Synthesis and Evaluation of Biological and Antitumor Activities of Tetrahydrobenzothieno[2,3-d]Pyrimidine Derivatives as Novel Inhibitors of FGFR1

Article abstract of DOI:10.1111/cbdd.12687

A series of tetrahydrobenzothieno[2,3-d]pyrimidine derivatives were designed, synthesized, and evaluated as inhibitors of FGFR1. These analogs were synthesized via Gewald's reaction under mild conditions. The structures of the synthesized compounds were characterized by spectroscopic data (IR, ¹H NMR and MS). Their antitumor activities were evaluated against H460, A549 and U251 cell lines in vitro. Results revealed that the tested compounds showed moderate antitumor activities. Structure-activity relationship analyses indicated that compounds with an aromatic ring substituted in the C-2 position or with larger molecules such as 3g, 4c, and 7 were more effective than others. The compound, 3g (78.8% FGFR1 inhibition at 10 μm), was identified to have the most potent antitumor activities, with IC₅₀ values of 7.7, 18.9, and 13.3 μm against the H460, A549, and U251 cell lines, respectively. Together, the results suggested that tetrahydrobenzothieno[2,3-d]pyrimidine derivatives may serve as a potential agent for the treatment of FGFR1-mediated cancers.

Full text of DOI:10.1111/cbdd.12687

Chem Biol Drug Des 2016; 87: 499–507
Research Article
Synthesis and Evaluation of Biological and Antitumor
Activities of Tetrahydrobenzothieno[2,3-d]Pyrimidine
Derivatives as Novel Inhibitors of FGFR1
Xuebao Wang1,†, Di Chen1,†, Shufang Yu1,
13.3 lM against H460, A549 and U251 cell lines, respec-
tively, along with 78.8% FGFR1 inhibition at 10 lM.
Zaikui Zhang1, Yu Wang1, Xiaolu Qi2, Weitao Fu1,
Zixin Xie1 and Faqing Ye1,*
ꢀ
Compound 3g could be a lead compound for design of
FGFR1 inhibitors.
1School of Pharmaceutical Sciences, Wenzhou Medical
University, Wenzhou 325035, China
2Yichang Humanwell Pharmaceutical Co., Ltd, Yichang
443005, China
*Corresponding author: Faqing Ye, yfq664340@163.com
Protein tyrosine kinases play an important role in the sig-
naling pathways that control cell proliferation and differenti-
ation. Among these, receptor tyrosine kinases (RTKs) have
been shown to be important mediators of signal transduc-
tion in cells (1–7). RTKs are important therapeutic targets
for cancer drug discovery and development.
†These authors contribute equally to this work.
A
series of tetrahydrobenzothieno[2,3-d]pyrimidine
derivatives were designed, synthesized, and evaluated
as inhibitors of FGFR1. These analogs were synthe-
sized via Gewald’s reaction under mild conditions. The
structures of the synthesized compounds were charac-
terized by spectroscopic data (IR, 1H NMR and MS).
Their antitumor activities were evaluated against H460,
A549 and U251 cell lines in vitro. Results revealed that
the tested compounds showed moderate antitumor
activities. Structure–activity relationship analyses indi-
cated that compounds with an aromatic ring substi-
tuted in the C-2 position or with larger molecules such
as 3g, 4c, and 7 were more effective than others. The
compound, 3g (78.8% FGFR1 inhibition at 10 lM), was
identified to have the most potent antitumor activities,
with IC50 values of 7.7, 18.9, and 13.3 lM against the
H460, A549, and U251 cell lines, respectively.
Together, the results suggested that tetrahydroben-
zothieno[2,3-d]pyrimidine derivatives may serve as a
potential agent for the treatment of FGFR1-mediated
cancers.
Fibroblast growth factor receptors (FGFRs) constitute a
major class of RTKs serving as high-affinity receptors.
FGFR1 is the founding member of the FGFR family and is
known to be overexpressed in prostate carcinoma cells,
breast carcinoma cells, lung cancer cells, and also other
cancer cells (8,9). Given the pivotal role of FGFR1 in
human cancers, the pathway of cancer development
through FGFR1 overexpression and activation was tar-
geted and analyzed. In the presence of tyrosine kinase
inhibitors, cell proliferation would be blocked by preventing
ATP from binding to the RTKs (10,11). Previous studies
had shown the importance of ATP-binding by FGFR1 for
its enzymatic activity (3,12). However, the in vivo efficacy
of these FGFR inhibitors was limited by their rapid blood
clearance (13). Furthermore, some FGFR1 inhibitors failed
to enter into the market because of their high toxicity, such
as PD173074, SU5402, and Brivanib (14–16). Based on
the above considerations, a new class of tyrosine kinase
inhibitors was designed in this study to mimic ATP and
block the constitutive activation of FGFR1, thereby block-
ing the FGFR1-dependent cancer signaling pathway.
