Discovery of diethyl 2,5-diaminothiophene-3,4-dicarboxylate derivatives as potent anticancer and antimicrobial agents and screening of anti-diabetic activity: Synthesis and in vitro biological evaluation. Part 1

Article abstract of DOI:10.1016/j.ejmech.2014.07.065

Series of diethyl 2,5-diaminothiophene-3,4-dicarboxylate (DDTD) derivatives: azomethines of DDTD (2a el) have been synthesized and screened for their anticancer, antimicrobial and anti-diabetic activities. The novel synthesized compounds were characterize

Full text of DOI:10.1016/j.ejmech.2014.07.065

European Journal of Medicinal Chemistry 84 (2014) 739e745
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of diethyl 2,5-diaminothiophene-3,4-dicarboxylate
derivatives as potent anticancer and antimicrobial agents and
screening of anti-diabetic activity: Synthesis and in vitro biological
evaluation. Part 1
,
Khurshed Bozorov a b, Hai-Rong Ma a, Jiang-Yu Zhao a, Hai-Qing Zhao a, Hua Chen a,
,
Khayrulla Bobakulov a b, Xue-Lei Xin a, Burkhon Elmuradov b,
,
*
Khusnutdin Shakhidoyatov b, Haji A. Aisa a
a Key Laboratory of Plant Resources and Chemistry in Arid Regions, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences,
South Beijing Road 40-1, Urumqi, Xinjiang 830011, PR China
b Institute of the Chemistry of Plant Substances, Academy of Sciences of Uzbekistan, 77, Mirzo Ulugbek Str., 100170 Tashkent, Uzbekistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of diethyl 2,5-diaminothiophene-3,4-dicarboxylate (DDTD) derivatives: azomethines of DDTD (2a
el) have been synthesized and screened for their anticancer, antimicrobial and anti-diabetic activities.
The novel synthesized compounds were characterized by 1H, 13C NMR, MS and FT-IR analyses. All
compounds were evaluated for their antiproliferative activity against three types of cancer cell line such
as T47D and MCF-7 (human breast cancer), Hela (human cervical cancer) and Ishikawa (human endo-
metrial cancer) lines. The results showed that most compounds exhibited significant antiproliferative
activity against breast cancer cells. The majority of azomethines DDTD influenced strongly against breast
Received 19 May 2014
Received in revised form
18 July 2014
Accepted 19 July 2014
Available online 21 July 2014
Keywords:
Diethyl 2,5-diaminothiophene-3,4-
dicarboxylate
Breast cancer cell line
Anticancer activity
Antimicrobial activity
PTP-1B inhibitor
cancer cells T47D and MCF-7, among them compounds 2b (2.3
(14.9 M), 2j (16.0 M), 2k (7.1 M), 2l (8.6 M) manifest potent anticancer activity against cancer cell
T47D than Doxorubicin (DOX, 15.5 M). Compound 2j has shown potent activity on all three types of
mM), 2c (12.1 mM), 2e (13.2 mM), 2i
m
m
m
m
m
cancer cells concurrently and IC50 values were considerably low in comparison with positive control
DOX. In addition, all compounds were tested for antimicrobial activity against Staphylococcus aureus
ATCC 6538 (Gram positive bacteria), Escherichia coli ATCC 11229 (Gram negative bacteria) and Candida
albicans ATCC 10231 (Fungi) strains and 2j which contains in the ring nitrofurfural fragment, showed the
highest effect on the three species of microbial pathogens simultaneously. Some compounds induced
enzymatic inhibition in a concentration-dependent manner on PTP-1B inhibitor.
© 2014 Published by Elsevier Masson SAS.
1. Introduction
block for the synthesis of thienopyrimidines (TP), we as previously
other chemists, found out that 2-AT is the source the imple-
2-Amino thiophenes (2-AT) are one of the important synthons
in organic chemistry and frequently used in medicinal chemistry in
recent years [1,2]. It should be noted that the substituted 2-AT
demonstrate a promising biological activities including antibacte-
rial [3], antifungal [4], anticancer [5,6], antitumor [7], anti-
inflammatory [8], antioxidant [9] anti-tubercular effects [10] and
were evaluated as potential allosteric modulators of the A1 aden-
osine receptor [11]. However, besides 2-AT being valuable building-
mentation of condensed thiophene and pyrimidine ring. It is of fact,
that the pyrimidine ring is included in composition of DNA, RNA
and other nucleotides. Synthesized TP from 2-AT possess important
biological properties, for example some of TP compound most
potent nonsteroidal 17b-HSD1 inhibitors reported to date-inhibited
the recombinant enzyme [12]. Other TP derivatives were combined
and their biochemical characterization has been evaluated to wild-
type HIV-1 RT, with rationally designed p66/p51 heterodimers
exhibiting high-level sensitivity or resistance [13]. Dai et al.
discovered novel thienopyrimidine-based receptor tyrosine kinase
inhibitors [14]. In addition, TP's are most bioactive heterocyclic
agents in medical chemistry and exert various important biological
* Corresponding author.
E-mail addresses: haji@ms.xjb.ac.cn, aisa@ms.xjb.ac.cn (H.A. Aisa).
http://dx.doi.org/10.1016/j.ejmech.2014.07.065
0223-5234/© 2014 Published by Elsevier Masson SAS.

