Design, synthesis and molecular mechanisms of novel dual inhibitors of heat shock protein 90/phosphoinositide 3-kinase alpha (Hsp90/PI3Kα) against cutaneous melanoma

Article abstract of DOI:10.1080/14756366.2019.1596903

Overexpression of heat shock protein 90 (Hsp90) is common in various types of cancer. In cutaneous melanoma, a cancer with one of the high levels of Hsp90 overexpression, such expression was correlated with a panel of protein kinases, thus offering an opportunity to identify Hsp90-based multi-kinase inhibitors for novel cancer therapies. Towards this goal, we utilized a 2,4-dihydroxy-5-isopropylbenzate-based Hsp90 inhibitor scaffold and thieno[2,3-d]pyrimidine-based kinase inhibitor scaffold to develop a Hsp90-inhibiting compound library. Our inhibitory compound named 8m inhibited Hsp90 and PI3Kα with an IC₅₀ value of 38.6 nM and 48.4 nM, respectively; it displayed improved cellular activity which could effectively induce cell cycle arrest and apoptosis in melanoma cells and lead to the inhibition of cell proliferation, colony formation, migration and invasion. Our results demonstrated 8m to be a promising lead compound for further therapeutic potential assessment of Hsp90/PI3Kα dual inhibitors in melanoma targeted therapy.

Full text of DOI:10.1080/14756366.2019.1596903

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and molecular mechanisms of
novel dual inhibitors of heat shock protein 90/
phosphoinositide 3-kinase alpha (Hsp90/PI3Kα)
against cutaneous melanoma
Feifei Qin, Yali Wang, Xian Jiang, Yujia Wang, Nan Zhang, Xiang Wen, Lian
Wang, Qinglin Jiang & Gu He
To cite this article: Feifei Qin, Yali Wang, Xian Jiang, Yujia Wang, Nan Zhang, Xiang Wen, Lian
Wang, Qinglin Jiang & Gu He (2019) Design, synthesis and molecular mechanisms of novel
dual inhibitors of heat shock protein 90/phosphoinositide 3-kinase alpha (Hsp90/PI3Kα) against
cutaneous melanoma, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 909-926, DOI:
10.1080/14756366.2019.1596903
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 909–926
https://doi.org/10.1080/14756366.2019.1596903
RESEARCH PAPER
Design, synthesis and molecular mechanisms of novel dual inhibitors of heat
shock protein 90/phosphoinositide 3-kinase alpha (Hsp90/PI3Ka) against
cutaneous melanoma
a,b
Feifei Qina,b , Yali Wang , Xian Jianga,b, Yujia Wanga,b, Nan Zhanga,b, Xiang Wena,b, Lian Wanga,b, Qinglin Jiangc
ꢀ
ꢀ
and Gu Hea,b
aDepartment of Dermatology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center
b
for Biotherapy, Chengdu, China; Department of Cardiology, West China Hospital, Sichuan University and Collaborative Innovation Center for
c
Biotherapy, Chengdu, China; School of Pharmacy and Sichuan Province College Key Laboratory of Structure-Specific Small Molecule Drugs,
Chengdu Medical College, Chengdu, China
ABSTRACT
ARTICLE HISTORY
Received 6 September 2018
Revised 9 March 2019
Accepted 14 March 2019
Overexpression of heat shock protein 90 (Hsp90) is common in various types of cancer. In cutaneous mel-
anoma, a cancer with one of the high levels of Hsp90 overexpression, such expression was correlated
with a panel of protein kinases, thus offering an opportunity to identify Hsp90-based multi-kinase inhibi-
tors for novel cancer therapies. Towards this goal, we utilized a 2,4-dihydroxy-5-isopropylbenzate-based
Hsp90 inhibitor scaffold and thieno[2,3-d]pyrimidine-based kinase inhibitor scaffold to develop a Hsp90-
inhibiting compound library. Our inhibitory compound named 8m inhibited Hsp90 and PI3Ka with an IC50
value of 38.6 nM and 48.4 nM, respectively; it displayed improved cellular activity which could effectively
induce cell cycle arrest and apoptosis in melanoma cells and lead to the inhibition of cell proliferation,
colony formation, migration and invasion. Our results demonstrated 8m to be a promising lead com-
pound for further therapeutic potential assessment of Hsp90/PI3Ka dual inhibitors in melanoma tar-
geted therapy.
KEYWORDS
Apoptosis; melanoma;
hsp90; PI3Ka;
kinase inhibitor
1. Introduction
approved for melanoma therapy9–12. However, there are still many
melanoma patients who do not response to these therapies.
Hsp90 is an important molecular chaperone protein that dir-
ectly binds to transition state conformations during the late stages
of protein assembly. It usually interacts with various client proteins
to construct multi-chaperone protein complexes and regulates
protein-protein interaction networks to carry out cellular functions
of oncogenic signaling transduction factors. Hsp90 is often overex-
pressed in tumor tissues because it maintains the correct folding
of various client oncoproteins.
Hsp90 inhibitors are structurally diversified by their scaffold,
e.g. benzoquinone from geldanamycin, resorcinol derivatives, pur-
ine-based Hsp90 inhibitors and 2-aminobenzamide derivatives.
These four major classes of Hsp90 inhibitors bind to the N-
terminal Hsp90 domain13–15. Marcu et al. reported coumarin-based
novobiocin binding at the second ATP site of Hsp90 chaperone C-
terminal domain16–19. There are more than twenty Hsp90 N-
terminal domain inhibitors at different stages of clinical trials,
however, no Hsp90 inhibitor has ever been approved by FDA. The
potential toxicities and knowledge gaps in how to classify patients
who can benefit from Hsp90 inhibitory therapy have limited the
Melanoma is a type of skin cancer with one of the highest moral-
ity rates. This disease accounts for over half of all cutaneous can-
cer-related deaths but is only one-tenth of newly diagnosed skin
cancers1,2. The incidence rate of melanoma has risen continually
in the past twenty years. Although early-stage melanoma patients
usually can be cured by dermatologic surgery, their prognosis
varies by tumor stages and mutation status3. In general, the
stages of melanoma are characterized using the TNM system from
the American Joint Committee on Cancer (AJCC). Here, we
referred to The Cancer Genome Atlas (TCGA) network to help
describe the genomic mutation landscape of cutaneous melanoma
using biopsies from 331 patients4–7
.
Most melanoma can be classified into four driver mutations
using biostatistical analysis: B-Raf mutated, Ras mutated, NF1
mutated and Triple-WT (wild-type)8. B-Raf, Ras or NF1 mutated
patients often also have activation of MAPK pathway, and Triple-
WT patients usually have activated receptor tyrosine kinase (RTK)
and serine/threonine kinase (STK), such as c-kit, CDK family, PI3K
family and Akt family, and so on5. Some targeted therapy
approaches, e.g. vemuragenib (B-Raf inhibitor), cobimetinib (MEK
inhibitor) and Yervoy (CTLA-4 mono-antibody) have been clinical applications of Hsp90 inhibitors.
CONTACT Qinglin Jiang
Chengdu Medical College, Chengdu 610500, China; Xian Jiang
jql_cmc@163.com
School of Pharmacy and Sichuan Province College Key Laboratory of Structure-Specific Small Molecule Drugs,
jennyxianj@163.com; Gu He hegu@scu.edu.cn Department of Dermatology, State Key
Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu 610041, China
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

