Synthesis and antitumor activity of α-aminophosphonate derivatives containing thieno[2,3-d]pyrimidines

Article abstract of DOI:10.1016/j.cclet.2015.03.026

Abstract Two series of thieno[2,3-d]pyrimidine derivatives were designed and synthesized, in which bioactive α-aminophosphonate subunits were introduced at the N3 position through an NN bond connection. The in vitro cytotoxic activity of the novel compounds was tested against human esophageal carcinoma cells (EC109), human hepatocarcinoma cells (HepG2), human gastric carcinoma cells (MGC-803), respectively, by the MTT method. The evaluation results revealed that compounds 6mb, 6mf, 6mg, 6nd and 6nh exerted the most potent inhibition against HepG2, MGC-803 and EC109 cells, respectively. In particular, compound 6mg presented excellent inhibitory effect against HepG2 (91.2%) and MGC-803 (94.4%) cells.

Full text of DOI:10.1016/j.cclet.2015.03.026

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CCLET-3255; No. of Pages 4
Chinese Chemical Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Synthesis and antitumor activity of
a-aminophosphonate derivatives
containing thieno[2,3-d]pyrimidines
Yan-Chun Guo a, Jing Li a, Jiao-Li Ma a, Zhi-Ran Yu a, Hai-Wei Wang a, Wen-Juan Zhu a,
a,b
Xin-Cheng Liao a, , Yu-Fen Zhao
*
a The College of Chemistry and Molecular Engineering, The Key Laboratory of Chemical Biology and Organic Chemistry of Henan Province, Zhengzhou
University, Zhengzhou 450052, China
b Department of Chemistry, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University,
Xiamen 361005, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Two series of thieno[2,3-d]pyrimidine derivatives were designed and synthesized, in which bioactive a-
Received 13 November 2014
Received in revised form 19 December 2014
Accepted 6 March 2015
Available online xxx
aminophosphonate subunits were introduced at the N3 position through an N–N bond connection. The
in vitro cytotoxic activity of the novel compounds was tested against human esophageal carcinoma cells
(EC109), human hepatocarcinoma cells (HepG2), human gastric carcinoma cells (MGC-803),
respectively, by the MTT method. The evaluation results revealed that compounds 6mb, 6mf, 6mg,
6nd and 6nh exerted the most potent inhibition against HepG2, MGC-803 and EC109 cells, respectively.
In particular, compound 6mg presented excellent inhibitory effect against HepG2 (91.2%) and MGC-803
(94.4%) cells.
Keywords:
a-Aminophosphonate derivatives
Thieno[2,3-d]pyrimidine
Synthesis
ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Antitumor
1. Introduction
or fluorine moiety containing a-aminophosphonates [24–27] were
proved to have high antitumor activity.
It is well known that fused pyridines and thienopyrimidines
have many valuable pharmacological properties, such as antiox-
idative [1,2], antimalarial [3,4], antibacterial [5], and antiviral
effects [6]. The pyrimidine-based derivatives, e.g. thieno[2,3-
d]pyrimidines, were proved to have pharmacological and thera-
peutic properties in medicinal chemistry, such as anti-inflamma-
tory [7], antibacterial [8,9], antifungal [10,11], antitumor [12],
antidepressant [13], antiplatelet [14], antihypertensive [15],
herbicidal [16] and plant growth regulatory [17] properties.
Based on the concept of bioisosterism and inspired by the above
observations, we were led to the proposal to incorporate
thieno[2,3-d]pyrimidine ring into the
pounds, to construct novel classes of thieno[2,3-d]pyrimidine
moiety containing -aminophosphonate derivatives, and to
evaluate their antitumor activity. Therefore, two series of such
compounds, incorporating the -aminophosphonate and the
a-aminophosphonate com-
a
a
thieno[2,3-d]pyrimidines, were designed and synthesized and
their antitumor properties were investigated.
As phosphorus analogs of
a
-amino acids and their esters,
a
-
Twenty-two new diethyl or diisopropyl [(3-substituted or 4-
substituted or 2,4-disubstituted) (2-methyl-6-ethyl-4-oxo-4-H-
thieno[2,3-d]pyrimidin-3-yl-amino) methyl] phosphonates were
synthesized and their in vitro antitumor activity under cell
membrane conditions were evaluated by the MTT method.
Compounds 6ma-6mk and 6na-6nk, as well as the intermediates
4a-4k, were tested for their anti-proliferation activity against
three human cancer cell lines (EC109, HepG2 and MGC-803) at
different concentrations. This represents the first report about
the synthesis and in vitro antitumor activity evaluation of
aminophosphonates have received much attention owing to their
potential biological activities such as antibacterial, anticancer, as
well as insecticidal and herbicidal properties, [18–22] and were
investigated as starting materials for the syntheses of phospho-
nopeptides. Moreover, if the aminophosphonate subunit was
suitably embedded into potential antitumor agents, it could
effectively improve their bioactivity [23], e.g. benzothiazole moiety
thieno[2,3-d]pyrimidines
derivatives.
containing
a-aminophosphonate
*
Corresponding author.
E-mail address: lxc666@zzu.edu.cn (X.-C. Liao).
http://dx.doi.org/10.1016/j.cclet.2015.03.026
1001-8417/ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Y.-C. Guo, et al., Synthesis and antitumor activity of
a-aminophosphonate derivatives containing
thieno[2,3-d]pyrimidines, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.026

