Trisubstituted purine inhibitors of PDGFRα and their antileukemic activity in the human eosinophilic cell line EOL-1

Article abstract of DOI:10.1016/j.bmc.2017.10.032

Inhibition of protein kinases is a validated concept for pharmacological intervention in cancers. Many kinase inhibitors have been approved for clinical use, but their practical application is often limited. Here, we describe a collection of 23 novel 2,6,9-trisubstituted purine derivatives with nanomolar inhibitory activities against PDGFRα, a receptor tyrosine kinase often found constitutively activated in various tumours. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leukemia cell line EOL-1, whereas several other cell lines were substantially less sensitive. The cytotoxicity in EOL-1, which is known to express the FIP1L1‐PDGFRA fusion gene encoding an oncogenic kinase, correlated significantly with PDGFRα inhibition. EOL-1 cells treated with the compounds also exhibited dose-dependent inhibition of PDGFRα autophosphorylation and suppression of its downstream signaling pathways with concomitant G₁ phase arrest, confirming the proposed mechanism of action. Our results show that substituted purines can be used as platforms for preparing tyrosine kinase inhibitors with specific activity towards eosinophilic leukemia.

Full text of DOI:10.1016/j.bmc.2017.10.032

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Trisubstituted purine inhibitors of PDGFR
a
and their antileukemic
activity in the human eosinophilic cell line EOL-1
b
b
a
b,
Veronika Malínková a,b, Eva Reznícková , Radek Jorda , Tomáš Gucky , Vladimír Kryštof
⇑
ˇ
ˇ
´
a Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacky´ University, Šlechtitelu˚ 27, 783
71 Olomouc, Czech Republic
b
´
Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacky University and Institute of Experimental Botany AS CR,
Šlechtitelu˚ 27, 783 71 Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of protein kinases is a validated concept for pharmacological intervention in cancers. Many
kinase inhibitors have been approved for clinical use, but their practical application is often limited.
Here, we describe a collection of 23 novel 2,6,9-trisubstituted purine derivatives with nanomolar inhibi-
Received 5 September 2017
Revised 12 October 2017
Accepted 20 October 2017
Available online xxxx
tory activities against PDGFRa, a receptor tyrosine kinase often found constitutively activated in various
tumours. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leu-
kemia cell line EOL-1, whereas several other cell lines were substantially less sensitive. The cytotoxicity
in EOL-1, which is known to express the FIP1L1-PDGFRA fusion gene encoding an oncogenic kinase, cor-
related significantly with PDGFR
dose-dependent inhibition of PDGFR
a
inhibition. EOL-1 cells treated with the compounds also exhibited
autophosphorylation and suppression of its downstream signaling
a
pathways with concomitant G1 phase arrest, confirming the proposed mechanism of action. Our results
show that substituted purines can be used as platforms for preparing tyrosine kinase inhibitors with
specific activity towards eosinophilic leukemia.
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
found in various tumors, arising from various mutations in gas-
trointestinal stromal tumors4 and melanoma,5 missense mutations
The development of protein kinase inhibitors has progressed
dramatically in the past years: over 30 kinase inhibitors have been
approved as drugs, and several others are undergoing advanced
clinical evaluation.1,2 Oncogenic kinases often arise as a result of
mutations, overexpression, or chromosomal translocations. More-
over, while chromosomal translocations in hematological malig-
nancies are rarer than in solid tumors, they are significant in
cancer therapy as well as in tumor initiation and progression.3
in childhood acute myeloid leukemia,6 reciprocal translocations in
chronic myeloid leukemia, and deletions that give rise to a fusion
protein in chronic eosinophilic leukemia.7 A typical fusion gene
encoding
a constitutively activated PDGFRa form is FIP1L1-
PDGFRA, which originates from an 800-kb cryptic interstitial
deletion in chromosome 4q12. The fusion gene encodes a ligand-
independent and constitutively active TK that confers growth
factor-independency to hematopoietic cells and is highly sensitive
to the kinase inhibitor imatinib.8,9 This fusion protein is estimated
to occur in 10–20% of eosinophilia cases. The FIP1L1-PDGFRA fusion
has been found in chronic eosinophilic leukemia, hypere-
osinophilic syndrome, eosinophilia-associated myeloproliferative
disorders, acute myeloid leukemia, and lymphoblastic T-cell non-
Hodgkin lymphoma.3 Other fusion partners include BCR, striatin
and ETV6.3
Platelet-derived growth factor receptor
a (PDGFRA, CD140A) is
a type III receptor tyrosine kinase (TK) that regulates cell prolifer-
ation, differentiation, adhesion and survival. Ligand binding acti-
vates the kinase, stimulating cellular signaling proteins including
mitogen-activated protein kinases (MAPKs), signal transducers
and activators of transcription (STATs), SRC and phosphatidylinos-
itol-3 kinases. Constitutively activated forms of PDGFRa have been
Various TK inhibitors have shown activity in FIP1L1-PDGFRA-
positive cells, including imatinib,9 sorafenib10 or ponatinib.11 These
compounds blocked proliferation and induced apoptosis, primarily
Abbreviations: PDGFRa, platelet derived growth factor receptor alpha; CDK2,
cyclin-dependent kinase 2; TK, tyrosine kinase; MAPK, mitogen-activated protein
kinase; STAT, signal transducers and activators of transcription.
as a result of direct FIP1L1-PDGFRa
inhibition.9 In clinical settings,
however, most patients require life-long treatment with these
⇑
Corresponding author.
E-mail address: vladimir.krystof@upol.cz (V. Kryštof).
agents, and resistance has developed in some cases.8,12 There is
https://doi.org/10.1016/j.bmc.2017.10.032
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
even a recent report of a patient with imatinib non-responsive
chronic eosinophilic leukemia, although in that patient’s tumor,
conditions to obtain a 2,6-dichloro-9-substituted-9H-purine with
good regioselectivity.14,15 The crude product was purified by crys-
tallization from methanol or column chromatography (petroleum
ether: ethyl acetate, 2:1).
PDGFR
of novel PDGFR
a
was fused with ETV6 rather than FIP1L1.13 Identification
a
inhibitors or thorough selectivity determination
for previously described compounds is therefore still a challenge.
We have previously prepared several series of 2,6,9-trisubsti-
tuted purines14,15 and related purine isosteres such as pyrazolo
[4,3-d]pyrimidines16 and pyrazolo[1,5-a]pyrimidines17 that selec-
tively inhibit cyclin-dependent kinases (CDK). Optimization of
these compounds yielded other potent CDK inhibitors as well as
new compounds with different kinase selectivities. Changes in
selectivity were achieved by using different substitution pat-
terns,18 or more simply, by replacing the 6-benzylamino moiety
with a 6-phenylamino group. Independent studies confirmed that
this shortening conferred potency against various other kinases,
including TKs, while retaining some degree of CDK inhibition. Such
compounds (some of them are illustrated in Fig. 1) include the
PAK4 inhibitor CGP74514A,19 the MER receptor inhibitor anilinop-
urine C52,20 the SRC and VEGFR2 inhibitor BDBM50451553,21 and
the SRC and BCR-ABL inhibitor AP23464.22 Importantly, the 6-
phenylaminopyrimidine motif present in all these compounds is
also present in several TK inhibitors such as erlotinib, gefitinib,
and lapatinib (Fig. 1), suggesting that substituted purines may also
serve as a source of TK inhibitors.
The second step was nucleophilic substitution at the C6 purine
position with a suitable substituted aniline or benzylamine. This
reaction was performed in n-propanol, using N,N-diisopropylethy-
lamine as the base. The reaction temperature was kept in the range
of 100–120 °C for 3–6 h, depending on aniline reactivity. Crude
products were purified by crystallization or column chromatogra-
phy (petroleum ether: ethyl acetate, 2:1).
The final step was nucleophilic aromatic substitution at the C2
purine position using a large excess of trans-1,4-diaminocyclohex-
ane at 160 °C for a few hours via solution-phase synthesis strategy
or via microwave heating. The use of microwave irradiation signif-
icantly reduced the time required for this reaction. Final com-
pounds
were
purified
by
crystallization
or
column
chromatography on silica (Table 1).
2.2. Biological and biochemical activity
All the prepared compounds were subjected to biochemical
assays to determine their activity against CDK2 and PDGFR , and
a
cytotoxicity assays using 5 cell lines with differing expression of
oncogenic TKs. We also performed phenotype screening to evalu-
ate cell cycle changes in treated cells. The results obtained are
summarized in Table 2. The previously reported structure–activity
relationships for 2,6,9-trisubstituted purines suggest that position
6 can host both phenyl and benzyl groups without significantly
altering activity against CDK2 (compare 4u with 4b, 4q with
4a).24,25 Further substitution of the phenyl or benzyl ring with
chlorine, hydroxy or methoxy groups also did not greatly affect
potency. On the other hand, CDK2 inhibition was clearly weakened
by increasing the size of the substituent in position 9. The most
potent CDK2 inhibitors bear a butyl chain in this position; bulkier
substituents (ranging from isopentyl in 4b to the bulkier benzyl
group in 4d or the longer geranyl unit in 4e) clearly reduced activ-
ity against CDK2.
In this paper we describe potent PDGFRa inhibitors with selec-
tive activity against the eosinophilic leukemia cell line EOL-1.
Structure-activity analyses and co-crystal structures of CDKs with
purine inhibitors indicated that the optimal substituents at posi-
tions 2 and 9 are branched alkyl groups with polar substituents
(e.g. aminocyclohexylamine) and a small alkyl or cycloalkyl group,
respectively.15,23,24 We expected that 2-(4-aminocyclohexyl)
amino-6-anilinopurines with enlarged substituents at position 9
would be weaker CDK inhibitors but more effective inhibitors of
TKs, and thus more active towards cell lines with aberrant TK
expression.
2. Results
2.1. Synthesis
In contrast to CDK2 inhibitors, the presence of an anilino func-
tion is necessary for PDGFR
a inhibition; while several anilino
The three-step synthesis of 2,6,9-trisubstituted purines started
from commercially available 2,6-dichloropurine, which was ini-
tially alkylated at N9 with a suitable alcohol under Mitsunobu
derivatives exhibited IC50 values below 0.1 mM, its replacement
with a homologous benzyl group completely eliminated this activ-
ity (compounds 4a and 4b). This is consistent with several studies
Fig. 1. Some inhibitors of cyclin-dependent kinases and tyrosine kinases.
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V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Scheme 1. General reaction scheme for synthesis of 2,6,9-trisubstituted purine derivatives. (a) The appropriate alcohol R1-OH (2 equiv), PPh3 (1.2 equiv), dry THF, DIAD (1.2
equiv), 0°C; (b) the appropriate amine or benzylamine (1.2 equiv), DIPEA (3 equiv), n-propanol, 100–120 °C (sealed tube), 3–6 h; (c) trans-1,4-diaminocyclohexane (15 equiv),
n-butanol, 160 °C (sealed tube), 4–10 h.
weakly to the compounds, with mid-micromolar GI50 values. Con-
Table 1
versely, the breast cancer cell line MCF7 responded strongly, with a
Structures of novel 2,6,9-trisubstituted purine analogues used in this study.
single-digit micromolar GI50 value, despite having no known tyro-
Compounda
R1
R2
sine kinase mutations. The leukemic cell line K562, which
expresses the BCR-ABL fusion kinase, was similarly responsive.
