Design, Synthesis and Anti-Proliferative Activities of 2,6-Substituted Thieno[3,2-d]pyrimidine Derivatives Containing ElectrophilicWarheads

Article abstract of DOI:10.3390/molecules22050788

Thieno[3,2-d]pyrimidine as an effective pharmacophore has been extensively studied. However, its 2,6-substituted derivatives are rarely reported. In the present study, eighteen 2,6-substituted thieno[3,2-d]pyrimidine derivatives containing electrophilic warheads were designed based on the first known Fibroblast growth factor receptor-4 (FGFR4) inhibitor Blu9931. Unexpectedly, all of the derivatives exhibited negligible activity against FGFR4. However, most of the target compounds exhibited antiproliferative activities against four human cancer cell lines, including A431, NCI-H1975, Ramos and SNU-16. Compound 12 showed the most potent antiproliferative activities on the above four cell lines with IC₅₀ values of 1.4 μM, 1.2 μM, 0.6 μM, and 2.6 μM, respectively. Additionally, the antiproliferative activity of 12 against MDA-MB-221 proved that 12 had the selectivity towards certain tumor cell lines. Furthermore, preliminary structure-Activity relationship analysis was discussed based on the experimental data.

Full text of DOI:10.3390/molecules22050788

molecules
Article
Design, Synthesis and Anti-Proliferative Activities of
2,6-Substituted Thieno[3,2-d]pyrimidine Derivatives
Containing Electrophilic Warheads
Qiumeng Zhang 1, Zonglong Hu 2, Qianqian Shen 2, Yi Chen 2,* and Wei Lu 1,
*
1
School of Chemistry and Molecular Engineering, East China Normal University,
3663 North Zhongshan Road, Shanghai 200062, China; qiumeng-zhang@foxmail.com
Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China;
zlhu@jding.dhs.org (Z.H.); shenqianqian@simm.ac.cn (Q.S.)
2
*
Correspondence: ychen@simm.ac.cn (Y.C.); wlu@chem.ecnu.edu.cn (W.L.);
Tel.: +86-21-5080-1552 (Y.C.); +86-21-6223-8771 (W.L.)
Academic Editor: Diego Muñoz-Torrero
Received: 23 April 2017; Accepted: 8 May 2017; Published: 12 May 2017
Abstract: Thieno[3,2-d]pyrimidine as an effective pharmacophore has been extensively studied.
However, its 2,6-substituted derivatives are rarely reported. In the present study, eighteen
2,6-substituted thieno[3,2-d]pyrimidine derivatives containing electrophilic warheads were designed
based on the first known Fibroblast growth factor receptor-4 (FGFR4) inhibitor Blu9931. Unexpectedly,
all of the derivatives exhibited negligible activity against FGFR4. However, most of the target
compounds exhibited antiproliferative activities against four human cancer cell lines, including
A431, NCI-H1975, Ramos and SNU-16. Compound 12 showed the most potent antiproliferative
activities on the above four cell lines with IC50 values of 1.4 µM, 1.2 µM, 0.6 µM, and 2.6 µM,
respectively. Additionally, the antiproliferative activity of 12 against MDA-MB-221 proved that
12 had the selectivity towards certain tumor cell lines. Furthermore, preliminary structure-activity
relationship analysis was discussed based on the experimental data.
Keywords: thieno[3,2-d]pyrimidines; antitumor activities; acrylamide warheads
1. Introduction
Cancer is one of the most formidable enemies to the public health worldwide [1]. Despite the
numerous achievements in the field of cancer pharmacotherapy, there is a continuous demand
for novel agents with improved therapeutic efficacy, selectivity, and safety. Over the past few
decades, more and more drugs with fused bicyclic pyrimidine scaffolds have been approved by
the Food and Drug Administration (FDA) with significant biological activities, especially antitumor
activities. These fused bicyclic pyrimidines exhibit anticancer function by targeting different kinases,
such as Epidermal growth factor receptor (EGFR), Brutons tyrosine kinase (BTK), Janus Kinase (JAK),
and Phosphatidylinositol 3 kinase (PI3K) (Figure 1) [2–6]. Considering broader applications of fused
bicyclic pyrimidines in anticancer drugs, chemical modifications of the bicyclic pyrimidine scaffolds
have been extensively studied. Previously, various thieno[3,2-d]pyrimidine derivatives have attracted
attention due to their broad spectrum activities, such as GDC-0941 and Olmutinib (Figure 2) [7–18].
Although a large number of studies on derivatives of thieno[3,2-d]pyrimidine have been documented,
the 2,6-substituted analogs are seldom reported.
Molecules 2017, 22, 788; doi:10.3390/molecules22050788
www.mdpi.com/journal/molecules

Molecules 2017, 22, 788
2 of 16
Figure 1. Structures of some Food and Drug Administration (FDA) approved drugs with bicyclic
pyrimidine scaffold.
Figure 2. A part of reported thieno[3,2-d]pyrimidine structures.
Recently, therapies using covalent drugs have been proved to be successful for various indications.
Although there have been concerns about the toxicity of covalent drugs, the high potencies and
prolonged effects might result in less frequent drug dosing and wide therapeutic margins for
patients [19–22]. Many covalent drugs have been reported for the treatment of cancer by inhibiting
different targets. Among these derivatives, Afatinib, an irreversible EGFR tyrosine kinase inhibitor
(TKI) [23], was approved by the FDA for the treatment of metastatic non-small cell lung cancer
in 2013 [24]. Ibrutinib, a selective irreversible BTK inhibitor, has been approved by FDA for

Molecules 2017, 22, 788
3 of 16
treatment of patients with chronic lymphocytic leukemia, mantle cell lymphoma and Waldenstrom’s
macroglobulinemia [25 26]. Blu9931, which contains a quinazoline core and an acrylamide electrophilic
,
warhead, is the first selective small molecule inhibitor of FGFR4 (Figure 3) [27]. Evidence suggests
that covalent drugs are a feasible antitumor strategy. It is worth mentioning that bicyclic pyrimidine
scaffolds are widely used in covalent drug design.
Figure 3. Structures of some covalent inhibitors.
Based on previously published results and our continued interest in studying thieno[3,2-d]
pyrimidine derivatives, we decided to design a series of 2,6-substituted thieno[3,2-d]pyrimidine
compounds containing electrophilic warheads based on the first known FGFR4 inhibitor Blu9931.
FGFR4 is a tyrosine kinase that regulates a wide range of important biological functions. The fibroblast
growth factor 19 (FGF19) is the first FGF member exhibiting selective binding to its receptor
FGFR4 [28]. Aberrant signaling through the FGF19/FGFR4 signaling complex has been shown to cause
hepatocellsular carcinoma in mice and has indicated a similar role in humans [27]. Therefore, FGFR4 is
a potential therapeutic target for anticancer drugs. In addition, FGFR1 is highly homologous to FGFR4.
Herein, we presented the design, synthesis and antitumor activities of the compounds related to the
general structure A (Figure 4). The preliminary structure-activity relationship was also discussed.
Figure 4. Structures and design strategy for target compounds.
2. Results and Discussion
2.1. Chemistry
Detailed synthetic strategy to key intermediate
synthesized according to published procedures [29] and then treated with urea at 170 ◦C to produce
compound . Subsequently, compound was treated with POCl3 followed by the treatment with
. The newly synthesized
was treated with NH3/EtOH (4 M) at 150 ◦C
8 was illustrated in Scheme 1. Compound 4 was
5
5
Pd/C/NaHCO3 to produce
7
7
in a steel tube to afford 8.

Molecules 2017, 22, 788
4 of 16
Scheme 1. Synthesis of key intermediate 8. Reagents and conditions: (a) i: thionyl chloride, reflux; ii: ethyl
potassium malonate, Et3N, MgCl2, anhydrous MeCN, r.t., overnight; (b) 3 M HCl, EtOH, reflux, 3 h, 79%;
(c) POCl3, anhydrous N,N-Dimethylformamide (DMF); (d) hydroxylamine hydrochloride, anhydrous
MeCN, r.t., overnight , 40% over two steps; (e) methyl 2-mercaptoacetate, MeONa, anhydrous MeOH,
◦
◦
0–80 C, 2 h, 96%; (f) urea, neat, 170 C, 4 h, 89%; (g) POCl3, reflux, overnight, 65%; (h) Pd/C, NaHCO3,
◦
Ethyl acetate (EA), EtOH, r.t., overnight, 79%; (i) 4 M NH3/EtOH, 150 C; 48 h, 67%.
The synthetic route for the synthesis of derivatives 12
chemical structures of 12 29 were shown in Table 1. The derivative
aromatic substitution reaction in the presence of 9a to obtain crude products. Furthermore, reduction
of the nitro group was carried out under basic conditions to give the compound 10a , which was
–
29 was depicted in Scheme 2 and the
–
8
was involved in a nucleophilic
–c
–
c
then treated with sulfonyl chloride to give 11a
–
c
. In addition, 10a
–
–
c
c
and 11a
and 11a
–
c
were mixed with
were treated with
acryloyl chloride to afford the targets 12 17. On the other hand, 10a
–
–
c
(E)-4-bromobut-2-enoyl chloride before reacting with 1-methylpiperazine or 30% dimethylamine
solution in water to afford targets 18–29.
Scheme 2. Synthesis of target compounds 12–29. Reagents and conditions: (a) NaH, anhydrous
N,N-Dimethylformamide (DMF), r.t.; (b) Fe(OH)3, 80% hydrazine hydrate, EtOH, reflux; (
c
) Sulfonyl
◦
chloride, Dichloromethane (DCM) or Tetrahydrofuran (THF),
or (E)-4-bromobut-2-enoyl chloride, N,N-Diisopropylethylamine (DIPEA), anhydrous DCM,
−
10 C, 20 min; (d) i: Acryloyl chloride
◦
−
10 C;
ii: DMF, NaI, 1-Methylpiperazine or 30% dimethylamine solution in water, r.t.

