Synthesis of new series of 1-Aryl-1,4-dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid as potential antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2005.05.012

New series of 1-aryl-1,4-dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid has been synthesized and the structures of the new compounds were established on the basis of ¹H-NMR, mass (ES/MS), elemental analysis and IR spectral data. In vitro antibacterial activity (MIC activity) was evaluated and compared with standard drugs ciprofloxacin, sparfloxacin and trovafloxacin. Most of the compounds in the series have shown very interesting antibacterial activity against both Gram-positive and Gram-negative organisms. In this paper, we describe studies leading to identification of antibacterial agents incorporating novel pyridazine ring surrogate. In a gratifying result, the initial pyridazine-3-carboxylic acid analogues prepared were found to exhibit in vitro antibacterial activity approaching that of corresponding fluoroquinolone progenitor.

Full text of DOI:10.1016/j.ejmech.2005.05.012

European Journal of Medicinal Chemistry 40 (2005) 1325–1330
www.elsevier.com/locate/ejmech
Short Communication
Synthesis of new series of 1-Aryl-1,4-dihydro-4-oxo-6-methyl
pyridazine-3-carboxylic acid as potential antibacterial agents
Rahul R. Nagawade a, Vijay V. Khanna b, Sachin S. Bhagwat c, Devanand B. Shinde a,*
a Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad-431004, India
b Post Graduate School for Biological Studies, Ahmednagar College, Ahmednagar-414001, India
c Department of Microbiology and Biotechnology, Barkatullah University, Bhopal-456010, India
Received 20 December 2004; received in revised form 17 May 2005; accepted 19 May 2005
Available online 29 August 2005
Abstract
New series of 1-aryl-1,4-dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid has been synthesized and the structures of the new com-
pounds were established on the basis of 1H-NMR, mass (ES/MS), elemental analysis and IR spectral data. In vitro antibacterial activity (MIC
activity) was evaluated and compared with standard drugs ciprofloxacin, sparfloxacin and trovafloxacin. Most of the compounds in the series
have shown very interesting antibacterial activity against both Gram-positive and Gram-negative organisms. In this paper, we describe studies
leading to identification of antibacterial agents incorporating novel pyridazine ring surrogate. In a gratifying result, the initial pyridazine-3-
carboxylic acid analogues prepared were found to exhibit in vitro antibacterial activity approaching that of corresponding fluoroquinolone
progenitor.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Pyridazine-3-carboxylic acids; Antibacterial activity; Minimum inhibitory concentration (µg/ml); Gram-positive and Gram-negative bacteria; Phar-
macophore; Quinolone antibiotic analogues
1. Introduction
penicillins and tetracyclines are scarcely used in clinical
therapy.
Quinolone antibiotics are widely prescribed drugs because
of their safety, good tolerance, broad antibacterial spectrum
and less resistance [1–4]. Macrolide antibiotics, including
erythromycin and related compounds, continue to be an
important therapeutic class against Gram-positive organ-
isms, with second generation macrolides such as clarithro-
mycin and azithromycin being widely prescribed due to their
efficacy, safety and lack of serious side effects [5]. Oxazoli-
dinone antibacterial agents [6] are newer class of synthetic
antibacterial agents with activity against Gram-positive bac-
teria. Linezolid [7] is well known as first promising candi-
date of oxazolidinone and works effectively against numer-
ous serious Gram-positive human pathogens caused by MRSA
and VRE.
Several antibiotics have been prescribed and found to be
effective on various infectious disorders. However, the appear-
ance of multidrug resistant Gram-positive bacteria, in particu-
lar, methicillin-resistant Staphylococcus aureus (MRSA) and
vancomycin-resistant Enterococci (VRE) is causing a seri-
ous menace. Moreover, the emergence of vancomycin-
resistant MRSA can be anticipated in foreseeable future. For
the treatment of these intractable infections, a new anti-
infectious agent is needed.
The synthetic antibiotics include the sulfa drugs, nitrofu-
ran derivative, pyridine-carboxylic acid analogues, fluoroqui-
nolones and oxazolidinones. The semi-synthetic antibiotics
include the penicillins, cephalosporins, tetracyclines and mac-
rolides. Among them, the sulfa drugs, nitrofuran derivatives,
Herein, we have described the synthesis of new series of
1-aryl-1,4-dihydro-4-oxo-6-methyl pyridazine-3-carboxylic
acid as novel class of synthetic antibacterials along with their
in vitro biological activity (minimum inhibitory concentra-
tion (MIC) activity).
