Efficient synthesis and biological evaluation of new benzopyran-annulated pyrano[2,3-c]pyrazole derivatives

Article abstract of DOI:10.1007/s11030-017-9734-y

Abstract: A one-pot method has been described to synthesize benzopyran-annulated pyrano[2,3-c]pyrazoles, effectively by combining O-alkenyloxy/alkynyloxy-acetophenones with various pyrazolones in triethylammonium acetate (TEAA) under microwave irradiation. While combination of O-allyloxy- or O-prenyloxy-acetophenones with pyrazolones occurred efficiently, that of O-propargyloxy-acetophenones was found effective in the presence of ZnO catalyst, via a domino Knoevenagel–hetero-Diels–Alder (DKHDA) reaction. Aminobenzopyran frameworks were also synthesized, after nitro-containing products were reduced in tandem with iron(II) in an acidic medium. The in vitro antiproliferative activity of these compounds was measured and discussed against gram-positive, gram-negative and M. tuberculosis bacteria, fungi, and various representative human solid tumor cell lines, in addition to their ferric reducing antioxidant capability.

Full text of DOI:10.1007/s11030-017-9734-y

Mol Divers
DOI 10.1007/s11030-017-9734-y
ORIGINAL ARTICLE
Efficient synthesis and biological evaluation of new
benzopyran-annulated pyrano[2,3-c]pyrazole derivatives
Balvantsingh M. Labana1 · Gaurangkumar C. Brahmbhatt1 · Tushar R. Sutariya1 ·
Narsidas J. Parmar1 · José M. Padrón2 · Rajni Kant3 · Vivek K. Gupta3
Received: 30 March 2016 / Accepted: 20 February 2017
© Springer International Publishing Switzerland 2017
Abstract A one-pot method has been described to synthe-
size benzopyran-annulated pyrano[2,3-c]pyrazoles, effec-
Alder (DKHDA) reaction. Aminobenzopyran frameworks
were also synthesized, after nitro-containing products were
tivelybycombining O-alkenyloxy/alkynyloxy-acetophenones reduced in tandem with iron(II) in an acidic medium. The in
with
various
pyrazolones
in
triethylammonium
vitro antiproliferative activity of these compounds was mea-
sured and discussed against gram-positive, gram-negative
and M. tuberculosis bacteria, fungi, and various represen-
tative human solid tumor cell lines, in addition to their ferric
reducing antioxidant capability.
acetate (TEAA) under microwave irradiation. While combi-
nation of O-allyloxy- or O-prenyloxy-acetophenones with
pyrazolones occurred efficiently, that of O-propargyloxy-
acetophenones was found effective in the presence of
ZnO catalyst, via a domino Knoevenagel–hetero-Diels–
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-017-9734-y) contains supplementary
material, which is available to authorized users.
Narsidas J. Parmar
njpchemdeptspu@yahoo.co.in
B
1
Department of Chemistry, Sardar Patel University, Vallabh
Vidyanagar, Dist. Anand, Gujarat 388120, India
2
BioLab, Instituto Universitario de Bio-Orgánica “Antonio
González” (IUBO-AG), Centro de Investigaciones
Biomédicas de Canarias (CIBICAN), Universidad de La
Laguna, C/ Astrofísico Francisco Sánchez 2, 38206 La
Laguna, Spain
3
Post-Graduate Department of Physics, University of Jammu,
Jammu Tawi 180006, India
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Graphical Abstract
Anticancer
Antioxidant
NO2
O2N
O
NO2
GI50 = 3.7-36 µM
FRAP value =
O2N
330-340 mM/100 g
O
O
H
O
H
N
H
N
N
N
N
N
O
O
Cl
O
Cl
H
N
N
6pc
6pc
O
6od
6ob
Cl
Cl
Cl
GI50 = 7.4-20µM
MIC = 62.5 µg/mL
GI50 = 3.3-6.2 µM
NO2
O2N
Inhibition = 92-93%
O
O
H
N
N
N
O
N
O
7pa
6oa
MIC = 62.5 µg/mL
Antitubercular
Antimicrobial
of such valuable compounds (i.e., isolation of taxol from
treebarks). The use of complicated, lengthy and expensive
procedures to isolate biomolecules from natural sources has
been another drawback. Synthetic chemists are therefore
inspired to develop molecular frameworks, analogous either
totally or partially to that of biomolecules, that can be syn-
thetically accessed.
Keywords DKHDA reaction · Ketone-based DKHDA
substrates · Benzopyran · Pyrano[2,3-c]pyrazole ·
Antiproliferative activity
Introduction
(Benzo) pyrano-fused heterocycles are interesting molec-
ular frameworks, constituting central skeletons of many
bioactive natural and unnatural products with potential appli-
cations in medicinal chemistry. Particularly, pyrano[2,3-
c]pyrazole derivatives have been found to exhibit antimi-
crobial [6], insecticidal [7], anti-inflammatory [8] and mol-
luscidal [9] activities. Moreover, some of these analogs have
shown promising human Chk1 kinase growth inhibition [10].
The development of new biomolecules of therapeutic inter-
est by searching and optimizing a simple, environmentally
benign and established synthetic route has been an inter-
esting part of contemporary medicinal chemistry research
[1–5]. Though nature is the best source of biomolecules,
its inadequacy in supplying desired quantities of material
sometimes becomes a limiting factor in the widespread use
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Considerable amount of work has been carried out in
microwave irradiation. All nitro-containing products were
reduced in tandem to access corresponding amino deriva-
tives. All final products were screened in vitro for their
antioxidant and antiproliferative activities.
the synthetic development of polyheterocycles, envisaging
the pyran fusion with a heterocyclic unit [11–13]. Benzopy-
rans, which exemplify the fusion of pyran with carbocyclics,
are useful materials for the development of photochromic
devices, such as data storage, optical switches, displays, sen-
sor protectors, waveguides and ophthalmic plastic lenses
[14] and [15]. Furthermore, aminobenzopyran is part of
many medicinally important compounds, such as antiangio-
genic 4-(N-imidazol-2-ylmethyl)-aminobenzopyran analogs
Results and discussion
Chemistry
[16], anti-ischemic ATP-sensitive potassium channel (KATP
)
Scheme 1 represents synthetic strategies for nitro-substituted
chromeno [4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazoles 5–7, and also
for respective amino products 8–9 derived from 5–6, start-
ing with five different pyrazolone units used in combination
with different O-alkenyloxy/alkynyloxy-acetophenones 2–
4. Nitro products 5–6 thus obtained were also treated further
with Fe/HCl in tandem to afford corresponding amino
analogs 8–9. Thus, ketone-based substrates were assembled
efficiently with pyrazolones by DKHDA strategy. The cen-
tral pyrano-pyranyl framework obtained here is relatively
more rigid (less flexible) than the one derived from using
an aldehyde substrate. All ketone-derived products are thus
anticipated to show improved receptor binding and biological
properties.
Reacting 5-pyrazolone 1 with nitroacetophenone 2 or 3
in TEAA under microwave irradiation at 460 W for 10 min
gave nitrobenzopyran-annulated pyrano[2,3-c]pyrazoles 5–
6. In the same way, polyheterocycles 7 were synthesized,
after heating nitroacetophenone 4 and 5-pyrazolone 1 in 25
mol% of ZnO. Finally, aminopolyheterocycles 8 and 9 were
alsosynthesizedbyreducingnitroproducts 5 and6inethanol
withironfilingsinthepresenceofafewdropsofconcentrated
HCl under refluxing water. Nitro-containing products 5oc,
5pc, 6oc and 6pc can also be synthesized using a solvent-
free tetrabutylammonium hydrogen sulfate medium [24].
The structure of all products was confirmed on the basis of
the respective spectral data. A singlet in the δ 1.67–1.84ppm
range in the 1H NMR spectra, as well as a signal in the δ 22–
28ppm range in 13C NMR spectra, is attributed to the methyl
bridgehead in all the products. Heterocycles 5 and 8 derived
from allyl substrates showed a ring-junction CH proton as
multiplets in the δ 2.10–2.29ppm range. This proton showed
a similar multiplate in the δ 2.0–2.2ppm range in hetero-
cycles 6 derived from prenyl substrates, with J = 4.0Hz.
Compound 7 did not show this signal due to the absence of
this proton. Instead, it gave characteristic doublets of pyranyl
O–CH protons in the δ 6.55–6.57ppm range. A signal in the
δ 34–38ppm range in the 13C NMR spectra is attributed to
a ring-junction tertiary carbon. The cis relationship between
bridgehead CH and Me protons was also established on the
basis of the single- crystal X-ray diffraction data of 5pa
(Fig. 2). Pyrazolone ring methyl protons resonate as a sin-
glet in the δ 2.39–2.79ppm range. A broad singlet in the
opener N-(6-aminobenzopyranyl)-N-benzyl-Nꢀ-cyanoguan
idine analogs [17], insulin-releasing process enhancer
aminobenzopyran derivatives [18] and potential Mycobac-
terium bovis BCG antagonists [19].
