Synthesis and immunomodulatory properties of selected oxazolone derivatives

Article abstract of DOI:10.1016/j.bmc.2004.02.034

Eleven oxazolone derivatives were synthesized and characterized by ¹H NMR, EI, IR and UV spectroscopic and CHN analysis. Three compounds, 4-[(E)-(4-nitrophenyl)methylidene]-2-phenyl-1,3-oxazol-5(4H)-one (11), 4-[(E)-(4-methoxyphenyl)methylidene]-2-methyl-1,3-oxazol-5-one (12) and 4-[(E)-(4-nitrophenyl)methylidene]-2-methyl-1,3-oxazol-5(4H)-one (13) were screened for phagocyte chemiluminescence, neutrophil chemotaxis, T-cell proliferation, cytokine production from mononuclear cells and cytotoxicity. 4-[(E)-(4-Nitrophenyl)methylidene]-2-methyl-1,3-oxazol-5(4H)-one (13) was found to be the most potent immunomodulator in the series.

Full text of DOI:10.1016/j.bmc.2004.02.034

Bioorganic & Medicinal Chemistry 12 (2004) 2049–2057
Synthesis and immunomodulatory properties of selected
oxazolone derivatives
Muhammad A. Mesaik,a Shagufta Rahat,b Khalid M. Khan,b,* Zia-Ullah,b
Muhammad I. Choudhary,b Shahnaz Murad,c Zakiah Ismail,c Atta-ur-Rahmanb
and Aqeel Ahmada,*
aDepartment of Microbiology, University of Karachi, Karachi 75270, Pakistan
bHEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan
cInstitute for Medical Research Kuala Lumpur, 50588 Kuala Lumpur, Malaysia
Received 21 January 2004; revised 24 February 2004; accepted 25 February 2004
1
Abstract—Eleven oxazolone derivatives were synthesized and characterized by H NMR, EI, IR and UV spectroscopic and CHN
analysis. Three compounds, 4-[(E)-(4-nitrophenyl)methylidene]-2-phenyl-1,3-oxazol-5(4H)-one (11), 4-[(E)-(4-methoxyphenyl)-
methylidene]-2-methyl-1,3-oxazol-5-one (12) and 4-[(E)-(4-nitrophenyl)methylidene]-2-methyl-1,3-oxazol-5(4H)-one (13) were
screened for phagocyte chemiluminescence, neutrophil chemotaxis, T-cell proliferation, cytokine production from mononuclear cells
and cytotoxicity. 4-[(E)-(4-Nitrophenyl)methylidene]-2-methyl-1,3-oxazol-5(4H)-one (13) was found to be the most potent immu-
nomodulator in the series.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
The immunomodulatory drugs such as cyclosporin
isolated from a soil fungus Trichoderma polysprm6
and tacrolimus (FK-506), a secondary metabolite of
Streptomyces tsukabaensis, are used to suppress immu-
nological rejection of the transplanted organs.7 These
unmodified natural products represent major break-
throughs in organ transplantation. Based on these nat-
ural products and their activities, new immunomodulator
drugs were synthesized, which are among the most
important anticancer drugs used today. Tamoxifen, an
antiestrogenic compound and methotrexate, which is a
folic acid antagonist, are two examples of synthetic drugs
that inhibits DNA synthesis and cell division.8–10
The knowledge of the inter-related and multiple levels of
interactions between various components of the immune
system have helped in understanding how the immune
system provides protection against the foreign invaders
and pathological conditions. This has also led to the
development of many drugs that modulate the host
natural defense mechanism and restore impaired
immune function.1 Immunomodulation denotes to any
change in the immune response and may involve
induction, expression, amplification or inhibition of
any part or phase in the immune response. Stimu-
lation of the immune response is required in certain
patients, whereas, suppression of the immune response
is needed in other conditions.2 Novel immunomodu-
lating agents are used for the treatment of various
conditions such as infections, organ transplantation,
cancer, rheumatoid arthritis, systemic lupus erythemat-
osus, etc.3–5
Oxazolone is a class of small heterocycles, which are
important intermediates in the synthesis of several small
molecules, including amino acids, peptides,11–16 anti-
microbial or antitumour compounds,17;18 heterocyclic
precursors19–22 as well as biosensors coupling and or
photosensitive composition devices for proteins.23 Some
oxazolones have shown a wide range of pharmaceutical
properties.24
Keywords: Oxazolone; Immunomodulation; Chemotaxis; T-Cell pro-
liferation.
In continuation of our drug discovery program,25–29 we
synthesized a variety of oxazolones and screened their
effects on different aspects of immune response. The
* Corresponding authors. Tel./fax: +92-21-4989018 (A.A.); fax: +92-
21-9243190-91 (K.M.K.); e-mail addresses: hassaan2@super.net.pk;
aahmad57@hotmail.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.034

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M. A. Mesaik et al. / Bioorg. Med. Chem. 12 (2004) 2049–2057
O
O
O
5
b, c
a
4
OH
OH
1
1
O
H2N
R
HN
RCHO
R1
Cl
R
N
O
2
O
3
R1
1
2
3-13
Scheme 1. Reagents and conditions: (a) BzCl, or AcCl, 10% NaOH, H2O; (b) Ac2O, NaOAc, reflux; (c) PPA, 80–90 ꢁC.
compounds were tested for phagocyte chemilumines-
cence, neutrophil chemotaxis, T-cell proliferation, cyto-
kine production from mononuclear cells and cytotoxicity.
