Synthesis, Antimicrobial Evaluations, and DNA Photo Cleavage Studies of New Bispyranopyrazoles

Article abstract of DOI:10.1002/jhet.2530

The bispyranopyrazoles (4a, 4b, 4c, 4d, 4e, 4f, 4g) were synthesized from the reactions of dibenzaldehydes (2a, 2b, 2c, 2d, 2e, 2f, 2g) with 3-methylpyrazole-5-one 3 and malononitrile by refluxing under alcoholic medium. The dibenzaldehydes were obtained from the O-alkylation of 3-hydroxybenzaldehyde with suitable 1,ω-dibromoalkanes under the alkaline conditions in the presence of dry EtOH/DMF. The structures of bisheterocyclics were determined from rigorous analysis of their spectral parameters (IR,¹H-NMR,¹³C-NMR, and ESI-MS). The newly prepared compounds were screened for their antimicrobial activity against seven bacterial and five fungal strains. The DNA photo cleavage potential of these compounds was also evaluated by using agarose gel electrophoresis, and bispyranopyrazoles 4b, 4d and 4e exhibited significant level of DNA photocleavage activity.

Full text of DOI:10.1002/jhet.2530

Month 2015 Synthesis, Antimicrobial Evaluations, and DNA Photo Cleavage Studies of
New Bispyranopyrazoles
a
a
b
*
Mohamad Yusuf, Manvinder Kaur, and Harvinder Singh Sohal
aDepartment of Chemistry, Punjabi University, Patiala, 147002 Punjab, India
bDepartment of Chemistry, M.M. University, Mullana, Ambala, Haryana, India
*
E-mail: yusuf_sah04@yahoo.co.in
Additional Supporting Information may be found in the online version of this article.
Received May 25, 2015
DOI 10.1002/jhet.2530
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
The bispyranopyrazoles 4(a–g) were synthesized from the reactions of dibenzaldehydes 2(a–g) with
3-methylpyrazole-5-one 3 and malononitrile by refluxing under alcoholic medium. The dibenzaldehydes
were obtained from the O-alkylation of 3-hydroxybenzaldehyde with suitable 1,ω-dibromoalkanes under
the alkaline conditions in the presence of dry EtOH/DMF. The structures of bisheterocyclics were
determined from rigorous analysis of their spectral parameters (IR, 1H-NMR, 13C-NMR, and ESI-MS).
The newly prepared compounds were screened for their antimicrobial activity against seven bacterial and
five fungal strains. The DNA photo cleavage potential of these compounds was also evaluated by using
agarose gel electrophoresis, and bispyranopyrazoles 4b, 4d, and 4e exhibited significant level of DNA
photocleavage activity.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
have moderate anticancer activity [15], and some of their
derivatives are also found to have broad spectrum biolog-
ical activities [16–25]. Substituted pyrano[2,3-c]pyrazoles
have been found to be effective antiplatelet molecules
[26] which affect K+ induced calcium dependent aortal con-
traction. Several pyrano[2,3-c]pyrazol-4-ones have demon-
strated an affinity toward A1 and A2a adenosine receptors
[27]. Also 6-amino-5-cyano-dihydropyranopyrazoles have
been identified as a screening hit for human Chk1 kinase in-
hibitors [28].
In recent years, attention has also been paid to evaluate
the DNA photocleavage potential of some azoles [29–31]
and heteroaryl-linked hydrazones [32] because of their
binding or interacting ability with the DNA structure.
DNA is a site where most of the chemotherapeutic drugs
act and interact which may result in DNA photocleavage
leading to inhibition of cancerous cells [33]. Therefore,
these nitrogen containing heterocyclic compounds could
Heterocyclic chemistry is an integral part of the chemi-
cal sciences and constitutes a considerable part of the
modern researches that are occurring presently throughout
the world. A wide range of heterocyclic compounds have
been synthesized in order to develop physiologically and
pharmacologically active products. Pyranopyrazoles are
the useful heterocycles where pyran and pyrazole moiety
coexist in the same structure, and these substances have
been prepared extensively in the past because of their var-
ious biological properties [1–3]. Synthesis of these com-
pounds has attracted the attentions of organic chemists in
the last decades on account of their prominent utilization
as analgesic, anti-inflammatory, ulcerogenic [4], antibacte-
rial, antifungal [5,6], antitubercular [7], antimalarial [8],
antitumor [5,9,10], antioxidant [10], antiproliferative
[11], antihypertensive [12], hypnotic [13], and vasodilator
[14]. Recently, some pyrazole derivatives are reported to
Copyright © 2015 HeteroCorporation

M. Yusuf, M. Kaur, and H. Singh Sohal
Vol 000
Scheme 1. Synthesis of bispyranopyrazoles.
be used as probes for DNA structure, and potential chemo-
therapeutic and diagnostic agents [34].
