Synthesis and biological evaluation of novel 2,4′-bis substituted diphenylamines as anticancer agents and potential epidermal growth factor receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.05.072

Four new series of 2,4′-bis diphenylamine hydrazones 14, 2,4′-bis aminothiadiazole 16, 2,4′-bis mercaptotriazole 17-18 and 2,4′-bis mercapto-oxadiazole diphenylamine derivatives 19-20 were synthesized and evaluated for their ability to inhibit EGFR tyrosine kinase. Compound N-ethyl-5-{2-[4-(5-(ethylamino)-1,3,4-thiadiazol-2-yl)- phenylamino]phenyl}-1,3,4-thiadiazol-2-amine 16a was the most active enzyme inhibitor (98% inhibition at 10 μM). Moreover, all compounds that showed enzyme inhibition activity were tested in vitro on human breast carcinoma cell line (MCF-7) in which EGFR is highly expressed. The tested compounds exploited potent antitumor activity with IC₅₀ values ranging 0.73-2.38 μM. Molecular modeling and docking of the synthesized compounds into the active site of EGFR kinase domain showed good agreement with the obtained biological results. The present work represents a novel class of diphenylamine based derivatives with potent cytotoxicity and promising EGFR PTK inhibition activity. Several analogues 2,4′-bis substituted diphenylamines were synthesized and evaluated as EGFR tyrosine kinase inhibitors as well as for their antiprolifertive properties on human breast cancer cell lines (MCF-7).

Full text of DOI:10.1016/j.ejmech.2010.05.072

European Journal of Medicinal Chemistry 45 (2010) 4113e4121
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel 2,40-bis substituted diphenylamines
as anticancer agents and potential epidermal growth factor receptor tyrosine
kinase inhibitors
*
Sahar Mahmoud Abou-Seri
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini street, Cairo, P.O. Box, 11562, Egypt
a r t i c l e i n f o
a b s t r a c t
Four new series of 2,40-bis diphenylamine hydrazones 14, 2,40-bis aminothiadiazole 16, 2,40-bis mer-
captotriazole 17e18 and 2,40-bis mercapto-oxadiazole diphenylamine derivatives 19e20 were synthe-
sized and evaluated for their ability to inhibit EGFR tyrosine kinase. Compound N-ethyl-5-{2-[4-
(5-(ethylamino)-1,3,4-thiadiazol-2-yl)- phenylamino]phenyl}-1,3,4-thiadiazol-2-amine 16a was the most
Article history:
Received 8 March 2010
Received in revised form
8 May 2010
Accepted 31 May 2010
Available online 9 June 2010
active enzyme inhibitor (98% inhibition at 10
mM). Moreover, all compounds that showed enzyme
inhibition activity were tested in vitro on human breast carcinoma cell line (MCF-7) in which EGFR is
highly expressed. The tested compounds exploited potent antitumor activity with IC50 values ranging
0.73e2.38 mM. Molecular modeling and docking of the synthesized compounds into the active site of
EGFR kinase domain showed good agreement with the obtained biological results. The present work
represents a novel class of diphenylamine based derivatives with potent cytotoxicity and promising EGFR
PTK inhibition activity.
Keywords:
2,40-Bis substituted diphenylamine
derivatives
EGFR tyrosine kinase inhibitors
Antitumor
Docking
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
signaling may not only has an antiproliferative and therapeutic
effects but also increases sensitivity to cytotoxic therapies [9].
The ErbB or epidermal growth factor (EGF) receptors are
members of subclass 1 of the protein tyrosine kinase (PTK) super
family. There are four ErbB receptor family members: ErbB1 (EGFR,
HER1), ErbB2 (HER2/neu), ErbB3 (HER3), ErbB4 (HER4) [1e4]. The
receptors are situated at the cell membrane and have an extracel-
Various approaches have been developed to target the ErbB
signaling pathways including: 1) Monoclonal antibodies like tras-
tuzumab and cetuximab directed against the receptor [10,11]; 2)
ATP-competitive small molecule inhibitors of the intrinsic tyrosine
kinase domain like geftinib 1 and erlotinib 2 [12]. Lapatinib 3 is
a potent dual EGFR/ErbB2 inhibitor, recently approved for treat-
ment of breast cancer [13]. These agents belong to the class of 4-
anilinoquinazoline (Fig. 1). Although the ATP binding site shows
high homology with other ATP driven receptor kinases, 4-anilino-
quinazolines have been proved as selective potent inhibitors of
EGFR binding site [14e18]. The quinazoline moiety fits into the ATP-
binding pocket in the kinase domain, while the aniline ring fills an
adjacent lipophilic pocket [19]. Since, 4-anilinoquinazolines have
very tight structure activity relationship, this emerged the need to
develop non-quinazoline EGFR inhibitors.
Hodge and Pierce revealed a bioisosteric relationship between
salicylic group and quinazoline. They suggested that the hydrogen
bond array of salicylamide could mimic the prymidine ring of
quinazolines [20]. Later, different salicylanilides 4 have been
reported as potent inhibitors of EGFR PTKs. This led to the
hypothesis, that salicylanilides may adopt the same binding mode
to EGFR PTK as quinazolines [21,22]. Moreover, 4-amino-6-aryla-
lular ligand binding domain,
a transmembrane domain and
a cytoplasmic tyrosine kinase domain. Ligand binding to the
receptors results in receptor homo- and hetero-dimers, activation
of the intrinsic kinase domain and phosphorylation of specific
tyrosine residues within the cytoplasmic tail. Protein dock on these
phosphorylated residues, leads to the activation of a variety of
intracellular signaling pathways that promote cell growth, prolif-
eration, differentiation, survival and migration [1e4]. Over-
expression of EGFR and ErbB2 has been associated with aggressive
diseases and poor prognosis in range of human tumors (e.g. breast,
lung, ovarian and squamous carcinoma of head and neck) [5e7].
The EGFR signaling is important for tumor cell proliferation, inhi-
bition of apoptosis, angiogenesis, metastasis and sensitivity to
chemotherapy and radiotherapy [8]. Therefore, inhibition of EGFR
* Tel.: þ2 02 25591683; fax: þ2 02 2362 8426.
E-mail address: saharshaarawy_69@yahoo.com
minopyrimidine-5-carbalehyde oximes
5 and 5-carbalehyde
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.072

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S.M. Abou-Seri / European Journal of Medicinal Chemistry 45 (2010) 4113e4121
O
F
COOH
CU/K2CO3
COOH
+
OH
NH
N
Cl
NH
N
O
SOCl2
O
O
N
H
O
O
N
N
O
O
H2N
Cl
N
CH3OH
10
9
11
HO
O
O
2
1
O
OCH3
NH2NH2
NHNH2
F
N
H
O
O
S
O
N
H
H3CO
O
12
13
HN
Cl
NH
N
H2NHN
O
O
N
Scheme 1. Synthetic pathway for the intermediates 12e13.
