Synthesis and Molecular Docking Studies of Novel 2-Phenyl-4-Substituted Oxazole Derivatives as Potential Anti-cancer Agents

Article abstract of DOI:10.1002/jhet.2422

(Figure presented) A novel series of 2,4-disubstituted oxazole derivatives were synthesized, screened for their anti-tumor activity against three cell lines MCF-7, TK-10, and UACC-62. Molecular docking study was carried out against epidermal growth factor receptor. A new series of 2-phenyl-4-substituted oxazole derivatives were synthesized. A series of chiral α-amino acid derivatives 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 were synthesized by coupling various l-acylated amino acid azide 3. The synthesized compounds were tested for their in vitro antitumor activity against MCF-7, TK-10, and UACC-62 cell lines. Compound 6 exhibited the strongest inhibitory activity against TK cell lines, while compound 12 showed the highest activity against MCF-7 cell lines. Compound 14 was the most active against UACC-62 cell lines. Furthermore, a molecular docking study of the most active compounds was carried out using epidermal growth factor receptor X-ray 3D structure (protein data bank ID 1 M17). Docking results revealed that compound 6 showed the highest binding energy of ΔG = -78.17 Kcal/mol.

Full text of DOI:10.1002/jhet.2422

Month 2015 Synthesis and Molecular Docking Studies of Novel 2-Phenyl-4-Substituted
Oxazole Derivatives as Potential Anti-cancer Agents
d
e
Ahmed O. H. El-Nezhawy,b,c Ahmad Farouk Eweas,a,b Mohamed A. A. Radwan, and Tarek B. A. El-Naggar
*
aMedicinal Chemistry Department, National Research Center, Dokki, Cairo, Egypt
bPharmaceutical Chemistry Department, College of Pharmacy, Taif University, Taif, Saudi Arabia
cDepartment of Chemistry of Natural and Microbial Products, National Research Center, Dokki, Cairo, Egypt
dDepartment of Applied Organic Chemistry, National Research Centre, Dokki, Giza, Egypt
ePharmacology Department, Faculty of Pharmacy, Complutense University of Madrid, Madrid, Spain
*
E-mail: eweas1@gmail.com
Received November 26, 2014
DOI 10.1002/jhet.2422
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
A novel series of 2,4-disubstituted oxazole derivatives were synthesized, screened for their anti-tumor ac-
tivity against three cell lines MCF-7, TK-10, and UACC-62. Molecular docking study was carried out against
epidermal growth factor receptor. A new series of 2-phenyl-4-substituted oxazole derivatives were synthe-
sized. A series of chiral α-amino acid derivatives 6–15 were synthesized by coupling various L-acylated
amino acid azide 3. The synthesized compounds were tested for their in vitro antitumor activity against
MCF-7, TK-10, and UACC-62 cell lines. Compound 6 exhibited the strongest inhibitory activity against
TK cell lines, while compound 12 showed the highest activity against MCF-7 cell lines. Compound 14 was
the most active against UACC-62 cell lines. Furthermore, a molecular docking study of the most active com-
pounds was carried out using epidermal growth factor receptor X-ray 3D structure (protein data bank ID 1 M17).
Docking results revealed that compound 6 showed the highest binding energy of ΔG = ꢀ78.17 Kcal/mol.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
in particular are common in nature. An increasing number
of biologically active compounds contain the 2,4-
disubstituted oxazole ring including the streptogramin A
antibiotics [9], the potent anti-tumor phorboxazoles [10],
the highly cytotoxic disorazoles [11], the secondary metab-
olite ajudazols [12], and the hennoxazole [13] (Fig. 1).
Recently, 2,4-disubstituted oxazoles derivatives have
been reported to have pronounced anti-tumor activities by
triggering apoptosis in human tumor cells [14].
The epidermal growth factor receptor (EGFR) is a cellu-
lar transmembrane tyrosine kinases that is over-expressed
in a significant number of human tumors (e.g., breast, ovar-
ian, colon, and prostate); their expression levels often cor-
relate with vascularity and are associated with poor
prognosis in patients [15–18]. Inhibitors of the EGFR
PTK are therefore expected to have great therapeutic poten-
tial in the treatment of malignant and nonmalignant epithe-
lial diseases. Therefore, EGFR tyrosine kinase represents
Cancer continues to be a major health problem in devel-
oping and nondeveloped countries [1–5]. Surpassing heart
diseases, it is the number one killer due to various world-
wide factors. Although major advances have been made
in the chemotherapeutic management of some patients,
the continued commitment and need for discovering new
anti-cancer drugs remains critically important.
