Novel dihydropyrazole derivatives linked with 4H-chromene: Microwave-promoted synthesis and antibacterial activity

Article abstract of DOI:10.2174/157017810791824847

Seven novel 6-(1-acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)-2-amino-4- substituted-phenyl-4H-chromene-3-carbonitrile derivatives were synthesized and characterized by ESI-MS, 1H NMR and 13C NMR. All of the compounds have been screened for their antibact

Full text of DOI:10.2174/157017810791824847

Letters in Organic Chemistry, 2010, 7, 487-490
487
Novel dihydropyrazole Derivatives Linked with 4H-Chromene:
Microwave-Promoted Synthesis and Antibacterial Activity
Xin-Hua Liu*, Jin-Xin Liu, Lin-Shan Bai, Guo-Lin Lan and Chu-Xiou Pan
School of Chemistry and Chemical Engineering, Anhui University of Technology, Maanshan 243002, China
Received March 23, 2009: Revised April 28, 2010: Accepted April 28, 2010
Abstract: Seven novel 6-(1-acetyl-3-methyl-4,5-dihydro-1H-pyrazol-5-yl)-2-amino-4-substituted-phenyl-4H-chromene-
1
3-carbonitrile derivatives were synthesized and characterized by ESI-MS, H NMR and 13C NMR. All of the compounds
have been screened for their antibacterial activity. The results show that compounds 7e and 7f displayed significant
activity with MIC of 1.562 μg/mL against B. subtilis ATCC 6633.
Keywords: Microwave-promoted, synthesis, characterize, dihydropyrazole, 4H-chromene, antibacterial activity.
INTRODUCTION
antidepressant, antihypertensive, anti-tubulin, antiviral,
antioxidative [10].
Now, multidrug-resistant Gram-positive bacteria have
started posing serious issues in medical science to deal with.
To overcome the limitations of the known inhibitors, it has
become imperative to identify new class of compound. Many
pyrazole derivatives are well acknowledged to possess a
wide range of antibacterial bioactivities [1-3]. Much
attention was paid to pyrazole as a potential antimicrobial
agent after the discovery of the natural pyrazole C-glycoside.
Hoffmann-La Roche’s group [4, 5] has developed a new lead
DNA gyrase inhibitor (compound 1, Fig. 1). Recently,
Motivated by the aforementioned findings, we anticipate
that the presence of 4H-chromene in the 4,5-dihydropyrazole
moiety may play an important role in the antimicrobial
activities. However, to date, few reports have been dedicated
to the synthesis and antimicrobial activity evaluation of 4H-
chromene -4,5-dihydropyrazole derivatives. Herein, in
continuation to extend our research on antibacterial
compounds containing 4,5-dihydropyrazole moiety, we
designed a series of novel 4,5-dihydropyrazole derivatives
H
O
H
N
O
O
N
N
N
CF3
S
NOC
N
N
N
N
HN
O
Ph
O
MeO
1
2
3
4
Fig. (1). Recently disclosed pyrazole as antibacterial inhibitors.
Tanitame, A. et al. [6] have found compound 2 (Fig. 1) as
potent and selective inhibitors of DNA gyrase. In our
previous work, we found that compound 3 (Fig. 1) possessed
potent antibacterial activity. We also reported that some 4,5-
dihydropyrazole derivatives and some 4,5-dihydropyrazole
ethanone oxime ester derivatives compound 4 (Fig. 1)
showed antibacterial bioactivities [7-9].
