Towards the development of chromone-based MEK1/2 modulators

Article abstract of DOI:10.1016/j.ejmech.2014.07.018

Inhibition or allosteric modulation of mitogen-activated protein kinase kinases MEK1 and MEK2 (MEK1/2) represent a promising strategy for the discovery of new specific anticancer agents. In this paper, structure-based design, beginning from the lead compound PD98059, was used to study potential structural modifications on the chromone structure in order to obtain highly potent derivatives that target the allosteric pocket in MEK1. Subsequently, a small series of PD98059 analogs were synthesized to provide a first generation of chromone-based derivatives that inhibit the activation of MEK1 with IC ₅₀ values as low as 30 nM in vitro. Complementary cellular studies also showed that two of the compounds in the series inhibit the activity of MEK1/2 with IC₅₀ values in the nanomolar range (73-97 nM). In addition, compounds in this series were found to inhibit the proliferation of a small panel of human cancer cell lines.

Full text of DOI:10.1016/j.ejmech.2014.07.018

European Journal of Medicinal Chemistry 85 (2014) 127e138
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Towards the development of chromone-based MEK1/2 modulators
Itedale Namro Redwan a, Christine Dyrager a, Carlos Solano a,
a
b
a
b
ꢀ
ꢀ
Guillermo Fernandez de Troconiz , Laure Voisin , David Bliman , Sylvain Meloche ,
,
*
Morten Grøtli a
a
€
Department of Chemistry and Molecular Biology, Medicinal Chemistry, University of Gothenburg, SE-41296 Goteborg, Sweden
Institute of Research in Immunology and Cancer, Universite de Montreal, Montreal, Quebec H3C 3J7, Canada
b
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition or allosteric modulation of mitogen-activated protein kinase kinases MEK1 and MEK2 (MEK1/
2) represent a promising strategy for the discovery of new specific anticancer agents. In this paper,
structure-based design, beginning from the lead compound PD98059, was used to study potential
structural modifications on the chromone structure in order to obtain highly potent derivatives that
target the allosteric pocket in MEK1. Subsequently, a small series of PD98059 analogs were synthesized
to provide a first generation of chromone-based derivatives that inhibit the activation of MEK1 with IC50
values as low as 30 nM in vitro. Complementary cellular studies also showed that two of the compounds
in the series inhibit the activity of MEK1/2 with IC50 values in the nanomolar range (73e97 nM). In
addition, compounds in this series were found to inhibit the proliferation of a small panel of human
cancer cell lines.
Received 10 February 2014
Received in revised form
30 June 2014
Accepted 6 July 2014
Available online 7 July 2014
Keywords:
Mitogen-activated protein kinase kinases
Chromones
Molecular modeling
Biochemical activity
Whole-cell assay
© 2014 Published by Elsevier Masson SAS.
Antiproliferative activity
Kinase selectivity profile
1. Introduction
We have for a long time been working on the synthesis and
functionalization of chromone derivatives [11e19]. Hence, in this
The highly conserved mitogen-activated protein kinases
(MAPKs) are essential components of signal transduction pathways
that play crucial roles in cellular processes such as transcription,
proliferation, differentiation and apoptosis [1e3]. Dysregulation of
MAPKs, in particular the extracellular-signal regulated kinases 1
and 2 (ERK1/2), the effector kinases of the Ras/Raf/MEK/ERK1/2
pathway, is strongly associated with human cancers and this
pathway therefore offers attractive targets for the development of
anticancer agents [4,5]. The dual specificity MEK1 and MEK2 ki-
nases (MEK1/2) are of special interest since they only have two
known substrates ERK1 and ERK2 (ERK1/2). Hence, the interest in
MEK1/2 has generated several small molecule inhibitors, e.g. highly
specific allosteric MEK1/2 modulators such as CI-1040 (PD184352),
PD0325901, U0126, PD98059, GSK1120212 and AZD2644 (Fig. 1.)
[4e9]. These allosteric MEK1/2 inhibitors are particularly discrim-
inating kinase inhibitors that bind to the inactive (dephosphory-
lated) form of MEK1/2 and are thus non-ATP competitive
modulators also called type III kinase inhibitors [10].
study we have investigated the use of PD98059 as a starting point
for designing chromone-based MEK1/2 inhibitors. Molecular
modeling was used to identify potential structural modifications on
the chromone scaffold and a series of chromone derivatives were
synthesized and evaluated using biochemical and cell-based assays
for their activity against MEK1/2.
2. Results and discussion
2.1. Molecular modeling
€
The docking study was performed using the Schrodinger Pack-
age (Glide XP Mode) [20]. The structure of MEK1 bound to ATPMg
in complex with the allosteric modulator PD0325901 (PDB 1S9J)
was used for the study [21,22]. The X-ray structure showed that
PD0325901 binds into the allosteric pocket of MEK1 via one
hydrogen bond between the hydroxamic acid oxygen and the side
chain of Lys97 (Fig. 2A and B). Additionally, the dihydroxypropoxy
moiety utilizes a hydrophobic cavity that connects the allosteric
pocket and the ATP-binding site. Thus, the diol fragment forms two
hydrogen bonds with the triphosphate group in ATP, one between
* Corresponding author.
E-mail address: grotli@chem.gu.se (M. Grøtli).
the primary alcohol and the a
-phosphate group (OeH…O]P) and
http://dx.doi.org/10.1016/j.ejmech.2014.07.018
0223-5234/© 2014 Published by Elsevier Masson SAS.

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did not affect the activity or the binding negatively. The chlorine
atom in the 6-position, as depicted in Fig. 2E, did not seem to affect
the docking or binding mode into the allosteric pocket. In fact, the
chlorine atom could possibly act as a handle for further modifica-
tions (e.g. via Pd-mediated coupling reactions) since the allosteric
pocket extends in the corresponding direction. We also observed
that the methoxy group in the 30-position of PD98059 could be
replaced with an ethoxy group, which could utilize the area more
efficiently (not shown).
2.2. Synthesis
The synthetic strategy was based on earlier work toward func-
tionalized chromone derivatives in our laboratory [11,12,17]. The
target compounds were prepared from 20-hydroxyacetophenones
1e3 and the appropriate acid chlorides, which were prepared in
situ from the corresponding carboxylic acids, 4 or 5, using oxalyl
chloride or thionyl chloride as chlorinating agents, which gave es-
ters 6e8 in 70e92% yield (Scheme 1) [17,23,24]. The 2-nitrobenzoic
acids 4e5 were used with the aim to later convert the nitro group to
the corresponding amine under reductive reaction conditions.
Base-promoted rearrangement of 6e8 to obtain diketones 9e11
was carried out in pyridine using conventional heating at 50 ꢀC or
microwave heating at 100 ꢀC for 30 min [11,12,17]. Subsequently,
acid-promoted cyclization gave the flavones 12e14 in high yields
(68e99%). Thereafter, the nitro functionality in 12e13 was suc-
cessfully converted to the corresponding amine under acidic con-
ditions using tin as a reducing agent in ethanol to afford 15e16 in
80% and 68% yield, respectively [25].
Regioselective introduction of N-Boc-protected allylamine in the
8-position of the dihalogenated flavone 14 was achieved using the
Heck reaction under inert conditions (Scheme 2). Compound 14
was treated with N-Boc-allylamine, PdCl2[P(o-Tol)3]2, and trie-
thylamine in acetonitrile at 75 ꢀC overnight to give an isomeric
mixture of 17 in 45% yield [27]. Subsequent reduction of the nitro
group to the corresponding amine and reduction of the olefin in 17
was performed simultaneously by catalytic hydrogenation over Pd/
C (10%) using an H-Cube® Continuous-flow Hydrogenation Reactor.
The product 18 was obtained in low yields (33%), which could be
explained by the observation of the formation of at least two
byproducts; dehalogenated product together with a product where
the olefin had been reduced but not the nitro group. Finally, acid-
promoted deprotection of the Boc-group using TFA in dichloro-
methane gave the target compound 19 in 78% yield.
Regioselective introduction of amines in the 8-position of the
dihalogenated flavone 14 was achieved using the Buch-
waldeHartwig reaction under inert conditions (Table 1). Com-
pound 14 was treated with the appropriate amine, Pd2(dba)3, R-
(þ)-BINAP and cesium carbonate in THF at 80 ꢀC overnight to give
compounds 20e25 in yields varying from 55 to 94% [27]. Subse-
quent reduction of the nitro group using tin in ethanol (and in-situ
deprotection of R groups for compounds 23, 24 and 26) afforded
29e37 in good yields [25]. Dechlorination of 32 and 33 with
Pd(OAc)2, 2-(di-t-butylphosphino)biphenyl and HCOONa in MeOH,
using microwave assisted heating (160 ꢀC, 40 min) afforded 38 and
39 in 73% and 77% yields respectively [26].
Fig. 1. Examples of non-ATP competitive MEK1/2 modulators that bind to an allosteric
pocket, adjacent to the ATP-binding site.
one between the secondary alcohol and the
g-phosphate
(OeH…O]P). Furthermore, the docking of PD98059 suggested that
it forms three hydrogen bonds with the protein backbone; one
between the chromone carbonyl oxygen and NH of Val211 in the
activation loop (C]O…HeN), a second hydrogen bond between the
chromone carbonyl oxygen and NH of Ser212 in the activation loop
(C]O…HeN) and a third between the aniline NH2 and the carbonyl
oxygen of Phe209 (NeH…O]C) (Fig. 2C and D). PD98059 does not
utilize the hydrophobic tunnel that links to the ATP-binding site
(Fig. 2C), as shown for PD0325901 (Fig. 2A). Nevertheless, the
docking study suggested that substituents in the 8-position on the
chromone structure could extend toward the ATP-binding site. For
example, introduction of an aminopropyl group in the 8-position
(e.g. via Pd-mediated cross coupling of the halogenated chro-
mone derivative) could tentatively reach down to the ATP-binding
site. According to the docking model, compound 19 can achieve
similar hydrogen bonding interactions as PD98059 (Val211, Ser212
and Phe209) in addition to the positively charged amino func-
tionality which can bind to the
g-phosphate group in ATP
(NeH…O]P) (Fig. 2E and F). Furthermore, compound 19 interacts
via an additional hydrogen bond to the side chain of Asp190
(NeH…O]C). Compound 32, can also achieve similar hydrogen
bonding interactions as PD98059 (Val211, Ser212 and Phe209) and
via an additional hydrogen bond between the hydroxyl function-
2.3. Biological evaluation
ality which can bind to the g
-phosphate group in ATP (OeH…O]P)
(Fig. 2G and H). Based on these results, we wanted to explore the
use of 30-bromo-50-chloro-20-hydroxyacetophenone as a starting
material for the synthesis of 8-substituted PD98059 analogs, due to
its commercial availability and low price in comparison with 30-
halogenated 20-hydroxyacetophenones. However, this required
that the chlorine atom in the 6-position on the chromone structure
The biochemical activity of the synthesized chromone-based
MEK1/2 modulators was first evaluated by measuring the ability
of recombinant active human MEK1 Ser218D/Ser222D (MEK1DD)
to increase the myelin basic protein (MBP) kinase activity of ERK2
(Table 2). The study showed that the tested compounds have IC50
values ranging from 0.03
mM (32) to 10.3 mM (35), which

