Tri-substituted imidazole analogues of SB203580 as inducers for cardiomyogenesis of human embryonic stem cells

Article abstract of DOI:10.1016/j.bmcl.2013.03.103

The p38α mitogen-activated protein kinase (MAPK) inhibitor SB203580 had been reported to enhance the cardiomyogenesis of human embryonic stem cells (hESCs). To investigate if tri-substituted imidazole analogues of SB203580 are equally effective inducers f

Full text of DOI:10.1016/j.bmcl.2013.03.103

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3300–3303
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tri-substituted imidazole analogues of SB203580 as inducers
for cardiomyogenesis of human embryonic stem cells
Joo-Leng Low a, Gerrit Jürjens a, Jayasree Seayad a, Jasmine Seow b, Sherwin Ting b, Filip Laco b,
Shaul Reuveny b, Steve Oh b, Christina L. L. Chai a,c,
⇑
a Institute of Chemical and Engineering Sciences, 8 Biomedical Grove, Neuros #07-01, Singapore 138665, Singapore
b Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore 138668, Singapore
c Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
The p38
a mitogen-activated protein kinase (MAPK) inhibitor SB203580 had been reported to enhance
Received 2 October 2012
Revised 10 March 2013
Accepted 26 March 2013
Available online 4 April 2013
the cardiomyogenesis of human embryonic stem cells (hESCs). To investigate if tri-substituted imidazole
analogues of SB203580 are equally effective inducers for cardiomyogenesis of hESCs, and if there is a cor-
relation between p38
novel tri-substituted imidazoles with a range of p38
a
MAPK inhibition and cardiomyogenesis, we designed and synthesized a series of
MAPK inhibitory activities. Our studies demon-
a
strated that suitably designed analogues of SB203580 can also be inducers of cardiomyogenesis in hESCs
and that cell growth is affected by changes in the imidazole structures.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Imidazoles
p38 MAPK
Human embryonic stem cells
Cardiomyocytes
Differentiation
In the past decade, significant advances have been made in the
field of regenerative medicine, especially that pertaining to stem
cell therapy. One such potential application is for treatment of
heart disease, which is associated with the death of cardiomyo-
cytes. Cardiac regeneration via differentiation of stem cells and
progenitor cells to cardiomyocytes offers exciting therapeutic pos-
sibilities.1 The differentiation of human embryonic stem cells
(hESCs) to cardiomyocytes has been extensively studied using var-
ious methods.2 Of these, the use of small molecules is particularly
advantageous as they can be manufactured easily, are reversible
with respect to their effects on cells, and can be administered tem-
porally.3,4 Additionally, the directed differentiation of hESCs using
small molecules with known targets may provide a better under-
standing of the mechanisms involved in cardiomyogenesis. One
such small molecule reported to enhance cardiomyogenesis of
potential therapeutic application in the treatment of inflammatory
diseases, numerous p38 MAPK inhibitors have been reported over
the years. However to the best of our knowledge, SB203580 is the
only p38 MAPK inhibitor that has been extensively studied for the
cardiomyogenesis of hESCs. Thus the generality of this observation
has not been reported nor verified. In this study, we set out to
investigate if tri-substituted imidazole analogues of SB203580
are equally effective inducers for cardiomyogenesis of hESCs, and
a
a
if there is a correlation between p38
cardiomyogenesis.
a MAPK inhibition and
The targeted analogues of SB203580 were designed with the aid
of molecular modelling (ICM-VLS) using the available crystallo-
graphic data of the SB203580 complexed to p38
a
MAPK (PDB code:
F
F
3
hESCs is SB203580 (Fig. 1), a p38
a mitogen-activated protein ki-
O
S
N
O
S
N
nase (MAPK) inhibitor.5–10
2
4
N
H
N
H
CH3
CH3
5
1
SB203580 was among the first few reported small molecule
inhibitors of p38 MAPK is one of the four iso-
MAPK.11 The p38
N
6
O
a
a
SB203580
CH3
forms of the p38 MAPK subfamily and has a central role in regulat-
CH3
ing the production of inflammatory cytokines such as tumour
necrosis factor
a (TNF-a
) and interleukin 1b (IL-1b).12 Due to their
R
O
S
N
N
N
H
N
CH3
H
⇑
Corresponding author. Tel.: +65 67998522, +65 66011061; fax: +65 64642102,
+65 67791554.
N
1 - 5
O
7
E-mail addresses: Christina_Chai@ices.a-star.edu.sg, phacllc@nus.edu.sg (C.L.L.
Chai).
Figure 1. Structures of SB203580 and the tri-substituted imidazoles reported in
this Letter.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.103

J.-L. Low et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3300–3303
3301
CH3
Lithiation of terminal alkyne 12 with n-butyllithium followed
by nucleophilic addition to tetrahydro-4H-pyran-4-one afforded
the 1,2-disubstituted alkyne 13. Oxidation of 13 with potassium
permanganate furnished the 1,2-diketone 14. Condensation of
diketone 14 with 4-(methylthio)benzaldehyde under conditions
analogous to that shown in Scheme 1 furnished imidazoles 15
and 16 in satisfactory yields. The 4-(methylthio)phenyl substituent
of imidazoles 15 and 16 was then oxidized with potassium persul-
fate to give the racemic 4-(methylsulfinyl)phenyl imidazoles 6 and
7 respectively.
CH3
CH3
O
O
c
a, b
Br
N
10
d or e
8
9
N
CH3
CH3
f
O
With the tri-substituted imidazoles in hand, the inhibitory
N
N
S
R
For
R = 4-(methylthio)phenyl
activities of the compounds against p38
a MAPK were evaluated
N
N
H
CH3
H
using commercially available, HitHunter™ p38 MAPK binding as-
say from DiscoveRx.14 The results are summarized in Table 1.
Compounds 1–5, in which the 4-fluorophenyl group of
SB203580 was replaced by a 3-tolyl group, were generally equipo-
tent or more potent than SB203580. Compound 1, with the 2,6-
difluorophenyl substituent at imidazole C-2, was the most potent
compound in the series and was almost sixfold more potent com-
pared to SB203580. Changing the C-2 substituent to the 2-fluoro-
phenyl (2) and 4-hydroxyphenyl (3) groups reduced the potency
of the compounds compared to 1. However compounds 2 and 3
were still more potent than SB203580. Compound 5, which con-
tains a 4-(methylsulfinyl)phenyl substituent at C-2 and hence is di-
rectly comparable to SB203580, had a comparable IC50 value as
SB203580. Compound 4 which contains the 1-fluoronaphthalen-
2-yl group at C-2 was also as potent as SB203580. Comparing the
IC50 values of 6 with that of SB203580 suggested that substitution
of the pyridin-4-yl group with a pyran-4-yl group at the C-5 posi-
tion of imidazole decreases the inhibitory potency of the com-
pound by 18-fold. This may be due to the weaker hydrogen-bond
accepting characteristic of the oxygen atom as compared to the
nitrogen atom.15 Consequently compounds containing the pyran-
4-yl substituent may have weaker hydrogen bonding interaction
5
N
N
1 - 4, 11
Scheme 1. Synthesis of tri-aryl imidazoles with pyridin-4-yl substituent. Reagents
and conditions: (a) TMS acetylene, triethylamine, Pd(PPh3)4, CuI, THF, 40 °C; (b)
TBAF, 4-bromopyridine HCl, triethylamine, 50 °C; (c) KMnO4, MgSO4, NaHCO3,
acetone/water (1:1), À10 °C; (d) R-CHO, NH4OAc, acetic acid, 115 °C; (e) R-CHO,
NH4OAc, Cu(OAc)2ÁH2O, acetic acid, 80 °C; (f) K2S2O8, acetic acid/water (3:2), rt.
1A9U).13 Tri-aryl imidazoles 1–5 (Fig. 1) retained the pyridin-4-yl
substituent at the C-5 position of imidazole as compared to
SB203580 but a 3-tolyl substituent replaced the 4-fluorophenyl
substituent at the C-4 position as docking studies suggest that this
replacement could maximize the ‘space’ available in the hydropho-
bic pocket of p38a MAPK. The C-2 substituent of the imidazole was
also varied in compounds 1–5. In contrast, imidazole 6 (Fig. 1) dif-
fers from SB203580 in that the C-5 pyridin-4-yl substituent has
been replaced with a pyran-4-yl group. From docking studies, this
substitution retains a critical H-bonding observed with SB203580
and p38a MAPK. Compound 7 (Fig. 1), possessing a 3-tolyl substi-
tuent at C4 and a pyran-4-yl group at C-5 retains the 4-(meth-
ylsulfinyl)phenyl substituent at C-2 present in SB203580.
Imidazoles 1–5 were synthesized using the route outlined in
Scheme 1.
to the ATP-binding site of p38
a MAPK compared to compounds
with the pyridin-4-yl substituent, leading to higher IC50 values in
the former. Interestingly, imidazole 7 was ca. sixfold more potent
than 6, indicating that replacing the 4-fluorophenyl group on the
C-4 of imidazole with a 3-tolyl group increases the inhibitory po-
tency of a compound.
With these imidazoles in hand, the compounds were tested for
cardiomyogenic activity at 5 lM, that is, the concentration at
which SB203580 (positive control) showed optimal cardiomyogen-
ic activity in previous reports.5,7,9 Nkx2.5eGFP/w hESCs, a transgenic
hESC line expressing enhanced green fluorescent protein (GFP) un-
der the control of the cardiac progenitor marker Nkx2.5 was
used.16 The compounds were added from Day 0, the day of embry-
oid body (EB) formation, to Day 8. EBs were harvested on Day 15
and the extent of cardiomyogenesis was analyzed by fluores-
A one-pot, two-step, double Sonogashira coupling reaction was
used for the rapid assembly of the key alkyne intermediate 9. The
diaryl alkyne 9 was oxidized to the 1,2-diketone 10 using potas-
sium permanganate under slightly basic conditions. With the dike-
tone 10 in hand, condensation with appropriate aryl aldehydes and
ammonium acetate in acetic acid afforded 2,4,5-trisubstituted imi-
dazoles 1–4 and 11 in reasonable yields. Oxidation of the 4-
(methylthio)phenyl substituent of imidazole 11 with potassium
persulfate gave the racemic 4-(methylsulfinyl)phenyl imidazole 5.
Tri-substituted imidazoles 6 and 7 containing the pyran-4-yl
substituent at imidazole C-5 were synthesized via the route out-
lined in Scheme 2.
cence-activated cell sorting (FACS) based on the Nkx2.5 and
a-
R1
myosin heavy chain ( -MHC) markers (Fig. 2). The effects of the
a
R1
O
O
compounds on cell growth and cardiomyogenesis were measured
and reported as fold over DMSO control. The results are summa-
rized in Figure 3.
R1
a, b
c
HO
OH
O
14
12
As shown in Figure 3A, only compound 5 resulted in improved
O
cell growth over the DMSO control at 5 lM. Compound 5 also had a
13
d or e
comparable effect on the growth as that of SB203580. Analysis of
the more effective compounds (i.e., 5 and 6) with regards to cell
expansion revealed that they have a common 4-(methylsulfi-
nyl)phenyl substituent at the imidazole C-2 position, similar to
SB203580. As such the cardiomyogenic activities of compounds
5–7, all of which have the 4-(methylsulfinyl)phenyl substituent,
were compared and the results are shown in Figures 3B and C.
Based on the results it was observed that replacing the 4-fluoro-
phenyl group of SB203580 with a 3-tolyl group in 5 had no adverse
effect on the cardiomyogenic activity of the compound. Compound
R1
R1
O
S
CH3
N
N
f
S
N
H
N
CH3
H
O
O
15, 16
6, 7
Scheme 2. Synthesis of tri-substituted imidazoles with pyran-4-yl substituent.
Reagents and conditions: (a) n-BuLi, LiBr, THF, RT; (b) tetrahydro-4H-pyran-4-one,
THF, À78 °C–rt; (c) KMnO4, dioxane/water (6:1), 0 °C–rt; (d) 4-(methylthio)benz-
aldehyde, NH4OAc, acetic acid, 115 °C;(e) 4-(methylthio)benzaldehyde, NH4OAc,
Cu(OAc)2ÁH2O, acetic acid, 80 °C; (f) K2S2O8, acetic acid/water (3:2), rt or 40 °C.

3302
J.-L. Low et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3300–3303
Table 1
IC50 values of the tri-substituted imidazole compounds
R2
N
2
R1
5
N
R3
H
b
Compounda
R2
R1
R
p38a
IC50 (nM)
SB203580
Pyridin-4-yl
Pyridin-4-yl
Pyridin-4-yl
Pyridin-4-yl
Pyridin-4-yl
Pyridin-4-yl
Pyran-4-yl
4-Fluorophenyl
3-Tolyl
3-Tolyl
3-Tolyl
3-Tolyl
3-Tolyl
4-Fluorophenyl
3-Tolyl
4-(Methylsulfinyl)phenyl
2,6-Difluorophenyl
2-Fluorophenyl
25
4
1
2
3
4
5
6
7
4.3 1.7
4.7 1.2
8.4 3.0
20
24
4-Hydroxyphenyl
1-Fluoronaphthalen-2-yl
4-(Methylsulfinyl)phenyl
4-(Methylsulfinyl)phenyl
4-(Methylsulfinyl)phenyl
7
9
454 67
73 21
Pyran-4-yl
a
Compounds were >95% pure by HPLC or elemental analysis (please refer to the Supplementary data).
IC50 values are the mean SEM of two runs in triplicate.
b
Figure 2. Representative FACS diagrams of hESCs treated with SB203580 at 5 lM. EBs on Day 15 were dissociated into single cells and analyzed for the Nkx2.5 marker (A)
through the detection of GFP expression and for the
a-MHC marker (B) through staining with MF20 primary antibody and detection with Fluorescein isothiocyanate-
conjugated secondary antibody.
Figure 3. Effect of tri-substituted imidazoles on cardiomyogenesis of hESCs at 5
(A) Effect of the compounds on the cell expansion as measured on Day 15; n P 3. (B) Percentage of cells expressing cardiomyocyte markers obtained on Day 15 as indicated
by FACS analysis of the markers Nkx2.5 and
-MHC; n P 3 except for DMSO (n = 1) and SB203580 (n = 2). (C) Cardiomyocyte yield per hESC seeded (=cell expansion fold Â
percentage of cell expressing cardiomyocyte markers) as indicated by the Nkx2.5 and -MHC markers; n = 3 except for DMSO (n = 1) and SB203580 (n = 2). Error bars
represent standard deviation. One-way analysis of variance (ANOVA) followed by Dunnett’s test between 5 and SB in (A), (B), and (C). P >0.05 (ns not significant).
lM. The results in the graphs are shown as fold over DMSO control. SB represents SB203580.
a
a
5 resulted in an average cardiomyocyte yield per hESC five to
eightfold greater than DMSO control, based on the cardiomyocyte
11-fold greater than DMSO control. However replacing the pyri-
din-4-yl group of SB203580 with the pyran-4-yl group (compound
6) significantly reduced the cardiomyogenic activity of the com-
pound as the yield of cardiomyocytes per hESC dropped to about
markers Nkx2.5 and
a-MHC. This is comparable to the effect of
SB203580 which resulted in cardiomyocyte yield almost 6- to

J.-L. Low et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3300–3303
3303
twofold compared to DMSO. Compound 7, which contains the
vicinal pyran-4-yl/3-tolyl motif instead of the vicinal pyridin-4-
yl/4-fluorophenyl motif of SB203580, also showed reduced
cardiomyogenic activity compared to SB203580 and resulted in
cardiomyocyte yield similar to that of DMSO.
docking studies and Ang Xiu Min and Dr. Hia Hui Ching for their
help in the p38 MAP kinase binding assays.
Supplementary data
We also examined the effect of varying the concentration of the
test compounds on cardiomyogenesis. As mentioned above and
based on previous reports, SB203580, with an IC50 value of
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03.
103.
25 nM for p38
at 5
M.5,7,9 We thus investigated if reducing the concentrations
of the more potent p38 MAPK inhibitors, and increasing the con-
a MAPK, showed optimal cardiomyogenic activity
l
References and notes
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among the compounds synthesized.
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In summary, we have designed and synthesized novel tri-
substituted imidazoles which are structural analogues of
SB203580, with a range of p38a MAPK inhibitory activities. Imida-
zoles possessing C-2 substituents other than the 4-(methylsulfi-
nyl)phenyl substituent had no effect on cell growth. Of the
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strated that suitably designed analogues of SB203580 can also be
inducers of cardiomyogenesis in hESCs and that embryoid bodies
are sensitive to changes in the imidazole structures. These observa-
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The authors would like to thank the Agency for Science, Tech-
nology and Research (A⁄STAR) for the generous funding of this pro-
ject. We are also grateful to Dr. Paul Bernardo for his advice in the