An ecofriendly synthesis and DNA binding interaction study of some pyrazolo [1,5-a]pyrimidines derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.10.027

The DNA molecule is a target for plethora of anticancer and antiviral drugs that forms covalent and non-covalent adducts with major or minor groove of DNA. In present study we synthesized series of novel Pyrazolo [1,5-a]pyrimidine derivatives. The newly s

Full text of DOI:10.1016/j.bmcl.2012.10.027

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7566–7572
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
An ecofriendly synthesis and DNA binding interaction study of some pyrazolo
[1,5-a]pyrimidines derivatives
Santosh S. Chobe a, Bhaskar S. Dawane a, , Khaled M. Tumbi b, Prajwal P. Nandekar b, Abhay T. Sangamwar b
⇑
a Organic Chemistry Research Laboratory, School of Chemical Sciences, Swami Ramanand Teerth Marathawada University, Nanded 431606, (M.S), India
b Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Punjab, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The DNA molecule is a target for plethora of anticancer and antiviral drugs that forms covalent and non-
covalent adducts with major or minor groove of DNA. In present study we synthesized series of novel Pyr-
azolo [1,5-a]pyrimidine derivatives. The newly synthesized compounds were characterized by elemental
analysis, IR, 1H NMR, and mass spectral data. The selected compounds were studied for interaction with
Calf thymus DNA (CT-DNA) using electronic spectra, viscosity measurement and thermal denaturation
studies. Further, molecular interactions were revealed for compound IIIa and IVa by computational
methodologies. The preferred mode of ligand binding with double helical DNA as well as preferable
DNA groove were explored by molecular docking in different DNA models.
Received 4 June 2012
Revised 2 October 2012
Accepted 4 October 2012
Available online 13 October 2012
Keywords:
Pyrazolo [1,5-a]pyrimidines
Green chemistry
5-Amino pyrazole
DNA binding interaction
Molecular docking
Ó 2012 Elsevier Ltd. All rights reserved.
Over the past decades, the design and synthesis of DNA binding
compounds have been an area of interest in cancer research.1–3
DNA acts as a significant intracellular receptor and many chemicals
bear their antitumor effects by binding to DNA. Pharmacological
actions of these compounds were mediated by means of change
in replication of DNA leading to inhibit growth of tumor cells
and their effectiveness depends upon the mode and affinity of their
DNA binding.4 A number of synthesized complexes and metal che-
lates, as agents for mediation of strand scission of duplex DNA and
as chemotherapeutic agents, have been used as probes of DNA
structure in solution.5,6 Furthermore, synthesis and kinetic study
of transition state analogs have been used as tools for understand-
ing DNA binding for mediation of DNA cleavage or as chemothera-
peutic agents.7
The fused pyrimidines are an important class of compounds as
chemotherapeutic agents in attracting attention of medicinal
chemists for their antibacterial8 antiviral9 and cytotoxic10 proper-
ties. Pyrazole ring also plays an important role in plethora of bio-
logical processes, as many therapeutic agents possess Pyrazole
moiety. Certain alkyl pyrazoles have shown significant antiallergic,
anti-inflammatory and anti-arthritic properties.11,12 Many pyra-
zole containing fused heterocyclic groups have exhibited biological
activities and widely studied in pesticide as well as medicine.13,14
Pyrazolopyrimidines are of considerable chemical and pharmaco-
logical importance as purine analogues,15,16 and have antitumor,
antileukemic activities. The role of pyrazolo [1,5-a]pyrimidines
are well documented as anti-metabolites and shown to have inhi-
bition of purine biochemical reactions.17,18 Pyrazolopyrimidine and
related heterocyclic compounds are found to own
a wide
important pharmacophore and privileged structure in medicinal
chemistry. Pyrazolopyrimidine derivatives exhibit diversified
pharmacological activities like CNS depressant19, neuroleptic20
,
tuberculostatic,10 antihypertensive,21 analgesic22 and antimicro-
bial activity.23 The Pyrazolo [1,5-a]pyrimidine frame work contain-
ing a number of fused bicyclic heterocycles has been a versatile
scaffold for various pharmacological studies.23,24 Apart from their
utility for several disease targets, pyrazolo [1,5-a]pyrimidine
derivatives has shown an enormous synthetic value in the prepara-
tion of various drugs and bio-active molecules. Prompted by the
varied biological activities of pyrazolopyrimidine derivatives, we
envisioned our approach towards the synthesis using the alterna-
tive green reaction mediums.
The literature survey reveals that the anti-tumor activity of
most drugs is due to their intercalations with base pairs of DNA
and interferences with normal functioning of enzyme topoisomer-
ase II, which is involved in breaking and releasing of DNA
strands.25–27 The anti-tumor drugs that intercalate DNA are of
growing interest in the field of anti-cancer drugs. Particularly, they
are characterized by planar chromospheres, which are often
constituted by three or four condensed rings, which can intercalate
into base pairs.28 Considering above mentioned fact the
⇑
Corresponding author.