Dihydropyrano[2,3-c]pyrazole was first synthesized by
a reaction between 3-methyl-1-phenylpyrazolin-5-one and
tetracyanoethylene [35]. H. Otto and co-workers reported a
base catalyzed two-component Michael type reaction
between 4-arylidiene-1-phenyl-1H-pyrazol-5-one and malono-
nitrile for the synthesis of various 4-aryl-4Hpyrano[2,3-c]
pyrazoles [36]. The most common and convenient approach
toward diverse pyranopyrazoles is
a base catalyzed
three-component reaction [37] of pyrazol-5-one, aldehydes,
and malononitrile.
Because of the importance of pyranopyrazole deriva-
tives considerable efforts have been made by several inves-
tigators, to prepare new compounds bearing a single
substituent or more complicated systems, but the synthesis
of the compounds containing two pyranopyrazoles in the
same molecule has not been much explored in the litera-
ture. Only one paper regarding the synthesis of two
bispyranopyran molecules was found [38]. Motivated by
these results, this protocol was evaluated for its suitability
to build new bispyranopyrazole scaffolds which were fur-
ther investigated for their DNA photocleavage study.
These compounds are formed by joining two pyrano-
pyrazole moieties together through the aliphatic carbon
chains of varying lengths. By keeping this aspect in view
and in continuation to our studies on the bisheterocycles
[39–41], the present researches are focussed upon the
transformations of dibenzaldehydes 2(a–g) to bispyrano-
pyrazoles 4(a–g) built around the alkyl chains consisting
of two to twelve methylene groups. The major interest be-
hind study was to investigate the effect of the central
spacer length upon the formation and antimicrobial behav-
ior of the bispyranopyrazoles 4(a–g).
C¼O group and two additional bands at 2874–2850
and 2759–2717 cmꢀ1 appeared because of aldehyde
C―H stretching.
In the 1H-NMR spectra (400MHz, DMSO-d6) of
dibenzaldehydes 2(a–g), the downfield signal at δ 9.98–
9.89 could be ascribed to CHO proton appeared. The hy-
drogens (H-2, 6, 5, and 4) of the phenyl ring were found
to be placed at d 7.86–7.37 (2H, s), 7.49–7.36 (2H, m),
7.46–7.30 (2H, d, Jo = 6.6 Hz), and 7.27–7.09 (2H, m) re-
spectively. The resonances of internal chain OCH2 group
were placed in the aliphatic region at δ 4.45–3.93.
RESULTS AND DISCUSSION
13C-NMR (100MHz, DMSO-d6) spectra of dibenzalde-
hydes 2(a–g) proved instrumental to corroborate their pro-
posed structures. The presence of aldehyde group was
confirmed by appearance of signal at δ 192.37–192.12
(C¼O). The aromatic carbon atoms showed suitable sig-
nals at δ 159.72–159.13 (C-3), 137.80–137.51 (C-1),
130.08–129.98 (C-6), 123.56–122.20 (C-5), 122.00–
121.16 (C-4), and 113.35–112.59 (C-2); the downfield
resonance of C-3 as compared to other carbon atoms
can be ascribed to its direct linkage to the electronegative
oxygen atom. The resonance placed at δ 68.30–67.60
could be generated by internal chain OCH2 group.
IR spectra of 4(a–g) were very helpful to corroborate their
structures which did not exhibit any absorption at 1698–
1679cmꢀ1 indicating the involvement of carbonyl group
during the formation of bispyranopyrazoles 4(a–g). In spite
of this, a significant absorption bands were observed at
3392–3350, 3228–3207 (NH2), 3158–3138 (NH), 2958–2933,
The bispyranopyrazoles 4(a–g) needed for the pres-
ent investigations were obtained starting from 3-
hydroxybenzaldehyde 1 which was reacted with suitable
1,ω-dibromoalkanes (1,2-dibromoethane, 1,4-dibromobutane,
1,5-dibromopropane, 1,6-dibromohexane, 1,8-dibromooctane,
1,10-dibromodecane, and 1,12-dibromododecane) under
refluxing conditions in the presence of dry EtOH/KOH/
DMF to yield dibenzaldehydes 2(a–g) [42]. The later were
treated with malononitrile and 3-methylpyrazole-5-one 3
[43,44] to furnish bispyranopyrazoles 4(a–g) in good
yields (Scheme 1).
The structures of the newly prepared compounds were
elucidated on the basis of their spectroscopic data (IR,
1H-NMR, 13C-NMR, and ESI-MS), and elemental analysis
results also confirmed the purity of these products.
IR spectra of dibenzaldehydes 2(a–g) showed strong ab-
sorption bands at 1698–1679 cmꢀ1 because of conjugated
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2888–2844 (methylene C―H), 2258–2250 (CN), 1606–1595
(C¼N), and 1252–1249, 1088–1032 cmꢀ1 (C―O).