740
K. Bozorov et al. / European Journal of Medicinal Chemistry 84 (2014) 739e745
and pharmacological properties including anticancer, antimicrobial
and anti-diabetic activities [15e25].
cancer cell TD47 with IC50 11.6 mM. The results showed that com-
pounds 2ael revealed potent anticancer activity against T47D,
MCF-7, and Hela (Fig. 1). The majority of DDTD azomethines
potently inhibited the growth of breast cancer cells T47D and MCF-
In the present work, we studied the synthesis and in vitro
anticancer, antimicrobial and anti-diabetic properties of a new class
of diethyl 2,5-diaminothiophene-3,4-dicarboxylate (DDTD) azo-
methines. It was interesting to find out and clarify the development
pharmacophore properties.
7: compounds 2b (2.3
2j (16.0 M), 2k (7.1
toxicity compared to doxorubicin (15.5
pounds 2a, 2b, 2c, 2h, 2j against breast cancer cell MCF-7 were
6.1 M, 1.3 M, 6.8 M, 5.7 M and 2.4 M accordingly, at this point
IC50 for DOX 6.75 M. When screening on Hela and Ishikawa cancer
cells, all compound showed high IC50 except for compound 2j. IC50
for 2j on Hela and Ishikawa cells was 4.7 M and 7.6 M respec-
m
M), 2c (12.1
m
m
M), 2e (13.2
M) all exerting stronger cyto-
M). IC50 values of com-
mM), 2i (14.9 mM),
m
mM), 2l (8.6
m
m
m
m
m
m
2. Chemistry
m
Chemistry of substituted 2-AT's has earlier started with Gewald
group [26,27]. The synthesis and characterization of DDTD 1 were
only investigated recently [28,29]. Furthermore, the biological ac-
tivities of DDTD 1 and DDTD substituted have not been studied yet.
Therefore, the aim of our current research was to implement syn-
thesis and obtain new bioactive agents of DDTD derivatives,
conducing anticancer, antimicrobial and anti-diabetic properties.
DDTD 1 was prepared as previously described [30]. Azomethines of
DDTD 2ael were prepared according to the procedure shown in
Scheme 1. Azomethines of DDTD 2ael were prepared from DDTD
and appropriate aromatic aldehydes in n-butanol for 40 min at
reflux. It should be noted that the reaction with the aldehydes
contained in the ring electron-withdrawing groups has formed
final products within 15 min with at least yield 65e70%. The
structures of the newly synthesized compounds were appropriately
confirmed by their spectral data. The 1H NMR spectra of the com-
m
m
tively. Additionally, all compounds 2ael displayed significant high
antiproliferative activity and a strong concentration-dependent
effect against breast cancer cell lines T47D, MCF-7 than cervical
cancer cell Hela and endometrial cancer cell Ishikawa.
3.2. In vitro antimicrobial screening
All the compounds 1 and 2ael were evaluated in vitro for their
antimicrobial activities at the Key Laboratory of Plant Resources
and Chemistry in Arid Regions, Xinjiang Technical Institute of
Physics and Chemistry, Chinese Academy of Sciences. Compounds
were tested for antimicrobial activity against Staphylococcus aureus
ATCC 6538 (Gram positive bacteria), Escherichia coli ATCC 11229
(Gram negative bacteria) and Candida albicans ATCC 10231 (Fungi)
strains. The obtained results suggest that the tested compounds
showed average and mild activity in comparison to the reference
drugs Ampicillin and Amphotericin B. Compound 2j which contains
in the ring nitrofurfural fragment, showed the highest effect on the
three species of microbial pathogens simultaneously (Table 1).
pounds 2ael showed
a characteristic singlets at a region
7.77e8.08 ppm accounting for N]CH proton and revealed carbon
signals for N]CH at the range of 137.65e153.86 ppm in 13C NMR
spectra. Moreover, in both 1Н and 13С NMR spectra of compounds
2ael shifted downfield signals of the one ethoxy group. The above
data confirms substitution of amino group protons in molecule of
all synthesized compounds.
3.3. PTP-1B inhibition screening
We also determined the effect of the corresponding compounds
for their in vitro inhibition of the enzyme PTP-1B. Compounds 2b,
2e, 2h, 2j and 2k induced a PTP-1B enzymatic inhibition in a
3. Results and discussion
3.1. In vitro anticancer screening
concentration-dependent manner with IC50 values 6.43
mM,
6.54 M, 7.32 M, 7.12 M and 14.76 M respectively. Other samples
have not shown any effect on PTP-1B inhibition. IC50 values of the
all compounds are outlined on Table 1.