E-mail address: dawanebhaskar@gmail.com (B.S. Dawane).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.027

S. S. Chobe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7566–7572
7567
Table 1
Absorption spectral properties of compounds IIIa and IVa bound to CT-DNA
R
N
N
O
O
O
Sr. No
Compound
kmax (nm)
Kb (Mꢁ1
)
Tm(°C)
PEG-400 / Sod.Acetate
R
Stirring, 60°C
N
N
+
H
1
2
IIIa
IVa
320
530
5.21 ꢀ 105
80
82
5.7 ꢀ 105
Scheme 2. Synthesis of 4-substituted benzylidene-3-methyl-1H-pyrazol-5(4H)-
one.
synthesized pyrazolo [1,5-a]pyrimidine derivatives have evaluated
for their DNA binding potential. The more efficient and eco-
friendly green reaction method for synthesis has been reported
in this Letter. The DNA binding ability of designed and synthesized
analogues were evaluated by absorption spectroscopy, viscosity
measurement, thermal denaturation and molecular docking stud-
ies. The DNA-binding studies indicate that compounds IIIa and
IVa have shown more binding with base pairs of Calf Thymes
DNA (CT-DNA). The preference of compounds towards major and
minor groove has also been supported by molecular docking
studies.
observed at 320 nm and 530 nm peak maximum in presence of
an excess CT-DNA. The lowest value observed in spectral changes
(including red shift and hypochromicity) were used to evaluate
intrinsic binding constant (Kb). The spectra results suggested that,
the compound IIIa binds strongly than the compound IVa with
DNA, as the calculated intrinsic binding constant for compound IIIa
and IVa was found to have 5.21 ꢀ 10ꢁ5 and 5.7 ꢀ 10ꢁ5 respectively.
The difference in binding constant was observed due to difference
in mode of binding with DNA. (Table 2).
In extension of our work on synthesis of bioactive heterocyclic
compounds,29–31 herein we report a novel series of pyrazolo [1,5-
a]pyrimidines by condensation of 4-substituted benzylidene-3-
methyl-1H-pyrazol-5(4H)-one with 5-aminopyrazole in PEG-400
(Table 1). The starting compound 5-aminopyrazole was prepared
in two steps from corresponding amine by diazotization and then
treated with malononitrile followed by reaction with hydrazine
hydrate (Scheme 1). The novel 4-substituted benzylidene-3-
methyl-1H-pyrazol-5(4H)-one compounds III(a–d) were prepared
in presence of sodium acetate and PEG-400 as a green reaction sol-
vent and hetero carbaldehyde (Scheme 2 and Fig. 1). Finally, the
synthesis of pyrazolo [1,5-a]pyrimidines IV(a–d) were attempted
by reacting 5-aminopyrazole with 4-substituted benzylidene-3-
methyl-1H-pyrazol-5(4H)-one in PEG-400 as reaction solvent
(Scheme 3 and Fig. 2). Formation of products were assumed to pro-
ceed through Michael-type addition of ring nitrogen in 5-amino-
pyrazole to activated double bond, followed by intra-molecular
cyclisation with elimination of water and then dehydrogenation.
Structures of compounds were appropriately established by spec-
troscopic and analytical methods.
Concentration of CT-DNA per nucleotide [C(P)] was measured
using its known extinction coefficient at 260 nm [6600 Mꢁ1
cmꢁ1 33
and absorbance at 260 nm (A260) and 280 nm (A280) for
]
CT-DNA was measure to check the purity. The ratio A260/A280
was found to be 1.8:1.9, indicating that CT-DNA was satisfactorily
free from protein. A phosphate buffer (5 Mm, pH 7.2), was used for
the absorption, viscosity and thermal denaturation experiments.
Absorption titration experiments were carried out by varying the
DNA concentration from 0 to 100
pound concentration constant at 30
l
M and maintaining the com-
M. Absorption spectra were
l
recorded after each successive addition of DNA and equilibrium
(approx. 10 min). For both compounds IIIa and IVa, the observed
data were fit into Eq. (1) in order to obtain the intrinsic binding
constant, Kb.29
½DNAꢂ=ðea
ꢁ
ef Þ ¼ ½DNAꢂ=ðeb
ꢁ
ef Þ þ 1=Kbððea
ꢁ
ef
Þ
ð1Þ
Where ea
,
e
f and b are the apparent, free and bound compound
e
extinction coefficient for compound IIIa at 290 nm and for com-
pound IVa at 305 nm respectively. A plot of [DNA]/(ea f) versus
[DNA] gave a slope of 1/(ea f) and an intercept y equal to 1/Kb
f), where Kb is the ratio of slope to y-intercept.
