Synthesis and: In vitro biological evaluation of novel diaminothiophene scaffolds as antitumor and anti-influenza virus agents. Part 2

Article abstract of DOI:10.1039/c7ra04808d

On the basis of high-throughput screening, fragment-based drug discovery, structure-activity relationships and building block analysis methods, herein we report the synthesis and biological evaluation of a novel series of diethyl 2,5-diaminothiophene-3,4-dicarboxylate derivatives. All of the prepared Schiff bases (with mono-, di- and poly-substituents at the aromatic portion), mono- and bis-amides of diethyl 2,5-diaminothiophene-3,4-dicarboxylate, were evaluated against various human cancer and non-cancerous (only for active compounds) cell lines, as well as influenza A (subtypes FM/1/47/H1N1, hanfang/359/95/H3N2) and B (subtype jifang/13/97) viruses. The obtained results suggest that some selected compounds revealed promising antitumor and antiviral activities and may serve as lead compounds for further drug discovery and development.

Full text of DOI:10.1039/c7ra04808d

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PAPER
Synthesis and in vitro biological evaluation of novel
diaminothiophene scaffolds as antitumor and anti-
influenza virus agents. Part 2†
Cite this: RSC Adv., 2017, 7, 31417
ab
Khurshed Bozorov,
Jiang yu Zhao,a Li Fei Nie,ac Hai-Rong Ma,a
Khayrulla Bobakulov,ab Rui Hu,ac Nigora Rustamova,ac Guozheng Huang,
a
Thomas Efferthd and Haji A. Aisa
a
*
On the basis of high-throughput screening, fragment-based drug discovery, structure–activity relationships
and building block analysis methods, herein we report the synthesis and biological evaluation of a novel
series of diethyl 2,5-diaminothiophene-3,4-dicarboxylate derivatives. All of the prepared Schiff bases
(with mono-, di- and poly-substituents at the aromatic portion), mono- and bis-amides of diethyl 2,5-
diaminothiophene-3,4-dicarboxylate, were evaluated against various human cancer and non-cancerous
(only for active compounds) cell lines, as well as influenza A (subtypes FM/1/47/H1N1, hanfang/359/95/
H3N2) and B (subtype jifang/13/97) viruses. The obtained results suggest that some selected compounds
revealed promising antitumor and antiviral activities and may serve as lead compounds for further drug
discovery and development.
Received 28th April 2017
Accepted 7th June 2017
DOI: 10.1039/c7ra04808d
rsc.li/rsc-advances
cancerous cells, however wide chemical modication of the
synthetic small molecules, as well as, their easy availability
Introduction
attracts more attention from pharmacists. In this way, small
molecules are priority candidates for treatment of diseases in
pharmacy. Among the prospectively using small molecule
family in medicinal chemistry is 2-aminothiophene (2-ATs)
scaffolds. Aside from their diverse biological properties,12–15
these classes of building blocks were reported as potential
anticancer16–18 and antiviral agents.19–21 Besides, cyclic 2-AT
derivatives, such as thieno[2,3-d]pyrimidines (TPs) also provide
anticancer properties,22 as well as, act as PI3K,23 FGFR1 24 and
EGFR25,26 inhibitors, indicating many more perspectives of 2-AT
libraries as initially estimated.
On the basis of these facts, we selected diethyl 2,5-
diaminothiophene-3,4-dicarboxylate (DDTD) building block for
the present investigation containing symmetrical NH2 at C-2
and C-5 and ester groups at C-3 and C-4 positions. Recently,
we described Schiff bases of DDTD with only 4-substituents at
the benzene ring (Chart 1) as potential anticancer and antimi-
crobial compounds and discussed the role of various substitu-
ents to DDTD under the aspect of structure activity
relationships (SARs).27 Among them, some promising nitro-
aromatic compounds were discovered which need further
modications. On the other hand, although nitroaromatic
drugs (Fig. 1) possess hepatotoxicity effects, latest reports have
asserted that the ability SARs to mechanism of actions for drugs
related nitro group indicates importance and perspectives to
continue this area in medicinal chemistry.28–32
Advances in high-throughput screening (HTS), fragment-based
drug discovery and building block analysis methods are
powerful tools for the discovery and optimization of new drug
leads in medicinal chemistry.1–4 Of particular importance are
building blocks, because the drug likeness of a compound can
be characterized by its building blocks.5 Likewise, this approach
represents a rapid way to prepare drug candidates in medicinal
chemistry.6 On the other hand, it is worth designing effective
small molecules with low IC50 values at this rst step of inves-
tigations. BBs containing various pharmacophore ring systems,
linkers or fragments,7 play a key role for HTS-based drug
discovery.8–10
Discoveries of new anticancer drug candidates are still of
tremendous challenging due to the complexity of cancer biology
and also of great emergency, since cancer is still the leading
cause of human death in all over the world.11 Natural products
remain a good arsenal for providing new entities to treat
aKey Laboratory of Plant Resources and Chemistry in Arid Regions, Xinjiang Technical
Institute of Physics and Chemistry, Chinese Academy of Sciences, 40-1 South Beijing
Rd, Urumqi, Xinjiang, 830011, PR China. E-mail: haji@ms.xjb.ac.cn
bInstitute of the Chemistry of Plant Substances, Academy of Sciences of Uzbekistan, 77,
Mirzo Ulugbek str., 100170, Tashkent, Uzbekistan
cUniversity of Chinese Academy of Sciences, 19 A Yuquan Rd, Beijing, 100049, PR
China
dDepartment of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry,
University of Mainz, 555128, Mainz, Germany
Thus, continuing our previous research on DDTD, we now
investigated novel synthesized Schiff base (mainly were selected
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra04808d
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Scheme
1
Synthesis of DDTD Schiff bases 3a–p. Reagents and
conditions: (a) aromatic aldehydes, toluene, reflux 30 min.
Chart 1
heated at reux in toluene for half an hour, and then evaporated
in vacuo. Recrystallization of the residue from ethanol provided
the target bases in excellent yield (79–94%).
If DDTD interacted with different aromatic acid chlorides
under basic conditions (methods a and b), only symmetrical
bis-amides of DDTD (compounds 4a–d) were obtained as nal
products. Particularly, this was observed with aromatic acid
chlorides related to methyl group at position C-3 (4a), tri-
uoromethyl in positions C-3 and C-5 (4b) as well as uoro
group in positions C-3, C-4, and C-5 (4c) in the benzene portion
of aromatic system. Because of planar structure of the
compounds 4a–d, it was found that by the enthalpy the optimal
conguration is Z forms for these compounds. Mono-amides of
DDTD (5a and 5c) were obtained in only two cases. It was also
possible to yield mono-(5b) and symmetrical bis-(4d) amides of
DDTD with 3,4,5-trimethoxybenzoyl chloride. For compound
5b, the product yield was 53% using method A. Applying
method B resulted in the symmetrical bis product (4d) in
excellent yield (92%) (Scheme 2).
Fig. 1 Biologically relevant nitroaromatic compounds.
aldehydes with di- and poly-substituents at the aromatic
portion), mono- and bis-amides of DDTD for their anti-
proliferative properties against cell lines of tumoral and normal
(here selected most active samples) types, and inuenza A and B
viruses. Additionally, we also investigated the effects of
compound 3j, 3n and 3p on cell apoptosis compared with
doxorubicin (DOX) which was used as reference drug in these
class derivatives.33,34
Biological evaluation
In vitro antiproliferative activity and SARs. All synthesized
novel samples (3a–p, 4a–d and 5a–c) were evaluated for their
antiproliferative activity towards four human cancer cell lines,
e.g. PC-3 prostate cancer cells, A549 lung cancer cells, HCT-15
colon cancer cells and T47D breast cancer cells, which are
considered as appropriate and relevant cell models in drug
discovery. The inhibition concentrations required for inhibition
of tumor cell proliferation of 50% (IC50 values) of the synthe-
sized compounds are shown in Table 1. Based on the results
Results and discussion
Chemistry
The target Schiff bases (3a–p) were prepared by a previously re- obtained, we drew our attention to di-substituted groups at the
ported synthetic route27,35 with minor changes (Scheme 1). In the aromatic and heteroaromatic rings (3b–h, 3m and 3o). Likewise,
present study, toluene was used as reaction solvent instead of n- we also prepared for investigation Schiff bases with diverse
butanol, enabling fast and clear workup procedure. The mixture mono-(3a, 3k, 3l and 3n), tri-(3i) and penta-(3j) substituents at
of DDTD and appropriate aryl- and heteroaryl-aldehydes was phenyl/heteryl portions, as well as the previously discovered
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Scheme 2 Synthesis of symmetrical bis-(4a–d) and mono-(5a–c) amides of DDTD. Reagents and conditions: (method a) aromatic acid chlo-
rides, dry benzene or chloroform, TEA, reflux 2–3 h. (Method b) aromatic acid chlorides, pyridine, r.t. 4 h.
