Ultrasound promoted green synthesis of spiro[pyrano[2,3-c]pyrazoles] as antioxidant agents

Article abstract of DOI:10.1007/s00044-013-0671-8

Ultrasound promoted, cerium ammonium nitrate catalyzed sustainable synthesis of spiro[indoline3,4′-pyrano[2,3-c]pyrazole] derivatives (4a-l) is reported herein. The synthesized compounds were screened for their antioxidant activities as free radical scave

Full text of DOI:10.1007/s00044-013-0671-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0671-8
ORIGINAL RESEARCH
Ultrasound promoted green synthesis
of spiro[pyrano[2,3-c]pyrazoles] as antioxidant agents
•
•
•
Anshu Dandia Deepti Saini Sumit Bhaskaran
Dinesh Kumar Saini
Received: 6 March 2013 / Accepted: 4 June 2013
ꢀ Springer Science+Business Media New York 2013
Abstract Ultrasound promoted, cerium ammonium nitrate
catalyzed sustainable synthesis of spiro[indoline3,40-pyr-
ano[2,3-c]pyrazole] derivatives (4a–l) is reported herein.
The synthesized compounds were screened for their anti-
oxidant activities as free radical scavenging effect on
diphenylpicryl hydrazine (DPPH•), 2,20-azino-bis(3-ethyl-
benzthiazoline-6-sulphonic acid) (ABTS•?) and nitric oxide
(NO) radicals. The screened compounds showed potent
scavenging activities against DPPH•, ABTS•? and NO
radicals. In order to further extend on studies and to obtain a
deep insight into structure activity relationship of this class
of compounds, we designed N-substitution of indole moiety
with the aim to study its antioxidant potential.
is more erratic, and assail by free radicals can generate
structural damage (i.e. strand breaks) and/or alteration of
the bases. Unrepaired DNA lesions might impair tran-
scription and protein synthesis (Hatahet et al., 1994).
Thus, an increasing interest in antioxidants, such as free
radicals and reactive oxygen species (ROS), has engrossed
substantial interest. The free radicals are also believed to be
associated with carcinogenesis, mutagenesis, arthritis, diabetes,
inflammation, cancer and genotoxicity (Kourounakis et al.,
1999; Buyukokuroglu et al., 2001) due to oxidative stress, which
arises as a result of imbalance between free radical generations.
Moreover, ROS are continuously generated in very low
amounts in active cells of aerobic organisms as byproducts
of metabolic processes, and they have found to be the key
players in the pathophysiological mechanisms associated
with various inflammatory disorders (Trouba et al., 2002).
So the significance of ROS in the pathogenesis of multi-
farious diseases has attracted considerable attention. Disease
progression may be retarded by administrating protective
compounds, which can act in several different ways as
inhibitors of ROS formation, free radical scavenging, chain
breaking antioxidants or transition metal chelators, and
therefore research on active antioxidant of natural or syn-
thetic origin received a great attention (Delles et al., 2008).
Moreover, the development of hybrid molecules having
different pharmacophores in one frame may lead to com-
pounds with interesting pharmacological profiles. In this
regards, substituted pyrano[2,3-c]pyrazoles are much sought
after class of heterocycles exhibiting wide range of biological
activities like antimicrobial (Mityurina et al., 1981; Lakshmi
et al., 2010), anticancer (Wang et al., 2009), anti-inflamma-
tory (Zaki et al., 2006), inhibitors of human Chk1 kinase
(Foloppe et al. 2006) and also as biodegradable agrochemicals
(Abdelrazek et al., 2007). Besides, indole derivatives consti-
tute an important class of therapeutic agents in medicinal
Keywords Indole-2,3-dione ꢀ
3-Methyl-1-phenyl-2-pyrazolin-5-one ꢀ
Active methylene group ꢀ Antioxidant activity
Introduction
Macromolecules like proteins, lipids and DNA are major
targets for free radical-induced damage. The oxidative
damage to proteins has been found to be high in specific
brain regions, and are elevated during ageing and in some
types of neurodegenerative disorders (Foster et al., 1996;
Floor and Wetzel, 1998). In particular, the damage to DNA
A. Dandia (&) ꢀ D. Saini ꢀ S. Bhaskaran
Center of Advanced Studies, Department of Chemistry,
University of Rajasthan, Jaipur 302004, India
e-mail: dranshudandia@yahoo.co.in
D. K. Saini
Radiation & Cancer Biology Lab, Department of Zoology,
University of Rajasthan, Jaipur 302004, India
123

Med Chem Res
chemistry, including anticancer (Suzen and Buyukbingol,
2000; Lakshmi et al., 2010), antioxidant (Lakshmi et al.,
2010), antirheumatoidal (Buyukbingol et al., 1994), anti- HIV
(Suzen and Buyukbingol, 1998), and also play a vital role in
the immune system (Lieberman et al., 1997; Page et al., 2007)
and as potent scavengers of free radicals (Chyan et al., 1999).
