Synthesis of a novel quinoline derivative, 2-(2-methylquinolin-4-ylamino)-N-phenylacetamide - A potential antileishmanial agent

Article abstract of DOI:10.1016/S0968-0896(02)00046-9

Some novel quinoline derivatives were prepared and tested for antileishmanial activity. 2-(2-Methylquinolin-4-ylamino)-N-phenylacetamide (2) was found to be significantly more active than the standard antileishmanial drug sodium antimony gluconate (SAG) i

Full text of DOI:10.1016/S0968-0896(02)00046-9

Bioorganic& Medicinal Chemistry 10 (2002) 1687–1693
Synthesis of a Novel Quinoline Derivative, 2-(2-Methylquinolin-4-
ylamino)-N-phenylacetamide—A Potential Antileishmanial Agent
Niranjan P. Sahu,a,* Chiranjib Pal,b,y Nirup B. Mandal,a Sukdeb Banerjee,a
Mausumi Raha,a Ashis P. Kundu,a Anirban Basu,b,{ Monidipa Ghosh,b
Keshab Royb and Santu Bandyopadhyayb,*
aSteroid and Terpenoid Chemistry Division, Indian Institute of Chemical Biology, 4 Raja SC Mullick Road,
Jadavpur, Kolkata 700 032, India
bImmunology Division, Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Jadavpur, Kolkata 700 032, India
Received 19 November 2001; accepted 19 January 2002
Abstract—Some novel quinoline derivatives were prepared and tested for antileishmanial activity. 2-(2-Methylquinolin-4-ylamino)-
N-phenylacetamide (2) was found to be significantly more active than the standard antileishmanial drug sodium antimony gluco-
nate (SAG) in reducing the parasite load both in the spleen and liver at a much lower concentration in hamster models. The results
suggest that the compound could be exploited as an antileishmanial drug. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
antimonials remain the mainstay of treatment for leish-
maniasis. The second line drugs, the bisamidines stilba-
midine or pentamidine, and the glycomacrolide
amphotericin B, although used clinically, display high
liver and heart toxicities,3À6 develop clinical resistance in
the subjects after few weeks of treatment, and con-
tribute to increased co-infections.7 Therefore, develop-
ment of more effective and safer chemotherapeutic
agents for treating leishmaniasis remains desirable, and
rational approaches are needed to identify novel drugs
against these protozoal cells. The present paper
describes the synthesis and antileishmanial activity eva-
luation of some quinoline derivatives. Among the quin-
oline derivatives tested, the compound 2-(2-methyl-
quinolin-4-ylamino)-N-phenylacetamide (2), easily pre-
pared in high yield, was found to be significantly more
active than the standard antileishmanial drug sodium
antimony gluconate both in vitro and in vivo. The
results suggest that the compound could be exploited as
an antileishmanial drug.
Leishmaniasis is a severe global publichealth problem,
especially in the tropical and sub-tropical countries.
Infection by various species and strains of Leishmania
causes a wide spectrum of disease in humans, with many
different clinical manifestations (cutaneous, mucocuta-
neous, and visceral). The asperity of the disease is lar-
gely ordained by the immunological status of the
infected individual and by the species of Leishmania
involved. Approximately 350 million people in 80
countries are estimated to be prone to the disease.1 The
visceral form of leishmaniasis, caused by the parasite
Leishmania donovani, is commonly known as Kala-azar
in which the phagocytic cells of the spleen, liver, and
bone marrow are invaded; it is often fatal in more than
90% of the untreated cases.2 There is still no effective
vaccine for Kala-azar, and chemotherapy remains the
most effective control measure.
In spite of the discovery of a number of potentially
useful antileishmanial compounds, the toxic pentavalent
Results and Discussion
Chemistry
*Corresponding author. Tel.: +91-33-473-3491; fax: +91-33-473-
0284/5197; e-mail: npsahu@iicb.res.in
yCurrent address: Department of Zoology, B.K.C. College, Kolkata
700 035, India.