m
m
m
m
The concentration required to inhibit tumor cell proliferation by
50% (IC50) for compounds 1 and 2ael were determined against the
following human cancer cell lines: T47D and MCF-7 (breast), Hela
(cervical), and Ishikawa (endometrial) by MTT ([3-(4,5-
Dimethylthiazol-yl)-diphenyl tetrazoliumbromide]) assay method
(Table 1). The potent anticancer drug doxorubicin (DOX) used as
positive control. The IC50 values of DDTD 1 against human cancer
cell lines T47D, MCF-7, Hela and Ishikawa is 11.6, 18.6, 40.3 and
3.4. Structure activity relationship
It is known that nitro compounds typically have different bio-
logical activity [31e33]. The study of anticancer activity of the
synthesized compounds (1, 2ae2l) displayed similar results. Along
with this, it was found that azomethines e containing electron
46.9 mM respectively. It is noteworthy that, when compared to DOX,
the antiproliferative activity of DDTD was strong against breast
S
S
H2N
H2N
O
NH2
O
N
O
i
ii
Ar
S8
+ NC
O
O
O
O
O
O
O
1
2a-l
2a Ar = C6H5, 2b Ar = 4-NO2C6H4
2c Ar = 4-ClC6H4, 2d Ar = 4-FC6H4
2e Ar = 4-BrC6H4, 2f Ar = 4-OCH3C6H4
2g Ar = 4-CH3C6H4, 2h Ar = 4-N(CH3)2C6H4
2i Ar = furfural, 2j Ar = 5-NO2 furfural
2k Ar = thiophene, 2l Ar = pyridine
Scheme 1. Synthesis of DDTD 1 and DDTD derivatives 2ael. Reagents and conditions: (i) DMF, TEA, 10e15 ꢀC, 72 h; (ii) aromatic aldehydes, n-BuOH, reflux, 40 min.

K. Bozorov et al. / European Journal of Medicinal Chemistry 84 (2014) 739e745
741
Table 1
In vitro comparison of IC50 values cancer cell lines, inhibition zone (mm) and enzyme inhibition activities of the tested compounds 1 and 2ael.
SD,b
m
M)
Inhibition zone
diameter (mm)
Enzyme inhibition IC50
a
Compounds
Structure
IC50
(
values (
mM)
T47Dd
MCF-7e
Helaf
Ishikawag
SAh
ECi
CAj
n.e.k
1
11.6 1.31
18.6 2.98
40.3 1.39
46.9 1.04
10
<5.5
<5.5
n.e.k
2a
30.3 2.70
6.1 0.66
31.2 0.52
37.9 1.76
9
<5.5
8
n.e.k
2b
2c
2.3 0.20
1.3 0.05
14.1 1.86
140.7 23.5
8.5
<5.5
<5.5
<5.5
<5.5
6.43 0.68
12.1 0.94
6.8 0.13
25.2 2.18
27.1 1.68
<5.5
n.e.k
2d
2e
22.5 1.78
22.5 2.47
30.8 1.78
32.1 0.87
10
<5.5
<5.5
8
n.e.k
13.2 1.03
12.5 1.74
25.9 0.98
46.1 2.25
<5.5
<5.5
6.54 0.61
2f
31.9 1.48
16.2 2.60
188.9 5.87
107.8 5.61
9
<5.5
<5.5
9
8
n.e.k
2g
37.2 2.58
8.9 1.52
41.9 2.72
52.5 2.44
10
n.e.k
2h
2i
16.7 1.00
14.9 1.04
16.0 1.41
5.7 0.15
62.2 5.53
2.4 0.07
131.1 1.41
46.7 0.70
4.7 0.10
73.0 2.82
69.3 2.08
7.6 0.10
11.5
10.5
22
<5.5
<5.5
16
8
7.32 0.59
<5.5
n.e.k
2j
>22
7.12 0.52
(continued on next page)

742
K. Bozorov et al. / European Journal of Medicinal Chemistry 84 (2014) 739e745
Table 1 (continued )
SD,b
m
M)
Inhibition zone
diameter (mm)
Enzyme inhibition IC50
a
Compounds
Structure
IC50
(
values (
mM)
T47Dd
MCF-7e
Helaf
Ishikawag
SAh
ECi
CAj
n.e.k
2k
2l
7.1 0.42
26.1 1.16
21.3 1.42
39.8 1.33
10
<5.5
<5.5
14.76 1.21
8.6 0.60
92.2 1.70
35.4 2.01
119 4.75
<5.5
<5.5
7
n.e.k
DOXc
Ampicillin
Amphotericin B
PTP1Bl
e
e
e
e
15.53 1.21
6.75 0.87
7.04 0.45
9.02 1.02
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
19
e
14
e
e
15
e
e
e
e
1.97 0.54
a
IC50 e concentration required to inhibit tumor cell proliferation by 50%.
SD e standard deviation when n ¼ 3.
DOX e Doxorubicin were used as positive control.
T47D e breast cancer.
MCF-7 e breast cancer.
Hela e cervical cancer.
b
c
d
e
f
g
h
i
Ishikawa e endometrial cancer.
SA e Staphylococcus aureus (ATCC 6538) bacteria strain.
EC e Escherichia coli (ATCC 11229) bacteria strain.
CA e Candida albicans (ATCC 10231) fungi strain.
n.e. e no effect.
j
k
l
PTP1B e Enzyme inhibitor.
withdrawing groups (EWG) or fragments: phenyl (2a), pyridine (2l)
rings with different halogens (2ce2e) or nitro group (2b) in the p-
position of the benzene ring show comparatively higher activity
than compounds containing the electron donating groups (EDG) e
methoxy (2f) or methyl (2g) groups. Similar results were observed
in the biological activity of Schiff bases, consisting of furyl (2i) and
nitrofuryl (2j) fragments. Since nitrofurfurals and similar com-
pounds have excellent antibacterial activity [33,34]. It should be
emphasized that of the synthesized compounds which showed the
highest activity were 4-nitrophenyl e (2b) and 5 nitrofuryl e (2f)
derivatives. From Table 1, we can notice two important factors.
