Hydroxyl alkyl ammonium ionic liquid assisted green and one-pot regioselective access to functionalized pyrazolodihydropyridine core and their pharmacological evaluation

Article abstract of DOI:10.1016/j.bioorg.2019.01.029

Herein our team explored a promising synthetic trail to Functionalized pyrazolodihydropyridine core using hydroxyl alkyl ammonium ionic liquid via one-pot fusion of 3-methyl-1-phenyl-1H-pyrazole-5-amine, different heterocyclic aldehydes and 1, 3-Cyclic diones. The aimed compounds were obtained by Domino-Knoevenagel condensation and Michael addition followed by cyclization. The reaction transformation involves the formation of two C–C and one C–N bond formation. The perspective of the present work is selectively approached to Functionalized pyrazolodihydropyridine core excluding other potential parallel reactions under environmentally benign reaction condition. The present protocol show features such as the low E-factor, ambiphilic behavior of ionic liquid during reaction transformation, scale-up to a multigram scale, reusability of the ionic liquid, mild reaction condition, and produce water as a byproduct. All newly derived compounds were evaluated for their in vitro biological activities. In preliminary biological studies compound, 4c showed better potency than the standard drug ampicillin against Gram-negative bacteria (E. coli); the compound 4i exhibited outstanding activity against S. aeruginosa which is far better than ampicillin, chloramphenicol, and ciprofloxacin. The compound 4m was found more potent against C. albicans, than that of griseofulvin and show equipotency to nystatin whereas, in preliminary antitubercular screening, compound 4o was exhibited more potency than rifampicin. Noteworthy compounds 4f and 4i were found most active in antiproliferative screening.

Full text of DOI:10.1016/j.bioorg.2019.01.029

Accepted Manuscript
Hydroxyl Alkyl Ammonium Ionic liquid assisted Green and One-pot regiose-
lective access to Functionalized Pyrazolodihydropyridine core and their phar-
macological evaluation
Divyang M. Patel, Mayank G. Sharma, Ruturajsinh M. Vala, Irene Lagunes,
Adrián Puerta, José M. Padrón, Dhanji P. Rajani, Hitendra M. Patel
PII:
S0045-2068(18)30961-1
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.029
YBIOO 2733
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
31 August 2018
25 December 2018
17 January 2019
Please cite this article as: D.M. Patel, M.G. Sharma, R.M. Vala, I. Lagunes, A. Puerta, J.M. Padrón, D.P. Rajani,
H.M. Patel, Hydroxyl Alkyl Ammonium Ionic liquid assisted Green and One-pot regioselective access to
Functionalized Pyrazolodihydropyridine core and their pharmacological evaluation, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.01.029
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Hydroxyl Alkyl Ammonium Ionic liquid assisted Green and One-pot
regioselective access to Functionalized Pyrazolodihydropyridine core
and their pharmacological evaluation
a
a
a
b
b
Divyang M. Patel, Mayank G. Sharma, Ruturajsinh M. Vala, Irene Lagunes, Adrián Puerta,
b
c
a,
José M. Padrón, Dhanji P. Rajani, and Hitendra M. Patel *
a
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar, 388120, Gujarat, India.
b
BioLab, Instituto Universitario de Bio-Orgánica Antonio González, CIBICAN, Universidad de
La Laguna, Avda. Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain.
c
Microcare Laboratory and Tuberculosis Diagnosis & Research Center, Surat, India
*
E-mail: hm_patel@spuvvn.edu
1

Abstract
Herein our team explored a promising synthetic trail to Functionalized
pyrazolodihydropyridine core using hydroxyl alkyl ammonium ionic liquid via
one-pot fusion of 3-methyl-1-phenyl-1H-pyrazole-5-amine, different heterocyclic
aldehydes and 1, 3-Cyclic diones. The aimed compounds were obtained by
Domino-Knoevenagel condensation and Michael addition followed by cyclization.
The reaction transformation involves the formation of two C-C and one C-N bond
formation. The perspective of the present work is selectively approached to
Functionalized pyrazolodihydropyridine core excluding other potential parallel
reactions under environmentally benign reaction condition. The present protocol
show features such as the low E-factor, ambiphilic behavior of ionic liquid during
reaction transformation, scale-up to a multigram scale, reusability of the ionic
liquid, mild reaction condition, and produce water as a byproduct. All newly
derived compounds were evaluated for their in-vitro biological activities. In
preliminary biological studies compound, 4c showed better potency than the
standard drug ampicillin against Gram-negative bacteria (E. coli); the compound 4i
exhibited outstanding activity against S. aeruginosa which is far better than
ampicillin, Chloramphenicol, and Ciprofloxacin. The compound 4m was found
more potent against C. albicans, than that of griseofulvin and show equipotency to
Nystatin whereas, in preliminary antitubercular screening, compound 4o was
2

exhibited more potency than rifampicin. Noteworthy compounds 4f and 4i were
found most active in antiproliferative screening.
Keywords
Pyrazolodihydropyridine, hydroxyl alkyl ammonium ionic liquids, task-specific,
cost-effective, ambiphilic behavior, E-factor, environmentally benign, recyclable.
1. Introduction
Multi-component reactions (MCRs) are the powerful tool for convenient synthesis
of biologically active scaffolds [1-4]. MCRs facilitate single step conversion over
the stepwise conversation of the organic molecules [5-7]. In synthetic chemistry,
MCRs proved as ideal synthetic tools for the construction of complex molecule
targets because of their inherent convergence power, ease of operation, atom-
economy, men power reduction and resource effectivity from the sustainability
point of view. The literature survey reveals some pioneering work for the
syntheses of some pyrazole fused polyheterocyclic compounds. [8-10]
Noteworthy, pyrazole fused N-heterocyclic derivatives exhibit potent biological
activity and some of them are celecoxib, sildenafil etc. [11-13]
pyrazolodihydropyridine and pyrazolopyridine have been studies for
antileishmanial activity and found to be potent as an adjunct therapy with
3

miltefosine [14]. Some pyrazole bearing biologically active molecules are shown
in figure 1.
Fig. 1 some biologically active molecules having pyrazole as a basic constituent.
These basic features of pyrazole bearing moieties attract the researchers for
development of the reaction methodology for pyrazolodihydropyridine core. As
MCRs involving three or more components, there are molecular interactions
between components sometimes generate the issue of selectivity which leads to
other potent parallel reactions [15-17]. Earlier reported protocols for the synthesis
of pyrazolo fused dihydropyridines was carried out using triethylamine under
4

microwave irradiation [18], imidazolium salts [19] polyethyleneglycol [20], amino
acid [21] glacial acetic acid [22] and sodium-1-dodecanesulfonate [23] catalyzed
access to pyrazolodihydropyridine derivatives. However, earlier reported protocols
faced drawbacks like the use of the expensive, hazardous, non-recoverable catalyst,
harsh reaction condition, relatively lower product yield, and tedious work up. As
per our literature survey is concerned, there is no reported methodology which
mainly involved the utilization of cost-effective, low environment factor,
ambiphilic catalytic behavior, atom-economic, less toxic and task-specific ionic
liquid. Earlier studies on hydroxyl alkyl ammonium ionic liquids show the wide
range of applications in organic syntheses [24-34] reclaiming phenolic compounds
from low-temperature coal tar [35] mobile phase modifier for reversed-phase
liquid chromatography [36] and also used as media for biocatalytic oxidations
[37]etc.
5

Scheme 1. Ionic liquid triggered selective one-pot access to functionalized
pyrazolodihydropyridine core.
Considering the highlighted pharmacological activities of pyrazolodihydropyridine
derivatives and in continuation to our work on the development of biologically
active heterocyclic scaffolds [38], here we reported hydroxyl alkyl ammonium
ionic liquid catalyzed one-pot regioselective synthesis of the functionalized
pyrazolodihydropyridine derivatives.
2
2
. Experimental section
.1. General Informations
All chemicals which are used in the present study were purchased from
commercially available sources and used without further purification. The
purification of all the synthesized compounds was made by the automated flash
chromatographic system (YAMAZEN AI-580S) with HI-FLASH silica gel
columns (230-400 µm mesh size) using 50% solution of ethyl acetate in petroleum
ether (60-80 °C) as a mobile phase. The Mass analysis of all newly synthesized
compounds was collected on MS - Agilent 6120 quadrupole. Melting points were
1
13
determined by open capillary tube method and are uncorrected. H-NMR and C-
APT spectral analysis were recorded on BRUKER AVANCE II 400 NMR
6

Spectrometer using CDCl and DMSO-d as the solvents. The chemical shifts are
3
6
expressed in parts per million and coupling constants (J) are provided in Hertz.
2.2. Single-crystal X-ray Diffraction Analysis
The single-crystal X-ray diffraction data of compounds 4b, 4h, 4i, and 4j were
collected on a Rigaku SCX mini diffractometer using graphite monochromated
Mo-Kα radiation at 293K temperature. The structures were solved by direct
methods [39] and expanded using Fourier techniques. Non-hydrogen atoms were
refined isotropically and hydrogen atoms were refined using the riding model. All
calculations were performed using the Crystal Structure [40] crystallographic
software package except for refinement, which was performed using SHELXL-97
[41].
2.3. General procedure for the synthesis of different hydroxyl alkyl
ammonium ionic liquids [42].
Hydroxyl alkyl ammonium ionic liquids were derived by neutralization reaction of
Ethanolamine derivatives in ethanol with different acid derivatives. For example,
here we described the synthetic procedure of 2-hydroxyethyl ammonium acetate
ionic liquid: 2-hydroxyethyl amine (3 mmol) was dissolved in 60 mL of absolute
alcohol to form a liquid mixture and charged in 500 mL two neck-flask equipped
with reflux condenser and dropping funnel and cooled in an ice bath(0-5 °C).
Under vigorous stirring the mixture of acetic acid (3 mmol) in 60 mL of absolute
7

ethanol was then added dropwise in the flask about 90 min by maintaining the
temperature below 5 °C. The stirring was continued for 24 h. The excess solvent
was evaporated under vacuum. The resulting viscous liquid was further used for
the synthesis of compounds.
2.4. General procedure for the synthesis of pyrazolodihydropyridine
derivatives 4a-o.
3-methyl-1-phenyl-5-aminopyrazole 1 (3 mmol), heterocyclic aldehyde 2a-e (3
mmol), 1, 3-cyclic dione 3a-c (3 mmol), were charged into 50ml round bottom
flask followed by addition of 2 mL of hydroxyl alkyl ammonium ionic liquid. The
content is then stirred at 90 °C until the TLC (50 % ethyl acetate in petroleum ether
(60-80 °C)) reveals the complete consumption of starting materials. After
completion of the reaction, the mixture was poured into ice water and filtered off.
The filtrate containing IL was then charged in Rota evaporator and recovered by
removing excess water under reduced pressure at 60-70 °C and was reused for
subsequent reactions. Furthermore, synthesized compounds were purified by
Automated Flash chromatographic system using 50% ethyl acetate in petroleum
ether (60-80 °C) and the crystal blocks of synthesized compounds were carefully
developed from methanol-ethanol (1:1, v/v) as a solvent system. The
characterization detail of all newly synthesized compounds was shown below.
8

