Synthesis and biological evaluation of novel formyl-pyrazoles bearing coumarin moiety as potent antimicrobial and antioxidant agents

Article abstract of DOI:10.1016/j.bmcl.2013.09.053

A series of coumarin appended formyl-pyrazoles 14-18 were synthesized by a simple and accessible approach. The reaction of 8-acetyl-4-methyl-7-hydroxy coumarin 3 and phenyl hydrazine hydrochlorides 4-8 produces the intermediate compounds 8-acetyl-4-methyl

Full text of DOI:10.1016/j.bmcl.2013.09.053

Accepted Manuscript
Synthesis and biological evaluation of novel formyl-pyrazoles bearing coumarin
moiety as potent antimicrobial and antioxidant agents
Renuka Nagamallu, Ajay Kumar Kariyappa
PII:
S0960-894X(13)01127-X
http://dx.doi.org/10.1016/j.bmcl.2013.09.053
BMCL 20905
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
6 July 2013
14 September 2013
18 September 2013
Please cite this article as: Nagamallu, R., Kariyappa, A.K., Synthesis and biological evaluation of novel formyl-
pyrazoles bearing coumarin moiety as potent antimicrobial and antioxidant agents, Bioorganic & Medicinal
Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.09.053
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Synthesis and biological evaluation of novel formyl-pyrazoles bearing
coumarin moiety as potent antimicrobial and antioxidant agents
Renuka Nagamallu and Ajay Kumar Kariyappa*
Post Graduate Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore
570005, India.
*Corresponding author: email: ajaykkchem@gmail.com
Abstract
A series of coumarin appended formyl-pyrazoles 14-18 were synthesized by a simple and
accessible approach. The reaction of 8-acetyl-4-methyl-7-hydroxy coumarin 3 and phenyl
hydrazine hydrochlorides 4-8 produces the intermediate compounds 8-acetyl-4-methyl-7-
hydroxy coumarin hydrazones 9-13. The reaction of compounds 9-13 and DMF in the presence
of POCl3 yielded formyl-pyrazoles bearing coumarin moiety 14-18 in good yield. The
synthesized new compounds 14-18 and the intermediates 8-acetyl-4-methyl-7-hydroxy coumarin
hydrazones 9-13 prepared were screened in vitro for their antibacterial, antifungal antioxidant
activities. The compounds 12 and 17 having chloro substitution exhibited promising antifungal
and antibacterial activity against the different organisms tested. The compound 17 showed
remarkable DPPH radical scavenging ability.
Key words: Antibacterial, antifungal, antioxidant, heterocycles, MIC.
Coumarins are chemically known as 2H-1-benzopyran-2-ones and were first identified in
1820’s as an oxygen heterocycle. Alternariol is chemically a 3,7,9-trihydroxy-1-methyl-6H-
benzo[c]chromen-6-one, a toxic metabolite of Alternaria fungi and is an important contaminant
in cereals and fruits exhibiting antifungal and phytotoxic activity.1 Coumarins are widely
distributed in plants, for example, umbelliferone (7-hydroxy coumarin) was found in Apiaceae,
osthole (7-methoxy-8-(3-methylbut-2-en-1-yl)coumarin) was found in Cnidium monnieri and

scoparone (6,7-dimethoxy coumarin) was found in Artemisia scoparia.2 In the recent years
coumarins have attracted great attention because of their synthetic utility as building blocks for
the synthesis of biologically potent molecules. Coumarin derivatives exhibit enormous amount of
biological activities such as antioxidant, antimicrobial, anti-HIV, antibiotic, anticancer, muscle
relaxant, anti-inflammatory and anti-coagulant activity.3 Further they are widely used as
perfumes, additives in food, chemical, laser dyes, optical brightening agents and cosmetics.
The discovery of antipyretic action of a pyrazole derivative in man by Knorr in 1884
created interest in the researchers. Pyrazoles represent a key motif in heterocyclic chemistry and
occupy a prime place in medicinal chemistry due to their competence to exhibit a wide range of
pharmacological activities such as antimicrobial,4 anticancer,5 anti-inflammatory,6
anticonvulsant,7 antipyretic activities.8 Pyrazoles having a functional group like aldehyde or
carboxylate C-4 position have shown promising antimicrobial properties.9 Pyrazole incorporated
with coumarin was synthesized and observed in the enhancement of pharmacological effect.10
When one biodynamic heterocyclic system was coupled with another heterocyclic system,
enhanced biological activity was observed.
With this in view, this project was undertaken to synthesize a series of new pyrazoles
bearing coumarin nucleus. We herein report the synthesis of series of novel heterocyclic
scaffolds formyl-pyrazoles bearing coumarin nucleus and in vitro screening of the synthesized
compounds for their antibacterial, antifungal and antioxidant activities.
In this work we intended to introduce the pyrazole fragment to the coumarin skeleton in
order to build a novel family of bioactive molecules. Thus a series of pyrazole derivatives 14-18
were synthesized starting from 4-methyl-7-hydroxy coumarin 1. Pechmann reaction has been
widely used for the synthesis of coumarins because of its preparative simplicity and inexpensive

starting material.11 4-Methyl-7-hydroxy coumarin 1 was prepared by the resorcinol with ethyl
acetoacetate in the presence of sulfuric acid as a catalyst.12
4-Methyl-7-hydroxy coumarin 1 was converted to an ester 2 with acetic anhydride by a
standard procedure; then the ester was subjected to Fries rearrangement at 140-160 ºC to get 8-
acetyl-4-methyl-7-hydroxy coumarin 3, the product characteristics are in good agreement with
the reported results.13 The condensation of the compound 3 with substituted phenylhydrazines 4-
8 in ethyl alcohol and a catalytic amount of acetic acid at water bath reflux conditions, produced
the corresponding hydrazones 9-13.14
The hydrazones 9-13 (0.0032 mol) were added to the Vilsmeier-Haack reagent prepared
by drop-wise addition of POCl3 (1.2 ml) in ice cooled DMF (10 ml). The mixture was stirred at
o
60-65 C for 6 hours. The progress of the reaction was monitored by TLC. After completion of
the reaction, the mixture was poured into ice cold water, neutralized with NaHCO3, the solid
separated was filtered, washed with water and recrystallized from ethanol to obtain target
molecules 1-aryl-3-(7-hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)-1H-pyrazole-4-carbaldehydes
14-18 in good yield.15 The reaction pathway is illustrated in Scheme 1.

3
3
3
3
3
2
2.
1
3
3
1
1
1
1
1
2
2
3
1
2
2
3
3
2
2
2
1
2
2
2
3
o
o
3
2
5
3
Designed series of molecules 14-18 were characterized by spectral and CHN analysis15
before being evaluated for in vitro antimicrobial and antioxidant activities. For instance, all new
compounds showed a sharp and strong absorption band in the region 1660-1671 cm-1 in the IR
spectrum, which is due to C=O str. of newly formed -CHO functional group. 1H NMR spectrum
of the compounds showed singlet in the region δ 10.70-10.82 ppm. These results supported the
presence of -CHO functional group in the products. Furthermore, all compounds showed signals
due to aromatic, substituent protons in the expected region. MS results and elemental analysis
data confirmed the formation of these compounds.
Minimum inhibitory concentrations (MICs) of the synthesized compounds 9-18 against
different bacterial and fungal strains were determined by broth dilution technique. Gram-
negative bacteria species Escherichia coli, Pseudomonas aeruginosa, Gram-positive bacteria
species Staphylococcus aureus, Streptococcus pyogenes were used as bacterial strains and

Cryptococcus neoformans, Aspergillus nigar, Aspergillus flavus, Candila albicans were used as
fungal strains. The antibiotics Ciprofloxacin and Amphotericin B were used as standard drugs
against bacteria and fungi species respectively. The experiments were carried out in triplicate;
the results were taken as a mean of three determinations. The results of MIC’s of the synthesized
compounds against bacteria species are summarized in Table 1; and against fungal species in
Table 2.
Table 1: MIC’s of the test compounds (9-18) against bacteria species
Compound
Minimum inhibitory concentration (MIC’s) in µg/mL
Staphylococcus Streptococcus Escherichia Pseudomonas
aureus
pyogenes
coli
aeruginosa
09
40
-
-
25
10
30
40
20
50
-
60
50
50
75
-
50
75
50
-
-
100
25
-
11
12
13
14
30
75
40
30
40
25
50
25
50
15
20
12.5
15
30
30
15
40
25
50
50
35
75
50
16
17
18
Ciprofloxacin
All the synthesized compounds hydrazones and formyl-pyrazoles exerted a wide range of
modest in vitro antibacterial activity against all the tested organisms. However, the compound 9
failed to inhibit the growth of S. pyogenes and E. coli even at a higher concentration of 200
µg/mL. Similarly, compound 10 failed to inhibit P. aeruginosa; 13 failed to inhibit E. coli and P.
aeruginosa; and 14 failed to inhibit S. aureus and S. pyogenes organisms.

The presence of chloro substitution in compounds 12, 17 influenced these molecules to
exhibit inhibition to greater extent against the organisms tested. Nitro substitution present in
compounds 13, 18 retarded the inhibitory effect of these compounds against the organism tested,
which may be due to electron withdrawing nature of this functional group. The presence of
electron donating groups at ortho position or no substitution resulted with moderate activity.
Table 2: MIC’s of the test compounds 9-18 against fungi species
Compound
Minimum inhibitory concentration (MIC) in µg/mL
Cryptococcus Aspergillus Aspergillus Candila
neoformans
niger
flavus
albicans
09
50
75
75
50
10
40
40
25
-
50
75
60
-
40
-
30
-
11
12
50
-
30
-
13
14
50
25
30
15
-
100
50
60
40
-
-
-
15
25
-
50
-
16
17
18
60
-
25
-
Amphotericin B
25
50
50
25
All the synthesized compounds 9-18 exerted a moderate to good in vitro antibacterial
activity against all the tested organisms, except 13, 18 which failed to exhibit inhibition against
all the organisms. However, the compounds 11, 14, 16 failed to inhibit the growth of A. flavus
and C. albicans even at a higher concentration of 200 µg/mL. The compounds 12, 17 having
chloro substitution exhibited remarkable activity against all the organisms tested, while the
remaining compounds exhibited moderate activity.

The antioxidant activity of synthesized compounds 14-18 was carried out by DPPH
radical scavenging assay using butylated hydroxyl toluene (BHT) as standard antioxidant.16 The
experiments were carried out at five different concentrations in triplicates; the results are
expressed as mean standard deviation (SD) and were summarised in Table 3.
Table 3: DPPH Radical Scavenging ability of the compounds 14-18 relative to the
standard antioxidant BHT
Test
% Radical Scavenging activity*
Samples
10 (µg/mL)
39.36 0.81
11.10 0.89
12.80 0.81
48.86 0.98
34.67 0.71
20 (µg/mL)
30 (µg/mL)
46.42 0.85
13.33 0.92
14.11 0.68
58.15 0.78
47.12 1.00
40 (µg/mL)
50 (µg/mL)
57.76 0.94
17.36 0.98
17.10 0.74
69.35 0.98
56.02 1.00
14
15
16
17
18
42.12 0.78
11.98 0.89
13.81 1.01
53.33 0.78
41.93 1.00
50.12 0.88
15.63 0.86
15.12 0.83
63.89 0.98
51.07 0.82
Control at a 0 µg/mL concentration 0.00 0.00
*Results are expressed as mean standard deviation (n=3)
The compounds 14-18 showed promising DPPH free radical scavenging ability, but of
lesser activity compared with the standard antioxidant. The results reveal that, all exhibited poor
radical scavenging ability at lower concentrations. However, the gradual increase in the activity
in all the cases was observed with increase in the concentrations of the test compounds. Among
the compounds tested, the compound 17 having chloro substituent in the aromatic ring exhibited
promising radical scavenging ability. The compounds 14 and 18 having no and two nitro
substituents in the aromatic ring showed moderate radical scavenging abilities; while 15 and 16
having methoxy and methyl substituent respectively in the aromatic ring showed poor radical
scavenging abilities in comparison with the standard antioxidant BHT. The presence of electron

donating methoxy and methyl groups; stereo chemical factors due to ortho substitution might be
the cause for lesser activity associated with the compounds 15 and 16.
The reactions described represent a simple access to the synthesis of coumarin based
pyrazoles derivatives of potential interest from the readily available materials. The antibacterial,
antifungal and antioxidant properties of the synthesized compounds show promising activity.
However, the structure-activity relationship remains of interest. From the results of biological
activity, it is concluded that, these molecules can be designed as potential drugs with a slight
modification in the structure of the molecules.
Acknowledgement: The authors are grateful to the University Grants Commission, New Delhi
for financial assistance, Dr. B.N. Mylarappa, Rangos Research Center, University of Pittsburgh,
PA 15201, USA, for help in biological activity studies.
References
1. (a) Davis, V. M.; Stack, M. E. Appl. Environ. Microbiol. 1994, 60, 3901. PMC 201908. ;
(b) Brugger, E.M.; Wagner, J.; Schumacher, D.M.; Koch, K.; Podlech, J.; Metzler, M.;
Lehmann,L. Toxicol. Lett. 2006, 164, 221. doi:10.1016/j.toxlet.2006.01.001.
2. (a) Raistrick, H.; Stilkings, C. E.; Thomas, R. Biochemistry. 1953, 55, 421; (b) Abaul, J.;
Philogene, E.; Bourgeois, P. J. Nat. Prod. 1994, 57, 846.
3. Murakami, A.; Gao, G.; Omura, M.; Yano, M.; Ito, C.; Furukawa, H.; Takahashi, D.;
Koshimizu, K.; Ohigashi, H. Bioorg. Med. Chem. Lett. 2000, 10, 59.
4. 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.
5. Magedov, I. V.; Manpadi, M.; Slambrouck, S. V.; Steelant, W. F. A.; Rozhkova, E.;
Przheval’skii, N. M.; Rogelj, S.; Kornienko, A. J. Med. Chem. 2007, 50, 5183.

6. (a) Szabo, G.; Fischer, J.; Kis-Varga, A.; Gyires, K. J. Med. Chem. 2008, 51, 142; (b)
Benaamane, N.; Nedjar-Kolli, B.; Bentarzi, Y.; Hammal, L.; Geron-ikaki, A.; Eleftheriou,
P.; Langunin, A. Bioorg. Med. Chem. 2008, 16, 3059.
7. Ozdemir, Z.; Kandilici, H. B.; Gumusel, B.; Calis, U.; Bilgin, A. A. Eur. J. Med. Chem.
2007, 42, 373.
8. Sener, A.; Sener, M. K.; Bildmci, I.; Kasimogullari, R.; Akcamur, Y.; J. Heterocycl. Chem.
2002, 39, 869.
9. Sridhar, R.; Perumal, P. T.; Etti, S.; Shanmugam, G.; Ponnuswamy, M. N.; Prabavathy, V.
R.; Mathivanan, N. Bioorg. Med. Chem. Lett. 2004, 14, 6035–6040.
10. Omaima, M. A.; Kamelia, M. A.; Rasha, Z. B.; Timothy, J. M.; Somaia, A. N.; Shalini, S.
Bioorg. Med. Chem, 2010, 18, 3378.
11. Sethna, S. M.; Phadke, R. Org. React. 1953, 7, 1.
12. Bose, D. S.; Rudradas, A. P.; Babu, M. H. Tetrahedron Lett. 2002, 43, 9195.
13. Valery, F. T. Molecules. 2004, 9, 50.
14. [Crystallized from ethyl alcohol, 9: yield 80%, mp 170-172 °C; 10: yield 78%, mp 98-100
°C; 11: yield 62%, mp 138-140°C; 12: yield 74%; 13: yield 70%, mp 116-118 °C].
15. [3-(7-Hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde
14: Obtained as light yellow solid in 75% yield, m.p. 220-222 ºC. IR (Nujol): 1660, 1745,
3210 cm-1 H1 NMR (DMSO-d6): δ 2.43 (s, 3H), 6.20 (s, 1H), 7.03 (d, 1H), 7.41 (d, 1H),
.
7.68-8.02 (m, 5H), 9.32 (s, 1H), 9.70 (s, 1H), 10.72 (s, 1H). MS (m/z): 347 (M+1), 319, 271,
250, 177 (base peak). Anal. Calcd. for C20H14N2O4: C, 69.36; H, 4.07; N, 8.09%; Found: C,
69.36; H, 4.15; N, 8.15%.

3-(7-Hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)-1-(2-methoxy-phenyl)-1H-pyrazole-4-
carbaldehyde 15: Obtained as orange solid in 78% yield; m.p. 108-110 ºC. IR (Nujol): 1671,
1740, 3228 cm-1 H1 NMR (DMSO-d6): δ 2.49 (s, 3H), 3.46 (s, 3H), 6.26 (s, 1H), 7.09 (d,
.
1H), 7.40 (d, 1H), 7.52-7.83 (m, 4H), 9.36 (s, 1H), 9.70 (s, 1H), 10.75 (s, 1H). MS (m/z):
361 (M+1), 333, 271, 255, 177 (base peak). Anal. Calcd. for C21H16N2O5: C, 67.02; H, 4.28;
N, 7.44%; Found: C, 67.08; H, 4.23; N, 7.35%.
3-(7-Hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)-1-(2-methylphenyl)-1H-pyrazole-4-
carbaldehyde 16: Obtained as light orange solid in 64% yield; m.p. 206-208 ºC. IR (Nujol):
1662, 1760, 3218 cm-1 H1 NMR (DMSO-d6): δ 2.12 (s, 3Η), 2.48 (s, 3H), 6.22 (s, 1H), 7.08
.
(d, 1H), 7.46 (d, 1H), 7.52-7.66 (m, 4H), 9.38 (s, 1H), 9.79 (s, 1H), 10.80 (s, 1H). Anal.
Calcd. for C21H16N2O4: C, 69.99; H, 4.48; N, 7.77%; Found: C, 69.83; H, 4.28; N, 7.43%.
1-(4-Chloro-phenyl)-3-(7-hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)-1H-pyrazole-4-
carbaldehyde 17: Obtained as light orange solid in 60% yield. IR (Nujol): 1668, 1752, 3220
cm-1 H1 NMR (DMSO-d6): δ 2.44 (s, 3H), 6.18 (s, 1H), 7.03 (d, 1H), 7.43 (d, 1H),7.56 (m,
.
4H), 9.35 (s, 1H), 9.71 (s, 1H), 10.77 (s, 1H). MS (m/z): 382 [M+, 37Cl], 380 [M+, 35Cl, 352,
270, 255, 177 (base peak). Anal. Calcd. for C20H13ClN2O4: C, 63.08; H, 3.44; N, 7.36%;
Found: C, 63.03; H, 3.44; N, 7.26%.
1-(2,4-Dinitro-phenyl)-3-(7-hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)-1H-pyrazole-4-
carbaldehyde 18: Obtained as brown solid in 71% yield; m.p. 130-132 ºC. IR (Nujol): 1665,
1748, 3215 cm-1 H1 NMR (DMSO-d6): δ 2.46 (s, 3H), 6.13 (s, 1H), 7.23 (d, 1H), 7.42 (d,
.
1H), 7.82 (d, 1H), 8.34 (d, 1H), 8.98 (s, 1H), 9.33 (s, 1H), 9.76 (s, 1H), 10.81 (s, 1H). Anal.
Calcd. for C20H12N4O8: C, 55.05; H, 2.77; N, 12.84%; Found: C, 55.12; H, 2.68; N,
12.96%.]

16. Ajay Kumar, K.; Lokanatha Rai, K. M.; Vasanth Kumar, G.; Mylarappa, B. N. Int. J. Pharm.
Pharm. Sci. 2012, 4 Suppl 4, 564.

Synthesis and biological evaluation of novel formyl-pyrazoles bearing
coumarin moiety as potent antimicrobial and antioxidant agents
Renuka Nagamallu and Ajay Kumar Kariyappa*
Post Graduate Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore, India.
CH3
CH3
2
3
2.
1
HO
O
O
HO
O
O
3
OHC
H
N
N
R1
o
N
CH3
N
3
R2
R1
R2