Synthesis and biological evaluation of pyrazoline derivatives bearing an indole moiety as new antimicrobial agents

Article abstract of DOI:10.1002/ardp.201200479

1-(p-Methylphenyl)-3,5-diaryl-2-pyrazoline derivatives (2a-f) were synthesized via the treatment of 1-(1H-indol-3-yl)-3-aryl-2-propen-1-ones (1a-f) with p-methylphenylhydrazine hydrochloride in hot acetic acid. The structures of these compounds were elucidated by IR, ¹H NMR, and mass spectral data and elemental analysis. These compounds were investigated for their antimicrobial activity. Brine-Shrimp lethality assay was carried out to determine the toxicity of the compounds. Compound 2e, which is the pyrazoline derivative bearing the 2,5-dichlorophenyl moiety, can be identified as the most promising agent against Klebsiella pneumoniae (ATCC 13883) and Candida glabrata (ATCC 36583) due to its inhibitory effects on K. pneumoniae and C. glabrata with a MIC value of 100 μg/mL as a non-toxic agent (LC₅₀ > 1000 μg/mL).

Full text of DOI:10.1002/ardp.201200479

Arch. Pharm. Chem. Life Sci. 2013, 000, 1–7
1
Full Paper
Synthesis and Biological Evaluation of Pyrazoline
Derivatives Bearing an Indole Moiety as
New Antimicrobial Agents
1
Ahmet Ozdemir , Mehlika Dilek Altintop , Zafer Asim Kaplancıklı , Gu¨lhan Turan-Zitouni ,
1
1
1
¨
2
Hu¨lya Karaca , and Yagmur Tunalı
2
˘
1 Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Anadolu University, 26470 Eskis¸ehir,
Turkey
2 Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Anadolu University, Eskis¸ehir, Turkey
1-(p-Methylphenyl)-3,5-diaryl-2-pyrazoline derivatives (2a–f) were synthesized via the treatment of
1-(1H-indol-3-yl)-3-aryl-2-propen-1-ones (1a–f) with p-methylphenylhydrazine hydrochloride in hot
1
acetic acid. The structures of these compounds were elucidated by IR, H NMR, and mass spectral
data and elemental analysis. These compounds were investigated for their antimicrobial activity.
Brine-Shrimp lethality assay was carried out to determine the toxicity of the compounds. Compound
2e, which is the pyrazoline derivative bearing the 2,5-dichlorophenyl moiety, can be identified as the
most promising agent against Klebsiella pneumoniae (ATCC 13883) and Candida glabrata (ATCC 36583)
due to its inhibitory effects on K. pneumoniae and C. glabrata with a MIC value of 100 mg/mL as a non-
toxic agent (LC50 > 1000 mg/mL).
Keywords: Antimicrobial activity / Brine-Shrimp lethality / Chalcone / Indole / Pyrazoline
Received: December 26, 2012; Revised: March 4, 2013; Accepted: March 8, 2013
DOI 10.1002/ardp.201200479
Introduction
importance in medicinal chemistry [9–13]. Chalcones and
pyrazolines have been reported to exhibit a wide spectrum
of biological effects including antimicrobial activity [12–18].
Medicinal chemists have also carried out considerable
research on indole and its derivatives. One of the essential
amino acids containing indole nucleus is tryptophan, which
acts as a biochemical precursor for serotonin (5-hydroxytrypt-
amine, 5-HT) [19–22]. Due to its crucial role in depression,
bipolar disorder and anxiety, serotonin is one of important
monoaminergic neurotransmitters as a drug target in several
major neuropsychiatric diseases [23, 24]. In addition, a con-
siderable number of currently available drugs carry an indole
moiety [19–22]. Indolmycin is an example of antibacterial
agents bearing an indole ring with anti-staphylococcal
activity [25, 26]. Furthermore, some researchers synthesized
indole derivatives and evaluated their antimicrobial activity
against bacterial and fungal strains [27–30].
The significant increase in resistance to conventional anti-
microbial agents has become a major concern for public
health. The high prevalence of multidrug-resistant patho-
gens has led to the failure of current treatments and deaths
in immunocompromised patients. In order to cope with
this serious problem, which is considered as the inevitable
consequence of the widespread use of these agents, the
development of new antimicrobial drugs has gained great
importance [1–5].
Chalcones are considered as precursors of open chain
flavonoids and isoflavonoids present in edible plants.