Compound 2b wherein the EWG NO2 at the 4-position of the
benzene ring, clearly has a strong effect against breast cancer cells
T47D and MSF-7. However, cervical cancer Hela and endometrial
cancer Ishikawa cell lines were not as sensitive. Such a selective
effect of samples is promising for further anticancer researches.
Compound 2j, which has the EWG NO2 at the 5-position of the
furfural ring has shown potent activity on all four three of cancer
cells concurrently and IC50 values were considerably low in com-
parison with the positive control DOX (Fig. 1 and Table 1). These
promising results build up a strong baseline for further targeted
research in this field.
A
140
120
100
80
60
40
20
0
1
2a 2b 2c 2e 2h
2i
2j 2k
2l DOX
Compounds
B
4. Conclusions
We have synthesized a series of novel DDTD derivatives con-
taining azomethine functional group in 2-position of thiophene
ring. All compounds 1,2ael have been investigated for their in vitro
anticancer, antimicrobial and anti-diabetic activities against human
breast, human cervical, human endometrial cancer cell lines, as
well as on S. aureus, E. coli and C. albicans types of microorganisms.
Furthermore, they inhibited to enzyme PTP-1B. The investigation of
these activities revealed that all the synthesized compounds
Fig. 1. Diagrammatic comparison of the IC50 values of tested compounds 1, 2a, 2b, 2c,
2e, 2h, 2i, 2j, 2k, 2l with Doxorubicin (DOX) against human breast T47D, MCF-7 and
human cervical Hela cancer cell lines (A) and IC50 values of potent active compounds
2b and 2j with DOX (B).

K. Bozorov et al. / European Journal of Medicinal Chemistry 84 (2014) 739e745
743
showed moderate to high inhibitory characteristics. Among all the
13 compounds screened, compound 2j showed potent anticancer
activity on cancer cell lines as T47D, MCF-7, Hela, and Ishikawa, also
the antibacterial and antifungal results of this compound displayed
strong activity against three species of microbial pathogens
simultaneously. Besides showing the highest cytotoxic effect,
compounds 2b and 2j showed average effect against PTP-1B. In
summary, based on these results, we believe on the probability
further purposeful synthesis in the field of AT derivatives as potent
candidate-drugs.
167e169 ꢀC; IR (KBr, cmꢁ1
)
y
: 3399, 3304 (NH2), 1688 (C]O), 1597
(C]N); 1H NMR (CDCl3, 400 MHz,
d
ppm): 8.25 (d, J ¼ 8.0 Hz, 2H,
AreH), 7.98 (s,1H, N]CH), 7.87 (d, J ¼ 8.0 Hz, 2H, AreH), 6.50 (s, 2H,
NH2), 4.44 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 4.27 (q, J ¼ 7.2 Hz, 2H,
OCH2CH3), 1.44 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 1.33 (t, J ¼ 7.2 Hz, 3H,
OCH2CH3); 13C NMR (CDCl3, 100 MHz,
d ppm): 165.23 (C]O),
164.39 (C]O), 160.88 (NH2CS), 149.08 (N]CH), 148.76 (C), 141.50
(C), 133.03 (C), 132.90 (C), 128.81 (2CH), 124.12 (2CH), 103.70 (C),
61.89 (CH2), 60.66 (CH2), 14.52 (CH3), 14.33 (CH3). MS (ESI): m/z 392
[MþH]þ.
5. Experimental
5.1.3.3. Diethyl
amino]thiophene-3,4-dicarboxylate (2c). Yield: 84%; green color
solid; m.p: 138e140 ꢀC; IR (KBr, cmꢁ1
: 3418, 3304 (NH2), 1673
(C]O), 1594 (C]N); 1H NMR (CDCl3, 400 MHz,
ppm): 7.90 (s, 1H,
2-amino-5-[(E)-[(4-chlorophenyl)methylidene]
5.1. Chemistry
) y
d
5.1.1. General methods
N]CH), 7.66 (d, J ¼ 8.4 Hz, 2H, AreH), 7.36 (d, J ¼ 8.4 Hz, 2H, AreH),
6.39 (br.s, 2H, NH2), 4.42 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 4.25 (q,
J ¼ 7.2 Hz, 2H, OCH2CH3), 1.42 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 1.31 (t,
Melting points were determined using a BUCHI Melting Point B-
540 apparatus (Germany) and were uncorrected. IR spectra were
recorded in KBr on a Shimadzu FT-IR 8400S spectrometer. The NMR
spectra were recorded on a Varian 400-MR spectrometer (400 MHz
for 1H and 100 MHz for 13C) using CDCl3 as a solvent and TMS as an
internal standard in 1H NMR spectra. In 13C NMR spectra chemical
shifts were internally referenced to the solvent signals (CDCl3
77.16 ppm related to TMS). Mass spectra were acquired on QSTAR
Elite LC-MS/MS system (Applied Biosystems/MDS Sciex Concord,
ON Canada) spectrometers.
J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR (CDCl3, 100 MHz,
d ppm): 165.59
(C]O), 164.52 (C]O), 160.02 (NH2CS), 151.33 (N]CH), 136.91 (C),
134.41 (C), 133.99 (C), 130.40 (C), 129.61 (2CH), 129.10 (2CH), 103.23
(C), 61.71 (CH2), 60.47 (CH2), 14.50 (CH3), 14.34 (CH3). MS (ESI): m/z
381 [MþH]þ.
5.1.3.4. Diethyl
amino]thiophene-3,4-dicarboxylate (2d). Yield: 90%; yellow color
solid; m.p: 116e118 ꢀC; IR (KBr, cmꢁ1
: 3457, 3324 (NH2), 1672
(C]O), 1601 (C]N); 1H NMR (CDCl3, 400 MHz,
ppm): 7.92 (s, 1H,
2-amino-5-[(E)-[(4-fluorophenyl)methylidene]
)
y
5.1.2. Synthesis of diethyl 2,5-diaminothiophene-3,4-dicarboxylate
(1)
d
N]CH), 7.73 (m, 2H, AreH), 7.08 (m, 2H, AreH), 6.39 (br.s, 2H,
NH2), 4.42 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 4.25 (q, J ¼ 7.2 Hz, 2H,
OCH2CH3), 1.43 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 1.31 (t, J ¼ 7.2 Hz, 3H,
Compound 1 was synthesized as previously stated [30] but with
minor changes. A mixture of elemental sulfur 5 g (156 mmol),
ethylcyanoacetate (35.6 g, 311.5 mmol), DMF (25 mL) and trie-
thylamine (TEA) (5 mL, 35.5 mmol) was stirred 3 days at 10e15 ꢀC.
The obtained mixture was added to 3 L cool water and stirred over
4 h. Further precipitate was filtered, dried and gave as a yellow solid
(12.9 g, 32%) which recrystallization from a solution of ethylacetate/
OCH2CH3); 13C NMR (CDCl3, 100 MHz,
d ppm): 165.77 (C]O),
164.54 (C]O), 163.26 (C), 159.81 (NH2CS), 151.52 (N]CH), 134.16
(C), 132.24 (C), 130.44 (2CH), 129.91 (C), 115.99 (2CH), 103.16 (C),
61.67 (CH2), 60.43 (CH2), 14.50 (CH3), 14.34 (CH3). MS (ESI): m/z 365
[MþH]þ.
heptane, m.p: 153e155 ꢀC; IR (KBr, cmꢁ1
) y: 3451, 3470 (NH2), 1674
(C]O); 1H NMR (CDCl3, 400 MHz,
d
ppm): 5.27 (br.s, 4H, NH2), 4.24
5.1.3.5. Diethyl
amino]thiophene-3,4-dicarboxylate (2e). Yield: 79%; yellow color
solid; m.p: 151e153 ꢀC; IR (KBr, cmꢁ1
: 3412, 3298 (NH2), 1669
(C]O), 1593 (C]N); 1H NMR (CDCl3, 400 MHz,
ppm): 7.88 (s, 1H,
2-amino-5-[(E)-[(4-bromophenyl)methylidene]
(q, J ¼ 7.2 Hz, 4H, OCH2CH3), 1.30 (t, J ¼ 7.2 Hz, 6H, OCH2CH3); 13C
NMR (CDCl3, 100 MHz,
d
ppm): 165.30 (C]O), 148.68 (SCNH2),
) y
104.15 (CCOO), 60.16 (CH2), 14.48 (CH3). MS (ESI): m/z 259 [MþH]þ.
d
N]CH), 7.59 (d, J ¼ 8.4 Hz, 2H, AreH), 7.53 (d, J ¼ 8.4 Hz, 2H, AreH),
6.39 (br.s, 2H, NH2), 4.42 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 4.25 (q,
J ¼ 7.2 Hz, 2H, OCH2CH3), 1.42 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 1.31 (t,
5.1.3. General procedure for synthesis azomethines of diethyl 2,5-
diaminothiophene-3,4-dicarboxylate derivatives (2ael)
Corresponding equimolar quantity of 3,4-diethyl 2,5-
diaminothiophene-3,4-dicarboxylate and aromatic aldehydes
were dissolved in n-butanol and refluxed for 40 min. After cooling,
the solution was evaporated at low pressure, and residue recrys-
tallized by appropriate solvent (ethanol) followed and obtained a
solid 2ael with high yields.
J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR (CDCl3, 100 MHz,
d ppm): 165.56
(C]O), 164.51 (C]O), 160.04 (NH2CS), 151.39 (N]CH), 134.82 (C),
133.96 (C), 132.06 (2CH), 130.50 (C), 129.80 (2CH), 125.41 (C), 103.27
(C), 61.70 (CH2), 60.47 (CH2), 14.50 (CH3), 14.34 (CH3). MS (ESI): m/z
426 [MþH]þ.
