Imatinib derivatives as inhibitors of K562 cells in chronic myeloid leukemia

Article abstract of DOI:10.1007/s00044-017-1993-8

Imatinib was the first representative of the class of Breakpoint cluster region-Abelson murine leukemia viral oncogene homolog (BCR-ABL) tyrosine kinase inhibitors used for the treatment of chronic myeloid leukemia. Second-generation and third-generation drugs have been introduced in this therapy, affording increased patient survival. However, all BCR-ABL tyrosine kinase inhibitors have been shown to induce resistance, necessitating a search for new therapeutic options. The sunitinib, another tyrosine kinase inhibitor used in the treatment of renal cell carcinoma and gastrointestinal stromal tumors is an isatin derivative. Isatin nucleus is highly versatile for the preparation of new substances, and several tyrosine kinase inhibitors examples have been obtained using it. This work aimed to design, synthesize, and biological evaluation of new compounds using the K562 cell line, which constitutively expresses the active BCR-ABL enzyme. Three new series of imatinib derivatives have been planned from the imatinib, and all have a phenylaminopyrimidine group as the main pharmacophore. Sunitinib was used as a structural prototype to planning the series 1 (8a–e) of hybrids between sunitinib and imatinib. Series 2 and 3 are 2-oxo-2-phenyacetamide and 2,2-difluoro-2-phenylacetamide derivatives, respectively. Isatins were used as the starting materials for all series. Compounds were synthesized using simple methodologies and were obtained in high purities. The compounds were tested against K562 cells, and four showed a reduction in cell viability, with EC₅₀ values ranging from 0.37 to 2.02 μM, some of which are close to the imatinib standard (0.21 μM).

Full text of DOI:10.1007/s00044-017-1993-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1993-8
ORIGINAL RESEARCH
Imatinib derivatives as inhibitors of K562 cells in chronic myeloid
leukemia
1
,2 ●
1,2 ●
4 ●
3 ●
Liviane D. Azevedo
Mônica M. Bastos
Flávia C. Vasconcelos
1 ●
4 ●
Lucas V. B. Hoelz Floriano P. S. Junior Rafael F. Dantas
4 ●
1,2 ●
1,5 ●
Ana C. M. de Almeida Andressa Paula de Oliveira
Raquel C. Maia Nubia Boechat
Larissa C. Gomes
3 ●
1,2
Received: 27 April 2017 / Accepted: 13 July 2017
Springer Science+Business Media, LLC 2017
©
Abstract Imatinib was the first representative of the class of
Breakpoint cluster region-Abelson murine leukemia viral
oncogene homolog (BCR-ABL) tyrosine kinase inhibitors
used for the treatment of chronic myeloid leukemia. Second-
generation and third-generation drugs have been introduced in
this therapy, affording increased patient survival. However, all
BCR-ABL tyrosine kinase inhibitors have been shown to
induce resistance, necessitating a search for new therapeutic
options. The sunitinib, another tyrosine kinase inhibitor used
in the treatment of renal cell carcinoma and gastrointestinal
stromal tumors is an isatin derivative. Isatin nucleus is highly
versatile for the preparation of new substances, and several
tyrosine kinase inhibitors examples have been obtained using
it. This work aimed to design, synthesize, and biological
evaluation of new compounds using the K562 cell line, which
constitutively expresses the active BCR-ABL enzyme. Three
new series of imatinib derivatives have been planned from the
imatinib, and all have a phenylaminopyrimidine group as the
main pharmacophore. Sunitinib was used as a structural
prototype to planning the series 1 (8a–e) of hybrids between
sunitinib and imatinib. Series 2 and 3 are 2-oxo-2-
phenyacetamide and 2,2-difluoro-2-phenylacetamide deriva-
tives, respectively. Isatins were used as the starting materials
for all series. Compounds were synthesized using simple
methodologies and were obtained in high purities. The com-
pounds were tested against K562 cells, and four showed a
reduction in cell viability, with EC50 values ranging from 0.37
to 2.02 μM, some of which are close to the imatinib standard
(0.21 µM).
●
●
●
Keywords Tyrosine kinase inhibitors Imatinib Sunitinib
Isatin Phenylacetamide
Electronic supplementary material The online version of this article
●
(
doi:10.1007/s00044-017-1993-8) contains supplementary material,
which is available to authorized users.
*
Nubia Boechat
nboechat@gmail.com
1
Departamento de Síntese de Fármacos, Fundação Oswaldo Cruz,
Instituto de Tecnologia em Fármacos, Farmanguinhos - Fiocruz,
Manguinhos, Rio de Janeiro, RJ CEP 21041-250, Brazil
Introduction
Chronic myeloid leukemia (CML) is a clonal myeloproli-
ferative disorder of pluripotent stem cells, initially described
by John Hughes Bennett in 1845 (Bennett 1845; Geary
2
3
4
5
Programa de Pós-Graduação em Farmacologia e Química
Medicinal – ICB-UFRJ, CCS, Bloco J, Ilha do Fundão, Rio de
Janeiro, RJ CEP 21941-902, Brazil
2000; Degos 2001; Joske 2008). In the late 1960s, Nowell
Laboratório de Hemato-Oncologia Celular e Molecular, Instituto
Nacional do Câncer, INCA, Hospital do Câncer I, Praça da Cruz
Vermelha, Centro, Rio de Janeiro, RJ 20230–130, Brazil
and Hungerford identified an abnormal chromosome pre-
sent in human leukemic cells (Geary 2000; Nowell and
Hungerford 1961; Majid and Ahmad 2015; Ali 2016). This
chromosome, called Philadelphia (Ph), was the first exam-
ple of a specific chromosomal abnormality related to
malignancies (Rowley 1973; Abraham et al. 2016), being
the result of a translocation between chromosome arms 9
Laboratório de Bioquímica Experimental e Computacional de
Fármacos, Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Rio
de Janeiro, RJ 21040-360, Brazil
PROBIN - Abeu - Centro Universitário UNIABEU, Belford Roxo,
RJ 26113-400, Brazil

Med Chem Res
and 22, which is found in 95% of patients with CML
Jabbour and Kantarjian 2016). This oncogene is respon-
sible for coding a protein with high tyrosine kinase activity
owing to the presence of the Breakpoint cluster region-
Abelson murine leukemia viral oncogene homolog (BCR-
ABL) domain (Nowell and Hungerford 1961; Druker et al.
with a resistance mechanism has been observed in some
patients (Iqbal and Iqbal 2014). This fact led to the devel-
opment of new TKIs, including second- (nilotinib, 2; and
dasatinib, 3) and third- (bosutinib, 4; and ponatinib, 5)
generation inhibitors (Fig. 1) (Azevedo et al. 2017). How-
ever, not all patients are successfully treated using those
drugs because of the development of resistance related to
mutations or serious side effects, including toxicity (Jabbour
et al. 2010; Rosti et al. 2012). Furthermore, the European
Leukemia net (ELN) and the USA National Comprehensive
Cancer Network (NCCN) recommend, for optimally
responding patients, following the TKI therapy indefinitely
(Baccarani et al. 2013; NCCN 2017) to prevent interruption,
which could cause cancer recurrence (Cortes et al. 2004).
For this reason, new anticancer drugs with higher efficacy
and lower toxicity are in constant demand (Liu et al. 2015).
In 2011, sunitinib (6), another TKI was used in the
treatment of renal cell carcinoma (RCC) and gastrointestinal
stromal tumors (Moreno et al. 2010). This anticancer drug is
an isatin derivative (7) (Fig. 2) presenting a benzoheterocycle
that is highly versatile for the preparation of various other
molecules with potential pharmacological activity (Silva
et al. 2001; Rane et al. 2016; Ibrahim et al. 2016). Moreover,
(
2
001) that is responsible for the cell transformation and the
disease pathogenesis (Lugo et al. 1990). Consequently, the
elucidation of its molecular mechanism was of great
importance for advancing CML chemotherapy, where the
inhibition of BCR-ABL tyrosine kinase represents an
interesting strategy, since it is present only in positive Ph
patients (Geary 2000; Rafiyath et al. 2013).
