PEG-mediated one-pot multicomponent reactions for the efficient synthesis of functionalized dihydropyridines and their functional group dependent DNA cleavage activity

Article abstract of DOI:10.1016/j.bioorg.2013.03.003

Polyethylene glycol (PEG) has been found to be an inexpensive, non-toxic and useful medium for the one pot synthesis of highly functionalized dihydropyridines using multicomponent reactions (MCRs) at room temperature under catalyst free conditions. The no

Full text of DOI:10.1016/j.bioorg.2013.03.003

Bioorganic Chemistry 48 (2013) 8–15
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
PEG-mediated one-pot multicomponent reactions for the efficient synthesis
of functionalized dihydropyridines and their functional group dependent DNA
cleavage activity
⇑
⇑
Suman Pal, Vandana Singh, Prolay Das , Lokman H. Choudhury
Department of Chemistry, Indian Institute of Technology Patna, Patna 800 013, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2012
Available online 9 April 2013
Polyethylene glycol (PEG) has been found to be an inexpensive, non-toxic and useful medium for the one
pot synthesis of highly functionalized dihydropyridines using multicomponent reactions (MCRs) at room
temperature under catalyst free conditions. The notable features of this protocol are: mild reaction con-
dition, applicability to wide range of substrates, reusability of the PEG and good yields. The interaction of
the synthesized compounds with pUC19 plasmid DNA was also analyzed. Some of the synthesized com-
pounds showed interesting functional group dependent nuclease activity for plasmid DNA cleavage
under physiological conditions.
Keywords:
Dihydropyridine derivatives
PEG
MCRs
Ó 2013 Elsevier Inc. All rights reserved.
pUC 19
DNA cutting activity
1. Introduction
various organic transformations under mild reaction conditions
[7]. PEG is considered as a non-ionic hydrophilic polymer which
Dihydropyridine (DHP) derivatives are abundant in various nat-
ural products as well as in synthetic pharmaceuticals. Substituted
DHPs exhibit diverse biological activities and have been used for
the treatment of Alzheimer’s disease, angina pectoris and cardio-
vascular diseases including hypertension [1]. They also demon-
strate anticancer, antitumor, antioxidant, antimutagenic and
antidiabetic activities [2]. In addition to these, substituted DHPs
exhibit other therapeutic properties including Platelet antiaggrega-
tory activities, neuroprotection, radioprotection, noncompetitive
inhibition of topoisomerase I and HIV protease inhibition [3]. DHPs
can also be used as hydrogen transfer reagents similar to coen-
zymes NADH and NADPH [4]. In view of the prominent pharma-
ceutical significance of DHPs, considerable attention have been
paid in recent times by organic as well as medicinal chemists for
the design and development of newer and greener methodologies
for the efficient synthesis of highly functionalized DHPs [5].
In continuation to our endeavor to develop multicomponent
reactions (MCRs) for the access to new functionalized small mole-
cules [6], we were interested to develop a greener protocol for the
synthesis of highly functionalized 6-amino DHP derivatives using
one-pot MCRs.
can be used in drug delivery and in bioconjugates as tools for diag-
nostics [8]. Most importantly, it is an inexpensive, non-toxic, ther-
mally stable, easy to handle and recyclable medium. Considering
its green chemical aspects, we were inspired to test the usefulness
of PEG in multicomponent reactions for the synthesis of highly
functionalized 6- amino DHPs.
Metal-free small molecules that can interact with DNA through
recognition, binding, modifying, cleaving or crosslinking have been
shown to have widespread applications [9]. Though DHPs are
widely used in the pharmaceutical and pharmacological fields, re-
port of interaction of these interesting small molecules with DNA is
scarce [10]. This prompted us to investigate the interaction be-
tween our synthesized DHPs with DNA. A number of simple
in vitro experiments [11], including evaluation of plasmid DNA-
compound interaction [12] can be performed to determine
whether the compounds physically interact with DNA. Many
chemical compounds show nuclease activity on supercoiled plas-
mid (Form I) by introducing nicks (relaxed circular plasmid, Form
II) or double strand breaks (linear plasmid, Form III) [13]. The com-
pounds recognize specific DNA sequences, intercalate into DNA
double strands or catalyze the hydrolysis of phosphodiester bonds
[14]. Hydrolytic cleavage of DNA by small molecules is less com-
mon than by transition metal ions, lanthanide ions, actinide ions
or their complexes [15]. Herein we report, a catalyst free synthesis
of 6-amino dihydropyridine derivatives and their functional group
dependent DNA cleaving activities [16].
Polyethylene glycol (PEG) has gained significant interest as a
powerful solvent system as well as a phase transfer catalyst for
⇑
Corresponding authors. Fax: +91 6122277383.
E-mail addresses: prolay@iitp.ac.in (P. Das), lokman@iitp.ac.in (L.H. Choudhury).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.03.003

S. Pal et al. / Bioorganic Chemistry 48 (2013) 8–15
9
Next, we checked the scope and general applicability of this PEG-
mediated MCR, through substrate variability of aldehyde, amine
and malononitrile derivatives under similar reaction conditions.
This methodology was found to be applicable to aromatic alde-
hydes tethered with both electron withdrawing as well as electron
releasing groups. Similarly, a wide range of substituted anilines
have been found to be pertinent for this methodology. Interest-
ingly, bulky amine such as b-naphthyl amine also reacted under
similar reaction condition to provide the corresponding DHP 2a
in 82% yield (Table 2, entry 2). As evident from Table 2, benzyl
amine offered moderate yield of the corresponding DHP (2e). Mal-
ononitrile derivatives such as 2-cyanoacetamide (Table 2, entry 3),
ethyl 2-cyanoacetate (Table 2, entries 9 and 10), and benzoyl ace-
tonitrile (Table 2, entries 4 and 11) were also found suitable for this
multicomponent reaction.
