Design, synthesis and biological evaluation of thienopyrimidine hydroxamic acid based derivatives as structurally novel histone deacetylase (HDAC) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.01.035

New thienopyrimidine hydroxamic acid derivatives as HDACs inhibitors were designed, synthesized and evaluated. All compounds were evaluated for their ability to inhibit recombinant human HDAC1, HDAC3, and HDAC6 isoforms and in vitro anti-proliferative act

Full text of DOI:10.1016/j.ejmech.2017.01.035

Accepted Manuscript
Design, synthesis and biological evaluation of thienopyrimidine hydroxamic acid
based derivatives as structurally novel histone deacetylase (HDAC) inhibitors
Jiang Wang, Mingbo Su, Tingting Li, Anhui Gao, Wei Yang, Li Sheng, Yi Zang, Jia Li,
Hong Liu
PII:
S0223-5234(17)30045-4
10.1016/j.ejmech.2017.01.035
EJMECH 9186
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 November 2016
Revised Date: 13 December 2016
Accepted Date: 21 January 2017
Please cite this article as: J. Wang, M. Su, T. Li, A. Gao, W. Yang, L. Sheng, Y. Zang, J. Li, H. Liu,
Design, synthesis and biological evaluation of thienopyrimidine hydroxamic acid based derivatives as
structurally novel histone deacetylase (HDAC) inhibitors, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.01.035.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Title:
Design, Synthesis and Biological Evaluation of Thienopyrimidine Hydroxamic
Acid Based Derivatives as Structurally Novel Histone Deacetylase (HDAC)
Inhibitors

ACCEPTED MANUSCRIPT
Graphical Abstract
By rational design and modification, compound 9m showed excellent HDACs
inhibitory activity, strong anti-proliferative activity against human cancer cell lines
including RMPI 8226 and HCT 116. Moreover, compound 9m noticeably
up-regulated the level of histone H3 acetylation at the low concentration of 0.3 µM.

ACCEPTED MANUSCRIPT
Highlights:
‹
Novel thienopyrimidine hydroxamic acid based derivatives were designed and
synthesized.
‹
Most of these compounds displayed good to excellent inhibitory activity against
HDACs with the IC values below 50 nM.
5
0
‹
Some compounds significantly up-regulated the levels of Histone H3 acetylation
and α-tubulin acetylation as well as exhibited powerful anti-proliferative
activities against cancer cell lines in vitro.

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Thienopyrimidine Hydroxamic
Acid Based Derivatives as Structurally Novel Histone Deacetylase (HDAC)
Inhibitors
†
†
†
Jiang Wang, Mingbo Su, Tingting Li, Anhui Gao, Wei Yang, Li Sheng, Yi Zang,
*
*
Jia Li, Hong Liu
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s
Republic of China.
Author Contributions
†
These authors contributed equally to this work.
Corresponding Author
*
H. Liu: Phone, 86-21-50807042; Fax, 86-21-50807042; E-mail, hliu@simm. ac.cn.
J. Li: Phone, 86-21-50801552; Fax, 86-21-50800721; E-mail, jli@simm.ac.cn.
*

ACCEPTED MANUSCRIPT
Abstract:
New thienopyrimidine hydroxamic acid derivatives as HDACs inhibitors were
designed, synthesized and evaluated. All compounds were evaluated for their ability
to inhibit recombinant human HDAC1, HDAC3, and HDAC6 isoforms and in vitro
anti-proliferative activity on tumor cell lines RMPI 8226 and HCT 116. Most of these
compounds displayed good to excellent inhibitory activities against HDACs. The IC50
values of compound 9m against HDAC1, HDAC3, and HDAC6 was 29.81 ± 0.52 nM,
2
4.71 ± 1.16 nM, and 21.29 ± 0.32 nM. Most of the compounds showed strong
anti-proliferative activity against human cancer cell lines including RMPI 8226 and
HCT 116. The IC50 values of compound 9m against RPMI 8226 and HCT 116
proliferation were 0.97 ± 0.072 µM and 1.01 ± 0.033 µM, respectively. In addition,
compound 9m noticeably up-regulated the level of histone H3 acetylation at the low
concentration of 0.3 µM.
Keywords: HDAC inhibitor, anti-proliferation, Western blot, thienopyrimidine,
Structure-activity relationship

ACCEPTED MANUSCRIPT
1
. Introduction
Histone acetylation and deacetylation, catalyzed by multisubunit complexes, play a
key role in the regulation of eukaryotic gene expression.[1] The acetylation status of
histones is determined by two sets of enzymes: histone acetyltransferases (HATs) and
histone deacetylases (HDACs).[2] HDACs also interact with retinoblastoma
tumor-suppressor protein and this complex is a key element in the control of cell
proliferation and differentiation. Together with metastasis-associated protein-2 MTA2,
HDACs deacetylates p53 and modulates its effect on cell growth and apoptosis.
HDAC inhibitors demonstrated prominent antitumor efficacy on broad spectrum
neoplasms in preclinical and clinical studies. [3-5] This concept has been well
validated by the approval of HDAC inhibitors vorinostat (suberoylanilide hydroxamic
acid, SAHA) [6] and romidepsin (depsipeptide) [7] for the treatment of cutaneous
T-cell lymphoma.
The thienopyrimidine fragment is widely present in antitumor agents, [8]
anti-inflammatory agents, [9] and anti-diabetic agents. [10] In our previous studies,
we reported 4-anilinothieno[2,3-d]pyrimidine derivatives as irreversible EGFR
inhibitors that displayed excellent inhibitory activities against wild type and mutant
EGFR (Fig. 1). [11] CUDC-101 has been reported as a multi inhibitors against HDAC,
EGFR and HER2 for the treatment of cancer, which contains a quinazoline moiety
within its structure. [12] On the basis of these observations, we attempted using
substituted 4-anilinothieno[3,2-d]pyrimidine moiety as a cap group (CAP) for protein
surface interactions, fixed carbon chain as a linker region, and hydroxamic acid group

ACCEPTED MANUSCRIPT
as a zinc binding group (ZBG) (Fig. 1). Herein, we presented the design, synthesis
and biological evaluation of thienopyrimidine hydroxamic acid based derivatives as
novel HDACs inhibitors. Most of these compounds showed excellent potencies
against HDACs and displayed powerful anti-proliferative activities against cancer cell
lines in vitro.
Fig. 1. Design strategy and modification of novel HDACs inhibitors.
2
2
. Results and discussion
.1. Chemistry
The synthetic route to obtain the desired thienopyrimidine-based hydroxamic
acid derivatives 9a-r are shown in Scheme 1. Heated by the microwave, the
thieno[3,2-d]pyrimidin-4(3H)-one 3 was obtained via the cyclization of methyl
3
-aminothiophene-2-carboxylate with formamidine acetate. Then, the nitration of
intermediate 3, followed by chlorination to afford 5. Reduction of nitro group using
iron/NH Cl, then coupling with various substituted anilines afforded the key
4