* Corresponding author. Tel.: +91 240 2400431 525.
E-mail address: devanandshinde@yahoo.com (D.B. Shinde).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.05.012

1326
R.R. Nagawade et al. / European Journal of Medicinal Chemistry 40 (2005) 1325–1330
2. Chemistry
followed by rearrangement to 1,4-dihydro-6-alkyl-1-aryl-4-
oxo-3-pyridazine carboxylic acid (4-pyridazones).
The synthesis of 1-aryl-1,4-dihydro-4-oxo-6-methyl
pyridazine-3-carboxylic acid is outlined in Scheme 1.
2.2. Synthesis of 1-aryl-1,4-dihydro-4-oxo-6-methyl
pyridazine-3-carboxylic acid
2.1. Synthesis of 4-pyridazones [8]
3-Acetyl-4-hydroxy-6-methyl-2H-pyran-2-one (dehy-
droacetic acid) (2) prepared from ethylacetoacetate (1) [9],
on refluxing with Conc. H2SO4 gives 4-hydroxy-6-methyl-2-
pyrone (3) [9]. This on coupling with diazonium chloride,
prepared by conventional diazotization technique from an
amine of the formula R-NH2 (where R is phenyl or substi-
tuted phenyl) gave hydrazone (4), which on treatment with
an aqueous acid yields pyridazine-3-carboxylic acid (5)
[8–10].
Azo dyes derived from substituted 1,2-pyran-2,4 (3) diones,
gradually fade in color when heated in dilute alkaline solu-
tion. For example, when an aqueous alkaline solution of
6-methyl-3-p-nitrophenyl-azo-1,2-pyran-2,4(3) dione was
refluxed for 1–2 h with equimolar amount of sodium hydrox-
ide, a good yield of yellow carboxylic acid resulted, as a result
of rearrangement of the original azo molecule.
The conversion of azo compounds to pyridazones may be
carried out about equally well in either aqueous alcohol or
water alone. The alkali used may be sodium hydroxide,
sodium carbonate or sodium bicarbonate. The reaction pro-
ceeds satisfactorily when excessive amounts of sodium bicar-
bonate are used though too much of sodium hydroxide may
have a harmful effect on yields.
The azo compounds required as starting material were eas-
ily prepared from 1,2-pyran-2,4(3) diones and the appropri-
ate diazo compounds in either alkaline or slightly acidic solu-
tion. The corresponding pyridazones are most conveniently
prepared without isolation or purification of azo intermedi-
ates.
The following new series of 1-(aryl substituted)-1,4-
dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid has
been synthesized and all the compounds were characterized
1
by H-NMR, electron spray ionization mass spectra (ES-
MS), elemental analysis, IR and MP (Table 1).
Also HPLC method development was established for
assessment of purity of each compound and each of the com-
pound showed excellent purity of about + 98% (Table 1).
3. Results and discussion
Thus alkaline hydrolysis of 6-alkyl-3-aryl-azo-1,2-pyran-
2,4(3)-dione results in cleavage of pyran-2,4(3) dione nucleus
The new 1-(aryl substituted)-1,4-dihydro-4-oxo-6-methyl
pyridazine-3-carboxylic acid analogues prepared above were
Scheme 1
.

R.R. Nagawade et al. / European Journal of Medicinal Chemistry 40 (2005) 1325–1330
1327
Table 1
Physical and spectral data of 1-(aryl substituted)-1,4-dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid (5a–i)
Serial number Compounds
R
Molecular
formula
Molecular weight
Yield
(%)
68
M.p.
