Microwave synthesis, characterization and bio-efficacy evaluation of novel chalcone based 6-carbethoxy-2-cyclohexen-1-one and 2H-indazol-3-ol derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.10.038

Novel chalcone based 6-carbethoxy-2-cyclohexen-1-one and 2H-indazol-3-ol derivatives were synthesized and characterized by using spectral techniques like IR, ¹H NMR, ¹³C NMR, COSY, DEPT, and GC-MS. All these compounds were screened for anti-fungal, anti-bacterial and anti-oxidant activity. Cyclohexenone derivatives, in general, showed better anti-fungal and anti-bacterial activity than parent chalcones. Whereas, all the Indazole derivatives showed very good anti-oxidant activity and some were also found to be active as anti-bacterial agent. Among the screened compounds, 15 was found to be most active as anti-fungal agent (against Rhizoctonia solani, LC ₅₀ = 2.36 μg mL^-1), 15b was found to be most active anti-bacterial agent (against Klebsiella pneumonia, MIC = 24.68 μg mL ^-1) and 14b emerged as most active anti-oxidant (IC₅₀ = 19.81 μg mL^-1).

Full text of DOI:10.1016/j.ejmech.2012.10.038

European Journal of Medicinal Chemistry 59 (2013) 120e131
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Microwave synthesis, characterization and bio-efficacy evaluation of novel
chalcone based 6-carbethoxy-2-cyclohexen-1-one and 2H-indazol-3-ol
derivatives
,
a,
N.A. Shakil a , Manish K. Singh b, M. Sathiyendiran b, J. Kumar a, Jasdeep C. Padaria c
*
a Division of Agricultural Chemicals, Indian Agricultural Research Institute, Room No. 256, LBS Building, New Delhi 110012, India
b Department of Chemistry, University of Delhi, Delhi 110007, India
c National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel chalcone based 6-carbethoxy-2-cyclohexen-1-one and 2H-indazol-3-ol derivatives were synthe-
sized and characterized by using spectral techniques like IR, 1H NMR, 13C NMR, COSY, DEPT, and GC-MS.
All these compounds were screened for anti-fungal, anti-bacterial and anti-oxidant activity. Cyclo-
hexenone derivatives, in general, showed better anti-fungal and anti-bacterial activity than parent
chalcones. Whereas, all the Indazole derivatives showed very good anti-oxidant activity and some were
also found to be active as anti-bacterial agent. Among the screened compounds, 15 was found to be most
Received 4 April 2012
Received in revised form
17 October 2012
Accepted 20 October 2012
Available online 20 November 2012
active as anti-fungal agent (against Rhizoctonia solani, LC50 ¼ 2.36
active anti-bacterial agent (against Klebsiella pneumonia, MIC ¼ 24.68
active anti-oxidant (IC50 ¼ 19.81
g mLꢀ1).
m
g mLꢀ1), 15b was found to be most
g mLꢀ1) and 14b emerged as most
Keywords:
Chalcone
Cyclohexenone
Indazole
m
m
Ó 2012 Elsevier Masson SAS. All rights reserved.
Anti-fungal
Anti-bacterial
Anti-oxidant
1. Introduction
Chalcone is a generic term for the compounds bearing the 1, 3-
diphenyl-prop-2-en-1-one framework [7]. Under homogeneous
conditions, these compounds are usually prepared by base or acid
catalyzed aldol condensation between aromatic aldehydes and
ketones. Chalcones represent an important class of compounds due
to their chemical flexibility, as synthons for the production of five-
and six-member ring systems [8,9] for example Pyrazoles [10],
Pyrazolines [11], isoxazolines [12], aurones [13], pyrimidine [14],
falvanones [15] and di-aryl cyclohexenones [16]. The biological
activities of chalcones are equally wide ranging [17e20]. In fact, not
many structural templates can claim association with such
a diverse range of pharmacological activities, among which anti-
microbial [21], anti-leishmanial [22], anti-malarial [23], anti-
fungal [24], anti-viral [25], anti-inflammatory [26], cytotoxicity
[27], anti-tumor [28], nematicidal [29] and anti-oxidant [30] are
widely cited.
Due to stringent and growing environmental regulations, the
chemical industry needs the development of more eco-compatible
synthetic methodologies and consequently
a
detailed re-
examination of the important synthetic processes [1]. In recent
decades, microwave heating has taken an incontestable place in
analytical and organic laboratories practice as a very effective and
non-polluting method of activation.
Microwave irradiation leads to large reduction in reaction time,
enhancement in conversion, and sometimes [2,3] in selectivity
with several advantages of the environmental approach, termed
green chemistry. The solvent-free Microwave assisted reactions [4]
have gained popularity as they provide potentialities to work with
open vessels and enhanced possibility of up-scaling the reactions
on preparative scale [4e6].
From a chemical point of view, an important feature of chal-
cones and their hetero-analogs is the ability to act as activated
unsaturated systems in conjugated addition reactions of carbanions
in the presence of basic catalysts [31,32]. This type of reaction
is more commonly used for the preparation of 3,5-diaryl-6-
carbethoxycyclohexenones via Michael addition of ethyl
* Corresponding author. Tel.: þ91 11 25843588; fax: þ91 11 25843272.
E-mail addresses: iamshakil@gmail.com (N.A. Shakil), mks.rahul@gmail.com
(M.K. Singh), msakhi@chemistry.du.ac.in (M. Sathiyendiran), jitendrakumar7@
yahoo.com (J. Kumar), jasdeep_kaur64@yahoo.co.in (J.C. Padaria).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.038

N.A. Shakil et al. / European Journal of Medicinal Chemistry 59 (2013) 120e131
121
acetoacetate [33e35]. The mentioned cyclohexenones are efficient
synthons in building spiranic compounds [36] or intermediates in
the synthesis of fused heterocycles such as benzoselenadiazoles
and benzothiadiazoles [37], benzopyrazoles and benzisoxazoles
[38,39], carbazole derivatives [40], and 2H-indazoles [41].
The anti-bacterial, anti-fungal, anti-cancer and anti-tubercular
activities of di-arylcyclohexenone derivatives were also reported
[42,43].
Wide spectrum of biological activities has been reported for
indazoles and they have increasingly attracted the attention in
pharmaceutical field due to various interesting bioactivity against
different targets [44,45].
In the quest for biologically more potent anti-microbial and
anti-oxidant agents and to increase the molecular diversity, we
envisioned to design and synthesize the chalcone based 6-
Carbethoxy-2-cyclohexen-1-one and 2H-Indazol-3-ol Derivatives
through a simple, efficient and environment friendly method
which utilizes the approach of “Green chemistry”.
and 120 respectively. Aromatic carbons appear at around
140. The IR spectrum supported the NMR data showing the char-
d
120e
acteristic band for C]O at 1655e1660 and C]C Ar at around 1600 and
1475 cmꢀ1
.
The cyclo-condensation of ethyl acetoacetate with chalcones
leads to the generation of two chiral centers at C-5 and C-6 in the
structure of cyclohexenones. As the explored reaction is not stereo
selective, both configurations of the chiral carbon atoms are ex-
pected to be noticed in the synthesized cyclohexenones, which
would result in a mixture of diastereomers. No attempt to separate
the diastereomeric cyclohexenones was undertaken, and the cyclo-
condensation products have been characterized in the form of the
mixture originated from the synthesis. The IR spectra of these
compounds revealed
a sharp strong absorption band above
1700 cmꢀ1 that can be correlated with the presence of the ester
function in the structure of cyclohexenones. Furthermore, another
sharp strong absorption band was noticed at approximately
1660 cmꢀ1 and was assigned to the carbonyl group conjugated with
a carbonecarbon double bond. No other absorption band could be
evidenced in the region of the IR spectrum associated with the
stretching vibrations of the carbonyl group, thus excluding the
intermediate Michael adduct having an extra carbonyl group.
The 1H NMR spectra substantiated the results of the IR analysis. The
characteristic signals of an ethyl ester moiety (a triplet at chemical
2. Results and discussion
2.1. Chemistry
Chalcones (1e16) were prepared from substituted acetophe-
nones and benzaldehydes using aq. NaOH under microwave irradi-
ation. The chalcones were then condensed with ethyl acetoacetate
using K2CO3, under microwave irradiation to give cyclohexenones
(1ae16a). These cyclohexenones were then condensed with
hydrazine hydrateinpresence of glacial aceticacid under microwave
irradiation to afford 2H-indazol-3-ols (1be16b) (Scheme 1). All the
cyclohexenone and indazole derivatives were characterized by
elemental analysis, IR, 1H NMR, 13C NMR, COSY, DEPT, and GC-MS.
Claisen-Schmidt condensation method for the synthesis of
chalcones is very attractive since it specifically generates the trans
(E)-isomer [8,46,47]. Signals for two vinylic protons in the structure
of chalcones appear as two doublets in aromatic region of 1H NMR
spectra, and their coupling constant (J) showed that all chalcones
were geometrically pure and with trans-configuration (JH
shift values of about d 1 and a quartet at around d 4) confirmed the
presence of the ester group in the structure of cyclohexenones. The
characteristic signal in the 1H NMR spectrum was however the
singlet of the vinylic proton of the cyclohexenone ring, that
appeared at approximately
d 6.5 and confirming that the intra-
molecular cyclo-condensation, subsequent to the Michael addition,
actually took place. The two protons at C-4, being non-equivalent,
appeared as two different signals, the axial proton appeared as
double of double-doublet at around
coupling, vicinal coupling and long range 1He1H coupling and the
equatorial proton appeared as double-doublet at around 3.05,
showing germinal and vicinal couplings. The signals for the protons
at C-5 and C-6 appeared as a multiplet at around 3.7. Vinylic
proton at C-2 appeared as doublet at around 6.5 and aromatic
d 2.95 showing both germinal
d
d
d
a
e
protons appeared between
d 6.7 and 7.8. COSY spectrum of cyclo-
¼ 15.50e16.50 Hz). Aromatic protons appear between
d
6.9
H
b
hexenones confirmed the long range 1He1H coupling between
and 8.1. In 13C NMR, a signal at around
d 190 confirms the presence
axial proton at C-4 and vinylic proton at C-2. 13C NMR also
of carbonyl group. Signal for
a
and
b
carbons appear at around
d
145
+
aq. NaOH
Ar`
Ar
Ar
Ar`
α
O
MWI
β
1-16
O
O
O
K2CO3
MWI
O
O
3`
3``
3``
3`
4`
5`
4``
5``
2`
1`
2`
1`
4`
2``
4``
5``
2``
Ar`
Ar
Ar`
Ar
4
5
3
5
4
5`
6
7
1``
2HN-NH2.H2O
1``
6``
6`
6`
6``
glacial acetic acid
MWI
3a
O
OH
1```
2
6
3```
1
7a
3
2```
1b-16b
O
O
N
NH
1a-16a
1
2
Scheme 1. General method for the synthesis of cyclohexenone and indazole derivatives.

122
N.A. Shakil et al. / European Journal of Medicinal Chemistry 59 (2013) 120e131
supported the above data showing two signals at around
195 due to carbonyl carbons and a signal at around
quaternary vinylic carbon (C-3).
Treatment of cyclohexenones 1ae16a with hydrazine hydrate in
presence of ethanolic acetic acid gave the corresponding indazole
derivatives 1be16b which can be tautomeric mixtures [48] (Fig. 1).
