Design, synthesis and biological evaluation of some 5-arylthieno[2,3-d]-pyrimidines as potential anti-cancer agents

Article abstract of DOI:10.1248/cpb.c16-00291

Structure-based design, synthesis and biological evaluation of new small molecules anti-cancer agents were described. On continuation of applying scaffold hopping theory, a series of 5-arylthieno[2,3-d]pyrimidines based on the structural features of lapat

Full text of DOI:10.1248/cpb.c16-00291

1172
Chem. Pharm. Bull. 64, 1172–1180 (2016)
Vol. 64, No. 8
Regular Article
Design, Synthesis and Biological Evaluation of Some 5-Arylthieno[2,3-d]-
pyrimidines as Potential Anti-cancer Agents
Afaf Kamal El-Ansary, Aliaa Mohammed Kamal,* and Mokhtar Abd-Hafiz Al-Ghorafi
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University; Cairo 11561, Egypt.
Received March 25, 2016; accepted May 1, 2016
Structure-based design, synthesis and biological evaluation of new small molecules anti-cancer agents
were described. On continuation of applying scaffold hopping theory, a series of 5-arylthieno[2,3-d]pyrimi-
dines based on the structural features of lapatinib was designed, synthesized and characterized using IR,
1H-NMR, 13C-NMR, mass and microanalyses. Biological evaluation of the cytotoxic activity against MCF-7
cell line and the inhibition of the enzymatic activity of epidermal growth factor tyrosine kinase were carried
out in vitro for the target compounds. Substituting the 4-anilino-5-arylthieno[2,3-d]pyrimidines with dif-
ferent pharmacophoric groups at ortho, meta and/or para positions led to discovery of two potent lead com-
pounds 3b and f with excellent cytotoxic activity and enzymatic inhibition activity.
Key words anti-tumor agent; nucleophilic substitution; Scaffold hopping theory; sulfonamide; thieno[2,3-
d]pyrimidine
Cancer is a leading cause of death worldwide, responsible C2 is hypothesized to form a unique CH–O type of hydrogen
for 8.2 million death in 2012. The design of new compounds bond with the carbonyl of Gln767.12) The side chain of Thr766
prototypes has been a good strategy for discovery of new in lapatinib–EGFR complex points away from the quinazoline
drugs.1,2) Small molecule kinase inhibitors—4-anilinoquin- and participated in hydrogen bond with the backbone carbonyl
azoline derivatives—have been successfully introduced to of Arg752.13)
the drug market as selective antitumor agents with low side
The 4-anilino group plays an important role in the bind-
effects.3–8) Many small molecules e.g., gefitinib and erlotinib ing and selectivity of these inhibitors. Examining the crystal
have been approved by the U.S. Food and Drug Administra- structure of lapatinib–EGFR complex confirmed that large
tion (FDA) for treatment of certain cancers over-expressing anilino substituents also entered the kinase specificity pock-
epidermal growth factor receptor (EGFR) and various ap- et.7) The large anilino substituents induced dramatic confor-
proaches were adopted to enhance the potency and selectivity mational changes in the ATP-binding site, the position of the
of these inhibitors. These efforts led to discovery of lapatinib, C-helix and the hydrogen binding pattern with quinazoline
a potent inhibitor of EGFR that approved by the FDA for ring.7,12,14) Molecular modeling studies have given insights
treatment of breast cancer.9) Kinase inhibitors that compete into structural features that enhanced inhibitory activity and
with the ATP can be classified into two major groups. Type 1 provided theoretical models to predict activities of this class
kinase inhibitors: such as gefitinib and erlotinib which target of compounds depending on the docking orientation of the
and induce the active conformation of kinase enzymes via ligands in the active site.15–17)
mimicking the binding mode of the ATP molecule. The mo-
Thienopyrimidines in general have become an interesting
lecular design strategies resulted in type 2 kinase inhibitors, structural element in development of pharmaceutical com-
such as lapatinib I (Fig. 1) which possesses extra structural pounds especially anticancer agents via various mechanisms
features that enable it to bind to the inactive form of the ki- e.g., thymidylate synthase inhibitors,18,19) Cyclin-dependent ki-
nases and subsequently lapatinib turned out to be more selec- nase inhibitors (CDKI),20,21) Gonadotropin releasing hormone
tive inhibitor than the corresponding type 1.10,11)
(GnRH) receptor antagonists,22) tyrosine kinase inhibitors
Lapatinib I shows its cytotoxic activity via a unique mecha- (TKIs)23–27) and as FGFR1 inhibitors.28)
nism of action and exhibits reversible, non-covalent inhibition
In our previous work,29) we replaced the benzene ring of
of EGFR, but it has a very slow off-rate compared with other
reversible 4-anilinoquinazoline compounds.7) Lapatinib
I
binds into the ATP-binding cleft where the quinazoline ring is
participating in hydrogen bond with the hinge region between
the NH2 and COOH terminal lobes of the kinase, whereas the
quinazoline ring of other inhibitors participated in hydrogen
bond with EGFR differently. The binding mode of lapatinib
to EGFR tyrosine kinase (TK) revealed that N1 of the quin-
azoline participated in hydrogen bond with the Met769 NH
while the N3 interacted with the side chain of Thr766 through
a water bridge. The C2 and the C8 of the quinazoline ring
were positioned close to the backbone carbonyls of Gln767
and Met769, respectively. The acidic hydrogen attached to
Fig. 1. Structure of Labitinib I
*To whom correspondence should be addressed. e-mail: Aliaa.kamal@pharma.cu.edu.eg; K_aliaa2511@hotmail.com
© 2016 The Pharmaceutical Society of Japan

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1173
the 4-anilinoquinazoline core with thiophene ring to result wards studying the effect of substituting the 4-anilino moiety
in 4-anilinothieno[2,3-d]pyrimidine core. Moreover, different of the 4-anilinothieno[2,3-d]pyrimidine core with small, me-
spacers between thieno[2,3-d]pyrimidine core and 4-anilino dium or large groups (benzyloxyphenyl, benzenesulfonamido,
group were introduced. Also, total replacement of the aniline methyl, carboxyl, nitro, or halide groups) on the cytotoxic
moiety with substituted heterocyclic rings was carried out. In activity of the newly synthesized compounds. Introduction of
the present work, we continued the strategy of the previous bulky groups such as benzyloxy and sulfonamide moieties at
work in modifying the structure of the core 4-anilinoquinazo- para position of the aniline ring to fill a pocket in the ATP
line by using 4-anilinothieno[2,3-d]pyrimidine core as well binding region as lapatinib does, was one of the possibilities,
as substituting the 4-anilino moiety with small, medium and so that the bulky group could be oriented deep into the back
large pharmacophores e.g., benzyloxy moiety (2a and b), sul- of the ATP binding site and makes predominantly hydropho-
fonamides (3a–j) and other small groups (4a–q) (Fig. 2).