For further clarification, the interaction modes of compound IIIa
ꢁe
ꢁe
Application of electronic absorption spectroscopy is considered
as one of the most important technique in CT-DNA binding stud-
ies.31 Binding of molecules to DNA has been well characterized
by the large hypochromism in UV spectrum. After intercalation,
(
eaꢁe
and IVa with DNA were investigated by viscosity measurement
technique. An increase in viscosity of native DNA is regarded as a
diagnostic feature of an intercalation process. During drug-DNA
intercalation, base pairs were separated to accommodate the inter-
calating ligand leads to increase in length of DNA helix usually
increasing viscosity of DNA.34,35 In order to elucidate the binding
mode of present compounds, we have measured the viscosity
changes in short, rod-like DNA fragments by varying the concen-
trations of added compounds. The effects of compounds on viscos-
ity of rod-like DNA were shown in Figure 4. The relative length
increase (L/L0) of the complex formed between IIIa and IVa with
DNA as shown in Figure 4. It is evident that the binding of com-
pounds IIIa and IVa with DNA leads to increase in viscosity of
DNA corresponding to an increase in contour length of DNA frag-
the
p
⁄ orbital of compounds could couple with
p-orbital of base
⁄– transition of energy and resulting
pairs, thus decreasing the
p
p
in bathochromism. Hence, decrease in absorption intensity and
significant red shift were observed due to stacking interaction be-
tween compound and CT-DNA.32 The DNA-binding studies were
characterized by absorbance maximum at 320 nm for compound
IIIa and 530 nm for compound IVa. Additionally, increasing con-
centration of DNA led to hypochromic and bathochromic (red shift)
changes in its visible absorption spectra as a result of formation of
more stable complex as shown in Figure 3A and B. The interactions
of compounds IIIa and IVa with CT-DNA resulted in decrease of
absorption intensity accompanied by a shift towards higher wave-
length. Around 12–9% reduction intensity of absorption was
H
N
CN
N C
NH2
N
NCl
CN
CH2(CN2)
NaNO2/HCl
CH2(CN2)
Cl
N
N
NH2
NH
H2N
N
Cl
Cl
Cl
Scheme 1. Synthesis of 5-aminopyrazole.

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S. S. Chobe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7566–7572
CH3
O
N
N
(IIIb)
CH3
R
Introduction of
different substituents
(IIIa) to (IIId)
N
H3C
N
N
NO2
Cl
HN
N
O
N
(IIIc)
(IIIa)
N
N
Cl
(IIId)
Figure 1. Structures of different substituted pyrrazole-5-one derivatives.
R
Cl
N
N
N
N
NH2 PEG-400/NaoH
Cl
N
N
R
O
N
N
N
+
Stirr, 60-80°C
NH
H2N
N
H2N
N
N
Scheme 3. Synthesis of pyrazolo [1,5-a]pyrimidines.
O2N
N
N
(IVb)
CH3
N
Cl
CH3
R
Introduction of
different substituents
(IVa) to (IVd)
N
N N
N
N
N
N
NH2
HN
N
N
Cl
(IVc)
(IVa)
Cl
A
CH3
O
N
(IVd)
Figure 2. Structures of different substituted pyrazolo [1,5-a]pyrimidines.

S. S. Chobe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7566–7572
7569
Figure 3. UV-absorption spectra in phosphate buffer upon addition of CT-DNA (IIIa) and (IVa).
Table 2
Physical and analytical data of synthesized compounds
⁄⁄Note: Grey background indicated pose in major groove of DNA.
Values in bracket indicates, (0) as no H-Bond, (B) as hydrogen bonding interactions with DNA backbone or name and residue number of nucleotide. The molecular docking
scores of most potent compounds are shown in Italics.
ments. The presence of compound had an obvious effect on the rel-
ative viscosity of CT-DNA with an increase in concentration of the
added compounds. This result further suggested an intercalative
binding mode of the complex with DNA and also parallel to the
above spectroscopic results, such as hypochromism and batho-
chromism of complexes in presence of DNA.
Another strong evidence for intercalative binding of compound
IIIa and IVa with double-helix DNA was obtained from DNA melt-
ing temperature (Tm), at which the DNA double-helix denatures
into a single-stranded DNA, owing to increased stability of helix
in presence of an intercalator ligand.28 The molar extinction coeffi-
cient of DNA bases at 260 nm in double helical form is much less
than the single-stranded form; hence, the melting of helix leads
to an increase in absorbance at 260 nm. DNA melting studies were
carried out with CT-DNA in presence as well as in absence of com-
pounds IIIa and IVa. The results reveal that melting temperature of
double helical CT-DNA was found to be 65 2 °C but in presence of
compound IIIa and IVa melting temperature increased by 10–15 °C
(Fig. 5). This variation in DNA melting temperature strongly sup-
ports the intercalation of compounds into DNA double-helix.36,37
Energetically favorable DNA-ligand complexes for compounds
III(a–d) and IV(a–d) were predicted by the systematic procedure
Figure 4. Effect of increasing amount of compound IIIa and IVa on the relative
as described in methods section, involving molecular docking
viscosity of CT-DNA.