The absence of carbonyl bands in the IR spectra of the products
ruled out lactam structures A and B. The 1H NMR spectra exhibited
three protons in the sp3 shift range (H-5eq, H-5ax and H-4). H-5eq
d
170 and
158 for
R. solani. The compounds i.e., 1-(4-Fluorophenyl)-3-phenyl-prope-
none (15) and 1,3-Diphenyl-propenone (16) were highly effective
as fungicide against R. solani with LC50 values 2.36 and 2.49 mg Lꢀ1
respectively, comparable with that of commercial fungicide Hex-
aconazole (LC50 1.27 mg Lꢀ1 and 1.12 mg Lꢀ1 against S. rolfsii and
R. solani respectively).
d
2.2.2. Anti-bacterial activity
All the synthesized compounds (100 mg/disk) were screened for
and H-4 appeared as double-doublets at around
respectively while, the signal for H-5ax appeared as double of
double-doublet at around 3.17 showing geminal coupling with
H-5eq, vicinal coupling with H-4 and long range proton coupling
with H-7. Protons that exchanged with D2O at around 9.7 were
assigned to OH and NH. Aromatic protons appeared between
d
2.90 and 4.18
their anti-bacterial activity using disk diffusion method, against
two gram positive beneficial bacteria, Bacillus thuringiensis and
Bacillus pumilis, two gram negative plant pathogens, Erwinia
chrysanthemi and Xanthomonas oryzae and three human pathogens,
Klebsiella pneumonia (gram negative), Staphylococcus aureus (gram
positive) and Pseudomonas aeruginosa (gram negative). Dimethyl
sulfoxide was used as control and Streptomycin, Ampicillin and
d
d
d
7
and 8 and a doublet at around d 6.7 was due to vinylic proton H-7. A
cross peak in the COSY spectrum confirmed the long range 1H-1H
Kanamycin (25 mg/disk) were used as positive control.
coupling between H-5ax and H-7. On the other hand, the 13C NMR
All the synthesized compounds were found to be inactive
against beneficial bacteria, B. thuringiensis and B. pumilis, giving an
idea that they were target specific and not toxic to beneficial
microbes. Also, all the compounds were found to be inactive against
X. oryzae (Table 3).
spectra of these compounds showed signal at around
C-3. Signals for other quaternary carbons and aromatic carbons
appeared at around 125e145. The signal for vinylic carbon C-7
appeared at around 98e
113. In 13C NMR of indazoles, a signal at
99 can be attributed to a sp2-hybridized carbon atom (C-3a) that
shifted to high field. DEPT experiments also support the 1H and 13
d 156 due to
d
d
d
Against E. chrysanthemi, compounds 3a and 11a showed good
activity (zone of inhibition ¼ 15e20 mm) where as compounds 14,
16, 9a, 13a, 14a and 15a showed moderate activity (zone of
inhibition ¼ 10e15 mm). Against Klebsiella pneumonia, compound
15b showed good anti-bacterial activity (zone of inhibition ¼ 15e
20 mm) and compounds 6a, 15a and 16a showed moderate activity
(zone of inhibition¼ 10e15 mm). Allthe synthesized chalcones were
found to be inactive against K. pneumonia. Against S. aureus,
compounds 16, 2b and 16b showed poor to moderate activity (zone
of inhibition ¼ 5e10 mm) and all the other synthesized compounds
were found to be inactive. Against P. aeruginosa, compounds 16, 6a,
2b and 6b showed moderate to good activity (zone of
inhibition ¼ 10e15 mm) where as compounds 1a and 1b showed
poor to moderate activity (zone of inhibition ¼ 5e10 mm). Strepto-
mycin was found to be most active antibiotic against all the test
bacteria (Table 3).
C
NMR data. The tautomer C was therefore assigned as the sole
component of indazole derivative formed.
2.1.1. Microwave method Vs conventional method
Chalcones (1e16), cyclohexenones (1ae16a) and indazoles (1be
16b) were synthesized using self designed microwave method and
already reported non-microwave method [43]. Table 1 summarizes
the comparison of two methods in terms of reaction time and yield
and emphasizes the advantages of microwave reaction in addition
to solvent less conditions, increased conversion etc.
2.2. Bio-efficacy evaluation
2.2.1. Fungicidal activity
Compounds 3a and 11a showed good activity against
E. chrysanthemi and 15b showed good activity against K. pneumonia.
These compounds were subjected to the anti-bacterial screening
using broth dilution technique. Streptomycin drug was used as the
standard. The minimum inhibitory concentration (MIC) was noted
byobserving thelowestconcentrationof thedrugat which therewas
more than 90% inhibition of bacteria. The results are listed in Table 4.
The investigation of anti-bacterial screening using broth dilution
All the synthesized compounds were tested for their fungicidal
activity against two phyto-pathogenic fungi Rhizoctonia solani and
Sclerotium rolfsii. Both are known pests on number of plants. The
inhibitory effects of synthesized compounds on the growth of test
fungi were expressed in terms of 50% lethal concentration (LC50).
None of the indazole derivative was found to be active against both
R. solani and S. rolfsii. LC50 values for indazoles ranged from 713e
1241 mg Lꢀ1 for S. rolfsii and 528e1034 mg Lꢀ1 for R. solani
(Table 2). Results for anti-fungal activity were summarized in
Table 2 which indicate that the synthesized compounds were
comparatively more active against R. solani than S. rolfsii. Also,
against S. rolfsii the cyclohexenones were more active than the
parent chalcones except for compounds 9 and 12. However, no such
generalization was possible when compounds were tested against
technique revealed that all the three compounds 3a (55.9
against E. chrysanthemi), 11a (73.6 g/mL against E. chrysanthemi)
and 15b (24.68 g/mL against K. pneumonia) showed moderate to
good bacterial inhibition as compared to the standard drug strep-
tomycin (4.6 g/mL against E. chrysanthemi and 3.1 g/mL against
K. pneumonia).
mg/mL
m
m
m
m
Fig. 1. Tautomeric forms of 2H-indazol-3-ol.

N.A. Shakil et al. / European Journal of Medicinal Chemistry 59 (2013) 120e131
123
Table 1
Comparison of microwave and conventional (non-microwave) methods used for the synthesis of chalcone, cyclohexenone and indazole derivatives.
Compound
Parameter
Reaction time
Yield (%)
Conventional method [43]
Microwave method
Conventional method
Microwave method
Increase
Chalcones
Cyclohexenones
Indazoles
24 h
5 h
6 h
5e7 min
6e8 min
3 min
55e89
60e91
54e82
71e96
62e95
69e93
6e14
3e7
9e16
2.2.3. Anti-oxidant activity
Table 7. Among all the indazoles tested, 6-(4-fluorophenyl)-4,5-
dihydro-4-(3-nitrophenyl)-2H-indazol-3-ol (14b) was highly
A simple method was used to determine the anti-oxidant
activity of synthesized compounds. It utilizes the stable 2,2-
diphenyl-1-picrylhydrazyl (DPPH) radical. The odd electron in the
DPPH free radical gave a strong absorption maximum at 517 nm
and was purple in color. The color turned from purple to yellow as
the molar absorptivity of the DPPH radical at 517 nm reduces when
the odd electron of DPPH radical becomes paired with hydrogen
from a free radical scavenging anti-oxidant to form the reduced
DPPH-H. The resulting decolorization is stoichiometric with respect
to number of electrons captured.
effective as anti-oxidant with IC50 value 19.81
m
g mLꢀ1 compared to
IC50 value 1.28
m
g mLꢀ1 for gallic acid.
3. Experimental
3.1. Chemicals and instruments
All the chemicals used were purchased from SigmaeAldrich
and were used without further purification. Analytical grade
reagents and solvents were locally procured and used as such.
Reactions were monitored by thin layer chromatography (TLC) on
All the synthesized compounds were first screened for their free
radical scavenging activity. Out of all the compounds tested, only
2H-indazol-3-ol series was found to be significantly active as anti-
oxidants (Table 5). Anti-oxidant activity of indazoles was then
tested at different concentration viz., 10, 20, 40, 60, 80, 100, 200,
pre-coated Merck silica gel 60F254, 200 mm thick aluminum sheets
and the spots were visualized either under UV or by iodine vapor.
Column chromatography (CC) was carried out on silica gel (Merck,
100e200 mesh). House-hold microwave, LG-MG607APR was used
at full power (900 W) for carrying out chemical reactions. Melting
points were determined on a JSW melting point apparatus and
were reported uncorrected. The 1H NMR, COSY, 13C NMR and DEPT
spectra were recorded on Bruker 400 MHz Spectrospin spec-
trometer, using tetramethylsilane (TMS) as an internal standard.
300, 400 and 500 m
g mLꢀ1 (Table 6, Fig. 2). The free radical scav-
enging power of indazoles can be attributed to the presence of eOH
and eNH groups in their structure.
It was very clear from Table 6 that with the increase in concen-
tration, the free radical scavenging activity of compounds also
increased. Also, once around ninety percent of inhibition was ach-
ieved, the increase in concentration ofcompounds hadno significant
effect on the free radical scavenging activity and it reached a plateau
(Table 6). This might be because the concentration of anti-oxidant in
the solution reaches a level where steric hindrance (intermolecular
interactions) comes into play and anti-oxidant compound was not
able to donate its electron to DPPH radical.
The chemical shift values were recorded on
d scale and the
coupling constants (J) are in Hz. Infrared (IR) spectra were recor-
ded on Bruker alpha FT-IR spectrophotometer using Attenuated
Total Reflectance (ATR) technique and values are expressed as nmax
cmꢀ1. Mass spectra were recorded on a Thermo Fisher GC-MS,
equipped with Focus GC, DSQ II mass spectrometer and
Triplus auto sampler. Thermo TR-5 MS capillary column
Free radical scavenging activity of synthesized indazoles in
terms of 50% inhibitory concentration (IC50) was summarized in
(30 m ꢁ 0.25 mm ꢁ 0.25
mm) was used in Focus GC. Elemental
analyses for all compounds were performed on a Carlo Erba Model
EA-1108 elemental analyzer. Anti-oxidant activity was done using
UVeVisible spectrophotometer Specord 200 Analytik Jena GmbH,
Germany. A computer based program, GWBASIC/Indostat, was
used to calculate LC50 and IC50 values.
Table 2
Anti-fungal activity of chalcone (1e16), cyclohexenone (1ae16a) and indazole (1be
16b) derivatives; LC50 (mg Lꢀ1).
Anti-fungal activitya
Chalcones
S.r.b
Cyclohexenones
Indazoles
S.r.
R.s b
S.r.
R.s.
198
R.s.