2.5. General procedure for the development of crystal blocks of 4b, 4h, 4i, and
4j.
After chromatographic purification of the synthesized compound, the dried and
pure compound (∼40 mg) was placed in neat and clean crystal developing vessel
followed by addition of ∼30 ml of methanol-ethanol (1:1, v/v) as a solvent system.
This whole solution is warmed for the preparation of the homogeneous solution
and then allowed to cool down gently and placed for slowly solvent evaporation.
2.6.
Characterizations
details
of
all
newly
synthesized
pyrazolodyhydropyridine derivatives.
3-methyl-4-(3-methylthiophen-2-yl)-1-phenyl-6,7,8,9-tetrahydro-1H-pyrazolo
[3,4-b] quinolin-5(4H)-one. 4a.
1
White solid; melting point: 261-264 °C; R = 0.32; H NMR (400 MHz, DMSO-
f
d6) (δ, ppm): 9.60 (s, 1H, NH),7.38-7.56 (m, 5H, ArH), 7.10 (d, J = 5.2 Hz, 1H,
Thiophene-CH), 6.66 (d, J = 5.2 Hz, 1H, Thiophene-CH), 5.32 (s, 1H, CH), 2.53-
2
.69 (m, 2H, CH ), 2.30 (s, 3H, CH ), 2.22 (t, J = 6.0 Hz, 2H, CH ), 1.91 (s, 3H,
2
3
2
13
CH ), 1.75-1.88 (m, 2H, CH ); C APT (100 MHz, DMSO-d ) (δ, ppm): 194.62,
3
2
6
1
1
52.90, 146.56, 146.46, 138.67, 136.27, 131.25, 129.88, 129.73, 127.32, 123.56,
22.38, 111.36, 104.62, 37.44, 29.35, 27.78, 21.39, 14.12, 12.36; MS (MM-
+
ES+APCI) 374.3(M+H) .
9

3,7,7-trimethyl-4-(3-methylthiophen-2-yl)-1-phenyl-6,7,8,9-tetrahydro-1H-
pyrazolo[3,4-b]quinolin-5(4H)-one. 4b.
1
White solid; melting point: 230-233 °C; R = 0.54; H NMR (400 MHz, CDCl )
f
3
(δ, ppm): 7.30-7.73 (m, 5H, ArH), 6.96 (d, J = 4.8 Hz, 1H, Thiophene-CH), 6.66
(
d, J = 5.2 Hz, 1H, Thiophene-CH), 5.41 (s, 1H, CH), 2.41 (s, 3H, CH ), 2.33 (d, J
3
=
5.2 Hz, 2H, CH), 2.16 (d, J = 9.2 Hz, 2H, CH), 2.09 (s, 3H, CH ), 1.03 (s, 6H,
3
13
CH ); C APT (100 MHz, CDCl ) (δ, ppm): 195.27, 148.67, 147.47, 145.15,
3
3
1
1
4
38.00, 135.14, 131.71, 131.71, 129.79, 129.47, 127.38, 122.98, 121.85, 111.74,
04.89, 50.72, 42.18, 32.46, 29.26, 27.24, 13.95, 12.10; MS (MM-ES+APCI)
+
02.3(M+H) .
3-methyl-4-(3-methylthiophen-2-yl)-1-phenyl-4,6,7,8-tetrahydrocyclopenta[b]
pyrazolo[4,3-e]pyridin-5(1H)-one. 4c.
1
White solid; melting point: 228-231 °C; R = 0.28; H NMR (400 MHz, DMSO-
f
d6) (δ, ppm): 9.97 (s, 1H, NH), 7.20-7.35 (m, 5H, ArH), 6.90 (d, J = 5.2 Hz, 1H,
Thiophene-CH), 6.51 (d, J = 5.2 Hz, 1H, Thiophene-CH), 4.97 (s, 1H, CH), 2.46 (t,
J = 4.0 Hz, 2H, CH ), 2.35 (t, 2.0 Hz, 2H, CH ), 2.13 (s, 3H, CH ), 1.67 (s, 3H,
2
2
3
13
CH ); C APT (100 MHz, CDCl + DMSO-d ) (δ, ppm): 200.23, 165.47, 146.76,
3
3
6
1
1
43.70, 137.95, 137.24, 131.60, 129.16, 129.12, 126.70, 123.03, 122.07, 115.30,
+
03.75, 33.63, 28.33, 24.23, 13.64, 11.77; MS (MM-ES+APCI) 360.2(M+H) .
1
0