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S. S. Chobe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7566–7572
Figure 5. Melting temperature curve of CT-DNA in presence and absence of compound IIIa and IVa.
Fig. 6. DNA ligand binding pose of compound IIIa and compound IVa in minor groove of DNA crystal structures (A) and (B) GC rich (PDBID 1QC1); (C) and (D) AT rich (PDBID
226D); in major groove of (E) and (F) mixed sequence (PDBID 2K0V).

S. S. Chobe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7566–7572
7571
using Glide software. The results obtained from molecular docking
of designed and synthesized analogues were summarized in Ta-
ble 1. The molecular docking analysis showed that all ligands pref-
erentially bind in minor groove of DNA in case of AT rich (PDB ID:
226D) and GC rich (PDB ID: 1QC1) nucleotide sequence, conversely
major groove binding is preferred for mixed (PDB ID: 2K0V) nucle-
otide sequence. The highest docking scores were observed for com-
pound IIIa (ꢁ7.644, ꢁ7.00 and ꢁ5.830 kcal/mol) and IVa (ꢁ6.441,
ꢁ6.65 and ꢁ5.170 kcal/mol) in all three DNA structures (PDB ID:
226D, 1QC1 and 2K0 V) respectively. The strong hydrogen bonding
interactions were observed with phosphate group of DNA back-
bone or adenine, guanine, thymine, cytosine nucleotides and li-
gands forming DNA intercalation as shown in Figure 6A–F. The
molecular electrostatic surface analysis of DNA shows that, the li-
gand prefers minor groove for AT rich (226D) and GC rich (1QC1)
nucleotide sequence. It is also clear that ligands bind preferentially
in major groove for mixed (2K0V) nucleotide sequence. The above
observation suggests that compound IIIa binds with DNA more
strongly than IVa forming intercalation complex in support with
previously described experimental results. Also the preference of
major and minor groove of DNA for binding is based on its nucle-
otide sequences.
concentration was found to be 496.2
l
mol/L, indicating that CT-
DNA was satisfactorily free from proteins. Buffer (0.2 M) phos-
phate, pH 7.2 was used for study. The absorbance measurement
was calibrated by several researchers using ethidium bromide.49
The previously calibrated results used in present study. Solution
of Pyrazolo [1,5-a]pyrimidine derivatives were prepared using
50% concentration of each DMSO and phosphate buffer. The UV–
visible absorption was measured in absences and in presences of
various concentration of DNA, while maintaining the compound
concentrations as constant.
The viscometer is cleaned thoroughly and DNA solution of
496.2 lmol/L is used to carry out viscometer titration, time of flow
measurements for DNA solution was carried out at 25 °C using
Oswald’s viscometer. Typically, 10 ml DNA solution is transferred
to measure the time required to flow. The increasing amount
of ethidium bromide was used by Raja et al. in previous DNA
biding study.50 Solution was allowed to thermally equilibrate for
30 min and time of flow is again measured for each time. Each
reading was taken three times and an average flow time was col-
lected. Similar method was repeated for all compounds in present
study.
DNA melting studies were carried out by monitoring the
The detailed analysis of DNA intercalation by compound IIIa
and IVa by UV–visible, viscosity measurements, thermal denatur-
ation studies shows the perfect binding to DNA. The designed
and synthesized compounds bind with DNA via interactions as
well as intercalation with the base pairs of CT-DNA. It also suggests
that the covalent binding of compound IIIa and IVa with DNA
cause change in conformation of DNA as increase in viscosity as
well as intensity of absorption band was generally observed with
increase in intercalation. Moreover, the results described in this
study showed that change in environment of molecule modulate
the binding property of compound with DNA. The relative binding
efficacy of the compound IIIa to DNA was much higher than that of
compound IVa as predicted by molecular docking exercise. The
preferential binding of compounds to minor groove of AT rich
(PDB ID: 226D) and GC rich (PDB ID: 1QC1) DNA sequence as well
as major groove of mixed (PDB ID: 2K0V) DNA sequence. The bind-
ing mode and molecular interactions of compounds with DNA will
provide necessary information on mechanism of antitumor/antibi-
otic compounds with DNA and benefit the drug discovery process
for designing of new molecules.