696
3.2. General procedure for the synthesis of chalcones (1e16) [49]
1
2
3
4
5
6
7
8
601
541
628
599
587
549
631
605
623
677
679
664
717
757
522
674
109
662
1369
421
107
635
1275
663
441
375
451
52
238
65
2.36
2.49
1.12
1a
2a
3a
4a
5a
6a
7a
8a
323
316
347
365
317
321
345
315
4308
380
372
6113
504
285
319
304
1b
2b
817
922
233
116
139
189
231
118
34
29
396
58
745
718
917
861
814
738
1034
931
643
587
776
611
528
669
802
Selected acetophenone (10 mmol) and selected benzaldehyde
(10 mmol) were added to 50 ml of 5% aqueous NaOH. The mixture was
placed in the centre of microwave oven besides a beaker with ice (50 g)
and irradiated for successive periods of 10 s, 5e7 min in total, with
cooling in between. After irradiation, color of the mixture changed to
crimson. The mixturewas thenpoured into 200 ml of ice cold waterand
neutralized with cold HCl. The precipitate formed was filtered and
recrystallized with ethanol to afford the chalcones namely (E)-3-(3,4-
Dimethoxyphenyl)-1-phenyl-propenone (1), (E)-1-Phenyl-3-p-tolyl-
propenone (2), (E)-3-(4-Methoxyphenyl)-1-phenyl-propenone(3), (E)-
3-(3,4,5-Trimethoxy-phenyl)-1-phenyl-propenone (4), (E)-3-(3,4-
Dimethoxyphenyl)-1-p-tolylprop-2-en-1-one (5), (E)-1,3-Di-p-tolyl-
prop-2-en-1-one (6), (E)-3-(3,4,5-Trimethoxyphenyl)-1-p-tolylprop-
2-en-1-one (7), (E)-3-(3,4-Dimethoxyphenyl)-1-(4-methoxyphenyl)-
propenone (8), (E)-3-Benzo[1,3]dioxol-5-yl-1-(4-nitrophenyl)-prope-
3b
713
4b
5b
1024
939
6b
876
7b
8b
9b
1173
1241
963
9
9a
10
11
12
13
14
15
16
Hxb
10a
11a
12a
13a
14a
15a
16a
10b
11b
12b
13b
14b
15b
16b
897
941
1031
894
796
1044
e
157
35
207
912
873
1.27
a
Anti-fungal activity of some chalcones and cyclohexenones were reproduced
from our previous publication [49].
b
S.r ¼ Sclerotium rolfsii and R.s ¼ Rhizoctonia solani; Hx. ¼ Hexaconazole.
none
(9),
(E)-1-Benzo[1,3]dioxol-5-yl-3-(4-methoxyphenyl)-

124
N.A. Shakil et al. / European Journal of Medicinal Chemistry 59 (2013) 120e131
Table 3
Anti-bacterial activity of chalcone (1e16), cyclohexenone (1ae16a) and indazole (1be16b) derivatives.
Compoundsa
Beneficial bacteria
Plant pathogen
Human pathogen
B.ta
B.pa
E.ca
X.oa
K.pa
S.aa
P.aa
Streptomycin
þ þ þ þ
ꢀ
þ þ þ
þ þ þ þ
ꢀ
þ þ þ þ
þ þ þ þ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ þ þ þ
þ þ
þ þ þ
ꢀ
þ þ þ þ
þ þ
þ þ
ꢀ
þ þ þ þ
þ þ
ꢀ
Ampicillin
Kanamycin
DMSO
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1a
2a
3a
4a
5a
6a
7a
þ þ þ þ
ꢀ
þ þ þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ þ
ꢀ
ꢀ
þ
þ þ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ
ꢀ
ꢀ
ꢀ
ꢀ
þ þ þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ
ꢀ
þ þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
8a
9a
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ þ
ꢀ
ꢀ
ꢀ
ꢀ
10a
11a
12a
13a
14a
15a
16a
1b
2b
3b
4b
5b
6b
7b
8b
9b
10b
11b
12b
13b
14b
15b
16b
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ þ þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ þ
þ þ
þ þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ þ
þ þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ
ꢀ
ꢀ
ꢀ
ꢀ
þ
þ þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ
þ þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ þ þ
þ þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
a
All the synthesized compounds and DMSO were tested at 100
m
g/disk. Streptomycin, Ampicillin and Kanamycin were tested at 25
m
g/disk. Zone of inhibition: (ꢀ) 0e5 mm,
inactive; (þ) 5e10 mm, poor activity; (þ þ) 10e15 mm, moderate activity; (þ þ þ) 15e20 mm, good activity and (þ þ þ þ) 20e25 mm, very good activity. B.t e Bacillus
thuringiensis, B.pe Bacillus pumilis, E.ce Erwinia chrysanthemi, X.o e Xanthomonasoryzae, K.p e Klebsiella pneumonia, S.ae Staphylococcusaureus andP.ae Pseudomonas aeruginosa.
propenone (10), (E)-1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-pro-
penone (11), (E)-1-(3,4-Dichlorophenyl)-3-(4-methoxyphenyl)-pro-
penone (12), (E)-1-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-
propenone (13), (E)-1-(4-Fluorophenyl)-3-(3-nitrophenyl)-propenone
(14), (E)-1-(4-Fluorophenyl)-3-phenyl-propenone (15) and (E)-1,3-
Diphenyl-propenone (16).
3.3. General procedure for the synthesis of cyclohexenones (1ae16a)
Table 4
[49]
Anti-bacterial screening of compounds 3a, 11a and 15b using Broth Dilution
Technique.
To a slurry of chalcone (10 mmol) and ethyl acetoacetate
(20 mmol) anhydrous K2CO3 (40 mmol) and silica (2 g) was added
and mixed thoroughly. The mixture was air dried and subjected to
microwave irradiation for 6e8 min with 30 s pulse and cooling in
between. After completion of reaction as indicated by TLC, the
reaction mixture was cooled to room temperature and extracted
with ethanol. The in-organics were filtered off. On standing the
Compound
Bacteria
MIC (mg/mL)
3a
11a
15b
Streptomycin
Streptomycin
E. chrysanthemi
E. chrysanthemi
K. pneumonia
E. chrysanthemi
K. pneumonia
55.9
73.6
24.68
4.6
3.1

N.A. Shakil et al. / European Journal of Medicinal Chemistry 59 (2013) 120e131
125
Table 5
Anti-oxidant activity of chalcone (1e16), cyclohexenone (1ae16a) and indazole
(1be16b) derivatives at 40 L of stock solution.
m
Anti-oxidant activity (%) (concentration ¼ 40
m
L stock)
Chalcones
Cyclohexenones
Indazoles
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
0.81
1.06
1.33
0.77
0.71
1.19
ꢀ0.65
0.56
0.68
0.73
0.65
0.48
0.81
0.77
4.03
9.11
21.4
1a
2a
3a
4a
5a
6a
7a
8a
4.23
1b
2b
3b
4b
5b
6b
7b
8b
69.87
18.21
50.25
67.1
4.17
2.69
1.69
7.38
3.54
4.52
4.71
3.60
4.26
4.74
2.92
11.30
4.23
3.86
3.33
62.60
18.15
56.86
65.33
19.81
56.09
43.16
48.55
12.80
67.86
70.48
40.27
9a
9b
10a
11a
12a
13a
14a
15a
16a
10b
11b
12b
13b
14b
15b
16b
Fig. 2. Anti-oxidant activity of Indazole derivatives (1be16b).
s, Ar-CH3), 2.87 (1H, ddd, H-4ax, J ¼ 2.0, 4.6 and 14.6 Hz), 3.03 (1H,
dd, H-4eq, J ¼ 4.8 and 14.8 Hz), 3.51 (2H, m, H-5 and H-6), 3.96 (2H,
q, H-2000, J ¼ 7.2 Hz), 6.57 (1H, d, H-2, J ¼ 2.0 Hz), 7.09 (2H, d, H-300
and H-500, J ¼ 8.4 Hz), 7.19 (2H, d, H-200 and H-600, J ¼ 8.4 Hz), 7.31e
Gallic acid
7.57 (5H, m, H-20-60). 13C NMR (CDCl3, 100 MHz): 14.4 (C-3000), 21.1
d
filtrate afforded crystals of cyclohexenones (1ae16a). All the
products were characterized by IR, NMR and mass spectra.
(Ar-CH3), 36.1 (C-4), 43.4 (C-5), 59.6 (C-6), 60.2 (C-2000), 126.7 (C-2),
159.7 (C-3), 169.8 (C-1000), 194.5 (C-1), 122.3, 127.8, 128.9, 129.8,
134.6, 136.5, 139.2 and 141.1 (Aromatic carbons). Mass (Mþ): 334.
Anal. Calculated for C22H22O3: C-79.02; H-6.03%. Observed: C-
79.44; H-6.00%.
3.3.1. 5-(3,4-Dimethoxyphenyl)-3-phenyl-6-carbethoxy-2-
cyclohexen-1-one (1a)
White solid. M.P.: 126 ꢂC. Yield: 88%. IR (ATR, cmꢀ1): 1743, 1659.
1H NMR (CDCl3, 400 MHz):
d
0.93 (3H, t, H-3000, J ¼ 6.8 Hz), 2.96
(1H, ddd, H-4ax, J ¼ 1.6, 4.8 and 14.2 Hz), 3.07 (1H, dd, H-4eq,
J ¼ 4.8 and 14.2 Hz), 3.66 (2H, m, H-5 and H-6), 3.85 and 3.87 (6H,
2s, Ar-OCH3), 3.92 (2H, q, H-2000, J ¼ 6.8 Hz), 6.56 (1H, d, H-2,
J ¼ 1.6 Hz), 6.80e6.87 (3H, m, H-200, H-500 and H-600), 7.19e7.25 (3H,
m, H-30, H-40 and H-50), 7.44 (2H, d, H-20 and H-60, J ¼ 8.0 Hz). 13C
3.3.3. 5-(4-Methoxyphenyl)-3-phenyl-6-carbethoxy-2-cyclohexen-
1-one (3a)
White solid. M.P.: 161 ꢂC. Yield: 92%. IR (ATR, cmꢀ1): 1746, 1651.
1H NMR (CDCl3, 400 MHz):
d
0.92 (3H, t, H-3000, J ¼ 7.2 Hz), 2.91
(1H, ddd, H-4ax, J ¼ 1.6, 4.8 and 14.2 Hz), 3.01 (1H, dd, H-4eq,
J ¼ 4.8 and 14.0 Hz), 3.50 (2H, m, H-5 and H-6), 3.72 (3H, s, Ar-
OCH3), 3.87 (2H, q, H-2000, J ¼ 7.2 Hz), 6.49 (1H, d, H-2, J ¼ 1.6 Hz),
6.86 (2H, d, H-300 and H-500, J ¼ 8.8 Hz), 7.23 (2H, d, H-200 and H-600,
J ¼ 8.0 Hz), 7.29e7.34 (3H, m, H-30, H-40 and H-50), 7.58 (2H, d, H-
NMR (CDCl3, 100 MHz):
d
14.1 (C-3000), 36.3 (C-4), 43.9 (C-5), 55.7
(eOCH3), 59.7 (C-6), 60.3 (C-2000), 126.1 (C-2), 157.8 (C-3), 168.7 (C-
1000), 194.6 (C-1), 111.1, 111.9, 119.5, 124.1, 130.4, 133.6, 139.4, 141.2,
148.5 and 148.9 (Aromatic carbons). Mass (Mþ): 380. Anal.
Calculated for C23H24O5: C-72.61; H-6.36%. Observed: C-72.34; H-
6.59%.
20 and H-60, J ¼ 8.0 Hz). 13C NMR (CDCl3, 100 MHz):
d
14.34 (C-3000),
35.8 (C-4), 43.5 (C-5), 55.5 (-OCH3), 59.5 (C-6), 60.3 (C-2000), 126.8
(C-2), 159.7 (C-3), 169.8 (C-1000), 194.8 (C-1), 114.2, 122.6, 129.1,
134.0, 140.9 and 158.7 (Aromatic carbons). Mass (Mþ): 350. Anal.
Calculated for C22H22O4: C-75.41; H-6.33%. Observed: C-75.64; H-
6.59%.
3.3.2. 3-Phenyl 5-(p-tolyl)-6-carbethoxy-2-cyclohexen-1-one (2a)
White solid. M.P.: 142 ꢂC. Yield: 91%. IR (ATR, cmꢀ1): 1739, 1652.
1H NMR (CDCl3, 400 MHz):
d
0.94 (3H, t, H-3000, J ¼ 7.2 Hz), 2.31 (3H,
Table 6
Anti-oxidant activity of Indazole derivatives (1be16b) at different concentrations.