Molecules 2017, 22, 788
5 of 16
Table 1. Structures of compounds 12–29.
Compounds
12
R
R1
R2
R3
Compounds
21
R
R1
R2
R3
H
H
H
Cl
Cl
Cl
H
H
H
H
H
Cl
H
H
13
14
15
16
17
18
19
20
Me
H
H
Me
H
22
23
24
25
26
27
28
29
Cl
Cl
H
Me
H
H
Me
H
H
H
Me
H
H
H
Me
H
H
Me
H
H
Me
H
H
Cl
Cl
Cl
H
Me
H
H
Me
H
H
Me
Me
2.2. Biological Evaluation
2.2.1. In Vitro Enzymatic Assays–FGFR1 and FGFR4
Firstly, the FGFR1 and FGFR4 enzymatic assays of compounds 12
structures are similar to the first small molecular FGFR4 inhibitor Blu9931. Unfortunately, all of the
compounds lost the enzyme activities against FGFR1 and FGFR4 with less than 50% inhibition at 1 M.
–29 were performed, since their
µ
The results summarized in Figures S1 and S2 (supplied in supplementary data) were expressed as
inhibition rates, which were derived from double independent determinations (the information of
positive controls can be found in supplementary data), suggesting that the interaction between Blu9931
and FGFR4 might be sensitive to the specific angle of two benzene rings.
2.2.2. In Vitro Cytotoxic Activities and SAR Analysis
The antiproliferative activity of compounds 12–29 against A431 (human skin squamous cell
carcinoma), NCI-H1975 (human lung adenocarcinoma cell), Ramos (human B lymphoma cell) and
SNU-16 (human gastric cancer cell) cell lines were tested. Using the approved drug Doxorubicin
(ADR) as positive controls, the results of the mean values of experiments from three independent
determinations, expressed as half-maximal inhibitory concentration (IC50) values, were summarized
in Table 2.
As presented in Table 2, most of the target compounds exhibited moderate antiproliferative
activities against Ramos cells. The different substituents of hydrogen or methyl in benzene ring were
first investigated. Compound 12 exhibited moderate activity (IC50 = 0.6
hydrogen with o-methyl group (compound 13) or p-methyl group (compound 14) resulted in a slight
loss of activity (IC50 = 3.9 M and 2.7 M, respectively), suggesting that the antiproliferative activity
is sensitive to small changes on the benzene ring adjacent to the pyrimidine side. To further identify
the effects of the benzene ring next to the thiophene side on the biological profiles, compounds 15 17
µM), while the replacement of
µ
µ
–

Molecules 2017, 22, 788
6 of 16
were synthesized and tested. Dramatically reduced antitumor activities were observed when 12
were transformed into their respective dichloride substituted derivatives 15 17 (IC50 = 8.0
14.1 M and 6.4 M, respectively). The result suggests that the benzene ring next to the thiophene
side plays an important role in antitumor activity. In addition, analogues 18 29 with different
Michael acceptors displayed significantly reduced activity against Ramos cells compared with the
corresponding compound with acryloyl group. Most of the compounds 12 29 also exhibited moderate
to great antiproliferative activity against the other three cell lines. It is interesting to note that the
structure-activity relationship analyses of 12 29 against the four tested cell lines were highly consistent.
–
14
–
µM,
µ
µ
–
–
–
Table 2. Antiproliferative activity of 12–29 against A431, NCI-H1975, Ramos, and SNU-16.
a
IC50 ± SD (µM)
Compounds
A431
NCI-H1975
Ramos
SNU-16
12
13
14
15
16
17
18
19
20
21
22
23
24
1.4 ± 0.2
4.1 ± 0.1
2.0 ± 0.2
16.3 ± 1.6
>20
10.3 ± 1.8
5.3 ± 0.4
5.9 ± 0.4
4.7 ± 0.1
4.4 ± 0.2
7.1 ± 3.6
4.5 ± 0.2
10.5 ± 0.2
>20
1.2 ± 0.1
5.4 ± 0.5
2.8 ± 0.2
12.6 ± 4.0
>20
9.7 ± 0.2
12.2 ± 0.6
9.4 ± 0.5
5.0 ± 1.3
7.2 ± 0.5
14.0 ± 3.7
6.8 ± 0.04
14.3 ± 3.1
>20
0.6 ± 0.3
3.9 ± 0.8
2.7 ± 0.8
8.0 ± 2.4
14.1 ± 4.4
6.4 ± 3.5
6.4 ± 1.9
9.9 ± 1.2
3.8 ± 0.4
4.6 ± 1.9
7.6 ± 2.2
7.5 ± 1.1
7.0 ± 1.9
>20
2.6 ± 0.2
6.7 ± 2.2
3.2 ± 0.4
19.2 ± 0.6
>20
14.2 ± 1.8
11.6 ± 1.2
16.3 ± 0.5
6.2 ± 2.0
9.1 ± 0.4
>20
6.1 ± 0.9
>20
25
>20
26
27
28
>20
>20
17.3 ± 4.1
11.9 ± 2.8
12.8 ± 4.3
10.2 ± 0.1
0.3 ± 0.01
>20
12.2 ± 1.1
12.1 ± 2.3
11.2 ± 0.6
11.3 ± 1.3
0.3 ± 0.03
14.8 ± 0.6
10.9 ± 4.0
>20
29
11.2
±
681.1
0.2 ± 0.05
12.2 ± 2.7
0.3 ± 0.05
ADR b
Data presented is the mean ± SD value of three independent determinations; b Used as positive control.
a
Furthermore, the antiproliferative activity of the most potent compound 12 against NCI-H1581
(Human non-small cell lung cancer cell line), MDA-MB-231 (Human breast cancer cell line) was
evaluated. The derivative 12 showed loss of antiproliferative activity against MDA-MB-221 (IC50 > 20
indicating that 12 exhibits selectivity towards certain tumor cell lines (Table 3).
µM)
Table 3. Antiproliferative activity of 12 against NCI-H1581, MDA-MB-231.
a
IC50 ± SD (µM)
Compounds
NCI-H1581
MDA-MB-231
12
1.32 ± 0.08
0.5 ± 0.03
>20
0.4 ± 0.05
ADR b
Data presented is the mean ± SD value of three independent determinations; b Used as positive control.
a
2.2.3. In Vitro Enzymatic Assay—BTK
BTK plays a crucial role in B cell lines growth and reproduction [30]. It has been well documented
that most of the BTK inhibitors formed a covalent bond between its electrophilic warhead and
a nucleophilic center in the BTK protein. As shown in Table 2, most of the target compounds displayed
potent anti-proliferative activity on Ramos cells (B cell lines). Therefore, the inhibition of 12
–29 against
BTK at 10 nM, 100 nM and 500 nM were tested. Using Ibrutinib as the positive control, the results of

Molecules 2017, 22, 788
7 of 16
the mean values of experiments from double independent determinations, expressed as inhibition
rates, were exhibited in Figure S3 (supplied in Supplementary Data). Compounds 12 29 demonstrated
–
less than 40% inhibition against BTK at 500 nM, and their inhibitory activities are much inferior to
Ibrutinib. These results indicated that BTK is not the biological target for these compounds.
2.2.4. In Vitro Enzymatic Assays
To investigate the molecular mechanisms, we screened compound 12 against twenty selected
tyrosine kinases. The results summarized in Table S3 (supplied in Supplementary Data) were expressed
as inhibition rates, derived from three independent determinations (details related to positive controls
can be found in Supplementary Data). Unfortunately, compound 12 was judged to have a negligible
impact on the tested kinases.
3. Materials and Methods
3.1. Materials and Methods
All reagents and solvents were purchased from commercial suppliers and were further purified
or dried if necessary. The 1H-NMR spectra and 13C-NMR spectra were recorded for CDCl3, MeOD,
or Dimethyl sulfoxide-d6 (DMSO-d6) by a Bruker DRX-400 (400 MHz) (Bruker Biospin AG, Fällanden,
Switzerland) with Tetramethylsilane (TMS) as an internal standard. Chemical shifts were reported
as
δ (ppm) and spin–spin coupling constants as J (Hz) values. The mass spectra were obtained
on a Waters -SDQ mass spectrometer (Manchester, UK) or Waters SYNAPT G2 ESI-TOF-MS
analyzer. Melting points were determined on an SGW X-4 melting point detector (Shanghai, China),
uncorrected and reported in degrees Centigrade. The derivatives synthesized were purified by column
chromatography using silica gel (200–300 mesh). The purity of all tested derivatives was established
by High Performance Liquid Chromatography (HPLC) to be >95%.
3.2. General Synthesis
3.2.1. 1-(3,5-Dimethoxyphenyl)ethanone (2)
Step 1. 3,5-dimethoxybenzoyl chloride
3,5-dimethoxybenzoic acid (27.3 g, 150 mmol, compound
1) was suspended in 120 mL thionyl
chloride, and the mixture was heated and held at reflux until
1
dissolved. Then, thionyl chloride
was evaporated under reduced pressure conditions and the residue was diluted with 150 mL
anhydrous acetonitrile.
Step 2. Ethyl 3,5-dimethoxybenzoate
Ethyl potassium malonate (51 g, 300 mmol) and magnesium chloride (36 g, 375 mmol) were
suspended in 600 mL anhydrous acetonitrile, and 63 mL Et3N was added slowly. The mixture stirred
at room temperature for 2 h. Then 3,5-dimethoxybenzoyl chloride in 50 mL anhydrous acetonitrile was
added dropwise. The reaction mixture stirred at room temperature overnight. Dilute hydrochloric
acid added to the mixture until pH < 5. The aqueous layer was extracted three times with ethyl acetate
(EA) and the combined organic layers were washed with brine and then dried over anhydrous sodium
sulfate, filtered and the solution was concentrated under reduced pressure to give the corresponding
crude product.
Step 3. 1-(3,5-dimethoxyphenyl)ethanone (2)
The crude product from step 2 dissolved in 150 mL 3 M HCl and 50 mL ethanol. The reaction
mixture was heated at reflux for 2 h. After cooling, the solvent was reduced under reduced pressure.
The residue was separated on a silica gel column, eluting with a petroleum ether:ethyl acetate (PE/EA)