Chalcones and their analogues are versatile and convenient
starting materials or intermediates for the synthesis of nat-
urally occurring flavonoids and various nitrogen-containing
heterocyclic compounds [6–8]. Among chalcone-derived het-
erocycles, pyrazolines, which are synthesized by the ring
closure reaction of chalcones with hydrazines, have attracted
a great deal of interest due to their synthetic and biological
On the basis of these findings and in the continuation of
our research on the synthesis and antimicrobial evaluation of
pyrazoline derivatives [31–35], herein we report the synthesis
and biological evaluation of chalcone and pyrazoline deriva-
tives bearing an indole moiety as new antimicrobial agents.
The compounds were also investigated for their toxicity.
¨
Correspondence: Dr. Ahmet Ozdemir, Faculty of Pharmacy, Anadolu
University, 26470 Eskis¸ehir, Turkey.
E-mail: ahmeto@anadolu.edu.tr
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¨
A. Ozdemir et al.
2
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–7
Results and discussion
unequivocally prove the 2-pyrazoline structure. The protons
belonging to the aromatic ring and the other aliphatic
groups were observed with the expected chemical shift
and integral values (cf. Experimental).
The mass spectra (MS-FABþ) of all compounds showed
[Mþ1] peaks, in agreement with their molecular formula.
All compounds gave satisfactory elemental analysis.
As described in the Introduction, both indoles and 2-pyrazo-
lines possess important biological activities which render
them useful substances in drug research. On this basis, it
appeared expedient to synthesize new heterocyclic com-
pounds bearing both an indole moiety and a 2-pyrazoline
unit. The reaction of (1H-indol-3-yl)chalcones, as easily avail-
able a,b-unsaturated ketones, with hydrazines seemed to be a
convenient route to fulfil this aim.
The compounds were tested in vitro against various
pathogenic bacteria and Candida species. Pyrazoline deriva-
tives (2a–f) were more effective than chalcone derivatives
(1a–f) against E. faecalis (ATCC 29212) and E. faecalis (ATCC
51299; Table 2). Among pyrazoline derivatives (2a–f), com-
pounds 2b, 2d, and 2f were found to be the most potent
derivatives against E. faecalis (ATCC 29212). These compounds
exhibited the inhibitory activity against E. faecalis (ATCC
29212) with a MIC value of 25 mg/mL, whereas chloramphe-
nicol exhibited its inhibitory activity with a MIC value of
12.5 mg/mL. Compound 2d was also the most effective deriva-
tive against E. faecalis (ATCC 51299). Compound 2d showed the
inhibitory activity against E. faecalis (ATCC 51299) with a MIC
value of 12.5 mg/mL, whereas chloramphenicol showed its
inhibitory activity with a MIC value of 6.25 mg/mL. This out-
come confirms that the position of chloro substituent may
have a considerable influence on antibacterial activity
against E. faecalis (ATCC 29212) and E. faecalis (ATCC 51299).
Compounds 1a, 2a, 2b, 2c, 2d, and 2e exhibited the highest
antibacterial activity against K. pneumoniae (ATCC 13883)
with a MIC value of 100 mg/mL. Compound 1a was the chal-
cone derivative bearing a 2,3-dichlorophenyl moiety, whereas
The treatment of 1-(1H-indol-3-yl)-3-aryl-2-propen-1-one
derivatives (1a–f) with p-methylphenylhydrazine hydrochlo-
ride in hot acetic acid afforded 1-(p-methylphenyl)-3,5-diaryl-
2-pyrazolines (2a–f) in good yields (74–82%; Scheme 1). Some
properties of the compounds are given in Table 1.
The structures of all new 1-(1H-indol-3-yl)-3-aryl-2-propen-1-
ones (1a–f) and 1-(p-methylphenyl)-3,5-diaryl-2-pyrazolines
(2a–f) were elucidated by IR, 1H NMR, FABþ-MS spectral data,
and elemental analyses.
In the IR spectra of compounds 1a–f, the characteristic
–
band due to the C O stretching vibration was observed in
–
the region 1704–1640 cmꢀ1. On the other hand, N–H and
–
–
–
C C, C N stretching bands of all compounds (1a–2f)
–
appeared in the regions 3433–3153 cmꢀ1 and 1614–
1436 cmꢀ1, respectively.
