Synthesis, Characterization, Solvatochromic Properties, and Antimicrobial-radical Scavenging Activities of New Diazo Dyes Derived from Pyrazolo[1,5-a]pyrimidine

Article abstract of DOI:10.1002/jhet.2977

5-Amino-3-methyl-4-phenylazo-1H-pyrazole and ethyl cyanoacetate reacted in solvent-free media at 150°C to produce 7-amino-3-phenylazo-2-methyl-4H-pyrazolo[1,5-a]pyrimidine-5-one (3). A series of aromatic amines was coupled using this compound (3) and nitrous acid to produce new pyrazolo[1,5-a] pyrimidine derivatives with two arylazo groups 4(a-m). The structures of these dyes were determined via UV–vis, Fourier transform infrared, proton nuclear magnetic resonance, high-resolution mass spectral data, and elemental analysis. After synthesis, the solvent and acid–base effects of the dyes were investigated within the UV–vis region. The antimicrobial properties of the dyes were also studied. All dyes exhibited activity against Gram-positive and Gram-negative bacteria, and even against fungi. The results were compared to conventional reference results from the antibiotics ciprofloxacin and ketoconazole. Antioxidant potentials were analyzed using in vitro antioxidant models on the basis of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activities. Most of the compounds exhibited excellent antioxidant activities. In particular, compound 4b had a higher activity than Vitamin C.

Full text of DOI:10.1002/jhet.2977

Month 2017
Synthesis, Characterization, Solvatochromic Properties, and Antimicro-
bial-radical Scavenging Activities of New Diazo Dyes Derived from
Pyrazolo[1,5-a]pyrimidine
b
c
d
Nesrin Şener,a
*
Selinay Erişkin, Serkan Yavuz, and İzzet Şener
aDepartment of Chemistry, Faculty of Science-Arts, Kastamonu University, 37200 Kastamonu, Turkey
bDepartment of Chemistry, Faculty of Science-Art, Pamukkale University, 20200 Denizli, Turkey
cDepartment of Chemistry, Faculty of Science, Gazi University, 06500 Ankara, Turkey
dDepartment of Food Engineering, Faculty of Engineering and Architecture, Kastamonu University, 37200 Kastamonu,
Turkey
*
E-mail: isener@kastamonu.edu.tr
Received May 30, 2017
DOI 10.1002/jhet.2977
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
5-Amino-3-methyl-4-phenylazo-1H-pyrazole and ethyl cyanoacetate reacted in solvent-free media at
150°C to produce 7-amino-3-phenylazo-2-methyl-4H-pyrazolo[1,5-a]pyrimidine-5-one (3). A series of aro-
matic amines was coupled using this compound (3) and nitrous acid to produce new pyrazolo[1,5-a] pyrim-
idine derivatives with two arylazo groups 4(a-m). The structures of these dyes were determined via UV–vis,
Fourier transform infrared, proton nuclear magnetic resonance, high-resolution mass spectral data, and ele-
mental analysis. After synthesis, the solvent and acid–base effects of the dyes were investigated within the
UV–vis region. The antimicrobial properties of the dyes were also studied. All dyes exhibited activity against
Gram-positive and Gram-negative bacteria, and even against fungi. The results were compared to conven-
tional reference results from the antibiotics ciprofloxacin and ketoconazole. Antioxidant potentials were
analyzed using in vitro antioxidant models on the basis of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical
scavenging activities. Most of the compounds exhibited excellent antioxidant activities. In particular, com-
pound 4b had a higher activity than Vitamin C.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
these molecules have pharmacological and biological
activities [1–3,5]. In addition, pyridyl-benzimidazole
analogues were evaluated in vitro for urease inhibitory
activity [11]. Indole-2-hydrazones were synthesized and
evaluated for their antileishmanial activities [12].
Oxindole derivatives were analyzed for their alpha-
glucosidase[13] and urease inhibitory activities [14], and
the molecular interactions of the active compounds within
the urease enzyme binding sites were studied via
molecular docking simulations.
Nitrogen-containing heterocyclic compounds are of
great interest to researchers owing to their biological
activities. Of these, heterocycles with pyrimidine
molecules represent a continuously expanding field of
research in heterocyclic chemistry because of their
syntheses, reactions, and biological activities. Pyrimidine
structures are seen in numerous pharmaceutical agents
and natural products [1–8].
Nitrogen heterocycles are well known for their special
value as pharmaceuticals. The pyrimidine nucleus is the
key feature of various drugs. Several studies of dyes that
include pyrimidine note that they have been used as
hypnotic drugs for the nervous system [9], to detect
cancer, as chemotherapeutic components, and within the
structures of nucleic acids in living cells [10]. Some of
Aminopyrazole compounds can be produced via
reactions between nitriles and hydrazine hydrate [15–21].
Pyrazole
derivatives
exhibit
biological
and
pharmacological activities [22–24]. Pyrazolopyrimidines
and related fused heterocycles are potential bioactive
molecules and are of great interest to researchers [25,26].
They have been proven to exhibit pharmacological
© 2017 Wiley Periodicals, Inc.

N. Şener, S. Erişkin, S. Yavuz, and İ. Şener
Vol 000
activities such as central nervous system depression,
reduction of nervous tension via depression of nerve
functions, and inhibition of tubercle mycobacteria
growth [27].
In this study, we synthesized various pyrazolo[1,5-a]
pyrimidine derivatives 4(a-m) and investigated their
spectral properties. The effects of solvents, acids, and
bases on their visible absorption spectra and antimicrobial
activities were evaluated.
NMR results include a singlet signal at δ 2.52 ppm from
–CH3 protons, as well as singlet peaks at δ 5.20 ppm and
at δ 5.75 ppm attributed to –CH and –NH2 protons,
respectively. The broad peak at δ 9.82 ppm was due to
–NH protons. The FT-IR spectra of dyes 4(a–m) exhibited
–NH bands at 3414–3265 cmꢀ1, as well as ═NH bands at
3328–3062 cmꢀ1. Compounds other than 4i and 4k
exhibited an intense band at 3569–3319 cmꢀ1 resulting
from –OH stretching vibrations. We think that this solid
state compound was present only in the imino-diazo-enol
form (T6). However, 4i and 4k exhibited bands from
RESULT AND DISCUSSION
carbonyl (C═O) groups at 1646 cmꢀ1 and 1636 cmꢀ1
,
respectively. We suggest that all of the compounds, except
for 4i and 4k, were in imino-diazo-keto (T1), imino-
hydrazo-keto-azo(T2), imino-diazo-keto (T3), imino-azo-
keto-hydrazo(T4), and amino-diazo-keto (T5) forms in
the solid state.
Chemistry.
This work is the continuation of our
previous studies [28,29]. We report here synthesis of
7-imino-3-phenylazo-6-arylazo-2-methyl-4H-pyrazolo[1,5-
a]pyrimidine-5-ole derivatives 4(a-m). Dye structures were
identified via several spectroscopic methods, including
proton nuclear magnetic resonance (1H-NMR), Fourier
1H-NMR spectra of azopyrazolo[1,5-a]pyrimidine
derivatives 4(b–m) exhibit a broad peak at δ 13.41–
9.36 ppm attributed to ═NH and –NH protons. The broad
peak at δ 11.61–16.09 ppm resulted from tautomeric
transform–infrared
(FT-IR)
spectroscopy,
mass
spectroscopy (MS), and elemental analysis. Compounds
1–2 were synthesized according to the previously reported
procedure [15,16,30], shown in Scheme 1.
7-Amino-3-phenylazo-2-methyl-4H-pyrazolo[1,5-a]
pyrimidine-5-one (3) was produced via a reaction between
5-amino-4-arylazo-3-methyl-1H-pyrazole
cyanoacetate at 150°C in solvent free media. The reaction
is depicted in Scheme 2.
