Synthesis, spectroscopic characterization and pharmacological evaluation of oxazolone derivatives

Article abstract of DOI:10.2298/JSC120917126F

A series of six 4-(arylmethylidene)-2-phenyl/methyl-5(4H)-oxazolone derivatives were synthesized using a reported method by condensation of aldehydes with N-benzoyl/N-acetyl glycine in the presence of zinc oxide as a catalyst and acetic anhydride at room temperature in ethanol. Five of the compounds are new derivatives. The structures of the compounds were evaluated based on 1H-NMR, 13C-NMR, EI-MS and FT-IR spectroscopy and elemental analysis. All the compounds were screened for their antibacterial and urease inhibition activity. The antibacterial activity was tested by the agar well diffusion method using Mueller-Hinton agar medium. Compound 2 showed excellent activity against Staphylococcus aureus exhibiting 16 mm (80 %) inhibition and above 24 mm (70 %) against Salmonella typhi. Compound 6 was the most active compound against Escherichia coli having 20 mm (80 %) inhibition followed by compound 5 having above 18 mm (70 %) inhibition. Urease inhibition activity of all the compounds was determined by the indophenol method. Compounds 3, 6 and 7 showed significant inhibition against Jack bean urease.

Full text of DOI:10.2298/JSC120917126F

J. Serb. Chem. Soc. 78 (8) 1127–1134 (2013)
UDC 547.78:542.913:615.281:612.064
JSCS–4484
Original scientific paper
Synthesis, spectroscopic characterization and pharmacological
evaluation of oxazolone derivatives
1
2
GHULAM FAREED1,2 , NIGHAT AFZA , MUHAMMAD ALI VERSIANI ,
NAZIA FAREED2, UZMA RASHEED MUGHAL3, MAHBOOB ALI KALHORO1,
LUBNA IQBAL1 and MEHREEN LATEEF1
*
1Pharmaceutical Research Center, PCSIR Laboratories Complex Karachi, Shahrah-e-Dr.
Salim-uz-Zaman Siddiqui Karachi-75280, Sindh, Pakistan, 2Department of Chemistry,
Federal Urdu University of Arts, Science and Technology, Ghulshan-e-Iqbal, Karachi-75300,
Sindh, Pakistan and 3HEJ Research Institute of Chemistry, ICCBS, University of Karachi,
Karachi-75280, Pakistan
(Received 17 September, revised 21 November 2012)
Abstract: A series of six 4-(arylmethylidene)-2-phenyl/methyl-5(4H)-oxazo-
lone derivatives were synthesized using a reported method by condensation of
aldehydes with N-benzoyl/N-acetyl glycine in the presence of zinc oxide as a
catalyst and acetic anhydride at room temperature in ethanol. Five of the
compounds are new derivatives. The structures of the compounds were eva-
luated based on 1H-NMR, 13C-NMR, EI-MS and FT-IR spectroscopy and
elemental analysis. All the compounds were screened for their antibacterial and
urease inhibition activity. The antibacterial activity was tested by the agar well
diffusion method using Mueller–Hinton agar medium. Compound 2 showed
excellent activity against Staphylococcus aureus exhibiting 16 mm (80 %) inhi-
bition and above 24 mm (70 %) against Salmonella typhi. Compound 6 was the
most active compound against Escherichia coli having 20 mm (80 %) inhi-
bition followed by compound 5 having above 18 mm (70 %) inhibition. Urease
inhibition activity of all the compounds was determined by the indophenol
method. Compounds 3, 6 and 7 showed significant inhibition against Jack bean
urease.
Keywords: aldehyde; antibacterial; urease inhibition activities; oxazolones;
synthesis; zinc oxide.
INTRODUCTION
For many decades, increasing resistance against human pathogens that cause
serious infections is one of the main topics of interest for medicinal chemists.
During the last century, many medicines were developed against bacterial infec-
tions. However, the available antibacterial drugs could cause undesirable effects
*Corresponding author. E-mail: fareedchm@yahoo.com
doi: 10.2298/JSC120917126F
1127
Available online at shd.org.rs/JSCS/
________________________________________________________________________________________________________________________
2013 Copyright (CC) SCS