1
In the H NMR spectra of compounds 2a–f, the three pro-
tons attached to the C-4 and C-5 carbon atoms of the 2-
pyrazoline unit gave an ABX spin system. Both the chemical
shifts and the coupling constant values (cf. Experimental)
O
O
O
NaOH
H
+
R
R
N
H
N
H
_
1a f
NHNH2. HCl
CH3COOH
H3C
H
N
N
N
R
Scheme 1. Synthesis of the compounds
1a–2f.
CH3
_
2a f
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Arch. Pharm. Chem. Life Sci. 2013, 000, 1–7
Pyrazoline Derivatives as New Antimicrobial Agents
3
Table 1. Some properties of compounds 1a–2f.
Compound
R
Yield
(%)
m.p.
(8C)
Molecular
formula
Molecular
weight
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
2,3-di(Cl)
2,4-di(Cl)
2,6-di(Cl)
3,4-di(Cl)
2,5-di(Cl)
3,5-di(Cl)
2,3-di(Cl)
2,4-di(Cl)
2,6-di(Cl)
3,4-di(Cl)
2,5-di(Cl)
3,5-di(Cl)
68
64
69
74
56
72
78
82
74
76
75
79
218–220
267–268
237–238
243–244
274–276
222–224
121–122
124–125
217–218
117–118
215–216
110–111
C17H11Cl2NO
C17H11Cl2NO
C17H11Cl2NO
C17H11Cl2NO
316.18
316.18
316.18
316.18
316.18
316.18
420.33
420.33
420.33
420.33
420.33
420.33
C17H11Cl2NO
C17H11Cl2NO
C24H19Cl2N3
C24H19Cl2N3
C24H19Cl2N3
C24H19Cl2N3
C24H19Cl2N3
C24H19Cl2N3
Table 2. Antibacterial activities of compounds 1a–2f as MIC values (mg/mL).
Compound
A
B
C
D
E
F
G
H
I
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
100
400
200
200
200
200
100
100
100
100
100
200
50
200
200
400
200
200
200
200
200
200
200
200
200
200
200
200
400
200
200
200
200
200
200
200
200
200
200
200
400
200
200
100
200
100
100
200
200
200
200
200
400
200
100
200
200
200
200
200
200
200
200
400
200
200
100
100
50
50
25
100
25
800
100
100
100
100
100
50
25
25
12.5
25
400
200
200
200
200
100
200
200
100
100
100
400
200
200
200
200
200
200
200
200
200
200
50
25
12.5
2f
Chloramphenicol
25
6.25
6.25
12.5
3.125
6.25
6.25
6.25
A: K. pneumoniae (ATCC 13883), B: L. monocytogenes (ATCC 7644), C: E. coli (ATCC 35218), D: Y. enterocolitica (clinical isolate, Osmangazi
University, Faculty of Medicine, Department of Microbiology, Eskis¸ehir, Turkey), E: S. typhimurium (NRRL B-4420), F: S. aureus (ATCC
25923), G: E. coli (ATCC 25922), H: E. faecalis (ATCC 29212), I: E. faecalis (ATCC 51299).
Table 3. Anticandidal activities of compounds 1a–2f as MIC values
compounds 2a, 2b, 2c, 2d, and 2e were the pyrazoline
(mg/mL).
derivatives.
Compounds 2d, 2e, and 2f showed their antifungal effects
Compound
A
B
C
D
on C. glabrata (ATCC 36583) with a MIC value of 100 mg/mL,
whilst ketoconazole showed its antifungal effect on
C. glabrata (ATCC 36583) with a MIC value of 50 mg/mL
(Table 3).
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
200
400
200
200
200
200
400
200
200
200
200
200
200
200
200
400
200
200
200
200
200
100
100
100
50
200
25
25
25
25
25
25
200
200
200
100
100
100
200
200
200
200
200
200
200
50
Brine-Shrimp toxicity test results were analyzed by the
LC50 computer program (Trimmed Spearman-Karber
Method, Version 1.5) so as to calculate LC50 values and 95%
confidence intervals [36] (Table 4). According to LC50 values of
the compounds (1a–2f), compounds 1a, 1b, 1c, 1d, 1e, and 2e
(LC50 > 1000 mg/mL) were determined as non-toxic.
Compounds 1f, 2b, 2c, 2d, and 2f were found to be
harmful with LC50 values of 178.18, 707.11, 428.71, 101.53,
50
50
50
2f
Ketoconazole
12.5
1.56
1.56
and 297.30 mg/mL, respectively. Compound 2a (LC50
56.12 mg/mL) was determined as toxic.
