Synthesis of new pyrazolines and their biological evaluation as antimicrobial agents

Article abstract of DOI:10.3184/174751912X13445835381909

2-Diazopropane and diazo compounds derived from aromatic aldehydes were reacted with aromatic esters of 3- hydroxyprop-1-yne to give the aromatic esters of 5-hydroxymethyl-3-substituted pyrazoles. Anti-microbial screening using the Gram positive (Staphylo

Full text of DOI:10.3184/174751912X13445835381909

JOURNAL OF CHEMICAL RESEARCH 2012
RESEARCH PAPER 563
OCTOBER, 563–565
Synthesis of new pyrazolines and their biological evaluation as
antimicrobial agents
Naoufel Ben Hamadi*, Amel Haouas, Mahbouba Methamem and Moncef Msaddek
Laboratory of Synthesis heterocyclic and Natural Substances. Department of Chemistry, Faculty of Sciences of Monastir, Boulevard of
Environment, 5000 Monastir, Tunisia
2-Diazopropane and diazo compounds derived from aromatic aldehydes were reacted with aromatic esters of 3-
hydroxyprop-1-yne to give the aromatic esters of 5-hydroxymethyl-3-substituted pyrazoles. Anti-microbial screening
using the Gram positive (Staphylococcus aureus and Enterococcus fecalis) and Gram negative (Escherichia coli
and Klebsiella pneumoniae) bacteria and the yeast Candida albicans, showed that these compounds had promising
activity against both Gram positive and Gram negative bacteria.
Keywords: diazo compounds, alkynes, cycloaddition 1,3-dipolar, pyrazoles, antimicrobial properties
We have been interested in the development of novel ring
systems because of their potential ability to influence the
pharmacokinetic properties of compounds in beneficial ways
for the drug discovery process.1,4 The pyrazole motif makes up
the core structure of numerous biologically active compounds.
Thus, some representatives of this heterocycle exhibit antivi-
ral/antitumour,5,6 antibacterial,7 antiinflamatory,8 analgesic,9
fungistatic,10 NO-donor antioxidants11 and XO inhibitory activ-
ity.12 In recent years, there has been a need for rapid reactions
that meet the three main criteria of an ideal synthesis: effi-
ciency, versatility, and selectivity. Such reactions would allow
medicinal chemistry to keep pace with the information derived
from modern biological screening techniques.13 In connection
with our medicinal chemistry project aimed at the discovery
of new antibacterial agents, we needed to prepare a series of
pyrazoles. A valid synthesis is represented by the 1,3-dipolar
cycloaddition of diazoalkanes with alkynes.14 The advantage
of this procedure is that it is often highly regioselective and, in
addition, the 1,3-dipole can react with an array of unsaturated
substrates to produce a number of different heterocyclic
entities.
p-toluenesulfonyl hydrazide with an aromatic aldehyde
followed by treatment with an aqueous solution of sodium
hydroxide led to a solution of aromatic aldehyde tosylhydra-
zone sodium salt, which upon warming to 50 °C gave a reddish
solution of diazo compounds.17 Prior to warming the reaction
mixture, the acetylenes 1b–d were added and the desired pyr-
azoles 6a–f were obtained in good yield as single regioisomers
(Scheme 2).
Antimicrobial activity
The antimicrobial activity of compounds 3 and 6 was deter-
mined by the agar dilution technique as recommended by the
Clinical and Laboratory Standard Institute (CLSI) 18 utilising a
variety of Gram-positive bacteria (Staphylococcus aureus and
Enterococcus fecalis), Gram-negative bacteria (Escherichia
coli and Klebsiella pneumoniae) and the yeast (Candida
albicans). From the results which were obtained (Table 1) it
has been noticed that all the tested compounds exhibit promis-
ing antimicrobial properties against both Gram-positive and
Gram-negative bacteria. However, all the tested compounds
seem completely inactive against the yeast (C. albicans) which
was used. It is also noted that, in general, compounds 6
are more effective antibacterial agents than compounds 3.
Compounds 6c and 6f seem to be the most effectively prepared
Gram-negative antibacterial agents. This may be attributed to
the role of the anisyl function attached to the 3-position of the
pyrazole ring system as it enhances antibacterial properties to
the greatest extent compared to the other residues.
We report here the synthesis of some new pyrazole deriva-
tives which have been found to possess an interesting profile of
antibacterial activity.
Results and discussion
The acetylenes 1a–d reacted with 2-diazopropane 2,15 to afford
the regioisomers 3a–d where the diazo carbon atom attacks
the terminal acetylenic carbon and the terminal diazo nitrogen
atom bonds to the central carbon atom of the acetylene
(Scheme 1).16
Conclusions
Other diazo compounds that have been investigated
include the 1-aryldiazomethanes 5a–c. Condensation of
In conclusion, our studies have revealed that the 1,3-dipolar
cycloaddition of diazo compounds with various alkynes gives
Scheme 1 Formation of pyrazoles using 2-diazopropane.
* Correspondent. E-mail: bh_naoufel@yahoo.fr