In a previous paper,8 we have reported the preparation
of several indolylquinoline derivatives from indole
by Friedel–Crafts reaction. One of the compounds,
{Department of Neuroscience and Anatomy, Penn State College of
Medicine, Hershey, PA, USA.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00046-9

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N. P. Sahu et al. / Bioorg. Med. Chem. 10 (2002) 1687–1693
2-(200-dichloroacetamidobenzyl)-3-(30-indolyl)-quinoline
(1), was found to be significantly active against L.
donovani both in vitro and in vivo.9,10 The carbon ske-
leton of the indolylquinoline moiety consists of three
entities, that is indole, quinoline, and acetanilide. To
study the structure–activity relationships, several analo-
gues were prepared and evaluated for their antileishma-
nial activity.
at a concentration of 5.0 mg/mL caused L. donovani
promastigotes to remain as resting G0/G1 cells and
inhibited their entry into the S phase. The percentage of
dead cells did not increase during this incubation per-
iod, when growth arrest was visible. After 2 days of cul-
ture, 2 caused substantial increase in cell death, compared
to DMSO treated cultures (Fig. 2). Of note, 2 induced
only barely detectable arrest of normal human T cell
blasts at the G0/G1 phase after incubation for 3 days.
2-Chloro-N-phenylacetamide (3), synthesized by reacting
chloroacetyl chloride with aniline, was condensed with
4-aminoquinaldine (4) in the presence of sodium hydride
and dimethyl sulfoxide, giving rise to four products. The
compounds were separated by column chromatography
over silica gel to furnish N-phenyl-2-phenylamino-
acetamide (5), 2,20-dimethyl-[3,30]biquinolinyl-4,400-diamine
(6), 1,4-diphenylpiperazine-2,5-dione (7), and 2-(2-methyl-
quinolin-4-ylamino)-N-phenylacetamide (2). The struc-
tures of 2, 5, 6, and 7 were deduced11 from their spectral
data (IR, 1H and 13C NMR, MS) and elemental analysis.
Compounds 2, 5, and 6 appear to be new. Although
synthesis of 7 has been reported by other workers,12,13
Treatment of established visceral leishmaniasis in ham-
sters with 2 (in vivo). Golden hamsters were infected
with L. donovani as described.9 After 1 month, groups
of four hamsters were treated with phosphate-buffered
saline (PBS), sodium antimony gluconate (SAG) or 2.
The parasite load in the spleen and in the liver was
determined as described.9 As shown in Figure 3, com-
pound 2 was significantly more effective than SAG in
reducing the parasitic burden in both spleen and liver,
lowering the parasite burden by almost 93.2% in both
the organs. On the other hand, SAG reduced the para-
site burden by 82.5 and 72.6% in the spleen and the
liver, respectively. Micrographs of Giemsa-stained liver
and splenicsmears of L. donovani infected golden ham-
sters after treatment with DMSO, SAG or 2 are shown
in Figure 4.
1
neither H nor 13C NMR spectral data was available in
the literature.
Preliminary investigation revealed that among the com-
pounds prepared, 2-(2-methylquinolin-4-ylamino)-N-
phenylacetamide (2) possesses very promising antil-
eishmanial activity against L. donovani both in vitro and
in vivo. We therefore sought to prepare 2 in better
yields. It was found that the desired compound 2 was
obtained quantitatively when the reaction was carried
out in an inert atmosphere keeping the other parameters
the same. This provided us with enough material to
carry out the biological activity tests described below.
Compound 2 appears non-toxic to liver as judged by spe-
cific enzyme levels. To check the liver function, the spe-
cific serum enzyme levels of hamsters undergoing
experimental visceral leishmaniasis and receiving ther-
apy with compound 2 were analyzed and the results are
shown in Figure 5. The level of serum alkaline phos-
phatase (ALP) was markedly higher in L. donovani
infected hamsters compared to uninfected controls. The
level of ALP decreased in infected hamsters upon treat-
ment with compound 2. However, the value was still
Antileishmanial activity
Effect of 2-(2-methylquinolin-4-ylamino)-N-phenylacet-
amide (2) on the growth of L. donovani promastigotes (in
vitro). To determine whether any of the above com-
pounds (2, 5–7) had any effect on the promastigotes,
various concentrations of these derivatives (dissolved in
DMSO) were added to the promastigote culture indivi-
dually. DMSO had no effect on the growth of L.