Compound
Anti-oxidant activity (%) at different concentrations (m )
g mLꢀ1
10
20
40
60
80
100
200
300
400
500
1b
2b
3b
4b
5b
6b
7b
8b
18.1
9.2
16.8
18.1
15.0
3.4
15.0
17.7
5.3
19.0
13.9
17.6
3.5
30.2
14.5
30.6
31.5
25.9
4.4
26.4
29.2
5.8
30.4
21.7
31.0
5.3
35.9
30.4
10.9
15.8 (0.2)
69.9
18.2
50.3
67.1
62.6
18.2
56.9
65.3
19.8
56.1
43.2
48.6
12.8
67.9
70.5
40.3
21.4 (0.4)
87.6
35.1
79.7
86.4
78.5
31.5
79.3
85.8
38.4
84.2
68.7
86.1
26.3
86.4
86.9
62.5
90.9
36.4
88.4
90.7
80.3
34.9
89.9
90.0
44.5
89.5
86.4
90.4
31.1
90.1
89.0
67.3
34.4 (0.8)
91.8
41.2
89.0
91.8
81.8
40.3
90.8
91.4
50.7
90.7
89.9
91.1
36.5
90.8
91.0
73.3
41.0 (1)
91.9
69.9
91.8
92.3
91.4
68.1
91.8
92.1
83.4
91.9
91.2
91.5
58.8
92.3
92.2
90.6
73.6 (2)
91.9
81.8
89.7
91.2
91.6
80.5
90.2
90.4
87.9
89.8
89.7
90.7
75.3
90.5
90.7
89.0
88.9 (3)
90.5
85.7
88.9
90.5
91.7
84.8
90.3
90.5
87.3
89.9
89.8
91.3
82.6
90.9
90.8
89.1
89.1 (4)
90.8
85.7
90.2
90.8
91.0
85.2
90.6
90.8
87.3
90.1
90.0
91.3
82.6
91.1
90.8
89.2
89.1 (5)
9b
10b
11b
12b
13b
14b
15b
16b
Gallic acida
22.6
23.5
9.1
13.9 (0.1)
28.0 (0.6)
a
Values in parenthesis are concentration at which Gallic acid is tested.

126
N.A. Shakil et al. / European Journal of Medicinal Chemistry 59 (2013) 120e131
Table 7
3.3.7. 5-(3,4,5-Trimethoxyphenyl)-3-(p-tolyl)-6-carbethoxy-2-
IC50 values for Indazole derivatives (1be16b).
cyclohexen-1-one (7a)
White solid. M.P.: 137 ꢂC. Yield: 86%. IR (ATR, cmꢀ1): 1742, 1657.
Compound
Inhibition concentration
50 percent (IC50) m
g mLꢀ1
1H NMR (CDCl3, 400 MHz):
d
0.95 (3H, t, H-3000, J ¼ 6.8 Hz), 2.32 (3H,
1b
2b
3b
4b
5b
6b
7b
8b
21.98
106.62
29.62
22.28
33.12
123.54
29.7
23.94
95.82
25.2
42.34
40.98
146.68
19.81
23.71
58.28
1.28
s, Ar-CH3), 2.95 (1H, ddd, H-4ax, J ¼ 1.6, 4.8 and 14.2 Hz), 3.02 (1H,
dd, H-4eq, J ¼ 4.8 and 14.6 Hz), 3.51 (2H, m, H-5 and H-6), 3.71e3.74
(9H, 3s, Ar-OCH3), 3.90 (2H, q, H-2000, J ¼ 7.2 Hz), 6.49 (1H, d, H-2,
J ¼ 1.6 Hz), 6.84e7.06 (3H, m, H-200, H-500 and H-600), 7.24 (2H, d, H-30
and H-50, J ¼ 8.0 Hz), 7.59 (2H, d, H-20 and H-60, J ¼ 8.4 Hz). 13C
NMR (CDCl3):
d
14.4 (C-3000), 21.3 (Ar-CH3), 35.8 (C-4), 43.9 (C-5),
55.98 (eOCH3), 56.01 (eOCH3), 59.4 (C-6), 60.3 (C-2000), 126.9 (C-2),
159.7 (C-3), 169.9 (C-1000), 194.8 (C-1), 112.0, 112.2, 120.0, 129.9,
134.6, 134.9, 140.9, 148.3 and 149.1 (Aromatic carbons). Mass (Mþ):
424. Anal. Calculated for C25H28O6: C-70.74; H-6.65%. Observed: C-
70.48; H-6.36%.
9b
10b
11b
12b
13b
14b
15b
16b
Gallic acid
3.3.8. 5-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-6-
carbethoxy-2-cyclohexen-1-one (8a)
Brownish white solid. M.P.: 129 ꢂC. Yield: 69%. IR (ATR, cmꢀ1):
1739, 1656. 1H NMR (CDCl3, 400 MHz):
d
0.98 (3H, t, H-3000,
J ¼ 6.8 Hz), 2.96 (1H, ddd, H-4ax, J ¼ 2.0, 4.8 and 14.2 Hz), 3.13 (1H,
dd, H-4eq, J ¼ 4.8 and 14.2 Hz), 3.55 (2H, m, H-5 and H-6), 3.72e
3.90 (9H, 3s, Ar-OCH3), 4.01 (2H, q, H-2000, J ¼ 6.8 Hz), 6.52 (1H, d,
H-2, J ¼ 2.0 Hz), 6.86e6.90 (2H, m, H-500 and H-600), 6.98 (2H, d, H-
30 and H-50, J ¼ 8.8 Hz), 7.06 (1H, d, H-200, J ¼ 1.8 Hz) and 7.69 (2H,
d, H-20 and H-60, J ¼ 8.8 Hz). 13C NMR (CDCl3, 100 MHz):
3.3.4. 5-(3,4,5-Trimethoxyphenyl)-3-phenyl-6-carbethoxy-2-
cyclohexen-1-one (4a)
White solid. M.P.: 119 ꢂC. Yield: 84%. IR (ATR, cmꢀ1): 1742, 1647.
1H NMR (CDCl3, 400 MHz):
d
0.92 (3H, t, H-3000, J ¼ 7.2 Hz), 2.95 (1H,
ddd, H-4ax, J ¼ 1.6, 4.8 and 14.2 Hz), 3.01 (1H, dd, H-4eq, J ¼ 4.8 and
14.2 Hz), 3.61 (2H, m, H-5 and H-6), 3.84e3.89 (9H, 3s, Ar-OCH3),
3.91 (2H, q, H-2000, J ¼ 7.2 Hz), 6.34 (2H, s, H-200 and H-600), 6.52 (1H,
d, H-2, J ¼ 1.6 Hz), 7.20e7.27 (3H, m, H-30, H-40 and H-50), 7.45 (2H,
d
14.3 (C-3000), 36.3 (C-4), 43.9 (C-5), 54.8 (eOCH3), 55.8 (eOCH3),
56.0 (eOCH3), 59.4 (C-6), 60.3 (C-2000), 127.3 (C-2), 161.7 (C-3),
170.0 (C-1000), 194.1 (C-1), 111.9, 112.2, 114.6, 120.0, 121.1, 129.7,
134.3, 148.2, 148.9 and 159.2 (Aromatic carbons). Mass (Mþ): 410.
Anal. Calculated for C24H26O6: C-70.23; H-6.38%. Observed:
C-70.04; H-6.12%.
m, H-20 and H-60). 13C NMR (CDCl3):
d
14.3 (C-3000), 35.8 (C-4), 43.6
(C-5), 55.8 (eOCH3), 56.0 (eOCH3), 59.5 (C-6), 60.4 (C-2000), 126.7 (C-
2), 159.6 (C-3), 169.4 (C-1000), 194.6 (C-1), 111.9, 112.1, 120.0, 122.6,
129.8, 134.9, 135.4, 140.8, 147.6 and 149.0 (Aromatic carbons). Mass
(Mþ): 410. Anal. Calculated for C24H26O6: C-70.23; H-6.38%.
Observed: C-70.44; H-6.47%.
3.3.9. 5-Benzo[1,3]dioxol-5-yl-3-(4-nitrophenyl)-6-carbethoxy-2-
cyclohexen-1-one (9a)
Pale yellow solid. M.P.: 162 ꢂC. Yield: 62%. IR (ATR, cmꢀ1): 1755,
3.3.5. 5-(3,4-Dimethoxyphenyl)-3-(p-tolyl)-6-carbethoxy-2-
cyclohexen-1-one (5a)
1641. 1H NMR (CDCl3, 400 MHz):
d
0.94 (3H, t, H-3000, J ¼ 6.9 Hz), 2.95
(1H, ddd, H-4ax, J ¼ 2.0, 4.8 and 14.2 Hz), 3.08 (1H, dd, H-4eq,
J ¼ 4.8 and 14.2 Hz), 3.65 (2H, m, H-5 and H-6), 4.05 (2H, q, H-2000,
J ¼ 6.9 Hz), 5.97 (2H, s, OeCH2eO), 6.64 (1H, d, H-2, J ¼ 2.0 Hz),
6.84e7.07 (3H, m, H-200, H-500 and H-600), 7.94 (2H, d, H-20 and H-60,
J ¼ 8.6 Hz) and 8.22 (2H, d, H-30 and H-50, J ¼ 8.8 Hz). 13C NMR
White solid. M.P.: 152 ꢂC. Yield: 90%. IR (ATR, cmꢀ1): 1748, 1649.
1H NMR (CDCl3, 400 MHz):
d
1.05 (3H, t, H-3000, J ¼ 7.2 Hz), 2.36 (3H, s,
Ar-CH3), 2.90 (1H, ddd, H-4ax, J ¼ 2.0, 4.8 and 14.6 Hz), 3.05 (1H, dd,
H-4eq, J ¼ 4.8 and 14.6 Hz), 3.70 (2H, m, H-5 and H-6), 3.85 and 3.86
(6H, 2s, Ar-OCH3), 4.02 (2H, q, H-2000, J ¼ 7.2 Hz), 6.53 (1H, d, H-2,
J ¼ 2.0 Hz), 6.80e6.87 (3H, m, H-200, H-500 and H-600), 7.19 (2H, d, H-30
and H-50, J ¼ 8.4), 7.43 (2H, d, H-20 and H-60, J ¼ 8.0 Hz). 13C NMR
(CDCl3, 100 MHz):
d
14.3 (C-3000), 35.8 (C-4), 43.9 (C-5), 59.4 (C-6),
60.5 (C-2000), 101.3 (eOeCH2eOe), 128.4 (C-2), 157.5 (C-3), 169.5 (C-
1000), 194.8 (C-1), 108.3, 108.5, 121.4, 124.2, 125.8, 135.6, 144.3, 146.6,
147.6 and 148.6 (Aromatic carbons). Mass (Mþ): 409. Anal. Calcu-
lated for C22H19NO7: C-64.54; H-4.68; N-3.42%. Observed: C-64.03;
H-4.14; N-3.26%.
(CDCl3,100 MHz): d
14.1 (C-3000), 21.3 (Ar-CH3), 36.3 (C-4), 43.7 (C-5),
55.9 (eOCH3), 59.7 (C-6), 60.9 (C-2),126.1 (C-2),158.6 (C-3),169.4 (C-
1000), 194.2 (C-1), 110.5, 111.3, 119.2, 123.2, 129.6, 133.7, 134.7, 141.1,
148.2 and 148.9 (Aromatic carbons). Mass (Mþ): 394. Anal. Calcu-
lated for C24H26O5: C-73.08; H-6.64%. Observed: C-73.54; H-6.09%.