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2. Experimental
spectrometry, respectively (see Supporting information for struc-
ture characterization, yields and melting points).
All starting materials were commercially available and
analytically pure. 1H NMR spectra were recorded on a BRUKER
DPX-400 spectrometer (Bruker Company, Germany), using TMS as
Fortunately, the single crystals of compound 6mi and 6mj were
obtained. The molecular structures of the two compounds are
illustrated in Figs. 1 and 2. Colorless single crystals of the
compound 6mi (0.35 mm ꢀ 0.30 mm ꢀ 0.30 mm) and 6mj
(0.30 mm ꢀ 0.25 mm ꢀ 0.22 mm) were selected for X-ray diffrac-
tion analysis. The data were collected on an Agilent xcalibur Eos-II-
CCD-diffractometer equipped with a graphite-monochromatic Cu
˚
Ka radiation (l = 1.5418 A) by using a v scan mode in the range of
4.46 < u < 72.288 for 6mi and 4.42 < u < 67.078 for 6mj at 291(2) K.
The entire structure was solved by the direct methods using the
SHELXS-97 program [31] and refined by the full-matrix least-
squares method on F2 with anisotropic thermal parameters for all
non-hydrogen atoms using SHELXL-97. [32] For compound 6mi, a
total of 8957 reflections were recorded and 4476 were unique
(R int. = 0.0225), among which 4245 (ꢁ13 ꢂ h ꢂ 13, ꢁ9 ꢂ k ꢂ 14,
ꢁ19 ꢂ l ꢂ 21) were observed. For compound 6mj, a total of 8872
reflections were recorded and 4183 were unique (R int. = 0.0232),
among which 3930 (ꢁ12 ꢂ h ꢂ 8, ꢁ14 ꢂ k ꢂ 13, ꢁ19 ꢂ l ꢂ 21)
were observed. X-ray diffraction analysis revealed that the thieno
[2,3-d]pyrimidine ring in two compounds did not exhibit good
coplanarity and the molecular structure is stabilized by intermolec-
ular N(1)–H(1). . .N(3) hydrogen bonds. Thereby, the structure of
6mi and 6mj were further confirmed.
an internal standard and CDCl3 as a solvent. Chemical shifts (
d
values) and coupling constants (J values) are given in ppm and Hz,
respectively. TLC analysis was carried out on silica gel plates GF254
(Qindao Haiyang Chemical, China). High-resolution mass spectra
were performed on an ESI Q-TOF MS spectrometer (Micromass,
England). Melting points were determined on an XT4A apparatus
(Beijing Keyi Electro-optic Instrument Plant, China) and were
uncorrected. The intensity data were collected on an Agilent
xcalibur Eos-II- CCD-diffractometer (Agilent Technologies).
Twenty-two
a-aminophosphonate derivatives (6ma-6mk and
6na-6nk) were prepared according to the reaction sequences
shown in Scheme 1. The starting materials, ethyl 2-amino-5-
ethylthiophene-3-carboxylate (1) [28] and dialkyl phosphite (5),
were prepared according to the literature methods. The target
analogs were easily obtained in high yields with the established
reaction conditions. The acetylation of compound (1) with acetyl
chloride in acetonitrile gave the ethyl ester of 2-acetylamino-5-
ethylthiophene-3-carboxylic acid (2) [29]. The acetylated inter-
mediate (2) then underwent condensation-cyclization reactions
with hydrazine hydrate to afford compound (3), 3-amino-6-ethyl-
2-methylthieno[2,3-d]pyrimidin-4-one, in good yield [30]. The
Schiff base type key intermediates (4), obtained by the reaction of
(3) with various substituted benzaldehydes, reacted with different
dialkyl phosphite (5) under nitrogen atmosphere to generate the
A
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay was performed to investigate in vitro cytotoxic
activity. Human esophageal carcinoma cells (EC109), human
hepatocarcinoma cells (HepG2) and human gastric carcinoma
corresponding
a
-aminophosphonate derivatives (6ma-6mk and
cells (MGC-803) seeded into the 96-well plate (100 mL each well)
were incubated at 37 8C under 5% CO2 and 95% O2 until cell
adherence was observed. Cells were exposed to suspension of
6na-6nk). All the new compounds were characterized using 1H
NMR and 13C NMR, mass spectrometry and high resolution mass
COOEt
CH3COCl
DMF
Et3N
CHO
+
NCCH2COOEt
+ S
CH3CN
Et
S
NH2
S
1 yield:74%
CHO
O
COOEt
NHCOCH3
R
.
NH2
NH2NH2 H2O
EtOH, reflux
N
Et
CH3COOH, reflux
Et
S
N
3 yield:85%
2 yield:76%
R
O
O
H
N
O
HP(OEt)2
N
Et
P
O
5m
S
O
N
O
R
6ma-6mk
N
C
H
N
yield:70%-95%
N2
ºC
Et
R
S
N
CH3
4a-4k
yield:55%-90%
O
H
O
N
N
O
HP(OPr-i)2
P
Et
O
5n
S
O
N
6na-6nk
yield:60%-87%
O
ºC
R'OH
+
PCl3
HP(OR')2
R' = CH2CH3 ( m ) , CH(CH3)2 ( n )
5m-5n
yield:90%
R = H ( a ), 3-CH3 ( b), 4-CH3 ( c ), 3-NO ( d ), 4-NO2 ( e ), 3-Cl ( f ), 4-Cl ( g ),
2
3-Br (h ), 4-Br ( i ), 4-OCH 3 ( j ), 2,4-Cl2 ( k )
Scheme 1. Synthetic routes for the targeted compounds.
Please cite this article in press as: Y.-C. Guo, et al., Synthesis and antitumor activity of
a-aminophosphonate derivatives containing
thieno[2,3-d]pyrimidines, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.026

G Model
CCLET-3255; No. of Pages 4
Y.-C. Guo et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
3
3. Results and discussion
The in vitro cytotoxic activity of compounds 6ma-6mk, 6na-
6nk, as well as the intermediates 4a-4k against human esophageal
carcinoma cells (EC109), human hepatocarcinoma cells (HepG2),
human gastric carcinoma cells (MGC-803) was evaluated by the
MTT method and the potency was expressed as inhibition rate. The
results are summarized in Table 1.
Results in Table 1 show that the changes of substituents
affected the antitumor activity. Among analogs 6ma-6mk and
6na-6nk, even intermediates Schiff bases 4a-4k, compounds 6ma-
6mk and 6na-6nk have a relatively higher antitumor activity than
that of Schiff bases 4a-4k. These observations indicate that the
introduction of phosphoryl group improves antitumor activity to
EC109, HepG2 and MGC-803 cells.
Fig. 1. X-ray structure of compound 6mi.
The antitumor data also indicate that the substitutes of
phosphonate has no apparent influence on antitumor activity to
EC109, HepG2 and MGC-803 cells. For example, when R was 4-NO2
and R0 was Et, compound 6me exhibited moderate antitumor
activity with an inhibition rate of 54.0% against EC109 cells at
50
mg/mL; the antitumor activity of 6ne, where the phosphonate
was substituted by an i-Pr, was little higher than the activity of
6me in EC109 with the inhibition rate of 58.5%. Compound 6mk
with 2-Cl (R), 4-Cl(R), and Et (R’) has antitumor activity in HepG2
cells, with an inhibition rate of 77.8% at the concentration of 50
mg/
mL. Compound 6nk with 2-Cl (R), 4-Cl(R), and i-Pr (R0) has a lower
antitumor activity (60.8%) than that of 6mk. This same trend was
observed for compounds 6md/6nd and 6mh/6nh against EC109,
HepG2 and MGC-803 cells.
Fig. 2. X-ray structure of compound 6mj.
compounds 6ma-6mk, 6na-6nk, as well as the intermediates 4a-
4k at different concentrations. After 48 h, 20
concentration of 5 g/mL was added into the 96-well plate and
incubated for 4 h at 37 8C. Then, the supernatant was aspirated,
150 L of DMSO was added to each well, and the absorbance was
m
L of MTT with a
Compounds 6mg (R:4-Cl, R0:Et) and 6nh (R:3-Br, R0:i-Pr)
m
showed potent inhibition against EC109 cells with the inhibition
rate of 86.8% and 85.4% at 50
mg/mL, respectively. Compounds
m
6mg (R:4-Cl, R0:Et) and 6nd (R:3-NO2, R0:i-Pr) showed potent
measured at 490 nm. The formula for calculating inhibition of cell
proliferation was as follows: inhibition of cell proliferation rate
(%) = (OD value of the control group - OD value of drug group)/
control OD value ꢀ 100%. This was used to plot an inhibition curve.
inhibition against HepG2 cells with the inhibition rate of 91.2% and
90.8% at 50
mg/mL, respectively. Compounds 6mb (R:3-CH3,
R0:Et),6mf (R:3-Cl, R0:Et),6mg (R:4-Cl, R0:Et) and 6nh (R:3-Br,
R0:i-Pr) showed potent inhibition against MGC-803 cells with the
Table 1
Antitumor activity of target compounds (inhibitory ratio/%).
Comp.
EC109
50 g/mL
HepG2
50 g/mL
MGC-803
m
10
mg/mL
5
mg/mL
m
10
m
g/mL
5
m
g/mL
50
m
g/mL
10
mg/mL
5 mg/mL
6ma
6mb
6mc
6md
6me
6mf
6mg
6mh
6mi
6mj
6mk
6nd
6ne
6nh
6nk
4a
66.3
79.8
64.4
43.3
54.0
81.4
86.8
40.4
76.5
58.2
73.6
45.3
58.5
85.4
51.0
51.4
49.5
29.2
18.8
17.0
32.0
19.3
43.1
26.1
43.7
8.0
27.5
11.5
1.0
66.0
81.6
62.3
72.9
61.1
86.7
91.2
53.8
74.3
70.3
77.8
90.8
58.1
48.7
60.8
57.0
38.7
39.6
37.8
40.7
74.8
33.1
64.8
77.0
33.1
35.4
41.9
12.8
43.3
15.3
43.7
39.7
23.7
12.3
42.4
64.4
40.3
24.3
39.7
48.1
19.5
25.9
29.1
20.9
18.9
55.2
9.0
32.4
31.4
6.6
50.3
91.3
64.6
49.6
59.8
94.4
94.4
46.3
65.4
65.4
75.3
63.6
84.3
93.7
64.5
74.6
35.5
33.3
55.9
28.2
80.7
30.6
63.4
74.8
41.1
29.0
53.1
8.2
20.6
44.7
1.1
16.4
10.8
36.1
37.6
44.8
15.3
23.0
57.7
11.2
5.3
17.2
4.4
32.9
9.5
29.2
ꢁ3.0
41.4
42.2
27.0
0.6
27.8
ꢁ10.4
41.5
25.8
18.2
ꢁ7.5
8.7
30.3
36.7
20.9
7.6
28.2
32.8
14.9
4.6
26.6
25.6
ꢁ2.0
ꢁ1.8
11.2
36.7
ꢁ8.9
ꢁ6.9
16.7
17.8
5.6
35.9
34.3
26.0
6.9
19.2
62.6
40.1
39.2
39.9
63.3
20.3
28.2
10.5
21.8
20.5
40.5
27.1
57.3
62.5
9.0
16.6
27.7
31.8
41.6
36.9
22.3
22.8
9.4
13.8
41.7
2.9
21.3
36.2
16.7
26.1
26.4
9.0
4b
14.7
17.7
14.9
9.3
4d
4e
2.8
4f
4.6
6.0
4g
24.4
19.7
8.5
5.4
43.1
22.8
17.7
37.3
6.1
ꢁ3.0
11.2
33.2
16.8
ꢁ4.6
4h
7.9
4i
3.4
39.9
66.2
23.8
4j
3.54
ꢁ7.0
0.2
64.7
4k
ꢁ4.5
ꢁ27.5
Results compared with the blank control group.
Please cite this article in press as: Y.-C. Guo, et al., Synthesis and antitumor activity of
a-aminophosphonate derivatives containing
thieno[2,3-d]pyrimidines, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.026

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In summary, two series of novel
a-aminophosphonate deriva-
tives containing thieno[2,3-d]pyrimidines were synthesized and
their antitumor activity in vitro against EC109, HepG2 and MGC-
803 cells has been evaluated. Biological assays revealed that the
substitutions on the phenyl ring influenced the antitumor activity
remarkably, while the substitutes of phosphonate had no apparent
influence on the antitumor activity. Analogs 6mf/6nf (3-Cl) and
6mg/6ng (4-Cl) with the substituents at meta- or para-position
showed better inhibition against all of the tested tumor cells.
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Acknowledgment
This work was supported by the National Natural Sciences
Foundations of China (Nos. 21171149, 21105091). We are grateful
to Pro. Yanbing Zhang’ group for performing in vitro antitumor
activity evaluation.
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Appendix A. Supplementary data
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Chem. 4 (2002) 551–554.
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Please cite this article in press as: Y.-C. Guo, et al., Synthesis and antitumor activity of
a-aminophosphonate derivatives containing
thieno[2,3-d]pyrimidines, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.03.026