Molecules 2017, 22, 788
8 of 16
(6:1 v/v) solvent system to give 2 (21.3 g, 79% over three steps); colorless oil; 1H-NMR (400 MHz, CDCl3)
δ 7.08 (s, 2H), 6.64 (s, 1H), 3.83 (s, 6H), and 2.56 (s, 3H).
3.2.2. (E)-3-Chloro-3-(3,5-dimethoxyphenyl)acrylonitrile (3)
In addition, 15 mL POCl3 was added dropwise into 25 mL anhydrous DMF, the mixture stirred at
room temperature for 15 min. Compound
2
(13.8 g, 78 mmol) in 30 mL anhydrous DMF was added
◦
to the mixture carefully and stirred for 20 min, and then the reaction was heated to 40 C for 1 h
(monitored by Thin Layer Chromatography, TLC). Then, the mixture was allowed to cool down to
25 ◦C, and 21.3 g hydroxylamine hydrochloride was added in batches slowly because of the intense
heat released. Another 150 mL of anhydrous acetonitrile was added and the mixture stirred at room
temperature for overnight. Then, the mixture poured into ice water, stirred for 5 h, filtered, and then
the solid was washed by water and ether. The crude product was purified by eluting through a silica
gel column with a 1:3 CH2Cl2/PE solvent system to give 3 (6.96 g, 40% over two steps); colorless oil;
1H-NMR (400 MHz, CDCl3) δ 6.76 (s, 2H), 6.58 (s, 1H), 6.00 (s, 1H), and 3.83 (s, 6H).
3.2.3. Methyl 3-Amino-5-(3,5-dimethoxyphenyl)thiophene-2-carboxylate (4)
Methyl 2-mercaptoacetate (3 mL, 33 mmol) was dissolved in 45 mL MeOH, MeONa
◦
(2.4 g, 45 mmol) was added slowly, and the mixture was stirred at 25 C for half an hour. Compound
3
(6.72 g, 10 mmol) was added and then heated to reflux for 1 h (monitored by TLC), and then the
mixture was allowed to cool down to 25 ◦C and poured into ice water, filtered, and then the filtrate
was extracted with EA twice, and the combined organic layers were washed with brine and then dried
over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by eluting
through a silica gel column with a 1:8 EA/PE solvent system to give 4
(8.4 g, 96%); yellow oil; 1H-NMR
(400 MHz, CDCl3) δ 6.74 (s, 1H), 6.72 (s, 2H), 6.46 (s, 1H), 5.46 (s, 2H), 3.84 (s, 3H), 3.82 (s, 6H).
3.2.4. 6-(3,5-Dimethoxyphenyl)thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (5)
Compound
The mixture was cooled to 120 ◦C, then added into 500 mL NaOH (1 M) solution, and stirred for 1 h.
After filtration, the solid was washed with water and dried by vacuum to obtain (7.6 g, 89%);
11.65 (s, 1H), 11.26 (s, 1H), 7.24 (s, 1H),
161.0, 158.8, 157.7, 151.5, 146.6,
4
(8.2 g, 28 mmol) was reacted with 40 g urea at 180 ◦C for 8 h (monitored by TLC).
5
yellow solid; M.p.: >250 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ
6.84 (s, 2H), 6.61 (s, 1H), 3.82 (s, 6H); 13C-NMR (101 MHz, DMSO-d6)
134.0, 113.5, 110.0, 104.2, 101.5, 55.5; ESI-MS: m/z 305.1 [M + H]+.
δ
3.2.5. 2,4-Dichloro-6-(3,5-dimethoxyphenyl)thieno[3,2-d]pyrimidine (6)
Compound
5
(7.5 g, 22 mmol) was dissolved in 30 mL POCl3. The solution was heated to reflux
◦
for overnight (monitored by TLC). The mixture was allowed to cool down to 40 C, and then added to
300 mL ice water slowly, white solid precipitated slowly. The precipitate was collected, washed with
water and dried to provide the desired product
(400 MHz, CDCl3)
δ 163.9, 161.5, 157.5, 156.4, 154.7, 133.7, 128.8, 119.4, 105.3, 102.5, 55.6; ESI-MS: m/z 341.0 [M + H]+.
6
(4.9 g, 65%); white solid; M.p.: >250 ◦C; 1H-NMR
δ
7.62 (s, 1H), 6.84 (d, 2H), 6.58 (s, 1H), 3.87 (s, 6H); 13C-NMR (101 MHz, CDCl3)
3.2.6. 2-Chloro-6-(3,5-dimethoxyphenyl)thieno[3,2-d]pyrimidine (7)
To a solution of
6 (4.8 g, 14 mmol) and NaHCO3 (2.35 g, 28 mmol) in 20 mL EtOH and 20 mL EA,
10% Pd/C was added (960 mg, 20% by wt). The suspension was stirred at room temperature under
an atmosphere of H2 for 23 h. A second portion of 10% Pd/C (980 mg, 20% by wt) was added after 12 h.
The reaction mixture was filtered through Celite® with EtOAc washings. The filtrate was washed with
H2O/brine (4:1), dried by MgSO4, filtered and concentrated under reduced pressure to provide
7
(3.4 g, 79%); light yellow solid; M.p.: >250 ◦C; 1H-NMR (400 MHz, CDCl3)
δ
9.04 (s, 1H), 7.62 (s, 1H),

