Synthesis of 5-arylated N-arylthiazole-2-amines as potential skeletal muscle cell differentiation promoters

Article abstract of DOI:10.1016/j.bmcl.2011.01.123

A series of N-arylthiazole-2-amines was prepared and their biological activity for the promotion of skeletal muscle cell differentiation was investigated, a process of significant importance in muscle regeneration. A versatile new synthetic route towards

Full text of DOI:10.1016/j.bmcl.2011.01.123

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2149–2154
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 5-arylated N-arylthiazole-2-amines as potential skeletal muscle
cell differentiation promoters
Michael Schnürch a, , Birgit Waldner a, Karlheinz Hilber b, Marko D. Mihovilovic a,
⇑
⇑
a Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-OC, A-1060, Vienna, Austria
b Center of Physiology and Pharmacology, Institute of Pharmacology, Medical University of Vienna, Währingerstraße 13, A-1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-arylthiazole-2-amines was prepared and their biological activity for the promotion of skel-
etal muscle cell differentiation was investigated, a process of significant importance in muscle regener-
ation. A versatile new synthetic route towards the target compounds was developed and the substrate
scope of this methodology was investigated. Introduction of the 2-aminoaryl substituent was carried
out via nucleophilic substitution reactions in excellent yields. Furthermore, the aryl in 5-position was
introduced applying a direct arylation reaction, a major improvement compared to reported synthetic
routes regarding atom efficiency and sustainability.
Received 10 December 2010
Revised 24 January 2011
Accepted 27 January 2011
Available online 2 February 2011
Keywords:
Heterocycles
CH-activation
Ó 2011 Elsevier Ltd. All rights reserved.
Cell differentiation
Bioactive compound
Molecules containing a thiazoleamine-moiety show a number
of interesting biological activities depending on the different sub-
stituents and the substitution pattern at the thiazole ring.1 Com-
pounds of type I (Fig. 1) have been reported as potent histone
deacetylase- and tyrosine kinase-inhibitors1b and N-phenylthiaz-
oleamine derivatives are known as selective aurora kinase inhibi-
tors.1c In addition, the ability to induce autophagic cell death in
renal cancer cells has been reported in a recent publication for
molecules of the general type II.1d A series of compounds (Fig. 1,
III) has been introduced previously as orally active glutamate
receptor antagonists (mGluRs), which are interesting targets for
regulating glutamate transmission, aiming at the treatment of neu-
rological and psychiatric diseases.1e Another interesting compound
is Neuropathiazole (IV), a thiazole-4-amine, which was reported to
induce neural differentiation of adult hippocampal neural progen-
itor cells.2 Approaches to influence cell differentiation processes
are of great potential and essentially two strategies have been de-
scribed:3 (i), de-differentiation of certain cell types back to multi-
potent progenitor cells which can then be differentiated again to
other cell types; (ii) trans-differentiation of a cell to another cell
type. The particular methods to influence differentiation processes
are highly diverse. For example, lineage conversions can be in-
duced through the introduction of certain transcription factors.4
Additionally, it was demonstrated that the transfer of nuclei from
either embryonic or somatic cell types can lead to the formation
of all three germ layers and—most remarkably—even to the gener-
ation of entire new animals.5 A recent development receiving
much attention was the reprogramming of somatic cells to induced
pluripotent stem cells (iPSCs) by the overexpression of a transcrip-
tion factor cocktail.6 These extremely remarkable results have cer-
tain disadvantages in view of future therapeutic application: a
retrovirus is required to insert genes into the genome to ultimately
stimulate de-differentiation into progenitor cells. As a consequence
there is an unacceptable risk of permanent transgene integration
into the genome. Additionally, two of the applied reprogramming
factors (Klf4 and c-Myc) were reported as oncogenic in the original
publication. However, this issue was already addressed by several
N
N
N
R1
R3
N
N
H
S
N
S
R2
N
O
I
F
R1, R2=Aryl, Hetaryl, Alkyl, H, Acyl
R3= Aryl, Hetaryl
III
R2
O
N
N
N
S
R1
N
COOEt
S
II
R1=Aryl, Hetaryl
R2= Aryl, Hetaryl
Neuropathiazole IV
Figure 1.
