Synthesis, pharmacological evaluation and molecular docking of pyranopyrazole-linked 1,4-dihydropyridines as potent positive inotropes

Article abstract of DOI:10.1007/s11030-017-9738-7

Abstract: 1,4-Dihydropyridines are well-known calcium channel blockers, but variations in the substituents attached to the ring have resulted in their role reversal making them calcium channel activators in some cases. We describe the microwave-assisted e

Full text of DOI:10.1007/s11030-017-9738-7

Mol Divers
DOI 10.1007/s11030-017-9738-7
ORIGINAL ARTICLE
Synthesis, pharmacological evaluation and molecular docking
of pyranopyrazole-linked 1,4-dihydropyridines as potent positive
inotropes
1
1
2
3
Rakesh Kumar · Neha Yadav · Rodolfo Lavilla · Daniel Blasi ·
3
4
4
5
Jordi Quintana · José Manuel Brea · María Isabel Loza · Jordi Mestres ·
1
1
1
1
Mamta Bhandari · Ritu Arora · Rita Kakkar · Ashok K. Prasad
Received: 14 September 2016 / Accepted: 9 April 2017
©
Springer International Publishing Switzerland 2017
Abstract 1,4-Dihydropyridines are well-known calcium
channel blockers, but variations in the substituents attached
to the ring have resulted in their role reversal making them
calcium channel activators in some cases. We describe the
microwave-assisted eco-friendly approach for the synthe-
sis of pyranopyrazole-1,4-dihydropyridines, a new class of
pounds 7c, 7g and 7i appear to be the most effective positive
inotropes, even at low doses, and bind with the calcium chan-
nels even more strongly than Bay K 8644, a well-known
calcium channel activator. The chronotropic effect for the
new compounds was also studied. The target and off-target
affinity profiling supported the in vivo results and revealed
that the hybridized pyranopyrazole dihydropyridine scaffold
has delivered new moderate hits, to be optimized, for the
cytochrome P450 3A4 enzymes, opening avenues for com-
bined pharmacological activity through standard structural
modification.
1,4-DHPs, under solvent-free conditions in good yield, and
screenthemforvariousinsilico, invitroandinvivoactivities.
The in vivo experimentation results show that the compounds
possess positive inotropic effect, and the docking results
validate their good binding with calcium channels. Com-
Graphical Abstract
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-017-9738-7) contains supplementary
material, which is available to authorized users.
B Rakesh Kumar
rakehskp@email.com
1
Bio-organic Laboratory, Department of Chemistry, University
of Delhi, Delhi 110 007, India
2
Laboratory of Organic Chemistry, Department of
Pharmacology and Therapeutical Chemistry, Faculty of
Pharmacy, University of Barcelona, Barcelona Science Park,
Baldiri Reixac 10-12, 08028 Barcelona, Spain
Keywords Pyranopyrazole-1 · 4-Dihydropyridines ·
Inotropic effect · Molecular docking · Structure–activity
analysis · CYP450 inhibition · Microwave synthesis · SeO
3
Drug Discovery Platform, Parc Científic de Barcelona, Baldiri
Reixac 10-12, 08028 Barcelona, Spain
2
4
USEF Screening Platform, Centre of Research on Molecular
Medicine and Chronic Diseases (CIMUS), Universidad de
Santiago de Compostela (USC), Santiago de Compostela,
Spain
Introduction
5
Systems Pharmacology, Research Program on Biomedical
Calcium ions are essential in various physiological signal-
ing pathways. The movement of calcium ions across the cell
occurs via the ligand-gated or voltage-gated calcium chan-
Informatics (GRIB), IMIM Hospital del Mar Medical
Research Institute and University Pompeu Fabra, Doctor
Aiguader 88 (PRBB), 08003 Barcelona, Catalonia, Spain
1
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Mol Divers
nels, leading to increased/decreased intracellular calcium
levels generating various physiological responses [1]. The
cell needs to be depolarized or excited for these channels to
open up and allow the influx of calcium into the cell [2,3].
Among the various known calcium channel activators
and blockers, 1,4-dihydropyridines have been observed to
possess most potent binding with L-type calcium channels.
thesize a new class of pharmacologically active scaffolds
by hybridizing two active pharmacophore units, namely
1,4-dihydropyridine analogues with the pyranopyrazole ring
(Fig. 1).
Herein, we report the MW-assisted solvent-free, greener
and eco-friendly approach for the synthesis of
pyranopyrazole-1,4-dihydropyridinederivatives, andwereport
the in vivo results describing their effect on blood pressure
and heart rate, supported by docking studies. We also report
the in silico and in vitro characterization of these compounds
interacting with adenosine and androgen receptors and with
cytochrome P450 enzymes.
1,4-DHPs behave both as calcium channel blockers and as
activators and are widely used in treating coronary heart dis-
eases. Nifedipine (A) and Bay K 8644 (B) are well-known
calcium channel blocker and activator, respectively [4].
Furthermore, 1,4-DHPs show a broad pharmacologi-
cal activity profile possessing antiviral [5], antitumor [6],
anti-inflammatory [7], antimicrobial [8], antitubercular [9]
and antihistaminic [10] activities. 1,4-DHPs are also being
explored for their binding with various receptors, namely
adenosine [11,12], cannabinoid [13], adrenergic [14] and
cytochrome P450 receptors [15].
Results and discussion
Synthesis of pyranopyrazole-1,4-dihydropyridines
In this work, we have hybridized the pyrazole moiety with
the pyran ring. Pyranopyrazoles are an important category
of heterocyclic compounds and are widely used as parental
compounds to synthesize molecules with medicinal bene-
fits. They possess vasodilator [17], anticancer [18], analgesic
[
19], anti-inflammatory [20], antimicrobial [21] activities.
These pyranopyrazoles are also of interest because of their
structural similarity to a wide variety of flavonoids that
exhibit interesting biological activities.
This vast pharmacological spectrum leads to the design
of new bioactive candidates by the hybridization of the
two active pharmacophore units, i.e., 1,4-dihydropyridine
derivatives linked with pyranopyrazoles, as an attractive drug
scaffold to evaluate their potency against hypertension and
other druggable targets.
With the aim to synthesize pyranopyrazole-1,4-
dihydropyridines, pyrano[2,3-c]pyrazole-4-carbaldehydes
were first synthesized. The reaction conditions for the
synthesis of the starting compounds pyrano[2,3-c]pyrazole-
With the “one-drug-one-target” paradigm, many diseases
remain inadequately treated today. The failure of this pro-
tocol to treat some diseases prompted us to explore the
“hybrid drugs” model, in which a compound interacts with
multiple targets [16]. This strategy leads to new chemi-
cal entities with modified selectivity profiles, different or
multiple modes of action and reduced side effects. The
concept of hybridization impelled us to design and syn-
4-carbaldehydes3a(Scheme1)wereoptimizedusingvarious
parameters as given in Table 1. Various solvents were
screened under microwave irradiation and conventional heat-
ing. The best yield was obtained for compounds 3a–b, in
the oxidation of compounds 2a–b using selenium diox-
ide in 1,4-dioxane at reflux. The compounds 2a–b were
in turn prepared by the modified Pechmann condensation
reaction of hydrazines 1a–b with ethyl acetoacetate (4a) at
◦
1
(
30−140 C via the in situ generation of pyrazolone [22]
Scheme 1).