Aniline derivatives were diazotized with nitrous acid
in the presence of sodium nitrite and hydrochloric acid
at 0–5°C, as shown in Scheme 3. Then a reaction
between diazonium salts and 7-amino-3-phenylazo-2-
1
hydroxyl –OH protons. H-NMR spectra of 4a showed a
singlet peak at δ 4.97 ppm resulting from –CH protons, a
broad peak at δ 10.12 ppm attributed to ═NH protons,
and a broad peak at δ 12.23 ppm from –NH protons. The
1H-NMR results show that the dyes 4(b-m) prefer imino-
diazo-enol form (T6). As seen in Scheme 4, the dye 4a
may exist in various tautomeric forms including imino-
diazo-keto (T1), imino-diazo-keto (T3), and imino-azo-
keto-hydrazo(T4) forms in DMSO-d6. The mass spectra
of the dyes match the molecular weights of the respective
compounds.
and
ethyl
methyl-4H-pyrazolo[1,5-a]pyrimidine-5-one
produced
Absorption spectra. All molecules have ground states
that are less polar than their excited states. Therefore,
polar solvents in excited states tend to stabilize more than
those in ground states. In addition, the relationship
between solvent polarity and dielectric constant is
proportional. [31,32]. For the UV measurements, we used
the following solvent dielectric constants (ε): DMSO
(46.45), DMF (36.71), acetonitrile (35.94), methanol
(32.66), acetic acid (6.17), and chloroform (4.89) [33].
The UV–vis absorption spectra of the synthesized dyes
were scanned from λ = 300 to λ = 700 nm at various
solvent concentrations (10ꢀ8 to 10ꢀ6 M). Spectral data
from all dyes are shown in Table 1.
corresponding 7-imino-3-phenylazo-6-arylazo-2-methyl-
4H-pyrazolo[1,5-a]pyrimidine-5-ole derivatives. In this
way, 13 new azo pyrazolo[1,5-a]pyrimidine derivatives
were synthesized.
Compounds 4(a–m) have six different tautomeric forms.
As shown in Scheme 4, the forms are imino-diazo-keto
(T1), imino-hydrazo-keto-azo(T2), imino-diazo-keto (T3),
imino-azo-keto-hydrazo(T4), amino-diazo-keto (T5),
imino-diazo-enol (T6). The FT-IR spectrum of compound
3 includes –OH stretching vibrations at 3580 cmꢀ1. While
a band situated at 3334 cmꢀ1 was assigned to –NH
stretching vibrations, ═NH stretching vibrations were also
seen at 3095 cmꢀ1. Another vibration at 1624 cmꢀ1
resulted from C═N stretching. No carbonyl (C═O)
stretching FT-IR band is observed from compound 3. Its
Generally, the visible absorption spectra of all dyes
except for chloroform correlate with solvent polarity. Dye
4m exhibits a bathochromic shift in chloroform relative
Scheme 1. Synthesis of compounds (1) and (2). [Color figure can be viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Pyrazolo[1,5-a]Pyrimidine Dyes
Scheme 2. Synthesis of compound (3). [Color figure can be viewed at wileyonlinelibrary.com]
Scheme 3. Synthesis of compounds 4(a–m).
Scheme 4. The tautomeric form of azo pyrazolo[1,5-a]pyrimidine derivatives.
to DMSO and DMF. For instance, dye 4m exhibited
absorption maxima at 397 nm in DMSO and 394 nm in
DMF but 398 nm in chloroform. Absorption
measurements showed that the λmax of the dye 4a was the
same in DMSO as in DMF. While the other dyes
exhibited bathochromic shifts in most polar solvents such
as DMSO and DMF, dyes 4f, 4g, and 4i did not. For
example, the maximum absorption of dye 4g was
observed at 373 nm in DMSO and 375 nm in DMF. Dye
4d exhibits absorption maxima at 402 nm in DMSO,
401 nm in DMF and acetonitrile, 392 nm in methanol,
396 nm in acetic acid, and 400 nm in chloroform. The
maximum absorption shifts of dye 4d in various solvents
are indicated in Figure 1.
The absorption spectrum of 4k exhibited two
absorbance peaks in acetonitrile. While dyes such as 4b
and 4m exhibited two absorption peaks with a shoulder
in DMF and acetonitrile, the others had either a single
absorption peak or a single absorption peak with
shoulders. Dyes 4a and 4g also had one absorption
maximum each but gave different results in acetic acid
and chloroform. With dyes 4c and 4e, a single absorption
peak was observed in all solvents but was not observed
for these compounds in chloroform and in acetonitrile,
Journal of Heterocyclic Chemistry
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N. Şener, S. Erişkin, S. Yavuz, and İ. Şener
Vol 000
Table 1
Influence of solvent on λmax (nm) of dyes.
Dye no.
DMSO
DMF
Acetonitrile
Methanol
Acetic acid
Chloroform
4a
4b
4c
4d
4e
4f
4g
4h
4i
374
373, 412a, 466a
371
374
371, 410, 457a
364
369
408, 362a, 452a
364
365
361, 405a
364
352, 430a
355, 403a, 448a
353
362, 438a
366, 407a, 458a
362, 370a
402, 430a
373
401, 434a
371
401, 435a
365
392, 438a
361
396, 337a, 438a
358, 467a
400, 426a, 444a
364
400, 473a
373
402, 441a
375
397, 441a
367
393, 433a
365
392, 435a
397, 441a
362, 461a
369, 406a
402, 443a
393, 441a
432, 360a
414, 337a, 438a
410, 437a
397, 448a
397, 447a
394, 439a
424, 360a
411, 435a, 460a, 337a
403, 433a, 453a
394, 448a
396, 438a
394, 437a
419, 350a
410, 433a, 338a
406, 428a, 448a
384, 443a
396, 437a
388, 435a
414, 348a
406, 430a, 455a
401, 441a
391, 442a
394, 433a
397, 423a, 443a
394, 442a
342, 392a, 435
419, 342a
4j
4k
4l
423, 348a
334, 485
411, 432a, 457a, 338a
403, 424a, 448a
398, 443a
401, 330a, 435a
350, 393, 437a
4m
aShoulder.
Table 2
Absorption maxima of dyes in acidic and basic solutions.
Methanol +
HCl
Methanol +
KOH
Dye no.
Methanol
4a
4b
4c
4d
4e
4f
4 g
4 h
4i
365, 433a
361, 405a
364
334, 453
341, 452
341, 455
332, 453
348, 453
334, 425
355, 461
333, 441
337, 455
341, 447
337, 478
385, 430a
391, 426a, 515a
388, 425a
394, 441
388, 430a
391, 447
393
391, 443
393, 438a
389, 461
444, 414a
392, 438a
361
393, 433a
365
396, 437a
388, 435a
414, 348a
406, 430a,
455a
4j
4k
4l
4m
401, 441a
391, 442a
338, 446
334, 458
398, 447
396, 435a
Figure 1. Absorption spectra of dye 4d in various solvents. [Color figure
can be viewed at wileyonlinelibrary.com]
aShoulder
single tautomeric form in this solution. However, the
situation is different for dyes such as 4a, 4b, 4d, 4f, 4h,
4i, 4j, 4k, 4l, and 4m, which are present more than one
tautomeric form. Adding HCl (0.1 M) to the methanol
solution causes a hypsochromic shift, and the formation
of two absorption maxima. It is thought that all dyes are
present primarily in two tautomeric forms. Upon adding
KOH (0.1 M) in methanol, a bathochromic shift in the
λmax values is detected and one absorption maximum
with a shoulder appears. Different behavior is observed
for 4d, 4f, 4g, 4h, 4j, and 4k. These results show that
dyes 4a, 4b, 4c, 4e, 4i, 4l, and 4m exist as a mixture of
tautomers. In a basic solution, dyes 4d, 4f, 4h, 4j, and 4l
are predominantly present in two tautomeric forms, but
dye 4g is present as a single tautomer. Spectral shifts of
compound 4d are seen in acidic and basic solutions and
are shown in Figure 2.
respectively. On the basis of these results, one can suggest
that dyes 4a and 4g exist predominantly in only one
tautomeric form in solvents other than acetic acid and
chloroform. Likewise, dyes 4c and 4e exist
predominantly in one tautomeric form in all solvents
except for chloroform and acetonitrile, respectively. It is
likely that other synthesized dyes exist in different
tautomeric forms in various solvents. The tautomeric
forms of dyes 4(a-m) are shown in Scheme 4.