1128
FAREED et al.
and might be toxic. In view of these facts, it is important to explore additional
drugs that have no adverse effects due to their different mode of action. In recent
years, there has also been an increased interest in urease inhibiting drugs. Urease
is a major cause of peptic and gastric ulcer. Compounds having urease inhibitory
1
potential play an important role in pharmaceutical companies. Activities of ure-
ase (E. C. 3.5.1.5) has been shown to be important virulence determinant in the
pathogenesis of many clinical conditions, which are detrimental for human and
animal health. Urease is directly involved in the formation of infection stones and
contributes to the pathogenesis of urolithiasis, pyelonephritis, ammonia and
2,3
heptic encephalopathy, hepatic coma and urinary catheter encrustation. For the
above reasons, a wide range of resistant drugs are important and required to treat
patients. Mostly nitrogen-, sulfur- and oxygen-containing five- and six-mem-
bered heterocyclic compounds have enormous significance in the field of medi-
4
cinal chemistry. Oxazolones are five-membered heterocyclic compounds con-
taining nitrogen and oxygen as hetero atoms. The C-2 and C-4 positions of oxa-
zolone are responsible for their various biological activities. Oxazolones perform
an important role in the synthesis of several organic molecules, including amino
5
6
7
acids, amino alcohols, thiamine and peptides. Certain natural and synthetic
oxazolones also possess important biological activities, such as anti-inflamma-
8
9
10
11
12
tory, antimicrobial, anticancer, anti-HIV, anti-angiogenic, anticonvul-
13
14–16
17
sant, antitumor, antagonistic, sedative
and cardiotonic activity. . Oxazo-
lone plays a very vital role in the manufacture of various biologically active
drugs, such as analgesic, anti-inflammatory, antidepressant, anticancer, antimic-
18,19
robial, antidiabetic and anti-obesity compounds.
In the literature, the syntheses of oxazolone derivatives were achieved by
condensation of aldehydes with N-benzoyl/N-acetyl glycine in the presence of
20
21
22
various reagents and catalysts, such as, ZnCl , KF/NaOAc, Al O –H BO ,
2
2
3
3
3
23
24
Bi(III) salts and Pb(OAc) . Herein, the synthesis of a series of 4-(arylmethyl-
2
idene)-2-phenyl/methyl-5(4H)-oxazolone derivatives (2–7) using a literature
25
method by the condensation of an aldehyde with N-benzoyl/N-acetyl glycine in
the presence of zinc oxide as a catalyst at room temperature in ethanol is reported.
Compounds 2–6 are new. All the synthesized compounds were tested for their
urease inhibition activity and antibacterial activity against Escherichia coli,
Bacillus subtilis, B. cereus, Staphylococcus aureus, Pseudomonas aeruginosa,
Salmonella typhi and Proteus mirabilis.
EXPERIMENTAL
All chemicals were of AR grade. The melting points were measured using Gallen Kemp
MF-370 apparatus. The IR spectra were recorded on Nicolet Avatar 300 DTGS instrument.
1
The H- and 13C-NMR spectra were recorded on a Bruker AV-300 instrument operating at
300 and 75 MHz, respectively, in dimethyl sulfoxide (DMSO) with trimethylsilane (TMS) as
an internal standard. The EI-MS were recorded on a Finnigan MAT-311 A. CHN analysis was
Available online at shd.org.rs/JSCS/
________________________________________________________________________________________________________________________
2013 Copyright (CC) SCS

