Divergent Synthesis and Evaluation of the in vitro Cytotoxicity Profiles of 3,4-Ethylenedioxythiophenyl-2-propen-1-one Analogues

Article abstract of DOI:10.1002/cmdc.201900225

A new series of 3,4-ethylenedioxythiophene (EDOT)-appended propenones were prepared by condensation reaction and their in vitro cytotoxicity effects were evaluated against five human cancer cell lines. Preliminary structure–activity relationships of EDOT-

Full text of DOI:10.1002/cmdc.201900225

DOI: 10.1002/cmdc.201900225
Full Papers
Divergent Synthesis and Evaluation of the in vitro
Cytotoxicity Profiles of 3,4-Ethylenedioxythiophenyl-2-
propen-1-one Analogues
Jayachandran Karunakaran,[a] Nachiappan Dhatchana Moorthy,[b, e]
Somenath Roy Chowdhury,[c] Saleem Iqbal,[d] Hemanta K. Majumder,[c]
Krishnasamy Gunasekaran,[d] Elangovan Vellaichamy,[b] and
Arasambattu K. Mohanakrishnan*[a]
A new series of 3,4-ethylenedioxythiophene (EDOT)-appended
propenones were prepared by condensation reaction and their
in vitro cytotoxicity effects were evaluated against five human
cancer cell lines. Preliminary structure–activity relationships of
EDOT-incorporated 2-propenone derivatives were also estab-
lished. The EDOT-appended enones demonstrated significant
cytotoxicity against human cancer cell lines. The most active
severely inhibited the clonogenic potential of cancer cells, and
induced cell-cycle arrest in the G2/M phase and caused an ac-
cumulation of HCT116 colon cancer cells with >4N DNA con-
tent. Also, 3p exhibited weak inhibition of the enzymatic activ-
ity of human topoisomerase I. Molecular docking studies indi-
cated preferential binding of the compounds to the ATP-bind-
ing pocket of the human checkpoint 2 kinase (Chk2) catalytic
domain, thus, identifying a novel diaryl 2-propenone chemo-
type for the development of potent inhibitors of Chk2.
analogue,
(E)-3-(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-1-
(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3p, GI50 =110 nm),
Introduction
For many years, diaryl chalcone derivatives have been exten-
sively studied for their diverse biological and pharmacological
properties.[1] The chalcones have a basic skeleton similar to fla-
vonoids and are also known as key intermediates in the syn-
thesis of various bioactive heterocycles.[2] In recent decades,
diaryl 2-propenones have been investigated for their biological
activities, including as antimalarial,[3] antibacterial,[4] antitu-
mor,[5] antioxidant,[6] antihyperglycemic,[7] and anti-HIV agents.[8]
Recently, Guo and co-workers developed an efficient synthesis
of a,b-unsaturated carbonyl compounds by FeII-mediated de-
carboxylative cross-coupling reactions,[9] whereas Correa et al.
established the synthesis of the g-butenolide natural product
framework with epoxidation of 2-propenone as a crucial
step.[10] Owing to their biological importance, nitrogen-contain-
ing six-membered pyrimidines[11] and five-membered isoxa-
zoles were synthesized from chalcones by cyclocondensation
reactions. Pyrimidines are basic components of nucleic acids,
and substituted pyrimidines have exhibited excellent biological
activity against various human cancer cell lines.[12] Isoxazoles
are also versatile building blocks for constructing numerous
natural products.[13] Substituted isoxazoles are well-known for
their promising and potent biological activities such as analge-
sic,[14] antiviral,[15] antimicrobial,[16] hypoglycemic,[17] and anti-
cancer properties.[15,18] Attar and co-workers reported that or-
ganic chalcones displayed better biological activity than organ-
ometallic chalcones.[19]
[a] Dr. J. Karunakaran, Dr. A. K. Mohanakrishnan
Department of Organic Chemistry, School of Chemistry, University of
Madras, Guindy Campus, Chennai 600025, Tamil Nadu (India)
E-mail: mohanakrishnan@unom.ac.in
[b] N. Dhatchana Moorthy, Dr. E. Vellaichamy
Department of Biochemistry, University of Madras, Guindy Campus, Chen-
nai 600025, Tamil Nadu (India)
Notably, the 3,4-ethylenedioxythiophene (EDOT) unit plays a
vital role in the field of material sciences[20] owing to the non-
covalent interaction between oxygen and a sulfur atom. How-
ever, reports describing the biological activity of EDOT deriva-
tives are few in number.[21] The direct CÀH functionalization of
EDOT has led to the synthesis of wide variety of EDOT ana-
logues.[22] In general, several thiophene-based chalcone ana-
logues exert cytotoxic activities through a variety of molecular
mechanisms involving disruption of the cell cycle, inhibition of
angiogenesis, tubulin polymerization and induction of apopto-
sis.[23] Also, several chalcones were reported to inhibit the activ-
ity of kinases associated with growth factor receptors essential
for the survival and proliferation of cancer cells, such as vascu-
[c] S. R. Chowdhury, H. K. Majumder
Division of Infectious Diseases & Immunology, Indian Institute of Chemical
Biology, 4, Raja S. C. Mallick Road, Jadavpur, Kolkata 700032, West Bengal
(India)
[d] S. Iqbal, Dr. K. Gunasekaran
Center for Advanced studies in Crystallography & Biophysics, University of
Madras, Guindy Campus, Chennai 600025, Tamil Nadu (India)
[e] N. Dhatchana Moorthy
Department of Biotechnology, Orchid Pharma Limited, Orchid Towers #313,
Valluvar Kottam High Road, Nungambakkam, Chennai 600034, Tamil Nadu
(India)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/cmdc.201900225.
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lar endothelial growth factor receptor-2 (VEGFR-2) and epider-
mal growth factor receptor (EGFR).[24] Recently, several chal-
cone inhibitors of human checkpoint kinase 2 (Chk2) were re-
ported to exhibit antitumor activity.[25] A series of 2-aminopyri-
dines containing thiophene dioxole rings were reported as
Chk2 inhibitors through interactions within the ATP-binding
pocket of Chk2 kinase.[26] The unrelated PV1019 exerted anti-
proliferative activity in cancer cells expressing high levels of
Chk2 kinase, whereas selective inhibition of Chk2 in p53-defi-
cient cells sensitizes them to DNA-damaging agents and radia-
tion.[27]
the corresponding 1-(2-thienyl)-3-aryl-propen-2-ones 3j–l, re-
spectively, in good yields (Scheme 2). Similarly, syntheses of
isomeric 2-thiophenyl, 3-methyl-2-thiophenyl and 2,2’-bithio-
phenyl-appended enones 3m--o were achieved by base-medi-
ated condensation of the respective thiophene aldehydes with
trimethoxyacetophenone (6a) in 84–88% yields (Scheme 2).
Next, base-mediated condensation of trisubstituted aceto-
phenone 6a and disubstituted 6b with EDOT-2-carboxalde-
hyde (5a) led to the formation of the corresponding prope-
nones 3p and 3q (Scheme 3). Furthermore, the condensation
of 3,4,5-trimethoxyacetophenone with 5-bromo-EDOT-2-car-
boxaldehyde as and EDOT-2,5-dicarboxaldehyde afforded de-
rivatives 3r and 3s, respectively. As a representative case, to
understand the significance of the vinyl spacer, trimethoxyben-
zoyl EDOT 3t was also prepared. As expected, Suzuki coupling
of 5-bromo-EDOT-2-carboxaldehyde (7) with 3,4-dimethoxy-
phenylboronic acid (8) in the presence of zero-valent Pd in
DMF heated at reflux furnished the 3,4-dimethoxyphenyl-sub-
stituted EDOT aldehyde, which upon condensation with 6a
using 10% NaOH solution heated at reflux for 1 h afforded 3,4-
dimethoxyphenyl-appended enone 3u in 82% yield
(Scheme 3).
Based on the above findings, we planned the synthesis of
new types of EDOT-based chalcone analogues and investigated
their in vitro cytotoxicity potential against human cancer cell
lines. Using an available crystal structure of Chk2 bound with a
small molecule, an attempt was made to determine the bind-
ing modes of the EDOT enones reported here using molecular
docking studies.
Results and Discussion
Finally, to investigate the importance of the enone, EDOT
compounds with masked enone moieties were synthesized.
Accordingly, the reaction of EDOT enone 3a with hydroxyla-
mine hydrochloride (9) in the presence of K2CO3 furnished the
isoxazole-containing EDOT 10 in 70% yield. Next, as an illustra-
tive case, the EDOT enone 3d reacted with guanidine hydro-
chloride (11) in 10% NaOH solution heated at reflux for 2 h to
afford EDOT-substituted pyrimidine 12 in a moderate yield
(Scheme 4).
Synthesis of novel thiophene-based derivatives 3a–u, 10,
and 12
As a typical example, the EDOT-based enone 3a can be
smoothly prepared by a condensation reaction between 2-
acetyl-EDOT (1) and veratraldehyde (2a) using 10% aqueous
NaOH solution heated at reflux for 1 h. Using similar proce-
dures, various EDOT-2-propenone derivatives 3b–f were also
synthesized. Next, condensation of 1 with 2,6-dichlorobenzal-
dehyde, thiophene-2-carboxaldehyde (2h), and EDOT-2-carbox-
aldehyde (5a) furnished respective enones 3g–i (Scheme 1).
