Synthesis, characterization and microbial activity of N-substituted pyrazolines

Article abstract of DOI:10.14233/ajchem.2016.19881

Acetic acid and propanoic acid were treated with thionyl chloride to give respective acid chlorides, which were then reacted with synthesized pyrazolines using triethylamine as catalyst in dry dichloromethane to give N-acetylated pyrazolines (1b-7b) and N

Full text of DOI:10.14233/ajchem.2016.19881

Asian Journal of Chemistry; Vol. 28, No. 9 (2016), 2031-2037
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2016.19881
Synthesis, Characterization and Microbial Activity of N-Substituted Pyrazolines
1,*
2
1
2
JYOTI GABA , SUNITA SHARMA , GEETIKA ARORA and POONAM SHARMA
1
2
Department of Chemistry, Punjab Agricultural University, Ludhiana-141 004, India
Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana-141 004, India
*
Corresponding author: E-mail: jyotgcw@gmail.com
Received: 2 February 2016; Accepted: 16 May 2016;
Published online: 1 June 2016;
AJC-17938
Acetic acid and propanoic acid were treated with thionyl chloride to give respective acid chlorides, which were then reacted with synthesized
pyrazolines using triethylamine as catalyst in dry dichloromethane to give N-acetylated pyrazolines (1b-7b) and N-propanoylated pyrazolines
1
13
(
1c-7c). Synthesized N-substituted pyrazolines were characterized by IR, H NMR and C NMR spectral studies. Prepared compounds
were screened for their microbial activity against Bacillus sp., Pseudomonas sp., Acinetobactor sp. and Klebsiella sp. N-acetylated (7b)
and N-propanoylated (7c) pyrazolines having substitution of methoxy group at meta position and hydroxy group at para position of
benzene ring were found effective against Bacillus sp., Acinetobactor sp. and Pseudomonas sp. but not against Klebsiella sp.All pyrazolines
and N-substituted pyrazolines exhibited less activity than standard ampicillin at all the tested concentrations.
1
13
Keywords: N-substituted pyrazolines, H NMR, C NMR, Microbial activity.
1
13
confirmed by routine spectrometric analysis. IR, H and C
NMR spectra were scanned from Sophisticated Analytical
Instrumentation Facility (SAIF), Central Instrument Laboratory
INTRODUCTION
Pyrazoles are well known important nitrogen containing
-membered hetrocyclic compounds. The two nitrogen atoms
5
(
CIL), Panjab University, Chandigarh. The IR spectra of com-
are present in the adjacent position with two endocyclic double
bonds. Pyrazoline is dihydropyrazole possessing only one
endocyclic double bond. These electron rich nitrogen hetero-
cycles play an important role in diverse biological activities.
Several pyrazoline derivatives possess important pharma-
cological activities and therefore these are useful materials in
drug research. Substituted pyrazolines are useful in pharma-
ceutical and agrochemical research. N-substituted pyrazoline
derivatives also exhibit biological activities like anti-inflamma-
tory, analgesic, antimicrobial, antitumor, antileukemia, anti-
depressant, angiotensin converting enzyme inhibitory activity
and hypertension. Several applications of N-arylpyrazoles in
medicines such as antitumor, antiviral, anti-inflammatory
agents, kinase inhibitors for the treatment of type-2 diabetes,
hyperlipidemia and obesity [1-4]. Keeping in view the potential
biological activities of pyrazolines, it was perceived that the
synergistic effect of heterocyclic moieties in single nucleus
might result in the formation of some worth-while molecules
from the biological point of view.
pounds were recorded using KBr discs on Perkin-Elmer FTIR
1
13
spectrophotometer. H NMR and C NMR spectra were recorded
on a Bruker Avance II 400 MHz instrument using TMS as an
internal standard.
General procedure for synthesis of N-substituted
pyrazolines (1b-7b, 1c-7c): To a solution of acetic acid or
propanoic acid (0.01 mol) in dry dichloromethane (50 mL)
was slowly added thionyl chloride (0.01 mol) at 0 °C, then the
solution was stirred at room temperature for 30 min and heated
to 40 °C for 1 h. The resulting solution was stirred in ice-cold
water. Already synthesized pyrazoline [5] (0.01 mol) was
added to above reaction mixture in small lots. Triethylamine
(
0.01 mol) was added drop-wise from a dropping funnel over
a period of 15 min. Then the reaction mixture was brought to
room temperature and stirred for another 2 h. The solution
was partitioned between dichloromethane and 2.7 N HCl (50
mL) and the two layers were separated. The aqueous layer
was again extracted with dichloromethane (50 mL). Saturated
aqueous sodium bicarbonate solution was added and the
resulting mixture was transferred to a 500 mL separatory
funnel. Layers were separated and aqueous phase was again
extracted with dichloromethane (50 mL). The combined
organic layers were dried over sodium sulphate (anhydrous)
EXPERIMENTAL
All the recorded melting points were determined in open
capillaries and are uncorrected. Structures of compounds were

2
032 Gaba et al.
Asian J. Chem.
and concentrated on rotary vacuum evaporator. The crude
product was recrystallized from methanol to yield different
N-substituted pyrazolines (1b-7b, 1c-7c).
Testing of microbial activity: The effect of N-substituted
pyrazolines on the growth of Bacillus sp., Pseudomonas sp.,
Acinetobacter sp. and Klebsiella sp. was assessed by bacterial
sensitivity-filter paper disc method. The plates were prepared
by pouring 15-20 mL of the sterilized nutrient agar media
1-[5-(2-Methoxyphenyl)-3-methyl-4,5-dihydro-1H-
pyrazol-1-yl]ethanone (4b): Yield 62 %, m.p.: 135-137 °C.
