6-Cinnamoyl-4-arylaminothienopyrimidines as highly potent cytotoxic agents: Design, synthesis and structure-activity relationship studies

Article abstract of DOI:10.1016/j.ejmech.2019.111786

In this paper, we described the synthesis and cytotoxic activities of two new series of thieno[2,3-d]pyrimidine and thieno[3,2-d] pyrimidine derivatives. Most of the synthesized compounds had significant antiproliferative activities against PC3, MDA-MB-23

Full text of DOI:10.1016/j.ejmech.2019.111786

Journal Pre-proof
6-Cinnamoyl-4-arylaminothienopyrimidines as highly potent cytotoxic agents: Design,
synthesis and structure-activity relationship studies
Mahsa Toolabi, Setareh Moghimi, Tayebeh Oghabi Bakhshaiesh, Somayeh
Salarinejad, Ayoub Aghcheli, Zaman Hasanvand, Elahe Nazeri, Ali Khalaj, Rezvan
Esmaeili, Alireza Foroumadi
PII:
S0223-5234(19)30938-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.111786
EJMECH 111786
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 June 2019
Revised Date: 10 October 2019
Accepted Date: 11 October 2019
Please cite this article as: M. Toolabi, S. Moghimi, T.O. Bakhshaiesh, S. Salarinejad, A. Aghcheli, Z.
Hasanvand, E. Nazeri, A. Khalaj, R. Esmaeili, A. Foroumadi, 6-Cinnamoyl-4-arylaminothienopyrimidines
as highly potent cytotoxic agents: Design, synthesis and structure-activity relationship studies, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111786.
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6-Cinnamoyl-4-arylaminothienopyrimidines as highly potent cytotoxic agents: design,
synthesis and structure-activity relationship studies
Mahsa Toolabi a, Setareh Moghimi b, Tayebeh Oghabi Bakhshaiesh c, Somayeh Salarinejad a,
Ayoub Aghcheli a, Zaman Hasanvand a, Elahe Nazeri c, Ali Khalaj a, Rezvan Esmaeili c,*, Alireza
Foroumadi a,**
a Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
bDrug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS),
Tehran University of Medical Sciences, Tehran, Iran
cGenetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR,
Tehran, Iran

6-Cinnamoyl-4-arylaminothienopyrimidines as highly potent cytotoxic agents: design,
synthesis and structure-activity relationship studies
Mahsa Toolabi a, Setareh Moghimi b, Tayebeh Oghabi Bakhshaiesh c, Somayeh Salarinejad a,
Ayoub Aghcheli a, Zaman Hasanvand a, Elahe Nazeri c, Ali Khalaj a, Rezvan Esmaeili c,*, Alireza
Foroumadi a,**
a Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
bDrug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS),
Tehran University of Medical Sciences, Tehran, Iran
cGenetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR,
Tehran, Iran
Corresponding authors:
* E-mail: esmaeili.rezvan@gmail.com (R. Esmaeili); Tel.: 98 21 88876869; Fax: 98 88796208.
** E-mail: aforoumadi@yahoo.com (A. Foroumadi); Tel.: 98 21 66406757; Fax: 98 21 66461178.

Abstract
In this paper, we described the synthesis and cytotoxic activities of two new series of
thieno[2,3-d]pyrimidine and thieno[3,2-d] pyrimidine derivatives. Most of the synthesized
compounds had significant antiproliferative activities against PC3, MDA-MB-231, A549, and
HeLa cell lines in comparison to the reference drug, erlotinib. Compounds
N-(4-((3,5-dichlorophenyl)amino)thieno[2,3-d]pyrimidin-6-yl)cinnamamide
8e
and
(E)-N-(4-((3,4-dichlorophenyl)amino)thieno[2,3-d]pyrimidin-6-yl)-3-(4-methoxyphenyl)acrylam
ide 8g with IC50 values of 4 nM and 33 nM, respectively, against HeLa cell line were chosen for
further studies. The apoptosis induced activity and cell cycle arrest were determined and the
results provided evidence that these compounds induced cell death via apoptosis and arrested cell
growth in G0 phase. In addition, western blot analysis manifested the promising result of
suppressing thes EGFR signaling pathway (p-EGFR/p-ERK1/2). The docking studies appreciated
the considerable potency of compound 8e based on hydrogen and covalent binding interactions.
Eventually, in silico pharmacokinetic prediction indicated the acceptable bioavailability of all
final compounds.
Keywords: Cytotoxic; thienopyrimidine; antiproliferative activity; α,β-Unsaturated carbonyl;
EGFR
1. Introduction
The field of anti-cancer drugs has seen advances in recent years, because of
disappointing outcomes of chemotherapy. The associated problems namely multidrug-resistant
tumors and systemic toxicity have hindered the efficacy of current chemotherapy strategies [1-3].
In light of this, the development of novel pharmacological agents for targeted therapy has been
pursued as a promising opportunity to increase the clinical effectiveness of chemotherapy drugs.
The principle of this approach is relied on targeting cancer cells through specific genes and
proteins without affecting normal ones. Monoclonal antibodies, immunotoxins, and small
molecule inhibitors including protein kinase inhibitors are three main types of targeted therapy,
trying to give patients more durable therapeutic effects.

Protein kinase is a protein with a crucial function in various intercellular processes
including cell growth and differentiation. The genetic mutation led to the abnormal enhancement
in the activity of this protein resulted in excessive cell proliferation [4-6]. Tyrosine kinases (TKs),
a large multigene family that can be grouped into transmembrane receptor tyrosine kinases
(RTKs) and cytoplasmic non-receptor tyrosine kinases (NRTKs), constitute an important cellular
signal transduction pathway affecting on many cellular functions. The activity of this kinase has
clear relationship with cancer development and progression, so, small molecule tyrosine kinase
inhibitors (TKI) have shown significant anti-cancer effects. Epidermal growth factor receptor
(EGFR), a member of TKs also called ErbB1/HER1, conformationally changes after ligand
binding and activates phosphorylation of downstream effectors such as RAS–RAF–MEK–ERK
and PI3K–AKT–mTOR via the homodimerization and heterodimerization. Considering the role
of EGFR over-expression in epithelial malignancies, enhanced tumor growth, invasion, and
metastasis, several tyrosine kinase inhibitors have been validated in cancer treatment [7-9].
Due to the prevalent presence of 4-arylaminoquinazoline nucleus in approved EGFR
inhibitor drugs such as gefitinib and erlotinib, the introduction of a bioisosteric form of this
nucleus is proposed as an alternative pathway for the design of new cytotoxic agents [10]. As
evidenced by previous studies, the thienopyrimidine core has shown a wide range of biological
effects, including antimicrobial, anti-inflammatory, anticancer, and EGFR inhibitory activity.
Apitolisib, a thienopyrimidine-based drug, is used in the clinical phases for the treatment of solid
cancers [11-15]. The presence of distinctive hydrophobic substitutions at meta position of the
4-arylamino ring (attached to the C-4 position of thienopyrimidine) and the substituents at the C-6
position of thienopyrimidine, interacted with the solvent region, is necessary to obtain potent
cytotoxic agents. Thererfore, according to the fundamental tactical approach in medicinal
chemistry, bioisosterism, hereby thienopyrimidine is chosen in light of its notable similarity with
4-arylaminoquinazoline.
α,β-Unsaturated carbonyl is a fascinating key structural motif in anticancer agents
exhibiting inhibitory activities against different targets. The irreversible Michael addition of the
sulfhydryl group of EGFR’s cysteine residue to this moiety increases the binding affinity and
introduces new compounds working through targeted covalent inhibition mechanism. By virtue of