The exact role of PEG in this multicomponent reaction is not yet
clear, however we believe that PEG acts as a promoter by its two
active sites (ethereal oxygen linkages and free hydroxyl group).
The free hydroxyl group can form hydrogen bonding with the alde-
hydes and make it more electrophilic, as a result it helps in the for-
mation of the alkene (A). At the same time, the ethereal linkages of
oxygen enhance the nucleophilicity of the amines for the formation
of B. In addition to this, we believe that these ethereal linkages of
oxygen also help in the cyclization process of D followed by tauto-
merization, leading to the formation of the final product F as
shown in Scheme 1.
2. Results and discussion
2.1. Synthesis
Initially, benzaldehyde, malononitrile, aniline and dimethyl-
acetylene dicarboxylate (DMAD) were chosen as model substrates
and reacted in presence of various solvents at room temperature
(Table 1). Under catalyst free condition, in presence of different or-
ganic solvents (Table 1, entries 1–5), as well as in water, the exper-
iments did not provide the desired product even after stirring at
room temperature for 24 h. We also failed to achieve the desired
DHP (1) under solvent free condition (Table 1, entry 7). Interest-
ingly, the same set of substrates provided 72% yield of the desired
DHP (1) in presence of PEG-200 as a reaction medium within 10 h.
This result prompted us to study this reaction with other higher
molecular weight PEGs. Both PEG 400 and 600 were tested under
the similar reaction condition and better yields were achieved (Ta-
ble 1, entries 9 and 10). Among all the screened solvents, PEG-400
provided the best result.
The structure of the compound 1 was established using usual
spectroscopic techniques (IR, 1H and 13C NMR) and CHN analysis.
Table 1
Optimization of reaction condition
MeO2C
N
CO2Me
CN
CO2Me
CO2Me
CHO
+
NH2
+
CN
Solvent
rt
2.2. Evaluation of nuclease activity
+
CN
H2N
Concentration dependent nuclease activity was observed for
2a–2d, as well as for the bromo derivatives, 3a and 3b as shown
in Fig. 1 (data not shown for 3b). Effect of concentration on nucle-
ase activity was moderate for 2a, 2b and the unsubstituted DHP
derivative 1, but considerable increase in nuclease activity was ob-
served for 2c, 2d, 3a and 3b with increase in compound concentra-
tion. However, 2e with a Benzyl group as R2 showed similar
nuclease activity as the unsubstituted analogue 1 (data not
shown). Replacement of –CN group by –CONH2 group in the DHP
ring (R3) and –C6H5 group by 4-OMe–C6H4 group (R1), as in 2b, de-
creases the nuclease activity considerably as compared to the
unsubstituted DHP 1. Comparison between 2c and 2d reveals that
–CN group has more contribution in the nuclease activity of the
.
Entry
Solvent
Ethanol
Reaction
time (h)
%
Yields
1
2
24
24
24
24
24
24
24
10
10
10
0
0
0
0
0
0
0
72
81
79
Methanol
3
4
5
Dichloromethane
Acetonitrile
DMF
6
Water
7
8
Solvent free
PEG-200
9
PEG-400
10
PEG-600
Table 2
PEG-mediated synthesis of substituted dihydropyridines with different aromatic aldehydes, anilines, malononitrile derivatives and dimethyl acetylenedicarboxylate
MeO2C
CO2Me
R1
CO2Me
PEG-400
rt
CN
R3
CHO
NH2
R2
N
+
+
R1
+
R2
CO2Me
H2N
R3
.
Entry
R1
R2
C6H5
R3
Product
Reaction time (h)
Yield (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
C6H5
CN
CN
CONH2
COPh
CN
CN
CN
CN
COOEt
COOEt
COPh
CN
CN
CN
1
2a
2b
2c
2d
2e
3a
3b
4a
4b
4c
4d
4e
5
10
8
81
82
52
55
80
50
78
80
83
77
60
85
83
81
4-Cl–C6H4
4-Cl–C6H4
4-Cl–C6H4
4-Cl–C6H4
4-Cl–C6H4
4-Br–C6H4
4-Br–C6H4
3-NO2–C6H4
3-NO2–C6H4
3-NO2–C6H4
3-NO2–C6H4
3-NO2–C6H4
4-OMe–C6H4
b-Naphthyl
4-OMe–C6H4
4-Me–C6H4
4-Me–C6H4
C6H5CH2
4-Me–C6H4
C6H5
C6H5
4-Cl–C6H4
C6H5
16
10
10
16
10
10
8
10
12
7
C6H5
4-Me–C6H4
4-OMe–C6H4
8
8

10
S. Pal et al. / Bioorganic Chemistry 48 (2013) 8–15
Scheme 1. Plausible reaction mechanism of PEG mediated MCRs for the synthesis of dihydropyridine derivatives.
Fig. 1. Effect of concentration of compounds 1, 2a–2d and 3a on the cleavage reaction of pUC19. Amount of plasmid relaxation are shown in agarose gels, and the
corresponding quantitative analysis in the bar chart below; numbers above the gels and below the bars indicate concentration of the compounds used during plasmid
incubation and the corresponding lane used in the gel electrophoresis.
compounds than –COPh group at the R3 position. Though both 2a
and 2d have –CN and –NH2 group in the DHP ring, in all probabil-
ity, the presence of Naphthyl moiety as R2, render 2a less reactive.