ACCEPTED MANUSCRIPT
intermediate 7. Treatment with different acyl chlorides to form amide 8. Finally, the
amide 8 was coupled with freshly prepared hydroxylamine hydrochloride to generate
the targeted products 9a-r.
Scheme 1. Reagents and conditions: a) microwave, 4-5 mins; b) H SO /HNO , 0
2
4
3
o
o
C-r.t; c) POCl , TEA, CH CN, 85 C; d) various substituted aniline, 1,4-dioxane, 85
3
3
o
o
C; e) Fe, NH Cl , EtOH, 50 C, 2 h; f) ROCO(CH ) COCl, DIPEA, THF, R =
4
(aq)
2 4
.
o
Methyl or Ethyl; g) NH OH HCl, KOH, MeOH-THF, 0 C-r.t.
2
2
.2. In vitro HDAC inhibitory activity
Firstly, in order to assess the efficacy of compounds 9a-r against recombinant human
HDAC activity, biochemical assays were performed as described previously. [13]
Compound potency against the recombinant human HDAC1, HDAC3, and HDAC6
isoforms was interrogated using SAHA as the positive control. The effects of
regiochemical substitution and electronics on the phenyl ring were explored. The

ACCEPTED MANUSCRIPT
results are presented in Table 1. Most of the compounds exhibited excellent inhibitory
activity against HDAC1, HDAC3, and HDAC6 isoforms which indicated the
thienopyrimidine scaffold was an outstanding capping group possessing strong
binding affinity with the HDACs surface. All of these inhibitors occupied a narrow
potency range, indicating tolerability for a wide variety of substituents on the phenyl
ring. A tendency in enzymatic potency of para-substitution < ortho-substitution <
meta-substitution was noted as shown in Table 1. Then, whether the introduced
halogen groups affect the inhibitory activity against HDACs is tested. We found that
most of these compounds (9c-9e and 9g-9i) showed strong potential activities against
HDACs with IC values < 100 nM. Both electron-withdrawing and electron-donating
5
0
groups on the phenyl ring were tolerated for HDACs Inhibition. For example,
compounds 9n (3-CN) and 9o (3-OMe) exhibited good inhibitory activities against
HDAC1, 3, and 6 with IC values of 45.02, 19.80, 20.05 and 62.25, 21.37, 20.90 nM,
50
respectively. Additionally, introducing steric hindrance at the 3-position and
-position of the phenyl ring (9j-9l and 9p-9r) decreased HDACs inhibitory activities.
4
Eventually, the SAR from this small group of compounds found compound 9m as a
potent HDACs inhibitor. The IC values of compound 9m against HDAC1, HDAC3,
5
0
and HDAC6 was 29.81 ± 0.52 nM, 24.71 ± 1.16 nM, and 21.29 ± 0.32 nM.
Table 1. Structures and in vitro inhibitory activity against HDACs of compounds 9a-r

ACCEPTED MANUSCRIPT
a
IC (nM) ± SD
5
0
Compd.
R
HDAC1
HDAC3
HDAC6
87.60 ± 1.39
119.94 ± 20.93
19.17 ± 1.07
36.98 ± 8.16
29.56 ±1.43
111.04 ± 9.50
42.52 ±1.88
33.84 ± 7.48
16.87 ± 0.15
58.54 ± 4.16
35.79 ±0.54
56.02 ±0.11
21.29 ± 0.32
20.05 ±1.25
20.90 ± 0.77
98.27 ± 1.36
9
a
2-Me
2-Me, 4-F
2-F, 4-F
2-F,4-Cl
137.85 ± 9.58
246.57 ± 4.92
91.99 ± 0.46
54.27 ± 21.44
75.12 ± 2.90
117.28 ± 7.70
73.73 ± 1.43
53.80 ± 5.37
54.46 ± 5.67
116.23 ± 5.09
99.91 ± 2.75
122.49 ± 8.49
29.81 ± 0.52
45.02 ± 0.94
62.25 ± 1.95
257.88 ± 47.48
98.79 ± 15.83
196.62 ± 3.13
99.87 ± 5.47
133.06 ± 11.79
67.54 ± 39.36
144.07 ± 5.25
64.51 ± 7.23
45.55 ± 1.88
33.77 ± 8.70
147.00 ± 0.90
65.05 ± 26.99
50.27 ± 8.03
24.71 ± 1.16
19.80 ± 4.31
21.37 ± 5.91
356.90 ± 9.93
9b
9
c
9d
9
e
2-F,5-CH
2-CH ,4-OCH
3
3
9
f
3
9g
3-F
9h
3-Cl
9i
3-Br
9j
3-ethyl
9
k
3-isopropyl
3-tertbutyl
3-enthynyl
3-CN
9
l
9
m
9
n
9o
3-OCH
3
9p
4-ethyl

ACCEPTED MANUSCRIPT
9
q
4-isopropyl
143.25 ± 40.11
174.73 ± 0.91
195.00 ± 16.12
152.46 ± 5.46
36.61 ± 0.81
71.38 ± 5.99
105.10 ± 25.46
9
r
4-OCH
3
215.48 ± 24.33
181.05 ± 28.92
SAHA
-
a
IC values were obtained based on three separate experiments and expressed as
5
0
means ± SD.
2
.3. Anti-proliferation of all compounds against RPMI 8226 and HCT 116 cells
Furthermore, the anti-proliferative activities of the new target compounds were
tested on human cancer cell lines RPMI 8226 and HCT 116. As summarized in Table
, most of the compounds exhibited good anti-proliferative activity against both
RPMI 8226 and HCT 116 cell lines. Compounds 9e (IC = 1.06 ± 0.193 µM) and 9o
2
5
0
(
IC = 1.26 ± 0.086 µM) showed similar anti-proliferative activities against RPMI
50
8
226 cells with the positive control SAHA, while the other compounds displayed less
anti-proliferative activity than SAHA. Compared to the RPMI 8226 cancer cells, the
HCT 116 cells appeared to be similar sensitive to our synthesized compounds.
Compounds 9e (IC = 1.33 ± 0.283 µM) and 9o (IC = 1.33 ± 0.255 µM) showed
5
0
50
similar anti-proliferative activities against HCT 116 cells with the reference
compound SAHA. Compound 9m possessed a slightly higher activity against both
RPMI 8226 and HCT 116 cells than SAHA, with the IC value of 0.97 ± 0.072 and
5
0
1
.01 ± 0.033 µM, respectively.
Table 2. In vitro anti-proliferative activity against RPMI 8226 and HCT 116 cells of
all compounds.

