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New Journal of Chemistry RSC

ARTICLE

New Journal of Chemistry Accepted Manuscript

Synthesis, characterization, antibacterial,
antioxidant, DNA binding and SAR study of novel
pyrazine moiety bearing 2-pyrazoline derivatives
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Bheru Singh Kitawat a and Man Singh* a

An environmentally safer method for synthesis of novel substituted 2-(5-(4-aryl)-1-phenyl-4,5-dihydro-

1H-pyrazol-3-yl)-3-alkylpyrazine, 1-(5-(4-aryl)-3-(3-alkylpyrazin-2-yl)-4,5-dihydro-1H-pyrazol-1-
yl)ethanone and 5-(4-aryl)-3-(3-alkylpyrazin-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide via
reactions of (E)-3-aryl-1-(3-alkyl-2-pyrazinyl)-2-propenones with phenyl hydrazine hydrochloride,
hydrazine monohydrate and thiosemicarbazide respectively has been reported. The reactions were carried
out using sodium acetate-aqueous acetic acid solution at room temperature, under ultrasound irradiation
(US). As compared to conventional method, the advantages of US method encompass milder condition,
operational simplicity, a higher yield up to ≈ 93%, safety and environment friendly protocol. Synthesized
compounds were evaluated in vitro for their antibacterial activities against gram-positive and negative
strains. Compounds 4a–4l showed 29–67% antioxidant activities, as determined through 2,2-diphenyl-1-
picrylhydrazyl (DPPH) free radical method. All the synthesized compounds were screened for their
DNA binding activities using spectrofluorometer, where those containing halogens exhibited significant
potential (upto 570 ng/mL) due to corresponding intercalation and groove binding mechanisms.

Introduction On the other hand, owing to an increasing environmental

Heterocyclic ring containing compounds have attracted
significant interest owing to their comprehensive biological
activities in the domain of bioorganic, biomedical,
pharmaceuticals and others. 1-3 Amongst these, pyrazoline is a
nitrogen containing five membered heterocyclic molecule and
plays an important role in the heterocyclic chemistry, a fact
well understood through its extensive usage as pharmacophore
and synthon.4 Interestingly, 2-pyrazoline, a significant
derivative of this heterocyclic compound, has been frequently
studied because of its relatively more stability and the
concurrent monoimino character. Numerous chemotherapeutic
agents possessing 2-pyrazoline moiety, have been studied for
their potent anticancer,2 antimicrobial,3 anti-inflammatory,5
antimalarial,2 antiproliferative6,7 analgesic,5 antidepressant8 and
insecticidal activities. 9,10
2-Pyrazolines have usually been synthesized by starting
from aldehydes or ketones, which have either actual or potential
α,β-unsaturation with substituted hydrazine under reflux
condition in the presence of base.11-14 However, these
conventional methods suffer from some crude drawbacks such
as those of longer reaction time, higher temperature, formation
of side product, typical workup procedure and environmental
anxieties.15-19
sustainability in the fields of chemical research and industry,
significant attention is being given towards the design and
development of sustainable and clean chemical procedures. 20
Such protocols minimize the use of expensive, toxic,
flammable, not recyclable organic solvents in chemical
processes. Recently, organic reactions in aqueous media have
attracted enormous interest because of environmental issues
and understanding of biochemical processes and termed as N-
synthesis.21,22
The ultrasound irradiation (US) has increasingly been
adopted in organic synthesis as a safer alternative technique to
accelerate various organic reactions.23 Varieties of organic
reactions could be carried out in high yield, short reaction time
or milder conditions under ultrasound irradiation,24 which is
based on cavitation effects leading to mass transfer
improvement.25 As compared to conventional methods, the US
is more suitable, easily controlled and is more appropriate in
the concern of green chemistry. 25,26 To our best understanding
based on a wider literature survey, it is revealed that the
synthesis of heterocyclic compounds has been accelerated by
ultrasound irradiation26 and no report was made on pyrazine
containing 2-pyrazolines.
Considering the advantages of ultrasonic irradiation and
continuing our investigations on new methodologies for the
synthesis of new heterocyclic ring bearing compounds 27, herein

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we wish to report an efficient and practical procedure for the
synthesis of 2-pyrazolines using heterocyclic analogues of
chalcones and substituted hydrazines in sodium acetate-acetic
acid aqueous solution at room temperature under ultrasound
irradiation (Scheme 1).
Initially, reaction between (E)-3-(4-bromophenyl)-1-(3-
methylpyrazin-2-yl) prop-2-en-1-one (3c) and phenyl hydrazine
hydrochloride was chosen as a model reaction for optimization
of reaction conditions. For this, equal molar ratio (1 mmol) of
reactants and 0.2 mmol sodium acetate were used and produced
79 % yield in 90 min at room temperature under ultrasonication
(Table 1, entry 1). The reaction using 2 mmol and 3 mmol of

New Journal of Chemistry Accepted Manuscript

phenyl hydrazine gave 82 % and 85 % yield respectively at the
same reaction conditions (Table 1, entries 2 and 3). The results
showed that increase in the molar ratio of phenyl hydrazine
from 1:1 to 1:3 had a significant effect on the yield and an
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optimum molar ratio of chalcone (3c): phenyl hydrazine was

found 1:3.

Table 1: Optimized conditions for 2-pyrazolines synthesis

under ultrasound irradiationa.