Amoxicillin and fluconazole were used as the standard
drug as positive control while the DMSO was used as neg-
ative control. This control did not show any activity against
the strains of micro-organisms used. Normal saline was
used to make a suspension of spore of bacterial and fungal
strain for lawning. A loopful of particular microbial strain
was transferred to 10-mL saline to get a suspension of cor-
responding species. After this, 1-mL volume of nutrient
broth was added to each tube successively, and all the
tubes were seeded with the bacterial and fungal strains.
The minimum inhibitory concentration (MIC-μg/mL) was
determined by using different dilutions of the concerned
compound. The lowest concentration required to arrest
the growth of bacterial and fungal strains was regarded as
minimum inhibitory concentration (MIC).
The results were compared with positive controls, the
standard drug Amoxicillin and Fluconazole. Serial dilution
of the test compounds previously dissolved in dimethyl
sulfoxide (DMSO) were prepared to final concentrations
of 128, 64, 32, 16, 8, 4, 2, and 1 μg/mL. All the bacteria
strains were grown at 37°C for 24h in nutrient broth, and
fungi were grown in malt extract at 28°C for 72h. Each test
compound was dissolved in DMSO and MIC (minimum
inhibitory concentration) thus obtained were compared
with control. The observed minimum inhibitory concentra-
tion (MIC-μg/mL) values are given in Tables 1 and 2. The
susceptibility of the bacteria and fungi to the test com-
pounds was determined by the appearance of turbidity after
the above said time period.
A comparison of the 1H-NMR spectra (400MHz,
DMSO-d6) of 2(a–g) and 4(a–g) showed that resonances
present at δ 9.98–9.89 in former were found missing alto-
gether in the later thereby pronouncing the involvement
of these hydrogens in the chemical transformation. A broad
singlet at δ 11.32–11.09 could be ascribed to because of
pyrazole ring NH protons. The significant feature of these
spectra was the signals of the two pyran ring protons (H-
4) which were clearly resonating as a sharp singlet at δ
5.31–5.03. Here, the aromatic ring protons H-6′, 5′, 4′,
and 2′ were centered at δ 7.32–7.09 (2H, d), 7.22–7.02
(2H, t), 7.13–6.95 (2H, t), and 7.03–6.82 (2H, brs) whereas
6-NH2 group protons appeared as a sharp singlet at δ 6.88–
6.65. In the aliphatic region, a triplet were also observed at
δ 4.24–3.80 which may be assigned to OCH2 group of al-
iphatic linker whereas other methylene groups were lo-
cated at δ 2.19–1.22 with appropriate multiplicities (vide
experimental). The singlets were also observed at δ 1.92–
1.77 which could be ascribed to C3―CH3 protons.
The carbon framework of the compounds 4(a–g) were
fully explained on the basis of their 13C-NMR spectra
(100 MHz, DMSO-d6). The downfield signals of C-6 were
resonating at δ 162.58–162.30 because of its direct bonding
to the electronegative nitrogen and oxygen atom and also its
presence in double bond. The carbon atoms C-3′ and C-3
directly connected to oxygen and nitrogen atom produced
suitable resonances at δ 157.91–157.69 and 139.15–
139.09 respectively. The carbon atoms associated to the py-
ran ring C-7a and C-3a were placed at δ 135.43–135.29 and
116.43–116.30, while other two carbon atoms C-5 and C-4
provided suitable signals at δ 55.77–57.60 and 35.63–35.49
respectively. Remaining aromatic carbon atoms were found
to be placed at the appropriate position in the aromatic re-
gion (see Experimental). The signals placed in the upfield
region at δ 65.95–64.39 and 9.69–9.54 were assignable to
internal chain OCH2 and C3―CH3 group respectively.
All the synthesized compounds 2(a–g) and 4(a–g), were
found to be potent antimicrobial agents (Tables 1 and 2)
against all tested strains.
It is evident from Table 1 that the compounds 2(a–g)
shows significant biological activity against various bacte-
rial and fungal strains at (MIC-8 μg/mL). The compound
2g showed significant activity against the most of strains
namely; E. coli, P. aeruginosa, S. aureus, B. subtilis
(bacteria strains) and P. glabrum, F. oxysporum, A.
sclerotiorum (fungi strains), while 2f displayed activity
against bacterial strains K. pneumoniae, P. fluorescens, B.
subtilis and A. janus, A. niger, A. sclerotiorum respec-
tively. Further 2c, 2d were also active with MIC-8 μg/mL
against the A. janus and E. coli respectively. Also, 2e
showed potent microbial activity against K. pneumoniae
and F. oxysporum.