Key words: antitumor, FGFR1, Gewald’s reaction, synthesis,
thieno[2,3-d]pyrimidine
Received 21 May 2015, revised 27 August 2015 and
accepted for publication 2 October 2015
In the present, indoles and quinolones are the most stud-
ied small molecule inhibitors toward FGFR1, while
thienopyrimidines occupy a special position because of
their broad spectrum of biological activities. The thieno
[2,3-d]pyrimidine ring was considered to show potential
antitumor activity (17–19). Compound 16 which also has
such a structure (Figure 1) was found to show inhibitory
activity toward FGFR1 kinase with an IC50 value of 50 lM
(20). Tetrahydrobenzothienopyrimidine has a good repre-
sentation of five- and six-membered heterocycles including
Highlights
ꢀ
Designed and synthesized a series of tetrahydrobenzoth-
ieno[2,3-d]pyrimidine derivatives.
ꢀ
Compound 3g was identified to have the most potent
antitumor activities, with IC50 values of 7.7, 18.9 and
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12687
499

Wang et al.
Chemistry
OCH3
OCH2COOH
O
S
NH
N
Synthesis of 2-amino-4,5,6,7-tetrahydrobenzo[2,3-
b]thiophene-3-carbonitrile (1)
2-Amino-4,5,6,7-tetrahydrobenzo[2,3-b]thiophene-3-carbon-
itrile (1) was prepared by means of Gewald’s reaction as
reported in the literature (20–22). Formation of 2-amino-
4,5,6,7- tetrahydrobenzo[b]thiophene-3-carbonitrile (1) was
characterized by the presence of a band at 2210 cmÀ1
due to a cyano group and N–H stretching bands at 3325
and 3209 cmÀ1 in their IR spectra.
Figure 1: The Chemical structure of compound 16.
six with fused polycyclic ring systems, the polycyclic cores
overlap the position of adenine of ATP when bound, and
meanwhile, the complexes feature hydrogen bonds in the
hinge region. In other words, the substitution of the C-2
position can occupy hydrophobic pocket II so that the
ATP cannot bind with receptors. Given these findings,
compound 16 was chosen as a lead compound, and
derivatives were synthesized to see if a simple analog in a
low lM range could be obtained.
Synthesis of 4-amino-2-chloromethyl-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (2)
A
mixture of 2-amino-4,5,6,7-tetrahydro-1-benzothio-
phene-3-carbonitrile 1 (1 g, 52 mmole) and chloroacetoni-
trile (1 mL) in dioxane (15 mL) was heated at 80 °C for
10 min; then, 20 mL of concentrated hydrochloric acid
was added and reacted for 6 h. On completion of reaction
(TLC), the mixture was cooled and poured onto cold
water. The mixture then basified with ammonium hydrox-
ide. The solid obtained was filtered, washed by methanol,
dried, and crystallized from DMF to give 2 as a brown
crystalline solid (62.4%).
The lactam structure of tetrahydrobenzothienopyrimidine
was considered to be a key group because of the ability
to form hydrogen bonds. Taking this into account, we
considered whether amino was better than carbonyl
group, as amino group could act both hydrogen bond
donor and hydrogen bond acceptor. Here, in this manu-
script, compounds with amino group in tetrahydrobenzoth-
ienopyrimidine were named series 1, while compounds
with carbonyl were named series 2. Series
designed to compare with series 1.
2 were
General procedure for synthesis of compounds
3a–g
A mixture of compound 2 (0.2 mmol) and the correspond-
ing amine (0.8 mmol) in dioxane (10 mL) was refluxed for
18 h. On completion of reaction (TLC), the solution was
concentrated under reduced pressure and the residue
was purified by column chromatography on silica gel (Ethyl
acetate/petroleum ether).
Methods and Materials
General
All organic chemicals were available commercially and used
without further purification. The reagents and anhydrous sol-
vents were purchased from Aladdin and Sinopharm Chemi-
cal Reagent Co., Ltd. All melting points (m.p.) were
uncorrected and measured using an XRC-1 micromelting
point apparatus. The thin layer chromatography (TLC, Rf val-
ues) was performed on F254 or silica gel plates F254 (0.2 mm
thick) and visualization of the spots was effected by expo-
sure to UV light. The IR spectra were recorded on a Bruker
4-Amino-2-[(diethylamino)methyl]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (3a)
Pale yellow solid, Yield: 50.6%; mp 178–181 °C; IR
(cmÀ1): 3,519 (NH2). 1H-NMR (CDCl3): d 1.10 (m, 6H, N
(CH2CH3)2), 1.87–1.92 (m, 4H, TBTP-H), 2.69–2.72 (m,
4H, N(CH2CH3)2), 2.78–2.81 (m, 2H, TBTP-H), 2.89–2.92
(m, 2H, TBTP-H), 3.75 (s, 2H, –CH2–N–), 5.44 (s, 2H, –
NH2). EI-MS: 290.9[M + H]+. Calcd. For C15H22N4S: C,
62.03; H, 7.64; N, 19.29; S, 11.04%. Found: C, 62.09; H,
7.55; N, 19.22; S, 11.14%.