4-(5-bromothiophen-2-yl)-3-methyl-1-phenyl-6,7,8,9-tetrahydro-1H-pyrazolo
[3,4-b] quinolin-5(4H)-one. 4d.
1
Yellowish white solid; melting point: 218-221 °C; R = 0.30; H NMR (400
f
MHz, DMSO-d ) (δ, ppm): 9.61 (s, 1H, NH), 7.35-7.5 (m, 5H, ArH), 6.83 (d, J =
6
3.6 Hz, 1H, Thiophene-CH), 6.54 (d, J = 3.6 Hz, 1H, Thiophene-CH), 5.33 (s, 1H,
CH), 2.53-2.68 (m, 2H, CH ), 2.28 (quint, J = 4.8 Hz, 2H, CH ), 2.05 (s, 3H, CH ),
2
2
3
13
(
m, 2H, CH ); C APT (100 MHz, CDCl + DMSO-d ) (δ, ppm): 194.39, 153.76,
2
3
6
1
1
4
53.18, 145.98, 138.03, 136.31, 129.14, 129.01, 126.78, 123.47, 123.20, 109.92,
08.78, 102.28, 36.79, 30.58, 27.28, 20.78, 11.87; MS (MM-ES+APCI)
+
40.3(M+H) .
4-(5-bromothiophen-2-yl)-3,7,7-trimethyl-1-phenyl-6,7,8,9-tetrahydro-1H-
pyrazolo[3,4-b] quinolin-5(4H)-one. 4e.
1
Yellowish white solid; melting point: 184-187 °C; R = 0.51; H NMR (400 MHz,
f
DMSO-d ) (δ, ppm): 9.54 (s, 1H, NH), 7.35-7.51 (m, 5H, ArH), 6.85 (d, J = 3.6
6
Hz, 1H, Thiophene-CH), 6.57 (d, J = 3.6 Hz, 1H, Thiophene-CH), 5.31 (s, 1H,
CH), 2.48 (s, 2H, CH ), 2.13 (m, 2H, CH ), 2.05 (s, 3H, CH ), 0.98 (s, 6H, CH );
2
2
3
3
13
C APT (100 MHz, CDCl + DMSO-d ) (δ, ppm): 194.05, 153.76, 151.41, 145.98,
3
6
1
5
38.00, 136.31, 129.14, 129.06, 126.78, 123.47, 123.16, 108.67, 108.58, 102.34,
+
0.33, 40.60, 31.85, 30.69, 26.86, 11.87; MS (MM-ES+APCI) 466.2(M+H) .
1
1

4-(5-bromothiophen-2-yl)-3-methyl-1-phenyl-4,6,7,8-tetrahydrocyclopenta[b]
pyrazolo[4,3-e]pyridin-5(1H)-one. 4f.
1
White solid; melting point: 222-225 °C; R = 0.26; H NMR (400 MHz, DMSO-
f
d6) (δ, ppm): 10.09 (s, 1H, NH), 7.22-7.36 (m, 5H, ArH), 6.71 (d, J = 4.0 Hz, 1H,
Thiophene-CH), 6.55 (d, J = 3.6 Hz, 1H, Thiophene-CH), 4.99 (s, 1H, CH), 2.35 (t,
13
J = 1.6 Hz, 2H, CH ), 2.17 (t, 2.4 Hz, 2H, CH ), 1.84 (s, 3H, CH ); C APT (100
2
2
3
MHz, CDCl + DMSO-d ) (δ, ppm): 200.32, 166.16, 152.08, 146.67, 137.79,
3
6
1
3
37.65, 129.17, 126.92, 124.06, 123.21, 120.88, 114.15, 109.20, 102.13, 33.67,
+
0.37, 24.20, 12.07; MS (MM-ES+APCI) 426.2(M+H) .
4
1
-(6-bromobenzo[d][1,3]dioxol-5-yl)-3-methyl-1-phenyl-6,7,8,9-tetrahydro-
H-pyrazolo [3,4-b] quinolin-5(4H)-one. 4g.
1
White solid; melting point: 255-258 °C; R = 0.33; H NMR (400 MHz, DMSO-
f
d6) (δ, ppm): 9.34 (s, 1H, NH), 7.31-7.49 (m, 5H, ArH), 6.88 (s, 1H, CH), 6.61 (s,
1
2
H, CH), 5.89 (s, 2H, CH ), 5.34 (s, 1H, CH), 2.65 (s, 2H, CH ), 2.2 (t, J = 4.8 Hz,
2
2
13
H, CH ), 1.93 (s, 3H, CH ) 1.76-1.91 (m, 2H, CH ). C APT (100 MHz, CDCl +
2
3
2
3
DMSO-d ) (δ, ppm): 194.25, 153.33, 153.10, 146.29, 145.95, 138.06, 136.10,
6
1
3
29.00, 128.23, 126.58, 123.15, 121.06, 115.87, 112.09, 103.04, 101.28, 36.99,
+
1.81, 27.42, 20.96, 12.46; MS (MM-ES+APCI) 476.2(M+H) .
4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3,7,7-trimethyl-1-phenyl-6,7,8,9-tetra-
hydro-1H-pyrazolo[3,4-b]quinolin-5(4H)-one. 4h.
1
2

1
Yellowish white solid; melting point: 252-255 °C; R = 0.65; H NMR (400
f
MHz, DMSO-d ) (δ, ppm): 7.32-7.44 (m, 5H, ArH), 6.95 (s, 1H, CH), 6.82 (s, 1H,
6
CH), 5.87 (s, 2H, CH ), 4.99 (s, 1H, CH), 2.35 (s, 2H, CH ), 2.14 (s, 2H, CH ),
2
2
2
13
2
.06 (s, 3H, CH ) 1.02 (s, 6H, CH ); C APT (100 MHz, CDCl + DMSO-d ) (δ,
3
3
3
6
ppm): 195.37, 166.86, 149.95, 147.89, 147.56, 146.47, 140.64, 137.84, 135.60,
1
4
35.58, 129.85, 127.56, 127.51, 123.04, 122.98, 113.22, 101.51 71.81, 50.77,
+
5.34, 42.35, 32.56, 12.72; MS (MM-ES+APCI) 504.3(M+H) .
4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3-methyl-1-phenyl-4,6,7,8-tetrahydro-
cyclopenta[b] pyrazolo[4,3-e]pyridin-5(1H)-one. 4i.
1
white solid; melting point: 261-264 °C; R = 0.28; H NMR (400 MHz, DMSO-
f
d6) (δ, ppm): 10.22 (s, 1H, NH), 7.40-7.55 (m, 5H, ArH), 7.09 (s, 1H, CH), 6.67 (s,
1
H, CH), 6.00 (d, J = 5.2 Hz, 2H, CH ), 5.21 (s, 1H, CH), 2.66 (dd, J = 4.8 Hz, 6.8
2
13
Hz, 2H, CH ), 2.26 (t, J = 4.8 Hz, 2H, CH ), 1.84 (s, 3H, CH ); C APT (100
2
2
3
MHz, DMSO-d ) (δ, ppm): 200.11, 166.80, 166.78, 146.43, 146.38, 137.88,
6
1
4
29.37, 127.07, 123.38, 112.42, 101.75, 33.67, 24.39, 12.26; MS (MM-ES+APCI)
+
64.2(M+H) .
4
1
-(2-butyl-5-chloro-1H-imidazol-4-yl)-3-methyl-1-phenyl-6,7,8,9-tetrahydro-
H-pyrazolo [3,4-b]quinolin-5(4H)-one. 4j.
1
Off white solid; melting point: 228-231 °C; R = 0.29; H NMR (400 MHz,
f
DMSO-d ) (δ, ppm): 11.81 (s, 1H, NH), 9.57 (s, 1H, NH), 7.39-7.55 (m, 5H, ArH),
6
1
3

5
2
.07 (s, 1H, CH), 2.55-2.68 (m, 2H, CH ), 2.47 (t, J = 7.6 Hz, 2H, CH ), 2.20-
2
2
.245 (m, 2H, CH ), 1.93(s, 3H, CH ), 1.82-1.89 (m, 2H, CH ), 1.55 (quint, J = 7.6
2
3
2
13
Hz, 2H, CH ), 1.55 (sext, J= 7.2 Hz, 2H, CH ), 0.86 (t, J = 7.6 Hz, 2H, CH ); C
2
2
2
APT (100 MHz, DMSO-d ) (δ, ppm): 194.25, 153.80, 145.93, 144.28, 138.16,
6
1
2
4
36.30, 129.35, 128.68, 126.86, 123.18, 122.13, 106.84, 101.16, 36.91, 29.95,
7.45, 27.37, 26.03, 21.58, 20.96, 13.60, 11.74; MS (MM-ES+APCI)
+
34.3(M+H) .
4-(2-butyl-5-chloro-1H-imidazol-4-yl)-3,7,7-trimethyl-1-phenyl-6,7,8,9-tetra-
hydro-1H-pyrazolo[3,4-b]quinolin-5(4H)-one. 4k.
1
White solid; melting point: 183-186 °C; R = 0.34; H NMR (400 MHz, DMSO-
f
d6) (δ, ppm): 11.75 (s, 1H, NH), 9.40 (s, 1H, NH), 7.33-7.49 (m, 5H, ArH), 5.06 (s,
1
H, CH), 2.46-2.51 (m, 2H, CH ), 2.03-2.16 (m, 2H, CH ), 1.92 (s, 3H, CH ), 1.56
2
2
3
(
quint, J = 7.6 Hz, 2H, CH ), 1.27 (t, J= 8 Hz, 2H, CH ), 1.23 (s, 2H, CH ), 1.01
2
2
2
13
(
d, J = 8 Hz, 6H, CH ), 0.87 (t, J = 3.6 Hz, 2H, CH ); C APT (100 MHz, DMSO-
3
2
d6) (δ, ppm): 193.97, 151.51, 146.07, 144.25, 138.20, 136.49, 129.04, 128.64,
1
2
26.50, 122.98, 122.32, 106.03, 101.04, 50.47, 40.69, 31.70, 30.03, 28.79, 27.61,
+
7.02, 26.03, 21.61, 13.52, 11.61; MS (MM-ES+APCI) 462.3(M+H) .
4-(2-butyl-5-chloro-1H-imidazol-4-yl)-3-methyl-1-phenyl-4,6,7,8-tetrahydro-
cyclopenta[b] pyrazolo[4,3-e]pyridin-5(1H)-one. 4l.
1
4