ACCEPTED MANUSCRIPT
a
IC (µM) ± SD
5
0
Compd.
RPMI 8226
1.75 ± 0.116
1.95 ± 0.141
1.74 ± 0.115
1.90 ± 0.108
1.06 ± 0.193
1.98 ± 0.069
1.63 ± 0.114
1.52 ± 0.104
1.75 ± 0.118
1.57 ± 0.119
2.08 ± 0.191
3.55 ± 0.403
0.97 ± 0.072
1.58 ± 0.066
1.26 ± 0.086
2.54 ± 0.135
2.79 ± 0.360
1.84 ± 0.129
1.25 ± 0.150
HCT 116
9
a
2.56 ± 0.386
2.64 ± 0.290
1.94 ± 0.334
1.79 ± 0.144
1.33 ± 0.283
2.48 ± 0.172
1.78 ± 0.351
1.82 ± 0.409
1.74 ± 0.170
1.44 ± 0.045
1.76 ± 0.339
2.93 ± 0.058
1.01 ± 0.033
2.09 ± 0.375
1.33 ± 0.255
2.36± 0.404
2.07 ± 0.442
2.18 ± 0.196
1.31 ± 0.085
9
b
9
c
9
d
9
e
9
f
9
g
9
h
9
i
9
j
9
k
9
l
9
m
9
n
9
o
p
q
9
9
9
r
SAHA
IC values were obtained based on three separate experiments and expressed as
a
5
0

ACCEPTED MANUSCRIPT
means ± SD.
2
.4. Analysis Ac-tubulin and Ac-histone H3 levels in RPMI 8226 cells by Western
boltting
With the cell proliferation data in hand for both RPMI 8226 and HCT 116 cells,
three representative compounds 9m, 9n, and 9o were selected to validate the observed
biochemical potencies by assessing their effects on the levels of tubulin and histone
H3 acetylation in RPMI 8226. For this assay, suberoylanilide hydroxamic aci d（ SAHA）
was used as the positive control. As shown in Fig. 2, Western blot analysis revealed
that 0.3 µM compound 9m induced significantly more acetylated tubulin and
acetylated histone H3 compared to that of 0.3 µM compounds 9n and 9o, which was
consistent with their strong inhibitory activities against HDAC and cancer cells. Even
0
.3 µM compound 9m can dramatically increase tubulin acetylation, which is much
better than that of SAHA. Additionally, the western blot analysis displayed that our
designed compounds, like the reference compound SAHA, were pan-HDACs
inhibitors.

ACCEPTED MANUSCRIPT
Fig. 2. Western blot analysis of Ac-tubulin and Ac-histone H3 from RPMI 8226 cell
cultured for 24 h with DMSO control and compounds 9m, 9n, 9o, and SAHA.
3
. Conclusion
In conclusion, based on the previous work in our laboratory, a series of
thienopyrimidine-based hydroxamic acid derivatives 9a-r were designed, synthesized
and evaluated as novel HDAC inhibitors. The in vitro biological evaluations indicated
that most compounds exhibited excellent inhibition against recombinant HDAC
enzyme activities and multiple cancer cells lines proliferation. Especially, the
compounds 9e, 9m, and 9o could significantly inhibit the proliferation of RPMI 8226
and HCT 116 cancer cell lines with low IC values. Western blot analysis revealed
5
0
that compounds 9m, 9n, and 9o caused a substantial increase in the level of acetylated
α-tubulin and histone H3 in a dose-dependent manner. The present results suggested
that thienopyrimidine-based hydroxamic acid derivatives could be lead compounds
for further optimization to develop novel HDAC inhibitors.

ACCEPTED MANUSCRIPT
4
4
. Experiment Section
.1. Chemistry
The reagents (chemicals) were commercially available and used without further
purification. Analytical thin-layer chromatography (TLC) was performed on HSGF
2
3
54 (0.15-0.2 mm thickness). Column chromatography was performed on silica gel
00-400 mesh to purify the compounds. Nuclear magnetic resonance (NMR) spectra
were performed on a Brucker AMX-400 (TMS as IS). Chemical shifts were reported
in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling
patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broad (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
4
.2 General procedures for synthesis of HDACs inhibitors
.2.1. Thieno[3,2-d]pyrimidin-4(3H)-one (3)
A mixture of compound 1 (20 g, 0.127 mol) and formimidamide acetate 2 (16 g,
.155 mol) was reacted in the condition of microwave for 4-5 mins. After cooled to
0
room temperature, 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
+
compound 3, 14.8 g as gray solid in 76% yield. ESI-MS m/z 153.0 [M+H] .
4
.2.2. 6-Nitrothieno[3,2-d]pyrimidin-4(3H)-one (4)
To a mixture of H SO (20 mL) and HNO (15 mL) was added compound 3 (9.7 g,
2
4
3

ACCEPTED MANUSCRIPT
0
.064 mol) slowly under ice-bath condition. Then, the reaction mixture was heated to
room tempreture. 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 water for three times, dried to afford the desired
1
compound 4 , 10.8 g as yellow solid in 86% yield. H NMR (400 MHz, DMSO-d ) δ
6
-
1
3.00 (s, 1H), 9.33 (s, 1H), 8.35 (s, 1H); MS (ESI) m/z 195.9 [M-H] .
.2.3. 4-Chloro-6-nitrothieno[3,2-d]pyrimidine (5)
4
To a solution of compound 4 (2 g, 0.01 mol) in CH CN (20 mL) was added POCl
3
3
(2.04 mL, 0.022 mol) and TEA (2.82 mL, 0.02 mol). Then the mixture was heated to
o
8
5 C. 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 ,
3
2
4
concentrated and purified by flash silica gel column (0-100% EA in PE gradient) to
+
obtain desired compound 5, 1.7 g in 78% yield. ESI-MS m/z 216.0 [M+H] .
4
4
.3. General process for synthesis of intermediates 6a-r
.3.1. 6-Nitro-N-(o-tolyl)thieno[3,2-d]pyrimidin-4-amine 6a
To a solution of compound 5 (1.7 g, 0.008 mol) in 1,4-dioxane (20 mL) was added
o-toluidine (1.1 mL, 0.01 mol) at room temperature. Subsequently, the mixture was
o
heated to 85 C. 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 6a, 2.05 g as yellow solid in 83% yield. ESI-MS m/z 287.0 [M+H] .
The other substituted aniline was treated with intermediate 5 for preparation of

