Synthesis, cytotoxic, and DNA binding studies of novel fluorinated condensed pyrano pyrazoles

Article abstract of DOI:10.1007/s00044-012-0239-z

One pot solvent-free synthetic method is developed for the synthesis of novel pyrano[2,3-c]pyrazole analogs. Cytotoxicity experiments conducted against a pair of cancerous, non-cancerous lung cell lines, and a cervical cell line is described. These compounds are selectively toxic against cancer cells and not normal cells. Molecular mechanism is established for the mode of DNA binding of these compounds using electrochemical and proton NMR methods.

Full text of DOI:10.1007/s00044-012-0239-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0239-z
ORIGINAL RESEARCH
Synthesis, cytotoxic, and DNA binding studies of novel fluorinated
condensed pyrano pyrazoles
•
S. Bhavanarushi V. Kanakaiah E. Yakaiah
•
•
•
V. Saddanapu A. Addlagatta J. Vatsala Rani
•
Received: 19 May 2012 / Accepted: 13 September 2012
Ó Springer Science+Business Media New York 2012
Abstract One pot solvent-free synthetic method is
developed for the synthesis of novel pyrano[2,3-c]pyrazole
analogs. Cytotoxicity experiments conducted against a pair
of cancerous, non-cancerous lung cell lines, and a cervical
cell line is described. These compounds are selectively
toxic against cancer cells and not normal cells. Molecular
mechanism is established for the mode of DNA binding of
these compounds using electrochemical and proton NMR
methods.
have been highly intensified with the recognition that they
constitute an essential pharmacophore in many naturally
occurring and biologically active agents (Dickinson, 1993).
Some pyrones are reported to possess cytotoxic effect
against a few human cancer cell lines (Marrison et al.,
2002). The substituted pyrans can bind to DNA through an
intercalative mode wherein the planar aromatic heterocy-
clic group inserts and stacks between the base pairs of
DNA (Barton, 1986). In addition, fluorine is intentionally
incorporated into inhibitors because of its lipophilicity and
also its comparable size with hydrogen atom. Fluo-
rine-containing molecules display better drug delivery
properties.
Keywords DBU Á Knoevenagel condensation Á
Pyrano[2,3-c]pyrazole Á HeLa cells Á Electrochemical Á
Intercalation
The use of conventional organic solvents in the syn-
thesis of 2-amino-3-cyano-4H-pyrans make the work up
procedure complicated and lead to poor yields of products
(Singh et al., 1996; Wang et al., 2003; Devi and Bhuyan,
2004). Lately, in addition to the usual synthetic methods,
2-amino-3-cyano-4H-pyrans have also been synthesized
under microwave (Lin et al., 2002; Tu et al., 2002) with
ultrasound irradiation (Li et al., 2004) in aqueous media
(Tong-Shou et al., 2004; Jin et al., 2005) two-component
(Kaupp et al., 2003) and three-component (Tong-Shou
et al., 2004) condensations, etc. Each of these methods
have some merits with at least one of the limitations of low
yields, long reaction time, effluent pollutions, harsh reac-
tion conditions, or tedious workup procedure.
Introduction
Potential pharmacologically active compounds have been
developed by exploring a wide variety of pyrazoles fused
with different heterocycles that are known to contribute to
various chemotherapeutic effects like antileukemic (Chou
et al., 2007), antitumor (Li et al., 2006), antimicrobial
(Holla et al., 2006), and antiviral (Yan et al., 2006) agents.
Investigations in the chemistry and biology of 2-pyrones
These findings have motivated us to explore a novel
method to synthesize fluorinated condensed pyrano[2,3-
c]pyrazole derivatives. An efficient one pot synthesis using
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base under
solvent-free condition was developed to synthesize fluori-
nated condensed pyrano[2,3-c]pyrazole derivatives. These
compounds were further tested for their cytotoxicity; few
of them displayed cytotoxicity toward cancer cell lines.
S. Bhavanarushi Á V. Kanakaiah Á E. Yakaiah Á J. V. Rani (&)
Fluoro Organic Division, Indian Institute of Chemical
Technology, Hyderabad 500 607, India
e-mail: j21_vat@rediffmail.com
V. Saddanapu Á A. Addlagatta (&)
Center for Chemical Biology, Indian Institute of Chemical
Technology, Hyderabad 500 607, India
e-mail: anthony@iict.res.in
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Med Chem Res
DNA binding studies (electrochemical) were carried out
with the compound which showed maximum cytotoxicity.
