Thermal solvent-free synthesis of chromonyl chalcones, pyrazolines and their in vitro antibacterial, antifungal activities

Article abstract of DOI:10.3109/14756366.2011.577035

A facile and ecofriendly synthesis of new chromonyl chalcones 3a-b from 3-formylchromone 1 and active methyl compounds 2a-b is reported under thermal solvent-free heating condition in good yields. The chromonyl chalcones 3a-b were used as intermediates under green condition for the synthesis of new bioactive pyrazoline derivatives 4a-f. The compounds were tested for antimicrobial activity by disk diffusion assay with slight modifications against Gram-positive, Gram-negative strains of bacteria as well as fungal strains. The investigation of antimicrobial screening revealed that compounds 3a-b and 4a-f showed antibacterial and antifungal activities.

Full text of DOI:10.3109/14756366.2011.577035

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(1): 84–91
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.577035
ReseaRch aRtIcle
ermal solvent-free synthesis of chromonyl chalcones,
pyrazolines and their in vitro antibacterial, antifungal activities
Zeba N. Siddiqui1, Shagufta Praveen1, Mohammed Musthafa. T.N1, Anis Ahmad2, and
Asad U. Khan2
1Department of Chemistry, Aligarh Muslim University, Aligarh, India and 2Interdisciplinary Biotechnology Unit, Aligarh
Muslim University, Aligarh, India
abstract
A facile and ecofriendly synthesis of new chromonyl chalcones 3a-b from 3-formylchromone 1 and active methyl
compounds 2a-b is reported under thermal solvent-free heating condition in good yields. The chromonyl chalcones
3a-b were used as intermediates under green condition for the synthesis of new bioactive pyrazoline derivatives
4a-f. The compounds were tested for antimicrobial activity by disk diffusion assay with slight modifications against
Gram-positive, Gram-negative strains of bacteria as well as fungal strains. The investigation of antimicrobial screening
revealed that compounds 3a-b and 4a-f showed antibacterial and antifungal activities.
Keywords: 3-Formylchromone, green synthesis, antimicrobial activity
Introduction
significant impact on huge demolition of host tissues
Human infections particularly those involving micro-
organisms like bacteria and fungi cause serious dam-
ages in tropical and subtropical countries of the world
and these infections are a major threat to public health
despite tremendous growth in human chemotherapeutic
medicine. ese infections can occur in invasive form,
and are an increasing problem due to the increase of
their incidence in hospitals, especially in patients who
are undergoing cancer treatment, transplantation or
are immune suppressed for other reasons1. Methicillin-
resistant Staphylococcus aureus (MRSA) is common
Gram-positive pathogen of nosocomial infections which
and severe diseases6,7. us, antibiotics provide the main
basis for the therapy of microbial (bacterial and fungal)
infections. However, overuse of antibiotics has become
the major factor for the emergence and dissemination of
multi-drug resistant strains of several groups of microor-
ganisms8. Furthermore, the drugs available are either too
expensive or have undesirable side effects or contrain-
dications9. e aim of this work is the synthesis of new
molecules able to inhibit the growth of Gram-positive,
Gram-negative bacteria and fungi.
Chromones comprise a vast array of oxygen con-
taining compounds ubiquitous in plants10. ey form
basic nucleus of flavones and have been recognized as
the essential component of pharmacophores of a large
number of bioactive molecules11. Interest in chromone
containing structures stems from their widespread occur-
renceinmoleculesthatexhibitsignificantantimicrobial12,
antioxidant13, neuroprotective14, HIV-inhibitory15 and
anticancer16 activities. In addition, chromone derivatives
account for outbreaks and are increasing in frequency2,3
.
Escherichia coli is responsible for the world’s most com-
mon and serious infectious diseases like invasive dys-
entery and diarrhea4,5. e different parasitic bacteria
such as S. aureus, S. pyogenes, S. typhimurium, E. coli
have important effect on the human’s mucosal health.
e infection with these microorganisms may have
Address for Correspondence: Zeba N. Siddiqui, Department of Chemistry, Aligarh Muslim University, Aligarh 202 002, India.
E-mail: siddiqui_zeba@yahoo.co.in
(Received 11 December 2010; revised 28 March 2011; accepted 29 March 2011)
84

Antimicrobial chromonyl chalcones and pyrazolines 85
are also active at benzodiazepine receptors, lipoxyge-
nases and cyclooxygenases17. Chalcones and pyrazolines
are another important class of compounds with wide
purified by recrystallization from chloroform-methanol
(1:3 v/v) mixture as shining yellow crystals.
spread biological applications18,19
.
General procedure for the synthesis of
Growing concern about environmental damage leads
to an urgent requirement for the development of efficient
and environmentally benign chemical processes for the
synthesis of new molecules. Solvent-free reactions are
gaining importance in this context as the procedure dem-
onstrates obvious and significant advantages20. However,
most of the reported solvent-free accomplishments focus
on reactions promoted by microwave irradiation, hand
grinding and mechanical milling21–23. In contrast, reports
on direct heating of the reagents under thermal heating
conditionswithoutanysolventarerelativelylessexplored.
To the best of our knowledge, no reports have been so far
made in the synthesis of chromonyl chalcones and deriv-
atives in solvent-free thermal heating condition. us, in
the present study, we wish to report the novel synthesis of
series of chromonyl chalcones and pyrazolines in excel-
lent yields, under thermal solvent-free heating condition
and their evaluation for antimicrobial activities.
chalcones (3a-b) under solvent-free condition
A mixture of 3-formylchromone 1 (1.00 g, 5.7 mmol),
dehydroacetic acid 2a (0.965 g, 5.7 mmol)/ 5-acetyl bar-
bituric acid 2b (0.970 g, 5.7 mmol) and pyridine (0.3 mL)
were mixed well and air dried. e reaction mixture was
then heated at 80 ˚C in a 100 mL beaker. After comple-
tion of the reaction (checked by TLC), the reaction
mixture was slurred in water (25 mL). e yellow solid
obtained was filtered, washed with methanol and dried.
e compounds were purified by recrystallization from
chloroform-methanol (1:3 v/v) mixture as shining yellow
crystals.
(2E)-1-(4-Hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-3-
(4-oxo-4H-1-benzopyran-3-yl)-2-propen-1-one (3a)
Pale yellow crystals; mp 235–237°C. 1H NMR (CDCl3,
300MHz) δ: 2.28 (3H, s, CH3), 5.96 (1H, s, C-5′), 7.36-7.73
(3H, m, Ar-H), 7.87 (1H, d, J=15.9 Hz, Ha), 8.32 (1H, s, C-2),
8.35 (1H, dd, J=7.8 Hz, 1.8 Hz, C-5), 8.77 (1H, d, J=15.9
Hz, Hb). IR (KBr) υ: 3437 (OH), 1721 (C=O), 1659 (C=O).
MS-FAB m/z (%): 324 (M+, 100), 323 (25), 309 (5), 308 (10),
279 (10), 198 (80), 171 (70), 120 (10), 92 (5). Anal. Cald for
C18H12O6: C, 66.67; H, 3.73. Found: C, 66.53; H, 3.69.
Materials and methods
Chemistry
MeltingpointsweretakeninRiechertermover(Austria)
instrument and are uncorrected. e IR spectra were
recorded on Perkin Elmer (Spectro Lab, United Kingdom)
RXI spectrometer in KBr, 1H NMR on Bruker DRX
300 MHz spectrometer and Bruker Avance (Switzerland)
II 400 MHz spectrometer using tetramethyl silane as the
internal standard and DMSO-d6/CDCl3 as solvent. Mass
spectra were obtained on Jeol-SX-102 (FAB) spectrom-
eter. e microanalytical data were collected on Carlo
Erba (Carlo Erba Instruments, Germany) analyzer model
1108. 3-Formylchromone 124, hydrazinobenzothiazole25
and 5-acetylbarbituric acid 2b26 were synthesized by
reported methods. Dehydroacetic acid 2a was purchased
from E. Merck (Merck, Darmstadt, Germany). Other
chemicals were of commercial grade and used without
further purification. e purity of the compounds was
checked by thin layer chromatography (TLC) on glass
plates coated with silica gel G254 (E. Merck) using chlo-
roform-methanol (3:1 v/v) mixture as mobile phase and
visualized by iodine vapours.
(2E)-3-(4-Oxo-4H-1-benzopyran-3-yl)-1-(2,4,6-
pyrimidinetrione-5-yl)-2-propen-1-one (3b)
Light yellow crystals; mp >300°C. H NMR (DMSO-d6,
300 MHz) δ: 7.40 (1H, d, J= 15.2 Hz, Ha), 7.55-7.91 (3H,
m, Ar-H), 8.17 (1H, dd, J= 8.0 Hz, 1.6 Hz, C-5), 8.95 (1H, s,
C-2), 9.09 (1H, d, J= 15.9 Hz, Hb), 11.06 (1H, s, NH), 11.76
(1H, s, NH). IR (KBr) υ: 3078 (NH), 1740 (C=O), 1643
(C=O). MS-FAB m/z (%): 326 (M+, 60), 206 (10), 198 (70),
171 (65), 145 (50), 120 (40). Anal. Cald for C16H10N2O6: C,
58.83; H, 3.06; N, 8.58. Found: C, 58.72; H, 3.14; N, 8.44.
1
General procedure for the synthesis of pyrazolines
(4a-e) and bipyrazole (4f) under conventional method
ecompound3a-b(1.00 g, 3mmol)wasdissolvedinace-
tic acid (8 mL) and hydrazines (hydrazine hydrate (0.15 g,
3 mmol)/hydrazinobenzothiazole (0.49 g, 3 mmol)/phe-
nylhydrazine (0.51 g, 3 mmol) added to it. e reaction
mixture was stirred at room temperature or refluxed in
a heating mantle for specified period of time. After that,
the reaction mixture was cooled and poured into ice-
cold water (20 mL). e solid, which precipitated out,
was filtered, washed with water and dried. Purification of
4a-f was achieved by recrystallization from chloroform-
methanol (1:4 v/v) mixture.
General procedure for the synthesis of
chalcones (3a-b) under conventional method
To a solution of 3-formylchromone 1(1.00g, 5.7mmol)and
dehydroacetic acid 2a (0.965g, 5.7 mmol)/5-acetyl barbi-
turic acid 2b (0.970g, 5.7 mmol) in methanol (12mL), pyri-
dine (0.5mL) was added. e reaction mixture was stirred
at room temperature/refluxed for a specified time. After
completion of the reaction (checked by TLC), the reaction
mixture was concentrated and cooled at room tempera-
ture. e yellow solid, which precipitated out, was filtered,
washed with methanol and dried. e compounds were
General procedure for the synthesis of pyrazolines
(4a-e) and bipyrazole (4f) under solvent-free heating
method
e compound 3a-b (1.00 g, 3 mmol) and hydra-
zines (hydrazine hydrate (0.15 g,
3
mmol)/
© 2012 Informa UK, Ltd.

86 Z.N. Siddiqui et al.
hydrazinobenzothiazole (0.49 g,
3 mmol)/phenylhy-
1-Benzothiazolyl-5-(4-oxo-4H-1-benzopyran-3-yl)-3-
(2,4,6-pyrimidinetrione-5-yl)pyrazoline (4e)
drazine (0.51 g, 3 mmol) were mixed well and heated at
80°C in a 100 mL beaker. After completion of the reaction
(checked by TLC), the reaction mixture was slurred in
water (20 mL). e solid, obtained was filtered, washed
with methanol and dried. e compounds were purified
by recrystallization from chloroform-methanol (1:4 v/v)
mixture to get 4a-f.
Brown crystals; mp >300°C. 1H NMR (DMSO-d6, 400 MHz)
δ: 3.81–3.87 (1H, m, He), 4.10–4.17 (1H, m, Hf), 5.49–5.53
(1H, m, Hd), 7.12–7.76 (7H, m, Ar-H), 8.15 (1H, dd, J= 8.0
Hz, 1.6 Hz, C-5), 8.27 (1H, s, C-2), 11.17 (1H, s, NH), 11.56
(1H, s, NH). IR (KBr) υ: 3069 (NH), 1701(C=O), 1644
(C=O). MS-FAB m/z (%): 473 (M+, 50), 326 (20), 198 (10),
145 (20), 120 (30). Anal. Cald for C23H15N5O5S: C, 58.35; H,
3.17; N, 14.79. Found: C, 58.27; H, 3.25; N, 14.71.
3-(4-Hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-5-(4-
oxo-4H-1-benzopyran-3-yl)pyrazoline (4a)
1
White solid; mp 260–262°C. H NMR (CDCl3, 300 MHz)
1-Phenyl-3-[4-hydroxy-6-methyl-2-oxo-2H-pyran-
3-yl]-5-[5-(2-hydroxyphenyl)-1-phenyl pyrazol-4-yl]
pyrazoline (4f)
δ: 2.27 (3H, s, CH3), 3.69-3.92 (2H, m, He, Hf), 5.20-5.26
(1H, m, Hd), 6.04 (1H, s, C-5′), 6.68 (1H, s, NH), 7.35-
7.68 (3H, m, Ar-H), 8.02 (1H, s, C-2), 8.16 (1H, d, J= 7.9
Hz, C-5), 12.78 (1H, s, OH, D2O exchangeable). IR (KBr)
υ: 1700 (C=O), 1665 (C=O). MS-FAB m/z (%): 338 (M+,
60), 337 (70), 309 (5), 281 (5), 217 (5), 193 (15), 189 (5),
185 (5), 163 (10), 120 (15), 93 (45), 92 (10). Anal. Cald for
C18H14N2O5: C, 63.90; H, 4.17; N, 8.28. Found: C, 63.83; H,
4.24; N, 8.22.
1
Red crystals; mp 250–253°C. H NMR (CDCl3, 300 MHz)
δ: 2.35 (3H, s, CH3), 3.38–3.50 (1H, m, He), 3.66–4.11 (1H,
m, Hf), 4.99–5.34 (1H, m, Hd), 6.01 (1H, s, C-5′), 6.90-7.13
(4H, m, Ar-H), 7.18–7.42 (14H, m, Ar-H), 7.68 (1H, s, Hc),
13.27 (1H, s, OH, D2O exchangeable). IR (KBr) υ: 3432
(OH), 1701 (C=O). MS-FAB m/z (%): 504 (M+, 70), 476 (5),
460 (15), 449 (8), 445 (10), 379 (5), 352 (20), 279 (15). Anal.
Cald for C30H24N4O4: C, 71.41; H, 4.79; N, 11.10. Found: C,
71.26; H, 4.84; N, 11.06.
1-Benzothiazolyl-3-(4-hydroxy-6-methyl-2-oxo-2H-
pyran-3-yl)-5-(4-oxo-4H-1-benzopyran-3-yl)pyrazoline
(4b)
Antibacterial studies
Pale yellow solid; mp >300°C. 1H NMR (DMSO-d6,
300MHz) δ: 2.48 (3H, s, CH3), 3.07–4.14 (2H, m, He, Hf),
5.44–5.50 (1H, m, Hd), 6.29 (1H, s, C-5′), 8.02 (1H, d, J=7.1
Hz, C-5), 7.05-7.81 (7H, m, Ar-H), 8.41 (1H, s, C-2) 12.23
(1H, s, OH, D2O exchangeable). IR (KBr) υ: 1727 (C=O),
1649 (C=O). MS-FAB m/z (%): 471 (M+, 100), 443 (5), 415
(5), 428 (5), 322 (5), 307 (50), 177 (5), 120 (20). Anal. Cald
for C25H17N3O5S: C, 63.68; H, 3.63; N, 8.91. Found: C, 63.61;
H, 3.67; N, 8.87.
e newly synthesized compounds were screened for
their antibacterial activity against Streptococcus pyogenes
(clinical isolate), MRSA (+ve), Pseudomonas aeruginosa
(ATCC-27853), Klebsiella pneumoniae (clinical isolate)
and E. coli (ATCC-25922) bacterial strains by disk diffu-
sion method27,28. A standard inoculums (1–2 × 107 c.f.u./
mL 0.5 McFarland standards) was introduced on to the
surface of sterile agar plates, and a sterile glass spreader
was used for even distribution of the inoculums. e
disks measuring 6 mm in diameter were prepared from
Whatman no. 1 filter paper and sterilized by dry heat
at 140°C for 1 h. e sterile disks previously soaked in a
known concentration of the test compounds were placed
in nutrient agar medium. Solvent and growth controls
were kept. Ciprofloxacin (30 µg) was used as positive con-
trol while the disk poured in DMSO was used as negative
control. e plates were inverted and incubated for 24 h
at 37°C. e susceptibility was assessed on the basis of
diameter of zone of inhibition against Gram-positive and
Gram-negative strains of bacteria. Inhibition zones were
measured and compared with the controls. e bacterial
zones of inhibition values are given in Table 1.
5-(4-Oxo-4H-1-benzopyran-3-yl)-3-(2,4,6-
pyrimidinetrione-5-yl)pyrazoline (4c)
1
White solid; mp >300°C. H NMR (DMSO-d6, 300 MHz)
δ: 3.43-3.49 (1H, m, He), 3.82–3.89 (1H, m, Hf), 4.69–4.75
(1H, m, Hd), 7.49–7.84 (3H, m, Ar-H), 6.69 (1H, s, NH),
8.09 (1H, dd, J= 8.0 Hz, 1.2 Hz, C-5), 8.29 (1H, s, C-2), 10.31
(1H, s, NH), 10.45 (1H, s, NH). IR (KBr) υ: 3214 (NH), 3069
(NH), 1721(C=O), 1628 (C=O). MS-FAB m/z (%): 340 (M+,
75), 326 (25), 322 (30), 206 (5), 198 (5), 171 (30), 145 (5),
120 (30). Anal. Cald for C16H12 N4 O5: C, 56.47; H, 3.52; N,
16.47. Found: C, 56.31; H, 3.46; N, 16.54.
1-Phenyl-5-(4-oxo-4H-1-benzopyran-3-yl)-3-(2,4,6-
pyrimidinetrione-5-yl)pyrazoline (4d)
Minimum inhibitory concentrations (MICs) were
determined by broth dilution technique. e nutrient
broth, which contained logarithmic serially two fold
diluted amount of test compound and controls were
inoculated with approximately 5 × 105 c.f.u/mL of actively
dividing bacteria cells. e cultures were incubated for
24 h at 37°C and the growth was monitored visually and
spectrophotometrically. e lowest concentration (high-
est dilution) required to arrest the growth of bacteria was
regarded as MIC. To obtain the minimum bactericidal
concentration (MBC), 0.1 mL volume was taken from
Pale yellow solid; mp >300°C. 1H NMR (DMSO-d6,
400 MHz) δ: 3.51–3.56 (1H, m, He), 3.73–3.78 (1H, m,
Hf), 4.42–4.44 (1H, m, Hd), 7.25–7.45 (8H, m, Ar-H), 7.55
(1H, s, C-2), 7.79 (1H, dd, J= 7.6 Hz, 1.8 Hz, C-5), 10.60
(1H, s, NH), 10.76 (1H, s, NH). IR (KBr) υ: 3068 (NH),
1737 (C=O), 1649 (C=O). MS-FAB m/z (%): 416 (M+, 70),
415(5), 326 (45), 198 (40) 171 (30), 120 (20). Anal. Cald for
C22H16N4O5: C, 63.46; H, 3.84; N, 13.46. Found: C, 63.50; H,
3.71; N, 13.38.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antimicrobial chromonyl chalcones and pyrazolines 87
© 2012 Informa UK, Ltd.

88 Z.N. Siddiqui et al.
each tube and spread on agar plates. e number of c.f.u
was counted after 18–24 h of incubation at 35°C. MBC was
defined as the lowest drug concentration at which 99.9%
of the inoculums were killed. e minimum inhibitory
concentration and minimum bactericidal concentration
are given in Table 1.
Results and discussion
Chemistry
3-Formylchromone has emerged as an important start-
ing material for incorporation of chromone moiety, ever
since its convenient synthesis was reported in 1970s24.
It has been used as an important synthon due to the
presence of three electron deficient centers viz C-2, C-4
carbonyl and C-3 formyl groups. In the present study,
we carried out the reaction of 3-formylchromone 1 and
active methyl compounds viz dehydroacetic acid 2a and
5-acetyl barbituric acid 2b under different reaction con-
ditions. Initially reactions were conducted at room tem-
perature in the presence of basic catalyst pyridine. e
reactions afforded the expected chromonyl chalcones
3a-b in 18–25 hours with 62–70% yields (Scheme 1). e
reactions carried using methanol as a solvent under con-
ventional heating condition were found to be completed
in 10–16 h, but there was no substantial increase in yields
of products. is prompted us to carry out reactions
under solvent-free heating technique by simply heating
the starting materials with the catalyst. To our pleasant
surprise, the reactions were completed in short span of
time (12–15 min) with considerable increase in yields
(88–92%) and were devoid of any toxic waste products.
us, the present protocol explains the superiority of
thermal solvent-free reactions over the conventional
methods. e products obtained were characterized by
elemental and spectral (IR, 1H NMR and mass spectrom-
etry) analysis.
Antifungal studies
Antifungal activity was also done by disk diffusion
method. For assaying antifungal activity Candida albi-
cans,Aspergillusfumigatus,Trichophytonmentagrophytes
and Penicillium marneffei were recultured in DMSO by
agar diffusion method29,30. Sabourauds agar media was
prepared by dissolving peptone (1g), D-glucose (4 g) and
agar (2 g) in distilled water (100 mL) and adjusting pH
to 5.7. Normal saline was used to make a suspension of
spore of fungal strain for lawning. A loopful of particular
fungal strain was transferred to 3 mL saline to get a sus-
pension of corresponding species. 20 mL of agar media
was poured into each Petri dish. Excess of suspension
was decanted and the plates were dried by placing in an
incubator at 37°C for 1 h. Using an agar punch, wells were
made and each well was labeled. A control was also pre-
pared in triplicate and maintained at 37°C for 3–4 days.
e fungal activity of each compound was compared
with griseofulvin as standard drug. Inhibition zones were
measured and compared with the controls. e fungal
zones of inhibition values are given in Table 2. e nutri-
ent broth, which contained logarithmic serially two fold
diluted amount of test compound and controls was inoc-
ulated with approximately 1.6 × 104–6 × 104 c.f.u/mL. e
cultures were incubated for 48 h at 35°C and the growth
was monitored. e lowest concentration (highest dilu-
tion) required to arrest the growth of fungi was regarded
as MIC. To obtain the minimum fungicidal concentration
(MFC), 0.1 mL volume was taken from each tube and
spread on agar plates. e number of c.f.u. was counted
after 48 h of incubation at 35°C. MFC was defined as the
lowest drug concentration at which 99.9% of the inocu-
lums were killed. e MIC and MFC are given in Table 2.
1
e H NMR spectrum of the compound 3b showed
trans olefinic protons Ha and Hb as ortho coupled dou-
blets at δ 7.40 (J= 15.2 Hz) and 9.09 (J= 15.9 Hz), respec-
tively. e value of spin-spin coupling constant Jab, in the
range of 15–16 Hz, is indicative of the E-configuration of
chalcone. e three aromatic protons of chromone moi-
ety were discernible in the form of multiplet at δ 7.55–7.91
whereas C-2 and C-5 protons appeared as a singlet and
doublet of doublet at δ 8.95 and 8.17 respectively. e NH
protons of barbituric acid moiety appeared as two broad
singlets at δ 11.06 and 11.76.
Table 2. Antifungal activity of compounds 3a-4f.
Candida albicans
Aspergillus fumigatus
Trichophyton mentagrophytes
Penicillium marneffei
Compounds
Zone
MIC
12.5
12.5
25
MFC
Zone
MIC
12.5
25
MFC
Zone
MIC
25
MFC
100
50
Zone
MIC MFC
3a
22.2 0.4
21.3 0.3
20.1 0.3
20.1 0.3
19.8 0.5
18.1 0.3
17.9 0.3
19.4 0.5
30.0 0.2
−
50
25
19.7 0.3
19.6 0.4
18.1 0.2
17.2 0.5
17.6 0.6
16.5 0.4
15.5 0.5
17.1 0.6
27.0 0.2
−
50
100
15.8 0.9
15.4 0.2
14.3 0.3
13.7 0.4
12.1 0.3
11.7 0.5
10.4 0.2
12.1 0.4
14.3 0.2
12.9 0.3
11.7 0.2
10.1 0.4
10.3 0.6
9.2 0.4
8.9 0.3
10.3 0.6
20.0 0.5
−
25
25
50
50
25
50
50
50
12.5
−
50
50
3b
12.5
25
4a
100
100
100
100
100
100
25
25
100
100
100
100
100
>100
100
25
100
100
100
100
100
>100
25
4b
25
50
100
50
4c
50
50
100
50
4d
25
25
100
50
4e
50
50
>100
>100
50
4f
50
100
50
Standard
DMSO
6.25
−
12.5
−
12.5 24.0 0.3
6.25
−
−
−
−
−
−
Diameter of zone of inhibition (mm), MIC and MFC results of compounds 3a-4f and positive control griseofulvin. Positive control
(griseofulvin) and negative control (DMSO) measured by the Halo Zone Test (Unit, mm). MIC (µg/mL), minimum inhibitory
concentration, i.e., the lowest concentration of the compound to inhibit the growth of fungi completely; MFC (µg/mL), minimum
fungicidal concentration, i.e., the lowest concentration of the compound for killing the fungi completely.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antimicrobial chromonyl chalcones and pyrazolines 89
results were comparable with reactions performed under
conventional procedures. However, in case of reaction of
3a with phenylhydrazine, the mixture did not afford the
desired pyrazoline, instead a bipyrazole 4f was obtained
due to the ring opening reaction of pyrone ring both
under conventional and thermal solvent-free methods.
All the newly synthesized compounds were character-
Nitrogen bases such as hydrazines react with α,β-
unsaturated carbonyl compounds to give pyrazolines31.
With the objective of synthesizing pyrazolines contain-
ing chromone moiety, chromonyl chalcones 3a-b were
allowed to react with different hydrazines in conventional
method using acetic acid as solvent and under solvent-
free heating conditions. As expected, pyrazolines 4a-e
were obtained from hydrazine hydrate, hydrazinoben-
zothiazole and phenylhydrazine (Scheme 1) and the
1
ized by elemental and spectral (IR, H NMR and mass
spectrometry) analysis.
Scheme 1. Formation of chalcones 3a-b and their reaction with different hydrazines to give compounds 4a-f.
© 2012 Informa UK, Ltd.

90 Z.N. Siddiqui et al.
1
e H NMR spectrum of compound 4f indicated the
than bacteria. us, the nature of heterocycles and basic
skeleton of molecule have significant influence on the
extent of antibacterial and antifungal activities. A com-
parative study of the activity results (Tables 1 and 2) with
standard drugs (ciprofloxacin, griseofulvin) revealed
that none of the compound exceeds the activity of com-
mercial drugs. However, compounds have produced the
marked enhancement in the potency of these analogues
as antibacterial and antifungal agents.
absence of resonances attributable to C-2 olefinic and C-5
protons of chromone moiety, whereas compound 4a/4b
showed these diagnostic signals of C-2 proton as singlet
at δ 8.02, 8.41 and C-5 proton as doublet at δ 8.16, 8.02
respectively. ese observations suggest the opening of
pyrone ring under the action of phenylhydrazine to lead
the formation of bipyrazole 4f. e other prominent peaks
as obtained are explained in the experimental part.
Moleculartargetsofthesynthesizedcompoundsonthe
bacteria and fungi were identified using BioSpec Module
of Raasi Suite. Every molecule has multiple targets on
bacteria and fungi. e synthesized molecules have the
following molecular targets: ATP-binding cassette, sub-
family C, member 1 isoform 1 inhibitor, Pyruvate kinase,
muscle isoform M2 activator, Eukaryotic translation ini-
tiation factor 4γ, 1 isoform 4 inhibitor.
Pharmacology
e investigation of antibacterial screening data revealed
that all the tested compounds 3a-4f showed moderate
to good bacterial inhibition. Among the tested bacterial
strains, good inhibitory results were obtained against S.
pyogenes and E. coli. e structural activity study showed
that chalcones 3a, 3b and their derivatives 4a-f have
varying degrees of microbial inhibition. e antimicro-
bial activity seemed to be dependent on the nature of
heterocyclic moieties. A comparative study (Table 1) also
revealed that chromonyl chalcones 3a-b, are more potent
antibacterial agents than chromonyl pyrazolines 4a-f.
Compounds 3a, 3b, 4a, 4b and 4c showed good inhibi-
tion (MIC = 25 µg/mL) against S. pyogenes. e maximum
inhibition was observed in 3a and 4c against S. aureus
(MRSA +ve). Except 3a and 3b all other compounds
showed moderate to less activity results against P. aerugi-
nosa, K. pneumoniae and E. coli bacterial strains. e
MICs i.e., the lowest concentration of the compounds
to inhibit the growth of bacteria completely were in
the range of 25–100 µg/mL. e minimum bactericidal
concentrations (MBCs) i.e., the lowest concentration of
the compounds for killing the bacteria completely were
found to be two, three or four folds higher than the cor-
responding MIC results.
conclusions
In summary, a clean and convenient synthesis of novel
chromone derivatives has been developed. e proce-
dure offers several advantages including mild reaction
conditions as well as simple experimental and product
isolation procedures, thus, making the current “green
protocol” as a useful and attractive methodology for
the synthesis of series of novel heterocycles in excellent
yields from cheap and readily available starting materi-
als. e antibacterial, antifungal screening data revealed
that newly generated compounds are good antimicrobial
agents. e newly generated compounds can be used as
template for future development through modification
and derivatization to design more potent and selective
antimicrobial agents.
e antifungal screening data of the compounds also
revealed good to moderate activity. Among the tested fun-
gal strains, good inhibitory results were obtained against
C. albicans, and A. fumigatus. Again the comparative
study (Table 2) revealed that chromonyl chalcones 3a-b,
were found to have better inhibitory action than pyrazo-
lines 4a-f. e compounds 3a, 3b, 4a, 4b and 4d showed
effective inhibition results (MIC = 12.5–25 µg/mL) against
C. albicans. e maximum inhibition was observed in 3a,
3b and 4a against A. fumigatus, and T. mentagrophytes
fungal cultures. e inhibitory activity against P. marneffei
was significantly higher for the compounds 3a, 3b and 4c
than the other tested compounds. Compounds 4c-f were
moderately active against most of the fungal strains. e
MICs, i.e., the lowest concentration of the compounds to
inhibit the growth of fungi completely were in the range
of 12.5–100 µg/mL. e MFCs, i.e., the lowest concentra-
tion of the compounds for killing the fungi completely
were found to be two, three or four folds higher than the
corresponding MIC results.
acknowledgments
eauthorsarethankfultoUniversityGrantsCommission,
New Delhi for providing financial assistance in the form
of major research project [F.No. 37-15/2009 (SR)], SAIF,
CDRI, Lucknow, India for providing analytical, spectral
data and Mr Sameer Chaudhary, MD RASA Life Science
Informatics, Pune, India for providing drug target identi-
fication using BioSpec Module of Raasi Suite.
Declaration of Interest
e authors have declared no conflicts of Interest.
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