(°C)
HPLC RT
(min)
11.72
10.34
11.46
11.69
12.42
12.31
12.52
9.03
HPLC purity
(%)
1
2
3
4
5
6
7
8
9
5-a
5-b
5-c
5-d
5-e
5-f
5-g
5-h
5-i
4-F
C
C
C
C
C
C
C
C
C
12H9FN2O3
248
255
298
248
266
266
266
274
273
158–60
218–20
210–12
190–91
181–83
220–22
207–09
217–18
214–15
99.68
98.97
99.37
99.95
98.91
99.85
99.67
97.10
99.92
4-CN
13H9N3O3
35
4-CF3
13H9F3N2O3
12H9FN2O3
12H8F2N2O3
12H8F2N2O3
12H8F2N2O3
13H10N2O5
13H11N3O4
40
3-F
76
2,4-Difluoro
3,5-Difluoro
3,4-Difluoro
4-COOH
4-CONH2
56
45
42
40
45
5.27
Serial number Compounds
1H-NMR (200 MHz, CDCl3)
Mass (ES/MS)
m/z (M + H)
249
1
2
3
4
5
6
7
8
9
5-a
5-b
5-c
5-d
5-e
5-f
5-g
5-h
5-i
d 2.40(s,3H), 6.95(s,1H), 7.35 (dd,2H), 7.50(dd,2H), 15.40(bs,1H)
d 2.35(s,3H), 6.90(s,1H), 7.60, (dd,2H), 7.90(dd,2H), 15.10(bs,1H)
d 2.35(s,3H), 6.90(s,1H), 7.60(dd,2H), 7.90(dd,2H), 15.20(bs,1H)
d 2.40(s,3H), 6.90(s,1H), 7.20(bm,3H), 7.60(m,1H), 15.30(bs,1H)
d 2.30(s,3H), 6.90(s,1H), 7.10(bm,2H), 7.50(m,1H), 15.20(bs,1H)
d 2.40(s,3H), 6.95(s,1H), 7.05(bm,3H), 15.10(bs,1H)
256
299
249
267
267
d 2.30(s,3H), 6.95(s,1H), 7.30(bm,3H), 15.20(bs,1H)
267
d 2.20(s,3H), 7.00(s,1H), 7.70(dd,2H), 8.20(dd,2H), 15.20(bs,1H)
d 2.30(s,3H), 7.10(s,1H), 7.70(dd,2H), 8.20(dd,2H), 15.20(bs,1H)
275
274
Elemental analysis data were obtained for all the compounds and was within the limit of 0.4% of the calculated value.
tested in vitro versus a panel of Gram-positive and Gram-
negative clinical isolates. MIC was determined by standard
agar dilution method as per NCCLS guidelines and the val-
ues are shown in Table 2. The data of ciprofloxacin, sparfloxa-
cin and trovafloxacin were used as reference standards.
Most of the compounds in the series exhibited excellent
and promising in vitro antibacterial activity against both
Gram-positive and Gram-negative strains, except for
Pseudomonas aeruginosa where all the tested compounds
were inactive (MIC > 32 µg/ml) in comparison with ciprof-
loxacin, sparfloxacin and trovafloxacin as reference stan-
dards.
Compounds 5-b and 5-h have shown MIC of 0.06–4 µg/ml
against S. aureus. Compounds 5-b and 5-g have shown MIC
of 0.03–4 and 0.06–8 µg/ml, respectively, against Escheri-
chia coli. Compounds 5-b and 5-h have shown MIC of 0.06–
4 µg/ml against S. epidermis. Compounds 5-b and 5-d have
shown MIC of 0.25–8 µg/ml against Klebsiella species while
compounds 5-e and 5-f have shown MIC of 0.06–16 and
0.125–16 µg/ml, respectively, against E. faecalis.All the tested
compounds have moderate activity (MIC = 1–32 µg/ml)
against E. faecium. Rest of the compounds in the series also
had excellent antibacterial activity against the strains tested
in comparison with ciprofloxacin, sparfloxacin and trovafloxa-
cin.
With the results in hand an extensive study survey of dif-
ferently substituted derivatives of above class was under-
taken in order to increase the spectrum of antibacterial activ-
ity.
4. Conclusion
In summary, we have described the genesis and synthesis
of antibacterially active 1-(aryl substituted)-1,4-dihydro-4-
oxo-6-methyl pyridazine-3-carboxylic acid derivatives. Nota-
bly, the analogues 5-b with a para cyano phenyl substituent,
5-e with a 2,4-difluoro phenyl substituent, 5-f with a 3,5-
difluoro phenyl substituent, 5-g with a 3,4-difluoro phenyl
substituent and 5-h with a para carboxy group phenyl sub-
stituent, in particular, have exhibited superior MIC activity
against S. aureus, E. coli, S. epidermis and E. faecalis as
compared to ciprofloxacin, sparfloxacin and trovafloxacin.