564 JOURNAL OF CHEMICAL RESEARCH 2012
Scheme 2 Proposed synthesis of pyrazoles using diazo compounds.
1
(N=N); 1740 (C=O); H NMR (300 MHz, CDCl3) δ: 1.47 (s, 6H,
pyrazoles with complete regioselectivity. These compounds
were tested for antimicrobial properties against both
Gram-positive and Gram-negative bacteria. From these studies
compounds 6c and 6f have emerged as the lead compound,
showing maximum antimicrobial activity. Thus, compound 6c
represents a fruitful matrix for development of a new class
of antimicrobial agent that deserve further investigation and
derivatisation.
CH3), 5.57 (s, 2H, CH2), 6.96 (s, 1H, CH), 8.24 (d, 2H, Harom, J =
9 Hz); 8.33 (d, 2H, Harom, J = 9 Hz); 13C{1H}NMR (75 MHz, CDCl3)
δ: 20.21 (CH3), 21.63 (CH3), 59.09 (CH2) 93.36 (C-5), 123.45–152.05
(Carom), 144.81 (C-3), 151.33 (C-4), 167.21 (C=O).
(3,3-Dimethyl-3H-pyrazol-5-yl)methyl 4-methoxybenzoate (3c): Yield,
75%; m.p. 157 °C. Anal. Calcd for C14H16N2O2: C, 68.83; H, 6.60;
N, 11.47. Found: C, 68.76; H, 6.55; N, 11.59%; IR (KBr) νcm−1; 1540
1
(N=N); 1735 (C=O); H NMR (300 MHz, CDCl3) δ: 1.44 (s, 6H,
CH3), 2.41 (s, 3H, CH3), 5.51 (s, 2H, CH2), 6.86 (s, 1H, CH), 7.23 (d,
2H, Harom, J = 7.8 Hz); 7.95 (d, 2H, Harom, J = 7.8 Hz); 13C{1H}NMR
(75 MHz, CDCl3) δ: 20.24 (CH3), 21.69 (CH3), 59.17 (CH2) 94.16
(C-5), 126.95–144.05 (Carom), 143.92 (C-3), 151.19 (C-4), 166.20
(C=O).
Experimental
All solvents were distilled prior to use. Chromatography was per-
formed with silica gel 60 (230–400 mesh), and silica gel F254 plates
were used for preparative TLC. The IR spectra frequencies are gives
in cm−1. NMR spectra were determined in CDCl3 solutions at 300 and
75.5 MHz for 1H and 13C NMR, respectively; chemical shifts (δ) were
reported in ppm and J values are given in Hz. Microanalyses were
performed using a Carlo Erba EA1108. All preparations involving
diazopropane were carried out in an efficient fume cupboard behind a
safety screen.
(3,3-Dimethyl-3H-pyrazol-5-yl)methyl 4-nitrobenzoate (3d): Yield,
85%; m.p. 148 °C. Anal. Calcd for C14H16N2O3: C, 64.60; H, 6.20;
N, 10.76. Found: C, 64.58; H, 6.15; N, 10.87%; IR (KBr) νcm−1; 1545
1
(N=N); 1740 (C=O); H NMR (300 MHz, CDCl3) δ: 1.45 (s, 6H,
CH3), 3.86 (s, 3H, OCH3), 5.49 (s, 2H, CH2), 6.86 (s, 1H, CH), 6.87
(d, 2H, Harom, J = 9 Hz); 8.02 (d, 2H, Harom, J = 9 Hz); 13C{1H}NMR
(75 MHz, CDCl3) δ: 20.25 (CH3), 55.47 (OCH3), 59.07 (CH2) 94.16
(C-5), 113.74–163.68 (Carom), 143.90 (C-3), 151.29 (C-4), 165.88
(C=O).