Scheme 1. Ring closure reaction of α, β-unsaturated ketones for

synthesis of substituted 2-pyrazolines using sodium acetate-acetic Entry Molar ratio Temp. Time Yield
acid aqueous medium under ultrasound irradiation. (3c:PhNHNH2:NaOAc) (0C)b (min) (%)c
1 1:1:0.2 rt 90 79
Further, the study on the interaction of small molecules 2 1:2:0.2 rt 90 82
3 1:3:0.2 rt 90 85
generally termed as drugs or ligands with DNA has been the
4 1:3:0.1 rt 90 78
focus of some recent research works in the scope of life 5 1:3:0.15 rt 90 81
science, chemistry and clinical medicines. Keeping such view 6 1:3:0.25 rt 90 89
in mind the synthesized compounds were screened for their 7 1:3:0.3 rt 90 92
8 1:3:0.4 rt 90 94
potential activities such as antibacterial, antioxidant and DNA 8 1:3:0.3 50 90 85
binding. 9 1:3:0.3 70 90 81
10 1:3:0.3 90 90 73
11 1:3:0.3 reflux 90 68
Results and discussion a
All reactions were carried out at 30 KHz,
b
Room temperature 30-33 0C
Synthesis and analytical characterizations c
Isolated yield
Aim of our study is to synthesize novel pyrazine moiety
containing substituted 2-pyrazolines in aqueous medium, After setting up the 1:3 molar ratios, we next optimize the
according to Scheme 1. In the first step, (E)-3-aryl-1-(3-alkyl-2- molar ratio of sodium acetate for better yield. For this 0.1, 0.15,
pyrazinyl)-2-propenones (3a-3g) were obtained by Claisen– 0.25, 0.30 and 0.40 mmol sodium acetate was used that had
Schmidt condensation reaction between 2-acetyl-3- produced 78, 81, 89, 92 and 94 % yield respectively (Table 1,
alkylpyrazine (1a-1b) and aromatic aldehydes (2a-2d) in entries 4-8). No better increment in yield was found at 0.4
solvent free K2CO3 mediated microwave environment with 90- mmol as comparisons to 0.3 mmol. Thus, the final ratio of
95 % yield as is depicted in our previously reported study.27 chalcone (3c): phenyl hydrazine: sodium acetate was 1:3:0.3
The spectral data and melting points of (3a–3g) were well mmol. Along with the molar ratio 1:3:0.3 mmol, we focused to
agreed with literature values.27 In second step, (3a-3g) were verify the effect of temperature on the reaction, and for this the
cyclized to substituted 2-pyrazolines (4a-4l) using phenyl reaction was conducted at room temperature (rt) to reflux
hydrazine, hydrazine monohydrate and thiosemicarbazide in (Table 1, entries 9-12), and the yield was decreased from 92 %
sodium acetate–acetic acid aqueous solution at room to 68 %, respectively. At higher temperature (reflux), the rate of
temperature under ultrasound irradiation. Optimized conditions reaction might have decrease due to higher kinetic energy with
for 2-pyrazolines synthesis using chalcones and phenyl increased numbers of successful collisions.
hydrazine hydrochloride are summarized in Table 1. The Further, to verify the effect of ultrasound irradiation, the
comparisons between conventional and ultrasound acceleration reaction of pyrazine bearing chalcone with phenyl hydrazine
methods are summarized in Table 2. Reaction in sodium was performed under conventional heating in ethanol for 8 h.
acetate-acetic acid aqueous medium proceeds faster and The yield of 2-pyrazolines was less as compared to US
produced good yield as compared to conventional method synthesis (Table 2). Thus, the effects of ultrasound irradiation
(Table 2). on rate of reactions might be due to cavitation which

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Journal Name ARTICLE

accelerates instantaneous high temperature and pressure. The
difference in yields and reaction times as comparison to
conventional method may be a consequence of the specific
effects of ultrasound on molecules attributed to cavitation, a
physical process that produces, extends, and collapses gaseous
and vaporous cavities in an irradiated liquid, thus enhancing the
mass transfer28 and allowing chemical reactions to occur.
>C=O stretching of (4d) and (4e) due to carbonyl group
attached to pyrazoline, observed at 1650 and 1668 respectively.
A band at 3344 and 3297 cm-1 owing to –NH2 of (4f-4l)
observed. Positive mode ESI-MS spectra of the synthesized
compounds (4a-4l) displayed the protonated molecular ion
peaks, [M+H]+ respectively, confirming their molecular masses.
The 1H NMR spectra showed an H abx pattern of protons of

New Journal of Chemistry Accepted Manuscript

Scope and versatility of our method was studied by
preparing a series of new 12 compounds (4a-4l) by using
different substitution -C6H5, COCH3 and –CSNH2 at 2-position
of pyrazoline, -F, Cl, Br, -N (CH3)2 at para position of phenyl
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pyrazoline ring caused by the coupling of three hydrogen atoms
(Ha, Hb and Hx) of 2-pyrazoline ring at δ 3.20-3.53 ppm, δ 3.86-
4.03 ppm and δ 5.27-6.0 ppm with coupling constant J = 5.0
Hz, 11.0-13.0 Hz and 5.0-6.0 Hz respectively. The existence of

ring and –CH3 and –C2H5 at 3-position of pyrazine. Probable
mechanism for 2-pyrazolines synthesis is illustrated in Fig. 1.
Figure 1. Plausible reaction mechanism of 2-pyrazolines synthesis
Table 2. Ultrasound assisted (US) synthesis of substituted 2-
pyrazolines in aqueous medium at room temperature a
methylenic protons (Ha and Hb) as dd, clearly postulates the
magnetic non-equivalence of these two protons, which have
chemical shift at δ 3.2-3.6 ppm and δ 3.4-4.0 ppm with J = 5.0-
6.0 Hz and 11.5-13.5 Hz, respectively. The H x proton as dd
appeared at δ 5.2-6.0 ppm with J = 5.0-6.0 Hz. The alkyl
protons (-CH3 and -C2H5) appear at upper filed due to higher
shielding effect. The two broad singlet of NH’s proton for
compounds (4f-4l) were observed at δ 6.17-6.25 ppm and δ
6.95-7.03 ppm. These spectral data unequivocally proved the 2-
pyrazolines structure. For compounds (4a-4l), the aromatic
protons appear as multiplet in the desired range.
The 13C NMR spectra of compounds provided final
structure of 2-pyrazolines. Chemical shift a value of the
methylpyrazine (4a-4i) carbons appears at δ 24.5-25.5 ppm,
while in ethylpyrazine (4j-l) appears at δ 12.1-12.2 ppm. The
chemical shifts at δ 22.05 ppm, δ 30.01 ppm and δ 44.39 ppm
are due to –CH2 of aliphatic carbon attached to pyrazine ring
(4j-4l), -CH3 of carbonyl (4d and 4e) and –CH3 of –N(CH3)2 of
compound (4i) respectively.

Comp. R X R2 Time Yieldb (%)

Conv. (h) US (min.) Conv. US
4a -CH3 -F -C6H5 8 90 58 88
4b -CH3 -Cl -C6H5 8 90 64 89
4c -CH3 -Br -C6H5 8 90 70 92
4d -CH3 -Cl -COCH3 8 95 55 85
4e -CH3 -Br -COCH3 8 95 59 88
4f -CH3 -F -CSNH2 8 75 68 86
4g -CH3 -Cl -CSNH2 8 75 71 89
4h -CH3 -Br -CSNH2 8 75 73 92
4i -CH3 -N(CH3)2 -CSNH2 8 75 70 89
4j -C2H5 -F -CSNH2 8 75 68 88
4k -C2H5 -Cl -CSNH2 8 75 72 89
4l -C2H5 -Br -CSNH2 8 75 75 93

a
room temperature 30-33 0C, bisolated yield.
Structures of 2-pyrazoline derivatives were elucidated with
their FTIR, mass spectra, 1H and 13C NMR. The ring closure
reaction of chalcones was established by the FTIR spectra of
(4a-4l), which clearly showed the absence of >C=O bond
stretching in region of 1650 cm-1 of chalcone backbone. A peak
at 1596 cm-1 is corresponding to –C=N confirms the formation
of –C=N bond and also due to pyrazine –C=N- bonds, while
peak at 1560 to 1568, 1522 and 1490 cm -1 are due to –C=C–
bond. In addition, the peaks at 1124 to 1211 cm-1 were
attributed to the (-C-N) vibrations, which also confirm the
formation of desired pyrazoline ring in all the compounds. The
The -C3 carbon of pyrazoline ring appears in the range of δ
62.0 ppm to δ 63.0 ppm of compounds (4a-4c and 4f-4l), while
in compounds (4d and 4e) appears at δ 58.86 ppm and δ 58.92
ppm. The -C4 and -C5 carbons of pyrazoline ring appears in the
range of δ 43.4 ppm to δ 44.7 ppm and δ 147 ppm to 156 ppm
respectively. The phenyl ring carbons of compounds (4a-4l)
appear in their desired aromatic region. The peaks at δ 169.32
ppm and δ 177.5 ppm are of >C=O group (4d and 4e) and
>C=S group (4f-4l) carbons respectively. (See ESI†)
Electronic spectra of the cyclized pyrazoline analogues (4a-
4l) were studied in the UV region at molar concentration of 40
μg/mL in ethanol and compounds exhibits different intense