3
addition, diphenylamine derivatives have been evaluated as orally
active antiproliferative agents that inhibit tyrosine kinase auto-
phosphorylation [25]. Also, hydrazones have been well acknowl-
edged to possess anticancer activity. Based on this knowledge,
a series of 2,40-bis substituted diphenylamine arylidene hydrazides
7 was synthesized. Such substitution pattern could target different
regions of the ATP binding site. The pseudo six-membered ring
formed by intramolecular hydrogen bond between the diphenyl-
amine NH and 2-hydrazide Oxygen (NH.O]C) would function to
mimic the pyrimidine ring of quinazolines. On the other hand, the
2-bulky substituent would interact deeply with an adjacent
hydrophobic pocket present in EGFR PTK. Moreover, the 40-sub-
tituent was expected to orient toward the solvent front, so that the
produce interaction might gain an additional potency.
Fig. 1. 4-Anilinoquinazolines EGFR-TK inhibitors, Gefitinib 1, Erlotinib 2, Lapitinib 3.
Ar
Ar
Ar
HN
O
NH
N
NH
N
R1
N
R1
R2
O
N
H
N
H
O
H
N
R
N
R2
N
H
N
H
4
6
5
R
N
O
X
N=CHAr
N
H
N
A number of reports have shown that fused tricyclic quinazoline
analogs as pyrrolo-, pyrazolo- and imidazo-quinazolines were very
potent EGFR PTKs inhibitors [26,27]. A large number of thiadiazole,
triazole and oxadiazole based compounds have been shown to
N
H
N
H
ArHC=N
N
H
O
7
X
N
8
N
R
Fig. 2. Salicylanilides 4, carbaldehyde 5-oxime 5, carbaldehyde 5-hydrazone 6, 2,40-bis
substituted diphenylamine arylidene hydrazides template 7, 2,40-bis-heterocyclic
substituted diphenylamines template 8 (X ¼ S, N or O).
O
H
NHN
R1
N
H
R1CHO
R2NCS
H
hydrazones 6 with dual EGFR/ErbB2 inhibitory properties have
been reported to inhibit tumor cell proliferation of breast carci-
noma in vitro [23,24]. These scaffolds effectively mimic the qui-
nazoline kinase template by forming pseudo-bicyclic structure
with the help of an intramolecular hydrogen bond (Fig. 2). In
14a-g
NHN
O
R1
13
O
S
C
NHNH
NHR2
N
H
S
C
R2HN
HNHN
O
15a-c
NaOH
PPA
N
N
N
SH
N
N
R2
NHR2
S
N
H
N
R2
H
N
N
S
N
N
N
17a-c
16a-c
HS
OCH3
OCH
R2HN
HO
,c=
3 ,d=
Cl ,e=
N
R1 a=
, f=
, b=
, =g
.
R2 a=C2H5, b=C6H5, c=C3H5
O
NO2
O
Fig. 3. Energy minimized structure of compound 16a, showing hydrogen bonding
between diphenylamine NH and N3 of thiadiazole ring.
Scheme 2. Synthetic pathway for compounds 14e17.

S.M. Abou-Seri / European Journal of Medicinal Chemistry 45 (2010) 4113e4121
4115
Table 2
N
N
In vitro cytotoxic activities of the synthesized compounds against human breast
cancer cell (MCF-7).
SH
O
CS2/KOH
Compound
Cytotoxicity IC50 (mmol)
13
N
H
14c
14e
16a
17a
19
0.81
1.23
0.94
2.38
2.00
0.73
1.05
19
O
N
R3X/
1-CS2/KOH
2- NH2NH2
K2CO3
N
HS
N
O
N
20b
20c
N
SR3
N
SH
N
NH2
N
H
N
phenylamino] benzoic acid hydrazide 13 was prepared by hydra-
zinolysis of the diester 12. Condensation of the dihydrazide 13
with different aldehydes afforded the hydrazones 14aeg, while
reaction with the appropriate isothiocyanate produced the thio-
semicarbazides derivatives 15aec. Compounds 16aec were
prepared by cyclodehydration of the corresponding thio-
semicarbazides 15aec with polyphosphoric acid. Alternatively, the
thiosemicarbazides 15aec were cyclized into the corresponding
N
N
H
O
H2N
20a-c
18
N
N
N
R3S
HS
R3: a=C2H5, b=CH2C5H5, c=C3H5
Scheme 3. Synthetic pathway for compounds 18e20.
1,2,4-triazol-5-thiones 17aec upon reflux with
2 N sodium
hydroxide solution. On the other hand the method of Reid and
Heindel was adopted for the synthesis of the 4-amino-5-mercapto-
1,2,4-triazole derivative 18 [33]. Finally, the intramolecular cycli-
zation of the diacid hydrazide 13 with carbon disulfide in presence
of sodium hydroxide, produced the 5-mercapto-1,3,4-oxadiazole
derivative 19, which was alkylated with different alkyl halides in
presence of potassium carbonate to yield compounds 20aec. The
reaction sequences are illustrated in Schemes 1e3.
exhibit antitumor activity against a wide variety of tumor cell lines
[28e31]. Therefore, different bis substituted 2,40-diphenylamines 8
were synthesized to explore the effect of incorporating various
heterocyclic bioisosters as 1,3,4-thiadiazole, 1,2,4-triazole or 1,3,4-
oxdiazole ring substituents on the diphenylamine core. Such
heterocyclic rings are capable of hydrogen bonding with the
diphenylamine NH and would mimic a pseudo fused tricyclic qui-
nazoline system (Figs. 2 and 3).
The present work reports the synthesis, EGFR inhibitory prop-
erties and in vitro antitumor activity against MCF-7 (breast) cell line
of some 2,40-bis substituted diphenylamines. This work represents
an attempt to find new structural type of tyrosine kinase inhibitors
that target EGFR.
3. Results and discussion
3.1. In vitro EGFR kinase inhibitory properties
Kinase inhibitory activity of all final compounds in Scheme 2
and 3 were evaluated using EGFR kinase activity assay by ELISA
[34]. The assay was based on the inhibition of the phosphorylation
of poly glutamic acid/tyrosine [Poly (Glu/Tyr), 4:1] by EGFR tyrosine
kinase. Results are summarized in Table 1. The inhibitory activities
2. Chemistry
2-{[4-(Methoxycarbonyl)phenyl]amino}benzoic acid methyl
ester 12 was obtained through esterification of 2,40-dipenylamine
dicarboxylic acid 11, obtained in turn through the condensation of
2-chlorbenzoic acid 9 and p-amino benzoic acid 10 under Ullmann
condition [32]. The key intermediate 2-[4-(hydrazinecarbonyl)
Table 1
EGFR tyrosine kinase inhibitory activity of the synthesized compounds at 10 mM.
Compound
Inhibitory activity [%] at 10 mM
14a
14b
14c
14d
14e
14f
0.4
1.2
47.1
2.4
50.0
0.8
14g
16a
16b
16c
17a
17b
17c
18
1.7
97.7
0.0
0.0
52.0
0.0
0.0
0.0
19
48.0
3.4
49.0
50.0
99.2
20a
20b
20c
Lapatinib
Fig. 4. The superposition of lapatinib (purple) and compound 14c (blue) docked in the
ATP binding site of EGFR PTK, showing perfect overlay of the quinazoline ring in
lapatinib and 2-benzoyl moiety in 14c (for interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article).

