Design, synthesis, molecular docking, and anticancer activity of benzoxazole derivatives as VEGFR-2 inhibitors

Article abstract of DOI:10.1002/ardp.201900113

Novel series of benzoxazoles 4_a-f-16 were designed, synthesized, and evaluated for anticancer activity against HepG2, HCT-116, and MCF-7 cells. HCT-116 was the most sensitive cell line to the influence of the new derivatives. In particular, compound 5_e was found to be the most potent against HepG2, HCT-116, and MCF-7 with IC₅₀ = 4.13 ± 0.2, 6.93 ± 0.3, and 8.67 ± 0.5 μM, respectively. Compounds 5_c, 5_f, 6_b, 5_d, and 6_c showed the highest anticancer activities against HepG2 cells with IC₅₀ of 5.93 ± 0.2, 6.58 ± 0.4, 8.10 ± 0.7, 8.75 ± 0.7, and 9.95 ± 0.9 μM, respectively; HCT-116 cells with IC₅₀ of 7.14 ± 0.4, 9.10 ± 0.8, 7.91 ± 0.6, 9.52 ± 0.5, and 12.48 ± 1.1 μM, respectively; and MCF-7 cells with IC₅₀ of 8.93 ± 0.6, 10.11 ± 0.9, 12.31 ± 1.0, 9.95 ± 0.8, and 15.70 ± 1.4 μM, respectively, compared with sorafenib as a reference drug with IC₅₀ of 9.18 ± 0.6, 5.47 ± 0.3, and 7.26 ± 0.3 μM, respectively. The most active compounds 5_c-f and 6_b,c were further evaluated for their vascular endothelial growth factor receptor-2 (VEGFR-2) inhibition. Compounds 5_e and 5_c potently inhibited VEGFR-2 at lower IC₅₀ values of 0.07 ± 0.01 and 0.08 ± 0.01 μM, respectively, compared with sorafenib (IC₅₀ = 0.1 ± 0.02 μM). Compound 5_f potently inhibited VEGFR-2 at low IC₅₀ value (0.10 ± 0.02 μM) equipotent to sorafenib. Our design was based on the essential pharmacophoric features of the VEGFR-2 inhibitor sorafenib. Molecular docking was performed for all compounds to assess their binding pattern and affinity toward the VEGFR-2 active site.

Full text of DOI:10.1002/ardp.201900113

Received: 13 April 2019
Revised: 12 July 2019
Accepted: 17 July 2019
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DOI: 10.1002/ardp.201900113
F U L L P A P E R
Design, synthesis, molecular docking, and anticancer activity
of benzoxazole derivatives as VEGFR‐2 inhibitors
Abdel‐Ghany A. El‐Helby1
Ahmed A. Al‐Karmalawy1
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Helmy Sakr1
Khaled El‐Adl1,3
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Ibrahim H. Eissa1
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Hamada Abulkhair2
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1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Al‐Azhar University,
Cairo, Egypt
Abstract
Novel series of benzoxazoles 4a‒f‒16 were designed, synthesized, and evaluated for
anticancer activity against HepG2, HCT‐116, and MCF‐7 cells. HCT‐116 was the most
sensitive cell line to the influence of the new derivatives. In particular, compound 5e
was found to be the most potent against HepG2, HCT‐116, and MCF‐7 with
IC50 = 4.13 0.2, 6.93 0.3, and 8.67 0.5 µM, respectively. Compounds 5c, 5f, 6b, 5d,
and 6c showed the highest anticancer activities against HepG2 cells with IC50 of
5.93 0.2, 6.58 0.4, 8.10 0.7, 8.75 0.7, and 9.95 0.9 µM, respectively; HCT‐116
cells with IC50 of 7.14 0.4, 9.10 0.8, 7.91 0.6, 9.52 0.5, and 12.48 1.1 µM,
respectively; and MCF‐7 cells with IC50 of 8.93 0.6, 10.11 0.9, 12.31 1.0,
9.95 0.8, and 15.70 1.4 µM, respectively, compared with sorafenib as a reference
drug with IC50 of 9.18 0.6, 5.47 0.3, and 7.26 0.3 µM, respectively. The most
active compounds 5c‒f and 6b,c were further evaluated for their vascular endothelial
growth factor receptor‐2 (VEGFR‐2) inhibition. Compounds 5e and 5c potently
inhibited VEGFR‐2 at lower IC50 values of 0.07 0.01 and 0.08 0.01 µM,
respectively, compared with sorafenib (IC50 = 0.1 0.02 µM). Compound 5f potently
inhibited VEGFR‐2 at low IC50 value (0.10 0.02 µM) equipotent to sorafenib. Our
design was based on the essential pharmacophoric features of the VEGFR‐2 inhibitor
sorafenib. Molecular docking was performed for all compounds to assess their binding
pattern and affinity toward the VEGFR‐2 active site.
2Department of Pharmaceutical Organic
Chemistry, Faculty of Pharmacy, Al‐Azhar
University, Cairo, Egypt
3Department of Pharmaceutical Chemistry,
Faculty of Pharmacy and Drug Technology,
Heliopolis University for Sustainable
Development, Cairo, Egypt
Correspondence
Khaled El‐Adl and Ibrahim H. Eissa,
Pharmaceutical Chemistry Department,
Faculty of Pharmacy, Al‐Azhar University,
1 Al‐Mukhayam Al‐Daem Street, 6th District,
Nassr City, Cairo 00202‐2262‐5796, Egypt.
Email: eladlkhaled74@yahoo.com;
eladlkhaled74@azhar.edu.eg (K. E‐A.) and
ibrahimeissa@azhar.edu.eg (I. H. E.)
K E Y W O R D S
anticancer agents, benzoxazole, molecular docking, VEGFR‐2 inhibitors
Sorafenib (Nexavar®) is a potent VEGFR‐2 inhibitor and has been
approved as antiangiogenic drug.[7–9] Study of the structure–activity
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1
INTRODUCTION
VEGF signaling pathway plays fundamental roles in regulating tumor
angiogenesis. VEGF as a therapeutic target has been validated in
various types of human cancers.[1] Vascular endothelial growth factor
receptor‐2 (VEGFR‐2) represents a major target within the angio-
genesis‐related kinases, hence considered the most important
transducer of VEGF‐dependent angiogenesis.[2] Thus, inhibition of
VEGF/VEGFR signaling pathway is regarded as an attractive
therapeutic target for inhibition of tumor angiogenesis and sub-
sequent tumor growth.[3–6]
relationships (SAR) and common pharmacophoric features shared
by sorafenib and various VEGFR‐2 inhibitors revealed that most
VEGFR‐2 inhibitors shared four main features as shown in
Figure 1:[10–12] (a) The core structure of most inhibitors consists of a
flat heteroaromatic ring system that contains at least one N‐atom,
which occupied the catalytic ATP‐binding domain. (b) A central aryl ring
(hydrophobic spacer), occupying the linker region between the ATP‐
binding domain and the DFG domain of the enzyme.[13] (c) A linker
containing a functional group acting as pharmacophore (e.g., amino or
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hybridization of benzoxazole and other effective antitumor moieties
was carried out in an attempt to get new molecules with promising
antitumor activities having the main pharmacophoric features of
VEGFR‐2 inhibitors.
The goal of our work was the synthesis of new agents with the
same essential pharmacophoric features of the reported and
clinically used VEGFR‐2 inhibitors (e.g., sorafenib). The main core
of our molecular design rationale comprised bioisosteric mod-
ification strategies of VEGFR‐2 inhibitors at four different
positions (Figure 2).
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2
RESULTS AND DISCUSSION
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2.1
Rationale and structure‐based design
FIGURE 1 The basic structural requirements for sorafenib as
reported vascular endothelial growth factor receptor‐2 inhibitor
Our target compounds were designed to have different spacers and
different linkers with HBA–HBD, the main pharmacophoric feature in
sorafenib, hoping to obtain more potent VEGFR‐2 inhibitors. First,
bioisosteric approach was adopted in the target benzoxazole to
replace pyridine ring. The second strategy is to use 2‐sulfanyl‐N‐
phenylacetamide and/or 3‐sulfanyl‐N‐phenylpropanamide ring sys-
tems to replace the central aryl ring of lead structure to increase the
space (linkers) between the pyridine ring and the central aryl ring to
impart more flexibility aiming to increase VEGFR‐2–binding affinity.
The third strategy is using HBA–HBD linkers containing functional
groups that possess H‐bond acceptors and/or donors, such as
urea) that possesses both H‐bond acceptor and donor to bind with two
crucial residues (Glu883 and Asp1044) in the DFG (Asp–Phe–Gly) motif,
an essential tripeptide sequence in the active kinase domain. The NH
motifs of the urea or amide moiety usually form one hydrogen bond
with Glu883, whereas the C═O motif forms another hydrogen bond
with Asp1044. (d) The terminal hydrophobic moiety of the inhibitors
occupies the newly created allosteric hydrophobic pocket, revealed
when the phenylalanine residue of the DFG loop flips out of its lipophilic
pocket defining DFG‐out or inactive conformation. Thus, hydrophobic
interactions are usually attained in this allosteric binding region.[14]
Furthermore, analysis of the X‐ray structure of various inhibitors bound
to VEGFR‐2 confirmed the sufficient space available for various
substituents around the terminal heteroaromatic ring.[15,16]
sulfonamide linker (SO2NH) in compounds 4a–f
(–SO2–NH–CO–NH–) in compounds 5a–f and sulfonylthiourea
(–SO2–NH–CS–NH–) in compounds 6a–c. Compounds 8a,b contain
only carbonyl (CO) group, whereas thiosemicarbazone
, sulfonylurea
,
Benzoxazole is a heterocyclic scaffold in many synthetic compounds,
so the chemistry of benzoxazole derivatives became increasingly
interesting due to their various biological and pharmacological
activities.[17,18] Benzoxazole nucleus is a core structure in many
synthetic compounds having different biological activities as anti‐
inflammatory[19] and anticancer.[20] In addition, the bis(benzoxazole)
natural product UK‐1 displayed a potent anticancer activity with an IC50
value 20 nM against certain solid tumors, leukemia, and lymphoma.[21]
VEGFR‐2 inhibitory activity of benzoxazole was also reported.[17,22] It is
suggested that benzoxazoles act as competitive inhibitors at the ATP‐
binding site of tyrosine kinases.[17]
(═N–NH–CS–NH2) in compounds 11a,b and hydrazone (═N–NH–CO)
in compounds 12a–d and 13a,b. Also oxime (═N–OH), chalcone
(COCH═CH–), and acetylpyrazoline linkers were used in compound
14, 15, and 16, respectively. Also, the hydrophobic phenyl tail of the
reported ligand was replaced in many compounds by other groups
such as thiazole in compound 4b, pyridine in compound 4c, cyclohexyl
in compounds 5a,c,e, ethyl in compounds 6a–c, and methyl in
compounds 8a,b, 11a,b, 13a,b, and 14. The fourth strategy focused
on the usage of different substituents on phenyl moiety as in
compounds 12b–d and methoxy group in compounds 15 and 16.
Benzoxazole nucleus is a privileged scaffold forming the most
promising class of heterocycles, which is well‐tolerated in humans
and possesses antitumor activity.[17,21,22] Moreover, they are the
backbone of many bioactive compounds that show potential
activities as VEGFR inhibitors.[17,22,23] In addition, several sulfona-
mide,[24] sulfonylurea,[25] sulfonylthiourea,[26] thiosemicarbazone,[27]
hydrazone,[28] oxime,[29] and pyrazoline[30] moieties were reported to
possess anticancer activities.
Depending on ligand‐based drug design, particularly a molecular
hybridization approach that involves the coupling of two or
more groups with relevant biological properties,[31] molecular
FIGURE 2 Summary for the possible modifications of vascular
endothelial growth factor receptor‐2 inhibitors

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Furthermore, the substitution pattern was selected to ensure
The essential pharmacophoric features[28,32–35] in the benzoxazole‐
derived VEGFR‐2 inhibitors (Figure 3) include: the presence of five‐
membered hetero ring, oxazole, fused with benzene ring, as hydrophobic
portion, forming aromatic system represented by benzoxazole ring linked
to different (un)substituted hydrophobic moieties through different
spacers and linkers (HBA–HBD), which interacts as H‐bond donors
through its NH with the side chain carboxylate of the essential amino acid
residue Glutamate883 and through its carbonyl group with Aspar-
tate1044, and also through hydrophobic interaction with its (un)–
substituted hydrophobic moieties with the hydrophobic pocket lined with
the hydrophobic side chains of Alanine864, Valine865 Lysine866,
Valine897, Valine914, Phenylalanine916, Cysteine917, Leucine1033,
different electronic and lipophilic environments, which could influence
the activity of the target compounds. On the contrary, the linker in
compound 16 was designed in a different way, where it constituted a
part of the rigid acetylpyrazoline ring structure to study the effect of
the free rotated open‐chain linkers and/or the ring structure on SAR.
These modifications were performed to carry out further elaboration
of the benzoxazole scaffold and to explore a valuable SAR. The
designed target benzoxazole derivatives were synthesized and
evaluated as potential VEGFR‐2 inhibitory and antitumor activities
against three human tumor cell lines, namely, hepatocellular carcinoma
(HepG2), breast cancer (MCF‐7), and colorectal carcinoma (HCT‐116).
FIGURE 3 Structural similarities and pharmacophoric features of vascular endothelial growth factor receptor‐2 inhibitors and selected
designed compounds

