffered saline (PBS), glucose (55 mM)/
EDTA (2 mM), lightly homogenized, macroscopic parti-
cles were allowed to settle. The turbid suspension was
decanted, centrifuged at 100 g for 10 min at 4 °C. The
amastigote-enriched suspension was centrifuged at
8009g for 10 min. The pellet was suspended in 45 %
Percoll (8.0 ml), and finally 25 % Percoll (4.0 ml) was
layered over the amastigote suspension and further centri-
fuged at 5,0009g for 1 h. The band containing amastigotes
was taken and washed with PBS (39) and finally resus-
pended in Medium-199 (Gibco Laboratories, New York,
NY, USA), supplemented with 20 % FBS. Promastigotes
were obtained by transforming amastigotes and were
maintained in vitro in medium-199 supplemented with 8 %
FBS.
In vivo assessment of antileishmanial activity
of compounds 1, 3, 4, 11, and 12
A group of five BALB/c mice (4–6 weeks) each were
injected intravenously with freshly transformed prom-
astigotes of L. donovani (2 9 107/mouse). After 1-month
postinfection, the infected BALB/c mice were treated with
11 at a dose of 4.5 and 10 mg/kg body weight (doses were
chosen as 1/22nd and 1/10th, respectively, of LD50 value
of compound 11, data not shown) intraperitoneally two
times weekly for 1 month. Each mouse received a total of
eight intraperitoneal administrations of compound 11 for
1 month. Mice in the untreated group were received
vehicle control (0.2 % Tween 80 in PBS) by the same
route. The activity was compared with the standard an-
tileishmanial drug sodium antimony gluconate, which also
treated with another group of mice at a dose of 250 mg/kg
body weight. All the mice in different groups were sacri-
ficed on 1 month posttreatment. The splenic and liver
parasites were determined by impression smear of Giemsa
staining. Levels of organ parasite load were determined and
expressed as the total parasite burden per organ, using the
formula (Stauber et al., 1958): (organ weight in mg 9 the
number of amastigotes per cell nucleus 9 2 9 105).
In vitro growth of L. donovani promastigotes
in the presence of diketopiperazines (1–12)
Promastigotes (2 9 106/ml) were incubated with or with-
out various concentrations of the compounds along with
standard antileishmanial drugs in Medium-199 (1.0 ml)
supplemented with 8 % FBS at 22 °C for 12 h. Growth of
promastigotes was monitored spectrophotometrically at
570 nm using MTT assay. The % of lysis of promastigotes
was calculated as described by Sahu et al. (2002).
In vitro infection of BALB/c mice peritoneal
macrophages and antileishmanial activity
of diketopiperazines (1–12) on intracellular amastigotes
Serum enzyme assay
Thioglycolate-elicited peritoneal exudate was used as the
source of macrophages for better recovery and easier
The blood sera of normal mice and mice receiving treat-
ment with compounds 1, 3, 4, 11, and 12 were collected
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Med Chem Res (2013) 22:3452–3458
3455
two times per week for 4 weeks, were subjected to estimate
for the enzymes serum glutamate pyruvate transaminase
(SGPT) and serum glutamate oxaloacetate transaminase
(SGOT) and blood urea, 1-month posttreatment. These
enzymes and blood urea were assayed using the kits from
Dr. Reddy’s Laboratories (Hyderabad, India) following the
manufacturer’s protocol. Blood urea activity was expressed
as mg/dl, whereas SGPT and SGOT activities were
expressed as IU/l.
Table 1 In vitro antileishmanial studies of the diketopiperazine
analogues (1–12) and the reference drugs pentamidine and ampho-
tericin-B; through MTT assay on promastigotes; and on intracellular
amastigotes within murine peritoneal macrophages
Compounds
IC50 lg/mla
(promastigotes)
through MTT assay
IC50 lg/mla M.T
(amastigotes) (lg/ml)b
(amastigotes)
1
1.4
NA
1.1
1.6
NA
1.8
0.61
4.3
6.4
6.6
2.2
2
[30
2
[100
[40
[25
[50
[35
[60
[60
[50
[70
[25
[50
[90
[4
3
4
1.02
2.2
5
Results and discussion
6
2.9
7
2.6
Antileishmanial activity
8
21.8
3.4
9
All the 12 DKP analogues were tested for cytotoxicity
against both extra cellular promastigotes and intracellular
amastigotes forms of the parasite L. donovani (MHOH/IN/
1983/AG83), originally obtained from Indian Kala-azar
patient (Ghosh et al., 1983) using the MTT assay and
giemsa staining counting method. Amphoteracin B was
used as the standard.