ACCEPTED MANUSCRIPT
intermediates 6b-r according to the same procedure described for 6a.
4
4
.4. General process for synthesis of intermediates 7a-r
4
.4.1. N -(o-tolyl)thieno[3,2-d]pyrimidine-4,6-diamine 7a
To a solution of intermediate 6a (2.05 g, 0.007 mol) in ethanol (30 mL) was added
iron powder (4 g, 0.07 mol) and saturated NH Cl solution (30 mL). The mixture was
4
o
stirred at 50 C for 2 h. 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 intermediate 7a, 1.32 g light green
+
solid in 61% yield. ESI-MS m/z 257.1 [M+H] .
The other nitro intermediates 6b-r was reduced to intermediates 7b-r according to the
same procedure described for 7a.
4
4
.5. General process for synthesis of intermediates 8a-r
.5.1. Methyl 7-oxo-7-((4-(o-tolylamino)thieno[3,2-d]pyrimidin-6-yl)amino)
heptanoate 8a
To a solution of 7-methoxy-7-oxoheptanoic acid (165 mg, 0.9 mmol) in SOCl (2 mL)
2
o
at 50 C, the mixture was stirred 2 h. After removal of the solvent, 7a (150 mg, 0.59
o
mmol) in dry THF was added, and DIPEA (193 µL, 1.18 mmol) at 0 C. Then the
mixture was stirred at room temperature and was monitored by TLC. After an
overnight time, the reaction was stopped and quenched by slowly addition of
saturated NH Cl solution. The mixture was then extracted by ethyl acetate three times.
4
The combined organic layer was washed with water and brine, respectively. dried
over anhydrous Na SO , concentrated in vacuo and purified by flash silica gel column
2
4

ACCEPTED MANUSCRIPT
(0-45% EA in PE gradient) to obtain desired production 8a as yellow solid in 71%
1
yield. H NMR (500 MHz, DMSO-d ) δ 10.31 (s, 1H), 9.67 (s, 1H), 8.69 (s, 1H), 8.52
6
(s, 1H), 8.33 (s, 1H), 7.48 – 7.26 (m, 4H), 3.96 (s, 3 H), 2.60 – 2.54 (m, 2H), 2.36 (s,
3
1
H), 1.88 (t, J = 7.4 Hz, 2H), 1.68 (p, J = 7.2 Hz, 2H), 1.51 (p, J = 7.2 Hz, 2H), 1.39 –
+
.33 (m, 4H). ESI-MS m/z 427.1 [M+H] .
The other intermediates 8b-r was prepared according to the same procedure described
for 8a.
4
4
.6. General process for synthesis of the targeted compounds 9a-r
.6.1.
1
7
N -hydroxy-N -(4-(o-tolylamino)thieno[3,2-d]pyrimidin-6-yl)heptanediamide 9a
To a solution of 8a (178 mg, 0.43 mmol) in anhydrous THF was added
hydroxylamine hydrochloride (5 mL). Then, the mixture was stirred at room
temperature and monitored by TLC. After 24 hours, the reaction was stopped and
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
o
1
give the targeted compound 9a as white solid in 57% yield. Mp 176.8-177.5 C; H
NMR (500 MHz, DMSO-d ) δ 10.36 (s, 1H), 10.11 (s, 1H), 9.57 (s, 1H), 8.74 (s, 1H),
6
8
.49 (s, 1H), 8.22 (s, 1H), 7.44 – 7.16 (m, 4H), 2.50 – 2.43 (m, 2H), 2.17 (s, 3H), 1.94
(
(
t, J = 7.4 Hz, 2H), 1.57 (p, J = 7.6, 7.1 Hz, 2H), 1.48 (p, J = 7.2 Hz, 2H), 1.34 – 1.20
1
3
m, 4H). C NMR (125 MHz, DMSO-d ) δ 171.6, 169.0, 156.7, 153.8, 152.5, 136.1,
6
1
1
36.0, 130.5, 130.2, 128.7, 127.2, 126.3, 115.8, 111.9, 35.3, 32.3, 28.4, 25.2, 25.1,
+
-
7.9. LRMS (ESI) m/z 428.1 [M+H] , HRMS (ESI) m/z for C H N O S [M-H]
2
1
25
5
3

ACCEPTED MANUSCRIPT
calcd 426.1605, found 426.1594.
4
.6.2.
1
8
N -(4-((4-fluoro-2-methylphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxy
octanediamide 9b
Compound 9b was prepared from 8b according the same process described for 9a in
o
1
5
1
5
7
1
1% yield. Mp 196.1-197.4 C; H NMR (400 MHz, DMSO-d ) δ 10.32 (s, 1H),
6
0.09 (s, 1H), 9.51 (s, 1H), 8.65 (s, 1H), 8.49 (s, 1H), 8.23 (s, 1H), 7.34 (dd, J = 8.7,
.6 Hz, 1H), 7.20 (dd, J = 9.7, 3.0 Hz, 1H), 7.09 (td, J = 8.5, 3.0 Hz, 1H), 2.46 (t, J =
.4 Hz, 2H), 2.16 (s, 3H), 1.93 (t, J = 7.4 Hz, 2H), 1.56 (p, 2H), 1.48 (p, 2H), 1.35 –
13
.19 (m, 4H). C NMR (125 MHz, DMSO-d ) δ 172.1, 169.5, 161.2 (d, J = 243.3
6
Hz), 157.3, 154.2, 152.9, 139.4 (d, J = 8.52 Hz), 132.9, 131.1 (d, J = 9.57 Hz), 130.7,
17.2 (d, J = 22.2 Hz), 116.3, 113.4 (d, J = 22.3 Hz), 112.3, 40.5, 35.7, 32.7, 28.9,
1
+
2
5.6, 25.5, 18.4. LRMS (ESI) m/z 446.1 [M+H] , HRMS (ESI) m/z for C H FN O S
21 24 5 3
-
[
M-H] calcd 444.1511, found 444.1516
4
.6.3.
1
8
N -(4-((2,4-difluorophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxyoctane
diamide 9c
Compound 9c was prepared from 8c according the same process described for 9a
o
1
in 40% yield. Mp 206.7-208.3 C; H NMR (400 MHz, DMSO-d ) δ 10.30 (s, 1H),
6
1
1
6
0.13 (s, 1H), 9.69 (s, 1H), 8.63 (s, 1H), 8.51 (s, 1H), 8.30 (s, 1H), 7.61 – 7.48 (m,
H), 7.44 – 7.30 (m, 1H), 7.22 – 7.06 (m, 1H), 1.92 (t, J = 7.4 Hz, 2H), 1.56 (p, J =
1
3
.4 Hz, 2H), 1.47 (p, J = 7.2 Hz, 2H), 1.34 – 1.18 (m, 6H). C NMR (125 MHz,