Oxazoles are considered an important class of nitrogen
heterocycles that exhibit diverse biological activities. A
large number of oxazole-containing natural products that
exhibit anti-cancer, antifungal, or antibacterial properties
have been isolated, and many of these compounds have
been synthesized [6]. Because of the sheer number of bio-
logically active oxazole natural products and pharmaceuti-
cals that have emerged in the last 20 years, this class of
compounds has begun to approach the forefront of impor-
tant nitrogen heterocycles [7,8]. 2,4-Disubstituted oxazoles
© 2015 HeteroCorporation

A O. H. El-Nezhawy, A. F. Eweas, M. A. A. Radwan, and T. B. A. E-Naggar
Vol 9999
Figure 1. Oxazole-containing marine natural products.
by cyclodehydration (TFAA/THF, 1:1v/v), afforded the
functionalized oxazoles 1 in good yield. Compound 1
reacted with hydrazine hydrate to give the corresponding
hydrazide 2, which in turn reacted with sodium nitrite to
afford 2-phenyloxazole-4-carbonyl azide 3. The hydrazide
2 then reacted with CS2 in the presence of KOH to yield
the sodium salt 4, which reacted with hydrazine hydrate to
give 4-amino-5-(2-phenyloxazol-4-yl)-4H-1, 2, 4-triazole-
3-thiol 5 (Scheme 1).
We extended our study on 2,4-disubstituted oxazoles
hoping to synthesize a new class of compounds that could
be used as an anti-tumor agent with improved cytotoxic ac-
tivity. One of the critical factors that affect cytotoxicity is
to enhance the drug delivery inside the cell by enhancing
the cell uptake. Amino acids are known to improve the cell
uptake of anti-tumor agents [25]. Therefore, a possible
strategy for increasing the transport of lipophilic com-
pounds of biological interest across the cell membrane
was the conjugation with amino acids. This motivated us
to synthesize 2,4-disubstituted oxazole moiety conjugated
with flexible amino acids as a side chain, hoping that the
produced compounds will have improved biological activ-
ity. At this point, a series of 2-phenyloxazole-L-amino acid
conjugates have been synthesized starting with 2-
phenyloxazole-4-carbonyl azide 3. The latter reacted with
the appropriate L-amino acid ester hydrochloride in the
presence of Et3N at ꢀ5°C to give 6–15 in good yields
(Scheme 2).
an attractive target for the development of novel anti-
cancer agents. In the past few years, large numbers of small
organic molecules have been employed to inhibit kinase
function by targeting the Adenosine Tri Phosphate (ATP)
binding site of EGFR tyrosine kinase. Gefitinib (Iressa,
AstraZeneca London, United Kingdom) and erlotinib
(Tarceva, OSI Pharmaceuticals, Long Island, NY) are the
representative drugs for this kind of inhibitors [19,20]. Har-
ris et al. reported that 2-anilino-5-aryloxazoles can be used
as a novel class of vascular endothelial growth factor recep-
tor 2 (also called the kinase insert domain containing recep-
tor) kinase inhibitors. The structure–activity relationships
as well as the enzyme-binding model were also illustrated.
In this model, 2-anilino-5-aryloxazoles bond to the ATP
binding site of kinase insert domain containing receptor.
The central oxazole ring comprises the core structure of
the compound and interacts with Cys919 of the peptide
backbone via hydrogen acceptor bonding with the oxazole
nitrogen [21].
Motivated by the previous findings and in continuation
of our ongoing program aimed at finding new structure
leads with potential chemotherapeutic activities [22,23], a
new series of novel 2,4-disubstituted 1,3-oxazole deriva-
tives are synthesized and screened for their anti-tumor ac-
tivity against three different cancer cell lines and
molecular docking of the most active compounds against
EGFR crystal structure. Protein data bank (PDB) code—
1 M17 is also demonstrated.
Importantly, this method is efficient, affordable, and re-
liable and can be generalized to link amino acids to differ-
ent moieties. The structure’s all new compounds were
RESULTS AND DISCUSSION
1
proved via H NMR, 13C NMR, mass spectrometry, and
Chemistry. The study started with preparation of the
important 2,4-disubstituted oxazole keystone building
block 1, which was achieved via a modified Hantzsch
synthesis [24]. The reaction of benzamide, with ethyl
bromopyruvate using CO3-buffered conditions, followed
correct elemental analysis compare with experimental part.
Anti-tumor activity.