I.N. Redwan et al. / European Journal of Medicinal Chemistry 85 (2014) 127e138
129
Fig. 2. Comparison of PD0325901 (A), PD98059 (C), compound 19 (E), and compound 32 (G) docked into the allosteric pocket of MEK1. Panels B, D, F and H show potential hydrogen
bonding interactions (black dashed lines) of PD0325901, PD98059, 19, or 32 respectively, with ATP and/or amino acid residues in the allosteric pocket.
corresponds up to 100 fold increased potency over PD98059 (IC50
2.0 M).
the activity (PD98059 vs. 16, IC50 2.0 and 6.4 mM, respectively).
m
Furthermore, introduction of terminal amino groups in the 8-
position as in 19, 34 and 35 did not increase the activity as ex-
pected. However, going back to the modeling study, this could be
explained by the positioning of the carbonyl and aniline amine of 19
(Fig. 2F) in the allosteric pocket. The structure is placed somewhat
further down towards the ATP-binding site, which could potentially
prevent the favored interaction between the chromone carbonyl
The results indicate that the replacement of the methoxy group
in the 30-position (PD98059) on the chromone structure to an
ethoxy group, as in 15, did not have any noticeable impact on the
in vitro activity (IC50 2.0 vs. 1.9 mM). Compound 16 having a chloride
in the 6-position, showed a slight decrease in the activity compared
to PD98059, which indicates that it seems to have minor effect on

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I.N. Redwan et al. / European Journal of Medicinal Chemistry 85 (2014) 127e138
Scheme 1. Synthesis of chromone-based allosteric MEK1/2 modulators 15 and 16.a
and Ser212. Alternatively, positively charged terminal amino
groups in the 8-position could be unfavorable for the inhibitory
activity due to charge repulsion of the adjacent Lys97 (Fig. 2B).
Interestingly, the introduction of an isopropyl, n-butyl, benzyl or a
tetrahydropyranyl substituent in the 8-position resulted in similar
activity for 29, 30, 36 and 37 respectively compared to PD98059
and the results could not directly be explained by our modeling
study.
In order to design an efficient inhibitor, in addition to strong and
fast inhibition of the kinase, the inhibitor should also bind selec-
tively to the target kinase. The selectivity of compound 32 (at a
concentration of 1.0 mM) was evaluated towards a panel of 97 ki-
(IC50 2.0 vs. 1.1e2.7
groups in the 8-position as in 32 and 33 resulted in a remarkable
increase in the activity (IC50 2.0 vs. 0.03 and 0.22 M respectively).
m
M). Strikingly, having terminal hydroxyl
nases distributed throughout the AGC, CAMK, CMGC, CK1, STE, TK,
TKL, lipid, and atypical kinase families, including important mutant
forms (Fig. A10) [28]. Only MEK1 was efficiently inhibited at 1 mM
m
Using the modeling study, these results can be explained by the
ability for these compounds to achieve the hydrogen bonding in-
teractions between the chromone carbonyl and Ser212, and also by
compound concentration. These results suggest that compound 32
has a very good selectivity profile towards MEK1 amongst other
human kinases and it is reasonable to believe that the selectivity for
the compounds are even higher at lower concentrations (below
achieving an additional hydrogen bond to the g-phosphate group in
ATP (Fig. 2H). However, having two terminal hydroxyl groups in the
8-position (compound 33) did not increase the activity as sus-
pected. In contrast, the activity for compound 32, with only one
1.0
m
M).
The whole cell activity of the analogs towards MEK1/2 was
investigated by monitoring the activating phosphorylation of their
downstream substrates ERK1/2 by immunoblotting analysis in IEC-
6 intestinal epithelial cells (Table 2). As hypothesized using our
terminal hydroxyl group, was 10 fold higher (IC50 0.03
mM) than for
33 (IC50 0.22 M). The reason for this is to our knowledge unknown
m

I.N. Redwan et al. / European Journal of Medicinal Chemistry 85 (2014) 127e138
131
Scheme 2. Synthesis of chromone-based allosteric MEK1/2 modulator 19.a
modeling study, the results obtained for compounds 32 and 33
activities (IC50 0.03e10.3
mM) against MEK1. The most potent de-
indicates an increase, although moderate, in the activity compared
to PD98059 in the whole cell assay (IC50 4.2 vs. 1.6 and 3.1 mM
respectively). A representative immunoblot is shown for compound
33 (Fig. 3A). Unfortunately, the whole cell activity for compounds
rivative in the series (compound 32, IC50 30 nM) also showed a high
selectivity profile against MEK1 in a panel of 97 different kinases.
Two of the synthesized compounds (38 and 39) efficiently inhibited
the MEK-ERK1/2 pathway in a whole cell assay with IC50 below
100 nM. Furthermore, compounds 32 and 33 demonstrated anti-
proliferative activity against both RAS and BRAF activated cancer
cell lines. The syntheses of chromone-based compounds represent
a promising starting point for the development of potent small
molecule inhibitors of the MEK1/2 kinases.
29e31 and 34e37 were either approximately or more than 100 mM.
Tentatively, these results indicate that lipophilic substituents in the
8-position interfere with the cellular uptake of these compounds
and that substituents with terminal hydroxyl groups are favorable
for cell permeability. Notably, permeability issues with the present
scaffold have previously been reported. Hence, clinical trials using
PD98059 as an MEK1 inhibitor was abandoned due to poor solu-
bility and bioavailability of the compound [29].
4. Experimental section
Interestingly the difference in activity between 15 (1.0
mM) and
4.1. Chemistry
16 (>100 M) suggested that the chlorine atom might affect the
m
compounds solubility and permeability. In order to test this hy-
pothesis the whole cell activity of the dechlorinated analogs of 32
and 33 (38 and 39) were determined. Compounds 38 and 39 gave
significant improvement of the inhibitory effect in the whole-cell
General All reagents and solvents were of analysis or synthesis
grade. PD98059 was purchased from a commercial supplier. 1H-
and 13C NMR-spectra were recorded on a Varian 400/54 spec-
trometer at 400 and 100 MHz, respectively, in CDCl3, CD3OD or
DMSO-d6. Chemical shifts are reported in ppm with the solvent
assay (IC50 0.097 and 0.073 mM respectively) compared with the
chlorinated counterparts 32 and 33 (IC50 1.6 and 3.1
mM
residual peak as reference; CDCl3 (dH 7.26, dC 77.0) DMSO-d6 (dH
respectively).
2.50, dC 39.5) and CD3OD (dH 3.31, dC 49.0). The reactions were
monitored by thin-layer chromatography (TLC), on silica plated
(Silica gel 60 F254, E. Merck) aluminum sheets, detecting spots by
UV (254 and 365 nm). Flash chromatography was performed
manually on Merck Silica gel 60 (0.040e0.063 mm) or using a
Biotage SP4 Flash instrument with prepacked columns. Analytical
high-performance liquid chromatography (HPLC) analysis was
carried out on a Waters separation module 2690 connected to a
To determine whether the inhibition of ERK1/2 phosphorylation
translates into an inhibitory effect on cell proliferation, we tested
the effect of compounds 32 and 33 on the proliferation of a small
panel of human cancer cell lines bearing either activated KRAS (HCT
116 and A549 cells) or BRAF (HT-29 and A-375 cells) mutations.
Compounds 32 and 33 were found to markedly inhibit the prolif-
eration of all cancer cell lines with IC50 values comparable to the
concentrations required to inhibit ERK1/2 phosphorylation (Fig. 4).
Waters photodiode array detector 996 using an Atlantis®
AQ (250*4.6 mm) column. Preparative HPLC was carried out on a
Waters 600 controller connected to a Waters 2487 Dual Absor-
bance detector using an Atlantis® Prep T3 5
m C-18 (250*19 mm)
5 mm C18
3. Conclusions
l
m
In conclusion, we have explored the use of chromone-based
PD98059 analogs as allosteric MEK1 modulators. Molecular
modeling and efficient synthesis provided a small series of chro-
mone derivatives that showed good to moderate biochemical
column, unless otherwise stated. Dry solvents: THF and toluene
were refluxed over sodium/benzophenone and distilled into 4 Å
MS. Microwave reactions were carried out in a Biotage Initiator
instrument with a fixed hold time using capped vials. Catalytic