this, novel EGFR inhibitors are discovered and used for the treatment of many types of cancers
[16-20].
Figure 1
Following our successful expertise in rationally design of molecules containing multiple
pharmacophores [21-26], herein, we report on the identification of new and highly potent
cytotoxic agents from thienopyrimidine family. A detailed in vitro and in silico pharmacokinetic
studies of final compounds are presented.
2. Chemistry
The procedures to synthesize the final compounds were outlined in Scheme 1 and 2.
Beginning with Gewald reaction, the reaction between 2,5-dihydroxy-1,4-dithiane and
malononitrile led to 2-aminothiophene-3-carbonitrile as a brown solid. The compound 2 was
cyclized with formic acid in the presence of sulfuric acid to yield
thieno[2,3-d]pyrimidin-4(3H)-one 3 [27]. Thieno[3,2-d]pyrimidin-4(3H)-one 11 was also
obtained from the reaction of commercially available methyl 3-aminothiophene-2-carboxylate 10
with formamide.
Further nitration of these compounds 3 and 11 with concentrated sulfuric acid and nitric
acid afforded compounds as orange solids. The chlorinated analogs, obtained by refluxing
compounds 4 and 12 in POCl3, were stirred with different anilines in isopropanol to afford
compounds 6a-e and 14a-e [28]. In the next step, reduction of the nitro group attached to the C-6
position of thienopyrimidine core was conducted with iron powder in the presence of ammonium
chloride in ethanol. At final, amidation was conducted with different cinnamoyl chlorides in the
presence of dimethylaminopyridine/pyridine in dry dichloromethane.
Scheme 1
Scheme 2

3. Results and discussion
3.1. Antiproliferative activity
The antiproliferative potential of the synthesized compounds was evaluated by
determining IC50 values against cell lines from multiple cancer origin with EGFR overexpression
including PC3 (prostate cancer), MDA-MB-231 and MCF7 (breast cancer), HepG2
(hepatocellular carcinoma), A549 (human lung carcinoma), and HeLa (cervical cancer) [26].
Herein, two series of compounds based on thieno[2,3-d]pyrimidine and
thieno[3,2-d]pyrimidine isomeric systems were synthesized. We explored different aniline and
cinnamamide substituents at the C-4 and C-6 positions of the thienopyrimidine core. The
corresponding results were summarized in Table 1.
The antiproliferative activity of compounds from thieno[2,3-d]pyrimidine series against PC3
indicated that all of the compounds had significant effects with IC50 values ranging from 0.1 to
0.79 µM compared to erlotinib (IC50 = 7 µM). The cytotoxicity results against MDA-MB-231
revealed that most of the compounds except 8e and 8g showed minimal cytotoxicity. In contrast,
compounds 8e and 8g exhibited the best cytotoxic effects with IC50 values of 4 and 33 nM against
HeLa cell line, respectively. These compounds also showed significant anticancer activities
against A549, while the other derivatives presented low activity on this cell line. Comparing to
erlotinib, all compounds presented lower toxicity on MCF7 cell, except 8c, 8d, 8e, and 8g.
All synthesized compounds from thieno[3,2-d]pyrimidine series 16a-f had significant effects on
PC3, MDA-MB-231, and HepG2 cell lines and moderate activities on A549. The most cytotoxic
agent against the PC3 cell line, compound 16f, from thieno[3,2-d]pyrimidine series was 70 times
more active than the positive control. All compounds were also active against HeLa cell line,
whereas, weak activities were observed against MCF7.
In both series, the presence of ethynyl group attached to the C-3 of the arylamine moiety
led to the favorable cytotoxic effects on PC3 and MDA-MB-231 cell lines compared to other cell
lines. In dihalo substituted derivatives, changing the position of fluorine from ortho to para
position (8b vs 8c) led to the almost 2-17 times increase in potency against MCF7, HepG2, A549,
and HeLa cell lines. The substitution of fluorine atom with chlorine provided almost lower IC50

amounts (8c vs 8d). 3,5-Dichloro substituted derivative 8e represented the preferred positioning in
terms of the highest activity against almost all examined cell lines.
It should be noted that the differently substituted cinnamamides have affected on the
activity of new compounds. The cytotoxic effects diminished upon substitution of methoxy group
at the C-4 position 8d and 8g, except the activity of 8g which was more potent than unsubstituted
analog against A549 and HeLa cell lines. This could be extended for compounds 8e and 8h, while
an exception was observed in MDA-MB-231 cell line.
Table 1
Figure 2
The cytotoxic activities of the most potent compounds were assessed on two human
colorectal cancer cell lines, SW480 and HCT116 with high levels of EGFR expression and
mutated KRAS gene. KRAS plays an important role in the signaling pathway known as
RAS-RAF-MEK-ERK, resulted in proliferation and differentiation of the cells. In the case of
mutations and because of this signaling pathway activation, resistance and subsequently low
response rates to anti-EGFR therapies were observed [29]. In addition, U87 (glioblastoma cell)
with EGFR overexpression [30], SKBR3 (breast cancer cell line), and SKOV3 (ovarian cancer
cell line) with lower levels of EGFR expression and high level of HER-2 were examined [31]. The
obtained results indicated that these compounds were active against these cell lines with IC50
values ranged from 3.83-11.94 µM, while, erlotinib had less potency against these cells (Table 2).
Table 2
For the selectivity study, the cytotoxicity of the most active compounds 8e and 8g was
determined against a normal lung cell line (MRC5) and compared with the related data presented
in Table 1. The compounds showed IC50 values of 28.8 and 18.7 µM, respectively. Based on the
obtained data, these compounds (8e and 8g) were selective to the lung cancer cell line, A549
(Table 3).

Table 3
3.2. Apoptosis-inducing activity
The apoptotic pathway, the programmed cell death, is an essential biological process for
the removal of unwanted and damaged cells. Any disruption in this system causes various
diseases, including cancer. To determine the cell death pathway created by our compounds, a
double staining flow cytometry assay using Annexin-V FITC- and Propidium iodide (PI) was
carried out. Dimethyl sulfoxide (DMSO) was used as the negative control and erlotinib was used
as the positive control.
To bring out this purpose, the activities of 8e and 8g at IC50 concentrations were
evaluated on HeLa cell line [32]. The different parts of the graph (Figure 3) represented the
percentage of live, apoptotic, and necrotic cells population. As shown, the percentage of early
apoptosis and late apoptosis for 8e and 8g was determined 11%, 15.5%, and 42.8%, 15.7%,
respectively. According to the obtained results and apoptotic cell population, compounds 8e and
8g induced cell death in the apoptotic pathway.
Figure 3
3.3. Cell cycle arrest
The effects of compounds 8e and 8g on cell cycle distribution were evaluated by flow
cytometer [33]. In this assay, the DNA content of cells was quantitated by binding to PI as a
fluorescent agent. In order to perform this experiment, HeLa cell line was incubated at IC50
concentrations of 8e and 8g, stained with PI. The results were illustrated as a histogram in Figure 4
and provided the following information. By comparing cell cycle analysis results, it can be
concluded that the cell growth was arrested and most of the cells remained in G0 phase.
Figure 4
3.4. Western blot analysis

