Concise synthesis and X-ray crystal structure of N -benzyl-2-(pyrimidin-4'- ylamino)-thiazole-4-carboxamide (Thiazovivin), a small-molecule tool for stem cell research

Article abstract of DOI:10.1080/00397911.2012.745567

Stem cell research is one of the most promising fields of modern biomedical research and regenerative medicine. Limited availability and ethical concerns suggest the renouncement of embryonic stem cells (ESCs), thus raising the need for more efficient pro

Full text of DOI:10.1080/00397911.2012.745567

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Concise Synthesis and X-Ray Crystal Structure of
N-Benzyl-2-(pyrimidin-4′-ylamino)-thiazole-4-
carboxamide (‘Thiazovivin’), a Small-Molecule Tool for
Stem Cell Research
Oliver Ries a , Markus Granitzka b , Dietmar Stalke b & Christian Ducho a c
a Georg-August-University Göttingen, Department of Chemistry, Institute of Organic and
Biomolecular Chemistry , Göttingen , Germany
b Georg-August-University Göttingen, Department of Chemistry, Institute of Inorganic
Chemistry , Göttingen , Germany
c University of Paderborn, Department of Chemistry , Paderborn , Germany
Accepted author version posted online: 31 May 2013.
To cite this article: Oliver Ries , Markus Granitzka , Dietmar Stalke & Christian Ducho (2013): Concise Synthesis and X-Ray
Crystal Structure of N-Benzyl-2-(pyrimidin-4′-ylamino)-thiazole-4-carboxamide (‘Thiazovivin’), a Small-Molecule Tool for Stem
Cell Research, Synthetic Communications: An International Journal for Rapid Communication of Synthetic Organic Chemistry,
DOI:10.1080/00397911.2012.745567
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Concise Synthesis and X-ray Crystal Structure of N-Benzyl-2-(pyrimidin-4'-
ylamino)-thiazole-4-carboxamide ('Thiazovivin'), a Small-Molecule Tool for Stem
Cell Research
Oliver Ries1, Markus Granitzka2, Dietmar Stalke2, Christian Ducho1,3
1Georg-August-University Göttingen, Department of Chemistry, Institute of Organic and
Biomolecular Chemistry, Göttingen, Germany, 2Georg-August-University Göttingen,
Department of Chemistry, Institute of Inorganic Chemistry, Göttingen, Germany,
3University of Paderborn, Department of Chemistry, Paderborn, Germany
Address correspondence to Christian Ducho, University of Paderborn, Department of
Chemistry, Warburger Str. 100, 33 098 Paderborn, Germany. Phone: +49-(0)5251-60-
5770. Fax: +49-(0)5251-60-3245. E-mail: christian.ducho@uni-paderborn.de
Abstract
Stem cell research is one of the most promising fields of modern biomedical research and
regenerative medicine. Limited availability and ethical concerns suggest the
renouncement of embryonic stem cells (ESCs), thus raising the need for more efficient
procedures for the generation of stem cells, ideally through reprogramming of
mammalian cells. The small molecule N-benzyl-2-(pyrimidin-4'-ylamino)-thiazole-4-
carboxamide ('Thiazovivin') is known to improve the generation of human induced
pluripotent stem cells (iPSCs) from human fibroblasts. We herein describe a highly
efficient procedure for the synthesis of Thiazovivin over just five steps, which should be
suitable for a large-scale application, and the first X-ray crystal structure of the target
compound.
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[Supplementary materials are available for this article. Go to the publisher’s online
edition of Synthetic Communications® for the following free supplemental resource(s):
Full experimental and spectral details.]
S
S
N
5 steps
H
N
H2N
NH2
NH2
H
N
N
N
N
N
CO2Et
14 %
O
Br
overall
O
KEYWORDS: Chemical biology; stem cells; heterocycles
INTRODUCTION
Within the last three decades, stem cell research has evolved to one of the most
promising, though controversially discussed, fields of biomedical research.[1] Stem cells
offer a great opportunity for the treatment of previously incurable diseases and novel
future therapies.[2] One major drawback is the need for at least pluripotent stem cells
(PSCs), which can form all of the human body's cell lineages.[3] Human embryonic stem
cells (hESCs) represent an ethically problematic source for PSCs. A preferred option is
the reprogramming of functionally heterogeneous cells of a multicellular organism to
induced pluripotent stem cells (iPSCs).[4] Several advances in the generation of iPSCs
have been made,[5–7] but the process takes up to four weeks and furnishes only a small
amount of heterogeneous cell populations.
In 2009, Ding and coworkers described a chemical method that boosted the efficiency of
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iPSC generation from human fibroblasts by a factor >200. They have achieved this vast
improvement by the application of N-benzyl-2-(pyrimidin-4'-ylamino)-thiazole-4-
carboxamide 1 ('Thiazovivin', Fig. 1).[8] Addition of Thiazovivin to iPSC colonies
promoted their survival after trypsinization and led to high levels of endogenous mRNA
and protein expression of pluripotent markers. The iPSC colonies created by this protocol
could be grown under conventional hESC culture conditions. In terms of morphology,
typical pluripotency marker expression and differentiation potential, the colonies likewise
come close to hESCs. Recently, Thiazovivin 1 has been employed for the generation of
fat-laden adipocytes from porcine embryonic fibroblasts (PEFs).[9] We herein describe an
efficient and concise synthesis of 1 which is expected to be suitable for the large scale.
Furthermore, an unprecedented X-ray crystal structure of 1 has been obtained.
RESULTS AND DISCUSSION
Thiazovivin has originally been obtained by a solid-phase supported synthesis involving
a Pd-catalyzed key step.[10] In contrast, our approach employs standard solution-phase
methodology and does not require transition metal catalysis. Using this novel route, the
condensation of thiourea 2 with ethyl 3-bromopyruvate 3 led to aminothiazole 4,[11,12]
which could be recrystallized from ethanol, in 83 % yield (Scheme 1). Subsequent
Sandmeyer-type bromination gave bromothiazole 5[11,13] in 68 % yield after column
chromatography. Saponification of the ethyl ester furnished carboxylic acid 6[11,14] in
62 % yield after recrystallisation from water, followed by amide coupling with benzyl
amine and EDC-HOBt to afford thiazole amide 7 (73 % yield). The final reaction
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providing thiazovivin 1 was accomplished by the nucleophilic aromatic substitution of 7
with 4-aminopyrimidine 8 in the presence of sodium hydride. Purification by column
chromatography and preparative HPLC in order to obtain highly pure material for cell
applications afforded thiazovivin 1 in 53 % yield. In summary, thiazovivin 1 was
prepared with an overall yield of 14 % from inexpensive starting materials over five steps
(Scheme 1).
A single crystal of 1 was obtained by crystallization of the purified material after
evaporation of residual solvent (dichloromethane). Thiazovivin 1 crystallizes in the
tetragonal space group I41/a with one molecule in the asymmetric unit (Fig. 2). The
hydrogen atoms H31 and H51 bonded to the nitrogen atoms N3 and N5 generate
hydrogen bonds to O1 and N1 of neighbouring molecules (Fig. 3). The hydrogen bond
distances are N3–H31"O1A 190.0(16) pm and N5–H51"N1A 221.0(17) pm,
respectively.
In summary, we have developed a convenient synthetic route providing thiazovivin 1
without the use of expensive heavy metal catalysts, thus making this concise synthesis
potentially useful for large-scale applications.
EXPERIMENTAL
Chemicals were purchased from Sigma-Aldrich, Alfa Aesar, Fluorochem, Merck, Roth,
Acros Organics and GL Biochem. Reactions involving moisture sensitive reagents were
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carried out under an atmosphere of argon using anhydrous solvents. Anhydrous solvents
were obtained in the following manner: acetonitrile was dried over P2O5 and distilled.
THF was dried over sodium/benzophenone and distilled. DMF was dried over activated
molecular sieves (4 Å). All other solvents were of technical quality and distilled prior to
their use, and deionized water was used throughout. Column chromatography was carried
out on silica gel 60 (0.040-0.063 mm, 230-400 mesh ASTM, VWR) under flash
conditions. TLC was performed on aluminum plates precoated with silica gel 60 F254
(VWR). Visualization of the spots was carried out using UV light (254 nm) and/or
staining under heating (staining solution: 1 g KMnO4, 6 g K2CO3 and 1.5 mL 5 %
NaOH(aq) (w/v), all dissolved in 100 mL H2O). Preparative HPLC was carried out on a
Jasco system equipped with PU-1587 Intelligent Prep. Pumps, a UV-1575 Intelligent
UV/Vis detector and a Merck LiChroCART column (250 x 10 mm, LiChrospher 100 RP
18, 10 µm). 300 MHz-1H as well as 75 MHz-, 76 MHz- and 126 MHz-13C NMR spectra
were recorded on Varian UNITY 300, MERCURY 300 and INOVA 500 spectrometers.
All 13C NMR spectra are 1H-decoupled. All spectra were recorded at room temperature or
at 35 °C (DMSO-d6) and were referenced internally to solvent reference frequencies.
Chemical shifts (δ) are quoted in ppm, and coupling constants (J) are reported in Hz.
Assignment of signals was carried out using 1H,1H-COSY, HSQC and HMBC spectra
obtained on the spectrometers mentioned above. Low resolution ESI mass spectrometry
was performed on a Varian MAT 311 A spectrometer operating in positive or negative
ionization mode. High resolution (HR) ESI mass spectrometry was carried out on a
Bruker microTOF spectrometer or a Bruker 7 T FTICR APEX IV spectrometer. Melting
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points (mp) were measured on a Büchi instrument and are not corrected. Infrared
spectroscopy (IR) was performed on a Perkin-Elmer Vektor 22 spectrometer with solids
being measured as KBr pills. Wavenumbers (ν) are quoted in cm-1. UV spectroscopy was
carried out on a Perkin-Elmer Lambda 2 spectrometer. Wavelengths of maximum
absorption (λmax) are reported in nm with the corresponding logarithmic molar extinction
coefficient (log ε, ε/dm3 mol-1 cm-1) given in parenthesis.
N-Benzyl-2-Bromothiazol-4-Carboxamide (7)
A solution of 6 (101 mg, 0.486 mmol) in THF (2.5 mL) was treated with HOBt (78 mg,
0.58 mmol), EDC hydrochloride (110 mg, 0.574 mmol) and DIPEA (0.10 mL,
0.59 mmol). The solution was stirred at room temperature for 30 min, and then a solution
of benzyl amine (80 µL, 0.73 mmol) in THF (1 mL) was added dropwise. The reaction
mixture was stirred at room temperature for 18 h and was then diluted with EtOAc
(2.5 mL) and water (2 mL). The organic layer was washed with 0.5 M HCl (1 x 5 mL),
sat. NaHCO3 solution (2 x 5 mL) and water (2 x 5 mL) and dried over Na2SO4. The
solvent was removed under reduced pressure, and column chromatography of the
resultant crude product (petroleum ether-EtOAc 4:1) gave 106 mg (0.357 mmol, 73 %) of
7 as a white solid. Rf = 0.41 (petroleum ether-EtOAc 1:1); mp = 78 °C; 1H NMR
(300 MHz, C6D6): δ = 4.32 (d, J = 6.3 Hz, 2H, benzyl-CH2), 6.96-7.17 (m, 5H, aryl-H),
7.19 (br s, 1H, NH), 7.51 (s, 1H, 5-H); 13C NMR (75 MHz, C6D6): δ = 43.25 (benzyl-
CH2), 127.08 (C-5), 127.41 (aryl-CH), 128.00 (aryl-CH), 128.72 (aryl-CH), 135.66 (C-2),
138.92 (aryl-C), 150.51 (C-4), 159.24 (C-6); IR (KBr): ν (cm-1) = 3388, 3092, 1665,
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1542, 1487, 1429, 1239, 1014, 764, 718, 605, 577; UV (MeOH): λmax (log ε) = 203
(4.42), 235 (4.07); MS (ESI): m/z = 319.0 [M+Na]+, 321.0 [M+Na]+; HRMS (ESI): m/z
calcd. for C11H9BrN2OS [M+Na]+ 320.9491, found 320.9487.
Thiazovivin (1)
Sodium hydride (60 % dispersion in mineral oil, 20 mg, 0.50 mmol) was added to a
solution of 7 (50 mg, 0.17 mmol) and 4-aminopyrimidine 8 (48 mg, 0.50 mmol) in DMF
(3 mL). The solution was stirred for 3 h at 140 °C and then cooled to room temperature.
The solvent was removed under reduced pressure and the residue was co-evaporated with
toluene and CH2Cl2. Column chromatography (EtOAc) and preparative HPLC
purification (MeOH/water 70:30, flow 8 mL/min, UV detection at 310 nm) gave 28 mg
(90 µmol, 53 %) of 1 as a yellow-brownish solid. Rf = 0.20 (EtOAc); mp = 216 °C;
1H NMR (300 MHz, DMSO-d6): δ = 4.49 (d, J = 6.2 Hz, 2H, benzyl-CH2), 7.15 (dd, J
= 5.9 Hz, J = 1.1 Hz, 1H, 5'-H), 7.20-7.38 (m, 5H, aryl-H), 7.79 (s, 1H, 5-H), 8.33 (t,
J = 6.2 Hz, 1H, amide-NH), 8.47 (d, J = 5.9 Hz, 1H, 6'-H), 8.84 (d, J = 1.1 Hz, 1H, 2'-H),
11.79 (br s, 1H, 2-NH); 13C NMR (126 MHz, DMSO-d6): δ = 42.20 (benzyl-CH2),
107.70 (C-5'), 117.06 (C-5), 126.62 (aryl-CH), 127.07 (aryl-CH), 128.11 (aryl-CH),
139.16 (aryl-C), 144.40 (C-4), 155.97 (C-6'), 156.38 (C-4'), 156.95 (C-2'), 158.00 (C-2),
160.53 (C-6); IR (KBr): ν (cm-1) = 3403, 3086, 1655, 1615, 1554, 1527, 1495, 1445,
1393, 1327, 765, 698, 565; UV (MeOH): λmax (log ε) = 210 (4.39), 225 (4.27), 258
(4.07), 299 (4.24), 345 (3.05). MS (ESI): m/z = 312.1 [M+H]+, 334.1 [M+Na]+; HRMS
(ESI): m/z calcd. for C15H13N5OS [M+Na]+ 334.0733, found 334.0733.
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X-RAY STRUCTURE DETERMINATION OF 1
Single crystals of 1 were selected from a flask and covered with perfluorated polyether
oil on a microscope slide. An appropriate crystal was selected using a polarize
microscope, mounted on the tip of a MITEGENMicroMount fixed to a goniometer head
and shock cooled by the crystal cooling device.
The data for 1 were collected from shock-cooled crystals at 100(2) K[15] on a INCOATEC
Mo Microsource[16] (used MoK radiation, = 71.073 pm) with mirror optics and APEX
II detector with a D8 goniometer. They were integrated with SAINT[17] and an empirical
absorption correction (SADABS)[18] was applied. The structure was solved by direct
methods (SHELXS-97)[19a] and refined by full-matrix least-squares methods against F2
(SHELXL-97)[19b,c] within the ShelXle GUI. All non-hydrogen-atoms were refined with
anisotropic displacement parameters. The hydrogen atoms, besides H31 and H51, were
refined isotropically on calculated positions using a riding model with their Uiso values
constrained to equal to 1.5 times the Ueq of their pivot atoms for terminal sp3 carbon
atoms and 1.2 times for all other carbon atoms. Only the hydrogen atoms of the nitrogen
atoms (H31 and H51) were refined freely and their positions were found from the
difference density map. The disordered dichloromethane solvent molecule was refined
using bond length and angle restraints and anisotropic displacement parameter restraints.
Data of the X-ray crystal structure of 1 were deposited at the Cambridge Crystallographic
Data Centre (excluding structure factors) and are available online. The CCDC numbers,
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crystal data and experimental details for the X-ray measurements are listed in Table S1
(Supplementary Material).
SUPPLEMENTARY MATERIAL
Supplementary Material: Syntheses of compounds 4-6, 1H and 13C NMR spectra of all
compounds, data on the X-ray crystal structure of 1. This material can be found via the
"Supplementary Content" section of this article's Web page. Copies of the
crystallographic data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif or from the
corresponding author.
ACKNOWLEDGMENTS
The authors thank Dr. Benjamin Groß (Hannover Medical School, Germany) for helpful
discussions and the Fonds der Chemischen Industrie (FCI, Sachkostenzuschuss) for
financial support. M.G. and D.S. thank the Danish National Research Foundation
(DNRF) funded Center for Materials Crystallography (CMC).
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Scheme 1. Synthesis of target compound 1.
t-BuONO
H2O
CuBr2, MeCN
S
N
S
N
S
COOEt
Br
+
H2N
Br
83 %
68 %
H2N
NH2
O
COOEt
COOEt
2
3
4
5
BnNH2
EDC, HOBt
DIPEA, THF
KOH, H2O
62 %
S
S
N
H
N
Br
Br
73 %
N
COOH
O
6
7
NH2
N
S
H
S
NaH, DMF
53 %
H
N
H
N
N
N
Br
+
N
N
N
N
O
O
7
8
1
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Figure 1. Structure of Thiazovivin 1, the target compound of this study.
S
N
H
N
H
N
N
N
O
1
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Figure 2. Molecular structure of Thiazovivin 1. Anisotropic displacement parameters are
depicted at the 50 % probability level. Atom color code: carbon = black, nitrogen = blue,
oxygen = red, sulfur = yellow and hydrogen = white. The other hydrogen atoms and the
disordered solvent molecule dichloromethane are omitted for clarity.
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Figure 3. Packing plot and hydrogen bonds of Thiazovivin 1. Atom color code:
carbon = black, nitrogen = blue, oxygen = red, sulfur = yellow and hydrogen = white.
Symmetry generated molecules are shown in wireframe projection for clarity.
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