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and Cysteine1043. In addition, oxazole moiety was designed to replace
the pyridine moiety of the reference ligand sorafenib. Compounds 5e,f
hydrate in the presence of acetic acid glacial produced the
,
corresponding acetylpyrazoline derivative (16; Scheme 3).
have SO2NHCONH while compounds 6b,c have SO2NHCSNH, which
resemble the urea of sorafenib and form H‐bond with the essential amino
acids Glutamate883 and Aspartate1044. They also have hydrophobic
distal cyclohexyl, phenyl, and/or ethyl moieties, which increase the affinity
toward VEGFR‐2 by hydrophobic interactions. These interactions may
explain the high anticancer activities of these compounds.
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2.3
Docking studies
In the present work, all modeling experiments were performed using
Molsoft software. Each experiment used VEGFR‐2 downloaded from
the Brookhaven Protein Databank (PDB ID 1YWN).[36]
Our target compounds were designed as hybrid molecules. These
molecules formed of benzoxazole ring system joined with different
(un)substituted moieties through different linkers offering various
electronic and lipophilic environments to study their impact on the
activity, hoping to obtain more potent anticancer agents. Molecular
docking studies were carried out to study the interaction of the
newly synthesized compounds with VEGFR‐2, their binding mode and
the ability to satisfy the pharmacophoric features required to induce
the desired inhibition.
The obtained results indicated that all studied ligands have
similar position and orientation inside the putative binding site of
VEGFR‐2, which revealed a large space bounded by a membrane‐
binding domain that served as an entry channel for substrate to
the active site (Figure 4). In addition, the affinity of any small
molecule can be considered as a unique tool in the field of drug
design. There is a relationship between the affinity of organic
molecules and the free energy of binding.[37–40] This relationship
can contribute in prediction and interpretation of the activity of
the organic compounds toward the specific target protein. The
obtained results of the free energy of binding (ΔG) explained that
most of these compounds had good binding affinity toward the
receptor and the computed values reflected the overall trend
(Table 1).
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2.2
Chemistry
The synthetic strategy for preparation of the target compounds (4–16)
is depicted in Schemes 1–3. Synthesis was initiated by reacting 2‐
amino‐4‐substituted phenol with carbon disulfide in the presence of
alcoholic potassium hydroxide to provide the corresponding 2‐
mercaptobenzoxazole derivatives (1a,b), respectively, which was
treated with alcoholic potassium hydroxide to afford the correspond-
ing potassium salts (2a,b). 4‐Amino‐N‐substituted phenylsulfonamides
were reacted with chloroacetyl chloride and/or chloropropionyl
chloride to afford the corresponding chloroamides (3a–d). The obtained
potassium salts (2a,b) were refluxed with the appropriate chloroamide
derivative (3a–c) to get the corresponding sulfonamides (4a–f). The
formed sulfanilamide derivatives (4a,d–g) underwent further reaction
with the appropriate isocyanates and/or isothiocyanates, namely,
cyclohexyl isocyanate, phenyl isocyanate, and/or ethyl isothiocyanate
to furnish the corresponding sulphonyl urea derivatives (5a–f) and/or
sulphonyl thiourea derivatives (6a–c), respectively (Scheme 1). 4‐
Aminoacetophenone reacted with chloroacetyl chloride to obtain the
corresponding chloroamide derivative (7), which underwent reaction
with the appropriate potassium salt (2a,b) to produce the correspond-
ing acetyl derivatives (8a,b), which underwent further condensation
with thiosemicarbazide to furnish the corresponding thiosemicarba-
zone derivatives (11a,b). Esterification of (un)substituted benzoic acid
with ethanol was carried out in the presence of conc. H2SO4 to get the
corresponding ethyl benzoate esters (9a–d), respectively, which was
allowed to react with hydrazine hydrate to afford the corresponding
benzohydrazide derivatives (10a–d), respectively. Condensation of the
acetyl derivative 8a with the appropriate benzohydrazide derivative
(10a–d) resulted in the corresponding benzoylhydrazone derivatives
(12a–d), respectively (Scheme 2). Condensation of the appropriate
acetyl derivative (8a,b) with 2‐cyanoacetohydrazide, hydroxylamine,
and/or 4‐methoxybenzaldehyde afforded the corresponding 2‐cyanoa-
cetyl)hydrazones (13a,b), oxime (14), and/or chalcone (15) derivatives,
respectively. Cyclocondensation of the chalcone (15) with hydrazine
The proposed binding mode of sorafenib revealed affinity value of
−100.87 kcal/mol and four H‐bonds. The urea linker formed one H‐
bond with the key amino acid Glutamate883 (2.13 Å) through its NH
group and one H‐bond with Aspartate1044 (1.65 Å) through its
carbonyl group. The central phenyl ring occupied the hydrophobic
pocket formed by Glutamate883, Isoleucine886, Leucine887, Iso-
leucine1042, Cysteine1043, and Aspartate1044. Moreover, the distal
hydrophobic 3‐trifluromethyl‐4‐chlorophenyl moiety attached to the
urea linker occupied the hydrophobic pocket formed by Cy-
steine1043, Leucine1033, Valine897, Valine914, Alanine864, Va-
line865, and Lysine866. Furthermore, the N‐methylpicolinamide
moiety occupied the hydrophobic groove formed by Arginine1025,
Histidine1024, Isoleucine1023, Cysteine1022, Leucine1017, Isoleu-
cine890, Histidine889, and Isoleucine886 while its carbonyl was
stabilized by formation of two H‐bonds with Arginine1025 (1.90 and
2.12 Å; Figure 5). The urea linker played an important role in the
binding affinity toward VEGFR‐2 enzyme, where it was responsible
for the higher binding affinity of sorafenib. This finding encouraged
us to use different linkers that resembled urea of sorafenib to obtain
potent VEGFR‐2 inhibitors.
As planned, the proposed binding mode of compound 5e is
virtually the same as that of sorafenib, which revealed affinity more
than that of sorafenib with value of −133.72 kcal/mol and five H‐
bonds. The NH group of the sulfonylurea linker formed one H‐bond
with Glutamate883 (2.39 Å) and one H‐bond with Aspartate1044
(1.26 Å) through its carbonyl group. The SCH2CH2CONH spacer was
stabilized by formation of H‐bond with Aspartate1044 (2.37 Å) and 2
H‐bonds with Arginine1025 (1.67 and 1.99 Å). The central phenyl
ring occupied the hydrophobic pocket formed by Glutamate883,
Isoleucine886, Leucine887, Leucine1017, Histidine1024, Isoleu-
cine1042, and Aspartate1044. The distal cyclohexyl moiety occupied

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SCHEME 1 Synthetic route for preparation of the target compounds 4a–f–6a–c

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SCHEME 2 Synthetic route for preparation of the target compounds 8–12a–d

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SCHEME 3 Synthetic route for preparation of the target compounds 13–16

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FIGURE 4 Superimposition of some
docked compounds inside the binding
pocket of 1YWN
the hydrophobic pocket formed by Alanine864, Valine865,
Lysine866, Valine897, Valine914, Phenylalanine916, Cysteine917,
Leucine1033, and Cysteine1043. Furthermore, the benzoxazole moiety
occupied the hydrophobic groove formed by Cysteine1022, Isoleu-
cine890, Histidine889, and Isoleucine886 (Figure 6). These interactions
of compound 5e may explain the highest anticancer activity.
Alanine864, Valine865 Lysine866, Valine897, Valine914, Phenylala-
nine916, Cysteine917, Leucine1033, and Cysteine1043. Further-
more, benzoxazole moiety occupied the hydrophobic groove formed
by Cysteine1022, Isoleucine890, Histidine889, and Isoleucine886
(Figure 7). These interactions of compound 5c may explain the
highest anticancer activity.
The proposed binding mode of compound 5c is virtually the same
as that of 5e, which revealed affinity value of −132.88 kcal/mol and
five H‐bonds. The sulfonylurea linker was stabilized by formation of
four H‐bonds. The NH group formed one H‐bond with Glutamate883
(2.78 Å) and three H‐bonds with Isoleucine886 (1.49, 1.70, and
1.99 Å). The SCH2CH2CONH spacer formed H‐bond with Aspar-
tate1044 (2.30 Å). The central phenyl ring occupied the hydrophobic
pocket formed by Glutamate883, Isoleucine886, Leucine887, Leu-
cine1017, Histidine1024, Isoleucine1042, and Aspartate1044. The
distal cyclohexyl moiety occupied the hydrophobic pocket formed by
From the obtained docking results (Table 1), we concluded
that sulfonylurea and/or sulfonylthiourea linkers impart higher
affinities toward VEGFR‐2 enzyme than urea linker. The longer
SCH2CH2CONH spacer showed higher binding affinities than the
SCH2CONH one. The open‐chain free rotating linkers exhibited
higher binding affinities than the rigid cyclic ones. Also lipophilicity
played an important role in their VEGFR‐2 inhibitory activities, which
may be due to higher hydrophobic interactions.
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2.4
Biological activity
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2.4.1
In vitro cytotoxic activity
TABLE 1 The calculated free energy of binding (ΔG in kcal/mol)
for the ligands
Antiproliferative activity of the newly synthesized benzoxazoles
a–f–16 was examined against three human tumor cell lines, namely,
4
hepatocellular carcinoma (HepG2), breast cancer (MCF‐7), and
colorectal carcinoma (HCT‐116) using 3‐(4,5‐dimethylthiazol‐2‐yl)‐
2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay as de-
scribed by Mosmann.[41–43] Sorafenib was included in the experi-
ments as a reference cytotoxic drug. The results were expressed as
growth inhibitory concentration (IC50) values, which represent the
compound concentrations required to produce a 50% inhibition of
cell growth after 72 hr of incubation calculated from the concen-
tration–inhibition response curve and summarized in Table 2. From
the obtained results, it was explicated that most of the prepared
compounds displayed excellent to modest growth inhibitory activity
against the tested cancer cell lines. Investigations of the cytotoxic
activity against HCT‐116 and HepG2 indicated that they were more
sensitive cell lines to the influence of the new derivatives,
respectively. In particular, compound 5e was found to be the most
potent derivative overall in the tested compounds against HepG2,
HCT‐116, and MCF‐7 cancer cell lines with IC50 = 4.13 0.2,
6.93 0.3, and 8.67 0.5 µM, respectively. It has nearly the half
Compound
ΔG (kcal/mol)
−84.17
Compound
8a
ΔG (kcal/mol)
−74.49
4a
4b
4c
4d
4e
4f
−105.60
−105.95
−95.40
8b
−73.48
11a
−105.99
−111.01
−78.88
11b
−93.34
12a
−93.87
12b
−72.61
5a
5b
5c
5d
5e
5f
−120.83
−118.31
−132.88
−129.92
−133.72
−131.03
−116.81
−128.05
−125.05
12c
−82.35
12d
−71.55
13a
−86.33
13b
−85.78
14
−66.11
15
−92.35
6a
6b
6c
16
−104.55
−100.87
Sorafenib

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FIGURE 5 Predicted binding mode for
sorafenib with 1WYN. H‐bonded atoms are
indicated by dotted lines
activity as sorafenib against HepG2 (IC50 = 9.18 0.6 µM) and nearly
the same activity against HCT‐116 (IC50 = 5.47 0.3 µM) and MCF‐7
cell lines (IC50 = 7.26 0.3 µM). With respect to the HepG2 hepato-
cellular carcinoma cell line, compounds 5c, 5f, 6b, 5d, and 6c displayed
the highest anticancer activities (with IC50 = 5.93 0.2, 6.58 0.4,
8.10 0.7, 8.75 0.7, and 9.95 0.9 µM, respectively). Compounds
4b, 4c, 4g, 5a, 5b, 6a, and 11b (with IC50 ranging from 11.48 1.0 to
24.84 1.8 µM) displayed good cytotoxicity. Compounds 4d, 4f, 11a,
12a, 12c, 15, and 16 (with IC50 ranging from 35.94 3.2
to 51.47 3.6 µM) exhibited moderate cytotoxicity. Other com-
pounds with (IC50 ranging from 67.42 4.1 to 88.97 4.8 µM)
exhibited low cytotoxicity.
Cytotoxicity evaluation against MCF‐7 cell line, revealed that
compounds 5c, 5d, and 5f with (IC50 = 8.93 0.6, 9.95 0.8, and
10.11 0.9 µM, respectively) exhibited the highest anticancer activ-
ities. Compounds 4b, 4c, 5a, 6b, 6c, and 11b (with IC50 ranging from
12.31 1.0 to 19.45 1.6 µM) displayed good cytotoxicity. Com-
pounds 4d, 4 g, 5b, 6a, 12a, 12c, 15, and 16 (with IC50 ranging from
26.86 1.9 to 45.62 2.7 µM) showed moderate cytotoxicity. On the
contrary, other compounds displayed low cytotoxicity.
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2.4.2
In vitro VEGFR‐2 kinase assay
The most active antiproliferative derivatives 5c–f and 6b,c were
selected to evaluate their inhibitory activities against VEGFR‐2 by
using an antiphosphotyrosine antibody with the Alpha Screen system
(PerkinElmer). The results were reported as IC50 value calculated
from the concentration–inhibition response curve and summarized in
Table 2. Sorafenib was used as positive control in this assay. The
tested compounds displayed high to good inhibitory activity with IC50
values ranging from 0.07 to 0.36 µM. Among them, compounds 5e
and 5c potently inhibited VEGFR‐2 at lower IC50 values of 0.07 0.01
and 0.08 0.01 µM, respectively, compared with sorafenib IC50 value
Cytotoxicity evaluation against colorectal carcinoma (HCT‐116)
cell line discovered that compounds 5c and 6b showed the highest
anticancer activities with IC50 = 7.14 0.4 and 7.91 0.6 µM, respec-
tively, and compounds 4b, 4c, 5a, 5d, 5f, 6c, and 11b (with IC50 ranging
from 9.10 0.8 to 18.33 1.7 µM) displayed good cytotoxicity.
Compounds 4d, 4f, 4g, 5b, 6a, 12a, and 12c (with IC50 ranging from
20.76 1.9 to 35.08 2.8 µM) exhibited moderate cytotoxicity.
Other compounds (with IC50 ranging from 44.3 0.49 to
78.24 4.7 µM) exhibited low cytotoxicity.
FIGURE 6 Predicted binding mode for
5e with 1WYN