Synthesis of the 1,3-dipolar cycloaddition of 2-diazopropane with
alkynes 1a–d
A 2.6 M ethereal solution of 2-diazopropane was added portionwise
to a solution of alkynes 1a–d (1.5 mmol) in diethyl ether, cooled at
–10 °C. The reaction was kept at the same temperature during 1h.
The solvent was removed and chromatography (SiO2; ethyl acetate/
petroleum ether, 1:3) afforded compounds 3a–d.
(3,3-Dimethyl-3H-pyrazol-5-yl)methyl benzoate (3a): Yield, 70%;
m.p. 133 °C. Anal. Calcd for C13H14N2O2: C, 67.81; H, 6.13; N, 12.17.
Found: C, 67.83; H, 6.17; N, 12.20%; IR (KBr) νcm−1; 1540 (N=N);
1745 (C=O); 1H NMR (300 MHz, CDCl3) δ: 1.46 (s, 6H, CH3), 5.49
(s, 2H, CH2), 6.98 (s, 1H, CH), 7.34–7.55 (m, 5H, Harom); 13C{1H}NMR
(75 MHz, CDCl3) δ: 20.19 (CH3), 21.65, 59.11 (CH2) 93.39 (C-5),
128.41–133.09 (Carom), 144.66 (C-3), 152.00 (C-4), 167.24 (C=O).
(3,3-Dimethyl-3H-pyrazol-5-yl)methyl 4-methylbenzoate (3b):Yield,
80%; m.p. 151 °C. Anal. Calcd for C13H13N3O4: C, 56.72; H, 4.76; N,
15.27. Found: C, 57.01; H, 4.68; N, 15.20%; IR (KBr) νcm−1; 1545
Synthesis of pyrazoles 6a–f; general procedure
The aromatic aldehydes were added to a solution of p-toluenesulfo-
nylhydrazide (1.5 mmol). After stirring for 3 h at room temperature, a
solution of 5N NaOH (300 µL, 1.5 mmol) was added and the mixture
was stirred for a further 20 min. The alkynes 1b or 1d (7.5 mmol)
were added and the mixture was stirred at 50 ºC for 48 h. The volatiles
were evaporated under reduced pressure and the residue was dissolved
in a 1:1 mixture of water–ethyl acetate (70 mL). The organic layer was
separated and dried over MgSO4. After filtration and removal of the
solvent under reduced pressure, the crude material was purified by
flash chromatography (eluent petroleum ether/ethyl acetate 4:1) to
afford a white solid.
(3-Phenyl-1H-pyrazol-5-yl)methyl 4-methylbenzoate (6a): Yield,
60%; m.p. 161 °C. Anal. Calcd for C18H16N2O2: C, 73.95; H, 5.52; N,
Table 1 Minimum inhibitory concentrations (MIC, mg mL−1) of the tested compounds against different organisms
Compounds
Organisms
3a
3b
3c
3d
6a
6b
6c
6d
6e
6f
CIP
AMP
Staphylococcus aureus
Enterococcus fecalis
Escherichia coli
Klebsiella pneumoniae
Candida albicans
10
2.5
10
10
10
5
10
10
5
10
(–)
10
5
10
10
(–)
5
0.6
1.3
2.5
5
2.5
5
0.6
1.3
(–)
10
2.5
1.3
1.3
1.3
(–)
5
0.3
0.3
0.3
1.3
(–)
(–)
(–)
(–)
(–)
4
2.5
2.5
2.5
(–)
0.6
2.5
5
2.5
0.6
2.5
(–)
10
5
(–)
(–)
(–)
(–)
CIP, Ciprofloxacin; AMP, Amphotericin B; (–), inactive.