A series of novel pyranopyrazole-1,4-dihydropyridines
7
a–l has been synthesized following the modified Hantzsch
synthesis.
The synthesis of symmetric pyranopyrazole-1,4-
Fig. 1 Hybridization of the pyrazole, pyrano and dihydropyridine
pharmacophores leading to the pyrano-pyrazole dihydropyridine com-
pounds
dihydropyridines 7a–h was accomplished by the reaction of
pyrano[2, 3-c]pyrazole-4-carbaldehydes 3a–b, β-keto esters
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Scheme 1 Synthesis of compounds 3a–b
Table 1 Optimization of the reaction conditions for the synthesis of
pyrano[2,3-c]pyrazole-4-carbaldehydes
Non-symmetric
The process is versatile and allows the synthesis of
symmetric DHPs and through mixed reaction of dime-
done/methyl 3-amino crotonate and acetoacetic esters leads
to non-symmetric derivatives as well. The transformations
are selective and afford the desired compounds in useful
◦
c
Entry
Solvent
Temp ( C)
Time
Yield (%)
1
2
3
4
5
6
7
8
a
Dioxanea
Xylenea
EtOHa
110
145
90
16 h
45
D
16 h
16 h
E
(high) yields and purities. The details of the various com-
Nonea
120
110
145
90
16 h
29
39
D
pounds synthesized are depicted in Table 2. The structures
of the compounds were unambiguously established based
Dioxaneb
Xyleneb
EtOHb
10 min
10 min
10 min
10 min
1
13
on their spectral data analysis such as H NMR, C NMR,
FTIR and HRMS. For all the new DHP derivatives, UPLC-
MS was also performed to check their purity (see supple-
mentary information). All the data were in good agreement
to our proposed structures.
E
Noneb
120
30
Conventional heating
Microwave irradiation at 90 W
Isolated yields
Reaction yielded multiple overlapping spots on TLC difficult to isolate
No reaction
b
c
d
e
Evaluation of samples for their effect on blood pressure
and heart rate
4
a–e and ammonium acetate via MW irradiation under
Since 1,4-dihydropyridines are primarily known to decrease
the blood pressure by acting as calcium channel blockers,
tracing of various hemodynamic parameters, such as HR,
MAP, DBP, SBP, Rat BP in spontaneously hypertensive rats
solvent-free conditions using barium nitrate as catalyst
Scheme 2), followed by a simple work-up to obtain the
pure compounds. Synthesis of asymmetric pyranopyrazole-
,4-dihydropyridines 7i–k was accomplished by the reaction
of pyrano[2, 3-c]pyrazole-4-carbaldehyde 3a, β-keto esters
a–c, dimedone 5 and ammonium acetate under similar con-
ditions. Similarly, synthesis of asymmetric pyranopyrazole-
,4-dihydropyridines 7l was accomplished by the reaction of
(
1
(SHR), was carried out. The effect of nifedipine (well-known
dihydropyridine calcium channel blocker) and all the synthe-
sized compounds on blood pressure and heart rate at different
doses, i.e., 1, 5 and 10 mg/kg, was measured (Table 3). How-
ever, upon monitoring, the standard, as expected decreased
the blood pressure, whereas, contrary to the expected results,
the newly synthesized compounds appeared to be increas-
ing the blood pressure instead of decreasing it. A graphical
representation of the effect on blood pressure of different
compounds at different doses is shown in Fig. 2. Compounds
4
1
pyrano[2,3-c]pyrazole-4-carbaldehyde 3a, methyl 3-amino
crotonate 6, β-keto ester 4c and ammonium acetate under
similar conditions (Scheme 3).
Symmetric
7c, 7g and 7i showed really good increase in BP at all three
doses. Compounds 7b, 7e, 7h, 7j, 7k and 7l showed moderate
to good increase in blood pressure at high doses (i.e., 5 and
10 mg/kg) in SHR rats. Some compounds 7c, 7d, 7e, 7g, 7h
and 7j showed moderate increase in heart rate as well.
The in vivo screening showed the compounds to be acting
as positive inotropes, as most of them lead to an increase in
arterial blood pressure (ABP). These results thus revealed
that the DHPs of this novel class were acting opposite to that
of nifedipine (calcium channel blocker) and that they might
Scheme 2 Synthesis of compounds 7a–h
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Scheme 3 Synthesis of compounds 7i–l
Table 2 Chemical structure of synthesized 1,4-dihydropyridines 7a–l
Comp.
No.
Aldehyde 3
ß-keto ester /
ß-diketone
Comp. No.
Aldehyde 3
ß-keto ester/ dimedone/
methyl 3-amino crotonate
7
a
b
7g
4
a
4c
3
b
b
3
3
3
3
a
a
a
a
7
7h
7i
4b
4d
3
7
c
4
c
4a
5
3a
3a
3a
7
d
e
7j
4d
4e
4b
4b
5
7
7k
7l
4c
4c
5
6
3
a
b
7
f
3
3
a
be acting as calcium channel activators, leading to positive
inotropic effect.
Binding mode of the best pose into the active site of
KVAP
These contradictory results prompted the exploration of
binding of these new 1,4-dihydropyridines with the calcium
channels. Hence, we carried out docking studies to support
the in vivo results.
For the theoretical studies, the experimental structures of
DHP-LTCC complexes would have been good starting points
for our calculations. However, owing to the large size and
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Table 3 Effect on BP and heart rate
Compound
Nifedipine
Effect on hemodynamic parameters
At the dose of 1 mg/kg, it showed maximum fall of 23.5 mmHg (29.5%) within 15 min before returning to baseline after
5 min. However, there was no significant change in heart rate
4
7
7
7
7
7
7
7
a
b
c
d
e
f
At 1 and 5 mg/kg, there was no significant change in BP, and at 10 mg/kg there was 10% rise in BP when compared to basal
BP. It did not affect heart rate. Labored respiration was observed in animal
It caused a rise of 13% at 1 mg/kg, 41% at 5 mg/kg and 48% at 10 mg/kg in BP after 15 min of drug administration. There
was 14% increase in heart rate at 5 mg/kg. Labored respiration was seen in animal
This compound turned milky when mixed with saline. There was 43, 53 and 73% increase in blood pressure at the doses of
1, 5 and 10 mg/kg, respectively. Increased heart rate was also observed at all three doses
When this compound was mixed with saline, it turned milky. There was no effect on BP, but there was 12% rise in heart rate
at 10 mg/kg
At 1 mg/kg, there was no significant change in BP, but it caused a rise of 15 and 30% in BP at the doses of 5 and 10 mg/kg,
respectively. There was 15% rise in heart rate at the dose of 10 mg/kg
When this compound was mixed with saline, it turned milky. There was no effect on BP. There was 38% rise in heart rate at
1
0 mg/kg
It caused a rise of 37% at 1 mg/kg, 52% at 5 mg/kg and 57% at 10 mg/kg in BP. Further, there was 11% fall in heart rate at
mg/kg
g
5
7
h
i
It caused a rise of 11% at 1 mg/kg, 27% at 5 mg/kg and 31% in BP. No effect on heart rate was seen
7
When this compound was mixed with saline, it became turbid. It caused a rise of 39% at 1 mg/kg, 101% at 5 mg/kg and
127% in BP. Heart rate at 1 mg/kg showed a fall of 5% after 15 min, but at 5 mg/kg there was 35% increase in heart rate.