Table 1 In vitro comparison of IC50 values human cancer cell lines of
the tested compounds 3a–p, 4a–d and 5a–c
proliferation of tumoral cell lines. Interestingly, the best result
was obtained with derivative 3n, i.e. by inserting 5-nitro-2-
thienyl fragment in DDTD provided excellent behavior against
Prostate
PC-3
Lung
A549
Colon
Breast
T47D
Histotype
Cell lines
all tumoral cell lines, which resulted high antiproliferative
activity. Among the phenyl substituted Schiff base, similar
behavior showed compound 3j, which derived by uorination
all position of phenyl ring. Contemporaneously uorine and
chlorine substitution in aromatic ring provided signicantly
less toxicity dates on cancer cells (3g, towards A549 and T47D)
as compared to tandem substitutions such as di-uorine (3c),
di-triuoro methyl (3d), chlorine/nitro (3e) and uorine/iodine
(3f), while compounds with di-chlorine (3b) and 3-chloro-2-u-
oro-5-(triuoromethyl)-(3i) phenyl fragments did not show
signicant concentration-dependent effects. As shown in Table
1, the majority of compounds were active against T47D, in
particular the compounds which contained furan/thiophene/
pyrazole linkers with various substituents (4-nitrophenyl or
methyl groups) at C-4 or C-5 positions such as 3k–m and 3p. An
interesting behavior was observed in case of derivatives (3c, 3g
and 3h) which containing 3,5-diuoro-, 4-chloro-3-uoro- or 1,4-
benzodioxin-substituents at the phenyl ring, these compounds
showed more effective antiproliferative properties than DOX.
These results conrm about many roles for uorine substitu-
ents in medicinal chemistry,36,37 particularly in cancer chemo-
therapy. One of the known examples is reference drug 5-FU (5-
uorouracil),38,39 which contains uorine in the own molecule.
It was observed that the majority of the selected Schiff bases
showed poor antiproliferative activity (IC50 $ 30 mM), except of
1,4-benzodioxin-(3h), 5-methylfuran-(3l) and 1-methyl-1H-pyr-
azole-(3p) derivatives.
HCT-15
No. Compounds IC50 (ꢀSD, mM)
1
2j
n.d.
22.8 ꢀ 0.6 10.2 ꢀ 0.6
4.6 ꢀ 0.8
n.d.
n.d.
20.6 ꢀ 2.2
51.0 ꢀ 5.2
n.d.
28.8 ꢀ 3.0
17.7 ꢀ 2.1
4.50 ꢀ 0.3
n.d.
6.40 ꢀ 0.4
20.6 ꢀ 2.3
22.9 ꢀ 2.4
23.6 ꢀ 3.2
21.4 ꢀ 2.2
2
3
4
5
6
7
3a
3b
3c
3d
3e
3f
33.3 ꢀ 0.3
n.d.
n.d.
34.0 ꢀ 0.3
n.d.
n.d.
69.8 ꢀ 5.9
65.0 ꢀ 4.8
55.0 ꢀ 0.5
45.4 ꢀ 4.9
42.1 ꢀ 5.4
29.6 ꢀ 0.3
n.d.
12.0 ꢀ 0.7
25.2 ꢀ 2.6
17.3 ꢀ 1.8
20.9 ꢀ 2.6
10.9 ꢀ 1.3
36.1 ꢀ 4.6
15.7 ꢀ 1.7
n.d.
17.9 ꢀ 2.1 40.9 ꢀ 4.8
33.0 ꢀ 3.5 40.2 ꢀ 4.2
n.d.
n.d.
28.0 ꢀ 2.6 57.7 ꢀ 5.8
11.0 ꢀ 1.0 37.3 ꢀ 3.6
60.1 ꢀ 0.9 22.3 ꢀ 0.4
8
9
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
4a
4b
4c
4d
5a
5b
5c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
n.d.
n.d.
4.60 ꢀ 0.7 2.59 ꢀ 0.3
28.3 ꢀ 2.9 31.9 ꢀ 4.1
31.3 ꢀ 3.4 25.9 ꢀ 2.7
16.1 ꢀ 2.0 30.9 ꢀ 3.3
1.30 ꢀ 0.2 6.10 ꢀ 0.3
14.8 ꢀ 1.5 101.6 ꢀ 8.7 n.d.
14.9 ꢀ 1.6 23.3 ꢀ 2.5
66.1 ꢀ 0.6 12.0 ꢀ 0.1
16.8 ꢀ 1.8
n.d.
11.6 ꢀ 0.8
9.38 ꢀ 0.2
n.d.
n.d.
n.d.
12.3 ꢀ 0.1
14.2 ꢀ 0.1
n.d.
62.9 ꢀ 0.3
12.1 ꢀ 0.4
n.d.
6.0 ꢀ 0.6
22.3 ꢀ 0.8
25.5 ꢀ 0.5
71.6 ꢀ 0.6 n.d.
11.5 ꢀ 0.6
n.d.
20.8 ꢀ 1.1
n.d.
128.6 ꢀ 1.4 n.d.
6.93 ꢀ 0.3
67.0 ꢀ 1.1 7.22 ꢀ 0.3
6.50 ꢀ 1.4 7.92 ꢀ 0.3
Doxorubicin 7.40 ꢀ 1.6
In general, Table 1 shows that among the all synthesized
active compound 2j. The set of heteroaromatics related Schiff Schiff bases, only 5-nitro-2-thienyl derivative 3n demonstrated
bases demonstrated more signicant activity results as antiproliferative activity on all tumoral cell lines. Perhaps,
compared to substituted phenyl related derivatives. For herein nitrothienyl group played a key role for increasing
example, introduction of 1-methyl-1H-pyrazole (3p) and 5-nitro- activity. In corresponding reports photochemical and acceptor
2-thienyl (3n) fragments in most cases slowed increasing behavior of the 5-nitro-2-thienyl group were well explained.40,41
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Moreover, nitrothienyl prodrugs may provide effective and that penta-uoro-derivative 3j which was most potent on cancer
selective delivery of the cytotoxic or cytostatic compound to cell lines, did not provide any selectivity.
a solid tumor. It was reported that in oxic liver homogenates,
Effects of 3j, 3n and 3p on human A549 cell cycle distribu-
nitrothienyl related compounds was metabolically stable, as tion and apoptosis induction. As shown in Table 1, the IC50
well as, when incubated in aerobic A549 whole-cell suspensions values of compounds 3j and 3n were signicantly lower than
also was observe stability, which suggest that such substitution other DDTD derivatives. Therefore, it was interesting to study
has also successfully inhibited undesired aerobic metabolism.42 the inuence of these two compounds on A549 cell growth
If bearing in mind that nitrothienyl containing scaffolds has regarding their cell cycle distribution and induction of
been shown to demonstrate modest activity in human xenogra apoptosis in comparison to DOX and compound 3p. If A549
multiple myeloma and B-cell leukemia in vivo models,43 further cells were treated with various concentrations of compounds 3j
developments with these class derivatives seem promising.
(0.1, 0.25, 0.5 mM) and 3p (2.5, 5, 15 mM), the cell cycle distri-
6-Chloronicotinamido-DDTD monoamide (5c) showed high bution did not change (Fig. 2A). However, annexin V/propidium
antiproliferative activity, than DOX towards PC-3, HCT-15, and iodide (PI) assays revealed percentages from 9.2 to 11.4% (3j)
on T47D cancer cell lines, among the synthesized mono-(5a–c) and 4.1 to 9.6% (3p) (Q2 + Q4) at different concentrations (p <
and symmetrical (4a–d) amide derivatives (Table 1), which 0.01) (Fig. 2B and C). The apoptosis rates of DOX ranged from
conrming anticancer drugs properties of discovered that 10.3 to 16.3% (Q2 + Q4) (p < 0.01). In addition, if A549 cells were
nicotinamide containing compositions.44 Moreover, nicotin- treated with compound 3n at low micromolar concentrations
amide is a member of vitamin B family, which has no serious (0.1, 0.25, 0.5 mM), and the induction of apoptosis increased
side effects in human organism.
from 8.4 to 12.6% (Q2 + Q4) (Fig. 2B, C).