Since this preliminary series of pyridazine-3-carboxylic
acid has shown good activity against the tested organisms,
the possibility remains that more divergent structural modi-
fications might improve the spectrum of antibacterial activ-
ity. In connection with this notion, we point out the pharma-
cophoric similarity of this pyridazine-3-carboxylic acid series
relative to fluoroquinolone counterpart and suggest that fur-
ther study of these agents is warranted.

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R.R. Nagawade et al. / European Journal of Medicinal Chemistry 40 (2005) 1325–1330
Table 2
MIC (µg/ml) of 1-(aryl substituted)-1,4-dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid (5a–i)
Compounds
P. aeruginosa (N = 26)a
S. aureus (N = 80)b
E. coli (N = 54)c
S. epidermis (N = 33)
MIC
50
MIC
90
Range
MIC
MIC
90
4
Range
MIC
50
MIC
Range
MIC
MIC
90
4
Range
50
90
50
5-a
5-b
5-c
5-d
5-e
Inactive (> 32)
2
0.25–16
0.06–4
0.5–32
0.12–8
0.12–2
0.25–16
0.25–32
0.06–4
0.12–16
0.5–8
0.25
0.06
0.5
4
0.06–8
0.03–4
0.25–8
0.06–8
0.06–8
0.12–8
0.06–8
0.25–16
0.25–16
0.003–
0.03
2
0.25–16
0.06–4
0.5–32
0.12–8
0.12–2
0.25–16
0.25–32
0.06–4
0.12–16
0.5–8
″
″
″
0.25
1
2
0.25
1
1
8
4
1
8
0.50
2
0.12
0.12
0.25
0.25
0.12
0.12
0.007
4
0.50
2
1
4
2
1
4
5-f
″
″
″
″
2
8
2
2
8
5-g
5-h
5-I
2
8
2
2
8
0.125
4
4
0.125
4
1
1
8
4
1
1
8
Cipro.
0.25
1
2
1
0.12–4
0.25–4
0.06–4
4
0.015
4
Spar.
1.0
1
2
1
0.5–4
0.007
0.002
0.03
0.003–
0.03
1
2
1
0.5–4
Trova.
0.25
0.5
0.25–4
0.015
0.003–
0.03
0.5
0.25–4
Compounds Klebsiella sp. (N = 24)
E. faecium (N = 16)
E. faecalis (N = 24)
MIC
MIC
90
8
Range
MIC
50
8
MIC
90
Range
MIC
50
1
MIC
90
8
Range
50
5-a
5-b
5-c
5-d
5-e
5-f
5-g
5-h
2
0.5–16
0.25–8
0.5–16
0.25–8
0.5–8
1–16
> 32
> 16
> 32
> 16
> 16
> 32
> 32
> 32
> 16
> 16
> 16
> 16
2–> 32
2–> 32
2–>32
1–> 32
2–> 32
4–> 32
4–> 32
8–> 32
2–> 32
NA
0.25–16
0.25–16
0.5–8
1
4
4
0.5
1
4
2
8
8
8
1
4
4
0.5
0.25
1
4
0.12–8
0.06–8
0.125–8
0.25–16
0.5–16
0.5–8
2
4
8
4
4
16
16
16
8
16
16
16
8
4
4
1–16
2
16
8
4
1–16
2
5-i
2
0.5–16
1
4
Cipro.
Spar.
Trova.
0.01
0.01
0.025
0.02
0.02
0.1
0.01–0.5 16
0.01–0.25 > 16
0.06–0.4 > 16
2
8
0.5–16
0.5–8
NA
1
4
NA
1
2
0.5–4
N = number of strains tested.
a P. aeruginosa ATCC 27853.
b S. aureus ATCC 25923.
c E. coli ATCC 25922.
5. Experimental section
5.1. Part A:- Preparation of 3-acetyl-4-hydroxy-6-methyl-
2H-pyran-2-one (dehydroacetic acid) (2) [9]
Melting points were determined on Quality Precise appa-
ratus and are uncorrected. H-NMR spectra were recorded
1
Hundred grams (780 mmol) of freshly vacuum distilled
ethylacetoacetate (1) and 0.5 g of sodium bicarbonate are
heated until the liquid has reached to 200–210 °C. The time
for heating was usually 7–8 h, during which period 27 g of
distillate boiling at 72 °C (mostly ethanol) was collected and
the color of the reaction mixture becomes dark brown. The
resulting dehydroacetic acid was distilled under reduced pres-
sure (the dehydroacetic acid was collected at 140 °C–
12 mm).
on Varian Gemini 200 MHz spectrometer. Chemical shifts
are reported in d units (ppm) relative to TMS as internal stan-
dard. ES-MS were recorded on Water-Micromass Quattro-II
spectrometer. HPLC analysis were carried out on Shimadzu
instrument with SPD-10A UV detector and YMC Pack
AM.25 cm column. IR spectra were recorded onVarian spec-
trometer.All the solvents and reagents used were ofAR grade
and were used without further purification.

