N-[2-methyl-5-(triazol-1-yl)phenyl]pyrimidin-2-amine as a Scaffold for the synthesis of inhibitors of Bcr-Abl

Article abstract of DOI:10.1002/cmdc.201100304

N-[2-Methyl-5-(triazol-1-yl)phenyl]pyrimidin-2-amine derivatives were synthesized and evaluated invitro for their potential use as inhibitors of Bcr-Abl. The design is based on the bioisosterism between the 1,2,3-triazole ring and the amide group. The synthesis involves a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) as the key step, with the exclusive production of anti-(1,4)-triazole derivatives. One of the compounds obtained shows general activity similar to that of imatinib; in particular, it was observed to be more effective in decreasing the fundamental function of cdc25A phosphatases in the K-562 cell line. Willing and Abl inhibitors! N-[2-Methyl-5-(triazol-1-yl)phenyl]pyrimidin-2-amine derivatives were synthesized by a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) as a key step, with anti-(1,4)-triazole derivatives as the exclusive products. One of these compounds shows general activity similar to that of imatinib, and in particular, it is more effective in decreasing the fundamental function of cdc25A phosphatases in the K-562 cell line.

Full text of DOI:10.1002/cmdc.201100304

DOI: 10.1002/cmdc.201100304
N-[2-Methyl-5-(triazol-1-yl)phenyl]pyrimidin-2-amine as a
Scaffold for the Synthesis of Inhibitors of Bcr-Abl
Federica Arioli,[a] Stella Borrelli,[a] Francesco Colombo,[a] Federico Falchi,[b] Irene Filippi,[c]
Emmanuele Crespan,[d] Antonella Naldini,[c] Giusy Scalia,[c] Alessandra Silvani,[a]
Giovanni Maga,[d] Fabio Carraro,[c] Maurizio Botta,[b] and Daniele Passarella*[a]
N-[2-Methyl-5-(triazol-1-yl)phenyl]pyrimidin-2-amine derivatives
were synthesized and evaluated in vitro for their potential use
as inhibitors of Bcr-Abl. The design is based on the bioisoster-
ism between the 1,2,3-triazole ring and the amide group. The
synthesis involves a copper(I)-catalyzed azide–alkyne cycloaddi-
tion (CuAAC) as the key step, with the exclusive production of
anti-(1,4)-triazole derivatives. One of the compounds obtained
shows general activity similar to that of imatinib; in particular,
it was observed to be more effective in decreasing the funda-
mental function of cdc25A phosphatases in the K-562 cell line.
Introduction
Chronic myelogenous leukemia
(CML) is a disease characterized
by the presence of the Philadel-
phia chromosome (discovered in
1960 by Nowell and Hunger-
ford)[1] that results from recipro-
cal translocation between chro-
mosomes 9 and 22.[2] This trans-
location fuses the breakpoint
cluster region (Bcr) and the Abel-
son kinase (Abl) genes, forming
the Bcr-Abl oncogene,[3] which
encodes the constitutively active
cytoplasmic tyrosine kinase (TK)
Bcr-Abl, present in >90% of
CML cases.[4] As a consequence
of this fusion, proliferation and
viability of myeloid lineage cells
is enhanced, and the disease
Figure 1. Some available inhibitors of Abl and our Abl inhibitor, FA030 (10).
progresses through its three phases—chronic proliferative, ac-
celerated, and blast crisis phase—becoming more resistant to
treatment in each successive phase. The findings that Bcr-Abl
is the cause of the leukemic phenotype and that the tyrosine
kinase activity of Abl is fundamental for Bcr-Abl-mediated
transformation have made this kinase an important target for
the development of specific therapies. In the recent past, ad-
vances in the selective inhibition of Bcr-Abl kinase activity led
to the development of several active compounds (Figure 1),[5]
and in particular, imatinib mesylate (Gleevec) is the one that
currently represents the first-line treatment of CML. However,
resistance to imatinib has been conferred by Bcr-Abl gene am-
plification leading to overexpression of the Bcr-Abl protein,
point mutations in the Bcr-Abl kinase domain that interfere
with imatinib binding, as well as point mutations outside the
kinase domain that allosterically inhibit imatinib binding to
Bcr-Abl. As a consequence, there is growing interest in the de-
[a] Dr. F. Arioli, Dr. S. Borrelli, Dr. F. Colombo, Dr. A. Silvani,
Prof. Dr. D. Passarella
Dipartimento di Chimica Organica e Industriale
Universitꢀ degli Studi di Milan
Via G. Venezian 21, 20133 Milano (Italy)
E-mail: daniele.passarella@unimi.it
[b] Dr. F. Falchi, Prof. Dr. M. Botta
Dipartimento Farmaco Chimico Tecnologico
Universitꢀ degli Studi di Siena, Via A. Moro, 53100 Siena (Italy)
[c] Dr. I. Filippi, Dr. A. Naldini, Dr. G. Scalia, Prof. Dr. F. Carraro
Dipartimento di Fisiologia
Universitꢀ degli Studi di Siena, Via A. Moro, 53100 Siena (Italy)
[d] Dr. E. Crespan, Dr. G. Maga
Istituto di Genetica Molecolare IGM-CNR
Via Abbiategrasso 207, 27100 Pavia (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100304.
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D. Passarella et al.
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velopment of second-generation small-molecule inhibitors that
are able to treat imatinib-resistant CML.[6]
route that partially follows the reported preparation of imati-
nib.[11]
We considered the importance of the (phenyl)pyrimidine-2-
amine scaffold in the inhibition of Bcr-Abl,[7] and we focused
our attention on the possibility of substituting the amide bond
present in the structure of imatinib with a 1,2,3-triazole ring.[8]
The bioisosterism between an amide group and a 1,2,3-triazole
ring is well known[9] and is supported by docking simulations.
Herein we report the versatile synthesis, biological evaluation,
and modeling studies of a small collection of triazole-based im-
atinib analogues. To the best of our knowledge, this is the first
time that the amide bond of any kinase inhibitor has been
substituted by a triazole ring.
The reaction of 3-acetylpyridine 1a and acetophenone 1b
with excess dimethylformamide dimethyl acetal (DMF-DMA) in
xylene gave the corresponding enaminones 2a–b. Compounds
2 reacted with guanidine nitrate and sodium hydroxide in n-
butanol, with subsequent formation of the pyrimidine ring,
leading to compounds 3a–b. The third step consists of an “Ull-
mann-type” coupling between 3 and o-bromo-p-nitrotoluene.
This reaction was performed in the presence of copper iodide,
N,N’-dimethylethane-1,2-diamine (DMEDA), and potassium car-
bonate in dioxane at reflux to furnish nitro derivatives 4a–b.
The reduction of the nitro group with hydrazine monohydrate/
iron(III) chloride in the presence of active carbon gave the cor-
responding amines 5a–b.
The production of the azide derivative was secured by the
formation of diazonium salt and subsequent reaction with a
solution of sodium azide in water, leading to the key inter-
mediates 6a–b. The reaction of 6 with various alkynes in the
presence of copper(II) sulfate pentahydrate and sodium ascor-
bate gave the corresponding 1–4 adducts 10–15 (6–76%
yield). Alkyne 8 is the one that contains the 1-benzyl-4-methyl-
piperazine framework, which characterizes imatinib. The other
four alkynes were selected to evaluate the efficacy of the
CuAAC reaction with various types of alkyne and the relevance
of the piperazine ring to biological activity. Three of the al-
kynes used are commercially available: methyl propiolate, 3-di-
methylamino-1-propyne, and phenylacetylene, whereas the re-
maining two—4-methylpiperazine derivative 8 and 2,4-dichlor-
oethynylbenzene 9—were prepared with 4-ethynylbenzalde-
hyde and 2,4-dichlorobenzaldehyde as starting materials
(Schemes 2 and 3). 4-Ethynylbenzaldehyde was reduced with
Results and Discussion
Chemistry
All the compounds were obtained by copper(I)-catalyzed
azide–alkyne cycloaddition (CuAAC) according to Sharpless
conditions,[10] between azides 6 and five different alkynes. The
azides were synthesized as shown in Scheme 1, by using a
Scheme 2. Reagents and conditions: a) NaBH4, MeOH, ꢀ788C, 30 min;
b) SOCl2, CH2Cl2, 608C, 4 h; c) N-methylpiperazine, 1408C, 40 min.
sodium borohydride to the corresponding benzyl alcohol 7a,
which, by reaction with excess thionyl chloride, gave the
chloro derivative 7b. Treatment with N-methylpiperazine at
reflux gave alkyne 8. Alkyne 9 was
obtained by homologation of
2,4-dichlorobenzaldehyde
Bestmann–Ohira
under
conditions
(Scheme 3).[12]
Scheme 1. Reagents and conditions: a) DMF-DMA, xylene; b) C(NH2)3NO3,
NaOH, nBuOH; c) o-Bromo-p-nitrotoluene, CuI, DMEDA, dioxane; d) N2H4,
FeCl3, active C, MeOH; e) NaNO2, H2SO4, NaN3, H2O; f) sodium ascorbate,
CuSO4·5H2O, tBuOH/H2O=1:1. See the Experimental for reaction tempera-
tures and times.
Biological evaluation
Scheme 3. a) Bestmann–Ohira
reagent, K2CO3, MeOH, 08C,
To assess the potency of our com-
pounds, we evaluated their effects then RT, 20 h.
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Bcr-Abl Inhibitors
on enzymatic activity, cell proliferation, phosphorylation,
cdc25A expression, and pro-apoptotic activity. Imatinib was
used as the reference compound.
812 cells. Recent studies with Src dominant-negative mutants
suggest that Src kinases play a role in proliferation of Bcr-Abl-ex-
pressing cell lines,[13] and overexpression of Src kinases is impli-
cated in Bcr-Abl-mediated leukemogenesis and in imatinib re-
sistance. As shown in Figure 2, after 3 h we observed a signifi-
cant decrease in the phosphorylation of all targets in the pres-
ence of selected compounds relative to control. The dose-de-
pendent inhibition of FA030 is similar to the effect of imatinib.
Enzymatic activity
The compound FA030 was tested in an in vitro assay against
the recombinant Abl kinase. It displayed potent anti-enzymatic
activity, with an IC50 value of 0.9ꢁ0.1 mm, while under our ex-
perimental conditions, imatinib showed a slightly lower IC50
value of 0.40ꢁ0.1 mm. When tested against the imatinib-resist-
ant mutant Abl T315I, however, FA030 activity was significantly
lower, showing an IC50 value of 10 mm. None of the other com-
pounds showed significant inhibitory activity toward Abl
(IC50 >100 mm). None of the tested compounds showed inhibi-
tory activity against Src kinase up to a final concentration of
100 mm. FA030 was also tested against two additional tyrosine
kinases: Hck and Pim1; the compound did not show significant
inhibition up to 100 mm.
Inhibition of cdc25A expression
Through the Ras pathways, Bcr-Abl controls the activity of cell
division cycle 25 (cdc25).[14] Cdc25 phosphatases are key regu-
lators of the cell cycle and its checkpoints. They are required
to dephosphorylate, and thus activate, the Cdk–cyclin com-
plexes that trigger progression through the various cell-cycle
phases. Cdc25A is required for the progression from G1 to the
S phase and G2M in the cell cycle, and has been shown to be
overexpressed in a number of cancer cells.[15] For this reason,
we examined the action of FA030 on cdc25A expression.
Figure 3 shows that this compound significantly inhibited
cdc25A mRNA levels after 3 h in K-562 cells, and this inhibition
was increased after 24 h exposure in the K-562 line (panel A). A
similar inhibitory effect at 24 h exposure was also observed in
MEG-01 cells (panel B). Notably, FA030 was observed to be
more active at 24 h than imatinib in the inhibitory effect,
toward both cell lines, even if these differences are not signifi-
cant. This may be due to a direct effect of FA030 on the Ras
Raf1 pathways.
Inhibition of proliferation
The decrease in the viability of K-562, MEG-01, and KU-812
cells treated with the compounds for 72 h was assessed with
the resazurin assay. Experiments performed to determine the
in vitro effects of such compounds revealed the significant an-
tiproliferative activity of FA030. In fact, activity data were in
the low micromolar range (Table 1). No similarly good biologi-
cal activity was observed among the other compounds. The
biological profile in terms of activity toward Abl and cell lines
encouraged the selection of FA030 (10) for further assays.
Pro-apoptotic activity: PARP assay and studies on Bax/Bcl-xL
expression
The new compound was also evaluated for its ability to induce
apoptosis in the human leukemia cell line incubated for 72 h
at the same concentration (1 mm) of the reference compound
(imatinib). First, we investigated the expression of pro-apoptot-
ic Bax and anti-apoptotic Bcl-xL genes, and we monitored the
ratio between Bax and Bcl-xl mRNA expression as a direct indi-
cator of the induction of apoptosis at the gene level. As a
result, incubation of cells for 24 h in the presence of FA030 or
imatinib led to an increase in the Bax/Bcl-xL ratio (Figure 4A,C)
in K-562 and MEG-01 cells. The same results were obtained in
KU-812 cells, but this effect was already present after a 3 h in-
cubation (Figure 4E). Finally, on the basis of the ability of this
compound to induce an increase in the ratio of the pro-apop-
totic versus anti-apoptotic genes in both cell lines, we also
evaluated the induction of caspase activity on the cleavage of
Poly(ADP-ribose) polymerase (PARP) after incubation for 72 h.
FA030 as well as imatinib potently induced apoptosis in K-562,
MEG-01, and KU-812 cells, as shown by the presence of the
cleaved form of PARP in immunoblots (Figure 4B,D,F).
Inhibition of Bcr-Abl, c-Abl, STAT-5, and Src phosphorylation
With the aim of an improved understanding of the antiprolifer-
ative activity of this new compound toward leukemia cells, we
decided to check, in each cell line, a direct link between the
antiproliferative effects of FA030 and the inhibition of Bcr-Abl
activity. Specifically, we evaluated the phosphorylation of Bcr-
Abl, c-Abl, and its downstream substrate STAT-5, in K-562 and
MEG-01 cells in addition to the phosphorylation of Src in KU-
Table 1. Antiproliferative activity of compounds 10–15 against the indi-
cated cell lines.
Compd
IC50 [mm][a]
K-562
MEG-01
KU-812
10
(FA030)
11
0.89ꢁ0.003
10.97ꢁ0.46
88.01ꢁ2.05
25.42ꢁ1.24
56.45ꢁ2.08
>100
>150
12
>100
>150
13
14
15
imatinib
24.22ꢁ0.28
>100
>150
0.37ꢁ0.09
68.64ꢁ0.9
>150
>150
10.77ꢁ0.41
77.36ꢁ3.11
>150
>150
1.89ꢁ0.14
Molecular modeling
[a] Values represent the mean ꢁSEM for separate assays (n=3), each per-
To better elucidate the structure–activity relationships (SAR), all
compounds, after protocol validation (see the Experimental
formed in triplicate.
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Figure 2. FA030 at various concentrations decreases phosphorylation of Bcr-Abl, c-Abl, and STAT-5 in A) K-562 and B) MEG-01 cells incubated for 3 h, with ima-
tinib used as reference compound. FA030 also decreases phosphorylation of Bcr-Abl and Src in C) KU-812 cells. Data were quantified by chemiluminescence,
and are expressed as percent phosphorylated protein. Statistical significance: *p<0.05 (Student’s t test).
Section below), were docked into the imatinib binding site of
Abl kinase in the inactive conformation. We selected the inac-
tive conformation of Abl because our compounds are strictly
similar, in terms of functionality and shape, to imatinib, which
binds to the inactive conformation.[16] For all compounds, the
binding mode is very similar to that of imatinib (Figure 5).
Compound 10 (FA030) seems to have six hydrogen bonds with
the protein, and the majority of contacts are mediated by
van der Waals interactions. Hydrogen bond interactions are
predicted between the pyridine nitrogen atom and the back-
bone NH group of Met318 in the hinge region, and the anilino
NH group with the side chain of the gatekeeper residue
Thr 315. In comparison with imatinib, FA030 loses a hydrogen
bond with the backbone NH group of Ala380, but two hydro-
gen bonds between the triazole ring and the side chain of
Asp381 are allowed. The N-methylpiperazine group is predict-
ed to have a strong interaction with the protein by hydrogen
bonds with the main-chain carbonyl groups of Asp381 and
His361, whereas imatinib interacts with the main-chain carbon-
yl groups of Ile360 and His361. The difference is most likely
due to the small difference in length between FA030 and ima-
tinib. These hydrogen bonds are complemented by extensive
hydrophobic interactions (Val256, Tyr253, Leu248, Leu370,
Phe317, Phe382, Gly321, Lys271, Thr315, Ile313, Val289,
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Bcr-Abl Inhibitors
Figure 3. FA030 inhibits cdc25A expression at 3 and 24 h in A) K-562 and
B) MEG-01 cells, with imatinib used as reference compound; both com-
pounds were used at 1 mm. The results were obtained by qRT-PCR analysis.
Statistical significance: *p<0.05 (Student’s t test).
Figure 4. Pro-apoptotic effect of FA030 relative to imatinib used at the same
concentration (1 mm). The ratio of Bax/Bcl-xL mRNA expression at 24 h in
A) K-562 and C) MEG-01 cells, and at 3 h in E) KU-812 cells was determined
by qRT-PCR. PARP cleavage at 72 h in B) K-562, D) MEG-01, and F) KU-812
cells was analyzed by immunoblots, and two forms of PARP were quantified
by chemiluminescence; results are expressed as the percentage of cleaved
over uncleaved PARP. Statistical significance: *p<0.05 (Student’s t test).
Met290, and Asp381) over the whole length of the inhibitor,
although there are fewer hydrophobic interactions for the N-
methylpiperazine, which is partially exposed to water. For all
remaining compounds, the binding mode is similar to that of
FA030 apart from the hydrogen bond with the hinge region
(the nitrogen atom of pyridine is missing). Concerning the
docking score, FA030 has a value similar to that of imatinib
(ꢀ12.7 versus ꢀ13.7), and all this confirms the good protein af-
finity. For all the other molecules, there is a significant loss in
score (values range from ꢀ7.0 to ꢀ8.4). This is due to the loss
of the hydrogen bond with the hinge region. Structures 12
and 13, in addition to this lost hydrogen bond, also set up
polar groups in the hydrophobic region.
Conclusions
Herein we have described the design and preparation of a
small collection of N-[2-methyl-5-(triazol-1-yl)phenyl]pyrimidin-
2-amine derivatives using a copper(I)-catalyzed azide–alkyne
cycloaddition. Preliminary antiproliferative activity in the micro-
molar range highlighted the efficacy of compound 10 (FA030).
This compound shows potent anti-enzymatic activity against
recombinant Abl kinase, but decreased activity against the im-
atinib-resistant mutant Abl T3151. FA030 is able to decrease
the phosphorylation of Bcr-Abl, c-Abl, STAT-5, and Src with a
dose-dependent behavior similar to the effect observed for im-
atinib; in addition, FA030 is more active than imatinib in the
inhibition of cdc25A in two different cell lines. The pro-apop-
totic activity of FA030 has been proven by the presence of the
cleaved form of PARP, after treatment of three different cell
lines. The ADME prediction suggests no significant difference
from the behavior of imatinib. Molecular modeling studies pre-
Regarding the T315I mutation, FA030 (with a very similar
shape to that of imatinib) undergoes the same problems of
steric hindrance and loss of a hydrogen bond[17] when Tyr315
is replaced by Ile315, thus diminishing its activity (10 mm for
T315I versus 0.9 mm for wild-type).
Finally, from the ADME point of view (calculated with Qik-
Prop), there is no significant difference between imatinib and
FA030. All the other compounds give similar properties to
FA030, except for 14 and 15, which have poor solubility (see
the Supporting Information for details).
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Figure 5. A) Ribbon representation of the Abl kinase domain (grey) in complex with FA030 (yellow). B) Schematic diagram of the interactions made by FA030
with Abl (from docking calculations); protein residues are labeled, and hydrogen bonds are indicated with dotted lines; residues making van der Waals inter-
actions with the inhibitor are shown in green. C) Comparison of the binding modes of FA030 (yellow) and imatinib (green).
1408C for 20 h. The solvent was removed in vacuo, and hexane
(200 mL) was added, with consequent formation of yellow crystals.
After complete crystallization, the product was collected by filtra-
tion in 81% yield. No further purification was needed. 1H NMR
(CDCl3, 300 MHz): d=9.02 (1H, s), 8.61 (1H, dd, J=5.3, 2.7 Hz),
8.15 (1H, dd, J=8.0 Hz, J=2.7 Hz), 7.80 (1H, d, J=12 Hz), 7.31 (1H,
dd, J=8.0 Hz, J=5.3 Hz), 5.67 (1H, d), 3.16 (3H, s), 2.94 ppm (3H,
s).
dict a similar binding mode to that of imatinib, with some
slightly different interactions.
Experimental Section
Chemistry
General. Thin-layer chromatography (TLC) was performed with
Merck pre-coated 60 F254 plates. Reactions were monitored by TLC
on silica gel, with detection by UV light (l 254 nm). Flash chroma-
tography was performed using silica gel (240–400 mesh, Merck).
1H NMR spectra were recorded with Bruker 300 and 400 MHz spec-
trometers using CDCl3 and [D6]DMSO. Chemical shifts (d) are re-
ported in ppm downfield from (CH3)4Si as an internal standard. EI
mass spectra were recorded at an ionizing voltage of 6 keV on a
VG 70–70 EQ instrument. ESI mass spectra were recorded on an
FT-ICR APEXII instrument (Bruker Daltonics).
(E)-3-(Dimethylamino)-1-phenylprop-2-en-1-one (2b).[10] A solu-
tion of acetophenone (25 mmol) and N,N-dimethylformamide di-
methylacetal (100 mmol) xylene (50 mL) was held at reflux at
1808C for 11 h. The solvent was removed in vacuo and hexane
(200 mL) was added, with consequent formation of yellow crystals.
After complete crystallization, the product was collected by filtra-
tion in 32% yield. No further purification was needed. 1H NMR
(CDCl3, 300 MHz): d=7.88 (2H, d, J=6.3 Hz), 7.79 (1H, d, J=
12.4 Hz), 7.46–7.36 (3H, m), 3.14 (3H, bs), 2.92 ppm (3H, bs).
(E)-3-(Dimethylamino)-1-(pyridine-3-yl)prop-2-en-1-one (2a).[10]
solution of 4-acetylpyridine (50 mmol) and N,N-dimethylformamide
A
4-(Pyridine-3-yl)pyrimidin-2-amine (3a).[10] C(NH2)3NO3 (40.3
mmol) and NaOH (40.3 mmol) were added to a solution of 2a
(40.3 mmol) in nBuOH (45 mL). The reaction mixture was held at
dimethylacetal (100 mmol) in xylene (50 mL) was held at reflux at
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Bcr-Abl Inhibitors
reflux at 1208C for 4 h, then cooled to room temperature. The pre-
cipitate was collected by filtration and washed with H2O (70 mL),
then dried in the oven at 75–808C. No further purification was
needed, and the desired product was obtained as white crystals in
60% yield. 1H NMR (CDCl3, 300 MHz): d=9.21 (1H, d, J=2.6 Hz),
8.63 (1H, d, J=3.3 Hz), 8.47 (1H, dt, J=8.3 Hz, J=1.7 Hz), 8.33 (1H,
d, J=5.9 Hz), 7.55 (1H, dd, J=8.3 Hz, J=3.3 Hz), 7.19 (1H, d, J=
5.9 Hz), 4.86 ppm (1H, s).
100 MHz): d=168.6, 161.1, 154.4, 147.9, 147.8, 147.5, 144.8, 134.0,
133.0, 130.6, 124.0, 119.0, 105.1, 103.3, 102.6, 17.6 ppm.
6-Methyl-N-(4-phenylpyrimidin-2-yl)benzene-1,3-diamine (5b).[10]
H2NNH2·H2O (25.6 mmol), FeCl3 (0.03 mmol), and active carbon
(0.010 g) were added to a solution of 4b (1.6 mmol) in MeOH
(20 mL), and the reaction mixture was held at reflux at 1008C for
4 h. The solvent was removed in vacuo, H2O (100 mL) was added,
and extracted with CH2Cl2 (3ꢁ30 mL). The organic layers were
dried over Na2SO4 and concentrated in vacuo. No further purifica-
tion was needed, and the desired product was collected as a
yellow powder in 93% yield. 1H NMR (CDCl3, 300 MHz): d=8.41
(1H, d, J=5.0 Hz), 8.06–8.02 (2H, m), 7.65 (1H, d, J=2.3 Hz), 7.52–
7.48 (3H, m), 7.41 (1H, bs), 7.14 (1H, d, J=5.8 Hz), 7.00 (1H, d, J=
8.0 Hz), 6.46 (1H, dd, J=7.9 Hz, J=2.1 Hz), 4.0–3.1 (2H, bs),
2.26 ppm (3H, s).
4-Phenylpyrimidin-2-amine (3b).[10] C(NH2)3NO3 (7.1 mmol) and
NaOH (7.1 mmol) were added to a solution of 2b (7.1 mmol) in
nBuOH (70 mL). The reaction mixture was held at reflux at 1308C
for 15 h, then cooled to room temperature. The precipitate was
collected by filtration, washed with H2O (70 mL) and dried in the
oven at 75–808C. No further purification was needed, and the de-
1
sired product was obtained as white crystals in 49% yield. H NMR
(CDCl3, 300 MHz): d=8.30 (1H, d, J=5.6 Hz), 8.00–7.97 (2H, m),
7.48–7.46 (3H, m), 7.04 (1H, d, J=5.6 Hz), 5.27 ppm (2H, bs).
N-(5-Azido-2-methylphenyl)-4-(pyridine-3-yl)pyrimidin-2-amine
(6a). Compound 5a (0.292 g, 1.06 mmol) was suspended in H2O
(15 mL) and was cooled to 08C, then concentrated H2SO4 was
added until 5a was completely dissolved. A solution of NaNO2
(1.59 mmol) in H2O (2 mL) was added, and the reaction mixture
was stirred at 08C for 30 min. Then a solution of NaN3 (2.12 mmol)
in H2O (2 mL) was added at 08C, and the reaction mixture was
stirred at room temperature for 3 h. K2CO3 was added until the so-
lution reached pH 8, and the reaction mixture was extracted with
CH2Cl2. The organic layers were dried over Na2SO4 and concentrat-
ed in vacuo. No further purification was needed, and the desired
product was collected as an orange amorphous solid in 81% yield.
1H NMR (CDCl3, 400 MHz): d=9.26 (1H, bs), 8.74 (1H, d, J=
3.4 Hz), 8.53 (1H, d, J=5.1 Hz), 8.42 (1H, d, J=7.8 Hz), 8.21 (1H, d,
J=2.1 Hz), 7.46 (1H, dd, J=7.9 Hz, J=7.8 Hz), 7.22 (1H, d, J=
5.1 Hz), 7.16–7.19 (2H, m), 6.68 (1H, dd, J=8.1 Hz, J=2.1 Hz),
2.34 ppm (3H, s); 13C NMR (CDCl3, 100 MHz): d=163.32, 160.99,
159.71, 152.20, 149.07, 139.38, 139.13, 135.25, 133.23, 132.03,
124.36, 114.29, 111.62, 109.28, 30.32, 18.13 ppm; Anal. calcd for
C16H13N7: C 63.36, H 4.32, N 32.32, found: C 63.41, H 4.29, N 32.35.
N-(2-Methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidin-2-amine
(4a).[10] o-Bromo-p-nitrotoluene (1.0 mmol), CuI (0.25 mmol),
DMEDA (0.25 mmol), and K2CO3 (2.0 mmol) were added to a solu-
tion of 3a (1.1 mmol) in dry dioxane (5 mL) under N2 atmosphere,
and the reaction mixture was held at reflux at 1208C for 15 h, then
cooled to room temperature. Concentrated NH3 (4 mL) and brine
(20 mL) were added, and extracted with EtOAc. The organic layers
were dried over Na2SO4 and concentrated in vacuo. The crude was
purified by column chromatography on silica gel (MeOH/CH2Cl2 =
1:80) to give the desired product as a pale-yellow powder in 40%
yield. 1H NMR (CDCl3, 300 MHz): d=9.47 (1H, d, J=1.8 Hz), 9.28
(1H, bs), 8.76 (1H, d, J=4.3 Hz), 8.59 (1H, d, J=6.5 Hz), 7.87 (1H,
dd, J=7.2 Hz, J=1.8 Hz), 7.54 (1H, dd, J=7.2 Hz, J=4.3 Hz), 7.38
(1H, s), 7.32 (1H, d, J=6.5 Hz), 7.23 (1H, bs), 2.44 ppm (3H, s);
13C NMR (CDCl3, 100 MHz): d=159.32, 158.76, 147.19, 146.94,
144.44, 139.02, 137.65, 134.58, 132.09, 131.03, 125.68, 118.14,
115.48, 109.03, 29.71, 18.42 ppm.
N-(2-Methyl-5-nitrophenyl)-4-phenylpyrimidin-2-amine (4b).[10] o-
Bromo-p-nitrotoluene (4.7 mmol), CuI (1.3 mmol), DMEDA
(1.3 mmol), and K2CO3 (10.5 mmol) were added to a solution of 3b
(5.2 mmol) in dry dioxane (23 mL) under N2 atmosphere, and the
reaction mixture was held at reflux at 1208C for 15 h, then cooled
to room temperature. Concentrated NH3 (12 mL) and brine (60 mL)
were added and extracted with EtOAc. The organic layers were
dried over Na2SO4 and concentrated in vacuo. The crude was puri-
fied by column chromatography on silica gel (EtOAc/hexane=1:1)
to give the desired product as pale-yellow powder in 31% yield.
1H NMR ([D6]DMSO, 300 MHz): d=9.16 (1H, s), 8.89 (1H, d, J=
2.6 Hz), 8.60 (1H, d, J=5.0 Hz), 8.20–8.17 (2H, m), 7.92–7.89 (1H,
m), 7.56–7.51 (5H, m), 4.46 ppm (3H, s).
N-(5-Azido-2-methylphenyl)-4-phenylpyrimidin-2-amine
(6b).
Compound 5b (1.5 mmol) was suspended in H2O (70 mL) and
cooled to 08C, then concentrated H2SO4 was added until 5b was
completely dissolved. A solution of NaNO2 (2.2 mmol) in H2O
(9 mL) was added and the reaction mixture was stirred at 08C for
30 min. Then a solution of NaN3 (2.9 mmol) in H2O (9 mL) was
added at 08C, and the reaction mixture was stirred at room tem-
perature for 3 h. K2CO3 was added until the solution reached pH 8,
and the reaction mixture was extracted with CH2Cl2 (4ꢁ30 mL).
The organic layers were dried over Na2SO4 and concentrated in va-
cuo. No further purification was needed, and the desired product
1
was collected as an orange amorphous solid in 92% yield. H NMR
(CDCl3, 300 MHz): d=8.46 (2H, d, J=5.2 Hz), 8.32 (1H, d, J=
2.1 Hz), 8.10–8.05 (2H, m), 7.51–7.49 (2H, m), 7.21–7.13 (3H, m),
6.64 (1H, dd, J=7.9 Hz, J=2.7 Hz), 2.33 ppm (3H, s); Anal. calcd for
C17H14N6: C 67.54, H 4.67, N 27.80, found: C 67.51, H 4.69, N 27.77.
6-Methyl-N-[4-(pyridine-3-yl)pyrimidin-2-yl]benzene-1,3-diamine
(5a).[10] H2NNH2·H2O (3.91 mmol), FeCl3 (0.02 mmol), and active
carbon (0.001 g) were added to a solution of 4a (0.98 mmol) in
MeOH (10 mL), and the reaction mixture was held at reflux at 808C
for 6 h. The solvent was removed in vacuo, H2O (30 mL) was
added, and extracted with CH2Cl2 (3ꢁ10 mL). The organic layers
were dried over Na2SO4 and concentrated in vacuo. No further pu-
rification was needed, and the desired product was collected as a
yellow powder in 99% yield. 1H NMR ([D6]DMSO, 300 MHz): d=
9.22 (1H, s), 8.67 (1H, d, J=4.4 Hz), 8.63 (1H, s), 8.44 (1H, d, J=
5.1 Hz), 8.39 (1H, d, J=8.0 Hz), 7.51 (1H, dd, J=7.9, 4.4 Hz), 6.85
(1H, d, J=8.0 Hz), 6.77 (1H, d, J=1.4 Hz), 6.32 (1H, dd, J=8.0,
2.0 Hz), 4.80 (2H, s), 2.04 ppm (3H, s); 13C NMR ([D6]DMSO,
(4-Ethynylphenyl) methanol (7a). A solution of 4-ethynylbenz-
aldehyde (1.5 mmol) in MeOH (10 mL) was cooled to ꢀ788C, and
NaBH4 (0.4 mmol) was added in small amounts. The reaction mix-
ture was stirred at ꢀ788C for 30 min and then warmed to room
temperature. HCl (1n) was added to quench unreacted NaBH4, and
the solvent was removed in vacuo. H2O (50 mL) was added and ex-
tracted with EtOAc (3ꢁ15 mL). The organic layers were dried over
Na2SO4 and concentrated in vacuo. The crude was purified by
column chromatography on silica gel (EtOAc/hexane=3:7), and
the desired product was obtained as a white amorphous solid in
ChemMedChem 2011, 6, 2009 – 2018
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2015

D. Passarella et al.
MED
44% yield. 1H NMR (CDCl3, 300 MHz): d=7.47 (2H, d, J=8.4 Hz),
7.33 (2H, d, J=8.4 Hz), 4.57 (2H, s), 3.09 ppm (1H, s); 13C NMR
(CDCl3, 100 MHz): d=141.6, 131.1 (2C), 126.9 (2C), 121.8, 83.6,
81.9, 64.1 ppm; Anal. calcd for C9H8O: C 93.06, H 6.94, found: C
93.12, H 7.01.
N-[2-Methyl-5-(4-{4-[(4-methylpiperazin-1-yl)methyl]phenyl}-1H-
1,2,3-triazol-1-yl)phenyl]-4-phenylpyrimidin-2-amine
(11).
Sodium ascorbate (0.007 mmol) and CuSO4·5H2O (0.001 g) were
added to a solution of 6b (0.07 mmol) and 8 (0.07 mmol) in H2O/
tBuOH=1:1 (2.5 mL), and the reaction mixture was stirred at 808C
for 1.5 h. The solvent was removed in vacuo, H2O (10 mL) and
brine (10 mL) were added, and extracted with CH2Cl2 (5ꢁ5 mL).
The organic layers were dried over Na2SO4 and concentrated in va-
cuo. The crude was purified by column chromatography on silica
gel (EtOAc/hexane=1:2) and the desired product was obtained as
4-Ethynyl chloromethylbenzene (7b). SOCl2 (2 mL) was added to
a solution of 7a (0.7 mmol) in CH2Cl2 (2 mL), and the reaction mix-
ture was held at reflux at 608C for 4 h. Then the volatiles were re-
moved in vacuo, and the crude was purified by column chroma-
tography on silica gel (Hex). The desired product was obtained as
pale-yellow oil in 47% yield. 1H NMR (CDCl3, 300 MHz): d=7.47
(2H, d, J=8.4 Hz), 7.33 (2H, d, J=8.4 Hz), 4.57 (2H, s), 3.09 ppm
(1H, s); 13C NMR (CDCl3, 100 MHz): d=138.1, 132.2 (2C), 128.9
(2C), 123.4, 81.8, 80.9, 47.5 ppm; Anal. calcd for C9H7Cl: C 71.78, H
4.68, found: C 71.81, H 4.65.
1
a yellow amorphous solid in 6% yield. H NMR (CDCl3, 300 MHz):
d=9.08 (1H, d, J=2.0 Hz), 8.54 (1H, bs), 8.21 (1H, s), 8.12–8.15
(2H, m), 7.85 (2H, d, J=7.8 Hz), 7.51–7.53 (3H, m), 7.34–7.48 (5H,
m), 7.16 (1H, bs), 3.63 (2H, s), 2.77 (8H, bs), 2.56 (3H, s), 2.44 ppm
(3H, s); 13C NMR (CDCl3, 100 MHz): d=164.87, 163.10, 157.25,
154.37, 150.22, 149.19, 142.89, 139.06, 137.54, 136.21, 135.01,
132.74, 131.89 (2C), 131.65 (2C), 129.01, 127.99 (2C), 124.80, 119.45,
116.51, 114.87, 109.12, 64.38, 56.39 (2C), 55.03 (2C), 46.12,
17.90 ppm; Anal. calcd for C31H32N8: C 72.07, H 6.24, N 21.69,
found: C 72.11, H 6.20, N 21.68.
1-(4-Ethynylbenzyl)-4-methylpiperazine (8). A solution of 7b
(0.3 mmol) and N-methylpiperazine was held at reflux at 1408C for
40 min. H2O (30 mL) was added and extracted with CH2Cl2 (3ꢁ
10 mL). The organic layers were dried over Na2SO4 and concentrat-
ed in vacuo. The crude was purified by column chromatography
on silica gel (CH2Cl2/MeOH=20:1), and the desired product was
Methyl 1-[4-methyl-3-(4-phenylpyrimidin-2-ylamino)phenyl]-1H-
1,2,3-triazole-4-carboxylate (12). Sodium ascorbate (0.016 mmol)
and CuSO4·5H2O (0.001 g) were added to a solution of 6b
(0.16 mmol) and methyl propiolate (0.16 mmol) in H2O/tBuOH=1:1
(3 mL), and the reaction mixture was stirred at 508C for 5 h. The
solvent was removed in vacuo, H2O (30 mL) was added and ex-
tracted with CH2Cl2 (4ꢁ10 mL). The organic layers were dried over
Na2SO4 and concentrated in vacuo. The crude was purified by
column chromatography on silica gel (EtOAc/hexane=1:1) and the
desired product was obtained as an orange amorphous solid in
1
obtained as a colorless oil in 75% yield. H NMR (CDCl3, 300 MHz):
d=7.42 (2H, d, J=8.3 Hz), 7.26 (2H, d, J=8.3 Hz), 3.49 (2H, s), 3.04
(1H, s), 2.49 (8H, bs), 2.31 ppm (3H, s); 13C NMR (CDCl3, 100 MHz):
d=138.8, 132.5 (2C), 127.9 (2C), 121.1, 85.5, 81.9, 63.7, 56.9 (2C),
54.1 (2C), 46.7 ppm; Anal. calcd for C14H18N2: C 78.46, H 8.47, N
13.07, found: C 78.49, H 8.41, N 13.12.
2,4-Dichloro-ethynylbenzene (9). A solution of 2,4-dichlorobenz-
aldehyde (0.6 mmol) in dry MeOH (20 mL) was cooled to 08C and
then K2CO3 (1.2 mmol) and dimethyl-1-diazo-2-oxopropylphospho-
nate (0.7 mmol) were added. The reaction mixture was stirred at
room temperature for 20 h. The solvent was removed in vacuo, sa-
turated NaHCO3 (70 mL) was added, and extracted with EtOAc (3ꢁ
20 mL). The organic layers were dried over Na2SO4 and the solvent
was removed in vacuo. The crude was purified by column chroma-
tography on silica gel (EtOAc/hexane=1:1) and the desired prod-
uct was obtained as a white amorphous solid in 41% yield.
1H NMR (CDCl3, 300 MHz): d=7.46–7.42 (2H, m), 7.20 (1H, dd, J=
8.6 Hz, J=1.7 Hz), 3.39 ppm (1H, s); 13C NMR (CDCl3, 100 MHz): d=
136.8, 135.3, 135.0, 129.8, 127.0, 115.8, 83.1, 81.2 ppm; Anal. calcd
for C8H4Cl2: C 56.18, H 2.36, found: C 56.23, H 2.31.
1
41% yield. H NMR (CDCl3, 400 MHz): d=9.14 (1H, bs), 8.58 (1H,
s), 8.51 (1H, d, J=5.1 Hz), 8.13–8.15 (2H, m), 7.53–7.60 (3H, m),
7.40 (1H, dd, J=8.3 Hz, J=1.9 Hz), 7.36 (1H, d, J=8.3 Hz), 7.31
(1H, bs), 7.26–7.28 (2H, m), 4.03 (3H, s), 2.46 ppm (3H, s); 13C NMR
(CDCl3, 100 MHz): d=165.97, 161.83, 160.37, 159.03, 141.00,
139.80, 137.22, 135.68, 132.03, 131.90, 129.73(2C), 127.97, 127.85
(2C), 126.15, 114.71, 112.52, 109.82 ppm; Anal. calcd for C21H18N6O2:
C 65.28, H 4.70, N 21.75, found: C 65.26, H 4.73, N 21.72; HRMS
(EI): m/z [M]+ calcd for C21H18N6O2: 386.1491, found: 386.1492.
N-[2-Methyl-5-{4-[(dimethylamino)methyl]-1H-1,2,3-triazol-1-yl}-
phenyl]-4-phenylpyrimidin-2-amine (13). Sodium ascorbate
(0.016 mmol) and CuSO4·5H2O (0.001 g) were added to a solution
of 6b (0.16 mmol) and 3-dimethylamino-1-propyne (0.16 mmol) in
H2O/tBuOH=1:1 (3 mL), and the reaction mixture was stirred at
room temperature for 30 h. The solvent was removed in vacuo,
H2O (30 mL) was added and extracted with CH2Cl2 (4ꢁ10 mL). The
organic layers were dried over Na2SO4 and concentrated in vacuo.
No further purification was needed, and the desired product was
obtained as a yellow amorphous solid in 16% yield. 1H NMR
(CDCl3, 400 MHz): d=9.06 (1H, d, J=1.8 Hz), 8.54 (1H, bs), 8.31
(1H, s), 8.13–8.15 (2H, m), 7.52–7.56 (3H, m), 7.39 (1H, dd, J=
8.2 Hz, J=1.8 Hz), 7.34 (1H, d, J=8.2 Hz), 7.25 (1H, d, J=4.7 Hz),
7.18 (1H, bs), 3.99 (2H, bs), 2.55 (6H, bs), 2.45 ppm (3H, s);
13C NMR (CDCl3, 100 MHz): d=166.51, 164.12, 157.31, 145.64,
138.95, 136.28, 133.34, 131.13 (2C), 129.18, 128.95, 127.41 (2C),
126.82, 125.09, 119.87, 105.75, 99.28, 64.08, 49.25 (2C), 18.39 ppm;
Anal. calcd for C22H23N7: C 68.55, H 6.01, N 25.44, found: C 68.58, H
5.99, N 25.47; HRMS (EI): m/z [M]+ calcd for C22H23N7: 385.2015,
found: 385.2005.
N-[2-Methyl-5-(4-{4-[(4-methylpiperazin-1-yl)methyl]phenyl}-1H-
1,2,3-triazol-1-yl)phenyl]-4-(pyridine-3-yl)pyrimidin-2-amine (10).
Sodium ascorbate (0.02 mmol) and CuSO4·5H2O (0.001 g) were
added to a solution of 6a (0.23 mmol) and 8 (0.23 mmol) in H2O/
tBuOH=1:1 (5 mL), and the reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was filtered and the
solid was washed with H2O and dried in the oven at 808C. The
crude was purified by column chromatography on silica gel
(MeOH/CH2Cl2 =1:10) and the desired product was obtained as a
yellow amorphous solid in 76% yield. 1H NMR (CDCl3, 400 MHz):
d=9.32 (1H, bs), 9.05 (1H, bs), 8.77 (1H, bs), 8.59 (1H, d, J=
5.0 Hz), 8.53 (1H, d, J=7.9 Hz), 8.22 (1H, s), 7.90 (2H, d, J=7.8 Hz),
7.51 (1H, t, J=7.9 Hz), 7.43–7.45 (3H, m), 7.37–7.39 (1H, d, J=
8.2 Hz), 7.28- 7.29 (2H, m), 3.59 (2H, s), 2.60 (8H, bs), 2.47 (3H, s),
2.38 ppm (3H, s); 13C NMR (CDCl3, 100 MHz): d=163.44, 160.94,
159.84, 152.41, 149.16, 148.80, 139.43, 138.94, 136.48, 135.32,
133.19, 131.94, 130.27 (2C), 130.08, 127.86, 126.49 (2C), 124.48,
118.07, 115.00, 113.14, 109.58, 63.22, 55.61 (2C), 53.22 (2C), 46.25,
18.34 ppm; Anal. calcd for C30H31N9: C 69.61, H 6.04, N 24.35,
found: C 69.65, H 6.10, N 24.31; MS (EI): m/z 517 [M]+.
N-[2-Methyl-5-(4-phenyl-1H-1,2,3-triazol-1-yl)phenyl]-4-phenyl-
pyrimidin-2-amine (14). Sodium ascorbate (0.016 mmol) and
CuSO4·5H2O (0.001 g) were added to a solution of 6b (0.16 mmol)
2016
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2009 – 2018

Bcr-Abl Inhibitors
and phenylacetylene (2.4 mmol) in H2O/tBuOH=1:1 (3.5 mL), and
the reaction mixture was stirred at room temperature for 28 h. The
solvent was removed in vacuo, H2O (60 mL) was added and ex-
tracted with CH2Cl2 (4ꢁ20 mL). The organic layers were dried over
Na2SO4 and concentrated in vacuo. The crude was purified by
column chromatography on silica gel (EtOAc/hexane=1:2) and the
desired product was obtained as a yellow amorphous solid in 19%
yield. 1H NMR (CDCl3, 400 MHz): d=9.10 (1H, d, J=2.1 Hz), 8.53
(1H, d, J=5.2 Hz), 8.25 (1H, s), 8.14–8.17 (2H, m), 7.92 (2H, dd, J=
8.4 Hz, J=1.3 Hz), 7.53–7.56 (3H, m), 7.46–7.50 (3H, m), 7.36–7.41
(3H, m), 7.26 (1H, d, J=5.2 Hz), 2.47 ppm (3H, s); 13C NMR (CDCl3,
100 MHz): d=166.04, 160.44, 158.90, 148.85, 139.52, 137.43,
136.40, 131.96, 131.83, 131.17, 129.70 (2C), 129.51 (2C), 128.94,
127.92 (2C), 127.52, 126.53 (2C), 118.25, 114.82, 112.64, 109.68,
18.46 ppm; Anal. calcd for C25H20N6: C 74.24, H 4.98, N 20.78,
found: C 74.26, H 5.02, N 20.75; HRMS (EI): m/z [M]+ calcd for
C25H20N6: 404.1749, found: 404.1763.
Imatinib ligand co-crystallized in the A-chain was chosen as cent-
roid to define the grid box. The option “dock ligands similar in size
to the workspace ligand” was selected for determining the grid
sizing. For docking, the extra precision mode was selected. The de-
fault settings for scaling the van der Waals radii were selected: a
scaling factor of 0.8 and a partial charge cutoff of 0.25. No con-
straints were defined for the docking runs. Finally, the option “Add
Epik state penalties to docking score” was selected. The molecules
were subjected to predict the pharmacokinetic or ADME properties
using the QikProp module (version 32212). All images were gener-
ated with PyMOL[21] software (version 1.2.x) and with PoseView
Web[22]
.
Biology
Enzyme assays: Recombinant human Abl was purchased from Up-
state Biotechnology (Waltham, MA, USA). Activity was measured in
a filter-binding assay using an Abl-specific peptide substrate (Abl-
tide, Upstate Biotechnology). Reaction were incubated for 15 min
at 308C, in 8 mm MOPS-NaOH pH 7.0, 1 mm EDTA, 25 mm MgOAc,
20 mm ATP, 0.012 mm [g-32P]ATP, 50 mm peptide, 0.005 mm c-Abl. The
apparent affinity (KM) values of the Abl preparation used for its
peptide and ATP substrates were determined separately and found
to be 1.5 mm and 10 mm, respectively. Each experiment was done in
triplicate, and mean values were used for the interpolation. Curve
fitting was performed with the program GraphPad Prism using
Equation (1):
N-[2-Methyl-5-(4-(2,4-dichlorophenyl)-1H-1,2,3-triazol-1-yl)phen-
yl]-4-phenylpyrimidin-2-amine (15). Sodium ascorbate (0.016
mmol) and CuSO4·5H2O (0.001 g) were added to a solution of 6b
(0.16 mmol) and 9 (2.4 mmol) in H2O/tBuOH=1:1 (4 mL), and the
reaction mixture was stirred at 708C for 6 h. The solvent was re-
moved in vacuo, H2O (50 mL) was added and extracted with CH2Cl2
(4ꢁ20 mL). The organic layers were dried over Na2SO4 and concen-
trated in vacuo. The crude was purified by column chromatogra-
phy on silica gel (EtOAc/hexane=2:3) and the desired product was
obtained as a yellow amorphous solid in 44% yield. 1H NMR
(CDCl3, 400 MHz): d=9.07 (1H, d, J=2.1 Hz), 8.62 (1H, s), 8.53
(1H, d, J=5.1 Hz), 8.33 (1H, d, J=8.5 Hz), 8.14–8.16 (2H, m), 7.51–
7.85 (4H, m), 7.45 (1H, dd, J=8.1 Hz, J=2.1 Hz), 7.42 (1H, dd, J=
8.5 Hz, J=2.1 Hz), 7.39 (1H, d, J=8.18 Hz), 7.26–7.28 (2H, m),
2.49 ppm (3H, s); 13C NMR (CDCl3, 100 MHz): d=165.36, 159.90,
158.44, 143.56, 138.98, 136.68, 135.62, 134.29, 131.78, 131.31,
131.15, 130.75, 129.98, 129.03 (2C), 127.87, 127.64, 127.21 (2C),
121.23, 114.38, 112.35, 109.13, 29.70, 17.86 ppm; Anal. calcd for
C25H18Cl2N6: C 63.43, H 3.83, N 17.75, found: C 63.45, H 3.79, N
17.76; HRMS (EI): m/z [M]+ calcd for C25H18Cl2N6: 472.0970, found:
472.0972.
v ¼ Vmax=ð1 þ ½Iꢂ=IC50Þ
ð1Þ
for which v is the reaction rate in the presence of the inhibitor,
Vmax is the apparent maximal reaction rate in the absence of inhibi-
tor, and [I] is the inhibitor concentration.
Cell culture: Human chronic myeloid leukemia (CML) K-562 cells in
blast crisis,[23] the human CML MEG-01 cell line in megakaryocytic
blast crisis,[24] and the human CML KU-812[25] cell line in myeloid
blast crisis were obtained from the American Type Culture Collec-
tion. The three cell lines were selected for their different origins, as
the K-562 line has been characterized as highly undifferentiated
cells of the granulocytic series, whereas the KU-812 line has some
characteristics of basophilic leukocytes, and the MEG-01 line is ori-
ginated from megakaryocytes. Nevertheless, all contain at least
one Philadelphia chromosome. Cells were grown in RPMI 1640
medium (Euroclone, Devon, UK) containing 10% fetal bovine
serum (FBS) or 20% FBS for KU-812, and antibiotics (100 UmLꢀ1
penicillin and 100 mgmLꢀ1 streptomycin). The cultures were free of
mycoplasma.
Molecular modeling
The computation was carried out in Schrçdinger[18] molecular mod-
eling software, which was installed on an Intel Xeon L5420
(2.50 GHz) workstation, with a Linux operating system (FC13).[19] All
the ligands were built using the builder interface of Maestro 9.0
and subjected to the LigPrep module (version 23211). LigPrep was
set to produce a single low-energy 3D structure with correct chiral-
ities (Force Field OPLS 2005) and multiple protonation/tautomeri-
zation states at pH 7.0ꢁ2.0 (Epik version 2.0211). The coordinates
for the Abl–imatinib complex (PDB ID: 1IEP)[13] were downloaded
from the RCSB Protein Data Bank.[20] The protein was then pre-
pared for subsequent grid generation and docking using the Pro-
tein Preparation Wizard tool.
Resazurin assay: The resazurin assay (Acros Organics, Geel, Belgium)
was used to determine cell proliferation.[26] Starved cells (1ꢁ104
cells per well) were plated in 96-well plates with RPMI containing
FBS, with or without various concentrations (0.05–150 mm) of the
studied compounds. Resazurin was dissolved in sterile water and
added to the wells 6 h before the term of 72 h incubation, in
amounts equal to 5% volume and at a concentration of 320 mm.
Resazurin reduction was determined by measuring fluorescence at
lex 530 nm and lem 590 nm using FLUOstar Optima (BMG Labtech,
Offenburg, Germany). Data analysis for IC50 calculations was per-
formed with the L5W Data Analysis Package plug-in for Excel (Mi-
crosoft). Results are reported as mean ꢁSEM.
Using this tool, the B-chain was removed. All hydrogen atoms
were added on the A-chain, and the protonation states for histi-
dine residues were optimized. All crystallographic waters were de-
leted, and the entire chain was minimized (RMSD=0.30 ꢂ, Force
Field OPLS 2005). Molecular docking studies were performed using
the Extra Precision Glide (XP) (version 55212). The docking protocol
was validated by reproducing the binding mode of imatinib in the
A-chain of the Abl crystal structure (PDB ID: 1IEP). A grid for dock-
ing was prepared using the Receptor Grid Generation tool in Glide.
Western blot analysis: The inhibitory effect of compounds toward
the phosphorylation of Bcr-Abl (Tyr245) and STAT-5 (Tyr694) were
tested using a PathScan Multiplex Western Detection Kit (Cell Sig-
ChemMedChem 2011, 6, 2009 – 2018
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2017

D. Passarella et al.
MED
naling Technology, Beverly, MA, USA). This kit was used to assay
the inhibition of multiple proteins on membrane without stripping
and re-probing. The inhibitory effect toward phosphorylation of
Src (Tyr416) was assessed using specific antibody (Cell Signaling
Technology). Cells were cultured at a concentration of 1ꢁ106 cells
per well and challenged with FA030 (50, 10, and 1 mm) and refer-
ence compound imatinib (1 mm). After 3 h, cells were harvested
and lysed in an appropriate buffer containing 1% Triton X-100. Fil-
ters were additionally re-probed with b-actin (control loading, Cell
Signaling Technology). The pro-apoptotic activity of the selected
compounds was also tested, using immunoblot analysis with poly-
(ADP-ribose) polymerase (PARP)-specific antibody, which reveals
both uncleaved (113 kDa) and cleaved (89 kDa) forms of PARP (Cell
Signaling Technology). Cell lines were cultured at a concentration
of 14ꢁ105 cells per well and challenged with various concentra-
tions of the studied compound or control compound, for 72 h. Pro-
teins were quantified by the BCA method (Pierce, Rockford, IL).
Equal amounts of total cellular protein were resolved by SDS-poly-
acrylamide gel electrophoresis, transferred to PVDF filters, and sub-
jected to immunoblot. Non-saturated, immunoreactive bands were
detected with a CCD camera gel documentation system (Chemi-
DocXRS, Bio-Rad Laboratories, Hercules, CA, USA) and then quanti-
fied with Quantity One (Bio-Rad Laboratories).
Keywords: Bcr-Abl · CuAAC · imatinib · inhibitors · leukemia ·
triazoles
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This research was developed under the umbrella of CM0602
COST Action “Inhibitors of Angiogenesis: Design, Synthesis and
Biological Exploitation”. This study was supported by the Associa-
zione Ricerche sul Cancro and Ministero dell’Istruzione, dell’Uni-
versitꢀ e della Ricerca (MIUR) PRIN 2007—Program “Sviluppo e
caratterizzazione di nuovi inibitori di tirosine chinasi cellulari con
attivitꢀ antiproliferativa e antiangiogenica nei confronti di differ-
enti tumori”. The authors express their gratitude to Novartis for
supplying imatinib as a reference compound.
Received: March 29, 2011
Revised: August 2, 2011
Published online on August 30, 2011
2018
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2009 – 2018