Both symmetrical and mono-DDTD amides 4d and 5b, which
Anti-inuenza activity. The target compounds (2j, 3a–p, 4a–
contained with 3,4,5-trimethoxy phenyl fragments, increased d and 5a–c) were screened against inuenza A virus of the
antiproliferative activity towards T47D cells. This nding once subtypes FM/1/47/H1N1, hanfang/359/95/H3N2 and inuenza B
more conrmed the importance of the 3,4,5-trimethoxy virus of the subtype jifang/13/97 (CPE) using MDCK cells with
substituent for drug discovery.45 Another symmetrical amides, oseltamivir and ribavirin (RBV) as positive controls. In our
which inserted substituents such as methyl at C-3 (4a), ditri- initial investigation, several thiophene-3,5-dicarboxalate deriv-
uoro methyl at C-3, C-5 (4b) and triuoro at C-3, C-4, C-5-(4c) atives (2j, 3a, 3g, 3j, 3k, 3n–p and 5a) displayed against the
also resulted in considerable cytotoxicity towards HCT-15 cells. H1N1 subtype potent activity with IC50 values ranging from 0.94
Selectivity of 2j, 3j, 3n and 5c on normal HEK-293 cells. to 10.69 mM. In particular compound 2j (IC50 ¼ 0.94 mM) was
Compounds 2j, 3j, 3n and 5c which showed less IC50 values on more active than oseltamivir and RBV (Table 3). Similar results
human cancer cell lines were selected towards selectivity were observed regarding inuenza subtype H3N2 and
screening on normal human embryonic kidney (HEK-293) cells. compounds 2j, 3a, 3b, 3d, 3g, 3h, 3k, 3l, 3n and 3p. Especially,
The SI calculated by the following formula:
4-chloro-3-uorophenyl related Schiff base (3g, IC50 ¼ 1.81)
showed similar anti-H3N2 activity as the positive control drugs.
SARs of selected compounds regarding inhibition of inuenza A
and B viruses suggest that introduction of diverse pharmaco-
phore substituents, such as nitro-furfuryl and uorine signi-
cantly increased inhibition activity. For instance, derivative 2j
with a 5-nitro-furanyl substituent showed the best anti-
inuenza activity against A and B subtypes. 4-Chloro-3-
uorophenyl-related compound 3g also showed high activity
against inuenza H3N2 virus. Among the mono- and bis amides
of DDTD, only mono amide 5a with a 3-nitrophenyl group
demonstrated good anti-inuenza activity. However, bis amides
of DDTD have demonstrated inactive on inuenza A and B
viruses. Although, several compounds showed potential anti-
viral activity, the most active compounds did not demonstrate
high selectivity indexes (SI) and 50% cytotoxic concentration
Selectivity indexðSIÞ ¼
IC50 of selected samples against HEK-293 cells
IC50 of selected samples against cancer cells
The results revealed that SI of these potentials was not high
for compare with known anticancer drugs (Table 2). However,
when SI between A549 and HEK-293 cells were calculated for
derivative 3n, satisfactory SI (50) have been obtained, which
deserves
further
investigation.
In
addition,
6-
chloronicotinamido-DDTD monoamide (5c) demonstrated
good selectivity when SI of PC-3 and HCT-25 cells were calcu-
lated between HEK-293 cells, while compound 2j showed
similar SI value between T47D and HEK-293 cells. It was found
Table 2 IC50 values on normal human embryonic kidney (HEK-293) cell and SI values of antiproliferative compounds
Compounds
IC50 (ꢀSD, mM)
SIPCꢁ3
SIA549
SIHCTꢁ15
SIT47D
2j
3j
3n
5c
111.3 ꢀ 2.8
31.4 ꢀ 1.7
65.0 ꢀ 2.2
160.0 ꢀ 2.3
nd
4.9 ꢀ 4.7
6.8 ꢀ 2.4
50 ꢀ 11
11 ꢀ 4.7
12.1 ꢀ 5.6
10.6 ꢀ 7.3
22 ꢀ 7.7
24.1 ꢀ 3.5
4.9 ꢀ 4.2
3.0 ꢀ 0.01
7.1 ꢀ 2.9
2.6 ꢀ 2.4
5.9 ꢀ 1.7
23 ꢀ 7.7
2.4 ꢀ 2.0
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Fig. 2 (A) Effects of compounds 3j, 3p and 3n on cell cycle distribution in A549 cells. (B) The induction of apoptosis in A549 cells of DOX, 3j, 3p
and 3n stained with PI and Annexin V-FITC after treatment at different concentrations for 12 h. Q1 [FITC(ꢁ)/PI(+)], necrotic cells; Q2 [FITC(+)/
PI(+)], late apoptotic cells; Q3 [Annexin V(+)/PI(+)], living cells; Q4 [FITC(+)/PI(ꢁ)], early apoptotic cells. (C) Percentage of apoptotic A549 cells
after treatment with samples DOX, 3j, 3p and 3n at different concentrations.
Table 3 Inhibitory activity of the compound 2j and novel compounds 3a–p, 4a–d and 5a–c against influenza A and B viruses
Anti-inuenza H1N1 virus
Anti-inuenza H3N2 virus
Anti-inuenza B virus
SIc
IC50
SI
IC50
SI
b
Compounds
TC50a (mM)
IC50
2j
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
4a
4b
4c
4d
5a
5b
5c
8.90
0.94 ꢀ 0.35d
6.28 ꢀ 0.08
>166.67
10.2
1.7
>3.0
nde
5.0
nd
6.0
9.9
6.9
>3.0
3.0
3.6
3.7
6.5
9.0
10.2
2.2
4.6
4.6
2.40 ꢀ 1.28
2.07 ꢀ 0.91
11.40 ꢀ 1.19
111.12 ꢀ 78.57
7.54 ꢀ 1.93
45.12 ꢀ 9.37
24.57 ꢀ 3.15
1.81 ꢀ 0.35
11.25 ꢀ 4.42
>166.67
26.73 ꢀ 3.73
2.63 ꢀ 0.01
14.07 ꢀ 0.01
97.00 ꢀ 9.58
3.72 ꢀ 1.51
28.30 ꢀ 2.20
9.81 ꢀ 1.31
79.60 ꢀ 8.62
67.34 ꢀ 7.45
>166.67
3.7
5.7
2.8
6.0
0.79 ꢀ 0.13
9.54 ꢀ 0.90
7.37 ꢀ 1.10
131.45 ꢀ 49.81
10.69 ꢀ 0.01
75.90 ꢀ 28.76
42.17 ꢀ 6.35
18.26 ꢀ 3.10
39.17 ꢀ 8.90
>166.67
14.40 ꢀ 0.04
14.30 ꢀ 0.01
18.50 ꢀ 0.03
14.61 ꢀ 5.54
10.52 ꢀ 2.80
24.82 ꢀ 1.90
8.29 ꢀ 0.90
55.52 ꢀ 5.75
59.11 ꢀ 3.50
>166.67
11.5
1.1
4.3
4.1
9.0
>7.1
3.1
4.0
3.3
>3.0
2.2
2.7
5.2
16.2
3.0
3.9
1.3
2.9
4.0
nd
10.69
32.08
>500
96.23
>500
129.34
73.12
129.34
>500
32.08
38.52
96.23
388.03
32.08
96.23
10.69
166.67
240.37
288.68
166.67
240.37
240.37
346.68
1260
>166.67
19.50 ꢀ 2.80
13.2
>11.4
5.3
41.2
12.5
>3.0
1.2
14.6
6.8
4.0
9.4
3.4
1.1
2.1
3.6
>3.0
2.7
>3.0
5.0
6.4
>166.67
21.32 ꢀ 3.21
7.41 ꢀ 1.94
18.75 ꢀ 2.45
>166.67
10.69 ꢀ 0.01
10.69 ꢀ 0.06
30.82 ꢀ 17.39
60.10 ꢀ 6.75
3.56 ꢀ 0.01
9.79 ꢀ 2.35
4.54 ꢀ 0.36
37.60 ꢀ 7.81
52.50 ꢀ 9.54
145.18 ꢀ 21.90
32.08 ꢀ 0.02
18.52 ꢀ 0.05
32.08 ꢀ 0.01
42.21 ꢀ 0.02
3.56 ꢀ 0.53
2.62 ꢀ 0.79
2.0
5.2
13
7.5
8.2
343.4
465.6
62.26 ꢀ 5.66
55.60 ꢀ 4.63
9.32 ꢀ 1.94
43.12 ꢀ 0.03
100.47 ꢀ 40.84
119.58 ꢀ 5.81
1.13 ꢀ 0.43
3.0
10.6
5.6
3.8
10.6
1116.9
>166.67
49.34 ꢀ 8.80
56.90 ꢀ 20.77
1.39 ꢀ 0.37
1.77 ꢀ 1.00
Oseltamivir
RBV
915.3
784.8
1164.1
a
b
c
50% cytotoxic concentration. 50% virus-inhibitory concentration, determined by the CPE inhibition assay. Selectivity index (TC50/IC50).
d
Results are expressed as mean values (ꢀSEM) derived from two replicates. e Not determined.
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(TC50) except of compound 3g (SI ¼ 41.2 on H3N2 virus, TC50
¼
further purication. Purities of the target compounds were
73.12). We thus investigated inuenza inhibition of DDTD established by analytical HPLC: Dionex Ultimate 3000 HPLC
derivatives and our obtained results suggest that further struc- system with DAD detection at 254 nm. The chromatography was
tural modication of these scaffolds against inuenza viruses performed on Sunre C18 column from Waters, USA, having the
may be promising. As conrm our assumption, recently it was following specications: internal diameter 4.6 mm, height
reported that, if cycloheptathiophene-3-carboxamide scaffold,20 250 mm, and particle size 5 mm. The method used had a ow of
when properly functionalized, emerged as particularly suitable 1 mL minꢁ1 and a gradient of water and acetonitrile from 90/10
to impart anti-Flu activity and these derivatives effectively to 0/100. All tested compounds are >95% pure.
inhibited all FluA strains tested, of both H1N1 and H3N2
subtypes, as well as, were suggested its peculiar mechanism of
action.20
General procedure for the synthesis of DDTD (1)
A mixture of elemental sulfur 10 g (312 mmol), ethyl-
cyanoacetate (71.2 g; 623 mmol), DMF (50 mL) and TEA (10 mL;
71 mmol) was allowed to react for 6 days with continuous stir-
ring at 15 ꢂC. The obtained mixture was added to 3 L cool water
and stirred over 4 h. The precipitate was ltered and dried
giving a yellow solid (59 g; 73%).
Conclusions
Recently, we had discovered Schiff bases of DDTD (with only
4-substituents at the benzene ring) as potential anticancer and
antimicrobial agents,27 now in this investigation we have
developed our previous work with additional assays, such as
anti-inuenza virus activities, cell cycle and apoptotic analysis,
General procedure for the synthesis of compounds 3a–p
also have been calculated SI of promising compounds by using Aromatic aldehydes (0.3 mmol) were added to a 0.2 mmol
human normal cell (HEK-293) as well as have synthesized novel solution of the DDTD (1) in toluene (10 mL). The mixture was
mono- and bis-amides and Schiff bases (in this time were selected reuxed for 0.5 h. Further, the solution was evaporated at low
aldehydes with di- and poly-substituents at the aromatic portion) pressure and the residue was puried and recrystallized from
of DDTD on the basis SARs. It was observed that, DDTD deriva- ethanol.
tives (3j, 3n, 3p, and 5c) showed signicant cytotoxicity against
different human tumor cells. In particular, 2-amino-5-[(E)-[(5- methylidene]amino]thiophene-3,4-dicarboxylate (3a). Yield:
nitrothiophen-2-yl)methylidene]amino]thiophene-derivative (3n) 82%; yellow solid, mp 127–129 ꢂC. HPLC purity 98.4% (tR
Diethyl
2-amino-5-[(E)-[(3-triuoromethoxyphenyl)-
¼
showed strong cytotoxic activity and selectivity. It signicantly 25.86 min). 1H NMR (CDCl3, 400 MHz, d ppm): 7.93 (s, 1H, N]
induced apoptosis in A549 cells at low micromolar concentra- CH), 7.62 (m, 2H, Ar-H), 7.41 (t, J ¼ 7.9 Hz, 1H, Ar-H), 7.23 (br.d,
tions. Regarding DDTD amides, 2-(6-chloronicotinamido-) con- J ¼ 8.0 Hz, 1H, Ar-H), 6.41 (br.s, 2H, NH2), 4.43 (q, J ¼ 7.1 Hz,
taining amide 5c displayed consistent antiproliferative activity 2H, OCH2CH3), 4.26 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 1.44 (t, J ¼
showing lower IC50 values than DOX in PC-3 prostate, HCT-15 7.1 Hz, 3H, OCH2CH3), 1.32 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 13C
colon and T47D breast cancer cell lines. Moreover, evaluation NMR (CDCl3, 100 MHz, d ppm): 165.46 (C]O), 164.49 (C]O),
of target compounds against inuenza A and B viruses showed 160.26 (NH2CS), 150.62 (N]CH), 149.79 (Ar-C), 138.04 (Ar-C),
moderate activity, in particular potential hit compound 2j and 3g 133.43 (C), 131.28 (C), 130.18 (Ar-CH), 127.00 (Ar-CH), 123.09
were more or similar active than or with control drugs oseltamivir (Ar-CH), 120.59 (q, J ¼ 257.7 Hz, CF3), 119.86 (Ar-CH), 103.34
and RBV. Thus, we concluded that our results are promising for (C), 61.78 (CH2), 60.52 (CH2), 14.45 (CH3), 14.31 (CH3). MS (ESI):
the future design of antitumor and antiviral activity of DDTD m/z 431 [M + H]+.
derivatives.
Diethyl 2-amino-5-[(E)-[(3,5-dichlorophenyl)methylidene]-
amino]thiophene-3,4-dicarboxylate (3b). Yield: 86%; yellow
solid, mp 223–225 ꢂC. HPLC purity 97.9% (tR ¼ 27.97 min). 1H
NMR (CDCl3, 400 MHz, d ppm): 7.80 (s, 1H, N]CH), 7.58 (d, J ¼
1.9 Hz, 2H, Ar-H), 7.35 (t, J ¼ 1.9 Hz, 1H, Ar-H), 6.44 (br.s, 2H,
Experimental section
Chemistry
Materials and methods. The NMR spectra were recorded on NH2), 4.44 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 4.26 (q, J ¼ 7.1 Hz, 2H,
a Varian 400-MR and Varian VNMRS-600 spectrometer (400 OCH2CH3), 1.45 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.32 (t, J ¼ 7.1 Hz,
MHz and 600 MHz for 1H) using CDCl3 as a solvent and TMS as 3H, OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm): 165.34 (C]
an internal standard in H NMR spectra. In 13C NMR spectra O), 164.41 (C]O), 160.52 (NH2CS), 149.04 (N]CH), 138.77 (C),
1
chemical shis were internally referenced to the solvent signals 135.50 (2Ar-C), 132.99 (Ar-C), 131.94 (C), 130.35 (Ar-CH), 126.46
(CDCl3 77.16 ppm related to TMS). Mass spectra were acquired (2Ar-CH), 103.43 (C), 61.86 (CH2), 60.57 (CH2), 14.50 (CH3),
on QSTAR Elite LC-MS/MS system (Applied Biosystems/MDS 14.32 (CH3). MS (ESI): m/z 416 [M + H]+.
Sciex Concord, ON Canada) spectrometers. Melting points
Diethyl 2-amino-5-[(E)-[(3,5-diuorophenyl)methylidene]-
were determined using a BUCHI Melting Point B-540 apparatus amino]thiophene-3,4-dicarboxylate (3c). Yield: 89%; yellow
1
ꢂ
(Germany) and were uncorrected. All reagents (aromatic alde- solid, mp 182–184 C. HPLC purity 100% (tR ¼ 24.08 min). H
hydes and acid chlorides) and solvents were purchased from NMR (CDCl3, 400 MHz, d ppm): 8.13 (s, 1H, N]CH), 7.98 (td, J ¼
Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), Energy 8.5; 6.6 Hz, 1H, Ar-H), 6.91 (td, J ¼ 8.5, 2.5 Hz, 1H, Ar-H), 6.81
Chemical Technology Co., Ltd., and Shanghai Qinghang (ddd, J ¼ 11.0, 8.5, 2.5 Hz, 1H, Ar-H), 6.50 (br.s, 2H, NH2), 4.41 (q,
Chemical Co., Ltd. (Shanghai, China) and were used without J ¼ 7.1 Hz, 2H, OCH2CH3), 4.25 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 1.42
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(t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.31 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 164.44 (C]O), 160.35 (NH2CS), 158.40 (d, J ¼ 249.4 Hz, Ar-C),
13C NMR (CDCl3, 100 MHz, d ppm): 165.66 (C]O), 164.60 (dd, J ¼ 149.81 (d, J ¼ 3.1 Hz, N]CH), 136.59 (d, J ¼ 6.7 Hz, Ar-C),
254.5, 12.2 Hz, Ar-CF), 164.44 (C]O), 162.50 (dd, J ¼ 256.0, 133.30 (C), 131.27 (C), 130.95 (Ar-CH), 124.97 (d, J ¼ 3.3 Hz,
11.9 Hz, Ar-CF), 160.36 (NH2CS), 144.40 (dd, J ¼ 4.6, 2.3 Hz, N] Ar-CH), 123.55 (d, J ¼ 18.0 Hz, Ar-C), 115.23 (d, J ¼ 22.4 Hz, Ar-
CH), 134.11 (C), 130.69 (C), 129.12 (dd, J ¼ 9.8, 4.2 Hz, Ar-C), CH), 103.30 (C), 61.79 (CH2), 60.52 (CH2), 14.49 (CH3), 14.32
120.39 (dd, J ¼ 9.4, 3.9 Hz, Ar-C), 112.38 (dd, J ¼ 21.9, 3.5 Hz, (CH3). MS (ESI): m/z 499.5 [M + H]+.
Ar-C), 104.08 (t, J ¼ 25.31 Hz, Ar-C), 103.00 (C), 61.70 (CH2), 60.41
Diethyl 2-amino-5-[(E)-[(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
methylidene]amino]thiophene-3,4-dicarboxylate (3h). Yield:
(CH2), 14.43 (CH3), 14.31 (CH3). MS (ESI): m/z 383 [M + H]+.
Diethyl 2-amino-5-[(E)-[(3,5-bis-triuoromethylphenyl)- 79%; yellow solid, mp 215–217 ꢂC. HPLC purity 97.9% (tR
¼
methylidene]amino]thiophene-3,4-dicarboxylate (3d). Yield: 24.42 min). 1H NMR (CDCl3, 400 MHz, d ppm): 7.81 (s, 1H, N]
85%; yellow solid, mp 228–230 ꢂC. HPLC purity 99.1% (tR
¼
CH), 7.29 (d, J ¼ 2.0 Hz, 1H, Ar-H), 7.20 (dd, J ¼ 8.2; 2.0 Hz, 1H,
1
27.99 min). H NMR (CDCl3, 400 MHz, d ppm): 8.16 (s, 2H, Ar- Ar-H), 6.85 (d, J ¼ 8.2 Hz, 1H, Ar-H), 6.44 (br.s, 2H, NH2), 4.41 (q,
H), 7.99 (s, 1H, N]CH), 7.86 (s, 1H, Ar-H), 6.45 (br.s, 2H, J ¼ 7.1 Hz, 2H, OCH2CH3), 4.25 (m, 6H, OCH2CH3, OCH2CH2O),
NH2), 4.45 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 4.27 (q, J ¼ 7.1 Hz, 2H, 1.42 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.30 (t, J ¼ 7.1 Hz, 3H,
OCH2CH3), 1.46 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.32 (t, J ¼ 7.1 Hz, OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm): 165.86 (C]O),
3H, OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm): 165.20 (C] 164.53 (C]O), 159.72 (NH2CS), 152.39 (N]CH), 146.35 (Ar-C),
O), 164.38 (C]O), 160.77 (NH2CS), 148.07 (N]CH), 137.92 (Ar- 143.73 (Ar-C), 134.65 (C), 129.70 (Ar-C), 128.69 (C), 122.68 (Ar-
C), 132.97 (C), 132.43 (C), 132.16 (q, J ¼ 32.4 Hz, 2Ar-C), 127.87 CH), 117.52 (Ar-CH), 116.86 (Ar-CH), 102.67 (C), 64.65 (OCH2),
(2Ar-CH), 123.65 (Ar-CH), 123.23 (q, J ¼ 272.8 Hz, 2CF3), 103.61 64.20 (OCH2), 61.60 (OCH2CH3), 60.26 (OCH2CH3), 14.45 (CH3),
(C), 61.93 (CH2), 60.67 (CH2), 14.45 (CH3), 14.28 (CH3). MS (ESI): 14.29 (CH3). MS (ESI): m/z 405 [M + H]+.
m/z 483 [M + H]+.
Diethyl 2-amino-5-[(E)-[(3-chloro-2-uoro-5-(triuoromethyl)-
Diethyl 2-amino-5-[(E)-[(2-chloro-5-nitrophenyl)methylidene]- phenyl)methylidene]amino]-thiophene-3,4-dicarboxylate (3i).
amino]thiophene-3,4-dicarboxylate (3e). Yield: 80%; brown Yield: 92%; yellow solid, mp 232–234 ꢂC. HPLC purity 97.2%
1
solid, mp 258–260 C. HPLC purity 97.5% (tR ¼ 24.98 min). H (tR ¼ 22.96 min). 1H NMR (CDCl3, 400 MHz, d ppm): 8.17 (dd, J ¼
ꢂ
NMR (CDCl3, 400 MHz, d ppm): 8.88 (d, J ¼ 2.7 Hz, 1H, Ar-H), 5.8; 2.3 Hz, 1H, Ar-H), 8.14 (s, 1H, N]CH), 7.67 (dd, J ¼ 6.5;
8.29 (s, 1H, N]CH), 8.13 (dd, J ¼ 8.8; 2.7 Hz, 1H, Ar-H), 7.54 2.3 Hz, 1H, Ar-H), 6.45 (br.s, 2H, NH2), 4.43 (q, J ¼ 7.1 Hz, 2H,
(d, J ¼ 8.8 Hz, 1H, Ar-H), 6.50 (br.s, 2H, NH2), 4.48 (q, J ¼ 7.1 Hz, OCH2CH3), 4.27 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 1.44 (t, J ¼ 7.1 Hz,
2H, OCH2CH3), 4.27 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 1.50 (t, J ¼ 3H, OCH2CH3), 1.32 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 13C NMR
7.1 Hz, 3H, OCH2CH3), 1.33 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 13C (CDCl3, 100 MHz, d ppm): 165.14 (C]O), 164.37 (C]O), 160.95
NMR (CDCl3, 100 MHz, d ppm): 165.12 (C]O), 164.40 (C]O), (NH2CS), 159.00 (d, J ¼ 259.6 Hz, Ar-CF), 141.93 (d, J ¼ 4.4 Hz,
161.08 (NH2CS),147.10 (Ar-C), 145.36 (N]CH), 140.61 (Ar-C), N]CH), 133.35 (C), 132.84 (C), 128.96 (d, J ¼ 3.8 Hz, Ar-CH),
134.62 (Ar-C), 133.66 (C), 132.75 (C), 131.11 (Ar-CH), 125.09 (Ar- 127.79 (d, J ¼ 33.5 Hz, Ar-C), 126.02 (d, J ¼ 10.8 Hz, Ar-C),
CH), 123.44 (Ar-CH), 103.78 (C), 62.11 (CH2), 60.70 (CH2), 14.59 123.03 (q, J ¼ 265.0 Hz, CF3), 123.44 (Ar-CH), 122.74 (d, J ¼
(CH3), 14.28 (CH3). MS (ESI): m/z 426.5 [M + H]+.
