Synthesis and molecular docking studies of 1-phenyl-4-glycosyl-dihydropyridines as potent antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2010.02.018

A series of 1-phenyl-4-glycosyl-dihydropyridines (4-17 and 19-21) were prepared by the one pot multicomponent reaction of glcosyl aldehyde, β-keto compounds and aniline or substituted aniline in the presence of TBAHS as catalyst. The compounds were screened in vitro and in vivo for their antileishmanial activities. Most of the compounds exhibited moderate to good activity against amastigotes and promastigotes of Leishmania donovani. The compounds 4, 11, 12, 13, and 17 exhibited potent in vivo activity with selectivity index (SI) values 7.43-18.93. Molecular docking studies with these compounds revealed L. donovani PTR1 as the possible target to show antileishmanial activities.

Full text of DOI:10.1016/j.ejmech.2010.02.018

European Journal of Medicinal Chemistry 45 (2010) 2381e2388
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and molecular docking studies of 1-phenyl-4-glycosyl-dihydropyridines
as potent antileishmanial agents
Vivek Parashar Pandey a, Surendra Singh Bisht a, Mridul Mishra a, Ashutosh Kumar b,
Mohammad Imran Siddiqi b, Aditya Verma c, Monika Mittal c, Shraddha A. Sane c, Suman Gupta c,
,
Rama P. Tripathi a
*
a Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226001,CSIR, India
b Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow 226001,CSIR, India
c Parasitology Division, Central Drug Research Institute, Lucknow 226001,CSIR, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1-phenyl-4-glycosyl-dihydropyridines (4e17 and 19e21) were prepared by the one pot
multicomponent reaction of glcosyl aldehyde, b-keto compounds and aniline or substituted aniline in the
Received 24 November 2009
Received in revised form
3 February 2010
Accepted 4 February 2010
Available online 12 February 2010
presence of TBAHS as catalyst. The compounds were screened in vitro and in vivo for their antileishmanial
activities. Most of the compounds exhibited moderate to good activity against amastigotes and pro-
mastigotes of Leishmania donovani. The compounds 4, 11, 12, 13, and 17 exhibited potent in vivo activity
with selectivity index (SI) values 7.43e18.93. Molecular docking studies with these compounds revealed
L. donovani PTR1 as the possible target to show antileishmanial activities.
Keywords:
Leishmaniasis
Ó 2010 Elsevier Masson SAS. All rights reserved.
Luciferase assay
Dihydropyridines
1-Phenyl-4-glycosyl-dihydropyridines
Tetrabutylammonium hydrogensulphate
1. Introduction
migrate to the vital organs of the body such as liver, spleen, bone
marrow and other lymphoid tissues and sometimes it is fatal. No
Leishmaniasis, a disease caused by protozoan parasite belonging
to the genus Leishmania is transmitted by the bite of mainly two
genera of sandflies e Lutzomyia in the New World and Phlebotomus
in the Old World [1]. The disease affects almost 2 million people
across the 88 countries throughout the world (www.who.int/tdr/
disease/leish).
The parasites have a digenetic life cycle, first residing in the gut
of phlebotomine sand fly where they replicate as procyclic pro-
mastigotes. After a blood meal, the parasites are transmitted and
engulfed by vertebrate mononuclear phagocytic system cells and
are transformed into the amastigote stage. These amastigotes
divide within the acidified phagolysosomes [2]. Human infection is
caused by about 21e30 species of Leishmania parasites and the two
forms of this disease cutaneous leishmaniasis (CL) and visceral
leishmanias (VL) are very important for health and socioeconomic
point of view. The VL is severe form of leishmaniasis as the parasites
effective vaccines are available [3] and the treatment of leishman-
iasis relies mainly on chemotherapy [4] with few drugs, which are
quite toxic. Chemotherapy is inadequate and drug resistance
against the known antileishmanials warrants the introduction of
newer and safer chemotherapeutic agents. A diverse group of
chemical structures have been reported as antileishmanial agents.
These include mostly the nitrogen heterocycles; quinolines [5],
acridines [6], phenothiazines [7], pyrimidines [8,9], purines [10],
and many other class of compounds including anilines [11], flavo-
noids [12], quinines [13,14], amino acid esters and amides [15,16],
amino alcohols [17], alkyl phospholipids [18], and certain Pt
complexes [19].
However, rational drug design approach involves identification
of selective drug targets and development of inhibitors of enzymes
present in the parasite but absent from their mammalian host. The
pteridine reductase 1 (PTR1, EC 1.5.1.33) of Leishmania is an excel-
lent drug target due to the unusual salvage of pterin from the host
while the host synthesizes pterin derivatives de novo from GTP and
lack PTR1 activity [20]. Biochemical studies indicate that this
enzyme is a NADPH dependent pterin reductase and exhibit its
enzymatic activity as a tetramer [21e23]. PTR1 reduces biopterin to
* Corresponding author. Tel.: þ91 0522 2612411; fax: þ91 522 2623405/
2623938/2629504.
E-mail address: rpt.cdri@gmail.com (R.P. Tripathi).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.018

2382
V.P. Pandey et al. / European Journal of Medicinal Chemistry 45 (2010) 2381e2388
Table 1
H2-biopterin and H4-biopterin; it is also capable of reducing folate
to 7,8-dihydrofolate and tetrahydrofolate. Recently we have shown
that dihydropyrimidinones and 1-phenyl-4-glycosyl-dihydropyr-
idines inhibit PTR1 of leishmania parasite and possess potent in
vitro activities also [24e26]. At least two of the dihydropyridine
class of compounds one bearing the phenyl and the other sugar
residue at the 4th-position of the dihydropyridine ring were
studied in great detail in our group for their possible mode of
antilieishmanial action [26,27]. In order to optimize this molecule
we have synthesized a series of 1-phenyl-4-glycosyl-dihydropyr-
idines and evaluated there in vitro and in vivo activities against
Leishmania donovani. Docking studies with 3D structure of PTR1
were also performed in order to find out essential features of this
skeleton responsible for binding with Leishmanial PTR1 and elic-
iting the biological response.
Synthesis of dihydropyridines (4e17 and 19e21) with different glycosyl aldoses,
anilines, and -keto esters.
b
Entry
Glycosyl
aldoses
R2
b
-keto
Product
Time
(h)
Isolated yield
(%)
esters
1
2
3
4
5
6
7
8
1a
1a
1a
1a
1a
1b
1b
1b
1c
1c
1c
1c
1c
1c
18
18
18
4-OMe
4-Cl
4-CH3
4-F
4-F
3,4-diCl
H
4-Cl
4-CH3
4-Br
4-OMe
4-Cl
4-CH3
2-OMe
4-Cl
3a
3a
3a
3a
3b
3a
3a
3a
3a
3a
3a
3a
3b
3b
3a
3a
3a
4
5
6
7
8
9
10
11
12
13
14
15
16
17
19
20
21
7
8
6
9
10
8
7
8
6
7
7
8
6
7
7
7
8
67
74
78
70
76
76
68
77
78
64
70
73
69
71
76
78
66
9
10
11
12
13
14
15
16
17
2. Results and discussion
4-OMe
NH2
2.1. Chemistry
The starting xylofuranosyl dialdoses, 3-O-benzyl-1,2-O-iso-
propylidene-
D
a
-
D
-xylofuran-1,5-dialdose (1a), 1,2-O-isopropylidene-
-xylofuran-1,5-dialdose (1b) and 1,2-O-isopropylidene-3-O-methyl-
-xylofuran-1,5-dialdose (1c) were prepared from -glucose according
a-
a-
In order to see the effect of pyranosyl sugar in the above
reaction, similarly condensation of galactopyranosyl dialdose 18
with methyl acetoacetate and three different anilines, 4-amino-,
4-methoxy- and 4-chloroanilines were carried out separately
as above to give good yields of 4-(10,20:30,40-di-O-isopropylidene-
D
D
to the procedure reported in literature [28e30]. Thus, reaction of 3-O-
benzyl-1,2-O-isopropylidene- -xylofuran-1,5-dialdose (1a) with
a-D
a-L
-arabinopyranos-50-yl)-1,4-dihydropyridines (19e21) (Scheme
methylacetoacetate and 4-methoxy aniline in the presence of tetrabu-
tylammonium hydrogensulphate (TBAHS) in diethylene glycol as our
earlier reported method [31] resulted in 2,6-dimethyl-4-(30-O-benz
2, Table 1).
yl-10,20-O-isopropylidene- -threo-pentofuranos-40-yl)-1-(4-methox-
b-L
yphenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid dimethyl ester (4)
in good yield. It was characterized on the basis of its spectroscopic data
and analyses. The IR, MS and NMR (1H and 13C) spectral data are
consistent with the proposed structure 4.
