Irreversible inhibitors of the epidermal growth factor receptor: Thienopyrimidine core with α,β-unsaturated amide side chain

Article abstract of DOI:10.1002/ardp.201400098

Overexpression of epidermal growth factor receptor (EGFR) tyrosine kinases has been found in a variety of cancers such as breast, ovarian, colon, and non-small-cell lung cancers, which is associated with poor prognosis in patients. In an effort to find effective irreversible inhibitors of the EGFR tyrosine kinase family (mainly HER2), two series of HER2 tyrosine kinase inhibitors with thieno[3,2-d]pyridine and thieno[2,3-d]pyridine as central part and with a basic α,β-unsaturated amide side chain were developed. The α,β-unsaturated amide side chain (the Michael acceptor) at the 6-position, which forms a covalent bond to Cys773 located in the ATP binding pocket of the EGFR enzyme, is a major factor in the generation of irreversible inhibition. In our study, thienopyrimidine instead of quinazoline was used as the central structure, and different substituents were introduced at the 4-position to investigate the structure-activity relationships. The thieno[2,3-d]pyrimidine derivatives 16a-d showed potent HER2 enzyme inhibition and anti-proliferative activity against SK-BR-3 cells. Especially, (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)thieno[2,3-d] pyrimidin-6-yl)-4-(dimethylamino)but-2-enamide 16d was identified as a potential irreversible HER2 inhibitor. Both its catalytic enzyme activity profile and its cellular efficacy were found to be superior to those of the marketed drug lapatinib.

Full text of DOI:10.1002/ardp.201400098

552
Arch. Pharm. Chem. Life Sci. 2014, 347, 552–558
Full Paper
Irreversible Inhibitors of the Epidermal Growth Factor
Receptor: Thienopyrimidine Core with a,b-Unsaturated
Amide Side Chain
Xiu L. Yang1, Tian C. Wang2, Sen Lin1, and Hou X. Fan2*
1
Department of Chemistry, Nanchang University, Nanchang, China
Shanghai Sun-sail Pharmaceutical Science & Technology Co., Ltd., Shanghai, China
2
Overexpression of epidermal growth factor receptor (EGFR) tyrosine kinases has been found in a
variety of cancers such as breast, ovarian, colon, and non-small-cell lung cancers, which is associated
with poor prognosis in patients. In an effort to find effective irreversible inhibitors of the EGFR
tyrosine kinase family (mainly HER2), two series of HER2 tyrosine kinase inhibitors with thieno[3,2-d]-
pyridine and thieno[2,3-d]pyridine as central part and with a basic a,b-unsaturated amide side chain
were developed. The a,b-unsaturated amide side chain (the Michael acceptor) at the 6-position, which
forms a covalent bond to Cys773 located in the ATP binding pocket of the EGFR enzyme, is a major
factor in the generation of irreversible inhibition. In our study, thienopyrimidine instead of
quinazoline was used as the central structure, and different substituents were introduced at the
4-position to investigate the structure–activity relationships. The thieno[2,3-d]pyrimidine derivatives
16a–d showed potent HER2 enzyme inhibition and anti-proliferative activity against SK-BR-3 cells.
Especially, (E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)thieno[2,3-d]pyrimidin-6-yl)-4-(dimethyl-
amino)but-2-enamide 16d was identified as a potential irreversible HER2 inhibitor. Both its catalytic
enzyme activity profile and its cellular efficacy were found to be superior to those of the marketed
drug lapatinib.
Keywords: HER2 / Irreversible inhibitor / Protein tyrosine kinases / Thieno[3,2-d]pyridine / Thieno[2,3-d]pyridine
Received: March 12, 2014; Revised: April 19, 2014; Accepted: April 22, 2014
DOI 10.1002/ardp.201400098
Introduction
(ErbB4). Overexpression of EGFR tyrosine kinases has been
found in a variety of cancers such as breast, ovarian, colon,
and non-small-cell lung cancers (NSCLC), which is associated
with poor prognosis in patients [3, 4].