donovani promastigotes at a final concentration of
0.1% (v/v). Of the four compounds (2, 5–7) tested,
compound 2 was found to inhibit the growth in a dose
dependent manner (Fig. 1). At a concentration as low as
5.0 mg/mL, 2 inhibited the growth of the organism by
approximately 66% on the second day, and 92 and 95%
on the fourth and seventh days of culture, respectively
(p<0.001 for each comparison). A lower concentration
(1.0 mg/mL) of 2 was also found to be growth inhibitory
to the organism. However, the differences were not sta-
tistically significant. The other compounds (5–7) were
virtually ineffective in inhibiting the growth up to a
concentration of 5.0 mg/mL (Fig. 1) (Scheme 1).
Arrest of cell-cycle progression in L. donovani promasti-
gotes by compound 2 leads to apoptosis, but has no
appreciable effect on normal human T cell blasts. Cell-
cycle analysis demonstrated that after 1 day of culture, 2
Figure 1. Effect of 2, 5, 6, and 7 on the growth of L. donovani pro-
mastigotes in vitro. Data are shown for one representative experiment
out of three with similar results. Media control (~); DMSO control
(*); 1.0 mg/mL (&); 5.0 mg/mL (~).

N. P. Sahu et al. / Bioorg. Med. Chem. 10 (2002) 1687–1693
1689
higher than normal. On the other hand, L. donovani
Conclusion
infection reduced glutamate pyruvate transaminase
(SGPT) drastically and glutamate oxaloacetate transa-
minase (SGOT) only marginally. The levels of both
SGPT and SGOT increased in infected hamsters upon
receiving compound 2 approaching almost normal
values. These results suggested that 2 is non-toxicto
liver up to a dose of 40 mg/kg body weight.
The results suggest that 2 mediates antileishmanial
activity by inducing cell-cycle arrest leading to apopto-
sis. It is worthy of mention that 2, which has a molec-
ular weight of 291, log P of 3.5 (calculated via ACD)
and only 3 H-bond donors and acceptors, is expected to
pass through the Lipinski filter.14 It therefore promises
Scheme 1.
Figure 2. Effect of 2 on the cell-cycle of L. donovani promastigotes: L. donovani promastigotes (5Â106/mL) were incubated with DMSO (0.2%),
compound 2 (5.0 mg/mL dissolved in DMSO) at 22 ꢀC and analyzed for DNA content by flow cytometry as described in the Experimental. DNA
content of T-cell blasts was analyzed in the same way, except that PHA (5.0 mg/mL) was added to normal human peripheral blood mononuclear cell
culture and incubation was carried out at 37 ꢀC. Gates were set to assess the percentages of dead (<2n DNA), G0/G1 (2n DNA), and S+G2+ M
(>2n DNA) cells.

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N. P. Sahu et al. / Bioorg. Med. Chem. 10 (2002) 1687–1693
to be the first antileishmanial drug that can be used
orally. Moreover, the preparation of 2 is very simple
and the yield is nearly quantitative.
aniline (4.56 g, 0.05 mol) in dry methylene chloride
(10 mL) at 0–5 ꢀC during 30 min. The reaction mixture
was stirred at the same temperature for another 30 min
and at ambient temperature (30 ꢀC) for a further hour.
It was then poured into crushed ice. The organic layer
was separated, washed successively with NaHCO3 soln
and water, and dried over anhydrous sodium sulphate.
Evaporation under reduced pressure gave a solid, which
was crystallized from ethylacetate–hexane to give 3
(7.61 g, 90%), mp 132 ꢀC (lit.12 mp 131–132 ꢀC); IR
(KBr, major peaks, cmÀ1) 3267, 1673, 752, 689; 1H
NMR (CDCl3) d 4.18 (2H,–COCH2Cl), 7.17 (1H, 40-H),
7.35 (2H, 30 and 50-H), 7.5 (2H, 20 and 60-H), 8.2 (1H,
br, NH); 13C NMR (CDCl3) d 43.6 (t, C-2), 120.8 (d, C-
20 and 60), 125.9 (d, C-40), 129.8 (d, C-30 and 50), 137.4 (s,
C-10) and 164.5 (s, C-1). (Found: C, 56.68; H, 4.71; N,
8.24; Cl, 20.87; calcd for C8H8ClNO, C, 56.65; H, 4.75;
N, 8.26; Cl, 20.90%).
Experimental
Melting points (uncorrected) were determined with a
capillary melting point apparatus. IR spectra were
recorded on a JASCO FT/IR (model 410) in KBr pel-
lets. MALDI-TOF MS (positive) was conducted using
Perseptive Bio System Voyager DE-STR Mass spectro-
meter with 2,5-dihydroxy benzoicaicd as matrix.
1H
and 13C NMR spectra were taken on a Bruker 300 MHz
DPX spectrometer at 300 and 74.99 MHz, respectively,
with tetramethylsilane as internal standard. Sodium
hydride, 4-aminoquinaldine, aniline and chloroacetyl
chloride were obtained from Aldrich. Organic solvents
are the products of E. Merck (India). Silica gel for col-
umn chromatography was obtained from SRL (India).
Thin layer chromatography was performed on pre-
coated silica gel 60 F254 aluminium sheets obtained from
E. Merck (Germany) using the solvent system 13%
MeOH in CHCl3 and spots were developed using
Drangendorff’s reagent. Medium 199 was purchased
from GIBCO BRL, USA.
Sodium hydride (400 mg) dispersed in oil was washed
with dry petroleum ether and then stirred in dry DMSO
(5 mL) at 75 ꢀC for 20 min. A solution of 4-aminoqui-
naldine15 (4) (158 mg, 1 mmol) in dry DMSO (3 mL) was
added dropwise to the mixture and stirring con-
tinued for 30 min at 75 ꢀC until olive green color
appears. 2-Chloro-N-phenylacetamide (3) (168 mg,
1 mmol) in DMSO was added to the mixture and stir-
ring continued at the same temperature for 8 h. After
completion of the reaction (monitored by tlc), the reac-
tion mixture was allowed to cool, poured into crushed
2-Chloro-N-phenylacetamide (3). A solution of chlor-
oacetyl chloride (5.6 g, 0.05 mol) in methylene chloride
(20 mL) was added dropwise to a stirred solution of
Figure 4. Micrographs of Giemsa-stained spleen and liver impression
smears of L. donovani infected hamsters after treatment with DMSO,
SAG or 2. Smears are from 10 weeks post-infected hamsters. Treat-
ment protocol was as described in the Experimental. Magnification,
Â1000. Note the presence of healthy amastigotes in smears of DMSO-
treated animals, reduced number of amastigotes in smears of SAG
treated animals and degraded amastigotes (!) or no amastigotes in
smears of compound 2 treated animals.
Figure 3. Treatment of established visceral leishmaniasis in hamsters
with compound 2. Indicated treatment was initiated at 4 weeks post-
infection, the parasite burden was determined at 10 weeks post-infec-
tion as described in the Experimental. Spleen ( ); liver (&). p<0.005
versus DMSO control (*); p<0.002 versus SAG (~).

N. P. Sahu et al. / Bioorg. Med. Chem. 10 (2002) 1687–1693
1691
ice, and then extracted with ethyl acetate (3Â100 mL).
The organiclayer was washed with water until free from
alkali, evaporated to dryness and chromatographed over
silica gel. Graded elution was effected with petroleum
ether (60–80 ꢀC), mixture of petroleum ether and chloro-
form (4:1, 3:2 and 1:4), chloroform and chloroform–
methanol (49:1, 19:1 and 9:1). A total of 45 fractions of
50 mL each were collected and mixed on the basis of tlc.
MALDI-TOF (positive ion) m/z 313 [(M+H)À2H]+,
1
311 [(M+H)À4H]+; H NMR (CDCl3) d 2.73 (6H, s,
CH3-2 and 20), 5.14 (4H, br s, NH2-4 and 40), 7.43 (t,
2H, J=7.5 Hz, H-6 and 60), 7.60 (m, 2H, H-7 and 70),
7.67 (m, 2H, H-5 and 50) and 7.92 (d, 2H, J=8.40Hz, H-
8 and 80); 13C NMR (CDCl3) d 24.0 (q, 2 and 2 -CH3),
110.2 (s, C-3 and 30), 117.7 (s, C-4a and 40a), 120.1 (d,
C-6 and 60), 124.9 (d, C-5 and 50), 129.2* (d, C-7 and 70),
129.3* (d, C-8 and 80), 144.9+ (s, C-8a and 80a), 145.9+
(s, C-4 and 40) and 156.2 (s, C-2 and 20) (assignments
marked with similar symbols may be interchanged).
(Found: C, 76.44; H, 5.73; N, 17.85; C20H18N4 requires,
C, 76.41; H, 5.77; N, 17.82%).
N-Phenyl-2-phenylaminoacetamide (5). Fractions eluted
with petroleum ether and chloroform (3:2) yielded a
residue which on crystallization from methanol furn-
ished needles (19 mg, 5.9%), mp 110 ꢀC; IR (KBr, major
1
peaks, cmÀ1) 3325, 1670, 1518, 755 and 695; H NMR
(CDCl3) d 3.9 (d, 2H, J=5.7 Hz, H-2), 4.36 (br s, 1H,
CH2NH), 6.69 (d, 2H, J=7.8 Hz, H-200 and 600), 6.86 (t,
1H, J=7.5 Hz, H-400), 7.11 (t, 1H, J=7.5 Hz, H-40), 7.23
(t, 2H, J=7.8 Hz, H-300 and 500), 7.31 (t, 2H, J=7.8 Hz,
H-30 and 50), 7.52 (d, 2H, J=7.8 Hz, H-20 and 60) and
8.59 (br s, 1H, CONH); 13C NMR (CDCl3) d 49.9 (t, C-
2), 113.6 (d, C-200 and 600), 119.8 (d, C-400), 119.9 (d, C-20
and 60), 124.5 (d, C-40), 129.0* (d, C-30 and 50), 129.6*
(d, C-300 and 500), 137.3 (s, C-10), 147.0 (s, C-100) and
168.8 (s, C-1) (*values may be interchanged). (Found:
C, 74.29; H, 6.27; N, 12.40; C14H14N2O requires, C,
74.31; H, 6.24; N, 12.38%).
1,4-Diphenylpiperazine-2,5-dione (7). Fractions eluted
with chloroform–methanol (49:1) on further chromato-
graphic purification and subsequent crystallization from
methanol gave 7 (15 mg, 5%), mp 267 ꢀC (lit.11 266–
267 ꢀC); IR (KBr, major peaks, cmÀ1) 1655, 1496, 1469,
1
756 and 693; H NMR (CDCl3) d 4.54 (s, 4H, H-3 and
6), 7.35 (m, 6H, H-30, 300, 40, 400 and 50, 500), 7.46 (m, 4H,
H-20, 200 and 60, 600); 13C NMR (C5D5N) d 53.4 (t, C-3,
6), 125.4 (d, C-20, 200 and 60, 600), 127.1 (d, C-40, 400),
129.2 (d, C-30, 300 and 50, 500), 140.4 (s, C-10 and 100),
164.7 (s, C-2 and 5). (Found: C, 72.19; H, 5.26; N,
10.49; calcd for C16H14N2O2, C, 72.16; H, 5.30; N,
10.52%).
2,20-Dimethyl-[3,30]biquinolinyl-4,40-diamine (6). Frac-
tions eluted with petroleum ether–chloroform mixture
(1:4) were combined and rechromatographed to give a
residue which on crystallization from methanol furn-
ished needles of 6 (27 mg, 8%), mp 215 ꢀC; IR (KBr,
major peaks, cmÀ1) 3458, 3292, 1642, 1578 and 759; MS
2-(2-Methylquinolin-4-ylamino)-N-phenylacetamide (2).