To see the scope of this reaction with different susbstrates,
condensation of the xylofuranosyl dialdoses 1a, 1b and 1c with
different anilines (2ae2h) and methyl acetoacetate (3a) or ethyl
acetoacetate (3b) were carried out under the above mentioned
2.2. Biology
The above synthesized compounds were evaluated in vitro
against transgenic L. donovani promastigotes and intracellular
amastigotes at 40 g/ml concentrations to identify the potent
compounds. Compounds showing >80% inhibition at this dose
against both the stages of L. donovani were further screened at
seven different concentrations to determine the IC50 values. The
m
condition give the respective 1-phenyl-4-(
b
-
L
-threo-pentofuranos-
compound with in vitro IC50 > 15
inactive and not pursued further for evaluation. Out of all the active
compounds with IC50 values ꢀ15 g/ml and better safety index (SI)
than the standard drugs Pentamidine and Miltefosine, only five
mg/ml was considered to be
40-yl)dihydropyridines (5e17) (Scheme 1, Table 1) in good yields.
The structures of these compounds were also established on the
basis of their spectroscopic data and microanalysis.
m
Scheme 1. Synthesis of 1-phenyl-4-glycosyl-dihydropyridines (4e17).

V.P. Pandey et al. / European Journal of Medicinal Chemistry 45 (2010) 2381e2388
2383
Scheme 2. Synthesis of 1-phenyl-4-glycosyl-dihydropyridines (19e21).
compounds were further evaluated for in vivo activity via intra-
peritoneal route at 50 mg/kg ꢁ 5 i.p dose against L. donovani/
Hamster model (Mesocricetus auretus).
The IC50 and SI values of 1-phenyl-4-glycosyl-dihydropyridines
against promastigotes and intracellular amastigotes of L. donovani
are depicted in Table 2. As evident from Table 2 most of the
1-phenyl-4-glycosyl-dihydropyridines displayed potent inhibition
of both the promastigotes and amastigotes of L. donovani. Among all
the compounds, only compounds 4, 11, 12, 13, and 17 have shown
better SI as compared to the standard drugs. Although no definite
conclusion of structure activity relationship could be drawn from
the above but these glycosyl dihydropyridines open the door to
synthesize better analogs as the possibility of optimization with
sugar as one of the components is highly enhanced.
2.3. Docking results of synthesized compounds
To explore their probable binding modes within the active site of
L. donovani PTR1, molecular docking of all the compounds was
performed by means of LigandFit program. Since no experimental
three-dimensional structure for L. donovani PTR1 is known till date,
all the compounds were docked into the active site of previously
reported L. donovani PTR1 homology model [25] using the same
protocol.
Molecular docking studies of dihydropyridine derivatives with
L. donovani PTR1 binding site revealed very clear preference for
most of the compounds. All the highly active dihydropyridine
derivatives occupy similar spatial position as shown for highly
active compound 11 in the L. donovani PTR1 binding site in Fig. 1.
The docking scores of 11 matches well with the biological activity
and compound 11 was among the top ranking compounds in
docking scored (Table 3). The docking results indicated that the
phenyl substituted dihydropyridine ring could fit well in a hydro-
phobic pocket formed by residues Ala 230, Tyr 191, Tyr 194, Phe 113,
Pro 224, Leu 18. Because the active site of L. donovani PTR1 was
IC50 in the range of 0.75e6.18 mg /ml with SI (selectivity index)
values of 18.93, 9.21, 7.43, 13.58, and 8.04, respectively, against
intracellular amastigotes. Further, it is also evident that SI values of
these glycosyl dihydropyrdines are several folds better than the
standard drugs Pentamidine and Miltefosine. The above five
compounds (4, 11, 12, 13, and 17) were further screened for their in
vivo activity against L. donovani in Hamster model intracellular.
Compound 12 have shown better inhibition (58.79%) as compared
to compounds 11, 13 and 17 which exhibited 44.9%, 43.9% and
49.73% inhibition, respectively. The compound
a marginal activity (26.78%).
4 has only
As evident from antileishmanial activity of the compounds, in
general 3,5-dimethyl ester of dihydropyridines are more active
than their counterparts the ethyl ester derivatives except
compound 17. Further, it is clear that compounds having 30-O-
methyl substituent are more potent than those with 30-O-benzyl
substituent. The glycosyl dihydropyridines 4, 11, 12, 13 and 17 have
Table 2
In vitro and in vivo antileishmanial activity of compounds (4e9, 11e17, and 19e21).
S. No.
Compound No.
In vitro assessment IC50
(
m
g/ml)
Anti-amastigote activity.
Cytotoxicity
CC50 g/ml)
Selective index (SI)
CC50/IC50
In vivo activity (dose-50 mg/kg ꢁ 5, i.p.)
(
m
percent inhibition
Anti-promastigote activity
1
2
3
4
5
6
7
8
4
5
6
7
8
9
11
12
13
14
15
16
17
19
20
21
1.94
1.86
3.62
3.67
1.65
3.72
0.86
1.74
0.88
12.41
1.94
Inactive
0.22
0.75
3.80
4.89
Inactive
8.08
10.13
3.85
4.45
3.57
4.42
19.05
Inactive
6.18
14.35
Inactive
Inactive
12.11
3.59
14.14
4.22
7.66
18.93
1.109
3.394
ND
7.42
2.96
9.21
7.43
13.58
4.21
4.32
ND
8.04
1.06
ND
ND
2.58
1.08
26.78 ꢂ 24.2
ND
ND
ND
ND
ND
ND
59.93
29.94
35.49
33.07
48.49
18.61
82.43
ND
49.73
15.23
ND
ND
31.31
3.91
44.90 ꢂ 9.8
58.79 ꢂ 23.5
9
43.90 ꢂ 10.3
10
11
12
13
14
15
16
17
18
ND
ND
ND
49.73 ꢂ 12.0
4.17
ND
Inactive
Inactive
0.643
3.23
ND
ND
Pentamidine
Miltefosine
92 ꢂ 2.8 (40 ꢁ 5, i.p.)
95.0 ꢂ 3.7 (30 ꢁ 5, p.o.)
ND ¼ Not done.