The compounds had similar cytotoxicity patterns in all tested
cell lines other than the eosinophilic leukemia cell line EOL-1
(Table 2). Most of them were much more active against this line
(with submicromolar to mid-nanomolar GI50 values) and had com-
pletely different relative activity levels, indicating a different
mechanism of action in this line. We hypothesized that this may
be due to the EOL-1 line’s expression of the FIP1L1-PDGFRA fusion
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
Butyl
3-Chlorobenzyl
3-Chlorobenzyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
2,4-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
4-Hydroxyphenyl
4-Chlorophenyl
4-Hydroxyphenyl
3,5-Dimethoxyphenyl
3-Chlorophenyl
3-Chlorophenyl
Phenyl
4-Chlorophenyl
3-Chlorophenyl
4-Hydroxyphenyl
Phenyl
3-Hydroxyphenyl
3-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
Isopentyl
4-Methoxybenzyl
Benzyl
3,7-Dimethylocta-2,6-dien-1-yl
Isopentyl
Butyl
Hexyl
Cyclohexyl
Cyclohexyl
Hexyl
Isopentyl
Hexyl
3-Methylbut-2-en-1-yl
Butyl
Hexyl
gene, which encodes an oncogenic variant of PDGFRa. This line has
been identified as an in vitro model for studying FIP1L1-PDGFRA-
positive chronic eosinophilic leukemia and for analysing FIP1L1-
PDGFR
toxicity in EOL-1 correlated strongly with PDGFR
a
inhibitors.9 Indeed, we found that the compounds’ cyto-
a
inhibition as
determined in a biochemical assay (Pearson coeff. = 0.76, p <
0.001, Fig. 2).
Butyl
Isopentyl
Isopentyl
Isopentyl
Isopentyl
Butyl
4s
4t
4u
4v
4w
To our knowledge, CDK inhibitors usually induce cell cycle
arrest preferentially in G2-M or in both G1 and G2-M, whereas
receptor tyrosine kinase inhibitors often produce clear G1 phase
arrest. We therefore performed cell cycle measurements in the
EOL-1, HCC827 and K562 cell lines after treatment with the new
compounds. For each compound, we measured the cell cycle
changes after 24 h treatment at a concentration corresponding to
the compound’s GI50, and determined the relative G1/G2-M ratio.
Illustrative cell cycle profiles for two potent compounds (4v and
4u) along with lapatinib and CGP74514A as controls (EGFR and
CDK inhibitors, respectively) are presented in Fig. 3. As shown in
this figure and Table 2, the most sensitive cell line EOL-1 entered
G1 cell cycle arrest (corresponding to a relative G1/G2-M ratio
above 1) upon treatment, in keeping with this line’s dependence
Isopentyl
a
See Scheme 1 for position of R1 and R2.
on kinase inhibitor selectivity, which reported that the 2,9-disub-
stituted-6-benzylpurine roscovitine also does not inhibit PDGFR
a
or other tyrosine kinases (IC50 > 10 mM).23,26,27 Other reports how-
ever showed that 6-anilino purines can inhibit tyrosine kinases. For
example, AP23464 was identified as a potent inhibitor of SRC and
BCR-ABL,28,22 and compound C52 inhibits MER.20
Analogues bearing chlorine- or hydroxyl-functionalized anilino
groups at the C6 position did not exhibit appreciably stronger
activity towards PDGFR
on the PDGFRa kinase. Importantly, we found that the compound’s
PDGFR inhibiting activity correlated strongly with their relative
a
a than the parent compound with a non-
G1/G2-M ratios (Pearson coefficient = ꢀ0.73, Fig. 2). Unlike EOL-1,
the other tested cell lines exhibited G2-M arrest. The different
effects observed in these cell lines suggest that the compounds
may target PDGFRa rather than CDK2 at equitoxic doses, but inter-
act with other kinases if their primary target is absent.
functionalized anilino group. However, dimethoxylated anilino
groups were usually detrimental to activity: compounds 4h, 4f,
4g exhibited only micromolar inhibition. The activities of 4f and
4l suggest that 3,5-dimethoxy substitution is preferred to the
2,4-configuration.
Because 4e showed some activity against the receptor kinase
HER4, which is related to EGFR (preliminary screening, see Supple-
mentary data), we were surprised that the HCC827 and K562 cell
lines entered G2-M arrest when treated with this compound. We
therefore expect that the compounds may preferentially target
kinases other than receptor tyrosine kinases, potentially including
CDKs. The weak correlation between cytotoxicity in HCC827 and
CDK2 inhibition may support this hypothesis (Pearson coeff. =
0.55, Fig. 2). While no clear relationship between cytotoxicity
and CDK inhibition has been found, targeting CDK inhibition may
at least partly contribute to the compounds’ mechanism of antipro-
liferative activity.
Our results also demonstrate that the substituent at position 9
of the purine should not be too bulky because benzyl derivatives
4c and 4d and geranyl derivative 4e were among the weakest inhi-
bitors, with IC50 values above 1 mM. PDGFRa was most sensitive to
derivatives with linear or branched chains of 4–5 carbon atoms at
the N9 position.
Cancer cell lines for cytotoxicity assays were selected with
respect to their expression of oncogenic kinases. The prepared
compounds were active against every cell line included in the
panel, but their activities were quite distinct (Table 2). The non-
small cell lung cancer cell line HCC827 and the breast cancer cell
line BT474 both have activating mutations in EGFR, and responded
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V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 2
Activity of novel compounds in cellular and biochemical assays.a
Compound
IC50 (mM)
GI50 (mM)
Relative G1/G2-M ratiob
CDK2
PDGFR
a
HCC827
BT474
MCF7
K562
EOL-1
EOL-1
HCC827
K562
CGP74514A
Lapatinib
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
0.017
>20
>20
>20
>20
>20
12.2
22.3
10.6
16.7
18.7
13.7
18.2
32.9
20.1
9.1
5.4
0.2
4.7
6.6
8.2
10.2
4.3
7.9
15.6
3.7
27.3
4.1
7.7
5.9
5.1
7.0
4.9
5.5
5.1
7.7
6.4
14.7
3.8
7.3
5.1
3.3
8.1
2.2
4.7
4.8
3.6
3.6
6.3
4.5
3.5
9.6
2.3
3.5
4.5
2.7
4.2
2.2
4.2
2.0
3.1
5.4
12.6
2.1
2.1
2.7
4.8
5.7
4.3
3.1
2.8
3.0
2.2
5.5
2.4
1.9
8.9
1.7
5.4
2.5
2.3
3.5
3.2
2.1
2.0
4.6
3.5
7.6
1.5
2.7
1.8
0.99
0.41
0.35
1.33
0.53
0.38
0.59
0.63
0.37
0.38
0.22
0.14
0.19
0.14
0.31
0.08
0.02
0.44
0.16
0.03
0.01
0.06
0.06
0.05
0.09
1.28
1.17
0.99
1.77
0.96
0.99
1.13
1.65
1.30
3.78
2.44
2.64
2.50
3.71
1.86
3.10
4.93
4.61
10.75
1.17
1.67
8.94
6.00
6.43
7.80
0.29
12.1
0.26
2.50
1.94
10.37
1.94
23.09
1.35
9.67
9.61
2.68
0.44
1.47
7.37
2.69
0.33
5.20
0.54
2.71
0.39
5.60
1.33
0.28
3.50
0.48
0.81
1.27
1.85
1.43
1.76
1.78
1.00
0.82
1.40
0.95
0.79
0.69
1.60
0.79
0.75
2.29
0.88
0.78
0.73
1.20
1.22
1.10
1.52
0.81
0.207
0.306
5.504
2.830
7.170
5.967
0.442
0.680
1.441
0.965
0.485
0.980
0.500
0.227
0.134
0.433
0.132
0.490
0.627
0.513
0.275
0.117
0.319
7.655
5.099
4.739
3.197
1.496
1.412
1.233
1.090
0.452
0.290
0.195
0.187
0.160
0.156
0.154
0.150
0.096
0.066
0.066
0.060
0.059
32.5
9.1
4k
4l
24.7
11.2
10.2
13.5
9.0
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
11.7
7.2
14.4
21.7
21.3
10.3
10.8
10.2
a
Data are averaged from at least three determinations.
Cell cycle effect at a dose corresponding to the GI50 value. Data were normalized against results for untreated controls, so a relative G1/G2-M ratio close to 1 means the
b
tested compound had no effect.
Fig. 2. Pearson correlation coefficients for measured parameters. Stars indicate
statistical significance (*P < 0.01, **P < 0.001).
2.3. Mechanism of cellular activity
The most sensitive cell line, EOL-1, bears an oncogenic activa-
tion of PDGFRA gene, which is consistent with our hypothesis that
inhibition of this kinase is linked to the compounds’ cellular activ-
ity. We therefore used immunoblotting to determine whether the
compounds’ cytotoxicity and the G1 phase arrest relate to PDGFR
a
inhibition. Several compounds with various activity levels were
applied to EOL-1 cells at two doses for 1 h, after which the cells
were harvested and analyzed for cellular signaling downstream
of PDGFR
and MEK1/2 and their phosphoforms, which are known to be
induced by PDGFR signaling (Fig. 4). There were clear changes
a. We immunoblotted the lysates for STAT3, ERK1/2,
a
in the abundance of the phosphoforms of all three proteins. Two
potent compounds, 4t and 4o, strongly suppressed the phosphory-
lation of STAT3 and ERK1/2 at both tested concentrations, whereas
the less active 4j and 4p only reduced the abundance of these
phosphoproteins at the higher concentration, and the least active
Fig. 3. Cell cycle measurements for EOL-1, HCC827 and K562 treated with 4u and
4v for 24 h. CGP74514A and lapatinib (CDK and EGFR inhibitor, respectively) were
used as positive controls. The compounds were used at doses corresponding to their
GI50 values.
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V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Fig. 4. Inhibition of PDGFR
a
downstream signaling by compounds with strong (4t and 4o), medium (4j and 4p) and weak (4a and 4b) inhibition of PDGFR
a
in EOL-1 cells
treated for 1 h.
compounds 4a and 4b did not affect these phosphoproteins at all.
Phosphorylation of MEK1/2 at serines 217/221 decreased slightly
only in cells treated with the most potent 4t and 4o. These obser-
vations correspond well with the kinase inhibition assay results.
In the next experiment, we treated EOL-1 cells for 1 h with one
of the most potent compounds, 4u, to confirm that its mechanism
These tyrosines are autophosphorylated upon external stimulation
by PDGF, but are constitutively phosphorylated independently of
cytokine presence in EOL-1 due to the formation of the FIP1L1-
PDGFR
PDGFRa 29
STAT3 and ERK1/2 phosphoforms. When the cells were treated
for periods longer than 4 h, they started to degrade FIP1L1-PDGFRa
a
fusion, which interrupts the juxtamembrane domain of
.
Corresponding downregulations were also seen for the
of action is specifically coupled to PDGFR
Immunoblotting revealed dose-dependent inhibition of PDGFR
autophosphorylation at tyrosines 754, 849 and 1018 (Fig. 5a).
a
inhibition.
a
(data not shown), ceasing to proliferate and showing markers of
apoptosis after 24 h (Fig. 5b).
Fig. 5. Cellular effects of compound 4u in EOL-1. (a) Inhibition of PDGFRa and its downstream signaling pathways after 1 h. (b) Cell cycle in EOL-1 cells treated for 24 h.
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V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
the expected values. Merck silica gel Kieselgel 60 (230–400 mesh)
3. Conclusion
was used for column chromatography. The purity of biologically
evaluated compounds was >95% as determined by HPLC-DAD-MS
and elemental analysis.
