Synthesis and anticancer activities of thieno[3,2-d]pyrimidines as novel HDAC inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.11.021

A series of thieno[3,2-d]pyrimidines bearing a hydroxamic acid moiety as novel HDAC inhibitors were designed and synthesized. The structures of the new synthesized compounds were confirmed using IR, ¹H, ¹³C NMR spectrum. Compounds 11-13 showed potent inhibitory activities against HDACs with IC₅₀ values at 0.38, 0.49 and 0.61 μM. Most of target compounds displayed strong anti-proliferative activity by a MTT assay on three human cancer cell lines including HCT-116, MCF-7 and HeLa. Compound 11, having potent inhibitory activities against HDACs, induced apoptosis and G2/M cell cycle arrest in HCT-116 cell line.

Full text of DOI:10.1016/j.bmc.2013.11.021

Bioorganic & Medicinal Chemistry 22 (2014) 358–365
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anticancer activities of thieno[3,2-d]pyrimidines
as novel HDAC inhibitors
⇑
Qiu Tan, Zhitao Zhang, Jie Hui, Yu Zhao, Li Zhu
Institute of Nautical Medicine, Nantong University, 19 Qixiu Road, Nantong 226001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thieno[3,2-d]pyrimidines bearing a hydroxamic acid moiety as novel HDAC inhibitors were
Received 4 September 2013
Revised 6 November 2013
Accepted 8 November 2013
Available online 21 November 2013
designed and synthesized. The structures of the new synthesized compounds were confirmed using IR,
1
13
H, C NMR spectrum. Compounds 11–13 showed potent inhibitory activities against HDACs with IC50
values at 0.38, 0.49 and 0.61 M. Most of target compounds displayed strong anti-proliferative activity
l
by a MTT assay on three human cancer cell lines including HCT-116, MCF-7 and HeLa. Compound 11, hav-
ing potent inhibitory activities against HDACs, induced apoptosis and G2/M cell cycle arrest in HCT-116
cell line.
Keywords:
Thieno[3,2-d]pyrimidines
HDAC
Ó 2013 Elsevier Ltd. All rights reserved.
Synthesize
Anti-proliferative
1
. Introduction
incorporating 5b into a hydroxamic acid moiety (Fig. 1). Compound
1
1, one of the synthesized compounds, showing potent HDAC
Protein function is regulated by the enzymatic addition and
inhibitory activity and antiproliferative activity, is a potential anti-
cancer drug candidate that should be further developed.
removal of acetyl groups at specific lysine residues. Lysine acetyla-
tion is mediated by two classes of enzymes with opposing
functions: histone acetyltransferase (HAT) and histone deacetylase
2
2
. Results and discussion
(
HDAC), which catalyze the addition and removal of acetyl groups,
1
–4
respectively.
HDACs are associated with many oncogenes and
.1. Synthesis
5,6
tumor suppressors, leading to altered expression patterns. HDAC
inhibitors have been shown to cause growth arrest, differentiation
and apoptosis in tumor cells and in animal models by inducing his-
The general synthetic routes used to prepare thieno[3,2-
d]pyrimidine/HDAC inhibitors are outlined in the Scheme 1.
Compounds 5a and 5b were prepared according to the procedure
waf1
7–10
tone hyperacetylation and p21
expression.
Several HDAC
2
9–32
inhibitors are currently under clinical trials on either monotherapy
reported previously.
ferent chain lengths of ethyl bromoalkanoate yielded ester products
a–6h. Hydrolysis of the ethyl group of compounds 6a–6h using
Alkylation of compound 5a–5b with dif-
or combination therapy for cancer treatment.1
1,12
The HDAC inhib-
itor vorinostat (SAHA) is a FDA-approved drug for the treatment of
6
cutaneous T-cell lymphoma (CTCL).13 Several small molecule
lithium hydroxide gave corresponding acids 7a–7h. Conversion of
carboxylic acid 7a–7h to hydroxamic acids by hydroxylamine
hydrochloride yielded targeted compounds 8–15 (Scheme 1).
HDAC inhibitors were used for clinical trials in a variety of haema-
tological and solid tumors.1
4–16
New scaffolds of small molecular
inhibitors against HDAC have been constantly reported.17–23
Another promising strategy of HDAC inhibition to overcome
limitations of targeting the PI3K pathway is to combine HDAC
2
.2. HDAC inhibition
2
4,25
and PI3K inhibitors to achieve synergistic antitumor activity.
We tested the HDAC inhibition activity of compounds 8–15
3
3
By regulating both histone and nonhistone substrates, HDAC inhib-
itors can affect a variety of cell functions and synergize with PI3K
using HDAC Fluorimetric Assay/Drug Discovery Kit. These com-
pounds showed inhibitory rate against HDACs from 18.7% to
2
6–28
inhibitors.
7
6.8% at a concentration of 1 lM (Table 1). Among these com-
On the basis of a widely accepted HDAC pharmacophore model,
we designed and synthesized a novel series of HDAC inhibitors by
pounds, compounds 8 and 9 showed the weakest HDAC inhibitory
activity, closely followed by compounds 14 and 15. Compounds
1
1
0–13 had potent HDAC inhibitory activity, especially compound
1. The HDAC inhibitory activities of compounds 11, 12 and 13
⇑
Corresponding author.
E-mail address: zhulili65@163.com (L. Zhu).
with IC50 were at 0.38, 0.49 and 0.61 lM, respectively (Table 1).
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.021

Q. Tan et al. / Bioorg. Med. Chem. 22 (2014) 358–365
359
Figure 1. Design of thieno[3,2-d]pyrimidine HDAC inhibitors.
Scheme 1. Reagents and conditions: (a) Br(CH
2
2 2
)nCO Et, K CO
3
, DMF, 44–65%; (b) LiOH, THF, 76–100%; (c) isobutyl chloroformate, Et
3
N, NH
2
OHꢀHCl, THF, 40–50% .
Table 1
In vitro HDAC inhibition and anti-proliferative activity
No.
Inhibition of HDACa (%)
IC50a
(l
M) HDAC
IC50c
(lM)
HCT-116
HeLa
MCF-7
8
9
18.8
18.7
61.7
76.8
73.8
72.0
25.4
40.6
87.8
—b
—
—
0.38
0.49
0.61
—
14
12
2.4
3
>20
9.9
5.2
4.8
10
11
18
>20
1.6
12.2
5.5
4
2.9
6.1
10.1
>20
2.8
1
1
1
1
1
1
0
1
2
3
4
5
4
7.4
11.9
19
20
2.7
—
SAHA
0.065d
a
b
c
HDAC inhibitory activity was measured by use of the HDAC Fluorimetric Assay/Drug Discovery Kit.
Not tested.
IC50 (lM) data for anti-proliferative activity in HCT-116, HeLa, and MCF-7 cell lines.
d
29
The data was obtained from the literature.
2
.3. Anti-proliferation
2.4. Structure–activity relationship
We evaluated the activity of compounds 8–15 in human colonic
From the IC50 values, it is clear that most of these derivatives
displayed potent anti-proliferative activities against three cancer
cell lines. However, these derivatives showed comparatively lesser
activity against MCF-7 cell line than HCT-116 and HeLa cell lines.
Compound 11 possessed the highest growth inhibitory effect on
HCT-116, HeLa and MCF-7 cancer cell lines with IC50 values of 4,
4 and 4.8 lM. Anti-proliferative activity of compounds bearing
four (8, 9) and seven (14, 15) methylene-linker decreased.
Compounds 9 and 11 subsitituted on meta-position were found
carcinoma cell line (HCT-116), human breast adenocarcinoma cell
line (MCF-7) and human cervical carcinoma cell line (HeLa) by a
MTT assay. Table 1 showed the IC50 values of each compound for
above three cell lines. Compounds 9–13 displayed strong anti-pro-
liferative activity. SAHA inhibited the proliferation of these tumor
cell lines with IC50 similar to the values.29 All compounds displayed
their anti-proliferative activities closely matched their HDAC
inhibitory activities except compound 15.