SYNTHESIS AND CHARACTERIZATION OF OXAZOLONE DERIVATIVES
1129
performed on a Carlo Erba Strumentazione-Mod-1106, Italy. Purity of the product was
determined by using silica gel 60F254 TLC, pre-coated cards (0.2 mm thickness) and visu-
alized under UV light (254 and 366 nm) and by iodine vapors.
Antibacterial assay
Antibacterial activity was tested by the agar well diffusion method using Mueller Hinton
Agar medium for the assay. The microorganisms were activated by inoculating a loopful of
the strains in the nutrient broth (25 mL) and incubating at room temperature on a rotary
shaker. Then 0.2 mL of inoculum (inoculum concentration was 108 cells mL-1 as per the
McFarland standard) was inoculated into the molten Mueller–Hinton agar media and after
proper homogenization, it was poured into 20 mm×100 mm Petri dishes. For the agar well
diffusion, a well was made in the seeded plates with the help of a cup-borer (8.5 mm). The
reference standard and test sample (1 %) 0.1 mL was introduced into the well and all the
plates were incubated at 37 °C for 24 h. The microbial growth was determined by measuring
the zone of inhibition (mm) and the mean values are presented. Percentage inhibitions of the
compounds were determined by comparing the zone of inhibition to that of standard drug
against each organism.
Urease inhibition assay
Reaction mixtures comprising 25 μL of enzyme (jack bean urease) solution and 55 μL of
buffers containing 100 mM urea were incubated with 5 μL of the test compounds (1 mM con-
centration) at 30 °C for 15 min in 96-well plates. The urease activity was determined by
measuring ammonia production using the indophenol method as described by Weatherburn.26
Briefly, 45 μL each of phenol reagent (1 % w/v phenol and 0.005 % w/v sodium nitroprus-
side) and 70 μL of alkali reagent (0.5 % w/v NaOH and 0.1 % (w/v) active chlorine NaOCl)
were added to each well. The increasing absorbance at 630 nm was measured after 50 min,
using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate
in a final volume of 200 μL. The results (change in absorbance per min) were processed using
Soft Max Pro software (Molecular Device, USA). All the assays were performed at pH 8.2
(0.01 M K2HPO4·3H2O, 1 mM EDTA and 0.01 M LiCl). The percentage inhibitions were
calculated from the formula 100–(ODtestwell/ODcontrol)×100. Thiourea was used as the standard
inhibitor of urease.
Synthesis of N-benzoylglycine (1a)
Glycine (10 g, 133 mmol) was dissolved in 10 % NaOH solution (100 mL) and benzoyl
chloride (21.6 ml, 186 mmol) was added in portions to this solution in 500mL conical flask.
After each addition, the flask was stoppered and shaken vigorously until all the acid chloride
had reacted. The solution was cooled by adding few grams of crushed ice and concentrated
hydrochloric acid (pH 2–3) was added. The so-formed precipitates were filtered and washed
several times with cold distilled water, dried, and recrystallized from hot water. The required
compound was obtained as a crystalline white solid.
Synthesis of N-acetylglycine (1b)
Glycine (10 g, 133 mmol) and water (30 mL) in a 500 mL conical flask was vigorously
stirred until the solid glycine had completely dissolved. Acetic anhydride (25 mL, 266 mmol)
was added in portions and stirred vigorously for 20 min. The exothermic reaction produced
solid N-acetylglycine, which showed the end-point of the reaction. The reaction mixture was
then placed in refrigerator overnight for the complete formation of the product. The solid was
filtered, washed with ice-cooled water and recrystallized from hot water.
Available online at shd.org.rs/JSCS/
________________________________________________________________________________________________________________________
2013 Copyright (CC) SCS

1130
FAREED et al.
Synthesis of the 4-(arylmethylidene)-2-phenyl/methyl-5-(4H)-oxazolones derivatives (2–7)
A mixture of the required aldehyde (1 mmol), N-benzoylglycine/N-acetylglycine (1 mmol),
acetic anhydride (3 mmol), ZnO as a catalyst (0.05 mmol) and 15 mL ethanol was stirred at
room temperature, 25 °C (Scheme 1). After a certain period, the syrupy reaction mixture was
solidified and the reaction was completed (Table I). After the final product had been obtained,
20 mL more of cold ethanol was added. The solid was filtered off, dried and washed with hot
water before recrystallization of the product.
Scheme 1. Synthesis of oxazolones 2–7; a) acetic anhydride or benzoyl chloride, 10 % NaOH,
H2O; b) ZnO, acetic anhydride, C2H5OH, reflux under stirring at r.t.
TABLE I. Synthesis of oxazolone derivatives (2–7) with their physical parameters
Yield Molecular weight Reaction time M.p.
Compound
R1
R2
%
g mol-1
328
min
°C
2
89
10
227
3
92
305
15
118
H3CO
4
5
80
70
266
360
12
19
160
166
6
7
82
70
423
293
15
10
149
197
Available online at shd.org.rs/JSCS/
________________________________________________________________________________________________________________________
2013 Copyright (CC) SCS