BIOLOGICAL EVALUATION
Antimicrobial activity. The synthesized compounds 2(a–g)
and 4(a–g) were screened for their in vitro antibacterial and
antifungal activity against seven bacterial and five fungal
species namely Klebsiella pneumoniae (MTCC 3384),
Pseudomonas aeruginosa (MTCC 424), Escherichia coli
(MTCC 443), Staphylococcus aureus (MTCC 96), Bacillus
subtilis (MTCC 441), Pseudomonas fluorescens (MTCC
103), Streptococcus pyogenes (MTCC 442) and Aspergillus
janus (MTCC 2751), Penicillium glabrum (MTCC 4951),
Fusarium oxysporum (MTCC 2480), Aspergillus
sclerotiorum (MTCC 1008), and Aspergillus niger (MTCC
281) respectively. Minimum inhibitory concentrations
(MICs) were determined by using serial dilution technique.
It is evident from Table 2 that the compounds 4a showed
significant biological activity against the bacterial and fun-
gal strains namely K. pneumoniae, A. janus, A. niger, and
A. sclerotiorum at MIC of 8 μg/mL. The compound 4b
displayed significant activity against P. aeruginosa, S.
pyogenes (bacteria strain), and P. glabrum (fungi strain).
The compound 4c displayed significant activity against E.
coli, S. pyogenes, and A. niger, F. oxysporum at MIC of
8 μg/mL. Compound 4d and 4e were found to most active
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Figure 1. DNA photo cleavage study of bispyranopyrazoles 4(a–g).
against E. coli, P. aeruginosa, K. pneumoniae, A. niger, A.
sclerotiorum, and P. glabrum with MIC of 8 and 4 μg/mL.
Interestingly the compound 4f showed MIC of 8 and
4 μg/mL against P. aeruginosa, S. aureus, S. pyogenes
and A. janus, P. glabrum and F. oxysporum. Improved re-
sult has been observed in the case of 4g which was found
to inhibit the growth of bacterial strains E. coli, P.
fluorescens, B. subtilis and fungal strains A. janus, P.
glabrum, A. niger, A. sclerotiorum effectively at MIC of
4 and 8 μg/mL. This study reveals that some of synthesized
compounds also showed MIC of 4 μg/mL against tested
strains which is comparable to standard drug Amoxicillin.
However, compounds 4c, 4f (Lane 4, 7) were responsi-
ble to decrease the intensity of Super coiled and Open cir-
cular forms of DNA.
No DNA cleavage was observed for negative control
(lane C). A significant change in intensity of DNA Forms
(I, II, and III) in case of synthesized compounds in compar-
ison with untreated DNA indicated some kinds of fragmen-
tations or interactions caused by the compounds. Decrease
in intensity of plasmid DNA in case of compounds 4b, 4c,
4d, 4e, and 4f (Lane 3, 4, 5, 6, and 7) as compared with
control (Lane 1) indicated the cleavage of DNA forms.
As shown in Figure 1, the compounds 4b, 4d, and 4e were
responsible for the complete disappearance of all the forms
(super coiled, open circular, and linear) of DNA.
DNA photocleavage study.
DNA photocleavage
experiment was performed by taking 10-μL solution
containing pBR322 DNA in TE (Tris 10 mM, EDTA
0.01mM, pH8.0) buffer in the presence of 40 μg of
synthesized compounds [45]. The sample solution held in
caps of polyethylene micro centrifuge tubes was placed
directly on the surface of a trans illuminator (8000mW/
cm) at 360nm and was irradiated for 30 min at room
temperature. After irradiation, samples were further
incubated at 37°C for 1 h. Irradiated samples were mixed
with 6× loading dye containing 0.25% bromophenol blue
and 30% glycerol. The samples were then analyzed by
electrophoresis on a 0.8% agarose horizontal slab gel in
Tris–acetate EDTA buffer (40 mM Tris, 20 mM acetic
acid, 1 mM EDTA, pH: 8.0). Untreated plasmid DNA
was maintained as a control in each run of gel
electrophoresis which was carried out at 5 V/cm for 2.0 h.
Gel was stained with ethidium bromide (1 μg/mL) and
photographed under UV light. To account the effect of
synthesized compounds on DNA, the band intensities
were analyzed using the GelQuant.NET software
provided by biochemlabsolutions.com.
The overall results observed from the present study have
indicated that bispyranopyrazoles 4(a–g) derivatives pos-
sessed significant potential for DNA photo cleavage study.
CONCLUSION
It may be concluded that this study describes the general
and efficient method for the synthesis of new
bispyranopyrazoles linked through the aliphatic chains un-
der the ordinary conditions. It is evident from in vitro anti-
microbial evaluations that prepared compounds showed
marked potency against antimicrobial agents as compared
to their intermediates bisaldehydes 2(a–g) Further, DNA
photo cleavage study reveals that compounds 4b, 4d, and
4e have emerged as the most active DNA photo cleaving
agents among all the synthesized compounds and some
modifications in the basic structure may lead to construct
some potential chemotherapeutic agents in future.
EXPERIMENTAL
The DNA photo cleavage study was performed using
agarose gel electrophoresis, and the overall pattern is
shown in Figure 1.