2384
V.P. Pandey et al. / European Journal of Medicinal Chemistry 45 (2010) 2381e2388
Fig. 1. Docked conformation (a) of compound 11 (atom color green) and (b) of compound 16 (atom color green), along with NADPH (yellow color), in the active site of Leishmania
donovani PTR1 (magenta).
mostly surrounded by hydrophobic side chains, hydrogen bonding
interactions with compound 11 were not prominent. However,
dihydropyridine core of these compounds was held in the pocket
by combination of van der Waals, hydrophobic, and edge to face
3. Conclusion
We have developed an efficient synthesis of glycosyl dihy-
dropyridines involving three component reactions of sugar alde-
p-stacking interactions with the protein. Major hydrophobic
hydes, b-keto esters and anilines. The compounds were evaluated
contacts occurred between the dihydropyridine core and
substituted phenyl rings with the side chain of Phe 113, Tyr 194 and
Ala 230. As seen from our docking studies, the Phe 113 is involved in
against L. donovani both in vitro and in vivo and few of the
compounds displayed potent activities. These compounds with
antileishmanial activities comparable to standard drugs and better
SI values have led to a new lead for further exploration and
development of safe and effective antileishmanial drugs.
edge to face p-stacking interaction with the pyridine core.
This edge to face or T shaped aromatic stacking interaction
seems very important for Leishmania donovani PTR1 inhibition and
may be responsible for the stability of the protein ligand complex
(Fig. 1). The docking scores for inactive compound 16 were also in
coherence with the biological activity, where it was among the low
ranking compounds (Table 3). The binding mode for compound 16
in L. donovani PTR1 binding site revealed the reason for inferior
activity of compound 16, where it binds in opposite orientation to
that of highly active compound 11. The possible reason for this type
of bound conformation may be the substitution of O-ethyl group at
3 and 5 position of the dihydropyridine ring which faces steric
clashes with the cofactor NADPH. Therefore, to avoid steric clashes
with the cofactor NADPH compound 16 prefers opposite orienta-
4. Experimental
4.1. Chemistry
Commercially available reagent grade chemicals were used as
received. All reactions were followed by TLC on E. Merck Kieselgel
60 F254, with detection by UV light, spraying a 20% KMnO4 aq.
solution and/or spraying a 4% H2SO4 ethanolic solution. Column
chromatography was performed on silica gel (100e200 mesh
E. Merck). IR spectra were recorded as thin films or on KBr pellets
with a Perkin Elmer Spectrum RX-1 (4000e450 cmꢃ1) spectro-
photometer. 1H and 13C NMR spectra were recorded on a Brucker
DRX-300/200 in CDCl3 and CDCl3 þ CCl4. Chemical shift values are
reported in ppm relative to TMS (tetramethylsilane) as internal
reference, unless otherwise stated; s (singlet), d (doublet), t
(triplet), m (multiplet); J in hertz. ESI mass spectra were performed
tion which lead to decrease in the hydrophobic and
p-stacking
interactions with the protein and hence lower the activity of the
compound (Fig. 1). Our docking studies were however unable to
explain the reason behind the high activity of compound 17 which
was also among the low ranking compound.
Table 3
LigandFit Docking scores of the compounds 4e6 and 10e17.
Compound
DOCK Score
Ligscore1
Ligscore 2
PLP1
PLP2
JAIN
2.78
0.06
0.26
PMF
73.01
LUDI
4
5
6
ꢃ11.89
24.90
51.21
42.31
48.56
32.49
30.24
20.38
27.68
ꢃ0.66
ꢃ5.01
ꢃ1.07
1.13
2.86
2.55
3.19
1.88
1.71
2.31
2.16
0.17
ꢃ0.51
ꢃ2.01
2.32
4.47
4.39
4.58
2.89
2.94
3.59
4.60
0.84
ꢃ0.5
23.22
52.04
86.78
71.18
80.38
67.04
59.86
61.03
71.96
8.2
32.21
48.42
68.45
54.47
65.73
55.31
48.33
45.78
52.58
18.53
44.31
621
476
479
392
507
441
388
343
290
280
455
124.52
138.89
98.21
122.29
106.94
91.35
58.27
83.89
74
10
11
12
13
14
15
16
17
ꢃ0.4
1.08
ꢃ0.49
ꢃ0.31
ꢃ0.67
ꢃ2.05
ꢃ0.83
0.81
48
124.51

V.P. Pandey et al. / European Journal of Medicinal Chemistry 45 (2010) 2381e2388
2385
using Quattro II (Micromass). Elemental analyses were performed
on a PerkineElmer 2400 II elemental analyzer. Optical rotations
were measured in a 1.0 dm tube with a Rudolph Autopol III polar-
imeter in CHCl3.
(50 MHz, CDCl3):
d
¼ 169.0, 168.7, 148.2, 148.0, 138.7, 138.5, 138.1,
130.4, 130.2, 28.6, 127.7, 127.1, 111.2, 105.0, 103.7, 102.6, 83.8, 82.7,
82.1, 71.4, 51.5, 51.4, 32.6, 27.2, 26.7, 21.5, 18.4, 18.1. ESMS: m/z ¼ 564
[M þ H]þ. Anal. For C32H37NO8: C, 68.19; H, 6.62; N, 2.49; Found C,
68.12; H, 6.67; N, 2.45.
4.1.1. General procedure for the synthesis of 1-phenyl-4-glycosyl-
dihydropyrdine (4e17 and 19e21)
4.1.5. 2,6-Dimethyl-4-(30-O-benzyl-10,20-O-isopropylidene-
b-L-
A mixture of the selected dialdose (1a or 1b or 1c or 18,
14.5 mmol), methyl/ethylacetoacetate (30.0 mmol) and selected
aniline (14.5 mmol) in diethylene glycol in the presence of catalytic
amount of tetrabutylammonium hydrogensulphate (20 mol%) was
magenetically stirred at 80e100 ꢄC till the disappearance of start-
ing material. After completion of reaction (TLC), the reaction
mixture was poured over crushed ice and precipitated solid was
filtered. The crude solid mass was purified by column chromatog-
raphy (SiO2, 100e200 mesh) using appropriate eluant to give the
respective dihydropyrdine derivative.
threo-pentofuranos-40-yl)-1-(4-flurophenyl)-1,4-dihydropyridine-
3,5-dicarboxylic acid dimethyl ester (7)
It was obtained as yellow solid in 70% yield; mp 78e81 ꢄC;
Rf ¼ 0.5 (7:3 hexaneeEtOAc); [
a
]D ¼ ꢃ4.63 (c 0.1, CHCl3, at 25 ꢄC); IR
(KBr) ymax in cmꢃ1 3020, 2926, 2361, 1691, 1509, 1215; 1H NMR
(300 MHz, CDCl3 þ CCl4), ¼ 7.38e7.20 (m, 5H, ArH), 7.20e7.10 (m,
d
4H, ArH), 5.84 (d, J ¼ 3.81 Hz, 1H, H-10), 4.63e4.51 (m, 3H, OCH2Ph
and H-4), 4.47 (d, J ¼ 3.87 Hz, 1H, H-20), 3.98 (dd, J1 ¼3.18 Hz,
J2 ¼ 6.72 Hz, 1H, H-40), 3.84 (d, J ¼ 3.15 Hz, 1H, H-30), 3.64 (s, 3H,
COOCH3), 3.51 (s, 3H, COOCH3), 2.07 (s, 3H, CH3), 2.04 (s, 3H, CH3),
1.46, 1.28, (each s, 6H, (CH3)2C); 13C NMR (75 MHz, CDCl3 þ CCl4),
4.1.2. 2,6-Dimethyl-4-(30-O-benzyl-10,20-O-isopropylidene-
threo-pentofuranos-40-yl)-1-(4-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylic acid dimethyl ester (4)
b
-
L
-
d
¼ 168.1, 147.1, 147.1, 138.0, 132.1, 132.0, 128.2, 127.3, 126.8, 116.3,
116.0, 110.8, 104.5, 103.8, 102.7, 83.5, 82.5, 81.8, 71.0, 51.0, 32.3, 32.3,
26.8, 26.3, 17.9, 17.8; ESI MS: 568.2 [M þ H]þ, 590 [M þ Na]þ, HRMS
(JOEL MS Route) m/z calc. for C31H35FNO8 [M þ H]þ 568.23467,
found 568.23490.