A study of the effects of acids and bases on dye
absorption has also been performed. The results are
shown in Table 2.
Addition of acids and bases to the dyes in methanol
caused some changes. Most dyes exhibited a single
absorption maximum with a shoulder in methanol except
for 4c, 4e, and 4g. It is possible that these dyes exist in a
Journal of Heterocyclic Chemistry
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Month 2017
Pyrazolo[1,5-a]Pyrimidine Dyes
Ciprofloxacin and ketoconazole were used as antibacterial
and antifungal references, respectively. All results were
collected and average inhibition zone diameters with no
bacterial or fungal growth were measured in millimeter.
All dyes obtained from this study exhibited excellent
antimicrobial and antifungal activities when compared
with the reference drugs. These dyes had inhibition
zones of various sizes when exposed to bacteria
(Table 3). In addition, the minimum inhibitory
concentrations (MICs) of these compounds were
determined to elucidate their inhibitory effects on various
bacterial and fungal strains as seen in Table 4.
The antibacterial screening data showed that that all
tested dyes exhibited significant and diverse activities
against the four human pathogenic bacteria. In general,
most of the target compounds exhibited better activities
against the Gram-negative bacteria than the Gram-
positive bacteria. Compounds 4b, 4d, 4e, 4j, and 4l
exhibited the highest potencies against the organisms
considered. The highest anti-bacterial activities were
achieved by compounds 4b, 4d, 4e, and 4j. This may
stem from the presence of aromatic rings, chlorine, and
nitro groups in para and ortho positions.
These compounds are better antibacterial agents than
antifungal agents. When potency against the various
organisms was compared with those of reference drugs,
good antimicrobial activity was observed with compound
4l. Dye 4d inhibited the growth of S. aureus ATCC
29213, B. subtilis ATCC6633, and K. pneumonia
Figure 2. Absorption spectra of dye 4d in acidic and basic solutions.
[Color figure can be viewed at wileyonlinelibrary.com]
Antimicrobial activity. In this study, 13 of the newly
synthesized compounds were tested for antimicrobial and
antifungal properties. We used concentrations of
100 μg/mL for each compound. In these analyses,
various microorganisms such as Staphylococcus aureus
ATCC 29213, Gram (+) Bacillus subtilis ATCC 6633,
Gram (ꢀ) Klebseilla pneumonia ATCC13883 and
Escherichia coli ATCC 25922, Saccharomyces cerevisiae,
and fungus Candida albicans NRRL Y-477 were used.
The agar-diffusion method was used to determine the
preliminary antibacterial and antifungal activities.
Table 3
Antimicrobial activity expressed as inhibition diameter zones in millimeters (mm) of chemical compounds against the pathological strains based on well-
diffusion assay.
Zone of inhibition (mm)
Bacteria
Fungi
Gram-positive
Staphylococcus
Gram-negative
Bacillus
subtilis
Klebseilla
Escherichia
coli
Saccharomyces
cerevisiae
Candida
albicans
Compound
aureus
pneumoniae
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
18 0.4
24 0.8
22 1.1
32 0.5
20 0.3
18 0.7
26 0.7
21 0.3
24 0.4
30 0.6
26 0.8
27 1.0
24 0.3
29 0.4
NT
23 0.6
24 0.3
26 0.4
29 0.6
23 0.9
16 1.2
21 0.4
17 0.7
18 0.5
23 0.2
21 0.5
29 0.6
16 0.4
28 0.3
NT
23 0.7
31 0.8
24 0.6
32 0.9
31 0.5
26 1.0
24 1.1
18 0.9
16 0.5
29 0.6
28 0.7
26 0.7
20 1.5
29 0.8
NT
20 1.1
26 0.6
23 0.4
27 0.4
15 0.7
24 0.6
20 0.3
16 1.4
15 0.3
28 0.6
21 0.4
29 0.9
16 0.7
25 0.6
NT
21 0.4
26 1.3
23 0.6
22 0.4
19 0.1
14 0.3
18 0.5
21 0.8
20 0.9
26 0.3
18 1.1
27 0.8
21 0.7
NT
19 0.6
28 0.7
23 0.2
20 0.4
21 0.3
19 0.5
15 1.3
24 0.6
23 0.9
20 0.4
23 0.3
30 0.6
25 0.3
NT
4m
Ciprofloxacin
Ketoconazole
27 0.4
29 0.6
Zone of inhibition values are presented as the mean SEM from at least three separated experiments NT not tested.
Journal of Heterocyclic Chemistry
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N. Şener, S. Erişkin, S. Yavuz, and İ. Şener
Vol 000
Table 4
Minimum inhibitory concentration (μg/mL) against the pathological strains based on two fold serial dilution technique.
MIC in μg/mL
Bacteria
Fungi
Gram-positive
Staphylococcus
Gram-negative
Bacillus
subtilis
Klebseilla
Escherichia
coli
Saccharomyces
cerevisiae
Candida
albicans
Compound
aureus
pneumoniae
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
66
33
33
8.25
66
132
33
33
33
16.5
8.25
33
132
66
132
132
33
33
8.25
33
66
16.5
66
16.5
132
33
66
16.5
33
66
16.5
33
66
66
66
132
33
33
66
33
8.25
33
NT
8.25
8.25
8.25
16.5
33
132
132
8.25
16.5
16.5
66
66
132
132
132
66
66
66
33
132
132
16.5
66
8.25
132
16.5
NT
66
8.25
16.5
16.5
33
8.25
NT
16.5
132
16.5
66
NT
16.5
66
16.5
132
16.5
NT
4m
Ciprofloxacin
Ketoconazole
8.25
NT
NT, not tested.
ATCC13883. In addition, compound 4j exhibited good
activity against S. aureus ATCC 29213 and
K. pneumonia ATCC13883. Compound 4 l showed
highest activities against S. cerevisiae and the C. albicans
NRRL Y-477 fungal strain.
(IC50 = 24.44 μg/mL) than the vitamin C used as a
reference (IC50 = 26.71 μg/mL).
The test compounds, such as 4c, 4e, 4h, 4i, 4j, 4l, and 4m
exhibit IC50s of 57.91, 63.85, 64.32, 57.69, 32.33, 42.87,
and 53.24 μg/mL, respectively. In addition, the remaining
test compounds exhibit IC50 values above 100 μg/mL.
Vitamin C was used as a positive control. The IC50
value was determined as the effective concentration at
which 50% of DPPH radicals were removed. The IC50
value was determined via interpolation after linear
regression analysis.
DPPH radical scavenging activity.
Free radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH) provides rapid,
a
simple, and inexpensive method of evaluating the
antioxidant capacities of various substances. In this
method, a stable radical is generated directly before
exposure to and reaction with putative antioxidants.
This method was used with all of the compounds
obtained. Ascorbic acid was used as a positive control
material. In Table 5, 50% inhibitory concentrations are
expressed as IC50s. All compounds exhibit DPPH
radical quenching activities in a concentration dependent
manner. Compound 4b exhibits better activity
CONCLUSION
In this work, new azopyrazolo[1,5-a]pyrimidine
derivatives 4(a-m) were synthesized. The resulting dyes
were evaluated for antimicrobial activity and compared to
various standards. Some of the compounds exhibited
significant antibacterial and antifungal activities.
Furthermore, the antioxidant activities of the dyes were
investigated. All of the compounds tested exhibited
DPPH radical quenching activities.
Table 5
DPPH radical scavenging activity of compounds 4(a-m).