The prepared compounds were
divided into two groups: The first group represents
carboxylate, carbohydrazide, and triazole-3-thiol derivatives,
while the second group represents amino acid derivatives
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Scheme 1. Reagents and conditions: (a) NaHCO3, THF, 60°C, 15 h. (b) NH2NH2, C2H5OH, reflux 3 h. (c) AcOH, 1 N HCl, NaNO2, ꢀ5°C at room
temperature, 15 min. (d) KOH, CH3OH, CS2, stirring overnight; and (e) NH2NH2, H2O, reflux 5 h.
and their activities were represented in Tables 1 and 2,
respectively. We examined the newly synthesized
compounds for in vitro activity against three human cancer
cell lines, namely, renal adenocarcinoma (TK-10), human
breast adenocarcinoma (MCF-7), and human melanoma
(UACC-62). Three response parameters (GI50, TGI, and
LC50) were calculated for each cell line. The growth
inhibitory activity (GI50) value corresponds to the
concentration of the compounds causing 50% decrease in
net cell growth. The total growth inhibition (TGI) value
(cytostatic activity) is the concentration of the compounds
resulting in total growth inhibition, and the lethal
concentration (LC50) value (cytotoxic activity) is the
concentration of the compounds causing net 50% loss of
initial cells at the end of the incubation period. Tables 1 and
2 list the GI50, TGI, and LC50 values for carboxylate,
carbohydrazide, and triazole-3-thiol derivatives (group I)
and amino acid derivatives (group II), respectively.
Regarding the activities of the carboxylate, carbohydrazide,
and triazole-3-thiol derivatives in Table 1, compounds 1, 2,
and 5 showed a GI50 on the three mentioned tumoral cell lines
at the tested doses. However, compound 1 showed only
growth inhibitory effect against the TK-10 and UACC-62
cells but did not possess any activity against the MCF-7 cell
line. Compound 2 only affected the renal adenocarcinoma
(TK-10) cell line. Compound 5 was the most cytotoxic against
TK-10 (GI50 =0.46μM). Moreover, the most effective com-
pound against the MCF-7 cell line was 5 (GI50 =22.36μM).
Compound 5 was the most effective against the UACC-62 cell
line (GI50 =4.23μM). The growth of UACC-62 cells was to-
tally inhibited by compound 1 (TGI =0.59 μM). The growth
of TK-10, MCF-7, and UACC-62 cells was totally inhibited
by compound 5 (TGI = 61.15, 77.71 and 36.61μM).
However, no tested compounds demonstrated cytostatic activ-
ity (LC50) against TK-10, MCF-7, and UACC-62 cell line. On
the other hand, regarding the activities of the amino acid deriv-
atives in Table 2, compounds 6, 9, 10, 12, 13, and 14 showed
GI50 against the three mentioned tumoral cell lines at the tested
doses. However, compounds 7, 8, 11, and 15 showed only an
inhibitory effect against the growth (GI50) of the TK-10 and
UACC-62 cells but did not possess any activity against the
MCF-7 cell line. Compound 6 was the most cytotoxic against
TK-10 (GI50 =0.78μM). Moreover, the most effective com-
pounds against the MCF-7 cell line were 9, 12, and 14
(GI50 = 5.30, 0.78 and 4.89μM). However, compounds 12,
13, and 14 were the most effective on the UACC-62 cell line
(GI50 = 0.66, 3.22 and 0.49μM). The growth of the TK-10 cell
line was totally inhibited by compounds 9, 10, and 13
(TGI=41.45, 78.93 and 13.02μM), while compounds 8, 9,
10, 12, and 13 showed a moderate cytostatic activity against
the UACC-62 cells with TGI values of 5.49, 46.63, 75.43,
64.33, and 4.44μM. Compounds 7 and 8 showed a cytostatic
activity against the MCF-7 cells with TGI values of 88.75 and
28.49μM.
Structure–activity relationship.
By observing the
activity of the two tested groups of compounds, the
relationship between the structure variation and the
activity can be concluded as follows.
The effect of cyclization by formation of triazolethiol on
the activity was established by comparing the activity of
group I compounds (Table 1) with their carboxylate,
carbohydrazide derivatives. It was obvious that the forma-
tion of triazolethiol resulted in an enhancement in the ac-
tivity against the three tumoral cell lines. By comparing
the activity of group II compounds (Table 2), it was obvi-
ous that the activity against the tested cell lines was
Journal of Heterocyclic Chemistry
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A O. H. El-Nezhawy, A. F. Eweas, M. A. A. Radwan, and T. B. A. E-Naggar
Vol 9999
Scheme 2. Reaction condition: appropriate amino acid methyl ester hydrochloride, ethyl acetate, Et3N, ꢀ5°C, 24 h.
Table 1
Group I concentrations (μM) required to cause different inhibitions.
Cell line
Inhibition
Compound number
parameter
TK-10
MCF-7
UACC-62
(1)
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
46.82 0.11
>100
>100
13.16 1.64
>100
>100
>100
>100
>100
>100
22.10 4.15
0.59 0.53
>100
(2)
(5)
>100
>100
>100
>100
>100
0.46 0.12
61.15 2.05
>100
22.36 2.60
77.71 14.21
>100
4.23 3.10
36.61 11.32
>100
LC50
sufficiently reduced. Compound 6 exhibited the strongest
inhibitory activity against TK cell lines, this may be due
to the presence of phenolic group, while compound 12
showed the highest activity against MCF-7 cell lines;
moreover, compound 14 was the most active against
UACC-62 cell lines.