The bulky naphthalene group in 2a might interrupt proper interac-
tion of the compound with the DNA [17]. The bromo derivative 3a
with a 4-Me–C6H4 group as R2 shows slightly greater overall nucle-
ase activity (ca. 38%) as compared to the non-methylated analogue
3b (ca. 36%, see supporting information). Comparison of nuclease
activity between 1 and 3b clearly reveals the effect of the presence
of a halogen atom in R1 in augmenting nuclease activity, while
slight differences in nuclease activity between 2d and 3a shows
that bromo has a slim edge over the chloro analogue. In summary,
nuclease activity of the DHP derivatives 2a–2e (R1 = 4-Cl–C6H4)
and 3a–3b (R1 = 4-Br–C6H4) were found to be greatly dependant
on the functional groups present in the 1st position (R2), 3rd-posi-
tion (R3) and as well as 4th position (R1) of the DHP ring.
For the compounds of the nitro series, nuclease activity for all
the compounds was found to be greater than the unsubstituted
DHP derivative 1 (Fig. 2). Comparison between 4a and 4c clearly
indicates that the presence of –COPh group at R3 in the DHP ring
enhances nuclease activity of the compound as compared to –COO-
Et group. Substitution of the –C6H5 group at R2 with 4-Cl–C6H4
does not change the nuclease activity as evident in 4a and 4b.
Nuclease activity increases by ꢀ7% when –C6H5 group as R2 was re-
placed by 4-Me–C6H4 (ca. 47% for 4e vs. 40% for 4d). This shows
that one methyl group in the R2 can have substantial influence in
increasing the nuclease activity in this class of compounds. As ob-
served earlier also, nuclease activity was on the higher side for
those compounds among all other entries from 4a–4e when the
DHP ring contains –CN group (R3) along with the usual –NH2 func-
tional group. However, no compound was found to have nuclease
activity beyond 47% or form linear DNA (form III) from the super-
coiled plasmid. Nevertheless, it is reasonable to infer that presence
of cyano (–CN) functional group was found to enhance the nucle-
ase activity of different dihydropyridine derivatives. In addition,
presence of –NO2 group in the phenyl ring of R1 supplemented
by a methyl group in the phenyl ring in R2 can have considerable
contribution in augmentation of nuclease activity of the com-
pounds. As observed, 4e showed highest nuclease activity among
all the entries (ca. 47%). The most important generalization that
might follow regarding the enhancement of nuclease activity in
the presence of –NO2 group is that the nitronium ions probably
neutralize the excess negative charge on phosphates of the DNA
backbone, which in turn enhances the cleaving activity of the ana-
logues [18]. Alternatively, nitro group can initiate the nuclease
activity of the compounds by producing DNA-Sugar damaged

S. Pal et al. / Bioorganic Chemistry 48 (2013) 8–15
11
Fig. 2. Effect of concentration of compounds 4a–4e on the cleavage reaction of pUC19. Convention and denotations followed in this figure is similar to Fig. 1.
Fig. 3. Effect of radical quenchers NaN3, Isopropanol and DMSO on the nuclease activity of the compounds (0.75 mM) and inhibition of endonucleases activity of EcoRI and
Hind III by the compounds 1, 2a–2d and 3a. In the gel lanes and the bar chart, P: pUC19 only (no compound, reagent or enzyme present), Pe: pUC19 + Eco RI (no compound
present), Ph: pUC19 + Hind III (no compound present).
products also [19]. Presence of electron donating groups like –OMe
on phenyl ring in the R1 and R2 position as in 5, shows nuclease
activity comparable to the unsubstituted DHP 1, while –Me groups
on phenyl ring at R2 was found to have important contribution in
increasing the potency of a compound’s nuclease activity.
NaN3, Isopropanol and DMSO (Fig. 4). Similar to the case of 2c,
2d and 3a, we also found that there is ꢀ8–10% decrease in nuclease
activity for 4e in presence of radical scavengers like NaN3, Isopro-
panol and DMSO, which may be attributed to the presence of –Me
group in R2. In general, in the presence of the nitro group, the dom-
inant mechanism responsible for creation of nicks in the plasmid
seems to follow a hydrogen abstraction route [23], where electron
are being shuttled from the functional groups present in the dihy-
dropyridine ring to initiate the process.
In order to assess whether the dihydropyridine derivatives
shows affinity towards any particular region (for example GC or
AT rich) in a plasmid, we carried out restriction endonuclease anal-
ysis [24,25]. Compound-pUC19 preincubated solutions were trea-
ted with restriction endonucleases EcoRI and Hind III, each
enzyme having single restriction site in pUC19. One interesting
observation was that while native pUC19 was totally converted
to the linear form (Form III), presence of any one of the compounds
from 2a–2e, 3a–3b including 1, completely inhibited the incision
The unsubstituted DHP, 1 along with 2a and 2b did not exhibit
reduction in nuclease activity either in presence of singlet oxygen
scavenger NaN3, or hydroxyl radical quenchers like Isopropanol
and DMSO. However, the relaxation of the plasmid during incuba-
tion with 2c, 2d and 3a (R2 = 4-Me–C6H4) was found to be notice-
ably reduced in the presence of NaN3, Isopropanol and DMSO
(Fig. 3). This suggest that hydroxyl radical or singlet oxygen med-
iated oxidative cleavage might occur in those reactions, leading to
DNA damage by oxidative pathway [20], in addition to nucleophilic
attack leading to hydrogen abstraction [21] and hydrolysis of phos-
phodiester bonds in the plasmid by those compounds [22].