It was obtained as yellow solid in 67% yield; mp 95e98 ꢄC;
Rf ¼ 0.5 (7:3 hexaneeEtOAc); [
a
]D ¼ 13.36 (c 0.1, CHCl3, at 25 ꢄC); IR
(KBr): ymax in cmꢃ1 2943, 1696, 1639, 1438, 1211, 1084; 1H NMR
(200 MHz, CDCl3):
d
¼ 7.40e7.24 (m, 5H, ArH), 7.08 (d, J ¼ 8.76 Hz,
4.1.6. 2,6-Dimethyl-4-(30-O-benzyl-10,20-O-isopropylidene-
b-L-
2H, ArH), 6.91 (d, J ¼ 8.76 Hz, 2H, ArH), 5.86 (d, J ¼ 3.94 Hz, 1H, H-
10), 4.67e4.54 (m, 3H, OCH2Ph, H-4), 4.48 (d, J ¼ 3.88 Hz, 1H, H-20),
4.01 (dd, J1 ¼3.24 Hz, J2 ¼ 7.78 Hz, 1H, H-40), 3.83e3.81 (m, 4H,
ArOCH3, H-30), 3.66, 3.46 (each s, 6H, 2ꢁCOOCH3), 2.07, 2.02 (each s,
6H, 2ꢁCH3), 1.46, 1.27 (each s, 6H, (CH3)2C); 13C NMR (50 MHz,
threo-pentofuranos-40-yl)-1-(4-flurophenyl)-1,4-dihydropyridine-
3,5-dicarboxylic acid diethyl ester (8)
It was obtained as yellow solid in 76% yield; m.p. ¼132e135 ꢄC;
Rf ¼ 0.5 (7:3 hexaneeEtOAc); [
a]
¼ 6.3 (c 0.1, CHCl3, at 25 ꢄC); IR
D
(KBr) ymax in cmꢃ1 3021, 2930, 1684, 1639, 1508, 1215; 1H NMR
CDCl3):
d
¼ 169.0,168.8,159.6,148.6,148.2,138.5,133.3,131.6,128.6,
(300 MHz, CDCl3 þ CCl4),
d
¼ 7.38e7.26 (m, 5H, ArH), 7.21e7.09
127.7, 127.2, 114.6, 111.2, 105.0, 103.6, 102.5, 83.8, 82.7, 82.1, 71.4,
55.7, 51.5, 32.6, 27.2, 26.7, 18.4, 18.1. ESMS: m/z ¼ 580 [M þ H]þ.
Anal. For C32H37NO9: C, 66.31; H, 6.43; N, 2.42; Found C, 66.28; H,
6.45; N, 2.40.
(m, 4H, ArH), 5.88 (d, J ¼ 3.90 Hz, 1H, H-10), 4.69e4.52 (m, 3H,
OCH2Ph and H-4), 4.50 (d, J ¼ 3.90 Hz, 1H, H-20), 4.14e4.00 (m, 5H,
OCH2CH3 and H-40), 3.88e3.84 (m, 1H, H-30), 2.07 (s, 3H, CH3), 2.02
(s, 3H, CH3), 1.29e1.21 (m, 9H, OCH2CH3 and (CH3)2C), 1.16e1.11
(t, J ¼ 7.10 Hz, OCH2CH3);13C NMR (75 MHz, CDCl3 þ CCl4),
¼ 168.1,
d
4.1.3. 2,4-Dimethyl-4-(30-O-benzyl-10,20-O-isopropylidene-
b
-
L
-
167.9, 146.9, 146.6, 138.1, 132.1, 132.0, 128.1, 127.3, 126.7, 116.2, 115.9,
110.9, 104.6, 104.0, 103.1, 83.5, 82.4, 81.8, 70.8, 59.8, 59.6, 32.6, 26.8,
26.3, 17.9, 17.7;ESI MS: 596.2 [M þ H]þ, 619.3 [M þ Na]þ, HRMS
(JOEL MS Route) m/z calc. for C33H39FNO8 [M þ H]þ 596.26597,
found 596.26815.
threo-pentofuranos-40-yl)-1-(4-chlorophenyl)-1,4-dihydropyridine-
3,5-dicarboxylic acid dimethyl ester (5)
It was obtained as yellow solid in 74% yield; mp 82e86 ꢄC;
Rf ¼ 0.5 (7:3 hexaneeEtOAc); [
a]
¼ ꢃ7.80 (c 0.1, CHCl3, at 25 ꢄC); IR
D
(KBr) ymax in cmꢃ1 2947, 1697, 1641, 1212, 1084; 1H NMR (200 MHz,
CDCl3)
d
¼ 7.42e7.20 (m, 7H, ArH), 7.15 (d, J ¼ 8.54 Hz, 2H, ArH),
4.1.7. 2,6-Dimethyl-4-(10,20-O-isopropylidene-
b-L-threo-
pentofuranos-40-yl)-1-(3,4-dichlorophenyl)-1,4-dihydropyridine-
3,5-dicarboxylic acid dimethyl ester (9)
5.85 (d, J ¼ 3.96 Hz, 1H, H-10), 4.65e4.53 (m, 3H, OCH2Ph, H-4), 4.47
(d, J ¼ 3.82 Hz, 1H, H-20), 3.99 (dd, J1 ¼3.28 Hz, J2 ¼ 7.18 Hz, 1H, H-
40), 3.84 (d, J ¼ 3.26 Hz, 1H, H-30), 3.63, 3.48 (each s, 6H,
2ꢁCOOCH3), 2.05, 2.02 (each s, 6H, 2ꢁCH3), 1.45, 1.27 (each s, 6H,
It was obtained as light yellow solid in 76% yield; mp
137e141 ꢄC; Rf ¼ 0.5 (8:2 hexaneeEtOAc); [
]D ¼ ꢃ15.06 (c 0.1,
a
(CH3)2C); 13C NMR (75 MHz, CDCl3)
d
¼ 168.7, 168.6, 147.9, 147.2,
CHCl3, at 25 ꢄC), IR (KBr) ymax in cmꢃ1, 3020, 1684, 1216, 1076; 1H
139.3, 138.4,134.9, 132.1, 129.8, 128.6, 127.8, 127.2, 111.3, 105.0, 104.3,
103.2, 83.9, 82.8, 82.1, 71.5, 51.5, 32.7, 32.0, 27.2, 26.6, 23.0,18.4,18.2,
14.5. ESMS: m/z ¼ 584 [M þ H]þ. Anal. For C31H34NClO8: C, 63.75; H,
5.87; N, 2.40; Found: C, 63.65; H, 5.90; N, 2.37.
NMR (200 MHz, CDCl3)
d
¼ 7.50e7.45 (m, 1H, ArH), 7.26e7.20 (m,
1H, ArH), 6.99e6.95 (m, 1H, ArH), 5.95 (d, J ¼ 3.08 Hz, 1H, H-10),
4.54 (dd, J1 ¼3.54 Hz, J2 ¼ 6.42 Hz, 1H, H-20), 4.22 (s, 1H, H-4), 4.00
(s, 1H, H-40), 3.73 (s, 1H, OH), 3.68, 3.66 (each s, 6H, 2ꢁCOOCH3),
3.18 (d, J ¼ 2.74 Hz,1H, H-30), 2.10,1.51 (each s, 6H, 2ꢁCH3),1.29,1.14
4.1.4. 2,6-Dimethyl-4-(30-O-benzyl-10,20-O-isopropylidene-
threo-pentofuranos-40-yl)-1-(4-methylphenyl)-1,4-
dihydropyridine-3,5-dicarboxylic acid dimethyl ester (6)
b
-
L
-
(s, 6H, (CH3)2C); 13C NMR (50 MHz, CDCl3):
d
¼ 171.4, 167.8, 156.0,
140.1, 133.4, 131.9, 130.1, 129.3, 112.3, 106.2, 96.5, 84.4, 83.7, 77.9,
77.8, 76.7, 74.3, 72.9, 72.6,52.1, 51.4, 42.4, 33.0, 30.1, 27.1, 26.5, 26.0,
19.5. ESMS: m/z ¼ 528 [M þ H]þ Anal. For C24H27NCl2O8: C, 54.55;
H, 5.15; N, 2.65; Found C, 54.52; H, 5.25; N, 2.67.