-1
IR (KBr, νmax, cm ): 2991 (aromatic C-H str.), 2933 νas(CH
1627 (C=O str.), 1597 (C=N str.), 1531 (N-N str.), 1457 (C=C
str.), 1369 (C-N str.), 1152 (νasC-O str.), 1046 (ν C-O str.),
870 (C-N bending) and 833 (C-H bending). H NMR (CDCl
δ (ppm): 2.09(s, 3H, CH ), 3.10(s, 3H, CH ), 3.75 (s, 3H, CH
2.59-2.65 (dd, 1H, CH ) (J = 18.2, 4.6), 3.29-3.36 (dd, 1H,
CH ) (J = 18.7, 11.2 Hz), 5.21-5.29 (dd, 1H, CH) (J = 11.7,
4.5 Hz), 7.39-7.56 (m, 4H, Ar-H). C NMR (CDCl
16.96 (CH ), 20.63 (CH -C=O), 45.19 (CH ), 56.71 (CH),
), 167.86 (C=O), 113.79-143.44
2
),
s
1
3
)
3
3
3
),
2
(
Bacillus sp., Acinetobacter sp. and Klebsiella sp.) and King’s
2
13
B (Pseudomonas sp.) on sterilized petriplates. Plates were then
allowed to solidify and stored for 2 days to ensure sterility.
Suspension of 3-4 days old broth of the test organism was
then spread on the required medium plates. Sterile filter paper
discs moistened with test compound solution in dimethyl
sulphoxide were carefully placed on the medium under aseptic
conditions inoculated with the respective bacterial suspension.
Sterilized filter paper discs dipped in dimethyl sulphoxide
served as control. Plates were incubated at 28 ± 1 °C and the
diameter of growth inhibition zone (mm) was measured after
3
) δ (ppm):
3
3
2
158.16 (C=N), 53.32 (OCH
(aromatic carbons).
3
1-[5-(3-Methoxyphenyl)-3-methyl-4,5-dihydro-1H-
pyrazol-1-yl]ethanone (5b): Yield 70 %, m.p.: 146-147 °C.
-
1
IR (KBr, νmax, cm ): 2995 (aromatic C-H str.), 2930 νas(CH
1633 (C=O str.), 1606 (C=N str.), 1528 (N-N str.), 1449 (C=C
str.), 1370 (C-N str.), 1157 νas(C-O str.), 1040 ν (C-O str.),
868 (C-N bending) and 836 (C-H bending). H NMR (CDCl
δ (ppm): 2.08(s, 3H, CH ), 3.12(s, 3H, CH ), 3.79 (s, 3H, CH
2.53-2.60 (dd, 1H, CH ) (J = 18.4, 4.1), 3.36-3.44 (dd, 1H,
CH ) (J = 18.5, 11.4 Hz), 5.21-5.27 (dd, 1H, CH) (J = 11.8,
4.5Hz), 7.37-7.62 (m, 4H, Ar-H). C NMR (CDCl
20.12 (CH ), 24.44 (CH -C=O), 42.81 (CH ), 55.66 (CH),
), 169.10 (C=O), 108.39-147.66
2
),
s
1
3
)
2
4 h. The growth of the organism on medium containing
3
3
3
),
the test compound was also compared with the growth on the
plates containing micro-organism without test compound as
control.
2
2
13
3
) δ (ppm):
1
-(3-Methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-
3
3
2
yl)ethanone (1b): Yield 60 %, m.p.: 156-158 °C. IR (KBr,
153.89 (C=N), 52.81 (OCH
(aromatic carbons).
3
-1
ν
max, cm ): 3038 (aromatic C-H str.), 2928 νas(CH
2
), 1631 (C=O
str.), 1593 (C=N str.), 1540 (N-N str.), 1458 (C=C str.), 1363
1-[5-(4-Hydroxyphenyl)-3-methyl-4,5-dihydro-1H-
pyrazol-1-yl]ethanone (6b): Yield 51 %, m.p.: 168-170. IR
1
(
(
2
(
C-N str.), 868 (C-N bending) and 834 (C-H bending). H NMR
CDCl ) δ (ppm): 2.06 (s, 3H, CH ), 3.17 (s, 3H, CH ), 2.55-
.62 (dd, 1H, CH ) (J = 18.1, 4.2 Hz), 3.33-3.40 (dd, 1H, CH
J = 18.5, 11.2 Hz), 5.25-5.29 (dd, 1H,CH) (J = 11.7, 4.6 Hz),
-1
3
3
3
(KBr, νmax, cm ): 3432 (O-H str.), 3069 (aromatic C-H str.),
2970 νas(CH ), 1632 (C=O str.), 1603 (C=N str.), 1517 (N-N
str.), 1439 (C=C str.), 1352 (C-N str.), 1176 νas(C-O str.), 1019
2
2
)
2
13
1
7
.10-7.29 (m, 5H, Ar-H). C NMR (CDCl
), 23.46 (CH -C=O), 47.22 (CH ), 52.19 (CH), 158.93
C=N), 170.32 (C=O) 118.39-149.43 (aromatic carbons).
-[5-(2-Chlorophenyl)-3-methyl-4,5-dihydro-1H-
pyrazol-1-yl]ethanone (2b): Yield 45 %, m.p.: 137-139 °C.
3
) δ (ppm): 20.21
ν
s
(C-O str.), 847 (C-N bending) and 830 (C-H bending). H
NMR (DMSO-d ) δ (ppm): 2.09 (s, 3H, CH ), 3.20 (s, 3H,
CH ), 2.53-2.60 (dd, 1H, CH ) (J = 18.1, 4.5), 3.36-3.342 (dd,
1H, CH ) (J = 18.7, 11.2 Hz), 5.23-5.27 (dd, 1H, CH) (J =
11.8, 4.6 Hz), 7.49-7.82 (m, 4H, Ar-H), 8.76 (s, 1H, OH). C
NMR (DMSO-d ) δ (ppm): 17.84 (CH ), 21.46 (CH -C=O),
46.71 (CH ), 57.11 (CH), 158.63 (C=N), 170.12 (C=O),
(CH
3
3
2
6
3
(
3
2
1
2
13
-1
IR (KBr, νmax, cm ): 3054 (aromatic C-H str.), 2918 νas(CH
630 (C=O str.), 1592 (C=N str.), 1529 (N-N str.), 1470 (C=C
str.), 1373 (C-N str.), 1044 (C-Cl str.), 871 (C-N bending) and
2
),
6
3
3
1
2
108.63-145.81 (aromatic carbons).
1
8
33 (C-H bending). H NMR (CDCl
CH ), 3.12 (s, 3H, CH ), 2.52-2.59 (dd, 1H, CH
.4), 3.35-3.41 (dd, 1H, CH ) (J = 18.6, 11.3 Hz), 5.26-5.30
dd, 1H, CH) (J = 11.4, 4.3 Hz), 7.44-7.69 (m, 4H, Ar-H). C
NMR (CDCl ) δ (ppm): 16.36 (CH ), 24.41 (CH -C=O), 42.18
CH ), 60.13 (CH), 157.92 (C=N), 168.55 (C=O), 112.68-
43.29 (aromatic carbons).
-[5-(4-Chlorophenyl)-3-methyl-4,5-dihydro-1H-
pyrazol-1-yl]ethanone (3b): Yield 60 %, m.p.: 152-154 °C.
3
) δ (ppm): 2.04 (s, 3H,
1-[5-(4-Hydroxyphenyl)-3-methyl-4,5-dihydro-1H-
pyrazol-1-yl]propan-1-one (7b):Yield 76 %, m.p.: 125-128.
3
3
2
) (J = 18.4,
-1
4
(
2
IR (KBr, νmax, cm ): 3322 (O-H str.), 2955 (aromatic C-H str.),
2931 νas(CH ), 1626 (C=O str.), 1590 (C=N str.), 1537 (N-N
str.), 1460 (C=C str.), 1368 (C-N str.), 1133 νas(C-O str.), 1032
13
2
3
3
3
1
(
1
2
ν
s
(C-O str.), 872 (C-N bending) and 837 (C-H bending). H
NMR (DMSO-d ) δ (ppm): 2.00 (s, 3H, CH ), 3.16 (s, 3H,
CH ), 3.75 (s, 3H, CH ), 2.58-2.64 (dd, 1H, CH ) (J = 18.2,
4.3), 3.32-3.40 (dd, 1H, CH ) (J = 18.5, 11.4 Hz), 5.22-5.26
(dd, 1H, CH) (J = 11.6, 4.4 Hz), 6.50-6.70 (m, 3H, Ar-H),
6
3
1
3
3
2
2
-1
IR (KBr, νmax, cm ): 3049 (aromatic C-H str.), 2919 νas(CH
630 (C=O str.), 1591 (C=N str.), 1526 (N-N str.), 1468 (C=C
str.), 1379 (C-N str.), 1041 (C-Cl str.), 863 (C-N bending) and
2
),
1
3
1
8.77 (s, 1H, OH). C NMR (DMSO-d
21.64 (CH -C=O), 45.98 (CH ), 58.61 (CH), 155.71 (C=N),
), 166.68 (C=O), 109.48-147.36 (aromatic
6
) δ (ppm): 15.58 (CH ),
3
3
2
1
8
39 (C-H bending). H NMR (CDCl
CH ), 3.19 (s, 3H, CH ), 2.57-2.66 (dd, 1H, CH
.4), 3.28-3.36 (dd, 1H, CH ) (J = 18.5, 11.5 Hz), 5.19-5.26
dd, 1H, CH) (J = 11.7, 4.3 Hz), 7.41-7.64 (m, 4H, Ar-H). C
NMR (CDCl ) δ (ppm): 18.32 (CH ), 24.81 (CH -C=O), 45.98
CH ), 54.89 (CH), 152.61 (C=N), 169.74 (C=O), 113.79-
43.44 (aromatic carbons).
3
) δ (ppm): 2.01(s, 3H,
55.43 (OCH
carbons).
3
3
3
2
) (J = 18.0,
4
(
2
1-(3-Methyl-5-phenyl-4,5-dihydro-1H-pyrazol-1-
yl)propan-1-one (1c):Yield 66 %, m.p.:178-180 °C. IR (KBr,
13
-1
3
3
3
ν
max, cm ): 3042 (aromatic C-H str.), 2915 νas(CH ), 1634 (C=O
2
(
2
str.), 1589 (C=N str.), 1533 (N-N str.), 1465 (C=C str.), 1371
1
1
(C-N str.), 870 (C-N bending) and 833 (C-H bending). H NMR

Vol. 28, No. 9 (2016)
Synthesis, Characterization and Microbial Activity of N-Substituted Pyrazolines 2033
(
(
3
CDCl
t, 3H, CH
.36 (dd, 1H, CH
CH) (J = 11.56, 4.4 Hz), 7.19-7.44 (m 5H, Ar-H). C NMR
3
) δ (ppm): 2.07 (s, 3H, CH
), 2.55-2.62 (dd, 1H, CH
) (J = 18.4, 11.6 Hz), 5.23-5.27 (dd, 1H,
3
), 3.22 (q, 2H, CH
2
), 2.20
CH
2
-C=O), 29.81 (CH
2
-C=O), 44.61 (CH
2
), 58.73 (CH),
3
2
) (J = 18.2, 4.4), 3.28-
155.97 (C=N), 54.42 (OCH
(aromatic carbons).
1-[5-(4-Hydroxyphenyl)-3-methyl-4,5-dihydro-1H-
pyrazol-1-yl]propan-1-one (6c): Yield 73 %, m.p.: 189-190
3
), 166.65 (C=O), 110.36-146.87
2
13
(CDCl
(CH -C=O), 45.18 (CH
(C=O), 120.44-153.81 (aromatic carbons).
-[5-(2-Chlorophenyl)-3-methyl-4,5-dihydro-1H-
pyrazol-1-yl]propan-1-one (2c): Yield 55 %, m.p.: 149-151 °C.
3
) δ (ppm): 19.43 (CH
3
), 23.98 (CH
3
-CH -C=O), 35.53
2
-1
2
2
), 57.77 (CH), 154.48 (C=N), 163.62
°C. IR (KBr, νmax, cm ): 3429 (O-H str.), 3072 (aromatic C-H
str.), 2968 νas(CH ), 1635 (C=O str.), 1608 (C=N str.), 1520
(N-N str.), 1445 (C=C str.), 1358 (C-N str.), 1166 νas(C-O str.),
1029 ν (C-O str.), 843 (C-N bending) and 835 (C-H bending).
H NMR (DMSO-d ) δ (ppm): 2.05 (s, 3H, CH ), 3.25 (q, 2H
CH ), 2.26 (t, 3H, CH ), 2.54-2.62 (dd, 1H, CH ) (J = 18.1,
4.4), 3.32-3.41 (dd, 1H, CH ) (J = 18.3, 11.3 Hz), 5.25-5.37
(dd, 1H, CH) (J = 11.7, 4.8 Hz), 7.52-7.85 (m, 4H, Ar-H),
2
1
s
-1
1
IR (KBr, νmax, cm ): 3049 (aromatic C-H str.), 2922 νas(CH
628 (C=O str.), 1598 (C=N str.), 1540 (N-N str.), 1467 (C=C
str.), 1379 (C-N str.), 1030 (C-Cl str.), 874 (C-N bending) and
2
),
6
3
1
2
3
2
2
1
8
CH
CH
35 (C-H bending). H NMR (CDCl
3
) δ (ppm): 2.10 (s, 3H,
), 2.16 (t, 3H, CH ), 2.49-2.56 (dd, 1H,
) (J = 18.1, 4.5), 3.38-3.45 (dd, 1H, CH ) (J = 18.3, 11.4
13
3
), 3.26 (q, 2H CH
2
3
8.80 (s, 1H, OH). C NMR (DMSO-d
21.68 (CH -CH -C=O), 31.65 (CH -C=O), 44.16 (CH
6
) δ (ppm): 18.88 (CH
3
),
2
2
3
2
2
2
), 57.79
Hz), 5.25-5.32 (dd, 1H, CH) (J = 11.8, 4.6 Hz), 7.46-7.59 (m
(CH), 158.81 (C=N), 167.38 (C=O), 111.39-149.36 (aromatic
carbons).
13
4
(
1
H, Ar-H). C NMR (CDCl
CH -CH -C=O), 34.69 (CH -C=O), 48.42 (CH
52.13 (C=N), 163.56 (C=O), 109.48- 145.19 (aromatic
3
) δ (ppm): 17.32 (CH
3
), 21.12
3
2
2
2
), 61.87 (CH),
1-[5-(4-Hydroxy-3-methoxyphenyl)-3-methyl-4,5-
dihydro-1H-pyrazol-1-yl]propan-1-one (7c): Yield 55 %,
-1
carbons).
m.p.: 154-155 °C. IR (KBr, νmax, cm ): 3323 (O-H str.), 2961
(aromatic C-H str.), 2926 νas(CH ), 1627 (C=O str.), 1599 (C=N
str.), 1527 (N-N str.),1452 (C=C str.), 1374 (C-N str.), 1124
as(C-O str.), 1037 ν (C-O str.), 868 (C-N bending) and 828
(C-H bending). H NMR (DMSO-d ) δ (ppm): 2.00 (s, 3H,
CH ), 3.75 (s, 3H, CH ), 3.35 (q, 2H CH ), 2.16 (t, 3H, CH ),
2.51-2.64 (dd, 1H, CH ) (J = 18.3, 4.4), 3.32-3.40 (dd, 1H,
CH ) (J = 18.5, 11.4 Hz), 5.22-5.26 (dd, 1H, CH) (J = 11.5,
4.4 Hz), 6.49-6.70 (m, 4H, Ar-H), 8.79 (s, 1H, OH). C NMR
(DMSO-d ) δ (ppm): 15.58 (CH ), 21.65 (CH -CH -C=O),
54.45 (CH -C=O), 45.97 (CH ), 58.61 (CH), 155.78 (C=N),
), 166.65 (C=O), 109.54-147.38 (aromatic
1
-[5-(4-Chlorophenyl)-3-methyl-4,5-dihydro-1H-
2
pyrazol-1-yl]propan-1-one (3c): Yield 62 %, m.p.: 160-162
-
1
°
C. IR (KBr, νmax, cm ): 3044 (aromatic C-H str.), 2917
ν
s
1
νas(CH
2
), 1634 (C=O str.), 1596 (C=N str.), 1530 (N-N str.),
6
1
464 (C=C str.), 1383 (C-N str.), 1032 (C-Cl str.), 862 (C-N
3
3
2
3
1
bending) and 836 (C-H bending). H NMR (CDCl
2
3
) δ (ppm):
), 2.14 (t, 3H, CH ), 2.56-
) (J = 18.5, 4.4), 3.36-3.42 (dd, 1H, CH ) (J
18.5, 11.3 Hz), 5.27-5.33 (dd, 1H, CH) (J = 11.6, 4.6 Hz),
2
.02 (s, 3H, CH
3
), 3.22 (q, 2H CH
2
3
2
13
2.65 (dd, 1H, CH
2
2
=
6
3
3
2
13
7
.38-7.54 (m, 4H, Ar-H). C NMR (CDCl
CH ), 22.56 (CH -CH -C=O), 31.82 (CH
CH ), 56.16 (CH), 153.87 (C=N), 162.59 (C=O), 114.76-
52.83 (aromatic carbons).
-[5-(2-Methoxyphenyl)-3-methyl-4,5-dihydro-1H-
pyrazol-1-yl]propan-1-one (4c): Yield 65 %, m.p.: 150-
3
) δ (ppm): 17.85
2
2
(
(
1
3
3
2
2
-C=O), 48.79
55.44 (OCH
carbons).
3
2
1
RESULTS AND DISCUSSION
-1
152 °C. IR (KBr, νmax, cm ): 2985 (aromatic C-H str.), 2923
ν ), 1630 (C=O str.), 1592 (C=N str.), 1527 (N-N str.),
1453 (C=C str.), 1372 (C-N str.), 1144 νas(C-O str.), 1050 ν (C-
Desired N-substituted pyrazolines were synthesized
according to steps depicted in Fig. 1. Pyrazolines are weak
bases, which can react with acids to form salts. Acids were
first changed to acid chlorides by reaction with thionyl chloride.
Addition of amine (pyrazoline) (1b-7b) in small lots to acid
chloride solution in dry dichloromethane in presence of
triethylamine was done to neutralize one equivalent of acid
formed. So triethylamine acted as a catalyst in preparation of
N-substituted pyrazolines (1b-7b and 1c-7c). As thionyl
chloride reacts explosively with water, use of dry solvent is
necessary. The physical parameters (colour, yield, melting
as(CH
2
s
1
O str.), 870 (C-N bending) and 825 (C-H bending). H NMR
(
CDCl
3
) δ (ppm): 2.02 (s, 3H, CH
), 2.19 (t, 3H, CH ), 2.52-2.58 (dd, 1H, CH
8.1, 4.5), 3.34-3.40 (dd, 1H, CH ) (J = 18.5, 11.2 Hz), 5.22-
.26 (dd, 1H, CH) (J = 11.4, 4.6 Hz), 7.44-7.66 (m, 4H, Ar-
3
), 3.78 (s, 3H, CH
3
), 3.25
(q, 2H, CH
2
3
2
) (J =
1
5
2
13
H). C NMR (CDCl
C=O), 29.89 (CH -C=O), 45.59 (CH
C=N), 54.53 (OCH ), 165.66 (C=O), 107.38-149.54 (aromatic
carbons).
-[5-(3-Methoxyphenyl)-3-methyl-4,5-dihydro-1H-
pyrazol-1-yl]propan-1-one (5c): Yield 69 %, m.p.: 151-153
3
3 3
) δ (ppm): 14.82 (CH ), 23.81 (CH -CH
2
-
2
2
), 56.18 (CH), 158.75
(
3
point, R
f
values) of synthesized compounds were determined
1
and are presented in Table-1.All compounds were characterized
1
13
by their spectral data (IR, H NMR and C NMR).
-
1
°
ν
C. IR (KBr, νmax, cm ): 2988 (aromatic C-H str.), 2923
), 1638 (C=O str.), 1610 (C=N str.), 1531 (N-N
str.),1451 (C=C str.), 1377 (C-N str.), 1150 νas(C-O str.), 1049
IR data: Infrared spectral data revealed the formation
of N-substituted pyrazolines. N-N, C=N and C-N stretching
were also observed in range of 1527-1514, 1606-1590 and
as(CH
2
1
-1
ν
s
(C-O str.), 863 (C-N bending) and 830 (C-H bending). H
NMR (CDCl ) δ (ppm): 2.00 (s, 3H, CH ), 3.81 (s, 3H, CH ),
.15 (q, 2H CH ), 2.17 (t, 3H, CH ), 2.52-2.62 (dd, 1H, CH
J = 18.3, 4.6), 3.39-3.47 (dd, 1H, CH ) (J = 18.6, 11.1 Hz),
.22-5.35 (dd, 1H, CH) (J = 11.3, 4.2 Hz), 7.40-7.66 (m, 4H,
1378-1348 cm , respectively which assured the formation
of five membered pyrazoline ring. The presence of C=C in
aromatic ring was confirmed due to presence of band in 1474-
3
3
3
3
(
5
2
3
2
)
-1
1431 cm , region. Band for O-H stretching was observed for
compounds 6b, 7b, 6c and 7c having hydroxy group on
aromatic ring.
2
13
Ar-H). C NMR (CDCl
3
) δ (ppm): 14.83 (CH
3
), 24.66 (CH -
3

2
034 Gaba et al.
Asian J. Chem.
CH3
N
4
5
CH3
H C
2
3
HC
N
N
2
N
O
1
X
R
CH3
O
CH
3
R1
X= CH , CH CH
3 2 3
H C
2
Fig. 2. Structure of pyrazoline ring
N
3
R
2
HC
N
(
1b-7b
)
13
C NMR data:A further more assurance to the formation
of desired compounds was added by C NMR data. Out of
three carbons of pyrazoline ring, C-3 of pyrazoline ring (Fig.
R
H
13
CH3
3
R1
2
2
) in contact with electronegative nitrogen atom corresponding
H C
2
R2
1a-7a
N
to C=N showed a maximum downfield shift. C-4 and C-5 of
pyrazoline ring appeared near 45.59 ppm and 51.13 ppm
respectively. Maximum absorption was observed for carbonyl
(
)
HC
N
O
R
13
carbon in the range of 162.59-170.32 ppm. The C NMR
spectra of N-acetylated pyrazolines exhibited one peak in range
of 20.62-24.44 ppm due to methyl carbon attached to carbonyl
carbon. Similarly spectra of N-propanoylated pyrazoline
exhibited peaks at 28.89-35.53 ppm and 21.12-24.66 ppm due
to methylene and methyl carbon bonded to carbonyl group.
CH2
H C
R
1
3
R2
1
2
3
4
5
6
7
a, 1b, 1c
a, 2b, 2c
a, 3b, 3c
a, 4b, 4c
a, 5b, 5c
a, 6b, 6c
a, 7b, 7b
R=H, R =H, R =H
1 2
R=Cl, R =H, R =H
(1c-7c)
1
2
R=H, R =H, R =Cl
1
2
R=OCH , R =H, R =H
R=H, R =OCH , R =H
3
1
2
13
1
3
2
The C NMR spectra of compounds 4b, 5b, 7b, 4c, 5c and 7c
showed peak at 52.81-57.34 ppm indicating the presence of
R=H, R =H, R =OH
1
2
R=H, R =OCH , R =OH
1
3
2
methoxy (OCH ) group on benzene ring.
3
Fig. 1. Scheme for the synthesis of N-substituted pyrazolines
Microbial activity
TABLE-1
Bacillus sp.: Table-2 showed that all the compounds
except 2c, 3c and 4b were active against Bacillus sp. at 250
µg/mL. N-propanoylated pyrazoline having methoxy group
at meta position of benzene ring (4c) was significantly higher
active than other compounds with an inhibition zone of 11
mm at 250 µg/mL.
PHYSICAL PARAMETERS OF N-SUBSTITUTED PYRAZOLINES
Yield
%)
m.p.
(°C)
Compd.
m.f.
Colour
Brown
Rf
(
1b
C
12
H
14
N
2
O
60
66
45
55
60
62
62
65
70
69
51
73
76
55
156-158 0.54
178-180 0.54
137-139 0.53
149-151 0.52
152-154 0.52
160-162 0.52
135-137 0.53
150-152 0.52
146-147 0.53
151-153 0.52
168-170 0.51
189-190 0.52
125-127 0.51
154-155 0.51
1c
C H N O
Brown
1
3
16
2
2b
C H N OCl Brown
1
2
13
2
2c
C H N OCl Brown
13 15 2
3b
C H N OCl Brown
TABLE-2
1
2
13
2
3c
C H N OCl Brown
MICROBIAL ACTIVITY OF DIFFERENT N-SUBSTITUTED
PYRAZOLINES ON THE GROWTH OF Bacillus sp.
1
3
15
2
4b
C H N O
2
Brown
1
3
16
2
4c
C H N O
Dark brown
Light brown
Brown
Yellow
Yellow
1
4
18
2
2
Diameter of growth inhibition zone (mm)
5b
C H N O
13 16 2 2
C H N O
14 18 2 2
Compd.
3000
2000
1000
500
250
5c
µg/mL
11.5
12.0
10.5
11.5
9.5
11.5
11.5
11.5
11.0
12.0
12.0
12.0
12.0
12.0
28.0
0.22
µg/mL
µg/mL
µg/mL
µg/mL
6b
C H N O
2
1
2
14
2
1
1
b
c
11.5
11.5
10.5
11.0
9.5
11.0
10.5
10.5
11.0
12.0
11.5
11.5
11.5
11.0
27.0
0.44
11.0
11.0
10.0
10.0
9.5
10.0
10.0
9.0
10.5
12.0
11.0
11.5
11.5
10.5
18.5
0.44
10.5
10.5
10.0
9.5
8.5
9.0
10.0
9.5
8.5
0
8.5
0
6c
C H N O
1
3
16
16
2
2
7b
C
13
H
N
2
O
3
Reddish brown
Off-white
2b
7c
C H N O
14 18 2 3
2
c
3b
1
H NMR data: The formation of desired N-substituted
3c
4b
4
1
pyrazolines was confirmed by H NMR spectrum. The two
hydrogens on C-4 of pyrazoline ring (Fig. 2) were not found
equivalent. Double doublets were obtained for both of these
protons at 2.51-2.65 ppm and 3.22-3.41 ppm. Proton at C-5
of pyrazoline also appeared as double doublet in the range of
8.0
8.5
0
c
8.5
10.0
11.0
9.5
10.0
9.5
9.5
15.5
0.44
5b
10.0
11.0
10.5
10.0
10.0
10.5
17.5
0.17
5
c
6b
6
c
5
.19-5.17 ppm. Also a singlet due to methyl group attached
7b
to carbonyl carbon appeared in spectra of N-acetylated pyra-
zolines. Similarly spectra of N-propanoylated pyrazolines
exhibited a triplet of three methyl protons and a quartet of two
methylene carbons attached to carbonyl carbon. Aromatic
protons appeared as multiplet in range of 6.50-7.71 ppm.
More confirmation to the formation of N-substituted pyrazo-
lines was provided by coupling constant values of double
doublets.
7c
Ampicillin
CD (p = 0.05)
Minimum inhibitory concentration (MIC) studies of
N-acetylated pyrazolines (Fig. 3) revealed that compound
5
b having methoxy group at meta position of benzene ring
was active against Bacillus sp. at concentration 190 µg/mL.

Vol. 28, No. 9 (2016)
Synthesis, Characterization and Microbial Activity of N-Substituted Pyrazolines 2035
3
2
2
1
1
00
50
00
50
00
TABLE-3
2
60
2
40
MICROBIAL ACTIVITY OF DIFFERENT N-SUBSTITUTED
PYRAZOLINES ON THE GROWTH OF Pseudomonas sp.
220
220
2
10
2
00
1
90
Diameter of growth inhibition zone (mm)
Compd.
3
000
2000
µg/mL
1000
µg/mL
500
µg/mL
11.5
9.5
11.0
8.5
9.5
8.5
9.0
11.0
10.0
10.0
10.0
11.0
11.0
10.0
16.5
0.17
250
µg/mL
11.0
0
9.0
0
8.0
0
9.0
0
9.5
9.5
10.0
10.5
10.0
9.5
15.0
0.44
µg/mL
12.0
14.5
12.5
10.5
11.0
12.0
11.5
15.0
11.5
13.5
13.0
13.0
12.5
12.0
24.0
0.44
1b
12.0
130
11.5
10.5
11.0
9.5
10.0
14.5
10.5
11.0
12.5
12.0
11.5
11.0
20.0
0.22
11.5
10.5
11.5
9.5
11.0
8.5
1c
2b
50
2c
0
3b
1b
2b
3b
4b
5b
6b
7b
3c
N-acetylated pyrazolines
4b
9.5
Fig. 3. Minimum inhibitory concentration (MIC) of different N-acetylated
4c
13.0
10.0
11.0
11.5
11.0
11.0
11.0
18.0
0.17
pyazolines against Bacillus sp.
5b
5c
N-propanoylated pyarzolines which were active against Bacillus
sp. were further studied for minimum inhibitory concentration
MIC) values (Fig. 4). Compound 6c having para hydroxy
group on benzene ring was most active with minimum inhibitory
concentration (MIC) value of 140 µg/mL. So it may be con-
cluded that with the introduction of spacer, MIC value of all
the compounds decreased except compounds 2c and 3c having
chloro group at ortho and at para position respectively of
benzene ring against Bacillus sp.
6b
6c
(
7b
7c
Ampicillin
CD (p = 0.05)
250
250
200
150
100
2
00
200
1
90
190
180
400
150
400
350
300
250
200
150
100
300
220
50
190
170
160
140
0
1
b
2b
3b
4b
5b
6b
7b
N-acetylated pyrazolines
50
Fig. 5. Minimum inhibitory concentration (MIC) of different N-acetylated
pyazolines against Pseudomonas sp.
0
1c
2c
3c
4c
5c
6c
7c
N-propanoylated pyrazolines
350
350
3
3
2
50
00
50
Fig. 4. Minimum inhibitory concentration (MIC) of different N-propanoy-
300
300
lated pyazolines against Bacillus sp.
Pseudomonas sp.: Table-3 revealed the inhibitory effect
of different N-substituted pyrazolines on Pseudomonas sp. At
200
170
170
160
1
000 µg/mL, compounds 1b, 2b, 4c and 6b exhibited higher
1
50
00
activity as compared to other compounds with inhibition zone
in range of 11.5-13.0 mm. N-propanoylated pyrazoline with
methoxy group at ortho position of benzene ring (compound
1
50
4
c) showed maximum inhibition zones of 15, 14.5 and 13.0
0
1c
2c
3c
4c
5c
6c
7c
mm at 3000, 2000 and 1000 µg/mL, respectively.
N-propanoylated pyrazolines
Minimum inhibitory concentration (MIC) studies of
N-acetylated pyrazolines (Fig. 5) revealed that compound 7b
Fig. 6. Minimum inhibitory concentration (MIC) of different N-propanoy-
lated pyazolines against Pseudomonas sp.
(
having both hydroxy and methoxy groups on benzene ring)
exhibited lowest MIC value of 150 µg/mL. Minimum inhibitory
concentration (MIC) studies of different N-propanoylated
pyrazolines (Fig. 6) suggested that presence of meta methoxy
group (compound 5c), para hydroxy group (compound 6c)
and both methoxy and hydroxy groups (compound 7c) lowered
the MIC values to a great extent as compared to substituted
N-propanoylated pyrazoline 1c. So it may be concluded that
both N-acetylated pyrazolines (7b) and N-propanoylated
pyrazolines having ortho methoxy group and para hydroxy
group (7c) exhibited considerably low MIC values (150 and
170 µg/mL) than unsubstituted pyrazolines (1b and 1c).
Compounds having electron donating groups like alkoxy
and hydroxy proved to be beneficial and exhibited excellent
antibacterial activity against Pseudomonas sp. [6,7].
Acinetobacter sp.: Perusal of data given in Table-4 showed
that N-propanoylated pyrazoline (7c) having both hydroxy and
methoxy group on benzene ring exhibited highest activity
against Acinetobacter sp. at all the concentrations. N-Acetylated

2
036 Gaba et al.
Asian J. Chem.
TABLE-4
Klebsiella sp.: Data presented in Table-5 exhibited
the inhibitory effect of different N-substituted pyrazolines on
Klebsiella sp. Compounds 3c (para chloro group on benzene
ring) and 6b (para hydroxy group on benzene ring) exhibited
highest inhibiton zones of 13.5 mm and 13.0 mm respectively
at 250 µg/mL. Minimum inhibitory concentration values (MIC)
of different N-acetylated pyrazolines against Klebsiella sp. are
presented in Fig. 9. Data revealed that compound 1b (no
substitution on benzene ring) and 6b (hydroxy group at para
position of benzene ring) exhibited least MIC value of 150
µg/mL. Minimum inhibitory concentration (MIC) studies of
different N-propanoylated pyrazolines (Fig. 10) exhibited that
MICROBIAL ACTIVITY OF DIFFERENT N-SUBSTITUTED
PYRAZOLINES ON THE GROWTH OF Acinetobacter sp.
Diameter of growth inhibition zone (mm)
Compd.
3
000
2000
µg/mL
1000
µg/mL
12.5
9.0
500
µg/mL
12.5
8.5
9.5
9.0
10.0
9.0
10.0
9.5
10.0
9.0
11.0
12.0
10.0
13.0
16.5
0.17
250
µg/mL
11.0
0
0
µg/mL
14.0
12.0
14.5
11.5
11.5
12.0
12.0
12.0
12.0
12.0
12.0
15.0
14.0
15.0
25.0
0.17
1b
13.0
11.5
11.5
11.5
11.0
12.0
11.5
12.0
11.0
11.5
12.0
13.5
12.0
14.0
23.0
0.17
1c
2b
11.0
10.5
11.0
11.0
10.0
11.5
11.0
10.5
11.0
13.0
11.0
14.0
17.0
0.44
2c
0
3b
9.0
9.0
9.0
9.0
10.0
0
3c
4b
4c
5b
TABLE-5
5c
MICROBIAL ACTIVITY OF DIFFERENT N-SUBSTITUTED
PYRAZOLINES ON THE GROWTH OF Klebsiella sp.
6b
0
6c
9.0
9.0
12.5
15.0
0.17
Diameter of growth inhibition zone (mm)
7b
Compd.
3000
2000
1000
500
250
7c
µg/mL
13.5
13.0
11.5
11.5
13.0
15.5
14.5
12.5
12.0
13.0
14.5
12.0
12.0
12.0
30.0
0.17
µg/mL
µg/mL
µg/mL
µg/mL
11.0
9.0
9.5
0
Ampicillin
CD (p = 0.05)
1
1
b
c
13.5
13.0
10.5
11.5
12.0
14.5
14.0
12.5
12.0
12.5
14.0
11.5
12.0
11.5
23.0
0.17
12.0
12.0
10.5
10.5
11.5
14.5
13.5
12.0
11.5
11.0
13.5
10.5
11.5
11.0
17.0
0.17
11.5
11.0
10.0
9.0
2b
pyrazolines with no substitution on benzene ring (1b) and ortho
chloro group (2b) were found more effective than respective
N-propanoylated pyrazolines from 500 to 3000 µg/mL. Inhibition
effect of standard was significantly higher than all tested
compounds at all concentrations. So all N-acetylated pyrazolines
exhibited lower MIC values as compared to all N-propanoylated
pyrazolines except compound 4c having chloro group at para
position. Both N-acetylated pyrazolines and N-propanoylated
pyrazolines having para chloro group registered lowest MIC
values of 190 µg/mL and 120 µg/mL, respectively (Figs. 7 and 8).
2
c
3b
10.0
13.5
13.5
11.0
11.0
10.5
13.5
10.5
11.0
10.5
16.5
0.17
9.0
3
4b
4c
c
13.5
12.5
10.0
10.0
9.5
13.0
9.0
11.0
10.5
15.0
0.17
5b
5
c
6b
6
c
7b
7
c
Ampicillin
CD (p = 0.05)
4
00
400
350
300
250
200
150
100
190
190
190
190
200
280
250
180
160
160
240
240
150
150
200
1
90
140
120
100
80
6
0
0
5
0
4
0
1b
2b
3b
4b
5b
6b
7b
20
0
N-acetylated pyrazolines
1
b
2b
3b
4b
5b
6b
7b
Fig. 7. Minimum inhibitory concentration (MIC) of different N-acetylated
N-acetylated pyrazolines
pyazolines against Acinetobacter sp.
Fig. 9. Minimum inhibitory concentration (MIC) of different N-acetylated
450
pyazolines against Klebsiella sp.
450
400
350
300
250
200
150
100
280
3
00
250
00
150
00
350
350
350
240
180
2
170
160
210
200
200
150
130
1
50
50
0
0
1c
2c
3c
4c
5c
6c
7c
1c
2c
3c
4c
5c
6c
7c
N-propanoylated pyrazolines
N-propanoylated pyrazolines
Fig. 8. Minimum inhibitory concentration (MIC) of different N-propanoy-
Fig. 10. Minimum inhibitory concentration (MIC) of different N-propanoy-
lated pyazolines against Acinetobacter sp.
lated pyazolines against Klebsiella sp.

Vol. 28, No. 9 (2016)
Synthesis, Characterization and Microbial Activity of N-Substituted Pyrazolines 2037
compound 3c (chloro group at para position of benzene ring)
and 4c (methoxy group at ortho position of benzene ring
recorded lowest minimum inhibitory concentration (MIC)
value of 130 µg/mL and 150 µg/mL respectively as compared
to compound 1c (no substitution on benzene ring).
ACKNOWLEDGEMENTS
The authors are thankful to Panjab University, Chandigarh
1
13
for providing facilities of IR, H NMR and C NMR recording.
REFERENCES
Conclusion
1
2
3
.
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synthesized by reacting different pyrazolines with acetic
acid and propanoic acid in the presence of triethylamine. N-
substituted pyrazolines exhibited less activity than standard
ampicillin at all the tested concentrations. All the compounds
showed significant differences among themselves at all the
concentrations. Compounds bearing substitution of methoxy
group at meta position and hydroxy group at para position of
benzene ring of all N-acetylated (7b) and N-propanoylated
4
4. D. Kim, Y. Lee,Y.W. Lee, P.M. Dewang,Y.Y. Sheen, Y.W. Kim, H. Park,
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5
6
7
.
.
.
9
(
7c) pyrazolines were found effective against Bacillus sp.,
(
2012).
Acinetobactor sp. and Pseudomonas sp. but not against Klebsiella
sp.