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Table 2
Group II concentrations (μM) required to cause different inhibitions.
Cell line
Inhibition
Compound number
parameter
TK-10
MCF-7
UACC-62
(6)
GI50
TGI
0.78 0.04
>100
26.67 3.69
>100
8.63 2.21
>100
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
>100
61.33 28.10
>100
>100
0.53 0.08
>100
>100
>100
88.57 18.28
>100
>100
52.27
>100
>100
(7)
(8)
>100
0.42 0.05
5.49 2.07
>100
25.43 2.12
46.63 10.72
>100
21.19 0.91
75.43 7.11
>100
50.33 8.27
>100
28.49 2.34
34.35 8.23
5.30 0.02
>100
>100
58.79 0.63
>100
>100
(9)
51.26 2.33
41.45 3.02
>100
41.75 7.21
78.93 21.13
>100
47.60 6.92
>100
>100
11.25 4.31
>100
(10)
(11)
(12)
(13)
(14)
(15)
>100
>100
>100
>100
0.78
>100
>100
87.23
>100
>100
4.89
>100
>100
>100
>100
>100
0.66 0.26
64.33 11.20
>100
3.22 0.45
4.44 2.11
27.37 6.69
0.49 0.19
>100
>100
18.39 0.30
13.02 2.49
38.28 12.33
22.67 2.02
>100
>100
7.29 0.19
>100
LC50
GI50
TGI
>100
4.27 3.22
>100
LC50
>100
>100
>100
The range of doses assayed was 10ꢀ4, 10ꢀ5, 10ꢀ6, 10ꢀ7, and 10ꢀ8 M. Results are mean S.E.M. (n = 3).
Molecular docking study.
The level of anti-tumor
the EGFR kinase forming four hydrogen bonds between
carbonyl oxygen O-2 of the ligand and NH of Gly-786 of
the receptor. Hydroxyl oxygen O-4 of the ligand and NH
H-22 of Glu-952 from the receptor, hydroxyl hydrogen
H-10 from the ligand with hydroxyl oxygen from Ser-
787 of the receptor, and amide hydrogen H-2 from the li-
gand with carbonyl oxygen at the α-carbon atom of Glu-
961 from the receptor. Compounds 6, 12, and 14 showed
a common hydrogen bonding with both Gly-786 and
Glu-961 of the EGFR kinase (Fig. 3), which suggests that
these two amino acids are critical in fitting the 2,4-oxazole
amino acid derivatives into the binding site of the EGFR
kinase receptor. From the previous results, we can con-
clude that the 2,4-disubstituted amino acid oxazole deriva-
tives prepared might inhibit the EGFR kinase enzyme,
especially in MCF-7 breast cancer cell lines.
activities of the proposed compounds over the MCF-7
breast cancer cell, in which EGFR is highly expressed,
prompted us to perform molecular docking into the ATP
binding site of EGFR to predict if these compounds have
analogous binding mode to the EGFR inhibitors.
Compounds 5, 6, 12, and 14 were used for the docking
study as representative examples of the most active and
moderate active compounds. Molecular docking studies
were carried out using Molsoft ICM 3.5-0a software
installed on 1.73G Core i7. The aim of the flexible docking
calculations is the prediction of correct binding geometry
for each binder. The scoring functions and hydrogen bonds
formed with the surrounding amino acids of the receptor.
The crystal structure of EGFR with Erlotinib (TarcevaTM
(1 M17) was retrieved from PDB. The docking energy
scores of tested compounds are listed in Table 3.
)
The docking results revealed the following facts: all
tested compounds showed moderate to high binding affin-
ity towards EGFR ranging from ꢀ60.79 to ꢀ78.17kcal/
mol. Compound 6 showed the highest binding energy of
ΔG =ꢀ78.17 Kcal/mol (Fig. 2) and was nicely bound to
EXPERIMENTAL
General data. All starting materials and reagents were
purchased from Sigma-Aldrich, BDH, and Fluka and used
without further purification. All solvents were either of
Journal of Heterocyclic Chemistry
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A O. H. El-Nezhawy, A. F. Eweas, M. A. A. Radwan, and T. B. A. E-Naggar
Vol 9999
Table 3
The docking energy scores of compounds 5, 6, 12, and 14 with the amino acid residues of the target enzyme EGFR forming hydrogen bonds.
