Synthesis and anti-tumor activities of N′-benzylidene-2-(4- oxothieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazone derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.09.061

A compound with a cyclic thienopyrimidine moiety and an aceto-hydrazone moiety in its chemical structure was discovered in a cell-based screening to have noticeable cytotoxicity on several tumor cell lines. A total of 38 derivatives of this compound were

Full text of DOI:10.1016/j.bmcl.2011.09.061

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6662–6666
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-tumor activities of N0-benzylidene-2-(4-oxothieno[2,3-d]
pyrimidin-3(4H)-yl)acetohydrazone derivatives
Jiangping Lou a, , Zhen Liu b, , Yan Li b, Mi Zhou b, Zhengxi Zhang b, Shu Zheng a, Renxiao Wang b,
,
⇑
Jian Li a,
⇑
a School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, People’s Republic of China
b State Key Laboratory of Bioorganic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2011
Revised 15 September 2011
Accepted 16 September 2011
Available online 21 September 2011
A compound with a cyclic thienopyrimidine moiety and an aceto-hydrazone moiety in its chemical
structure was discovered in a cell-based screening to have noticeable cytotoxicity on several tumor cell
lines. A total of 38 derivatives of this compound were synthesized at five steps with high yields. These
compounds were tested in standard MTT assays, and several compounds exhibited improved cytotoxic
activities. The most potent compounds have IC50 values of 10–20 lM on A549, HeLa, and MBA-MD-
231 tumor cells. Flow cytometry analysis of several active compounds and subsequent examination of
caspase activation indicate that they induce caspase-dependent apoptosis in tumor cells. In addition,
these compounds do not have obvious effect on a normal cell line HEK-293T, demonstrating the desired
selectivity against tumor cells. Results from a fluorescence polarization-based in vitro binding assay indi-
cate that this class of compounds does not significantly interrupt the interactions between Mcl-1 and Bid.
Their cytotoxicity is achieved presumably through other mechanisms.
Keywords:
Thienopyrimidine
Acetohydrazone
Cytotoxicity
Apoptosis inducer
Anti-tumor compound
Ó 2011 Elsevier Ltd. All rights reserved.
The discovery and development of effective anti-cancer thera-
pies has been accelerated in recent years by various molecular tar-
geted techniques and strategies.1 Nevertheless, in vitro screening
with tumor cell lines is still an essential approach to the identifica-
tion of compounds with desired potency.2 One famous example is
the NCI-60 platform, which is a panel of 60 human tumor cell lines
made available by the US National Cancer Institute.3 Millions of
compounds have been screened on the NCI-60 platform so far,
and such efforts have led to the discovery of many compounds
with clear pharmaceutical implications.
As part of our efforts on discovering new potential anti-cancer
compounds, we screen the compounds in our inventory, which
are either purchased from a commercial source or synthesized by
ourselves, whenever enough samples are available. In our cell-
based screening, each compound is tested at a single concentration
MB-231 cells, respectively, but its effect on A549 cells was rather
low ( Fig. 1). Considering the biological potency and synthetic
accessibility of compound 1, it may serve as a reasonable lead com-
pound for further developments.
From the viewpoint of a medicinal chemist, two distinctive moi-
eties are included in the chemical structure of this compound: a
cyclic thienopyrimidine moiety and an acetohydrazone moiety
(2 in Fig. 2). Our literature survey indicates that some known com-
pounds containing such moieties are indeed associated with
anti-cancer activities. A number of tetrahydrobenzothieno-pyrimi-
dine (3 in Fig. 2) and arylthienopyrimidine (4 in Fig. 2) derivatives
were reported as anti-cancer, anti-bacterial and anti-microbial
agents.4–6 The molecular targets of these compounds are often
unclear. At present, two possible molecular targets of these
compounds have been reported, that is, 5-HT1A receptor7 and
17-b-hydroxysteroid dehydrogenase.8,9 Both of them are related
to cancers. The thienopyrimidine moiety is therefore a valuable
scaffold for developing new anti-cancer compounds. Our literature
survey also found several acetohydrazone derivatives that exhibit
broad-spectrum anti-tumor and anti-microbial activities. The
oxoquinoxaline derivatives (5 in Fig. 2) are reported to show
anti-tubercular, anti-bacterial and anti-fungal activities.10 Such
compounds with an aryl group instead of oxoquinoxaline will
cause distinct cytotoxic activities.11 In particular, an arylpiperazi-
of 20 lM in a standard MTT assay on three selected tumor cell
lines, including A549 (human alveolar epithelial cell), HeLa (hu-
man cervical tumor cell), and MBA-MD-231(human breast tumor
cell). Recently, a compound, that is, 1 in Figure 1, was discovered
in our screening, which exhibited notable cytotoxicity at the tested
concentration. This compound was originally purchased from the
Specs catalog (Specs ID = AF-399/40713992). The IC50 values of this
compound in the MTT assay were 40 and 16 lM on HeLa and MDA-
nyl aceto-hydrazone derivative (PAC-1,
6 in Fig. 2) was
⇑
Corresponding authors.