Compound
IC50 (μM)
Compound
IC50 (μM)
4a
4b
4c
4d
4e
4f
94.56 1.41
24.45 0.96
57.91 2.24
145.32 1.55
63.85 1.39
81.24 0.67
110.87 2.01
4h
4i
4j
4k
4l
64.32 1.53
57.69 0.62
32.33 1.90
108.41 1.66
42.87 1.47
53.24 1.23
26.71 1.62
EXPERIMENTAL
General. All chemicals were purchased and were used
as received. Solvents were spectroscopic grade. A melting
point apparatus (Stuart SMP 30, UK) was used to
4m
Vitamin C
4g
Journal of Heterocyclic Chemistry
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Month 2017
Pyrazolo[1,5-a]Pyrimidine Dyes
1
determine the dye melting points. H-NMR spectra were
recorded using a Bruker (Germany) Ultra Shield Plus
400-MHz spectrometer at room temperature in deuterated
dimethyl sulphoxide (DMSO-d6). Chemical shifts are (δ)
given in ppm. FT-IR spectra were recorded on a Perkin
Elmer Mattson (USA) 1000 FT-IR spectrometer. A Waters
LCT Premier XE LTOF (TOFMS) instrument (Waters Cor-
poration, Milford MA, USA) was used for MS. A Leco
CHNS-932 analyzer (USA) was used for elemental analy-
sis. UV–vis absorption spectra were recorded on a
Shimadzu (Japan) UV-1601 UV–visible spectrophotometer
over the λ range of 300 to 700 nm. The wavelengths of
maximum absorption (λmax) were determined with DMSO,
DMF, acetonitrile, methanol, acetic acid, and chloroform as
solvents using concentrations that ranged from 1 × 10ꢀ8 to
1 × 10ꢀ6 M. Changes in absorption maxima were investi-
gated by adding 0.1 mL of hydrochloric acid and 0.1 mL
of potassium hydroxide to 1 mL of the dye solution in
methanol.
decomp > 130°C; FT–IR (cmꢀ1) νmax: 3580 (ꢀOH), 3334
(ꢀNH), 3095 and 3073 (Ar–H), 2927 (Aliphatic C–H),
1624 (C═N), 1560 (N═N); 1H-NMR (400 MHz, CDCl3) δ
(ppm): 2.52 (3H, s, ꢀCH3), 5.20 (1H, s, ꢀCH), 5.75 (2H,
s, NH2), 7.31 (1H, t, J = 7.3 Hz, H-18), 7.38 (2H, t,
J = 7.4 Hz, H-17 and H-19), 7.68 (2H, d, J = 7.5 Hz, H-16
and H-20), 9.82 (b, pyrimido –NH); 13C-NMR (100 MHz,
DMSO-d6) δ:12.37 (CH3, C-12), 83.49 (CH, C-6), 109.84
(C, C-9), 125.66 (CH, C-16, C-20), 127.34 (CH, C-17, C-
19), 129.37 (CH, C-18), 143.35 (C, C-8), 146.91 (C, C-7),
150.32 (C, C-15), 162.21 (C, C-5), 169.90 (C, C-3); HR-
MS: 268.2816 [M + H]+, calcd 268.2819. Anal. Calcd for
C13H12N6O: C, 58.20%; H, 4.51%; N, 31.33%. Found: C,
58.22%; H, 4.50%; N, 31.32%.
General procedure for the synthesis of azo dyes 4(a–m). 7-
Imino-2-methyl-3, 6-diphenylazo-6, 7-dihydro-1H-pyrazolo[1,5-
a]pyrimidine-5-one (4a).
A solution of aniline (1.04 g,
11.19 mmol) in hydrochloric acid (7.5 mL) was reacted
with a solution of sodium nitrite (1.16 g, 16.80 mmol) in
water (10 mL) at 0–5°C. This mixture was stirred over 1 h.
The resulting diazonium solution was added to a
vigorously stirred solution of 7-amino-3-phenylazo-2-
methyl-4H-pyrazolo[1,5-a]pyrimidine-5-one (3.00 g,
11.19 mmol), which was dissolved in pyridine (10 mL).
The reaction was stirred for 2 h at 0–5°C. The product
separated upon dilution with water (50 mL). It was
filtered, dried, and crystallized from a mixture of DMSO
and water (2:3) to obtain 7-imino-2-methyl-3,6-
diphenylazo-6,7-dihydro-1H-pyrazolo[1,5-a]pyrimidine-5-
one (4a) as a brown crystals, yield (75%), mp: 262–264°C;
FT–IR (cmꢀ1) νmax: 3319 (ꢀOH), 3265 (ꢀNH), 3062
(═NH), 3062 (Ar–H), 2926 (Aliphatic C–H), 1597 (C═N),
Synthesis. Synthesis of 2-phenylhydrazone-3-ketimino
butyronitrile (1). A solution of aniline (0.93 g, 10 mmol)
in hydrochloric acid (10 mL) reacted with a sodium
nitrite (10 mmol) solution in water (3 mL) at 0–5°C for
2 h. The resulting diazonium salt solution was then added
to 3-aminocrotononitrile (0.82 g, 10 mmol) in sodium
acetate (2 g, dissolved in 10 mL of 1:1 ethanol/water)
under stirring, at the same temperature. Stirring continued
for 2 h and cold water (50 mL) was added. Solids were
filtered off, dried and recrystallized from aqueous
ethanol. Greenish-yellow crystals were obtained (1.45 g,
78% yield), mp: 166–167°C.
Synthesis of 5-amino-3-methyl-4-phenylazo-1H-pyrazole
(2). Compound 1 (1.86 g, 10 mmol) was dissolved in
1
1563 (N═N); H-NMR (400 MHz, DMSO-d6) δ (ppm):
2.58 (3H, s, ꢀCH3), 4.97 (1H, s, ꢀCH),7.42 (1H, t,
J = 7.3 Hz, H-25), 7.47-7.57 (5H, m, H-16, H-15, H-17,
H-24, H-26), 7.83 (1H, d, J = 7.3 Hz, H-14), 7.94 (2H, d,
J = 7.3 Hz, H-18, H-27), 8.01 (1H, d, J = 7.3 Hz, H-23),
10.12 (1H, b, pyrimido ═NH), 12.23 (1H, b, pyrazole–
NH); 13C-NMR(100 MHz, DMSO-d6) δ:12.37 (CH3,
C-28), 92.78 (CH, C-4), 118.22 (C, C-8), 123.24 (CH,
C-14, C-18), 125.66 (CH, C-23, C-27), 127.34 (CH, C-24,
C-26), 129.37 (CH, C-25), 129.64 (C, C-16), 130.01 (CH,
C-15, C-17), 140.92 (C, C-3), 146.90 (C, C-9), 151.41 (C,
C-13), 151.44 (C, C-22), 162.51 (C, C-7), 163.72 (C, C-
5); HR-MS: 372.3954 [M + H]+, calcd 372.3955. Anal.
Calcd for C19H16N8O: C, 61.28%; H, 4.33%; N, 30.09%.
Found: C, 61.24%; H, 4.36%; N, 30.11%.
30 mL of ethanol and hydrazine hydrate (0.5 g, 10 mmol)
was added. The reaction balloon was heated under reflux
for 4 h, and then cooled to room temperature. The
precipitated products separated upon dilution with water
(50 mL) and were filtered off, dried, and recrystallized
from aqueous ethanol. Yellow crystals were obtained
(1.52 g, 76% yield), mp: 165–166°C.
Synthesis of 7-amino-3-phenylazo-2-methyl-4H-pyrazolo
[1,5-a]pyrimidine-5-one (3).
Compound (3) was
synthesized via a reaction between 5-amino-3-methyl-4-
phenylazo-1H-pyrazole and ethyl cyanoacetate. 5-Amino-
3-methyl-4-phenylazo-1H-pyrazole (2.01 g, 10 mmol)
reacted with ethyl cyanoacetate 6.0 mL (50 mmol)
without a solvent. The reaction was heated for 2 h at
150°C and then allowed to cool to 60–70°C. It was then
heated under reflux in 20–30 mL of acetic acid. The
mixture was cooled and added to a 100 mL mixture of
ethanol and water (1:1). The precipitated brown product
was filtered off, washed with water several times, and
dried. The obtained product was crystallized from
DMSO : water mixture as brown crystals, yield (82%),
The above procedure was also used to synthesize dye
4(b–m). The general route of synthesized dyes is outlined
in Scheme 3.
7-Imino-3-phenylazo-6-(40-nitrophenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4b).