Labored respiration and gnawing of teeth were seen in rats
7j
After administration, this compound caused an increase of 13, 32 and 45% in BP of at the dose 1, 5 and 10 mg/kg,
respectively. After the administration of 5 mg/kg, rate of respiration increased in animal. Further, there was 29% increase
in heart rate
7
k
l
When this compound was mixed with saline, it turned milky. It caused a rise of 10% at 1 mg/kg, 25% at 5 mg/kg, and there
was no effect on BP at 10 mg/kg. There was 13% increase in heart rate at 1 mg/kg
7
When this compound was mixed with saline, it became turbid. There was 24% increase at 5 mg/kg in BP. There was no
effect on heart rate
structure of voltage-gated Ca2 channels is not available to
+
2
+
date. Fortunately, voltage-dependent Ca channels (KVAP)
belong to the class of voltage-dependent cation (Na , K
+
+
and Ca2 ) channels which allow conduction of these ions
with cell membrane voltage changes. All members of this
class contain six hydrophobic segments, S1–S6, per subunit,
out of which the four subunits surrounding the central ion-
conduction pore are identical in all these channels [23–25].
+
+
In fact, many small-molecule ligands of K channels have
been identified, but only a few co-crystallized with KVAP.
Therefore, we first focused on determining the active site for
the binding of DHPs to KVAP and then carried out dock-
ing studies using extra precision Glide (XP) available in the
Schrödinger suite [26,27]. Various possible binding states
were predicted by SiteMap [28], but based on their Site score
and other important parameters, the active site was found to
be at the interface of the two chains (A and B) of KVAP
(Fig. 3). The point to note here is that this is the same site as
Fig. 2 Effect on blood pressure with different drug dosage. (Color
figure online)
that of binding of its inhibitor [29–31].
The Glide XP docking results are summarized in Table 4.
Out of the five poses obtained, the docking score of pose
hydrophobic nature of calcium channels, the crystallographic
studies on these proteins have always been a challenging
task for experimentalists. Hence, a complete X-ray crystal
7i is found to be the best. This suggests that the ligand 7i
activates the receptor more efficiently than the reference, in
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Fig. 3 Active site (white dots in protein) obtained from SiteMap
Table 4 Glide docking results of DHPs in the active site of KVAP
Fig. 4 Van der Waals contacts (orange dashed lines) and hydrogen-
bonding interactions (yellow dashed-line) of 7i with the residues of
KVAP. (Color figure online)
L#
Docking
score
Emodel/
kcal/mol
Glide energy/
kcal/mol
7
7
7
7
7
i
−6.29
−6.21
−5.82
−5.54
−5.42
−56.42
−44.61
−65.47
−57.08
−50.69
−30.14
−43.15
−41.89
−47.13
−45.62
−40.40
−28.08
c
i
g
g
(S)-BAY K 8644 −5.73
accord with our in vivo results. This fact is also supported
by the calculated values (Table 4) of other important scoring
functions, like Emodel and Glide energy. Therefore, the best
scoring pose of 7i was used for further analysis.
The orientation of the best pose of the most potent cal-
cium channel activator, compound 7i, in the active site of
KVAP is shown in Fig. 4 along with its van der Waals con-
tacts and hydrogen-bonding interactions. As can be clearly
seen, the active site of the receptor comprises residues LEU
Fig. 5 L#7i (shown in green color) inside the hydrophobic (orange)
and hydrophilic (blue) protein environment of KVAP. (Color figure
online)
2
6, VAL 27, LEU 29, VAL 31 and TYR 33 of chain A
and VAL 56, TYR 59, LEU 60, LEU 159, LEU 162, TYR
63 and PHE 166 of chain B, which make good con-
For the synthesized compounds, in silico and in vitro
experiments to analyze their activity against various other
targets (such as adenosine, androgen and Cyp 450 receptor)
were also carried out as follows.
1
tacts with ligand 7i. A hydrogen bond with the amino acid
residues of the receptor is also formed, i.e., between the
hydroxyl group of TYR 163 and carbonyl oxygen of the
ligand.
Structure–activity discussion
The active site is completely buried in a hydrophobic envi-
ronment. The hydrophobic (orange) and hydrophilic (blue)
maps around the active site are displayed in Fig. 5.
The observations discussed above are sufficient to esti-
mate the potential of our compounds as activators of L-type
calcium channels. Thus, by combining the results obtained
by our in vivo and molecular docking studies, we conclude
that our compound 7i is a novel calcium channel activator
and should be explored further.
The interesting biological activity results impelled the anal-
ysis of the point of attachment of the pyranopyrazole ring
in combination with the varying ester group substituents at
C-3 and C-5 position of the DHP ring. The comparison of
the activity results of the newly synthesized pyranopyrazole-
1,4-dihydropyridines showed that the compound 7i with
nonidentical ester substitution at C-3 and C-5 and a bulky
group at 4-position is the most potent out of all, in increasing
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Table 5 Target and off-target
affinity profiles of nifedipine
Compound
Nifedipine
CACNA1C
CACNA1D
CACNA1S
AR
A1
A3
CYP3A4
(ChEMBL193) and compounds
8.1a
8.3a
8.2a
5.4a
5.2a
5.0a
–
7a–c, 7f–g, and 7i–j
8.8
–
6.2
–
6.4
–
6.2
–
7.1
–
6.7
–
6.7
–
6.8
7.2
–
7.2
–
<6.0
<6.0
–
<6.0
2%b
<6.0
1%b
<6.0
2%b
<6.0
–
<6.0
37%b
6.0
–
7
7
7
7
7
7
7
a
b
c
f
–
7.3
–
7.7
–
<6.0
<6.0
6%b
<6.0
–
7.2
–
6.3
–
–
–
–
50%b
6.0
–
7.2
–
7.8
–
<6.0
16% /28%c
<6.0
–
b
7.2
–
8.1
–
<6.0
17% /31%c
–
–
6.2
–
b
g
i
–
–
7.4
–
8.2
–
6.0
–
–
–
2%b
<6.0
–
6%b
6.1
11%b
6.3
6.7
–
7.6
–
–
6.5
<6.0
–
j
–
13%b
6.7
7.4
7.6
–
–
Experimental values are highlighted in bold, whereas predicted pK values (in μM) (obtained from CT-link
i
[
32,33]) are given in italics
a
Average pK values from ChEMBL [34]
i
b
c
%Inhibition at 10 μM
%Inhibition at 100 μM
Target abbreviations: CACNA1C, Voltage-dependent L-type calcium channel subunit alpha-1C; CACNA1D,
Voltage-dependent L-type calcium channel subunit alpha-1D; CACNA1S, Voltage-dependent L-type calcium
channel subunit alpha-1S; AR, androgen receptor; A1, adenosine A1 receptor; A3, adenosine A3 receptor;
CYP3A4, cytochrome P450 3A4
the BP. This is followed by compounds 7c and 7g. DHPs
c and 7g with identical ester substituent showed maxi-
alpha-1C (CACNA1C), alpha-1D (CACNA1D), and alpha-
1S (CACNA1S) [34]. For these three targets, affinities (pKi )
were predicted within 1 log unit of the respective experimen-
tal values. In addition, nifedipine is also known to have weak
off-targetaffinitiesfortheadenosineA1andA3receptorsand
the cytochrome P450 3A4 (CYP3A4) [34]. Indeed, predicted
7
mum activity with the presence of i-propyl ester substituent
as compared to the less bulky ethyl and methyl ester sub-
stituents. However, the activity decreased or was completely
lost when the i-propyl ester was replaced with the benzyl
esterin7hand7d. Whenthedimedoneringin 7iwasreplaced
with the ethyl ester substituent yielding a symmetrical DHP
pK values for those three off-targets were above 1 micro-
i
molar (pK < 6.0). Finally, nifedipine was also predicted
i
(
7a), the complete loss of activity was observed.