¼
A: C. albicans (ATCC 90028), B: C. glabrata (ATCC 36583), C: C. krusei
(NRRL Y-7179), D: C. parapsilosis (NRRL Y-12696).
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¨
A. Ozdemir et al.
4
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–7
Table 4. Brine-Shrimp toxicity results of compounds 1a–2f.
nol (50 mL) was stirred at room temperature for 16 h. The result-
ing solid was washed, dried, and crystallized from ethanol [37].
Compound
LC50
(mg/mL)
Lower
95% limit
Upper
95% limit
Toxicity
1-(p-Methylphenyl)-3-(1H-indol-3-yl)-5-(disubstituted-
phenyl)-2-pyrazoline derivatives (2a–f)
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
>1000
>1000
>1000
>1000
>1000
178.18
56.12
707.11
428.71
101.53
>1000
297.30
–
–
–
–
–
–
–
–
–
–
Non-toxic
Non-toxic
Non-toxic
Non-toxic
Non-toxic
Harmful
Toxic
Harmful
Harmful
Harmful
Non-toxic
Harmful
A mixture of 1-(1H-indol-3-yl)-3-aryl-2-propen-1-one derivatives
(1a–f) (10.0 mmol) and p-methylphenylhydrazine hydrochloride
(30.0 mmol) in the presence of acetic acid (50 mL) was refluxed
for 6 h, then poured onto crushed ice. The precipitate was
separated by filtration, washed with water and crystallized from
methanol to afford 2-pyrazolines [10].
77.96
25.55
176.78
179.18
57.97
–
407.25
123.28
2828.43
1025.72
177.81
–
1-(1H-Indol-3-yl)-3-(2,3-dichlorophenyl)-2-propen-
1-one (1a)
IR (KBr) nmax (cmꢀ1): 3421 (N–H), 1699 (C O), 1521, 1444 (C C),
–
–
–
–
127.66
692.38
1242, 1153, 1099 (C–N), 746 (1,2,3-trisubstituted benzene).
1H NMR (400 MHz, DMSO-d6) d (ppm): 7.19–7.23 (2H, m), 7.36–
7.46 (1H, m), 7.54–7.57 (1H, m), 7.57–7.62 (2H, d, J ¼ 8.02 Hz),
7.66–7.71 (2H, d, J ¼ 8.03 Hz), 8.34–8.40 (1H, m), 8.53 (1H, s),
11.92 (1H, brs, NH).
Conclusion
For C17H11Cl2NO, calculated: C, 64.58; H, 3.51; N, 4.43; found:
C, 64.65; H, 3.55; N, 4.39.
MS (FAB) [Mþ1]þ: m/z 317.
In the present paper, we synthesized 1-(1H-indol-3-yl)-3-aryl-2-
propen-1-ones (1a–f) and 1-(p-methylphenyl)-3,5-diaryl-2-pyra-
zolines (2a–f), which were tested for their antimicrobial
activity against pathogenic bacteria and Candida species.
Brine-Shrimp lethality assay was carried out to determine
the toxicity of the compounds. Among these compounds,
compound 2e can be identified as the most promising agent
against K. pneumoniae (ATCC 13883) and C. glabrata (ATCC
36583) due to its inhibitory effects on K. pneumoniae and
C. glabrata with a MIC value of 100 mg/mL as a non-toxic agent
(LC50 > 1000 mg/mL). In the view of this study, further
research can be carried out on the development of new
effective antimicrobial agents bearing the pyrazoline moiety
by the modification of compound 2e.
1-(1H-Indol-3-yl)-3-(2,4-dichlorophenyl)-2-propen-
1-one (1b)
IR (KBr) nmax (cmꢀ1): 3176 (N–H), 1640 (C O), 1515, 1506, 1471
–
–
–
(C C), 1240, 1157 (C–N), 750 (1,2,4-trisubstituted benzene).
–
1H NMR (400 MHz, DMSO-d6) d (ppm): 7.25–7.28 (2H, m), 7.52–
7.56 (2H, m), 7.71–7.72 (1H, d, J ¼ 2.04 Hz), 7.93–7.92 (2H, m),
8.22–8.25 (1H, d, J ¼ 8.83 Hz), 8.35–8.37 (1H, m), 8.79 (1H, s),
12.20 (1H, s, NH).