Dark brown solid
crystals, (78%), decomp > 287°C; FT–IR (cmꢀ1) νmax
3371 (ꢀOH), 3371 (ꢀNH), 3063 (═NH), 3063 (Ar–H),
:
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

N. Şener, S. Erişkin, S. Yavuz, and İ. Şener
Vol 000
2925(Aliphatic C–H), 1641 (C═N), 1599 (N═N), 1505 and
1315 (ꢀNO2); 1H-NMR (400 MHz, DMSO-d6) δ
(ppm):2.57 (3H, s, ꢀCH3), 7.41 (1H, t, J = 7.3 Hz, H-25),
7.52 (2H, t, J = 7.4 Hz, H-24, H-26), 7.83 (2H, d,
J = 7.3 Hz, H-23, H-27), 7.92 (2H, d, J = 8.0 Hz, H-14,
H-18), 8.10 (2H, d, J = 8.0 Hz, H-15, H-17), 9.82 (1H, b,
pyrimido ═NH), 11.05 (1H, b, pyrimido –NH), 12.32
(1H, b, ꢀOH); 13C-NMR(100 MHz, DMSO-d6) δ:12.40
(CH3, C-28), 92.75 (C, C-4), 118.22 (C, C-8), 123.24
(CH, C-14, C-18), 125.66 (CH, C-23, C-27), 125.77 (CH,
C-15, C-17), 127.34 (CH, C-24, C-26), 129.37 (CH, C-
25), 140.92 (C, C-3), 146.89 (C, C-9), 148.40 (C, C-16),
151.44 (C, C-22), 154.60 (C, C-13), 162.52 (C, C-7),
163.72 (C, C-5); HR-MS: 417.3951 [M + H]+, calcd
417.3955. Anal. Calcd for C19H15N9O3: C, 54.68%; H,
3.62%; N, 30.20%. Found: C, 54.65%; H, 3.63%; N,
30.24%.
160.53 (C, C-7), 194.63 (C, C-5); HR-MS:407.1132
[M
H]+, calcd 407.1136. Anal. Calcd for
C19H15ClN8O: C, 56.09%; H, 3.72%; N, 27.54%. Found:
+
C, 56.07%; H, 3.75%; N, 27.52%.
7-Imino-3-phenylazo-6-(40-methylphenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4e). Dark mustard yellow
solid crystals, (73%), mp: 213°C; FT–IR (cmꢀ1) νmax
:
3332 (ꢀOH), 3332 (ꢀNH), 3094 (═NH), 3071 (Ar–H),
2926 (Aliphatic C–H), 1623 (C═N), 1597 (N═N); 1H-
NMR (400 MHz, DMSO-d6) δ (ppm): 2.60 (3H, s, Ar–
CH3), 2.69 (3H, s, pyrazole–CH3), 7.18 (1H, t,
J = 7.3 Hz, H-25), 7.47 (2H, d, J = 7.5 Hz, H-15, H-17),
7.52 (2H, t, J = 7.3 Hz, H-24, H-26), 7.65 (2H, d,
J = 8.0 Hz, H-14, H-18), 7.98 (2H, d, J = 7.3 Hz, H-23,
H-27),11.76 (2H, b,pyrimido ═NH, pyrimido –NH),
16.09 (1H, b, ꢀOH); 13C-NMR (100 MHz, DMSO-d6) δ:
12.37 (CH3, C-28), 22.21 (CH3, C-29), 101.71 (C, C-4),
112.04 (CH, C-14, C-18), 121.83 (C, C-8), 124.85 (CH,
C-23, C-27), 127.34 (CH, C-24, C-26), 129.37 (CH, C-
25), 130.70 (CH, C-15, C-17), 132.32 (C, C-16), 140.52
(C, C-3), 144.36 (C, C-9),149.61 (C, C-13),151.48 (C, C-
22), 160.53 (C, C-7), 194.63 (C, C-5); HR-MS: 386.4223
[M + H]+, calcd 386.4227. Anal. Calcd for C20H18N8O:
C, 62.17%; H, 4.70%; N, 29.00%. Found: C, 62.12%; H,
4.72%; N, 29.04%.
7-Imino-3-phenylazo-6-(40-methoxyphenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4c).
Brick red solid
crystals, (74%), mp: 255–257°C; FT–IR (cmꢀ1) νmax
:
3569 (ꢀOH), 3318 (ꢀNH), 3068 (═NH), 3068 (Ar–H),
2927 (Aliphatic C-H), 1661 (C═N), 1622 (N═N); 1H-
NMR (400 MHz, DMSO-d6) δ (ppm): 2.57 (3H, s,
ꢀCH3), 3.77 (3H, s, ꢀOCH3), 7.19 (2H, d, J = 7.5 Hz,
H-15, H-17), 7.43 (1H, t, J = 7.3 Hz, H-25),7.56 (2H, t,
J = 7.3 Hz, H-24, H-26), 7.74 (2H, d, J = 8.0 Hz, H-14,
H-18), 7.91 (2H, d, J = 7.3 Hz, H-23, H-27), 12.18 (2H,
b, pyrimido ═NH, pyrimido –NH), 15.83 (1H, b, ꢀOH);
13C-NMR(100 MHz, DMSO-d6) δ:12.37 (CH3, C-28),
55.40 (OCH3, C-30), 101.71 (C, C-4), 116.20 (CH, C-15,
C-17), 121.83 (C, C-8) 122.00 ppm (CH, C-14, C-18),
124.85 (CH, C-23, C-27), 127.34 (CH, C-24, C-26),
129.37 (CH, C-25), 140.92 (C, C-3), 144.36 (C, C-9),
147.93 (C, C-13), 156.83 (C, C-22), 159.08 (C, C-
16),160.53 (C, C-7), 194.63 (C, C-5); HR-MS: 402.4231
[M + H]+, calcd 402.4236. Anal. Calcd for C20H18N8O2:
C, 59.69%; H, 4.51%; N, 27.85%. Found: C, 59.72%; H,
4.50%; N, 27.81%.
7-Imino-3-phenylazo-6-(30-nitrophenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4f).
Bright dark brown
solid crystals, (74%), mp: 308–309°C; FT–IR (cmꢀ1
max: 3427 (ꢀOH), 3341 (ꢀNH), 3265 (═NH), 3090 and
)
ν
3058 (Ar–H), 2924 (Aliphatic C–H), 1640 (C═N), 1595
(N═N), 1524 and 1348 (ꢀNO2); 1H-NMR (400 MHz,
DMSO-d6) δ (ppm): 2.59 (3H, s, ꢀCH3), 7.44 (1H, t,
J = 7.3 Hz, H-25), 7.53 (2H, t, J = 7.5 Hz, H-24, H-26),
7.76 (1H, t, J = 8.0 Hz, H-17), 7.98 (2H, d, J = 7.5 Hz,
H-23, H-27), 8.18 (1H, d, J = 7.8 Hz, H-18), 8.23 (1H, d,
J = 8.0 Hz, H-16), 8.61 (1H, s, H-14), 9.66 (1H, b,
pyrimido ═NH), 10.84 (1H, b, pyrimido –NH), 12.12
(1H, b, ꢀOH); 13C-NMR (100 MHz, DMSO-d6) δ:12.37
(CH3, C-28), 101.71 (C, C-4), 111.05 (CH, C-18), 117.00
(CH, C-16),118.07 (CH, C-14),121.83 (C, C-8), 124.85
(CH, C-23, C-27), 127.34 (CH, C-24, C-26), 129.37 (CH,
C-25),133.42 (CH, C-15), 140.52 (C, C-3), 144.36 (C, C-
9), 150.50 (C, C-13), 150.83 (C, C-22), 151.50 (C, C-
17), 160.53 (C, C-7), 194.63 (C, C-5); HR-MS:
417.3951[M + H]+, calcd 417.3958. Anal. Calcd for
C19H15N9O3: C, 54.68%; H, 3.62%; N, 30.20%. Found:
C, 54.63%; H, 3.64%; N, 30.24%.
7-Imino-3-phenylazo-6-(40-chlorophenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4d).