Nuclease activity of the compounds of the Nitro series 4a–4d
was not appreciably reduced in presence of radical quenchers
Fig. 4. Effect of radical quenchers NaN3, Isopropanol and DMSO on the nuclease activity of the compounds (0.75 mM) and inhibition of endonucleases activity of EcoRI and
Hind III by the compounds 4a–4d. In the gel lanes and the bar chart, P: pUC19 only (no compound, reagent or enzyme present), Pe: pUC19 + Eco RI (no compound present),
Ph: pUC19 + Hind III (no compound present).

12
S. Pal et al. / Bioorganic Chemistry 48 (2013) 8–15
activity on the plasmid by both EcoRI (cutting site G;A, recognition
sequence 50-AATT) and Hind III (cutting site A;A, recognition se-
quence 50-AGCT) (Fig. 3). Inhibitions of endonuclease activity of
EcoRI and Hind III in the compound-pUC19 preincubated solutions
suggest that the compounds can interact with recognition sites of
both the restriction endonucleases in the plasmid. Thus, site spec-
ificity or selectivity for any nucleoside or sequence in compound-
DNA interaction was not observed. With Eco RI and Hind III restric-
tion enzymes, similar results were observed for 4a–4e and 5 (Fig. 4,
4e and 5 not shown), where none of them showed presence of the
linear form (Form III) of the plasmid upon incubation with the
restriction endonucleases.
To see whether Dihydropyridine compounds interact directly
with restriction endonuclease and inhibit their enzymatic activity,
plasmid DNA was incubated with restriction enzyme that were
pretreated with one of the compound. 100 units of Hind III enzyme
was incubated with 0.75 mM of 4d (one of the nitro derivatives
having high nuclease activity) at 37 °C overnight. The enzyme com-
pound mixture was subjected to dialysis for 24 h in a dialysis cas-
sette (MWCO 7 K, Slide-A-Lyzer™, Thermofisher Scientific). After
completion of the dialysis, the enzyme was recovered from the
dialysis membrane. Plasmid pUC19 was incubated following the
usual procedure with calculated amount of the recovered enzyme.
Agarose gel electrophoresis with the DNA incubated with recov-
ered Hind III showed complete restriction digestion of the plasmid
(see Supplementary section). This proves that compound has no ef-
fect on the activities of the restriction enzymes, and inhibition of
endonuclease activity of the restriction enzyme is due to interac-
tion of the compounds with the recognition sites of the enzyme
and not due to compound–enzyme interaction.
To summarize the interaction of the compounds 1, 2a–2e, 3a–
3b, 4a–4e and 5, from the experiments performed herein, it is rea-
sonable to say that the dominant mechanism responsible for plas-
mid relaxation by the compounds is electron transfer from DHP
ring substituents followed by hydrolysis of phosphodiester bonds
in DNA. However, single electron oxidative pathway has been
found to be operative also in few entries particularly with R2 as
4-Me–C6H4. Efficiency of electron donation and hence nuclease
activity is dependent on the functional group present in the DHP
ring where –CN was found to be the most effective among all oth-
ers tested. In addition, R1 groups can have important role in dictat-
ing nuclease activity, whereby it was found that 3-NO2–C6H4,
where –NO2 can exist as a nitronium ion are more efficient to pro-
mote nuclease activity than halides. Presence of methyl group in R2
tends to enhance nuclease activity. Also, bulky substituents at R2
probably tend to decrease nuclease activity by increasing the dis-
tance from the nucleophilic centre in the compound and the elec-
trophilic centres in the DNA. Overall, few DHP derivatives showed
appreciable nuclease activity in absence of any transition or other
heavy metal ions, and the nuclease activity can be tuned by appro-
priate substitution at R1, R2 and R3 positions.
tion of the DHP ring can also play significant role in deciding the
efficiency of the compound as a catalyst in nuclease activity lead-
ing to plasmid DNA damage. In particular, 3a, 3b, 4d and 4e were
found to be very effective nucleases, yielding DNA cleavage prod-
uct (nicked, open circular form of plasmid) from supercoiled
plasmid.
4. Experimental
4.1. General
All the reagents were procured from commercial sources and
used as received. All the reactions were monitored by TLC on
Merck GF 254 plates and spots were visualized by UV light and/
or using iodine vapor chamber. IR spectra were recorded on a Shi-
madzu Affinity 1, FTIR spectrophotometer. 1H and 13C NMR was
either recorded on a Jeol 500 or Bruker 500/400 MHz spectrometer
in CDCl3 using TMS as internal reference. CHN analysis was carried
out either on an Elementar Vario EL III or Perkin–Elmer 2400 II ele-
mental analyzers. Melting points were obtained manually by cap-
illary methods and uncorrected.
4.2. Preparation of substituted DHPs
A mixture of aromatic aldehyde (1.0 mmol), malononitrile or its
derivative (1.0 mmol) in 2.0 mL PEG-400 was stirred at room tem-
perature for half an hour. A solution of arylamine (1.0 mmol) and
dimethylacetylenedicarboxylate (1.0 mmol) in 2.0 mL PEG was
then added to the mixture. The whole solution was stirred at room
temperature for appropriate time. After completion of the reaction,
as indicated by TLC, the reaction mixture was extracted with
diethyl ether and PEG was separated from the products. The com-
bined organic layers were evaporated under reduced pressure, and
the crude product was either purified by column chromatography
(silica gel, 60–120 mesh and hexane: ethyl acetate (8:2) as eluent)
or by recrystalisation technique to obtain the desired DHPs. The
recovered PEG was dried under vacuum and reused for 3 cycles
without significant loss of activity.