It was obtained as yellow solid in 78 % yield; mp 78e82 ꢄC;
Rf ¼ 0.5 (7:3 hexaneeEtOAc); [
a
]D ¼ 6.58 (c 0.1, CHCl3, at 25 ꢄC); IR
(KBr) ymax in cmꢃ1 2986, 2946, 1696, 1211, 1058; 1H NMR (200 MHz,
CDCl3)
d
¼ 7.32e7.24 (m, 5H, ArH), 7.18 (d, J ¼ 8.08 Hz, 2H, ArH), 7.05
4.1.8. 2,6-Dimethyl-4-(10,20-O-isopropylidene-
b-L-threo-
(d, J ¼ 8.22 Hz, 2H, ArH), 5.86 (d, J ¼ 3.96 Hz, 1H, H-10), 4.68e4.53
(m, 3H, OCH2Ph, H-4), 4.48 (d, J ¼ 3.92 Hz, 1H, H-20), 4.02 (dd,
J1 ¼3.24 Hz, J2 ¼ 7.98 Hz, 1H, H-40), 3.83 (d, J ¼ 3.22 Hz, 1H, H-20)
3.66, 3.43 (each s, 6H, 2ꢁCOOCH3), 2.40 (s, 3H, ArCH3), 2.07, 2.02
(each s, 6H, 2ꢁCH3), 1.46, 1.27 (each s, 6H, (CH3)2C); 13C NMR
pentofuranos-40-yl)-1-phenyl-1,4-dihydropyridine-3,5-dicarboxylic
acid dimethyl ester (10)
It was obtained as light yellow solid in 68% yield; mp 85e89 ꢄC;
Rf ¼ 0.5 (8:2, hexaneeEtOAc); [
a
]D ¼ ꢃ22.97 (c 0.1, CHCl3 at 25 ꢄC);
IR (KBr) ymax in cmꢃ1 3020, 1676, 1216, 1089; 1H NMR (300 MHz,

2386
V.P. Pandey et al. / European Journal of Medicinal Chemistry 45 (2010) 2381e2388
CDCl3)
d
¼ 7.48e7.31 (m, 4H, ArH), 7.07e7.03 (m, 1H, ArH), 5.98
3419, 2950, 1705, 1654, 1212, 1090; [
25 ꢄC); 1H NMR (300 MHz, CDCl3)
a
]
¼ ꢃ38.05 (c 0.1, CHCl3, at
D
(d, J ¼ 3.66 Hz, 1H, H-10), 4.52 (d, J ¼ 3.60 Hz, 1H, H-4), 4.27
(t, J ¼ 3.03 Hz, 1H, H-20), 4.05 (d, J ¼ 1.80 Hz, 1H, H-40), 3.77 (s, 1H,
OH), 3.69, 3.68 (each s, 6H, 2ꢁCOOCH3), 3.20 (d, J ¼ 2.76 Hz, 1H, H-
30), 2.10, 1.54 (each s, 6H, 2ꢁCH3), 1.30, 1.27 (s, 6H, (CH3)2C); 13C
d
¼ 7.09 (d, J ¼ 8.85 Hz, 2H, ArH),
6.91 (d, J ¼ 8.85 Hz, 2H, ArH), 5.90 (d, J ¼ 3.96 Hz, 1H, H-10),
4.51e4.48 (m, 2H, H-4, H-20), 3.97 (dd, J1 ¼3.18 Hz, J2 ¼ 7.08 Hz, 1H,
H-40), 3.85 (s, 3H, ArOCH3), 3.76, 3.75 (each s, 6H, 2ꢁCOOCH3), 3.58
(d, J ¼ 2.12 Hz, 1H, H-30), 3.32 (s, 3H, OCH3), 2.08, 2.02 (each s, 6H,
2ꢁCH3), 1.48, 1.27 (each s, 6H, (CH3)2C); 13C NMR (50 MHz, CDCl3):
NMR (50 MHz, CDCl3):
d
¼ 171.4, 156.6, 139.2, 134.5, 133.1, 131.8,
129.6, 111.9, 106.2, 94.1, 84.2, 83.7, 74.3, 52.1, 51.3, 42.5, 33.0, 27.1,
26.5, 25.9, 19.5 ESMS: m/z ¼ 460 [M þ H]þ; Anal. For C24H29NO8: C,
62.73; H, 6.36; N, 3.05; Found: C, 62.53; H, 6.51; N, 3.10.
d
¼ 169.0, 168.8, 159.6, 148.4, 148.1, 133.3, 131.6, 114.6, 111.2, 105.1,
103.4, 101.9, 84.7, 83.6, 81.2, 78.0, 77.3, 76.7, 57.2, 55.7, 51.5, 33.2,
27.2, 26.6, 18.3, 18.1; ESMS: m/z ¼ 504 [M þ H]þ Anal. For
C26H33NO9: C, 62.02; H, 6.61; N, 2.78; Found: C, 62.03; H, 6.59; N,
2.59.
4.1.9. 2,6-Dimethyl-4-(10,20-O-isopropylidene-
b-L-threo-
pentofuranos-40-yl)-1-(4-chlorophenyl)-1,4-dihydropyridine-3,5-
dicarboxylic acid dimethyl ester (11)
It was obtained as light yellow solid in 77% yield; mp 88e92 ꢄC;
4.1.13. 2,6-Dimethyl-4-(10,20-O-isopropylidene-30-O-methyl-
b-L-
Rf ¼ 0.5 (7:3 hexaneeEtOAc); [
a
]D ¼ ꢃ9.13 (c 0.1, CHCl3, at 25 ꢄC); IR
threo-pentofuranos-40-yl)-1-(4-chlorophenyl)-1,4-dihydropyridine-
3,5-dicarboxylic acid dimethyl ester (15)
(KBr) ymax in cmꢃ1 3020, 1698, 1597, 1216, 1043; 1H NMR (200 MHz,
CDCl3)
d
¼ 7.40e7.34 (m, 2H, ArH), 7.06e6.93 (m, 2H, ArH), 5.73
It was obtained light yellow solid in 73%; mp 190e194 ꢄC;
(d, J ¼ 3.42 Hz, 1H, H-10), 4.57 (d, J ¼ 3.66 Hz, 1H, H-4), 4.19e4.15
(m, 2H, H-20 and H-40), 3.94 (s, 1H, OH), 3.69 (s, 6H, 2ꢁCOOCH3),
2.56 (d, J ¼ 2.86 Hz, 1H, H-30), 2.08, 1.49 (each s, 6H, 2ꢁCH3), 1.30
Rf ¼ 0.5 (7: 3 hexaneeEtOAc); [
a
]
¼ ꢃ48.47 (c 0.1, CHCl3, at 25 ꢄC);
D
IR (KBr) ymax in cmꢃ1 3020, 1692, 1652, 1316, 1216, 1046; 1H NMR
(300 MHz, CDCl3)
d
¼ 7.40 (d, J ¼ 6.69 Hz, 2H, ArH), 7.15
(s, 6H, (CH3)2C); 13C NMR (50 MHz, CDCl3):
d
¼ 170.3, 167.6, 155.2,
(d, J ¼ 6.81 Hz, 2H, ArH), 5.87 (d, J ¼ 3.96 Hz, 1H, H-10), 4.49e4.46
(m, 2H, H-4, H-20), 3.94 (dd, J1 ¼3.18 Hz, J2 ¼ 6.33 Hz, 1H, H-40),
3.75, 3.74 (each s, 6H, 2ꢁCOOCH3), 3.57 (d, J ¼ 3.18 Hz, 1H, H-30),
3.29 (s, 3H, OCH3), 2.38 (s, 3H, ArCH3), 2.05, 2.00 (each s, 6H,
2ꢁCH3), 1.45, 1.30 (each s, 6H, (CH3)2C); 13C NMR (75 MHz, CDCl3):
138.8, 134.6, 133.4, 131.9, 130.1, 129.3, 112.3, 106.2, 97.0, 84.4, 84.1,
76.9, 74.7, 51.9, 51.3, 43.2, 33.1, 27.4, 26.7, 25.4, 19.5 ESMS: m/z ¼ 494
[M þ H]þ. Anal. For C24H28NClO8: C, 58.36; H, 5.71; N, 2.84; Found C,
58.26; H, 5.78; N, 2.85.