By finding interesting behavior of compound with DNA,
we have further studied liquid-state proton NMR to
delineate the mechanism of interaction of pyrazole deriv-
atives and DNA nucleotides.
explained by the generation of stable DBU–H? species in
the reaction, which suppresses the formation of these side
products. The a-cyanocinnamonitrile formed initially by
Knoevenagel condensation undergoes subsequent reaction
with 5-hydroxy pyrazoles in the presence of DBU to give
the desired products.
Cytotoxicity
Results and discussion
Inhibitory efficiency was tested for some of these fluori-
nated condensed pyrano pyrazoles against lung cancer
(A549), non-lung cancer cells (MRC-5) and cervical cancer
(HeLa) cells (Table 1). The 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide (MTT) assay was per-
formed following the previously reported protocol in the
96-well plate (Myadaraboina et al., 2010). The IC50 values
for the compounds 4a–4k are summarized in the Table 1.
Doxorubcin was used as the control. In general, A549 cells
were sensitive against the compounds tested in the current
study than HeLa cells. Compound 4h was the best against
A549 cells followed by 4d and 4i. Incidentally, compound
4d also displayed equal activity against HeLa cells. Sur-
prisingly, none of the compounds have displayed toxicity
against the normal lung cells. Pyrano pyrazoles are known
to bind to DNA (Barton, 1986). To understand the mech-
anism of inhibition displayed by compound 4h, we have
carried out DNA binding cyclic voltametry studies.
Synthesis of pyrano[2,3-c]pyrazole derivatives
Substituted condensed pyrano[2,3-c]pyrazole derivatives
(Table 1) were prepared by mechanical grinding of the
three components—substituted aromatic aldehyde, malon-
onitrile, and 1-phenyl-3-[trifluoromethyl]-1H-pyrazol-5-ol
in one pot by using DBU base at room temperature. The
reactions lead to completion in 5 min with good yields and
easy work up. The synthetic protocol is shown as following
in Scheme 1.
R: C6H5, 4-FC6H4, 3-ClC6H4.
R1: C6H5, 4-NO2C6H4, 3-OCH3C6H4, 2-furyl, 2-thionyl,
4-CH3C6H4, 4-OCH3C6H4, 4-FC6H4.
According to the proposed mechanism, the first step of
this reaction is the formation of Knoevenagel product
by the condensation of an aldehyde with malononitrile
(Goujon et al., 2002; Costa et al., 2008). Generally, base
catalyzed three-component reaction is accompanied by the
formation of many side products such as enaminonitrile,
higher adducts, reduced products, and malononitrile which
reduce the overall yield of the final product (Khurana et al.,
2010). The better yield observed in Scheme 1 can be
Cyclic voltammetry (CV) of compound 4h and DNA
interaction
The cyclic voltammogram of 1 mM DNA and 1 mM 4h in
sodium acetate buffer (pH 4.6) at graphite electrode at scan
rate of 10 mV/s is shown in (Fig. 1). The CV of plain DNA
in sodium acetate buffer is shown as inset in (Fig. 1a)—as
seen from the figure, graphite electrode provides a stable
background. The CV of 4h alone in acetate buffer did not
show any peaks at graphite electrode.
Table 1 Inhibitory efficiency of synthesized fluorinated condensed
pyrano pyrazole derivatives against lung cancer (A549) and non-lung
cancer cells (MRC-5) and cervical cancer (HeLa) cells
Compound
R
R1
A549
(lM)
MRC-5 HeLa
(lM) (lM)
The CV of DNA mixed with 4h shows two prominent
peaks (Fig. 1b) at potential 0.45 V and 1.3 V versus Ag/
AgCl. The peaks are due to the oxidation of guanine and
adenine residues, respectively (Wang et al., 2001). Upon
the interaction of 4h with DNA, double stranded DNA
separates partly into two single stranded DNA by the
rupture of the hydrogen bonds. It means that adenine and
guanine bases are exposed to electrode surface when the 4h
interacts with DNA so the oxidative reaction occurs easily.
Successive CVs of the DNA and 4h show gradual decrease
in the two peak currents due to the adsorption of the DNA
which forms a DNA multilayer on the graphite electrode.