Compound number
Docking score (Kcal/mol)
ꢀ62.71
Number of hydrogen bonds
Amino acid residues forming hydrogen bonds in A0
5
6
2
4
T766 hg1–m M n2: 2.63 A
M769 hn–m M o1: 2.27 A
G786 hn–m M o2: 1.05 A
Q952 he21–m M o4: 1.95 A
S787 og–m M h10: 2.52 A
E961 o–m M h2: 1.79 A
I785 hn–m M n2: 2.73 A
G786 hn–m M o3: 1.12 A
G786 hn–m M o2: 2.10 A
E961 o–m M h5: 1.66 A
G786 hn–m M o1: 1.58 A
E961 hn–m M o3: 2.37 A
ꢀ78.17
12
14
ꢀ60.79
ꢀ76.36
4
2
Figure 2. Molecular docking of compound 6 with EGFR kinase shows intermolecular hydrogen bonds with Gly-786, Glu-952, Ser-787, and Glu-961.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 3. Molecular docking of compound 14 with EGFR kinase shows intermolecular hydrogen bonds with Gly-786 and Glu-961. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
analytical grades or dried and distilled immediately prior to
use. Melting points were measured on a Stuart-SMP10
melting point apparatus and are uncorrected. TLC was
performed using Merck pre-coated Silica gel 60F254
aluminum sheets, and spots were visualized by UV
(254nm), KMnO4 solution and/or charring with H2SO4/
EtOH (5% v/v). Column chromatography was carried out
on Silica Gel 60 (70–230meshes ASTM, Merck) using the
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specified eluents. NMR spectra were recorded with JEOL
ECA-500, JEOL EX-270, and Varian Mercury-200BB
spectrometers at room temperature in solvents given. The
infrared spectra were carried out in a potassium bromide
disk on JASCO FT/IR spectrophotometer. The mass
spectra were recorded on mass spectrometer MS 50
equipment of (AEI) (Kratos, FRG) and Finnegan SSQ
7000 at 70eV. The elemental analyses were carried out at
Cairo University in an elemental analyzer Vario EI—0.5%.
NaNO2 (0.069 g, 1.0 mmol) in cold water (3mL) was
added. After stirring at ꢀ5°C for 15min, a yellow syrup
was formed. The crude product was extracted with cold
ethyl acetate (30 mL), washed with cold 3% NaHCO3
then water and finally, dried over anhydrous Na2SO4 to
afford 3 (1.85 g, 75%). Recrystallization from EtOH gave
analytically pure material mp 116–119°C. 1H NMR
(500 MHz, CDCl3): δ (ppm): 7.37–7.45 (m, 3H, Ar),
7.91–8.00 (m, 2H, Ar), 8.55 (s, 1H, Ar). 13C NMR
(50 MHz, CDCl3): δ 125.9, 128.3, 129.2, 131.6, 136.4,
140.3, 165.9, 166.7; MS (m/z, %) 214 (M+, 12), 172 (M+
ꢀ31, 100). Anal. Calcd. for C10H6N4O2 (214.18): C,
Chemistry
Ethyl 2-phenyloxazole-4-carboxylate (1) [26]. To a stirred
suspension of benzamide (2.41 g, 33.9mmol) and
powdered NaHCO3 (11.4 g, 136 mmol) in THF (130 mL)
under nitrogen, ethyl bromopyruvate (5.2 mL, 37mmol)
was added. The reaction mixture was heated at 55–60°C
for 15h; additional ethyl bromopyruvate (5.2 mL,
37mmol) was added and heating continued for an
additional 8h. The reaction mixture was then cooled at
room temperature; solid impurities were removed by
filtration through celite. The filtrate was concentrated under
reduced pressure. The crude residue was immediately
dissolved in THF (15mL), cooled to 0°C, and treated with
trifluoroacetic anhydride (15mL). This stirred solution was
warmed at room temperature and stirred overnight. The
reaction mixture was slowly quenched with saturated
aqueous NaHCO3 (30mL). Layers were separated, and the
aqueous layer was extracted with ethyl acetate (375mL).
The combined organic layers were dried over (MgSO4) and
concentrated under reduced pressure. The resultant product
was then purified by column chromatography (SiO2,
hexanes/ethyl acetate, 3:1) to afford 1 as a white solid
(4.34g, 77%) mp 65–66°C. The spectral data of isolated 1
were in good agreement with the literature values.
56.08 H, 2.82; N, 26.16. Found: C, 55.97; H, 2.97; N,
26.11.
Potassium 2-(2-phenyloxazole-4-carbonyl)hydrazinecarbodithioate
(4).
Carbohydrazide 2 (0.083mol) was treated with a
solution of potassium hydroxide (6.4g, 0.125mol) then
dissolved in methanol (50mL) at 0–5°C during stirring.
Carbon disulfide (9.6g, 0.125mol) was then added slowly,
and the reaction mixture was allowed to stir overnight at
room temperature. The solid product of potassium
dithiocarbazinate 4 was filtered off, washed with chilled
methanol, and air-dried. The product was directly used for
the next step without further purification.
4-Amino-5-(2-phenyloxazol-4-yl)-4H-1,2,4-triazole-3-thiol (5).