d
¼ 168.4, 168.4, 147.5, 146.6, 138.9, 134.5, 131.7, 129.4, 110.8, 104.7,
4.1.10. 2,6-Dimethyl-4-(10,20-O-isopropylidene-30-O-methyl-
threo-pentofuranos-40-yl)-1-(4-methylphenyl)-1,4-
dihydropyridine-3,5-dicarboxylic acid dimethyl ester (12)
b-
L
-
103.7, 102.0, 84.4, 83.2, 80.8, 56.8, 51.2, 51.1, 32.9, 26.7, 26.1, 17.9, 17.7
ESMS: m/z ¼ 508 [M þ H]þ Anal. For C25H28NClO8: C, 59.11; H, 5.95;
N, 2.76; Found: C, 59.10; H, 5.97; N, 2.80.
It was obtained as light yellow solid in 78% yield; mp
185e188 ꢄC; Rf ¼ 0.5 (7:3 hexaneeEtOAc); [
a
]
¼ ꢃ59.69 (c 0.1,
4.1.14. 2,6-Dimethyl-4-(10,20-O-isopropylidene-30-O-methyl-
threo-pentofuranos-40-yl)-1-(4-methylphenyl)-1,4-
dihydropyridine-3,5-dicarboxylic acid diethyl ester (16)
b-L-
D
CHCl3, at 25 ꢄC); IR (KBr) ymax in cmꢃ1 3020, 1689, 1216, 1039; 1H
NMR (200 MHz, CDCl3)
d
¼ 7.20 (d, J ¼ 8.14 Hz, 2H, ArH), 7.03
(d, J ¼ 8.24 Hz, 2H, ArH), 5.86 (d, J ¼ 3.98 Hz, 1H, H-10), 4.47e4.44
(m, 2H, H-4, H-20), 3.95 (dd, J1 ¼3.18 Hz, J2 ¼ 7.30 Hz,1H, H-40), 3.73,
3.72 (each s, 6H, 2ꢁCOOCH3), 3.55 (d, J ¼ 3.20 Hz, 1H, H-30), 3.29
(s, 3H, OCH3), 2.38 (s, 3H, ArCH3), 2.05, 1.98 (each s, 6H, 2ꢁCH3),
It was obtained as light yellow solid in 70% yield; mp
180e183 ꢄC; Rf ¼ 0.5 (6:4 hexaneeEtOAc); [
a]
¼ ꢃ36.48 (c 0.1,
D
CHCl3, at 25 ꢄC); IR (KBr) ymax in cmꢃ1 3020, 1687, 1215, 1085, 1034;
1H NMR (200 MHz, CDCl3)
d
¼ 7.19 (d, J ¼ 8.18 Hz, 2H, ArH), 7.03
1.45,1.28 (each s, 6H, (CH3)2C); 13C NMR (50 MHz, CDCl3):
d
¼ 169.0,
(d, J ¼ 8.20 Hz, 2H, ArH), 5.88 (d, J ¼ 4.00 Hz, 1H, H-10), 4.55e4.47
(m, 2H, H-4, H-20), 4.26e4.12 (m, 4H, 2ꢁCOOCH2CH3), 3.99 (dd,
J1 ¼3.22 Hz, J2 ¼ 7.12 Hz, 1H, H-40), 3.57(d, J ¼ 3.20 Hz, 1H, H-30),
3.29 (s, 3H, OCH3), 2.38 (s, 3H, ArCH3), 2.04, 1.98 (each s, 6H,
2ꢁCH3), 1.46 (s, 3H, (CH3)2C), 1.34e1.25 (m, 9H, (CH3)2C and
168.8, 148.0, 147.9, 138.6, 138.1, 130.4, 130.1, 111.1, 105.1, 103.4, 102.0,
84.6, 83.6, 81.2, 57.2, 51.5, 33.1, 27.2, 26.6, 21.54, 18.3, 18.1; ESMS: m/
z ¼ 488 [M þ H]þ; Anal. For C26H33NO8: C, 64.05; H, 6.82; N, 2.87;
Found: C, 64.10; H, 6.89; N, 2.79.
2ꢁCOOCH2CH3) 13C NMR (50 MHz CDCl3):
d
¼ 168.8, 168.5, 147.7,
4.1.11. 2,6-Dimethyl-4-(10,20-O-isopropylidene-30-O-methyl-
b
-
L
-
147.5, 138.6, 138.2, 130.5, 130.1, 111.1, 105.1, 103.7, 102.3, 84.7, 83.6,
81.3, 60.1, 60.0, 57.1, 33.2, 27.1, 26.6, 21.5, 18.4, 18.1, 14.8, 14.7 ESMS:
m/z ¼ 516 [M þ H]þ; Anal. For C28H37NO8: C, 65.23; H, 7.23; N, 2.72;
Found: C, 65.18; H, 7.30; N, 2.80.
threo-pentofuranos-40-yl)-1-(4-bromophenyl)-1,4-dihydropyridine-
3,5-dicarboxylic acid dimethyl ester (13)
It was obtained as light yellow solid in 64% yield; mp
209e212 ꢄC; Rf ¼ 0.5 (6:4 hexaneeEtOAc); [
a]
¼ ꢃ33.56 (c 0.1,
D
CHCl3, at 25 ꢄC). IR (KBr) ymax in cmꢃ1 3020, 1697, 1215, 1082; 1H
4.1.15. 2,6-Dimethyl-4-(10,20-O-isopropylidene-30-O-methyl-
threo-pentofuranos-40-yl)-1-(2-methoxyphenyl)1,4-
dihydropyridine-3,5-dicarboxylic acid diethyl ester (17)
b-L-
NMR (200 MHz, CDCl3)
d
¼ 7.55 (d, J ¼ 6.64 Hz, 2H, ArH), 7.09
(d, J ¼ 6.62 Hz, 2H, ArH), 5.86 (d, J ¼ 4.00 Hz, 1H, H-10), 4.47e4.42
(m, 2H, H-4, H-20), 3.92 (dd, J1 ¼3.24 Hz, J2 ¼ 6.22 Hz, 1H, H-40),
3.74, 3.73 (each s, 6H, 2ꢁCOOCH3), 3.56 (d, J ¼ 3.22 Hz, 1H, H-30),
3.28 (s, 3H, OCH3), 2.04, 2.00 (each s, 6H, 2ꢁCH3), 1.44, 1.25 (each s,
It was obtained as light yellow solid in 71% yield; mp
160e163 ꢄC; Rf ¼ 0.5 (6:4 hexaneeEtOAc); [
a]
¼ ꢃ26.43 (c 0.1,
D
CHCl3, at 25 ꢄC); IR (KBr) ymax in cmꢃ1 3020, 1682, 1216, 1042; 1H
6H, (CH3)2C) 13C NMR (50 MHz, CDCl3):
d
¼ 168.7,147.8,146.9,139.9,
NMR (200 MHz, CDCl3)
d
¼ 7.40e7.31 (m, 1H, ArH), 7.08e6.91
132.8, 132.5, 122.9, 111.2, 105.1, 104.1, 102.5, 84.9, 83.6, 81.2, 77.9,
77.3, 76.7, 57.2, 55.7, 51.6, 33.3, 27.2, 26.6, 18.3, 18.2, ESMS: m/
z ¼ 552 [M þ H]þ, 574 [M þ Na]þ Anal. For C25H30NBrO9: C, 54.36;
H, 5.47; N, 2.54; Found: C, 54.43; H, 5.44; N, 2.57.