On comparing the effect of N-phenyl pyranopyrazole and
to have weak off-target affinities for several nuclear recep-
tors, the androgen receptor (AR) being among them. Even
though experimental binding affinities between nifedipine
and nuclear receptors are currently lacking in publicly avail-
able databases [34], the antagonistic effect of nifedipine on
the mineralocorticoid receptor has been reported [35], and
thus, nuclear receptors also deserve some consideration as
weak off-targets of this drug.
unsubstituted pyranopyrazole at 4-position, it appeared that
N-substituted pyranopyrazole yielded compounds with bet-
ter activity, with exception of benzyl ester substituents at the
3
rd and 5th position, presumably because of the steric hin-
drance due to bulky groups.
Off-target pharmacology: computational prediction and
experimental validation
Taking the target and off-target pharmacology of nifedip-
ine as reference, the corresponding predicted affinities for
compounds 7a–c, 7f–g, and 7i–j werealsoobtained(Table 5).
As can be observed, all seven 1,4-DHPs are predicted to bind
to the same three L-type calcium channel targets of nifedip-
ine, which would substantiate their effect on blood pressure
and heart rate (vide supra).
An integrated combination of ligand-based approaches, as
implemented in the software CT-link [32,33], were used to
estimate the target and off-target pharmacology of nifedipine
andcompounds7a–c, 7f–g, and7i–j. Theresultsarecollected
in Table 5.
Moreover, weakmicromolaraffinitiesfortheoff-targetsof
nifedipine were also predicted for some of the compounds.
Nifedipine is known to bind with potent affinity (<10 nM)
to the voltage-dependent L-type calcium channel subunits
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Mol Divers
For example, compounds 7a–c and 7i–j were predicted to
have affinity for the adenosine A1 and A3 receptors. Unfor-
tunately, in vitro testing of the five compounds on those two
off-targets did not result in any relevant affinity. Also, com-
pounds 7a, 7c and 7f–g were predicted to have affinity for the
androgen receptor. However, in vitro testing of compounds
some local suppliers, and were used as such without any prior
purification. Homogeneity of all the products was analyzed
by thin-layer chromatography (TLC) on alumina-coated
plates (Merck). Product samples in MeOH were loaded
on TLC plates and developed in CHCl3–MeOH (9.5:0.5,
v/v). UV radiation was used as the visualizing agent. Com-
pounds were purified by column chromatography on silica
gel columns (100–200 mesh size, CDH), using petroleum
ether-ethyl acetate (3:2, v/v) as the eluent. Melting points
were determined in open glass capillary tubes on a Buchi
M-560 instrument and are uncorrected. Infrared (IR) spec-
tra were recorded in KBr medium using a PerkinElmer
7f–g on that off-target returned only very weak affinities.
Finally, compounds 7a, 7c, 7f–g and 7i were predicted to be
weakbindersofCYP3A4. Inthiscase, invitrotestingofcom-
pounds 7a and 7c on this key enzyme for drug metabolism
did confirm weak micromolar affinity binding.
1
13
Fourier Transform-IR spectrometer, whereas H and
C
Conclusion
nuclear magnetic resonance (NMR) spectra were recorded
in DMSO-d6 and CDCl3 medium on a JNM ECX-400P
A new class of 1,4-dihydropyridines containing the pyra-
nopyrazole moiety was successfully synthesized and
screened for various in silico, in vitro and in vivo activi-
ties. The in vivo experimentation results show that most of
the compounds of this novel class of 1,4-DHPs act as potent
positive inotropes, as they lead to an increase in arterial blood
pressure (ABP). Compounds 7c, 7g and 7i appear to be the
most effective positive inotropes, even at low doses.
Some of them caused moderate increase in the heart rate
as well. The docking results for these compounds were also
in agreement with the in vivo results. Based on the in vivo
and docking experiments, compound 7i appeared to be the
most potent positive inotrope. The docking results showed
that compound 7i possesses stronger binding than the stan-
dard BAY K 8644 with the calcium channel. The positive
inotropic effect of these compounds might be attributed to
the attachment of pyranopyrazole ring at the 4-position of
(JEOL, USA) spectrometer with tetramethylsilane (TMS)
as internal reference and recorded in ppm. IR and NMR
spectra were recorded at the Department of Chemistry and
USIC, University of Delhi, India. HRMS data were collected
on high-resolution EI-mass spectrometer with a resolution
of 10,000 on 6530 QTOF LCMS, at University Scientific
Instrument Center (USIC), University of Delhi, Delhi, India.
−1
Absorption frequencies (ν) are expressed in cm , chemi-
cal shifts in ppm (δ-scale) and coupling constants (J) in Hz.
Splitting patterns are described as singlet (s), doublet (d) and
multiplet (m). All the experiments in microwave were car-
ried out in a closed vial applying a dedicated CEM-Discover
monomode microwave apparatus operating at a frequency of
2.45 GHz with continuous irradiation power from 0 to 300 W
(CEM Corporation).
Synthesis
1,4-DHP ring. The compounds can be further investigated
for their effects on BP in normotensive rats and their binding
with calcium channel, to confirm the mechanistic pathway
for the positive inotropic activity.
General procedure for the synthesis of
3,4-dimethylpyrano[2,3-c]pyrazol-6(1H)-one, 2a–b
The target and off-target affinity profiles of nifedip-
ine (ChEMBL193) and compounds 7a–c, 7f–g, and 7i–j
show that the compounds are predicted to bind with the
three L-type calcium channel targets quite strongly like
nifedipine, hence altering the blood pressure and heart rate.
Also, these affinity profiles revealed that the hybridized
dihydropyridine–pyranopyrazole scaffold has delivered new
moderate hits, to be optimized, for the cytochrome P450 3A4
receptors, opening avenues for combined pharmacological
activity through standard structural modification.
A well-stirred mixture of phenylhydrazine 1a/hydrazine
hydrate 1b (0.14 mol) and ethyl acetoacetate 4a (0.28 mol)
◦
was heated for 12 h at 130−140 C with continuous stirring,
and the progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was
allowed to cool down to room temperature and washed with
diethyl ether (5×150 mL). A yellowish crystalline solid was
obtained as pure solid.