All the chemicals used in this study were purchased
from E. Merck, S. D. Fine Chem. Ltd., Mumbai and
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M. Yusuf, M. Kaur, and H. Singh Sohal
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Sigma-Aldrich. The melting points were determined by
using the open capillary method and are uncorrected.
The Infrared (IR) spectra were scanned in KBr pellets on
a Perkin Elmer RXIFT Infrared spectrophotometer. The
1H-NMR and 13C-NMR spectra were recorded in
CDCl3/DMSO-d6 solvent on a 400-MHz Bruker spectro-
photometer. The purity of the compounds was checked by
TLC plates coated with silica gel (suspended in
chloroform-methanol, 1:1), and iodine vapors were used as
visualizing agent.
125.34 (C-5′), 117.30 (C≡N), 116.41 (C-3a), 113.47
(C-4′), 111.40 (C-2′). 65.95 (OCH2), 57.77 (C-5), 35.63
(C-4), 9.71 (CH3); ESI-MS: m/z 585 (M + Na, 10%), 563
(M + 1, 15%), 511 (10%), 489 (15%), 371 (9%), 294
(13%), 243 (19%), 242 (100%), 107 (8%), 93 (11%); Anal.
Calcd. For C30H26N8O4: C, 64.05; H, 4.66; N, 19.92; found
C, 64.09; H, 4.61; N, 19.96%.
Synthesis of 4,4′-(3,3′-(butane-1,4-diylbis(oxy))bis(3,1-
phenylene))bis(6-amino-3-methyl-1,4-dihydropyrano[2–45]pyrazole-
5-carbonitrile) 4b. The compound 4b was synthesized from
the reaction of 2b (0.01mol) with 3-methylpyrazole-5-one 3
(0.02mol) and malononitrile (0.02mol) under the same
conditions as described earlier for 4a.
Synthesis of 3,3′-[alkane-1,n-diylbis (oxy)] dibenzaldehydes
2(a–g). 3-Hydroxybenzaldehyde 1 (0.01 mol) and KOH
(0.01 mol) was dissolved in alcohol (100 mL), and then
solvent was removed under vacuum. The residue was
dissolved in DMF (25 mL) and 1,ω-dibromoalkanes
(0.005 mol) was added slowly. The reaction mixtures
were refluxed for 4h, during which KBr was separated
out. The solvent was removed under vacuum, and the
remaining material was poured into iced HCl to give
solid substances which were filtered under suction and
thoroughly washed with water. The crude product thus
obtained was crystallized from MeOH to yield pure
compounds 2(a–g). The physical and spectral data of
2(a–g) were found to be consistent as reported in
literature [41].
4b: Yellow solid; Yield 74%; m.p.: 116–118°C; IR
(KBr) cmꢀ1 3392 and 3207 (NH2), 3138 (NH), 2953,
2850 (methylene C―H), 2256 (CN), 1600 (C¼N), 1252,
1
1061 (C―O); H-NMR (400MHz, CDCl3): δ 11.29 (2H,
s, NH), 7.32 (2H, d, J = 7.1 Hz, H-6′), 7.22 (2H, t,
Jo = 7.0 Hz, H-5′), 7.12 (2H, d, Jo =7.5 Hz, H-4′), 7.00
(2H, brs, H-2′), 6.81 (4H, s, NH2), 5.29 (1H, s, H-4),
4.24 (4H, t, Jvic = 6.3 Hz, OCH2), 2.19 (2H, q,
Jvic = 6.0 Hz, OCH2CH2), 1.81 (6H, s, CH3); 13C-NMR
(400 MHz, CDCl3): δ 162.46 (C-6), 157.81 (C-3′),
139.11 (C-3), 136.11 (C-1′), 135.40 (C-7a), 129.28
(C-6′), 125.29 (C-5′), 117.26 (C≡N), 116.40 (C-3a),
113.33 (C-4′), 111.30 (C-2′). 64.42 (OCH2), 57.70
(C-5), 35.54 (C-4), 29.23 (OCH2CH2), 9.67 (CH3);
ESI-MS: m/z 613 (M + Na, 28%), 591 (M+ 1, 13%), 569
(15%), 543 (23%), 489 (16%), 301 (8%), 294 (21%),
242 (100%), 107 (9%), 94 (11%); Anal. Calcd. For
C32H30N8O4: C, 65.07; H, 5.12; N,18.97; Found C,
Synthesis of 3-methyl-1H-pyrazol-5(4H)-one 3.
Ethyl
acetoacetate (0.5 mol) was taken in a 100-mL conical
flask with absolute alcohol (20 mL), and then hydrazine
hydrate (0.5 mol) was added drop wise with continuous
shaking. The temperature of the reaction mixture was
maintained throughout at 40°C. After the completion of
reaction, a solid was separate which was filtered under
suction and further crystallized from MeOH to yield pure
65.11; H, 5.16; N, 18.93%.