All melting points were uncorrected and determined in an open
capillary tube. The chemicals and solvents used were of analytical
grade. The completion of reaction was monitored by thin layer
chromatography on precoated sheets of silica gel-G (Merck, Ger-
many) using iodine vapor for detection. IR spectra were recorded
in matrix of KBr on a Shimadzu spectrometer (Japan). 1H NMR
spectra was recorded in DMSO-d6 on Advance spectrometer
(300 MHz) using TMS as an internal standard. Mass spectra were
recorded on an EI-Shimadzu QP 2010 PLUS GC–MS system (Shima-
dzu, Japan). Elemental analyses were performed on a Carlo Erba
106 Perkin-Elmer model 240 analyzer (Perkin-Elmer, USA).
General procedure for synthesis of 4-substituted benzylidene-
3-methyl-1H-pyrazol-5(4H)-one III(a–d)38 and of pyrazolo [1,5-a]
pyrimidines IV(a–d)39 used are mentioned in references. The spec-
tral details of compounds synthesized viz III(a–d)and IV(a–d) are
mentioned in reference footnotes.40–47
absorption of CT-DNA (50
in the presence (5–10 M) and in absence of compound IIIa and
IVa. The melting temperature (Tm) at which 50% of double stranded
DNA becomes single stranded and the curve width T (tempera-
lM) at 260 nm at various temperatures
l
r
ture range where 10–90% of the absorption increase occurred)
were calculated as previously described.51–53
The DNA-ligand binding affinity and DNA intercalating pose
were predicted using molecular docking methodology in Glide
software included in Schrodinger suite 9.0.211.54,55 The molecular
docking of designed and synthesized analogues was performed in
three different DNA crystal structures having PDB ID: 226D,
1QC1 and 2K0V which were retrieved from protein data bank.
(PDB).56–58 The three crystal structures were selected on the basis
of AT rich, GC rich and mixed nucleotide sequence, as it has signif-
icance in ligand binding.59 DNA structures were prepared using
preparation wizard for molecular docking as follows; hydrogen
atoms were added, hydrogen bonding network was optimized,
and protein was minimized to RMSD (Root Mean Square Deviation)
0.30 Å using OPLS (Optimized Potential for Liquid Simulations)
2005 force field. The designed ligands were prepared in LigPrep
and minimized using OPLS 2005 force field using set of default
parameters.60 The whole DNA structure was defined as macromo-
lecular grid for molecular docking. The ligand docking calculations
were done in the standard precision mode of glide software. Li-
gands were passed through a scaling factor of 0.80 and partial
charge cutoff of 0.15.
Acknowledgments
One of the authors (BSD) is sincerely thankful to University
Grant Commission, New Delhi for Post-Doctoral Research Award
(F. 30-1/2009, SA-II). Authors gratefully acknowledge to Director,
School of chemical Sciences Swami Ramanand Teerth Marathwada
University for providing laboratory facilities and IICT Hyderabad
for spectral analysis. ATS acknowledge financial support from
Department of Biotechnology (DBT), New Delhi.
DNA-Binding assay: CT-DNA was purchase from Hi-Media,
Mumbai, India. Pyrazolopyrimidine derivatives chemically were
synthesized in our laboratory and anhydrous disodium hydrogen
phosphate and monosodium hydrogen phosphate, used were of
analytical grade for the preparation of buffer. The concentration
of CT-DNA per nucleotide was measured using its known extinc-
tion coefficient at 260 nm for CT-DNA.48 Absorbance at 260 nm
for CT-DNA was measured in order to check the purity level. The
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
10.027. These data include MOL files and InChiKeys of the most
important compounds described in this article.

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S. S. Chobe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7566–7572
corresponding product IVa. Similarly, IVb, IVc, IVd were synthesized
References and notes
following the same procedure.
40. 3-(2-Butyl-4-chloro-1H-imidazol-5-yl-methylene)-4-methyl-1-phenyl-1H-
pyrrol-2(3H)one (IIIa): Colour: Yellowish, mp (°C): 178–182, yield-88, IR (KBr,
1. Hannon, M. J. Chem. Soc. Rev. 2006, 36, 280.
2. MacMillan, A. M. Pure Appl. Chem. 2004, 76, 1521.
3. Zhang, R.; Wu, X.; Yalowich, J. C.; Hasinoff, B. B. Bioorg. Med. Chem. 2011, 19,
7023.
4. Munde, M.; Kumar, A.; Nhili, R.; Depauw, S.; David-Cordonnier, M. H.; Ismail,
M. A.; Stephens, C. E.; Farahat, A. A.; Batista-Parra, A.; Boykin, D. W. J. Mol. Biol.
2010, 402, 847.
5. Lippard, S. J. Acc. Chem. Res. 1978, 11, 211.
6. Hecht, S. M. Acc. Chem. Res. 1986, 19, 383.
7. Holla, B. S.; Mahalinga, M.; Karthikeyan, M. S.; Akberali, P. M.; Shetty, N. S.
Bioorg. Med. Chem. 2006, 14, 2040.
8. Holla, B. S.; Kalluraya, B.; Sridhar, K. R.; Drake, E.; Thomas, L. M.; Bhandary, K.
K.; Levine, M. J. Eur. J. Med. Chem. 1994, 29, 301.
9. Molina, P.; Aller, E.; Lorenzo, Á.; López-Cremades, P.; Rioja, I.; Ubeda, A.;
Terencio, M. C.; Alcaraz, M. J. J. Med. Chem. 2001, 44, 1011.
10. Di Parsia, M. T.; Suarez, C.; Vitolo, M. J.; Marquez, V. E.; Beyer, B.; Urbina, C.;
Hurtado, I. J. Med. Chem. 1981, 24, 117.