E-mail addresses: wangrx@mail.sioc.ac.cn (R. Wang), jianli@ecust.edu.cn (J. Li).
These authors made equal contributions to this work.
demonstrated to induce apoptosis in cancer cells by activating
procaspase-3 to caspase-3.12 Given these facts, it is reasonable to
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.061

J. Lou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6662–6666
6663
O
N
H
N
N
N
O
S
HO
1
Figure 1. The lead compound (1) identified in our screening and its dose-dependent cytotoxicity on three selected tumor cell lines.
O
R1
is close to compound 1. According to the record in the PubChem
BioAssay database (CID: 5711212),13 this compound inhibits the
interactions between Mcl-1 and Bid proteins and thus may induce
apoptosis in tumor cells as other effective Mcl-1 inhibitors.
The above analysis provides additional supports that the active
compound identified in our screening, that is, compound 1, is worth
further developments. In this study, we have synthesized a total of
38 derivatives (12–49) of compound 1. Chemical synthesis of this
class of compounds can be completed relatively efficiently in high
yields (10–30% total yield over five steps). The synthetic methods
for the preparation of compounds 1 and 12–49 is illustrated in
Scheme 1. Using our method described previously,14 ketones, cya-
noacetates, and sulfur were mixed and subjected to microwave
irradiation for several minutes to produce 2-amino-thiophene (8).
In this reaction, basic aluminum oxide was used as solid support
and morpholine as base catalyst. Compound 8 was cyclized to
thieno[2,3-d]pyrimidine (9) by condensation with formamide.15
Compound 10 was the obtained by reacting ethyl bromoacetate
and 9, which then was converted to the hydrazide 11, the key inter-
mediate, by refluxing with 85% hydrazine monohydrate in ethanol.
Various aromatic aldehydes were reacted with compound 11 to
produce the target compounds 1 and 12–49.
H
N
N
N
R3
R2
O
O
2
S
N
R3
R1
R2
O
R1
R2
N
N
S
N
N
3
S
N
4
N
O
5
N
O
6
N
R1
N
H
N
N
N
H
O
R2
OH
O
N
O
N
H
N
N
N
H
O
7
S
All of the synthesized compounds (Table 1) were then evaluated
at a single concentration of 20 lM on A549, HeLa, and MDA-MB-231
cells. Inhibitions of cell growth were determined using a standard
MTT assay after 48-hour treatment. Among them, compounds 24,
33, 35, 37 and 45 exhibited inhibition ratio over 50% on at least
one tumor cell line (Table 1). These compounds were then tested
at multiple concentrations to determine their cytotoxicity quantita-
tively. In this experiment, cell line HEK-293T, that is, human
embryonic kidney cells transformed with adenovirus 5 DNA and
Figure 2. The structural scaffold of the compounds synthesized in this study (2)
and some structurally similar compounds reported in literature (3–7).
expect that a compound combining these two moieties may have
anti-tumor activities. In fact, one known compound (7 in Fig. 2)

6664
J. Lou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6662–6666
O
O
R1
O
N
R1
R2
O
O
O
O
a
H
NH2
NH
R1
Br
R2
+
S
+
b
40-60%
NH2
R2
O
NC
S
S
80-90%
O
9
8
R1
R1
O
OEt
NHNH2
OEt
d
N
N
R2
R2
80-90%
O
c
O
S
S
N
N
60-70%
10
11
R1
O
O
R3
H
N
N
N
R2
R3
e
O
S
N
and
70-80%
1
12 49
-
Scheme 1. The synthetic route for obtaining the class of compounds reported in this study. Reagents and conditions: (a) MWI, morpholine, basic Al2O3, 140 W, 15 min; (b)
200 °C, 1.5 h; (c) K2CO3, acetone, 55 °C; (d) N2H4.H2O (85%), C2H5OH, reflux; (e) n-butanol, reflux.
Table 1
Chemical structures of compounds 1 and 12–49 and their cytotoxicity on three tumor cell lines
Compd
R1
R2
R3
Inhibition ratioa (%) at 20
HeLa
lM
A549
MDA-MB-231
1
–(CH2)4–
2-OH
18
34
29
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)3–
–(CH2)5–
3-F, 4-Cl
4-CN
3-NO2, 4-Cl
4-CF3
2-OH
4-CN
3-F, 4-Cl
3-NO2, 4-Cl
3-OH
2-OH
4-CN
3-F, 4-Cl
3-NO2, 4-Cl
3-OH
4-OH
4-OH
3-F, 4-Cl
3-F, 4-Cl
2-OH
2-OH
4-CN
3-NO2, 4-Cl
4-CN
3-NO2, 4-Cl
3-F, 4-Cl
3-F, 4-Cl
2-OH
4-CN
3-NO2, 4-Cl
2-OH
3-F, 4-Cl
2-OH
4-CN
3-NO2, 4-Cl
3-NO2
3-OH, 4-OH
3-NO2
26
41
N.A
27
34
23
N.A
54
34
N.A
36
11
39
N.A
38
30
36
N.A
29
41
N.A
40
14
N.Ab
10
23
22
N.A
47
N.A
N.A
29
10
43
50
96
28
66
N.A
N.A
27
N.A
N.A
N.A
48
26
N.A
58
N.A
66
N.A
51
N.A
N.A
13
35
N.A
44
N.A
69
N.A
N.A
N.A
N.A
N.A
29
N.A
N.A
N.A
43
31
N.A
74
N.A
64
N.A
52
N.A
N.A
18
33
N.A
22
N.A
69
N.A
N.A
N.A
N.A
N.A
25
N.A
N.A
N.A
39
30
N.A
78
N.A
29
N.A
74
30
N.A
13
37
N.A
36
N.A
55
N.A
N.A
N.A
N.A
Phenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Cl-phenyl
4-Cl-phenyl
Phenyl
4-Cl-phenyl
4-Cl-phenyl
Phenyl
Phenyl
CH3
3-CH3-4-Cl-phenyl
CH3
CH3
CH3
3-CH3-4-Cl-phenyl
Naphthalen-2-yl
Naphthalen-2-yl
Naphthalen-2-yl
Naphthalen-2-yl
4-Cl-phenyl
4-Cl-phenyl
Naphthalen-2-yl
Naphthalen-2-yl
3-OH, 4-OH
a
Cell growth inhibition ratio were measured after 48-hour treatment.
No significant activity observed at the tested concentration.
b
SV40 large T-antigen, was added as a model of normal cell. The
concentrations at 50% inhibition (IC50) of these compounds on all
four cell lines are summarized in Table 2. As one can see here,
compounds 33, 35, 37 and 45 exhibited dose-dependent cytotoxic
profiles on all three tumor cell lines with IC50 values ranging from
(IC50 >50 lM). It is encouraging to observe that all five selected
compounds did not exhibit obvious cytotoxicity on HEK-293T cells,
demonstrating the desired selectivity between tumor cells and
normal cells.
Some typical apoptotic phenotypes, such as blebbing and cell
shrinkage, were observed on tumor cells after treated with the
selected compounds. Thus, the pro-apoptotic activity of the most
11 to 32
lM. Compound 24 also exhibited lM-level potency on HeLa
and MDA-MB-231 cells, but it was not effective on A549 cells

J. Lou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6662–6666
6665
Table 2
Cytotoxicity profiling of the most potent compounds
a
Compd
IC50
(lM)
A549
HeLa
MDA-MB-231
HEK-293T
1
>50
>50
20
16
17
40
6
11
11
15
24
16
23
32
20
16
20
>50
>50
>50
>50
>50
>50
24
33
35
37
45
13
a
Cytotoxicity was determined in MTT assay after 48-s treatment.
potent compounds identified in our MTT assays (Table 2) were fur-
ther examined in a flow cytometry assay. After treated by the se-
lected compounds at 20 lM for 24 h, HeLa cells were stained
Figure 4. Activation of caspase-3 and PARP by three selected compounds. HeLa
cells were treated by these compounds at 20 lM for 12 h, and then the expression
with propidium iodide, and then were analyzed by flow cytometry.
The sub-diploid DNA contents of cells (apoptotic sub-G1 area) in-
creased from 6% in the control cells to 22%, 27%, 20% upon the
treatment of compounds 33, 37, and 45 (Fig. 3), indicating the obvi-
ous apoptotic activity of these compounds. In contrast, compounds
24 and 35 induced 6% and 8% cells to apoptosis, which was not sig-
nificantly different from the negative control in this experiment.
Activation of caspases and the consequent cleavage of PARP is
one of the indicators of cell apoptosis. The three compounds exhib-
iting apparent apoptotic signals in the flow cytometric analysis,
that is, 33, 37, and 45, were further examined in such an assay
on HeLa cells. As shown by the immunoblotting results (Fig. 4),
all three compounds clearly increased the level of cleaved cas-
pase-3 and cleaved PARP as compared with a negative control. This
observation confirms the results of flow cytometry analysis that
compounds 33, 37, and 45 are able to induce caspase-dependent
apoptosis.
levels of caspase-3 and PARP were examined by western blot.
between Mcl-1 and Bid proteins and thus has a potential to induce
apoptosis in tumor cells with Mcl-1 over-expression. In order to ver-
ify if our compounds are also potential Mcl-1 inhibitors, compounds
1 and 12–49 were tested in a fluorescence polarization-based
in vitro binding assay to measure their binding affinity to the
Mcl-1 protein. Here, a FAM-labeled Bid-BH3 peptide was used as
the fluorescence tracer. Competitive binding of a given compound
was characterized quantitatively by monitoring the resulting
changes in FP signals. This assay has been successfully applied in
our previous study of small-molecule inhibitors of the Bcl-2 family
proteins.16
As a preliminary screening, all compounds (1 and 12–49) were
tested at three typical concentrations, that is, 1, 10, and 50
None of these compounds was observed to achieve a significant
binding to Mcl-1 even at 50 M (data not shown here). Thus, these
lM.
l
As mentioned earlier, we noticed one compound in literature
(7 in Fig. 1) which is close to the compounds obtained and tested
in our study. This compound is reported to interrupt the interactions
compounds were not examined at other concentrations. Only
compounds 37, 38, and 49 exhibited inhibition ratio around 40%
Figure 3. Results of flow cytometric analysis on HeLa cells for five selected compounds. Cells were treated by these compounds at 20 lM for 24 h. Apoptosis was analyzed by
propidium iodide staining. DMSO was used as the negative control in this assay.

6666
J. Lou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6662–6666
at 50
l
M. Note that this level of binding affinity to Mcl-1 of 37
interrupt the interactions between Mcl-1 and Bid. Their cytotoxic-
ity is achieved presumably through other mechanisms.
apparently cannot explain the potency of this compound observed
in the MTT assay (IC50 = 10–20 M). In addition, compounds 38
l
and 49 were basically inactive on the tumor cell lines used in our
study (Table 1). Thus, we conclude that cytotoxicity of this class
of compounds is not the consequence of Mcl-1 inhibition but
rather some other unknown mechanisms.
Acknowledgments
The authors are grateful to the financial supports from the
Chinese National Natural Science Foundation (Grants No.
20772149, No. 90813006, No. 21072213, No. 21002117 & No.
20921091), the Chinese Ministry of Science and Technology (Grants
No. 2009ZX09501-002 and 2011ZX09102-005-02), and in particu-
lar, a supporting grant to Professor Jian Li by the State Key Laboratory
of Bioorganic Chemistry at the Shanghai Institute of Organic
Chemistry.
It is difficult to give a clear structure–activity relationship for
this class of compounds based on the results obtained so far. Nev-
ertheless, some preliminary conclusions can be drawn here. First of
all, cyclization of R1 and R2 (2 in Fig. 2) into an aliphatic ring will
almost abolish the biological potency of the resulting compounds
(1 and 12–27). The lead compound pursued in our study, that is,
compound 1, has this cyclic moiety in its structure, and thus the
first batch of compounds synthesized by us (12–27) followed this
idea. Unfortunately, they were not very successful in terms of
achieving the desired cytotoxicity on tumor cells. One such
compound, that is, 24, exhibited noticeable cytotoxicity on HeLa
and MDA-MB-231 cells but not on A549 cells just as compound 1
(Table 2). On our second batch of compounds (28–49), R1 is a sep-
arate branch group. Interestingly, this modification leads to the
cytotoxicity on A549 cells at least on some of the corresponding
compounds. Second, it seems that R1 has to be a bulky aromatic
group to maintain the biological potency. Several compounds with
a methyl group as R1, such as 36, 38, 39, and 40, are not active.
Third, the best option for substituent group R3 for maintaining
the desired cytotoxicity is a combination of 3-NO2 and 4-Cl, such
as 33, 35, and 45. It seems to be important that the meta- and
the para-position on this phenyl ring should be occupied simulta-
neously. The role of R3 needs to be explored more thoroughly in the
future.
Supplementary data
Supplementary data (the synthetic methods and spectroscopic
data of the compounds as well as the details of the MTT assay, flow
cytometry analysis, western blot detection, and fluorescence polar-
ization-based binding assay) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl. 2011.09.061.
References and notes
1. Ovaa, H.; Kuijl, C.; Neefjes, J. Drug Discovery Today: Technologies 2009, 6, e3.
2. Sharma, S. V.; Haber, D. A.; Settleman, J. Nat. Rev. Cancer 2010, 10, 241.
3. Shoemaker, R. H. Nat. Rev. Cancer 2006, 6, 813.
4. Ghorab, M. M.; Abdel-Hamide, S. G. Indian J. Heterocycl. Chem. 1994, 4, 103.
5. Hozien, Z. A.; Atta, F. M.; Hassan, Kh. M.; Abdel-Wahab, A. A.; Ahmed, S. A.
Synth. Commun. 1996, 26, 3733.
6. Ashalatha, B. V.; Narayana, B.; Vijaya Raj, K. K.; Suchetha Kumari, N. Eur. J. Med.
Chem. 2007, 42, 719.
7. Modica, M.; Santagati, M.; Russo, F.; Parotti, L.; De Gioia, L.; Selvaggini, C.;
Salmona, M.; Mennini, T. J. Med. Chem. 1997, 40, 574.
8. Lilienkampf, A.; Karkola, S.; Alho-Richmond, S.; Koskimies, P.; Johansson, N.;
Huhtinen, K.; Vihko, K.; Wähälä, K. J. Med. Chem. 2009, 52, 6660.
9. Wähälä, K.; Lilienkampf, A.; Alho, S.; Huhtinen, K.; Johansson, N.; Koskimies, P.;
Wihko, K. PCT. Int. Appl. 2004, 70pp, CODEN: PIXXD2 WO2004110459A1.
10. Rao, G. K.; Kotnal, R. B.; Pai, P. N. E. -J. Chem. 2010, 7, 1435.
11. Nakka, M.; Begum, M. S.; Varaprasad, B. F.; Reddy, L. V.; Bhattacharya, A.;
Helliwell, M.; Mukherjee, A. K.; Beevi, S. S.; Mangamoori, L. N.; Mukkanti, K.;
Pal, S. J. Chem. Pharm. Res. 2010, 2, 393.
In conclusion, we noticed the cytotoxicity of a compound con-
taining a thienopyrimidine and an aceto-hydrazone moiety (1)
on several tumor cell lines. Based on this compound, a total of 38
derivatives of this compound were synthesized in five steps. All ob-
tained compounds were tested in MTT assays, and several com-
pounds exhibit improved cytotoxicity as compared to the lead
compound. The most potent compounds have IC50 values ranging
from 10–20 lM on A549, HeLa, and MBA-MD-231 cells. Flow
12. Putt, K. S.; Chen, G. W.; Pearson, J. M.; Sandhorst, J. S.; Hoagland, M. S.; Kwon, J.-
T.; Hwang, S.-K.; Jin, H.; Churchwell, M. I.; Cho, M.-H.; Doerge, D. R.; Helferich,
W. G.; Hergenrother, P. J. Nat Chem. Biol. 2006, 2, 543.
13. Wang, Y.; Bolton, E.; Dracheva, S.; Karapetyan, K.; Shoemaker, B. A.; Suzek, T.
O.; Wang, J.; Xiao, J.; Zhang, J.; Bryant, S. H. Nucleic Acids Res. 2010, 38, D255.
14. Huang, W.; Li, J.; Tang, J.; Liu, H.; Shen, J.; Jiang, H. Synth. Commun. 2005, 35,
1351.
15. Bhaskar, V. H.; Kumar, P. P.; Sangameshwaran, B. Asian J. Chem. 2007, 19, 5187.
16. Zhou, B.; Li, X.; Li, Y.; Xu, Y.; Zhang, Z.; Zhou, M.; Zhang, X.; Liu, Z.; Zhou, J.; Cao,
C.; Yu, B.; Wang, R. ChemMedChem 2011, 6, 904.
cytometry analysis and detection of caspase activation of several
active compounds indicate that they induce caspase-dependent
apoptosis in tumor cells. In addition, these compounds do not have
obvious effects on a normal cell line HEK-293T (IC50 >50 lM), dem-
onstrating the desired selectivity against tumor cells. The results
from a fluorescence polarization-based in vitro binding assay
indicate that this class of compounds does not significantly