To understand the mechanism of DNA and 4h interaction,
4a
4b
4c
4d
4e
4f
C6H5
C6H5
C6H5
C6H5
4-NO2C6H4 [100
3-OCH3C6H4 [100
[100 [100
[100 [100
[100 [100
2-furyl
[100
2-thionyl
75.5 ( 1.90) [100
74.07 ( 1.40)
4-FC6H4 4-CH3C6H4 [100
4-FC6H4 2-thionyl [100
4-FC6H4 4-NO2C6H4 [100
[100 [100
[100 [100
[100 [100
4g
4h
4i
4-FC6H4 4-OCH3C6H4 10.3 ( 0.89) [100 [100
3-ClC6H4 4-OCH3C6H4 89.4 ( 1.05) [100 [100
4j
3-ClC6H4 4-NO2C6H4 [100
3-ClC6H4 2-furyl [100
[100 [100
[100 [100
4k
123

Med Chem Res
Scheme 1 Synthesis of
pyrano[2,3-c]pyrazole
derivatives
R1
O
F3C
N
F3C
CN
DBU
O
CN
CN
H
N
OH
R1
N
R
N
Grinding at r.t
NH2
R
1 a-c
2 a-n
3
4 a-n
R: C6H5, 4-FC6H4, 3-ClC6H4
R1: C6H5, 4-NO2C6H4, 3-OCH3C6H4, 2-furyl, 2-thionyl, 4-CH3C6H4, 4-OCH3C6H4, 4-FC6H4.
Fig. 1 Cyclic voltammogram of DNA
mixed with compound ‘‘4h’’ in sodium
acetate buffer at scan rate of 10 mV/s.
a DNA alone in the buffer solution, b DNA
mixed with 1 mM compound ‘‘4h’’ in the
buffer solution
CV studies were further extended by adding cytosine to the
mixture of DNA and 4h.
Interaction of cytosine and compound 4h with DNA
Cyclic voltammogram of 1 mM DNA and 1 mM 4h in the
presence of 1 mM cytosine in sodium acetate buffer at a
scan rate of 10 mV/s is shown in (Fig. 2). The CV indi-
cated only one peak at 0.45 V; the peak at 1.3 V was not
observed immediately. However, it appeared after about
30 min. When the concentration of cytosine was increased
to 3 mM, the peak at 0.45 V was persistent, but the peak at
1.3 V did not reappear even after 3 h.
1H NMR spectra
To examine the compound 4h’s interaction with cytosine,
NMR studies of 4h, cytosine, and cytosine mixed with 4h
were carried out (Fig. 3). The NMR spectra of 4h mixed
with cytosine shows the peak broadening (0.15 ppm) and
field shift of the N–H protons of the 4h. A similar NMR
peak broadening was observed in DNA and indole deriv-
ative complexes (Sartorius and Schneider, 1995), which
Fig. 2 Cyclic voltammogram of DNA mixed with 1 mM compound
‘‘4h’’ and 1 mM cytosine in sodium acetate buffer at scan rate of
10 mV/s
was suggested to be because of intercalation between DNA
and indole molecule. Apart from DNA intercalation, we
propose possible hydrogen bonding between 4h-cytosine
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Med Chem Res
and 4h-adenine as shown in (Fig. 4) cytosine at equal con-
centrations. Note that broadening and also shift in peak
position is observed in solution that contains the mixture of
4h and cytosine suggesting their interaction.
Intercalators contain planar heterocyclic groups which stack
between adjacent DNA base pairs.
It is clear that compound 4h is able to induce unwinding
of the dsDNA at both G–C and A–T centers. In the pres-
ence of equal concentrations of 4h and cytosine, there
could be three situations: interaction of 4h with G–C and
A–T pairs in DNA; interaction of 4h with free cytosine;
interaction of free cytosine with G–C and A–T in DNA.
From the CV experimental observation oxidation of only
G–C pair occurs readily when compared with A–T pair.
This may be due to the preferential interaction of cytosine
with only G–C pair and 4h than with A–T pair. The slow
interaction of cytosine with A–T pair may be due to narrow
A/T regions than G/C groove regions and also because of
the steric hindrance in the latter presented by the C2 amino
group of the guanine base. However, given enough time
(about 30 min), a part of the 4h becomes free from cyto-
sine and promotes the A–T pair unwinding. In the presence
of excess of cytosine, no free 4h is available even in the
extended time resulting in complete disappearance of peak
Mechanism of interaction of compound 4h with G–C
and A–T base pairs
Drug bind to DNA both covalently as well as non-covalently
(Chaires, 1998; Hurley, 2002; Neidle, 2001; Wemmer and
Dervan, 1997). Covalent binding in DNA is irreversible
and invariably leads to complete inhibition of DNA pro-
cesses. Non-covalently bound drugs mostly fall under the
following two class (a) Minor groove binders and (b) in-
tercalators. Minor groove binding drugs are usually cres-
cent shaped, which complement the shape of the groove
and facilitates binding by promoting Van der Waals
interactions. In addition, these drugs can form hydrogen
bonds to bases, typically to N3 of adenine and O2 of thymine.