As complex fused-ring systems, many polycyclic com-
pounds bear an angular shape with a bridgehead methyl
group, representing common structural features of number
of bioactive compounds. Naturally occurring cannabicyclols,
mahanimbine and steroids are examples of this class, bear-
ing angular-shaped rigid frameworks. Thyrsiferyl 23-acetate
(TF23A), a potential cytotoxic agent with a specific ser-
ine/threonine protein phosphatase (PP2A) inhibitory action,
has similar angular molecular frameworks [20]. Medicinally
important heterosteroids are other potential candidates of this
class [21]. In view of this, angular molecular framework is
worth studying to accessing important new biomaterials.
We have developed a conventional synthesis [22] and [23]
as well as tetrabutylammonium hydrogen sulfate [24], ionic
liquid [25–28] and glycerol mediated [29] synthetic routes to
afford polyheterocycles of biological interest. In the present
work, we describe the synthesis of benzopyran-annulated
pyrano[2,3-c]pyrazoles, all of which contain angularly fused
central pyranyl rings (Fig. 1), via a domino Knoevenagel–
hetero-Diels–Alder (DKHDA) reaction. The reaction was
studied under different conditions to optimize the reac-
tion output. The desired conversion took place optimally
while heating in triethylammonium acetate (TEAA) under
Fig. 1 Benzopyran-annulated pyrano[2,3-c]pyrazole framework with
a methyl bridgehead
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Scheme 1 Synthesis of nitro (5–7) and amino (8–9) derivatives of chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazoles 5–9. TEAA triethylammonium acetate,
MW microwave
δ 4.76–4.94ppm range can be attributed to amino group
protons in compounds 8–9. All aromatic protons showed
1H NMR signals in the δ 6.35–8.49ppm range. An FTIR
band in the 1200–1240cm−1 range can be attributed to the
pyranyl cyclic ether linkage. Characteristic bands of amino
groups were clearly detected in the 3480–3430 and 3380–
3330cm−1 ranges. Finally, a band in the 1575–1630cm−1
range can be assigned to V sp2 C–N functionality.
and 3. Many of the compounds displayed better bioactiv-
ity than standard reference drugs (Table 1). The majority
of nitro compounds 5–7 have good antibacterial activity,
as their antifungal test results are very poor except for few
cases where griseofulvin-equivalent antifungal activity was
found against C. albicans fungi. Further analysis of antibac-
terial results revealed most of the compounds active against
gram-positive B. subtilis and C. tetani bacteria. While com-
pounds 5oa, 5oc, 5pb, 6ob, 6od, 6pa and 6pc were ampicillin
equivalent against B. Subtilis bacteria, compounds 5pc, 6oc,
6pb, 6pd, 7oa and 7pb were higher in potency than ampi-
cillin against the same bacterial type. Candidates 5pd, 7ob
and 7pa that revealed similar potency against same bacteria
have recorded MIC values close to that of norfloxacin. On the
other hand, compound 5pa resembled both the standard ref-
Biological studies
The biological evaluation of all the synthesized compounds
was carried out by screening them against different bacteria,
fungi and human solid tumor cell lines as listed in Tables 2
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Fig. 2 ORTEP view of compound 5pa and packing arrangement
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Table 1 Antimicrobial, anti-tubercular and antioxidant activities of 5oa–oc, 5pa–pd, 6oa–od, 6pa–pd, 7oa–ob and 7pa–pb
Compound
Antimicrobial activity (MIC µg/mL)
Gram +ve bacteria Gram –ve bacteria
Anti-TBa activity
(% inhibition)
Antioxidant activityb
FRAP valuec
Fungi
B.S.
C.T.
S.P.
E.C.
S.T.
V.C.
A.F.
C.A.
5oa
5ob
5oc
5pa
5pb
5pc
5pd
6oa
6ob
6oc
6od
6pa
6pb
6pc
6pd
7oa
7ob
7pa
7pb
[A]
[B]
250
500
250
62.5
250
200
125
500
250
200
250
250
200
250
200
200
125
100
200
250
50
250
250
200
125
100
100
200
62.5
200
250
200
100
250
200
250
250
250
100
125
250
50
200
200
100
100
250
200
100
250
250
125
500
250
500
500
200
250
250
500
250
100
50
125
100
250
250
250
200
500
250
62.5
200
500
200
500
125
125
250
100
500
100
100
50
200
250
500
250
250
250
500
250
100
250
500
200
500
200
200
500
100
500
200
100
50
250
250
200
100
100
125
250
100
125
500
250
125
250
250
200
250
200
200
250
100
50
>500
>500
250
>500
>500
>500
500
>500
500
500
500
200
>500
>500
>500
>500
500
>500
250
–
>500
500
91%
78%
38%
74%
14%
36%
12%
92%
57%
39%
23%
46%
25%
84%
88%
56%
87%
93%
12%
–
220.5
254.2
287.3
246.9
263.6
234.5
219.1
237.1
339.5
219.3
310.4
247.5
288.7
331.7
202.4
220.4
214.8
217.8
253.0
>500
200
>500
>500
>500
>500
>500
500
>500
>500
>500
250
500
>500
500
500
>500
–
–
–
–
[C]
50
100
50
50
25
25
25
–
–
–
[D]
[E]
100
–
10
10
10
10
–
–
–
–
–
–
–
–
100
100
–
100
–
[F]
–
–
–
–
–
–
500
–
[G]
–
–
–
–
–
–
–
99%
These bold values indicate they are either near to 10 or not too much far from that at least one of the reference drugs has registered
S.P. Streptococcus pneumoniae, C.T. Clostridium tetani, B.S. Bacillus subtilis, S.T. Salmonella typhi , V.C. Vibrio coleare, E.C . Escherichia coli,
A.F. Aspergillus fumigates, C.A. Candida albicans, [A] Ampicillin, [B] Chloramphenicol, [C] Ciprofloxacin, [D] Norfloxacin, [E] Nystatin, [F]
Griseofulvin, [G] Isoniazide
a Concentration of compounds used against M. tuberculosis H37RV bacteria = 250 µg/mL, standard antimicrobials used: Isoniazide (0.20 µg/mL)
b
Concentration of compounds = 200µg/mL and standard: A.A. (ascorbic acid) = 176µg/mL.
A.A. mM/100 g sample
c
erence drugs chloramphenicol and ciprofloxacin in activity
against Bacillus subtilis bacteria. Compounds 5oa, 5ob, 6pb,
6pd, 7oa and 7ob are as potent as ampicillin reference drug,
against C. tetani bacteria. Compounds 5oc, 5pd, 6ob, 6od
and 6pc, on the other hand, are more potent than ampicillin
againstthesamebacterialstrain. Inthesameway, compounds
5pa, 5pb, 5pc, 6pa, 7pa and 7pb exhibited activity com-
parable to or more potent than reference drug ciprofloxacin.
Finally, compound 6oa resembles two reference drugs, chlo-
ramphenicol and norfloxacin, in activity which is better than
ampicillin. Screening results against gram-negative E. coli
bacteria showed majority of compounds ampicillin equiva-
lent in the activity. A similar trend was observed for most
of these compounds against V. cholerae bacteria. For exam-
ple, 6oa–oc, 5pa–pc and 6pa exhibited ampicillin-equivalent
activity. Only two candidates revealed ampicillin-equivalent
activity against gram-negative S. typhi bacteria, such as 6ob
and 7ob.
Amino products 8–9 derived from nitro compounds 5–6
were anticipated to have new bioprofiles. In many cases, the
activity of nitro products (Table 2) was found better than their
correspondingaminoproducts. Forexample, 8oband9pbare
better than corresponding 5ob and 6ob derivatives in activity
against four bacterial strains. Same was the observation for
compounds 8ob, 8oc, 8pa, 8pc, 8pd, 9oa, 9oc, 9od 9pa and
9pb derived from nitro derivatives 5ob, 5oc, 5pa, 5pc, 5pd,
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Table 2 Antimicrobial, anti-tubercular and antioxidant activities of 8oa–oc, 8pa–pb, 9oa–od and 9pa–pd (MIC, µg/mL)
Compound
Antimicrobial activity (MIC µg/mL)
Gram-positive bacteria Gram-negative bacteria
Anti-TBa
Inhibition
%
Antioxidant activityb
FRAP valuec
Fungi
B.s.
C.t.
S.p.
E.c.
S.t.
V.c.
A.f.
C.a.