SYNTHESIS AND CHARACTERIZATION OF OXAZOLONE DERIVATIVES
1131
RESULTS AND DISCUSSION
Chemistry
Oxazolone derivatives were synthesized by condensation of substituted aro-
matic aldehydes with N-benzoyl/N-acetyl glycine using zinc oxide as a catalyst in
ethanol at room temperature. All the synthesized compounds were characterized
using different spectroscopic techniques. The IR spectrum of compound 3, as
typical, showed characteristic band of carbonyl group at 1781 and C=N at 1652
–1
1
cm . Also, typical H-NMR spectrum showed characteristic pattern of peaks.
The methyl protons appeared in the region of 3.84 ppm, whereas the aromatic
protons appeared at 6.89–8.12 ppm. The electron ionization mass spectrometric
fragmentation patterns of the compounds were the same.
The complete analytic and spectral data of the obtained products are given in
the Supplementary material to this paper.
Antibacterial activity
All the compounds (2–7) were tested for their antibacterial activity against
Escherichia coli, Bacillus subtilis, B. cereus, Staphylococcus aureus, Pseudomo-
nas aeruginosa, Salmonella typhi and Proteus mirabilis. The results were com-
pared with those of the standard 0.3 % gentamycin. The antibacterial activity of
compounds 2–7 are shown in Fig 1. The bacterial zones of inhibition values are
summarized in Table II. Compounds 5 and 6 showed excellent activities against
E. coli, which is very difficult to treat with traditionally used antibiotics. The
most active compound against E. coli was compound 6 having 20 mm inhibition
zone (80 % inhibition), followed by compound 5 having above 18 mm inhibition
zone (70 %). Thus, compounds 5 and 6 would be the better choice for the
treatment of infections caused by E. coli than the derivatives 1–4 or 7. All the
screened compounds showed low to moderate activities against B. cereus, except
Fig. 1. Antibacterial activity of compounds 2–7.
Available online at shd.org.rs/JSCS/
________________________________________________________________________________________________________________________
2013 Copyright (CC) SCS

1132
FAREED et al.
compound 4 that showed 50 % inhibition. S. aureus is responsible for various
throat infections and cholic diseases. Therefore, compound 2 would be the best
choice to treat infections caused by S. aureus as it had a 16 mm inhibition zone
(80 %). None of the compounds showed inhibition against P. aeruginosa except
compound 2 that had less than 30 % inhibition. S. typhi, responsible for typhoid,
could be inhibited by compound 2, showing an inhibition zone of 24 mm (70 %).
Compound 4 also showed 50 % inhibition followed by compounds 5–7. B.
subtilis was only inhibited by compound 6, which showed an inhibition zone of
15 mm (75 %). Compound 6 also showed ≈20 mm inhibition zone (50 %) against
P. mirabilis, followed by compounds 4, 5 and 7.
TABLE II. Antibacterial activity (concentration used: 1 % of each compound); zone of inhi-
bition was measured in mm; SD – standard drug, 0.3 % gentamycin
Compound
Bacterium
SD
2
3
4
5
6
7
E. coli
10
00
16
10
24
00
12
00
00
05
00
08
00
00
12
15
00
00
16
00
10
18
10
00
05
12
00
15
20
12
12
00
10
15
20
00
09
00
00
00
00
10
25
30
20
35
34
20
39
B. cereus
S. aureus
P. aeruginosa
S typhi
B. subtilis
P. mirabilis
Compounds 5 and 6 proved themselves to be very effective antibacterial
agents. Compound 5 showed inhibition against four and compound 6 showed
inhibitions against six different bacterial strains. The C-2 and C-4 positions of the
oxazolones are responsible for their crucial biological activities (Fig. 2). A phe-
nyl group instead of a methyl group at C-2 plays a vital role in the enhancement
of the activities, as shown by compounds 5 and 6. Compared to compound 5,
compound 6 showed a greater tendency of inhibition against different bacterial
strains due to the phenyl group at C-2. Electron withdrawing substituents at C-4
position are also a good choice for the enhancement of the activities.
Fig. 2. Structure of oxazolones.
Available online at shd.org.rs/JSCS/
________________________________________________________________________________________________________________________
2013 Copyright (CC) SCS