Literature survey reveals that synthesis of pyran system
has been accomplished by many workers (Saundane et al.,
2013; Mandha et al., 2012) employing various catalysts as
ammonium chloride (Dabiri et al., 2009), ethylenediamine
diacetate (Lee and Hari, 2010), triethylbenzyl ammonium
(TEBA) salt (Zhu et al., 2007), L-proline (Yuling et al.,
2010), surfactant metal carboxylates (Wang et al., 2010), b-
cyclodextrin (Sridhar et al., 2009), ionic liquids (Moghadam
and Miri, 2011) and reaction conditions (Shanthi et al.,
2007; Elinson et al., 2008). We have also reported the
synthesis of spirooxindole derivatives by a multi-step pro-
cess catalyzed by Et3N in ethanol under refluxing condition
Dandia et al., (2003a, b; Joshi et al., 1989). Although most
of the recent methods have their own merits, but some
methods are weakened by at least one limitation such as low
yield, (especially when bulky substituent on substrates lead
to low solubility in water) complicated workup procedure,
chromatographic separation and technical intricacy.
different substitutions and their evaluated for their antiox-
idant potential.
Initially, we tested the reaction of isatin, malanonitrile
and 3-methyl-1-phenyl-2-pyrazolin-5-one as a simple
model substrate under various reaction conditions
(Scheme 1) for synthesis of spiro[indoline3,40-pyrano[2,3-
c]pyrazole] derivative (4a). The results are shown in
Table 1. With reference to the crucial utility of solvent in
chemical transformation, a variety of solvents were
employed for the synthesis of spiro[indoline3,40-pyr-
ano[2,3-c]pyrazole] derivatives. As can be seen, that
among all the solvents, water seems to be the solvent of
choice both in terms of time and yields of the spiro[indo-
line3,40-pyrano[2,3-c]pyrazole] derivatives (4a–l).
It was observed that when the reaction was carried out at
room temperature stirring without any catalyst, the yield of
product was very low (Table 1, 35 %, entry 1) even after pro-
long time. Increasing the temperature does not seem to affect the
yieldsof the product. Afterwards, evaluation ofvariouscatalysts
was carried out for the synthesis of spirooxindole derivatives in
aqueous medium under ultrasonic irradiation (Table 1, entry
4–9). It can be seen that a mixture of isatin, ethylcyanoacetate
and 3-methyl-1-phenyl-2-pyrazolin-5-one in the presence of
catalytic amount of cerium ammonium nitrate afforded ethyl 60-
amino-30-methyl-2-oxo-10-phenyl-10H-spiro[indoline3,40-pyr-
ano[2,3-c]pyrazole]-50-carboxylate (4a) in excellent yields
(90 %), while with other catalysts, the product formed with
yields ranging between 54 and 76 %. Although, ultrasound
irradiation decreased the reaction time, but failed to increase
the yield of the final product (Table 1, entry 3).
In the above regards and on the basis of pharmacological
indications that show the existence of two or more different
heterocyclic moieties in a single molecule often remarkably
enhances the biocidal profile, we intended the synthesis of a
series of spiro[indoline3,40-pyrano[2,3-c]pyrazole] deriva-
tives (4a–l) through a three component reaction of substi-
tuted isatin, active methylene reagent, and 3-methyl-1-
phenyl-2-pyrazolin-5-one in the presence of catalytic
amount of cerium ammonium nitrate as a green and efficient
catalyst in water, thus utilizing the multipurpose promoter
efficacy of CAN (Sridharan and Menendez, 2010).
Further, the catalyst loading was optimized by using
different concentration of CAN in the model reaction. It
was found that with increasing the amount of CAN from
5 mol% to 10 and 20 mol%, the yields increased from
74 % to 90 and 92 %, respectively (Table 1, entries
10–12). Further increase in amount of catalyst does not
seem to affect the overall yields of the product. 10 mol%
CAN in water under ultrasonic irradiation, was found to the
best combination for synthesis of spiro[indoline3,40-pyr-
ano[2,3-c]pyrazole] derivatives (4a–l) in excellent yields.
Under the optimized reaction conditions, a series
of spiro[indoline3,40-pyrano[2,3-c]pyrazole] derivatives
Results and discussion
Chemistry
We approached the synthesis of a library of spiro[indo-
line3,40-pyrano[2,3-c]pyrazole] derivatives (4a–l), bearing
Scheme 1 Model reaction
C6H5
C6H5
N
COOEt
+
NC
O
N
O
C
N
H2N
N
H2
(2)
Solvent
Catalyst
EtOOC
O
(3)
O
NH
O
N
(4)
H
(1)
123

Med Chem Res
Table 1 Optimization of
reaction conditions
Entry
Catalyst
Method
Time (min)
Yield (%)
1.
–
Stirr., r.t.
Stirr., 80 ꢁC
))))
480
300
70
55
52
60
68
50
40
15
10
14
35
45
50
58
61
63
54
68
76
74
90
92
2.
–
3.
–
4.
K2CO3
))))
5.
NaHCO3
TBAB
))))
6.
))))
7.
CTAC
))))
8.
P-TSA
))))
9.
L-Proline
CAN (5 mol%)
CAN (10 mol%)
CAN (20 mol%)
))))
10.
11.
12.
