Synthesis of some new urea and thiourea derivatives and evaluation of their antimicrobial activities

Article abstract of DOI:10.3906/kim-1106-54

In the present study, 18 new compounds were synthesized, 9 of which were urea (9a-e, 10a-d) while the others were thiourea (11a-e, 12a-d) derivatives. These derivatives were prepared by the reaction of 6-amino-5-nonsubstituted/ chloro-3-methyl-2(3H) -benzoxazolones used as key intermediates with the appropriate isocyanates and isothiocyanates. The chemical structures of new compounds were confirmed by 1H-NMR, mass, and elemental analysis. The synthesized compounds were screened for their antibacterial and antifungal activities against some pathogenic strains. Compounds 9a, 9b, 9e, 10a, and 10c, urea derivatives, and compounds 12a and 12d, thiourea derivatives, exhibited a relatively good inhibitory profile against E. coli, with a MIC value of 32 ig/mL when compared with the other target compounds.

Full text of DOI:10.3906/kim-1106-54

Turk J Chem
36 (2012) , 279 – 291.
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˙
c
ꢀ TUBITAK
doi:10.3906/kim-1106-54
Synthesis of some new urea and thiourea derivatives and
evaluation of their antimicrobial activities
1
1
2
2
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˙
˘
¨
Yıldız GULKOK , Tuba BIC¸ER , Fatma KAYNAK ONURDAG , Selda OZGEN
3
3,∗
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Mustafa Fethi S¸AHIN , Deniz Songu¨l DOGRUER
1
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Deva Ila¸c, C¸erkezk¨oy, Tekirda˘g-TURKEY
2Department of Microbiology, Faculty of Pharmacy, Gazi University,
06330 Etiler, Ankara-TURKEY
3Department of Pharmaceutical Chemistry, Faculty of Pharmacy,Gazi University,
06330 Etiler, Ankara-TURKEY
e-mail: denizdogruer2002@yahoo.com
Received: 22.06.2011
In the present study, 18 new compounds were synthesized, 9 of which were urea (9a-e, 10a-d) while
the others were thiourea (11a-e, 12a-d) derivatives. These derivatives were prepared by the reaction of
6-amino-5-nonsubstituted/chloro-3-methyl-2(3H)-benzoxazolones used as key intermediates with the ap-
propriate isocyanates and isothiocyanates. The chemical structures of new compounds were confirmed by
1 H-NMR, mass, and elemental analysis. The synthesized compounds were screened for their antibacte-
rial and antifungal activities against some pathogenic strains. Compounds 9a, 9b, 9e, 10a, and 10c, urea
derivatives, and compounds 12a and 12d, thiourea derivatives, exhibited a relatively good inhibitory profile
against E. coli, with a MIC value of 32 μg/mL when compared with the other target compounds.
Key Words: Urea, thiourea, 2(3H)-benzoxazolone, antimicrobial activity
Introduction
Antimicrobial drugs (antibacterial and antifungal) have been successfully used in the treatment of infectious
diseases caused by bacteria and fungi since the first half of the 20th century. At the same time, antimicrobial
therapy has been effective in prolonging of the average life expectancy by reducing the deaths caused by infectious
diseases. However, the increasing use and misuse of antimicrobial drugs have led to the development of more
∗Corresponding author
279

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Synthesis of some new urea and thiourea derivatives and..., Y. GULKOK, et al.
resistant pathogens to commonly used antimicrobials. In particular, the emergence of multidrug resistant
bacterial strains has made the treatment of bacterial diseases more complex when compared with the first half
of last century.1
As for fungi, systemic fungal infections have become increasingly common in individuals with suppressed
immune systems, such as patients undergoing anticancer chemotherapy or organ transplants and patients with
AIDS.2 In addition, antifungal drugs used in fungal infections have also become less effective owing to the
development of fungal resistance.3
As a consequence, in recent decades, the incidence of microbial infections has increased at alarming levels
in many countries around the world because of antimicrobial resistance. In order to overcome this challenging
problem, there is the urgent need for the development of new antimicrobial compounds.
2(3H)-Benzoxazolone heterocyclic has received attention due to its capacity to mimic a phenol or a
catechol.4,5 Many 2(3H)-benzoxazolone-containing compounds have been reported to possess various bio-
logical activities such as analgesic-anti-inflammatory,6−8 anticonvulsant,9 antioxidant,10 and antimicrobial
properties.11,12 Moreover, some authors have reported that the introduction of halogen atoms to the phar-
macophore structure can be beneficial for antimicrobial activity.13,14 Therefore, 2(3H)-benzoxazolone and its
5-chloro derivative were chosen to synthesize our target compounds. In addition, some urea and thiourea deriva-
tives are known to be associated with a wide range of biological activities such as analgesic,15 antitumor,16,17
anti-HIV,18,19 and antimicrobial properties.20−23
In view of these observations, we have designed new compounds incorporating the above pharmacophores
together in order to prepare molecules having enhanced antimicrobial activity.
Therefore, we synthesized new compounds containing urea (9a-e, 10a-d) and thiourea (11a-e, 12a-d)
groups at position 6 of 3-methyl-2(3H)-benzoxazolone and 5-chloro-3-methyl-2(3H)-benzoxazolone rings in
order to investigate their antibacterial and antifungal activities.
Experimental
Chemistry
All the chemicals used for the synthesis of the compounds were purchased from Aldrich (Steinheim, Germany)
and Merck (Darmstadt, Germany). Melting points of the compounds were recorded on an Electrothermal-
9200 digital melting points apparatus (Southend, Great Britain) and the values are uncorrected. Microwave
irradiation was carried out in a microwave oven (Milestone-MicroSYNTH, Italy). Thin-layer chromatography
(TLC) was performed on Merck 60F254 plates. Reactions were monitored by TLC on silica gel, with detection by
UV light (254 nm). Flash column chromatography was performed on Kieselgel 60 (0.063-0.2 mm) (Merck). The
1 H-NMR spectra (400 MHz) and 13 C-NMR (100 MHz) spectra were recorded employing a Varian Mercury
400 MHz FT spectrometer (Varian Inc., Palo Alto, CA, USA), in DMSO-d6 at the Faculty of Pharmacy,
Ankara University, Ankara, Turkey. The mass spectra were obtained on a Waters ZQ micromass LC-MS
spectrometer (Waters Corporation, Milford, MA, USA) using the ESI(+) method at the Faculty of Pharmacy,
Ankara University. Elemental analysis was performed on a Leco 932 CHNS instrument (St. Joseph, MI,
USA) at the Faculty of Pharmacy, Ankara University, and the results were within 0.4% of the theoretical
280

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Synthesis of some new urea and thiourea derivatives and..., Y. GULKOK, et al.
values. The synthesis of 2(3H)-benzoxazolone (1),24 3-methyl-2(3H)-benzoxazolone (3),24 5-chloro-3-methyl-
2(3H)-benzoxazolone (4),25 3-methyl-6-nitro-2(3H)-benzoxazolone (5),26 5-chloro-3-methyl-6-nitro-2(3H)-
benzoxazolone (6),27 6-amino-3-methyl-2(3H)-benzoxazolone (7),26 and 6-amino-5-chloro-3-methyl-2(3H)-
benzoxazolones (8)27 was previously reported.
General procedure for the synthesis of 5-nonsubstituted/chloro-3-methyl-6-nitro-2(3H)-benzoxa-
zolones (5, 6)
3-Methyl-2(3H)-benzoxazolone (0.1 mol) or 5-chloro-3-methyl-2(3H)-benzoxazolone (0.1) was added in por-
tions to 100 mL of HNO3 by keeping the internal temperature of flask at 40-50 ◦ C. At the end of addition, the
reaction mixture was poured into ice water. The forming precipitate was filtered, dried, and used for the next
step without further purification. Yield 91%, mp: 182 ◦ C for compound 5 (Ref. 26; mp: 181-182 ◦ C). Yield
91%, mp: 157 ◦ C for compound 6.
General procedure for the synthesis of 6-amino-5-nonsubstituted/chloro-3-methyl-2(3H)-benzoxa-
zolones (7, 8)
SnCl2 (0.05 mol) was added to the solution of 3-methyl-6-nitro-2(3H)-benzoxazolone (0.01 mol) or 5-chloro-
3-methyl-6-nitro-2(3H)-benzoxazolone (0.01 mol) in 40 mL of ethanol and 10 mL of 6 N HCl and reaction
mixture was refluxed for 1.5 h. At the end of this period, the reaction mixture was cooled, and made alkaline
with 1 M NaOH. The organic layer was extracted with CH2 Cl2 , dried with MgSO4 , and the residue obtained
after the solvent evaporation was washed with methanol. The forming precipitate was filtered, dried, and used
for next step without further purification. Yield 96%, mp: 154 ◦ C for compound 7 (Ref. 26; mp: 153-154 ◦ C).
Yield 86%, mp: 228 ◦ C for compound 8.
General procedure for the synthesis of urea and thiourea derivatives obtained from 6-amino-3-
methyl-2(3H)-benzoxazolone (9a-e, 11a-e)
Substituted isocyanate or isothiocyanate derivatives (1 mmol) were added to the solution of 6-amino-3-methyl-
2(3H)-benzoxazolone (1 mmol) in 10 mL of THF at room temperature. The resulting mixture was stirred at
room temperature for 1 h. At the end of this period, the forming precipitate was filtered, dried, and crystallized
from appropriate solvent.
General procedure for the synthesis of urea and thiourea derivatives obtained from 6-amino-5-
chloro-3-methyl-2(3H)-benzoxazolone:
Method A for compounds (10b, 10d)
Substituted isocyanate derivatives (1 mmol) were added to a solution of 6-amino-5-chloro-3-methyl-2(3H)-
benzoxazolone (1 mmol) in 10 mL of THF at room temperature. The resulting mixture was stirred at room
temperature until the starting material had been consumed. At the end of this period, the forming precipitate
was filtered, dried, and crystallized from appropriate solvent.
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Synthesis of some new urea and thiourea derivatives and..., Y. GULKOK, et al.
Method B for compounds (10a, 10c, 12b, 12c)
A mixture of substituted isocyanate or isothiocyanate derivatives (3 mmol) and 6-amino-5-chloro-3-methyl-
2(3H)-benzoxazolone (2.5 mmol) was heated at 65 ◦ C until the amino derivative had been consumed. At the
end of this period, the forming precipitate was filtered and either purified by flash column chromatography
using ethyl acetate-hexane (50:50) as eluent or crystallized from appropriate solvent.
Method C for compounds (12a, 12d)
A mixture of substituted isothiocyanate derivatives (15 mmol) and 6-amino-5chloro-3-methyl-2(3H)-benzoxazo-
lone (2.5 mmol) was heated at 100 ◦ C until the amino derivative had been consumed. At the end of this period,
methanol-water was added to reaction medium and the forming precipitate was filtered, dried, and purified by
flash column chromatography using ethyl acetate-hexane (50:50) as eluent.
1-Phenyl-3-(3-methyl-2-oxo-3H-benzoxazole-6-yl)urea (9a)
Recrystallized from acetic acid, yield 92%, mp: 258 ◦ C. 1 H-NMR (DMSO-d6) δ: 8.77 (s, 1H, -NH), 8.70 (s,
ꢀ
ꢀ
ꢀ
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1H, -NH), 7.65(s, 1H, H7), 7.50 (m, 2H, H2 , H6 ), 7.32 (m, 2H, H3 , H5 ), 7.19 (s, 2H, H5 , H4), 7.02 (m, 1H,
ꢀ
H4 ), 3.35 (s, 3H, -CH3). MS ESI(+) m/e284 (M+H, 100). Anal. (C15 H13 N3 O3): C, H, N calc. 63.60, 4.63,
14.83 found 63.52, 4.82, 14.65.
1-(4-Chlorophenyl)-3-(3-methyl-2-oxo-3H-benzoxazole-6-yl)urea (9b)
Recrystallized from isopropanol, yield 89%, mp: 338 ◦ C. 1 H-NMR (DMSO-d6 -RT) δ: 8.78 (s, 1H, -NH), 8.74
ꢀ
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(s, 1H, -NH), 7.58 (s, 1H, H7), 7.45 (d, 2H, H2 , H6 ), 7.29 (d, 2H, H3 , H5 ), 7.13 (s, 2H, H5 , H4), 3.26 (s,
3H, -CH3). 13 C-NMR (DMSO-d6) δ: 154.83, 153.22, 142.69, 139.36, 135.42, 129.29, 127.17, 126.01, 120.44,
144.69, 109.58, 101.85, 28.70. MS ESI(+) m/e318 (M+H, 100). Anal. (C15 H12 ClN3 O3): C, H, N calc. 56.70,
3.81, 13.23 found 56.75, 3.86, 13.34.
ꢀ
ꢀ
(DMSO-d6 55 ◦ C) δ: 8.70 (s, 1H, -NH), 8.65 (s, 1H, -NH), 7.57 (d, 1H, H7), 7.47 (d, 2H, H2 , H6 ),
ꢀ
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7.30 (d, 2H, H3 , H5 ), 7.14 (m, 2H, H5 , H4), 3.35 (s, 3H, -CH3).
(Acetone-d6 RT) δ: 8.24 (s, 1H, -NH), 7.68 (d, 1H,Jm = 2 Hz, H7), 7.56 (d, 2H, H2 , H6 ), 7.29 (d,
ꢀ
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2H, H3 , H5 ), 7.21 (dd, 1H, Jm = 2 Hz, Jo = 8.8 Hz, H5), 7.08 (d, 1H, Jo = 8.4 Hz, H4), 3.26 (s, 3H, -CH3).
1-(4-Methoxyphenyl)-3-(3-methyl-2-oxo-3H-benzoxazole-6-yl)urea (9c)
Recrystallized from isopropanol, yield 91%, mp: 334 ◦ C. 1 H-NMR (DMSO-d6) δ: 8.71 (s, 1H, -NH), 8.53 (s,
ꢀ
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1H, -NH), 7.64 (s, 1H, H7), 7.37 (d, 2H, H2 , H6 ), 7.16 (s, 2H, H5 , H4), 6.89 (s, 2H, H3 , H5 ), 3.74 (s, 3H,
-OCH3), 3.26 (s, 3H, CH3). MS ESI(+) m/e314 (M+H, 100). Anal. (C16 H15 N3 O4): C, H, N calc. 61.34,
4.83, 13.41 found 61.30, 4.98, 13.33.
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Synthesis of some new urea and thiourea derivatives and..., Y. GULKOK, et al.
1-Benzyl-3-(3-methyl-2-oxo-3H-benzoxazole-6-yl)urea (9d)
Recrystallized from isopropanol, yield 92%, mp: 260 ◦ C. 1 H-NMR (DMSO-d6) δ: 8.70 (s, 1H, -NH), 7.64-7.12
(m, 8H, H7 , H5 , H4 , phenyl protons), 6.67 (s, 1H, NH), 4.32 (s, 2H, -CH2), 3.26 (s, 3H, -CH3). MS ESI(+)
m/e298 (M+H, 100). Anal. (C16 H15 N3 O3): C, H, N calc. 64.64, 5.09, 14.13 found 64.39, 4.90, 13.69.
1-(2-Phenylethyl)-3-(3-Methyl-2-oxo-3H-benzoxazole-6-yl)urea (9e)
Recrystallized from methanol, yield 88%, mp: 238 ◦ C. 1 H-NMR (DMSO-d6) δ: 8.57 (s, 1H, -NH), 7.60 (s,
1H, H7), 7.33-7.02 (m, 7H, H5 , H4 , phenyl protons), 6.12 (s, 1H, -NH), 3.35 (t, 2H, -CH2), 3.3 (s, 3H, -CH3),
2.75 (t, 2H, -CH2) MS ESI(+) m/e312 (M+H, 100). Anal. (C17 H17 N3 O3): C, H, N calc. 65.58, 5.50, 13.50
found 65.24, 5.44, 13.25.
1-Phenyl-3-(5-chloro-3-methyl-2-oxo-3H-benzoxazole-6-yl)urea (10a)
Purification by column chromatography on silica gel using ethyl acetate-hexane (50:50) as eluent, yield 20%, mp:
366 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.36 (s, 1H, -NH), 8.33 (s, 1H, -NH), 8.06 (s, 1H, H7), 7.52 (s, 1H, H4), 7.50-
6.99 (m, 5H, phenyl protons), 3.32 (s, 3H, -CH3). MS ESI(+) m/e318 (M+H, 100). Anal. (C15 H12 ClN3 O3 .
H2 O): C, H, N calc. 53.66, 4.20, 12.52 found 53.76, 3.88, 2.13.
1-(4-Chlorophenyl)-3-(5-chloro-3-methyl-2-oxo-3H-benzoxazole-6-yl)urea (10b)
Recrystallized from DMF-H2 O, yield 51%, mp: 262 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.47 (s, 1H, -NH), 8.34 (s,
ꢀ
ꢀ
ꢀ
ꢀ
1H, -NH), 8.02 (s, 1H, H7), 7.51 (s, 1H, H4), 7.49 (d, 2H, H2 , H6 ), 7.34 (d, 2H, H3 , H5 ), 3.32 (s, 3H, -CH3).
13 C-NMR (DMSO-d6) δ: 180.76, 154.82, 142.11, 139.12, 134.63, 129.66, 128.97, 126.10, 121.09, 109.20, 107.79,
28.78.
MS ESI(+) m/e353 (M+H, 100). Anal. (C15 H11 Cl2 N3 O3 .0.5 H2 O): C, H, N calc. 49.88, 3.34, 11.63
found 49.94, 3.26, 11.75.
1-(4-Methoxyphenyl)-3-(5-chloro-3-methyl-2-oxo-3H-benzoxazole-6-yl)urea (10c)
Purification by column chromatography on silica gel using ethyl acetate-hexane (50:50) as eluent, yield 15%,
mp: 338 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.13 (s, 1H, -NH), 8.18 (s, 1H, -NH), 8.00 (s, 1H, H7), 7.44 (s, 1H, H4),
ꢀ
ꢀ
ꢀ
ꢀ
7.31 (d, 2H, H2 , H6 ), 6.83 (d, 2H, H3 , H5 ), 3.66 (s, 3H, -OCH3), 3.32 (s, 3H,-CH3). MS ESI(+) m/e348
(M+H, 100). Anal. (C16 H14 ClN3 O4): C, H, N calc. 55.26, 4.06, 12.08 found 54.80, 4.17, 11.90.
1-Benzy-3-(5-chloro-3-methyl-2-oxo-3H-benzoxazole-6-yl)urea (10d)
Recrystallized from methanol, yield 17%, mp: 254 ◦ C. 1 H-NMR (DMSO-d6) δ: 8.20 (s, 1H, -NH), 8.10 (s,1H,
-NH), 7.50-7.20 (m, 7H, H4 , H7 , phenyl protons), 4.32 (s, 2H, -CH2), 3.34 (s, 3H, -CH3). MS ESI(+) m/e332
(M+H, 100). Anal. (C16 H14 ClN3 O3): C, H, N calc. 57.93, 4.25, 12.67 found 57.95, 4.39, 12.50.
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1-Phenyl-3-(3-methyl-2-oxo-3H-benzoxazole-6-yl)thiourea (11a)
Recrystallized from acetic acid, yield 92%, mp: 252 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.81 (s, 1H, -NH), 9.79 (s,
ꢀ
ꢀ
ꢀ
ꢀ
1H,-NH), 7.56 (s, 1H, H7), 7.42 (d, 2H, H2 , H6 ), 7.32 (t, 2H, H3 , H5 ), 7.19 (s, 2H, H5 , H4), 7.10 (t, 1H,
ꢀ
H4 ), 3.26 (s, 3H, -CH3) MS ESI(+) m/e300 (M+H, 100). Anal. (C15 H13 N3 O2 S): C, H, N, S calc. 60.18,
4.38, 14.04, 10.71 found 59.98, 4.47, 13.99, 10.56.
1-(4-Chlorophenyl)-3-(3-methyl-2-oxo-3H-benzoxazole-6-yl)thiourea (11b)
Recrystallized from methanol, yield 93%, mp: 242 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.89 (s, 1H, -NH), 9.83 (s, 1H,
-NH), 7.53-7.22 (m, 7H, H7 , H5 , H4 , phenyl protons), 3.35 (s, 3H, CH3). 13 C-NMR (DMSO-d6) δ: 154.73,
152.90, 141.41, 139.09, 131.08, 129.40, 128.49, 126.31, 120.39, 118.66, 110.22, 104.86, 28.94. MS ESI(+) m/e334
(M+H, 100). Anal. (C15 H12 ClN3 O2 S): C, H, N, S calc. 53.97, 3.62, 12.59, 9.61 found 53.89, 3.86, 12.19, 9.36.
1-(4-Methoxyphenyl)-3-(3-methyl-2-oxo-3H-benzoxazole-6-yl)thiourea (11c)
Recrystallized from methanol, yield 91%, mp: 235 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.65 (s, 1H, -NH), 9.60 (s,
ꢀ
ꢀ
ꢀ
ꢀ
1H, -NH), 7.56 (s, 1H, H7), 7.35 (d, 2H, H2 , H6 ), 7.23 (s, 2H, H5 , H4), 6.95 (d, 2H, H3 , H5 ), 3.80 (s, 3H,
-OCH3), 3.26 (s, 3H, CH3). MS ESI(+) m/e330 (M+H, 100). Anal. (C16 H15 N3 O3 S): C, H, N, S calc. 58.34,
4.59, 12.76, 9.74 found 57.94, 4.66, 12.68, 9.40.
1-Benzyl-3-(3-methyl-2-oxo-3H-benzoxazole-6-yl)thiourea (11d)
Recrystallized from methanol, yield 91%, mp: 191.5 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.68 (s, 1H, -NH), 8.16 (s,
1H, -NH), 7.51 (s, 1H, H7), 7.35-7.15 (m, 7H, H5 , H4 , phenyl protons), 4.75 (s, 2H, -CH2), 3.37 (s, 3H, -CH3).
MS ESI(+) m/e314 (M+H, 100). Anal. (C16 H15 N3 O2 S): C, H, N, S calc. 61.32, 4.82, 13.41, 10.23 found
61.23, 5.05, 13.43, 10.24.
1-(2-Phenylethyl)-3-(3-methyl-2-oxo-3H-benzoxazole-6-yl)thiourea (11e)
Recrystallized from acetic acid, yield 90%, mp: 211 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.50 (s, 1H, -NH), 7.58 (s,
1H, -NH), 7.30-7.10 (m, 8H, H7 , H5 , H4 , phenyl protons), 3.60 (m, 2H, -CH2), 3.26 (s, 3H, -CH3), 2.78 (t,
2H, -CH2). MS ESI(+) m/e328 (M+H, 100). Anal. (C17 H17 N3 O2 S): C, H, N, S calc. 62.36, 5.23, 12.83,
9.79 found 62.16, 5.37, 12.81, 9.79.
1-Phenyl-3-(5-chloro-3-methyl-2-oxo-3H-benzoxazole-6-yl)thiourea (12a)
Purification by column chromatography on silica gel using ethyl acetate-hexane (50:50) as eluent, yield 17%,
mp: 202 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.92 (s, 1H, -NH), 9.46 (s, 1H, -NH), 7.53-7.15 (m, 7H, H4 , H7 , phenyl
protons), 3.35 (s, 3H, -CH3). MS ESI(+) m/e334 (M+H, 100). Anal. (C15 H12 ClN3 O2 S): C, H, N, S calc.
53.97, 3.62, 12.59, 9.61 found 54.04, 3.80, 12.20, 9.20.
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1-(4-Chlorophenyl)-3-(5-chloro-3-methyl-2-oxo-3H-benzoxazole-6-yl)urea (12b)
Recrystallized from acetone, yield, 18%, mp: 189 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.88 (s, 1H, -NH), 9.51 (s,
1H, -NH), 7.48-7.32 (m, 6H, H4 , H7 , phenyl protons), 3.30 (s, 3H, -CH3). 13 C-NMR (DMSO-d6) δ: 180.75,
153.91, 140.15, 138.13, 131.20, 130.32, 128.44, 128.25, 126.35, 125.39, 111.60, 109.43, 28.25. MS ESI(+) m/e369
(M+H, 100). Anal. (C15 H11 Cl2 N3 O2 S): C, H, N, S calc. 48.93, 3.01, 11.41, 8.71 found 48.80, 3.10, 11.42,
8.72.
1-(4-Methoxyphenyl)-3-(5-chloro-3-methyl-2-oxo-3H-benzoxazole-6-yl)urea (12c)
Recrystallized from methanol, yield 20%, mp: 188 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.73 (s, 1H, -NH), 9.30 (s, 1H,
ꢀ
ꢀ
ꢀ
ꢀ
-NH), 7.52 (s, 2H, H4 , H7), 7.33 (d, 2H, H2 , H6 ), 6.92 (d, 2H, H3 , H5 ), 3.35 (s, 3H, -CH3). MS ESI(+)
m/e364 (M+H, 100). Anal. (C16 H14 ClN3 O3 S): C, H, N, S calc. 52.82, 3.88, 11.55, 8.81 found 52.39, 3.98,
11.56, 8.53.
1-Benzyl-3-(5-chloro-3-methyl-2-oxo-3H-benzoxazole-6-yl)thiourea (12d)
Purification by column chromatography on silica gel using ethyl acetate-hexane (50:50) as eluent, yield 10%,
mp: 185 ◦ C. 1 H-NMR (DMSO-d6) δ: 9.34 (s, 1H, -NH), 8.20 (s, 1H, -NH), 7.56-7.25 (m, 7H, H4 , H7 , phenyl
protons), 4.72 (s, 2H, -CH2), 3.34 (s, 3H, -CH3). MS ESI(+) m/e 348 (M+H, 100). Anal. (C16 H14 ClN3 O2 S.
H2 O): C, H, N, S calc. 52.53, 4.41, 11.49, 8.76 found 52.29, 4.14, 11.48, 8.89.
Antimicrobial activity
The in vitro minimum inhibitory concentrations (MICs) of the synthesized compounds were determined using
the 2-fold serial dilution technique in 96-well microtest plates according to the methods recommended by the
Clinical Laboratory Standards Institute (CLSI).28,29 Standard and the isolated strains of bacteria, S. aureus
ATCC 29213, MRSA, E. faecalis ATCC 29212, E. faecalis isolate, E. coli ATCC 35218, E. coli producing
extended spectrum β-lactamase (ESβL), and P. aeruginosa ATCC 27853, and its isolate were used to determine
antibacterial activity. As for antifungal activity, standard strain of Candida albicans ATCC 10231 was used.
Ampicillin, amoxicillin/clavulonic acid, gentamicin, and fluconazole were used as references.
Clinical isolates were obtained from Gazi University Hospital, Microbiology Laboratory.
Standard powders of ampicillin (Mustafa Nevzat Pharma), amoxicillin/clavulonic acid (Deva Pharma),
gentamicin sulfate (Paninkret Chem. Pharm.), and fluconazole (Sigma) were dissolved in appropriate solvents
recommended by CLSI guidelines.28,29 Stock solutions of the tested compounds were dissolved in DMSO. Stock
solutions of the tested compounds and reference drugs were diluted 2-fold in microplate wells. All solvents and
diluents, pure microorganisms, and pure media were used in the control wells.
Bacteria were subcultured in Mueller Hinton Agar (MHA) (Merck) plates and incubated overnight at 37
◦ C and Candida was subcultured in Sabouraud Dextrose Agar (SDA; Merck) plates at 35 ◦ C for 24-48 h.
28
Bacterial susceptibility testing was performed according to the guidelines of CLSI M100-S18.
Mueller
Hinton Broth (MHB; Merck) was added to each microplate well. Then the tested compounds and reference
drugs were added to this medium in wells by 2-fold serial dilution to obtain the required concentrations of 512,
285

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Synthesis of some new urea and thiourea derivatives and..., Y. GULKOK, et al.
256, 128. . . . . . . . . .0.5 μg/mL. The bacterial suspensions used for inoculation were prepared at 105 CFU/mL by
diluting fresh cultures at McFarland 0.5 density (107 CFU/mL). Suspensions of the bacteria at 105 CFU/mL
concentrations were inoculated with a 2-fold diluted solution of the compounds. A 10 μL bacteria inoculum
was added to each microplate well. There were 104 CFU/mL bacteria in the wells after inoculations.
Fungal susceptibility testing was performed according to the guidelines of CLSI M27-A.29 Roswell Park
Memorial Institute (RPMI)-1640 medium with L-glutamine (Sigma) buffered to pH 7 with 3-(N-morpholino)pro-
panesulfonic acid (MOPS) (Sigma) was added to each microplate well. Then the tested compounds and reference
drugs were added to this medium in wells by 2-fold serial dilution to obtain the required concentrations of 512,
256, 128. . . 0.125 μg/mL. The yeast suspensions used for inoculation were prepared at 104 CFU/mL by diluting
fresh cultures at McFarland 0.5 density (106 CFU/mL). Suspensions of the yeast at 104 CFU/mL concentrations
were inoculated to the 2-fold-diluted solution of the compounds. A 10 μL yeast inoculum was added to each
well of the microplates. There were 103 CFU/mL yeast in the wells after inoculations.
Microplates were incubated at 37 ◦ C for 24 h for antibacterial activity and at 37 ◦ C for 48 h for antifungal
activity. After incubation, the lowest concentration of the compounds that completely inhibits macroscopic
growth was determined and reported as MICs. All the experiments were done in 3 parallel series.
Results and discussion
Chemistry
The new compounds (9a-e, 10a-d, 11a-e, and 12a-d) were synthesized by a series of reactions as shown in the
Scheme. 2(3H)-Benzoxazolone 1,24 which was prepared by the solvent-free reaction of urea and o-aminophenol
under microwave irradiation, and commercially available 5-chloro-2(3H)-benzoxazolone 2 were used as starting
compounds. The treatment of compounds 1 and 2 with dimethyl sulfate yielded 5-nonsubstituted/chloro-3-
methyl-2(3H)-benzoxazolones, 324 and 4,25 respectively. The reaction of compounds 3 and 4 with nitric acid
yielded 5-nonsubstituted/chloro-3-methyl-6-nitro-2(3H)-benzoxazolones, 526 and 6,27 respectively. Then the
reduction of the nitro derivatives with SnCl2 gave the corresponding amine derivatives, compounds 726 and 8,27
respectively. Finally, 3-methyl-2(3H)-benzoxazolone ring-containing urea 9a-e and thiourea 11a-e derivatives
were readily prepared in very high yields by the reaction of 6-amino-3-methyl-2(3H)-benzoxazolone (7) with the
appropriate isocyanates and isothiocyanates, respectively. However, 5-chloro-3-methyl-2(3H)-benzoxazolone
ring-containing urea 10a-d and thiourea 12a-d derivatives were prepared with 3 different methods in very low
yields by the reaction of 6-amino-5-chloro-3-methyl-2(3H)-benzoxazolone (8) with the appropriate isocyanates
and isothiocyanates, respectively.
The chemical structures of the newly synthesized compounds were elucidated by 1 H-NMR, mass, and
elemental analysis. The 1 H-NMR, mass spectra, and elemental analysis data of the compounds are in agreement
with the proposed structures.
In the 1 H-NMR spectra, the N-H protons of the urea (9a-e and 10a-d) and thiourea (11a-e and 12a-d)
derivatives were observed as singlets at 9.47-6.12 ppm and 9.92-7.58 ppm, respectively, which can exchange with
D2 O. All other aromatic protons were observed in the expected regions. However, in the 1 H-NMR spectra in
DMSO-d6 at room temperature of 3-methyl-2(3H)-benzoxazolone ring-containing compounds 9a, 9b, 9c, 11a,
286

¨
Synthesis of some new urea and thiourea derivatives and..., Y. GULKOK, et al.
and 11c, the splitting of H7 , H5 , and H4 protons of 2(3H)-benzoxazolone was not seen as expected. That is,
H7 proton was seen as a singlet instead of a doublet and H5 proton was observed as a singlet together with H4
proton instead of a doublet of doublet. Therefore, compound 9b was selected for explaining this situation. In
the 1 H-NMR spectrum in DMSO-d6 at 55 ◦ C of this compound, H7 proton was seen as a doublet at 7.57 ppm.
H5 and H4 protons were seen as a multiplet at 7.14 ppm. In the 1 H-NMR spectrum in acetone-d6 at room
temperature of this compound, the splitting of H7 , H5 , and H4 protons was observed as expected. That is, H7 ,
H5 , and H4 protons were seen as a doublet at 7.68 ppm, a doublet of doublet at 7.21 ppm, and a doublet at
7.08 ppm, respectively. This may be attributed to the retention of relaxation time in DMSO-d6 . In addition, 4
H
N
H
N
Cl
OH
H2N
H2N
MW
+
O
O
C
O
O
O
NH2
(CH3)2SO4
NaOH/H2O
1
2
CH3
N
CH3
R
R
N
O
O
HNO3
O
O
O2N
5, 6
3, 4
SnCl2
CH3
N
CH3
N
R
R
R'
(CH2)n N=C=X
X
O
O
(CH2)n
O
O
N
N
H
H2N
H
R'
7, 8
9, 10, 11, 12
Comp.
Comp.
n
R'
R'
n
X
X
R
R
9a
9b
0
0
0
1
2
0
0
0
11a
11b
11c
11d
11e
12a
12b
12c
12d
0
0
0
1
2
0
0
0
-H
O
O
O
O
O
O
O
O
O
-H
S
S
S
S
S
S
S
S
S
-H
-H
-H
-H
-H
-Cl
-Cl
-Cl
-Cl
-H
-H
-H
-H
-H
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
9c
-OCH3
-OCH3
9d
9e
10a
10b
10c
10d
-H
-H
-H
-H
-H
-Cl
-OCH3
-H
-H
-Cl
-OCH3
-H
1
1
Scheme. Synthetic route of the title compounds.
287

¨
Synthesis of some new urea and thiourea derivatives and..., Y. GULKOK, et al.
of the synthesized compounds (9b, 10b, 11b, 12b) were selected for structure elucidation by 13 C-NMR. The
13 C-NMR spectral data of the compounds are in agreement with the proposed structures.
Antimicrobial activity
As shown in the Table, the synthesized compounds were tested for their antibacterial activity against 4
gram-positive bacteria (Staphylococcus aureus, methicillin-resistant S. aureus (MRSA, isolate), Enterococcus
faecalis, E. faecalis isolate), 4 gram-negative bacteria (Escherichia coli, E. coli producing extended spectrum
β-lactamase, Pseudomonas aeruginosa, P. aeruginosa isolate), and for their antifungal activity against Candida
albicans using microdilution. Ampicillin, amoxicillin/clavulonic acid, gentamicin, and fluconazole were used as
references.
Regarding the antibacterial activity of the target compounds against gram-positive bacteria, the com-
pounds had no significant activity against S. aureus, its isolate, or E. faecalis isolate. However, compounds
10a and 10b, urea derivatives, showed the best inhibitory activity against E. faecalis with a MIC value of 16
μg/mL. The higher activity of these compounds might be attributed to chloro substituent at the 5-position
of 3-methyl-2(3H)-benzoxazolone ring. The rest of the compounds showed activity against E. faecalis, with
MIC values ranging from 32 to 256 μg/mL. In terms of the antibacterial activity of the target compounds
against gram-negative bacteria, the compounds had no significant activity against P. aeruginosa, its isolate,
or E. coli isolate. However, compounds 9a, 9b, 9e, 10a, and 10c, urea derivatives, and compounds 12a and
12d, thiourea derivatives, exhibited a relatively good inhibitory profile against E. coli, with a MIC value of
32 μg/mL when compared with the other compounds. In other words, their antibacterial activity was 25% of
that of amoxicillin/clavulonic acid against E. coli. These compounds may be modified for further studies with
the hope of obtaining better antibacterial agents against E. coli. The rest of the compounds showed activity
against E. coli, with MIC values ranging from 64 to 128 μg/mL.
As for antifungal activity of the synthesized derivatives, compounds 9b, 9e, 10b, and 10d, urea
derivatives, and compounds 11a, 11e, 12a and 12d, thiourea derivatives, displayed activity against C. albicans
with a MIC value of 64 μg/mL.
As regards the relationships between the structure and the detected antibacterial activity, one might say
that gram-negative E. coli and gram-positive E. faecalis were generally more sensitive to the title compounds
in comparison with the other used Gram bacteria. When the chemical structures of the active compounds were
taken into consideration, one can say that the urea derivatives were generally more active than the thiourea
derivatives against E. faecalis and E. coli and that chloro substitution at the 5-position of the 3-methyl-2(3H)-
benzoxazolone ring did not cause any remarkable change in antibacterial activity of the target compounds,
except for 10a and 10b. In addition, one can say that structural differences do not play a critical role in
antifungal activity.
288

¨
Synthesis of some new urea and thiourea derivatives and..., Y. GULKOK, et al.
Table. Antimicrobial activity of the synthesized compounds (MICs, μg/mL).
Compounds
512
256
128
128
32
64
256
256
32
32
256
256
128
128
128
128
128
64
9a
9b
512
512
256
128
128
128
32
64
64
256
256
256
64
64
32
256
256
256
128
128
128
128
128
128
128
128
64
9c
9d
9e
256
256
128
128
16
16
256
256
32
256
256
128
128
128
128
128
64
10a
10b
128
512
256
256
256
256
256
256
256
128
128
128
128
128
128
128
128
64
64
256
256
256
256
256
256
256
256
32
64
256
256
256
256
256
256
256
256
256
256
128
128
128
128
128
128
256
128
128
128
128
128
128
128
256
64
10c
10d
11a
11b
11c
11d
11e
12a
128
32
128
64
64
128
128
128
64
128
128
64
64
128
64
256
32
64
512
512
128
128
128
256
256
256
64
256
256
128
128
256
256
256
128
12b
12c
128
256
0.5
0.5
128
-
128
2
256
0.5
32
32
-
256
1024
1024
128
128
-
64
-
12d
Ampicillin
-
Gentamicin
Amoxicillin:
16
16
-
1
256
-
0.125
-
8
-
0.25
-
0.5
-
8
-
4
-
-
-
-
-
-
Clavulanic Acid (2:1)
Fluconazole
1
289

¨
Synthesis of some new urea and thiourea derivatives and..., Y. GULKOK, et al.
Acknowledgments
This study was supported financially with a grant from the Research Foundation of Gazi University (EF 02/2009-
24).
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291

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