Fractions eluted with chloroform–methanol mixture
(9:1) were mixed, purified by rechromatography and
crystallized from methanol to furnish needles of 2
(74 mg, 22%), mp 220 ꢀC; IR (KBr, major peaks, cmÀ1
)
3265, 1673, 1596, 1569, 1540, 1439, 1255, and 659; MS
(MALDI-TOF, positive ion) m/z 292 [M+H]+; 1H
NMR (CDCl3) d 2.60 (3H, s, 200-CH3), 4.08 (2H, d,
J=5 Hz, 2-H2), 5.9 (1H, br s, CH2NH), 6.23 (1H, s, H-
300), 7.13 (1H, t, J=8 Hz, H-40), 7.32 (2H, t, J=8 Hz, H-
30 and 50), 7.42 (1H, br t, J=8 Hz, H-600), 7.53 (2H, br d,
J=8 Hz, H-20 and 60), 7.64 (1H, t, J=8.5 Hz, H-700),
7.83 (1H, br d, J=8.5 Hz, H-500), 7.95 (1H, br d,
J=8.5 Hz, H-800) and 8.5 (1H, s, CONH); 13C NMR
(CDCl3) d 25.6 (q, 200-CH3), 47.8 (t, C-2), 100.2 (d, C-
300), 117.5 (s, C-400a), 119.2 (d, C-600), 120.1 (d, C-20, 60),
124.7* (d, C-500), 125.0* (d, C-40), 129.2+ (d, C-30, 50),
129.6+ (d, C-700, 800), 137.1 (s, C-10), 148.2, (s, C-400),
148.9, (s, C-800a), 159.8 (s, C-200) and 167.1 (s, C-1)
(assignments having similar symbols may be inter-
changed). (Found: C, 74.21; H, 5.86; N, 14.39;
C18H17N3O requires, C, 74.20; H, 5.88; N, 14.42%).
When the same reaction was carried out in dry nitrogen
atmosphere keeping all the parameters the same as
described above, only the biologically active compound
2 was obtained (312 mg, 95%).16
Figure 5. Specific enzyme levels in sera of hamsters undergoing
experimental visceral leishmaniasis. Enzyme assays were performed
using the kits from Dr. Reddy’s Laboratories following the manufac-
turer’s instructions. ALP activity was expressed as KA units/dL; 1 KA
unit/dL=7.1 U/L.19 SGPT and SGOT activities were expressed as
units/mL. Animals received indicated treatment four weeks post-
infection, sacrificed 10 weeks post-infection for collection of sera as
described in the Experimental. Data represent meanꢁSD of 3–5 ani-
mals per group. Normal (&); infected animals treated with DMSO
(&); infected animals treated with 2 [10.0 mg/kg body weight ( )];
infected animals treated with 2 [20 mg/kg body weight ( )]; infected
animals treated with 2 [40 mg/kg body weight ( )].
Determination of antileishmanial activity in vitro. Antil-
eishmanial activity of 2, 5–7 was tested on a pathogenic
strain of L. donovani (strain AG 83), originally obtained
from an Indian Kala-azar patient17 and maintained in
golden hamsters. Promastigotes were obtained by
transforming amastigotes (isolated from spleens of
infected hamsters) and were maintained in vitro in

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N. P. Sahu et al. / Bioorg. Med. Chem. 10 (2002) 1687–1693
Medium-199 supplemented with 10% fetal bovine
serum (FBS). Promastigotes (1Â106) were incubated
with or without various concentrations of different syn-
thesized compounds in Medium-199 supplemented with
10% FBS at 22 ꢀC. Growth of promastigotes was mon-
itored by counting the number of motile promastigotes
microscopically.
Government of India for financial support. We also
thank Mr. S. Sahu and D. Das for the art work.
References and Notes
1. World Health Organization. Fact Sheet No. 116; World
Health Organization: Geneva, Switzerland, 2000.
2. Berkow, R. Leishmaniasis: Infectious Disease: Parasitic
Infections. The Merck Manual, 16th ed.; Merck Research
Laboratory: Rahway, NJ, USA, 1992; p 232.
Analysis of cell-cycle progression by flow cytometry. In
vitro grown L. donovani promastigotes and normal
human T-cell blasts were analyzed for cell-cycle pro-
gression. T-cell blasts were prepared by culturing nor-
mal human peripheral blood mononuclear cells
(PBMC), isolated from freshly drawn heparinized blood
by Ficoll/Hypaque density gradient centrifugation, in
RPMI-1640 medium containing 10% FBS and 5.0 mg/
mL phytohemagglutinin (PHA) for 3 days at 37 ꢀC in
5% CO2. Cells were fixed with 40% ethanol, treated
with 500 mg/mL RNase A, and then with 69 mM propi-
dium iodide (PI) for analysis of DNA content by flow
cytometry as described18 using FACS Calibur (Becton
Dickinson, USA).
3. Olliaro, P.; Bryceson, A. D. M. Parasitol. Today 1993, 9,
323.
4. Berman, J. D. Clinical Infect. Dis. 1997, 24, 684.
5. Croft, S. L. Trends Pharmacol. Sci. 1988, 9, 376.
6. Berman, J. D. Rev. Infect. Dis. 1988, 10, 560.
7. Murray, H. W.; Burman, J. D.; Wright, S. D. J. Infect. Dis.
1988, 157, 973.
8. Mahato, S. B.; Mandal, N. B.; Chattopadhyay, S.; Nandi,
G.; Luger, P.; Weber, M. Tetrahedron 1994, 50, 10803.
9. Chakrabarti, G.; Basu, A.; Manna, P. P.; Mahato, S. B.;
Mandal, N. B.; Bandyopadhyay, S. J. Antimicrob. Chemother.
1999, 43, 359.
10. Pal, C.; Raha, M.; Basu, A.; Roy, K. C.; Gupta, A.;
Ghosh, M.; Sahu, N. P.; Banerjee, S.; Mandal, N. B.; Ban-
dyopadhyay, S. Antimicrob. Agents Chemother. 2002, 46, 259.
11. It appears that 7 originated from self-condensation of two
units of the anion of 3, but the origin of 5 and 6 is less
obvious. It is, however, possible that 5 originated from an
intermediate like 8 through cleavage of the tertiary amide
bond via a nucleophilic attack, while 6 could have formed by
the dimerisation of two anion radicals derived from 4. The
dimericnature of 6 was evident from its NMR spectra. The
FAB-MS of 6 did not show the expected protonated molecular
ion peak at m/z 315, but displayed prominent peaks at m/z 313
and 311 corresponding to loss of two or four hydrogen atoms,
respectively, from the parent ion. It is presumed that the
hydrogen atoms are lost from the two amino groups with the
formation of a six-membered ring.
Determination of antileishmanial activity of 2 in vivo.
Golden hamsters (4–6 weeks old) were injected with
freshly transformed promastigotes of L. donovani
(2Â107/hamster) by intra-cardiac route. Therapy with 2
and sodium antimony gluconate (SAG) started 1 month
after infection. Compound 2 or SAG was injected at the
same dose (40 mg/kg body weight) via the same route
(intra-muscular) following the same protocol (once a
week for 4 weeks). Animals in the control group
received 100 mL DMSO intra-muscularly once a week
for 4 weeks. Animals of all groups were sacrificed 2
weeks after the last treatment. Parasite load in the
spleen and in the liver was determined from impression
smears after Giemsa staining. Results were expressed as
the total parasite load per organ, using the formula19
(organ weight in mgÂthe number of amastigotes per cell
nucleusÂ2Â105).
.
Serum enzyme assays. The blood sera of normal ham-
sters and L. donovani-infected hamsters receiving treat-
ment were subjected to assay for the enzymes alkaline
phosphatase (ALP), glutamate pyruvate transaminase
(SGPT) and glutamate oxaloacetate transaminase
(SGOT). These enzymes were assayed using the kits
from Dr. Reddy’s Laboratories, Hyderabad, India fol-
lowing the manufacturer’s instructions. ALP activity
was expressed as KA units/dL,20,21 whereas SGPT and
SGOT activities were expressed as units/mL.21,22
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Statistical analysis. Statistical analyses were performed
by Student’s t-test with the program Tadpole III.23
Acknowledgements
Thanks are due to Dr. Kazuo Koike, Department of
Pharmacognosy, Toho University, Japan for the mass
spectra, Director, CDRI, Lucknow for elemental ana-
lysis of the compounds, Council of Scientific & Indus-
trial Research and the Department of Biotechnology,
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