Fig. 3 Interaction of compound
‘‘4h’’ with cytosine in solution.
a Proton NMR spectra of the
compound ‘‘4h,’’ b Proton
NMR spectra of cytosine,
c Proton NMR spectra of
compound ‘‘4h’’ mixed with
cytosine
123

Med Chem Res
H
not normal cells. Molecular mechanism was established for
the mode of DNA binding of these compounds by elec-
trochemical and proton NMR methods. Based on these
encouraging results, we are in the process of redesigning
the pyrano[2,3-c]pyrazole moiety for a better efficacy.
H
R1
N
N
F
3C
N
N
H
N
N
H
O
N
O
H
R
Experimental
4
C
All the substituted pyrazoles were prepared by the known
method (Liu et al., 2006) which involves reaction of the
corresponding ethyltrifluoroaceto acetate with phenyl
hydrazine in the presence of aqHCl catalyst using EtOH as
the solvent. The chemicals used were of analytical grade
and the solutions were prepared using double distilled
water. 4-F-phenyhydrazine hydrochloride, DBU, and ethyl
trifluoroacetoacetate (ETFAA) were purchased from M/s
Sigma Aldrich (St. Louise, USA) and malanonitrile, para-
methoxy benzaldehyde, and cytosine from M/s Lancaster
(India). Miniprep kit was obtained from M/s Qaigen
(USA). Melting points of all the pyrano[2,3-c]pyrazoles
prepared were recorded on Casiae–Siamia (VMP-AM)
melting point apparatus and are uncorrected. IR spectra
were recorded on a Perkine-Elmer FT-IR 240-C spectro-
photometer using KBr optics. 1H NMR and 13C NMR
spectra were recorded on Bruker AV 300 MHz; 19F NMR
spectra were recorded on Inova 400 MHz spectrometer in
DMSO using TMS as internal standard. Electron Spray
Ionization (ESI) and high-resolution spectra was recorded
on QSTARXL hybrid MS/MS system (Applied Biosys-
tems, USA) under electrosprayionization. All the reactions
were monitored by thin layer chromatography (TLC) on
precoated silica gel 60 F254 (mesh); spots were visualized
using UV light. Merck silica gel (60–120 mesh) was used
for column chromatography.
H
H
R1
N
F
3C
N
N
N
N
H
N
N
O
N
H
R
N
H
4
A
Fig. 4 Model of interaction of compound ‘‘4h’’ with DNA. The top
two panels indicate possible hydrogen bonding between the basic
skeleton of molecules and DNA bases. Unwinding of double stranded
DNA in the presence of compound ‘‘4h’’ depicted as spheres
related to A–T pair. A model of interaction of 4h with
DNA and the unwinding of double stranded DNA in the
presence of compound 4h that leads to base pair oxidation
during CV experiments is shown in (Fig. 4). Cyclic vol-
tammetric results specify the interaction of the drug and
DNA is non-covalent, and it is an intercalator. Intercalators
contain planar heterocyclic groups which stack between
adjacent DNA base pairs and it is thought to be stabilized
by p–p stacking interactions between the drug and DNA
bases. Non-covalent binding is reversible and is typically
preferred over the covalent adduct formation keeping the
drug metabolism and toxic side effects in mind.
General procedure for the preparation of pyrano[2,3-
c]pyrazole derivatives (4a–n)
A mixture of aromatic aldehyde (2) (2 mmol), malononit-
rile (3) (132 mg, 2 mmol), 1-phenyl-3-(trifluoromethyl)-
1H-pyrazol-5-ol (1) (2 mmol), and DBU (0.072 mg,
0.47 mmol) were taken in a mortar and ground thoroughly
using pestle at room temperature (25 °C). The reaction was
monitored by TLC. The product was isolated by passing
through a column packed with silica gel (60–120 mesh)
using n-hexane:ethylacetate (4:1) as eluents.