3-Methyl-6-oxo-1-phenyl-1,6-dihydropyrano[2,3-
c]pyrazole-4-carbaldehyde, 3a
Experimental
To a stirred solution of 3,4-dimethyl-1-phenylpyrano[2,3-
c]pyrazol-6(1H)-one 2a (20 g, 0.08 mol) in 1,4-dioxane was
added SeO2 (0.14 mol) and the reaction mixture was refluxed
for 16 h. The progress of the reaction was monitored by
TLC, and it was allowed to attain room temperature after
Materials and methods
All chemicals used in the synthesis of 1,4-dihydropyridine
analogues were purchased from Sigma-Aldrich, Fluka, and
1
23

Mol Divers
the complete conversion of the reactant. The deposited sele-
nium metal was filtered off, and the residue was washed with
ethyl acetate (2–3 times). The filtrate was concentrated in
vacuo, and the obtained residue was purified using silica gel
column chromatography petroleum ether-ethyl acetate (3:2,
v/v) to obtain pure product as a yellow colored solid in 45 %
Dimethyl 2,6-dimethyl-4-(3-methyl-6-oxo-1-phenyl-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-1,4-dihydropyridine-
3,5-dicarboxylate, 7b
Compound 7b was prepared from 3a (1 g, 0.004 mol),
methyl acetoacetate 4b (0.008 mol) using the procedure
described for 7a. Yield 752%; off-white solid; melting point
◦
−1
)
yield. Melting Point 154−156 C; IR (KBr) νmax: (cm
1
◦
−1
1
9
7
709, 1688, 1604, 1478; H NMR (400 MHz, CDCl3) δ
288−290 C; IR (KBr) ν max: (cm ) 3200, 2947, 1748,
1
.99 (1H, s, –CHO), 7.82 (2H, d, J = 7.32 Hz, Ar–H),
1709, 1549, 1381, 1210, 768; H NMR (400 MHz, CDCl3)
.51–7.47 (2H, m, Ar–H), 7.37–7.33 (1H, m, Ar–H), 6.49
δ 7.86 (2H, d, J = 7.79 Hz, Ar–H), 7.48–7.44 (2H, m,
1
3
ꢀ
(
1H, s, H–5), 2.63 (3H, s, −CH ); C NMR (100 MHz,
Ar–H), 7.32–7.28 (1H, m, Ar–H), 6.00 (1H, s, H-5 ), 5.87
3
DMSO) 191.85, 159.68, 159.10, 151.10, 145.39, 136.48,
(1H, br, –NH), 5.12 (1H, s, H-4), 3.59 (6H, s, −OCH ), 2.76
3
ꢀ
13
1
36.22, 129.52, 127.54, 120.99, 114.22, 15.90; HRMS: Cal-
(3H, s, H-3 ), 2.34 (6H, s, H-2 & 6); C NMR (100 MHz,
+
culated for [M + H] 255.0770, found 255.0761.
DMSO) δ 168.61, 167.48, 160.71, 149.52, 147.09, 145.94,
1
36.77, 129.95, 127.70, 121.05, 105.44, 102.20, 101.86,
+
3-Methyl-6-oxo-1,6-dihydropyrano[2,3-c]pyrazol-4-
51.25, 38.87, 18.73, 14.97; HRMS: Calculated for [M + H]
450.1660, found 450.1651.
carbaldehyde, 3b
Compound 3b was prepared from 3,4-dimethylpyrano[2,3-
c]pyrazol-6(1H)-one 2a (20 g, 0.08 mol) using the procedure
described for 3a. Yield 55 %; Yellow colored solid; melt-
Diisopropyl 2,6-dimethyl-4-(3-methyl-6-oxo-1-phenyl-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-1,4-dihydropyridine-
3,5-dicarboxylate, 7c
◦
−1
ing point 162−164 C; IR (KBr) νmax: (cm ) 3222, 1701,
1
1
692, 1601; H NMR (400 MHz, DMSO) δ 13.15 (1H, s,
Compound 7c was prepared from 3a (1 g, 0.004 mol),
isopropyl acetoacetate 4c (0.008 mol) using the procedure
described for 7a. Yield 70%; off-white solid; melting point
–
NH), 9.86 (1H, s, –CHO), 6.69 (1H, s, H-5), 2.44 (3H,
1
3
s, −CH ); C NMR (100 MHz, DMSO) δ 192.63, 161.92,
3
◦
−1
161.32, 155.58, 145.08, 137.63, 117.86, 13.05; HRMS: Cal-
268−270 C; IR (KBr) νmax: (cm ) 3286, 2980, 1774,
+
1
culated for [M + H] 179.0457, found 179.0459.
1743, 1551, 1384, 1221, 772; H NMR (400 MHz, DMSO)
δ 8.85 (1H, s, –NH), 7.72 (2H, d, J = 7.93 Hz, Ar–
Diethyl 2,6-dimethyl-4-(3-methyl-6-oxo-1-phenyl-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-1,4-dihydropyridine-
H), 7.54–7.50 (2H, m, Ar–H), 7.37–7.34 (1H, m, Ar–H),
ꢀ
5.76 (1H, s, H-5 ), 4.95 (1H, s, H-4), 4.83–4.80 (2H, m,
ꢀ
3,5-dicarboxylate, 7a
−CH(CH3)2), 2.69 (3H, s, H-3 ), 2.22 (6H, s, H-2 & 6), 1.08
(
6H, d, J = 6.10 Hz, −OCH(CH )2), 0.87 (6H, d, J =
3
1
3
Amixtureof3-methyl-6-oxo-1-phenyl-1,6-dihydropyrano[2,
-c]pyrazole-4-carbaldehyde 3a (1 g, 0.004 mol), ethyl ace-
6.10 Hz, −OCH(CH )2); C NMR (100 MHz, DMSO) δ
3
3
168.68, 166.33, 160.39, 149.06, 146.07, 136.56, 129.68,
127.29, 120.72, 104.96, 102.67, 101.85, 66.83, 36.24, 21.74,
toacetate 4a (0.008 mol), ammonium acetate (0.006 mol)
◦
+
and barium nitrate (2 mol%) was irradiated at 90 C for
21.27, 18.89, 18.69, 15.77; HRMS: Calculated for [M + H]
20 min at 100 W power in a microwave synthesizer. The
506.2286, found 506.2283.
reaction mixture was allowed to attain room temperature
after the complete conversion of the reactant as monitored
by TLC. The reaction mixture was poured into ethanol,
and the solid obtained was washed with cold water. The
desired product was obtained as an off-white solid in 75%
Dibenzyl 2,6-dimethyl-4-(3-methyl-6-oxo-1-phenyl-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-1,4-dihydropyridine-
3,5-dicarboxylate, 7d
◦
−1
)
yield. Melting Point 297−299 C; IR (KBr) νmax: (cm
Compound 7d was prepared from 3a (1 g, 0.004 mol),
benzyl acetoacetate 4d (0.008 mol) using the procedure
described for 7a. Yield 75%; off-white solid; melting point
1
3
241, 2963, 1754, 1698, 1574, 1383, 1220, 774; H NMR
(400 MHz, DMSO) δ 8.94 (1H, s, –NH), 7.74 (2H, d,
◦
−1
J = 7.69 Hz, Ar–H), 7.54–7.51 (2H, m, Ar–H), 7.37–7.34
252−254 C; IR (KBr) νmax: (cm ) 3293, 2957, 1711,
ꢀ
1
(
3
1H, m, Ar–H), 5.77 (1H, s, H-5 ), 5.03 (1H, s, H-4), 3.97–
1676, 1599, 1382, 1210, 758; H NMR (400 MHz, DMSO)
ꢀ
.91 (4H, m, −OCH CH3), 2.66 (3H, s, H-3 ), 2.25 (6H, s,
δ 9.05 (1H, s, –NH), 7.70 (2H, d, J = 7.93 Hz, Ar–H),
7.59–7.55 (2H, m, Ar–H), 7.41–7.37 (1H, m, Ar–H), 7.12–
7.10 (6H, m, Ar–H), 7.01–6.99 (4H, m, Ar–H), 5.76 (1H,
2
1
3
H-2 & 6), 0.96 (6H, m, −OCH2CH ); C NMR (100 MHz,
3
DMSO) δ 168.34, 166.64, 166.13, 149.12, 146.35, 145.71,
ꢀ
136.53, 129.57, 127.03, 120.66, 105.11, 102.17, 101.65,
s, H-5 ), 5.17 (2H, d, J = 12.21 Hz, −OCH ), 5.10 (1H,
2
5
9.45, 36.00, 18.64, 15.15, 13.99; HRMS: Calculated for
s, H-4), 4.81 (2H, d, J = 12.21 Hz, −OCH ), 2.45 (3H, s,
2
+
ꢀ
13
[
M + H] 478.1976, found 478.1970.
H-3 ), 2.28 (6H, s, H-2 & 6); C NMR (100 MHz, DMSO)
1
23

Mol Divers
+
δ 168.47, 166.32, 160.10, 148.77, 146.95, 145.32, 136.21,
18.80, 11.40; HRMS: Calculated for [M + H] 430.1979,
found 430.1970.
129.47, 128.07, 127.67, 126.97, 120.38, 105.12, 102.41,
1
01.41, 64.82, 35.95, 18.65, 14.78; HRMS: Calculated for
+
[
M + H] 602.2286, found 602.2278.
Dibenzyl 2,6-dimethyl-4-(3-methyl-6-oxo-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-1,4-dihydropyridine-
1
,1-(2,6-Dimethyl-4-(3-methyl-6-oxo-1-phenyl-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-1,4-dihydropyridine-
,5-diyl)diethanone, 7e
3,5-dicarboxylate, 7h
3
Compound 7h was prepared from 3b (1 g, 0.004 mol),
benzyl acetoacetate 4d (0.008 mol) using the procedure
described for 7a. Yield 72%; off-white solid; melting point
Compound 7e was prepared from 3a (1 g, 0.004 mol), acety-
lacetone 4e (0.008 mol) using the procedure described for
◦
7
a. Yield 70%; off-white solid; melting point 283−285 C;
◦
−1
2
03−205 C; IR (KBr) νmax (cm ) 3221, 3031, 2359, 1698,
−1
IR (KBr) νmax: (cm ) 3200, 2840, 1710, 1577, 1380, 1220,
93; H NMR (400 MHz, DMSO) δ 8.99 (1H, s, –NH), 7.74
1
1601, 1498, 1213; H NMR (400 MHz, DMSO) δ 12.95 (1H,
1
7
s, –NH), 9.01 (1H, s, –NH), 7.23–7.21 (6H, m, Ar-H), 7.04–
(
7
2H, d, J = 7.32 Hz, Ar–H), 7.56–7.52 (2H, m, Ar–H),
ꢀ
7
.02 (4H, m, Ar–H), 5.69 (1H, s, H-5 ), 5.05–4.90 (5H, m,
ꢀ
.39–7.35 (1H, m, Ar–H), 5.66 (1H, s, H-5 ), 5.03 (1H, s,
ꢀ
−
OCH & H-4), 2.47 (3H, s, H-3 ), 2.26 (6H, s, H-2 & 6);
C NMR (100 MHz, DMSO) δ 166.85, 166.57, 162.66,
ꢀ
2
H-4), 2.84 (3H, s, H-3 ), 2.36 (6H, s, −COCH ), 2.26 (6H,
3
13
s, H-2 & 6); 13C NMR (100 MHz, DMSO) δ 195.53, 168.43,
1
1
1
61.45, 159.12, 152.77, 146.82, 137.12, 136.35, 128.55,
28.50, 128.35, 128.19, 127.96, 127.77, 107.46, 106.90,
160.24, 149.04, 146.62, 145.70, 136.60, 129.52, 127.11,
1
20.79, 114.97, 103.96, 102.61, 36.03, 31.51, 19.65, 15.61;
01.55, 100.62, 65.24, 35.70, 18.80, 11.43; HRMS: Calcu-
+
HRMS: Calculated for [M + H] 418.1768, found 418.1762.
+
lated for [M + H] 526.1977, found 526.1977.
Dimethyl 2,6-dimethyl-4-(3-methyl-6-oxo-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-1,4-
dihydropyridine-3,5-dicarboxylate, 7f
Ethyl 2,7,7-trimethyl-4-(3-methyl-6-oxo-1-phenyl-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate, 7i
Compound 7f was prepared from 3b (1 g, 0.004 mol),
methyl acetoacetate 4b (0.008 mol) using the procedure
described for 7a. Yield 72%; off-white solid; melting point
Amixtureof3-methyl-6-oxo-1-phenyl-1,6-dihydropyrano[2,
3-c]pyrazole-4-carbaldehyde 3a (1 g, 0.004 mol), dimedone
5, ethyl acetoacetate 4a (0.008 mol), ammonium acetate
(0.006 mol) and barium nitrate (2 mol%) was irradiated
◦
−1
2
1
8
3
2
1
5
3
69−271 C; IR (KBr) νmax (cm ) 3229, 2952, 1688, 1599,
1
502; H NMR (400 MHz, DMSO) δ 12.97 (1H, s, –NH),
.96 (1H, s, –NH), 5.69 (1H, s, H-5 ), 4.98 (1H, s, H-4),
ꢀ
ꢀ
◦
.46 (6H, s, −OCH ), 2.64 (3H, s, H-3 ), 2.26 (6H, s, H-
at 90 C for 20 min at 100 W power in a microwave syn-
3
& 6); 13C NMR (100 MHz, DMSO) δ 167.08, 166.79,
thesizer. The reaction mixture was allowed to attain room
temperature after the complete conversion of the reactant
as monitored by TLC. The reaction mixture was poured
into ethanol, and the solid obtained was washed with cold
water. The desired product was obtained as an off-white
62.50, 158.86, 146.42, 137.68, 107.42, 101.56, 100.18,
0.85, 35.69, 18.42, 11.82; HRMS: Calculated for [M + H]
74.1360, found 374.1359.
+
◦
Diisopropyl 2,6-dimethyl-4-(3-methyl-6-oxo-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-1,4-dihydropyridine-
solid in 71% yield. Melting point 308−310 C; IR (KBr)
−1
νmax: (cm ) 3193, 2976, 1739, 1694, 1549, 1375, 1221,
770; H NMR (400 MHz, DMSO) δ 9.27 (1H, s, –NH), 7.77
1
3,5-dicarboxylate, 7g
(
2H, d, J = 7.79 Hz, Ar–H), 7.56–7.52 (2H, m, Ar–H), 7.39–
ꢀ
Compound 7g was prepared from 3b (1 g, 0.004 mol),
isopropyl acetoacetate 4c (0.008 mol) using the procedure
described for 7a. Yield 73%; off-white solid; melting point
7.35 (1H, m, Ar–H), 5.67 (1H, s, H-5 ), 5.05 (1H, s, H-4),
ꢀ
4.00–3.89 (2H, m, −OCH CH3), 2.77 (3H, s, H-3 ), 2.45–
2
2.35 (2H, m, −CH ), 2.32 (3H, s, H-2), 2.20 (1H, d, J =
2
◦
−1
2
1
–
4
36−238 C; IR (KBr) νmax (cm ) 3222, 2979, 2933, 1696,
16.03 Hz, −CH ), 2.01 (1H, d, J = 16.03 Hz, −CH ), 1.01
2
2
1
674, 1598,; H NMR (400 MHz, DMSO) δ 12.95 (1H, s,
NH), 8.81 (1H, s, –NH), 5.67 (1H, s, H-5 ), 4.89 (1H, s, H-
(3H, s, –C(CH )2), 0.95–0.92 (3H, m, −OCH2CH ), 0.89
3
3
ꢀ
13
(3H, s, –C(CH )2); C NMR (100 MHz, DMSO) δ 194.86,
3
ꢀ
), 4.86–4.80 (2H, m, −CH(CH3)2), 2.69 (3H, s, H-3 ), 2.24
167.72, 166.48, 160.25, 150.27, 149.15, 146.46, 146.23,
136.58, 129.61, 127.23, 120.72, 109.96, 104.55, 103.13,
102.63, 59.56, 49.97, 33.49, 32.30, 28.90, 26.56, 18.63,
(
6H, s, H-2 & 6), 1.08 (6H, d, J = 6.10 Hz, −OCH(CH )2),
3
1
3
0
.87 (6H, d, J
=
6.10 Hz, −OCH(CH )2); C NMR
3
+
(
100 MHz, DMSO) δ 166.50, 162.63, 161.40, 152.72,
15.49, 13.87; HRMS: Calculated for [M + H] 488.2193,
145.75, 138.10, 137.09, 107.30, 106.87, 66.72, 21.72, 21.26,
found 488.2194.
1
23

Mol Divers
Methyl 2,7,7-trimethyl-4-(3-methyl-6-oxo-1-phenyl-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate, 7j
The reaction mixture was allowed to attain room temperature
after the complete conversion of the reactant as monitored by
TLC. The reaction mixture was poured into ethanol, and the
solid obtained was washed with cold water. The desired prod-
uct was obtained as an off-white solid in 62% yield. Melting
Compound 7j was prepared from 3a (1 g, 0.004 mol),
methyl acetoacetate 4b (0.008 mol) using the procedure
described for 7i. Yield 75%; off-white solid; melting point
◦
−1
Point 253−255 C; IR (KBr) νmax: (cm ) 3210, 2981, 1748,
1
1689, 1550, 1335, 1220, 772; H NMR (400 MHz, DMSO)
◦
−1
2
1
95−297 C; IR (KBr) νmax (cm ) 3184, 2975, 1746, 1701,
δ 9.11 (1H, s, –NH), 7.77 (2H, d, J = 5.49 Hz, Ar–H), 7.57–
1
563, 1381, 1238, 768; H NMR (400 MHz, DMSO) δ 9.28
7.53 (2H, m, Ar–H), 7.40–7.38 (1H, m, Ar–H), 5.80 (1H, s,
ꢀ
(
1H, s, –NH), 7.78 (2H, d, J = 7.33 Hz, Ar–H),7.56–7.52
H-5 ), 5.03 (1H, s, H-4), 4.88–4.82 (1H, m, −OCH(CH3)2),
ꢀ
ꢀ
(2H, m, Ar–H), 7.39–7.36 (1H, m, Ar–H), 5.67 (1H, s, H-5 ),
3.48 (1H, s, H-4), 2.70 (3H, s, H-3 ), 2.27 (6H, s, H-2), 1.11
ꢀ
5
.07 (1H, s, H-4), 3.47 (3H, s, −OCH ), 2.76 (3H, s, H-3 ),
(3H, d, J = 6.10 Hz, −OCH(CH )2), 0.87 (3H, d, J =
3
3
1
3
2
.46–2.35 (2H, s, −CH ), 2.32 (3H, s, H-2), 2.20 (1H, d,
6.10 Hz, −OCH(CH )2); C NMR (100 MHz, DMSO) δ
2
3
J = 16.03 Hz, −CH ), 2.02 (1H, d, J = 16.03 Hz, −CH ),
168.56, 167.26, 166.39, 160.39, 149.26, 146.71, 146.26,
145.86, 136.61, 129.73, 127.41, 120.82, 105.17, 102.35,
102.17, 101.38, 66.89, 50.94, 36.12, 21.80, 21.25, 18.82,
2
2
1
3
1.01 (3H, s, –C(CH )2), 0.90 (3H, s, –C(CH )2); C NMR
3 3
(100 MHz, DMSO) δ 195.13, 167.69, 167.13, 160.44,
+
1
1
3
50.49, 149.31, 146.70, 146.23, 136.68, 129.72, 127.35,
20.79, 110.21, 104.70, 103.10, 102.54, 51.07, 50.04, 33.56,
18.66, 15.37; HRMS: Calculated for [M + H] 478.1978,
found 478.1972.
2.44, 29.09, 26.57, 18.66, 15.31; HRMS: Calculated for
+
[
M + H] 474.2030, found 474.2035.
Biological evaluation
Isopropyl 2,7,7-trimethyl-4-(3-methyl-6-oxo-1-phenyl-1,6-
dihydropyrano[2,3-c]pyrazol-4-yl)-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate, 7k
Blood pressure and heart rate monitoring
Male spontaneously hypertensive rats (200–300 g), obtained
from animal house, CSIR-Central Drug Research Institute
(CSIR-CDRI), were maintained under standard conditions
Compound 7k was prepared from 3a (1 g, 0.004 mol),
isopropyl acetoacetate 4c (0.008 mol) using the proce-
dure described for 7i. Yield 68%; off-white solid; melting
◦
of 12-h/12-h light-dark cycle and temperature (23 ± 2 C).
The animals were housed in cages and provided free access
to food (standard pellet chow) and water. The animals were
handledaccordingtotheguidelinesofCommitteeforthePur-
pose of Control and Supervision of Experiments on Animals
(CPCSEA), India, and Institutional Animal Ethics Commit-
tee of CSIR-CDRI. These animals were used to study the
antihypertensive potential of the compounds.
◦
−1
point 292−294 C; IR (KBr) νmax (cm ) 3215, 2991, 1789,
1
1
711, 1589, 1393, 1241, 772; H NMR (400 MHz, DMSO)
δ 9.23 (1H, s, –NH), 7.76 (2H, d, J = 7.93 Hz, Ar-
H), 7.56–7.52 (2H, m, Ar–H), 7.39–7.36 (1H, m, Ar–H),
ꢀ
5
.67 (1H, s, H-5 ), 5.01 (1H, s, H-4), 4.86–4.80 (1H, m,
ꢀ
−
CH(CH3)2), 2.78 (3H, s, H-3 ), 2.44–2.34 (2H, m, −CH ),
2
2
2
6
.32 (3H, s, H-2), 2.18 (1H, d, J = 15.87 Hz, −CH ),
2
.01 (1H, d, J = 16.03 Hz, −CH ), 1.09 (3H, d, J =
Measurement of hemodynamic parameters
2
.10 Hz, −OCH(CH )2), 1.00 (3H, s, –C(CH )2), 0.89 (3H,
3
3
s, –C(CH )2), 0.76 (3H, d, J = 6.10 Hz, −OCH(CH )2);
Theanimalswereweighedandanaesthetizedforsurgerywith
urethane (Sigma, USA) administered intraperitoneally (i.p.)
at the dose of 1.25 g/kg. The trachea was cannulated with
polyethylene tube for respiration. For measurement of blood
pressure (BP) and heart rate (HR), the left carotid artery was
cannulated with a pressure transducer which was connected
with a data acquisition system (AD Instruments, Australia).