(m, 3H, ArH), 5.88 (d, J ¼ 3.94 Hz, 1H, H-10), 4.58e4.47 (m, 2H, H-4,
H-20), 4.25e4.15 (m, 4H, 2ꢁCOOCH2CH3), 3.98 (dd, J1 ¼3.18 Hz,
J2 ¼ 6.98 Hz, 1H, H-40), 3.82 (s, 3H, ArOCH3), 3.56 (d, J ¼ 4.00 Hz, 1H,
H-30), 3.28 (s, 3H, OCH3), 2.02, 1.96 (each s, 6H, 2ꢁCH3), 1.46 (s, 6H,
(CH3)2C), 1.34e1.24 (m, 9H, (CH3)2C and 2ꢁCOOCH2CH3), 13C NMR
4.1.12. 2,6-Dimethyl-4-(10,20-O-isopropylidene-30-O-methyl-
threo-pentofuranos-40-yl)-1-(4-methoxyphenyl)-1,4-
dihydropyridine-3,5-dicarboxylic acid dimethyl ester (14)
b
-L
-
(50 MHz, CDCl3):
d
¼ 168.9, 168.7, 157.0, 148.6, 148.2, 131.2, 130.5,
29.8, 121.7, 111.7, 111.1, 105.2, 102.8, 101.3, 84.8, 83.4, 81.3, 60.0, 59.9,
57.1, 55.5, 33.2, 27.2, 26.6, 16.5, 16.2, 14.8, 14.8 ESMS: m/z ¼ 532
[M þ H]þ, 554 [M þ Na]þ Anal. For C28H37NO9: C, 63.26; H, 7.02; N,
2.63; Found : C, 63.30; H, 7.17; N, 2.59.
It was obtained as light yellow solid in 70% yield; mp
214e217 ꢄC; Rf ¼ 0.5 (6:4 hexaneeEtOAc); IR (KBr) ymax in cmꢃ1

V.P. Pandey et al. / European Journal of Medicinal Chemistry 45 (2010) 2381e2388
2387
4.1.16. 2,6-Dimethyl-4-(10,20,30,40-di-O-isopropylidene-
a
-l-arabino-
The in vitro effect of the compounds on the growth of promas-
tigotes was assessed by monitoring the luciferase activity of viable
cells after treatment. The transgenic promastigotes of late log phase
ml medium 199 well in 96-well flat-
bottomed microtiter (MT) plates (CELLSTAR) and incubated for 72 h
in medium alone or in the presence of serial dilutions of drugs
pyranos-50-yl)-1-(4-chlorophenyl)-1,4-dihydropyridine-3,5-
dicarboxylic acid dimethyl ester (19)
It was obtained as yellow solid in 76% yield; mp 190e194 ꢄC;
were seeded at 5 ꢁ 105/100
Rf ¼ 0.5 (7:3 hexaneeEtOAc); [
a
]D ¼ ꢃ73.83(c 0.1, CHCl3, at 25 ꢄC),
IR (KBr) ymax in cmꢃ1 3020, 1689, 1216, 1068; 1H NMR (200 MHz,
CDCl3)
d
¼ 7.40 (dd, J1 ¼ 2.00 Hz, J2 ¼ 6.58 Hz, 2H, ArH), 7.19 (dd,
(1e40
m
g/ml) in DMSO. Parallel dilutions of DMSO were used as
l) of promastigote
J1 ¼2.08 Hz, J2 ¼ 6.64 Hz, 2H, ArH); 5.48 (d, J ¼ 4.86 Hz, 1H, H-10),
4.51 (dd, J1 ¼2.16 Hz, J2 ¼ 8.03 Hz, 1H, H-3), 4.40 (d, J ¼ 2.12 Hz, 1H,
H-4), 4.27 (dd, J1 ¼1.38 Hz, J2 ¼ 7.90 Hz, 1H, H-40), 4.20 (dd,
J1 ¼2.16 Hz, J2 ¼ 4.88 Hz, 1H, H-20), 3.71, 3.68 (each s, 6H,
2ꢁCOOCH3), 3.60 (dd, J1 ¼1.28 Hz, J2 ¼ 3.76 Hz, 1H, H-50), 2.00
(s, 6H, 2ꢁCH3), 1.46, 1.39, 1.27, 1.25 (each s, 12H, 2ꢁ(CH3)2C); 13C
controls. After incubation, an aliquot (50
m
suspension was aspirated from each well of a 96-well plate and
mixed with an equal volume of Steady GloÒ reagent (Promega) and
luminescence was measured by a luminometer. The values were
expressed as relative luminescence unit (RLU) [32]. The inhibition
of parasitic growth is determined by comparison of the luciferase
activity of drug treated parasites with that of untreated controls by
the general formula:
NMR (50 MHz CDCl3):
d
¼ 168.9, 168.8, 149.7, 147.3, 139.9, 134.6,
132.1, 129.7, 125.2, 109.9, 109.4, 108.6, 103.1, 101.2, 97.2, 74.7, 73.3,
71.7, 71.5, 71.2, 51.5, 51.2, 35.6, 26.2, 25.5, 25.2, 18.5, 18.3 ESMS: m/
z ¼ 564 [M þ H]þ Anal. For C28H34ClNO9 : C, 59.63; H, 6.08; N, 2.48;
Found : C, 59.61; H, 6.12; N,2.50.
%Inhibition ¼ ðN ꢃ n ꢁ 100Þ=N
where N is average relative luminescence unit (RLU) of control
wells; and n is average RLU of treated wells.