For C17H11Cl2NO, calculated: C, 64.58; H, 3.51; N, 4.43; found:
C, 64.62; H, 3.56; N, 4.41.
MS (FAB) [Mþ1]þ: m/z 317.
1-(1H-Indol-3-yl)-3-(2,6-dichlorophenyl)-2-propen-
1-one (1c)
IR (KBr) nmax (cmꢀ1): 3245 (N–H), 1704 (C O), 1593, 1442 (C C),
–
–
–
–
Experimental
1242, 1157 (C–N), 736 (1,2,3-trisubstituted benzene).
1H NMR (400 MHz, DMSO-d6) d (ppm): 7.26–7.29 (2H, m), 7.38–
7.44 (1H, t, J ¼ 8.41 Hz), 7.52–7.56 (1H, m), 7.57–7.62 (2H, d,
J ¼ 8.02 Hz), 7.63–7.73 (2H, d, J ¼ 8.80 Hz), 8.34–8.40 (1H, m),
8.60 (1H, s), 12.18 (1H, br, NH).
Chemistry
All reagents were purchased from commercial suppliers and
were used without further purification. Melting points were
determined on an Electrothermal 9100 melting point apparatus
(Weiss-Gallenkamp, Loughborough, UK) and were uncorrected.
IR spectra were recorded on a Shimadzu 8400 FT-IR spectropho-
tometer (Shimadzu, Tokyo, Japan). 1H NMR spectra were
recorded on a Bruker 400 MHz spectrometer (Bruker, Billerica,
USA). Mass spectra were recorded on a VG Quattro mass spec-
trometer (Agilent, Minnesota, USA). Elemental analyses were
performed on a Perkin Elmer EAL 240 elemental analyzer
(Perkin-Elmer, Norwalk, USA). The TLC was performed on
Kieselgel 60 F254 (Merck) layer using toluene/ethyl acetate
(4:1 v/v) as eluents.
For C17H11Cl2NO, calculated: C, 64.58; H, 3.51; N, 4.43; found:
C, 64.59; H, 3.56; N, 4.42.
MS (FAB) [Mþ1]þ: m/z 317.
1-(1H-Indol-3-yl)-3-(3,4-dichlorophenyl)-2-propen-
1-one (1d)
IR (KBr) nmax (cmꢀ1): 3153 (N–H), 1643 (C O), 1571, 1517, 1458
–
–
–
(C C), 1157 (C–N), 752 (1,2,4-trisubstituted benzene).
–
1H NMR (400 MHz, DMSO-d6) d (ppm): 7.22–7.26 (2H, m), 7.51–
7.62 (2H, m), 7.70–7.72 (1H, d, J ¼ 8.80 Hz), 7.82–7.85 (1H, m),
7.91–7.95 (1H, d, J ¼ 15.61 Hz), 8.23–8.24 (1H, m), 8.32–8.34 (1H,
m), 8.78 (1H, s), 12.16 (1H, brs, NH).
General procedure for the synthesis of the compounds
1-(1H-Indol-3-yl)-3-aryl-2-propen-1-one derivatives (1a–f)
A mixture of 3-acetylindole (0.04 mol), aromatic aldehyde
(0.04 mol) and 10% aqueous sodium hydroxide (10 mL) in etha-
For C17H11Cl2NO, calculated: C, 64.58; H, 3.51; N, 4.43; found:
C, 64.63; H, 3.56; N, 4.40.
MS (FAB) [Mþ1]þ: m/z 317.
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Arch. Pharm. Chem. Life Sci. 2013, 000, 1–7
Pyrazoline Derivatives as New Antimicrobial Agents
5
1-(1H-Indol-3-yl)-3-(2,5-dichlorophenyl)-2-propen-
1-one (1e)
1-(p-Methylphenyl)-3-(1H-indol-3-yl)-5-(2,6-
dichlorophenyl)-2-pyrazoline (2c)
IR (KBr) nmax (cmꢀ1): 3429 (N–H), 1649 (C O), 1624, 1442 (C C),
IR (KBr) nmax (cmꢀ1): 3433 (N–H), 1623, 1616, 1517, 1463 (C N and
–
–
–
–
–
–
–
1155, 1097 (C–N), 744 (1,2,4-trisubstituted benzene).
C C), 1245, 1126 (C–N), 786, 730 (1,2,3-trisubstituted benzene).