8oa
8ob
8oc
8pa
8pb
8pc
8pd
9oa
9ob
9oc
9od
9pa
9pb
9pc
9pd
[A]
[B]
250
250
200
250
200
250
250
200
125
100
125
200
200
500
500
250
50
125
250
200
250
100
125
250
62.5
125
250
200
250
200
100
250
250
50
250
250
100
250
100
250
250
250
250
200
100
250
200
500
200
100
50
250
62.5
200
200
250
100
250
100
250
200
100
125
250
125
250
100
50
250
100
100
200
500
125
200
125
200
200
250
100
250
200
250
100
50
250
125
250
200
250
100
250
100
125
250
250
100
250
200
125
100
50
500
>500
>500
>500
500
200
250
>500
>500
>500
500
200
>500
>500
>500
–
>500
500
500
>500
>500
>500
>500
250
>500
200
250
>500
500
500
250
–
23%
55%
47%
62%
34%
62%
41%
35%
84%
35%
68%
58%
36%
58%
77%
–
263.3
213.1
200.4
201.0
217.0
254.8
250.2
263.8
243.1
218.8
223.9
202.8
224.1
252.2
243.5
–
–
–
[C]
50
100
50
50
25
25
25
–
–
–
[D]
[E]
100
–
10
10
10
10
–
–
–
–
–
–
–
–
100
100
–
100
500
–
–
[F]
–
–
–
–
–
–
–
[G]
–
–
–
–
–
–
99%
These bold values indicate they are either near to 10 or not too much far from that at least one of the reference drugs has registered
S.P. Streptococcus pneumoniae, C.T. Clostridium tetani, B.S. Bacillus subtilis, S.T.Salmonella typhi, V.C.Vibrio coleare, E.C. Escherichia coli,
A.F. Aspergillus fumigates, C.A. Candida albicans, [A] Ampicillin, [B] Chloramphenicol, [C] Ciprofloxacin, [D] Norfloxacin, [E] Nystatin, [F]
Griseofulvin, [G] Isoniazide
a Concentration of compounds used against M. tuberculosis H37RV bacteria = 250 µg/mL,standard antimicrobials used: Isoniazide (0.20 µg/mL).
bConcentration of compounds = 200µg/mL and standard: A.A. (ascorbic acid) = 176µg/mL
cA.A. mM/100 g sample
6oa, 6oc, 6od,6pa and 6pb against gram-negative E. coli
and S. typhi, and gram-positive B. subtilis bacteria. A similar
trend was seen for compounds 7oa–ob and 7pa–pb.
panel of six different human solid tumor cell lines: A549
(non-small cell lung), HBL-100 (breast), HeLa (cervix),
SW1573 (non-small cell lung), T-47D (breast) and WiDr
(colon). The in vitro antiproliferative activity study was car-
ried out after 48h of drug exposure to the cell line using
sulforhodamine B (SRB). According to NCI guidelines, only
compounds soluble in DMSO at 40mM concentration were
tested. The results are expressed in GI50 (50% growth inhibi-
tion) and shown in Table 3. From the set of tested compounds,
only compound 5pb was inactive against all tested cell lines.
The most active compound of the series was 6od, which
showed GI50 values in the 3.3–6.2µM range against all tested
cell lines. The results on the antiproliferative activity did not
provide clear structure–activity relationships. Overall, the
results show that nitro derivatives 5–6 are more active than
the corresponding amino analogs 8–9. However, the activity
The Lowenstein–Jensen (L–J) MIC method was used
to determine antitubercular activity of compounds at a
250µg/mL concentration against M. tuberculosis H37Rv
bacteria [30]. The screening test results are summarized
in Tables 1 and 2 as % growth inhibition relative to stan-
dard drugs. From the results, compounds 5oa, 6oa and
7pa showed excellent antitubercular activity with inhibition
growth recorded in the 90–100% range. On the other hand,
compounds 6pc, 6pd and 7ob showed moderate antituber-
cular activity with a percent inhibition growth around 90%.
Antiproliferative assays were performed in 96-well plate
using the National Cancer Institute (NCI) protocol [31] to
explore antitumor activity. For this study, we employed a
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Table 3 Antiproliferative
activity of selected candidates
Cell line (origin)
Compound
A549
HBL-100
HeLa
(Cervix)
SW1573
(Lung)
T-47D
(Breast)
WiDr
(Colon)
5–9 (GI50
)
(Lung)
(Breast)
5pa
5pb
6ob
6od
6pc
8oa
8pa
8pb
8pc
9pa
9pb
9pc
9pd
[A]
[B]
7.6 ( 1.8)
>100
31 ( 9.9)
>100
8.3b
3.9b
7.4b
7.4( 3.0)
>100
16 ( 0.6)
>100
5.9 ( 4.0)
>100
6.0 ( 0.5)
>100
7.9 ( 2.0)
4.5 ( 0.7)
7.9 ( 0.9)
35 ( 3.6)
28 ( 1.8)
34 ( 0.1)
27 ( 3.0)
34 ( 2.1)
30 ( 0.3)
60 ( 0.1)
33 ( 0.2)
–
4.8( 1.7)
3.5( 0.5)
11 ( 2.7)
27 ( 7.5)
12 ( 1.5)
21 ( 5.7)
19 ( 3.2)
25 ( 15)
19 ( 9.5)
27 ( 5.1)
27 ( 20)
2.0 ( 0.3)
3.3 ( 1.6)
0.6 ( 0.4)
36 ( 16)
6.2 ( 0.8)
9.6 ( 0.7)
35 ( 0.7)
16 ( 2.1)
35 ( 1.4)
28 ( 6.3)
36 ( 0.9)
29 ( 0.5)
45 ( 1.1)
43 ( 4.1)
3.0 ( 0.4)
14 ( 1.5)
0.25 ( 0.12)
3.7 ( 0.7)
3.3 ( 0.3)
20 ( 0.9)
33 ( 4.8)
23 ( 7.9)
30 ( 2.8)
26 ( 6.6)
31 ( 3.8)
23 ( 4.1)
38 ( 5.6)
32 ( 14)
15 ( 2.3)
22 ( 5.5)
2.0 (0.5)
4.0 ( 0.4)
4.5 ( 0.9)
13 ( 5.8)
47 ( 17)
31 ( 6.3)
48 ( 8.7)
28 ( 3.8)
42 ( 23)
26 ( 3.2)
57 ( 25)
37 ( 2.9)
26 ( 5.3)
23 ( 3.1)
1.8 ( 0.7)
36 ( 0.2)
21 ( 0.9)
37 ( 4.8)
19 ( 2.6)
52 ( 8.4)
31 ( 5.8)
35 ( 1.8)
39 ( 5.5)
1.9 ( 0.2)
1.4 ( 0.1)
0.23 ( 0.05)
–
[C]
–
These bold values indicate they are either near to 10 or not too much far from that at least one of the reference
drugs has registered
[A] Cisplatin, [B] Etoposide, [C] Camptothecin
a Values are given in µM and are means of two to four experiments; standard deviation is given in parentheses.
b
Only one experiment was done
is not correlated with the location (R2 or R3) of the nitro
group attached to the aromatic ring. Additionally, the effect
of substituent R1 on the modulation of the antiproliferative
activity is not clear.
For comparison, we have included the antiproliferative
data of the standard anticancer drugs cisplatin, etoposide and
camptothecin against the same panel of solid tumor cell lines.
When compared to the standard anticancer drugs, the lead
compound 6od showed similar activity profile. This is of
relevant interest in the drug-resistant cell lines T-47D and
WiDr.
Experimental
All microwave heating experiments were performed in a
closed system using a Catalyst Scientific Microwave Oven
CATA-R (2.45GHz, 140 to 700Watts) by CatalystTM Sys-
tems, Pune, India. All solvents and reagents were used as
supplied from commercial sources. Melting points were
determined on a TEMPO melting point apparatus in an open
capillary tube and are uncorrected. IR spectra were recorded
in KBr on a Shimadzu FTIR 8401 spectrometer and are
reported in wave numbers (cm−1). 1H NMR and 13C NMR
spectra were recorded on a Bruker Avance 400 spectrom-
1
eter operating at 400MHz for H NMR and 100MHz for
13C NMR using CDCl3 as solvent unless otherwise indi-
cated. Chemical shifts are reported as parts per million (ppm,
δ) and referenced to the residual protic solvent. Coupling
constants are reported in Hertz (Hz). Splitting patterns are
designated as s, singlet; d, doublet; t, triplet; q, quartet; br,
broad; m, multiplet. The degree of substitution (C, CH, CH2,
and CH3) was determined using the DEPT-135 method. ESI
mass spectra were measured on a Shimadzu LCMS-2010
spectrometer. Elemental analysis (% C, H, N) was carried
out using a Perkin-Elmer 2400 series-II elemental analyzer
(Perkin-Elmer, USA). TLCwasperformedonMerck60F254
pre-coated silica plates, and spots were detected either by UV
(254, 366nm) or dipping into a permanganate [KMnO4 (3 g),
Conclusions
In conclusion, we have demonstrated an efficient synthesis
of nitro benzopyrano-annulated pyrano[2,3-c]pyrazoles and
their analogous amino frameworks employing ionic liquid
TEAA as an efficient reaction medium under microwave
irradiation. The methodology is efficient, environmentally
benign and eco-friendly in nature. The majority of com-
pounds are poor in antifungal potential, against A. fumigates.
Compounds 5ob, 6oc, 6pd, 7ob, 7pc, 8ob, 8oc, 9pc and 9pb
registered griseofulvin-equivalent potency against a fungi C.
albicans. Compounds 5pa and 6od showed a promising anti-
cancer activity profile.
123

Mol Divers
K2CO3 (20 g), NaOH (5mL, 5% in H2O), H2O (300mL)] or
an anisaldehyde solution [3% p-methoxybenzaldehyde and
1%H2SO4 inMeOH]or2,4-dinitrophenylhydrazinesolution
[2,4-DNP (12g), conc. H2SO4 (6mL), water (8mL), EtOH
(20mL)] followed by heating.
Synthesisofamines8–9(reductionofnitrochromeno-
fused pyranopyrazoles)
In a round-bottom flask attached to a reflux condenser,
the corresponding nitro compound 5–6 (1 equiv.) was dis-
solved in water (10mL) containing 1–2 drops of concentrated
hydrochloric acid. Iron filings (6 equiv.) in ethanol were
added drop-wise. The mixture was refluxed for 1–2h. TLC
was used to monitor the reaction progress. After completion,
the reaction mixture was cooled to room temperature, filtered
and extracted with chloroform (3×5mL). The combined
chloroform extracts were dried over anhydrous MgSO4.
A solid was obtained upon removal of chloroform under
reduced pressure. Further purification of the products was
achieved through column chromatography using a 4:6 ethyl
acetate:n-hexane mixture as an eluent. Microwave irradiation
was also used to promote the reduction of a representative
of nitro products 6pa into amino product 9pa at 460W
(126◦C).
General procedures for the synthesis of O-allylated-
/prenylated-/propargylated acetophenones 2–4
2-Hydroxy-3/5-nitroacetophenone (1.0mmol) was added to
a suspension of anhydrous K2CO3 (1.5mmol) in DMF
(10mL), and the resulting mixture was stirred mechanically
for few minutes. A DMF solution of allyl-/prenyl-/propargyl
bromide (1.3mmol in 2mL of DMF) was then added drop-
wise to that resulted mixture. The mixture was stirred
mechanically at room temperature until the reaction was
complete as indicated and confirmed by TLC (8–15h). The
reaction mixture was then poured onto 100g of ice-cooled
water with constant stirring. Solid products 2p, 3p and 4o–
4p were isolated by filtration and liquid products 2o and 3o
by solvent extraction using three 25-mL anhydrous diethyl
ether portions. Combinedether extracts yielded colorless oily
products in the 93–97% range, after ether evaporation.
Spectroscopic data of selected products
1,11b-Dimethyl-8-nitro-3-phenyl-3,5a,6,11b-tetrahydro-
5H-chromeno[4,3ꢀ:4,5]pyrano[2,3-c]pyrazole (5oa)
White solid, yield 81%; m.p.: 162–164◦C; IR (KBr, cm−1):
2983, 2928, 1597, 1518, 1488, 1444, 1340, 1242, 1089, 1045,
831, 757, 695; 1H NMR (400MHz; CDCl3) 1.82 (s, 3H, CH3
attached to 3o ring juncture carbon), 2.28 (m, 1H, C-H5a),
2.60 (s, 3H, CH3 of pyrazolone), 4.26 (t, J = 11.2Hz, 1H,
H6), 4.44 (dd, J =12.4, J = 3.2Hz, 1H, H5), 4.57 (dd, J =
Synthesis of nitrochromeno-pyrano[2,3-c]pyrazoles
5–6
In a round-bottom flask, a mixture containing 5-pyrazolone
1a–d (3.0mmol, 0.522g of 1a or 0.624 of 1b or 0.729g
of 1c or 0.565g of 1d), O-allyl (0.529g) or O-prenyl-
hydroxyacetophenone (0.613g) (3.0mmol) and TEAA (0.75
mmol, 25 mol%) was heated under microwave irradiation
at 460W (126◦C) for 10min. The reaction was monitored
by TLC. After completion, the reaction mixture was poured
onto ice-cooled water. The crude solid products 5–6 were
collected quantitatively and purified further by column chro-
matography using a 2:8 ethyl acetate–n-hexane mixture as
an eluent.
ꢀ
11.6, J = 3.6Hz, 1H, H6 ), 4.70 (dd, J = 12.0, J = 3.2Hz,
1H, H5 ), 6.98–7.71 (m, 8H, Ar–H); 13C NMR (100 MHz;
ꢀ
CDCl3): 16.40 (CH3 of pyrazolone), 29.30 (CH3 attached to
3o ring juncture carbon), 34.38 (C-11b), 37.00 (C-5a), 63.77
(C-5), 68.64 (C-6), 102.05, 120.09, 120.77, 123.96, 125.98,
128.96, 132.09, 134.21, 138.17, 139.44, 145.22, 146.10,
148.45; ESI-MS (m/z): 378.43 [M+H+]; Anal Calcd for
C21H19N3O4: C, 66.83; H, 5.07; N, 11.13; Found: C, 66.68;
H, 5.19; N, 11.01.
General procedure for the synthesis of 7o–p
3-(2,5-Dichlorophenyl)-1,11b-dimethyl-8-nitro-3,5a,6,11b-
tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazole
(5oc)
A mixture containing 5-pyrazolone 1a–b (3.0mmol), O-
propargyl-hydroxyacetophenone 4o–p (3.0mmol), TEAA
(0.75mmol, 25%) and zinc oxide (0.75mmol, 25mol% in a
round bottom flask) was heated under microwave irradiation
at 460W (126◦C) for 10min. The reaction was monitored
by TLC. After completion, the reaction mixture was poured
onto ice-cooled water. The crude solid products 7oa–7ob and
7pa–7pb were collected quantitatively and purified further
by column chromatography using a 2:8 ethyl acetate–n-
hexane mixture as an eluent.
◦
Off-white solid, yield 61%; m.p.: 204–206 C; IR (KBr,
cm−1): 2968, 2934,1594, 1516, 1493, 1436, 1336, 1234,
1
1092, 1041, 864, 758, 687; H NMR (400MHz; CDCl3):
1.81(s, 3H, CH3 attached to 3o ring-junction carbon), 2.29
(m, 1H, H5a), 2.59 (s, 3H, CH3 of pyrazolone), 4.24 (m, 2H,
ꢀ
H5 and H6), 4.43 (dd, J = 11.2, J= 3.6Hz, 1H, H6 ), 4.58
ꢀ
(dd, J = 12.0,J = 2.8Hz, 1H, H5 ), 6.98–7.78 (m, 7H, Ar–H);
13C NMR (100MHz; CDCl3): 16.40 (CH3 of pyrazolone),
123

Mol Divers
29.62 (CH3 attached to 3o ring junction carbon), 34.09 (C-
11b), 38.04 (C-5a), 62.98 (C-5), 69.06 (C-6), 102.11, 117.10,
121.04, 127.90, 128.78, 129.48, 129.60, 129.68, 129.98,
130.98, 132.86, 136.24, 147.75, 149.40, 151.29 (Ar–C); ESI-
MS (m/z): 446.2 [M+H]+; Anal Calcd for C21H17Cl2N3O4:
C 56.52, H 3.84, N 9.42; Found: C 56.68, H 3.46, N 9.31.
1.84 (s, 3H, CH3 attached to 3o ring-junction carbon), 2.17
(m, 1H, H5a), 2.65 (s, 3H, CH3of pyrazolone), 4.20 (m, 2H,
ꢀ
H5 and H6), 4.37 (dd, J = 11.2, J = 3.6 Hz, 1H, H6 ), 4.58 (dd,
ꢀ
J = 12.0, J = 2.8 Hz, 1H, H5 ), 6.89–8.45 (m, 7H, Ar–H);
13C NMR (100MHz; CDCl3) 16.43 (CH3 of pyrazolone),
29.64 (CH3 attached to 3o ring junction carbon), 34.09 (C-
11b), 38.04 (C-5a), 62.98 (C-5), 69.06 (C-6), 102.11, 117.10,
121.04, 127.90, 128.78, 129.48, 129.60, 129.68, 129.98,
130.98, 132.86, 136.24, 147.75, 149.40, 151.29 (Ar-C); ESI-
MS (m/z): 446.1[M+H]+; Anal Calcd for C21H17Cl2N3O4:
C 56.52, H 3.84, N 9.42; Found: C 56.42, H 3.56, N 9.49.
1,11b-Dimethyl-10-nitro-3-phenyl-3,5a,6,11b-tetrahydro-
5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazole (5pa)
White solid, yield 75%; m.p.: 172–174◦C; IR (KBr, cm−1):
2983, 2928,1597, 1518, 1488, 1444, 1341, 1242, 1089, 1045,
831, 757, 695; 1H NMR (400MHz; CDCl3) 1.83 (s, 3H, CH3
attached to 3o ring juncture carbon), 2.26 (m, 1H, C–H5a),
2.66 (s, 3H, CH3 of pyrazolone), 4.21 (t, J = 10.4Hz, 1H,
H6), 4.38 (dd,J =12.0, J = 3.6Hz, 1H, H5), 4.56 (dd, J =
1,5,5,11b-Tetramethyl- 8-nitro-3-phenyl -3,5a,6,11b-
tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazole
(6oa)
ꢀ
11.2, J = 3.2Hz, 1H, H6 ), 4.63 (dd, J = 12.0, J = 3.2Hz,
1H, H5 ), 6.88–8.48 (m, 8H, Ar–H); 13C NMR(100MHz;
ꢀ
White solid, yield 78%; m.p.: 220–222◦C; IR (KBr, cm−1):
CDCl3) 16.34 (CH3 of pyrazolone), 29.38 (CH3 attached to
3o ring juncture carbon), 34.26 (C-11b), 37.18 (C-5a), 62.78
(C-5), 68.49 (C-6), 101.93, 117.87, 120.77, 123.77, 125.95,
126.36, 128.93, 129.51, 138.19, 141.73, 146.19, 148.29,
156.78 (Ar–C); ESI-MS (m/z): 378.2 [M+H+]; Anal Calcd
for C21H19N3O4: C, 66.83; H, 5.07; N, 11.13; Found: C,
66.68; H, 5.19; N, 11.01.
2976, 2931, 1586, 1515, 1436, 1339, 1245, 1043, 1034, 848,
1
758, 668; H NMR (400MHz; CDCl3) 1.10 (s, 3H, CH3
attached to 3o ring juncture carbon), 1.66 (s, 3H, CH3-5),
1.78 (s, 3H, CH3-5’), 2.02 (d, J = 3.6, 1H, H5a), 2.76 (s,
3H, CH3 of pyrazolone), 4.56 (d, J = 11.6, Hz, 1H, H6),
ꢀ
4.77 (dd, J = 12.8, J = 4.0, Hz, 1H, H6 ), 6.72–7.78 (m,
8H, Ar–H); 13C NMR (100MHz; CDCl3), 16.24 (CH3 of
pyrazolone), 22.13 (CH3 attached to 3o ring juncture car-
bon), 28.88 (CH3-5), 32.43 (CH3-5ꢀ), 34.06 (C-11b), 46.43
(C-5a), 62.65 (C-6), 82.64 (C-5), 101.56, 120.26, 120.85,
123.67, 125.79, 128.36, 131.94, 134.46, 138.33, 139.87,
145.46, 146.57, 148.94; ESI-MS (m/z): 406.12 [M+H+];
Anal Calcd for C23H23N3O4;Calcd: C, 68.13; H, 5.72; N,
10.36; Found: C, 68.51; H, 5.46; N, 10.12.
3-(3-Chlorophenyl)-1,11b-dimethyl-10-nitro-3,5a,6,11b-
tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5] pyrano[2,3-c] pyrazole
(5pb)
White solid, yield 79%; m.p.: 196–198◦C; IR (KBr, cm−1):
2978, 2923,1594, 1518, 1482, 1432, 1339, 1240, 1089, 1048,
1
849, 783, 742, 676; H NMR (400MHz; CDCl3) 1.81 (s,
3H, CH3 attached to 3o ring juncture carbon), 2.24 (m, 1H,
C-H5a), 2.65 (s, 3H, CH3 of pyrazolone), 4.22 (t, J = 11.2 Hz,
1H, H6), 4.38 (dd, J =12.4, J = 3.6 Hz, 1H, H5), 4.55 (dd, J
3-(2,5-Dichlorophenyl)- 1,5,5,11b -tetramethyl-8- nitro-3,
5a,6,11b- tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5] pyrano [2,3-
c]pyrazole (6oc)
ꢀ
= 12.0, J = 3.2 Hz, 1H, H6 ), 4.63 (dd, J = 12.0, J= 3.6 Hz,
1H, H5 ), 6.75–8.42 (m, 7H, Ar–H); 13C NMR (100MHz;
ꢀ
CDCl3) 16.10 (CH3 of pyrazolone), 29.07 (CH3 attached to
3o ring juncture carbon), 34.68 (C-11b), 37.86 (C-5a), 62.78
(C-5), 69.13 (C-6), 101.65, 116.78, 121.43, 124.02, 128.05,
129.21, 129.54, 129.79, 130.32, 131.54, 135.84, 141.13,
147.34, 149.43, 155.87; ESI-MS (m/z): 412.88 [M+H+];
Anal Calcd for C21H18ClN3O4:Calcd. C, 61.24; H, 4.41; N,
10.20; Found: C, 61.38; H, 4.52; N, 10.08.
Off-white solid, yield 68%; m.p.: 242–244◦C; IR (KBr,
cm−1): 2983, 2933, 1593, 1517, 1443, 1342, 1239, 1084,
1
1037, 858, 757, 679; H NMR (400MHz; CDCl3):1.07 (s,
3H, angular CH3), 1.66 (s, 3H, 5-CH3), 1.78 (s, 3H, 5’-
CH3), 1.98 (d, J= 3.6, 1H, 5a-H), 2.76 (s, 3H, pyrazolone
CH3), 4.57 (d, J= 10.8Hz, 1H, 6-H), 4.78 (dd, J= 12.4,
J= 4.0Hz, 1H, 6ꢀ-H), 6.73–7.84 (m, 6H, Ar–H); 13C NMR
(100MHz; CDCl3): 16.69 (pyrazolone CH3), 22.84 (angular
CH3), 29.46 (CH3-5), 32.63 (CH3-5ꢀ), 33.30 (C-11b), 46.84
(C-5a), 63.16 (C-6), 82.56 (C-5), 100.80, 120.53, 121.68,
121.99, 123.76, 125.68, 127.84, 129.60, 133.45, 134.79,
138.03, 139.15, 145.73, 146.90, 150.20 (Ar–C); ESI-MS
(m/z): 475.2 [M+H]+; Anal Calcd for C23H21Cl2N3O4: C
58.24, H 4.46, N 8.86; Found: C 58.36, H 4.53, N 8.49.
3-(2,5-Dichlorophenyl)-1,11b-dimethyl-10-nitro-3,5a,6,
11b-tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c] pyra-
zole (5pc)
Off-white solid, yield 68%; m.p.: 228–230◦C; IR (KBr,
cm−1): 2978, 2924,1594, 1517, 1488, 1432, 1339, 1240,
1089, 1048, 849, 783, 742, 676; 1H NMR(400MHz; CDCl3)
123

Mol Divers
1,5,5,11b-Tetramethyl-10-nitro-3-phenyl-3,5a,6,11b-
tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazole
(6pa)
bon), 29.65 (CH3-5), 32.06 (CH3-5ꢀ), 34.02 (C-11b), 46.65
(C-5a), 62.43 (C-6), 83.12 (C-5), 102.32, 117.43, 121.21,
127.97, 128.88, 129.15, 129.68, 129.93, 130.92, 131.16,
132.95, 136.54, 147.86, 149.67, 151.52 (Ar–C); ESI-MS
(m/z): 475.5 [M+H]+; Anal Calcd for C23H21Cl2N3O4: C
58.24, H 4.46, N 8.86; Found: C 58.59, H 4.59, N 8.76.
Off-white solid, yield 79%; m.p.: 232–234◦C; IR (KBr,
cm−1): 2981, 2929, 1598, 1512, 1444, 1339, 1250, 1089,
1
1041, 832, 756, 694; H NMR (400MHz; CDCl3) 1.08 (s,
3H, CH3 attached to 3o ring juncture carbon), 1.67 (s, 3H,
CH3-5), 1.79 (s, 3H, CH3-5ꢀ), 2.05 (d, J = 4.0, 1H, H5a),
2.79 (s, 3H, CH3 of pyrazolone), 4.58 (d, J = 13.2, Hz, 1H,
1,11b-Dimethyl-3 -phenyl-3,5a,6,11b-tetrahydro-5H-
chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazol-8-amine (8oa)
H6), 4.78 (dd, J = 13.0, J = 4.4, Hz, 1H, H6 ), 6.84–8.49
Pink solid, yield 74%; m.p.: 116–118◦C; IR (KBr, cm−1):
ꢀ
(m, 8H, Ar–H); 13C NMR (100MHz; CDCl3), 16.73 (CH3
of pyrazolone), 22.71 (CH3 attached to 3o ring juncture car-
bon), 29.12 (CH3-5), 32.03 (CH3-5ꢀ), 33.77 (C-11b), 46.31
(C-5a), 62.75 (C-6), 82.21 (C-5), 99.99, 116.94, 120.79,
123.74, 124.24, 125.71, 128.88, 129.88, 138.50, 141.73,
146.36, 148.00, 158.04; ESI-MS (m/z): 406.17[M+H+];
Found: 406.15 Anal Calcd for C23H23N3O4: Calcd.C, 68.13;
H, 5.72; N, 10.36; Found: C, 68.41; H, 5.40; N, 10.42.
3330, 3207, 2928, 1518, 1488, 1444, 1360, 1242, 1089,
1
1045, 831, 757, 695; H NMR(400MHz; DMSO-d6) 1.83
(s, 3H, CH3 attached to 3o ring juncture carbon), 2.28 (m,
1H, C-H5a), 2.60 (s, 3H, CH3 of pyrazolone), 4.26 (t, J
= 11.2 Hz, 1H, H6), 4.44 (dd,J =12.4, J = 3.2 Hz, 1H,
ꢀ
H5), 4.57 (dd, J = 11.6, J = 3.6 Hz, 1H, H6 ), 4.70 (dd,
ꢀ
J = 12.0, J = 3.2 Hz, 1H, H5 ), 4.73(s, 2H, NH2), 6.98–
7.71 (m, 8H, Ar–H); 13C NMR (100MHz; DMSO-d6) 16.40
(CH3 of pyrazolone), 29.30 (CH3 attached to 3o ring juncture
carbon), 34.38 (C-11b), 37.00 (C-5a), 63.77 (C-5), 68.64 (C-
6), 102.05, 120.09, 120.77, 123.96, 125.98, 128.96, 132.09,
134.21, 138.17, 139.44, 145.22, 146.10, 148.45; ESI-MS
(m/z): 348.28 [M+H+];AnalCalcdforC21H21N3O2:Calcd:
C, 72.60; H, 6.09; N, 12.10; Found: C, 72.68; H, 5.99; N,
12.19.
3-(3-Chlorophenyl)-1,5,5,11b-tetramethyl-10-nitro-3,5a,6,
11b-tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]
pyrazole (6pb)
White solid, yield 72%; m.p.: 208–210◦C; IR (KBr, cm−1):
2972, 2931, 1604, 1516, 1498, 1437, 1336, 1233, 1091, 1039,
853, 764, 683; 1H NMR (400MHz; CDCl3) 1.09 (s, 3H, CH3
attachedto3o ringjuncturecarbon), 1.67(s, 3H, CH3-5), 1.78
(s, 3H, CH3-5ꢀ), 2.01 (d, J = 4.0, 1H, H5a), 2.76 (s, 3H, CH3
of pyrazolone), 4.57 (d, J = 13.2, Hz, 1H, H6), 4.78 (dd,
3-(2,5- Dichlorophenyl)-1,11b-dimethyl-3,5a,6,11b-
tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazol-8-
amine (8oc)
ꢀ
J = 13.0, J = 4.4, Hz, 1H, H6 ), 6.78–8.46 (m, 7H, Ar-H);
13C NMR (100MHz; CDCl3), 17.13 (CH3 of pyrazolone),
22.56 (CH3 attached to 3o ring juncture carbon), 29.72 (CH3-
5), 32.73 (CH3-5ꢀ), 33.76 (C-11b), 46.62 (C-5a), 63.31 (C-6),
82.29 (C-5), 100.55, 117.13, 120.43, 123.87, 128.65, 129.28,
129.64, 129.81, 130.54, 131.73, 136.24, 141.26, 148.01,
149.77, 156.6; ESI-MS (m/z):440.89 [M+H+]; Anal Calcd
for C23H22ClN3O4: Calcd: C, 62.80; H, 5.04; N, 9.55;Found:
C, 62.59; H, 5.16; N, 9.49.
Yellow solid, yield 61%; m.p.: 167–168◦C IR (KBr, cm−1):
3334, 3210, 2931, 1518, 1436, 1360,1338, 1234, 1092, 1041,
863, 756, 684; 1H NMR (400MHz; DMSO-d6): 1.82 (s,
3H, CH3 attached to 3o ring junction carbon), 2.17 (m, 1H,
H5a), 2.59 (s, 3H, CH3 of pyrazolone), 4.26 (m, 2H, H5and
ꢀ
H6), 4.47 (dd, J = 11.2, J = 3.6Hz, 1H, H6 ), 4.55 (dd, J
ꢀ
= 12.0, J = 2.8 Hz, 1H, H5 ), 4.57 (s, 2H, NH2), 6.80–
7.52 (m, 7H, Ar–H); 13C NMR (100 MHz; DMSO-d6):16.40
(CH3 of pyrazolone), 29.62 (CH3 attached to 3o ring-junction
carbon), 34.09 (C-11b), 38.04 (C-5a), 62.98 (C-5), 69.06 (C-
6), 102.11, 117.10, 121.04, 127.90, 128.78, 129.48, 129.60,
129.68, 129.98, 130.98, 132.86, 136.24, 147.75, 149.40,
151.29 (Ar–C); ESI-MS (m/z): 416.2 [M+H]+; Anal Calcd
for C21H19Cl2N3O2: C 60.59, H 4.60, N 10.09; Found: C
60.34, H 4.86, N 10.30
3-(2,5-Dichlorophenyl)-1,5,5,11b-tetramethyl-10-nitro-3,
5a,6,11b-tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]
pyrazole (6pc)
White solid, yield 65%; m.p.: 130–132◦C; IR (KBr, cm−1):
2970, 2925, 1590, 1512, 1481, 1445, 1228, 1088, 1039, 851,
1
759, 675; H NMR (400MHz; CDCl3):1.08 (s, 3H, CH3
attached to 3o ring-junction carbon), 1.67 (s, 3H, CH3-5),
1.78 (s, 3H, CH3-5ꢀ), 1.96 (d, J= 3.2, 1H, H5a), 2.70 (s, 3H,
CH3 of pyrazolone), 4.41 (td, J= 12.4, J = 1.6Hz, 1H, H6),
1,11b-Dimethyl-3-phenyl-3,5a,6,11b-tetrahydro-5H-
chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazol-10-amine (8pa)
4.66 (ddd, J = 12.6, J = 4.2, J = 2.0Hz, 1H, H6 ), 6.69–7.56
Brown solid, yield 72%); m.p.: 118–120◦C; IR (KBr, cm−1):
ꢀ
(m, 7H, Ar–H); 13C NMR (100MHz; CDCl3): 16.77 (CH3
3454, 3336, 2971, 2925, 1632, 1594, 1578, 1521, 1497, 1443,
of pyrazolone), 22.53 (CH3 attached to 3o ring-junction car-
1227, 1095, 1049, 159,681; H NMR (400MHz; CDCl3)
1
123

Mol Divers
1.78 (s, 3H, CH3 attached to 3o ring juncture carbon), 2.12
(m, 1H, C-H5a), 2.64 (s, 3H, CH3 of pyrazolone), 4.28 (t,
J = 10.4 Hz, 1H, H6), 4.26 (dd,J =12.0, J = 3.6 Hz, 1H,
416.4[M+H]+; Anal Calcd for C21H19Cl2N3O2: C 60.59,
H 4.60, N 10.09; Found: C 60.68, H 5.02, N 10.28.
ꢀ
H5), 4.56 (dd, J = 11.2, J = 3.2 Hz, 1H, H6 ), 4.63 (dd,
1,5,5,11b-Tetramethyl-3-phenyl-3,5a,6,11b-tetrahydro-5H-
chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazol-8-amine (9oa)
ꢀ
J = 12.0, J = 3.2 Hz, 1H, H5 ), 4.77(s, 2H, NH2),6.50–
7.71 (m, 8H, Ar–H); 13C NMR (100 MHz; CDCl3) 16.53
(CH3 of pyrazolone), 29.44 (CH3 attached to 3o ring juncture
carbon), 34.26 (C-11b), 38.03(C-5a), 62.67 (C-5), 69.34 (C-
6), 103.18, 115.84,115.87, 117.66, 120.79, 125.72, 128.89,
129.89, 138.36, 139.95, 143.94, 146.41,148.70; ESI-MS
(m/z):348.53 [M+H+]; AnalCalcdforC21H21N3O2: Calcd:
C, 72.60; H, 6.09; N, 12.10; Found: C, 72.45; H, 6.54; N,
11.16.
Pink solid, yield 73%; m.p.: 115–117◦C; IR (KBr, cm−1):
3328,3211,2973, 2926,1486, 1440, 1338, 1241, 1089, 1032,
852, 758, 667; 1H NMR (400MHz; DMSO-d6) 1.10 (s, 3H,
CH3 attached to 3o ring juncture carbon), 1.66 (s, 3H, CH3-
5), 1.78 (s, 3H, CH3-5ꢀ), 2.02 (d, J = 3.6, 1H, H5a), 2.76
(s, 3H, CH3 of pyrazolone), 4.56 (d, J = 11.6Hz, 1H, H6),
ꢀ
4.77 (dd, J = 12.8, J = 4.0Hz, 1H, H6 ), 4.80 (s, 2H, NH2),
6.72–7.78 (m, 8H, Ar–H); 13C NMR (100MHz; DMSO-
d6), 16.24 (CH3 of pyrazolone), 22.13 (CH3 attached to 3o
ring juncture carbon), 28.88 (CH3-5), 32.43 (CH3-5ꢀ), 34.06
(C-11b), 46.43 (C-5a), 62.65 (C-6), 82.64 (C-5), 101.56,
120.26, 120.85, 123.67, 125.79, 128.36, 131.94, 134.46,
138.33, 139.87, 145.46, 146.57, 148.94; ESI-MS (m/z):
376.50 [M+H+]; Anal Calcd for C23H25N3O2: Calcd: C,
73.57; H, 6.71; N, 11.19; Found: C, 73.68; H, 6.34; N, 11.08.
3-(3-Chlorophenyl)-1,11b-dimethyl-3,5a,6,11b-tetrahydro-
5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazol-10-amine
(8pb)
Pink solid, yield 86%; m.p.: 110–112◦C; IR (KBr, cm−1):
3387, 3336, 2971, 2924, 1615, 1593, 1526, 1497, 1443,
1395, 1228, 1097, 1044, 865, 759, 645; 1H NMR (400MHz;
DMSO-d6) 1.67(s, 3H, CH3 attached to 3o ring juncture
carbon), 2.10 (m, 1H, C-H5a), 2.53 (s, 3H, CH3 of pyra-
zolone), 4.02 (t, J = 10.4Hz, 1H, H6), 4.36 (dd, J =12.0, J
3-(2,5-Dichlorophenyl)-1,5,5,11b-tetramethyl-3,5a,6,11b-
tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazol-8-
amine (9oc)
ꢀ
= 3.6Hz, 1H, H5), 4.58 (dd, J = 11.2, J = 3.2Hz, 1H, H6 ),
ꢀ
4.62 (dd, J = 12.0, J = 3.2Hz, 1H, H5 ), 4.94(s, 2H, NH2),
Pink solid, yield 69%; m.p.: 171–173◦C; IR (KBr, cm−1):
3334, 3210, 2976, 2934, 1465, 1445, 1340, 1249, 1085, 1031,
860, 754, 689; 1H NMR (400MHz; DMSO-d6): 1.09 (s, 3H,
angular CH3), 1.67 (s, 3 H, 5-CH3), 1.78 (s, 3H, 5ꢀ-CH3),
1.99 (d, J= 3.6, 1H, 5a-H), 2.77 (s, 3H, pyrazolone CH3),
4.57 (d, J= 10.8Hz, 1H, 6-H), 4.74 (s, 2H, NH2), 4.78 (dd,
J= 12.4, J= 4.0 Hz, 1 H, 6’-H), 6.73–7.86 (m, 6H, Ar–
H); 13C NMR (100MHz; DMSO-d6): 16.71 (pyrazolone
CH3), 22.85 (angular CH3), 29.48 (CH3-5), 32.65 (CH3-
5ꢀ), 33.34 (C-11b), 46.89 (C-5a), 63.25 (C-6), 82.58 (C-5),
100.65, 120.61, 121.70, 121.86, 123.79, 125.70, 127.84,
129.65, 133.54, 134.85, 137.76, 139.40, 145.65, 146.56,
149.89 (Ar–C); ESI-MS (m/z): 444.3 [M+H]+; Anal Calcd
for C23H23Cl2N3O2: C 62.17, H 5.22, N 9.46; Found: C
62.26, H 5.18, N 9.57.
6.35–7.54 (m, 8H, Ar–H); 13C NMR(100MHz; DMSO-d6)
16.59 (CH3 of pyrazolone), 29.34 (CH3attached to 3o ring
juncture carbon), 34.31 (C-11b), 38.09(C-5a), 62.70 (C-5),
69.39 (C-6), 103.21, 115.89, 115.92, 117.63, 120.76, 125.78,
128.86, 129.87, 138.38, 139.85, 143.91, 146.51, 148.76;ESI-
MS (m/z): 382.83 [M+H+]; Anal Calcd for C21H20ClN3O2:
Calcd: C, 66.05; H, 5.28; N, 11.00; Found: C, 66.38; H, 5.12;
N, 11.08.
3-(2,5-Dichlorophenyl)-1,11b-dimethyl-3,5a,6,11b-
tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazol-
10-amine (8pc)
Yellow solid, yield 61%; m.p.: 152–154◦C; IR (KBr, cm−1):
3387, 3336, 2971, 2924, 1615,1593,1526, 1497, 1443, 1395,
1228, 1097, 1044, 865, 759, 645; 1H NMR (400MHz;
DMSO-d6): 1.81 (s, 3H, CH3 attached to 3o ring-junction
carbon), 2.17 (m, 1H, H5a), 2.59 (s, 3H, CH3 of pyrazolone),
4.26 (m, 2H, H5 and H6), 4.47 (dd, J= 11.2,J= 3.6Hz, 1H,
1,5,5,11b-Tetramethyl-3-phenyl-3,5a,6,11b-tetrahydro-5H-
chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazol-10-amine (9pa)
Pink solid, yield 89%; m.p.: 162–164◦C; IR (KBr, cm−1):
ꢀ
ꢀ
H6 ), 4.55 (dd, J = 12.0, J = 2.8Hz, 1H, H5 ), 4.59 (s,
2H, NH2), 6.80–7.52 (m, 7H, Ar–H); 13C NMR (100MHz;
DMSO-d6): 16.40 (CH3 of pyrazolone), 29.62 (CH3 attached
to 3o ring-junction carbon), 34.09 (C-11b), 38.04 (C-5a),
62.98 (C-5), 69.06 (C-6), 102.11, 117.10, 121.04, 127.90,
128.78, 129.48, 129.60, 129.68, 129.98, 130.98, 132.86,
136.24, 147.75, 149.40, 151.29 (Ar–C); ESI-MS (m/z):
3432, 3215, 2984, 2930„1523, 1488, 1447, 1361, 1241,
1
1088, 1044, 839, 751, 693; H NMR (400MHz; DMSO-
d6) 1.09 (s, 3H, CH3 attached to 3o ring juncture carbon),
1.68 (s, 3H, CH3-5), 1.78 (s, 3H, CH3-5’), 1.99 (d, J = 3.4,
1H, H5a), 2.78 (s, 3H, CH3 of pyrazolone), 4.56 (d, J =
ꢀ
12.7Hz, 1H, H6), 4.78 (dd, J = 12.6, J = 4.0Hz, 1H, H6 ),
4.80 (s, 2H, NH2), 6.79–8.47 (m, 7H, Ar–H); 13C NMR
123

Mol Divers
(100MHz; DMSO-d6), 16.67 (CH3 of pyrazolone), 22.75
(CH3 attached to 3o ring juncture carbon), 29.65 (CH3-5),
32.87 (CH3-5ꢀ), 33.68 (C-11b), 47.34 (C-5a), 62.24 (C-6),
82.07 (C-5), 102.13, 116.35, 120.57, 123.61, 128.12, 129.72,
130.01, 135.64, 136.23, 141.86, 146.63, 149.20, 157.14;ESI-
MS (m/z): 376.46 [M+H+]; Anal Calcd for C23H25N3O2:
Calcd: C, 73.57; H, 6.71; N, 11.19; Found: C, 73.48; H, 6.89;
N, 11.28.
Biological assay
In vitro evaluation of antimicrobial activity
In vitro antimicrobial studies were conducted to measure
growth inhibition of a test compound against Streptococ-
cus pneumoniae (MTCC 1936),Clostridium tetani (MTCC
449) and Bacillus subtilis (MTCC 441); i.e., three gram-
positive bacteria, Salmonella typhi (MTCC 98), Vibrio
cholerae (MTCC 3906) and Escherichia coli (MTCC 443);
i.e., three gram-negative bacteria, and two fungal species
such as Aspergillus fumigatus (MTCC 3008) and Candida
albicans (MTCC 227), employing the NCCLS-accorded
broth microdilution MIC (minimum inhibitory concentra-
tion) method [32]. The values are given in Tables 1 and 2. The
microbial strains employed in the study were all procured
from (MTCC) Institute of Microbial Technology, Chandi-
garh, India. As a nutrient medium, Mueller Hinton broth
was used to grow microbial species and also for diluting
the compound suspension. Sabouraud dextrose broth was in
the same way used for the nutrition of fungal strains. Ampi-
cillin, norfloxacin, chloramphenicol and ciprofloxacin were
used as standards in the evaluation of antibacterial activity
of the test compounds. The antifungal potential of the com-
pounds was evaluated by using griseofulvin and nystatin as
standards to set a baseline. Bacterial strains were primar-
ily inoculated into Mueller–Hinton agar, and after overnight
growth, a number of colonies were suspended directly in
saline solution until the turbidity matched the turbidity of
the McFarland standard (approximately 108 CFU/mL), i.e.,
inoculum size for test strain was adjusted to 108 CFU/mL
(colony- forming unit) per milliliter well by comparing the
turbidity (turbidimetric method). Similarly, fungus species
were inoculated on Sabouraud dextrose broth; the procedures
of inoculum standardization were similar to described above.
DMSO was employed as diluents or vehicle to obtain desired
concentration of compounds to test upon standard microbial
strains, i.e., the compounds were dissolved in DMSO and
resulted solutions were diluted with a culture medium. All
compounds as well as standard drug were diluted to achieve
a 2000µg/mL stock solution. The required concentrations
were then obtained for primary and secondary screening,
by progressive dilutions of this sock solution with the test
medium. Primary screening test was run with three differ-
ent compound concentrations: 1000, 500 and 250µg/mL.