18.2 Hz, Ar-C), 103.67 (C), 61.92 (CH2), 60.68 (CH2), 14.44 (CH3),
Diethyl 2-amino-5-[(E)-[(2-uoro-5-iodophenyl)methylidene]- 14.28 (CH3). MS (ESI): m/z 467.5 [M + H]+.
amino]thiophene-3,4-dicarboxylate (3f). Yield: 81%; yellow
Diethyl 2-amino-5-[(E)-[(peruorophenyl)methylidene]amino]-
solid, mp 206–208 ꢂC. HPLC purity 98.7% (tR ¼ 26.86 min). 1H thiophene-3,4-dicarboxylate (3j). Yield: 87%; yellow solid, mp
NMR (CDCl3, 400 MHz, d ppm): 8.26 (dd, J ¼ 6.7; 2.3 Hz, 1H, Ar- 192–194 ꢂC. HPLC purity 98.6% (tR ¼ 25.16 min). 1H NMR (CDCl3,
H), 8.06 (s, 1H, N]CH), 7.61 (ddd, J ¼ 8.6; 4.9; 2.3 Hz, 1H, Ar-H), 400 MHz, d ppm): 7.95 (s, 1H, N]CH), 6.54 (br.s, 2H, NH2), 4.39
6.83 (dd, J ¼ 10.2; 8.6 Hz, 1H, Ar-H), 6.50 (br.s, 2H, NH2), 4.45 (q, (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 4.25 (q, J ¼ 7.1 Hz, 2H, OCH2CH3),
J ¼ 7.1 Hz, 2H, OCH2CH3), 4.25 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 1.41 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.31 (t, J ¼ 7.1 Hz, 3H,
1.47 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.32 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm): 165.15 (C]O),
OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm): 165.46 (C]O), 164.39 (C]O), 161.31 (NH2CS), 145.87 (d, J ¼ 257.5 Hz, 2Ar-C),
164.39 (C]O), 161.98 (d, J ¼ 255.0 Hz, Ar-C), 160.69 (NH2CS), 141.99 (d, J ¼ 258.1 Hz, Ar-C), 139.05 (d, J ¼ 3.4 Hz, N]CH),
143.29 (d, J ¼ 4.6 Hz, N]CH), 140.66 (d, J ¼ 8.0 Hz, Ar-CH), 137.68 (d, J ¼ 235.5 Hz, 2Ar-C), 133.73 (C), 132.79 (C), 111.50 (Ar-
136.44 (d, J ¼ 2.4 Hz, Ar-CH), 133.62 (C), 131.81 (C), 125.94 (d, C), 103.49 (C), 61.97 (CH2), 60.63 (CH2), 14.31 (CH3), 14.26 (CH3).
J ¼ 10.2 Hz, Ar-C), 118.01 (d, J ¼ 21.9 Hz, Ar-CH), 103.23 (C), MS (ESI): m/z 437 [M + H]+.
87.83 (d, J ¼ 3.6 Hz, Ar-C), 61.84 (CH2), 60.50 (CH2), 14.56 (CH3),
Diethyl 2-amino-5-[(E)-[(5-(4-nitophenyl)furan-2-yl)methylidene]-
amino]thiophene-3,4-dicarboxylate (3k). Yield: 90%; dark red
14.29 (CH3). MS (ESI): m/z 491 [M + H]+.
Diethyl 2-amino-5-[(E)-[(4-chloro-3-uorophenyl)methyl- solid, mp 197–199 ꢂC. HPLC purity 96.7% (tR ¼ 22.28 min). 1H
idene]amino]thiophene-3,4-dicarboxylate (3g). Yield: 83.5%; NMR (CDCl3, 400 MHz, d ppm): 8.23 (d, J ¼ 8.9 Hz, 2H, Ar-H),
orange solid, mp 208–210 ꢂC. HPLC purity 96.2% (tR ¼ 25.68 7.81 (d, J ¼ 8.9 Hz, 2H, Ar-H), 7.76 (s, 1H, N]CH), 6.95 (s,
1
min). H NMR (CDCl3, 400 MHz, d ppm): 7.84 (s, 1H, N]CH), 2H, furan-H), 6.54 (br.s, 2H, NH2), 4.44 (q, J ¼ 7.1 Hz, 2H,
7.54 (m, 1H, Ar-H), 7.40 (m, 2H, Ar-H), 6.45 (br.s, 2H, NH2), 4.42 OCH2CH3), 4.25 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 1.47 (t, J ¼ 7.1 Hz,
(q, J ¼ 7.1 Hz, 2H, OCH2CH3), 4.25 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 3H, OCH2CH3), 1.32 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 13C NMR
1.43 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.32 (t, J ¼ 7.1 Hz, 3H, (CDCl3, 100 MHz, d ppm): 165.51 (C]O), 164.38 (C]O), 160.23
OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm): 165.45 (C]O), (NH2CS), 153.77 (furan-C), 153.30 (furan-C), 146.93 (Ar-C),
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139.70 (N]CH), 135.44 (furan-C), 134.05 (C), 130.69 (C), 124.61
(2Ar-CH), 124.43 (2Ar-CH), 116.52 (furan-CH), 111.64 (furan-
CH), 103.35 (C), 61.74 (CH2), 60.48 (CH2), 14.47 (CH3), 14.29
(CH3). MS (ESI): m/z 458 [M + H]+.
Diethyl
2-amino-5-[(E)-[(1-methyl-1H-pyrazol-4-yl)methyl-
idene]amino]thiophene-3,4-dicarboxylate (3p). Yield: 91%;
yellow solid, mp 175–177 ꢂC. HPLC purity 99.7% (tR ¼ 16.29
1
min). H NMR (CDCl3, 400 MHz, d ppm): 7.88 (s, 1H, N]CH),
Diethyl
2-amino-5-[(E)-[(5-methylfuran-2-yl)methylidene]-
7.79 (s, 1H, pyrazole-H), 7.71 (s, 1H, pyrazole-H), 6.40 (br.s, 2H,
NH2), 4.38 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 4.24 (q, J ¼ 7.1 Hz, 2H,
OCH2CH3), 3.90 (s, 3H, N-CH3), 1.40 (t, J ¼ 7.1 Hz, 3H,
OCH2CH3), 1.30 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 13C NMR (CDCl3,
100 MHz, d ppm): 165.81 (C]O), 164.55 (C]O), 159.26
(NH2CS), 145.73 (N]CH), 139.82 (pyrazole-CH), 135.08 (C),
130.89 (pyrazole-CH), 127.69 (C), 121.24 (pyrazole-C), 102.75 (C),
61.52 (CH2), 60.26 (CH2), 39.33 (N–CH3), 14.42 (CH3), 14.32
(CH3). MS (ESI): m/z 351 [M + H]+.
amino]thiophene-3,4-dicarboxylate (3l). Yield: 89%; yellow
solid, mp 152–154 ꢂC. HPLC purity 99.0% (tR ¼ 21.06 min). 1H
NMR (CDCl3, 400 MHz, d ppm): 7.71 (s, 1H, N]CH), 6.75 (d, J ¼
3.3 Hz, 1H, furan-H), 6.42 (br.s, 2H, NH2), 6.10 (d, J ¼ 3.3 Hz,
1H, furan-H), 4.39 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 4.23 (q, J ¼
7.1 Hz, 2H, OCH2CH3), 2.36 (s, 3H, furan-CH3), 1.41 (t, J ¼
7.1 Hz, 3H, OCH2CH3), 1.30 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 13C
NMR (CDCl3, 100 MHz, d ppm): 165.69 (C]O), 164.53 (C]O),
159.45 (NH2CS), 156.52 (furan-C), 150.56 (furan-C), 141.37 (N]
CH), 135.09 (C), 128.41 (C), 117.00 (furan-CH), 109.20 (furan-
CH), 103.11 (C), 61.61 (CH2), 60.28 (CH2), 14.31 (furan-CH3,
CH3), 14.09 (CH3). MS (ESI): m/z 351 [M + H]+.
General procedure for the synthesis of compounds 4a–d and
5a–c
Diethyl 2-amino-5-[(E)-[(4,5-dimethylfuran-2-yl)methylidene]-
Method (a). DDTD (1 mmol) and aromatic acid anhydride
amino]thiophene-3,4-dicarboxylate (3m). Yield: 86%; brown (1.2 mmol) were dissolved in 10 mL dry benzene or chloroform.