–
1H NMR (400 MHz, DMSO-d6) d (ppm): 7.23–7.29 (2H, m),
7.46–7.53 (2H, m), 7.60–7.61 (1H, d, J ¼ 5.02 Hz), 7.87–7.90
(1H, d, J ¼ 15.59 Hz), 7.97–8.00 (1H, d, J ¼ 15.58 Hz), 8.32–8.34
(2H, m), 8.84 (1H, s), 12.23 (1H, brs, NH).
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.15 (3H, s, CH3), 3.29
(1H, dd, JAM ¼ 16.73 Hz, JAX ¼ 5.78 Hz, C4–HA pyrazoline), 3.93
(1H, dd, JMA ¼ 16.73 Hz, JMX ¼ 11.58 Hz, C4–HM pyrazoline), 5.83
(1H, dd, JMX ¼ 11.58 Hz, JAX ¼ 5.80 Hz, C5–HX pyrazoline), 6.78
(2H, d, J ¼ 8.76 Hz, phenyl C2,6–H), 6.95 (2H, d, J ¼ 8.78 Hz,
phenyl C3,5–H), 7.16–7.46 (5H, m, phenyl and indole protons),
7.54–7.60 (1H, m, indole C2–H), 7.63 (1H, d, J ¼ 5.03 Hz,
indole C4–H), 8.26–8.28 (1H, m, phenyl or indole proton),
11.44 (1H, s, NH).
For C17H11Cl2NO, calculated: C, 64.58; H, 3.51; N, 4.43; found:
C, 64.54; H, 3.50; N, 4.45.
MS (FAB) [Mþ1]þ: m/z 317.
1-(1H-Indol-3-yl)-3-(3,5-dichlorophenyl)-2-propen-
1-one (1f)
For C24H19Cl2N3, calculated: C, 68.58; H, 4.56; N, 10.00; found:
C, 68.55; H, 4.57; N, 9.96.
IR (KBr) nmax (cmꢀ1): 3172 (N–H), 1689 (C O), 1554, 1452 (C C),
MS (FAB) [M þ 1]þ: m/z 421.
–
–
–
–
1153 (C–N), 746 (1,3,5-trisubstituted benzene).
1H NMR (400 MHz, DMSO-d6) d (ppm): 7.18–7.27 (2H, m), 7.51–
7.65 (4H, m), 7.96–8.01 (2H, m), 8.31–8.33 (1H, m), 8.82 (1H, s),
12.20 (1H, s, NH).
1-(p-Methylphenyl)-3-(1H-indol-3-yl)-5-(3,4-
dichlorophenyl)-2-pyrazoline (2d)
IR (KBr) nmax (cmꢀ1): 3396 (N–H), 3056 (aromatic C–H), 2916
For C17H11Cl2NO, calculated: C, 64.58; H, 3.51; N, 4.43; found:
C, 64.55; H, 3.43; N, 4.47.
–
–
(aliphatic C–H), 1622, 1616, 1515, 1456 (C N and C C), 1245,
–
–
1029 (C–N), 819 (1,2,4-trisubstituted benzene).
MS (FAB) [Mþ1]þ: m/z 317.
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.17 (3H, s, CH3), 3.22
(1H, dd, JAM ¼ 16.77 Hz, JAX ¼ 5.71 Hz, C4–HA pyrazoline), 3.89
(1H, dd, JMA ¼ 16.72 Hz, JMX ¼ 11.53 Hz, C4–HM pyrazoline), 5.91
(1H, dd, JMX ¼ 11.50 Hz, JAX ¼ 5.78 Hz, C5–HX pyrazoline), 6.76
(2H, d, J ¼ 8.70 Hz, phenyl C2,6–H), 6.98 (2H, d, J ¼ 8.74 Hz,
phenyl C3,5–H), 7.19–7.44 (5H, m, phenyl and indole protons),
7.56–7.65 (1H, m, indole C2–H), 7.69 (1H, d, J ¼ 5.30 Hz,
indole C4–H), 8.27–8.29 (1H, m, phenyl or indole proton),
11.51 (1H, s, NH).
1-(p-Methylphenyl)-3-(1H-indol-3-yl)-5-(2,3-
dichlorophenyl)-2-pyrazoline (2a)
IR (KBr) nmax (cmꢀ1): 3404 (N–H), 3062 (aromatic C–H), 2918
–
–
(aliphatic C–H), 1616, 1515, 1463 (C N and C C), 1245, 1099
–
–
(C–N), 746 (1,2,3-trisubstituted benzene).