Molecules 2017, 22, 788
9 of 16
6.87 (s, 2H), 6.58 (s, 1H), 3.88 (s, 6H); 13C-NMR (101 MHz, CDCl3)
δ
163.6, 161.4, 157.7, 157.2, 152.7,
134.1, 129.7, 119.0, 105.4, 102.2, 77.3, 77.0, 76.7, 55.6; ESI-MS: m/z 307.0 [M + H]+.
3.2.7. 6-(3,5-Dimethoxyphenyl)thieno[3,2-d]pyrimidin-2-amine (8)
Compound 7 (3.1 g, 10 mmol) was dissolved in 80 mL 4 M NH3/EtOH. The solution was heated to
◦
150 C for 48 h in sealed tube. The reaction mixture was cooled to room temperature and then poured
into ice water, filtered through Celite® with EtOAc washings. The solid dried by vacuum to obtain
compound
8 δ 8.89
(1.9 g, 67%); Off-white solid; M.p.: 249.4–250.2 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(s, 1H), 7.65 (s, 1H), 6.96 (s, 2H), 6.62 (s, 1H), 6.57 (s, 2H), 3.83 (s, 6H); 13C-NMR (101 MHz, DMSO-d6)
δ 162.7, 161.8, 160.9 152.8, 152.3, 134.7, 119.5, 119.2, 104.5, 101.5, 55.5; ESI-MS: m/z 288.1 [M + H]+.
3.2.8. General Procedure 1 of the Synthesis of Compounds 10a–c
The solution of compound
DMF was stirred for 30 min, corresponding o-fluoronitrobenzene (9a
8
(287 mg, 1 mmol) and NaH (60%, 80 mg, 2 mmol) in 10 mL anhydrous
, 1.05 mmol) in 5 mL anhydrous
–c
DMF was added to the mixture dropwise. The reaction mixture was stirred for overnight and then
poured into 100 mL water, filtered through Celite with water washings. The solid dried by vacuum
to give corresponding crude products. To a solution of corresponding crude products (0.8 mmol) in
15 mL EtOH, Fe(OH)3 (43mg, 0.4 mmol) and Hydrazine hydrate (80%, 0.5 mL, 8 mmol) were added.
◦
The mixture was heated to 70 C for about 4 h (monitored by TLC). The reaction mixture was
cooled to room temperature and then filtered through Celite® with EtOAc washings. The filtrate was
concentrated under reduced pressure to provide crude products and then the residue was purified
through a silica gel column to give the corresponding 10a–c.
Preparation of 10a. From compound 9c (131 mg, 1.05 mmol), as that described in procedure 1,
gave pure 10a (298 mg, 96%); off-white solid; M.p.: 209.2–210.2 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ
9.02 (s, 1H), 8.51 (s, 1H), 7.80 (s, 1H), 7.42 (d, J = 7.1 Hz, 1H), 6.99 (d, J = 2.1 Hz, 2H), 6.93–6.86 (m, 1H),
6.75 (d, J = 6.9 Hz, 1H), 6.61 (dd, J = 7.0, 5.0 Hz, 2H), 4.88 (s, 2H), 3.83 (s, 6H); 13C-NMR (101 MHz,
DMSO-d6) δ 162.3, 161.0, 159.4, 152.8, 142.3, 134.6, 125.3, 124.8, 120.7, 119.6, 116.2, 115.6, 104.4, 101.7,
55.5; ESI-MS: m/z 379.1 [M + H]+.
Preparation of 10b. From compound 9b (131 mg, 1.05 m◦mol), as that described in procedure 1,
gave pure 10b (275 mg, 88%); yellow solid; M.p.: 180.0–181.2 C; 1H-NMR (400 MHz, DMSO-d6)
δ 8.96
(s, 1H), 8.28 (s, 1H), 7.76 (s, 1H), 6.95 (s, 2H), 6.88 (t, J = 7.6 Hz, 1H), 6.60 (s, 2H), 6.46 (d, J = 7.3 Hz, 1H), 4.71
(s, 2H), 3.81 (s, 6H), 2.06 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
162.6, 160.9, 160.1, 152.9, 152.5, 145.6,
136.5, 134.7, 126.4, 123.4, 120.3, 119.7, 117.8, 112.8, 104.4, 101.6, 55.5, 18.3; ESI-MS: m/z 393.2 [M + H]+
δ
.
Preparation of 10c. From compound 9b (131 mg, 1.05 mmol), as that described in procedure 1,
gave pure 10c (282 mg, 90%); yellow solid; M.p.: 192.0–193.2 ◦C; 1H-NMR (400 MHz, CDCl3)
8.80
(s, 1H), 7.40 (s, 1H), 7.28 (s, 1H), 6.84 (d, J = 1.8 Hz, 2H), 6.82 (s, 1H), 6.68 (s, 1H), 6.65 (d, J = 8.0 Hz, 1H), 6.53
δ
(s, 1H), 3.85 (s, 7H), 2.30 (s, 3H); 13C-NMR (101 MHz, CDCl3)
δ 163.0, 161.2, 159.8, 154.5, 152.4, 141.9,
136.8, 135.1, 126.3, 123.2, 122.2, 120.0, 119.2, 117.7, 105.0, 101.7, 55.5, 21.2; ESI-MS: m/z 393.2 [M + H]+
.
3.2.9. General Procedure 2 of the Synthesis of Compounds 11a–c
Corresponding 10a
–c (0.35 mmol) was dissolved in anhydrous THF or anhydrous DCM (5 mL),
◦
10 C. The resulting
and sulfonyl chloride (290 mg, 2.1 mmol) was added to the solution in portions at
−
mixture stirred at 0 ◦C for 1 h (monitored by TLC). EA (20 mL) and Na2CO3 solution (20 mL) were
added, and the layers were partitioned and separated. The organic layers were washed with water
and brine, dried over anhydrous sodium sulfate, filtered and then the filtration was concentrated in
the vacuum to give corresponding 11a–c.
Preparation of 11a. From compound 10a (133 mg, 0.35 mmol), as that described in procedure 2,
gave pure 11a (128 mg, 82%); yellow solid; M.p.: >250 ◦C; 1H-NMR (400 MHz, CDCl3)
δ 8.89 (s, 1H),

Molecules 2017, 22, 788
10 of 16
7.55–7.39 (m, 1H), 7.13 (s, 1H), 7.07 (m, 1H), 6.92–6.81 (m, 2H), 6.74 (s, 1H), 6.68 (s, 1H), 3.97 (s, 6H),
3.93–3.78 (m, 2H); 13C-NMR (101 MHz, CDCl3)
δ 162.2, 159.3, 154.7, 152.7, 148.4, 141.4, 133.4, 126.5,
126.1, 125.6, 124.7, 123.7, 119.4, 117.2, 115.3, 98.2, 56.7; ESI-MS: m/z 447.0 [M + H]+.
Preparation of 11b. From compound 10b (138 mg, ◦0.35 mmol), as that described in procedure 2,
gave pure 11b (127 mg, 79%); yellow solid; M.p.: >250 C; 1H-NMR (400 MHz, DMSO-d6)
δ 9.02 (s, 1H),
8.32 (s, 1H), 7.14 (s, 1H), 7.07 (s, 1H), 6.87 (t, J = 7.7 Hz, 1H), 6.59 (d, J = 7.8 Hz, 1H), 6.46 (d, J = 7.3 Hz, 1H),
4.72 (s, 2H), 3.97 (s, 6H), 2.07 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
δ 161.8, 160.1, 154.5, 153.2, 145.7,
136.5, 132.6, 126.5, 124.2, 123.3, 121.5, 117.7, 113.4, 112.8, 99.4, 56.9, 18.3; ESI-MS: m/z 461.0 [M + H]+.
Preparation of 11c. From compound 10c (138 mg, 0.35 mmol), as that described in procedure 2,
gave pure 11c (131 mg, 81%); yellow solid; M.p.: 229.6–231.4 ◦C; 1H-NMR (400 MHz, CDCl3)
δ
8.87
(s, 1H), 7.27 (m, 1H), 7.11 (s, 1H), 6.80 (s, 1H), 6.67 (s, 2H), 3.97 (s, 6H), 2.29 (s, 3H); 13C-NMR (101 MHz,
CDCl3)
δ 162.3, 159.7, 154.7, 152.7, 148.3, 141.9, 136.8, 133.4, 126.3, 124.7, 123.4, 123.2, 120.0, 117.7, 115.3,
98.2, 56.1, 21.2; ESI-MS: m/z 461.0 [M + H]+.
3.2.10. General Procedure 3 of the Synthesis of Compounds 12–17
One of corresponding 10a
(0.04 mL) and 0.1 mL 10% Acryloyl chloride (anhydrous THF) were added to the solution dropwise at
10 ◦C. The resulting mixture was stirred for 1 h (monitored by TLC), DCM (20 mL) and NaHCO3
–c and 11a–c (0.1 mmol) was dissolved in anhydrous DCM. DIPEA
−
solution (20 mL) were then added, and the layers were partitioned and separated. The organic layers
were washed with NH4Cl solution, washed with water and brine, dried over anhydrous sodium
sulfate, filtered and the solution was then concentrated under reduced pressure to give corresponding
crude product 12–17, which was furthermore purified through a silica gel column.
Preparation of 12. From compound 10a (38 mg, 0.1 mmol), as that described in procedure 3, gave pure
◦
12 (21 mg, 50%); yellow solid; M.p.: 217.1–218.0 C; 1H-NMR (400 MHz, DMSO-d6)
δ 9.88 (s, 1H), 9.08
(s, 1H), 8.64 (s, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.84 (s, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H),
7.12 (t, J = 7.6 Hz, 1H), 7.00 (s, 2H), 6.63 (s, 1H), 6.51 (dd, J = 16.6, 10.2 Hz, 1H), 6.28 (d, J = 17.0 Hz, 1H),
5.77 (d, J = 10.5 Hz, 1H), 3.83 (s, 6H); 13C-NMR (101 MHz, DMSO-d6)
δ 163.8, 162.1, 161.0, 158.4, 153.4,
153.0, 134.5, 132.8, 131.5, 129.7, 127.2, 125.3, 124.6, 124.0, 123.4, 121.8, 119.5, 104.5, 101.8, 55.5; HPLC:
method 1, room temperature, tg = 11.72 min, UV254 = 98%; HRMS (ESI) m/z calcd for C23H20N4O3S
[M + H]+: 433.1329, found: 433.1350.
Preparation of 13. From compound 10b (39 mg, 0.1 mmol), as that described in procedure 3, gave
pure 13 (18 mg, 40%); yellow solid; M.p.:184.5–185.6 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ 9.52 (s, 1H),
8.99 (s, 1H), 8.33 (s, 1H), 7.77 (s, 1H), 7.71 (d, J = 7.0 Hz, 1H), 7.18 (t, J = 7.9 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H),
6.96 (d, J = 1.8 Hz, 2H), 6.60 (s, 1H), 6.51 (dd, J = 16.8, 10.1 Hz, 1H), 6.21 (d, J = 16.7 Hz, 1H),
5.69 (d, J = 10.3 Hz, 1H), 3.81 (s, 6H), 2.16 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
δ 168.8, 163. 5, 162.4,
160.9, 159.5, 152.9, 150.8, 136.9, 134.9, 134.6, 131.8, 126.8, 126.5, 125.9, 121.0, 120.0, 104.4, 101.7, 55.5,
18.5; HPLC: method 2, room temperature, tg = 11.67 min, UV254 = 97%; HRMS (ESI) m/z calcd for
C24H22N4O3S [M + H]+: 447.1485, found: 447.1495.
Preparation of 14. From compound 10c (39 mg, 0.1 mmol), as that described in procedure 3, gave
pure 14 (16 mg, 36%); light yellow solid; M.p.: 239.0–240.1 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ 9.82
(s, 1H), 9.05 (s, 1H), 8.55 (s, 1H), 7.82 (s, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.40 (s, 1H), 7.04 (d, J = 8.3 Hz,
1H), 6.99 (d, J = 2.1 Hz, 2H), 6.62 (s, 1H), 6.50 (dd, J = 17.0, 10.2 Hz, 1H), 6.28 (d, J = 17.0 Hz, 1H),
5.76 (t, J = 5.0 Hz, 1H), 3.83 (s, 6H), 3.34 (s, 4H), 2.50 (s, 4H), 2.31 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
δ
163.6, 162.2, 161.0, 158.6, 153.2, 153.0, 134.5, 132.7, 131.5, 130.1, 129.8, 127.1, 125.8, 124.7, 124.3, 121.5, 119.6,
104.5, 101.8, 55.5, 20.5; HPLC: method 2, room temperature, tg = 12.05 min, UV254 = 98%; HRMS (ESI) m/z
calcd for C24H22N4O3S [M+Na]+: 469.1305, found: 469.1299.