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ARTICLE New Journal of Chemistry

absorption band due to different functionality on 2-position of Table 3. Onset temperature, enthalpy and melting point of
pyrazolines as illustrated in Fig. 2. Compounds (4a-4c) showed synthesized compounds.
two spectral bands, whose maxima were located in the range Comp. Heat flow endothermic m.p.
between 240 nm to 260 nm, and a broad band larger than 350 Onset (0C) ΔH(J/g) (0C)
4a 114.01 38.32 113-115
nm.
4b 150.66 90.54 149-151
3.0 4c 152.66 67.81 151-153
4a 4b 4d 107.65 63.27 108-111

New Journal of Chemistry Accepted Manuscript

4c 4d 4e 148.58 68.49 148-150
2.5 4e 4f
4g 4h 4f 221.23 179.66 220-222
4i 4j 4g 229.49 110.69 228-230
2.0 4k 4l 4h 240.11 99.03 239-241
Absorbance

4i 190.62 195.70 189-191

4j 176.92 119.74 176-178
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1.5
4k 179.28 104.18 179-181
4l 181.12 131.79 182-184
1.0

Antibacterial study
0.5
The in vitro antibacterial activities of compounds (4a–4l)
0.0 were investigated against the human pathogenic gram-positive
200 250 300 350 400 450 500 S. aureus (NCIM 2079) and gram-negative E. coli (NCIM
Wavelength (nm) 2109) bacterial strain. The minimum inhibitory concentration
(MIC) was evaluated by the broth tube dilution method.32
Figure 2. UV absorption spectra of compounds (4a-l) in ethanol Nutrient agar microbiological media used for bacteria (S.
aureus and E. coli), obtained from Hi-media (India) and its
The shorter bands were attributed to →* transition, while composition (grams per litre) has sodium chloride, 5.0; beef
the longer wavelength band were correspondence to extract 10.0; peptone 10.0 (pH 7.2). Stock solution of 1000 μg
intramolecular charge transfer transition owing to two nitrogen mL-1 of each compound prepared in DMSO. Serial dilutions of
of pyrazoline29 and additional phenyl ring at 2-position of the test compounds, already dissolved in DMSO, were prepared
pyrazolines. Compounds (4d) and (4e) showed a broad band in to final concentrations of 512, 256, 128, 126, 64 and 32 μg mL-
the range of 290 nm to 340 nm due to →* transitions. In 1
. The MIC which inhibits the visible growth was determined
carbothioamide containing pyrazolines (4f-4l) three absorption visually after incubation for 24 h, at 37 0C and pH 7.4.
bands in range 210-230 nm, 240-260 nm and 335-355 nm were
owing to n→σ*, →* and n→* transitions, respectively. The Table 4. MIC values (μg/mL) for (4a–4l) against S. aureus, and
band at 335-355 nm assigned to the n→* transition involving E. coli bacterial strains.a,b
the thione portion (-C=S) of thiocarboxamide group. The two Comp. S. aureus E. coli
other absorption bands at 240-260 and 210-230 nm were owing 4a >512 >512
to →* transition of phenyl ring and n→σ* transition of 4b >512 >512
pyrazine ring nitrogen respectively.30 4c >512 >512
4d >512 >512
4e >512 >512
Thermal stability of 2-pyrazolines was studied with DSC, 4f 256 >512
their onset and enthalpy data are given Table 3. The DSC 4g 256 >512
measurements provide quantitative and qualitative information 4h >512 >512
4i 256 >512
about physical and chemical changes that involve endothermic 4j 256 >512
or exothermic processes, or changes in heat capacity. This may 4k 256 >512
result to provide a significant impact on the chemical stability 4l 256 >512
Chloramphenicol <16 <16
and shelf life of the drug products. During the standard heating a
Data are the mean values of three replicates for each concentration.
run, we have observed one endothermic thermograms and the
Concentration in μg mL-1 in DMSO.
b
onset point of endothermic thermograms is noted as solid–
liquid transition or melting transition (T M) and the peak as
liquid-liquid transition (TL-L) for each compounds.31 The The lowest concentration, which showed no visible growth, was
enthalpy (ΔH) values of compounds (4a-e) were found in the considered as an end point for MIC. The MIC level of
range of ΔH = 38.32 J/g to 90.54 J/g, while compounds compounds (4a–4l) against these organisms is given in Table 4.
containing thiocarboxamide groups (4f-l) fall in the range of It is evident from the MIC data that the compounds (4a-4e) and
99.03 J/g to 195 J/g. Compounds having thiocarboxamide (4h) showed MIC value more than 512 μg mL -1 having phenyl
group are more thermally stable than of the phenyl and and ethanone functionality at 2-position of pyrazoline ring,
ethanone functionality containing pyrazolines. while rest of the compounds having –CSNH2 functionality on 2-
position of pyrazoline increase 50% activity against S. aureus
as compared to phenyl and ethanone group with MIC 256 μg

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mL-1. All compounds against gram-negative strain showed noted as AA>4a>4h>4g>4d>4b>4f>4j>4k>4i>4c>4e>4l.

resistance activity (Table 4) with MIC more than 512 μg mL -1.
The higher MIC of compounds can be attributed to their weak
ability to irritate the cell membranes or inhibition of bacterial
growth could be dependent on the ability of the synthesized
compounds to cross the outer membrane in gram-negative
bacteria and the cell wall in gram-positive bacteria. 33 Of course,
Compound (4a) showed superior activity (68.86%) over other
compounds, while compound (4l) (45.41%) showed least
activity at concentration100 μg mL-1. In general, most of the
compounds decrease antioxidant activity on increasing halogen
size, while ethylpyrazine containing -CSNH2 group increase
activity on halogen size increase. Thus, ethylpyrazine

New Journal of Chemistry Accepted Manuscript

the higher MIC of the reported compounds was noted and to
further enhance its level, the structural modifications are being
pursued in the laboratory and soon would be communicated for
publication.
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containing compounds due to increase in alkyl chain lower
scavenging activity, which could be due to hydrophobic effect.
DNA Binding Study