))))
))))
))))
Scheme 2 Synthesis of
spiro[indoline3,40-pyrano[2,3-
c]pyrazole derivatives (4a–l)
X
NC
C6H5
C
H2
(2)
O
N
N
H2N
NC
))))
O
H2O / CAN
C6H5
O
R1
O
N
N
R1
O
NR
N
R
(4)
(1)
(3)
Table 2 Synthesis of
spiro[indoline3,40-pyrano[2,3-
c]pyrazole] derivatives (4a–l)
Entry
R
R1
H
X
Time (min)
Yield (%)
Mp (ꢁC)
4a
4b
4c
4d
4e
4f
H
H
H
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
CN
20
18
18
20
15
10
10
12
10
18
10
8
94
92
91
84
92
97
95
92
98
90
84
97
238–240
246–248
260–262
182–184
208–210
244–246
237–238
242–244
288–290
218–220
210–212
232–234
5-Cl
5,7-diCH3
CH2–CH=CH2
H
C2H5
H
CH2–C6H5
H
4g
4h
4i
H
H
H
5-Br
5-CH3
H
CN
H
CN
4j
CH2–CH=CH2
C2H5
CN
4k
4l
H
CN
CH2–C6H5
H
CN
(4a–l) were synthesized (Scheme 2). The results are sum-
marized in Table 2.
(activated by CAN) facilitates the formations of the
corresponding isatiylidene malanonitrile intermediate
(c) under sonic condition, which in turn reacts with the
active methylene site of 3-methyl-1-phenyl-2-pyrazolin-5-
one. This C-4 alkylation of 3-methyl-1-phenyl-2-pyrazolin-
5-one with electrophilic C=C of intermediate (c) followed
by nucleophilic addition of the -OH group on the cyano
moiety subsequently results in formation of the desired
product (e).
The multicomponent synthesis of spiro[indoline3,40-
pyrano[2,3-c]pyrazole] derivatives can be explained by a
plausible mechanism presented in Scheme 3. The ultra-
sonic cavitation induced shear forces and the jets produced
near the surface of the vessel, and the catalyst may activate
malanonitrile through sonolysis of the C–H bond. The
reaction between the activated malanonitrile and the isatin
123

Med Chem Res
Scheme 3 A probable
mechanism of formation of
spiro[indoline3,40-pyrano
[2,3-c]pyrazole derivatives
CN
Ce
O
Ce
OH
Ph
Ce
C
CN
H
H
CN
CN
CN
C
H
CN
C
N
N
N
H
O
O
O
H
(Hc)
(b)
(a)
N
O
Ph
HO
C
N
N
Ce
C
O
N
N
CN
CH3
CH3
N
H
(d)
C6H5
N
O
NH
O
NH
O
H2N
NC
N
C
C
Ce
Ce
C
C
H
H
NC
NC
Ph
Ph
O
N
N
CH3
N
N
N
H
O
N
O
NH
H
H3C
H3C
(e)
Antioxidant activity
substitution in indole ring and carboxylate group at 5th
position of pyran ring showed good activity, while compound
4g without any substitution in indole ring and carbonitrile
group showed least activity. In compound 4d, with N-allyl
substitution and carboxylate group showed moderate activity,
while same indole ring with carbonitrile group, i.e. com-
pound 4j, showed least activity. In compound 4i, incorpo-
ration of CH3 group at 5th position of isatin showed electron
donating effect, but carbonitrile group with electron with-
drawing effect turned it to give moderate activity, while in
compound 4c two electron donating groups are present at 5th
and 7th position of isatin with carboxylate group, but this
compound is only moderately active whereas, compound 4b
and 4h, i.e. Cl and Br substitution in indole ring, have not
shown any effect and gives least activity. The N-ethyl sub-
stitution in indole ring with carboxylate group, i.e. compound
4e, showed good activity and with carbonitrile group showed
least activity. The results are shown in Table 3 and Fig. 1.
The results show that compound 4a and 4f showed highest
activity. In compound 4a, there is presence of an electron
donating groups (hydrogen in form of NH of indole ring and
carboxylate group at 5th position of pyran ring), which sta-
bilized the compound after donating the hydrogen to DPPH•
radical. In compound 4f, there is presence of an electron
donating groups (N-benzyl, a bulky group substitution on
indole ring and carboxylate group at 5th position of pyran
ring) which stabilized the compound after donating the
hydrogen to DPPH• radical.
The antioxidant activities of spiroindolinones were deter-
mined as an index of pharmacological efficacy. Three
model systems were used namely DPPH•, ABTS•? and NO
scavenging activity. In the assessment of antioxidant
activity, only synthetic relevant free radicals were used.
The synthetic nitrogen-centred DPPH•, ABTS•? and NO
radicals were used as indicator compounds in testing
hydrogen transfer capacity that are related to the antioxi-
dant activity. Antioxidant activities data were compared
with standard drug ascorbic acid. In results, we have found
correlation between substitution in indole ring and substi-
tution of pyran ring at 5th position. Overall, all compounds
exhibited good DPPH• and NO scavenging activity
whereas, moderate ABTS•? scavenging activity. The
antioxidant properties were expressed as EC50 values.
DPPH radical scavenging activity
Although the DPPH radical scavenging abilities of all the
spiroindoline derivatives were moderately lower than those
of ascorbic acid (409.75 0.288) lg/ml, it was evident that
they show reducing ability (possibly by hydrogen transfer)
and could serve as free radical scavengers. It was anticipated
that compounds possessing carboxylate group (an electron
donating group) at 5th position of pyran ring showed higher
activity except compound 4b and 4c. The combination of
carboxylate group with N-benzyl substitution in indole ring
(i.e. compound 4f) showed good activity. This revealed to be
important for activity, since the N-benzyl substituted indole
ring with carbonitrile group (i.e. compound 4l) showed
decreased potency. The compound 4a, i.e. without any
ABTS radical scavenging activity
In ABTS assay, among the tested compounds, compound 4f
with combination of carboxylate group at 5th position of
123

Med Chem Res
Table 3 EC50 values of compounds in antioxidant properties
C6H5
N
O
H2N
X
N
O
R1
NR
EC50
Compound
R
R1
X
DPPH
ABTS
NO
671.95 0.230
4a
H
H
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
CN
539.53 0.345
811.20 0.236
920.20 0.257
619.45 0.347
600.66 0.191
456.91 0.148
1054.45 0.259
959.28 0.239
727.42 0.282
974.25 0.311
969.48 0.178
922.98 0.193
409.75 0.288
1010.20 0.346
3678.57 0.246
2763.61 0.269
1597.09 0.257
1076.53 0.242
488.60 0.358
3324.66 0.256
2941.47 0.358
1147.95 0.159
3345.33 0.512
3340.00 0.347
4167.91 0.567
550.00 0.134
4b
H
5-Cl
1372.50 0.345
1122.33 0.236
891.09 0.185
730.86 0.202
560.49 0.173
1188.57 0.132
1131.47 0.218
793.73 0.239
898.09 0.198
877.28 0.241
892.67 0.188
544.21 0.132
4c
H
5,7-diCH3
4d
CH2–CH=CH2
H
4e
C2H5
H
4f
CH2–C6H5
H
4g
H
H
4h
H
5-Br
5-CH3
H
CN
4i
H
CN
4j
CH2–CH=CH2
C2H5
CN
4k
H
CN
4l
CH2–C6H5
H
CN
Ascorbic acid
EC50 value: The effective concentration at which the antioxidant activity was 50 %; DPPH•, ABTS•? and NO radicals were scavenged by 50 %,
respectively. EC50 values were obtained by interpolation from linear regression analysis. Values were the mean of three replicates SE
Fig. 1 DPPH radical
scavenging activity of indole
derivatives at different
concentrations
pyran ring and N-benzyl substitution at indole ring, showed
good activity which is equipotent to ascorbic acid, which we
took as standard drug. Besides, with N-substitution and car-
boxylate group at 5th position of pyran ring (i.e. compound
4d) and with carbonitrile group (i.e. compounds 4j–l) showed
least activity, but compound 4e, having N-ethyl substitution
at indole ring and carboxylate group at 5th position of pyran
ring showed moderate activity. Incorporation of Cl, H and Br
(i.e. compound 4b, 4g and 4h) have not showed any effect
and shows least activity. It was observed that unsubstituted in
indole ring with carboxylate group (i.e. compound 4a) shown
moderate activity. Whereas, incorporation of CH-3 group at
5th position in indole ring with carbonitrile group (i.e. com-
pound 4i) also gave moderate activity, but the 5,7-diCH3
substitution with carboxylate group (i.e. compound 4c) dis-
played least activity (Table 3; Fig. 2). The results show that
compound 4f showed highest activity. In compound 4f, there
is presence of an electron donating groups (in form of benzyl
group substituted on nitrogen of indole ring and carboxylate
at 5th position of pyran ring) which stabilized the compound
after donating the hydrogen or lone pair to ABTS•? radical
cation.
123

Med Chem Res
Fig. 2 ABTS scavenging
activity of indole derivatives at
different concentrations
Fig. 3 NO scavenging activity
of indole derivatives at different
concentrations
Nitric oxide scavenging activity
Conclusion
In NO assay, among the tested compounds, compounds 4e
and 4f having N-substituted indole ring with carboxylate
group at 5th position of pyran ring, showed good activity.
Other N-substituted indole with carboxylate group or with
carbonitrile group (i.e. compounds 4d, 4j, 4k, 4l, respec-
tively) showed moderate activity. Free NH functionality of
indole ring with carboxylate group (i.e. compound 4a)
showed moderate activity, while with carbonitirle group
(i.e. compound 4g) showed least activity. The incorpora-
tion of CH-3 group at 5th position in indole ring with
carbonitrile group (i.e. compound 4i) gives moderate
activity, but the incorporation of 5,7-diCH3, Cl with car-
boxylate group (i.e. compounds 4b and 4c), and Br with
carbonitrile group (i.e. compound 4h) have not shown any
effect and gives least activity (Table 3; Fig. 3). The results
show that compound 4f possess highest activity. In com-
pound 4f, there is presence of an electron donating groups
(in form of benzyl group substituted on nitrogen of indole
ring and carboxylate at 5th position of pyran ring) which
stabilized the compound after donating the hydrogen or
lone pair to NO radical.
This work describes for the first time the in vitro antioxidant
activities of spirooxindole derivatives, as a new class of
potential antioxidants. The results showed that all the spiro-
oxindole derivatives had demonstrated strong activity in
DPPH• and NO scavenging method and moderate activity in
ABTS•? scavenging procedure. The compound 4f (Ethyl60-
amino-30-methyl-2-oxo-10-phenyl-10H-spiro[1-benzylindo-
line-3,40-pyrano[2,3-c]pyrazole]-50-carboxylate) has shown
strong activity in DPPH•, ABTS•? and NO scavenging
screening. Overall, compound 4a having electron donating
carboxylate group and compound 4f having both N-benzyl
and carboxylate group having electron donating effect were
found to bethe mostpotentantioxidant described inthisstudy.
Experimental
Chemistry
The melting points of all the compounds were determined
on a Toshniwal apparatus. The purity of compounds was
123

Med Chem Res
Ethyl 60-amino-5-chloro-30-methyl-2-oxo-10-phenyl-10H-
spiro[indoline-3,40-pyrano[2,3-c]pyrazole]-50-carboxylate
(4b)
checked on thin layers of silica gel G-coated glass plates
with n-hexane ethyl acetate (7:3) as eluent. Infrared (IR)
spectra were recorded on a Shimadzu Fourier transform
(FT)-IR 8400 S spectrophotometer using KBr pellets.
Sonication was carried out with the help of a standard
ultrasonic irradiation instrument SonaprosPR-1000 MP
(Oscar Ultrasonics Pvt. Ltd.) operating at 750 W and
generating 23 KHz output frequency. It has the following
characteristics: Standard Titanium horn with a diameter of
6 mm/12 mm, replaceable flat stain less steel tip and dig-
ital thermometer to determine temperature. The glass
reactor was designed and made from borosil glass.
White crystalline solid; YIELD: 92 %; m.p. 246–248 ꢁC;
IR (KBr): 3396, 3228, 3172, 1698, 1644, 1608, 1566,
1
1160 cm-1; H NMR (DMSO-d6, 300 MHz) : d 0.77 (t,
3H, CH3), 1.61 (s, 3H, CH3), 3.75 (q, J = 6.6 Hz, 2H,
CH2), 6.89 (d, J = 8.4 Hz, 1H), 7.08 (s, 1H), 7.23 (d,
J = 8.1 Hz, 1H), 7.33 (t, J = 7.2 Hz, 1H), 7.50 (t,
J = 7.8 Hz, 2H), 7.80 (d, J = 8.1 Hz, 2H), 8.25 (brs, 2H,
NH2, D2O exchangeable), 10.68 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6, 75 MHz): d 11.8,
13.2, 47.8, 59.2, 74.1, 97.6, 110.4, 120.2, 123.5, 125.9,
126.5, 127.7, 129.5, 137.3, 137.9, 141.1, 144.1, 161.5,
167.8, 179.2; MS (m/z) : 450 (M?); Anal. calcd. for
C23H19ClN4O4 : C, 61.27, H, 4.25, N, 12.43. Found : C,
61.30, H, 4.31, N, 12.42.
Synthesis of spiro[indoline3,40-pyrano[2,3-c]pyrazole
derivatives (4a–l)
An equimolar mixture of isatin (1 mmol, 0.147 g), eth-
ylcyanoacetate (1 mmol, 0.113 g), 3-methyl-1-phenyl-2-
pyrazolin-5-one (1 mmol, 0.174 g) and 10 mol% CAN in
5 ml water was introduced in a 20 mL, heavy-walled,
pear-shaped, two-necked flask with non standard taper
outer joint. The flask was attached to a 12 mm tip
diameter probe, and the reaction mixture was sonicated at
ambient temperature for the specified period at 50 %
power of the processor and 230 W output in a 4 s pulse
mode. At the end of the reaction period, thin-layer
chromatography (TLC) was checked, and the flask was
detached from the probe. The contents were transferred to
a beaker. The formed solid was filtered off, washed
thoroughly with warm water (2 9 20 ml), and then dried
to obtain crude products which were purified by crystal-
lization from mixture of methanol acetone (6:4) to give
compound 4, giving satisfactory spectral and elemental
analysis.
Ethyl 60-amino-30,5,7-trimethyl-2-oxo-10-phenyl-10H-
spiro[indoline-3,40-pyrano[2,3-c]pyrazole]-50-carboxylate
(4c)
White crystalline solid; YIELD: 91 %; m.p. 260–262 ꢁC;
IR (KBr): 3398, 3228, 3170, 1702, 1648, 1611, 1570,
1
1168 cm-1; H NMR (DMSO-d6, 300 MHz) : d 0.75 (t,
3H, CH3), 1.58 (s, 3H, CH3), 2.12 (s, 3H, CH3), 2.19 (s,
3H, CH3), 3.73 (q, J = 6.90 Hz, 2H, CH2), 6.57 (s, 1H),
6.78 (s, 1H), 7.32 (t, J = 7.2 Hz, 1H), 7.50 (t, J = 7.8 Hz,
2H), 7.80 (d, J = 7.8 Hz, 2H), 8.17 (brs, 2H, NH2, D2O
exchangeable), 10.47 (s, 1H, NH, D2O exchangeable); 13C
NMR (DMSO-d6, 75 MHz): d 11.8, 12.9, 16.3, 18.6, 20.6,
47.8, 56.2, 59.2, 75.0, 98.6, 117.9, 120.0, 121.2, 126.4,
129.4, 129.5, 130.6, 135.5, 137.4, 138.3, 144.0, 144.4,
161.3, 168.1, 179.9; MS (m/z) : 444 (M?); Anal. calcd. for
C25H24N4O4 : C, 67.55, H, 5.44, N, 12.60. Found: C, 67.49,
H, 5.39, N, 12.63.