ACCEPTED MANUSCRIPT
DMSO-d ) δ 172.2, 169.5, 160.8 (dd, J = 246.7, 11.1 Hz), 158.0 (dd, J = 249.5, 12.7
6
Hz), 156.6, 154.1, 152.9, 130.8, 130.7 (d, J = 10.0 Hz), 122.7(dd, J =13.0, 2.8 Hz),
1
4
4
4
11.9 (d, J = 22.7 Hz), 116.3, 113.1, 35.7, 32.7, 28.9, 25.6, 25.5. LRMS (ESI) m/z
+
-
50.1 [M+H] , HRMS (ESI) m/z for C H ClFN O S [M-H] calcd 464.0965, found
2
0
21
5
3
64.0962.
.6.4.
1
8
N -(4-((4-chloro-2-fluorophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxyo
ctanediamide 9d
Compound 9d was prepared from 8d according the same process described for
o
1
9
1
2
a in 42% yield. Mp 209.1-211.0 C; H NMR (400 MHz, DMSO-d ) δ 10.30 (s, 1H),
6
0.15 (s, 1H), 9.78 (s, 1H), 8.63 (s, 1H), 8.53 (s, 1H), 8.32 (s, 1H), 7.56 (t, J = 8.5 Hz,
H), 7.33 (d, J = 9.7 Hz, 1H), 1.92 (t, J = 7.4 Hz, 2H), 1.56 (p, J = 7.5 Hz, 2H), 1.47
1
3
(
p, J = 8.1, 7.5 Hz, 2H), 1.35 – 1.16 (m, 6H). C NMR (125 MHz, DMSO-d ) δ 172.2,
6
1
1
2
69.5, 157.3 (d, J = 251.0 Hz), 156.2, 154.0, 152.9, 131.0 (d, J = 7.6 Hz), 130.8,
30.1, 125.8 (d, J = 10.2 Hz), 125.1, 117.1 (d, J = 24.0 Hz).116.3, 113.5, 35.7, 32.7,
+
8.9, 25.6, 25.5. LRMS (ESI) m/z 466.0 [M+H] , HRMS (ESI) m/z for
-
C H ClFN O S [M-H] calcd 464.0965, found 464.0962.
20
21
5
3
4
.6.5.
1
8
N -(4-((2-fluoro-5-methylphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxy
octanediamide 9e
Compound 9e was prepared from 8e according the same process described for 9a
o
1
in 56% yield. Mp 200.0-201.8 C; H NMR (400 MHz, DMSO-d ) δ 10.32 (s, 1H),
6

ACCEPTED MANUSCRIPT
10.15 (s, 1H), 9.73 (s, 1H), 8.68 (s, 1H), 8.53 (s, 1H), 8.30 (s, 1H), 7.32 (dd, J = 7.7,
2.2 Hz, 1H), 7.20 (dd, J = 10.1, 8.4 Hz, 1H), 7.17 – 7.08 (m, 1H), 2.48 (d, J = 8.0 Hz,
2H), 2.32 (s, 3H), 1.94 (t, J = 7.3 Hz, 2H), 1.59 (p, J = 7.2 Hz, 2H), 1.49 (p, J = 7.2
1
3
Hz, 2H), 1.35 – 1.20 (m, 4H). C NMR (125 MHz, DMSO-d ) δ 172.1, 169.6, 156.0
6
(d, J = 245.5 Hz), 156.6, 154.10, 152.9, 134.1 (d, J = 3.5 Hz), 130.8, 129.6, 128.6 (d,
J = 7.5 Hz), 125.5 (d, J = 12.6 Hz), 116.2, 116.1 (d, J = 20.2 Hz), 113.1, 35.7, 32.7,
+
2
8.9, 25.6, 25.5, 20.7. LRMS (ESI) m/z 446.1 [M+H] , HRMS (ESI) m/z for
-
C H FN O S [M-H] calcd 444.1511, found 444.1504.
21
24
5
3
4
.6.6.
1
8
N -hydroxy-N -(4-((4-methoxy2-methylphenyl)amino)thieno[3,2-d]pyrimidin-6-y
l)octanediamide 9f
Compound 9f was prepared from 8f according the same process described for 9a
o
1
in 34% yield. Mp 190.0-191.8 C; H NMR (400 MHz, DMSO-d ) δ 10.31 (s, 1H),
6
1
0.03 (s, 1H), 9.39 (s, 1H), 8.64 (d, J = 1.7 Hz, 1H), 8.45 (s, 1H), 8.15 (s, 1H), 7.19 (d,
J = 8.6 Hz, 1H), 6.89 (d, J = 2.9 Hz, 1H), 6.80 (dd, J = 8.6, 2.9 Hz, 1H), 3.77 (s, 3H),
2
1
1
.44 (t, J = 7.4 Hz, 2H), 2.10 (s, 3H), 1.91 (t, J = 7.4 Hz, 2H), 1.53 (p, J = 7.2 Hz, 2H),
1
3
.45 (p, J = 7.1 Hz, 2H), 1.32 – 1.20 (m, 4H). C NMR (125 MHz, DMSO-d ) δ
6
72.0, 169.6, 158.9, 157.8, 154.3, 152.9, 138.6, 130.9, 130.5, 129.0, 116.5, 115.9,
+
1
12.0, 55.7, 35.7, 32.7, 28.9, 25.6, 25.5, 18.5. LRMS (ESI) m/z 458.1 [M+H] , HRMS
-
(
ESI) m/z for C H N O S [M-H] calcd 456.1711, found 456.1713.
22 27 5 4
4
.6.7.
1
8
N -(4-((3-fluorophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxyoctanedia

ACCEPTED MANUSCRIPT
mide 9g
Compound 9g was prepared from 8g according the same process described for 9a
o
1
in 56% yield. Mp 213.1-214.8 C; H NMR (400 MHz, DMSO-d ) δ 10.35 (d, 1H),
6
1
0.24 (s, 1H), 9.95 (s, 1H), 8.72 (s, 1H), 8.68 (d, J = 1.7 Hz, 1H), 8.38 (s, 1H), 7.91
dt, J = 12.0, 2.3 Hz, 1H), 7.62 (ddd, J = 8.3, 2.0, 0.9 Hz, 1H), 7.40 (td, J = 8.3, 6.9
Hz, 1H), 6.93 (tdd, J = 8.4, 2.6, 0.9 Hz, 1H), 2.48 (d, J = 4.2 Hz, 2H), 1.93 (t, J = 7.4
(
1
3
Hz, 2H), 1.58 (p, J = 7.3 Hz, 2H), 1.48 (p, J = 7.2 Hz, 2H), 1.34 – 1.22 (m, 4H). C
NMR (125 MHz, DMSO-d ) δ 172.2, 169.6, 162.5 (d, J = 240.9 Hz), 155.3, 153.7,
6
1
52.9, 141.5 (d, J = 11.3 Hz), 130.9, 130.5 (d, J = 9.6 Hz), 117.6, 117.6, 116.4, 114.3,
110.2 (d, J = 21.1 Hz), 108.7 (d, J = 26.2 Hz), 35.7, 32.7, 28.9, 25.6, 25.5. LRMS
+
-
(
ESI) m/z 432.1 [M+H] , HRMS (ESI) m/z for C H FN O S [M-H] calcd
20 22 5 3
4
4
30.1355, found 430.1358.
.6.8.
1
8
N -(4-((3-chlorophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxyoctanedia
mide 9h
Compound 9h was prepared from 8h according the same process described for
o
1
9
1
1
2
2
1
a in 56% yield. Mp 191.8-193.0 C; H NMR (400 MHz, DMSO-d ) δ 10.33 (s, 1H),
6
0.21 (s, 1H), 9.92 (s, 1H), 8.72 (s, 1H), 8.66 (s, 1H), 8.38 (s, 1H), 8.09 (t, J = 2.1 Hz,
H), 7.77 (ddd, J = 8.3, 2.1, 1.0 Hz, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.22 – 7.04 (m, 1H),
.50 (t, 2H), 1.94 (t, J = 7.4 Hz, 2H), 1.58 (p, J = 7.2 Hz, 2H), 1.49 (p, J = 7.2 Hz,
1
3
H), 1.37 – 1.22 (m, 4H). C NMR (125 MHz, DMSO-d ) δ 172.2, 169.6, 155.2,
6
53.7, 152.9, 141.2, 133.3, 130.9, 130.6, 123.4, 121.4, 120.3, 116.4, 114.3, 35.7, 32.7,

ACCEPTED MANUSCRIPT
+
2
8.9, 25.6, 25.5. LRMS (ESI) m/z 448.1 [M+H] , HRMS (ESI) m/z for
-
C H ClN O S [M-H] calcd 446.1059, found 446.1066.
20
22
5
3
4
.6.9.
1
8
N -(4-((3-bromophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxyoctanedia
mide 9i
Compound 9i was prepared from 8i according the same process described for 9a
o
1
in 59% yield. Mp 204.8-206.0 C; H NMR (400 MHz, DMSO-d ) δ 10.34 (s, 1H),
6
1
0.22 (s, 1H), 9.91 (s, 1H), 8.72 (s, 1H), 8.67 (s, 1H), 8.38 (s, 1H), 8.22 (t, J = 2.0 Hz,
H), 7.87 – 7.77 (m, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.28 (dt, J = 8.1, 1.4 Hz, 1H), 2.49
1
(t, J = 7.2 Hz, 2H), 1.94 (t, J = 7.4 Hz, 2H), 1.64 – 1.54 (m, 2H), 1.53 – 1.43 (m, 2H),
13
1
.33 – 1.22 (m, 4H). C NMR (125 MHz, DMSO-d ) δ 172.2, 169.6, 155.2, 153.7,
6
1
52.9, 141.3, 130.9, 126.3, 124.2, 121.7, 120.7, 116.4, 114.3, 35.7, 32.7, 28.9, 25.6,
+
+
2
5.5. LRMS (ESI) m/z 492.0 [M+H] , HRMS (ESI) m/z for C H BrN O S [M+H]
20 23 5 3
calcd 492.0699, found 492.07134.
4
.6.10.
1
8
N -(4-((3-ethylphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxyoctanediam
ide 9j
Compound 9j was prepared from 8j according the same process described for 9a
o
1
in 70% yield. Mp 184.9-186.1 C; H NMR (400 MHz, DMSO-d ) δ 10.33 (d, J = 1.8
6
Hz, 1H), 10.16 (s, 1H), 9.72 (s, 1H), 8.66 (d, J = 1.7 Hz, 1H), 8.63 (s, 1H), 8.32 (s,
1
H), 7.66 – 7.61 (m, 1H), 7.59 (t, J = 1.9 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 6.99 (dt, J
7.5, 1.3 Hz, 1H), 2.48 (t, J = 7.4 Hz, 2H), 1.94 (t, J = 7.4 Hz, 2H), 1.58 (p, 2H), 1.49
=

ACCEPTED MANUSCRIPT
1
3
(
p, 2H), 1.35 – 1.24 (m, 4H), 1.21 (t, J = 7.6 Hz, 3H). C NMR (125 MHz, DMSO-d )
6
δ 172.2, 169.6, 155.7, 153.9, 152.7, 144.6, 139.3, 130.9, 128.9, 123.8, 122.2, 120.4,
+
1
16.0, 113.8, 35.7, 32.7, 28.9, 28.7, 25.6, 25.5, 16.0. LRMS (ESI) m/z 442.1 [M+H] ,
-
HRMS (ESI) m/z for C H N O S [M-H] calcd 440.1762, found 440.1761.
2
2
27
5
3
4
.6.11.
1
8
N -hydroxy-N -(4-((3-isopropylphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)octane
diamide 9k
Compound 9k was prepared from 8k according the same process described for
o
1
9
1
7
1
1
1
2
a in 74% yield. Mp 182.8-184.2 C; H NMR (400 MHz, DMSO-d ) δ 10.33 (s, 1H),
6
0.16 (s, 1H), 9.73 (s, 1H), 8.72 – 8.55 (m, 2H), 8.32 (s, 1H), 7.68 (d, J = 7.7 Hz, 1H),
.58 (s, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 7.4 Hz, 1H), 2.97 – 2.81 (m, 1H),
.94 (t, J = 7.2 Hz, 2H), 1.66 – 1.54 (m, 2H), 1.54 – 1.41 (m, 2H), 1.37 – 1.12 (m,
1
3
2H). C NMR (125 MHz, DMSO-d ) δ 172.2, 169.6, 155.7, 153.9, 152.7, 149.2,
6
39.2, 130.9, 128.8, 122.4, 120.8, 120.5, 116.0, 113.8, 35.7, 33.9, 32.7, 28.9, 25.6,
+
5.5, 24.3. LRMS (ESI) m/z 456.1 [M+H] , HRMS (ESI) m/z for C H N O S
23
29
5
3
-
[
M-H] calcd 454.1918, found 454.1924.
4
.6.12.
1
8
N -(4-((3-tert-butylphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxyoctane
diamide 9l
Compound 9l was prepared from 8l according the same process described for 9a
o
1
in 71% yield. Mp 159.6-160.3 C; H NMR (400 MHz, DMSO-d ) δ 10.34 (s, 1H),
6
1
0.16 (s, 1H), 9.73 (s, 1H), 8.65 (d, J = 14.4 Hz, 2H), 8.33 (s, 1H), 7.84 – 7.57 (m,

