Ethyl 2-((4-Chlorophenyl)amino)thiazole-4-carboxylate and Derivatives Are Potent Inducers of Oct3/4

Article abstract of DOI:10.1021/acs.jmedchem.5b00226

The octamer-binding transcription factor 4 (Oct3/4) is a master gene in the transcriptional regulatory network of pluripotent cells. Repression of Oct3/4 in embryonic stem cells (ESCs) is associated with cell differentiation and loss of pluripotency, wher

Full text of DOI:10.1021/acs.jmedchem.5b00226

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Article
Ethyl 2-((4-chlorophenyl)amino)thiazole-4-carboxylate
and derivatives are potent inducers of Oct3/4
Xinlai Cheng, Hiroki Yoshida, Dena Raoofi, Sawsan Saleh, Hamed Alborzinia, Frank Wenke,
Alex Goehring, Stefanie Reuter, Nancy Mah, Heiko Ralph Fuchs, Miguel A Andrade-
Navarro, James Adjaye, Sheraz Gul, Jochen Utikal, Ralf Mrowka, and Stefan Wölfl
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00226 • Publication Date (Web): 05 Jul 2015
Downloaded from http://pubs.acs.org on July 10, 2015
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Ethyl 2-((4-chlorophenyl)amino)thiazole-4-
carboxylate and derivatives are potent inducers
of Oct3/4
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Xinlai Cheng,1 Hiroki Yoshida,1 Dena Raoofi,1 Sawsan Saleh,1 Hamed Alborzinia,1 Frank
Wenke,2 Axel Göhring,2 Stefanie Reuter,2 Nancy Mah,3 Heiko Fuchs,4 Miguel A. Andrade-
Navarro,3 James Adjaye,4 Sheraz Gul,5+ Jochen Utikal,6 Ralf Mrowka,2* Stefan Wölfl1*
1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer
Feld 364, Dꢀ69120 Heidelberg, Germany
2Experimentelle Nephrologie. KIM III, Universitätsklinikum Jena, Am Nonnenplan 2ꢀ4, Dꢀ
07743 Jena, Germany
3Max Delbrück Center for Molecular Medicine, Berlin, Germany
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Institute for Stem cell Research and Regenerative Medicine. Medical Faculty, Heinrich Heine
University, Duesseldorf, Germany
5European Screening Port GmbH, Hamburg, Germany
6Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg University, Mannheim,
Germany
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+Current address: FraunhoferꢀIME SP, Schnackenburgallee 114, Hamburg, Germany
*Equal contribution
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KEYWORDS.
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iPSCs, Oct3/4 activator, stem cell, pluripotent, highꢀthroughput screening, small molecule,
chemical reprogramming, 2ꢀaminothiazole
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ABSTRACT: The Octamerꢀbinding transcription factor 4 (Oct3/4) is a master gene in the
transcriptional regulatory network of pluripotent cells. Repression of Oct3/4 in embryonic stem
cells (ESCs) is associated with cell differentiation and loss of pluripotency, whereas forced
overexpression in cooperation with other transcriptional factors, such as Nanog, Sox2 and Lin28,
can reprogram somatic cells back into pluripotent cells, termed induced pluripotent stem cells
(iPSCs). However, random integration and potential tumorigenic transformation caused by viral
transduction limit the clinical application of iPSCs. Performing a cellꢀbased High Throughput
Screening (HTS) campaign, we identified several potential small molecules as inducers of
Oct3/4 expression. Here we report a lead structure ethyl 2ꢀ((4ꢀchlorophenyl)amino)ꢀthiazoleꢀ4ꢀ
carboxylate, termed O4I2, showing high activity in enforcing Oct3/4 expression. Based on
chemical expansion we further identified derivatives having increased activities towards Oct3/4
induction. Thus O4I2 and its derivatives should provide a new class of small molecules suitable
for iPSC generation.
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Introduction
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Oct3/4 (Octamerꢀbinding transcription factor 4), also referred to POU5F1, Oct4A, Oct3 or
Oct4, is a member of the POU family of transcription factors1 expressed specifically in
pluripotent cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells
(iPSCs).2 Evidence is given that lossꢀofꢀfunction mutation of Oct3/4 leads to differentiation of
ESCs into trophoblast,3 while gainꢀofꢀfunction or overꢀexpression leads to differentiation into
primitive endoderm and mesoderm,4 supporting the important role of Oct3/4 in regulation of the
cellular pluripotency core network. It had been shown by the groups of Yamanaka and Thomson
that terminally differentiated cells can be deꢀdifferentiated back to pluripotency by viral
transduction with a set of defined factors, Oct3/4, Sox2, KLF4 and cꢀMyc,5, 6 or alternatively
Oct3/4, Sox2, Nanog and Lin28.7 However, the low efficiency and risks associated with the
genetic transformation handicap the wide use of iPSCs in the clinic,8 where iPSCs could be an
infinite source for patientꢀspecific cells and tissue. The desire for improved protocols for the
generation of iPSCs boosts researches into reprogramming technology to increase efficacy and
address safety concerns.9, 10 Of particular interest are small molecules that can replace defined
factors and enhance reprogramming efficiency.11ꢀ13 Recently, an azaindole derivative was
reported to enhance Oct3/4 activation and thereby promote reprogramming efficiency of mouse
embryonic fibroblast transfected with four defined factors.14 Moreover, scientists successfully
generated mouse iPSCs from mouse embryonic fibroblasts (MEFs) cells by using a combination
of seven small molecules, indicating that small molecule driven induction of defined factors can
be sufficient in reprogramming.15 However, human fibroblasts still seem to be more refractory,
and so far researchers had to overꢀexpress at least one transcription factor, namely Oct3/4, to
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achieve generation of human iPSCs, in combination with different small molecules. Thus, Oct3/4
seems to be a nonꢀredundant defined factor in human cell reprogramming.16, 17
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Results and discussion
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Initially, we performed a High Throughput Screening (HTS) campaign using HEK293 cells
stably expressing luciferase reporter under the control of a larger human Oct3/4ꢀpromoter region
(HEKꢀOct3/4) and identified hundreds of compounds from a pool of 250,000 small molecules
able to elevate luminescent intensity in the threshold of 150% effect relative to cells treated with
DMSO alone. Interestingly, analysis of the hit compounds using Instant JChem (ChemAxon,
Hungary) revealed a cluster of dozens of compounds containing a thiazoleꢀring. Among them,
ethyl 2ꢀ(4ꢀchlorophenylamino)thiazoleꢀ4ꢀcarboxylate (Fig. 1A), referred to as Oct4 inducing
compound 2 (O4I2) to distinguish from O4I1 we reported recently,18 and others with 2ꢀ
(phenylamino)thiazoleꢀ4ꢀcarboxylate moiety exhibited approximate 2ꢀfold higher activity at 20
µM. Although in general HTS is an efficient approach to identify desired small molecule(s)
using a well designed assay, it suffers from classes of small molecules, that appear as frequent
hitters in divers assays in a drugꢀunlike fashion, called panꢀassay interference compounds
(PAINS).19, 20 Very recently, 2ꢀaminothiazoles (in concentrations above 200 µM) have been
identified as one of the PAINS in biological protein binding assays.21 Nevertheless the authors
emphasized that 2ꢀaminothiazoles at low concentrations (like 20 µM) could still show specific
activities in HTSs.21 Considering that our results were obtained from a cellꢀbased HTS at a
recommended concentration21 and numerous approved drugs, such as Talipexole and
Pramipexole, are based on 2ꢀaminothiazole as pharmacophore,22 we were curious to investigate
O4I2 in detail.