R.R. Nagawade et al. / European Journal of Medicinal Chemistry 40 (2005) 1325–1330
1329
The yield of dehydroacetic acid melting at 104–110 °C is
34 g (53%). A purer product m.p. 108 °C was secured in 80%
by recrystallization from ethanol using 2 ml/g of material.
M.p. 107–108 °C; 1H-NMR (200 MHz, CDCl3) d 2.05 (s,
3H), 2.25 (s, 3H), 5.90 (dd, 1H); MS (ES) m/z 169 (M + H,
100%).
5.3.2. Compound 5-b:- 1-(4-Cyano phenyl)-1,4-dihydro-4-
oxo-6-methyl pyridazine-3-carboxylic acid (5-b)
1H-NMR (200 MHz, CDCl3) d 2.35 (s, 3H), 6.90 (s, 1H),
7.60 (dd, 2H), 7.90 (dd, 2H), 15.10 (bs, 1H); MS (ES) m/z
256 (M + H, 100%).
5.3.3. Compound 5-c:- 1-(4-Trifluoromethyl phenyl)-1,4-
dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid
1H-NMR (200 MHz, CDCl3) d 2.35 (s, 3H), 6.90 (s, 1H),
7.60 (dd, 2H), 7.90 (dd, 2H), 15.20 (bs, 1H); MS (ES) m/z
299 (M + H, 100%).
5.2. Part B:- De-acetylation of dehydroacetic acid to
6-methyl-2H-pyran-2,4-(3H)-dione (3) [9]
Fifty grams (290 mmol) of dehydroacetic acid (2) was dis-
solved in 150 g of dilute 90% H2SO4. The mixture was then
heated upto 130 °C and maintained at the same temperature
for about 15 min. The flask was then rapidly cooled and the
contents were poured into 200 ml of ice-cold water. The pre-
cipitated solid was collected by filtration, washed with 2 ×
15 ml cold water and dried completely to get off-white solid
as desired compound with yield of 36 g (90%).
5.3.4. Compound 5-d:- 1-(3-Fluoro phenyl)-1,4-dihydro-4-
oxo-6-methyl pyridazine-3-carboxylic acid
1H-NMR (200 MHz, CDCl3) d 2.40 (s, 3H), 6.90 (s, 1H),
7.20 (bm, 3H), 7.60 (m, 1H), 15.30 (bs, 1H); MS (ES) m/z
249 (M + H, 100%).
M.p. 188–190 °C; 1H-NMR (200 MHz, CDCl3) d 2.20 (s,
3H), 5.60 (dd, 1H), 5.90 (dd, 1H); MS (ES) m/z 127 (M + H,
100%); IR (cm−1) 2900, 1680, 1600, 1310, 1265, 1005, 850,
530.
5.3.5. Compound 5-e:- 1-(2,4-Difluoro phenyl)-1,4-
dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid
1H-NMR (200 MHz, CDCl3) d 2.30 (s, 3H), 6.90 (s, 1H),
7.10 (bm, 2H), 7.50 (m, 1H), 15.20 (bs, 1H); MS (ES) m/z
267 (M + H, 100%).
5.3. Part C:- General procedure for synthesis of 1-(aryl
substituted)-1,4-dihydro-4-oxo-6-methyl
pyridazine-3-carboxylic acid (compound 5a–5i) [8–10]
5.3.6. Compound 5-f:- 1-(3,5-Difluoro phenyl)-1,4-
dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid
1H-NMR (200 MHz, CDCl3) d 2.40 (s, 3H), 6.95 (s, 1H),
7.05 (bm, 3H), 15.10 (bs, 1H); MS (ES) m/z 267 (M + H,
100%).
4-Hydroxy-6-methyl-2-pyrone (3) (0.5 g, 3.96 mmol) was
dissolved in 20 ml water and 2.24 g (21.03 mmol) of sodium
carbonate was added to the suspension to make the solution
alkaline.