Protein tyrosine kinases (PTKs) play important roles in signal
transduction pathways that regulate numerous cellular
functions, including proliferation, differentiation, migration,
apoptosis, and angiogenesis. Because signal transduction
pathways are up-regulated in many tumor cells, inhibitors of
PTKs that target these up-regulated pathways are attractive
candidates for cancer treatment [1, 2]. Targeting the epidermal
growth factor receptor (EGFR) family of receptor tyrosine
kinases (RTKs) represents one such therapeutic approach. The
EGFR tyrosine kinase family consists of four members: EGFR
(HER1 or ErbB1), HER2 (ErbB2 or neu), HER3 (ErbB3), and HER4
The EGFR inhibitors gefitinib [5] (IressaTM) and erlotinib [6]
(TarcevaTM
) as well as the dual EGFR/HER2 inhibitor
lapatinib [7] (TykerbTM) (Fig. 1) are FDA-approved cancer
drugs that are effective against multiple solid tumor
cancers [8]. However, early trials of these drugs usually have
been conducted using extended multiple-dosing schedules
to achieve and maintain the required plasma levels. In order
to achieve highly effective cancer drugs targeting the EGFR
tyrosine kinase family, many irreversible EGFR tyrosine
kinase inhibitors have been discovered in recent years, such
as canertinib [9] (CI-1033), afatinib [10, 11] (BIBW-2992), and
Correspondence: Prof. Sen Lin, Department of Chemistry, Nanchang
University, Nanchang 330031, China
E-mail: lin.596286@163.com
Fax: þ86 21 50278493
aAdditional correspondence: Prof. Hou X. Fan, E-mail: hxfan@sun-sail.cn
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2014, 347, 552–558
Thienopyridine EGFR Inhibitors
553
Figure 1. Reversible and irreversible inhibitors targeting the EGFR tyrosine kinase family.
neratinib [12] (HKI-272) (Fig. 1). Three significant advantages
of the second-generation irreversible EGFR TKIs over the first-
generation reversible EGFR TKIs are summarized as follows.
First, an irreversible inhibitor has prolonged circulating
blood levels in order to achieve a sustained antitumor effect.
Second, when the target enzyme is deactivated by covalent
bond formation, the biological effects should persist even
after the drug leaves the circulation. Third, irreversible
inhibition resulting from a covalent interaction with an
active-site cysteine residue could serve as a mechanism to
achieve better selectivity [13].
EGFR enzyme, the Michael acceptor, which is a major factor in
the production of irreversible inhibition [14]. In an effort to
find effective irreversible inhibitors of EGFR tyrosine kinase
family (mainly HER2), two series of HER2 tyrosine kinase
inhibitors, thieno[3,2-d]pyridine and thieno[2,3-d]pyridine as
central part, with basic a,b-unsaturated amide side chain
were developed (Fig. 2). In our study, thienopyrimidine
instead of quinazoline was used as the central structure,
and different substituents were introduced at 4-position to
investigate the structure–activity relationships (SAR).
Looking into the chemical structure of BIBW-2992 and HKI- Results and discussion
272 (Fig. 1), each of them has a a,b-unsaturated amide side
chain (the Michael acceptor) at the 6-position, which forms a
covalent bond to Cys773 located in the ATP binding pocket of
To examine the potential of thienopyrimidine derivatives for
use as a HER2 kinase inhibitor, target compounds were
Figure 2. Design of substituted thienopyr-
imidine derivatives as irreversible inhibitors of
the EGFR tyrosine kinase family.
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X. L. Yang et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 552–558
evaluated in HER2 kinase activity assays with lapatinib as
reference compound. As shown in Table 1, thieno[2,3-d]-
pyrimidine derivatives 16a–d showed much more potent
in vitro enzymatic inhibition than thieno[3,2-d]pyrimidine
derivatives 14a–g, maybe because thieno[2,3-d]pyrimidine
scaffold has a stronger binding affinity with the receptor
than thieno[3,2-d]pyrimidine scaffold. Compound 16d
exhibited the most potent inhibition among the target
compounds with IC50 values of 7 nM, while changing pyridyl
(16d) to fluorinephenyl (16c) decreased the activity. In
addition, compounds 16c and 16d displayed more potent
activity than compounds 16a and 16b, suggesting that a
long side chain at 4-position of thieno[2,3-d]pyrimidine
scaffold is superior to a short side chain in HER2 inhibition
activity.
Compounds 16a–d were further subjected to in vitro anti-
proliferative activity assay against breast cancer cell line
SK-BR-3. As shown in Table 2, all compounds showed more
potent activity than lapatinib. On the other hand, a good
correlation was observed between the catalytic enzyme
activity profile and the cellular efficacy of these derivatives.