bic interaction with the protein mimicking the 3′-chloro-4-
Accordingly, the strategy of this work was the usage of the [(3-fluorobenzyl)oxy]aniline group of lapatinib I. Also sub-
potent inhibitors’ common features to design a series of po- stitution of the anilino moiety with small groups at ortho,
tential anti-breast cancer thieno[2,3-d]pyrimidine derivatives. meta and/or para positions e.g., 4a–q took place to evaluate
The synthesis of the target thieno[2,3-d]pyrimidine derivatives the effect of both the position and the size of the substituting
was represented in Chart 1.
group on the biological activity of the target compounds. To
justify the suggested compounds as good cytotoxic agents, the
binding model of EGFR with some thieno[2,3-d]pyrimidine
Results and Discussion
Designing the Target Compounds For continuation of derivatives 2a and b, 3a–j and 4a–q was generated (Fig. 3).
our previous work in designing a series of potential anti-breast
The binding model suggested that, N1 of the thieno[2,3-
cancer thieno[2,3-d]pyrimidine derivatives by using the potent d]pyrimidine interacted with the main chain NH group of
inhibitors’ common features29); this work was directed to- Met793 in the hinge region and N3 interacted with the side
chain OH group of Thr854 through a water mediated hy-
drogen bonding. The substituents at the para position of the
aniline moiety extended toward the back hydrophobic pocket.
Thus, the compounds were designed to possess both nitrogen
atoms for binding and a lipophilic group (R1) at the para
position of the aniline moiety, via an appropriate linker (X,
Y) (Fig. 3). Secondary amino group at C4 of thieno[2,3-d]-
pyrimidine derivatives 2a and b, 3a–j and 4a–q was acting as
Fig. 2. General Formula of the Target Compounds 2a and b, 3a–j and
4a–q
conformational lock and extending substituted aniline portion
into the hydrophobic pocket of EGFR-TK, making predomi-
nantly hydrophobic interactions with the protein. Generally,
Ar=4-chlorophenyl, 4-nitrophenyl, R=benzyloxy (2a and b), different sulfon-
amides (3a–j) and small groups (4a–q).
Chart 1

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Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
the thieno[2,3-d]pyrimidine core of the suggested compounds
The sulfonamide moieties of compounds 3a–j were deeply
was sandwiched between the side chains of Ala743 and oriented in the hydrophobic pocket and made a predominant
Leu844, respectively, bound to the adenine binding pocket and hydrophobic interaction with the adjacent lipophilic pocket
mimicked the quinazoline ring of lapatinib I. Upon docking lined by Met766, Leu777, Thr790, and Phe856. The additional
the target compounds into ATP of the EGFR, they were bound binding energy provided by the binding of the substituted
to the ATP catalytic domain with two or more hydrogen sulfonamides of compounds 3a–j in this deep pocket may be
bonds. One was formed between N1 of the thienopyrimidine the reason for the high binding affinity of these compounds. It
core and main chain NH of Met793, another was formed be- was also found out that both the p-chlorophenyl and the p-ni-
tween N3 and side chain OH of Thr854 through a water medi- trophenyl at C5 of the thieno[2,3-d]pyrimidine were directed
ated hydrogen bond. The benzyloxyaniline of compound 2b toward the solvent interface, we envisioned that aromatic ring
for example mimicked the 3-chloro-4-[(3-fluorobenzyl)oxy]- at this position, could be utilized to further improve the cellu-
aniline moiety of lapatinib I. The saved pose (Fig. 4) for the lar activity and pharmacokinetic properties of the synthesized
ligand–enzyme complex of compound 3b revealed that several compounds.
molecular interactions were considered to be responsible for
With the guidance of the above findings, a series of thieno-
the observed affinity.
pyrimidine derivatives were designed, docked into EGFR-TK,
Moreover, Fig. 4 showed perfect overlay of lapatinib and synthesized and evaluated for their anti-breast cancer activ-
compounds 2b and 3b as they both connected to the same ity and the EGFR-TK enzymatic inhibition activity. The sug-
amino acids residues with different hydrogen bond lengths gested compounds showed good binding to the receptor, good
(Table 1).
superposition and good docking scores. Their good fitting to
Fig. 3. Binding Model of Some Thieno[2,3-d]pyrimidine Scaffold to EGFR Kinase Receptor
Fig. 4. (a) The Superposition of Lapatinib (White) and Compound 2b (Dark Yellow) Docked in the ATP Binding Site of EGFR PTK; (b) The Super-
position of Lapatinib (White) and Compound 3b (Dark Yellow) Docked in the ATP Binding Site of EGFR PTK

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1175
the active site pocket and good interactions with the amino amides or suitable aromatic amines afforded compounds 2a
acid residues (Table 1) suggested that they might show good and b, 3a–j (Table 2) and 4a–q sequentially (Chart 1).
enzymatic inhibition activity.
Confirmatory evidences for structure of all the newly syn-
Synthesis of the Target Compounds Synthesis of com- thesized compounds (2a, b, 3a–j and 4a–q) were provided by
1
pounds 1a and b was achieved via chlorination of thieno[2,3- elemental analysis, IR, H-NMR, 13C-NMR and mass spec-
d]pyrimidin-2-ones according to a reported method.29) Nu- troscopy.
cleophilic substitution of the 4-chloro group of compounds 1a
Correlation between Cytotoxic Activity and Docking Re-
and b with 4-benzyloxyphenylamine, 4-aminobenzenesulfon- sults A series of thieno[2,3-d]pyrimidine derivatives based
Table 1. Docking Data of the Target Compounds and Lapatinib into EGFR-TK Enzyme
Binding energy
Compound number Number of H-bonds
Amino acid residues forming H-bonds (H-bond length in Å)
scoreb)
Lapatiniba)
2
3
2
2
2
4
3
3
3
2
3
3
2
3
2
2
3
Met793 (3.1), Thr854 (2.79),
Met793 (3.2), Thr854 (3.1), π-cation (Lys745)
Met793 (3.1), Thr854 (3.1)
−35.2
−34.0
−33.0
−32.0
−36.0
−25.0
−22.0
−19.0
−36.0
−12.0
−33.0
−31.0
−29.0
−26.4
−32.0
−23.0
−26.0
2a
2b
3a
3b
3c
3d
3e
3f
Met793 (3.2), Thr854 (2.9)
Met793 (3.2), Thr854 (2.9)
Met793 (3.2), Thr854 (2.0), Thr854 (2.0), π-cation (Lys745)
Met793 (3.1), Thr854 (2.0), π-cation (Lys745)
Met793 (3.2), Thr854 (2.0), π-cation (Lys745)
Met793 (3.2), Thr854 (2.0), π-cation (Lys745)
Met793 (3.2), Thr854 (2.9)
3g
3h
3i
Met793 (3.3), Thr854 (3.2), π-cation (Lys745)
Met793 (3.3), Thr854 (3.0), Thr854 (2.5)
Met793 (3.3), Thr854 (2.9)
4a
4g
4h
4j
Met793 (3.2), Thr854 (3.0), π-cation (Lys745)
Met793 (3.3), Thr854 (2.9)
Met793 (3.1), Thr854 (2.9)
4p
Met793 (3.2), Thr854 (2.9), π-cation (Lys745)
a) Lapatinib (Tykerb™) was used as a standard in the docking study. b) Binding energy score (kcal/mol): energy of interaction of the ligand and organic compounds in the
active site.