A detailed literature survey revealed that the biological activ-
ity of enone derivatives could be varied by changing either
group or individual atoms, that is, the positions of the group
play an essential role in the activity of diaryl-2-propenone ana-
logues.[19,28] Hence, to better understand the structure–activity
relationships of EDOT enones, derivatives lacking the EDOT
unit were synthesized. As expected, 2-acetylthiophene (4a) un-
derwent a similar type of base-mediated condensation reaction
with substituted aromatic aldehydes 2a, 2c, and 2e to afford
Cytotoxicity screening
The synthesized compounds 3a–u were evaluated for their cy-
totoxic potential using a sulforhodamine B assay against five
different human cancer cell lines of diverse tumor origin:
HCT116 (colon), NCI-H460 (lung), U251 (glioma), MCF7 (breast),
and HeLa (cervical). The in vitro concentration for 50% growth
inhibition (GI50) values of test compounds against individual
cell lines are presented in Table 1. Detailed structure–activity
Scheme 1. Synthesis of EDOT-based 2-propenones 3a–i.
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Scheme 2. Synthesis of thiophene-based 2-propenones 3j–o.
Scheme 3. Synthesis of EDOT-based ketones 3p–u.
atoms in ortho positions to the vinyl ketone spacer (GI50 >
8 mm). However, in the case of 3e (Figure 1A), having para-
fluoro and meta-bromo substitution on the phenyl unit with
respect to the 2-propen-1-one spacer, the average GI50 value
across the five cell lines was below 2.5 mm.
The growth inhibition of 3a was moderately compromised
upon introduction of a thiophene group (3h) but was com-
pletely abolished if a second EDOT moiety was introduced at
the 3-position (3i). Also, if the EDOT unit was replaced with
thiophene (3j–m), the activity was marginally decreased (GI50 >
4 mm). However, a similar replacement of the EDOT unit with 3-
methylthiophene resulted in 3n exhibiting potent cytotoxic ac-
tivity (average GI50 <2.5 mm). Again, the activity was decreased
for the compound containing bis-thiophene and 3,4,5-trime-
thoxyphenyl moieties (3o).
Scheme 4. Synthesis of masked-enone EDOT derivatives 10 and 12.
relationships were examined by varying the position of the
ketone functional group in the linker and by changing the sub-
stituents on the thiophene and phenyl groups. Firstly, among
the 2-propen-1-ones containing an EDOT moiety, varying the
di- or trisubstitution on the 3’-phenyl ring resulted in promis-
ing cytotoxic profiles for compounds 3a–g, with GI50 values in
the range of 2–20 mm, except for 3g having two chlorine
Interestingly, changing the position of the vinyl ketone func-
tion in the spacer, with a trimethoxyphenyl group attached to
the 1-position of 2-propen-1-one (3p), resulted in superior cy-
totoxicity relative to the other compounds tested, with GI50
values between 40 and 110 nm (Figure 1B and Table 1). Similar
results were obtained with 3r (Figure 1C and Table 1), which,
with a bromine substituent on the EDOT ring, has both its 2-
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Table 1. In vitro cytotoxicity data for EDOT-based enones.
Compound
GI50 [mm][a]
HCT116 (colon)
NCI-H460 (lung)
U251 (glioma)
HeLa (cervix)
MCF7 (breast)
3a
3b
3c
3d
3e
3 f
3g
3h
3i
3j
3k
5.0Æ1.0
20.0Æ0.0
4.9Æ0.42
5.0Æ1.80
2.1Æ0.33
5.0Æ0.71
>20
5.7Æ0.58
5.5Æ0.71
6.0Æ1.0
5.5Æ0.71
5.0Æ1.0
6.3Æ1.06
4.2Æ1.35
7.0Æ1.41
2.4Æ0.56
2.9Æ1.27
1.8Æ0.24
2.4Æ0.85
8.0Æ0.0
7.5Æ0.71
>20
6.4Æ1.48
4.6Æ0.64
2.8Æ0.28
6.0Æ1.41
3.5Æ2.12
12.5Æ0.71
0.09Æ0.21
>20
3.7Æ0.76
3.4Æ1.52
3.0Æ0.0
2.1Æ0.14
7.5Æ0.71
1.0Æ0.14
1.5Æ0.46
1.3Æ0.35
1.7Æ0.14
11.0Æ1.41
3.5Æ0.42
5.0Æ0.0
3.2Æ0.42
4.2Æ0.58
2.8Æ0.35
3.5Æ0.71
1.5Æ0.35
4.0Æ1.41
0.11Æ0.014
>20
3.23Æ1.63
2.1Æ0.32
4.3Æ1.13
11.0Æ1.41
5.6Æ0.64
20.0
>20
9.0Æ1.41
>20
8.5Æ0.71
>20
7.7Æ0.58
5.8Æ0.35
4.6Æ0.57
9.5Æ2.12
2.8Æ1.06
9.5Æ0.71
0.07Æ0.0
>20
7.5Æ0.71
7.3Æ0.35
6.5Æ0.71
7.0Æ0.0
6.5Æ0.71
5.0Æ0.0
3l
5.3Æ1.06
7.0Æ1.41
1.6Æ0.07
9.5Æ0.71
0.04Æ0.008
3m
3n
3o
3p
3q
3r
2.1Æ0.14
1.8Æ0.35
0.10Æ0.021
>20
>20
0.3Æ0.20
1.3Æ0.14
>20
0.5Æ0.07
1.9Æ0.0
0.5Æ0.21
2.3Æ0.35
>20
0.31Æ0.01
1.0Æ0.07
0.1Æ0.01
ND
>20
3s
3t
>20
>20
3u
10
12
>20
>20
>20
ND
4.8Æ1.06
>20
>20
15.0Æ0.0
1.7Æ0.92
>20
>20
>20
>20
ND
>20
0.060Æ0.0
nocodazole
0.075Æ0.007
0.075Æ0.007
0.080Æ0.014
0.061Æ0.035
[a] Values are the meanÆSD of at least two independent experiments; ND: not determined.
Figure 1. Growth inhibition (dose–response) profiles of EDOT derivatives A) 3e, B) 3p, and C) 3r in a panel of five cancer cell lines after treatment for 48 h.
HCT116 (green), NCI-H460 (blue), U251 (cyan), HeLa (magenta), and MCF7 (yellow).
Effect of EDOT derivatives on the clonogenicity of cancer
cells
and 5-positions blocked. Also, symmetrical substitution of
EDOT at the 2- and 5-positions with trimethoxyphenyl vinyl
ketone moieties (3s) maintained the antiproliferative potential
(GI50 <2 mm).
Having established the cytotoxic potential of EDOT derivatives,
the long-term impact of 3e, 3p and 3r on the colony-forming
ability of HCT116 and NCI-H460 cells was studied using a clo-
nogenic cell survival assay, in which the anticancer alkaloid
camptothecin served as a standard (Figure 2). The derivatives
3p and 3r effectively abrogated colony formation even at con-
centrations as low as 0.5 mm. However, 3p was more potent,
inhibiting colony growth at a concentration of 0.25 mm. Com-
pound 3e, which showed moderate cytotoxicity, could attenu-
ate the formation of colonies, albeit at concentrations above
The cytotoxicity was completely abolished if the 3,4,5-trime-
thoxyphenyl group were replaced with a 3,4-dimethoxyphenyl
group (3q) or with the absence of vinyl group in the spacer
(3t). Also, the EDOT derivatives in which the cyclized vinyl
ketone functionality was converted into heterocycles (10 and
12) were inactive analogues, thus highlighting the importance
of the vinyl ketone spacer for anticancer properties.
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Figure 2. Effect of EDOT derivatives on the clonogenicity of A) HCT116 and B) NCI-H460 cells. Cells were seeded in six-well plates, allowed to adhere over-
night, then treated with various concentrations (0.25–10 mm) of enones 3e, 3p, and 3r. After 48 h, the cells were washed with Dulbecco’s phosphate-buffered
saline and grown for 14 days in drug-free medium. Cell colonies were stained with crystal violet and photographed.
1 mm in both cell lines. Thus, the results of the preliminary cy-
totoxicity assay were confirmed by the long-term clonogenic
cell survival assay.
Effect of EDOT derivatives 3p and 3r on human topoisomer-
ase activity
DNA topoisomerases are class of enzymes involved in catalyz-
ing changes in DNA topology, thus playing an essential role in
DNA metabolism. In humans, there are two classes of topoiso-
merases causing either single-strand DNA cleavage (topoiso-
merase I) or cleavage of both strands (topoisomerase II) to re-
lease topological strain. Many small molecules are reported to
influence the catalytic activities of topoisomerases, causing
cell-cycle arrest or cell death and are recognized as antitumor
drugs.[30] Several chalcone derivatives have been shown to in-
hibit different enzymes crucial to cell replication.[31] Also, it is
reported that tricyclic planar pyrroloquinoline-conjugated chal-
cones inhibit the relaxation of supercoiled DNA by topoisomer-
ase II.[32] Hence, the ability of the promising chalcone-like 3p
and 3r to interfere with DNA topoisomerase activity was ex-
amined in a plasmid relaxation assay. Compound 3p could par-
tially inhibit hTopI activity at 200 mm, whereas no inhibition of
hTopII was observed at this concentration (Figure 4). In con-
trast, 3r failed to inhibit the relaxation activities of either top-
oisomerase at 200 mm.