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To independently verify the results obtained from HTS, we followed Hantzsch thiazole
synthesis23 and successfully synthesized O4I2 in a reaction of 1ꢀ(4ꢀchlorophenyl)thiourea with
ethyl 3ꢀbromoꢀ2ꢀoxopropanoate in ethanol in a good yield of 89%. The detailed reaction protocol
as well as compound characterization is described in the experimental section.
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Using the independently newly synthesized, purified O4I2, we analyzed the response to O4I2
on the level of luminescence and found the positive correlation to concentrations at 72 hrs of
treatment, showing the highest activation in 3.5ꢀfold at 40 µM, while we could not detect clear
difference within the signals after 48 hrs treatment at the tested concentrations, suggesting that a
longꢀterm exposure might be required to achieve a higher activation (Fig. 1B). Previous reports
illustrated the importance of the Oct3/4 protein in completely accomplishing conversion from
somatic cells into iPSCs by binding to the corresponding promoters of other pluripotency related
genes and thereby facilitating their expressions.24 Hence, we immunoblotted nonꢀinfected
HEK293 cells after 72 hrs treatment to determine the protein level of Oct3/4. The results
demonstrated a clear accumulation of Oct3/4 in a concentrationꢀdependent manner (Fig. 1C).
Moreover, the level of Nanog protein was increased (Fig. 1C). Analysis by qRTꢀPCR revealed a
significant induction of stemnessꢀassociated genes after stimulation with O4I2, including Oct3/4,
Nanog and Sox2 in HEK293 cells, while the level of Lin28 remained unaffected and Rex1 was
even reduced (Fig. 1D).
Our results showed that O4I2 might not only promote Oct3/4 expression by activating its
promoter site, but also stabilize the Oct3/4 protein in HEK293 cells. Next, we directly assayed
the transcriptional activity of Oct3/4 using a luciferase reporter driven by an Oct3/4
transcriptional response element in embryonal carcinoma NCCIT cells (NCCITꢀOct3/4) and
found an up to 4ꢀfold increased luminescence intensity after 72 hrs treatment (Fig. 2A). In good
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agreement, the results obtained from an online luciferase assay, in which the signal of
luminescence was constantly recorded in NCCITꢀOct3/4 cells treated with increasing
concentrations of O4I2, also interestingly showed transcriptional activation induced not only by
Oct3/4 but also Nanog over the tested period (SI. 1 and 2). Moreover, we accordingly observed
that O4I2 markedly stimulated Oct3/4 even at 5 µM after 72 hrs treatment on the translational
level (Fig. 2B). Of note, the accumulation of Oct3/4 in NCCIT occurred already 2 hrs after
incubation with 20 µM O4I2 (Fig. 2C), while the detectable Sox2 increase appeared much later,
implicating compoundꢀmediated activation of Oct3/4 might be different between embryonic
kidney HEK293 cells and embryonal carcinoma NCCIT cells. Next, we examined the inducible
ability of O4I2 in HeLa cells, which do not express Oct3/4.25 The results clearly showed the
concentrationꢀdependent activation of Oct3/4, as well as its downstream target Nanog, thus
implying that O4I2ꢀmediated induction was independent of the cellular intrinsic endogenous
Oct3/4 level (Fig. 2D).
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Human fibroblasts are terminally differentiated, accessible cells, which have been shown to be
amenable to reprogramming with relatively high efficiency and thus have emerged as a
favourable donor source in iPSCs generation. To test if our newly discovered small molecule
could induce pluripotent marker activation in fibroblasts, we investigated the expression of
several reprogrammingꢀassociated markers measured by qRTꢀPCR after 48 hrs treatment with
O4I2 in human primary skin fibroblasts (HFs) and detected a marked increase of Oct3/4
expression (15ꢀfold), as shown in Fig. 3A. Moreover, the induction of Lin28 and Rex1 was even
higher than Oct3/4, showing 20ꢀ and 30ꢀfold increase in expression, respectively. We further
detected a moderate activation of Nanog (2ꢀfold). Interestingly, a mesenchymal marker Nꢀ
Cadherin was notably repressed. We next examined the status of stemness markers at the protein
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level in HFs, which were treated with O4I2 for 72 hrs. Immunoblot analysis revealed a
considerable accumulation of Lin28 and Nanog (Fig. 3B). We performed immunocytochemistry
and successfully detected coꢀexpression of Oct3/4 and Sox2 (Fig. 3C; upper), as well as Nanog
and Klf4 (Fig. 3C; lower) in HF cells treated with O4I2 for 5 days. However the level of Oct3/4
induced by small molecule was still much lower than that in nonꢀtreated embryonal NCCIT cells
analyzed by immunoblotting (Fig 3D). It is wellꢀknown that levels of alkaline phosphatase
(ALP) activity are elevated in ESCs and iPSCs.4, 5, 7, 9, 11, 12, 15, 24 Therefore, we examined activity
of ALP in O4I2ꢀtreated fibroblasts after 5 days, and included propidium iodide (PI) staining to
visualize dead cells. In comparison to DMSO (mock) treatment, a slight but clear increase in the
ALPꢀFITC signal could be detected indicating that an alternation of endogenous cellular
properties occurred (SI. 3).
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To further elucidate the activity, we next studied the influence of various substituents on the
aminoꢀgroup of O4I2 through chemical modification illustrated in SI. 4. The derivatives are
listed in Tab. 1 and grouped as three subꢀfamilies, namely variant in 4ꢀposition of Nꢀphenyl
(indicated in pink), halogenꢀ (indicated in violet) and disubstitutedꢀvariants (indicated in orange),
whose respective activities were measured in HEKꢀOct3/4 reporter cells and illustrated in Fig. 4.
Regarding the paraꢀvariants, we found that the depletion of chlorophenylꢀgroup (2), or
replacement with butylꢀ (3), allylꢀ (4), acetylꢀ (5), benzylꢀgroup (7) or rigid, bulk substituents (8)
resulted in a significant reduction (3, 4, 7) or a complete loss (2, 5, 8) of activity, whereas
removal of chloride (6) led to a slight functional enhancement, suggesting that the phenylꢀgroup
may be preferable in this position. Therefore, we developed another series of O4I2 derivatives
based on 2ꢀ(phenylamino)ꢀthiazoleꢀ4ꢀcarboxylate to investigate the influence of various halogens
at different substituent position on the activity. As shown in Fig. 4, highlighted in violet, the
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results demonstrated that compounds with chloroꢀ and fluoroꢀgroups in particular at mꢀposition
(compounds 9 and 15) showed more activity in the luciferase assay. Moreover, we synthesized
various dichloroꢀsubstituted derivatives and found that only m,pꢀ or o,pꢀdisubstituted could hold
(17) or slightly enhance (18) luciferase activity. Finally, we examined if the aqueous solubility
played a role in the activity. Thus we converted 4 compounds (O4I2, 9, 18 and 20) to their
corresponding carboxylic acids (22-25) by heating with 4 N HCl, indicated as acidꢀvariant in Fig.
4. To our surprise, no activity was observed with any of these four compounds. Further
conversion of 22 with N,Nꢀdimethylpropaneꢀ1,3ꢀdiamine to its corresponding amide (26) under
dicyclohexylcarbodiimid (DCC) and Nꢀhydroxybenzotriazol (HOBt) in dried CH2Cl2 also did not
result in activity enhancement in HEKꢀOct3/4 (SI. 4). These results might indicate a requirement
of the short hydrophobic group in the thiazole moiety for sufficient activation, and further
substantiate that the 2ꢀaminothiazole derivatives do not act as PAINS in our cellꢀbased luciferase
reporter assay. For a more detailed analysis, other carboxamides should be synthesized and
studied in the near future.
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Conclusion
Oct3/4 is an essential element in the maintenance and generation of pluripotent cells and small
molecule to activate Oct3/4 is still scant. In this work, we present a class of compounds carrying
2ꢀaminothiazoles as potential Oct3/4 inducers identified from a cellꢀbased HTS. Given that in
very recent papers, 2ꢀaminothiazole based compounds had been described to be panꢀactive in
biological assays and therefore had been termed "PAINS",19ꢀ21, 26 we carefully validated their
specific activity. Using a chemical approach, we structurally verified the lead compound O4I2
and showed that (i) O4I2 activates Oct3/4 in HEK293 and embryonal NCCIT cells, and also in
Octꢀdeficient HeLa and terminallyꢀdifferentiated human fibroblasts, and (ii) that O4I2 activates
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also other pluripotencyꢀassociated genes such as Lin28 and Nanog in a drugꢀlike manner.
Studying the structureꢀactivity relationship of O4I2 based on 25 derivatives revealed an
indispensable role of the phenylꢀgroup and that its chloroꢀ or fluoroꢀmꢀsubstituted derivatives
further enhanced activity, while conversion to carboxylic acids led to a completed loss of activity
Thus, the results obtained in our study provide clear evidence that a small molecule, namely
O4I2, acts as a potent Oct3/4 inducer in various human cell lines including human fibroblasts.
Importantly, the basic 2ꢀaminothiazole structure itself, as well as its carboxylic acid derivatives,
showed no activity. We completely agree that this basic 2ꢀaminothiazole structure should have
the potential to interact with many biological target molecules, based on its ability to form
hydrogen bonds and aromatic interactions. Considering the complexity of the control of Oct3/4
expression, this could be even beneficial for the desired biological effect. The careful validation
of our results using reꢀsynthesis, chemical expansion of the basic structural motif and highly
purified compounds for activity evaluation confirm the highly specific activity of the compounds
described here. Interestingly, the very low concentration used in our initial screening, has been in
the concentration range recommended by Devine et al.,21 to avoid false positive results. Taken
together, our results provide evidence, that this class of compounds could be a useful tool for
chemicalꢀmediated induction of pluripotency in somatic cells.
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Experimental section
Cell culture
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HEK293 and HeLa cells were cultured in DMEM (Gibco, Germany) containing 10% FBS (PAA,
Germany) and 1% Penicillin/Streptomycin (Pen/Strep, Gibco, Germany) under 5% CO2 at 37 °C
in a humidified atmosphere. HF and HFF cells were under the same conditions, but with 20%
FBS and 15% FBS, respectively. Oct3/4 stable reporter HEK293 cells were maintained in
DMEM medium (10% FCS, 1% 1 M Hepes – Buffer, 0.5% Pen/Strep and 0.5% 200 mM
glutamine with fresh addition of 100 µg/mL Hygromycin before use). NCCIT cells were
maintained in RPMI1640 (Gibco, 10% FBS and 1% PS). NCCIT cells were infected Cignal
Lenti Oct3/4 (SA Bioscience, Qiagen, Germany) according to the manufacturer’s instructions
and cultured in RPMI 1640 containing 10% FBS and 1% Pen/Strep with fresh addition of 400
ng/mL Puromycin (Santa Cruz, Germany).
Isolation and culture of primary human fibroblasts
Experiments with primary human fibroblasts were conducted with patients’ informed consent
and were approved by the Medical Ethics Committee of the Medical Faculty Mannheim,
Heidelberg University (2009ꢀ350NꢀMA). The procedure was published previously.18 Briefly,
freshly retained human skin samples from redundant operation material containing the dermis
and adipose tissue were cut into small pieces. In the presence of 0.25% trypsin in PBS the tissue
was then cut with scalpels in order to dissociate the tissue until reaching a pasty consistency.
These tissue fragments were then resuspended in fibroblast cell culture medium (DMEM
containing 10% FBS, 1x nonꢀessential amino acids, 1% penicillin/streptomycin and 50 µM ßꢀ
mercaptoethanol), plated on tissue culture dishes and kept at 37°C in a humidified atmosphere at
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5% CO2 until fibroblasts grew out. These fibroblasts were further expanded and used for
experiments.
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Luciferase reporter assay
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Beetle juice kit (PJK, Germany) was used to determine the luciferase activity as previously
reported.18 Briefly, HEK293ꢀOct3/4 or NCCITꢀOct3/4 reporter cells were seeded into 24ꢀwell
tissue culture plates at a density of 100,000 per well for 24h. After treatment with compounds as
indicated in the text, the cells were harvested with 80 µL Beetle lysisꢀjuice at 37°C for 10 min.
The protein concentration was determined by Bradford assay (Sigma, Germany). 100 µL
reaction mixture containing luciferin and ATP was added to 20 µL cell lysis in a white plate
(Gibco, Germany), incubated for 3 min and measured by a luminometer plateꢀreader. The
activity was determined as percent luminous intensity of treated cells over control cells from at
least fiveꢀindependent experiments.
qRT-PCR
Total RNA was isolated by using GenElute™ Mammalian Total RNA Miniprep Kit (Sigmal,
Germany) to reverseꢀtranscriptional synthesize cDNA by using random primers. Quantitative
reverseꢀtranscription realꢀtimeꢀPCR was performed using a Light Cycler 480 (Roche, Germany)
following a manufacturer’s protocol using the following primer pairs (MWG, Eurofins,
Germany):
(Oct3/4)
5s:
GAAGTTGGAGAAGGTGGAAC;
3as:
GGTGATCCTCTTCTGCTTCAG; (Nanog) 5s: GAACTGTGTTCTCTTCCACC; 3as:
CACCTGTTTGTAGCTGAGGTTC; (Sox2): 5s: CAAGACGCTCATGAAGAAGG; 3as:
CATGTGCGCGTAACTGTCCATG; (Rex1) 5s: GATCTTCAACGAGTCCACCAG; 3as:
GAAAGGTGGGAGATCCTCCTCTTC; (Lin28A) 5s: GTGGATGTCTTTGTGCACCAG; 3as:
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GACACGGATGGATTCCAGAC. ßꢀActin 5s: CTGACTACCTCATGAAGATCCTC; 3as:
CATTGCCAATGGTGATGACCTG was used as an endogenous control. Data were normalized
to the value of DMSOꢀtreated cells showing the mean ± SD of quadruplicates and are
representative of two independent experiments.
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Immunoblotting
The immunoblotting was performed as previously described.18,
Briefly, the cells were
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incubated with the compounds as indicated in the text and lysed using ureaꢀlysis buffer (1 mM
EDTA, 0.5% Triton Xꢀ100, 5 mM NaF, 6 M Urea, 1 mM Na3VO4, 10 µg/mL Pepstatin, 100 µM
PMSF and 3 µg/mL Aprotinin in PBS). Enhanced chemiluminescence (ECL) immunoblot
analysis was performed. 40µg of total protein was resolved on 10% SDSꢀPAGE gels and
immunoblotted with Oct3/4 (Cell Signaling, NEB, Germany), Nanog (Abcam, USA), Lin28
(Epitomics, Biomol, Germany), Vinculin (Cell Signaling, NEB, Germany) or ßꢀActin antibodies
(Sigma Aldrich, Germany). Primary antibodies were incubated at a 1:1000 dilution in TBS (pH
7.5) with 0.1% Tweenꢀ20 and 5% BSA/milk with gentle agitation overnight at 4°C. Secondary
antibodies (Cell Signaling) were incubated in TBS (pH 7.5) with 5% BSA/Milk and 0.1%
Tweenꢀ20 at a 1:10,000 dilution for 1 hr at room temperature.
Immunofluorescence
As we reported,18 human fibroblasts were seeded in a 96ꢀwell plate coated with Geltrex
(Invitrogen, Germany) at a density of 10,000 cells/well. After 24 hrs the cells were incubated
with O4I2 for 5 days in DMEM/F12 Glutamax containing 20% knockout serum replacement, 10
ng/mL bFGF and 1 mM ßꢀmercaptoethanol. Afterwards, the cells were fixed with 4% PFA at RT
for 15 min, and blocked with blocking buffer (5% goat serum and 0.3% Triton Xꢀ100 in PBS)
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for 1 hr. Blocking solution was aspirated and incubated with Oct3/4 (1:400, Cell Signaling),
Nanog (1:1000, Cell Signaling), Sox2 (1:100, Cell Signaling) and Klf4 (1:100, Biomol) in
antibody dilution buffer (1% BSA and 0.2% Triton Xꢀ100 in PBS) at 4°C overnight. The
secondary antibody (Goat antiꢀmouse Alexa Flour® 594 or 488, Dianova, Germany) was added
and incubated for 1 hr. Hoechst 33258 (1 µg/mL in PBS) was used to visualize nuclei.
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Statistics
Statistical and graphical data analyses were performed using Microsoft Excel 2007 software. The
statistical significance of compared measurements was performed by using the Student’s oneꢀ
tailed tꢀtest.
Chemistry
Reagents
Solvents and reagents obtained from commercial suppliers were at least of reagent grade and
were distilled or dried according to prevailing methods prior to use, if necessary. For monitoring
the reactions, Merck silica gel plastic plates 60 F254 (detection by UV) for TLC (thinꢀlayer
chromatography) were used. Chromatography was accomplished on silica (0.060−0.200 mm, 60
Å). HPLC analysis confirmed the purity of each compound was at least >96%.
Analytical Methods
1H and 13C NMR spectra were recorded on a Varian 300 MHz NMR system (1H: 300 MHz, 13C:
75 MHz). Chemical shifts are reported in ppm from tetramethylsilane with solvent as the
internal standard (1H DMSOꢀd6: δ 2.50; 13C DMSOꢀd6: δ 39.5). The following abbreviations
were used to explain the multiplicities in NMR spectra: s =singlet, d = doublet, t = triplet, q =
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quartet, m = multiplet. Highꢀresolution mass spectra (HRMS) were recorded on a Bruker
ApexQe hybrid 9.4 T FTꢀICR (ESI). The purity of the compounds was determined by HPLC
(Agilent 1200 HPLC system, detection by UV at 277 nm) on the column Poroshell 120 SBꢀC18
(4.6×100mm 2.7 Micron) and ≥95% unless otherwise indicated. Elemental analyses were
performed on an Element Analyzer Perkin Elmer EA 240 or 2400 CHN.
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General procedure for the syntheses
Method: A solution of ethyl bromopyruvate (1.1 mmol) and thiourea (1.0 mmol) in absolute
ethanol 20 mL was stirred under reflux monitored by TLC. Afterwards the mixture was diluted
in sodium bicarbonate (saturated) and extracted with ethyl acetate and dried over the sodium
sulfate. The combined organic phase was evaporated and the residue was purified by
chromatography if required to get the product in high yield (>70%). The carboxylic acids were
obtained from conversion of corresponding ester by incubation with 4 N HCl under reflux.
Ethyl 2-((4-chlorophenyl)amino)thiazole-4-carboxylate (O4I2, 1) 89%
3
3
1HꢀNMR (300 MHz; DMSOꢀd6): 1.29 (t, J=6.9, 3H), 4.27 (q, J=7.2, 2H), 7.35ꢀ7.40 (m, 2H),
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7.65ꢀ7.70 (m, 2H), 7.79(s, 1H), 10.52 (s, 1H). CꢀꢀNMR (DMSOꢀd6): 14.2, 60.4, 118.4, 118.9,
124.9, 128.8, 139.6, 142.3, 160.7, 162.8. HRMS (ESI) Calculated m/z: 283.0303; found m/z:
283.0303 [M+H]+. Anal. Calcd for C12H11ClN2O2S: C, 50.98; H, 3.92; N, 9.91. Found: C, 50,84;
H, 4.22; N, 9.83. IR (cmꢀ1): 2495, 2161, 2022, 1729, 1543, 1436, 1199, 1086, 1017, 792, 706.
Ethyl 2-aminothiazole-4-carboxylate (2) 75%
1HꢀNMR (300 MHz; DMSOꢀd6): 1.25 (t, 3J=6.9, 3H), 4.19 (q, 3J=7.2, 2H), 7.21 (s, 2H), 7.45 (s,
1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 14.2, 60.1, 116.9, 142.3, 161.0, 168.1. HRMS (ESI)
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Calculated m/z: 195.0199; found m/z:195.0199 [M+Na]+. Anal. Calcd for C6H8N2O2S.0.25H2O:
C, 40.78; H, 4.85; N, 15.85. Found: C, 41.01; H, 4.79; N, 15.85. IR (cmꢀ1): 3437, 3257, 3126,
2981, 2512, 2159, 2031, 1976, 1687, 1615, 1535, 1515, 1367, 1335, 1234, 1117, 1074, 1020,
961, 875, 784, 746, 665
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Ethyl 2-(butylamino)thiazole-4-carboxylate (3) 79%
1HꢀNMR (300 MHz; DMSOꢀd6): 0.89 (t, 3J=7.5, 3H), 1.23ꢀ1.38 (m, 5H), 1.49ꢀ1.55 (m, 2H), 3.19
(q, 3J=6.9, 2H), 4.20 (q, 3J=7.2, 2H), 7.47(s, 1H), 7.77 (s, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 13.6,
14.2, 19.5, 30.6, 44.2, 60.1, 116.1, 142.5, 161.0, 168.3. HRMS (ESI) Calculated m/z: 251.0825;
found m/z:251.0825 [M+Na]+. Anal. Calcd for C10H16N2O2S: C, 52.61; H, 7.06; N, 12.27.
Found: C, 52.44.; H, 7.01; N, 12.20. IR (cmꢀ1): 3190, 3128, 2956, 2930, 2516, 2159, 2030, 1976,
1722, 1590, 1227, 1203, 1092, 713
Ethyl 2-(allylamino)thiazole-4-carboxylate (4) 91%
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1HꢀNMR (300 MHz; DMSOꢀd6): 1.26 (t, J=6.9, 3H), 3.86ꢀ3.90 (m, 2H), 4.21 (q, J=7.2, 2H),
5.10ꢀ5.14 (m, 1H), 5.20ꢀ5.27 (m, 1H), 5.83ꢀ5.95 (m, 1H), 7.51 (s, 1H), 7.96 (t, J=5.7, 1H). 13Cꢀ
3
{1H}ꢀNMR (DMSOꢀd6): 14.2, 46.5, 60.1, 115.9, 116.7, 134.6, 142.3, 161.0, 168.2. HRMS (ESI)
Calculated m/z: 213.0692; found m/z: 213.0692 [M+H]+. Anal. Calcd for C8H10N2O2S: C, 48.57;
H, 5.08; N, 14.13. Found: C, 48.87; H, 5.31; N, 14.28. IR (cmꢀ1): 3179, 3123, 2985, 2520, 2159,
2030, 1976, 1724, 1577, 1415, 1200, 1141, 1089, 1028, 1001, 916, 729, 700
Ethyl 2-acetamidothiazole-4-carboxylate (5)
1HꢀNMR (300 MHz; DMSOꢀd6): 1.28 (t, 3J=6.9, 3H), 2.13 (s, 3H), 4.26 (q, 3J=6.9, 2H), 8.01 (d, ,
5J=0.9, 1H), 12.49 (s, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 14.2, 22.4, 60.5, 122.4, 140.8, 158.0,
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161.0, 169.0. HRMS (ESI) Calculated m/z: 237.0304; found m/z:237.0304 [M+Na]+. Anal.
Calcd for C8H10N2O3S: C,44.85; H,4.71; N,13.08. Found: C, 44.65; H, 4.47; N, 13.21. IR (cmꢀ1):
3223, 2991, 2513, 2159, 2031, 1976, 1726, 1700, 1613, 1552, 1293, 1203, 1013, 754, 734, 687
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Ethyl 2-(phenylamino)thiazole-4-carboxylate (6) 81%
1HꢀNMR (300 MHz; DMSOꢀd6): 1.29 (t, 3J=6.9 Hz, 3H), 4.27 (q, 3J=6.9 Hz, 2H), 6.98 (t, 3J=7.5,
3
1H), 7.33 (t, J=7.8 Hz, 2H), 7.61ꢀ7.63 (m, 2H), 7.76 (s, 1H), 10.34 (s, 1H). 13Cꢀ{1H}ꢀNMR
(DMSOꢀd6): 14.2, 60.4, 117.0, 118.4, 121.7, 129.0, 140.7, 142.4, 160.8, 163.2. HRMS (ESI)
Calculated m/z: 271.0512; found m/z:271.0512 [M+Na]+. Anal. Calcd for C12H12N2O2S.0.5 H2O:
C, 56.01; H, 5.09; N, 10.89. Found: C, 56.41; H, 5.03; N, 10.65. IR (cmꢀ1): 3281, 3058, 1714,
1685, 1597, 1527, 1454, 1377, 1319, 1206, 1089, 1018, 966, 889, 843, 752, 692, 667.
Ethyl 2-(benzylamino)thiazole-4-carboxylate (7) 75%
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1HꢀNMR (300 MHz; DMSOꢀd6): 1.25 (t, J=7.2, 3H), 4.20 (q, J=7.2, 2H), 4.45 (d, J=6, 2H),
3
7.26ꢀ7.27 (m, 5H), 7.51 (s, 1H), 8.30 (t, J=6, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 14.2, 47.7,
60.1, 116.8, 127.0, 127.4, 128.3, 138.7, 142.3, 161.0, 168.3. HRMS (ESI) Calculated m/z:
263.0849; found m/z: 263.0849 [M+H]+. Anal. Calcd for C13H14N2O2S.0.5H2O: C, 57.55; H,
5.57; N, 10.32. Found: C, 57.66; H, 5.26; N, 10.09. IR (cmꢀ1): 3178, 3112, 2982, 2886, 1516,
2159, 1031, 1976, 1726, 1588, 1576, 1423, 1195, 1090, 1067, 1018, 972, 760, 726, 695
Diethyl 2,2'-(1,4-phenylenebis(azanediyl))dithiazole-4-carboxylate (8) 75%
1HꢀNMR (300 MHz; DMSOꢀd6): 1.29 (t, 3J=7.2, 6H), 4.26 (q, 3J=6.9, 4H), 7.59 (s, 4H), 7.71 (s,
13
2H), 10.37 (bs, 2H). Cꢀ{1H}ꢀNMR (DMSOꢀd6): 14.3, 60.4, 118.0, 118.3, 135.4, 142.1, 160.8,
163.7. HRMS (ESI) Calculated m/z: 419.0842; found m/z: 419.0842 [M+H]+. Anal. Calcd for
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C18H18N4O4S2: C, 51.66; H, 4.34; N, 13.39. Found: C, 51.41; H, 4.27; N, 13.13. IR (cmꢀ1): 3056,
2519, 2159, 2023, 1976, 1817, 1591, 1517, 1494, 1254, 1196, 1088, 1016, 757, 736, 669
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Ethyl 2-((3-chlorophenyl)amino)thiazole-4-carboxylate (9) 81%
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1HꢀNMR (300 MHz; DMSOꢀd6): 1.30 (t, J=7.2, 3H), 4.27 (q, J=6.9, 2H), 7.01 (dd, J=7.8,
4J=1.2, 1H), 7.34 (t, 3J=7.8, 1H), 7.45 (dd, 3J=8.1, 4J=0.9, 1H), 7.83 (s, 1H), 7.93 (t, 4J=2.1, 1H),
10.60 (s, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 14.2, 60.5, 115.3, 116.3, 119.1, 121.1, 130.6,
133.4, 142.0, 142.3, 160.6, 162.5. HRMS (ESI) Calculated m/z: 283.0303; found m/z: 283.0303
[M+H]+. Anal. Calcd for C12H11ClN2O2S: C, 50.98; H, 3.92; N, 9.91. Found: C, 50.59; H, 4.29;
N, 10.26. IR (cmꢀ1): 3267, 3097, 2986, 2517, 2163, 2024, 1702, 1593, 1538, 1475, 1448, 1380,
1335, 1211, 1091, 1023, 864, 762, 731, 682.
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Ethyl 2-((2-cholorphenyl)amino)thiazole-4-carboxylate (10) 72%
1HꢀNMR (300 MHz; DMSOꢀd6): 1.28 (t, J=6.9, 3H), 4.25 (q, J=7.2, 2H), 7.06ꢀ7.12 (m, 1H),
7.33ꢀ7.38 (m, 1H), 7.47ꢀ7.50 (m, 1H), 7.80 (s, 1H), 8.27ꢀ8.30 (m, 1H), 9.84 (s, 1H).13Cꢀ{1H}ꢀ
NMR (DMSOꢀd6): 14.2, 60.4, 119.8, 121.7, 123.1, 124.1, 127.8, 129.7, 137.2, 141.9, 160.7,
164.0. HRMS (ESI) Calculated m/z: 301.0122; found m/z: 305.0122 [M+Na]+. Anal. Calcd for
C12H11ClN2O2S.0.25 H2O: C, 50.18; H, 4.04; N, 9.75. Found: C, 50.32; H, 4.02; N, 9.73. IR (cmꢀ
1): 3135, 3066, 2935, 2833, 1704, 1549, 1469, 1436, 1375, 1327, 1297, 1205, 1083, 1067, 1007,
949, 869, 806, 734, 658.
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Ethyl 2-((4-bromophenyl)amino)thiazole-4-carboxylate (11) 80%
1HꢀNMR (300 MHz; DMSOꢀd6): 1.29 (t, 3J=6.9 Hz, 3H), 4.27 (q, 3J=7.2 Hz, 2H), 7.48ꢀ7.52 (m,
2H), 7.59ꢀ7.64 (m, 2H), 7.80(s, 1H), 10.52 (s, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 14.2, 60.4,
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112.8, 118.8, 118.9, 131.7, 140.0, 142.3, 160.6, 162.7. HRMS (ESI) Calculated m/z: 326.9797;
found m/z: 326.797 [M+H]+. Anal. Calcd for C12H11BrN2O2S: C, 44.05; H, 3.39; N, 8.56. Found:
C, 43.87; H, 3.71; N, 8.27. IR (cmꢀ1): 3641, 3265, 2979, 2514, 2162, 2023, 1729, 1590, 1544,
1484, 1435, 1199, 1084, 1015, 792, 703.
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Ethyl 2-(3-bromophenylamino)thiazole-4-carboxylate (12) 77%
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1HꢀNMR (300 MHz; DMSOꢀd6): 1.30 (t, J=7.2, 3H), 4.27 (q, J=6.9, 2H), 7.13ꢀ7.16 (m, 1H),
7.25ꢀ7.31 (m, 1H), 7.48ꢀ7.52 (m, 1H), 7.82 (s, 1H), 8.06ꢀ8.07 (m, 1H), 10.57 (s, 1H). Cꢀ{1H}ꢀ
13
NMR (DMSOꢀd6): 14.6, 60.5, 115.7, 119.1, 121.9, 124.0, 130.9, 142.2, 160.6, 162.5. HRMS
(ESI) Calculated m/z: 326.9797; found m/z: 326.9797 [M+H]+. Anal. Calcd for
C12H11BrN2O2S.0.5H2O: C, 42.87; H, 3.60; N, 8.33. Found: C, 43.18; H, 3.34; N, 8.17. IR (cmꢀ
1): 3337, 3112, 2979, 2514, 2159, 2030, 1976, 1699, 1581, 1547, 1481, 1451, 1370, 1220, 1158,
1117, 1094, 1075, 1026, 989, 972, 862, 799, 771, 754, 715, 682, 652
Ethyl 2-(2-bromophenylamino)thiazole-4-carboxylate (13) 87%
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1HꢀNMR (300 MHz; DMSOꢀd6): 1.27 (t, J=6.9, 3H), 4.24 (q, J=6.9, 2H), 7.03ꢀ7.09 (m,1H),
7.34ꢀ7.42 (m, 1H), 7.64ꢀ7.68 (m, 1H), 7.78 (s, 1H), 8.05ꢀ8.08 (m,1H), 9.71 (s, 1H). Cꢀ{1H}ꢀ
13
NMR (DMSOꢀd6): 14.2, 60.4, 114.9, 119.5, 123.3, 125.3, 128.5, 133.1, 138.6, 142.0, 160.7,
164.7. HRMS (ESI) Calculated m/z: 326.9797; found m/z: 326.9797 [M+H]+. Anal. Calcd for
C12H11BrN2O2S.0.25 H2O: C, 43.05; H, 3.49; N, 8.45. Found: C, 43.34; H, 3.54; N, 8.06. IR (cmꢀ
1): 3642, 3143, 2934, 2515, 2162, 2024, 1975, 1702, 1548, 1327, 1297, 1204, 1081, 1038, 1007,
867, 729.
Ethyl 2-((4-fluorophenyl)amino)thiazole-4-carboxylate (H14) 99%
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1HꢀNMR (300 MHz; DMSOꢀd6): 1.29 (t, J=6.9, 3H), 4.26 (q, J=7.2, 2H), 7.14ꢀ7.20 (m, 2H),
7.63ꢀ7ꢀ67 (m, 2H), 7.75 (s, 1H), 10.40(s, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 14.2, 60.4, 115.5
(2J=22 Hz), 118.4, 118.6 (3J=7.7 Hz), 137.2 (4J=2.3 Hz), 142.3, 165.6 (1J=237 Hz), 160.7, 163.3.
HRMS (ESI) Calculated m/z: 267.0598; found m/z: 267.0598 [M+H]+. Anal. Calcd for
C12H11FN2O2S.0.67H2O: C, 51.79; H, 4.47; N, 10.07. Found: C, 51.33; H, 4.28; N, 9.74. IR (cmꢀ
1): 3102, 2517, 2159, 2030, 1976, 1712, 1623, 1565, 1527, 1502, 1445, 1411, 1368, 1294, 1251,
1207, 1172, 1156, 1092, 1015, 871, 859, 838, 797, 772, 760
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Ethyl 2-((3-fluorophenyl)amino)thiazole-4-carboxylate (15) 76%
1HꢀNMR (300 MHz; DMSOꢀd6): 1.30 (t, 3J=7.2 Hz, 3H), 4.27 (q, 3J=7.2 Hz, 2H), 6.75ꢀ6.81 (m,
1H), 7.24ꢀ7.38 (m, 2H), 7.73ꢀ7.78 (m, 1H), 7.82(s, 1H), 10.62 (s, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀ
2
2
4
d6): 14.2, 60.5, 103.7 (d, J = 26.7 Hz), 107.8 (d, J = 21.2 Hz), 112.8 (d, J = 2.5 Hz), 119.1,
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130.5 (d, J = 9.8 Hz), 102.3, 142.3 (d, J = 11.4 Hz), 160.7, 162.5 (d, J = 240.0 Hz), 162.6.
HRMS (ESI) Calculated m/z: 289.0417; found m/z: 289.0417 [M+Na]+. Anal. Calcd for
C12H11FN2O2S.0.25 H2O: C, 53.23; H, 4.28; N, 10.35. Found: C, 53.42; H, 4.56; N, 10.02. IR
(cmꢀ1): 3263, 3102, 2988, 2517, 2163, 2024, 1976, 1696, 1609, 1541, 1486, 1458, 1378, 1333,
1210, 1146, 1091, 1018, 954, 850, 756, 681.
Ethyl 2-((2-fluorophenyl)amino)thiazole-4-carboxylate (16) 41%
3
3
1HꢀNMR (300 MHz; DMSOꢀd6): 1.29 (t, J=7.2, 3H), 4.26 (q, J=7.2, 2H), 7.02ꢀ7.04 (m, 1H),
13
7.17ꢀ7.29 (m, 2H), 7.79 (S, 1H), 8.39ꢀ8.45 (m, 1H), 10.17 (s, 1H). Cꢀ{1H}ꢀNMR (DMSOꢀd6):
14.2, 60.4, 115.2 (2J=18.6 Hz), 119.6, 120.1 (4J=1.5 Hz), 122.7 (3J=7.4 Hz), 124.6 (3J=3.5 Hz),
128.6 (2J=10.7 Hz), 142.0, 151.8 (1J=242.2 Hz), 160.7, 163.3. HRMS (ESI) m/z: [M+H]+. HRMS
(ESI) Calculated m/z: 267.0598; found m/z: 267.0598 [M+H]+. Anal. Calcd for
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C12H11FN2O2S2 0.5H2O: C, 52.35; H, 4.39; N, 10.18. Found: C, 52.68; H, 3.98; N, 9.98. IR (cmꢀ
1): 3249, 3119, 2978, 1516, 2031, 1976, 1719, 1704, 1620, 1530, 1503, 1488, 1458, 1326, 1312,
1255, 1217, 1187, 1173, 1087, 1173, 1087, 1022, 959, 771, 751, 732, 683
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Ethyl 2-((3,4-dichlorophenyl)amino)thiazole-4-carboxylate (17) 84%
1HꢀNMR (300 MHz; DMSOꢀd6): 1.30 (t, 3J=7.2, 3H), 4.27 (q, 3J=7.2 Hz, 2H), 7.47 (dd, 3J=9 Hz,
4J=2.4 Hz, 1H), 7.56 (d, 3J=8.7 Hz, 1H), 7.85 (s, 1H), 8.15 (d, 4J=2.4 Hz, 1H), 10.70(s, 1H). 13Cꢀ
{1H}ꢀNMR (DMSOꢀd6): 14.2, 60.5, 117.0, 117.9, 119.5, 122.6, 130.7, 131.2, 140.6, 142.3,
160.6, 162.3. HRMS (ESI) Calculated m/z: 316.9913; found m/z: 316.9913 [M+H]+. Anal. Calcd
for C12H10Cl2N2O2S.0.2 H2O: C, 44.93; H, 3.27; N, 8.73. Found: C, 45.29; H, 3.61; N, 8.68. IR
(cmꢀ1): 2159, 2024, 1976, 1719, 1698, 1590, 1538, 1473, 1442, 1216, 1132, 1216, 1132, 1092,
1023, 813, 791.
Ethyl 2-((2,4-dichlorophenyl)amino)thiazole-4-carboxylate (18) 82%
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1HꢀNMR (300 MHz; DMSOꢀd6): 1.28 (t, J=7.8, 3H), 4.24 (q, J=6.9, 2H), 7.42ꢀ7.46 (m, 1H),
7.62ꢀ7.63 (m, 1H), 7.84 (s, 1H), 8.39ꢀ8.42 (m, 1H), 9.94 (s, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6):
14.2, 60.4, 120.4, 122.0, 122.0, 126.4, 127.8, 128.9, 136.4, 141.8, 160.6, 163.4. HRMS (ESI)
Calculated m/z: 316.9913; found m/z: 316.9913 [M+H]+. Anal. Calcd for C12H10Cl2N2O2S.0.2
H2O: C, 44.93; H, 3.27; N, 8.73. Found: C, 45.11; H, 3.62; N, 8.45. IR (cmꢀ1): 3347, 3117, 2981,
2921, 1704, 1587, 1520, 1469, 1381, 1302, 1202, 1084, 1025, 976, 849, 818, 785, 737.
Ethyl 2-((2,6-dicholorphenyl)amino)thiazole-4-carboxylate (19) 82%
3
3
1HꢀNMR (300 MHz; DMSOꢀd6): 1.25 (t, J=7.2, 3H), 4.20 (q, J=6.9, 2H), 7.36ꢀ7ꢀ41 (m, 2H),
7.59ꢀ7.62 (m, 1H), 7.68 (s, 1H), 9.95 (s, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 14.2, 60.3, 118.8,
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118.8, 118.8, 129.2, 129.3, 133.7, 160.7, 166.1. HRMS (ESI) Calculated m/z: 338.9722; found
m/z: 338.9722 [M+Na]+. Anal. Calcd for C12H10Cl2N2O2S.0.2 H2O: C, 44.93; H, 3.27; N, 8.73.
Found: C, 45.11; H, 3.63; N, 8.46. IR (cmꢀ1): 2974, 2827, 1697, 1552, 1434, 1377, 1328, 1282,
1210, 1082, 1011, 953, 876, 782, 716.
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Ethyl 2-((2,5-dichlorophenyl)amino)thiazole-4-carboxylate (20) 84%
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1HꢀNMR (300 MHz; DMSOꢀd6): 1.30 (t, J=7.2, 3H), 4.27 (q, J=7.2, 2H), 7.08ꢀ7.11 (m, 1H),
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7.48ꢀ7.51 ( m, 1H), 7.87 (s, 1H), 8.70ꢀ8.71(m, 1H), 10.03 (s, 1H). Cꢀ{1H}ꢀNMR (DMSOꢀd6):
14.1, 60.5, 119.5, 120.0, 120.8, 122.6, 130.8, 132.0, 138.2, 141.6, 160.6, 162.7. HRMS (ESI)
Calculated m/z: 338.9722; found m/z: 338.9722 [M+Na]+. Anal. Calcd for C12H10Cl2N2O2S.0.2
H2O: C, 44.93; H, 3.27; N, 8.73. Found: C, 45.18; H, 3.50; N, 8.60. IR (cmꢀ1): 3319, 3108, 2500,
2163, 2024, 1975, 1695, 1586, 1528, 1404, 1372, 1331, 1251,1166, 1094, 1033, 870, 787, 717.
Ethyl 2-(3,5-dichlorophenylamino)thiazole-4-carboxylate (21) 83%
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1HꢀNMR (300 MHz; DMSOꢀd6): 1.30 (t, J=7.2,3H), 4.27 (q, J=6.9, 2H), 7.14 (t,4J=1.8, 1H),
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7.73 (d, J=1.8, 1H), 7.88 (s, 1H), 10.77 (s, 1H). Cꢀ{1H}ꢀNMR (DMSOꢀd6): 14.1, 60.5, 114.9,
119.8, 120.4, 134.3, 142.2, 142.7, 160.5, 162.0. HRMS (ESI) Calculated m/z: 338.9722; found
m/z: 338.9722 [M+Na]+. Anal. Calcd for C12H10Cl2N2O2S.0.2 H2O: C, 44.93; H, 3.27; N, 8.73.
Found: C, 45.23; H, 3.34; N, 8.82. IR (cmꢀ1): 3347, 3117, 2981, 2921, 1704, 1587, 1520, 1469,
1381, 1302, 1202, 1084, 1025, 976, 849, 818, 785, 737.
2-(4-Chlorophenylamino)thiazole-4-carboxylic acid (22)
1HꢀNMR (300 MHz; DMSOꢀd6): 7.35ꢀ7.38 (m, 2H), 7.69ꢀ7.74 (m, 3H), 10.48 (s, 1H). 13Cꢀ{1H}ꢀ
NMR (DMSOꢀd6): 118.4, 118.6, 124.8, 128.8, 139.7, 143.4, 162.1, 162.5. HRMS (ESI)
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Calculated m/z: 276.9809; found m/z: 276.9809 [M+Na]+. Anal. Calcd for C10H7ClN2O2S: C,
47.16; H, 2.77; N, 11.00. Found: C, 47.33; H, 2.70; N, 10.87. IR (cmꢀ1): 2501, 2172, 2032, 1732,
1539, 1432, 1197, 1085, 1011, 793, 711.
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2-(3-Chlorophenylamino)thiazole-4-carboxylic acid (23) 67%
1HꢀNMR (300 MHz; DMSOꢀd6): 6.99ꢀ7.02 (m, 1H), 7.34 (t, 3J=8.1, 1H), 7.46ꢀ7.50 (m, 1H), 7.77
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(s, 1H), 7.92 (t, J=2.1, 1H), 10.54 (s, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 115.3, 116.2, 118.9,
121.0, 130.5, 133.4, 142.1, 143.3, 162.0, 162.3. HRMS (ESI) Calculated m/z: 276.9809; found
m/z: 276.9809 [M+Na]+. Anal. Calcd for C10H7ClN2O2S: C, 47.16; H, 2.77; N, 11.00. Found: C,
47.23; H, 2.72; N, 10.62. IR (cmꢀ1): 2522, 2160, 2027, 1700, 1536, 1478, 1450, 1377, 1334,
1210, 1093, 866, 767, 729, 683.
2-(2,4-Dichlorophenylamino)thiazole-4-carboxylic acid (24) 56%
1HꢀNMR (300 MHz; DMSOꢀd6): 7.43 (dd, 3J=9.0, 4J=2.7, 1H), 7.62 (d, 3J=2.4, 1H), 7.79 (s, 1H),
8.47 (d, 3J=9.0, 1H), 9.89 (s, 1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 120.5, 122.3, 123.3, 126.6, 128.2,
129.8, 136.9, 143.3, 162.5, 163.5. HRMS (ESI) Calculated m/z: 310.9419; found m/z: 310.9419
[M+Na]+. Anal. Calcd for C10H6Cl2N2O2S: C, 41.54; H, 2.09; N, 9.69. Found: C, 41.88; H, 2.02;
N, 9.54. IR (cmꢀ1): 3350, 3114, 2980, 2917, 1703, 1585, 1522, 1471, 1380, 1300, 1198, 1083,
1027, 974, 845, 813.
2-(2,5-Dichlorophenylamino)thiazole-4-carboxylic acid (25) 59%
1HꢀNMR (300 MHz; DMSOꢀd6): 7.08 (dd, 3J=8.7, 4J=2.7, 1H), 7.49 (d, 3J=8.4, 1H), 7.83 (s, 1H),
8.69 (d, 4J=2.4, 1H), 9.98 (s, 1H), 12.84(s,1H). 13Cꢀ{1H}ꢀNMR (DMSOꢀd6): 119.8, 120.3, 121.0,
123.0, 131.2, 132.5, 138.8, 143.2, 162.5, 163.0. HRMS (ESI) Calculated m/z: 310.9419; found
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m/z: 310.9419 [M+Na]+. Anal. Calcd for C10H6Cl2N2O2S: C, 41.54; H, 2.09; N, 9.69. Found: C,
41.89; H, 1.98; N, 9.66. IR (cmꢀ1): 3300, 3121, 2504, 2163, 1695, 1587, 1523, 1377, 1336, 1259,
1169, 1090, 1031, 870, 719.
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Synthesis
of
2-((4-Chlorophenyl)amino)-N-(3-(dimethylamino)propyl)thiazole-4-
carboxamide (26)
To a solution of 22 (1 mmol) in dried CH2Cl2 (20 mL) DCC (1.1 mmol) and HOBt (1.1 mmol)
were added and stirred under N2 for 2 hrs. After adding N,Nꢀdimethylpropaneꢀ1,3ꢀdiamine (4
mmol) the mixture was further stirred for 3 days. The mixture was diluted with 200 mL CH2Cl2
and extracted with saturated Na2CO3 aqueous solution. The organic phase was dried over
Na2SO4. After removal of solvent, the residue was purified by column chromatography (ethyl
acetate : cyclohexan = 1:1) to obtain pure compound. 1HꢀNMR (300 MHz; DMSOꢀd6): 10.52 (s,
1H), 10.01 (s, 1H), 8.12 (d, 3J=8.5, 1H), 7.82 (d, 3J=8.4, 1H), 7,69 (m, 1H), 7.52 (m, 1H), 6.56 (s,
1H), 3.18 (m, 2H), 1.81 (m, 10H). HRMS (ESI) Calculated m/z: 339.1041; found m/z: 339.1041
[M+H]+.
FIGURES.
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Figure 1. O4I2 activates Oct3/4 in HEK293 cells. A) Structure of O4I2. B) Concentration
dependent activation of Oct3/4 detected in HEKꢀOct3/4 reporter cells. C) Accumulation of
Oct3/4 and Nanog in HEK293 cells detected by immunoblot (72 hrs treatment). D) Increased
expression of pluripotency related genes in HEK293 cells treated with O4I2 for 72 hrs quantified
by qRTꢀPCR. Bars indicate standard deviation (*: p<0.05; **: p<0.01)
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Figure 2. O4I2 activates Oct3/4 in NCCIT and HeLa cells. A) O4I2 induced transcriptional
activity of luciferase reporter gene in NCCITꢀOct3/4 reporter cells after 72 hrs treatment. B) The
level of Oct3/4 was stabilized in a concentrationꢀdependent study (72 hrs), as well as C) in a
timeꢀdependent study. D) Expression of Oct3/4ꢀprotein in HeLa cells after 48 hrs treatment.
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Figure 3. O4I2 activates Oct3/4 on the transcriptional and translational level in human
fibroblasts. A) O4I2 promoted the expression of pluripotencyꢀassociated genes as analyzed by
qRTꢀPCR in human neonatal foreskin fibroblasts (HFFs) after 48 hrs. B) Nanog and Lin28 were
stimulated in response to O4I2 in primary human fibroblasts (HFs) after 72 hrs treatment. C) The
coꢀexpression of Oct3/4 (red) and Sox2 (green), as well as Nanog (red) and KLF4 (green) in
HFF after 5 days incubation detected by the respective antibodies and nuclei were stained by
Hoechst 33258. Bar 20 µm. D) Induction of Oct3/4 in the HFF cells by immunoblot; NCCIT was
used as positive control. (**: p<0.01; ***: p<0.001)
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Figure 4: The effect of O4I2 derivatives on Oct3/4 expression in HEKꢀOct3/4 cells.
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