Table 2. The Structural Formulae of Compounds 3a–j
Cpd. No.
R
H
Ar
Cpd. No.
R
Ar
3a
3f
3b
3g
3c
3h
3i
H
3d
3e
3j

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Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
on the structure feature of lapatinib had been synthesized and Activity Using Anti-phosphotyrosine Antibodies The PTK
evaluated for their biological activity in vitro. Molecular dock- assay was intended for the measurement of EGFR-TK activity.
ing studies helped in designing novel inhibitors and thereby Protein tyrosine phosphorylation is a central mechanism that
helped to understand the antitumor activity against MCF-7 mediates signal transduction events involved in a wide range
cell line and the enzymatic inhibition activity. Almost all of of cellular processes. PTKs are considered to play a critical
the test compounds exhibited distinguished antitumor activity role in signal transduction pathways. Several of the PTKs
against MCF-7 breast cancer cell with IC50 (concentration of have been identified as oncogenes. Measurement of EGFR-
compounds that is required for 50% inhibition) ranging from PTK activity is an essential requirement in development of
9.1 to 59.6nM, and using lapatinib as a reference drug with EGFR-TKI in vitro. This method depends on the direct- and
IC50 11.0 nM (Table 3). Compound 3b was the most active com- specific detection of only phosphorylated EGFR-TK residue
pound with IC50 9.1 nM.
using monoclonal anti-phosphotyrosine–peroxidase conjugate.
In addition most tested compounds revealed good binding The PTK activity is based on enzyme-linked immunosolvent
mode with binding energy score ranged from −36.0 to −12.0 assay (ELISA) assay. The inhibition activity of EGFR-TK was
(lapatinib binding energy score −35.2) (Table 1). This may be measured for the doses of the target compounds that caused
attributed to that they possess the key binding features with 50% inhibition of MCF-7 cell line activity. Generally, com-
EGFR-TK which represented in N1 and N3 of the thienopy- pounds 2a and b and 3a–j showed very good PTK inhibition
rimidine core. Also the different pharmacophores at the para activity (even better than the activity against MCF-7 cell line)
position of aniline moiety in case of compounds 2a and b and compared to compounds 4a–q and this was attributed to that
3a–j extended toward the back of ATP binding site and made they possess the key binding features with EGFR-TK spe-
a predominantly hydrophobic interaction with the protein. cially they had bulky substitution at C4 position (para) of the
When the substituent at the C4 of aniline moiety was ben- anilino moiety. Compound 3b showed the highest inhibition
zyloxy group in compounds 2a and b, they displayed good activity of EGFR-TK enzyme with the lowest phosphorylated
activity (IC50 45.1 and 31.7nM, respectively) (Table 3).
tyrosine kinase concentration 1.83359ng/mL even more potent
The introduction of larger groups such as heterocyclic than the reference drug that showed phosphorylated tyrosine
sulfonamides at position C4 of the aniline moiety e.g., com- kinase concentration 2.43252ng/mL (Table 3). Compounds 2a
pounds 3c–g showed potent activity (IC50 28.5–46.0nM). Upon and b showed potent enzymatic inhibition activity in com-
investigating the effect of the substitution of the sulfonamide parison to the reference drug with phosphorylated tyrosine
group itself, it was found that, the extra hydrophobic interac- kinase concentration 3.99583 and 3.12059ng/mL, respectively.
tion of the five membered ring provided by 4,5 dimethyl isox- Regarding compounds 3a, c–i, compound 3f showed excellent
azolyl moiety in compound 3f (IC50 28.5nM) showed better enzymatic inhibition activity with phosphorylated tyrosine
anti-breast cancer activity compared to the unsubstituted
five membered ring 3e (IC50 46.0nM). The cytotoxic activity
of compounds 3c, d and g (substituted and unsubstituted six
membered heterocyclic sulfonamide derivatives) were nearly
the same (IC50 31.3, 30.9, 30.9nM, respectively) and this was
in contrast to the substituted and unsubstituted five membered
ring derivatives 3f and e. This may be attributed to the con-
formational structures of both five and six membered rings.
Substitutions of the anilino moiety with small electron with-
drawing or electron donating groups e.g., compounds 4a–q
showed moderate activity (IC50 45.5–59.6 nM) but still lower
than compounds 2a and b and 3a–j which were substituted
with larger moieties at the para position of the anilino group.
Compounds 4a, g, h, j and 4p with ortho or meta substitution
showed the least anti-breast cancer activity.
kinase concentration 2.63671ng/mL and nearly as active as
the reference drug followed by compounds 3g, d and c with
phosphorylated tyrosine kinase concentration 2.92053, 3.00051
and 3.12058ng/mL, respectively. Compound 3e revealed a
moderate inhibition activity with phosphorylated tyrosine ki-
nase concentration 4.00082ng/mL while compound 3a showed
the least TK inhibition activity of this group of compounds
with phosphorylated tyrosine kinase concentration 4.89956ng/
mL. Moreover, compounds 4a, g, h, j and p showed the least
EGFR-TK inhibition activity with phosphorylated tyrosine
kinase concentrations ranging from 3.99583–6.99995ng/mL
(Table 3).
Conclusion
A series of thieno[2,3-d]pyrimidine derivatives based on
Nonradioactive Assay of Protein-Tyrosine Kinase (PTK) the structure feature of lapatinib have been designed, synthe-
Table 3. Results of in Vitro Cytotoxic Activity of Tested Compounds and Lapatinib on Breast Cancer Cell Line (MCF-7) and Inhibition Activity of
EGFR-TK Enzyme
Tyrosine kinase conc.
(ng/mL)
Tyrosine kinase conc.