The first selective BCR-ABL tyrosine kinase inhibitor
(
TKI), imatinib (1; Fig. 1), was approved for use in 2001 by
the Food and Drug Administration, revolutionizing the
treatment of CML (Jabbour et al. 2007). Imatinib has a
phenylaminopyrimidine group (PAP, Fig. 1) as its pharma-
cophore, which prevents ATP from binding to the Abl
domain, via hydrogen bonds and van der Waals interactions
(
2
Manley et al. 2002; Asaki et al. 2006; Eck and Manley
009; Mughal et al. 2013; Iqbal and Iqbal 2014). However,
despite all its benefits, failure of the treatment associated
Fig. 1 Three generations of
tyrosine kinase inhibitors
(
imatinib, 1; nilotinib, 2;
dasatinib, 3; bosutinib, 4;
ponatinib, 5) used in the
treatment of chronic myeloid
leukemia (CML). The
phenylaminopyrimidine group
(
PAP) is highlighted in red in 1
and 2 (color figure online)

Med Chem Res
several derivatives of isatin with CML activity have been
described (Sabet et al. 2010; Aboul-Fadl et al. 2012).
Material and methods
Chemistry
Hence, in the present work, we have carried out the
synthesis of three series of imatinib derivatives 8a–e, 9a–e,
and 10a–e followed by their cytotoxic activity evaluation on
the K562 cell line, which constitutively expresses the active
BCR-ABL enzyme. The three series of new derivatives
have been planned from the imatinib, and the sunitinib was
been used as a structural prototype to planning the series 1
Reagents utilized in most cases were purchased from
Sigma-Aldrich Co. and used without further purification.
Solvents were purchased from Tedia and Vetec, dried as
described using appropriate techniques for each type of
solvent, and stored under nitrogen atmosphere.
(
8a–e); all of them have the PAP group as their main
Analyzes were monitored by thin layer chromatography
(TLC) using silica gel sheets supported on aluminum indi-
cators by ultraviolet light (254 and 366 nm)—Kieselgel 60
F254 Merck.
pharmacophore fragment. Series 1 (8a–e) has hybrids
between sunitinib and imatinib. Isatins were used as starting
materials for all series; series
2
has 2-oxo-2-
phenylacetamides (9a–e), and series 3 has 2,2-difluoro-2-
phenylacetamides (10a–e) (Fig. 3).
Mass spectra from a system coupled to an electron
impact gas chromatograph (GC-MS) were obtained at 70 eV
on an Agilent 6890 apparatus with an Agilent 5973 mass
spectrometer. Fragmentation values and molecular ions
were described by the relation between the atomic mass unit
and the load thereof (m/z), and relative abundances were
expressed as percentages. The column used was an Agilent
122-5532 DB-5MS (5% diphenyl: 95% dimethyl poly-
siloxane), and the runs were conducted using a temperature
ramp from 50 to 350 °C.
Melting points were determined in a Büchi B-545
apparatus, and the values were not corrected.
Low-resolution mass spectra were achieved by electro-
spray ionization (MS-ESI) in a Micromass ZQ4000 appa-
ratus. The molecular ion was described by the relation
between the atomic mass unit and the load thereof (m/z),
and relative abundances were expressed as percentages.
Fig. 2 Sunitinib (6), a tyrosine kinase inhibitor used in the treatment
of renal cell carcinoma (RCC) and gastrointestinal stromal tumors,
which has an isatin nucleus (7), highlighted in blue (color figure
online)
Fig. 3 The three series of new
derivatives planned from the
imatinib and sunitinib structures;
all of them have the
phenylaminopyrimidine group
(
PAP) highlighted in red and
isatin derivatives highlighted in
blue or pink. Series 1 (8a–e) has
hybrids between sunitinib and
imatinib, series 2 has 2-oxo-2-
phenylacetamides (9a–e), and
series 3 has 2,2-difluoro-2-
phenylacetamides (10a–e) (color
figure online)

Med Chem Res
Infrared spectra were recorded in a Thermo Scientific
spectrophotometer, Nicolet 6700 model. The values of the
absorptions are reported in wave numbers, using reciprocal
centimeters (cm ) as a unit.
Nuclear magnetic resonance (NMR) spectra were
obtained at 400 MHz for hydrogen, 100 MHz for carbon,
1.8 Hz, 1 H, H-10); 7.35 (m, 3 H, H-13, H-14, H-27); 7.42
(d, J = 5.1 Hz, 1 H, H-20); 8.31 (dt, J = 1.8 e 8.0 Hz, 1 H,
H-28); 8.49 (d, J = 5.2 Hz, 1 H, H-21); 8.65 (dd, J = 1.4 e
4.7 Hz, 1 H, H-26); 8.99 (s, 1 H, H-16); 9.21 (d, J = 1.7 Hz,
−
1
1
3
1 H, H-24); 10.96 (s, 1 H, H-1), C NMR (100 MHz;
DMSO-d , δ, ppm): 17.59 (CH , C-15); 107.65 (C-20);
6
3
4
00 MHz for phosphorus and 376 MHz for fluorine. Tri-
111.30 (C-7); 113.52 (C-10); 113.72 (C-3a); 115.64 (C-5);
121.55 (C-14); 123.55 (C-27); 125.64 (C-4); 128.47 (C-12);
131.16 (C-13); 131.99 (C-6); 133.99 (C-23); 134.25 (C-28);
138.72 (C-11); 146.82 (C-7a); 147.95 (C-9); 148.26 (C-3);
151.29 (C-24); 154.74 (C-19); 159.34 (C-26); 160.98 (C-
17); 161.41 (C-21); 163.52 (C-2), MS-ESI ([M + Na] + ,
m/z, %): 443 (100), HRMS—theoretical value
(C H N O): 406.1542, value obtained: 406.1540, HPLC
methylsilane (TMS) was used as an internal reference
standard for hydrogen and carbon (0 ppm). Chemical shifts
were reported in dimensionless units (δ) representing parts
per million (ppm). The relative areas of the signals were
obtained by electronic integration, and their multiplicities
were described as single signal (s), double signal (d), triple
signal (t), multiple signal (m), large signal (ls) and double
signal (dd).
2
4 18 6
(%, nm): 97 (261).
High-resolution mass spectra (HRMS) were registered
using a mass spectrometer by electron ionization (EI-MS,
digitalizing ES + capillary), in a Maxis 3 G apparatus.
Analytical high-performance liquid chromatography
5-methyl-3-((4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)
amino)phenyl)imino)indolin-2-one (8b) Yield: 62%, mp:
−
1
195–197 °C, IR (cm ): 2984 (C-H sp2); 1727 (C=C);
1
(
HPLC) analyzes were obtained in a Shimadzu device (VP).
1616 (C=N); 798; 749; 705 (aromatic ring), H NMR (400
Data acquisition and control were performed using Shi-
madzu CLASS-VP software version 6.13 SP2. Chromato-
graphic runs were from 190 to 800 nm. The eluents used
were (A) methanol:water (1:2) and (B) acetonitrile with
isocratic elution of 45 min of 25% (B); the flow of the
mobile phase was 1.7 mL/min, and the injected volume was
MHz; DMSO-d , δ, ppm): 1.88 (s, 3 H, 5-CH ); 2.31 (s, 3
6
3
H, H-15); 6.55 (s, 1 H, H-4); 6.73 (dd, J = 2.1 e 7.8 Hz, 1 H,
H-6); 6.78 (d, J = 8,1 Hz, 1 H, H-7); 7.13 (dd, J = 1.0 e 8.6
Hz, 1 H, H-13); 7.22 (d, J = 2.0 Hz, 1 H, H-27); 7.35 (d, J
= 8.1 Hz, 1 H, H-14); 7.39 (m, 2 H, H-20 e H-10); 8.31 (dt,
J = 1.9 e 8.0 Hz, 1 H, H-28); 8.48 (d, J = 5.1 Hz, 1 H, H-
21); 8.65 (dd, J = 1.6 e 4.7 Hz, 1 H, H-26); 9.05 (s, 1 H, H-
16); 9.21 (d, J = 1.6 Hz, 1 H, H-24); 10.85 (s, 1 H, H-1),
2
0 µL. Separation was obtained in a Shimpack MRC-C8
column, 250 × 6 mm, with particle diameters of 5 µm.