SYNTHESIS AND CHARACTERIZATION OF OXAZOLONE DERIVATIVES
1133
Urease inhibition activity
All the synthesized compounds were screened for their urease inhibition
activity. Compounds 3, 6 and 7 showed strong inhibition against jack bean
urease, having IC values near to the standard inhibitor thiourea as determined
50
by the indophenol method. The results are listed in Table III.
TABLE III. Urease inhibition activity of compounds 2–7; Standard IC50 = 21.4 µM
Compound
Urease inhibition, %
IC50 / µM
2
3
4
5
6
7
21
85.2
44
49
82.5
73.2
Not determined
62
NA
NA
65.3
58
CONCLUSION
Based on the biological activities of the synthesized oxazolone derivatives, it
could be concluded that they are therapeutic antibacterial agents and urease inhi-
bitors.
SUPPLEMENTARY MATERIAL
Analytic and spectral data for the synthesized compounds are available electronically
from http://www.shd.org.rs/JSCS/, or from the corresponding author on request.
Acknowledgement. All the authors wish to thank the Pharmaceutical Research Center,
PCSIR Laboratories Complex, Karachi, Pakistan, for financial and technical support for this
research.
И З В О Д
СИНТЕЗА, СПEКТРАЛНА КАРАКТЕРИЗАЦИЈА И ФАРМАКОЛОШКА ИСПИТИВАЊА
ДЕРИВАТА ОКСАЗОЛОНА
1,2
1
2
2
GHULAM FAREED , NIGHAT AFZA , MUHAMMAD ALI VERSIANI , NAZIA FAREED ,
3
1
1
1
UZMA RASHEED MUGHAL , MAHBOOB ALI KALHORO , LUBNA IQBAL и MEHREEN LATEEF
1
Pharmaceutical Research Center, PCSIR Laboratories Complex Karachi, Shahrah-e-Dr. Salim-uz-Zaman
2
Siddiqui Karachi-75280, Sindh, Pakistan, Department of Chemistry, Federal Urdu University of Arts,
3
Science and Technology, Ghulshan-e-Iqbal, Karachi-75300, Sindh, Pakistan и HEJ Research Institute of
Chemistry, ICCBS, University of Karachi, Karachi-75280, Pakistan
Синтетисана је серија деривата 4-(арилметилиден)-2-фенил/метил-5(4H)-оксазо-
лона (2–7) од којих су деривати 2–6 синтетисани први пут. Једињења су синтетисана
кондензацијом алдехида и N-бензоил/N-ацетил-глицина у присуству цинк-оксида као
катализатора и анхидрида сирћетне киселине, на собној температури у етанолу као рас-
тварачу. Структура деривата одређена је спектралном (1H-NMR, 13C-NMR, EI-MS и FT-
-IR) и елементалном анализом. Свим једињењима одређена је антибактеријска актив-
ност и инхибиторна активност према уреази. Антибактеријска активност је тестирана
према методи дифузије у агару помоћу Mueller–Hinton агар медијума. Једињење 2 пока-
зује одличну активност према Staphylococcus aureus ≈16 mm (80 %) инхибиције и преко
Available online at shd.org.rs/JSCS/
________________________________________________________________________________________________________________________
2013 Copyright (CC) SCS