Conclusion
In summary, we have reported an efficient method to
synthesize novel pyrano[2,3-c]pyrazole molecules in one
pot using DBU as a base under solvent free conditions. We
also reported cytotoxic and DNA binding studies of novel
fluorinated condensed pyrano pyrazoles cytotoxicity
experiments were conducted against a pair of cancerous,
non-cancerous lung cell lines, and a cervical cell line. Our
compounds were selectively toxic against cancer cells and
6-Amino-4-(4-nitrophenyl)-1-phenyl-3-(trifluoromethyl)-
1,4-dihydropyrano[2,3-c]pyrazole-5-carobonitrile (4a)
Yield 0.76 g (87 %); m. p. 242–244 °C; 1H NMR (DMSO-
d6, 300 MHz): d 4.91 (s, 1H, CH), 7.28 (s, 2H, NH2),
123

Med Chem Res
7.38–7.58 (m, 5H, Ar), 7.89 (d, J = 7.7 Hz, 2H), 8.19 (d,
J = 8.5 Hz, 2H). 13C NMR (DMSO-d6, 75 MHz): d 36.1,
57.1, 97.6, 119.0, 121.9, 123.6, 128.3, 129.2, 129.5, 136.5,
145.2, 146.7, 150.8, 158.8. 19F NMR (DMSO-d6, 376 MHz):
d -61.01 (s, 3F, CF3). I.R (KBr, cm-1): 3,424, 3,328 (NH2),
2,203 (CN), 1667 (C=N), 1,519, 1,351 (NO2), 1,143 (C–F).
HRMS m/z calcd for C20H13F3N5O3 (M ? H?): 428.097;
found: 428.096.
(DMSO-d6, 376 MHz): d -60.96 (s, 3F, CF3). I.R (KBr,
cm-1): 3,462, 3,316 (NH2), 2,198 (CN), 1,660 (C=N),
1,143 (C–F). HRMS m/z calcd for C18H12F3N4OS
([M ? H]?): 389.068 found: 389.067.
6-Amino-1-(4-fluorophenyl)-4-p-tolyl-3-(trifluoromethyl)-
1,4-dihydropyrano[2,3-c]pyrazole-5-carobonitrile (4e)
Yield 0.69 g (84 %); m. p. 177–179 °C; 1H NMR (DMSO-
d6, 300 MHz): d 2.33 (s, 3H, CH3), 4.66 (s, 1H, CH),
6.90–6.97 (br, 2H, NH2), 7.03–7.13 (m, 4H), 7.25 (d,
J = 8.9 Hz, 2H), 7.88–7.94 (m, 2H). 13C NMR (DMSO-d6,
75 MHz): d 20.5, 36.3, 59.0, 95.5, 98.8, 115.5, 115.8,
122.7, 122.8, 122.8, 126.9, 128.6, 135.9, 139.7, 144.4,
158.2. 19F NMR (DMSO-d6, 376 MHz): d -60.8 (s, 3F,
CF3); -113.01 to -112.92 (m, 1F, Ar–F). I.R (KBr,
cm-1): 3,469, 3,320 (NH2), 2,203 (CN), 1,660 (C=N),
1,141 (C–F). HRMS m/z calcd for C21H15F4N4O
([M ? H]?): 415.118 found: 415.117.
6-Amino-4-(3-methoxyphenyl)-1-phenyl-3-
(trifluoromethyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-
carobonitrile (4b)
Yield 0.67 g (81 %); m. p. 137–139 °C; 1H NMR (DMSO-
d6, 300 MHz): d 3.78 (s, 3H, OCH3), 4.68 (s, 1H, CH),
6.69–6.79 (m, 3H, Ar), 6.91 (s, 2H, NH2), 7.17–7.26 (m,
2H, Ar), 7.34–7.43 (m, 1H, Ar), 7.47–7.57 (m, 1H, Ar),
7.88 (d, J = 7.7, 2H, Ar). 13C NMR (DMSO-d6, 75 MHz):
d 36.5, 54.9, 58.3, 98.7, 112.0, 113.7, 119.3, 119.8, 121.8,
123.0, 128.2, 129.2, 129.4, 133.3, 136.3, 144.9, 145.0,
158.6, 159.1. 19F NMR (DMSO-d6, 376 MHz): d -59.97
to -60.07 (m, 3F, CF3). I.R (KBr, cm-1): 3,414, 3,327
(NH2), 2,200 (CN), 1,661 (C=N), 1,146 (C–F). HRMS m/z
calcd for C21H16F3N4O2(M ? H?): 413.11 found: 413.10.
6-Amino-1-(4-fluorophenyl)-4-(thiophen-2-yl)-3-
(trifluoromethyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-
carobonitrile (4f)
Yield 0.66 g (82 %); m. p. 162–164 °C; 1H NMR (DMSO-
d6, 300 MHz): d 5.10 (s, 1H, CH), 6.90–7.02 (m, 2H),
7.23–7.36 (m, 5H, Ar & NH2), 7.85–7.94 (m, 2H). 13C
NMR (DMSO-d6, 75 MHz): d 36.5, 57.4, 95.4, 115.5,
115.8, 118.2, 122.7, 122.8, 123.3, 128.1, 146.6, 149.5,
158.6, 162.6. 19F NMR (DMSO-d6, 376 MHz): d -60.95
(s, 3F, CF3); -112.85 to -112.95 (m, 1F, Ar–F). I.R (KBr,
cm-1): 3,451, 3,320 (NH2), 2,200 (CN), 1,659 (C=N),
1,134 (C–F). HRMS m/z calcd for C18H11F4N4OS
([M ? H]?): 407.048 found: 407.047.
6-Amino-4-(furan-2-yl)-1-phenyl-3-(trifluoromethyl)-1,4-
dihydropyrano[2,3-c]pyrazole-5-carobonitrile (4c)
Yield 0.60 g (83 %); m. p. 180–182 °C; 1H NMR (DMSO-
d6, 300 MHz): d 4.89 (s, 1H, CH), 6.2 (d, J = 3.2 Hz, 1H,
furan), 6.29–6.32 (m, 1H, furan), 6.87 (s, 2H, NH2),
7.32–7.40 (m, 2H, furan & Ar), 7.45–7.53 (m, 2H, Ar);
7.84 (d, J = 7.9 Hz, 2H, Ar). 13C NMR (DMSO-d6,
75 MHz): d 30.5, 56.0, 95.6, 96.3, 106.0, 110.0, 118.7,
121.0, 127.4, 128.4, 128.9, 136.4, 141.7, 144.6, 153.5,
159.4. 19F NMR (DMSO-d6, 376 MHz): d -61.26 (s, 3F,
CF3). I.R (KBr, cm-1): 3,443, 3,323 (NH2), 2,202 (CN),
1,661 (C=N), 1,145 (C–F). HRMS m/z calcd for
C18H12F3N4O2 ([M ? H]?): 373.091 found: 373.090.
6-Amino-1-(4-fluorophenyl)-4-(4-nitrophenyl)-3-
(trifluoromethyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-
carobonitrile (4g)
Yield 0.75 g (85 %); m. p. 240–242 °C; 1H NMR (DMSO-
d6, 300 MHz): d 4.87 (s, 1H, CH), 7.04 (s, 2H, NH2),
7.19–7.28 (m, 2H), 7.43 (d, J = 8.7 Hz, 2H), 7.87–7.96
(m, 2H), 8.18 (d, J = 8.7 Hz, 2H). 13C NMR (DMSO-d6,
75 MHz): d 31.9, 58.6, 95.4, 98.8, 115.7, 116.0, 118.7,
123.1, 124.4, 124.8, 126.1, 132.5, 144.1, 147.5, 158.4,
162.6. 19F NMR (DMSO-d6, 376 MHz): d -61.06 (s, 3F,
CF3); -112.79 to -112.89 (m, 1F, Ar–F). I.R (KBr,
cm-1): 3,423, 3,328 (NH2), 2,203 (CN), 1,666 (C=N),
1,519, 1,349 (NO2), 1,142 (C–F). HRMS m/z calcd for
C20H12F4N5O3 ([M ? H]?): 446.081 found: 446.080.