2.2. Biological activity
In order to test the immunomodulating effects of the test
compounds, we used a number of assays in different
immune-responsive cells. We investigated oxidative
burst activity of neutrophils and monocytes, chemotoxis
in neutrophils, T-cell proliferation and cytokine release
by lymphocytes. We used prednisolone a known
immune suppressive drug as a positive control in these
studies. We initially tested 11 oxazolones derivatives for
their possible immunomodulatory activities using inhi-
bition of zymosan-induced phagocytosis. Three com-
pounds 11, 12 and 13 showed varying degrees of
suppressive activities (96%, 32% and 78%, respectively),
whereas rest of the compounds 3–10 either showed
moderate or no activities (Table 1). We, therefore, used
compounds 11, 12 and 13 for further evaluation and
the results are summarized as follows.
2. Results
2.1. Chemistry
The compounds 3–13 were synthesized from commer-
cially available glycine with acetic anhydride/benzoyl
chloride in the presence of anhydrous sodium acetate
followed by Erlenmeyer condition with different alde-
hydes in very high yields.30–32 All the oxazolones were
isomerized by heating them with polyphosphoric acid on
a water bath 80–90 ꢁC for 2 h according to literature
procedure.33 The structures were determined by using
1
different spectroscopic methods like H NMR, EI, IR
and UV and confirmed by CHN analysis (Scheme 1).
R1
2.2.1. Effect on MDBK cell proliferation. The test com-
pounds were tested for possible cellular toxic effects by
determining cell proliferation on Mavin Darby Bovine
Kidney (MDBK) cells (Table 2). Results presented in
Table 1 indicate that the test compounds as high as
50 lg/mL did not exhibit any toxic effect after 3 days
incubation with MDBK cells. Therefore various doses
up to 50 lg/mL were used during present investigation.
Compound
R
3
OMe
NO2
OMe
4
5
6
7
8
9
N
H
2.2.2. Effect on phagocytes oxidative burst. Phagocytic
cells on activation induce release of reactive oxygen free
Cl
Isobutyl
Table 1. Effect of compounds on oxidative burst (chemiluminescence
assay)
N
H
Compound
50 lg/mL
25 lg/mL
3
24
2
19
12
17
10
9
4
5
11
14
35
10
46
32
85
93
99
NH2
Br
6
7
10
11
8
0
9
12
7
10
11
12
13
78
32
96
NO2
Two concentrations of compounds 3–13 were incubated with neutro-
phil for 30 min. After the addition of serum treated zymosan (STZ)
and luminol, neutrophils were scanned at 37 ꢁC for their CL activity
for 50 min. Compounds activity was compared with the control. The
percentage effect was calculated as ¼ 100 ) {(CL in presence of com-
pound/CL count in the absence of compound) · 100}. The reading
represents a triplicate result of each concentration.
12
13
–CH3
–CH3
OCH3
NO2

M. A. Mesaik et al. / Bioorg. Med. Chem. 12 (2004) 2049–2057
2051
Table 2. Compounds IC50 value from the MTT cytotoxicity studies
70
60
50
40
30
20
10
0
Compound
IC50 (lg/mL)
5 days MTT results
6.04
20.6
6.0
3 days MTT results
11
12
13
P50.0
Nil
Nil
Serial compounds dilution (0.195–50 lg/mL) were incubated with
Mavin Darby bovine kidney (MDBK) cells for 72 h and cells viability
was evaluated by MTT reduction to the blue coloured formazan in
living cells. All values were means of 3–5 replicate. Nil ¼ no cytotox-
icity detected.
11
12
13
Prednisolone
-10
5
µ
g
2.5
µ
g
Figure 2. Effect of test compounds on chemotoxis. Effect of the test
compounds on the PMNs chemotoxis was evaluated using Boyden
chamber (Neuro Probe, USA). The chemoattractant (fMLP 10ꢀ8) was
added into the lower chamber. Compounds and cells were introduced
simultaneously into the upper chamber at 37 ꢁC. The movement of
cells in the filter membranes was observed under the light microscope.
fMLP ¼ formyl-methionyl-leucyl-phenylalanine. Each plot and error
bar represents reading SD of three repeats.
radicals (oxidative burst), which is then quantified by a
luminol-enhanced chemiluminescence assay. Results
presented in Figure 1 indicate that the zymosan-induced
oxidative burst in polymorphoneutrophils (PMNs) was
inhibited potently by compound 13 at a concentration of
1.56 lg/mL. However, compound 13 was less active in
suppressing the activity of the monocytes, 20% inhibi-
tion was obtained at 3.125 lg/mL. Compound 12 was
found to be less active than compounds 11 and 13. The
inhibition of the oxidative burst was not due to cyto-
toxic effects as none of these compounds were found to
exhibit cytotoxicity (Table 2).
inhibited only 7–12% fMLP-induced chemotoxis,
whereas at 50 lg/mL the inhibition of 32% and 17%, was
observed, respectively. Compound 13 exhibited the most
potent suppressive activity on fMLP-induced PMNs
movement and caused about 40% inhibition of chemo-
toxis at 25 lg/mL concentration, which did not change
significantly at 50 lg/mL concentration.