ACCEPTED MANUSCRIPT
2
H), 7.30 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 7.8 Hz, 1H), 1.94 (t, J = 7.4 Hz, 2H), 1.65 –
1
3
1
.54 (m, 2H), 1.54 – 1.44 (m, 2H), 1.39 – 1.19 (m, 15H). C NMR (125 MHz,
DMSO-d ) δ 172.2, 169.6, 155.7, 153.9, 152.7, 151.5, 139.0, 130.9, 128.6, 121.2,
6
1
4
4
4
20.1, 119.8, 116.0, 113.8, 35.7, 34.9, 32.7, 31.6, 28.9, 25.6, 25.5. LRMS (ESI) m/z
+
-
70.1 [M+H] , HRMS (ESI) m/z for C H N O S [M-H] calcd 468.2075, found
2
4
31
5
3
68.2079.
.6.13.
1
8
N -(4-((3-ethynylphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxyoctanedi
amide 9m
Compound 9m was prepared from 8m according the same process described for
o
1
9
1
1
7
a in 50% yield. Mp 214.6-216.1 C; H NMR (400 MHz, DMSO-d ) δ 10.32 (s, 1H),
6
0.19 (s, 1H), 9.83 (s, 1H), 8.68 (s, 1H), 8.65 (d, J = 1.4 Hz, 1H), 8.35 (d, J = 0.5 Hz,
H), 8.03 (t, J = 1.8 Hz, 1H), 7.81 (ddd, J = 8.3, 2.2, 1.0 Hz, 1H), 7.37 (dd, J = 8.3,
.5 Hz, 1H), 7.20 (dt, J = 7.7, 1.3 Hz, 1H), 4.19 (s, 1H), 2.47 (t, J = 4.9 Hz, 2H), 1.92
1
3
(
t, J = 7.4 Hz, 2H), 1.63 – 1.53 (m, 2H), 1.52 – 1.42 (m, 2H), 1.35 – 1.20 (m, 4H). C
NMR (125 MHz, DMSO-d ) δ 172.2, 169.6, 155.4, 153.8, 152.8, 139.8, 130.9, 129. 5,
6
1
27.1, 125.0, 122.8, 122.3, 116.3, 114.2, 84.0, 81.1, 35.7, 32.7, 28.9, 25.6, 25.5.
+
-
LRMS (ESI) m/z 438.1 [M+H] , HRMS (ESI) m/z for C H N O S [M-H] calcd
2
2
23
5
3
4
36.1449, found 436.1447.
4
.6.14.
1
8
N -(4-((3-cyanophenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxyoctanedia
mide 9n

ACCEPTED MANUSCRIPT
Compound 9n was prepared from 8n according the same process described for
o
1
9
a in 48% yield. Mp 199.1-200.8 C; H NMR (400 MHz, DMSO-d ) δ 10.36 (s, 1H),
6
1
0.25 (s, 1H), 10.08 (s, 1H), 8.75 (s, 1H), 8.69 (s, 1H), 8.43 (d, J = 12.8 Hz, 2H), 8.10
(d, J = 7.8 Hz, 1H), 7.65 – 7.48 (m, 2H), 1.95 (t, J = 7.7 Hz, 2H), 1.60 (p, J = 7.5 Hz,
1
3
2
H), 1.50 (p, J = 7.4 Hz, 2H), 1.39 – 1.18 (m, 6H). C NMR (125 MHz, DMSO-d ) δ
6
1
72.2, 169.5, 155.2, 153.7, 153.0, 140.6, 131.0, 130.4, 127.1, 126.4, 124.6, 119.3,
-
1
16.6, 114.4, 111.8, 35.7, 32.7, 28.9, 25.6, 25.5. LRMS (ESI) m/z 437.1 [M-H] ,
-
HRMS (ESI) m/z for C H N O S [M-H] calcd 437.1401, found 437.1409.
2
1
22
6
3
4
.6.15.
1
8
N -hydroxy-N -(4-((3-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)octaned
iamide 9o
Compound 9o was prepared from 8o according the same process described for
o
1
9
1
7
1
a in 60% yield. Mp 188.6-189.7 C; H NMR (400 MHz, Methanol-d ) δ 10.33 (s,
4
H), 10.17 (s, 1H), 9.75 (s, 1H), 8.66 (s, 2H), 8.34 (s, 1H), 7.62 – 7.46 (m, 1H), 7.46 –
.37 (m, 1H), 7.28 (t, J = 8.1 Hz, 1H), 6.78 – 6.63 (m, 1H), 3.78 (s, 3H), 2.48 (t, 2H),
.94 (t, J = 7.4 Hz, 2H), 1.59 (p, J = 7.2 Hz, 2H), 1.49 (p, 2H), 1.38 – 1.21 (m, 4H).
1
3
C NMR (125 MHz, DMSO-d ) δ 172.2, 169.6, 155.7, 153.9, 152.7, 139.2, 138.2,
6
1
30.9, 128.8, 125.0, 123.4, 120.1, 116.0, 113.9, 35.7, 32.7, 28.9, 25.6, 25.5, 21.7.
+
-
LRMS (ESI) m/z 444.1 [M+H] , HRMS (ESI) m/z for C H N O S [M-H] calcd
2
1
25
5
4
4
42.1554, found 442.1551.
4
.6.16.
1
8
N -(4-((4-ethylphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)-N -hydroxyoctanediam