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FIGURE 7 Predicted binding mode for
5c with 1WYN
TABLE 2 In vitro cytotoxic activities of the newly synthesized compounds against HepG2, MCF‐7, and HCT‐116 cell lines and VEGFR‐2
kinase assay
IC50 (µM)a
Compound
HepG2
HCT‐116
MCF‐7
VEGFR‐2
4a
88.97 4.8
24.84 1.8
15.34 1.3
44.71 3.1
67.42 4.1
18.02 1.4
13.15 2.1
14.12 1.1
5.93 0.2
8.75 0.7
4.13 0.2
6.58 0.4
21.37 1.6
8.10 0.7
9.95 0.9
35.94 3.2
11.48 1.0
42.96 2.8
NA
48.39 3.4
17.18 1.5
16.38 1.4
25.38 2.2
62.82 3.9
26.41 2.3
18.33 1.7
20.76 1.9
7.14 0.4
9.52 0.5
6.93 0.3
9.10 0.8
29.38 2.5
7.91 0.6
12.48 1.1
45.10 3.3
10.35 0.9
31.49 2.6
75.33 4.5
30.49 2.6
78.24 4.7
64.86 4.8
46.10 3.4
NA
59.19 3.3
16.42 1.4
18.30 1.5
26.86 1.9
73.25 3.8
34.72 2.3
19.45 1.6
28.13 2.0
8.93 0.6
9.95 0.8
8.67 0.5
10.11 0.9
36.80 2.4
12.31 1.0
15.70 1.4
54.85 3.2
13.79 1.2
45.62 2.7
78.25 4.0
38.48 2.5
81.32 4.2
75.23 5.3
55.85 3.1
NA
NT
4b
NT
4c
NT
4d
NT
4e
NT
4f
NT
5a
NT
5b
NT
5c
0.08 0.01
0.19 0.02
0.07 0.01
0.10 0.02
NT
5d
5e
5f
6a
6b
0.27 0.02
0.36 0.03
NT
6c
11a
11b
12a
12b
12c
12d
13a
13b
14
NT
NT
NT
41.93 2.6
NA
NT
NT
68.22 5.1
73.94 4.2
NA
NT
NT
NT
15
48.5 3.26
36.25 2.45
9.18 0.6
45.4 3.44
44.3 0.49
5.47 0.3
42.21 3.12
40.91 3.06
7.26 0.3
NT
16
NT
Sorafenib
0.10 0.02
Abbreviations: NA, compounds having IC50 value > 100 µM; NT, compounds not tested for their VEGFR‐2 inhibitory activity; VEGFR‐2, vascular
endothelial growth factor receptor‐2.
aIC50 values are the mean standard deviation (SD) of three separate experiments.

EL‐HELBY ET AL.
11 of 19
|
(0.1 0.02 µM). Compound 5f potently inhibited VEGFR‐2 at low IC50
value (0.10 0.02 µM) equipotent as sorafenib. Also, compounds 5d,
6c, and 6b possessed good VEGFR‐2 inhibition with IC50 values of
0.19 0.02, 0.27 0.02, and 0.36 0.03 µM, respectively.
shorter spacer as in compound 5a. Furthermore, the more lipophilic
cyclohexyl moiety as in compounds 5e and 5c displayed higher
anticancer potency against all cell lines than the distal phenyl group
as in compounds 5f and 5d, respectively. In the third group, the
unsubstituted benzoxazole derivative 6b displayed higher activity
than the 5‐methylbenzoxazole derivative 6c. The long‐chain spacer as
in compounds 6b and 6c exhibited higher activities than the shorter
spacer as in compound 6a. The fourth group 11a,b revealed that the 5‐
methylbenzoxazole derivative 11b exhibited higher activity than the
unsubstituted one 11a. In the fifth group, 12a–d, the 4‐chloro
substituted distal phenyl moiety as in compound 12c exhibited
higher activity than the unsubstituted one 12a. Contrarily, the
electron withdrawing Cl group at position 2 as compound 12b
showed lower activity than the unsubstituted one 12a. On the other
The preliminary SAR study has focused on the effect of replace-
ment of the urea linkers of sorafenib with different linkers, which
interacted as H‐bond donor through its NH with the side chain
carboxylate of the essential amino acid residue Glutamate883 and with
Aspartate1044 through its carbonyl group. Also, hydrophobic interac-
tions through the attached (un)substituted hydrophobic moieties. The
effect of replacement of pyridine moiety of sorafenib by the
benzoxazole scaffold of the synthesized compounds on the antitumor
activities also was noticed. The benzoxazole scaffold occupied the same
hydrophobic pocket, which was occupied by the pyridine moiety of the
standard ligand. On the contrary, different hydrophobic groups were
introduced instead of the phenyl moiety of the reference ligand.
Moreover, different substitutions were introduced to the phenyl group
with different lipophilicity and electronic nature to study their effect in
the anticancer activity. The data obtained revealed that the tested
compounds displayed different levels of anticancer activity and
possessed a distinctive pattern of selectivity against the HCT‐116
and HepG2 cell lines, respectively. Generally, the spacers, linkers
(HBA–HBD), lipophilicity, and electronic nature exhibited an important
role in the anticancer activity. The sulfonylurea linkers of compounds
hand, the electron withdrawing group Cl at position
2 as in
compound 12b exhibited more activity than the less electron
withdrawing Br group as in compound 12d, which enabled us to
deduce that the lipophilic and electron deficient groups at position
4 increased the activity while at position 2 decreased the activity. In
the other group, the 5‐methylbenzoxazole derivative 13b exhibited
higher activity than the unsubstituted one 13a. The cyclic pyrazoline
structure 16 showed higher activity than its parent chalcone 15 and
the oxime 14, respectively.
5
a–f and sulfonylthiourea linkers as in compounds 6a,b were found to be
essential for the higher anticancer activity than that of sulfonamide
linker (SO2NH) as in compounds 4a–f The thiosemicarbazone
|
3
CONCLUSION
.
(═N–NH–CS–NH2) linker in compounds 11a,b exhibited higher activ-
ities than the hydrazone (═N–NH–CO) linker in compounds 12a–d and
The molecular design was performed to investigate the binding mode
of the proposed compounds with VEGFR‐2 receptor. The data
obtained from the docking studies were fitted with that obtained
from the biological screening. This higher effect may be due to the
change of the linkers, which resemble that of sorafenib. All the tested
compounds showed variable anticancer activities. Novel series of
benzoxazole derivatives 4a–f–16 were designed, synthesized, and
evaluated for their anticancer activity against three human tumor cell
lines hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT‐
116), and breast cancer (MCF‐7) targeting VEGFR‐2 enzyme. HCT‐
116 was the most sensitive cell line to the influence of the new
derivatives. In particular, compound 5e was found to be the most
potent derivative overall in the tested compounds against HepG2,
HCT‐116, and MCF‐7 cancer cell lines with IC50 = 4.13 0.2,
6.93 0.3, and 8.67 0.5 µM, respectively. Compounds 5c, 5f, 6b, 5d,
and 6c showed the highest anticancer activities against all HepG2
with IC50 of 5.93 0.2, 6.58 0.4, 8.10 0.7, 8.75 0.7, and
9.95 0.9 µM, respectively; HCT‐116 with IC50 of 7.14 0.4,
9.10 0.8, 7.91 0.6, 9.52 0.5 and 12.48 1.1 µM, respectively;
and MCF‐7 with IC50 of 8.93 0.6, 10.11 0.9, 12.31 1.0,
9.95 0.8, and 15.70 1.4 µM, respectively, in comparison with
sorafenib as a reference drug with IC50 of 9.18 0.6, 5.47 0.3,
and 7.26 0.3 µM, respectively. The most active six compounds in
this series 5c–f and 6b,c were further evaluated for their inhibitory
activity against VEGFR‐2. Compounds 5e and 5c potently inhibited
VEGFR‐2 at lower IC50 values of 0.07 0.01 and 0.08 0.01 µM,
13a,b, respectively. Also acetylpyrazoline linker in compound 16
showed higher activity than its parent chalcone (COCH═CH–) and
oxime (═N–OH), as in compounds 15 and 14, respectively. The free
rotating open‐chain HBA–HBD linkers exhibited higher activity than
that of the cyclic one. The SCH2CH2CONH spacers resulted in higher
activities than the SCH2CONH one. The 5‐methylbenzoxazole deriva-
tives, for example, compound 5e, showed higher activities than the
unsubstituted ones, for example, compound 5c. Alternatively, the distal
phenyl group is not essential for the activity where the more lipophilic
cyclohexyl moiety as in compounds 5e and 5c displayed the highest
anticancer potency against all cell lines. Also, the more lipophilic
electron‐releasing cyclohexyl moiety as in compound 5e and 5c
exhibited higher anticancer activity than the propyl moiety as in 6b,c
.
From the structure of the synthesized derivatives and the
data shown in Table 2 we can divide these tested compounds into
six groups. The first group is compounds 4a–f, where the
5‐methylbenzoxazole derivatives for example, compound 4d and 4f
showed higher activities than the unsubstituted ones for example,
compound 4a and 4e, respectively. The distal pyridine moiety as in
compound 4c exhibited higher activity than the thiazole one as in
compound 4b. In the second group 5a–f, the 5‐methylbenzoxazole
derivatives for example compound 5e showed higher activities than
the unsubstituted ones for example compound 5c. Also the long‐chain
spacer as in compounds 5e and 5c exhibited higher activities than the

