New 2,6,9-trisubstituted purine derivatives as Bcr-Abl and Btk inhibitors and as promising agents against leukemia

Article abstract of DOI:10.1016/j.bioorg.2019.103361

Bcr-Abl and Btk kinases are among the targets that have been considered for the treatment of leukemia. Therefore, several strategies have focused on the use of inhibitors as chemotherapeutic tools to treat these types of leukemia, such as imatinib (for Bcr-Abl) or ibrutinib (for Btk). However, the efficacy of these drugs has been reduced due to resistance mechanisms, which have motivated the development of new and more effective compounds. In this study, we designed, synthesized and evaluated 2,6,9-trisubstituted purine derivatives as novel Bcr-Abl and Btk inhibitors. We identified 5c and 5d as potent inhibitors of both kinases (IC₅₀ values of 40 nM and 0.58/0.66 μM for Abl and Btk, respectively). From docking and QSAR analyses, we concluded that fluorination of the arylpiperazine system is detrimental to the activity against two kinases, and we also validated our hypothesis that the substitution on the 6-phenylamino ring is important for the inhibition of both kinases. In addition, our studies indicated that most compounds could suppress the proliferation of leukemia and lymphoma cells (HL60, MV4-11, CEM, K562 and Ramos cells) at low micromolar concentrations in vitro. Finally, we preliminarily demonstrated that 5c inhibited the downstream signaling of both kinases in the respective cell models. Therefore, 5c or 5d possessed potency to be further optimized as anti-leukemia drugs by simultaneously inhibiting the Bcr-Abl and Btk kinases.

Full text of DOI:10.1016/j.bioorg.2019.103361

Journal Pre-proofs
New 2,6,9-trisubstituted purine derivatives as Bcr-Abl and Btk inhibitors and
as promising agents against leukemia
Jeanluc Bertrand, Hana Dostálová, Vladimír Kryštof, Radek Jorda, Alejandro
Castro, Jaime Mella, Christian Espinosa-Bustos, Ana María Zarate, Cristian
O. Salas
PII:
S0045-2068(19)31549-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.103361
YBIOO 103361
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Accepted Date:
17 September 2019
12 October 2019
Please cite this article as: J. Bertrand, H. Dostálová, V. Kryštof, R. Jorda, A. Castro, J. Mella, C. Espinosa-
Bustos, A. María Zarate, C.O. Salas, New 2,6,9-trisubstituted purine derivatives as Bcr-Abl and Btk inhibitors
and as promising agents against leukemia, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103361
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New 2,6,9-trisubstituted purine derivatives as Bcr-Abl and Btk
inhibitors and as promising agents against leukemia
Jeanluc Bertrand,1 Hana Dostálová,2 Vladimír Kryštof,2,* Radek Jorda,2 Alejandro Castro,3 Jaime Mella,4
Christian Espinosa-Bustos,5 Ana María Zarate,1 and Cristian O. Salas1,*
1 Departamento de Química Orgánica, Facultad de Química y de Farmacia, Pontificia Universidad Católica de
Chile, 702843, Santiago de Chile, Chile.
2 Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany, The Czech Academy
of Sciences, Slechtitelu 27, 783 71 Olomouc, Czech Republic.
3
Laboratorio de Bioproductos Farmacéuticos y Cosméticos, Centro de Excelencia en Medicina Traslacional,
Facultad de Medicina, Universidad de La Frontera, Av. Francisco Salazar 01145, Temuco, 4780000, Chile
4 Instituto de Química y Bioquímica, Facultad de Ciencias, Universidad de Valparaíso, 2360102, Av. Gran Bretaña
1111, Playa Ancha, Valparaíso, Casilla 5030, Chile.
5
Departamento de Farmacia, Facultad de Química y de Farmacia, Pontificia Universidad Católica de Chile,
702843, Santiago de Chile, Chile.
1

Abstract
Bcr-Abl and Btk kinases are among the targets that have been considered for the treatment of leukemia.
Therefore, several strategies have focused on the use of inhibitors as chemotherapeutic tools to treat these
types of leukemia, such as imatinib (for Bcr-Abl) or ibrutinib (for Btk). However, the efficacy of these
drugs has been reduced due to resistance mechanisms, which have motivated the development of new
and more effective compounds. In this study, we designed, synthesized and evaluated 2,6,9-trisubstituted
purine derivatives as novel Bcr-Abl and Btk inhibitors. We identified 5c and 5d as potent inhibitors of
both kinases (IC50 values of 40 nM and 0.58/0.66 M for Abl and Btk, respectively). From docking and
QSAR analyses, we concluded that fluorination of the arylpiperazine system is detrimental to the activity
against two kinases, and we also validated our hypothesis that the substitution on the 6-phenylamino ring
is important for the inhibition of both kinases. In addition, our studies indicated that most compounds
could suppress the proliferation of leukemia and lymphoma cells (HL60, MV4-11, CEM, K562 and
Ramos cells) at low micromolar concentrations in vitro. Finally, we preliminarily demonstrated that 5c
inhibited the downstream signaling of both kinases in the respective cell models. Therefore, 5c or 5d
possessed potency to be further optimized as anti-leukemia drugs by simultaneously inhibiting the Bcr-
Abl and Btk kinases.
Graphical Abstract
Keywords
Purine derivatives, Bcr-Abl inhibitors, Btk inhibitors, leukemia, docking, QSAR.
2

1. Introduction
Leukemia is a group of hematologic malignancies within a very large number of genetically diverse
diseases, including an array of cancer types [1, 2] Today, numerous therapies exist to treat leukemia,
such as the use of tyrosine kinase inhibitors (TKIs), which have been very successful.[3-5] The
therapeutic potential of protein kinases has been widely accepted, and over 37 kinase inhibitor drugs
have received approval for clinical use in certain cancers. [6] In fact, the kinase domain is the most
common domain in known oncogenes. Mutations and other genetic aberrations often cause
hyperactivation or changes in expression levels.[7] For example, the vast majority of chronic myelocytic
leukemia (CML) cases and 20–30 % of acute lymphocytic leukemia (ALL) cases are caused by a
reciprocal chromosomal translocation between chromosomes 9 and 22—t(9, 22)—thus forming the so-
called Philadelphia chromosome (Ph).[8] This translocation results in the break-point cluster region-
Abelson (Bcr-Abl) fusion gene, encoding a constitutively active protein tyrosine kinase, the Bcr-Abl
fusion protein, which can deregulate tyrosine kinase activity. The dysregulated activity of Bcr-Abl is
considered both a necessary and sufficient inducer of CML because it drives survival and proliferation
through multiple downstream pathways.[9] Considering the role of Bcr-Abl in leukemia, in the 1980s, a
therapeutic effort was directed towards the development of those specific inhibitors.[10, 11] Imatinib
(Figure 1) was discovered as the first selective Bcr-Abl TKI, which elicited a high response rate and low
toxicity, and the long-term survival of patients treated with imatinib currently makes it the frontline agent
of choice for CML. Second-line treatments include dose escalation of imatinib and the second-generation
TKIs dasatinib and nilotinib.[12] Despite the increase in overall survival promoted by imatinib,[13] more
than 25 % of patients with CML will switch TKIs during their lifetime because of resistance or
intolerance. Most resistance is due to amino acid substitutions in Bcr-Abl, mainly within the kinase
domain.[14, 15] One of the most frequent mutations, ranging from 2 to 20 % of CML cases, is T315I (Ile
replaces Thr at position 315 of Bcr-Abl), which is also the deadliest case since it leads to resistance to
second-generation TKIs, such as nilotinib and dasatinib.[16, 17]
Another tyrosine kinase aberrantly activated in some hematological malignancies is Bruton’s tyrosine
kinase (Btk), which is a nonreceptor cytoplasmic protein tyrosine kinase usually expressed in immune
cells such as B cells, mast cells and macrophages.[18, 19] The deregulation of Btk has been observed in
mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), acute myelocytic leukemia (AML)
and activated B-cell-diffuse large B-cell lymphoma. Btk is both a key regulator and a major kinase of the
3