According to the results of HER2 kinase enzyme inhibition
and SK-BR-3 cellular anti-proliferative assay, compound 16d
was proved to be a potent HER2 inhibitor, with IC50 ¼ 7 nM for
HER2 enzyme inhibition and IC50 ¼ 0.13 mM against SK-BR-3
cellular proliferation.
Table 1. Enzymatic inhibition assay results of target compounds.
a)
Structure
(R)
Molecular
weight
HER2 IC50
Compound
14a
(mM)
450.33
501.58
>3
>3
14b
14c
484.58
>3
14d
14e
405.88
501.58
>3
2.191
14f
512.00
495.00
2.469
Conclusion
14g
>3
In conclusion, two series of HER2 tyrosine kinase inhibitors,
thieno[3,2-d]pyridine and thieno[2,3-d]pyridine as central
part, with basic a,b-unsaturated amide side chain were
developed. Thieno[2,3-d]pyrimidine derivatives 16a–d showed
potent HER2 enzyme inhibition and anti-proliferative activity
against SK-BR-3 cells. Especially, compound (E)-N-(4-((3-chloro-
4-(pyridin-2-ylmethoxy)phenyl)amino)thieno[2,3-d]pyrimidin-
6-yl)-4-(dimethylamino)but-2-enamide 16d was identified as a
potential irreversible HER2 inhibitor. Both catalytic enzyme
activity profile and the cellular efficacy were found to be
superior to marketed drug lapatinib. Further, pharmaco-
kinetic profiles and in vivo anti-tumor studies of compound 16d
are ongoing in our laboratory.
16a
16b
450.33
405.88
0.227
0.029
16c
16d
512.00
0.024
495.00
581.06
0.007
0.017
Experimental
Lapatinib
Compound (E)-4-bromobut-2-enoyl chloride 7 was synthesized
from (E)-but-2-enoic acid according to the known method [15,
16], and the synthesis of compound thieno[3,2-d]pyrimidin-
4(3H)-one 8 has also been reported [17]. As shown in Scheme 1,
treatment of compound 8 with concentrated nitric acid and
sulfuric acid provided nitro product 6-nitrothieno[3,2-d]pyrimi-
din-4(3H)-one 9, chlorination of 9 with phosphoryl chloride
afforded the chloride 4-chloro-6-nitrothieno[3,2-d]pyrimidine
a)
IC50 values were obtained by Logit method based on the data
obtained from three separate experiments and expressed as
means ꢀ SD.
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Table 2. Anti-proliferative effects of compounds 16a–d on cell line
of SK-BR-3.
6-Nitrothieno[3,2-d]pyrimidin-4(3H)-one (9)
A solution of thieno[3,2-d]pyrimidin-4(3H)-one 8 (3.6 g, 24 mmol)
in concentrated sulfuric acid (47 mL) was stirred at 0°C; mixed
acid (fuming nitric acid/concentrated sulfuric acid ¼ 1/5, 5.7 mL)
was slowly added dropwise, then stirred for 20min for additional
mixed acid solution (1.9 mL). The reaction solution was poured into
ice water (130mL) after stirring for 40min and pH was adjusted
to 3–4 with saturated sodium carbonate solution. The solid was
washed with water to give a white solid 6-nitrothieno[3,2-d]-
pyrimidin-4(3H)-one 9 (3.64 g, 78%). 1H NMR (400MHz, CDCl3):
d 8.87 (s, 1H), 8.31 (s, 1H).
a)
SK-BR-3 IC50
Compound
16a
Structure (R)
(mM)
0.45
4-Chloro-6-nitrothieno[3,2-d]pyrimidine (10)
A
mixture of 6-nitrothieno[3,2-d]pyrimidin-4(3H)-one 9 (7 g,
35.5 mmol) and POCl3 (50 mL) was stirred at refluxing for 3 h.
Excess POCl3 was removed on the rotoevaporator and the residue
was added to ice water; the solvent of the filtrate was removed
and dried in vacuo to give chloride 4-chloro-6-nitrothieno[3,2-d]-
pyrimidine 10 (6.67 g, 87%) as a yellow solid. The compound was
used directly in the next step without further purification.