Brick red solid
crystals, (77%), mp: 276–278°C; FT-IR (cmꢀ1) νmax
:
3507 (ꢀOH), 3414 (ꢀNH), 3328 (═NH), 3062 (Ar–H),
2927 (Aliphatic C–H), 1624 (C═N), 1576 (N═N); 1H-
NMR (400 MHz, DMSO-d6) δ (ppm): 2.61 (3H, s,
ꢀCH3), 7.44 (1H, t, J = 7.3 Hz, H-–25), 7.51–7.56 (4H,
m, H-23, H-24, H-26, H-27), 7.81 (2H, d, J = 8.8 Hz, H-
14, H-18), 7.99 (2H, d, J = 7.5 Hz, H-15, H-17), 9.51
(1H, b, pyrimido ═NH), 10.82 (1H, b, pyrimido –NH),
12.10 (1H, b, ꢀOH); 13C-NMR(100 MHz, DMSO-d6)
δ:12.37 (CH3, C-28), 101.71 (C, C-4), 121.83 (C, C-8),
124.82 (CH, C-14, C-18), 124.85 (CH, C-23, C-27),
127.34 (CH, C-24, C-26), 130.08 (CH, C-25),130.40
(CH, C-15, C-17), 139.74 (C, C-16), 140.52 (C, C-3),
144.36 (C, C-9), 150.83 (C, C-22), 153.02 (C, C-13),
7-Imino-3-phenylazo-6-(30-methoxyphenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4g).
Dark brown solid
1crystals, (80%), decomp > 292°C; FT-IR (cmꢀ1
max: 3354 (ꢀOH), 3281 (ꢀNH), 3144 (=NH), 3033
(Ar–H), 2978 and 2939 (Aliphatic C–H), 1646 (C═N),
1602 (N═N); 1H-NMR (400 MHz, DMSO-d6)
(ppm): 2.65 (3H, s, ꢀCH3), 5.00 (3H, s, ꢀOCH3),
)
ν
δ
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Pyrazolo[1,5-a]Pyrimidine Dyes
7.35 (1H, d, J = 8.0 Hz, H-16), 7.37 (1H, s, H-14), 7.41
(1H, t, J = 7.3 Hz, H-25), 7.46 (1H, t, J = 8.0 Hz, H-
17), 7.56 (2H, t, J = 7.3 Hz, H-24, H-26), 7.84 (1H,
d, J = 7.5 Hz, H-18), 7.93 (2H, d, J = 7.5 Hz, H-23,
H-27), 11.26 (3H, b, pyrimido ═NH, pyrimido –NH,
ꢀOH); 13C-NMR(100 MHz, DMSO-d6) δ: 12.37 (CH3,
C-28), 55.71 (CH3, C-30),101.71 (C, C-4), 106.36
(CH, C-18), 109.62 (CH, C-16), 112.08 (CH, C-14),
121.83 (C, C-8),124.85 (CH, C-23, C-27), 127.34
(CH, C-24, C-26), 129.37 (CH, C-25), 133.32 (CH, C-
15), 140.52 (C, C-3), 144.36 (C, C-9),150.83 (C, C-
22), 151.98 (C, C-13), 160.53 (C, C-7), 162.88 (CH,
C-17), 194.63 (C, C-5); HR-MS: 402.4231 [M + H]+,
calcd 402.4339. Anal. Calcd for C20H18N8O2: C,
59.69%; H, 4.51%; N, 27.85%. Found: C, 59.72%; H,
4.50%; N, 27.81%.
(CH, C-25), 137.13 (CH, C-17), 139.72 (C, C-15),
140.52 (C, C-3), 144.36 (C, C-9), 150.83 (C, C-22),
158.29 (C, C-13), 160.53 (C, C-7), 194.63 (C, C-5); HR-
MS: 386.4223 [M + H]+, calcd 386.4228. Anal. Calcd for
C20H18N8O: C, 62.17%; H, 4.70%; N, 29.00%. Found:
C, 62.20%; H, 4.71%; N, 28.97%.
7-Imino-3-phenylazo-6-(20-nitrophenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4j).
Burgundy solid
crystals, (72%), mp: 298–299°C; FT–IR (cmꢀ1) νmax
:
3495 (ꢀOH), 3394 (ꢀNH), 3278 (═NH), 3127 (Ar–H),
2920 (Aliphatic C–H), 1643 (C═N), 1579 (N═N), 1507
and 1348 (ꢀNO2); 1H-NMR (400 MHz, DMSO-d6) δ
(ppm): 2.61 (3H, s, ꢀCH3), 7.45 (1H, t, J = 7.3 Hz, H-
25), 7.52–7.60 (3H, m, H-17, H-24, H-26), 7.82 (1H, t,
J = 7.6 Hz, H-16), 7.90 (1H, d, J = 7.8 Hz, H-18), 8.00
(2H, d, J = 7.3 Hz, H-23, H-27), 8.09 (1H, d, J = 8.0 Hz,
H-15), 10.05 (1H, b, pyrimido ═NH), 11.02 (1H, b,
pyrimido –NH), 12.08 (1H, b, ꢀOH); 13C-
NMR(100 MHz, DMSO-d6) δ:12.37 (CH3, C-28), 101.71
(C, C-4), 121.83 (C, C-8), 121.96 (CH, C-18), 122.96
(CH, C-16), 124.85 (CH, C-23, C-27), 127.34 (CH, C-24,
C-26), 129.08 (CH, C-15), 129.37 (CH, C-25), 133.64
(C, C-14), 136.42 (CH, C-17), 140.52 (C, C-3), 144.36
(C, C-9), 144.58 (C, C-13), 150.83 (C, C-22); 160.53 (C,
C-7), 194.63 (C, C-5); HR-MS: 417.3951 [M + H]+,
calcd 417.3963. Anal. Calcd for C19H15N9O3: C, 54.68%;
H, 3.62%; N, 30.20%. Found: C, 54.68%; H, 3.62%; N,
30.20%.
7-Imino-3-phenylazo-6-(30-chlorophenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4h). Reddish brown solid
crystals, (77%), mp: 282–284°C; FT-IR (cmꢀ1) νmax: 3428
(ꢀOH), 3337 (ꢀNH), 3242 (═NH), 3064 (Ar–H), 2927,
and 2853 (Aliphatic C–H), 1645 (C═N), 1595 (N═N);
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 2.62 (3H, s,
ꢀCH3), 7.41 (2H, m, H-25, H-16), 7.53 (3H, t,
J = 7.4 Hz, H-17, H-24, H-16), 7.77 (1H, d, J = 8.0 Hz,
H-18), 7.85 (1H, s, H-14), 7.99 (2H, d, J = 7.5 Hz, H-23,
H-27), 9.53 (1H, b, pyrimido ═NH), 10.81 (1H, b,
pyrimido –NH), 12.22 (1H, b, ꢀOH); 13C-
NMR(100 MHz, DMSO-d6) δ:12.37 (CH3, C-28), 101.71
(C, C-4), 114.13 (CH, C-18), 114.37 (CH, C-14), 121.83
(C, C-8), 122.93 (CH, C-16), 124.85 (CH, C-23, C-27),
127.34 (CH, C-24, C-26), 129.37 (CH, C-25), 131.67
(CH, C-17), 138.84 (C, C-15), 140.52 (C, C-3), 144.36
(C, C-9), 150.83 (C, C-22), 151.98 (C, C-13), 160.53 (C,
C-7), 194.63 (C, C-5); HR-MS: 407.1132 [M + H]+,
calcd 407.1143. Anal. Calcd for C19H15ClN8O: C,
56.09%; H, 3.72%; N, 27.54%. Found: C, 56.05%; H,
3.75%; N, 27.55%.