3.3.10. 3-Benzo[1,3]dioxol-5-yl-5-(4-methoxyphenyl)-6-
carbethoxy-2-cyclohexen-1-one (10a)
3.3.6. 3,5-Di-(p-tolyl)-6-carbethoxy-2-cyclohexen-1-one (6a)
Electric white solid. M.P.: 153 ꢂC. Yield: 90%. IR (ATR, cmꢀ1):
White solid. M.P.: 148 ꢂC. Yield: 94%. IR (ATR, cmꢀ1): 1737, 1647.
1744, 1659. 1H NMR (CDCl3, 400 MHz): 0.93 (3H, t, H-3000
d ,
1H NMR (CDCl3, 400 MHz):
d
0.92 (3H, t, H-3000, J ¼ 7.2 Hz), 2.24 and
J ¼ 7.0 Hz), 2.98 (1H, ddd, H-4ax, J ¼ 1.6, 4.4 and 14.6 Hz), 3.11
(1H, dd, H-4eq, J ¼ 4.8 and 14.6 Hz), 3.56 (2H, m, H-5 and H-6),
3.86 (3H, s, Ar-OCH3), 3.96 (2H, q, H-2000, J ¼ 7.0 Hz), 5.97 (2H, s,
OeCH2eO), 6.52 (1H, d, H-2, 1.6 Hz), 6.81/7.04 (3H, m, H-20, H-50
and H-60), 6.97 (2H, d, H-300 and H-500, J ¼ 8.8 Hz) and 7.68 (2H,
2.32 (6H, 2s, Ar-CH3), 2.92 (1H, ddd, H-4ax, J ¼ 1.6, 4.8 and 14.2 Hz),
3.00 (1H, dd, H-4eq, J ¼ 4.8 and 14.2 Hz), 3.53 (2H, m, H-5 and H-6),
3.87 (2H, q, H-2000, J ¼ 7.2 Hz), 6.49 (1H, d, H-2, J ¼ 1.6 Hz), 7.11 (2H, d,
H-300 and H-500, J ¼ 8.0 Hz), 7.23 (2H, d, H-200 and H-600, J ¼ 8.0 Hz),
7.27 (2H, d, H-30 and H-50, J ¼ 8.0 Hz) and 7.58 (2H, d, H-20 and H-60,
d, H-200 and H-500, J ¼ 8.8 Hz). 13C NMR (CDCl3, 100 MHz):
d 14.4
J ¼ 8.0 Hz). 13C NMR (CDCl3, 100 MHz):
d
14.3 (C-3000), 21.1 (Ar-CH3),
(C-3000), 35.6 (C-4), 44.0 (C-5), 55.3 (eOCH3), 59.3 (C-6), 60.4 (C-
21.3 (Ar-CH3), 35.7 (C-4), 43.9 (C-5), 59.3 (C-6), 60.4 (C-2000), 126.8
(C-2), 159.7 (C-3), 169.8 (C-1000), 194.7 (C-1), 122.5, 127.9, 129.4,
129.9, 134.8, 136.6, 139.1 and 141.0 (Aromatic carbons). Mass (Mþ):
348. Anal. Calculated for C23H24O3: C-79.28; H-6.94%. Observed: C-
79.34; H-7.07%.
2
000), 101.3 (eOeCH2eOe), 129.3 (C-2), 159.2 (C-3), 169.8 (C-1000),
194.6 (C-1), 108.3, 108.5, 114.7, 121.40, 121.44, 123.7, 136.0, 146.5,
147.6 and 161.7 (Aromatic carbons). Mass (Mþ): 394. Anal.
Calculated for C23H22O6: C-70.04; H-5.62%. Observed: C-70.44;
H-6.01%.

N.A. Shakil et al. / European Journal of Medicinal Chemistry 59 (2013) 120e131
127
3.3.11. 3-(4-Chlorophenyl)-5-(4-methoxyphenyl)-6-carbethoxy-2-
cyclohexen-1-one (11a)
NMR (CDCl3, 100 MHz):
d
14.1 (C-3000), 35.6 (C-4), 44.2 (C-5), 59.2
(C-6), 60.7 (C-2000), 159.0 (C-3), 169.9 (C-1000), 195.0 (C-1), 127.1 (C-2),
116.1, 123.1, 127.9, 128.9, 129.1, 134.2, 143.6 and 162.6 (Aromatic
carbons). Mass (Mþ): 338. Anal. Calculated for C21H19FO3: C-74.54;
H-5.66%. Observed: C-74.12; H-5.16%.
White solid. M.P.: 138 ꢂC. Yield: 81%. IR (ATR, cmꢀ1): 1747, 1651.
1H NMR (CDCl3, 400 MHz):
d
0.92 (3H, t, H-3000, J ¼ 6.8 Hz), 2.91 (1H,
ddd, H-4ax, J ¼ 1.6, 4.8 and 14.2 Hz), 3.02 (1H, dd, H-4eq, J ¼ 4.8 and
14.2 Hz), 3.52 (2H, m, H-5 and H-6), 3.72 (3H, s, Ar-OCH3), 3.88 (2H,
q, H-2000, J ¼ 7.2 Hz), 6.54 (1H, d, H-2, J ¼ 1.6 Hz), 6.87 (2H, d, H-300
and H-500, J ¼ 6.8 Hz), 7.30 (2H, d, H-30 and H-50, J ¼ 8.8 Hz), 7.48 (2H,
d, H-200 and H-500, J ¼ 6.8 Hz) and 7.72 (2H, d, H-20 and H-60,
3.3.16. 3,5-(Diphenyl)-6-carbethoxy-2-cyclohexen-1-one (16a)
White solid. M.P.: 155 ꢂC. Yield: 95%. IR (ATR, cmꢀ1): 1734, 1652.
1H NMR (CDCl3, 400 MHz):
d
1.04 (3H, t, H3000, J ¼ 6.8 Hz), 2.95 (1H,
J ¼ 8.8 Hz). 13C NMR (CDCl3, 100 MHz):
d
14.3 (C-3000), 35.8 (C-4),
ddd, H-4ax, J ¼ 1.6, 4.8 and 14.2 Hz), 3.06 (1H, dd, H-4eq, J ¼ 4.8 and
14.2 Hz), 3.80 (2H, m, H-5 and H-6), 4.04 (2H, q, H-2000, J ¼ 7.2 Hz),
6.57 (1H, d, H-2, J ¼ 2.0 Hz), 7.27e7.57 and (10H, m, H-200-600 and
43.5 (C-5), 55.5 (eOCH3), 59.5 (C-6), 60.4 (C-2000), 128.7 (C-2), 158.7
(C-3),169.7 (C-1000), 194.8 (C-1),114.2,129.1,129.3,133.8,135.6,136.7
and 158.5 (Aromatic carbons). Mass (Mþ): 384. Anal. Calculated for
C22H21ClO4: C-68.66; H-5.50%. Observed: C-69.24; H-5.12%.
H-20-60). 13C NMR (CDCl3, 100 MHz):
d
13.9 (C-3000), 36.1 (C-4), 44.1
(C-5), 59.6 (C-6), 60.9 (C-2000), 124.1 (C-2), 158.5 (C-3), 169.2 (C-1000),
194.0 (C-1), 126.2, 127.3, 127.5, 128.8, 128.9, 130.5, 137.7 and 141.0
(Aromatic carbons). Mass (Mþ): 320. Anal. Calculated for C21H20O3:
C-78.73; H-6.29%. Observed: C-79.14; H-6.38%.
3.3.12. 3-(3,4-Dichlorophenyl)-5-(4-methoxyphenyl)-6-
carbethoxy-2-cyclohexen-1-one (12a)
White solid. M.P.: 145 ꢂC. Yield: 69%. IR (ATR, cmꢀ1): 1744, 1660.
1H NMR (CDCl3, 400 MHz):
d
0.93 (3H, t, H-3000, J ¼ 6.7 Hz), 2.50 (1H,
3.4. Generalprocedureforthesynthesisof2H-indazol-3-ols(1be16b)
ddd, H-4ax, J ¼ 2.0, 4.8 and 14.2 Hz), 3.01 (1H, dd, H-4eq, J ¼ 4.8 and
14.2 Hz), 3.64 (2H, m, H-5 and H-6), 3.84 (3H, s, Ar-OCH3), 3.94 (2H,
q, H-2000, J ¼ 6.7 Hz), 6.67 (1H, d, H-2, J ¼ 2.0 Hz), 7.01 (2H, d, H-300
and H-500, J ¼ 8.8 Hz), 7.24 (2H, m, H-50 and H-60) and 7.38e7.67 (3H,
To a mixture of cyclohexenone (1 mmol) and glacial acetic acid
(1.5 mL) in ethanol (1.5 mL) was added hydrazine hydrate (2 mmol).
The mixture was then placed in the center of a house-hold micro-
wave oven, beside a beaker containing 50 g ice, and irradiated for
3 min in total with 30 s pulse and cooling in between. After irra-
diation, ice cold distilled water (20 mL) was added to the reaction
mixture. This aq. suspension was then centrifuged and supernatant
was discarded. Precipitate collected was washed thrice, using
centrifugation, with distilled water (20 mL) to remove acetic acid.
The wet solid was lyophilized to get 98% pure powdery solid of 2H-
indazol-3-ols (1be16b). Further purification, if required, was done
by washing it with cold ethanol. All the products were character-
ized by IR, NMR and mass spectra.
m, H-0, H-200 and H-600). 13C NMR (CDCl3, 100 MHz):
d
14.2 (C-3000),
37.9 (C-4), 44.0 (C-5), 55.4 (eOCH3), 59.8 (C-6), 60.3 (C-2000), 128.2
(C-2), 158.7 (C-3), 169.6 (C-1000), 194.6 (C-1), 114.3, 125.9, 128.9,
129.8, 131.0, 131.7, 133.2, 134.6, 137.5 and 160.0 (Aromatic carbons).
Mass (Mþ): 418. Anal. Calculated for C22H20Cl2O4: C-63.02; H-4.81%.
Observed: C-63.56; H-4.23%.
3.3.13. 3-(3,4-Dimethoxyphenyl)-5-(4-methoxyphenyl) -6-
carbethoxy-2-cyclohexen-1-one (13a)
White solid. M.P.: 161 ꢂC. Yield: 91%. IR (ATR, cmꢀ1): 1745,1663.1H
NMR (CDCl3):
d
0.95 (3H, t, H-3000, J ¼ 7.0 Hz), 2.79 (1H, ddd, H-4ax,
J ¼ 1.6, 4.8 and 14.2 Hz), 3.01 (1H, dd, H-4eq, J ¼ 4.8 and 14.2 Hz), 3.54
(2H, m, H-5 and H-6), 3.73e3.81 (9H, 3s, Ar-OCH3), 3.98 (2H, q, H-2000,
J ¼ 6.9 Hz), 6.51 (1H, d, H-2, J ¼ 1.6 Hz), 6.88e7.06 (5H, m, H-20, H-50,
H-60, H-300 and H-500) and 7.43 (2H, d, H-200 and H-600, J ¼ 8.4 Hz). 13C
3.4.1. 4,5-Dihydro-4-(3,4-dimethoxyphenyl)-6-phenyl-2H-indazol-
3-ol (1b)
White solid. M.P.: 168 ꢂC. Yield: 83%. IR (ATR, cmꢀ1): 3321, 3212.
1H NMR (DMSO-d6, 400 MHz):
d
2.81 (1H, dd, H-5eq, J ¼ 3.4 and
NMR (CDCl3):
d
14.2 (C-3000), 35.6 (C-4), 43.5 (C-5), 55.4 (eOCH3), 55.9
17.2 Hz), 3.09 (1H, ddd, H-5ax, J ¼ 2.4, 8.2 and 17.2 Hz), 3.85 & 3.86
(6H, 2s, Ar-OCH3), 4.07 (1H, dd, 4-H, J ¼ 3.4 and 8.2 Hz), 6.61 (1H, d,
H-7, J ¼ 2.4 Hz), 6.80e6.87 (3H, m, H-200, H-500 and H-600), 7.18e7.35
(5H, m, H-20-60) and 9.70 (2H, brs, OH and NH). 13C NMR (DMSO-d6,
(eOCH3), 59.6 (C-6), 60.6(C-2000),127.0(C-2),159.9(C-3),170.0 (C-1000),
194.9 (C-1), 109.8, 111.9, 114.2, 120.4, 121.6, 129.9, 133.8, 134.5, 149.1,
151.4 and 158.6 (Aromatic carbons). Mass (Mþ): 410. Anal. Calculated
for C24H26O6: C-70.23; H-6.38%. Observed: C-70.54; H-6.49%.
100 MHz): d 33.2 (C-4), 36.8 (C-5), 55.1 (eOCH3), 55.4 (eOCH3), 98.6
(C-3), 113.6 (C-7), 136.1 (C-3a), 139.3 (C-7a), 156.8 (C-6), 111.4, 122.2,
125.5, 128.0, 128.9, 129.2, 133.5, 145.2, 149.3 and 150.1 (Aromatic
carbons). Mass (Mþ): 348. Anal. Calculated for C21H20N2O3:
C-72.40; H-5.79; N-8.04%. Observed: C-72.44; H-6.05; N-8.26%.
3.3.14. 3-(4-Fluorophenyl)-5-(3-nitrophenyl)-6-carbethoxy-2-
cyclohexen-1-one (14a)
White solid. M.P.: 134 ꢂC. Yield: 72%. IR (ATR, cmꢀ1): 1739, 1661.
1H NMR (CDCl3, 400 MHz):
d
0.86 (3H, t, H-3000, J ¼ 7.2 Hz), 2.96e3.11
(2H, m, H-4eq and H-4ax), 3.58 (2H, m, H-5 and H-6), 4.01 (2H, q, H-
2000, J ¼ 7.2 Hz), 6.51 (1H, d, H-2, J ¼ 2.0 Hz), 7.21e7.43 (4H, m, H-20, H-
30, H-50 and H-60) and 7.58e7.72 (4H, m, H-200, H-400, H-500 and H-600).
3.4.2. 4,5-Dihydro-6-phenyl-4-p-tolyl-2H-indazol-3-ol (2b)
White solid. M.P.: 227 ꢂC. Yield: 73%. IR (ATR, cmꢀ1): 3318, 3204.
1H NMR (DMSO-d6, 400 MHz):
d 2.28 (3H, s, Ar-CH3), 2.81 (1H, dd,
13C NMR (CDCl3, 100 MHz):
d
14.1 (C-3000), 34.4 (C-4), 42.2 (C-5), 57.9
H-5eq, J ¼ 3.6 and 17.2 Hz), 3.14 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and
17.2 Hz), 4.09 (1H, dd, 4-H, J ¼ 3.6 and 8.6 Hz), 6.67 (1H, d, H-7,
J ¼ 2.4 Hz), 7.08 (2H, d, H-200 and H-600, J ¼ 7.8 Hz), 7.29e7.54 (7H, m,
H-20-60, H-300 and H-500) and 9.73 (2H, brs, OH and NH). 13C NMR
(C-6), 60.8 (C-2000), 128.1 (C-2), 158.3 (C-3), 169.4 (C-1000), 194.7 (C-1),
116.2, 123.1, 129.3, 130.0, 133.2, 138.5, 162.6 and 165.1 (Aromatic
carbons). Mass (Mþ): 383. Anal. Calculated for C21H18FNO5: C-65.79;
H-4.73; N-3.65%. Observed: C-65.27; H-4.23; N-3.26%.
(DMSO-d6, 100 MHz):
d 21.2 (Ar-CH3), 33.4 (C-4), 36.7 (C-5), 98.5
(C-3),114.2 (C-7),136.3 (C-3a),139.4 (C-7a),156.4 (C-6),125.8,127.9,
128.6, 129.3, 129.7, 134.5, 138.7 and 144.3 (Aromatic carbons). Mass
(Mþ): 302. Anal. Calculated for C20H18N2O: C-79.44; H-6.00;
N-9.26%. Observed: C-79.03; H-6.53; N-9.46%.
3.3.15. 3-(4-Fluorophenyl)-5-phenyl-6-carbethoxy-2-cyclohexen-
1-one (15a)
Greenish white solid. M.P.: 178 ꢂC. Yield: 74%. IR (ATR, cmꢀ1):
1733, 1659. 1H NMR (CDCl3, 400 MHz):
d
0.89 (3H, t, H-3000,
J ¼ 7.2 Hz), 2.93 (1H, ddd, H-4ax, J ¼ 1.6, 4.8 and 14.2 Hz), 3.02 (1H,
dd, H-4eq, J ¼ 4.8 and 14.2 Hz), 3.53 (2H, m, H-5 and H-6), 3.95 (2H,
q, H-2000, J ¼ 7.2 Hz), 6.49 (1H, d, H-2, J ¼ 1.6 Hz), 7.20e7.37 (7H, m,
H-200-600, H-30 and H-50) and 7.69e7.73 (2H, m, H-20 and H-60). 13C
3.4.3. 4,5-Dihydro-4-(4-methoxyphenyl)-6-phenyl-2H-indazol-3-
ol (3b)
Brownish white solid. M.P.: 232 ꢂC. Yield: 81%. IR (ATR, cmꢀ1):
3308, 3210. 1H NMR (DMSO-d6, 400 MHz):
d 2.79 (1H, dd, H-5eq,

128
N.A. Shakil et al. / European Journal of Medicinal Chemistry 59 (2013) 120e131
J ¼ 3.6 and 17.2 Hz), 3.00 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and 17.2 Hz),
3.64 (3H, s, Ar-OCH3), 4.10 (1H, dd, 4-H, J ¼ 3.6 and 8.6 Hz), 6.68 (1H,
d, H-7, J ¼ 2.4 Hz), 6.72e7.38 (9H, m, Aromatic protons) and 9.70
(C-6), 111.9, 118.9, 125.9, 129.7, 132.0, 138.2, 147.4 and 148.7
(Aromatic carbons). Mass (Mþ): 392. Anal. Calculated for
C23H24N2O4: C-70.39; H-6.16; N-7.14%. Observed: C-70.68; H-5.87;
N-7.38%.
(2H, brs, OH and NH). 13C NMR (DMSO-d6, 100 MHz):
d 33.8 (C-4),
36.7 (C-5), 55.3 (eOCH3), 98.4 (C-3), 113.9 (C-7), 136.6 (C-3a), 137.8
(C-7a), 156.4 (C-6), 125.4, 126.0, 128.2, 129.60, 129.63, 129.8, 137.3
and 157.9 (Aromatic carbons). Mass (Mþ): 318. Anal. Calculated for
C20H18N2O2: C-75.45; H-5.70; N-8.80%. Observed: C-75.07; H-6.09;
N-9.16%.
3.4.8. 4,5-Dihydro-4-(3,4-dimethoxyphenyl)-6-(4-methoxyphenyl)-
2H-indazol-3-ol (8b)
Brownish white solid. M.P.: 186 ꢂC. Yield: 69%. IR (ATR, cmꢀ1):
3318, 3217. 1H NMR (DMSO-d6, 400 MHz):
d 2.88 (1H, dd, H-5eq,
J ¼ 3.6 and 17.2 Hz), 3.06 (1H, ddd, H-5ax, J ¼ 2.4, 8.8 and 17.6 Hz),
3.63e3.73 (9H, 3s, Ar-OCH3), 4.08 (1H, dd, 4-H, J ¼ 3.6 and 8.8 Hz),
6.54 (1H, dd, H-600, J ¼ 2.0 and 8.4 Hz), 6.62 (1H, d, H-7, J ¼ 2.4), 6.72
(1H, d, H-500, J ¼ 8.4 Hz), 6.82 (1H, d, H-200, J ¼ 2.0 Hz), 6.89 (2H, d, H-
30 and H-50, J ¼ 6.8 Hz), 7.40 (2H, d, H-20 and H-60, J ¼ 6.8 Hz) and
3.4.4. 4,5-Dihydro-4-(3,4,5-trimethoxyphenyl)-6-phenyl-2H-
indazol-3-ol (4b)
White solid. M.P.: 179 ꢂC. Yield: 86%. IR (ATR, cmꢀ1): 3319, 3216.
1H NMR (DMSO-d6, 400 MHz):
d
2.81 (1H, dd, H-5eq, J ¼ 3.4 and
16.8 Hz), 3.08 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and 16.8 Hz), 3.88e3.97
(9H, 3s, Ar-OCH3), 4.09 (1H, dd, 4-H, J ¼ 3.4 and 8.6 Hz), 6.54 (1H, d,
H-7, J ¼ 2.4 Hz), 6.67 (2H, m, H-200 and H-600), 7.08e7.35 (5H, m, H-
20-60) and 9.72 (2H, brs, OH and NH). 13C NMR (DMSO-d6, 100 MHz):
9.70 (2H, brs, OH and NH). 13C NMR (DMSO-d6, 100 MHz):
d 34.3
(C-4), 36.6 (C-5), 55.6 (eOCH3), 55.8 (eOCH3), 56.0 (eOCH3), 98.4
(C-3), 112.1 (C-7), 133.1 (C-3a), 136.6 (C-7a), 156.3 (C-6), 111.7, 114.4,
118.9, 126.8, 127.1, 127.6, 138.4, 147.5, 148.8 and 159.3 (Aromatic
carbons). Mass (Mþ): 378. Anal. Calculated for C22H22N2O4: C-
69.83; H-5.86; N-7.40%. Observed: C-69.43; H-6.17; N-7.29%.
d
33.9 (C-4), 36.5 (C-5), 55.7 (eOCH3), 55.9 (eOCH3), 98.3 (C-3),
113.4 (C-7), 137.5 (C-3a), 138.2 (C-7a), 156.4 (C-6), 111.9, 118.9, 125.9,
129.7, 132.0, 134.5, 147.4 and 148.7 (Aromatic carbons). Mass (Mþ):
378. Anal. Calculated for C22H22N2O4: C-69.83; H-5.86; N-7.40%.
Observed: C-70.29; H-6.15; N-7.26%.
3.4.9. 4-(Benzo[d][1,3]dioxol-5-yl)-4,5-dihydro-6-(4-nitrophenyl)-
2H-indazol-3-ol (9b)
Pale yellow solid. M.P.: 198 ꢂC. Yield: 82%. IR (ATR, cmꢀ1): 3324,
3.4.5. 4,5-Dihydro-4-(3,4-dimethoxyphenyl)-6-p-tolyl-2H-indazol-
3-ol (5b)
3221. 1H NMR (DMSO-d6, 400 MHz):
d
2.84 (1H, dd, H-5eq, J ¼ 3.8
and 17.4 Hz), 3.14 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and 17.4 Hz), 4.13 (1H,
dd, 4-H, J ¼ 3.8 and 8.6 Hz), 5.85 (2H, s, OeCH2eO), 6.53 (1H, dd, H-
600, J ¼ 1.6 and 8.0 Hz), 6.62 (1H, d, H-200, J ¼ 1.6), 7.01 (1H, d, H-7,
J ¼ 2.4 Hz), 7.12 (2H, d, H-20 and H-60, J ¼ 8.8 Hz), 8.14 (2H, d, H-20
and H-60, J ¼ 8.8 Hz) and 9.69 (2H, brs, OH and NH). 13C NMR
White solid. M.P.: 193 ꢂC. Yield: 81%. IR (ATR, cmꢀ1): 3321, 3215.