10
9.4
[10
0.53
1.1
2.8
0.1
11
1.0
12
0.7
Pentamidine
2.1
Amphotericin-
B
0.16
MTT assay for Leishmania donovani promastigotes (MHOH/IN/
1983/AG83) lysis by different compounds; % of lysis on promastig-
otes was evaluated using the MTT assay with comparison to untreated
0.2 % DMSO control. All assays were carried out in triplicate. Thi-
oglycolate-elicited peritoneal macrophages of BALB/c mice were
infected in vitro with L. donovani promastigotes and after conversion
into amastigotes, the macrophage culture was incubated with graded
concentrations of the compounds (dissolved in 0.2 %DMSO) for
2 days at 37 °C for the determination of anti-leishmanial activity on
intracellular amastigotes
Effects of diketopiperazine derivatives on the growth
of L. donovani promastigotes in vitro
The Logarithm phase promastigotes (2 9 106/ml) were
incubated with or without various compounds. The com-
pounds were dissolved in 0.2 % DMSO and given to the
culture at graded dose and treatment was made for 2 h. The
% of lysis was calculated as described by Tim Mosmann
(1983). The IC50 value of the compounds was evaluated
by linear regression analysis method using Graphpad prism
3 software. The results are summarized in Table 1. The
IC50 values (Table 1) indicated that most of the compounds
are more or less cytotoxic to the promastigote forms of
the parasite except compound 2. However, compounds 1, 3,
4, 11, and 12 showed strong cytotoxic effect on the
promastigotes.
NA Not active
a
IC50 are the sample concentrations that kill 50 % cells compared to
solvent controls
b
MT are the sample concentrations that destroy the host macro-
phage, indicates the toxic dose
compounds 4, 11, and 12, at a concentration of 10 lg/ml,
reduced the parasite burden by 92, 92.19, and 84.66 %,
respectively, after 48 h where as, compounds 1, 3, 8, and 9
showed less efficacy, and compound 2 was found to be
ineffective in reducing intracellular parasite burden at the
same dose, compared with medium or 0.2 % DMSO. The
IC50 values were calculated by linear regression analysis
method using Graphpad prism 3 software. The results are
summarized in Table 1.
Effects of diketopiperazine derivatives in destroying
intracellular amastigotes of L. donovani in vitro
The protozoan parasites L. donovani survive and multiply
within mammalian macrophages. It is, therefore, of interest
to test the efficacy of the compounds on intracellular am-
astigotes. Peritoneal macrophages of BALB/c mice were
infected with L. donavani AG 83 promastigotes (Dey et al.,
2000) in vitro. Infected macrophages after subsequent
washings were incubated with different concentrations of
the compounds. Similar to their effect on promastigotes,
The results suggested that 4, 11, and 12 exhibited
promising activity against pathogenic strain L. donovani
AG 83 promastigotes and amastigotes in a dose dependent
manner. The IC50 values of 4, 11, 12, and amphotericin-B
against intracellular amastigotes were 0.61, 0.53, 1.1, and
0.1 lg/ml, respectively, indicating the compounds could be
exploited as antileishmanial chemotherapeutic agents.
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Med Chem Res (2013) 22:3452–3458
In vivo antilishmanial activity in established visceral
leishmaniasis on BALB/C mice model
Table 2 Effect of compounds 1, 3, 4, 11, 12 on weight of spleen and
liver after 1 month intraperitoneal treatment
Compounds Dose (mg/kg Spleen wt (mg) after Liver wt (mg) after
body wt)
treatment ( SE)
treatment ( SE)
The in vitro studies results of compounds 1, 3, 4, 11, and 12
prompted us to investigate the antileishmanial activity in
established VL on BALB/c mice model through intraperi-
toneal administration. BALB/c mice were infected with
L. donovani as described (Sahu et al., 2002). After 1 month,
groups of four mice were treated with phosphate-buffered
saline (PBS), sodium antimony gluconate (SAG) or
compounds 1, 3, 4, 11, and 12 (intraperitoneally). The
parasite load in the spleen and in the liver was determined
as described (Chakrabarti et al., 1999). The treatment led
to significant reduction of parasite burden in spleen
91.35 % (P \ 0.0001) and 67.97 % (P \ 0.0001) and in
liver 94.75 % (P \ 0.0001) and 69.73 % (P \ 0.0001),
compared to untreated controls, 30 days post intraperito-
neal treatment with 11 at a dose of 10 and 4.5 mg/kg body
weight, respectively. Compounds 4 and 12 had also ren-
dered appreciable results by reducing 71.81 and 80.95 %
splenic and 80.37 and 81.74 % liver parasite load,
respectively, in experimental mice. Dose of 250-mg/kg
body weight of SAG was able to reduce the parasite
burden from spleen and liver only by 60.16 and 57.73 %,
respectively.
