Synthesis, in silico and in vitro Evaluation of Novel Oxazolopyrimidines as Promising Anticancer Agents

Article abstract of DOI:10.1002/hlca.202000169

New potential bioactive oxazolopyrimidines have been synthesized using two main approaches: the pyrimidine ring annulation on a functionalized oxazole and the benzoyl bromide trimerization followed by rearrangement and formation of the oxazolo[5,4-d]pyrim

Full text of DOI:10.1002/hlca.202000169

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chimica acta
Accepted Article
Title: Synthesis, In silico and In vitro Evaluation of Novel
Oxazolopyrimidines as Promising Anticancer Agents
Authors: Steven Patrick Nolan, Thomas Scattolin, Flavio Rizzolio,
Isabella Caligiuri, Yevgen Karpichev, Yevheniia Velihina,
Denys Bondar, Stepan Pil’o, Nataliya Obernikhina, Olesksiy
Kachkovskyi, Volodymyr Brovarets, and Ivan Semenyuta
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To be cited as: Helv. Chim. Acta 10.1002/hlca.202000169
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10.1002/hlca.202000169
Helvetica Chimica Acta
HELVETICA
Synthesis, In silico and In vitro Evaluation of Novel Oxazolopyrimidines as
Promising Anticancer Agents
Yevheniia Velihina,a,b Thomas Scattolin,c Denys Bondar,a Stepan Pil’o,b Nataliya Obernikhina,d
Olesksiy Kachkovskyi,b Ivan Semenyuta,b Isabella Caligiuri,e Flavio Rizzolio,e,f
Volodymyr Brovarets*,b Yevgen Karpichev*,a and Steven P. Nolan*c
a Department of Chemistry and Biotechnology, School of Science, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia,
yevgen.karpichev@taltech.ee
b V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Science of Ukraine, Murmanska str. 1, 02094 Kyiv-94, Ukraine,
brovarets@bpci.kiev.ua
c Department of Chemistry, Ghent University, Krijgslaan 281, 9000 Gent, Belgium, Steven.Nolan@UGent.be
d O.O. Bogomolets National Medical University, T. Shevchenko Blvd. 13, 01601, Kyiv, Ukraine
e Pathology Unit, Department of Molecular Biology and Translational Research, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS ,
via Franco Gallini 2, 33081, Aviano, Italy
f Department of Molecular Sciences and Nanosystems, Università Ca’ Foscari, Campus Scientifico Via Torino 155, 30174, Venezia-Mestre, Italy
Dedicated to Prof. Antonio Togni on the occasion of his 65th birthday and retirement.
New potential bioactive oxazolopyrimidines have been synthesized using two main approaches: the pyrimidine ring annulation on a functionalized oxazole
and the benzoyl bromide trimerization followed by rearrangement and formation of the oxazolo[5,4-d]pyrimidine scaffold. The docking analyzes have
shown that 7-piperazine substituted oxazolo[4,5-d]pyrimidines 8a-c could be potential VEGFR2 inhibitors with high free energy of ligand–protein complex
formation (ΔG: –10.1, –9.6, –9.8 kcal/mol, respectively). In vitro antitumor assays confirmed theoretical predictions that oxazolo[4,5-d]pyrimidines 8a-c
containing positively charged piperazine moiety should demonstrate significantly higher cytotoxic effects. 5-(4-Chlorophenyl)-2-phenyl-oxazolo[4,5-
d]pyrimidin-7-yl)-piperazin-1-ium trifluoroacetate 8c exhibited a slightly higher antiproliferative effect (IC50 = 0.21 µM) than doxorubicin (IC50 = 0.36 µM) on
MDA-MB-231 cell line and has relatively good results on OVCAR-3 (IC50 = 1.7 µM) and HCT-116 (IC50 = 0.24 µM) cells.
Keywords: oxazolopyrimidines, Suzuki-Miyaura reaction, anticancer activity, molecular docking, donor-acceptor properties.
survival. Anti-angiogenic therapies target angiogenesis by two main
Introduction
mechanisms: blocking the receptor tyrosine kinases intracellularly or
neutralizing angiogenic factors such as VEGF or its receptors.[7]
The oxazolopyrimidine architecture represents an universal scaffold used in
the design of bioactive ligands against enzymes or receptors such as
adenosine kinase (AK) inhibitors,[1,2] cannabinoid receptor 2 (CB2) neutral
antagonists,[3] fatty acid amide hydrolase (FAAH) inhibitors,[4] endothelial
differentiation gene receptor 1 (EDG-1) agonist[5] and purinergic G protein-
coupled (P2Y1) receptor antagonist.[6] Thus, among the numerous
heterocyclic moieties of biological and pharmacological interest, this
pharmacophore represents an important structural motif in oncology and
infectious disease treatments.
The VEGF family consists of 6 secreted glycoproteins, VEGF-A, VEGF-
B, VEGF-C, VEGF-D, VEGF-E and placenta growth factor.[8] VEGF mediates
angiogenesis via several different tyrosine kinase domain receptors.
VEGFR1 (Flt-1) is critical in developmental angiogenesis, as well as other
processes including monocyte migration, recruitment of endothelial cell
(EC) progenitors, and inducing growth factors from liver sinusoidal ECs.[9,10]
VEGFR2 (KDR and the murine homologue Flk-1) mediates the majority of
the downstream effects of VEGF-A in angiogenesis, including
microvascular permeability, EC proliferation, and survival.[9,11] TheVEGFR3
is expressed throughout the embryonic vasculature and later is limited to
lymphatic ECs.[12] The various members of the VEGF family have differing
binding affinities for each of these receptors, which have helped in
elucidating the functions of each of these receptors.
Cancer is a general term for malignant diseases characterized by
uncontrolled abnormal cell growth. There is substantial preclinical and
clinical evidence that angiogenesis plays a role in tumor development and
growth/progression. A large number of cancers have been reported to be
dependent on angiogenesis and respond well to anti-angiogenic therapies.
These include cancers of the breast, colon, lung, and bladder as well as renal
cell carcinoma and non-small cell lung cancer. Additionally, some of these
cancers require the vascular endothelial growth factor (VEGF) for their
Three major classes of agents which target VEGF have been developed:
monoclonal antibodies (Bevacizumab), VEGF decoy receptor (Aflibercept),
and small molecule tyrosine kinase inhibitors (Imatinib, Sorafenib, Sunitinib,
1
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10.1002/hlca.202000169
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Pazopanib, Axitinib).[7] Among tyrosine kinase inhibitors (TKIs), there are
bicyclic N-containing heterocycles that bind with the hinge region of the
enzyme through H-bonds as mimic of the purine ring of ATP.[13,14] In this
context, oxazolo[5,4-d]pyrimidines, which can be considered as 9-
oxapurine analogs of nucleobases, have been reported as potent VEGFR2
inhibitors.[15,16] Therefore, synthesis and modification of drugs based on the
oxazolopyrimidine scaffold as well as other organic and metal compounds
with anticancer properties is currently a pressing issue.[17]
4a and 5a were reliably established earlier,[25-26] the structures of congeners
3b, 4b and 5b were confirmed by 1H, 13C-NMR, IR spectra and elemental
analysis. NH signals of 3b are appear at 12.08 and 9.29 ppm in the 1H-NMR
spectra and at 3427 – 3204 cm-1 in the IR spectra, while the NH signal of 4b
can be observed at 13.15 ppm and 3064 cm-1, respectively. 1H-NMR
spectrum of 6 in (D6)-DMSO revealed a singlet at 1.48 ppm due to the 1-
Boc-piperazine moiety.
Cl
1-Boc-piperazine,
Cl
We have recently reported in vitro studies of 7-piperazine- and 7-
diazepan substituted [1,3]oxazolo[4,5-d]pyrimidines against a panel of
different cancer cell lines, which have exhibited a growth inhibitory,
cytostatic and cytotoxic effects at submicromolar and micromolar
concentrations.[18,19] To further explore the structure-biological activity
relationship of this scaffold, oxazolo[4,5-d]pyrimidines and oxazolo[5,4-
d]pyrimidines with phenyl group at C(2), aryl or biaryl substituents at C(5)
and electron donating (piperazine) or withdrawing groups (aryl residues) at
C(7) were developed as potential VEGFR2 inhibitors. Since this receptor is a
mediator of growth and proliferation of the colon, ovarian, and breast
cancer cells,[20,21] their biological activity were investigated on HCT-116,
OVCAR-3 and MDA-MB-231 lines of the above-mentioned cancer cells.
N
N
N
O
N
O
Ph
Ph
TEA
N
N
O
dioxane,
reflux, 6 h
Cl
N
N
5b
6 (95%)
O
CF3CO2H
N2, 0 OC, 6 h
R
Ar
N
N
N
N
O
N
Ph
Ph
N
N
O
O
CF3CO2H
ArB(OH)2
N2, 0 OC, 6 h
N
N
F
0.5 mol % [Pd(IPr)Cl2]2,
3 K2CO3, EtOH,
60 OC, 18 h
O
F
N+
H2
F
O
O
8a R = Ph (80%)
7a Ar = Ph (91%)
7b Ar = MeO2CC6H4 (81%)
8b R = MeO2CC6H4 (81%)
8c R = Cl (85%)
Results and Discussion
Scheme 2. Synthesis of oxazolo[4,5-d]pyrimidines 8a-c.
Synthesis of Target Oxazolopyrimidines
Compounds 7a-b were obtained by Suzuki-Miyaura cross coupling
reaction of 7-N-Boc-piperazin-substituted oxazolo[4,5-d]pyrimidine 6 with
the corresponding arylboronic acids in the presence of [Pd(μ-Cl)Cl(IPr)]2
(0.5 mol %),EtOH and K2CO3 (Scheme 2).[27] N-Boc-protection was removed
by reacting 6 or 7a-b with CF3COOH under an inert atmosphere resulting
in the formation of oxazolo[4,5-d]pyrimidin-7-yl)-piperazin-1-ium
trifluoroacetates 8a-c. Compounds 8a-c reveal in their 1H-NMR spectra a
resonance at 9.04–9.16 ppm corresponding to H2N+ functionality in the
piperazine moiety, and their 19F-NMR spectra in (D6)DMSO revealed a
singlet at -73.5 ppm.
The synthesis of new oxazolo[4,5-d]pyrimidine 6 was accomplished in
analogy to previously reported derivatives as depicted in Schemes 1,2.[22,23]
Compound 6 is obtained by the sequence of reactions starting from
available 2-aryl-4-dichloromethylene-1,3-oxazol-5(4H)-one 1.[24] Treating
of 1 with amidine hydrochlorides 2a-b in the presence of triethylamine led
to substituted imidazol-5(4)-ones 3a-b, which on heating in pyridine
underwent cyclo-condensation followed by recyclization affording the
corresponding oxazolo[4,5-d]pyrimidin-7-ones 4a-b.
H
Functionalization of 7-chloro-substituted oxazolo[4,5-d]pyrimidine 5a
was also carried out by Suzuki-Miyaura reaction with phenyl- (9a) or 4-
methoxycarbonylphenylboronic acids (9b) in the presence of [Pd(μ-
Cl)Cl(IPr)]2, EtOH and K2CO3 (Scheme 3) affording products 10a-b in high
yield.
Cl
Cl
Cl
O
O
Cl
H2N
TEA
Py
N
Ar
+
Ph
N
THF, r.t.,
48 h
reflux,
10 h
N
NH
H
Ph
O
H2N+ Cl
O
Ar
1
2a Ar = Ph
2b Ar = 4-ClC6H4
3a Ar = Ph (71%)
3b Ar = 4-ClC6H4 (73%)
Ar
N
O
Ar
N
POCl3,
Me2NPh
N
N
O
Ph
N
Ph
N
N
O
N
O
HO
HO
Ph
Ph
Ph
Ph
NH
N
B
R
+
N
N
O
reflux, 3 h
0.5 mol % [Pd(IPr)Cl2]2,
3 K2CO3, EtOH,
THF, 60 OC, 18 h
Cl
Cl
4a Ar = Ph (90%)
4b Ar = 4-ClC6H4 (82%)
5a Ar = Ph (92%)
5b Ar = 4-ClC6H4 (96%)
R
10a R = H (70%)
10b R = CO2Me (79%)
Scheme 1. Synthesis of 7-chloro-substituted oxazolo[4,5-d]pyrimidines 5a-b.
5a
9a R = H
9b R = CO2Me
Scheme 3. Synthesis of 7-aryl-substituted oxazolo[4,5-d]pyrimidines 10.
The reaction of compounds 4a-b with trichlorophosphate in the
presence of N,N-dimethylaniline proceeded 2,5-diaryl-7-chlor-oxazolo[4,5-
d]pyrimidines 5a-b. Compound 5b was converted into desired 7-N-Boc-
piperazin-substituted oxazolo[4,5-d]pyrimidine 6 by reaction with tert-
butyl piperazine-1-carboxylate. Since the structures of the intermediates 3a,
2
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Figure 1. Molecular docking of compounds 8a (a) and 8b (b) into the active site VEGFR2.
The isomeric oxazolo[5,4-d]pyrimidin-7-one 13 was synthesized using the
route improved by Dounchis (Scheme 4).[28] The reaction of benzoyl
bromide 11 with silver cyanide led to oxazolo[3,4-d][1,3]oxazine 12.
Treatment of the trimer 12 with sodium methoxide followed by stirring with
acetic acid afforded 2,5-diphenyl-oxazolo[5,4-d]pyrimidin-7-one 13.
The reaction of compound 13 with trichlorophosphate in the presence
of N,N-dimethylaniline proceeded 2,5-diphenyl-7-chlor-oxazolo[5,4-
d]pyrimidine 14. 7-Aryl-substituted oxazolo[5,4-d]pyrimidines 15a-b were
obtained under the same conditions as their isomers 10a-b described above.
Number of H-bonds and
electrostatic interactions
Compound
∆G, kcal/mol
Sunitinib
5a
̶
̶
̶
̶
̶
̶
̶
̶
̶
̶
9.8
9.1
10.1
9.6
9.8
9.5
9.6
8.8
9.2
9.1
3
0
3
8a
8b
4
3
8c
10a
10b
14
0
0
0
0
0
O
1) MeONa
N
Ph
Ph
O
MeOH
r.t., 16 h
O
AgCN
N
O
N
O
O
NH
Ph
Br
THF, reflux
21 h
2) MeCO2H
r.t., 1 h
Ph
Ph
Ph
N
N
15a
15b
11
12 (90%)
13 (61%)
Ar
N
Cl
N
POCl3,
Me2NPh
ArB(OH)2
N
O
N
O
N
Ph
Ph
0.5 mol % [Pd(IPr)Cl2]2,
3 K2CO3, EtOH,
reflux, 3 h
Each piperazine group of compounds 8a and 8b (Figure 1) forms two H–
bonds (2.46-3.24 Å) with amino acid Leu840. The phenyl ring at C(2) of each
compound forms the electrostatic interactions (4.13-4.57 Å) with amino
acid Lys838. The phenyl rings at C(5) of compounds 8a and 8b are
characterized by forming of the many hydrophobic π-alkyl interactions
(3.56-5.47 Å) with amino acids Leu840, Val848, Ala866, Val899, Cys919,
Leu1035 and Cys1045. Moreover, 8b forms an additional hydrogen bond
between the carboxyl group and the amino acid Glu885.
N
Ph
Ph
60 OC, 18 h
15 a Ar = Ph (60%)
15 b Ar = 4-MeO2CC6H4 (65%)
14 (97%)
Scheme 4. Synthesis of oxazolo[5,4-d]pyrimidines 15.
Molecular Docking
It is known that oxazolopyrimidine-containing compounds are inhibitors of
the VEGFR2 and interact with ATP binding site.[15,16] The docking validation
was conducted by redocking sunitinib-ligand into the active site VEGFR2
(PDB ID: 4AGD). The obtained ligand-protein complex showed ΔG = –9.8
kcal/mol (Table 1). RMSD value for all atoms did not exceed 2Ǻ. It was
assumed that all studied compounds containing the oxazolopyrimidine
moiety have a similar molecular mechanism of action. Therefore, the
molecular docking of the studied compounds was performed similarly using
the VEGFR2 crystal structure (Table 1).
Docking analysis of potential biologically active compounds 8a-c with
the highest binding energy
̶ 10.1, ̶ 9.6, ̶ 9.8 kcal/mol respectively is
presented in Figures 1-2.
Figure 2. Molecular docking of compounds 8c into the active site VEGFR2.
Table 1. Molecular docking results.
3
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Piperazine moiety of ligand 8c forms the H–bonds (2.48 Å) with amino
acids Leu840. The phenyl group at C(2) forms the electrostatic interaction
(4.68 Å) with amino acid Lys838. The phenyl group at C(5) is characterized
by forming of the five hydrophobic π-alkyl interactions (3.46-4.46 Å) with
amino acids Leu840, Val848, Ala866 and Leu1035. Also, the chlorine atom
forms one H–bond (3.77 Å) with amino acids Lys868 and two π-alkyl
hydrophobic interactions (4.39-4.42 Å) with amino acids Val916, Val 899.
Thus, the docking results show a similar binding mode of compounds
8a-c in the active site VEGFR2 with the formation of stable protein-ligand
complexes with predicted ΔG= –10.1, –9.6, –9.8 kcal/mol, respectively (see
Table 1).
Figure 4 shows that the formed ligand-protein complex is stabilized
mainly by hydrophobic interactions (3.71-5.46 Ǻ) with no evidence of
hydrogen bonds and electrostatic interactions. The localization of ligand
10a was significantly different in comparison with docking position of the
highly effective ligands 8a-c in the VEGFR2 active site.
Electron Structure of the oxazolopyrimidine derivatives
As can be seen from Figure 4 careful analysis of molecular docking
calculations illustrates essential contribution of -system of the studied
compounds, interaction of the -electrons can additionally stabilize the
generated complex of compounds with the suitable fragments of
biomolecules (proteins). In addition, N-heterocycles with di-coordinated
nitrogen atoms (and hence with the lone electron pair - LEP) could generate
hydrogen bonds with amino acids.
We may optimistically suppose that the biological activity (or affinity)
of conjugated heterocycles is primarily determined by its donor/acceptor
properties; their complex with protein fragments can be stabilized by --
stack interactions and H-bond formation. Donor-acceptor properties of any
conjugated molecule can be estimated quantitatively by changes of the
relative positions of their frontier molecular orbitals: highest occupied
molecular orbital (HOMO) and lowest unoccupied molecular orbital
[29,30]
(LUMO) by the following index 0
.
When 0 = 0.5, the donor and acceptor properties are mutually
balanced; an increase of the parameter (0 > 0.5) indicates a predominately
donor nature; if 0 < 0.5, the frontier molecular orbitals are shifted
downward, indicating a predominantly acceptor character. Here, the
compounds were optimized before calculation by DFT/6-31 (d,p)/wB97XD
method using package GAUSSIAN 03;[31] obtained values of the substituted
oxazolopyrimidines are collected in Table 2.
Figure 3. The docking position of compounds 8a-c and sunitinib (co-crystallized with
VEGFR2) in VEGFR2 active site. Compounds 8a – light blue, 8b – violet, 8c – yellow,
sunitinib – orange.
Figure 3 shows the similar binding mode of compounds 8a-c and
VEGFR2 inhibitor sunitinib in the active site of the protein.
To investigate the structure-biological activity relationship of the
oxazolopyrimidine scaffold, the ligand 10a with an acceptor group at C(7)
and a binding energy lower than that of the reference molecule sunitinib
was docked to the active center of the VEGFR2 (Figure 4).
Table 2. Electron characteristics of oxazolopyrimidines (5a, 8a-c, 10a-b, 15a-b).
(MO), eV
b
a
0
Compound
HOMO
LUMO
Model moleculec
-8.963
-8.320
-7.520
-7.599
-7.786
-8.053
-8.148
-7.833
-7.943
0.544
-0.457
0.121
9.507
7.862
7.399
7.149
7.772
7.617
7.742
7.401
7.269
0.43
0.39
0.48
0.44
0.46
0.41
0.39
0.42
0.40
5a
8a
8b
-0.450
-0.014
-0.436
-0.686
-0.432
-0.674
8c
10a
10b
15a
15b
[a]  = LUMO - HOMO is energy gap
[b] 0 = (LUMO-)/ [11,12]
Figure 4. Molecular docking of compound 10a into the active site VEGFR2.
[c] 2,5,7-trimethyloxazolo[4,5-d]pyrimidine
4
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Table 4. Effects of the studied compounds on proliferation of different cancer cell lines
after 96 h of incubation.
It follows from Table 2 that:
1) introduction of the phenyl substituents (compounds 5a, 10a-b,
15a-b) decreases their donor ability compared to the model
molecule;
IC50 (μM)
Compound
OVCAR3
HCT-116
MDA-MB-231
2) introduction of the donor methoxycarbonyl group (compounds
10b and 15b) additionally increases donor properties of the
molecules;
Doxorubicin
0.0021± 0.0004
0.10 ± 0.01
0.36 ± 0.03
5a
>100
>100
>100
5.3 ± 0.3
3.9 ± 0.4
1.4 ± 0.3
8a
3) replacement of the phenyl ring by the piperazine fragment
(compounds 8a-c) is accompanied by an increase of the
parameter 0.
1.8 ± 0.3
1.7 ± 0.2
3.9 ± 0.3
0.24 ± 0.05
0.21 ± 0.03
8b
0.24 ± 0.03
8c
10a
10b
14
>100
43 ± 4
>100
>100
>100
>100
>100
>100
>100
>100
>100
22 ± 2
>100
>100
>100
The calculations of the electron structure shows that the studied
compounds can generate a stable complex with the aromatic fragments of
phenylalanine, tyrosine or tryptophan. Additionally, the aryl substituents
could act as the additional centers for the formation of complexes with the
fragments of the proteins.
15a
15b
Since the LEPs at the nitrogen atoms in the pyrimidine heterocycle can
form hydrogen bonds (see Figs. 1-3), the binding energies of these bonds
are undoubtedly dependent on the position of the corresponding n-level
(LEP level). The performed calculations show that n-level is positioned
relatively high. This causes the some mixing of n* - and *
transitions (see in detail supplementary part). Consequently, LEP can
effectively generate the hydrogen bonds, which is consistent with the
molecular docking modeling.
7-(4-Methoxycarbonylphenyl)-substituted oxazolo[4,5-d]pyrimidine
10b, with the same free energy of ligand–protein complex as 8c (ΔG = –9.6
kcal/mol, see Table 1), also showed cytotoxic effect on OVCAR-3 and MDA-
MB-231 only at 43 ± 4 µM and 22 ± 2 µM, respectively.
It can be concluded that piperazine fragment containing a positively
charged nitrogen atom play a significant role in the cytotoxicity of
oxazolo[4,5-d]pyrimidines 8a-c.
In vitro Anticancer Activity
Conclusions
The ability of all new synthesized oxazolopyrimidines (8a-c, 10a-b, 15a-b)
and their precursors (5a, 14) to inhibit cell proliferation of ovarian (OVCAR-
3), colon (HCT-116) and breast (MDA-MB-231) cancer cells was analyzed
after 96 h treatment. All oxazolopyrimidines are sparingly soluble in water,
and for this reason the stock solutions for biological investigations were
prepared in dimethylsulfoxide. The data obtained on these three cell lines
In order to investigate the structure-biological activity relationship of the
oxazolopyrimidine framework, new oxazolo[4,5-d]pyrimidines and
isomeric oxazolo[5,4-d]pyrimidines containing acceptor groups at C(2) and
C(5), and electron donating (piperazine) or withdrawing substituents (aryl
residues) at C(7) have been synthesized.
The docking results show that oxazolo[4,5-d]pyrimidines 8a-c with
nitrogen atom at the piperazine moiety bearing positive charge could be
potential VEGFR2 inhibitors with the highest binding energy (ΔG: –10.1, –
9.6, –9.8 kcal/mol, respectively). In the other cases, the nature of the ligand-
protein interaction differs: the formed ligand-protein complexes are
stabilized mainly by hydrophobic interactions, without any hydrogen bonds
and electrostatic interactions.
along with data for doxorubicin,
a known antineoplastic drug, are
summarized in Table 4. Oxazolo[4,5-d]pyrimidines 8a-c exhibited cytotoxic
effect against all three cancer cell lines and compound 10b showed activity
against two of them. Other compounds, including precursors, exhibiting ΔG
≤ –9.5 kcal/mol (see Table 1), have not demonstrated an effect on the
growth of the studied cancer cell lines.
It is worth noting that oxazolo[4,5-d]pyrimidines 8b-c displayed a
slightly higher antiproliferative activity (IC50: 0.24 ± 0.05 µM and 0.21 ± 0.03
µM, respectively) than doxorubicin (IC50 = 0.36 ± 0.03 µM) on MDA-MB-231
and have relatively good results on OVCAR-3 (IC50: 1.8 ± 0.3 µM and 1.7 ± 0.2
µM) and HCT-116 (IC50: 3.9 ± 0.3 µM and 0.24 ± 0.03 µM). At the same time,
compound 8a exhibited lower activity than the previous two, but also
inhibited the growth of OVCAR-3, HCT-116, and MDA-MB-231 at
micromolar concentrations (IC50: 5.3 ± 0.3 µM, 3.9 ± 0.4 µM and 1.4 ± 0.3 µM,
respectively).
The introduction of piperazine moiety increases the donor-acceptor
parameter (0) of oxazolopyrimidines 8a-c in the range: 0.48, 0.44, 0.46,
respectively, which is inversely proportional to the obtained results of their
anticancer activity on all studied cancer cell lines.
Oxazolo[4,5-d]pyrimidines 8a-c containing positively charged
piperazine moiety displayed the best cytotoxicity effects showing in some
cases similar or even better activity than doxorubicin. Prospects for these
molecules as potential anticancer agents are very attractive and will be the
subject of future studies.
5
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10.1002/hlca.202000169
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of 1 Å were used. The visualization of protein-ligand interactions was
performed by Accelrys DS program.
Experimental Section
Materials and Methods
Cell Viability Assays
Commercially available reagents were used without purification. The
reagents and the solvents were purchased from Sigma Aldrich or Eurisotop.
The solvents used were purified by distillation over the drying agents
indicated: THF (Na/benzophenone), DMF, MeCN (CaH2), MeOH (Mg).
Reaction monitoring by thin layer chromatography was performed on pre-
coated alumina plates SiO2 60 F254 from Merck. Melting points were
determined on a Fisher-Johns (compounds 3b, 4b, 5b, 6) or a Stuart SMP40
apparatus (compounds 7a-b, 8a-c, 10a-b, 15a-b) and are uncorrected.
Infrared (IR) spectra were recorded on a Bruker Tensor 27 (compounds 7a-b,
Cell viability inhibition assays were carried out using human colon HCT-116,
ovarian OVCAR-3, breast MDA-MB-231 cancer cells. Cells were obtained
from ATCC (Manassas, VA). Cells were grown in accordance with the
supplier and maintained at 37°C in a humidified atmosphere of 5% carbon
dioxide. Five hundred cells were plated in 96 wells and treated with six
different concentrations (0.001, 0.01, 0.1, 1, 10 and 100 µM) of the
synthesized compounds. After 96 hours from the treatment cell viability
was measured with Cell Titer glow assay (Promega, Madison, WI, USA) with
a Tecan M1000 instrument. IC50 values were calculated from logistical dose
response curves. Averages were obtained from triplicates, and error bars
are standard deviations.
8a-c, 10a-b, 15a-b) or
a Vertex-70 spectrometer from KBr pellets
(compounds 3b, 4b, 5b, 6). 1H and 13C NMR spectra were recorded on a
Bruker Avance II (296.2 K, 400 and 101 MHz, respectively) or 500 MHz Agilent
DD2 (353.2 K, 500 and 126 MHz, respectively; only for compounds 4b, 6, 8c)
spectrometers, with tetramethylsilane (TMS) as internal standard. All 1H
signals are reported in ppm with the internal chloroform signal at 7.26 ppm
or dimethyl sulfoxide at 2.50 ppm as internal reference. Data are reported
as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q
= quartet, m = multiplet), integration, and assignment. Broad signals are
indicated as br. Coupling constants (J) are given in Hertz [Hz]. All 13C-NMR
signals are reported in ppm with the internal chloroform signal at 77.16 ppm
or dimethyl sulfoxide at 39.52 ppm as internal references. The LC- or HR-
MS identification of compounds was conducted on an Agilent 1200 Series
system equipped (compounds 3b, 4b, 5b, 6) with a diode array and a
G6130A mass-spectrometer (atmospheric pressure electrospray ionization)
or on an Agilent 6540 UHD Accurate-Mass Q-TOF LC/MS G6540A mass-
spectrometer (compounds 7a-b, 8a-c, 10a-b, 15a-b). Elemental analysis
were carried out on a Vario Macro Cube/Elementar. DFT calculations were
performed by DFT/6-31 (d,p)/wB97XD method using package GAUSSIAN
03.[30]
General Procedure for the Synthesis of 4(5)-Benzoylamino-4(5)-
dichloromethyl-4,5-dihydro-1H-imidazol-5(4)-ones 3a,b
To a solution of 1,3-oxazol-5(4H)-one 1 (62-66 mmol, 1.00 equiv.), prepared
by the literature procedure,[24] in dry THF (100 mL) was added one of the
amidine hydrochlorides 2a,b (62-66 mmol, 1.00 equiv.) followed by Et3N
(64-68 mmol, 1.03 equiv.). The mixture was stirred at r.t. for 48 h. The
precipitate that formed was collected by filtration, washed with H2O, and
dried.
4(5)-(Benzoylamino)-4(5)-(dichloromethyl)-2-phenyl-4,5-dihydro-1H-
imidazol-5(4)-one (3a)
Compound 3a was prepared as described above in the general procedure,
starting from 1 (15.01 g, 62 mmol, 1.00 equiv.), benzamidine hydrochloride
2a (9.71 g, 62 mmol, 1.00 equiv.), and Et3N (9 mL, 64 mmol, 1.03 equiv.).
Yield: 15.94 g, 71 %. Spectral and elemental analysis data are identical to
literature reports.[26]
4(5)-(Benzoylamino)-4(5)-(dichloromethyl)-2-(4-chlorophenyl)-4,5-
dihydro-1H-imidazol-5(4)-one (3b)
Molecular docking
Compound 3b was prepared as described above in the general procedure,
starting from 1 (16.00 g, 66 mmol, 1.00 equiv.), 4-chlorobenzamidine
hydrochloride 2b (12.63 g, 66 mmol, 1.00 equiv.), and Et3N (9.50 mL, 68
mmol, 1.03 equiv.). 3b was obtained as light grey solid. Yield: 19.11 g, 73 %.
M.p. 144 – 146 °C. IR (KBr): 3427 – 3204 (NH, assoc.), 1785 (C=O), 1649
(NC=O), 1614, 1475, 1285, 1093, 923, 841, 712, 689, 645, 466. 1H-NMR (400
MHz, (D6)DMSO): 12.08 (s, 1H, NH); 9.29 (s, 1H, NH); 8.06 (d, J = 8.4, 2H,
ArH); 7.83-7.81 (m, 2H, ArH); 7.66 (d, J = 8.6, 2H, ArH); 7.59 (dd, J = 8.4, 6.3,
1H, ArH); 7.51 (t, J = 7.5, 2H, ArH); 6.46 (s, 1H, CHCl2). 13C-NMR (101 MHz,
(D6)DMSO): 178.13; 166.16; 137.66; 132.63; 132.21; 130.12; 129.26; 129.15;
128.50; 127.74; 126.65; 81.29; 71.99. MS: 398.0 ([M + H]+, C17H13Cl3N3O2+; calc.
397.7). Anal. calc. for C17H12Cl3N3O2 (396.66): C 51.48, H 3.05, N 10.59; found:
C 51.40, H 3.12, N 10.45.
The docking preparation of the protein with ligands was done by AutoDock
Tools (ADT) (ver. 1.5.6).[32] The crystal structure (PDB ID: 4AGD) of VEGFR-
2 was used for the docking of nine studied compounds. Water molecules
and ligand were removed from the 4AGD crystal structure using Accelrys
DS (ver. 4.0).[33] The protein was prepared by adding all hydrogens using the
“no Bond Order method” (ADT). Gasteiger partial charge calculation
method was used and the created files were saved in PDBQT format. The
created ligand structures were saved in Mol2 format using ChemAxon
Marvin Sketch 5.3.735.[34] All ligands optimization and energy minimization
were performed by Avogadro v1.1.1[35] using Auto Optimization Tool by
applying the MMFF94s force field. Partial charges and torsion angles of
ligands were changed by ADT and saved in PDBQT format. AutoDock Vina
1.1.2[36] program was applied to docking. The box center (x = 45.434, y =
̶
3.366, z = 13.177) and the grid map (40*40*40 points) with a grid spacing
̶
6
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10.1002/hlca.202000169
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HELVETICA
5-(4-Chlorophenyl)-7-(1-Boc-piperazin-4-yl)-2-phenyl-oxazolo[4,5-
d]pyrimidine 6.
General Procedure for the Synthesis of 2-Phenyl-oxazolo[4,5-d]-
pyrimidin-7(6H)-ones 4a,b
The synthesis of compound 6 was accomplished in analogy to previously
reported derivatives.[18,19] A mixture of compound 5b (5.50 g, 16 mmol, 1.00
equiv.), 1-Boc-piperazine (2.98 g, 16 mmol, 1.00 equiv.), and Et3N (2 mL, 16
mmol, 1.00 equiv.) in dioxane (50 ml) was refluxed for 6 h. After removal of
the solvent, the residue was triturated with water, filtered off, dried, and
recrystallized from MeCN obtaining white solid. Yield: 7.48 g, 95%. M.p. 248
– 250 °C. IR (KBr): 3065 – 2880 (CH2 (piperazine)), 1693, 1620, 1547, 1479,
1364, 1289, 1173, 1131, 1084, 1011, 930, 848, 776, 686. 1H-NMR (500 MHz,
353.2 K, (D6)DMSO): 8.40-8.37 (m, 2H, ArH); 8.28 (dd, J = 5.2, 3.4, 2H, ArH);
7.72-7.63 (m, 3H, ArH), 7.53-7.50 (m, 2H, ArH); 4.07 (dd, J = 6.2, 4.4, 4H,
piperazine); 3.63 (dd, J = 6.2, 4.5, 4H, piperazine); 1.48 (s, 9H, Me3CO2C). 13C-
NMR (126 MHz, (D6)DMSO): 164.24; 161.42; 158.19; 153.57; 147.58; 136.35;
134.54; 132.23; 129.07; 128.73; 127.79; 127.70; 127.39; 125.28; 78.80; 44.19;
42.67; 27.67. MS: 492.0 ([M + H]+, C26H26ClN5O3+; calc. 493.0). Anal. calc. for
C26H26ClN5O3 (491.98): C 63.48, H 5.33, N 14.23; found: C 63.44, H 5.30, N
14.18.
A solution of one of the compounds 3a,b (48 mmol) in pyridine (60 mL) was
refluxed for 10 h. The pyridine was removed in vacuo. The residue was
treated with H2O, filtered, dried, and recrystallized.
2,5-Diphenyl-oxazolo[4,5-d]pyrimidin-7(6H)-one (4a)
Compound 4a was prepared according to the above general procedure,
starting from 3a (17.38 g, 48 mmol). Purification by recrystallization (DMF)
gave white crystals. Yield: 12.50 g, 90 %. Spectral and elemental analysis
data are identical to literature reports.[27]
5-(4-Chlorophenyl)-2-phenyl-oxazolo[4,5-d]pyrimidin-7(6H)-one (4b)
Compound 4b was prepared according to the above general procedure,
starting from 3b (19.10 g, 48 mmol). Purification by recrystallization (DMF)
gave white crystals. Yield: 12.74 g, 82 %. M.p. 294 – 296 °C. IR (KBr): 3064
(NH), 1690 (NC=O), 1601, 1484, 1342, 1262, 920, 837, 717, 683, 545, 462. 1H-
NMR (500 MHz, 353.2 K, (D6)DMSO): 8.19-8.13 (m, 4H, ArH); 7.69-7.56 (m,
5H, ArH). 13C-NMR (126 MHz, (D6)DMSO): 164.59; 158.23; 154.59; 151.52;
136.13; 132.10; 131.66; 130.68; 129.32; 128.80; 128.14; 127.06; 125.29. MS:
324.0 ([M + H]+, C17H11ClN3O2+; calc. 324.7). Anal. calc. for C17H10ClN3O2
(323.74): C 63.07, H 3.11, N 12.98; found: C 63.00, H 3.17, N 12.91.
General Procedure for the Synthesis of 5-Biaryl-substituted
oxazolo[4,5-d]pyrimidin-7-yl)-piperazine-1-carboxylic acid tert-butyl
ester 7a-b
A mixture of compound 6 (1.00 g, 2 mmol, 1.00 equiv.), arylboronic acid (4
mmol, 2.00 equiv.), K2CO3 (6 mmol, 3.00 equiv.), [Pd(IPr)Cl2]2 (10 µmol,
0.005 equiv.) and absolute EtOH (20 mL) was stirred at 60 ˚C for 18 h. The
precipitate was collected by filtration, washed with water and ethanol and
then dissolved in DCM and passed through a microfilter. The solvent was
removed in vacuo.
General Procedure for the Synthesis of 7-Chloro-2-phenyl-
oxazolo[4,5-d]pyrimidines 5a,b
A mixture of one of the compounds 4a,b (39 mmol, 1.00 equiv.), POCl3 (75
mL), and Me2NPh (78 mmol, 2.00 equiv.) was refluxed for 3 h. After
evaporation of POCl3 excess the solid residue was recrystallized from 1,4-
dioxane.
4-(5-Biphenyl-4-yl-2-phenyl-oxazolo[4,5-d]pyrimidin-7-yl)-piperazine-
1-carboxylic acid tert-butyl ester 7a
Compound 7a was prepared as described above in the general procedure,
starting from 6 (1.00 g, 2 mmol, 1.00 equiv.), phenylboronic acid (0.49 g, 4
mmol, 2.00 equiv.), K2CO3 (0.83 g, 6 mmol, 3.00 equiv.), [Pd(IPr)Cl2]2 (11 mg,
10 µmol, 0.005 equiv.) and absolute EtOH (20 mL). White solid. Yield: 0.97
g, 91%. M.p. 288.3 °C. IR (KBr): 3451-2973 (CH2 (piperazine)), 1698, 1617,
1547, 1474, 1375, 1251, 1169, 1056, 1013, 930, 854, 769, 696. 1H-NMR (400
MHz, CDCl3): 8.56 – 8.52 (m, 2H, ArH); 8.26 – 8.22 (m, 2H, ArH); 7.71 – 7.65
(m, 4H, ArH); 7.62 – 7.51 (m, 3H, ArH); 7.50 – 7.43 (m, 2H, ArH); 7.40 – 7.34
(m, 1H, ArH); 4.08 (dd, J = 6.2, 4.3 Hz, 4H, piperazine); 3.68 (dd, J = 10.3, 5.3
Hz, 4H, piperazine); 1.53 (s, 9H, Me3CO2C). 13C-NMR (101 MHz, CDCl3):
165.15; 162.69; 160.51; 154.79; 148.03; 142.82; 140.84; 137.02; 132.70;
129.77; 129.21; 128.93; 128.90; 128.16; 127.66; 127.26; 127.06; 126.26;
80.50; 45.07; 28.58. HR-MS: 534.2493 ([M + H]+, C32H32N5O3+; calc. 534.2500).
Anal. calc. for C32H31N5O3 (533.64): C 72.03, H 5.86, N 13.12; found: C 71.99,
H 5.84, N 13.02.
7-Chloro-2,5-diphenyl-oxazolo[4,5-d]pyrimidine (5a)
Compound 5a was prepared as described above in the general procedure,
starting from 4a (11.28 g, 39 mmol, 1.00 equiv.), POCl3 (75 mL), and Me2NPh
(10 mL, 78 mmol, 2.00 equiv.). Yield: 11.04 g, 92 %. Spectral and elemental
analysis data are identical to literature reports.[27]
7-Chloro-5-(4-chlorophenyl)-2-phenyl-oxazolo[4,5-d]pyrimidine (5b)
Compound 5b was prepared as described above in the general procedure,
starting from 4b (12.63 g, 39 mmol, 1.00 equiv.), POCl3 (75 mL), and Me2NPh
(10 mL, 78 mmol, 2.00 equiv.). Light pink solid. Yield: 12.81 g, 96 %. M.p.
241 – 243 °C. IR (KBr): 1609, 1538, 1487, 1378, 1221, 1086, 908, 842, 708, 682,
481.1H-NMR (400 MHz, CDCl3): 8.44 (dd, J = 39.4, 7.8, 4H, ArH); 7.71-7.46 (m,
5H, ArH). 13C-NMR (101 MHz, CDCl3): 169.03; 163.96; 161.26; 141.58; 138.29;
137.64; 134.78; 134.20; 130.17; 129.50; 129.20; 129.05; 125.20. MS: 344.0 ([M
+ H]+, C17H10Cl2N3O+; calc. 343.2). Anal. calc. for C17H9Cl2N3O (342.19): C
59.67, H 2.65, N 12.28; found: C 59.56, H 2.66, N 12.20.
4-[5-(4'-Methoxycarbonyl-biphenyl-4-yl)-2-phenyl-oxazolo[4,5-
d]pyrimidin-7-yl]-piperazine-1-carboxylic acid tert-butyl ester 7b
7
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10.1002/hlca.202000169
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HELVETICA
Compound 7b was prepared as described above in the general procedure,
1482, 1376, 1278, 1208, 1132, 1024, 935, 833, 773, 720, 693. 1H-NMR (400
MHz, (D6)DMSO): 9.16 (br s, 2H, NH2+ (piperazine)); 8.46 (d, J = 8.4 Hz, 2H,
ArH); 8.29 (d, J = 7.1 Hz, 2H, ArH); 8.06 (d, J = 8.4 Hz, 2H, ArH); 7.89 (d, J =
8.4 Hz, 2H, ArH); 7.84 (d, J = 8.5 Hz, 2H, ArH); 7.76 – 7.70 (m, 1H, ArH); 7.69
– 7.61 (m, 2H, ArH); 4.26 (s, 4H, piperazine); 3.89 (s, 3H, MeO2C); 3.40 (s, 4H,
piperazine). 13C-NMR (101 MHz, (D6)DMSO): 166.01; 164.66; 161.93; 160.98;
158.81; 147.40; 143.92; 140.29; 137.32; 133.08; 129.83; 129.54; 129.37;
128.71; 128.48; 127.95; 126.98; 125.40; 52.21; 42.63. 19F-NMR (376 MHz,
(D6)DMSO): 73.56. HR-MS: 492.2017 ([M + H]+, C29H27N5O3+; calc. 492.2030).
Anal. calc. for C31H26F3N5O5 (605.58): C 61.49, H 4.33, N 11.56; found: C 61.4,
H 4.29, N 11.50.
starting from
6
(1.00 g,
2
mmol, 1.00 equiv.), 4-
(methoxycarbonyl)phenylboronic acid (0.72 g, 4 mmol, 2.00 equiv.), K2CO3
(0.83 g, 6 mmol, 3.00 equiv.), [Pd(IPr)Cl2]2 (11 mg, 10 µmol, 0.005 equiv.) and
absolute EtOH (20 mL). White solid. Yield: 0.96 g, 81%. M.p. 280.3 °C. IR
(KBr): 3450-2973 (CH2 (piperazine)), 1720(C=O), 1693, 1608, 1545, 1426,
1377, 1278, 1172, 1058, 1026, 931, 844, 774, 693. 1H-NMR (400 MHz, CDCl3):
8.55 (d, J = 8.2 Hz, 2H, ArH); 8.24 (d, J = 7.5 Hz, 2H, ArH); 8.12 (d, J = 8.3 Hz,
2H, ArH); 7.73 – 7.69 (m, 4H, ArH); 7.61 – 7.52 (m, 3H, ArH); 4.11 – 4.06 (m,
4H, piperazine); 3.94 (s, 3H, MeO2C); 3.72 – 3.65 (m, 4H, piperazine); 1.53 (s,
9H, Me3CO2C). 13C-NMR (101 MHz, CDCl3): 167.11; 165.22; 162.67; 160.24;
154.78; 148.04; 145.22; 141.47; 137.85; 132.76; 130.24; 129.22; 129.18;
129.01; 128.22; 128.17; 127.21; 127.13; 126.20; 80.53; 52.28; 45.06; 28.57.
HR-MS: 592.2535 ([M + H]+, C34H34N5O5+; calc. 592.2554). Anal. calc. for
C34H33N5O5 (591.67): C 69.02, H 5.62, N 11.84; found: C 68.98, H 5.60, N 11.78.
5-(4-chlorophenyl)-2-phenyl-oxazolo[4,5-d]pyrimidin-7-yl)-piperazin-1-
ium trifluoroacetate 8c
Compound 8c was prepared as described above in the general procedure,
starting from 6 (0.98 g, 2 mmol), 1,4-dioxane (5 mL), and an excess of
trifluoroacetic acid (5 mL). White solid. Yield: 0.86 g, 85%. M.p. 258.6 °C. IR
(KBr): 3417 – 2509 (NH2+, CH2 (piperazine)), 1718, 1674, 1622, 1545, 1452,
1358, 1283, 1207, 1127, 1021, 930, 832, 767, 694. 1H-NMR (500 MHz, 353.2 K,
(D6)DMSO): 9.04 (br s, 2H, NH2+ (piperazine)); 8.40 (dd, J = 8.9, 2.1 Hz, 2H,
ArH); 8.33 – 8.29 (m, 2H, ArH); 7.73 (dd, J = 8.4, 6.3 Hz, 1H, ArH); 7.70 – 7.65
(m, 2H, ArH); 7.55 (dd, J = 8.9, 2.3 Hz, 2H, ArH); 4.31 – 4.24 (m, 4H,
piperazine); 3.42 – 3.36 (m, 4H, piperazine). 13C-NMR (126 MHz, 80 ˚C,
(D6)DMSO): 164.59; 161.77; 158.26; 147.29; 136.18; 134.83; 132.60; 129.22;
128.92; 128.02; 127.84; 127.60; 125.20; 42.38; 41.56. 19F-NMR (376 MHz,
(D6)DMSO): 73.52. HR-MS: 392.1264 ([M + H]+, C21H20ClN5O+; calc. 392.1273).
Anal. calc. for C23H19F3ClN5O3 (505.89): C 54.61, H 3.79, N 13.84; found: C
54.56, H 3.81, N 13.86.
General Procedure for the Synthesis of [1,3]oxazolo[4,5-d]pyrimidin-
7-yl)-piperazin-1-ium Trifluoroacetates 8a-c.
To a mixture of 6 (2 mmol) or 7a-b (1 mmol) and 1,4-dioxane (5 mL) while
stirring at 0 ˚C under an inert atmosphere (Ar) was added an excess of
trifluoroacetic acid (5 mL). The solution was stirred at r.t. for 5 h. The excess
of trifluoroacetic acid and 1,4-dioxane were removed under reduced
pressure at 70 ˚C. The solid was washed with water, dried and recrystallized
from MeCN/DMF.
4-(5-Biphenyl-4-yl-2-phenyl-oxazolo[4,5-d]pyrimidin-7-yl)-piperazin-1-
ium trifluoroacetate 8a
Compound 8a was prepared as described above in the general procedure,
starting from 7a (0.53 g, 1 mmol), 1,4-dioxane (5 mL), and an excess of
trifluoroacetic acid (5 mL). White solid. Yield: 0.44 g, 80%. M.p. 288.5 °C. IR
(KBr): 3427 – 3009 (NH2+, CH2 (piperazine)), 1681, 1620, 1548, 1452 1379,
1201, 1130, 1025, 930, 769, 720, 695. 1H-NMR (400 MHz, (D6)DMSO): 9.16
(br s, 2H, NH2+ (piperazine)); 8.45 (d, J = 8.5 Hz, 2H, ArH); 8.29 (d, J = 7.1 Hz,
2H, ArH); 7.81 – 7.69 (m, 5H, ArH); 7.68 – 7.61 (m, 2H, ArH); 7.55 – 7.47 (m,
2H, ArH); 7.41 (ddd, J = 6.5, 4.4, 1.1 Hz, 1H, ArH); 4.33 – 4.21 (m, 4H,
piperazine); 3.47 – 3.36 (m, 4H, piperazine). 13C-NMR (101 MHz, (D6)DMSO):
164.66; 161.97; 159.06; 158.34; 147.42; 141.81; 139.49; 136.49; 133.07;
129.38; 129.04; 128.43; 127.96; 127.93; 126.76; 126.68; 125.43; 42.62. 19F-
NMR (376 MHz, (D6)DMSO): - 73.54. HR-MS: 434.1962 ([M + H]+, C27H25N5O+;
calc. 434.1975). Anal. calc. for C29H24F3N5O3 (546.55): C 63.62, H 4.42, N
12.79; found: C 63.59, H 4.47, N 12.76.
General Procedure for the Synthesis of 2,5,7-Triaryl-oxazolo[4,5-
d]pyrimidines 10a-b
To a solution of 7-chloro-substituted [1,3]oxazolo[4,5-d]pyrimidine 5a (0.31
g, 1 mmol, 1.00 equiv.) in THF (7 mL) and EtOH (1 mL) were added one of
the arylboronic acids 9a-b (2 mmol, 2.00 equiv.), K2CO3 (3 mmol, 3.00
equiv.), followed by [Pd(IPr)Cl2]2 (0.005 equiv.). The reaction mixture was
stirred at 60 ˚C for 18 h. The precipitate was collected by filtration, washed
with water, ethanol, and dried.
2,5,7-Triphenyl-oxazolo[4,5-d]pyrimidine 10a.
Compound 10a was prepared as described above in the general procedure,
starting from 5a (0.31 g, 1 mmol, 1.00 equiv.) in THF (7 mL) and EtOH (1 mL),
phenylboronic acid 9a (0.25 g, 2 mmol, 2.00 equiv.), K2CO3 (0.42 g, 3 mmol,
3.00 equiv.), and [Pd(IPr)Cl2]2 (6 mg, 5 µmol, 0.005 equiv.). White solid.
Yield: 0.25 g, 70%. M.p. 268.5 °C. IR (KBr): 1613, 1546, 1485, 1448, 1396,
1363, 1216, 1021, 922, 763, 689. 1H-NMR (400 MHz, CDCl3): 8.70 (dd, J = 7.9,
1.7 Hz, 2H, ArH); 8.63 (d, J = 7.0 Hz, 2H, ArH); 8.42 (d, J = 7.1 Hz, 2H, ArH);
7.69 – 7.56 (m, 6H, ArH); 7.56 – 7.46 (m, 3H, ArH). 13C-NMR (101 MHz, CDCl3):
168.16; 164.32; 161.53; 146.76; 138.48; 137.78; 133.86; 133.55; 131.74; 130.59;
129.37; 129.18; 129.04; 128.91; 128.68; 128.63; 125.89. HR-MS: 350.1279 ([M
5-(4'-Methoxycarbonylbiphenyl-4-yl)-2-phenyl-oxazolo[4,5-
d]pyrimidin-7-yl)-piperazin-1-ium trifluoroacetate 8b
Compound 8b was prepared as described above in the general procedure,
starting from 7b (0.59 g, 1 mmol), 1,4-dioxane (5 mL), and an excess of
trifluoroacetic acid (5 mL). White solid. Yield: 0.49 g, 81%. M.p. 243.9 °C. IR
(KBr): 3422 – 2522 (NH2+, CH2 (piperazine)), 1721(C=O), 1667, 1625, 1548,
8
This article is protected by copyright. All rights reserved.

10.1002/hlca.202000169
Helvetica Chimica Acta
HELVETICA
+ H]+, C23H16N3O+; calc. 350.1288). Anal. calc. for C23H15N3O (349.40): C 79.07,
General Procedure for the Synthesis of 2,5,7-Triaryl-oxazolo[5,4-
H 4.33, N 12.03; found: C 79.03, H 4.36, N 11.95.
d]pyrimidines 15a-b.
A mixture of 7-chloro-substituted [1,3]oxazolo[5,4-d]pyrimidine 14 (0.31 g,
1 mmol, 1.00 equiv.), arylboronic acid (2 mmol, 2.00 equiv.), K2CO3 (3 mmol,
3.00 equiv.), EtOH (6 mL) and [Pd(IPr)Cl2]2 (5 µmol, 0.005 equiv.) was stirred
at 60 ˚C for 18 h. The precipitate was collected by filtration, washed with
water, ethanol, and dried.
2,5-Diphenyl-7-(4-methoxycarbonylphenyl)-oxazolo[4,5-d]pyrimidine
10b.
Compound 10b was prepared as described above in the general procedure,
starting from 5a (0.31 g, 1 mmol, 1.00 equiv.) in THF (7 mL) and EtOH (1 mL),
4-(methoxycarbonyl)phenylboronic acid 9b (0.36 g, 2 mmol, 2.00 equiv.),
K2CO3 (0.42 g, 3 mmol, 3.00 equiv.), [Pd(IPr)Cl2]2 (6 mg, 5 µmol, 0.005
equiv.) White solid. Yield: 0.32 g, 79%. M.p. 272.2 °C. IR (KBr): 1718 (C=O),
1602, 1544 1394, 1358, 1283, 1217, 1115, 1020, 918, 767, 695. 1H-NMR (400
MHz, CDCl3): 8.69 – 8.62 (m, 4H, ArH); 8.40 (dd, J = 5.3, 3.3 Hz, 2H, ArH);
8.29 – 8.26 (m, 2H, ArH); 7.70 – 7.65 (m, 1H, ArH); 7.64 – 7.57 (m, 2H, ArH);
7.56 – 7.47 (m, 3H, ArH); 3.98 (s, 3H, MeO2C). 13C-NMR (101 MHz, CDCl3):
168.45; 166.59; 164.65; 161.67; 145.36; 138.60; 137.83; 137.48; 133.77;
132.64; 130.76; 130.29; 129.43; 128.98; 128.89; 128.67; 125.65; 52.57. HR-
MS: 408.1331 ([M + H]+, C25H18N3O3+; calc. 408.1343). Anal. calc. for
C25H17N3O3 (407.43): C 73.70, H 4.21, N 10.31; found: C 73.65, H 4.20, N 10.35.
2,5,7-Triphenyl-oxazolo[5,4-d]pyrimidine 15a.
Compound 15a was prepared as described above in the general procedure,
starting from 14 (0.31 g, 1 mmol, 1.00 equiv.), EtOH (6 mL), phenylboronic
acid 9a (0.25 g, 2 mmol, 2.00 equiv.), K2CO3 (0.42 g, 3 mmol, 3.00 equiv.),
and [Pd(IPr)Cl2]2 (6 mg, 5 µmol, 0.005 equiv.). White solid. Yield: 0.21 g, 60%.
M.p. 288.7 °C. IR (KBr): 1615, 1569, 1450, 1392, 1365, 1271, 1022, 963, 916,
760, 687. 1H-NMR (400 MHz, (CDCl3): 8.96 (d, J = 6.7 Hz, 2H, ArH); 8.66 (dd,
J = 7.7, 1.8 Hz, 2H, ArH); 8.38 (dd, J = 7.3, 1.1 Hz, 2H, ArH); 7.65 – 7.49 (m, 9H,
ArH). 13C-NMR (101 MHz, CDCl3): 167.26; 162.52; 160.08; 154.84; 137.47;
135.32; 132.74; 131.60; 130.89; 129.84; 129.24; 128.89; 128.74; 128.65;
128.31; 128.05; 126.22. HR-MS: 350.1279 ([M + H]+, C23H16N3O+; calc.
350.1288). Anal. calc. for C23H15N3O (349.40): C 79.07, H 4.33, N 12.03; found:
C 79.00, H 4.33, N 11.98.
7-Benzoylimino-2,5-diphenyl-oxazolo[3,4-d][1,3]oxazine 12.
Compound 12 was prepared by a literature procedure[13]. A solution of
benzoyl bromide 11 (7 mL, 59 mmol, 3.00 equiv.) in anhydrous ether (35 mL),
in which was suspended silver cyanide (7.96 g, 59 mmol, 3.00 equiv.), was
stirred and refluxed for 21 h. The solids were collected and washed with cold
ether. Yellow product was dissolved in hot chloroform and separated from
the silver salts. The solvent was removed in vacuo. Yield: 6.96 g, 90%. 1H-
NMR (400 MHz, (D6)DMSO): 8.14-8.02 (m, 6H, ArH); 7.72-7.54 (m, 9H, ArH).
13C-NMR (100 MHz, (D6)DMSO): 174.70; 160.95; 160.20; 159.53; 145.84;
133.96; 133.48; 133.42; 133.23; 129.51; 129.36; 128.80; 128.69; 128.08;
126.74; 125.28; 117.70.
2,5-Diphenyl-7-(4-methoxycarbonylphenyl)-oxazolo[5,4-d]pyrimidine
15b.
Compound 15b was prepared as described above in the general procedure,
starting from 14 (0.31 g,
1 mmol, 1.00 equiv.), EtOH (1 mL), 4-
(methoxycarbonyl)phenylboronic acid 9b (0.36 g, 2 mmol, 2.00 equiv.),
K2CO3 (0.42 g, 3 mmol, 3.00 equiv.), and [Pd(IPr)Cl2]2 (6 mg, 5 µmol, 0.005
equiv.) White solid. Yield: 0.27 g, 65%. M.p. 277.5 °C. IR (KBr): 1722 (C=O),
1615, 1538, 1392, 1361, 1278, 1107, 1020, 962, 767, 700. 1H-NMR (400 MHz,
(CDCl3): 9.04 – 9.01 (m, 2H, ArH); 8.66 – 8.61 (m, 2H, ArH); 8.42 – 8.33 (m,
2H, ArH); 8.29 – 8.21 (m, 2H, ArH); 7.68 – 7.48 (m, 6H, ArH); 3.99 (s, 3H,
MeO2C). 13C-NMR (101 MHz, CDCl3): 167.43; 166.88; 163.03; 160.23; 153.43;
139.33; 137.22; 133.00; 132.41; 131.08; 130.03; 129.69; 129.33; 128.81; 128.67;
128.58; 128.44; 126.01; 52.51. HR-MS: 408.1331 ([M + H]+, C25H18N3O3+; calc.
408.1343). Anal. calc. for C25H17N3O3 (407.43): C 73.70, H 4.21, N 10.31; found:
C 73.66, H 4.22, N 10.20.
2,5-Diphenyl-oxazolo[5,4-d]pyrimidin-7-one 13. Compound 13 was
obtained by a published procedure.[13] Trimer 12 (7.00 g, 18 mmol, 1.00
equiv.) was stirred at r.t. for 16 h in anhydrous methanol (70 mL) containing
sodium methoxide (2.16 g, 36 mmol, 2.00 equiv.). The solid was filtered,
washed with methanol and dried. The resulting sodium salt was converted
into 13 by stirring in 50% aqueous acetic acid (50 mL) at r.t. for 1 h. Yield:
3.18 g, 61%. 1H-NMR (400 MHz, (D6)DMSO): 13.12 (s, 1H, NH); 8.18-8.11 (m,
4H, ArH); 7.65-7.56 (m, 6H, ArH). 13C-NMR (100 MHz, (D6)DMSO): 163.90;
158.50; 155.63; 132.07; 131.81; 129.40; 128.83; 128.12; 126.70; 125.98.
7-Chloro-2,5-diphenyl-oxazolo[5,4-d]pyrimidine 14
Acknowledgements
Authors thank Indrek Reile for assistance with NMR studies. Support from
COST Action CA15106: “C-H Activation in Organic Synthesis” (CHAOS) is
gratefully acknowledged. Ye.V. thanks the European Regional
Development Fund (Dora Pluss programme) for financial support. SPN
acknowledges the financial support from the BOF fund (starting and senior
grants).
A mixture of compound 13 (4.12 g, 14 mmol, 1.00 equiv.), POCl3 (50 ml), and
Me2NPh (4 mL, 28 mmol, 2.00 equiv.) was refluxed for 3 h. After
evaporation of POCl3 excess the solid residue was recrystallized from 1,4-
dioxane. Yield: 4.18 g, 97%. M.p. 209 – 211 °C.[13] 1H-NMR (400 MHz,
(D6)DMSO): 8.43-8.39 (m, 2H, ArH); 8.30-8.28 (m, 2H, ArH); 7.78-7.73 (m, 1H,
ArH); 7.71-7.67 (m, 2H, ArH); 7.62-7.56 (m, 3H, ArH). 13C-NMR (100 MHz,
(D6)DMSO): 166.19; 162.99; 159.36; 148.96; 135.29; 133.51; 131.70; 129.63;
129.46; 129.08; 128.11; 128.06; 125.00.
Author Contribution Statement
9
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10.1002/hlca.202000169
Helvetica Chimica Acta
HELVETICA
Tyrosine Kinase Inhibitor Targeting Vascular Endothelial and Platelet-Derived
Growth Factor Receptor Tyrosine Kinase’, J. Med. Chem., 2003, 46, 1116–1119.
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Taylor, W. Antonios- McCrea, C. M. McBride, K. Frazier, M. Wiesmann, G. R.
Lapointe, P. H. Feucht, R. L. Warne, C. C. Heise, D. Menezes, K. Aardalen, H.
Ye, M. He, V. Le, J. Vora, J. M. Jansen, M. E. Wernette-Hammond, A. L. Harris,
‘Design, Structure-Activity Relationships and in Vivo Characterization of 4-
amino-3-benzimidazol-2-ylhydroquinolin-2-ones: A Novel Class of Receptor
Tyrosine Kinase Inhibitors’, J. Med. Chem., 2009, 52, 278–292.
The project and experiments were designed by Ye. V., T. S., S. P., Y. K., V.
B., and S. P. N. The experimental work was performed by Ye. V., D. B. and
T. S., the biological research was carried out by I.C. and F.R., and the
computational work was conducted by I. S., O. K. and N. O. All authors
contributed to the manuscript writing and review process.
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