1
ꢂ
solid, mp 182–184 C. HPLC purity 97.4% (tR ¼ 22.59 min). H The mixture was reuxed for 2–3 hours aer the addition of TEA
NMR (CDCl3, 400 MHz, d ppm): 7.68 (s, 1H, N]CH), 6.67 (s, 1H, (2 mmol). 5 mL water was added to the cooled mixture and
furan-H), 6.34 (br.s, 2H, NH2), 4.39 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), extracted with chloroform (3 ꢃ 10 mL). The combined organic
4.23 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 2.27 (s, 3H, furan-CH3), 1.96 layers were washed with brine, dried over Na2SO4 and evapo-
(s, 3H, furan-CH3), 1.40 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.30 (t, J ¼ rated under reduced pressure. The obtained solid was ltered,
7.1 Hz, 3H, OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm): washed with petroleum ester, dried and recrystallized from
165.67 (C]O), 164.59 (C]O), 159.23 (NH2CS), 152.54 (furan-C), ethanol.
149.29 (furan-C), 141.56 (N]CH), 135.41 (C), 128.02 (C), 119.08
Method (b). DDTD (1 mmol) and aromatic acid anhydride
(furan-CH), 117.96 (furan-C), 103.24 (C), 61.59 (CH2), 60.30 (1.2 mmol) were dissolved in 10 mL dry pyridine at 0 ꢂC and
(CH2), 14.35 (2CH3), 12.00 (furan-CH3), 9.91 (furan-CH3). MS stirred for 4 hours at room temperature. Water (5 mL) and 5%
(ESI): m/z 365 [M + H]+.
NH4OH were added to the reaction mixture. The solid formed
Diethyl 2-amino-5-[(E)-[(5-nitrothiophen-2-yl)methylidene]- was ltered, dried and recrystallized from ethanol.
amino]thiophene-3,4-dicarboxylate (3n). Yield: 94%; dark red Diethyl 2,5-bis(3-methylbenzamido)thiophene-3,4-dicarboxylate
solid, mp 187–189 ꢂC. HPLC purity 99.8% (tR ¼ 22.70 min). 1H (4a). Yield: 93%; green solid, mp 158–160 ꢂC. HPLC purity
NMR (CDCl3, 400 MHz, d ppm): 7.92 (s, 1H, N]CH), 7.84 (d, J ¼ 96.9% (tR ¼ 28.16 min). 1H NMR (CDCl3, 600 MHz, d ppm):
4.3 Hz, 1H, thiophene-H), 7.19 (d, J ¼ 4.3 Hz, 1H, thiophene-H), 11.36 (s, 2H, NH), 7.83 (s, 2H, Ar-H), 7.76 (d, J ¼ 6.4 Hz, 2H, Ar-
6.54 (br.s, 2H, NH2), 4.43 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 4.26 (q, H), 7.41 (m, 4H, Ar-H), 4.40 (q, J ¼ 7.2 Hz, 4H, OCH2CH3), 2.46
J ¼ 7.1 Hz, 2H, OCH2CH3), 1.46 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), (s, 6H, Ar-CH3), 1.39 (t, J ¼ 7.2 Hz, 6H, OCH2CH3). 13C NMR
1.32 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 13C NMR (CDCl3, 100 MHz, (CDCl3, 100 MHz, d ppm): 166.17 (2C]O), 164.06 (2HNC]O),
d ppm): 164.87 (C]O), 164.29 (C]O), 161.09 (NH2CS), 153.19 142.63 (2HNCS), 139.08 (2Ar-C), 133.60 (2Ar-CH), 132.20 (2Ar-
(thiophene-C), 148.93 (thiophene-C), 142.84 (N]CH), 133.49 C), 128.96 (2Ar-CH), 128.52 (2Ar-CH), 124.50 (2Ar-CH), 108.58
(C), 132.07 (C), 129.01 (thiophene-CH), 128.20 (thiophene-CH), (2C), 61.39 (2CH2), 21.60 (2Ar-CH3), 14.43 (2CH3). MS (ESI): m/
103.88 (C), 62.04 (CH2), 60.74 (CH2), 14.62 (CH3), 14.28 (CH3). z 495 [M + H]+.
MS (ESI): m/z 398 [M + H]+.
Diethyl 2,5-bis(3,5-bis(triuoromethyl)benzamido)thiophene-
Diethyl 2-amino-5-[(E)-[(4,5-dimethylthiophen-2-yl)methyl- 3,4-dicarboxylate (4b). Yield: 95%; white solid, mp 203–
1
ꢂ
idene]amino]thiophene-3,4-dicarboxylate (3o). Yield: 88%; 205 C. HPLC purity 97.6% (tR ¼ 32.53 min). H NMR (CDCl3,
brown solid, mp 210–212 ꢂC. HPLC purity 98.9% (tR ¼ 24.64 600 MHz, d ppm): 11.72 (s, 2H, NH), 8.43 (s, 4H, Ar-H), 8.12 (s,
1
min). H NMR (CDCl3, 400 MHz, d ppm): 7.94 (s, 1H, N]CH), 2H, Ar-H), 4.44 (q, J ¼ 7.2 Hz, 4H, OCH2CH3), 1.41 (t, J ¼ 7.2 Hz,
7.05 (s, 1H, thiophene-H), 6.24 (br.s, 2H, NH2), 4.40 (q, J ¼ 6H, OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm): 165.97
7.1 Hz, 2H, OCH2CH3), 4.23 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 2.36 (2C]O), 161.11 (2HNC]O), 141.83 (2HNCS), 134.39 (2Ar-C),
(s, 3H, thiophene-CH3), 2.11 (s, 3H, thiophene-CH3), 1.43 (t, J ¼ 132.96 (q, J ¼ 34.0 Hz, 4Ar-C), 127.86 (4Ar-CH), 126.33 (2Ar-
7.1 Hz, 3H, OCH2CH3), 1.30 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 13C CH), 122.90 (d, J ¼ 272.9 Hz, 4CF3), 110.08 (2C), 61.96 (2CH2),
NMR (CDCl3, 100 MHz, d ppm): 165.57 (C]O), 164.59 (C]O), 14.36 (2CH3). MS (ESI): m/z 739 [M + H]+.
159.17 (NH2CS), 146.50 (N]CH), 139.98 (thiophene-C), 137.58
Diethyl
2,5-bis(3,4,5-triuorobenzamido)thiophene-3,4-
(thiophene-C), 134.72 (C), 134.63 (thiophene-CH), 134.51 dicarboxylate (4c). Yield: 95%; green solid, mp 164–166 ꢂC.
(thiophene-C), 128.30 (C), 103.18 (C), 61.57 (CH2), 60.34 (CH2), HPLC purity 99.1% (tR ¼ 30.82 min). 1H NMR (CDCl3, 600 MHz,
14.59 (CH3), 14.30 (CH3), 14.03 (thiophene-CH3), 13.69 (thio- d ppm): 11.52 (s, 2H, NH), 7.65 (d, J ¼ 6.4 Hz, 2H, Ar-H), 7.64 (d,
phene-CH3). MS (ESI): m/z 381 [M + H]+.
J ¼ 6.4 Hz, 2H, Ar-H), 4.42 (q, J ¼ 7.2 Hz, 4H, OCH2CH3), 1.40 (t,
31424 | RSC Adv., 2017, 7, 31417–31427
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J ¼ 7.2 Hz, 6H, OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm):
166.20 (2C]O), 160.80 (2HNC]O), 151.57 (dd, J ¼ 253.3;
10.3 Hz, 4Ar-CF), 142.99 (dt, J ¼ 259.0; 11.4 Hz, 2Ar-CF), 142.11
(2HNCS), 128.14 (Ar-C), 112.45 (dd, J ¼ 16.3; 6.6 Hz, 4Ar-CH),
109.43 (2C), 61.84 (2CH2), 14.39 (2CH3). MS (ESI): m/z 575
[M + H]+.
Diethyl 2,5-bis(3,4,5-trimethoxybenzamido)thiophene-3,4-
dicarboxylate (4d). Yield: 92%; white solid, mp 216–218 ꢂC.
HPLC purity 98.0% (tR ¼ 27.92 min). 1H NMR (CDCl3, 600 MHz,
d ppm): 11.42 (s, 2H, NH), 7.23 (s, 4H, Ar-H), 4.38 (q, J ¼ 7.2 Hz,
4H, OCH2CH3), 3.97 (s, 12H, OCH3), 3.94 (s, 6H, OCH3), 1.38 (t,
J ¼ 7.2 Hz, 6H, OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm):
166.29 (2C]O), 163.26 (2HNC]O), 153.58 (4Ar-C), 142.64
(2HNCS), 142.17 (2Ar-C), 127.46 (2Ar-C), 108.58 (2C), 105.05
(4Ar-CH), 61.39 (2CH2), 61.15 (2OCH3), 56.53 (4OCH3), 14.41
(2CH3). MS (ESI): m/z 647 [M + H]+.