The new age cancer therapy effectively concentrates on

Antioxidant Study
In general, antioxidant compounds are capable of inhibiting
the oxidation of other molecules and resultant preventing the
cell death that occurs owing to the free radicals and for this
purpose, antioxidant therapy is one of the most recent options.34
Antioxidant activity of (4a–4l) was determined by 2,2-
diphenyl-1-picrylhydrazyl (DPPH) free radical method. 34 Stock
solution of DPPH free radical in ethanol (2.16 mg/50 mL) was
prepared and the absorbance was recorded at 517 nm. Then, 20,
40, 60, 80, 100 μg mL-1 of (4a–4l) and L-ascorbic acid (AA) as
standard antioxidant, were prepared in ethanol by dilution
method of 100 μg mL-1 stock solution. For analysis 1 mL
sample solution and 1 mL of stock solution of DPPH were
added and put into dark conditions up to 30 min. Then, an
absorbance was recorded at 517 nm and the percentage
selectively targeting the DNA associated processes, which
interfere or inhibit the DNA replication. The present study was
performed for the DNA binding activity using dsDNA and
dsDNA specific Molecular Probes® dye having green and blue
fluorescence provided by Applied Bio-systems along with
Qubit 2.0 Spectroflurometer. The chalcones (3a-3g) and new
series of 2-pyrazolines (4a-4l) are tested as potential targets for
cancer therapy. These compounds have structural binding
affinity towards the DNA molecules, while they produce
negligible fluorescence in conjugation with dye as illustrated in
Figure 4.
600
(4a) y = 198.5x - 197.5
R² = 0.9999
dsDNA (ng/mL)

scavenging activity was calculated with the following relation 400

and the results are plotted in Figure 3.
Scavenging activity (%) = (A c - As/Ac) ×100 200
Where, Ac = absorbance of DPPH, As = absorbance of test
STD dsDNA
sample.
0
STD 1 STD 2 STD 3 STD 4
100
4a 4b 4c
90 4d 4e 4f
4g 4h 4i
Comp.+DNA+Dye
Scavenging activity (%)

80 4j 4k 4l 600 Comp.+Dye
AA (4b)
70
500
60
DNA binding (ng/mL)

50 400
40
300
30

20 200
20 40 60 80 100
Concentration (μg mL-1 ) 100
Figure 3. Percentage antioxidant or scavenging activities of
compounds (4a–4l) in μg mL-1 determined with DPPH free 0
DD
3b

3d

3g

4b

4d

4g
4h
4i
3a

3c

3e

4a

4c

4e

4j
4k
4l
3f

4f

radical method at different concentrations

Compounds

DPPH radical scavenging activity of the compounds was found
to be good-to-moderate as compared to the standard AA.
Compounds (4a-4l) showed an increase in % antioxidant
activity within the range of 29.26% to 68.86% with an increase
in concentration. An increasing trend of antioxidant activity is
Figure 4. (a) Standard DNA calibration curve and (b) dsDNA
binding affinity of compounds in the presence of fluorescence
dye
From the data it was evident that chalcones and pyrazolines
have structural binding affinity towards the DNA molecule

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(Figure 4b) and increase two to three folds concentration of
DNA, while they produce negligible fluorescence in
conjugation with dye (Figure 4b). When the compounds were
added to the DNA-Dye (DD) conjugate, increase in
fluorescence was observed, which on result increase DNA
concentration in the range of 229-570 ng/mL, due to
intercalation and minor groove binding mechanisms with
The results revealed that all compounds are within the range set by
Lipinski rule of five as per Table 5. It was also found that none of
the compounds have toxicity risk in terms of mutagenic,
tumorigenic, irritant and reproductive effect. Thus, synthesized
compounds could be considered as potent lead compounds in drug
discovery.

New Journal of Chemistry Accepted Manuscript

DNA.35,36 The intercalation binding involves the aromatic ring Table 5. Molecular weight, ClogP, drug likeness, drug score
portion of compounds positioning itself between base pairs of values and toxicity risk data of 4a–4l.
DNA due to π-π stacking interaction as proposed by Lerman to Mol. Clog Drug Drug Toxicity riska
Comp. Wt. P likeness Score M b
Tc Id Re
explain the strong affinity of certain heterocyclic aromatic dyes
4a 332.37 3.60 4.49 0.76 + + + +
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such as acridines for DNA. 35,36 If small molecules intercalate

4b 348.83 4.16 6.03 0.68 + + + +
into DNA base pairs, the double helix of DNA would protect 4c 393.28 4.24 2.88 0.62 + + + +
the bound molecules from the anionic quencher owing to the 4d 314.77 2.27 6.03 0.86 + + + +
base above and below the intercalator. 37 As a result of 4e 359.22 2.36 2.93 0.81 + + + +
4f 315.37 1.28 3.91 0.87 + + + +
compound-DNA interaction raised structural complexity and 4g 331.82 1.83 5.47 0.84 + + + +
affinity to bind with dye. 4h 376.27 1.92 2.29 0.76 + + + +
The data revealed that ethylpyrazine chalcone (3g) has 4i 340.44 1.22 2.54 0.51 + + + +
4j 329.39 1.64 3.71 0.85 + + + +
highest binding affinity (570 ng/mL) to dsDNA which is an 4k 345.85 2.19 5.26 0.81 + + + +
anticancer active compound having GI 50 <0.1 µM as reported in 4l 390.30 2.28 2.1 0.73 + + + +
our previous study.27 It was also found that on increasing the
a
size of halogens at para position and alkyl chain of pyrazine Ranking as (+) no bad effect, (±) medium bad effect, (-) bad effect.
b
M (mutagenic effect)
ring, dsDNA binding activity increased but in the case of (4j- c
T (tumorigenic effect)
4l) having –CSNH2 functionality at 2-pyrazolines showed d
I (irritant effect)
reverse binding trends. Also, compound (3d) and (4i) having – e
R (reproductive effect)
N(CH3) at para position lower the fluorescence intensity. On the basis of reported biological activities, the structural
Chalcones (3a-3g) and compounds (4a-4c) showed good activity relationship has been proposed which agreed well with
binding activity as comparison to –COCH3 and –CSNH2 the effect of substitution at the para position on the phenyl ring,
containing pyrazolines. From the above study or the reaction alkyl substitution on pyrazine ring and 2-position of pyrazoline,
mechanism between compounds, dye and DNA, it can be which seems responsible for lower or higher activities. Groups
concluded that the compounds have high binding affinity such as –F, –Cl, Br and -N(CH3)2 were introduced at the para
towards DNA. This property of the compounds can be utilized position on the phenyl ring along with introduction of methyl
to inhibit the DNA replication at the stage of single stranded and ethyl chain at the 2-position on the pyrazine ring to
structure by attachment of the compounds. Thus, the newly introduce structural diversity. Results from the biological assay
synthesized DNA strand with a few molecules of compound showed that, in general, thiocarboxamide group at pyrazoline
will not be capable of coding the m-RNA strand and render the ring is better for antibacterial activity, while 2-phenyl
cell incapable of producing essential proteins which will lead to pyrazolines containing floro at para position showed better
improper cellular metabolism and ultimately apoptosis or cell antioxidant activity. For DNA binding on increasing size of
death. halogens binding activity increased.

In silico pharmacology and structural activity relationship Conclusions

(SAR) study
Substantially, we have reported an efficient, convenient,
Drug likeliness is a qualitative analysis to check drug like
clean, and green synthetic method using sodium acetate-acetic
properties of compounds which are defined as a complex
acid aqueous solution under ultrasonication at room
balance of various molecular properties and structural features,
temperature. It affords 78−94% yield of pyrazolines with
and to determine whether a molecule is similar to the known
minimal environmental impact. This method was extended for a
drugs. The hydrophobicity, electronic density distribution,
series of structurally different 2-pyrazolines synthesis in
hydrogen bonding, molecule size and presence of
aqueous medium as a new option for synthesis of various
pharmacophoric features influence the behaviour of a molecule
compounds. The work-up is simple and the obtained results
in a living organism, including bioavailability, transport
indicate that, unlike classical heating, ultrasonic irradiation
properties, binding affinity to DNA or proteins, reactivity,
results in higher yields, shorter reaction times and milder
toxicity, metabolic stability and many others. Drug like
conditions. The UV absorption study will provide an insight to
properties were analyzed by the parameters set by Lipinski’s
study organic light emitting diode (OLED) properties, hole
rule of five (ClogP, solubility, molecular weight, drug likeness
transport tendency, photoluminescence in their solid state and
and drug score) using Osiris property explorer 38 and
fluorescence properties of pyrazolines. Further, compounds
summarized in Table 5.