Cell-cycle effects of EDOT derivatives in HCT116 cells
The time-dependent cellular effects of the promising ana-
logues 3p and 3r on HCT116 cells was studied using flow cy-
tometry (Figure 3). The cell-cycle analysis with 2 mm of the pro-
penone 3e at 24 and 48 h showed only marginal changes in
the population of cells in different phases. The compound 3p,
which emerged as a potent analogue from the cytotoxicity
analysis, caused a significant arrest of HCT116 cells in the G2/M
phase (60%) and the subsequent appearance of polyploid cells
with >4N DNA content (15.8%) after 24 h treatment.
However, an increase in cell death as evident from the ap-
pearance of a sub-G1 population of cells (16.5%) was seen in
3p-treated cells at 48 h. Similarly, in the case of 3r, there was
arrest of cells in the G2/M phase with an increase in the
number of polyploid cells (10.5%) at 24 h. Compound 3p was
found to be more potent at causing cell death both at 24 and
48 h, whereas 3r produced stronger G2/M arrest than 3p in
HCT116 cells. The occurrence of G2/M arrest following treat-
ment with 3r and 3p suggests the activation of the mitotic
checkpoint, whereas an increase in the numbers of polyploid
cells indicates abnormal chromosomal segregation during mi-
tosis, which is characteristic of agents that interfere with tubu-
lin dynamics.[29]
Molecular docking of EDOT derivatives and inhibiting inter-
actions with Chk2 kinase
Many bifunctional 2-aminopyridine analogues have been
widely reported as kinase inhibitors. Crystallographic studies of
Chk2 kinase and such inhibitors indicated binding of the 2-
aminopyridine group into the ribose-binding pocket, with 3,5-
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Figure 4. Effects of EDOT derivatives on recombinant human topoisomerases
I and II. Relaxation of negatively supercoiled pBS (SK+) DNA with either pu-
rified A) hTopI or B) hTopII at a molar ratio of 3:1 under identical assay condi-
tions. Lanes 1: 90 fmol pBS (SK+) DNA; lanes 2: same as lanes 1, but with
30 fmol hTopI or hTopII incubated for 30 min at 378C; lanes 3: same as lanes
2, but in the presence of DMSO (2% v/v); lanes 4: same as lanes 2, but in
the presence of 25 mm camptothecin (A) or 25 mm etoposide (B) as positive
controls; lanes 5 and 6: same as lanes 2, but in the presence of 200 mm 3p
and 3r, respectively. Positions of supercoiled monomer (SM) and relaxed
and nicked monomer (RL/NM) are indicated. All experiments were carried
out in triplicate. The images shown here are representative of one set of
those experiments.
Figure 3. Time-dependent arrest of cells in EDOT-derivative-treated HCT116
cells. Cell-cycle analysis of human colon HCT116 cells treated with EDOT de-
rivatives 3e, 3p, and 3r (2 mm) for 24 and 48 h. Each panel shows the mean
percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle; fig-
ures are representative of two independent experiments.
Figure 5. General structures of EDOT derivatives 3a–i and 3m–u and diaryl
2-aminopyrimidine analogue 12.
diaryl substituents serving as hinge-binding motifs.[26a] In the
present study, the resemblance of aminopyrimidine 12 and the in ring of 2-aminopyrimidine 12 reached the surface defined
isoxazole 10 to the ATP ligand raises the possibility of their ac- by residues Leu303 and Met304 to provide a better fit to this
commodation into the ribose-binding site of the Chk2 kinase, region of the Chk2 kinase, which was not observed with the
and their substituents might serve as hinge-binding motifs. corresponding five-membered isoxazole derivative 10.
A
Thus, to examine the orientation of 4,6-diaryl-substituted 2- common striking feature of the vinyl ketones was the interac-
aminopyrimidine 12 and the 3,5-diaryl-substituted isoxazole 10 tion of the carbonyl group with the Met304 residue in the
in the active ATP-binding site of the Chk2 kinase, an induced- hinge region of the kinase. The symmetrical bis-trimethoxy-
fit docking protocol was adopted using the coordinates from phenyl EDOT derivative 3s emerged as the most promising an-
the reported[26a] co-crystal structure of a related EDOT-substi- alogue with the lowest Glide score, and its methoxy functional
tuted 2-aminopyrimidine bound to the Chk2 kinase (PDB ID: group forms hydrogen bonds with the key amino acid residues
2WTJ). We investigated whether the EDOT-substituted diaryl of the ATP-binding site. The compounds 3r and 3p, which dis-
propenone interacts with the ATP-binding pocket of the Chk2 played potent growth inhibition activity in the preliminary cy-
kinase as their structural contours resemble those of diaryl- totoxicity assay, exhibited low binding free energy
substituted 2-aminopyridines (Figure 5).
(À54.952 kcalmolÀ1
)
with their trimethoxyphenyl groups
The binding energy, glide score, and the key interacting resi- making hydrophobic interactions with the binding pocket. In
dues of selected compounds are displayed in Table 2, and contrast, the 3-phenylpropenone analogues 3b, 3n, and 3e
Figure 6 shows their corresponding 2D plots. The dihydrodiox- displayed comparatively weak binding within the active site, al-
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Table 2. Induced-fit docking of EDOT analogues with Chk2 kinase.
Compound
DG [kcalmolÀ1
]
Glide energy
H-bonding
interactions
Hydrophobic interactions
[kcalmolÀ1
]
3s
À9.578
À72.022
NÀH···O (Met304) Ile251, Ile250, Leu301, Ile286, Leu226, Ala247, Leu354, Met304, Leu303, Leu236, Val234
NÀH···O (Lys224)
NÀH···O (Lys245)
NÀH···O (Asp368)
3r
10
À8.215
À6.113
À54.952
À53.767
NÀH···O (Met304) Leu354, Leu301, Ile286, Val234, Met304, Leu303, Ala247, Leu226
NÀH···O (Thr367)
NÀH···O (Met304) Ile286, Leu301, Val234, Ala247, Met304, Leu226, Leu303, Leu354
NÀH···O (Lys249)
3p
3b
À8.118
À6.603
À52.025
À51.692
NÀH···O (Met304) Ile299, Ala247, Leu301, Ile248, Met304, Leu303, Leu226, Leu354, Val234
NÀH···O (Met304) Leu354, Ile286, Leu301, Ala247, Leu303, Met304, Leu226, Val234
NÀH···O (Lys249)
12
À6.951
À50.152
NÀH···O (Met304) Val234, Leu354, Leu301, Ile286, Ala247, Met304, Leu236, Val246, Leu303, Leu226
CÀO···H (Glu308)
3n
3e
À7.762
À7.021
À9.031
À49.620
À48.784
À62.126
NÀH···O (Met304) Leu354, Ile299, Ala247, Ile286, Leu301, Val300, Ile248, Met304, Leu303, Leu226, Val234
NÀH···O (Met304) Leu301, Ala247, Val234, Met304, Leu303, Leu236, Leu226, Leu354
NÀH···N (Met304) Val234, Leu301, Ile286, Ala247, Leu354, Phe310, Met304, Leu303, Leu226
CÀO···H (Met304)
Co-crystal[26a]
CÀO···H (Glu308)
Figure 6. Key interacting residues of the ATP-binding domain of Chk2 kinase with the reported analogues.
though the carbonyl group of the enone maintains an essen-
tial hydrogen bond with the hinge region residue Met304.
Thus, from these docking studies, it is clear that the reported
derivatives also interacted with the ribose-binding domain of
Chk2 kinase in a similar manner to 2-aminopyridines, which are
known to inhibit the kinase activity of Chk2.
lationships determined with 23 compounds showed the func-
tional requirement of the phenyl ring and the importance of
the relative positions of the ketone and vinyl spacer for opti-
mal cytotoxicity. Among the investigated compounds, 3p and
3r emerged as potent analogues displaying cytotoxicity at
sub-micromolar concentrations on the tested tumor cell lines.
In particular, 3p exhibited antiproliferative activity similar to
that of the reference antimitotic agent nocodazole. Another
important observation was the 2,5-disubstituted EDOT 3r
having a cytotoxicity profile similar to that of 2-substituted
EDOT 3p, thus providing the possibility of avoiding the toxico-
logical issues associated with unsubstituted thiophene ana-
logues.[33] Consistent with the cytotoxicity assay, 3p severely
Conclusions
In summary, we synthesized various types of thiophene deriva-
tives (3a–u) in good to excellent yields. The cytotoxic poten-
cies of the reported EDOT derivatives were investigated in a
panel of five human cancer cell lines. The structure–activity re-
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Full Papers
(E)-1-(2,3-Dihydrothieno[3,4-b][1,4]dioxin-5-yl)-3-(3,4,5-trime-
thoxyphenyl)prop-2-en-1-one (3b): Condensation of 1 (0.155 g,
0.93 mmol) with 3,4,5-trimethoxybenzaldehyde (2b, 0.20 g,
0.93 mmol) according to the general procedure afforded 3b as a
yellow solid (0.298 g, 88%). Rf =0.25 (20% EtOAc/hexane); mp:
affected the clonogenic potential of HCT116 cells with no
colony growth observed at concentrations as low as 250 nm.