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EL‐HELBY ET AL.
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|
General method for the synthesis
respectively, compared with sorafenib IC50 value (0.11 0.02 µM).
Compound 5f potently inhibited VEGFR‐2 at low IC50 value
(0.10 0.02 µM) equipotent as sorafenib. Also, compounds 5d, 6c,
and 6b possessed good VEGFR‐2 inhibition with IC50 values of
0.19 0.02, 0.27 0.02, and 0.36 0.03 µM, respectively. The ob-
tained results showed that the most active compounds could be
useful as a template for future design, optimization, adaptation, and
investigation to produce more potent and selective VEGFR‐2
inhibitors with higher anticancer analogs.
4.1.3
of 2‐[(5‐(un)substituted benzoxazol‐2‐yl)thio]‐N‐{4‐[N‐
(un)substituted sulfamoyl]phenyl}acetamide (4a–d
and 2‐[(5‐(un)substituted benzoxazol‐2‐yl)thio]‐N‐(4‐
sulfamoylphenyl)propanamide (4e–f
)
)
To a mixture of compound 2a and/or 2b (0.002 mol) in dry N,N‐
dimethylformamide anhydrous (DMF; 50 ml), the appropriate chlor-
oamide derivatives 3a–d (0.002 mol) were added. The reaction
mixture was heated using a water bath for 12 hr. After cooling to
room temperature, the reaction mixture was poured onto crushed
ice. The precipitated solids were filtered, dried, and crystallized from
|
4
EXPERIMENTAL
Chemistry
ethanol to give the corresponding compounds 4a–g
.
|
4.1
2‐(Benzoxazol‐2‐ylthio)‐N‐(4‐sulfamoylphenyl)acetamide (4a)
Yield, 79%; m.p. 150–152°C; IRνmax (cm−1): 3,339, 3,240 (NH2), 3,119
(NH), 3,060 (CH aromatic), 2,972 (CH aliphatic), 1,689 (C═O), 1,601
(C═N), 1,309, 1,150 (SO2); 1H NMR (400 MHz, dimethyl sulfoxide
[DMSO]‐d6): 4.42 (s, 2H, –SCH2), 7.25 (s, 2H, NH2; D2O exchange-
able), 7.31 (dd, 1H, J = 5.7, 5.4 Hz, Ar‐H, H‐6 of benzoxazole), 7.33
(dd, 1H, J = 5.7, 4.8 Hz, Ar‐H, H‐5 of benzoxazole), 7.60 (d, 1H,
J = 4.8 Hz, Ar‐H, H‐4 of benzoxazole), 7.62 (d, 1H, J = 5.4 Hz, Ar‐H, H‐
7 of benzoxazole), 7.72 (d, 2H, J = 9 Hz, Ar‐H, H‐2, H‐6 of phenyl),
7.76 (d, 2H, J = 9 Hz, Ar‐H, H‐3, H‐5 of phenyl), 10.75 (s, 1H, NH; D2O
exchangeable); 13C NMR (400 MHz, DMSO‐d6): δ = 20.03, 79.95,
114.30 (2C), 116.04 (2C), 117.46, 118.87, 126.99, 127.41, 128.50,
129.21 (2C), 160.95, and 162.98; MS (m/z): 363 (M+, 100%, base
peak), 225 (22.99%), 151 (22.16%), 132 (22.77%), 57 (21.79%); Anal.
calcd. for C15H13N3O4S2 (m.w. 363.41): C, 49.58; H, 3.61; N, 11.56; S,
17.64. Found: C, 49.96; H, 3.28; N, 11.40; S, 17.45.
|
4.1.1
General
All melting points (m.p.) were carried out by open capillary method
on a Gallenkamp melting‐point apparatus at the Faculty of Pharmacy,
Al‐Azhar University, and were uncorrected. The infrared spectra
were recorded on Pye Unicam SP 1000 IR spectrophotometer at
Pharmaceutical Analytical Unit, Faculty of Pharmacy, Al‐Azhar
University, using potassium bromide disc technique. Proton magnetic
resonance 1H NMR spectra were recorded on a Bruker 400 Mhz‐
NMR spectrometer at Faculty of Sciences, Cairo University, Cairo,
Egypt. 13C NMR spectra were recorded on an Mercury 400 Mhz‐
NMR spectrometer at Chemical Laboratory, Ministry of Defense,
Cairo. TMS was used as internal standard and chemical shifts were
measured in δ scale (ppm). The mass spectra were carried out on
Direct Probe Controller Inlet part to Single Quadropole mass
analyzer in Thermo Scientific GCMS model ISQ LT using Thermo X‐
Calibur software at the Regional Center for Mycology and
Biotechnology, Al‐Azhar University. Elemental analyses (C, H, N)
were performed on a CHN analyzer at Regional Center for Mycology
and Biotechnology, Al‐Azhar University. All compounds were within
0.4 of the theoretical values. The reactions were monitored by thin‐
layer chromatography (TLC) using TLC sheets precoated with UV
fluorescent silica gel Merck 60 F254 plates and were visualized using
UV lamp and different solvents as mobile phases.
3‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(N‐(thiazol‐2‐yl)sulfamoyl)phenyl)‐
acetamide (4b)
Yield, 75%; m.p. 217–219°C; IRνmax (cm−1): 3,365, 3,150 (2 NH),
3,097 (CH aromatic), 2,888 (CH aliphatic), 1,664 (C═O), 1,593 (C═N),
1,398, 1,182 (SO2); 1H NMR (400 MHz, DMSO‐d6): 4.39 (s, 2H,
–SCH2), 6.78 (d, 1H, J = 4 Hz, Ar‐H, H‐5 of thiazole), 7.21 (d, 1H,
J = 4 Hz, Ar‐H, H‐4 of thiazole), 7.28 (dd, 1H, J = 8.4, 9 Hz, Ar‐H, H‐6
of benzoxazole), 7.45 (dd, 1H, J = 8.4, 8 Hz, Ar‐H, H‐5 of benzoxazole),
7.58 (d, 1H, J = 8 Hz, Ar‐H, H‐4 of benzoxazole), 7.60 (d, 1H, J = 9 Hz,
Ar‐H, H‐7 of benzoxazole), 7.70 (d, 2H, J = 8 Hz, Ar‐H, H‐2, H‐6 of
phenyl), 7.74 (d, 2H, J = 8 Hz, Ar‐H, H‐3, H‐5 of phenyl), 10.75 (s, 1H,
–CONH; D2O exchangeable), 12.67 (s, 1H, –SO2NH; D2O exchange-
able); MS (m/z): 446 (M+, 11.20%), 372 (69.39%), 359 (100%, base
peak), 151 (12.65%), and 63 (25.83%); Anal. calcd. for C18H14N4O4S3
(m.w. 446.51): C, 48.42; H, 3.16; N, 12.55; S, 21.54. Found: C, 48.01;
H, 3.12; N, 12.89; S, 21.85.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
|
4.1.2
8
Synthesis of compounds 1a,b, 2a,b, 3a‒d, 6,
a‒d, and 9a‒d
Benzoxazole‐2‐thiol (1a), 5‐methylbenzoxazole‐2‐thiol (1b), and their
corresponding potassium salts (2a,b), 2‐chloro‐N‐(4‐sulfamoylphenyl)‐
acetamide (3a), 2‐chloro‐N‐(4‐(N‐(thiazol‐2‐yl)sulfamoyl)phenyl)aceta-
mide (3b), 2‐chloro‐N‐(4‐(N‐(pyridin‐2‐yl)sulfamoyl)phenyl)acetamide
(3c), 3‐chloro‐N‐(4‐sulfamoylphenyl)propanamide (3d), N‐(4‐acetylphe-
nyl)‐2‐chloroacetamide (6), ethyl substituted benzoate (8a–d), and
substituted benzohydrazide (9a–d) were obtained according to the
reported procedures.[19]
2‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(N‐(pyridin‐2‐yl)sulfamoyl)phenyl)‐
acetamide (4c)
Yield, 78%; m.p. 200–202°C; IRνmax (cm−1): 3,350, 3,114 (2 NH),
3,050 (CH aromatic), 2,936 (CH aliphatic), 1,675 (C═O), 1,600
(C═N), 1,379, 1,141 (SO2); 1H NMR (400 MHz, DMSO‐d6): 4.36

EL‐HELBY ET AL.
13 of 19
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(s, 2H, –SCH2), 6.94 (dd, 1H, J = 7.6, 8.4 Hz, Ar‐H, H‐4 of pyridine),
7.00 (dd, 1H, J = 7.6, 8.4 Hz, Ar‐H, H‐5 of pyridine), 7.09 (dd, 1H, J = 8,
8.4 Hz, Ar‐H, H‐6 of benzoxazole), 7.34 (dd, 1H, J = 7.6, 8.4 Hz, Ar‐H,
H‐5 of benzoxazole), 7.47 (d, 1H, J = 7.6 Hz, Ar‐H, H‐4, of
benzoxazole), 7.66 (d, 1H, J = 8.4 Hz, Ar‐H, H‐6 of pyridine), 7.76 (d,
1H, J = 8 Hz, Ar‐H, H‐7 of benzoxazole), 7.80 (d, 2H, J = 8 Hz, Ar‐H, H‐
2, H‐6 of phenyl), 7.84 (d, 2H, J = 8 Hz, Ar‐H, H‐3, H‐5 of phenyl), 7.96
(d, 1H, J = 8.4 Hz, Ar‐H, H‐3 of pyridine), 10.74 (s, 1H, –CONH; D2O
exchangeable), 11.13 (s, 1H, –SO2NH; D2O exchangeable); Anal.
calcd. for C20H16N4O4S2 (m.w. 440.49): C, 54.53; H, 3.66; N, 12.72; S,
14.56. Found: C, 54.75; H, 3.39; N, 12.94; S, 14.81.
(400 MHz, DMSO‐d6): δ = 20.38, 33.01, 41.12, 109.07, 110.53,
118.14 (2C), 124.29, 126.09 (2C), 130.97, 134.17, 137.86, 141.13,
144.10, 168.44, and 178.64; MS (m/z): 391 (M+, 100%, base peak),
358 (12.47%), 226 (1.29%), 107 (11.57%), and 55 (47.19%); Anal.
Calcd. for C17H17N3O4S2 (m.w. 391.46): C, 52.16; H, 4.38; N, 10.73; S,
16.38. Found: C, 52.47; H, 4.20; N, 10.64; S, 16.33.
|
4.1.4
General method for the synthesis of
2‐(benzoxazol‐2‐ylthio)‐N‐(4‐(N‐
(substitutedcarbamoyl)sulfamoyl)phenyl)acetamide
(5a–b) and 3‐((5‐(un)substituted benzoxazol‐2‐yl)‐
thio)‐N‐(4‐(N‐(substitutedcarbamoyl)sulfamoyl)‐
2‐((5‐Methylbenzoxazol‐2‐yl)thio)‐N‐(4‐sulfamoylphenyl)acetamide
(4d)
phenyl)propanamide (5b–c
)
Yield, 75%; m.p. 183–185°C; IRνmax (cm−1): 3,326, 3,237 (NH2), 3,124
(NH), 3,070 (CH aromatic), 2,930 (CH aliphatic), 1,673 (C═O), 1,597
(C═N), 1,329, 1,155 (SO2); 1H NMR (400 MHz, DMSO‐d6): 2.38 (s,
3H, CH3), 4.40 (s, 2 H, –SCH2), 7.11 (d, 1H, J = 8.4 Hz, Ar‐H, H‐6 of
benzoxazole), 7.27 (s, 2H, NH2; D2O exchangeable), 7.41 (s, 1H, Ar‐H,
H‐4 of benzoxazole), 7.49 (d, 1H, J = 8.4 Hz, Ar‐H, H‐7 of benzox-
azole), 7.73 (d, 2H, J = 9 Hz, Ar‐H, H‐2, H‐6 of phenyl), 7.77 (d, 2H,
J = 9 Hz, Ar‐H, H‐3, H‐5 of phenyl), 10.77 (s, 1H, NH; D2O
exchangeable); MS (m/z): 377 (M+, 100%), 303 (37.05%), 297
(3.17%), 136 (3.63%), and 78 (10.91%); Anal. calcd. for C16H15N3O4S2
(m.w. 377.43): C, 50.92; H, 4.01; N, 11.13; S, 16.99. Found: C, 50.71;
H, 3.94; N, 11.33; S, 17.19.
A mixture of the appropriate sulfanilamide derivative 4a and/or 4d–f
(0.01 mol) and anhydrous potassium carbonate (2.76 g, 0.02 mol) in
100 ml of dry acetone was refluxed while stirring for about 1.5 hr.
The appropriate isocyanates, namely, cyclohexyl isocyanate and/or
phenyl isocyanate (0.01 mol) were added drop‐wise to the reaction
mixture. Refluxing and stirring were continued during the course of
the addition and for an additional 24 hr. The acetone was removed by
evaporation under reduced pressure, and about 50 ml of water was
added to dissolve the resulting residue. The solution was filtered.
Acidification of the filtrate with diluted hydrochloric acid caused the
precipitation of the product, which was filtered. Crystallization of the
filter cake from 90% aqueous ethanol yielded the corresponding
sulfonylurea derivatives (5a–f).
3‐(Benzoxazol‐2‐ylthio)‐N‐(4‐sulfamoylphenyl)propanamide (4e)
Yield, 70%; m.p. 190–192°C; IRνmax (cm−1): 3,304, 3,240 (NH2), 3,190
(NH), 3,304 (CH aromatic), 2,927 (CH aliphatic), 1,661 (C═O), 1,602
(C═N), 1,338, 1,154 (SO2); 1H NMR (400 MHz, DMSO‐d6): 2.96 (t,
2H, J = 6.8 Hz, CH2CO–), 4.49 (t, 2H, J = 6.8 Hz, –SCH2), 7.23 (s, 2H,
NH2; D2O exchangeable), 7.28 (dd, 1H, J = 8, 8.4 Hz, Ar‐H, H‐6 of
benzoxazole), 7.35 (dd, 1H, J = 8, 7.6 Hz, Ar‐H, H‐5 of benzoxazole),
7.54 (d, 1H, J = 7.6 Hz, Ar‐H, H‐4 of benzoxazole), 7.57 (d, 1H,
J = 8.4 Hz, Ar‐H, H‐7 of benzoxazole), 7.65 (d, 2H, J = 8.8 Hz, Ar‐H, H‐
2, H‐6 of phenyl), 7.72 (d, 2H, J = 8.8 Hz, Ar‐H, H‐3, H‐5 of phenyl),
10.40 (s, 1H, NH; D2O exchangeable); MS (m/z): 377 (M+, 100%, base
peak), 226 (6.82%), 151 (26.71%), 64 (31.66%), and 55 (83.65%);
Anal. calcd. for C16H15N3O4S2 (m.w. 377.43): C, 50.92; H, 4.01; N,
11.13; S, 16.99. Found: C, 51.26; H, 3.90; N, 11.56; S, 16.72.
2‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(N‐(cyclohexylcarbamoyl)sulfamoyl)‐
phenyl)acetamide (5a)
Yield, 79%; m.p. 163–165°C; IRνmax (cm−1): 3,333, 3,300, 3,240 (3
NH), 3,099 (CH aromatic), 2,930 (CH aliphatic), 1,687 (C═O), 1,335,
1,156 (SO2); 1H NMR (400 MHz, DMSO‐d6): 1.06–1.13 (m, 4H, H‐3
and H‐5 of cyclohexyl), 1.16–1.29 (m, 2H, H‐4 of cyclohexyl),
1.49–1.72 (m, 4H, H‐2 and H‐6 of cyclohexyl), 3.41 (m, 1H, H‐1 of
cyclohexyl), 5.56 (s, 2H, –SCH2), 6.32 (s, 1H, NH‐cyclohexyl; D2O
exchangeable), 7.24 (dd, 1H, J = 8, 8.4 Hz, Ar‐H, H‐6 of benzoxazole),
7.27 (dd, 1H, J = 8, 7.6 Hz, Ar‐H, H‐5 of benzoxazole), 7.54 (d, 1H,
J = 7.6 Hz, Ar‐H, H‐4 of benzoxazole), 7.75 (d, 1H, J = 8.4 Hz, Ar‐H,
H‐7 of benzoxazole), 7.80 (d, 2H, J = 8 Hz, Ar‐H, H‐2 and H‐6 of
phenyl), 7.90 (d, 2H, J = 8 Hz, Ar‐H, H‐3 and H‐5 of phenyl), 10.24 (s,
1H, –NHph; D2O exchangeable), 10.56 (s, 1H, –SO2NH; D2O
exchangeable); MS (m/z): 488 (M+, 8.73%), 368 (67.97%), 359
(100%, base peak), 106 (20.25%), and 55 (80.53%); Anal. calcd. for
C22H24N4O5S2 (m.w. 488.58): C, 54.08; H, 4.95; N, 11.47; S, 13.12.
Found: C, 54.54; H, 5.08; N, 11.72; S, 12.82.
3‐((5‐Methylbenzoxazol‐2‐yl)thio)‐N‐(4‐sulfamoylphenyl)‐
propanamide (4f
)
Yield, 77%; m.p. 237–239°C; IRνmax (cm−1): 3,373, 3,291 (NH2), 3,134
(NH), 3,050 (CH aromatic), 2,970 (CH aliphatic), 1,661 (C═O), 1,599
(C═N), 1,386, 1,158 (SO2); 1H NMR (400 MHz, DMSO‐d6): 2.34 (s,
3H, CH3), 2.92 (t, 2H, J = 6.8, CH2CO–), 4.47 (t, 2H, J = 6.8, –SCH2),
7.09 (d, 1H, J = 8.4 Hz, Ar‐H, H‐6 of benzoxazole), 7.24 (s, 2H, NH2;
D2O exchangeable), 7.35 (s, 1H, Ar‐H, H‐4 of benzoxazole), 7.41 (d,
1H, J = 8.4 Hz, Ar‐H, H‐7 of benzoxazole), 7.65 (d, 2H, J = 8.8 Hz, Ar‐
H, H‐2 and H‐6 of phenyl), 7.72 (d, 2H, J = 8.8 Hz, Ar‐H, H‐3 and
H‐5 of phenyl), 10.40 (s, 1H, NH; D2O exchangeable); 13C NMR
2‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(N‐(phenylcarbamoyl)sulfamoyl)phenyl)‐
acetamide (5b)
Yield, 70%; m.p. 146–148°C; IRνmax (cm−1): 3,350, 3,340, 3,275 (3
NH), 3,090 (CH aromatic), 2,980 (CH aliphatic), 1,637 (C═O), 1,321,
1,030 (SO2); 1H NMR (400 MHz, DMSO‐d6): 3.52 (s, 2H, –SCH2), 6.90
(s, 1H, –NHph′; D2O exchangeable), 6.98 (dd, 1H, J = 8, 8 Hz, Ar‐H,