4.2.1. Dimethyl 6-amino-5-cyano-1,4-diphenyl-1,4-dihydropyridine-
2,3-dicarboxylate (1)
Yield 81%; Light yellowish solid; mp 161–163 °C; IR (KBr): 3465,
3376, 3336, 2950, 2175, 1746, 1707, 1650, 1568, 1492, 1413, 1353,
1247, 1212, 1109, 1076 cmꢁ1; 1H NMR (CDCl3, 500 MHz): d = 7.48–
7.49 (m, 3H, Ar–H), 7.34–7.38 (m, 6H, Ar–H), 7.25–7.27 (m, 1H, Ar–
H), 4.65 (s, 1H, CH), 4.11 (bs, 2H, NH2), 3.58 (s, 3H, OMe), 3.42 (s,
3H, OMe); 13C NMR (CDCl3, 125 MHz): d = 165.9, 163.6, 149.7,
144.8, 141.9, 135.2, 130.6, 130.3, 130.0, 128.9, 127.3, 127.1,
120.6, 105.3, 62.9, 52.7, 52.1, 38.5; Anal. calcd for C22H19N3O4
(389.40): C, 67.86; H, 4.92; N, 10.79; Found: C, 67.94; H, 4.95; N,
10.88.
3. Conclusions
4.2.2. Dimethyl 6-amino-4-(4-chlorophenyl)-5-cyano-1-(naphthalen-
2-yl)-1,4-dihydro pyridine-2,3-dicarboxylate (2a)
We have described a highly atom economic multicomponent
reaction for the efficient synthesis of functionalized amino dihy-
dropyridine derivatives under mild and neutral catalyst free condi-
tions using PEG-400 as promoter. The notable advantages of this
protocol are: (a) applicability to a wide range of aromatic alde-
hydes, amines as well as malononitrile derivatives, (b) reusability
of PEG and its low cost and (c) simple reaction conditions with
good yields. Some of the DHPs demonstrate appreciable metal-free
nuclease activity, whereby –CN group on the heterocyclic ring has
been found to be very efficient in initiating the cleavage of pUC19
DNA. Additionally, functional groups attached to the 1 and 4 posi-
Yield 82%; Light yellowish solid; mp 171–173 °C; IR (KBr): 3455,
3327, 3220, 2945, 2178, 1753, 1712, 1651, 1571, 1422, 1351, 1223,
1120, 1009 cmꢁ1; 1H NMR (CDCl3, 400 MHz): d = 7.95 (d, J = 8.4 Hz,
2H, Ar–H), 7.89 (t, J = 6.8 Hz, 2H, Ar–H), 7.83 (s, 1H, Ar–H), 7.58–
7.64 (m, 2H, Ar–H), 7.31–7.36 (m, 4H, Ar–H), 4.68 (s, 1H, CH),
4.12 (s, 2H, NH2), 3.58 (s, 3H, OMe), 3.33 (s, 3H, OMe); 13C NMR
(CDCl3, 100 MHz): d = 165.7, 163.5, 149.9, 143.4, 142.2, 133.5,
133.0, 132.3, 130.3, 129.8, 129.1, 128.5, 128.4, 128.0, 127.7,
126.5, 120.4, 104.9, 62.6, 52.8, 52.2, 38.2; Anal. calcd for C26H20-
ClN3O4 (473.90): C, 65.89; H, 4.25; N, 8.87; Found: C, 65.93; H,
4.29; N, 8.96.

S. Pal et al. / Bioorganic Chemistry 48 (2013) 8–15
13
4.2.3. Dimethyl 6-amino-5-carbamoyl-4-(4-chlorophenyl)-1-(4-
methoxyphenyl)-1,4-dihydropyridine-2,3-dicarboxylate (2b)
Yield 52%; Light yellowish solid; mp 221–223 °C; IR (KBr): 3451,
3327, 3182, 2950, 2843, 1748, 1685, 1656, 1571, 1480, 1436, 1404,
121.1, 120.4, 104.5, 62.1, 52.6, 52.1, 38.1, 21.3; Anal. calcd for C23-
H20BrN3O4 (482.33): C, 57.27; H, 4.18; N, 8.71. Found: C, 57.37; H,
4.24; N, 8.83.
1335, 1298, 1255, 1218, 1185, 1109, 1062 cmꢁ1 1H NMR (CDCl3,
;
400 MHz): d = 7.38 (d, J = 8.4 Hz, 2H, Ar–H), 7.26–7.32 (m, 4H,
Ar–H), 6.97 (d, J = 8.8 Hz, 2H, Ar–H), 6.74 (bs, 2H, NH2), 4.95 (s,
2H, NH2), 4.74 (s, 1H, CH), 3.85 (s, 3H, OMe), 3.68 (s, 3H, OMe),
3.42 (s, 3H, OMe); 13C NMR (CDCl3, 125 MHz): 171.9, 165.9,
163.7, 160.6, 151.4, 144.2, 141.5, 132.8, 131.7, 129.1, 128.6,
127.2, 114.9, 105.9, 79.2, 55.6, 52.4, 51.9, 38.1. Anal. calcd for C23-
H22ClN3O6 (471.89): C, 58.54; H, 4.70; N, 8.90. Found: C, 58.43; H,
4.76; N, 8.78.
