Synthesis of pyrazole encompassing 2-pyridone derivatives as antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2013.02.077

A series of novel compounds 6-amino-1-((1,3-diphenyl-1H-pyrazole-4-yl) methyleneamino)-4-(aryl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitriles (4a-t) were synthesized and characterized by IR, ¹H NMR, ¹³C NMR and mass spectral data. Th

Full text of DOI:10.1016/j.bmcl.2013.02.077

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2714–2717
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of pyrazole encompassing 2-pyridone derivatives
as antibacterial agents
⇑
Nisheeth C. Desai , Kiran M. Rajpara, Vivek V. Joshi
Division of Medicinal Chemistry, Department of Chemistry, (DST-FIST Sponsored Department) Mahatma Gandhi Campus, Maharaja Krishnakumarsinhji Bhavnagar University,
Bhavnagar 364 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel compounds 6-amino-1-((1,3-diphenyl-1H-pyrazole-4-yl)methyleneamino)-4-(aryl)-2-
oxo-1,2-dihydropyridine-3,5-dicarbonitriles (4a–t) were synthesized and characterized by IR, 1H NMR,
13C NMR and mass spectral data. These compounds were screened for their in vitro antibacterial activity
against Staphylococcus aureus, Streptococcus pyogenes (Gram positive), Escherichia coli, Pseudomonas aeru-
ginosa (Gram negative) by serial broth dilution and cytotoxic activity (NIH 3T3 & HeLa) by MTT assay. The
results indicated that compounds 4g, 4i, 4m, 4o, 4r and 4t exhibit potent antibacterial activity against
bacterial strains at non-cytotoxic concentrations.
Received 16 January 2013
Revised 6 February 2013
Accepted 15 February 2013
Available online 26 February 2013
Keywords:
Pyrazole
2-Pyridone
Ó 2013 Elsevier Ltd. All rights reserved.
Antibacterial activity
Cytotoxicity
Since resistance of pathogenic bacteria towards available anti-
biotics is rapidly becoming a major worldwide problem, the design
of new compounds to deal with resistant bacteria has become one
of the most important areas of antibacterial research today.1 De-
spite the development of several new antibacterial agents, their
clinical value is limited to treating an increasing array of life-
threatening systemic infections because of their relatively high risk
of toxicity, emergence of drug resistant strains, pharmacokinetic
differences, and/or insufficiencies in their activity.
Pyridone and their derivatives play an essential role in several
biological processes and have considerable chemical and pharma-
cological importance.2–4 2-Pyridones represent a unique class of
pharmacophores, which are observed in various therapeutic
agents5 and antibiotics.6 In recent years, 2-pyridones have capti-
vated much importance as these compounds have been found to
exhibit several biological activities, such as HIV-1 non-nucleoside
reverse transcriptase inhibitors (NNRTIs),7 antifungals,8 sedatives9
and cardiotonic agents.10–12 Moreover, 2-pyridones are a class of
recently discovered potent antibacterial agents that are of particu-
lar interest due to their in vitro and in vivo antibacterial potencies
against the bacterial type II DNA topoisomerases, which includes
two highly homologous enzymes DNA gyrase and topoisomerase
IV.13 Furthermore, pyrazoles occupy a distinct place in heterocyclic
chemistry and represent a key motif in medicinal chemistry due to
their capability to exhibit an array of bioactivities such as antimi-
crobial,14–16 anticancer,17 anti-inflammatory,18 antidepressant,19
anticonvulsant,20 antipyretic21 and selective enzyme inhibitory
activities.22 Recently, we have reported pyrazole containing com-
pounds as antibacterial agents (NCD1–17) (Fig. 1).23 Recognizing
these facts and in continuation of our previous work24,25 to look
for new antibacterial agents with possible novel mechanisms of ac-
tion, it was thought worthwhile to synthesize some new congeners
of 2-pyridones by incorporating 2-pyridones and pyrazoles in a
single molecular framework.
The quest for synthesis of 6-amino-1-((1,3-diphenyl-1H-pyra-
zole-4-yl)methyleneamino)-4-(aryl)-2-oxo-1,2-dihydropyridine-
3,5-dicarbonitriles (4a–t) was accomplished in two steps as out-
lined in Scheme 1. In the first step, 1,3-diphenylpyrazole-4-carbox-
aldehyde (1), prepared according to previously published
methods26 was condensed with cyanoacetic acid hydrazide using
1,4-dioxane as a solvent to yield the intermediate 2-cyano-N-
((1,3-diphenyl-1H-pyrazol-4-yl)methylene)acetohydrazide (2).27
In the second step, the intermediate 2 was further reacted with
(2-arylidene)malononitriles (3a–t) in the presence of catalytic
amount of piperidine utilizing ethanol (95%) as a solvent to yield
the desired products 6-amino-1-((1,3-diphenyl-1H-pyrazole-4-
yl)methyleneamino)-4-(aryl)-2-oxo-1,2-dihydropyridine-3,5-
dicarbonitriles (4a–t).28 The yields of the products were obtained
in the range of 54–65%. Designed series of molecules 4a–t were
characterized by IR, 1H NMR, 13C NMR, and mass spectrometry
techniques before evaluating for in vitro antibacterial and cytotox-
icity essays.
IR spectrum of compound 4o showed stretching band of car-
bonyl group at 1684 cmÀ1 and absorption bands of NH2 group at
3439 and 3448 cmÀ1
. Strong intense absorption bands were
observed at 2210 and 2214 cmÀ1 due to stretching vibration of
⇑
–CBN groups. In 1H NMR spectra of 4o, characteristic signal of
Corresponding author. Tel./fax: +91 278 2439852.
E-mail address: dnisheeth@rediffmail.com (N.C. Desai).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.077

N. C. Desai et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2714–2717
2715
NO2) displayed strong inhibition action at MIC = 12.5
lg/mL
O
S
F
against E. coli. Compounds 4i (4-F) and 4o (4-NO2) showed appre-
ciable activity at MIC = 25 g/mL against P. aeruginosa. Compounds
4g (2-F), 4m (2-NO2) and 4t (4-CF3) exhibited significant potential
at MIC = 25 g/mL while compounds 4i (4-F), 4p (2-Br) and 4r
(4-Br) exerted highest inhibition at MIC = 12.5 g/mL against S.
aureus. Compound 4g (2-F) indicated good inhibition at MIC =
25 g/mL and compounds 4f (4-Cl) and 4o (4-NO2) showed
remarkable activity at MIC = 12.5 g/mL against S. pyogenes.
N
N
N
N
N
l
O
N
N
N
R
N
l
N
H2N
l
R
N
l
Present work
l
NCD1-17
As shown in our results, some analogues of this series were
found to have more potency than the standard drug ciprofloxacin
while some of them have comparable potency. The antimicrobial
activity was considerably affected by substitution pattern on the
phenyl ring and the most active compounds contained an electron
withdrawing substituent at ortho and para positions of the phenyl
ring. The role of electron withdrawing group in improving antimi-
crobial activity is very well supported by previous studies.30,31
Compounds 4g, 4i, 4m, 4o, 4r and 4t bearing F, Cl, NO2, Br, and
CF3 groups were most potent. Thus, compounds bearing substitu-
ents at 2- or 4-position of the terminal benzene ring of 2-pyridone
part were found to have higher potency than compounds bearing
groups at 3-position or at both. Moreover all active compounds
were effective against Gram negative bacterial strain S. aureus. Rest
of the compounds bearing these substituents at 3-position showed
moderate activity with respect to standard drug against the test
strains.
R = Differentsubstituents
Figure 1. Structural similarity between newly synthesized compounds and previ-
ously synthesized compounds.
primary amine showed a broad singlet at d = 5.93 ppm which is
exchangable with D2O. The aromatic and CH@N protons appeared
at d = 7.29–8.50 ppm. The 13C NMR spectrum of compound 4o dis-
played characteristic signal of carbonyl carbon at d = 171.2 ppm.
Carbons of –CBN group showed
a
chemical shift at
d = 115.6 ppm. Carbon of nitrophenyl ring showed a chemical shift
at d = 148.2 ppm. The mass spectrum revealed a molecular ion
peak at m/z = 526. In mass spectra, molecular ion peak is in agree-
ment with proposed molecular weight and elemental analysis.
A plausible mechanism for the formation of compounds (4a–t)
is suggested in Scheme 2. Firstly, the Michael addition of Schiff
base cyanoacetic hydrazide a to Knovenagel product b yielded
the intermediate c, which further underwent intramolecular nucle-
ophilic attack on cyano carbon followed by annulation to give
intermediate e. The compound e was converted to final compound
by intramolecular electron transfer to nitrogen atom.
All the newly synthesized compounds 4a–t were tested for
cytotoxic activity on Mouse embryonic fibroblasts cell line (NIH
3T3) and human cervical cancer cell line (HeLa) using MTT colori-
metric assay.32 The IC50 values obtained for these compounds are
shown in Table 2. Pyrazole analogues 4g, 4i, 4m, 4r and 4t showed
All the newly synthesized compounds (4a–t) were initially
screened for their in vitro antibacterial activity against Gram posi-
tive bacteria (Staphylococcus aureus (MTCC-96), Streptococcus pyog-
enes (MTCC-442)) and Gram negative bacteria (Escherichia coli
(MTCC-443), Pseudomonas aeruginosa (MTCC-1688)) using conven-
tional broth-dilution method.29 The MIC (minimum inhibitory con-
centration) values were determined by using ciprofloxacin as
reference drug. The results of antibacterial studies of newly syn-
thesized compounds revealed that these compounds exhibited sig-
nificant antibacterial activity. From antibacterial activity data
(Table 1), it was observed that compounds 4f (2-Cl), 4g (2-F), 4i
(4-F), 4m (2-NO2), 4o (4-NO2), 4p (2-Br), 4r (4-Br) and 4t (4-CF3)
were most active compounds. Compound 4m (2-NO2) exhibited
no toxicity at concentration of 200 lg/mL (IC50 >200 lg/mL), while
other compounds showed moderate toxicity against NIH 3T3 cells.
Moreover, none of the derivatives showed cytotoxicity against
HeLa cells (IC50 >200 lg/mL).
In conclusion, our attempts at exploring pyrazole based 2-pyri-
done derivatives have unexpectedly led to the identification of a
novel chemotype with substantial antibacterial activity against
various bacterial strains. Among the newly synthesized com-
pounds 4a–t, analogues 4g (2-F), 4i (4-F), 4m (2-NO2), 4o (4-
NO2), 4r (4-Br) and 4t (4-CF3) showed the highest inhibition
against nearly all of the tested bacteria, superior to the reference
drugs, and displayed antibacterial activity at non-cytotoxic con-
centrations. Our preliminary results suggest that the mechanism
of action may be distinct from that of known antibacterial agents.
inhibition at MIC = 25
lg/mL and compounds 4i (4-F) and 4o (4-
H
N
O
O
CNCH2CONHNH2
N
N
N
N
R
N
+
1,4-Dioxane
N
N
H
Reflux, 3h
N
(3a-t)
(1)
(2)
Yield 73%
Ethanol
Piperidine
Reflux, 3h
O
N
N
N
N
N
H2N
R
R = Different substituents
Scheme 1. Synthesis of title compounds 4a–t.
(4a-t)
N

2716
N. C. Desai et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2714–2717
N
N
N
R
O
+
H
N
R
(3a-t)
N
R
R
N
N
H
H
H
N
N
..
N
R1
N
R1
N
+
R1
N
O
N
N
O
O
C
a
N
d
N
c
N
N
R
..
b
N
H
N
O
R
O
N
R
R1
N
N
R1
N
NH
N
NH2
(4a-t)
N
e
R1
= 1,3-Diphenyl-1H-pyrazol-4-yl
R = Different substituents
Scheme 2. Plausible mechanistic pathway for the synthesis of compounds 4a–t.
Table 1
Results of antibacterial screening of compounds 4a–t
Entry
–R
Minimum inhibitory concentration (MIC) in lg/mL SD
E. coli
P. aeruginosa
S. aureus
S. pyogenes
MTCC 443
MTCC 1688
MTCC 96
MTCC 442
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
41
4m
4n
4o
4p
–H
250 3.05
500 2.64c
500 3.21
100 1.60b
50 3.61c
500 3.60
250 1.20b
100 1.78c
12.5 3.78c
500 2.44c
500 4.40
500 5.19c
25 1.60a
100 1.52c
12.5 3.60
100 2.52
100 1.24a
500 2.14c
100 1.38
250 3.46c
25 2.05
500 1.00
500 1.00b
500 2.88
100 2.04b
250 4.04
500 1.60b
50 3.60c
50 2.21b
25 3.78
500 4.04c
500 3.21
500 2.05b
100 3.05
500 1.16c
100 1.05a
25 2.04c
50 4.16
500 3.46c
250 3.60b
500 2.05
100 3.21c
100 3.21
12.5 4.16°
25 1.92c
50 3.78b
100 3.46
250 1.78
250 4.61c
250 3.78c
250 2.64b
50 2.05a
12.5 3.60c
250 2.84c
500 2.48a
100 3.64c
250 1.52
250 2.10c
50 2.06
–2-OH
–4-OH
–2-C1
–3-CI
–4-C1
–2-F
–3-F
–4-F
12.5 2.04b
500 4.16
500 3.78b
500 4.60c
25 2.64
–4-CH3
–4-OCH3
–3,4,5-(OCH3)3
–2-NO2
–3-NO2
–4-NO2
–2-Br
–3-Br
–4-Br
–2-CF3
–4-CF3
250 1.21c
500 3.26
250 1.04c
100 3.78c
100 2.04b
25 1.16c
250 2.98a
250 2.16c
100 1.28
250 1.84c
25 1.24c
25 1.0
250 4.04c
50 4.50a
12.5 1.86b
250 2.48c
12.5 2.28
100 1.34a
25 1.68c
50 1.52
4q
4r
4s
4t
Ciprofloxacin
SD = standard deviation.
a
P <0.001 extremely significant.
P <0.01 moderately significant.
b
c
P <0.05 significant. All values are presented as mean of 6 experiments (n = 6). All significant differences are considered from control value 0.00.
Structure–activity relationship studies have led to identification of
positions of pharmacophores on the scaffold for additional
structural modifications. These modifications would allow the
introduction of probes designed to examine cognate binding
partners and molecular targets, at the same time not significantly
compromising antibacterial potency.
Acknowledgment
We would like to express our sincere gratitude to the Depart-
ment of Chemistry, (DST-FIST Sponsored), Mahatma Gandhi
Campus, Maharaja Krishnakumarsinhji Bhavnagar University,
Bhavnagar for providing research and library facilities.

N. C. Desai et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2714–2717
2717
Table 2
7. Goldman, M. E.; Nunberg, J. H.; O’Brien, J. A.; Quintero, J. C.; Schleif, W. A.;
Freund, K. F.; Gaul, S. L.; Saari, W. S.; Wai, J. S.; Hoffman, J. M., et al Proc. Natl.
Acad. Sci. U.S.A. 1991, 88, 6863.
Cytotoxic activity of compounds 4a–t against NIH 3T3 and HeLa cell line
Entry
Cytotoxicity (IC50)(l
g/mL)a
8. Gupta, A. K.; Plott, T. Int. J. Dermatol. 2004, 43, 3.
9. Collins, I.; Moyes, C.; Davey, W. B.; Rowley, M.; Bromidge, F. A.; Quirk, K.; Atack,
J. R.; McKernan, R. M.; Thompson, S. A.; Wafford, K.; Dawson, G. R.; Pike, A.;
Sohal, B.; Tsou, N. N.; Ball, R. G.; Castro, J. L. J. Med. Chem. 1887, 2002, 45.
10. Endoh, M.; Hori, M. Expert Opin. Pharmacother. 2006, 7, 2179.
11. Robert, N.; Verrier, C.; Hoarau, C.; Celanire, S.; Marsais, F. ARKIVOC 2008, 7, 92.
12. Lo Presti, E.; Boggia, R.; Feltrin, A.; Menozzi, G.; Dorigo, P.; Mosti, L. Farmaco
1999, 54, 465.
13. Li, Q.; Mitscher, L. A.; Shen, L. L. Med. Res. Rev. 2000, 20, 231.
14. Sullivan, T. J.; Truglio, J. J.; Boyne, M. E.; Novichenok, P.; Zhang, X.; Stratton, C.
F.; Li, H.-J.; Kaur, T.; Amin, A.; Johnson, F.; Slayden, R. A.; Kisker, C.; Tonge, P. J.
ACS Chem. Biol. 2006, 1, 43.
15. Gilbert, A. M.; Failli, A.; Shumsky, J.; Yang, Y.; Severin, A.; Singh, G.; Hu, W.;
Keeney, D.; Petersen, P. J.; Katz, A. H. J. Med. Chem. 2006, 49, 6027.
16. Isloor, A. M.; Kalluraya, B.; Shetty, P. Eur. J. Med. Chem. 2009, 44, 3784.
17. Magedov, I. V.; Manpadi, M.; Slambrouck, S. V.; Steelant, W. F.; Rozhkova, E.;
Przheval’skii, N. M.; Rogelj, S.; Kornienko, A. J. Med. Chem. 2007, 50, 5183.
18. Szabó, G.; Fischer, J.; Kis-Varga, Á.; Gyires, K. J. Med. Chem. 2007, 51, 142.
19. Rajendra Prasad, Y.; Lakshmana Rao, A.; Prasoona, L.; Murali, K.; Ravi Kumar, P.
Bioorg. Med. Chem. Lett. 2005, 15, 5030.
NIH 3T3b
HeLac
4a
4b
4c
4d
4e
4f
4g
4h
4i
180 2.4
162 4.8
110 4.4
84 2.4
52 1.2
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
64
3
>200
154 3.2
>200
4j
4k
4l
98 2.6
50 1.4
64 2.4
>200
124 1.8
84 3.6
174 2.8
42 3.2
>200
4m
4n
4o
4p
4q
4r
4s
4t
20. Ozdemir, Z.; Kandilci, H. B.; Gumusel, B.; Calis, U.; Bilgin, A. A. Eur. J. Med. Chem.
2007, 42, 373.
21. Sßener, A.; Kasim Sßener, M.; Bildmci, I.; Kasimogullari, R.; Akçamur, Y. J.
56 2.6
>200
Heterocycl. Chem. 2002, 39, 869.
22. Wachter, G. A.; Hartmann, R. W.; Sergejew, T.; Grun, G. L.; Ledergerber, D. J.
Med. Chem. 1996, 39, 834.
23. Desai, N. C.; Joshi, V. V.; Rajpara, K. M.; Vaghani, H. V.; Satodiya, H. M. J. Fluorine
Chem. 2012, 142, 67.
24. Desai, N. C.; Rajpara, K. M.; Joshi, V. V.; Vaghani, H. V.; Satodiya, H. M. Anti
Infect. Agents 2012, 10, 75.
The values represent mean SD.
a
IC50 is the concentration required to inhibit 50% of cell growth.
NIH 3T3: Mouse embryonic fibroblasts cell line.
HeLa: Human cervical cancer cell line.
b
c
25. Desai, N. C.; Rajpara, K. M.; Joshi, V. V.; Vaghani, H. V.; Satodiya, H. M. Med.
Chem. Res. 2013, 22, 1172.
26. Prakash, O.; Pannu, K.; Naithani, R.; Kaur, H. Synth. Commun. 2006, 36, 3479.
Supplementary data
27. Synthesis
of
2-cyano-N-((1,3-diphenyl-1H-pyrazol-4-
yl)methylene)acetohydrazide (2): A mixture containing 1,3-diphenylpyrazole-
4-carbaldehyde (1) (0.01 mol) and 2-cyanoacetohydrazide (0.01 mol) in 1,4-
dioxane (30 mL) was refluxed for 1 h. After cooling, the crystals formed were
filtered, washed with water and recrystallized from ethanol (95%) to give
compound (2).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.02.077.
28. General procedure for the synthesis of 6-amino-1-((1,3-diphenyl-1H-pyrazole-4-
yl)methyleneamino)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3,5-
References and notes
dicarbonitriles (4a–t):
pyrazol-4-yl)methylene)acetohydrazide
(arylidene)malononitriles (3a–t) (0.01 mol) and catalytic amount of
piperidine in ethanol (95%) (30 mL) was refluxed for 3 h. After cooling, the
crystals formed were filtered, washed with water and recrystallized from
aqueous DMF to give compounds 4a–t.
A
mixture containing 2-cyano-N-((1,3-diphenyl-1H-
(2) (0.01 mol), 2-
1. Grare, M.; Mourer, M.; Fontanay, S.; Regnouf-de-Vains, J. B.; Finance, C.; Duval,
R. E. J. Antimicrob. Chemother. 2007, 60, 575.
2. Choi, W.-B.; Houpis, I. N.; Churchill, H. R. O.; Molina, A.; Lynch, J. E.; Volante, R.
P.; Reider, P. J.; King, A. O. Tetrahedron Lett. 1995, 36, 4571.
3. Bhupathy, M.; Conlon, D. A.; Wells, K. M.; Nelson, J. R.; Reider, P. J.; Rossen, K.;
Sager, J. W.; Volante, R. P.; Dorsey, B. D.; Hoffman, J. M.; Joseph, S. A.; McDaniel,
S. L. J. Heterocycl. Chem. 1995, 32, 1283.
4. Kappe, C. O.; Kappe, T. Monatsh. Chem. 1989, 120, 1095.
5. Saiki, A. Y.; Shen, L. L.; Chen, C. M.; Baranowski, J.; Lerner, C. G. Antimicrob.
Agents Chemother. 1999, 43, 1574.
29. Hannan, P. C. Vet. Res. 2000, 31, 373.
30. Sharma, P.; Rane, N.; Gurram, V. K. Bioorg. Med. Chem. Lett. 2004, 14, 4185.
31. Sharma, D.; Narasimhan, B.; Kumar, P.; Judge, V.; Narang, R.; De Clercq, E.;
Balzarini, J. Eur. J. Med. Chem. 2009, 44, 2347.
32. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
6. Brickner, S. Chem. Ind. (London) 1997, 131.