1134
FAREED et al.
24 mm (70 %) према Salmonella typhi. Према Escherichia coli најактивније је једињење 6 са
20 mm (80 %) инхибиције, а затим једињење 5 са преко 18 mm (70 %) инхибиције.
Инхибиторна активност према уреази за сва једињења одређена је индофенолном мето-
дом. Једињења 3, 6 и 7 показују значајну активност према уреази пасуља сорте Jack.
(Примљено 17. септембра, ревидирано 21. новембра 2012)
REFERENCES
1. A. Hussain, S. Perveen, A. Malik, N. Afza, L. Iqbl, R. B. Tareen, Pol. J. Chem. 83 (2009)
1329
2. H. L. Mobley, M. D. Island, R. P. Hausinger, Microb. Rev. 59 (1989) 451
3. H. L. Mobley, R. P. Hausinger, Microb. Rev. 53 (1989) 85
4. M. J. Aaglawe, S. S. Dhule, S. S. Bahekar, P. S Wakte, D. B. Shinde. J. Korean Chem.
Soc. 47 (2003) 133
5. J. Lamb, W. Robson. Biochem. J. 25 (1931) 1231
6. M. I. Ismail. Can. J. Chem. 69 (1991) 1886
7. M. Palcut. Acta Chim. Slov. 56 (2009) 362.
8. U. S. Goksen, N. G. Kelekci, O. Goktas, Y. Koysal, E. Kilic, S. Isik, G. Aktay, M. Ozalp,
Bioorg. Med. Chem. 15 (2007) 5738
9. G. Brownlee, M. Woodbine. Br. J. Pharmacol. 3 (1948) 305
10. C. Sanchez, C. Mendez, J. A. Salas. Nat. Prod. Rep. 23 (2006) 1007
11. M. Witvrouw, C. Pannecouque, E. D. Clercq, E. Fernandez-Alvarez, J. L Marco. Arch.
Pharm. Med. Chem. 352 (1999) 163
12. F. M. P Sierra, A. Pierre, M. Burbridge, N. Guilbaud. Bioorg. Med. Chem. Lett. 12 (2002)
1463
13. H. M. F. Madkour. Chem. Pap. 56 (2002) 313
14. K. M. Khan, U. R. Mughal, M. T. H. Khan, Z. Ullah, S. Perveen, M. I. Choudhary.
Bioorg. Med. Chem. 14 (2006) 6027
15. A. M. Tikdari, S. Fozooni, H. Hamidian. Molecules 13 (2008) 3246
16. M. A. Mesaik, S. Rahat, K. M. Khan, Z. Ullah, M. I. Choudhary, S. Murad, Z. Ismail, A.
Rahman, A. Ahmad. Bioorg. Med. Chem. 12 (2004) 2049
17. R. A. Schnettler, W. D. Jones Jr., G. P. Claxton (Merrell Dow Pharmaceuticals Inc.),
United States, Patent No.: US 4698353 (1987)
18. P. A. Conway, K. Devine, F. Paradisi. Tetrahedron 65 (2009) 2935
19. V. Taile, K. Hatzade, P. Gaidhane, V. Ingle. Turk. J. Chem. 33 (2009) 295
20. P. S. Rao, R. V. Venkataraman. Indian. J. Chem., B 33 (1994) 984
21. F. M. Baustista, J. M. Campelo, A. Garcia, D. Luna, J. M. Marinas, A. A. Romero. J.
Chem. Soc., Perkin Trans. 2 (2002) 227
22. H. J. Ringold, B. Loeken, G. Rosenkranz, F. Sondheimer. J. Am. Chem. Soc. 78 (1956)
816
23. M. M. Khodaei, A. R. Khosropour, S. J. H. Jomor. J. Chem. Res. Synop. 10 (2003) 638
24. H. Hamidian, A. M. Tikari. Heterocycl. Commun. 12 (2006) 29
25. M. A. Pasha, V. P. Jayashankara, K. N. Venugopala, G. K. Rao. J. Pharmacol. Toxicol. 2
(2007) 264.
Available online at shd.org.rs/JSCS/
________________________________________________________________________________________________________________________
2013 Copyright (CC) SCS

Copyright of Journal of the Serbian Chemical Society is the property of National Library of
Serbia and its content may not be copied or emailed to multiple sites or posted to a listserv
without the copyright holder's express written permission. However, users may print,
download, or email articles for individual use.