910
F. QIN ET AL.
There have been some advances achieved by combining 2.3. The synthesis and characterization of key intermediates
Hsp90 inhibitors and other targeted therapeutics, e.g. HER2 mono- and compounds 8a-n
antibody trastuzumab, ALK inhibitor crizotinib, and so on20–29. It is
6-(tert-butyl) 3-ethyl 2-amino-4,7-dihydrothieno[2,3-c]pyridine-
therefore reasonable to discover novel inhibitors targeting Hsp90
and the chaperone protein simultaneously, we shall focus on the
discovery multitarget small molecular inhibitors of Hsp90 and its
oncogenic client kinase proteins.
3,6(5H)-dicarboxylate (1)
To a mixture of N-Boc-4-piperidone (10.00 g, 50 mmol), ethyl cya-
noacetate (5.66 g, 50.0 mmol), and sulfur (1.60 g, 50.0 mmol) in
absolute ethanol (100 ml), triethylamine (10 ml) was added and
refluxed for 12 h; the reaction mixture was concentrated and the
residue was partitioned between water and ethyl acetate. The
organic layer was separated, and concentrated, and the crude
product was recrystallized with ethanol (150 ml), to give 1. Yield
86%, white powder. 1H NMR (400 MHz, Chloroform-d) d 6.02 (s,
2H), 4.35 (s, 2H), 4.26 (q, J ¼ 7.0 Hz, 2H), 3.61 (t, J ¼ 5.7 Hz, 2H),
2.80 (t, J ¼ 5.7 Hz, 2H), 1.48 (s, 9H), 1.34 (t, J ¼ 7.0 Hz, 3H).
In the current study, we report the design, synthesis and
molecular mechanism studies of Hsp90/protein kinases multi-
inhibitors. Considering the structural scaffolds of previously
reported Hsp90 inhibitors and kinase inhibitors in clinical study or
application studies, we employed the 2,4-dihydroxy-5-isopropyl-
benzate fragment for Hsp90 inhibition and the thieno[2,3-d]pyr-
imidine scaffold for kinase inhibition, respectively. The association
between 4-substituted groups on the thieno[2,3-d]pyrimidine scaf-
fold and in vitro kinase inhibition, cell proliferation, apoptosis,
migration and invasion was measured as well as evaluation of an
in vivo xenograft model and the molecular mechanisms of these
novel Hsp90/PI3K dual inhibitors as determined by protein
immunoblotting and immunoprecipitation assays.
tert-butyl 4-oxo-3,5,6,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-d]pyr-
imidine-7(4H)-carboxylate (2)
A mixture of (1) (3.26 g, 10.0 mmol) and formamidine acetate
(1.56 g, 15.0 mmol) was stirred in DMF for 12 h at 100 ꢁC. The reac-
tion mixture was cooled, water was added, and the precipitate
formed was collected by filtration, and washed thoroughly with
water to give 2, Yield, 90%. 1H NMR (400 MHz, Chloroform-d) d
11.99 (s, 1H), 7.99 (s, 1H), 4.66 (s, 2H), 3.74 (t, J ¼ 5.8 Hz, 2H), 3.13
(t, J ¼ 5.8 Hz, 2H), 1.50 (s, 9H).
2. Materials and methods
2.1. Cell lines and reagents
B16, HepG2, A549, SW620, MCF-7 and Hela cells were obtained
from ATCC and maintained by State Key Laboratory of
Biotherapy, Sichuan University in DMEM medium supplemented
with 10% fetal bovine serum (FBS, GIBCO, Australia). MTT was
tert-butyl 4-chloro-5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimi-
dine-7(6H)-carboxylate (3)
A
mixture of (2) (16.32 g, 50.0 mmol), SOCl2 (100 ml) and
purchased from Sigma-Aldrich. Antibodies against hsp90a, DMF(2 ml) was refluxed for 4 h. The reaction mixture was cooled
stat3 were purchased from Genetex. Antibodies against Bcl-2, in an ice bath and then carefully neutralized by the addition of
Bim, caspase-3, caspase-8, caspase-9, FasL, p27, c-myc, N-cad- aqueous sodium bicarbonate solution. The resulting mixture was
herin, EGFR, pEGFR(T1068), pEGFR(T1173), C-Raf, pC-Raf, ERK1/2, extracted with ethyl acetate, the organic layer was separated,
pERK1/2, Akt, pAkt(S308), pAkt(S473), P90RSK, pP90RSK, Met, dried overMgSO4, and concentrated, and the crude product was
pMet, PI3K, B-Raf, pB-Raf, hif1-a were purchased from Cell purified by silica gel column chromatography using a mixture of
Signaling Technology. Antibodies against poly polymerase petroleum ether: dichloromethane (5:1), to give 3, Yield, 85%,
(PARP), FasL, JNK, pJNK, Met, pMet were purchased from Abcam. white powder. 1H NMR (400 MHz, Chloroform-d) d 8.77 (s, 1H),
Antibodies against p21, b-actin, cytochrome c, Bad, Bax, E-cad- 4.74 (s, 2H), 3.80 (t, J ¼ 5.8 Hz, 2H), 3.21 (t, J ¼ 5.8 Hz, 2H), 1.51
herin, vimentin, MMP2, MMP9, ZEB1, b-catenin, hsp90b, hsp70, (s, 9H).
p53, mdm2, cdk2, cdk4, cdk6, cdc37, MNK1, ERK5, GAPDH
and Secondary antibodies (HRP-conjugated sheep anti-rabbit
tert-butyl
4-((3-ethylphenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]-
antibodies or HRP-conjugated sheep anti-mouse antibodies) for
western blot were obtained from Proteintech. Protein A/G Mix
Magnetic Beads for Immunoprecipitation were purchased from
Millipore. The detailed synthesis, characterization and in vitro
biological procedures of compounds 8a–n were described in
Supplementary materials.
thieno[2,3-d]pyrimidine-7(6H)-carboxylate (4a)
To a suspension of 60% NaH(120 mg, 3 mmol) in anhydrous 1,4-
dioxane(10 ml), was added, a solution of 2-ethylaniline (242 mg,
2 mmol) in anhydrous 1,4-dioxane(5 ml), the reaction mixture was
stirred for 20 min at room temperature and then compound (3)
(325 mg, 1 mmol) in 1,4-dioxane (5 ml) was added to the above
reaction mixture, the mixture was stirred for another 2 h at 90 ꢁC,
at the end of the reaction, the excessive NaH was quenched by
H2O, the mixture was concentrated and the residue was parti-
tioned between water and ethyl acetate. The organic layer was
2.2. Molecular docking
The molecular docking was performed by the CDOCKER modules separated, and concentrated, and then the crude product was
embedded in Accelrys Discovery Studio 3.5 Software, with purified by silica gel column chromatography using a mixture
CHARMm27 force field and MMFF94 charge. The protein structures solvent of petroleum ether: ethylacetate (5:1), to give 4a. Yield
of the Hsp90 and PI3Ka were retrieved from PDB database. The 55%, light yellow powder, m.p. 159.0–160.4 ꢁC. 1H NMR (400 MHz,
residues within 1.0 nm around the co-crystallized ligands at the Chloroform-d) d 8.45 (s, 1H), 7.34 – 7.27 (m, 2H), 7.21 (td, J ¼ 7.4,
ATP-binding sites were considered as interacted residues for dock- 1.4 Hz, 1H), 4.72 (t, J ¼ 2.0 Hz, 2H), 3.86 (t, J ¼ 5.7 Hz, 2H), 3.13 (t,
ing sphere definition. The other parameters were set to J ¼ 5.7 Hz, 2H), 2.66 (q, J ¼ 7.6 Hz, 2H), 1.51 (s, 9H), 1.26 (t,
default values.
J ¼ 7.6 Hz, 3H).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
911
tert-butyl 4-((4-bromophenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]- tert-butyl 4-((3-isopropylphenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]-
thieno[2,3-d]pyrimidine-7(6H)-carboxylate (4h)
thieno[2,3-d]pyrimidine-7(6H)-carboxylate (4b)
Compound (4h) was synthesized from 3-isopropylaniline and com-
pound (3), in a manner similar to (4a). Yield: 56%, White powder,
m. p. 115.7–117.2 ꢁC, 1H NMR (400 MHz, Chloroform-d) d 8.51 (s,
1H), 7.53 (dt, J ¼ 8.1, 1.6 Hz, 1H), 7.38 (t, J ¼ 1.9 Hz, 1H), 7.32 (t,
J ¼ 7.9 Hz, 1H), 7.04 (dt, J ¼ 7.7, 1.3 Hz, 1H), 6.96 (s, 1H), 4.71 (s,
2H), 3.86 (t, J ¼ 5.7 Hz, 2H), 3.16 (d, J ¼ 5.6 Hz, 2H), 2.94 (hept,
J ¼ 6.9 Hz, 1H), 1.51 (s, 9H), 1.28 (d, J ¼ 7.0 Hz, 6H).
Compound (4b) was synthesized from 4-bromoaniline and com-
pound (3), in a manner similar to (4a). Yield 66%, Light yellow
powder, m. p. 178.9–181.7 ꢁC, 1H NMR (400 MHz, Chloroform-d) d
8.52 (s, 1H), 7.58 – 7.46 (m, 4H), 6.94 (s, 1H), 4.71 (s, 2H), 3.85 (t,
J ¼ 5.7 Hz, 2H), 3.14 (d, J ¼ 5.7 Hz, 2H), 1.51 (s, 8H).
tert-butyl
4-((3-chlorophenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]-
thieno[2,3-d]pyrimidine-7(6H)-carboxylate (4c)
tert-butyl 4-((2,4-dimethylphenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]-
thieno[2,3-d]pyrimidine-7(6H)-carboxylate (4i)
Compound (4c) was synthesized from 3-chloroaniline and com-
pound (3), in a manner similar to (4a). Yield 65%, Light yellow
powder, m. p. 180.4–181.5 ꢁC, 1H NMR (400 MHz, Chloroform-d) d
8.55 (s, 1H), 7.82 (s, 1H), 7.48 (s, 1H), 7.29 (t, J ¼ 8.1 Hz, 1H), 7.11
(ddd, J ¼ 8.0, 2.1, 1.0 Hz, 1H), 6.98 (s, 1H), 4.72 (s, 2H), 3.86 (t,
J ¼ 5.7 Hz, 2H), 3.14 (t, J ¼ 5.7 Hz, 2H), 1.51 (s, 9H).
Compound (4i) was synthesized from 2,4-dimethylaniline and
compound (3), in a manner similar to (4a). Yield: 61%, Yellow
powder, m. p. 159.6–161.3 ꢁC, 1H NMR (400 MHz, Chloroform-d) d
8.44 (s, 1H), 7.58 (s, 1H), 7.16 – 7.02 (m, 2H), 6.70 (s, 1H), 4.71 (s,
2H), 3.85 (t, J ¼ 5.7 Hz, 2H), 3.13 (t, J ¼ 5.7 Hz, 2H), 2.34 (s, 3H), 2.27
(s, 3H), 1.51 (s, 9H).
tert-butyl 4-(m-tolylamino)-5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidine-7(6H)-carboxylate (4d)
tert-butyl 4-(piperidin-1-yl)-5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidine-7(6H)-carboxylate (4k)
Compound (4d) was synthesized from m-toluidine and compound
(3), in a manner similar to (4a). Yield 59%, Light yellow powder,
m. p. 150.7–152.4 ꢁC, 1H NMR (400 MHz, Chloroform-d) d 8.51 (s,
1H), 7.45 (dd, J ¼ 8.0, 2.2 Hz, 1H), 7.41 (d, J ¼ 1.8 Hz, 1H), 7.28 (d,
J ¼ 7.8 Hz, 1H), 6.97 (ddt, J ¼ 7.6, 1.7, 0.9 Hz, 1H), 6.94 (s, 1H), 4.70
(s, 2H), 3.85 (t, J ¼ 5.7 Hz, 2H), 3.13 (t, J ¼ 5.7 Hz, 2H), 2.39 (s, 3H),
1.51 (s, 9H).
Compound 3 (325 mg, 1.0 mmol) was dissolved in piperidine
(15 ml), the mixture was stirred at 100ꢁ C for 1 h. after the reaction
solution was cooled, ethyl acetate (100 ml) were added, and then
the organic layer was washed with water, and then dried over
MeSO4, filtered and evaporated and then the crude product was
purified by silica gel column chromatography using a mixture of
petroleum ether: ethyl acetate (3:1), to give 4k, yield: 91%, White
1
powder, H NMR (400 MHz, CDCl3) d 8.52 (s, 1H), 4.71 (s, 2H), 3.67
(s, 2H), 3.41 – 3.31 (m, 4H), 3.05 (s, 2H), 1.78 – 1.65 (m, 6H), 1.52
tert-butyl
4-((3-fluorophenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]-
(s, 9H).
thieno[2,3-d]pyrimidine-7(6H)-carboxylate (4e)
Compound (4e) was synthesized from 3-fluoroaniline and com-
pound (3), in a manner similar to (4a). Yield: 69%, Light yellow
powder, m. p. 164.4–165.0 ꢁC, 1H NMR (400 MHz, Chloroform-d) d
8.55 (s, 1H), 7.73 (d, J ¼ 11.0 Hz, 1H), 7.32 (td, J ¼ 8.1, 6.3 Hz, 1H),
7.23 (d, J ¼ 8.3 Hz, 1H), 7.03 (s, 1H), 6.83 (tdd, J ¼ 8.2, 2.5, 1.0 Hz,
1H), 4.72 (s, 2H), 3.86 (t, J ¼ 5.7 Hz, 2H), 3.14 (t, J ¼ 5.7 Hz, 2H), 1.51
(s, 9H).
tert-butyl 4-(pyrrolidin-1-yl)-5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidine-7(6H)-carboxylate (4l)
Compound (4l) was synthesized from pyrrolidine and compound
1
(3), in a manner similar to (4k). Yield: 93%, White powder, H NMR
(400 MHz, CDCl3) d 8.36 (s, 1H), 4.68 (s, 2H), 3.69 (t, J ¼ 6.6 Hz, 4H),
3.62 (s, 2H), 2.99 (s, 2H), 1.99 – 1.87 (m, 4H), 1.52 (s, 9H).
tert-butyl
d]pyrimidine-7(6H)-carboxylate (4m)
4-morpholino-5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-
tert-butyl
4-((4-chlorophenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]-
thieno[2,3-d]pyrimidine-7(6H)-carboxylate (4f)
Compound (4m) was synthesized from morpholine and com-
pound (3), in a manner similar to (4k). Yield: 95%, White powder,
1H NMR (400 MHz, CDCl3) d 8.56 (s, 1H), 4.72 (s, 2H), 3.93 – 3.83
(m, 4H), 3.69 (s, 2H), 3.50 – 3.41 (m, 4H), 3.04 (s, 2H), 1.52 (s, 9H).
Compound (4f) was synthesized from 4-chloroaniline and com-
pound (3), in a manner similar to (4a). Yield: 68%, Light yellow
powder, m. p. 183.7–185.6 ꢁC, 1H NMR (400 MHz, Chloroform-d) d
8.52 (s, 1H), 7.64 – 7.57 (m, 2H), 7.40 – 7.32 (m, 2H), 6.95 (s, 1H),
4.71 (s, 2H), 3.86 (t, J ¼ 5.7 Hz, 2H), 3.15 (t, J ¼ 5.7 Hz, 2H), 1.51
(s, 9H).
tert-butyl 4-(dimethylamino)-5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidine-7(6H)-carboxylate (4n)
Compound (4n) was synthesized from 30% dimethylamine in
methanol and compound (3), in a manner similar to (4k). Yield:
89%, White powder, 1H NMR (400 MHz, CDCl3) d 8.47 (s, 1H), 4.71
(s, 2H), 3.65 (t, J ¼ 4.7 Hz, 2H), 3.06 (s, 6H), 3.04 (s, 2H), 1.52 (s, 9H).
tert-butyl 4-((3-methoxyphenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]-
thieno[2,3-d]pyrimidine-7(6H)-carboxylate (4g)
Compound (4g) was synthesized from 3-methoxyaniline and com-
pound (3), in a manner similar to (4a). Yield: 58%, Light yellow
powder, m. p. 136.3–139.6 ꢁC, 1H NMR (400 MHz, Chloroform-d) d
8.52 (s, 1H), 7.41 (t, J ¼ 2.3 Hz, 1H), 7.29 (t, J ¼ 8.1 Hz, 1H), 7.10
(ddd, J ¼ 8.0, 2.1, 0.9 Hz, 1H), 6.97 (s, 1H), 6.70 (ddd, J ¼ 8.3, 2.5,
0.9 Hz, 1H), 4.71 (s, 2H), 3.89 – 3.81 (m, 5H), 3.14 (d, J ¼ 5.7 Hz, 2H),
1.51 (s, 9H).
Methyl 2,4-dihydroxy-5-isopropylbenzoate (5)
A mixture of methyl 2,4-dihydroxybenzoate (1.68 g, 10.0 mmol),
2-bromopropane (1.87 ml, 20.0 mmol), and aluminum chloride
(2.65 g, 20.0 mmol) in CH2Cl2 was stirred at 50ꢁ C for 24 h under
argon. 2-bromopropane (1.87 ml, 20.0 mmol) was added to the

912
F. QIN ET AL.
reaction mixture two times every 8 h. The mixture was neutralized 157.6 – 159.6 ꢁC, 1H NMR (400 MHz, Chloroform-d) d 8.54 (s, 1H),
with 15% NaOH to pH 5, concentrated under reduced pressure,
and then extracted with ethyl acetate. The organic layer was
washed with saturated NaHCO3 solution, dried over MeSO4, con-
7.83 (t, J ¼ 2.1 Hz, 1H), 7.50 (ddd, J ¼ 8.2, 2.2, 1.0 Hz, 1H), 7.30 (t,
J ¼ 8.1 Hz, 1H), 7.10 (ddd, J ¼ 8.0, 2.0, 0.9 Hz, 1H), 7.04 (s, 1H), 4.13
(t, J ¼ 2.0 Hz, 2H), 3.31 (t, J ¼ 5.7 Hz, 2H), 3.07 (tt, J ¼ 5.7,
centrated under reduced pressure, and purified by column to
2.0 Hz, 2H).
1
afford 5, yield 45%, colorless oil. H NMR (400 MHz, Chloroform-d)
d10.8 (s, 1H), 7.64 (s, 1H), 6.34 (s, 1H), 5.50 (s, 1H), 3.90 (s, 3H),
3.15–3.08 (m, 1H), 1.25 (d, J ¼ 10.8 Hz, 6H).
N-(m-tolyl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimi-
din-4-amine (7d)
Compound (7d) was synthesized from compound (4d), in a man-
ner similar to (7a). Yield: 89%, White powder, m. p. 174.9–178.0 ꢁC,
1H NMR (400 MHz, Chloroform-d) d 8.51 (s, 1H), 7.47 (dd, J ¼ 8.0,
2.2 Hz, 1H), 7.43 (d, J ¼ 1.8 Hz, 1H), 7.29 (d, J ¼ 7.8 Hz, 1H), 7.00 (s,
1H), 6.97 (ddt, J ¼ 7.6, 1.7, 0.8 Hz, 1H), 4.12 (t, J ¼ 2.0 Hz, 2H), 3.31
(t, J ¼ 5.7 Hz, 2H), 3.08 (tt, J ¼ 5.7, 2.0 Hz, 2H), 2.39 (s, 3H)
2,4-bis(benzyloxy)-5-isopropylbenzoic acid (6)
To a solution of methyl 2,4-dihydroxy-5-isopropylbenzoate (2.10 g,
10.0 mmol) and potassium carbonate (4.14 g, 30.0 mmol) in DMF
(50 ml) was added benzyl chloride (2.53 ml, 22.0 mmol). The sus-
pension was heated to 80 ꢁC for 16 h under a nitrogen atmos-
phere. Ethyl acetate (100 ml) and water (100 ml) were added, and
then the organnic layer was washed with water, and then dried
over MeSO4, filtered and evaporated and then the crude product
was purified by silicagel column chromatography using a mixture
of petroleum ether:ethyl acetate (8:1), to give methyl 2,4-bis (ben-
N-(3-fluorophenyl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidin-4-amine (7e)
Compound (7e) was synthesized from compound (4e), in a man-
ner similar to (7a). Yield: 93%, White powder, m. p. 146.6–150.9 ꢁC,
1H NMR (400 MHz, Chloroform-d) d 8.54 (s, 1H), 7.74 (dt, J ¼ 11.1,
2.3 Hz, 1H), 7.35 – 7.27 (m, 1H), 7.25 (s, 1H), 7.09 (s, 1H), 6.82 (tdd,
J ¼ 8.2, 2.5, 1.1 Hz, 1H), 4.12 (t, J ¼ 2.2 Hz, 2H), 3.31 (td, J ¼ 5.8,
3.0 Hz, 2H), 3.07 (tt, J ¼ 5.8, 1.9 Hz, 2H).
zyloxy)-5-isopropylbenzoate; and then
a
solution of THF:
Methanol: Water 100 ml, (3:1:1) mixed by methyl 2,4-bis (benzy-
loxy)-5-isopropylbenzoate (3.90 g, 10.0 mmol) and LiOHꢂH2O
(1.26 g, 30.0 mmol) were stirred for 12 h. Ethyl acetate (100 ml) and
water (100 ml) were added, then the organic layer was washed
with water, dried over MeSO4, filtered and evaporated, the crude
product was purified by silica gel column chromatography using a
mixture of petroleum ether: ethyl acetate (2:1), to give 6, Yield
N-(4-chlorophenyl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidin-4-amine (7f)
1
81%, white powder. H NMR (400 MHz, DMSO-d6) d 10.55 (brs, lH),
8.04 (s, lH), 7. 46–7.34 (m,10H), 6.57 (s,1H), 5.20 (s,2H), 5.16–5.13
(m, 2H), 5.11 (s, 2H), 2.06 (s, 3H).
Compound (7f) was synthesized from compound (4f), in a manner
similar to (7a). Yield: 92%, Light yellow powder, m. p.
171.3–176.0 ꢁC, 1H NMR (400 MHz, Chloroform-d) d 8.50 (s, 1H),
7.69 – 7.51 (m, 2H), 7.40 – 7.28 (m, 2H), 7.00 (s, 1H), 4.11 (t,
J ¼ 2.0 Hz, 2H), 3.30 (t, J ¼ 5.7 Hz, 2H), 3.06 (tt, J ¼ 5.9, 2.0 Hz, 2H).
N-(3-ethylphenyl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidin-4-amine (7a)
Compound 4a (410 mg, 1.0 mmol) was dissolved in ethylacetate
(10 ml) and then 9 N HCl (5 ml) was added, the mixture was stirred
at room temperature for 30 min. The reaction solution was neu-
tralized with sodium hydrogen carbonate, then alkalized by NaOH
to pH ¼ 9. Fifty millilitres of ethylacetate and 30 ml H2O was
added, after stirred adequately, the organic layer was separated,
and concentrated, and then the crude product was purified by sil-
ica gel column chromatography using a mixture solvent of
dichloromethane: methanol (20–50:1), to give 7a, yield 90%, White
powder, m. p. 178.8–180.9 ꢁC, 1H NMR (400 MHz, Chloroform-d) d
8.44 (s, 1H), 7.83 (dd, J ¼ 8.4, 1.4 Hz, 1H), 7.22 – 7.18 (m, 2H), 7.20
(td, J ¼ 7.3, 1.4 Hz, 1H), 6.86 (s, 1H), 4.13 (t, J ¼ 2.0 Hz, 2H), 3.31 (t,
J ¼ 5.7 Hz, 2H), 3.08 (tt, J ¼ 5.8, 2.0 Hz, 2H), 2.67 (q, J ¼ 7.6 Hz, 2H),
1.25 (d, J ¼ 7.6 Hz, 3H).
N-(3-methoxyphenyl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidin-4-amine (7g)
Compound (7g) was synthesized from compound (4g), in a man-
ner similar to (7a). Yield: 90%, White powder, m. p. 179.2–180.5 ꢁC,
1H NMR (400 MHz, Chloroform-d) d 8.52 (s, 1H), 7.44 (t, J ¼ 2.3 Hz,
1H), 7.29 (d, J ¼ 8.1 Hz, 1H), 7.11 (ddd, J ¼ 8.1, 2.1, 0.9 Hz, 1H), 7.04
(s, 1H), 6.69 (ddd, J ¼ 8.3, 2.5, 0.9 Hz, 1H), 4.11 (t, J ¼ 2.0 Hz, 2H),
3.85 (s, 3H), 3.30 (t, J ¼ 5.7 Hz, 2H), 3.07 (tt, J ¼ 5.7, 2.0 Hz, 2H).
N-(3-isopropylphenyl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidin-4-amine (7h)
Compound (7h) was synthesized from compound (4h), in a man-
ner similar to (7a). Yield: 90%, Light yellow powder, m. p.
144.7–151.6 ꢁC, 1H NMR (400 MHz, Chloroform-d) d 8.50 (s, 1H),
7.53 (ddd, J ¼ 8.1, 2.3, 1.1 Hz, 1H), 7.40 (t, J ¼ 2.0 Hz, 1H), 7.32 (t,
J ¼ 7.8 Hz, 1H), 7.03 (dt, J ¼ 8.0, 1.5 Hz, 2H), 4.12 (s, 2H), 3.31 (t,
J ¼ 5.7 Hz, 2H), 3.09 (tt, J ¼ 5.7, 2.0, 2H), 2.94 (hept, J ¼ 7.0 Hz, 1H),
1.29 (d, J ¼ 7.0 Hz, 6H).
N-(4-bromophenyl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidin-4-amine (7b)
Compound (7b) was synthesized from compound (4b), in a man-
ner similar to (7a). Yield: 92%, Light yellow powder, m. p.
178.9–181.7 ꢁC, 1H NMR (400 MHz, Chloroform-d) d 8.52 (s, 1H),
7.58 – 7.46 (m, 4H), 6.94 (s, 1H), 4.71 (s, 2H), 3.85 (t, J ¼ 5.7 Hz, 2H),
3.14 (d, J ¼ 5.7 Hz, 2H), 1.51 (s, 8H).
N-(2,4-dimethylphenyl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]-
thieno[2,3-d]pyrimidin-4-amine (7i)
N-(3-chlorophenyl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidin-4-amine (7c)
Compound (7c) was synthesized from compound (4c), in a
Compound (7i) was synthesized from compound (4i), in a manner
similar to (7a). Yield: 89%, Light yellow powder, m. p.
164.6–169.0 ꢁC, 1H NMR (400 MHz, Chloroform-d) d 8.43 (s, 1H),
manner similar to (7a). Yield: 90%, White powder, m. p. 7.67 – 7.60 (m, 1H), 7.09 – 7.07 (m, 2H), 6.76 (s, 1H), 4.12 (t,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
913
J ¼ 2.0 Hz, 2H), 3.30 (t, J ¼ 5.7 Hz, 2H), 3.07 (tt, J ¼ 5.7, 3.6, 2.0 Hz, (4-((4-bromophenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-
2H), 2.33 (s, 3H), 2.26 (s, 3H).
d]pyrimidin-7(6H)-yl)(2,4-dihydroxy-5-isopropylphenyl)metha-
none (8b)
Compound (4b) (270 mg, 0.5 mmol) and Compound (6) (188 mg,
0.5 mmol) were dissolved in CH2Cl2 (15 ml) and then EDCI
(144 mg,0.5 mmol) and HOBt (101 mg, 0.5 mmol) was added, the
mixture was stirred overnight. CH2Cl2 (50 ml) and H2O (50 ml) was
added, the organic layer was separated and concentrated, the
crude product was purified by silica gel column chromatography
using dichloromethane: acetone (100:1), yield: 73%; The obtained
compound (334 mg, 0.5 mmol) was dissolved in CH2Cl2 (10 ml) and
then (CH3)3SiI (1.00g, 0.68 ml, 0.5 mmol) was added, the reaction
mixture was stirred for 24 h at room temperature. Excessive
(CH3)3SiI was quenched by MeOH, the mixture was concentrated,
and the residue was purified by silica gel column chromatography
using a mixture of dichloromethane: acetone (30:1), to give 8b,
4-(piperidin-1-yl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidine (7k)
Compound (7k) was synthesized from compound (4k), in a man-
ner similar to (7a). Yield: 87%,White powder, 1H NMR (400 MHz,
CDCl3) d 8.50 (s, 1H), 4.72 (s, 2H), 3.66 (s, 2H), 3.40 – 3.31 (m, 4H),
3.03 (s, 2H), 1.77 – 1.64 (m, 6H).
4-(pyrrolidin-1-yl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidine (7l)
Compound (7l) was synthesized from compound (4l), in a manner
similar to (7a). Yield: 88%, White powder, 1H NMR (400 MHz,
CDCl3) d 8.33 (s, 1H), 4.65 (s, 2H), 3.70 (t, J ¼ 6.6 Hz, 4H), 3.62 (s,
2H), 2.97 (s, 2H), 1.97 – 1.84 (m, 4H).
1
yield: 33%, Brown powder, m. p. 248.8–250.2 ꢁC, H NMR (400 MHz,
DMSO-d6) d 9.58 (s, 2H), 8.44 (s, 1H), 8.35 (s, 1H), 7.62 (d,
J ¼ 8.6 Hz, 2H), 7.53 (d, J ¼ 8.7 Hz, 2H), 6.91 (s, 1H), 6.41 (s, 1H),
4.79 (s, 2H), 3.77 (s, 2H), 3.26 (t, J ¼ 5.7 Hz, 2H), 3.08 (hept,
J ¼ 6.9 Hz, 1H), 1.12 (d, J ¼ 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-
d6) d 168.87, 166.63, 157.10, 155.12, 153.30, 152.62, 138.92,
131.67(2C), 130.55, 126.58, 126.37, 126.05, 124.60(2C), 116.91,
115.65, 113.93, 102.68, 26.56, 26.35, 23.10(2C). HRMS (ESI) calcu-
lated for C25H23BrN4O3S [M þ H]þ 539.0747, found 539.0742
4-(pyrrolidin-1-yl)-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidine (7m)
Compound (7m) was synthesized from compound (4m), in a man-
ner similar to (7a). Yield: 89%, White powder, 1H NMR (400 MHz,
CDCl3) d 8.61 (s, 1H), 4.71 (s, 2H), 3.90 – 3.81 (m, 4H), 3.67 (s, 2H),
3.51 – 3.40 (m, 4H), 3.02 (s, 2H).
(4-((3-chlorophenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidin-7(6H)-yl)(2,4-dihydroxy-5-isopropylphenyl)metha-
none (8c)
N,N-dimethyl-5,6,7,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimi-
din-4-amine (7n)
Compound (7n) was synthesized from compound (4n), in a man-
Compound (8c) was synthesized from compound (4c), in a manner
1
ner similar to (7a). Yield: 85%, White powder, 1H NMR (400 MHz, similar to (8b). Yield: 35%, White powder, m. p. 189.9–190.6 ꢁC, H
NMR (400 MHz, DMSO-d6) d 9.58 (s, 2H), 8.48 (s, 1H), 8.37 (s, 1H),
CDCl3) d 8.50 (s, 1H), 4.73 (s, 2H), 3.66 (t, J ¼ 4.7 Hz, 2H), 3.04 (s,
7.81 (t, J ¼ 2.0 Hz, 1H), 7.62 (dd, J ¼ 7.5, 1.8Hz, 1H), 7.37 (t,
6H), 3.02 (s, 2H).
J ¼ 8.1 Hz, 1H), 7.13 (dd, J ¼ 7.9, 1.9 Hz, 1H), 6.92 (s, 1H), 6.41 (s, 1H),
4.80 (s, 2H), 3.78 (s, 2H), 3.25 (t, J ¼ 5.7 Hz, 2H), 3.08 (hept,
J ¼ 7.0 Hz, 1H), 1.12 (d, J ¼ 6.9 Hz, 6H). 13 C NMR (100MHz, DMSO-d6)
(2,4-dihydroxy-5-isopropylphenyl)(4-((3-ethylphenyl)amino)-5,8-
d 168.88, 167.14, 157.14, 155.01, 153.35, 152.84, 141.22, 133.19,
130.67, 130.45, 126.61, 126.30, 126.07, 123.27, 121.62, 120.67,
117.01, 113.94, 102.71, 26.55, 26.38(2C), 23.10. HRMS (ESI) calculated
for C25H23ClN4O3S [M þ H]þ 495.1252, found 495.1253.
dihydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)metha-
none (8a)
Compound (4a) (205 mg, 0.5 mmol) and Compound (6) (188 mg,
0.5 mmol) were dissolved in CH2Cl2 (15 ml) and then EDCI
(144 mg, 0.5 mmol) and HOBt (101 mg, 0.5 mmol) was added, the
mixture was stirred overnight. CH2Cl2 (50 ml) and H2O (50 ml) was
added, the organic layer was separated and concentrated, the
crude product was purified by silica gel column chromatography
using dichloromethane: acetone (100:1), yield: 73%; The obtained
compound (334 mg, 0.5 mmol) was dissolved in THF (10 ml) and
then 10% Pd/C (35 mg, 10%mol) was added, the reaction mixture
was catalytic hydrogenated for 24 h at room temperature in
hydrogen atmosphere. The solvent was concentrated, the residue
was purified by silica gel column chromatography using a mixture
of dichloromethane: acetone (30:1), to give 8a, yield: 66%, White
(2,4-dihydroxy-5-isopropylphenyl)(4-(m-tolylamino)-5,8-dihydropyr-
ido[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)methanone (8d)
Compound (8d) was synthesized from compound (4d), in a man-
ner similar to (8a). Yield: 65%, White powder, m. p. 157.6–160.2 ꢁC,
1H NMR (400 MHz, DMSO-d6) d 9.58 (s, 2H), 8.41 (s, 1H), 8.15 (s,
1H), 7.48 (dd, J ¼ 7.9, 2.1 Hz, 1H), 7.43 (t, J ¼ 1.9 Hz, 1H), 7.23 (t,
J ¼ 7.8 Hz, 1H), 6.92 – 6.90 (m, 2H), 6.41 (s, 1H), 4.79 (s, 2H), 3.77 (s,
2H), 3.26 (t, J ¼ 5.7 Hz, 2H), 3.08 (hept, J ¼ 6.9 Hz, 1H), 2.31 (s, 3H),
1.12 (d, J ¼ 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) d 168.87,
166.80, 157.12, 155.49, 153.35, 153.02, 139.50, 138.10, 130.06,
128.75, 126.60, 126.36, 126.06, 124.61, 123.04, 119.73, 116.67,
113.96, 102.70, 26.62, 26.38, 23.10(2C), 21.59. HRMS (ESI) calculated
for C26H26N4O3S [M þ H]þ 475.1798, found 475.1796.
1
powder, m. p. 227.2–230.1 ꢁC, H NMR (400 MHz, DMSO-d6) d 9.59
(s, 2H), 8.24 (s, 1H), 8.05 (s, 1H), 7.45 – 7.37 (m, 1H), 7.34 – 7.28
(m, 1H), 7.27 – 7.21 (m, 2H), 6.92 (s, 1H), 6.41 (s, 1H), 4.78 (s, 2H),
3.79 (s, 2H), 3.23 (t, J ¼ 5.8 Hz, 2H), 3.08 (hept, J ¼ 6.9 Hz, 1H), 2.56
(q, J ¼ 7.6 Hz, 2H), 1.12 – 1.09 (m, 9H). 13 C NMR (100 MHz, DMSO-
d6) d 168.92, 166.54, 157.13, 156.70, 153.45, 153.30, 140.39, 137.20,
(2,4-dihydroxy-5-isopropylphenyl)(4-((3-fluorophenyl)amino)-5,8-
129.40, 128.85, 128.10, 126.72, 126.56, 126.51, 126.33, 126.04, dihydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)metha-
115.77, 113.88, 102.74, 26.84, 26.34, 24.51, 23.11(2C), 14.44.
HRMS (ESI) calculated for C27H28N4O3S [M þ H]þ 489.1955,
found 489.1953.
none (8e)
Compound (8e) was synthesized from compound (4e), in a man-
ner similar to (8b). Yield: 34%, White powder, m. p.218.1–219.2 ꢁC,

914
F. QIN ET AL.
1H NMR (400 MHz, DMSO-d6) d 9.58 (s, 2H), 8.50 (s, 1H), 8.44 (s, (2,4-dihydroxy-5-isopropylphenyl)(4-((2,4-dimethylphenyl)amino)-
1H), 7.63 (dt, J ¼ 11.7, 2.2 Hz, 1H), 7.45 (dd, J ¼ 8.1, 1.9 Hz, 1H), 7.38
5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)metha-
(td, J ¼ 8.1, 6.6 Hz, 1H), 6.94 – 6.89 (m, 2H), 6.41 (s, 1H), 4.80 (s,
none (8i)
2H), 3.78 (s, 2H), 3.27 (t, J ¼ 5.7 Hz, 2H), 3.08 (hept, J ¼ 6.9 Hz, 1H),
Compound (8i) was synthesized from compound (4i), in a manner
1.12 (d, J ¼ 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) d 168.91,
1
similar to (8a). yield: 63%, White powder, m. p. 254.3–255.4 ꢁC, H
165.42, 162.47 (d, J ¼ 240 Hz, 1C), 157.14, 155.04, 153.32, 151.96,
NMR (400 MHz, DMSO-d6) d 9.58 (s, 1H), 9.57 (s, 1H), 8.24 (s, 1H),
141.00 (d, J ¼ 11 Hz, 1C), 131.06, 130.50(d, J ¼ 9 Hz, 1C), 126.54 (d, 7.98 (s, 1H), 7.32 (d, J ¼ 8.0 Hz, 1H), 7.09 (s, 1H), 7.03 (d, J ¼ 7.4 Hz,
1H), 6.92 (s, 1H), 6.40 (s, 1H), 4.77 (s, 2H), 3.79 (s, 2H), 3.23 (t,
J ¼ 13 Hz, 1C), 126.08, 118.51, 118.50, 117.05, 113.88, 110.65 (d,
J ¼ 5.7 Hz, 2H), 3.08 (hept, J ¼ 6.9 Hz, 1H), 2.29 (s, 3H), 2.13 (s, 3H),
1.12 (d, J ¼ 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) d 168.89,
166.45, 157.10, 156.43, 153.42, 153.30, 135.31, 135.22, 134.22,
131.29, 129.28, 127.12, 127.07, 126.54, 126.39, 126.04, 115.82,
113.91, 102.71, 26.85, 26.35, 23.10(2C), 21.04, 18.45. HRMS (ESI) cal-
culated for C27H28N4O3S [M þ H]þ 489.1955, found 489.1953.
J ¼ 21 Hz, 1C), 109.48 (d, J ¼ 25 Hz, 1C), 102.69, 26.55, 26.37,
23.11(2C). HRMS (ESI) calculated for C25H23FN4O3S [M þ H]þ
479.1548, found 479.1550.
(4-((4-chlorophenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-
d]pyrimidin-7(6H)-yl)(2,4-dihydroxy-5-isopropylphenyl)metha-
none (8f)
(2,4-dihydroxy-5-isopropylphenyl)(4-((3-hydroxyphenyl)amino)-5,8-
dihydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)metha-
none (8j)
Compound (8f) was synthesized from compound (4f), in a manner
1
similar to (8b). Yield: 36%, White powder, m. p. 178.0–182.0 ꢁC, H
Compound (8j) was synthesized from compound (4g), in a man-
ner similar to (8b) 48 h. yield: 25%, Yellow powder, m. p.
NMR (400 MHz, DMSO-d6) d 9.64 (s, 2H), 8.43 (s, 1H), 8.34 (s, 1H),
7.75 – 7.62 (m, 2H), 7.46 – 7.32 (m, 2H), 6.92 (s, 1H), 6.44 (s, 1H),
4.80 (s, 2H), 3.78 (s, 2H), 3.27 (t, J ¼ 5.8 Hz, 2H), 3.08 (hept,
J ¼ 6.9 Hz, 1H), 1.12 (d, J ¼ 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-
d6) d 168.91, 167.02, 157.18, 155.20, 153.40, 152.86, 138.63, 130.43,
128.72(2C), 127.47, 126.58, 126.34, 126.03, 124.11(2C), 116.87,
113.91, 102.74, 26.59, 26.37, 23.11(2C). HRMS (ESI) calculated for
C25H23ClN4O3S [M þ H]þ 495.1252, found 495.1249.
1
146.4–148.2 ꢁC, H NMR (400 MHz, DMSO-d6) d 9.58 (s, 2H), 9.37 (s,
1H), 8.42 (s, 1H), 8.12 (s, 1H), 7.18 (s, 1H), 7.11 (t, J ¼ 8.0 Hz, 1H),
7.02 (d, J ¼ 8.0 Hz, 1H), 6.92 (s, 1H), 6.50 (d, J ¼ 7.9 Hz, 1H), 6.41 (s,
1H), 4.78 (s, 2H), 3.76 (s, 2H), 3.24 (s, 2H), 3.08 (p, J ¼ 7.0 Hz, 1H),
1.12 (d, J ¼ 7.0 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) d 168.85,
166.81, 157.94, 157.11, 155.44, 153.35, 152.98, 140.62, 130.10,
129.52, 126.58, 126.41, 126.05, 116.79, 113.95, 113.16, 111.01,
109.48, 102.70, 26.57, 26.36, 23.11(2C). HRMS (ESI) calculated for
C25H24N4O4S [M þ H]þ 477.1591, found 477.1591.
(2,4-dihydroxy-5-isopropylphenyl)(4-((3-methoxyphenyl)amino)-5,8-
dihydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)metha-
none (8g)
(2,4-dihydroxy-5-isopropylphenyl)(4-(piperidin-1-yl)-5,8-dihydropyr-
ido[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)methanone (8k)
Compound (8k) was synthesized from compound (4k), in a man-
ner similar to (8a). yield: 67%, White powder, m. p. 196.1–198.5 ꢁC,
1H NMR (400 MHz, DMSO) d 9.60 (s, 1H), 9.58 (s, 1H), 8.47 (s, 1H),
6.96 (s, 1H), 6.42 (s, 1H), 4.81 (s, 2H), 3.64 (s, 2H), 3.33 (s, 4H), 3.11
– 3.03 (m, 3H), 1.64 (s, 6H), 1.12 (d, J ¼ 6.7 Hz, 6H). 13 C NMR
(101 MHz, DMSO) d 169.12, 168.16, 162.38, 157.04, 153.45, 152.21,
130.84, 127.14, 126.43, 126.03, 119.85, 114.07, 102.74, 51.41 (s, 2C),
33.12, 29.47, 27.60, 26.29, 25.65 (s, 2C), 24.26 (s), 23.10 (s, 2C).
HRMS (ESI) calculated for C25H24N4O4S [M þ H] þ 452.1882,
found 452.1879.
Compound (8g) was synthesized from compound (4g), in a man-
ner similar to (8a). Yield: 67%, White powder, m. p. 220.4–221.2 ꢁC,
1H NMR (400 MHz, DMSO-d6) d 9.58 (s, 2H), 8.43 (s, 1H), 8.20 (s,
1H), 7.34 – 7.28 (m, 1H), 7.25 (d, J ¼ 4.9 Hz, 2H), 6.92 (s, 1H), 6.67
(td, J ¼ 4.7, 2.6 Hz, 1H), 6.41 (s, 1H), 4.79 (s, 2H), 3.86 – 3.66 (m,
5H), 3.26 (t, J ¼ 5.7 Hz, 2H), 3.08 (hept, J ¼ 6.8 Hz, 1H), 1.12 (d,
J ¼ 6.8 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) d 168.88, 166.89,
159.91, 157.14, 155.36, 153.37, 152.94, 140.77, 130.24, 129.61,
126.60, 126.33, 126.07, 116.82, 114.73, 113.94, 109.16, 108.25,
102.71, 55.55, 26.58, 26.38, 23.10(2C). HRMS (ESI) calculated for
C26H26N4O4S [M þ H]þ 491.1748, found 491.1744.
(2,4-dihydroxy-5-isopropylphenyl)(4-(pyrrolidin-1-yl)-5,8-dihydro-
pyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)methanone (8l)
Compound (8l) was synthesized from compound (4l), in a manner
(2,4-dihydroxy-5-isopropylphenyl)(4-((3-isopropylphenyl)amino)-
5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)metha-
none (8h)
1
similar to (8a). yield: 69%, White powder, m. p. 185.6–188.9 ꢁC, H
NMR (400 MHz, DMSO) d 9.60 (s, 1H), 9.56 (s, 1H), 8.27 (s, 1H), 6.95
(s, 1H), 6.41 (s, 1H), 4.77 (s, 2H), 3.63 (s, 6H), 3.13 – 3.04 (m, 1H),
3.00 (s, 2H), 1.85 (s, 4H), 1.12 (d, J ¼ 6.7 Hz, 6H). 13 C NMR
(101 MHz, DMSO) d 168.96, 167.85, 158.16, 157.02, 153.32, 151.68,
128.42, 127.17, 126.56, 126.03, 116.45, 114.18, 102.69, 50.91 (s, 2C),
31.75, 29.90, 29.48, 26.32, 25.56 (s, 2C), 23.09 (s, 2C). HRMS (ESI)
calculated for C25H24N4O4S [M þ H]þ 438.1726, found 438.1730.
Compound (8h) was synthesized from compound (4h), in a man-
ner similar to (8a). Yield: 60%, White powder, m. p. 209.5–212.4 ꢁC,
1H NMR (400 MHz, DMSO-d6) d 9.58 (s, 2H), 8.40 (s, 1H), 8.18 (s,
1H), 7.53 (d, J ¼ 9.0 Hz, 1H), 7.44 (t, J ¼ 2.0 Hz, 1H), 7.26 (t,
J ¼ 7.8 Hz, 1H), 6.98 (dt, J ¼ 7.7, 1.3 Hz, 1H), 6.92 (s, 1H), 6.41 (s,
1H), 4.79 (s, 2H), 3.78 (s, 2H), 3.24 (t, J ¼ 6.5 Hz, 2H), 3.08 (hept,
J ¼ 6.9 Hz, 1H), 2.87 (hept, J ¼ 6.9 Hz, 1H), 1.22 (d, J ¼ 6.9 Hz, 6H),
1.12 (d, J ¼ 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) d 168.86,
166.79, 157.12, 155.54, 153.36, 153.01, 149.25, 139.50, 130.01, (2,4-dihydroxy-5-isopropylphenyl)(4-morpholino-5,8-dihydropyri-
128.73, 126.59, 126.39, 126.05, 121.97, 120.59, 120.40, 116.63,
113.95, 102.71, 33.94, 26.64, 26.37, 24.35(2C), 23.10(2C). HRMS (ESI)
calculated for C28H30N4O3S [M þ H]þ 503.2111, found 503.2112.
do[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)methanone (8m)
Compound (8m) was synthesized from compound (4m), in a man-
ner similar to (8a). yield: 65%, White powder, m. p. 183.4–185.1 ꢁC,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
915
1H NMR (400 MHz, DMSO) d 9.60 (s, 1H), 9.58 (s, 1H), 8.53 (s, 1H), 2.6. Western blotting analysis
6.97 (s, 1H), 6.42 (s, 1H), 4.82 (s, 2H), 3.75 (t, J ¼ 3.7 Hz, 4H), 3.66 (s,
2H), 3.38 (t, J ¼ 3.7 Hz, 4H), 3.16 – 3.02 (m, 3H), 1.13 (d, J ¼ 6.9 Hz,
6H). 13 C NMR (101 MHz, DMSO) d 169.10, 168.40, 161.91, 157.07,
153.49, 152.22, 131.54, 126.87, 126.47, 126.04, 119.99, 114.02,
102.74, 66.26 (s, 2C), 50.90 (s, 2C), 31.13, 29.48, 27.50, 26.30, 23.09
(s, 2C). HRMS (ESI) calculated for C25H24N4O4S [M þ H]þ 454.1675,
found 454.1677.
The total protein was extracted from B16 cells, separated on SDS
PAGE gel (7.5–15%, according to the molecular weight of protein)
and then transferred to PVDF membranes (Millipore, MA, USA).
The transferred PVDF membranes were blocked by 5% BSA and
then incubated with primary antibodies under ice bath overnight,
then the secondary antibodies were added for one hour incuba-
tion at room temperature. Specific protein bands were visualized
using ECL Western Blotting Substrate (Millipore, MA, USA) and
imaged by Chemi Scope 3300 Mini (Clinx Science Instruments,
Shanghai, China).
(2,4-dihydroxy-5-isopropylphenyl)(4-(dimethylamino)-5,8-dihydro-
pyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-7(6H)-yl)methanone (8n)
Compound (8n) was synthesized from compound (4n), in a man-
ner similar to (8a). Yield: 66%, White powder, m. p. 189.1–192.2 ꢁC,
1H NMR (400 MHz, DMSO) d 9.59 (s, 1H), 9.56 (s, 1H), 8.40 (s, 1H),
6.95 (s, 1H), 6.40 (s, 1H), 4.80 (s, 2H), 3.60 (s, 2H), 3.14 – 2.99 (m,
9H), 1.12 (d, J ¼ 6.9 Hz, 6H). 13 C NMR (101 MHz, DMSO) d 169.05,
168.07, 161.89, 157.01, 153.34, 151.85, 130.02, 127.21, 126.52,
126.03, 118.42, 114.20, 102.70, 41.95 (s, 2C), 31.15, 29.48, 28.43,
26.31, 23.10 (s, 2C). HRMS (ESI) calculated for C25H24N4O4S
[M þ H]þ 412.1569, found 412.1566.
2.7. Subcutaneous tumor xenograft model
Female C57BL/6J mice with 8–12 weeks old were purchased from
Beijing Vitalriver Co. Ltd. (Beijing, China). All animals were housed
in the SPF animal facility at State Key Laboratory of Biotherapy in
individually ventilated cages. The experimental protocols were in
compliance with the guidelines of the Animal Ethics Committee
(Sichuan University) and were approved by the Animal Ethics
Committee of West China Hospital of Sichuan University. Tumor
cells lines B16 were injected subcutaneously in the dorsal flank of
C57BL/6J mice as single cell suspensions (5 ꢃ 105 in 100 lL) in
PBS. The tumor sizes were measured every two days from the day
after injection of cancer cells. Calculation of tumor volume was
according to Vol ¼ aꢃb2ꢃ0.5, where a is the large diameter tumor
tissue and b is the small diameter tumor tissue. The tumor tissues
were stripped, fixed in formalin, then embedded in paraffin, and
sectioned. Tumor tissue sections were stained with TUNEL, EGFR,
Akt, pAkt473 and Ki67 for further histologic examination.
2.4. Cell proliferation assay
Cells were grown as monolayers at 37 ꢁC in a humidified atmos-
phere of 5% CO2. To remove adherent cells from the flask for sub-
culturing and counting, cells were washed with PBS without
calcium or magnesium, incubated in a small volume of 0.25%
trypsin–EDTA solution for 5–10 min, resuspended with culture
medium, and centrifuged. All experiments were carried out using
cells in the exponential growth phase. Growth inhibition was
assessed using an MTT assay as described previously. Cells were
seeded (3–6 ꢃ 103 cells/well) in 96-well plates with 200 lL com-
plete culture medium for 24 h, followed by the treatments of
either DMSO (control) or various concentrations of 8m for 24 or
48 h. After each time point, 200 lL of the culture medium contain-
ing 0.5 mg/mL MTT was added to each well and incubated for 3 h
at 37 ꢁC. After removal of the supernatant, DMSO was added to
the wells and the absorbance was read at 570 nm. All experiments
were performed three times in triplicate.
3. Results and discussion
3.1. Hsp90 and protein kinases are overexpressed in melanoma
We first observed an Hsp90 mRNA expression pattern in 33 cancer
subtypes using the TCGA database30. Hsp90 was significantly over-
expressed in ten cancers (fold change > 2.0, p < .01), including
cutaneous melanoma (Figure 1(A)). In total, 461 HCC cutaneous
melanoma tissues and 558 adjacent or normal skin tissues from
the TCGA and GTEx databases were included in this study4. Hsp90
expression levels were significantly upregulated in melanoma tis-
sues compared with normal skin tissues (p < .001, Figure 1(B)).
Somatic mutation and expression analysis of Hsp90 in 470 cutane-
ous melanoma patients suggested that Hsp90 expression dysregu-
lation occurred in about 25% of melanoma patients, only four
patients had Hsp90 copy number alterations (one for amplification
and three for deep deletion, Figure 1(C))31.
Although there was no difference in expression between T1 to
T4 stage melanoma tissues, both had higher expression compared
to T0 melanoma tissues (Figure 1(D)). Moreover, there were no
significant differences observed in total survival ratios between
Hsp90 groups with high expression vs low expression (Figure
1(E)). In order to further validate the protein expression levels of
Hsp90 (Figure 1(F)) and protein kinases (Figure 2(A)), expression of
Hsp90 and several protein kinases were determined by IHC immu-
nohistochemistry (IHC) of melanoma tissue microarray from the
Protein Atlas database32. Relative expression levels of Hsp90 and
protein kinases (such as c-Kit, VEGFR2, EGFR and PI3Ka) in both
melanoma and normal skin tissues are illustrated in Figure 2(C,D).
Both Hsp90 and these protein kinases were overexpressed in mel-
anoma tissues compared to normal skin tissues.
2.5. Cell migration and invasion assays
Cell migration assay was performed using the agarose wound-
healing method. In brief, B16 cells were seeded in 6-well plate
with DMEM for 1 ꢃ 105 cells per well and was further grown over-
night. Three lines were cut in each well using a 200 ml pipette tip.
Cells were then washed with PBS and serum-free DMEM including
various concentrations of drugs or DMSO were added. Evaluation
of the cell migration was determined under a light microscope
between 0 h and 24 h of treatment.
For the invasion assay, we used Transwell chambers (8-lm
pore size, Corning). Specifically, 1 ꢃ 105 of B16 cells were sus-
pended in serum-free medium, and then loaded into the Matrigel
precoated top-chambers (BD Bioscience, San Jose, CA, USA) and
incubated with DMEM culture in the bottom chamber. Crystal vio-
let staining was used to measure the invasiveness of B16 cells.
Cell counts were performed in triplicate under a microscope.

916
F. QIN ET AL.
Figure 1. The mRNA expression pattern of Hsp90 in TCGA (The Cancer Genome Atlas) database, its genetic alteration from cBioPortal web server and protein expres-
sion changes from proteinatlas database. (A) The logarithmic data of the Hsp90 mRNA expression TPM (transcripts per million reads) values presented by GEPIA (Gene
Expression Profiling Interactive Analysis) database; (B) The mRNA levels in melanoma or normal skin tissues in TCGA and GTEx database; (C) The genetic alterations of
Hsp90 were analyzed in 470 melanoma samples based on cBioPortal web server, Hsp90 DNA copy number amplified in one patient and deep deleted in three
patients, and Hsp90 mRNA upregulated in 38 cases but downregulated in 74 cases; (D) The Hsp90 mRNA levels in melanoma patients with different stages; (E) The
Kaplan–Meier plot of overall survival in melanoma patients with different Hsp90 mRNA levels; (F) The representative Hsp90 protein levels between melanoma and nor-
mal skin tissues detected by IHC.
Considering that the oncogenic function of Hsp90 is often
related to its chaperone proteins, survival analysis was further per-
formed in Hsp90 combining with several protein kinases (Figure
2(B)). The total survival ratio of those with high levels of Hsp90-
3.2. Design and synthesis of novel Hsp90/protein
kinases inhibitors
Some representative chemical structures of Hsp90 inhibitors, PI3Ka
inhibitors and multi-kinase inhibitors are shown in Figure 3(A).
The 2,4-dihydroxy-5-isopropylbenzate fragment was observed to
be common in a panel of synthetic Hsp90 inhibitors. A 4-substi-
tuted pyrimidine fragment is a known scaffold for multi-kinase
inhibitors. A2 or 4-alkylation substituted pyrimidine was common
in PI3K inhibitors, and the 4-aromatic substituted pyrimidine was
kinase expression were significantly worse than those with lower
expression.33,34 Correlated expression profile of Hsp90 and protein
kinases were also analyzed, PI3Ka was utilized as an example kin-
ase to calculate the Pearson correlation coefficient with Hsp90. A
high coefficient of 0.51 was determined to be suggestive of a
strong relationship between PI3Ka and Hsp90 (Figure 2(C)).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
917
Figure 2. Identification of overexpressed protein kinases and their relationship to Hsp90 in melanoma tissues. (A) The representative IHC images of VEGFR2, c-Kit,
EGFR and PI3Ka in melanoma and normal skin tissues; (B) Kaplan–Meier analysis of overall survival in melanoma patients, the median expression levels of Hsp90 and
protein kinases were used as cutoff; (C) The correlation of Hsp90 and PI3Ka mRNA expressions in melanoma patients.
suited for tyrosine protein kinases, such as EGFR, VEGFR, c-Kit, etc. dioxane(20 ml) for 2 h under 90 ꢁC to achieve tert-butyl 4-((2-ethyl-
Therefore, the role of the 2,4-dihydroxy-5-isopropylbenzate frag- phenyl)amino)-5,8-dihydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidine-
ment was to maintain binding specificity to Hsp90, and the role 7(6H)-carboxylate (intermediates 4a). For synthesis of 2,4-dihy-
of the 4-aromatic substituted pyrimidine fragment was to achieve droxy-5-isopropylbenzoic acid fragment, we used methyl 2,4-dihy-
the potent binding to protein kinases, respectively (Figure 3(B)). droxybenzoate (1.68 g, 10 mmol) as start compound, after the
The piperido[4’,3’:4,5]thieno[2,3-d]pyrimidine scaffold utilized con- protection of hydroxyl group by benzyl chloride (2.53 ml,
jugated Hsp90 and protein kinase binding fragments. The Hsp90 22 mmol), and potassium carbonate (3.20 g, 22 mmol), the 5-iso-
binding fragment could easily form an amide bind to the nitrogen propyl group was introduced by isopropyl bromide and DIBAL-H
atom of piperidine ring, and the 4-substituted thieno[2,3-d]pyrimi- to form intermediates 6. The N-Boc group of intermediates 4a
dine was a known scaffold to protein kinase inhibitors35–38
.
was removed by TFA in dichloromethane, and then conjugated to
intermediates 6 by the catalyzed by EDC (1.5M) and HOBt (1.5M)
in THF under room temperature. After removal of benzyl protec-
tion groups of 2,4-dihydroxybenzoate by hydrogen atmosphere
and 10% Pd-C or by Me3SiI (8M) in dichloromethane (especially
for the structures contained halogen) at room temperature for
48 h., ultimately the targeted compounds 8a–n were successfully
synthesized, and characterized by 1H NMR, 13 C NMR and mass
spectrum to validate their chemical structures.
The synthetic process for novel Hsp90/protein kinase inhibitors
is depicted in Scheme 1. In brief, 6-(tert-butyl) 3-ethyl
2-amino-4,7-dihydrothieno[2,3-c] pyridine-3,6(5H)-dicarboxylate (1)
was synthesized from sulfur (3.20 g, 100.0 mmol) and ethyl cyana-
cetate (11.32 g, 100.0 mmol), into N-Boc-piperidin-4-one (19.90 g,
100.0 mmol) and refluxed for 12 h, then form amidine acetate
(6.24 g, 60.0 mmol) and compound 1 (13.00 g, 40.0 mmol) was
stirred in DMF for 12 h at 100 ꢁC to give the pyrimidine-4-one ring
2. The pyrimidine-4-one (15.35 g, 50.0 mmol) was transformed to
Reagents and conditions: (a) NCCH2CO2Et, S8, Et3N, EtOH,
4-Cl-pyrimidine 3 by SO2Cl2 (100 ml) and DMF (2 ml), and then the reflux, 12h; (b) formamidine acetate, DMF, 100 ꢁC,16h; (c) SO2Cl2,
2-ethylaniline (242.39 mg, 2.0 mmol), compound 3 (327.82 mg, reflux, 4h; (d) Aromatic amine, NaH, 1,4-dioxane, 90 ꢁC, 2 h or ali-
1.0 mmol) and NaH (72 mg, 3 mmol) were stirred in anhydrous 1,4- phatic amine, self-solvent, 100 ꢁC, 1 h; (e) 9 N HCl, ethyl acetate,

918
F. QIN ET AL.
Figure 3. (A) Representative structures of Hsp90 inhibitors, PI3K inhibitors and multikinase inhibtors; (B) Depiction of the design method of Hsp90/protein kinase
dual inhibitors.
O
O
N
R1
R2
N
Cl
N
N
N
O
Boc
O
Boc
N
a
N
c
b
d
Boc
NH
N
N
N
N
Boc
Boc
S
NH2
S
S
S
2
3
1
4a-4n
e
Ph
O
O
O
OH
O
R1
R2
N
OH
O
g
f
OH
OMe
+
HN
OMe
N
HO
S
HO
N
Ph
7a-7n
5
6
h
OH
O
R1
N
R2
N
HO
N
S
N
8a-8n
Scheme 1. Synthesis of Compounds 8a–8n.

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919
25 ꢁC, 30 min; (f) aluminum chloride, CH2Cl2, 50 ꢁC, 24 h; (g) benzyl 3.3. Structure-activity relationship (SAR) analysis and binding
chloride, K2CO3, DMF; LiOHꢂH2O, 25 ꢁC, 12h; (h) EDCI, HOBt, DCM, modes of novel Hsp90/PI3Ka dual inhibitors
overnight and then trimethyliodosilane (TMIS), 25 ꢁC, 24 h or H2,
Protein kinases and the cytotoxicity of B16 melanoma inhibitory
potencies of compounds 8a–8n are shown in Table 1. These data
10% Pd/C, 25 ꢁC, 24 h.
Table 1. Kinase inhibition activities and cytotoxicity of compounds 8a–8m.
No.
R1
R2
H
Hsp90a IC50(nM)a,b
EGFR IC50(nM)a,b
60.7
VEGFR2 IC50(nM)a,b
PI3Ka IC50(nM)a,b
B16 GI ratio (%)b
26.8
8a
375.3
>10000
N.D.
8b
8c
8d
8e
8f
H
H
H
H
H
H
H
H
H
289.6
196.3
318.2
229.0
318.5
210.4
468.2
1637.8
207.4
49.9
37.1
>10000
>10000
>10000
>10000
>10000
>10000
>10000
169.5
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
33.6
51.2
41.7
38.9
29.5
37.5
39.1
24.8
36.0
51.8
40.2
76.6
8g
8h
8i
71.0
380.1
578.4
146.5
8j
715.9
8k
8l
51.9
65.0
38.6
79.5
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
132.6
168.9
48.4
65.2
60.4
72.5
46.7
8m
8n
Me
Me
567.5
aIC50 values are determined from KinaseProfiler of Eurofins for Hsp90 and HTRF based method for PI3Ka. The data represent the mean values of three independent
experiments.
bEach compound is tested in triplicate; the data are presented as the mean values.

920
F. QIN ET AL.
Figure 4. The binding modes of compound 8m to kinase domains of Hsp90 and PI3Ka, respectively. (A) the surface of binding pocket contoured by the atomic charge
of Hsp90; (B) The 2D diagrams of interaction residues between compound 8m and Hsp90; (C) the surface of binding pocket contoured by the atomic charge of PI3Ka;
D. The 2D diagrams of interaction residues between compound 8m and PI3Ka.
include the kinase activity of Hsp90 and several kinases including 3.4. Novel Hsp90/PI3Ka dual inhibitors 8m suppressed cell B16
EGFR, VEGFR2 and PI3Ka, as well as the cellular viability of the B16 melanoma cell proliferation via inducing apoptosis
melanoma cell line. The half of the maximal inhibitory concentra-
tions (IC50) values for these compounds on Hsp90 suggested that
the 4-alkylation substitution was a more favorable mechanism than
of 4-aromatic substitution. For compounds 8a–8j, different substi-
tuted groups on the 4-phenyl ring resulted different kinase binding
potencies and selectivity. Specifically, the 2,4-dimethylphenyl or 3-
The cytotoxic activity of 8m was initially evaluated in several can-
cer cells and was most potent in melanoma cells (Figure 5(A)).
Then we treated a B16 melanoma cell line with 8m for a 24 or
48-h incubation. Our results suggested that IC50 were around 0.56
and 0.47 lg/mL, respectively (Figure 5(B)). Furthermore, results of
clone formation assay in 8m-incubated B16 cells indicated a dose-
hydroxylphenyl group at 4-position of thieno[2,3-d]pyrimidine scaf-
dependent decline manner compared to control group in both
fold displayed moderate VEGFR2 inhibitory activities, but did not
the size and number of the colonies (Figure 5(C)).
contribute to B16 cytotoxicity. In general, the Hsp90/PI3Ka dual
inhibitor based on 4-alkylation thieno[2,3-d]pyrimidine scaffold
demonstrated higher inhibition in Hsp90 and B16 cell proliferation
In order to assess the possibility of subroutine cell death under-
lying 8m treatment, flow cytometry with annexinV/PI dual staining
was used to assess the activation of apoptosis39–45. Hoechst 33258
assays than that of Hsp90/RTKs inhibitors. Finally, compound 8m
displayed the strongest inhibitory capacity of Hsp90 and PI3Ka with
IC50 values of 38.6 and 48.4 nM, respectively.
staining also clearly showed that nuclear morphological changes in
apoptotic cells, including chromatin condensation and apoptotic
bodies, mediated apoptosis (Figure 5(D)). There was a significant
increase in the apoptotic positive cells under 24h compound 8m
incubation (Figure 5(E)). As western blot analysis results shown,
apoptosis was initiated through the intrinsic pathway and the
extrinsic pathway by the treatment of 8m in B1 cells with the
cleavage of Caspase-3, Caspase-8, Caspase-9 and PARP (Figure 5(F)).
Compound 8m also induced cell cycle arrest in B16 cells. Cell
counts in G2 phase were significantly increased after 8m treatment
8m for 24 h, and cell counts in G1 phase demonstrated a corre-
sponding descent (Figure 6(A)). These results were confirmed by
overexpression of p53, p21, p27 proteins and decrease of mdm2, c-
myc, CDK2 and CDK6 proteins compared to NS group (Figure 6(D)).
The possible binding conformations of 8m to the binding sites
of Hsp90 and PI3Ka are depicted in Figure 4. From Figure 4(A,B), it
can be observed that 2,4-dihydroxybenzoate could form a stable
hydrogen bond with Asp93 of Hsp90, and the thieno[2,3-d]pyrimi-
dine scaffold formed a pi-cation interaction to Lys58. Moreover, 8m
formed three stable hydrogen bonds to PI3Ka, at Lys802, Ala805
and Val882. Additionally, 2,4-dihydroxyl-5-isopropyl benzoate formed
a pi-cation interaction with Lys890. It was notable that compound
8m also formed stable hydrogen bonds and pi-cation interactions
to both Hsp90 and PI3Ka, which might be the source of the better
inhibition activity on Hsp90 and PI3Ka than the other inhibitors.

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921
Figure 5. The Hsp90/PI3Ka dual inhibitor 8m inhibits the proliferation and induces apoptosis of cancer cell lines (A and B); compound 8m potently inhibited colony
formation (B); induced apoptotic cell death (D and E) and the changes on expression levels of apoptosis related proteins (F).
3.5. 8m inhibited cell migration and invasion in B16 cells
decrease in B16 cells after incubation with 8m in a dose-
dependent manner (Figure 6(B)). The wound areas wounds in 6-
well culture plates had minimal changes after applying 8m in a
dose-dependent manner after 24 h of exposure. Contrastingly,
control plates were significantly healed (Figure 6(C)).
Cellular migration and invasion inhibitory potencies of 8m were
determined by Transwell and agarose wound healing assays
using B16 cells. For cell invasion assays assays based on
Matrigel, Transwells were used to observe
a
remarkable

922
F. QIN ET AL.
Figure 6. Compound 8m-induced changes of cell cycle arrest, cell invasion and migration and changes on related proteins. (A) The cell cycle analysis under treatment
of various doses of 8m; (B) Changes on cell invasion under treatment of various doses of 8m by transwell assay; (C) Changes on cell migration under treatment of vari-
ous doses of 8m by wound healing assay; (D) The changes on expression levels of cell cycle related proteins; (E) The changes on expression levels of EMT
related proteins.
Furthermore, the expression levels of tumor migration- and cadherin, b-catenin, ZEB1 and up-regulated E-cadherin expres-
invasion-associated proteins at different concentrations of 8m sions through phenotypic analysis in B16 cells (Figure 6(E)).
(20, 40 lg/mL) were examined by western blot. We observed Taken together, the above results indicated that 8m markedly
that 8m remarkably down-regulated MMPs, vimentin, N- inhibited the migration and invasion of B16 cells.

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923
Figure 7. The molecular mechanisms of compound 8m to Hsp90 and PI3Ka kinase and downstream signaling pathways. (A) The changes on expression or phosphoryl-
ation levels of PI3Ka or Hsp90 protein family; (B) The changes on expression or phosphorylation levels of Hsp90 client proteins; (C) The changes on expression or phos-
phorylation levels of proteins in downstream MAPK signaling pathway; (D) The changes on expression or phosphorylation levels of proteins in downstream PI3K-Akt
signaling pathway.
3.6 8m function via PI3K-Akt pathway and signaling pathways
associated to Hsp90 client proteins
treatment without significantly altering their phosphorylation
ratio (Figure 7(B)).
MAPK and PI3K-Akt signaling pathways were important sig-
naling pathways associated with Hsp90 client proteins, which
stimulated cell proliferation, oncogene transcription, cell migra-
tion and invasion. Western blot analysis showed that 8m inhib-
ited the expression of c-Myc, ERK1/2 and JNK, and suppressed
the activation of ERK-MNK/p90RSK signaling axis (Figure 7(C)). In
addition, 8m downregulated p-Akt (Ser473), p-Akt (Thr308) and
total Akt expression as the results of PI3Ka inhibition, which
caused significant expression changes of downstream proteins,
such as Bim and MDM2-p53 complex. Taken together, these
results indicated that 8m could potently regulate PI3K-Akt sig-
naling as well as Hsp90 client proteins and their downstream
In order to further validate the dual inhibitory effects on
Hsp90/PI3Ka and molecular mechanisms of compound 8 m, the
expression levels of total and phosphorylated PI3Ka and Hsp90
family proteins were determined by western blotting of B16
melanoma cell extracts (Figure 7(A)). As expected, 8m indeed
inhibited the autophosphorylation of PI3Ka in B16 cells without
influencing levels of total PI3Ka. However, the expression levels
of Hsp90a and Hsp70 declined with 8m incubation, which par-
tially contributed to 8m inhibition of the interaction between
Hsp90 complexed to its client proteins46–48. Western blot ana-
lysis of the Hsp90 client proteins demonstrated that the expres-
sion levels of many canonical clients, such as cdc37, CDK4,
EGFR, bRaf and cRaf were remarkably suppressed by 8m effectors so as to inhibit overall melanoma progression.

924
F. QIN ET AL.
Figure 8. 8m has an antitumor effect in mouse xenograft models via inducing apoptosis. (A) Mice bearing B16 (n ¼ 8 per group) melanoma xenograft were treated
with NS control, PI-103, Ganetespib or 8m once a day. Tumor volume data were presented as mean SD. (B) Tumor weight change of mice in different group: (*)
p < 0.05; (**) p < 0.01. (C) Immunohistochemical analysis of EGFR, Akt, p-Akt473, proliferative marker Ki-67 and apoptosis marker TUNEL. Representative images of posi-
tive staining are shown.
3.7. 8m suppressed tumor growth by targeting Hsp90 and PI3Ka
in vivo
Moreover, there were remarkable declines in weight decline in all
groups (p < .01) (Figure 8(B)).
To further validate whether 8m-mediated tumor growth inhib-
ition was associated with suppressed cell proliferation and Hsp90/
PI3Ka dual inhibition, tumor tissues from all groups were proc-
essed for the immunofluorescence and IHC analysis using TUNEL
and Ki-67, EGFR, Akt and pAkt473, respectively. In all therapeutic
In order to evaluate the antitumor activity of 8m in vivo, a B16
subcutaneous xenograft model was used. We performed this in
vivo study using three agents: PI3K inhibitor PI-103, Hsp90 inhibi-
tor Ganetespib and 8m. It was clear that 8m possessed superior
tumor growth inhibition as 73%, 57% and 29% tumor growth
inhibition ratio were observed, respectively (Figure 8(A)). groups, we found that 8m achieved had the highest number of

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
925
apoptotic positive cells, most significant suppression of Ki-67
(p < .01) positive cells, degradation of Hsp90 client protein EGFR,
as well as a decline in both expression and phosphorylation levels
of PI3Ka downstream Akt protein (p < .01) (Figure 8(C)). In brief,
the in vivo results were almost consistent with in vitro antitumor
assays and signaling pathway analysis.
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4. Conclusion
In summary, we reported on the design, synthesis and biological
evaluation of
a
series of 2,4-dihydroxy-5-isopropylphenyl-
thieno[2,3-d]pyrimidin derivatives as novel Hsp90/PI3Ka dual
inhibitors in B16 melanoma cells. The most potent compound,
8m, inhibited Hsp90 and PI3Ka with IC50 values at nanomolar lev-
els, suppressing melanoma cell migration, invasiveness and prolif-
eration, induced apoptosis and cell cycle arrest. In addition, the
western blotting analysis validated the kinase inhibitory activities
of 8m on B16 cells. These results suggested that Hsp90/PI3Ka
dual inhibition might be an attractive approach to melanoma tar-
geted therapy.
Detailed binding modes of 8m to Hsp90 and PI3Ka were probed
using a molecular docking method. WB analysis results of the Hsp90
and PI3Ka downstream signaling pathways as indicated that both
MAPK pathway regulated by Hsp90 client proteins and PI3K-Akt
pathway are involved in the novel Hsp90/PI3Ka dual inhibitor 8m
for melanoma cell apoptotic death. These results also were vali-
dated in subcutaneous B16 xenograft murine model. Collectively,
our results provide valuable insights about our library of novel
Hsp90/PI3Ka inhibitors which allows us to explore a further integra-
tive translational medicine paradigm. This future direction includes
transcriptomic analysis, chemical synthesis and molecular mecha-
nisms to study synthesized Hsp90/PI3Ka dual inhibitors as an
attractive lead compound for melanoma targeted therapy.
identification of
a novel napthalamide-isoselenocyanate
compound NISC-6 as a dual topoisomerase-II alpha and Akt
pathway inhibitor, and evaluation of its anti-melanoma
activity. Eur J Med Chem 2017;135:282–95.
Acknowledgements
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dinyl acyl thioureas as novel Hsp90 inhibitors in invasive
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The characterization and NMR spectrum of synthesized com-
pounds are supplied in Supplementary material. This material is
available free of charge via the Internet.
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chloro-2,4-dihydroxybenzoyl)-(R)-1,2,3,4-tetrahydroisoquino-
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Disclosure statement
No potential conflict of interest was reported by the authors.
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pharmacological evaluation of 4,5-diarylisoxazols bearing
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Funding
We are grateful for financial support from the National Natural
Science Foundation of China (21772131 and 81872535), the
Fundamental Research Funds of Science
Department of Sichuan Province (2019YFSY0004 and 2017JY0123),
China Postdoctoral Science Foundation (No. 2016M602696 and
2016M592694) and the Fundamental Research Funds for the
Central Universities and Distinguished Young Scholars of Sichuan
University (2015SCU04A13).
&
Technology
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ORCID
Gu He
http://orcid.org/0000-0002-1536-8882
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