1
3
C NMR (100 MHz; DMSO-d , δ, ppm): 17.67 (CH , C-
6
3
General procedure for the synthesis of 8 (a–e)
15); 20.29 (CH C-5); 107.64 (C-20); 111.07 (C-7); 113.66
3
,
(C-10); 113.73 (C-3a); 115.62 (C-5); 123.54 (C-14); 126.00
To a 100-mL flask equipped with a condenser, Dean-Stark
apparatus and mechanical stirrer were added 1 mmol of the
corresponding isatins (7a–e) and 1 mmol (277 mg) PAP
(C-27); 128.70 (C-4); 130.30 (C-12); 131.13 (C-13); 132.00
(C-6); 134.00 (C-23); 134.60 (C-28); 138.64 (C-11); 144.55
(C-7a); 147.93 (C-9); 148.13 130 (C-3); 151.27 (C-24);
154.81 (C-19); 159.31 (C-26); 160.94 (C-17); 161.45 (C-
21); 163.61 (C-2), MS-ESI ([M − 1] + , m/z, %): 405 (100),
HRMS-theoretical value (C H N O): 420.1699, value
(
11) in 50 mL of toluene. The reaction mixture was main-
tained under reflux for 24 h. The completion of the reaction
was monitored by TLC, and then the medium was cooled to
room temperature. A solid was obtained, filtered and
washed with cold toluene.
2
5 20 6
obtained: 420.1702, HPLC (%, nm): 88 (261).
5-fluoro-3-((4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)
amino)phenyl)imino)indolin-2-one (8c) Yield: 68%, mp:
−
1
2
1
37–239 °C, IR (cm ): 3452; 3256 (N–H); 2853 (CH );
3
1
739 (C=O); 1620 (C=N); 867; 822; 784 (aromatic ring), H
NMR (400 MHz; DMSO-d , δ, ppm): 2.32 (s, 3 H, H-15);
6
6
1
.52 (dd, J = 2.4 e 8.5 Hz, H-4); 6.75 (dd, J = 2.1 e 7.9 Hz,
H, H-10); 6.89 (dd, J = 4.3 e 8.6 Hz, 1 H, H-7); 7.20 (td, J
Preparation of 3-((4-methyl-3-((4-(pyridin-3-yl)pyrimidin-
= 2.5 e 8.9 Hz, 1 H, H-6); 7.27 (d, J = 1.8 Hz, 1 H, H-27);
7.37 (m, 3 H, H-13, H-14 e H-20); 8.31 (dt, J = 1.8 e 8.0 Hz,
1 H, H-28); 8.44 (d, J = 5.1 Hz, 1 H, H-21); 8.64 (dd, J = 1.1
e 4.6 Hz, 1 H, H-26); 9.05 (s, 1 H, H-16); 9.20 (d, J = 1.6 Hz,
2
-yl)amino)phenyl)imino)indolin-2-one (8a) Yield: 96%,
−
1
mp: 184–185 °C, infrared (IR) (cm ): 3444 (N–H); 3068
(
(
(
C-H sp2); 1727 (C=O); 1614 (C=N); 832; 795; 755
aromatic ring), H NMR (400 MHz; dimethyl sulfoxide
1
13
1 H, H-24); 10.99 (s, 1 H, H-1), C NMR (100 MHz;
DMSO)-d , δ, ppm): 2.32 (s, 3 H, H-15); 6.73 (m, 3 H, H-
DMSO-d , δ, ppm): 17.72 (CH , C-15); 107.86 (C-20)
6
6
3
4
, H-6, H-7); 6.90 (d, J = 7.8 Hz, 1 H, H-5); 7.27 (d, J =
112.43 (d, J = 12.8 Hz, C-7); 112.52 (d, J = 20.4 Hz, C-4);

Med Chem Res
1
1
1
1
1
2
2
13.88 (C-10); 113.97 (C-27); 116.07 (d, J = 8.0 Hz, C-3a);
20.76 (d, J = 23.6 Hz, C-6) 123.63 (C-14); 129.10 (C-12);
31.37 (C-13); 132.07 (C-23); 134.09 (C-28); 138.76 (C-11);
43.28 (d, J = 1.1 Hz, C-7a); 147.69 (C-9); 148.06 (C-19);
51.37 (C-24); 154.33 (d, J = 2.3 Hz, C-3); 156.87 (d, J =
36.05 Hz, C-5); 159.27 (C-26); 161.07 (C-17); 161.66 (C-
General procedure for the synthesis of 12 (a–e)
To a 100-mL flask equipped with a reflux condenser were
added 5-substituted isatin (7a–e) (1 mol) and 20 moles of
previously distilled acetic anhydride. The reaction mixture
was kept under magnetic stirring at reflux for 2–4 h until the
reaction was complete. The completion of the reaction was
monitored by TLC. The reaction mixture was cooled in the
freezer for 2 h, and after this time, the precipitate was fil-
tered under vacuum, washed with hexane and dried at room
temperature. The solids were recrystallized with hexane and
ethyl acetate (1:1). The derivatives were characterized by
GC-MS, and melting points were compared with the lit-
erature (Boechat et al. 2008; James et al. 1989).
1
9
1); 163.67 (C-2), NMR F (376 MHz; DMSO-d , δ, ppm):
6
−
121.15 (Ar–F), MS-ESI ([M − 1] + , m/z, %): 423 (100),
HRMS—theoretical value (C H FN O): 424.1448, value
obtained: 424.1447, HPLC (%, nm): 96 (261).
2
4
17
6
5
-chloro-3-((4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)
amino)phenyl)imino)indolin-2-one (8d) Yield: 75%, mp:
47–248 °C, IR (cm− ): 3449 (N–H); 3115 (C–H sp2);
1
2
1
740 (C=O); 1623 (C=N); 819; 792; 747 (aromatic ring),
1
1-acetylindoline-2,3-dione (12a) Yield: 82%, Mp:
H NMR (400 MHz; DMSO-d , δ, ppm): 2.34 (s, 3 H, H-
6
139–140 °C (Lit. 143 °C), GC-MS (70 eV, m/z, %): 189
1
5); 6.73 (d, J = 2.0 Hz, 1 H, H-4); 6.76 (dd, J = 7.9 e 2.0
(14); 147 (23); 146 (100); 119 (6); 90 (23).
Hz, 1 H, H-7); 6.92 (d, J = 8.4 Hz, 1 H, H-6); 7.31 (d, J =
1
2
.9 Hz, 1 H, H-10); 7.39 (m, 4 H, H-13, H-14, H-20 e H-
7); 8.32 (dt, J = 8.0 e 1.8 Hz, 1 H, H-28); 8.49 (d, J = 5.1
1
-acetylb-5-methylindoline-2,3-dione (12b) Yield: 89%,
Mp: 173–174 °C (Lit. 173 °C), GC-MS (70 eV, m/z, %):
Hz, 1 H, H-21); 8.65 (dd, J = 4.6 e 1.2 Hz, 1 H, H-26); 9.07
s, 1 H, H-16); 9.22 (d, J = 1.4 Hz,1 H, H-24); 11.12 (s, 1 H,
2
1
03 (17); 161 (51); 160 (100); 133 (20); 104 (26).
(
1
3
H-1), C NMR (100 MHz; DMSO-d , δ, ppm): 17.79
6
-acetyl-5-fluoroindoline-2,3-dione (12c) Yield: 72%,
(
CH , C-15); 107.90 (C-20); 112.99 (C-7); 113.58 (C-10);
3
Mp: 148–150 °C (Lit. 149 °C), GC-MS (70 eV, m/z, %):
1
1
1
7
1
13.66 (C-3a); 116.86 (C-5); 123.63 (C-14); 125.15 (C-27);
25.40 (C-4); 128.98 (C-12); 131.35 (C-13); 132.11 (C-6);
33.70 (C-23); 134.14 (C-28); 138.84 (C-11); 145.67 (C-
a); 147.75 (C-9); 148.09 (C-3); 151.36 (C-24); 153.83 (C-
9); 159.40 (C-26); 161.00 (C-17); 161.66 (C-21); 163.36
207 (14); 165 (50); 164 (100); 137 (16); 108 (64).
1-acetyl-5-chloroindoline-2,3-dione (12d) Yield: 87%,
Mp: 242–243 °C (Lit. 245 °C), GC-MS (70 eV, m/z, %):
23 (9); 182 (35); 181 (53); 180 (100); 153 (19); 124 (41).