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H‐4′ of phenyl), 7.21 (dd, 2H, J = 7.6, 8 Hz, Ar‐H, H‐3′ and H‐5′ of
phenyl), 7.25 (dd, 1H, J = 8, 8 Hz, Ar‐H, H‐6 of benzoxazole), 7.31 (dd,
1H, J = 8, 8.2 Hz, Ar‐H, H‐5 of benzoxazole), 7.35 (d, 2H, J = 7.6 Hz,
Ar‐H, H‐2′ and H‐6′ of phenyl), 7.53 (d, 1H, J = 8.2 Hz, Ar‐H, H‐4 of
benzoxazole), 7.55 (d, 1H, J = 8 Hz, Ar‐H, H‐7 of benzoxazole), 7.69 (d,
2H, J = 8.4 Hz, Ar‐H, H‐2 and H‐6 of phenyl), 7.85 (d, 2H, J = 8.4 Hz,
Ar‐H, H‐3, and H‐5 of phenyl), 8.76 (s, 1H, –NHph–; D2O exchange-
able), 10.55 (s, 1H, –SO2NH; D2O exchangeable); Anal. calcd. for
C22H18N4O5S2 (m.w. 482.53): C, 54.76; H, 3.76; N, 11.61; S, 13.29.
Found: C, 54.30; H, 3.90; N, 11.72; S, 13.30.
and H‐5 of cyclohexyl), 1.20–1.25 (m, 2H, H‐4 of cyclohexyl),
1.45–1.64 (m, 4H, H‐2 and H‐6 of cyclohexyl), 2.31 (s, 3H, CH3 of
benzoxazole), 2.93 (t, 2H, J = 6.8 Hz, CH2CO–), 3.17 (m, 1H, H‐1 of
cyclohexyl), 4.47 (t, 2H, J = 6.8 Hz, –SCH2), 6.29 (s, 1H, –NH‐
cyclohexyl; D2O exchangeable), 7.08 (d, 1H, J = 8.4 Hz, Ar‐H, H‐6 of
benzoxazole), 7.33 (s, 1H, Ar‐H, H‐4 of benzoxazole), 7.40 (d, 1H,
J = 8.4 Hz, Ar‐H, H‐7 of benzoxazole), 7.67 (d, 2H, J = 8.8 Hz, Ar‐H, H‐
2 and H‐6 of phenyl), 7.79 (d, 2H, J = 8.8 Hz, Ar‐H, H‐3 and H‐5 of
phenyl), 10.22 (s, 1H, –NHph; D2O exchangeable), 10.47 (s, 1H,
–SO2NH; D2O exchangeable); MS (m/z): 516 (M+, 3.02%), 422
(62.83%), 265 (35.22%), 102 (99.01%), and 54 (100%, base peak);
Anal. calcd. for C24H28N4O5S2 (m.w. 516.63): C, 55.80; H, 5.46; N,
10.84; S, 12.41. Found: C, 55.64; H, 5.29; N, 10.68; S, 12.59.
3‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(N‐(cyclohexylcarbamoyl)sulfamoyl)‐
phenyl)propanamide (5c)
Yield, 75%; m.p. 204–206°C; IRνmax (cm−1): 3,329, 3,265, 3,150 (3
NH), 3,070 (CH aromatic), 2,929 (CH aliphatic), 1,678 (C═O), 1,330,
1,166 (SO2); 1H NMR (400 MHz, DMSO‐d6): 1.05–1.16 (m, 4H, H‐3
and H‐5 of cyclohexyl), 1.19–1.26 (m, 2H, H‐4 of cyclohexyl),
1.48–1.72 (m, 4H, H‐2 and H‐6 of cyclohexyl), 2.99 (t, 2H, J = 7.6,
CH2CO–), 3.31 (m, 1H, H‐1 of cyclohexyl), 4.48 (t, 2H, J = 7.6, –SCH2),
6.30 (s, 1H, –NH‐cyclohexyl; D2O exchangeable), 7.30 (dd, 1H, J = 7.6,
8 Hz, Ar‐H, H‐6 of benzoxazole), 7.34 (dd, 1H, J = 7.6, 7.2 Hz, Ar‐H, H‐
5 of benzoxazole), 7.54 (d, 1H, J = 7.2 Hz, Ar‐H, H‐4 of benzoxazole),
7.57 (d, 1H, J = 8 Hz, Ar‐H, H‐7 of benzoxazole), 7.68 (d, 2H,
J = 8.4 Hz, Ar‐H, H‐2 and H‐6 of phenyl), 7.79 (d, 2H, J = 8.4 Hz, Ar‐
H, H‐3 and H‐5 of phenyl), 10.26 (s, 1H, –CONH; D2O exchangeable),
and 10.48 (s, 1H, –SO2NH; D2O exchangeable); Anal. calcd. for
3‐((5‐Methylbenzoxazol‐2‐yl)thio)‐N‐(4‐(N‐(phenylcarbamoyl)sulfa-
moyl)phenyl)propanamide (5f
)
Yield, 77%; m.p. 217–219°C; IRνmax (cm−1): 3,380, 3,317, 3,118 (3
NH), 3,090 (CH aromatic), 2,980 (CH aliphatic), 1,670 (C═O), 1,392,
1,155 (SO2); 1H NMR (400 MHz, DMSO‐d6): 2.29 (s, 3H, CH3), 2.92 (t,
2H, J = 6.8 Hz, –CH2CO), 4.47 (t, 2H, J = 6.8 Hz, –SCH2), 6.99 (dd, 1H,
J = 7.6, 7.6 Hz, Ar‐H, H‐4′ of phenyl), 7.05 (d, 1H, J = 8.4 Hz, Ar‐H, H‐6
of benzoxazole), 7.23 (dd, 2H, J = 7.6, 8 Hz, Ar‐H, H‐3′ and H‐5′ of
phenyl), 7.31 (d, 2H, J = 8 Hz, Ar‐H, H‐2′ and H‐6′ of phenyl), 7.35 (s,
1H, Ar‐H, H‐4 of benzoxazole), 7.39 (d, 1H, J = 8.4 Hz, Ar‐H, H‐7 of
benzoxazole), 7.70 (d, 2H, J = 8.8 Hz, Ar‐H, H‐2 and H‐6 of phenyl),
7.87 (d, 2H, J = 8.8 Hz, Ar‐H, H‐3 and H‐5 of phenyl), 8.77 (s, 1H,
–NHph′; D2O exchangeable), 10.49 (s, H, –NHph–; D2O exchange-
able), and 10.63 (s, 1H, –SO2NH; D2O exchangeable); Anal. calcd. for
C23H26N4O5S2 (m.w. 502.60): C, 54.96; H, 5.21; N, 11.15; S, 12.76.
Found: C, 54.99; H, 5.28; N, 10.98; S, 12.95.
C24H22N4O5S2 (m.w. 510.58): C, 56.46; H, 4.34; N, 10.97; S, 12.56.
3‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(N‐(phenylcarbamoyl)sulfamoyl)phenyl)‐
propanamide (5d)
Found: C, 56.67; H, 4.18; N, 10.58; S, 12.37.
Yield, 77%; m.p. 187–189°C; IRνmax (cm−1): 3,324, 3,300, 3,150 (3
NH), 3,050 (CH aromatic), 2,922 (CH aliphatic), 1,637 (C═O), 1,326,
1,152 (SO2); 1H NMR (400 MHz, DMSO‐d6): 2.96 (t, 2H, J = 6.8
Hz, –CH2CO), 4.49 (t, 2H, J = 6.8 Hz, –SCH2), 6.98 (s, 1H, –NHph′;
D2O exchangeable), 7.21 (dd, 1H, J = 8, 8 Hz, Ar‐H, H‐4′ of phenyl),
7.27 (dd, 1H, J = 7.2, 8.4 Hz, Ar‐H, H‐6 of benzoxazole), 7.31 (dd, 1H,
J = 7.2, 6.4 Hz, Ar‐H, H‐5 of benzoxazole), 7.35 (dd, 2H, J = 8, 8 Hz, Ar‐
H, H‐3′ and H‐5′ of phenyl), 7.37 (d, 2H, J = 8 Hz, Ar‐H, H‐2′ and H‐6′
of phenyl), 7.53 (d, 1H, J = 6.4 Hz, Ar‐H, H‐4 of benzoxazole), 7.55 (d,
1H, J = 8.4 Hz, Ar‐H, H‐7 of benzoxazole), 7.69 (d, 2H, J = 8.4 Hz, Ar‐
H, H‐2 and H‐6 of phenyl), 7.85 (d, 2H, J = 8.4 Hz, Ar‐H, H‐3 and H‐5
of phenyl), 8.73 (s, 1H, –NHph–; D2O exchangeable), 10.47 (s, 1H,
–SO2NH; D2O exchangeable); MS (m/z): 495 (M+−1, 0.93%), 339
(10.50%), 298 (10.90%), 64 (40.88%), and 55 (100%, base peak); Anal.
calcd. for C23H20N4O5S2 (m.w. 496.56): C, 55.63; H, 4.06; N, 11.28; S,
12.91. Found: C, 55.71; H, 4.38; N, 11.57; S, 13.01.
|
4.1.5
General method for the synthesis of
N‐(4‐(N‐(ethylcarbamothioyl)sulfamoyl)phenyl)‐2‐((5‐
methylbenzoxazol‐2‐yl)thio)acetamide (6a) and
N‐(4‐(N‐(ethylcarbamothioyl)sulfamoyl)phenyl)‐3‐((5‐(un)‐
substituted benzoxazol‐2‐yl)thio)propanamide (6b–c
)
Ethyl isothiocyanate (0.87 g, 0.01 mol) was added drop‐wise to a
mixture of the appropriate sulfanilamide derivative 4d–f (0.01 mol)
and anhydrous potassium carbonate (2.76 g, 0.02 mol) in 100 ml of
dry acetone. The reaction mixture was refluxed while stirring for
24 hr. The acetone was removed by evaporation under reduced
pressure, and about 50 ml of water was added to dissolve the
resulting residue. The solution was filtered. Acidification of the
filtrate with diluted hydrochloric acid caused the precipitation of the
product, which was filtered. Crystallization of the filter cake from
90% aqueous ethanol yielded the corresponding sulfonylthiourea
derivatives (6a–c).
N‐(4‐(N‐(Cyclohexylcarbamoyl)sulfamoyl)phenyl)‐3‐((5‐methylben-
zoxazol‐2‐yl)thio)propanamide (5e)
N‐(4‐(N‐(Ethylcarbamothioyl)sulfamoyl)phenyl)‐2‐((5‐methylbenzoxa-
Yield, 80%; m.p. 232–234°C; IRνmax (cm−1): 3,342, 3,300, 3,102 (3
NH), 3,060 (CH aromatic), 2,930 (CH aliphatic), 1,686 (C═O), 1,335,
1,156 (SO2); 1H NMR (400 MHz, DMSO‐d6): 1.04–1.18 (m, 4H, H‐3
zol‐2‐yl)thio)acetamide (6a)
Yield, 78%; m.p. 220–222°C; IRνmax (cm−1): 3,450, 3,363, 3,260
(3 NH), 3,080 (CH aromatic), 2,979 (CH aliphatic), 1,628 (C═O),