ACCEPTED MANUSCRIPT
ide 9p
Compound 9p was prepared from 8p according the same process described for
o
1
9
1
2
a in 71% yield. Mp 180.4-181.6 C; H NMR (400 MHz, DMSO-d ) δ 10.33 (s, 1H),
6
0.14 (s, 1H), 9.70 (s, 1H), 8.65 (s, 1H), 8.59 (s, 1H), 8.31 (s, 1H), 7.64 (d, J = 8.5 Hz,
H), 7.21 (d, J = 8.5 Hz, 2H), 2.61 (q, J = 7.6 Hz, 2H), 2.50 – 2.45 (m, 2H), 1.94 (t, J
=
7.4 Hz, 2H), 1.64 – 1.54 (m, 2H), 1.52 – 1.43 (m, 2H), 1.34 – 1.24 (m, 4H), 1.20 (t,
13
J = 7.6 Hz, 3H). C NMR (125 MHz, DMSO-d ) δ 172.1, 169.6, 155.7, 153.9, 152.7,
6
1
39.9, 136.8, 130.9, 128.2, 123.2, 115.9, 113.7, 35.7, 32.7, 28.9, 28.1, 25.6, 25.5, 16.2.
+
-
LRMS (ESI) m/z 442.1 [M+H] , HRMS (ESI) m/z for C H N O S [M-H] calcd
2
2
27
5
3
4
40.1762, found 440.1762.
4
.6.17.
1
8
N -hydroxy-N -(4-((4-isopropylphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)octane
diamide 9q
Compound 9q was prepared from 8q according the same process described for
o
1
9
1
2
2
a in 68% yield. Mp 154.0-156.3 C; H NMR (400 MHz, DMSO-d ) δ 10.33 (s, 1H),
6
0.13 (s, 1H), 9.70 (s, 1H), 8.65 (s, 1H), 8.59 (s, 1H), 8.31 (s, 1H), 7.69 – 7.61 (m,
H), 7.28 – 7.21 (m, 2H), 2.94 – 2.85 (m, 1H), 2.50 – 2.46 (m, 2H), 1.94 (t, J = 7.4 Hz,
H), 1.65 – 1.54 (m, 2H), 1.53 – 1.43 (m, 2H), 1.35 – 1.25 (m, 2H), 1.22 (d, J = 6.9
1
3
Hz, 6H). C NMR (125 MHz, DMSO-d ) δ 172.1, 169.5, 155.7, 153.9, 152.6, 144.5,
6
1
36.9, 130.9, 126.7, 115.9, 113.7, 35.7, 33.41, 32.7, 28.9, 25.6, 25.5, 24.5. LRMS
+
-
(
ESI) m/z 456.1 [M+H] , HRMS (ESI) m/z for C H N O S [M-H] calcd 454.1918,
23 29 5 3
found 454.1919.

ACCEPTED MANUSCRIPT
4
.6.18.
1
8
N -hydroxy-N -(4-((4-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-6-yl)octaned
iamide 9r
Compound 9r was prepared from 8r according the same process described for 9a
o
1
in 59% yield. Mp 222.2-223.9 C; H NMR (400 MHz, DMSO-d ) δ 10.35 (s, 1H),
6
1
2
1
0.12 (s, 1H), 9.65 (s, 1H), 8.75 (s, 1H), 8.55 (s, 1H), 8.28 (s, 1H), 7.69 – 7.46 (m,
H), 7.11 – 6.78 (m, 2H), 3.77 (s, 3H), 2.50 – 2.42 (m, 2H), 1.93 (t, J = 7.4 Hz, 2H),
13
.64 – 1.53 (m, 2H), 1.53 – 1.43 (m, 2H), 1.36 – 1.19 (m, 4H). C NMR (125 MHz,
DMSO) δ 172.1, 169.5, 156.7, 156.1, 154.0, 152.6, 131.8, 130.8, 125.5, 115.9, 114.2,
+
1
13.3, 55.7, 35.7, 32.7, 28.9, 25.6, 25.5. LRMS (ESI) m/z 444.1 [M+H] , HRMS (ESI)
-
m/z for C H N O S [M-H] calcd 442.1554, found 442.1539.
21
24
5
4
4
4
.7. Biological evaluation
.7.1. Materials
The antibodies for Histone H , α-tubulin and GAPDH were purchased from Cell
3
Signaling Technology. Acetyl-Histone H was purchased from Merck Millipore.
3
Acetyl-α-Tubulin was purchased from Sigma-aldrich. Both anti-Rabbit and anti-mouse
IgG (H+L), DyLight 800 labeled secondary antibodies were purchased from KPL
(Kirkegaard & Perry Laboratories. Inc), PVDF transfer membrane was purchased
from Amersham Biosciences corporation
.
4
.7.2. Cell culture and Cell Viability Assay.

ACCEPTED MANUSCRIPT
Human multiple myeloma (MM) cell line RPMI 8226 was gifted from
Professor Jian Hou (Chang Zheng Hospital, Shanghai, China). RPMI 8226 cells
were cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine
Serum (FBS). Human HCT 116 colon cancer cells were obtained from the Cell
Bank (Chinese Academy of Sciences, Shanghai, China). HCT 116 cells were
cultured in 5A medium supplemented with 10% Fetal Bovine Serum (FBS). Cells
were maintained in a humidified 37°C/5% CO incubator.
2
4
.7.3. In vitro HDAC activity assay
All three full-length recombinant human HDACs (rhHDACs)1, 3 and 6 were
expressed in insect High5 cells using a baculoviral expression system, and all
His -tagged and GST-fusion proteins was purified using Ni-NTA (QIAGEN). The
6
deacetylase activity of rhHDACs 1 and 3 were assayed with a HDAC substrate
(
Ac-Lys-Tyr-Lys(ε-acetyl)-AMC), and HDAC6 was assayed with another HDAC
substrate (Boc-Lys(ε-acetyl)-AMC). The total HDAC assay volume was 25 µL and all
the assay components were diluted in Hepes 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
2
3
84-well plates (OptiPlateTM-384F, PerkinElmer). In brief, the HDAC assay mixture
contained the substrate (5-50 µM, 5 µL), rhHDAC isoforms (20-200 nM) and
inhibitors (1 µL). Positive controls contained all the above components except the
inhibitors. The negative controls contained neither enzymes nor inhibitors. The
HDAC6 assay components were incubated at room temperature for 3 h, and HDAC1
or 3 were incubated for 24 h. The reaction was quenched with the addition of 25 µL.