(ng/mL)
Compound
IC50 (nM)
Compound
IC50 (nM)
Lapatinib
11 0.1
45.1 0.0
31.7 2.0
47.5 0.0
9.1 0.0
2.43252
3.99583
3.12059
4.89956
1.83359
3.12058
3.00051
4.00082
2.63671
3g
3h
30.9 0.1
35.1 0.0
35.8 2.0
45.5 2.0
55.6 2.0
48.2 0.0
59.6 0.0
45.7 2.0
—
2.92053
3.34586
3.57895
3.99585
5.89966
4.22382
6.99995
3.99583
26.55291
2a
2b
3a
3b
3c
3d
3e
3f
3i
4a
4g
31.3 0.1
30.9 0.1
46.0 2.0
28.5 0.1
4h
4j
4p
Control

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1177
sized and evaluated for their anti-breast cancer activity as well thieno[2,3-d]pyrimidine 1a or b (1mmol) and 4-benzyloxy-
as enzymatic inhibition activity of EGFR-TK in vitro. Molecu- phenylamine (1.2mmol) in isopropanol (for compound 1a)
lar docking studies further helped in designing novel potent or in isopropanol/2–3 drops of conc. HCl (for compound 1b)
inhibitors. As a result of the docking studies, it was expected (20mL) was heated under reflux for 16–18h. The product was
that compounds 2a and b, the sulfonamide derivatives 3a–j either filtered, dried and crystallized from ethanol (compound
and compounds 4a–q would show good cytotoxic activity 2a) or the reaction mixture was left at ambient temperature
due to their very close resemblance to lapatinib in the binding overnight then the solid formed was triturated with sodium
mode to EGFR-TK. In general the PTK assay showed better carbonate solution (1%, 0.5mL), stirred, filtered, and crystal-
cytotoxic activity of the target compounds. Most of the tested lized from ethanol (compound 2b).
compounds showed excellent to good biological activity in
comparison with lapatinib. Broadly, compounds 2a and b and pyrimidin-4-yl]amine (2a)
3a–j showed very good-excellent EGFR-TK inhibition activity
Yield, 75%. mp 144–146°C. IR (KBr) cm−1: 3402, 3059,
(4-Benzyloxyphenyl)-[5-(4-chlorophenyl)thieno[2,3-d]-
1
due to the very close resemblance to the small molecule ki- 3028, 1604, 2924. H-NMR (DMSO-d6) δ: 5.08 (2H, s), 6.95
nase inhibitors—4-anilinoquinazolines derivatives’ structure. (2H, d, J=9.3Hz), 7.29 (2H, d, J=8.7Hz), 7.31–7.42 (9H, m),
The variation in inhibition activity between the same deriva- 7.67 (1H, s), 7.68 (1H, s), 8.49 (1H, s). GC-MS (electrospray
+·
+·
tives owed to the conformational variations of the substituents ionization (ESI)) m/z: 445 (M+2¬ ), 443 (M¬ ), 352 (M−
+·
at C4 position of the 4-anilino group e.g., compounds 3b–g. C6H5−CH2¬ ). Anal. Calcd for C25H18ClN3OS: C, 67.64; H,
Compounds 3b and f were the most biologically active com- 4.09; N, 9.47. Found: C, 67.56; H, 4.08; N, 9.78.
pounds with IC50 9.1 and 28.5nM and this was in accordance
with their EGFR-TK inhibition activity with phosphorylated pyrimidin-4-yl]amine (2b)
tyrosine kinase concentrations 1.83359 and 2.63671ng/mL, Yield, 68%. mp 192–194°C. H-NMR (DMSO-d6) δ: 5.07
(4-Benzyloxy-phenyl)-[5-(4-nitrophenyl)thieno[2,3-d]-
1
respectively due to the bulky well-fit para substituents of the (2H, s), 6.93 (2H, d, J=9Hz), 7.30–7.44 (8H, m), 7.67 (1H,
4-anilino group. Moreover, the importance of the para bulky s), 7.82 (2H, d, J=9.3Hz), 8.32 (2H, d, J=8.7Hz), 8.49 (1H,
substitution of the 4-anilino group was confirmed when com- s). 13C-NMR (DMSO-d6) δ: 167.7, 157.1, 152.5, 148.5, 142.6,
pounds 4a, g, h, j and p that have small substitutions showed 142.3, 140.9, 139.0, 128.7, 128.2, 127.9, 127.5, 127.3, 124.4,
the least anti-breast cancer activity as well as the EGFR-TK 124.1, 121.8, 116.1, 114.9, 77.8. IR (KBr) cm−1: 3417, 3028,
+·
inhibitory activity.
3062, 3101, 1600, 2916. GC-MS (ESI) m/z: 455 (M+1¬ ).
Anal. Calcd for C25H18N4O3S: C, 66.07; H, 3.99; N, 12.33.
Found: C, 66.12; H, 4.04; N, 12.45.
Experimental
Chemistry Melting points (mp) were determined on Stu-
5-(4-Aryl)-4-[4-(N-(substituted)sulfomyl)]anilino-
art apparatus and the values given are uncorrected. IR spectra thieno[2,3-d]pyrimidine (3a–j) A stirred mixture of either
were determined on Shimadzu IR 8400s spectrophotometer thieno[2,3-d]pyrimidine 1a or b (1mmol) and the appropriate
(KBr, cm−1) at Faculty of Pharmacy, Cairo University, Egypt. sulfonamide derivative (1.2mmol) in dry pyridine (for com-
1H-NMR spectra were carried out using a Mercury, a Gemini pounds 3b and i) or isopropanol (for the rest of compounds)
300-BB 300MHz and Joel (eca) 500MHz spectrophotometers (20mL) was heated under reflux for 18–24h. The reaction
using tetramethylsilane (TMS) as internal standard. Chemical mixture was cooled and the formed solid was filtered, dried
shift values were recorded in ppm on δ scale, Microanalytical and crystallized from the appropriate solvent.
Center, Cairo University, Egypt, Main laboratory of the War
Chemical, Chemical War Department, Ministry of Defense benzenesulfonamide (3a)
and National Research Centre. 13C-NMR spectra were car-
Yield, 80% (ethanol). mp 202–204°C. H-NMR (DMSO-d6)
4-[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-
1
ried out using Mercury and Gemini 300-BB 75MHz spec- δ: 7.24 (2H, s), 7.57–7.59 (4H, m), 7.72–7.78 (4H, m), 7.69 (1H,
trophotometers using TMS as internal standard. Chemical s), 7.79 (1H, s), 8.65 (1H, s). 13C-NMR (DMSO-d6) δ: 167.5,
shift values were recorded in ppm on δ scale, Microanalytical 157.3, 149.9, 142.4, 134.7, 133.9, 129.4, 129.1, 128.4, 128.2,
Center, Cairo University, Egypt. Mass spectra were recorded 126.5, 124.5, 121.9, 115.6. IR (KBr) cm−1: 3402, 3305, 3232,
+·
on Hewlett Packard 5988 spectrometer, Microanalytical Cen- 3066, 1604, 1330. GC-MS (ESI) m/z: 418 (M+2¬ ), 416
+·
+·
ter, Cairo University, Egypt. Elemental analyses were car- (M¬ ), 415 (M−1¬ ). Anal. Calcd for C18H13ClN4O2S2: C,
ried out at the Microanalytical Center, Faculty of Pharmacy, 51.86; H, 3.14; N, 13.44. Found: C, 51.93; H, 3.17; N, 13.71.
