Azastilbenes: A cut-off to p38 MAPK inhibitors

Article abstract of DOI:10.1039/c3ob27449g

Inhibitors with vicinal 4-fluorophenyl/4-pyridine rings on a five- or six-membered heterocyclic ring are known to inhibit the p38 mitogen-activated protein kinase (MAPK), which is a potential target for rheumatoid arthritis and several different types of cancer. Several substituted azastilbene-based compounds with vicinal 4-fluorophenyl/4-pyridine rings were designed using computational docking, synthesized, and evaluated in a cell-free radiometric p38α assay. The biochemical evaluation shows that the best inhibition (down to 110 nM) is achieved for azastilbene-based compounds having an isopropylamine substituent in the 2-position of the pyridine ring. The inhibition of p38 signaling in human breast cancer cells was observed for two of the compounds. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob27449g

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PAPER
Azastilbenes: a cut-off to p38 MAPK inhibitors†
Jia-Fei Poon,a John Patrick Alao,b Per Sunnerhagenb and Peter Dinér*a
Cite this: Org. Biomol. Chem., 2013, 11,
4526
Inhibitors with vicinal 4-fluorophenyl/4-pyridine rings on a five- or six-membered heterocyclic ring
are known to inhibit the p38 mitogen-activated protein kinase (MAPK), which is a potential target
for rheumatoid arthritis and several different types of cancer. Several substituted azastilbene-based
compounds with vicinal 4-fluorophenyl/4-pyridine rings were designed using computational docking,
synthesized, and evaluated in a cell-free radiometric p38α assay. The biochemical evaluation shows that
the best inhibition (down to 110 nM) is achieved for azastilbene-based compounds having an isopropyl-
amine substituent in the 2-position of the pyridine ring. The inhibition of p38 signaling in human breast
cancer cells was observed for two of the compounds.
Received 18th December 2012,
Accepted 14th May 2013
DOI: 10.1039/c3ob27449g
www.rsc.org/obc
Introduction
The mitogen-activated protein kinases (MAPKs) are essential
regulators for signal transduction pathways and play crucial
roles in cellular processes such as transcription, apoptosis and
differentiation.1 The MAP kinase p38 responds to environ-
mental stress (e.g. UV radiation, osmotic shock and mechan-
ical stress) and is involved in the production of cytokines (IL-1
and TNFα), which are implicated in chronic inflammatory dis-
eases.2 Consequently, inhibition of the p38 MAP kinase with
small organic molecules could provide an effective therapy for
the chronic treatment of these autoimmune diseases, and
therefore, large efforts have been made in order to synthesize
and evaluate different inhibitors of p38 kinase.3 Further,
recently the p38 kinase has also been suggested to be a target
for treatments of several different cancers, e.g. such as breast
cancer, colon cancer, and ovarian cancer.3f,4
Fig. 1 p38 inhibitors with vicinal 4-fluorophenyl/4-pyridine rings.
One class of selective p38 inhibitors is the pyridinylimida-
zole-based compounds that are recognized by their vicinal
4-fluoro-phenyl/pyridyl rings.5 Since then, several different p38
inhibitors with the 4-fluoro-phenyl/pyridinyl motif have been
developed using different ring scaffolds, such as five-membered
pyrazoles,6 isoxazoles,7 triazoles,8 and six-membered pyrimi-
dines,9 quinolinones,10 and chromones11 and several of these
compounds have shown to be highly potent and inhibit the
p38 kinase activity at nanomolar concentrations (Fig. 1).
Fig. 2 A cut-off of the core ring leads to azastilbene compounds.
aDepartment of Chemistry – BMC, Uppsala University, Box 576, SE-75123 Uppsala,
Sweden. E-mail: peter.diner@kemi.uu.se; Fax: +46 18 4713818; Tel: +46 18 4713755
bDepartment of Chemistry and Molecular Biology, University of Gothenburg,
In order to determine the importance of the core ring
system in the scaffold, we wanted to explore if it is possible to
reduce the core of the scaffold from a five- or six-membered
ring to a double bond and still keep the strong inhibitory
effect observed for previous inhibitors with the 4-fluoro-
phenyl/pyridinyl motif (Fig. 2).
Box 462, SE-405 30 Göteborg, Sweden
†Electronic supplementary information (ESI) available: General experimental
procedure, characterization of synthesized compounds, 1H NMR and 13C NMR
spectra. See DOI: 10.1039/c3ob27449g
4526 | Org. Biomol. Chem., 2013, 11, 4526–4536
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Here we wish to present the design, synthesis, and biologi- azastilbene (R2 in Fig. 1B). Further, the introduction of an
cal evaluation of p38α inhibitors based on the azastilbene amino group in the 2-position (R1 in Fig. 1B) of the pyridine
scaffold with a 4-fluoro-phenyl/pyridinyl motif.
ring can also increase the binding affinity of the p38 inhibitors
as seen in previous studies.8a,13 Several azastilbenes were
docked into the ATP binding site of p38α kinase (PDB: 1a9u)
using Glide (XP)14 in the Schrödinger Suite15 in order to ident-
ify different compounds that bind to the ATP binding site in a
similar way to SB203580.
Results and discussion
Design
Several inhibitors with a 4-fluoro-phenyl/pyridinyl motif are
potent inhibitors of the p38α kinase and have been co-crystal-
lized with the p38α kinase (for example with the SB203580
inhibitor, IC50 = 48 nM). The binding of the inhibitor
SB203580 to the p38α kinase involves hydrogen bonding to the
hinge region with the nitrogen of the pyridine ring and
between the imidazole nitrogen and the amino group of Lys53
(Fig. 3A). In addition, the 4-fluorophenyl ring of the inhibitor
interacts with an additional hydrophobic pocket and the other
aryl group extends into the phosphate-binding region where a
π–π-stacking with Tyr35 is possible (Fig. 3A). The aim of the
present work is to investigate if it is possible to deconstruct
the inner core ring of the imidazole scaffold and to replace it
with a CvC double bond motif and still obtain binding
affinity, and thus, inhibition of the p38α kinase. However, the
removal of the imidazole ring also removes the possibility to
hydrogen bond to the Lys53 in the active site, which is impor-
tant for the activity as shown in previous work.8b,12 Therefore,
The docking result suggests that several different azastil-
benes (yellow in Fig. 3C) mimic the binding mode of the
known imidazole-based SB203580 inhibitor (blue in Fig. 3C),
i.e. the inhibitor hydrogen bonds to the hinge region and the
phenyl ring interacts with the hydrophobic pocket (Fig. 3D).
The docking result shows that compounds with a hydrogen
bond acceptor group in the double bond of the azastilbene,
such as an ester or a carboxylic acid group, can form a hydro-
gen bond to Lys53. The result also suggests that the introduc-
tion of an amine (–NH2 or –NH-i-Pr) in the 2-position of the
pyridine ring could lead to increased inhibition capacity due
to the formation of an extra hydrogen bond to Met109, which
has previously been observed experimentally.8a,13 These prom-
ising docking results prompted us to synthesize and evaluate
several different azastilbenes as p38α inhibitors.
Chemistry
we also wanted to introduce a hydrogen bond acceptor group The synthesis of the azastilbenes is depicted in Scheme 1. The
(e.g. ester or carboxylic acid) in the double bond of the azastilbenes 6a–c were obtained in moderate yields (32–50%)
Fig. 3 (A) Schematic picture of the binding of the SB203580 inhibitor to the ATP pocket. (B) Schematic binding of azastilbenes to the ATP pocket. (C) The docking
results suggest a similar binding mode for the known imidazole-based SB203580 inhibitor (blue) and several different azastilbenes (yellow), i.e. hydrogen bonding
to the hinge region and interaction of a phenyl ring in the hydrophobic pocket. (D) Binding mode of compound 16c into the ATP pocket. The docking suggests
hydrogen bonding to the hinge region and to Lys53 and an interaction of the aromatic group into the hydrophobic pocket.