6 | New J. Chem., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 2012

Page 7 of 10 New Journal of Chemistry
Journal Name
showed good to moderate antioxidant (36-69%) and dsDNA
binding activities. Thus, synthesized compounds could be
considered as potent lead compounds in bioorganic chemistry.
Experimental
Hydrazine monohydrate, phenylhydrazine hydrochloride,
thiosemicarbazide and acetic acid were used as received from
Sigma Aldrich chemicals. Sodium acetate and solvents were of
Rankem, India. Compounds prior to use were stored in
moisture-free environment. The ultrasonic irradiation was
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performed at 30 KHz using an Oscar ultrasonic cleaner (model
FP-108FX). The reaction progress was monitored with TLC
(Merck, silica GF257), and spots were visualized under UV
light (RICO Scientific Industries, Model RSUV-5). Melting
points were determined on a Stuart SMP 40 apparatus and
found uncorrected. Elemental analysis was made with Euro
Vector elemental analyser. FT-IR spectra were recorded in KBr
pellets with a Perkin Elmer spectrum-65 FT-IR
spectrophotometer, and wavenumbers are given in cm −1. Mass
spectral analysis (m/z) was obtained using Agilent
Technologies G6520B LCMS-QTOF mass spectrometry with
positive electron spray ionization (+ESI) method. The mobile
phase containing 2% trifloroacetic acid in water and acetonitrile
(30:70 v/v) was run on Agilent zorbax 300 SB-C18 column (3.5
μm, 4.6×50 mm) with flow rate 0.6 mL/min. 1H and 13C NMR
spectra were recorded at r.t. in 5 mm tube using Bruker Avance
III 500 MHz spectrometer in deuterated chloroform (CDCl3) or
DMSO-d6 and tetramethylsilane (TMS) as internal standard.
The chemical shifts are given in  ppm and coupling constants
J in Hz. Splitting patterns are described as singlet (s), doublet
(d), double doublet (dd), triplet (t), quartet (q), broad (br) and
multiplet (m). The absorption transition (λmax) was recorded in
ethanol at r.t. ranging from 200–600 nm using Analytical UV
spectro 2060 plus. The equilibrium transition temperatures and
enthalpies were noted using differential scanning calorimetry
(DSC) with DSC-6000 Perkin Elmer instrument under nitrogen
(flow rate 20 mL/min) and at heating rate of 5 0C/min. for 30-
300 0C and aluminum pan was used as reference. The DSC was
calibrated with indium provided by Perkin Elmer as standard
calibration sample. DNA interaction study was performed using
dsDNA HS assay kit with the Qubit 2.0 Fluorometer.
Synthesis
Microwave assisted solvent free synthesis of (E)-3-aryl-1-(3-
alkyl-2-pyrazinyl)-2-propenones (3a-3g)27
A mixture of 2-acetyl-3-alkylpyrazine (1a-1b, 0.01 mol),
and substituted aromatic aldehyde (2a–2d, 0.01 mol), was
mixed in ethyl acetate (5 mL), absorbed on anhy. K2CO3 and
taken in teflon reaction vessels, dried in air and irradiated at
300 W, infrared red (IR) temperature 70C and a maximum
pressure of 20 bar inside the vessels for completion of
reactions. The progress of the reaction was monitored by TLC
using ethyl acetate: hexane (2:8 v/v) as eluents after each 30 sec
This journal is © The Royal Society of Chemistry 2012
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DOI: 10.1039/C4NJ00594E
ARTICLE
interval. On completion of the reaction (5-7 min), the mixture
was cooled to ambient temperature and the product was
extracted with ethyl acetate and concentrated under vacuum
using a Buchi rotary evaporator or poured over the crushed ice
and filter, washed with water and dried under vacuum. The
overall yields of synthesized compounds were obtained as 90–
95%. The compounds were recrystallized in ethanol/methanol
New Journal of Chemistry Accepted Manuscript
to afford 3a–3g. The spectral data and melting point of
compounds 3a–3g were reliable with literature values.27
Method A: General procedure for the synthesis of
compounds 4a–4l by conventional method
In a 50 mL round bottom flask, a mixture of pyrazine
bearing chalcones (4a-g, 1 mmol) and substituted hydrazines (2
mmol) were charged into a 10 mL ethanol and dissolved on
reflux. To this clear solution, a solution of potassium hydroxide
(2 mmol dissolved in 10 mL ethanol) added drop wise, and
refluxed for a completion of the reaction (Table 2). The
obtained solid was filtered, washed with water; dry under
vacuum and gives 58-75% yields (Table 2). For compound (4d)
and (4e) catalytic amount of acetic acid was added. The
substituted 2-pyrazolines (4a-l) were recrystallized in ethanol
or methanol.
Method B: General procedure for the synthesis of
compounds 4a–4l by ultrasound irradiation method
A mixture of respective pyrazine bearing chalcones (3a-g, 1
mmol) and substituted hydrazines (3 mmol) were taken into a
50 mL capacity Elmer flask. To this mixture added a solution of
sodium acetate (0.3 mmol) in 15 mL acetic acid–water (10:5
v/v), and sonicated for a completion of the reaction. The
progress of the reaction was monitored by TLC. After
completion of the reaction (Table 2), the mixture was poured on
crushed ice and stirred vigorously. The obtained solid was
filtered, washed with water and recrystallize in ethanol or
methanol to afford compound 4a-l with 85 to 93 % yield. Their
structures were confirmed with 1H NMR, 13C NMR, FTIR,
mass and elemental analysis.
2-(5-(4-fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-
yl)-3-methylpyrazine (4a)
Chemical formula: C20H17FN4; Color: Pale yellow; Rf : 0.53;
m.p.: 113-115 0C; Elemental analysis (%): Calculated: C 72.27,
H 5.16, N 16.86; Found: C, 72.24, H 5.18, N 16.90; FTIR (KBr,
cm-1): νmax 3051, 2999 (Ar, -CH str.); 2961, 2928, 2856 (-CH
str., -CH3); 1585 (-C=N), 1596, 1569, 1551, 1524 (-C=C); 1H
NMR (500 MHz, CDCl3): δ 3.05 (s, 3H, -CH3, pyrazine ring),
3.36 (dd, 1H, J = 5.0, 5.0 Hz, Hb), 4.00 (dd, 1H, J = 13.0, 13.0
Hz, Ha), 5.32 (dd, 1H, J = 7.0, 6.5 Hz, H x), 7.27-6.85 (m, 9H,
Ar rings), 8.32 (s, 1H, pyrazine), 8.35 ppm (s, 1H, pyrazine);
13
C NMR (125 MHz, CDCl3): δ 24.63 (-CH3, pyrazine ring),
43.41, 61.94 (pyrazoline ring), 112.63, 115.15, 119.10, 126.44,
128.08, 136.81, 139.81(Ar rings), 142.69, 144.87, 146.46
New J.Chem., 2012, 00, 1-3 | 7

New Journal of Chemistry
ARTICLE
(pyrazine ring), 152.20 (pyrazoline), 160.13 (pyrazine ring),
162.09 ppm (Ar ring); +ESI-MS (m/z): calculated for
C20H18FN4+ 333.1437 [M+1]+, found 333.1521.
2-(5-(4-chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-
yl)-3-methylpyrazine (4b)
Chemical formula: C20H17ClN4; Color: Pale yellow; Rf : 0.55;
m.p.: 149-151 0C; Elemental analysis (%): Calculated C 68.86,
H 4.91, N 16.06; Found: C 68.81, H 4.95, N 16.01; FTIR (KBr,
cm-1): νmax 3044, 2993 (Ar, -CH str.), 2965, 2925 (-CH str., -
CH3), 1594 (-C=N), 1566, 1522 (-C=C); 1H NMR (500 MHz,
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DMSO-d6): δ 2.95 (s, 3H, -CH3, pyrazine ring), 3.23 (dd, 1H, J
= 5.0, 5.0 Hz, Hb), 4.01 (dd, 1H, J = 12.5, 12.0 Hz, Ha), 5.60
(dd, 1H, J = 5.5, 6.0 Hz, Hx), 7.41-6.79 (m, 9H, Ar rings), 8.42
(d, 1H, J = 2.0 Hz, pyrazine ring), 8.49 ppm (s, 1H, pyrazine);
13
C NMR (125 MHz, DMSO-d6): δ 25.26 (-CH3, pyrazine ring),
43.78, 61.52 (pyrazoline ring), 113.33, 119.68, 127.84, 129.01,
129.11, 132.00, 141.06, 141.22 (Ar rings), 141.31, 143.15,
144.95 (pyrazine ring), 147.72 (pyrazoline ring), 152.12 ppm
(pyrazine ring), +ESI-MS (m/z): Calculated for C20H18ClN4+
349.1142 [M+1]+, found 349.1242.
2-(5-(4-bromophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-
yl)-3-methylpyrazine (4c)
Chemical formula: C20H17BrN4; Color: Pale yellow; Rf : 0.56;
m.p.: 151-153 0C; Elemental analysis (%): Calculated C 61.08,
H 4.36, N 14.25; Found: C 61.12, H 4.31, N 14.21; FTIR (KBr,
cm-1): νmax 3052, 2992 (Ar, -CH str.), 2948, 2912 (-CH str., -
CH3), 1595 (-C=N), 1567, 1523 (-C=C); 1H NMR (500 MHz,
CDCl3): δ 3.04 (s, 3H, -CH3, pyrazine ring), 3.36 (dd, 1H, J =
5.0, 5.0 Hz, Hb), 4.00 (dd, 1H, J = 12.5, 12.0 Hz, Ha), 5.29 (dd,
1H, J = 5.0, 7.0 Hz, Hx), 7.47-6.85 (m, 9H, Ar rings), 8.32 (s,
1H, pyrazine ring), 8.35 ppm (s, 1H, pyrazine); 13C NMR (125
MHz, CDCl3): δ 25.72 (-CH3, pyrazine ring), 44.33, 63.07
(pyrazoline ring), 113.65, 120.21, 121.49, 127.58, 129.16,
132.34, 140.83, 140.99, (Ar rings), 141.15, 143.68, 145.78
(pyrazine ring), 147.58 (pyrazoline ring), 153.31 ppm (pyrazine
ring), +ESI-MS (m/z): Calculated for C20H18BrN4+ 393.0637
[M+1]+, found 393.0697.
1-(5-(4-chlorophenyl)-3-(3-methylpyrazin-2-yl)-4,5-dihydro-
1H-pyrazol-1-yl)ethanone (4d)
Chemical formula: C16H15ClN4O, Rf : 0.57, Color: Pale yellow,
m.p.: 108-111 0C; Elemental analysis (%): calculated: C, 61.05;
H, 4.80; N, 17.80; found: C, 61.09; H, 4.83; N, 17.76; FTIR
(KBr, cm-1): νmax 3052, 2997 (Ar, -CH str.), 2965, 2937 (-CH st,
-CH3); 1650 (-C=O), 1594 (-C=N, st), 1530, 1490 (C=C, st);
1
H NMR (500 MHz, CDCl3): δ 2.43 (s, 3H, -CH3, carbonyl),
2.97 (s, 3H, -CH3, pyrazine ring), 3.42 (dd, J = 5.0, 5.0 Hz, 1H,
Hb), 3.89 (dd, J = 10.0, 12.0 Hz, 1H, H a), 5.52 (dd, J = 5.0, 5.0
Hz, 1H, Hx,), 7.31-7.17 (m, 4H, Ar), 8.44 ppm (d, J = 10.0 Hz,
2H, pyrazine ring); 13C NMR (125 MHz, CDCl3): δ 22.05 (-
CH3, carbonyl), 25.19 (-CH3-pyrazine ring), 43.48, 58.86,
8 | New J. Chem., 2012, 00, 1-3
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DOI: 10.1039/C4NJ00594E
New Journal of Chemistry
(pyrazoline), 127.16, 129.06, 133.48, 140.06 (-CH=CH-, Ar
ring), 141.19, 142.91, 144.52 (pyrazine ring), 153.98
(pyrazoline), 154.99 (pyrazine ring), 169.32 ppm (>C=O);
+ESI-MS (m/z): calculated for C16H16ClN4O+ 315.0934
[M+1]+, found 315.1029.
1-(5-(4-bromophenyl)-3-(3-methylpyrazin-2-yl)-4,5-
New Journal of Chemistry Accepted Manuscript
dihydro-1H-pyrazol-1-yl)ethanone (4e)
Chemical formula: C16H15BrN4O, Rf : 0.59; Color: Pale yellow,
m.p.: 148-150; Elemental analysis (%): calculated: C, 53.50; H,
4.21; N, 15.60; found: C, 53.52; H, 4.17; N, 15.64; FTIR (KBr,
cm-1):- νmax 3082, 3046 (Ar, -CH str.), 2963, 2927 (-CH str., -
CH3); 1668 (>C=O), 1597 (-C=N, st), 1554, 1529, 1485 (C=C,
st); 1H NMR (500 MHz, CDCl3): δ 2.43 (s, 3H, -CH3,
carbonyl), 2.97 (s, 3H, -CH3, pyrazine ring), 3.42 (dd, J = 5.0,
5.0 Hz, 1H, Hb), 3.89 (dd, J = 12.0, 12.0 Hz, 1H, H a), 5.50 (dd,
J = 5.0, 5.0 Hz, 1H, H x), 7.46-7.11 (m, 4H, Ar), 8.44 ppm (d, J
= 10.0 Hz, 2H, pyrazine ring); 13C NMR (125 MHz, CDCl3): δ
22.05 (-CH3, carbonyl), 25.19 (-CH3, pyrazine ring), 43.42,
58.92, (pyrazoline), 121.61, 127.48, 132.02, 140.59 (-CH=CH-,
Ar ring), 141.19, 142.93, 144.52 (-CH=CH-, pyrazine ring),
153.99 (pyrazoline), 154.95 (pyrazine ring), 169.28 ppm
(>C=O); +ESI-MS (m/z): calculated for C16H16BrN4O+
359.0429 [M+1]+, found 359.0513.
5-(4-fluorophenyl)-3-(3-methylpyrazin-2-yl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (4f)
Chemical formula: C15H14FN5S; Color: white; Rf : 0.61; m.p.:
220-222 0C; Elemental analysis (%): Calculated: C 57.13, H
4.47, N 22.21; Found: C 57.10, H 4.51, N 22.26; FTIR (KBr,
cm-1): νmax 3244, 3141 (-NH2), 3049 (Ar, -CH str.); 2984, 2916
(-CH str., -CH3) 1584 (-C=N-), 1596, 1552, 1528, 1512 (-C=C-
), 1367, 852 (-C=S); 1H NMR (500 MHz, CDCl3): δ 2.97 (s,
3H, -CH3, pyrazine ring), 3.51 (dd, 1H, J = 5.0, 5.0 Hz, Hb),
4.00 (dd, 1H, J = 11.5, 12.0 Hz, Ha), 6.01 (dd, 1H, J = 5.0, 5.0
Hz, Hx), 6.29, 7.03 (br 2s, 2H, -NH2), 7.24-7.04 (m, 4H, Ar
rings), 8.47, 8.50 ppm (2s, 2H, pyrazine ring); 13C NMR (125
MHz, CDCl3): δ 25.31 (-CH3, pyrazine ring), 44.45, 62.28
(pyrazoline ring), 115.89, 127.33, 137.46, (Ar rings), 141.50,
143.47, 143.98 (pyrazine ring), 153.91 (pyrazoline ring),
156.94 (pyrazine ring), 161.15 (-C-F, Ar ring), 177.57 ppm (-
C=S); +ESI-MS (m/z): Calculated for C15H15FN5S+ 316.0954
[M+1]+, found 316.1061.
5-(4-chlorophenyl)-3-(3-methylpyrazin-2-yl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (4g)
Chemical formula: C15H14ClN5S; Color: white; Rf : 0.61; m.p.:
228-230 0C; Elemental analysis (%): Calculated: C 54.29, H
4.25, N 21.11; Found: C 54.33, H 4.19, N 21.16; FTIR (KBr,
cm-1): νmax 3251, 3144 (-NH2), 3044 (Ar, -CH str.); 2968, 2909
(-CH str., -CH3); 1588 (-C=N-), 1553, 1529, 1481 (-C=C-),
1370, 851 (-C=S); 1H NMR (500 MHz, CDCl3): δ 2.94 (s, 3H, -
CH3, pyrazine ring), 3.47 (dd, 1H, J = 5.0, 5.0 Hz, Hb), 3.98
This journal is © The Royal Society of Chemistry 2012