1
Nicolet 670FT-IR spectrophotometer using KBr pellets. H-
NMR spectra were determined on a Brucker Avance III
Spectrometer (600 MHz). Samples were dissolved in
DMSO-d6 or CDCl3 while TMS was used as internal stan-
dard. Chemical shifts were recorded in (ppm) values relative
to TMS and J values are expressed in Hertz. Splitting pat-
terns were designated as follows: s, singlet; d, doublet; t, tri-
plet. Mass spectra were recorded using an Agilent-1200
LC–MS Spectrometer. Elemental analyses were performed
on a Thermo Scientific Elemental Analyzer Flash EA 1112
and were found within Æ0.4% of the theoretical values.
4-Amino-2-[(piperidin-1-yl)methyl]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (3b)
Pale yellow solid, Yield: 53.1%; mp 175–177 °C; IR
1
(cmÀ1): 3380 (NH2). H-NMR (CDCl3): d 1.50–1.55 (m, 6H,
piperidin-H), 1.71–1.95 (m, 4H, TBTP-H), 2.73–2.84 (m,
2H, TBTP-H), 2.86–2.93 (m, 2H, TBTP-H), 3.62–3.69 (m,
4H, N(CH2)2), 3.81 (s, 2H, –CH2-piperidin), 5.45 (s, 2H, –
NH2). EI-MS: 303.2[M + H]+. Calcd. For C16H22N4S: C,
For convenience in the manuscript, TBTP was used to
represent
5,6,7,8-tetrahydrobenzo-
[4,5]thieno[2,3-d]
pyrimidine.
500
Chem Biol Drug Des 2016; 87: 499–507

Tetrahydrobenzothieno[2,3-d]Pyrimidines Antitumor
63.54; H, 7.33; N, 18.53; S, 10.60%. Found: C, 63.50; H,
7.37; N, 18.45; S, 10.68%.
N’-[(4-amino-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-2-yl)methyl]isonicotinohydrazide
(3g)
Yellow solid, Yield: 43.5%; mp >250 °C; IR (cmÀ1): 3436
(NH2), 1639 (C=O). 1H-NMR (DMSO-d6): d 1.73–1.89 (m,
4H, TBTP-H), 2.73–2.84 (m, 2H, TBTP-H), 2.86–2.93 (m,
2H, TBTP-H), 3.91 (s, 2H, –CH2NH–), 5.74 (s, 1H,
NHNHCO), 6.83 (s, 2H, –NH2), 7.72 (d, 2H, Ar-H), 8.71 (d,
2H, Ar-H), 10.45 (s, 1H, NHNHCO). EI-MS: 354.7
[M + H]+. Calcd. For C17H18N6OS: C, 57.61; H, 5.12; N,
23.71; S, 9.05%. Found: C, 57.66; H, 5.14; N, 23.65; S,
9.04%.
4-Amino-2-[(morpholino-1-yl)methyl]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (3c)
Pale yellow solid, Yield: 51.7%; mp 195–198 °C; IR
(cmÀ1): 3,499 (NH2). 1H-NMR (CDCl3): d 1.75–1.95 (m,
4H, TBTP-H), 2.60 (m, 4H, morpholine-H), 2.73–2.84 (m,
2H, TBTP-H), 2.86–2.93 (m, 2H, TBTP-H), 3.65–3.72
(m, 4H, morpholine-H), 3.81 (s, 2H, –CH2-morpholine),
5.46 (s, 2H, –NH2). EI-MS: 305.1[M + H]+. Calcd. For
C
15H20N4OS: C, 59.18; H, 6.62; N, 18.41; O, 5.26; S,
10.53%. Found: C, 59.24; H, 6.64; N, 18.36; S,
10.52%.
General procedure for synthesis of compounds
4a–d
A mixture of compound 2 (0.2 mmol) and the correspond-
ing nitrile (0.4 mmol) in dioxane (10 mL) was heated at
80 °C for 10 min; then, 15 mL of concentrated hydrochlo-
ric acid was added and reacted for 6 h. On completion of
reaction (TLC), the mixture was cooled and poured onto
cold water. The mixture was then basified with ammonium
hydroxide. The solid obtained was filtered and purified by
column chromatography on silica gel (Ethyl acetate/petro-
leum ether) to give 4a–d as a solid.
4-Amino-2-[(4-methylpiperazin-1-yl)methyl]-
5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidine (3d)
Pale yellow solid, Yield: 61.3%; mp 210–212 °C; IR
(cmÀ1): 3440 (NH2). 1H-NMR (CDCl3): d 1.75–1.95 (m,
4H, TBTP-H), 2.28 (s, 3H, N-CH3), 2.45 (m, 4H, piper-
azin-H), 2.62 (m, 4H, piperazin-H), 2.73–2.84 (m, 2H,
TBTP-H), 2.86–2.93 (m, 2H, TBTP-H), 3.81 (s, 2H, –
CH2-piperazin), 5.60 (s, 2H, –NH2). EI-MS: 318.0
[M + H]+. Calcd. For C16H23N5S: C, 60.54; H, 7.30; N,
22.06; S, 10.10%. Found: C, 60.50; H, 7.34; N, 22.06;
S, 10.10%.
4-Amino-2-methyl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidine (4a)
Yellow solid, Yield: 59.1%; mp 224–225 °C; IR (cmÀ1):
3470 (NH2). 1H-NMR (CDCl3): d 1.71–1.88 (m, 4H, TBTP-
H), 2.41 (s, 3H, –CH3), 2.73–2.84 (m, 2H, TBTP-H), 2.85–
2.95 (m, 2H, TBTP-H), 5.01 (s, 2H, –NH2). EI-MS: 220.3
[M + H]+. Calcd. For C11H13N3S: C, 60.24; H, 5.97; N,
19.16; S, 14.62%. Found: C, 60.27; H, 5.90; N, 19.21; S,
14.58%.
4-Amino-2-[(cyclohexylamino)methyl]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (3e)
Pale yellow solid, Yield: 46.9%; mp 221–223 °C; IR
(cmÀ1): 3443 (NH2). 1H-NMR (CDCl3): d 1.12–1.20 (m,
4H, cyclohexyl-H), 1.23–1.35 (m, 4H, cyclohexyl-H),
1.56–1.61 (m, 2H, cyclohexyl-H), 1.73–1.89 (m, 4H,
TBTP-H), 2.02 (s, 1H, –NH–), 2.25–2.27 (m, 1H, cyclo-
hexyl-H), 2.73–2.84 (m, 2H, TBTP-H), 2.86–2.93 (m, 2H,
TBTP-H), 3.89 (s, 2H, –CH2NH–), 5.38 (s, 2H, –NH2).
EI-MS: 317.2[M + H]+. Calcd. For C17H24N4S: C, 64.52;
H, 7.64; N, 17.70; S, 10.13%. Found: C, 64.55; H,
7.60; N, 17.73; S, 10.12%.
4-Amino-2-phenyl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidine (4b)
Dark brown solid, Yield: 57.6%; mp 195–197 °C; IR
(cmÀ1): 3515 (NH2). 1H-NMR (DMSO-d6): d 1.71–1.87 (m,
4H, TBTP-H), 2.73–2.84 (m, 2H, TBTP-H), 2.85–2.94 (m,
2H, TBTP-H), 5.53 (s, 2H, –NH2), 7.41–8.37 (m, 5H, Ph-
H). EI-MS: 282.3[M + H]+. Calcd. For C16H15N3S: C,
68.30; H, 5.37; N, 14.93; S, 11.40%. Found: C, 68.32H,
5.34; N, 14.90; S, 11.44%.
4-Amino-2-[N-(3-carboxyphenyl)aminomethyl]-
5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidine (3f)
White solid, Yield: 35.7%; mp 185–187 °C; IR (cmÀ1):
3457 (NH2). 1H-NMR (DMSO-d6): d 1.73–1.89 (m, 4H,
TBTP-H), 2.73–2.84 (m, 2H, TBTP-H), 2.86–2.93 (m, 2H,
TBTP-H), 3.90 (s, 2H, –CH2NH–), 5.44 (s, 2H, –NH2), 6.89
(s, 1H, –NH–), 6.83–7.20 (m, 4H, Ph-H), 11.45 (s, 1H, –
COOH). EI-MS: 354.8[M + H]+. Calcd. For C18H18N4O2S:
C, 61.00; H, 5.12; N, 15.81; S, 9.05%. Found: C, 61.06;
H, 5.04; N, 15.83; S, 9.01%.
4-Amino-2-(4-hydroxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (4c)
Brown solid, Yield: 36.6%; mp >250 °C; IR (cmÀ1): 3529
(NH2), 3100 (OH). 1H-NMR (DMSO-d6): d 1.80–1.83 (m,
4H, TBTP-H), 2.71–2.80 (m, 2H, TBTP-H), 2.87–2.98 (m,
2H, TBTP-H), 6.71 (s, 2H, –NH2), 6.81–6.87 (d, 2H, Ph-H,
J = 7.4 Hz), 8.16–8.20 (d, 2H, Ph-H, J = 7.4 Hz), 9.76 (s,
1H, –OH). EI-MS: 297.6[M + H]+. Calcd. For C16H15N3OS:
Chem Biol Drug Des 2016; 87: 499–507
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Wang et al.
C, 64.62; H, 5.08; N, 14.13; S, 10.78%. Found: C, 64.65;
H, 5.04; N, 14.17; S, 10.77%.
CH2NH–), 7.84–7.85 (m, 2H, Ar-H), 8.16 (s, 1H,
CHNHNH), 8.82 (m, 2H, Ar-H), 12.01 (s, 1H, NHNHCO),
12.61 (s, 1H, –OH). EI-MS: 356.2[M + H]+. Calcd. For
C
17H17N5O2S: C, 57.45; H, 4.82; N, 19.70; S, 9.02%.
4-Amino-2-(4-methoxyphenyl)-5,6,7,8-
Found: C, 57.41; H, 4.89; N, 19.63; S, 8.97%.
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (4d)
Yellow solid, Yield: 39.7%; mp 200–202 °C; IR (cmÀ1):
3491 (NH2). 1H-NMR (DMSO-d6): d 1.70–1.87 (m, 4H,
TBTP-H), 2.73–2.86 (m, 2H, TBTP-H), 2.87–2.96 (m, 2H,
TBTP-H), 3.91 (s, 3H, –OCH3), 6.78 (s, 2H, –NH2), 7.01–
7.09 (d, 2H, Ph-H, J = 7.4 Hz), 7.92–7.98 (d, 2H, Ph-H,
Synthesis of 2-(4-hydroxyphenyl)-4-hydroxy-
5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyri-
midine (8)
A mixture of compound 5 (1 g) and 4-hydroxybenzonitrile
(2 g) in dioxane (15 mL) was heated at 80 °C for 10 min;
then, 20 mL of concentrated hydrochloric acid was added
and reacted for 6 h. On completion of reaction (TLC), the
mixture was cooled and poured onto cold water. The mix-
ture was then basified with ammonium hydroxide. The
obtained solid was filtered and purified by column chro-
matography on silica gel (Ethyl acetate/petroleum ether) to
give 8 as a white solid (35.1%). Mp >250 °C; IR (cmÀ1):
3100 (NH), 3095 (OH). 1H-NMR (DMSO-d6): d 1.70–1.78
(m, 4H, TBTP-H), 2.71–2.80 (m, 2H, TBTP-H), 2.85–2.90
(m, 2H, TBTP-H), 6.84–6.89 (d, 2H, Ph-H, J = 7.2 Hz),
7.96–8.04 (d, 2H, Ph-H, J = 7.2 Hz), 10.21 (s, 1H, Ph-
OH), 12.30 (s, 1H, TBTP-NH2). EI-MS: 299.4[M + H]+.
Calcd. For C16H14N2O2S: C, 64.41; H, 4.73; N, 9.39; S,
10.75%. Found: C, 64.35; H, 4.67; N, 9.49; S, 10.83%.
J = 7.3 Hz).
EI-MS:
311.8[M + H]+.
Calcd.
For
C
17H17N3OS: C, 65.57; H, 5.50; N, 13.49; S, 10.30%.
Found: C, 65.63; H, 5.52; N, 13.40; S, 10.32%.
Synthesis of ethyl 2-amino-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carboxylate (5)
Cyclohexanone (0.020 mol), sulfur (0.020 mol), ethyl
cyanoacetate (0.020 mol), and ethanol (4 mL) were mixed
and stirred together. To this mixture, diethylamine
(0.025 mol) was added dropwise for up to half an hour
and stirred continuously for another 3 h at room tempera-
ture. The reaction mixture was kept in a refrigerator over-
night. The separated solid was filtered the next day, and
washed with 4 mL of chilled 50% methanol to give 5 as
light yellow crystals. Yield: 89.8%; mp 113–115 °C; IR
(cmÀ1): 3165 (NH), 2988 (CH), 1649 (C=O). 1H-NMR
(CDCl3): d 6.01 (s, 2H), 4.25 (q, 2H, J = 7.2 Hz), 2.71–
2.69 (m, 2H), 2.48–2.46 (m, 2H), 1.75–1.73 (m, 4H), 1.33
(t, 3H, J = 7.2 Hz) (23,24).