4.2.8. Dimethyl 6-amino-4-(4-bromophenyl)-5-cyano-1-phenyl-1,4-
dihydropyridine-2,3-dicarboxylate (3b)
Yield 80%; Light yellowish solid; mp 194–196 °C; IR (KBr): 3486,
3386, 3065, 2954, 2185, 1746, 1710, 1652, 1575, 1491, 1421, 1355,
1326, 1251, 1112, 1010 cmꢁ1; 1H NMR (CDCl3, 400 MHz): d = 7.48–
7.51 (m, 5H, Ar–H), 7.32–7.34 (m, 2H, Ar–H), 7.23–7.25 (m, 2H, Ar–
H), 4.64 (s, 1H, CH), 4.18 (s, 2H, NH2), 3.59 (s, 3H, OMe), 3.43 (s, 3H,
OMe); 13C NMR (CDCl3, 100 MHz): 165.5, 163.4, 149.7, 143.8,
141.9, 134.9, 131.9, 130.7, 130.2, 130.0, 128.8, 121.1, 120.3,
4.2.4. Dimethyl 6-amino-5-benzoyl-4-(4-chlorophenyl)-1-p-tolyl-1,4-
dihydropyridine-2,3-dicarboxylate (2c)
104.6, 62.1, 52.6, 52.1, 38.1; Anal. calcd for C22H18BrN3O4
(468.30): C, 56.42; H, 3.87; N, 8.97. Found: C, 56.35; H, 3.83; N,
8.89.
Yield 55%; Yellow solid; mp 184–186 °C; IR (KBr): 3420, 3049,
2951, 1736, 1703, 1639, 1596, 1450, 1355, 1224, 1111,
1048 cmꢁ1 1H NMR (CDCl3, 500 MHz): d = 7.35–7.39 (m, 3H, Ar–
;
H), 7.32 (d, J = 8.3 Hz, 2H, Ar–H),7.22–7.25 (m, 4H, Ar–H), 7.14 (d,
J = 8.6 Hz, 2H, Ar–H), 6.84 (d, J = 8.6 Hz, 2H, Ar–H), 4.99 (s, 1H,
CH), 3.62 (s, 3H, OMe), 3.45 (s, 3H, OMe), 2.43 (s, 3H, Me); 13C
NMR (CDCl3, 125 MHz): d = 194.5, 165.7, 163.7, 154.4, 144.7,
141.5, 141.2, 132.2, 131.8, 131.0, 130.2, 129.9, 129.5, 129.2,
128.6, 128.0, 126.8, 108.7, 89.5, 52.7, 52.2, 38.0, 21.4; Anal. calcd
for C29H25ClN2O5 (516.97): C, 67.38; H, 4.87; N, 5.42; Found: C,
67.43; H, 4.82; N, 5.51.
4.2.9. 5-Ethyl 2,3-dimethyl 6-amino-4-(3-nitrophenyl)-1-phenyl-1,4-
dihydropyridine-2,3,5-tricarboxylate (4a)
Yield 83%; Light yellowish solid; mp 172–173 °C; IR (KBr): 3472,
3264, 2979, 2951, 1749, 1709, 1667, 1639, 1600, 1530, 1506, 1436,
1354, 1213, 1123, 1095, 1017 cmꢁ1 1H NMR (CDCl3, 500 MHz):
;
d = 8.54 (s, 1H, Ar–H), 8.25 (d, J = 8.0 Hz, 1H, Ar–H), 7.94 (d,
J = 7.5 Hz, 1H, Ar–H), 7.70–7.73 (m, 3H, Ar–H), 7.64–7.67 (m, 3H,
Ar–H), 6.50 (bs, 2H, NH2), 5.31 (s, 1H, CH), 4.24–4.30 (m, 2H,
OCH2), 3.61 (s, 3H, OMe), 3.82 (s, 3H, OMe), 1.41–1.44 (m, 3H,
Me); 13C NMR (CDCl3, 125 MHz): 169.1, 165.7, 163.6, 151.4,
149.3, 148.3, 142.2, 134.9, 134.0, 130.7, 130.5, 130.0, 128.8,
123.0, 121.5, 106.6, 79.4, 61.9, 59.6, 52.5, 52.0, 51.9, 37.3, 14.4;
Anal. calcd for C24H23N3O8 (481.45): C, 59.87; H, 4.82; N, 8.73.
Found: C, 59.96; H, 4.90; N, 8.84.
4.2.5. Dimethyl 6-amino-4-(4-chlorophenyl)-5-cyano-1-p-tolyl-1,4-
dihydropyridine-2,3-di carboxylate (2d)
Yield 80%; Light yellowish solid; mp 186–188 °C; IR (KBr): 3472,
3360, 3221, 2950, 2175, 1742, 1699, 1646, 1574, 1508, 1413, 1355,
1301, 1248, 1216, 1176, 1016 cmꢁ1 1H NMR (CDCl3, 500 MHz):
;
d = 7.33 (d, J = 8.5 Hz, 2H, Ar–H), 7.29 (d, J = 7.9 Hz, 2H, Ar–H),
7.27 (d, J = 7.6 Hz, 2H, Ar–H), 7.20 (d, J = 7.9 Hz, 2H, Ar–H), 4.65
(s, 1H, CH), 4.09 (bs, 2H, NH2), 3.58 (s, 3H, OMe), 3.46 (s, 3H,
OMe), 2.40 (s, 3H, Me); 13C NMR (CDCl3, 125 MHz): 165.6, 163.4,
149.9, 143.5, 142.2, 141.1, 132.9, 132.2, 130.6, 129.9, 128.8,
128.5, 120.4, 104.6, 62.1, 52.6, 52.1, 38.1, 21.3; Anal. calcd for C23-
H20ClN3O4 (437.87): C, 63.09; H, 4.60; N, 9.60. Found: C, 63.18; H,
4.67; N, 9.72.