5.3.7. Compound 5-g:- 1-(3,4-Difluoro phenyl)-1,4-
dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid
1H-NMR (200 MHz, CDCl3) d 2.30 (s, 3H), 6.95 (s, 1H),
7.30 (bm, 3H), 15.20 (bs, 1H); MS (ES) m/z 267 (M + H,
100%).
In a separate flask, 4.20 mmol of required substituted
aniline was dissolved in 10 ml 6 N HCl and cooled to 0 °C. A
solution of 0.35 g (5.04 mmol) of sodium nitrite in 5 ml water
was added at 0 °C and the resulting diazonium chloride solu-
tion was added dropwise to stirred pyrone solution while
maintaining the temperature at 5–10 °C and pH at 10–12.
The resulting hydrazone was refluxed for 3 h at 110 °C
and then neutralized with acetic acid upto pH 7. A small
amount of charcoal was added and again refluxed for 30 min.
The solid was filtered in hot, the filtrate cooled to 0–5 °C and
treated with conc. HCl. The precipitated solid was collected
by filtration, washed well with water and dried completely to
get white to off-white solid as desired compound.
5.3.8. Compound 5-h:- 1-(4-Carboxy-phenyl)-1,4-dihydro-
4-oxo-6-methyl pyridazine-3-carboxylic acid
1H-NMR (200 MHz, DMSO-d6) d 2.20 (s, 3H), 7.00 (s,
1H),7.70 (dd, 2H), 8.20 (dd, 2H), 15.20 (bs, 1H); MS (ES)
m/z 275 (M + H, 100%).
5.3.9. Compound 5-i:- 1-(4-Carbamoyl-phenyl)-1,4-
dihydro-4-oxo-6-methyl pyridazine-3-carboxylic acid
1H-NMR (200 MHz, DMSO-d6) d 2.30 (s, 3H), 7.10 (s,
1H), 7.70 (dd, 2H), 8.20 (dd, 2H), 15.20 (bs, 1H); MS (ES)
m/z 274 (M + H, 100%).
1
All the compounds were characterized by H-NMR, IR,
ES-MS and MP. The purity was determined by HPLC (YMC
Pack AM.25 cm column, Mobile phase as 0.05% TFA in
acetonitrile, flow rate of 1.25 ml/min, run time of 30 min and
UV detection at 254 nm) (Table 1).
5.4. Part D:- MIC determination
5.4.1. Bacterial isolates
5.3.1. Compound 5-a:- 1-(4-Fluoro phenyl)-1,4-dihydro-4-
oxo-6-methyl pyridazine-3-carboxylic acid
The strains were collected from major hospitals of India
during the period 2003–2004 and were identified by standard
laboratory procedures.
MIC was determined by standard agar dilution method as
per NCCLS guidelines (M7-A5 January 2000). Strains were
1H-NMR (200 MHz, CDCl3) d 2.40 (s, 3H), 6.95 (s, 1H),
7.35 (dd, 2H), 7.50 (dd, 2H), 15.40 (bs, 1H); MS (ES) m/z
249 (M + H, 100%).

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R.R. Nagawade et al. / European Journal of Medicinal Chemistry 40 (2005) 1325–1330
[2] Y. Mizuki, I. Fujawara, T. Yamaguchi, J. Antimicrob. Chemother. 37
(Suppl. A) (1996) 41–55.
grown in tryptic soya broth (TSB, HiMedia, India) for
18–24 h. The overnight grown cultures were diluted appro-
priately so that the final density is approximately 107 CFU/ml.
A portion of this diluted broth was transferred to seed block
of a multipoint inoculator (AQS Manufacturing, UK). The
inoculating pins were standardized to inoculate 1–2 µl of this
broth, so that the final CFU was 1 × 104–5 × 104 CFU per
spot. The inoculated plates were allowed to stand until the
moisture in the inoculum spot has been absorbed in the media.
The inoculated plates were inverted and incubated for
18–24 h at 35 °C in an ambient air incubator (Newtronic,
India). After the completion of incubation period, the plates
were read visually.
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MIC was defined as the lowest concentration which inhib-
ited the growth of strain completely. Drug free plates were
used to ensure the growth of strains.ATCC strain of S. aureus,
E. coli and P. aeruginosa were used as quality control.
[8] J.F. Morgan, et al., J. Am. Chem. Soc. 70 (1948) 2253.
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