4.1.17. 2,6-Dimethyl-4-(10,20,30,40-di-O-isopropylidene-
a-L-
arabinopyranos-50-yl)-1-(4-methoxyphenyl)-1,4-dihydropyridine-
3,5-dicarboxylic acid dimethyl ester (20)
It was obtained as yellow solid in 78% yield; mp 190e193 ꢄC;
4.2.2. Anti-amastigote assay
Rf ¼ 0.5 (7:3 hexaneeEtOAc); [
a
]
¼ ꢃ73.94 (c 0.1, CHCl3, at 25 ꢄC);
D
For assessing the activity of compounds against the amastigote
stage of the parasite, mouse macrophage cell line (J-774A.1) infec-
ted with promastigotes expressing luciferase firefly reporter gene
IR (KBr) ymax in cmꢃ1 2984, 2934, 1697, 1637, 1206, 1073; 1H NMR
(200 MHz, CDCl3)
d
¼ 7.10 (dd, J1 ¼ 2.72 Hz, J2 ¼ 6.72 Hz, 2H, ArH),
was used. Cells were seeded in a 96-well plate (4 ꢁ104cell/100
ml/
6.88 (dd, J1 ¼ 2.14 Hz, J2 ¼ 6.86 Hz, 2H, ArH), 5.47 (d, J ¼ 2.94 Hz, 1H,
H-10), 4.48 (dd, J1 ¼ 2.12 Hz, J2 ¼ 7.90 Hz, 1H, H-30), 4.43
(d, J ¼ 9.28 Hz, 1H, H-4), 4.24 (dd, J1 ¼1.24 Hz, J2 ¼ 7.84 Hz, 1H, H-
40), 4.17 (dd, J1 ¼ 2.16 Hz, J2 ¼ 4.96 Hz, 1H, H-20), 3.81 (s, 3H,
ArOCH3), 3.69, 3.65 (each s, 6H, 2ꢁCOOCH3), 3.57 (dd, J1 ¼1.18 Hz,
J2 ¼ 4.16 Hz, 1H, H-20), 2.00, 1.98 (each s, 6H, 2ꢁCH3), 1.46, 1.41, 1.26,
well) in RPMI-1640 containing 10% fetal calf serum and the plates
were incubated at 37 ꢄC in a CO2 incubator. After 24 h, the medium
was replaced with fresh medium containing stationary phase
promastigotes (4 ꢁ105/100
ml/well). Promastigotes invade the
macrophages and are transformed into amastigotes. The test
compounds are added at two fold dilutions up to 7 points in
1.24 (each s, 12H, 2ꢁ(CH3)2C); 13C NMR (50 MHz, CDCl3):
¼ 168.8,
d
168.7, 167.7, 159.4, 153.1, 150.1, 148.0, 145.7, 134.0, 132.6, 131.5, 126.2,
116.6, 115.0, 114.5, 110.3, 109.7, 109.3, 109.1, 109.0, 108.4, 108.2, 102.5,
100.8, 74.7, 73.3, 71.7, 71.5, 71.2, 51.5, 51.2, 35.6, 26.2, 25.5, 25.2, 18.5,
18.3 ESMS: m/z ¼ 560 [M þ H]þ 582 [M þ Na]þ; Anal. For
C29H37NO10: C, 62.24; H, 6.66; N, 2.50; Found : C, 62.30; H, 6.70; N,
2.47.
complete medium starting from 40 mg/ml conc. after replacing the
previous medium and the plates were incubated at 37 ꢄC in a CO2
incubator for 72 h. After incubation, the drug containing medium
was decanted and 50 ml PBS was added in each well and mixed with
an equal volume of steady Glo reagent. After gentle shaking for
1e2 min, the reading was taken in a luminometer as repoeted
earlier [33,34]. The values are expressed as relative luminescence
units (RLU). Data are transformed into a graphic program (Excel).
IC50 of antileishmanial activity was calculated by nonlinear
regression analysis of the concentrationeresponse curve using the
four parameter Hill equations.
4.1.18. 2,6-Dimethyl-4-(10,20,30,40-di-O-isopropylidene-
a-L-
arabinopyranos-50-yl)-1-(4-aminophenyl)-1,4-dihydropyridine-3,5-
dicarboxylic acid dimethyl ester (21)
It was obtained as yellow solid in 66% yield; mp 189e192 ꢄC;
Rf ¼ 0.5 (7: 3 hexaneeEtOAc); [
a
]D ¼ ꢃ73.94 (c 0.1, CHCl3, at 25 ꢄC);
IR (KBr) ymax in cmꢃ1 3021, 2929, 1683, 1587, 1216; 1H NMR
(200 MHz, CDCl3)
d
¼ 6.94 (d, J ¼ 8.12 Hz, 2H, ArH), 6.67
4.2.3. Cytotoxicity assay
(d, J ¼ 8.18 Hz, 2H, ArH); 5.51 (d, J ¼ 2.94 Hz, 1H, H-10), 4.51e4.44
(m, 2H, H-30 and H-4), 4.26e4.22 (m, 2H, H-40 and H-20), 3.70e3.61
(m, 9H, 2ꢁCOOCH3, H-50, NH2), 2.05, 2.02 (each s, 6H, 2ꢁCH3), 1.39,
1.38, 1.27, 1.25 (each s, 12H, 2ꢁ(CH3)2C); 13C NMR (50 MHz, CDCl3):
The cell viability was determined using the MTT assay
developed by Mossman et al. [35] Exponentially growing cells
(J-774A.1) (1 ꢁ105cells /100
ml/well) were incubated with
different drug concentrations for 72 h and were incubated at
37 ꢄC in a humidified mixture of CO2 and 95% air in an incubator.
Stock solutions of compounds prepared in DMSO were further
d
¼ 169.5, 150.2, 148.7, 146.6, 131.6, 131.3, 115.4, 109.5, 108.6, 102.2,
100.7, 72.8, 71.7, 71.6, 71.4, 51.4, 51.2, 35.5, 30.1, 29.7, 26.3, 25.5, 25.2,
18.4, 18.3; ESMS: m/z ¼ 567 [M þ Na]þ Anal. For C28H36N2O9: C,
61.75; H, 6.66; N, 5.14; Found: C, 61.77; H, 6.62; N, 5.13.
diluted with fresh complete medium. After incubation, 25 ml of
MTT reagent (5 mg/ml) in PBS medium, followed by syringe
filtration were added to each well and incubated at 37 ꢄC for 2 h.
At the end of the incubation period, the supernatant was
removed by tilting plate completely without disturbing cell layer
4.2. Biology
and 150 ml of pure DMSO are added to each well. After 15 min of
4.2.1. Anti-promastigote assay
The L. donovani promastigotes (MHOM IN/Dd8; originally
obtained from Imperial college, London) were transfected with fire
fly luciferase gene, and the transfectants were maintained in
medium 199 (Sigma chemical Co., USA) supplemented with 10%
shaking the readings were recorded as absorbance at 544 nm on
a micro plate reader. The cytotoxic effect were expressed as 50%
lethal dose, i.e., as the concentration of a compound which
provoked a 50% reduction in cell viability compared to cell in
culture medium alone. IC50 values were estimated as described
by Huber and Koella [36].
fetal calf serum (GIBCO) and gentamycin (4
under pressure of G 418 (Sigma) [26].
mg/ml) solution (Sigma)

2388
V.P. Pandey et al. / European Journal of Medicinal Chemistry 45 (2010) 2381e2388
4.2.4. In vivo assay
Appendix A. Supplementary data
The method of Beveridge [37], as modified by Bhatnagar et al.
[38] and Gupta et al. [39] was used for in vivo screening. Golden
hamsters (inbred strain) of either sex weighing 40e45 g were
infected intracardially with 1 ꢁ107 amastigotes per animal. The
infection is well adapted to the hamster model and establishes
itself in 15e20 days. Meanwhile, hamsters gain weight (85e95 g)
and can be subjected to repeated spleen biopsies. Pre-treatment
spleen biopsy in all the animals is carried out to assess the
degree of infection. The animals with þ1 infection (5e15
amastigotes/100 spleen cell nuclei) are included in the chemo-
therapeutic trials. The infected animals are randomized into
several groups on the basis of their parasitic burdens. Five to six
animals are used for each test sample. Drug treatment by
intraperitoneal (i.p.) route is initiated after 2 days of biopsy and
continued for 5 consecutive days. Post-treatment biopsies are
done on day 7 of the last drug administration and amastigote
counts are assessed by Giemsa staining. Intensity of infection in
both, treated and untreated animals, as also the initial count in
treated animals is compared and the efficacy is expressed in
terms of % inhibition of amastigotes multiplication using the
following formula:
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2010.02.018.
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Acknowledgments
Authors thank DRDO, ICMR and DBT New Delhi for financial
assistance. VPP, SSB and MM are thankful to CSIR New Delhi for SRF.
It is a CDRI Communication No. 7815.