Molecules 2017, 22, 788
11 of 16
Preparation of 15. From compound 11a (45 mg, 0.1 mmol), as that described in procedure 3, gave pure
◦
15 (22 mg, 44%); white solid; M.p.: 236.6–236.9 C; 1H-NMR (400 MHz, CDCl3)
7.93 (s, 1H), 7.52 (s, 1H), 7.38 (s, 1H), 7.22 (d, J = 8.2 Hz, 1H), 7.15 (s, 1H), 6.68 (s, 1H), 6.35 (d, J = 16.9 Hz, 1H),
6.20 (dd, J = 16.8, 10.3 Hz, 1H), 5.69 (d, J = 10.2 Hz, 1H), 3.98 (s, 6H); 13C-NMR (101 MHz, DMSO-d6)
δ 8.91 (s, 1H), 8.63 (s, 1H),
δ
163.7, 161.2, 158.3, 154.5, 153.3, 148.0, 132.6, 132.4, 131.5, 130.1, 127.1, 125.3, 124.5, 124.4, 124.2, 123.7,
123.0, 113.3, 99.5, 56.9; HPLC: method 2, room temperature, tg = 11.37 min, UV254 = 97%; HRMS (ESI)
m/z calcd for C23H18Cl2N4O3S [M+Na]+: 523.0369, found: 523.0371.
Preparation of 16. From compound 11b (46 mg, 0.1 mmol), as that described in procedure 3, gave pure
16 (24 mg, 47%); light yellow solid; M.p.: 142.5–143.8 ◦C; 1H-NMR (400 MHz, CDCl3)
δ 8.89 (s, 1H),
8.49 (s, 1H), 8.05 (s, 1H), 7.24 (m, 1H), 7.13 (s, 1H), 7.09 (d, J = 7.5 Hz, 1H), 6.68 (s, 1H), 6.60 (s, 1H),
6.30 (d, J = 17.0 Hz, 1H), 6.14 (dd, J = 16.8, 10.2 Hz, 1H), 5.65 (d, J = 10.2 Hz, 1H), 3.98 (s, 6H), 2.31 (s, 3H);
13C-NMR (101 MHz, DMSO-d6)
δ 163.5, 161.6, 159.5, 154.5, 153.2, 147.4, 136.9, 135.0, 132.5, 131.9, 126.8,
126.4, 125.9, 124.2, 122.2, 120.8, 113.4, 99.4, 56.9, 18.5; HPLC: method 2, room temperature, tg = 11.12 min,
UV254 = 98%; HRMS (ESI) m/z calcd for C24H20Cl2N4O3S [M + H]+: 515.0706, found: 515.0715.
Preparation of 17. From compound 11c (46 mg, 0.1 mmol), as that described in Procedure 3, gave
pure 17 (22 mg, 44%); white solid; M.p.: 258.2–259.6 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ 9.81 (s, 1H),
9.11 (s, 1H), 8.59 (s, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.41 (s, 1H), 7.23 (s, 1H), 7.08 (s, 1H), 7.03 (d, J = 8.3 Hz, 1H),
6.50 (dd, J = 17.0, 10.2 Hz, 1H), 6.27 (dd, J = 17.0, 1.7 Hz, 1H), 5.76 (dd, J = 10.1, 1.7 Hz, 1H), 3.98 (s, 6H),
2.30 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
δ 163.6, 161.3, 158.6, 154.5, 153.3, 147.7, 133.0, 132.4,
131.5, 130.2, 129.9, 127.0, 125.8, 124.7, 124.6, 124.2, 122.7, 113.3, 99.4, 56.9, 20.5; HPLC: method 2, room
temperature, tg = 11.54 min, UV254 = 98%; HRMS (ESI) m/z calcd for C24H20Cl2N4O3S [M + H]+:
515.0706, found: 515.0723.
3.2.11. General Procedure 4 of the Synthesis of Compounds 18–29
Step 1. One of corresponding 10a–c and 11a–c (0.2 mmol) was dissolved in anhydrous DCM.
DIPEA (0.07 mL) and (E)-4-br◦omobut-2-enoyl chloride (0.01 g/mL in anhydrous THF, 3 mL) were
added to the solution at
−
5 C. The resulting mixture was stirred for 2 h (monitored by TLC),
DCM (10 mL) and water (10 mL) were added, the layers were partitioned and separated. The organic
layers were washed with NaHCO3 solution, water and brine, dried over anhydrous sodium sulfate,
filtered and the solution was concentrated under reduced pressure, used directly in the next step.
Step 2. One of corresponding crude product in the Step 1 (0.1 mmol) was dissolved in anhydrous
DMF. NaI (46 mg, 0.3 mmol) and 0.07 mL (40%) dimethylamine solution in water were added.
The resulting mixture was stirred at room temperature (monitored by TLC). EA (10 mL) and water
(10 mL) were added, and the layers were partitioned and separated. The organic layers were washed
with water and brine, dried over anhydrous sodium sulfate, filtered and concentrated, and then the
residue was purified through a silica gel column to give corresponding 18–23.
Preparation of 18. From compound 10a (38 mg, 0.1 mmol), as that described in step 1 and step 2
of procedure 4, gave pure 18 (24 mg, 49%); yellow solid; M.p.: 175.1–175.9 ◦C; 1H-NMR (400 MHz,
CDCl3)
6.92 (dt, J = 12.2, 5.9 Hz, 1H), 6.84 (d, J = 2.1 Hz, 2H), 6.53 (t, J = 2.1 Hz, 1H), 6.06 (d, J = 15.4 Hz, 1H),
3.85 (s, 6H), 3.05 (d, J = 5.7 Hz, 2H), 2.22 (s, 6H); 13C-NMR (101 MHz, CDCl3)
172.5, 164.0, 162.6,
δ 8.81 (s, 1H), 8.55 (s, 1H), 7.88 (s, 1H), 7.58 (s, 2H), 7.40 (s, 1H), 7.22 (d, J = 4.1 Hz, 2H),
δ
161.3, 159.1, 155.3, 152.3, 141.8, 134.9, 131.7, 126.2, 125.6, 124.9, 124.6, 122.9, 118.9, 105.0, 101.9, 60.2, 55.6,
45.3; HPLC: method 1, room temperature, tg = 11.94 min, UV280 = 96%; HRMS (ESI) m/z calcd for
C26H27N5O3S [M + H]+: 490.1907, found: 490.1940.
Preparation of 19. From compound 10b (39 mg, 0.1 mmol), as that de◦scribed in step 1 and step 2 of
procedure 4, gave pure 19 (20 mg, 40%); yellow solid; M.p.: 151.2–152.9 C; 1H-NMR (400 MHz, CDCl3)
δ
8.79 (s, 1H), 8.37 (s, 1H), 8.08 (s, 1H), 7.36 (s, 1H), 7.24 (m, J = 8.0 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H),
6.91–6.84 (m, 2H), 6.83 (d, J = 2.2 Hz, 2H), 6.53 (t, J = 2.1 Hz, 1H), 5.99 (d, J = 15.3 Hz, 1H), 3.85 (s, 6H),