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I.N. Redwan et al. / European Journal of Medicinal Chemistry 85 (2014) 127e138
Table 1
Synthesis of chromone-based allosteric MEK1/2 modulators 29e39.a
Compound
R
Yield (%)
93
Compound
R
Yield (%)
74
20
29
21
22
23
94
80
81
30
31
32
83
74
51
24
76
33
77
25
26
82
55
34
35
67
61
27
28
78
59
36
58
37
38
39
54
73
77
a
Reagents and conditions: (a) Pd2(dba)3, R-(þ)- BINAP, Cs2CO3, THF, 80 ꢀC, overnight. (b) Sn powder, conc. HCl, EtOH, reflux, 1 h; (c) Pd(OAc)2, 2-(di-t-butylphosphino)
biphenyl, HCOONa, MeOH, 160 ꢀC (MW), 40 min.
hydrogenation was performed in a H-Cube® Continuous-flow Hy-
drogenation Reactor from ThalesNano using sealed cartridges
(Thales'CarCart®) with a heterogeneous palladium catalyst. A purity
Table 2
Biochemical and whole cell inhibitory activity against MEK1/2 of compounds
of more than 95% was determined by analytical HPLC for all com-
PD98059, 15, 16, 19 and 29e39.
pounds evaluated for biological activity.
Compound
Biochemical assay
Whole cell assay
IC50
(m
M)a MEK1
IC50 (m
M)b MEK1/2
4.1.1. 3-Ethoxy-2-nitrobenzoic acid (4)
PD98059
15
16
19
29
30
31
32
33
34
35
36
37
38
39
2.0
1.9
6.4
4.4
2.7
1.9
9.1
0.03
0.22
6.8
10.3
1.4
4.2
1.0
Ethyl iodide (1.1 mL, 13.7 mmol) was added to a suspension of 3-
hydroxy-2-nitrobenzoic acid (0.5 g, 2.73 mmol) and K2CO3 (2.45 g,
17.75 mmol) in DMF (6 mL). The reaction mixture was stirred at
60 ꢀC for 2 h, then diluted with water (50 mL) and extracted with
ethyl acetate (3 ꢁ 40 mL). The combined organic layers was washed
with water (3 ꢁ 50 mL) and brine (50 mL), dried with Na2SO4,
filtered and concentrated under reduced pressure. The crude res-
idue was purified by column chromatography (heptane/ethyl ace-
tate, gradient 20 / 40% ethyl acetate) to give ethyl 3-ethoxy-2-
nitrobenzoate (0.61 g, 93%) as a white solid. 1H NMR (CDCl3):
>100
NDc
>100
>100
>100
1.6
3.1
>100
>100
>100
>100
0.097
0.073
1.1
d
1.35 (t, J ¼ 7.1 Hz, 3H), 1.42 (t, J ¼ 6.0 Hz, 3H), 4.16 (q, J ¼ 7.0 Hz,
NDc
NDc
2H), 4.36 (q, J ¼ 7.1 Hz, 2H), 7.23 (d, J ¼ 8.4 Hz, 1H), 7.44e7.49 (m,
1H), 7.58 (d, J ¼ 7.6 Hz, 1H). Ethyl 3-ethoxy-2-nitrobenzoate (0.59 g,
2.47 mmol) was suspended in aqueous NaOH (0.1 M) (100 mL) and
the mixture was refluxed for 1 h. The solution was acidified with
a
Duplicate determination.
Triplicate determination.
ND ¼ Not determined.
b
c

I.N. Redwan et al. / European Journal of Medicinal Chemistry 85 (2014) 127e138
133
7.44 (dd, J ¼ 1.4, 8.1 Hz, 1H), 7.51e7.60 (m, 2H). Notably, the car-
boxylic acid hydrogen was not observed in the spectrum due to
solvent exchange.
4.1.2. General procedure for the synthesis of compounds 6e7
Oxalyl chloride (1.2 equiv) followed by DMF (0.1 equiv) were
added to an ice-cooled stirred suspension of the appropriate car-
boxylic acid (1.0 equiv) in DCM (1.5 mL/mmol carboxylic acid). The
reaction mixture was allowed to reach room temperature, and was
stirred for 2 h. It was then concentrated under reduced pressure to
give a crude residue of the corresponding acid chloride. The
appropriate 20-hydroxyacetophenone (1 equiv) was added to an
ice-cooled solution of the crude residue dissolved in pyridine
(4 mL/mmol). The reaction mixture was stirred at 0 ꢀC for 15 min
and was then stirred at room temperature for 45 min. The mixture
was poured over aqueous HCl (1 M) and ice and the formed pre-
cipitate was filtered off, washed with water and dried under
reduced pressure.
4.1.2.1. 2-Acetylphenyl 3-ethoxy-2-nitrobenzoate (6). The title
compound was synthesized according to the general procedure. 20-
Hydroxyacetophenone 1 (255 mg, 1.87 mmol) gave 6 (508 mg, 82%)
as a beige powder. 1H NMR (CDCl3):
d
1.44 (t, J ¼ 7.0 Hz, 3H), 2.54 (s,
Fig. 3. Compounds 33 and 39 inhibits the activating phosphorylation of ERK1/2 in
intact cells. Quiescent IEC-6 cells were incubated with the indicated concentration of
compound 33 or 39 for 30 min prior to stimulation with serum for 5 min. Total lysates
were analyzed by immunoblotting using antibodies specific for phospho-ERK1/2, total
ERK1/2 and HSC70. The results are representative of two independent experiments.
3H), 4.20 (q, J ¼ 7.0 Hz, 2H), 7.22 (dd, J ¼ 1.2, 8.0 Hz, 1H), 7.31 (dd,
J ¼ 1.2, 8.4 Hz, 1H), 7.36e7.41 (m, 1H), 7.53e7.61 (m, 2H), 7.80 (dd,
J ¼ 1.0, 7.8 Hz, 1H), 7.85 (dd, J ¼ 1.7, 7.8 Hz, 1H); 13C NMR (CDCl3):
d
14.4, 29.2, 65.8, 118.7, 122.5, 123.3, 123.7, 126.6, 130.4, 130.6, 131.0,
133.6, 136.5, 148.4, 150.6, 161.7, 197.1.
HCl (4 M) and extracted with ethyl acetate (3 ꢁ 60 mL). The com-
bined organic layers were dried (Na2SO4), filtered and concentrated
under reduced pressure to give 4 (0.46 g, 88%) as a pale brown solid.
4.1.2.2. 2-Acetyl-4-chlorophenyl 3-methoxy-2-nitrobenzoate (7).
The title compound was synthesized according to the general pro-
cedure. 50-Chloro-20-hydroxyacetophenone 2 (285 mg, 1.17 mmol)
1H NMR (CD3OD):
d
1.37 (t, J ¼ 7.0 Hz, 3H), 4.18 (q, J ¼ 7.0 Hz, 2H),
Fig. 4. Antiproliferative activity of compounds PD98059, 32 and 33 in four different human cancer cell lines. (A) Antiproliferation activity of PD98059, 32 and 33 in HT-29 cells. (B)
Antiproliferative activity of compound 32 in A-375, A549, HT-29 and HCT 116 cells. (C) Antiproliferative activity of compound 33 in A-375, A549, HT-29 and HCT 116 cells. The cancer
cell lines were incubated with increasing concentrations of the given compounds for 5 days. Cell proliferation was measured using a colorimetric WST-1 assay (mean SEM values
from triplicate wells are presented and are expressed as percentage of the DMSO control.