5.1.3.6. Diethyl
amino]thiophene-3,4-dicarboxylate (2f). Yield: 77%; brown color
solid; m.p: 138e140 ꢀC; IR (KBr, cmꢁ1
: 3407, 3306 (NH2), 1674
(C]O), 1602 (C]N); 1H NMR (CDCl3, 400 MHz,
ppm): 7.91 (s, 1H,
2-amino-5-[(E)-[(4-methoxyphenyl)methylidene]
5.1.3.1. Diethyl
phene-3,4-dicarboxylate (2a). Yield: 79%; yellow color solid; m.p:
126e128 ꢀC; IR (KBr, cmꢁ1
: 3409, 3314 (NH2), 1677 (C]O), 1598
(C]N); 1H NMR (CDCl3, 400 MHz,
ppm): 7.97 (s, 1H, N]CH), 7.74
2-amino-5-[(E)-(phenylmethylidene)amino]thio-
)
y
)
y
d
d
N]CH), 7.69 (d, J ¼ 8.8 Hz, 2H, AreH), 6.91 (d, J ¼ 8.8 Hz, 2H, AreH),
6.32 (br.s, 2H, NH2), 4.41 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 4.24 (q,
J ¼ 7.2 Hz, 2H, OCH2CH3), 3.84 (s, 3H, OCH3), 1.43 (t, J ¼ 7.2 Hz, 3H,
OCH2CH3), 1.31 (t, J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR (CDCl3,
(m, 2H, AreH), 7.40 (m, 3H, AreH), 6.35 (br.s, 2H, NH2), 4.42 (q,
J ¼ 7.2 Hz, 2H, OCH2CH3), 4.25 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 1.43 (t,
J ¼ 7.2 Hz, 3H, OCH2CH3), 1.31 (t, J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR
(CDCl3, 100 MHz,
d
ppm): 165.67 (C]O), 164.58 (C]O), 159.86
100 MHz, d ppm): 165.83 (C]O), 164.61 (C]O), 162.11 (C), 159.41
(NH2CS), 153.02 (N]CH), 135.90 (2C), 134.44 (C), 131.04 (CH),
128.77 (2CH), 128.59 (2CH), 103.15 (C), 61.65 (CH2), 60.41 (CH2),
14.51 (CH3), 14.34 (CH3). MS (ESI): m/z 347 [MþH]þ.
(NH2CS), 152.80 (N]CH), 135.09 (C), 130.31 (2CH), 128.91 (C),
128.53 (C), 114.28 (2CH), 103.03 (C), 61.57 (CH2), 60.33 (CH2), 55.52
(OCH3), 14.50 (CH3), 14.34 (CH3). MS (ESI): m/z 377 [MþH]þ.
5.1.3.2. Diethyl 2-amino-5-[(E)-[(4-nitrophenyl)methylidene]amino]
5.1.3.7. Diethyl
2-amino-5-[(E)-[(4-methylphenyl)methylidene]
thiophene-3,4-dicarboxylate (2b). Yield: 94%; red color solid; m.p:
amino]thiophene-3,4-dicarboxylate (2g). Yield: 74%; green color

744
K. Bozorov et al. / European Journal of Medicinal Chemistry 84 (2014) 739e745
solid; m.p: 134e136 ꢀC; IR (KBr, cmꢁ1
)
y
: 3404, 3306 (NH2), 1675
ppm): 7.95 (s, 1H,
5.1.3.12. Diethyl
thiophene-3,4-dicarboxylate (2l). Yield: 92%; orange color solid;
m.p: 146e148 ꢀC; IR (KBr, cmꢁ1
: 3418, 3307 (NH2), 1677 (C]O),
1585 (C]N); 1H NMR (CDCl3, 400 MHz,
ppm): 8.66 (d, J ¼ 5.6 Hz,
2-amino-5-[(E)-(pyridin-4-ylmethylidene)amino]
(C]O), 1596 (C]N); 1H NMR (CDCl3, 400 MHz,
d
N]CH), 7.64 (d, J ¼ 8.0 Hz, 2H, AreH), 7.20 (d, J ¼ 8.0 Hz, 2H, AreH),
6.32 (br.s, 2H, NH2), 4.42 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 4.25 (q,
J ¼ 7.2 Hz, 2H, OCH2CH3), 2.38 (s, 3H, AreCH3), 1.43 (t, J ¼ 7.2 Hz, 3H,
OCH2CH3), 1.31 (t, J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR (CDCl3,
) y
d
2H, AreH), 7.87 (s, 1H, N]CH), 7.57 (d, J ¼ 5.6 Hz, 2H, AreH), 6.65
(br.s, 2H, NH2), 4.43 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 4.27 (q, J ¼ 7.2 Hz,
2H, OCH2CH3), 1.44 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 1.32 (t, J ¼ 7.2 Hz,
100 MHz,
d ppm): 165.73 (C]O), 164.60 (C]O), 159.64 (NH2CS),
153.20 (N]CH), 141.59 (C), 134.80 (C), 133.33 (C), 129.55 (2CH),
128.61 (2CH), 103.11 (C), 61.60 (CH2), 60.37 (CH2), 21.77 (AreCH3),
14.51 (CH3), 14.34 (CH3). MS (ESI): m/z 361 [MþH]þ.
3H, OCH2CH3); 13C NMR (CDCl3, 100 MHz,
d ppm): 165.27 (C]O),
164.41 (C]O),161.09 (NH2CS),150.37 (2CH),149.31 (N]CH),142.71
(C), 132.86 (C), 132.77 (C), 121.81 (2CH), 103.43 (C), 61.84 (CH2),
60.59 (CH2), 14.50 (CH3), 14.32 (CH3). MS (ESI): m/z 348 [MþH]þ.
5.1.3.8. Diethyl 2-amino-5-[(E)-{[4-(dimethylamino)phenyl]methyl-
5.2. Biology
idene}amino] thiophene-3,4-dicarboxylate (2h). Yield: 72%; brown
color solid; m.p: 164e166 ꢀC; IR (KBr, cmꢁ1
)
y
: 3437, 3330 (NH2),
Doxorubicin and trypsin were purchased from BBI Inc.
(Shanghai, China), Ampicillin from Sigma Chemicals Co., Ampho-
tericin B. from AMRESCO LLC, PTP1B Inhibitor from Merck spe-
cialties private limited. The human cancer cell lines breast (T47D,
MCF-7), cervical (Hela), endometrial (Ishikawa) were obtained
from Chinese Type Culture Collection, CAS (Shanghai, China).
1662 (C]O), 1587 (C]N); 1H NMR (CDCl3, 400 MHz,
d
ppm): 7.87
(s, 1H, N]CH), 7.63 (d, J ¼ 8.4 Hz, 2H, AreH), 6.68 (d, J ¼ 8.4 Hz, 2H,
AreH), 6.24 (br.s, 2H, NH2), 4.40 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 4.24
(q, J ¼ 7.2 Hz, 2H, OCH2CH3), 3.03 (s, 6H, N-(CH3)2),1.42 (t, J ¼ 7.2 Hz,
3H, OCH2CH3), 1.30 (t, J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR (CDCl3,
100 MHz,
d ppm): 166.03 (C]O), 164.70 (C]O), 158.84 (NH2CS),
153.80 (N]CH), 152.26 (C), 136.30 (C), 130.39 (2CH), 126.68 (C),
124.16 (C), 111.79 (2CH), 102.97 (C), 61.43 (CH2), 60.22 (CH2), 40.34
(N-(CH3)2), 14.52 (CH3), 14.35 (CH3). MS (ESI): m/z 390 [MþH]þ.
5.2.1. MTT assay
All compounds were dissolved in DMSO in a stock concentration
of 10 mM. 200 mL of the breast cells T47D and MCF-7, the cervical
cancer cells Hela, and the endometrial cancer cells Ishikawa all at
the logarithmic phase, were separately seeded in 96-well plates at
the density of 3e5 ꢂ 103 cells/well. The cells were grown for 24 h in
a humidified incubator (Binder, Germany) at 37 ꢀC with 95% hu-
midity and 5% CO2. Thereafter, the cells were treated with 1, 5, 10,
5.1.3.9. Diethyl 2-amino-5-[(E)-(furan-2-ylmethylidene)amino]thio-
phene-3,4-dicarboxylate ꢁ(21i). Yield: 81%; yellow color solid; m.p:
150e151 ꢀC; IR (KBr, cm
) y: 3402, 3302 (NH2), 1670 (C]O), 1593
(C]N); 1H NMR (CDCl3, 400 MHz,
d ppm): 7.82 (s, 1H, N]CH), 7.54
(d, J ¼ 1.6 Hz, 1H, furaneH), 6.87 (d, J ¼ 3.6 Hz, 1H, furaneH), 6.50
(dd, J ¼ 3.6; 1.6 Hz, 1H, furaneH), 6.33 (br.s, 2H, NH2), 4.40 (q,
J ¼ 7.2 Hz, 2H, OCH2CH3), 4.25 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 1.41 (t,
J ¼ 7.2 Hz, 3H, OCH2CH3), 1.31 (t, J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR
15, 20, 25 and 30 mM of compounds (1 and 2) for 48 h. 20 mL MTT
(5 mg/mL) was then added to each well and the plates were incu-
bated at 37 ꢀC. Four hours later, the supernatant was removed and
200 mL DMSO was added to each well and the multiwell plates were
(CDCl3, 100 MHz,
d
ppm): 165.50 (C]O), 164.56 (C]O), 159.56
shaken for 10 min to dissolve the crystallines. Absorbance was read
at a wavelength of 540 nm using an enzyme-linked immunosor-
bent assay reader (SpectraMax M5, Molecular Devices, U.S.). The
IC50 values were calculated with the inhibition rate. Inhibition
rate ¼ (OD value of control group ꢁ experiment group)/(OD value
of control group ꢁ OD value of blank group).
(NH2CS), 152.18 (C), 145.54 (N]CH), 141.38 (CH), 129.17 (C), 128.36
(C), 114.63 (CH), 112.55 (CH), 103.42 (C), 61.69 (CH2), 60.42 (CH2),
14.40 (CH3), 14.34 (CH3). MS (ESI): m/z 337 [MþH]þ.
5.1.3.10. Diethyl
amino]thiophene-3,4-dicarboxylate (2j). Yield: 83%; dark red color
solid; m.p: 162e164 ꢀC; IR (KBr, cmꢁ1
: 3443, 3292 (NH2), 1678
(C]O), 1597 (C]N); 1H NMR (CDCl3, 400 MHz,
ppm): 7.77 (s, 1H,
2-amino-5-[(E)-[(5-nitrofuran-2-yl)methylidene]
5.2.2. Antimicrobial inhibition of compounds 1,2ael
)
y
Antimicrobial activity of compounds 1,2ael was measured using
the agar well diffusion method. Bacterial and fungal pathogens
such as S. aureus (ATCC 6538), E. coli (ATCC 11229) and C. albicans
(ATCC 10231) were used as indicator strains for this analysis. These
microorganisms were aseptically inoculated into appropriate liquid
media and incubated at 37 ꢀC. After 16 h, the cells were centrifuged
at 6000 rpm for 10 min and then suspended in sterile water. The
different cells (1 mL) were added to appropriate agar media
(100 mL) prior to plating, and the wells were made using an agar
well borer. To these wells, different concentrations of 1 and 2ael
were added and subsequently incubated at 37 ꢀC for 24 h. Zone of
inhibitions were estimated by measuring the diameter of the mi-
crobial growth inhibition zone. Values were averaged from three
independent experiments.
d
N]CH), 7.38 (d, J ¼ 3.6 Hz, 1H, furaneH), 7.00 (d, J ¼ 3.6 Hz, 1H,
furaneH), 6.63 (br.s, 2H, NH2), 4.42 (q, J ¼ 7.2 Hz, 2H, OCH2CH3),
4.26 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 1.43 (t, J ¼ 7.2 Hz, 3H, OCH2CH3),
1.31 (t, J ¼ 7.2 Hz, 3H, OCH2CH3); 13C NMR (CDCl3, 100 MHz,
d ppm):
164.98 (C]O), 164.27 (C]O), 161.42 (NH2CS), 153.87 (C), 152.36 (C),
137.65 (N]CH), 134.04 (C), 132.63 (C), 113.68 (CH), 113.30 (CH),
103.97 (C), 61.99 (CH2), 60.74 (CH2), 14.45 (CH3), 14.29 (CH3). MS
(ESI): m/z 382 [MþH]þ.
5.1.3.11. Diethyl 2-amino-5-[(E)-(thiophen-2-ylmethylidene)amino]
thiophene-3,4-dicarboxylate (ꢁ21k). Yield: 80%; yellow color solid;
m.p: 115e117 ꢀC; IR (KBr, cm
) y: 3404, 3308 (NH2), 1676 (C]O),
1590 (C]N); 115e117 ꢀC; 1H NMR (CDCl3, 400 MHz,
d ppm): 8.08 (s,
1H, N]CH), 7.43 (d, J ¼ 5.2 Hz, 1H, thiopheneH), 7.34 (d, J ¼ 3.2 Hz,
1H, thiopheneH), 7.06 (dd, J ¼ 5.2; 3.2 Hz, 1H, thiopheneH), 6.34
(br.s, 2H, NH2), 4.41 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 4.24 (q, J ¼ 7.2 Hz,
2H, OCH2CH3), 1.44 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 1.30 (t, J ¼ 7.2 Hz,
5.2.3. Enzymatic assay and IC50 determination
All 13 compounds were tested for the measurement of PTP-1B
activity using pNPP (p-nitrophenyl phosphate disodium salt) as
substrate. Compounds were pre-incubated with the enzyme at
room temperature for 5 min. Buffer (20 mM Hepes, 150 mM NaCl,
3H, OCH2CH3); 13C NMR (CDCl3, 100 MHz,
d ppm): 165.50 (C]O),
164.53 (C]O), 159.54 (NH2CS), 146.05 (N]CH), 142.72 (C), 134.05
(C), 131.18 (CH), 130.31 (CH), 129.44 (C), 127.90 (CH), 103.18 (C),
61.64 (CH2), 60.40 (CH2), 14.58 (CH3), 14.28 (CH3). MS (ESI): m/z 353
[MþH]þ.
1 mM EDTA) at a volume of 178 mL was added in 96-well plates. 1 mL
of PTP-1B protein solution (0.115 mg/mL) was added subsequently.
After that, the test and positive control sample were added at a
volume of 1 mL. Thereafter 20 mL of the substrate pNPP (35 mM) was

K. Bozorov et al. / European Journal of Medicinal Chemistry 84 (2014) 739e745
745
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added and mixed for 10 min. The plate was incubated in the dark
for 30 min and the reaction was terminated by adding 10 L (3 M)
m
NaOH. Then, the absorbance was determined at 405 nm wave-
length. The system does not contain the enzyme solution in a blank,
using a micro plate reader SpectraMax MD5 (USA Molecular De-
vices) absorption was measured at 405 nm. Inhibition
rate ¼ (OD405 ꢁ OD405 blank sample)/OD405 blank ꢂ 100% (IC50
calculation software).
Acknowledgments
This work was financially supported by the Funds for Interna-
tional Cooperation and Exchange of the National Natural Science
Foundation of China (No. 31110103908), the CAS Postdoctoral
Fellowship for Dr. Khurshed Bozorov (No. 2013FFGB0002) and the
Central Asia Drug Research and Development Center of Chinese
Academy of Sciences (No. CAM 2013-01)
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Appendix A. Supplementary data
[19] S.E. Abbas, N.M.A. Gawad, R.F. George, Y.A. Akar, Eur. J. Med. Chem. 65 (2013)
195e204.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.065. These data include MOL
files and InChiKeys of the most important compounds described in
this article.
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