Biology
Materials. Doxorubicin and trypsin were purchased from BBI
Inc. (Shanghai, China), ampicillin from Sigma Chemicals Co.,
amphotericin B. from AMRESCO LLC, ribavirin from Xinxiang
Pharmaceutical Co., Ltd. (batch 20081227) and oseltamivir from
Chinese Academy of Food and Drug (batch 101096-200901). The
human cancer cell prostate (PC-3), lung (A549), colon (HCT-15),
breast (T47D) and human embryonic kidney (HEK-293) lines
were obtained from Chinese Type Culture Collection, CAS
(Shanghai, China). Madin-Darby canine kidney cells (MDCK)
were purchased from the American Type Culture Collection
(ATCC).
Cell cultures. Human PC-3 cells and HCT-15 cells were
grown in DMEM/F12 medium (Gibco), human T47D cells and
A549 cells were grown in Dulbecco's modied Eagle's medium
(DMEM) with 4.5 g Lꢁ1 glucose and 0.37% sodium bicarbonate
(Gibco, Rockville, MD, USA). All cell culture media contained
with 10% FBS and antibiotic mix (1 ꢃ 100 mM penicillin A and
100 mM of streptomycin) and were grown at 37 ꢂC in a humidi-
ed incubator (Binder, Germany) containing 95% air/5% CO2
and have been fed every 3–4 days.27
Proliferation assays. All compounds were dissolved in DMSO
in a stock concentration of 10 mM. PC-3, A549, HCT-15 or T47D
cells grown in the logarithmic phase were separately seeded in
aliquots of 200 mL in 96-well plates at a density of 5 ꢃ 103 cells
per well. The cells were grown for 24 h in a humidied incubator
(Binder, Germany) at 37 ꢂC with 95% humidity and 5% CO2.
Thereaer, the cells were treated with 1, 10, 25 and 30 mM of
compounds (1–5) for 48 h. Then 20 mL MTT (5 mg mLꢁ1) was
added to each well and the plates incubated at 37 ꢂC. Four hours
later, the supernatant was removed and 200 mL of DMSO were
added to each well and the multiwell plates shaken for 10 min
to dissolve the crystals. Absorbance was read at a wavelength of
540 nm using an enzyme-linked immunosorbent 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).27
Diethyl
2-amino-5-(3-nitrobenzamido)thiophene-3,4-
dicarboxylate (5a). Yield: 96%; yellow solid, mp 198–200 ꢂC.
HPLC purity 98.5% (tR ¼ 22.35 min). 1H NMR (CDCl3, 600 MHz,
d ppm): 11.65 (s, 1H, NH), 8.83 (t, J ¼ 2.0 Hz, 1H, Ar-H), 8.44
(ddd, J ¼ 8.0; 2.0; 0.9 Hz, 1H, Ar-H), 8.24 (dt, J ¼ 8.0; 1.1 Hz, 1H,
Ar-H), 7.74 (t, J ¼ 8.0 Hz, 1H, Ar-H), 5.56 (br.s, 2H, NH2), 4.37 (q,
J ¼ 7.1 Hz, 2H, OCH2CH3), 4.30 (q, J ¼ 7.1 Hz, 2H, OCH2CH3),
1.37 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.34 (t, J ¼ 7.1 Hz, 3H,
OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm): 166.20 (C]O),
165.24 (C]O), 161.36 (HNC]O), 154.73 (NH2CS), 148.82
(HNCS), 134.63 (Ar-C), 134.05 (Ar-C), 132.73 (Ar-CH), 130.37 (Ar-
CH), 127.14 (Ar-CH), 122.97 (Ar-CH), 111.22 (C), 102.37 (C),
61.51 (CH2), 60.48 (CH2), 14.57 (CH3), 14.34 (CH3). MS (ESI): m/z
408 [M + H]+.
Diethyl 2-amino-5-(3,4,5-trimethoxybenzamido)thiophene-
3,4-dicarboxylate (5b). Yield: 53%; green solid, mp 138–
140 ꢂC. HPLC purity 99.6% (tR ¼ 21.95 min). 1H NMR (CDCl3,
400 MHz, d ppm): 11.41 (s, 1H, NH), 7.18 (s, 2H, Ar-H), 5.54
(br.s, 2H, NH2), 3.95 (s, 6H, OCH3), 3.92 (s, 3H, OCH3), 4.34 (q,
J ¼ 7.1 Hz, 2H, OCH2CH3), 4.28 (q, J ¼ 7.1 Hz, 2H, OCH2CH3),
1.35 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.33 (t, J ¼ 7.1 Hz, 3H,
OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm): 166.20 (C]O),
165.38 (C]O), 163.48 (HNC]O), 154.50 (NH2CS), 153.56 (2Ar-
C), 142.14 (HNCS), 135.61 (Ar-C), 127.34 (Ar-C), 110.14 (C),
104.88 (2Ar-CH), 102.20 (C), 61.18 (CH2), 61.13 (OCH3), 60.34
(CH2), 56.49 (2OCH3), 14.55 (CH3), 14.35 (CH3). MS (ESI): m/z
453 [M + H]+.
Cell cycle assay. For cell cycle analysis, approximately 5 ꢃ 105
log phase A549 cells were seeded in six-well plates in triplicate
and treated with various concentrations of selected compounds
(DOX, 3j, 3n, 3p) for 12 h. Then, samples were suspended in
absolute ice-cold ethanol and kept overnight. The next day,
samples were stained with 30 mL PI and 0.3 mL RNase A at room
temperature in the absence of light for at least 0.5 h. The
stained cells were ltered through a 30 mm nylon mesh (Spec-
trum, Los Angeles, CA, U.S.).
Cellular apoptosis analysis by ow cytometry. A549 cells
were seeded in six-well plates at a density of 5 ꢃ 105 cells per
well and allowed to attach for 24 h. Further, cells were incubated
with DOX (1, 2.5 and 5 mM) or with selected compounds 3j (1,
2.5, 5 mM), 3n (0.75, 1.5, 2 mM) and 3p (5, 10, 20 mM) for 12 h.
Treated cells were collected, washed twice with ice-cold PBS,
centrifuged at 200 ꢃ g for 5 min. and re-suspended in 100 mL of
Annexin-V-FLOUS (Roche, Germany) for 15 min. Incubation at
RT in the dark followed according to the manufacturer's
Diethyl 2-amino-5-(6-chloronicotinamido)thiophene-3,4-
ꢂ
dicarboxylate (5c). Yield: 97.5%; yellow solid, mp 204–206 C.
HPLC purity 98.9% (tR ¼ 21.24 min). 1H NMR (CDCl3, 600 MHz,
d ppm): 11.57 (s, 1H, NH), 8.98 (d, J ¼ 2.7 Hz, 1H, Ar-H), 8.17 (dd,
J ¼ 8.4; 2.7 Hz, 1H, Ar-H), 7.49 (d, J ¼ 8.4 Hz, 1H, Ar-H), 5.52
(br.s, 2H, NH2), 4.35 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 4.29 (q, J ¼
7.1 Hz, 2H, OCH2CH3), 1.36 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.34 (t,
J ¼ 7.1 Hz, 3H, OCH2CH3). 13C NMR (CDCl3, 100 MHz, d ppm):
166.25 (C]O), 165.24 (C]O), 162.15 (HNC]O), 160.95
(NH2CS), 155.58 (HNCS), 154.70 (Ar-C), 149.11 (Ar-CH), 137.63
(Ar-CH), 134.52 (Ar-C), 126.95 (C), 124.73 (Ar-CH), 102.30 (C),
61.49 (CH2), 60.47 (CH2), 14.56 (CH3), 14.33 (CH3). MS (ESI): m/z
398.5 [M + H]+.
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Acknowledgements
This work was funded by the National Natural Science Foun-
dation of China (NSFC) (Grant No. 21550110495) and Chinese
Academy of Sciences President's International Fellowship
Initiative (Grant No. 2016PT014).
`
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