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I.N. Redwan et al. / European Journal of Medicinal Chemistry 85 (2014) 127e138
gave 7 (480 mg, 70%) as white crystals. 1H NMR (CDCl3):
3H), 3.96 (s, 3H), 7.17 (d, J ¼ 8.6 Hz, 1H), 7.34 (d, J ¼ 8.4 Hz, 1H),
7.49e7.64 (m, 2H), 7.74e7.82 (m, 2H); 13C NMR (CDCl3):
29.4, 57.0,
d
2.52 (s,
d6)
14.6, 66.0, 76.8, 77.2, 77.5, 111.4, 116.6, 118.3, 120.6, 123.9, 125.8,
125.9, 126.6, 131.7134.4, 151.2, 156.3, 160.4, 178.0.
d
3.97 (s, 3H), 6.92 (s, 1H), 7.40e7.79 (m, 6H); 13C NMR (CDCl3):
d
d
118.0,122.8,123.2,125.3,130.4,131.4,132.0,133.5,146.9,151.4,161.7,
196.0.
4.1.6. 8-Bromo-6-chloro-2-(3-methoxy-2-nitrophenyl)chromone
(14)
4.1.3. 2-Acetyl-6-bromo-4-chlorophenyl 3-methoxy-2-
nitrobenzoate (8)
KOH (0.36 g, 6.43 mmol) was added to a solution of 8 (0.92 g,
2.14 mmol) in pyridine (20 mL). The reaction was heated in a mi-
crowave cavity at 100 ꢀC for 30 min. The solution was poured into
water (20 mL) and neutralized with HCl (1 M). The yellow precip-
itate was filtered off and dried in vacuo to give 1-(3-Bromo-5-
chloro-2-hydroxyphenyl)-3-(3-methoxy-2-nitrophenyl)propane-
1,3-dione (11) (0.66 g, 72%) as a yellow powder. 11 was used in the
next reaction step without any further purification. 1H NMR
SOCl2 (5 mL, 68.8 mmol) was added to a solution of 3-methoxy-
2-nitrobenzoic acid (0.14 g, 0.71 mmol) in toluene (5 mL). The re-
action mixture was refluxed for 20 h, allowed to reach room tem-
perature and was then concentrated under reduced pressure. The
crude residue was dissolved in pyridine (0.5 mL) and the mixture
was added to an ice-cooled solution of 20-hydroxyacetophenone 3
(90 mg, 0.355 mmol) in pyridine (2.5 mL). The reaction mixture was
allowed to reach room temperature and was stirred for 2.5 h. The
crude mixture was concentrated under reduced pressure and pu-
rified by column chromatography (toluene/ethyl acetate 4:1) to
(CDCl3): d 3.96 (s, 3H), 6.55 (s, 1H), 7.22e7.28 (m, 1H), 7.30e7.34 (m,
1H), 7.52e7.62 (m, 2H), 7.74 (d, J ¼ 2.2 Hz, 1H), 12.43 (s, 1H).
Concentrated H2SO4 (0.01 mL, 0.21 mmol) was added to a solution
of 11 (0.03 g, 0.07 mmol) in EtOH (2 mL). The reaction was heated in
a microwave cavity at 100 ꢀC for 30 min. The solution was allowed
to reach room temperature and the white precipitate was filtered
off to give 14 (28 mg, 99%) as white fluffy crystals. 1H NMR (CDCl3):
give 8 (0.14 g, 92%) as offwhite crystals. 1H NMR (CDCl3):
d 2.53 (s,
3H), 3.96 (s, 3H), 7.34 (dd, J ¼ 0.7, 8.4 Hz,1H), 7.37e7.64 (m,1H), 7.73
(d, J ¼ 2.2 Hz, 1H), 7.79 (d, J ¼ 2.2 Hz, 1H), 7.88 (dd, J ¼ 1.0, 7.9 Hz,
1H); 13C NMR (CDCl3):
d
29.3, 57.0, 118.5, 119.4, 121.8, 123.0, 128.3,
d 3.99 (s, 3H), 6.64 (s,1H), 7.27e7.32 (m, 2H), 7.59e7.69 (m,1H), 7.89
129.3, 131.2, 133.0, 133.5, 136.4, 144.8, 151.3, 160.3, 195.1.
(d, J ¼ 2.5 Hz, 1H), 8.12 (d, J ¼ 2.5 Hz, 1H); 13C NMR (CDCl3):
d 56.9,
11.7, 113.0, 116.0, 121.2, 124.5, 125.5, 126.6, 131.9, 132.0, 137.4, 139.2,
151.7, 152.0, 161.0, 176.
4.1.4. 2-(3-Ethoxy-2-nitrophenyl)chromone (12)
Pulverized KOH (97 mg, 1.75 mmol) was added to a solution of 6
(385 mg, 1.17 mmol) in pyridine (8 mL). The reaction mixture was
stirred at 50 ꢀC for 30 min. The mixture was allowed to reach room
temperature and was then acidified with aqueous acetic acid (5%).
The precipitate was filtered off, washed with water dried which
4.1.7. General procedure for the synthesis of chromones 15e16
Tin powder (5 equiv) was added to a suspension of the appro-
priate nitroflavone (12 or 13) (1 equiv) in EtOH (9 mL/mmol) and
the reaction mixture was heated to reflux. Concentrated HCl
(6 equiv) was added to the heated mixture and the reaction was
refluxed for one hour. The reaction was allowed to reach room
temperature and was then basified with aqueous NaOH (5%). The
mixture was diluted with water and extracted with ethyl acetate.
The combined organic phases were dried with Na2SO4, filtered and
concentrated under reduced pressure to give the pure product.
gave
1-(3-Ethoxy-2-nitrophenyl)-3-(2-hydroxyphenyl)propane-
1,3-dione (9). (285 mg, 74%) as a solid powder. 9 was used in the
next reaction step without any further purification. 1H NMR
(CDCl3):
d
1.44 (t, J ¼ 7.0 Hz, 3H), 4.19 (q, J ¼ 7.0 Hz, 2H), 6.61 (s, 1H),
6.88e6.94 (m, 1H), 7.00 (dd, J ¼ 1.2, 8.4 Hz, 1H), 7.21 (d, J ¼ 8.2 Hz,
1H), 7.31 (dd, J ¼ 1.2, 7.8 Hz, 1H), 7.45e7.54 (m, 2H), 7.66 (dd, J ¼ 1.2,
8.4 Hz,1H),11.81 (s,1H). Concentrated H2SO4 (70 ml,1.31 mmol) was
added to a suspension of 9 (250 mg, 0.76 mmol) in acetic acid
(4 mL). The reaction mixture was refluxed for 45 min and was then
allowed to reach room temperature. The generated precipitate was
filtered off and washed with water. The crude residue was purified
by column chromatography (heptane/ethyl acetate 3:2) and 12
4.1.7.1. 2-(2-Amino-3-ethoxyphenyl)chromone (15). The title com-
pound was synthesized according to the general procedure
described above. Compound 12 (0.1 g, 0.32 mmol) gave 15 (72 mg,
80%) as a yellow solid. 1H NMR (CDCl3):
d
1.47 (t, J ¼ 7.0 Hz, 3H), 4.10
(q, J ¼ 7.0 Hz, 2H), 4.70 (bs, 2H), 6.66 (s, 1H), 6.72e6.79 (m, 1H), 6.86
(d, J ¼ 7.9 Hz, 1H), 7.09 (d, J ¼ 7.9 Hz, 1H), 7.37e7.44 (m, 1H), 7.48 (d,
J ¼ 8.4 Hz, 1H), 7.62e7.70 (m, 1H), 8.22 (d, J ¼ 7.9 Hz, 1H); 13C NMR
(160 mg, 68%) was isolated as a beige solid. 1H NMR (CDCl3):
d 1.46
(t, J ¼ 6.9 Hz, 3H), 4.22 (q, J ¼ 7.0 Hz, 2H), 6.65 (s, 1H), 7.20e7.31 (m,
2H), 7.41e7.46 (m, 2H), 7.51e7.58 (m, 1H), 7.69 (dd, J ¼ 7.2, 8.4 Hz,
(CDCl3):
d 14.9, 64.2, 110.3, 112.9, 116.3, 117.4, 117.8, 120.9, 123.8,
1H), 8.20 (d, J ¼ 8.1 Hz, 1H); 13C NMR (DMSO-d6):
d
14.6, 66.0, 111.4,
125.2, 125.7, 133.6, 136.0, 146.7, 156.2, 165.0, 178.2. HRMS (ESI): m/z
calculated for C17H15NO3: 282.1046. Found: 282.1128.
116.6,118.3,120.6,120.7,123.9,125.8,125.9,126.6,131.7,134.4,151.2,
156.3, 160.4, 178.0.
4.1.7.2. 2-(2-Amino-3-methoxyphenyl)-6-chlorochromone
(16).
4.1.5. 6-Chloro-2-(3-methoxy-2-nitrophenyl)chromone (13)
The title compound was synthesized according to the general
Pulverized KOH (92 mg, 1.67 mmol) was added to a solution of 7
(390 mg, 1.11 mmol) in pyridine (7 mL). The reaction mixture was
stirred at 50 ꢀC for 30 min. The mixture was allowed to reach room
temperature and was then acidified with aqueous acetic acid (5%).
The precipitate was filtered off, washed with water dried which
gave 1-(5-Chloro-2-hydroxyphenyl)-3-(3-methoxy-2-nitrophenyl)
propane-1,3-dione (10). (280 mg, 72%). as a solid powder. 10 was
used in the next reaction step without any further purification. 1H
procedure. Compound 13 (67 mg, 0.20 mmol) gave 16 (41 mg, 68%)
as a yellow solid. 1H NMR (CDCl3):
d 3.91 (s, 3H), 4.67 (bs, 2H), 6.68
(s, 1H), 6.76e6.82 (m, 1H), 6.90 (d, J ¼ 7.9 Hz, 1H), 7.10 (d, J ¼ 7.9 Hz,
1H), 7.46 (d, J ¼ 8.9 Hz, 1H), 7.62 (dd, J ¼ 2.5, 8.9 Hz, 1H), 8.19 (d,
J ¼ 2.5 Hz, 1H); 13C NMR (CDCl3):
d 55.9, 110.2, 112.1, 115.9, 117.5,
119.5, 121.0, 124.8,125.2, 131.2, 133.8, 136.0, 147.5, 154.5, 165.1, 176.9.
HRMS (ESI): m/z calculated for C16H12ClNO3: 302.0584. Found:
302.0568.
NMR (CDCl3):
d
3.97 (s, 3H), 6.55 (s, 1H), 6.90e7.06 (m, 1H),
l,
7.09e7.79 (m, 5H), 11.73 (s, 1H). Concentrated H2SO4 (30
m
4.1.8. (E)-8-(3-tert-Butoxycarbonylamino-1-propenyl)-6-chloro-2-
(3-methoxy-2-nitrophenyl)chromone (17)
The reaction was performed under inert conditions using dry
evacuated glassware, which were backfilled with nitrogen. N-Boc-
allylamine (191 mg, 1.20 mmol), PdCl2[P(o-Tol)3]2 (48 mg,
0.1 mmol) and triethylamine (0.17 mL, 1.22 mmol) were added to a
0.32 mmol) was added to a suspension of the 10 (95 mg,
0.27 mmol) in acetic acid (1.9 mL). The reaction mixture was
refluxed for 45 min and was then allowed to reach room temper-
ature. The generated precipitate was filtered off and washed which
gave 13 (70 mg, 78%) as a white solid after drying. 1H NMR (DMSO-