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S.M. Abou-Seri / European Journal of Medicinal Chemistry 45 (2010) 4113e4121
Fig. 5. Docking of compound 16a in the ATP binding site of EGFR PTK in 2D diagram (a) and in 3D style (b).
are given as percentage inhibition at concentration of 10
inhibitor.
m
M of the
the aryl moiety with furan 14f or 5-nitrofuran 14g, probably due to
the electron-withdrawing property of the furan ring.
The results showed that, for a series of 2,40-bis-(N-substituted)
diphenylamine hydrazones 14aeg, only the arylidene analogues
with lipophilic electron-donating group on the benzylidene moiety
showed moderate inhibitory activity. e.g. 14c and 14e resulted in
47% and 50% inhibition, respectively. The unsubstituted analogue
14a, the 2-OH derivative 14b and the 4-Cl derivative 14d were
inactive. Also, a similar behavior has been noted on replacement of
In the 2,40-bis-aminothiadiazole series 16aec, it was found that
the substituent on amino greatly affects the kinase inhibitory
activity. The N-ethyl analogue 16a was a potent EGFR PTK inhibitor
(98% inhibition). However, the activity was abolished if a phenyl or
allyl group was attached to the amino (16b and 16c). This proves
poor bulk tolerance at this class of compounds. A similar trend was
observed with respect to N-4 substitution of the triazole ring in the

S.M. Abou-Seri / European Journal of Medicinal Chemistry 45 (2010) 4113e4121
4117
to determine the effectiveness of EGFR-TK inhibition. The IC50 of the
tested compounds in mM are presented in Table 2.
The results indicate that, the 2,40-bis-(N-substituted)diphenyl-
amine hydrazones 14c and 14e were potent antiproliferative
agents (IC50 0.81 and 1.23
EGFR inhibitor 16a showed remarkable antitumor activity
(IC50 ¼ 0.94 M). The replacement of the 5-ethylamino-1,3,4-
mM, respectively). The most potent
m
thiadiazole moiety in 16a with 4-ethyl-5-mercopto-1,2,4-triazole
moiety in 17a or 5-mercapto-1,3,4-oxadiazole moiety in 19 led to
two fold decrease in activity. It is worth to note that both
compounds possess free polar SH group that might lead to
decreased cellular penetration. The elaboration of the 5-mercapto
in 19 with allyl in 20c resulted in two fold increase of activity,
while substitution with the more lipophilic benzyl moiety
produced the most active antiproliferative agent 20b
(IC50 ¼ 0.73
mM).
3.3. Molecular modeling
To rationalize the obtained biological results and to describe the
binding mode of the active compounds with their predicted
intracellular target, docking analysis was carried out. Docking of the
inhibitors into the crystal structure of EGFR kinase domain with
lapatinib (PDB ID code 1XKK) [36] were performed using Molecular
Operating Environment MOE version 2008.10 [37]. The X-ray
crystal structure illustrates that lapatinib binds to the ATP binding
cleft, where the quinazoline ring binds to the narrow hydrophobic
pocket in the N-terminal of EGFR-TK, which is the binding site of
the adenine base of ATP. The N-1 of the quinazoline ring is
hydrogen bonded to the main chain NH of Met 793 whereas the N-3
makes water mediated hydrogen bond to the side chain of Thr 854.
The 30-chloro-4-[(3-fluorobenzyl)oxy]aniline lies in a deep hydro-
phobic pocket at the back of the ATP binding site [36]. In consis-
tency with the design rational, docking of the hydrazone derivative
14c showed that the benzoyl group of 2-benzohydrazide moiety
binds in the adenine binding pocket and mimics the quinazoline
ring of lapatinib, but with no possibility of intramolecular hydrogen
bonding (Fig. 4). Both the carbonyl oxygen and NH of the hydrazide
function are involved in water bridged hydrogen bond with Thr
854. The 2-(3,4-dimethoxybenzylidene)hydrazine is deeply
oriented in the hydrophobic pocket occupied by the 30-chloro-4-
[(3-fluorobenzyl)oxy]aniline moiety of lapatinib. The 40-(3,4-
dimethoxybenzylidene)benzohydrazide is directed toward the
entrance of the active site, where 3,4-dimethoxybenzylidene is
positioned in a secondary pocket made of Ala 722, Arg 841 and Asn
842. It is worthy to note that, the extra hydrophobic interaction
provide by para and meta methoxy groups in 14c and the para
dimethyl amino substituent in 14e allowed more deep interaction
with the hydrophobic pocket at the back of ATP binding site and
more perfect fitting of the benzoyl moiety in the adenine binding
pocket compared to the inactive hydrazone analogues 14a, b, d, f
and g.
Fig. 6. Docking of compound 19 in the ATP binding site of EGFR PTK in 3D style.
2,40-bis-5-mercapto-1,2,4-triazole series 17aec. The utility of small
lipophilic ethyl group in 17a resulted in 52% inhibition. Increasing
the size of N-4 substituent on the triazole vanquished the activity in
17b and 17c. Also, changing the ethyl in 17a with small hydrophilic
amino function in 18 destroyed the enzyme inhibitory activity. The
bis-5-mercapto-1,3,4-oxadiazole derivative 19 revealed 48% inhi-
bition. Appending benzyl (20b) or allyl (20c) at 5-SH in 19 produced
slight increase in the kinase inhibition. Interestingly, adding an
ethyl group as in 20a dramatically decreased the inhibitory potency
against EGFR PTK.
3.2. In vitro cytotoxicity
Compounds 14c,14e,16a,17a,19, 20b and 20c that showed EGFR
inhibitory activity were evaluated for their antiproliferative activity
on human breast cancer cell line (MCF-7) by Skehan’s method [35].
MCF-7 is known to over-express EGFR and provides a good measure
Due to geometry and structural difference between the hydra-
zones 14 and the 2,40-bis-heterocyclic diphenylamines 16e201, the
latter compounds were expected to adopt a slightly different
binding mode. The details of interaction between the protein and
the most potent 2,40-bis-aminothiadiazole derivative 16a are
shown in Fig. 5aeb. The model reveals that, the 40-(5-ethylamino-
1,3,4-thiadiazol-2-yl)phenyl moiety binds the adenine binding
pocket. The thiadiazole ring nitrogen N-4 interact with the
1
Fig. 7. The superposition of lapatinib (purple) and compound 20b (blue) docked in the
ATP binding site of EGFR PTK in 3D style (for interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article).
To facilitate the interpretation of docking results, compounds 16e20 are
considered as derivatives of 2,40-bis substituted diphenylamine regardless of the
IUPAC nomenclature adopted in the experimental part.

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S.M. Abou-Seri / European Journal of Medicinal Chemistry 45 (2010) 4113e4121
ꢀ
backbone NH of Met 793 via hydrogen bond (2.7 A) and the
5-ethylamino NH is engaged in a second hydrogen bond with the
5. Experimental
ꢀ
side chain OH of Thr 790 (2.8 A). The ethyl group is projecting in
5.1. Chemistry
this narrow pocket near by Thr 790 side chain (Fig. 5b). This may
explain the lack of activity of the phenyl and allyl analogues 16b
and 16c. The steric clash caused by such bulky groups pushed the
thiadiazole ring away leading to loss of the key hydrogen bond with
Met 793. Hydrogen bonding with this amino acid helps to fix the
adenine base of ATP in the binding pocket and is considered crucial
for the activity of the classical quinazoline inhibitors [27,36].