6-Amino-1-phenyl-4-(thiophen-2-yl)-3-(trifluoromethyl)-
1,4-dihydropyrano[2,3-c]pyrazole-5-carobonitrile (4d)
Yield 0.64 g (84 %); m. p. 182–184 °C; 1H NMR (DMSO-
d6, 300 MHz): d 5.09 (s, 1H, CH), 6.90–6.94 (m, 1H), 6.98
(d, J = 3.2 Hz, 1H), 7.02–714 (br, 2H, NH2), 7.21–7.27
(m, 1H), 7.35–7.43 (m, 1H), 7.47–7.57 (t, J = 7.4, 2H),
7.87 (d, J = 8.1 Hz, 2H). 13C NMR (DMSO-d6, 75 MHz):
d 32.0, 58.7, 95.5, 98.8, 118.8, 120.9, 124.4, 124.7, 126.1,
127.5, 128.6, 129.0, 136.3, 144.1, 147.6, 158.5. 19F NMR
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Med Chem Res
6-Amino-1-(4-fluorophenyl)-4-(4-methoxyphenyl)-3-
(trifluoromethyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-
carobonitrile (4h)
6.32 (s, 1H), 7.14 (s, 2H, NH2), 7.37 (d, J = 7.554 Hz, 2H),
7.44–7.53 (m, 1H), 7.81–7.92 (d, J = 12.6 Hz, 2H). 13C
NMR (DMSO-d6, 300 MHz): d 30.3, 55.6, 95.5, 106.1,
110.0, 118.9, 120.5, 122.0, 127.3, 129.8, 130.3, 134.3,
137.5, 141.8, 144.9, 151.3, 153.4, 159.3. 19F NMR (DMSO-
d6, 376 MHz): d -61.42(s, 3F, CF3). I.R (KBr, cm-1):
3,479, 3,331 (NH2), 2,204 (CN), 1,664 (C=N), 1,142 (C–F).
HRMS m/z calcd for C18H11F3ClN4O2 ([M ? H]?):
407.0522 found: 407.0521.
Yield 0.72 g (85 %); m. p. 208–210 °C; 1H NMR (DMSO-
d6, 300 MHz): d 3.74 (s, 3H, OCH3), 4.73 (s, 1H, CH), 6.88
(d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H), 7.27 (s, 2H,
NH2), 7.39–7.47 (m, 2H), 7.85–7.92 (m, 2H). 13C NMR
(DMSO-d6, 75 MHz): d 35.6, 54.7, 58.6, 98.8, 113.4,
115.8, 116.2, 119.1, 124.0, 124.1, 128.4, 132.4, 135.2,
144.6, 157.9, 158.1, 162.8. 19F NMR (DMSO-d6,
376 MHz): -60.61 (s, 3F, CF3); -112.81 to -112.92 (m,
1F, Ar–F). I.R (KBr, cm-1): 3,388, 3,324 (NH2), 2,196
(CN), 1,661 (C=N), 1,143 (C–F). HRMS m/z calcd for
C21H15F4N4O2 ([M ? H]?): 431.113 found: 431.114.
6-Amino-1-(3-chlorophenyl)-4-phenyl-3-(trifluoromethyl)-
1,4-dihydropyrano[2,3-c]pyrazole-5-carobonitrile (4l)
Yield 0.73 g (88 %); m. p. 181–183 °C; 1H NMR (DMSO-
d6, 300 MHz): d 4.71 (s, 1H, CH), 6.86 (br, 2H, NH2),
7.16–7.39 (m, 5H), 7.45–7.51 (m, 2H), 7.80–7.93 (m, 2H).
13C NMR (DMSO-d6, 75 MHz): d 36.6, 58.4, 95.4, 99.0,
118.9, 119.1, 120.6, 121.8, 122.2, 127.1, 128.0, 130.5,
133.4, 134.1, 137.5, 142.9, 158.4, 164.9. 19F NMR
(DMSO-d6, 376 MHz): -61.69 (s, 3F, CF3). I.R (KBr,
cm-1): 3,468, 3,326 (NH2), 2,200 (CN), 1,664 (C=N),
1,136 (C–F). HRMS m/z calcd for C20H13F3Cl N4O
([M ? H]?): 417.068 found: 417.067.
6-Amino-1-(3-chlorophenyl)-4-(4-methoxyphenyl)-3-
(trifluoromethyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-
carobonitrile (4i)
1
Yield: 0.72 g (82 %); mp 189–191 °C; H NMR (DMSO-
d6, 300 MHz): d 3.78 (s, 3H, OCH3), 4.66 (s,1H, CH), 6.81
(d, J = 8.687 Hz, 2H), 6.86 (s, 2H, NH2), 7.10 (d,
J = 8.687 Hz, 2H), 7.32–7.40 (m, 1H), 7.44–7.52 (m, 1H),
7.84–7.92 (m, 2H). 13C NMR (DMSO-d6, 300 MHz): d
30.5, 35.7, 54.9, 58.8, 99.3, 113.6, 119.3, 120.3, 121.4,
128.0, 128.7, 135.3, 131.1, 133.8, 137.4, 145.1, 158.2. 19F
NMR (DMSO-d6, 400MHZ): -61.09 (s, 3F, CF3). I.R
(KBr, cm-1): 3,392, 3,323 (NH2), 2,193 (CN), 1,659
(C=N), 1,144 (C–F). HRMS m/z calcd for
C21H15F3ClN4O2 ([M ? H]?): 447.079 found: 447.078.