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1,332, 1,152 (SO2); 1H NMR (400 MHz, DMSO‐d6): 1.04 (t, 3H,
J = 7.4 Hz, –CH2CH3), 2.16 (s, 3H, CH3 of benzoxazole), 3.35
(s, 2H, –SCH2), 3.97 (q, 2H, J = 7.4 Hz, –CH2CH3), 6.98 (d, 1H,
J = 8 Hz, Ar‐H, H‐6 of benzoxazole), 7.32 (s, 1H, Ar‐H, H‐4
of benzoxazole), 7.39 (d, 1H, J = 8 Hz, Ar‐H, H‐7 of benzoxazole),
7.82 (d, 2H, J = 8.4 Hz, Ar‐H, H‐2 and H‐6 of phenyl), 7.92 (d, 2H,
J = 8.4 Hz, Ar‐H, H‐3 and H‐5 of phenyl), 8.47 (s, 1H, –NH‐ethyl;
D2O exchangeable), 11.16 (s, 1H, –CONH; D2O exchangeable),
11.39 (s, 1H, –SO2NH; D2O exchangeable); MS (m/z): 464 (M+,
5.74%), 379 (66.79%), 373 (100%, base peak), 111 (43.35%,
base peak), and 73 (49.19%); Anal. calcd. for C19H20N4O4S3
(m.w. 464.57): C, 49.12; H, 4.34; N, 12.06; S, 20.70. Found: C,
49.45; H, 4.57; N, 12.37; S, 20.98.
4.1.6
acetylphenyl)‐2‐((5‐(un)substituted benzoxazol‐2‐yl)‐
thio)acetamide (8a,b
General method for the synthesis of N‐(4‐
|
)
A mixture of the appropriate potassium salt of benzoxazole‐2‐thiol
2a,b (0.01 mol) and N‐(4‐acetylphenyl)‐2‐chloroacetamide 7 (2.12 g,
0.01 mol) in DMF (50 ml) was heated using a water bath for 8 hr.
After cooling to room temperature, the reaction mixture was poured
onto crushed ice. The beige and brown precipitates, respectively,
were collected by filtration, dried and crystallized from absolute
ethanol to give the target compounds 8a,b, respectively.
N‐(4‐Acetylphenyl)‐2‐(benzoxazol‐2‐ylthio)acetamide (8a
)
Yield, 79%; m.p. 155–157°C; IRνmax (cm−1): 3,324 (NH), 3,060 (CH
aromatic), 2,913 (CH aliphatic), 1,671 (C═O); 1H NMR (400 MHz,
DMSO‐d6): 2.42 (s, 3H, CH3), 4.44 (s, 2H, CH2), 7.31 (dd, 1H, J = 7.6,
9.6 Hz, Ar‐H, H‐6 of benzoxazole), 7.60 (dd, 1H, J = 7, 7.6 Hz, Ar‐H, H‐
5 of benzoxazole), 7.63 (d, 1H, J = 7 Hz, Ar‐H, H‐4 of benzoxazole),
7.65 (d, 1H, J = 9.6 Hz, Ar‐H, H‐7 of benzoxazole), 7.72 (d, 2H,
J = 8.4 Hz, Ar‐H, H‐2 and H‐6 of phenyl), 7.93 (d, 2H, J = 8.4 Hz, Ar‐H,
H‐3 and H‐5 of phenyl), 10.78 (s, 1H, NH; D2O exchangeable); MS (m/
z): 326 (M+, 13.87%), 313 (96.03%), 183 (37.60%), 77 (100, base peak
%), and 42 (65.35%); Anal. calcd. for C17H14N2O3S (326.37): C, 62.56;
H, 4.32; N, 8.58; S, 9.82. Found: C, 62.25; H, 4.16; N, 8.99; S, 10.02.
3‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(N‐(ethylcarbamothioyl)sulfamoyl)‐
phenyl)propanamide (6b
)
Yield, 75%; m.p. 177–179°C; IRνmax (cm−1): 3,360, 3,296, 3,114
(3 NH), 3,058 (CH aromatic), 2,982 (CH aliphatic), 1,670 (C═O),
1,393, 1,151 (SO2); 1H NMR (400 MHz, DMSO‐d6): 1.16 (t, 3H,
J = 8.4 Hz, CH3), 2.79 (t, 2H, J = 8 Hz, CH2CO), 3.12 (q, 2H,
J = 8.4 Hz, –CH2CH3), 4.48 (t, 2H, J = 8 Hz, –SCH2), 7.02 (s, 1H,
–NH‐ethyl; D2O exchangeable), 7.33 (dd, 1H, J = 7.8, 8 Hz,
Ar‐H, H‐6 of benzoxazole), 7.38 (dd, 1H, J = 7.8, 7.6 Hz, Ar‐H, H‐
5
of benzoxazole), 7.43 (d, 1H, J = 7.6 Hz, Ar‐H, H‐4 of
benzoxazole), 7.53 (d, 1H, J = 8 Hz, Ar‐H, H‐7 of benzoxazole),
7.68 (d, 2H, J = 7.4 Hz, Ar‐H, H‐2 and H‐6 of phenyl), 7.80 (d, 2H,
J = 7.4 Hz, Ar‐H, H‐3 and H‐5 of phenyl), 10.12 (s, 1H, –CONH;
D2O exchangeable), and 10.46 (s, 1H, –SO2NH; D2O exchange-
able); Anal. calcd. for C19H20N4O4S3 (m.w. 464.57): C, 49.12;
H, 4.34; N, 12.06; S, 20.70. Found: C, 49.39; H, 4.17; N, 12.52;
S, 20.16.
N‐(4‐Acetylphenyl)‐2‐(5‐methylbenzoxazol‐2‐ylthio)acetamide (8b
)
Yield, 70%; m.p. 183–185°C; IRνmax (cm−1): 3,330 (NH), 3,103 (CH
aromatic), 2,996 (CH aliphatic), 1,686 (C═O); 1H NMR (400 MHz,
DMSO‐d6): 2.36 (s, 3H, CH3 of benzoxazole), 2.50 (s, 3H, COCH3),
4.39 (s, 2H, –SCH2), 7.09 (d, 1H, J = 8.4 Hz, Ar‐H, H‐6 of benzoxazole),
7.39 (s, 1H, Ar‐H, H‐4 of benzoxazole), 7.48 (d, 1H, J = 8.4 Hz, Ar‐H,
H‐7 of benzoxazole), 7.68 (d, 2H, J = 8.8 Hz, Ar‐H, H‐2 and H‐6 of
phenyl), 7.91 (d, 2H, J = 8.8 Hz, Ar‐H, H‐3 and H‐5 of phenyl), 10.74 (s,
1H, NH; D2O exchangeable); 13C NMR (400 MHz, DMSO‐d6):
δ = 21.26, 26.88, 37.25, 110.00, 110.99, 118.88 (2 C), 125.61,
130.02 (2 C), 132.48, 134.54, 141.83, 143.41, 150.05, 164.08,
166.10, 196.96; MS (m/z): 340 (M+, 78%), 232 (21.84%), 191
(28.55%), 62 (42.95%), and 43 (100%, base peak); Anal. calcd. for
C18H16N2O3S (340.40): C, 63.51; H, 4.74; N, 8.23; S, 9.42. Found: C,
63.56; H, 4.97; N, 7.94; S, 9.61.
N‐(4‐(N‐(Ethylcarbamothioyl)sulfamoyl)phenyl)‐3‐((5‐methylbenzoxa-
zol‐2‐yl)thio)propanamide (6c
)
Yield, 72%; m.p. 203–205°C; IRνmax (cm−1): 3,322, 3,222, 3,190
(3 NH), 3,070 (CH aromatic), 2,975 (CH aliphatic), 1,690 (C═O),
1,340, 1,165 (SO2); 1H NMR (400 MHz, DMSO‐d6): 1.01 (t, 3H,
J = 7.2 Hz, –CH2CH3), 2.32 (s, 3, CH3 of benzoxazole), 2.94 (t, 2H,
J = 6.8 Hz, CH2CO–), 3.32 (q, 2H, J = 7.2 Hz, –CH2CH3), 4.48
(t, 2H, J = 6.8 Hz, –SCH2), 7.08 (d, 1H, J = 8 Hz, Ar‐H, H‐6 of
benzoxazole), 7.36 (s, 1H, Ar‐H, H‐4 of benzoxazole), 7.40 (d, 1H,
J = 8 Hz, Ar‐H, H‐7 of benzoxazole), 7.73 (d, 2H, J = 8.8 Hz, Ar‐H,
H‐2 and H‐6 of phenyl), 7.81 (d, 2H, J = 8.8 Hz, Ar‐H, H‐3 and H‐5
of phenyl), 8.67 (s, 1H, –NH‐ethyl; D2O exchangeable), 10.74 (s,
1H, CONH; D2O exchangeable), and 11.47 (s, 1H, SO2NH; D2O
exchangeable); 13C NMR (400 MHz, DMSO‐d6): δ = 13.68, 21.38
(2C), 34.13, 42.16, 110.07, 111.56, 119.02, 119.12, 125.29,
127.05, 129.13, 131.96, 133.38, 135.19, 143.76, 145.09,
169.74, 178.28, and 179.63; Anal. calcd. for C20H22N4O4S3
(m.w. 478.60): C, 50.19; H, 4.63; N, 11.71; S, 20.10. Found: C,
49.17; H, 4.59; N, 11.38; S, 19.85.
|
4.1.7
General method for the synthesis
of N‐(4‐(1‐(2‐carbamothioylhydrazono)ethyl)‐
phenyl)‐2‐((5‐(un)substituted benzoxazol‐2‐yl)thio)‐
acetamide (11a,b
)
The appropriate acetyl derivative 8a,b (0.001 mol) was treated with
thiosemicarbazide (0.001 mol) in ethanol (50 ml) in the presence of
catalytic amount of glacial acetic acid and refluxed for 8 hr.
Resulting solids were filtered, washed with water, dried, and
recrystallized from ethanol to afford the corresponding derivatives
11a,b, respectively.