Molecules 2017, 22, 788
12 of 16
3.10–2.90 (m, 2H), 2.27 (s, 3H), 2.19 (s, 6H); 13C-NMR (101 MHz, CDCl3)
δ
163.7, 162. 9, 161.3, 159.7,
155.4, 152.5, 141.7, 135.5, 135.2, 134.9, 127.2, 126.6, 126.3, 122.9, 120.8, 118.9, 105.0, 101.9, 60.2, 55.7, 45.4,
18.7; HPLC: method 1, room temperature, tg = 11.87 min, UV254 = 98%; HRMS (ESI) m/z calcd for
C27H29N5O3S [M + H]+: 504.2064, found: 504.2082.
Preparation of 20. From compound 10c (39 mg, 0.1 mmol), as that described in step 1 and step 2
of procedure 4, gave pure 20 (17 mg, 34%); yellow solid; M.p.: 170.8–171.5 ◦C; 1H-NMR (400 MHz,
DMSO-d6)
7.04 (d, J = 8.5 Hz, 1H), 7.00 (s, 2H), 6.84–6.71 (m, 1H), 6.64 (s, 1H), 6.39 (d, J = 15.9 Hz, 1H), 3.84 (s, 6H),
3.27 (s, 2H), 2.32 (s, 9H); 13C-NMR (101 MHz, DMSO-d6)
163.5, 162.2, 161.0, 158.6, 153.2, 153.0, 134.5,
δ 9.78 (s, 1H), 9.06 (s, 1H), 8.56 (s, 1H), 7.82 (s, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.41 (s, 1H),
δ
132.7, 130.0, 126.3, 125.7, 124.6, 124.4, 121.5, 119.6, 104.5, 101.8, 59.3, 55.5, 44.7, 20.5; HPLC: method 1,
room temperature, tg = 12.46 min, UV254 = 99%; HRMS (ESI) m/z calcd for C27H29N5O3S [M + H]+:
504.2064, found: 504.2092.
Preparation of 21. From compound 11a (45 mg, 0.1 mmol), as that described in step 1 and step 2
of procedure 4, gave pure 21 (21 mg, 38%); yellow solid; M.p.: 231.7–232.5 ◦C; 1H-NMR (400 MHz,
DMSO-d6)
δ 9.79 (s, 1H), 9.14 (s, 1H), 8.70 (s, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H),
7.26 (s, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.08 (s, 1H), 6.76 (dt, J = 15.2, 5.8 Hz, 1H),
6.34 (d, J = 15.5 Hz, 1H), 3.98 (s, 6H), 3.05 (d, J = 5.5 Hz, 2H), 2.16 (s, 6H); 13C-NMR (101 MHz, DMSO-d6)
δ
163.8, 161.3, 158.4, 154.5, 153.4, 147. 8, 141.8, 132.4, 130.2, 125.4, 125.1, 124.4, 124.2, 123.6, 123.0, 113.3,
99.5, 59.7, 56.9, 45.2; HPLC: method 1, room temperature, tg = 14.20 min, UV254 = 98%; HRMS (ESI)
m/z calcd for C26H25Cl2N5O3S [M + H]+: 558.1128, found: 558.1158.
Preparation of 22. From compound 11b (46 mg, 0.1 mmol), as that described in step 1 and step 2 of
◦
procedure 4, gave pure 22 (24 mg, 42%); yellow solid; M.p.: >250 C; 1H-NMR (400 MHz, DMSO-d6)
δ
9.50 (s, 1H), 9.04 (s, 1H), 8.40 (s, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.18 (t, J = 7.8 Hz, 2H), 7.08 (d, J = 8.2 Hz, 2H),
6.70 (dt, J = 15.3, 6.2 Hz, 1H), 6.41 (d, J = 15.4 Hz, 1H), 3.97 (s, 6H), 3.21 (d, J = 4.4 Hz, 2H), 2.28 (s, 6H),
2.16 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
δ 163.2, 161.6, 159.5, 154.5, 153.2, 147.4, 136.9, 135.2,
132.5, 130.3, 126.3, 125.9, 124.2, 122.2, 120.6, 113.3, 99.4, 58.9, 56.9, 44.0, 18.5; HPLC: method 2,
room temperature, tg = 11.35 min, UV254 = 99%; HRMS (ESI) m/z calcd for C27H27Cl2N5O3S [M + H]+
:
572.1284, found: 572.1327.
Preparation of 23. From compound 11c (46 mg, 0.1 mmol), as that described in step 1 and step 2
of procedure 4, gave pure 23 (19 mg, 33%); yellow solid; M.p.: 214.0–214.6 ◦C; 1H-NMR (400 MHz,
CDCl3)
7.00 (d, J = 7.8 Hz, 1H), 6.91 (dt, J = 15.0, 6.0 Hz, 1H), 6.68 (s, 1H), 6.03 (d, J = 15.4 Hz, 1H), 5.30 (s, 1H),
3.98 (s, 6H), 3.07 (d, J = 5.8 Hz, 2H), 2.36 (s, 3H), 2.24 (s, 6H); 13C-NMR (101 MHz, CDCl3)
163.8, 161.9,
δ 8.89 (s, 1H), 8.45 (s, 1H), 7.80 (s, 1H), 7.36 (d, J = 7.2 Hz, 1H), 7.16 (s, 1H), 7.14 (s, 1H),
δ
159.3, 154.7, 152.8, 149.0, 141.5, 133.2, 126.4, 126.3, 125.2, 124.7, 124.4, 124.1 115.2, 98.2, 60.2, 56.7, 45.3,
21.2; HPLC: method 1, room temperature, tg = 12.66 min, UV254 = 95%; HRMS (ESI) m/z calcd for
C27H27Cl2N5O3S [M + Na]+: 594.1104, found: 594.1091.
Step 3. Corresponding crude product in the step 1 (0.1 mmol) was dissolved in anhydrous DMF.
NaI (46 mg, 0.3 mmol) and 0.02 mL N-Methylpiperazine were added. The resulting mixture was stirred
at room temperature (monitored by TLC). EA (10 mL) and water (10 mL) were added, and the layers
were partitioned and separated. The organic layers were washed with water and brine, and dried over
anhydrous sodium sulfate. Filtered and the solution concentrated under reduced pressure, and then
the residue was purified through a silica gel column to give corresponding 24–29.
Preparation of 24. From compound 10a (38 mg, 0.1 mmol), as that described in step 1 and step 3
of procedure 4, gave pure 24 (23 mg, 43%); yellow solid; M.p.: 182.9–183.7 ◦C; 1H-NMR (400 MHz,
CDCl3)
6.91 (d, J = 9.3 Hz, 1H), 6.84 (d, J = 2.0 Hz, 2H), 6.53 (s, 1H), 6.04 (d, J = 15.3 Hz, 1H), 3.85 (s, 6H),
3.09 (d, J = 5.5 Hz, 2H), 2.42 (m, 8H), 2.23 (s, 3H); 13C-NMR (101 MHz, CDCl3)
163.9, 162.5, 161.3,
159.1, 155.4, 152.3, 141.7, 134.8, 131.7, 126.2, 125.7, 124.9, 124.7, 122.8, 118.8, 105.0, 101.8, 59.2, 55.6, 54.9,
δ 8.82 (s, 1H), 8.50 (s, 1H), 7.88 (s, 1H), 7.55 (s, 1H), 7.48 (s, 1H), 7.41 (s, 1H), 7.21 (s, 2H),
δ