1
Off white solid; melting point: 179-182 °C; R = 0.20; H NMR (400 MHz,
f
DMSO-d ) (δ, ppm): 11.73 (s, 1H, NH), 10.23 (s, 1H, NH), 7.40-7.58 (m, 5H,
6
ArH), 4.88 (s, 1H, CH), 2.64 (s, 2H, CH ), 2.45-2.49(t, 2H, CH ), 2.29 (d, J = 2.0
2
2
Hz, 2H, CH ), 1.86 (s, 3H, CH ), 1.55 (quint, J = 7.6 Hz, 2H, CH ), 1.26 (sext, J =
2
3
2
13
7
.2 Hz, 2H, CH ), 0.86 (t, J = 7.2 Hz, 3H, CH ); C APT (100 MHz, DMSO-d )
2
3
6
(δ, ppm): 200.66, 167.50, 146.95, 145.73, 138.44, 138.37, 129.92, 127.55, 127.02,
1
1
23.72, 123.16, 112.29, 101.58, 34.17, 30.40, 28.00, 25.73, 24.88, 22.09, 14.10,
+
2.25; MS (MM-ES+APCI) 420.3(M+H) .
3-methyl-1-phenyl-4-(thiophen-2-yl)-6,7,8,9-tetrahydro-1H-pyrazolo[3,4-
b]quinolin-5(4H)-one. 4m.
1
Off white solid; melting point: 212-215 °C; R = 0.31; H NMR (400 MHz,
f
CDCl ) (δ, ppm): 7.32-7.46 (m, 5H, ArH), 7.15 (s, 1H, Thiophene-CH), 7.08 (s,
3
1
2
H, Thiophene-CH), 6.87 (s, 1H, Thiophene-CH), 5.53 (s, 1H, CH), 2.46-2.52 (m,
H, CH ), 2.27-2.40 (m, 2H, CH ), 2.16 (s, 3H, CH ), 1.94-1.99 40 (m, 2H, CH );
2
2
3
2
13
C APT (100 MHz, CDCl ) (δ, ppm): 195.73, 151.38, 151.03 147.51, 137.96,
3
1
3
35.66, 129.83, 127.44, 126.43, 123.67, 123.54, 123.01, 112.79, 104.03, 37.08,
+
0.57, 28.66, 21.10, 12.18; MS (MM-ES+APCI) 360.2(M+H) .
3,7,7-trimethyl-1-phenyl-4-(thiophen-2-yl)-6,7,8,9-tetrahydro-1H-
pyrazolo[3,4-b]quinolin-5 (4H)-one. 4n.
1
5

1
Off white solid; melting point: 226-229 °C; R =0.51; H NMR (400 MHz,
f
CDCl ) (δ, ppm): 7.35-7.48 (m, 5H, ArH), 7.06 (d, J = 5.2 Hz ,1H, Thiophene-
3
CH), 6.84 (d, J = 3.6 Hz ,1H, Thiophene-CH), 6.69 (s,1H, Thiophene-CH), 5.52 (s,
1
H, CH), 2.35 (s, 2H, CH ), 2.24 (s, 2H, CH ), 2.16 (s, 3H, CH ), 1.05 (d, J = 7.2
2
2
3
13
Hz , 6H, CH ); C APT (100 MHz, CDCl ) (δ, ppm): 195.42, 151.11, 148.87,
3
3
1
5
3
47.56, 135.64, 129.95, 127.53, 126.40, 123.66, 123.53, 122.90, 111.74, 104.10,
0.80, 42.39, 32.57, 30.61, 29.13, 27.35, 12.16; MS (MM-ES+APCI)
+
88.2(M+H) .
3-methyl-1-phenyl-4-(thiophen-2-yl)-4,6,7,8-tetrahydrocyclopenta[b]pyrazolo-
[4,3-e] pyridin-5(1H)-one. 4o.
1
White solid; melting point: 233-236 °C; R = 0.22; H NMR (400 MHz, DMSO-
f
d6) (δ, ppm): 10.01 (s, 1H, NH), 7.21-7.36 (m, 5H, ArH), 6.99 (d, J = 5.2 Hz, 1H,
Thiophene-CH), 6.75 (d, J = 3.2 Hz, 1H, Thiophene-CH), 6.68 (d, J = 3.6 Hz, 1H,
Thiophene-CH), 5.03 (s, 1H, CH), 2.36(t, J = 1.6 Hz 2H, CH ), 2.16(m, 2H, CH ),
2
2
13
2
.16 (t, J = 3.6 Hz 2H, CH ), 1.81(s, 3H, CH ); C APT (100 MHz, DMSO-d ) (δ,
2
3
6
ppm): 200.31, 165.94, 150.36, 146.71, 137.88, 137.50, 129.15, 128.82, 126.79,
126.11, 123.51, 123.10, 114.89, 103.13, 33.71, 29.90, 24.17, 12.06; MS (MM-
+
ES+APCI) 346.2(M+H) .
2.7. Biological evaluation
1
6

2.7.1. In vitro antimicrobial activity
This study was screened against 24 hr. old bacterial cultures which include two
Gram-positive, two Gram-negative bacteria and three fungi via the use of disc
diffusion method according to the National Committee for Clinical Laboratory
Standards (NCCLS) [43]. Muller-Hinton Broth was used as a nutrient medium to
grow and dilute the drug suspension for the test bacteria and sabouraud dextrose
broth was used for nutrition of the fungi. All the synthesized compounds were
screened at 1000 ppm in DMSO solution. The strains which were used for this
study were attained from MTCC, Institute of Microbial Technology, Chandigarh.
The synthesized compounds 4(a-o) were screened for their antibacterial activity
against Gram-negative bacteria like E. Coli (MTCC 443), P. Aeruginosa (MTCC
96), and against Gram-positive bacteria such as S. aureus(MTCC 96) and S.
Pyogenus(MTCC 442) and their antifungal screening was performed against C.
Albicans (MTCC 227), A. Niger (MTCC 282) and A. Clavatus (MTCC 1323).In
this study, the standard drugs used for the comparison of antibacterial activity were
ampicillin, chloramphenicol, ciprofloxacin, norfloxacin whereas nystatin and
griseofulvin were used as standard drugs for the antifungal activity.
2.7.2. In vitro antituberculosis activity
1
7

The encouraging results from the antibacterial studies impelled us for preliminary
screening of all newly synthesized compounds against Mycobacterium
tuberculosis. In this screening, 1,000, 500, and 250 µg/ml concentrations of the
compounds were taken. The active compounds found in this screening were further
tested in a secondary screening against M. tuberculosis H37Rv in L. J. Medium
(conventional method). The compounds found active in primary screening were
similarly diluted to obtain 200, 100, 50, 25, 12.5, 6.250, and 3.50 µg/ml
concentrations. The antitubercular activity data were compared with rifampicin at a
-
1
4
0 µg/ml concentration. All the values of MIC (µg. mL ) are provided for 99%
inhibition of M. tuberculosis growth. As shown in table 5.
2.7.3. In vitro Antiproliferative activity
The anti-proliferative activity of the reported compounds was studied using our
implementation of the NCI protocol [44] against human solid tumor cell lines
(
A549, HBL-100, HeLa, SW1573, T-47D, WiDr) and the human fibroblast cell
line BJ-hTert kindly provided by Dr. G. J. Peters (The Netherlands) and Dr.
Raimundo Freire (Canary Islands). The results are expressed as GI (dose that
50
produces 50% growth inhibition) and were determined for an exposure time of 48
h. Cell line suspensions were counted with Moxi Z and diluted to reach the
appropriate cell densities (A549, HBL-100, HeLa and SW1573 2,500 cells/well; T-
47D and WiDr 5,000 cells/well; BJ-hTert 4,000 cells/well) for inoculation onto 96-
1
8

well plates. After 24 hours, pure compounds were added. Each agent was initially
dissolved in DMSO at 400 times the desired final maximum test concentration
(
100 µM). Control cell samples were exposed to an equivalent concentration of
DMSO (0.25% v/v, negative control). Compounds were tested at serial dilutions
0.01-100 µM).
(
3
3
. Results and Discussion
.1. Chemistry
In the present protocol, we selected one-pot fusion of 3-methyl-1-phenyl-1H-
pyrazole-5-amine 1 (3 mmol), 3-methylthiophene-2-carboxaldehyde 2a (3 mmol)
and 5, 5-Dimethyl-1, 3-dione 3b (3 mmol) in the presence of different hydroxyl
alkyl ammonium ionic liquids as a model reaction. As shown in figure 2.
Fig. 2 Different hydroxyl alkyl ammonium ionic liquids used in this study.
During the optimization of reaction conditions we made attempt to scale down the
amount of ionic liquid 0.5 and 1.0 mL which equal to ∼4.7 mmol and 9.5 mmol
-
3
-1
respectively (density =1.149039 g . cm ; M
121.14 g . mol ) [37].
w
=
1
9

Unfortunately, we observed solidification of the reaction mass and no complete
consumption of starting materials. This problem was overcome by loading a
minimum 1.5 mL or more amount of ionic liquid. Here we carried out the synthesis
of 4b using different hydroxyl alkyl ammonium ionic liquids under different
reaction conditions as shown in Table-1(1-12 entries). Our first attempt was to
synthesize 4b at ambient temperature condition using different hydroxyl alkyl
ammonium ionic liquids. But at ambient temperature condition, we observed that
(
HE) AAc IL shows no reaction transformation, and satisfactory reaction
3
transformation observed in case of (HE) AAc, HEALac, HEAF, and HEAAc
2
isolated yields are 48%, 55%, 62%, and 67% respectively. Among all ILs best
result obtained with HEAAc (2 mL, ∼6.3 equiv.). So we selected HEAAc IL to
synthesize desired products. In order to reduce the reaction time and achieving the
complete conversion of starting materials into desired products, we go further for
the screening of temperature and different solvents. Here we carried out the
reaction using different hydroxyl alkyl ammonium ionic liquids at different
temperatures (Table-1) and also compared the catalytic efficiency of HEAAc IL
with some reported and non-reported catalysts (Table-2, entries 1-16) as well.
After analyzing all the results collected from optimization of the reaction
parameters, the excellent yield is obtained when the reaction is carried out using
HEAAc (2 mL, ∼ 6.3 equiv.) at 90 °C under neat condition. We did not find any
2
0

significant increment in the yield of the desired product for the temperature range
beyond 90 °C. Therefore, we selected it as the optimal reaction temperature for all
these reactions.
Table. 1. Optimization of the reaction conditions for the synthesis of 4b.
2
1

Temp Time
b
a
Yield
%)
Entry
1
Ionic liquids
(
(
°C ) (min.)
tris(hydroxyethyl)ammoniumacetate
3600 NR
35
[
(HE) AAc]
3
bis(2-hydroxyethyl)ammoniumacetate
(HE) AAc]
35
35
35
35
50
70
90
95
90
3600 48
3600 55
3600 62
3600 67
2400 70
2
3
4
5
6
7
8
9
[
2
2
2
2
2
2
2
2
2
-hydroxyethylammonium lactate [HEALac]
-hydroxyethylammonium formate [HEAF]
-hydroxyethylammonium acetate [HEAAc]
-hydroxyethylammonium acetate [HEAAc]
-hydroxyethylammonium acetate [HEAAc]
-hydroxyethylammonium acetate [HEAAc]
-hydroxyethylammonium acetate [HEAAc]
-shtydroxyethylammonium acetate [HEAAc]
180
120
120
120
76
84
84
82
1
1
1
0
1
2
(1 run)
2
-nhdydroxyethylammonium acetate [HEAAc]
90
90
180
180
81
77
(2 run)
2
-rhdydroxyethylammonium acetate [HEAAc]
(3 run)
a
Reaction conditions: 3-methyl-1-phenyl-1H-pyrazole-5-amine
1
(3 mmol), 5,5-
dimethylcyclohexane-1,3-dione 3b (3 mmol), 3-methylthiophene-2-carboxaldehyde 2a (3
b
mmol), and HEAAc IL(2 mL, ∼ 6.3 equiv.). Isolated yield of products.
2
2

Table 2. The comparison of the catalytic efficiency of Ionic liquid with other
catalysts.
b
References
for reported
catalysts
a
Time Yield
min) (%)
Entry Catalyst
Solvent
--
Temp
°C )
(
(
L-Proline
1
MW/110 15
75
17
(10 mol%)
2
3
PEG-400
--
100-110 240
MW/150 15
68
70
16
14
Et N
Ethanol
3
(
3.6 mmol)
SDS
0.3 gm)
Water
90
420
30
60
12
58
45
38
19
--
4
5
6
7
(
Ethanol
Ethanol
Ethanol
Ethanol
Reflux
Reflux
Reflux
Reflux
--
CAN
180
180
180
--
(
20 mol%)
AlCl3
20 mol%)
--
(
--
--
FeCl3
8
9
(
20 mol%)
Citric acid Ethanol
20 mol%)
Reflux
180
46
70
(
Glacial
acetic
acid
RT (35) 180
18
1
0
--
HEAAc
Water
90
180
120
NR
66
--
--
1
1
1
2
(2 ml)
HEAAc
Methanol
Reflux
(2 ml)
2
3

HEAAc
2ml)
Ethanol
Reflux
90
120
120
80
60
--
--
1
1
3
4
(
HEAAc
2ml)
Aqueous
ethanol
(
(1:1, v/v)
HEAAc
2ml)
Aqueous
ethanol
90
90
120
120
55
84
--
1
1
5
6
(
(1:1, v/v)
HEAAc
2ml)
Neat
(IL only)
This
work
(
a
Reaction conditions: 3-methyl-1-phenyl-1H-pyrazole-5-amine
1
(3 mmol), 5,5-
dimethylcyclohexane-1,3-dione 3b (3 mmol), 3-methylthiophene-2-carboxaldehyde 2a (3
b
mmol). Isolated yield of 4b.
Procedure for Catalyst Recycling:
After completion of the reaction, the mixture was poured into ice cold water and
filtered off. The filtrate containing IL was then charged in Rota evaporator and
recovered by removing excess water under reduced pressure at 60-70 °C and was
reused up to three more runs for subsequent reactions (without considering fresh
run). Up to the second run (without considering fresh run), there is no significant
change in the yield of the desired product but product yield significantly decreases
in the third run.
2
4

Fig. 3 Recycling of HEAAc Ionic liquid.The synthesis of pyrazolodihydropyridine
derivatives was carried out under the optimized reaction conditions of HEAAc
with 3-methyl-1-phenyl-1H-pyrazole-5-amine 1, 1,3-diones 2(a-c), and different
aldehydes 3 (a-e). To our delight, all the reactions proceed smoothly under
optimized reaction conditions and the isolated yields of desired products were in
the range of 60-89 % and as shown in table 3.
2
5

a
Table 3. Synthesis of functionalized pyrazolodihydropyridine derivatives 4a-o .
2
6

2
7

As an efficient and economic protocol, the scale-up and operational simplicity are
also important parameters thus the different scale reactions of 3-methyl-1-phenyl-
1H-pyrazole-5-amine 1 (1 equiv.), 5,5-dimethylcyclohexane-1,3-dione 3b (1
equiv.), 3-methylthiophene-2-carboxaldehyde 2a (1 equiv.) under optimized
reaction conditions were carried out. The scale-up outcomes are shown in table 4.
Table 4. Gram-scale synthesis of 4b.
a
b
b
Entry
Batch amount
mmol)
Purification Technique
Yield
(%)
Yield
(gm)
(
1
2
3
3.0
Column Chromatography
Column Chromatography
Column Chromatography
84
82
80
1.01
1.67
3.23
5.0
10.0
2
8

a
Reaction conditions: 3-methyl-1-phenyl-1H-pyrazole-5-amine
1 (1 equiv.), 5,5-
dimethylcyclohexane-1,3-dione 3b (1 equiv.), 3-methylthiophene-2-carboxaldehyde 2a
b
(
1 equiv.). Isolated yield of 4b.
In the preliminary investigation desired product formation was confirmed by
thin layer chromatographic Technique using 50% ethyl acetate in petroleum
ether (60-80 °C) as a mobile phase. The structure of all desired compounds was
1
13
unambiguously confirmed by H-NMR, C- NMR, and MS spectroscopic
techniques. For further verification of the desired products 4b, 4h, 4i, and 4j
were selected as representative compounds and were confirmed by single-crystal
X-ray diffraction analysis, as shown in Figures 4, 5, 6, and 7.
2
9