Cell-cycle analysis demonstrated the capacity of 3p to arrest
cells in the G2/M phase with a significant increase in the
number of cells with >4N DNA content, indicating interfer-
ence with mitotic function.[34] Interestingly, experiments with
human topoisomerases identified 3p as weakly inhibiting the
hTopI-mediated relaxation of supercoiled DNA, thus represent-
ing an opportunity for the development of EDOT-enone-based
topoisomerase inhibitors for cancer therapy. Finally, docking re-
sults suggest that the reported EDOT analogues interfere with
the ATP binding pocket of Chk2 kinase catalytic domain in the
manner reported for 2-aminopyrimidines. Furthermore, this
study identified trimethoxyphenyl EDOT derivative 3p as a
lead compound with the potential for future drug develop-
ment.
1
204–2068C; H NMR (300 MHz, CDCl3): d=7.74 (d, J=15.3 Hz, 1H,
vinylic CH), 7.51 (d, J=15.3 Hz, 1H, vinylic CH), 6.86 (s, 2H, ArH),
6.74 (s, 1H, ArH), 4.43–4.29 (m, 4H, OCH2), 3.92 (s, 6H, OCH3),
3.90 ppm (s, 3H, OCH3); 13C NMR (75 MHz, CDCl3): d=180.9 (CO),
153.4 (C), 144.3 (C), 143.1 (CH), 141.9 (C), 140.4 (C), 130.7 (C), 123.2
(CH), 121.3 (C), 109.1 (CH), 105.9 (CH), 65.5 (OCH2), 63.9 (OCH2),
60.9 (OCH3), 56.3 ppm (OCH3); HRMS (ESI): m/z: calcd for C18H19O6S:
363.0902 [M+H]+, found: 363.0916; elemental analysis calcd (%)
for C18H18O6S: C 59.66, H 5.01, found: C 59.83, H 4.88.
(E)-3-(4-Chlorophenyl)-1-(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-
yl)prop-2-en-1-one (3c): Condensation of 1 (0.236 g, 1.42 mmol)
with 4-chlorobenzaldehyde (2c, 0.20 g, 1.42 mmol) according to
the general procedure afforded 3c as a yellow solid (0.352 g, 86%).
1
Rf =0.32 (15% EtOAc/hexane); mp: 152–1548C; H NMR (300 MHz,
CDCl3): d=7.69 (d, J=15.6 Hz, 1H, vinylic CH), 7.53 (d, J=15.9 Hz,
1H, vinylic CH), 7.48 (d, J=8.4 Hz, 2H, ArH), 7.29 (d, J=8.4 Hz, 2H,
ArH), 6.68 (s, 1H, ArH), 4.38–4.21 ppm (m, 4H, OCH2); 13C NMR
(75 MHz, CDCl3): d=180.8 (CO), 144.5 (C), 141.9 (C), 141.4 (CH),
136.1 (C), 133.7 (C), 129.7 (CH), 129.1 (CH), 124.3 (CH), 121.3 (C),
109.5 (CH), 65.5 (OCH2), 63.9 ppm (OCH2); HRMS (ESI): m/z: calcd
for C15H12ClO3S: 307.0196 [M+H]+, found: 307.0209; elemental
analysis calcd (%) for C15H11ClO3S: C 58.73, H 3.61, found: C 58.93,
H 3.76.
Experimental Section
Chemistry
All melting points are reported uncorrected. Progression of the re-
actions was monitored by thin-layer chromatography using
hexane/ethyl acetate as an eluent. Column chromatography was
carried out on silica gel (230–400 mesh, Merck) by using an eluent
1
of increasing polarity. The H, 13C, and DEPT-135 NMR spectra were
recorded in CDCl3 using tetramethylsilane as an internal standard
on a 300 MHz (Bruker Avance II) spectrometer at room tempera-
ture. Chemical shift values are quoted in parts per million (ppm)
and coupling constants J are expressed in hertz (Hz). Elemental
analysis data were recorded with a Elementar Vario Series Analyser
instrument. Commercially available EDOT was purchased from
Sigma–Aldrich and used as received in synthetic transformations.
EDOT-2-carboxaldehyde (5a) and 2-acetyl-EDOT (1) were prepared
using published procedures.[35]
(E)-3-(4-Bromophenyl)-1-(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-
yl)prop-2-en-1-one (3d): Condensation of 1 (0.179 g, 1.08 mmol)
with 4-bromobenzaldehyde (2d, 0.20 g, 1.08 mmol) according to
the general procedure afforded 3d as a yellow solid (0.303 g,
84%). Rf =0.30 (15% EtOAc/hexane); mp: 170–1728C; 1H NMR
(300 MHz, CDCl3): d=7.73 (d, J=15.9 Hz, 1H, vinylic CH), 7.61 (d,
J=15.6 Hz, 1H, vinylic CH), 7.54–7.46 (m, 4H, ArH), 6.74 (s, 1H,
ArH), 4.46–4.43 (m, 2H, OCH2), 4.29–4.26 ppm (m, 2H, OCH2);
13C NMR (75 MHz, CDCl3): d=180.8 (CO), 144.5 (C), 141.8 (C), 141.4
(CH), 134.1 (C), 132.0 (CH), 129.8 (CH), 124.4 (C), 124.3 (CH), 121.2
(C), 109.5 (CH), 65.5 (OCH2), 63.9 ppm (OCH2); HRMS (ESI): m/z:
calcd for C15H12BrO3S: 350.9691 [M+H]+, found: 350.9689; elemen-
tal analysis calcd (%) for C15H11BrO3S: C 51.30, H 3.16, found: C
51.48, H 3.28.
General procedure for the synthesis of EDOT derivatives: The
substituted benzaldehydes (1.0 mmol) were added to stirred solu-
tions of 2-acetyl-EDOT (1, 1.0 mmol) in 10% NaOH in ethanol/
water (2:1, 15 mL), and the reaction mixtures were heated at reflux
for 1 h. Solid products precipitated and were filtered and washed
with ethanol (10 mL) to give the respective 1,3-diaryl-2-propenone
derivatives in good to excellent yields.
(E)-3-(3-Bromo-4-fluorophenyl)-1-(2,3-dihydrothieno[3,4-b]
[1,4]dioxin-5-yl)prop-2-en-1-one (3e): Condensation of 1 (0.18 g,
0.98 mmol) with 3-bromo-4-fluorobenzaldehyde (2e, 0.20 g,
0.98 mmol) according to the general procedure afforded 3e as a
yellow solid (0.317 g, 87%). Rf =0.27 (15% EtOAc/hexane); mp:
(E)-1-(2,3-Dihydrothieno[3,4-b][1,4]dioxin-5-yl)-3-(3,4-dimethoxy-
phenyl)prop-2-en-1-one (3a): Condensation of
1
(0.18 g,
1.08 mmol) with veratraldehyde (2a, 0.20, 1.08 mmol) according to
the general procedure afforded 3a as a yellow solid (0.302 g,
84%). Rf =0.28 (15% EtOAc/hexane); mp: 142–1468C; 1H NMR
(300 MHz, CDCl3): d=7.77 (d, J=15.6 Hz, 1H, vinylic CH), 7.50 (d,
J=15.6 Hz, 1H, vinylic CH), 7.28–7.22 (m, 1H, ArH), 7.13 (s, 1H,
ArH), 6.89 (d, J=8.4 Hz, 1H, ArH), 6.72 (s, 1H, ArH), 4.44–4.41 (m,
2H, OCH2), 4.29–4.27 (m, 2H, OCH2), 3.94 (s, 3H, OCH3), 3.92 ppm
(s, 3H, OCH3); 13C NMR (75 MHz, CDCl3): d=181.1 (CO), 151.2 (C),
149.1 (C), 144.2 (C), 143.1 (CH),141.9 (C), 128.2 (C), 122.7 (CH), 121.8
(CH), 121.5 (C),111.2 (CH), 110.9 (CH), 108.9 (CH), 65.5 (OCH2), 63.9
(OCH2), 55.9 ppm (OCH3); DEPT-135 NMR (75 MHz, CDCl3): d=
143.1, 122.7, 121.8, 111.2, 110.9, 108.9, 65.5, 63.9, 55.9 ppm; HRMS
(ESI): m/z: calcd for C17H17O5S: 333.0797 [M+H]+, found:
333.0783l; elemental analysis calcd (%) for C17H16O5S: C 61.43, H
4.85, found: C 61.62, H 4.67.
1
176–1788C; H NMR (300 MHz, CDCl3): d=7.83–7.81 (m, 1H, ArH),
7.70 (d, J=15.6 Hz, 1H, vinylic CH), 7.57–7.52 (m, 2H, ArH), 7.17–
7.12 (m, 1H, ArH), 6.76 (s, 1H, ArH), 4.47–4.45 (m, 2H, OCH2), 4.28–
4.27 ppm (s, 2H, OCH2); 13C NMR (75 MHz, CDCl3): d=180.6 (CO),
4
143.3 (d, 1JCF =204.8 Hz), 140.1 (CH), 133.1 (CH), 133.0 (d, JCF
=
2.6 Hz), 129.3 (d, 2JCF =7.1 Hz), 124.7 (d, 3JCF =2.7 Hz), 121.2 (C),
116.9 (d, 2’JCF =22.7 Hz), 109.7 (d, 3’JCF =5.3 Hz), 65.6 (OCH2),
63.9 ppm (OCH2); DEPT-135 NMR (75 MHz, CDCl3): d=140.1, 133.1,
2’
129.3 (d, 3’JCF =7.1 Hz), 124.7 (d, 3JCF =2.7 Hz), 116.9 (d,
J =
CF
22.7 Hz), 109.7, 65.6, 64.0 ppm; HRMS (ESI): m/z: calcd for
C15H11BrFO3S: 368.9596 [M+H]+, found: 368.9610; elemental analy-
sis calcd (%) for C15H10BrFO3S: C 48.80, H 2.73, found: C 48.96, H
2.90.