Potassium dithiocarbazinate 4 was dissolved in water (8 mL)
and hydrazine hydrate (2mmol), the reaction mixture was
refluxed for 5h. During progress of the reaction, the
reaction mixture turned to green with evolution of hydrogen
sulfide and finally, it became homogeneous. The reaction
mixture was then diluted with cold water and acidified with
conc. hydrochloric acid. The white precipitate was filtered
off, washed with cold water, and recrystallized from
aqueous methanol to afford compound 5. Yield 85%, mp
2-Phenyloxazole-4-carbohydrazide
(2).
Hydrazine
1
199–203°C. H NMR (500MHz, CDCl3): δ (ppm): 5.24
hydrate (1.25 g, 25 mmol) was added to a mixture of ethyl
2-phenyloxazole-4-carboxylate 1, (10 mmol), in (30 ml)
absolute ethanol. The reaction mixtures underwent reflux
for 3 h. The reaction mixture was then left to cool at room
temperature and poured over an ice/water mixture; the
formed solid product was filtered off and washed with
ethanol to afford 2 as white solid (98%). The crude
product was recrystallized from EtOH/DMF to afford
(br, s, 2H, NH2), 7.46–7.53 (m, 3H, Ar), 7.95–8.02 (m, 2H,
Ar), 8.63 (s, 1H, Ar) 13.20 (s, 1H, SH); MS (m/z, %) 259
(M+, 23), 226 (M+ ꢀ33, 10), 210 (M+ ꢀ49, 48), 145 (M+
ꢀ114, 100). Anal. Calcd. for C11H9N5OS (259.29): C,
50.95; H, 3.50; N, 27.01; S, 12.37. Found: C, 51.05; H,
3.59; N, 27.16; S, 12.29.
General procedure for the preparation of chiral α-amino acid
derivatives conjugated with 2-phenyloxazole (6–15).
To a
analytically pure material mp 158–160°C. IR (cmꢀ1
)
solution of the appropriate amino acid methyl ester
hydrochloride (1.0 mmol) in ethyl acetate (20 mL)
containing Et3N (0.2 mL), the azide 3 was added. The
reaction mixture was kept at ꢀ5°C for 24h, then at 25°C
for another 24h, followed by washing with 0.5 N HCl,
water, 3% solution of NaHCO3, and finally, dried over
anhydrous Na2SO4. The solution was evaporated to
dryness under reduced pressure to give the desired
3420–3320 (NH, NH2), 1680 (C¼O). 1H NMR
(500 MHz, CDCl3): δ (ppm): 4.20 (br s, 2H, NH2),
7.47–7.66 (m, 3H, Ar), 8.01–8.02 (m, 2H, Ar), 8.10 (br, s,
1H, NH), 8.75 (s, 1H, Ar). 13C NMR (50 MHz, CDCl3): δ
120.3, 123.9, 124.1, 127.3, 130.5, 135.1, 136.9, 143.8,
163.6, 165.9; MS (m/z, %) 203 (M+, 20), 172 (M+ ꢀ31,
100). Anal. Calcd. for C10H9N3O2 (203.2): C, 59.11; H,
4.46; N, 20.68. Found: C, 58.91; H, 4.53; N, 20.47.
2-Phenyloxazole-4-carbonyl azide (3). A mixture of the
hydrazide 2 (0.203 g, 1.0 mmol) in AcOH (6mL), 1 N HCl
(3 mL), and water (25 mL) was cooled to ꢀ5°C, then
products, 6–15, in 75–85% yields.
Methyl 3-(4-hydroxyphenyl)-2-(2-phenyloxazole-4-carboxamido)-
propanoate (6). Azide 3 and L-tyrosine methyl ester HCl.
1
Yield: (84%) of white crystals mp 191–193°C. H NMR
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A O. H. El-Nezhawy, A. F. Eweas, M. A. A. Radwan, and T. B. A. E-Naggar
Vol 9999
(DMSO-d6) δ (ppm): 3.02 (d, 2H, CH2), 3.61 (s, 3H, OCH3),
4.61 (t, 1H, NC*H), 6.62–6.64 (d, 2H, Ar), 6.99–7.00 (d, 2H,
Ar), 7.54–7.55 (m, 3H, Ar), 7.98–7.99 (m, 2H, Ar), 8.39 (br,
s, 1H, NH), 8.65 (s, 1H, Ar). Anal. Calcd. for C20H18N2O5
(366.37): C, 65.57; H, 4.95; N, 7.65. Found: C, 65.70; H,
1.37 (3H, d, J = 7.0, Me), 3.66 (s, 3H, OCH3), 4.45–4.60
(1H, m, CH), 7.51–7.56 (m, 3H, Ar), 8.08–8.12 (m, 2H,
Ar), 8.65 (br, s, 1H, NH), 8.76 (s, 1H, Ar). Anal. Calcd.
for C14H14N2O4 (274.27): C, 61.31; H, 5.14; N, 10.21.
Found: C, 61.39; H, 5.26; N, 10.11.