Al Azhar University, Egypt. Progress of the reactions was
N-Carbamimidoyl-4-{[5-(4-chlorophenyl)thieno[2,3-d]-
monitored using TLC sheets precoated with UV fluorescent pyrimidin-4-yl]amino}benzenesulfonamide (3b)
1
silica gel Merck 60F 254. The solvent system was benzene,
Yield, 69% (acetone). mp 208–210°C. H-NMR (DMSO-d6)
chloroform and methanol with different ratios and spots were δ: 7.47–7.56 (4H, m), 7.49–7.51 (5H, m), 7.55–7.56 (4H, m),
+·
visualized using UV lamp. The docking was performed using 7.58 (1H, s), 8.34 (1H, s). GC-MS (ESI) m/z: 459 (M¬ ). IR
Molecular Operating Environment (MOE 2008.10). Evaluation (KBr) cm−1: 3448, 3383, 3197, 3089, 1631, 1300. Anal. Calcd
of the cytotoxic activity was performed at the Egyptian Na- for C19H15ClN6O2S2: C, 49.72; H, 3.29; N, 18.31. Found: C,
tional Cancer Institute. Enzyme assay testing was performed 49.84; H, 3.34; N, 18.52.
at VACSERA-Egypt using SIGMA Protein Tyrosine Kinase
4-[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-N-
Assay Kit (specific for EGFR-TK) and ROBONIK P2000 pyridin-2-ylbenzenesulfonamide (3c)
1
spectrophotometer wl 450nm reader. Compounds 1a and b
Yield, 64% (ethanol). mp 220–222°C. H-NMR (DMSO-
were synthesized according to reported method.29)
d6) δ: 6.85 (1H, t, J=12.6, 12.6Hz), 7.13 (1H, d, J=8.7Hz),
5-(4-Aryl)-N-[4-(benzyloxy)phenyl]thieno[2,3-d]pyrimi- 7.53–7.60 (4H, m), 7.68–7.71 (1H, t, J=12, 12Hz), 7.73–7.78
din-4-amine (2a and b) A well-stirred mixture of either (4H, m), 7.81 (1H, s), 7.97 (1H, s), 8.03 (1H, d, J=4.5Hz), 8.64

1178
Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
(1H, s), 12.01 (1H, s). 13C-NMR (DMSO-d6) δ: 167.9, 161.2, 127.7, 126.6, 124.5, 123.9, 121.8, 115.4. IR (KBr) cm−1: 3414,
+·
157.3, 150.0, 148.8, 142.6, 138.2, 134.5, 133.7, 129.6, 129.2, 3325, 3105, 1345. MS (ESI) m/z: 469 (M¬ ). Anal. Calcd for
128.8, 128.2, 126.4, 124.6, 121.8, 115.4, 113.1, 108.4. IR (KBr) C19H15N7O4S2: C, 48.61; H, 3.22; N, 20.88. Found: C, 48.58; H,
cm−1: 3390, 3047, 1597, 1354. Anal. Calcd for C23H16ClN5O2S2: 3.25; N, 20.98.
C, 55.92; H, 3.26; N, 14.18. Found: C, 56.01; H, 3.32; N, 14.32.
N-(4,6-Dimethylpyrimidin-2-yl)-4-[5-(4-nitrophenyl)-
4-[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-N- thieno[2,3-d]pyrimidin-4-ylamino]benzenesulfonamide (3j)
1
pyrimidin-2-yl-benzenesulfonamide (3d)
Yield, 55% (ethanol). mp 270–272°C. H-NMR (DMSO-d6)
Yield, 63% (acetone). mp 310–312°C. 1H-NMR (DMSO- δ: 2.23 (6H, s), 6.73 (1H, s), 7.58 (1H, d, J=8.7Hz), 7.72 (1H,
d6) δ: 5.37 (2H, brs), 6.33–6.36 (4H, m), 6.42 (1H, t, J=9.3, s), 7.78–7.82 (3H, m), 7.88 (1H, d, J=8.7Hz), 7.91 (1H, s), 8.16
9.3Hz), 7.43–7.45 (5H, m,), 8.07 (2H, d, J=4.8Hz), 8.09 (1H, (1H, s), 8.20–8.26 (3H, m), 8.67 (1H, s). 13C-NMR (DMSO-d6)
s). 13C-NMR (DMSO-d6) δ: 169.4, 167.8, 157.3, 150.1, 142.6, δ: 168.5, 167.6, 166.1, 157.1, 149.9, 148.4, 142.6, 142.3, 129.3,
134.4, 133.9, 129.5, 129.2, 128.5, 128.2, 126.2, 124.5, 121.7, 128.2, 127.9, 126.3, 124.4, 124.2, 121.8, 115.4, 109.6, 21.2. IR
115.2, 110.7. IR (KBr) cm−1: 3418, 3298, 1628, 1357. Anal. (KBr) cm−1: 3371, 3085, 1355. Anal. Calcd for C24H19N7O4S2:
Calcd for C22H15ClN6O2S2: C, 53.38; H, 3.05; N, 16.98. Found: C, 54.02; H, 3.59; N, 18.38. Found: C, 54.10; H, 3.63; N, 18.51.
C, 53.52; H, 3.02; N, 17.12.
4-[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-N- amine (4a–q) A well-stirred mixture of either thieno[2,3-
thiazol-2-yl-benzenesulfonamide (3e) d]pyrimidine 1a or b (1mmol) and appropriate aniline de-
5-(4-Aryl)-N-substituted Arylthieno[2,3-d]pyrimidin-4-
1
Yield, 72% (ethanol). mp 223–225°C. H-NMR (DMSO-d6) rivatives (1.2mmol) in either isopropanol for compound 1a or
δ: 6.81 (1H, d, J=4.5Hz), 7.24 (1H, d, J=5.4Hz), 7.53–7.61 isopropanol/2–3 drops conc. HCl for compound 1b (20mL)
(4H, m), 7.70–7.73 (4H, m), 7.77 (1H, s), 7.98 (1H, s), 8.64 (1H, was heated under reflux for 12–16h. The product was either
s), 12.68 (1H, s). 13C-NMR (DMSO-d6) δ: 171.5, 167.8, 157.1, cooled and the separated solid was filtered, dried and crystal-
149.8, 142.3, 138.8, 134.9, 133.9, 129.7, 129.3, 128.5, 126.2, lized from the appropriate solvent for compounds 4a–i or the
124.2, 121.7, 115.4, 108.7. IR (KBr) cm−1: 3390, 3093, 1598, reaction mixture was left at ambient temperature overnight,
+·
+·
1327. GC-MS (ESI) m/z: 502 (M+2¬ ), 500 (M¬ ), 336 then the formed solid was triturated in sodium carbonate solu-
+·
(M−C3H3N2O2S2¬ ). Anal. Calcd for C21H14ClN5O2S3: C, tion (1%, 0.5mL), stirred, filtered, and crystallized from the
50.44; H, 2.82; N, 14.01. Found: C, 50.49; H, 2.78; N, 14.22.
4-[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-N-
(4,5-dimethylisoxazol-3-yl)benzenesulfonamide (3f)
appropriate solvent for compounds 4j–q.
[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-yl]m-tolyl-
amine (4a)
1
Yield, 60% (ethanol). mp 178–180°C. 1H-NMR (DMSO-
Yield, 73% (acetone). mp 125–127°C. H-NMR (DMSO-d6)
d6) δ: 1.60 (3H, s), 2.08 (3H, s), 6.20 (1H, s), 6.57 (2H, d, δ: 2.26 (3H, s), 6.86 (1H, d, J=6.3Hz), 7.11–7.18 (2H, m), 7.21
J=8.7Hz), 7.33 (2H, d, J=8.7Hz), 7.36–7.53 (4H, m), 8.06 (1H, s), 7.30 (1H, s), 719–7.59 (4H, m), 7.66 (1H, s), 8.56 (1H,
(1H, s), 8.98 (1H, s), 10.49 (1H, s). IR (KBr) cm−1: 3444, 3379, s). 13C-NMR (DMSO-d6) δ: 167.6, 157.1, 146.4, 142.3, 138.6,
3089, 1628, 1346. Anal. Calcd for C23H18ClN5O3S2: C, 53.95; 134.5, 133.8, 129.4, 129.2, 128.4, 128.2, 124.4, 121.8, 119.4,
H, 3.54; N, 13.68. Found: C, 54.01; H, 3.83; N, 13.40.
4-[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-N- (ESI) m/z: 353 (M+2¬ ), 351 (M¬ , 46), 350 (M−1¬ ).
(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide (3g) Anal. Calcd for C19H14ClN3S: C, 64.86; H, 4.01; N, 11.94.
Yield, 60% (ethanol). mp 230–232°C. H-NMR (DMSO- Found: C, 65.15; H, 4.07; N, 12.22.
115.8, 112.1, 20.9. IR (KBr) cm−1: 3410, 3082, 1610. GC-MS
+·
+·
+·
1
d6) δ: 2.24 (6H, s), 6.76 (1H, s), 7.52–7.60 (6H, m), 7.78 (1H,
[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-yl]p-tolyl-
s), 7.89 (2H, d, J=8.7Hz), 7.92 (1H, s), 7.98 (1H, s), 8.65 (1H, amine (4b)
1
s). 13C-NMR (DMSO-d6) δ: 168.5, 167.6, 166.1, 149.9, 142.3,
Yield, 75% (acetone). mp 188–190°C. H-NMR (DMSO-d6)
134.6, 133.8, 129.4, 129.3, 128.4, 128.2, 126.3, 124.4, 121.8, δ: 2.31 (3H, s), 7.10 (2H, d, J=8.7Hz), 7.27 (2H, d, J=8.7Hz),
115.4, 109.7, 21.2. IR (KBr) cm−1: 3371, 3062, 1597, 1327. 7.38 (1H, s), 7.56–7.62 (4H, m), 7.70 (1H, s), 8.54 (1H, s).
Anal. Calcd for C24H19ClN6O2S2: C, 55.11; H, 3.66; N, 16.07. 13C-NMR (DMSO-d6) δ: 167.6, 157.1, 143.4, 142.3, 134.6,
Found: C, 54.77; H, 3.37; N, 15.93.
4-[5-(4-Nitrophenyl)thieno[2,3-d]pyrimidin-4-ylamino]- (KBr) cm−1: 3414, 3082, 3051, 1608, 2925. Anal. Calcd for
benzenesulfonamide (3h) C19H14ClN3S: C, 64.86; H, 4.01; N, 11.94. Found: C, 64.97; H,
Yield, 74% (ethanol). mp 263–265°C. H-NMR (DMSO-d6) 4.12; N, 12.21.
133.8, 130.0, 129.4, 128.4, 128.2, 127.6, 124.4, 115.0, 20.8. IR
1
δ: 7.24 (3H, s), 7.58 (2H, d, J=9Hz), 7.70 (2H, d, J=9Hz), 7.82
4-[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-
(2H, d, J=9Hz), 7.98 (1H, s), 8.30 (2H, d, J=8.7Hz), 8.68 (1H, phenol (4c)
1
s). 13C-NMR (DMSO-d6) δ: 167.8, 157.1, 149.8, 148.4, 142.6,
Yield, 70% (ethanol). mp 300–302°C. H-NMR (DMSO-
142.2, 129.3, 128.2, 127.9, 126.3, 124.4, 124.2, 121.8, 115.4. d6) δ: 6.69 (2H, d, J=8.7Hz), 7.14 (2H, d, J=9Hz), 7.18 (1H,
IR (KBr) cm−1: 3387, 3302, 3190, 3062, 1600, 1348. MS (ESI) s), 7.46–7.51 (4H, m), 7.64 (1H, s), 8.45 (1H, s), 9.30 (1H, s).
+·
m/z: 427 (M¬ ). Anal. Calcd for C18H13N5O4S2: C, 50.58; H, 13C-NMR (DMSO-d6) δ: 167.6, 157.1, 147.4, 142.3, 139.3,
3.07; N, 16.38. Found: C, 50.62; H, 3.11; N, 16.43.
134.6, 133.8, 129.4, 128.4, 128.2, 124.4, 121.8, 116.4. IR (KBr)
N-Carbamimidoyl-4-{[5-(4-nitrophenyl)thieno[2,3-d]- cm−1: 3444, 3402, 3097, 3026, 1608, 2920. Anal. Calcd for
pyrimidin-4-yl]amino}benzenesulfonamide (3i) C18H12ClN3OS: C, 61.10; H, 3.42; N, 11.88. Found: C, 61.06; H,
Yield, 51% (acetone). mp 240–242°C. H-NMR (DMSO- 3.49; N, 12.02.
1
d6) δ: 6.65 (4H, brs), 7.53 (2H, d, J=7.8Hz), 7.64 (2H, d,
(4-Chlorophenyl)[5-(4-chlorophenyl)thieno[2,3-d]-
J=8.7Hz), 7.82 (2H, d, J=8.4Hz), 7.96 (1H, s), 8.20 (1H, s), pyrimidin-4-yl]amine (4d)
8.31 (2H, d, J=9Hz), 8.34 (1H, s). 13C-NMR (DMSO-d6) δ:
Yield, 82% (isopropanol). mp 162–164°C. 1H-NMR (DMSO-
167.3, 158.2, 149.9, 148.3, 142.9, 142.1, 130.62, 129.7, 128.2, d6) δ: 7.34 (2H, d, J=9Hz), 7.37 (2H, d, J=9Hz), 7.45–7.63

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1179
(4H, m), 7.66 (1H, s), 7.74 (1H, s), 8.58 (1H, s). IR (KBr) cm−1: 2930. Anal. Calcd for C19H14N4O2S: C, 62.79; H, 3.89; N,
+·
3406, 3066, 1604. GC-MS (ESI) m/z: 374 (M+2¬ ), 372 15.46. Found: C, 63.03; H, 3.91; N, 15.62.
+·
+·
(M¬ ), 370 (M−2¬ ). Anal. Calcd for C18H11Cl2N3S: C,
58.07; H, 2.98; N, 11.29. Found: C, 58.14; H, 3.02; N, 11.38.
2-[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-
4-[5-(4-Nitrophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-
phenol (4l)
Yield, 64% (ethanol). mp 270–272°C. H-NMR (DMSO-d6)
1
benzoic Acid (4e)
δ: 6.67 (2H, d, J=8.7Hz), 7.16 (2H, d, J=8.7Hz), 7.51 (1H, s),
1
Yield, 69% (acetone). mp 262–264°C. H-NMR (DMSO- 7.82–7.85 (3H, m), 8.33 (2H, d, J=8.7Hz), 8.46 (1H, s), 9.30
d6) δ: 7.42–7.46 (4H, m), 7.53 (1H, s), 7.54–7.57 (4H, m), 8.14 (1H, s). 13C-NMR (DMSO-d6) δ: 167.6, 157.1, 148.4, 147.4,
(1H, s), 8.15 (1H, s), 12.51 (1H, s). IR (KBr) cm−1: 3402, 3360, 142.7, 142.3, 139.3, 128.2, 127.9, 124.4, 124.2, 121.8, 116.5.
3067, 1685, 1581. GC-MS (ESI) m/z: 383 (M+2¬ ), 382 IR (KBr) cm−1: 3444, 3402, 3066, 1604. GC-MS (ESI) m/z
+·
+·
+·
+·
(M+1¬ ), 381(M¬ ). Anal. Calcd for C19H12ClN3O2S: C, 365(M+1¬ ). Anal. Calcd for C18H12N4O3S: C, 59.33; H, 3.32;
59.76; H, 3.17; N, 11.00. Found: C, 59.73; H, 3.22; N, 11.12.
4-[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-
benzoic Acid (4f)
N, 15.38. Found: C, 59.32; H, 3.34; N, 15.44.
(4-Chlorophenyl)-[5-(4-nitrophenyl)thieno[2,3-d]-
pyrimidin-4-yl]amine (4m)
1
1
Yield, 72% (acetic acid). mp 303–305°C. H-NMR (DMSO-
Yield, 73% (acetone). mp 210–212°C. H-NMR (DMSO-d6)
d6) δ: 7.50 (2H, d, J=8.4Hz), 7.58–7.66 (4H, m), 7.74 (1H, s), δ: 7.32 (2H, d, J=8.7Hz), 7.46 (2H, dd, J=8.7, 8.7Hz), 7.82
7.86 (2H, d, J=8.4Hz), 7.89 (1H, s), 8.66 (1H, s), 12.50 (1H, (2H, dd, J=8.7, 8.7Hz), 7.93 (1H, s), 8.02 (1H, s), 8.31 (2H,
s). 13C-NMR (DMSO-d6) δ: 172.0, 167.6, 157.1, 151.8, 142.3, d, J=8.7Hz), 8.60 (1H, s). IR (KBr) cm−1: 3425, 3105, 3062,
+·
+·
134.6, 133.7, 130.9, 129.3, 128.5, 128.2, 124.4, 121.8, 120.6, 1604. GC-MS (ESI) m/z: 384 (M+2¬ ), 382 (M¬ ), 381
+·
115.0. IR (KBr) cm−1: 3483, 3402, 3074, 1689, 1605. MS (ESI) (M−1¬ ). Anal. Calcd for C18H11ClN4O2S: C, 56.47; H, 2.90;
+·
+·
+·
m/z: 383 (M+2¬ ), 382 (M+1¬ ), 337 (M−COOH¬ ). N, 14.64. Found: C, 56.51; H, 2.93; N, 14.81.
Anal. Calcd for C19H12ClN3O2S: C, 59.76; H, 3.17; N, 11.00.
Found: C, 59.92; H, 3.23; N, 11.31.
[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-yl]-(3-
2-[5-(4-Nitrophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-
benzoic Acid (4n)
Yield, 55% (isopropanol). mp 300–302°C. 1H-NMR
(DMSO-d6) δ: 7.77–7.83 (5H, m), 8.16 (1H, s), 8.19–8.26 (4H,
nitrophenyl)amine (4g)
Yield, 70% (acetone). mp 160–162°C. H-NMR (DMSO- m), 12.60 (2H, s). IR (KBr) cm−1: 3356, 3290, 3101, 3059,
1
+·
d6) δ: 7.54–7.64 (5H, m), 7.72 (1H, d, J=7.5Hz), 7.80 (1H, s), 1732, 1643. GC-MS (ESI) m/z: 393 (M+1¬ ). Anal. Calcd for
7.87 (1H, d, J=7.8Hz), 8.20 (1H, s), 8.52 (1H, s), 8.67 (1H, C19H12N4O4S: C, 58.16; H, 3.08; N, 14.28. Found: C, 58.28; H,
s). IR (KBr) cm−1: 3394, 3070, 1605. GC-MS (ESI) m/z: 384 3.11; N, 14.43.
+·
+·
+·
(M+2¬ ), 382 (M¬ ), 381 (M−1¬ ). Anal. Calcd for
C18H11ClN4O2S: C, 56.47; H, 2.90; N, 14.64. Found: C, 56.19; benzoic Acid (4o)
H, 2.98; N, 14.60. Yield, 69% (acetic acid). mp 240–242°C. H-NMR (DMSO-
[5-(4-Chlorophenyl)thieno[2,3-d]pyrimidin-4-yl]-(3,4- d6) δ: 6.52 (1H, d, J=6.3Hz), 7.55–7.86 (4H, m), 7.98 (1H, s),
4-[5-(4-Nitrophenyl)thieno[2,3-d]pyrimidin-4-ylamino]-
1
dichloro-phenyl)amine (4h)
7.82–8.32 (4H, m), 8.69 (1H, s), 12.63 (1H, s). GC-MS (ESI)
Yield, 69% (acetone). mp 164–166°C. H-NMR (DMSO-d6) m/z 392 (M¬ ). IR (KBr) cm−1: 3414, 3055, 1705, 1608. Anal.