Page 9 of 10 New Journal of Chemistry
Journal Name
(dd, 1H, J = 12.0, 11.5 Hz, Ha), 5.98 (dd, 1H, J = 5.0, 5.0 Hz,
Hx), 6.25, 6.99 (br 2s, 2H, -NH2), 7.32-7.16 (m, 4H, Ar rings),
8.45, 8.47 ppm (2s, 2H, pyrazine ring); 13C NMR (125 MHz,
CDCl3): δ 25.30 (-CH3, pyrazine ring), 44.36, 62.33 (pyrazoline
ring), 126.96, 129.11, 133.45, 140.14 (Ar ring), 141.50, 143.50,
143.92 (pyrazine ring), 153.91 (pyrazoline ring), 156.92
(pyrazine ring), 177.58 ppm (-C=S); +ESI-MS (m/z):
Calculated for C15H15ClN5S+ 332.0658 [M+1]+, found
332.0761.
5-(4-bromophenyl)-3-(3-methylpyrazin-2-yl)-4,5-dihydro-
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1H-pyrazole-1-carbothioamide (4h)
Chemical formula: C15H14BrN5S; Color: white; Rf : 0.60; m.p.:
239-241 0C; Elemental analysis (%): Calculated: C 47.88, H
3.75, N 18.61; Found: C 47.90, H 3.78, N 18.64; FTIR (KBr,
cm-1): νmax 3250, 3137 (-NH2), 3051 (Ar, -CH str.); 2987, 2913
(-CH str., -CH3); 1584 (-C=N), 1524, 1487, 1480 (-C=C-),
1364, 832 (-C=S); 1H NMR (500 MHz, CDCl3): δ 2.94 (s, 3H, -
CH3, pyrazine ring), 3.47 (dd, 1H, J = 5.0, 5.0 Hz, Hb), 3.97
(dd, 1H, J = 12.0, 11.5 Hz, Ha), 5.98 (dd, 1H, J = 5.0, 5.0 Hz,
Hx), 6.25, 6.99 (br 2s, 2H, -NH2), 7.47-7.10 (m, 4H, Ar rings),
8.44 (s, 1H, pyrazine ring), 8.48 ppm (s, 1H, pyrazine ring); 13C
NMR (125 MHz, CDCl3): δ 25.29 (-CH3, pyrazine ring), 44.31,
62.39 (pyrazoline ring), 121.55, 127.29, 132.05, 140.68 (Ar
ring), 141.50, 143.50, 143.91 (pyrazine ring), 153.91
(pyrazoline ring), 156.91 (pyrazine ring), 177.58 ppm (-C=S);
+ESI-MS (m/z): Calculated for C15H15BrN5S+ 376.0153
[M+1]+, found 376.0262.
5-(4-(dimethylamino)phenyl)-3-(3-methylpyrazin-2-yl)-4,5-
dihydro-1H-pyrazole-1-carbothioamide (4i)
Chemical formula: C17H20N6S; Color: pale yellow; Rf : 0.58;
m.p.: 189-191 0C; Elemental analysis (%): Calculated: C 59.97,
H 5.92, N 24.69; Found: C 59.94, H 5.96, N 24.65; FTIR (KBr,
cm-1): νmax 3270, 3149 (-NH2), 3069, 3028 (Ar, -CH str.); 2992,
2891 (-CH str., -CH3), 1589 (-C=N), 1617, 1553, 1528, 1472 (-
C=C), 1359, 869 (-C=S); 1H NMR (500 MHz, CDCl3): δ 2.92
(s, 6H, -N(CH3)2, 2.94 (s, 3H, -CH3), 3.50 (dd, 1H, J = 5.0, 5.0
Hz, Hb), 3.92 (dd, 1H, J = 11.5, 11.5 Hz, Ha), 5.93 (dd, 1H, J =
5.0, 5.0 Hz, Hx), 6.17, 6.95 (br 2s, 2H, -NH2), 6.67-7.11 (m,
4H, Ar rings), 8.44 (s, 1H, pyrazine ring), 8.45 ppm (s, 1H,
pyrazine ring); 13C NMR (125 MHz, CDCl 3): δ 25.34 (-CH3,
pyrazine ring), 40.53 (pyrazoline ring), 44.39 (-N(CH3)2), 62.61
(pyrazoline ring), 112.59, 126.54, 129.27 (Ar rings), 141.47,
143.23, 144.34 (pyrazine ring), 150.02 (Ar ring), 153.82
(pyrazoline ring), 157.20 (pyrazine ring), 177.45 ppm (-C=S);
+ESI-MS (m/z): Calculated for C17H21N6S+ 341.1470 [M+1]+,
found 341.1601.
3-(3-ethylpyrazin-2-yl)-5-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazole-1-carbothioamide (4j)
Chemical formula: C16H16FN5S; Color: white; Rf : 0.58; m.p.:
176-178 0C; Elemental analysis (%): Calculated: C 58.34, H
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DOI: 10.1039/C4NJ00594E
ARTICLE
4.90, N 21.26; Found: C 58.30, H 4.95, N 21.30; FTIR (KBr,
cm-1): νmax 3250, 3145 (-NH2), 3045, 3028 (Ar, -CH str.); 2988,
2939, 2879 (-CH str., -CH2CH3), 1584 (-C=N), 1551, 1532,
1510, 1480 (-C=C), 1374, 872 (-C=S); 1H NMR (500 MHz,
CDCl3): δ 1.40 (t, 3H, J = 7.5 Hz, -CH2CH3), 3.32 (q, 2H, J =
7.5 Hz, -CH2CH3), 3.47 (dd, 1H, J = 5.0, 5.0 Hz, Hb), 3.98 (dd,
1H, J = 12.0, 11.5 Hz, Ha), 5.99 (dd, 1H, J = 5.0, 5.0 Hz, Hx),