6-Amino-1-(4-fluorophenyl)-4-phenyl-3-(trifluoromethyl)-
1,4-dihydropyrano[2,3-c]pyrazole-5-carobonitrile (4m)
Yield 0.66 g (83 %); m. p. 173–175 °C; 1H NMR (DMSO-
d6, 300 MHz): d 4.71 (s, 1H, CH), 7.10–7.63 (m, 9H, Ar &
NH2), 7.87–7.98 (m, 2H, Ar). 13C NMR (DMSO-d6,
75 MHz): d 36.7, 58.5, 95.4, 98.7, 115.7, 116.0, 118.9,
123.0, 123.1, 126.8, 127.1, 128.0, 132.7, 142.8, 144.6,
158.4. 19F NMR (DMSO-d6, 376 MHz): -61.25 (s, 3F,
CF3); -113.05 to -113.13 (m, 1F, Ar–F). I.R (KBr,
cm-1): 3,467, 3,325 (NH2), 2,193 (CN), 1,659 (C=N),
1,142 (C–F). HRMS m/z calcd for C20H13F4N4O
([M ? H]?): 401.102 found: 401.103.
6-Amino-1-(3-chlorophenyl)-4-(4-nitrophenyl)-3-
(trifluoromethyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-
carobonitrile (4j)
Yield 0.80 g (88 %); m. p. 224–226 °C; 1H NMR (DMSO-
d6, 300 MHz): d 4.89 (s, 1H, CH), 7.26–7.34 (br, 2H, NH2),
7.37–7.55 (m, 4H), 7.86–7.95 (m, 2H), 8.19 (d, J = 8.7 Hz,
2H). 13C NMR (DMSO-d6, 75 MHz): d 36.4, 57.2, 95.5,
97.6, 118.1, 118.4, 119.1, 120.7, 123.3, 127.4, 128.4, 130.3,
134.3, 137.4, 145.0, 146.6, 149.8, 158.6. 19F NMR (DMSO-
d6, 376 MHz): d -61.15 (s, 3F, CF3). I.R (KBr, cm-1):
3,414, 3,329 (NH2), 2,205 (CN), 1,667 (C=N), 1,520, 1,350
(NO2), 1,143 (C–F). HRMS m/z calcd for C20H12F3ClN5O3
([M ? H]?): 462.048 found: 462.047.
6-Amino-4-(4-fluorophenyl)-1-phenyl-3-(trifluoromethyl)-
1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (4n)
Yield 0.61 g (85 %); m. p. 221–223 °C; 1H NMR (DMSO-
d6, 300 MHz): d 4.75 (s, 1H, CH), 7.06 (t, J = 8.7 Hz,
2H), 7.20–7.29 (m, 4H, Ar & NH2), 7.42 (t, J = 7.4 Hz,
1H), 7.51–7.58 (m, 2H), 7.87 (d, J = 7.7 Hz, 2H). 13C
NMR (DMSO-d6, 75 MHz): d 35.9, 58.2, 95.4, 98.5, 114.6,
114.9, 118.8, 121.0, 127.5, 129.0, 136.3, 139.0, 144.6,
158.3, 159.5, 162.7. 19F NMR (DMSO-d6, 376 MHz): d -
61.44 (s, 3F, CF3); -112.95 to -113.03 (m, 1F, Ar–F). I.R
(KBr, cm-1): 3,414, 3,327 (NH2), 2,200 (CN), 1,661
(C=N), 1,142 (C–F). HRMS m/z calcd for C20H13F4N4O
([M ? H]?): 401.098 found: 401.097.
6-Amino-1-(3-chlorophenyl)-4-(furan-2-yl)-3-
(trifluoromethyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-
carobonitrile (4k)
Yield: 0.64 g (82 %); m. p. 161–163 °C; 1H NMR (DMSO-
d6, 300 MHz): d 4.88 (s, 1H), 6.21 (d, J = 2.644 Hz, 1H),
123

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system consisted of natural graphite sheet working elec-
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plate and cultured in the medium containing 10 % serum.
Incubated for 24 h in a 5 % CO2 humid chamber at 37 °C
so that the cells adhere to the surface. MTT was dissolved
in PBS at 5 mg/ml and sterile filtered.
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