16 of 19
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2‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(1‐(2‐carbamothioylhydrazono)ethyl)‐
phenyl)acetamide (11a)
(d, 1H, J = 7.2 Hz, Ar‐H, H‐7 of benzoxazole), 7.82 (d, 2H, J = 9.2 Hz,
Ar‐H, H‐2 and H‐6 of phenyl), 7.85 (d, 2H, J = 9.2 Hz, Ar‐H, H‐3 and H‐
5 of phenyl), 7.88 (d, 2H, J = 7.2 Hz, Ar‐H, H‐2′ and H‐6′ of phenyl),
10.58 (s, 1H, CONH; D2O exchangeable), 10.70 (s, 1H, NHCO; D2O
exchangeable); MS (m/z): 444.99 (M+, 25.22%), 443.98 (74.46%),
370.02 (100%, base peak), 104.66 (12.51%), and 76.99 (11.17%);
Anal. calcd. for C24H20N4O3S (m.w. 444.51): C, 64.85; H, 4.54; N,
12.60; S, 7.21. Found: C, 64.98; H, 4.71; N, 12.89; S, 7.32.
Yield, 79%; m.p. 191–193°C; IRνmax (cm−1): 3,449, 3,370 (NH2), 3,263,
3,310 (2 NH), 3,050 (CH aromatic), 2,927 (CH aliphatic), 1,689
(C═O); 1H NMR (400 MHz, DMSO‐d6): 2.27 (s, 3H, CH3), 4.41 (s, 2H,
–SCH2), 7.31 (dd, 1H, Ar‐H, J = 8, 8 Hz, H‐6 of benzoxazole), 7.34 (dd,
1H, Ar‐H, J = 8.8, 8 Hz, H‐5 of benzoxazole), 7.57 (d, 1, J = 8.8 Hz, Ar‐
H, H‐4 of benzoxazole), 7.63 (d, 1H, J = 8 Hz, Ar‐H, H‐7 of
benzoxazole), 7.66 (d, 2H, J = 8.4 Hz, Ar‐H, H‐2 and H‐6 of phenyl),
7.91 (d, 2H, J = 8.4 Hz, Ar‐H, H‐3 and H‐5 of phenyl), 8.25 (s, 2H, NH2;
D2O exchangeable), 10.17 (s, 1H, CONH; D2O exchangeable), 10.57
(s, 1H, NHCS; D2O exchangeable); 13C NMR (400 MHz, DMSO‐d6):
δ = 24.90, 33.79, 110.54, 111.54 (2 C), 118.98 (2 C), 124.77, 125.46,
128.89 (2 C), 132.05, 146.91, 157.06 (2 C), 169.56 (2 C), and 179.55;
Anal. calcd. for C18H17N5O2S2 (m.w. 399.49): C, 54.12; H, 4.29; N,
17.53; S, 16.05. Found: C, 54.57; H, 4.49; N, 17.48; S, 16.26.
2‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(1‐(2‐(2‐chlorobenzoyl)hydrazono)‐
ethyl)phenyl)acetamide (12b
)
Yield, 77%; m.p. 190–192°C; IRνmax (cm−1): 3,299, 3,187 (2 NH),
3,053 (CH aromatic), 2,929 (CH aliphatic), 1,660 (C═O); 1H NMR
(400 MHz, DMSO‐d6): 2.26 (s, 3H, CH3), 4.35 (s, 2H, –SCH2), 7.30 (dd,
1H, Ar‐H, J = 7.6, 8 Hz, H‐6 of benzoxazole), 7.32 (dd, 1H, Ar‐H,
J = 7.4, 8 Hz, H‐5 of benzoxazole), 7.34–7.62 (m, 4H, Ar‐H, H‐3′, H‐4′,
H‐5′ and H‐6′ of phenyl), 7.63 (d, 1H, J = 7.4 Hz, Ar‐H, H‐4 of
benzoxazole), 7.65 (d, 1H, J = 7.6 Hz, Ar‐H, H‐7 of benzoxazole), 7.80
(d, 2H, J = 8.4 Hz, Ar‐H, H‐2 and H‐6 of phenyl), 7.87 (d, 2H,
J = 8.4 Hz, Ar‐H, H‐3 and H‐5 of phenyl), 10.47, 10.61 (s, s, 1H,
CONH; D2O exchangeable), 10.91, 11.16 (s, s, 1H, NHCO; D2O
exchangeable); 13C NMR (400 MHz, DMSO‐d6): δ = 14.86, 37.21,
110.69, 118.70, 119.18, 124.81 (2 C), 125.14 (2 C), 126.95, 127.55,
128.98, 129.25, 130.00, 130.91, 133.50, 137.17, 140.35, 148.45,
151.75, 154.64, 163.35, 164.29, and 169.96; MS (m/z): 481 (M++2,
11.30%), 479 (M+, 44.90%), 404 (100%, base peak), 314 (14.21%),
139 (82.24%), 77 (5.50%); Anal. calcd. for C24H19ClN4O3S (m.w.
478.95): C, 60.19; H, 4.00; N, 11.70; S, 6.69. Found: C, 60.42; H, 4.02;
N, 11.56; S, 6.57.
N‐(4‐(1‐(2‐Carbamothioylhydrazono)ethyl)phenyl)‐2‐((5‐methylben-
zoxazol‐2‐yl)thio)acetamide (11b)
Yield, 70%; m.p. 211–213°C; IRνmax (cm−1): 3,369, 3,307 (NH2), 3,245,
3,172 (2 NH), 3,091 (CH aromatic), 2,961 (CH aliphatic), 1,687
(C═O); 1H NMR (400 MHz, DMSO‐d6): 2.27 (s, 3H, CH3), 2.39 (s, 3H,
CH3 of benzoxazole), 4.39 (s, 2H, –SCH2), 7.10 (d, 1H, Ar‐H, J = 8 Hz,
H‐6 of benzoxazole), 7.37 (s, 1H, Ar‐H, H‐4 of benzoxazole), 7.45
(d, 1H, J = 8 Hz, Ar‐H, H‐7 of benzoxazole), 7.57 (d, 2H, J = 8.6 Hz,
Ar‐H, H‐2 and H‐6 of phenyl), 7.85 (d, 2H, J = 8.6 Hz, Ar‐H, H‐3 and
H‐5 of phenyl), 8.25 (s, 2H, NH2; D2O exchangeable), 10.17 (s, 1H,
CONH; D2O exchangeable), and 10.56 (s, 1H, NHCS; D2O exchange-
able); Anal. calcd. for C19H19N5O2S2 (m.w. 413.51): C, 55.19; H, 4.63;
N, 16.94; S, 15.51. Found: C, 55.56; H, 4.43; N, 16.97; S, 15.64.
2‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(1‐(2‐(4‐chlorobenzoyl)hydrazono)‐
ethyl)phenyl)acetamide (12c
)
|
4.1.8
General method for the synthesis of 2‐
Yield, 75%; m.p. 247–249°C; IRνmax (cm−1): 3,327, 3,255 (2 NH),
3,056 (CH aromatic), 2,924 (CH aliphatic), 1,651 (C═O); 1H NMR
(400 MHz, DMSO‐d6): 2.31 (s, 3H, CH3), 4.39 (s, 2H, –SCH2), 7.28 (dd,
1H, Ar‐H, J = 7, 8 Hz, H‐6 of benzoxazole), 7.30 (dd, 1H, Ar‐H, J = 7,
8.4 Hz, H‐5 of benzoxazole), 7.32 (d, 2H, J = 7.2 Hz, Ar‐H, H‐3′ and H‐
5′ of phenyl), 7.55 (d, 2H, J = 8.4 Hz, Ar‐H, H‐4 of benzoxazole), 7.57
(d, 2H, J = 8 Hz, Ar‐H, H‐7 of benzoxazole), 7.60 (d, 2H, J = 8 Hz, Ar‐H,
H‐2 and H‐6 of phenyl), 7.63 (d, 2H, J = 8 Hz, Ar‐H, H‐3 and H‐5 of
phenyl), 7.82 (d, 2H, J = 7.2 Hz, Ar‐H, H‐2′ and H‐6′ of phenyl), 10.59
(s, 1H, CONH; D2O exchangeable), 10.77 (s, 1H, NHCO; D2O
exchangeable); 13C NMR (400 MHz, DMSO‐d6): δ = 21.37, 37.21,
110.06 (2 C), 118.64 (2 C), 119.15 (2 C), 125.61 (2 C), 127.70, 128.82,
130.24, 133.52, 134.54 (2 C), 141.86 (2 C), 150.05 (2 C), 164.15, and
165.65 (2 C); Anal. calcd. for C24H19ClN4O3S (m.w. 478.95): C, 60.19;
H, 4.00; N, 11.70; S, 6.69. Found: C, 60.07; H, 3.84; N, 11.50; S, 6.68.
(benzoxazol‐2‐ylthio)‐N‐(4‐(1‐(2‐((un)substituted
benzoyl)hydrazono)ethyl)phenyl)acetamide (12a‐d
)
N‐(4‐Acetylphenyl)‐2‐(benzoxazol‐2‐ylthio)acetamide (8a; 0,33 gm,
0.001 mol) was treated with the appropriate benzohydrazide
derivative, namely, benzohydrazide, 2‐chlorobenzohydrazide, 4‐
chlorobenzohydrazide, and/or 2‐bromobenzohydrazide 10a–d
(0.001 mol) in ethanol (20 ml) in the presence of catalytic amount
of glacial acetic acid and refluxed for 8 hr. The resulting solids were
filtered, washed with water, dried, and recrystallized from ethanol to
afford the corresponding compounds 12a–d, respectively.
2‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(1‐(2‐benzoylhydrazono)ethyl)phenyl)‐
acetamide (12a)
Yield, 79%; m.p. 260–262°C; IRνmax (cm−1): 3,320, 3,235 (2 NH),
3,070 (CH aromatic), 2,936 (CH aliphatic), 1,664 (C═O); 1H NMR
(400 MHz, DMSO‐d6): 2.31 (s, 3H, CH3), 4.39 (s, 2H, –SCH2), 7.30 (dd,
1H, Ar‐H, J = 5.6, 7.2 Hz, H‐6 of benzoxazole), 7.47 (dd, 1H, Ar‐H,
J = 9.2, 5.6 Hz, H‐5 of benzoxazole), 7.54 (dd, 2H, J = 6.8, 7.2 Hz, Ar‐H,
H‐3′ and H‐5′ of phenyl), 7.62 (dd, 1H, J = 6.8, 6.8 Hz, Ar‐H, H‐4′
of phenyl), 7.64 (d, 1H, J = 9.2 Hz, Ar‐H, H‐4 of benzoxazole), 7.67
2‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(1‐(2‐(2‐bromobenzoyl)hydrazono)‐
ethyl)phenyl)acetamide (12d
Yield, 75%; m.p. 186–188°C; IRνmax (cm−1): 3,311, 3,210 (2 NH),
3,070 (CH aromatic), 2,930 (CH aliphatic), 1,660 (C═O); 1H NMR
(400 MHz, DMSO‐d6): 2.27 (s, 3H, CH3), 4.36 (s, 1H, –SCH2), 7.29 (dd,
)

EL‐HELBY ET AL.
17 of 19
|
1H, Ar‐H, J = 8.4, 8 Hz, H‐6 of benzoxazole), 7.32 (dd, 1H, Ar‐H,
J = 7.4, 8 Hz, H‐5 of benzoxazole), 7.34 (dd, 1H, Ar‐H, J = 8.8, 8.8 Hz,
H‐5′ of phenyl), 7.41 (d, 1H, J = 7.4 Hz, Ar‐H, H‐4 of benzoxazole),
7.43 (d, 1H, J = 8.4 Hz, Ar‐H, H‐7 of benzoxazole), 7.45 (d, 2H,
J = 8.4 Hz, Ar‐H, H‐2 and H‐6 of phenyl), 7.51 (d, 1H, J = 7.2 Hz, Ar‐H,
H‐3′ of phenyl), 7.57 (dd, 1H, J = 7.2, 8.8 Hz, Ar‐H, H‐4′ of phenyl),
7.64 (d, 1H, J = 8.8 Hz, Ar‐H, H‐6′ of phenyl), 7.81 (d, 2H, J = 8.4 Hz,
Ar‐H, H‐3 and H‐5 of phenyl), 10.48, 10.60 (s, s, 1H, CONH; D2O
exchangeable), 10.91, 11.16 (s, s, 1H, NHCO; D2O exchangeable); MS
(m/z): 525 (M++2, 13.38%), 523 (M+, 33.27%), 358 (100%, base peak),
326 (76.72%), 184 (32.96%), and 63 (75.27%); Anal. calcd. for
C24H19BrN4O3S (m.w. 523.41): C, 55.07; H, 3.66; N, 10.70; S, 6.13.
Found: C, 54.81; H, 3.95; N, 10.36; S, 5.99.
Anal. calcd. for C21H19N5O3S (m.w. 421.48): C, 59.84; H, 4.54; N,
16.62; S, 7.61. Found: C, 59.91; H, 4.59; N, 16.40; S, 7.55.
|
4.1.10
Synthesis of 2‐(benzoxazol‐2‐ylthio)‐N‐(4‐
(1‐(hydroxyimino)ethyl)phenyl)acetamide (14)
A mixture of equimolar amounts of the acetyl derivative 8a (0.33 g,
0.001 mol) and hydroxylamine hydrochloride (0.07 g, 0.001 mol) in
absolute ethanol (50 ml) was heated under reflux for 20 hr and then
left to cool. The separated solid was filtered, washed with water,
dried, and recrystallized from ethanol.
Yield, 65%; m.p. 205–207°C; IRνmax (cm−1): 3,310 (NH), 3,065 (CH
aromatic), 2,915 (CH aliphatic), 1,672 (C═O); 1H NMR (400 MHz,
DMSO‐d6): 2.12 (s, 3H, CH3), 3.75 (s, 2H, –SCH2), 7.53 (dd, 1H, Ar‐H,
J = 8, 8.4 Hz, H‐6 of benzoxazole), 7.56 (dd, 1H, Ar‐H, J = 8, 8.8 Hz,
H‐5 of benzoxazole), 7.65 (d, 1H, J = 8.8 Hz, Ar‐H, H‐4 of benzox-
azole), 7.70 (d, 2H, J = 8.4 Hz, Ar‐H, H‐7 of benzoxazole), 7.87 (d, 2H,
J = 8.4 Hz, Ar‐H, H‐2 and H‐6 of phenyl), 7.91 (d, 2H, J = 8.4 Hz, Ar‐H,
H‐3 and H‐5 of phenyl), 10.26 (s, 1H, CONH; D2O exchangeable), and
11.07 (s, 1H, NOH; D2O exchangeable); Anal. calcd. for C17H15N3O3S
(m.w. 341.39): C, 59.81; H, 4.43; N, 12.31; S, 9.39. Found: C, 59.81; H,
4.43; N, 12.31; S, 9.39.
|
4.1.9
General method for the synthesis of N‐(4‐(1‐
(2‐(2‐cyanoacetyl)hydrazono)ethyl)phenyl)‐2‐(((un)‐
substituted benzoxazol‐2‐yl)thio)acetamide (13a,b
)
A mixture of the appropriate acetyl derivative 8a,b (0.001 mol) and 2‐
cyanoacetohydrazide (0.01 g, 0.001 mol) in ethanol (25 ml) was
refluxed for 3 hr, then left to cool. The solid product formed upon
pouring onto ice/water was filtered and recrystallized from ethanol
to give the corresponding derivatives 13a,b, respectively.
|
4.1.11
Synthesis of 2‐(benzoxazol‐2‐ylthio)‐N‐(4‐
2‐(Benzoxazol‐2‐ylthio)‐N‐(4‐(1‐(2‐(2‐cyanoacetyl)hydrazono)ethyl)‐
phenyl)acetamide (13a)
(3‐(4‐methoxyphenyl)acryloyl)phenyl)acetamide (15)
Yield, 79%; m.p. 207–209°C; IRνmax (cm−1): 3,350, 3,179 (2 NH),
3,057 (CH aromatic), 2,925 (CH aliphatic), 2,260 (CN), 1,673 (C═O);
1H NMR (400 MHz, DMSO‐d6): 2.20 (s, 3H, CH3), 4.20 (s, 2H, –SCH2),
4.39 (s, 2H, J = 6.8 Hz, –CH2CN), 7.30 (dd, 1H, Ar‐H, J = 8.8, 9 Hz, H‐6
of benzoxazole), 7.33 (dd, 1H, Ar‐H, J = 8.8, 9.2 Hz, H‐5 of
benzoxazole), 7.60 (d, 1H, J = 9.2 Hz, Ar‐H, H‐4 of benzoxazole),
7.64 (d, 1H, J = 9 Hz, H‐7 of benzoxazole), 7.76 (d, 2H, J = 8.4 Hz, Ar‐
H, H‐2 and H‐6 of phenyl), 7.87 (d, 2H, J = 8.4 Hz, Ar‐H, H‐3 and H‐5
of phenyl), 10.60 (s, 1H, CONHph; D2O exchangeable), and 10.96 (s,
1H, NHCO; D2O exchangeable); Anal. calcd. for C20H17N5O3S (m.w.
407.45): C, 58.96; H, 4.21; N, 17.19; S, 7.87. Found: C, 59.25; H, 4.35;
N, 16.92; S, 7.62.
To a mixture of the acetyl derivative 8a (0.65 g, 0.002 mol) in ethyl
alcohol (50 ml) and 5% NaOH in ethyl alcohol (10 ml), 4‐methox-
ybenzaldehyde (0.27 g, 0.002 mol) was added drop‐wise within
15 min. The reaction mixture was refluxed for 3 hr. The formed
precipitate was cooled, filtered, air dried, and then recrystallized
from ethanol to give the corresponding chalcone 15.
Yield, 83%; m.p. 146–148°C; IRνmax (cm−1): 3,114 (NH), 3,080
(CH aromatic), 2,905 (CH aliphatic), 1,665 (C═O); 1H NMR
(400 MHz, DMSO‐d6): 3.02 (s, 3H, COCH3), 4.65 (s, 2H, –SCH2),
6.70 (d, 1H, J = 9.2 Hz, COCH═), 6.81 (d, 2H, J = 8.8 Hz, Ar‐H, H‐3′
and H‐5′ of phenyl′), 7.28 (dd, 1H, Ar‐H, J = 8.4, 8.8 Hz, H‐6 of
benzoxazole), 7.35 (dd, 1H, Ar‐H, J = 8.4, 8 Hz, H‐5 of benzoxazole),
7.41 (d, 1H, J = 8 Hz, Ar‐H, H‐4 of benzoxazole), 7.43 (d, 1H,
J = 8.8 Hz, Ar‐H, H‐7 of benzoxazole), 7.47 (d, 2H, J = 8.8 Hz, Ar‐H,
H‐2′ and H‐6′ of phenyl′), 7.74 (d, 2H, J = 8 Hz, Ar‐H, H‐2 and H‐6
of phenyl), 7.96 (d, 2H, J = 8 Hz, Ar‐H, H‐3 and H‐5 of phenyl), 8.08
(d, 1H, J = 9.2 Hz, = CH‐ph′), and 12.81 (s, 1H, NH; D2O exchange-
able); Anal. calcd. for C25H20N2O4S (m.w. 444.51): C, 67.55; H,
4.54; N, 6.30; S, 7.21. Found: C, 67.33; H, 4.45; N, 6.50; S, 7.33.
N‐(4‐(1‐(2‐(2‐Cyanoacetyl)hydrazono)ethyl)phenyl)‐2‐((5‐methylben-
zo[d]oxazol‐2‐yl)thio)‐acetamide (13b
)
Yield, 70%; m.p. 241–243°C; IRνmax (cm−1): 3,320, 3,183 (2 NH),
3,056 (CH aromatic), 2,925 (CH aliphatic), 2,258 (CN), 1,685 (C═O);
1H NMR (400 MHz, DMSO‐d6): 2.20 (s, 3H, CH3), 2.37 (s, 3H, CH3 of
benzoxazole), 4.20 (s, 2H, –SCH2), 4.37 (s, 2H, J = 6.8 Hz, –CH2CN),
7.10 (d, 1H, Ar‐H, J = 8.4 Hz, H‐6 of benzoxazole), 7.40 (s, 1H, Ar‐H,
H‐4 of benzoxazole), 7.48 (d,1H, J = 8.4 Hz, Ar‐H, H‐7 of benzox-
azole), 7.60 (d, 2H, J = 8 Hz, Ar‐H, H‐2 and H‐6 of phenyl), 7.76 (d, 2H,
J = 8 Hz, Ar‐H, H‐3 and H‐5 of phenyl), 10.57 (s, 1H, CONH; D2O
exchangeable), and 10.96 (s, 1H, CONHph; D2O exchangeable); 13C
NMR (400 MHz, DMSO‐d6): δ = 14.11, 21.36, 25.33, 37.20, 110.06,
116.72, 118.62, 119.08, 119.17, 125.61, 127.46, 127.73, 133.26,
134.54, 140.16, 141.84, 149.10, 150.04, 164.15, 165.63, and 166.17;
|
4.1.12
Synthesis of N‐(4‐(1‐acetyl‐5‐(4‐
methoxyphenyl)‐4,5‐dihydro‐1H‐pyrazol‐3‐yl)phenyl)‐
2‐(benzoxazol‐2‐ylthio)acetamide (16)
A mixture of the chalcone 15 (0.89 g, 0.002 mol) and hydrazine
hydrate (1 ml, 98%) in ethanol (50 ml) in the presence of glacial acetic
acid (5 ml) was refluxed for 6 hr. After cooling, the separated