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Scheme 1 (a) Boc2O, DMAP, t-BuOH, RT. (b) NaBH4, CaCl2, EtOH, RT. (c) Dess–
Martin periodinane, DCM, RT. (d) LiOH, 2-propanol, MW (60–80 °C). (e) Com-
pound 6c, TFA, DCM. (f) NaH, 2-iodopropane, DCM, 0 °C. (g) TFA, DCM.
via a Wittig reaction between the aldehydes 4a–b and two
different triphenylphosphonium salts 5a–b using lithium
hydroxide monohydrate as a base in 2-propanol.16 In the reac-
tion, both the (Z)- and the (E)-isomers were formed and the
pure (Z)-isomers could be separated and isolated using flash
chromatography. The (Z)-stereochemistry of the products 6a–c
was determined by comparison of the size of the coupling con-
stants of the two protons in the double bond (see ESI†).
Scheme 2 (a) 2-Chloro-2-oxoacetate, AlCl3, DCM,
0 °C. (b) NaBH4, EtOH,
−30 °C. (c) PBr3, CHCl3, RT. (d) P(OEt)3, reflux. (e) 4a–b, NEt3, LiCl, MeCN, RT. (f)
LiOH, THF–water (4 : 1), RT. (g) TFA, DCM. (h) NaH, 2-iodopropane, 0 °C. (i) TFA,
DCM.
Compound 7 was obtained via the removal of the Boc-
protecting group by treatment with TFA in DCM (92%). Com-
pound 9 was synthesized via alkylation of 6c using 2-iodopro-
pane yielding 8 followed by subsequent deprotection by TFA.
The aldehyde 4b, used in the Wittig reaction, was prepared
in a three-step procedure, starting with the Boc-protection of
the amino group17 (97%), followed by the reduction of the
ester to the corresponding benzylic alcohol using an NaBH4–
CaCl2-mixture in ethanol (74%).18 The alcohol 3 was then
converted to the aldehyde 4b using Dess–Martin periodinane
in dichloromethane in good yield (73%).19
DBU was employed as a base for the synthesis of 2-aryl-substi-
tuted cinnamic acid esters from 2-aryl-substituted phosphono-
acetates with excellent (E)-selectivity. However, using this
procedure for aldehydes 4a–b in the reaction, no stereo-
selectivity was observed.‡ Several different organic amine
bases, such as DIPEA, (−)-quinine, 1-methylpiperidine, and
triethylamine, were tested in the HWE reaction (Table 1,
entries 2–4). Among these bases, triethylamine showed the
best stereoselectivity (3 : 1) in a small test scale reaction.
The azastilbenes 14a–d, having an ester group or a car-
boxylic acid group in the double bond, were prepared via a
Horner–Wadsworth–Emmons reaction using two different
phosphonates 13a–b and the aldehydes 4a–b using triethyl-
amine as a base in the presence of lithium chloride in aceto-
‡The E/Z stereochemistry of the azastilbene-based compounds 14a–17 was deter-
mined by COSY and NOESY NMR experiments for compound 14b (see ESI†).
Further, the E/Z ratio was determined by comparing the characteristic peaks of
the alkenyl proton, which are located around 7.7–7.9 ppm and 6.8 ppm for the
nitrile (Scheme 2). In the original procedure by Ianni et al.,20 (E)-isomers and (Z)-isomers, respectively.
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Table 1 Stereoselectivity of the HWE reaction for different organic basesa
Table 2 IC50-values for synthesized compounds 6a–b, 7, 9, 14a–b, 15a–b, and
16a–c in p38α MAP kinase inhibition
Entry
Prod.
X
R1
R2
Base
E : Z
Conv
1
2
3
4
5
14a
14a
14a
14a
14a
H
H
H
H
H
H
H
H
H
H
COOEt
COOEt
COOEt
COOEt
COOEt
DBU
1 : 1
Full
Full
Full
Full
Full
DIPEA
Quinine
1-MPb
Et3N
2.3 : 1
2.7 : 1
2.4 : 1
3 : 1
a The reaction was performed in small scale (0.1 mmol aldehyde)
according to general procedure and the stereoselectivity and
A
1
conversion were determined by H NMR-analysis of the crude reaction
mixture. b 1-Methylpiperidine.
Entry
Cmpd.
X
R1
R2
IC50-value (μM)
1
2
6a
6b
H
F
H
H
H
H
4.8
2.0
Under the modified conditions, compounds 14a–d were
afforded in moderate yields (38–64%) with a stereoselectivity
ranging from 1.6 to 2.2. The phosphonate 13b was prepared in
a 4-step procedure starting with a Friedel–Crafts acylation of
fluorobenzene with ethyl 2-chloro-2-oxoacetate in the presence
of anhydrous aluminium trichloride (76%), followed by the
reduction of the ketone carbonyl group to an alcohol using
sodium borohydride (92%).20 The alcohol 11 was converted to
the alkyl halide 12b using phosphorus tribromide (54%). The
phosphonates 13a–b were prepared from the alkylhalides
12a–b via a Michaelis–Arbuzov reaction in the presence of triethyl
phosphite (87% and 78%, respectively).20 Compounds 14a–b
were hydrolyzed to the corresponding carboxylic acids 15a–b
using LiOH in a THF–water solution in good yields (91% and
75%, respectively). Compounds 16a–b were obtained by
removal of the Boc-protecting group using TFA in DCM. Com-
pound 16c was obtained via a base-promoted alkylation using
2-iodopropane yielding 17, followed by a deprotection by TFA.
3
4
5
6
7
8
9
10
11
7
9
F
F
H
F
H
F
H
F
F
NH2
i-Pr-NH
H
H
H
H
H
1.7
0.11
5.1
2.0
18.3
8.2
2.4
1.5
0.14
14a
14b
15a
15b
16a
16b
16c
COOEt
COOEt
COOH
COOH
COOEt
COOEt
COOEt
H
NH2
NH2
i-Pr-NH
16a–b. The hydrolysis of the ester group to a carboxylic acid
leads to a 4-fold decrease in inhibition for compounds 15a–b.
This shows that the ester group or carboxylic acid at the
double bond does not contribute to binding affinity and
reveals that the hydrogen bond interaction to Lys53 does not
play an important role in the azastilbene series. Compound
16c, having an ester group in the double bond, a fluorine atom
in the 4-position of the phenyl ring, and an isopropylamine in
the 2-position of the pyridine ring also inhibits the p38α
kinase more strongly with an IC50-value of 140 nM. These inhi-
bition results for compounds 9 and 16c suggest that the core
ring system of the inhibitors having a 4-fluoro-phenyl/pyridinyl
motif can be replaced with the structurally more simple
double bond and still obtain good inhibitory capacity.
However, it should be noted that the inhibition capacity for
these azastilbene compounds is lower compared to other
known imidazole-based p38 inhibitors with vicinal isopropyl-
aminopyridine and fluorobenzene substituents (down to 2 nM
IC50-value).5d
Compounds 9 and 16c were also evaluated in a cell-based
assay with human derived MCF7 breast cancer cells as
previously described.11
Anisomycin-induced activation of p38 and phosphorylation
of its downstream target, heat shock protein 27 (Hsp27), were
inhibited at doses as low as 0.1 μM and maximal inhibition
was observed at 1–5 μM (Fig. 4). The result shows that the azas-
tilbenes 9 and 16c inhibit the downstream signaling of p38
despite their slightly decreased activity in the radiometric
assay compared to the other known p38 inhibitors.