⇑
Corresponding author.
E-mail address: mmihovil@pop.tuwien.ac.at (M.D. Mihovilovic).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.123

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M. Schnürch et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2149–2154
research groups and methods which avoid use of these two genes
have been published.6f,7 Still, both facts make the approval of such
a method for medicinal applications problematic and eventually
unlikely. Besides the aforementioned problems, iPSC reprogram-
ming is still a slow and inefficient process.
established Hantzsch synthesis14 usually give high yields, but re-
quire the preparation of suitable reaction partners for cyclization
for every single compound. Therefore, a modular synthetic route
which allows for the synthesis of a large number of derivatives
from only a small set of building blocks is highly attractive. Within
this contribution such a modular synthesis is reported and the
scope of the method is demonstrated together with initial results
from biological screening as differentiation accelerators.
2-Arylamino-5-aryl-thiazoles were considered as target com-
pounds, since this compound class showed the potential to influ-
ence cell differentiation in a preliminary biological screening.
The conventional synthetic route to polysubstituted N-phenyl-
2-thiazoleamines is outlined in Scheme 1.1c–f,15 The first step con-
sists of a cyclization reaction of N-phenylthiourea derivatives (V)
An interesting alternative to influence cell differentiation is the
application of synthetic small molecules. Some synthetic small
molecules have been reported to induce cardiomyogenesis,8 neu-
rogenesis,2,9 as well as de-differentiation,10 and a series of publica-
tions has been dedicated to this topic in recent years.11 The use of
synthetic small molecules in therapies involving cell differentia-
tion processes would be highly desirable since most approved
drugs originate from the class of small chemical entities, and meth-
ods to test for activity and toxicology are well established. This
suggests that processes leading to approval of these compounds
for treatment in patients will be more facile compared to other
methods such as inserting genes into the cells via a retrovirus.
Until now synthetic small molecules played only a minor role in
accelerating cell differentiation processes. For example, adhes-
amine was reported to facilitate differentiation of primary cultured
hippocampal neurons12 and derivatives of wrenchnolol have been
shown to activate gene expression in cells when tethered to a DNA
binding molecule.13 As outlined in Figure 2, these compounds rep-
resent quite complex molecules and substituting them with sim-
pler scaffolds would be of great value.
and
a-halocarbonyl compounds to form N-phenylthiazoleamine
(VI). As the introduction of a halogen is required to perform
cross-coupling reactions, a protecting group has to be installed
prior to the bromination reaction avoiding formation of complex
product mixtures.16 However, bromination in 5-position of com-
pounds of type VII usually proceeds efficiently.16,17 Subsequently,
several types of cross-coupling reactions (Suzuki–Miyaura, Stille,
Negishi) can be performed to introduce different substituents on
the thiazole ring; removal of the protecting group ultimately leads
to target compounds X. This route has several disadvantages:
Introduction of a substituent on the phenyl ring requires the syn-
thesis of the corresponding thiourea derivative, which can be quite
tedious or even impossible for some substituents. Additionally, the
introduction of bromine for subsequent cross-coupling reactions is
not desirable from the viewpoint of atom efficiency.
Within this contribution, aryl-thiazole-amines are introduced
as a class of compounds which displays the ability to significantly
promote the differentiation process of C2C12 skeletal muscle cells.
Conventional synthetic routes to access these products are often
cumbersome and do not allow the efficient synthesis of com-
pound-libraries that can be screened for biological activity. For
example, cyclization reactions towards thiazoles such as the long
Therefore, an alternative route was envisioned to synthesize
target compounds of general structure X. As first step, amination
in position 2 of a 2-halothiazole was envisioned, followed by direct
O
R
SMe
N
N
OH
O
N
Cl
N
CHO
CHO
SMe
N
N
O
Cl
N
N
S
O
(CF3COO-)2
O
adhesamine
wrenchnolol derivatives
Figure 2.