Untreated
infected
control
_
509 25
1,746 56
1
10
10
10
10
4
166 12.7
184 19.5
106 9.5
87 8.5
999 25.9
1,127 30.4
976 19.7
3
4
11
11
12
89 32.6
155 10.5
139 24.6
303 14.67
1,134 47.6
1,076 46.05
1,317 45.1
10
250
Sodium
antimony
gluconate
Statistical significance compared to untreated mice is indicated as follows:
*** P \ 0.001 significant versus untreated controls (analyzed by unpaired
student t test). Values are mean standard error of results for mice
Table 3 Serum clinical chemistry evaluations
Dose
(mg/kg)
SGPT
(IU/liter)
SGOT
(IU/liter)
Blood urea
(mg/dl)
Vehicle
28 (5)
34 (2)
33 (6)
28 (5)
28 (4)
29 (7)
30 (3)
71 (4)
74 (3)
75 (2)
72 (5)
67 (5)
69 (6)
73 (4)
29 (6)
32 (5)
34 (4)
30 (7)
29 (3)
28 (1)
31 (5)
Hence, both the doses of compounds 4, 11, and 12 were
much more effective compared to the standard drug sodium
antimony gluconate and other DKP analogues in eradicat-
ing the parasite load from the spleen and liver. Moreover,
treatment with 4, 11, and 12 showed significant decrease in
weights of the spleen and liver too, compared to untreated
controls (Table 2).
1
0
10
10
10
4
3
4
11
11
12
10
Values are means (standard deviations) of four animals
To check the liver and kidney function, the specific
serum enzyme and blood urea levels of normal mice
receiving treatment with the active compounds were ana-
lyzed and the results are shown in Fig. 2.
SGPT serum glutamate pyruvate transaminase, SGOT serum gluta-
mate oxaloacetate transaminase
The levels of both SGPT and SGOT in mice upon the
active compounds (Table 3) approach more or less normal
values with respect to untreated control. Blood urea level
after treatment with different active compounds is within
the normal acceptable range. It was evident that the values
of serum enzyme levels of treated mice are almost lower
than the untreated normal mice. The results revealed that
compound 11 is nontoxic to kidney and liver up to a dose
of 10 mg/kg body weight intraperitoneally.
remarkable activity of compound 12, which lacks of
aromaticity and also of any substituent effect, is obscure to
us.
Conclusion
In conclusion, DKPs are new additions in the domain of
antileishmanial chemotherapeutic agents. The antileish-
manial activity on intracellular amastigotes of compounds
4, 11, and 12 was quite promising in a dose-dependent
manner. The indicated effective dose of the above com-
pounds was proved to be nontoxic by MTT assays. The
DKP nuclei could serve as a lead molecule for further
modification to obtain a clinically useful novel class of
antileishmanial agents.
The structure–activity relationship of the diketo piper-
azines as revealed from the studies indicate that the cyto-
toxicity was strongly influenced by the nature of the
substituent on the phenyl rings. The presence of halogen in
the aromatic rings has a great impact on cytotoxicity in
both forms of parasites. But surprisingly, the presence of
more than one halogen in aromatic rings (5, 6, 7, and 10)
has decreased the efficacy. However, the reason of
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Med Chem Res (2013) 22:3452–3458
3457
Fig. 2 Antileishmanial activity of compounds 1, 3, 4, 11, and 12
in vivo against established VL infection by L. donovani AG83 in
BALB/c mice. One-month postinfection, infected mice were treated
with compound 11, intraperitoneally at a dose of 4.5 and 10 mg/kg
body weight and compounds 1, 3,4, and 12 at 10 mg/kg body weight
in 0.2 % Tween 80 in PBS (0.02 M), two times weekly for 1 month.
Another group of mice were treated with standard antileishmanial
drug sodium antimony gluconate at a dose of 250 mg/kg body weight
intraperitoneally two times weekly for 1 month. Control untreated
group received only 0.2 % Tween 80 in PBS (0.02 M). Mice were
sacrificed 4 weeks posttreatment. Levels of parasite burden in spleen
and liver were counted from impression smears after Giemsa staining
and expressed as the total parasite load per organ, using the formula
(organ weight in mg 9 the number of amastigotes per cell
nucleus 9 2 9 105). Values represent the mean SE of five animals
Acknowledgments We gratefully acknowledge funding from the
Department of Biotechnology, Government of India and Council of
Science & Industrial Research for the award of fellowships to A.
Maity, A. Hazra, S. Mondal and P Palit is thankful to ICMR for his
fellowship. We are also thankful to Dr. B. Achari, Emeritus Scientist,
CSIR, for his helpful suggestions and encouragements.
Doemling A (2005) Organic Chemistry highlights. http://www.organic
chemistryhighlights.org//2005/05July.shtm. Accessed 5 July 2005
Ghosh AK, Rakshit MM, Ghosh DK (1983) Effect of berberine
chloride on Leishmania donovani. Indian J Med Res 78:407–416
Hazra A, Paira P, Palit P, Banerjee S, Mondal NB, Sahu NP (2007)
Synthesis of symmetrically 1,4-disubstituted piperazine-2,5-
diones: a new class of antileishmanial agents. J Chem Res 381–83
Ikeda Y, Matsuki T, Ogawa T, Munaka T (1983) Safracins, new
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