3
60
Q. Tan et al. / Bioorg. Med. Chem. 22 (2014) 358–365
to be more potent than compounds 8 and 10 subsitituted on
para-position. Apparently, the meta-position substituted on
benzene moiety was critical for their anti-proliferative activties.
Furthermore, compounds bearing five (10, 11) and six (12, 13)
methylene-linker exhibited better HDAC inhibitory properties than
four (8, 9) and seven (14, 15) methylene-linked compounds. Com-
pound 11, bearing five methylene-linker, was found more potent
than 10, which indicated meta-position substitute was favorable
to HDAC inhibition. Taken together, the results suggested that
the inhibition of HDAC played a major role in the anti-proliferative
activity of these compounds.
compounds exhibited potent HDACs inhibitory activity and anti-
proliferative activity in vitro. Compound 11 induced HCT-116 cell
cycle arrest at G2/M phase and apoptosis, suggesting it should be
further developed as anticancer drug candidate.
4. Experimental part
4.1. Chemical procedures
Solvents were purified in the usual way. TLC was performed on
precoated Merck silica Gel 60 F254 plates. Flash chromatography
1
was performed on silica gel (100–200 mesh, Qingdao, China).
H
1
3
2
.5. Cell cycle arrest and apoptosis
NMR and C NMR spectra were taken on a Bruker 400 MHz spec-
trometer with tetramethylsilane (TMS) as an internal standard, and
chemical shifts were recorded in ppm values. The high resolution
spectra were obtained on a Q-TOF Global Mass (ESIMS).
As compound 11 displayed the best anti-proliferative activity
and HDAC inhibitory activity, we examined the effects of com-
pound 11 on cell cycle by flow cytometry in HCT-116 cells
(Fig. 2). Our results demonstrated that the treatment of HCT-116
4.1.1. Thieno[3,2-d]pyrimidines-2,4(1H,3H)-dione (2)
cells with compound 11 at 0, 5 and 10 M for 24 h induced G2/
l
A mixture of 3-amino-2-thiophene carboxylic acid methyl ester
(20.8 g, 132.3 mmol) and urea (45.9 g, 661.7 mmol) was heated at
190 °C. After 2 h, 7.5% NaOH solution (100 mL) was poured into the
reaction mixture and a white solid precipitated. The solid filtered
and the filtrate washed with 2 M HCl. A large amount of white solid
was precipitated, filtered and the filter cake was dried to give the
white solid product 2 (23.8 g). Yield 98%.
M cell cycle arrest. A concentration-dependent increase of cell per-
centage in subG1 phase was observed from 0% to 44.5%, indicating
apoptosis induction. The flow cytometric data clearly indicated
that compound 11 induced cell cycle arrest at G2/M phase and
apoptosis in HCT-116 cell line.
3
. Conclusion
4
.1.2. 2,4-Dichlorothieno[3,2-d]pyrimidines (3)
In summary, a series of thieno[3,2-d]pyrimidines bearing a
Compound 2 (23.8 g, 140.7 mmol) was dissolved in POC1
3
hydroxamic acid moiety were synthesized effectively. These
(200 mL), the mixture was heated under reflux for 6 h. After
Figure 2. HCT116 cells treated with various concentrations of compound 11 for 24 h. (A) Control, (B) 2.5 lM, (C) 5 lM, (D) 10 lM. Cells were stained by propidium iodide,
and cell cycle phases were analyzed by flow cytometry.

Q. Tan et al. / Bioorg. Med. Chem. 22 (2014) 358–365
361
removal of most POC1
3
under pressure, the remaining reaction
4.1.7. 1-Hydroxyamino-6-[4-(4-morpholin-4-yl-thieno[3,2-
solution was slowly poured into ice/water while vigorously stirred
yielding a gray precipitate. This was collected by filtration and
air-dried to yield a gray solid 3 (16.9 g). Yield 58.3%.
d]pyrimidines-2-yl)phenoxy]hexanoate (12)
A solution of 7a (0.4 g, 0.9 mmol), isobutyl chloroformate (0.1 g,
1 mmol), Et N (0.2 g, 1.8 mmol) and DMF (10 mL) was added at
°C. After 10 min, the reaction was added hydroxylamine hydro-
3
0
4
.1.3. 2-Chloro-4-(morpholin-4-yl)-thieno[3,2-d]pyrimidines (4)
To a stirred solution of compound 3 (16.9 g, 83.2 mmol) in dry
chloride (0.1 g, 1.8 mmol) solution in methanol (4 mL), allowed
to warm naturally to room temperature and stirred. After 2 h, the
reaction was diluted with ethyl acetate, washed with water, dried
over anhydrous sodium sulfate, filtered, and concentrated under
reduced pressure. The product was purified by column chromatog-
methanol (300 mL) and morphine (15.9 g, 183.1 mmol) was added.
After the addition, the reaction mixture was stirred at room
temperature for 1 h. Separated gray solid was filtered and washed
with water and methanol. The gray solid was dried and filtered.
The crude product was found to be pure and taken to the next step
without purification, yield a solid 4 (15.6 g). Yield 73.5%.
f
raphy to give a white solid. Yield 50.5%. R = 0.3 (acetone/petro-
1
leumther = 1:1). Mp: 176–178 °C. H NMR (400 Hz, DMSO): d
10.37 (s, 1H, –OH), 8.69 (s, 1H, –NH), 8.35 (d, J = 8.8 Hz, 2H, ArH),
8
.23 (d, J = 5.6 Hz, 1H, 6-H), 7.49 (d, J = 5.6 Hz, 1H, 7-H), 7.02 (d,
0
0
4
.1.4. 2-Phenol-4-(morpholin-4-yl)-thieno[3,2-d]pyrimidines
5a)
A mixture of 4 (1.5 g, 5.9 mmol), 4-hydroxyphenyl boronic acid
1.4 g, 9.9 mmol), K CO (1.2 g, 8.8 mmol), Pd (PPh (0.2 g,
.2 mmol) and DMF (20 mL) was heated at 85 °C under nitrogen.
After 6 h, the reaction was was poured to H O (30 mL) and
J = 8.8 Hz, 2H, ArH), 4.03 (m, 6H, –CH
2
–, 2 -CH
), 2.00 (t, J = 7.2 Hz, 2H, –CH
2
, 6 -CH
2
), 3.81 (t,
–), 1.77
–). 13
): d 169.50, 162.98, 160.86, 159.47, 158.26, 134.18,
130.77, 129.76, 125.22, 114.58, 112.01, 67.91, 66.47. HRMS (ESI)
0
0
(
J = 4.8 Hz, 4H, 3 -CH
(m, 2H, –CH –), 1.60 (m, 2H, –CH
NMR (CDCl
2
, 5 -CH
2
2
2
2
–), 1.44 (m, 2H, –CH
2
C
(
2
3
3
)
4
3
0
+
2
calcd for C22
H
26
N
4
O
4
S (M+H) 443.1675, found 443.1755. IR (KBr)
ꢁ1
extracted with ethyl acetate. The combined organic was washed
with brine, dried over anhydrous sodium sulfate, filtered and evap-
orated under vacuum. The residue was recrystallized from ethanol
to give a yellowed solid 5a. Yield 80%.
m (cm ) = 3436, 2241, 2937, 2861, 1657, 1584, 1533, 1400, 1245,
650.
4.1.8. 3-Phenol-4-(morpholin-4-yl)-thieno[3,2-d]pyrimidines
(
5b)
Compound 5b was prepared from 4 according to the same pro-
4
2
.1.5. Ethyl-6-[4-(4-morpholin-4-yl-thieno[3,2-d]pyrimidines-
-yl)phenoxy]hexanoate (6a)
cedure described for 5a using 3-hydroxyphenyl boronic acid. Yield
73%. H NMR (400 Hz, DMSO): d 9.51 (s, 1H, 6-H), 8.25 (d,
1
A mixture of 5a (1.5 g, 4.8 mmol), K
-bromohexanoate (1.6 g, 7.2 mmol) and DMF (10 mL) was heated
O (30 mL)
2
CO
3
(1.6 g, 11.5 mmol) and
6
J = 5.6 Hz, 1H, ArH), 7.87 (dd, J = 1.6 Hz, 6.8 Hz, 2H, 7-H, ArH),
7.51 (d, J = 5.6 Hz, 1H, ArH), 7.29 (t, J = 7.6 Hz, 1H, ArH), 6.87 (m,
at 60 °C. After 12 h, the reaction was was poured to H
2
0
0
and extracted with ethyl acetate. The combined organic was
washed with brine, dried over anhydrous sodium sulfate, filtered
and evaporated under vacuum. The residues were purified by flash
column chromatography (petroleum/EtOAc = 3:1) to give com-
2 2
1H, –OH), 4.01 (t, J = 4.8 Hz, 4H, 2 -CH , 6 -CH ), 3.82 (t, J = 4.8 Hz,
0
0
+
2 2
4H, 3 -CH , 5 -CH . ESI-MS: m/z, 314.0 [M+H] .
4.1.9. Ethyl-6-[3-(4-morpholin-4-yl-thieno[3,2-d]pyrimidines-
2-yl)phenoxy]hexanoate (6b)
1
pound 6a (1.4 g) as yellow Oil. Yield 65%. Mp: 86–88 °C. H NMR
(
1
4
400 Hz, DMSO): d 8.35 (d, J = 8.8 Hz, 2H, ArH), 8.23 (d, J = 5.2 Hz,
H, 6-H), 7.49 (d, J = 5.6 Hz, 1H, 7-H), 7.02 (d, J = 8.8 Hz, 2H, ArH),
Compound 6b was prepared from 5b according to the same
procedure described for 6a to give a white solid. Yield 64.2%. Mp:
80–83 °C. H NMR (400 Hz, DMSO): d 8.26 (d, J = 5.6 Hz, 1H, ArH),
1
.08 (m, 2H, –CH
2
–), 4.04 (d, J = 14.1 Hz, 2H, –CH
2
–), 4.00 (t,
0
0
0
0
J = 4.8 Hz, 4H, 2 -CH
2
, 6 -CH
2
), 3.81 (t, J = 5.2 Hz, 4H, 3 -CH
–), 1.78 (m, 2H, –CH –), 1.63 (m,
–), 1.19 (t, J = 7.2 Hz, 3H, –CH
NMR (DMSO): d 173.30, 162.99, 160.84, 159.47, 158.25, 148.44,
2
, 5 -
8.00 (d, J = 8.0 Hz, 1H, 6-H), 7.93 (m, 1H, ArH), 7.54 (d, J = 5.6 Hz,
CH
2
2
), 2.33 (t, J = 7.2 Hz, 2H, –CH
–), 1.47 (m, 2H, –CH
2
2
1H, ArH), 7.41 (t, J = 8.0 Hz, 7-H), 7.05 (dd, J = 1.6 Hz, 7.2 Hz, 1H,
1
3
0
0
0
H, –CH
2
2
3
).
C
ArH), 4.08 (m, 8H, 2 -CH
2
, 6 -CH
2
), 3.82 (t, J = 5.2 Hz, 4H, 3 -CH
–), 1.79 (m, 2H, –CH –), 1.65
–), 1.19 (t, J = 7.2 Hz,3H, –CH ).
C NMR (DMSO): d 173.32, 162.86, 159.33, 159.24, 158.28,
2
,
0
5 -CH
2
), 2.34 (t, J = 7.6 Hz, 2H, –CH
–), 1.49 (m, 2H, –CH
2
2
1
6
1
4
1
34.14, 130.79, 129.75, 125.23, 114.55, 114.32, 112.01, 67.84,
(m, 2H, –CH
2
2
3
1
3
7.09, 66.47, 60.14, 40.40, 40.20, 33.92, 28.83, 25.53, 24.71,
+
4.60. HRMS (ESI) calcd for C24
29
H N
3
O
4
S (M+H) 456.1952, found
139.95, 134.38, 129.88, 125.31, 120.57, 116.53, 113.95, 112.78,
ꢁ1
56.1931. IR (KBr)
m
(cm ) = 3447, 2935, 2862, 1731, 1605,
67.77, 66.46, 60.13, 46.33, 40.61, 40.40, 33.94, 28.89, 25.58,
+
585, 1306, 1246, 1114, 758.
24.74, 16.60. HRMS (ESI) calcd for C24
found 456.1934. IR (KBr)
H
29
3
N O
4
S (M+H) 456.1952,
ꢁ1
m (cm ) = 3433, 2970, 2870, 1726,
4
.1.6. 6-[4-(4-Morpholin-4-yl-thieno[3,2-d]pyrimidines-2-
1595, 1471, 1311, 1233, 1116, 794.
yl)phenoxy]hexanoa-te (7a)
To a solution of 6a (0.5 g, 1.1 mmol) in THF (20 mL), LiOHꢀH
2
O
4.1.10. 6-[3-(4-Morpholin-4-yl-thieno[3,2-d]pyrimidines-2-
yl)phenoxy]hexanoate (7b)
(
0.3 g, 6.6 mmol) was added with stirring at 55 °C. After 3 h, the
mixture was cooled to 0 °C and acidified with 2 M HCl. The solid
was filtered, washed with H O, and dried to give the product,
which was taken to next step without purification (0.4 g). Yield
Compound 7b was prepared from 6b according to the same
procedure described for 7a to give a white solid. Yield 76%. Mp:
169–171 °C. H NMR (400 Hz, DMSO): d 8.50 (d, J = 5.6 Hz, 1H, 6-
2
1
1
8
1.7%. Mp: 148–151 °C. H NMR (400 Hz, DMSO): d 8.48 (d,
H), 8.02 (d, J = 7.2 Hz, 2H, ArH), 7.83 (d, J = 5.2 Hz, 1H, 7-H), 7.51
J = 5.6 Hz, 1H, 6-H), 8.43 (d, J = 9.2 Hz, 2H, ArH), 7.77(d, J = 5.2 Hz,
(t, J = 8.0 Hz, 1H, ArH), 7.21 (d, J = 9.2 Hz, 1H, ArH), 4.16 (t,
0
0
0
1
H, 7-H), 7.13 (d, J = 8.8 Hz, 2H, ArH), 4.15 (d, J = 4.4 Hz, 4H, 2 -
J = 4.4 Hz, 4H, 2 -CH
(t, J = 4.8 Hz, 5H, 3 -CH
2
0
, 6 -CH
2
0
-), 4.12 (t, J = 6.4 Hz, 2H, –CH
2
–), 3.87
0
CH
2
, 6 -CH
2
), 4.10 (t, J = 6.4 Hz, 2H, –CH
), 2.27 (t, J = 7.2 Hz, 2H, –CH
–), 1.49 (m, 2H, –CH –). C NMR (DMSO): d
74.92, 161.00, 158.83, 158.18, 148.82, 132.44, 129.84, 128.05,
23.90, 114.62, 112.41, 112.05, 109.80, 67.93, 67.17, 66.45, 40.60,
0.40, 34.10, 28.90, 25.63, 24.76. HRMS (ESI) calcd for C22
2
–), 3.86 (t, J = 4.8 Hz, 4H,
2
, 5 -CH2, –OH), 2.27 (t, J = 7.2 Hz, 2H, –
0
0
3
-CH
2
, 5 -CH
2
2
–), 1.80 (m, 2H, –CH
2
–
CH
CH
2
–), 1.80 (m, 2H, –CH
2
–), 1.63 (m, 2H, –CH
2
–), 1.50 (m, 2H, –
13
13
)
, 1.62 (m, 2H, –CH
2
2
2
–). C NMR (DMSO): d 174.89, 159.42, 157.08, 155.32, 152.81,
1
1
4
138.59, 130.56, 121.50, 120.29, 119.44, 114.62, 113.34, 105.2,
68.25, 66.23, 47.22, 40.60, 40.39, 34.13, 28.89, 25.66, 24.77. HRMS
+
25
H N
3
O
4
S
(ESI) calcd for C22
H
25
N
3
O
4
S (M+H) 428.1639, found 428.1632. IR
+
ꢁ1
ꢁ1
(
M+H) 428.1639, found 428.1628. IR (KBr)
m
(cm ) = 3446, 2939,
(KBr)
m
(cm ) = 3447, 2952, 2873, 1727, 1571, 1542, 1499, 1453,
2
360, 2341, 1607, 1585, 1306, 1247, 1172, 935.
1267, 1045.

3
62
Q. Tan et al. / Bioorg. Med. Chem. 22 (2014) 358–365
4
.1.11. 1-Hydroxyamino-6-[3-(4-morpholin-4-yl-thieno[3,2-
H
28
N
4
1
O
4
S
(M+H)+ 457.1831, found 457.1911. IR (KBr)
m
ꢁ
d]pyrimidines-2-yl)phenoxy]hexanoate (13)
(cm ) = 3424, 2926, 2853, 1652, 1608, 1585, 1302, 1245, 1112,
654.
Compound 13 was prepared from 7b according to the same pro-
cedure described for 12 to give a white solid. Yield 41%. R
f
= 0.3
NMR
1
(
(
acetone/petroleumether = 1:1). Mp: 168–170 °C.
400 Hz, DMSO): d 8.38 (d, J = 8.8 Hz, 2H, ArH), 8.27 (d, J = 5.2 Hz,
H
4.1.15. Ethyl-7-[3-(4-morpholin-4-yl-thieno[3,2-d]pyrimidines-
2-yl)phenoxy]hexanoate (6d)
1
H, 6-H), 8.11(s, 1H, –NH), 8.01 (d, J = 5.6 Hz, 1H, 7-H), 7.02 (d,
Compound 6d was prepared from 5b according to the same pro-
cedure described for 6a using 7-bromoheptanoate ethyl to give a
white solid. Yield 48%. Mp: 88–90 °C. H NMR (400 Hz, DMSO): d
8.26 (d, J = 5.6 Hz, 1H, 6-H), 8.00 (d, J = 7.6 Hz, 1H, ArH), 7.93 (m,
1H, ArH), 7.54 (d, J = 5.6 Hz, 1H, 7-H), 7.41 (t, J = 8.0 Hz, 1H, ArH),
7.05 (dd, J = 2.8, 8.4 Hz, 1H, ArH), 4.07 (dd, J = 7.2, 14.4 Hz, 4H,
0
0
J = 8.8 Hz, 2H, ArH), 4.03 (m, 6H, –CH
2
–, 2 -CH
), 2.00 (t, J = 7.2 Hz, 2H, –CH
2
, 6 -CH
2
), 3.81 (t,
–) 1.77
0
0
1
J = 4.8 Hz, 4H, 3 -CH
m, 2H, –CH –), 1.60 (m, 2H, –CH
NMR (CDCl ): d 162.85, 159.31, 159.24, 158.26, 139.93, 134.43,
29.90, 125.30, 120.56, 116.51, 113.89, 112.76, 67.81, 66.45,
3.51, 40.46, 40.35, 32.66, 28.96, 25.68, 25.43. HRMS (ESI) calcd
S (M+H)+ 443.1675, found 443.1759. IR (KBr)
2
, 5 -CH
2
2
1
3
(
2
2
–), 1.44 (m, 2H, –CH
2
–).
C
3
1
6
0
0
–CH
J = 4.8 Hz, 4H, 3 -CH
(m, 2H, –CH –), 1.59 (m, 2H, –CH
(m, 2H, –CH –), 1.18 (t, J = 7.2 Hz, 3H, –CH
73.35, 162.86, 159.34, 159.26, 158.28, 139.95, 134.39, 129.87,
2
, –CH
2
), 4.01 (t, J = 4.0 Hz, 4H, 2 -CH
2
, 6 -CH
), 2.31 (t, J = 7.2 Hz, 2H, –CH
–), 1.48 (m, 2H, –CH
2
), 3.82 (t,
–), 1.78
–), 1.38
3
). C NMR (DMSO): d
0
0
for C22
H
26
N
4
O
4
m
2
, 5 -CH
2
2
ꢁ1
(
cm ) = 3405, 2931, 2860, 1609, 1540, 1489, 1409, 1275, 1232,
2
2
2
1
3
6
56.
2
1
4
2
.1.12. Ethyl-7-[4-(4-morpholin-4-yl-thieno[3,2-d]pyrimidines-
-yl)phenoxy]hexanoate (6c)
Compound 6c was prepared from 5a according to the same pro-
125.30, 120.56, 116.55, 113.93, 112.78, 67.84, 66.46, 60.11, 46.33,
40.61, 40.40, 33.91, 29.03, 28.68, 25.71, 24.88, 14.60. HRMS (ESI)
+
calcd for C25
H
31
N
3
O
4
S (M+H) 470.2108, found 470.2091. IR (KBr)
ꢁ
1
cedure described for 6a using 7-bromoheptanoate ethyl to give a
yellow solid. Yield 44.5%. Mp: 90–91 °C. H NMR (400 Hz, DMSO):
m (cm ) = 3446, 3090, 2936, 2589, 1731, 1601, 1538, 1352, 1229,
1
1116, 656.
d 8.35 (d, J = 9.2 Hz, 2H, ArH), 8.23 (d, J = 5.2 Hz, 1H, 6-H), 7.49 (d,
J = 5.6 Hz, 1H, 7-H), 7.02 (d, J = 9.2 Hz, 2H, ArH), 4.07 (dd, J = 7.2,
4.1.16. 7-[3-(4-Morpholin-4-yl-thieno[3,2-d]pyrimidines-2-
yl)phenoxy]hexanoate (7d)
0
0
1
3
–
–
4.4 Hz, 4H, –CH
.81 (t, J = 4.8 Hz, 4H, 3 -CH
2
–, –CH
2
–), 4.0 (t, J = 4.4 Hz, 4H, 2 -CH
2
, 6 -CH
), 2.31 (t, J = 7.2 Hz, 2H,
–), 1.59 (m, 2H, –CH –), 1.46 (m, 2H,
–), 1.19 (t, J = 7.2 Hz, 3H, –CH
2
),
0
0
2
, 5 -CH
2
Compound 7d was prepared from 6d according to the same
procedure described for 7a to give a white solid. Yield 98%. Mp:
CH
CH
2
–), 1.76 (m, 2H, –CH
–), 1.37 (m, 2H, –CH
2
2
1
3
1
2
2
3
).
C
130–132 °C. H NMR (400 Hz, DMSO): d 8.27 (d, J = 5.2 Hz, 1H,
NMR (DMSO): d 173.34, 162.99, 160.86, 159.27, 158.25, 134.15,
6-H), 8.01 (d, J = 7.6 Hz, 1H, ArH), 7.94 (t, J = 2.0 Hz, 1H, ArH),
1
30.17, 129.75, 125.22, 114.56, 112.01, 112.00, 102,21, 67.91,
7.55 (d, J = 5.6 Hz, 1H, 7-H), 7.41 (t, J = 8.0 Hz, 1H, ArH), 7.06 (dd,
6
2
6.47, 60.12, 46.32, 40.61, 40.40, 33.90, 28.98, 28.66, 25.67,
2
J = 2.4, 8 Hz, 1H, ArH), 4.06 (t, J = 6.4 Hz, 2H, –CH –), 4.02 (t,
+
0
0
0
0
4.87, 14.60. HRMS (ESI) calcd for C25
H
31
N
3
O
4
S (M+H) 470.2108,
J = 4.8 Hz, 4H, 2 -CH
CH ), 2.24 (t, J = 7.2 Hz, 2H, –CH
2H, –CH –), 1.49 (m, 2H, –CH –), 1.39 (m, 2H, –CH
DMSO): 174.99, 162.86, 159.34, 159.26, 158.28, 139.94,
2
, 6 -CH
2
), 3.83 (t, J = 5.2 Hz, 4H, 3 -CH
2
, 5 -
ꢁ1
found 470.2089. IR (KBr)
m
(cm ) = 2946, 2863, 1731, 1608,
2
2
–), 1.79 (m, 2H, –CH –), 1.58 (m,
2
13
1
537, 1488, 1302, 1278, 1115, 656.
2
2
2
–). C NMR
(
d
4
.1.13. 7-[4-(4-Morpholin-4-yl-thieno[3,2-d]pyrimidines-2-
134.38, 129.88, 125.31, 120.55, 116.55, 113.93, 112.77, 67.87,
yl)phenoxy]hexanoate (7c)
Compound 7c was prepared from 6c according to the same
procedure described for 7a to give a white solid. Yield 98%. Mp:
66.45, 46.33, 40.60, 40.39, 34.13, 29.08, 28.82, 25.78, 24.95. HRMS
+
(ESI) calcd for C23
(KBr) m (cm ) = 3474, 3093, 2936, 2863, 1671, 1604, 1538, 1352,
H
27
N
3
O
4
S (M+H) 442.1795, found 442.1779. IR
ꢁ1
1
1
6
7
4
5
28–130 °C. H NMR (400 Hz, DMSO): d 8.52 (d, J = 5.6 Hz, 1H,
1228, 1117, 656.
-H), 8.47 (d, J = 9.2 Hz, 2H, ArH), 7.84 (d, J = 5.6 Hz, 1H, 7-H),
.15 (d, J = 8.8 Hz, 2H, ArH), 4.18 (t, J = 4.0 Hz, 4H, 2 -CH
.11 (t, J = 6.8 Hz, 2H, –CH
0
0
2
, 6 -CH
2
),
4.1.17. 1-Hydroxyamino-7-[3-(4-morpholin-4-yl-thieno[3,2-
d]pyrimidines-2-yl)phenoxy]hexanoate (15)
0
2
–), 3.87 (t, J = 5.2 Hz, 4H, 3 -CH
2
,
0
-CH
2
), 2.24 (t, J = 7.2 Hz, 2H, –CH
–), 1.48 (m, 2H, –CH
2
–), 1.79 (m, 2H, –CH
–), 1.38 (m, 2H, –CH
2
–), 1.57
–).
Compound 15 was prepared from 7d according to the same pro-
1
3
(
m, 2H, –CH
2
2
2
C
cedure described for 12 to give a white solid. Yield 40.3%. R
f
= 0.3
NMR
1
NMR (DMSO): d 174.93, 166.65, 158.10, 157.30, 149.98, 137.34,
(acetone/petroleumether = 1:1). Mp: 180–185 °C.
H
1
6
35.73, 130.88, 128.18, 115.03, 113.11, 112.48, 102.20, 68.26,
(400 Hz, DMSO): d 8.38 (d, J = 8.8 Hz, 2H, ArH), 8.27 (d, J = 5.2 Hz,
1H, 6-H), 8.01 (s, 1H, –NH), 8.01 (d, J = 5.6 Hz, 1H, 7-H), 7.02 (d,
6.29, 46.93, 40.57, 40.36, 34.09, 28.90, 28.75, 25.68, 24.90. HRMS
+
0
0
(
ESI) calcd for C23
27
H N
3
O
4
S (M+H) 442.1795, found 442.1783. IR
J = 8.8 Hz, 2H, ArH), 4.03 (m, 6H, –CH
2
–, 2 -CH
2
, 6 -CH
2
), 3.81 (t,
–), 1.77
–), 1.35
): d 171.57, 162.00, 160.23,
ꢁ1
0
0
(
KBr)
m
(cm ) = 3424, 2926, 2853, 1652, 1608, 1585, 1302, 1245,
J = 4.8 Hz, 4H, 3 -CH
(m, 2H, –CH –), 1.60 (m, 2H, –CH
m, 2H, –CH
2
, 5 -CH
2
), 2.00 (t, J = 7.2 Hz, 2H, –CH
2
1
112, 654.
2
–), 1.44 (m, 2H, –CH
2 2
1
3
(
2
–).
C NMR (CDCl
3
4
.1.14. 1-Hydroxyamino-7-[4-(4-morpholin-4-yl-thieno[3,2-
159.29, 158.14, 139.45, 132.79, 130.95, 128.98, 124.01, 116.16,
113.69, 112.79, 77.51, 67.59, 66.43, 47.91, 47.70, 32.38, 29.38,
d]pyrimidines-2-yl)phenoxy]hexanoate (14)
28 4 4
28.91, 25.51, 25.34. HRMS (ESI) calcd for C23H N O
S (M+H)+
Compound 14 was prepared from7c according to the same pro-
ꢁ
1
cedure described for 12 to give a white solid. Yield 42%. R
f
= 0.3
NMR
457.1831, found 457.1910. IR (KBr) m (cm ) = 3424, 2930, 2853,
1
(
(
acetone/petroleumether = 1:1). Mp: 179–183 °C.
400 Hz, DMSO): d 10.40 (s, 1H, –OH), 8.68 (s, 1H, –NH), 8.35 (d,
H
2360, 1633, 1540, 1351, 1278, 1115, 656.
J = 8.8 Hz, 2H, ArH), 8.23 (d, J = 5.6 Hz, 1H, 6-H), 7.49 (d,
J = 5.6 Hz, 1H, 7-H), 7.02 (d, J = 8.8 Hz, 2H, ArH), 4.03 (m, 6H, –
4.1.18. Ethyl-4-[4-(4-morpholin-4-yl-thieno[3,2-d]pyrimidines-
2-yl)phenoxy]hexanoate (6e)
0
0
0
0
CH
t, J = 7.2 Hz, 2H, –CH
CH –), 1.45 (m, 2H, –CH
d 169.58, 169.47, 162.98, 160.87, 159.48, 158.25, 134.21, 130.45,
2
–, 2 -CH
2
, 6 -CH
2
), 3.81 (t, J = 4.8 Hz, 4H, 3 -CH
–), 1.76 (m, 2H, –CH –), 1.55 (m, 2H, –
–), 1.35 (m, 2H, –CH –). C NMR (CDCl
2
, 5 -CH
2
), 1.98
Compound 6e was prepared from 5a according to the same
procedure described for 6a using 4-bromobutyrate to give a white
(
2
2
13
1
2
2
2
3
):
solid. Yield 85%. Mp: 87–88 °C. H NMR (400 Hz, DMSO): d 8.36 (d,
J = 8.8 Hz, 2H, ArH), 8.23 (d, J = 5.6 Hz, 1H, 6-H), 7.50 (d, J = 5.6 Hz,
1
4
29.77, 128.1, 125.22, 114.69, 112.01, 67.96, 66.47, 49.05, 46.31,
5.80, 32.67, 29.03, 28.81, 25.72, 25.54. HRMS (ESI) calcd for C23-
1H, 7-H), 7.03 (d, J = 8.8 Hz, 2H, ArH), 4.11 (m, 4H, –CH
2
, –CH
2
),
0
0
4.00 (t, J = 4.8 Hz, 4H, 2 -CH , 6 -CH ), 3.82 (t, J = 5.2 Hz, 4H,
2 2

Q. Tan et al. / Bioorg. Med. Chem. 22 (2014) 358–365
363
0
0
3
1
1
1
3
4
2
-CH
2
, 5 -CH
2
), 2.50 (t, J = 8.8 Hz, 2H, –CH
2
–), 2.04 (m, 2H, –CH
2
–),
131.24, 128.28, 118.27, 115.22, 114.95, 112.37, 102.34, 67.38,
13
.21 (t, J = 7.2 Hz, 3H, ––CH
60.64, 159.44, 158.26, 145.88, 134.16, 130.95, 129.77, 125.23,
14.58, 114.27, 112.04, 67.05, 66.47, 64.28, 60.36, 40.61, 40.41,
25 3 3
0.61, 24.7, 14.58. HRMS (ESI) calcd for C22H N O S (M+H)
28.1639, found 428.1634. IR (KBr)
906, 2850, 1735, 1608, 1364, 1248, 1114, 762.
3
). C NMR (DMSO): d 173.01, 162.98,
67.36, 66.34, 40.59, 40.38, 30.54, 24.62. HRMS (ESI) calcd for
+
C
20
H
21
N
3
O
4
S (M+H) 400.1339, found 400.1321. IR (KBr)
m
ꢁ
1
(cm ) = 3454, 2922, 2867, 1731, 1608, 1590, 1307, 1262, 1182,
778.
+
ꢁ1
m (cm ) = 3097, 3076, 2957,
4.1.23. 1-Hydroxyamino-4-[3-(4-morpholin-4-yl-thieno[3,2-
d]pyrimidines-2-yl)phenoxy]hexanoate (9)
4
.1.19. 4-[4-(4-Morpholin-4-yl-thieno[3,2-d]pyrimidines-2-
Compound 9 was prepared from 7f according to the same pro-
yl)phenoxy]hexanoate (7e)
cedure described for 12 to give a white solid. Yield 89.6%. R
f
= 0.3
NMR
1
Compound 7e was prepared from 6e according to the same
(acetone/petroleumether = 1:1). Mp: 175–177 °C.
H
procedure described for 7a to give a white solid. Yield 94%. Mp:
(400 Hz, DMSO): d 10.35 (s, 1H, –OH), 8.55 (d, J = 5.6 Hz, 1H, 6-
H), 8.47 (d, J = 8.4 Hz, 2H, ArH), 7.89 (d, J = 5.6 Hz, 1H, 7-H), 7.13
1
1
83–185 °C. H NMR (400 Hz, DMSO): d 8.53 (d, J = 5.6 Hz, 1H,
0
0
ArH), 8.45 (d, J = 8.8 Hz, 2H, 6-H), 7.82 (d, J = 5.6 Hz, 1H, 7-H),
(d, J = 8.8 Hz, 2H, ArH), 4.18 (s, 4H, 2 -CH
2
0
, 6 -CH
2
), 4.10 (t,
0
0
0
7
4
CH
(
1
6
H
.16 (d, J = 8.8 Hz, 2H, ArH), 4.19 (t, J = 4.4 Hz, 4H, 2 -CH
2
0
, 6 -CH
2
0
),
J = 6.0 Hz, 2H, –CH
2
–), 3.86 (s, 4H, 3 -CH
2
, 5 -CH
2
), 3.08 (m,1H,
.14 (t, J = 6.4 Hz, 2H, –CH
), 2.44 (t, J = 7.2 Hz, 2H, –CH
DMSO): 174.50, 162.77, 156.98, 156.69, 138.50, 136.66,
31.23, 128.29, 118.29, 115.23, 113.57, 112.66, 102.39, 67.58,
7.37, 66.24, 40.60, 40.39, 30.50, 24.56. HRMS (ESI) calcd for C20-
2
), 3.87 (t, J = 4.8 Hz, 4H, 3 -CH
2
, 5 -
–NH–), 2.19 (t, J = 7.2 Hz, 2H, –CH
2
–), 2.00 (t, J = 6.8 Hz, 2H,
3
C NMR (CDCl ): d 174.74, 169.31, 158.93, 157.34,
1
3
13
2
2
–), 2.03 (m, 2H, –CH
2
–). C NMR
2
–CH –).
d
147.86, 136.18, 130.99, 129.22, 115.18, 114.02, 106.15, 99.99,
69.37, 67.58, 66.29, 40.56, 40.35, 29.07, 25.72. HRMS (ESI) calcd
+
for C20
H
22
N
4
O
4
S (M+H) 414.1459, found 414.1362. IR (KBr)
m
N
O
3 4
1
S
(M+H)+ 400.1339, found 400.1325. IR (KBr)
m
(cm ) = 3416, 3200, 2924, 2873, 1726, 1677, 1592, 1264, 1196,
777.
ꢁ1
21
ꢁ
(
cm ) = 3460, 3299, 2909, 2867, 1731, 1590, 1306, 1263, 1115,
7
59.
4
.1.24. Ethyl-5-[4-(4-morpholin-4-yl-thieno[3,2-d]pyrimidines-
4
.1.20. 1-Hydroxyamino-4-[4-(4-morpholin-4-yl-thieno[3,2-
2-yl)phenoxy]hexanoate (6g)
d]pyrimidines-2-yl)phenoxy]hexanoate (8)
Compound 8 was prepared from 7e according to the same pro-
cedure described for 12 to give a white solid. Yield 66%. R
Compound 6g was prepared from 5b according to the same pro-
cedure described for 6a using 5-bromo-pentanoate ethyl to give a
white solid. Yield 96%. Mp 96–98 °C. H NMR (400 Hz, DMSO):
1
f
= 0.3
NMR
1
(
(
acetone/petroleumether = 1:1). Mp: 165–168 °C.
400 Hz, DMSO): d 8.53 (d, J = 5.6 Hz, 1H, 6-H), 8.45 (d, J = 8.8 Hz,
H
d 8.36 (d, J = 8.8 Hz, 2H, ArH), 8.22 (d, J = 5.6 Hz, 1H, 6-H), 7.50
(d, J = 5.6 Hz, 1H, 7-H), 7.02 (d, J = 8.8 Hz, 2H, ArH), 4.09 (m, 6H,
0
0
2
4
4
H, ArH), 7.83 (d, J = 5.6 Hz, 1H, 7-H), 7.15 (d, J = 8.8 Hz, 2H, ArH),
–CH
2
, –CH
2
, –CH
2
), 4.00 (t, J = 4.8 Hz, 4H, 2 -CH
2
, 6 -CH
2
), 3.82 (t,
–), 2.40
). C NMR (DMSO): d
0
0
0
0
.18 (s, 4H, 2 -CH
2
, 6 -CH
2
), 4.12 (t, J = 6.4 Hz, 2H, –CH
2
–), 3.86 (s,
J = 4.8 Hz, 4H, 3 -CH
(t, J = 7.2 Hz, 2H, –CH
2
, 5 -CH
2
), 2.51(d, J = 1.6 Hz, 2H, –CH
2
0
0
13
2
H, 3 -CH , 5 -CH
2
), 3.62 (s, 1H, –NH), 2.19 (t, J = 7.2 Hz, 2H, –
2
–), 1.20 (m, 3H, –CH
3
13
CH
2 2
–), 2.00 (t, J=6.4 Hz, 2H, –CH –). C NMR (CDCl
3
): d 169.02,
173.23, 162.98, 160.78, 159.46, 158.26,134.14, 133.51, 130.83,
130.66, 129.75, 125.22, 114.57, 112.02, 67.62, 66.47, 60.20, 46.31,
1
9
61.81, 157.9, 157.5, 131.11, 130.32, 115.27, 114.76, 112.62,
9.99, 67.10, 66.27, 56.61, 40.49, 40.38, 29.07, 25.12. HRMS (ESI)
40.61, 40.41, 33.62, 28.49, 21.67, 14.60. HRMS (ESI) calcd for
+
+
calcd for C20
H
22
N
4
O
4
S (M+H) 414.1445, found 414.1362. IR (KBr)
C
22
H
25
ꢁ1
N
3
O
3
S
(M+H) 442.1795, found 442.1779. IR (KBr)
m
ꢁ1
m
1
(cm ) = 3417, 3201, 2924, 2873, 1726, 1676, 1592, 1309, 1263,
196, 777.
(cm ) = 3449, 3090, 2984, 2869, 1736, 1606, 1306, 1252, 1118,
740.
4
2
.1.21. Ethyl-4-[3-(4-morpholin-4-yl-thieno[3,2-d]pyrimidines-
-yl)phenoxy]hexanoate (6f)
4.1.25. 5-[4-(4-Morpholin-4-yl-thieno[3,2-d]pyrimidines-2-
yl)phenoxy]hexanoate (7g)
Compound 6f was prepared from 5a according to the same
Compound 7g was prepared from 6g according to the same
procedure described for 7a to give a white solid. Yield 94%. Mp:
195–197 °C. H NMR (400 Hz, DMSO): d 8.52 (d, J = 5.6 Hz, 1H, 6-
procedure described for 6a using 4-bromobutyrate to give a white
1
1
solid. Yield 80%. Mp: 90–92 °C. H NMR (400 Hz, DMSO): d 8.36 (d,
J = 8.8 Hz, 2H, ArH), 8.23 (d, J = 5.2 Hz, 1H, 6-H), 7.50 (d, J = 5.6 Hz,
H), 8.43 (d, J = 8.8 Hz, 2H, ArH), 7.79 (d, J = 5.6 Hz, 1H, 7-H), 7.16
0
0
1
4
CH
1
1
1
3
4
2
H, 7-H), 7.03 (d, J = 9.2 Hz, 2H, ArH), 4.11 (m, 4H, –CH
2
, –CH
2
0
),
(d, J = 8.8 Hz, 2H, ArH), 4.19 (t, J = 5.2 Hz, 4H, 2 -CH
2
0
, 6 -CH
2
0
), 4.13
0
0
.01 (t, J = 4.8 Hz, 4H, 2 -CH
2
, 6 -CH
2
), 3.82 (t, J = 5.2 Hz, 4H, 3 -
–), 2.04 (m, 2H, –CH –),
). C NMR (DMSO): d 173.02, 162.98,
2
(t, J = 6.4 Hz, 2H, –CH ), 3.87 (t, J = 4.8 Hz, 4H, 3 -CH
2
, 5 -CH
2
),
0
2
, 5 -CH
2
), 2.50 (t, J = 8.4 Hz, 2H, –CH
2
2
2.33 (t, J = 7.6 Hz, 2H, –CH
2
–), 1.82 (m, 2H, –CH
2
–), 1.71 (m, 2H, –
13
13
.21 (t, J = 6.8 Hz, 3H, –CH
3
2
CH –).
C NMR (DMSO): d 174.81, 167.03, 162.83, 159.46,
60.64, 159.44, 158.26, 147.93, 134.18, 130.94, 129.77, 128.08,
157.05, 140.07, 136.38, 131.19, 124.82, 115.22, 113.20, 112.63,
25.23, 114.58, 112.04, 74.96, 67.06, 66.47, 60.36, 40.61, 40.40,
92.26, 68.13, 67.05, 66.25, 40.59, 40.38, 33.74, 28.44, 21.62. HRMS
+
+
0.61, 24.7, 14.58. HRMS (ESI) calcd for C22
H
25
N
3
O
3
S (M+H)
(ESI) calcd for C21
H
23
N
3
O
4
S (M+H) 414.1482, found 414.1480. IR
ꢁ1
ꢁ1
28.1639, found 428.1630. IR (KBr)
m
(cm ) = 3435, 3097, 2962,
(KBr)
m
(cm ) = 3466, 3100, 2933, 2874, 1721, 1608, 1591, 1306,
897, 2858, 1725, 1608, 1302, 1251, 1119, 762.
1263, 1115, 776.
4
.1.22. 4-[3-(4-Morpholin-4-yl-thieno[3,2-d]pyrimidines-2-
4.1.26. 1-Hydroxyamino-5-[4-(4-morpholin-4-yl-thieno[3,2-
d]pyrimidines-2-yl)phenoxy]hexanoate (10)
yl)phenoxy]hexanoate (7f)
Compound 7f was prepared from 6f according to the same
Compound 10 was prepared from 7g according to the same
procedure described for 7a to give a white solid. Yield 80%. Mp:
procedure described for 12 to give a white solid. Yield 50.6%.
1
1
1
79–180 °C. H NMR (400 Hz, DMSO): d 8.51 (d, J = 5.6 Hz, 1H,
R
f
= 0.3 (acetone/petroleumether = 1:1). Mp: 144–147 °C. H NMR
ArH), 8.43 (d, J = 8.8 Hz, 2H, 6-H), 7.78 (d, J = 5.6 Hz, 1H, 7-H),
(400 Hz, DMSO): d 10.45 (s, 1H, –OH), 8.54 (d, J = 5.6 Hz, 1H, 6-
0
0
7
.16 (d, J = 9.2 Hz, 2H, ArH), 4.18 (t, J = 4.4 Hz, 4H, 2 -CH
2
0
, 6 -CH
2
0
),
H), 8.45 (d, J = 8.8 Hz, 2H, ArH), 7.83 8(d, J = 5.6 Hz, 1H, 7-H), 7.16
0
0
4
.14 (t, J = 6.4 Hz, 2H, –CH
), 2.44 (t, J = 7.2 Hz, 2H, –CH
NMR (DMSO): d 174.53, 172.38, 157.60, 150.35, 130.54, 136.65,
2
), 3.87 (t, J = 4.8 Hz, 4H, 3 -CH
2
, 5 -
(d, J = 8.8 Hz, 2H, ArH), 4.18 (s, 4H, 2 -CH
2
0
, 6 -CH
2
), 4.12 (t,
1
3
0
CH
2
2
–), 2.03 (m, 2H, –CH
2
–).
C
J = 6.0 Hz, 2H, –CH
2
–), 3.87 (t, J = 8.8 Hz, 5H, 3 -CH
2
, 5 -CH
2
, –NH–
), 2.51 (t, J = 1.6 Hz, 4H, –CH
2 2
–, –CH –), 3.08 (m, 1H, –NH), 2.08

3
64
Q. Tan et al. / Bioorg. Med. Chem. 22 (2014) 358–365
(
1
6
m, 2H, –CH
2
–). 13C NMR (CDCl
57.06, 153.70, 136.13, 131.14, 124.61, 119.35, 115.28, 112.63,
8.09, 67.48, 66.25, 40.59, 40.38, 32.33, 28.50, 22.20. HRMS (ESI)
3
): d 171.94, 169.35, 164.34, 162.86,
stopped by addition of a developer containing TSA. Fluorescence
was monitored after 15 min at excitation and emission wave-
lengths of 360 and 460 nm, respectively.
+
24 4 4
calcd for C21H N O S (M+H) 428.1601, found 428.1518. IR (KBr)
m
7
ꢁ1
(cm ) = 3423, 3170, 2923, 2869, 1651, 1560, 1373, 1278, 1182,
76.
4.2.2. In vitro antiproliferative assays
Antiproliferative cellular assays were conducted as described
using HCT-116, MCF-7 and HeLa cells. These cancer cells were a
generous gift from Jiangsu University. All cell lines were cultured
4
2
.1.27. Ethyl-5-[3-(4-morpholin-4-yl-thieno[3,2-d]pyrimidines-
-yl)phenoxy]hexanoate (6h)
2
in a 37 °C incubator with a 5% CO environment. Compounds were
Compound 6h was prepared from 5b according to the same pro-
dissolved in DMSO with a concentration of 10 mM. Cells were
3
cedure described for 6a using 5-bromo-pentanoate ethyl to give a
seeded into 96-well plates at a concentration of 2 ꢂ 10 per well,
1
white solid. Yield 80%. Mp: 75–77 °C. H NMR (400 Hz, DMSO): d
cultured for 24 h, exposed to the compounds at various concentra-
tions for cancer cell viability experiments, cells were cultured for
72 h and viability was determined through the use of the MTT
assay.
8
1
7
.25 (d, J = 5.6 Hz, 1H, ArH), 8.01 (d, J = 7.6 Hz, 1H, 6-H), 7.93 (s,
H, ArH), 7.54 (d, J = 5.2 Hz, 1H, 7-H), 7.41 (t, J = 8 Hz, 1H, ArH),
.05 (dd, J = 2.4, 8 Hz, 1H, ArH), 4.09 (m, 5H, –CH
2
, –CH
), 3.82 (t, J = 4.8 Hz, 4H,
), 2.51 (d, J = 1.6 Hz, 1H, –CH –), 2.40 (t, J = 7.2 Hz,
–), 2.34 (t, J = 7.6 Hz, 1H, –CH –), 1.20 (t, J = 7.2 Hz, 4H,
): d 173.26, 162.83, 159.31, 159.18,
2 2
, –CH ),
0
0
4
3
2 2
.01 (t, J = 4.4 Hz, 4H, 2 -CH , 6 -CH
0
0
-CH
H, –CH
CH –, –CH
2
, 5 -CH
2
2
4.2.3. Cell cycle analysis by FACS
2
–
1
2
2
HCT-116 cells were seeded into six-well plates at a concentra-
1
3
5
2
3
). C NMR (CDCl
3
tion of 2 ꢂ 10 per well, exposed to the compounds at various
58.27, 139.95, 134.36, 129.87, 125.29, 120.61, 116.52, 113.97,
concentrations, cultured for 24 h, collected, fixed with 70% cold
1
2
12.78, 67.54, 66.46, 60.19, 46.33, 40.41, 40.20, 33.65, 28.56,
ethanol,stained with PI-solution (50
l
g/mL PI from 50ꢂ stock
+
1.72, 14.59. HRMS (ESI) calcd for C22
H
25
N
3
O
3
S (M+H) 442.1793,
solution (2.5 mg/mL), 0.1 mg/mL RNase A, 0.05% Tritin X-100),
incubated for 40 min at 37 °C and analyzed by FACS using the
Gallios flow cytometry system (Beckman Coulter, Brea, CA, USA).
ꢁ
1
found 442.1779. IR (KBr)
m (cm ) = 3101, 2974, 2935, 2868,
1
713, 1537, 1304, 1268, 1187, 746.
4
.1.28. 5-[3-(4-Morpholin-4-yl-thieno[3,2-d]pyrimidines-2-
Acknowledgments
yl)phenoxy]hexanoate (7h)
Compound 7h was prepared from 6h according to the same
The studies in the laboratory were supported by the Natural
Science Foundation of Jiangsu Province (BK2011390), Natural Sci-
ence Foundation of Nantong City (K2010024), Priority Academic
Program Development of Jiangsu Higher Education Institutions
(PAPD), National Natural Science Foundation of China (Grant Nos.
30971197 and 31171143), and 973 Program (2011CB510004).
procedure described for 7a to give a white solid. Yield 80%. Mp:
1
1
6
80–182 °C. H NMR (400 Hz, DMSO): d 8.47 (d, J = 5.2 Hz, 1H,
-H), 8.00 (m, 2H, ArH), 7.74 (d, J = 5.6 Hz, 1H, 7-H), 7.21 (dd,
0
0
J = 2.8 Hz, 1H, ArH), 4.15 (t, J = 4.8 Hz, 4H, 2 -CH
2
, 6 -CH
2
), 4.11
), 2.34
0
0
(
t, J = 6 Hz, 2H, –CH
2
), 3.86 (t, J = 4.8 Hz, 4H, 3 -CH
2
, 5 -CH
2
(
t, J = 7.2 Hz, 2H, –CH
2
–), 1.80 (m, 2H, –CH
2
–), 1.73 (m, 2H,
1
3
–
1
9
2
CH –).
C NMR (DMSO): d 174.70, 167.03, 162.83, 159.44,
References and notes
57.05, 140.07, 136.38, 130.01, 121.19, 114.42, 113.16, 112.63,
2.26, 68.12, 67.84, 66.41, 40.90, 40.60, 33.80, 28.62, 21.73 HRMS
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3. Johnstone, R. W. Nat. Rev. Drug Disc. 2002, 1, 287.
+
(
ESI) calcd for C21
H
23
N
3
O
4
S (M+H) 414.1486, found 414.1485.
ꢁ1
IR (KBr)
m (cm ) = 3424, 3106, 2939, 2864, 1716, 1586, 1570,
4
.
Somech, R.; Izraeli, S.; Simon, A. J. Cancer Treat. Rev. 2004, 30, 461.
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323, 1263, 1113, 776.
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7
.
.
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4
.1.29. 1-Hydroxyamino-5-[3-(4-morpholin-4-yl-thieno[3,2-
d]pyrimidines-2-yl)phenoxy]hexanoate (11)
8. Glick, R. D.; Swindemen, S. L.; Coffey, D. C.; Rifkind, R. A.; Marks, P. A.; Richon,
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Compound 11 was prepared from 7h according to the same pro-
9.
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,
5165.
cedure described for 12 to give a white solid. Yield 25.4%, R
f
= 0.3
1
(
acetone/petroleumether =1:1). Mp 144–146 °C. H NMR (400 Hz,
DMSO): d 10.45 (s, 1H, –OH), 8.54 (d, J = 5.6 Hz, 1H, 6-H), 8.45 (d,
J = 8.8 Hz, 2H, ArH), 7.83 (d, J = 5.6 Hz, 1H, 7-H), 7.16 (d,
J = 8.8 Hz, 2H, ArH), 4.18 (s, 4H, 2 -CH
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1. Lane, A. A.; Chabner, B. A. J. Clin. Oncol. 2009, 27, 5459.
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0
0
2
, 6 -CH
2
0
), 4.12 (t, J = 6.0 Hz,
0
2
H, –CH
2
–), 3.87 (t, J = 8.8 Hz, 5H, 3 -CH
2
, 5 -CH
2
, –NH), 2.51 (t,
–), 3.08 (m, 1H, –NH), 2.08 (m, 2H, –
169.38, 165.94, 159.38, 157.21,
13. Erlich, R. B.; Kherrouche, Z.; Rickwood, D.; Endo-Munoz, L.; Cameron, S.;
Dahler, A.; Rethinam, M. H.; De-Long, L. M.; Wooley, K.; Guminski, A.;
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CH
2
–, –CH
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2
13
2
–).
C
3
):
d
14. Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505.
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6
39.81, 132.92, 130.62, 120.91, 117.61, 114.63, 113.29, 106.80,
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+
calcd for C21
H
24
N
4
O
4
S (M+H) 428.1604, found 428.1518. IR (KBr)
ꢁ1
m
7
(cm ) = 3422, 3205, 2928, 2866, 1715, 1648, 1587, 1375, 1228,
78.
1
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1
5
l
L of HeLa nuclear extract was mixed with 10
pound. Fluorogenic substrate (25 L) was added, and reaction
was allowed to proceed for 15 min at room temperature and then
lL of 5ꢂ com-
2
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2