S
PG
N
H
N
Cl
S
Protection
S
H
NH
H2N
+
N
N
S
R
R
O
VII
R
V
VI
Bromination
PG
N
PG
N
H
S
S
Cross-coupling
R
Deprotection
R
Ar
N
Ar
Br
N
N
N
R
VIII
X
IX
PG = Protecting group
Ar = Aryl, Hetaryl
Scheme 1.

M. Schnürch et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2149–2154
2151
arylation in 5-position. Starting from either 2-chloro- or commer-
cially available 2-bromothiazole, nitrogen incorporation in 2-posi-
tion can be carried out via palladium catalyzed Buchwald–Hartwig
amination reaction, a common method for the preparation of aryl-
and hetarlyamines,18 or by classical nucleophilic substitution.
Since considerable efforts to optimize the Buchwald–Hartwig reac-
tion did not provide good yields of the desired compounds due to
by-product formation, nucleophilic substitution was favored
(Scheme 2).19
ole 2 as halide component. The protocol enabled access to substi-
tuted thiazoleamines in good to excellent yields for electron-poor
as well as electron-rich aniline-derivatives (Table 1). Typically,
yields >90% were obtained and only in two cases the isolated yield
was <80% (entries 5 and 9). Regarding the halide leaving group, the
initial reaction with simple aniline displayed only a negligible dif-
ference in yield comparing Cl and Br; both experiments required
the same reaction time until completion (entries 1 and 2). Increas-
ing the temperature to shorten the reaction time was avoided since
then by-products were formed (although only in low amounts).
Electron-rich aniline-derivatives (entries 3–6) reacted significantly
slower, but only in the case of m-anisidine significant formation of
secondary products was observed reducing the yield of 7 (48%; en-
try 5). The method tolerates a wide range of different functional
groups (esters, nitro, chloro, methoxy, morpholino) and also steri-
cally demanding ortho-substituted anilines were well tolerated
with no loss in yield (entries 6 and 8). Also N-methylaniline gave
an excellent yield as an example for a secondary amine.
Reactions were performed using 0.5 equiv of p-TsOH as catalyst
in i-PrOH at 80 °C. A series of substituted anilines (2.0 equiv) was
used as nucleophile utilizing 2-bromothiazole 1 or 2-chlorothiaz-
N
N
p-TsOH (0.5 equiv.)
i-PrOH, 80°C
Ar
Ar NH2
+
X
N
H
S
S
X=Br (1), Cl (2)
IV
Direct arylation in 5-position of thiazoles has been reported
previously20 and gave superior results compared to Suzuki cross-
coupling reactions in some cases.21 This method is especially
attractive since it avoids introduction of a leaving group for subse-
quent cross-coupling and increases the atom- and time- efficiency
of a given synthetic route by concomitantly avoiding one reaction
step. Several protocols have been reported for the direct arylation
reaction of heterocyclic systems utilizing a conventional Pd(0) spe-
cies/ligand combination as catalyst,20b,22 in some cases with very
low catalyst loadings (0.001 mol %) in the absence of a ligand using
N,N-dimethylacetamide (DMAc) at 130 °C or 150 °C.23
In order to avoid competitive arylation of the amino-functional-
ity in 2-position a protective group had to be installed as direct
arylation conditions are quite similar (Pd catalyst, base, aryl donor)
to Buchwald–Hartwig amination conditions. This does not repre-
sent a disadvantage compared to a conventional cross coupling
reaction route since for the introduction of, for example, bromine
as leaving group a protective group has to be introduced as well.
For our purpose, the benzyl-group was chosen due to its simple
introduction and usually equally simple removal.24 In this case
the introduction of the benzyl-protecting group led to somewhat
unexpected results: depending on the reaction conditions different
products were formed starting from precursor 3 (Scheme 3). Using
benzylbromide and 1.0 equiv of NaH as base in DMF at room tem-
perature, the reaction was complete within 2–4 h and N-benzyl-N-
phenylthiazoleamine 1325 was formed in the reaction as expected.
However, when the protection was carried out with triethylamine
as base in dioxane at 120 °C, the reaction took three days to com-
plete and the resulting compound was isomeric N-(3-benzylthiaz-
ole-2(3H)-ylidene)aniline 1426 originating from benzylation of the
endocyclic nitrogen atom. Similar behavior was recently reported
in particular for aromatic electrophiles27 and is significantly af-
fected by substituents on the exocyclic nitrogen. In particular,
strong electron-withdrawing substituents on the exocyclic hetero-
atom shift the equilibrium towards the imino-product. In addition,
solvents with high dipole-moment favor formation of the more
polarized imine product.27c
Scheme 2.
Table 1
Nucleophilic substitution towards 2-amino-thiazoles
Entry
1
X
Time
3 d
Ar
Product
Yield (%)
90
N
H
Br
3
N
H
2
3
4
Cl
Cl
Cl
3 d
3
4
5
84
87
97
N
H
N
O
3 w
3 w
N
H
OMe
N
H
5
Cl
3 w
6
48
OMe
N
H
6
7
Cl
Cl
Cl
Cl
Cl
Br
3 w
7
8
85
97
95
76
98
94
MeO
N
H
COOEt
1.5 d
2 d
N
H
8
9
Cl
N
H
Cl
9
2 d
10
11
12
N
H
10
11
1.5 d
1.5 d
NO2
N
N
BnBr (1.3 equiv.)
BnBr (1.3 equiv.)
TEA (1.0 equiv.)
NaH (1.0 equiv.)
N
N
S
N
N
H
S
dry DMF, rt, 3h
dry dioxane,120°C, 3d
N
S
76%
66%
14
3
13
Scheme 3.

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M. Schnürch et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2149–2154
Table 2
The present case is a unique situation for the benzyl protecting
Direct arylation of thiazoles 13 and 14
group. Using the weak base triethylamine deprotonation is a slow
process and at high temperatures the equilibrium is shifted to the
imino-product. When sodium hydride is used as a base, deprotona-
tion occurs practically instantaneously and now the nucleophilicity
of the deprotonated exocyclic nitrogen is much higher upon attack
at benzyl bromide.
Entry
Time (h)
Ar
Product
Yield (%)
85
1
48
15
2
3
4
16
17
94
60
With precursor 13 in hand, direct arylation conditions were
tested (Scheme 4; Table 2). When using iodobenzene as the halide
component, best results were obtained using 1.0 equiv of 13,
2.0 equiv of iodobenzene, 2.0 equiv KOAc as base and 1 mol % of
Pd(OAc)2 as catalyst in DMAc at 120 °C leading to an isolated yield
of 85% of 15 (entry 1).28 As similar transformation with bromoben-
zene displayed only low conversion to 15, iodoaryls and iodohete-
roaryls were used as aryl donors in the following reactions. Good
yields were obtained using electron deficient aryliodides (entries
2, 3 and 9). However, nitro-substituents were generally not toler-
ated well resulting in decreased yields (entries 6 and 7). Also, 4-
iodoanisole gave a lower yield even upon increasing catalyst load-
ing to 10 mol % (entry 8); this goes in line with results obtained by
Roger et al. when using p-bromoanisole in direct arylation reac-
tions.23b Electronically neutral aryliodides also gave good yields
(entries 1 and 5). Heterocyclic systems gave good results (entries
1 and 10) with the exception of 5-iodopyrazole (entry 11); this
can be attributed to the presence of a free NH group and is in line
with previous reports by Roger et al. on similar reactants contain-
ing free –NH groups.23a On the other hand, ortho substituents were
well tolerated in direct arylation reactions (entries 2 and 4).
N
F
24
EtOOC
4
5
20
20
18
19
80
77
6
3
20
41
NO2
O2N
7
8
9
18
18
20
21
22
23
20
47a
78
MeO
F
KOAc (2.0 equiv.)
Pd(OAc)2 (0.01 equiv.)
N
N
10
11
20
48
24
25
85
0b
+
Ar
I
S
N
Bn
Ar
N
Bn
S
S
dry DMAc, 120°C
3
NH
N
15-24 (20-94%)
a
10 mol% of catalyst had to be applied.
No conversion.
Scheme 4.
b
Figure 3.

M. Schnürch et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2149–2154
2153
Table 3
Target compounds were then tested regarding their biological
Morphological effect of test compounds on cell differentiation towards myotubes
activities in context of influencing the differentiation of C2C12
skeletal muscle cells. This cell line represents a well established
model to study myogenic differentiation, which impacts muscle
regeneration.29 Within this preliminary study, only changes in
cell morphology were monitored under the microscope at differ-
ent time points (3, 5 and 7 days) after induction of differentiation
by serum reduction,30 whereby control cells were compared with
single compound-treated cells. All C2C12 cells were incubated for
two days in growth medium to reach a cell confluence of about
70%. Thereafter, differentiation medium without any compound
was applied to one part of the cells (control group). The other
cells were incubated in differentiation medium containing the
Entry
Compound
Differentiation
induction
N
N
1
Low
N
Bn
15
16
17
S
S
2
Moderate
N
Bn
N
F
compounds to be tested in a concentration of 5 lM (experimental
N
groups). Prior to application, each single compound was
dissolved in DMSO to obtain 20 mM stock solutions. Appropriate
volumes of these stock solutions were then added to the differen-
tiation medium to reach the desired final compound concentra-
3
4
5
Moderate
None
N
Bn
S
EtOOC
N
tion of 5 lM. The differentiation medium applied to control
18
19
20
cells contained equal amounts of DMSO. Each experiment was
carried out as doublet, and four control experiments were
performed.
N
Bn
S
After beginning of treatment with the compounds, cell cultures
were incubated for 7 days at 37 °C. The medium was changed twice
(after 3 days and 5 days of incubation). Morphological changes of
cell cultures were documented by taking pictures after 3, 5, and
7 days (Fig. 3). Normally, 2–3 days after the induction of differen-
tiation, undifferentiated C2C12 myoblasts begin to fuse to each
other and form small multinuclear myotubes.30 Larger longitudinal
myotubes containing tens of nuclei usually appear after 5–6 days.
Already after 3 days of treatment it could be observed that several
of the tested compounds considerably promoted the differentia-
tion process of muscle cells in comparison with the controls. Espe-
cially compound 24 proved to be highly active (Fig. 3). It can be
observed that in cultures treated with 24 the number of large
and well developed myotubes is considerably higher than in the
control culture. In comparison to the pictures taken from the
shown control experiment, the cells treated with 24 showed visible
myotubes already after 3 days and a further enhancement of differ-
entiation after 5 and 7 days. Morphological changes generated by
all the tested compounds are summarized in Table 3. Except for
compounds 18, 19 and 21, all the other compounds seemed to pro-
mote C2C12 cell differentiation whereby compound 24 was most
effective.
N
None
N
S
Bn
N
N
6
Moderate
S
Bn
O2N
O2N
N
21
23
24
7
8
9
None
N
Bn
S
N
Moderate
High
N
S
F
Bn
N
N
S
S
Bn
In summary, an alternative, more practical pathway is pre-
sented for the synthesis of N,5-substituted thiazoleamine deriva-
tives. The nucleophilic substitution reaction represents a high
yielding and more practical alternative to the cyclization reaction
to form N-phenylthiazoleamine 3 and derivatives thereof. After
protection of the exocyclic nitrogen introduction of an aryl or het-
eroaryl substituent in 5-position was achieved by direct arylation.
The particular reaction protocol utilized a ligand free system with
Pd(OAc)2 as catalyst and gave good to excellent yields in most
cases with low catalyst loading. This route presents a three-
step synthesis for 5-arylated-N-protected-N-phenylthiazoleamine
derivatives, which is a significant improvement to already estab-
lished strategies requiring 5+ steps.
With respect to biological activity of target compounds, a
significant promotion of cell differentiation in C2C12 skeletal
muscle cells was observed. Since enhanced myogenic differentia-
tion in vitro goes along with improved muscle regeneration
in vivo29b,31 the active compounds identified in the present study
could be useful to promote healing of muscles damaged by injury
or disease. Further investigations regarding structure activity rela-
tionships aiming at the improvement of these results is currently
underway in our laboratory.
Acknowledgment
The authors thank Vienna University of Technology for support-
ing this project by an internal Micro Fund.
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24. Greene, T. W. W., 3rd ed. In Protective groups in Organic Synthesis; Wiley-
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25. N-Benzyl-N-phenylthiazoleamine 13: N-Phenylthiazoleamine
3
(30 mg,
0.17 mmol. 1.0 equiv), benzyl bromide (38 mg, 0.22 mmol, 1.3 equiv), and
NaH (4.1 mg, 0.17 mmol, 1.0 equiv) were dissolved in dry dimethylformamide
(2 mL) and stirred at room temperature for 5 h. The reaction mixture was
diluted with ethyl acetate, silica gel was added, and this suspension was
concentrated in vacuo, followed by MPLC purification (light petroleum/ethyl
acetate 85:15 + 5% of triethylamine). Compound 13 was obtained as a beige
solid (30 mg, 66%). Mp: 79–80 °C; Rf (light petroleum/ethyl acetate 5:2) 0.20;
1H NMR (200 MHz, CDC13) d = 5.12 (s, 2H), 6.40 (d, J = 3.7 Hz), 7.15–7.33 (m,
10H); 13C NMR (50 MHz, CDC13) d = 56.6 (t), 107.7 (d), 126.2 (d), 126.8 (d),
127.2 (d), 127.7 (d), 128.5 (d), 129.8 (d), 137.7 (s), 139.3 (d), 145.3 (s), 170.9 (s);
HRMS: MH+, found 267.0955. C16H14N2S requires 267.0950.
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26. N-(3-Benzylthiazol-2(3H)-ylidene)aniline 14: N-Phenylthiazoleamine 3 (264 mg,
1.50 mmol, 1.0 equiv.), benzyl bromide (334 mg, 1.96 mmol, 1.3 equiv), and
triethylamine (152 mg, 1.50 mmol, 1.0 equiv) were dissolved in dry dioxane
(4 mL) at 0 °C. The reaction mixture was slowly warmed to room temperature
before it was heated to 120 °C for 3 days. The mixture was then diluted with
ethyl acetate, silica gel was added, and this suspension was concentrated in
vacuo, followed by MPLC purification (light petroleum/ethyl acetate 85:15 + 4%
of triethylamine). Compound 14 was obtained as a light yellow solid (287 mg,
72%). Mp: 94–95 °C; Rf (light petroleum/ethyl acetate 5:2) 0.20; 1H NMR
(200 MHz, CDC13) d = 5.06 (s, 2H), 5.85 (d, J = 4.9 Hz, 1H), 6.50 (d, J = 4.9 Hz,
1H), 7.00–7.10 (m, 3H), 7.33–7.37 (m, 7H); 13C NMR (50 MHz CDCl3) d = 49.8
(t), 97.6 (d), 121.4 (d), 123.2 (d), 126.3 (s), 128.1 (s), 128.6 (d), 129.4 (d), 136.8
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0.01 equiv of Pd(OAc)2 were charged into the reaction vessel in that same
order. Dimethylacetamide was added and the reaction vial was evacuated and
purged with argon three times. The reaction mixture was then stirred at 120 °C
until the reaction was complete. The solution was diluted with ethyl acetate,
silica gel was added, and this mixture was concentrated in vacuo, followed by
MPLC purification.
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19. General procedure for nucleophilic substitution reactions towards compounds
3–12: 1.0 equiv of the halothiazole, 1.5 equiv of the amino-substrate and
0.5 equiv of p-toluenesulfonic acid were dissolved in i-propanol and the