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(E)-3-(4-Chloro-3-fluorophenyl)-1-(2,3-dihydrothieno[3,4-b]
[1,4]dioxin-5-yl)prop-2-en-1-one (3 f): Condensation of 1 (0.23 g,
1.26 mmol) with 4-chloro-3-fluorobenzaldehyde (2 f, 0.20 g,
1.26 mmol) according to the general procedure afforded 3 f as a
yellow solid (0.348 g, 85%). Rf =0.29 (15% EtOAc/hexane); mp:
(E)-3-(3,4-Dimethoxyphenyl)-1-(thiophen-2-yl)prop-2-en-1-one
(3j): Condensation of 2-acetylthiophene (4a, 0.15 g, 1.20 mmol)
with 2a (0.20 g, 1.20 mmol) according to the general procedure af-
forded 3j as a yellow solid (0.28 g, 85%). Rf =0.24 (10% EtOAc/
1
hexane); mp: 76–788C (lit.102–1038C[37]); H NMR (300 MHz, CDCl3):
1
>3008C; H NMR (300 MHz, CDCl3): d=7.82 (d, J=15.6 Hz, 1H, vi-
nylic CH), 7.51 (d, J=15.9 Hz, 1H, vinylic CH), 7.36–7.19 (m, 3H,
d=7.87 (d, J=3.6 Hz, 1H, ArH), 7.79 (d, J=15.3 Hz, 1H, vinylic CH),
7.66 (d, J=4.8 Hz, 1H, ArH), 7.32 (s, 1H, ArH), 7.25–7.15 (m, 3H,
ArH), 6.89 (d, J=8.1 Hz, 1H, ArH), 3.95 (s, 3H, OCH3), 3.92 ppm (s,
3H, OCH3); 13C NMR (75 MHz, CDCl3): d=182.0 (CO), 151.5 (C),
149.2 (C), 145.7 (C), 144.2 (CH), 133.6 (CH), 131.6 (CH), 128.2 (CH),
127.7 (C), 123.2 (CH), 119.5 (CH), 111.2 (CH), 110.3 (CH), 56.0 (OCH3),
55.9 ppm (OCH3); HRMS (ESI): m/z: calcd for C15H15O3S: 275.0742
[M+H]+, found: 275.0750; elemental analysis calcd (%) for
C15H14O3S: C 65.67, H 5.14, found: C 65.80, H 5.07.
ArH), 6.68 (s, 1H, ArH), 4.22–4.19 (m, 2H, OCH2), 4.39–4.37 ppm (m,
1
2H, OCH2); 13C NMR (75 MHz, CDCl3): d=180.5 (CO), 158.3 (d, JCF
=
247.7 Hz), 144.7 (C), 141.9 (C), 140.3 (d, 4JCF =2.5 Hz), 135.9 (d,
3
3’JCF =6.8 Hz), 131.0 (CH), 125.4 (C), 125.3 (d, JCF =3.4 Hz), 122.7 (d,
2JCF =17.9 Hz), 121.2 (C), 115.4 (d, 2’JCF =21.4 Hz), 109.8 (CH), 65.6
(OCH2), 63.9 ppm (OCH2); DEPT-135 NMR (75 MHz, CDCl3): d=140.3
4
3
2’
(d, JCF =2.5 Hz), 131.0, 125.3 (d, JCF =3.4 Hz), 125.2, 115.4 (d,
J =
CF
21.4 Hz), 109.9, 65.6, 63.9 ppm; HRMS (ESI): m/z: calcd for
C15H11ClFO4S: 325.0101 [M+H]+, found: 325.0119; elemental analy-
sis calcd (%) for C15H10ClFO3S: C 55.48, H 3.10, found: C 55.63, H
3.28.
(E)-3-(4-Chlorophenyl)-1-(thiophen-2-yl)prop-2-en-1-one
(3k):
Condensation of 4a (0.18 g, 1.42 mmol) with 2c (0.20 g,
1.42 mmol) according to the general procedure afforded 3k as a
colorless solid (0.295 g, 83%). Rf =0.34 (10% EtOAc/hexane); mp:
1
112–1148C (lit. 110–1118C[38]); H NMR (300 MHz, CDCl3): d=7.88 (d,
(E)-3-(2,6-Dichlorophenyl)-1-(2,3-dihydrothieno[3,4-b][1,4]dioxin-
5-yl)prop-2-en-1-one (3g): Condensation of 1 (0.21 g, 1.14 mmol)
with 2,6-dichlorobenzaldehyde (2g, 0.20 g, 1.14 mmol) according
to the general procedure afforded 3g as a yellow solid (0.32 g,
82%). Rf =0.32 (15% EtOAc/hexane); mp: >3008C; 1H NMR
(300 MHz, CDCl3): d=7.89 (d, J=15.6 Hz, 1H, vinylic CH), 7.80 (d,
J=15.9 Hz, 1H, vinylic CH), 7.37–7.18 (m, 3H, ArH), 6.76 (s, 1H,
ArH), 4.38 (s, 2H, OCH2), 4.26 ppm (m, 4H, OCH2); 13C NMR
(75 MHz, CDCl3): d=180.7 (CO), 144.9 (C), 141.9 (C), 136.0 (CH),
135.3 (C), 132.9 (C), 131.8 (CH), 129.6 (CH), 128.8 (CH), 121.0 (C),
109.8 (CH), 65.4 (OCH2), 63.9 ppm (OCH2); HRMS (ESI): m/z: calcd
for C15H11Cl2O3S: 340.9806 [M+H]+, found: 340.9818; elemental
analysis calcd (%) for C15H10Cl2O3S: C 52.80, H 2.95, found: C 53.01,
H 2.79.
J=3.3 Hz, 1H, ArH), 7.78 (d, J=15.6 Hz, 1H, vinylic CH), 7.69 (d, J=
4.8 Hz, 1H, ArH), 7.57 (d, J=8.4 Hz, 1H, ArH), 7.41–7.36 (m, 3H,
ArH), 7.18 ppm (t, J=4.2 Hz, 1H, ArH); 13C NMR (75 MHz, CDCl3):
d=181.8 (CO), 145.4 (C), 142.6 (CH), 136.5 (C), 134.1 (CH), 133.2 (C),
131.9 (CH), 129.7 (CH), 129.3 (CH), 128.4 (CH), 122.1 ppm (CH);
HRMS (ESI): m/z: calcd for C13H10ClOS: 249.0141 [M+H]+, found:
249.0135; elemental analysis calcd (%) for C13H9ClOS: C 62.78, H
3.65, found: C 62.89, H 3.54.
(E)-3-(3-Bromo-4-fluorophenyl)-1-(thiophen-2-yl)prop-2-en-1-one
(3l): Condensation of 4a (0.12 g, 0.98 mmol) with 2e (0.20 g,
0.98 mmol) according to the general procedure afforded 3l as a
colorless solid (0.245 g, 80%). Rf =0.29 (10% EtOAc/hexane); mp:
142–1448C; 1H NMR (300 MHz, CDCl3): d=7.79 (d, J=3.6 Hz, 1H,
ArH), 7.76–7.73 (m, 1H, ArH), 7.65–7.60 (m, 2H, ArH), 7.47–7.42 (m,
1H, ArH), 7.25 (d, J=15.6 Hz, 1H, vinylic CH), 7.11–7.04 ppm (m,
(E)-1-(2,3-Dihydrothieno[3,4-b][1,4]dioxin-5-yl)-3-(thiophen-2-
yl)prop-2-en-1-one (3h): Condensation of 1 (0.33 g, 1.78 mmol)
with thiophene-2-carboxaldehyde (2h, 0.20 g, 1.78 mmol) accord-
ing to the general procedure afforded 3h as a red solid (0.413 g,
83%). Rf =0.38 (15% EtOAc/hexane); mp: 134–1368C (lit. 133–
1
2H, ArH); 13C NMR (75 MHz, CDCl3): d=181.4 (CO), 160.7 (d, JCF
=
251.1 Hz), 145.1 (C), 141.1 (CH), 134.2 (CH), 133.0 (CH), 132.4 (d,
3JCF =3.9 Hz), 132.0 (CH), 129.3 (d, 3’JCF =7.6 Hz), 128.3 (CH), 122.4
2
(d, 4JCF =2.3 Hz), 117.0 (d, 2’JCF =22.7 Hz), 109.8 ppm (d, JCF
=
1
1348C[36]); H NMR (300 MHz, CDCl3): d=7.93 (d, J=15.3 Hz, 1H, vi-
21.5 Hz); DEPT 135-NMR (75 MHz, CDCl3): d=141.1, 134.4, 133.1,
132.1, 129.3 (d, 3’JCF =7.6 Hz), 128.4, 122.4 (d, 3JCF =2.3 Hz),
117.0 ppm (d, 2’JCF =22.7 Hz); HRMS (ESI): m/z: calcd for
C13H9BrFOS: 310.9542 [M+H]+, found: 310.9549; elemental analy-
sis calcd (%) for C13H8BrFOS: C 50.18, H 2.59, found: C 50.29, H
2.48.
nylic CH), 7.44 (d, J=15.6 Hz, 1H, vinylic CH), 7.39 (d, J=4.8 Hz,
1H, ArH), 7.33 (d, J=3.3 Hz, 1H, ArH), 7.09–7.06 (m, 1H, ArH), 6.72
(s, 1H, ArH), 4.45–4.42 (m, 2H, OCH2), 4.28–4.26 ppm (s, 2H, OCH2);
13C NMR (75 MHz, CDCl3): d=180.6 (CO), 144.3 (C), 141.9 (C), 140.7
(C), 135.4 (CH), 131.6 (CH), 128.4 (CH), 128.3 (CH), 122.9 (CH), 121.3
(C), 109.1 (CH), 65.5 (OCH2), 63.9 ppm (OCH2); HRMS (ESI): m/z:
calcd for C13H11O3S2: 279.0150 [M+H]+, found: 279.0165; elemental
analysis calcd (%) for C13H10O3S2: C 56.10, H 3.62, found: C 56.30, H
3.52.
(E)-3-(Thiophen-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(3m): Condensation of 2b (0.38 g, 1.78 mmol) with 2h (0.20 g,
1.78 mmol) according to the general procedure afforded 3m as a
red solid (0.455 g, 84%). Rf =0.30 (20% EtOAc/hexane); mp: 82–
848C (lit. 69–718C[39]); 1H NMR (300 MHz, CDCl3): d=7.95 (d, J=
15.3 Hz, 1H, vinylic CH), 7.43 (d, J=5.1 Hz, 1H, ArH), 7.38 (d, J=
3.6 Hz, 1H, ArH), 7.31–7.26 (m, 3H, ArH), 7.11 (t, J=4.2 Hz, 1H,
ArH), 3.95 ppm (s, 9H, OCH3); 13C NMR (75 MHz, CDCl3): d=188.4
(CO), 153.0 (C), 142.5 (C), 140.2 (C), 136.9 (CH), 133.3 (C), 131.8 (CH),
128.6 (CH), 128.2 (CH), 120.3 (CH), 106.0 (CH), 60.8 (OCH3),
56.3 ppm (OCH3); HRMS (ESI): m/z: calcd for C16H17O4S: 305.0848
[M+H]+, found: 305.0860; elemental analysis calcd (%) for
C16H16O4S: C 63.14, H 5.30, found: C 63.33, H 5.11.
(E)-1,3-Bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)prop-2-en-1-
one (3i): Condensation of 1 (0.22 g, 1.17 mmol) with EDOT-2-car-
boxaldehyde (5a, 0.20 g, 1.17 mmol) according to the general pro-
cedure afforded 3i as a yellow solid (0.308 g, 78%). Rf =0.25 (20%
1
EtOAc/hexane); mp: 196–1988C; H NMR (300 MHz, CDCl3): d=7.86
(d, J=15.3 Hz, 1H, vinylic CH), 7.36 (d, J=15.3 Hz, 1H, vinylic CH),
6.67 (s, 1H, ArH), 6.42 (s, 1H, ArH), 4.41–4.22 ppm (m, 8H, OCH2);
13C NMR (75 MHz, CDCl3): d=180.7 (CO), 144.0 (C), 143.6 (C), 142.3
(C), 141.9 (C), 132.1 (CH), 121.7 (C), 120.6 (CH), 115.7 (C), 108.3 (CH),
102.7 (CH), 65.4 (OCH2), 65.1 (OCH2), 64.6 (OCH2), 64.1 ppm (OCH2);
HRMS (ESI): m/z: calcd for C15H13O5S2: 337.0204 [M+H]+, found:
337.0221; elemental analysis calcd (%) for C15H12O5S2: C 53.56, H
3.60, found: C 53.71, H 3.73.
(E)-3-(3-Methylthiophen-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (3n): Condensation of 2b (0.33 g, 1.59 mmol) with 3-
methyl-thiophene-2-carboxaldehyde (0.20 g, 1.59 mmol) according
to the general procedure afforded 3n as a red oil (0.435 g, 86%).
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Rf =0.33 (20% EtOAc/hexane); 1H NMR (300 MHz, CDCl3): d=8.04
(d, J=15.0 Hz, 1H, vinylic CH), 7.31 (d, J=5.1 Hz, 1H, ArH), 7.26–
7.20 (m, 3H, ArH), 6.92 (d, J=4.8 Hz, 1H, ArH), 3.96 (s, 6H, OCH3)
3.94 (s, 3H, OCH3), 2.41 ppm (s, 3H, CH3); 13C NMR (75 MHz, CDCl3):
d=188.6 (CO), 153.1 (C), 142.8 (C), 142.4 (C), 135.5 (CH), 134.5 (C),
133.6 (C), 131.5 (CH), 127.3 (CH), 119.5 (CH), 106.0 (CH), 60.9
(OCH3), 56.4 (OCH3), 14.3 ppm (CH3); HRMS (ESI): m/z: calcd for
C17H19O4S: 319.1004 [M+H]+, found: 319.1018; elemental analysis
calcd (%) for C17H18O4S: C 64.13, H 5.70, found: C 64.32, H 5.86.
(0.17 g, 0.80 mmol) with crude 7 (0.20 g, 0.80 mmol) according to
the general procedure afforded 3r as a green solid (0.275 g, 78%).
Rf =0.23 (25% EtOAc/hexane); mp: 168–1708C; H NMR (300 MHz,
1
CDCl3): d=7.97 (d, J=15.3 Hz, 1H, vinylic CH), 7.23 (s, 2H, ArH),
7.15 (d, J=15.3 Hz, 1H, vinylic CH), 4.34 (s, 4H, OCH2), 3.95 (s, 6H,
OCH3), 3.93 ppm (s, 3H, OCH3); 13C NMR (75 MHz, CDCl3): d=188.5
(CO), 153.1 (C), 142.9 (C), 142.4 (C), 140.6 (C), 133.7 (C), 132.7 (CH),
118.3 (CH), 115.4 (C), 106.0 (CH), 92.2 (CBr), 65.0 (OCH2), 64.9
(OCH2), 60.9 (OCH3), 56.4 ppm (OCH3); HRMS (ESI): m/z: calcd for
C18H18BrO6S: 441.0007 [M+H]+, found: 441.0019; elemental analy-
sis calcd (%) for C18H17BrO6S: C 48.99, H 3.88, found: C 49.18, H
3.74.
(E)-3-[(2,2’-Bithiophen)-5-yl]-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (3o): Condensation of 2b (0.22 g, 1.03 mmol) with 2,2’-
bithiophene-5-carboxaldehyde (0.20 g, 1.03 mmol) according to
the general procedure afforded 3o as a yellow solid (0.35 g, 88%).
(2E,2’E)-3,3’-(2,3-Dihydrothieno[3,4-b][1,4]dioxine-5,7-diyl)bis[1-
(3,4,5-trimethoxyphenyl)prop-2-en-1-one] (3s): Condensation of
6a (0.45 g, 2.12 mmol) with EDOT-2,5-dicarboxaldehyde (0.20 g,
1.01 mmol) according to the general procedure afforded 3s as a
yellow solid (0.435 g, 74%). Rf =0.29 (40% EtOAc/hexane); mp:
1
Rf =0.30 (20% EtOAc/hexane); mp: 132–1348C; H NMR (300 MHz,
CDCl3): d=7.90 (d, J=15.0 Hz, 1H, vinylic CH), 7.31–7.26 (m, 6H,
ArH), 7.18 (d, J=3.9 Hz, 1H, ArH), 7.07–7.05 (m, 1H, ArH), 3.97 (s,
6H, OCH3) 3.94 ppm (s, 3H, OCH3); 13C NMR (75 MHz, CDCl3): d=
188.3 (CO), 153.2 (C), 142.5 (C), 140.8 (2C), 138.9 (C), 136.9 (CH),
136.7 (C), 133.5 (CH), 128.2 (CH), 125.9 (CH), 124.9 (CH), 124.6 (CH),
119.9 (CH), 106.0 (CH), 60.9 (OCH3), 56.4 ppm (OCH3); HRMS (ESI):
m/z: calcd for C20H19O4S2: 387.0725 [M+H]+, found: 387.0740; ele-
mental analysis calcd (%) for C20H18O4S2: C 62.16, H 4.69, found: C
62.36, H 4.59.
1
246–2488C; H NMR (300 MHz, CDCl3): d=7.86 (d, J=15.0 Hz, 2H,
vinylic CH), 7.34–7.26 (m, 6H, ArH), 4.41 (s, 4H, OCH2), 3.96 ppm (s,
18H, OCH3); 13C NMR (75 MHz, CDCl3): d=188.4 (CO), 153.2 (C),
143.7 (C), 142.6 (C), 133.5 (C), 132.7 (CH), 120.3 (CH), 117.8 (C),
106.2 (CH), 65.0 (OCH2), 60.9 (OCH2), 56.5 (OCH3), 56.4 ppm (OCH3);
HRMS (ESI): m/z: calcd for C30H31O10S: 583.1638 [M+H]+, found:
583.1650; elemental analysis calcd (%) for C30H30O10S: C 61.85, H
5.19, found: C 61.99, H 5.08.
(E)-3-(2,3-Dihydrothieno[3,4-b][1,4]dioxin-5-yl)-1-(3,4,5-trime-
thoxyphenyl)prop-2-en-1-one (3p): Condensation of 6a (0.25 g,
1.18 mmol) with EDOT-2-carboxaldehyde (5a, 0.20 g, 1.18 mmol)
according to the general procedure afforded 3p as a yellow solid
(0.365 g, 86%). Rf =0.25 (20% EtOAc/hexane); mp: 148–1508C;
1H NMR (300 MHz, CDCl3): d=7.85 (d, J=15.3 Hz, 1H, vinylic CH),
7.27–7.23 (m, 3H, ArH), 6.50 (s, 1H, ArH), 4.36–4.34 (m, 2H, OCH2),
4.26–4.24 (m, 2H, OCH2), 3.95 (s, 6H, OCH3), 3.93 ppm (s, 3H,
OCH3); 13C NMR (75 MHz, CDCl3): d=188.9 (CO), 153.1 (C), 143.8
(2C), 142.2 (C), 133.9 (CH), 133.9 (C), 118.3 (CH), 115.2 (C), 106.0
(CH), 103.4 (CH), 65.1 (OCH2), 64.5 (OCH2), 60.9 (OCH3), 56.4 ppm
(OCH3); HRMS (ESI): m/z: calcd for C18H19O6S: 363.0902 [M+H]+,
found: 363.0919; elemental analysis calcd (%) for C18H18O6S: C
59.66, H 5.01, found: C 59.80, H 5.11.
(2,3-Dihydrothieno[3,4-b][1,4]dioxin-5-yl)(3,4,5-trimethoxyphe-
nyl)methanone (3t): N,N-Dimethylformamide (1 drop) was added
to a solution of EDOT-2-carboxylic acid (0.25 g, 1.34 mmol) in thio-
nyl chloride (0.15 mL, 2.0 mmol) at 08C. The reaction mixture was
then heated in a water bath (908C) for 30 min. Subsequent remov-
al of excess thionyl chloride under reduced pressure gave the
crude acid chloride as a colorless liquid (0.24 g), which was used in
the next step without further purification.
Tin(IV) chloride (0.14 mL, 1.19 mmol) was slowly added to a solu-
tion of the crude EDOT acid chloride (0.24 g) in anhydrous CH2Cl2
(10 mL) at 08C. To this, a solution of 3,4,5-trimethoxybenzene
(0.225 g, 1.34 mmol) in anhydrous CH2Cl2 (5.0 mL) was added and
the mixture was stirred at room temperature for 3 h. After comple-
tion of the reaction (monitored by TLC), the mixture was poured
into ice-water (15 mL) containing conc. HCl (5.0 mL). The organic
layer was separated and the aqueous layer was extracted with
CH2Cl2 (2ꢁ5 mL). The combined organic layers were washed with
water and dried (Na2SO4). Purification of the crude product by
column chromatography (silica gel, 20% ethyl acetate in hexane)
afforded 3t as a green oil (0.315 g, 70%). Rf =0.40 (20% EtOAc/
hexane); 1H NMR (300 MHz, CDCl3): d=8.10 (s, 1H, ArH), 6.96 (s,
2H, ArH), 4.30–4.28 (m, 4H, OCH2), 3.94 (s, 3H, OCH3) 3.89 ppm (s,
6H, OCH3); 13C NMR (75 MHz, CDCl3): d=184.3 (CO), 155.9 (C),
152.2 (C), 144.3 (C), 141.8 (C), 138.3 (C), 127.4 (C), 123.8 (CH), 106.6
(CH), 65.1 (OCH2), 64.4 (OCH2), 62.2 (OCH3), 60.9 (OCH3), 56.1 ppm
(OCH3); HRMS (ESI): m/z: calcd for C16H17O6S: 337.0746 [M+H]+,
found: 337.0752; elemental analysis calcd (%) for C16H16O6S: C
57.13, H 4.79, found: C 57.31, H 4.60.
(E)-3-(2,3-Dihydrothieno[3,4-b][1,4]dioxin-5-yl)-1-(3,4-dimethoxy-
phenyl)prop-2-en-1-one (3q): Condensation of 6b (0.21 g,
1.17 mmol) with 5a (0.20 g, 1.17 mmol) according to the general
procedure afforded 3q as a yellow solid (0.332 g, 85%). Rf =0.28
(15% EtOAc/hexane); mp: 190–1928C; 1H NMR (300 MHz, CDCl3):
d=7.84 (d, J=15.3 Hz, 1H, vinylic CH), 7.67–7.61 (m, 2H, ArH), 7.34
(d, J=15.3 Hz, 1H, ArH), 6.92 (d, J=8.4 Hz, 1H, ArH), 6.48 (s, 1H,
ArH), 4.36–4.34 (m, 2H, OCH2), 4.27–4.24 (m, 2H, OCH2), 3.96 ppm
(s, 6H, OCH3); 13C NMR (75 MHz, CDCl3): d=188.2 (CO), 153.0 (C),
149.2 (C), 143.5 (C), 142.2 (C), 133.2 (CH), 131.6 (C), 122.8 (CH),
118.3 (CH), 115.4 (C), 110.8 (CH), 109.9 (CH), 103.0 (CH), 65.1 (OCH2),
64.5 (OCH2), 56.1 (OCH3), 56.0 ppm (OCH3); HRMS (ESI): m/z: calcd
for C17H17O5S: 333.0797 [M+H]+, found: 333.0787; elemental anal-
ysis calcd (%) for C17H16O5S: C 61.43, H 4.85, found: C 61.63, H 4.73.
7-Bromo-2,3-dihydrothieno[3,4-b][1,4]dioxine-5-carboxaldehyde
(7): N-Bromosuccinimide (0.46 g, 2.59 mmol) was added to a stirred
solution of 5a (0.40 g, 2.35 mmol) in DMF (10 mL), and the mixture
was stirred (shielded from light) at room temperature for 14 h.
After completion of reaction, the mixture was poured over crushed
ice (50 g) and stirred for 10 min. The resulting solid was filtered,
washed with water, and dried. The crude 7 (0.45 g) was used in the
next step without further purification.
7-(3,4-Dimethoxyphenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxine-5-
carboxaldehyde: 3,4-Dimethoxyphenylboronic acid (0.145 g,
0.80 mmol), Pd(PPh3)4 (0.093 g, 0.08 mmol), K2CO3 (0.22 g,
1.60 mmol), and H2O (2 drops) were added to a solution of 5-
bromo-EDOT-2-carboxaldehyde (0.20 g, 0.80 mmol) in DMF (15 mL).
The reaction mixture was degassed and stirred at 808C for 2 days.
The resulting black solution was passed through a Celite pad and
the pad was washed with CH2Cl2 (10 mL). The filtrate was washed
(E)-3-(7-Bromo-2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (3r): Condensation of 6a
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Full Papers
with cold water (20 mL) and the aqueous layer was extracted with
CH2Cl2 (2ꢁ5 mL). The combined organic layers were washed with
water and dried (Na2SO4). Evaporation of solvent followed by
column chromatographic purification (silica gel, 25% ethyl acetate
in hexane) afforded 5-(3,4-dimethoxyphenyl)EDOT-2-carboxalde-
hyde as a red solid (0.184 g, 75%). Rf =0.36 (15% EtOAc/hexane);
mp: 190–1928C; 1H NMR (300 MHz, CDCl3): d=9.91 (s, 1H, CHO),
7.39 (d, J=8.4 Hz, 1H, ArH), 7.31 (s, 1H, ArH), 6.90 (d, J=8.4 Hz,
1H, ArH), 4.41–4.39 (m, 4H, OCH2), 3.92 ppm (s, 6H, OCH3);
13C NMR (75 MHz, CDCl3): d=179.4 (CHO), 149.7 (C), 149.1 (C),
149.1 (C), 136.9 (C), 129.3 (C), 124.6 (C), 120.1 (CH), 114.9 (C), 111.3
(CH), 110.2 (CH), 65.2 (OCH2), 64.6 (OCH2), 56.0 ppm (OCH3); HRMS
(ESI): m/z: calcd for C15H15O5S: 307.0640 [M+H]+, found: 307.0653.
for C16H12BrN3O2S: C 49.24, H 3.10, N 10.77, found: C 49.42, H 3.29,
N 10.94.
Biological studies
Cell lines and cytotoxicity assays: Human cancer cell lines select-
ed for the study—HCT116 (colon cancer), NCI-H460 (lung cancer),
U251 (glioblastoma), HeLa (cervical cancer), and MCF7 (breast
cancer)—were purchased from ATCC (Manassas, VA, USA) and cul-
tured following the manufacturer’s protocol. The above tumor cell
lines were grown in DMEM medium containing 10% fetal bovine
serum supplemented with l-glutamine (2 mm) and penicillin–strep-
tomycin antibiotic solution at 378C with 5% CO2. For cytotoxicity
assays, cells were seeded at densities of 1500–3000 cells per well
in 96-well cell culture plates. Stock concentrations of EDOT ana-
logues (20 mm) were made in DMSO and diluted in the culture
media to working concentrations starting from 20 mm. Thus, test
compounds at concentrations ranging from 20 mm to 1.2 nm were
added to overnight adhered cells and incubated for a further 48 h
at 378C. The antiproliferative effects of the test compounds were
assessed using a sulforhodamine B assay[40] following the protocol
developed by the NCI.[41] The growth inhibitory parameter GI50
value represents the time-zero-corrected drug concentration re-
quired to cause 50% growth inhibition relative to that of vehicle
control. The standard deviation from at least two independent ex-
periments were calculated using Microsoft Excel.
(E)-3-[7-(3,4-Dimethoxyphenyl)-2,3-dihydrothieno[3,4-b]
[1,4]dioxin-5-yl]-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3u):
Condensation of 6a (0.10 g, 0.49 mmol) with 5-(3,4-dimethoxyphe-
nyl)EDOT-2-carboxaldehyde (0.15 g, 0.49 mmol) according to the
general procedure afforded 3u as a red solid (0.20 g, 82%). Rf =
0.29 (25% EtOAc/hexane); mp: 172–1748C; 1H NMR (300 MHz,
CDCl3): d=7.93 (d, J=14.7 Hz, 1H, vinylic CH), 7.31–7.18 (m, 5H,
ArH), 6.90 (d, J=7.8 Hz, 1H, ArH), 4.39 (m, 4H, OCH2), 3.96 ppm (s,
15H, OCH3); 13C NMR (75 MHz, CDCl3): d=188.6 (CO), 153.1 (C),
149.1 (C), 144.7 (2C), 142.2 (C), 137.7 (C), 134.1 (C), 133.6 (CH),
125.2 (C), 121.7 (C), 119.5 (CH), 117.3 (CH), 112.3 (C), 111.4 (CH),
109.9 (CH), 106.0 (CH), 64.9 (OCH2), 64.7 (OCH2), 60.9 (OCH3), 56.4
(OCH3), 56.0 ppm (OCH3); HRMS (ESI): m/z: calcd for C26H27O8S:
499.1427 [M+H]+, found: 499.1443; elemental analysis calcd (%)
for C26H26O8S: C 62.64, H 5.26, found: C 62.84, H 5.12.
Clonogenic cell survival assay: Clonogenic assays were performed
on HCT116 and NCI-H460 cells according to the reported proce-
dure.[42] In brief, EDOT analogues at different concentrations were
incubated with cells seeded at a very low density (100 cells per
well of a six-well plate), followed by washing with Dulbecco’s
phosphate-buffered saline (DPBS) and culturing the drug-treated
cells for a further 12 days in DMEM culture medium. The emerging
colonies at the end of the assay were stained with crystal violet
and photographed.
3-(2,3-Dihydrothieno[3,4-b][1,4]dioxin-5-yl)-5-(3,4-dimethoxy-
phenyl)isoxazole (10): Hydroxylamine hydrochloride (9, 0.05 g,
0.72 mmol) and K2CO3 (0.17 g, 1.23 mmol) were added to a solu-
tion of enone 3a (0.20 g, 0.60 mmol) in THF (15 mL). Then, the re-
action mixture was heated at reflux for 2 h. After completion of re-
action, the mixture was poured into ice-water (20 mL) and stirred
for 10 min. The resulting solid was filtered and triturated with
methanol (10 mL) to give isoxazole 10 as a yellow solid (0.146 g,
70%). Rf =0.24 (10% EtOAc/hexane); mp: 110–1128C; 1H NMR
(300 MHz, CDCl3): d=7.38 (s, 1H, ArH), 7.29 (d, J=8.1 Hz, 1H, ArH),
6.89 (d, J=8.1 Hz, 1H, ArH), 6.67 (s, 1H, ArH), 6.43 ppm (s, 1H,
ArH); 13C NMR (75 MHz, CDCl3): d=163.2 (C), 162.6 (C), 150.6 (C),
149.3 (C), 141.7 (C), 140.3 (C), 121.9 (C), 120.0 (CH), 111.1 (CH),
109.5 (CH), 104.8 (C), 101.9 (CH), 97.3 (CH), 65.2 (OCH2), 64.5
(OCH2), 56.1 (OCH3), 55.9 ppm (OCH3); HRMS (ESI): m/z: calcd for
C17H16NO5S: 346.0749 [M+H]+, found: 346.0762; elemental analysis
calcd (%) for C17H15NO5S: C 59.12, H 4.38, N 4.06, found: C 59.31, H
4.49, N 4.19.
Flow cytometry: Cell-cycle analysis was performed on HCT116
cells collected at the end of 24 and 48 h post-treatment with EDOT
enones (2 mm). The collected cells were washed with DPBS and
fixed using 70% ethanol solution. Immediately prior to staining,
the cells were washed twice with DPBS and stained with propidi-
um iodide (20 mgmLÀ1) in DPBS containing Triton X-100 (0.1% v/v)
and RNase A (0.2 mgmLÀ1) for 1 h at room temperature. The popu-
lations of cells in different phases was analyzed using a Beckman–
Coulter MoFlo XDP Cell Sorter flow cytometer.
Plasmid relaxation assay: The activities of human topoisomerase-
s I and II were assayed by analyzing the retarded mobility of the re-
laxed isomers of supercoiled pBS (SK+)[pBluescript(SK+)] DNA on
a 1.2% agarose gel. Recombinant human topoisomerase I (hTopI)
was expressed in Baculoviral Sf9 insect cell system and purified
using a reported procedure,[43] whereas the human topoisomerase I
(hTopII) was purchased from TopoGEN Inc. (Human Topo IIa, Topo-
GEN Inc.). The plasmid relaxation assay was carried out in relaxa-
tion buffer (25 mm Tris-HCl, pH 7.5, 5% glycerol, 0.5 mm
DTT,10 mm MgCl2, 50 mm KCl, 25 mm EDTA, and 150 mgmLÀ1 BSA)
for hTopI,[44] and by following the manufacturer’s protocol for
hTopII. Prior to the addition of enzyme to the reaction mixture, the
temperature of the DNA and buffer mixture was adjusted to 378C.
The concentration of the EDOT analogues 3p and 3r was 200 mm.
After 30 min, the enzyme reaction was stopped using stop solution
(5% SDS, 15% Ficoll, and 0.25% bromophenol blue) and the
sample was kept on ice until the resolution of plasmid DNA in
4-(4-Bromophenyl)-6-(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-
pyrimidin-2-amine (12): Guanidine hydrochloride (11, 0.08 g,
0.84 mmol) was added to
a solution of enone 3d (0.20 g,
0.57 mmol) in 10% NaOH in ethanol/water (2:1, 30 mL), and the re-
action mixture was heated at reflux for 2 h. The resulting precipi-
tate was filtered and washed with ethanol (5.0 mL) to give pyrimi-
dine 12 as a yellow solid (0.12 g, 60%). Rf =0.30 (25% EtOAc/
hexane); mp: >3008C; 1H NMR (300 MHz, CDCl3): d=7.84 (d, J=
8.4 Hz, 2H, ArH), 7.57 (s, 1H, ArH), 7.53 (d, J=8.7 Hz, 2H, ArH), 6.46
(s, 1H, ArH), 5.0 (s, 2H, NH2), 4.37–4.34 (m, 2H, OCH2), 4.23–
4.20 ppm (m, 2H, OCH2); 13C NMR (75 MHz, CDCl3): d=164.5 (C),
162.9 (C), 159.5 (C), 142.6 (C), 142.0 (C), 136.8 (C), 131.8 (CH), 128.7
(CH), 124.8 (C), 117.1 (C), 103.8 (CH), 103.7 (CH), 65.3 (OCH2),
64.3 ppm (OCH2); HRMS (ESI): m/z: calcd for C16H13BrN3O2S:
389.9912 [M+H]+, found: 389.9926; elemental analysis calcd (%)
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Acknowledgements
We thank the Department of Science and Technology Funds for
the Improvement of Science and Technology (DST-FIST) New
Delhi for the use of NMR and HRMS facilities. J.K. thanks CSIR
New Delhi for a CSIR-SRF fellowship. E.V. greatly acknowledges a
UPE-Phase II (India) grant (C3/UPE-Phase II/Theme A/2013/338)
for financial support.
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Conflict of interest
The authors declare no conflict of interest.
Keywords: Chk2 inhibitors · cytotoxicity · G2/M cell-cycle
arrest · thiophenes · topoisomerase I inhibitors
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Manuscript received: April 11, 2019
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Accepted manuscript online: && &&, 0000
Version of record online: && &&, 0000
ChemMedChem 2019, 14, 1 – 14
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13
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
J. Karunakaran, N. Dhatchana Moorthy,
S. R. Chowdhury, S. Iqbal, H. K. Majumder,
K. Gunasekaran, E. Vellaichamy,
A. K. Mohanakrishnan*
Better on vinyl: A series of thiophene-
containing vinyl ketones inspired by
bioactive chalcone derivatives was syn-
thesized and evaluated for their anti-
cancer activity. Molecular docking with
Chk2 kinase and a variety of cell-based
and biochemical assays showed the
most promising compound to be a
vinyl ketone flanked by 3,4-ethylene-
dioxythiophene and 3,4,5-trimethoxy-
phenyl moieties.
&& – &&
Divergent Synthesis and Evaluation of
the in vitro Cytotoxicity Profiles of 3,4-
Ethylenedioxythiophenyl-2-propen-1-
one Analogues
&
ChemMedChem 2019, 14, 1 – 14
www.chemmedchem.org
14
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!