linked with 4H-chromene. The structures of novel
compounds were confirmed by spectral analysis. The
antibacterial activity of the synthesized compounds was
tested against B. subtilis ATCC 6633, E. coli ATCC 35218,
P. aeruginosa ATCC 13525 and S. aureus ATCC 6538.
RESULTS AND DISCUSSION
4H-Chromenes are important classes of oxygenated
heterocyclic compounds. More recently, there has been
increased interest in the synthesis of 4H-chromene since
bioactive natural product containing 4H-chromene ring
system possesses wide spectrum of activities including as
The synthetic route to target compound was shown in
Scheme 1. Compounds 5, 6 (Scheme 1) were synthesized
according to the previously published report [11]. Phenol
with malononitrile and substituted aldehyde proceeding in
ethanolic piperidine to form chromenes was a classical
procedure. This procedure employs piperidine as a hazardous
organic base, refluxing for long hours and gives low yields
of products. In order to optimize the reaction conditions and
to greenify the classical method to a clean, economical
*Address correspondence to this author at the School of Chemistry and
Chemical Engineering, Anhui University of Technology, Maanshan 243002,
China; Tel: +86 555 2311551; Fax: +86 555 2311552;
E-mail: xhliuhx@163.com
1570-1786/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

488 Letters in Organic Chemistry, 2010, Vol. 7, No. 6
Liu et al.
H3C
CHO
O
NH2-NH2.H2O
CH3COOH
CH3COCH3
N
CH3
N
OH
HO
H3C
O
5
OH
6
H3C
O
N
RCHO
Microwave
NH2
N
NaHCO3
O
CH3
CN
R
CH2(CN)2
7
Scheme 1. Synthesis of dihydropyrazole derivatives Linked with 4H-Chromene.
7a: R= benzenyl; 7b: R= 4-methoxy-benzenyl; 7c: R= 2,4-dichloro-benzenyl; 7d: R= 4-trifluoromethyl -benzenyl; 7e: R= 2-furanyl; 7f: R=
3-pyridinyl; 7g: R= 2-naphthalenyl.
method for preparation of chromene, the syntheses were
carried out in presence of various bases, for example:
NaHCO3, KHCO3, Na2CO3, NaCH3CO2, pyridine. It was
found that a yield up to 72% when the reaction mixture was
refluxed for 20 min in ethanol under microwave irradiation
catalyzed by NaHCO3. Using the above optimal condition,
compounds 7a-7g were prepared.
positive control penicillin. The compounds 7a, 7c, 7e and 7f
showed moderate antibacterial activities against S. aureus
ATCC 6538 with MIC of 6.25 μg/mL. All the compounds
showed poor antibacterial activities against P. fluorescens
ATCC 13525 and E. coli ATCC 35218.
EXPERIMENTAL
The activities of synthesized compounds were tested
against B. subtilis ATCC 6633, E. coli ATCC 35218, P.
fluorescens ATCC 13525 and S. aureus ATCC 6538 which
may be the cause agents of some serious infections in
humans using MH medium (Mueller-Hinton medium: casein
hydrolysate 17.5 g, soluble starch 1.5 g, beef extract 1000
mL). The MICs of the compounds against four bacteria are
presented in Table 1. Also included are the activities of
reference compounds kanamycin, penicillin and novobiocin
(Nanjing Zhuyan Biotechnology Co. Ltd, Amresco
060D0504, Nanjing 210002, China). The compounds 7e and
7f showed antibacterial activities against B. subtilis ATCC
6633 with the MIC of 1.562 μg/mL, comparable to that of
Analysis and Instruments
1
Melting points were measured and not corrected. The H
NMR spectra were recorded on a Varian INOVA300 (300
MHz) pulse Fourier-transform NMR spectrometer in CDCl3.
ESI mass spectra were obtained on a Mariner System 5303
mass spectrometer. The studies were carried out in a
microwave reactor (Discover, CEM-SP1245). Analytical
TLC (E.Merck, Type 5554 plates) was performed on silica
gel GF254. Column chromatographic purification was
carried out using silica gel. All reagents were of analytical
grade or chemically pure.
Table 1. Minimum Inhibitory Concentrations (MIC-μg/mL) of the Title Compounds
Compounds
Microorganisms
Gram Positive
Staphylococs aureus
Gram Negative
Pseudomonas aeruginosa
Bacillus Subtilis
Escherichia coli
7a
6.25
6.25
12.5
25.0
1.562
1.562
50
6.25
25.0
6.25
25.0
6.25
6.25
25
50
50
>50
50
7b
7c
50
>50
50
7d
7e
50
>50
>50
50
50
7f
50
7g
25
Novobiocina
Penicillina
Kanamycina
0.78
1.562
0.78
3.125
1.562
1.562
6.25
6.25
3.125
6.25
6.25
3.125
aUsed as a positive control.
Negative control DMSO, no activity.

Novel dihydropyrazole Derivatives Linked
Letters in Organic Chemistry, 2010, Vol. 7, No. 6
489
Syntheses: general synthetic procedure for 6-(1-acetyl-3-
methyl-4,5-dihydro-1H-pyrazol-5-yl)-2-amino-4-substitu-
ted-phenyl-4H-chromene-3-carbonitrile derivatives (7)
(s, 1H, 4H-chromene), 5.68 (dd, J=11.1 and 3.0 Hz, 1H,
pyrazole, 5-H), 5.93 (d, 1H, J=3.2 Hz, furan-H), 6.22 (dd,
1H, J=3.2 and 1.7 Hz, furan-H), 6.41 (s, 2H, NH2), 6.67-
7.29 (m, 4H, ArH and furan-1H); 13C NMR (CDCl3, 125
MHz): ꢀ 17.0, 22.3, 31.5, 43.9, 53.8, 59.9, 107.2, 110.5,
115.8, 117.2, 117.8, 123.5, 128.0, 138.5, 142.0, 151.0, 152.2,
158.3, 169.9, 178.0; ESI-MS: 363.0 (C20H18N4O3, [M+H]+).
To a solution of 1-(5-(4-hydroxyphenyl)-3-methyl-4,5-
dihydropyrazol-1-yl)ethanone 6 (2 mmoL) and aqueous
o
Na2HCO3 (2 mmoL) in C2H5OH (30 mL) at 20 C for 30
min. Then the respective aldehyde (2 mmol) and
malononitrile (2 mmol) were added, the reaction mixture
was microwave irradiation for 20 min at 60 oC. Upon
completion, the reaction mixture was allowed to cool to
room temperature and then maintained at 0~5oC for 10 h.
The product was collected by filtration, and the crude residue
was purified by recrystallization (acetone/petroleum, V:V =
1:2) to give 7 as colorless solids. Their spectra are provided
in the supporting information.
o
1
7f: Colorless crystal, yield 62%. mp 219~221 C, H
NMR (CDCl3, 300 MHz): ꢀ 2.18 (s, 3H, Me), 2.37 (s, 3H,
Me-amide), 3.00 (dd, J=18.2 and 3.0 Hz, 1H, pyrazole, 4-
Ha), 3.36 (dd, J=18.2 and 11.1 Hz, pyrazole, 1H, 4-Hb), 4.86
(s, 1H, 4H-chromene), 5.69 (dd, J=11.1 and 3.0 Hz, 1H,
pyrazole, 5-H), 6.49 (s, 2H, NH2), 6.73-8.69 (m, 7H, ArH
and pyridine-H); 13C NMR (CDCl3, 125 MHz): ꢀ 16.8, 21.4,
30.9, 43.5, 53.8, 59.9, 118.0, 118.5, 122.1, 124.0, 130.9,
132.0, 136.2, 137.5, 147.0, 149.4, 151.7, 153.3, 158.0, 169.2,
178.4; ESI-MS: 374.0 (C21H19N5O2, [M+H]+).
o
1
7a: Colorless crystal, yield 70%. mp 201~203 C, H
NMR (CDCl3, 300 MHz): ꢀ 2.12 (s, 3H, Me), 2.36 (s, 3H,
Me-amide), 3.01 (dd, J=18.2 and 3.0 Hz, 1H, pyrazole, 4-
Ha), 3.49 (dd, J=18.2 and 11.1 Hz, pyrazole, 1H, 4-Hb), 4.82
(s, 1H, 4H-chromene), 5.73 (dd, J=11.1 and 3.0 Hz, 1H,
pyrazole, 5-H), 6.30 (s, 2H, NH2), 6.68-7.27 (m, 8H, ArH);
13C NMR (CDCl3, 125 MHz): ꢀ 17.8, 22.7, 31.3, 42.9, 54.5,
62.3, 118.8, 119.2, 120.6, 126.2, 127.1, 128.3, 128.9, 130.7,
136.8, 143.1, 150.7, 157.5, 170.1, 179.2; ESI-MS: 371.5
(C22H20N4O2, [M+H]+).
o
1
7g: Colorless crystal, yield 67%. mp 226~227 C, H
NMR (CDCl3, 300 MHz): ꢀ 2.15 (s, 3H, Me), 2.35 (s, 3H,
Me-amide), 2.98 (dd, J=18.2 and 3.0 Hz, 1H, pyrazole, 4-
Ha), 3.39 (dd, J=18.2 and 11.1 Hz, pyrazole, 1H, 4-Hb), 4.81
(s, 1H, 4H-chromene), 5.69 (dd, J=11.1 and 3.0 Hz, 1H,
pyrazole, 5-H), 6.41 (s, 2H, NH2), 6.66-7.86 (m, 10H, ArH);
13C NMR (CDCl3, 125 MHz): ꢀ 17.3, 21.9, 31.2, 43.9, 54.6,
60.1, 117.8, 118.0, 121.3, 125.1, 125.3, 127.2, 128.1, 128.4,
128.6, 128.7, 129.4, 131.0, 132.3, 134.5, 134.9, 137.6, 151.0,
153.5, 169.0, 178.0; ESI-MS: 422.1 (C26H22N4O2, [M+H]+).
o
1
7b: Colorless crystal, yield 52%. mp 216~217 C, H
NMR (CDCl3, 300 MHz): ꢀ 2.10 (s, 3H, Me), 2.31 (s, 3H,
Me-amide), 3.00 (dd, J=18.2 and 3.0 Hz, 1H, pyrazole, 4-
Ha), 3.55 (dd, J=18.2 and 11.1 Hz, pyrazole, 1H, 4-Hb), 3.78
(s, 3H, OMe), 4.87 (s, 1H, 4H-chromene), 5.69 (dd, J=11.1
and 3.0 Hz, 1H, pyrazole, 5-H), 6.37 (s, 2H, NH2), 6.68-7.02
(m, 7H, ArH); 13C NMR (CDCl3, 125 MHz): ꢀ 17.2, 22.2,
31.0, 43.3, 54.2, 61.8, 62.0, 115.4, 116.6, 116.9, 121.1,
125.1, 129.3, 130.5, 131.3, 140.0, 152.0, 157.8, 159.3, 169.4,
179.0; ESI-MS: 401.7 (C23H22N4O3, [M+H]+).
Bioassay Conditions: In Vitro Antibacterial Activity
The antibacterial activities of the synthesized compounds
were tested against B. subtilis, E. coli, P. fluorescens and S.
aureus using MH medium (casein hydrolysate 17.5g, soluble
starch 1.5 g, beef extract 1000 mL). The MICs of the test
compounds were determined by a colorimetric method using
the dye MTT [12]. A stock solution of the synthesized
compound (100 ꢁg/mL) in DMSO was prepared and graded
quantities of the test compounds were incorporated in
specified quantity of sterilized liquid MH medium. A
specified quantity of the medium containing the compound
was poured into microtitration plates. Suspension of the
microorganism was prepared to contain approximately 105
cfu/mL (colony forming units) and applied to microtitration
plates with serially diluted compounds in DMSO for testing
and incubation at 37 °C for 24 h. After the MICs were
visually determined on each of the microtitration plates, 50
μL of PBS (Phosphate Buffered Saline 0.01 mol/L, pH 7.4:
Na2HPO4.12H2O 2.9 g, KH2PO4 0.2 g, NaCl 8.0 g, KCl 0.2
g, distilled water 1000 mL) containing 2 mg of MTT/mL
was added to each well. Incubation was continued at room
temperature for 4-5 h. The content of each well was
removed, and 100 μL of isopropanol containing 5% 1 mol/L
HCl was added to extract the dye. After 12 h of incubation at
room temperature, the optical density was measured with a
microplate reader at 550 nm. The MICs were observed.
o
1
7c: Colorless crystal, yield 72%. mp 225~227 C, H
NMR (CDCl3, 300 MHz): ꢀ 2.19 (s, 3H, Me), 2.36 (s, 3H,
Me-amide), 3.03 (dd, J=18.2 and 3.0 Hz, 1H, pyrazole, 4-
Ha), 3.50 (dd, J=18.2 and 11.1 Hz, pyrazole, 1H, 4-Hb), 4.82
(s, 1H, 4H-chromene), 5.76 (dd, J=11.1 and 3.0 Hz, 1H,
pyrazole, 5-H), 6.48 (s, 2H, NH2), 6.71-7.26 (m, 6H, ArH);
13C NMR (CDCl3, 125 MHz): ꢀ 17.2, 20.2, 22.7, 43.8, 54.3,
62.1, 117.0, 117.5, 122.7, 125.0, 128.3, 128.9, 132.4, 133.0,
133.5, 136.1, 140.1, 149.7, 151.0, 157.9, 169.0, 178.2; ESI-
MS: 440.9 (C22H18Cl2N4O2, [M+H]+).
o
1
7d: Colorless crystal, yield 51%. mp 208~209 C, H
NMR (CDCl3, 300 MHz): ꢀ 2.17 (s, 3H, Me), 2.39 (s, 3H,
Me-amide), 3.01 (dd, J=18.2 and 3.0 Hz, 1H, pyrazole, 4-
Ha), 3.37 (dd, J=18.2 and 11.1 Hz, pyrazole, 1H, 4-Hb), 4.89
(s, 1H, 4H-chromene), 5.62 (dd, J=11.1 and 3.0 Hz, 1H,
pyrazole, 5-H), 6.48 (s, 2H, NH2), 6.69-7.44 (m, 7H, ArH);
13C NMR (CDCl3, 125 MHz): ꢀ 17.9, 22.8, 30.8, 43.5, 54.7,
61.1, 118.2, 118.4, 121.5, 125.2, 126.4, 127.0, 128.7, 128.9,
130.1, 139.6, 145.0, 152.4, 159.1, 169.3, 178.7; ESI-MS:
439.7 (C23H19F3N4O2, [M+H]+).
o
1
CONCLUSION
7e: Colorless crystal, yield 59%. mp 208~209 C, H
NMR (CDCl3, 300 MHz): ꢀ 2.12 (s, 3H, Me), 2.33 (s, 3H,
Me-amide), 3.04 (dd, J=18.2 and 3.0 Hz, 1H, pyrazole, 4-
Ha), 3.31 (dd, J=18.2 and 11.1 Hz, pyrazole, 1H, 4-Hb), 5.05
A series of novel 6-(1-acetyl-3-methyl-4,5-dihydro-1H-
pyrazol-5-yl)-2-amino-4-substituted-phenyl-4H-chromene-3-
carbonitrile derivatives 7 were synthesized. The compounds

490 Letters in Organic Chemistry, 2010, Vol. 7, No. 6
Liu et al.
biased needle screening, hit validation by biophysical methods, and
were evaluated and assayed for their antibacterial (B. subtilis
ATCC 6633, E. coli ATCC 35218, P. fluorescens ATCC
13525 and S. aureus ATCC 6538) activity by MTT method.
The results showed that compounds 7e and 7f possess potent
antibacterial activity.
3D guided optimization:
a promising alternative to random
screening. J. Med. Chem., 2000, 43, 2664.
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ACKNOWLEDGEMENTS
The authors wish to thank the National Natural Science
Foundation of China (No.20902003); the Key Research
Projects of University Natural Science, Anhui Province
(No.KJ2009A011Z); the Young College Teachers Research
Projects of Anhui Province (No. 2008JQ1030); the Young
College Teachers Research Projects of Anhui University of
Technology (No. QZ200809, QZ200919), SRTP of Anhui
university of technology (No.09018).
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