Molecules 2017, 22, 788
13 of 16
53.2, 45.9; HPLC: method 1, room temperature, tg = 11.04 min, UV280 = 96%; HRMS (ESI) m/z calcd for
C29H32N6O3S [M+Na]+: 567.2149, found: 567.2169.
Preparation of 25. From compound 10b (39 mg, 0.1 mmol), as that described in step 1 and step 3
of procedure 4, gave pure 25 (20 mg, 36%); yellow solid; M.p.: 156.5–157.8 ◦C;1H-NMR (400 MHz,
CDCl3)
6.84 (s, 2H), 6.82–6.69 (m, 2H), 6.54 (s, 1H), 5.99 (d, J = 15.0 Hz, 1H), 3.86 (s, 6H), 3.32 (s, 2H), 3.16 (s, 2H),
2.83 (s, 6H), 2.68 (s, 3H), 2.29 (s, 4H); 13C-NMR (101 MHz, MeOD)
166.3, 164.0, 162.9, 160.7, 156.5,
δ 8.83 (s, 1H), 8.58 (s, 1H), 8.00 (s, 1H), 7.39 (s, 1H), 7.23 (s, 1H), 7.10 (d, J = 7.0 Hz, 1H),
δ
153.9, 141.6, 138.4, 136.1, 135.9, 132.1, 128.9, 127.9, 127.7, 123.3, 119.7, 105.8, 102.7, 59.5, 56.1, 55.2, 52.4,
44.8, 18.8; HPLC: method 1, room temperature, tg = 11.26 min, UV280 = 97%; HRMS (ESI) m/z calcd for
C30H34N6O3S [M + H]+: 559.2486, found: 559.2494.
Preparation of 26. From compound 10c (39 mg, 0.1 mmol), as that described in step 1 and step 3 of
◦
procedure 4, gave pure 26 (19 mg, 34%); yellow solid; M.p.: 182.0–183.2 C; 1H-NMR (400 MHz, CDCl3)
δ
8.80 (s, 1H), 7.73 (s, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.37 (s, 1H), 7.02 (d, J = 7.3 Hz, 1H), 6.83 (d, J = 2.0 Hz,
2H), 6.82–6.72 (m, 1H), 6.53 (t, J = 1.9 Hz, 1H), 6.08 (d, J = 15.0 Hz, 1H), 3.85 (s, 6H), 3.17 (s, 2H),
3.14 (d, J = 5.5 Hz, 2H), 2.80 (s, 6H), 2.63 (s, 3H), 2.36 (s, 3H); 13C-NMR (101 MHz, CDCl3)
163.5,
δ
161.3, 155.3, 139.8, 135.6, 134.8, 131.4, 129.0, 126.5, 125.0, 118.8, 105.0, 101.8, 58.1, 55.6, 53.8, 49.7, 43.5,
21.1; HPLC: method 1, room temperature, tg = 11.87 min, UV254 = 99%; HRMS (ESI) m/z calcd for
C30H34N6O3S [M+Na]+: 581.2305, found: 581.2278.
Preparation of 27. From compound 11a (45 mg, 0.1 mmol), as that described in step 1 and step 3 of
procedure 4, gave pure 27 (21 mg, 34%); off-white solid; M.p.: 160.1–160.7 ◦C; 1H-NMR (400 MHz,
CDCl3)
6.93 (dt, J = 15.1, 6.1 Hz, 1H), 6.68 (s, 1H), 6.05 (d, J = 15.4 Hz, 1H), 3.98 (s, 6H), 3.11 (d, J = 5.8 Hz,
2H), 2.45 (s, 8H), 2.27 (s, 3H); 13C-NMR (101 MHz, CDCl3)
163.9, 161.8, 159.0, 154.7, 152.7, 149.1,
δ 8.91 (s, 1H), 8.46 (s, 1H), 7.88 (s, 1H), 7.56 (s, 1H), 7.29 (s, 1H), 7.22 (s, 2H), 7.16 (s, 1H),
δ
141.9, 133.1, 131.6, 126.2, 125.7, 124.9, 124.7, 124.4, 124.2, 115.1, 98.2, 59.3, 56.7, 55.0, 53.3, 46.0; HPLC:
method 1, room temperature, tg = 11.63 min, UV254 = 97%; HRMS (ESI) m/z calcd for C29H30Cl2N6O3S
[M + H]+: 613.1550, found: 613.1577.
Preparation of 28. From compound 11b (46 mg, 0.1 mmol), as that described in step 1 and step 3 of
◦
procedure 4, gave pure 28 (18 mg, 29%); white solid; M.p.: 225.0–226.2 C; 1H-NMR (400 MHz, CDCl3)
δ
8.85 (s, 1H), 8.46 (s, 1H), 8.01 (s, 1H), 7.23 (t, J = 7.9 Hz, 1H), 7.09 (s, 1H), 7.07 (d, J = 7.4 Hz, 1H)
6.96 (s, 1H), 6.87 (dt, J = 15.3, 6.2 Hz, 1H), 6.68 (s, 1H), 6.01 (d, J = 15.3 Hz, 1H), 3.97 (s, 6H),
3.07 (d, J = 6.1 Hz, 2H), 2.43 (s, 8H), 2.28 (s, 3H), 2.26 (s, 3H); 13C-NMR (101 MHz, CDCl3)
172.6,
,
δ
163.7, 162.1, 159.5, 154.7, 152.9, 149.1, 141.5, 135.6, 135.1, 133.1, 127.1, 126.7, 124.4, 124.0, 115.2, 98.3, 59.2,
56.7, 55.0, 53.2, 45.9, 18.8; HPLC: method 1, room temperature, tg = 11.50 min, UV254 = 99%; HRMS
(ESI) m/z calcd for C30H32Cl2N6O3S [M + H]+: 627.1706, found: 627.1736.
Preparation of 29. From compound 11c (46 mg, 0.1 mmol), as that described in step 1 and step 3 of
◦
procedure 4, gave pure 29 (22 mg, 35%); white solid; M.p.: 212.4–212.9 C; 1H-NMR (400 MHz, CDCl3)
δ
8.82 (s, 1H), 8.39 (s, 1H), 7.69 (s, 1H), 7.30 (d, J = 6.6 Hz, 1H), 7.14 (d, J = 13.1 Hz, 1H), 7.07 (s, 1H),
6.93 (d, J = 7.6 Hz, 1H), 6.85 (dd, J = 13.8, 7.4 Hz, 1H), 6.61 (s, 1H), 5.95 (d, J = 15.3 Hz, 1H), 3.90 (s, 6H),
3.03 (d, J = 5.9 Hz, 2H), 2.40 (s, 8H), 2.29 (s, 3H), 2.22 (s, 3H); 13C-NMR (101 MHz, DMSO-d6)
163.7,
δ
161.3, 158.6, 154.5, 153.3, 147.7, 141.1, 133.1, 132.4, 130.3, 125.8, 125.7, 124.7, 125.6, 124.2, 122.7, 113.3,
99.4, 58.3, 56.8, 54.4, 52.4, 45.4, 20.5; HPLC: method 1, room temperature, tg = 12.03 min, UV254 = 96%;
HRMS (ESI) m/z calcd for C30H32Cl2N6O3S [M + Na]+: 649.1526, found: 649.1482.
3.2.12. General Procedure for In Vitro Cell-Proliferation Assays
NCI-H1975, Ramos, and SNU-16 cells were maintained in Revolutions-Per-minute Indicator
1640 (RPMI 1640) medium (Gibco, Grand Island, NY, USA), A431 was maintained in Dulbecco’s
Modified Eagle Medium (DMEM) medium (Gibco), MDA-MB-231 was cultured in L-15 medium
(Gibco), and NCI-H1581 was cultured in ACL-4 (Gibco), and all of them were supplemented with

Molecules 2017, 22, 788
14 of 16
◦
10% heat-inactivated fetal calf serum (FBS; Gibco) at 37 C in a 5% CO2 humidified environment.
◦
Cells in 96-well plates were treated with gradient concentrations of compounds at 37 C for 72 h.
Cell proliferation assays in NCI-H1975, A431, MDA-MB-231 and NCI-H1581 were determined by using
sulforhodamine B (SRB; Sigma, St. Louis, MO, USA). The antiproliferative activity of compounds,
examined in Ramos and SUN-16 cell lines, was determined by using CCK-8 (Dojindo Laboratories,
Kamimashiki-gun, Japan). The dosages corresponding to the half-maximal inhibition (IC50) were
calculated using SoftMax pro-based nonlinear 4-parameter regression analysis (n = 3).
4. Conclusions
In summary, a series of 2,6-substituted thieno[3,2-d]pyrimidine containing electrophilic warheads
derivatives were designed and synthesized. The derivatives possessed moderate antiproliferative
properties against four tested cancer cell lines (A431, NCI-H1975, Ramos and SNU-16). Compound 12
showed the most potent cytotoxic activity on the four cell lines above with IC50 values of 1.4
µM,
1.2 M, 0.6 M, and 2.6 M, respectively. However, the antiproliferative activity of 12 against
µ
µ
µ
MDA-MB-221 indicated that 12 had the selectivity towards certain tumor cell lines. The preliminary
investigation showed that the acrylamide electrophilic warhead was more active than the other
two warheads. It was interesting to note that an o-Me substituent on the benzene ring attached to
the electrophilic warhead weakens the activity slightly while p-Me substituent results in a significant
loss of activity. Another interesting phenomenon was that a dramatic loss in antitumor activity was
observed when 12
–14 were transformed into their dichloride substituted derivatives 15–17. In addition,
compounds 12 29 demonstrated less than 40% inhibition against BTK at 500 nM, indicating that BTK
–
might not be the biological target for these compounds. The enzymatic screen assays suggested that
the selected twenty kinases might also not be the biological targets for the synthesized compounds.
Thus, future efforts will focus on investigating the molecular mechanisms of the target compounds,
especially 12. In summary, compound 12 is worth further research as a new potential anticancer agent.
Supplementary Materials: The following are available online. The general HPLC method, ESI-TOF-MS spectrums
of 12
FGFR1 inhibition rate of 12
in FGFR1 enzymatic assay, Figure S2. FGFR4 inhibition rate of 12
and 1000 nM, Table S2: Positive controls in FGFR4 enzymatic assay, Figure S3. BTK inhibition rate of 12
–29, FGFR1 and FGFR4 enzymatic assay, BTK enzymatic assay, and in vitro enzymatic assays, Figure S1.
–
29 at the concentration of 10 nM, 100 nM and 1000 nM, Table S1: Positive controls
29 at the concentration of 10 nM, 100 nM
29 at
–
–
the concentration of 10 nM, 100 nM and 1000 nM, Table S3–S6: Some positive controls in in vitro enzymatic
screen assays.
Author Contributions: Q.Z. and W.L. participated in designing the target compounds. Q.Z. was the chief
experimenter of chemistry, analyzed the experimental data and wrote the paper. Y.C., Z.H., and Q.S. performed
all of the biological assays and participated in analyzing the data. W.L. and Y.C. participated in the corrections of
the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
FDA
EGFR
TKI
BTK
JAK
Food and Drug Administration
Epidermal growth factor receptor
Tyrosine kinase inhibitors
Brutons tyrosine kinase
Janus kinase
PI3K
FGFR4
FGF19
EA
Phosphatidylinositol 3 kinase
Fibroblast growth factor receptor-4
Fibroblast growth factor 19
Ethyl acetate
DMF
N,N-Dimethylformamide

Molecules 2017, 22, 788
15 of 16
DIPEA
ADR
N,N-Diisopropylethylamine
Doxorubicin
DMSO
HPLC
TLC
Dimethyl sulfoxide
High Performance Liquid Chromatography
Thin Layer Chromatography
Petroleum ether
PE
ESI MS
HRMS
TMS
Electrospray ionization mass spectrometry
High resolution mass
Tetramethylsilane
RPMI 1640
DMEM
ELISA
Revolutions-Per-minute Indicator 1640
Dulbecco’s Modified Eagle Medium
Enzyme-linked immunosorbent assay
References
1.
Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2017. CA Cancer J. Clin. 2017, 67, 7–30. [CrossRef]
[PubMed]
2.
Vitaku, E.; Smith, D.T.; Njardarson, J.T. Analysis of the structural diversity, substitution patterns, and
frequency of nitrogen heterocycles among U.S. Fda approved pharmaceuticals. J. Med. Chem. 2014, 57,
10257–10274. [CrossRef] [PubMed]
3.
4.
Wu, P.; Nielsen, T.E.; Clausen, M.H. Small-molecule kinase inhibitors: An analysis of fda-approved drugs.
Drug Discov. Today 2016, 21, 5–10. [CrossRef] [PubMed]
Tracy, S.; Mukohara, T.; Hansen, M.; Meyerson, M.; Johnson, B.E.; Jänne, P.A. Gefitinib Induces Apoptosis
in the EGFRL858R Non–Small-Cell Lung Cancer Cell Line H3255. Cancer Res. 2004, 64, 7241. [CrossRef]
[PubMed]
5.
6.
7.
Li, Z.; Xu, M.; Xing, S.; Ho, W.T.; Ishii, T.; Li, Q.; Fu, X.; Zhao, Z.J. Erlotinib effectively inhibits jak2v617f
activity and polycythemia vera cell growth. J. Biol. Chem. 2007, 282, 3428–3432. [CrossRef] [PubMed]
Burris, H.A., 3rd. Dual kinase inhibition in the treatment of breast cancer: Initial experience with the
egfr/erbb-2 inhibitor lapatinib. Oncologist 2004, 9, 10–15. [CrossRef] [PubMed]
Zhu, W.; Liu, Y.; Zhai, X.; Wang, X.; Zhu, Y.; Wu, D.; Zhou, H.; Gong, P.; Zhao, Y. Design, synthesis
and 3d-qsar analysis of novel 2-hydrazinyl-4-morpholinothieno[3,2-d]pyrimidine derivatives as potential
antitumor agents. Eur. J. Med. Chem. 2012, 57, 162–175. [CrossRef] [PubMed]
8.
9.
Folkes, A.J.; Ahmadi, K.; Alderton, W.K.; Alix, S.; Baker, S.J.; Box, G.; Chuckowree, I.S.; Clarke, P.A.;
Depledge, P.; Eccles, S.A.; et al. The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-
1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (gdc-0941) as a potent, selective, orally bioavailable
inhibitor of class i pi3 kinase for the treatment of cancer. J. Med. Chem. 2008, 51, 5522–5532. [CrossRef]
[PubMed]
Mathieu, S.; Gradl, S.N.; Li, R.; Wen, Z.; Aliagas, I.; Gunznertoste, J.; Lee, W.; Pulk, R.; Zhao, G.;
Alicke, B. Potent and selective aminopyrimidine-based b-raf inhibitors with favorable physicochemical and
pharmacokinetic properties. J. Med. Chem. 2014, 55, 2869–2881. [CrossRef] [PubMed]
10. Perspicace, E.; Marchais-Oberwinkler, S.; Hartmann, R.W. Synthesis and biological evaluation of
thieno[3,2-d]pyrimidinones, thieno[3,2-d]pyrimidines and quinazolinones: Conformationally restricted
17b-hydroxysteroid dehydrogenase type 2 (17b-hsd2) inhibitors. Molecules 2013, 18, 4487–4509. [CrossRef]
[PubMed]
11. Liu, Z.; Wang, Y.; Lin, H.; Zuo, D.; Wang, L.; Zhao, Y.; Gong, P. Design, synthesis and biological evaluation
of novel thieno[3,2-d]pyrimidine derivatives containing diaryl urea moiety as potent antitumor agents.
Eur. J. Med. Chem. 2014, 85, 215–227. [CrossRef] [PubMed]
12. Disch, J.S.; Evindar, G.; Chiu, C.H.; Blum, C.A.; Dai, H.; Jin, L.; Schuman, E.; Lind, K.E.; Belyanskaya, S.L.;
Deng, J. Discovery of thieno[3,2-d]pyrimidine-6-carboxamides as potent inhibitors of sirt1, sirt2, and sirt3.
J. Med. Chem. 2014, 56, 3666–3679. [CrossRef] [PubMed]
13. Tan, Q.; Zhang, Z.; Hui, J.; Zhao, Y.; Zhu, L. Synthesis and anticancer activities of thieno[3,2-d]pyrimidines as
novel hdac inhibitors. Bioorg. Med. Chem. 2014, 22, 358–365. [CrossRef] [PubMed]

Molecules 2017, 22, 788
16 of 16
14. Liu, Z.; Wu, S.; Wang, Y.; Li, R.; Wang, J.; Wang, L.; Zhao, Y.; Gong, P. Design, synthesis and biological
evaluation of novel thieno[3,2-d]pyrimidine derivatives possessing diaryl semicarbazone scaffolds as potent
antitumor agents. Eur. J. Med. Chem. 2014, 87, 782–793. [CrossRef] [PubMed]
15. Temburnikar, K.W.; Zimmermann, S.C.; Kim, N.T.; Ross, C.R.; Gelbmann, C.; Salomon, C.E.; Wilson, G.M.;
Balzarini, J.; Seley-Radtke, K.L. Antiproliferative activities of halogenated thieno[3,2-d]pyrimidines.
Bioorg. Med. Chem. 2014, 22, 2113–2122. [CrossRef] [PubMed]
16. Refat, H.M.; Fadda, A.A.; El-Mekawy, R.E.; Sleat, A.M. Synthesis of some novel thieno[3,2-d]pyrimidine
derivatives of pharmaceutical interest. Heterocycles 2015, 91, 2271. [CrossRef]
17. Ross, C.R.; Temburnikar, K.W.; Wilson, G.M.; Seley-Radtke, K.L. Mitotic arrest of breast cancer mda-mb-231
cells by a halogenated thieno[3,2-d]pyrimidine. Bioorg. Med. Chem. Lett. 2015, 25, 1715–1717. [CrossRef]
[PubMed]
18. Kim, E.S. Erratum to: Olmutinib: First global approval. Drugs 2016, 76, 1233. [CrossRef] [PubMed]
19. Baillie, T.A. Targeted covalent inhibitors for drug design. Angew. Chem. Int. Ed. Engl. 2016, 55, 13408–13421.
[CrossRef] [PubMed]
20. Bauer, R.A. Covalent inhibitors in drug discovery: From accidental discoveries to avoided liabilities and
designed therapies. Drug Discov. Today 2015, 20, 1061–1073. [CrossRef] [PubMed]
21. Barf, T.; Kaptein, A. Irreversible protein kinase inhibitors: Balancing the benefits and risks. J. Med. Chem.
2012, 55, 6243–6262. [CrossRef] [PubMed]
22. Paola, V.; Botrugno, O.A.; Anna, C.; Giuseppe, C.; Paola, D.; Antonello, M.; Andrea, M.; Giuseppe, M.;
Saverio, M.; Florian, T. Synthesis, biological activity and mechanistic insights of 1-substituted cyclopropylamine
derivatives: A novel class of irreversible inhibitors of histone demethylase kdm1a. Eur. J. Med. Chem. 2014
86, 352.
,
23. Minkovsky, N.; Berezov, A. Bibw-2992, a dual receptor tyrosine kinase inhibitor for the treatment of
solid tumors. Curr. Opin. Investig. Drugs 2008, 9, 1336–1346. [PubMed]
24. Boehringer Ingelheim. US Fda Approves Gilotrif™ (afatinib)* as First-Line Treatment for Lung Cancer
Patients with Egfr Mutations. 2013. Available online: http://us.boehringer-ingelheim.com/news_events/
press_releases/press_release_archive/2013/07-12-13-fdaapproves-gilotrif-afatinib-first-line-treatment-
metastatic-non-smallcell-lung-cancer-common-egfr-mutations.html (accessed on 17 May 2016).
25. Brown, J.R. Ibrutinib (pci-32765), the first btk (bruton’s tyrosine kinase) inhibitor in clinical trials.
Curr. Hematol. Malig. Rep. 2013, 8, 1–6. [CrossRef] [PubMed]
26. Li, J.J.; Johnson, D.S. 8. Ibrutinib (Imbruvica): The First-in-Class Btk Inhibitor for Mantle Cell Lymphoma, Chronic
Lymphocytic Leukemia, and Waldenstrom’s Macroglobulinemia; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2015.
27. Hagel, M.; Miduturu, C.; Sheets, M.; Rubin, N.; Weng, W.; Stransky, N.; Bifulco, N.; Kim, J.L.; Hodous, B.;
Brooijmans, N. First selective small molecule inhibitor of fgfr4 for the treatment of hepatocellular carcinomas
with an activated fgfr4 signaling pathway. J. Hepatol. 2015, 5, 424–437. [CrossRef] [PubMed]
28. Xie, M.H.; Holcomb, I.; Deuel, B.; Dowd, P.; Huang, A.; Vagts, A.; Foster, J.; Liang, J.; Brush, J.; Gu, Q.
Fgf-19, a novel fibroblast growth factor with unique specificity for fgfr4. Cytokine 1999, 11, 729–735. [CrossRef]
[PubMed]
29. Ho, H.K.; Németh, G.; Ng, Y.R.; Pang, E.; Szántaikis, C.; Zsákai, L.; Breza, N.; Greff, Z.; Horváth, Z.; Pató, J.
Developing fgfr4 inhibitors as potential anti-cancer agents via in silico design, supported by in vitro and
cell-based testing. Curr. Med. Chem. 2013, 20, 1203–1217. [CrossRef] [PubMed]
30. Harrison, C. Trial watch: Btk inhibitor shows positive results in b cell malignancies. Nat. Rev. Drug Discov.
2012, 11, 96. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 12–29 are available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).