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.20 (3H, s, CH3), 3.18
(1H, dd, JAM ¼ 16.87 Hz, JAX ¼ 5.58 Hz, C4–HA pyrazoline), 4.06
(1H, dd, JMA ¼ 16.88 Hz, JMX ¼ 11.57 Hz, C4–HM pyrazoline), 5.61
(1H, dd, JMX ¼ 11.59 Hz, JAX ¼ 5.59 Hz, C5–HX pyrazoline), 6.83
(2H, d, J ¼ 8.81 Hz, phenyl C2,6–H), 6.91 (2H, d, J ¼ 8.83 Hz,
phenyl C3,5–H), 7.04 (1H, m, indole C6–H), 7.21–7.58 (5H, m,
phenyl and indole protons), 7.71 (1H, d, J ¼ 2.53 Hz,
indole C4–H), 8.31–8.34 (1H, m, phenyl or indole proton),
11.53 (1H, s, NH).
For C24H19Cl2N3, calculated: C, 68.58; H, 4.56; N, 10.00; found:
C, 68.57; H, 4.55; N, 9.98.
MS (FAB) [Mþ1]þ: m/z 421.
1-(p-Methylphenyl)-3-(1H-indol-3-yl)-5-(2,5-
dichlorophenyl)-2-pyrazoline (2e)
IR (KBr) nmax (cmꢀ1): 3415 (N–H), 3029 (aromatic C–H), 2914
–
–
(aliphatic C–H), 1616, 1558, 1506, 1456 (C N and C C), 1245,
–
–
For C24H19Cl2N3, calculated: C, 68.58; H, 4.56; N, 10.00; found:
C, 68.54; H, 4.54; N, 9.96.
1087 (C–N), 810 (1,2,4-trisubstituted benzene).
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.15 (3H, s, CH3), 3.28
(1H, dd, JAM ¼ 16.77 Hz, JAX ¼ 5.68 Hz, C4–HA pyrazoline), 3.94
(1H, dd, JMA ¼ 16.79 Hz, JMX ¼ 11.56 Hz, C4–HM pyrazoline), 5.44
(1H, dd, JMX ¼ 11.58 Hz, JAX ¼ 5.65 Hz, C5–HX pyrazoline), 7.01
(2H, d, J ¼ 8.73 Hz, phenyl C2,6–H), 7.24 (2H, d, J ¼ 8.70 Hz,
phenyl C3,5–H), 7.31–7.61 (5H, m, phenyl and indole protons),
7.70 (1H, m, indole C2–H), 7.97 (1H, m, indole C4–H), 8.29–8.35
(1H, m, phenyl or indole proton), 11.55 (1H, s, NH).
For C24H19Cl2N3, calculated: C, 68.58; H, 4.56; N, 10.00; found:
C, 68.61; H, 4.58; N, 10.04.
MS (FAB) [M þ 1]þ: m/z 421.
1-(p-Methylphenyl)-3-(1H-indol-3-yl)-5-(2,4-
dichlorophenyl)-2-pyrazoline (2b)
IR (KBr) nmax (cmꢀ1): 3404 (N–H), 3031 (aromatic C–H), 2918
–
–
(aliphatic C–H), 1614, 1514, 1456, 1436 (C N and C C), 1245,
–
–
1099 (C–N), 808, 746 (1,2,4-trisubstituted benzene).
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.13 (3H, s, CH3), 3.05
(1H, dd, JAM ¼ 16.80 Hz, JAX ¼ 5.60 Hz, C4–HA pyrazoline), 3.96
(1H, dd, JMA ¼ 16.80 Hz, JMX ¼ 11.60 Hz, C4–HM pyrazoline), 5.43
(1H, dd, JMX ¼ 11.60 Hz, JAX ¼ 6.00 Hz, C5–HX pyrazoline),
6.80 (2H, d, J ¼ 8.40 Hz, phenyl C2,6–H), 6.96 (2H, d,
J ¼ 8.80 Hz, phenyl C3,5–H), 7.07 (1H, d, J ¼ 8.40 Hz,
indol C6–H), 7.16–7.48 (5H, m, phenyl and indole protons),
7.64 (1H, d, J ¼ 2.42 Hz, indole C4–H), 8.28–8.30 (1H, m,
phenyl C3–H), 11.45 (1H, s, NH).