2.2.3. Effect on polymorphoneutrophil cells chemotoxis.
Effect of the test compounds on PMN chemotoxis in
response to a chemotactic peptide formyl-methionyl-
leucyl-phenylalanine (fMLP 10ꢀ8) was assayed using
Boyden chambers. PMNs were incubated with 25 and
50 lg/mL of each compound for 1 h and the difference in
the cell movement was studied. Prednisolone was run as
a positive control. The data presented in Figure 2 show
that all tested compounds exhibited chemotoxis sup-
pressive activity. Compounds 11 and 12 at 25 lg/mL
2.2.4. Effect on T-cell proliferation. The anti-prolifera-
tion effect of the test compounds was determined by
measuring the inhibition of phytohemagglutinin (PHA)-
induced T-cell proliferation by determining radioactive
thymidine incorporation. Results shown in Figure 3
indicate that all three test compounds significantly
suppressed T-cell proliferation in dose dependent man-
ner. However, the compounds 11 and 13 showed the
0.78
1.56
3.125
6.25
12.5
25
140
120
100
80
PMNs 11
PBMC 11
PMNs 12
PBMC 12
PMNs 13
PBMC 13
PMNs C
PBMC C
60
40
20
0
-20
-40
compound concentration (µg/ml)
Figure 1. Effect of compounds on oxidative burst by phagocyte cells.
Various concentrations of compounds 11, 12 and 13 were incubated
with polymorphoneutrophil (PMNs) or peripheral blood mononuclear
cells (PBMC) for 30 min. After the addition of serum treated zymosan
(STZ) and luminol, phagocytic cells were scanned at 37 ꢁC for their
chemiluminescence (CL) activity. The compounds activity was com-
pared with the control (C). The percentage effect was calculated
as ¼ 100 ) {(CL in presence of compound/CL count in the absence of
compound) · 100}. Each plot and error bar represents reading SD of
three repeats.
Figure 3. Effect of compounds on phytohemagglutinin (PHA) T-cell
proliferation. Cells were incubated with different concentrations of the
test compounds in RPMI-1640 along with PHA for 72 h at 37 ꢁC in
CO2 environment. Cells were further incubated for 18 h after the
addition of thymidine [3H] and the radioactivity count as CPM reading
was recorded using beta scintillation counter. The effect of compounds
on the T-cell proliferation is compared with control. Each bar repre-
sents the mean value of triplicate reading SD.

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M. A. Mesaik et al. / Bioorg. Med. Chem. 12 (2004) 2049–2057
compounds. In contrast compound 12 has no effect on
the inhibition of any cytokine release from activated
lymphocytes.
3. Statistics
All data are presented as means standard deviation of
the mean. Statistical analysis for all the results was
compared using student’s t-test. Significance was
attributed to probability values P 6 0:05 (P 6 0:005 in
some cases). The IC50 values were calculated using Excel
based program.
4. Discussion
A number of assays were used to investigate the
immunomodulating effect of oxazolone derivatives on
different immune cells. Early in the phagocytosis pro-
cess, neutrophils undergo a respiratory burst, generating
free radicals, which react with luminol and emit light.34
Thus, chemiluminescence enhanced by the presence of
luminol may be used as an indicator of phagocytic
activity.35 The present data show that compounds exert
inhibitory effect on phagocytic activity of PMNs and
PBMC. It was also found to interfere with the phyto-
hemagglutinin (PHA) T-cell activation in a dose
dependant manner. The impaired incorporation of tri-
tiated thymidine into cellular DNA may not be due to
toxic effect since the compound was found to be non-
toxic against MDBK cell line with a similar incubation
time. The effect of compound 11 on T-cell proliferation
was further confirmed by suppression of IL-2 and IFN-c
production. These two compounds 11 and 13 are clearly
exerting immunomodulatory activity on the system used
and could be suitable immunomodulatory lead com-
pounds for future research.
Figure 4. Effect of the test compounds on cytokine production. Four
concentrations (0.5, 1, 4 and 16 lg/mL) from each compound were
incubated with the peripheral blood mononuclear cells (PBMC) cells
for 18 h along with the stimulant (PHA). Level of IL-2, INF-c and IL-4
was determined by cytokine ELISA kits (Diaclone, Besancon Cedex,
France) compare to control (PHA). Each bar represents the mean
value of triplicate reading SD level. IL-2 level ¼ 528.5 34.52, INF-c
level ¼ 4033.7 180, IL-4 level ¼ 120.5 18.1.
The results of preliminary assays and structures of these
compounds indicated that oxazolones with a suitably
substituted exocyclic phenyl ring possess immunosup-
pressive activity. The possible structural factor, which
influences the activity, is exocyclic substitution on oxa-
zolone at C-4. The compounds having exocyclic
p-substituted nitrophenyl rings showed varying degrees
of activities as in case of compounds 3, 11, 12 and 13.
The most active compound of the series was 13, which
fulfil essential structural requirements, described above.
most significant suppressive activity. A dose as low as
7.5 lg/mL of compounds 11 and 13, caused approxi-
mately 40–50% reduction in T-cell proliferation com-
pared to control. The anti-proliferation effect of these
compounds was further increased with concentration
and the cell proliferation was reduced by 75–85% at
50 lg/mL. Compound 12 had least potent anti-prolifer-
ation activity and only 10–15% inhibition was observed
with 16–50 lg/mL concentrations.
Furthermore weak activities were observed in case of
compounds 12 and 3 (Table 1). However, both com-
pounds contain para-methoxy group on exocyclic phe-
nyl ring. The activity difference between compounds 12
and 3 might be due to the presence of an additional
methoxy group at meta-position of compound 3, which
possibly restrict the molecule to interact with cells.
2.2.5. Effect on cytokine release by peripheral blood
mononuclear cells (PBMC). The suppressive effect of the
test compounds was tested on the release of selected
cytokine including IL-2, IFN-c and IL-4 by PHA-
induced lymphocytes. Results shown in Figure 4 indi-
cate that compounds 11 and 13 apparently inhibited
IL-2, IL-4 and INF-c release in a dose dependent
manner from PHA-activated lymphocytes. A dose as
low as 0.5 lg/mL was found to be very effective for these
In case of compounds 11, 12 and 13, which were active
at the preliminary chemiluminescence (CL) screening, a

M. A. Mesaik et al. / Bioorg. Med. Chem. 12 (2004) 2049–2057
2053
detailed study of these compounds was carried out,
which includes phagocyte chemiluminescence, neutro-
phils chemotaxis, T-cell proliferation assay, cytokine
production from mononuclear cells and cytotoxicity
evaluation.
ian MAT 711 (70 eV) spectrometer, and data are tabu-
lated as m=z. Elemental analyses were carried out on a
Perkin Elmer 2400 CHN Elemental Analyzer. 1H NMR
spectra were recorded, using tetramethylsilane as an
€
internal standard, on a Bruker AC 250 (300 and
400 MHz) spectrometer, respectively. Chemical shifts
are reported in d (ppm) and coupling constants are given
in Hz. The progress of all reactions was monitored by
TLC on 2 · 5 cm pre-coated plates with silica gel 60 F254
to a thickness of 0.25 mm (Merck). The chromatograms
were visualized under ultraviolet light (254–366 nm)
and iodine vapours.
The chemotoxis studies were carried out on compounds,
11, 12 and 13. The compound 13 was found most active
(38.6%) at a 50 lg/mL concentration. However, at the
lower concentration (25 lg/mL) level, it showed slight
increase (40%) in the inhibition. The other tested com-
pounds 11 and 12 showed weak activity in this assay at
both concentrations (Fig. 2). The results suggested that
methyl chain and exocyclic substituted phenyl group
might participate in interaction of these compounds
with phagocytic cells movement.
6.1.1. N-Benzoylglycine (2a). Glycine (10 g, 133.0 mmol)
was dissolved in 10% sodium hydroxide solution
(100 mL), and then benzoyl chloride (21.6 mL,
186.0 mmol) was added in portions to this solution,
stirred vigorously after each addition. Crushed ice
(100 g) was added to the solution and then concentrated
HCl was added dropwise until the mixture was acidified
(pH 2–3). The resulting compound 2 was obtained as
white crystalline solid 23.0 g (96%); 1H NMR (300 MHz,
CD3OD) d: 4.09 (s, 2H, CH2), 7.46 (d, 2H, J ¼ 8:1 Hz,
Ar-H), 7.52 (d, 1H, J ¼ 7:2 Hz, Ar-H), 7.85 (dd, 2H,
J ¼ 1:5, 8.4 Hz, Ar-H), 8.17 (br s, 1H, NH); IR (KBr):
3457, 3029, 1771, 1721; UV kmax (CHCl3): 268, 209 nm;
EI-MS m=z (rel abund %): 179 (Mþ, 29), 134 (100), 102
(59), 77 (83). Anal. Calcd for C9H9NO3: C, 60.33; H,
5.06; N, 7.82. Found: C, 60.39; H, 5.00; N, 7.85.
In case of T-cell activation, the percentage inhibition of
compounds 11 and 13 showed that compound 13 was
more potent at all concentrations (Fig. 3). The results
also suggested that the activity might be due to the
presence of a methyl and exocyclic nitro substituted
phenyl group.
The result on IL-2, IFN-c and IL-4 release suggests that
compounds 13 and 11 were the most potent compounds,
whereas compound 12 was not suppressing the pro-
duction of any of the tested cytokine, rather a slight
increase is observed on IL-2 production (Fig. 4). Effect
of compound 13 again can be attributed to methyl group
and exocyclic nitro substituted phenyl group. The IL-4
studies on the aforementioned compounds showed that
compound 13 is the most potent in series (Fig. 4), which
might be because of the earlier described reasons.
6.1.2. N-Acetylglycine (2b). A solution of glycine (10 g,
133.0 mmol) in water (135 mL) was dissolved, then ace-
tic anhydride (24 mL, 266.0 mmol) was added to it in
one portion and vigorously stirred for 30 min, cooled
overnight, solid was collected and washed with ice cold
water, dried to afford compound 2b as white solid 7.14 g
5. Conclusions
1
(46%). Mp: 208 ꢁC; H NMR (300 MHz, CD3OD) d:
1.99 (s, 3H, CH3), 3.89 (s, 2H, CH2), 8.22 (br s, 1H,
NH); IR (KBr): 3461, 1765, 1732; UV kmax (CHCl3):
209 nm; EI-MS m=z (rel abund %):117 (Mþ, 8), 82 (100),
100 (78). Anal. Calcd for C4H7NO3: C, 41.03; H, 6.03;
N, 11.96. Found: C, 41.00; H, 6.06; N, 11.99.
The data presented above indicate that a C-4 para-nitro
substituted exocyclic phenyl group on oxazolone moiety
greatly influences the immunosuppressive activity. The
nitro substitution at para-position contributes in activ-
ity, while a methoxy group at the same position
decreases the activity. These studies also suggest that
para-nitro substituted exocyclic phenyl ring at C-4 might
enhance the interaction of the molecule with cell.
However, further mechanism-based studies are required
for a better understanding of the mechanism of action
of oxazolones on immune response.
6.1.3. 4-[(E)-(3,4-Dimethoxyphenyl)methylidene]-2-phen-
yl-1,3-oxazol-5(4H)-one (3). A solution of 3,4-dimethoxy
benzaldehyde (1.50 g, 9.0 mmol), N-benzoyl- glycine
(2.0 g,
11.0 mmol),
acetic
anhydride
(3.0 mL,
33.6 mmol), fused sodium acetate (1.0 g, 12.1 mmol) was
heated, after liquification, heating was continued for
additional 2 h. After the completion of reaction (TLC
analysis) 4.4 mL ethanol was added and kept at room
temperature for 18 h. It was filtered, solid thus obtained
was isomerized by mixing and then heating with
polyphosphoric acid on a steam bath 80–90 ꢁC for 2 h
and then poured into water.33 The resultant solid
product was collected, washed with cold ethanol, hot
water, ethanol and then small amount of hexane and
then dried to afford compound 3 as pale yellow solid;
Yield: 1.96 g (70%). Mp: 149.3 ꢁC; Rf : 0.87 (n-butanol–
6. Experimental section
6.1. General
€
Melting points were determined with a Buchi SMP-20
apparatus and are uncorrected. The ultraviolet spectra
were measured in chloroform on a Lambda 5 UV/vis
spectrometer (Perkin–Elmer). IR spectra (KBr discs)
€
were recorded on a Bruker FT-IR IFS 48 spectrometer.
EI and FD mass spectral data were recorded with Var-
1
acetic acid–H2O 3:3:1); H NMR (300 MHz, CDCl3) d:

2054
M. A. Mesaik et al. / Bioorg. Med. Chem. 12 (2004) 2049–2057
3.92 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 7.19 (br s, 1H,
H-6), 6.91 (s, 1H, H-60), 7.40 (t, 1H, J ¼ 7:2 Hz, H-400),
7.50 (d, 1H, J ¼ 7:5 Hz, H-60), 7.57 (d, 1H, J ¼ 7:2 Hz,
H-50), 8.10 (d, 2H, J ¼ 6:9 Hz, H-200,600), 8.15 (dd, 2H,
J ¼ 1:8, J ¼ 6:9 Hz, H-300,500); IR (KBr): 1783, 1771,
1666, 1497, 1462, 1329, 1286, 1160, 1112, 1013, 994, 917,
889, 867 cmꢀ1; UV kmax (CHCl3): 396, 260, 201 nm; EI-
MS m=z (rel abund %): 309 (Mþ, 43), 176 (6) 131 (2), 105
(100), 51 (5). Anal. Calcd for C18H15NO4: C, 69.89; H,
4.89; N, 4.52. Found C, 69.87; H, 4.88; N, 4.50.
1641, 1563, 1486, 1388, 1268, 1184, 1066, 982, 743 cmꢀ1
;
UV kmax (CHCl3): 378, 372, 359, 259, 255, 251, 243, 230,
203, 198 nm; EI-MS m=z (rel abund %): 283 (Mþ, 63),
141 (9), 104 (100), 51 (39). Anal. Calcd for C16H10NO2: C,
67.74; H, 3.55; N, 4.94. Found: C, 67.73; H, 3.54; N, 4.96.
6.1.7.
4-[(E)-3-Methylbutylidene]-2-phenyl-1,3-oxazol-
5(4H)-one (7). Isovaleraldehyde (1.63 mL, 15.3 mmol),
N-benzoylglycine (2.5 g, 13.96 mmol), acetic anhydride
(3.93 mL, 36.3 mmol) and fused sodium acetate (1.14 g,
13.9 mmol) were treated in the same way as described
for compound 3. The orange yellow product obtained
was 2.0 g (63%). Mp: 136 ꢁC; Rf : 0.56 (n-butanol–acetic
6.1.4. 4-[(E)-(3-Nitrophenyl)methylidene]-2-phenyl-1,3-
oxazol-5(4H)-one (4). The compound 4 was prepared
in the same way as described for compound 3 from
3-nitro benzaldehyde (1.20 g, 7.94 mmol). The product
was obtained as a light orange solid 1.55 g (66%). Mp:
1
acid–H2O 3:3:1); H NMR (300 MHz, CDCl3) d: 1.50
(d, 6H, J ¼ 7:5 Hz, (CH3)2), 2.95 (m, 1H, CH), 7.24 (s,
1H, H-6), 7.35 (d, 2H, J ¼ 7:9 Hz, H-30,50), 7.46 (t, 1H,
J ¼ 7:6 Hz, H-40), 7.54 (d, 2H, J ¼ 7:9 Hz, H-20,60); UV
kmax (CHCl3): 382, 378, 274, 237, 202, 194 nm; EI-MS
m=z (rel abund %): 229 (Mþ, 45), 105 (100), 77 (45).
Anal. Calcd for C14H15NO2: C, 73.34; H, 6.60; N, 6.10.
Found: C, 73.33; H, 6.59; N, 6.11.
1
162 ꢁC; Rf : 0.64 (n-butanol–acetic acid–H2O 3:3:1); H
NMR (300 MHz, CDCl3) d: 7.24 (br s, 1H, H-6), 7.54 (t,
1H, J ¼ 8:2 Hz, H-30), 7.59–7.65 (m, 1H, H-400, Ar-H),
7.67 (dd, 2H, J ¼ 3:8, J ¼ 8:0 Hz, H-300,500), 8.18 (dd,
2H, J ¼ 1:3, J ¼ 8:0 Hz, H-200,600), 8.28 (dd, 1H, J ¼ 1:3,
J ¼ 8:2 Hz, H-200), 8.40 (d, 1H, J ¼ 8:2 Hz, H-40), 9.30
(s, 1H, H-60, Ar-H); IR (KBr): 1788, 1657, 158, 1349,
1294, 1163, 994, 889 cmꢀ1; UV kmax (CHCl3): 3576, 268,
225 nm; EI-MS m=z (rel abund %): 294 (Mþ, 46), 105
(100), 77 (50). Anal. Calcd for C16H10N2O4: C, 65.30; H,
3.43; N, 9.52. Found: C, 65.28; H, 3.42; N, 9.58.
6.1.8. 4-[(E)-Pyrrol-3-ylmethylidene]-2-phenyl-1,3-oxa-
zol-5(4H)-one (8). Compound 8 was prepared in the
same way as described for compound 3, from pyrrole
carbaldehyde (1.5 g, 15.77 mmol). The product was
obtained as yellow solid 2.26 g (60%); Rf : 0.66 (n-butanol–
1
acetic acid–H2O 3:3:1); H NMR (300 MHz, CDCl3) d:
7.19 (br s, 1H, H-6), 7.3–7.8 (m, 3H, H-20,30,40), 8.5–8.8
(m, 5H, H-200,300,400,500,600, Ar-H); FD MS m=z: 238 (Mþ);
IR (KBr): 1795, 1775, 1665; 1555, 1515, 1480, 1460,
1410, 1380, 1360, 1315, 1245, 1125, 1095, 965, 875, 865,
765 cmꢀ1; UV kmax (CHCl3): 374, 273, 227, 262, 262 nm.
Anal. Calcd for C14H10N2O2: C, 70.58; H, 4.23; N,
11.76. Found: C, 70.55; H, 4.22; N, 11.77.
6.1.5.
4-[(E)-1H-Indol-3-ylmethylidene]-2-phenyl-1,3-
oxazol-5(4H)-one (5). Indole-3-carbaldehyde (1.50 g,
10.34 mmol), N-benzoylglycine (2.5 g, 13.97 mmol),
acetic anhydride (3.75 mL) and fused sodium acetate
were treated in the same way as described for compound
3. The product was obtained as yellow solid 1.81 g
(61%). Mp: 206 ꢁC; Rf : 0.65 (n-butanol–acetic acid–H2O
3:3:1); 1H NMR (300 MHz, CDCl3) d: 7.49 (s, 1H, H-6),
7.42 (dd, 2H, J ¼ 1:7, J ¼ 7:4 Hz, H-300,500), 7.54 (t, 2H,
J ¼ 7:4 Hz, H-200,600), 7.62 (t, 1H, J ¼ 7:4 Hz, H-400), 7.84
(dd, 1H, J ¼ 6:8, J ¼ 7:8 Hz, H-50), 8.13 (d, 2H,
J ¼ 7:2 Hz, H-2, H-60,70), 8.47 (d, 1H, J ¼ 7:4 Hz, H-80),
8.75 (s, 1H, CH); IR (KBr): 1781, 1646, 1581, 1339,
1291, 1153, 987, 875 cmꢀ1; UV kmax (CHCl3): 400, 377,
296, 203, 194 nm; EI-MS m=z (rel abund %): 288 (Mþ,
51), 105 (100), 77 (34). Anal. Calcd for C18H12N2O2: C,
74.99; H, 4.20; N, 9.72. Found: C, 74.89; H, 4.25; N,
9.82.
6.1.9. 4-[(E)-(3-Aminophenyl)methylidene]-2-phenyl-1,3-
oxazol-5(4H)-one (9). 3-Amino benzaldehyde (1.85 g,
15.3 mmol), N-benzoylglycine (2.5 g, 13.9 mmol), acetic
anhydride (3.93 mL, 36.3 mmol) and fused sodium ace-
tate (1.14 g, 13.89 mmol) were treated in the same way as
described for compound 3. The yellow solid product
obtained was 1.85 g (50%). Mp: 179 ꢁC; Rf : 0.65 (n-
butanol–acetic acid–H2O 3:3:1); 1H NMR (300 MHz,
CDCl3) d: 4.24 (s, 2H, NH2), 7.24 (br s, 1H, H-6), 7.4 (t,
1H, J ¼ 7:8 Hz, H-20), 7.51 (t, 1H, J ¼ 7:8 Hz, H-30),
7.60 (d, 1H, J ¼ 7:2 Hz, H-40), 7.69 (d, 2H, J ¼ 8:0 Hz,
H-200,600), 7.70 (d, 2H, J ¼ 8:0 Hz, H-300), 8.17 (t, 1H,
J ¼ 7:2 Hz, H-400), 8.30 (s, 1H, H-60); EI-MS m=z (rel
abund %): 264 (Mþ, 13), 248 (39), 171 (56), 127 (63), 105
(100), 77 (83); IR (KBr): 3458, 1769, 1751, 1655, 1576,
1486, 1367, 1278, 1163, 1061, 980, 739 cmꢀ1; UV kmax
(CHCl3): 313, 269, 227, 215, 200, 197, 193 nm. Anal.
Calcd for C16H12N2O2: C, 72.72; H, 4.58; N, 10.60.
Found: C, 72.72; H, 4.57; N, 10.60.
6.1.6. 4-[(E)-(3-Chlorophenyl)methylidene]-2-phenyl-1,3-
oxazol-5(4H)-one (6). 3-Chlorobenzaldehyde (1.98 g,
14.1 mmol), N-benzoylglycine (2.5 g, 13.97 mmol), acetic
anhydride (3.93 mL, 41.7 mmol) and fused sodium ace-
tate (1.14 g, 13.9 mmol) were treated in the same way as
described for compound 3. The product was obtained as
yellow solid 1.58 g (40%). Mp: 166 ꢁC; Rf : 0.67 (n-buta-
nol–acetic acid–H2O 3:3:1); 1H NMR (400 MHz,
CDCl3) d: 7.16 (s, 1H, H-6), 7.40–7.42 (m, 3H,
H-300,400,500, Ar-H), 7.52 (dd, 1H, J ¼ 0:93, J ¼ 8:2 Hz,
H-20, H-30), 7.56 (d, 2H, J ¼ 6:4 Hz, H-200,600), 7.61 (d,
1H, J ¼ 8:2 Hz, H-200), 8.18 (dd, 1H, J ¼ 2:2, J ¼ 7:1 Hz,
H-40, Ar-H), 8.28 (s, 1H, H-60, Ar-H); IR (KBr): 1786,
6.1.10. 4-[(E)-(2-Bromophenyl)methylidene]-2-phenyl-1,3-
oxazol-5(4H)-one (10). 2-Bromobenzaldehyde (1.79 mL,
15.29 mmol), N-benzoylglycine (2.5 g, 13.96 mmol),
acetic anhydride (3.93 mL, 36.3 mmol) and fused sodium

M. A. Mesaik et al. / Bioorg. Med. Chem. 12 (2004) 2049–2057
2055
acetate (1.25 g, 15.23 mmol) were treated in the same
way as described for compound 3. The product was
obtained as yellow solid 2.54 g (55%). Mp: 144 ꢁC; Rf :
0.55 (n-butanol–acetic acid–H2O 3:3:1); 1H NMR
(400 MHz, CDCl3) d: 7.13 (br s, 1H, H-6), 7.26 (d, 1H,
J ¼ 7:4 Hz, H-20), 7.44 (t, 1H, J ¼ 7:4 Hz, H-30), 7.52 (t,
1H, J ¼ 7:4 Hz, H-40), 7.60 (d, 1H, J ¼ 7:4 Hz, H-50),
8.16 (d, 2H, J ¼ 7:6 Hz, H-200,600), 8.87 (d, 2H,
J ¼ 7:5 Hz, H-300,500), 8.37 (s, 1H, H-60); EI-MS m=z (rel
abund %): 330 (M+2, 19), 328 (Mþ, 20), 105 (100), 77
(54); IR (KBr): 3127, 1807, 1763, 1673, 644; UV
(CHCl3): 311, 262, 231, 211, 201, 197, 194 nm. Anal.
Calcd for C16H10BrNO2: C, 58.56; H, 3.07; N, 4.27.
Found: C, 58.59; H, 3.04, N, 4.29.
(dd, 2H, J ¼ 2:1, J ¼ 7:8 Hz, H-20,60); IR (KBr): 1783,
1757, 1666, 1587, 1561, 1468, 1356, 1152, 974, 866 cmꢀ1
;
UV kmax (CHCl3): 423, 282, 261 nm; EI-MS m=z (rel
abund %): 295 (Mþ, 44.97), 105 (100), 77 (75.68). Anal
Calcd for C16H10N2O4: C, 65.30; H, 3.43; N, 9.52.
Found C, 65.28; H, 3.45; N, 9.50.
6.2. Phagocyte chemiluminescence
Luminol-enhanced chemiluminescence assay was per-
formed using Helfand et al. protocol.36 Briefly 5 · 105
neutrophils, suspended in modified Hank’s solution
(MHS), were incubated with varying concentrations of
compounds (0.78–25 lg/mL) for 30 min. Zymosan (Sig-
ma Chemical Co., USA) 100 lL (20 mg/mL), followed
by 100 lL (7 · 105 M) luminol (Sigma Chemical Co.,
USA) was added to make a final volume of 0.25 mL.
MHS alone was run as a control. Peak chemilumines-
cence was recorded with the luminometer (Labsystem
Luminoskan RS, Finland). The luminometer was set
with repeated scan mode, 50 scans with 30 s intervals
and one second point measuring time.
6.1.11. 4-[(E)-(4-Nitrophenyl)methylidene]-2-phenyl-1,3-
oxazol-5(4H)-one (11). Compound 11 was prepared in
the same way as described for compound 3, from 4-
nitrobenzaldehyde (2.31 g, 15.28 mmol), N-benzoylgly-
cine (2.0 g, 11.1 mmol), acetic anhydride (3.0 mL,
33.6 mmol), fused sodium acetate (1.0 g, 12.1 mmol).
The product was obtained as yellow solid 2.0 g (61%).
1
Rf : 0.68 (n-butanol–acetic acid–H2O 3:3:1); H NMR
(300 MHz, CDCl3) d: 7.24 (br s, 1H, H-6), 7.40–7.42 (m,
1H, H-400, Ar-H), 7.50 (d, 2H, J ¼ 6:0 Hz, H-300,500), 7.57
(d, 2H, J ¼ 6:0 Hz, H-200,600), 8.01 (dd, 2H, J ¼ 1:5,
J ¼ 7:8 Hz, H-30,50), 8.08 (dd, 2H, J ¼ 2:1, J ¼ 7:8 Hz,
H-20,60); IR (KBr): 1790, 1765, 1665, 1590, 1555, 1465,
1355, 1150, 975, 860 cmꢀ1; UV kmax (CHCl3): 423, 282,
261 nm; EI-MS m=z (rel abund %): 294 (Mþ, 45), 105
(100), 77 (75). Anal. Calcd for C16H10N2O4: C, 65.30; H,
3.43; N, 9.52. Found: C, 65.28; H, 3.45; N, 9.50.
6.3. Neutrophil chemotaxis
Chemotaxis was measured using a modified Boyden
chamber (Neuro Probe, Cabin John, MD, USA), as
previously described.37 Briefly to the lower compartment
25 lL of the formyl-methionyl-leucyl-phenylalanine
(fMLP) (10ꢀ8 M) chemoattractant (Sigma Chemical Co.,
USA) was added. The upper compartment was sepa-
rated from the lower compartment by a cellulose filter
(2 lm pore size; Poretics Co., USA). To the upper
compartment 5 · 104 cells in a 50 lL HBSS (with cal-
cium and magnesium) was added. Compounds (25 and
50 lg/mL) and control were added each in duplicate
following the addition of cells. The chemoattractant
buffer (DMSO and HBSS without calcium and magne-
sium, 1:1 ratio) was added as a control. The chamber
was incubated at 37 ꢁC in CO2 environment for 60 min.
Cells within the filter were stained with hematoxylin
and their travelling distance was measured with the
help of light microscope in five randomly chosen fields
per filter.
6.1.12. 4-[(E)-(4-Methoxyphenyl)methylidene]-2-methyl-
1,3-oxazol-5(4H)-one (12). Compound 12 was prepared
in the same way as described for compound 3 from
4-methoxy benzaldehyde (1.20 g, 12.28 mmol), N-acet-
ylglycine (2.0 g, 11.1 mmol), acetic anhydride (3.0 mL,
33.6 mmol) and fused sodium acetate (1.0 g, 12.1 mmol).
The product was obtained as light orange solid 2.0 g
(64%). Mp: 158 ꢁC; Rf : 0.66 (n-butanol–acetic acid–H2O
3:3:1); 1H NMR (300 MHz, CDCl3) d: 2.18 (s, 3H, CH3),
3.88 (s, 3H, OCH3), 7.0 (d, 2H, J ¼ 8:8 Hz, H-30,50), 7.22
(s, 1H, H-6), 8.17 (t, 2H, J ¼ 8:8 Hz, H-20,60); IR (KBr):
1793, 1764, 1651, 1314, 1306, 1166, 984, 896 cmꢀ1; UV
kmax (CHCl3): 385, 259, 201 nm; EI-MS m=z (rel abund
%): 279 (Mþ, 27), 105 (100) 51 (6). Anal. Calcd for
C17H13NO3: C, 73.10; H, 4.70; N, 5.01. Found: C, 73.11;
H, 4.69; N, 5.00.
6.4. T-Cell proliferation assay
Peripheral blood mononuclear cells (PBMC) were iso-
lated from heparinized venous blood of healthy adult
donor by Ficoll–Hypaque gradient centrifugation.38
Cells were proliferated following a method reported by
Nielsen et al.39 Briefly cells were cultured at a concen-
tration of 5 · 105/mL in a 96 well round bottom tissue
culture plate (Nalge Nunc. Inter.). Cells were stimulated
with 1.25 lg/mL of phytohemagglutinin (Sigma Co.,
USA). Various concentrations of oxazolone compounds
were added to give final concentrations of 7.5, 16, 25 and
50 lg/mL each in triplicate. The plate was incubated for
72 h at 37 ꢁC in 5% CO2 incubator. After 72 h, cells were
6.1.13. 4-[(E)-(4-Nitrophenyl)methylidene]-2-methyl-1,3-
oxazol-5(4H)-one (13). Compound 13 was prepared in
the same way as described for compound 3, from 4-
nitrobenzaldehyde (2.31 g, 15.28 mmol), N-acetylglycine
(2.0 g,
11.1 mmol),
acetic
anhydride
(3.0 mL,
33.6 mmol), fused sodium acetate (1.0 g, 12.1 mmol).
The product was obtained as yellow solid 2.0 g (61%).
1
Rf : 0.68 (n-butanol–acetic acid–H2O 3:3:1); H NMR
(300 MHz, CDCl3) d: 2.18 (s, 3H, CH3), 7.24 (br s, 1H,
H-6), 8.01 (dd, 2H, J ¼ 1:5, J ¼ 7:8 Hz, H-30,50), 8.08

2056
M. A. Mesaik et al. / Bioorg. Med. Chem. 12 (2004) 2049–2057
pulsed with (0.5 lCi/well) tritiated thymidine, and fur-
ther incubated for 18 h. Cells were harvested onto a glass
fibre filter (Cambridge Technology, USA) using cell
harvester (SKATRON A.S. Flow Lab. Norway). The
tritiated thymidine incorporation into the cells was
measured by a liquid scintillation counter (1211 LKB
WALLAC). CPM results were recorded after 120 s.
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This work was supported by a grant from the Allergy
and Immunology Research Center and Herbal Medicine
Research Center, Institute for Medical Research, Kuala
Lumpur, Malaysia. A travelling grant to Dr. M. Ahmad
for visit to Institute of Medical Research and a research
grant to Dr. Khalid M. Khan (grant no. 01-310 RG/
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Sciences) is hereby gratefully acknowledged. Mr. Zia-
Ullah and Ms. Shagufta Rahat are thankful to the
Higher Education Commission (HEC), Pakistan for
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to Dr. Rafat A. Siddiqui, Senior Investigator and
Director, Cellular Biochemistry, Methodist Research
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