B-cell receptor (BCR) signaling pathway and plays an important role in the regulation of B-cell
activation, survival, proliferation and differentiation.[18] Thus, Btk mediates B-cell proliferation and
apoptosis and can be considered a prospective target for the treatment of autoimmune diseases and
cancer.[20, 21] In recent years, many small-molecule Btk inhibitors that bind to Btk catalytic domains
have been reported.[4, 22] Ibrutinib (Figure 1) is an irreversible Btk inhibitor and has an acrylamide
group in its structure, which can form a covalent bond by Michael addition with the conserved
noncatalytic cysteine residue (C481) of Btk to achieve strong binding.[23] To capitalize on the efficacy
of ibrutinib, several second-generation Btk inhibitors have been developed, such acalabrutinib, which
has demonstrated significant activity in patients with CLL and MCL.[24] However, similar to imatinib,
Btk inhibitors have also elicited resistance, limiting their clinical efficacy. This mechanism is associated
with the C481M Btk mutation.[25, 26]
N
O
Cl
N
H
N
NH
N
N
S
O
N
OH
N
O
N
N
N
N
N
N
N
N
N
N
N
NH
NH2
NH2
Dasatinib
O
Aproved by FDA in 2006
Second generation Bcr-Abl
Inhibitor
N
N
H
NH
O
O
N
N
Imatinib
Aproved by FDA in 2001
First generation Bcr-Abl Inhibitor
N
H
N
Ibrutinib
Aproved by FDA in 2013
First generation Btk Inhibitor
Acalabrutinib
Aproved by FDA in 2017
Second generation Btk Inhibito
N
N
N
N
H
r
O
N
CF3
Nilotinib
Aproved by FDA in 2007
Second generation Bcr-Abl Inhibitor
Figure 1. Chemical structures of some TKIs used in the treatment of leukemia.
Most of the Bcr-Abl and Btk inhibitors reported are heterocyclic compounds, and several of them are
nitrogen heterocycles.[4, 10] These compounds share nitrogen heterocycle scaffolds as part of their
structures (Figure 1), which in most cases is a pyrimidine ring. Therefore, in the search for new Bcr-Abl
and Btk inhibitors, an extension of the pyrimidine system, such as the purine ring or isostere derivatives,
has been studied.[27, 28] From these results, key information about the substitution patterns of the purine
ring has been collected. For example, Wang et al.,[29], developed compound AP-23464 (Figure 2),
which has a high affinity for Bcr-Abl. The aniline substitution on C-6 (together with the N7 atom) favored
the interaction by hydrogen bonding with M321, and alkylation on N-7 favored the interaction with the
hydrophobic pocket. Both of these interactions are important for the binding mode with Bcr-Abl.
4

Likewise, Qing Shi et al.,[30] optimized a heterocyclic fragment for new Btk inhibitors. Their results
indicate that the purine core elicited the best performance on Btk (Figure 2, compound I). Once again,
NH (from the aniline fragment) together with N7 favored the interaction by hydrogen bonding, in this
case, with M477 in the Btk pocket. In addition, purvalanol B, a JAK3 inhibitor,[31] also elicited slight
biochemical potency against Bcr-Abl. Crystallographic studies[32] demonstrated that this purine
derivative had an interaction between the purine nitrogen (N7) and the N-H of M318 in the binding
pocket (PDB:6BL8).
O
P
CN
Met 341
H
N
N
N
F
N
N
N
N
N
N
N
N
H
H
F
New purine derivatives
Bcr-Abl or Btk inhibitors
Cl
SGI-7079
N
R = EWG, To increase the
strength the hydrogen bond
between NH and M318 (Bcr-Abl)
or M477 (Btk)
OH
N
F
N
AP-23464
O
N
N
Met 477
H
H
N
N
R
O
N
N
N
H
N
II
N
Cl
OH
N
O
N
HN
O
HN
N
N
F
O
N
N
N
N
Met 318
N
N
Other TKIs with
anticancer
H
NH
N
N
HO
N
N
H
N
I
properties
N
N
N
N
H
Fragment presented in cytotoxic
TKIs
Milciclib
Purvalanol B
Figure 2. Chemical structures of 2,6,9-trisubstituted purines with biological properties and the design of the
proposed compounds.
As mentioned, specific resistance often reduces the sensitivity of oncogenic kinases to drugs during
therapy; therefore, novel molecules or approaches are intensively sought. In this work, during our
programs to develop 2,6,9-trisubstituted purine derivatives with antitumor activity,[33] we report a new
collection of 13 purine derivatives with interesting effects on Bcr-Abl and Btk activities. First, in the
design of our compounds, the aniline fragment of the aforementioned kinase inhibitors (Figure 2, left
side), was substituted with some electron-withdrawing groups (EWG), with the goal of increasing the
strength of the hydrogen bond in both binding pockets. Second, considering that the chemical structures
of some other kinase inhibitors that exhibit anticancer properties (including the CDK inhibitor
milciclib[34] and the Axl inhibitors SGI-7079[35] and II[36] (Figure 2, right side), contains the N-
methyl-piperazinyl-phenylamine moiety, we used this fragment in our compounds. In addition, the
cytotoxic effect of these new purine derivatives on some cancer cell lines related to hematological
5

malignancies was assayed, and in silico studies were carried out to understand the structure-activity
relationship on both kinases.
2. Results and discussions
2.1
Synthesis
We synthesized new 2,6,9-trisubstituted purines 5a-m using short, simple and efficient synthetic
methods, as described in Scheme 1.[33, 37, 38] We obtained fourteen compounds using 2,6-
dichloropurine (1) as a starting material. The first step was the alkylation of 1 with the respective alkyl
halides under basic conditions to give a mixture of N-9- and N-7-alkylated purine regioisomers 2a-a' at
a proportion of 4:1.[33] The second step was a regioselective nucleophilic substitution (SNAr) at position
C-6 with several anilines using n-butanol as the solvent, N,N-diisopropylethylamine as the base, at 110
ºC for 12 h to obtain high yields of compounds 3a-g. Finally, a Buchwald-Hartwig coupling reaction at
C-2 of 3a-g with 4-(4-methylpiperazin-1-yl)aniline or 3-fluoro-4-(4-methylpiperazin-1-yl)aniline (4a or
4b) promoted by MW and catalyzed by palladium (II) yielded the purine derivatives 5a-m in moderate
to high yields. 4a and 4b were previously synthesized from the respective fluoronitrobenzene compounds
(6a-b). We purified all compounds by column chromatography and established their structures based on
1
their spectral properties (IR, MS, H NMR and 13C NMR, see the experimental section and
Supplementary Information, SI).
6

R
R
Cl
N
2
HN
N
HN
Cl
N
N
N
N
N
N
N
N
N
N
i
iii
ii
N
N
Cl
Cl
N
R1
N
N
N
H
H
Cl
N
1
5a-f
5g-m
(R1 = H)
(R1 = F)
3a-g
Cl
N
N
N
N
Cl
2´
NH2
NO2
NO2
v
iv
R1
R1
R1
N
N
F
N
N
6a
6b
(R1 = H)
(R1 = F)
4a
4b
(R1 = H)
(R1 = F)
7a
7b
(R1 = H)
(R1 = F)
Scheme 1. Reagents and conditions: i) (Bromomethyl)cyclopropane, K2CO3, DMF, 12 h, RT. ii) Anilines, DIPEA,
n-butanol, reflux, 12 h. iii) 4-(4-Methylpiperazin-1-yl)aniline 4a or 3-fluoro-4-(4-methylpiperazin-1-yl)aniline 4b,
2M K2CO3, Pd(OAc)2, xantphos, dioxane, MW, 100 °C, 2 h. iv) N-Methylpiperazine, K2CO3, DMF, 2 h. v)
Hydrazine, Pd-C, ethanol, 70 °C, 2 h.
2.2. Kinase inhibition
Due to the known inhibitory effect of related 6-anilino purines on tyrosine kinases,[39] we screened all
prepared compounds for inhibitory activity against Abl and Btk. As expected, the Abl kinase was the
most sensitive kinase to inhibition by several compounds at submicromolar concentrations; the most
active compounds, 5c and 5d, displayed IC50 values of 40 nM (Table 1). The latter compound was also
most active against the Btk kinase, with an IC50 of 0.58 µM. The serine/threonine kinase Cdk-2 was
assayed in parallel and was approximately two orders of magnitude less sensitive, confirming selectivity
towards tyrosine kinases.
7

Our observation suggests that fluorination of the N-methyl-piperazinyl-phenylamine at purine position 2
is detrimental for activity against Abl; its presence reduces inhibitory activity by approximately ten-fold
(e.g., 5a vs 5g, 5b vs 5h, 5c vs 5i, 5d vs 5j and 5f vs 5m). We also found that the substitution on the 6-
phenylamino ring markedly influenced the inhibitory potency in both directions, depending both on its
type and position. For example, with respect to 5a or 5g (without substitution on the aniline ring), the
presence of fluorine in meta slightly increased or maintained the potency towards Abl (5b or 5i,
respectively), whereas para-fluorine substitution (5b, 5h) reduced the potency. Interestingly, the
comparison of the pair 5b and 5d or 5h and 5i indicated that the positive effect of meta-fluorine was
stronger than the negative effect of para-fluorine, difluorine derivatives. However, when disubstitution
was performed with fluorine at the meta/para position on the 6-phenylamino ring, the increased potency
of 5d and 5j towards Abl was similar to the reference compound. Likewise, a methoxy group in the para
position slightly increased the potency towards Abl (5k). We also noted that a combination of chlorine
with one or two methoxy groups lead to a decrease in potency (5e, 5f, 5l and 5m). Similar but much
weaker effects of this substitution were also observed on Btk inhibition.
8