Synthesis of 4,4′-(3,3′-(pentane-1,3-diylbis(oxy))bis(3,1-
phenylene))bis(6-amino-3-methyl-1,4-dihydropyrano[2–45]pyrazole-
5-carbonitrile) 4c. The compound 4c was prepared from the
3-methylpyrazole-5-one as a white solid [42,43].
reaction of 2c (0.01mol) with 3-methylpyrazole-5-one 3
(0.02mol) and malononitrile (0.02mol) under the same
conditions as described earlier for 4a.
Synthesis of 4,4′-(3,3′-(ethane-1,2-diylbis(oxy))bis(3,1-
phenylene))bis(6-amino-3-methyl-1,4-dihydropyrano[2–45]pyrazole-
5-carbonitrile) 4a. A mixture of compound 2a (0.01mol),
3-methylpyrazole-5-one 3 (0.02 mol), and malononitrile
(0.02 mol) was stirred in dry EtOH (25 mL) at 45°C for
3 h, and progress of reaction was monitored by TLC. The
resulting reaction mixture was cooled to yield light yellow
solid that was filtered, thoroughly washed with water and
dried. The crude product thus obtained was crystallized
from MeOH to yield a pure compound 4a.
4a: Light yellow solid; Yield 72%; m.p.: 152–154°C; IR
(KBr) cmꢀ1 3392 and 3210 (NH2), 3158 (NH), 2956, 2888
(methylene C―H), 2259 (CN), 1606 (C¼N), 1250, 1032
(C―O); 1H-NMR (400 MHz, CDCl3): δ 11.30 (2H, s,
NH), 7.30 (2H, d, J= 7.1 Hz, H-6′), 7.21 (2H, t, Jo =7.5 Hz,
H-5′), 7.13 (2H, d, Jo = 7.4 Hz, H-4′), 7.03 (2H, s, H-2′),
6.88 (4H, s, NH2), 5.31 (1H, s, H-4), 4.09 (4H, t,
Jvic =6.1 Hz, OCH2), 1.92 (6H, s, CH3); 13C-NMR
(400 MHz, CDCl3): δ 162.58 (C-6), 157.91 (C-3′), 139.15
(C-3), 136.15 (C-1′), 135.43 (C-7a), 129.30 (C-6′),
4c: light yellow solid; Yield 73%; m.p.: 138–140°C; IR
(KBr) cmꢀ1 3380 and 3219 (NH2), 3147 (NH), 2958, 2870
(methylene C―H), 2254 (CN), 1598 (C¼N), 1250, 1065
(C―O); 1H-NMR (400MHz, CDCl3): δ 11.28 (2H, s,
NH), 7.23 (2H, d, J =7.0 Hz, H-6′), 7.19 (2H, t, Jo = 7.3Hz,
H-5′), 7.11 (2H, d, Jo = 7.6 Hz, H-4′), 7.01 (2H, brs, H-2′),
6.85 (4H, s, NH2), 5.27 (1H, s, H-4), 4.08 (4H, t,
Jvic = 6.4 Hz, OCH2), 2.18 (4H, q, Jvic =6.0 Hz,
OCH2CH2), 1.98 (2H, m, OCH2CH2CH2), 1.83 (6H, s,
CH3); 13C-NMR (400MHz, CDCl3): δ 162.49 (C-6),
157.85 (C-3′), 139.12 (C-3), 136.13 (C-1′), 135.41 (C-7a),
129.30 (C-6′), 125.32 (C-5′), 117.29 (C≡N), 116.43 (C-3a),
113.43 (C-4′), 111.39 (C-2′). 64.43 (OCH2), 57.72 (C-5),
35.59 (C-4), 29.29 (OCH2CH2), 21.32 (OCH2CH2CH2),
9.69 (CH3); ESI-MS: m/z 627 (M+ Na, 100%), 604 (M + 1,
3%), 590 (13%), 576 (9%), 548 (14%), 496 (5%), 386 (4%),
372 (7%), 344 (7%), 294 (11%), 238 (11%), 162 (13%), 121
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Synthesis of New Bispyranopyrazoles
(14%), 107 (11%), 94 (18%); Anal. Calcd. For C31H28N8O4:
C36H38N8O4: C, 66.86; H, 5.92; N, 17.33; Found C,
C, 64.57; H, 4.89; N,19.43; Found C, 64.62; H, 4.93; N,
19.39%.
66.90; H, 5.88; N, 17.37%.
Synthesis of 4,4′-(3,3′-(decane-1,10-diylbis(oxy))bis(3,1-
phenylene))bis(6-amino-3-methyl-1,4-dihydropyrano[2–45]pyrazole-
5-carbonitrile) 4f. The compound 4f was synthesized from
the reaction of 2f (0.01mol) with 3-methylpyrazole-5-one 3
(0.02mol) and malononitrile (0.02 mol) under the same
conditions as described earlier for 4a.
Synthesis of 4,4′-(3,3′-(hexane-1,6-diylbis(oxy))bis(3,1-
phenylene))bis(6-amino-3-methyl-1,4-dihydropyrano[2–45]pyrazole-
5-carbonitrile) 4d. The compound 4d was obtained by
treating 2d (0.01mol) with 3-methylpyrazole-5-one 3
(0.02mol) and malononitrile (0.02mol) under the same
conditions as used earlier for 4a.
4f: Light brown solid; Yield 76%; m.p.: 96–98°C. IR
(KBr) cmꢀ1 3350 and 3222 (NH2), 3149 (NH), 2933, 2844
(methylene C―H), 2253 (CN), 1599 (C¼N), 1250, 1042
(C―O); 1H-NMR (400MHz, CDCl3): δ 11.14 (2H, s, NH),
7.10 (2H, d, Jo =7.5Hz, H-6′), 7.04 (2H, t, Jo =7.3Hz, H-
5′), 6.98 (2H, dd, Jo = 7.4, 2.3Hz, H-4′), 6.86 (2H, brs, H-
2′), 6.69 (4H, s, NH2), 5.09 (1H, s, H-4), 4.13 (4H, t,
Jvic =5.9Hz, OCH2), 2.02 (2H, q, Jvic =4.6Hz, OCH2CH2),
1.79 (6H, s, CH3), 1.51 (12H, m, OCH2CH2CH2CH2CH2);
13C-NMR (400MHz, CDCl3): δ 162.41 (C-6), 157.72
(C-3′), 139.09 (C-3), 136.16 (C-1′), 135.35 (C-7a), 129.21
(C-6′), 125.26 (C-5′), 117.18 (C≡N), 116.40 (C-3a), 113.13
(C-4′), 111.25 (C-2′). 65.12 (OCH2), 57.60 (C-5), 35.59
(C-4), 28.61 (OCH2CH2), 25.31 (OCH2CH2CH2), 22.10
(OCH2CH2CH2CH2), 19.11 (OCH2sCH2CH2CH2CH2); 9.54
(CH3); ESI-MS: m/z 697 (M +Na, 100%), 675 (M+1,
56%), 642 (17%), 584 (9%), 568 (12%), 493 (14%), 472
(11%), 398 (29%), 302 (14%), 214 (4%), 121 (21%), 93
(12%); Anal. Calcd. For C38H42N8O4: C, 67.64; H, 6.27; N,
4d: Brown solid; Yield 80%; m.p.: 122–124°C; IR
(KBr) cmꢀ1 3391 and 3214 (NH2), 3140 (NH), 2935,
2865 (methylene C―H), 2257 (CN), 1595 (C¼N), 1251,
1
1088 (C―O); H-NMR (400 MHz, CDCl3): δ 11.32 (2H,
s, NH), 7.28 (2H, d, Jo = 7.4 Hz, H-6′), 7.19 (2H, t,
Jo = 7.1Hz, H-5′), 7.11 (2H, d, Jo = 7.2 Hz, H-4′), 6.93
(2H, brs, H-2′), 6.78 (4H, s, NH2), 5.20 (1H, s, H-4),
4.19 (4H, t, Jvic = 6.6 Hz, OCH2), 2.11 (4H, q,
Jvic =6.4 Hz, OCH2CH2), 1.92 (4H, m, OCH2CH2CH2),
1.73 (6H, s, CH3); 13C-NMR (400 MHz, CDCl3): δ
162.46 (C-6), 157.80 (C-3′), 139.11 (C-3), 136.21 (C-1′),
135.40 (C-7a), 129.28 (C-6′), 125.29 (C-5′), 117.20
(C≡N), 116.38 (C-3a), 113.33 (C-4′), 111.30 (C-2′). 64.39
(OCH2), 57.68 (C-5), 35.52 (C-4), 29.13 (OCH2CH2),
21.78 (OCH2CH2CH2), 9.67 (CH3); ESI-MS: m/z 641 (M
+Na, 24%), 619 (M+1, 26%), 590 (19%), 538 (12%), 493
(14%), 462 (11%), 398 (29%), 270 (14%), 242 (100%),
122 (21%), 108 (4%); Anal. Calcd. For C34H34N8O4: C,
66.01; H, 5.54; N,18.11; Found C, 65.97; H, 5.59; N,
16.61; Found C, 67.69; H, 6.31; N, 16.65%
Synthesis of 4,4′-(3,3′-(dodecane-1,12-diylbis(oxy))bis(3,1-
phenylene))bis(6-amino-3-methyl-1,4-dihydropyrano[2–45]pyrazole-
18.15%.
Synthesis of 4,4′-(3,3′-(octane-1,8-diylbis(oxy))bis(3,1-phenylene))
bis(6-amino-3-methyl-1,4-dihydropyrano[2–45]pyrazole-5-car
bonitrile) 4e. The compound 4e was prepared from the
reaction of 2e (0.01 mol) with 3-methylpyrazole-5-one 3
(0.02 mol) and malononitrile (0.02 mol) under the same
conditions as described above for 3a.