A collection of 23 novel 2,6,9-trisubstituted purine derivatives
with nanomolar inhibitory activities against PDGFRa has been pre-
pared. The compounds demonstrated strong and selective cytotox-
icity in the human eosinophilic leukemia cell line EOL-1, which
expresses the oncogenic kinase FIP1L1-PDGFRA. Their cytotoxicity
4.2. Protocol for preparation of compounds 2a–2h
in EOL-1 correlated significantly with PDGFR
over, they exhibited dose-dependent inhibition of PDGFR
a inhibition. More-
2,6-Dichloro-9H-purine (15.8 mmol), an appropriate alcohol
(31.7 mmol), and triphenylphosphine (19.0 mmol) were dissolved
in dry tetrahydrofuran (100 mL) and cooled to 0 °C. Diisopropyl
azodicarboxylate (19.0 mmol) was added dropwise to the stirred
solution under an argon atmosphere, and the temperature was
kept between 0 and 20 °C. The reaction mixture was stirred under
an argon atmosphere at 20 °C for a further 2–4 h. The reaction was
monitored by TLC until completion. The reaction mixture was then
evaporated under reduced pressure, and the residue was dissolved
in boiling toluene (100 mL). After cooling to room temperature, the
solution was inoculated with a small amount of triphenylphos-
phine oxide and the solution was kept at 4 °C for 24 h. The triph-
enylphosphine oxide was then filtered off and the filtrate was
evaporated under reduced pressure. The residue was crystallized
from ethanol or purified by column chromatography on silica (pet-
roleum ether: ethyl acetate, 2:1) to afford the pure product.
a
autophosphorylation and suppressed its downstream signaling
pathways in treated cells, confirming the proposed cellular mech-
anism of action. Our results suggest that substituted purines are
versatile platforms for preparing tyrosine kinase inhibitors with
specific activity towards eosinophilic leukemia and other cancers
expressing constitutively activated PDGFRa mutants such as hepa-
tocellular carcinoma, gastrointestinal stromal tumors or
melanoma.4,5,30
4. Experimental section
4.1. Chemistry and general information
All microwave irradiation experiments were carried out in a
dedicated CEM-Discover mono-mode microwave apparatus. The
reactor was used in the standard configuration as delivered,
including proprietary software. The reactions were carried out in
10 mL glass vials sealed with a silicone/PTFE top, which can be
exposed to a maximum of 250 °C and 21 bar internal pressure.
The temperature was measured with an IR sensor on the outer sur-
face of the process vial. After the irradiation period, the reaction
vessel was cooled to ambient temperature by gas jet cooling. Melt-
ing points were determined on Buchi Melting Point B-540 appara-
tus and were uncorrected. 1H and 13C NMR spectra were recorded
on a Jeol 500 ECA instrument operating at 500 MHz for 1H and 126
MHz for 13C at ambient temperature in DMSO d6. Chemical shifts
are reported in ppm. Coupling constants (J) are reported in Hertz
(Hz), and the following abbreviations are used: singlet (s), doublet
(d), doublet of doublet (dd), triplet (t), doublet of triplet (dt), quar-
tet (q), quintet (qui), sextet (sex), nonet (n), multiplet (m). Mass
spectra were recorded using an LCQ ion trap mass spectrometer
(Finnigan MAT). The chromatographic purity of the compounds
was determined using HPLC-DAD-MS. An Alliance 2695 separa-
4.2.1. 9-Butyl-2,6-dichloro-9H-purine (2a)
Yield: 86%; M.p. = 79–80 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.71 (s, 1H), 4.22 (t, J = 7.0, 2H), 1.85–1.70 (m, 2H), 1.32–
1.16 (m, 2H), 0.85 (t, J = 7.4, 3H). 13C NMR (126 MHz, DMSO d6) d
(ppm): 153.89, 151.34, 150.00, 148.84, 130.88, 44.25, 31.43,
19.72, 13.81. HPLC-MS (ESI+): 245.13 (99.8%). Elemental analysis
Calcd. for C9H10Cl2N4 (245.11): C, 44.10; H, 4.11; N, 22.86. Found:
C, 43.97; H, 4.05; N, 22.53.
4.2.2. 2,6-Dichloro-9-isopentyl-9H-purine (2b)
Yield: 83%; M.p. = 80–81 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.73 (s, 1H), 4.22 (t, J = 7.5, 2H), 1.70 (q, J = 7.1, 2H), 1.44
(n, J = 6.5, 1H), 0.88 (d, J = 6.6, 6H). 13C NMR (126 MHz, DMSO d6)
d (ppm): 156.68, 153.93, 151.33, 148.95, 130.93, 42.91, 38.14,
25.57, 22.57. HPLC-MS (ESI+): 259.32 (99.8%). Elemental analysis
Calcd. for C10H12Cl2N4 (259.14): C, 46.35; H, 4.67; N, 21.62. Found:
C, 46.02; H, 4.58; N, 21.39.
4.2.3. 2,6-Dichloro-9-(3-methylbut-2-en-1-yl)-9H-purine (2c)
Yield: 96%; M.p. = 95–96 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.66 (s, 1H), 4.80 (d, J = 7.2, 2H), 1.76 (t, J = 7.2, 1H), 1.77
(s, 3H), 1.68 (s, 3H). 13C NMR (126 MHz, DMSO d6) d (ppm):
153.71, 151.41, 149.97, 148.68, 138.97, 131.03, 118.34, 42.36,
25.89, 18.42. HPLC-MS (ESI+): 257.63 (96.50%). Elemental analysis
Calcd. for C10H10Cl2N4 (257.12): C, 46.71; H, 3.92; N, 21.79. Found:
C, 46.59; H, 3.95; N, 21.63.
tions module (Waters) linked simultaneously to
a PDA 996
(Waters) and a Q-Tof micro (Waters) benchtop quadrupole orthog-
onal acceleration time-of-flight tandem mass spectrometer were
used. Samples were dissolved in methanol and diluted to a concen-
tration of 10
10
mm; 3.5
l
gꢁmL–1 in the mobile phase (initial conditions). Then,
l
L of the solution were injected on a RP-column (150 mm ꢂ 2.1
l
m; Symmetry C18, Waters). The column was kept in a
thermostat at 25 °C. Solvent (A) consisted of 15 mM formic acid
adjusted to pH 4.0 with ammonium hydroxide. Methanol was used
as the organic modifier (solvent B). At flow rate of 0.2 mLꢁmin–1
,
4.2.4. 2,6-Dichloro-9-hexyl-9H-purine (2d)
Yield: 75%; M.p. = 82–84 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.70 (s, 1H), 4.19 (t, J = 7.2, 2H), 1.78 (qui, J = 7.2, 2H),
1.27–0.91 (m, 6H), 0.76 (t, J = 7.1, 3H). 13C NMR (126 MHz,
DMSO d6) d (ppm): 156.69, 153.99, 151.35, 149.03, 130.97, 44.49,
31.09, 29.29, 26.05, 22.44, 14.34. HPLC-MS (ESI+): 273.19 (99.8%).
Elemental analysis Calcd. for C11H14Cl2N4 (273.16): C, 48.37; H,
5.17; N, 20.51. Found: C, 48.21; H, 4.96; N, 20.33.
the following binary gradient was used: 0 min, 10% B; 0–24 min,
a linear gradient to 90% B, followed by 10 min isocratic elution of
90% B. At the end of the gradient, the column was re-equilibrated
to the initial conditions for 10 min. The effluent was introduced
into the DAD (scanning range 210–400 nm, with 1.2 nm resolu-
tion) and an electrospray source was applied (source temperature
110 °C, capillary voltage +3.0 kV, cone voltage +20 V, desolvation
temperature 250 °C). Nitrogen was used as both the desolvation
gas (500 Lꢁh–1) and the cone gas (50 Lꢁh–1). The mass spectrometer
was operated in positive (ESI+) ionization mode, and data were
acquired in the 50–1000 m/z range. Elemental analyses were per-
formed using an EA1112 CHN analyser (Thermo Finnigan); the
results obtained for C, H, and N were within acceptable limits of
4.2.5. 2,6-Dichloro-9-cyclohexyl-9H-purine (2e)
Yield: 79%; M.p. = 111–112 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.82 (s, 1H), 4.41 (qui, J = 6.3, 1H), 2.04–1.95 (m, 2H),
1.93–1.77 (m, 4H), 1.69–1.65 (m, 1H), 1.49–1.35 (m, 2H), 1.30–
1.16 (m, 1H). HPLC-MS (ESI+): 271.17 (99.7%). Elemental analysis
Please cite this article in press as: Malínková V., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.032

V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Calcd. for C11H12Cl2N4 (271.15): C, 48.73; H, 4.46; N, 20.66. Found:
C, 48.67; H, 4.39; N, 20.44.
127.34, 126.54, 118.70, 43.18, 42.05, 38.63, 25.58, 22.63. HPLC-
MS (ESI+): 364.80 (99.53%). Elemental analysis Calcd. for C17H19
-
Cl2N5 (364.27): C, 56.05; H, 5.26; N, 19.23. Found: C, 56.18; H,
5.23; N, 19.24.
4.2.6. 2,6-Dichloro-9-(4-methoxybenzyl)-9H-purine (2f)
Yield: 91%; M.p. = 121–123 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.77 (s, 1H), 7.28 (d, J = 8.5, 2H), 6.86 (d, J = 8.5, 2H), 5.36
(s, 2H), 3.67 (s, 3H). 13C NMR (126 MHz, DMSO d6) d (ppm):
159.63, 153.75, 151.60, 150.25, 148.78, 131.01, 129.90, 128.02,
114.66, 60.16, 47.05. HPLC-MS (ESI+): 309.74 (95.4%). Elemental
analysis Calcd. for C13H10Cl2N4O (309.15): C, 50.51; H, 3.26; N,
18.12. Found: C, 50.42; H, 3.35; N, 18.03.
4.3.3. 2-Chloro-N-(3-chlorophenyl)-9-(4-methoxybenzyl)-9H-purin-
6-amine (3c)
Yield: 71%; M.p. = 115–117 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.47 (s, 1H), 8.40 (s, 1H), 8.01 (t, J = 2.5, 1H), 7.79 (d, J =
8.5, 1H), 7.34 (t, J = 8.0, 1H), 7.27 (d, J = 8.5, 2H), 7.10 (d, J = 7.5,
1H), 6.88 (d, J = 8.5, 2H), 5.29 (s, 2H), 3.68 (s, 3H). 13C NMR (126
MHz, DMSO d6) d (ppm): 159.50, 152.75, 152.62, 151.38, 143.12,
140.97, 133.37, 130.70, 129.72, 128.88, 123.44, 120.85, 119.85,
4.2.7. (E)-2,6-Dichloro-9-(3,7-dimethylocta-2,6-dien-1-yl)-9H-purine
(2g)
114.72, 55.62. Elemental analysis Calcd. for
C19H15Cl2N5O
Yield: 51%; M.p.= 101–102 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.21 (s, 1H), 5.22 (t, J = 6.8, 1H), 4.99–4.86 (m, 3H), 2.05–
1.87 (m, 4H), 1.78 (s, 3H), 1.52 (s, 3H), 1.45 (s, 3H). HPLC-MS (ESI
+): 325.22 (96.20%). Elemental analysis Calcd. for C15H18Cl2N4
(325.24): C, 55.39; H, 5.58; N, 17.23 Found: C, 55.32; H, 5.60; N,
17.21.
(400.26): C, 57.01; H, 3.78; N, 17.50. Found: C, 57.06; H, 3.84; N,
17.17. HPLC-MS (ESI+): 400.71 (95.10%).
4.3.4. 9-Benzyl-2-chloro-N-(3-chlorophenyl)-9H-purin-6-amine (3d)
Yield: 65%; M.p. = 124–125 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.49 (s, 1H), 8.43 (s, 1H), 8.00 (s, 1H), 7.79 (d, J = 8.1,
1H), 7.42–7.21 (m, 6H), 7.09 (d, J = 7.7, 1H), 5.37 (s, 2H). 13C NMR
(126 MHz, DMSO d6) d (ppm): 154.92, 154.57, 153.24, 144.13,
140.79, 137.47, 134.43, 130.17, 128.68, 128.48, 127.74, 122.58,
122.54, 120.89, 120.18, 48.86. HPLC-MS (ESI+): 370.76 (96.26%).