4.2.10. 5-Ethyl 2,3-dimethyl 6-amino-1-(4-chlorophenyl)-4-(3-
nitrophenyl)-1,4-dihydropyridine-2,3,5-tricarboxylate (4b)
Yield 77%; Light yellowish solid; mp 186–188 °C; IR (KBr): 3442,
3221, 3107, 2985, 2954, 1751, 1710, 1663, 1602, 1523, 1440, 1401,
1345, 1236, 1095, 1042 cmꢁ1 1H NMR (CDCl3, 400 MHz): d = 8.31
;
(s, 1H, Ar–H), 8.06 (d, J = 7.6 Hz, 1H, Ar–H), 7.71 (d, J = 7.6 Hz, 1H,
Ar–H), 7.51 (d, J = 8.4 Hz, 2H, Ar–H), 7.45 (t, J = 7.6 Hz, 1H, Ar–H),
7.39 (d, J = 8.4 Hz, 2H, Ar–H), 6.25 (bs, 2H, NH2), 5.10 (s, 1H, CH),
4.05–4.13 (m, 2H, OCH2), 3.67 (s, 3H, OMe), 3.49 (s, 3H, OMe),
1.23 (t, J = 6.8 Hz, 3H, Me); 13C NMR (CDCl3, 125 MHz): 169.0,
165.6, 163.6, 151.1, 149.0, 148.4, 141.8, 136.9, 133.9, 133.4,
132.1, 130.3, 128.9, 122.9, 121.5, 107.1, 79.8, 62.2, 59.7, 52.7,
52.1, 37.2, 14.4; Anal. calcd for C24H22ClN3O8 (515.90): C, 55.87;
H, 4.30; N, 8.15. Found: C, 55.94; H, 4.35; N, 8.29.
4.2.6. Dimethyl 6-amino-1-benzyl-4-(4-chlorophenyl)-5-cyano-1,4-
dihydropyridine-2,3-dicarboxylate (2e)
Yield 50%; Yellow solid; mp: 146–148 °C; IR (KBr): 3466, 3346,
3038, 2955, 2188, 1743, 1705, 1645, 1569, 1493, 1434, 1343, 1290,
1215, 1116, 1018 cmꢁ1
;
1H NMR (CDCl3, 400 MHz): d = 7.34–
7.41(m, 3H, Ar–H), 7.22–7.29 (m, 4H, Ar–H), 7.10 (d, J = 8.4 Hz,
2H, Ar–H), 4.78 (d, J = 17.2 Hz, 1H, Ar–CH2), 4.66 (d, J = 17.2 Hz,
1H, Ar–CH2), 4.57 (s, 1H, CH), 4.15 (s, 2H, NH2), 3.77 (s, 3H,
OMe), 3.63 (s, 3H, OMe); 13C NMR (CDCl3, 125 MHz): d = 165.6,
164.6, 151.3, 142.6, 142.2, 135.0, 132.9, 131.9, 130.1, 129.3,
128.8, 128.4, 126.7, 120.3, 106.9, 65.9, 53.3, 52.2, 50.8, 37.9; Anal.
calc. for C23H20ClN3O4 (437.87): C, 63.09; H, 4.60; N, 9.60. Found: C,
63.01; H, 4.67; N, 9.72.
4.2.11. Dimethyl 6-amino-5-benzoyl-4-(3-nitrophenyl)-1-phenyl-1,4-
dihydropyridine-2,3-dicarboxylate (4c)
Yield 60%; Yellow solid; mp 156–157 °C; IR (KBr): 3424, 3068,
2951, 2928, 2853, 1745, 1706, 1643, 1608, 1530, 1452, 1354,
1220, 1123, 1076, 1053, 1025 cmꢁ1 1H NMR (CDCl3, 500 MHz):
;
4.2.7. Dimethyl 6-amino-4-(4-bromophenyl)-5-cyano-1-p-tolyl-1,4-
dihydropyridine-2,3-dicarboxylate (3a)
d = 7.90–7.91 (m, 1H, Ar–H), 7.80–7.81 (m, 1H, Ar–H), 7.46–7.51
(m, 3H, Ar–H), 7.36–7.39 (m, 2H, Ar–H), 7.33–7.35 (m, 1H, Ar–H),
7.28–7.31 (m, 2H, Ar–H), 7.22 (t, J = 8.0 Hz, 1H, Ar–H), 7.13 (d,
J = 8.5 Hz, 2H, Ar–H), 7.00 (d, J = 7.5 Hz, 1H, Ar–H), 5.02 (s, 1H,
CH), 3.40 (s, 3H, OMe), 3.37 (s, 3H, OMe); 13C NMR (CDCl3,
125 MHz): d = 194.2, 165.3, 163.4, 154.2, 148.4, 148.3, 141.4,
141.2, 134.3, 133.3, 130.9, 130.4, 130.3, 129.4, 129.1, 128.5,
128.4, 126.5, 122.3, 121.7, 108.2, 89.2, 52.7, 52.2, 38.6; Anal. calcd
for C28H23N3O7 (513.50): C, 65.49; H, 4.51; N, 8.18. Found: C, 65.57;
H, 4.57; N, 8.30.