The right external jugular vein was cannulated for the admin-
istration of drugs.
3
3
1
3
C NMR (100 MHz, DMSO) δ 194.71, 167.82, 165.89,
60.16, 150.14, 149.05, 146.28, 146.22, 136.51, 129.55,
27.16, 120.68, 109.79, 104.43, 103.30, 102.81, 66.55,
1
1
4
9.95, 33.49, 32.18, 28.72, 26.60, 21.63, 20.91, 18.65, 15.73;
HRMS: Calculated for [M + H] 502.2349, found 502.2344.
+
Isopropyl 5-acetyl-2,6-dimethyl-4-(3-methyl-6-oxo-1-
phenyl-1,6-dihydropyrano[2,3-c]pyrazol-4-yl)-1,4-
dihydropyridine-3-carboxylate, 7l
Preparation and administration of nifedipine and
compounds
Amixtureof3-methyl-6-oxo-1-phenyl-1,6-dihydropyrano[2,
3-c]pyrazole-4-carbaldehyde 3a (1 g, 0.004 mol), methyl 3-
amino crotonate 6 (0.006 mol), isopropyl acetoacetate 4c
Standard calcium channel blocker nifedipine was dissolved
in polyethylene glycol 400 and was administered at the dose
of 1 mg/kg intravenously (i.v.) in a constant volume of 0.5 mL
(
0.008 mol) and barium nitrate (2 mol%) was irradiated at
◦
90 C for 20 min at 100 W power in a microwave synthesizer.
1
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Mol Divers
3
over a period of 10–15 s. The venous catheter was then
flushed with an additional 0.2 mL of saline. The compounds
were solubilized in 100% DMSO and were administered at
the doses of 1, 5 and 10 mg/kg through i.v. route in a constant
volume of 0.5 mL over a period of 10–15 s, and the venous
catheter was then flushed with an additional 0.2 mL of saline.
size 80 × 80 × 80 Å with coordinates X = 24.9501 Å, Y =
41.5458 Å and Z = −8.5647 Å was generated at the centroid
oftheligand. Togaininsightintotheprotein–ligandcomplex,
all the ligands prepared above were docked into the active site
using the “extra precision” XP algorithm. The docking algo-
rithm in Glide utilizes the OPLS-2005 molecular mechanics
potential energy function for the final minimization.
Molecular docking studies
Predicted off-target pharmacology
The work is divided into three steps as follows:
The off-target pharmacology of molecules from this work
was predicted with CT-link [32,33]. Using as input two-
dimensional chemical structures, CT-link applies ligand-
based methods to predict the binding affinity of small
molecules to a list of 3492 proteins for which pharmaco-
logical data are available in the public domain [34]. The five
independent ligand-based methods implemented in CT-link
version 2016 include descriptor-based similarity approaches
[32,38], fuzzy fragment-based mapping [35], and target
cross-pharmacology [36]. Detailed information on those
methods is provided elsewhere [32,33,38,39]. The results
from CT-link have been validated prospectively in a variety
of applications [40–44].
Step 1: Preparation of ligand library of synthesized acti-
vators
Out of the twelve DHPs (Table 2) that we synthesized and
evaluated for their biological activities, we took the best five
compounds (7a, 7b, 7c, 7g and 7i) for our theoretical cal-
culations. The geometries of these five ligands were refined
using the LigPrep module (Schrödinger, Inc.). All possible
ionization states and tautomers were generated at target pH
7
± 2. Since our ligand molecules contain chiral centers, we
also generated various chirality combinations in order to see
which enantiomeric form provides the best binding results.
After generating all possible ionization states, tautomers and
stereoisomers, our ligand library comprised of a total of eight
states, i.e., seven states of the best five compounds obtained
after LigPrep refinement, plus one reference compound, (S)-
BAY K 8644.
Binding at human androgen receptor
Assays were carried out at endogenous androgen receptor
5
expressed at T47D cell line [45]. Briefly 3 × 10 cells/well
Step 2: Protein preparation and identification of binding
site
were seeded in a poly-D-lysine-treated 24-well plate in
RPMI-1640 medium supplemented with 10% FBS and main-
The structure of KVAP-33H1 FV complex (PDB ID: 2A0L)
was downloaded from the RCSB Protein Data Bank (www.
rcsb.in) and refined for our further calculations using the pro-
tein preparation workflow in Schrödinger. The imported pro-
tein is a multimeric chain containing monoclonal fragments
of FV chains, which were deleted, and the protein chains
◦
tained at 37 C in a 5% CO2 atmosphere for 24 h. Medium
was replaced by assay medium containing RPMI-1640, 0.1%
3
BSA, 1 μM Triamcinolone, 2 nM [ H]R1881 and the test
◦
compounds. Cells were incubated for 2 h at 37 C and then
◦
washed twice with HBSS at 4 C. Then 500 μL of HBSS
containing 0.5% sodium dodecyl sulfate and 20% glycerol
were added to all the wells. 350 μL was transferred to a
scintillation vial, and radioactivity was measured in a Beck-
man LS6500 scintillation detector. Nonspecific binding was
defined with 100 μM testosterone.
(A and B) of KVAP were retained. Employing the OPLS-
2005 force field [36], the resultant structure was minimized
untiltheroot-mean-squaredeviation(RMSD)reachedamax-
imum cutoff of 0.30 Å. This prepared protein, along with its
well-known activator (S)-BAY K 8644, was used for finding
the probable binding sites using the SiteMap module [37]
developed by Schrödinger, Inc. SiteMap is Schrödinger’s
program for identifying and characterizing ligand binding
sites. Various possible binding states were predicted by
SiteMap, and based on their Site score and other important
parameters, the active site was found to be at the interface of
the two chains (A and B) of KVAP. The point to note here is
that this is the same site as that of binding of its inhibitor.
CYP3A4 inhibition
Activity of the compounds at human CYP3A4 inhibition was
measured by using Vivid fluorogenic cytochrome inhibition
kit from Invitrogen following supplier recommendations.
Radioligand binding assays
Step 3: Glide docking
Radioligand binding competition assays were performed in
vitrousingA1, A2A, A2BandA3humanreceptorsexpressed
in transfected CHO (hA1), HeLa (hA2A and hA3) and HEK-
293 (hA2B) cells [46].
After ensuring that the protein and the ligand were in the cor-
rect form for docking, a receptor grid was generated using an
advanced molecular docking program, Glide. A grid box of
1
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Mol Divers
Acknowledgements This work was partially supported by Chem-
Biobank, the Chemical Biology infrastructure initiative in Spain,
carrying out the predictive and the biological activity of the synthe-
sized compounds. The authors would like to thank CDRI, Lucknow, for
carrying out the in vivo studies, Defence Research and Development
Organization (DRDO) for financial support and University of Delhi,
Delhi, for providing the laboratory and instrumentation facility.
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