In an effort to assess the kinase inhibition of 8e and its function on phosphorylation of
EGFR and downstream signaling proteins, ERK1/2 and AKT, in HeLa cells, western blot analysis
was carried out [34]. In this experiment, the results were evaluated after 0, 3, 12, 24, and 48 h
treatment of HeLa cells at 8e and erlotinib IC50 concentrations. According to the obtained data
(Figure 5), compound 8e inhibited EGFR and downstream molecule ERK1/2 phosphorylation,
while the protein levels of AKT phosphorylation remained unchanged.
Figure 5
3.5. In silico pharmacokinetics prediction
The pharmacokinetic profile of synthesized compounds was computed by SwissADME
web-based tool and presented in Table 4 [35]. The calculated drug-likeness values forecasted the
overall potential of compounds to pass the drug development process and become
drug-candidates. Bioavailability is one of the most considerable pharmacokinetic properties of
drugs, defined as the percentage of unchanged medicine that reaches the body’s circulatory
system. According to Abbott bioavailability score, all of the compounds had the acceptable
bioavailability score and were distinguished to be orally bioavailable. The water solubility of
compounds was predicted by two topological methods on SwissADME and the data indicated the
moderate to poor solubility of the synthesized compounds. According to the lipophilicity
descriptor, expressed as log P, the partition coefficient between an organic and aqueous phase, all
analyzed compounds showed the approved amounts of log P ranged between 3.5 and 5 (average of
four log P amounts were presented based on different methods including iLOGP, WLOGP,
MLOGP, and SILICOS-IT).
Table 4
3.6. Molecular docking study
Docking analysis was performed using AutoDoc 4.2.1 [36,37]. The co-crystal structure of
EGFR and erlotinib (1M17) with 2.6 Ao resolution was selected and the most potent compound 8e
was docked into the active site of the receptor (ATP binding site). According to the docking results

(Figure 6), compound 8e formed two main types of interactions involving the hydrogen binding
interactions with Met 769, Asp 831, and Lys 721 and the pi-pi and pi-alkyl interactions with Phe
699 and Val 702 in a hydrophobic pocket. As predicted, cinnamamide moiety posed into the
solvent region and made Van der Waals interactions with Gly 772 and Pro 770. In a brief
summary, the docking score and interactions were in good agreement with biological
investigations.
Figure 6
4. Conclusion
In summary, a series of compounds based on thienopyrimidine were synthesized and
evaluated as cytotoxic agents. The MTT assay revealed that most of the synthesized compounds
had considerable antiproliferative activities. Compound 8e with IC50 = 4 nm against HeLa cell line
was the most potent agent. The obtained results from the most potent compounds 8e and 8g
against overexpressing EGFR and mutant KRAS cell lines revealed the high potency of these
compounds. These compounds also showed selectivity for A549 lung cancerous cell line over
MRC5 normal cell. Flow cytometry assay indicated the induction of apoptosis in the cell death
pathway and arrested the cell growth at G0-phase. Moreover, the western blot experiment
confirmed the probable mechanism of action for 8e working by diminishing EGFR and ERK1/2
autophosphorylation in HeLa cell lins. This study may help with optimization and development of
new thienopyrimidine-based anticancer agents.
5. Experimental
5.1. Chemistry
All chemical compounds were purchased from commercial sources such as Sigma, Merck, and
Flukka or synthesized. Infrared spectra were recorded on a Nicolet FT-IR Magna 550
1
spectrometer. H NMR spectra and 13C NMR were carried out with a Bruker FT-500 MHz
spectrometer using CDCl3 or DMSO-d6 as the solvent and TMS as the internal standard. The
detailed characterization data and numbering of atoms were presented in Figure 7. Elemental
analyses were measured with a Perkin-Elmer 240-C apparatus (Perkin-Elmer, Beaconsfield, UK)

and were within ± 0.4% of the theoretical values for C, H, and N. Synthesized compounds were
purified by column chromatography with silica gel 230-400 mesh. Mass spectra were obtained by
HP Agilent Technologies 5937 at ionization potential of 70ꢀeV.
Figure 7
5.2. Synthesis of 2-aminothiophene-3-carbonitrile (2)
To the mixture of 2,5-dihydroxy-1,4-dithiane (6.5 mmol, 990 mg), malononitrile (6.5 mmol, 429
mg) in DMF (5 mL), Et3N (0.8 ml) was added dropwise and the mixture was heated at 50 oC for 2
h. After that, the solution was diluted with water and neutralized with acetic acid. Then, the
mixture was extracted with diethyl ether and dried over sodium sulfate. The solvent was
evaporated to obtain the product as a brown solid.
5.3. Synthesis of thieno[2,3-d]pyrimidin-4(3H)-one (3)
To the solution of compound 2 (3 mmol, 372 mg) in formic acid (2.5 mL), sulfuric acid (0.3 ml)
was added and the reaction was heated at 90 oC for 5 h. After cooling, the solution was diluted with
water (50 mL) and the resulting solid was separated to obtain purple solid.
Caution! Preparation of thieno[2,3-d]pyrimidin-4(3H)-one (3) must be carried out in an efficient
and well-ventilated hood, because carbon monoxide is evolved.
5.4. Synthesis of thieno[3,2-d]pyrimidin-4(3H)-one (11)
Methyl 3-aminothiophene-2-carboxylate (1 mmol, 157 mg) in excess amounts of formamide (5
mL) was refluxed overnight. After completion, the mixture was cooled, filtered and washed with
water to give the corresponding product.
5.5. General procedure for the preparation of 4 and 12

The solution of nitric acid (0.9 mL) and sulfuric acid (1.2 mL) was slowly added via dropping
funnel to the compound 3 or 11 (3.9 mmol, 594 mg) at 0-5 oC. Then, the mixture was heated at 50
oC for 1.5 h. After completion, the mixture was poured into ice/water (approx. 100 mL) and the
resultant precipitate was filtered.
5.6. General procedure for the preparation of 5 and 13
o
The mixture of compound 4 or 12 (2.3 mmol, 454 mg) and POCl3 (5 mL) was heated at 90 C
overnight. After that, POCl3 was evaporated under vacuum and the resultant was slowly added to
the ice/water (approx. 100 mL) with constant stirring until the precipitation completed. The
precipitate was filtered and purified using column chromatography (1:9 ethyl acetate /petroleum
ether).
5.7. General procedure for the preparation of 6a-e, 14a-e
Compound 5 or 13 (1.5 mmol, 327 mg) and aniline derivatives (1.7 mmol) were dissolved in
isopropanol and refluxed for about 3 h. Then, the reaction was cooled and the resulting precipitate
was filtered and washed with ethyl acetate to obtain the product as a yellow solid.
5.8. General procedure for the preparation of 7a-e, 15a-e
Compound 6 or 14 (1 mmol), NH4Cl (3 mmol, 160 mg), and iron (3 mmol, 167 mg) in H2O/EtOH
(5:10 mL) were stirred at 50 oC for 1 h. After completion, the mixture was filtered through celite
and washed with ethanol (30 mL). The organic layer was removed under vacuum and then water
was added. The resulting precipitate was filtered and recrystalized from ethanol.
5.8.1. N4-(3-ethynylphenyl)thieno[2,3-d]pyrimidine-4,6-diamine (7a)
Gray solid; yield: 63%; IR (KBr, cm-1): 3404 (N-H), 3339 (N-H), 2132
(
)
, 1641, 1289; 1H
NMR (500 MHz, DMSO-d6) δ: 4.21 (s, 1H), 5.74 (s, 2H), 6.75 (s, 1H), 7.24 (d, J = 7.8 Hz, 1H),
7.40 (t, J = 7.8 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 8.08 (s, 1H), 8.67 (s, 1H), 9.25 (s, 1H);13C-NMR
(DMSO-d6, 125 MHz) δ: 78.53, 80.33, 102.31, 118.18, 122.97, 123.69, 124.33, 127.46, 130.14,
141.90, 143.78, 151.17, 153.68, 158.30.
5.8.2. N4-(3-chloro-2-fluorophenyl)thieno[2,3-d]pyrimidine-4,6-diamine (7b)