5-carbonitrile) 4g.
The compound 4g was obtained by
reacting 2g (0.01mol) with 3-methylpyrazole-5-one 3
(0.02mol) and malononitrile (0.02mol) under the same
conditions used earlier for 4a.
4g: Grey solid; Yield 68%; m.p.: 104–106°C; IR (KBr)
cmꢀ1 3359 and 3228 (NH2), 3145 (NH), 2938, 2851 (meth-
ylene C―H), 2257 (CN), 1598 (C¼N), 1252, 1044 (C―O);
1H-NMR (400MHz, CDCl3): δ 11.09 (2H, s, NH), 7.09 (2H,
d, J=7.0Hz, H-6′), 7.02 (2H, t, Jo =7.3Hz, H-5′), 6.95
(2H, d, Jo =7.6Hz, H-4′), 6.82 (2H, brs, H-2′), 6.65
(4H, s, NH2), 5.03 (1H, s, H-4), 3.80 (4H, t, Jvic =6.1Hz,
OCH2), 1.68 (2H, q, Jvic =6.6Hz, OCH2CH2), 1.77 (3H, s,
CH3), 1.22 (16H, m, CH2CH2CH2CH2CH2CH2); 13C-
NMR (400MHz, CDCl3): δ 162.40 (C-6), 157.69 (C-3′),
139.08 (C-3), 136.10 (C-1′), 135.31 (C-7a), 129.19 (C-6′),
125.21 (C-5′), 117.15 (C≡N), 116.40 (C-3a), 113.11
(C-4′), 111.30 (C-2′). 64.42 (OCH2), 57.70 (C-5), 35.54
(C-4), 2925.45 (OCH2CH2), 25.34 (OCH2CH2CH2), 23.21
(OCH2CH2CH2CH2), 19.11 (OCH2CH2CH2CH2CH2),
15.01 (OCH2CH2CH2CH2CH2CH2), 9.67 (CH3); ESI-MS:
m/z 725 (M+Na, 21%), 703 (M+1, 13%), 629 (4%), 581
(9%), 527 (8%), 428 (5%), 372 (4%), 372 (7%), 313
(19%), 294 (15%), 239 (11%), 162 (13%). 107 (11%); Anal.
Calcd. For C40H46N8O4: C, 68.35; H, 6.60; N, 15.94; Found
C, 68.39; H, 6.56; N, 15.89%.
4e: Brown solid; Yield 65%, m.p.: 130–132°C; IR
(KBr) cmꢀ1 3364 and 3210 (NH2), 3145 (NH), 2938,
2867 (methylene C―H), 2250 (CN), 1598 (C¼N), 1249,
1
1086 (C―O); H-NMR (400 MHz, CDCl3): δ 11.21 (2H,
s, NH), 7.22 (2H, d, Jo = 7.8 Hz, H-6′), 7.13 (2H, t,
Jo = 7.1Hz, H-5′), 7.09 (2H, d, Jo = 7.0 Hz, H-4′), 6.92
(2H, s, H-2′), 6.81 (4H, s, NH2), 5.29 (1H, s, H-4), 4.24
(4H, t, Jvic = 6.5 Hz, OCH2), 1.86 (4H, quintet,
Jvic =6.5 Hz, OCH2CH2), 1.46 (4H, quintet, Jvic = 5.3 Hz,
OCH2CH2CH2), 1.35 (4H, m, OCH2CH2CH2CH2), 1.81
(6H, s, CH3); 13C-NMR (400 MHz, CDCl3): δ 162.30
(C-6), 157.72 (C-3′), 139.1 (C-3), 136.02 (C-1′), 135.29
(C-7a), 129.20 (C-6′), 125.18 (C-5′), 117.09 (C≡N),
116.33 (C-3a), 113.32 (C-4′), 111.23 (C-2′). 64.36
(OCH2), 57.61 (C-5), 35.49 (C-4), 29.15 (OCH2CH2),
24.21 (OCH2CH2CH2), 17.10 (OCH2CH2CH2CH2), 9.58
(CH3); ESI-MS: m/z 668 (M+ Na, 19%), 647 (M +1,
16%), 642 (17%), 628 (13%), 586 (9%), 572 (12%), 516
(11%), 474 (14%), 473 (11%), 398 (29%), 214 (5%), 121
(10%), 108 (16%), 94 (12%); Anal. Calcd. For
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

M. Yusuf, M. Kaur, and H. Singh Sohal
Vol 000
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Acknowledgment. The authors are highly thankful to UGC, New
Delhi for providing the financial support for this research work.
[25] Samb, I.; Pellegrini-Moïse, P.; Chapleur, Y. Tetrahedron Lett
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet