Biological evaluation of 2-methylpyrimidine derivatives as active pan Bcr-Abl inhibitors

Article abstract of DOI:10.1007/s11426-013-5011-9

We designed a series of 2-methylpyrimidine derivatives as new BCR-ABL inhibitors using scaffold-hopping strategy. These synthetic compounds exhibited significant inhibition against a broad spectrum of Bcr-Abl mutants including the gatekeeper T315I mutant. Compound 7u showed very potent kinase inhibitory activities against Bcr-Abl WT, Bcr-Abl E255K, Bcr-Abl Q252H, Bcr-Abl G250E and Bcr-Abl T315I, with IC50 values of 0.13 nM, 0.17 nM, 0.24 nM, 0.19 nM and 0.65 μM, respectively. This compound also displayed anti-proliferation activity against K562 cell line with an IC50 value of 1.1 nM, thus representing a new lead for further optimization.

Full text of DOI:10.1007/s11426-013-5011-9

SCIENCE CHINA
Chemistry
June 2014 Vol.57 No.6: 823–832
doi: 10.1007/s11426-013-5011-9
• ARTICLES •
Biological evaluation of 2-methylpyrimidine derivatives as active
pan Bcr-Abl inhibitors
ZOU DingBiao1†, QIU YaTao1†, TU ZhengChao1, LIAO ChenZhong2, LUO JinFeng1, MENG QingQing3,
YAO RiSheng2, LI Zheng3* & JIANG Sheng1*
1Laboratory of Medicinal Chemistry, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences,
Guangzhou 510663, China
2School of Medical Engineering, Hefei University of Technology, Hefei 230009, China
3Department of Radiology, the Methodist Hospital Research Institute, Houston TX 77030, USA
Received August 28, 2013; accepted September 25, 2013; published online November 22, 2013
We designed a series of 2-methylpyrimidine derivatives as new BCR-ABL inhibitors using scaffold-hopping strategy. These
synthetic compounds exhibited significant inhibition against a broad spectrum of Bcr-Abl mutants including the gatekeeper
T315I mutant. Compound 7u showed very potent kinase inhibitory activities against Bcr-Abl WT, Bcr-Abl E255K, Bcr-Abl
Q252H, Bcr-Abl G250E and Bcr-Abl T315I, with IC50 values of 0.13 nM, 0.17 nM, 0.24 nM, 0.19 nM and 0.65 M, respec-
tively. This compound also displayed anti-proliferation activity against K562 cell line with an IC50 value of 1.1 nM, thus rep-
resenting a new lead for further optimization.
chronic myeloid leukemia (CML), anticancer agents, Bcr-Abl, imatinib resistance
this deregulated gene is considered to be an effective and
1 Introduction
non-cytotoxic strategy to treat CML [3]. Although great
clinical success has been achieved for the treatment of CML
by the first generation Bcr-Abl inhibitor imatinib (1), many
patients eventually develop intrinsic or acquired resistance
to imatinib. The resistance rates were reported to be 3%4%
per year in newly diagnosed chronic phase CML patients,
and up to 40%–50% or 80%, respectively, in accelerated or
blastic phase disease [4]. Mutations in the kinase domain of
Bcr-Abl are the primary mechanism associated with imat-
inib resistance, and more than 100 different point mutations
have been identified in CML patients who are resistant to
imatinib [5].
To overcome the imatinib resistance in CML, scientists
have developed a number of second generation inhibitors
[6], which include nilotinib [7] and bafetinib [8], and dual
SRC/ABL inhibitors dasatinib [9] and bosutinib [10]. Most
mutants can be effectively inhibited by dasatinib and ni-
lotinib, which have been approved for the treatment of
Chronic myelogenous leukemia (CML) is a clonal stem-cell
disorder caused by excessive proliferation of the myeloid
lineage cells and their subsequent differentiation into pe-
ripheral white blood cells. CML is characterized by the
presence of the Philadelphia chromosome resulted from a
balanced reciprocal translocation between chromosomes 9
and 22, leading to the expression of a chimeric protein
BCR-ABL [1]. The fusion protein Bcr-Abl contains a GRB2
binding site, SH2 and SH3 domains, and a tyrosine-kinase
(TK) domain and can activate several signal transduction
pathways, such as RAS, the STAT family of transcription
factors, and the phosphatidylinositol-3′ kinase (PI3K) [2].
Because the TK activity of BCR-ABL is necessary for its
transforming function, blocking the enzymatic activity of
†These two authors contributed equally to this work
*Corresponding authors (email: ZLi@HoustonMethodist.org; jiang_sheng@gibh.ac.cn)
© Science China Press and Springer-Verlag Berlin Heidelberg 2013
chem.scichina.com link.springer.com

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Zou DB, et al. Sci China Chem June (2014) Vol.57 No.6
adults in all phases of CML with resistance or intolerance to
imatinib. However, most second generation inhibitors can-
not inhibit the T315I mutant at the “gatekeeper” position,
which represents about 15%–20% of all clinically observed
mutants [11]. To solve this problem, a number of “third
generation” Bcr-Abl inhibitors have been developed to in-
hibit almost the full range of Bcr-Abl kinase domain muta-
tions as well as the native enzyme itself (Figure 1) [12–17].
In this report, we describe the design, synthesis and biolog-
ical evaluation of new 2-methylpyrimidine derivatives as
pan Bcr-Abl inhibitors.
Dasatinib is a dual SRC/ABL kinase inhibitor, which has
demonstrated promising efficacy for the treatment of
imatinib-resistant CML patients. But it cannot inhibit the
T315I mutant at the “gatekeeper” position 1 [18]. Co-crystal
structure of Bcr-Abl with dasatinib revealed that the pyrim-
idine and thiazolo-5-carboxamide segments of dasatinib
form three hydrogen bonds with protein [19]. However,
2-(piperazin-1-yl)ethanol moiety of dasatinib cannot bind to
the pocket induced by the DFG-out conformation of T315I
mutant. We assumed that replacement of 2-(piperazin-1-
yl)ethanol moiety in dasatinib with more hydrophobic group
may result in favorable and well defined Van der Waals
interaction in the tail region of T315I mutant. In the present
study, we decided to keep the pyrimidine/thiazole system,
as well as the amide substitution, intact and focus our ef-
forts to replace the (2-hydroxyethyl)-piperazine moiety of
dasatinib with more hydrophobic groups (Figure 2).
matography was carried on silica gel (300–400 mesh). All
reactions were monitored by TLC. All products were char-
1
acterized by NMR and LC/MS. H NMR and 13C NMR
spectra were recorded on Bruker Avance ARX-400. Mass
spectra were performed on Kompact Axima-CFR MALDI
mass spectrometers. The first-order peak patterns are indi-
cated as s (singlet), d (doublet), t (triplet), q (quadruplet).
Complex non first-order signals are indicated as m (multi-
plet). HPLC methods: an isotropic program using 80%
methanol (with NH3·H2O, pH 8), 20% water (with
NH3·H2O, pH 8) that was employed on a Chromollth Per-
formance RP-18e 100 × 4.6 mm column. Flow rate was 0.6
mL/min, and UV detection was set to 315 nm. The LC
column was maintained at room temperature.
2.2 Synthesis of 7a7u
The detailed synthesis of 7a7u is illustrated in Scheme 1.
The key intermediate, 2-((6-chloro-2-methylpyrimidin-4-
yl)amino)-N-(2-chloro-6-methylphenyl)thiazole-5-carbox
amide (6), was prepared from N-(2-aminothiazol-5-yl)-2-
chloro-6-methylbenzamide 5 and 4,6-dichloro-2-methylpyr-
imidine as follows. To a solution of N-(2-aminothiazol-5-
yl)-2-chloro-6-methylbenzamide 5 (10.0 g, 37.36 mmol) in
anhydrous THF (124 mL) was added 4,6-dichloro-2-methy-
lpyrimidine (7.3 g, 44.82 mmol) at room temperature, and
the mixture was stirred for 15 min. After the mixture was
stirred for an additional 30 min at 5 °C, NaOtBu (12.6 g,
130.72 mmol) was added slowly. The mixture was stirred at
the temperature for 15 min and for other 2 h at 10 °C. Then
the mixture was cooled to 0 °C for 30 min and neutralized
by 1 mol/L HCl to pH 6 within 1.5 h. The mixture were
filtered and the cake washed by 2 × 5 mL CH3OH, 5 mL
2 Experimental
2.1 Materials and methods
1
All reagents were commercial available and were used
without further purification only if specially noted. Chro-
H2O, dried, afford pure 6 (white solid, 7.8 g, 97%). H
NMR (400 MHz, DMSO-d6): δ 12.23 (s, 1H), 10.01 (s, 1H),
Figure 1 Chemical structures of BCR-ABL inhibitors.

Zou DB, et al. Sci China Chem June (2014) Vol.57 No.6
825
Scheme 1 General method for the synthesis of compounds 7a–7u.
N-(2-Chloro-6-methylphenyl)-2-((6-((4-chlorophenyl)amino)-
2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (7b)
1
White solid, yield: 73%. H NMR (400 MHz, DMSO-d6):
δ 11.66 (s, 1H), 9.89 (s, 1H), 9.56 (s, 1H), 8.24 (s, 1H),
7.67 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 1.2 Hz, 1H), 7.417.24
(m, 4H), 6.30 (s, 1H), 2.49 (s, 3H), 2.25 (s, 3H). 13C NMR
(125 MHz, DMSO-d6):  165.7, 162.4, 160.6, 159.8, 156.4,
140.8, 139.3, 138.8, 133.5, 132.4, 129.0, 128.5, 128.1,
127.0, 125.8, 125.4, 121.1, 85.9, 25.5, 18.3. HRMS (EI):
C22H18Cl2N6OS: calculated [M + H]+ 485.0713; found:
485.0716. Purity: 99.5% (tR = 8.16 min).
2-((6-((3-Chloro-4-fluorophenyl)amino)-2-methylpyrimidin-
4-yl)amino)-N-(2-chloro-6-methylphenyl)thiazole-5-carbox
amide (7c)
1
White solid, yield: 46%. H NMR (400 MHz, DMSO-d6):
Figure 2 Design of new Bcr-Abl inhibitors with conformation con-
strained right part.
δ 11.70 (s, 1H), 9.91 (s, 1H), 9.62 (s, 1H), 8.25 (s, 1H),
7.98 (d, J = 2.4 Hz, 1H), 7.55 (m, 1H), 7.427.26 (br, 4H),
6.29 (s, 1H), 2.50 (s, 3H), 2.25 (s, 3H). 13C NMR (125 MHz,
DMSO-d6):  165.7, 162.4, 160.5, 159.8, 156.4, 153.3,
151.3, 140.8, 138.8, 137.7, 137.7, 133.5, 132.4, 129.0,
128.2, 127.0, 125.8, 120.8, 119.7, 119.7, 119.1, 118.9,
116.9, 116.7, 85.9, 25.5, 18.3. HRMS (EI):
C22H17Cl2FN6OS: calculated [M + H]+ 503.0618; found:
506.0616. Purity: 97.4% (tR = 8.65 min).
8.32 (s, 1H), 7.41 (m, 1H), 7.29 (m, 2H), 6.95 (s, 1H), 2.59
(s, 3H), 2.25 (s, 3H), (see the Supporting Information
online).
Then various aniline derivatives reacted with 6 under
acidic conditions to afford 7a7u in good yields by the sim-
ilar method as compound 7a synthesized. 2-chloro-N-(2-
((6-chloro-2-methyl-pyrimidin-4-yl)amino)thiazol-5-yl)-6-
methylbenzamide 6 (100 mg, 0.254 mmol) and 4-chlor-
oaniline (48 mg, 0.382 mmol) was dissolved in 0.7 mL of
1,4-dioxane at room temperature, Concd. HCl (37%, 0.1
mL) was added under stirring, and the mixture was heated
at 110 °C for 12 h. Cooled to room temperature and 5 mol/L
NaOH was added to pH 10, evaporation of the solvent gave
the crude product, which was purified by flash column
chromatography (dichloromethane/methanol, v/v = 20:1, 1%
triethylamine) and recrystallization in methanol to afford
the product 7a (90 mg, 73%). 1H NMR (400 MHz,
DMSO-d6): δ 11.66 (s, 1H), 9.89 (s, 1H), 9.56 (s, 1H), 8.24
(s,1H), 7.67 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 1.2 Hz, 1H),
7.417.24 (m, 4H), 6.30 (s, 1H), 2.49 (s, 3H), 2.25 (s, 3H).
13C NMR (125 MHz, DMSO-d6):  165.7, 162.4, 160.6,
159.8, 156.4, 140.8, 139.3, 138.8, 133.4, 132.4, 123.0,
128.5, 128.1, 127.0, 125.8, 125.4, 121.1, 85.9, 25.5, 18.3.
HRMS (EI): C22H18Cl2N6OS: calculated [M + H]+ 485.0713;
found: 485.0716. Purity: 99.5% (tR = 8.16 min).
N-(2-Chloro-6-methylphenyl)-2-((6-((4-fluorophenyl)amino)-
2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (7d)
1
White solid, yield: 57%. H NMR (400 MHz, DMSO-d6):
δ 11.60 (s, 1H), 9.89 (s, 1H), 9.43 (s, 1H), 8.23 (s, 1H),
7.67 (m, 2H), 7.40 (d, J = 8.0 Hz, 1H), 7.287.25 (m, 2H),
7.15 (t, J = 8.8 Hz, 2H), 6.24 (s, 1H), 2.46 (s, 3H), 2.24 (s,
3H). 13C NMR (125 MHz, DMSO-d6):  172.1, 165.7, 162.5,
160.9, 159.9, 158.5, 156.6, 156.4, 140.9, 138.8, 136.5,
133.5, 132.4, 129.0, 128.2, 127.0, 125.7, 121.9, 121.8,
115.4, 115.2. HRMS (EI): C22H18ClFN6OS: calculated [M +
H]+ 469.1008; found: 469.1008. Purity: 100% (tR = 6.12
min).
N-(2-Chloro-6-methylphenyl)-2-((6-((3-fluorophenyl)amino)-
2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (7e)
1
White solid, yield: 55%. H NMR (400 MHz, DMSO-d6):
δ 11.69 (s, 1H), 9.90 (s, 1H), 9.65 (s, 1H), 8.24 (s, 1H),
7.70 (d, J = 12.0 Hz, 1H), 7.39 (d, J = 6.4 Hz, 1H),
7.397.23 (br, 4H), 6.779 (m, 1H), 6.34 (s, 1H), 2.50 (s,

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Zou DB, et al. Sci China Chem June (2014) Vol.57 No.6
(s, 3H), 1.62 (m, 4H), 1.53 (t, J = 5.6 Hz, 2H). 13C NMR
(125 MHz, DMSO-d6):  165.4, 162.5, 161.5, 159.8, 156.3,
147.9, 140.7, 138.7, 133.5, 132.4, 131.0, 128.8, 127.9,
126.8, 125.4, 122.4, 116.3, 83.9, 48.4, 25.2, 25.1, 23.7, 18.1.
HRMS (EI): C27H28ClN7OS: calculated [M + H]+ 534.1837;
found: 534.1837. Purity: 95.9% (tR = 8.45 min).
3H), 2.24 (s, 3H). 13C NMR (125 MHz, DMSO-d6):  165.7,
163.2, 162.4, 161.3, 160.6, 159.9, 156.4, 142.3, 142.2,
140.9, 138.8, 133.5, 132.4, 130.2, 130.1, 129.0, 128.2,
127.0, 125.8, 115.2, 108.1, 107.9, 106.2, 105.9, 86.3, 25.5,
18.3. HRMS (EI): C22H18ClFN6OS: calculated [M + H]+
469.1008; found: 469.1009. Purity: 100% (tR = 6.11 min).
2-((6-((4-Bromo-2-fluorophenyl)amino)-2-methylpyrimidin-
4-yl)amino)-N-(2-chloro-6-methylphenyl)thiazole-5-carbox
amide (7f)
N-(2-Chloro-6-methylphenyl)-2-((2-methyl-6-((4-(pyrrolidin-
1-yl)phenyl)amino)pyrimidin-4-yl)amino)thiazole-5-carbox
amide (7j)
White solid, yield: 59%. 1H NMR (400 MHz, DMSO-d6): 
11.64 (s, 1H), 9.90 (s, 1H), 9.28 (s, 1H), 8.23 (s, 1H), 7.86 (t,
J = 8.8 Hz, 1H), 7.58 (d, J = 1.6 Hz, 1H), 7.307.23 (br,
4H), 6.29 (s, 1H), 2.44 (s, 3H), 2.24 (s, 3H). 13C NMR (125
MHz, DMSO-d6):  165.6, 162.4, 160.9, 159.9, 156.6, 155.4,
153.4, 140.9, 138.8, 133.5, 132.4, 129.0, 128.2, 127.4 (d, J
= 3.4 Hz, 1C), 127.0 (t, J = 5.1 Hz, 1C), 126.3, 125.8, 119.0,
118.9, 114.9, 114.9, 85.9, 25.4, 18.3. HRMS (EI):
C22H17BrClFN6OS: calculated [M + H]+ 547.0113; found:
547.0114. Purity: 95.8% (tR = 6.11 min).
White solid, yield: 49%. 1H NMR (400 MHz, DMSO-d6): 
11.37 (s, 1H), 9.85 (s, 1H), 8.91 (s, 1H), 8.20 (s, 1H), 7.39 (t,
J = 6.0 Hz, 1H), 7.307.20 (br, 4H), 6.54 (d, J = 8.8 Hz,
2H), 6.06 (s, 1H), 3.21 (t, J = 6.0 Hz, 4H), 2.41 (s, 3H), 2.24
(s, 3H), 1.95 (t, J = 6.4 Hz, 4H). 13C NMR (125 MHz,
DMSO-d6):  165.5, 162.6, 162.0, 159.9, 156.3, 144.8,
140.8, 138.8, 133.5, 132.4, 129.0, 128.1, 127.0, 125.5,
123.9, 111.8, 83.3, 47.5, 25.3, 24.9, 18.3. HRMS (EI):
C26H26ClN7OS: calculated [M + H]+ 520.1681; found:
520.1682. Purity: 96.7% (tR = 5.96 min).
N-(2-Chloro-6-methylphenyl)-2-((6-((4-methoxyphenyl)
amino)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxa
mide (7g)
N-(2-Chloro-6-methylphenyl)-2-((2-methyl-6-((4-(4-methyl
piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)thiazole-
5-carboxamide (7k)
1
White solid, yield: 67%. H NMR (400 MHz, DMSO-d6):
White solid, yield: 35%. 1H NMR (400 MHz, DMSO-d6): 
11.47 (s, 1H), 9.88 (s, 1H), 9.11 (s, 1H), 8.22 (s, 1H), 7.40
(d, J = 6.4 Hz, 1H), 7.337.23 (br, 4H), 6.92 (d, J = 8.8 Hz,
2H), 6.16 (s, 1H), 3.08 (d, J = 4.8 Hz, 4H), 2.472.43 (br,
7H), 2.24 (s, 3H), 2.22 (s, 3H). 13C NMR (125 MHz,
DMSO-d6):  165.5, 162.5, 161.5, 159.9, 156.3, 147.2,
140.8, 138.8, 133.5, 132.4, 131.4, 128.9, 128.1, 126.9,
125.6, 122.4, 115.9, 84.0, 54.6, 48.6, 45.7, 25.3, 18.3.
HRMS (EI): C27H29ClN8OS: calculated [M+H]+ 549.1946;
found: 549.1941. Purity: 95.4% (tR = 4.33 min).
δ 11.49 (s, 1H), 9.89 (s, 1H), 9.18 (s, 1H), 8.22 (s, 1H),
7.40 (br, 3H), 7.26 (d, J = 11.2 Hz, 2H), 6.91 (d, J = 8.4 Hz,
2H), 6.17 (s, 1H), 3.74 (s, 3H), 2.47 (s, 3H), 2.24 (s, 3H).
13C NMR (125 MHz, DMSO-d6):  165.7, 162.9, 161.4,
159.9, 156.4, 155.2, 140.9, 138.9, 133.5, 132.9, 132.5,
129.1, 128.2, 127.0, 125.7, 122.8, 114.2, 84.2, 55.3, 25.4,
18.3. HRMS (EI): C23H21ClN6O2S: calculated [M + H]+
481.1208; found: 481.1203. Purity: 98.9% (tR = 5.50 min).
N-(2-Chloro-6-methylphenyl)-2-((6-((3,4-dimethoxyphenyl)
amino)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxami
de (7h)
2-Chloro-6-methyl-N-(2-((2-methyl-6-((4-((4-methylpiper-
azin-1-yl)methyl)-3-(trifluoromethyl)phenyl)amino)pyrimi-
din-4-yl)amino)thiazol-5-yl)benzamide (7l)
1
White solid, yield: 69%. H NMR (400 MHz, DMSO-d6):
δ 11.53 (s, 1H), 9.88 (s, 1H), 9.23 (s, 1H), 8.22 (s, 1H),
7.40 (d, J = 7.2 Hz, 1H), 7.27 (m, 3H), 6.98 (d, J = 7.6 Hz,
1H), 6.92 (d, J = 8.4 Hz, 1H), 6.23 (s, 1H), 3.76 (s, 3H),
3.73 (s,3H), 2.45 (s, 3H), 2.24 (s, 3H). 13C NMR (125 MHz,
DMSO-d6):  165.5, 162.5, 161.2, 159.9, 156.3, 148.7,
144.6, 140.8, 138.8, 133.5, 133.4, 132.4, 128.9, 128.1,
126.9, 125.6, 112.6, 112.4, 106.1, 84.4, 55.8, 55.4, 25.4,
18.3. HRMS (EI): C24H23ClN6O3S: calculated [M + H]+
511.1314; found: 511.1317. Purity: 95.2% (tR = 3.85 min).
White solid, yield: 43%. 1H NMR (400 MHz, DMSO-d6): 
11.71 (s, 1H), 9.01 (s, 1H), 9.74 (s, 1H), 8.25 (s, 1H), 8.12(s,
1H), 7.88 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.40
(d, J = 7.2 Hz, 1H), 7.307.24 (m, 2H), 6.33 (s, 1H), 3.53 (s,
2H), 2.39 (br, 11H), 2.25 (s, 3H), 2.19 (s, 3H). 13C NMR
(125 MHz, DMSO-d6):  165.7, 162.4, 160.6, 159.9, 140.8,
140.8, 139.6, 138.8, 133.5, 132.4, 131.3, 129.7, 129.0,
128.2, 127.6, 127.4, 127.0, 125.8, 125.5, 123.3, 122.5,
116.2, 86.2, 57.4, 54.6, 52.5, 45.5, 25.5, 18.3. HRMS (EI):
C29H30ClF3N8OS: calculated [M + H]+ 631.1977; found:
631.1974. Purity: 98.8% (tR = 3.91 min).
N-(2-Chloro-6-methylphenyl)-2-((2-methyl-6-((4-(piperidin-
1-yl)phenyl)amino)pyrimidin-4-yl)amino)thiazole-5-carbox
amide (7i)
White solid, yield: 43%. 1H NMR (400 MHz, DMSO-d6): 
11.45 (s, 1H), 9.87 (s, 1H), 9.08 (s, 1H), 8.21 (s, 1H), 7.40
(d, J = 6.4 Hz, 1H), 7.317.25 (br, 4H), 6.91 (d, J = 8.8 Hz,
2H), 6.16 (s, 1H), 3.07 (t, J = 5.2 Hz, 4H), 2.42 (s, 3H), 2.24
N-(2-Chloro-6-methylphenyl)-2-((2-methyl-6-((4-(morpholino
methyl)phenyl)amino)pyrimidin-4-yl)amino)thiazole-5-
carboxamide (7m)
White solid, yield: 43%. 1H NMR (400 MHz, DMSO-d6): 

Zou DB, et al. Sci China Chem June (2014) Vol.57 No.6
827
11.58 (s, 1H), 9.89 (s, 1H), 9.40 (s, 1H), 8.23 (s, 1H), 7.51
(d, J = 8.4 Hz, 2H), 7.39 (t, J = 6.4 Hz, 1H), 7.287.22 (br,
4H), 6.29 (s, 1H), 3.56 (t, J= 4.0 Hz, 4H), 3.40 (s, 2H), 2.50
(s, 3H), 2.34 (br, 4H), 2.24 (s, 3H). 13C NMR (125 MHz,
DMSO-d6):  165.5, 162.5, 160.9, 156.4, 140.7, 138.8,
138.7, 133.5, 132.4, 131.5, 129.3, 128.9, 128.0, 126.8,
119.9, 85.0, 66.1, 61.9, 53.0, 25.2, 18.1. HRMS (EI):
C27H28ClN7O2S: calculated [M + H]+ 550.1787; found:
550.1784. Purity: 100% (tR = 4.71 min).
N-(2-Chloro-6-methylphenyl)-2-((2-methyl-6-((4-((4-methyl
piperazin-1-yl)methyl)phenyl)amino)pyrimidin-4-yl)amino)
thiazole-5-carboxamide (7q)
White solid, yield: 54%. 1H NMR (400 MHz, DMSO-d6): 
11.59 (s, 1H), 9.690 (s, 1H), 9.40 (s, 1H), 8.24 (s,1H), 7.50
(d, J = 8.0 Hz, 2H), 7.40 (d, J = 7.2 Hz, 1H), 7.307.21 (br,
4H), 6.30 (s, 1H), 3.36 (s, 2H), 2.47 (s, 3H), 2.362.24 (br,
11H), 2.15 (s, 3H). 13C NMR (125 MHz, DMSO-d6): 
166.1, 162.9, 161.3, 160.3, 156.8, 141.3, 139.3, 139.2,
133.9, 132.9, 132.5, 129.7, 129.4, 128.6, 127.42, 126.1,
120.4, 85.8, 62.1, 55.1, 52.8, 46.1, 25.9, 18.7. HRMS (EI):
C28H31ClN8OS: calculated [M + H]+ 563.2103; found:
563.2102. Purity: 99.3% (tR = 4.73 min).
N-(2-Chloro-6-methylphenyl)-2-((6-((3-fluoro-4-((4-methyl
piperazin-1-yl)methyl)phenyl)amino)-2-methylpyrimidin-4-
yl)amino)thiazole-5-carboxamide (7n)
White solid, yield: 40%. 1H NMR (400 MHz, DMSO-d6): 
11.68 (s, 1H), 9.90 (s, 1H), 9.63 (s, 1H), 8.25 (s, 1H), 7.65
(d, J = 12.4 Hz, 1H), 7.40 (d, J = 6.8 Hz, 1H), 7.307.24 (br,
4H), 6.33 (s, 1H), 3.44 (s, 2H), 2.502.36 (br, 11H), 2.25 (s,
3H), 2.14 (s, 3H). 13C NMR (125 MHz, DMSO-d6):  165.7,
162.4, 161.6, 160.5, 159.8, 159.7, 156.4, 140.9, 138.8,
133.5, 132.4, 131.6, 131.6, 128.9, 128.1, 126.9, 125.8,
117.3, 117.2, 114.9, 106.1, 105.9, 86.0, 54.6, 54.29, 52.2,
45.6, 25.4, 18.2. HRMS (EI): C28H30ClFN8OS: calculated
[M + H]+ 581.2009; found: 581.2005. Purity: 100% (tR =
6.11 min).
N-(2-Chloro-6-methylphenyl)-2-((2-methyl-6-((4-(pyrrolidin-
1-ylmethyl)phenyl)amino)pyrimidin-4-yl)amino)thiazole-5-c
arboxamide (7r)
White solid, yield: 37%. 1H NMR (400 MHz, DMSO-d6): 
11.58 (s, 1H), 9.90 (s, 1H), 9.39 (s, 1H), 8.24 (s,1H), 7.49 (d,
J = 8.0 Hz, 2H), 7.40 (d, J = 7.2 Hz, 1H), 7.307.23 (br,
4H), 6.29 (s, 1H), 3.54 (s, 2H), 2.47 (s, 3H), 2.44 (br, 4H),
2.24 (s, 3H), 1.69 (br, 4H). 13C NMR (125 MHz, DMSO-d6):
 165.6, 162.5, 161.0, 159.91, 156.4, 140.8, 138.8, 138.8,
133.5, 132.4, 129.0, 128.1, 127.0, 125.7, 120.0, 85.1, 59.1,
53.4, 25.4, 23.1, 18.3. HRMS (EI): C27H28ClN7OS:
calculated [M + H]+ 534.1837; found: 534.1831. Purity:
98.9% (tR = 3.9 min).
2-((6-((3-Chloro-4-((4-methylpiperazin-1-yl)methyl)phenyl)
amino)-2-methylpyrimidin-4-yl)amino)-N-(2-chloro-6-methyl
phenyl)thiazole-5-carboxamide (7o)
N-(2-Chloro-6-methylphenyl)-2-((2-methyl-6-((4-(piperidin-
1-ylmethyl)phenyl)amino)pyrimidin-4-yl)amino)thiazole-5-
carboxamide (7s)
White solid, yield: 56%. 1H NMR (400 MHz, DMSO-d6): 
11.68 (s, 1H), 9.89 (s, 1H), 9.59 (s, 1H), 8.24 (s, 1H), 7.82(s,
1H), 7.49 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 6.0 Hz, 1H),
7.30-7.23 (br, 3H), 6.30 (s, 1H), 3.49(s, 2H), 2.50-2.25 (br,
14H), 2.15 (s, 3H). 13C NMR (125 MHz, DMSO-d6): 
165.6, 162.4, 160.5, 159.8, 156.4, 140.8, 140.3, 138.8,
133.5, 133.2, 132.4, 131.1, 129.0, 128.5, 128.1, 126.9,
119.6, 118.0, 86.0, 58.2, 54.7, 52.5, 45.7, 25.5, 18.3, 7.2.
HRMS (EI): C27H30ClN7OS: calculated [M + H]+ 597.1713;
found: 591.1714. Purity: 97.2% (tR = 4.65 min).
White solid, yield: 50%. 1H NMR (400 MHz, DMSO-d6): 
11.58 (s, 1H), 9.89 (s, 1H), 9.39 (s, 1H), 8.24 (s, 1H), 7.49
(d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 1H), 7.307.20 (br,
4H), 6.30 (s, 1H), 3.36 (s, 2H), 2.47 (s, 3H), 2.30 (br, 4H),
2.24 (s, 3H), 1.491.46 (br, 4H), 1.37 (d, J = 4.0 Hz, 2H).
13C NMR (125 MHz, DMSO-d6):  166.1, 162.9, 161.4,
160.3, 156.8, 141.3, 139.3, 139.2, 133.9, 132.9, 129.7,
129.4, 128.6, 127.4, 126.1, 120.4, 85.5, 62.7, 54.2, 26.6,
25.9, 24.5, 16.7. HRMS (EI): C28H30ClN7OS: calculated [M
+ H]+ 548.1994; found: 548.1993. Purity: 95.7% (tR = 7.24
min).
N-(2-Chloro-6-methylphenyl)-2-((6-((3-methoxy-4-(pyrrolid
in-1-ylmethyl)phenyl)amino)-2-methylpyrimidin-4-yl)amino)
thiazole-5-carboxamide (7p)
White solid, yield: 37%. 1H NMR (400 MHz, DMSO-d6): 
11.65(s, 1H), 9.97 (s, 1H), 9.50 (s, 1H), 8.29 (s, 1H), 7.41 (t,
J = 5.6 Hz, 2H), 7.337.23 (br, 3H), 7.08 (d, J = 8.0 Hz,
1H), 6.36 (s, 1H), 3.80 (s, 3H), 3.68 (s, 2H), 2.61 (br, 4H),
2.48 (s, 3H), 2.24 (s, 3H), 1.73 (br, 4H). 13C NMR (125
MHz, DMSO-d6):  165.6, 162.5, 160.8, 159.9, 157.3, 156.4,
140.9, 138.8, 133.5, 132.4, 130.6, 129.0, 128.1, 127.0,
125.7, 111.4, 103.0, 84.3, 62.6, 55.2, 53.2, 52.4, 52.0, 25.5,
22.9, 18.3. HRMS (EI): C28H30ClN7O2S: calculated [M +
H]+ 520.1681; found: 520.1682. Purity: 98.75% (tR = 4.64
min).
2-((6-((4-((4-Benzylpiperazin-1-yl)methyl)phenyl)amino)-2-
methylpyrimidin-4-yl)amino)-N-(2-chloro-6-methylphenyl)
thiazole-5-carboxamide (7t)
White solid, yield: 41%. 1H NMR (400 MHz, DMSO-d6): 
11.58 (s, 1H), 9.86 (s, 1H), 9.39 (s, 1H), 8.23 (s, 1H), 7.50
(d, J = 8.0 Hz, 2H), 7.39 (d, J = 4.0 Hz, 1H), 7.317.20 (br,
9H), 6.29 (s, 1H), 3.45 (s, 2H), 3.40 (s, 2H), 2.50 (s, 3H),
2.452.25 (br, 8H), 2.24 (s, 3H). 13C NMR (125 MHz,
DMSO-d6):  165.6, 162.5, 160.9, 159.9, 156.4, 146.5,
140.9, 138.8, 138.2, 132.4, 132.0, 129.3, 129.0, 128.7,

828
Zou DB, et al. Sci China Chem June (2014) Vol.57 No.6
128.5, 128.1, 127.0, 126.8, 125.7, 119.9, 113.8, 85.1, 62.1,
61.6, 52.6, 52.5, 25.4, 18.3. HRMS (EI): C34H35ClN8OS:
calculated [M + H]+ 639.2416; found: 639.2412. Purity:
100% (tR = 9.68 min).
(Graph- Pad Software Inc., USA).
2.5 Molecular imaging
NU/NU mice received subcutaneous injections with 5 × 106
K562 myeloid leukemia cells in Matrigel in the right thigh
and was used for imaging study when the tumor reached a
diameter of 10–15 mm. Positron emission tomography (PET)
of tumor-bearing mice was performed on a Siemens Inveon
PET/CT system. Sixty minutes after an intraperitoneal (ip)
injection of 18F-FDG (3.7 MBq), small-animal PET/CT was
performed (10 min) at a single-bed position. Therapeutic
doses of compound 1 (50 mg/kg, 1 mg in 100 μL of 80%
PBS 20% Tween 20) were administered ip following initial
PET-CT scan at 0, 3 h, respectively. After 6 h from initial
therapeutic dose, 18F-FDG was administrated by ip and
PET-CT scan was performed at 60 min postinjection. For
each microPET scan, regions of interest (ROIs) were drawn
over the tumor on the whole-body coronal images. Assum-
ing a tissue density of 1 g/mL, the units of ROIs were then
converted from Bq/mL to Bq/g. After decay-correction, the
voxel intensity of ROI was divided by the administered ra-
dioactivity to obtain an imaging ROI-derived percentage
administered radioactivity per gram of tissue (%ID/g).
N-(2-Chloro-6-methylphenyl)-2-((6-((4-((diethylamino)methyl)
phenyl)amino)-2-methylpyrimidin-4-yl)amino)thiazole-5-
carboxamide (7u)
White solid, yield: 37%. 1H NMR (400 MHz, DMSO-d6): 
11.58 (s, 1H), 9.89 (s, 1H), 9.38 (s, 1H), 8.24 (s,1H), 7.49 (d,
J = 8.4 Hz, 2H), 7.41 (d, J = 6.0 Hz, 1H), 7.307.23 (br,
4H), 6.30 (s, 1H), 3,49(s, 2H), 2.50-2.44 (br, 7H), 2.25 (s,
3H), 0.98 (t, J = 6.8 Hz, 6H). 13C NMR (125 MHz,
DMSO-d6):  165.6, 162.5, 160.9, 159.9, 156.3, 140.8,
138.8, 138.6, 133.5, 132.4, 128.9, 128.1, 126.9, 125.7,
112,0, 85.0, 56.4, 45.9, 25.4, 18.3, 11.6. HRMS (EI):
C27H30ClN7OS: calculated [M + H]+ 536.1994; found:
536.1999. Purity: 99.0% (tR = 3.86 min).
2.3 In vitro ABL activity assay
Inhibition activities of inhibitors against Bcr-Abl and its
mutants were performed in 384-well plates using the
FRET-based Z’-Lyte assay system according to the manu-
facturer’s instructions (Invitrogen, Carlsbad, USA). Briefly,
10 L per well reactions contained ATP concentration at 10
μM (for Bcr-Abl wild-type, and mutants Y253F, Q252H,
M351T and H396P) or 5 μM (for mutants E255K, G250E,
T315I), 2 M Tyr2 Peptide substrate in 50 mM HEPES (PH
7.5), 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.0247
µg/mL Bcr-Abl and inhibitors as appropriate. The reaction
was performed at room temperature for 2.0 hours, and then
5 L development reagent was added for further 2 hours
room temperature incubation followed by the addition of 5
L of stop solution. Fluorescence signal ratio of 445 nm
(Coumarin)/520 nm (fluorescin) was examined on EnVision
Multilabel Reader (Perkin Elmer, Inc., USA). The data were
analyzed using Graphpad Prism5 (Graphpad Software, Inc.,
USA). The data was the mean value of 3 experiments.
2.6 Molecular modeling
Compound 7l was docked into the active site of BCR-ABL
kinase domain using the program of MOE-Dock. The crys-
tal structure employed is 2QGQ (ref: Cancer Research,
2006, 66, 5790), in which Dasatinib binds to the activated
ABL kinase domain. Very similar to the binding mode of
Dasatinib, the 2-chloro-6-methyl phenyl ring of this com-
pound is orthogonal to the thiazole carboxamide group and
inserts into a hydrophobic pocket comprised of Thr 315, Ile
313, Met 290, Val 299, Phe 382 and Ala 380. A hydrogen
of this phenyl ring may has a heteroaromatic CH···OC
interaction with Glu 286, the only polar residue in this
pocket. Three notable hydrogen bonds can be found be-
tween the complexs: two of them are formed in the hinge
region of the ATP-binding site by interacting with the am-
ide nitrogen and carbonyl oxygen atoms of Met 318 respec-
tively; another hydrogen bond is formed between amide
nitrogen of compound 7l and the side chain hydroxyl oxy-
gen of Thr 315. The aminothiazole moiety of 7l is in the
channel normally bound by the adenine group of ATP. The
thiazole ring is sandwiched by Ala 269 and Leu 370 and the
substituted pyrimidine is sandwiched by Leu 248 and Gly
321, indicating hydrophobic interactions exist between
these two rings and the protein. Two heteroaromatic CH···
O=C interactions with the hinge region backbone can be
observed: the c4 thiazole carbon and the C5 pyrimidine
carbon interact with the carbonyl oxygen atoms of Glu 316
and Met 318 respectively. Different from Dasatinib, a weak
2.4 Cell proliferation and growth inhibition assay
The leukemia cell lines K562 were maintained at 37 °C in a
5% CO2 incubator in medium RPMI 1640 (Gibco, Invitro-
gen) containing 10% fetal bovine serum (Gibco, Invitrogen).
Cell proliferation assay was determined by the CCK8
(DOjinDo, Japan) assay. Cells were seeded at a density of
800–1000 cells/well in 384 well plates and treated with
various concentration of compounds or solvent control. Af-
ter 72 h incubation, CCk8 reagent was added, and absorb-
ance was measured at 450 nm using Envision 2104 multi-
label Reader (Perkin Elmer, Inc., USA). All experiments
were repeated at least three times. Dose response curves
were plotted to determine the IC50 values using Prism 5.0

Zou DB, et al. Sci China Chem June (2014) Vol.57 No.6
829
hydrogen bond between the nitrogen atom in the aniline
group of compound 7l and the carbonyl group of Thr 319
may be found since the distance between these two atoms is
3.60 Å. The phenyl ring and the piperazine group are ex-
posed to the solvent, however, a strong hydrogen bond can
be found between the positively charged piperazine ring and
the negatively charged residue of Glu 329.
ide nitrogen and carbonyl oxygen atoms of Met318 respec-
tively; another hydrogen bond is formed between amide
nitrogen of compound 7q and the side chain hydroxyl oxy-
gen of Thr315. The thiazole ring is sandwiched by Ala269
and Leu370 and the substituted pyrimidine is sandwiched
by Leu248 and Gly321, indicating hydrophobic interactions
exist between these two rings and the protein. Two het-
eroaromatic CH···OC interactions with the hinge region
backbone are observed: the C4 thiazole carbon and the C5
pyrimidine carbon interact with the carbonyl oxygen atoms
of Glu316 and Met318, respectively. Different from Da-
satinib, a weak hydrogen bond between the nitrogen atom in
the aniline group of 7q and the carbonyl group of Thr319
may be formed since the distance between these two atoms
is 3.60 Å. The phenyl ring and the piperazine group are ex-
posed to the solvent, with a strong hydrogen bond formed
between the positively charged piperazine ring and the neg-
atively charged residue of Glu329 (Figure 3). When small
substituents, such as fluoro, chloro or trifluoromethyl were
introduced in the R2 position of 7q, the kinase inhibitory
activities decreased slightly. For instance, compound 7l
3 Results and discussion
The direct binding affinities of 7a7u with the native Abl
kinase and the Abl mutants were tested by using dasatinib
as the positive control. The results are summarized in Table
1. Initially, we replaced the 2-(piperazin-1-yl)ethanol of
dasatinib with 4-chlorobenzene ring. The resulting com-
pound 7a inhibited the Bcr-Abl WT with IC50 value of 25
nM, and accordingly showed potent inhibition for the pro-
liferation of Bcr-Abl WT -positive K562 CML cells with
IC50 value of 77 nM. However, this compound poorly inhib-
ited Bcr-Abl T315I mutant (IC50 > 10 μM). When fluoro,
bromo and chloro groups were introduced at R1 or R2 posi-
tion of compound 7a, respectively, the inhibitory activities
of 7b7f against Bcr-Abl WT changed very little compared
with compound 7a. To our delight, compound 7h with
methoxy group at both R1 and R2 positions displayed IC50
values of 1.2, 2.1, 2.7, and 2.1 nM against AblWT,
AblE255K, AblG250E, and AblQ252, respectively, which
were about 2093 times more potent than the original ana-
log 7a. The results suggested that R1 and R2 positions
could be used for further optimization. Subsequently, a va-
riety of functional groups such as piperidine, pyrrolidine, or
1-methylpiperazine groups were introduced at R1 position
of compound 7a, respectively. All of them (7i7k) showed
significantly better inhibitory activities against full range of
Bcr-Abl kinase domain mutations as well as Bcr-Abl WT.
Among them, compound 7k with 1-methylpiperazine at
para-position was about 192750 times more potent than
compound 7a, with IC50 values of 0.13, 1380, 0.21, 0.26,
and 0.26 nM against AblWT, AblT315I, AblE255K,
AblG250E, and AblQ252H, respectively. These promising
results encouraged us to further explore the potential impact
of R1 and R2 on Abl kinase inhibition. Introduction of
methylene group between benzene and 1-methylpiperazine
group did not provide any enhancement (7q vs. 7k). Molec-
ular modeling indicated that 2-chloro-6-methyl phenyl ring
of compound 7q is orthogonal to the thiazole carboxamide
group and stays in the hydrophobic pocket comprised of
Thr315, Ile313, Met290, Val299, Phe382 and Ala380. One
hydrogen atom of this phenyl ring may have a hetero aro-
matic CH···OC interaction with Glu286, the only polar
residue in this pocket. Three notable hydrogen bonds exist
within the complex: two of them are formed in the hinge
region of the ATP-binding site by interacting with the am-
with trifluromethyl at R2 position displayed IC50
values of
4.4, 5580, 4.5, 5.2, and 7.4 nM against AblWT, AblT315I,
AblE255K, AblG250E, and AblQ252H, respectively. When
1-methylpiperazine group of compound 7q was replaced
with a variety of functional groups such as pyrrolidine, pi-
Figure 3 (a) Binding mode of compound 7q to the BCR-ABL kinase do-
main. Hydrogen bonds are dashed by magenta lines. (b) Part of compound
7q inserts into the active site of BCR-ABL; part of compound 7q exposes
to the solvent, and the methylpiperazine ring of 7q has a hydrogen bond
interaction with Glu329, a residue in the rim of the pocket.

830
Zou DB, et al. Sci China Chem June (2014) Vol.57 No.6
Table 1 Inhibitory activities of 7a7u against Abl WT and different mutants a)
Kinase
E255K
Cell line
K562
77
Compound
R1
R2
ABL
25
T315I b)
>10
G250E
184
147
39
Q252H
195
76
7a
7b
7c
7d
7e
7f
Cl
H
H
Cl
152
204
58
50
>10
91
F
Cl
16
>10
32
121
28
F
H
12
>10
46
42
42
H
F
14
>10
25
53
16
39
Br
F
22
>10
29
43
38
44
7g
7h
OCH3
OCH3
H
3.0
1.2
>10
12
25
15
11
OCH3
>10
2.1
2.7
2.1
8.6
7i
7j
7k
7l
H
H
1.7
0.85
0.13
4.4
0.89
0.88
1.38
5.6
3.9
2.6
6.4
3.3
3.0
2.0
30
31
H
0.21
4.5
0.26
5.2
0.26
7.4
1.1
6.8
CF3
7m
7n
7o
H
F
1.2
1.4
1.1
>10
4.7
5.8
0.95
0.76
1.7
1.8
1.0
3.3
1.4
1.2
2.9
3.3
2.4
15
Cl
7p
7q
7r
OCH3
H
0.14
0.20
0.68
0.88
1.0
0.18
0.30
0.56
0.25
0.038
0.83
0.23
0.30
0.81
3.6
1.7
1.5
H
1.3
7s
7t
H
H
H
0.14
1.5
0.65
>10
0.25
1.7
0.19
3.5
0.29
0.93
1.9
14
7u
0.13
0.11
0.65
>10
0.17
0.08
0.19
>1
0.24
0.20
1.1
Dasatinib
0.80
a) IC50 values (nM) are means of three experiments, and standard error of the IC50 was generally less than 10%; b) IC50, μM.
peridine, or diethylamine groups, respectively, their inhibi-
tory activities against Bcr-Abl WT and Bcr-Abl mutants
increased (7r, 7s and 7u). Among them, compound 7u dis-
played IC50 values of 0.65 µM and 1.1 nM against AblT315I
and K562 cells, respectively, which was about 192968
times more potent than the original compound 7a. Although
introduction of appropriate more hydrophobic groups in the
R1 position of 7a is a useful strategy to improve anticancer
activities of 7a, such improvement should be controlled in a
proper range. Under some circumstances, this enhancement
can be overweighed by increasing exposed hydrophobic
surface area. For instance, when 1-methylpiperazine group
of compound 7q was replaced by a larger substituent
1-benzylpiperazine, the resulting compound 7t completely
lost its inhibitory activity against AblT315I, most likely due
to the steric clash between benzylpiperazine and the pocket
induced by the DFG-out conformation of T315I mutant.
As one of the most potent compounds shown in Table 1,
7l was selected for further investigation in human chronic
myelogenous leukemia K562 xenograft model using molec-
ular imaging method. Positron emission tomography (PET)
with 18F-fluorodeoxyglucose (FDG) is capable to identify
tumor glucose metabolism and proliferation, which makes it
a powerful tool in monitoring therapeutical response in
clinic [20, 21]. We performed in vivo 18F-FDG microP-
ET/CT imaging (3.7 Mbq, 60 min post injection) before and
after treatment with compound 7l (2 × 50 mg/kg, 1 mg in
100 μL of 80% PBS 20% Tween 20, intraperitoneal injec-
tion) (Figure 4(a)). As expected, the region of interest (ROI)
analysis on microPET showed the tumor uptake of 18F-FDG

Zou DB, et al. Sci China Chem June (2014) Vol.57 No.6
831
1
a) Heisterkamp N, Stam K, Groffen J, de Klein, A, Grosveld G.
Structural organization of the bcr gene and its role in the Ph8 trans-
location. Nature, 1985, 315: 758–761; b) Goldman JM, Melo JV.
Chronic myeloid leukemia-advances in biology and new approaches
to treatment. New Engl J Med, 2003, 349: 1451–1464; c) Melo JV.
The diversity of BCR-ABL fusion proteins and their relationship to
leukemia phenotype. Blood, 1996, 88: 2375–2384
2
a) Cortez D, Kadlec L, Pendergast AM. Structural and signaling re-
quirements for BCR-ABL-mediated transformation and inhibition of
apoptosis. Mol Cell Biol, 1995, 15: 5531–5541; b) Goga A,
McLaughlin J, Afar DEH, Saffran DC, Witte ON. Alternative signals
to RAS for hematopoietic transformation by the BCR-ABL oncogene.
Cell, 1995, 82: 981–988; c) Carlesso LN, Frank DA, Griffin JD.
Tyrosyl phosphorylation and DNA binding activity of signal trans-
ducers and activators of transcription (STAT) proteins in hematopoi-
etic cell lines transformed by Bcr/Abl. J Exp Med, 1996, 183:
811–820; d) Shuai K, Halpner J, ten Hoeve J, Rao X, Sawyers CL.
Constitutive activation of STAT5 by BCR-ABL oncogene in chronic
myelogenous leukemia. Oncogene, 1996, 13: 247–254; e) Sattler M,
Salgia R, Okuda K, Uemura N, Durstin M, Pisick E, Xu G, Li JL,
Prasad KV, Griffin JD. The protooncogene product p120CBL and the
adaptor proteins CRKL and c-CRK link c-ABL, p190BCR/ ABL and
p210BCR/ABL to the phosphatidylinositol-38 kinase pathway. On-
cogene, 1996, 12: 839–846
3
4
Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S,
Zimmermann J, Lydon NB. Effects of a selec-tive inhibitor of the
Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nature
Med, 1996, 2: 561–566
Druker BJ, Guilhot F, O’Brien SG, Gathmann I, Kantarjian H, Gat-
termann N, Deininger MW, Silver RT, Goldman JM, Stone RM,
Cervantes F, Hochhaus A, Powell BL, Gabrilove JL, Rousselot P,
Reiffers J, Cornelissen JJ, Hughes T, Agis H, Fischer T, Verhoef G,
Shepherd J, Saglio G, Gratwohl A, Nielsen JL, Radich JP, Simonsson
B, Taylor K, Baccarani M, So C, Letvak L, Larson RA. Five-year
follow-up of patients receiving imatinib for chronic myeloid leuke-
mia. N Engl J Med, 2006, 355: 2408–2417
Figure 4 (a) PET-CT scan of K562 tumor xenograft before and after
treatment with the compound 7l (2 × 50 mg/kg, 1 mg in 100 μL of 80%
PBS 20% Tween 20, intraperitoneal injection) as measured with 18F-FDG.
(b) Decay-Corrected region-of interest (ROI) analysis on microPET images
with the tumor uptake of 18F-FDG before and after treatment with 7l. The
radioactivity uptakes in the tumor and normal tissues are expressed as a
percentage of the injected radioactive dose per gram of tissue (%ID/g). P =
0.0019.
5
6
a) O’Hare T, Eide CA, Deininger MW. BCR-ABL kinase domain
mutations, drug resistance, and the road to a cure for chronic- mye-
loid leukemia. Blood, 2007, 110: 2242–2249; b) Melo JV, Chuah C.
Resistance to imatinib mesylate in chronic myeloid leukaemia. Can-
cer Lett, 2007, 249: 121–132
a) Weisberg E, Manley PW, Cowan-Jacob SW, Hochhaus A, Griffin
JD. Second generation inhibitors of BCR-ABL for the treatment of
imatinib-resistant chronic myeloid leukaemia. Nat Rev Cancer, 2007,
7: 345–356; b) Quintas-Cardama A, Kantarjian H, Cortes J. Flying
under the radar: The new wave of BCR-ABL inhibitors. Nat Rev
Drug Discov, 2007, 6: 834–848
decreased from 1.71 ± 0.03 to 1.03 ± 0.08 %ID/g (40% de-
creasing, Figure 4(b)) after intraperitoneal injection two
single doses of compound 7l treatment. These results indi-
cated that compound 7l has high potential to effectively
treat chronic myelogenous leukemia. Further therapy evalu-
ation is ongoing.
7
8
9
Weisberg E, Manley PW, Breitenstein W, Brueggen J, Cowan-Jacob
SW, Ray A, Huntly B, Fabbro D, Fendrich G, Hall-Meyers E, Kung
AL, Mestan J, Daley GQ, Callahan L, Catley L, Cavazza C, Mo-
hammed A, Neuberg D, Wright RD, Gilliland DG, Griffin JD. Char-
acterization of AMN107, a selective inhibitor of native andmutant
Bcr-Abl. Cancer Cell, 2005, 7: 129–141
Kimura S, Naito H, Segawa H, Kuroda J, Yuasa T, Sato K, Yokota A,
Kamitsuji Y, Kawata E, Ashihara E, Nakaya Y, Naruoka H, Waka-
yama T, Nasu K, Asaki T, Niwa T, Hirabayashi K, Maekawa T.
NS-187, a potent and selective dual Bcr-Abl/Lyn tyrosine kinase in-
hibitor, is a novel agent for imatinib resistant leukemia. Blood, 2005,
106: 3948–3954
a) Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers CL. Over-
riding imatinib resistance with a novel Abl kinase inhibitor. Science,
2004, 305: 399–401; b) Lombardo LJ, Lee FY, Chen P, Norris D,
Barrish JC, Behnia K, Castaneda S, Cornelius LAM, Das J, Doweyko
AM, Fairchild C, Hunt JT, Inigo I, Johnston K, Kan D, Pang S, Pe-
terson R, Pitt S, Schieven GL, Schmidt RJ, Tokarski J, Wen ML,
Wityak J, Borzilleri RM. Discovery of N-(2-chloro-6-methyl-
phenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-
4-ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl
kinase inhibitor with potent antitumor activity in preclinical assays. J
4 Conclusions
In summary, a series of new 2-methylpyrimidine derivatives
as BCR-ABL inhibitors have been designed, synthesized
and evaluated in this work. Among them, compounds 7l and
7u strongly inhibited full range of Bcr-Abl mutants includ-
ing the “gatekeeper” T315I mutant as well as Bcr-Abl WT.
Both of them also displayed anti-proliferation of K562 cells
with IC50 values of 6.8 and 1.1 nM, respectively. The in vivo
study with 18F-FDG-PET imaging demonstrated compound
7l reduced FDG uptake in K562 xenograft tumor. These
compounds may serve as new promising leads for further
investigation of BCR-ABL inhibitors associated with CML.
This work was supported by the National Natural Science Foundation of
China (21172220), and the National Basic Research Program of China
(2009CB940900).

832
Zou DB, et al. Sci China Chem June (2014) Vol.57 No.6
Med Chem, 2004, 47: 6658–6661
(BCR-ABL) kinase including the T315I gatekeeper mutant. J Med
Chem, 2010, 53: 4701–4719
10 a) Boschelli DH, Ye F, Wang YD, Dutia M, Johnson SL, Wu B, Mil-
ler K, Powell DW, Yaczko D, Young M, Tischler M, Arndt K, Dis-
cafani C, Etienne C, Gibbons J, Grod J, Lucas J, Weber JM, Boschel-
li F. Optimization of 4-phenylamino-3-quinolinecarbonitriles as po-
tent inhibitors of Src kinase activity. J Med Chem, 2001, 44:
3965–3977; b) Puttini M, Coluccia AM, Boschelli F, Cleris L, Mar-
chesi E, Donella-Deana A, Ahmed S, Redaelli S, Piazza R, Magistro-
ni V, Andreoni F, Scapozza L, Formelli F, Gambacorti-Passerini C.
In vitro and in vivo activity of SKI-606, a novel Src-ABL inhibitor,
against imatinib-resistant BCRABL neoplastic cells. Cancer Res,
2006, 66: 11314–11322
11 a) O’Hare T, Walters DK, Stoffregen EP, Jia T, Manley PW, Mestan
J, Cowan-Jacob SW, Lee FY, Heinrich MC, Deininger MW, Druker
BJ. In vitro activity of BCR-ABL inhibitors AMN107 and BMS-
354825 against clinically relevant imatinib-resistant ABL kinase do-
main mutants. Cancer Res, 2005, 65: 4500–4505; b) Deguchi Y, Ki-
mura S, Ashihara E, Niwa T, Hodohara K, Fujiyama Y, Maekawa T.
Comparison of imatinib, dasatinib, nilotinib and INNO-406 in
imatinib-resistant cell lines. Leuk Res, 2008, 32: 980–983; c) Redaelli
S, Piazza R, Rostagno R, MagistroniV, Perini P, Marega M,
Gambacorti-Passerini C, Boschelli F. Activity of bosutinib, dasatinib,
and nilotinib against 18 imatinib resistant BCR/ABL mutants. J Clin
Oncol, 2009, 27: 469–471
12 Choi HG, Ren P, Adrian F, Sun F, Lee HS, Wang X, Ding Q, Zhang
G, Xie Y, Zhang J, Liu Y, Tuntland T, Warmuth M, Manley PW,
Mestan J, Gray NS, Sim T. A type-II kinase inhibitor capable of in-
hibiting the T315I “gatekeeper” mutant of Bcr-Abl. J Med Chem,
2010, 53: 5439–5448
13 Huang WS, Metcalf CA, Sundaramoorthi R, Wang Y, Zou D, Thom-
as RM, Zhu X, Cai L, Wen D, Liu S, Romero J, Qi J, Chen I, Banda
G, Lentini SP, Das S, Xu Q, Keats J, Wang F, Wardwell S, Ning Y,
Snodgrass JT, Broudy MI, Russian K, Zhou T, Commodore L, Nara-
simhan NI, Mohemmad QK, Iuliucci J, Rivera VM, Dalgarno DC,
Sawyer TK, Clackson T, Shakespeare WC. Discovery of 3-[2-(imid-
azo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpiperazin-
1-yl)-methyl]-3-(trifluoromethyl)phenyl}benzamide (AP24534), a po-
tent, orally active pan-inhibitor of breakpoint cluster regionabelson
14 O'Hare T, Shakespeare WC, Zhu XT, Eide CA, Rivera VM, Wang F,
Adrian LT, Zhou TJ, Huang WS, Xu QH, Metcalf C, Tyner JW, Lo-
riaux MM, Corbin AS, Wardwell S, Ning YY, Keats JA, Wang YH,
Sundaramoorthi R, Thomas M, Zhou D, Snodgrass J, Commodore L,
Sawyer TK, Dalgarno DC, Deininger MWN, Druker BJ, Clackson T.
AP24534, a Pan-BCR-ABL inhibitor for chronic myeloid leukemia,
potently inhibits the T315I mutant and overcomes mutation-based re-
sistance. Cancer Cell, 2009, 16: 401–412
15 Eide CA, Adrian LT, Tyner JW, Mac Partlin M, Anderson DJ, Wise
SC, Smith BD, Petillo PA, Flynn DL, Deininger MW, O'Hare T,
Druker BJ. The ABL switch control inhibitor DCC-2036 is active
against the chronic myeloid leukemia mutant BCR-ABLT315I and
exhibits a narrow resistance profile. Cancer Res, 2011, 71: 3189–
3195
16 Deng X, Okram B, Ding Q, Zhang J, Choi Y, Adrian FJ, Wojciech-
owski A, Zhang G, Che J, Bursulaya B, Cowan-Jacob SW, Rummel
G, Sim T, Gray NS. Expanding the diversity of allosteric Bcr-Abl in-
hibitors. J Med Chem, 2010, 53: 6934–6946
17 Ren X, Pan X, Zhang Z, Wang D, Lu X, Li Y, Wen D, Long H, Luo J,
Feng Y, Zhuang X, Zhang F, Liu J, Leng F, Lang X, Bai Y, She M,
Tu Z, Pan J, Ding K. Identification of GZD824 as an orally bioavail-
able inhibitor that targets phosphorylated and nonphosphorylated
breakpoint cluster region-abelson (Bcr-Abl) kinase and overcomes
clinically acquired mutation-induced resistance against imatinib. J
Med Chem, 2013, 56: 879–894
18 O’Hare T, Walters DK, Deininger MW, Druker BJ. AMN107: tight-
ening the grip of imatinib. Cancer Cell, 2005, 7: 117–119
19 Tokarski JS, Newitt JA, Chang CYJ, Cheng JD, Wittekind M, Kiefer
SE, Kish K, Lee FYF, Borzillerri R, Lombardo LJ, Xie D, Zhang Y,
Klei HE. The structure of dasatinib (BMS-354825) bound to activat-
ed ABL kinase domain elucidates its inhibitory activity against
imatinib-resistant ABL mutants. Cancer Research, 2006, 66: 5790–
5797
20 Wolfgang A, Weber WA. Use of PET for monitoring cancer therapy
and for predicting outcome. J Nucl Med, 2005, 46: 983–995