1
Gray solid; yield: 64%; IR (KBr, cm-1): 3425 (N-H), 3365 (N-H), 1639, 1279; H NMR (500
MHz, DMSO-d6) δ: 5.34 (s, 2H), 7.50-7.53 (m, 1H), 7.64-7.67 (m, 1H), 7.83-7.85 (m, 1H), 8.69
(s, 1H), 9.17 (s, 1H), 10.83 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ: 103.22, 118.94, 121.91,
125.07, 126.45 (d, J = 20.1 Hz), 128.96, 134.94 (d, J = 20.1 Hz), 135.02, 144.73, 150.04 (d, J =
251.1 Hz), 152.11, 155.05, 159.44.
5.8.3. N4-(3-chloro-4-fluorophenyl)thieno[2,3-d]pyrimidine-4,6-diamine (7c)
1
Gray solid; yield: 65%; IR (KBr, cm-1): 3495 (N-H), 3307 (N-H), 1651, 1281; H NMR (500
MHz, DMSO-d6) δ: 5.35 (s, 2H), 6.97 (s, 1H), 7.48 (t, J = 9.0 Hz, 1H), 7.79-7.80 (m, 1H),
8.21-8.23 (m, 1H), 8.75 (s, 1H), 9.14 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ: 100.89, 116.56
(d, J = 21.2 Hz), 116.77, 118.80, 121.03 (d, J = 21.2 Hz), 122.59, 137.91, 142.36, 149.75, 152.29,
155.10 (d, J = 252.5 Hz), 156.86.
5.8.4. N4-(3,4-dichlorophenyl)thieno[2,3-d]pyrimidine-4,6-diamine (7d)
Off-white solid; yield: 67%; IR (KBr, cm-1): 3435 (N-H), 3365 (N-H), 1648, 1269; 1H NMR (500
MHz, DMSO-d6) δ: 5.43 (s, 2H), 6.84 (s, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.75 (dd, J = 8.9, 2.5 Hz,
1H), 8.23 (d, J = 2.5 Hz, 1H) 8.72 (s, 1H), 9.01 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ:
102.71, 118.58, 120.56, 122.72, 130.59, 132.74, 134.15, 142.47, 144.18, 151.56, 154.07, 158.67.
5.8.5. N4-(3,5-dichlorophenyl)thieno[2,3-d]pyrimidine-4,6-diamine (7e)
Off-white solid; yield: 75%; IR (KBr, cm-1): 3404 (N-H), 3378 (N-H), 1645, 1291; 1H NMR (500
MHz, DMSO-d6) δ: 5.51 (s, 2H), 6.57 (s, 1H), 7.32 (t, J = 2.0 Hz, 1H), 8.22 (d, J = 2.0 Hz, 2H),
8.73 (s, 1H), 9.43 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ: 104.05, 119.92, 121.10 (2C),
125.55, 137.66 (2C), 145.52, 146.20, 152.90, 155.40, 160.01.
5.8.6. N4-(3-ethynylphenyl)thieno[3,2-d]pyrimidine-4,6-diamine (15a)
Off-white solid; yield: 70%; IR (KBr, cm-1): 3414 (N-H), 3335 (N-H), 2125
(
), 1631, 1305;
1H NMR (500 MHz, DMSO-d6) δ: 4.17 (s, 1H), 5.97 (s, 2H), 6.87 (s, 1H), 7.12 (d, J = 8.0 Hz, 1H),
7.45 (t, J = 8.1 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 8.02 (s, 1H), 9.17 (s, 1H); 13C-NMR (125 MHz,
DMSO-d6) δ: 78.92, 80.73 , 106.55, 112.43, 123.34, 124.09, 124.77, 127.85, 130.53, 141.45,
154.58, 156.15, 158.70, 160.08.

5.8.7. N4-(3-chloro-4-fluorophenyl)thieno[3,2-d]pyrimidine-4,6-diamine (15b) [38]
Off-white solid; yield: 72%; IR (KBr, cm-1): 3450 (N-H), 3388 (N-H), 1647, 1273; 1H NMR (500
MHz, DMSO-d6) δ: 5.25 (s, 2H), 6.61 (s, 1H), 7.41 (t, J = 9.1 Hz, 1H), 7.78 (dt, J = 9.1, 3.6 Hz,
1H), 8.18 (dd, J = 6.9, 2.7 Hz, 1H), 8.61 (s, 1H), 9.67 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ:
106.15, 112.04, 117.87 (d, J = 20.1 Hz), 120.22, 122.45 (d, J = 20.1 Hz), 123.98, 138.78, 154.15,
156.08, 156.52 (d, J = 251.5 Hz), 158.30, 159.83.
5.8.8. N4-(3,4-dichlorophenyl)thieno[3,2-d]pyrimidine-4,6-diamine (15c)
Off-white solid; yield: 77%; IR (KBr, cm-1): 3454 (N-H), 3373 (N-H), 1651, 1301; 1H NMR (500
MHz, DMSO-d6) δ: 5.58 (s, 2H), 6.97 (s, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.57 (dd, J = 8.6, 2.3 Hz,
1H), 8.07 (d, J = 2.5 Hz, 1H), 8.54 (s, 1H), 9.65 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ:
105.72, 111.60, 119.73, 121.84, 129.76, 131.71, 133.32, 140.96, 153.72, 155.53, 157.88, 159.22.
5.8.9. N4-(3,5-dichlorophenyl)thieno[3,2-d]pyrimidine-4,6-diamine (15d)
Off-white solid; yield: 80%; IR (KBr, cm-1): 3415 (N-H), 3358 (N-H), 1644, 1282; 1H NMR (500
MHz, DMSO-d6) δ: 5.74 (s, 2H), 6.92 (s, 1H), 7.29 (t, J = 2.1 Hz, 1H), 7.78 (d, J = 2.2 Hz, 2H),
8.45 (s, 1H), 9.63 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ: 107.25, 113.14, 120.46 (2C),
124.92, 137.03 (2C), 144.82, 155.25, 156.97, 159.41, 160.75.
5.8.10. 3-((6-Aminothieno[3,2-d]pyrimidin-4-yl)amino)benzonitrile (15e)
Gray solid; yield: 72%; IR (KBr, cm-1): 3494 (N-H), 3379 (N-H), 2243 (
), 1649, 1327; 1H
NMR (500 MHz, DMSO-d6) δ: 6.05 (s, 2H), 6.75 (s, 1H), 7.58-7.64 (m, 2H), 8.15-8.17 (m, 1H),
8.45-8.46 (m, 1H), 8.57 (s, 1H), 9.12 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) δ: 107.49, 113.38,
114.68, 121.01, 124.87, 125.82, 129.32, 132.20, 142.77, 155.52, 157.31, 159.64, 161.03.
5.9. General procedure for the preparation of 8a-e, 16a-e
The mixture of cinnamic acid derivatives (1.2 mmol) and thionyl chloride (5 mL) was refluxed for
2 h and then the thionyl chloride was evaporated under vacuum to obtain corresponding
cinnamoyl chloride. The solution of cinnamoyl chloride (1 mmol) in dry dichloromethane (1 mL)
was added dropwise to the solution of 4-dimethylaminopyridine (0.1 mmol, 12 mg) and pyridine
o
(1 mmol, 0.08 mL) in dry dichloromethane (5 mL) at 0 C under argon atmosphere. After 15

minutes, the solution of compound 7 or 15 (0.9 mmol) in dry dichloromethane (1 mL) was slowly
added. The reaction was stirred for another 2 h at r.t. Then, dichloromethane was added and
washed with HCl (1 M). The organic layer was dried over sodium sulfate and concentrated under
reduced pressure. The residue was recrystallized from methanol and water to give the final
products.
5.9.1. N-(4-((3-ethynylphenyl)amino)thieno[2,3-d]pyrimidin-6-yl)cinnamamide (8a)
Off-white solid; yield: 72%; mp: 274-276 °C; IR (KBr, cm-1): 3474 (N-H), 3387 (CON-H), 3041,
2124
(
)
, 1669 (C=O), 1614, 1339; 1H NMR (500 MHz, DMSO-d6) δ: 4.20 (s, 1H, CH), 6.87
(d, J = 15.8 Hz, 1H, H-A), 7.39-7.46 (m, 5H, H-3'', H-4'',H-5'', H-2' and H-4' ), 7,67 (d, J = 7.2 Hz,
2H, H-2'' and H-6''), 7.69 (d, J = 15.8 Hz, 1H, H-B), 7.96-8.03 (m, 1H , H-5'), 8.18-8.19 (m, 1H,
H-6'), 8.46 (s, 1H, H-2), 9.64 (s, 1H, NH), 11.90 (s, 1H, NHCO); 13C-NMR (125 MHz, DMSO-d6)
δ: 77.14, 78.23, 100.84, 114.98, 116.62, 118.70, 118.84, 119.75, 121.18, 122.21, 127.91 (2C),
129.01 (2C), 130.16, 134.31, 136.84, 141.85, 151.85, 152.67, 153.78, 161.71, 162.65; Anal.
Calcd. For C23H16N4OS: C, 69.68; H, 4.07; N, 14.13. Found: C, 69.45; H, 4.12; N, 14.27; ESI-MS
m/z: 397.5 [M+H]+.
5.9.2. N-(4-((3-chloro-2-fluorophenyl)amino)thieno[2,3-d]pyrimidin-6-yl)cinnamamide (8b)
Off-white solid; yield: 69%; mp: 255-257 °C; IR (KBr, cm-1): 3411 (N-H), 3374 (CON-H), 3072,
1649 (C=O), 1615, 1347; 1H NMR (500 MHz, DMSO-d6) δ: 6.88 (d, J = 15.8 Hz, 1H, H-A), 7.27
(t, J = 8.1 Hz, 1H, H-5'), 7.30 (s, 1H, H-5), 7.44-7.48 (m, 4H, H-3'', H-4'',H-5'' and H-6'), 7.58 (t, J
= 8.1 Hz, 1H, H-4'), 7.68 (d, J = 7.9 Hz, 2H, H-2'' and H-6''), 7.71 (d, J = 15.8 Hz, 1H, H-B), 8.33
(s, 1H, H-2), 9.63 (s, 1H, NH), 11.92 (s, 1H, NHCO); 13C-NMR (125 MHz, DMSO-d6) δ: 102.16,
110.92 (d, JC-F = 20.1 Hz), 118.64, 121.61, 121.67, 126.14, 128.01, 128.57 (2C), 129.06 (2C),
129.90, 134.30 (d, JC-F = 20.1 Hz), 135.01, 137.80, 140.76, 147.36, 149.76 (d, JC-F = 251.2 Hz),
153.59, 160.90, 162.59; Anal. Calcd. For C21H14ClFN4OS: C, 59.37; H, 3.32; N, 13.19. Found: C,
59.24; H, 3.17; N, 13.34; ESI-MS m/z: 425.3 [M+H]+.
5.9.3. N-(4-((3-chloro-4-fluorophenyl)amino)thieno[2,3-d]pyrimidin-6-yl)cinnamamide (8c)
Off-white solid; yield: 75%; mp: 270-272 °C; IR (KBr, cm-1): 3420 (N-H), 3387 (CON-H), 3025,
1669 (C=O), 1614, 1271; 1H NMR (500 MHz, DMSO-d6) δ: 6.87 (d, J = 15.7 Hz, 1H, H-A), 7.39

(s, 1H, H-5), 7.40-7.49 (m, 4H, H-3'', H-4'',H-5'' and H-5'), 7.68 (d, J = 7.9 Hz, 2H, H-2'' and
H-6''), 7.71 (d, J = 15.7 Hz, 1H, H-B), 7.78-7.81 (m, 1H, H-6'), 8.19 (dd, J = 6.8, 2.6 Hz, 1H,
H-2''), 8.46 (s, 1H, H-2), 9.65 (s, 1H, NH), 11.91 (s, 1H, NHCO); 13C-NMR (125 MHz, DMSO-d6)
δ: 103.26, 109.12 (d, JC-F = 20.0 Hz), 117.88 (d, JC-F = 20.0 Hz), 120.06, 120.76, 122.78, 128.03,
129.67 (2C), 130.16 (2C), 131.00, 136.11, 138.90, 139.80, 141.86, 148.63, 154.69, 155.79 (d, JC-F
= 252.5 Hz), 162.00, 163.24; Anal. Calcd. For C21H14ClFN4OS: C, 59.37; H, 3.32; N, 13.19.
Found: C, 59.49; H, 3.15; N, 13.23; ESI-MS m/z: 425.5 [M+H]+.
5.9.4. N-(4-((3,4-dichlorophenyl)amino)thieno[2,3-d]pyrimidin-6-yl)cinnamamide (8d)
Off-white solid; yield: 77%; mp: 322-324 °C; IR (KBr, cm-1): 3462 (N-H), 3322 (CON-H), 3012,
1670 (C=O),1620, 1338; 1H NMR (500 MHz, DMSO-d6) δ: 6.88 (d, J = 15.8 Hz, 1H, H-A), 7.42
(s, 1H, H-5), 7.44-7.49 (m, 3H, H-3'', H-4'' and H-5''), 7.61 (d, J = 8.8 Hz, 1H, H-5'), 7.67-7.69 (m,
2H, H-2'' and H-6''), 7.71 (d, J = 15.8 Hz, 1H, H-B), 8.19 (dd, J = 8.9, 2.6 Hz, 1H, H-6'), 8.31 (d, J
= 2.6, 1H, H-2'), 8.51 (s, 1H, H-2), 9.73 (s, 1H, NH), 11.93 (s, 1H, NHCO); 13C-NMR (125 MHz,
DMSO-d6) δ: 101.35, 115.77, 120.29, 121.05, 122.12, 124.31, 128.42 (2C), 129.51 (2C), 130.65,
130.71, 131.15, 134.84, 137.57, 140.44, 142.38, 151.87, 152.97, 162.46, 163.21; Anal. Calcd. For
C21H14Cl2N4OS: C, 57.15; H, 3.20; N, 12.70. Found: C, 57.42; H, 2.97; N, 12.59; ESI-MS m/z:
441.2 [M+H]+.
5.9.5. N-(4-((3,5-dichlorophenyl)amino)thieno[2,3-d]pyrimidin-6-yl)cinnamamide (8e)
Off-white solid; yield: 79%; mp: 366-368 °C; IR (KBr, cm-1): 3474 (N-H), 3337 (CON-H), 3042,
1653 (C=O), 1613, 1353; 1H NMR (500 MHz, DMSO-d6) δ: 6.86 (d, J = 15.7 Hz, 1H, H-A), 7.23
(s, 1H, H-4'), 7.23 (s, 1H, H-5), 7.45-7.47 (m, 3H, H-3'', H-4'' and H-5''), 7.67-7.69 (m, 2H, H-2''
and H-6''), 7.71 (d, J = 15.7 Hz, 1H, H-B), 8.06-8.08 (m, 2H, H-2' and H-6'), 8.53 (s, 1H, H-2),
9.72 (s, 1H, NH), 11.93 (s, 1H, NHCO); 13C-NMR (125 MHz, DMSO-d6) δ: 100.75, 115.47,
118.38 (2C), 119.75, 121.49, 128.03 (2C), 129.16 (2C), 130.35, 133.85 (2C), 134.38, 137.33,
142.11, 142.31, 151.45, 152.43, 162.16, 162.84; Anal. Calcd. For C21H14Cl2N4OS: C, 57.15; H,
3.20; N, 12.70. Found: C, 57.32; H, 3.31; N, 12.47; ESI-MS m/z: 441.5 [M+H]+.
5.9.6.(E)-N-(4-((3,4-dichlorophenyl)amino)thieno[2,3-d]pyrimidin-6-yl)-3-(4-hydroxyphenyl)acr
ylamide (8f)

Off-white solid; yield: 77%; mp: 353-355 °C; IR (KBr, cm-1): 3573 (O-H), 3453 (N-H), 3312
(CON-H), 3021, 1668 (C=O), 1615, 1287; 1H NMR (500 MHz, DMSO-d6) δ: 6.75 (d, J = 15.9 Hz,
1H, H-A), 6.84 (d, J = 8.5 Hz, 2H, H-3'' and H-5''), 7.36-7.39 (m, 3H, H-5', H-2'' and H-6''), 7.44
(s, 1H, H-5), 7.50-7.56 (m, 2H, H-B and H-6'), 7.95 (d, J = 1.8 Hz, 1H, H-2'), 8.61 (s, 1H, H-2),
13
8.72 (s, 1H, OH), 9.02 (s, 1H, NH), 11.74 (s, 1H, NHCO); C-NMR (125 MHz, DMSO-d6) δ:
104.55, 115.33, 115.88 (2C), 119.48, 119.82, 120.81, 125.02, 125.21, 125.31, 130.72 (2C),
133.29, 135.65, 140.89, 141.15, 148.83, 149.92, 152.35, 157.01, 163.12; Anal. Calcd. For
C21H14Cl2N4O2S: C, 55.15; H, 3.09; N, 12.25. Found: C, 54.89; H, 3.24; N, 12.07; ESI-MS m/z:
458.4 [M+H]+.
5.9.7.(E)-N-(4-((3,4-dichlorophenyl)amino)thieno[2,3-d]pyrimidin-6-yl)-3-(4-methoxyphenyl)ac
rylamide (8g)
Off-white solid; yield: 69%; mp: 331-323 °C; IR (KBr, cm-1): 3425 (N-H), 3352 (CON-H), 3042,
1656 (C=O), 1612, 1339; 1H NMR (500 MHz, DMSO-d6) δ: 3.81 (s, 3H, CH3), 6.72 (d, J = 15.6
Hz, 1H, H-A), 7.02 (d, J = 8.3 Hz, 2H, H-3'' and H-5''), 8.38 (s, 1H, H-5), 7.59-7.67 (m, 4H, H-B,
H-5', H-2'' and H-6''), 7.74 (dd, J = 8.9, 2.6 Hz, 1H, H-6'), 8.31 (d, J = 2.6 Hz, 1H, H-2'), 8.49 (s,
1H, H-2), 9.71 (s, 1H, NH), 11.84 (s, 1H, NHCO); 13C-NMR (125 MHz, DMSO-d6) δ: 55.33,
100.50, 114.52 (2C), 115.31, 117.09, 120.57, 121.62, 123.80, 126.94, 129.71 (2C), 130.28,
130.68, 137.29, 139.99, 141.78, 151.34, 152.43, 160.96, 161.95, 163.06; Anal. Calcd. For
C22H16Cl2N4O2S: C, 56.06; H, 3.42; N, 11.89. Found: C, 56.31; H, 3.29; N, 11.95; ESI-MS m/z:
471.7 [M+H]+.
5.9.8.(E)-N-(4-((3,5-dichlorophenyl)amino)thieno[2,3-d]pyrimidin-6-yl)-3-(4-methoxyphenyl)ac
rylamide (8h)
Off-white solid; yield: 69%; mp: 341-343 °C; IR (KBr, cm-1): 3452 (N-H), 3315 (CON-H), 3031,
1672 (C=O), 1617, 1337; 1H NMR (500 MHz, DMSO-d6) δ: 3.72 (s, 3H, CH3), 6.78 (d, J = 15.9
Hz, 1H, H-A), 6.86 (d, J = 8.5 Hz, 2H, H-3'' and H-5''), 7.14 (s, 1H, H-5), 7.35-7.45 (m, 4H, H-B,
H-4', H-2'' and H-6''), 7.48 (s, 2H, H-2' and H-6' ), 8.59 (s, 1H, H-2), 9.23 (s, 1H, NH), 11.57 (s,
1H, NHCO); 13C-NMR (125 MHz, DMSO-d6) δ: 55.35, 99.97, 100.26, 104.56, 114.28 (2C),
115.34, 119.54 (2C), 127.70, 130.73 (2C), 135.65, 141.13 (2C), 144.58, 149.91, 152.49, 157.01,

160.10, 161.74, 163.26; Anal. Calcd. For C22H16Cl2N4O2S: C, 56.06; H, 3.42; N, 11.89. Found: C,
55.91; H, 3.57; N, 11.72; ESI-MS m/z: 471.4 [M+H]+.
5.9.9. N-(4-((3-ethynylphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)cinnamamide (16a)
Off-white solid; yield: 75%; mp: 188-190 °C; IR (KBr, cm-1): 3423 (N-H), 3395 (CON-H), 3028,
2134
(
)
, 1664 (C=O), 1614, 1286; 1H NMR (500 MHz, DMSO-d6) δ: 4.20 (s, 1H, CH), 6.53
(d, J = 15.8 Hz, 1H, H-A), 6.68 (d, J = 7.6, 1H, H-4'), 6.90 (d, J = 7.8, 1H, H-6'), 7.03 (t, J = 7.8 Hz,
1H, H-5'), 7.23-7.27 (m, 4H, H-5, H-3'', H-4'' and H-5''), 7.32 (s, 1H, H-2'), 7.36 (d, J = 15.9 Hz,
1H, H-B), 7.41-7.43 (m, 2H, H-2'' and H-6''), 8.46 (s, 1H, H-5), 9.14 (s, 1H, NH), 11.32 (s, 1H,
13
NHCO); C-NMR (125 MHz, DMSO-d6) δ: 77.14, 78.92, 107.99, 111.86, 119.63, 121.65,
122.30, 123.07, 126.04, 128.80 (2C), 129.13 (2C), 129.49, 132.30, 133.80, 139.67, 140.96,
144.95, 152.92, 155.04, 156.94, 162.83; Anal. Calcd. For C23H16N4OS: C, 69.68; H, 4.07; N,
14.13. Found: C, 69.82; H, 4.15; N, 13.78; ESI-MS m/z: 397.2 [M+H]+.
5.9.10. N-(4-((3-chloro-4-fluorophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)cinnamamide (16b)
Off-white solid; yield: 75%; mp: 224-226 °C; IR (KBr, cm-1): 3470 (N-H), 3382 (CON-H), 3052,
1
1680 (C=O), 1614, 1281; H NMR (500 MHz, DMSO-d6) δ: 7.27 (d, J = 15.8 Hz, 1H, H-A),
7.39-7.48 (m, 5H, H-5, H-3'', H-4'', H-5'' and H-5'), 7.62-7.66 (m, 3H, H-B, H-2'' and H-6''),
7.72-7.75 (m, 1H, H-6'), 8.12 (dd, J = 6.8, 2.6 Hz, 1H, H-2''), 8.60 (s, 1H, H-2), 8.86 (s, 1H, NH),
13
10.95 (s, 1H, NHCO); C-NMR (125 MHz, DMSO-d6) δ: 109.41, 113.24, 117.43 (d, JC-F = 20
Hz), 120.39, 121.10, 122.59 (d, JC-F = 20 Hz), 124.01, 129.43 (2C), 130.30 (2C), 131.49, 135.42,
138.77, 142.42, 146.33, 154.29, 155.51 (d, JC-F = 252.5 Hz), 156.57, 157.35, 164.25; Anal. Calcd.
For C21H14ClFN4OS: C, 59.37; H, 3.32; N, 13.19. Found: C, 59.14; H, 3.49; N, 13.35; ESI-MS
m/z: 425.7 [M+H]+.
5.9.11. N-(4-((3,4-dichlorophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)cinnamamide (16c)
Off-white solid; yield: 75%; mp: 186-188 °C; IR (KBr, cm-1): 3410 (N-H), 3352 (CON-H), 3014,
1
1664 (C=O), 1620, 1341; H NMR (500 MHz, DMSO-d6) δ: 6.82 (d, J = 16.0 Hz, 1H, H-A),
7.27-7.39 (m, 4H, H-5, H-3'', H-4'' and H-5''), 7.41 (d, J = 8.4 Hz, 1H, H-5'), 7.51-7.57 (m, 4H,
H-B, H-6', H-2'' and H-6''), 7.97-7.99 (m, 1H, H-2'), 8.61 (s, 1H, H-2), 9.87 (s, 1H, NH), 11.45 (s,
1H, NHCO); 13C-NMR (125 MHz, DMSO-d6) δ: 108.81, 112.64, 119.65, 120.50, 121.69, 128.83

(2C), 129.16, 129.71 (2C), 130.98, 131.11, 131.77, 134.83, 140.60, 141.82, 145.73, 153.70,
155.96, 156.75, 163.65; Anal. Calcd. For C21H14Cl2N4OS: C, 57.15; H, 3.20; N, 12.70. Found: C,
57.27; H, 3.52; N, 12.47; ESI-MS m/z: 441.2 [M+H]+.
5.9.12. N-(4-((3,5-dichlorophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)cinnamamide (16d)
Off-white solid; yield: 78%; mp: 217-219 °C; IR (KBr, cm-1): 3412 (N-H), 3385 (CON-H), 3017,
1659 (C=O), 1614, 1336; 1H NMR (500 MHz, DMSO-d6) δ: 6.80 (d, J = 16.1 Hz, 1H, H-A), 7.46
(d, J = 2.6 Hz, 1H, H-4'), 7.60-7.62 (m, 4H, H-5, H-3'', H-4'' and H-5''), 7.74-7.80 (m, 5H, H-2',
H-6', H-B, H-2'' and H-6''), 8.79 (s, 1H, H-2), 9.82 (s, 1H, NH), 11.68 (s, 1H, NHCO); 13C-NMR
(125 MHz, DMSO-d6) δ: 114.30, 116.69, 119.00 (2C), 121.70, 122.03, 127.74 (2C), 128.97 (2C),
129.78, 130.52, 133.79 (2C), 134.87, 140.64, 141.80, 152.56, 153.20, 154.51, 163.84; Anal.
Calcd. For C21H14Cl2N4OS: C, 57.15; H, 3.20; N, 12.70. Found: C, 56.95; H, 3.08; N, 12.92;
ESI-MS m/z: 441.5 [M+H]+.
5.9.13. N-(4-((3-cyanophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)cinnamamide (16e)
Off-white solid; yield: 70%; mp: 282-284 °C; IR (KBr, cm-1): 3417 (N-H), 3327 (CON-H), 3047,
2258 (
), 1681 (C=O), 1624, 1347; 1H NMR (500 MHz, DMSO-d6) δ: 7.33 (d, J = 15.9 Hz,
1H, H-A), 7.41-7.47 (m, 3H, H-3'', H-4'' and H-5''), 7.60-7.66 (m, 6H, H-5, H-4', H-5', H-2'', H-6''
and H-B), 8.09-8.10 (m, 1H, H-6'), 8.40 (s, 1H, H-2'), 8.60 (s, 1H, H-5), 10.56 (s, 1H, NH), 10.78
(s, 1H, NHCO); 13C-NMR (125 MHz, DMSO-d6) δ: 111.42, 114.29, 115.61, 118.15, 118.74,
121.43, 124.98, 126.68, 127.41, 127.81, 129.02, 130.07, 134.76, 139.51, 140.85, 152.19, 155.17,
163.82, 171.57; Anal. Calcd. For C22H15N5OS: C, 66.48; H, 3.80; N, 17.62. Found: C, 66.32; H,
3.69; N, 17.87; ESI-MS m/z: 398.1[M+H]+.
5.9.14.(E)-N-(4-((3-chloro-4-fluorophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-3-(4-methoxyphe
nyl)acrylamide (16f)
Off-white solid; yield: 74%; mp: 200-202 °C; IR (KBr, cm-1): 3415 (N-H), 3321 (CON-H), 3037,
1674 (C=O), 1618, 1270; 1H NMR (500 MHz, DMSO-d6) δ: 3.80 (s, 3H, CH3), 6.89 (d, J = 15.9
Hz, 1H, H-A), 6.96 (d, J = 8.7 Hz, 2H, H-3'' and H-5''), 7.11-7.15 (m, 1H, H-6'), 7.50-7.55 (m, 4H,
H-5, H-5', H-2'' and H-6''), 7.57 (d, J = 15.9 Hz, 1H, H-B), 7.90 (dd, J = 4.9, 1.9 Hz, 1H, H-2''),
8.64 (s, 1H, H-2), 9.18 (s, 1H, NH), 11.67 (s, 1H, NHCO); 13C-NMR (125 MHz, DMSO-d6) δ:

57.63, 106.53, 116.59 (2C), 117.60, 118.56 (d, JC-F = 21.2 Hz), 121.32, 121.71, 123.74 (d, JC-F
21.2 Hz), 124.96, 129.58, 132.83 (2C), 137.93, 140.19, 143.45, 152.22, 154.75, 157.03 (d, JC-F
=
=
251.25 Hz), 159.25, 163.78, 165.16; Anal. Calcd. For C22H16ClFN4O2S: C, 58.09; H, 3.55; N,
12.32. Found: C, 58.24; H, 3.62; N, 12.14; ESI-MS m/z: 455.1 [M+H]+.
5.10. MTT assay
Cytotoxicity assays were performed using the MTT assay method. To evaluate the cytotoxic
effects of the final compounds eleven cancer cell lines were used provided by the National Cell
Bank of Iran (Pastor Institute, Tehran, Iran). Other materials and reagents are purchased from
Sigma. To perform this assay, firstly the cells were cultured in RPMI-1640 medium and DMEM
containing 10% FBS (Gibco, Milano, Italy).
Then a cell suspension of 8,000 cells was poured into 96 well plate and incubated in a humidified
incubator containing 5% CO2 at 37 °C for 24 hours. Subsequently, different concentrations of
compounds dissolved in DMSO were added to these plates and incubated for 48 hours under the
above
conditions.
After
incubation,
the
solution
containing
5%
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was added to all the
wells and then incubated for another 4 hours.
Color intensity was recorded at 570 nm by Bio-Rad microplate reader (Model 680) and the
percentage of live cells was obtained. IC50 was determined based on the drawn curve (cell survival
percentage in different concentrations of the compounds). In this assay, 0.1% DMSO and erlotinib
were used as negative and positive controls, respectively.
5.11. Apoptosis assay
To determine the induction of apoptosis, the most effective concentration IC50 of (8e and 8g) was
selected and their activities were evaluated on HeLa cell line. At first, the cells were incubated in a
6-well plate at 37 °C for 24 hours, then treated with the IC50 concentration of (8e and 8g)
compounds and incubated again for 48 hours, the following steps were carried out after
incubation. The cells were initially trypsinized and then rinsed with Phosphate buffered saline
(pbs), then centrifuged at 1200 rpm for 3 minutes and 500 µL of the binding buffer was added to
the resulting deposits (cells), followed by slow addition of 5 µL of Annexin V-APC and PI to it

and gently mixed. In the next step, the samples were incubated for 10 to 15 minutes at room
temperature in dark, and finally, the cellular analysis was done by flow cytometer (FACS Calibur
Bectone-Dickinson). In this experiment, erlotinib was used as the positive control.
5.12. Cell-Cycle Analysis
The Hela cells were treated with IC50 concentration of compounds (8e and 8g) for 48 h. After
treatment, the cells were trypsinized and rinsed twice with PBS, then centrifuged at 1000 rpm for 5
minutes and the resulting cells were incubated with PBS and fixed in ice-cold 70% ethanol. After
that the cells were washed with PBS, resuspended in RNase A (0.1 mg/ml) and incubated for 5h.
In the following, the cells were stained with PI (50 mg/ml) and incubated for 15 minutes. The cells
were analyzed by Novocyte flow cytometer (ACEA Biosciences) and the cell cycle distributions
were calculated by NovoExpress 1.1.0 software.
5.13. Western blotting
To perform western blot analysis firstly, HeLa cells were treated with IC50 concentrations of 8e
and erlotinib then incubated for 0, 3, 12, 24, and 48 h. The cells were lysed by 100 mL of Lysis
buffer and centrifuged at 1200 rpm for 10 min at 4°C , then the obtained total proteins (20 µg)
were loaded and electrophoresed on 12% SDSPAGE and transferred to a nitrocellulose membrane
(0.45 mm) for 1.5 h at 100 V and blocked with 5% blocking buffer. After that the membrane was
incubated with antibodies at 4°C for 12 h. Beta-actin was used as a western blot loading control.
The proteins were detected using enhanced chemiluminescence (ECL) method.
5.14. Docking studies
To examine the best poses and interactions of compounds in the active site of the receptor, the
AutoDoc 4.2.1 program was used. The crystallographic structure of EGFR receptor with erlotinib
(1M17) was taken from PDB. According to the biological results, the most potent compound (8e)
was selected for evaluation. In order to prepare and optimize the ligand, MarvinSketch 15.8.1,
2015 was used. In the next step, for preparation of macromolecules, the molecules of water and
ligand were removed using the discovery studio software. Subsequent steps were taken on the
resulting PDB file including the addition of polar hydrogens and electric charges in the auto dock

program and the grid box was determined around ATP binding site. The results of docking are
analyzed based on the best binding mode and energy by the discovery studio program and are
shown in Figures 2 and 3.
Acknowledgement
This work was supported and funded by Tehran University of Medical Sciences (TUMS) Grant
no.; 96-04-33-36978; and National Institute for Medical Research Development (NMIAD) Grant
no. 962567.
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Captions:
Figure 1. Design strategy of final compounds 8a-h and 16a-f.
Figure 2. In vitro cell cytotoxicity: MTT assay result of a panel of cancer lines.
Figure 3. Flow cytometry analysis of HeLa cells treated with compounds 8e and 8g. (A) Nontreated cells as negative
control group; (B) treated with erlotinib as the positive control; (C) treated with 8e at IC50 concentration; (D) treated
with 8g at IC50 concentration; (E) treated with DMSO.
Figure 4. Effect of compounds 8e and 8g on cell cycle analysis of HeLa cells by flow cytometry analysis. (A)
Non-treated cells as control group; (B) treated with 8e at IC50 concentration; (C) treated with 8g at IC50 concentration;
(D) quantitative measurement of cell cycle analysis.
Figure 5. Western blot analysis of compound (8e) on EGFR and downstream effectors ERK1/2 and AKT
autophosphorylation in HeLa cell line and compared with the positive control (Erlotinib).
Figure 6. Binding mode of compound 8e with EGFR
Figure 7. Atom numbering of target compounds.
Scheme 1. a) Malononitrile, DMF, Et3N, 50 oC, 2 h; b) formic acid, H2SO4, 90 oC, 5 h; c) H2SO4, HNO3, 50 oC, 1.5 h;
o
o
d) POCl3, 90 C, overnight; e) isopropanol, aniline derivatives, reflux, 3 h; f) Fe, NH4Cl, EtOH, 50 C, 1 h; g) (i)
different cinnamic acid, SOCl2, (ii) DMAP, pyridine, DCM, 2 h, r.t.
Scheme 2. a) Formamide, reflux; b) H2SO4, HNO3, 50 oC, 1.5h; c) POCl3, 90 oC, overnight; d) isopropanol, aniline
o
derivatives, reflux, 3h; e) Fe, NH4Cl, EtOH, 50 C, 1h; f) (i) different cinnamic acid, SOCl2, (ii) DMAP, pyridine,
DCM, 2h, r.t.

Table 1
In vitro antiproliferative effects (IC50, µM) of compounds 8a-h and 16a-f against PC3,
MDA-MB-231, MCF7, HepG2, A549 and HeLa cell lines.a
MDA-MB
Compound
X
Y
PC3
MCF7
HepG2
A549
HeLa
-231
0.40±0.4
5.05±0.1
38.40±0.3
58.10±0.9
10.05±0.5
4.64±0.1
8a
0.40±0.6
3.93±0.1
93.90±0.1
44.60±0.7
26.24±0.3
34.85±0.7
8b
0.70±0.2
0.48±0.4
7.60±0.8
4.40±0.4
14.30±0.5
15.30±0.2
22.80±0.5
30.10±0.5
11.20±0.8
7.60±0.5
2.14±0.7
0.43±0.1
8c
8d
0.39±0.4
11.50±1.1
1.98±0.3
5.80±1.0
21.80±0.3
0.04±0.05
0.004±0.001
8e
0.30±0.4
0.79±0.4
60.60±0.3
42.90±0.1
30.60±0.2
34.31±0.6
3.32±0.5
24.82±0.3
8f
16.60±0.1 20.10±0.1
0.033±0.01
8g
0.45±0.9
0.70±0.3
1.97±0.3
2.34±1.1
75.6±0.1
75.6±0.3
41.80±0.8
15.40±0.2
21.94±0.3
26.24±0.2
43.26±0.8
20.53±0.7
8h
16a

0.20±0.6
0.26±0.2
2.10±0.1
2.41±0.8
30.90±0.1
>100
10.60±0.3
13.40±0.8
6.67±0.1
2.81±0.3
16b
16c
19.45±0.2
10.66±0.6
0.22±0.5
0.54±0.2
1.00±0.6
1.04±0.6
>100
>100
10.70±0.8
15.90±0.1
12.54±0.3
17.17±0.1
3.21±0.4
7.22±0.5
16d
16e
0.10±0.2
7.00±0.1
2.60±0.3
8.30±0.1
29.10±0.4
11.70±0.1
13.9±0.5
28.8±0.3
22.74±0.2
3.80±0.7
20.58±0.5
7.48±0.2
16f
-
-
Erlotinib
aValues were the means of three replicates±standard deviation (SD).