Methyl 2-(2-phenyloxazole-4-carboxamido)acetate (13).
4.89; N, 7.55.
Methyl 3-methyl-2-(2-phenyloxazole-4-carboxamido)butanoate
(7). Azide 3 and L-valine methyl ester HCl. Colorless
Azide 3 and glycine methyl ester HCl. White solid,
mp 168–171°C yield: (81%). 1H NMR (DMSO-d6) δ
(ppm): 3.56 (s, 3H, OCH3), 4.20 (2H, d, J = 5.6 CH2),
7.45–7.53 (m, 3H, Ar), 8.00–8.08 (m, 2H, Ar), 8.66
(br, s, 1H, NH), 8.79 (s, 1H, Ar). Anal. Calcd. for
C13H12N2O4 (260.25): C, 60.00; H, 4.65; N, 10.76.
oil yield: (83%). 1H NMR (DMSO-d6) δ (ppm): 0.85
(3H, d, J = 6.2 Hz, Me), 0.92 (3H, d, J = 6.2 Hz, Me),
1.82 (3H, m, CH), 3.69 (s, 3H, OCH3), 4.41–4.29
(1H, m, CH) 2.18 (m, 1H, CH), 3.64 (s, 3H, OCH3),
4.34 (m, 1H, NCH), 7.47–7.66 (m, 3H, Ar), 8.01–8.02
(m, 2H, Ar), 8.45 (br, s, 1H, NH), 8.75 (s, 1H, Ar).
Found: C, 59.90; H, 4.72; N, 10.64.
Methyl 4-(methylthio)-2-(2-phenyloxazole-4-carboxamido)
butanoate (14). Azide 3 and L-methionine methyl ester
Anal. Calcd. for C16H18N2O4 (302.33): C, 63.56; H,
6.00; N, 9.27. Found: C, 63.63; H, 5.91; N, 9.39.
Dimethyl 2-(2-phenyloxazole-4-carboxamido)pentanedioate
1
HCl. Colorless oil yield: (81%). H NMR (DMSO-d6) δ
(ppm): 1.97 (3H, s, SMe), 2.19–2.03 (2H, m, CH2), 2.40
(2H, t, J = 7.2 Hz, CH2), 3.68 (s, 3H, OCH3), 4.55–4.64
(1H, m, CH), 7.52–7.59 (m, 3H, Ar), 8.10–8.16 (m, 2H,
Ar), 8.45 (br, s, 1H, NH), 8.59 (s, 1H, Ar). Anal. Calcd.
for C16H18N2O4S (334.39): C, 57.47; H, 5.43; N, 8.38; S,
9.59. Found: C, 57.61; H, 5.57; N, 8.19; S, 9.71.
(8).
Azide 3 and L-glutamic acid dimethyl ester HCl.
Colorless oil yield: (78%). 1H NMR (DMSO-d6) δ
(ppm): 1.00–1.08 (m, 2H, CH2), 1.18–1.23 (m, 2H, CH2),
3.54 (s, 3H, OCH3), 3.62 (s, 3H, OCH3), 4.49 (m, 1H,
NC*H), 7.55–7.56 (m, 3H, Ar), 8.01–8.02 (m, 2H, Ar),
8.63 (br, s, 1H, NH), 8.72 (s, 1H, Ar). Anal. Calcd. for
C17H18N2O6 (346.33): C, 58.96; H, 5.24; N, 8.09. Found:
Methyl 2-phenyl-2-(2-phenyloxazole-4-carboxamido)acetate
(15).
Azide 3 and L-phenylglycine methyl ester HCl.
Colorless oil yield: (75%). 1H NMR (DMSO-d6) δ
(ppm): 3.58 (s, 3H, OCH3), 4.71 (1H, d, J = 5.4 CH),
7.33–7.58 (m, 8H, Ar), 8.01–8.11 (m, 2H, Ar), 8.30 (br,
s, 1H, NH), 8.62 (s, 1H, Ar). Anal. Calcd. for
C19H16N2O4 (336.34): C, 67.85; H, 4.79; N, 8.33. Found:
C, 59.02; H, 5.16; N, 8.17.
Methyl 3-methyl-2-(2-phenyloxazole-4-carboxamido)pentanoate
(9). Azide 3 and L-isoleucine methyl ester HCl. Colorless
1
oil yield: (82%). H NMR (DMSO-d6) δ (ppm): 0.84 (t,
J=2.99Hz, CH3), 0.87 (t, J=3.00Hz, 3H, CH3), 1.55–1.58
(m, 2H, CH2), 2.00–2.01 (m, 1H, CH), 3.56 (s, 3H, OCH3),
3.52 (d, J=1.00 Hz, 1H, NC*H), 7.50–7.51 (m, 3H, Ar),
7.98–7.99 (m, 2H, Ar), 8.49 (br, s, 1H, NH), 8.67 (s, 1H,
Ar). Anal. Calcd. for C17H20N2O4 (316.35): C, 64.54; H,
6.37; N, 8.86. Found: C, 64.70; H, 6.22; N, 8.79.