In vitro FGFR-1 kinase assays
The ability of all target compounds to inhibit the activation
of FGFR1 kinase domain was detected by Caliper Mobility
Shift Assay on EZ Reader (Caliper Life Sciences, Hopking-
ton, MA, USA). According to the instructions provided, the
concentration of compounds was 10 lM, and the reference
inhibitor staurosporine (Sigma, St.Louis, MO, USA) was
used as the control.
Synthesis of 2-(chloromethyl)-4-hydroxy-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (6)
A mixture of compound 5 (1 g) and chloroacetonitrile
(1 mL) in dioxane (15 mL) was heated at 80 °C for
10 min; then, 20 mL of concentrated hydrochloric acid
was added and reacted for 6 h. On completion of reaction
(TLC), the mixture was cooled and poured onto cold
water. The mixture was then basified with ammonium
hydroxide. The obtained solid was filtered, washed by
methanol, dried, and crystallized from DMF to give 6 as a
pale white crystalline solid (54.6%).
MTT Assay for antitumor activity at the cellular
level
The MTT assay was performed to evaluate the cytotoxic and
antiproliferative activities of all compounds. Three cell lines
(A549, H460 and U251) were seeded (2000–20 000 cells per
well) in 96-well plates, respectively. After incubation for 24 h
in serum-containing media, the cells were treated with inhibi-
tors (50, 10, 2, and 0.4 lM), diluted with a culture medium for
24 h at 37 °C under a 5% CO2 atmosphere. Thereafter,
20 lL of the MTT reagent (5 mg/mL; Sigma) was added to
each well, and the plates were incubated for 4 h at 37 °C.
Finally, the absorbance at 490 nm was read using an
ELX800 microplate reader. Compound 16 was used as the
positive control, and DMSO was used as negative control. All
experiments were performed parallel in triplicate.
Synthesis of N’-[(4-hydroxy-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)-
methyl] isonicotin ohydrazide (7)
A
mixture of compound 6 (0.2 mmol) and isoniazid
(0.8 mmol) in dioxane (10 mL) was refluxed overnight. On
completion of reaction (TLC), the solution was concen-
trated under reduced pressure and the residue was puri-
fied by column chromatography on silica gel (Ethyl
acetate/petroleum ether) to give 7 as a yellow solid (66%).
mp >250 °C; IR (cmÀ1): 3300 (NH), 1655 (C=O). 1H-NMR
(DMSO-d6): d 1.74–1.82 (m, 4H, TBTP-H), 2.72–2.78 (m,
2H, TBTP-H), 2.88–2.90 (m, 2H, TBTP-H), 3.91 (s, 2H,
Docking
To gain further insight into the interaction mode between
these derivatives and FGFRs, molecular docking analysis
502
Chem Biol Drug Des 2016; 87: 499–507

Tetrahydrobenzothieno[2,3-d]Pyrimidines Antitumor
was performed by AutoDock version 4.2 (25). A crystal
structure (Protein Data Bank code: 3JS2) was selected for
the construction of the docking template (26). To better
prepare the target protein as a template, the ligand and
crystallographic water were removed. Subsequently, com-
pounds 3g and 4c were chosen to dock with the validated
template.
Results and Discussion
Chemistry
Based on the core scaffold of 5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidine, 13 compounds 3a–g, 4a–d, 7,
and 8 were designed and synthesized. Among these, 7
compounds (3a, 3b, 3e, 3f, 3g, 4c, and 7) are first
reported here. These target compounds were further char-
acterized by physicochemical and spectral studies (IR, 1H
NMR, ESI-MS). IR spectra showed characteristic bands of
amide (3200–3500 cmÀ1). The 1H spectrum was carried
out at 600 MHz and showed a characteristic peak pattern.
Synthetic pathways of the designed compounds were
shown in Schemes 1 and 2, respectively.
Statistical analysis
Data were presented as mean Æ SD of three parallel mea-
surements. Statistical significance was analyzed using a
two-tailed Student’s t-test for comparison of two groups. In
all cases, post hoc comparisons of the means of individual
groups were performed using one-way analysis of variance
(ANOVA). p < 0.05 was considered as significant. All statistical
analyses were performed by GraphPad Prism version 5.0
(GraphPad Inc., San Diego, CA, USA) or Statistical Product
and Service Solutions version 16.0 (SPSS Inc., Chicago, IL,
USA). For data analysis: IC50 values were obtained using
GraphPad Prism version 5.0 (GraphPad Inc.).
Structural optimization was performed by focusing on the
C-2 position of the tetrahydrobenzothienopyrimidine
scaffold. The influence of key factors, such as reaction
temperature, and the yield of the target compounds were
investigated. The key intermediate of 2-amino-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carbonitrile (1) was pre-
H2N
H2N
O
Cl
R
N
N
CN
iii
i
ii
N
N
NH2
S
S
S
3a-g
2
1
v
i
H2N
N
R
N
S
4a-d
N
R=
R=
N
R=
N
N
3d
N
O
R=
R=
3a
3b
3c
HN
3f
O
H N
3e
R=
HN
R=
HN
COOH
R=
N
3g
CH3
4a
OCH3
R=
OH
R=
R=
4b
4d
4c
Scheme 1: Route for the synthesis of 4-amino-tetrahydrobenzothieno[2,3-d]pyrimidine derivatives 3a–g, 4a–d. Reagents and conditions:
(i) sulfur, malononitrile, morpholine, ethanol, 60 °C, 12 h; (ii) chloroacetonitrile, conc HCl, dioxane, 80 °C, 6 h; (iii) amine, dioxane, reflux,
20 h; (iv) RCN, HCl, dioxane, 80 °C,12 h.
Chem Biol Drug Des 2016; 87: 499–507
503

Wang et al.
HO
HO
O
Cl
R
N
N
t
COOE
N
N
NH2
S
S
S
7
6
5
Scheme 2: Route for the synthesis of
tetrahydrobenzothieno[2,3-d]pyrimine-4-one
derivatives 7 and 8. Reagents and
conditions: (i) sulfur, ethyl cyanoacetate,
morpholine, ethanol, 60 °C, 12 h; (ii)
chloroacetonitrile, conc HCl, dioxane, 80 °C,
6 h; (iii) amine, dioxane, reflux, 20 h; (iv)
RCN, HCl, dioxane, 80 °C,12 h.
O
HO
N H
H N
N
=
R
OH
=
R
R
N
N
S
7
8
8
pared by means of a Gewald’s reaction (19,26). 4-amino-
2-chloromethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyr-
imidine (2) was prepared with an excellent yield by treating
1 with chloroacetonitrile in dioxane at 80 °C. The reaction
Antitumor activities in vitro based on MTT assay
All synthesized compounds were further screened for their
antitumor activities in vitro. Three cancer cell lines known
to express high levels of FGFR1 were tested in MTT assay
including H460, A549, and U251 (27,28). The data were
summarized in Table 1. The results indicated that
compounds showed different antitumor activities, which
corresponded to the inhibition of FGFR1. Compound 3g
displayed good activities against all three cells (H460,
A549, and U251) with IC50 values of 7.7, 18.9, and
13.3 lM, respectively.
of
4-amino-2-chloromethyl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidine (2) with various nitriles afforded 4-
amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine
(3a–g) at 35–61% yields. Similarly, compounds (4a–d)
were prepared by 2-amino-4,5,6,7-tetrahydrobenzo[b]thio-
phene-3-carbonitrile (1) with various nitriles. Spectral data
of all synthesized compounds were in full agreement with
the proposed structures.
Docking
Inhibition of FGFR1 kinase activity in vitro
The docking results revealed that compound 4c formed
two hydrogen bonds with the hydroxyl oxygen of Lys-514
All compounds were screened by Caliper Mobility Shift
Assay on EZ Reader (Caliper Life Sciences) on kinase
FGFR1 in vitro while staurosporine was used as the refer-
ence compound. As shown in Figure 2, the general trend
showed that the majority of the analogs tested appeared
to show concentration-dependent inhibition to FGFR1.
Compounds 3g, 4c, and 7 showed the highest potency
whether at 10, 1, or 0.1 lM. One suggestion put forward
was that the results could be due to the space structure
of the ATP site of FGFR1, which was large enough to con-
tain larger molecules and formed more bonds with these
molecules. Among the tested compounds, 3g exhibited
the best performance at a concentration of 10 lM.
ꢀ
and Asp-64, the distance between is 3.1 and 2.0 A,
respectively (Figure 3A); while compound 3g formed six
hydrogen bond, its carbonyl oxygen being hydrogen-
bonded by the side chains of Asn-568 and Glu-571,
hydrogen to nitrogen being hydrogen-bonded by the side
chains of Ala-564, Leu484, and Gly-487 (Figure 3B). It
was different to the reference, and ligands were bind in
the hinge region and featured two hydrogen bonds with
Ala564 via the amido fragments (O=C–NH) in the pseu-
dothiohydantoin and pyrimidinone rings (26). Although
compound 3g formed more hydrogen bonds than
compound 4c, the distance of the hydrogen bonds were
100
10 μM
1 μM
0.1 μM
80
60
40
20
0
Figure 2: Inhibition of FGFR1 kinase activity
by the tested compounds in vitro. The
activity of all synthesized compounds toward
in inhibiting FGFR1 kinase activity was
determined by Caliper Mobility Shift Assay
on EZ Reader.
504
Chem Biol Drug Des 2016; 87: 499–507

Tetrahydrobenzothieno[2,3-d]Pyrimidines Antitumor
Table 1: The IC50 values for the tested compounds on cell proliferation based on MTT assay
IC50 values/lM
Compounds
H460
A549
U251
Compound 16
32.6 Æ 1.9
260.6 Æ 10.5***
236.4 Æ 0.2***
>1000***
161.3 Æ 13.7***
87.2 Æ 2.5***
25.1 Æ 0.8###
7.7 Æ 2.1###
>1000***
98.3 Æ 8.4***
41.3 Æ 3.4***
84.3 Æ 1.5***
19.1 Æ 1.3###
69.5 Æ 1.4***
47.1 Æ 1.2
86.3 Æ 2.1
361.4 Æ 8.2***
>1000***
287.0 Æ 12.5***
149.3 Æ 1.5***
102.4 Æ 5.3***
27.6 Æ 2.3###
13.3 Æ 2.4###
>1000***
3a
3b
3c
3d
3e
3f
328.6 Æ 0.7***
131.2 Æ 1.7***
347.7 Æ 12.0***
288.2 Æ 8.7***
75.6 Æ 2.6***
38.3 Æ 2.5###
18.9 Æ 2.3###
346.7 Æ 11.0***
256.8 Æ 18.9***
89.6 Æ 6.2***
74.6 Æ 1.1***
23.1 Æ 1.8###
86.9 Æ 2.1***
3g
4a
4b
4c
4d
7
87.1 Æ 8.2
37.2 Æ 2.3***
56.4 Æ 1.7***
21.0 Æ 1.6###
85.6 Æ 1.5
8
Each value represents the mean Æ SEM from three experiments significantly different from compound 16 at ***p < 0.001 (Student’s t-test).
### stands for compounds which show significantly higher activity than compound 16. Bold value shows the most active compound.
A
B
Figure 3: Computed binding geometry of the new inhibitors Compound 4c and 3g, with the FGFR1 ATP-binding pocket.(A: Compound
4c formed 2 hydrogen bonds. B: Compound 3g formed 6 hydrogen bonds).
farther away. Also the free energies of binding were
À7.22 kcal/mol (3g) and À8.97 kcal/mol (4c). It was obvi-
ous that docking results were consistent with the activity.
phenyl (see 4c). For all, 3g showed a significantly affect on
inhibiting the FGFR than any other compounds.
Conclusion
Structure–activity relationship
In summary, a new class of tetrahydrobenzothienopyrim-
idine derivatives were designed, synthesized, and evalu-
ated for their antitumor activity in various cancer cells with
high FGFR1 kinase activity. Although most compounds
showed a different inhibition of FGFR1, the compound 3g
showed better activity toward FGFR1 in this research, sug-
gesting that compounds with side chains substituted with
larger groups exhibited better activity. This could be due
to the space structure of the FGFR1 ATP pocket, which
was large enough to accommodate larger molecules and
could form more bonds with these molecules. The results
Considering the whole results, we here analyze the struc-
ture–activity relationship (SAR). Firstly, compare 3g with 7,
we found –NH2 is more favorable than –C=O in the C-4
position, this was opposite to the original intention. Sec-
ondly, the activities were slightly affected by the type of
the substituents on the C-2 position (see 3a, 3b, 3c, 3d,
and 4a). Thirdly, compared 4a with 4b, one can see aro-
matic substituted showed more positive activities than
methyl. Furthermore, activities appeared significantly
increase when C-2 position is substituted by 4-hydroxy-
Chem Biol Drug Des 2016; 87: 499–507
505

Wang et al.
of this article has broaden the application of tetrahy-
drobenzothieno[2,3-d]pyrimidine core.
focal FGFR1 amplification associates with therapeuti-
cally tractable FGFR1 dependency in squamous cell
lung cancer. Sci Transl Med, 2, 62ra93.
10. Ye F., Chen L., Hu L., Xiao T., Yu S., Chen D., Wang
S. (2015) Design, synthesis and preliminary biological
evaluation of C-8 substituted guanine derivatives as
small molecular inhibitors of FGFRs. Bioorg Med Chem
Lett;25:1556–1560.
11. Ye F., Wang Y., Nian S., Wang Y., Chen D., Yu S.,
Wang S. (2015) Synthesis and evaluation of biological
and antitumor activities of 5,7-dimethyl-oxazolo [5,4-d]
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Acknowledgments
This work was supported by the National Natural Science
Foundation of China (81373262) for financial support. The
IR, 1H-NMR, and ESI-MS were recorded by the Analysis
and Test Center of Wenzhou Medical University.
Conflict of Interest
bitors of FGFR1. J Enzyme Inhib Med Chem.
doi:10.3109/14756366.2014.1002401
The authors confirm that this article content has no conflict
of interests.
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