18 of 19
EL‐HELBY ET AL.
|
precipitate was filtered, air dried, and recrystallized from ethanol to
give the corresponding N‐acetylpyrazoline derivative 16.
Cells then were incubated in a humidified atmosphere at 37°C for
24 hr. Then, cells were exposed to different concentrations of
compounds (0.1, 10, 100, and 1,000 µM) for 72 hr. Then the viability
of treated cells was determined using MTT technique as follows. Media
were removed, cells were incubated with 200 μl of 5% MTT solution/
well (Sigma‐Aldrich, MO) and were allowed to metabolize the dye into
colored‐insoluble formazan crystals for 2 hr. The remaining MTT
solution were discarded from the wells and the formazan crystals were
dissolved in 200 µl/well acidified isopropanol for 30 min, covered with
aluminum foil and with continuous shaking using a MaxQ 2000 plate
shaker (Thermo Fisher Scientific Inc, MI) at room temperature.
Absorbance was measured at 570 nm using a Stat Fax 4200 plate
reader (Awareness Technology, Inc., FL). The cell viability was expressed
as percentage of control and the concentration that induces 50%
of maximum inhibition of cell proliferation (IC50) were determined
using GraphPad Prism version 5 software (GraphPad software Inc,
CA).[41–43]
Yield, 65%; m.p. 281–283°C; IRνmax (cm−1): 3,323 (NH), 3,090 (CH
aromatic), 2,978 (CH aliphatic), 1,675 (C═O); 1H NMR (400 MHz,
DMSO‐d6): 1.66 (s, 3H, COCH3), 3.70 (d, 2H, J = 12 Hz, CH2 of
pyrazol), 3.75 (s, 2H, OCH3), 3.76 (s, 3H, SCH2), 6.35 (t, 1H, CH of
pyrazol), 6.77 (dd, 1H, J = 8.4, 7 Hz, H‐6 of benzoxazole), 6.89 (d, 2H,
J = 8.4 Hz, Ar‐H, H‐2 and H‐6 of phenyl), 7.05 (dd, 1H, Ar‐H, J = 8.4,
8.8 Hz, H‐5 of benzoxazole), 7.16 (d, 1H, J = 8.8 Hz, Ar‐H, H‐4 of
benzoxazole), 7.41 (d, 2H, J = 8.4 Hz, Ar‐H, H‐3 and H‐5 of phenyl),
7.81 (d, 1H, J = 7 Hz, H‐7 of benzoxazole), and 9.70 (s, 1H, NH; D2O
exchangeable); 13C NMR (400 MHz, DMSO‐d6): δ = 14.11, 21.36,
25.33, 37.20, 110.06, 116.72, 118.62, 119.08, 119.17, 125.61,
127.46, 127.73, 133.26, 134.54, 140.16, 141.84, 149.10, 150.04,
164.15, 165.63, 166.17; 26.80, 41.09, 55.79 (2 C), 108.29, 114.82
(2 C), 114.97 (2 C), 115.46, 119.44, 120.46, 123.38 (2 C), 128.21 (2 C),
130.29 (2 C), 130.81 (2 C), 132.26, 134, 142.21, 147.65, 158.6, 161.39,
and 186.73; Anal. calcd. for C27H24N4O4S (m.w. 500.57): C, 64.79; H,
4.83; N, 11.19; S, 6.40. Found: C, 64.71; H, 4.89; N, 10.99; S, 6.51.
|
4.3.2
In vitro VEGFR‐2 kinase assay
The kinase activity of VEGFR‐2 was carried out in Pharmacology and
Toxicology Department, Faculty of Pharmacy, Al‐Azhar University,
Cairo, Egypt, and measured by use of an antiphosphotyrosine antibody
with the Alpha Screen system (PerkinElmer) according to manufac-
turer’s instructions.[6] Enzyme reactions were performed in 50 mM
Tris‐HCl, pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01% Tween‐20, and
2 mM DTT, containing 10 μM adenosine triphosphate (ATP), 0.1 μg/ml
biotinylated poly‐GluTyr (4:1) and 0.1 nM of VEGFR‐2 (Millipore, UK).
Before catalytic initiation with ATP, the tested compounds at final
|
4.2
Docking studies
In the present work, all the target compounds were subjected to
docking study to explore their binding mode toward VEGFR‐2
enzyme. All modeling experiments were performed using molsoft
program, which provides a unique set of tools for the modeling of
protein/ligand interactions. It predicts how small flexible molecule
such as substrates or drug candidates bind to a protein of known
three‐dimensional structure represented by grid interaction poten-
tials (http://www.molsoft.com/icm_pro.html). Each experiment used
the biological target VEGFR‐2 downloaded from the Brookhaven
Protein Databank (http://www.rcsb.org/pdb/explore/explore.do?
structureId=1YWN). To qualify the docking results in terms of
accuracy of the predicted binding conformations in comparison with
the experimental procedure, the reported VEGFR‐2 inhibitor drugs
vatalanib and sorafenib were used as reference ligands.
concentrations ranging from
0 to 300 μg/ml and enzyme were
incubated for 5 min at room temperature. The reactions were
quenched by the addition of 25 μl of 100 mM EDTA (ethylenediami-
netetraacetic acid), 10 μg/ml AlphaScreen streptavidine cytotoxicity
evaluation donor beads and 10 μg/ml acceptor beads in 62.5 mM
HEPES (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid) pH 7.4,
250 mM NaCl, and 0.1% bovine serum albumin. Plate was incubated in
the dark overnight and then read by ELISA reader (PerkinElmer). Wells
containing the substrate and the enzyme without compounds were
used as reaction control. Wells containing biotinylated poly‐GluTyr
(4:1) and enzyme without ATP were used as basal control. Percent
inhibition was calculated by the comparison of compounds treated to
control incubations. The concentration of the test compound causing
50% inhibition (IC50) was calculated from the concentration–inhibition
response curve (triplicate determinations) and the data were
compared with sorafenib (Sigma‐Aldrich) as standard VEGFR‐2
inhibitor.
|
4.3
Biological assays
|
4.3.1
In vitro cytotoxic activity
The cytotoxicity assays were performed at Pharmacology & Toxicol-
ogy Department, Faculty of Pharmacy, Al‐Azhar University, Cairo,
Egypt. Cancer cells from different cancer cell lines hepatocellular
carcinoma (HepG2), breast cancer (MCF‐7), and colorectal carcinoma
(HCT‐116), were purchased from American Type Cell Culture
Collection (ATCC; Manassas) and grown on the appropriate growth
medium Roswell Park Memorial Institute medium (RPMI 1640)
supplemented with 100 mg/ml of streptomycin, 100 U/ml of penicillin
and 10% of heat‐inactivated fetal bovine serum in a humidified, 5% (v/
v) CO2 atmosphere at 37°C. Cytotoxicity assay by MTT.
ACKNOWLEDGMENTS
The authors extend their appreciation and thanks to Dr. Tamer
Abdel‐Ghany, Pharmacology & Toxicology Department, Faculty of
Pharmacy, Al‐Azhar University, Cairo, Egypt for helping with the
pharmacological part.
Exponentially growing cells from different cancer cell lines were
trypsinized, counted, and seeded at the appropriate densities
(2,000–1,000 cells/0.33 cm2 per well) into 96‐well microtiter plates.

EL‐HELBY ET AL.
19 of 19
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CONFLICT OF INTERESTS
[23] X. Yuan, Q. Yang, T. Liu, K. Li, Y. Liu, C. Zhu, Z. Zhang, L. Li, C. Zhang,
M. Xie, J. Lin, J. Zhang, Y. Jin, Eur. J. Med. Chem. 2019, 179, 147–165.
https://doi.org/10.1016/j.ejmech.2019.06.054
The authors declare that there are no conflicts of interests.
[24] A. Kamal, D. Dastagiri, M. J. Ramaiah, J. S. Reddy, E. V. Bharathi, M.
K. Reddy, M. V. P. Sagar, T. L. Reddy, S. N. Pushpavalli, M. Pal‐
Bhadra, Eur. J. Med. Chem. 2011, 46, 5817. https://doi.org/10.1016/j.
ejmech.2011.09.039
ORCID
Khaled El‐Adl
http://orcid.org/0000-0002-8922-9770
[25] C. Wu, M. Wang, Q. Tang, R. Luo, L. Chen, P. Zheng, W. Zhu, Molecules
2015, 20, 19361. https://doi.org/10.3390/molecules201019361
[26] Ö. Yılmaz, S. Özbaş Turan, J. Akbuğa, P. M. Tiber, O. Orun, C. T.
Supuran, Ş. G. Küçükgüzel, J. Enzyme Inhib. Med. Chem. 2015, 30, 967.
https://doi.org/10.3109/14756366.2014.1001756
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How to cite this article: El‐Helby A‐GA, Sakr H, Eissa IH,
Abulkhair H, Al‐Karmalawy AA, El‐Adl K. Design, synthesis,
molecular docking, and anticancer activity of benzoxazole
derivatives as VEGFR‐2 inhibitors. Arch Pharm Chem Life Sci.
2019;e1900113. https://doi.org/10.1002/ardp.201900113