Inhibition of p38α MAPK for azastilbene compounds
The inhibitory potencies of the compounds 6a–b, 7, 9, 14a–b,
15a–b, and 16a–c were evaluated using a commercial radio-
metric p38 assay.21 The result shows that of the parent azastil-
benes compound 6a only moderately inhibits the p38 activity
(4.8 μM). The result also indicates that the fluorine atom in
the 4-position of the phenyl ring and the NH2-group of the pyr-
idine ring increases the inhibitory activity for compounds 6b
and 7 (Table 2, entries 2 and 3). The introduction of an amine
in the 2-position of the pyridine ring may result in another
possible hydrogen bond interaction to the hinge region, which
is seen from the docking studies (see the ESI†).
Especially, the introduction of an isopropylamine in the
2-position of the pyridine ring, leading to compound 9, gives a
20-fold increase of the inhibitory capacity (110 nM). The iso-
propyl substituent influences the basicity of the amino group
and thereby also increases the strength of the hydrogen bond
to the hinge region and could be one reason for the large
increase in binding affinity of compound 9 compared to 7. Sec-
ondly, the sterically more demanding isopropyl group also
interacts with the hydrophobic region II (see ESI†), which also
may be a reason for the increased binding. The addition of the
Conclusions
ester functionality in the double bond did not lead to any We have developed two synthetic routes yielding substituted
increase of the inhibition of p38α for compounds 14a–b and azastilbenes and the synthesized compounds have been
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TMS. Chemical shifts and literature NMR shifts were used to
assign the NMR spectra of the synthesised compounds. Flash
column chromatography was performed using Merck silica
gel (0.04–0.06 mm). Thin layer chromatography (TLC) was
performed on ALUGRAM® SIL G/UV254 plates (0.2 mm), using
UV-light (254 nm) for visualization. IR spectra were recorded
on a Perkin-Elmer Spectrum One (ATR Technique). HPLC ana-
lysis was performed using a Young Lin (YL-9100) system
equipped with a Kromasil 5-CellCoat column and using
hexane and isopropanol (flow: 0.5 ml min−1) as eluents and
UV-detection (220 nm and 254 nm).
Synthesis
2-tert-Butoxycarbonylamino-isonicotinate (2).18 To a solu-
tion of methyl 2-aminopyridin-4-carboxylate (2.5 g, 16.7 mmol)
in acetone (10 ml) and tert-butanol (30 ml), DMAP (115 mg,
0.94 mmol) and di-tert-butyl dicarbonate (10.5 mg, 48.1 mmol)
were added under vigorous stirring. The reaction mixture was
stirred at room temperature for 15 hours. The solution was
diluted with pentane (36 ml), cooled in the refrigerator for
3 hours and filtered to yield 2 as a white powder (4.1 g, 97%).
1H NMR (DMSO-d6): δ 10.11 (s, 1H), 8.43 (d, J = 5.1 Hz, 1H),
8.33 (s, 1H), 7.45 (d, J = 5.1 Hz, 1H), 3.89 (s, 3H), 1.48 (s, 9H).
13C NMR (DMSO-d6): δ 165.2, 153.5, 152.7, 149.0, 138.6, 117.0,
111.2, 80.0, 52.7, 28.0.
Fig. 4 Compounds 9 and 16c inhibit p38 activation and signaling in human
breast cancer cells. MCF7 cells were preincubated with the indicated doses of 9
and 16c for 30 min and then exposed to 10 μg ml−1 anisomycin for another
30 min. Total cell lysates were resolved by SDS–PAGE. Membranes were probed
with antibodies directed against phosphorylated and total p38 and phosphoryl-
ated Hsp27 (Ser82). A polyclonal antibody directed against PARP was used as a
loading control.
N-[4-Hydroxymethyl)-2-pyridinyl]-1,1,1-dimethylethylester
(3).22 To a solution of compound 2 (1.20 g, 4.76 mmol) in
ethanol (18 ml) were added sodium borohydride (540 mg,
14.3 mmol) and calcium chloride (792 mg, 7.14 mmol). After
stirring at room temperature for 7 hours, the solution was
quenched with water (20 ml) and extracted with dichloro-
methane (20 ml). The precipitate was filtered through Celite,
washed with dichloromethane (4 × 20 ml). The organic phase
was washed with brine (20 ml) and evaporated under reduced
pressure. The mixture was purified by flash column chromato-
graphy using pentane–ethyl acetate (1 : 1) as an eluent, yielding
evaluated as p38α inhibitors in a radiometric cell-free assay
and in MCF7 breast cancer cells. The first strategy was based
on the Wittig reaction and gave (Z)-azastilbenes and the
second synthetic approach yielded (E)-diarylacrylates via a
Horner–Wadsworth–Emmons reaction. The inhibition data
show that the two best inhibitors, having a fluorine atom in
the 4-position of the phenyl ring, and an isopropylamine in
the 2-position of the pyridine ring strongly reduce the activity
of p38α (111 nM and 137 nM, respectively). This suggests that
the core ring system of the 4-fluoro-phenyl/pyridinyl inhibitors
can be replaced with the structurally more simple double bond
and still obtain good inhibitory capacity. In spite of a decrease
in IC50-values due to the truncation of the inhibitors, the two
best azastilbene inhibitors decreased the downstream signal-
ing of p38α in human breast cancer cells.
1
3 as a colorless solid (789 mg, 74%). H NMR (CDCl3): δ 9.65
(s, 1H), 8.25 (d, J = 4.8 Hz, 1H), 7.93 (s, 1H), 6.96 (d, J = 4.8 Hz,
1H), 4.68 (s, 2H), 3.44 (s, 1H), 1.50 (s, 9H). 13C NMR (CDCl3):
δ 153.1, 153.0, 152.9, 147.7, 115.8, 109.8, 81.0, 63.7, 28.4.
2-[N,N-Mono-(tert-butoxycarbonyl)amino]-pyridyl-4-aldehyde
(4b).22 To a solution of Dess–Martin periodinane (2.7 g,
6.4 mmol) in dichloromethane (24 ml), compound 3 (1.3 g,
5.8 mmol) in dichloromethane (24 ml) was added. After stir-
ring for 15 hours, the solution was quenched by saturated
NaHCO3 (aq., 30 ml) and saturated Na2S2O3 (aq., 30 ml), fol-
lowed by extraction with dichloromethane (3 × 50 ml). The
combined organic phases were washed with water (40 ml),
dried with magnesium sulfate, filtered, and evaporated under
reduced pressure. The mixture was purified by flash column
Experimental section
General
All reagents were purchased from Sigma-Aldrich and were chromatography using pentane–ethyl acetate (1 : 1) as an
1
used without further purification. H NMR (399.97 MHz) and eluent, yielding 4b as a white solid (939 mg, 73%). 1H NMR
13C NMR (100.58 MHz) spectra were recorded on a Varian (CDCl3): δ 10.32 (s, 1H), 10.00 (s, 1H), 8.52 (d, J = 4.8 Hz, 1H),
Unity 400 spectrometer, using solvent residual peaks (1H: 8.44 (s, 1H), 7.50 (dd, J = 1.2 Hz, J = 4.8 Hz, 1H), 1.53 (s, 9H).
CDCl3: δ 7.26 ppm, DMSO-d6: δ 2.50 ppm; 13C: CDCl3: 13C NMR (CDCl3): δ 191.6, 154.6, 152.8, 148.7, 144.0, 114.8,
δ 77.0 ppm, DMSO-d6: δ 39.52 ppm) as an indirect reference to 113.8, 81.5, 28.3.
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(Z)-4-Styryl-pyridine (6a). To a solution of benzyltriphenyl- 147.6, 147.4, 132.5, 132.4 (d, JCF = 14.1 Hz), 130.8 (d, JCF
=
phosphonium chloride 5a (350 mg, 0.90 mmol) in isopropanol 8.0 Hz), 128.3, 117.8, 115.4 (d, JCF = 21.1 Hz), 112.6, 80.8, 28.4.
(3 ml) was added lithium hydroxide monohydrate (35 mg, IR (cm−1): 3193, 2965, 1716, 1603, 1565, 1529, 1506. HRMS
1.4 mmol) under vigorous stirring. The solution was stirred (ESI) Calcd for C18H20FN2O2+: 315.1503, found: 315.1521.
at room temperature for 15 min. 4-Pyridinecarboxaldehyde
(Z)-4-(4-Fluorostyryl)-pyridin-2-amine (7). To a solution of
(0.078 ml, 0.82 mmol) was added and the solution was heated compound 6c (210 mg) in dry dichloromethane (1.5 ml), tri-
by microwave irradiation at 80 °C for 3 hours. The solution fluoroacetic acid (1.5 ml) was added. The solution was stirred
was quenched with water (30 ml) and extracted with ethyl for 2 hours at room temperature. The solvent was removed by
acetate (50 ml × 3). The combined organic phase was dried flushing nitrogen gas to the solution and NaOH (2 M, 1 ml)
with anhydrous Na2SO4, filtered and evaporated under was added to basify the solution. The solution was extracted
reduced pressure. The mixture was purified through flash with dichloromethane (8 ml × 5) and the organic layer was
column chromatography using pentane–ethyl acetate (1 : 1) as dried with magnesium sulfate, filtered and concentrated
an eluent to yield 6a as a yellow oil (52 mg, 34%). 1H NMR under reduced pressure. The target molecule was obtained
(CDCl3): δ 8.49 (d, J = 6.0 Hz, 2H), 7.2–7.25 (m, 5H, Ar), 7.14 (d, through silica gel using dichloromethane–methanol (9 : 1) as
J = 5.98 Hz, 2H), 6.82 (d, J = 12.2 Hz, 1H), 6.53 (d, J = 12.2 Hz, an eluent, yielding 7 as a powder (132 mg, 92%). 1H NMR
1H). 13C NMR (CDCl3): δ 149.9, 144.9, 136.2, 134.0, 128.8, (CDCl3): δ 7.82 (d, J = 5.2 Hz, 1H), 7.16–7.20 (m, 2H), 6.88–6.93
128.5, 127.9, 127.6, 123.5. IR (cm−1): 3054, 3022, 1631, 1594. (m, 2H), 6.62 (d, J = 12.5 Hz, 1H), 6.31–6.43 (m, 3H), 4.56
HRMS (ESI) Calcd for C13H12N+: 182.0964, found: 182.0963.
(broad s, 2H). 13C NMR (CDCl3): δ 162.1 (d, JCF = 248.7 Hz),
(Z)-4-(4-Fluorostyryl)pyridine (6b). To a solution of (4-fluoro- 158.8, 147.9, 146.9, 132.4 (d, JCF = 4.0 Hz), 132.0, 130.7 (d, JCF
=
benzyl)-triphenylphosphonium bromide 5b (407 mg, 18.1 Hz), 128.1 (JCF = 6.0 Hz), 115.3 (d, JCF = 21.1 Hz), 114.1,
0.90 mmol) in isopropanol (3 ml) was added lithium hydroxide 108.2. IR (cm−1): 3453, 3298, 3132, 2924, 1638, 1597, 1541,
+
monohydrate (35 mg, 1.4 mmol) under vigorous stirring. The 1505. HRMS (ESI) Calcd for C13H12FN2 : 215.0979, found:
solution was stirred at room temperature for 15 min. 4-Pyridi- 215.0962.
necarboxaldehyde (0.078 ml, 0.82 mmol) was added and the
(Z)-tert-Butyl 4-(4-fluorostyryl)pyridin-2-yl(isopropyl)carbamate
solution was heated by microwave at 80 °C for 3 hours. The (8). To a solution of compound 6c (400 mg, 1.3 mmol) in DMF
solution was quenched with water (30 ml) and extracted with (3.9 ml) at 0 °C, sodium hydride (69 mg, 2.9 mmol) was added
ethyl acetate (50 ml × 3). The combined organic layer was under vigorous stirring in an inert atmosphere. After stirring
dried with anhydrous Na2SO4, filtered, and evaporated under for 30 min at 0 °C, 2-iodopropane (0.3 ml, 3.0 mmol) was
reduced pressure. The mixture was purified through flash added dropwise. The solution was stirred for 30 min at 0 °C
column chromatography using pentane–ethyl acetate (1 : 1) as and brought to room temperature over 4 hours, followed
an eluent to yield 6b as a yellow oil (82 mg, 50%). 1H NMR by addition of water (50 ml), extraction of diethyl ether
(CDCl3): δ 8.42 (dd, J = 1.6 Hz, J = 4.4 Hz, 2H), 7.10–7.14 (m, (5 × 50 ml) and washing with diluted hydrochloric acid (0.1 M,
2H), 7.03 (dd, J = 1.6 Hz, J = 4.4 Hz, 2H), 6.86–6.90 (m, 2H), 50 ml), saturated sodium hydrogen carbonate (50 ml) and
6.67 (d, J = 12.4 Hz, 1H), 6.43 (d, J = 12.4 Hz, 1H). 13C NMR brine (50 ml). The organic layer was dried with magnesium
(CDCl3): δ 162.2 (d, JCF = 247.4 Hz), 149.9, 144.7, 132.7, 132.1 sulfate, and concentrated under reduced pressure. The
(d, JCF = 3.1 Hz), 130.5 (d, JCF = 8.0 Hz), 127.5, 123.4, 115.4 (d, mixture was purified by flash column chromatography using
JCF = 21.1 Hz). IR (cm−1): 3070, 3048, 3023, 2981, 1630, 1594, pentane–ethyl acetate (9 : 1) as an eluent, yielding 8 as a yellow
1
1506. HRMS (ESI) Calcd for C13H11FN+: 200.0870, found: liquid (240 mg, 76%). H NMR (CDCl3): δ 8.31 (d, J = 5.2 Hz,
200.0894.
1H), 7.15–7.18 (m, 2H), 6.92–6.90 (m, 4H), 6.71 (d, J = 12.2 Hz,
tert-Butyl [(Z)-4-(4-fluorostyryl)pyridin-2-yl] carbamate (6c). 1H), 6.49 (d, J = 12.2 Hz, 1H), 4.40–4.47 (m, 1H), 1.39 (s, 1H),
To
a
solution of (4-fluoro-benzyl)-triphenylphosphonium 1.17 (d, J = 6.8 Hz, 6H). 13C NMR (CDCl3): δ 162.1 (d, JCF
=
=
bromide 5b (564 mg, 2.54 mmol) in isopropanol (4.5 ml), 246.3 Hz), 154.3, 154.2, 148.4, 146.2, 132.6, 132.0 (d, JCF
lithium hydroxide monohydrate (107 mg, 2.56 mmol) was 3.0 Hz), 130.5 (d, JCF = 7.6 Hz), 127.3, 123.6, 121.3, 115.4 (d, JCF
added under vigorous stirring. The solution was stirred at = 21.4 Hz), 80.2, 49.2, 28.3, 21.3. IR (cm−1): 2974, 1696, 1596,
room temperature for 15 min. Compound 4b (1.25 g, 1540, 1507. HRMS (ESI) Calcd for C21H26FN2O2+: 357.1973,
2.76 mmol) was added and the solution was heated by micro- found: 357.1976.
wave irradiation at 60 °C for 1 hour. The solution was
(Z)-4-(4-Fluorostyryl)-N-isopropylpyridin-2-amine (9). To a
quenched with water (50 ml) followed by extraction with ethyl solution of compound 8 (180 mg, 0.5 mmol) in anhydrous
acetate (100 ml × 3). The organic layer was dried with mag- dichloromethane (0.8 ml), trifluoroacetic acid (0.8 ml,
nesium sulfate, filtered, and evaporated under reduced 9.7 mmol) was added. After stirring for 3 hours at room temp-
pressure. The mixture was purified through silica gel using erature, the excess reagent was removed by flushing nitrogen
pentane–ethyl acetate (1 : 1) as an eluent, yielding 6c as a white gas to the solution, followed by addition of saturated sodium
1
powder (310 mg, 39%). H NMR (CDCl3): δ 10.21 (s, 1H), 8.18 hydrogen carbonate for basification. The solution was
(d, J = 5.25 Hz, 1H), 7.94 (s, 1H), 7.17–7.21 (m, 2H), 6.89–6.93 extracted with dichloromethane (5 × 10 ml) and the combined
(m, 2H), 6.67–6.72 (m, 2H), 6.52 (d, J = 12.2 Hz, 1H), 1.52 (s, organic layer was dried with magnesium sulfate, filtered, and
9H). 13C NMR (CDCl3): δ 162.3 (d, JCF = 247.4 Hz), 153.7, 153.1, evaporated under reduced pressure. The target molecule was
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obtained by flash column chromatography using pentane– (s, 1H), 4.23 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3):
ethyl acetate (8 : 2) as an eluent, yielding 9 as a yellow oil δ 168.2, 163.1 (d, JCF = 248.1 Hz), 131.9 (d, JCF = 3.8 Hz), 130.7
(60 mg, 46%).1H NMR (CDCl3): δ 7.93 (d, J = 5.2 Hz, 1H), (d, JCF = 9.1 Hz), 115.9 (d, JCF = 22.0 Hz), 62.1, 45.9, 13.8. IR
7.20–7.26 (m, 2H), 6.90–6.95 (m, 2H), 6.91 (d, J = 8.8 Hz, 1H), (cm−1): 2984, 1738, 1508, 1222, 1139, 1023, 839.
6.36–6.43 (m, 2H), 6.17 (s, 1H), 4.42 (broad s, 1H), 3.66–3.70
(m, 1H), 1.14 (d, J = 6.0 Hz, 6H). 13C NMR (CDCl3): δ 162.0 (d, alpha-bromophenylacetate 12a (3.0 ml, 17.1 mmol) was heated
JCF = 247.2 Hz), 158.4, 148.2, 146.2, 132.5, 131.5, 130.7 (d, JCF to reflux with triethyl phosphite (1.65 ml, 9.62 mmol) for
Ethyl 2-(diethylphosphoryl)-2-phenylacetate (13a).20 Ethyl
=
7.6 Hz), 128.6 (d, JCF = 1.5 Hz), 115.2 (d, JCF = 21.1 Hz), 112.6, 4 hours, followed by addition of water (40 ml) and extraction
106.0, 43.0, 23.0. IR (cm−1): 3256, 2967, 1599, 1547, 1505. with ethyl acetate (5 × 80 ml). The organic layer was washed
+
HRMS (ESI) Calcd for C16H18FN2 : 257.1449, found: 257.1445.
with brine (2 × 40 ml), dried with magnesium sulfate, filtered
Ethyl 2-(4-fluorophenyl)-2-oxoacetate (10).23 To a solution of and evaporated under reduced pressure. The mixture was puri-
fluorobenzene (1.87 ml, 20.0 mmol) in anhydrous dichloro- fied by flash column chromatography using pentane–ethyl
methane (40 ml) at 0 °C, ethyl oxalylchloride (3.35 ml, acetate (1 : 1) as an eluent, yielding 13a as a colorless oil (4.5 g,
1
30.0 mmol) was added under nitrogen. The solution was 87%). H NMR (DMSO-d6): δ7.51 (d, J = 7.2 Hz, 2H), 7.31–7.37
stirred for 10 min under inert conditions followed by addition (m, 3H), 4.60 (d, JPH = 24.0 Hz, 1H), 3.83–4.17 (m, 6H), 1.20 (t,
of anhydrous aluminum chloride (5.10 g, 38.2 mmol) in small J = 18.1 Hz, 6H), 1.06 (t, J = 18.1 Hz). 13C NMR (DMSO-d6): δ
portions over 15 min. After stirring for 4 hours, crushed ice 167.4, 129.7 (d, JCP = 6.1 Hz), 131.5, 128.2 (d, JCP = 2.3 Hz),
(150 g) and concentrated hydrochloric acid (100 ml) were 127.6 (d, JCP = 2.2 Hz), 62.6 (d, JCP = 6.9 Hz), 62.4 (d, JCP = 6.8
added. The solution was extracted with dichloromethane Hz), 61.2, 50.5 (d, JCP = 131.2 Hz), 16.1 (d, JCP = 5.3 Hz), 16.0
(3 × 25 ml) and the organic layer was washed with sodium (d, JCP = 6.0 Hz), 13.9.
hydroxide (0.1 M, 30 ml) and brine (2 × 40 ml), dried with
Ethyl
2-(diethoxyphosphoryl)-2-(4-fluorophenyl)acetate
magnesium sulfate, filtered and evaporated under reduced (13b).25 Compound 12b (650 mg, 2.5 mmol) and triethyl phos-
pressure. The mixture was purified by flash column chromato- phite (0.48 ml, 2.8 mmol) were heated under reflux for
graphy using pentane–ethyl acetate (9 : 1) as an eluent, yielding 5 hours, followed by addition of water (6 ml) and extraction
10 as a yellow oil (2.9 g, 74%). 1H NMR (CDCl3): δ 8.04–8.08 with ethyl acetate (5 × 12 ml). The organic layer was washed
(m, 2H), 7.15–7.19 (m, 2H), 4.43 (q, J = 7.2 Hz, 2H), 1.41 (t, with brine (2 × 6 ml), dried with magnesium sulfate, filtered
J = 7.2 Hz, 3H). 13C NMR (CDCl3): δ 184.7, 166.9 (d, JCF
259.0 Hz), 163.5, 133.1 (d, JCF = 9.8 Hz), 129.1, 116.4 (d, JCF
22.1 Hz), 62.6, 14.2.
=
=
and evaporated under reduced pressure. The mixture was
purified by flash column chromatography using pentane–
ethylacetate (1 : 1) as an eluent, yielding 13b as a colorless oil
Ethyl 2-(4-fluorophenyl)-2-hydroxyacetate (11).23 Sodium (620 mg, 78%). 1H NMR (CDCl3): δ 7.44–7.46 (m, 2H),
borohydride (330 mg, 8.7 mmol) was added to a solution of 6.96–7.00 (m, 2H), 3.89–4.20 (m, 7H), 1.13–1.24 (m, 9H).
compound 10 (3.4 g, 17.3 mmol) in ethanol (31 ml) under 13C NMR (CDCl3): δ 167.5, 162.5 (dd, JCF = 246.0 Hz, JCP
inert conditions at −30 °C. After stirring for 1 hour, the solu- 3.0 Hz), 131.3 (dd, JCF = 8.3 Hz, JCP = 6.8 Hz), 126.7 (dd, JCF
=
=
tion was quenched with hydrochloric acid (10%) until the evol- 3.0 Hz, JCP = 8.3 Hz), 115.4 (dd, JCF = 21.3 Hz, JCP = 2.2 Hz),
ution of gas stopped. Water (100 ml) was added and the 63.3 (d, JCP = 6.9 Hz), 63.1 (d, JCP = 6.8 Hz), 51.4 (d, JCP
solution was extracted with ethyl acetate (5 × 30 ml). The 135.1 Hz), 16.3 (d, JCP = 6.1 Hz), 16.2 (d, JCP = 6.1 Hz), 14.0.
organic layer was washed with brine (2 × 45 ml), dried with
=
General procedure for synthesis of α,β-disubstituted
magnesium sulfate, filtered, and evaporated under reduced
α,β-unsaturated esters (procedure A)
pressure. The mixture was purified by flash column chromato-
graphy using pentane–ethyl acetate (6 : 4) as an eluent, To a solution of anhydrous lithium chloride (3 eq.) in aceto-
1
affording 11 as a colorless oil (3.2 g, 92%). H NMR (CDCl3): nitrile, phosphonate (1 eq.) and triethylamine (3 eq.) were
δ 7.35–7.38 (m, 2H), 6.96–7.00 (m, 2H), 5.11 (s, 1H), 4.12–4.18 added. The solution was stirred for 10 min at room tempera-
(m, 3H), 1.15 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3): δ 173.4, ture, followed by addition of aldehyde (1.1 eq.). After stirring
162.9 (d, JCF = 246.4 Hz), 134.3 (d, JCF = 7.8 Hz), 128.3 (d, JCF
8.4 Hz), 115.3 (d, JCF = 21.4 Hz), 72.2, 62.1, 13.8.
=
for 3 hours, DBU (0.3 eq.) was added and the solution was
quenched with an aqueous solution of citric acid (13 ml, 10%)
Ethyl 2-bromo-2-(4-fluorophenyl)acetate (12b).24 To a solu- for 10 min and extracted with ethyl acetate (5 × 5 ml). The
tion of compound 11 (1.2 g, 6.1 mmol) in chloroform (25 ml), organic layer was washed with brine (3 × 5 ml), dried with
phosphorus tribromide (0.35 ml, 3.7 mmol) was added under magnesium sulfate, filtered and evaporated under reduced
inert conditions at 0 °C. After stirring for 7 hours, the solution pressure. The target molecule was obtained by flash column
was quenched with water (12 ml) and extracted with ethyl chromatography using pentane–ethyl acetate as an eluent.
acetate (5 × 17 ml). The organic layer was washed with brine
(E)-Ethyl 2-phenyl-3-(pyridin-4-yl)acrylate (14a). Lithium
(2 × 17 ml), dried with magnesium sulfate, filtered, and evap- chloride (720 mg, 17 mmol), compound 13a (1.7 g, 5.7 mmol),
orated under reduced pressure. The mixture was purified by triethylamine (2.4 ml, 17 mmol), and isonicotinaldehyde
flash column chromatography using pentane–ethyl acetate (0.6 ml, 6.3 mmol) were reacted in acetonitrile (18 ml) accord-
(9 : 1) as an eluent, yielding 12b as a yellow oil (850 mg, 54%). ing to the general procedure A. The mixture was purified by
1H NMR (CDCl3): δ 7.53–7.56 (m, 2H), 7.02–7.06 (m, 2H), 5.32 flash column chromatography using pentane–ethyl acetate
4532 | Org. Biomol. Chem., 2013, 11, 4526–4536
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(1 : 1) as an eluent, yielding 14a as a yellow oil (764 mg, 53%).
(E)-2-Phenyl-3-(pyridin-4-yl)acrylic acid (15a). To a solution
1H NMR (CDCl3): δ 8.35 (d, J = 5.6 Hz, 2H), 7.67 (s, 1H), of compound 14a (220 mg, 0.87 mmol) in water (2 ml) and
7.15–7.67 (m, 3H), 7.13–7.15 (m, 2H), 6.81 (d, J = 6.4 Hz, 2H), THF (8 ml), LiOH (167 mg, 7.0 mmol) was added. After stirring
4.23 (q, J = 7.2 Hz, 2H), 1.24 (t, J = 7.2 Hz, 3H). 13C NMR for 2 days, the solvent was evaporated under reduced pressure,
(CDCl3): δ 167.3, 145.0, 142.3, 137.3, 137.0, 134.8, 129.6, 128.7, purified by flash column chromatography using chloroform–
128.4, 124.2, 61.7, 14.3. IR (cm−1): 2981, 1709, 1592, 1245, MeOH–acetic acid (90 : 10 : 1) as an eluent. The residue acetic
1175. HRMS (ESI) Calcd for C16H16NO2+: 254.1176, found acid was removed by washing the solid material with
254.1178.
hot ethanol, yielding 15a as a white powder (179 mg, 91%).
(E)-Ethyl 3-(pyridin-4-yl)-2-(4-fluorophenyl)acrylate (14b). 1H NMR (DMSO-d6): δ 13.0 (bs, 1H), 8.39 (d, J = 5.2, 1H), 7.70
Lithium chloride (115 mg, 2.7 mmol), compound 13b (300 mg, (s, 1H), 7.37–7.39 (m, 3H), 7.16–7.18 (m, 2H), 6.95 (d, 2H).
0.9 mmol), triethylamine (0.38 ml, 2.7 mmol), and isonicoti- 13C NMR (DMSO-d6): δ167.7, 149.6, 142.0, 137.7, 136.1, 135.3,
naldehyde (0.1 ml, 1.2 mmol) were reacted in acetonitrile 129.2, 128.5, 128.0, 123.8. IR (cm−1): 2396, 1702, 1598, 1233,
+
(4 ml) according to the general procedure A. The mixture was 1207. HRMS (ESI) Calcd for C14H12NO2 : 226.0863, found:
purified by flash column chromatography using pentane–ethyl 226.0870.
acetate (9 : 1) as an eluent, yielding 14b as a yellow powder
(E)-2-(4-Fluorophenyl)-3-(pyridin-4-yl)acrylic acid (15b). To a
(156 mg, 64%). 1H NMR (CDCl3): δ 8.40 (d, J = 6.0 Hz, 2H), solution of compound 14b (40 mg, 0.14 mmol) in water (2 ml)
7.69 (s, 1H), 7.11–7.15 (m, 2H), 7.00–7.04 (m, 2H), 6.85 (d, J = and THF (8 ml), LiOH (28 mg, 1.7 mmol) was added. After stir-
6.0 Hz, 2H), 4.26 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). ring for 2 days, the solvent was evaporated under reduced
13C NMR (CDCl3): δ 166.8, 162.7 (d, JCF = 228.9 Hz), 150.0, pressure, purified by flash column chromatography using
142.1, 137.3, 136.2, 131.5 (d, JCF = 7.5 Hz), 130.6 (d, JCF
=
chloroform–MeOH–acetic acid (90 : 10 : 1) as an eluent. The
3.8. Hz), 124.0, 115.8 (d, JCF = 22.0 Hz), 61.7, 14.2. IR (cm−1): residue acetic acid was removed by washing the solid material
2982, 1711, 1594, 1509, 1233, 1176. HRMS (ESI) Calcd for with hot ethanol, yielding 15b as a white powder (27 mg,
+
1
C16H15FNO2 : 272.1081, found: 272.1089.
75%). H NMR (DMSO-d6): δ 13.04 (broad s, 1H), 8.43 (d, J =
(E)-Ethyl 3-(2-(tert-butoxycarbonylamino)pyridin-4-yl)-2- 4.8 Hz, 2H), 7.73 (s, 1H), 7.20–7.22 (m, 4H), 6.98 (d, J = 4.8 Hz,
phenyl-acrylate (14c). Lithium chloride (157 mg, 3.7 mmol), 2H). 13C NMR (DMSO-d6): δ 167.5, 161.8 (d, JCF = 245.2 Hz),
ethyl compound 13a (370 mg, 1.2 mmol), triethylamine 149.7, 141.9, 136.6, 136.4, 131.6 (d, JCF = 8.4 Hz), 131.5 (d, JCF
=
(0.52 ml, 3.7 mmol), and aldehyde 4b (300 mg, 1.3 mmol) 3.1 Hz), 123.8, 115.4 (d, JCF = 21.4 Hz). IR (cm−1): 2382, 1698,
+
were reacted in acetonitrile (4 ml) according to the general pro- 1602, 1512, 1227, 1211. HRMS (ESI) Calcd for C14H11FNO2 :
cedure A. The mixture was purified by flash column chromato- 244.0768, found: 244.0770.
graphy using pentane–ethyl acetate (9 : 1) as an eluent,
(E)-Ethyl 3-(2-aminopyridin-4-yl)-2-phenyl acrylate (16a). To
yielding 14c as a yellow powder (390 mg, 38%). 1H NMR a solution of compound 14c (175 mg, 0.5 mmol) in anhydrous
(CDCl3): δ 9.65 (s, 1H), 7.97 (d, J = 5.2 Hz, 1H), 7.93 (s, 1H), dichloromethane (1.5 ml), trifluoroacetic acid (0.7 ml,
7.75 (s, 1H), 7.19–7.33 (m, 3H), 7.17–7.19 (m, 2H), 6.29 (dd, J = 9.1 mmol) was added. After stirring for 7 hours at room temp-
1.2 Hz, J = 5.2 Hz, 1H), 4.28 (q, J = 7.2 Hz, 2H), 1.50 (s, 9H), erature, the excess reagent was removed by flushing nitrogen
1.31 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3): δ 167.0, 153.2, 152.8, gas over the solution, followed by addition of saturated
146.9, 145.1, 137.6, 137.1, 134.8, 129.7, 128.6, 128.3, 117.9, sodium hydrogen carbonate for basification. The solution was
114.2, 80.9, 61.4, 28.4, 14.3. IR (cm−1): 2976, 1724, 1709, 1568, extracted with dichloromethane (5 × 10 ml) and the combined
+
1531, 1250, 1158. HRMS (ESI) Calcd for C21H25N2O4 : organic layer was dried with magnesium sulfate, filtered and
369.1809, found: 369.1816.
evaporated under reduced pressure. The mixture was purified
(E)-Ethyl 3-(2-(tert-butoxycarbonylamino)pyridin-4-yl)-2-(4- by flash column chromatography using dichloromethane–
fluorophenyl) acrylate (14d). Lithium chloride (115 mg, methanol (9 : 1) as an eluent, yielding 16a as a yellow powder
2.7 mmol), compound 13b (300 mg, 0.9 mmol), triethylamine (109 mg, 86%). 1H NMR (CDCl3): δ 7.85 (d, J = 5.6 Hz, 1H),
(0.38 ml, 2.7 mmol), aldehyde 4b (230 mg, 1.1 mmol) and 7.61 (s, 1H), 7.34–7.36 (m, 3H), 7.18–7.20 (m, 2H), 6.20 (d, J =
DBU (0.1 ml, 0.63 mmol) were reacted in acetonitrile (4 ml) 5.6 Hz, 1H), 6.13 (s, 1H), 4.28 (q, J = 7.2 Hz, 4H), 1.30 (t, J =
according to the general procedure A. The mixture was purified 7.2 Hz, 3H). 13C NMR (CDCl3): δ 167.2, 158.6, 148.0, 144.0,
by flash column chromatography using pentane–ethyl acetate 137.7, 136.5, 135.1, 129.6, 128.5, 128.2, 114.6, 109.5, 61.5, 14.3.
(9 : 1) as an eluent, yielding 14d as a white powder (184 mg, IR (cm−1): 3428, 3159, 2986, 1703, 1633, 1597, 1246, 1169.
+
50%). 1H NMR (CDCl3): δ 9.60 (broad s, 1H), 8.05 (d, J = HRMS (ESI) Calcd for C16H17N2O2 : 269.1285, found: 269.1279.
5.2 Hz, 1H), 7.87 (s, 1H), 7.75 (s, 1H), 7.14–7.18 (m, 2H),
(E)-Ethyl 3-(2-aminopyridine-4-yl)-2-(4-fluorophenyl)acrylate
7.00–7.04 (m, 2H), 6.34 (dd, J = 1.2 Hz, J = 5.2 Hz, 1H), 4.28 (q, (16b). To a solution of compound 14d (120 mg, 0.3 mmol) in
J = 7.2 Hz, 2H), 1.49 (s, 9H), 1.33 (t, J = 7.2 Hz, 3H). 13C NMR anhydrous dichloromethane (1.5 ml), trifluoroacetic acid
(CDCl3): δ 166.9, 162.8 (d, JCF = 247.9 Hz), 153.3, 152.8, 147.4, (0.5 ml, 21 mmol) was added. After stirring for 4 hours at
144.8, 138.1, 136.0, 131.6 (JCF = 7.6 Hz), 130.7 (d, JCF = 3.0 Hz), room temperature, the excess reagent was removed by flushing
118.0, 115.7 (d, J = 21.1 Hz), 113.8, 81.0, 61.7, 28.4, 14.3. IR nitrogen gas to the solution, followed by addition of saturated
(cm−1): 2975, 1722, 1707, 1567, 1530, 1511, 1224, 1156. HRMS sodium hydrogen carbonate for basification. The solution was
(ESI) Calcd for C21H24FN2O4+: 387.1715, found: 387.1718.
extracted with dichloromethane (5 × 10 ml) and the combined
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organic layer was dried with magnesium sulfate, filtered and 28.5, 21.4, 14.3. IR (cm−1): 2976, 1698, 1596, 1510, 1222, 1162.
+
evaporated under reduced pressure. The mixture was purified HRMS (ESI) Calcd for C24H30FN2O4 : 429.2184, found:
by flash column chromatography using dichloromethane– 429.2204.
methanol (9 : 1) as an eluent, yielding 16b as a yellow powder
1
Biological evaluation
(62 mg, 70%). H NMR (CDCl3): δ 7.87 (d, J = 5.2 Hz, 1H), 7.62
(s, 1H), 7.15–7.18 (m, 2H), 7.02–7.06 (m, 2H), 6.20 (d, J = 5.2
Cell lines. MCF7 breast cancer cells were cultured in Dul-
Hz, 1H), 6.15 (s, 1H), 4.32 (broad s, 2H), 4.27 (q, J = 7.2 Hz, becco’s modified eagle medium (DMEM) supplemented with
2H), 1.30 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3): δ 167.1, 162.7 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100
(d, JCF = 248.1 Hz), 158.7, 148.3, 144.0, 138.2, 135.5, 131.6 (d, units ml−1 penicillin and 100 μg ml−1 streptomycin at 37 °C in
JCF = 8.4 Hz), 131.0 (d, JCF = 3.8 Hz), 115.7 (d, JCF = 22.0 Hz), humidified 5% CO2.
114.6, 109.3, 61.7, 14.3. IR (cm−1): 3426, 3165, 2990, 1699,
Reagents. p38 inhibitors were diluted in DMSO (10 mM)
1641, 1594, 1508, 1246, 1155. HRMS (ESI) Calcd for and stored at 4 °C. Stock solutions were freshly diluted to the
C16H16FN2O2+: 287.1190, found: 287.1190.
appropriate concentration prior to every experiment. Anisomy-
(E)-Ethyl 2-(4-fluorophenyl)-3-(2-(isopropylamino)pyridin-4- cin was from Sigma (Sigma-Aldrich, Stockholm, Sweden).
yl)acrylate (16c). To a solution of compound 17 (100 mg, 10 mg ml−1 stock solutions in DMSO were stored at −20 °C.
0.23 mmol) in anhydrous dichloromethane (0.7 ml), trifluoro- Antibodies directed against phospho-Hsp27 (Ser82), phospho-
acetic acid (0.36 ml, 4.7 mmol) was added. After stirring for p38 (Thr180/Tyr182) and p38 were from Cell signalling Tech-
3 hours at room temperature, the excess reagent was removed nology (In Vitro Sweden AB, Stockholm, Sweden). Antibodies
by flushing nitrogen gas to the solution, followed by addition directed against PARP were from Santa Cruz Biotechnology
of saturated sodium hydrogen carbonate for neutralization. (Heidelberg, Germany).
The solution was extracted with dichloromethane (5 × 20 ml)
Cell treatments. MCF7 cells were seeded to approximately
and the combined organic layer was dried with magnesium 80% confluency and allowed to recover for at least 16 h. The
sulfate, filtered and evaporated under reduced pressure. The cultures were then pre-treated for 30 min with the desired
target molecule was obtained by flash column chromatography inhibitor concentration, followed by the addition of 10 μg ml−1
using pentane–ethyl acetate (9 : 1) as an eluent, yielding 17 as anisomycin for another 30 min.
1
a yellow oil (68 mg, 89%). H NMR (CDCl3): δ 7.89 (d, J = 5.6
Immunoblotting. Cells treated as indicated were washed
Hz, 1H), 7.64 (s, 1H), 7.17–7.20 (m, 2H), 7.03–7.17 (m, 2H), with phosphate buffered saline (PBS) and lysed directly in ice-
6.16 (d, J = 5.6 Hz, 1H), 5.96 (s, 1H), 4.30 (broad s, 1H), 4.27 (d, cold HEPES buffer [50 mM HEPES (pH 7.5), 10 mM NaCl,
J = 7.2 Hz, 2H), 3.46–3.54 (m, 1H), 1.30 (t, J = 7.2 Hz, 3H), 1.10 5 mM MgCl2, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton
(d, J = 6.4 Hz, 6H). 13C NMR (CDCl3): δ 167.0, 162.5 (d, JCF
=
X-100 and a cocktail of protease inhibitors (Roche Diagnostics
247.1 Hz), 148.4, 143.2, 138.7, 134.9, 131.5 (d, JCF = 8.4 Hz), Scandinavia AB, Bromma, Sweden)] at 4 °C for 30 min. Lysates
115.5 (d, JCF = 21.4 Hz), 113.3, 107.0, 61.5, 43.1, 22.9, 14.2. IR were clarified by centrifugation (13 000 rpm for 15 min at 4 °C)
(cm−1): 3254, 2969, 1710, 1598, 1508, 1232, 1158. HRMS (ESI) and the supernatants then either analyzed immediately or
+
Calcd for C19H22FN2O2 : 329.1660, found: 329.1677.
stored at −80 °C. Equivalent amounts of protein (20–50 μg)
3-[2-(tert-Butoxycarbonyl-isopropyl-amino)-pyridin-4-yl]-2-(4- from total cell lysates were resolved by SDS-PAGE and trans-
fluoro-phenyl)-acrylic acid ethyl ester (17). To a solution of ferred onto nitrocellulose membranes. Membranes were
compound 14d (100 mg, 0.26 mmol) in DMF at 0 °C (1.0 ml), blocked in blocking buffer [5% (w/v) nonfat dried milk,
sodium hydride (14 mg, 0.58 mmol) was added under vigorous 150 mM NaCl, 10 mM Tris (pH 8.0) and 0.05% (v/v) Tween 20].
stirring in an inert atmosphere. After stirring for 20 min at Proteins were detected by incubation with primary antibodies
0 °C, 2-iodopropane (0.06 ml, 0.60 mmol) was added dropwise. at appropriate dilutions in blocking buffer overnight at 4 °C.
The solution was stirred for 30 min at 0 °C and brought to Blots were then incubated at room temperature with
room temperature over 4 hours, followed by addition of water horseradish peroxidase-conjugated secondary antibody. Bands
(10 ml) and extraction of diethyl ether (5 × 10 ml). The organic were visualized by enhanced chemiluminescence (Supersignal
phase was washed with diluted hydrochloric acid (0.1 M, West Pico; Pierce, Nordic Biolabs AB, Täby, Sweden) followed
10 ml), saturated sodium hydrogen carbonate (10 ml) and by exposure to autoradiography film (General Electric Bio-
brine (10 ml). The organic layer was dried with magnesium Sciences, Uppsala, Sweden).
sulfate and concentrated under reduced pressure. The mixture
was purified by flash column chromatography using pentane–
Docking of ligands
ethyl acetate (9 : 1) as an eluent, yielding 17 as a yellow liquid Molecular modeling was performed using the Schrödinger
(70 mg, 63%). 1H NMR (CDCl3): δ 8.27 (d, J = 5.2 Hz, 1H), Package, Maestro interface version 9.0 (r211).15 The structure
7.71 (s, 1H), 7.14–7.18 (m, 2H), 7.01–7.06 (m, 2H), 6.84 (d, J = of p38 in complex with the inhibitor SB203580 (PDB 1A9U)
0.8 Hz, 1H), 6.15 (d, J = 0.8 Hz, J = 5.2 Hz, 1H), 4.39–4.42 (m, was utilized for the study. The p38 structure and compounds
1H), 4.28 (q, J = 7.2 Hz, 2H), 1.29–1.40 (m, 12H). 13C NMR 6a–b, 7, 9, 14a–b, 15a–b, and 16a–c were prepared and energy
(CDCl3): δ 166.8, 162.8 (d, JCF = 248.1 Hz), 154.6, 154.2, 148.5, minimized according to the standard procedure. Docking was
143.6, 137.3, 136.2, 131.6 (d, JCF = 8.3 Hz), 130.7 (d, JCF
=
performed by using Glide v. 5.5 (r211)14 with extra precision
4.6 Hz), 124.5, 121.6, 115.9 (d, JCF = 21.4 Hz), 80.5, 61.8, 49.5, (XP) settings and standard parameters for ligand docking.
4534 | Org. Biomol. Chem., 2013, 11, 4526–4536
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Acknowledgements
P.D. is grateful for financial support from Vetenskapsrådet
(621-2009-4018), The Magnus Bergvalls Foundation, and the
Royal Swedish Academy of Sciences and the National Super-
computer centre (NSC, Linköping). P.S. is grateful for a project
grant from the Swedish Cancer Fund (2010-0633).
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