2
(
C-2), MS-ESI ([M − 1] + , m/z, %): 439 (100), HRMS—
theoretical value (C H ClN O): 440.1152, value obtained:
1-acetyl-5-bromoindoline-2,3-dione (12e) Yield: 85%,
Mp: 169–170 °C (Lit. 173 °C), GC-MS (70 eV, m/z, %):
2
4
17
6
4
5
40.1173. HPLC (%. nm): 98 (261).
268 (22); 267 (22); 227 (64); 226 (100); 225 (64); 224
-bromo-3-((4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)
(100); 170 (33).
amino)phenyl)imino)indolin-2-one (8e) Yield: 77%, mp:
63–164 °C, IR (cm− ): 3450; 3223 (N–H); 3115 (C–H sp2);
1
General procedure for the synthesis of 9 (a–e)
1
1
1
741 (C=O); 1606 (C=N) 817; 787; 740 (aromatic ring), H
To a 100-mL flask were added 1 mmol of the corresponding
N-acetylisatins (12a–e) and 1 mmol (277 mg) of PAP (11)
in 50 mL of acetonitrile. The reaction mixture was main-
tained under mechanical stirring for 24 h. The completion of
the reaction was monitored by TLC, and the product was
precipitated in the medium. The solid was isolated by fil-
tration and washed with cool acetonitrile.
NMR (500 MHz; DMSO-d , δ, ppm): 2.32 (s, 3 H, H-15);
6
6
(
=
(
.73 (d, J = 6.3 Hz, 1 H, H-4); 6.85 (m, 2 H, H-6 e H-7); 7.30
s, 1 H, H-10); 7.39 (m, 3 H, H-13, H-14 e H-20); 7.48 (d, J
8.4 Hz, 1 H, H-27); 8.32 (d, J = 7.8 Hz, 1 H, H-28); 8.49
d, J = 4.9 Hz, 1 H, H-21); 8.64 (d, J = 2.9 Hz, 1 H, H-26);
9
.05 (s, 1 H, H-16); 9.21 (s,1 H, H-24); 11.11 (s, 1 H, H-1),
1
3
C NMR (100 MHz; DMSO-d , δ, ppm): 17.79 (CH , C-
6
3
1
3
4
2
5); 107.90 (C-20); 113.04 (C-7); 113.45 (C-10); 117.34 (C-
a); 123.61 (C-5); 125.30 (C-14); 127.92 (C-27); 128.16 (C-
); 128.88 (C-12); 131.29 (C-13); 132.11 (C-6); 134.13 (C-
3); 134.46 (C-28); 138.82 (C-11); 146.01 (C-7a); 147.72
(
(
(
C-9); 148.09 (C-3); 151.34 (C-24); 153.67 (C-19); 159.42
C-26); 160.96 (C-17); 161.65 (C-21); 163.18 (C-2), MS-ESI
[M + Na] + , m/z, %): 508 (99); 506 (100), HRMS—theo-
retical value (C H BrN O): 484.0647, value obtained:
2
4
17
6
4
84.0634, HPLC (%, nm): 89 (261).

Med Chem Res
1
2
-(2-acetamidophenyl)-N-(4-methyl-3-((4-(pyridin-3-yl)
787 (aromatic ring), H NMR (400 MHz; DMSO-d , δ,
6
pyrimidin-2-yl)amino)phenyl)-2-oxoacetamide (9a) Yield:
5
ppm): 1.95 (s, 3 H, H-9); 2.24 (s, 3 H, H-16); 7.22 (d, J =
8.2 Hz, 1 H, H-18); 7.43 (d, J = 5.1 Hz, 1 H, H-24); 7.48
(m, 5 H, H-14, H-31, H-6, H-5 e H-3); 8.19 (s, 1 H, H-19);
8.47 (d, J = 7.8 Hz, 1 H, H-32); 8.52 (d, J = 5.4 Hz, 1 H, H-
25); 8.70 (d, J = 4.1 Hz, 1 H, H-30); 8.99 (s, 1 H, H-20);
9.30 (s, 1 H, H-28); 10.49 (s, 1 H, H-7); 10.62 (s, 1 H, H-
8%, mp: 211–214 °C, IR (cm− ): 3246 (N–H); 3061 (C–H
1
sp2); 2249 (CH ); 1691; 1670 (C=O); 875; 821; 763
3
1
(
aromatic ring), H NMR (400 MHz; DMSO-d , δ, ppm):
6
2
7
.03 (s, 3 H, H-9); 2.24 (s, 3 H, H-16); 8.15 (s, 3 H, H-19);
.23 (d, J = 8.4 Hz, 1 H, H-18); 7.28 (td, J = 1.0 e 7.7 Hz, 1
1
3
H, H-4); 7.44 (d, J = 5.12 Hz, 1 H, H-14); 7.51 (dd, J = 4.7
e 7.9 Hz, 1 H, H-31); 7.79 (m, 1 H, H-6); 7.64 (d, J = 1.4 e
12), C NMR (100 MHz; DMSO-d , δ, ppm): 17.59 (CH ,
6 3
C-16); 23.05 (CH3, C-9); 107.51 (C-24); 136.17 (d, J =
23.8 Hz, C-5); 116.38 (C-14); 116.72 (C-31); 119.66 (d, J
= 22.3 Hz, C-3); 123.71 (C-19); 124.59 (d, J = 7.5 Hz, C-
2); 128.06 (C-17); 128.82 (d, J = 6.5 Hz, C-6); 130.11 (C-
18); 132.11 (C-27); 133.04 (d, J = 2.3 Hz, C-1); 134.40 (C-
32); 135.87 (C-15); 137.81 (C-13); 148.08 (C-28); 151.27
(C-30); 157.96 (d, J = 241.1 Hz, C-4); 159.40 (C-25);
160.32 (C-21); 160.98 (C-11); 161.45 (C-23); 168.74 (C-8);
7
=
.8 Hz, 1 H, H-5); 7.72 (d, J = 7.7 Hz, 1 H, H-3); 8.46 (dt, J
2.0 e 7.9 Hz, 1 H, H-32); 8.52 (d, J = 5.16 Hz, 1 H, H-
2
2
1
5); 8.70 (dd, J = 1.4 e 4.7 Hz, 1 H, H-30); 9.00 (s, 1 H, H-
0); 9.29 (d, J = 1.7 Hz, 1 H, H-28); 10.64 (s, 1 H, H-7);
1
3
0.69 (s, 1 H, H-12), C NMR (100 MHz; DMSO-d , δ,
6
ppm): 17.69 (C-16); 23.82 (C-9); 107.65 (C-24); 116.30 (C-
1
3
4); 116.64 (C-19); 121.79 (C-6); 123.79 (C-2); 123.81 (C-
1); 124.51 (C-4); 128.18 (C-17); 130.30 (C-18); 131.41
1
9
187.26 (C-10), NMR F (376 MHz; DMSO-d , δ, ppm):
6
(
(
C-3); 132.19 (C-27); 134.03 (C-5); 134.47 (C-1); 135.90
C-32); 137.99 (C-13); 138.26 (C-15); 148.19 (C-28);
−118,00 (Ar–F), MS-ESI ([M + Na] + , m/z, %): 523 (100),
HRMS—theoretical value (C H FN O ): 484.1659, value
2
6
21
6 3
1
1
51.36 (C-30); 159.52 (C-25); 161.05 (C-23); 161.57 (C-
1); 161.76 (C-21); 168.95 (C-8); 190.23 (C-10), MS-ESI
obtained: 484.1656, HPLC (%, nm): 98 (261).
(
(
(
[M + 1] + , m/z, %): 465 (100), HRMS—theoretical value
2-(2-acetamido-5-chlorophenyl)-N-(4-methyl-3-((4-(pyri-
din-3-yl)pyrimidin-2-yl)amino)phenyl)-2-oxoacetamide
C H N O ): 466.1753, value obtained: 446.1754, HPLC
2
6 22 6 3
−1
%, nm): 100 (261).
(9d) Yield: 54%, mp: 217–218 °C, IR (cm ): 3442; 3252
N–H); 3110 (C–H sp2); 1687; 1672; 1662 (C=O); 829;
(
1
2
-(2-acetamido-5-methylphenyl)-N-(4-methyl-3-((4-(pyri-
818; 797 (aromatic ring), H NMR (400 MHz; DMSO-d , δ,
6
din-3-yl)pyrimidin-2-yl)amino)phenyl)-2-oxoacetamide
ppm): 1.97 (s, 3 H, H-9); 2.23 (s, 3 H, H-16); 7.22 (d, J =
5.7 Hz, 1 H, H-18); 7.43 (d, J = 5.1 Hz, 1 H, H-24); 7.50
(m, 3 H, H-5, H-31 e H-14); 7.66 (m, 2 H, H-3 e H-6); 8.17
(d, J = 1.1 Hz, 1 H, H-19); 8.47 (dt, J = 1.8 e 8.0 Hz, 1 H,
H-32); 8.51 (d, J = 5.1 Hz, 1 H, H-25); 8.69 (dd, J = 1.4 e
4.7 Hz, 1 H, H-30); 8.97 (s, 1 H, H-20); 9.28 (d, J = 1.7 Hz,
−
1
(
(
(
9b) Yield: 51%, mp: 226–228 °C, IR (cm ): 3242
N–H); 3066 (C–H sp2); 1687; 1670 (C=O); 827; 800; 787
1
aromatic ring), H NMR (400 MHz; DMSO-d , δ, ppm):
6
2
.00 (s, 3 H, H-9); 2.24 (s, 3 H, H-16); 2.31 (CH , C-4);
3
7
.23 (d, J = 8.3 Hz, 1 H, H-18); 7.44 (m, 3 H, H-31, H-24 e
1
3
H-14); 7.50 (m, 2 H, H-3 e H-5); 7.66 (d, J = 8.2 Hz, 1 H,
H-6); 8.15 (d, J = 1.6 Hz, 1 H, H-19); 8.46 (dt, J = 1.4 e
1 H, H-28); 10.55 (s, 1 H, H-7); 10.60 (s, 1 H, H-12),
C
NMR (100 MHz; DMSO-d , δ, ppm): 17.63 (C-16); 23.31
6
6
.2 Hz, 1 H, H-32); 8.51 (d, J = 5.1 Hz, 1-H, H-25); 8.70
(C-9); 107.58 (24); 116.46 (C-14); 116.79 (C-19); 123.75
(C-6); 123.97 (C-31); 127.72 (C-4); 128.16 (C-17); 129.66
(C-18); 130.18 (C-27); 132.17 (C-3); 132.55 (C-5); 134.44
(C-1); 135.67 (C-32); 138.88 (C-15); 137.90 (C-13); 148.13
(C-28); 151.28 (C-30); 159.44 (C-25); 160.23 (C-21);
161.05 (C-11); 161.54 (C-23); 168.84 (C-8); 187.23 (C-10),
MS-ESI ([M + 1] + , m/z, %): 479 (100), HRMS—theore-
tical value (C H ClN O ): 500.1364, value obtained:
(
(
dd, J = 1.4 e 4.7 Hz, 1 H, H-30); 8.98 (s, 1 H, H-20); 9.29
d, J = 1.7 Hz, 1 H, H-28); 10.52 (s, 1 H, H-7); 10.65 (s, 1
1
3
H, H-12), C NMR (100 MHz; DMSO-d , δ, ppm): 17.65
6
(
C-16); 20.16 (C-9); 23.70 (CH , C-4); 107.63 (C-24);
3
1
1
1
1
3
16.29 (C-14); 116.63 (C-19); 122.02 (C-6); 123.76 (C-31);
24.68 (C-4); 128.15 (C-17); 130.26 (C-18); 131.18 (C-27);
32.18 (C-3); 133.03 (C-5); 134.43 (C-1); 134.55 (C-32);
35.91 (C-15); 137.97 (C-13); 148.17 (C-28); 151.30 (C-
0); 159.47 (C-25); 161.04 (C-23); 161.56 (C-11); 161.82
2
6
21
6 3
500.1362, HPLC (%, nm): 98 (261).
(
C-21); 168.78 (C-8); 190.31 (C-10), MS-ESI ([M + 1] + ,
2-(2-acetamido-5-bromophenyl)-N-(4-methyl-3-((4-(pyri-
din-3-yl)pyrimidin-2-yl)amino)phenyl)-2-oxoacetamide
(9e) Yield: 64%, mp: 211–212 °C, IR (cm ): 3247; 3110
m/z, %): 479 (100), HRMS—theoretical value
−
1
(
C H N O ): 480.1910, value obtained: 480.1897, HPLC
2
7 24 6 3
(
%, nm): 97 (261).
(N–H); 3071 (C–H sp2); 1688; 1674; 1662 (C=O); 852;
1
8
18; 797 (aromatic ring), H NMR (400 MHz; DMSO-d , δ,
6
2
-(2-acetamido-5-fluorophenyl)-N-(4-methyl-3-((4-(pyri-
ppm): 1.96 (s, 3 H, H-9); 2.23 (s, 3 H, H-16); 7.21 (d, J =
8.4 Hz, 1 H, H-18); 7.47 (m, 4 H, H-14, H-24, H-32 e H-5);
7.77 (m, 2 H, H-6 e H-3); 8.16 (d, J = 1.6 Hz, 1 H, H-19);
8.46 (dt, J = 2.0 e 8.0 Hz, 1 H, H-32); 8.51 (d, J = 5.2 Hz, 1
din-3-yl)pyrimidin-2-yl)amino)phenyl)-2-oxoacetamide
−1
(
(
9c) Yield: 63%, mp: 231–233 °C, IR (cm ): 3329
N–H); 3065 (C–H sp2); 1687; 1669 (C=O); 832; 815; 801;

Med Chem Res
H, H-25); 8.69 (dd, J = 1.6 e 4.8 Hz, 1 H, H-30); 8.97 (s, 1
H, H-20); 9.27 (d, J = 1.6 Hz, 1 H, H-28); 10.54 (s, 1 H, H-
General procedure for the synthesis of 10 (a–e)
1
3
7
); 10.29 (s, 1 H, H-12), C NMR (100 MHz; DMSO-d , δ,
To a 100-mL flask were added 1 mol of (13a–e) and 1 mmol
(277 mg) of PAP (11) in 50 mL of acetonitrile. The reaction
mixture was maintained at room temperature under
mechanical stirring for 24 h. The completion of the reaction
was monitored by TLC, and the product was precipitated in
the reaction mixture. The solid was filtered and washed with
cool acetonitrile.
6
ppm): 17.64 (C-16); 23.34 (C-9); 107.59 (C-24); 115.55 (C-
1
3
4); 116.47 (C-19); 116.79 (C-6); 123.77 (C-2); 124.20 (C-
1); 128.18 (C-17); 128.44 (C-4); 130.19 (C-18); 132.18
(
(
C-27); 132.49 (C-27); 134.45 (C-5); 135.45 (C-1); 135.88
C-32); 136.08 (C-13); 137.91 (C-15); 148.13 (C-28);
1
1
51.30 (C-30); 159.44 (C-25); 160.23 (C-21); 161.06 (C-
1); 161.55 (C-23); 168.84 (C-8); 187.15 (C-10), MS-ESI
(
(
[M − 1] + , m/z, %): 543 (100), HRMS—theoretical value
C H BrN O ): 544.0859, value obtained: 544. 0852,
2
6
21
6 3
HPLC (%, nm): 98 (261).
General procedure for the synthesis of 13 (a–e)
To a 250-mL flask were added 1 mol of (12a–e) and
5
0 mL of fresh distilled dichloromethane (CH Cl ). Next, 4
2 2
moles of DAST were added, and the reaction mixture was
maintained under magnetic stirring at room temperature for
2
-(2-acetamidophenyl)-2,2-difluoro-N-(4-methyl-3-((4-
(
(
3
pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)acetamide
10a) Yield: 58%, mp: 211–214 °C, IR (cm ): 3392;
307(N–H); 1689; 1662 (C=O); 862; 794; 765 (aromatic
4
h. The completion of the reaction was monitored by
−
1
TLC, and the mixture was poured onto ice. The organic
layer was washed with distilled water (3 × 30 mL), and the
dichloromethane solution was dried with anhydrous sodium
sulfate, dried and filtered. The solvent was evaporated in a
rotavapor, and the product was vacuum dried. Derivatives
were characterized by GC-MS and melting points are
compared with the literature (Cheah et al. 2008).
1
ring), H NMR (400 MHz; DMSO-d , δ, ppm): 1.96 (s, 3 H,
H-9); 2.22 (s, 3 H, H-16); 7.22 (d, J = 8.3 Hz, 1 H, H-18);
6
7
7
.37 (m, 2 H, H-14 e H-4); 7.43 (d, J = 5.1 Hz, 1 H, H-24);
.46 (dd, J = 4.8 e 7.9 Hz, 1 H, H-31); 7.56 (t, J = 7.4 Hz, 1
H, H-5); 7.62 (d, J = 7.8 Hz, 1 H, H-3); 7.68 (d, J = 7.6 Hz,
H, H-6); 7.99 (d, J = 1.7 Hz, 1 H, H-19); 8.43 (dt, J = 1.8
e 8.0 Hz, 1 H, H-32); 8.50 (d, J = 5.1 Hz, 1 H, H-25); 8.68
1
1-acetyl-3,3-difluoroindolin-2-one (13a) Yield: 91%, Mp:
108–111 °C (Lit. 109–111 °C), GC-MS: (70 eV, m/z, %):
211 (13); 169 (100); 168 (10);141 (63); 114 (10).
(
(
(
dd, J = 1.4 e 4.7 Hz, 1 H, H-30); 8.97 (s, 1 H, H-20); 9.26
d, J = 1.8 Hz, 1 H, H-28); 9.38 (s, 1 H, H-12); 10.68
1
3
s, 1 H, H-7), C NMR (100 MHz; DMSO-d , δ, ppm):
6
17.59 (CH3, C-16); 23.31 (C-9); 107.60 (C-24); 114.31
(
t, J = 252.3 Hz, C-10); 117.22 (C-19); 117.61 (C-14);
1
9
-acetyl-3,3-difluoro-5-methylindolin-2-one (13b) Yield:
5%, Mp: 70–73 °C (Lit. 73–76 °C), GC-MS: (70 eV,
1
1
1
1
23.66 (C-31); 125.44 (C-3); 126.33 (t, J = 7.5 Hz, C-1);
27.42 (C-5); 128.96 (C-2); 130.14 (C-17); 131.47 (C-18);
32.06 (C-4); 134.30 (C-27); 134.82 (C-32); 135.73 (C-6);
37.91 (C-15); 148.09 (C-13); 151.29 (C-28); 159.39
m/z, %): 225 (12); 184 (9); 183 (100); 182 (8); 155 (73).
1
-acetyl-3,3,5-trifluoroindolin-2-one (13c) Yield: 95%,
(C-30); 160.97 (C-25); 161.52 (C-23); 161.79 (C-11);
162.09 (C-21); 168.68 (C-8), NMR F (376 MHz; DMSO-
1
9
Mp: 72–75 °C (Lit. 73–76 °C), GC-MS: (70 eV, m/z, %):
2
29 (13); 187 (100); 159 (64); 131 (13).
d , δ, ppm): −99.00 (CF ), MS-ESI ([M + Na] + , m/z, %):
6
2
5
4
9
12 (100), HRMS—theoretical value (C H F N O ):
88.1772, value obtained: 488.1754, HPLC (%, nm):
9 (261).
26 22 2 6 2
1
8
-acetyl-5-chloro-3,3-difluoroindolin-2-one (13d) Yield:
5%, Mp: 133–135 °C (Lit. 134–136 °C), GC-MS: (70 eV,
m/z, %): 245 (14); 205 (32); 203 (100); 175 (55).
2-(2-acetamido-5-methylphenyl)-2,2-difluoro-N-(4-methyl-
3
-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)acetamide
−1
1
7
-acetyl-5-bromo-3,3-difluoroindolin-2-one (13e) Yield:
0%, Mp: 143–145 °C (Lit. 144–145 °C), GC-MS: (70 eV,
(10b) Yield: 45%, mp: 225–227 °C, IR (cm ): 3222
(N–H); 2984 (C–H sp2); 1693; 1682 (C=O); 823; 804; 790
1
m/z, %): 290 (13); 288 (13); 248 (99); 246 (100); 220 (34);
18 (35); 74 (15).
(aromatic ring), H NMR (400 MHz; DMSO-d , δ, ppm);
6
2
1.96 (s, 3 H, H-9); 2.22 (s, 3 H, H-16); 7.22 (d, J = 8.3 Hz,

Med Chem Res
1
1
H, H-18); 7.37 (m, 2 H, H-14 e H-4); 7.43 (d, J = 5.1 Hz,
H, H-24); 7.46 (dd, J = 4.8 e 7.9 Hz, 1 H, H-31); 7.56 (t,
3338; 3263 (N–H); 1714; 1668 (C=O); 812; 801; 776
1
(aromatic ring), H NMR (400 MHz; DMSO-d , δ, ppm);
6
J = 7.4 Hz, 1 H, H-5); 7.62 (d, J = 7.8 Hz, 1 H, H-3); 7.68
d, J = 7.6 Hz, 1 H, H-6); 7.99 (d, J = 1.7 Hz, 1 H, H-19);
.43 (dt, J = 1.8 e 8.0 Hz, 1 H, H-32); 8.50 (d, J = 5.1 Hz, 1
1.95 (s, 3 H, H-9); 2.22 (s, 3 H, H-16); 7.23 (d, J = 8.0 Hz,
1 H, H-18); 7.36 (dd, J = 2.8 e 8.2 Hz, 1 H, H-5); 7.44 (d, J
= 5.1 Hz, 1 H, H-24); 7.48 (dd, J = 4.8 e 7.9 Hz, 1 H, H-
31); 7.65 (s, 2 H, H-3 e H-14); 7.71 (s, 1 H, H-6); 7.97 (d, J
= 1.7 Hz, 1 H, H-19); 8.43 (dt, J = 1.7 e 8.0 Hz, 1 H, H-32);
8.51 (d, J = 5.1 Hz, 1 H, H- 25); 8.69 (dd, J = 1.4 e 4.7 Hz,
1 H, H-30); 8.97 (s, 1 H, H-20); 9.26 (d, J = 1.8 Hz, 1 H, H-
(
8
H, H-25); 8.68 (dd, J = 1.4 e 4.7 Hz, 1 H, H-30); 8.97 (s, 1
H, H-20); 9.26 (d, J = 1.8 Hz, 1 H, H-28); 9.38 (s, 1 H, H-
1
3
1
2); 10.68 (s, 1 H, H-7), C NMR (100 MHz; DMSO-d , δ,
6
ppm): 17.68 (CH , C-16); 20.52 (CH , C-9); 23.29
3
3
1
3
(
1
(
1
CH3, C-4); 107.66 (C-24); 114.35 (t, J = 251.9 Hz, C-10);
17.26 (C-19); 117.66 (C-14); 123.76 (C-31); 126.59
t, J = 7.8 Hz, C-1); 126.85 (C-5); 127.78 (C-3); 128.99 (C-
7); 130.22 (C-18); 132.07 (d, J = 13.1 Hz, C-4); 133.11
28); 9.41 (s, 1 H, H-12); 10.67 (s, 1 H, H-7), C NMR
(100 MHz; DMSO-d , δ, ppm): 17.65 (CH , C-16); 23.28
6
3
(CH3, C-9); 107.68 (C-24); 113.44 (t, J = 254.0 Hz, C-10);
117.27 (C-19); 117.66 (C-14); 123.72 (C-31); 126.21
(t, J = 8.0 Hz, C-1); 128.37 (t, J = 23.4 Hz, C-2) 129.10 (C-
5); 129.33 (C-3); 129.64 (C-17); 130.24 (C-18); 131.50 (C-
4); 132.11 (C-27); 134.38 (C-32); 134.77 (C-6); 134.82 (C-
15); 138.00 (C-13); 148.13 (C-28); 151.34 (C-30); 159.44
(C-25); 161.02 (C-23); 161.20 (t, J = 30.0 Hz, C-11);
(
(
C-27); 134.39 (C-32); 134.98 (C-6); 135.09 (C-15); 137.96
C-13); 148.17 (C-28); 151.37 (C-30); 159.48 (C-25);
1
61.05 (C-23); 161.58 (C-21); 161.83 (t, J = 30.3 Hz,
1
9
C-11); 168.82 (C-8), NMR F (376 MHz; DMSO-d6,
δ, ppm): −99.01 (CF ), MS-ESI ([M−1] + , m/z, %):
2
1
9
5
5
9
01 (100), HRMS—theoretical value (C H F N O ):
02.1929, value obtained: 502.1925, HPLC (%, nm):
8 (261).
161.58 (C-21); 168.85 (C-8), NMR F (376 MHz; DMSO-
2
7 24 2 6 2
d , δ, ppm): −99.00 (CF ), MS-ESI ([M + Na] + , m/z, %):
6
2
546 (100), HRMS—theoretical value (C H ClF N O ):
2
6
21
2 6 2
522.1383, value obtained: 522.1383, HPLC (%, nm):
2
-(2-acetamido-5-fluorophenyl)-2,2-difluoro-N-(4-methyl-
99 (261).
3
-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)acetamide
−
1
(
(
10c) Yield: 76%, mp: 133–135 °C, IR (cm ): 2073
C–H sp2); 1693; 1678 (C=O); 835; 809; 789 (aromatic
2-(2-acetamido-5-bromophenyl)-2,2-difluoro-N-(4-methyl-
1
ring), H NMR (400 MHz; DMSO-d , δ, ppm): 1.96 (s, 3 H,
3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)acetamide
6
−1
H-9); 2.22 (s, 3 H, H-16); 7.22 (d, J = 8.3 Hz, 1 H, H-18);
(10e) Yield: 72%, mp: 237–238 °C, IR (cm ): 3441;
7
7
.37 (m, 2 H, H-14 e H-4); 7.43 (d, J = 5.1 Hz, 1 H, H-24);
.46 (dd, J = 4.8 e 7.9 Hz, 1 H, H-31); 7.56 (t, J = 7.4 Hz, 1
3278 (N–H); 1688; 1683 (C=O); 806; 792 (aromatic ring),
1
H NMR (400 MHz; DMSO-d , δ, ppm); 1.94 (s, 3 H, H-9);
6
H, H-5); 7.62 (d, J = 7.8 Hz, 1 H, H-3); 7.68 (d, J = 7.6 Hz,
H, H-6); 7.99 (d, J = 1.7 Hz, 1 H, H-19); 8.43 (dt, J = 1.8
e 8.0 Hz, 1 H, H-32); 8.50 (d, J = 5.1 Hz, 1 H, H-25); 8.68
2.22 (s, 3 H, H-16); 7.22 (d, J = 8.4 Hz, 1 H, H-18); 7.33
(dd, J = 1.8 e 8.0 Hz, 1 H, H-6); 7.43 (d, J = 4.8 Hz, 1 H, H-
24); 7.45 (dd, J = 8.0 Hz e J = 4.6 Hz, 1 H, H-31); 7.59 (d,
J = 8.4 Hz, 1 H, H-5); 7.76 (dd, J = 1.6 e 2.72 Hz, 1 H, H-
3); 7.82 (d, J = 2.4 Hz, 1 H, H-14); 7.97 (d, J = 1.6 Hz, 1 H,
H-19); 8.42 (d, J = 8.0 Hz, 1 H, H-32); 8.50 (d, J = 4.8 Hz,
1 H, H-25); 8.68 (dd, J = 0.8 e 1.2 Hz, 1 H, H-30); 8.96 (s,
1 H, H-20); 9.25 (d, J = 1.6 Hz, 1 H, H-28), 9.41 (s, H, H-
1
(
(
dd, J = 1.4 e 4.7 Hz, 1 H, H-30); 8.97 (s, 1 H, H-20); 9.26
d, J = 1.8 Hz, 1 H, H-28); 9.38 (s, 1 H, H-12); 10.68 (s, 1
1
3
H, H-7), C NMR (100 MHz; DMSO-d , δ, ppm): 17.69
6
(
C-16); 23.87 (C-9); 108.73 (C-24); 114.46 (t, J = 254.2
Hz, C-10); 116.38 (d, J = 5.3 Hz, C-2); 116.45 (C-14);
1
3
1
1
1
3
1
4
17.04 (C-19); 118.97 (d, J = 22.7 Hz, C-3); 124.24 (C-31);
28.27 (C-17); 128.55 (d, J = 8.4 Hz, C-6); 128.27 (C-18);
32.42 (d, J = 21.9 Hz, C-5); 133.09 (C-27); 133.72(d, J =
.6 Hz, C-1); 135.01 (C-32); 135.62 (C-15); 139.09 (C-13);
49.20 (C-28); 152.13 (C-30); 159.58 (d, J = 242.7 Hz, C-
); 160.12 (C-25); 160.24 (C-11); 161.76 (C-23); 162.91
12); 10.69 (s, 1 H, H-7), C NMR (100 MHz; DMSO-d , δ,
6
ppm): 17.65 (CH , C-16); 23.32 (CH , C-9); 107.67
3
3
(C-24); 113.37 (t, J = 253.3 Hz, C-10); 117.25 (C-19);
117.64 (C-14); 123.73 (C-31); 128.45 (t, J = 23.7 Hz, C-2);
128.00 (t, J = 9.0 Hz, C-3); 129.10 (C-5); 129.44 (C-17);
130.24 (C-18); 132.10 (C-27); 134.46 (C-32); 134.75
(C-6); 135.25 (C-15); 137.99 (C-13); 148.12 (C-28); 151.33
(C-30); 159.45 (C-25); 161.01 (C-23); 161.22 (t, J = 30.3
1
9
(
C-21); 168.97 (C-8), NMR F (376 MHz; DMSO-d , δ,
6
ppm): −99.11 (CF ); −115.28 (Ar–F), MS-ESI ([M − 1] + ,
2
1
9
m/z, %): 505 (100), HRMS—theoretical value
Hz, C-11); 161.56 (C-21); 168.78 (C-8), NMR
F
(
C H F N O ): 506.1678, value obtained: 506.1677,
26 21 3 6 2
(376 MHz; DMSO-d , δ, ppm): −99.01 (CF ), MS-ESI
6
2
HPLC (%, nm): 97 (261).
([M − 1] + , m/z, %): 565 (100), HRMS—theoretical value
C H BrF N O ): 566.0877, value obtained: 566.0872,
(
2
6
21
2 6 2
2
-(2-acetamido-5-chlorophenyl)-2,2-difluoro-N-(4-methyl-
HPLC (%, nm): 99 (261).
3
-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)acetamide
−
1
(
10d) Yield: 45%, mp: 218–220 °C, IR (cm ): 3442;

Med Chem Res
Biological evaluation
Statistical analysis
Cell lines
The EC50 values were calculated by a four-parameter logistic
curve function using GraphPad Prism v. 6.01 (GraphPad
Software, La Jolla, CA, USA, www.graphpad.com).
The K562 human chronic myeloid cell line expressing the
BCR-ABL protein was grown in RPMI-1640 culture med-
ium (SIGMA) supplemented with 10% heat-inactivated
fetal bovine serum (FBS; Gibco). Human kidney epithelial
cells WSS-1 [WS-1] (ATCC CRL-2029™) were growth
in high glucose DMEM (VitroCel, Campinas, SP, Brazil)
supplemented with 10% FBS (Vitrocel). Both cell lineages
Results and discussion
Chemistry
®
were maintained at 37 °C under 5% CO in a water jacket
Fifteen new molecules presenting the PAP group, as the
pharmacophore moiety of imatinib, (Choi et al. 2010;
Druker and Lydon 2000) and isatin derivatives were syn-
thesized and are discussed in three groups according to their
structural similarities (Scheme 1).
2
CO incubator (Forma series II incubator from Thermo
2
Scientific).
Cytotoxicity assay in K562 cells
The first series planned through molecular hybridization
from imatinib and sunitinib (8a–e) was synthesized by
reacting isatins (7a–e) with a PAP derivative 11. The
intermediate 11 was obtained through a donation from
Cristália S.A. with 99% purity. However, we have pre-
viously described a variation of the classical synthetic route
(Zimmermann et al. 1996a, b; Rewcastle et al. 2000;
Manley et al. 2002; Feng et al. 2013; Boechat et al. 2013).
The imine products were formed through a substitution
reaction at the C-3 carbonyl group of 7a–e, with yields
between 77 and 96%. The NMR spectra show signal
duplication in the ratio of 9:1 due to the formation of both E
and Z isomers, where the E isomer is the thermo-
dynamically most stable and, consequently, more abundant
(supplementary material, Figure S). Furthermore, similar
results are described in the literature (Subari et al. 2010;
Ikotun et al. 2012; Aslam et al. 2015).
The cell viability was measured using the colorimetric
MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide; GE Healthcare, Buckinghamshire, UK).
4
The cells (2 × 10 cells/well) were incubated with the
compounds at concentrations ranging from 0.05 to 5.0 µM
for 72 h at 37 °C. The MTT solution (5 mg/mL) was
added in the last four hours of incubation. After dissolving
the formazan product using DMSO, the plates were read
using a microtiter plate reader (SpectraMAX® 190 Micro-
plate Reader; Molecular Devices) at 570 nm. The optical
density (OD) of cells with no compound (control) was
referred to as 100% viability. The OD of cells incubated
with the compounds was referred to as a percentage of
viability in relation to the control. Tests were performed in
triplicate.
Compounds of the second series 9a–e were prepared in
two steps, using 5-substituted isatins (7a–e) as starting
materials (Scheme 1). The N-acetylations of 7a–e were
obtained in good yield (72–89%) through a solvolysis
process using acetic anhydride. The compounds 12a–e are
already described in the literature (Popp and Piccirilli 1971;
Boechat and Pinto 2000; Cheah et al. 2008; Obafemi et al.
2012; Jeankumar et al. 2014). The preceding acetylation
step is required to produce the series 9a–e, leaving the C-2
carbonyl group susceptible to nucleophilic attack by amines
(Silva et al. 2001; Boechat et al. 2007). Consequently, the
reaction between the N-acetylisatins 12a–e with 6-methyl-
N-1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine
(11) via nucleophilic addition to the C-2 carbonyl provides
the respective products in yields ranging from 51 to 64%.
The series 10a–e was also obtained from N-acetylisatin
derivatives 12a–e, using methodology described by
our group. The intermediates 12a–e were treated with
diethylaminosulfur trifluoride (DAST) to produce the
gem-difluorinated compounds 13a–e in yields of 70–95%.
Cytotoxicity assay in WSS-1 cells
The cytotoxic effects of imatinib and its derivatives on
WSS-1 control cells were evaluated by a resazurin-based
viability assay (Neves et al. 2016). Briefly, WSS-1 cells
suspended in DMEM were seeded into 96-well microplates
4
at 1 × 10 cells/well. 20 h later, cells were incubated with
0
.1–1.000 µM of compounds and kept under a humidified
atmosphere (5% CO , 37 °C) for 72 h. Four hours before
2
the end of the experiment, resazurin (0.01 mg/mL) was
added to each well. The fluorescence of resorufin was
registered (λ = 560 nm; λ = 590 nm) immediately and 4
ex
em
h after resazurin addition in a FlexStation 3 Benchtop multi-
mode microplate reader (Molecular Devices, Sunnyvale,
CA, USA). The cell viability was calculated by subtracting
the initial from the final fluorescence reading and was
expressed as percentage of control (DMSO 1%). All assays
were performed in triplicate.

Med Chem Res
a) R = H
b) R = CH
c) R = F
d) R = Cl
e) R = Br
3
Scheme 1 Synthetic route for the preparation of compounds 8a–e, 9a–e, and 10a–e
These compounds have already been obtained by Boechat
and Coworkers (2008). Then, the reactions between inter-
mediates 13a–e and 11 provided the products 10a–e in
yields of 45–72% (Scheme 1) (Boechat and Pinto 2000;
Boechat et al. 2008, 2013).
similar fashion to imatinib, followed by 9b (R=CH ), 9e
3
(R=Br), 9a (R=H), and 9c (R=F). In addition, comparable
effects to imatinib were also observed for the compounds 9d
and 9e, with EC50 values of 0.37 and 0.56 μM, respectively
(Table 1). However, compounds 9a (R=H), and 9c (R=F),
even at higher concentrations (5 µM), did not achieve the
effectiveness of imatinib, presenting decreases of cell viability
of approximately 50 and 10%, correspondingly.
Compounds 10a–e presented no effect in reducing the
cellular viability of the K562 cell line when they were
incubated at concentrations ranging from 0.05 to 5 µM. In
that series, the highest drop in cell viability was observed
for compound 10a (40%), followed by 10b, 10d, 10e, and
The chemical structures of series 8a–e, 9a–e, and 10a–e
1
13
were elucidated by H and C NMR, IR spectroscopy, high-
performance liquid chromatography (HPLC), mass spectro-
metry by electrospray ionization (MS-ESI), and HRMS.
Biological evaluation
Cytotoxic effects in K562 and WSS-1 cells
10c. However, when compared with imatinib (80%), that
Cell viability was analyzed by the MTT method after
incubation with all compounds 8a–e, 9a–e, and 10a–e, and
the results were compared to imatinib activity (Fig. 4).
The K562 cellular viability results for the series 8a–e,
series was not active (Fig. 4).
Consequently, the most active compounds (9d, 9b, 9e,
and 9a) were tested on the WSS-1 lineage (human cells) to
establish the selectivity index. For those compounds, which
were able to reduce cell viability by over 50%, EC50 values
were determined on K562 and WSS-1 cells, and thus, the
selectivity index was calculated (Table 1). Compounds 9a,
9b, 9d, and 9e showed EC50 values between 0.56 and 2.02.
Nevertheless, those molecules showed lower selectivity
indexes when compared to imatinib, mostly due to a higher
toxicity to the WSS-1 lineage, with the notable exception of
compound 9b (Table 1).
9
a–e, and 10a–e are described in Fig. 4. Interestingly, none
of the imine derivatives 8 (a–e) could reduce cell viability
significantly, even at higher concentrations (Fig. 4). Among
those compounds, 8a was the most active but still inhibited
only 20% of cell viability.
Among the synthesized compounds, the series 9a–e was
the most active against the viability of K562 cells. 9d (R=Cl)
had the best cytotoxic activity, reducing the cell viability in a

Med Chem Res
Fig. 4 Cells K562 inhibition assays of derivatives 8a–e (a), 9a–e (b), and 10a–e (c). (d) The structures of compounds 8a–e, 9a–e, and 10a–e
Table 1 EC50 values on K562
Compounds
EC50 (mean ± standard error) (µM)
Selectivity index: WSS-1/K562
and WSS-1 cells and the
selectivity index of compounds
K562
WSS-1
9
a, 9b, 9d, and 9e
9
9
9
9
a
b
d
e
2.02 ± 0.11
0.86 ± 0.06
0.37 ± 0.05
0.56 ± 0.06
0.21 ± 0.03
6.88 ± 0.65
34.87 ± 1.72
10.71 ± 0.88
10.83 ± 0.59
33.93 ± 0.79
3.39
40.78
29.18
19.20
160.80
Imatinib
Overall, the comparison between the 9a–e and 10a–e
series shows that the presence of the carbonyl group instead
cellular viability reduction. These results provide a solid
basis for conducting further studies aimed at identifying
new antiproliferative drug candidates.
of –CF as a spacer between the phenyl ring and the PAP
2
amide group drastically increases the activity of the mole-
cules. In addition, the substitution of the PAP amide group for
the ring of the isatin derivatives 7a–e provides inactive imines
Acknowledgements We are grateful to Coordination for the
Improvement of Higher Education Personnel (CAPES), National
Council for Scientific and Technological Development (CNPq) and
Foundation for Research Support of the State of Rio de Janeiro
8a–e, indicating the importance of that moiety for activity.
(FAPERJ) for funding this work.
Compliance with ethical standards
Conclusions
Conflict of interest The authors declare that they have no competing
Fifteen new imatinib derivatives were synthesized via an
effective synthetic route with high purity. Biological eva-
luations emphasized the importance of the 2-oxo-2-
phenylacetamide group in imatinib derivatives (9a–e).
However, the 2,2-difluoro-2-phenylacetamide series (10a–e)
and the imines 8a–e presented no effect in reducing the
cellular viability of the K562 cell line. Compound 9d (0.37
µM) is equipotent to imatinib (0.21 µM), followed by 9e
interests.
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