1H NMR (DMSO-d6, 400 MHz):
d 2.37 (3H, s, Ar-CH3), 2.71 (1H, dd,
H-5eq, J ¼ 3.4 and 17.2 Hz), 3.11 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and
17.2 Hz), 3.81 & 3.85 (6H, 2s, Ar-OCH3), 4.14 (1H, dd, 4-H, J ¼ 3.6 and
8.6 Hz), 6.67 (1H, d, H-7, J ¼ 2.4 Hz), 6.77e6.89 (3H, m, H-200, H-500
and H-600), 7.19e7.24 (2H, m, H-30 and H-50), 7.31 (2H, d, H-20 and H-
6, J ¼ 8 Hz) and 9.70 (2H, brs, OH and NH). 13C NMR (DMSO-d6,
(DMSO-d6,100 MHz): d 34.1 (C-4), 36.4 (C-5), 99.3 (C-3), 101.0 (eOe
CH2eOe), 108.3 (C-7), 136.1 (C-3a), 139.4 (C-7a), 156.8 (C-6), 107.8,
120.1, 124.3, 126.5, 134.6, 141.7, 145.8, 146.5, 147.1 and 147.4
(Aromatic carbons). Mass (Mþ): 377. Anal. Calculated for
C20H15N3O5: C-63.66; H-4.01; N-11.14%. Observed: C-63.14; H-4.09;
N-10.39%.
100 MHz): d 21.2 (Ar-CH3), 33.6 (C-4), 36.7 (C-5), 55.0 (eOCH3), 55.2
(eOCH3), 98.6 (C-3), 112.9 (C-7), 136.4 (C-3a), 148.1 (C-7a), 156.9 (C-
6), 111.3, 121.2, 122.4, 125.6, 128.9, 135.2, 136.1, 137.6, 150.3 and 151.1
(Aromatic carbons). Mass (Mþ): 362. Anal. Calculated for
C22H22N2O3: C-72.91; H-6.12; N-7.73%. Observed: C-72.34; H-6.28;
N-7.31%.
3.4.10. 6-(Benzo[d][1,3]dioxol-5-yl)-4,5-dihydro-4-(4-methoxyphenyl)
-2H-indazol-3-ol (10b)
White solid. M.P.: 203 ꢂC. Yield: 89%. IR (ATR, cmꢀ1): 3307, 3198.
3.4.6. 4,5-Dihydro-4,6-di-p-tolyl-2H-indazol-3-ol (6b)
1H NMR (DMSO-d6, 400 MHz):
d
2.80 (1H, dd, H-5eq, J ¼ 3.6 and
White solid. M.P.: 211 ꢂC. Yield: 79%. IR (ATR, cmꢀ1): 3321, 3210.
17.2 Hz), 3.02 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and 17.2 Hz), 3.88 (3H, s, Ar-
OCH3), 4.08 (1H, dd, 4-H, J ¼ 3.6 and 8.6 Hz), 5.85 (2H, s, OeCH2eO),
6.54 (1H, dd, H-60, J ¼ 1.60 and 8.0 Hz), 6.62 (1H, d, H-20, J ¼ 1.60 Hz),
6.64 (1H, d, H-7, J ¼ 2.4), 6.68 (1H, d, H-50, J ¼ 8.0 Hz), 6.87 (2H, d, H-
300 and H-500, J ¼ 8.8 Hz), 7.39 (2H, d, H-200 and H-600, J ¼ 8.8 Hz) and
1H NMR (DMSO-d6, 400 MHz):
d 2.26 & 2.31 (6H, 2s, Ar-CH3), 2.60
(1H, dd, H-5eq, J ¼ 3.6 and 17.2 Hz), 2.72 (1H, ddd, H-5ax, J ¼ 2.4, 8.6
and 17.2 Hz), 4.01 (1H, dd, 4-H, J ¼ 3.6 and 8.6 Hz), 6.71 (1H, d, H-7,
J ¼ 2.4 Hz), 6.96e7.38 (4H, m, H-200, H-300, H-500 and H-600), 7.54 (2H,
d, H-30 and H-50, J ¼ 8.0 Hz), 7.60 (2H, d, H-20 and H-60, J ¼ 8.0 Hz)
9.73 (2H, brs, OH and NH). 13C NMR (DMSO-d6, 100 MHz):
d 34.1 (C-
and 9.70 (2H, brs, OH and NH). 13C NMR (DMSO-d6):
d
21.1 (Ar-CH3),
4), 36.6 (C-5), 55.6 (eOCH3), 98.7 (C-3), 101.0 (eOeCH2eOe), 107.8
(C-7),136.6 (C-3a),139.8 (C-7a),156.2 (C-6),108.2,114.4,120.0,126.8,
132.6, 132.8, 145.7, 147.3, 148.0, and 159.3 (Aromatic carbons). Mass
(Mþ): 362. Anal. Calculated for C21H18N2O4: C-69.60; H-5.01; N-
7.73%. Observed: C-69.41; H-5.58; N-7.16%.
21.2 (Ar-CH3), 33.7 (C-4), 36.5 (C-5), 98.4 (C-3), 114.0 (C-7), 136.9 (C-
3a), 140.5 (C-7a), 156.9 (C-6), 125.9, 127.3, 127.5, 129.2, 129.3, 135.6,
136.1, 136.4 and 141.2 (Aromatic carbons). Mass (Mþ): 316. Anal.
Calculated for C21H20N2O: C-79.72; H-6.37; N-8.85%. Observed: C-
79.44; H-6.49; N-8.56%.
3.4.11. 6-(4-Chlorophenyl)-4,5-dihydro-4-(4-methoxyphenyl)-2H-
indazol-3-ol (11b)
3.4.7. 4,5-Dihydro-4-(3,4,5-trimethoxyphenyl)-6-p-tolyl-2H-
indazol-3-ol (7b)
Greenish white solid. M.P.: 169 ꢂC. Yield: 87%. IR (ATR, cmꢀ1):
White solid. M.P.: 181 ꢂC. Yield: 78%. IR (ATR, cmꢀ1): 3312, 3211.
3315, 3191. 1H NMR (DMSO-d6, 400 MHz):
d 2.76 (1H, dd, H-5eq,
1H NMR (DMSO-d6, 400 MHz):
d
2.28 (3H, s, Ar-CH3), 2.81 (1H, dd,
J ¼ 3.6 and 16.8 Hz), 3.06 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and 16.8 Hz),
3.89 (3H, s, Ar-OCH3), 4.03 (1H, dd, 4-H, J ¼ 3.6 and 8.6 Hz), 6.71 (2H,
d, H-300 and H-500, J ¼ 8.8 Hz), 6.77 (1H, d, H-7, J ¼ 2.4 Hz), 6.98 (2H, d,
H-200 and H-600, J ¼ 8.8 Hz), 7.34 (2H, d, H-30 and H-50, J ¼ 8.8 Hz), 7.45
(2H, d, H-20 and H-60, J ¼ 8.8 Hz) and 9.68 (2H, brs, OH and NH). 13C
H-5eq, J ¼ 3.4 and 16.8 Hz), 3.09 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and
16.8 Hz), 3.88e3.97 (9H, 3s, Ar-OCH3), 4.09 (1H, dd, 4-H, J ¼ 3.4 and
8.6 Hz), 6.54 (1H, d, H-7, J ¼ 2.4 Hz), 6.69e6.79 (2H, m, H-200 and H-
600), 7.11 (2H, d, H-30 and H-50, J ¼ 8.0 Hz), 7.32 (2H, d, H-20 and H-60,
J ¼ 8.0 Hz) and 9.70 (2H, brs, OH and NH). 13C NMR (DMSO-d6,
NMR (DMSO-d6, 100 MHz):
d 33.5 (C-4), 36.6 (C-5), 55.4 (eOCH3),
100 MHz):
d
21.1 (Ar-CH3), 33.9 (C-4), 36.5 (C-5), 55.7 (eOCH3), 55.9
99.1 (C-3), 113.9 (C-7), 135.5 (C-3a), 139.4 (C-7a), 156.3 (C-6), 127.3,
128.2.128.9,129.3,132.4,134.5,144.5, and 157.9 (Aromatic carbons).
(eOCH3), 98.3 (C-3), 111.4 (C-7), 134.5 (C-3a), 137.5 (C-7a), 156.3

N.A. Shakil et al. / European Journal of Medicinal Chemistry 59 (2013) 120e131
129
Mass (Mþ): 352. Anal. Calculated for C20H17ClN2O2: C-68.09; H-4.86;
N-7.94%. Observed: C-67.92; H-4.91; N-7.26%.
4-H, J ¼ 3.2 and 8.4 Hz), 6.74 (1H, d, H-7, J ¼ 2.4), 7.10e7.46 (10H, m,
Aromatic protons) and 9.71 (2H, brs, OH and NH). 13C NMR (DMSO-
d6,100 MHz): d 34.6 (C-4), 36.7 (C-5), 98.7 (C-3),113.8 (C-7),136.6 (C-
3.4.12. 6-(3,4-Dichlorophenyl)-4,5-dihydro-4-(4-methoxyphenyl)-
2H-indazol-3-ol (12b)
3a), 140.6 (C-7a), 156.8 (C-6), 125.5, 126.3, 127.3, 127.9, 128.5, 129.0,
141.6 and 145.7 (Aromatic carbons). Mass (Mþ): 288. Anal. Calcu-
lated for C19H16N2O: C-79.14; H-5.59; N-9.72%. Observed: C-79.24;
H-6.01; N-9.43%.
Greenish white solid. M.P.: 173 ꢂC. Yield: 71%. IR (ATR, cmꢀ1):
3318, 3209. 1H NMR (DMSO-d6):
d
2.54 (1H, dd, H-5eq, J ¼ 3.6 and
17.2 Hz), 3.07 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and 17.2 Hz), 3.78 (3H, s, Ar-
OCH3), 4.07 (1H, dd, 4-H, J ¼ 3.6 and 8.6 Hz), 6.44 (1H, d, H-7, J ¼ 2.4),
6.73 (2H, d, H-300 and H-500, J ¼ 8.8 Hz), 6.99 (2H, d, H-200 and H-600,
J ¼ 8.8 Hz), 7.16 (1H, d, H-50, J ¼ 8.4 Hz), 7.33 (1H, dd, H-60, J ¼ 1.0 and
8.4 Hz), 7.51 (1H, d, H-20, J ¼ 1.0 Hz) and 9.75 (2H, brs, OH and NH).13C
3.5. In vitro anti-fungal activity (food poisoning method)
Phyto-pathogenic test fungi, Rhizoctonia solani ITCC 5563 and
Sclerotium rolfsii ITCC 6181 were procured from Indian Type Culture
Collection (ITCC) center, Division of Plant Pathology, Indian Agri-
cultural Research Institute, New Delhi-110 012, India. Cultures of
the test fungi were maintained on Potato Dextrose Agar (PDA) slant
at 27 ꢂC for at least 4e7 days and were sub-cultured in Petri dishes
prior to testing. The test fungi were routinely grown on fresh slant
tubes of PDA and stored at 4 ꢂC.