7-Imino-3-phenylazo-6-(20-methoxyphenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4k). Dark burgundy solid
crystals, (76%), mp: 275–276°C; FT-IR (cmꢀ1) νmax: 3300
(ꢀNH), 3166 (═NH), 3066 and 3010 (Ar–H), 2942
(Aliphatic C–H), 1636 (C═O), 1580 (C═N), 1523
1
(N═N); H-NMR (400 MHz, DMSO-d6) δ (ppm): 2.63
(3H, s, CH3), 3.96 (3H, s, ꢀOCH3), 7.07 (1H, t,
J = 7.6 Hz, H-16), 7.25 (1H, d, J = 7.5 Hz, H-15), 7.40
(1H, t, J = 7.8 Hz, H-25), 7.45 (1H, t, J = 7.3 Hz, H-17),
7.54 (2H, t, J = 7.5 Hz, H-24, H-26), 7.65 (1H, d,
J = 7.5 Hz, H-18), 7.99 (2H, d, J = 7.5 Hz, H-23, H-27),
9.46 (1H, s, pyrimido ═NH), 11.43 (1H, s, pyrimido
–NH), 12.01 (1H, b, ꢀOH); 13C-NMR(100 MHz,
DMSO-d6) δ:12.37 (CH3, C-28), 56.10 (CH3, C-30),
101.71 (C, C-4), 112.58 (CH, C-15), 117.08 (CH, C-18),
121.83 (C, C-8), 121.96 (CH, C-16), 124.68 (CH, C-17),
124.85 (CH, C-23, C-27), 127.34 (CH, C-24, C-26),
129.37 (CH, C-25), 140.52 (C, C-3), 140.60 (C, C-13),
144.36 (C, C-9), 150.73 (C, C-14), 150.83 (C, C-22),
160.53 (C, C-7), 194.63 (C, C-5); HR-MS: 402,4231
[M + H]+, calcd 402.4335. Anal. Calcd for C20H18N8O2:
C, 59.69%; H, 4.51%; N, 27.85%. Found: C, 59.67%; H,
4.54%; N, 27.82%.
7-Imino-3-phenylazo-6-(30-methylphenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4i). Bright dark red solid
crystals, (75%), mp: 252–254°C; FT-IR (cmꢀ1) νmax
:
3312 (ꢀNH), 3259 (═NH), 3060 (Ar–H), 2921
(Aliphatic C–H),1646 (C═O),1610 (C═N), 1592 (N═N);
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 2.39 (3H, s,
Ar–CH3), 2.63 (3H, s, pyrazole–CH3), 7.21 (1H, d,
J = 7.5 Hz, H-16), 7.39 (1H, t, J = 7.6 Hz, H-25), 7.45
(1H, t, J = 7.1 Hz, H-17), 7.52 (1H, d, J = 7.8 Hz, H-24),
7.57 (1H, d, J = 7.8 Hz, H-26), 7.61 (1H, s, H-14), 7.93
(1H, d, J = 7.5 Hz, H-18), 7.99 (2H, d, J = 7.5 Hz, H-23,
H-27), 9.36 (1H, b, pyrimido ═NH), 10.86 (1H,s,
pyrimido –NH), 11,79 (1H, b, ꢀOH); 13C-
NMR(100 MHz, DMSO-d6) δ:12.37 (CH3, C-28), 21.02
(CH3, C-29), 101.71 (C, C-4), 118.63 (CH, C-18), 121.78
(CH, C-14), 121.83 (C, C-8), 124.85 (CH, C-23, C-27),
126.54 (CH, C-16), 127.34 (CH, C-24, C-26), 129.37
7-Imino-3-phenylazo-6-(20-chlorophenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4l).
Bright light brown
solid crystals, (75%), mp: 300–301°C; FT–IR (cmꢀ1
)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

N. Şener, S. Erişkin, S. Yavuz, and İ. Şener
Vol 000
ν
max: 3446 (ꢀOH), 3322 (ꢀNH), 3145 (═NH), 3064 (Ar–
48 h at 28οC for fungi. Negative controls were
prepared by using DMSO to dissolve the tested
compound. Ciprofloxacin (50 μg/mL) and ketoconazole
(50 μg/mL) were used as references for antibacterial
and antifungal activities, respectively. Antimicrobial
activities were measured as inhibition diameter zones
in millimeters (mm). The results are shown in Table 3.
The dyes produced significant growth inhibition zones
(>14 mm) using the twofold serial dilution technique.
Their MICs were measured as well.
H), 2926 (Aliphatic C–H), 1637 (C═N), 1578 (N═N); 1H-
NMR (400 MHz, DMSO-d6) δ (ppm): 2.63 (3H, s, ꢀCH3),
7.41 (1H, t, J = 7.5 Hz, H-25), 7.47 (2H, m, H-16, H-17),
7.54 (2H, t, J = 7.6 Hz, H-24, H-26), 7.67 (1H, d,
J = 7.8 Hz, H-18), 7.80 (1H, d, J = 7.8 Hz, H-15), 8.01
(2H, d, J = 7.8 Hz, H-23, H-27), 9.91 (1H, s, pyrimido
═NH), 11.25 (1H, b, pyrimido –NH), 12.11 (1H, b,
ꢀOH); 13C-NMR(100 MHz, DMSO-d6) δ:12.37 (CH3,
C-28), 101.71 (C, C-4), 116.62 (CH, C-18), 117.26 (C,
C-14),120.86 (CH, C-16), 121.83 (C, C-8), 124.85 (CH,
C-23, C-27), 127.34 (CH, C-24, C-26), 128.17 (CH,
C-17), 129.37 (CH, C-25), 130.82 (CH, C-15), 140.52
(C, C-3), 143.35 (C, C-13), 144.36 (C, C-9), 150.83 (C,
C-22), 160.53 (C, C-7), 194.63 (C, C-5); HR-MS: 407,
1132 [M + H]+, calcd 407.1137. Anal. Calcd for
C19H15ClN8O: C, 56.09%; H, 3.72%; N, 27.54%. Found:
Minimal inhibitory concentration measurement.
The
micro dilution susceptibility test in Müueller Hinton
Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid)
were used to determine antibacterial and antifungal
activities, respectively. Ciprofloxacin and ketoconazole,
as well as stock solutions of the tested compounds,
were prepared at concentrations of 1000 mg/mL in
DMF. Dye solutions were prepared by using the
proper nutrient broth twice, and the final solution
concentrations were 132, 66, 33, 16.5, and 8.25 μg/mL.
Test tubes were prepared with the target bacteria
grown in appropriate broths for 24 h at 37οC (about
1 × 106 CFU/mL). Each 5 mL of broth received
0.1 mL of the previous inoculum. The samples were then
incubated for 24 h at 37οC. The lowest concentration that
showed no growth was taken to be the MIC. Control
experiments with DMF and uninoculated media were run
parallel and in the same conditions as the test compounds.
The MICs (mg/mL) and inhibition zone diameters are
shown in Table 4.
C, 56.12%; H, 3.73%; N, 27.52%.
7-Imino-3-phenylazo-6-(20-methylphenylazo)-2-methyl-4H-
pyrazolo[1,5-a]pyrimidine-5-one (4m). Bright dark brown
solid crystals, (79%), mp: 269–271°C; FT-IR (cmꢀ1
)
ν
max: 3415 (ꢀOH), 3294 (ꢀNH), 3146 (═NH), 3063
(Ar–H), 2962 and 2925 (Aliphatic C–H), 1634 (C═N),
1581 (N═N); H-NMR (400 MHz, DMSO-d6) δ (ppm):
1
2.56 (3H, s, Ar–CH3), 2.67 (3H, s, pyrazole–CH3), 7.34
(1H, t, J = 8.0 Hz, H-16), 7.41 (1H, t, J = 7.3 Hz, H-25),
7.45 (1H, d, J = 7.8 Hz, H-15), 7.51 (2H, t, J = 7.5 Hz,
H-24, H-26), 7.62 (1H, t, J = 8.0 Hz, H-17), 7.74 (1H, d,
J = 7.8 Hz, H-18), 7.96 (2H, d, J = 7.5 Hz, H-23, H-27),
9.57 (1H, s, pyrimido ═NH), 10.95 (1H, b, pyrimido
–NH), 12.01 (1H, b, ꢀOH); 13C-NMR(100 MHz,
DMSO-d6) δ:12.37 (CH3, C-28), 17.70 (CH3, C-29),
101.71 (C, C-4), 110.51 (CH, C-18), 121.22 (CH, C-16),
121.83 (C, C-8), 124.85 (CH, C-23, C-27), 127.17 (CH,
C-17), 127.34 (CH, C-24, C-26), 128.14 (C, C-14),
129.37 (CH, C-25), 132.07 (CH, C-15), 140.52 (C, C-3),
144.36 (C, C-9), 150.14 (C, C-13), 150.83 (C, C-22),
160.53 (C, C-7), 194.63 (C, C-5); HR–MS: 386.4223
[M + H]+, calcd 386.4231. Anal. Calcd for C20H18N8O:
C, 62.17%; H, 4.70%; N, 29.00%. Found: C, 62.21%; H,
4.72%; N, 28.95%.
DPPH radical scavenging activity.
The antioxidant
activities of the test compounds were determined using
a previously published diphenylpicrylhydrazyl (DPPH)
radical scavenging method [36]. Various concentrations
of the test compounds (10–1000 μg/mL) in methanol
were mixed with a methanolic DPPH solution (1 mM)
to produce final volume of 2.0 mL. The mixtures were
incubated at room temperature in the dark, and their
absorbances were recorded at 517 nm. A control was
tested under the same conditions without
a test
compound, and ascorbic acid was used as a reference.
The experiment was performed in triplicate, and the
percentage of radicals scavenged was determined as
follows;
Antimicrobial evaluation. The compounds were tested
against a panel of Gram-positive and Gram-negative
bacterial pathogens, fungi, and yeast. Antimicrobial
experiments were performed using the agar diffusion
method [34,35] with 100 μL 1 × 106 CFU/mL of
pathological organisms tested and 1 × 106/mL of yeast
spread on nutrient agar and Sabouraud dextrose agar,
respectively. After cooling and solidification, the media
(10 mm in diameter) were placed in the solidified agar
and loaded with a solution of the compound to be
tested (100 μL) prepared by dissolving the chemical
(100 mg) in DMSO (1 mL). The inoculcated plates
were then incubated for 24 h at 37οC for bacteria and
½ðAbsorbance of blank ꢀ Absorbance of testÞ=
Absorbance of blankꢁꢂ100:
The radical-scavenging activities of the compounds
were presented as IC50s. The IC50 is the concentration
in micrometer required to produce a 50% decrease in
absorbance by DPPH radicals.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Pyrazolo[1,5-a]Pyrimidine Dyes
[18] Karci, F. Color Technol 2005, 121, 275.
[19] Ho, Y. W. Dyes Pigments 2005, 64, 223.
[20] Zvilichovsky, G.; David, M. J Chem Soc Perk T1 1983 11.
[21] Kandeel, Z. E.; Abdelrazek, F. M.; Eldin, N. E. M. S.; Elnagdi,
M. H. J Chem Soc Perk T1 1985 1499.
Acknowledgments. The authors are grateful to the Scientific
Research Projects Council of Pamukkale University (PAU. BAP,
2012FBE066 and Kastamonu University).
[22] Singh, S. P.; Kumar, D. Heterocycles 1990, 31, 855.
[23] Jaiswal, N.; Jaiswal, R.; Barthwal, J.; Kishor, K. Indıan J
Chem 1981, 20B, 252.
REFERENCES
[1] Zeng, H.; Lin, Z. P.; Sartorelli, A. C. Biochem Pharmacol
2004, 68, 911.
[24] Küçükgüzel, S. G.; Rollas, S.; Erdeniz, H.; Kiraz, M.; Ekinci,
A. C.; Vidin, A. J Med Chem 2000, 35, 761.
[2] Sharma, P.; Rane, N.; Gurram, V. K. Bioorg Med Chem Lett
2004, 14, 4185.
[25] Ghozlan, S. A. S.; Abdelrazek, F. M.; Mohamed, M. H.; Azmy,
K. E. J Heterocyclic Chem 2010, 47, 1379.
[3] Huang, C. Q.; Wilcoxen, K. M.; Grigoriadis, D. M.; McCarthy,
J. R.; Chen, C. Bioorg Med Chem Lett 2004, 14, 3943.
[4] Dhavale, D. D.; Matin, M. M.; Sharma, T.; Sabharwal, S. G.
Bioorg Med Chem Lett 2004, 12, 4039.
[5] West, T. P. Microbiol Res 2004, 159, 29.
[6] Devesa, I.; Alcaraz, M. J.; Riguera, R.; Ferrandiz, M. L. Eur J
Pharmacol 2004, 488, 225.
[7] Kumar, A. K. A.; Bodke, Y. D.; Gowda, A. N.; Sambasivam,
G.; Bhat, K. G. J Heterocyclic Chem 2017, 54, 1904.
[8] Saundane, A. R.; Walmik, P.; Yarlakatti, M.; Katkar, V.;
Verma, V. A. J. Heterocyclic Chem. 2014, 51, 303.
[9] Cutting, W. C. Handbook of Pharmacology, 3rd ed.; Meredith
Company: New York, 1967.
[10] Izatt, R. M.; Christensen, J. H.; Rytting, J. H. Chem Rev 1972,
71, 439.
[11] Saify, Z. S.; Kamil, A.; Akhtar, S.; Taha, M.; Khan, A.; Khan,
K. M.; Jahan, S.; Rahim, F.; Perveen, S.; Choudhary, M. I. Med Chem Res
2014, 23, 4447.
[26] Shamroukh, A. H.; Rashad, A. E.; Ali, H. S.; Abdel-Megeida,
F. M. E. J Heterocyclic Chem 2013, 50, 758.
[27] Holla, B. S.; Mahalinga, M.; Karthikeyan, M. S.; Akberal,
P. M.; Shetty, N. S. Bioorg Med Chem 2006, 14, 2040.
[28] Erişkin, S.; Şener, N.; Yavuz, S.; Şener, İ. Med Chem Res
2014, 23, 3733.
[29] Şener, N.; Şener, İ.; Yavuz, S.; Karcı, F. Asıan J Chem 2015,
27, 3003.
[30] Elnagdi, M. H.; Elmoghaya, M. R. H.; Fleita, D. H.; Hafez,
E. A. A.; Fahmy, S. M. J Org Chem 1976, 41, 3781.
[31] Jain, K. J.; Kanaiya, P. H.; Bhojak, N. Fiber Polym 2008, 9,
720.
[32] Harikrishnan, U.; Menon, S. K. Dyes Pigments 2008, 77, 462.
[33] Reichart, C.; Welton, T. Solvent and Solvent Effect in
Organic Chemistry, Fourth ed.; Willey-VCH Verlag&Co.KgaA:
Weinheim, 2011.
[34] Yavuz, S.; Yıldırım, H. J Chem 2013, 2013, 1.
[35] Scott, A. C. Laboratory control of antimicrobial therapy; In
Mackie and Mac-Cartney Practical Medical Microbiology, Collee, J. G.;
Duguid, J. P.; Fraser, A. G.; Marmion, B. P. Eds., 13th ed.; Churchill
Livingstone, 1989, pp. 161–181.
[12] Taha, M.; Ismail, N. H.; Ali, M.; Khan, K. M.; Jamil, W.;
Kashif, S. M.; Asraf, M. Med Chem Res 2014, 23, 5282.
[13] Taha, M.; İsmail, N. H.; İmran, S.; Rokei, M. Q. B.; Saad, S. M.;
Khan, K. M. Bioorg Med Chem 2015, 23, 4155.
[14] Taha, M.; Ismail, N. H.; Khan, A.; Shah, S. A. A.; Anwar, A.;
Halim, S. A.; Fatmi, M. Q.; Imran, S.; Rahim, F.; Khan, K. M. Bioorg
Med Chem Lett 2015, 25, 3285.
[36] Rajakumar, D. V.; Rao, M. N. A. Free Radic Res 1995, 22,
309.
[15] Elnagdi, M. H.; Fahmy, S. M.; Elmoghayar, M. R. H.; Elias,
M. A. M. Z.; Naturforsch, B. J Chem Sci 1975, 30b, 778.
[16] Elnagdi, M. H.; Sallam, M. M. M.; Fahmy, H. M.; Ibrahim,
S. A. M.; Elias, M. A. M. Helv Chım Acta 1976, 59, 551.
[17] Ghozlan, S. A. S.; Abdelhamid, I. A.; Abdelshaft, G. H.;
Elnagdi, M. H. J Chem Res 2004, 12, 789.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet