Unsymmetrical 1,3-disubstituted urea derivatives as α-chymotrypsin inhibitors

Article abstract of DOI:10.1007/s00044-014-0930-3

The objective of this study was to synthesize potent and/or novel inhibitors for α-chymotrypsin activity. Eighteen derivatives of N-methylphenyl-N′-(alkyl/aryl) urea (1-18) were synthesized, and their inhibitory effects on α-chymotrypsin enzyme were evalu

Full text of DOI:10.1007/s00044-014-0930-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0930-3
ORIGINAL RESEARCH
Unsymmetrical 1,3-disubstituted urea derivatives
as a-chymotrypsin inhibitors
•
Shahnaz Perveen Sana Mustafa Mehreen Latif
•
•
•
•
•
Lubna Iqbal Tanzil H. Usmani Khalid Mohammed Khan
Wolfgang Voelter
Received: 22 January 2013 / Accepted: 27 January 2014
Ó Springer Science+Business Media New York 2014
Abstract The objective of this study was to synthesize
potent and/or novel inhibitors for a-chymotrypsin activity.
Eighteen derivatives of N-methylphenyl-N0-(alkyl/aryl) urea
(1–18) were synthesized, and their inhibitory effects on
a-chymotrypsin enzyme were evaluated. Two compounds
exhibited potent inhibitory activities. The most potent,
N-(2-methylphenyl)-2-oxo-1-pyrrolidinecarboxamide (15)
having a methyl group at ortho position was the most active
inhibitor with an IC50 value of 8.10 0.14 lM, which was
comparable to standard chymostatin (IC50 = 8.24
0.11 lM). A slightly less potent, N-(2-acetylphenyl)-N0-(3-
methylphenyl) urea (10), exhibited an IC50 of 13.6
0.23 lM. Compounds 3, 4, 7, 11, and 13 exhibited moderate
activities. The results demonstrated that a-chymotrypsin
inhibition is related to the position of the methyl group and
the presence of substituent at the nitrogen of the urea bridge.
The inhibitory trend suggests that a-chymotrypsin inhibitory
activity declines with ortho [ meta [ para substitution
order. In conclusion, our data suggest that the compound 15
may serve as a lead compound for further designing of other
potent or novel a-chymotrypsin inhibitors.
Keywords Amines ꢀ a-Chymotrypsin ꢀ Tolyl isocyanate ꢀ
Unsymmetrical urea derivatives
Introduction
Urea is a class of compounds constituted of a carbonyl
group flanked by two amino residues (Finar, 2003).
Substituted urea derivatives have been the subject of con-
siderable attention due to their applications, i.e., dyeing of
hair, cellulose fibers, antioxidants in gasoline for automo-
biles, additives in detergents to prevent carbon deposits,
inhibit corrosion, and intermediates for the production of
carbamates, which represent raw materials for agrochemi-
cals (Katritzky et al., 1997). The N,N0-disubstituted sulfo-
nylurea and sulfonylthiourea pharmacophores have shown
antitumor activity in cell cultures (Sherif, 2006). Deriva-
tives of urea that contain piperazine rings and additional
substituted phenyl residues were effective in blocking
calcium ion channel activity (Hossein et al., 2010). Sub-
stantial phenylurea derivatives have been used for con-
trolling plant growth (Fischer et al., 1972). N,N0-
Diphenylurea was shown to have cytokinin activity (Bruce
and Zwar, 1966). Urea derivatives are useful as active
ingredients for antimicrobial, and algaecides or antiperi-
phytic agents for industrial use (Igarashi et al., 1998).
Compounds having a diphenylurea moiety have been
described for their serotonergic antagonistic properties and
as Raf kinases inhibitors (Gilbert et al., 2006). Several
unsymmetrical urea derivatives are also known as active
pharmaceuticals, herbicides (Groszek, 2002; Yonova and
Stoilkova, 2005), pesticides, and insecticides (Akira et al.,
S. Perveen (&) ꢀ M. Latif ꢀ L. Iqbal ꢀ T. H. Usmani
PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr.
Salimuzzaman Siddiqui, Karachi 75280, Pakistan
e-mail: dr_shahnaz_perveen@yahoo.com
S. Mustafa
Department of Chemistry, University of Karachi, Karachi 75270,
Pakistan
K. M. Khan
International Center for Chemical and Biological Sciences,
H. E. J. Research Institute of Chemistry, University of Karachi,
Karachi 75270, Pakistan
W. Voelter
¨
¨
¨
¨
Interfakultares Institut fur Biochemie der Universitat Tubingen,
Hoppe-Seyler-Straße 4, 72076 Tu¨bingen, Germany
e-mail: wolfgang.voelter@uni-tuebingen.de
123

Med Chem Res
Table 1 Results of synthesized unsymmetrical 1,3-disubstituted urea derivatives 1–18
Compound
CH3
R
R0
Compound
CH3
R
R0
O
1
m-CH3
–
10
m-CH3
H
N
O
CH3
CH3
2
3
4
m-CH3
m-CH3
m-CH3
H
H
H
11
12
13
p-CH3
o-CH3
o-CH3
H
H
H
N
S
CH3
CH3
CH3
CH3
CH
CH3
CH3
CH3
CH
OCH3
5
6
p-CH3
p-CH3
–
14
15
o-CH3
o-CH3
H
–
N
O
OCH3
CH3
H
N
CH3
O
CH3
CH3
CH
7
8
9
p-CH3
p-CH3
m-CH3
H
H
H
16
17
18
o-CH3
p-CH3
o-CH3
H
H
H
N
S
O
CH3
OCH3
O
N
S
CH3
1988). These observations clearly indicate the versatile use
of urea derivatives.
Recently, we have reported urea and its derivatives for
inhibition of urease, and a-chymotrypsin activities (Perv-
een et al., 2008; Perveen, 2008, 2010, 2011). The objective
of present investigation was to synthesize novel urea
derivatives for a-chymotrypsin inhibitory activities. We
synthesized 1,3-disubstituted urea derivatives as an novel
inhibitors for a-chymotrypsin activity. Evaluation of these
synthetic compounds identified two potent inhibitors
against a-chymotrypsin, which may have therapeutic
potential in the future.
Previous studies have shown that urea derivatives can be
used as effective inhibitors for enzymatic activities. The sym-
metric urea derivative N,N0-bis(4-nitrophenyl) urea strongly
inhibited urease (IC50 = 1.25 lM) and a-chymotrypsin
(IC50 = 3.15 lM) activities (Perveen et al., 2008). Urea and its
derivatives are also reported as components for HIV protease
inhibitors, and CCK-b receptor and endothelin antagonists
(Lam et al., 1994; Castro et al., 1996; Perveen et al., 2008;
Patick and Potts, 1998; Perveen, 2008, 2010, 2011).
Serine proteases (i.e., elastase, collagenase, cathepsin G,
and chymotrypsin) play an important role in extracellular
matrix degradation, and digesting fibrous proteins such as
elastin and collagen (Berquin and Sloane, 1996). Further-
more, serine protease inhibitors are also involved in phago-
cytosis, coagulation, complement activation, fibrinolysis,
and blood pressure regulation. A selective inhibition of
serine protease, therefore, could potentially be used to reg-
ulate several cellular processes.
Results and discussion
Chemistry
In a typical reaction, ortho, meta, and para tolyl isocya-
nates were treated with different amines in the presence of
1,4-dioxane at room temperature. The progress of reaction
was monitored by means of TLC. After completion of the
123

Med Chem Res
reaction, as shown by TLC, the reaction mixture was
poured into ice cold water with continuous stirring. The
solid material obtained was filtered, and after purification,
the desired product was obtained (Table 1). The structures
of the synthetic derivatives were confirmed by mass, NMR,
and IR spectroscopy, and the melting points were
determined.
methylphenyl) urea (8). These results demonstrated that
meta-substitution of a phenyl group at the nitrogen of urea
bridge exerted the most potent a-chymotrypsin inhibitory
activity. Furthermore, ortho-, meta-, and para- substitution
of a mesityl group at the nitrogen of urea bridge resulted in
ineffective inhibition of a-chymotrypsin activity.
In case of urea derivatives bearing a 1,3-benzothiazol-2-
yl group at the nitrogen of the urea bridge, it is noted that
its para-substituted N-(1,3-benzothiazol-2-yl)-N0-(4-
methylphenyl) urea (11) derivative substantially inhibited
a-chymotrypsin activity (IC50 = 20.6 0.06 lM). Due to
the presences of the bulky 1,3-benzothiazol-2-yl group at
one of the nitrogen of the urea bridge, the activity enhances
by the reduction of the steric hindrance in the order
ortho [ meta [ para. This suggests that the presence of
various substituted groups and the presences of a methyl
group either at ortho, meta, or para position of the phenyl
a-Chymotrypsin inhibitory activities
Activities of a-chymotrypsin were determined using an
enzymatic assay as described previously. Chymostatin was
used as standard inhibitor to evaluate the activity of the
synthesized urea derivatives in search of new a-chymo-
trypsin inhibitors. The IC50 values of the synthesized o-,
m-, p-methylphenyl urea derivatives (3, 4, 7, 10, 11, 13,
and 15) were found in the range of 8.10–27.41 lM against
a-chymotrypsin (Table 2; Fig. 1).
The most potent inhibitor of the series was N-(2-methyl-
phenyl)-2-oxo-1-pyrrolidinecarboxamide (15) with an
IC50 = 8.10 0.14 lM, which is compared to the standard
chymostatin (IC50 = 8.24 0.11 lM). While comparing its
activity with the meta N-(3-methylphenyl)-2-oxo-1-pyrroli-
dinecarboxamide (1) and para N-(4-methylphenyl)-2-oxo-1-
pyrrolidinecarboxamide (5) analogs, the SAR (Structure
Activity Relation) showed interesting results that both, meta,
and para compounds have insignificant a-chymotrypsin
inhibition. Similarly, substitution of 1-phenylethyl group at
the nitrogen of the urea bridge and a methyl substitution at
ortho position of the phenyl ring, as in N-(2-methylphenyl)-
N0-(1-phenylethyl) urea (13) (IC50 19.0 0.08 lM), resulted
in improved inhibition of a-chymotrypsin as compared to its
Table 2 a-Chymotrypsin activity of unsymmetrical 1,3-disubstituted
urea derivatives 1–18
Compound
IC50 (lM) SD Compound IC50 (lM) SD
1
2
3
4
5
6
7
8
9
NS
10
11
12
13
14
15
16
17
18
13.6 0.23
20.6 0.06
NS
NS
24.02 0.12
22.6 0.08
19.0 0.08
NS
NS
NS
8.10 0.14
NS
27.41 0.26
NS
NS
NS
NS
Chymostatin** 8.24 0.11 lM
meta-substituted
[N-(3-methylphenyl)-N0-(1-phenylethyl)
NS not significant, SD standard deviation
Chymostatin** = Standard
urea(3) (IC50 24.02 0.12 lM)] and para-substituted[N-(4-
methylphenyl)-N0-(1-phenylethyl)urea (7) (IC50 27.41
0.26 lM)] analogs. By comparing the a-chymotrypsin inhi-
bition activities of compounds 1, 3, 5, 7, 13, and 15, a decline
was observed in reducing the steric hindrance of the com-
pounds in the order of ortho[ meta[ para substitution.
Contrary, when 2-acetylphenyl group is attached to the
nitrogen of the urea bridge, the meta-substituted N-(2-acet-
ylphenyl)-N0-(3-methylphenyl)urea (10) significantly inhib-
ited the a-chymotrypsin activity (IC50 13.6 0.23 lM),
whereas, its ortho-substituted N-(2-acetylphenyl)-N0-(2-
methylphenyl) urea (18) and para-substituted N-(2-acetyl-
phenyl)-N0-(4-methylphenyl) urea (17) were ineffective on
a-chymotrypsin activity. Similarly, when a 3-methoxy-
phenyl group was present at the nitrogen of the urea bridge,
the meta-substituted N-(3-methoxyphenyl)-N0-(3-methyl-
phenyl)urea (4) with an IC50 value of 22.6 0.08 lM sig-
nificantly inhibited a-chymotrypsin activity as compared to its
ortho-substituted N-(3-methoxyphenyl)-N0-(2-methylphenyl)
urea (14) and para-substituted N-(3-methoxyphenyl)-N0-(4-
Fig. 1 a-Chymotrypsin activity of compounds 1–18
123

Med Chem Res
ring play an important role in inhibiting a-chymotrypsin
activity (Chart 1).
Conclusion
Chymotrypsins contain serine 195, histidine 57 and
aspartic acid 102 at the active sites, which are directly
involved in the enzymatic reaction. The serine 195 residue
can potentially form a covalent bond with the substrate by a
strong nucleophilic attack of serine on the carbonyl group of
urea or its derivatives (substrate) resulting in an enzyme-
substrate intermediate, resulting ultimately in the hydrolysis
of peptide bonds in a-chymotrypsin causing its inactivation.
In conclusion, compounds 3, 4, 7, 10, 11, 13, and 15 signifi-
cantly inhibited a-chymotrypsin activity with IC50 values of
24.02 0.12, 22.6 0.08, 27.41 0.26, 13.6 0.23,
20.6 0.06, 19.0 0.08, and 8.10 0.14 lM, respec-
tively. The activity of all these compounds depends upon the
position of the methyl group at the phenyl ring and the pre-
sence of substituents at the nitrogen of the urea bridge.
Compound 15 with a methyl group at ortho position of the
Chart 1 Structures of active compounds
123

Med Chem Res
phenyl ring and an electron withdrawing group (Derek et al.,
2003) pyrrolidine moiety attached to the nitrogen of the urea
bridgewasfound to bethe mostactive compoundofthe series.
This compound may serve as a lead compound for further
designing of other potent or novel a-chymotrypsin inhibitors
in the future.
enzyme reaction. The absorbance of released p-nitroaniline
was continuously monitored at 410 nm until a considerable
color change was achieved. The final DMSO concentration
in the reaction mixture was 7 %. Best inhibition was found at
concentration 3.02 9 10-5 g/100 lL.
The percentage (%) inhibition was calculated as follows:
ðE ꢁ SÞ=E ꢂ 100Þ
where E is the activity of the enzyme without test com-
pound, and S is the activity of enzyme in the presence of
the test compound. The concentrations of test compounds
that inhibited the hydrolysis of substrate up to 50 % (IC50)
were determined by monitoring the effect of various con-
centrations of these compounds in the assays. The IC50
values were then calculated using the EZ-Fit Enzyme
Kinetics program (Version Perrella Scientific Inc.,
Amherst, USA).
Experimental
Melting points of all compounds were taken on a Gal-
lenkamp melting point apparatus. Thin layer chromatog-
raphy was performed on pre-coated silica gel plates
(Kieselgel 60 F254, E. Merck, Germany), and chromato-
grams were visualized under UV (Dual range, UK, Ger-
many) at 254 and 365 nm. IR spectra were taken on a
Thermo Nicolet Avatar 370 DTGS FT-IR Spectrometer. EI
MS were performed using a MAT-312 and a JEOLJMS-
General procedure for the preparation of N,N0-(1,3-
1
HX 110 instrument. H NMR spectra were recorded using
disubstituted)urea
a Bruker AVANCE 400, 500 MHz instrument, d in ppm
are related to SiMe4 (0 ppm) as internal standard. Ele-
mental analyses were carried out on a Perkin Elmer 2400
CHN elemental analyzer. All solvents used were of reagent
grade. Bioscreening and syntheses of compounds were
carried out in Pharmaceutical Research Centre, PCSIR
Laboratories Complex, Karachi-75280, Pakistan.
For the synthesis of unsymmetrical 1,3-disubstituted urea,
3.18 mM of primary/secondary amine was taken in a
round-bottomed flask containing 5–10 mL of 1,4-dioxane,
and 1.55–1.61 mM tolyl isocyanate was added drop wise at
30–40 °C with continuous stirring. The reaction was
completed in 3 h. The reaction mixture was then poured
into ice cold water with continuous stirring. The solids
obtained were filtered and washed several times with dis-
tilled water and purified by means of crystallization with
ethanol to obtain the desired products as described (Khan
et al., 2009) (Scheme 1).
Materials and methods
In vitro a-chymotrypsin assay
The a-chymotrypsin inhibitory activity was performed using
a previously described method (Cannell et al., 1988). Briefly,
a-chymotrypsin (9 units/mL in 50 mM Tris–HCl buffer, pH
7.6; Sigma Chemical Co. USA) was pre-incubated with the
test compounds at the concentration varying from 5 to
500 lM for 20 min at 25 °C. 100 lL of substrate solution
(N-succinyl-phenylalanine-p-nitroanilide, 0.01–0.06 mM in
50 mM Tris–HCl buffer, pH 7.6) was added to start the
N-(3-Methylphenyl)-2-oxo-1-pyrrolidinecarboxamide (1) (Yield
68 %). Rf = 0.68 (CH2Cl2/CH3OH, 9:1). mp 229 °C. IR
(solid): mmax: 3,233, 1,638, 1,552, 1,491, 1,290, 1,233,
1
882 cm-1. H NMR (400 MHz, DMSO) d; 8.56 (1H, br.s,
NH), 7.28 (1H, br.s, H2), 7.19 (1H, d, J6,5 = 7.2 Hz, H6),
7.13 (1H, t, J5,(4,6) = 7.2 Hz, H5), 6.76 (1H, d,
J4,5 = 7.2 Hz, H4), 3.28 (2H, br.s, CH2), 2.49 (4H, br.s,
2CH2), 2.26 (3H, s, CH3)—EI MS: m/z (rel. abund. %), 218
Scheme 1 Synthetic route for the synthesis of unsymmetrical 1,3-disubstituted urea derivatives
123

Med Chem Res
(M?, 2), 161 (4), 133 (8), 134 (4), 107 (100), 106 (38), 91
(10), 85 (22), 65 (11)—Anal.: Calcd for C12H14N2O2:
(218): C, 66.11; H, 6.47; N, 12.85 %. Found: C, 66.10; H,
6.43; N, 12.81 %.
56 (18)—Anal.: Calcd for C12H14N2O2: (218): C, 66.11; H,
6.47; N, 12.85 %. Found: C, 66.10; H, 6.43; N, 12.81 %.
N-Mesityl-N0-(4-methylphenyl)urea (6) (Yield 69 %).
Rf = 0.55 (CH2Cl2/CH6H14, 7:3). mp 270 °C. IR (solid):
m
max: 3,293, 1,646, 1,601, 1,560, 1,433, 1,290, 1,041 cm-1
.
N-Mesityl-N0-(3-methylphenyl)urea (2) (Yield 69 %).
Rf = 0.66 (CH2Cl2/C12H14, 7:3). mp 271 °C. IR (solid):
1H NMR (400 MHz, DMSO) d; 7.57 (1H, br.s, NH), 7.30
0
0
(2H, d, J2 ,3 =6 ,5 =0 80.0 Hz, H2 ,6 ), 7.03 (2H, d,
0
0
0
0
max: 3,293, 1,646, 1,601, 1,560, 1,433, 1,290, 1,041 cm-1
.
J3 ,2 =5 ,6 = 8.0 Hz, H3 ,5 ), 6.85 (2H, br.s, H3,5), 4.76 (1H,
br.s, NH), 2.28 (6H, s, 2CH3), 2.21 (3H, s, CH3), 2.14 (3H,
s, CH3)—EI MS: m/z (rel. abund. %), 268 (M?, 12), 146
(1), 135 (55), 107 (100), 91 (20), 65 (9)—Anal.: Calcd for
C17H20N2O: (268): C, 76.18; H, 7.52; N, 10.45 %. Found:
C, 76.15; H, 7.51; N, 10.47 %.
m
0
0
0
0
1H NMR (400 MHz, DMSO) d; 8.26 (2H, br.s, NH), 7.31
0
(2H, d, J3,5=5,3 = 4.8 Hz,0 H3,5), 7.19 (1H, s, H2 ), 7.07 (1H,
t, J5 ,(4 ,6 ) = 7.2 Hz, H5 ), 6.68 (1H, d, J4 ,5 = 7.2 Hz,
0
0
0
0
0
0
0
H4 ), 6.56 (1H, d, J6 ,5 = 7.2 Hz, H6 ), 3.31 (6H, s, 2CH3),
3.28 (3H, s, CH3), 2.21 (3H, s, CH3)—EI MS: m/z (rel.
abund. %), 268 (M?, 21), 161 (6), 135 (85), 107 (100), 91
(26), 65 (11)—Anal.: Calcd for C17H20N2O: (268): C,
76.18; H, 7.52; N, 10.45 %. Found: C, 76.15; H, 7.51; N,
10.47 %.
0
0
N-(4-Methylphenyl)-N0-(1-phenylethyl)urea (7) (Yield 69 %).
Rf = 0.58 (CH2Cl2/CH6H14, 7:3). mp 164 °C. IR (solid):
m
max: 3,293, 1,629, 1,576, 1,323, 1,245, 1,037, 886 cm-1
.
1H NMR (400 MHz, DMSO) d; 8.23 (2H, br.s, NH), 7.30
N-(3-Methylphenyl)-N0-(1-phenylethyl)urea (3) (Yield 69 %).
Rf = 0.73 (CH2Cl2/CH3OH, 9:1). mp 123 °C. IR (solid):
(4H, s, Ar–H), 7.22 (2H, d, J2,3=6,5 = 8.0 Hz, H2,6), 6.99
(2H, d, J3,2=5,6 = 8.0 Hz, H3,5), 6.53 (1H, d,
0
max: 3,317, 1,634, 1,560, 1,491, 1,442, 1,233, 882 cm-1
.
J2 ,3 = 7.6 Hz, H2 ), 4.78 (1H, qin, J1 ,(2 , NH) = 7.2 Hz,
m
0
0
00
00
1H NMR (400 MHz, DMSO) d; 7.56 (2H, br.s, NH), 7.26
(1H, br.s, H2), 7.19 (1H, br.d, J6,5 = 8.0 Hz, H6), 7.10 (1H,
t, J5,(4,6) = 8.0 Hz, H5), 6.86 (5H, br.s, Ar–H), 6.72 (1H,
br.d, J4,5 = 8.0 Hz, H4), 2.23 (1H, br.s, CH), 2.21 (3H,
br.s, CH3), 2.14 (3H, s, CH3)—EI MS: m/z (rel. abund. %),
254 (M?, 10), 196 (2), 120 (3), 107 (100), 77 (19), 51 (5)—
Anal.: Calcd for C16H18N2O: (254): C, 75.65; H, 7.14; N,
11.02 %. Found: C, 75.67; H, 7.13; N, 11.04 %.
00 00
CH), 2.19 (3H, s, CH3), 1.36 (3H, d, J2 ,1 = 7.2 Hz,
CH3)—EI MS: m/z (rel. abund. %), 254 (M?, 8), 149 (1),
132 (2), 107 (100), 79 (31), 51 (5)—Anal.: Calcd for
C16H18N2O: (254): C, 75.65; H, 7.14; N, 11.02 %. Found:
C, 75.67; H, 7.13; N, 11.04 %.
N-(3-Methoxyphenyl)-N0-(4-methylphenyl)urea (8) (Yield
°
88 %). Rf = 0.91 (CH2Cl2/CH3OH, 9:1). mp 183 C. IR
(solid): mmax: 3,293, 2,345, 1,638, 1,564, 1,491, 1,413,
N-(3-Methoxyphenyl)-N0-(3-methylphenyl)urea (4) (Yield
88 %). Rf = 0.80 (CH2Cl2/CH3OH, 9:1). mp 202 °C. IR
(solid): mmax: 3,284, 2,913, 1,629, 1,560, 1,294, 1,229,
1,274, 1,188, 1,037, 845 cm-1 1H NMR (400 MHz,
.
DMSO) d; 8.60 (1H, br.s, NH),0 8.51 (1H, br.s, NH), 7.31
0
(2H, d, J2 ,3 =6 ,5 = 8.0 Hz, H2 ,6 ), 7.16 (1H, br.s, H20 ), 7.14
0
0
0
0
0
1
845 cm-1. H NMR (400 MHz, DMSO) d; 8.63 (1H, br.s,
(1H, m, H5), 7.06 (1H, d, J3 ,2 =5 ,6 = 8.0 Hz, H3 ,5 ), 6.89
(1H, d, J4,5 = 8.0 Hz, H4), 6.52 (1H, d, J6,5 = 8.0 Hz, H6),
3.71 (3H, s, OCH3), 2.23 (3H, s, CH3)—EI MS: m/z (rel.
abund. %), 256 (M?, 15), 257 (M?1, 1), 149 (5), 123 (44),
107 (100), 79 (27), 65 (17)—Anal.: Calcd for C15H16N2O2:
(256): C, 70.37; H, 6.30; N, 10.94 %. Found: C, 70.35; H,
6.31; N, 10.95 %.
0
0
0
0
NH), 8.54 (1H, br.s, NH), 7.28 (1H, br.s, H2), 7.18 (2H, m,
0
0
H2 ,6), 7.15 (2H, t, J5 ,(4 ,6 )=5,(4,6) = 8.0 Hz, H5 ,5), 6.90
0
0
0
0
(1H, dd, J6 ,5 = 8.0 Hz, J6 ,2 = 1.6 Hz, H6 ), 6.77 (1H, d,
0
0
0
0
J4,5 = 8.0 Hz, H40), 6.53 (1H, dd, J4 ,5 = 8.0 Hz,
0
0
J4 ,6 = 2.4 Hz, H4 ), 3.71 (3H, s, OCH3), 2.26 (3H, s,
CH3)—EI MS: m/z (rel. abund. %), 256 (M?, 22), 257
(M?1, 5), 149 (4), 123 (65), 107 (100), 94 (18), 65 (10)—
Anal.: Calcd for C15H16N2O2: (256): C, 70.37; H, 6.30; N,
10.94 %. Found: C, 70.35; H, 6.31; N, 10.95 %.
0
0
N-(1,3-Benzothiazol-2-yl)-N0-(3-methylphenyl)urea (9) (Yield
88 %). Rf = 0.57 (CH2Cl2/CH3OH, 8:2). mp 319 °C; IR
(solid): mmax: 3,396, 3,256, 1,695, 1,634, 1,568, 1,499,
1,278, 1,213, 1,037, 826 cm-1 1H NMR (400 MHz,
.
N-(4-Methylphenyl)-2-oxo-1-pyrrolidinecarboxamide (5) (Yield
68 %). Rf = 0.81 (CH2Cl2/CH3OH, 9:1). mp 263 °C; IR
(solid): mmax: 3,309, 2,908, 1,634, 1,601, 1,560, 1,527,
DMSO) d; 9.07 (1H, br.s,0 NH), 8.81 (1H, br.s, NH), 8.13
(1H, d, J6 ,5 = 8.0 Hz, H6 ), 7.90 (1H, br.s, H5), 7.81 (1H,
0
0
0
0
1,413, 1,311, 1,298, 816 cm-1 1H NMR (300 MHz,
.
d, J4 ,5 = 8.0 Hz, H4 ), 7.65 (1H, br.s, H2 ), 7.38 (1H, d,
J2,3 = 7.6 Hz, H2), 7.25 (1H, t, J3,(2,4) = 7.6 Hz, H3), 7.20
0
0
CDCl3) d; 7.18 (2H, d, J2,3=6,5 = 8.1 Hz, H2,6), 7.08 (2H,
d, J3,2=5,6 = 8.1 Hz, H3,5), 6.32 (1H, br.s, NH), 4.19 (2H,
br.s, CH2), 2.29 (2H, d, J = 7.8 Hz, CH2), 1.77 (2H, br.s,
CH2), 1.52 (3H, s, CH3)—EI MS: m/z (rel. abund. %), 218
(M?, 21), 167 (1), 149 (10), 133 (100), 107 (15), 86 (39),
(1H, t, J4,(3,5) = 7.6 Hz, H4), 6.85 (1H, t, J5 ,(4 ,6 )
0
0
0
=
0
8.0 Hz, H5 ), 2.29 (3H, s, CH3)—EI MS: m/z (rel. abund.
%), 283 (M?, 4), 240 (1), 176 (20), 150 (100), 123 (13),
107 (46), 77 (14), 69 (10)—Anal.: Calcd for C15H13N3OS:
123

Med Chem Res
(283): C, 63.65; H, 4.63; N, 14.84 %. Found: C, 63.67; H,
4.61; N, 14.86 %.
C16H18N2O: (254): C, 75.65; H, 7.14; N, 11.02 %. Found:
C, 75.67; H, 7.13; N, 11.04 %.
N-(2-Acetylphenyl)-N0-(3-methylphenyl)urea (10) (Yield
88 %). Rf = 0.60 (CH2Cl2/CH3OH, 8:2). mp 182 °C; IR
(solid): mmax: 3,297, 1,683, 1,634, 1,568, 1,356, 1,225,
N-(3-Methoxyphenyl)-N0-(2-methylphenyl)urea (14) (Yield
88 %). Rf = 0.84 (CH2Cl2/CH3OH, 9:1). mp 163 °C. IR
(solid): mmax: 3,301, 1,638, 1,601, 1,552, 1,458, 1,294,
1
1
886 cm-1. H NMR (400 MHz, DMSO) d; 8.87 (1H, br.s,
1,160, 1,053 cm-1. H NMR (400 MHz, DMSO) d; 9.00
NH), 8.62 (1H, br.s, NH), 8.06 (1H, br.s, H2), 7.65 (1H, d,
(2H, br.s, NH), 7.88 (1H, s, H2), 7.81 (1H, d,
J4,5 = 8.0 Hz, H4), 7.17 (1H, br.s, H6), 7.15 (1H, t,
0
J4,5 = 8.0 Hz, H4), 7.57 (1H, d, J6 ,5 = 7.6 Hz, H6 ), 7.43
0
0
0
0
(1H, t, J5,(4,6) = 8.0 Hz, H5), 7.31 (1H, br.s, H2 ), 7.21 (1H,
d, J6,5 = 8.0 Hz, H6), 7.16 (1H, t, J5 ,(4 ,6 ) = 7.6 Hz, H5 ),
6.79 (1H, d, J4 ,5 = 7.6 Hz, H4 ), 2.56 (3H, s, OCCH3),
2.27 (3H, s, CH3)—EI MS: m/z (rel. abund. %), 268 (M?,
16), 146 (9), 135 (44), 120 (59), 107 (100), 91 (26), 65
(21)—Anal.: Calcd for C16H16N2O2: (268): C, 71.70; H,
6.01; N, 10.45 %. Found: C, 71.68; H, 6.02; N, 10.42 %.
J5,(4,6) = 8.0 Hz, H5), 7.10 (1H, d, J6 ,5 = 7.6 Hz, H6 ),
0
0
0
0
0
6.93 (2H, t, J4 ,(3 ,5 )=5 ,(4 ,6 ) = 7.6 Hz, H4 ,5 ), 6.53 (1H, d,
0
0
0
0
0
0
0
0
0
0
0
J3 ,4 = 7.6 Hz, H3 ), 3.72 (3H, s, OCH3), 2.22 (3H, s,
CH3)—EI MS: m/z (rel. abund. %), 256 (M?, 42), 257
(M?1, 7), 149 (7), 123 (84), 107 (100), 79 (20), 65 (10)—
Anal.: Calcd for C15H16N2O2: (256): C, 70.37; H, 6.30; N,
10.94 %. Found: C, 70.35; H, 6.31; N, 10.95 %.
0
0
0
0
N-(1,3-Benzothiazol-2-yl)-N0-(4-methylphenyl)urea (11) (Yield
88 %). Rf = 0.58 (CH2Cl2/CH3OH, 8:2). mp 328 °C. IR
(solid): mmax: 3,440, 3,211, 1,691, 1,617, 1,564, 1,519,
N-(2-Methylphenyl)-2-oxo-1-pyrrolidinecarboxamide (15) (Yield
68 %). Rf = 0.90 (CH2Cl2/CH3OH, 9:1). mp 263 °C. IR
(solid): mmax: 3,313, 2,357, 1,646, 1,548, 1,458, 1,249,
1
1
1,278, 1,204, 836 cm-1. H NMR (400 MHz, DMSO) d;
1,176, 1,119 cm-1. H NMR (400 MHz, DMSO) d; 8.22
9.04 (1H, br.s, NH), 8.46 (1H, br.s, NH), 7.89 (1H, br.s,
H2 ), 7.65 (1H, br.s, H6 ), 7.38 (1H, t, J3,(2,4) = 7.6 Hz, H3),
7.30 (1H, d, J2,3 = 7.6 Hz, H2), 7.24 (1H, t,
(1H, br.s, NH), 7.78 (1H, d, J6,5 = 7.6 Hz, H6), 7.16 (1H,
d, J3,4 = 7.6 Hz, H3), 7.11 (1H, t, J5,(4,6) = 7.6 Hz, H5),
6.93 (1H, t, J4,(3,5) = 7.6 Hz, H4), 3.28 (2H, br.s, CH2),
2.49 (4H, br.s, 2CH2), 2.26 (3H, s, CH3)—EI MS: m/z (rel.
abund. %), 218 (M?, 1), 161 (1), 133 (2), 107 (100), 91
(13), 77 (9), 65 (8)—Anal.: Calcd for C12H14N2O2: (218):
C, 66.11; H, 6.47; N, 12.85 %. Found: C, 66.10; H, 6.43;
N, 12.81 %.
0
0
J4,(3,5) = 7.6 Hz, H4), 7.13 (2H, d, J3 ,2 =5 ,6 = 7.2 Hz,
0
0
0
0
0
0
H3 ,5 ), 7.06 (1H, d, J5,6 = 7.8 Hz, H5), 2.23 (3H, s, CH3)—
EI MS: m/z (rel. abund. %), 283 (M?, 4), 176 (25), 150
(100), 133 (14), 106 (38), 77 (15), 69 (13)—Anal.: Calcd
for C15H13N3OS: (283): C, 63.65; H, 4.63; N, 14.84 %.
Found: C, 63.67; H, 4.61; N, 14.86 %.
N-(1,3-Benzothiazol-2-yl)-N0-(2-methylphenyl)urea (16) (Yield
88 %). Rf = 0.72 (CH2Cl2/CH3OH, 9:1). mp 329 °C. IR
(solid): mmax: 3,472, 3,358, 2,982, 1,695, 1,597, 1,560,
1,450, 1,307, 1,254, 1,213, 834 cm-1. 1H NMR (400 MHz,
DMSO) d; 11.11 (1H, br.s,0 NH), 8.65 (1H, br.s, NH), 7.91
N-Mesityl-N0-(2-methylphenyl)urea
Rf = 0.74 (CH2Cl2/CH3OH, 9:1). mp 279 °C; IR (solid):
(12) (Yield
69 %).
m
max: 3,297, 2,913, 2,361, 1,642, 1,593, 1,552, 1,495,
1,298, 1,123, 1,025 cm-1. 1H NMR (400 MHz, DMSO) d;
0
7.95 (2H, br.s, NH), 7.77 (1H, d, J6 ,5 = 7.6 Hz, H6 ), 7.14
(1H, d, J6 ,5 = 7.6 Hz, H6 ), 7.84 (1H, d, J3 ,4 = 7.6 Hz,
0
0
0
0
0
0
0
0
(1H, d, J3 ,4 = 7.6 Hz, H3 ), 7.09 (1H, t, J5 ,(4 ,6 ) = 7.6 Hz,
H3 ), 7.65 (2H, d, J2,3=5,4 = 8.0 Hz, H2,5), 7.38 (1H, t,
J5 ,(4,6) = 7.6 Hz, H5 ), 7.22 (2H, m, H3,4), 7.02 (1H, t,
J4 ,(3 ,5 ) = 7.6 Hz, H4 ), 2.27 (3H, s, CH3)—EI MS: m/z
(rel. abund. %), 283 (M?, 6), 176 (12), 150 (100), 123 (11),
107 (16), 77 (6), 51 (6)—Anal.: Calcd for C15H13N3OS:
(283): C, 63.65; H, 4.63; N, 14.84 %. Found: C, 63.67; H,
4.61; N, 14.86 %.
0
0
0
0
0
0
0
0
H5 ), 6.90 (1H, t, J4 ,(3 ,5 ) = 7.6 Hz, H4 ), 6.87 (2H, s, H3,5),
2.23 (3H, s, CH3), 2.22 (3H, s, CH3), 2.16 (6H, s, 2CH3)—
EI MS: m/z (rel. abund. %), 268 (M?, 23), 161 (1), 135
(69), 107 (100), 91 (17), 65 (3)—Anal.: Calcd for
C17H20N2O: (268): C, 76.18; H, 7.52; N, 10.45 %. Found:
C, 76.15; H, 7.51; N, 10.47 %.
0
0
0
0
0
0
0
0
N-(2-Methylphenyl)-N0-(1-phenylethyl)urea (13) (Yield
69 %). Rf = 0.65 (CH2Cl2/CH3OH, 9:1). mp 179 °C. IR
(solid): mmax: 3,374, 3,203, 2,933, 1,703, 1,646, 1,605,
N-(2-Acetylphenyl)-N0-(4-methylphenyl)urea (17) (Yield
88 %). Rf = 0.66 (CH2Cl2/CH3OH, 9:1). mp 235 °C. IR
(solid): mmax: 3,378, 1,719, 1,674, 1,605, 1,544, 1,482,
1
1,535, 1,491, 1,376, 1,180, 1,119, 1,049, 837 cm-1. H
1,290, 1,262, 1,217, 886 cm-1 1H NMR (400 MHz,
.
NMR (400 MHz, DMSO) d; 7.82 (1H, d, J6,5 = 8.0 Hz,
H6), 7.60 (2H, br.s, NH), 7.34 (5H, s, Ar–H), 7.09 (1H, t,
J5,(4,6) = 7.6 Hz, H5), 7.04 (1H, d, J3,4 = 7.6 Hz, H3), 6.83
DMSO) d; 8.84 (1H, br.s, NH), 8.58 (1H, br.s, NH), 8.04
(1H, br.s, H2), 7.65 (1H, d, J4,5 = 8.0 Hz, H4), 7.56 (1H, d,
J6,5 = 8.0 Hz, H6), 7.42 (1H, t, J5,(4,6) = 8.0 Hz, H5), 7.33
0
0
(1H, t, J4,(3,5) = 7.6 Hz, H4), 4.80 (1H, q, J1 ,2 = 7.2 Hz,
(2H, d, J2 ,3 =6 ,5 =0 8.0 Hz, H2 ,6 ), 7.08 (1H, d,
00 00
0
0
0
0
0
J3 ,2 =5 ,6 = 8.0 Hz, H3 ,5 ), 2.55 (3H, s, OCCH3), 2.24 (3H,
s, CH3)—EI MS: m/z (rel. abund. %), 268 (M?, 21), 146
(7), 135 (33), 120 (46), 107 (100), 79 (34), 65 (22)—Anal.
00 00
CH), 2.16 (3H, s, CH3), 1.38 (3H, d, J2 ,1 = 7.2 Hz,
CH3)—EI MS: m/z (rel. abund. %), 254 (M?, 11), 196 (1),
0
0
0
0
120 (3), 107 (100), 77 (15), 51 (3)—Anal. Calcd for
123

Med Chem Res
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(1998) Urea derivatives, and industrial antibacterial and anti-
fungal agents, algaecides and antiperiphytic agents containing
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unsymmetrical tetrasubstituted ureas. J Org Chem 62:4155–4158
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Calcd for C16H16N2O2: (268): C, 71.70; H, 6.01; N,
10.45 %. Found: C, 71.68; H, 6.02; N, 10.42 %.
N-(2-Acetylphenyl)-N0-(2-methylphenyl)urea (18) (Yield
88 %). Rf = 0.80 (CH2Cl2/CH3OH, 9:1). mp 184 °C. IR
(solid): mmax: 3,297, 1,683, 1,634, 1,568, 1,356, 1,225,
1
886 cm-1. H NMR (400 MHz, DMSO) d; 9.24 (1H, br.s,
NH), 8.04 (1H, br.s, NH), 7.94 (1H, br.s, H2), 7.81 (1H, d,
0
J4,5 = 7.6 Hz, H4), 7.68 (1H, d, J6 ,5 = 8.0 Hz, H6 ), 7.56
(1H, d, J6,5 = 7.6 Hz, H6), 7.43 (1H, t, 0J5,(4,6) = 7.6 Hz,
0
0
H5), 7.17 (1H, d, J3 ,4 = 8.0 Hz, H3 ), 7.13 (1H, t,
J5 ,(4 ,6 ) = 8.0 Hz, H5 ), 6.96 (1H, t, J4 ,(3 ,5 ) = 7.6 Hz,
H4 ), 2.55 (3H, s, OCCH3), 2.23 (3H, s, CH3)—EI MS m/z
(rel. abund. %), 268 (M?, 32), 146 (8), 135 (52), 120 (57),
107 (100), 78 (23), 65 (21)—Anal.: Calcd for C16H16N2O2:
(268): C, 71.70; H, 6.01; N, 10.45 %. Found: C, 71.68; H,
6.02; N, 10.42 %.
0
0
0
0
0
0
0
0
0
0
Acknowledgments The authors gratefully acknowledge the Higher
Education Commission (HEC) for granting fellowship to Ph. D. stu-
dent Ms. Sana Mustafa. Authors are also grateful to Dr. Rafat Ali
Siddiqui, Cellular Biochemistry Laboratory, Methodist Research
Institute, Room E504, Indianapolis, IN 46202, USA for his kind
guidance in the preparation of the manuscript.
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123