NMR (DMSO-d6,100 MHz): d 34.0 (C-4), 37.1 (C-5), 55.4 (-OCH3), 98.2
(C-3), 113.8 (C-7), 137.0 (C-3a), 139.9 (C-7a), 156.7 (C-6), 127.9, 128.5,
129.6, 130.3, 131.7, 132.6, 133.1, 133.4, 145.1, and 158.0 (Aromatic
carbons). Mass (Mþ): 386. Anal. Calculated for C20H16Cl2N2O2: C-
62.03; H-4.16; N-7.23%. Observed: C-61.76; H-5.01; N-7.21%.
3.4.13. 4,5-Dihydro-6-(3,4-dimethoxyphenyl)-4-(4-
methoxyphenyl)-2H-indazol-3-ol (13b)
The above synthesized compounds were tested for their ability to
inhibit soil borne pathogenic fungi against the standard fungicide
hexaconazole. The fungicidal activity of synthesized compounds
was evaluated at various concentrations by the poisoned food
technique using PDA (potato dextrose agar) media. The readymade
PDA medium (39 g) was suspended in distilled water (1000 mL) and
heated to boiling until completely dissolved. The medium and petri
dishes were autoclaved at 15 mm Hg for 30 min. These compounds
weretested at concentrations of 500, 250,125, 62.5, 31.25,15.63, 7.81
White solid. M.P.: 192 ꢂC. Yield: 90%. IR (ATR, cmꢀ1): 3324, 3217.
1H NMR (DMSO-d6, 400 MHz):
d
2.79 (1H, dd, H-5eq, J ¼ 3.2 and
16.8 Hz), 3.13 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and 16.8 Hz), 3.73e3.79
(9H, 3s, Ar-OCH3), 4.09 (1H, dd, 4-H, J ¼ 3.2 and 8.6 Hz), 6.56 (1H, d,
H-7, J ¼ 2.4), 6.83 (1H, d, H-50, J ¼ 8.8 Hz), 6.94 (2H, d, H-300 and H-
500, J ¼ 8.8 Hz), 7.24 (1H, d, H-20, J ¼ 2.2 Hz), 7.29 (1H, dd, H-60, J ¼ 2.2
and 8.8 Hz), 7.31 (2H, d, H-200 and H-600, J ¼ 8.8 Hz) and 9.73 (2H, brs,
OH and NH). 13C NMR (DMSO-d6, 100 MHz):
d 34.4 (C-4), 36.9 (C-5),
and 3.90 m m
g mLꢀ1. A stock solution of 5000 g mLꢀ1 was prepared in
55.5 (eOCH3), 55.9 (eOCH3), 56.1 (eOCH3), 98.6 (C-3), 112.2 (C-7),
136.5 (C-3a), 138.4 (C-7a), 156.4 (C-6), 111.4, 114.3, 119.0, 126.4,
127.3. 127.7, 133.2, 148.3, 148.8, and 158.2 (Aromatic carbons). Mass
(Mþ): 378. Anal. Calculated for C22H22N2O4: C-69.83; H-5.89; N-
7.40%. Observed: C-69.64; H-6.03; N-7.82%.
Acetone or DMSO. Acetone or DMSO was used as a control. These
solutions were added to the media (50 mL) in conical flasks to obtain
the desired concentrations of the test compounds in the media. The
medium was poured into a set of two petri dishes (90 cm in diam-
eter) under aseptic conditions in a laminar flow hood. The plates
were kept in the laminar flow chamber for solidification of the
media. After solidification, a 5 mm (diameter) mycelia plug cut from
the actively growing front of a 2 week old colony of the desired
pathogenic fungus was thenplaced with the inoculums side down in
the center of each treatment plate, aseptically. Treated petri dishes
were then incubated at 27 ꢂC till the fungal growth was almost
complete in the control plates. All experiments were in quadruplet
for each treatment against each fungus.
3.4.14. 6-(4-Fluorophenyl)-4,5-dihydro-4-(3-nitrophenyl)-2H-
indazol-3-ol (14b)
White solid. M.P.: 169 ꢂC. Yield: 89%. IR (ATR, cmꢀ1): 3305, 3201.
1H NMR (DMSO-d6, 400 MHz):
d
2.72 (1H, dd, H-5eq, J ¼ 3.6 and
17.2 Hz), 3.15 (1H, ddd, H-5ax, J ¼ 2.4, 8.4 and 17.2 Hz), 4.21 (1H, dd,
4-H, J ¼ 3.2 and 8.4 Hz), 6.75 (1H, d, H-7, J ¼ 2.4), 6.79 (2H, d, H-30
and H-50, J ¼ 7.2 Hz), 7.08e7.15 (4H, m, H-20, H-60, H-500 and H-600),
7.37e7.41 (2H, m, H-200 and H-400) and 9.70 (2H, brs, OH and NH). 13C
NMR (DMSO-d6, 100 MHz): d 31.5 (C-4), 35.3 (C-5), 97.3 (C-3), 115.7
3.5.1. Recording of observations
(C-7), 136.9 (C-3a), 141.6 (C-7a), 155.7 (C-6), 115.9, 125.5, 127.3,
127.4, 128.4, 129.9, 132.4, 135.5, 160.8 and 162.4 (Aromatic carbons).
Mass (Mþ): 351. Anal. Calculated for C19H14FN3O3: C-64.95; H-4.02;
N-11.61%. Observed: C-65.17; H-4.61; N-11.03%.
The mycelia growth of fungus (mm) in both treated (T) and
control (C) petri dishes was measured diametrically. The mean and
standard errors were calculated from the four replicates of each
treatment and the percentage inhibition of growth (I) was calcu-
lated using the following formula
3.4.15. 6-(4-Fluorophenyl)-4,5-dihydro-4-phenyl-2H-indazol-3-ol
(15b)
Inhibition ð% IÞ : ðC ꢀ TÞ ꢁ 100=C
Pale white solid. M.P.: 220 ꢂC. Yield: 84%. IR (ATR, cmꢀ1): 3311,
3209. 1H NMR (DMSO-d6, 400 MHz):
d
2.80 (1H, dd, H-5eq, J ¼ 3.6
3.5.2. Calculation of LC50 values
and 17.2 Hz), 3.11 (1H, ddd, H-5ax, J ¼ 2.4, 8.6 and 17.4 Hz), 4.17 (1H,
dd, H-4, J ¼ 3.6 and 8.6 Hz), 6.72 (1H, d, H-7, J ¼ 2.4), 7.07e7.18 (7H, m,
H-30, H-50, H-200,-600), 7.46 (2H, d, H-20 and H-60, J ¼ 8.4 Hz) and 9.70
For calculation of LC50 values (lethal concentration for 50%
inhibition; m
g mLꢀ1), the percent inhibition was converted to cor-
rected percent inhibition by using Abbott’s formula:
Corrected inhibition ð%Þ : ðI ꢀ CFÞ ꢁ 100=ð100 ꢀ CFÞ
where CF is the correction factor obtained by the equation:
Correction factor ðCFÞ : ð9 ꢀ CÞ ꢁ 100=C
(2H, brs, OH and NH). 13C NMR (DMSO-d6, 100 MHz):
d 34.3 (C-4),
36.6 (C-5), 98.3 (C-3),115.7 (C-7),136.9 (C-3a),140.3 (C-7a),157.2 (C-
6), 125.9, 126.5, 127.4, 128.6, 132.0, 141.7, 145.5, and 161.4 (Aromatic
carbons). Mass (Mþ): 306. Anal. Calculated for C19H15FN2O: C-74.5;
H-4.94; N-9.14%. Observed: C-74.32; H-5.35; N-9.26%.
3.4.16. 4,5-Dihydro-4,6-diphenyl-2H-indazol-3-ol (16b)
White solid. M.P.: 215 ꢂC. Yield: 93%. IR (ATR, cmꢀ1): 3321, 3212.
where 9 is the diameter of the petri dish in cm and C is the diameter
1H NMR (DMSO-d6, 400 MHz):
16.8 Hz), 3.15 (1H, ddd, H-5ax, J ¼ 2.4, 8.4 and 16.6 Hz), 4.16 (1H, dd,
d
2.86 (1H, dd, H-5eq, J ¼ 3.2 and
of growth of the fungus in control plates. From the concentration
(m
g mLꢀ1) and corresponding corrected percentage inhibition data

130
N.A. Shakil et al. / European Journal of Medicinal Chemistry 59 (2013) 120e131
of each compound, the LC50
(m
g mLꢀ1) values were calculated
contents were shaken vigorously. The tubes were allowed to stand
statistically by computer programme (Indostat Services, Hyder-
abad) on a personal computer.
at room temperature for 20 min in the dark. The control was
prepared as above without any compound (only DMSO), and
methanol was used for the baseline correction. The absorbance of
the samples was measured at 517 nm. Radical scavenging activity
was calculated using the following formula:
3.6. In vitro anti-bacterial activity (disk diffusion method)
Two phytopathgenic bacteria Xanthomonas oryzae ITCC B-47 and
Erwinia crysanthemi ITCC B-40 were procured from Indian Type
Culture Collection (ITCC) center, Division of Plant Pathology, Indian
Agricultural Research Institute, New Delhi-110 012, India. Two
beneficial bacteria Bacillus thuringiensis MTCC 6941 and Bacillus
pumilis MTCC 2466 and three human pathogens Staphylococcus
aureus MTCC 3160, Pseudomonas aeruginosa MTCC 2581 and Kleb-
siella pneumoneae MTCC 7028 were procured from Microbial Type
Culture Collection and gene bank, Institute of Microbial Technology,
sector-39A, Chandigarh 110 036, India. Cultures of the test bacteria
were maintained on nutrient Agar (NA) slant at 37 ꢂC for 1e3 days,
depending upon the growth period of bacteria, and were sub-
cultured in nutrient broath culture tubes prior to testing. The test
bacteria were routinely grown on fresh slant tubes of NA and stored
at 4 ꢂC.
Percent radical scavenging activity ¼ðControlODꢀsampleODÞ=
ControlODꢁ100:
4. Conclusion
We report the green synthesis of chalcone, cyclohexenone and
indazole derivatives (sixteen each), using microwave irradiation for
activation under solvent less condition, their characterization and
bio-efficacy evaluation. Microwave heating, which was used for
synthesis, showed several advantages viz., solvent less reaction,
reduced reaction time (from hours to minutes), increased yield (3e
16%) and high purity. Biological activity data showed promising
results as the compounds were very selective in nature and results
for some compounds are very much comparable with the control/
standard used. The microwave method, being very simple and cost
effective, can be used for the synthesis of more potent analogues of
mentioned compounds that too in large (gram) quantities.
All the synthesized compounds were tested for their ability to
inhibit above mentioned bacteria against the standard antibiotics
Streptomycin, Ampicillin and Kanamycin. The concentrations of the
latter were used as recommended manufacturer. The anti-bacterial
activity of synthesized compounds was evaluated at single
Acknowledgment
concentration (100 mg/disc) by disk diffusion technique using NA
(Nutrient agar) media. The readymade NA medium (37 g) was
suspended in distilled water (1000 mL) and heated to boiling until
completely dissolved. The medium and petri dishes were auto-
The authors thank the Head, Division of Agricultural Chemicals,
IARI, for carrying out the research work. Author (MKS) is thankful to
Indian Council of Agricultural Research, New Delhi, India for
financial support. The assistance provided by Deepika parmar and
Neetika Parmar is acknowledged.
claved at 15 mm Hg for 30 min. A stock solution of 20,000
for all the compounds was prepared in DMSO. DMSO (5
m
m
g mLꢀ1
L) was
used as control. The medium (25 mL) was poured into petri dishes
(90 cm in diameter) under aseptic conditions in a laminar flow
hood. The plates were kept in the laminar flow chamber for
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