Elemental analysis Calcd. for C18H13Cl2N5 (370.24): C, 58.39; H,
3.54; N, 18.92. Found: C, 58.23, H, 3.59, N, 18.73.
4.2.8. 9-Benzyl-2,6-dichloro-9H-purine (2h)
Yield: 93%; M.p. = 148–149 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.81 (s, 1H), 7.39–7.16 (m, 5H), 5.45 (s, 2H). 13C NMR
(126 MHz, DMSO d6) d (ppm): 153.90, 151.66, 150.33, 148.95,
136.16, 131.01, 129.34, 128.63, 128.14, 47.64. HPLC-MS (ESI+):
279.72 (99.8%). Elemental analysis Calcd. for
(279.12): C, 51.64; H, 2.89; N, 20.07. Found: C, 51.56; H, 2.95; N,
19.90.
C12H8Cl2N4
4.3.5. (E)-2-Chloro-N-(3-chlorophenyl)-9-(3,7-dimethylocta-2,6-dien-
1-yl)-9H-purin-6-amine (3e)
4.3. Protocol for preparation of compounds 3a–3w
Yield: 73%; 1H NMR (500 MHz, DMSO d6) d (ppm): 10.44 (s, 1H),
8.26 (s, 1H), 8.02 (t, J = 1.5, 1H), 7.80 (d, J = 8.0, 1H), 7.34 (t, J = 8.5,
1H), 7.09 (d, J = 7.5, 1H), 5.37 (t, J = 6.0, 1H), 4.97 (t, J = 6.0, 1H), 4.73
(d, J = 7.0, 2H), 2.07–1.97 (m, 4H), 1.76 (s, 3H), 1.54 (s, 3H), 1.48 (s,
3H). 13C NMR (126 MHz, DMSO d6) d (ppm): 152.54, 151.30,
142.80, 141.53, 141.04, 133.37, 131.59, 130.72, 124.23, 123.39,
120.77, 119.77, 119.48, 118.95, 41.56, 39.31, 26.16, 25.94, 18.05,
16.68. HPLC-MS (ESI+): 416.14 (93.30%). Elemental analysis Calcd.
for C21H23Cl2N5 (416.35): C, 60.58; H, 5.57; N, 16.82. Found: C,
60.51; H, 5.63; N, 16.77.
To a suspension of 9-substituted-2,6-dichloro-9H-purine (6.25
mmol) in a mixture of n-propanol (40 mL) and N,N-diisopropyl-
N-ethylamine (18.75 mmol), the appropriate amine or benzy-
lamine (7.5 mmol) was added. The suspension was heated with
stirring in a sealed tube under an argon atmosphere at 120 °C until
completion of the reaction was indicated by TLC. After cooling to
room temperature, the reaction mixture was evaporated under
reduced pressure and the residue was partitioned between water
(50 mL) and dichloromethane (50 mL). In addition, the water phase
was extracted with dichloromethane (2 ꢂ 30 mL). The combined
organic phases were washed with water (30 mL) and brine (30
mL), dried over anhydrous Na2SO4, filtered, and evaporated under
reduced pressure. If necessary, the crude product was purified by
column chromatography on silica (petroleum ether: ethyl acetate,
2:1).
4.3.6. 2-Chloro-N-(2,4-dimethoxyphenyl)-9-isopentyl-9H-purin-6-
amine (3f)
Yield: 71%; M.p. = 144–145 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.01 (s, 1H), 8.22 (s, 1H), 7.51 (d, J = 7.1, 1H), 6.64 (d, J =
2.6, 1H), 6.53 (dd, J = 8.7, J0 = 2.6, 1H), 4.11 (t, J = 7.4, 2H), 3.75 (d,
J = 4.3, 6H), 1.66 (q, J = 7.0, 2H), 1.45 (n, J = 6.5, 1H), 0.88 (d, J =
6.6, 6H). 13C NMR (126 MHz, DMSO-d6) d (ppm): 158.53, 153.14,
153.08, 142.55, 129.29, 120.23, 118.95, 104.94, 99.70, 56.31,
55.89, 42.11, 38.60, 25.62, 22.66. HPLC-MS (ESI+): 376.43
(99.98%). Elemental analysis Calcd. for C18H22ClN5O2 (375.15): C,
57.52; H, 5.90; N, 18.63. Found: C, 57.38; H, 5.72; N, 18.58.
4.3.1. 9-Butyl-2-chloro-N-(3-chlorobenzyl)-9H-purin-6-amine (3a)
Yield: 61%; M.p. = 120–123 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.79 (t, J = 5.9, 1H), 8.15 (s, 1H), 7.35 (s, 1H), 7.32–7.21
(m, 3H), 4.59 (d, J = 6.0, 2H), 4.05 (t, J = 7.0, 2H), 1.74–1.65 (m,
2H), 1.23–1.13 (m, 2H), 0.82 (t, J = 7.4, 3H). 13C NMR (126 MHz,
DMSO d6)
d (ppm): 155.29, 153.41, 150.55, 142.41, 142.15,
133.43, 130.72, 127.69, 127.32, 126.55, 118.65, 43.34, 43.16,
31.82, 19.72, 13.87. HPLC-MS (ESI+): 350.82 (99.98%). Elemental
analysis Calcd. for C16H17Cl2N5 (350.25): C, 55.19; H, 4.34; N,
20.11. Found: C, 55.02; H, 4.38; N, 19.98.
4.3.7. 9-Butyl-2-chloro-N-(3,5-dimethoxyphenyl)-9H-purin-6-amine
(3g)
Yield: 80%; M.p. = 100–102 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.18 (s, 1H), 8.32 (s, 1H), 7.20 (d, J = 2.1, 2H), 6.22 (t, J =
2.2, 1H), 4.12 (qui, J = 6.5, 2H), 3.70 (s, 6H), 1.75 (qui, J = 7.0, 2H),
1.22 (sex, J = 7.0, 2H), 0.87 (t, J = 7.4, 3H). 13C NMR (126 MHz,
4.3.2. 2-Chloro-N-(3-chlorobenzyl)-9-isopentyl-9H-purin-6-amine
(3b)
Yield: 63%; M.p. = 120–121 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.80 (s, 1H), 8.17 (s, 1H), 7.48–7.13 (m, 4H), 4.69–4.51 (s
(br), 2H), 4.08 (t, J = 6.5, 2H), 1.63 (q, J = 6.5, 2H), 1.42 (n, J = 6.5,
1H), 0.85 (d, J = 6.6, 6H). 13C NMR (126 MHz, DMSO d6) d (ppm):
155.30, 153.40, 150.53, 142.50, 142.10, 133.47, 130.69, 127.73,
DMSO d6)
141.09, 119.42, 99.82, 95.53, 55.57, 43.52, 31.77, 19.75, 13.90.
HPLC-MS (ESI+): 362.32 (96.69%). Elemental analysis Calcd. for C17
20ClN5O2 (361.83): C, 56.43; H, 5.57; N, 19.36. Found: C: 56.36; H:
5.59; N: 19.17.
d (ppm): 160.84, 155.29, 153.57, 152.71, 143.05,
-
H
Please cite this article in press as: Malínková V., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.032

8
V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.3.8. 2-Chloro-N-(3,5-dimethoxyphenyl)-9-hexyl-9H-purin-6-amine
(3h)
(96.99%). Elemental analysis Calcd. for C17H19Cl2N5 (364.27): C,
56.05; H, 5.26; N, 19.23. Found: C, 55.88; H, 5.14; N, 18.97.
Yield: 54%; M.p. = 90–101 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.16 (s, 1H), 8.30 (s, 1H), 7.20 (d, J = 2.2, 2H), 6.22 (t, J =
2.2, 1H), 4.10 (t, J = 7.1, 2H), 3.70 (s, 6H), 1.76 (qui, J = 6.5, 2H),
1.29–1.14 (m, 6H), 0.84–0.74 (t, J = 7.0, 3H). 13C NMR (126 MHz,
4.3.14. 2-Chloro-N-(3-chlorophenyl)-9-(3-methylbut-2-en-1-yl)-9H-
purin-6-amine (3n)
Yield: 72%; M.p. = 115–116 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.43 (s, J = 6.2, 1H), 8.27 (s, 1H), 8.01 (t, J = 2.1, 1H), 7.79
(dd, J = 8.5, J0 = 2.1, 1H), 7.33 (t, J = 8.5, 1H), 7.08 (dd, J = 8.5, J0 =
2.1, 1H), 5.38–5.34 (m, 1H), 4.71 (d, J = 7.2, 2H), 1.76 (s, 3H), 1.68
(s, 3H). 13C NMR (126 MHz, DMSO d6) d (ppm): 152.53, 151.23,
142.83, 140.98, 138.22, 133.35, 130.68, 123.36, 120.74, 119.74,
119.43, 119.05, 41.60, 25.81, 18.44. HPLC-MS (ESI+): 348.30
(97.37%). Elemental analysis Calcd. for C16H15Cl2N5 (348.22): C,
55.19; H, 4.34; N, 20.11. Found: C, 55.02; H, 4.38; N, 19.98.
DMSO d6)
d (ppm): 160.81, 152.69, 152.52, 151.32, 143.00,
141.11, 119.41, 99.79, 95.53, 55.56, 43.77, 31.11, 29.67, 26.10,
22.46, 14.35. HPLC-MS (ESI+): 390.79 (98.27%). Elemental analysis
Calcd. for C19H24ClN5O2 (389.88): C, 58.53; H, 6.20; N, 17.96.
Found: C, 56.77; H, 6.18; N, 16.79.
4.3.9. 4-((2-Chloro-9-cyclohexyl-9H-purin-6-yl)amino)phenol (3i)
Yield: 58%; M.p. = 156–157 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.97 (s, 1H), 9.27 (s, 1H), 8.33 (s, 1H), 7.47 (d, J = 8.5, 2H),
6.71 (d, J = 8.5, 2H), 4.28 (qui, J = 6.6, 1H), 1.99–1.95 (m, 2H),
1.86–1.79 (m, 4H), 1.67–1.65 (m, 1H), 1.48–1.32 (m, 2H), 1.26–
1.15 (m, 1H). 13C NMR (126 MHz, DMSO d6) d (ppm): 154.45,
153.23, 152.78, 150.47, 140.53, 130.59, 124.10, 119.09, 115.47,
54.25, 32.84, 25.55, 25.27. HPLC-MS (ESI+): 344.59 (95.16%). Ele-
mental analysis Calcd. for C17H18ClN5O (343.82): C, 59.39; H,
5.28; N, 20.37. Found: C, 59.76; H, 5.42; N, 20.16.
4.3.15. 9-Butyl-2-chloro-N-phenyl-9H-purin-6-amine (3o)
Yield: 83%; M.p. = 133–134 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.25 (s, 1H), 8.31 (s, 1H), 7.80 (d, J = 8.5, 2H), 7.32 (t, J =
7.0, 2H), 7.06 (t, J = 7.5, 1H), 4.13 (t, J = 7.0, 2H), 1.76 (qui, J = 7.0,
2H), 1.24 (sep, J = 7.0, 2H), 0.87 (t, J = 7.4, 3H). 13C NMR (126
MHz, DMSO d6) d (ppm): 152.89, 152.74, 151.34, 142.92, 139.36,
129.05, 123.95, 121.74, 119.31, 43.51, 31.79, 19.76, 13.91. HPLC-
MS(ESI+): 302.31 (98.58%). Elemental analysis Calcd. for C15H16
-
4.3.10. 2-Chloro-N-(4-chlorophenyl)-9-cyclohexyl-9H-purin-6-amine
(3j)
ClN5 (301.78): C, 59.70; H, 5.34; N, 23.21. Found: C, 59.51; H,
5.12; N, 23.14.
Yield: 59%. M.p. = 134–136 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): d 10.39 (s, 1H), 8.43 (s, 1H), 7.85 (d, J= 7.0, 2H), 7.38 (d, J=
7.0, 2H), 4.38–4.27 (m, 1H), 1.98 (d, J = 8.3, 2H), 1.91–1.77 (m,
4H), 1.67 (d, J = 8.3, 1H), 1.50–1.36 (m, 2H), 1.30–1.16 (m, 2H).
13C NMR (126 MHz, DMSO d6) d (ppm): 152.67, 152.39, 150.88,
141.31, 138.42, 128.95, 127.56, 123.10, 119.53, 54.39, 32.81,
25.54, 25.27. HPLC-MS (ESI+): 362.38 (95.45%). Elemental analysis
Calcd. for C17H17Cl2N5 (362.26): C, 56.37; H, 4.73; N, 19.33. Found:
C, 56. 20; H, 4.79; N, 19.28.
4.3.16. 2-Chloro-N-(4-chlorophenyl)-9-hexyl-9H-purin-6-amine (3p)
Yield: 84%; M.p. = 149–151 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.38 (s, 1H), 8.33 (s, 1H), 7.85 (d, J = 9.0, 2H), 7.37 (d, J =
9.0, 2H), 4.12 (t, J = 7.0, 2H), 1.82–1.72 (m, 2H), 1.29–1.17 (m,
6H), 0.80 (t, J = 6.4, 3H). 13C NMR (126 MHz, DMSO d6) d (ppm):
156.55, 152.62, 151.45, 143.18, 138.44, 128.95, 127.56, 123.08,
119.38, 43.79, 31.11, 29.67, 26.09, 22.46, 14.36. HPLC-MS (ESI+):
364.61 (99.49%). Elemental analysis Calcd. for
C17H19Cl2N5
(364.27): C, 56.05; H, 5.26; N, 19.23. Found: C, 55.85; H, 5.41; N,
19.49.
4.3.11. 4-((2-Chloro-9-hexyl-9H-purin-6-yl)amino)phenol (3k)
Yield: 63%; M.p. = 195–196 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.96 (s, 1H), 9.25 (s, 1H), 8.23 (s, 1H), 7.47 (d, J = 8.0 Hz,
2H), 6.70 (d, J = 8.0, 2H), 4.08 (t, J = 7.5, 2H), 1.76 (qui, J = 6.5,
2H), 1.15–1.30 (m, 6H), 0.79 (t, J = 6.9, 3H). 13C NMR (126 MHz,
4.3.17. 9-Butyl-2-chloro-N-(3-chlorophenyl)-9H-purin-6-amine (3q)
Yield: 84%; M.p. = 106–107 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.44 (s, 1H), 8.34 (s, 1H), 8.02 (d, J = 1.7, 1H), 7.81 (d, J =
8.5, 1H), 7.35 (t, J = 8.0, 1H), 7.09 (t, J = 8.0, 1H), 4.13 (t, J = 7.1,
2H), 1.81–1.72 (m, 2H), 1.29–1.18 (m, 2H), 0.86 (t, J = 7.4, 3H).
13C NMR (126 MHz, DMSO d6) d (ppm): 152.56, 152.50, 151.55,
143.31, 141.03, 133.36, 130.65, 123.37, 120.79, 119.79, 119.48,
43.57, 31.77, 19.76, 13.90. HPLC-MS (ESI+): 336.20 (98.57%). Ele-
mental analysis Calcd. for C15H15Cl2N5 (336.22): C, 53.59; H,
4.50; N, 20.83. Found: C, 53.84; H, 4.75; N, 21.09.
DMSO d6)
d (ppm): 154.43, 153.16, 152.98, 150.98, 142.43,
130.56, 124.04, 118.93, 115.47, 43.68, 31.12, 29.71, 26.10, 22.47,
14.37. HPLC-MS (ESI+): 346.42 (95.72%). Elemental analysis Calcd.
for C17H20ClN5O (345.83): C, 59.04; H, 5.83; N, 20.25. Found: C,
58.78; H, 5.79; N, 20.03.
4.3.12. 2-Chloro-N-(3,5-dimethoxyphenyl)-9-isopentyl-9H-purin-6-
amine (3l)
Yield: 68%; M.p. = 103–104 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.18 (s, 1H), 8.35 (s, 1H), 7.20 (d, J = 2.2, 2H), 6.22 (t, J =
2.2, 1H), 4.13 (t, J = 7.0, 2H), 3.70 (s, 6H), 1.69 (q, J = 7.0, 2H), 1.46
(n, J = 6.6, 1H), 0.89 (d, J = 6.6, 6H). 13C NMR (126 MHz, DMSO d6)
d (ppm): 160.82, 152.70, 152.52, 151.31, 142.98, 141.10, 119.41,
99.72, 95.50, 55.56, 42.19, 38.56, 25.62, 22.65. HPLC-MS (ESI+):
4.3.18. 4-((2-Chloro-9-isopentyl-9H-purin-6-yl)amino)phenol (3r)
Yield: 61%; M.p. = 234–235 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.97 (s, 1H), 9.26 (s, 1H), 8.26 (s, 1H), 7.48 (d, J = 8.5, 2H),
6.71 (d, J = 8.5 Hz, 2H), 4.11 (t, J = 7.3, 2H), 1.68 (q, J = 7.0, 2H),
1.44 (non, J = 6.6, 1H), 0.88 (d, J = 6.6 Hz, 6H). 13C NMR (126 MHz,
DMSO d6)
d (ppm): 154.43, 153.16, 152.98, 142.34, 130.61,
376.32 (97.14%). Elemental analysis Calcd. for
(375.86): C, 57.52; H, 5.90; N, 18.63. Found: C, 57.21; H, 5.98; N,
18.50.
C
18H22ClN5O2
124.07, 118.96, 115.48, 42.10, 38.61, 25.62, 22.66. HPLC-MS (ESI
+): 332.59 (95.45%). Elemental analysis Calcd. for C16H18ClN5O
(331.80): C, 57.92; H, 5.47; N, 21.11. Found: C, 57.70; H, 5.48; N,
20.80.
4.3.13. 2-Chloro-N-(3-chlorophenyl)-9-hexyl-9H-purin-6-amine (3m)
Yield: 78%; M.p. = 108–110 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.48 (s, 1H), 8.39 (s, 1H), 8.02 (t, J = 2.5, 1H), 7.80 (d, J =
8.5, 1H), 7.35 (t, J = 8.0, 1H), 7.08 (d, J = 8.0, 1H), 4.12 (t, J = 7.0,
2H), 1.78 (qui, J = 6.5 2H), 1.25–1.18 (m, 6H), 0.80 (t, J = 6.9, 3H).
13C NMR (126 MHz, DMSO d6) d (ppm): 152.78, 152.25, 151.33,
143.12, 140.90, 133.40, 130.75, 123.49, 120.71, 119.71, 118.59,
44.01, 31.11, 29.58, 26.06, 22.46, 14.36. HPLC-MS (ESI+): 364.48
4.3.19. 2-Chloro-9-isopentyl-N-phenyl-9H-purin-6-amine (3s)
Yield: 60%; M.p. = 124–126 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.26 (s, 1H), 8.33 (s, 1H), 7.80 (d, J = 7.7, 2H), 7.32 (t, J =
7.5, 2H), 7.06 (t, J = 7.5, 1H), 4.14 (t, J = 7.4, 2H), 1.68 (q, J = 7.0,
2H), 1.47 (n, J = 6.5, 1H), 0.89 (d, J = 6.5, 6H). 13C NMR (126 MHz,
DMSO d6)
d (ppm): 152.90, 152.73, 151.32, 142.88, 139.35,
129.06, 123.96, 121.75, 119.29, 42.18, 38.58, 25.63, 22.67. HPLC-
Please cite this article in press as: Malínková V., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.032

V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
MS (ESI+): 316.26 (98.37%). Elemental analysis Calcd. for C16H18
-
ClN5 (315.80): C, 60.85; H, 5.75; N, 22.18. Found: C, 60.25; H,
5.79; N, 21.86.
4.4.1. N2-(4-Aminocyclohexyl)-9-butyl-N6-(3-chlorobenzyl)-9H-
purine-2,6-diamine (4a)
Yield: 78%; M.p. = 149–150 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 7.87 (s(br), 1H), 7.66 (s, 1H), 7.33 (s, 1H), 7.30–7.25 (m,
2H), 7.24–7.20 (m, 1H), 6.10 (s(br), 1H), 4.60–4.46 (m, 2H), 3.89
(t, J = 5.9, 2H), 3.57–3.46 (m, 1H), 2.63–2.60 (m, 1H), 1.88–1.61
(m, 4H), 1.60–1.57 (m, 2H), 1.45–1.40 (m, 1H), 1.20–1.14 (m,
5H), 0.89–0.80 (m, 6H). 13C NMR (126 MHz, DMSO d6) d (ppm):
159.10, 154.78, 144.09, 137.88, 133.29, 130.49, 127.50, 126.91,
126.49, 50.07, 49.87, 33.58, 31.82, 31.43, 29.55, 25.52, 22.73.
4.3.20. 3-((2-Chloro-9-isopentyl-9H-purin-6-yl)amino)phenol (3t)
Yield: 84%; M.p. = 156–157 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.11 (s, 1H), 8.34 (s, 1H), 7.30 (t, J = 1.5, 1H), 7.22 (d, J =
8.1, 1H), 7.07 (t, J = 8.1 Hz, 1H), 6.47 (dd, J = 8.1, J0 = 2.3, 1H), 4.13
(t, J = 7.0, 2H), 1.68 (q, J = 7.0, 2H), 1.47 (n, J = 6.0, 1H), 0.88 (d, J
= 6.6, 6H). 13C NMR (126 MHz, DMSO d6)
d (ppm): 157.97,
HPLC-MS (ESI+): 428.23 (99.45%). Elemental analysis Calcd. for C22
-
152.80, 151.19, 142.74, 140.32, 129.64, 119.11, 113.70, 112.60,
111.18, 108.89, 42.22, 38.56, 25.63, 22.67. Elemental analysis
Calcd. for C16H18ClN5O (331.12): C, 57.92; H, 5.47; N, 21.11. Found:
C, 57.87; H, 5.50; N, 21.09. HPLC-MS (ESI+): 332.30 (99.16%).
H30ClN7 (427.98): C, 61.74; H, 7.07; N, 22.91. Found: C, 61.55; H,
7.31; N, 22.73.