I.N. Redwan et al. / European Journal of Medicinal Chemistry 85 (2014) 127e138
135
solution of 14 (250 mg, 0.61 mmol) in acetonitrile (1.1 mL) and the
reaction mixture was heated at 75 ꢀC overnight. The mixture was
concentrated under reduced pressure and the crude residue was
purified by column chromatography (heptane/ethyl acetate 6:4) to
give 17 (32 mg, 11%) as a pure product. In addition, fractions con-
taining 17 and the E/Z isomers of the corresponding enamine
(105 mg, 34%) were combined and used in the next reaction step. 1H
13C NMR (CDCl3):
d 22.4, 44.5, 57.0, 109.7, 111.3, 113.2, 115.6, 120.8,
124.7, 126.3, 132.0, 132.7, 143.6, 151.6, 159.4, 177.3.
4.1.11.2. 8-(Butylamino)-6-chloro-2-(3-methoxy-2-nitrophenyl)-4H-
chromen-4-one (21). The title compound was synthesized accord-
ing to the general procedure using n-butylamine (27
m
l, 0.27 mmol)
gave 21 (92 mg, 94%) as a beige solid. 1H NMR (CDCl3):
d
0.97 (t,
NMR (CDCl3):
d
1.42 (s, 9H), 3.93e4.04 (m, 5H), 4.96 (bs, 1H),
J ¼ 7.4 Hz, 3H), 1.42e1.55 (m, 2H), 1.68e1.79 (m, 2H), 3.07e3.18 (m,
2H), 3.95 (s, 3H), 4.56 (t, J ¼ 5.0 Hz, 1H), 6.63 (s, 1H), 6.69 (d,
J ¼ 2.4 Hz, 1H), 7.20e7.27 (m, 3H), 7.53 (t, J ¼ 8.2 Hz, 1H); 13C NMR
6.23e6.32 (m, 1H), 6.63e6.65 (m, 1H), 6.77 (d, J ¼ 15.9 Hz, 1H),
7.22e7.25 (m, 2H), 7.54e7.60 (m, 1H), 7.68e7.72 (m, 1H), 8.00e8.03
(m, 1H); 13C NMR (CDCl3):
d
28.3 (3C), 42.5, 56.8, 79.4, 111.4 (2C),
(CDCl3): d 13.9, 20.3, 30.9, 43.5, 56.9, 109.7, 111.1, 112.6, 115.6, 120.6,
115.7, 121.0, 121.4, 123.8, 124.9, 126.7, 129.8, 130.7, 131.7, 132.0, 132.5,
151.4, 151.8, 155.8, 160.5, 176.6.
124.4, 126.0, 131.9, 132.6, 139.7, 143.4, 151.5, 159.3, 177.1.
4.1.11.3. 6-Chloro-2-(3-methoxy-2-nitrophenyl)-8-((3-
methoxypropyl)amino)-4H-chromen-4-one (22). The title com-
pound was synthesized according to the general procedure using 3-
4.1.9. 2-(2-Amino-3-methoxyphenyl)-8-(3-tert-butoxycarbonyl-
aminopropyl-6-chlorochromone (18)
An isomeric mixture of 17 (75 mg, 0.15 mmol) in THF (6 mL) was
injected through a cartridge of Pd/C (10%) using an H-cube reactor
with a flow rate of 1 mL/min at 20 ꢀC (atm. pressure). The hydrogen
gas pressure was increased to 30 bar and the mixture was looped
again (twice) through the catalyst. The solution was concentrated
and the crude residue was purified by column chromatography
(heptane/ethyl acetate 65:35) to give 18 (23 mg, 33%). 1H NMR
Methoxypropylamine (27
m
l, 0.27 mmol) gave 22 (82 mg, 80%) as a
beige solid. 1H NMR (CDCl3):
d
1.92e2.08 (m, 2H), 3.17e3.42 (m,
5H), 3.45e3.62 (m, 2H), 3.90e3.99(m, 3H), 4.67 (bs, 1H), 6.61e6.85
(m, 2H), 7.18e7.35 (m, 3H), 7.56 (t, J ¼ 8.2 Hz, 1H); 13C NMR (CDCl3):
d
26.8, 42.2, 45.6, 57.0, 57.4, 110.0, 111.3, 112.9, 115.6, 120.7, 124.5,
126.3, 131.9, 132.7, 139.2, 139.7, 143.6, 151.6, 159.3, 177.2.
(CDCl3):
d
1.42 (s, 9H), 1.83e1.95 (m, 2H), 2.88e2.95 (m, 2H),
4.1.11.4. 8-((3-((Tert-butyldimethylsilyl)oxy)propyl)amino)-6-chloro-
2-(3-methoxy-2-nitrophenyl)-4H-chromen-4-one (23). The title
compound was synthesized according to the general procedure
using 3-((tert-butyldimethylsilyl)oxy)propan-1-amine (51 mg,
0.27 mmol) gave 23 (102 mg, 81%) as a beige solid. 1H NMR (CDCl3):
3.13e3.30 (m, 2H), 3.91 (s, 3H), 4.60e4.82 (m, 3H), 6.67 (s, 1H),
6.77e6.83 (m, 1H), 6.90 (dd, J ¼ 1.4, 8.0 Hz, 1H), 7.09 (dd, J ¼ 1.4,
8.0 Hz, 1H), 7.47 (d, J ¼ 2.5 Hz, 1H), 8.02 (d, J ¼ 2.5 Hz, 1H); 13C NMR
(CDCl3):
d 27.1, 28.5 (3C), 30.4, 40.3, 56.0, 79.4, 110.3, 112.2, 116.3,
117.8, 121.1, 123.2, 125.0, 131.0, 133.3, 133.8, 136.2, 147.7, 153.0, 156.1,
164.8, 177.4.
d 0.03 (s, 6H), 0.87 (s, 9H),1.89e2.03 (m, 2H), 3.21e3.32 (m, 2H),
3.80 (t, J ¼ 5.9 Hz, 2H), 3.95 (s, 3H), 4.62 (bs, 1H), 6.64 (s, 1H), 6.75
(d, J ¼ 2.4 Hz, 1H), 7.19e7.30 (m, 3H), 7.54 (t, J ¼ 8.0 Hz, 1H); 13C
4.1.10. 2-(2-Amino-3-methoxyphenyl)-8-(3-aminopropyl)-6-
chloro-chromone (19)
NMR (CDCl3): d 5.3 (2C), 18.4, 26.0 (3C), 31.9, 40.6, 56.9, 60.6, 109.8,
111.1, 112.7, 115.6, 120.6 (2C), 124.4, 126.1, 131.9, 132.6, 139.7, 143.4,
1515, 159.3, 177.1.
Trifluoroacetic acid (0.2 mL, 2.61 mmol) was added to a solution
of 18 (23 mg, 0.05 mmol) in dichloromethane (0.5 mL) and the
reaction mixture was stirred at room temperature overnight. The
mixture was concentrated under reduced pressure and the crude
residue was purified by column chromatography (heptane/ethyl
acetate 4:6) to give 19 (14 mg, 78%) as a yellow solid. 1H NMR
4.1.11.5. (S)-6-Shloro-8-((2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethyl)
amino)-2-(3-methoxy-2-nitrophenyl)-4H-chromen-4-one
(24).
The title compound was synthesized according to the general
procedure using (S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethan-1-
(CDCl3):
d
1.64 (bs, 2H), 1.79e1.91 (m, 2H), 2.70e2.85 (m, 2H),
amine (70
ml, 0.27 mmol) gave 24 (87 mg, 76%) as a beige solid.
2.89e3.01 (m, 2H), 3.92 (s, 3H) 4.68 (bs, 2H), 6.69 (s, 1H), 6.75e6.83
(m, 1H), 6.90 (dd, J ¼ 1.4, 7.9 Hz, 1H), 7.10 (dd, J ¼ 1.4, 7.9 Hz, 1H),
7.48 (d, J ¼ 2.5 Hz, 1H), 8.03 (d, J ¼ 2.5 Hz, 1H): 13C NMR (CDCl3):
1H NMR (CDCl3):
d
1.33 (s, 3H), 1.39 (s, 3H), 1.99 (dd, J ¼ 13.5, 6.7 Hz,
2H), 3.23e3.37 (m, 2H), 3.55e3.66 (m, 1H), 3.95 (s, 3H), 4.04e4.15
(m, 1H), 4.20e4.35 (m, 1H), 4.68 (t, J ¼ 5.1 Hz, 1H), 6.63 (s, 1H), 6.76
(d, J ¼ 2.3 Hz, 1H), 7.15e7.33 (m, 3H), 7.55 (t, J ¼ 8.2 Hz, 1H); 13C
d
26.8, 29.7, 41.5, 55.8, 110.2, 112.0, 116.2, 117.6, 120.9, 122.8, 124.9,
130.8, 133.6, 133.7, 136.0, 147.5, 152.9, 164.5, 177.3. HRMS (ESI): m/z
NMR (CDCl3): d 25.7, 27.0, 33.0, 40.7, 56.9, 69.4, 73.9, 109.2, 110.1,
calculated for C19H19ClN2O3: 359.1162. Found: 359.1146.
111.2, 112.9, 115.6, 120.6,124.4, 126.1, 131.9, 132.6, 139.0, 139.4, 143.4,
151.5, 159.2, 177.0.
4.1.11. General procedure for the synthesis of chromones 20ꢂ28
The reactions were performed under inert conditions using dry
evacuated glassware, which were backfilled with nitrogen.
Appropriate amine (1.10 mmol), Pd2(dba)3 (11 mg, 0.05 mmol), R-
(þ)-BINAP (18 mg, 0.12 mmol), and Cesium carbonate (111 mg,
1.40 mmol) were added to a solution of 14 (100 mg, 0.24 mmol) in
THF (3 mL) and the reaction mixtures were heated at 80 ꢀC over-
night. The mixtures were concentrated under reduced pressure and
the crude residues purified by column chromatography (heptane/
ethyl acetate 7:3).
4.1.11.6. 6-Chloro-8-((3-(dimethylamino)propyl)amino)-2-(3-
methoxy-2-nitrophenyl)-4H-chromen-4-one (25). The title com-
pound was synthesized according to the general procedure using
N,N-Dimethyl-1,3-propanediamine (34
m
l, 0.27 mmol) gave 25
(86 mg, 82%) as a beige solid. 1H NMR (CDCl3):
d
1.87e2.06 (m, 2H),
2.29 (s, 6H), 2.53 (t, J ¼ 7.0 Hz, 2H), 3.14e3.37 (m, 2H), 3.97 (s, 2H),
4.85 (bs, 1H), 6.64 (s, 1H), 6.77 (d, J ¼ 2.3 Hz, 1H), 7.20e7.34 (m, 3H),
7.57 (t, J ¼ 8.2 Hz, 1H); 13C NMR (CDCl3):
d 26.5, 42.1, 45.3 (2C), 57.0,
57.3, 110.0, 111.3, 112.9, 115.7, 120.7, 124.5, 126.2, 131.9, 132.7, 139.6,
143.5, 151.6, 159.2, 177.1.
4.1.11.1. 6-Chloro-8-(isopropylamino)-2-(3-methoxy-2-nitrophenyl)-
4H-chromen-4-one (20). The title compound was synthesized ac-
4.1.11.7. Tert-butyl (3-((6-chloro-2-(3-methoxy-2-nitrophenyl)-4-
cording to the general procedure using isopropyl amine (23
0.27 mmol) gave 20 (88 mg, 93%) as a beige solid. 1H NMR (CDCl3):
1.33 (bs, 3H), 1.35 (bs, 3H), 3.60e3.70 (m, 1H), 3.99 (s, 3H), 4.53
m
l,
oxo-4H-chromen-8-yl)amino)propyl)carbamate
compound was synthesized according to the general procedure
using tert-butyl (3-aminopropyl)carbamate (47 l, 0.27 mmol) gave
26 (67 mg, 55%) as a beige solid. 1H NMR (CDCl3):
1.43 (s, 9H),
1.88e2.11 (m, 2H), 3.14e3.39 (m, 4H), 3.98 (s, 3H), 4.60 (bs, 1H),
(26). The
title
d
m
(bs, 1H), 6.69, (s, 1H), 6.76 (dd, J ¼ 2.4, 0.5 Hz, 1H), 7.24 (m, 2H), 7.29
d
(dd, J ¼ 7.9,1.0 Hz,1H), 7.33 (d, J ¼ 2.4 Hz,1H), 7.59 (t, J ¼ 8.3 Hz,1H);

136
I.N. Redwan et al. / European Journal of Medicinal Chemistry 85 (2014) 127e138
4.75 (bs, 1H), 6.69 (s, 1H), 6.75 (d, J ¼ 2.1 Hz, 1H), 7.21e7.38 (m, 3H),
73%) as a yellow solid. 1H NMR (CDCl3):
d 1.86e2.02 (m, 2H),
7.58 (t, J ¼ 8.2 Hz, 1H); 13C NMR (CDCl3):
d
28.5, 29.2, 41.0, 57.0,
3.21e3.38 (m, 5H), 3.54 (t, J ¼ 5.4 Hz, 2H), 3.91 (s, 3H), 5.10 (bs, 2H),
110.4, 111.3, 112.9, 115.7, 120.7, 124.6, 126.2, 128.5, 129.1, 130.3, 132.0,
132.7, 139.4, 143.7, 151.7, 156.2, 159.2, 177.1.
6.65 (s, 1H), 6.72e6.94 (m, 3H), 7.08 (d, J ¼ 7.8 Hz, 1H), 7.35 (d,
J ¼ 1.9 Hz, 1H); 13C NMR (CDCl3):
d 28.9, 42.2, 56.0, 58.9, 71.7, 110.4,
110.5, 112.2, 112.3, 116.9, 118.0, 121.1, 124.3, 132.0, 135.4, 139.5, 143.8,
147.8, 163.7, 177.8. HRMS (ESI): m/z calculated for C20H21ClN2O4:
389.1268. Found: 389.1259.
4.1.11.8. 8-(Benzylamino)-6-chloro-2-(3-methoxy-2-nitrophenyl)-
4H-chromen-4-one (27). The title compound was synthesized ac-
cording to the general procedure using Benzylamine (29
0.27 mmol) gave 27 (83 mg, 78%) as a beige solid. 1H NMR (CDCl3):
3.94 (s, 3H), 4.45 (d, J ¼ 5.8 Hz, 2H), 5.16 (t, J ¼ 5.7 Hz, 1H), 6.66 (s,
1H), 6.73 (d, J ¼ 2.4 Hz, 1H), 7.19e7.47 (m, 8H), 7.56 (t, J ¼ 8.2 Hz,
1H); 13C NMR (CDCl3):
47.7, 56.9, 110.7, 111.4, 113.4, 115.7, 120.7,
ml,
4.1.12.4. 2-(2-Amino-3-methoxyphenyl)-6-chloro-8-((3-
hydroxypropyl)amino)-4H-chromen-4-one (32). The title com-
pound was synthesized according to the general procedure
described above. Compound 23 (90 mg, 0.17 mmol) gave 32 (33 mg,
d
d
124.6, 126.2, 127.5 (2C), 127.7, 128.9 (2C), 131.9, 132.6, 137.6, 139.1,
143.6, 151.6, 159.4, 177.0.
51%) as a yellow solid. 1H NMR (CDCl3):
d 1.98 (m, 2H), 3.37 (t,
J ¼ 6.4 Hz, 2H), 3.85e3.94 (m, 5H), 4.67 (bs, 2H), 6.66 (s, 1H),
6.73e6.80 (m, 2H), 6.86 (d, J ¼ 7.1 Hz, 1H), 7.04 (d, J ¼ 8.0 Hz, 1H),
4.1.11.9. 6-Chloro-2-(3-methoxy-2-nitrophenyl)-8-((tetrahydro-2H-
pyran-4-yl)amino)-4H-chromen-4-one (28). The title compound
was synthesized according to the general procedure using Ben-
7.34 (d, J ¼ 2.2 Hz,1H); 13C NMR (CDCl3):
d 31.1, 41.9, 56.0, 61.6,110.1,
110.6, 112.3, 112.6, 116.4, 118.1, 121.2, 124.0, 132.2, 135.8, 139.4, 143.8,
147.8, 178.2. HRMS (ESI): m/z calculated for C19H19ClN2O4: 375.1111.
Found: 375.1099.
zylamine (28
m
l, 0.27 mmol) gave 28 (62 mg, 59%) as a beige solid.
1.65e1.84 (m, 2H), 2.00e2.19 (m, 2H), 3.56 (t,
1H NMR (CDCl3):
d
J ¼ 11.3 Hz, 3H), 3.99 (s, 2H), 4.02e4.11 (m, 2H), 4.62 (d, J ¼ 7.3 Hz,
4.1.12.5. (S)-2-(2-Amino-3-methoxyphenyl)-6-chloro-8-((3,4-
dihydroxybutyl)amino)-4H-chromen-4-one (33). The title com-
pound was synthesized according to the general procedure
described above. Compound 24 (50 mg, 0.11 mmol) gave 33 (33 mg,
1H), 6.69 (s, 1H), 6.78 (d, J ¼ 1.8 Hz, 1H), 7.21e7.38 (m, 3H), 7.59 (t,
J ¼ 8.2 Hz, 1H). 13C NMR (CDCl3):
d 32.7 (2C), 49.1, 57.0, 66.7 (2C),
110.4, 111.3, 113.1, 115.7, 120.7 (2C), 124.8, 126.2, 132.0, 138.2, 143.6,
151.7, 159.4, 177.1.
77%) as a yellow solid. 1H NMR (DMSO-d6):
d 1.49e1.63 (m, 1H),
1.77e1.89 (m, 1H), 3.20e3.45 (m, 4H), 3.53e3.63 (m, 1H), 3.84 (s,
3H), 6.03 (bs, 1H), 6.56 (s, 1H), 6.71 (t, J ¼ 8.0 Hz, 1H), 6.89 (d,
J ¼ 2.4 Hz, 1H), 7.00 (d, J ¼ 7.1 Hz, 1H), 7.08 (d, J ¼ 2.4 Hz,1H), 7.11 (d,
4.1.12. General procedure for the synthesis of chromones 29ꢂ37
Tin powder (5 equiv) was added to a suspension of the appro-
priate nitroflavone (29¡37) (1 equiv) in EtOH (9 mL/mmol) and the
reaction mixture was heated to reflux. Concentrated HCl (6 equiv)
was added to the heated mixture and the reaction was refluxed for
one hour. The reaction was allowed to reach room temperature and
was then basified with aqueous NaOH (5%). The mixture was
diluted with water and extracted with ethyl acetate. The combined
organic phases were dried with Na2SO4, filtered and concentrated
under reduced pressure. The crude residue was purified using
preparative HPLC (H2O:AcCN (0.1%TFA) 100:0 to 0:100 for 45 min,
then at 0:100 for 15 min, with a flow of 14 ml/min).
J ¼ 1.1 Hz, 1H), 7.13 (d, J ¼ 1.1 Hz, 1H): 13C NMR (DMSO-d6):
d 31.9,
55.3, 55.8, 65.8, 69.9, 107.9, 109.9, 111.0, 112.4, 116.4, 121.3, 124.0,
130.5, 136.5, 140.2, 143.6, 147.1, 150.1, 163.6, 176.2. HRMS (ESI): m/z
calculated for C20H21ClN2O5: 405.1217. Found: 405.1199.
4.1.12.6. 2-(2-Amino-3-methoxyphenyl)-6-chloro-8-((3-(dimethyla-
mino)propyl)amino)-4H-chromen-4-one (34). The title compound
was synthesized according to the general procedure described
above. Compound 25 (80 mg, 0.19 mmol) gave 34 (50 mg, 67%) as a
yellow solid. 1H NMR (DMSO-d6):
d 1.80e2.02 (m, 2H), 3.05e3.19
(m, 2H), 3.31 (t, J ¼ 6.5 Hz, 2H), 3.85 (s, 3H), 6.06 (bs, 1H), 6.56 (s,
4.1.12.1. 2-(2-Amino-3-methoxyphenyl)-6-chloro-8-(iso-
propylamino)-4H-chromen-4-one (29). The title compound was
synthesized according to the general procedure described above.
Compound 20 (80 mg, 0.21 mmol) gave 29 (55 mg, 75%) as a yellow
1H), 6.67e6.76 (m, 1H), 6.96 (d, J ¼ 2.3 Hz, 1H), 7.01 (d, J ¼ 8.0 Hz,
1H), 7.08e7.16 (m, 1H); 13C NMR (DMSO-d6):
d 23.0, 40.2, 42.3, 54.7,
55.8, 108.4, 110.1, 111.3, 112.4, 115.9, 116.3, 121.5, 124.2, 130.5, 136.5,
139.7, 143.8,147.1, 163.7, 176.2. HRMS HRMS (ESI): m/z calculated for
C21H24ClN3O3: 402.1584. Found: 402.1565.
solid. 1H NMR (CDCl3):
d
1.29 (s, 3H), 1.30 (s, 3H), 3.61e3.74 (m, 1H),
3.92 (s, 3H), 4.36 (s, 3H), 6.64 (s, 1H), 6.91 (dd, J ¼ 8.0, 1.3 Hz, 1H),
7.06 (dd, J ¼ 8.0, 1.3 Hz, 1H), 7.26 (s, 1H), 7.38 (d, J ¼ 2.4 Hz, 1H); 13
C
4.1.12.7. 2-(2-Amino-3-methoxyphenyl)-8-((3-aminopropyl)amino)-
6-chloro-4H-chromen-4-one (35). The title compound was synthe-
sized according to the general procedure described above. Com-
pound 26 (40 mg, 0.08 mmol) gave 35 (18 mg, 61%) as a yellow
NMR (CDCl3): d 22.8, 44.4, 56.0, 110.4,110.5, 112.2, 112.8, 116.5, 117.9,
121.1, 124.5,132.1, 135.9, 138.4, 143.6, 147.7, 164.0, 177.8. HRMS (ESI):
m/z calculated for C19H19ClN2O3: 356.1162. Found: 356.1174.
solid. 1H NMR (DMSO-d6):
d 1.74e1.94 (m, 2H), 2.82e3.03 (m, 2H),
4.1.12.2. 2-(2-Amino-3-methoxyphenyl)-8-(butylamino)-6-chloro-
4H-chromen-4-one (30). The title compound was synthesized ac-
cording to the general procedure described above. Compound 21
(70 mg, 0.17 mmol) gave 30 (54 mg, 83%) as a yellow solid. 1H NMR
3.24e3.39 (m, 2H), 3.84 (s, 3H), 6.04 (bs, 1H), 6.55 (s, 1H), 6.96 (d,
J ¼ 2.4 Hz, 1H), 7.00 (dd, J ¼ 8.1, 1.3 Hz, 1H), 7.16e7.04 (m, 1H), 7.66
(bs, 2H); 13C NMR (DMSO-d6):
d 25.8, 36.9, 40.2, 55.8, 108.3, 110.1,
111.3, 112.3, 115.8, 116.2, 121.4, 124.2, 130.5, 136.6, 139.8, 143.8, 147.0,
163.7, 176.2. HRMS (ESI): m/z calculated for C19H20ClN3O3: 374.1271.
Found: 374.1259.
(CDCl3):
d
0.99 (t, J ¼ 7.3 Hz, 3H), 1.40e1.53 (m, 2H), 1.63e1.75 (m,
2H), 3.21 (t, J ¼ 6.8 Hz, 2H), 3.94 (s, 3H), 6.95e6.71 (m, 6H), 7.09 (d,
J ¼ 7.2 Hz, 1H), 7.32 (s, 1H); 13C NMR (CDCl3):
d 13.9, 20.4, 31.1, 43.4,
56.2, 109.6, 110.4, 112.8, 113.1, 116.9, 119.2, 121.2, 123.5, 132.6, 134.6,
4.1.12.8. 2-(2-Amino-3-methoxyphenyl)-8-(benzylamino)-6-chloro-
4H-chromen-4-one (36). The title compound was synthesized ac-
cording to the general procedure described above. Compound 27
(100 mg, 0.23 mmol) gave 36 (54 mg, 58%) as a yellow solid. 1H
139.2, 143.9, 148.3, 165.1, 179.0. HRMS (ESI): m/z calculated for
C
20H21ClN2O3: 373.1319. Found: 373.1321.
4.1.12.3. 2-(2-Amino-3-methoxyphenyl)-6-chloro-8-((3-
methoxypropyl)amino)-4H-chromen-4-one (31). The title com-
pound was synthesized according to the general procedure
described above. Compound 22 (50 mg, 0.12 mmol) gave 31 (34 mg,
NMR (DMSO-d6):
d
4.03 (s, 2H), 4.40 (s, 2H), 6.39 (d, J ¼ 2.2 Hz, 1H),
6.98 (d, J ¼ 2.2 Hz, 1H), 7.24 (d, J ¼ 7.2 Hz, 1H), 7.29e7.40 (m, 7H),
7.49 (d, J ¼ 8.0 Hz, 1H), 7.71 (d, J ¼ 8.3 Hz, 1H); 13C NMR (DMSO-d6):
d
46.0, 56.1, 99.9, 109.5, 110.0, 111.3, 113.4, 116.3, 120.5, 122.2, 122.2,

I.N. Redwan et al. / European Journal of Medicinal Chemistry 85 (2014) 127e138
137
125.8, 126.7, 127.0, 128.35, 128.35, 136.4, 139.7, 140.0, 147.8, 152.9,
156.1, 162.9. 174.0. HRMS (ESI): m/z calculated for C23H19ClN2O3:
407.1162. Found: 407.1163.
the allosteric modulator PD0325901 (PDB 1S9J) was used for the
study [7,22]. The MEK1-ATPMg complex, PD0325901, PD98059 and
various chromone derivatives were prepared and energy mini-
mized using ligand preparation. Molecular docking was performed
using GLIDE with extra precision (XP) settings and standard pa-
rameters for ligand docking.
4.1.12.9. 2-(2-Amino-3-methoxyphenyl)-6-chloro-8-((tetrahydro-
2H-pyran-4-yl)amino)-4H-chromen-4-one (37). The title com-
pound was synthesized according to the general procedure
described above. Compound 28 (30 mg, 0.07 mmol) gave 37 (15 mg,
4.3. Biological assays
54%) as a yellow solid. 1H NMR (DMSO-d6):
d 1.35e1.64 (m, 2H),
1.77e2.01 (m, 2H), 3.45 (t, J ¼ 10.9 Hz, 2H), 3.56 (s, 2H), 3.88 (d,
J ¼ 11.3 Hz, 3H), 4.02 (s, 3H), 6.70 (s, 1H), 7.05 (s, 1H), 7.31 (d,
J ¼ 7.9 Hz, 1H), 7.40 (s, 1H), 7.49 (t, J ¼ 8.1 Hz, 1H), 7.71 (d, J ¼ 8.3 Hz,
4.3.1. Biochemical analysis of MEK1 kinase activity
Recombinant purified active human GST-tagged MEK1DD
(S218D/S22D) protein was purchased from Jena Bioscience, Ger-
many. Recombinant His6-ERK2 (plasmid kindly provided by Dr
Melanie Cobb, University of Texas Southwestern Medical Center)
was expressed and purified from E. coli [30]. The enzymatic activity
of MEK1 was assayed by measuring its ability to increase the MBP
kinase activity of ERK2 in vitro. GST-MEK1DD (50 ng) was incubated
for 30 min at 30 ꢀC with vehicle or indicated MEK1/2 inhibitor and
1H); 13C NMR (DMSO-d6):
d 32.2, 48.0, 55.8, 65.8, 108.5, 109.5, 111.9,
112.4, 115.7, 116.3, 121.4, 124.2, 130.4, 138.9, 143.6, 147.2, 156.2, 164.1,
176.2. HRMS (ESI): m/z calculated for C21H21ClN2O4: 401.1268.
Found: 401.1254.
4.1.13. General procedure for the synthesis of chromones 38ꢂ39
Using a modified literature procedure [26], a stock solution in
ATP (mix of 50 mM ATP and 5 mCi [g-
32P]ATP) in kinase assay buffer
MeOH (100
m
l) of the catalyst complex containing palladiumacetate
(20 mM Hepes, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.4) [31].
Then, recombinant His6-ERK2 (300 ng) was added and incubated
for 30 min. Finally, bovine myelin basic protein (MBP) (0.2 mg/mL)
was added to the reaction and the incubation was continued for an
additional 10 min. Control incubations were performed in the
absence of ERK2. The reaction was stopped by addition of 5ꢁ
Laemmli's sample buffer. The samples were analyzed by SDS-gel
electrophoresis on 12% acrylamide gels and the band correspond-
ing to MBP was excised and counted in a liquid scintillation counter.
Dose-response curves were analyzed according to a three- or four-
parameter logistic equation using the SigmaPlot software.
(0.04 eqviv) and 2-(di-t-butylphosphino)biphenyl (0.075 equiv)
was diluted in MeOH (1.4 ml) and sodium formate (3 equiv) was
added. The vial was flushed with nitrogen and stirred at room
temperature for 5 min followed by addition of starting material
(1 equiv). The vial was capped, flushed again with nitrogen and
heated in a microwave reactor at 160 ꢀC for 40 min. Full con-
sumption of starting material was confirmed by TLC (10% MeOH in
CHCl3) and the reaction mixture was diluted with ethyl acetate
(2 ml) and filtered through a plug of celite. The celite plug was
washed with ethyl acetate (8 ml) and the organic solution was
washed with water (4 ml). The aqueous phase was extracted with
ethyl acetate (2 ꢁ 4 ml), the organic phases were pooled, washed
with brine and dried over Na2SO4. The solvents were removed
under reduced pressure and the crude product purified by flash
column chromatography (2e5% MeOH in CH2Cl2).
DiscoveRx scan EDGE selectivity profiling was conducted by
DiscoveRx Bioscience with KinomeScan™ Technology.
4.3.2. Cell culture
All cell lines [IEC-6 (rat epithelial), A-375 (human malignant
melanoma), A549 (human lung carcinoma), HCT 116 and HT-29
(human colorectal adenocarcinoma)] were cultured in Dulbecco's
Modified Eagle Medium (DMEM) containing 10% fetal bovine
serum, 2 mM glutamine and antibiotics. Quiescent IEC-6 were ob-
tained by incubation of 95% confluent cell cultures in serum-free
DMEM-Ham's F-12 (1:1) supplemented with 15 mM Hepes (pH
7.4) and 0.1% bovine serum albumin for 24 h.
4.1.13.1. 2-(2-Amino-3-methoxyphenyl)-8-((3-hydroxypropyl)
amino)-4H-chromen-4-one (38). The title compound was synthe-
sized from 32 according to the general procedure described above
and was obtained as a yellow solid (8 mg, 73%). 1H NMR (CDCl3):
1.64 (br s, 1H), 2.02e1.92 (m, 2H), 3.39 (t, J 6.4 Hz, 2H), 3.86 (t, J 5.7
Hz, 2H), 3.91 (s, 3H), 4.63 (br s, 2H), 4.95 (br s, 1H), 6.61 (s, 1H), 6.79
(t, J 8.0 Hz, 1H), 6.92e6.86 (m, 2H), 7.07 (dd, J 8.0, 1.4 Hz, 1H), 7.24 (t,
J 7.9 Hz, 1H), 7.47 (dd, J 8.0, 1.4 Hz, 1H); 13C NMR (CDCl3): 31.6, 41.7,
56.0, 61.4, 110.4, 111.9, 112.0, 112.8, 116.9, 117.8, 121.3, 123.7, 125.7,
135.8,138.2,145.2,147.7,163.9,179.0. HRMS (ESI): m/z calculated for
4.3.3. Whole cell analysis of MEK1/2 activity
The cellular activity of MEK1/2 was assayed by monitoring the
activation loop phosphorylation of ERK1/2. Compounds dissolved
in DMSO were added to the culture medium 30 min prior to
stimulation of quiescent IEC-6 cells with 10% serum for 5 min. Cell
were lysed as previously reported and the activation loop phos-
phorylation of ERK1/2 was analyzed by immunoblotting with anti-
phospho-ERK1/2(Thr202/Tyr204) (Cell Signaling Technology) [31].
Total ERK1/2 expression and protein loading were controlled by
immunoblotting with anti-ERK1/2 (Cell Signaling Technology) and
anti-HSC70 (Santa Cruz Biotechnology) antibodies. Immunoblot-
ting results were quantified by densitometry analysis using Multi
Gauge software.
C
19H20N2O4: 340.1423. Found: 340.1429.
4.1.13.2. (S)-2-(2-Amino-3-methoxyphenyl)-8-((3,4-dihydroxybutyl)
amino)-4H-chromen-4-one (39). The title compound was synthe-
sized from 33 according to the general procedure described above
and was obtained yellow solid (6 mg, 77%). 1H NMR (CDCl3):
1.90e1.79 (m, 2H), 3.47e3.33 (m, 2H), 3.54 (dd, J 11.0, 7.2 Hz, 1H),
3.70 (dd, J 11.0, 3.3 Hz, 1H), 3.90 (s, 3H), 3.99e3.91 (m, 1H), 4.62 (br
s, 2H), 5.02 (br s, 1H), 6.59 (s, 1H), 6.78 (t, J 8.0 Hz, 1H), 6.87 (ddd, J
8.0, 4.7, 1.4 Hz, 2H), 7.06 (dd, J 7.9, 1.4 Hz, 1H), 7.21 (t, J 7.9 Hz, 1H),
7.44 (dd, J 8.0, 1.4 Hz, 1H); 13C NMR (CDCl3): 32.0, 41.1, 56.0, 66.9,
71.2, 110.5, 111.9, 112.1, 112.9, 117.0, 117.9, 121.3, 123.7, 125.7, 135.7,
138.1, 145.2, 147.7, 164.0, 179.0. HRMS (ESI): m/z calculated for
4.3.4. Cell proliferation assays
Cell proliferation was measured by the colorimetric WST-1
assay. Briefly, cells were seeded in 96-well plates at 2 ꢁ 103 (HCT
116 and HT-29) or 1.2 ꢁ 103 (A-375 and A549) cells per well in
C
20H22N2O5: 370.1529. Found: 370.1534.
4.2. Molecular modeling
100 ml of medium (DMEM containing 10% fetal bovine serum, 2 mM
glutamine and antibiotics). The compounds dissolved in DMSO
were added to the plates and the culture medium was changed
daily for 5 days. Then, 5 ml of WST-1 (Roche) was added to each well,
€
The docking study was performed using the Schrodinger Pack-
age, MAESTRO interface [6]. The structure of MEK1 in complex with

138
I.N. Redwan et al. / European Journal of Medicinal Chemistry 85 (2014) 127e138
[14] C. Dyrager, A. Friberg, K. Dahlen, M. Friden-Saxin, K. Borjesson,
L.M. Wilhelmsson, M. Smedh, M. Grøtli, K. Luthman, 2,6,8-trisubstituted 3-
hydroxychromone derivatives as fluorophores for live-cell imaging, Chem.
Eur. J. 15 (2009) 9417e9423.
the plates were incubated for 1 h, and absorbance was measured at
450 nm with reference at 620 nm. Proliferation assays were per-
formed on triplicate wells.
[15] M. Friden-Saxin, N. Pemberton, K.D. Andersson, C. Dyrager, A. Friberg,
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Acknowledgments
We thank Edith Giasson and Maude David for technical assis-
tance during the biological experiments. For the financial support
we thank the Knut and Alice Wallenberg Foundation, the Swedish
Research Council (Grant 62120083533) and the Cancer Research
Society (S.M.).
€
€
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.018.
€
€
[20] Maestro [9.0], Schrodinger, LLC, Portland, 2009. Glide [2.1], Schrodinger, LLC,
Portland, 2009.
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