1H NMR (400 MHz, DMSO-d6): d 9.72 (s, 1H), 9.26 (s, 1H).
16b
16c
0.20
0.45
N-(3-Chloro-4-fluorophenyl)-6-nitrothieno[3,2-d]pyrimidin-
4-amine (11d)
A mixture of 4-chloro-6-nitrothieno[3,2-d]pyrimidine 10 (2.0 g,
9.28 mmol) and 3-chloro-4-fluoroaniline (1.62 g, 11.13 mmol)
in isopropanol (30 mL) was stirred at reflux for 3.5 h. The solid
was collected and washed with ether to give N-(3-chloro-4-
fluorophenyl)-6-nitrothieno[3,2-d]pyrimidin-4-amine 11d (2.91,
94%) as a yellow powder. The compound was used directly in
the next step without further purification. 1H NMR (300 MHz,
DMSO-d6): d 10.67 (s, 1H), 9.47 (s, 1H), 8.80 (s, 1H), 8.13
(dd, J1 ¼ 2.7 Hz, J2 ¼ 3.9 Hz, 1H), 7.73–7.78 (m, 1H), 7.47 (t,
J ¼ 9.2 Hz, 1H).
16d
0.13
0.49
Lapatinib
–
a)
IC50 values were obtained by Logit method based on the data
obtained from three separate experiments and expressed as
means ꢀ SD.
N-4-(3-Chloro-4-fluorophenyl)thieno[3,2-d]pyrimidine-4,6-
diamine (12d)
A mixture of N-(3-chloro-4-fluorophenyl)-6-nitrothieno[3,2-d]pyr-
imidin-4-amine 11d (3.24 mg, 1 mmol), iron (279 mg, 5 mmol),
and acetic acid (2 mL) in CH3CH2OH (30 mL) was mechanically
stirred at reflux for 3 h. The boiling mixture was filtered, the solid
was washed with hot CH3CH2OH, the solvent was removed from
filtrate, and evaporation gave a solid, which was chromato-
graphed on silica gel, eluting with CH2Cl2/MeOH (100:1) to give a
yellow solid N-4-(3-chloro-4-fluorophenyl)thieno[3,2-d]pyrimidine-
4,6-diamine 12d (124 mg, 42%). 1H NMR (400 MHz, DMSO-d6):
d 9.66 (s, 1H), 8.59 (s, 1H), 8.16 (t, J ¼ 3.4 Hz, 1H), 7.75 (t, J ¼ 3.8 Hz,
1H), 7.39 (t, J ¼ 9.2 Hz, 1H), 6.59 (s, 1H), 5.24 (s, 2H); ESI-MS:
m/z ¼ 295.1 [MþH]þ.
10, which coupled with appropriate anilines to give compounds
11a–g, and further reduction of 11a–g with iron/ammonium
chloride gave the corresponding amines 12a–g as important
intermediates. Condensation of 12a–g with 7 yielded amines
13a–g; target compounds 14a–g were obtained by the treatment
of 13a–g with dimethylamine. Compounds 16a–d were synthe-
sized according to the similar method to get compounds 14a–g.
Structures of the desired compounds were confirmed by 1H NMR
and LC–MS or HR-MS.
General method for chemistry
All reagents and solvents were used as purchased from
commercial sources. Flash chromatography was performed using
200–300 mesh silica gel. All reactions were monitored by TLC,
ESI-MS and recorded on a Kratos MS-80 spectrometer. HRMS
was performed on a Bruker Maxis mass spectrometer. Nuclear
magnetic resonance (NMR) spectroscopy was performed on a
Varian Mercury 400 or 300 MHz and referenced to tetramethyl-
silane (Si(CH3)4) (TMS). Chemical shifts were reported in parts per
million (ppm, d) downfield from TMS. Proton coupling patterns
were described as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), and broad (br).
(E)-4-Bromo-N-(4-((3-chloro-4-fluorophenyl)amino)-
thieno[3,2-d]pyrimidin-6-yl)but-2-enamide (13d)
Oxalyl chloride (0.5 mL, 4.68 mmol) was added to (E)-4-bromobut-
2-enoyl chloride 7 (579 mg, 3.51 mmol) which was suspended
in dichloromethane (8 mL). To this, three drops of N,N-dimethyl-
formamide were added. After stirring for about 2 h, the solvents
were removed in vacuo, and the residual oil was dissolved
in tetrahydrofuran (10 mL). This solution was cooled in an ice
bath, and a solution of 690 mg (2.34 mmol) of (E)-4-bromo-N-(4-
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Scheme 1. Reagents and conditions: (a) NBS, AIBN, CCl4, reflux, 98%; (b) (COCl)2, DMF, CH2Cl2, r.t.; (c) HCOOH, Ac2O, r.t., 4 h, 79%;
(d) NH4COOH, NH2CHO, 150°C, 4 h, 86%; (e) HNO3/H2SO4, 0°C, 1 h, 78%; (f) POCl3, 80°C, 3 h, 87%; (g) Ar–NH2, i-PrOH, reflux, 3 h,
80–99%. (h) Fe, NH4Cl, EtOH/H2O, 27–50%; (i) 7, (i-Pr)2NEt, THF, 0°C, 49–90%; (j) (CH3)2NH, THF, r.t., 10–54%. The structures of a–g
are listed in Table 1.
((3-chloro-4-fluorophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)but-2-
enamide 12d in tetrahydrofuran (10 mL) was added dropwise.
This was followed, still with cooling, by the dropwise addition of
0.3 mL (3.51 mmol) of diisopropylethylamine in tetrahydrofuran
(5 mL). After cooling and stirring for an hour, ethyl acetate (80 mL)
and water (100 mL) were added. The layers were separated, and
the aqueous layer was extracted with tetrahydrofuran and ethyl
acetate (1:1, 10 mL). The combined organic layers were washed
with brine (20 mL), then dried over sodium sulfate. Evaporation
gave a solid, which was chromatographed on silica gel, eluting
with cyclohexane/ethyl acetate (3:1) to give a yellow solid (E)-
4-bromo-N-(4-((3-chloro-4-fluorophenyl)amino)thieno[3,2-d]pyrimi-
din-6-yl)but-2-enamide 13d (500 mg, 49%). 1H NMR (DMSO-d6,
400 MHz): d 10.71 (s, 1H), 9.97 (s, 1H), 8.72 (d, J ¼ 6.4 Hz, 1H), 8.50
(t, J ¼ 5.4 Hz, 1H), 8.18 (t, J ¼ 3.3 Hz, 1H), 7.75 (t, J ¼ 4.3 Hz, 1H), 7.43
(t, J ¼ 9.3 Hz, 1H), 6.80–6.89 (m, 2H), 4.40 (q, J ¼ 7.13 Hz, 2H).
(d, J ¼ 15.2 Hz, 1H), 6.66 (d, J ¼ 14.8 Hz, 1H), 3.81 (d, J ¼ 7.2 Hz, 2H),
2.77 (s, 6H); ESI-MS: m/z¼ 406.2 [MþH]þ.
(E)-N-(4-((4-Bromo-2-fluorophenyl)amino)thieno[3,2-d]-
pyrimidin-6-yl)-4-(dimethylamino)but-2-enamide (14a)
1H NMR (400 MHz, CDCl3): d 8.72 (s, 1H), 8.68 (s, 1H), 8.38 (s, 1H),
8.21 (t, J ¼ 9.2 Hz, 1H), 7.33–7.36 (m, 2H), 7.01–7.08 (m, 2H), 6.25
(d, J ¼ 15.3 Hz, 1H), 3.14 (d, J ¼ 6.1 Hz, 2H), 2.28 (s, 6H); ESI-MS:
m/z ¼ 452.2 [MþH]þ.
(E)-4-(Dimethylamino)-N-(4-((2-(3-fluorobenzyl)-2H-
indazol-5-yl)amino)thieno[3,2-d]pyrimidin-6-yl)but-2-
enamide (14b)
1H NMR (600 MHz, CD3OD): d 8.51 (s, 1H), 8.33 (s, 1H), 8.31 (s, 1H),
8.01 (s, 1H), 7.62 (d, J ¼ 9.0 Hz, 1H), 7.45 (d, J ¼ 9.0 Hz, 1H), 7.35 (dd,
J ¼ 14.0, 8.0 Hz, 1H), 7.10 (d, J ¼ 7.6 Hz, 1H), 7.02 (dd, J ¼ 14.0,
8.5 Hz, 2H), 6.89–6.94 (m, 1H), 6.60 (d, J ¼ 15.0 Hz, 1H), 5.64 (s, 2H),
3.74 (d, J ¼ 6.8 Hz, 2H), 2.72 (s, 6H); ESI-MS: m/z ¼ 502.5 [MþH]þ.
(E)-N-(4-((2-Bromo-4-fluorophenyl)amino)thieno[3,2-d]-
pyrimidin-6-yl)-4-(dimethylamino)but-2-enamide (14d)
A
solution of (E)-4-bromo-N-(4-((3-chloro-4-fluorophenyl)amino)-
thieno[3,2-d]pyrimidin-6-yl)but-2-enamide 13d (500 mg, 1.13 mmol)
in tetrahydrofuran (5mL) was stirred at 0°C, and a solution of
dimethylamine in tetrahydrofuran (50mL, 2 M) was added
dropwise. After the addition was complete, the mixture was stirred
at room temperature overnight. Then evaporation gave a solid,
which was chromatographed on silica gel, eluting with CH2Cl2/
MeOH (20:1) to give a white solid (E)-N-(4-((2-bromo-4-fluorophenyl)-
amino)thieno[3,2-d]pyrimidin-6-yl)-4-(dimethylamino)but-2-enamide
14d. 1H NMR (CDCl3, 400MHz): d 8.63 (s, 1H), 8.44 (s, 1H), 8.03 (dd,
J1 ¼ 2.8 Hz, J2 ¼ 3.6 Hz, 1H), 7.65 (m, 1H), 7.25 (t, J ¼ 8.8 Hz, 1H), 6.95
(E)-4-(Dimethylamino)-N-(4-((2-(pyridin-2-ylmethyl)-2H-
indazol-5-yl)amino)thieno[3,2-d]pyrimidin-6-yl)but-2-
enamide (14c)
1H NMR (300 MHz, CD3OD): d 8.55 (s, 1H), 8.53 (s, 1H), 8.38 (s, 1H),
8.34 (s, 1H), 8.06 (s, 1H), 7.78–7.84 (m, 1H), 7.61 (d, J ¼ 9.3 Hz, 1H),
7.47 (dd, J1 ¼ 1.8 Hz, J2 ¼ 9.2 Hz, 1H), 7.36 (dd, J1 ¼ 7.2 Hz, J2 ¼ 5.0
Hz, 1H), 7.20 (d, J ¼ 8.0 Hz, 1H), 6.90–6.99 (m, 1H), 6.68 (d,
J ¼ 15.4 Hz, 1H), 5.76 (s, 2H), 3.95 (d, J ¼ 6.9 Hz, 2H), 2.87 (s, 6H); ESI-
MS: m/z ¼ 485.4 [MþH]þ.
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(E)-4-(Dimethylamino)-N-(4-((1-(3-fluorobenzyl)-1H-
indazol-5-yl)amino)thieno[3,2-d]pyrimidin-6-yl)but-2-
(m, J ¼ 1.2 Hz, 1H), 7.38–7.43 (m, 1H), 7.25 (s, 1H), 7.38–7.42 (m,
1H), 7.14 (d, J ¼ 3.6 Hz, 1H), 7.05–7.08 (m, 1H), 6.90–7.00 (m, 1H),
6.58 (d, J ¼ 6.2 Hz, 1H), 5.21 (s, 2H), 3.80 (d, J ¼ 2.8 Hz, 2H), 2.79 (s,
6H); ESI-MS: m/z ¼ 497.5 [MþH]þ; HR-MS (ESIþ) calcd. 495.1370 for
enamide (14e)
1H NMR (CD3OD, 400 MHz): d 8.53 (s, 1H), 8.38 (s, 1H), 8.11 (s, 1H),
8.07 (s, 1H), 7.59 (m, 2H), 7.35–7.30 (m, 1H), 7.05–6.89 (m, 4H), 6.65
(d, J ¼ 15.2 Hz, 1H), 5.69 (s, 2H), 3.84 (d, J ¼ 7.2 Hz, 2H), 2.80 (s, 6H);
ESI-MS: m/z ¼ 502.4 [MþH]þ.
C
24H24N6O2SCl, found m/z 495.1360 [MþH]þ.
The author has declared no conflict of interest.
(E)-N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-
thieno[3,2-d]pyrimidin-6-yl)-4-(dimethylamino)but-2-
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1633–1637.
(E)-N-(4-((3-Chloro-4-(pyridin-3-ylmethoxy)phenyl)amino)-
thieno[3,2-d]pyrimidin-6-yl)-4-(dimethylamino)but-2-
enamide (14g)
1H NMR (400 MHz, CD3OD): d 3.01 (s, 6H), 4.07 (d, J¼ 7.3 Hz, 2H), 5.67
(d, J ¼ 1.0 Hz, 2H), 6.74 (dd, J¼ J1 ¼ 1.0 Hz, J2 ¼ 15.4 Hz, 1H), 6.99–7.04
(m, 1H), 7.43 (dd, J¼ 8.8, 1.0 Hz, 1H), 7.67 (dd, J1 ¼ 2.5 Hz, J2 ¼ 8.8 Hz,
1H), 7.91 (d, J ¼ 2.3 Hz, 1H), 8.13 (dd, J1 ¼ 6.0 Hz, J2 ¼ 7.5 Hz, 1H), 8.32
(d, J¼ 8.3 Hz, 1H), 8.49 (s, 1H), 8.71–8.76 (m, 1H), 8.81 (d, J¼ 1.0 Hz,
1H), 8.95 (d, J¼ 6.0 Hz, 1H); ESI-MS: m/z¼ 495.4 [MþH]þ.
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(E)-N-(4-((2-Bromo-4-fluorophenyl)amino)thieno[2,3-d]-
pyrimidin-6-yl)-4-(dimethylamino)but-2-enamide (16a)
1H NMR (300 MHz, CD3OD): d 8.33 (s, 1H), 8.41 (s, 1H), 7.70
(t, J ¼ 8.5 Hz, 1H), 7.47 (dd, J1 ¼ 2.0 Hz, J2 ¼ 10.0 Hz, 1H), 7.37–7.42
(m, 1H), 7.28 (s, 1H), 6.92–7.01 (m, 1H), 6.61 (d, J ¼ 15.4 Hz, 1H),
4.03 (d, J ¼ 7.5 Hz, 2H), 2.93 (s, 6H); ESI-MS: m/z ¼ 452.3 [MþH]þ.
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pyrimidin-6-yl)-4-(dimethylamino)but-2-enamide (16b)
1H NMR (400 MHz, CD3OD): d 8.41 (s, 1H), 8.06 (dd, J1 ¼ 2.8 Hz,
J2 ¼ 6.4 Hz, 1H), 7.66–7.70 (m, 1H), 7.37 (m, 1H), 7.23 (t, J ¼ 9.2 Hz,
1H), 6.92–6.98 (m, 1H), 6.62 (d, J ¼ 15.2 Hz, 1H), 4.01 (d, J ¼ 7.2 Hz,
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(E)-N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-
thieno[2,3-d]pyrimidin-6-yl)-4-(dimethylamino)but-2-
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1H NMR (400 MHz, CD3OD): d 8.57 (s, 1H), 8.41 (s, 1H), 7.83
(d, J ¼ 2.4 Hz, 1H), 7.52 (dd, J1 ¼ 2.4 Hz, J2 ¼ 8.8 Hz, 1H), 7.38–7.43
(m, 1H), 7.30 (d, J ¼ 7.6 Hz, 1H), 7.25 (d, J ¼ 9.6 Hz, 1H), 7.14
(d, J ¼ 8.8 Hz, 1H), 7.03–7.07 (m, 1H), 6.90–6.97 (m, 1H), 6.71
(d, J ¼ 15.6 Hz, 1H), 5.21 (s, 2H), 4.01 (dd, J1 ¼ 1.2 Hz, J2 ¼ 7.6 Hz,
2H), 2.93 (s, 6H); ESI-MS: m/z ¼ 512.5 [MþH]þ.
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(E)-N-(4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-
thieno[2,3-d]pyrimidin-6-yl)-4-(dimethylamino)but-2-
enamide (16d)
1H NMR (400 MHz, CD3OD): d (ppm) 8.62 (m, J ¼ 1.6 Hz, 1H),
8.41 (s, 1H), 7.84–7.88 (m, 1H), 7.66 (d, J ¼ 2.8 Hz, 1H), 7.57
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