MS (FAB) [Mþ1]þ: m/z 421.
1-(p-Methylphenyl)-3-(1H-indol-3-yl)-5-(3,5-
dichlorophenyl)-2-pyrazoline (2f)
IR (KBr) nmax (cmꢀ1): 3404 (N–H), 3060 (aromatic C–H), 2918
–
–
(aliphatic C–H), 1614, 1568, 1514, 1436 (C N and C C), 1245,
–
–
1099 (C–N), 796, 746 (1,3,5-trisubstituted benzene).
For C24H19Cl2N3, calculated: C, 68.58; H, 4.56; N, 10.00; found:
C, 68.54; H, 4.52; N, 9.95.
1H NMR (400 MHz, DMSO-d6) d (ppm): 2.19 (3H, s, CH3), 3.20
(1H, dd, JAM ¼ 16.77 Hz, JAX ¼ 5.87 Hz, C4–HA pyrazoline), 3.90
(1H, dd, JMA ¼ 16.79 Hz, JMX ¼ 11.57 Hz, C4–HM pyrazoline), 5.37
MS (FAB) [Mþ1]þ: m/z 421.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

¨
A. Ozdemir et al.
6
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–7
(1H, dd, JMX ¼ 11.56 Hz, JAX ¼ 5.86 Hz, C5–HX pyrazoline), 6.93
(2H, d, J ¼ 8.79 Hz, phenyl C2,6–H), 7.03 (2H, d, J ¼ 8.79 Hz,
phenyl C3,5–H), 7.22–7.34 (4H, m, phenyl and indole protons),
7.44–7.48 (1H, m, phenyl C4–H), 7.51–7.53 (1H, m, indole C2–H),
7.66 (1H, d, J ¼ 2.44 Hz, indole C4–H), 8.29–8.31 (1H, m, phenyl
or indole proton), 11.49 (1H, brs, NH).
ing 1 mg of test compound in 10 mL DMSO and completing to
1000 mL with artificial seawater [39]. Pure DMSO was used as a
positive control for the toxicity assay. The eggs of Brine-Shrimp
hatched in a conical flask containing 300 mL artificial seawater
made by dissolving a commercial marine salt in deionized water.
The flasks were well aerated with the aid of an air pump, and
kept in a water bath at 25–308C. The larvae hatched within 48 h.
Ten larvae were transferred with pipetter into each vial contain-
ing test compound and artificial seawater. A check count was
performed after 24 h of exposure at room temperature and the
number of dead larvae, exhibiting no internal or external move-
ment during several seconds of observation, was noted. Three
independent experiments were performed for each concen-
tration of compounds 1a–2f.
For C24H19Cl2N3, calculated: C, 68.58; H, 4.56; N, 10.00; found:
C, 68.54; H, 4.52; N, 10.05.
MS (FAB) [Mþ1]þ: m/z 421.
Microbiology
The study was designed to compare MICs obtained by the CLSI
reference M7-A7 broth microdilution method as described in the
previous study [38]. MIC readings were performed twice for each
chemical agent. For both the antibacterial and antifungal assays,
the compounds were dissolved in DMSO. Further dilutions of the
compounds and standard drugs in test medium were prepared at
the required quantities of 800, 400, 200, 100, 50, 25, 12.5, 6.25,
3.125, 1.5625 mg/mL concentrations with Mueller–Hinton broth
and Sabouraud dextrose broth. In order to ensure that the
solvent per se had no effect on bacteria or yeast growth, a control
test was also performed containing inoculated broth supple-
mented with only DMSO at the same dilutions used in our
experiments and found inactive in culture medium.
Final products were tested for their in vitro growth inhibitory
activity against K. pneumoniae (ATCC 13883), L. monocytogenes
(ATCC 7644), E. coli (ATCC 35218), Y. enterocolitica (clinical isolate,
Osmangazi University, Faculty of Medicine, Department of
Microbiology, Eskis¸ehir, Turkey), S. typhimurium (NRRL B-4420),
S. aureus (ATCC 25923), E. coli (ATCC 25922), E. faecalis (ATCC
29212), E. faecalis (ATCC 51299), C. albicans (ATCC 90028), C. glabrata
(ATCC 36583), C. krusei (NRRL Y-7179), C. parapsilosis (NRRL
Y-12696).
The authors have declared no conflict of interest.
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www.archpharm.com