11. Nugent, R. A.; Murphy, M.; Schlachter, S. T.; Dunn, C. J.; Smith, R. J.; Staite, N. D.;
Galinet, L. A.; Shields, S. K.; Aspar, D. G.; Richard, K. A.; Rohloff, N. A. J. Med.
Chem. 1993, 36, 134.
12. Antonini, I.; Polucci, P.; Magnano, A.; Martelli, S. J. Med. Chem. 2001, 44, 3329.
13. Silva, A. M. G.; Tome, A. C.; Neves, M.; Cavaleiro, J. A. S. Synlett 2002, 1155.
14. Kapplinger, C.; Beckert, R. Synlett 2000, 1679.
15. Bendich, A.; Russell, P. J.; Fox, J. J. J. Am. Chem. Soc. 1954, 76, 6073.
16. Alexander, J. A.; Wheeler, G. P.; Hill, D. D.; Morris, H. P. Biochem. Pharmacol.
1966, 15, 881.
17. Elion, G. B.; Callahan, S.; Nathan, H.; Bieber, S.; Rundles, R. W.; Hitchings, G. H.
Biochem. Pharmacol. 1963, 12, 85.
18. Earl, R. A.; Pugmire, R. J.; Revankar, G. R.; Townsend, L. B. J. Org. Chem. 1822,
1975, 40.
19. Abdou, I. M.; Saleh, A. M.; Zohdi, H. F. Molecules 2004, 9, 109.
20. Stanoeva, E.; Varbanov, S.; Alexieva, V.; Sergiev, I.; Vasileva, V.; Rashkova, M.;
Georgieva, A. Phosphorus Sulfur 2000, 165, 117.
21. Zhang, Y.; Jin, G.; Illarionov, B.; Bacher, A.; Fischer, M.; Cushman, M. J. Org.
Chem. 2007, 72, 7176.
cmꢁ1): 3404, 3300, 3188, 2926, 1616,1564, 690 cmꢁ1 1H NMR (DMSO-d6): d
;
0.93 (t, 3H, –CH3), d 1.31 (m, 2H, –CH2–), d 1.65 (m, 2H, –CH2–), d 2.76 (t, 2H, –
CH2), d 1.93 (t, 3H, –CH3), d 7.24–7.86 (m, 5H, Ar-H + s,1H,CH), d 8.19 (s, 1H, –
NH, D2O exchangeable), ppm;. EIMS (m/z) MS (m/z): 341 (M+), 343 (M+2), Anal.
Calcd for C19H20N3ClO: C, 66.76; H, 5.90; N, 12.29%. Found: C, 66.82; H, 5.78; N,
12.16%.
41. 3-[(4-Methoxyphenyl)-1-phenyl-1H-pyrrol-4-yl]methylene-4-methyl-1-
phenyl-1H-pyrrol-2(3H)one (IIIb): Color: Yellowish orange, mp(°C): 167–169,
yield-82, IR (KBr, cmꢁ1): 3325, 3158, 1620, 1574, 1435 cmꢁ1 1H NMR (DMSO-
;
d6): d 1.94 (t, 3H, CH3), d 3.76 (t, 3H, OCH3), d 7.1–7.98 (m, 15H, ArH + s,1H,CH),
ppm; EIMS (m/z) MS (m/z): 433 (M+), Anal. Calcd for C28H23N3O2: C, 77.58; H,
5.35; N, 9.69%. Found: C, 77.64; H, 5.28; N, 9.62%.
42. 3-[(4-Chlorophenyl)-1-phenyl-1H-pyrrol-4-yl]methylene-4-methyl-1-phenyl-
1H-pyrrol-2(3H)one (IIIc): Color: Orange solid, mp (°C): 167–169, yield-84, IR
(KBr, cmꢁ1): 3108, 3072, 2902, 1624, 1573,743 cmꢁ1 1H NMR (DMSO-d6): d
;
1.89 (t, 3H, CH3), d 7.2–7.98 (m, 15H, ArH + s,1H,CH), ppm; EIMS (m/z) MS (m/
z): 438(M+), 440(M+2), Anal. Calcd for C28H23ClN3O: C, 74.05; H, 4.60; N, 9.60%.
Found: C, 73.92; H, 4.46; N, 9.65%.
43. 3-[(4-Nitrophenyl)-1-phenyl-1H-pyrrol-4-yl]methylene-4-methyl-1-phenyl-
1H-pyrrol-2(3H)one (IIId): Colour: brown, mp (°C): 178–182, yield-85, IR (KBr,
cmꢁ1): 3539, 312, 3152, 1625, 1590, 778 cmꢁ1 1H NMR (DMSO-d6): d 1.90 (t,
;
3H, –CH3), d 7.18–7.96 (m, 15H, Ar-H + s,1H,CH), ppm;. EIMS (m/z) MS (m/z):
448(M+), Anal. Calcd for C19H20N3ClO: C, 72.31; H, 4.49; N, 12.49%. Found: C,
72.18; H, 4.42; N, 12.38%.