δ: 7.23 (1H, d, J=2.4Hz), 7.26 (1H, d, J=2.4Hz), 7.52–7.61 Calcd for C19H12N4O4S: C, 58.16; H, 3.08; N, 14.28. Found: C,
(4H, m), 7.77 (1H, s), 7.91 (1H, s), 7.92 (1H, s), 8.63 (1H, s). IR 58.38; H, 3.10; N, 14.50.
1
+·
(KBr) cm−1: 3402, 3040, 1600. Anal. Calcd for C18H10Cl3N3S:
(3-Nitrophenyl)-[5-(4-nitro-phenyl)thieno[2,3-d]-
C, 53.16; H, 2.48; N, 10.33. Found: C, 53.28; H, 2.54; N, 10.52. pyrimidin-4-yl]amine (4p)
1
(4-Bromophenyl)-[5-(4-chloro-phenyl)thieno[2,3-d]-
pyrimidin-4-yl]amine (4i)
Yield, 80% (acetone). mp 160–162°C. H-NMR (DMSO- (3H, m), 7.99 (1H, s), 8.22–8.32 (2H, m), 8.40 (1H, s), 8.49
d6) δ: 7.39 (2H, d, J=8.2Hz), 7.47 (2H, d, J=9Hz), 7.59–7.65 (1H, s), 8.68 (1H, s). IR (KBr) cm−1: 3421, 3078, 1601. Anal.
(4H, m), 7.65 (1H, s), 7.74 (1H, s), 8.59 (1H, s). IR (KBr) cm−1: Calcd for C18H11N5O4S: C, 54.96; H, 2.82; N, 17.80. Found: C,
Yield, 63% (acetone). mp 220–222°C. H-NMR (DMSO-d6)
δ: 7.54 (1H, t, J=7.8, 7.8Hz), 7.74 (1H, d, J=9Hz), 7.80–7.85
1
+·
3406, 3050, 1600. GC-MS (ESI) m/z: 418 (M+2¬ ), 416 55.05; H, 2.80; N, 17.93.
+·
(M¬ ). Anal. Calcd for C18H11BrClN3S: C, 51.88; H, 2.66; N,
10.08. Found: C, 51.91; H, 2.63; N, 10.21.
(4-Bromophenyl)-[5-(4-nitrophenyl)thieno[2,3-d]-
pyrimidin-4-yl]amine (4q)
Yield, 70% (ethanol). mp 206–208°C. H-NMR (DMSO-
1
[5-(4-Nitrophenyl)thieno[2,3-d]pyrimidin-4-yl]m-tolyl-
amine (4j)
d6) δ: 7.40–7.47 (3H, m), 7.48 (1H, s), 7.82–7.85 (3H, m), 7.91
1
Yield, 68% (acetone). mp 130–132°C. H-NMR (DMSO- (1H, s, NH), 8.30 (2H, d, J=8.7Hz), 8.60 (1H, s). 13C-NMR
d6) δ: 2.24 (3H, s), 6.85 (1H, t, J=5.4, 5.4Hz), 7.14 (2H, d, (DMSO-d6) δ: 167.6, 157.1, 148.4, 145.8, 142.6, 142.3, 132.6,
J=4.8Hz), 7.23 (1H, s), 7.80 (2H, d, J=8.7Hz), 7.84 (1H, s), 128.2, 127.9, 124.4, 124.2, 121.8, 117.4, 113.1. IR (KBr) cm−1:
7.89 (1H, s), 8.31 (2H, d, J=8.7Hz), 8.57 (1H, s). IR (KBr) 3417, 3383, 3097, 3059, 1608. GC-MS (ESI) m/z: 429, 427
cm−1: 3398, 3050, 1608, 2920. Anal. Calcd for C19H14N4O2S: (M¬ ), 425. Anal. Calcd for C18H11BrN4O2S: C, 50.60; H,
+·
C, 62.79; H, 3.89; N, 15.46. Found: C, 63.08; H3.91; N, 15.63.
2.59; N, 13.11. Found: C, 50.61; H, 2.60; N, 13.01.
[5-(4-Nitrophenyl)thieno[2,3-d]pyrimidin-4-yl]p-tolylamine
Docking Studies The X-ray crystallographic structure of
EGFR complexed with Lapatinib (PDB ID: 1XKK) was ob-
(4k)
1
Yield, 70% (acetone). mp 210–212°C. H-NMR (DMSO-d6) tained from protein data bank available at the RCSB Protein
δ: 2.25 (3H, s), 7.08 (2H, d, J=8.7Hz), 7.29 (2H, d, J=8.7Hz), Data Bank, http://www.rcsb.org.
7.66 (1H, s), 7.82 (2H, d, J=8.7Hz), 7.86 (1H, s), 8.32 (2H,
Compounds were built using Molecular Operating Environ-
d, J=8.7Hz), 8.54 (1H, s). IR (KBr) cm−1: 3417, 3043, 1608, ment (MOE) molecule builder. The structures were energy

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Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
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tool. Poses generated by the placement methodology were
scored and then subjected to MMFF94x energy minimiza-
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based on number of binding interactions, superposition with
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Cytotoxic Activity The antitumor activity was deter-
mined for the newly synthesized compounds in Egyptian
National Cancer Institute (NCI) for in-vitro detection of IC50
of their antitumor activity and was repeated three times (n=3).
The breast tumor cell line (MCF-7) was obtained frozen in
liquid nitrogen (−180°C) from the American Type Culture
Collection (ATCC) and was maintained in the National
Cancer Institute, Cairo, Egypt, by serial sub-culturing. All
chemicals used in this study are of high analytical grade.
They were obtained from (either Sigma-Aldrich or Bio-Rad).
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SRB assay applying the method of Monk et al.30) The Cells
were plated in 96-multiwell plate (104 cells/well) for 24h be-
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of cells to the wall of the plate. Different concentrations of
each compound (0, 1, 2.5, 5, 10µg/mL) were added to the cell
monolayer triplicate wells were prepared for each individual
dose. Monolayer cells were incubated with the compounds
for 48h at 37°C and in atmosphere of 5% CO2. After 48h,
cells were fixed, washed, and stained with sulforhodamine B
stain. Excess stain was washed with acetic acid and attached
stain was recovered with tris ethylenediaminetetraacetic acid
(EDTA) buffer. Color intensity was measured in an ELISA
reader. The relation between surviving fraction and drug con-
centration was plotted to get the survival curve of each tumor
cell line. The IC50 value was calculated using sigmoidal dose
response curve-fitting models (Graph Pad, Prizm Software In-
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carried out three times for every IC50 dose of each target com-
pound.
Acknowledgments The authors are grateful to all mem-
bers of the Cancer Biology Department, National Cancer
Institute, Cairo, Egypt, for carrying out the cytotoxicity test-
ing. The authors are grateful to all members of confirmatory
diagnostic unit, VACSERA-Egypt.
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Conflict of Interest The authors declare no conflict of
interest.
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