Ethyl 60-amino-30-methyl-2-oxo-10-phenyl-10H-
spiro[indoline-3,40-pyrano[2,3-c]pyrazole]-50-carboxylate
(4a)
Ethyl 60-amino-30-methyl-2-oxo-10-phenyl-10H-spiro[1-
allylindoline-3,40-pyrano[2,3-c]pyrazole]-50-carboxylate
(4d)
White crystalline solid; YIELD: 94 %; m.p. 238–240 ꢁC;
IR (KBr) : 3392, 3230, 3172, 1716, 1652, 1600, 1554,
1160 cm-1; 1H NMR (DMSO-d6, 300 MHz): d 0.71 (t, 3H,
CH3), 1.55 (s, 3H, CH3), 3.72 (q, J = 6.80 Hz, 2H, CH2),
6.84–6.93 (m, 3H), 7.18 (t, J = 5.40 Hz, 1H), 7.32 (t,
J = 07.5 Hz, 1H), 7.49(t, J = 8.4 Hz, 2H), 7.75 (d,
J = 7.5 Hz, 2H), 8.14 (brs, 2H, NH2, D2O exchangeable),
10.53(s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-
d6, 75 MHz): d 12.0, 13.4, 48.0, 59.6, 75.1, 98.7, 109.4,
120.5, 122.3, 123.5, 126.9, 128.3, 129.9, 136.1, 137.9,
142.4, 144.7, 161.7, 168.3, 179.9; MS (m/z): 416 (M?).
Anal. calcd. for C23H20N4O4: C, 66.34, H, 4.84, N, 13.45.
Found: C, 66.30, H, 4.81, N, 13.38.
White crystalline solid; YIELD: 84 %; m.p. 182–184 ꢁC;
IR (KBr): 3398, 3228, 3170, 1702, 1648, 1611, 1570,
1
1168 cm-1; H NMR (DMSO-d6, 300 MHz) : d 0.67 (t,
3H, CH3), 1.50 (s, 3H, CH3), 3.73 (q, J = 6.90 Hz, 2H,
CH2), 4.29 (dd, 1H, CH2), 4.58 (dd, 1H, CH2), 5.25 (d, 1H,
CH2), 5.43 (d, 1H, CH2), 5.84 (m, 1H, CH), 6.95–7.83 (m,
9H, ArH), 8.24 (brs, 2H, NH2, D2O exchangeable). 13C
NMR (DMSO-d6, 75 MHz): d 11.7, 13.4, 47.0, 58.9, 74.6,
97.6, 118.7, 119.8, 119.8, 122.2, 126.0, 127.4, 129.0,
131.4, 134.7, 137.4, 142.3, 143.9, 144.0, 167.6, 177.1; MS
123

Med Chem Res
60-Amino-5-bromo-30-methyl-2-oxo-10-phenyl-10H-
spiro[indoline-3,40-pyrano[2,3-c]pyrazole]-50-carbonitrile
(m/z) : 457 (M?). Anal. calcd. for C26H24N4O4 : C, 68.40,
H, 5.32, N, 12.10. Found: C, 68.49, H, 5.39, N, 12.13.
(4h)
Ethyl 60-amino-30-methyl-2-oxo-10-phenyl-10H-spiro[1-
ethylindoline-3,40-pyrano[2, 3-c]pyrazole]-50-carboxylate
(4e)
White crystalline solid; YIELD: 92 %; m.p. 242–244 ꢁC;
IR (KBr): 3436, 3269, 3168, 2198, 1706, 1650, 1576,
1168 cm-1; H NMR (DMSO-d6, 300 MHz): d 1.58 (s,
1
3H, CH3), 6.91 (d, J = 7.6 Hz, 1H), 7.42–7.54(4H, m),
7.28 (t, J = 7.9 Hz, 1H), 7.64 (brs, 2H, NH2, D2O
exchangeable), 7.79 (d, J = 7.8 Hz, 2H), 10.90 (s, 1H, NH,
D2O exchangeable); 13C NMR (DMSO-d6, 75 MHz): d
12.1, 47.9, 56.4, 96.7, 110.6, 118.2, 120.9, 125.8, 126.6,
129.5, 129.6, 131.7, 132.3, 137.3, 139.2, 144.1, 145.0,
161.0, 178.5; MS (m/z): 447(M?); Anal. calcd. for
C21H14BrN5O2: C, 56.27, H, 3.15, N, 15.62. Found: C,
56.17, H, 3.21, N, 15.60.
White crystalline solid; YIELD: 92 %; m.p. 208–210 ꢁC;
IR (KBr): 3398, 3228, 3170, 1702, 1648, 1611, 1570,
1
1168 cm-1; H NMR (DMSO-d6, 300 MHz) : d 0.63 (t,
3H, CH3), 1.33 (t, 3H, J = 6.8 Hz), 1.50 (s, 3H, CH3), 3.73
(q, 2H, J = 6.8 Hz), 6.39–7.24 (m, 9H, ArH), 8.29 (brs,
2H, NH2, D2O exchangeable); 13C NMR (DMSO-d6,
75 MHz): d 10.2, 11.7, 47.5, 58.7, 72.6, 95.6, 114.2, 117.9,
118.2, 120.0, 121.2, 124.4, 127.4, 129.5, 130.6, 134.3,
137.4, 142.5, 143.3, 144.0, 166.1, 176.9; MS (m/z) : 445
(M?); Anal. calcd. For C25H24N4O4 : C, 67.50, H, 5.41, N,
12.57. Found: C, 67.55, H, 5.44, N, 12.60.
60-Amino-30,5-dimethyl-2-oxo-10-phenyl-10H-
spiro[indoline-3,40-pyrano[2,3-c]pyrazole]-50-carbonitrile
Ethyl 60-amino-30-methyl-2-oxo-10-phenyl-10H-spiro[1-
benzylindoline-3,40-pyrano[2, 3-c]pyrazole]-50-
carboxylate (4f)
(4i)
White crystalline solid; YIELD: 98 %; m.p. 288–290 ꢁC;
IR (KBr): 3426, 3269, 3178, 2192, 1696, 1650, 1576,
White crystalline solid; YIELD: 97 %; m.p. 244–246 ꢁC;
IR (KBr): 3398, 3228, 3170, 1702, 1648, 1611, 1570,
1
1174 cm-1; H NMR (DMSO-d6, 300 MHz): d 1.56 (s,
1
1168 cm-1. H NMR (DMSO-d6, 300 MHz) : d 0.63 (t,
3H, CH3), 2.26 (s, 3H, CH3),6.84 (d, J = 7.8 Hz, 1H), 7.08
(s, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.35 (t, J = 8 Hz, 1H),
7.52 (t, J = 8.4 Hz, 2H), 7.55 (brs, 2H, NH2, D2O
exchangeable), 7.78 (d, J = 8.3 Hz, 2H), 10.64 (s, 1H, NH,
D2O exchangeable); 13C NMR (DMSO-d6,75 MHz): 11.9,
20.4, 48.2, 56.7, 96.6, 110.1, 118.4, 119.9, 125.3, 126.8,
129.9, 130.5, 131.9, 132.6, 137.4, 139.8, 144.3, 145.7,
161.2, 177; MS(m/z):383(M?); Anal. calcd. for
C22H17N5O2: C, 68.92, H, 4.47, N, 18.27. Found: C, 68.82,
H, 4.51, N, 18.11.
3H, CH3), 1.50 (s, 3H, CH3), 3.73 (q, J = 6.90 Hz, 2H,
CH2), 4.91 and 5.13 (2H, AB system J = 15.5 Hz),
6.57–7.89 (m, 14H, ArH), 8.20 (brs, 2H, NH2, D2O
exchangeable); 13C NMR (DMSO-d6, 75 MHz): d 11.5,
13.6, 46.9, 58.9, 73.6, 96.6, 117.9, 118.2, 120.0, 122.2,
126.4, 127.4, 129.5, 131.6, 134.3, 137.4, 142.3, 143.8,
144.0, 161.6, 165.5, 176.9; MS (m/z) : 507 (M?); Anal.
calcd. for C30H26N4O4 : C, 71.04, H, 5.13, N, 11.01.
Found: C, 71.13, H, 5.17, N, 11.06.
60-amino-30-methyl-2-oxo-10-phenyl-10H-spiro[indoline-
3,40-pyrano[2,3-c]pyrazole]-50-carboxylate (4g)
60-amino-30-methyl-2-oxo-10-phenyl-10H-spiro[1-
allylindoline-3,40-pyrano[2,3-c]pyrazole]-50-carbonitrile
(4j)
White crystalline solid; YIELD: 95 %; m.p. 237–238 ꢁC;
IR (KBr): 3412, 3280, 3174, 2200, 1692, 1650, 1526,
1
1132 cm-1; H NMR (DMSO-d6, 300 MHz) : d 1.55 (s,
White crystalline solid; YIELD: 90 %; m.p. 218–220 ꢁC; IR
(KBr): 3408, 1708, 1660, 1605, 1540, 1166 cm-1 1H
;
3H, CH3), 6.94 (d, J = 7.4 Hz, 1H), 7.03 (t, J = 7.6 Hz,
1H), 7.18 (d, J = 7.2 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H),
7.36 (t, J = 7.6 Hz, 1H), 7.52 (t, J = 7.8 Hz, 2H), 7.58
(brs, 2H, NH2, D2O exchangeable), 7.79 (d, J = 7.9 Hz,
2H), 10.76 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6, 75 MHz): d 12.3, 48.3, 56.6, 96.9, 110.4,
118.6, 120.7, 123.2, 125.4, 127.1, 129.8, 130.8, 132.6,
138.2, 142.1, 144.5, 145.5, 162.3, 178.8; MS (m/z) : 369
(M?); Anal. calcd. for C21H15N5O2 : C, 68.28, H, 4.09, N,
18.96. Found: C, 68.26, H, 4.04, N, 18.88.
NMR (DMSO-d6, 300 MHz): 1.42 (s, 3H, CH3), 4.29 (dd,
1H, CH2), 4.58 (dd, 1H, CH2), 5.25 (d, 1H, CH), 5.43
(d, 1H, CH), 5.84 (m, 1H, CH), 6.94–7.50 (m, ArH, 14H),
8.28 (brs, 2H, NH2, D2O exchangeable); 13C NMR (DMSO-
d6, 75 MHz): 11.7, 43.4, 55.9, 109.2, 117.8, 120.0, 123.2,
124.5, 126.2, 127.3, 128.3, 129.0, 131.2, 135.5, 137.2, 141.9,
143.8, 144.9, 161.1, 176.0; MS (m/z): 410 (M?); Anal. calcd.
for C24H19N5O2 : C, 70.35, H, 4.59, N, 17.15. Found: C,
70.40, H, 4.68, N, 17.10.
123

Med Chem Res
60-amino-30-methyl-2-oxo-10-phenyl-10H-spiro[1-
ethylindoline-3,40-pyrano[2,3-c]pyrazole]-50-carbonitrile
(4k)
ABTS•? scavenging activity
ABTS•? scavenging activity of synthesized compounds
were measured by the method, appeared in the literature
(Re et al., 1999). First, ABTS•? free radicals were gener-
ated through the oxidation of ABTS with potassium per-
sulphate. For this purpose, ABTS was dissolved in
deionized water to 7 mM concentration, and potassium
persulphate was added to a concentration of 2.45 mM. The
reaction mixture was kept in dark at room temperature for
12–16 h before final use. Lastly, the ABTS•? solution was
diluted with absolute ethanol till the absorbance was read
0.700 0.020 at 734 nm. Synthesized compounds in eth-
anol were added to 3 ml of ABTS•? solution and the
absorbance was read after 6 min.
White crystalline solid; YIELD: 84 %; m.p. 210–212 ꢁC;
1
IR (KBr): 3406, 1705, 1656, 1608, 1542, 1162 cm-1; H
NMR (DMSO-d6, 300 MHz): d 1.33 (t, 3H, J = 6.8 Hz),
1.42 (s, 3H, CH3), 3.85 (q, 2H, J = 6.8 Hz), 6.92–7.52 (m,
ArH, 14H), 8.23 (brs, 2H, NH2, D2O exchangeable); 13C
NMR (DMSO-d6, 75 MHz): 11.7, 43.4, 55.9, 109.2, 117.8,
120.0, 123.2, 124.5, 126.2, 127.3, 128.3, 129.0, 131.2,
135.5, 137.2, 141.9, 143.8, 144.9, 161.1, 176.0; MS (m/z):
398 (M?); Anal. calcd. for C23H19N5O2 : C, 73.29, H,
4.67, N, 15.27. Found: C, 73.19, H, 4.61, N, 15.24.
60-amino-30-methyl-2-oxo-10-phenyl-10H-spiro[1-
benzylindoline-3,40-pyrano[2,3-c]pyrazole]-50-carbonitrile
(4l)
Nitric oxide scavenging activity
The interaction of synthesized compounds with nitric oxide
(NO) was assessed by the nitrate ion detection method
(Sreejayan, 1997). Sodium nitropruside (5 mM) in phos-
phate buffer spontaneously generates NO in an aqueous
solution. NO interacts with oxygen and produces nitrate
ions, which can be estimated by the use of Greiss reagent
(1 % sulphanilamide, 2 % H3PO4 and 0.1 % napthyleth-
ylene diamine dihydrochloride). Sodium nitroprusside
(5 mM) in phosphate buffer was mixed with synthesized
compounds and incubated at 25 ꢁC for 150 min. Prepared
samples were allowed to react with Greiss reagent. The
absorbance of chromophore formed during the diazotiza-
tion of nitrite with sulphanilamide and subsequent coupling
with napthylethylene diamine was read at 546 nm. The
same reaction mixture without the synthesized compound,
but with equal amount of distilled water served as control.
White crystalline solid; YIELD: 97 %; m.p. 232–234 ꢁC;
1
IR (KBr): 3406, 1705, 1656, 1608, 1542, 1162 cm-1; H
NMR (DMSO-d6, 300 MHz): d 1.42 (s, 3H, CH3), 4.89 (d,
1H, J = 15.6 Hz, benzylic proton), 5.09 (d, 1H,
J = 15.6 Hz, benzylic proton), 6.97–7.42 (m, ArH, 14H),
8.13 (brs, 2H, NH2, D2O exchangeable); 13C NMR
(DMSO-d6, 75 MHz): 11.7, 43.4, 55.9, 109.2, 117.8, 120.0,
123.2, 124.5, 126.2, 127.3, 128.3, 129.0, 131.2, 135.5,
137.2, 141.9, 143.8, 144.9, 161.1, 176.0; MS (m/z): 460
(M?); Anal. calcd. for C28H21N5O2 : C, 68.92, H, 4.47, N,
18.27. Found: C, 68.82, H, 4.51, N, 18.11.
Antioxidant activity
Antioxidant activities of test compounds were measured by
estimating DPPH•, ABTS•? and NO scavenging activity
in vitro using ascorbic acid as standard drug.
Acknowledgments Regional Sophisticated Instrumentation Centre,
Chandigarh and Central Drug Research Institute, Lucknow for the
elemental and spectral analyses. Department of Zoology, University
of Rajasthan for their help in biological studies.
DPPH• scavenging activity
Ability of synthesized compounds to scavenge the stable free
radical, DPPH• is measured by the method, appeared in the
literature (Mensor et al., 2001). To 1 ml methanolic solution of
DPPH• (0.25 mM), 1 ml of ethanolic solution of synthesized
compounds was added. To prepare control, 1 ml of methanol
was added to the 1 ml methanolic solution of DPPH•
(0.25 mM). After 20 min, absorbance was recorded at 517 nm
in a UV–Vis doublebeam spectrophotometer. The inhibition (%)
of free radicals was calculated by using the following formula:
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