Methyl 3-phenyl-2-(2-phenyloxazole-4-carboxamido)propanoate
(10). Azide 3 and L-phenylalanine methyl ester HCl.
C, 67.97; H, 4.71; N, 8.26.
Anti-tumor activity
Assay for cytotoxic activity: human cell lines.
The
following human cancer cell lines were used in these
experiments: the human renal adenocarcinoma (TK-10), the
human breast adenocarcinoma (MCF-7), and the human
melanoma (UACC-62) cell lines. The human tumors
cytotoxicity was determined following protocols established
by National Cancer Institute (NCI) [27]. TK-10, MCF-7,
and UACC-62 cell lines were cultured in RPMI 1640
medium (BioWhittaker®) containing 20% fetal calf serum,
2mmol/L L-glutamine, 100 U/mL penicillin, and 100 μg/mL
streptomycin. All cell lines were maintained at 37°C in a 5%
CO2 atmosphere with 95% humidity. Maintenance cultures
were passaged weekly, and the culture medium was changed
twice a week. According to their growth profiles, the
optimal plating densities of each cell line were determined
(15× 103, 5×103, and 100× 103 cells/well for TK-10, MCF-
7, and UACC-62, respectively) to ensure exponential growth
throughout the experimental period and to ensure a linear
relationship between absorbance at 492nm and cell number
White solid, mp 221–223°C yield: (83%). 1H NMR
(DMSO-d6) δ (ppm): 3.44 (dd, J = 0.93, 2H, CH2), 3.69
(s, 3H, OCH3), 4.79 (t, J = 1.02 Hz, 1H, NC*H),
7.29–7.35 (m, 5H, Ar), 7.60–7.61 (m, 3H, Ar), 8.04–8.05
(m, 2H, Ar), 8.56 (br, s, 1H, NH), 8.73 (s, 1H, Ar). Anal.
Calcd. for C20H18N2O4 (350.37): C, 68.56; H, 5.18; N,
8.00. Found: C, 68.48; H, 5.30; N, 8.11.
Methyl 4-methyl-2-(2-phenyloxazole-4-carboxamido)pentanoate
(11). Azide 3 and L-leucine methyl ester HCl. Colorless
oil yield: (85%). 1H NMR (DMSO-d6) δ (ppm): 0.87
(3H, d, J = 6.4 Hz, Me), 0.94 (3H, d, J = 6.4 Hz, Me),
1.42–1.82 (3H, m, CH2, CH), 3.69 (s, 3H, OCH3),
4.41–4.29 (1H, m, CH), 7.48–7.69 (m, 3H, Ar), 8.08–8.14
(m, 2H, Ar), 8.77 (br, s, 1H, NH), 8.76 (s, 1H, Ar). Anal.
Calcd. for C17H20N2O4 (316.35): C, 64.54; H, 6.37; N,
when analyzed by the sulphorhodamine B (SRB) assay.
Testing procedure and data processing. The SRB assay
8.86. Found: C, 64.45; H, 6.28; N, 8.95.
Methyl 2-(2-phenyloxazole-4-carboxamido)propanoate (12).
was used in this study to assess growth inhibition. This
colorimetric assay estimates cell number indirectly by
staining total cellular protein with the SRB dye. For the
Azide 3 and L-alanine methyl ester HCl. White solid, mp
1
181–183°C yield: (85%). H NMR (DMSO-d6) δ (ppm):
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
2,4-Disubstituted Oxazoles, L-Amino Acids, Anti-tumor, Molecular Docking
assay, cells were detached with 0.1% trypsin-EDTA
(Sigma Chemical Co.) to make single-cell suspensions,
and viable cells were counted using a Coulter counter
and diluted with medium to give final densities of
15× 104, 5× 104, and 100× 104 cells/mL for TK-10,
MCF-7, and UACC-62, respectively. One hundred
microliters per well of these cell suspensions was seeded
in 96-well microliter plates and incubated to allow for
cell attachment. After 24 h, the cells were treated with the
serial concentrations of the synthesized compounds. They
were initially dissolved in an amount of 100% DMSO
(40 mmol/L) and further diluted in medium to produce
five concentrations. One hundred microliters per well of
each concentration was added to the plates to obtain final
concentration of 10ꢀ4, 10ꢀ5, 10ꢀ6, 10ꢀ7, and 10ꢀ8 M for
the synthesized compounds. The DMSO concentration
for the tested dilutions was not greater than 0.25% (v/v),
the same as in solvent control wells. The final volume in
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Acknowledgments. This work was financially supported by Taif
University, Taif, Saudi Arabia grant no. 1782-433-1.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet