Design, synthesis and biological evaluation of 4-anilinothieno[2,3-d]pyrimidine-based hydroxamic acid derivatives as novel histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.08.030

A series of 4-anilinothieno[2,3-d]pyrimidine-based hydroxamic acid derivatives as novel HDACs inhibitors were designed, synthesized and evaluated. Most of these compounds displayed good to excellent inhibitory activities against HDAC1, 3, 6. The IC₅₀ values of compound 10r against HDAC1, HDAC3, HDAC6 was 1.14 ± 0.03 nM, 3.56 ± 0.08 nM, 11.43 ± 0.12 nM. Compound 10r noticeably up-regulated the level of histone H3 acetylation compared to the SAHA. Most of the compounds showed the strong anti-proliferative activity against human cancer cell lines including RMPI8226 and HCT-116. The IC₅₀ values of Compounds 10r and 10t against RPMI8226 was 2.39 ± 0.20 μM, 1.41 ± 0.44 μM, respectively, and the HCT-116 was sensitive to the compounds 10h, 10m, 10r, 10w with the IC₅₀ values <1.9 μM.

Full text of DOI:10.1016/j.bmc.2014.08.030

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation
of 4-anilinothieno[2,3-d]pyrimidine-based hydroxamic
acid derivatives as novel histone deacetylase inhibitors
Wei Yang , Lixuan Li b,c, , Xun Ji , Xiaowei Wu , Mingbo Su , Li Sheng , Yi Zang , Jia Li
a,
a
a
c
c
c
b,c,
⇑
, Hong Liu a,
⇑
a
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, PR China
East China Normal University, Institutes for Advanced Interdisciplinary Research, North Zhongshan Road Campus: 3663 N. Zhongshan Rd., Shanghai 200062, PR China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, SIBS, Chinese Academy of Sciences, Shanghai 201203, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-anilinothieno[2,3-d]pyrimidine-based hydroxamic acid derivatives as novel HDACs inhibi-
tors were designed, synthesized and evaluated. Most of these compounds displayed good to excellent
inhibitory activities against HDAC1, 3, 6. The IC50 values of compound 10r against HDAC1, HDAC3, HDAC6
was 1.14 ± 0.03 nM, 3.56 ± 0.08 nM, 11.43 ± 0.12 nM. Compound 10r noticeably up-regulated the level of
histone H3 acetylation compared to the SAHA. Most of the compounds showed the strong anti-prolifer-
ative activity against human cancer cell lines including RMPI8226 and HCT-116. The IC50 values of Com-
Received 11 June 2014
Revised 22 August 2014
Accepted 26 August 2014
Available online xxxx
Keywords:
HDACs
HDACs inhibitor
Hydroxamic acid
Thieno[2,3-d]pyrimidines
pounds 10r and 10t against RPMI8226 was 2.39 ± 0.20
was sensitive to the compounds 10h, 10m, 10r, 10w with the IC50 values <1.9
Ó 2014 Elsevier Ltd. All rights reserved.
lM, 1.41 ± 0.44
l
M, respectively, and the HCT-116
l
M.
+
1
. Introduction
1–7) are NAD -dependent enzymes. Given the critical role that
5
HDACs play in tumor cell biology, they have become a major field
The deacetylation and acetylation of histones are important epi-
of research interest as an attractive potential anticancer target.6
Many HDACs inhibitors have been developed and exhibited excel-
lent therapeutic efficacy in both preclinical and clinical trials
genetic modifications, which play a crucial role in controlling chro-
matin conformation. The acetylation status of histones is
1
7
8
determined by two sets of enzymes: histone deacetylases (HDACs)
and histone acetyltransferases (HATs). HDACs catalyze the removal
of acetyl groups from lysine residues in the N-terminal tails of core
histones in the nucleosomes and increase the number of proton-
ated lysines that bind closely to the negatively charged DNA phos-
phate groups leading to chromatin compaction, inaccessibility of
(Fig. 1). For example, SAHA and FK228 have been approved by
FDA for the treatment of cutaneous T-cell lymphoma. Additionally,
compounds such as PXD101, LBH589, MS275, and CUDC-
101 are currently undergoing clinical trials. HDACs inhibitors
induce histone hyperacetylation and (or) increase the acetylation
9
10
11
1
2
2
transcription factors to DNA and alteration in gene expression.
There is growing evidence showing the gene expression controlled
by epigenetic modifications is pivotal to the onset and progression
of cancer; furthermore, the aberrant overexpression of HDACs
have been found in many solid tumors and hematological
CAP
Linker
ZBG
O
O
H
O
N
H
NH2
H
N
N
OH
N
H
N
3
O
malignancies.
O
SAHA
MS-275
To date, 18 HDACs isoforms have been found in humans. Based
on their homology to yeast HDACs, they can be divided into four
O
S
O
4
NH HN
O
classes. Class I (HDAC1-3 and 8), II (HDAC 4–7, 9 and 10), and IV
O
2
+
S
(
HDAC11) are Zn -dependent enzymes, whereas class III (sirtuins
NH
N
HN
HN
H
N
O
O
N
OH
O
O
⇑
Corresponding authors. Tel.: +86 21 50801552; fax: +86 21 50800721 (J.L.); tel./
fax: +86 21 50807042 (H.L.).
O
FK228
CUDC-101
E-mail addresses: jli@mail.shcnc.ac.cn (J. Li), hliu@mail.shcnc.ac.cn (H. Liu).
These authors contributed equally to this work.
Figure 1. Approved and clinical HDACs inhibitors.
http://dx.doi.org/10.1016/j.bmc.2014.08.030
0
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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2
W. Yang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
F
accepted pharmacophore depicted by SAHA, that includes a cap
A
B
F
group (CAP) for protein surface interactions, a zinc binding group
(ZBG) that chelates with the zinc ion to repress the hydrolysis of
acetyl group in the lysine residue, and a linker region that connects
HN
N
Cl
H
N
HN
N
Cl
N
O
N
N
N
O
17
HN
the CAP and ZBG (Fig. 1).
O
S
The thieno[2,3-d]pyrimidine fragment is widely present in anti-
EGFR wt: IC50 =0.024 µM
EGFR T790M/L858R: IC50 = 0.324 µM
18
19
20
bacterial agents, antioxidant agents and antitumor agents. In
our previous studies, we have reported the replacement of the 4-
anilinoquinazolin scaffold in afatinib structure with the 4-anilino-
thieno[2,3-d]pyrimidine fragment to design and synthesis of 6-
alkenylamides substituted of 4-anilinothieno[2,3-d]pyrimidine
derivatives as irreversible EGFR inhibitors that displayed excellent
inhibitory activities against wild type and mutant EGFR21 (Fig. 2A).
Furthermore, many literatures have reported modifications of the
O
Afatinib
R
NH
H
N
NH
N
5
O
N
OH
O
N
O
NH
O
N
O
NH
HO
S
n
22
CUDC-101
quinazoline fragment with a thienopyrimidine structure. CUDC-
10
1
01 has been reported as a dual EGFR and HDACs inhibitor which
Figure 2. Design strategy and modification of novel HDACs inhibitors.
contains a quinazoline moiety within its structure. On the basis of
these observations, we attempted to substitute the 4-anilinoqui-
nazoline fragment with a 4-anilinothieno[2,3-d]pyrimidine scaf-
fold to design compounds 10 as novel HDACs inhibitors (Fig. 2B).
In this paper, we presented the design, synthesis of thieno[2,3-
d]pyrimidine-based novel HDACs inhibitors and their preliminary
biological evaluation against HDACs and cancer cells. Most of these
compounds showed excellent potencies against HDACs. Further-
more, some compounds displayed powerful antiproliferative activ-
ities against cancer cell lines in vitro.
of nonhistone proteins, leading to cell growth arrest, differentia-
1
3
tion and apoptosis. An extensive number of studies have shown
that HDACs inhibitors have additional promising potential
therapeutic applications in the treatment of neurodegenerative
1
4
15
16
diseases, inflammation and malaria. Although diversity in
the structures of HDACs inhibitors, they broadly conform to an
O
O
O
N
O
NH
NH
2
+
a
NH
b
NH
c
AcOH
2
2
O N
NH
2
S
S
N
S
N
1
3
4
R
e
R
Cl
N
HN
N
HN
N
d
f
N
N
O
2
N
O
2
N
H N
2
S
S
S
5
6a-y
7a-y
R
R
NH
N
O
NH
O
g
O
h
N
NH
OH
O
N
n
NH
S
O
n
N
S
8a-y
9a-y
R
NH
N
O
HN OH
O
N
NH
n
S
1
0a-y
Cl
N
Cl
Cl
O
N
e
N
f
O
O
O
2
N
H
2
N
N
NH
S
S
N
N
S
5
11
12
Cl
O
Cl
N
O
OH
O
N
h
g
HN OH
O
NH
N
NH
N
S
13
S
14
Scheme 1. Reagents and conditions: (a) microwave, 4–5 mins; (b) H
2
SO
4
/HNO
3
, RT-90 °C, 1 h; (c) POCl
3 3
, 110 °C or POCl , TEA, CH
3
CN, 70 °C; (d) various substituted anilines,
i-PrOH, 80 °C; (e) Fe, NH Cl(aq), EtOH, 45 °C; (f) MeOCO(CH )nCOCl, TEA, THF, n = 4, 5, 6; (g) LiOH, MeOH-THF, RT; (h) NH
4
2
2
OH.HCl, DMAP, BOP, DMF, RT.
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W. Yang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Table 1
Structures and in vitro inhibitory activity against HDACs of compound 10 derivatives and compound 14
a
3
2
R
4
1
H
N
H
N
NH
N
OH
n
5
6
S
O
O
N
1
0a-y
Compd
SAHA
R
n
IC50 (nM) ± SD
HDAC3
HDAC1
HDAC6
—
—
2
3
4
2
3
4
4
4
4
4
4
4
3
4
3
4
3
4
4
4
4
93.34 ± 2.78
131.30 ± 25.21
35.89 ± 16.34
13.36 ± 1.48
218.10 ± 55.35
40.84 ± 8.23
21.67 ± 5.65
10.07 ± 0.53
12.50 ± 1.11
13.54 ± 3.08
6.24 ± 1.59
7.78 ± 2.14
3.24 ± 0.77
11.77 ± 0.50
1.90 ± 0.39
14.01 ± 1.32
2.01 ± 0.29
158.17 ± 6.66
126.56 ± 9.04
37.67 ± 1.61
17.91 ± 0.17
607.03 ± 52.61
48.26 ± 1.78
23.53 ± 2.00
16.85 ± 0.30
14.47 ± 0.08
11.88 ± 0.86
13.52 ± 0.74
14.63 ± 1.20
6.46 ± 0.01
20.77 ± 0.64
3.47 ± 0.26
9.33 ± 0.10
2.92 ± 0.06
14.74 ± 0.03
3.56 ± 0.08
78.98 ± 13.19
568.76 ± 45.38
23.99 ± 0.72
21.96 ± 1.42
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
0g
0h
0i
3-Cl, 4-F
3-Cl, 4-F
3-Cl, 4-F
3-CF
3-CF
3-CF
2-F, 4-F
2-F, 4-Cl
2-F, 4-Br
2-F, 4-I
2-F, 3-Cl
3-Cl
b
3
3
3
, 4-Cl
, 4-Cl
, 4-Cl
ND
30.00 ± 1.14
11.49 ± 0.01
7.88 ± 0.22
5.58 ± 0.06
8.87 ± 0.99
6.55 ± 0.13
8.46 ± 0.23
8.58 ± 0.04
26.99 ± 4.95
12.04 ± 0.04
19.68 ± 1.96
5.37 ± 0.72
16.87 ± 3.02
11.43 ± 0.12
43.16 ± 0.73
3.44 ± 0.06
2.69 ± 0.01
0j
0k
0l
0n
0m
0o
0p
0q
0r
0s
0t
H
H
4-CH
4-CH
3-CH
3-CH
4- N(Et)
3-F, 4-N(Et)
3
3
3
3
, 4-CH
, 4-CH
3
3
29.82 ± 11.51
1.14 ± 0.03
32.87 ± 7.35
4.57 ± 0.04
2
39.72 ± 1.62
3.52 ± 0.02
3.22 ± 0.10
2
0u
3-F, 4-N(CH
3
)
2
4.51 ± 0.03
O
1
0v
3-Cl, 4-
4
2.97 ± 0.07
3.58 ± 0.00
1.90 ± 0.03
O
1
1
0w
0x
3-F,4-
N
4
4
5.39 ± 0.07
8.19 ± 0.24
3.42 ± 0.07
9.28 ± 0.03
N
14.49 ± 0.85
14.05 ± 0.74
3-F, 4-
1
1
0y
4
4-
N
4
4
13.85 ± 0.55
25.53 ± 0.15
14.10 ± 0.34
26.53 ± 0.80
7.19 ± 0.08
—
15.10 ± 0.28
a
IC50 values were obtained based on three separate experiments and expressed as means ± SD.
ND indicated no data was obtained.
b
2
2
. Results and discussion
.1. Chemistry
Table 2
Antiproliferative activities of representative compounds against RPMI 8826 and
HCT116 cell linesa
The route for synthesizing compounds 10 and compound 14 is
depicted in Scheme 1 The thieno[2,3-d]pyrimidine scaffold 3 was
obtained via the cyclization of methyl 2-aminothiophene-3-car-
boxylate with formamidine acetate assisted by microwave. Then,
the nitrification of intermediate 3, followed by chlorination, treat-
ment with various substituted anilines and reduction of the nitro
group afforded the key intermediate 7, which reacted with acyl
chloride to form intermediate 8. Hydrolysis with lithium hydroxide
in a methanol solution gave the acid intermediate 9, which subse-
quently was coupled with hydroxylamine hydrochloride in the
presence of BOP and DMAP to afford the targeted compounds 10.
Reduction of intermediate 5, followed by treatment with acyl chlo-
ride, formed the intermediate 12 which was subsequently hydro-
lyzed by lithium hydroxide to give acid intermediate 13.
Intermediate 13 was coupled with hydroxylamine hydrochloride
to give the targeted compound 14.
Compd
IC50 (lM) ± SD
RPMI8226
HCT-116
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0g
0h
0i
0k
0l
0m
0p
0r
0s
0t
0u
0v
0w
0x
0y
4
7.49 ± 1.54
3.50 ± 0.29
8.12 ± 0.36
8.17 ± 0.45
4.49 ± 0.41
3.79 ± 0.22
3.84 ± 0.03
2.39 ± 0.20
5.95 ± 0.62
1.41 ± 0.44
3.49 ± 0.28
4.20 ± 0.50
3.28 ± 0.53
4.63 ± 0.47
5.42 ± 0.62
2.18 ± 0.98
2.39 ± 0.08
NDb
1.70 ± 0.56
b
ND
ND
ND
b
b
1.82 ± 0.76
2.34 ± 0.29
1.61 ± 0.48
4.44 ± 0.96
2.55 ± 0.22
4.03 ± 0.62
2.24 ± 0.70
1.44 ± 0.39
4.09 ± 0.80
3.74 ± 0.41
2.92 ± 0.15
3.45 ± 0.20
2
.2. In vitro HDAC inhibitory activity
SAHA
a
IC50 values were obtained based on three separate experiments and expressed
as means ± SD.
We initially evaluated the inhibitory activities of compounds
10a–10y and 14 against recombinant human HDAC1, HDAC3,
b
ND indicated no data was obtained.
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4
W. Yang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
2
.3. In vitro inhibition against cancer cells
1
0m
10r
1
10p
1
SAHA
(µM)
0
.3
1
3
9
0.3
3
9 0.3
3
9
3
To further characterize these compounds, the antiproliferative
GAPDH
activities of the thieno[2,3-d]pyrimidine-based HDAC inhibitors
were assessed in two cancer cell lines: the human multiple mye-
loma cell line, RPMI8226 and the colon cancer cell line, HCT116.
The results are summarized in Table 2. Compounds 10g, 10i and
Ac-tubulin
Tubulin
Ac-H3
1
0k showed poor antiproliferative activities probably due to
potentially limited membrane permeability. On the contrary, com-
H3
pound 10h showed good activities against both RPMI 8226 and
HCT-116 cell lines. Compounds 10r (IC50 = 2.39 ± 0.20
lM) and
1
0t (IC50 = 1.41 ± 0.44 M) showed similar antiproliferative activi-
l
1
0t
10u
1
14
SAHA
3 (µM)
ties against RPMI 8226 cells with SAHA, while the other com-
pounds displayed less antiproliferative activities than SAHA.
Compared to the RPMI 8226 cancer cells, the HCT116 cells
appeared to be more sensitive to our synthesized compounds.
Most compounds such as 10h, 10m, 10r, 10p, 10t, 10v and 10w
exhibited higher antiproliferative potency against HCT116 cell
than SAHA. Compound 10w inhibited the HCT-116 cell prolifera-
0
.3
1
3
9 0.3
3
9
0.3 1
3 9
GAPDH
Ac-tubulin
Tubulin
tion most effectively with the IC50 value of 1.44 ± 0.39 lM. Com-
pound 14 possessed a slightly higher activities against both
RPMI8226 and HCT116 cells than SAHA.
Ac-H3
H3
2
.4. Analysis of the levels of Ac-tubulin and Ac-histone H3 in
RPMI8226 cells by western blotting
Figure 3. Western blot analysis of Ac-tubulin and Ac-histone H3 from RPMI 8226
cell cultured for 24 h with DMSO control and compounds 10m, 10r, 10p, 10t, 10u,
Next, some representative compounds were selected to validate
the observed biochemical potencies by assessing their effects on
1
4and SAHA.
and HDAC6 isoforms using SAHA as positive control. As shown in
Table 1, most of the compounds exhibited excellent inhibitory
activities against HDAC1, HDAC3 and HDAC6 which indicated the
4
-anilinothieno[2,3-d]pyrimidine scaffold was an outstanding cap-
ping group possessing strong binding affinity with the HDACs sur-
face. Furthermore, these findings confirmed that the design
strategy was rational and successful. Increasing the length of the
carbon side chain between the two amides increased the inhibitory
activities against HDACs. The good to excellent inhibitory activities
were obtained when the carbon chain length reached five or six
carbons (n = 3 or 4, 10b–c, 10e–f vs 10a, 10d); moreover, the opti-
mal carbon chain length was six (n = 4) which gave the best inhibi-
tion with the minimum IC50 values of 1.14 ± 0.03 nM in HDAC1
(
1
compound 10r), 2.92 ± 0.06 nM in HDAC3 (compound 10p), and
.90 ± 0.03 nM in HDAC6 (compound 10v), respectively. Based on
the previous report that halogen-containing substituents on the
phenyl ring in the CUDC-101 displayed excellent inhibitory activi-
1
2
ties, we introduced halogen or halogen-containing groups into
the phenyl ring to test their effects on inhibitory activities against
HDACs. We found that most of these compounds (10b–c, 10e–l)
showed strong potential activities against HDACs with IC50 values
<50 nM. While, compared to compound 10m which had an IC50
R275
value in the single-digit nanomolar range, these compounds had
weaker inhibition on HDACs indicating that the halogen substitu-
ents were detrimental to potency. Introduction of a methyl group
into the phenyl ring (compounds 10p and 10r) displayed similar
activities with compound 10m. In order to improve the aqueous
solubility, some polar functional groups were introduced into the
phenyl ring (compounds 10s–y). Most of these compounds had
excellent HDACs inhibitory activities for the IC50 values, of which
compounds 10t, 10u, 10v and 10w against HDAC1, HDAC3, and
HDAC6 were all below 9 nM. When removal of the 4-aniline frag-
ment from the 10 derivatives, compound 14 showed poor inhibi-
tory activity than compounds 10m, 10p, 10r, 10v. This observed
result proved that the presence of the 4-aniline fragment could
dramatically increase the lipophilic interaction with HDACs to
afford excellent inhibitory activities against HDACs.
H33
Zn
D104
P106
F115
F210
Figure 4. Molecular docking of compound 10m (green) and SAHA (purple) in the
active site of HDAC2.
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W. Yang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
the levels of
a
-tubulin and histone H3 acetylation in the multiple
microwave for 4–5 min. After cooled to RT, water was added and
the solid was precipitate, then filtered, and the cake was washed
with water, dried under infrared light to obtain the desired com-
myeloma cancer cell line RPMI8226 (Fig. 3). These proteins are
important biomarkers that are associated with intracellular HDACs
inhibition. Western blot analysis revealed that compounds 10m,
+
pound 3, 17.2 g as gray solid in 89% yield. ESI-MS m/z 153 [M+H] .
1
0r, 10p, 10t, 10u and 14 dramatically increased the acetylation
of -tubulin and histone H3 in a dose-dependent manner, which
a
4.2.2. 6-Nitrothieno[2,3-d]pyrimidin-4-ol (4)
was consistent with their inhibitory activities against HDACs and
cancer cells. Compound 10r noticeably up-regulated the level of
histone H3 acetylation compared to SAHA at the same concentra-
tion. Based on their ability to up-regulate histone H3 and a-tubulin
acetylation, our designed compounds, like the reference compound
2 4 3
To a mixture of H SO (20 mL) and HNO (20 mL) was added
compound 3 (20 g, 0.131 mol) slowly under ice-bath condition
within 50 min. Then, the reaction mixture was heated to 90 °C
and stirred for 2 h. When the staring material was consumed, the
mixture was poured into the ice-water carefully, and the yellow
solid was formed, followed by filtration. The cake was washed with
SAHA, were pan-HDACs inhibitors.
water for three times, dried to afford the desired compound 4,
+
2
.5. Molecular docking
2
0.5 g as yellow solid in 80 yield. ESI-MS m/z 198 [M+H] .
In order to understand the interaction of these inhibitors and
4
.2.3. 4-Chloro-6-nitrothieno[2,3-d]pyrimidine (5)
To a solution of compound 4 (10 g, 0.051 mol) in CH CN
3
HDACs, we docked the compound 10m and SAHA in the active site
of HDAC2 (PDB code: 4LXZ) using AutoDock4.22
3,24
(Fig. 4). The
(
100 mL) was added POCl
3
(14 mL, 0.15 mol) and TEA (21 mL,
results showed that compound 10m displayed a similar binding
mode to SAHA in the active site of HDAC2. The hydroxamic acid
of the compound 10m could chelate Zn2 and the nitrogen atom
in the pyrimidine ring could form hydrogen bond with R275. In
addition, the phenyl moiety could hydrophobically interact with
P106 and H33 that could enhance the binding affinity.
0
.15 mol). Then the mixture was heated to 70 °C for 3 h. After the
start material was completed, the mixture was poured into
ice-water, and extracted with EA, washed by NaHCO solution,
brine, dried by Na SO , concentrated and purified by flash silica
gel column (0–100% EA in PE gradient) to obtain desired compound
5
4
4
+
3
2
4
+
, 8.22 g in 75% yield. ESI-MS m/z 216 [M+H] .
3
. Conclusion
.3. General process for synthesis of intermediate 6 derivatives
In summary, we used 4-anilinothieno[2,3-d]pyrimidine scaffold
.3.1. N-(3-Chloro-4-fluorophenyl)-6-nitrothieno[2,3-d]pyrimi-
to modify the dual inhibitor CUDC-101 to design, synthesize and
evaluate compound 10 derivatives and compound 14 as novel
HDAC inhibitors. The biological evaluations indicated that when
the length of the carbon side chain reached six carbon units, most
compounds exhibited excellent inhibitory potencies against
HDACs. Some representative compounds could markedly inhibit
the proliferation of RPMI 8226 and HCT116 cancer cell lines with
low IC50 values. These results proved the 4-anilinothieno[2,3-
d]pyrimidine segment to be a good capping group. Compounds
din-4-amine 6a
To a solution of compound 5 (10 g, 0.046 mol) in i-PrOH (50 mL)
was added 3-chloro-4-fluoroaniline (7.43 g, 0.051 mol) at room
temperature. Subsequently, the mixture was heated to 80 °C and
stirred for 2 h. When the start material was consumed monitored
by TLC, the reaction was cooled to room temperature. After
removal of the solvent, the residue was suspended in ethyl acetate,
filtered and washed with ethyl acetate to afford the intermediate 6,
+
1
4.3 g as yellow solid in a 95% yield. ESI-MS m/z 325 [M+H] .
The other substituted anilines was treated with intermediate 5
1
0t and 10w showed the strongest antiproliferative activity
against RPMI8226 and HCT116 cancer cells lines respectively.
These present results suggested that 4-anilinothieno[2,3-d]pyrim-
idine-based hydroxamic acid derivatives could be lead compounds
for further optimization to develop novel anticancer agents.
for preparation of intermediates 6b–y according to the same proce-
dure described for 6a.
4
.4. General process for synthesis of intermediates 7a–y
4
4
4
. Experimental section
4.4.1. N -(3-Chloro-4-fluorophenyl)thieno[2,3-d]pyrimidine-
4
,6-diamine 7a
.1. Chemistry
To a solution of intermediate 6a (10 g, 0.03 mol) in ethanol
(
50 mL) was added iron powder (8.4 g, 0.15 mol) and saturated
The reagents (chemicals) were commercially available and used
NH Cl solution (50 mL). The mixture was stirred at 50 °C for 1 h.
4
without further purification. Analytical thin-layer chromatography
TLC) was performed on HSGF 254 (0.15–0.2 mm thickness). Col-
Then, the mixture was filtered through celite, washed with ethanol
(
followed by the water was added to the mixture to give the solid.
Then the solid was filtered, washed and dried to afford the intermedi-
umn chromatography was performed on silica gel 300–400 mesh
to purify the compounds. Nuclear magnetic resonance (NMR) spec-
tra were performed on a Brucker AMX-400 (TMS as IS). Chemical
shifts were reported in parts per million (ppm, d) downfield from
tetramethylsilane. Proton coupling patterns were described as sin-
glet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad
+
ate 7a, 6.35 g as off-white solid in 70% yield. ESI-MS m/z 295 [M+H] .
The other nitro intermediates 6b–y was reduced to intermedi-
ates 7b–y according to the same procedure described for 7a.
4.5. General process for synthesis of intermediates 8a–y
(
br). Low- and high-resolution mass spectra (LRMS and HRMS)
were given with electric, electrospray and matrix-assisted laser
desorption ionization (EI, ESI and MALDI) produced by Finnigan
MAT-95, LCQ-DECA spectrometer and IonSpec4.7 T.
4.5.1. Methyl-6-((4-((4-chloro-3-fluorophenyl)amino)thieno[2,
3-d]pyrimidin-6-yl)-amino)-6-oxohexanoate 8a
To a solution of 7a (147 mg, 0.5 mmol) in dry THF was added
methyl-6-chloro-6-oxohexanoate (133 mg, 0.75 mmol) and DIPEA
(129 mg, 1 mmol) at 0 °C. Then the mixture was stirred at room tem-
perature overnight. After the start material was consumed, satu-
4
4
.2. General procedures for synthesis of HDACs inhibitors
.2.1. Thieno[2,3-d]pyrimidin-4-ol (3)
rated NH Cl solution was added to quench the reaction and
4
A mixture of compound 1 (20 g, 0.127 mol) and formimidamide
extracted by EA three times. The organic phase was washed by water
acetate 2 (16 g, 0.153 mol) was reacted in the condition of
and NaCl(aq) respectively, dried by Na SO , concentrated and
2
4
Please cite this article in press as: Yang, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.030

6
W. Yang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
purified by flash silica gel column (0–45% EA in PE gradient) to
152.49, 151.86, 137.47, 137.40, 122.69, 121.68, 121.63, 119.31,
119.19, 117.12, 116.98, 115.42, 100.60, 35.57, 32.69, 28.88,
obtain desired production 8a as an oil in 75% yield. ESI-MS m/z
+
+
4
37 [M+H] .
28.82, 25.48, 25.20. ESI-MS m/z 466 [M+H] . HRMS calcd for
+
The other intermediates 8b–y was prepared according to the
20 5
C H22ClFN O
3
S [M+H] 466.1116, found 466.1120.
same procedure described for 8a.
1
4
.7.4. N -(4-((4-Chloro-3-(trifluoromethyl)phenyl)amino)thieno
6
4
.6. General process for synthesis of intermediates 9a–y
[2,3-d]pyrimidin-6-yl)-N -hydroxyadipamide 10d
Compound 10d was prepared from 9d according the same pro-
1
4
.6.1. 6-((4-((4-Chloro-3-fluorophenyl)amino)thieno[2,3-d]pyr-
cess described for 10a in 78% yield. H NMR (500 MHz, DMSO) d
imidin-6-yl)amino)-6-oxohexanoic acid 9a
To a solution of 8a (218 mg, 0.5 mmol) in MeOH was added
2
LiOH H O (105 mg, 2.5 mmol), the mixture was stirred at room
temperature overnight, followed by concentrated. The residue dis-
solved in water, extracted by EA. Acetic acid was added to the
water phase to form precipitate. Filter and wash the solid to give
desired product 9a in 50% yield. The crude product was used
11.66 (s, 1H), 10.40 (s, 1H), 9.87 (s, 1H), 8.64 (s, 1H), 8.48 (s, 1H),
8.44 (s, 1H), 8.27 (dd, J = 8.8,1.6 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H),
7.34 (s, 1H), 2.45 (t, J = 6.8 Hz, 2H), 1.99 (t, J = 6.9 Hz, 2H), 1.67–
1
3
1.49 (m, 4H).
C NMR (126 MHz, DMSO) d 171.00, 169.36,
162.15, 152.89, 151.68, 139.83, 137.68, 132.18, 127.03, 126.78,
125.64, 124.41, 123.31, 122.27, 119.60, 115.76, 100.49, 35.40,
+
32.57, 25.27, 24.95. ESI-MS m/z 488 [M+H] . HRMS calcd for
+
+
directly to next step without purification. ESI-MS m/z 423 [M+H] .
C
19
H
18ClF
3
N
5
O
3
S [M+H] 488.0771, found 488.0775.
The other intermediates 9b–y was prepared according to the
same procedure described for 9a.
1
4.7.5. N -(4-((4-Chloro-3-(trifluoromethyl)phenyl)amino)thie-
7
no[2,3-d]pyrimidin-6-yl)-N -hydroxyheptanediamide 10e
4
1
.7. General process for synthesis of the targeted compounds
0a–y
Compound 10e was prepared from 9e according the same pro-
cess described for 10a in 89% yield. H NMR (500 MHz, DMSO) d
1
1
1.64 (s, 1H), 10.39 (s, 1H), 9.85 (s, 1H), 8.62 (s, 1H), 8.46 (s, 1H),
1
4
.7.1. N -(4-((4-chloro-3-fluorophenyl)amino)thieno[2,3-d]pyr-
8.44 (s, 1H), 8.23 (dd, J = 8.8,1.6 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H),
7.34 (s, 1H), 2.43 (t, J = 6.8 Hz, 2H), 2.00 (t, J = 6.9 Hz, 2H), 1.64–
1.62 (m, 2H), 1.58–1.49 (m, 2H), 1.33–1.31 (m, 2H). 13C NMR
(126 MHz, DMSO) d 171.00, 169.36, 162.15, 152.89, 151.68,
139.83, 137.68, 132.18, 127.03, 126.78, 125.64, 124.41, 123.31,
6
imidin-6-yl)-N -hydroxyadipamide 10a
To a solution of 9a (211 mg, 0.5 mmol) in DMF solution was
added Hydroxylamine hydrochloride (52 mg, 0.75 mmol), DMAP
(
122 mg, 1 mmol), BOP (442 mg, 1 mmol). Then, the mixture was
stirred at room temperature until the start material was consumed
completely. Water was added into the mixture, filtered the formed
solid and washed with water. The crude product was purified by
flash silica gel column (0–10% MeOH in DCM) to give the targeted
122.27, 119.60, 115.76, 100.49, 35.48, 32.59, 28.66, 25.36, 25.01.
+
S [M+H]+
ESI-MS m/z 502 [M+H] . HRMS calcd for C20
H
20ClF
3
N
5
O
3
502.0927, found 502.0928.
1
1
compound 10a as solid in 86% yield. H NMR (600 MHz, DMSO) d
4.7.6. N -(4-((4-Chloro-3-(trifluoromethyl)phenyl)amino)thie-
8
1
8
1.62 (s, 1H), 10.40 (s, 1H), 9.66 (s, 1H), 8.70 (s, 1H), 8.45 (s, 1H),
.20 (s, 1H), 7.80 (s, 1H), 7.49–7.36 (m, 1H), 7.32 (s, 1H), 2.45 (t,
no[2,3-d]pyrimidin-6-yl)-N -hydroxyoctanediamide 10f
Compound 10f was prepared from 9f according the same pro-
1
3
1
J = 6.6 Hz, 2H), 2.00 (t, J = 6.6 Hz, 2H), 1.55–1.60 (m, 4H).
C
cess described for 10a in 86% yield. H NMR (500 MHz, DMSO) d
NMR (151 MHz, DMSO) d 170.95, 169.35, 161.87, 154.11, 153.16,
11.62 (s, 1H), 10.35 (s, 1H), 9.86 (s, 1H), 8.67 (s, 1H), 8.48 (s, 1H),
1
1
52.50, 151.89, 137.46, 137.37, 122.71, 121.69, 121.65, 119.33,
19.21, 117.14, 116.99, 115.41, 100.59, 35.40, 32.58, 25.28, 24.97.
8.44 (s, 1H), 8.28 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.33
(s, 1H), 2.43 (t, J = 7.3 Hz, 2H), 1.94 (t, J = 7.2 Hz, 2H), 1.65–1.56
+
+
13
ESI-MS m/z 438 [M+H] . HRMS calcd for C18
H
18ClFN
5
O
3
S [M+H]
(m, 2H), 1.50–1.48 (m, 2H), 1.30–1.10 (m, 4H).
C NMR
4
38.0803, found 438.0808.
(126 MHz, DMSO) d 171.11, 169.54, 162.15, 152.89, 151.69,
39.87, 137.79, 132.10, 127.04, 126.76, 125.65, 124.45, 123.32,
122.28, 119.55, 115.77, 100.43, 35.61, 32.70,28.85, 28.83, 25.47,
1
1
4
.7.2. N -(4-((4-Chloro-3-fluorophenyl)amino)thieno[2,3-d]pyr-
7
+
imidin-6-yl)-N -hydroxyheptanediamide 10b
25.10. ESI-MS m/z 516 [M+H] . HRMS calcd for C21
[M+H] 516.1084, found 516.1088.
H22ClF
3
N
5
O
3
S
+
Compound 10b was prepared from 9b according the same pro-
cess described for 10a in 87% yield. 1H NMR (600 MHz, DMSO) d
1
1
8
1.62 (s, 1H), 10.36 (s, 1H), 9.67 (s, 1H), 8.68 (s, 1H), 8.45 (s, 1H),
.20 (s, 1H), 7.81 (s, 1H), 7.42 (t, J = 8.8 Hz, 1H), 7.32 (s, 1H), 2.44
4.7.7. N -(4-((2,4-Difluorophenyl)amino)thieno[2,3-d]pyrimid-
8
in-6-yl)-N -hydroxyoctanediamide 10g
(
t, J = 6.6 Hz, 2H), 1.97 (t, J = 6.6 Hz, 2H), 1.63–1.62 (m, 2H), 1.54–
Compound 10g was prepared from 9g according the same pro-
cess described for 10a in 86% yield. H NMR (500 MHz, DMSO) d
.52 (m, 2H), 1.32–1.28 (m, 2H). 13C NMR (151 MHz, DMSO) d
71.05, 169.48, 161.87, 154.10, 153.16, 152.47, 151.88, 137.47,
37.40, 122.70, 121.68, 121.64, 119.32, 119.20, 117.13, 116.99,
15.42, 100.57, 35.48, 32.59, 28.66, 25.36, 25.01. ESI-MS m/z 452
1
1
1
1
1
11.62 (s, 1H), 10.36 (s, 1H), 9.44 (s, 1H), 8.67 (s, 1H), 8.26 (s, 1H),
7.57 (m, 1H), 7.35 (m, 1H), 7.17 (s, 1H), 7.13 (m, 1H), 2.42 (t,
J = 7.0 Hz, 2H), 1.95 (t, J = 7.1 Hz, 2H), 1.60–1.58 (m, 2H), 1.55–
+
+
13
[
M+H] . HRMS calcd for C19
H20ClFN
5
O
3
S [M+H] 452.0959, found
1.43 (m, 2H), 1.33–1.23 (m, 4H). C NMR (126 MHz, DMSO) d
4
52.0962.
171.01, 169.55, 161.79, 159.22, 159.13, 158.39, 158.29, 156.41,
1
56.31, 154.43, 152.28, 137.19, 129.77, 129.69, 123.46, 123.37,
1
4
.7.3. N -(4-((4-chloro-3-fluorophenyl)amino)thieno[2,3-d]pyr-
114.80, 111.81, 111.61, 105.12, 104.91, 104.71, 100.39, 35.57,
32.70, 28.83, 28.82, 25.48, 25.20. ESI-MS m/z 450 [M+H] . HRMS
calcd for C20
8
+
imidin-6-yl)-N -hydroxyoctanediamide 10c
+
Compound 10c was prepared from 9c according the same pro-
H
22
F
2
5
N O
3
S [M+H] 450.1411, found 450.1409.
cess described for 10a in 84% yield. 1H NMR (600 MHz, DMSO) d
1
1
8
1.66 (s, 1H), 10.36 (s, 1H), 9.69 (s, 1H), 8.69 (s, 1H), 8.45 (s, 1H),
.21–8.16 (m, 1H), 7.86–7.81 (m, 1H), 7.41 (t, J = 9.1 Hz, 1H), 7.34
4.7.8. N -(4-((4-Chloro-2-fluorophenyl)amino)thieno[2,3-d]
pyrimidin-6-yl)-N -hydroxyoctanediamide 10h
8
(
(
(
s, 1H), 2.44 (t, J = 7.3 Hz, 2H), 1.95 (t, J = 7.3 Hz, 2H), 1.67–1.57
Compound 10h was prepared from 9h according the same pro-
cess described for 10a in 84% yield. H NMR (500 MHz, DMSO) d
11.55 (s, 1H), 10.35 (s, 1H), 9.47 (s, 1H), 8.68 (s, 1H), 8.29 (s, 1H),
13
1
m, 2H), 1.55–1.46 (m, 2H), 1.35–1.27 (m, 4H).
C
NMR
151 MHz, DMSO) d 171.11, 169.54, 161.87, 154.09, 153.16,
Please cite this article in press as: Yang, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.030

W. Yang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
1
7
7
.63 (t, J = 8.5 Hz, 1H), 7.53 (dd, J = 10.2, 1.9 Hz, 1H), 7.32 (d,
4.7.13. N -Hydroxy-N -(4-(phenylamino)thieno[2,3-
d]pyrimidin-6-yl)heptanediamide 10n
J = 8.4 Hz, 1H), 7.18 (s, 1H), 2.41 (t, J = 7.3 Hz, 2H), 1.95 (t,
J = 7.3 Hz, 2H), 1.61 (m, 2H), 1.55–1.45 (m, 2H), 1.33–1.24 (s, 4H).
Compound 10n was prepared from 9n according the same pro-
cess described for 10a in 80% yield. H NMR (600 MHz, DMSO) d
11.61 (s, 1H), 10.38 (s, 1H), 9.51 (s, 1H), 8.70 (s, 1H), 8.39 (s, 1H),
7.83 (d, J = 7.3 Hz, 2H), 7.39–7.34 (m, 3H), 7.07 (t, J = 6.3 Hz, 1H),
2.43 (t, J = 6.3 Hz, 2H), 1.97 (t, J = 6.3 Hz, 2H), 1.67–1.57 (m, 2H),
1
3
1
C NMR (126 MHz, DMSO) d 171.02, 169.56, 161.98, 157.74,
55.75, 153.99, 152.18, 137.34, 130.12, 130.05, 129.21, 126.24,
26.15, 125.00, 117.09, 116.90, 115.05, 100.34, 35.59, 32.70,
1
1
2
+
8.84, 28.82, 25.48, 25.18. ESI-MS m/z 466 [M+H] . HRMS calcd
+
13
for C20
5
H22ClFN O
3
S [M+H] 466.1116, found 466.1112.
1.58–1.48 (m, 2H), 1.36–1.25 (m, 2H). C NMR (151 MHz, DMSO)
d 171.00, 169.51, 161.72, 153.60, 152.07, 140.05, 137.00, 128.93,
1
123.30, 121.76, 115.32, 100.85, 35.47, 32.59, 28.66, 25.36, 25.04.
4
.7.9. N -(4-((4-Bromo-2-fluorophenyl)amino)thieno[2,3-
+
S [M+H]+
8
ESI-MS m/z 400[M+H] . HRMS calcd for C19
H
22
N
5
O
3
d]pyrimidin-6-yl)-N -hydroxyoctanediamide 10i
4
00.1443, found 400.1440.
Compound 10h was prepared from 9h according the same pro-
cess described for 10a in 90% yield. 1H NMR (500 MHz, DMSO) d
1
8
4
.7.14. 21N -Hydroxy-N -(4-(phenylamino)thieno[2,3-
1
7
1
1
(
1
1
1.60 (s, 1H), 10.36 (s, 1H), 9.50 (s, 1H), 8.68 (s, 1H), 8.29 (s, 1H),
.64 (d, J = 9.5 Hz, 1H), 7.57 (t, J = 8.1 Hz, 1H), 7.44 (d, J = 8.1 Hz,
H), 7.19 (s, 1H), 2.42 (t, J = 6.5 Hz, 2H), 1.96 (t, J = 6.5 Hz, 2H),
.62 (m, 2H), 1.56–1.48 (m, 2H), 1.33–1.25 (m, 4H). C NMR
126 MHz, DMSO) d 170.98, 169.49, 161.99, 157.78, 155.79,
53.91, 152.16, 137.33, 137.32, 129.51, 127.92, 126.69, 126.59,
19.82, 119.63, 115.08, 100.37, 35.47, 32.58, 28.66, 25.35, 25.00.
5 3
ESI-MS m/z 510[M+H] . HRMS calcd for C20H22BrFN O S [M+H]
d]pyrimidin-6-yl)octanediamide 10m
Compound 10m was prepared from 9m according the same
1
1
3
process described for 10a in 82% yield. H NMR (600 MHz, DMSO)
d 11.55 (s, 1H), 10.35 (s, 1H), 9.49 (s, 1H), 8.68 (s, 1H), 8.39 (s, 1H),
7
.83 (d, J = 7.8 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.32 (s, 1H), 7.08 (t,
J = 7.0 Hz, 1H), 2.43 (t, J = 7.0 Hz, 2H), 1.95 (t, J = 6.9 Hz, 2H), 1.68–
1.56 (m, 2H), 1.54–1.46 (m, 2H), 1.35–1.27 (m, 4H). 13C NMR
+
+
(
151 MHz, DMSO) d 171.07, 169.55, 161.72, 153.51, 152.08,
5
10.0611, found 510.0612.
1
3
40.05, 137.00, 128.94, 123.37, 121.75, 115.32, 100.74, 35.59,
+
2.71, 28.85, 28.83, 25.49, 25.21. ESI-MS m/z 414 [M+H] . HRMS
1
+
4
.7.10. N -(4-((2-Fluoro-4-iodophenyl)amino)thieno[2,3-
d]pyrimidin-6-yl)-N -hydroxyoctanediamide 10j
calcd for C20
H
24
N
5
O
3
S [M+H] 414.1600, found 414.1604.
8
1
7
Compound 10j was prepared from 9j according the same pro-
4
.7.15. N -Hydroxy-N -(4-(p-tolylamino)thieno[2,3-
cess described for 10a in 86% yield. 1H NMR (500 MHz, DMSO) d
d]pyrimidin-6-yl)heptanediamide 10o
1
7
1.54 (s, 1H), 10.34 (s, 1H), 9.44 (s, 1H), 8.67 (s, 1H), 8.28 (s, 1H),
.72 (dd, J = 9.8, 1.7 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.41 (t,
J = 8.3 Hz, 1H), 7.18 (s, 1H), 2.41 (t, J = 7.4 Hz, 2H), 1.94 (t,
J = 7.3 Hz, 2H), 1.68–1.56 (m, 2H), 1.55–1.44 (m, 2H), 1.34–1.24
m, 4H). C NMR (126 MHz, DMSO) d 171.01, 169.54, 162.00,
57.62, 155.62, 153.88, 152.16, 137.33, 133.81, 129.71, 127.13,
27.04, 125.18, 125.00, 115.10, 100.36, 35.59, 32.70, 28.84,
8.82, 25.48, 25.18. ESI-MS m/z 558 [M+H] . HRMS calcd for
Compound 10m was prepared from 9m according the same
1
process described for 10a in 87% yield. H NMR (600 MHz, DMSO)
d 11.55 (s, 1H), 10.36 (s, 1H), 9.42 (s, 1H), 8.69 (s, 1H), 8.36 (s, 1H),
7
.69 (d, J = 8.0 Hz, 2H), 7.30 (s, 1H), 7.17 (d, J = 8.0 Hz, 2H), 2.43 (t,
1
3
(
J = 7.2 Hz, 2H), 2.29 (s, 3H), 1.97 (t, J = 7.2 Hz, 2H), 1.67–1.58 (m,
1
3
1
1
2
C
2
H), 1.58–1.48 (m, 2H), 1.35–1.26 (m, 2H). C NMR (151 MHz,
DMSO) d 170.94, 169.48, 161.56, 153.70, 152.16, 137.42, 136.84,
+
1
2
32.40, 129.36, 121.95, 115.14, 100.84, 35.47, 32.59, 28.66, 25.36,
+
+
20
H
22FIN
5
O
3
S [M+H] 558.0472, found 558.0473.
24 5 3
5.03, 20.97. ESI-MS m/z 414[M+H] . HRMS calcd for C20H N O S
+
[
M+H] 414.1600, found 414.1598.
1
4
.7.11. N -(4-((3-Chloro-2-fluorophenyl)amino)thieno[2,3-d]
pyrimidin-6-yl)-N -hydroxyoctanediamide 10k
8
1
8
4
.7.16. N -Hydroxy-N -(4-(p-tolylamino)thieno[2,3-
Compound 10k was prepared from 9k according the same pro-
d]pyrimidin-6-yl)octanediamide 10p
cess described for 10a in 70% yield. 1H NMR (500 MHz, DMSO) d
Compound 10p was prepared from 9p according the same pro-
1
1
7
1
1
1.65 (s, 1H), 10.37 (s, 1H), 9.63 (s, 1H), 8.68 (s, 1H), 8.31 (s, 1H),
.56 (t, J = 6.3 Hz, 1H), 7.44 (t, J = 6.2 Hz, 1H), 7.25 (t, J = 7.7 Hz,
H), 7.21 (s, 1H), 2.43 (t, J = 6.3 Hz, 2H), 1.95 (t, J = 6.3 Hz, 2H),
.66–1.58 (m, 2H), 1.56–1.48 (m, 2H), 1.32–1.22 (m, 4H).
NMR (126 MHz, DMSO) d 171.01, 169.50, 162.05, 153.91, 153.60,
52.19, 151.62, 137.41, 128.67, 128.58, 127.29, 126.94, 125.34,
25.30, 120.64, 120.51, 115.11, 100.38, 35.47, 32.58, 28.65,
8.63, 25.35, 25.01. ESI-MS m/z 466 [M+H] . HRMS calcd for
cess described for 10a in 86% yield. H NMR (600 MHz, DMSO) d
1
7
1.52 (s, 1H), 10.35 (s, 1H), 9.41 (s, 1H), 8.67 (s, 1H), 8.36 (s, 1H),
.68 (d, J = 8.3 Hz, 2H), 7.29 (s, 1H), 7.17 (d, J = 8.2 Hz, 2H), 2.42
1
3
C
(
(
(
t, J = 7.4 Hz, 2H), 2.30 (s, 3H), 1.95 (t, J = 7.3 Hz, 2H), 1.64–1.59
13
m, 2H), 1.56–1.47 (m, 2H), 1.33–1.25 (m, 4H).
C NMR
1
1
2
C
151 MHz, DMSO) d 170.98, 169.54, 161.56, 153.70, 152.16,
1
3
37.42, 136.85, 132.41, 129.37, 122.00, 115.14, 100.82, 35.59,
+
+
2.70, 28.85, 28.83, 25.48, 25.21, 20.97. ESI-MS m/z 428 [M+H] .
+
+
20
H
22ClFN
5
O
3
S [M+H] 466.1116, found 466.1118.
HRMS calcd for C21
H
26
N
5
O
S [M+H] 428.1756, found 428.1760.
3
1
1
4.7.17. N -(4-((3,4-Dimethylphenyl)amino)thieno[2,3-
4
6
.7.12. N -(4-((3-Chlorophenyl)amino)thieno[2,3-d]pyrimidin-
7
8
d]pyrimidin-6-yl)-N -hydroxyheptanediamide 10q
Compound 10q was prepared from 9q according the same
process described for 10a in 79% yield. H NMR (600 MHz,
DMSO) d 11.56 (s, 1H), 10.36 (s, 1H), 9.36 (s, 1H), 8.71 (s, 1H),
.36 (s, 1H), 7.55 (m, 2H), 7.29 (s, 1H), 7.11 (d, J = 7.9 Hz, 1H),
.43 (t, J = 7.2 Hz, 2H), 2.24 (s, 3H), 2.21 (s, 3H), 1.97 (t,
J = 7.2 Hz, 2H), 1.61 (m, 2H), 1.54 (m, 2H), 1.30 (m, 2H).
NMR (151 MHz, DMSO) d 170.94, 169.49, 161.52, 153.77,
-yl)-N -hydroxyoctanediamide 10l
Compound 10l was prepared from 9l according the same pro-
1
cess described for 10a in 88% yield. 1H NMR (600 MHz, DMSO) d
1
8
(
1.59 (s, 1H), 10.35 (s, 1H), 9.63 (s, 1H), 8.68 (s, 1H), 8.47 (s, 1H),
.10 (s, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.38 (t, J = 8.1 Hz, 1H), 7.35
8
2
s, 1H), 7.11 (d, J = 7.9 Hz, 1H), 2.43 (t, J = 7.3 Hz, 2H), 1.95 (t,
J = 7.3 Hz, 2H), 1.64–1.60 (m, 2H), 1.53–1.48 (m, 2H), 1.35–1.25
13
C
1
3
(
1
1
m, 4H). C NMR (151 MHz, DMSO) d 171.10, 169.55, 161.98,
1
1
52.20, 137.64, 136.78, 136.46, 131.28, 129.84, 123.17, 119.57,
15.10, 100.86, 35.46, 32.59, 28.66, 25.36, 25.04, 20.12, 19.29.
ESI-MS m/z 428 [M+H] . HRMS calcd for C21H N O S [M+H]
26 5 3
53.12, 151.87, 141.76, 137.41, 133.27, 130.58, 122.68, 120.54,
19.58, 115.60, 100.61, 35.60, 32.70, 28.84, 28.83, 25.48, 25.19.
ESI-MS m/z 448[M+H] . HRMS calcd for C20
+
+
+
+
H23ClN
5
O
3
S [M+H]
4
28.1756, found 428.1760.
4
48.1210, found 448.1212.
Please cite this article in press as: Yang, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.030

8
W. Yang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
1
4
.7.18. N -(4-((3,4-Dimethylphenyl)amino)thieno[2,3-
153.76, 152.20, 137.64, 136.79, 136.50, 131.28, 129.84, 123.17,
119.56, 115.10, 100.84, 80.3, 79.6, 67.5, 35.61, 32.69, 32.2, 28.85,
8
d]pyrimidin-6-yl)-N -hydroxyoctanediamide 10r
+
Compound 10r was prepared from 9r according the same pro-
28.80, 25.45, 25.09. ESI-MS m/z 534 [M+H] . HRMS calcd for
cess described for 10a in 79% yield. 1H NMR (600 MHz, DMSO) d
C
24
H
29ClN
O
5
S [M+H] 534.1578, found 534.1572.
+
5
1
7
1.52 (s, 1H), 10.35 (s, 1H), 9.34 (s, 1H), 8.68 (s, 1H), 8.36 (s, 1H),
.55 (m, 2H), 7.28 (s, 1H), 7.11 (d, J = 8.0 Hz, 1H), 2.42 (t,
1
4.7.23. N -(4-((3-Fluoro-4-(piperidin-1-yl)phenyl)amino)thie-
8
J = 7.4 Hz, 2H), 2.24 (s, 3H), 2.21 (s, 3H), 1.95 (t, J = 7.3 Hz, 2H),
.61 (m, 2H), 1.55–1.44 (m, 2H), 1.35–1.26 (m, 4H). 13C NMR
151 MHz, DMSO) d 170.97, 169.55, 161.52, 153.76, 152.20,
no[2,3-d]pyrimidin-6-yl)-N -hydroxyoctanediamide 10w
Compound 10w was prepared from 9w according the same pro-
cess described for 10a in 70% yield. H NMR (500 MHz, DMSO) d
1
(
1
1
1
37.64, 136.79, 136.50, 131.28, 129.84, 123.17, 119.56, 115.10,
00.84, 35.58, 32.70, 28.85, 28.83, 25.48, 25.21, 20.12, 19.29. ESI-
11.56 (s, 1H), 10.35 (s, 1H), 9.47 (s, 1H), 8.67 (s, 1H), 8.39 (s, 1H),
7.78 (m, 1H), 7.46 (m, 1H), 7.29 (m, 1H), 7.02 (t, J = 9.0 Hz, 1H),
2.92 (m, 4H), 2.46 (t, J = 7.2 Hz, 2H), 1.99 (t, J = 7.2 Hz, 2H), 1.71–
1.56 (m, 4H), 1.55–1.44 (m, 6H), 1.35–1.20 (m, 4H). 13C NMR
+
+
MS m/z 442 [M+H] . HRMS calcd for
22 28 5 3
C H N O S [M+H]
4
42.1913, found 442.1910.
(
126 MHz, DMSO) d 171.02, 169.55, 161.58, 155.83, 153.37,
1
4
.7.19. N -(4-((4-(Diethylamino)phenyl)amino)thieno[2,3-
152.06, 137.06, 136.68, 136.61, 134.87, 134.78, 119.66, 119.64,
117.51, 115.23, 109.88, 109.68, 100.74, 52.28, 35.59, 32.70, 28.85,
28.82, 26.23, 25.47, 25.20, 24.26. ESI-MS m/z 515[M+H] . HRMS
8
d]pyrimidin-6-yl)-N -hydroxyoctanediamide 10s
+
Compound 10s was prepared from 9s according the same pro-
cess described for 10a in 81% yield. 1H NMR (600 MHz, DMSO) d
calcd for C25
S [M+H] 515.2241, found 515.2243.
+
6
H32FN O
3
1
7
1.50 (s, 1H), 10.37 (s, 1H), 9.22 (s, 1H), 8.70 (s, 1H), 8.26 (s, 1H),
.46 (d, J = 8.6 Hz, 2H), 7.18 (s, 1H), 6.54 (d, J = 8.8 Hz, 2H), 3.11
1
4.7.24. N -(4-((3-Fluoro-4-(pyrrolidin-1-yl)phenyl)amino)thie-
8
(
q, J = 7.0 Hz, 4H), 2.40 (t, J = 7.4 Hz, 2H), 1.97 (t, J = 7.3 Hz, 2H),
no[2,3-d]pyrimidin-6-yl)-N -hydroxyoctanediamide 10x
1
.65–1.58 (m, 2H), 1.54–1.45 (m, 2H), 1.34–1.20 (m, 4H), 1.09 (t,
Compound 10x was prepared from 9x according the same pro-
cess described for 10a in 75% yield. H NMR (500 MHz, DMSO) d
1
3
1
J = 7.0 Hz, 6H).
C NMR (151 MHz, DMSO) d 170.88, 169.57,
1
1
1
61.08, 154.23, 152.32, 145.20, 136.30, 128.05, 124.53, 114.63,
11.82, 100.93, 46.80, 35.60, 32.70, 28.85, 28.83, 25.48, 25.20,
11.48 (s, 1H), 10.34 (s, 1H), 9.35 (s, 1H), 8.66 (s, 1H), 8.35 (s, 1H),
7.67 (m, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.24 (s, 1H), 6.75 (t,
J = 9.3 Hz, 1H), 3.28 (m, 4H), 2.45 (t, J = 7.0 Hz, 2H), 1.98 (t,
J = 7.0 Hz, 2H), 1.92–1.86 (m, 4H), 1.63–1.57 (m, 2H), 1.54–1.45
+
33 6 3
2.70. ESI-MS m/z 485 [M+H] . HRMS calcd for C24H N O S
+
[
M+H] 485.2335, found 485.2330.
1
3
(
m, 2H), 1.33–1.25 (m, 4H). C NMR (126 MHz, DMSO) d 170.95,
1
4
.7.20. N -(4-((4-(Diethylamino)-3-fluorophenyl)amino)thie-
169.55, 161.32, 153.51, 152.20, 150.28, 136.81, 133.58, 133.53,
130.60, 130.45, 118.37, 115.73, 115.68, 114.98, 110.78, 110.40,
8
no[2,3-d]pyrimidin-6-yl)-N -hydroxyoctanediamide 10t
Compound 10t was prepared from 9t according the same pro-
100.75, 50.03, 35.59, 32.70, 28.85, 28.83, 25.48, 25.20, 25.01. ESI-
cess described for 10a in 86% yield. 1H NMR (500 MHz, DMSO) d
MS m/z 501 [M+H] . HRMS calcd for C24
+
S [M+H]+
6
H30FN O
3
1
7
3
2
0
1
1
3
1.53 (s, 1H), 10.34 (s, 1H), 9.47 (s, 1H), 8.67 (s, 1H), 8.40 (s, 1H),
.77 (m, 1H), 7.46 (d, J = 6.9 Hz, 1H), 7.28 (s, 1H), 7.05 (s, 1H),
.13 (q, J = 7.0 Hz, 4H), 2.42 (t, J = 7.4 Hz, 2H), 1.94 (t, J = 7.3 Hz,
H), 1.65–1.58 (m, 2H), 1.54–1.45 (m, 2H), 1.35–1.22 (m, 4H),
.99 (t, J = 7.0 Hz, 6H). 13C NMR (126 MHz, DMSO) d 171.01,
69.55, 161.63, 156.48, 154.59, 153.34, 152.03, 137.09, 122.00,
17.43, 117.41, 115.26, 110.00, 109.76, 100.70, 46.20, 35.60,
501.2084, found 501.2080.
1
8
4.7.25. N -Hydroxy-N -(4-((4-(pyrrolidin-1-yl)phenyl)amino)th-
ieno[2,3-d]pyrimidin-6-yl)octanediamide 10y
Compound 10y was prepared from 9y according the same pro-
cess described for 10a in 79% yield. H NMR (600 MHz, DMSO) d
11.50 (s, 1H), 10.37 (s, 1H), 9.22 (s, 1H), 8.70 (s, 1H), 8.26 (s, 1H),
7.46 (d, J = 8.6 Hz, 2H), 7.18 (s, 1H), 6.54 (d, J = 8.8 Hz, 2H), 3.38
(m, 4H), 2.41 (t, J = 7.3 Hz, 2H), 1.98–1.93 (m, 6H), 1.64–1.57 (m,
1
+
2.70, 28.85, 28.83, 25.48, 25.20, 12.70. ESI-MS m/z 503 [M+H] .
+
HRMS calcd for C24
6
H32FN O
3
S [M+H] 503.2241, found 503.2243.
1
3
2
H), 1.53–1.47 (m, 2H), 1.32–1.25 (m, 4H). C NMR (151 MHz,
1
4
.7.21. N -(4-((4-(Dimethylamino)-3-fluorophenyl)amino)thie-
DMSO) d 170.88, 169.57, 161.08, 154.23, 152.32, 145.20, 136.30,
128.05, 124.53, 114.63, 111.82, 100.93, 47977, 35.56, 32.70,
28.86, 28.83, 25.49, 25.41, 25.23. ESI-MS m/z 483 [M+H] . HRMS
8
no[2,3-d]pyrimidin-6-yl)-N -hydroxyoctanediamide 10u
Compound 10u was prepared from 9u according the same pro-
cess described for 10a in 86% yield. 1H NMR (500 MHz, DMSO) d
+
+
calcd for C24
H
31
N
6
O
3
S [M+H] 483.2178, found 483.2180.
1
7
1.51 (s, 1H), 10.35 (s, 1H), 9.44 (s, 1H), 8.67 (s, 1H), 8.38 (s, 1H),
.76 (m, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.27 (s, 1H), 6.97 (t,
4.7.26. 4-Chlorothieno[2,3-d]pyrimidin-6-amine 11
J = 9.5 Hz, 1H), 2.74 (s, 6H), 2.41 (t, J = 7.2 Hz, 2H), 1.95 (t,
J = 7.2 Hz, 2H), 1.68–1.55 (m, 2H), 1.54–1.45 (m, 2H), 1.35–1.23
To a solution of 5 (215 mg, 1 mmol) in EtOH was added Iron
power (280 mg, 5 mmol) and saturated NH Cl solution (4 mL).
4
1
3
(
m, 4H). C NMR (126 MHz, DMSO) d 171.00, 169.50, 161.46,
The mixture was stirred at 50 °C for 1 h. Then, the mixture was fil-
tered through celite, washed with ethanol, concentrated. The resi-
due was purified by flash silica gel column (0–25% EA in PE
gradient) to afford the intermediate 11 as an oil in 75% yield. ESI-
1
1
3
55.18, 153.42, 153.25, 152.08, 137.01, 136.39, 136.32, 133.95,
33.86, 118.64, 117.65, 115.17, 110.13, 109.98, 100.71, 43.12,
+
5.60, 32.70, 28.85, 28.83, 25.48, 25.19. ESI-MS m/z 475[M+H] .
+
+
HRMS calcd for C22
6
H28FN O
3
S [M+H] 475.1928, found 475.1930.
MS m/z 186 [M+H] .
1
4
.7.22. (S)-N -(4-((3-Chloro-4-((tetrahydrofuran-3-yl)oxy)phe
4.7.27. Methyl 8-((4-chlorothieno[2,3-d]pyrimidin-6-yl)amino)-
8-oxooctanoate 12
8
nyl)amino)thieno[2,3-d]pyrimidin-6-yl)-N -hydroxyoctanedia
mide 10v
Compound 12 was prepared according to the same procedure
+
Compound 10v was prepared from 9v according the same pro-
described for 8a–y to give an oil, yield 78%. ESI-MS m/z 356 [M+H] .
cess described for 10a in 70% yield. 1H NMR (600 MHz, DMSO) d
1
7
4
1.52 (s, 1H), 10.35 (s, 1H), 9.34 (s, 1H), 8.68 (s, 1H), 8.36 (s, 1H),
.55 (m, 2H), 7.28 (s, 1H), 7.11 (d, J = 8.0 Hz, 1H), 5.32 (m, 1H),
.18–4.03 (m, 2H), 4.07–3.92 (m, 2H), 2.47–2.27 (m, 4H), 1.95 (t,
4.7.28. 8-((4-Chlorothieno[2,3-d]pyrimidin-6-yl)amino)-8-oxo
octanoic acid 13
Compound 13 was prepared according to the same procedure
described for 9a–y to give an white solid in 59% yield which was
J = 7.3 Hz, 2H), 1.61–1.58 (m, 2H), 1.55–1.44 (m, 2H), 1.35–1.26
1
3
+
(
m, 4H). C NMR (151 MHz, DMSO) d 170.97, 169.55, 161.52,
used without purification for next step. ESI-MS m/z 341 [M+H] .