4.4.2. N2-(4-Aminocyclohexyl)-N6-(3-chlorobenzyl)-9-isopentyl-9H-
purine-2,6-diamine (4b)
4.3.21. 2-Chloro-N-(3-chlorophenyl)-9-isopentyl-9H-purin-6-amine
(3u)
Yield: 67%; M.p. = 94–96 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 7.68 (s, 1H), 7.33 (s, 1H), 7.31–7.18 (m, 3H), 6.10 (s(br),
1H), 4.54 (s(br), 2H), 3.99–3.87 (m, 2H), 1.90–1.69 (m, 3H), 1.67–
1.55 (m, 2H), 1.43 (non, J = 6.5, 1H), 1.28–1.07 (m, 6H), 0.85 (d, J
Yield: 81%; M.p. = 86–88 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.43 (s, 1H), 8.36 (s, 1H), 8.01 (t, J = 2.0, 1H), 7.79 (dd, J
= 8.5, J0 = 2.0, 1H), 7.34 (t, J = 8.1, 1H), 7.12–7.05 (m, 1H), 4.14 (t,
J = 6.5, 2H), 1.68 (q, J = 6.5, 2H), 1.46 (n, J = 7.0, 1H), 0.88 (d, J =
6.6, 6H). 13C NMR (126 MHz, DMSO d6) d (ppm): 152.55, 152.49,
151.52, 143.25, 141.02, 133.36, 130.68, 123.37, 120.79, 119.79,
119.46, 42.24, 38.55, 25.63, 22.66. HPLC-MS (ESI+): 350.39
(97.85%). Elemental analysis Calcd. for C16H17Cl2N5 (350.25): C,
54.87; H, 4.89; N, 20.00. Found: C, 54.94; H, 4.99; N, 20.13.
= 6.5, 6H). 13C NMR (126 MHz, DMSO d6)
d (ppm): 159.04,
154.76, 144.07, 138.02, 137.82, 133.30, 130.49, 127.50, 126.90,
126.48, 50.06, 49.86, 41.13, 38.85, 33.57, 31.82, 31.42, 29.54,
25.51, 22.72. HPLC-MS (ESI+): 442.17 (97.30%). Elemental analysis
Calcd. for C23H32ClN7 (442.01): C, 62.50; H, 7.30; N, 22.18. Found:
C, 62.35; H, 7.41; N, 22.11.
4.4.3. N2-(4-Aminocyclohexyl)-N6-(3-chlorophenyl)-9-(4-
methoxybenzyl)-9H-purine-2,6-diamine (4c)
4.3.22. 9-Butyl-2-chloro-N-(4-chlorophenyl)-9H-purin-6-amine (3v)
Yield: 94%; M.p. = 156–157 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.38 (s, 1H), 8.32 (s, 1H), 7.83 (d, J = 8.5, 2H), 7.37 (d, J =
8.5, 2H), 4.12 (t, J = 7.1, 2H), 1.75 (qui, J = 7.0, 2H), 1.22 (sex, J =
7.0, 2H), 0.85 (t, J = 7.4, 3H). 13C NMR (126 MHz, DMSO d6) d
(ppm): 152.63, 152.54, 151.43, 143.23, 138.40, 128.91, 127.55,
122.98, 119.36, 43.54, 31.58, 19.69, 13.96. HPLC-MS (ESI+):
Yield: 71%; M.p. = 125–126 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 7.88 (s(br), 1H), 7.65 (s, 1H), 7.38–7.32 (m, 2H), 7.28–7.24
(m, 4H), 7.22–7.19 (m, 2H), 6.08 (s, 1H), 4.53 (s, 2H), 3.89 (s, 3H),
3.58–3.48 (m, 1H), 1.82–1.74 (m, 1H), 1.72–1.62 (m, 4H), 1.16–
1.00 (m, 4H). 13C NMR (126 MHz, DMSO d6) d (ppm): 159.27,
158.92, 152.27, 142.51, 133.30, 130.34, 129.79, 121.68, 119.60,
118.70, 114.47, 114.13, 65.50, 56.60, 55.63, 50.07, 33.59, 31.47,
24.08, 19.10, 15.63. HPLC-MS (ESI+): 478.54 (99.99%). Elemental
analysis Calcd. for C25H28ClN7O (478.00): C, 62.82; H, 5.90; N,
20.51. Found: C, 62.66; H, 6.01; N, 20.64.
336.50 (95.66%). Elemental analysis Calcd. for
C15H15Cl2N5
(336.22): C, 53.59; H, 4.50; N, 20.83. Found: C, 53.45; H, 4.41; N,
20.78.
4.3.23. 2-Chloro-N-(4-chlorophenyl)-9-isopentyl-9H-purin-6-amine
(3w)
4.4.4. N2-(4-Aminocyclohexyl)-9-benzyl-N6-(3-chlorophenyl)-9H-
purine-2,6-diamine (4d)
Yield: 81%; M.p. = 156–158 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 10.39 (s, 1H), 8.35 (s, 1H), 7.84 (d, J = 8.5, 2H), 7.36 (d, J =
8.5, 2H), 4.15 (t, J = 7.25, 2H), 1.68 (q, J = 6.5, 2H), 1.46 (n, J = 6.6,
1H), 0.89 (d, J = 6.6, 6H). 13C NMR (126 MHz, DMSO d6) d (ppm):
152.62, 151.43, 143.10, 138.41, 128.94, 127.55, 123.07, 119.36,
42.20, 38.55, 25.61, 22.65. HPLC-MS (ESI+): 350.46 (95.99%). Ele-
mental analysis Calcd. for C16H17Cl2N5 (350.25): C, 54.87; H,
4.89; N, 20.00. Found: C, 54.68; H, 4.74; N, 19.94.
Yield: 65%; M.p. = 110–111 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.67 (s(br), 1H), 8.30 (s, 1H), 7.93 (s, 1H), 7.84 (s, 1H),
7.42–7.17 (m, 6H), 6.97 (d, J = 7.5, 1H), 6.56 (t, J = 6.75, 1H), 5.19
(s, 2H), 3.70–3.57 (m, 1H), 1.93–1.86 (m, 2H), 1.80–1.71 (m, 2H),
1.26–1.13 (m, 5H). 13C NMR (126 MHz, DMSO d6)
d (ppm):
159.02, 152.28, 142.49, 138.95, 137.85, 133.27, 130.35, 129.12,
128.13, 127.61, 121.71, 119.59, 118.69, 114.01, 50.52, 35.69,
32.36, 31.83. HPLC-MS (ESI+): 448.68 (99.99%). Elemental analysis
Calcd. for C24H26ClN7 (447.97): C, 64.35; H, 5.85; N, 21.89. Found:
C, 64.15; H, 5.99; N, 21.81.
4.4. Protocol for preparation of compounds 4a–4w
A 2-chloro-6,9-disubstituted-9H-purine (5.0 mmol) and trans-
1,4-diaminocyclohexane (75.0 mmol) were mixed in n-butanol.
The reaction mixture was heated in a sealed vial with a Teflon sep-
tum using a CEM Discover reactor at 140–170 °C for 0.5–1.5 h (con-
trolled by TLC – chloroform: methanol, 9:1; see Supplementary
data) or in a sealed tube under an argon atmosphere at 160 °C
for 4–10 h. After cooling to room temperature, water (50 mL)
was added to the reaction mixture and the resulting suspension
was extracted three times with ethyl acetate (3 ꢂ 50 mL). The
combined organic phases were washed with water (50 mL) and
brine (50 mL), dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The crude product was purified by column chro-
matography on silica (chloroform: methanol, 19:1 > 4:1) to yield
the final compound.
4.4.5. (E)-N2-(4-Aminocyclohexyl)-N6-(3-chlorophenyl)-9-(3,7-
dimethylocta-2,6-dien-1-yl)-9H-purine-2,6-diamine (4e)
Yield: 49%; M.p. = 142–144 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.61 (s(br), 1H), 8.29 (s(br), 1H), 7.77 (s, 1H), 7.24 (t, J =
8.0, 1H), 6.96 (d, J = 7.6, 1H), 6.61–6.48 (m, 1H), 5.33 (t, J = 6.6,
1H), 4.99 (t, J = 6.2, 1H), 4.57 (s, 2H), 3.71–3.60 (m, 1H), 2.68–
2.56 (m, 1H), 2.06–1.90 (m, 6H), 1.86–1.71 (m, 5H), 1.55 (s, 3H),
1.48 (s, 3H), 1.35–1.16 (m, 4H). 13C NMR (126 MHz, DMSO d6) d
(ppm): 158.82, 152.22, 142.57, 140.34, 138.43, 133.28, 131.56,
130.34, 124.26, 121.62, 119.79, 119.53, 118.65, 114.14, 50.26,
34.41, 31.62, 26.25, 25.98, 18.06, 16.63. HPLC-MS (ESI+): 493.89
(98.60%). Elemental analysis Calcd. for C27H36ClN7 (494.08): C,
65.64; H, 7.34; N, 19.84. Found: C, 65.60; H, 7.36; N, 19.79.
Please cite this article in press as: Malínková V., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.032

10
V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.4.6. N2-(4-Aminocyclohexyl)-N6-(2,4-dimethoxyphenyl)-9-
isopentyl-9H-purine-2,6-diamine (4f)
4.4.11. 4-((2-((4-Aminocyclohexyl)amino)-9-hexyl-9H-purin-6-yl)
amino)phenol (4k)
Yield: 52%; M.p. = 108–109 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 8.40 (d, J = 8.8, 1H), 7.77 (s, 1H), 7.74 (s, 1H), 6.63 (d, J =
2.3, 1H), 6.45 (d, J = 8.8, 2H), 3.97 (t, J = 6.5, 2H), 3.89–3.82 (m,
3H), 3.75–3.70 (m, 3H), 3.57 (d, J = 8.3, 1H), 1.94–1.90 (m, 2H),
1.79–1.75 (m, 2H), 1.63 (d, J = 6.7, 2H), 1.44 (t, J = 6.6, 1H), 1.32–
1.04 (m, 4H), 0.87 (d, J = 6.6, 6H). 13C NMR (126 MHz, DMSO d6) d
(ppm): 158.99, 158.21, 155.75, 152.19, 150.22, 138.71, 122.44,
113.87, 112.79, 104.62, 99.07, 56.48, 55.86, 50.36, 41.31, 38.80,
35.00, 31.58, 25.50, 22.70. HPLC-MS (ESI+): 454.00 (98.30%). Ele-
mental analysis Calcd. for C24H35N7O2 (453.59): C, 63.55; H, 7.78;
N, 21.62. Found: C, 63.45; H, 7.85; N, 21.56.
Yield: 40%; M.p. = 151–153 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.54 (s(br), 1H), 8.05 (d, J = 8.9, 2H), 7.82 (s, 1H), 7.26 (d,
J = 8.9, 2H), 6.51 (s(br), 1H), 3.95 (t, J = 6.1, 1H), 3.64–3.52 (m,
2H), 2.54–2.48 (m, 1H), 1.92 (d, J = 10.5, 2H), 1.80–1.68 (m, 4H),
1.29–1.12 (m, 10H), 0.80 (t, J = 6.8, 3H). 13C NMR (126 MHz,
DMSO d6)
d (ppm): 158.82, 152.21, 140.13, 128.92, 128.67,
128.37, 128.10, 125.55, 121.79, 114.06, 50.61, 42.93, 35.81, 31.84,
31.17, 29.66, 26.18, 22.47, 14.41. HPLC-MS (ESI+): 424.87
(98.95%). Elemental analysis Calcd. for C23H33N7O (423.57): C,
65.22; H, 7.85; N, 23.15. Found: C, 64.98; H, 7.95; N, 22.89.
4.4.12. N2-(4-Aminocyclohexyl)-N6-(3,5-dimethoxyphenyl)-9-
isopentyl-9H-purine-2,6-diamine (4l)
4.4.7. N2-(4-Aminocyclohexyl)-9-butyl-N6-(3,5-dimethoxyphenyl)-
9H-purine-2,6-diamine (4g)
Yield: 81%; M.p. = 118–121 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.19 (s(br), 1H), 7.81 (s, 1H), 7.26 (s, 2H), 6.38 (s, 1H),
6.11–6.09 (m, 1H), 3.98 (t, J = 4.6, 2H), 3.79–3.60 (m, 6H), 1.91–
1.88 (m, 2H), 1.74–1.72 (m, 2H), 1.63 (q, J = 6.6 Hz, 2H), 1.45 (n, J
= 8.0, 1H), 1.33–1.04 (m, 4H), 0.87 (d, J = 6.6, 6H). 13C NMR (126
MHz, DMSO d6) d (ppm): 160.80, 158.90, 152.50, 142.44, 138.62,
114.94, 99.06, 94.09, 55.58, 50.47, 50.32, 41.24, 38.78, 35.59,
31.79, 25.57, 22.73. HPLC-MS (ESI+): 453.90 (98.73%). Elemental
analysis Calcd. for C24H35N7O2 (453.59): C, 63.28; H, 7.96; N,
19.24. Found: C, 63.45; H, 7.78; N, 19.31.
Yield: 72%; M.p. = 87–88 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.20 (s (br), 1H), 7.79 (s, 1H), 7.26 (d, J = 2.0, 2H), 6.39 (s,
1H), 6.10 (s, 1H), 3.95 (t, J = 6.6, 2H), 3.69 (s, 6H), 2.55–2.47 (m,
1H), 1.89 (d, J = 10.9, 2H), 1.77–1.68 (m, 4H), 1.30–1.16 (m, 4H),
1.14–1.07 (m, 2H), 0.85 (t, J = 7.4, 3H). 13C NMR (126 MHz,
DMSO d6)
d (ppm): 160.81, 158.92, 152.51, 142.44, 138.74,
116.85, 114.17, 99.05, 94.11, 55.58, 50.41, 50.28, 42.56, 35.44,
31.82, 19.77, 13.93. HPLC-MS (ESI+): 440.00 (99.37%). Elemental
analysis Calcd. for C23H33N7O2 (439.56): C, 62.85; H, 7.57; N,
22.31. Found: C, 62.59; H, 7.69; N, 22.26.
4.4.13. N2-(4-Aminocyclohexyl)-N6-(3-chlorophenyl)-9-hexyl-9H-
purine-2,6-diamine (4m)
4.4.8. N2-(4-Aminocyclohexyl)-N6-(3,5-dimethoxyphenyl)-9-hexyl-
9H-purine-2,6-diamine (4h)
Yield: 59%; M.p. = 99–102 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.61 (s(br), 1H), 8.28 (s(br), 1H), 7.84 (s, 1H), 7.24 (t, J =
8.1, 1H), 6.96 (d, J = 8.1, 1H), 6.55 (d, J = 5.6, 1H), 3.95 (t, J = 6.4,
2H), 3.67–3.60 (m, 1H), 2.59–2.50 (m, 1H), 1.94–1.88 (m, 2H),
1.82–1.69 (m, 5H), 1.29–1.10 (m, 12H), 0.80 (t, J = 6.8, 3H). 13C
Yield: 69%; M.p. = 108–110 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.19 (s(br), 1H), 7.78 (s, 1H), 7.25 (d, J = 1.8, 2H), 6.38 (s,
1H), 6.12–6.09 (m, 1H), 3.95 (t, J = 7.0, 2H), 3.68 (s, 6H), 1.90–
1.88 (m, 2H), 1.71–1.68 (m, 4H), 1.32–1.17 (m, 9H), 1.15–1.04
(m, 1H), 0.79 (t, J = 6.7, 3H). 13C NMR (126 MHz, DMSO d6) d
(ppm): 160.81, 158.96, 152.50, 142.45, 138.75, 114.20, 99.06,
94.08, 55.59, 50.46, 50.29, 42.91, 35.58, 31.86, 31.18, 29.67,
26.19, 22.47, 14.40. HPLC-MS (ESI+): 468.10 (97.78%). Elemental
analysis Calcd. for C25H37N7O2 (467.62): C, 64.21; H, 7.98; N,
20.97. Found: C, 64.19; H, 8.05; N, 20.83.
NMR (126 MHz, DMSO d6)
d (ppm): 153.59, 142.57, 139.56,
139.12, 133.29, 131.87, 130.34, 121.62, 119.55, 118.65, 114.12,
50.39, 42.95, 35.18, 31.78, 31.20, 29.67, 26.20, 22.48, 14.40.
HPLC-MS (ESI+): 442.16 (99.99%). Elemental analysis Calcd. for C23
32ClN7 (442.01): C, 62.50; H, 7.30; N, 22.18. Found: C, 62.42; H,
7.33; N, 22.12.
-
H
4.4.9. 4-((2-((4-Aminocyclohexyl)amino)-9-cyclohexyl-9H-purin-6-
yl)amino)phenol (4i)
4.4.14. N2-(4-Aminocyclohexyl)-N6-(3-chlorophenyl)-9-(3-methylbut-
2-en-1-yl)-9H-purine-2,6-diamine (4n)
Yield: 57%; M.p. = 137–138 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.06 (s, 1H), 7.81 (s, 1H), 7.69 (d, J = 8.8, 2H), 6.63 (dd, J =
8.8, J0 = 3.5, 2H), 6.31 (s, 1H), 4.15–4.03 (m, 1H), 3.58–3.50 (m,
1H), 2.53 (t, J = 6.5, 1H), 1.98–1.87 (m, 4H), 1.82–1.75 (m, 4H),
1.64 (d, J = 12.4, 1H), 1.55 (d, J = 10.3, 1H), 1.40–1.27 (m, 3H),
Yield: 73%; M.p. = 100–101 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.61 (s(br), 1H), 8.28 (s(br), 1H), 7.80 (s, 1H), 7.24 (t, J =
8.1, 1H), 6.96 (d, J = 7.9, 1H), 6.52 (d, J = 8.2 1H), 5.34 (t, J = 7.0,
1H), 4.56 (d, J = 6.5, 2H), 3.69–3.59 (m, 1H), 1.94–1.90 (m, 2H),
1.82–1.72 (m, 5H), 1.68 (s, 3H), 1.32–1.10 (m, 4H). 13C NMR (126
MHz, DMSO d6) d (ppm): 158.81, 152.11, 142.48, 138.71, 137.07,
133.37, 130.47, 121.73, 119.98, 119.48, 118.59, 114.03, 50.24,
49.11, 40.73, 34.44, 31.61, 25.85, 18.34. HPLC-MS (ESI+): 426.81
(95.58%). Elemental analysis Calcd. for C22H28ClN7 (425.96): C,
62.03; H, 6.63; N, 23.02. Found: C, 61.90; H, 6.85; N, 22.83.
1.26–1.05 (m, 5H). 13C NMR (126 MHz, DMSO d6)
d (ppm):
164.00, 158.72, 152.88, 152.52, 151.72, 136.13, 132.57, 122.22,
115.15, 58.85, 50.46, 35.31, 33.07, 32.77, 31.75, 25.80, 25.41,
18.16. HPLC-MS (ESI+): 422.82 (99.40%). Elemental analysis Calcd.
for C23H31N7O (421.55): C, 65.53; H, 7.41; N, 23.26. Found: C,
65.49; H, 7.45; N, 23.25.
4.4.10. N2-(4-Aminocyclohexyl)-N6-(4-chlorophenyl)-9-cyclohexyl-
9H-purine-2,6-diamine (4j)
4.4.15. N2-(4-Aminocyclohexyl)-9-butyl-N6-phenyl-9H-purine-2,6-
diamine (4o)
Yield: 73%; M.p. = 118–120 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.57 (s(br), 1H), 8.05 (d, J = 8.6, 2H), 7.90 (s, 1H), 7.26 (d,
J = 8.3, 2H), 6.52 (s, 1H), 4.13 (t, J = 6.5, 1H), 3.62–3.51 (m, 1H),
1.98–1.88 (m, 4H), 1.86–1.75 (m, 4H), 1.69–1.62 (m, 1H) 1.60–
1.58 (m, 3H), 1.41–1.06 (m, 6H).13C NMR (126 MHz, DMSO d6) d
(ppm): 158.62, 152.24, 140.13, 136.89, 128.51, 125.57, 121.89,
121.78, 58.87, 50.48, 35.35, 32.71, 31.73, 31.23, 25.80, 25.42.
Yield: 68%; M.p. = 183–184 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.34 (s(br), 1H), 7.98 (d, J = 8.5, 2H), 7.79 (s, 1H), 7.22 (t, J
= 8.5, 2H), 6.92 (t, J = 7.5, 1H), 6.44 (s(br), 1H), 3.96 (t, J = 5.5, 2H),
3.66–3.55 (m, 1H), 3.38–3.30 (m, 1H), 1.92 (d, J = 10.4, 2H), 1.80–
1.64 (m, 4H), 1.29–1.00 (m, 6H), 0.86 (t, J = 7.4, 3H). 13C NMR
(126 MHz, DMSO d6) d (ppm): 221.80, 158.94, 152.56, 141.06,
138.60, 128.74, 122.11, 120.41, 114.06, 65.46, 50.60, 42.59, 36.07,
31.85, 19.78, 15.70, 13.94. HPLC-MS (ESI+): 380.10 (99.67%). Ele-
mental analysis Calcd. for C21H29N7 (379.51): C, 66.46; H, 7.70;
N, 25.84. Found: C, 66.26; H, 7.83; N, 25.69.
HPLC-MS (ESI+): 439.90 (99.05%). Elemental analysis Calcd. for C23
30ClN7 (439.99): C, 62.79; H, 6.87; N, 22.28. Found: C, 62.64; H,
7.01; N, 22.23.
-
H
Please cite this article in press as: Malínková V., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.032

V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
4.4.16. N2-(4-Aminocyclohexyl)-N6-(4-chlorophenyl)-9-hexyl-9H-
purine-2,6-diamine (4p)
C22H31N7O (409.54): C, 64.52; H, 7.63; N, 23.94. Found: C, 64.47;
H, 7.68; N, 23.92.
Yield: 76%; M.p. = 131–133 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.54 (s(br), 1H), 8.05 (d, J = 8.9, 2H), 7.82 (s, 1H), 7.26 (d,
J = 8.9, 2H), 6.51 (s(br), 1H), 3.95 (t, J = 6.1, 1H), 3.64–3.52 (m,
2H), 2.54–2.48 (m, 1H), 1.92 (d, J = 10.5, 2H), 1.80–1.68 (m, 4H),
1.29–1.12 (m, 10H), 0.80 (t, J = 6.8, 3H). 13C NMR (126 MHz,
4.4.21. N2-(4-Aminocyclohexyl)-N6-(3-chlorophenyl)-9-isopentyl-9H-
purine-2,6-diamine (4u)
Yield: 79%; M.p. = 115–117 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.59 (s(br), 1H), 8.27 (s(br), 1H), 7.84 (s, 2H), 7.23 (t, J =
8.1, 1H), 6.94 (d, J = 7.8, 1H), 6.52 (d, J = 7.4, 1H), 4.01–3.93 (m,
2H), 3.66–3.57 (m, 1H), 2.54 (t, J = 10.0, 1H), 1.91 (d, J = 10.8, 2H),
1.76 (d, J = 10.3, 2H), 1.64–1.60 (m, 2H), 1.44 (non, J = 6.5, 1H),
1.31–1.13 (m, 4H), 0.86 (d, J = 6.6, 6H). 13C NMR (126 MHz,
DMSO d6)
d (ppm): 158.82, 152.21, 140.13, 128.92, 128.67,
128.37, 128.10, 125.55, 121.79, 114.06, 50.61, 42.93, 35.81, 31.84,
31.17, 29.66, 26.18, 22.47, 14.40. HPLC-MS (ESI+): 442.85
(98.39%). Elemental analysis Calcd. for C23H32ClN7 (442.01): C,
62.50; H, 7.30; N, 22.18. Found: C, 62.48; H, 7.33; N, 22.14.
DMSO d6)
d (ppm): 158.80, 157.84, 152.68, 152.20, 142.55,
138.93, 133.26, 130.31, 121.60, 119.53, 118.64, 114.09, 50.37,
41.28, 38.73, 35.05, 31.72, 25.61, 22.72. HPLC-MS (ESI+): 428.00
(97.00%). Elemental analysis Calcd. for C22H30ClN7 (427.98): C,
61.74; H, 7.07; N, 22.91. Found: C, 61.55; H, 7.12; N, 22.83.
4.4.17. N2-(4-Aminocyclohexyl)-9-butyl-N6-(3-chlorophenyl)-9H-
purine-2,6-diamine (4q)
Yield: 41%; M.p. = 128–130 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.61 (s(br), 1H), 8.29 (s(br), 1H) 7.83 (s, 1H), 7.25 (t, J =
8.5, 1H), 6.95 (d, J = 7.5, 1H), 6.54 (s, J = 7.0, 1H), 3.95 (t, J = 6.4,
2H), 3.67–3.56 (m, 1H), 2.57–2.47 (m, 1H), 1.92–1.87 (m, 2H),
1.81–1.66 (m, 4H), 1.22 (m, 6H), 0.89–0.82 (m, 3H). 13C NMR
(126 MHz, DMSO d6) d (ppm): 158.82, 152.78, 152.23, 142.57,
139.13, 133.30, 130.34, 127.05, 121.63, 119.57, 118.66, 114.15,
50.31, 50.10, 42.66, 33.57, 31.82, 19.80, 13.95. HPLC-MS (ESI+):
4.4.22. N2-(4-Aminocyclohexyl)-9-butyl-N6-(4-chlorophenyl)-9H-
purine-2,6-diamine (4v)
Yield: 61%; M.p. = 95–96 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.57 (s(br), 1H), 8.05 (d, J = 8.8, 2H), 7.82 (s, 1H), 7.27 (d,
J = 7.4, 2H), 6.54 (s(br), 1H), 3.96 (t, J = 6.5, 2H), 3.58 (s(br), 2H),
2.58–2.53 (m, 1H), 1.94–1.92 (m, 2H), 1.85–1.64 (m, 4H), 1.30–
1.10 (m, 6H), 0.86 (t, J = 7.2, 3H). 13C NMR (126 MHz, DMSO d6) d
(ppm): 158.81, 152.72, 152.22, 140.12, 138.92, 128.52, 125.58,
121.78, 114.05, 50.28, 42.60, 34.70, 31.82, 31.62, 19.78, 13.94.
HPLC-MS (ESI+): 414.00 (98.17%). Elemental analysis Calcd. for
413.90 (96.68%). Elemental analysis Calcd. for
C21H28ClN7
(413.95): C, 60.93; H, 6.82; N, 23.69. Found: C, 60.85; H, 6.88; N,
23.57.
C
21H29N7 (413.95): C, 60.93; H, 6.82; N, 23.69. Found: C, 60.85;
4.4.18. 4-((2-((4-Aminocyclohexyl)amino)-9-isopentyl-9H-purin-6-
yl)amino)phenol (4r)
H, 7.01; N, 23.57.
Yield: 81%; M.p. = 123–125 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.05 (s(br), 1H), 7.76 (s, 1H), 7.70 (d, J = 8.8, 2H), 6.64 (d,
J = 8.8, 2H), 6.28 (s, 1H), 3.97 (t, J = 6.5, 2H), 3.62–3.50 (m, 1H),
1.92–1.88 (m, 2H), 1.78–1.73 (m, 2H), 1.67–1.59 (m, 2H), 1.45 (n,
J = 6.5, 1H), 1.27–0.97 (m, 4H), 0.88 (d, J = 6.6, 6H). 13C NMR (126
MHz, DMSO d6) d (ppm): 158.95, 152.90, 150.97, 138.04, 132.56,
122.27, 115.18, 113.78, 50.55, 41.20, 38.83, 35.79, 31.83, 25.57,
22.75. HPLC-MS (ESI+): 410.00 (95.83%). Elemental analysis Calcd.
for C22H31N7O (409.54): C, 64.52; H, 7.63; N, 23.94. Found: C,
64.33; H, 7.75; N, 23.86.
4.4.23. N2-(4-Aminocyclohexyl)-N6-(4-chlorophenyl)-9-isopentyl-9H-
purine-2,6-diamine (4w)
Yield: 83%; M.p. = 94–97 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.56 (s(br), 1H), 8.05 (d, J = 8.0, 2H), 7.82 (s, 1H), 7.25 (d,
J = 8.0, 2H), 6.51 (s(br), 1H), 4.11–3.87 (m, 2H), 2.58–2.54 (m,
1H), 1.94 (m, 2H), 1.79 (m, 2H), 1.62 (m, 2H), 1.43 (n, J = 6.5, 1H),
1.31–1.08 (m, 5H), 0.86 (d, J = 6.5, 6H). 13C NMR (126 MHz,
DMSO d6)
d (ppm): 158.79, 152.60, 152.22, 140.12, 138.74,
128.52, 125.56, 121.79, 114.05, 50.47, 41.27, 38.79, 35.39, 31.73,
25.56, 22.73. HPLC-MS (ESI+): 427.90 (98.20%). Elemental analysis
Calcd. for C22H30ClN7 (427.23): C, 61.74; H, 7.07; N, 22.91. Found:
C, 61.55; H, 7.19; N, 22.73.
4.4.19. N2-(4-Aminocyclohexyl)-9-isopentyl-N6-phenyl-9H-purine-
2,6-diamine (4s)
Yield: 81%; M.p. = 150–152 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.33 (s(br), 1H), 7.98 (d, J = 7.8, 2H), 7.81 (s, 1H), 7.22 (t, J
= 7.8, 2H), 6.91 (t, J = 7.3, 1H), 6.44 (s, 1H), 4.01–3.93 (m, 2H),
3.65–3.53 (m, 1H), 2.55–2.51 (m, 1H), 1.93 (d, J = 11.0, 2H), 1.77
(d, J = 11.7, 2H), 1.63 (sex, J = 6.5, 2H), 1.44 (non, J = 6.5, 1H),
4.5. Cancer cell lines and cytotoxicity assay
Human cancer cell lines were obtained from the American Type
Culture Collection or the German Collection of Microorganisms and
Cell Cultures, and were cultivated according to the provider’s
instructions. Briefly, MCF-7, BT474 and K562 cell lines were main-
tained in DMEM medium supplemented with 10% fetal bovine
serum, penicillin (100 U/ml), and streptomycin (100 mg/ml).
HCC827 and EOL-1 cell lines were maintained in RPMI-1640 med-
ium supplemented with 20% fetal bovine serum, penicillin (100 U/
ml), and streptomycin (100 mg/ml). All cell lines were cultivated at
37 °C in 5% CO2. The cell lines display various alterations in tyro-
sine kinase genes: HCC827 has an acquired mutation in the EGFR
tyrosine kinase domain (E746-A750 deletion), BT474 has an ampli-
fication of EGFR, and EOL-1 and K562 express the fusion kinases
1.32–1.05 (m, 4H), 0.87 (d, J = 6.6, 6H).13
DMSO d6)
C
NMR (126 MHz,
d
(ppm): 158.86, 152.48, 141.01, 138.50, 128.74,
122.10, 120.40, 114.03, 50.45, 41.24, 38.79, 35.38, 32.54, 31.73,
25.54, 22.73. HPLC-MS (ESI+): 394.00 (98.82%). Elemental analysis
Calcd. for C22H31N7 (393.54): C, 67.15; H, 7.94; N, 24.91. Found: C,
66.98; H, 8.01; N, 24.83.
4.4.20. 3-((2-((4-Aminocyclohexyl)amino)-9-isopentyl-9H-purin-6-
yl)amino)phenol (4t)
Yield: 86%; M.p. = 144–145 °C. 1H NMR (500 MHz, DMSO d6) d
(ppm): 9.17 (s(br), 1H), 7.81 (s, 1H), 7.40 (d, J = 7.8, 1H), 6.97 (t, J
= 8.1, 1H), 6.34 (d, J = 7.8, 1H), 4.02–3.91 (m, 2H), 3.65–3.55 (m,
2H), 2.59 (s, 1H), 1.94–1.90 (m, 2H), 1.76 (d, J = 9.5, 2H), 1.62 (d,
J = 6.7, 2H), 1.44 (non, J = 6.6, 1H), 1.30–1.07 (m, 6H), 0.87 (d, J =
FIP1L1-PDGFR
a and BCR-ABL, respectively. For the cytotoxicity
assays, cells were treated in triplicate with six different doses of
each compound for 72 h. After treatment, Calcein AM solution
was added for 1 h, and the fluorescence from live cells was mea-
sured at 485 nm/538 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 that resulted from the assays.
6.6, 6H). 13C NMR (126 MHz, DMSO d6)
d (ppm): d 158.83,
157.88, 152.44, 152.44, 141.90, 138.50, 129.33, 114.01, 111.51,
109.52, 107.71, 57.12, 50.13, 33.65, 31.41, 29.53, 25.56, 22.73.
HPLC-MS (ESI+): 410.00 (98.80%). Elemental analysis Calcd. for
Please cite this article in press as: Malínková V., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.032

12
V. Malínková et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.6. Protein kinase inhibition
Acknowledgements
CDK2/Cyclin E was produced in Sf9 insect cells via baculoviral
infection and purified on a NiNTA column (Qiagen). PDGFR was
purchased from ProQinase. The kinase reactions were assayed with
The authors wish to acknowledge the institutional support of
the Ministry of Education, Youth and Sports of the Czech Republic
(National Program of Sustainability I, LO1204), the Ministry of
Health of the Czech Republic (15-28951A) and Palacky University
in Olomouc (IGA_PrF_2017_014).
a
suitable substrates (1 mg/mL AGLT (poly(Ala,Glu,Lys,Tyr) 6:2:5:1
hydrobromide) for PDGFR
the presence of 1 and 15 mM ATP for PDGFR
tively, 0.05 mCi [
33P]ATP, and the test compound in a final volume
of 10 mL, all in a reaction buffer (60 mM HEPES-NaOH, pH 7.5, 3
mM MgCl2, 3 mM MnCl2, 3 M Na-orthovanadate, 1.2 mM DTT,
2.5 g/50 l PEG20.000). The reactions were stopped by adding 5
a
and 1 mg/mL histone H1 for CDK2) in
a
and CDK2, respec-
c
-
A. Supplementary data
l
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2017.10.032.
l
l
mL of 3% aq. H3PO4. Aliquots were spotted onto P-81 phosphocellu-
lose (Whatman), washed 3ꢂ 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 kinase activity by 50% was determined from dose-
response curves and recorded as their IC50. The protein kinase
selectivity of compound 4e was preliminarily evaluated at a single
concentration (1 mM) by screening against 50 enzymes as
described previously.31
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4.7. Flow cytometry
Asynchronous cells were seeded into a 96 well plate and then,
after a preincubation period, treated with the tested compounds
for 24 h. Adherent cells were first washed with PBS, trypsinized,
and finally treated with a solution of trypsin inhibitor (0.1%). After
incubation, 5ꢂ staining solution (17 mM trisodium citrate dihy-
drate, 0.5% IGEPALÒ CA-630, 7.5 mM spermine tetrahydrochloride,
2.5 mM Tris; pH 7.6 containing 50 mg/ml propidium iodide) was
added. Leukemic cells were stained directly with the 5ꢂ staining
solution (i.e. without trypsinization). The cells’ DNA content was
analyzed by flow cytometry using a 488 nm laser (BD FACS Verse
with software BD FACSuiteTM, version 1.0.6.). Cell cycle distribution
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