44. 4-(2-Butyl-5-chloro-4,5-dihydro-1H-imidazol-4-yl)-8(4-chlorophenyl)-
diazenyl)-3-methyl-1-phenyl-1H-dipyrazolo(1,5-a:30,40-d)pyrimidin-7-amine
(IVa): Colour: Yellowish powder, mp (°C): 180–182, yield-88, IR (KBr, cmꢁ1):
3541, 3355, 3162, 2315 1640, 1556, 758 cmꢁ1 1H NMR (DMSO-d6): d 0.90(t,
;
3H, –CH3), d 1.34 (m, 2H, –CH2–), d 1.62 (m, 2H, CH2), d 2.78 (t, 2H, –CH2), d 1.96
(t, 3H, CH3), d 3.43 (dd, 1H, HA), d 4.04 (dd, 1H, HB) d 4.92 (br s, 2H, NH2) d
7.14–7.92 (m, 9H, Ar-H), d 8.17 (s, 1H, –NH, D2O exchangeable), ppm; EIMS (m/
z) MS (m/z): 560(M+), 562(M+2), 564(M+4) Anal. Calcd for C27H26Cl2N10: C,
57.95; H, 4.29; N, 25.03%. Found: C, 57.86; H, 4.24; N, 25.12%.
45. 8(4-Chlorophenyl)-diazenyl)-4,5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
22. Vijaya Raj, K. K.; Narayan, B. V.; Ashalatha, N.; Suchita Kumari, J. J. Pharmacol.
Toxicol. 2006, 1, 559.
23. Peet, N. P.; Lentz, N. L.; Sunder, S.; Dudley, M. W.; Ogden, A. M. L. J. Med. Chem.
1992, 35, 3263.
24. Baguley, B. Anti-Cancer Drug Des. 1991, 6, 1.
25. Denny, W. Anti-Cancer Drug Des. 1989, 4, 241.
yl)-dipyrazolo(1,5-a:30,40-d)pyrimidin-7-amine (IVb):Colour: Orange, mp (°C):
167–169, yield-84, IR (KBr, cmꢁ1): 3567, 3390, 3180, 2338, 1610, 1545 cmꢁ1
;
1H NMR (DMSO-d6): d 1.90 (t, 3H, CH3), d 3.39 (dd, 1H, HA), d 3.89 (t, 3H, –
OCH3), d 4.10 (dd, 1H, HB) d 4.89 (br s, 2H, NH2) d 7.02–8.21 (m, 18H, ArH),
ppm; EIMS (m/z) MS (m/z): 653(M+), 655(M+2) Anal. Calcd for C36H29ClN10O:
C, 66.38; H, 4.16; N, 21.50%. Found: C, 66.32; H, 4.10; N, 21.36%.
26. Liu, L. F. Annu. Rev. Biochem. 1989, 58, 351.
46. 8(4-Chlorophenyl)-diazenyl)-4,5-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-
yl)-dipyrazolo(1,5-a:30,40-d)pyrimidin-7-amine (IVc): Colour: Yellowish, mp
(°C): 156–159, yield-86, IR (KBr, cmꢁ1): 3447, 3344, 3153, 2934, 1648,
27. Wolfe, A.; Shimer, G. H.; Meehan, T. Biochemistry 1987, 26, 6392.
28. Bhaskar, S. D.; Baseer, M. S.; Namdev, T. K.; Gajanan, G. M.; Santosh, S. C. K. S. G.
Asian J. Res. Chem. 2010, 3, 90.
29. Dawane, B. S.; Konda, S. G.; Mandawad, G. G.; Shaikh, B. M. Eur. J. Med. Chem.
2010, 45, 387.
30. Dawane, B. S.; Shaikh, B. M.; Khandare, N. T.; Kamble, V. T.; Chobe, S. S.; Konda,
S. G. Green Chem. Lett. Rev. 2010, 3, 205.
31. Li, T. R.; Yang, Z. Y.; Wang, B. D.; Qin, D. D. Eur. J. Med. Chem. 2008, 43, 1688.
32. Kumar, C.; Asuncion, E. H. J. Am. Chem. Soc. 1993, 115, 8547.
33. Reichmann, M. E.; Rice, S. A.; Thomas, C. A.; Doty, P. J. Am. Chem. Soc. 1954, 76,
3047.
34. Arjmand, F.; Mohani, B.; Ahmad, S. Eur. J. Med. Chem. 2005, 40, 1103.
35. Nandeshwarappa, B. P.; Aruna Kumar, D. B.; Bhojya Naik, H. S.; Mahadevan, K.
M. J. Sulfur Chem. 2005, 26, 373.
36. Prakash Naik, H. R.; Bhojya Naik, H. S.; Ravikumar Naik, T. R.; Naik, H. R.;
Lamani, D. S.; Aravinda, T. J. Sulfur Chem. 2008, 29, 583.
37. McCoubrey, A.; Latham, H. C.; Cook, P. R.; Rodger, A.; Lowe, G. FEBS Lett. 1996,
380, 73.
854,709 cmꢁ1 1H NMR (DMSO-d6): d 2.14 (t, 3H, CH3),d 3.22(dd, 1H, HA), d
;
4.70(dd, 1H, HB), d 3.86 (br s, 2H, –NH2) d 7.2–8.10 (m, 18H, ArH), ppm; EIMS
(m/z) MS (m/z): 656(M+), 658(M+2), 660(M+4) Anal. Calcd for C35H26Cl2N10: C,
64.12; H, 3.68; N, 21.36%. Found: C, 64.06; H, 3.57; N, 21.28%.
47. 8(4-Chlorophenyl)-diazenyl)-4,5-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-
dipyrazolo(1,5-a:30,40-d)pyrimidin-7-amine (IVd): Colour: Yellowish orange,
mp (°C): 161–163, yield-85, IR (KBr, cmꢁ1): 3478, 3150, 2929, 1649, 834 cmꢁ1
;
1H NMR (DMSO-d6): d 1.89 (t, 3H, CH3), d 3.47(dd, 1H, HA), d 4.15 (dd, 1H, HB),
4.86 (br s, 2H, –NH2) d 7.10–8.24 (m, 18H, ArH), ppm; EIMS (m/z) MS (m/z):
667(M+), Anal. Calcd for C35H26ClN11O2: C, 63.09; H, 3.62; N, 23.11%. Found: C,
62.95; H, 3.54; N, 22.98%.
48. Marmur, J. J. Mol. Biol. 1961, 3, 208.
49. Wu, P. K.; Kharatishvili, M.; Qu, Y.; Farrell, N. J. Inorg. Biochem. 1996, 63, 9.
50. Raja, A.; Rajendiran, V.; Uma Maheswari, P.; Balamurugan, R.; Kilner, C. A.;
Halcrow, M. A.; Palaniandavar, M. J. Inorg. Biochem. 2005, 99, 1717.
51. Kelly, J. M.; Tossi, A. B.; McConnell, D. J.; OhUigin, C. Nucleic Acids Res. 1985, 13,
6017.
38. General procedure for synthesis of 4-substituted benzylidene-3-methyl-1H-
pyrazol-5(4H)-one III(a–d):
A mixture of 3-methyl-1-phenyl-1H-pyrazol-
52. Marmur, J.; Doty, P. J. Mol. Biol. 1962, 5, 109.
5(4H)-one (1 mmol), hetero carbaldehyde (1 mmol) and sodium acetate
(4 mmol) was stirred in PEG-400 (10 ml) at 60–80 °C for 1–2 h. The
completion of reaction was checked by TLC and the reaction mixture was
extracted with diethyl ether (2 ꢀ 20 ml). The combined organic layers were
dried over anhydrous Na2SO4, and solvent was evaporated under reduced
pressure. The crude product was recrystallized from proper solvent to furnish
the desired product.
53. Lamani, D. S.; Reddy, K. R. V.; Naik, H. S. B. Afr. J. Pure Appl. Chem. 2010, 4, 247.
54. Srivastava, H. K.; Chourasia, M.; Kumar, D.; Sastry, G. N. J. Chem. Inf. Model.
2011, 51, 558.
55. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Daniel, T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K. J. Med. Chem. 2004, 47, 1739.
56. Thompson, A. S.; Hurley, L. H. J. Mol. Biol. 1995, 252, 86.
57. Timsit, Y.; Moras, D. EMBO J. 1994, 13, 2737.
58. Bhattacharyya, D.; Ramachandran, S.; Sharma, S.; Pathmasiri, W.; King, C. L.;
Baskerville-Abraham, I.; Boysen, G.; Swenberg, J. A.; Campbell, S. L.; Dokholyan,
N. V. PLoS ONE 2011, 6, e23582.
59. Andac, C. A.; Miandji, A. M.; Hornemann, U.; Noyanalpan, N. Int. J. Biol.
Macromol. 2011, 48, 531.
39. Synthesis of pyrazolo [1,5-a]pyrimidines IV(a–d): A mixture of IIIa (0.340 g,
1 mmol), 5-amino pyrazole (0.236 g, 1 mmol) and 1–2 pallets of NaOH were
dissolved in Polyethylene glycol (PEG-400 (15 mL). The reaction mixture was
stirred for the period as shown in Table 1. The progress of the reaction was
monitored by TLC. After completion, the reaction mixture was extracted with
diethyl ether (2 ꢀ 20 mL). The combined organic layers were dried over
anhydrous Na2SO4, and the solvent was evaporated under reduced pressure.
The crude product was recrystallized from proper solvent to give the
60. Shelke, S. M.; Bhosale, S. H.; Dash, R. C.; Suryawanshi, M. R.; Mahadik, K. R.
Bioorg. Med. Chem. Lett. 2011, 21, 2419.