Moreover, the 2-(5-ethylamino-1,3,4-thiadiazol-2-yl)phenyl is
facing the entrance of the pocket with the phenyl is extended to the
solvent exposed region. The thiadiazole N-4 and the ethylamino NH
are involved in H-bonding with the side chain of Lys 745 and Asp
Melting points were determined on a Gallenkamp melting point
apparatus and are uncorrected. 1H NMR spectra were recorded in
DMSO-d6, either on Varian XL-300 MHz or Jeol FX 90Q-90 MHz
spectrometer (chemical shifts are given in parts per million PPM).
13C NMR spectra were scanned on Varian XL-300 MHz spectrom-
eter. The IR spectra were measured as potassium bromide pellets
using a Pruker-FT IR spectrophotometer or Schimadzu IR spectro-
photometer. Mass spectra were obtained at Fennigan MAT, SSQ
7000 spectrophotometer at 70 eV. Microanalytical data (C, H, N) of
the synthesized compounds are within ꢀ0.4 of the calculated
values. Elemental analysis was carried out at the Microanalytical
Unit, Faculty of Science, Cairo University. The intermediate 2,
40-dipenylamine dicarboxylic acid 11 was prepared by reported
procedure [32].
ꢀ
ꢀ
855 (3.4 A and 2.8 A respectively).
Docking of 2,40-bis-5-mercaptotriazoles 17aec showed
almost similar orientations and binding behavior. On the other
hand compounds 18 and 19 revealed a backward orientation. It
should be mentioned that hydrogen bonding with Met 793
residue can only be observed with the active analogues 17a and
19 (Fig. 6).
5.1.1. 2-{[4-(Methoxycarbonyl)phenyl]amino}benzoic acid methyl
ester (12)
The 2,40-bis-1,3,4-oxadiazle series 20aec illustrates good bulk
tolerance. This leads to the suggestion that, these molecules
could interact deeply in the back of ATP binding site similar to
lapatinib. The additional binding energy provided by the binding
of the benzyl and allyl substituents in 20b and 20c in this deep
pocket may be the reason for the high binding affinity of these
molecules. Fig. 7 represents the superposition of the binding of
lapatinib and 20b. The 40-[5-(benzylthio)-1,3,4-oxadiazol-2-yl] in
20b is located in the back of the ATP binding site and makes
predominant hydrophobic interaction with the protein
mimicking the 30-chloro-4-[(3-fluoro-benzyl)oxy]aniline in
lapatinib. Moreover, the other benzyl group is involved in are-
neearene interaction with Arg 841 side chain. In comparison, the
much less potent analogue 20a does not project deep in the
pocket and binds near the entrance of the active site making
fewer hydrophobic contacts.
To an ice cold, stirred solution of 11 (3.9 mmol) in methanol
(20 ml), thionyl chloride (1 ml) was added dropwise. Stirring was
continued for 20 min at room temperature. The reaction mixture
was heated under reflux for 4 h, then concentrated and left to cool.
The crystalline product was filtered to give 0.85 g (77%). The
product was recrystallized from ethanol, m.p. 74e75 ꢁC. IR (KBr,
cmꢂ1) 3300 (NH), 2950, 2900 (CH3)s,1720 (C]O)s. 1H NMR (DMSO-
d6) (ppm) 3.81 (s, 3H, CH3), 3.83 (s, 3H, CH3), 7.00e7.93 (m, 8H, Ar-
n
d
H), 9.39 (s, 1H, NH, D2O exchangeable). Anal. Calc. for C16H15NO4: C,
67.36; H, 5.30; N, 4.91. Found C, 67.40; H, 5.16; N, 4.98.
5.1.2. 2-[4-(Hydrazinecarbonyl)phenylamino]benzoic acid
hydrazide (13)
A mixture of 12 (3.5 mmol) and hydrazine hydrate (10 ml) was
heated under reflux for 6 h, then concentrated under reduced
pressure. After cooling the separated solid was filtered, washed
with ethanol and dried to give 0.73 g (72%). The product was
4. Conclusion
recrystallized from DMF, m.p. 245e247 ꢁC. IR (KBr, cmꢂ1) 3289
n
(NH), 3219, 3182 (NH þ NH2)s, 1663 (C]O)s. 1H NMR (DMSO-d6)
This study reported the synthesis of 2,40-(bis substituted)
diphenylamine derivatives as a novel class of EGFR PTK inhibitors.
Derivatives 14c, 14e, 16a, 17a, 19, 20b and 20c exhibited promising
enzyme inhibitory activity. The most active compound was 16a that
d (ppm) 6.87e7.81 (m, 8H, Ar-H), 9.38 (s, 1H, NH, D2O exchange-
able), 9.57 (s, 4H, NHNH2, D2O exchangeable), 9.93 (s, 2H, NHNH2,
D2O exchangeable). Anal. Calc. for C14H15N5O2: C, 58.94; H, 5.30; N,
24.55. Found: C, 59.10; H, 5.01; N, 24.32.
revealed 98% inhibition at concentration of 10 mM. SAR was dis-
cussed in terms of the enzyme inhibitory activity and was sup-
ported by docking studies and analysis of the binding mode of the
new compounds. The electronic factor seem to be the most crucial
factor affecting the activity of a series of 2,40-bis-(N-substituted)
diphenylamine hydrazones 14aeg. Meanwhile, the steric effect
seems to be the most important factor affecting the activity of 2,40-
bis-heterocyclic diphenylamines 16e20. On the other hand, cyto-
toxicity results indicate that all evaluated compounds showed
5.1.3. General procedure for the synthesis of compounds (14)
To a refluxing solution of 13 (1.7 mmol) and the appropriate
aldehyde (3.5 mmol) in absolute ethanol, 1 ml glacial acetic acid
was added. The reaction mixture was heated under reflux for 4 h,
then concentrated. After cooling, the separated solid was filtered
and washed with ethanol. The crude product was recrystallized
from the appropriate solvent.
potent antitumor activity, IC50 ranging from 0.73 (20b) to 2.38
mM
5.1.3.1. 2,40-Azanediylbis(N0-benzylidenebenzohydrazide) (14a).
Recrystallization from ethanol (yield 95%), m.p. 260e262 ꢁC.
(17a). There is no definite relationship was found between the
enzyme inhibitory activity and cellular efficiency, which suggests
that this class of compounds might function by inhibiting multiple
key proteins involved in the EGFR signaling pathways, not only
targeting the tyrosine kinase. Thus, the new compounds led to
significant antiproliferative effect against EGFR dependent tumor
cell line, which over-express EGFR. The definite mechanism is to be
investigated.
IR (KBr,
n
cmꢂ1) 3359, 3216 (NH)s, 3032 (aromatic CH), 1635 (C]O)
s. 1H NMR (DMSO-d6)
d
(ppm) 6.70e7.67 (m, 18H, Ar-H), 8.39 (s, 2H,
2ꢃ eN¼CH), 9.30 (s, NH), 11.68 and 11.90 (s, 2ꢃ CONH cis and trans
conformers). Anal. Calc. for C28H23N5O2: C, 72.87; H, 5.02; N, 15.1.
Found: C, 72.81; H, 4.82; N, 15.23.
5.1.3.2. 2,40-Azanediylbis[N0-(2-hydroxybenzylidene)benzohydrazide]
In summary, the newly synthesized compounds provide
a promising new template for further development of potent EGFR
inhibitors.
(14b). Recrystallization from DMF/H2O (yield 95%), m.p.
212e214 ꢁC. IR (KBr,
n
cmꢂ1) 3400, 3350 (OH)s, 3200 (NH)s,
1640,1620 (C]O)s. 1H NMR (DMSO-d6)
d (ppm) 6.89e7.91 (m, 16H,

S.M. Abou-Seri / European Journal of Medicinal Chemistry 45 (2010) 4113e4121
4119
Ar-H), 8.60 (s, 2H, 2ꢃ eN¼CH), 9.41 (s, NH, D2O exchangeable),
11.24 and 11.42 (s, 2ꢃ CONH cis and trans conformers, D2O
exchangeable), 11.95 and 12.15 (s, 2ꢃ OH, D2O exchangeable). Mass
(%): Mþ 493 (12.75). Anal. Calc. for C28H23N5O4.2H2O: C, 63.51; H,
5.14; N, 13.23. Found: C, 63.39; H, 5.37; N, 13.05.
9.6 (s, NH, D2O exchangeable), 10.11 and 10.37 (s, 2ꢃ, CONH, D2O
exchangeable). Anal. Calc. for C20H25N7O2S2: C, 52.27; H, 5.48; N,
21.33. Found: 52.68; 4.83; 21.33.
5.1.4.2. N-Phenyl-2-{2-[4-(2-(phenylcarbamothioyl)hydrazinecarb-
onyl)- phenylamino]benzoyl}hydrazinecarbothioamide (15b). Recrys-
5.1.3.3. 2,40-Azanediylbis[N0-(3,4-dimethoxybenzylidene)benzohy-
drazide] (14c). Recrystallization from DMF (yield 92%), m.p
tallization from ethanol (yield 76%), m.p. 162-4 ꢁC. IR (KBr, cmꢂ1
n )
3238 (NH)s, 3059 (aromatic CH), 1656 (C]O)s, 1254 (C]S)s. 1H
NMR (DMSO-d6) (ppm) 7.17e7.91 (18H, m, Ar-H), 9.52 (s, NH),
9.76 (d, 2ꢃ NHph and 2ꢃ NHC ¼ S), 10.33 and 10.60 (s, 2ꢃ CONH).
13C NMR (DMSO-d6)
116.79,119.66,120.68,124.52,125.07,127.99,
>300 ꢁC. IR (KBr,
n
cmꢂ1) 3313, 3184 (NH)s, 3001 (aromatic CH),
d
1636 (C]O)s. 1H NMR (DMSO-d6)
d
(ppm) 3.73 and 3.82 (s, 12H, 4ꢃ
OCH3), 6.97e7.82 (m, 14H, Ar-H), 8.32 (s, 2H, 2ꢃ eN¼CH), 9.38 (s,
NH), 11.64 and 11.86 (s, 2ꢃ CONH cis and trans conformers). Anal.
Calc. for C32H31N5O6: C, 66.08; H, 5.37; N, 12.04. Found: C, 66.13; H,
5.06; N, 11.93.
d
129.55,132.45, 139.10 (CeNHC]S), 139.26 (CeNHC]S), 145.16
(CeNH), 149.48 (CeNH), 165.51 (C]O), 181.38 (C]S). Mass (%):
M-1 554 (4.26). Anal. Calc. for C28H25N7O2S2: C, 60.52; H, 4.53; N,
17.64. Found: C, 60.24; H, 4.61; N, 17.64.
5.1.3.4. 2,40-Azanediylbis[N0-(4-chlorobenzylidene)benzohydrazide]
(14d). Recrystallization from DMF/H2O (yield 83%), m.p.
5.1.4.3. N-Allyl-2-{2-[4-(2-(allylcarbamothioyl)hydrazinecarbonyl)-
270e272 ꢁC. IR (KBr,
n
cmꢂ1) 3200 (NH)s, 3050 (aromatic CH), 1650
phenylamino]benzoyl}hydrazinecarbothioamide (15c). Recrystalli-
(C]O)s, 750 (CeCl). 1H NMR (DMSO-d6)
d
(ppm) 7.10e7.87 (m, 16H,
zation from DMF/H2O (yield 90%), m.p. 214e216 ꢁC. IR (KBr, cmꢂ1
n )
Ar-H), 8.40 and 8.70 (s, 2H, 2ꢃ eN]CH cis and trans conformers),
9.57 (s, NH), 11.90 and 12.10 (s, 2ꢃ CONH cis and trans conformers).
Anal. Calc. for C28H21Cl2N5O2: C, 63.40; H, 3.99; N, 13.37. Found: C,
63.41; H, 4.26; N, 13.20.
3251, 3167 (NH)s, 1648 (C]O)s, 1258 (C]S)s. 1H NMR (DMSO-d6)
d
(ppm) 4.05 (d, 4H, 2ꢃ eCH2eCH]CH2), 4.89 (d, 4H, 2ꢃ
eCH2eCH]CH2), 5.6e6.00 (m, 2H, 2ꢃ eCH2eCH]CH2), 7.15e7.81
(m, 8H, Ar-H), 8.30 (s, 2ꢃ NH-allyl), 9.3 (s, 2ꢃ NHC]S), 9.60 (s, NH),
10.18 and 10.60 (s, 2ꢃ, CONH). Anal. Calc. for C22H25N7O2S2: C,
54.64; H, 5.21; N, 20.27. Found: C, 54.33; H, 5.00; N, 20.10.
5.1.3.5. 2,40-Azanediylbis{N0-[4-(dimethylamino)benzylidene]ben-
zohydrazide} (14e). Recrystallization from DMF/H2O (yield 93%),
m.p. 224e226 ꢁC. IR (KBr,
n
cmꢂ1) 3200 (NH)s, 3050 (aromatic CH),
5.1.5. General procedure for the synthesis of compounds (16)
2900 (aliphatic CH),1640,1650 (C]O)s. 1H NMR (DMSO-d6)
d
(ppm)
A suspension of the appropriate thiosemicarbazide 15
2.97 (s, 12H, 4ꢃ CH3), 6.70e7.58 (m, 16H, Ar-H), 8.27 and 8.51 (s, 2H,
2ꢃ eN¼CH cis and trans conformers), 9.40 (s, NH), 11.40 and 11.66
(s, 2ꢃ CONH cis and trans conformers). Anal. Calc. for C32H33N7O2:
C, 70.18; H, 6.07; N, 17.90. Found: C, 70.06; H, 6.28; N, 17.96.
(1.0 mmol) in 2 equivalent amount of freshly prepared PPA solution
was stirred at room temperature till homogenous, then for 1 h at
110 ꢁC. The reaction mixture was cooled, then poured over crushed
ice. The precipitated product was filtered, washed with water and
crystallized from an appropriate solvent.
5.1.3.6. 2,40-Azanediylbis[N0-(furan-2-ylmethylene)benzohydrazide]
(14f). Recrystallization from DMF/H2O (yield 78%), m.p.
5.1.5.1. N-Ethyl-5-{2-[4-(5-(ethylamino)-1,3,4-thiadiazol-2-yl)phe-
246e248 ꢁC. IR (KBr,
n
cmꢂ1) 3200 (NH)s, 3050 (aromatic CH),
nylamino]-phenyl}-1,3,4-thiadiazol-2-amine
tion from DMF/ethanol (yield 75%), m.p. 150e152 ꢁC. IR (KBr,
cmꢂ1) 3267 (NH)s, 2972 (C2H5)s, 1589 (C]N)s. 1H NMR (DMSO-d6)
(ppm) 1.04e1.18 (t, 3H, CH2CH3), 1.22e1.23 (t, 3H, CH2CH3),
(16a). Recrystalliza-
1640,1650 (C]O)s. 1H NMR (DMSO-d6)
d
(ppm) 6.63e7.83 (14H, m,
n
Ar-H þ furan protons), 8.28 (s, 2H, 2ꢃ eN¼CH), 9.31 (s, NH), 11.65
and 11.88 (s, 2ꢃ CONH cis and trans conformers). Anal. Calc. for
C24H19N5O4: C, 65.30; H, 4.34; N, 15.86. Found: C, 65.02; H, 4.62; N,
15.70.
d
3.31e3.34 (q, 2H, CH2CH3) 3.42e3.51 (m, 2H, CH2CH3, overlaped),
6.93e7.84 (m, 8H, Ar-H), 9.19 (s, NH), 10.36 (s, 2ꢃ NHCH2CH3). 13
C
NMR (DMSO-d6)
d 14.14 (CH3), 14.39 (CH3), 38.52 (CH2), 116.52,
5.1.3.7. 2,40-Azanediylbis{N0-[(5-nitrofuran-2-yl)methylene]benzohy-
118.28, 119.25, 120.63, 123.19, 127.63, 129.58, 132.39, 143.28
(CeNH), 155.61 (C]N), 167.48 (C]N). Mass (%): Mþ 423 (75.50).
Anal. Calc. for C20H21N7S2: C, 56.71; H, 5.00; N, 23.15; Found C,
56.56; H, 4.72; N, 23.15.
drazide} (14g). Recrystallization from DMF/H2O (yield 70%), m.p.
288e290 ꢁC. IR (KBr, cmꢂ1) 3320,3227 (NH)s, 3060 (aromatic CH),
n
1647 (C]O)s. 1H NMR (DMSO-d6)
d (ppm) 7.11e7.84 (m, 12H, Ar-
H þ furan protons), 8.39 (s, 2H, 2ꢃ eN¼CH), 9.31 (s, NH), 12.04 and
12.30 (s, 2ꢃ CONH cis and trans conformers). Anal. Calc. for
C24H17N7O8: C, 54.24; H, 3.22; N, 18.45. Found: C, 54.15; H, 3.51; N,
18.36.
5.1.5.2. N-Phenyl-5-{2-[4-(5-(phenylamino)-1,3,4-thiadiazol-2-yl)
phenylamino]-pheny])-1,3,4-thiadiazol-2-amine (16b). Recrystalli-
zation from DMF/ethanol (yield 65%), m.p. 178e180 ꢁC. IR (KBr,
n
cmꢂ1) 3275 (NH)s, 3029 (aromatic CH), 1594 (C]N)s. 1H NMR
(DMSO-d6) (ppm) 7.26e8.41 (m, 18H, Ar-H), 8.60 (s. NH), 11.96
5.1.4. General procedure for the synthesis of compounds (15)
A mixture of the acid hydrazide 13 (1.0 mmol) and the appro-
priate isothiocyanate (3.0 mmol) was refluxed in absolute ethanol
for 8 h the reaction mixture was concentrated and left to cool. The
separated solid was filtered and recrystallized from the appropriate
solvent.
d
(s, 2ꢃ NH-ph). Anal. Calc. for C28H21N7S2: C, 64.72; H, 4.07; N, 18.87;
Found C, 64.55; H, 3.86; N, 18.73.
5.1.5.3. N-Allyl-5-{2-[4-(5-(allylamino)-1,3,4-thiadiazol-2-yl)phenyl-
amino]-phenyl}-1,3,4-thiadiazol-2-amine
(16c). Recrystallization
from DMSO (yield 81%), m.p. 167e169 ꢁC. IR (KBr,
n
cmꢂ1) 3271
5.1.4.1. N-Ethyl-2-{2-[4-(2-(ethylcarbamothioyl)hydrazinecarbonyl)-
(NH)s, 1589 (C]N)s. 1H NMR not done due to solubility problems.
Anal. Calc. for C22H21N7S2: C, 59.04; H, 4.73; N, 21.91. Found: C,
59.25; H, 4.56; N, 21.95.
phenylamino]benzoyl}hydrazinecarbothioamide (15a). Recrystalli-
zation from DMF/H2O (yield 85%), m.p. 220e222 ꢁC. IR (KBr, cmꢂ1
n )
3250, 3177 (NH)s, 2969 (CeH), 1647 (C]O)s, 1254 (C]S)s. 1H NMR
(DMSO-d6)
d
(ppm) 1.04e1.09 (t, 6H, 2ꢃ CH2CH3), 3.45e3.49 (q, 4H,
5.1.6. General procedure for the synthesis of compounds (17)
A solution of appropriate thiosemicarbazide 15 (1.0 mmol) in
2 N NaOH was refluxed for 3 h. The resulting solution was filtered.
2ꢃ CH2CH3), 6.98e7.87 (m, 8H, Ar-H), 8.01e8.06 (d, 2ꢃ NHCH2CH3,
D2O exchangeable), 9,14e9.19 (d, 2ꢃ, NHC]S, D2O exchangeable),

4120
S.M. Abou-Seri / European Journal of Medicinal Chemistry 45 (2010) 4113e4121
The filtrate was cooled and acidified with dilute HCl to pH ¼ 5e6.
The precipitate formed was filtered, washed with water and crys-
tallized from an appropriate solvent.
The precipitated product was filtered, washed with water (yield
81%). The product was recrystallized from ethanol, m.p. 270e272 ꢁC.
IR (KBr,
n
cmꢂ1) 3335 (NH), 2740 (w, SH),1600 (C]N),1173 (CeOeC).
1H NMR (DMSO-d6)
d
(ppm) 7.15e7.81 (m, 8H, Ar-H), 8.59 (s, NH, D2O
5.1.6.1. 4-Ethyl-3-{2-[4-(4-ethyl-5-thioxo-4,5-dihydro-1H-1,2,4-
triazol-3-yl)phenylamino]phenyl}-1H-1,2,4-triazole-5(4H)-thione
(17a). Recrystallization from aqueous ethanol (yield 93%), m.p.
exchangeable), 14.60 (s, br, 2ꢃ thioxo-oxadiazole NH D2O
exchangeable). Mass (%): Mþ 369 (8.90) Anal. Calc. for C16H11N5O2S2:
C, 52.02; H, 3.00; N, 18.9. Found C, 52.30; H, 3.21; N, 18.72.
223e225 ꢁC. IR (KBr, cmꢂ1) 3308 (NH), 3084 (aromatic CH), 2933
n
(C2H5)s, 2747 (w, SHs), 1608 (C]N)s. 1H NMR (DMSO-d6)
d
(ppm)
5.1.9. General procedure for the synthesis of compounds (20)
1.01e1.05 and 1.15e1.21 (t, 3H, 2ꢃ CH2CH3), 3.76e3.80 and
4.02e4.05 (q, 2H, 2ꢃ CH2CH3), 7.10e7.53 (m, 8H, Ar-H), 8.23 (s, NH,
D2O exchangeable), 13.78 and 13.81 (s, 2ꢃ, thioxotriazole NH, D2O
exchangeable). Mass (%): Mþ 423 (85.65). Anal. Calc. for
C20H21N7S2: C, 56.71; H, 5.00; N, 23.15; Found C, 56.90; H, 4.90; N,
23.08.
To a mixture of the mercapto-oxadiazole derivative 19
(2.5 mmol) and anhydrous potassium carbonate (10 mmol) in dry
acetone, a solution of the appropriate alkyl halide (10 mmol) in dry
acetone was added. The mixture was heated under reflux for 24 h,
filtered while hot and the residue was washed with hot acetone.
The combined filtrate and wash were evaporated under vacuum.
The solid product was collected and washed with water.
5.1.6.2. 4-Phenyl-3-{2-[4-(4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-
triazol-3-yl)-phenylamino]phenyl}-1H-1,2,4-triazole-5(4H)-thione
(17b). Recrystallization from aqueous ethanol (yield 83%), m.p.
5.1.9.1. 2-[5-(Ethylthio)-1,3,4-oxadiazol-2-yl]-N-{4-[5-(ethylthio)-
1,3,4-oxadiazol-2-yl]phenyl}aniline (20a). Recrystallization from
192e194 ꢁC. IR (KBr,
n
cmꢂ1) 3200 (NH), 3064 (aromatic CH), 2741
acetone (yield 90%), m.p. 124e126 ꢁC. IR (KBr, cmꢂ1) 3295 (NH),
n
(w, SHs), 1591 (C]N)s. 1H NMR (DMSO-d6)
(m,18H, Ar-H), 8.03 (s, NH), 13.94 and 14.08 (s, 2ꢃ thioxotriazole
NH). 13C NMR (DMSO-d6)
115.14, 116.15, 117.15, 119.63, 120.64,
d
(ppm) 6.44e7.60
2969 (C2H5)s, 1602 (C]N), 1175 (CeOeC). 1H NMR (CDCl3)
1.47e1.53 (t, 6H, 2ꢃ CH2CH3), 3.25e3.34 (m, 4H, 2ꢃ CH2CH3),
6.93e7.93 (m, 8H, Ar-H), 9.44 (s, NH). 13C NMR (CDCl3)
14.69
d (ppm)
d
d
121.64, 127.39,128.09, 128.34, 128.77, 129.29, 131.29, 132.23, 133.74
(CeN), 134.58 (CeN), 140.52 (CeNH), 144.93 (CeNH), 149.53 (C-3
thioxotriazole), 150.73 (C-3 thioxotriazole), 167.62 (C-5 thioxo-
triazole), 168.24 (C-5 thioxotriazole). Anal. Calc. for C28H21N7S2: C,
64.72; H, 4.07; N, 18.87; Found C, 64.50; H, 3.92; N, 18.85.
(CH3),14.76 (CH3), 27.05 (CH2),108.88,115.49,117.24,119.65,128.00,
128.34, 132.23, 141.86 (CeNH), 144.24 (CeNH), 163.47 (C-2 oxa-
diazole), 1644.97(C-5 oxadiazole), 165.50 (C-5 oxadiazole). Anal.
Calc. for C20H19N5O2S2: C, 56.45; H, 4.50; N, 16.46. Found: C, 56.50;
H, 4.60; N, 16.54.
5.1.6.3. 4-Allyl-3-{2-[4-(4-allyl-5-thioxo-4,5-dihydro-1H-1,2,4-
triazol-3-yl)phenylamino]phenyl}-1H-1,2,4-triazole-5(4H)-thi-
one (17c). Recrystallization from aqueous ethanol (yield 95%), m.p.
5.1.9.2. 2-[5-(Benzylthio)-1,3,4-oxadiazol-2-yl]-N-{4-[5-(benzylthio)-
1,3,4-oxadiazol-2-yl]phenyl}aniline (20b). Recrystallization from
acetone, (yield 85%), m.p. 138e140 ꢁC. IR (KBr,
n
cmꢂ1) 3303 (NH),
129e131 ꢁC. IR (KBr,
n
cmꢂ1) 3358 (NH), 3096 (aromatic CH), 2750
3063, 3030 (aromatic CH), 2978 (CH2)s, 1594 (C]N), 1186 (CeOeC).
(w, SHs), 1606 (C]N)s. 1H NMR (DMSO-d6)
d
(ppm) 4.42 (d, 4H, 2ꢃ
1H NMR (DMSO-d6)
d
(ppm) 4.55 (s, 4H, 2ꢃ CH2), 7.11e7.86 (m, 18H,
CH2eCH]CH2), 4.66 (d, 4H, 2ꢃ CH2eCH]CH2), 5.46e5.90 (m, 2H,
2ꢃ CH2eCH]CH2), 7.03e7.46 (m, 8H, Ar-H), 8.28 (s. NH),
13.90e13.93 (d, 2ꢃ thioxotriazole NH). Anal. Calc. for C22H21N7S2: C,
59.04; H, 4.73; N, 21.91. Found: C, 59.40; H, 4.90; N, 21.85.
Ar-H), 9.12 (s, NH D2O exchangeable). Mass (%): Mþ 549 (19.30),.
Anal. Calc. for C30H23N5O2S2: C, 65.55; H, 4.22; N, 12.74. Found: C,
65.90; H, 4.50; N, 12.60.
5.1.9.3. 2-[5-(Allylthio)-1,3,4-oxadiazol-2-yl]-N-{4-[5-(allylthio)-
5.1.7. 4-Amino-5-{2-[4-(4-amino-5-mercapto-4H-1,2,4-triazol-3-
yl)phenylamino]-phenyl}-4H-1,2,4-triazole-3-thiol (18)
1,3,4-oxadiazol-2-yl]phenyl}aniline (20c). Recrystallization from
acetone (yield 85%), m.p. 90e92 ꢁC. IR (KBr,
n
cmꢂ1) 3289 (NH),
To an ice-cooled mixture of the hydrazide 13 (10 mmol) and
potassium hydroxide (30 mmol) in absolute ethanol (20 ml), carbon
disulfide (12 ml) was added dropwise. After addition was complete,
absolute ethanol (15 ml) was added and the reaction mixture was
allowed to stir at room temperature overnight. After the addition of
dry ether (50 ml), the obtained product was treated with hydrazine
hydrate (99%; 40 mmol) and water (4 ml) and heated under reflux
for 2 h. The reaction mixture was diluted with water and neutral-
ized with concentrated hydrochloric acid. The separated solid was
filtered, wash with water and recrystallized from ethanol (yield
3072 (aromatic CH), 2977, 2934 (aliphatic CH), 1602 (C]N),1173
(CeOeC). 1H NMR (DMSO-d6)
d
(ppm) 3.93e4.00 (d, 4H, 2ꢃ
CH2eCH]CH2), 5.24e5.44 (d, 4H, 2ꢃ CH2eCH]CH2), 5.87e6.30
(m, 2H, 2ꢃ CH2eCH]CH2), 7.28e7.91 (m, 8H, Ar-H), 9.17 (s, NH).
Anal. Calc. for C22H19N5O2S2: C, 58.78; H, 4.26; N, 15.58. Found: C,
58.90; H, 4.10; N, 15.33.
5.2. In vitro EGFR inhibitory activity assays by ELISA
The assay was performed in 96-well plates pre-coated with
95%), m.p. 160e162 ꢁC. IR (KBr,
n
cmꢂ1) 3300, 3153 (NH)s, 2749 (w,
(ppm) 5.67e5.74 (d, 2ꢃ
20
m
g/mL Poly (Glu, Tyr) 4:1 (Sigma) as a substrate. In each well,
L of 8 M ATP solution and 100 L of title compounds were
M concentrations. PD153035 was used as a positive
control for EGFR. The reaction was initiated by adding 5 L of EGFR
kinase. After incubation for 1 h at 37 ꢁC, the plate was washed three
times with PBS containing 0.1% Tween 20 (T-PBS). Next, 100
SH), 1594 (C]N)s. 1H NMR (DMSO-d6)
d
85
m
m
m
NH2, D2O exchangeable), 7.03e7.73 (m, 8H, Ar-H), 8.32 (s, NH, D2O
exchangeable),13.8 (s, br, 2ꢃ thioxotriazole NH, D2O exchangeable).
Mass (%): Mþ 397 (0.25). Anal. Calc. for C16H15N9S2: C, 48.35; H,
3.80. Found: C, 48.70; H, 4.00.
added at 10 m
m
mL
HRP-conjugate antiphospho-tyrosine antibody was added. After 1 h
of incubation at room temperature, the plate was washed three
times. TMB substrate solution (100 mL) diluted in T-PBS containing
5.1.8. 5-{2-[4-(5-Mercapto-1,3,4-oxadiazol-2-yl)phenylamino]
phenyl}-1,3,4-oxadiazole-2-thiol (19)
To an ice-cooled mixture of the hydrazide 13 (10 mmol) and
potassium hydroxide (20 mmol) in absolute ethanol (20 ml), carbon
disulfide (100 mmol) was added dropwise. The reaction mixture was
heated under reflux for 24 h, then distilled under vacuum. The
residue was diluted with water and acidified with hydrochloric acid.
5 mg/mL BSA was added. The plate was reincubated at room
temperature (18e25 ꢁC) for 15 min, and washed as before. The
reaction was terminated by the addition of 100 mL of 1 M H2SO4, and
A492 was measured using an ELISA reader. Results should be read
immediately after addition of the stop solution.

S.M. Abou-Seri / European Journal of Medicinal Chemistry 45 (2010) 4113e4121
4121
The inhibition rate (%) was calculated using the equation: The
inhibition % ¼ [1 ꢂ (A492/A492 control)] ꢃ 100%
thanks the National Cancer Institute, Cairo University, for per-
forming the in vitro cytotoxicity.
5.3. In vitro cytotoxic assay
References
[1] M.A. Olayioye, R.M. Neve, H.A. Lane, N.E. Hynes, EMBO. J. 19 (2000)
3159e3167.
[2] Y. Yarden, Eur. J. Cancer 37 (2001) S3eS8.
[3] J. Baselga, C.L. Arteag, J. Clin. Oncol. 23 (2005) 2445e2459.
[4] A. Reid, L. Vidal, H. Shaw, J. De Bono, Eur. J. Cancer 43 (2007) 481e489.
[5] A.A. El-Zayat, T.F. Pingree, P.M. Mock, G.M. Clark, R.A. Otto, D.D. Von Hoff,
Cancer J. 4 (1991) 375e380.
The human tumor cell lines (MCF-7) were obtained as a gift
from NCI, MD, USA. All chemicals and solvents were purchased
from SigmaeAldrich. The cytotoxic activity was measured in vitro
using the Sulfo-Rhodamine-B stain (SRB) assay by the method of
Skehan [35].
Cells were inoculated in 96-well microtiter plate (104 cells/well)
for 24 h before treatment with the compound(s) to allow attach-
ment of cell to the wall of the plate. Test compounds were dissolved
in DMSO and diluted with saline to the appropriate volume.
Different concentrations of the compound under test (0.1, 2.5, 5,
and 10 nmol/ml) were added to the cell monolayer. Triplicate wells
were prepared for each individual dose. Monolayer cells were
incubated with the compound(s) for 48 h at 37 ꢁC and in atmo-
sphere of 5% CO2. After 48 h, cells were fixed, washed, and stained
for 30 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Unbound
dye was removed by four washes with 1% acetic acid, and attached
stain was recovered with Tris-EDTA buffer. Color intensity was
measured in an ELISA reader. The relation between surviving
fraction and drug concentration is plotted to get the survival curve
for breast tumor cell line after the specified time. The concentration
required for 50% inhibition of cell viability (IC50) was calculated and
the results are given in Table 2.
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All molecular modeling calculation and docking studies were
carried out using Molecular Operating Environment MOE version
2008.10 [37]. The target compounds were drawn on MOE. The
structures were subjected to energy minimization using Hamilto-
nian-Force Field-MMFF94x and partial charges were calculated.
The X-ray crystal structure of the kinase domain of EGFR in complex
with GW572016 (lapatinib) PDB ID code 1XKK was recovered RSCB
protein data bank [36]. The enzyme was prepared for docking as
follows: (1) The Co-crystallized ligand, phosphate ions and water
molecules outside the binding pocket were removed. (2) The
enzyme was 3D protonated, where hydrogen atoms were added at
their standard geometry, the partial charges were computed and
the system was optimized. Flexible ligand e rigid receptor docking
was done with MOE-DOCK using triangle matcher as placement
method and affinity dG as a scoring function. The obtained poses
were subjected to force field refinement using the same scoring
function. 30 conformers of the ligand were retained with the
highest and best score. In order to validate the docking procedure,
lapatinib was docked into the active site of 1XKK. The docking
results showed that the compound exhibited similar interaction to
that reported in literature [36]. Ligandereceptor docking was
demonstrated by 2D and 3D ligandereceptor interaction.
Acknowledgment
[36] E.R. Wood, A.T. Truesdale, O.B. McDonald, D. Yuan, A. Hassell, S.H. Dickerson,
B. Ellis, C. Pennisi, E. Horne, K. Lackey, K.J. Alligood, D.W. Rusnak, T.M. Gilmer,
L. Shewchuk, Cancer Res. 64 (2004) 6652e6659.
[37] MOE. Chemical computing Group, Inc., Montereal http://www.chemcomp.
com.
The author is grateful for Dr. Nadia Hamdy, Department of
Biochemistry, Faculty of Pharmacy, Ain Shams University, for con-
ducting the in vitro EGFR inhibitory activity assays. The author