Table 1. In vitro inhibition of compounds 5a-m on the kinases Abl, Btk and Cdk-2/Cyclin E.
Ar
HN
N
N
N
HN
N
R
N
N
Compound
IC50 (µM)a
Btk
Ar
R
H
H
H
H
H
H
F
Abl
Cdk-2/Cyclin E
21.64 ± 3.80
11.03 ± 1.83
24.00 ± 6.00
18.49 ± 6.76
28.61 ± 6.23
16.80 ± 3.12
14.93 ± 1.88
9.01 ± 2.96
5a
5b
5c
5d
5e
5f
0.09 ± 0.01
0.11 ± 0.01
0.04 ± 0.01
0.04 ± 0.01
36.00 ± 3.86
0.80 ± 0.07
0.58 ± 0.10
1.07 ± 0.24
0.60 ± 0.07
0.58 ± 0.05
0.34 ± 0.06
>100
0.90 ± 0.18
1.30 ± 0.29
0.66 ± 016
0.58 ± 0.19
>100
F
F
F
F
O
Cl
O
Cl
O
21.68 ± 9.66
2.01 ± 0.60
3.46 ± 0.94
1. 81 ± 0.46
2.65 ± 0.02
3.17 ± 0.00
>100
5g
5h
5i
F
F
F
F
22.10 ± 4.75
13. 73 ± 3.54
10.90 ± 1.97
29.42 ± 6.80
22.18 ± 4.82
F
5j
F
F
O
5k
5l
F
O
Cl
O
F
Cl
5m
O
F
8.43 ± 1.27
>100
a IC50 values were determined in at least three independent experiments in the range of 0.01-100 µM.
9

2.3 Quantitative structure-activity relationship (QSAR)
From the results shown in Table 1 and to propose a rational series of structural modifications that give
rise to a new family of compounds, we carried out analyses of quantitative structure-activity relationships
(QSARs) to guide the synthesis of the new molecules. We found an equation for Bcr-Abl (Eq. 1) and
one for Btk (Eq. 2). In Tables S1 and S2, we report the experimental versus predicted activity for each
equation (Bcr-Abl and Btk, respectively). Figures S1 and S2 show the regression graph for each equation
(supplementary material).
(Eq. 1)
(Eq. 2)
푝퐼퐶50 = 355.46 ― 65.55퐶푀푅퐵 ―1.072퐶푙푝푎푟푎퐴 ―1.630푀푒푂표푟푡ℎ표퐴
푟2 = 0.9742;푀푆 = 0.0259;퐹 = 100.73;푝 = 1 ∗ 10 ―6
푝퐼퐶50 = 9.065 ― 1.426푝푖퐵 + 0.201퐹푚푒푡푎퐴 ― 1.322퐶푙푝푎푟푎퐴
푟2 = 0.9527;푀푆 = 0.597;퐹 = 40.29;푝 = 0.000227
A
HN
N
N
N
HN
N
B
N
N
The analysis of equation 1 for Bcr-Abl showed that:
i.
Abl inhibition is inversely proportional to the molar refractivity of fragment B (CMR B). This
means that the insertion of high refractivity groups in this portion of the molecule is not favorable
(e.g., Cl, Br or I).
ii.
It is not favorable to insert a chlorine atom in a para position on ring A (Clpara A). Additionally,
the insertion of a methoxy group in the ortho position on ring A (MeOortho A) is not favorable.
Therefore, the addition of these functional groups to these positions should be avoided. The meta
position of the ring could be explored more freely.
10

The analysis of equation 2 for Btk shows us that:
i.
Btk inhibition decreases as the lipophilicity of the "B" ring system increases. Therefore, it is not
favorable to increase lipophilicity in this region. Preferably, substitutions that reduce lipophilicity
and that do not prevent the protonation of the piperazine system should be inserted.
Activity towards Btk is increased by the presence of a fluorine atom in the A-ring (FmetaA).
Considering both this information and the previously obtained Abl equation, an insertion at the
meta position would probably generate the most bioactive compounds.
ii.
iii.
The activity is drastically reduced if chlorine atoms are inserted in para position at ring A
(ClparaA). The insertion of groups with electronic properties other than chlorine at this position
or that reduce the size of the group is encouraged. The most active compounds have no
substitutions at the para position on ring A or have a fluorine atom.
2.4 Docking studies
With the goal of rationalizing the inhibition of Abl and Btk and the differences in the inhibition of both
kinases by these purine derivatives, we carried out docking studies. From Abl and Btk analysis, the lowest
energy 5d-kinase complexes are shown in Figure 4 (see SI for the values of all compounds in both
kinases), using the respective reported crystal structures (Bcr-Abl, PDB code: 2GQG[40] and Btk, PDB
code: 4OT5[41]). As we expected, the interactions that stabilized both complexes involved the same
parts of the ligands, a hydrogen bond between the NH/N7 (purine moiety) and NH/CO (M318 or M477
residue). In addition, the interactions between the NH+-piperazine ring and the respective residue (salt
bridge for Abl and hydrogen bond for Btk) contributed to the stabilization of these complexes. In
addition, compound 5d established interactions of the π-π type between the difluorophenyl portion and
the amino acids F317 and Y320 at the binding site of the Abl. This fact could explain the higher potency
of 5d towards Abl (IC50 = 40 nM) than towards Btk (IC50 = 0.58 µM), since in latter kinase, the aromatic
portion would not establish a similar interaction. However, an interesting result is shown on the right
side of Figure 4. In both cases, the position of the ligand was away from some amino acids that have
been reported as point of molecular mutations related to resistance to imatinib (T315) or ibrutinib (C481).
11

a)
T315
b)
Figure 4. Different views of the molecular docking modes of 5d at the binding site: a) for Abl and b) for Btk. On
the left-side, 2D representations show the main interactions in the binding pocket; in the middle, 3D view; and on
the right-side, a representation of 5d with the amino acids that are observed in the mutant forms of these kinases.
The compounds that showed low activity (5e, 5l and 5m) against both kinases contain bulky functional
groups in the phenyl ring linked to position 6 of the purine. This structure causes steric limitations, which
increase the distance between the NH fragment and the N7 of the purine derivatives to form hydrogen
bonds with M318 or M477 in Abl and Btk, respectively (see supplementary information).
2.5 Cytotoxic studies
We screened all prepared compounds for cytotoxic activity against the cancer cell lines HL-60 (acute
promyelocytic leukemia), MV4-11 (acute myelogenous leukemia), CEM (acute lymphoblastic
leukemia), K562 (chronic myelogenous leukemia), Ramos (non-Hodgkin lymphoma) and MCF7 (breast
adenocarcinoma). All cell lines yielded micromolar GI50 values (Table 2). MV4-11 was the most
sensitive cell line, and CEM was the least sensitive. Overall, compounds 5c and 5d were the most
cytotoxic among the series of compounds with single digit micromolar GI50 values in all cell lines. In
12

general, the cytotoxicity of the prepared compounds was consistent with enhanced Abl inhibition,
especially in the K562 cell line, with similar structure-activity relationships to those described above.
Table 2. In vitro cytotoxicity of compounds 5a-m on cancer cell lines.
Compound
GI50 (µM)a
HL-60
MV4-11
Ramos
MCF-7
CEM
K562
5a
5b
5c
3.65 ± 0.76
3.06 ± 0.03
1.23 ± 0.20
1.72 ± 0.02
1.46 ± 0.18
1.65 ± 0.23
1.67 ± 0.19
4.38 ± 0.37
2.74 ± 0.67
1.35 ± 0.02
1.44 ± 0.16
1.37 ±0.13
1.48 ± 0.11
1.39 ± 0.11
4.09 ± 0.40
3.37 ± 0.39
12.58 ± 0.56
10.65 ± 2.09
8.89 ± 0.18
7.62 ± 0.80
7.14 ± 0.71
6.89 ± 0.15
7.78 ± 0.02
8.00 ± 0.60
5.98 ± 0.03
7.27 ± 0.61
8.20 ± 1.21
7.10 ± 0.24
7.38 ± 1.05
1.53 ± 0.04
1.71 ± 0.00
0.77 ± 0.06
1.24 ± 0.27
4.70 ± 0.23
2.95 ± 0.01
3.61 ± 0.79
3.50 ± 0.16
2.89 ± 0.21
2.17 ± 0.12
3.14 ± 0.86
4.55 ± 0.19
4.27 ± 0.30
9.60 ± 0.65
7.56 ± 0.34
5.77 ± 0.32
4.63 ± 0.59
4.72 ± 0.15
4.53 ± 0.41
5.17 ± 0.88
4.73 ± 0.36
4.33 ± 0.48
4.10 ± 0.37
7.24 ± 0.49
5.01 ± 0.27
3.91 ± 0.08
6.84 ± 0.99
5.17 ± 0.37
4.17 ± 0.13
3.41 ± 0.42
9.03 ± 0.61
8.71 ± 0.54
4.95 ± 0.56
4.64 ± 0.20
3.90 ± 0.06
4.12 ± 0.02
6.04 ± 0.14
5d
5e
5f
1.17 ± 0.47
0.38 ± 0.05
0.71 ± 0.03
2.49 ± 0.52
1.60 ± 0.26
1.48 ± 0.05
1.29 ± 0.17
1.39 ± 0.18
0.54 ± 0.01
0.68 ± 0.31
5g
5h
5i
5j
5k
5l
9.25 ± 0.02
7.26 ± 1.81
5m
a
IC50 values were determined in three independent experiments in the range 0.1 to 25 µM, depending of the compound
solubility.
Next, we wanted to demonstrate that 5c, a representative of the most potent compounds, is able to inhibit
Abl and Btk in cells. We therefore analyzed signaling pathways downstream of these kinases in cultured
cells treated with various doses of 5c for 1 h. In K562 cells, western blotting revealed decreased levels
of phosphorylated Stat5 and CrkL, which are both well-established substrates of Bcr-Abl (Figure 5a).
Btk inhibition was detected in Ramos cells, in which the Btk signaling pathway requires activation by
immunoglobulin M. Cells exposed to 5c responded by reducing the level of phospho-Btk (Y223) in a
dose-dependent manner as a result of blocked autophosphorylation, and by decreasing the
phosphorylation of its downstream targets, the kinases Erk1/2 and Akt and phospholipase PLC2, which
together confirms the efficient suppression of the pathway (Figure 5b).
13

Figure 5. Compound 5c inhibits Bcr-Abl and Btk activity in cells. a) K562 cells were treated with vehicle or the
indicated concentration of 5c for 1 h; imatinib (IM, 10 µM) was used as a positive control. b) Ramos cells were
treated with vehicle or the indicated dose of 5c for 1 h and stimulated with anti-IgM (5 μg/ml) for 10 minutes
before harvesting. Cells were lysed, and proteins were detected by immunoblotting.
3. Conclusions
This study on the effect of new 2,6,9-trisubstituted purines on two tyrosine kinases involved in leukemia,
Bcr-Abl and Btk, showed that certain chemical modifications of the aniline moiety increased the
inhibitory activity against both proteins. Two of these compounds, 5c and 5d, were highly potent and
exhibited cytotoxicity against six cancer cell lines. Our results confirm a previous conclusion that the
purine core is a privilege scaffold to develop new Bcr-Abl/Btk inhibitors, and structure-activity
relationships indicated that the substitution of a fluorine atom on the aniline fragment at meta- or para-
positions enhanced the activity towards Abl and Btk. However, fluorination of the N-methyl-piperazinyl-
phenylamine moiety decreased the potency of both kinases. The docking studies identified the main
interactions of inhibitors with Abl and Btk and helped to show the difference in potency against both
kinases. Likewise, 5c was able to inhibit Abl and Btk in cells, which was demonstrated by monitoring
some kinases that are in the downstream signaling pathways of both kinases. These results showed that
the 5c and 5d compounds are simpler than the TKIs in clinical trials and, according to our results, could
be considered promising leads for the development of new anti-leukemia drugs.
14

4. Experimental
4.1 Materials and measurements
Melting points were determined on a Kofler Thermogerate apparatus and were uncorrected. Infrared
spectra were recorded on a JASCO FT/IR-400 spectrophotometer. Nuclear magnetic resonance spectra
were recorded, unless otherwise specified, on a Bruker AM-400 instrument using deuterated chloroform
or dimethylsulfoxide solutions containing tetramethylsilane as an internal standard. Mass spectra were
obtained on an HP 5988A mass spectrometer. HRMS-ESI-MS experiments were performed using a
Thermo Scientific Exactive Plus Orbitrap spectrometer with a constant nebulizer temperature of 250 °C.
The experiments were carried out in positive or negative ion mode, with a scan range of m/z 300.00-
1510.40 with a resolution of 140 000. The samples were infused directly into the ESI source via a syringe
pump at flow rates of 5 µL·min-1 through the instrument’s injection valve. Thin layer chromatography
(TLC) was performed using Merck GF-254 type 60 silica gel. Column chromatography was carried out
using Merck type 9385 silica gel. The purity of the compounds was determined by TLC and high-
resolution mass spectrometry (HRMS).
4.2 Synthesis
To a solution of 2,6-dichloro-9H-purine (1, 2.0 g, 1.058 mol), (bromomethyl)cyclopropane (2.14 g, 1.58
mol) and K2CO3 (3.36 mg, 3.174 mol) in DMF (20 mL) were added, and the mixture was stirred at room
temperature for 12 h. The mixture was filtrated and evaporated. The residue was purified by silica gel
chromatography using a polarity mobile phase (acetone/dichloromethane, 5:100) to give products 2 and
2´.
2,6-Dichloro-9-(cyclopropylmethyl)-9H-purine, (2)
White solid, yield 51 %, mp 88-89 °C. 1H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H), 4.10 (d, J = 7.4 Hz,
2H), 1.33 (ddd, J = 12.4, 7.8, 4.8 Hz, 1H), 0.72 (q, J = 5.6 Hz, 2H), 0.48 (q, J = 5.2 Hz, 2H). 13C NMR
(101 MHz, CDCl3) δ 153.31, 153.02, 151.81, 145.52, 130.88, 77.16, 49.48, 11.03, 4.64 ppm. ESI/MS for
(C9H8Cl2N4 [M+H]+). Calcd: 243.0. Found: 242.9.
2,6-Dichloro-7-(cyclopropylmethyl)-7H-purine, (2´)
White solid, yield 18 %, mp 90-91 °C. 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 4.33 (d, J = 7.3 Hz,
2H), 1.47 – 1.31 (m, 1H), 0.85 – 0.66 (m, 2H), 0.48 (q, J = 5.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ
163.63, 153.01, 149.90, 143.77, 121.80, 52.50, 11.56, 4.67. ESI/MS for (C9H8Cl2N4 [M+H]+). Calcd:
243.0. Found: 242.9.
15

General procedures for the synthesis compounds 3a-g
To a solution of 2 (100 mg, 0.411 mmol), aniline (154 mg, 0.411 mmol) and DIPEA (0.15 mL, 0.822
mmol) in n-butanol (20 mL) were added, and the mixture was stirred at 110 °C for 12 h. After cooling to
room temperature, the reaction mixture was concentrated. The residue was purified by silica gel
chromatography using a polarity mobile phase (acetone/dichloromethane, 10:100) to give product 3a.
2-Chloro-9-(cyclopropylmethyl)-N-phenyl-9H-purin-6-amine (3a) White solid, yield 85 %, mp 158-
160 °C. 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H, N-H), 7.89 (s, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.38 (t,
J = 7.7 Hz, 2H), 7.13 (t, J = 7.2 Hz, 1H), 4.02 (d, J = 7.3 Hz, 2H), 1.39 – 1.24 (m, 1H), 0.68 (q, J = 5.4
Hz, 2H), 0.45 (q, J = 5.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 154.18, 152.46, 150.85, 140.56, 138.27,
129.22, 124.10, 120.38, 119.25, 48.78, 11.17, 4.49 ppm. ESI/MS for (C15H14ClN5 [M+H]+). Calcd:
300.1. Found: 299.7.
2-Chloro-9-(cyclopropylmethyl)-N-(4-fluorophenyl)-9H-purin-6-amine (3b) White solid, yield 89
%, mp 143-144 °C. 1H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H, N-H), 7.87 (s, 1H), 7.71 (dd, J = 8.9, 4.7
Hz, 2H), 7.06 (t, J = 8.6 Hz, 2H), 4.02 (d, J = 7.3 Hz, 2H), 1.40 – 1.20 (m, 1H), 0.68 (q, J = 5.5 Hz, 2H),
0.44 (q, J = 5.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 160.51, 158.09, 154.06, 152.33, 150.75, 140.51,
134.15, 134.12, 122.21, 122.14, 118.96, 115.84, 115.61, 48.68, 11.04, 4.36. 19F NMR (376 MHz, CDCl3)
δ -118.39 (s, 1F) ppm. ESI/MS for (C15H13ClFN5 [M+H]+). Calcd: 318.1. Found: 317.8.
2-Chloro-9-(cyclopropylmethyl)-N-(3-fluorophenyl)-9H-purin-6-amine (3c) White solid, yield 76
%, mp 140-142 °C.1H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H, N-H), 7.92 (s, 1H), 7.79 (d, J = 11.1 Hz,
1H), 7.41 (d, J = 8.2 Hz, 1H), 7.31 (dd, J = 14.8, 8.0 Hz, 1H), 6.81 (td, J = 8.3, 2.2 Hz, 1H), 4.04 (d, J =
7.3 Hz, 2H), 1.32 (qd, J = 7.6, 3.7 Hz, 1H), 0.69 (q, J = 5.4 Hz, 2H), 0.46 (q, J = 5.2 Hz, 2H). 13C NMR
(101 MHz, CDCl3) δ 164.28, 161.85, 153.95, 152.04, 150.88, 140.71, 139.83, 139.72, 130.18, 130.08,
119.18, 115.39, 115.36, 110.59, 110.38, 107.63, 107.36, 48.73, 11.04, 4.38. 19F NMR (376 MHz, CDCl3)
δ -111.18 (s, 1F) ppm. ESI/MS for (C15H13ClFN5 [M+H]+). Calcd: 318.1. Found: 317.8.
2-Chloro-9-(cyclopropylmethyl)-N-(3,4-difluorophenyl)-9H-purin-6-amine (3d) White solid, yield
77 %, mp 138-139 °C. 1H NMR (400 MHz, CDCl3) δ 8.04 (s, 1H, N-H), 7.91 (s, 1H), 7.90 – 7.84 (m,
1H), 7.35 – 7.28 (m, 1H), 7.14 (q, J = 9.2 Hz, 1H), 4.04 (d, J = 7.3 Hz, 2H), 1.39 – 1.24 (m, 1H), 0.69
13
(q, J = 5.4 Hz, 2H), 0.45 (q, J = 5.2 Hz, 2H). C NMR (101 MHz, CDCl3) δ 154.05, 152.11, 151.54,
151.41, 151.06, 149.09, 148.95, 148.22, 148.10, 145.79, 145.66, 140.99, 134.85, 134.82, 134.76, 134.73,
119.23, 117.51, 117.34, 116.02, 115.98, 115.96, 115.93, 110.18, 109.96, 77.16, 48.88, 11.17, 4.51. 19F
NMR (376 MHz, CDCl3) δ -135.31 (d, J = 21.8 Hz), -143.20 (d, J = 21.8 Hz) ppm. ESI/MS for
(C15H12ClF2N5 [M+H]+). Calcd: 336.1. Found: 336.0.
16

2-Chloro-9-(cyclopropylmethyl)-N-(4-methoxyphenyl)-9H-purin-6-amine (3e) White solid, yield
81 %, mp 183-185 °C. 1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H, N-H), 7.84 (s, 1H), 7.62 (d, J = 8.9
Hz, 2H), 6.91 (t, J = 6.1 Hz, 2H), 4.00 (d, J = 7.3 Hz, 2H), 3.81 (s, 3H), 1.37 – 1.23 (m, 1H), 0.66 (q, J
= 5.4 Hz, 2H), 0.43 (q, J = 5.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 156.58, 154.28, 152.70, 150.74,
140.45, 131.15, 122.58, 119.06, 114.39, 77.16, 55.63, 48.70, 11.18, 4.45 ppm. ESI/MS for (C16H16ClN5O
[M+H]+). Calcd: 330.1. Found: 329.7.
2-Chloro-N-(4-chloro-2,5-dimethoxyphenyl)-9-(cyclopropylmethyl)-9H-purin-6-amine (3f) White
solid, yield 62 %, mp 148-151 °C.1H NMR (400 MHz, DMSO) δ 8.42 (s, 1H, N-H), 8.32 (s, 1H), 8.20
(s, 1H), 6.99 (s, 1H), 4.00 (d, J = 7.3 Hz, 2H), 3.85 (s, 3H), 3.83 (s, 3H), 1.36 – 1.23 (m, 1H), 0.58 – 0.52
(m, 2H), 0.43 (q, J = 5.0 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 152.17, 151.29, 150.42, 148.17, 141.97,
126.59, 118.90, 114.65, 112.36, 105.76, 56.37, 56.00, 47.78, 39.52, 10.93, 3.74 ppm. ESI/MS for
(C17H17Cl2N5O2 [M+H]+). Calcd: 394.1. Found: 394.0.
2-Chloro-N-(4-chloro-3-methoxyphenyl)-9-(cyclopropylmethyl)-9H-purin-6-amine (3g) White
solid, yield 76 %, mp 216-218 °C. 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H, N-H), 7.87 (s, 1H), 7.86
(d, J = 2.1 Hz, 1H), 3.99 (d, J = 7.3 Hz, 2H), 3.92 (s, 3H), 1.28 (ddd, J = 12.4, 7.8, 4.8 Hz, 1H), 0.65 (q,
J = 5.5 Hz, 2H), 0.42 (q, J = 5.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 155.30, 153.90, 152.07, 150.93,
140.78, 138.24, 130.09, 119.26, 116.92, 112.38, 104.78, 77.16, 56.23, 48.85, 11.14, 4.50 ppm. ESI/MS
for (C16H15Cl2N5O [M+H]+). Calcd: 364.1. Found: 363.9.
General procedures for the synthesis compounds 7a-b
To a solution of 6a (1000 mg, 7.1 mmol), 1-methylpiperazine (708 mg, 7.1 mmol) and K2CO3 (2226 mg,
21 mmol) in DMF (20 mL) were added, and the mixture was stirred at room temperature for 2 h. Then,
the reaction mixture was poured into 400 mL of cold water and filtered, and the solid was dried in the
oven at 80 °C for 2 h to give product 7a. The same procedure was used for compound 7b.
1-Methyl-4-(4-nitrophenyl)piperazine (7a)
Orange solid, yield 98 %, mp 105-107 °C. 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 9.4 Hz, 2H), 6.80
(d, J = 9.4 Hz, 2H), 3.48 – 3.37 (m, 4H), 2.58 – 2.50 (m, 4H), 2.35 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 154.91, 138.55, 126.03, 112.79, 77.16, 54.59, 47.02, 46.10. ESI/MS for (C11H15N3O2 [M+H]+). Calcd:
222.1. Found: 221.4.
1-(2-Fluoro-4-nitrophenyl)-4-methylpiperazine (7b)
Red solid, yield 95 %, mp 98-100 °C. 1H NMR (400 MHz, CDCl3) δ 7.93 (dd, J = 8.9, 1.7 Hz, 1H), 7.85
(dd, J = 13.2, 2.4 Hz, 1H), 6.87 (t, J = 8.8 Hz, 1H), 3.35 – 3.19 (m, 4H), 2.66 – 2.45 (m, 4H), 2.32 (s,
17

13
3H). C NMR (101 MHz, CDCl3) δ 154.35, 151.87, 145.72, 145.65, 121.12, 121.09, 117.23, 117.19,
112.79, 112.53, 77.16, 54.85, 49.64, 49.59, 46.15. 19F NMR (188 MHz, CDCl3) δ -126.45. ESI/MS for
(C11H14FN3O2 [M+H]+). Calcd: 240.1. Found: 240.3.
General procedures for the synthesis compounds 4a-b
To a solution of 7a (300 mg, 1.35 mmol), Pd-C (30 mg) in ethanol (20 mL) was added, and the mixture
was stirred under reflux for 1 h. Then, hydrazine (2.6 mL) was added dropwise to the mixture and stirred
over reflux for an additional 2 h. After the completion of the reaction, the reaction mixture was filtered
in Celite and concentrated to give 4a.
4-(4-Methylpiperazin-1-yl)aniline (4a)
Black solid, yield 99 %, mp 128-130 °C. 1H NMR (400 MHz, CDCl3) δ 6.80 (t, J = 9.1 Hz, 1H), 6.48 –
6.33 (m, 2H), 3.53 (s, 2H), 3.10 – 2.83 (m, 4H), 2.66 – 2.54 (m, 4H), 2.34 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 144.55, 140.23, 118.67, 116.29, 77.16, 55.39, 50.89, 46.17. ESI/MS for (C11H17N3 [M+H]+).
Calcd: 192.2. Found: 191.9.
3-Fluoro-4-(4-methylpiperazin-1-yl)aniline (4b)
Black solid, yield 98 %, mp 120-125 °C. 1H NMR (400 MHz, CDCl3) δ 6.80 (t, J = 9.1 Hz, 1H), 6.48 –
6.33 (m, 2H), 3.53 (s, 2H), 3.10 – 2.83 (m, 4H), 2.66 – 2.54 (m, 4H), 2.34 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 159.14, 154.27, 142.94, 142.73, 131.59, 131.39, 120.56, 120.47, 110.65, 110.59, 104.06,
103.59, 55.13, 50.81, 50.77, 45.73. 19F NMR (376 MHz, CDCl3) δ -122.56 (s, 1F). ESI/MS for
(C11H16FN3 [M+H]+). Calcd: 210.1. Found: 210.3.
General procedures for the synthesis of final compounds 5a-m
To a solution of 3a (150 mg, 0.519 mmol) and 4a (110 mg, 0.57 mmol) or 4b (120 mg, 0.57 mmol) in
dioxane (2 mL), Pd(OAc)2 (24 mg, 0.1 mmol), xantphos (120 mg, 0.2 mmol) and 2 M K2CO3 (aq) (1
mL, 0.2 mmol) were added, and the mixture was stirred at 100 °C for 1 h in a microwave reactor. After
cooling to room temperature, the reaction mixture was filtrated in celite, and the filtrate was diluted with
H2O (100 mL). The mixture was extracted with EtOAc, and the organic layer was dried by anhydrous
Na2SO4 and concentrated. The residue was purified by silica gel chromatography using a polarity mobile
phase (methanol/dichloromethane, 10:100) to give a final product.
9-(Cyclopropylmethyl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)-N6-phenyl-9H-purine-2,6-diamine
(5a) White solid, yield 60 %, mp 88-89 °C. 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.68 (d, J = 8.1
Hz, 2H), 7.61 (s, 1H), 7.50 (d, J = 8.9 Hz, 2H), 7.23 (t, J = 7.8 Hz, 2H), 7.19 (s, 1H), 6.99 (t, J = 7.3 Hz,
18

1H), 6.87 (d, J = 8.9 Hz, 2H), 3.84 (d, J = 7.2 Hz, 2H), 3.17 – 3.07 (m, 4H), 2.61 – 2.50 (m, 4H), 2.31
(s, 3H), 1.22 (qd, J = 7.4, 2.5 Hz, 1H), 0.58 (q, J = 5.4 Hz, 2H), 0.36 (q, J = 5.1 Hz, 2H). 13C NMR (101
MHz, CDCl3) δ 156.84, 152.21, 151.27, 146.68, 139.18, 137.78, 133.41, 128.74 (2C), 122.94, 120.91
(2C), 120.36 (2C), 116.97 (2C), 115.21, 55.19 (2C), 50.03, 48.07 (2C), 46.11, 11.04, 4.28 (2C) ppm.
ESI/MS for (C26H30N8 [M+H]+). Calcd: 455.2666. Found: 455.2671.
9-(Cyclopropylmethyl)-N6-(4-fluorophenyl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)-9H-purine-
2,6-diamine (5b) White solid, yield 52 %, mp 148-149 °C. 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H),
7.91 (t, J = 6.7 Hz, 3H), 7.76 (d, J = 8.7 Hz, 2H), 7.23 (t, J = 8.7 Hz, 2H), 7.20 (s, 1H), 7.16 (d, J = 8.8
Hz, 2H), 4.17 (d, J = 7.1 Hz, 2H), 3.47 – 3.38 (m, 4H), 2.90 – 2.77 (m, 4H), 2.60 (s, 3H), 1.54 (ddd, J =
15.3, 10.2, 6.1 Hz, 1H), 0.90 (q, J = 5.3 Hz, 2H), 0.68 (q, J = 5.1 Hz, 2H). 13C NMR (101 MHz, CDCl3)
δ 159.97, 157.57, 156.78, 152.13, 151.31, 146.79, 137.79, 135.11, 135.08, 133.22, 122.05, 121.97 (2C),
120.92 (2C), 116.94 (2C), 115.46 (2C), 115.24, 115.07, 55.20 (2C), 50.00 (2C), 48.12, 46.11, 11.07,
19
4.29. F NMR (376 MHz, CDCl3) δ -119.90 (s, 1F) ppm. ESI/MS for (C26H29FN8 [M+H]+). Calcd:
473.2572. Found: 473.2571.
9-(Cyclopropylmethyl)-N6-(3-fluorophenyl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)-9H-purine-
2,6-diamine (5c) White solid, yield 45 %, mp 79-81 °C. 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 8.15
(d, J = 11.6 Hz, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.7 Hz, 2H), 7.54 – 7.43 (m, 2H), 7.27 (s, 1H), 7.22 (d, J
= 8.7 Hz, 2H), 6.99 (t, J = 7.7 Hz, 1H), 4.20 (d, J = 7.2 Hz, 2H), 3.52 – 3.40 (m, 4H), 2.95 – 2.83 (m,
4H), 2.63 (s, 3H), 1.61 – 1.53 (m, 1H), 0.93 (q, J = 5.3 Hz, 2H), 0.71 (q, J = 5.1 Hz, 2H). 13C NMR (101
MHz, CDCl3) δ 164.28, 161.86, 156.86, 151.84, 151.44, 147.01, 140.87, 140.75, 137.94, 132.95, 129.77,
129.67, 121.32 (2C), 117.04(2C), 115.26, 115.05, 115.03, 109.39, 109.18, 107.45, 107.18, 55.21(2C),
49.95(2C), 48.14, 46.13, 11.05, 4.29. 19F NMR (376 MHz, CDCl3) δ -111.75 (s, 1F) ppm. ESI/MS for
(C26H29FN8 [M+H]+). Calcd: 473.2572. Found: 473.2578.
9-(Cyclopropylmethyl)-N6-(3,4-difluorophenyl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)-9H-
purine-2,6-diamine (5d) White solid, yield 61 %, mp 125-126 °C. 1H NMR (400 MHz, CDCl3) δ 8.09
(s, 1H), 7.97 (ddd, J = 12.8, 7.3, 2.5 Hz, 1H), 7.68 (s, 1H), 7.51 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.9 Hz,
1H), 7.08 (s, 1H), 7.02 (dd, J = 18.5, 9.1 Hz, 1H), 6.95 (d, J = 8.9 Hz, 2H), 3.92 (d, J = 7.2 Hz, 2H), 3.26
– 3.11 (m, 4H), 2.68 – 2.53 (m, 4H), 2.36 (s, 3H), 1.30 (ddd, J = 10.2, 7.5, 3.7 Hz, 1H), 0.66 (q, J = 5.4
Hz, 2H), 0.44 (q, J = 5.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 156.88, 151.75, 151.47, 151.24, 151.11,
148.67, 147.09, 144.89, 144.76, 137.99, 135.87, 135.78, 132.85, 121.41 (2C), 116.97 (2C), 116.73,
115.27, 115.06, 109.71, 109.49, 55.20 (2C), 49.90 (2C), 48.14, 46.12, 11.03, 4.29. 19F NMR (376 MHz,
19

CDCl3) δ -136.18 (d, J = 22.1 Hz, 1F), -145.05 (d, J = 22.1 Hz, 1F) ppm. ESI/MS for (C26H28F2N8
[M+H]+). Calcd: 491,2478. Found: 491.2482.
N6-(4-chloro-2,5-dimethoxyphenyl)-9-(cyclopropylmethyl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)-
1
9H-purine-2,6-diamine (5e) White solid, yield 38 %, mp 103-104 °C. H NMR (400 MHz, CDCl3) δ
8.18 (s, 1H), 7.81 (s, 1H), 7.47 (s, 1H), 7.24 (d, J = 8.8 Hz, 2H), 6.73 (s, 1H), 6.66 (d, J = 8.9 Hz, 2H),
6.63 (s, 1H), 3.68 (d, J = 7.1 Hz, 2H), 3.61 (s, 3H), 3.38 (s, 3H), 3.03 – 2.83 (m, 4H), 2.46 – 2.36 (m,
13
4H), 2.15 (s, 3H), 1.16 – 0.94 (m, 1H), 0.41 (q, J = 5.4 Hz, 2H), 0.26 – 0.10 (m, 2H). C NMR (101
MHz, CDCl3) δ 156.67, 151.69, 151.33, 148.95, 146.87, 142.67, 138.14, 133.11, 128.03, 121.47 (2C),
120.79, 117.15, 117.00 (2C), 115.84, 114.32, 111.97, 105.90, 56.98, 56.35, 55.06, 49.68, 48.04, 45.91,
11.08, 4.25 ppm. ESI/MS for (C28H33ClN8O2 [M+H]+). Calcd: 549.2488. Found: 549.2490.
N6-(4-chloro-3-methoxyphenyl)-9-(cyclopropylmethyl)-N2-(4-(4-methylpiperazin-1-yl)phenyl)-
9H-purine-2,6-diamine (5f) White solid, yield 42 %, mp 96-98 °C. 1H NMR (400 MHz, CDCl3) δ 7.99
(s, 1H), 7.70 (s, 1H), 7.51 (d, J = 8.8 Hz, 2H), 7.42 (s, 1H), 7.23 (s, 2H), 7.04 (s, 1H), 6.92 (d, J = 8.9
Hz, 2H), 3.93 (d, J = 7.2 Hz, 2H), 3.72 (s, 3H), 3.25 – 3.13 (m, 4H), 2.66 – 2.58 (m, 4H), 2.38 (s, 3H),
1.39 – 1.23 (m, 1H), 0.67 (q, J = 5.4 Hz, 2H), 0.45 (q, J = 5.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ
156.76, 155.02, 151.96, 151.44, 146.94, 138.94, 138.00, 133.04, 129.84, 121.20 (2C), 116.93 (2C),
116.08, 115.29, 112.88, 104.80, 56.06, 55.17, 49.90, 48.13, 46.09, 11.06, 4.29 ppm. ESI/MS for
(C27H31ClN8O [M+H]+). Calcd: 519.2382. Found: 519.2386.
9-(Cyclopropylmethyl)-N2-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-N6-phenyl-9H-purine-2,6-
diamine (5g) White solid, yield 61 %, mp 93-94 °C. 1H NMR (400 MHz, CDCl3) δ 7.69 (s, 1H), 7.66 –
7.62 (m, 1H), 7.61 (d, J = 5.5 Hz, 3H), 7.23 (t, J = 7.9 Hz, 2H), 7.03 – 6.99 (m, 1H), 6.97 (d, J = 4.1 Hz,
2H), 6.80 (t, J = 9.1 Hz, 1H), 3.84 (d, J = 7.2 Hz, 2H), 2.99 (s, 4H), 2.52 (s, 4H), 2.26 (s, 3H), 1.29 –
1.15 (m, 1H), 0.56 (q, J = 5.6 Hz, 2H), 0.35 (q, J = 5.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 156.98,
156.11, 154.56, 152.24, 151.06, 138.81, 138.11, 136.01, 135.90, 134.39, 134.29, 128.91, 123.33, 120.49,
119.11, 119.07, 115.57, 114.51, 114.48, 108.03, 107.77, 55.28, 50.95, 50.93, 48.22, 46.13, 11.08, 4.31.
19F NMR (376 MHz, CDCl3) δ -121.75 (s, 1F) ppm. ESI/MS for (C26H29FN8 [M+H]+). Calcd: 473.2572.
Found: 473.2578.
9-(Cyclopropylmethyl)-N2-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-N6-(4-fluorophenyl)-9H-
purine-2,6-diamine (5h) White solid, yield 63 %, mp 96-103 °C. 1H NMR (400 MHz, CDCl3) δ 8.43 (s,
1H), 7.96 (d, J = 17.6 Hz, 2H), 7.87 (dd, J = 8.3, 4.8 Hz, 2H), 7.55 (s, 1H), 7.32 (d, J = 8.2 Hz, 1H), 7.23
(t, J = 8.5 Hz, 2H), 7.13 (t, J = 9.1 Hz, 1H), 4.18 (d, J = 7.1 Hz, 2H), 3.33 (s, 4H), 2.87 (s, 4H), 2.61 (s,
3H), 1.61 – 1.51 (m, 1H), 0.91 (q, J = 5.1 Hz, 2H), 0.68 (t, J = 4.9 Hz, 2H). 13C NMR (101 MHz, CDCl3)
20

δ 160.15, 157.74, 156.92, 156.14, 154.49, 152.26, 151.08, 138.09, 135.99, 135.88, 134.86, 134.83,
134.36, 134.27, 122.49 (2C), 122.41, 119.06, 119.02, 115.50 (2C), 115.28, 115.25, 114.47, 114.44,
108.00, 107.74, 55.23 (2C), 50.91 (2C), 50.89, 48.20, 46.10, 11.03, 4.29. 19F NMR (376 MHz, CDCl3) δ
-119.37 (s, 1F), -121.69 (s, 1F) ppm. ESI/MS for (C26H28F2N8 [M+H]+). Calcd: 491.2478. Found:
491.2484.
9-(Cyclopropylmethyl)-N2-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-N6-(3-fluorophenyl)-9H-
purine-2,6-diamine (5i) White solid, yield 55 %, mp 93-95 °C. 1H NMR (400 MHz, CDCl3) δ 8.07 (s,
1H), 7.63 (d, J = 11.4 Hz, 1H), 7.57 (s, 1H), 7.52 (dd, J = 14.8, 2.3 Hz, 1H), 7.23 (s, 1H), 7.11 – 6.99 (m,
3H), 6.77 (t, J = 9.1 Hz, 1H), 6.58 (dd, J = 10.4, 5.0 Hz, 1H), 3.79 (d, J = 7.2 Hz, 2H), 2.96 (s, 4H), 2.48
(s, 4H), 2.22 (s, 3H), 1.20 – 1.09 (m, 1H), 0.52 (q, J = 5.5 Hz, 2H), 0.30 (q, J = 5.0 Hz, 2H). 13C NMR
(101 MHz, CDCl3) δ 164.20, 161.78, 156.92, 156.13, 154.49, 151.91, 151.23, 140.66, 140.55, 138.31,
135.78, 135.68, 134.59, 134.49, 129.81, 129.71, 119.16, 119.12, 115.52, 115.38, 115.35, 114.93, 114.90,
109.63, 109.41, 108.25, 108.00, 107.62, 107.35, 55.25 (2C), 50.87, 50.84 (2C), 48.24, 46.11, 11.01, 4.31.
19F NMR (376 MHz, CDCl3) δ -111.86 (s, 1F), -121.72 (s, 1F) ppm. ESI/MS for (C26H28F2N8 [M+H]+).
Calcd: 491.2478. Found: 491.2481.
9-(Cyclopropylmethyl)-N6-(3,4-difluorophenyl)-N2-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-
9H-purine-2,6-diamine (5j) White solid, yield 68 %, mp 94-97 °C. 1H NMR (400 MHz, CDCl3) δ 8.08
(s, 1H), 7.87 (ddd, J = 12.6, 7.2, 2.5 Hz, 1H), 7.71 (s, 1H), 7.65 (dd, J = 14.8, 2.4 Hz, 1H), 7.23 (s, 1H),
7.15 (dd, J = 7.4, 5.3 Hz, 2H), 7.04 (dd, J = 18.5, 9.1 Hz, 1H), 6.92 (t, J = 9.1 Hz, 1H), 3.95 (d, J = 7.2
Hz, 2H), 3.09 (s, 4H), 2.62 (s, 4H), 2.36 (s, 3H), 1.34 – 1.27 (m, 1H), 0.70 – 0.62 (m, 2H), 0.45 (q, J =
13
5.0 Hz, 2H). C NMR (101 MHz, CDCl3) δ 156.91, 156.11, 154.48, 151.84, 151.25, 151.13, 148.82,
148.69, 147.52, 147.39, 145.09, 144.97, 138.32, 135.63, 135.52, 134.90, 134.73, 134.63, 119.15, 119.11,
117.05, 116.87, 115.70, 115.67, 115.35, 114.95, 109.93, 109.71, 108.32, 108.17, 108.07, 55.26 (2C),
53.43, 50.88 (2C), 50.85, 48.27, 46.12, 31.57, 22.64, 14.10, 11.03, 4.31. 19F NMR (376 MHz, CDCl3) δ
-121.65 (s, 1F), -136.18 (d, J = 22.0 Hz, 1F), -144.55 (d, J = 22.0 Hz, 1F) ppm. ESI/MS for (C26H27F3N8
[M+H]+). Calcd: 509.2384. Found: 509.2390.
9-(Cyclopropylmethyl)-N2-(3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)-N6-(4-methoxyphenyl)-
9H-purine-2,6-diamine (5k) White solid, yield 55 %, mp 85-86 °C. 1H NMR (400 MHz, CDCl3) δ 8.03
(s, 1H), 7.75 (dd, J = 15.0, 2.3 Hz, 1H), 7.68 (s, 1H), 7.58 (d, J = 8.9 Hz, 2H), 7.23 (s, 1H), 7.08 (dd, J =
8.6, 2.1 Hz, 1H), 6.88 (dt, J = 9.4, 4.6 Hz, 3H), 3.94 (d, J = 7.2 Hz, 2H), 3.80 (s, 3H), 3.09 (s, 4H), 2.64
(s, 4H), 2.37 (s, 3H), 1.30 (dd, J = 10.2, 5.1 Hz, 1H), 0.66 (q, J = 5.5 Hz, 2H), 0.44 (q, J = 5.1 Hz, 2H).
13C NMR (101 MHz, CDCl3) δ 156.97, 156.18, 156.04, 154.54, 152.57, 150.90, 137.86, 136.25, 136.14,
21

134.08, 133.99, 131.81, 122.86 (2C), 119.11, 119.07, 115.23, 114.23 (2C), 114.20, 114.09, 107.77,
107.51, 55.51, 55.23 (2C), 50.89, 50.86 (2C), 48.16, 46.06, 31.57, 22.64, 14.11, 11.05, 4.28. 19F NMR
(376 MHz, CDCl3) δ -121.55 (s, 1F) ppm. ESI/MS for (C27H31FN8O [M+H]+). Calcd: 503.2678. Found:
503.2677.
N6-(4-chloro-2,5-dimethoxyphenyl)-9-(cyclopropylmethyl)-N2-(3-fluoro-4-(4-methylpiperazin-1-
yl)phenyl)-9H-purine-2,6-diamine (5l) White solid, yield 40 %, mp 97-98 °C. 1H NMR (400 MHz,
CDCl3) δ 8.35 (s, 1H), 8.00 (s, 1H), 7.70 (s, 1H), 7.55 (dd, J = 14.6, 2.3 Hz, 1H), 7.13 (dd, J = 8.6, 2.1
Hz, 1H), 6.87 (dd, J = 10.9, 4.7 Hz, 3H), 3.92 (d, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.71 (s, 3H), 3.14 – 3.02
(m, 4H), 2.63 (s, 4H), 2.36 (s, 3H), 1.28 (dd, J = 9.9, 5.2 Hz, 1H), 0.64 (q, J = 5.4 Hz, 2H), 0.42 (q, J =
13
5.2 Hz, 2H). C NMR (101 MHz, CDCl3) δ 156.91, 155.88, 154.48, 151.74, 151.08, 148.94, 142.88,
138.42, 135.82, 135.72, 134.54, 134.45, 127.79, 119.23, 119.19, 116.17, 114.98, 114.95, 114.78, 112.13,
108.27, 108.02, 106.03, 57.03, 56.37, 55.17 (2C), 50.69 (2C), 48.22, 45.97, 11.11, 4.29. 19F NMR (376
MHz, CDCl3) δ -121.55 (s, 1F) ppm. ESI/MS for (C28H32ClFN8O2 [M+H]+). Calcd: 567.2394.
Found:567.2396.
N6-(4-chloro-3-methoxyphenyl)-9-(cyclopropylmethyl)-N2-(3-fluoro-4-(4-methylpiperazin-1-
yl)phenyl)-9H-purine-2,6-diamine (5m) White solid, yield 60 %, mp 100-101 °C. 1H NMR (400 MHz,
CDCl3) δ 8.44 (s, 1H), 7.79 (s, 1H), 7.74 (d, J = 14.8 Hz, 1H), 7.62 (d, J = 4.5 Hz, 1H), 7.36 (d, J = 8.5
Hz, 1H), 7.24 (dd, J = 17.5, 9.1 Hz, 3H), 6.96 (t, J = 9.1 Hz, 1H), 4.01 (d, J = 7.1 Hz, 2H), 3.78 (s, 3H),
3.18 (s, 4H), 2.71 (s, 4H), 2.45 (s, 3H), 1.36 (dd, J = 7.3, 4.9 Hz, 1H), 0.75 (d, J = 7.5 Hz, 2H), 0.53 (d,
J = 4.5 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 156.87, 156.09, 154.89, 154.44, 152.01, 151.16, 138.72,
138.22, 135.89, 135.79, 134.47, 134.37, 129.77, 119.09, 119.05, 116.29, 115.40, 114.71, 114.68, 113.30,
108.10, 107.84, 104.93, 55.92, 55.21 (2C), 50.86, 50.83 (2C), 48.22, 46.08, 11.01, 4.31. 19F NMR (376
MHz, CDCl3) δ -121.59 (s, 1F) ppm. ESI/MS for (C27H30ClFN8O [M+H]+). Calcd: 537.2288. Found:
537.2292.
4.3 Kinase assays
Abl1 and Cdk-2/cyclin E kinases were produced in-house, and Btk was purchased from ProQinase. The
kinase reactions were assayed with a suitable substrate (500 µM peptide GGEAIYAAPFKK for Abl, 1
mg/mL histone H1 for CDK2, 100 µM poly(Glu:Tyr; 4:1)) in the presence of ATP (1 µM for Abl, 15
µM for Cdk-2 and Btk), 0.05 µCi [γ-33P]ATP, and the test compound in a final volume of 10 µL of
reaction buffer (60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate,
1.2 mM DTT, 2.5 μg/50 μL PEG20.000). The reactions were stopped by adding 5 µL of 3 % aq. H3PO4.
22

Aliquots were spotted onto P-81 phosphocellulose (Whatman), washed 3× with 0.5 % aq. H3PO4 and
finally air-dried. Kinase inhibition was quantified using a FLA-7000 digital image analyzer. The
concentration of the test compounds required to reduce the activity by 50 % was determined from dose-
response curves and reported as the IC50 value.
4.4 2D-QSAR
For the 2D-QSAR study, the molar refractivity (CMR) and lipophilicity (CLogP) parameters were
calculated using ChemBioDraw software (15.1.0, PerkinElmer, Waltham, MA, USA). The multilinear
regression analysis was performed with Statistica software (8.0, StatSoft, Tulsa, OK, USA). The
biological activity was converted to a logarithmic scale (pIC50 = -LogIC50) and was used as a dependent
variable. ClogP, ClogP2, CMR, and CMR2 were used as independent variables for both the whole
molecule and its fragments. Free-Wilson descriptors (Xi) for the presence (Xi = 1) or absence (Xi = 0) of
-F, -Cl and -OCH3 on the different rings were also considered (20 independent variables). All
combinations among the independent variables with biological activity were evaluated. For the sequential
search of combinations, the “best subsets” option was used in the “General Regression Models” menu of
Statistica. The best model presented here contained the fewest number of independent variables to avoid
overfitting,[42] and chance correlation[43] and to obtain the highest correlation coefficient.
4.5 Docking
The 3D structures of compounds synthetized were built using OECHEM followed by protonation states
were adjusted to pH 7.2 using FixpKa from the QUACPAC package. The conformers were generated
using OMEGA software. The crystalline structures of enzymes (Abl and Btk) were downloaded from the
RCSB PDB Protein Data Bank (2GQG[40] and 4OT5[41], respectively). All water, ions and cofactors
were removed. Next, hydrogen atoms and protons were added, and partial charges (in accordance with
the protonation state at the physiological pH) were assigned. The preparation of the proteins was
performed using the Chimera USCF program.[44] This was followed by a local minimization to relieve
potential bad contacts. The minimization was performed in the presence of restraints to maintain the
protein conformation very similar to that observed in the experimental model. The molecular docking
studies were carried out by the FRED program, and docking runs were performed using the fast-rigid
exhaustive docking approach implemented in the software.[45] In this case, the ChemGauss4 scoring
function was used to evaluate and score the resulting docked poses. Next, the best poses were optimized
using SZYBKI, and the energy of binding was estimated by taking into account the solvation energies of
23

the interacting molecules, in addition to the molecular mechanics (MM) energies. The contribution of
polar solvation energies was computed by the generalized Born (GB) implicit solvent model, while the
nonpolar contribution of the solvation energy was dependent on the solvent accessible surface area
(SA).[46] The interactions of residues in the active site were identified using a 6 Å radius around the
docked position as a reference. The pictures were obtained with PyMol software.
4.6 Cell Cultures
Human cell lines were obtained from the German Collection of Microorganisms and Cell Cultures and
were cultivated according to the provider’s instructions. MV4-11 cells were maintained in RPMI-1640
medium supplemented with 10 % fetal bovine serum, penicillin (100 U/mL), and streptomycin (100
µg/mL). CCRF-CEM cells were maintained in RPMI-1640 medium supplemented with 20 % fetal bovine
serum, penicillin (100 U/mL), and streptomycin (100 µg/mL). The cell lines K562 and MCF-7 were
cultivated in DMEM supplemented with 10 % fetal bovine serum, penicillin (100 U/mL), and
streptomycin (100 µg/ml). Ramos cells (obtained from DSMZ) were maintained in IMDM supplemented
with 10 % fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL). All cell lines were
kept at 37 °C in 5 % CO2. BTK activation in cells was induced by treatment with goat F(ab')2 anti-human
IgM purchased from Southern Biotech.
4.7 Cytotoxicity Assays
Cytotoxicity assays were performed using resazurin, an indicator dye to measure oxidation-reduction
reactions occurring in the mitochondria of live cells. The cells were treated in triplicate with six different
doses of each compound for 72 h. After treatment, a resazurin (Sigma Aldrich) solution was added for 4
h, and the fluorescence of resorufin corresponding to live cell quantity was measured at 544 nm/590 nm
(excitation/emission) using a Fluoroskan Ascent microplate reader (Labsystems). The GI50 value, the
drug concentration lethal to 50 % of the cells, was calculated from the dose response curves.
4.8 Immunoblotting
Cell lysates were separated on SDS-polyacrylamide gels and electroblotted onto nitrocellulose
membranes. After blocking, overnight incubation with specific primary antibodies, and incubation with
peroxidase-conjugated secondary antibodies, peroxidase activity was detected with SuperSignal West
Pico reagents (Thermo Scientific) using a CCD camera LAS-4000 (Fujifilm). The following specific
antibodies (Cell Signaling) were used: anti-BTK (#3533), anti-phospho-BTK Y223 (#5082),
24

anti-ERK1/2 (#9101), anti-phospho-ERK1/2 T202/Y204 (#9102), anti-AKT (#4691), anti-phospho-
AKT S473 (#4060), anti-PLCγ2 (#3872), anti-phospho-PLCγ2 Y1217 (#3871), anti-STAT5 (#94205),
anti-phospho-STAT5 Y694 (#9351), anti-Crkl (#3182), and anti-phospho-Crkl Y207 (#3181). Anti-
PCNA (clone PC-10) was generously gifted by Dr. B. Vojtěšek.
Acknowledgments
The authors wish to acknowledge the support from FONDECYT (COS, Research Grant N° 1161816),
the Ministry of Health of the Czech Republic (17-31834A), Palacký University in Olomouc
(IGA_PrF_2019_013) and the European Regional Development Fund (Project ENOCH, No.
CZ.02.1.01/0.0/0.0/16_019/0000868). JLB is grateful to CONICYT for a PhD fellowship (N°
21180975).
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