New Journal of Chemistry Accepted Manuscript
6.22, 6.97 (br 2s, 2H, -NH2), 7.22-7.01 (m, 4H, Ar rings), 8.44
(s, 1H, pyrazine ring), 8.52 ppm (s, 1H, pyrazine ring); 13C
NMR (125 MHz, CDCl3): δ 12.20 (-CH3, -CH2CH3), 30.02 (-
CH2, -CH2CH3), 44.67 (pyrazoline ring), 62.22 (pyrazoline
ring), 115.91, 127.32, 137.44 (Ar ring), 141.30, 143.67, 156.65
(pyrazine ring), 158.37 (pyrazoline ring), 161.15 (pyrazine
ring), 163.11 (-C-F, Ar ring), 177.54 ppm (-C=S); +ESI-MS
(m/z): Calculated for C16H17FN5S+ 330.1110 [M+1]+, found
330.1230.
5-(4-chlorophenyl)-3-(3-ethylpyrazin-2-yl)-4,5-dihydro-1H-
pyrazole-1-carbothioamide (4k)
Chemical formula: C16H16ClN5S; Color: white; Rf : 0.60; m.p.:
179-181 0C; Elemental analysis (%): Calculated: C 55.56, H
4.66, N 20.25; Found: C 55.51, H 4.70, N 20.30; FTIR (KBr,
cm-1): νmax 3250, 3145 (-NH2), 3040, 3025 (Ar, -CH str.); 2987,
2947, 2909 (-CH str., -CH2CH3), 1584 (-C=N), 1551, 1528,
1494, 1476 (-C=C), 1375, 843 (-C=S); 1H NMR (500 MHz,
CDCl3): δ 1.40 (t, 3H, J = 7.5 Hz, -CH2CH3), 3.31 (q, 2H, J =
7.5 Hz, -CH2CH3), 3.45 (dd, 1H, J = 5.0, 5.0 Hz, Hb), 3.99 (dd,
1H, J = 12.0, 11.5 Hz, Ha), 5.98 (dd, 1H, J = 5.0, 5.0 Hz, Hx),
6.25, 6.97 (br 2s, 2H, -NH2), 7.32-7.16 (m, 4H, Ar ring), 8.44
(s, 1H, pyrazine ring), 8.52 ppm (s, 1H, pyrazine ring); 13C
NMR (125 MHz, CDCl3): δ 12.17 (-CH3, -CH2CH3), 30.00 (-
CH2, -CH2CH3), 44.58 (pyrazoline ring), 62.26 (pyrazoline
ring), 126.94, 129.10, 133.44, 140.12 (Ar ring), 141.28, 143.48,
143.68, (pyrazine ring), 156.62 (pyrazoline ring), 158.36
(pyrazine ring), 177.53 ppm (-C=S); +ESI-MS (m/z):
Calculated for C16H17ClN5S+ 346.0815 [M+1]+, found
346.0938.
5-(4-bromophenyl)-3-(3-ethylpyrazin-2-yl)-4,5-dihydro-1H-
pyrazole-1-carbothioamide (4l)
Chemical formula: C16H16BrN5S; Color: white; Rf : 0.57; m.p.:
183-185 0C; Elemental analysis (%): Calculated: C 49.24, H
4.13, N 17.94; Found: C 49.20, H 4.15, N 17.97; FTIR (KBr,
cm-1): νmax 3254, 3142 (-NH2), 3045, 3023 (Ar, -CH str.); 2982,
2941, 2907 (-CH str., -CH2CH3), 1586 (-C=N), 1548, 1530,
1493, 1474 (-C=C), 1373, 845 (-C=S); 1H NMR (500 MHz,
CDCl3): δ 1.40 (t, 3H, J = 7.5 Hz, -CH2CH3), 3.31 (q, 2H, J =
7.5 Hz, -CH2CH3), 3.45 (dd, 1H, J = 5.0, 5.0 Hz, Hb), 3.99 (dd,
1H, J = 12.0, 11.5 Hz, Ha), 5.96 (dd, 1H, J = 5.0, 5.0 Hz, Hx),
6.23, 6.97 (br 2s, 2H, -NH2), 7.47-7.10 (m, 4H, Ar ring), 8.44
(s, 1H, pyrazine ring), 8.52 ppm (s, 1H, pyrazine ring); 13C
NMR (125 MHz, CDCl3): δ 12.18 (-CH3, -CH2CH3), 30.01 (-
CH2, -CH2CH3), 44.54 (pyrazoline ring), 62.33 (pyrazoline
ring), 121.56, 127.28, 132.06 140.65 (Ar ring), 141.30, 143.48,
New J.Chem., 2012, 00, 1-3 | 9
 View Article Online
New Journal of Chemistry Page 10 of 10
DOI: 10.1039/C4NJ00594E
ARTICLE New Journal of Chemistry

143.70 (pyrazine ring), 156.62 (pyrazoline ring), 158.37
(pyrazine ring), 177.54 ppm (-C=S); +ESI-MS (m/z):
Calculated for C16H17BrN5S+ 390.0310 [M+1]+, found
390.0418.
Acknowledgements
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New Journal of Chemistry Accepted Manuscript

Authors are thankful to Vice Chancellor Central University of
Gujarat, Gandhinagar, for financial, infrastructural support and
instrumental facility. Also thanks to Dr. MS Dahiya, Director,
The Institute of Forensic Science, Gujarat Forensic Sciences
Published on 28 May 2014. Downloaded by York University on 06/06/2014 12:57:46.
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