On being found active in primary screening, it was diluted
further to get 200, 100, 62.5µg/mL concentrations for the
secondary screening. Briefly, the control tube containing no
antibiotic was immediately sub-cultured, i.e., before inocu-
lation, by spreading a loopful evenly over a quarter of plate
of medium suitable for the growth of the test organisms. The
tubes were then put under incubation at 37◦C for 24h for
bacteria and 48h for fungi. Growth or a lack of growth in
3-(3-Chlorophenyl)-1,5,5,11b-tetramethyl-3,5a,6,11b-
tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazol-
10-amine (9pb)
Yellow solid, yield 71%; m.p.: 132–134◦C; IR (KBr, cm−1):
3430, 3204, 2986, 2930, 1521, 1485, 1443, 1362, 1243, 1085,
1
1036, 832, 732, 689; H NMR (400MHz; DMSO-d6) 1.08
(s, 3H, CH3 attached to 3o ring juncture carbon), 1.66 (s,
3H, CH3-5), 1.76 (s, 3H, CH3-5ꢀ), 1.96 (d, J = 3.8, 1H,
H5a), 2.75 (s, 3H, CH3 of pyrazolone), 4.53 (d, J = 12.7Hz,
ꢀ
1H, H6), 4.76 (dd, J = 12.6, J = 4.2Hz, 1H, H6 ), 4.78(s,
2H, NH2),6.78–8.46 (m, 7H, Ar–H); 13C NMR (100MHz;
DMSO-d6), 16.63 (CH3 of pyrazolone), 22.76(CH3 attached
to 3o ring juncture carbon), 29.66 (CH3-5), 32.91 (CH3-
5ꢀ), 33.70 (C-11b), 47.36 (C-5a), 62.36 (C-6), 82.05 (C-5),
102.16, 116.30, 120.59, 123.59, 128.16, 129.80, 130.10,
135.70, 136.26, 141.89, 146.66, 149.28, 157.20; ESI-MS
(m/z): 410.91 [M H+]; Anal Calcd for C23H24ClN3O2: C,
67.39; H, 5.90; N, 10.25; Found: C, 67.86; H, 6.01; N, 10.32.
3-(2,5-Dichlorophenyl)-1,5,5,11b-tetramethyl-3,5a,6,11b-
tetrahydro-5H-chromeno[4ꢀ,3ꢀ:4,5]pyrano[2,3-c]pyrazol-
10-amine (9pc)
Pink solid, yield 75%; m.p.: 160–162◦C; IR (KBr, cm−1):
3430, 3204, 2986, 2930,1521, 1485, 1443, 1362, 1243, 1085,
1036, 832, 732, 689; 1H NMR (400MHz; DMSO-d6): 1.08
(s, 3H, CH3 attached to 3o ring-junction carbon), 1.67 (s,
3H, CH3-5), 1.78 (s, 3H, CH3-5ꢀ), 1.96 (d, J= 3.2, 1H, H5a),
2.70 (s, 3H, CH3 of pyrazolone), 4.41 (td, J = 12.4, J =
1.6Hz, 1H, H6), 4.66 (ddd, J= 12.6, J= 4.2, J= 2.0Hz, 1H,
H6ꢀ), 4.56 (s, 2H, NH2), 6.69–7.56 (m, 6H, Ar–H); 13C NMR
(100MHz; DMSO-d6): 16.77 (CH3 of pyrazolone), 22.53
(CH3 attached to 3o ring-junction carbon), 29.65 (CH3-5),
32.06 (CH3-5ꢀ), 34.02 (C-11b), 46.65 (C-5a), 62.43 (C-6),
83.12 (C-5), 102.32, 117.43, 121.21, 127.97, 128.88, 129.15,
129.68, 129.93, 130.92, 131.16, 132.95, 136.54, 147.86,
149.67, 151.52 (Ar–C); ESI-MS (m/z): 444.2[M+H]+; Anal
Calcd for : C23H23Cl2N3O2 : C 62.17, H 5.22, N 9.46; Found:
C 62.46, H 5.51, N 9.12.
123

Mol Divers
the tubes containing the antimicrobial agent was determined
by comparison with the growth control, indicated by tur-
bidity. The lowest concentration that completely inhibited
visiblegrowthoftheorganismswasrecordedastheminimum
inhibitory concentration (MIC, µg/mL), i.e., the amount of
growth from the control tube before incubation (which rep-
resents the original inoculums) is compared. A set of tubes
containing only seeded broth and the solvent controls was
maintained under identical condition so as to make sure that
the solvent had no influence on strain growth. All bioassays
were repeated at least three times, and cultured error margin
value was 2 µg/mL.
and WiDr (colon). The in vitro antiproliferative activity was
evaluated after 48 h of drug exposure using the sulforho-
damine B (SRB) assay. According to NCI protocol, only
compounds soluble in DMSO at 40mM were tested. The
results expressed as GI50 (50% growth inhibition) are given
in Table 3.
In vitro antioxidant activity study [33]
The FRAP assay was used to display antioxidant capac-
ity of all the compounds, in which the resistance of these
compounds toward reduction of ferric tripyridyl triazine (Fe
(III)-TPTZ) complex into a blue color ferrous tripyridyl
triazine (Fe (II)-TPTZ) complex was determined spectropho-
tometrically at 593 nm at low pH.
In vitro evaluation of antituberculosis activity
Reagents:
Lowenstein–Jensen (L–J) medium method was used to deter-
mine in vitro antituberculosis activity, using M. tuberculosis
H37Rv strain. Table 2 reveals % inhibition of the strain by
the compound. A principal screen was conducted at 6.25
mg/mL concentration of the test compound. After adding
it to liquid Lowenstein–Jensen (L–J) medium, the resulted
content was sterilized by inspissation method. The culture of
bacteria grown on L-J medium was then harvested in bijou
bottles with 0.85% saline. DMSO was used as vehicle to get a
desired concentration. The tubes were incubated at 37◦C for
24h followed by streaking of M. tuberculosis H37Rv bacteria
(5×104 bacilli per tube). These tubes were then incubated at
37◦C. Growth of bacilli was seen after 12, 22, and finally
28-day incubation period. Tubes having compounds were
compared with control tubes where the medium alone was
incubated with M. tuberculosis H37Rv bacteria. The strain
of M. tuberculosis H37Rv bacteria was tested against known
drugs, namely isoniazide, fluconazole and miconazole. Only
those compounds which had M. tuberculosis H37Rv inhibi-
tion above or at least equal to 90% determined in the primary
screen were re-tested at lower concentration (MIC) using the
L–J medium.
Buffer solution Acetate buffer, pH 3.6, 300mmol/L (3.1g
sodium acetate trihydrate and 16mL glacial acetic acid per
litter of buffer solution).
TPTZ 10mmol/L, 2,4,6-tripyridyl-s-triazine (TPTZ) (MW
312.34) solution in 40mmol/L HCl.
FeCl3 solution: 20mmol/L, FeCl3 · 6H2O (MW 270.30) in
distilled water (DW).
Standard Ascorbic acid: It can be prepared by mixing of
25mL acetate buffer, 2.5mL TPTZ solution and 2.5mL
FeCl3 · 6H2O solution. The working solution must always
be a freshly prepared.
Sample solution: 0.005g dissolved in 25mL DMF.
Standard solution: 1mmol of ascorbic acid (MW 176.13
g/mol) in 100mL DW. 400µL sample or 200µL standard
was mixed with 3.0mL FRAP working solution and incu-
bated the resulted mixture at 37◦C for 10min. Its absorbance
was measured at 593 nm using respective blank solutions.
The antioxidant activity is expressed as ascorbic acid equiv-
alent (mM/100g of dried compound).
Ascorbic acid taken = 3.99 × 10−4 mm
Sample taken = 0.08 mg
Acknowledgements The authors are thankful to the Head, Depart-
ment of Chemistry, Sardar Patel University, for providing necessary
research facilities. We are also thankful to the University Grants Com-
mission (UGC), New Delhi, for financial support under the UGC
Scheme of Major Research Project (F. No: 39-822/2010 (SR) dated
11.01.2011). Authors (BML, TRS and GCB) are grateful to the UGC,
New Delhi, for research fellowship. Co-financed by the EU Research
Potential (FP7-REGPOT-2012-CT2012-31637-IMBRAIN), the Euro-
pean Regional Development Fund (FEDER), and the Spanish Instituto
de Salud Carlos III (PI11/00840).
In vitro antiproliferative activity
Antiproliferative assays for antitumor activity were per-
formed in 96-well plates using the National Cancer Institute
(NCI) protocol. As a model to study the anticancer activity of
the synthetic compounds, we used six human solid tumor cell
lines A549 (non-small cell lung), HBL-100 (breast), HeLa
(cervix), SW1573 (non-small cell lung), T-47D (breast)
123

Mol Divers
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