Yield 78%; Light yellowish solid; mp 185–187 °C; IR (KBr): 3475,
3364, 3033, 2952, 2174, 1741, 1701, 1646, 1575, 1509, 1414, 1356,
1326, 1250, 1113, 1071 cmꢁ1 1H NMR (CDCl3, 500 MHz): d = 7.48
;
(d, J = 8.6 Hz, 2H, Ar–H), 7.27 (d, J = 8.0 Hz, 2H, Ar–H), 7.23 (d,
J = 8.0 Hz, 2H, Ar–H), 7.19 (d, J = 8.0 Hz, 2H, Ar–H), 4.62 (s, 1H,
CH), 4.12 (bs, 2H, NH2), 3.58 (s, 3H, OMe), 3.45 (s, 3H, OMe), 2.39
(s, 3H, Me); 13C NMR (CDCl3, 125 MHz): 165.6, 163.4, 149.9,
143.9, 142.2, 141.1, 133.1, 132.2, 132.0, 131.8, 129.9, 129.6,

14
S. Pal et al. / Bioorganic Chemistry 48 (2013) 8–15
4.2.12. Dimethyl 6-amino-5-cyano-4-(3-nitrophenyl)-1-phenyl-1,4-
dihydropyridine-2,3-dicarboxylate (4d)
0.75 mM of the compounds at 37 °C for 24 h. For restriction endo-
nucleases studies, plasmid DNA was preincubated with 0.75 mM of
the compounds for 24 h and then the reaction mixture was sub-
jected to 8 units of either EcoRI or Hind III restriction enzymes
along with the appropriate enzyme buffer for 1 h at 37 °C.
Nuclease activity was calculated as follows (1.42 is the correc-
tion factor for supercoiled DNA):
Yield 85%; Yellow solid; mp 217–219 °C; IR (KBr): 3446, 3336,
3229, 2951, 2178, 1755, 1704, 1651, 1577, 1521, 1421, 1349,
1250, 1213, 1112, 926 cmꢁ1 1H NMR (CDCl3, 500 MHz): d = 7.84
;
(s, 1H, Ar–H), 7.73 (d, J = 8.2 Hz, 1H, Ar–H), 7.34 (d, J = 7.9 Hz, 1H,
Ar–H), 7.20 (t, J = 7.9 Hz, 1H, Ar–H), 7.12–7.14 (m, 3H, Ar–H),
6.96–6.98 (m, 2H, Ar–H), 4.75 (bs, 2H, NH2), 4.36 (s, 1H, CH),
3.20 (s, 3H, OMe), 3.01 (s, 3H, OMe); 13C NMR (CDCl3, 125 MHz):
d = 165.0, 162.9, 150.6, 148.3, 147.3, 142.4, 134.7, 133.2, 130.4,
130.1, 129.7, 129.7, 121.9, 121.7, 120.4, 103.8, 59.7, 52.4, 51.9,
38.6; Anal. calcd for C22H18N4O6 (434.40): C, 60.83; H, 4.18; N,
12.90; Found: C, 60.89; H, 4.24; N, 13.03.
% Activity
Amount of nicked plasmid ꢂ 100
¼
ðAmount of supercoiled plasmid ꢂ 1:42Þ þ amount of nicked Plasmid
Acknowledgments
L.H.C is thankful to Department of Science and Technology, In-
dia for financial support with sanction No. SR/FT/CS-042/2009 for
carrying out this study. S.P. and V.S. thank UGC and CSIR New Delhi
respectively for their research fellowships. The authors are also
grateful to IIT Patna for providing basic research facilities as well
as SAIF, CDRI Lucknow and SAIF, IIT Madras for providing analytical
facilities.
4.2.13. Dimethyl 6-amino-5-cyano-4-(3-nitrophenyl)-1-p-tolyl-1,4-
dihydropyridine-2,3-dicarboxylate (4e)
Yield 83%; Yellow solid; mp 212–214 °C; IR (KBr): 3424, 3374,
3180, 2960, 2185, 1752, 1703, 1653, 1574, 1525, 1423, 1351,
1250, 1218, 1109, 1052 cmꢁ1 1H NMR (CDCl3, 400 MHz): d = 8.26
;
(s, 1H, Ar–H), 8.14 (dd, J = 8.2 and 1.4 Hz, 1H, Ar–H), 7.71 (d,
J = 7.6 Hz, 1H, Ar–H), 7.54 (t, J = 7.9 Hz, 1H, Ar–H), 7.31 (d,
J = 8.1 Hz, 2H, Ar–H), 7.26 (d, J = 8.3 Hz, 2H, Ar–H), 4.78 (s, 1H,
CH), 4.29 (bs, 2H, NH2), 3.61 (s, 3H, OMe), 3.46 (s, 3H, OMe), 2.42
(s, 3H, Me); 13C NMR (CDCl3, 100 MHz): d = 165.2, 163.1, 150.4,
148.7, 147.1, 142.5, 141.3, 133.4, 131.8, 130.8, 129.8, 129.7,
122.4, 122.2, 120.1, 104.2, 61.3, 52.7, 52.2, 38.6, 21.3; Anal. calcd
for C23H20N4O6 (448.43): C, 61.60; H, 4.50; N, 12.49; Found: C,
61.66; H, 4.53; N, 12.56.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2013.
03.003.
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