JOURNAL OF CHEMICAL RESEARCH 2012 565
9.58. Found: C, 73.91; H, 5.55; N, 9.59%; IR (KBr) νcm−1; 1635 (C=N);
1740 (C=O); 1H NMR (300 MHz, CDCl3) δ: 2.39 (s, 3H, CH3), 5.49
(s, 2H, CH2), 6.83 (s, 1H, CH), 6.82–7.45 (m, 5H, Harom); 7.63 (d, 2H,
of about 1.5–108 colony forming unit per spot was applied to the sur-
faces of Mueller–Hinton agar plates containing graded concentrations
of the respective compound; plates were incubated at 37 °C for 18 h.
The spot with the lowest concentration of compound showing no
growth was defined as the minimum inhibitory concentration (MIC).
All organisms used in this study were standard strains obtained from
American Type Culture Collection. The organisms included repre-
sentatives of Gram-positive bacteria (S. aureus 25923 and E. fecalis
29212), Gram-negative bacteria (E. coli 25922 and K. pneumoniae
33495) and yeast (C. albicans 20260). The MIC of ciprofloxacin and
amphotericin B was determined concurrently as reference for antibac-
terial activities, respectively (Table 1). Control DMSO was carried out
with each experiment.
H
arom, J = 8.1 Hz); 7.76 (d, 2H, Harom, J = 8.1 Hz); 13C{1H}NMR
(75 MHz, CDCl3) δ: 21.31 (CH3), 59.21 (CH2), 101.10 (C-4), 125.47–
142.07 (Carom), 148.16 (C-5), 153.02 (C-3), 167.21 (C=O).
(3-p-Tolyl-1H-pyrazol-5-yl)methyl 4-methylbenzoate (6b): Yield,
70%; m.p. 173 °C. Anal. Calcd for C19H18N2O2: C, 74.49; H, 5.92; N,
9.14. Found: C, 74.51; H, 5.96; N, 9.17%; IR (KBr) νcm−1; 1630 (C=N);
1740 (C=O); 1H NMR (300 MHz, CDCl3) δ: 2.41 (s, 3H, CH3), 2.43
(s, 3H, CH3), 5.52 (s, 2H, CH2), 6.67 (s, 1H, CH), 7.23–7.98 (m, 8H,
Harom); 13C{1H}NMR (75 MHz, CDCl3) δ: 21.23 (CH3), 21.53 (CH3),
59.21 (CH2), 101.21 (C-4), 126.32–144.17 (Carom), 148.23 (C-5),
152.98 (C-3), 168.22 (C=O).
(3-(4-Methoxyphenyl)-1H-pyrazol-5-yl)methyl 4-methylbenzoate
Received 1 June 2012; accepted 13 July 2012
Paper 1201345 doi: 10.3184/174751912X13445835381909
Published online: 28 September 2012
(6c): Yield, 80%; m.p. 161 °C. Anal. Calcd for C19H18N2O3: C, 70.79;
−1
H, 5.63; N, 8.69. Found: C, 70.73; H, 5.66; N, 8.72%; IR (KBr) νcm
;
1645 (C=N); 1745 (C=O); 1H NMR (300 MHz, CDCl3) δ: 2.41 (s, 3H,
CH3), 3.80 (s, 3H, OCH3), 5.56 (s, 2H, CH2), 6.69 (s, 1H, CH), 6.85 (d,
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(C=N); 1745 (C=O); H NMR (300 MHz, CDCl3) δ: 5.46 (s, 2H,
CH2), 6.79 (s, 1H, CH), 6.83–7.44 (m, 5H, Harom); 8.25 (d, 2H, Harom
,
J = 9 Hz); 8.33 (d, 2H, Harom, J = 9 Hz); 13C{1H}NMR (75 MHz,
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(C-5), 152.97 (C-3), 166.09 (C=O).
(3-p-Tolyl-1H-pyrazol-5-yl)methyl 4-nitrobenzoate (6e): Yield,
75%; m.p. 135 °C. Anal. Calcd for C18H15N3O4: C, 64.09; H, 4.48; N,
12.46. Found: C, 64.11; H, 4.52; N, 12.50%; IR (KBr) νcm−1; 1640
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(C=N); 1745 (C=O); H NMR (300 MHz, CDCl3) δ: 2.41 (s, 3H,
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7.8 Hz); 7.91 (d, 2H, Harom, J = 7.8 Hz); 8.21 (d, 2H, Harom, J = 9 Hz);
8.29 (d, 2H, Harom, J = 9 Hz); 13C{1H}NMR (75 MHz, CDCl3) δ: 21.17
(CH3), 59.23 (CH2), 102.09 (C-4), 119.44–152.17 (Carom), 149.10
(C-5), 152.99 (C-3), 166.22 (C=O).
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(3-(4-Methoxyphenyl)-1H-pyrazol-5-yl)methyl 4-nitrobenzoate (6f):
Yield, 80%; m.p.139 °C. Anal. Calcd for C18H15N3O5: C, 61.19; H,
−1
4.28; N, 11.89. Found: C, 61.21; H, 4.22; N, 11.90%; IR (KBr) νcm
;
1640 (C=N); 1745 (C=O); 1H NMR (300 MHz, CDCl3) δ: 5.39 (s, 2H,
CH2), 3.78 (s, 3H, OCH3), 6.61 (s, 1H, CH), 6.88 (d, 2H, Harom, J =
9 Hz); 8.01 (d, 2H, Harom, J = 9 Hz); 8.23 (d, 2H, Harom, J = 9 Hz);
8.28 (d, 2H, Harom, J = 9 Hz);13C{1H}NMR (75 MHz, CDCl3) δ: 55.79
(OCH3), 59.24 (CH2), 102.21 (C-4), 123.08–160.11 (Carom), 149.15
(C-5), 153.01 (C-3), 166.29 (C=O).
Antimicrobial activity
The antimicrobial activity of the synthesised compounds 3 and 6 was
determined by the agar dilution technique as recommended by the
Clinical and Laboratory Standard Institute (CLSI).18 The tested com-
pounds were dissolved in dimethyl sulfoxide (DMSO). An inoculum
18 Clinical and Laboratory Standards Institute (Formerly NCCLS), Methods
for Dilution Antimicrobial Susceptibility Tests for Bacteria Grow
Aerobically. Approved Standard M7–A4, Clinical and Laboratory
Standards Institute, Wayne, Pennsylvania, USA, 2005.

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