Please cite this article in press as: Yang, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.030

W. Yang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
1
8
4
.7.29. N -(4-Chlorothieno[2,3-d]pyrimidin-6-yl)-N -
4.8.4. Western blot analysis
20–100 g of protein per lane was loaded onto a 10% SDS-poly-
hydroxyoctanediamide 14
l
Compound 14 was prepared according to the same process
described for 10a in 86% yield. H NMR (600 MHz, DMSO) d 11.58
s, 1H), 10.34 (s, 1H), 8.67 (s, 1H), 8.56 (s, 1H), 6.76 (s, 1H), 2.41
acrylamide gel and then transferred to a PVDF membrane. The
membrane was incubated with the following primary antibodies:
anti-tubulin, anti-ac tubulin, anti-actin, anti-H3 and anti-ac H3 at
4 °C overnight. Then, the membrane was washed with TBST three
times and incubated with Anti-Rabbit or mouse IgG (H+L), DyLight
800 labeled secondary antibody for 60 min at room temperature.
After three times washing with TBST, The immunoblots were
visualized by OdysseyÒ Infrared Imaging System (LI-COR
Biosciences).
1
(
(
1
1
3
t, J = 7.2 Hz, 2H), 1.95 (t, J = 7.2 Hz, 2H), 1.64–1.58 (m, 2H), 1.54–
.46 (m, 2H), 1.34–1.26 (m, 4H). 13C NMR (151 MHz, DMSO) d
71.05, 169.55, 163.10, 162.26, 151.97, 138.99, 116.71, 98.75,
+
5.61, 32.69, 28.85, 28.80, 25.45, 25.09. ESI-MS m/z 357 [M+H] .
+
HRMS calcd for C14
4
H18ClN O
3
S [M+H] 357.0788, found 357.0784.
4
4
.8. Biological evaluation
Acknowledgements
.8.1. Materials
Cell counting kit-8 was purchased from Dojindo, anti-histone
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (21021063, 91229204,
H3 primary antibody (#4499) was purchased from Cell Signaling
Technology Corporation, anti-tubulin primary antibody (#1878-
8
(
0
1125023, 81173033 and 81025017), National S&T Major Projects
2012ZX09103101-072, 2012ZX09301001-005, and 2013ZX09507-
01), Chinese Academy of Science ‘strategic leader in science and
1
) was purchased from epitomics, anti-acetyl-histone H3primary
antibody (#06-599) was purchased from Millipore, anti-acetyl-
tubulin primary antibody (#T7451) was purchased from Sigma-
Aldrich. Both anti-Rabbit and anti-mouse IgG (H+L), DyLight 800
labeled secondary antibodies were purchased from KPL (Kirkeg-
aard & Perry Laboratories. Inc.), PVDF transfer membrane was pur-
chased from Amersham Biosciences Corporation.
technology projects’ (XDA01040303), Program of Shanghai Subject
Chief Scientist (12XD1407100, 13XD1404300), the National
Science & Technology Major Project ‘Key New Drug Creation and
Manufacturing Program’ of China (2014ZX09507-002).
References and notes
4
.8.2. In vitro HDAC activity assay
All three full-length recombinant human HDACs (rhHDACs) 1, 3
1. (a) Gallinari, P.; Di Marco, S.; Jones, P.; Pallaoro, M.; Steinkuhler, C. Cell. Res.
2
2
007, 17, 195; (b) Grunstein, M. Nature 1997, 389, 349; (c) Kouzarides, T. Cell
007, 128, 693; (d) Li, E. Nat. Rev. Genet. 2002, 3, 662.
and 6 were expressed in insect High5 cells using a baculoviral
expression system, and all His -tagged and GST-fusion proteins
6
2
.
Peserico, A.; Simone, C. J. Biomed. Biotechnol. 2011, 2011, 371832.
3. (a) Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev. Drug. Discov. 2006, 5, 769;
b) Pandolfi, P. P. Cancer Chemother. Pharmcol. 2001, 48, S17; (c) Weichert, W.
was purified using Ni-NTA (QIAGEN). The deacetylase activity of
rhHDACs 1 and 3 were assayed with a HDAC substrate (Ac-Lys-
Tyr-Lys(-acetyl)-AMC), and HDAC6 was assayed with another
HDAC substrate (Boc-Lys(-acetyl)-AMC). The total HDAC assay vol-
(
Cancer Lett. 2009, 280, 168; (d) Weichert, W.; Roske, A.; Gekeler, V.; Beckers, T.;
Stephan, C.; Jung, K.; Fritzsche, F. R.; Niesporek, S.; Denkert, C.; Dietel, M.;
Kristiansen, G. Br. J. Cancer 2008, 98, 604.
4.
(a) De Ruijter, A. J. M.; Van Gennip, A. H.; Caron, H. N.; Kemp, S.; Van
Kuilenburg, A. B. P. Biochem. J. 2003, 370, 737; (b) Gregoretti, I. V.; Lee, Y. M.;
Goodson, H. V. J. Mol. Biol. 2004, 338, 17; (c) Michan, S.; Sinclair, D. Biochem. J.
2007, 404, 1.
ume was 25
buffer (25 mM Hepes, 137 mM NaCl, 2.7 mM KCl and 4.9 mM
MgCl , pH 8.0). The reaction was carried out in black 384-well
plates (OptiPlateTM-384F, PerkinElmer). In brief, the HDAC assay
mixture contained the substrate (5–50 M, 5 l), rhHDAC isoforms
20–200 nM) and inhibitors (1 l). Positive controls contained all
ll and all the assay components were diluted in Hepes
2
5. Witt, O.; Deubzer, H. E.; Milde, T.; Oehme, I. Cancer Lett. 2009, 277, 8.
6.
(a) Johnstone, R. W. Nat. Rev. Drug Discov. 2002, 1, 287; (b) Ropero, S.; Esteller,
M. Mol. Oncol. 2007, 1, 19.
l
l
(
l
7.
Butler, L. M.; Agus, D. B.; Scher, H. I.; Higgins, B.; Rose, A.; Cordon-Cardo, C.;
Thaler, H. T.; Rifkind, R. A.; Marks, P. A.; Richon, V. M. Cancer Res. 2000, 60,
the above components except the inhibitors. The negative controls
contained neither enzymes nor inhibitors. The HDAC6 assay com-
ponents were incubated at room temperature for 3 h, and HDAC1
or 3 were incubated for 24 h. The reaction was quenched with
5165.
8
9
.
.
(a) Furumai, R.; Matsuyama, A.; Kobashi, N.; Lee, K. H.; Nishiyama, N.;
Nakajima, I.; Tanaka, A.; Komatsu, Y.; Nishino, N.; Yoshida, M.; Horinouchi, S.
Cancer Res. 2002, 62, 4916; (b) Li, K. W.; Wu, J.; Xing, W. N.; Simon, J. A. J. Am.
Chem. Soc. 1996, 118, 7237.
Steele, N. L.; Plumb, J. A.; Vidal, L.; Tjornelund, J.; Knoblauch, P.; Rasmussen, A.;
Ooi, C. E.; Buhl-Jensen, P.; Brown, R.; Evans, T. R. J.; DeBono, J. S. Clin. Cancer Res.
2008, 14, 804.
the addition of 25
ll Trypsin with the final concentration of
0
.3125%. After 30 min incubation at room temperature, the 384
micro-well plates were read at wavelengths 355 nm (excitation)
and 460 nm (emission) using Envision (PerkinElmer). Each experi-
ment was done in triplicate.
1
1
0. Prince, H. M.; Bishton, M. J.; Johnstone, R. W. Future Oncol. 2009, 5, 601.
1. (a) Parodi, S.; Santi, I.; Marani, E.; Casella, C.; Puppo, A.; Vercelli, M.; Stagnaro,
E. Arch. Environ. Occup. Health 2014, 69, 139; (b) Saito, A.; Yamashita, T.;
Mariko, Y.; Nosaka, Y.; Tsuchiya, K.; Ando, T.; Suzuki, T.; Tsuruo, T.; Nakanishi,
O. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4592; (c) Suzuki, T.; Ando, T.; Tsuchiya,
K.; Fukazawa, N.; Saito, A.; Mariko, Y.; Yamashita, T.; Nakanishi, O. J. Med.
Chem. 1999, 42, 3001.
4
.8.3. Cell proliferation assay
Cell proliferation assay was determined with the Cell Counting
Kit-8 (CCK-8) which utilized Dojindo’s highly water-soluble tetra-
zolium salt. WST-8. WST-8 is reduced by dehydrogenases in cells
to give an orange colored product (formazan) which is soluble in
the tissue culture medium. The amount of the formazan dye gener-
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